Toxicology at Environmentally Relevant Concentrations in Caenorhabditis elegans 9789811667466, 9811667462

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Dayong Wang

Toxicology at Environmentally Relevant Concentrations in Caenorhabditis elegans

Toxicology at Environmentally Relevant Concentrations in Caenorhabditis elegans

Dayong Wang

Toxicology at Environmentally Relevant Concentrations in Caenorhabditis elegans

Dayong Wang Medical School Southeast University Nanjing, Jiangsu, China

ISBN 978-981-16-6745-9 ISBN 978-981-16-6746-6 https://doi.org/10.1007/978-981-16-6746-6

(eBook)

© The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Singapore Pte Ltd. The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721, Singapore

Preface

Whether the condition of environmentally relevant concentrations (ERCs) can open a new window for the field of toxicology? So far, most of the toxicity assessment data on environmental toxicants or stresses is obtained based on the exposure at high concentrations or doses. In addition, in most cases, the studies on molecular toxicology have also been performed for toxicants or stresses after exposure at high concentrations or doses. However, in the realistic environment, the exposure concentrations for environmental toxicants or pollutants are relatively low. The ERCs can refer to both the predicted environmental concentrations and the realistic concentrations of toxicants in the polluted environment. Thus, the establishment of knowledge system on the toxicology at ERCs (especially the molecular toxicology at ERCs) is necessary and urgent. Nematode Caenorhabditis elegans has been proven to be susceptible to the toxicity of various environmental toxicants or stresses. As a classic model animal, C. elegans can provide the powerful platform for the studies of both molecular toxicology and target organs toxicology at the whole animal level. Mainly based on previous studies on toxicants at high concentrations, the knowledge system on molecular toxicology organized by different signaling pathways has been raised in nematodes. These backgrounds made it possible to systematically perform study on the toxicology (especially the molecular toxicology) at ERCs in nematodes. In this book, we have raised these four important concerns: 1. Under what conditions the toxicity of toxicants at ERCs can be detected? 2. What are the molecular networks formed in different tissues in regulating the response to toxicants at ERCs? 3. What are the signaling communications among different tissues in regulating the response to toxicants at ERCs? 4. How are the molecular networks formed during the control of response to toxicants at ERCs? Based on these concerns, in Chap. 1, we introduced and discussed the conditions useful for detecting the toxicity of toxicants. In Chaps. 2–10, we introduced the v

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detailed signaling pathways and molecular regulations required for the control of response to toxicants at ERCs. In Chap. 11, we summarized the signaling networks formed in different tissues in regulating the response to toxicants at ERCs. In this chapter, we further raised several basic conclusions for the molecular response to toxicants at ERCs in nematodes. Nanjing, Jiangsu, China

Dayong Wang

Contents

1

Toxicity Induction of Toxicants at Environmentally Relevant Concentrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 Toxicity Induction by Long-Term Exposure to Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2.1 Toxicity of Toxicants at ERCs After Chronic Exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2.2 Toxicity of Toxicants at ERCs After Prolonged Exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3 Toxicity Induction of Toxicants at ERCs Under Oxidative Stress Condition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4 Toxicity Induction of Toxicants at ERCs Under Environmental Stress Condition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4.1 Toxicity Induction of Toxicants at ERCs Under Heat Stress Condition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4.2 Toxicity Induction of Toxicants at ERCs Under Simulated Microgravity Stress Condition . . . . . . . . . . . 1.5 Toxicity Induction of Toxicants at ERCs During the Aging Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.6 Toxicity Induction of Toxicants at ERCs Under Susceptible Genetic Backgrounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.7 Toxicity Induction of Toxicants at ERCs in Nematodes with Deficit in Intestinal Barrier . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.8 Toxicity Induction of Toxicants with Certain Surface Modifications at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.9 Toxicity Induced by Combinational or Sequential Exposure to Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.9.1 Toxicity Induced by Combinational Exposure to Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.9.2 Toxicity Induced by Sequential Exposure to Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1.10

Cellular Basis for the Toxicity Induction of Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.10.1 Activation of Oxidative Stress . . . . . . . . . . . . . . . . . . . 1.10.2 Enhancement in Intestinal Permeability . . . . . . . . . . . . 1.10.3 Suppression in Innate Immune Response . . . . . . . . . . . 1.10.4 Suppression in Mitochondrial Unfolded Protein Response (mt UPR) . . . . . . . . . . . . . . . . . . . . . . . . . . 1.10.5 Prolonged Defecation Cycle Length . . . . . . . . . . . . . . . 1.11 Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Response of Oxidative Stress-Related Molecular Signals to Toxicants at Environmentally Relevant Concentrations . . . . . . 2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Alteration in Molecular Basis for Oxidative Stress Induced by Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1 Alteration in Molecular Basis for Oxidative Stress Induced by Toxicants After Long-Term Exposure . . . 2.2.2 Alteration in Molecular Basis for Oxidative Stress Induced by Toxicants in Nematodes with Deficit in Intestinal Barrier . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.3 Alteration in Molecular Basis for Oxidative Stress Induced by Combinational Exposure to Different Toxicants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Oxidative Stress-Related Molecular Signals Regulating the Response to Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . 2.3.1 SOD-2 and SOD-3 . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.2 GST-5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.3 CLK-1 and ISP-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4 Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Response of Insulin Signaling Pathway to Toxicants at Environmentally Relevant Concentrations . . . . . . . . . . . . . . . . . . 3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Alteration in Expression of Genes Encoding Insulin Signaling Pathway Induced by Toxicants at ERCs . . . . . . . . . . . . . . . . . 3.3 Function of Insulin Signaling Pathway in Regulating the Toxicity of Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . 3.4 Tissue-Specific Activity of DAF-16 in Regulating the Toxicity of Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5 Identification of Downstream Targets of DAF-16 in Regulating the Toxicity of Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . 3.5.1 SOD-3, MTL-1, and GPB-2 . . . . . . . . . . . . . . . . . . . 3.5.2 LGG-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Insulin Peptides Involved in Regulating the Toxicity of Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7 Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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4

Response of MAPK Signaling Pathways to Toxicants at Environmentally Relevant Concentrations . . . . . . . . . . . . . . . . . . . 4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Response of p38 MAPK Signaling Pathway to Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.1 Alteration in Expression of Genes Encoding p38 MAPK Signaling Pathway Induced by Toxicants at ERCs . . . . 4.2.2 Functional Analysis of p38 MAPK in Regulating the Response to Toxicants at ERCs . . . . . . . . . . . . . . . . . . 4.2.3 Tissue-Specific Activity of PMK-1 in Regulating the Response to Toxicants at ERCs . . . . . . . . . . . . . . . . . . 4.2.4 Identification of Downstream Targets of PMK-1 in Regulating the Response to Toxicants at ERCs . . . . . . . 4.3 Response of JNK MAPK Signaling Pathway to Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.1 Alteration in Expression of Genes Encoding JNK MAPK Signaling Pathway Induced by Toxicants at ERCs . . . . 4.3.2 Functional Analysis of JNK MAPK Signaling Pathway in Regulating the Response to Toxicants at ERCs . . . . . 4.3.3 Identification of Downstream Targets for JNK-1 in Regulating the Response to Toxicants at ERCs . . . . . . . 4.4 Response of ERK MAPK Signaling Pathways to Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4.1 Alteration in Expression of Genes Encoding ERK MAPK Signaling Pathway Induced by Toxicants at ERCs . . . . 4.4.2 Functional Analysis of ERK MAPK Signaling Pathway in Regulating the Response to Toxicants at ERCs . . . . . 4.4.3 Tissue-Specific Activity of MPK-1/ERK MAPK in Regulating the Response to Toxicants at ERCs . . . . . . . 4.4.4 Identification of Downstream Targets of Neuronal MPK-1 in Regulating the Response to Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5 Genetic Interaction Between p38 MAPK Signaling Pathway and Insulin Signaling Pathway in Regulating the Toxicity of Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.6 Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

57 57 59 63 63 64 64 64 66 67 72 72 75 75 77 77 77 77

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Response of Development-Related Signaling Pathways to Toxicants at Environmentally Relevant Concentrations . . . . . . . . . . . . . . . . . 5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2 Response of Intestinal Development-Related Molecular Signals to Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.1 Alteration in Expression of Intestinal DevelopmentRelated Genes Induced by Toxicants at ERCs . . . . . . . 5.2.2 ELT-2 Signal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.3 ACS-22 Signal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.4 IFC-2 Signal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3 Response of Cell Death and DNA Damage-Related Signaling Pathways to Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . 5.4 Response of Wnt Signaling Pathway to Toxicants at ERCs . . . . 5.4.1 Alteration in Expression of Genes Encoding Wnt Signaling Pathway Induced by Toxicants at ERCs . . . . 5.4.2 Functional Analysis of BAR-1 and GSK-3 in Regulating the Response to Toxicants at ERCs . . . . . 5.4.3 Tissue-Specific Activity of BAR-1 in Regulating the Response to Toxicants at ERCs . . . . . . . . . . . . . . . . . . 5.4.4 Identification of Downstream Targets of Intestinal BAR-1 in Regulating the Response to Toxicants at ERCs . . . . . 5.5 Response of DBL-1-Mediated TGF-β Signaling Pathway to Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5.1 Alteration in Expression of DBL-1 Induced by Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . 5.5.2 Tissue-Specific Activity of DBL-1 in Regulating the Response to Toxicants at ERCs . . . . . . . . . . . . . . . . . . 5.5.3 Identification of Upregulators of DBL-1 in Regulating the Response to Toxicants at ERCs . . . . . . . . . . . . . . . 5.5.4 Identification of TGF-β Receptors in Regulating the Response to Toxicants at ERCs . . . . . . . . . . . . . . . . . . 5.5.5 Identification of Cytoplasmic Smads in Regulating the Response to Toxicants at ERCs . . . . . . . . . . . . . . . . . . 5.5.6 Identification of Transcriptional Factors in Regulating the Response to Toxicants at ERCs . . . . . . . . . . . . . . . 5.5.7 Identification of Downstream Targets of Intestinal SMA-9 and MAB-31 in Regulating the Response to Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . 5.6 Response of DAF-7-Mediated TGF-β Signaling Pathway to Toxicants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.6.1 Role of DAF-7 in Regulating the Response to Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.6.2 DAF-1, DAF-8, DAF-5, and DAF-3 Acted in the Intestine to Regulate the Response to Toxicants at ERCs . . . . . .

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Genetic Interaction DAF-12 and DAF-3 or DAF-5 in Regulating the Response to Toxicants at ERCs . . . . . . . 125 5.7 Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128 6

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Response of Metabolism-Related Signaling Pathways to Toxicants at Environmentally Relevant Concentrations . . . . . . . . . . . . . . . . . 6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2 Response of MDT-15-Mediated Signaling Pathway to Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2.1 Increase in Fat Storage . . . . . . . . . . . . . . . . . . . . . . . . 6.2.2 Alteration in Expression of MDT-15 and SBP-1 by Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2.3 Tissue-Specific Activity of MDT-15 and SBP-1 in Regulating the Response to Toxicants at ERCs . . . . . . . 6.2.4 Identification of Downstream Targets of Intestinal SBP-1 in Regulating the Response to Toxicants at ERCs . . . . . 6.3 Response of FAT-6-Mediated Signaling Pathway to Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4 Response of NHR-8-Mediated Signaling Pathway to Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4.1 Alteration in NHR-8 Expression by Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4.2 Functional Analysis of NHR-8 in Regulating the Response to Toxicants at ERCs . . . . . . . . . . . . . . . . . . 6.4.3 Identification of Downstream Targets of NHR-8 in Regulating the Response to Toxicants at ERCs . . . . . . . 6.5 Response of Heme Homeostasis-Related Signaling Pathway to Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5.1 Functional Analysis of GLB-10 in Regulating Toxicity of Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . 6.5.2 Downstream Neuronal Targets of GLB-10 in Regulating Toxicity of Toxicants at ERCs . . . . . . . . . . . . . . . . . . 6.5.3 Downstream Intestinal Targets of GLB-10 in Regulating Toxicity of Toxicants at ERCs . . . . . . . . . . . . . . . . . . 6.5.4 Interaction Between HRG-7 and HRG-5 in Controlling the Toxicity of Toxicants at ERCs . . . . . . . . . . . . . . . . 6.6 Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

133 133 134 134 135 135 137 139 139 139 141 142 147 147 147 149 150 153 154

Response of Protective Response-Related Signaling Pathways to Toxicants at Environmentally Relevant Concentrations . . . . . . . 159 7.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159 7.2 Response of mt UPR-Related Signaling Pathway to Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160

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7.2.1

Dynamic Alteration in mt UPR Induced by Toxicants at ERCs During the Aging . . . . . . . . . . . . . . . . . . . . . 7.2.2 Dynamic Alteration in mt UPR Induced by Toxicants at ERCs Under Simulated Microgravity Stress Condition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2.3 Activation of Intestinal mt UPR Response in Nematodes Exposed to Toxicants at ERCs . . . . . . . . . . . . . . . . . . 7.2.4 Tissue-Specific Activity of HSP-6 in Regulating the Response to Toxicants at ERCs . . . . . . . . . . . . . . . . . . 7.2.5 Identification of Upstream Regulators of Intestinal mt UPR Activation Induced by Toxicants at ERCs . . . . . . 7.2.6 Downstream Targets for Intestinal Insulin, Wnt, or ELT-2 Signaling in Controlling Intestinal mt UPR Activation Induced by Toxicants at ERCs . . . . . . . . . . . . . . . . . . 7.2.7 Interactions Among Intestinal ATFS-1, DVE-1, and UBL-5 in Controlling the Toxicity of Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3 Response of ER UPR-Related Signaling Pathway to Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3.1 Activation of ER UPR by Toxicants at ERCs . . . . . . . . 7.3.2 Upstream Regulators of ER UPR Activation Induced by Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . 7.4 Response of Antimicrobial Proteins to Toxicants at ERCs . . . . . 7.4.1 Alteration in Expressions of Antimicrobial Proteins by Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . 7.4.2 Upstream Regulators of Antimicrobial Protein Activation Induced by Toxicants at ERCs . . . . . . . . . . 7.4.3 Involvement of Antimicrobial Proteins in Regulating the Response to Toxicants at ERCs . . . . . . . . . . . . . . . 7.5 Response of Autophagy-Related Signaling Pathway to Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.5.1 Alteration in Autophagy Activation Induced by Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . 7.5.2 Upstream Regulators of Autophagy Activation Induced by Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . 7.5.3 Involvement of Autophagy-Related Signaling Pathway in Regulating the Response to Toxicants at ERCs . . . . . 7.6 Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

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Response of Neurotransmission-Related Molecular Signals to Toxicants at Environmentally Relevant Concentrations . . . . . . . . . 185 8.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185 8.2 Requirement of Neurotransmission to the Response to Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186

Contents

Genes Required for Neurotransmitter Biosynthesis or Transport Regulate the Response to Toxicants at ERCs . . . . . . . . . . . . . 8.3.1 Functions of TBH-1 and CAT-2 in Regulating Response to Toxicants at ERCs . . . . . . . . . . . . . . . . . 8.3.2 Functions of TDC-1 and EAT-4 in Regulating Response to Toxicants at ERCs . . . . . . . . . . . . . . . . . 8.4 Neurotransmitter Receptors Regulating the Response to Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.4.1 Intestinal Neurotransmitter Receptors Regulating the Response to Toxicants at ERCs . . . . . . . . . . . . . . . . . 8.4.2 Neuronal Neurotransmitter Receptors Regulating the Response to Toxicants at ERCs . . . . . . . . . . . . . . . . . 8.5 Downstream Signals of Neurotransmitter Receptors in Regulating the Response to Toxicants at ERCs . . . . . . . . . . 8.5.1 Downstream Signals of Intestinal Neurotransmitter Receptors in Regulating the Response to Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.5.2 Downstream Signals of Neuronal Neurotransmitter Receptors in Regulating the Response to Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.6 Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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8.3

9

Response of G Protein-Coupled Receptors and Ion Channels to Toxicants at Environmentally Relevant Concentrations . . . . . . 9.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2 Response of G Protein-Coupled Receptors to Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.1 Response of Intestinal G Protein-Coupled Receptors to Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . 9.2.2 Response of Neuronal G Protein-Coupled Receptors to Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . 9.2.3 Response of Germline G Protein-Coupled Receptors to Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . 9.3 Response of G Proteins to Toxicants at ERCs . . . . . . . . . . . . . 9.3.1 Response of Intestinal Gα Subunits to Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.3.2 Response of Neuronal Gα Subunits to Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.3.3 Response of Germline Gα Subunits to Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4 Response of Ion Channels to Toxicants at ERCs . . . . . . . . . . . 9.4.1 Response of Ion Channels to Toxicants at ERCs . . . . . 9.4.2 Ion Channels Involved in Controlling Toxicity of Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . .

. 187 . 187 . 190 . 193 . 193 . 196 . 199

. 199

. 199 . 201 . 202 . 207 . 207 . 208 . 208 . 215 . 223 . 229 . 229 . 237 . 245 . 248 . 249 . 250

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Contents

9.4.3

Intestinal Ion Channels Involved in Controlling Toxicity of Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . 9.4.4 Neuronal Ion Channels Involved in Controlling Toxicity of Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . 9.4.5 Targets for Intestinal and Neuronal Ion Channels in Controlling Toxicity of Toxicants at ERCs . . . . . . . . . . 9.5 Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Epigenetic Control of Response to Toxicants at Environmentally Relevant Concentrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2 miRNAs Control of Response to Toxicants at ERCs . . . . . . . . . 10.2.1 Response of miRNAs to Toxicants at ERCs . . . . . . . . . 10.2.2 Functional Analysis of miRNAs in Regulating the Response to Toxicants at ERCs . . . . . . . . . . . . . . . . . . 10.2.3 Molecular Basis for mir-35 in Regulating the Response to Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . 10.2.4 Molecular Basis for mir-794 in Regulating the Response to Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . 10.2.5 Molecular Basis for mir-354 in Regulating the Response to Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . 10.2.6 Molecular Basis for mir-38 in Regulating the Response to Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . 10.2.7 Molecular Basis for mir-76 in Regulating the Response to Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . 10.3 lncRNAs Control of Response to Toxicants at ERCs . . . . . . . . . 10.3.1 Response of lncRNAs to Toxicants at ERCs . . . . . . . . 10.3.2 Functional Analysis of lncRNAs in Regulating the Response to Toxicants at ERCs . . . . . . . . . . . . . . . . . . 10.3.3 Intestinal lncRNAs Required for the Control of Response to Toxicants at ERCs . . . . . . . . . . . . . . . . 10.3.4 Downstream Targets for Intestinal lncRNAs in Controlling the Response to Toxicants at ERCs . . . . . . 10.4 circRNAs Control of Response to Toxicants at ERCs . . . . . . . . 10.4.1 Response of circRNAs to Toxicants at ERCs . . . . . . . . 10.4.2 Functional Analysis of circRNAs in Regulating the Response to Toxicants at ERCs . . . . . . . . . . . . . . . . . . 10.4.3 Functional Analysis of circ_0000115 in Regulating the Response to Toxicants at ERCs . . . . . . . . . . . . . . . 10.5 Epigenetic Control of Response to Toxicants at ERCs by Histone Methylation-Related Signals . . . . . . . . . . . . . . . . . . 10.5.1 Response of Histone Methylation-Related Signals to Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . .

250 252 254 255 256 263 263 264 264 265 267 273 278 281 289 291 291 293 296 298 303 303 303 304 307 307

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10.5.2

Functional Analysis of MET-2 in Regulating the Response to Toxicants at ERCs . . . . . . . . . . . . . . . . . . 10.5.3 Tissue-Specific Activity of MET-2 in Regulating the Toxicity of Toxicants at ERCs . . . . . . . . . . . . . . . . . . 10.5.4 Targets for Intestinal MET-2 in Regulating the Toxicity of Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . 10.5.5 Targets for Germline MET-2 in Regulating the Toxicity of Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . 10.6 Epigenetic Control of Response to Toxicants at ERCs by Acetylation-Related Signals . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.6.1 Response of Acetylation-Related Signals to Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.6.2 Functional Analysis of Acetylation-Related Signals in Regulating the Response to Toxicants at ERCs . . . . . 10.6.3 Tissue-Specific Activity of CBP-1 in Regulating Toxicity of Toxicants at ERCs . . . . . . . . . . . . . . . . . . 10.6.4 Targets of Intestinal CBP-1 in Regulating Toxicity of Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . 10.6.5 Targets of Neuronal CBP-1 in Regulating Toxicity of Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . 10.6.6 Targets of Germline CBP-1 in Regulating Toxicity of Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . 10.7 Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Molecular Networks in Different Tissues in Response to Toxicants at Environmentally Relevant Concentrations . . . . . . . . . . . . . . . . 11.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2 Molecular Network in the Intestine in Regulating Response to Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2.1 Molecular Signaling Network in the Intestine in Response to Toxicants at ERCs . . . . . . . . . . . . . . . 11.2.2 G Protein-Coupled Receptor (GPCR)-Mediated Molecular Signaling Network in the Intestine in Response to Toxicants at ERCs . . . . . . . . . . . . . . . 11.2.3 Ion Channel-Mediated Molecular Signaling Network in the Intestine in Response to Toxicants at ERCs . . . 11.2.4 Epigenetic Control-Mediated Molecular Signaling Network in the Intestine in Response to Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.3 Molecular Network in the Neurons in Regulating Response to Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.3.1 Molecular Signaling Network in the Neurons in Response to Toxicants at ERCs . . . . . . . . . . . . . . .

308 309 310 310 313 313 313 314 316 316 317 321 322

. 329 . 329 . 330 . 330

. 333 . 335

. 336 . 338 . 338

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11.3.2

GPCR-Mediated Molecular Signaling Network in the Neurons in Response to Toxicants at ERCs . . . . . . . . . 11.3.3 Ion Channel-Mediated Molecular Signaling Network in the Neurons in Response to Toxicants at ERCs . . . . . 11.3.4 Epigenetic Control-Mediated Molecular Signaling Network in the Neurons in Response to Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.4 Molecular Network in the Germline in Regulating Response to Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.4.1 Molecular Signaling Network in the Germline in Response to Toxicants at ERCs . . . . . . . . . . . . . . . . . . 11.4.2 GPCR-Mediated Molecular Signaling Network in the Germline in Response to Toxicants at ERCs . . . . 11.4.3 Epigenetic Control-Mediated Molecular Signaling Network in the Germline in Response to Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.5 Signaling Communications Between Different Tissues in Regulating the Response to Toxicants at ERCs . . . . . . . . . . . . . 11.5.1 Signaling Communications Between Neurons and Intestine in Regulating the Response to Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.5.2 Signaling Communications Between Germline and Intestine in Regulating the Response to Toxicants at ERCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.5.3 Signaling Communications Between Different Neurons in Regulating the Response to Toxicants at ERCs . . . . . 11.6 Basic Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.7 Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

340 342

344 345 345 346

347 348

348

351 351 352 354 356

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359

Chapter 1

Toxicity Induction of Toxicants at Environmentally Relevant Concentrations

Abstract The environmentally relevant concentrations (ERCs) refer to both the predicted environmental concentrations and the realistic concentrations of toxicants in the polluted environment. Due to the sensitivity to environmental exposures, Caenorhabditis elegans has the potential to assess the toxicity of environmental toxicant at ERCs. We here introduced and discussed several possibilities to detect the toxicity of environmental toxicant at ERCs in nematodes. The cellular basis for the toxicity induction of toxicants at ERCs in nematodes was also discussed. Keywords Environmentally relevant concentrations · Toxicity assessment · Environmental exposure · Caenorhabditis elegans

1.1

Introduction

Caenorhabditis elegans is a classic model animal for life science. Meanwhile, C. elegans has been proven to be a powerful animal model for both toxicity assessment and toxicological study of environmental toxicants or stresses at the whole animal level [1–4]. C. elegans can provide the contribution to the studies of both molecular toxicology and target organ toxicology at the whole animal level [2, 3]. C. elegans has been widely used for toxicity assessment of different environmental toxicants, including heavy metals, organic pollutants, engineered nanomaterials (ENMs), and fine particulate matter (PM2.5) [5–18]. Meanwhile, C. elegans has also been frequently used for toxicity assessment of various stresses, such as heat stress, UV irradiation, simulated microgravity, and pathogen infection [19–28]. Due to the sensitivity to environmental exposures, C. elegans has the potential to detect the toxicity of environmental toxicant at environmentally relevant concentrations (ERCs) [29–35]. The ERCs refer to both the predicted environmental concentrations and the realistic concentrations of toxicants in the polluted environment. In this chapter, we first discussed under what conditions the toxicity of toxicants at ERCs can be potentially detected in nematodes. Moreover, we introduced and discussed the cellular basis for the toxicity induction of toxicants at ERCs in nematodes. © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 D. Wang, Toxicology at Environmentally Relevant Concentrations in Caenorhabditis elegans, https://doi.org/10.1007/978-981-16-6746-6_1

1

2

1.2

1 Toxicity Induction of Toxicants at Environmentally Relevant Concentrations

Toxicity Induction by Long-Term Exposure to Toxicants at ERCs

In nematodes, long-term exposures (such as prolonged exposure and chronic exposure) can be performed in order to assess the toxicity of environmental toxicants at low concentrations [1–4]. The detailed information for long-term exposures has been described in Chapter 1 of “Exposure Toxicology in Caenorhabditis elegans” (2020) [4].

1.2.1

Toxicity of Toxicants at ERCs After Chronic Exposure

1.2.1.1

Chronic Exposure from Adult Day-1 to Day-8

In nematodes, exposure to titanium dioxide nanoparticles (TiO2-NPs) could cause multiple aspects of toxicity [36–39]. The TiO2-NPs were selected as the example of environmental toxicants, and the nematodes were exposed to TiO2-NPs from adult Day-1 to Day-8 [39]. The predicted environmental concentrations of TiO2-NPs have been reported as 16 or 24.5 μg/L [40, 41]. Intestinal reactive oxygen species (ROS) production was selected as an endpoint [39]. After the chronic exposure, 1–100 μg/L TiO2-NPs (60 or 90 nm) could cause the significant induction of intestinal ROS production (Fig. 1.1) [39]. Moreover, chronic exposure to 0.01–100 μg/L TiO2-NPs

Fig. 1.1 Comparison of ROS production in nematodes acutely or chronically exposed to different sizes of TiO2-NPs (reprinted with permission from [39]). (a) Pictures showing the ROS production in nematodes acutely exposed to different sizes of TiO2-NPs. (b) Comparison of ROS production in nematodes acutely exposed to different sizes of TiO2-NPs. (c) Pictures showing the ROS production in nematodes chronically exposed to different sizes of TiO2-NPs. (d) Comparison of ROS production in nematodes chronically exposed to different sizes of TiO2-NPs. Bars represent mean
S.E. M. **p < 0.01

1.2 Toxicity Induction by Long-Term Exposure to Toxicants at ERCs

3

(4 or 10 nm) could induce the significant induction of intestinal ROS production (Fig. 1.1) [39]. These observations have indicated the potential of chronic exposure to TiO2-NPs at ERCs in inducing toxicity in nematodes.

1.2.1.2

Chronic Exposure from Adult Day-1 to Day-10

Exposure to heavy metals, such as chromium (Cr), could cause several aspects of toxicity in nematodes [42–47]. The Cr(VI) was selected as the example of environmental toxicants, and the nematodes were exposed to Cr(VI) from adult Day-1 to Day-10 [47]. It was reported that the naturally occurring Cr(VI) in the ground and the surface waters at values could exceed the World Health Organization limit for drinking water of 50 μg/L [48]. Intestinal ROS production was selected as one of the endpoints [47]. After the chronic exposure, 13–260 μg/L Cr(VI) could cause the significant induction of intestinal ROS production (Fig. 1.2) [47], which suggested the potential of chronic exposure to Cr(VI) at ERCs in causing toxicity in nematodes.

1.2.1.3

Chronic Exposure from L1-Larvae to Adult Day-8

Graphene oxide (GO) is a carbon-based ENM, and exposure to GO could cause multiple aspects of toxicity in nematodes [49–56]. GO was selected as the example of environmental toxicants, and the nematodes were exposed to GO from L1-larvae to adult Day-8 [56]. Lethality, locomotion behavior reflected by head thrash and body bend, intestinal autofluorescence, and intestinal ROS production were selected as the toxicity assessment endpoints [56]. After the chronic exposure, only 1 mg/L GO could induce the obvious lethality (Fig. 1.3) [56]. In contrast, chronic exposure to 10–1000 μg/L GO could cause the significant decrease in locomotion behavior, induction of intestinal autofluorescence, and induction of intestinal ROS production (Fig. 1.3) [56]. Considering that the environmental concentrations of nanomaterials are normally considered in the range of ng/L or μg/L, this observation implied the potential of chronic exposure to GO at ERCs in inducing the adverse effects in nematodes.

1.2.2

Toxicity of Toxicants at ERCs After Prolonged Exposure

1.2.2.1

Prolonged Exposure from L1-Larvae to Adult Day-1

Multi-walled carbon nanotubes also belong to carbon-based ENMs, and exposure to MWCNTs could cause the severe toxicity at various aspects in nematodes [57– 62]. The MWCNTs were selected as the example of environmental toxicants, and the nematodes were exposed to MWCNTs from L1-larvae to adult Day-1 [62]. Intestinal

4

1 Toxicity Induction of Toxicants at Environmentally Relevant Concentrations

Fig. 1.2 ROS production of nematodes exposed to Cr for 10 days (reprinted with permission from [47]). (a) Pictures showing the ROS production as detected by CM-H2DCFDA labeling in nematodes exposed to Cr for 10 days. (b) Comparison of ROS production in nematodes exposed to Cr at different concentrations for 10 days. Bars represent means
S.E.M. *p < 0.05; **p < 0.01

autofluorescence, reproduction reflected by the brood size, and locomotion behavior reflected by the head thrash and the body bend were selected as the toxicity assessment endpoints [62]. After the prolonged exposure, 10–1000 μg/L MWCNTs could cause the significant induction of intestinal autofluorescence (Fig. 1.4) [62]. Moreover, prolonged exposure to 0.1–1000 μg/L MWCNTs could even cause the significant reduction in brood size and decrease in locomotion behavior (Fig. 1.4) [62], which suggested that the prolonged exposure to MWCNTs in the range of ng/L could even cause the toxicity in nematodes.

1.2.2.2

Prolonged Exposure from L1-Larvae to Adult Day-3

In the recent several years, C. elegans has been frequently used to determine the toxicity and the underlying molecular mechanisms of nanoplastics, such as nanopolystyrene [63–76]. The predicted environmental concentrations of

1.2 Toxicity Induction by Long-Term Exposure to Toxicants at ERCs

5

Fig. 1.3 Chronic toxicity assessment of GO using two different assay systems (reprinted with permission from [56]). (a) Diagram of two assay systems for chronic GO exposure. (b) Effects of chronic GO exposure on survival of nematodes at the stage of adult Day-8. (c) Effects of chronic GO exposure on head thrash. (d) Effects of GO chronic exposure on body bend. (e) Effects of chronic GO exposure on intestinal autofluorescence. (f) Effects of chronic GO exposure on intestinal ROS production. GO exposure was performed from L1-larvae to adult Day-8 or from adult Day-1 to adult Day-8. Bars represent means
S.E.M. *p < 0.05, **p < 0.01

6

1 Toxicity Induction of Toxicants at Environmentally Relevant Concentrations

Fig. 1.4 Effects of MWCNTs and MWCNTs-PEG exposure from L1-larvae to adult at predicted environmental relevant concentrations on functions of the primary and secondary targeted organs in nematodes (reprinted with permission from [62]). (a) Pictures showing intestinal autofluorescences of MWCNTs and MWCNT-PEG-exposed nematodes. (b) Comparison of intestinal autofluorescences in MWCNT-PEG-exposed nematodes from those in MWCNT-exposed nematodes. (c) Comparison of brood sizes in MWCNT-PEG-exposed nematodes from those in MWCNT-exposed nematodes. (d) Comparison of head thrashes in MWCNT-PEG-exposed nematodes from those in MWCNT-exposed nematodes. (e) Comparison of body bends in MWCNTPEG-exposed nematodes from those in MWCNT-exposed nematodes. Bars represent mean
S.E. M. *p < 0.05, **p < 0.01

nanoplastics (100 nm) have been considered in the range 1 μg/L [77]. The nanopolystyrene (100 nm) was selected as the example of environmental toxicants [78]. Intestinal ROS production, locomotion behavior reflected by head thrash and body bend, and reproduction reflected by brood size were selected as the endpoints [78]. It was observed that prolonged exposure (from L1-larvae to adult Day-1) to nanopolystyrene (100 nm) at concentrations 10 μg/L could significantly induce the intestinal ROS production, decrease the locomotion behavior, and reduce the brood size [78]. Different from this, it was found that prolonged exposure (from L1-larvae to adult Day-3) to nanopolystyrene (100 nm) at concentrations 1 μg/L could further cause the severe toxicity in nematodes [79–81]. After the prolonged exposure from L1-larvae to adult Day-3, nanopolystyrene (100 nm) at concentrations 1 μg/L could result in the significant induction of intestinal ROS production and decrease in

1.3 Toxicity Induction of Toxicants at ERCs Under Oxidative Stress Condition

7

Fig. 1.5 Effects of nanopolystyrene exposure on wild-type nematodes (reprinted with permission from [81]). (a) Effects of nanopolystyrene exposure on induction of ROS production in wild-type nematodes. (b) Effects of nanopolystyrene exposure on locomotion behavior in wild-type nematodes. Nanopolystyrene exposure was performed from L1-larvae to adult Day-3. Bars represent means
SD. **p < 0.01 vs. control

locomotion behavior (Fig. 1.5) [81], which highlighted the potential of nanoplastic exposure at predicted ERCs in causing toxicity in nematodes.

1.3

Toxicity Induction of Toxicants at ERCs Under Oxidative Stress Condition

CeO2 nanoparticles (CeO2-NPs, 8.5 nm) were selected as the example of environmental toxicants, and the nematodes were exposed to CeO2-NPs from L1-larvae for 3 days [82]. Lifespan was selected as a toxicity assessment endpoint [82]. The juglone (600 μM) was used as an oxidative stress generator [82]. Under the oxidative stress condition, exposure to 5–100 nM CeO2-NPs could significantly reduce the lifespan of nematodes [82], which indicated the potential toxicity of CeO2-NPs at ERCs under oxidative stress condition in nematodes.

8

1.4 1.4.1

1 Toxicity Induction of Toxicants at Environmentally Relevant Concentrations

Toxicity Induction of Toxicants at ERCs Under Environmental Stress Condition Toxicity Induction of Toxicants at ERCs Under Heat Stress Condition

CeO2-NPs (8.5 nm) were further selected as the example of environmental toxicants, and the nematodes were exposed to CeO2-NPs from L1-larvae for 10 h at 35  C [82]. Viability was selected as a toxicity assessment endpoint [82]. Under the heat stress condition, exposure to 1–100 nM CeO2-NPs could also significantly reduce the viability [82], which suggested the potential toxicity of CeO2-NPs at ERCs under heat stress condition in nematodes.

1.4.2

Toxicity Induction of Toxicants at ERCs Under Simulated Microgravity Stress Condition

In the recent years, the simulated microgravity stress can be evaluated in nematodes [83–89]. The nanopolystyrene (30 nm) was selected as an example of environmental toxicants [67]. The young adult nematodes were exposed to nanopolystyrene (30 nm) for 24 h under the simulated microgravity stress condition [67]. The predicted environmental concentrations of nanopolystyrene (50 nm) have been considered in the range 15 μg/L [90]. Intestinal ROS production was selected as an endpoint [61]. In nematodes, mutation of sod-3 encoding a mitochondrial Mn-SOD could induce a susceptibility to the toxicity of environmental toxicants or stress [1– 3]. In wild-type nematodes, exposure to 1–100 μg/L nanopolystyrene (30 nm) could not induce the significant induction of intestinal ROS production in both wild-type and sod-3 mutant nematodes [67]. After the exposure, 1 μg/L nanopolystyrene (30 nm) did not affect the induction of intestinal ROS production in sod-3 nematodes treated with simulated microgravity stress (Fig. 1.6) [67]. Different from these, it was observed that 10 or 100 μg/L nanopolystyrene (30 nm) could obviously enhance the induction of intestinal ROS production in sod-3 nematodes treated with simulated microgravity stress (Fig. 1.6) [67]. These observations suggested the possible toxicity of nanopolystyrene at ERCs under the simulated stress condition in nematodes.

1.6 Toxicity Induction of Toxicants at ERCs Under Susceptible Genetic Backgrounds

9

Fig. 1.6 Toxicity of nanopolystyrene in inducing ROS production in wild-type or sod-3 mutant nematodes under microgravity stress condition (reprinted with permission from [67]). Nanopolystyrene exposure was performed from young adults for 24-h. “+”, addition and/or treatment; “–”, without addition and treatment. Control, without both nanopolystyrene exposure and microgravity treatment. Bars represent means
SD. **p < 0.01 vs. control (if not specially indicated)

1.5

Toxicity Induction of Toxicants at ERCs During the Aging Process

Nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [91]. Intestinal ROS production and locomotion behavior reflected by head thrash and body bend were selected as the toxicity assessment endpoints [91]. After the exposure, nanopolystyrene (100–1000 μg/L) could significantly reduce the lifespan [91]. In contrast, exposure to nanopolystyrene at concentration of 1 or 10 μg/L did not obviously affect the lifespan [91]. Nevertheless, 8 days after exposure (adult Day-11, during the aging), nanopolystyrene (1 or 10 μg/L) could cause the more severe induction of intestinal ROS production and decrease in locomotion behavior compared with controls (Fig. 1.7) [91]. These observations implied the toxicity induction of nanopolystyrene at ERCs during the aging process in nematodes.

1.6

Toxicity Induction of Toxicants at ERCs Under Susceptible Genetic Backgrounds

TiO2-NPs (10 nm) were selected as the example of environmental toxicants, and the nematodes were exposed to TiO2-NPs from L1-larvae to adult Day-1 [92]. Lethality, development reflected by body length, reproduction reflected by brood size, locomotion behavior reflected by head thrash and body bend, and intestinal autofluorescence were selected as the toxicity assessment endpoints [92]. Using lethality as the endpoint, exposure to 0.01–1 μg/L TiO2-NPs could induce the obvious lethality in sod-2, sod-3, or mtl-2 mutant nematodes (Fig. 1.8) [92]. Using

Fig. 1.7 Effect of nanopolystyrene exposure on intestinal ROS production and locomotion behavior during the aging process (reprinted with permission from [91]). (a) Effect of nanopolystyrene exposure on intestinal ROS production during the aging process. Bars represent means
SD. **p < 0.01 vs. control (if not specially indicated). (b) Effect of nanopolystyrene exposure on locomotion behavior during the aging process. Bars represent means
SD. **p < 0.01 vs. control. Day 0, adult Day-3. Nanopolystyrene exposure was performed from L1-larvae to adult Day-3

10 1 Toxicity Induction of Toxicants at Environmentally Relevant Concentrations

1.6 Toxicity Induction of Toxicants at ERCs Under Susceptible Genetic Backgrounds

11

Fig. 1.8 Toxicity assessment of TiO2-NPs using the strains with mutations of susceptible genes (reprinted with permission from [92]). (a) Toxicity assessment with the aid of lethality as endpoint. (b) Toxicity assessment with the aid of body length as endpoint. (c) Toxicity assessment with the aid of brood size as endpoint. (d) Toxicity assessment with the aid of head thrash as endpoint. (e) Toxicity assessment with the aid of body bend as endpoint. (f) Toxicity assessment with the aid of intestinal autofluorescence as endpoint. Bars represent means
S.E.M. **p < 0.01

body length as the endpoint, exposure to 0.01–1 μg/L TiO2-NPs could induce the significant reduction in body length in mtl-2 mutant nematodes (Fig. 1.8) [92]. Using brood size as the endpoint, exposure to 0.001–1 μg/L TiO2-NPs could induce the significant reduction in brood size in sod-2, sod-3, hsp-16.48, or mtl-2 mutant nematodes (Fig. 1.8) [92]. Using intestinal autofluorescence as the endpoint, exposure to 0.01–1 μg/L TiO2-NPs could cause the significant induction of intestinal autofluorescence in sod-2, sod-3, or mtl-2 mutant nematodes (Fig. 1.8) [92]. Using locomotion behavior as the endpoint, exposure to 0.0001–1 μg/L TiO2-NPs could further induce the significant decrease in locomotion behavior in sod-2, sod-3, hsp-16.48, or mtl-2 mutant nematodes (Fig. 1.8) [92]. That is, certain genetic mutation backgrounds can enhance the susceptibility of nematodes to exposure to toxicants at ERCs. Meanwhile, the sensitive endpoints (such as locomotion behavior) can further help us detect the potential toxicity of toxicants at low concentrations under certain genetic mutation backgrounds in nematodes.

12

1.7

1 Toxicity Induction of Toxicants at Environmentally Relevant Concentrations

Toxicity Induction of Toxicants at ERCs in Nematodes with Deficit in Intestinal Barrier

Nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-1 [93]. Mutation of acs-22 encoding a protein homologous to mammalian fatty acid transport protein can cause the deficit in functional state of intestinal barrier as indicated by the enhanced intestinal permeability [93]. Locomotion behavior reflected by head thrash and body bend and intestinal ROS production were selected as the endpoints [93]. Exposure to 10–100 μg/L nanopolystyrene could cause the significant decrease in locomotion behavior and induction of intestinal ROS production in wild-type nematodes (Fig. 1.9) [93]. In contrast, exposure to 1–100 μg/L nanopolystyrene could cause the significant decrease in locomotion behavior and induction of intestinal ROS production in acs-22 mutant nematodes (Fig. 1.9) [93]. Therefore, both mutation of genes required for the control of stress response and deficit in biological barriers (such as intestinal barrier) will enhance the susceptibility of nematodes to toxicity of toxicants at ERCs.

1.8

Toxicity Induction of Toxicants with Certain Surface Modifications at ERCs

Amino surface modification was firstly selected as the example of surface modifications [94]. Both pristine and amino modified nanopolystyrene particles (35 nm) were examined [94]. The nematodes were exposed to pristine or amino modified nanopolystyrene particles from L1-larvae to adult Day-1 [94]. Using number of total germline cells as the endpoint, the significant reduction in the number of total germline cells could be detected in nematodes exposed to 1–1000 μg/L pristine or amino modified nanopolystyrene (Fig. 1.10) [94]. Moreover, exposure to 1–1000 μg/L amino modified nanopolystyrene could cause the more severe reduction in the number of total germline cells than exposure to 1–1000 μg/L pristine nanopolystyrene in nematodes (Fig. 1.10) [94]. Sulfonate surface modification was selected another example of surface modifications [95]. Both pristine and sulfonate modified nanopolystyrene particles (35 nm) were examined [95]. The nematodes were exposed to pristine or sulfonate modified nanopolystyrene particles from L1-larvae to adult Day-3 [95]. The locomotion behavior reflected by head thrash, body bend, forward movement, and backward movement was selected as the toxicity assessment endpoint [95]. After the exposure, only 10–1000 μg/L pristine or sulfonate modified nanopolystyrene could significantly decrease the forward turns and increase the backward turns (Fig. 1.11) [95]. In contrast, exposure to 1–1000 μg/L pristine or sulfonate modified nanopolystyrene could significantly decrease both the head thrash and the body bend (Fig. 1.11) [95]. Moreover, exposure to 1–1000 μg/L sulfonate modified nanopolystyrene could

1.8 Toxicity Induction of Toxicants with Certain Surface Modifications at ERCs

13

Fig. 1.9 Effect of acs-22 mutation on toxicity of nanopolystyrene particles in nematodes after prolonged exposure (reprinted with permission from [93]). (a) Effect of acs-22 mutation on toxicity of nanopolystyrene particles in decreasing locomotion behavior. (b) Effect of acs-22 mutation on toxicity of nanopolystyrene particles in inducing intestinal ROS production. Two-way ANOVA was performed for the comparison between wild-type and acs-22 mutant. Prolonged exposure to nanopolystyrene particles was performed from L1-larvae to adult Day-1. Bars represent means
SD. **p < 0.01 vs. control (if not specially indicated)

cause the more severe decrease in head thrash or body bend than exposure to 1–1000 μg/L pristine nanopolystyrene (Fig. 1.11) [95]. Therefore, certain surface modifications (such as amino or sulfonate modification) can enhance the susceptibility of nematodes to the toxicity of toxicants at ERCs in nematodes.

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1 Toxicity Induction of Toxicants at Environmentally Relevant Concentrations

Fig. 1.10 Amino modification enhanced the reproductive toxicity on gonad development in nematodes (reprinted with permission from [94]). (a) DAPI staining results. n ¼ 50. (b) Comparison of effect of pristine and amino modified nanopolystyrene particles on the number of germline cells. n ¼ 50. (c) Comparison of effect of pristine and amino modified nanopolystyrene particles on the length of gonad arm. n ¼ 50. (d) Comparison of effect of pristine and amino modified nanopolystyrene particles on the relative area of gonad arm. n ¼ 50. Exposure to nanopolystyrenes was performed from L1-larvae to adult Day-1. Bars represent means
SD. **p < 0.01 vs. control (if not specially indicated)

1.9 1.9.1

Toxicity Induced by Combinational or Sequential Exposure to Toxicants at ERCs Toxicity Induced by Combinational Exposure to Toxicants at ERCs

Nanopolystyrene (100 nm) and microcystine-LR (MC-LR) were selected as the examples of environmental toxicants [96]. The concentrations of MCs in some marine environment were reported in the range 0.1–20 μg/L [29]. The combinational exposure to nanopolystyrene and MC-LR was performed from L1-larvae to adult Day-1 [96]. In nematodes, exposure to MC-LR (0.1–10 μg/L) could reduce the brood size and decrease the locomotion behavior, whereas exposure to nanopolystyrene (0.1 or 1 μg/L) could not alter the brood size and the locomotion

1.9 Toxicity Induced by Combinational or Sequential Exposure to Toxicants at ERCs

15

Fig. 1.11 Comparison of neurotoxicity on locomotion behaviors between pristine and sulfonate modified nanopolystyrene particles in nematodes (reprinted with permission from [95]). (a) Effects of pristine or sulfonate modified nanopolystyrene exposure on head thrash. (b) Effects of pristine or sulfonate modified nanopolystyrene exposure on body bend. (c) Effects of pristine or sulfonate modified nanopolystyrene exposure on forward movement. (d) Effects of pristine or sulfonate modified nanopolystyrene exposure on backward movement. Exposure was performed from L1-larvae to adult Day-3. Bars represent means
SD. **p < 0.01 vs. control (if not specially indicated)

behavior [96]. After the combinational exposure, the nanopolystyrene (0.1 μg/L) did not obviously affect the MC-LR (0.1 μg/L) toxicity in reducing the brood size and in inhibiting the locomotion behavior (Fig. 1.12) [96]. Exposure to nanopolystyrene (0.1 μg/L) also did not obviously influence the MC-LR (1 or 10 μg/L) toxicity in reducing the brood size and in inhibiting the locomotion behavior [96]. Different from these, exposure to nanopolystyrene (1 μg/L) could significantly increase the MC-LR (0.1, 1, or 10 μg/L) toxicity in reducing the brood size and in inhibiting the locomotion behavior in nematodes (Fig. 1.12) [96]. Nanopolystyrene (100 nm) and TiO2-NPs were further selected as the examples of environmental toxicants [97]. The combinational exposure to nanopolystyrene and TiO2-NPs was performed from L1-larvae to adult Day-1 [97]. Locomotion behavior reflected by head thrash and body bend and reproduction reflected by brood size were selected as the endpoints [97]. After the prolonged exposure, TiO2-NPs (1 μg/L) could significantly decrease the locomotion behavior and reduce the brood size, whereas nanopolystyrene (0.01, 0.1, or 1 μg/L) did not obviously affect both the locomotion behavior and the brood size (Fig. 1.13) [97]. In nematodes, exposure to nanopolystyrene (0.01 or 0.1 μg/L) did not significantly influence the toxicity of TiO2-NPs (1 μg/L) in decreasing the locomotion behavior and in

16

1 Toxicity Induction of Toxicants at Environmentally Relevant Concentrations

Fig. 1.12 Combinational effects between MC-LR and nanopolystyrene particles on locomotion behavior (a) and brood size (b) in nematodes (reprinted with permission from [96]). Prolonged exposure was performed from L1-larvae to adult Day-1. “+”, addition; “–”, without addition. Control, without MC-LR and nanopolystyrene particle exposure. Bars represent means
SD. ** p < 0.01 vs. control (if not specially indicated); NS, no significant difference

1.9 Toxicity Induced by Combinational or Sequential Exposure to Toxicants at ERCs

17

Fig. 1.13 Combinational effects of TiO2-NPs and nanopolystyrene particles on locomotion behavior (a) and brood size (b) in wild-type nematodes (reprinted with permission from [97]). Prolonged exposure was performed from L1-larvae to adult Day-1. Exposure concentration of TiO2-NPs was 1 μg/L. Control, without TiO2-NPs and nanopolystyrene particle exposure. Bars represent means
SD. **p < 0.01 vs. control (if not specially indicated); ns, no significant difference

18

1 Toxicity Induction of Toxicants at Environmentally Relevant Concentrations

reducing the brood size (Fig. 1.13) [97]. In contrast, exposure to nanopolystyrene (1 μg/L) could significantly enhance the toxicity of TiO2-NPs (1 μg/L) in decreasing the locomotion behavior (Fig. 1.13) [97]. Nevertheless, different from the combinational effect between TiO2-NPs (1 μg/L) and nanopolystyrene (1 μg/L) on locomotion behavior, it was found that exposure to nanopolystyrene (1 μg/L) did not obviously affect the toxicity of TiO2-NPs (1 μg/L) in reducing the brood size (Fig. 1.13) [97]. That is, the combinational exposure to nanopolystyrene can potentially enhance the toxicity of other toxicants at ERCs at least at certain aspects in nematodes.

1.9.2

Toxicity Induced by Sequential Exposure to Toxicants at ERCs

Nanopolystyrene (30 nm) was selected as the example of environmental toxicant, and fungal infection was selected as the example of environmental stress [98]. The nematodes were first infected with C. albicans for 4 h and then exposed to nanopolystyrene for 24 h [98]. Lifespan and locomotion behavior were used as the endpoints [98]. It was observed that C. albicans infection (4 h) could reduce the lifespan and decrease the locomotion behaviors, such as head thrash and body bend (Fig. 1.14) [98]. After the C. albicans infection, exposure to nanopolystyrene (0.1 or 1 μg/L) for 24 h did not affect both the lifespan and the locomotion behavior in C. albicans-infected nematodes (Fig. 1.14) [98]. Different from this, exposure to 10 μg/L nanopolystyrene for 24 h could cause the more severe reduction in lifespan and decrease in locomotion behavior in C. albicans-infected nematodes compared with C. albicans infection alone (Fig. 1.14) [98]. The 10 μg/L was the predicted environmental concentration of nanoplastics (30 nm) [77, 78]. Therefore, exposure to nanopolystyrene at ERCs potentially strengthened the adverse effects of fungal infection on nematodes.

1.10

Cellular Basis for the Toxicity Induction of Toxicants at ERCs

1.10.1 Activation of Oxidative Stress Nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-1 [83]. After the exposure, nanopolystyrene at concentrations 1 μg/L caused the significant induction of intestinal ROS production in acs-22 mutant nematodes with the deficit in functional state of intestinal barrier [99]. In nematodes, SKN-1 encoding an Nrf protein plays a pivotal role in regulating the oxidative stress [99],

1.10

Cellular Basis for the Toxicity Induction of Toxicants at ERCs

19

Fig. 1.14 Effect of nanopolystyrene exposure on lifespan (a) and locomotion behavior (b) of nematodes after fungal infection (reprinted with permission from [98]). The nematodes were first infected with C. albicans for 4 h and then exposed to nanopolystyrene for 24 h. Control, without both fungal infection and nanopolystyrene exposure. NP nanopolystyrene. Bars represent means
SD. **p < 0.01. Two-way ANOVA analysis followed by post hoc test was performed

and GST-4 encoding a putative glutathione-requiring prostaglandin D synthase acts as one of the direct targets of SKN-1 [100]. Meanwhile, it was observed that exposure to nanopolystyrene (1 μg/L) caused the obvious translocation of SKN-1:: GFP into the nucleus in acs-22 mutant nematodes (Fig. 1.15) [100], which was different from the expression pattern of SKN-1::GFP in acs-22 mutant nematodes under the normal conditions (Fig. 1.15) [93]. Additionally, exposure to nanopolystyrene (1 μg/L) also induced the significant increase in expressions of both SKN-1::GFP and GST-4::GFP in acs-22 mutant nematodes (Fig. 1.15) [93]. These observations suggested the association of oxidative stress activation with the toxicity induction of nanopolystyrene at ERCs in acs-22 mutant nematodes.

1.10.2 Enhancement in Intestinal Permeability Nanopolystyrene (100 nm) was further selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-1 [93]. After the exposure, nanopolystyrene (1 μg/L) could cause the significant induction of intestinal ROS production and decrease in locomotion behavior in acs-22 mutant nematodes with the deficit in functional state of intestinal

20

1 Toxicity Induction of Toxicants at Environmentally Relevant Concentrations

Fig. 1.15 Effect of acs-22 mutation on expressions of SKN-1::GFP (a) and GST-4::GFP (b) (reprinted with permission from [93]). Arrowheads indicate the distribution of SKN-1::GFP in the nucleus. Prolonged exposure to nanopolystyrene particles was performed from L1-larvae to adult Day-1. Exposure concentration of nanopolystyrene particles was 1 μg/L

barrier [93]. Using the technique of erioglaucine disodium (5.0% wt/vol in water, a blue dye) staining, it was also observed that the more severe dye leakage from the intestinal lumen into the intestinal cells and the body cavity could be observed in nanopolystyrene (1 μg/L)-exposed acs-22 mutant nematodes compared with those in acs-22 mutant nematodes under the control conditions (Fig. 1.16) [93], suggesting the enhancement in intestinal permeability. In control or nanopolystyrene (1 μg/L)exposed wild-type nematodes, the dye was mainly accumulated within the intestinal lumen (Fig. 1.16) [93]. At least partially due to this enhancement in intestinal permeability, not only the accumulation of nanopolystyrene in pharynx and in intestinal lumen but also the accumulation of certain amount of nanopolystyrene in gonad and in intestinal cells could be observed in nanopolystyrene (1 μg/L)-exposed acs-22 mutant nematodes [93]. In contrast, only a moderate accumulation of nanopolystyrene could be detected in the intestinal lumen in nanopolystyrene (1 μg/L)-exposed wild-type nematodes [93]. Therefore, the enhancement in intestinal permeability may contribute to the toxicity induction of nanopolystyrene at

1.10

Cellular Basis for the Toxicity Induction of Toxicants at ERCs

21

Fig. 1.16 Effect of acs-22 mutation on intestinal permeability (reprinted with permission from [93]). Arrowheads indicate the dye leakage from the intestinal lumen into the body cavity. The intestinal lumen (*) and the intestinal cells (**) were indicated by asterisks. Prolonged exposure to nanopolystyrene particles was performed from L1-larvae to adult Day-1. Exposure concentration of nanopolystyrene particles was 1 μg/L

ERCs and the increase in accumulation and translocation of nanopolystyrene in nematodes.

1.10.3 Suppression in Innate Immune Response Nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [91]. Exposure to 1 μg/L nanopolystyrene could cause the severe induction of intestinal ROS production and decrease in locomotion behavior compared with control during the aging process (at adult Day-11) in nematodes [91]. Meanwhile, it was observed that exposure to nanopolystyrene (1 μg/L) led to a more severe OP50::GFP accumulation in the intestine compared with control at 8 days after exposure (Fig. 1.17) [91]. Moreover, the expressions of some immune response genes were obviously altered by exposure to nanopolystyrene (1 μg/L) during the

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1 Toxicity Induction of Toxicants at Environmentally Relevant Concentrations

Fig. 1.17 Effects of nanopolystyrene exposure on OP50::GFP accumulation and expressions of immune response genes (reprinted with permission from [91]). (a) Effect of nanopolystyrene exposure on intestinal OP50::GFP accumulation. Exposure concentrations of nanopolystyrene were 1–10 g/L. Bars represent means
SD. **p < 0.01 vs. control (if not specially indicated). (b) Effect of exposure to nanopolystyrene (1 μg/L) on expressions of immune response genes. Bars represent means
SD. **p < 0.01 vs. control (Day 0). (c) Effect of exposure to nanopolystyrene (10 μg/L) on expressions of immune response genes. Bars represent means
SD. ** p < 0.01 vs. control (Day 0). Nanopolystyrene exposure was performed from L1-larvae to adult Day-3. Day 0, adult Day-3

aging process [91]. Eight immune response genes (lys-1, lys-7, lys-8, spp-1, spp-12, F55G11.4, dod-6, and dod-22) expressed in the intestine were used to reflect the alteration in intestinal innate immune response [52]. Among these genes, at adult Day-3 (Day 0 after the exposure), expressions of lys-1, lys-7, lys-8, and spp-1 were significantly increased by exposure to nanopolystyrene (1 μg/L) (Fig. 1.17) [91]. In control nematodes, the expressions of lys-1, lys-7, lys-8, and spp-1 were significantly decreased at 8 days after nanopolystyrene exposure (Fig. 1.17) [91]. Compared with the expression in nanopolystyrene (1 μg/L)-exposed nematodes at adult Day-3, the expressions of lys-1, lys-7, lys-8, and spp-1 were more severely decreased in nanopolystyrene (1 μg/L)-exposed nematodes at adult Day-11 (Fig. 1.17) [91]. That is, the observed toxicity induction of nanopolystyrene at ERCs during the aging process was largely due to the suppression in innate immune response in nematodes.

1.11

Perspectives

23

1.10.4 Suppression in Mitochondrial Unfolded Protein Response (mt UPR) Nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [91]. The mt UPR is an important protection strategy for nematodes against the toxicity of environmental toxicants or stresses [2–4]. HSP-6 was employed as a marker for mt UPR, and the expression of hsp-6 was significantly increased by exposure to nanopolystyrene (1 μg/L) at adult Day-3 [91]. At 8 days after exposure, the hsp-6 expression could be significantly inhibited in control nematodes [91]. Moreover, compared with the expression in nanopolystyrene (1 μg/L)-exposed nematodes at adult Day-3, the expression of hsp-6 was more severely decreased in nanopolystyrene (1 μg/L)-exposed nematodes at adult Day-11 [91]. Therefore, the suppression in mt UPR response may also contribute to the toxicity induction of nanopolystyrene at ERCs in nematodes.

1.10.5 Prolonged Defecation Cycle Length TiO2-NPs (10 nm) were selected as the example of environmental toxicants, and the nematodes were exposed to TiO2-NPs from L1-larvae to adult Day-1 [92]. The susceptibility of sod-2, sod-3, mtl-2, and hsp-16.48 mutant nematodes to the toxicity of TiO2-NPs in inducing lethality, in reducing body length, in reducing brood size, in decreasing locomotion behavior, in inducing intestinal autofluorescence, and/or in inducing intestinal ROS production could be detected [92]. Prolonged exposure to 1 μg/L TiO2-NPs could further increase the mean defecation cycle length in wildtype nematodes (Fig. 1.18) [92]. Moreover, the more severe increase in the mean defecation cycle length could be detected in 1 μg/L TiO2-NP-exposed sod-2, sod-3, mtl-2, or hsp-16.48 mutant nematodes compared with that in 1 μg/L TiO2-NPs exposed wild-type nematodes (Fig. 1.18) [92]. Therefore, both the enhancement in intestinal permeability and the increase in defecation cycle length contribute to the toxicity induction and accumulation of toxicants at ERCs in nematodes.

1.11

Perspectives

C. elegans has been shown to be a very sensitive animal model to assess the toxicity of environmental toxicants or stresses [1–4, 101–109]. In this chapter, we mainly introduced and discussed under what conditions the toxicity of toxicants at ERCs can be successfully assessed in nematodes. With the concern on this aspect, we discussed eight possibilities in nematodes. These possibilities are (1) toxicity induction by long-term exposure to toxicants, (2) toxicity induction of toxicants under

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1 Toxicity Induction of Toxicants at Environmentally Relevant Concentrations

Fig. 1.18 Pharyngeal pumping, defecation, and intestinal development in nematodes exposed to 1 μg/L of TiO2-NPs (reprinted with permission from [92]). (a) Pumping rate in nematodes. (b) Mean defecation cycle length in nematodes. (c) Nile Red staining results in nematodes. (d) Intestinal ROS production in nematodes. Bars represent means
S.E.M. **p < 0.01

oxidative stress condition, (3) toxicity induction of toxicants under environmental stress condition, (4) toxicity induction of toxicants during the aging process, (5) toxicity induction of toxicants under susceptible genetic backgrounds, (6) toxicity induction of toxicants in nematodes with deficit in intestinal barrier, (7) toxicity induction of toxicants with certain surface modifications, and (8) toxicity induced by combinational or sequential exposure to toxicants. That is, any possibilities to create the susceptible property will be useful to detect the possible toxicity of toxicants at ERCs in nematodes. In nematodes, for the cellular basis of toxicity induction of toxicants at ERCs, we discussed five cellular contributors. These five cellular contributors include the activation of oxidative stress, the enhancement in intestinal permeability, the suppression in innate immune response, the suppression in mt UPR, and the prolonged defecation cycle length. At least so far, no specific cellular contributors to the toxicity induction of toxicants at ERCs have been found in nematodes.

References

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Chapter 2

Response of Oxidative Stress-Related Molecular Signals to Toxicants at Environmentally Relevant Concentrations

Abstract Oxidative stress is an important cellular basis to toxicity induction of toxicants or stresses. Meanwhile, the oxidative stress-related signaling pathways play an important function in regulating the response to toxicants or stresses. In this chapter, we first introduced and discussed the alteration in molecular basis for oxidative stress by toxicants at environmentally relevant concentrations (ERCs) in nematodes. Moreover, we introduced and discussed the oxidative stress-related molecular signals involved in regulating the response to toxicants at ERCs in nematodes. Keywords Environmentally relevant concentrations · Oxidative stress-related molecular signals · Response · Caenorhabditis elegans

2.1

Introduction

Caenorhabditis elegans is a powerful animal model for toxicity assessment and toxicological study of different toxicants or stresses [1–12]. During the toxicity induction of environmental toxicants or stresses, activation of oxidative stress is usually an important cellular contributor in nematodes [13–18]. In Chapter 1 of “Molecular Toxicology in Caenorhabditis elegans,” we have introduced the molecular basis for oxidative stress induced by environmental toxicants or stresses in nematodes [2]. Moreover, in Chapter 3 of “Molecular Toxicology in Caenorhabditis elegans,” we have introduced the roles of oxidative stress-related molecular signals in the regulation of toxicity induced by environmental toxicants or stresses in nematodes [2]. In this chapter, we focus on the introduction and discussion of response of oxidative stress-related molecular signals to toxicants at environmentally relevant concentrations (ERCs) in nematodes. For this aim, we first introduced the alteration in molecular basis for oxidative stress induced by toxicants at ERCs. Again, we introduced the oxidative stress-related molecular signals required for the regulation of response to toxicants at ERCs.

© The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 D. Wang, Toxicology at Environmentally Relevant Concentrations in Caenorhabditis elegans, https://doi.org/10.1007/978-981-16-6746-6_2

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2.2 2.2.1

2 Response of Oxidative Stress-Related Molecular Signals to Toxicants at. . .

Alteration in Molecular Basis for Oxidative Stress Induced by Toxicants at ERCs Alteration in Molecular Basis for Oxidative Stress Induced by Toxicants After Long-Term Exposure

In nematodes, exposure to titanium dioxide nanoparticles (TiO2-NPs) can cause several aspects of toxicity [19–25]. The TiO2-NPs were selected as the example of environmental toxicants, and the nematodes were exposed to TiO2-NPs from L1-larvae to adult Day-1 [25]. Intestinal reactive oxygen species (ROS) production was employed as an endpoint to reflect the activation of oxidative stress [25]. After the exposure, the significant induction of intestinal ROS production could be detected in nematodes exposed to 4 or 10 nm TiO2-NPs at concentrations of 0.01–10 μg/L (Fig. 2.1) [25]. Exposure to 60 or 90 nm TiO2-NPs at concentrations of 1–10 μg/L also induced a significant induction of intestinal ROS production (Fig. 2.1) [25]. In nematodes, the activation of oxidative stress is associated with the functions of some genes, such as sod-1, sod-2, sod-3, sod-4, sod-5, ctl-1, ctl-2, ctl-3, clk-1, clk-2, isp-1, gas-1, and mev-1 [2]. Among these 13 genes examined, the expressions of sod-2 and sod-3 could be noticeably altered (Fig. 2.1) [25]. The expressions of sod-2 and sod-3 were significantly increased after exposure to 4, 10, 60, or 90 nm TiO2-NPs at the concentration of 10 μg/L (Fig. 2.1) [25]. With the increase in exposure concentrations of different diameters of TiO2-NPs, the expressions of sod-2 or sod-3 increased gradually compared with those in controls (Fig. 2.1) [25]. Additionally, the expressions of sod-2 and sod-3 were significantly increased by exposure to 4 or 10 nm TiO2-NPs at concentrations of 0.001–10 μg/L, as well as by exposure to 60 or 90 nm TiO2-NPs at concentrations of 1–10 μg/L (Fig. 2.1) [25]. These observations suggested the expressional alteration in genes encoding molecular basis of oxidative stress in nematodes exposed to TiO2-NPs at ERCs. In nematodes, sod-2 and sod-3 encode mitochondrial Mn-SODs.

2.2.2

Alteration in Molecular Basis for Oxidative Stress Induced by Toxicants in Nematodes with Deficit in Intestinal Barrier

In the recent years, C. elegans has been frequently used for toxicity assessment and toxicological study of nanoplastics (such as nanopolystyrene) at ERCs [26–49]. The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-1 [38]. In nematodes, acs-22 encodes a protein homologous to mammalian fatty acid transport protein, and mutation of acs-22 caused the deficit in functional state of intestinal barrier [38]. Intestinal ROS production was used as an endpoint to reflect

2.2 Alteration in Molecular Basis for Oxidative Stress Induced by Toxicants at. . .

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Fig. 2.1 Effects of different sizes of TiO2-NPs on ROS production and expression patterns of genes controlling oxidative stress in nematodes (reprinted with permission from [25]). (a, b) Comparison of ROS production in nematodes exposed to different sizes of TiO2-NPs. (c) Expression patterns of genes controlling oxidative stress in nematodes exposed to different sizes of TiO2NPs at the concentration of 10 μg/L. (d) Expression patterns of sod-2 gene in nematodes exposed to different sizes of TiO2-NPs. (e) Expression patterns of sod-3 gene in nematodes exposed to different sizes of TiO2-NPs. Relative expression ratios (between target genes and act-1 reference gene) in treatments were normalized to the control. Exposure of TiO2-NPs was performed from L1-larvae, and the endpoints were examined when nematodes developed into the adults. Ti-NPs, TiO2-NPs. Bars represent mean  S.E.M. *p < 0.05, **p < 0.01

the activation of oxidative stress [25]. The 1 μg/L is a predicted environmental concentration for nanopolystyrene, and exposure to nanopolystyrene at concentrations 1 μg/L could cause the significant induction of intestinal ROS production in nematodes [38]. To determine the mechanism for the observed induction of intestinal ROS production in nanopolystyrene-exposed acs-22 mutant nematodes, the effect of acs-22 mutation on molecular basis of oxidative stress was examined. Under the control conditions, mutation of acs-22 could not affect the transcriptional expressions of all the examined genes required for the control of oxidative stress

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2 Response of Oxidative Stress-Related Molecular Signals to Toxicants at. . .

Fig. 2.2 Effect of acs-22 mutation on expressions of genes required for the control of oxidative stress (reprinted with permission from [38]). Two-way ANOVA was performed for the comparison between wild-type and acs-22 mutant. The data was expressed the ratio between examined genes and reference tba-1 gene. Prolonged exposure to nanopolystyrene particles was performed from L1-larvae to adult Day-1. Exposure concentration of nanopolystyrene particles was 1 μg/L. Bars represent means  SD. **p < 0.01 vs. wild type

(Fig. 2.2) [38]. After the exposure to nanopolystyrene (1 μg/L), it was found that mutation of acs-22 could significantly increase the transcriptional expressions of sod-2, ctl-1, ctl-2, ctl-3, clk-1, isp-1, skn-1, and gst-4 (Fig. 2.2) [38]. In nematodes, sod-2 encodes a mitochondrial Mn-SOD, ctl-1, ctl-2, and ctl-3 encode catalases, clk1 encodes a demethoxyubiquinone hydroxylase, isp-1 encodes a subunit of mitochondrial complex III, skn-1 encodes a Nrf protein, and gst-4 encodes a putative glutathione-requiring prostaglandin D synthase (a direct target of SKN-1). That is, the expressional alteration in genes encoding molecular basis of oxidative stress could also be detected in nematodes with deficit in intestinal barrier after exposure to nanopolystyrene at ERCs.

2.2.3

Alteration in Molecular Basis for Oxidative Stress Induced by Combinational Exposure to Different Toxicants

One example is the combinational exposure to nanopolystyrene (100 nm) and microcystin-LR (MC-LR) [50]. The nematodes were subjected to the combinational exposure to nanopolystyrene and MC-LR from L1-larvae to adult Day-1 [50]. Intestinal ROS production was used as an endpoint to reflect the activation of oxidative stress [50]. After the exposure, nanopolystyrene (0.1 or 1 μg/L) did not cause the significant induction of intestinal ROS production (Fig. 2.3) [50]. Meanwhile, exposure to the nanopolystyrene (0.1 μg/L) could not affect the toxicity of MC-LR (0.1 μg/L) in inducing the intestinal ROS production (Fig. 2.3) [50]. Additionally, exposure to the nanopolystyrene (0.1 μg/L) also did not influence the toxicity of MC-LR (1 or 10 μg/L) in inducing the intestinal ROS production [50]. However, exposure to the nanopolystyrene (1 μg/L) could increase the induction of intestinal

2.2 Alteration in Molecular Basis for Oxidative Stress Induced by Toxicants at. . .

37

Fig. 2.3 Combinational effects between nanopolystyrene particles and MC-LR in inducing intestinal ROS production in nematodes (reprinted with permission from [50]). (a) Combinational effects between nanopolystyrene particles and MC-LR in inducing intestinal ROS production. “+”, addition; “–”, without addition. Semi-quantification of intestinal ROS signals was examined in comparison to autofluorescence. (b) Combinational effects between nanopolystyrene particles and MC-LR on expression of genes required for the control of oxidative stress. Prolonged exposure was performed from L1-larvae to adult Day-1. Control, without MC-LR and nanopolystyrene

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2 Response of Oxidative Stress-Related Molecular Signals to Toxicants at. . .

ROS production in MC-LR (0.1, 1, or 10 μg/L)-exposed nematodes (Figs. 2.3) [50]. The effect of combinational exposure to nanopolystyrene and MC-LR on molecular basis of oxidative stress was further examined. ISP-1, CLK-1, MEV-1, and GAS-1 are electron transport chain or mitochondrial complex components [2]. Catalases (CTL-1-3) and superoxide dismutases (SOD-1-5) can provide antioxidation defense system for nematodes against the oxidative stress [2]. Among the genes required for the control of oxidative stress, exposure to the MC-LR (0.1 μg/L) increased sod-3, sod-4, clk-1, isp-1, and ctl-1 expressions; however, exposure to nanopolystyrene (1 μg/L) did not affect expressions of all the examined genes (Fig. 2.3) [50]. Nevertheless, exposure to the nanopolystyrene (1 μg/L) could significantly enhance the increase in sod-3, sod-4, clk-1, isp-1, and ctl-1 expressions in MC-LR (0.1 μg/L)-exposed nematodes (Fig. 2.3) [50]. Moreover, exposure to the nanopolystyrene (1 μg/L) could even result in the significant increase in sod-2 and ctl-3 expressions in MC-LR (0.1 μg/L)-exposed nematodes (Fig. 2.3) [50]. Another example is the combinational exposure to nanopolystyrene (100 nm) and TiO2-NPs [51]. The combinational exposure to nanopolystyrene and TiO2-NPs was performed from L1-larvae to adult Day-1 [51]. Intestinal ROS production was used as an endpoint to reflect the activation of oxidative stress [51]. In nematodes, exposure to the nanopolystyrene (0.01 or 0.1 μg/L) could not noticeably affect the toxicity of TiO2-NPs (1 μg/L) in inducing the intestinal ROS production [51]. Different from this, exposure to the nanopolystyrene (1 μg/L) significantly enhanced the induction of intestinal ROS production in TiO2-NP (1 μg/L)-exposed nematodes [51]. The effect of combinational exposure to nanopolystyrene and TiO2-NPs on molecular basis of oxidative stress was further examined. Exposure to the TiO2-NPs (1 μg/L) significantly increased expressions of sod-2 and sod-3, whereas exposure to the nanopolystyrene (1 μg/L) did not alter expressions of all the examined genes (Fig. 2.4) [51]. Nevertheless, exposure to nanopolystyrene (1 μg/L) significantly enhanced sod-2 and sod-3 expressions in nematodes exposed to TiO2-NPs (1 μg/L) (Fig. 2.4) [51]. Using the transgenic strain carrying SOD-3::GFP, it was further observed that exposure to the nanopolystyrene (1 μg/L) could obviously strengthen the expression of SOD-3::GFP in nematodes exposed to TiO2-NPs (1 μg/L), although exposure to the nanopolystyrene (1 μg/L) alone did not affect the SOD-3::GFP expression (Fig. 2.4) [51]. In nematodes, skn-1 acts as a regulator for antioxidation or xenbiotic defense [52]. Using transgenic strain carrying SKN-1:: GFP, it was also found that exposure to the nanopolystyrene (1 μg/L) obviously enhanced the SKN-1::GFP expression and the SKN-1::GFP translocation into the nucleus in TiO2-NP (1 μg/L)-exposed nematodes (Fig. 2.4) [51]. Therefore, the combinational exposure to nanopolystyrene and other toxicant at ERCs potentially alter the molecular basis for oxidative stress in nematodes.

Fig. 2.3 (continued) particle exposure. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated); NS, no significant difference

2.2 Alteration in Molecular Basis for Oxidative Stress Induced by Toxicants at. . .

39

Fig. 2.4 Combinational exposure to TiO2-NPs and nanopolystyrene particles altered the molecular basis for oxidative stress in wild-type nematodes (reprinted with permission from [51]). (a) Combinational exposure to TiO2-NPs and nanopolystyrene particles altered transcriptional expressions of genes required for the control of oxidative stress. (b) Combinational exposure to TiO2-NPs and nanopolystyrene particles affected the expression of SOD-3::GFP. (c) Combinational exposure to TiO2-NPs and nanopolystyrene particles affected the expression of SKN-1::GFP. Arrowheads indicate the signal of SKN-1::GFP in the nucleus. Prolonged exposure was performed from L1-larvae to adult Day-1. Control, without TiO2-NPs and nanopolystyrene particles exposure. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated)

40

2.3 2.3.1

2 Response of Oxidative Stress-Related Molecular Signals to Toxicants at. . .

Oxidative Stress-Related Molecular Signals Regulating the Response to Toxicants at ERCs SOD-2 and SOD-3

In nematodes, SOD-2 and SOD-3 are mitochondrial Mn-SODs [2]. The TiO2-NPs were selected as the example of environmental toxicants, and the nematodes were exposed to TiO2-NPs from L1-larvae to adult Day-1 [20]. Reproduction reflected by brood size and locomotion behavior reflected by head thrash and body bend were used as the toxicity assessment endpoints [20]. In nematodes, exposure to 0.01 μg/L TiO2-NPs could cause the significant reduction in brood size and decrease in locomotion behavior in wild-type nematodes (Fig. 2.5) [20]. Moreover, it was observed that the more severe reduction in brood size and decrease in locomotion behavior could be detected in TiO2-NP-exposed sod-2 or sod-3 mutant nematodes compared with those in TiO2-NP-exposed wild-type nematodes (Fig. 2.5) [20], suggesting the susceptibility of sod-2 or sod-3 mutant nematodes to the toxicity of TiO2-NPs. These observations suggested the involvement of SOD-2 and SOD-3 signals in regulating the response of nematodes to TiO2-NPs at ERCs.

2.3.2

GST-5

Nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [33]. In nematodes, exposure (from L1-larvae to adult Day-3) to nanopolystyrene (100 nm) at concentrations 1 μg/L could cause the significant induction of intestinal ROS production and decrease in locomotion behavior reflected by head thrash and body bend [34]. Exposure to nanopolystyrene (1 μg/L) could further significantly increase the transcriptional expression of gst-5 [33]. The intestinal ROS production was used as a toxicity assessment endpoint [33]. Meanwhile, the more severe induction of intestinal ROS production could be detected in nanopolystyreneexposed nematodes with intestine-specific RNAi knockdown of gst-5 compared with that in nanopolystyrene-exposed VP303 nematodes [33], suggesting the susceptibility of nematodes with intestine-specific RNAi knockdown of gst-5 to the toxicity of nanopolystyrene. In nematodes, gst-5 encodes a glutathione-S-transferase. That is, the GST-5 is required for the control of response to nanopolystyrene at ERCs.

2.3 Oxidative Stress-Related Molecular Signals Regulating the Response to. . .

41

Fig. 2.5 Comparison of reproduction and locomotion behavior between wild-type and mutants exposed to 0.01 μg/L of TiO2-NPs (reprinted with permission from [20]). Bars represent means  S. E.M. **p < 0.01

2.3.3

CLK-1 and ISP-1

In nematodes, exposure to 2.5–10 mg/L CdTe quantum dots (CdTe QDs) from L1-larvae to young adult could cause the significant decrease in locomotion behavior

2 Response of Oxidative Stress-Related Molecular Signals to Toxicants at. . .

42

and induction of intestinal ROS production [53]. Meanwhile, exposure to 20 mg/L CdTe QDs induced the severe accumulation and translocation of CdTe QDs in the pharynx, intestine, gonad, and even the embryos in the body [53]. In contrast, lossof-function mutation of clk-1 or isp-1 could effectively prevent the toxicity induction of CdTe QDs (20 mg/L) in decreasing locomotion behavior and in inducing intestinal ROS production [53]. Moreover, intestine-specific RNAi knockdown of clk-1 or isp-1 could also effectively prevent the toxicity induction of CdTe QDs (20 mg/L) in inducing intestinal ROS production [53]. Similarly, loss-of-function mutation of clk-1 or isp-1 could effectively prevent the toxicity of exposure (from L1-larvae to young adults) to 100 mg/L graphene oxide (GO) in decreasing locomotion behavior and in inducing intestinal ROS production [54]. Considering that the mutation or intestine-specific RNAi knockdown of clk-1 or isp-1 could prevent the toxicity of CdTe QDs or GO at high concentrations, mutation or intestine-specific RNAi knockdown of clk-1 or isp-1 has the potential to prevent the toxicity of toxicants (such as CdTe QDs and GO) at ERCs.

2.4

Perspectives

C. elegans is an important animal model to determine the molecular basis of oxidative stress [2, 3, 55–62]. In this chapter, we first introduced the alteration in molecular basis for oxidative stress induced by toxicants at ERCs under several different conditions. The alteration in molecular basis for oxidative stress could be detected by toxicants at ERCs in nematodes after long-term exposure or with the deficit in functional state of intestinal barrier. Additionally, the alteration in molecular basis for oxidative stress could also be induced by combinational exposure to different toxicants at ERCs. Thus, the alteration in molecular basis for oxidative stress can be induced by toxicants at ERCs under different conditions in nematodes. Moreover, we discussed the involvement of some oxidative stress-related molecular signals in regulating the response to toxicants at ERCs. We here mainly discussed the roles of SOD-2, SOD-3, GST-5, CLK-1, and ISP-1. That is, both the signals mediating the protective response to oxidative stress and the signals required for the activation of oxidative stress were involved in the control of response to toxicants at ERCs. Surrounding this aspect, more efforts are still needed to identify the other potential oxidative stress-related genes (especially those in the mitochondrion) required for the control of response to toxicants at ERCs in nematodes.

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Chapter 3

Response of Insulin Signaling Pathway to Toxicants at Environmentally Relevant Concentrations

Abstract Insulin signaling pathway is a conserved and important signaling pathway involved in the control of response to toxicants or stresses. We first discussed the alteration in expression of genes encoding insulin signaling pathway induced by toxicants at environmentally relevant concentrations (ERCs). We further introduced and discussed the function and the tissue-specific activity of insulin signaling pathway in regulating the toxicity of toxicants at ERCs. Moreover, we introduced and discussed the downstream targets of DAF-16 and insulin peptides involved in regulating the toxicity of toxicants at ERCs. Keywords Environmentally relevant concentrations · Insulin signaling pathway · Response · Caenorhabditis elegans

3.1

Introduction

Caenorhabditis elegans is useful for the toxicological study of various environmental toxicants or stresses [1–13]. C. elegans is a powerful animal model for the study of molecular toxicology at the whole animal level [2, 14–25]. Among the signaling pathways, the insulin signaling pathway plays an important function in regulating the response to environmental toxicants or stresses in nematodes [26–35]. In Chapter 5 of “Molecular Toxicology in Caenorhabditis elegans,” we have introduced the function of insulin and the related signaling pathways in the regulation of toxicity of environmental toxicants or stresses in nematodes [2]. In the insulin signaling pathway, after binding by the insulin ligands, DAF-2/IGF-1 receptor (InR) will activate a kinase cascade of AGE-1-PDK-1-AKT-1/2-SGK-1, which will phosphorylate and inactivate the transcription factor DAF-16 [2]. Meanwhile, the DAF-18 can dephosphorylate the AGE-1 [2]. The insulin signaling pathway also plays an important function in regulating the response to toxicants at environmentally relevant concentrations (ERCs). We here focused on the introduction and the discussion of the role of insulin signaling pathway in regulating the response to toxicants at ERCs in nematodes.

© The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 D. Wang, Toxicology at Environmentally Relevant Concentrations in Caenorhabditis elegans, https://doi.org/10.1007/978-981-16-6746-6_3

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3.2

3

Response of Insulin Signaling Pathway to Toxicants at Environmentally. . .

Alteration in Expression of Genes Encoding Insulin Signaling Pathway Induced by Toxicants at ERCs

C. elegans can be employed as an animal model to detect the possible toxicity of nanopolystyrene at ERCs [36–46]. The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [47]. Exposure to nanopolystyrene at concentrations
1 μg/L could cause the significant induction of intestinal reactive oxygen species (ROS) production and decrease in locomotion behavior [47]. In the insulin signaling pathway, exposure to nanopolystyrene (1 μg/L, a predicted environmental concentration) decreased expressions of daf-2, age-1, and akt-1 and increased expression of daf-16 (Fig. 3.1) [47]. In addition, a significant increase in DAF-16:GFP expression in the nucleus was observed in nanopolystyrene (1 μg/L)exposed nematodes (Fig. 3.1) [47]. Under normal condition, the DAF-16:GFP is mainly located in the cytoplasm (Fig. 3.1) [47]. Thus, exposure to the nanopolystyrene (1 μg/L) may potentially alter both the transcriptional activities of

Fig. 3.1 Effects of nanopolystyrene exposure on the expression of genes encoding insulin signaling pathway in wild-type nematodes (reprinted with permission from [47]). (a) Nanopolystyrene exposure altered expression levels of some genes encoding insulin signaling pathway in wild-type nematodes. (b) Nanopolystyrene exposure influenced the nucleus translocation of DAF-16:GFP. Arrowheads indicate the nucleus translocation of DAF-16 in intestinal cells. Nanopolystyrene exposure concentration was 1 μg/L. Nanopolystyrene exposure was performed from L1-larvae to adult Day-3. Bars represent means  SD. **p < 0.01 vs. control

3.4 Tissue-Specific Activity of DAF-16 in Regulating the Toxicity of Toxicants. . .

49

genes encoding the insulin signaling pathway and the nucleus-cytoplasm translocation of DAF-16. The triadimenol was selected as another example of environmental toxicants [48]. After the chronic exposure, the triadimenol (300 μg/L) could also induce the significant increase in DAF-16:GFP expression in the nucleus [48], suggesting the induction of nucleus-cytoplasm translocation of DAF-16 in triadimenol (300 μg/L)exposed nematodes.

3.3

Function of Insulin Signaling Pathway in Regulating the Toxicity of Toxicants at ERCs

Mutation of daf-16 induced a more severe induction of intestinal ROS production and decrease in locomotion behavior compared with those in wild-type nematodes after nanopolystyrene (1 μg/L) exposure (Fig. 3.2) [47]. In contrast, mutation of daf2, age-1, or akt-1 could significantly suppress the induction of intestinal ROS production and the decrease in locomotion behavior induced by exposure to nanopolystyrene (1 μg/L) (Fig. 3.2) [47]. These observations suggested the involvement of insulin signaling in regulating the response to nanopolystyrene at ERCs. After the exposure, RNAi knockdown of daf-16 could significantly suppress the resistance of daf-2(e1370), age-1(hx546), or akt-1(ok525) mutant nematodes to the toxicity of nanopolystyrene in inducing the intestinal ROS production and in decreasing the locomotion behavior (Fig. 3.3) [47], suggesting the requirement of insulin signaling pathway during the control of response to nanopolystyrene at ERCs in nematodes.

3.4

Tissue-Specific Activity of DAF-16 in Regulating the Toxicity of Toxicants at ERCs

In nematodes, daf-16 is expressed in almost all tissues. After exposure to nanopolystyrene (1 μg/L), intestinal RNAi knockdown of daf-16 resulted in the more severe intestinal ROS production compared with that in VP303 nematodes (Fig. 3.4) [47]. In contrast, after exposure to nanopolystyrene (1 μg/L), epidermal, neuronal, muscle, or germline RNAi knockdown of daf-16 did not significantly affect the toxicity of nanopolystyrene (Fig. 3.4) [47]. These data suggest that DAF-16 acted in the intestinal cells to regulate the toxicity of nanopolystyrene at ERCs. Intestinal RNAi knockdown of daf-2, age-1, or akt-1 resulted in the resistance of nematodes to the toxicity of nanopolystyrene in inducing the intestinal ROS production (Fig. 3.4) [47], suggesting that DAF-2, AGE-1, and AKT-1 also acted in the intestinal cells to regulate the toxicity of nanopolystyrene at ERCs.

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Fig. 3.2 Effects of daf-2, daf-16, age-1, or akt-1 mutation on nematodes exposed to nanopolystyrene particles (reprinted with permission from [47]). (a) Effects of daf-2, daf-16, age1, or akt-1 mutation on induction of ROS production in nematodes exposed to nanopolystyrene particles. (b) Effects of daf-2, daf-16, age-1, or akt-1 mutation on locomotion behavior in nematodes exposed to nanopolystyrene particles. Nanopolystyrene exposure concentration was 1 μg/L. Nanopolystyrene exposure was performed from L1-larvae to adult Day-3. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated)

3.5 3.5.1

Identification of Downstream Targets of DAF-16 in Regulating the Toxicity of Toxicants at ERCs SOD-3, MTL-1, and GPB-2

daf-16 has many potential targeted genes during the control of various biological processes, and some of them can be expressed in the intestine (http://www. wormbase.org). Among these intestinal genes, exposure to nanopolystyrene particles (1 μg/L) only significantly increased expressions of sod-3, mtl-1, gpb-2, fat-7, and sodh-1 (Fig. 3.5) [47]. daf-16 mutation further significantly decreased the transcriptional expressions of sod-3, mtl-1, gpb-2, fat-7, and sodh-1 after exposure to nanopolystyrene (1 μg/L) (Fig. 3.5) [47]. Intestine-specific RNAi knockdown of sod-3, mtl-1, or gpb-2 induced a susceptibility to the toxicity of nanopolystyrene, whereas intestine-specific RNAi knockdown of fat-7 or sodh-1 did not obviously

3.5 Identification of Downstream Targets of DAF-16 in Regulating the Toxicity. . .

51

Fig. 3.3 Genetic interaction between DAF-16 and DAF-2, AGE-1, or AKT-1 in the regulation of toxicity caused by exposure to nanopolystyrene particles (reprinted with permission from [47]). (a) Genetic interaction between DAF-16 and DAF-2, AGE-1, or AKT-1 in the regulation of toxicity in inducing ROS production caused by exposure to nanopolystyrene particles. (b) Genetic interaction between DAF-16 and DAF-2, AGE-1, or AKT-1 in the regulation of toxicity in decreasing locomotion behavior caused by exposure to nanopolystyrene particles. Nanopolystyrene exposure concentration was 1 μg/L. Nanopolystyrene exposure was performed from L1-larvae to adult Day-3. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated)

affect the toxicity of nanopolystyrene (Fig. 3.5) [47]. These results suggest that sod3, mtl-1, and gpb-2 may act as the targeted genes of daf-16 in the regulation of toxicity of nanopolystyrene at ERCs. sod-3 encodes a manganese superoxide dismutase, mtl-1 encodes a metallothionein, and gpd-2 encodes a glyceraldehyde3-phosphate dehydrogenase. Moreover, after the exposure, RNAi knockdown of sod-3, mtl-1, or gpd-2 could significantly suppress the resistance of transgenic strain overexpressing intestinal DAF-16 to the toxicity of nanopolystyrene (Fig. 3.6) [47], which further conformed the role of SOD-3, MTL-1, and GPD-2 as the downstream targets of intestinal DAF-16 in regulating the toxicity of nanopolystyrene at ERCs.

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Fig. 3.4 Tissue-specific activity of genes in the insulin signaling pathway in the regulation of response to nanopolystyrene particles (reprinted with permission from [47]). (a) Tissue-specific activity of DAF-16 in the regulation of response to nanopolystyrene particles. (b) Intestine-specific activity of DAF-2, AGE-1, and AKT-1 in the regulation of response to nanopolystyrene particles. Nanopolystyrene exposure concentration was 1 μg/L. Nanopolystyrene exposure was performed from L1-larvae to adult Day-3. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated)

3.5.2

LGG-1

The nanopolystyrene (30 nm) was selected as the example of environmental toxicants [49]. After the exposure from adult Day-1 for 8-day, nanopolystyrene (30 nm) at concentrations
1 μg/L caused the significant decrease in locomotion behavior and induction of intestinal ROS production [49]. Autophagy induction mediates a protective response to environmental toxicants [2, 3], and adIs2122[LGG-1:: GFP + rol-6(su1006)] can be used a marker of autophagy induction. lgg-1 encodes an ortholog of Atg8/LC3 during the control of autophagy. After the exposure, at the

3.5 Identification of Downstream Targets of DAF-16 in Regulating the Toxicity. . .

53

Fig. 3.5 Identification of targeted genes for daf-16 in the regulation of response to nanopolystyrene particles (reprinted with permission from [47]). (a) Effects of exposure to nanopolystyrene particles on gene expression in wild-type nematodes. (b) Effects of daf-16 mutation on expression of intestinal targeted genes of daf-16 after exposure to nanopolystyrene particles. (c) Effect of intestine-specific RNAi knockdown of sod-3, mtl-1, gpb-2, fat-7, or sodh-1 on induction of ROS production in nematodes exposed to nanopolystyrene particles. Nanopolystyrene exposure concentration was 1 μg/L. Nanopolystyrene exposure was performed from L1-larvae to adult Day-3. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated)

adult Day-9, the LGG-1::GFP positive puncta was significantly increased compared with that in nematodes at adult Day-1 (Fig. 3.7) [49], suggesting the induction of autophagy at the adult Day-9. Moreover, exposure to 10 μg/L nanopolystyrene further significantly increased the LGG-1::GFP positive puncta compared with that

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Fig. 3.6 Genetic interaction between DAF-16 and its targets of SOD-3, MTL-1, and GPD-2 in the regulation of response to nanopolystyrene particles (reprinted with permission from [47]). (a) Genetic interaction between DAF-16 and its targets of SOD-3, MTL-1, and GPD-2 in regulating the toxicity in inducing ROS production caused by exposure to nanopolystyrene particles. (b) Genetic interaction between DAF-16 and its targets of SOD-3, MTL-1, and GPD-2 in regulating the toxicity in decreasing locomotion behavior caused by exposure to nanopolystyrene particles. (c) A model for the function of insulin signaling in regulating the response of animals to nanopolystyrene particles. Nanopolystyrene exposure concentration was 1 μg/L. Nanopolystyrene exposure was performed from L1-larvae to adult Day-3. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated)

in control nematodes at adult Day-9 (Fig. 3.7) [49]. RNAi knockdown of daf-2 significantly increased the LGG-1::GFP positive puncta in nanopolystyrene (10 μg/ L)-exposed nematodes at adult Day-9 (Fig. 3.7) [49]. In contrast, RNAi knockdown

3.5 Identification of Downstream Targets of DAF-16 in Regulating the Toxicity. . .

55

Fig. 3.7 Effect of nanopolystyrene exposure on autophagy induction in nematodes (reprinted with permission from [49]). (a) Autophagy induction reflected by the LGG-1::GFP positive puncta in intestinal cells. L4440, empty vector. Arrowheads indicate the LGG-1::GFP positive puncta. (b) Effect of 10 μg/L nanopolystyrene exposure on autophagy induction. Bars represent means  SD. ** p < 0.01 vs. control (if not specially indicated). (c) Effect of RNAi knockdown of daf-2 or daf-16 on autophagy induction in 10 μg/L nanopolystyrene-exposed nematodes. L4440, empty vector. Bars represent means  SD. **p < 0.01 vs. L4440. The nematodes were exposed to nanopolystyrene from adult Day-1 for 8 days

of daf-16 significantly decreased the LGG-1::GFP positive puncta in nanopolystyrene (10 μg/L)-exposed nematodes at adult Day-9 (Fig. 3.7) [49]. That is, RNAi knockdown of daf-2 enhanced the autophagy induction, whereas RNAi knockdown of daf-16 suppressed the autophagy induction in 10 μg/L nanopolystyrene-exposed nematodes. Therefore, LGG-1 acted as a downstream target of DAF-2-DAF-16 signaling cascade during the control of toxicity of nanopolystyrene at ERCs in nematodes. In nematodes, RNAi knockdown of lgg-1 caused the more severe induction of ROS production and decrease in locomotion behavior in nanopolystyrene (10 μg/L)exposed nematodes compared with those in nanopolystyrene (10 μg/L)-exposed wild-type nematodes at adult Day-9 (Fig. 3.8) [49], suggesting the susceptibility of lgg-1(RNAi) nematodes to nanopolystyrene toxicity on aging-related endpoints. Moreover, it was observed that RNAi knockdown of lgg-1 could further cause the significant induction of ROS production and decrease in locomotion behavior in nanopolystyrene (10 μg/L)-exposed Is(Pges-1-daf-16) nematodes overexpressing intestinal DAF-16 (Fig. 3.8) [49]. Therefore, DAF-16 was confirmed to act upstream of LGG-1, an ortholog of Atg8/LC3, to regulate the toxicity of nanopolystyrene at ERCs at adult Day-9 in nematodes.

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Fig. 3.8 Genetic interaction between LGG-1 and DAF-16 in regulating the nanopolystyrene toxicity in nematodes (reprinted with permission from [49]). (a) Genetic interaction between LGG-1 and DAF-16 in regulating the nanopolystyrene toxicity in inducing ROS production. The nematodes were exposed to nanopolystyrene from adult Day-1 for 8 days. Bars represent means  SD. **p < 0.01 vs. wild-type (if not specially indicated). (b) Genetic interaction between LGG-1 and DAF-16 in regulating the nanopolystyrene toxicity in decreasing locomotion behavior. The nematodes were exposed to nanopolystyrene from adult Day-1 for 8 days. Bars represent means  SD. **p < 0.01 vs. wild type (if not specially indicated). (c) A diagram showing the role of insulin signaling pathway in regulating the autophagy induction and nanopolystyrene toxicity in nematodes

3.7 Perspectives

3.6

57

Insulin Peptides Involved in Regulating the Toxicity of Toxicants at ERCs

The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [50]. Among the genes (ins-1, ins-4, ins-7, ins-39, and daf-28) encoding neuronal insulin peptides, exposure to nanopolystyrene (1 μg/L) could significantly decrease the expressions of ins-4, ins-39, and daf-28 [50]. Meanwhile, under the TU3401 background, the decrease in intestinal ROS production and the increase in locomotion behavior could be observed in nanopolystyrene-exposed nematodes with neuronal RNAi knockdown of ins-4, ins-39, or daf-28 compared with those in nanopolystyrene-exposed TU3401 nematodes, suggesting that the neuronal RNAi knockdown of ins-4, ins-39, or daf-28 induced a resistance to nanopolystyrene toxicity [50]. Moreover, in nanopolystyrene-exposed nematodes, the neuronal RNAi knockdown of mpk-1 encoding ERK MAPK in ERK MAPK signaling pathway could only significantly increase the expressions of ins-4, ins-39, and daf28 [50]. These observations implied the potential role of INS-4, INS-39, and DAF-28 as the downstream targets of neuronal MPK-1 in regulating the response to nanopolystyrene at ERCs in nematodes. In nematodes, DAF-2 acted in the intestine to regulate the response to nanopolystyrene by suppressing the DAF-16 function [47]. After the nanopolystyrene exposure, it was further found that neuronal RNAi knockdown of ins-4, ins-39, or daf-28 could significantly decrease the intestinal daf-2 expression (Fig. 3.9) [50]. Moreover, the neuronal RNAi knockdown of ins-4, ins-39, or daf-28 could significantly increase the intestinal daf-16 expression in nanopolystyreneexposed nematodes (Fig. 3.9) [50]. These observations suggested that the neuronal ERK MAPK signaling pathway mediated the protective response to nanopolystyrene by modulating insulin signaling-mediated communication between neurons and intestine in nematodes (Fig. 3.9) [50]. That is, the decrease in expressions of INS-4, INS-39, and DAF-28 activated the communication between neurons and intestine in nanopolystyrene-exposed nematodes (Fig. 3.9) [50].

3.7

Perspectives

C. elegans has been shown to be very sensitive to different environmental toxicants or stresses [1–4, 51–59]. The response of insulin signaling pathway to environmental toxicants (such as nanopolystyrene) at ERCs could be observed in nematodes. During the response to environmental toxicants at ERCs, the insulin receptor DAF-2 in intestinal cells could be suppressed, which suggested that the signaling cascade in insulin signaling pathway may be modulated by environmental toxicants at ERCs via binding to the insulin receptor in intestinal cells in nematodes. Nevertheless, it was found that only the signaling cascade of DAF-2-AGE-1-

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Fig. 3.9 Effect of neuronal RNAi knockdown of ins-4, ins-39, or daf-28 on expressions of daf-2 and daf-16 in nanopolystyrene-exposed nematodes (reprinted with permission from [50]). (a) Effect of neuronal RNAi knockdown of ins-4, ins-39, or daf-28 on intestinal daf-2 expression in nanopolystyrene-exposed nematodes. qRT-PCR was performed in isolated intact intestines (n ¼ 40). L4440, empty vector for RNAi. Exposure concentration of nanopolystyrene was 1 μg/ L. Exposure was performed from L1-larvae to adult Day-3. Bars represent means  SD. ** p < 0.01 vs. TU3401. (b) Effect of neuronal RNAi knockdown of ins-4, ins-39, or daf-28 on intestinal daf-16 expression in nanopolystyrene-exposed nematodes. qRT-PCR was performed in isolated intact intestines (n ¼ 40). L4440, empty vector for RNAi. Exposure concentration of nanopolystyrene was 1 μg/L. Exposure was performed from L1-larvae to adult Day-3. Bars represent means  SD. **p < 0.01 vs. TU3401. (c) A diagram showing the molecular basis for neuronal ERK MAPK signaling in regulating the response to nanopolystyrene in nematodes

AKT-1-DAF-16 in the insulin signaling pathway responded to the nanopolystyrene at ERCs. This observation implied that exposure to toxicants at ERCs may induce a different signaling cascade in insulin signaling pathway from that in nematodes exposed to high concentrations of toxicant. During the control of response to nanoplastic exposure at ERCs, at least SOD-3, MTL-1, GPB-2, and LGG-1 were identified as the downstream targets of FOXO transcriptional factor DAF-16 in the insulin signaling pathway. Although we still do not exclude the existence of other downstream targets of DAF-16 in regulating the response to toxicants at ERCs, only very limited downstream targets may be required to be activated or inhibited for DAF-16 in regulating the response to toxicants at ERCs in nematodes. More recently, it was found that the intestinal DAF-16 could

References

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also regulate the toxicity of nanopolystyrene at ERCs by affecting the molecular basis for mitochondrial unfolded protein response (mt UPR) in nematodes [25]. Moreover, some insulin peptides (INS-4, INS-39, and DAF-28) have been identified to be altered by toxicants at ERCs. More importantly, these insulin peptides could act in the neurons to regulate the response to toxicant ERCs. Meanwhile, the signaling cascade of DAF-2-AGE-1-AKT-1-DAF-16 acted in the intestine to regulate the response to toxicants at ERCs. Therefore, during the control of response to toxicants at ERCs, the insulin signaling pathway at least mediated an important neuron-intestine communication in nematodes. More recently, it was found that the insulin peptide of DAF-28 could also act in the germline to regulate the toxicity of nanopolystyrene at ERCs by mediating an important germlineintestine communication in nematodes [24].

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48. How, C. M., Li, S., & Liao, V. H. (2018). Chronic exposure to triadimenol at environmentally relevant concentration adversely affects aging biomarkers in Caenorhabditis elegans associated with insulin/IGF-1 signaling pathway. Science of the Total Environment, 640–641, 485–492. 49. Qiu, Y.-X., Liu, Y.-Q., Li, Y.-H., Li, G.-J., & Wang, D.-Y. (2020). Effect of chronic exposure to nanopolystyrene on nematode Caenorhabditis elegans. Chemosphere, 256, 127172. 50. Qu, M., Li, D., Qiu, Y.-X., & Wang, D.-Y. (2020). Neuronal ERK MAPK signaling in response to low-dose nanopolystyrene exposure by suppressing insulin peptide expression in Caenorhabditis elegans. Science of the Total Environment, 724, 138378. 51. Sun, L.-M., Li, D., Yuan, Y.-J., & Wang, D.-Y. (2021). Intestinal long non-coding RNAs in response to simulated microgravity stress in Caenorhabditis elegans. Scientific Reports, 11, 1997. 52. Wang, S.-T., Zhang, R.-J., & Wang, D.-Y. (2021). Induction of protective response to polystyrene nanoparticles associated with methylation regulation in Caenorhabditis elegans. Chemosphere, 271, 129589. 53. Liu, H.-L., Qiu, Y.-X., & Wang, D.-Y. (2021). Alteration in expressions of ion channels in Caenorhabditis elegans exposed to polystyrene nanoparticles. Chemosphere, 273, 129686. 54. Zhao, Y.-Y., Xu, R.-R., Chen, X., Wang, J., Rui, Q., & Wang, D.-Y. (2021). Induction of protective response to polystyrene nanoparticles associated with dysregulation of intestinal long non-coding RNAs in Caenorhabditis elegans. Ecotoxicology and Environmental Safety, 212, 111976. 55. Liu, H.-L., Zhao, Y.-Y., Bi, K., Rui, Q., & Wang, D.-Y. (2021). Dysregulated mir-76 mediated a protective response to nanopolystyrene by modulating heme homeostasis related molecular signaling in nematode Caenorhabditis elegans. Ecotoxicology and Environmental Safety, 212, 112018. 56. Yang, Y.-H., Dong, W.-T., Wu, Q.-L., & Wang, D.-Y. (2021). Response of G protein-coupled receptor CED-1 in germline to polystyrene nanoparticles in Caenorhabditis elegans. Nanoscale Advances, 3, 1997–2006. 57. Yang, Y.-H., Dong, W.-T., Wu, Q.-L., & Wang, D.-Y. (2021). Induction of protective response associated with expressional alterations in neuronal G protein-coupled receptors in polystyrene nanoparticle exposed Caenorhabditis elegans. Chemical Research in Toxicology, 34, 1308. https://doi.org/10.1021/acs.chemrestox.0c00501 58. Yang, Y.-H., Wu, Q.-L., & Wang, D.-Y. (2021). Dysregulation of G protein-coupled receptors in the intestine by nanoplastic exposure in Caenorhabditis elegans. Environmental Science. Nano, 8, 1019–1028. 59. Wang, C., Chen, H., Li, H., Zhang, Y., Ren, L., Chen, C., Wang, X., Yu, J., Li, Z., & Liu, Y. (2020). Tris(1,2-dichloro-2-propyl)phosphate reduces the lifespan via activation of an unconventional insulin/insulin-like growth factor-1 signaling pathway. Environmental Science & Technology, 54, 10783–10796.

Chapter 4

Response of MAPK Signaling Pathways to Toxicants at Environmentally Relevant Concentrations

Abstract The p38 mitogen-activated protein kinase (MAPK), extracellular signalregulated kinase (ERK) MAPK, and c-Jun N-terminal kinase (JNK) MAPK signaling pathways have been frequently found to be involved in the control of toxicity of various environmental toxicants or stresses. We here focused on introduction and discussion on the response of these three MAPK signaling pathways to toxicants at environmentally relevant concentrations (ERCs) in nematodes. The obtained data suggested the role of signaling cascade of p38 MAPK signaling pathway in the intestine to regulate the response to toxicants at ERCs. Moreover, both ERK MAPK signaling pathway and JNK MAPK signaling pathway mediated important neuronintestine communication in regulating the response to toxicants at ERCs. Keywords Environmentally relevant concentrations · p38 MAPK signaling pathway · ERK MAPK signaling pathway · JNK MAPK signaling pathway · Caenorhabditis elegans

4.1

Introduction

The model animal of nematode Caenorhabditis elegans is a powerful model for the toxicological study of environmental toxicants or stresses [1–12]. Especially, due to the well-described genetic and molecular backgrounds, C. elegans is a useful animal model for the molecular toxicology at the whole animal level [1–3, 13–26]. In Chapter 4 of “Molecular Toxicology in Caenorhabditis elegans,” we have introduced and discussed the functions of mitogen-activated protein kinase (MAPK) signaling pathways in the regulation of toxicity of environmental toxicants or stresses in nematodes [2]. The MAPK signaling pathways mainly contain p38 MAPK, extracellular signal-regulated kinase (ERK) MAPK, and c-Jun N-terminal kinase (JNK) MAPK signaling pathways. In nematodes, the p38 MAPK signaling pathway was involved in the control of response to different environmental toxicants or stresses [27–31]. The ERK MAPK signaling pathway and the JNK MAPK signaling pathway were also required for the control of response to environmental toxicants, such as graphene oxide (GO), in nematodes [32–34]. In this chapter, we © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 D. Wang, Toxicology at Environmentally Relevant Concentrations in Caenorhabditis elegans, https://doi.org/10.1007/978-981-16-6746-6_4

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further introduced and discussed the involvement of these three MAPK signaling pathways in regulating the response to toxicants at environmentally relevant concentrations (ERCs) in nematodes.

4.2 4.2.1

Response of p38 MAPK Signaling Pathway to Toxicants at ERCs Alteration in Expression of Genes Encoding p38 MAPK Signaling Pathway Induced by Toxicants at ERCs

C. elegans has been frequently used as an animal model to assess the possible toxicity of nanopolystyrene at ERCs [35–45]. The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [46]. Exposure to nanopolystyrene at concentrations 1 μg/L caused the significant induction of intestinal reactive oxygen species (ROS) production and decrease in locomotion behavior [45]. After the exposure, the nanopolystyrene (1 μg/L, a predicted environmental concentration) induced a significant increase in transcriptional expression of pmk-1, and the expression of pmk-1 was concentration dependent (Fig. 4.1) [46]. Exposure to nanopolystyrene (1 μg/L) significantly increased the phosphorylation level of PMK-1 (Fig. 4.1) [46]. Thus, exposure to nanopolystyrene at ERCs potentially altered both the transcriptional expressions of pmk-1 and the phosphorylation level of PMK-1.

4.2.2

Functional Analysis of p38 MAPK in Regulating the Response to Toxicants at ERCs

After the exposure, the more severe induction of intestinal ROS production and decrease in locomotion behavior could be detected in nanopolystyrene (1 μg/L)exposed pmk-1(km25) mutant nematodes compared with those in nanopolystyrene (1 μg/L)-exposed wild-type nematodes (Fig. 4.1) [46], suggesting the susceptibility of pmk-1 mutant nematodes to the toxicity of nanopolystyrene. Therefore, the PMK-1/p38 MAPK is involved in the control of response to nanopolystyrene at ERCs.

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Fig. 4.1 Effect of pmk-1 mutation on response to nanopolystyrene particles (reprinted with permission from [46]). (a) Effect of nanopolystyrene exposure on transcriptional levels of nsy-1, sek-1, and pmk-1 in wild-type nematode. Bars represent means  SD. **p < 0.01 vs. control. (b) Effect of nanopolystyrene exposure on expression level of phosphorylated PMK-1 in wild-type nematode. Bars represent means  SD. **p < 0.01 vs. control. (c) Effect of pmk-1 mutation on toxicity of nanopolystyrene particles in inducing intestinal ROS production. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). (d) Effect of pmk-1 mutation on toxicity of nanopolystyrene particles in decreasing locomotion behavior. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). Prolonged exposure was from L1-larvae to adult Day-3. Exposure concentration of nanopolystyrene was 1 μg/L

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4.2.3

4 Response of MAPK Signaling Pathways to Toxicants at Environmentally. . .

Tissue-Specific Activity of PMK-1 in Regulating the Response to Toxicants at ERCs

PMK-1 can be expressed in both intestine and neurons. After the exposure to nanopolystyrene (1 μg/L), intestinal expression of PMK-1 could suppress the susceptibility of pmk-1(km25) mutant nematodes to the toxicity of nanopolystyrene (Fig. 4.2) [46]. In contrast, neuronal expression of PMK-1 did not obviously affect the susceptibility of pmk-1(km25) mutant nematodes to the toxicity of nanopolystyrene (Fig. 4.2) [46]. These observations suggested that the PMK-1 acted in the intestine to regulate the response to nanopolystyrene at ERCs.

Fig. 4.2 Tissue-specific activity of PMK-1 in regulating the toxicity of nanopolystyrene particles (reprinted with permission from [46]). (a) Tissue-specific activity of PMK-1 in regulating the toxicity of nanopolystyrene particles in inducing ROS production. (b) Tissue-specific activity of PMK-1 in regulating the toxicity of nanopolystyrene particles in decreasing locomotion behavior. Prolonged exposure was from L1-larvae to adult Day-3. Exposure concentration of nanopolystyrene was 1 μg/L. Bars represent means  SD. **p < 0.01 vs. wild type (if not specially indicated)

4.2 Response of p38 MAPK Signaling Pathway to Toxicants at ERCs

4.2.4

Identification of Downstream Targets of PMK-1 in Regulating the Response to Toxicants at ERCs

4.2.4.1

SKN-1 and ATF-7

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Both atf-7 and skn-1 encode transcription factors, which are normally considered as downstream targets of PMK-1 during the control of stress response [2, 3]. Exposure to nanopolystyrene (1 μg/L) could significantly increase the transcriptional expressions of both atf-7 and skn-1, and expressions of atf-7 and skn-1 were also concentration dependent in nematodes exposed to nanopolystyrene (1–100 μg/L) (Fig. 4.3) [46]. Meanwhile, pmk-1 mutation significantly decreased the transcriptional expressions of both atf-7 and skn-1 in nanopolystyrene (1 μg/L)-exposed nematodes (Fig. 4.3) [46]. Intestine-specific RNAi knockdown of skn-1 or atf-7 induced a susceptibility to the toxicity of nanopolystyrene (Fig. 4.3) [46]. Moreover, RNAi knockdown of atf-7 or skn-1 significantly suppressed the resistance of nematodes overexpressing intestinal PMK-1 to the toxicity of nanopolystyrene (Fig. 4.3) [46], suggesting that ATF-7 and SKN-1 acted downstream of intestinal PMK-1 to regulate the response to nanopolystyrene at ERCs.

4.2.4.2

ATF-7-Mediated Signaling Cascade

During the control of various biological processes, atf-7 has some potential targeted genes. Among the candidate targeted genes, eya-1, hif-1, klf-1, nhr-111, smo-1, unc62, and xbp-1 can be expressed in the intestine (http://www.wormbase.org). After the exposure, the nanopolystyrene (1 μg/L) significantly increased the transcriptional expression of xbp-1 (Fig. 4.4) [46]. xbp-1 encodes a bZIP transcription factor required for control of unfolded protein response (UPR) in endoplasmic reticulum (ER) [2, 3]. Intestine-specific RNAi knockdown of atf-7 could evidently decrease the transcriptional expression of xbp-1 in nanopolystyrene (1 μg/L)-exposed nematodes (Fig. 4.4) [46]. Intestine-specific RNAi knockdown of xbp-1 induced a susceptibility to the toxicity of nanopolystyrene (Fig. 4.4) [46]. Moreover, RNAi knockdown of xbp-1 could significantly inhibit the resistance of nematodes overexpressing intestinal ATF-7 to the toxicity of nanopolystyrene (Fig. 4.4) [46]. These results suggested that XBP-1 acted as a downstream target of ATF-7 in regulating the response to nanopolystyrene at ERCs.

4.2.4.3

SKN-1-Mediated Signaling Cascade

skn-1 regulates the biological processes by affecting the expressions of some potential targeted genes, and some of them (xbp-1, gcs-1, ptps-1, pal-1, med-1, gst-4, gst-5, gst-7, gst-10, gst-14, gst-38, dod-17, F55G11.2, dct-1, ugt-16, vha-6, vha-8, vha-16, vha-17, and dhs-23) are expressed in the intestine (http://www.

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Fig. 4.3 ATF-7 and SKN-1 act as downstream targets of intestinal PMK-1 in regulating the response to nanopolystyrene particles (reprinted with permission from [46]). (a) Effect of nanopolystyrene particles on transcriptional expressions of atf-7 and skn-1 in wild-type nematodes. Bars represent means  SD. **p < 0.01 vs. control. (b) Effect of pmk-1 mutation on transcriptional expressions of atf-7 and skn-1 in nematodes exposed to nanopolystyrene particles. Bars represent means  SD. **p < 0.01 vs. wild-type (nanopolystyrene). (c) Effect of intestine-specific RNAi knockdown of atf-7 or skn-1 on toxicity of nanopolystyrene particles in inducing ROS production. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). (d) Genetic interaction between intestinal PMK-1 and ATF-7 or SKN-1 in regulating the toxicity of nanopolystyrene particles in inducing ROS production. Bars represent means  SD. ** p < 0.01 vs. wild-type (if not specially indicated). Prolonged exposure was from L1-larvae to adult Day-3. Exposure concentration of nanopolystyrene was 1 μg/L

wormbase.org). After the exposure, the nanopolystyrene (1 μg/L) significantly increased the transcriptional expressions of xbp-1 and gst-5 (Fig. 4.5) [46]. Intestine-specific RNAi knockdown of skn-1 could significantly decrease the transcriptional expressions of xbp-1 and gst-5 in nanopolystyrene (1 μg/L)-exposed

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Fig. 4.4 Identification of downstream targets of intestinal ATF-7 in regulating the response to nanopolystyrene particles (reprinted with permission from [46]). (a) Effect of nanopolystyrene particles on transcriptional expressions of eya-1, hif-1, klf-1, nhr-111, smo-1, unc-62, and xbp-1 in wild-type nematodes. Bars represent means  SD. **p < 0.01 vs. control. (b) Effect of intestinespecific RNAi knockdown of atf-7 on transcriptional expression of xbp-1 in nematodes exposed to nanopolystyrene particles. Bars represent means  SD. **p < 0.01 vs. VP303 (L4440). (c) Effect of intestine-specific RNAi knockdown of xbp-1 on the toxicity of nanopolystyrene particles in inducing ROS production. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). (d) Genetic interaction between intestinal ATF-7 and XBP-1 in regulating the toxicity of nanopolystyrene particles in inducing ROS production. Bars represent means  SD. ** p < 0.01 vs. wild type (if not specially indicated). Prolonged exposure was from L1-larvae to adult Day-3. Exposure concentration of nanopolystyrene was 1 μg/L

nematodes (Fig. 4.5) [46]. gst-5 encodes a glutathione-S-transferase, and intestinespecific RNAi knockdown of gst-5 induced a susceptibility to the toxicity of nanopolystyrene (Fig. 4.5) [46]. Moreover, RNAi knockdown of xbp-1 or gst-5

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Fig. 4.5 Identification of downstream targets of intestinal SKN-1 in regulating the response to nanopolystyrene particles (reprinted with permission from [46]). (a) Effect of nanopolystyrene particles on transcriptional expressions of xbp-1, gcs-1, ptps-1, pal-1, med-1, gst-4, gst-5, gst-7, gst10, gst-14, gst-38, dod-17, F55G11.2, dct-1, ugt-16, vha-6, vha-8, vha-16, vha-17, and dhs-23 in wild-type nematodes. Bars represent means  SD. **p < 0.01 vs. control. (b) Effect of intestinespecific RNAi knockdown of skn-1 on transcriptional expressions of xbp-1 and gst-5 in nematodes exposed to nanopolystyrene particles. Bars represent means  SD. **p < 0.01 vs. VP303. (c) Effect of intestine-specific RNAi knockdown of gst-5 on the toxicity of nanopolystyrene particles in inducing ROS production. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). (d) Genetic interaction between intestinal SKN-1 and XBP-1 or GST-5 in regulating the toxicity of nanopolystyrene particles in inducing ROS production. Bars represent means  SD. ** p < 0.01 vs. wild type (if not specially indicated). Prolonged exposure was from L1-larvae to adult Day-3. Exposure concentration of nanopolystyrene was 1 μg/L

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could significantly suppress the resistance of nematodes overexpressing intestinal SKN-1 to the toxicity of nanopolystyrene (Fig. 4.5) [46]. These results demonstrated that both the XBP-1 and the GST-5 acted as downstream targets of intestinal SKN-1 in regulating the response to nanopolystyrene at ERCs.

4.2.4.4

MDT-15 and SBP-1

The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [35]. MDT-15 and SBP-1 are two lipid metabolic sensors and required for the control of response to nanopolystyrene (100 nm) at ERCs in nematodes [35]. Intestinal ROS production was used as a toxicity assessment endpoint [35]. It was found that RNAi knockdown of mdt-15 or sbp-1 could suppress the resistance of transgenic strain overexpressing intestinal PMK-1 to the toxicity of nanopolystyrene in inducing the intestinal ROS production (Fig. 4.6) [35]. Similarly, RNAi knockdown of mdt-15 or sbp-1 also inhibited the resistance of transgenic strain overexpressing intestinal SKN-1 to the toxicity of nanopolystyrene in inducing the intestinal ROS production (Fig. 4.6) [35]. Different from these, RNAi knockdown of mdt-15 or sbp-1 did not influence the resistance of nematodes overexpressing intestinal ATF-7 to the toxicity of nanopolystyrene in inducing the intestinal ROS production (Fig. 4.6) [35]. Therefore, MDT-1 and SBP-1 further acted as downstream targets of intestinal signaling cascade of PMK-1-SKN-1 to regulate the toxicity of nanopolystyrene at ERCs in nematodes.

Fig. 4.6 Genetic interaction of MDT-15/SBP-1 with PMK-1, SKN-1, or ATF-7 in regulating the nanopolystyrene toxicity in inducing intestinal ROS production (reprinted with permission from [35]). Exposure concentration of nanopolystyrene was 1 μg/L. Bars represent means  SD. ** p < 0.01 vs. control (if not specially indicated)

4 Response of MAPK Signaling Pathways to Toxicants at Environmentally. . .

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4.2.4.5

NHR-8

Sterol-sensing nuclear hormone receptor NHR-8 functions to regulate several aspects of metabolisms, including the fat metabolism. Meanwhile, NHR-8 was also required for the control of response to nanopolystyrene (100 nm) at ERCs in nematodes [36]. The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [36]. In nematodes, intestinal insulin signaling pathway is involved in the control of response to nanopolystyrene (1 μg/L) [45]. DAF-2 is the insulin receptor in the insulin signaling pathway. Intestinal ROS production and locomotion behavior reflected by head thrash and body bend were employed as the endpoints [36]. It was observed that RNAi knockdown of nhr-8 could not obviously affect the resistance of daf-2 mutant nematodes to nanopolystyrene toxicity in inducing the intestinal ROS production and in decreasing the locomotion behavior (Fig. 4.7) [36], suggesting that NHR-8 did not act downstream of the insulin signal to regulate the response to nanopolystyrene at ERCs in nematodes. Intestinal overexpression of PMK-1 induced a resistance to the nanopolystyrene toxicity in inducing the intestinal ROS production and in decreasing the locomotion behavior (Fig. 4.7) [36]. RNAi knockdown of nhr-8 could significantly suppress the resistance of nematodes overexpressing intestinal PMK-1 to nanopolystyrene toxicity in inducing the intestinal ROS production and in decreasing the locomotion behavior (Fig. 4.7) [36], suggesting that the NHR-8 could act downstream of intestinal p38 MAPK signal to regulate the response to nanopolystyrene at ERCs in nematodes. After nanopolystyrene exposure, it was further observed that RNAi knockdown of both nhr-8 and skn-1 caused a more severe induction of intestinal ROS production compared with that in nhr-8(RNAi) or in skn-1(RNAi) nematodes [36]. Similarly, after nanopolystyrene exposure, RNAi knockdown of both nhr-8 and atf-7 also resulted in a more severe induction of intestinal ROS production compared with that in nhr-8(RNAi) or in atf-7(RNAi) nematodes [36]. These observations suggested the synergistic effect between NHR-8 and SKN-1 or ATF-7 in regulating the response to nanopolystyrene at ERCs in nematodes.

4.3 4.3.1

Response of JNK MAPK Signaling Pathway to Toxicants at ERCs Alteration in Expression of Genes Encoding JNK MAPK Signaling Pathway Induced by Toxicants at ERCs

The JNK MAPK signaling pathway mainly contains JNK-1, homolog of human JNK, and two MAP kinase kinases (MEK-1 and JKK-1). MEK-1 and JKK-1 act as activators of JNK. The nanopolystyrene (100 nm) was selected as the example of

4.3 Response of JNK MAPK Signaling Pathway to Toxicants at ERCs

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Fig. 4.7 Genetic interaction between NHR-8 and DAF-2 or PMK-1 in regulating the response to nanopolystyrene (reprinted with permission from [36]). (a) Genetic interaction between NHR-8 and DAF-2 in regulating the nanopolystyrene toxicity in inducing intestinal ROS production. (b) Genetic interaction between NHR-8 and DAF-2 in regulating the nanopolystyrene toxicity in decreasing locomotion behavior. (c) Genetic interaction between NHR-8 and PMK-1 in regulating the nanopolystyrene toxicity in inducing intestinal ROS production. (d) Genetic interaction between NHR-8 and PMK-1 in regulating the nanopolystyrene toxicity in decreasing locomotion behavior. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). Exposure concentration of nanopolystyrene was 1 μg/L

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Fig. 4.8 Response of JNK MAPK signaling pathway to nanopolystyrene exposure in nematodes (reprinted with permission from [47]). (a) Effect of nanopolystyrene exposure on expression of genes encoding the JNK MAPK signaling pathway. Bars represent means  SD. ** p < 0.01 vs. control. (b) Effect of jkk-1, mek-1, or jnk-1 RNAi knockdown on nanopolystyrene toxicity in inducing intestinal ROS production. Exposure concentration of nanopolystyrene was 1 μg/L. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). (c) Effect of jkk-1, mek-1, or jnk-1 RNAi knockdown on nanopolystyrene toxicity in decreasing locomotion behavior. Exposure concentration of nanopolystyrene was 1 μg/L. Bars represent means  SD. ** p < 0.01 vs. control (if not specially indicated). Exposure was performed from L1-larvae to adult Day-3

environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [47]. After the exposure, nanopolystyrene (0.1 μg/L) did not affect the expressions of jkk-1, mek-1, and jnk-1, and nanopolystyrene (1 μg/L) also did not influence the expressions of jkk-1 and mek-1 (Fig. 4.8) [47]. In contrast, exposure to nanopolystyrene (1 μg/L) significantly increased the jnk-1 expression (Fig. 4.8) [47]. Exposure to nanopolystyrene at concentrations of 10 and 100 μg/L further significantly increased the expressions of jkk-1, mek-1, and jnk-1 (Fig. 4.8) [47]. These observations suggested the potential of response of JNK MAPK to nanopolystyrene at ERCs in nematodes.

4.3 Response of JNK MAPK Signaling Pathway to Toxicants at ERCs

4.3.2

75

Functional Analysis of JNK MAPK Signaling Pathway in Regulating the Response to Toxicants at ERCs

The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [47]. Intestinal ROS production and locomotion behavior reflected by head thrash and body bend were used as the endpoints [47]. The more severe induction of intestinal ROS production and decrease in locomotion behavior were observed in nanopolystyrene-exposed jkk-1(RNAi), mek-1(RNAi), or jnk-1(RNAi) nematodes compared with those in nanopolystyrene-exposed wild-type nematodes (Fig. 4.8) [47]. That is, RNAi knockdown of jkk-1, mek-1, or jnk-1 caused a susceptibility of nematodes to the nanopolystyrene toxicity. Thus, the JNK MAPK signaling pathway was involved in the regulation of response to nanopolystyrene at ERCs in nematodes.

4.3.3

Identification of Downstream Targets for JNK-1 in Regulating the Response to Toxicants at ERCs

The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [47]. Previous studies have raised some potential downstream neuronal targets (such as UNC-16, SHC-1, and SNB-1) of JNK-1 [48, 49]. UNC-16 is a JNK-signaling scaffold protein, SHC-1 is a p52Shc, and SNB-1 is a synaptobrevin. Exposure to nanopolystyrene (1 μg/L) did not alter expressions of unc-16 and shc-1 (Fig. 4.9) [47]. Different from this, nanopolystyrene exposure (1 μg/L) significantly increased the snb-1 expression (Fig. 4.9) [47]. In nematodes, jnk-1 encodes a neuronal protein. Meanwhile, in nanopolystyrene-exposed nematodes, RNAi knockdown of jnk-1 could significantly decrease the snb-1 expression (Fig. 4.9) [47]. In nematodes, the more severe induction of intestinal ROS production and decrease in locomotion behavior were observed in nanopolystyrene-exposed snb-1(RNAi) nematodes compared with those in nanopolystyrene-exposed wild-type nematodes (Fig. 4.9) [47]. These results suggested the potential role of SNB-1 as the downstream target of neuronal JNK-1 in regulating the response to nanopolystyrene at ERCs in nematodes. To determine the genetic interaction between SNB-1 and JNK-1 in regulating the response to nanopolystyrene, transgenic strain Is(Punc-14-jnk-1) overexpressing neuronal JNK-1 was generated. Neuronal overexpression of JNK-1 could prevent the toxicity of nanopolystyrene in inducing intestinal ROS production and in decreasing locomotion behavior (Fig. 4.9) [47], suggesting the resistance of nematodes with neuronal overexpression of JNK-1 to nanopolystyrene toxicity. Moreover, RNAi knockdown of snb-1 could induce the significant induction of intestinal ROS production and decrease in locomotion behavior in nanopolystyrene-exposed

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Fig. 4.9 Identification of targets of JNK-1 in regulating the response to nanopolystyrene (reprinted with permission from [47]). (a) Effect of nanopolystyrene exposure on expressions of unc-16, shc1, and snb-1. Bars represent means  SD. **p < 0.01 vs. control. (b) Effect of jnk-1 RNAi knockdown on snb-1 expression in nanopolystyrene-exposed nematodes. Bars represent means  SD. **p < 0.01 vs. wild type. (c) Effect of snb-1 RNAi knockdown on nanopolystyrene toxicity in inducing intestinal ROS production. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). (d) Effect of snb-1 RNAi knockdown on nanopolystyrene toxicity in inducing intestinal ROS production in nematodes overexpressing neuronal JNK-1. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). Exposure concentration of nanopolystyrene was 1 μg/L. Exposure was performed from L1-larvae to adult Day-3

nematodes overexpressing neuronal JNK-1 (Fig. 4.9) [47]. That is, RNAi knockdown of snb-1 could suppress the resistance of nematodes overexpressing neuronal JNK-1 to nanopolystyrene toxicity. Therefore, SNB-1 acted as a downstream target of neuronal JNK-1 to regulate the response to nanopolystyrene at ERCs in nematodes.

4.4 Response of ERK MAPK Signaling Pathways to Toxicants at ERCs

4.4 4.4.1

77

Response of ERK MAPK Signaling Pathways to Toxicants at ERCs Alteration in Expression of Genes Encoding ERK MAPK Signaling Pathway Induced by Toxicants at ERCs

In the core ERK MAPK signaling pathway, lin-45 encodes a Raf, mek-2 encodes a MEK, and mpk-1 encodes a ERK. The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [50]. After the exposure, nanopolystyrene (0.1 μg/L) did not affect the expressions of lin-45, mek-2, and mpk-1 (Fig. 4.10) [50]. Nanopolystyrene (1 μg/L) exposure also did not affect the expressions of lin-45 and mek-2 (Fig. 4.10) [50]. In contrast, nanopolystyrene (1 μg/ L) exposure significantly increased the mpk-1 expression (Fig. 4.10) [50]. Exposure to nanopolystyrene at concentrations of 10 and 100 μg/L further significantly increased the expressions of lin-45, mek-2, and mpk-1 (Fig. 4.10) [50]. These observations suggested the alteration in expression of MPK-1/ERK MAPK induced by exposure to nanopolystyrene at ERCs in nematodes.

4.4.2

Functional Analysis of ERK MAPK Signaling Pathway in Regulating the Response to Toxicants at ERCs

The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [50]. Using intestinal ROS production and locomotion behavior as endpoints, the more severe induction of intestinal ROS and decrease in locomotion behavior were detected in nanopolystyrene-exposed lin-45(RNAi), mek-2(RNAi), or mpk-1(RNAi) nematodes compared with those in nanopolystyrene-exposed wildtype nematodes (Fig. 4.10) [50]. That is, RNAi knockdown of lin-45, mek-2, or mpk1 could result in a susceptibility of nematodes to toxicity of nanopolystyrene at ERCs.

4.4.3

Tissue-Specific Activity of MPK-1/ERK MAPK in Regulating the Response to Toxicants at ERCs

The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [50]. MPK-1 is expressed in germline and neurons in adult nematodes.

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Fig. 4.10 Effect of nanopolystyrene exposure on expressions of genes encoding ERK MAPK signaling pathway (reprinted with permission from [50]). (a) Effect of nanopolystyrene exposure on expressions of lin-45, mek-2, and mpk-1. Bars represent means  SD. **p < 0.01 vs. control. (b) Effect of lin-45, mek-2, or mpk-1 RNAi knockdown on nanopolystyrene toxicity in inducing intestinal ROS production. Exposure concentration of nanopolystyrene was 1 μg/L. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). (c) Effect of lin-45, mek-2, or mpk-1 RNAi knockdown on nanopolystyrene toxicity in decreasing locomotion behavior. Exposure concentration of nanopolystyrene was 1 μg/L. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). Exposure was performed from L1-larvae to adult Day-3. L4440, empty vector for RNAi

Using intestinal ROS production as an endpoint, it was observed that neuronal RNAi knockdown of mpk-1 induced the more severe intestinal ROS production in nanopolystyrene-exposed nematodes compared with nanopolystyrene-exposed TU3401 nematodes (Fig. 4.11) [50]. Moreover, neuronal RNAi knockdown of mpk-1 could cause the more severe decrease in locomotion behavior in nanopolystyrene-exposed nematodes compared with nanopolystyrene-exposed TU3401 nematodes (Fig. 4.11) [50]. Therefore, MPK-1 could act in the neurons to regulate the response to nanopolystyrene at ERCs in nematodes.

4.4 Response of ERK MAPK Signaling Pathways to Toxicants at ERCs

79

Fig. 4.11 Tissue-specific activity of MPK-1 in regulating the response to nanopolystyrene (reprinted with permission from [50]). (a) Tissue-specific activity of MPK-1 in regulating the nanopolystyrene toxicity in inducing intestinal ROS production. (b) Neuronal activity of MPK-1 in regulating the nanopolystyrene toxicity in decreasing locomotion behavior. L4440, empty vector for RNAi. Exposure concentration of nanopolystyrene was 1 μg/L. Exposure was performed from L1-larvae to adult Day-3. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated)

4.4.4

Identification of Downstream Targets of Neuronal MPK-1 in Regulating the Response to Toxicants at ERCs

The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [50]. During the regulation of various biological processes, a large amount of genes have been raised as the potential downstream targeted genes of mpk-1, and some of them can be expressed in the neurons (https://wormbase.org/). Among these potential neuronal targets of MPK-1, nanopolystyrene (1 μg/L) exposure could significantly increase the expressions of lin-12, gck-3, dpl-1, lin-31, and tra-1 (Fig. 4.12) [50]. In nematodes, lin-12 encodes the Notch, gck-3 encodes an ortholog of STK39/SPAK, dpl-1 encodes an ortholog of mammalian E2F heterodimerization partner DP, lin-31 encodes a putative transcriptional factor, and tra-1 encodes a Gli transcriptional repressor. Considering the fact that MPK-1 can regulate biological processes, such as DNA damage response, via insulin signaling pathway [51], the effects of nanopolystyrene exposure on expressions of genes (ins1, ins-4, ins-7, ins-39, and daf-28) encoding neuronal insulin peptides were also

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Fig. 4.12 Identification of downstream targets of neuronal MPK-1 in regulating the response to nanopolystyrene (reprinted with permission from [50]). (a) Effect of nanopolystyrene exposure on gene expressions. Bars represent means  SD. **p < 0.01 vs. control. (b) Effect of neuronal RNAi knockdown of mpk-1 on gene expressions in nanopolystyrene-exposed nematodes. Bars represent means  SD. **p < 0.01 vs. TU3401. (c) Effect of neuronal RNAi knockdown of ins-4, ins-39, or daf-28 on nanopolystyrene toxicity in inducing intestinal ROS production. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). (d) Effect of neuronal RNAi knockdown of ins-4, ins-39, or daf-28 on nanopolystyrene toxicity in decreasing locomotion behavior. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). L4440, empty vector for RNAi. Exposure concentration of nanopolystyrene was 1 μg/L. Exposure was performed from L1-larvae to adult Day-3

determined. Among the genes encoding neuronal insulin peptides, nanopolystyrene (1 μg/L) exposure could significantly decrease the expressions of ins-4, ins-39, and daf-28 (Fig. 4.12) [50]. Moreover, in nanopolystyrene (1 μg/L)-exposed nematodes, neuronal RNAi knockdown of mpk-1 could only significantly increase the expressions of ins-4, ins-39, and daf-28 (Fig. 4.12) [50]. These observations implied the potential role of INS-4, INS-39, and DAF-28 as the downstream targets of neuronal MPK-1 in regulating the response to nanopolystyrene at ERCs in nematodes.

4.4 Response of ERK MAPK Signaling Pathways to Toxicants at ERCs

81

Fig. 4.13 Genetic interaction between MPK-1 and INS-4, INS-39, or DAF-28 in regulating the response to nanopolystyrene (reprinted with permission from [50]). (a) Genetic interaction between MPK-1 and INS-4, INS-39, or DAF-28 in regulating nanopolystyrene toxicity in inducing intestinal ROS production. (b) Genetic interaction between MPK-1 and INS-4, INS-39, or DAF-28 in regulating nanopolystyrene toxicity in decreasing locomotion behavior. L4440, empty vector for RNAi. Exposure concentration of nanopolystyrene was 1 μg/L. Exposure was performed from L1-larvae to adult Day-3. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated)

Under the TU3401 background, the decrease in intestinal ROS production and the increase in locomotion behavior could be further observed in nanopolystyreneexposed nematodes with neuronal RNAi knockdown of ins-4, ins-39, or daf-28 compared with those in nanopolystyrene-exposed TU3401 nematodes (Fig. 4.12) [50]. Therefore, neuronal RNAi knockdown of ins-4, ins-39, or daf-28 induced a resistance to toxicity of nanopolystyrene at ERCs in nematodes. To examine the genetic interaction between MPK-1 and INS-4, INS-39, or DAF-28 in regulating the response to nanopolystyrene, the double neuronal RNAi knockdown between mpk-1 and ins-4, ins-39, or daf-28 was generated [50]. Under the TU3410 background, RNAi knockdown of ins-4, ins-39, or daf-28 could significantly suppress the intestinal ROS production and increase the locomotion behavior in nanopolystyrene-exposed mpk-1(RNAi) nematodes (Fig. 4.13) [50]. Therefore, RNAi knockdown of ins-4, ins-39, or daf-28 could inhibit the susceptibility of mpk-1(RNAi) nematodes to toxicity of nanopolystyrene, which

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confirmed the role of INS-4, INS-39, and DAF-28 as downstream targets of neuronal MPK-1 during the control of response to nanopolystyrene at ERCs in nematodes.

4.5

Genetic Interaction Between p38 MAPK Signaling Pathway and Insulin Signaling Pathway in Regulating the Toxicity of Toxicants at ERCs

The genetic interaction between DAF-16 in the insulin signaling pathway and SKN-1 or MDT-15 in the p38 MAPK signaling pathway in regulating the response to nanopolystyrene (100 nm) was investigated [52]. Intestine-specific RNAi knockdown of daf-16, skn-1, or mdt-15 caused the more severe induction of ROS production and reduction in brood size in nanopolystyrene-exposed nematodes compared with those in nanopolystyrene-exposed VP303 nematodes (Fig. 4.14) [52], suggesting the susceptibility of daf-16(RNAi), skn-1(RNAi), and mdt-15(RNAi) nematodes to nanopolystyrene toxicity. Double RNAi knockdown of daf-16 and skn-1 caused the more severe induction of ROS production and reduction in brood size in nanopolystyrene-exposed nematodes compared with those in nanopolystyreneexposed daf-16(RNAi) or skn-1(RNAi) nematodes (Fig. 4.14) [52], suggesting the synergistic effect between DAF-16 and SKN-1 in the intestine to regulate the response to nanopolystyrene. Similarly, double RNAi knockdown of daf-16 and mdt-15 resulted in the more severe induction of ROS production and reduction in brood size in nanopolystyrene-exposed nematodes compared with those in nanopolystyrene-exposed daf-16(RNAi) or mdt-15(RNAi) nematodes (Fig. 4.14) [52], suggesting the synergistic effect between DAF-16 and MDT-15 in the intestine to regulate the response to nanopolystyrene. Therefore, in the intestine, the insulin signaling pathway may function synergistically with the p38 MAPK signaling pathway to regulate the nanopolystyrene at ERCs in nematodes.

4.6

Perspectives

C. elegans is an important model for the study of in vivo toxicology of various toxicants or stresses [1–4, 53–60]. With the nanopolystyrene as the example of toxicants, exposure to nanopolystyrene at ERCs only increased the PMK-1/p38 MAPK in the p38 MAPK signaling pathway, but did not further alter the expressions of NSY-1 and SEK-1 in the p38 MAPK signaling pathway. This implied that a simple signaling cascade in the p38 MAPK signaling pathway will be activated in response to toxicants at ERCs. That is, the p38 MAPK signaling may be activated through previously unknown upregulators and G protein-coupled receptors (GPCRs) in nematodes exposed to toxicants at ERCs. Moreover, besides the GST proteins

4.6 Perspectives

83

Fig. 4.14 Genetic interaction between DAF-16 and SKN-1 or MDT-15 in the intestine to regulate the response to nanopolystyrene (reprinted with permission from [52]). (a) Genetic interaction between DAF-16 and SKN-1 or MDT-15 in the intestine to regulate the toxicity of nanopolystyrene in inducing ROS production. L4440, empty vector. Exposure concentration of nanopolystyrene was 1 μg/L. Exposure was performed from L1-larvae to adult Day-3. Bars represent means  SD. ** p < 0.01 vs. control (if not specially indicated). (b) Genetic interaction between DAF-16 and SKN-1 or MDT-15 in the intestine to regulate the toxicity of nanopolystyrene in reducing brood size. L4440, empty vector. Exposure concentration of nanopolystyrene was 1 μg/L. Exposure was performed from L1-larvae to adult Day-3. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). (c) A diagram showing the molecular basis of intestinal mir-794 in regulating the response to nanopolystyrene in nematodes

(such as GST-5), PMK-1 also regulated the response to nanopolystyrene at ERCs through activating ER UPR in the intestine [46]. With the nanopolystyrene as the example of toxicants, in the ERK MAPK signaling pathway, nanopolystyrene at ERCs only significantly increased the MPK-1/ERK expression, but did not obviously affect the expressions of upstream LIN-45 and MEK-2. Therefore, the ERK MAPK signaling could be activated by some unknown upstream regulators and GPCRs in nematodes exposed to toxicants at ERCs. In nematodes, MEK-1 acted in the neurons and functioned upstream of several insulin peptides (INS-4, INS-39, and DAF-28) to regulate the response to

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nanopolystyrene at ERCs [47]. These three insulin peptides further regulated the response to nanopolystyrene at ERCs by activating intestinal insulin receptor DAF-2. Therefore, the neuronal ERK signaling regulated the response to toxicants at ERCs by modulating the functional state of intestinal insulin signaling pathway. With the nanopolystyrene as the example of toxicants, in the JNK MAPK signaling pathway, nanopolystyrene at ERCs only significantly increased the JNK-1/JNK expression, but did not obviously affect the expressions of JKK-1 and MEK-1. Therefore, the JNK MAPK signaling may also be activated through some unknown GPCRs in nematodes exposed to toxicants at ERCs. In nematodes, JNK-1 also acted in the neurons and functioned upstream of TBH-1 and CAT-2 to regulate the response to nanopolystyrene at ERCs [50]. TBH-1 and CAT-2 control the synthesis of neurotransmitters of octopamine and dopamine, respectively, and regulated the response to nanopolystyrene by modulating the corresponding octopamine receptors of SER-6 and OCTR-1 and dopamine receptor of DOP-1 in the intestine [50]. More importantly, it was found that the intestinal GPCR DOP-1 is just the corresponding upstream GPCR to activate the signaling cascade of PMK-1MDT-15-SBP-1 in the p38 MAPK signaling pathway in regulating the response to nanopolystyrene at ERCs [50]. Therefore, both the ERK signaling and the JNK signaling mediated the important neuron-intestine communication in regulating the toxicants at ERCs.

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Chapter 5

Response of Development-Related Signaling Pathways to Toxicants at Environmentally Relevant Concentrations

Abstract Some development-related signaling pathways are required for the control of response to environmental toxicants or stresses. We focused on the introduction and discussion of identification of development-related signaling pathways involved in the control of response to toxicants at environmentally relevant concentrations (ERCs). We first introduced the intestinal development-related molecular signals required for the control of response to toxicants at ERCs. The response of cell death and DNA damage-related signaling pathways to toxicants at ERCs was further discussed. Moreover, we introduced and discussed the involvement of Wnt and transforming growth factor-β (TGF-β) signaling pathways in regulating the response to toxicants at ERCs. Keywords Environmentally relevant concentrations · Development-related signaling pathways · Response · Caenorhabditis elegans

5.1

Introduction

Caenorhabditis elegans is a powerful animal model for the study of both molecular toxicology and target organ toxicology at the whole animal level [1–4]. Meanwhile, due to the sensitivity to environmental exposure, C. elegans can be used to detect the potential toxicity of different environmental toxicants or stresses [1–15]. Some development-related signaling pathways, such as Wnt and transforming growth factor-β (TGF-β) signaling pathways, play an important function in regulating the toxicity of environmental toxicants or stresses, and we have introduced this in Chapters 6 and 7 in “Molecular Toxicology in Caenorhabditis elegans” [2]. In this chapter, we focused on the introduction and the discussion of some development-related signaling pathways in regulating the response to toxicants at environmentally relevant concentrations (ERCs) in nematodes.

© The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 D. Wang, Toxicology at Environmentally Relevant Concentrations in Caenorhabditis elegans, https://doi.org/10.1007/978-981-16-6746-6_5

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5.2

5 Response of Development-Related Signaling Pathways to Toxicants at. . .

Response of Intestinal Development-Related Molecular Signals to Toxicants at ERCs

In nematodes, some intestinal development-related molecular signals are involved in the regulation of toxicity of environmental toxicants, such as graphene oxide (GO) and multi-walled carbon nanotubes (MWCNTs) [16–18]. Some intestinal development-related molecular signals are also involved in the regulation of toxicity of environmental stresses, such as simulated microgravity stress [19].

5.2.1

Alteration in Expression of Intestinal Development-Related Genes Induced by Toxicants at ERCs

The nanopolystyrene (100 nm) and microcystin-LR (MC-LR) were selected as the examples of environmental toxicants [20]. The combinational exposure to nanopolystyrene and MC-LR was performed from L1-larvae to adult Day-1 in nematodes [20]. It was observed that exposure to nanopolystyrene (1 μg/L, a predicted environmental concentration) could enhance the MC-LR (0.1 μg/L) toxicity in reducing the brood size, in decreasing the locomotion behavior, and in inducing the intestinal reactive oxygen species (ROS) production [20]. Meanwhile, it was found that the adsorption of MC-LR by nanopolystyrene particles played an important role in inducing the enhancement in MC-LR toxicity by nanopolystyrene particles, and only exposure to resuspension of nanopolystyrene (1 μg/L) caused the increased intestinal permeability in MC-LR (0.1 μg/L)-exposed nematodes based on the erioglaucine disodium (a blue dye) staining (Fig. 5.1) [20]. In nematodes, some important proteins, such as ACS-22, PKC-3, ERM-1, and HMP-2, provide the important molecular basis for the intestinal barrier against environmental toxicants [17, 18]. It was further observed that exposure to nanopolystyrene (1 μg/L), MC-LR (0.1 μg/L), or suspension of nanopolystyrene (1 μg/L) alone did not alter the expressions of acs-22, pkc-3, erm-1, and hmp-2 (Fig. 5.1) [20]. Combinational exposure to nanopolystyrene (1 μg/L) and MC-LR (0.1 μg/L), as well as combinational exposure to suspension of nanopolystyrene (1 μg/L) and MC-LR (0.1 μg/L), also could not alter the erm-1 expression (Fig. 5.1) [20]. Different from these, a combinational exposure to nanopolystyrene (1 μg/L) and MC-LR (0.1 μg/L) significantly decreased the acs-22, pkc-3, and hmp-2 expressions (Fig. 5.1) [20]. Similarly, combinational exposure to suspension of nanopolystyrene (1 μg/L) and MC-LR (0.1 μg/L) significantly decreased the acs-22, pkc-3, and hmp-2 expressions (Fig. 5.1) [20]. These data suggested the possible alteration in expression of intestinal development-related genes in nematodes exposed to toxicants at ERCs.

5.2 Response of Intestinal Development-Related Molecular Signals to Toxicants. . .

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Fig. 5.1 Contributions of supernatant and particulate resuspension to toxicity in enhancing intestinal permeability in nematodes exposed to both nanopolystyrene particles and MC-LR (reprinted with permission from [20]). (a) Resuspension of nanopolystyrene particulate contributed to toxicity in enhancing intestinal permeability in nematodes exposed to both nanopolystyrene particles and MC-LR. Arrowheads indicate the body cavity. The intestinal lumen (*) and the intestinal cells (**) were indicated with asterisks. (b) Contributions of resuspension of nanopolystyrene particulate to expressional alterations of genes required for function of intestinal barrier. Semi-quantification of intestinal ROS signals was examined in comparison to autofluorescence. Prolonged exposure was performed from L1-larvae to adult Day-1. Control, without MC-LR and nanopolystyrene particle exposure. Bars represent means
SD. **p < 0.01 vs. control

5.2.2

ELT-2 Signal

5.2.2.1

Alteration in ELT-2 Expression Induced by Toxicants at ERCs

In the recent years, C. elegans has been frequently employed to detect the possible toxicity of nanopolystyrene at ERCs [21–32]. Using intestinal ROS production and locomotion behavior reflected by head thrash and body bend as the endpoints,

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Fig. 5.2 Function of ELT-2 in regulating the response of nematodes to nanopolystyrene (reprinted with permission from [34]). (a) Effect of nanopolystyrene on transcriptional expressions of elt-2. Bars represent means
SD. **p < 0.01 vs. control. (b) Mutation of elt-2 induced a susceptibility to the toxicity of nanopolystyrene in inducing intestinal ROS production. Exposure concentration of nanopolystyrene was 1 μg/L. Bars represent means
SD. **p < 0.01 vs. control (if not specially indicated). (c) Mutation of elt-2 induced a susceptibility to the toxicity of nanopolystyrene in decreasing locomotion behavior. Exposure concentration of nanopolystyrene was 1 μg/L. Bars represent means
SD. **p < 0.01 vs. control (if not specially indicated)

exposure (L1-larvae to adult Day-3) to nanopolystyrene (100 nm) at concentrations 1 μg/L could cause the significant induction of intestinal ROS production and decrease in locomotion behavior in nematodes [33]. The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [34]. In nematodes, ELT-2, a GATA transcriptional factor, is required for the regulation of functional state of intestinal barrier [34]. After the exposure, although 0.1 μg/L nanopolystyrene could not significantly affect the elt-2 expression, 1 μg/L nanopolystyrene increased the elt-2 expression (Fig. 5.2) [34]. Moreover, the elt-2 expression was concentration dependent in nematodes exposed to nanopolystyrene at concentrations of 1–100 μg/L (Fig. 5.2) [34].

5.2 Response of Intestinal Development-Related Molecular Signals to Toxicants. . .

5.2.2.2

93

Functional Analysis of ELT-2 in Regulating the Response to Toxicants at ERCs

The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [34]. Considering the fact that the elt-2 null mutants have the severe abnormality in intestinal development and are difficult to develop into the adults, RNAi strain was used to investigate the function of ELT-2 in regulating the response to nanopolystyrene at ERCs [34]. Intestinal ROS production and locomotion behavior were used as the endpoints [34]. Compared with toxicity induction of nanopolystyrene in wild-type nematodes, the more severe decrease in locomotion behavior and induction of intestinal ROS production were detected in nanopolystyrene-exposed elt-2(RNAi) nematodes (Fig. 5.2) [34], suggesting that the RNAi knockdown of elt-2 potentially caused the susceptibility to toxicity of nanopolystyrene. Thus, the ELT-2 was required for the control of response to nanopolystyrene at ERCs in nematodes.

5.2.2.3

Tissue-Specific Activity of ELT-2 in Regulating the Response to Toxicants at ERCs

The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [34]. In nematodes, ELT-2 can be expressed in neurons and intestine. VP303 is a strain for intestinal RNA interference (RNAi) knockdown, and TU3401 is a strain for neuronal RNAi knockdown. It was observed that RNAi knockdown of elt-2 in neurons did not significantly influence nanopolystyrene toxicity in decreasing the locomotion behavior and in inducing the intestinal ROS production (Fig. 5.3) [34]. Different from this, intestinal RNAi knockdown of elt-2 resulted in the susceptibility to nanopolystyrene toxicity in inducing the intestinal ROS production (Fig. 5.3) [34]. Therefore, ELT-2 acted in intestinal cells to regulate the response to nanopolystyrene at ERCs in nematodes.

5.2.2.4

Identification of Downstream Targets of Intestinal ELT-2 in Regulating the Response to Toxicants at ERCs

In nematodes, ELT-2 has many potential targeted genes during the regulation of biological processes, and some of them are expressed in the intestine (https://www. wormbase.org). The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [34]. Among these intestinal targeted genes, nanopolystyrene (1 μg/L) could increase expression of daf-16a, daf-16d/f, elt-4, hrg-1, ftn-2, ref-1, mtl-1, F55G11.2, T24B8.5, erm-1, acs-2, bli-3, clec-63, dhs-21,

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Fig. 5.3 Tissue-specific activity of ELT-2 in regulating the response to nanopolystyrene (reprinted with permission from [34]). (a) Tissue-specific activity of ELT-2 in regulating the toxicity of nanopolystyrene in inducing intestinal ROS production. (b) Effect of neuronal RNAi knockdown of elt-2 on toxicity of nanopolystyrene in decreasing locomotion behavior. L4440, empty vector. Exposure concentration of nanopolystyrene was 1 μg/L. Bars represent means
SD. ** p < 0.01 vs. control (if not specially indicated)

skn-1, ugt-29, pqm-1, pop-1, atf-7, mdt-15, and clec-85 compared with control (Fig. 5.4) [34]. Among these 21 candidate genes, intestinal RNAi knockdown of elt-2 could only significantly decrease the expressions of daf-16d/f, elt-4, hrg-1, F55G11.2, erm-1, clec-63, and clec-85 in nanopolystyrene exposed nematodes (Fig. 5.4) [34]. Moreover, under the normal conditions (without the nanopolystyrene exposure), it was found that only the expressions of hrg-1, erm-1, clec-63, and clec85 could be significantly decreased by intestinal RNAi knockdown of elt-2 (Fig. 5.4) [34]. In nematodes, intestinal RNAi knockdown of erm-1, clec-63, or clec-85 caused susceptibility to nanopolystyrene toxicity in inducing the intestinal ROS production (Fig. 5.4) [34]. However, intestinal RNAi knockdown of hrg-1 did not affect nanopolystyrene toxicity in inducing the intestinal ROS production (Fig. 5.4) [34]. Therefore, ERM-1, CLEC-63, and CLEC-85 acted as potential targets for intestinal ELT-2 in regulating the response to nanopolystyrene at ERCs. In

5.2 Response of Intestinal Development-Related Molecular Signals to Toxicants. . .

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Fig. 5.4 Identification of downstream targets of ELT-2 in regulating the response to nanopolystyrene (reprinted with permission from [34]). (a) Effect of nanopolystyrene exposure on gene expressions. Bars represent means
SD. **p < 0.01 vs. control. (b) Effect of intestinal RNAi knockdown of elt-2 on gene expressions in nematodes exposed to nanopolystyrene. Bars represent means
SD. **p < 0.01 vs. VP303. (c) Effect of intestinal RNAi knockdown of elt-2 on gene expressions in nematodes under the normal conditions. Bars represent means
SD. ** p < 0.01 vs. VP303. (d) Effect of intestinal RNAi knockdown of erm-1, hrg-1, clec-63, or clec-85 on toxicity of nanopolystyrene in inducing intestinal ROS production. Bars represent means
SD. **p < 0.01 vs. control (if not specially indicated). Exposure concentration of nanopolystyrene was 1 μg/L

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Fig. 5.5 Genetic interaction between intestinal ELT-2 and ERM-1, CLEC-63, or CLEC-85 in regulating the response of nematodes to nanopolystyrene (reprinted with permission from [34]). (a) Genetic interaction between intestinal ELT-2 and ERM-1, CLEC-63, or CLEC-85 in regulating the toxicity of nanopolystyrene in inducing intestinal ROS production. (b) Genetic interaction between intestinal ELT-2 and ERM-1, CLEC-63, or CLEC-85 in regulating the toxicity of nanopolystyrene in decreasing locomotion behavior. Exposure concentration of nanopolystyrene was 1 μg/L. Bars represent means
SD. **p < 0.01 vs. wild type (if not specially indicated)

nematodes, ERM-1 is an Ezrinradixin-moesin protein, and CLEC-63/85 are two C-type lectin (CLEC) proteins. In nematodes, the intestinal ELT-2 overexpression significantly inhibited the nanopolystyrene toxicity in decreasing the locomotion behavior and in inducing the intestinal ROS production (Fig. 5.5) [34], suggesting that the intestinal ELT-2 overexpression induced the resistance to nanopolystyrene toxicity. Genetic interaction analysis further indicated that RNAi knockdown of clec-63, clec-85, or erm-1 caused the significant decrease in locomotion behavior and induction of intestinal ROS production in nanopolystyrene-exposed nematodes with intestinal overexpression of ELT-2 (Fig. 5.5) [34]. That is, RNAi knockdown of clec-63, clec-85, or erm-1 could inhibit the resistance of nematodes overexpressing intestinal ELT-2 to the nanopolystyrene toxicity. These observations further confirmed the role of CLEC-63, CLEC-85, and ERM-1 as the downstream targets of intestinal ELT-2 in regulating the response to nanopolystyrene at ERCs in nematodes. It was further found that the double RNAi knockdown of erm-1 and clec-63 induced the more severe intestinal ROS production in nanopolystyrene-exposed nematodes compared with that in nanopolystyrene-exposed erm-1(RNAi) or

5.2 Response of Intestinal Development-Related Molecular Signals to Toxicants. . .

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Fig. 5.6 Synergistic interaction between ERM-1 and CLEC-63 or CLEC-85 in regulating the response of nematodes to nanopolystyrene (reprinted with permission from [34]). (a) Synergistic interaction between ERM-1 and CLEC-63 or CLEC-85 in regulating the toxicity of nanopolystyrene in inducing intestinal ROS production. Exposure concentration of nanopolystyrene was 1 μg/L. Bars represent means
SD. **p < 0.01 vs. VP303 (if not specially indicated). (b) A diagram showing the molecular basis for ELT-2 in regulating the intestinal response in nematodes after long-term and low-dose nanopolystyrene exposure

clec-63(RNAi) nematodes (Fig. 5.6) [34]. Similarly, the double RNAi knockdown of erm-1 and clec-85 also caused the more severe intestinal ROS production in nanopolystyrene-exposed nematodes compared with that in nanopolystyreneexposed erm-1(RNAi) or in clec-85(RNAi) nematodes (Fig. 5.6) [34]. Therefore, ERM-1 and CLEC-63 or CLEC-85 functioned synergistically to regulate the response to nanopolystyrene at ERCs in nematodes.

5.2.3

ACS-22 Signal

The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to

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Fig. 5.7 Distribution and translocation of nanopolystyrene particles in wild-type and acs-22 mutant nematodes after prolonged exposure (reprinted with permission from [35]). The pharynx (*) and the intestinal lumen (**) were indicated by asterisks. White arrowheads indicate the gonad, and yellow heads indicate the intestinal cells. Prolonged exposure to nanopolystyrene particles was performed from L1-larvae to adult Day-1. Two-way ANOVA was performed for the comparison between wild-type and acs-22 mutant. Bars represent means
SD. **p < 0.01 vs. 1 μg/L (if not specially indicated)

adult Day-1 [35]. In nematodes, ACS-22 is a protein homologous to mammalian fatty acid transport protein and required for the control of functional state of intestinal barrier [35]. After the exposure, although nanopolystyrene (1 μg/L) did not cause the significant induction of intestinal ROS production and decrease in locomotion behavior, nanopolystyrene at concentrations 1 μg/L could cause the toxicity in inducing the intestinal ROS production and in decreasing the locomotion behavior in acs-22 mutant nematodes [35]. Using Rho B-labeled nanopolystyrene, distribution and translocation of nanopolystyrene were further examined in acs-22 mutant nematodes [35]. After exposure to nanopolystyrene (1 μg/L), the nanopolystyrene was not only accumulated in the pharynx and the intestinal lumen, but also translocated and accumulated in the gonad and the intestinal cells in acs-22 mutant nematodes (Fig. 5.7) [35]. In contrast, after exposure to nanopolystyrene (1 μg/L), only a moderate accumulation of nanopolystyrene was observed in the intestinal lumen in wild-type nematodes (Fig. 5.7) [35]. These results suggested the requirement of ACS-22 in regulating the response to nanopolystyrene at ERCs in nematodes.

5.2 Response of Intestinal Development-Related Molecular Signals to Toxicants. . .

5.2.4

99

IFC-2 Signal

In nematodes, exposure to GO could cause multiple aspects of toxicity [36–42]. GO was selected as the example of environmental toxicants, and the nematodes were exposed to GO from L1-larvae to adult Day-1 [43]. IFC-2 is a protein in the cytoplasm of intestinal cells. The intestinal lumen of ifc-2 (RNAi) nematodes was considerably widened, suggesting that the IFC-2 plays an important role in maintaining the intestinal morphological structure (Fig. 5.8) [43]. Exposure to GO (100 μg/L) could significantly decrease the ifc-2 expression [43]. Meanwhile, after the GO exposure, intestinal RNAi knockdown of ifc-2 induced a more significant change of irregularly widened intestinal lumen (Fig. 5.8) [43]. Nevertheless, the intestine-specific RNAi knockdown of ifc-2 did not affect both the intestinal permeability and the defecation behavior [43]. Using intestinal ROS production as the

Fig. 5.8 Genetic interactions between circ_0000115 and IFC-2 in regulating the GO toxicity (reprinted with permission from [43]). (a) Intestinal morphology in nematodes with ifc-2 RNAi knockdown. Arrowheads indicate the altered intestinal lumen. (b) Effect of intestinal RNAi knockdown of ifc-2 on GO toxicity in inducing ROS production. (c) Genetic interactions between circ_0000115 and IFC-2 in regulating GO toxicity in inducing ROS production. GO concentration is 100 μg/L. Prolonged exposure to GO was performed from L1-lavae to adult Day-1. Bars represent means
SD. **p < 0.01 vs. control (if not specially indicated)

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endpoint, it was further observed that intestinal RNAi knockdown of ifc-2 caused the more significant induction of intestinal ROS production in GO-exposed nematodes compared with that in GO-exposed VP303 nematodes (Fig. 5.8) [43], suggesting the formation of a susceptibility of ifc-2(RNAi) nematodes to the GO toxicity. Moreover, it was found that the induction of intestinal ROS production in GO-exposed circ_0000115(RNAi);ifc-2(RNAi) nematodes was similar to that in GO-exposed ifc-2(RNAi) nematodes (Fig. 5.8) [43] suggesting that IFC-2 functions downstream of circ_0000115 to regulate the response to GO in nematodes. In nematodes, insulin signaling and p38 MAPK signaling acted in the intestine to regulate the GO toxicity [1–3, 37]. DAF-16 is a FOXO transcriptional factor in the insulin signaling pathway, and PMK-1 is a p38 MAPK in the p38 MAPK signaling pathway. It was found that mutation of daf-16, but not the mutation of pmk-1, could suppress the resistance of circ_0000115(RNAi) nematodes to the GO toxicity in inducing intestinal ROS production and in decreasing locomotion behavior (Fig. 5.9) [43], suggesting that the circ_0000115 regulated the GO toxicity by acting upstream of insulin signaling. Intestinal overexpression of IFC-2 could suppress the induction of intestinal ROS production and the decrease in locomotion behavior in GO-exposed nematodes (Fig. 5.9) [43], suggesting that the intestinal overexpression of IFC-2 induced a resistance to GO toxicity. Moreover, the daf-16 mutation could further inhibit the resistance of nematodes overexpressing intestinal IFC-2 to the GO toxicity in inducing intestinal ROS production and in decreasing locomotion behavior (Fig. 5.9) [43]. Therefore, DAF-16 in the insulin signaling pathway could act downstream of IFC-2 to regulate the response to GO in nematodes.

5.3

Response of Cell Death and DNA Damage-Related Signaling Pathways to Toxicants at ERCs

In nematodes, the cell death and the DNA damage-related signaling pathways were involved in the regulation of toxicity of environmental toxicants, such as GO [44]. The pristine and amino modified nanopolystyrene particles (35 nm) were selected as the examples of environmental toxicants, and the nematodes were exposed to pristine and amino modified nanopolystyrene particles from L1-larvae to adult Day-1 [45]. After the exposure, the pristine nanopolystyrene (10 μg/L) induced the obvious germline apoptosis (Fig. 5.10) [45]. Different from this, the noticeable germline apoptosis was observed in nematodes exposed to amino modified nanopolystyrene at concentrations 1 μg/L (Fig. 5.10) [45]. Meanwhile, exposure to amino modified nanopolystyrene (10–1000 μg/L) induced the more severe induction of germline apoptosis than pristine nanopolystyrene (10–1000 μg/L) (Fig. 5.10) [45]. In nematodes, CED-3, CED-4, and CED-9 constitute the core molecular basis for apoptosis [2, 3]. Exposure to the pristine nanopolystyrene (10 μg/L, a predicted environmental concentration) significantly increased the ced3 expression and decreased the ced-9 expression (Fig. 5.10) [45]. In contrast,

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Fig. 5.9 IFC-2 acts upstream of DAF-16 in the insulin signaling pathway to regulate the GO toxicity (reprinted with permission from [43]). (a) Genetic interaction between circ_0000115 and DAF-16 or PMK-1 in regulating the GO toxicity in inducing ROS production. (b) Genetic interaction between circ_0000115 and DAF-16 or PMK-1 in regulating the GO toxicity in

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exposure to amino modified pristine nanopolystyrene (10 μg/L) not only significantly decreased the ced-9 expression but also significantly increased expressions of both ced-3 and ced-4 (Fig. 5.10) [45]. Additionally, amino modified nanopolystyrene caused the more severe increase in ced-3 expression and decrease in ced-9 expression compared with those in pristine nanopolystyrene-exposed nematodes (Fig. 5.10) [45]. Moreover, RNAi knockdown of ced-3 or ced-4 suppressed the germline apoptosis induced by nanopolystyrene exposure, whereas RNAi knockdown of ced-9 enhanced the germline apoptosis induced by nanopolystyrene exposure [45]. These observations suggested the involvement of CED-3, CED-4, and CED-9 in regulating the induction of germline apoptosis in nematodes exposed to nanopolystyrene at ERCs in nematodes. In nematodes, HUS-1::GFP is a marker to reflect the induction of germline DNA damage [44]. After the exposure, the pristine nanopolystyrene (10 μg/L) could induce the obvious germline DNA damage (Fig. 5.11) [45]. Different from this, the noticeable germline DNA damage signals could be detected in amino modified nanopolystyrene (1 μg/L)-exposed nematodes (Fig. 5.11) [45]. Moreover, amino modified nanopolystyrene (10–1000 μg/L) could cause the more severe induction of germline DNA damage than pristine nanopolystyrene (10–1000 μg/L) (Fig. 5.11) [45]. In nematodes, CLK-2, CEP-1, EGL-1, and HUS-1 constitute the important molecular basis for DNA damage by forming the signaling cascade of HUS-1/CLK2-CEP-1-EGL-1, and this signaling cascade further acts upstream of CED-9 and CED-4-CED-3 to regulate the germline apoptosis [2, 3]. Exposure to the pristine nanopolystyrene (10 μg/L) significantly increased expressions of cep-1 and egl-1 (Fig. 5.11) [45]. In contrast, exposure to amino modified pristine nanopolystyrene (10 μg/L) could further significantly increase the expressions of all the examined three genes (Fig. 5.11) [45]. Meanwhile, amino modified nanopolystyrene exposure led to the more severe increase in expressions of clk-2, cep-1, and egl-1 compared with those in pristine nanopolystyrene-exposed nematodes (Fig. 5.11) [45]. Moreover, RNAi knockdown of clk-2, cep-1, or egl-1 inhibited the germline DNA damage induced by nanopolystyrene exposure [45]. These observations suggested the involvement of CLK-2, CEP-1, and EGL-1 in regulating the induction of reproductive toxicity in nematodes exposed to nanopolystyrene at ERCs.

Fig. 5.9 (continued) decreasing locomotion behavior. (c) Genetic interaction between IFC-2 and DAF-16 in regulating the GO toxicity in inducing ROS production. (d) Genetic interaction between IFC-2 and DAF-16 in regulating the GO toxicity in decreasing locomotion behavior. GO concentration is 100 μg/L. Prolonged exposure to GO was performed from L1-lavae to adult Day-1. Bars represent means
SD. **p < 0.01 vs. control (if not specially indicated)

5.4 Response of Wnt Signaling Pathway to Toxicants at ERCs

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Fig. 5.10 Amino modification enhanced the reproductive toxicity in inducing germline apoptosis in nematodes (reprinted with permission from [45]). (a) Images showing the induction of germline apoptosis. Asterisk indicates the germline apoptosis signal. Exposure concentrations were 1–1000 μg/L. n ¼ 50. (b) Comparison of effect of pristine and amino modified nanopolystyrene particles in inducing germline apoptosis. Exposure concentrations were 1–1000 μg/L. n ¼ 50. (c) Comparison of effect of pristine and amino modified nanopolystyrene particles in affecting expressions of genes required for the control of germline apoptosis. Exposure concentration was 10 μg/L. Exposure to nanopolystyrenes was performed from L1-larvae to adult Day-1. Bars represent means
SD. **p < 0.01 vs. control (if not specially indicated)

5.4

Response of Wnt Signaling Pathway to Toxicants at ERCs

In nematodes, the Wnt signaling pathway can regulate the toxicity induction of environmental toxicants, such as GO [46, 47]. In addition, the Wnt signaling pathway is also required for the control of response to environmental stresses (such as simulated microgravity stress) in nematodes [48, 49].

5.4.1

Alteration in Expression of Genes Encoding Wnt Signaling Pathway Induced by Toxicants at ERCs

The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [50]. In nematodes, the major components of canonical Wnt signaling pathway contain β-catenin BAR-1 (a transcriptional factor), the APC complex containing APR-1/Axin, PRY-1/CK1a, KIN-19, and GSK-3, Dishevelled proteins (MIG-5, DSH-1, and DSH-2), and Frizzled receptors (LIN-17, MOM-5, MIG-1, and

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Fig. 5.11 Amino modification enhanced the reproductive toxicity in inducing DNA damage in nematodes (reprinted with permission from [45]). (a) Images showing the induction of germline DNA damage. Asterisk indicates the germline HUS-1::GFP signal. Exposure concentrations were 1–1000 μg/L. n ¼ 50. (b) Comparison of effect of pristine and amino modified nanopolystyrene particles in inducing germline DNA damage. Exposure concentrations were 1–1000 μg/L. n ¼ 50. (c) Comparison of effect of pristine and amino modified nanopolystyrene particles in affecting expressions of genes required for the control of DNA damage. Exposure concentration was 10 μg/L. Exposure to nanopolystyrenes was performed from L1-larvae to adult Day-1. Bars represent means
SD. **p < 0.01 vs. control (if not specially indicated)

CFZ-2) [2, 3]. After the exposure, nanopolystyrene (0.1 μg/L) did not obviously alter expressions of all the examined genes, and exposure to nanopolystyrene (1 μg/L) also did not significantly affect the expressions of genes (lin-17, mom-5, mig-1, and cfz-2) encoding Frizzled receptors and genes (mig-5, dsh-1, and dsh-2) encoding Dishevelled proteins (Fig. 5.12) [50]. Among the genes encoding the APX complex, exposure to nanopolystyrene (1 μg/L) also did not significantly alter the expressions of apr-1, pry-1, and kin-19 (Fig. 5.12) [50]. Different from these, exposure to nanopolystyrene (1 μg/L) significantly decreased the expression of the gsk-3 encoding an ortholog of human GSK3A, a component of the APC complex, and increased the expression of the bar-1 encoding the canonical β-catenin (Fig. 5.12) [50]. Moreover, exposure to nanopolystyrene (1 μg/L) significantly increased the expression of intestinal BAR-1::mcherry (Fig. 5.12) [50]. Exposure to nanopolystyrene (10–1000 μg/L) did not affect the expressions of lin-17, mig-1, cfz-2, mig-5, pry-1, and kin-19 (Fig. 5.12) [50]. In contrast, the expressions of mom5, dsh-1, and dsh-2 could be increased by exposure to nanopolystyrene (10–1000 μg/ L) (Fig. 5.12) [50]. Expressions of apr-1 and gsk-3 could also be decreased by

5.4 Response of Wnt Signaling Pathway to Toxicants at ERCs

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Fig. 5.12 Effect of nanopolystyrene on expressions of genes encoding the canonical Wnt/β-catenin signaling pathway (reprinted with permission from [50]). (a) TEM image of nanopolystyrene in K medium. (b) Raman spectrum of nanopolystyrene. (c) Effect of nanopolystyrene on transcriptional expressions of genes encoding the canonical Wnt/β-catenin signaling pathway. Control, without nanopolystyrene exposure. Bars represent means
SD. **p < 0.01 vs. control. (d) Effect of nanopolystyrene on expression of intestinal BAR-1::mcherry. Control, without nanopolystyrene exposure. Exposure to nanopolystyrene was performed from L1-larvae to adult Day-3. Exposure concentration of nanopolystyrene was 1 μg/L. Bars represent means
SD. **p < 0.01 vs. control

exposure to nanopolystyrene (10–1000 μg/L) (Fig. 5.12) [50]. These results suggested the response of BAR-1 and GSK-3 to nanopolystyrene at ERCs in nematodes.

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5.4.2

5 Response of Development-Related Signaling Pathways to Toxicants at. . .

Functional Analysis of BAR-1 and GSK-3 in Regulating the Response to Toxicants at ERCs

The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [50]. Intestinal ROS production and locomotion behavior reflected by head thrash and body bend were employed as the endpoints [50]. Exposure to 1 μg/L nanopolystyrene induced the significant intestinal ROS production and the decrease in locomotion behavior (Fig. 5.13) [50]. In nanopolystyrene-exposed bar-1 mutant nematodes, the more severe induction of intestinal ROS production and decrease in locomotion behavior were observed compared with those in nanopolystyreneexposed wild-type nematodes (Fig. 5.13) [50]. Therefore, bar-1 mutation potentially induces a susceptibility to the nanopolystyrene toxicity. Moreover, in nanopolystyrene-exposed gsk-3 mutant nematodes, the suppression in intestinal ROS production and the increase in locomotion behavior were observed compared with those in nanopolystyrene exposed wild-type nematodes (Fig. 5.14) [50]. That is, compared with toxicity induction of nanopolystyrene in wild-type nematodes, the toxicity of nanopolystyrene could be obviously suppressed in gsk-3 mutant nematodes, which suggested that the gsk-3 mutation potentially induced a resistance to nanopolystyrene toxicity. Using the strain of gsk-3(tm1020);bar-1 (RNAi), it was further observed that the RNAi knockdown of bar-1 significantly

Fig. 5.13 Mutation of bar-1 induced a susceptibility to the nanopolystyrene toxicity in inducing intestinal ROS production (a) and in decreasing locomotion behavior (b) (reprinted with permission from [50]). Control, without nanopolystyrene exposure. Exposure concentration of nanopolystyrene was 1 μg/L. Exposure to nanopolystyrene was performed from L1-larvae to adult Day-3. Bars represent means
SD. **p < 0.01 vs. control (if not specifically indicated)

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Fig. 5.14 Genetic interaction between BAR-1 and GSK-3 in regulating the response to nanopolystyrene (reprinted with permission from [50]). (a) Mutation of gsk-3 induced a resistance to the nanopolystyrene toxicity in inducing intestinal ROS production. (b) Mutation of gsk-3 induced a resistance to the nanopolystyrene toxicity in decreasing locomotion behavior. (c) Genetic interaction between BAR-1 and GSK-3 in regulating the nanopolystyrene toxicity in inducing intestinal ROS production. (d) Genetic interaction between BAR-1 and GSK-3 in regulating the nanopolystyrene toxicity in decreasing locomotion behavior. Control, without nanopolystyrene exposure. Exposure concentration of nanopolystyrene was 1 μg/L. Exposure to nanopolystyrene was performed from L1-larvae to adult Day-3. Bars represent means
SD. **p < 0.01 vs. control (if not specifically indicated). NS no significance

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induced the intestinal ROS production and decreased the locomotion behavior in nanopolystyrene-exposed gsk-3 mutant nematodes (Fig. 5.14) [50]. That is, the RNAi knockdown of bar-1 could effectively suppress the resistance of gsk-3 (tm1020) mutant nematodes to the nanopolystyrene toxicity. Therefore, GSK-3 acted upstream of BAR-1 to regulate the response to nanopolystyrene at ERCs in nematodes.

5.4.3

Tissue-Specific Activity of BAR-1 in Regulating the Response to Toxicants at ERCs

In nematodes, BAR-1 is expressed in many tissues, including the intestine. The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [50]. Intestinal ROS production was used as the endpoint [50]. In nanopolystyreneexposed nematodes with the RNAi knockdown of bar-1 in epidermis, neurons, muscle, or germline, the similar intestinal ROS production to that in nanopolystyrene-exposed NR222, TU3401, WM118, or rrf-1 nematodes was detected (Fig. 5.15) [50]. That is, the RNAi knockdown of bar-1 in the epidermis, neurons, muscle, or germline did not significantly affect the nanopolystyrene toxicity. In contrast, the more severe intestinal ROS production and reduction in brood size in nanopolystyrene-exposed nematodes with the intestinal RNAi knockdown of bar-1 were observed compared with those in nanopolystyrene-exposed VP303 nematodes (Fig. 5.15) [50]. That is, the intestinal RNAi knockdown of bar-1 induced a susceptibility to the nanopolystyrene toxicity. Using VP303 as a genetic tool, it was further found that the suppression in intestinal ROS production and the increase in brood size could be detected in nanopolystyrene-exposed gsk-3(RNAi) nematodes compared with those in nanopolystyrene-exposed VP303 nematodes (Fig. 5.15) [50]. That is, the intestinal RNAi knockdown of gsk-3 induced a resistance to the nanopolystyrene toxicity. Thus, BAR-1 and GSK-3 could act in the intestine to regulate the response to nanopolystyrene at ERCs in nematodes.

5.4.4

Identification of Downstream Targets of Intestinal BAR-1 in Regulating the Response to Toxicants at ERCs

5.4.4.1

POP-1, DAF-16, and PRX-5

In nematodes, BAR-1 has a series of potential targets in regulating various biological processes, and some of them can be expressed in the intestine (https://www. wormbase.org). Among these possible intestinal targeted genes, exposure to

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Fig. 5.15 Tissue-specific activity of BAR-1 and GSK-3 in regulating the response to nanopolystyrene (reprinted with permission from [50]). (a) Tissue-specific activity of BAR-1 in regulating the response to nanopolystyrene. (b) GSK-3 acted in the intestine to regulate the response to nanopolystyrene. Control, without nanopolystyrene exposure. L4440, empty vector. Exposure concentration of nanopolystyrene was 1 μg/L. Exposure to nanopolystyrene was performed from L1-larvae to adult Day-3. Bars represent means
SD. **p < 0.01 vs. control (if not specifically indicated)

nanopolystyrene (1 μg/L) could significantly increase the expressions of pop-1, daf16, clec-60, clec-52, F53A9.8, sta-2, grd-12, mrp-5, prx-5, and daf-41 (Fig. 5.16) [50]. Moreover, intestinal RNAi knockdown of bar-1 could significantly decrease the expressions of pop-1, daf-16, and prx-5 in nematodes exposed to nanopolystyrene (1 μg/L) (Fig. 5.16) [50]. The increase in expression of bar-1, pop-1, or prx-5 was concentration dependent in nanopolystyrene (1–100 μg/L)exposed nematodes [50]. In nematodes, daf-16 encoding a FOXO transcriptional factor in the insulin signaling pathway acted in the intestine to regulate the response of nematodes to nanopolystyrene by affecting the functions of its targeted genes (sod-3, mtl-1, and gpd-2) [33]. Using VP303 as a genetic tool, intestinal RNAi knockdown of pop-1 or prx-5 also induced a susceptibility to the nanopolystyrene toxicity in inducing intestinal ROS production (Fig. 5.16) [50]. Therefore, POP-1,

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Fig. 5.16 Identification of downstream targets of BAR-1 in regulating the response to nanopolystyrene (reprinted with permission from [50]). (a) Effect of nanopolystyrene exposure on gene expressions. Bars represent means
SD. **p < 0.01 vs. control. (b) Effect of the intestinal RNAi knockdown of bar-1 on gene expressions in nematodes exposed to nanopolystyrene. Bars represent means
SD. **p < 0.01 vs. VP303. (c) Effect of the intestinal RNAi knockdown of pop-1 or prx-5 on nanopolystyrene toxicity in inducing intestinal ROS production. Bars represent means
SD. **p < 0.01 vs. control (if not specifically indicated). (d) Effect of the intestinal RNAi knockdown of pop-1 or daf-16 on gene expressions. Bars represent means
SD. ** p < 0.01 vs. VP303. (e) Genetic interaction between DAF-16 and PRX-5 in regulating the nanopolystyrene toxicity in inducing intestinal ROS production. Bars represent means
SD. ** p < 0.01 vs. control (if not specifically indicated). Control, without nanopolystyrene exposure. Exposure concentration of nanopolystyrene was 1 μg/L. Exposure to nanopolystyrene was performed from L1-larvae to adult Day-3

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DAF-16, and PRX-5 may act as downstream targets for intestinal BAR-1 in regulating the response to nanopolystyrene at ERCs in nematodes. In nematodes, BAR-1 mediates the canonical Wnt signaling by forming a BAR-1/ POP-1 physical complex to activate the expression of its downstream target genes. After the exposure to nanopolystyrene, the intestinal RNAi knockdown of pop-1 significantly decreased the expressions of daf-16 and prx-5 (Fig. 5.16) [50], which confirmed the role of POP-1 in mediating the function of BAR-1 in regulating the stress response. In nematodes, prx-5 encodes an ortholog of human receptor PEX (peroxisomal biogenesis factor). After the exposure to nanopolystyrene, the intestinal RNAi knockdown of daf-16 did not obviously alter the expression of prx-5 (Fig. 5.16) [50], which implied that PRX-5 and DAF-16 did not act in the same genetic pathway to regulate the response to nanopolystyrene. Meanwhile, the more severe induction of intestinal ROS production was observed in nanopolystyrene-exposed daf-16(RNAi);prx-5(RNAi) nematodes compared with that in nanopolystyrene-exposed daf-16(RNAi) or prx-5 (RNAi) nematodes (Fig. 5.16) [50], which suggested that DAF-16 and PRX-5 functioned synergistically to regulate the response to nanopolystyrene at ERCs in nematodes.

5.4.4.2

PRX-5-Mediated Signaling Cascade

During the regulation of various biological processes, PRX-5 has some potential targets, and at least four of them (ACS-1, PNK-1, ELO-5, and ELO-6) can be expressed in the intestine (https://www.wormbase.org). Receptor PEX5, an ortholog of PRX-5, can interact directly with proteins containing type 1 peroxisomal targeting signal (PTS1), and peroxisomal PTS1 proteins contain KAT-1, T02G5.7, ACOX-1.1, ACOX-1.2, ACOX-1.3, ACOX-1.4, ACOX-1.5, ACOX-1.6, and ACOX-3 in nematodes. Among these possible targeted genes, exposure to nanopolystyrene (1 μg/L) could only significantly decrease the expressions of kat1 and acox-1.6 (Fig. 5.17) [50]. Both KAT-1 and ACOX-1.6 can be expressed in the intestine (https://www.wormbase.org). Moreover, intestinal RNAi knockdown of prx-5 could significantly increase the expressions of kat-1 and acox-1.6 in nematodes exposed to nanopolystyrene (1 μg/L) (Fig. 5.17) [50]. Meanwhile, intestinal RNAi knockdown of prx-5 could not obviously affect the expression of daf-16 (Fig. 5.17) [50]. Using VP303 as a genetic tool, it was further observed that the intestinal RNAi knockdown of kat-1 or acox-1.6 induced a resistance to the nanopolystyrene toxicity in inducing intestinal ROS production (Fig. 5.17) [50], suggesting that KAT-1 and ACOX-1.6 may act as potential downstream targets for intestinal PRX-5 in regulating the response to nanopolystyrene. Genetic interaction analysis further demonstrated that the intestinal RNAi knockdown of kat-1 or acox-1.6 could effectively suppress the susceptibility of prx-5(RNAi) nematodes to the nanopolystyrene toxicity in inducing intestinal ROS production (Fig. 5.17) [50]. Therefore, KAT-1 and ACOX-1.6 acted downstream of intestinal PRX-5 in the peroxisome to regulate the response to nanopolystyrene at ERCs in nematodes.

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Fig. 5.17 KAT-1 and ACOX-1.6 acted downstream of PRX-5 to regulate the response to nanopolystyrene (reprinted with permission from [50]). (a) Effect of nanopolystyrene exposure on gene expressions. Control, without nanopolystyrene exposure. Exposure concentration of nanopolystyrene was 1 μg/L. Exposure to nanopolystyrene was performed from L1-larvae to adult Day-3. Bars represent means
SD. **p < 0.01 vs. control. (b) Effect of the intestinal RNAi knockdown of prx-5 on gene expressions in nematodes exposed to nanopolystyrene. Exposure concentration of nanopolystyrene was 1 μg/L. Exposure to nanopolystyrene was performed from L1-larvae to adult Day-3. Bars represent means
SD. **p < 0.01 vs. VP303. (c) Genetic interaction between PRX-5 and KAT-1 or ACOX-1.6 in regulating the nanopolystyrene toxicity in inducing intestinal ROS production. Control, without nanopolystyrene exposure. Exposure concentration of nanopolystyrene was 1 μg/L. Exposure to nanopolystyrene was performed from L1-larvae to adult Day-3. Bars represent means
SD. **p < 0.01 vs. control (if not specifically indicated). (d) A diagram showing the molecular basis for intestinal canonical Wnt/β-catenin signaling in regulating the response to nanopolystyrene at a predicted environmental concentration

5.5 Response of DBL-1-Mediated TGF-β Signaling Pathway to Toxicants at ERCs

5.5 5.5.1

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Response of DBL-1-Mediated TGF-β Signaling Pathway to Toxicants at ERCs Alteration in Expression of DBL-1 Induced by Toxicants at ERCs

DBL-1 is a TGF-β ligand in the TGF-β signaling pathway. The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [51–55]. In 1–1000 μg/L nanopolystyrene-exposed nematodes, the dbl-1 expression was significantly increased (Fig. 5.18) [52]. The dbl-1(RNAi) nematodes with RNAi knockdown of dbl-1 showed the more severe ROS production and decrease in locomotion behavior compared with those in wild-type nematodes after nanopolystyrene exposure (Fig. 5.18) [52], suggesting the susceptibility of dbl-1(RNAi) nematodes to nanopolystyrene toxicity. These observations suggested that the increase in TGF-β ligand DBL-1 mediated a protective response to nanopolystyrene at ERCs in nematodes.

5.5.2

Tissue-Specific Activity of DBL-1 in Regulating the Response to Toxicants at ERCs

In nematodes, DBL-1 is expressed in both the muscle and the neurons. Strains of WM118 and TU3410 were used to perform the RNAi knockdown of dbl-1 in muscle or neurons, respectively. The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [52]. Muscle RNAi knockdown of dbl-1 did not influence the nanopolystyrene toxicity in inducing ROS production in WM118 nematodes (Fig. 5.18) [52]. In contrast, neuronal RNAi knockdown of dbl-1 could cause the susceptibility to nanopolystyrene toxicity in inducing ROS production and in decreasing locomotion behavior in TU3401 nematodes (Fig. 5.18) [52]. Therefore, DBL-1 acted in the neurons to regulate the toxicity of nanopolystyrene at ERCs in nematodes.

5.5.3

Identification of Upregulators of DBL-1 in Regulating the Response to Toxicants at ERCs

Some possible upregulator genes have been raised for dbl-1, and some of the possible upregulator genes can be expressed in the neurons in nematodes (https:// www.wormbase.org). The nanopolystyrene (100 nm) was selected as the example of

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Fig. 5.18 Tissue-specific activity of DBL-1 in regulating the response to nanopolystyrene in nematodes (reprinted with permission from [52]). (a) Effect of nanopolystyrene exposure on dbl1 expression. (b) Tissue-specific activity of DBL-1 in regulating the toxicity of nanopolystyrene in inducing ROS production. The nanopolystyrene exposure concentration was 1 μg/L. L4440, empty vector. (c) Tissue-specific activity of DBL-1 in regulating the toxicity of nanopolystyrene in decreasing locomotion behavior. The nanopolystyrene exposure concentration was 1 μg/L. L4440, empty vector. The nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3. Bars represent means
SD. **p < 0.01 vs. control (if not specially indicated)

environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [52]. After the exposure, 1–1000 μg/L nanopolystyrene did not obviously alter the dpy-17, dpy-19, dpy-23, dpy-31, mua-3, and ceh-28 expressions (Fig. 5.19) [52]. However, 1–1000 μg/L nanopolystyrene could significantly increase the expressions of zag-1 and adt-2 and decrease the expression of smoc-1 (Fig. 5.19) [52]. In nanopolystyrene-exposed TU3401 nematodes, neuronal RNAi knockdown of smoc-1 significantly increased the dbl-1 expression, whereas neuronal RNAi knockdown of zag-1 or adt-2 significantly decreased the dbl-1 expression (Fig. 5.19) [52]. Neuronal RNAi knockdown of smoc-1 caused the resistance to the

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Fig. 5.19 Identification of SMOC-1, ZAG-1, and ADT-2 as upregulators of DBL-1 in regulating the response to nanopolystyrene in nematodes (reprinted with permission from [52]). (a) Effect of nanopolystyrene exposure on expressions of dpy-17, dpy-19, dpy-23, dpy-31, smoc-1, zag-1, adt-2, mua-3, and ceh-28. Bars represent means
SD. **p < 0.01 vs. control. (b) Effect of neuronal RNAi knockdown of smoc-1, zag-1, or adt-2 on dbl-1 expression in nanopolystyrene-exposed nematodes. The nanopolystyrene exposure concentration was 1 μg/L. L4440, empty vector. Bars represent means
SD. **p < 0.01 vs. TU3401. (c) Effect of neuronal RNAi knockdown of smoc-1, zag-1, or adt-2 on toxicity of nanopolystyrene exposure in inducing ROS production. The nanopolystyrene exposure concentration was 1 μg/L. L4440, empty vector. Bars represent means
SD. ** p < 0.01 vs. control (if not specially indicated). (d) Effect of neuronal RNAi knockdown of smoc-1, zag-1, or adt-2 on toxicity of nanopolystyrene exposure in decreasing locomotion behavior. The nanopolystyrene exposure concentration was 1 μg/L. L4440, empty vector. Bars represent means
SD. **p < 0.01 vs. control (if not specially indicated). The nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3

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nanopolystyrene toxicity in inducing ROS production and in decreasing locomotion behavior, and neuronal RNAi knockdown of zag-1 or adt-2 induced the susceptibility to the nanopolystyrene toxicity in inducing ROS production and in decreasing locomotion behavior (Fig. 5.19) [52]. These observations suggested that SMOC-1, ZAG-1, and ADT-2 acted as the upregulators of neuronal DBL-1 to regulate the nanopolystyrene toxicity at ERCs in nematodes. SMOC-1 is a secreted modular calcium-binding protein-1, ZAG-1 is a ZEB-family factor, and ADT-2 is an ADAMTS protein. The genetic interaction among SMOC-1, ZAG-1, and ADT-2 in regulating the nanopolystyrene toxicity was further determined. Neuronal RNAi knockdown of zag-1 or adt-2 inhibited the resistance of smoc-1(RNAi) nematodes to the nanopolystyrene toxicity in inducing ROS production and in decreasing locomotion behavior [52], suggesting that ZAG-1 and ADT-2 acted downstream of SMOC-1 to regulate the nanopolystyrene toxicity. Meanwhile, after the nanopolystyrene exposure, the zag-1(RNAi);adt-2(RNAi) nematodes showed the more severe toxicity as reflected by the induction of ROS production and decrease in locomotion behavior than zag-1(RNAi) or adt-2(RNAi) nematodes [52], suggesting that ZAG-1 and ADT-2 acted in different pathways to regulate the nanopolystyrene toxicity. Therefore, two signaling cascades (SMOC-1-ZAG-1 and SMOC-1-ADT-2) were formed to act upstream of neuronal DBL-1 to control the response to nanopolystyrene at ERCs in nematodes.

5.5.4

Identification of TGF-β Receptors in Regulating the Response to Toxicants at ERCs

DAF-4 and SMA-6 act as the receptors of DBL-1 in receiving cells. The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [52]. Nanopolystyrene (1–1000 μg/L) did not obviously alter daf-4 expression, whereas sma-6 expression was significantly increased by exposure to nanopolystyrene (1–1000 μg/L) (Fig. 5.20) [52]. Meanwhile, the sma-6(RNAi) nematodes showed the susceptibility to nanopolystyrene toxicity in inducing ROS production and in decreasing locomotion behavior (Fig. 5.20) [52]. SMA-6 is expressed in epidermis, intestine, neurons, and muscle. Strains of VP303, TU3401, WM118, and NR222 were employed to perform the RNAi knockdown of sma-6 in intestine, neurons, muscle, or epidermis, respectively. Neuronal, muscle, or epidermal RNAi knockdown of sma-6 did not influence nanopolystyrene toxicity in inducing ROS production in TU3401, WM118, and NR222 nematodes, respectively (Fig. 5.20) [52]. In contrast, after the nanopolystyrene exposure, intestinal RNAi knockdown of sma-6 resulted in the more severe induction of ROS production than that in VP303 nematodes (Fig. 5.20) [52]. Therefore, SMA-6 acted in the intestine to regulate the response to nanopolystyrene at ERCs in nematodes.

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Fig. 5.20 Tissue-specific activity of SMA-6 in regulating the response to nanopolystyrene in nematodes (reprinted with permission from [52]). (a) Effect of nanopolystyrene exposure on daf4 and sma-6 expressions. (b) Tissue-specific activity of SMA-6 in regulating the toxicity of nanopolystyrene in inducing ROS production. The nanopolystyrene exposure concentration was 1 μg/L. L4440, empty vector. (c) Effect of RNAi knockdown of sma-6 on locomotion behavior in nanopolystyrene-exposed wild-type nematodes. The nanopolystyrene exposure concentration was 1 μg/L. L4440, empty vector. The nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3. Bars represent means
SD. **p < 0.01 vs. control (if not specially indicated)

Therefore, an important neuron-intestine communication mediated by DBL-1/TGFβ signaling pathway was identified in regulating the nanopolystyrene toxicity. This identified neuron-intestine communication will be helpful for our understanding the transbiological tissue response to nanopolystyrene in organisms.

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Identification of Cytoplasmic Smads in Regulating the Response to Toxicants at ERCs

In the TGF-β signaling pathway, R-Smads (SMA-2 and SMA-3) and Co-Smad (SMA-4) function downstream of SMA-6 in the cytoplasm to regulate various biological processes. The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [52]. Nanopolystyrene (1–1000 μg/L) did not obviously alter sma-2 and sma-3 expressions; however, sma-4 expression was significantly increased by exposure to nanopolystyrene (1–1000 μg/L) (Fig. 5.21) [52]. The sma-4(RNAi) nematodes also showed the susceptibility to nanopolystyrene toxicity in inducing ROS production and in decreasing locomotion behavior (Fig. 5.21)

Fig. 5.21 Function of Co-Smad/SMA-4 in regulating the response to nanopolystyrene in nematodes (reprinted with permission from [52]). (a) Effect of nanopolystyrene exposure on sma-2, sma3, and sma-4 expressions. (b) SMA-4 could act in the intestine to regulate the toxicity of nanopolystyrene in inducing ROS production. The nanopolystyrene exposure concentration was 1 μg/L. L4440, empty vector. (c) Effect of RNAi knockdown of sma-4 on locomotion behavior in nanopolystyrene-exposed wild-type nematodes. The nanopolystyrene exposure concentration was 1 μg/L. L4440, empty vector. The nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3. Bars represent means
SD. **p < 0.01 vs. control (if not specially indicated)

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[52]. Moreover, intestinal RNAi knockdown of sma-4 induced the susceptibility to nanopolystyrene toxicity in inducing ROS production (Fig. 5.21) [52]. That is, the Co-Smad/SMA-4 could function in the cytoplasm of intestine to control the response to nanopolystyrene at ERCs in nematodes.

5.5.6

Identification of Transcriptional Factors in Regulating the Response to Toxicants at ERCs

In the TGF-β signaling pathway, transcriptional factors of SMA-9, MAB-31, and LIN-31 function downstream of SMA-4 in the nucleus to regulate biological processes. The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [52]. Exposure to nanopolystyrene (1–1000 μg/L) did not obviously alter lin-31 expression; however, sma-9 and mab-31 expressions were significantly increased by exposure to nanopolystyrene (1–1000 μg/L) (Fig. 5.22) [52]. The sma-9 (RNAi) or mab-31(RNAi) nematodes showed the susceptibility to nanopolystyrene toxicity in inducing ROS production and in decreasing locomotion behavior (Fig. 5.22) [52]. Moreover, intestinal RNAi knockdown of sma-9 or mab-31 could induce the susceptibility to nanopolystyrene toxicity in inducing ROS production (Fig. 5.22) [52]. Therefore, the transcriptional factors of SMA-9 and MAB-31 could act in the intestine to regulate the toxicity of nanopolystyrene at ERCs in nematodes. That is, a very simple signaling cascade of DBL-1-SMA-6-SMA-4-MAB-31/SMA9 in the DBL-1/TGF-β signaling pathway was raised in response to nanopolystyrene at ERCs in nematodes.

5.5.7

Identification of Downstream Targets of Intestinal SMA-9 and MAB-31 in Regulating the Response to Toxicants at ERCs

Some intestinal signaling pathways have been previously identified to be required for the control of nanopolystyrene toxicity [8, 22, 23, 25, 28, 33, 34, 50]. The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [52]. Among the genes in these intestinal signaling pathways, intestinal RNAi knockdown of sma-9 decreased elt-2 and hsp-6 expressions, and intestinal RNAi knockdown of mab-31 decreased daf-16 and sod-3 expressions (Fig. 5.23) [52]. Considering that the elt-2(RNAi) nematodes showed the deficit in intestinal barrier [34], erioglaucine disodium staining was performed to determine the intestinal permeability in sma-9(RNAi) nematodes. Nevertheless, the sma-9(RNAi) nematodes did not exhibit obvious alteration in intestinal permeability, and the blue dye was mainly

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Fig. 5.22 Transcriptional factors of SMA-9 and MAB-31 were involved in the regulation of response to nanopolystyrene in nematodes (reprinted with permission from [52]). (a) Effect of nanopolystyrene exposure on sma-9, mab-31, and lin-31 expressions. (b) SMA-9 and MAB-31 could act in the intestine to regulate the toxicity of nanopolystyrene in inducing ROS production. The nanopolystyrene exposure concentration was 1 μg/L. L4440, empty vector. (c) Effect of RNAi knockdown of sma-9 or mab-31 on locomotion behavior in nanopolystyrene-exposed wild-type nematodes. The nanopolystyrene exposure concentration was 1 μg/L. L4440, empty vector. The nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3. Bars represent means
SD. **p < 0.01 vs. control (if not specially indicated)

distributed in the intestinal lumen in sma-9(RNAi) nematodes as observed in VP303 nematodes (Fig. 5.23) [52]. Considering that the HSP-6 is a marker of mitochondrial unfolded protein response (mt UPR) [8], the mt UPR activation in nanopolystyreneexposed sma-9(RNAi) or elt-2(RNAi) nematodes was also investigated. Nanopolystyrene exposure activated a pronounced mt UPR, and this activated mt UPR could be suppressed in nanopolystyrene-exposed sma-9(RNAi) or elt-2(RNAi) nematodes (Fig. 5.23) [52]. Therefore, the intestinal transcriptional factor SMA-9 acted upstream of signaling cascade of ELT-2-HSP-6 to control the toxicity of nanopolystyrene at ERCs in nematodes. Meanwhile, the intestinal transcriptional factor MAB-31 acted upstream of signaling cascade of DAF-16-SOD-3 to control

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Fig. 5.23 Identification of downstream targets of SMA-9 and MAB-31 in the intestine to regulate the response to nanopolystyrene in nematodes (reprinted with permission from [52]). (a) Effect of intestinal RNAi knockdown of sma-9 or mab-31 on gene expressions in nanopolystyrene-exposed nematodes. The nanopolystyrene exposure concentration was 1 μg/L. L4440, empty vector. The nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3. Bars represent means
SD. **p < 0.01 vs. VP303. (b) Effect of intestinal RNAi knockdown of sma-9 on intestinal permeability based on the erioglaucine disodium staining. The nematodes were stained by 5% erioglaucine disodium (a blue dye) for 3 h. (c) Effect of RNAi knockdown of sma-9 or elt-2 on HSP-6::GFP expression in nanopolystyrene-exposed nematodes. The nanopolystyrene exposure concentration was 1 μg/L. L4440, empty vector. The nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3. Bars represent means
SD. **p < 0.01 vs. L4440. (d) A diagram showing the molecular basis of DBL-1-mediated TGF-β signaling pathway in regulating the response to nanopolystyrene in nematodes

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the toxicity of nanopolystyrene at ERCs in nematodes. The raised signaling cascades of SMA-9-ELT-2-HSP-6 and MAB-31-DAF-16-SOD-3 further suggested the formation of important nucleus-mitochondria communication activated by DBL-1/ TGF-β signaling pathway in controlling the toxicity of nanopolystyrene at ERCs in nematodes.

5.6 5.6.1

Response of DAF-7-Mediated TGF-β Signaling Pathway to Toxicants Role of DAF-7 in Regulating the Response to Toxicants at ERCs

DAF-7 is another TGF-β ligand in the TGF-β signaling pathway. The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [51–55]. Exposure to nanopolystyrene (1 μg/L) could significantly increase the daf-7 expression (Fig. 5.24) [56]. In nanopolystyrene-exposed nematodes, RNAi knockdown of daf-7 significantly decreased the daf-1 expression (Fig. 5.24) [49]. Moreover, after the nanopolystyrene exposure, RNAi knockdown of daf-7 caused the susceptibility to the toxicity of nanopolystyrene in inducing ROS production and in decreasing locomotion behavior (Fig. 5.24) [56], suggesting the important role of DAF-7 in regulating the response to nanopolystyrene at ERCs in nematodes.

5.6.2

DAF-1, DAF-8, DAF-5, and DAF-3 Acted in the Intestine to Regulate the Response to Toxicants at ERCs

In the ligand DAF-7-mediated TGF-β signaling pathway, DAF-1 and DAF-4 are two TGF-β receptors, DAF-8 and DAF-14 are two R-Smads, and DAF-3 and DAF-5 are two transcriptional factors. The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [56]. Exposure to nanopolystyrene (1 μg/L) did not obviously affect the expressions of daf-4 and daf-14 in wild-type nematodes (Fig. 5.25) [56]. In contrast, exposure to nanopolystyrene (1 μg/L) significantly increased the expressions of daf-1 and daf-8 and decreased the daf-5 and daf-3 expressions in wild-type nematodes (Fig. 5.25) [56]. Meanwhile, intestinal RNAi knockdown of daf-1 or daf-8 caused the susceptibility to the toxicity of nanopolystyrene in VP303 nematodes (Fig. 5.25) [56]. Different from this, intestinal RNAi knockdown of daf-5 or daf-3 suppressed the toxicity in nanopolystyreneexposed VP303 nematodes (Fig. 5.25) [56], suggesting the resistance of daf-5(RNAi)

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Fig. 5.24 Role of DAF-7 in regulating the response to nanopolystyrene (reprinted with permission from [56]). (a) Effect of nanopolystyrene exposure on daf-7 expression. Bars represent means
SD. ** p < 0.01 vs. control. (b) Effect of daf-7 RNAi knockdown on daf-1 expression in nanopolystyrene-exposed nematodes. L4440, empty vector. Bars represent means
SD. ** p < 0.01 vs. wild type. (c) Effect of daf-7 RNAi knockdown on toxicity of nanopolystyrene in inducing ROS production. L4440, empty vector. Bars represent means
SD. **p < 0.01 vs. control (if not specially indicated). (d) Effect of daf-7 RNAi knockdown on toxicity of nanopolystyrene in decreasing locomotion behavior. L4440, empty vector. Bars represent means
SD. ** p < 0.01 vs. control (if not specially indicated). The nanopolystyrene exposure concentration was 1 μg/L. The nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3

or daf-3(RNAi) nematodes to the toxicity of nanopolystyrene. Therefore, in the DAF-7-mediated TGF-β signaling pathway, DAF-1, DAF-8, DAF-5, and DAF-3 could act in the intestine to regulate the toxicity of nanopolystyrene at ERCs in nematodes. In nanopolystyrene-exposed VP303 nematodes, intestinal RNAi knockdown of daf-1 significantly decreased the daf-8 expression (Fig. 5.25) [56]. Intestinal overexpression of DAF-1 (Is(Pges-1-daf-1)) obviously inhibited the toxicity in nanopolystyrene-exposed wild-type nematodes, suggesting the resistance of Is

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Fig. 5.25 DAF-1, DAF-8, DAF-5, and DAF-3 acted in the intestine to regulate the response to nanopolystyrene (reprinted with permission from [56]). (a) Effect of nanopolystyrene exposure on expressions of daf-4, daf-1, daf-8, daf-14, and daf-5. Bars represent means
SD. ** p < 0.01 vs. control. (b) Intestinal RNAi knockdown of daf-1, daf-8, daf-5, or daf-3 on toxicity of nanopolystyrene in inducing ROS production. L4440, empty vector. Bars represent means
SD. ** p < 0.01 vs. control (if not specially indicated). (c) Effect of intestinal RNAi knockdown of daf-1 on daf-8 expression in nanopolystyrene-exposed VP303 nematodes. L4440, empty vector. Bars represent means
SD. **p < 0.01 vs. VP303. (d) Effect of intestinal RNAi knockdown of daf-8 on expressions of daf-3 and daf-5 in nanopolystyrene-exposed VP303 nematodes. L4440, empty vector. Bars represent means
SD. **p < 0.01 vs. VP303. The nanopolystyrene exposure concentration was 1 μg/L. The nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3

(Pges-1-daf-1) nematodes to the toxicity of nanopolystyrene [56]. After the nanopolystyrene exposure, RNAi knockdown of daf-8 caused the susceptibility of daf-8(RNAi) nematodes to the toxicity of nanopolystyrene [56]. Moreover, RNAi knockdown of daf-8 could cause the significant induction of ROS production and decrease in locomotion behavior in nanopolystyrene-exposed Is(Pges-1-daf-1)

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nematodes [56]. Therefore, intestinal DAF-1 regulated the toxicity of nanopolystyrene at ERCs by activating the downstream DAF-8 in nematodes. In nanopolystyrene-exposed VP303 nematodes, intestinal RNAi knockdown of daf-8 significantly increased the expressions of daf-3 and daf-5 (Fig. 5.25) [56]. After the exposure, intestinal RNAi knockdown of daf-5 suppressed the susceptibility of daf-8(RNAi) nematodes to the toxicity of nanopolystyrene [56]. Similarly, intestinal RNAi knockdown of daf-3 also suppressed the susceptibility of daf8(RNAi) nematodes to the toxicity of nanopolystyrene [56]. Therefore, the intestinal DAF-8 regulated the toxicity of nanopolystyrene at ERCs by antagonizing the activity of downstream two transcriptional factors (DAF-3 and DAF-5) in nematodes. In the intestine, a signaling cascade of DAF-1-DAF-8-DAF-3/DAF-5 in TGF-β signaling pathway was raised to be required for the control of toxicity of nanopolystyrene at ERCs in nematodes. These observations further suggested that the alteration in signaling cascade of DAF-1-DAF-8-DAF-3/DAF-5 in TGF-β signaling pathway mediated a protective response to nanopolystyrene exposure. In nematodes, DAF-7 is a neuronal expressed protein. That is, the protective response of intestinal signaling cascade of DAF-1-DAF-8-DAF-3/DAF-5 in TGF-β signaling pathway to nanopolystyrene at ERCs was activated by the increase in neuronal DAF-7 in nanopolystyrene nematodes. During this process, the neuron-intestine communication may be an important mechanism for nematodes in response to nanoplastic exposure at ERCs in nematodes.

5.6.3

Genetic Interaction DAF-12 and DAF-3 or DAF-5 in Regulating the Response to Toxicants at ERCs

During the control of biological processes, nuclear hormone receptor DAF-12 acts as the downstream target of both DAF-3 and DAF-5. The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [56]. Intestinal overexpression of DAF-5 (Is(Pges-1-daf-5)) or DAF-3 (Is(Pges-1-daf-3)) enhanced the toxicity in nanopolystyrene-exposed wild-type nematodes (Fig. 5.26) [56], suggesting the susceptibility of Is(Pges-1-daf-5) and Is(Pges-1-daf-3) nematodes to the toxicity of nanopolystyrene. RNAi knockdown of daf-12 significantly suppressed the toxicity in nanopolystyrene-exposed wild-type nematodes (Fig. 5.26) [56], suggesting the resistance of daf-12(RNAi) nematodes to the toxicity of nanopolystyrene. Moreover, RNAi knockdown of daf-12 inhibited the toxicity induction in nanopolystyrene-exposed Is(Pges-1-daf-5) or Is(Pges-1-daf-3) nematodes (Fig. 5.26) [56], suggesting that RNAi knockdown of daf-12 could suppress the resistance of Is(Pges-1-daf-5) and Is(Pges-1-daf-3) nematodes to the toxicity of nanopolystyrene. Previous study has suggested that DAF-12 regulated the toxicity of nanopolystyrene by suppressing the activity of downstream FAT-6, a fatty acyl CoA

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Fig. 5.26 Genetic interaction DAF-12 and DAF-3 or DAF-5 in regulating the response to nanopolystyrene (reprinted with permission from [56]). (a) Genetic interaction DAF-12 and DAF-3 or DAF-5 in regulating the toxicity of nanopolystyrene in inducing ROS production. The nanopolystyrene exposure concentration was 1 μg/L. The nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3. Bars represent means
SD. **p < 0.01 vs. control (if not specially indicated). (b) Genetic interaction DAF-12 and DAF-3 or DAF-5 in regulating the toxicity of nanopolystyrene in decreasing locomotion behavior. The nanopolystyrene exposure concentration was 1 μg/L. The nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3. Bars represent means
SD. **p < 0.01 vs. control (if not specially indicated). (c) A diagram showing the underlying mechanism for intestinal mir-354 in regulating the response to nanopolystyrene in nematodes

5.7 Perspectives

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desaturase [23]. These findings suggested that the DAF-7-mediated TGF-β signaling pathway may regulate the response to nanopolystyrene at ERCs by modulating the fat metabolism-related signals in nematodes.

5.7

Perspectives

With the aid of high sensitivity of C. elegans to environmental exposures [1–4, 57– 64], we here first introduced and discussed the intestinal development-related molecular signals involved in the control of toxicants at ERCs. For this aspect, we focused on the introduction and discussion of functions of ELT-2, ACS-22, and IFC-2. These three proteins reflect two aspects of functions. ELT-2 and ACS-22 are required for the control of functional state of intestinal barrier, and IFC-2 is required for the development of intestinal lumen. During the control of response to toxicants at ERCs, ELT-2 acted upstream of two antimicrobial proteins (CLEC-63 and CLEC85) and ERM-1, another protein required for the control of functional state of intestinal barrier. In contrast, the downstream targets of ACS-22 and IFC-2 in regulating the response to toxicants at ERCs are still largely unclear. The response of cell death and DNA damage-related signaling pathways to toxicants at ERCs was also introduced and discussed. During the control of response to toxicants at ERCs, the cell death-related signaling pathway constituted by CED-9, CED-4, and CED-3 was identified. During the control of response to toxicants at ERCs, the signaling cascade of HUS-1/CLK-2-CEP-1-EGL-1 in the DNA damagerelated signaling pathway was further identified. Nevertheless, besides the germline, whether the cell death- and DNA damage-related signaling pathways act in other tissues to regulate the response to toxicants at ERCs remains largely unclear. In the canonical Wnt signaling pathway, exposure to toxicants (such as nanopolystyrene) at ERCs only significantly altered expressions of GSK-3 and BAR-1/β-catenin, but did not affect the expressions of Frizzled receptors, Dishevelled proteins, and other components in the APC complex. This observation suggested that, in nematodes exposed to toxicants at ERCs, some unknown upstream regulators and GPCRs activated the BAR-1/β-catenin signaling in the intestine. Moreover, in the intestine, the peroxisomal biogenesis factor PRX-5-mediated signaling cascade of PRX-5-KAT-1/ACOX-1.6 was identified to act downstream of signaling cascade of BAR-1-POP-1 to regulate the response to toxicants at ERCs. That is, the canonical Wnt signaling pathway mediated an important nucleusperoxisome communication in the intestine in regulating the response to toxicants at ERCs. In nematodes, both the DBL-1-mediated TGF-β signaling pathway and the DAF-7-mediated TGF-β signaling pathway were involved in the control of response to toxicants at ERCs. With the DBL-1-mediated TGF-β signaling pathway as an example, exposure to toxicants (such as nanopolystyrene) at ERCs only significantly altered the expressions of DBL-1, SMA-6, SMA-4, MAB-31, and SMA-9, but did not obviously affect the expressions of TGF-β receptor DAF-4, R-Smads of SMA-2

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and SMA-3, and transcriptional factor LIN-31. Therefore, a simple signaling cascade of DBL-1-SMA-6-SMA-4-MAB-31/SMA-9 was formed in the DBL-1/TGF-β signaling pathway to regulate the response to toxicants at ERCs. In this signaling pathway, DBL-1 acted in the neurons to regulate the response to toxicants at ERCs, and its function was under the control of two signaling cascades of SMOC1-ZAG-1 and SMOC-1-ADT-2. Meanwhile, the signaling cascade of SMA-6-SMA4-MAB-31/SMA-9 acted in the intestine to regulate the response to toxicants at ERCs. In the intestine, the transcriptional factor of SMA-9 regulated the response to toxicants at ERCs by affecting the activity of signaling cascade of ELT-2-HSP-6, which suggested that the ELT-2 signaling and the mt UPR signaling were under the control of TGF-β receptor in the intestine in regulating the response to toxicants at ERCs. Meanwhile, the transcriptional factor of MAB-31 regulated the response to toxicants at ERCs by affecting the activity of signaling cascade of DAF-16-SOD-3, which suggested that the FOXO transcriptional factor DAF-16 in the insulin signaling pathway can be activated by the alterations in expressions of both insulin receptor and TGF-β receptor in regulating the response to toxicants at ERCs.

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Chapter 6

Response of Metabolism-Related Signaling Pathways to Toxicants at Environmentally Relevant Concentrations

Abstract Exposure to environmental toxicants potentially induces alterations in some metabolic events. Meanwhile, some metabolism-related signaling pathways are required for the control of response to environmental toxicants. We here introduced and discussed the involvement of metabolism-related signaling pathways in regulating the response to toxicants at environmentally relevant concentrations (ERCs). In nematodes, the requirement of MDT-15, FAT-6, and NHR-8-mediated signaling pathways in regulating the response to toxicants at ERCs are first introduced and discussed. Moreover, the requirement of heme homeostasis-related signaling pathway in regulating the response to toxicants at ERCs was also discussed. Keywords Environmentally relevant concentrations · Metabolism-related signaling pathways · Response · Caenorhabditis elegans

6.1

Introduction

Caenorhabditis elegans is helpful for detecting the potential toxicity of various environmental toxicants or stresses [1–13]. Moreover, C. elegans is important for the toxicological study of different toxicants or stresses [14–22]. Especially, C. elegans has been proven to be a powerful model for the study of molecular toxicology at the whole animal level [2, 3, 23–27]. In nematodes, on the one hand, exposure to environmental toxicants or stresses can alter some metabolic events, such as fat storage [28]. On the other hand, some metabolism-related signaling pathways are involved in the regulation of toxicity of environmental toxicants or stresses in nematodes. In Chapter 8 of “Molecular Toxicology in Caenorhabditis elegans,” we have introduced the functions of metabolism-related signaling pathways in the regulation of toxicity of environmental toxicants or stresses in nematodes [2]. In this chapter, we further focused on the introduction and the discussion of metabolism-related signaling pathways in regulating the response to toxicants at environmentally relevant concentrations (ERCs) in nematodes.

© The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 D. Wang, Toxicology at Environmentally Relevant Concentrations in Caenorhabditis elegans, https://doi.org/10.1007/978-981-16-6746-6_6

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Response of MDT-15-Mediated Signaling Pathway to Toxicants at ERCs Increase in Fat Storage

In the recent years, C. elegans has been frequently used to assess the toxicity of nanopolystyrene at ERCs [29–40]. Using intestinal reactive oxygen species (ROS) production and locomotion behavior reflected by head thrash and body bend as the endpoints, exposure (L1-larvae to adult Day-3) to nanopolystyrene (100 nm) at concentrations 1 μg/L could cause the significant induction of intestinal ROS production and decrease in locomotion behavior in nematodes [41]. The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [42]. The concentration of 1 μg/L is a predicted environmental concentration for nanopolystyrene. Based on Sudan black staining, exposure to 0.1 μg/L nanopolystyrene did not influence the lipid accumulation (Fig. 6.1) [42]. Different

Fig. 6.1 Effect of nanopolystyrene exposure on lipid accumulation (reprinted with permission from [42]). (a) TEM image of nanopolystyrene particles in K medium. (b) Raman spectroscopy of nanopolystyrene particles. (c) Sudan black staining showing the effect of nanopolystyrene exposure on lipid accumulation. (d) Effect of nanopolystyrene on transcriptional expressions of sbp-1, nhr49, nhr-80, and mdt-15. Bars represent means  SD. **p < 0.01 vs. control

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from this, exposure to 1–100 μg/L nanopolystyrene caused the noticeable increase in lipid accumulation (Fig. 6.1) [42], suggesting the increase in fat storage in nematodes exposed to nanopolystyrene at ERCs.

6.2.2

Alteration in Expression of MDT-15 and SBP-1 by Toxicants at ERCs

The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [42]. In nematodes, NHR-80, NHR-49, SBP-1, and MDT-15 are four lipid metabolic sensors. It was observed that the exposure to nanopolystyrene (0.1–100 μg/L) did not obviously influence nhr-49 and nhr-80 expressions (Fig. 6.1) [42]. In contrast, exposure to nanopolystyrene (0.1–100 μg/L) increased sbp-1 and mdt-15 expressions (Fig. 6.1) [42], suggesting the response of MDT-15 and SBP-1 to nanopolystyrene at ERCs in nematodes.

6.2.3

Tissue-Specific Activity of MDT-15 and SBP-1 in Regulating the Response to Toxicants at ERCs

The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [42]. Locomotion behavior reflected by head thrash and body bend and intestinal ROS production were employed as the endpoints [42]. Using these endpoints, it was observed that the loss-of-function mutation of mdt-15 caused more severe toxicity in decreasing locomotion behavior and in inducing intestinal ROS production in nanopolystyrene (1 μg/L)-exposed nematodes compared with nanopolystyrene (1 μg/L)-exposed wild-type nematodes (Fig. 6.2) [42], suggesting the involvement of MDT-15 in regulating the response to nanopolystyrene at ERCs in nematodes. In nematodes, MDT-15 is expressed in neurons, intestine, and reproductive organs. Nevertheless, expression of mdt-15 in neurons or in germline did not obviously influence the susceptibility of mdt-15 mutant nematodes to nanopolystyrene toxicity in decreasing locomotion behavior and in inducing intestinal ROS production (Fig. 6.2) [42]. Different from this, expression of mdt-15 in intestine suppressed the toxicity of nanopolystyrene in decreasing locomotion behavior and in inducing intestinal ROS production in mdt-15 mutant nematodes (Fig. 6.2) [42]. Therefore, intestinal MDT-15 was involved in the control of response to nanopolystyrene at ERCs in nematodes. In nematodes, SBP-1 is exclusively expressed in the intestine. Using VP303 strain, the more severe intestinal ROS production was found in

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Fig. 6.2 Tissue activity of MDT-15 in regulating nanopolystyrene toxicity (reprinted with permission from [42]). (a) Tissue activity of MDT-15 in regulating nanopolystyrene toxicity in inducing intestinal ROS production. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). (b) Tissue activity of MDT-15 in regulating nanopolystyrene toxicity in decreasing locomotion behavior. Considering that the mdt-15 mutant shows deficit in locomotion behavior, the locomotion behavior was expressed as the ratio between nanopolystyrene treatment and control. Bars represent means  SD. **p < 0.01 vs. wild type (if not specially indicated). Exposure concentration of nanopolystyrene was 1 μg/L

nanopolystyrene-exposed nematodes with intestine-specific RNAi knockdown of sbp-1 compared with that in nanopolystyrene-exposed VP303 strain (Fig. 6.3) [42], suggesting that intestine-specific RNAi knockdown of sbp-1 caused the susceptibility to nanopolystyrene toxicity. Meanwhile, after the nanopolystyrene exposure, mutation of mdt-15 could significantly decrease the sbp-1 expression (Fig. 6.3) [42]. In nematodes, intestinal overexpression of MDT-15 caused a resistance to nanopolystyrene toxicity (Fig. 6.3) [42]. It was further observed that RNAi knockdown of sbp-1 effectively suppressed this resistance in nematodes overexpressing intestinal MDT-15 to nanopolystyrene toxicity in inducing intestinal ROS production (Fig. 6.3) [42], which suggested that MDT-15 acted upstream of SBP-1 to regulate the response to nanopolystyrene at ERCs in nematodes.

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Fig. 6.3 Genetic interaction between MDT-15 and SBP-1 in the intestine to regulate the response to nanopolystyrene (reprinted with permission from [42]). (a) Effect of intestine-specific RNAi knockdown of sbp-1 on nanopolystyrene toxicity in inducing intestinal ROS production. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). (b) Effect of mdt-15 mutation on sbp-1 expression in nanopolystyrene-exposed nematodes. Bars represent means  SD. ** p < 0.01 vs. wild type. (c) Effect of RNAi knockdown of sbp-1 on nanopolystyrene toxicity in inducing intestinal ROS production in nematodes overexpressing intestinal MDT-15. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). Exposure concentration of nanopolystyrene was 1 μg/L

6.2.4

Identification of Downstream Targets of Intestinal SBP-1 in Regulating the Response to Toxicants at ERCs

The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [42]. During the control of various biological processes, SBP-1 has some potential targets, and some of them are expressed in the intestine (https://www. wormbase.org). Among these intestinal targeted genes, exposure to nanopolystyrene (1 μg/L) significantly increased the expressions of fat-7, fat-6, fat-4, fat-2, hsp-4, and sod-3 and decreased elo-5, acs-2, and hpl-2 expressions in wild-type nematodes (Fig. 6.4) [42]. Meanwhile, intestinal RNAi knockdown of sbp-1 only decreased fat2, fat-6, fat-7, and hsp-4 expressions and increased the acs-2 expression in nanopolystyrene-exposed nematodes (Fig. 6.4) [42]. Moreover, intestine-specific

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Fig. 6.4 Identification of downstream targets of intestinal SBP-1 in regulating the response to nanopolystyrene (reprinted with permission from [42]). (a) Effect of nanopolystyrene exposure on gene expressions in wild-type nematodes. Bars represent means  SD. **p < 0.01 vs. control. (b) Effect of intestinal RNAi knockdown of sbp-1 on gene expressions in nanopolystyrene-exposed nematodes. Bars represent means  SD. **p < 0.01 vs. VP303. (c) Effect of intestinal RNAi knockdown of fat-2, fat-6, fat-7, acs-2, or hsp-4 on nanopolystyrene toxicity in inducing intestinal ROS production. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). (d) Genetic interaction between SBP-1 and FAT-6 or HSP-4 in regulating the nanopolystyrene toxicity in inducing intestinal ROS production. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). Exposure concentration of nanopolystyrene was 1 μg/L

RNAi knockdown of fat-6 or hsp-4 caused susceptibility to nanopolystyrene toxicity in inducing intestinal ROS production (Fig. 6.4) [42]. In contrast, intestine-specific RNAi knockdown of fat-2, fat-7, or acs-2 did not influence the nanopolystyrene toxicity in inducing intestinal ROS production (Fig. 6.4) [42].

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To confirm the role of FAT-6 and HSP-4 as the downstream targets of intestinal SBP-1 in regulating nanopolystyrene toxicity, the transgenic strain overexpressing intestinal SBP-1 was generated [42]. Intestinal overexpression of SBP-1 caused the resistance to nanopolystyrene toxicity in inducing intestinal ROS production (Fig. 6.4) [42]. Moreover, RNAi knockdown of fat-6 or hsp-4 could effectively inhibit the resistance of transgenic strain overexpressing intestinal SBP-1 to nanopolystyrene toxicity in inducing intestinal ROS production (Fig. 6.4) [42], which confirmed that FAT-6 and HSP-4 acted downstream of intestinal SBP-1 to regulate the response to nanopolystyrene at ERCs in nematodes.

6.3

Response of FAT-6-Mediated Signaling Pathway to Toxicants at ERCs

The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [42]. In nematodes, the fatty acyl CoA desaturase FAT-6 controls the synthesis of monounsaturated fatty acid acyl CoAs from saturated fatty acid acyl CoAs. Some antimicrobial proteins can act as potential downstream targets of FAT-6 in controlling the stress response [43], and some of them (irg-4, F49F1.7, cyp-35A3, cyp-35B1, dsh-23, cdr-1, oac-6, clec-67, and lys-7) are expressed in the intestine (https://www.wormbase.org). Among these nine intestinal antimicrobial genes, nanopolystyrene (1 μg/L) exposure could increase cyp-35A3, clec-67, and lys-7 expressions (Fig. 6.5) [42]. Meanwhile, intestinal RNAi knockdown of fat-6 could significantly decrease the expressions of cyp-35A3, clec-67, and lys-7 in nanopolystyrene-exposed nematodes (Fig. 6.5) [42]. Furthermore, intestine-specific RNAi knockdown of cyp-35A3, clec-67, or lys-7 could induce a susceptibility to nanopolystyrene toxicity in inducing intestinal ROS production (Fig. 6.5) [42], suggesting that CYP-35A3, CLEC-67, and LYS-7 acted as the potential targets of intestinal FAT-6 to regulate the response to nanopolystyrene at ERCs in nematodes.

6.4 6.4.1

Response of NHR-8-Mediated Signaling Pathway to Toxicants at ERCs Alteration in NHR-8 Expression by Toxicants at ERCs

The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [44]. In nematodes, NHR-8, a homolog of sterol-sensing nuclear hormone receptor, functions to regulate several aspects of metabolisms, including the fat metabolism. After the exposure, nanopolystyrene (1 μg/L) could induce a

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Fig. 6.5 Identification of several antimicrobial proteins as downstream targets of intestinal FAT-6 in regulating the response to nanopolystyrene (reprinted with permission from [42]). (a) Effect of nanopolystyrene exposure on gene expressions in wild-type nematodes. Exposure concentration of nanopolystyrene was 1 μg/L. Bars represent means  SD. **p < 0.01 vs. control. (b) Effect of intestinal RNAi knockdown of fat-6 on gene expressions in nanopolystyrene-exposed nematodes. Exposure concentration of nanopolystyrene was 1 μg/L. Bars represent means  SD. ** p < 0.01 vs. VP303. (c) Effect of intestinal RNAi knockdown of cyp-35A3, clec-67, or lys-7 on nanopolystyrene toxicity in inducing intestinal ROS production. Exposure concentration of nanopolystyrene was 1 μg/L. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). (d) A diagram showing the molecular basis for lipid metabolic response and its association with toxicity regulation in nanopolystyrene-exposed nematodes

significant increase in nhr-8 expression (Fig. 6.6) [44]. The increase in nhr-8 expression was concentration dependent in nanopolystyrene-exposed nematodes at the concentrations of 1–100 μg/L (Fig. 6.6) [44]. Moreover, exposure to nanopolystyrene (1 μg/L) also caused a significant increase in intestinal NHR-8::

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Fig. 6.6 Effect of nanopolystyrene on NHR-8 expression (reprinted with permission from [44]). (a) Effect of nanopolystyrene on transcriptional expressions of nhr-8. (b) Effect of nanopolystyrene (1 μg/L) on expression of intestinal NHR-8::mcherry. Bars represent means  SD. ** p < 0.01 vs. control

mcherry (Fig. 6.6) [44]. These observations suggested the response of NHR-8 to nanopolystyrene at ERCs in nematodes.

6.4.2

Functional Analysis of NHR-8 in Regulating the Response to Toxicants at ERCs

The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [44]. Intestinal ROS production and locomotion behavior reflected by head thrash and body bend were employed as the endpoints [44]. The more severe intestinal ROS production and decrease in locomotion behavior were observed in nanopolystyrene (1 μg/L)-exposed nhr-8 mutant nematodes compared with those in nanopolystyrene (1 μg/L)-exposed wild-type nematodes (Fig. 6.7) [44]. That is, mutation of nhr-8 induced a susceptibility to the nanopolystyrene toxicity. In nematodes, NHR-8 is exclusively expressed in intestinal cells. Using VP303 as a genetic tool, the more severe intestinal ROS production was observed in nanopolystyrene (1 μg/L)-exposed nematodes with intestine-specific RNAi knockdown of nhr-8 compared with that in nanopolystyrene (1 μg/L)-exposed VP303 nematodes (Fig. 6.7) [44], suggesting that intestine-specific RNAi knockdown of nhr-8 also induced a susceptibility to the nanopolystyrene toxicity. These observations suggested the involvement of intestinal NHR-8 in regulating the response to nanopolystyrene at ERCs in nematodes.

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Fig. 6.7 Mutation of nhr-8 induced a susceptibility to the toxicity of nanopolystyrene (reprinted with permission from [44]). (a) Mutation of nhr-8 induced a susceptibility to the toxicity of nanopolystyrene in inducing intestinal ROS production. (b) Mutation of nhr-8 induced a susceptibility to the toxicity of nanopolystyrene in decreasing locomotion behavior. (c) Intestine-specific RNAi knockdown of nhr-8 induced a susceptibility to the toxicity of nanopolystyrene in inducing intestinal ROS production. Exposure concentration of nanopolystyrene was 1 μg/L. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated)

6.4.3

Identification of Downstream Targets of NHR-8 in Regulating the Response to Toxicants at ERCs

The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [44]. During the control of various biological processes, NHR-8 has some potential targets. Some of them can be expressed in the intestine (https://www. wormbase.org). Among these intestinal targeted genes, exposure to nanopolystyrene (1 μg/L) could only significantly increase the expressions of daf-16, fat-2, fat-6, fat7, ugt-18, and pgp-6 and decrease the expression of daf-12 (Fig. 6.8) [44]. Meanwhile, intestinal RNAi knockdown of nhr-8 could further significantly increase the daf-12 expression and decrease the expressions of fat-2, fat-6, fat-7, ugt-18, and

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Fig. 6.8 Identification of downstream targets of intestinal NHR-8 in regulating the response to nanopolystyrene (reprinted with permission from [44]). (a) Effect of nanopolystyrene exposure on gene expressions. Bars represent means  SD. **p < 0.01 vs. control. (b) Effect of intestinal RNAi knockdown of nhr-8 on gene expressions in nanopolystyrene exposed nematodes. Bars represent means  SD. **p < 0.01 vs. VP303. (c) Effect of intestinal RNAi knockdown of daf-12, fat-2, fat-6, fat-7, ugt-18, or pgp-6 on nanopolystyrene toxicity in inducing intestinal ROS production. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). Exposure concentration of nanopolystyrene was 1 μg/L

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pgp-6 in nematodes exposed to nanopolystyrene (1 μg/L) (Fig. 6.8) [44]. Intestinal RNAi knockdown of nhr-8 did not obviously affect the daf-16 expression in nematodes exposed to nanopolystyrene (1 μg/L) (Fig. 6.8) [44]. Using intestinal ROS production as an endpoint, intestine-specific RNAi knockdown of daf-12 induced a resistance to nanopolystyrene toxicity, and intestinespecific RNAi knockdown of fat-6, ugt-18, or pgp-6 induced a susceptibility to nanopolystyrene toxicity (Fig. 6.8) [44]. In contrast, intestine-specific RNAi knockdown of fat-2 or fat-7 did not obviously affect the nanopolystyrene toxicity (Fig. 6.8) [44]. These observations suggest that the DAF-12, FAT-6, UGT-18, and PGP-6 acted as the potential downstream targets of intestinal NHR-8 in regulating the response to nanopolystyrene at ERCs in nematodes. In nematodes, DAF-12 is a nuclear hormone receptor, FAT-6 is a fatty acyl CoA desaturase, UGT-18 is a UDP-glucuronosyl transferase, and PGP-6 is a Pglycoprotein. Using VP303 as the genetic tool, it was found that intestine-specific RNAi knockdown of daf-12 could suppress the susceptibility of nhr-8(RNAi) nematodes to nanopolystyrene toxicity in inducing intestinal ROS production (Fig. 6.9) [44], suggesting that DAF-12 acted as a downstream target of intestinal NHR-8 in regulating the response to nanopolystyrene. Meanwhile, intestine-specific RNAi knockdown of fat-6 could further inhibit the resistance of daf-12(RNAi) nematodes to nanopolystyrene toxicity in inducing intestinal ROS production (Fig. 6.9) [44], suggesting that FAT-6 acted as a downstream target of intestinal DAF-12 in regulating the response to nanopolystyrene. Therefore, an intestinal signaling cascade of NHR-8-DAF-12-FAT-6 was identified to be involved in the control of response to nanopolystyrene at ERCs in nematodes. In nanopolystyrene-exposed nematodes, intestine-specific RNAi knockdown of fat-6 could significantly decrease the expressions of ugt-18 and pgp-6 (Fig. 6.10) [44]. Using intestinal ROS production and locomotion behavior as endpoints, intestinal FAT-6 overexpression caused a resistance to the nanopolystyrene toxicity in inducing intestinal ROS production and in decreasing locomotion behavior (Fig. 6.10) [44]. Moreover, RNAi knockdown of ugt-18 or pgp-6 could suppress the resistance of nematodes overexpressing intestinal FAT-6 to nanopolystyrene toxicity in inducing intestinal ROS production and in decreasing locomotion behavior (Fig. 6.10) [44]. These observations suggest that UGT-18 and PGP-6 acted as the downstream targets of intestinal FAT-6 in regulating the response to nanopolystyrene at ERCs in nematodes.

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Fig. 6.9 Intestinal signaling cascade of NHR-8-DAF-12-FAT-6 required for the response to nanopolystyrene (reprinted with permission from [44]). (a) Genetic interaction between NHR-8 and DAF-12 in the intestine to regulate the response to nanopolystyrene. Exposure concentration of nanopolystyrene was 1 μg/L. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). (b) Genetic interaction between DAF-12 and FAT-6 in the intestine to regulate the response to nanopolystyrene. Exposure concentration of nanopolystyrene was 1 μg/L. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). (c) Effect of nanopolystyrene exposure on fat storage. Bars represent means  SD. **p < 0.01 vs. control

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Fig. 6.10 Intestinal FAT-6 acted upstream of UGT-18 and PGP-6 to regulate the response to nanopolystyrene (reprinted with permission from [44]). (a) Effect of intestine-specific RNAi of fat6 on expressions of ugt-18 and pgp-6. Bars represent means  SD. **p < 0.01 vs. VP303. (b) Genetic interaction between FAT-6 and UGT-18 or PGP-6 in regulating the nanopolystyrene toxicity in inducing intestinal ROS production. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). (c) Genetic interaction between FAT-6 and UGT-18 or PGP-6 in regulating the nanopolystyrene toxicity in decreasing locomotion behavior. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). Exposure concentration of nanopolystyrene was 1 μg/L

6.5 Response of Heme Homeostasis-Related Signaling Pathway to Toxicants at ERCs

6.5 6.5.1

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Response of Heme Homeostasis-Related Signaling Pathway to Toxicants at ERCs Functional Analysis of GLB-10 in Regulating Toxicity of Toxicants at ERCs

The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [45]. In nematodes, glb-10 encodes a globin protein. In wild-type nematodes, exposure to 1–100 μg/L nanopolystyrene increased the expression of glb-10 [45]. Meanwhile, neuronal RNAi knockdown of glb-10 induced the susceptibility to the nanopolystyrene toxicity in inducing ROS production and in decreasing locomotion behavior [45], suggesting that the GLB-10 functioned in neuronal cells to control the toxicity of toxicants (such as nanopolystyrene) at ERCs in nematodes.

6.5.2

Downstream Neuronal Targets of GLB-10 in Regulating Toxicity of Toxicants at ERCs

The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [45]. In nematodes, aspartic protease HRG-7 acted in the neurons to regulate the heme homeostasis [46]. Meanwhile, hypoxia-inducible transcriptional factor HIF-1 regulates hypoxia stress, and EGL-9 affects HIF-1 activity during hypoxia stress [2, 47, 48]. HIF-1 and EGL-9 can be expressed in neuronal cells (https://www.wormbase.org). In wild-type nematodes, exposure to 1–100 μg/L nanopolystyrene increased the expressions of hrg-7, hif-1, and egl-9 (Fig. 6.11) [45]. In nematodes, JNK-1/JNK and MPK-1/ERM signals acted in the neurons to regulate the nanopolystyrene toxicity [49, 50]. Meanwhile, neuronal RNAi knockdown of glb-10 decreased the expressions of hrg-7 and hif-1 in nanopolystyreneexposed nematodes, but could not alter the expressions of egl-9, jnk-1, and mpk-1 in nanopolystyrene-exposed nematodes (Fig. 6.11) [45]. Nevertheless, neuronal RNAi knockdown of hif-1 could not affect the nanopolystyrene toxicity in inducing ROS production and in decreasing locomotion behavior (Fig. 6.11) [45]. In contrast, neuronal RNAi knockdown of hrg-7 caused the susceptibility to the nanopolystyrene toxicity in inducing ROS production and in decreasing locomotion behavior (Fig. 6.11) [45], suggesting that HRG-7 acted in the neurons to control the nanopolystyrene toxicity. That is, aspartic protease HRG-7 was identified as the target of neuronal GLB-10 in controlling nanopolystyrene toxicity. Therefore, during the control of nanopolystyrene toxicity, a signaling cascade of mir-76GLB-10-HRG-7 was formed in the neurons. Besides the neurons, HRG-7 can also be expressed in the intestine and muscle (https://www.wormbase.org). However,

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Fig. 6.11 Identification of downstream neuronal targets of GLB-10 in regulating the response to nanopolystyrene (reprinted with permission from [45]). (a) Effect of nanopolystyrene exposure on expressions of hrg-7, hif-1, and egl-9 in wild-type nematodes. Bars represent means  SD. ** p < 0.01 vs. control. (b) Effect of neuronal RNAi knockdown of glb-10 on expressions of hrg7, hif-1, egl-9, jnk-1, and mpk-1 in nanopolystyrene-exposed TU3401 nematodes. L4440, empty vector. Exposure concentration of nanopolystyrene was 1 μg/L. Bars represent means  SD. ** p < 0.01 vs. TU3401. (c) Effect of neuronal RNAi knockdown of hif-1 or hrg-7 on toxicity of nanopolystyrene in inducing ROS production. L4440, empty vector. Exposure concentration of nanopolystyrene was 1 μg/L. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). (d) Effect of neuronal RNAi knockdown of hrg-7 on toxicity of nanopolystyrene in decreasing locomotion behavior. L4440, empty vector. Exposure concentration of nanopolystyrene was 1 μg/L. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). Exposure was performed from L1-larvae to adult Day-3

intestinal or muscle RNAi knockdown of hrg-7 could not affect the nanopolystyrene toxicity in inducing ROS production and in decreasing locomotion behavior [45], suggesting that the HRG-7 did not function in the intestine or muscle to control the nanopolystyrene toxicity.

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6.5.3

149

Downstream Intestinal Targets of GLB-10 in Regulating Toxicity of Toxicants at ERCs

The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [45]. Besides the HRG-7, the heme homeostasis also require involvement of HRG-6, HRG-5, HRG-4, HRG-3, and HRG-1, and they functioned in the intestine to control heme homeostasis [51]. In wild-type nematodes, exposure to nanopolystyrene did not alter the expressions of hrg-1, hrg-3, hrg-4, and hrg-6; however, the hrg-5 expression was decreased by exposure to nanopolystyrene (Fig. 6.12) [45]. HRG-5 can be expressed in intestinal cells (https://www. wormbase.org). Neuronal RNAi knockdown of glb-10 further increased the hrg-5 expression in nanopolystyrene-exposed worms (Fig. 6.12) [45]. Meanwhile, intestinal RNAi knockdown of hrg-5 suppressed the toxicity of nanopolystyrene in

Fig. 6.12 Identification of downstream intestinal targets of GLB-10 in regulating the response to nanopolystyrene (reprinted with permission from [45]). (a) Effect of nanopolystyrene exposure on expressions of hrg-1, hrg-3, hrg-4, hrg-5, and hrg-6 in wild-type nematodes. Bars represent means  SD. **p < 0.01 vs. control. (b) Effect of neuronal RNAi knockdown of glb-10 on expressions of hrg-5, daf-16, pmk-1, bar-1, and elt-2 in nanopolystyrene-exposed TU3401 nematodes. L4440, empty vector. Exposure concentration of nanopolystyrene was 1 μg/L. Bars represent means  SD. **p < 0.01 vs. TU3401. (c) Effect of intestinal RNAi knockdown of hrg-5 or hif-1 on toxicity of nanopolystyrene in inducing ROS production. L4440, empty vector. Exposure concentration of nanopolystyrene was 1 μg/L. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). Exposure was performed from L1-larvae to adult Day-3

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inducing ROS production (Fig. 6.12) [45], indicating the resistance of hrg-5(RNAi) nematodes to the nanopolystyrene toxicity. HIF-1 can also be expressed in intestinal cells (https://www.wormbase.org). Intestinal RNAi knockdown of hif-1 induced the susceptibility to the nanopolystyrene toxicity in inducing ROS production (Fig. 6.12) [45]. That is, both HRG-5 and HIF-1 could act in intestinal cells to control the nanopolystyrene toxicity. The p38 MAPK, insulin, Wnt, and ELT-2 signaling pathways acted in intestinal cells to regulate the nanopolystyrene toxicity [23, 24, 31, 41]. DAF-16 is a FOXO transcriptional factor in the insulin signaling pathway, BAR-1 is β-catenin in the Wnt signaling pathway, and PMK-1 is p38 MAPK in the p39 MAPK signaling pathway. After the nanopolystyrene exposure, neuronal RNAi knockdown of glb-10 did not alter the expressions of daf-16, pmk-1, and bar-1, whereas neuronal glb-10 RNAi knockdown further decreased the elt-2 expression (Fig. 6.12) [45]. Therefore, HRG-5, HIF-1, and ELT-2 might act as downstream targets in the intestine of GLB-10 to regulate the toxicity of toxicants (such as nanopolystyrene) at ERCs in nematodes. Moreover, after the nanopolystyrene exposure, intestinal RNAi knockdown of hrg-5 increased the expressions of hif-1 and elt-2 (Fig. 6.13) [45]. Meanwhile, using ROS production as the endpoint, intestinal RNAi knockdown of hif-1 or elt-2 inhibited the resistance of hrg-5(RNAi) nematodes to the nanopolystyrene toxicity (Fig. 6.13) [45], which confirmed the role of HIF-1 and ELT-2 as the downstream targets of intestinal HRG-5 in regulating the nanopolystyrene toxicity. In nematodes, ELT-2 regulated nanoplastic toxicity by activating both ERM-1 signaling and CLEC-63/85 signaling [23]. Both ELT-2 and ERM-1 are required for the maintenance of intestinal barrier [23]. CLEC-63 and CLEC-85 are two antimicrobial proteins involved in the control of innate immunity. That is, the expression of intestinal HRG-5 may enhance the nanopolystyrene toxicity (such as the ROS production) by suppressing the functional state of intestinal barrier and the innate immune response. The detailed mechanism for intestinal HIF-1 in controlling the nanopolystyrene toxicity needs the further elucidation. Moreover, after the exposure to nanopolystyrene, double intestinal RNAi knockdown of hif-1 and elt-2 resulted in the more severe ROS production in VP303 nematodes compared with that in hif-1 (RNAi) or elt-2(RNAi) nematodes (Fig. 6.13) [45], which suggested that HIF-1 and ELT-2 functions in different pathways to regulate the nanoplastic toxicity.

6.5.4

Interaction Between HRG-7 and HRG-5 in Controlling the Toxicity of Toxicants at ERCs

The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [45]. In wild-type nematodes, RNAi knockdown of hrg-7 resulted in the

6.5 Response of Heme Homeostasis-Related Signaling Pathway to Toxicants at ERCs

151

Fig. 6.13 Genetic interactions among HRG-5, HIF-1, and ELT-2 in the intestine to regulate the response to nanopolystyrene (reprinted with permission from [45]). (a) Effect of intestinal RNAi knockdown of hrg-5 on expressions of hif-1 and elt-2 in nanopolystyrene-exposed VP303 nematodes. L4440, empty vector. Exposure concentration of nanopolystyrene was 1 μg/L. Exposure was performed from L1-larvae to adult Day-3. Bars represent means  SD. **p < 0.01 vs. VP303. (b) Genetic interaction between HRG-5 and HIF-1 or ELT-2 in the intestine to regulate the toxicity of nanopolystyrene in inducing ROS production. L4440, empty vector. Exposure concentration of nanopolystyrene was 1 μg/L. Exposure was performed from L1-larvae to adult Day-3. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). (c) Genetic interaction between HIF-1 and ELT-2 in the intestine to regulate the toxicity of nanopolystyrene in inducing ROS production. L4440, empty vector. Exposure concentration of nanopolystyrene was 1 μg/L. Exposure was performed from L1-larvae to adult Day-3. Bars represent means  SD. ** p < 0.01 vs. control (if not specially indicated). (d) A diagram showing the molecular mechanism of neuronal mir-76 in regulating the response to nanopolystyrene in nematodes

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Fig. 6.14 Genetic interaction between HRG-7 and HRG-5 in regulating the response to nanopolystyrene (reprinted with permission from [45]). (a) Genetic interaction between HRG-7 and HRG-5 in regulating the toxicity of nanopolystyrene in inducing ROS production. (b) Genetic interaction between HRG-7 and HRG-5 in regulating the toxicity of nanopolystyrene in decreasing locomotion behavior. L4440, empty vector. Exposure concentration of nanopolystyrene was 1 μg/ L. Exposure was performed from L1-larvae to adult Day-3. Bars represent means  SD. ** p < 0.01 vs. control (if not specially indicated)

susceptibility to the nanopolystyrene toxicity in inducing ROS production and in decreasing locomotion behavior, and RNAi knockdown of hrg-5 led to the resistance to the nanopolystyrene toxicity in inducing ROS production and in decreasing locomotion behavior (Fig. 6.14) [45]. Moreover, RNAi knockdown of hrg-5 inhibited the susceptibility of hrg-7(RNAi) nematodes to the nanopolystyrene toxicity in inducing ROS production and in decreasing locomotion behavior (Fig. 6.14) [45]. Therefore, a signaling cascade of HRG-7-HRG-5 required for the control of heme homeostasis was confirmed to be involved in controlling nanopolystyrene toxicity. For this signaling cascade, HRG-5 acted in the intestine to regulate the nanopolystyrene toxicity, and its function in intestinal cells was antagonized by the neuronal HRG-7 (Fig. 6.14) [45]. The signaling cascade of HRG-7-HRG-5 mediated an important neuron-intestine communication in regulating the toxicity of toxicants (such as nanopolystyrene) at ERCs in nematodes.

6.6 Perspectives

6.6

153

Perspectives

Based on the well-elucidated molecular backgrounds [2, 3, 52–57], C. elegans can provide a useful platform to determine the molecular basis for the involvement of metabolism-related signals in regulating the toxicity of toxicants at ERCs. In nematodes, although there are four lipid metabolic sensors, exposure to toxicants (such as nanopolystyrene) at ERCs only induced the significant increase in expressions of MDT-15 and SBP-1, but did not obviously affect the expressions of NHR-80 and NHR-49. These observations suggested that only limited number of genes required for the control of fat storage were involved in the regulation of response to toxicants at ERCs. In the intestine, the MDT-15 and the SBP-1 formed a signaling cascade of MDT-15-SBP-1 to regulate the response to toxicants at ERCs. Moreover, in the intestine, both FAT-6 and HSP-4 were identified as the downstream targets of transcriptional factor SBP-1 in regulating the response to toxicants at ERCs. Therefore, besides the FAT-6-mediated molecular signaling, the HSP-4-mediated ER UPR activation was under the control of signaling cascade of MDT-15-SBP-1 in the intestine in regulating the response to toxicants at ERCs. Among the genes encoding fatty acyl CoA desaturases required for the fatty acid synthesis, so far only the FAT-6 was found to be required for the control of response to toxicants at ERCs. More importantly, three antimicrobial proteins of CYP-35A3, CLEC-67, and LYS-7 were identified as the downstream targets of FAT-6 in the intestine in regulating the response to toxicants at ERCs. Therefore, the activation of innate immune response in the intestine was further under the control of FAT-6 in regulating the response to toxicants at ERCs. That is, in the intestine, the signaling cascade of MDT-15-SBP-1 regulated the response to toxicants at ERCs by modulating both the ER UPR activation and the activation of innate immune response. In nematodes, the nuclear hormone receptor NHR-8 controls several aspects of metabolisms by inhibiting the activity of another nuclear hormone receptor DAF-12. The signaling cascade of NHR-8-DAF-12 further acted in the intestine to regulate the response to toxicants at ERCs. Moreover, the FAT-6 was identified as the downstream target of DAF-12 in regulating the response to toxicants at ERCs, suggesting the function of NHR-8-DAF-12 signaling cascade in controlling the lipid metabolism was also required for the response to toxicants at ERCs. Besides the antimicrobial proteins (CYP-35A3, CLEC-67, and LYS-7), two detoxification proteins (UGT-18 and PGP-6) were also identified as the downstream targets of FAT-6 in the intestine in regulating the response to toxicants at ERCs. Therefore, in the intestine, both the activation of innate immune response and the detoxification system were under the control of FAT-6 during the regulation of response to toxicants at ERCs. In nematodes, the heme homeostasis-related signaling pathway was also required for the control of toxicity of toxicants at ERCs. Different from the roles of MDT-15/ SBP-1 signaling and NHR-8 signaling, the heme homeostasis-related signaling acted in the neurons to regulate the toxicity of nanopolystyrene. More importantly, in this signaling pathway, the signaling cascade of HRG-7-HRG-5 mediated the

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neuron-intestine communication in regulating the toxicity of nanopolystyrene at ERCs. So far, at least JNK MAPK signaling, ERK MAPK signaling, DBL-1/TGFβ signaling, DAF-7/TGF-β signaling, and heme homeostasis-related signaling can mediate the neuron-intestine communication in regulating the toxicity of toxicants at ERCs [20, 37, 45, 49, 50].

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Chapter 7

Response of Protective Response-Related Signaling Pathways to Toxicants at Environmentally Relevant Concentrations

Abstract After the exposure to environmental toxicants or stresses, some protective molecular responses will be activated. Meanwhile, these protective response-related signaling pathways are potentially involved in the control of toxicity of toxicants or stresses. We here introduced and discussed the involvement of mitochondrial unfolded protein response (mt UPR), endoplasmic reticulum UPR (ER UPR), innate immune response, and autophagy-related signaling pathways in the regulation of response to toxicants at environmentally relevant concentrations (ERCs). The detailed protective response-related signaling pathways involved in the control of response to toxicants at ERCs were further discussed. Keywords Environmentally relevant concentrations · Protective response related signaling pathways · Response · Caenorhabditis elegans

7.1

Introduction

Caenorhabditis elegans is helpful to detect the toxicity of environmental toxicants or stresses at various aspects [1–15]. Especially, C. elegans can provide a powerful assay system for the study of molecular toxicology [2, 3, 16–24]. In nematodes, on the one hand, exposure to environmental toxicants or stresses can activate some protective response-related signaling pathways, such as those related to innate immune response, mitochondrial unfolded protein response (mt UPR), endoplasmic reticulum UPR (ER UPR), and autophagy. On the other hand, these protective response-related signaling pathways are involved in the control of toxicity of environmental toxicants or stresses. In Chapter 9 of “Molecular Toxicology in Caenorhabditis elegans,” we have introduced the functions of protective responserelated signaling pathways in the regulation of toxicity of environmental toxicants or stresses [2]. In this chapter, we mainly introduced and discussed the response of protective response-related signaling pathways to toxicants at environmentally relevant concentrations (ERCs) in nematodes.

© The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 D. Wang, Toxicology at Environmentally Relevant Concentrations in Caenorhabditis elegans, https://doi.org/10.1007/978-981-16-6746-6_7

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Response of Protective Response-Related Signaling Pathways to Toxicants at. . .

Response of mt UPR-Related Signaling Pathway to Toxicants at ERCs

In nematodes, the mt UPR could be activated or altered by different environmental toxicants or stresses [25, 26].

7.2.1

Dynamic Alteration in mt UPR Induced by Toxicants at ERCs During the Aging

In the recent years, the assay system of C. elegans has been frequently used to detect the potential toxicity of nanopolystyrene at ERCs [27–31]. Using intestinal reactive oxygen species (ROS) production and locomotion behavior reflected by head thrash and body bend as the endpoints, exposure (L1-larvae to adult Day-3) to nanopolystyrene (100 nm) at concentrations 1 μg/L could cause the significant induction of intestinal ROS production and decrease in locomotion behavior in nematodes [32]. The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [33]. Exposure to nanopolystyrene (1 or 10 μg/L) could further induce the more severe intestinal ROS production and decrease in locomotion behavior during the aging process (at adult Day-11) compared with control [33]. Meanwhile, a dynamic alteration in mt UPR was induced by nanopolystyrene during the aging process [33]. HSP-6 was employed as a marker for mt UPR [2– 4]. After the exposure (at adult Day-3), the expressions of hsp-6 was significantly increased by exposure to nanopolystyrene (1 μg/L, a predicted environmental concentration) [33], suggesting the activation of mt UPR induced by nanopolystyrene at ERCs. Moreover, it was observed that the hsp-6 expression in nanopolystyrene (1 μg/L)-exposed nematodes during the aging process (at adult Day-11) was lower than that in nanopolystyrene (1 μg/L)-exposed nematodes at adult Day-3 [33], suggesting the suppression in mt UPR activation in nanopolystyrene (1 μg/L)-exposed nematodes during the aging process.

7.2.2

Dynamic Alteration in mt UPR Induced by Toxicants at ERCs Under Simulated Microgravity Stress Condition

The nanopolystyrene (30 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from young adults for 24 h under the simulated microgravity stress condition [34]. Using brood size and locomotion behavior reflected by head thrash and body bend as endpoints, it was

7.2 Response of mt UPR-Related Signaling Pathway to Toxicants at ERCs

161

Fig. 7.1 Effect of nanopolystyrene in inducing mt UPR activation in nematodes under microgravity stress condition (reprinted with permission from [34]). Nanopolystyrene exposure was performed from young adults for 24 h. “+”, addition and/or treatment; “–”, without addition and treatment. Control, without both nanopolystyrene exposure and microgravity treatment. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated)

found that exposure to nanopolystyrene (10 μg/L, a predicted environmental concentration) enhanced the toxicity of simulated microgravity stress in nematodes with mutation of sod-3 encoding a Mn-SOD protein [34]. Under the microgravity stress condition, RNAi knockdown of sod-3 caused the more severe activation of mt UPR (Fig. 7.1) [34]. Moreover, in sod-3(RNAi) nematodes, exposure to nanopolystyrene (10 μg/L) further significantly enhanced the mt UPR activation induced by simulated microgravity stress (Fig. 7.1) [34]. These observations suggested the response of mt UPR to nanopolystyrene at ERCs in nematodes under the simulated microgravity stress condition.

7.2.3

Activation of Intestinal mt UPR Response in Nematodes Exposed to Toxicants at ERCs

The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [35]. HSP-6::GFP was used as a marker of mt UPR. Exposure to nanopolystyrene (0.1 μg/L) could not obviously alter expressions of both intestinal HSP-6::GFP and intestinal hsp-6 (Fig. 7.2) [35]. However, exposure to nanopolystyrene (1–100 μg/L) caused the significant increase in expressions of both intestinal HSP-6::GFP and intestinal hsp-6 (Fig. 7.2) [35]. Similarly, an increase in neuronal HSP-6::GFP expression was also observed in nanopolystyrene (1–100 μg/L)-exposed animals [35]. These observations suggested the potential

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Response of Protective Response-Related Signaling Pathways to Toxicants at. . .

Fig. 7.2 Activation of intestinal mt UPR response in nanopolystyrene-exposed nematodes (reprinted with permission from [35]). (a) Effect of nanopolystyrene exposure on hsp-6 expression in the intestine. Forty intact intestines were isolated for the qRT-PCR assay. (b) Effect of nanopolystyrene exposure on expression of HSP-6::GFP in the intestine. Exposure was performed from L1-larvae to adult Day-3. Bars represent means  SD. **p < 0.01 vs. control

activation of intestinal or neuronal mt UPR by toxicants (such as nanopolystyrene) at ERCs in nematodes.

7.2.4

Tissue-Specific Activity of HSP-6 in Regulating the Response to Toxicants at ERCs

The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [35]. HSP-6 can be expressed in several tissues, including the intestine (https://www.wormbase.org). ROS production and locomotion behavior were used as toxicity assessment endpoints. Strains of TU3401, VP303, DCL569, or WM118 are genetic tools for neuronal, intestinal, germline, or muscle RNAi knockdown of gene(s). Induction of ROS production, as well as decrease in locomotion behavior, could be observed in nanopolystyrene (1 μg/L)-exposed TU3401, VP303, DCL569, or WM118 nematodes (Fig. 7.3) [35]. Using these two endpoints, muscle or germline RNAi knockdown of hsp-6 did not alter the toxicity induction of nanopolystyrene (Fig. 7.3) [35]. However, neuronal or intestinal RNAi knockdown of hsp-6 resulted in the more severe nanoplastic toxicity compared with that in TU3401 or VP303 nematodes (Fig. 7.3) [35]. Thus, HSP-6 could function in both the intestine and the neurons to control the response to toxicants (such as nanoplastics) at ERCs in nematodes.

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Fig. 7.3 Tissue-specific activity of HSP-6 in regulating the response to nanopolystyrene (reprinted with permission from [35]). (a) Tissue-specific activity of HSP-6 in regulating the toxicity of nanopolystyrene in inducing ROS production. (b) Tissue-specific activity of HSP-6 in regulating the toxicity of nanopolystyrene in decreasing locomotion behavior. L4440, empty vector. Exposure concentration of nanopolystyrene was 1 μg/L. Exposure was performed from L1-larvae to adult Day-3. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated)

7.2.5

Identification of Upstream Regulators of Intestinal mt UPR Activation Induced by Toxicants at ERCs

Previous studies have identified some upstream upregulators for the control of mt UPR activation [2, 36–41]. Some of them can be expressed in the intestine, such as UBL-5, ATFS-1, HAF-1, CLPP-1, DVE-1, and NDUF-7. The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [35]. In wild-type nematodes, exposure to nanopolystyrene (1–100 μg/L) could not alter expressions of clpp-1, haf-1, and nduf-7 (Fig. 7.4) [35]. In contrast, exposure to nanopolystyrene (1–100 μg/L) increased expressions of atfs-1, dve-1, and ubl-5 (Fig. 7.4) [35]. Intestinal HSP-6::GFP expression was further inhibited in nanopolystyrene-exposed atfs1(RNAi), dve-1(RNAi), and ubl-5(RNAi) nematodes (Fig. 7.4) [35], suggesting the role of ATFS-1, DVE-1, and UBL-5 as the upstream regulators of intestinal mt UPR activation in nanopolystyrene-exposed worms. In nematodes, ATFS-1 is a leucine

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Fig. 7.4 Identification of upstream regulators of intestinal mt UPR activation in nanopolystyreneexposed nematodes (reprinted with permission from [35]). (a) Effect of nanopolystyrene exposure on expressions of clpp-1, haf-1, atfs-1, dve-1, ubl-5, and nduf-7 in wild-type nematodes. Bars represent means  SD. **p < 0.01 vs. control. (b) Effect of RNAi knockdown of atfs-1, dve-1, or ubl-5 on expression of intestinal HSP-6::GFP in nanopolystyrene-exposed nematodes. L4440, empty vector. Exposure concentration of nanopolystyrene was 1 μg/L. Bars represent means  SD. ** p < 0.01 vs. control (if not specially indicated). (c) Effect of RNAi knockdown of daf-2, daf-16, pmk-1, or bar-1 on expression of intestinal HSP-6::GFP in nanopolystyrene-exposed nematodes. L4440, empty vector. Exposure concentration of nanopolystyrene was 1 μg/L. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). Exposure was performed from L1-larvae to adult Day-3

zipper (bZIP) transcriptional factor, DVE-1 is a homeodomain-containing transcriptional factor, and UBL-5 is small ubiquitin-like protein. Previously studies have identified that insulin, p38 MAPK, and Wnt signaling pathways functioned in the intestine to regulate the nanoplastic toxicity [16, 17, 32], suggesting the crucial role of intestine in response to nanoplastic exposure. DAF-16 is a FOXO transcriptional factor, DAF-2 is an insulin receptor, PMK-1 is p38 MAPK, and BAR-1 is a β-catenin transcriptional factor. RNAi knockdown of pmk-1 could not affect intestinal HSP-6::GFP expression in nematodes after nanoplastic exposure (Fig. 7.4) [35]. Different from this, an enhanced induction of intestinal HSP-6::GFP expression was formed in daf-2(RNAi) animals after

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nanoplastic exposure, and RNAi knockdown of daf-16 or bar-1 suppressed intestinal HSP-6::GFP expression activated by exposure to nanopolystyrene (Fig. 7.4) [35]. Therefore, both the insulin signaling and the Wnt signaling could also act as upstream regulators of intestinal mt UPR response to toxicants (such as nanoplastics) at ERCs in nematodes. The nanopolystyrene (100 nm) was further selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [42]. It was further found that the activation of pronounced mt UPR by nanoplastic exposure could also be suppressed in nanopolystyreneexposed nematodes with RNAi knockdown of elt-2 encoding a GATA transcriptional factor [42]. Therefore, DAF-16, BAR-1, and ELT-2 were identified as upstream regulators of mt UPR activation induced by exposure to toxicants at ERCs in nematodes.

7.2.6

Downstream Targets for Intestinal Insulin, Wnt, or ELT-2 Signaling in Controlling Intestinal mt UPR Activation Induced by Toxicants at ERCs

The nanopolystyrene (100 nm) was further selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [35]. In VP303 animals exposed to nanoplastics, the increase in ubl-5 expression was observed in daf-2(RNAi) animals, and the decrease in ubl-5 expression was observed in daf-16(RNAi) animals (Fig. 7.5) [35]. In VP303 animals after nanoplastic exposure, the decrease in dve-1 expression could be detected in bar-1 (RNAi) animals (Fig. 7.5) [35]. In nanoplastic-exposed VP303 animals, the decrease in ubl-5 expression was formed in elt-2(RNAi) animals (Fig. 7.5) [35]. Intestinal overexpression of DAF-16, BAR-1, or ELT-2 caused an enhancement in intestinal mt UPR response to nanoplastic exposure (Fig. 7.5) [35]. Moreover, RNAi knockdown of ubl-5 suppressed this intestinal mt UPR activation in animals overexpressing intestinal DAF-16 after nanoplastic exposure (Fig. 7.5) [35], suggesting that UBL-5 functioned downstream of DAF-16 to control intestinal mt UPR response to nanoplastic exposure. Similarly, RNAi knockdown of dve-1 suppressed the intestinal mt UPR activation in animals overexpressing intestinal BAR-1 after nanoplastic exposure (Fig. 7.5) [35], suggesting that DVE-1 acted downstream of BAR-1 to control intestinal mt UPR response to nanoplastic exposure. Additionally, RNAi knockdown of atfs-1 inhibited the intestinal mt UPR activation in animals overexpressing intestinal ELT-2 after nanoplastic exposure (Fig. 7.5) [35], suggesting that ATFS-1 functioned downstream of ELT-2 to control intestinal mt UPR response to nanoplastic exposure. These observations demonstrated that the ATFS-1-, DVE-1-, and UBL-5-mediated activations of intestinal mt UPR were under the control of ELT-2, BAR-1, and DAF-16, respectively. That is, three signaling cascades of ELT-2-ATFS-1, BAR-1-DVE-1, and DAF-16-UBL-5

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Fig. 7.5 Identification of downstream targets of intestinal insulin, Wnt, or ELT-2 signaling in regulating the activation of intestinal mt UPR (reprinted with permission from [35]). (a) Effect of intestinal RNAi knockdown of daf-2, daf-16, bar-1, or elt-2 on expressions of atfs-1, dve-1, and ubl-5 in nanopolystyrene-exposed VP303 nematodes. L4440, empty vector. Exposure concentration of nanopolystyrene was 1 μg/L. Exposure was performed from L1-larvae to adult Day-3. Bars represent means  SD. **p < 0.01 vs. VP303. (b) Genetic interaction between DAF-16 and UBL-5 in regulating the intestinal mt UPR activation in nanopolystyrene-exposed nematodes. L4440, empty vector. Exposure concentration of nanopolystyrene was 1 μg/L. Exposure was performed from L1-larvae to adult Day-3. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). (c) Genetic interaction between BAR-1 and DVE-1 in regulating the intestinal mt UPR activation in nanopolystyrene-exposed nematodes. L4440, empty vector. Exposure concentration of nanopolystyrene was 1 μg/L. Exposure was performed from L1-larvae to adult Day-3. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). (d) Genetic interaction between ELT-2 and ATFS-1 in regulating the intestinal mt UPR activation in nanopolystyreneexposed nematodes. L4440, empty vector. Exposure concentration of nanopolystyrene was 1 μg/L. Exposure was performed from L1-larvae to adult Day-3. Bars represent means  SD. ** p < 0.01 vs. control (if not specially indicated)

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were formed in the intestine to regulate the activation of intestinal mt UPR in nematodes exposed to toxicants at ERCs in nematodes.

7.2.7

Interactions Among Intestinal ATFS-1, DVE-1, and UBL-5 in Controlling the Toxicity of Toxicants at ERCs

The nanopolystyrene (100 nm) was further selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [35]. Nanoplastic exposure could induce the significant induction of ROS production in VP303 animals [35]. Intestinal RNAi knockdown of atfs-1, dve1, or ubl-5 caused more severe toxicity in VP303 animals after nanoplastic exposure [35], indicating the susceptibility of atfs-1(RNAi), dve-1(RNAi), and ubl-5(RNAi) animals to nanoplastic toxicity. Intestinal overexpression of DAF-16, BAR-1, or ELT-2 induced a resistance to nanoplastic toxicity in inducing ROS production and in decreasing locomotion behavior in wild-type nematodes [35]. The resistance to nanoplastic toxicity in animals overexpressing intestinal DAF-16 could be inhibited by RNAi knockdown of ubl-5 [35], suggesting that UBL-5 functioned downstream of DAF-16 to control the nanoplastic toxicity. Similarly, the resistance to nanoplastic toxicity in animals overexpressing intestinal BAR-1 could be suppressed by RNAi knockdown of dve-1 [35], suggesting that DVE-1 acted downstream of BAR-1 to regulate the nanoplastic toxicity. Additionally, the resistance to nanoplastic toxicity in animals overexpressing intestinal ELT-2 could be further inhibited by RNAi knockdown of atfs-1 [35], suggesting ATFS-1 functioned downstream of ELT-2 to regulate the nanoplastic toxicity. Moreover, it was found that double intestinal RNAi knockdown of atfs-1 and dve1 caused more severe toxicity compared with that in atfs-1(RNAi) or dve-1(RNAi) animals after nanopolystyrene exposure (Fig. 7.6) [35]. Double intestinal RNAi knockdown of atfs-1 and ubl-5 resulted in more severe toxicity compared with that in animals with atfs-1 RNAi or ubl-5 RNAi after nanoplastic exposure (Fig. 7.6) [35]. Additionally, double intestinal RNAi knockdown of dve-1 and ubl5 induced more severe toxicity compared with that in dve-1(RNAi) or ubl-5(RNAi) animals after nanoplastic exposure (Fig. 7.6) [35]. Therefore, in the intestine, ATFS1, DVE-1, and UBL-5 acted in parallel pathways to control the toxicity of toxicants (such as nanopolystyrene) at ERCs in nematodes.

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Fig. 7.6 Genetic interactions among intestinal ATFS-1, DVE-1, and UBL-5 in regulating the toxicity of nanopolystyrene (reprinted with permission from [35]). (a) Genetic interactions among intestinal ATFS-1, DVE-1, and UBL-5 in regulating the toxicity of nanopolystyrene in inducing ROS production. L4440, empty vector. Exposure concentration of nanopolystyrene was 1 μg/L. Exposure was performed from L1-larvae to adult Day-3. Bars represent means  SD. ** p < 0.01 vs. control (if not specially indicated). (b) A diagram showing the molecular basis for the activation of mt UPR in the intestine in nanopolystyrene-exposed nematodes

7.3 7.3.1

Response of ER UPR-Related Signaling Pathway to Toxicants at ERCs Activation of ER UPR by Toxicants at ERCs

The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [43]. HSP-4::GFP is a fluorescent marker to reflect the induction of ER UPR caused by certain environmental or physiological changes [2, 3]. In nematodes, it was found that exposure to nanopolystyrene (1 μg/L) induced a significant increase in expression of intestinal HSP-4::GFP (Fig. 7.7) [43], suggesting the response of ER UPR to nanopolystyrene at ERCs in nematodes.

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Fig. 7.7 Genetic interaction between XBP-1 and GST-5 or HSP-4 in regulating the response to nanopolystyrene particles (reprinted with permission from [43]). (a) Induction of endoplasmic reticulum unfolding protein response in nematodes exposed to nanopolystyrene particles. Bars represent means  SD. **p < 0.01 vs. L4440 (if not specially indicated). (b) Effect of intestine-

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7.3.2

Upstream Regulators of ER UPR Activation Induced by Toxicants at ERCs

7.3.2.1

XBP-1 Signaling and p38 MAPK Signaling

The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [43]. It was first observed that RNAi knockdown of pmk-1, atf-7, skn-1, or xbp-1 significantly suppressed this induction of ER UPR in nematodes exposed to nanopolystyrene (1 μg/L) (Fig. 7.7) [43]. Different from this, RNAi knockdown of gst-5 did not obviously affect the induction of ER UPR in nematodes exposed to nanopolystyrene (1 μg/L) (Fig. 7.7) [43]. Therefore, PMK-1, ATF-7, SKN-1, and XBP-1 were required for the response of ER UPR to nanopolystyrene at ERCs in nematodes. PMK-1, ATF-7, and SKN-1 are components of p38 MAPK signaling pathway, and we have introduced and discussed the function of p38 MAPK signaling pathway in regulating the response to toxicants at ERCs in Chap. 4. XBP-1 is a transcriptional factor governing the induction of ER UPR in nematodes.

7.3.2.2

MDT-15 and SBP-1

In nematodes, MDT-15 and SBP-1 are two lipid metabolic sensors. Using transgenic strain of SJ4005/zcIs4[HSP-4::GFP] as a ER UPR marker, it was found that the activation of HSP-4::GFP induced by nanopolystyrene exposure was also significantly inhibited by RNAi knockdown of mdt-15 or sbp-1 (Fig. 7.8) [44]. Different from this, RNAi knockdown of fat-6 did not obviously influence this activation of HSP-4::GFP in nanopolystyrene-exposed nematodes (Fig. 7.8) [44]. These observations suggested that MDT-15 and SBP-1 also acted as upstream regulators of ER UPR activation in nanopolystyrene-exposed nematodes.

Fig. 7.7 (continued) specific RNAi knockdown of hsp-4 on the toxicity of nanopolystyrene particles in inducing ROS production. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). (c) Genetic interaction between XBP-1 and GST-5 or HSP-4 in regulating the toxicity of nanopolystyrene particles in inducing ROS production. Bars represent means  SD. ** p < 0.01 vs. wild type (if not specially indicated). (d) A diagram showing the molecular basis for intestinal p38 MAPK signaling pathway in regulating the toxicity of nanopolystyrene particles in nematodes. Prolonged exposure was from L1-larvae to adult Day-3. Exposure concentration of nanopolystyrene was 1 μg/L

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Fig. 7.8 MDT-15 and SBP-1 were required for the activation of ER UPR in nanopolystyreneexposed nematodes (reprinted with permission from [44]). (a) Effect of RNAi knockdown of mdt15, sbp-1, or fat-6 on activation of HSP-4::GFP in nanopolystyrene-exposed nematodes. (b) Genetic interaction between FAT-6 and HSP-4 in the intestine to regulate the nanopolystyrene toxicity in inducing intestinal ROS production. Exposure concentration of nanopolystyrene was 1 μg/L. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated)

7.3.2.3

Involvement of ER UPR Signaling Pathway in Regulating the Response to Toxicants at ERCs

The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [43]. In nematodes, hsp-4 encodes one of the two homologues of ER chaperone. Intestinal ROS production was used as the toxicity assessment endpoint. It was observed that intestine-specific RNAi knockdown of hsp-4 could induce a susceptibility to the toxicity of nanopolystyrene in inducing intestinal ROS production (Fig. 7.7) [43]. Intestinal overexpression of XBP-1 could induce a resistance to the toxicity of nanopolystyrene in inducing intestinal ROS production (Fig. 7.7) [43]. Moreover, it was observed that RNAi knockdown of hsp-4 significantly suppressed the resistance of nematodes overexpressing intestinal XBP-1 to the toxicity of nanopolystyrene in inducing intestinal ROS production (Fig. 7.7) [43]. In contrast, RNAi knockdown of gst-5 did not affect the resistance of nematodes overexpressing intestinal XBP-1 to the toxicity of nanopolystyrene in inducing intestinal ROS production (Fig. 7.7) [43]. These results suggest that HSP-4 acted

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downstream of XBP-1 to regulate the response to nanopolystyrene at ERCs in nematodes. Using intestinal ROS production as an endpoint, it was further found that RNAi knockdown of both hsp-4 and fat-6 caused the more severe toxicity compared with RNAi knockdown of hsp-4 or fat-6 alone in nanopolystyrene-exposed nematodes (Fig. 7.8) [44], which suggested that HSP-4 and FAT-6 functioned synergistically to regulate the response to nanopolystyrene at ERCs in nematodes.

7.4

Response of Antimicrobial Proteins to Toxicants at ERCs

In nematodes, the expression of antimicrobial proteins can be activated by environmental toxicants, such as graphene oxide (GO) [45, 46]. The expression of antimicrobial proteins can be also activated by environmental stresses (such as pathogen infection) in nematodes [47–51].

7.4.1

Alteration in Expressions of Antimicrobial Proteins by Toxicants at ERCs

7.4.1.1

CLEC-63 and CLEC-85

The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [52]. In nematodes, clec-63 and clec-85 encode two C-type lectin (CLEC) proteins with carbohydrate binding activity. After the exposure, nanopolystyrene (1 μg/L) could increase the expressions of clec-63 and clec-85 in nematodes [52].

7.4.1.2

CYP-35A3, CLEC-67, and LYS-7

The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [43]. In nematodes, CYP-35A3, CLEC-67, and LYS-7 are antimicrobial proteins expressed in the intestine. After the exposure, it was found that the nanopolystyrene (1 μg/L) exposure could also increase cyp-35A3, clec-67, and lys7 expressions in nematodes [44].

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Intestinal Antimicrobial Proteins

The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [33]. In nematodes, eight innate immune response genes (lys-1, lys-7, lys-8, spp-1, spp-12, F55G11.4, dod-6, and dod-22) are expressed in the intestine [45]. Among these genes, expressions of lys-1, lys-7, lys-8, and spp-1 were further significantly increased by exposure to nanopolystyrene (1 or 10 μg/L) at adult Day-3 [33]. Moreover, exposure to nanopolystyrene (1 μg/L) induced the severe decrease in expressions of lys-1, lys-7, lys-8, and spp-1 during the aging process (at adult Day-11) compared with those in nanopolystyrene (1 μg/L)-exposed nematodes at adult Day-3 [33]. Thus, a dynamic alteration in expression of antimicrobial genes was induced by nanopolystyrene at ERCs during the aging of nematodes.

7.4.2

Upstream Regulators of Antimicrobial Protein Activation Induced by Toxicants at ERCs

7.4.2.1

ELT-2

The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [52]. It was found that the intestinal RNAi knockdown of elt-2 could significantly decrease the expressions of F55G11.2, clec-63, and clec-85 in nanopolystyrene (1 μg/L)-exposed nematodes [52]. Moreover, under the normal conditions (without the nanopolystyrene exposure), the expressions of clec-63 and clec-85 could also be significantly decreased by intestinal RNAi knockdown of elt-2 [52]. These observations suggested that ELT-2 acted as an important upstream regulator of antimicrobial proteins activation induced by nanopolystyrene at ERCs in nematodes.

7.4.2.2

FAT-6

The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [44]. It was found that the intestinal RNAi knockdown of fat-6 could significantly decrease the expressions of cyp-35A3, clec-67, and lys-7 in nanopolystyrene (1 μg/L)-exposed nematodes [44], which suggested that the FAT-6 also acted as an important upstream regulator of antimicrobial proteins activation induced by nanopolystyrene at ERCs.

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7.4.3

Involvement of Antimicrobial Proteins in Regulating the Response to Toxicants at ERCs

7.4.3.1

CLEC-63 and CLEC-85

The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [52]. Intestinal ROS production was employed as an endpoint [52]. It was observed that the intestinal RNAi knockdown of clec-63 or clec-85 caused the susceptibility to toxicity of nanopolystyrene (1 μg/L) in inducing the intestinal ROS production [52], which suggested the involvement of clec-63 and clec-85 in regulating the response to nanopolystyrene at ERCs in nematodes.

7.4.3.2

CYP-35A3, CLEC-67, and LYS-7

The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [44]. Intestinal ROS production was employed as an endpoint [44]. It was observed that intestine-specific RNAi knockdown of cyp-35A3, clec-67, or lys-7 could also induce the susceptibility to toxicity of nanopolystyrene (1 μg/L) in inducing intestinal ROS production [44], which suggested the involvement of cyp35A3, clec-67, and lys-7 in regulating the response to nanopolystyrene at ERCs in nematodes.

7.5 7.5.1

Response of Autophagy-Related Signaling Pathway to Toxicants at ERCs Alteration in Autophagy Activation Induced by Toxicants at ERCs

Exposure to nanopolystyrene at ERCs could potentially cause multiple aspects of toxicity in nematodes [53–56]. The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-1 [57]. In nematodes, the LGG-1:: GFP is a marker of autophagy induction, and a significant increase in the number of LGG-1::GFP positive puncta was observed in wild-type nematodes exposed to nanopolystyrene particles (10 μg/L) compared with the controls (Fig. 7.9) [57]. In contrast, the observed increase in the number of LGG-1::GFP positive puncta in nanopolystyrene (10 μg/L)-exposed nematodes was significantly inhibited by exposure to higher levels of nanopolystyrene (1000 μg/L) (Fig. 7.9) [57]. In nematodes, LGG-1, LGG-2, ATG-18, and BEC-1 are key regulators of autophagy induction,

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Fig. 7.9 Dynamic autophagy induction in wild-type and mutant nematodes exposed to nanopolystyrene particles (reprinted with permission from [57]). (a) LGG-1::GFP positive puncta in intestinal cells of nematodes exposed to nanopolystyrene particles. Exposure concentrations of nanopolystyrene particles were 10 and 1000 μg/L. Bars represent means  SD. **p < 0.01 vs. control. (b) Effects of nanopolystyrene exposure on the expression of several genes required for autophagy control. Exposure concentrations of nanopolystyrene particles were 10 and 1000 μg/L. Bars represent means  SD. **p < 0.01 vs. control (if not specifically indicated). (c) Expression of several genes required for autophagy control in wild-type and mutant nematodes exposed to nanopolystyrene particles. Exposure concentration of nanopolystyrene particles was 1000 μg/L. Bars represent means  SD. **p < 0.01 vs. wild-type. (d) LGG-1::GFP positive puncta in intestinal cells of sod-3 or acs-22 mutant nematodes exposed to nanopolystyrene particles. Exposure concentration of nanopolystyrene particles was 1000 μg/L. Bars represent means  SD. ** p < 0.01 vs. control (if not specifically indicated). Prolonged exposure was performed from L1-larvae to adult Day-1

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and a significant increase in the expression of lgg-1, lgg-2, atg-18, and bec-1 was also detected in wild-type nematodes exposed to nanopolystyrene particles (10 μg/L) compared with the controls (Fig. 7.9) [57]. Additionally, the increase in the expression of these four genes in nanopolystyrene (10 μg/L)-exposed nematodes was further significantly suppressed by exposure to higher concentrations of nanopolystyrene (1000 μg/L) (Fig. 7.9) [57]. Thus, a dynamic alteration in autophagy activation existed in nanopolystyrene-exposed nematodes.

7.5.2

Upstream Regulators of Autophagy Activation Induced by Toxicants at ERCs

The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-1 [57]. In nematodes, mutation of sod-3 encoding a Mn-SOD induced the susceptibility to the toxicity of environmental toxicants, and mutation of acs-22 caused the deficit in functional state of intestinal barrier [2, 3]. The more severe decrease in the expressions of lgg-1, lgg-2, atg-18, and bec-1 was detected in nanopolystyrene-exposed sod-3 or acs-22 mutant nematodes compared with those in nanopolystyrene-exposed wild-type nematodes (Fig. 7.9) [57]. Additionally, exposure to nanopolystyrene also induced a more severe decrease in the number of LGG-1::GFP positive puncta in sod-3 or acs-22 mutant nematodes compared with that in wild-type nematodes (Fig. 7.9) [57]. Therefore, SOD-3 and ACS-22 acted as upstream regulators of autophagy activation in nanopolystyrene-exposed nematodes.

7.5.3

Involvement of Autophagy-Related Signaling Pathway in Regulating the Response to Toxicants at ERCs

The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-1 [57]. To determine the association between the autophagy induction and the neurotoxicity of nanopolystyrene, an RNAi knockdown of lgg-1 was performed in a transgenic strain of oxIs12 labeling the GABAergic D-type motor neurons [57]. RNAi knockdown of lgg-1 induced the severe formation of gaps on both the ventral and the dorsal cords and neuronal loss in nanopolystyrene-exposed nematodes (Fig. 7.10) [57]. Therefore, the RNAi knockdown of lgg-1 induced a susceptibility to neurotoxicity of nanopolystyrene on the development of D-type motor neurons in nematodes. Moreover, RNAi knockdown of lgg-1 also resulted in a more severe decrease in head thrash, body bend, and forward movement frequency and an increase in backward movement frequency compared with those in nematodes

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Fig. 7.10 Susceptibility to the neurotoxic effects of nanopolystyrene particles on the development and function of D-type GABAergic motor neurons in lgg-1(RNAi) nematodes (reprinted with permission from [57]). (a) Effect of RNAi knockdown of lgg-1 on the development of D-type motor neurons in nanopolystyrene-exposed nematodes. Asterisks indicate the neuronal loss, and arrowheads indicate the gap formation on the ventral or dorsal cord. Bars represent means  SD. ** p < 0.01 vs. control (if not specifically indicated). (b) Effect of RNAi knockdown of lgg-1 on locomotion behaviors in nanopolystyrene-exposed nematodes. Bars represent means  SD. ** p < 0.01 vs. control (if not specifically indicated). Exposure concentration of nanopolystyrene particles was 100 μg/L. Prolonged exposure was performed from L1-larvae to adult Day-1

without lgg-1 RNAi knockdown after nanopolystyrene exposure (Fig. 7.10) [57]. Therefore, RNAi knockdown of lgg-1 also induced the susceptibility to neurotoxicity of nanopolystyrene on the function of D-type motor neurons in

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nematodes. These observations suggested the role of LGG-1 in regulating the response to nanopolystyrene at ERCs in nematodes. In nematodes, HLH-30/TFEB is a MiT transcription factor governing the autophagy induction [2, 3]. HLH-30 overexpression effectively suppressed the neurodegeneration of D-type motor neurons induced by exposure to nanopolystyrene in Is(Phlh-30-hlh-30) nematodes (Fig. 7.11) [57]. Additionally, HLH-30 overexpression also effectively inhibited the damage of nanopolystyrene on locomotion behaviors (such as the decrease in head thrash, body bend, and forward movement frequency and the increase in backward movement frequency) in Is(Phlh-30-hlh-30) nematodes (Fig. 7.11) [57]. Meanwhile, it was further observed that, in the nanopolystyrene-exposed nematodes, overexpression of HLH-30 obviously prevented the decrease in LGG-1::GFP expression [57]. Thus, the HLH-30 was also involved in the control of response to nanopolystyrene at ERCs in nematodes.

7.6

Perspectives

Because of the high sensitivity to environmental exposures [1–4, 58–65], C. elegans is helpful for detecting the multiple protective responses to toxicants at ERCs. Mitochondrion is usually considered as a very sensitive organelle to exposure to various environmental toxicants or stresses. For the response of mt UPR-related signaling pathway to toxicants at ERCs, the dynamic alteration in mt UPR could be at least induced by toxicants at ERCs during the aging process or under the simulated microgravity stress condition. Similarly, under other stress conditions, exposure to toxicants at ERCs may also induce the activation of mt UPR. Meanwhile, long-term exposure to toxicants at ERCs may also activate the mt UPR response for nematodes against the induced toxicity. In nematodes, long-term exposure to toxicants (such as nanopolystyrene) at ERCs could induce the ER UPR response for nematodes against the toxicity. During the activation of ER UPR response, some upstream regulators have been identified. It has been found that at least XBP-1, p38 MAPK signaling, MDT-15, and SBP-1 acted as the upregulators for ER UPR activation in nematodes exposed to toxicants at ERCs. These upregulators for ER UPR activation acted in the intestine to exhibit their functions. Moreover, it was found that the intestinal ER UPR signaling pathway was also required for the control of response to toxicants at ERCs in nematodes. For example, the intestinal signaling cascade of XBP-1-HSP-4 was involved in the control of response to toxicants at ERCs. Normally, the innate immune response will be activated by nematodes against the toxicity induced by pathogen infection. Meanwhile, long-term exposure to toxicants (such as nanopolystyrene) at ERCs could further induce the innate immune response for nematodes against the toxicity. Exposure to different toxicants at ERCs may activate different expression patterns of antimicrobial genes. In nematodes, so far at least the ELT-2 and the FAT-6 have been identified as the upstream regulators of

7.6 Perspectives

179

Fig. 7.11 Resistance to the neurotoxic effects of nanopolystyrene particles on the development and function of D-type GABAergic motor neurons in nematodes overexpressing HLH-30 (reprinted with permission from [57]). (a) Effect of HLH-30 overexpression on the development of D-type motor neurons in nanopolystyrene-exposed nematodes. Asterisks indicate the neuronal loss, and arrowheads indicate the gap formation on the ventral or dorsal cord. Bars represent means  SD. ** p < 0.01 vs. control (if not specifically indicated). (b) Effect of HLH-30 overexpression on locomotion behaviors in nanopolystyrene-exposed nematodes. Bars represent means  SD. ** p < 0.01 vs. control (if not specially indicated). Exposure concentration of nanopolystyrene particles was 1000 μg/L. Prolonged exposure was performed from L1-larvae to adult Day-1

innate immune response induced by toxicants at ERCs. Meanwhile, the antimicrobial proteins (such as CLC-63, CLEC-85, CYP-35A3, CLEC-67, and LYS-7) were also involved in the control of response to toxicants at ERCs.

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In nematodes, besides the mt UPR, the ER UPR, and the innate immune response, the dynamic alteration in autophagy could also be detected after exposure to difference concentrations of toxicants. Usually, the toxicants at ERCs will activate the mt UPR, the ER UPR, the innate immune response, and the autophagy. So far, only the SOD-3 and the ACS-22 were identified as the potential upstream regulators for autophagy activation in nematodes exposed to toxicants at ERCs. More efforts are needed to identify the other potential upstream regulators for the activation of mt UPR, ER UPR, innate immune response, and autophagy after exposure to toxicants at ERCs. Moreover, the autophagy signaling pathway was also involved in the control of response to toxicants at ERCs in nematodes. For example, the signaling cascade of HLH-30-LGG-1 was involved in the control of response to toxicants at ERCs.

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Chapter 8

Response of Neurotransmission-Related Molecular Signals to Toxicants at Environmentally Relevant Concentrations

Abstract In nematodes, the genes required for the control of neurotransmission can also participate in the regulation of toxicity of environmental toxicants or stresses. We here introduced and discussed the response of neurotransmission-related molecular signals to toxicants at environmentally relevant concentrations (ERCs). We first discussed the requirement of neurotransmission to the response to toxicants at ERCs. Moreover, we introduced and discussed the neurotransmitter receptors and their downstream signals in different tissues to regulate the response to toxicants at ERCs. We also introduced and discussed the genes required for the neurotransmitter biosynthesis or transport in regulating the response to toxicants at ERCs. Keywords Environmentally relevant concentrations · Neurotransmission-related molecular signals · Response · Caenorhabditis elegans

8.1

Introduction

Caenorhabditis elegans has been frequently used to assess the potential toxicity of different environmental toxicants or stresses [1–16]. C. elegans is a powerful animal model for the study of molecular toxicology of various environmental toxicants, such as nanopolystyrene [17–28]. In nematodes, exposure to certain environmental toxicants (such as graphene oxide (GO)) could potentially affect the neurotransmission process as indicated by the alteration in genes required for the control of neurotransmitter or neuropeptide release [29, 30]. Meanwhile, the genes required for the control of neurotransmitter or neuropeptide release could be involved in the control of response to environmental toxicants in nematodes [29–31]. We have introduced the involvement of neurotransmission-related molecular signals in regulating the environmental toxicants or stresses in Chapter 9 of “Molecular Toxicology in Caenorhabditis elegans” and Chapter 1 of “Target Organ Toxicology in Caenorhabditis elegans” [2, 3]. In this chapter, we focused on the introduction and the discussion of response of neurotransmission-related molecular signals to toxicants at environmentally relevant concentrations (ERCs) in nematodes.

© The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 D. Wang, Toxicology at Environmentally Relevant Concentrations in Caenorhabditis elegans, https://doi.org/10.1007/978-981-16-6746-6_8

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Requirement of Neurotransmission to the Response to Toxicants at ERCs

In the recent years, C. elegans has been frequently employed as an animal model to detect the possible toxicity of nanopolystyrene at ERCs [32–44]. Using intestinal reactive oxygen species (ROS) production and locomotion behavior reflected by head thrash and body bend as the endpoints, exposure (L1-larvae to adult Day-3) to nanopolystyrene (100 nm) at concentrations
1 μg/L could cause the significant induction of intestinal ROS production and decrease in locomotion behavior in nematodes [45]. The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [46]. In nematodes, SNB-1/synaptobrevin is a vesicleassociated protein, which mediates the biological process of neurotransmitter release [47]. After the exposure, the nanopolystyrene (1 μg/L, a predicted environmental concentration) significantly increased the snb-1 expression [46]. In nematodes, jnk-1 encodes a neuronal JNK MAPK. Meanwhile, in nanopolystyrene-exposed nematodes, RNAi knockdown of jnk-1 could significantly decrease the snb-1 expression [46]. The more severe induction of intestinal ROS production and decrease in locomotion behavior could be observed in nanopolystyrene-exposed snb-1(RNAi) nematodes compared with those in nanopolystyrene-exposed wild-type nematodes (Fig. 8.1) [46], suggesting the potential role of SNB-1 in regulating the response to nanopolystyrene at ERCs. These observations have implied the involvement of neurotransmission in the regulation of response to nanopolystyrene at ERCs in nematodes. In nematodes, neuronal overexpression of JNK-1 could prevent the toxicity of nanopolystyrene in inducing intestinal ROS production and in decreasing locomotion behavior (Fig. 8.1) [46]. Moreover, it was further observed that RNAi knockdown of snb-1 could cause the significant induction of intestinal ROS production and decrease in locomotion behavior in nanopolystyrene-exposed nematodes overexpressing neuronal JNK-1 (Fig. 8.1) [46]. Therefore, SNB-1 acted as a downstream target of neuronal JNK-1/JNK MAPK to regulate the response to nanopolystyrene at ERCs in nematodes.

8.3 Genes Required for Neurotransmitter Biosynthesis or Transport Regulate the. . .

187

Fig. 8.1 Effect of snb-1 RNAi knockdown on nanopolystyrene toxicity in nematodes overexpressing neuronal JNK-1 (reprinted with permission from [46]). (a) Effect of snb-1 RNAi knockdown on nanopolystyrene toxicity in decreasing locomotion behavior. (b) Effect of snb-1 RNAi knockdown on nanopolystyrene toxicity in decreasing locomotion behavior in nematodes overexpressing neuronal JNK-1. Exposure concentration of nanopolystyrene was 1 μg/L. Exposure was performed from L1-larvae to adult Day-3. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated)

8.3

8.3.1

Genes Required for Neurotransmitter Biosynthesis or Transport Regulate the Response to Toxicants at ERCs Functions of TBH-1 and CAT-2 in Regulating Response to Toxicants at ERCs

The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [46]. In nematodes, the octopamine biosynthesis requires tyramine β-hydroxylase TBH-1 to convert tyramine into octopamine [48], and the dopamine biosynthesis requires tyrosine hydroxylase CAT-2 [49]. It was found that the nanopolystyrene exposure could significantly increase the tbh-1 expression and decrease the cat-2 expression (Fig. 8.2) [46], suggesting the potential involvement of octopamine and dopamine neurotransmissions in the regulation of response to nanopolystyrene at ERCs in nematodes. Meanwhile, RNAi knockdown of jnk-1 could cause the significant decrease in tbh-1 expression and increase in cat-2

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Fig. 8.2 Involvement of TBH-1 and CAT-2 in the control of response to nanopolystyrene (reprinted with permission from [46]). (a) Effect of nanopolystyrene exposure on expressions of tbh-1 and cat-2. Bars represent means  SD. **p < 0.01 vs. control. (b) Effect of jnk-1 RNAi knockdown on expressions of tbh-1 and cat-2 in nanopolystyrene-exposed nematodes. Bars represent means  SD. **p < 0.01 vs. wild type. (c) Effect of tbh-1 or cat-2 RNAi knockdown on nanopolystyrene toxicity in inducing intestinal ROS production. Bars represent means  SD. ** p < 0.01 vs. control (if not specially indicated). (d) Effect of tbh-1 or cat-2 RNAi knockdown on nanopolystyrene toxicity in decreasing locomotion behavior. Bars represent means  SD. ** p < 0.01 vs. control (if not specially indicated). Exposure concentration of nanopolystyrene was 1 μg/L. Exposure was performed from L1-larvae to adult Day-3

expression in nanopolystyrene-exposed nematodes (Fig. 8.2) [46]. Moreover, the more severe induction of intestinal ROS production and decrease in locomotion behavior were observed in nanopolystyrene-exposed tbh-1(RNAi) nematodes compared with nanopolystyrene-exposed wild-type nematodes (Fig. 8.2) [46]. In contrast, RNAi knockdown of cat-2 significantly inhibited the nanopolystyrene toxicity in inducing intestinal ROS production and in decreasing locomotion behavior (Fig. 8.2) [46]. Therefore, both TBH-1 and CAT-2 were involved in the regulation of response to nanopolystyrene at ERCs in nematodes.

8.3 Genes Required for Neurotransmitter Biosynthesis or Transport Regulate the. . .

189

Fig. 8.3 Genetic interaction between JNK-1 and TBH-1 or CAT-2 in regulating the nanopolystyrene toxicity in inducing intestinal ROS production (a) and in decreasing locomotion behavior (b) (reprinted with permission from [46]). Exposure concentration of nanopolystyrene was 1 μg/L. Exposure was performed from L1-larvae to adult Day-3. Bars represent means  SD. ** p < 0.01 vs. control (if not specially indicated)

In nematodes, it was further observed that RNAi knockdown of tbh-1 could induce the significant ROS production and decrease in locomotion behavior in nanopolystyrene-exposed nematodes overexpressing neuronal JNK-1 (Fig. 8.3) [46], which suggested that RNAi knockdown of tbh-1 could suppress the resistance of nematodes overexpressing neuronal JNK-1 to nanopolystyrene toxicity. Meanwhile, RNAi knockdown of cat-2 inhibited the induction of ROS production and the decrease in locomotion behavior in nanopolystyrene-exposed jnk-1(RNAi) nematodes (Fig. 8.3) [46], suggesting that the RNAi knockdown of cat-2 could suppress the susceptibility of jnk-1(RNAi) nematodes to nanopolystyrene toxicity. Thus, TBH-1 and CAT-2 acted downstream of JNK-1/JNK MAPK to regulate the response to nanopolystyrene at ERCs in nematodes.

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Response of Neurotransmission-Related Molecular Signals to Toxicants at. . .

Functions of TDC-1 and EAT-4 in Regulating Response to Toxicants at ERCs

In C. elegans, tyrosine decarboxylase TDC-1 is required for the tyramine synthesis, tryptophan hydroxylase TPH-1 is required for the serotonin synthesis, EAT-4 is a glutamate transporter, choline acetyltransferase CHA-1 is required for the acetylcholine synthesis, and glutamic acid decarboxylase UNC-25 is required for γ-aminobutyric acid (GABA) synthesis. The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [46]. In wild-type nematodes, exposure to nanopolystyrene (1–1000 μg/L) did not obviously affect the expressions of tph-1, cha-1, and unc-25 (Fig. 8.4) [50], which implied that no obvious responses of serotonin, acetylcholine, and GABA-related signals to nanopolystyrene exposure could be detected. In contrast, in nanopolystyrene (1–1000 μg/L)-exposed wild-type nematodes, the tdc-1 expression was significantly increased, and the eat-4 expression was significantly decreased (Fig. 8.4) [50]. That is, after exposure to nanopolystyrene at ERCs in nematodes, the tyramine neurotransmission might be enhanced, whereas the glutamate neurotransmission might be inhibited. Moreover, the more severe induction of ROS production was detected in tdc-1 (RNAi) nematodes, and the suppression in ROS production was observed in eat-4 (RNAi) nematodes after nanopolystyrene exposure (Fig. 8.4) [50]. Similarly, RNAi knockdown of tdc-1 enhanced the toxicity of nanopolystyrene in decreasing locomotion behavior, and RNAi knockdown of eat-4 inhibited the toxicity of nanopolystyrene in decreasing locomotion behavior in wild-type nematodes (Fig. 8.4) [50]. These observations suggested the susceptibility of tdc-1(RNAi) nematodes to the toxicity of nanopolystyrene and the resistance of eat-4(RNAi) nematodes to the toxicity of nanopolystyrene. Therefore, the alterations in expressions of tdc-1 and eat-4 mediated a protective response to nanopolystyrene at ERCs in nematodes. In nematodes, TDC-1 is expressed in both the neurons and the germline (https:// wormbase.org/). Exposure to nanopolystyrene (1 μg/L) could result in the significant induction of ROS production and/or decrease in locomotion behavior in TU3401 or DCL569 nematodes (Fig. 8.5) [50]. Germline RNAi knockdown of tdc-1 did not obviously affect the toxicity of nanopolystyrene in inducing ROS production in DCL569 nematodes (Fig. 8.5) [50]. In contrast, after the nanopolystyrene exposure, neuronal RNAi knockdown of tdc-1 caused the more severe induction of ROS production and decrease in locomotion behavior compared with those in TU3401 nematodes (Fig. 8.5) [50]. Thus, TDC-1 acted in the neurons to regulate the toxicity of nanopolystyrene at ERCs in nematodes. In nematodes, EAT-4 is expressed in both the neurons and the intestine (https:// wormbase.org/). Meanwhile, the intestinal RNAi knockdown of eat-4 could not obviously influence the toxicity of nanopolystyrene in inducing ROS production in VP303 nematodes (Fig. 8.5) [50]. Different from this, neuronal RNAi knockdown of eat-4 suppressed the toxicity of nanopolystyrene in inducing ROS production and in

8.3 Genes Required for Neurotransmitter Biosynthesis or Transport Regulate the. . .

191

Fig. 8.4 Involvement of TDC-1 and EAT-4 in the regulation of nanopolystyrene toxicity (reprinted with permission from [50]). (a) Effect of nanopolystyrene exposure on expressions of tdc-1, tph-1, eat-4, cha-1, and unc-25 in wild-type nematodes. Exposure was performed from L1-larvae to adult Day-3. Exposure concentrations of nanopolystyrene was 1–1000 μg/L. Bars represent means  SD. ** p < 0.01 vs. control. (b) Effect of RNAi knockdown of tdc-1 or eat-4 on toxicity of nanopolystyrene in inducing ROS production in wild-type nematodes. L4440, empty vector. Exposure was performed from L1-larvae to adult Day-3. Exposure concentration of nanopolystyrene was 1 μg/L. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). (c) Effect of RNAi knockdown of tdc-1 or eat-4 on toxicity of nanopolystyrene in decreasing locomotion behavior in wild-type nematodes. L4440, empty vector. Exposure was performed from L1-larvae to adult Day-3. Exposure concentration of nanopolystyrene was 1 μg/ L. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated)

decreasing locomotion behavior in nanopolystyrene-exposed TU3401 nematodes (Fig. 8.5) [50], which suggested that EAT-4 also acted in the neurons to regulate the toxicity of nanopolystyrene at ERCs in nematodes.

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Fig. 8.5 Tissue-specific activities of TDC-1 and EAT-4 in regulating the response to nanopolystyrene (reprinted with permission from [50]). (a) Effect of neuronal or germline RNAi knockdown of tdc-1 on toxicity of nanopolystyrene in inducing ROS production. (b) Effect of neuronal RNAi knockdown of tdc-1 on toxicity of nanopolystyrene in decreasing locomotion behavior. (c) Effect of neuronal or intestinal RNAi knockdown of eat-4 on toxicity of nanopolystyrene in inducing ROS production. (d) Effect of neuronal RNAi knockdown of eat-4 on toxicity of nanopolystyrene in decreasing locomotion behavior. L4440, empty vector. Exposure concentration of nanopolystyrene was 1 μg/L. Exposure was performed from L1-larvae to adult Day-3. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated)

8.4 Neurotransmitter Receptors Regulating the Response to Toxicants at ERCs

8.4 8.4.1

193

Neurotransmitter Receptors Regulating the Response to Toxicants at ERCs Intestinal Neurotransmitter Receptors Regulating the Response to Toxicants at ERCs

The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [46]. After the exposure, a large amount of nanopolystyrene particles were translocated and accumulated in the intestinal cells [8, 35]. It was assumed that the SNB-1-mediated neuronal neurotransmission may regulate the nanopolystyrene toxicity by activating the corresponding intestinal neurotransmitter receptors. Among the neurotransmitter receptors in nematodes, two octopamine receptors (OCTR-1 and SER-6), one tyramine receptor (TYRA-3), three serotonin receptors (SER-1, SER-3, and SER-5), three acetylcholine receptors (ACR-9, ACR-14, and ACR-17), and two dopamine receptors (DOP-1 and DOP-4) can be expressed in the intestinal cells (Table 8.1) [46]. Among the genes encoding these intestinal neurotransmitter receptors, exposure to nanopolystyrene could significantly decrease the expression of octr-1 and increase the expressions of tyra-3, ser-6, and dop-1 Table 8.1 Information for neurotransmitter receptors in nematodes (reprinted with permission from [46]) Receptor type Octopamine receptors Tyramine receptors Serotonin receptors Acetylcholine receptors Dopamine receptors GABA receptors Glutamate receptors Neuropeptide receptors

FMRFamide peptide receptors

Receptor genes octr-1, ser-6

Expressed in the intestine octr-1, ser-6

tyra-2, tyra-3, lgc-55

tyra-3

ser-1, ser-2, ser-3, ser-4, ser-5, ser-7

ser-1, ser-3, ser-5 acr-9, acr-14, acr-17

acr-2, acr-3, acr-5, acr-6, acr-7, acr-8, acr-9, acr-10, acr11, acr-12, acr-14, acr-15, acr-16, acr-17, acr-18, acr-19, acr-20, acr-21, acr-23, acr-24, acr-25 dop-1, dop-6, dop-4, dop-2, dop-5, dop-3 avr-14, gab-1, ggr-3, ggr-2, ggr-1 glr-1, glr-2, glr-3, glr-4, glr-5, glr-6, glr-7, glr-8 npr-1, npr-2, npr-3, npr-4, npr-5, npr-6, npr-7, npr-8, npr9, npr-10, npr-11, npr-12, npr-13, npr-14, npr-15, npr-16, npr-17, npr-18, npr-19, npr-20, npr-21, npr-22, npr-23, npr-24, npr-25, npr-26, npr-27, npr-28, npr-29, npr-30, npr-31, npr-32, npr-33, npr-34, npr-35 frpr-1, frpr-2, frpr-3, frpr-4, frpr-5, frpr-6, frpr-7, frpr-8, frpr-9, frpr-10, frpr-11, frpr-12, frpr-13, frpr-14, frpr-15, frpr-16, frpr-17, frpr-18, frpr-19

dop-1, dop-4

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Fig. 8.6 Identification of intestinal neurotransmitter receptors involved in the regulation of response to nanopolystyrene (reprinted with permission from [46]). (a) Effect of nanopolystyrene exposure on expressions of genes encoding intestinal neurotransmitter receptors. Bars represent means  SD. **p < 0.01 vs. control. (b) Effect of octr-1, tyra-3, ser-6, or dop-1 RNAi knockdown on nanopolystyrene toxicity in inducing intestinal ROS production. Bars represent means  SD. ** p < 0.01 vs. control (if not specially indicated). Exposure concentration of nanopolystyrene was 1 μg/L. Exposure was performed from L1-larvae to adult Day-3

(Fig. 8.6) [46]. Using VP303 as a genetic tool for intestine-specific RNAi knockdown of gene(s), it was found that intestine-specific RNAi knockdown of tyra-3 did not significantly affect the nanopolystyrene toxicity in inducing intestinal ROS production (Fig. 8.6) [46]. In contrast, intestine-specific RNAi knockdown of octr1 suppressed the nanopolystyrene toxicity in inducing intestinal ROS production, and intestine-specific RNAi knockdown of ser-6 or dop-1 caused the more severe induction of intestinal ROS production in nanopolystyrene-exposed nematodes compared with that in nanopolystyrene-exposed VP303 nematodes (Fig. 8.6) [46]. These observations suggested that intestinal octopamine receptors (OCTR-1 and SER-6) and intestinal dopamine receptor (DOP-1) were involved in the control of response to nanopolystyrene at ERCs in nematodes. To determine the octopamine and the dopamine-mediated communication between neurons and intestine, the effect of RNAi knockdown of cat-2 or tbh-1 on expression of intestinal genes encoding the corresponding neurotransmitter receptors in nanopolystyrene-exposed nematodes was examined [46]. In nanopolystyreneexposed nematodes, RNAi knockdown of cat-2 could significantly increase the expression of intestinal dop-1 (Fig. 8.7) [46]. Meanwhile, in nanopolystyreneexposed nematodes, RNAi knockdown of tbh-1 could significantly decrease the expression of intestinal ser-6 and increase the expression of intestinal octr-1

8.4 Neurotransmitter Receptors Regulating the Response to Toxicants at ERCs

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Fig. 8.7 Involvement of octopamine and dopamine signals in the regulation of response to nanopolystyrene (reprinted with permission from [46]). (a) Effect of RNAi knockdown of cat-2 on expression of intestinal dop-1 in nanopolystyrene exposed nematodes. qRT-PCR was performed in isolated intact intestines (n ¼ 40). Exposure concentration of nanopolystyrene was 1 μg/L. Exposure was performed from L1-larvae to adult Day-3. Bars represent means  SD. ** p < 0.01 vs. wild type. (b) Effect of RNAi knockdown of tbh-1 on expressions of intestinal ser-6 and octr-1 in nanopolystyrene-exposed nematodes. qRT-PCR was performed in isolated intact intestines (n ¼ 40). Exposure concentration of nanopolystyrene was 1 μg/L. Exposure was performed from L1-larvae to adult Day-3. Bars represent means  SD. **p < 0.01 vs. wild type. (c) Effect of intestinal RNAi knockdown of dop-1, octr-1, or ser-6 on expressions of pmk-1, mdt-15, or sbp-1 in nanopolystyrene-exposed nematodes. Exposure concentration of nanopolystyrene was 1 μg/L. Exposure was performed from L1-larvae to adult Day-3. Bars represent means  SD. ** p < 0.01 vs. VP303. (d) Effect of intestinal RNAi knockdown of dop-1, octr-1, or ser-6 on expression of daf-16 in nanopolystyrene-exposed nematodes. Exposure concentration of nanopolystyrene was 1 μg/L. Exposure was performed from L1-larvae to adult Day-3. Bars represent means  SD. **p < 0.01 vs. VP303. (e) A diagram showing the molecular basis of neuronal JNK MAPK signaling in regulating the response to nanopolystyrene in nematodes

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(Fig. 8.7) [46]. These observations suggested the interaction between CAT-2 or TBH-1 and their corresponding intestinal receptor(s) in regulating the response to nanopolystyrene at ERCs in nematodes.

8.4.2

Neuronal Neurotransmitter Receptors Regulating the Response to Toxicants at ERCs

8.4.2.1

Tyramine Receptors Involved in the Control of Response to Toxicants at ERCs

The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [46]. In nematodes, tyramine receptors contain TYRA-2, TYRA-3, and LGC-55. TYRA-2, TYRA-3, and LGC-55 are expressed in the neurons, and TYRA-3 can also be expressed in the intestine. Nevertheless, as indicated above, TYRA-3 did not act in the intestine to regulate the toxicity of nanopolystyrene [46]. In wild-type nematodes, exposure to nanopolystyrene (1–1000 μg/L) did not obviously affect the lgc-55 expression, but could significantly increase the expressions of tyra-2 and tyra-3 (Fig. 8.8) [50]. Neuronal RNAi knockdown of tyra-3 did not obviously affect the toxicity of nanopolystyrene in inducing ROS production in TU3401 nematodes (Fig. 8.8) [50]. In contrast, in TU3401 nematodes, neuronal RNAi knockdown of tyra-2 enhanced the toxicity of nanopolystyrene in inducing ROS production and in decreasing locomotion behavior (Fig. 8.8) [50]. Therefore, TYRA-2 acted as the corresponding receptor of tyramine to regulate the toxicity of nanopolystyrene at ERCs in nematodes.

8.4.2.2

Glutamate Receptors Involved in the Control of Response to Toxicants at ERCs

The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [46]. In nematodes, glutamate receptors contain GLR-1, GLR-2, GLR-3, GLR-4, GLR-5, GLR-6, GLR-7, and GLR-8, and all these glutamate receptors are expressed in the neurons. In wild-type nematodes, exposure to nanopolystyrene (1–1000 μg/L) did not obviously affect the expressions of glr-2, glr-5, glr-6, and glr-7 (Fig. 8.9) [50]. In contrast, exposure to nanopolystyrene (1–1000 μg/L) significantly increased the expressions of glr-1, glr-3, and glr-4 and decreased the glr-8 expression (Fig. 8.9) [50]. In nematodes, neuronal RNAi knockdown of glr-1 or glr-3 did not significantly influence the toxicity of nanopolystyrene in inducing ROS production in TU3401 nematodes (Fig. 8.9) [50]. Different from this, neuronal RNAi knockdown of glr-4

8.4 Neurotransmitter Receptors Regulating the Response to Toxicants at ERCs

197

Fig. 8.8 Identification of tyramine receptors involved in the control of response to nanopolystyrene (reprinted with permission from [50]). (a) Effect of nanopolystyrene exposure on expressions of tyra-2, tyra-3, and lgc-55 in wild-type nematodes. Exposure was performed from L1-larvae to adult Day-3. Exposure concentrations of nanopolystyrene were 1–1000 μg/L. Bars represent means  SD. ** p < 0.01 vs. control. (b) Effect of neuronal RNAi knockdown of tyra-2 or tyra-3 on toxicity of nanopolystyrene in inducing ROS production in TU3401 nematodes. L4440, empty vector. Exposure was performed from L1-larvae to adult Day-3. Exposure concentration of nanopolystyrene was 1 μg/L. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). (c) Effect of neuronal RNAi knockdown of tyra-2 on toxicity of nanopolystyrene in decreasing locomotion behavior in TU3401 nematodes. L4440, empty vector. Exposure was performed from L1-larvae to adult Day-3. Exposure concentration of nanopolystyrene was 1 μg/L. Bars represent means  SD. ** p < 0.01 vs. control (if not specially indicated)

enhanced the toxicity of nanopolystyrene in inducing ROS production and in decreasing locomotion behavior, and neuronal RNAi knockdown of glr-8 suppressed the toxicity of nanopolystyrene in inducing ROS production and in decreasing locomotion behavior in TU3401 nematodes (Fig. 8.9) [50]. Therefore, GLR-4 and GLR-8 functioned as the corresponding receptors of glutamate to regulate the toxicity of nanopolystyrene at ERCs in nematodes.

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Fig. 8.9 Identification of glutamate receptors involved in the control of response to nanopolystyrene (reprinted with permission from [50]). (a) Effect of nanopolystyrene exposure on expressions of glr-1, glr-2, glr-3, glr-4, glr-5, glr-6, glr-7, and glr-8 in wild-type nematodes. Exposure was performed from L1-larvae to adult Day-3. Exposure concentrations of nanopolystyrene were 1–1000 μg/L. Bars represent means  SD. **p < 0.01 vs. control. (b) Effect of neuronal RNAi knockdown of glr-1, glr-3, glr-4, or glr-8 on toxicity of nanopolystyrene in inducing ROS production in TU3401 nematodes. L4440, empty vector. Exposure was performed from L1-larvae to adult Day-3. Exposure concentration of nanopolystyrene was 1 μg/L. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). (c) Effect of neuronal RNAi knockdown of glr-4 or glr-8 on toxicity of nanopolystyrene in decreasing locomotion behavior in TU3401 nematodes. L4440, empty vector. Exposure was performed from L1-larvae to adult Day-3. Exposure concentration of nanopolystyrene was 1 μg/L. Bars represent means  SD. ** p < 0.01 vs. control (if not specially indicated)

8.5 Downstream Signals of Neurotransmitter Receptors in Regulating the. . .

8.5 8.5.1

199

Downstream Signals of Neurotransmitter Receptors in Regulating the Response to Toxicants at ERCs Downstream Signals of Intestinal Neurotransmitter Receptors in Regulating the Response to Toxicants at ERCs

The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [46]. In nematodes, p38 MAPK and insulin signaling pathways could act in the intestine to regulate the response to nanopolystyrene at ERCs [17, 35]. pmk-1 encodes the p38 MAPK in p38 MAPK signaling pathway, and daf-16 encodes the FOXO transcriptional factor in insulin signaling pathway. In nematodes, it has been found that the signaling cascade of mdt-15-sbp-1 encoding the lipid metabolic signaling acted downstream of p38 MAPK signaling to regulate the response to nanopolystyrene [17]. In the intestine, the effect of RNAi knockdown of dop-1, octr-1, or ser-6 on expression of genes encoding p38 MAPK and insulin signaling pathways in nanopolystyrene-exposed nematodes was examined [46]. In nanopolystyrene-exposed nematodes, intestinal RNAi knockdown of dop-1 could significantly decrease the expressions of pmk-1, mdt-15, and sbp-1 (Fig. 8.7) [46]. In contrast, in nanopolystyrene-exposed nematodes, intestinal RNAi knockdown of octr-1 or ser-6 did not affect the expressions of pmk-1, mdt-15, and sbp-1 (Fig. 8.7) [46]. Additionally, in nanopolystyrene-exposed nematodes, intestinal RNAi knockdown of dop-1, octr-1, or ser-6 did not influence the expression of daf-16 (Fig. 8.7) [46]. Thus, in the intestine, the DOP-1 regulated the response to nanopolystyrene at ERCs by activating the downstream signaling cascade in p38 MAPK signaling pathway in nematodes. The downstream targets of SER-6 and OCTR-1 in the intestine during the control of response to nanopolystyrene are still unclear in nematodes.

8.5.2

Downstream Signals of Neuronal Neurotransmitter Receptors in Regulating the Response to Toxicants at ERCs

The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [46]. The previous studies have demonstrated the role of JNK signaling, ERK signaling, and TGF-β signaling in the neurons to regulate the toxicity of nanopolystyrene [26, 30, 41, 46]. jnk-1 encodes a JNK, mpk-1 encodes a ERK, and dbl-1 encodes a TGF-β. In nanopolystyrene-exposed TU3401 nematodes, neuronal RNAi knockdown of tyra-2 significantly decreased the mpk-1 expression

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Fig. 8.10 Identification of downstream targets of TYRA-2, GLR-4, and GLR-8 in the neurons to regulate the toxicity of nanopolystyrene (reprinted with permission from [50]). (a) Effect of neuronal RNAi knockdown of tyra-2, glr-4, or glr-8 on expressions of jnk-1, mpk-1, and dbl-1 in nanopolystyrene-exposed TU3401 nematodes. L4440, empty vector. Exposure was performed from L1-larvae to adult Day-3. Exposure concentration of nanopolystyrene was 1 μg/L. Bars represent means  SD. **p < 0.01 vs. TU3401. (b) Effect of RNAi knockdown of jnk-1 or mpk-1 on toxicity

8.6 Perspectives

201

(Fig. 8.10) [50]. The (Is(Punc-14-tyra-2)) showed the resistance to toxicity of nanopolystyrene in inducing ROS production and in decreasing locomotion behavior (Fig. 8.10) [50]. Moreover, RNAi knockdown of mpk-1 could suppress the resistance of nematodes overexpressing neuronal TYRA-2 (Is(Punc-14-tyra-2)) nematodes to toxicity of nanopolystyrene in inducing ROS production and in decreasing locomotion behavior (Fig. 8.10) [50]. Therefore, in the neurons, TYRA-2 acted upstream of MPK-1/ERK to regulate the toxicity of nanopolystyrene at ERCs in nematodes. In nanopolystyrene-exposed TU3401 nematodes, neuronal RNAi knockdown of glr-4 could cause the significant decrease in jnk-1 expression (Fig. 8.10) [50]. Moreover, the resistance of nematodes overexpressing neuronal GLR-4 (Is(Punc-14-glr4)) to toxicity of nanopolystyrene in inducing ROS production and in decreasing locomotion behavior could be further inhibited by RNAi knockdown of jnk-1 (Fig. 8.10) [50]. These observations suggested the role of JNK-1/JNK as the downstream target of neuronal GLR-4 in regulating the toxicity of nanopolystyrene at ERCs in nematodes. Neuronal RNAi knockdown of glr-8 could further resulted in the significant increase in dbl-1 expression in nanopolystyrene-exposed TU3401 nematodes (Fig. 8.10) [50]. Meanwhile, the resistance of glr-8(RNAi) nematodes to toxicity of nanopolystyrene in inducing ROS production and in decreasing locomotion behavior could be inhibited by neuronal RNAi knockdown of dbl-1 (Fig. 8.10) [50]. These observations suggested that neuronal TYRA-2 acted upstream of DBL-1/TGF-β to regulate the toxicity of nanopolystyrene at ERCs in nematodes.

8.6

Perspectives

C. elegans is a powerful model animal for the study of molecular toxicology [2, 3, 51–57]. In nematodes, based on the expression and the function of SNB-1, a synaptobrevin, the requirement of neurotransmission to the response to toxicants at ERCs was confirmed. On the one hand, exposure to toxicants (such as nanopolystyrene) at ERCs could increase SNB-1 expression. On the other hand, RNAi knockdown of snb-1 caused a susceptibility to toxicity of toxicants. Besides these, exposure to toxicants (such as nanopolystyrene) at ERCs could increase the  ⁄ Fig. 8.10 (continued) of nanopolystyrene in inducing ROS production in nematodes overexpressing neuronal TYRA-2 or GLR-4. Exposure was performed from L1-larvae to adult Day-3. Exposure concentration of nanopolystyrene was 1 μg/L. Bars represent means  SD. ** p < 0.01 vs. control (if not specially indicated). (c) Genetic interaction between GLR-8 and DBL-1 in the neurons in regulating the toxicity of nanopolystyrene in inducing ROS production. L4440, empty vector. Exposure was performed from L1-larvae to adult Day-3. Exposure concentration of nanopolystyrene was 1 μg/L. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). (d) A diagram showing the molecular basis of tyramine- and glutamate-related signals in regulating the response to nanopolystyrene in nematodes

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expressions of TBH-1 required for the octopamine synthesis and TDC-1 required for the tyramine synthesis and decrease the expressions of CAT-2 required for the dopamine synthesis and EAT-4 required for the transport of glutamate, which further indicated the involvement of release of some neurotransmitters in regulating the response to toxicants at ERCs. In nematodes, both TBH-1 and CAT-2 acted in the neurons to regulate the response to toxicants at ERCs. Meanwhile, in the intestine, the octopamine receptors (OCTR-1 and SER-6) and dopamine receptor (DOP-1) were involved in the control of response to toxicants at ERCs. Therefore, some neurotransmitters (such as octopamine and dopamine) and their receptors mediated an important neuronintestine communication in regulating the response to toxicants at ERCs. Both TDC-1 and EAT-4 acted in the neurons to regulate the response to toxicants at ERCs. Meanwhile, the tyramine receptor TYRA-2 also functioned in the neurons to control the response to toxicants at ERCs. Additionally, the glutamate receptors of GLR-4 and GLR-8 acted in the neurons to control the response to toxicants at ERCs. Therefore, different from octopamine and dopamine and their receptors, tyramine and glutamate and their receptors mediated an important neuron-neuron communication in regulating the response to toxicants at ERCs. In nematodes, the SNB-1 acted downstream of JNK-1/JNK MAPK in the JNK MAPK signaling pathway to regulate the response to toxicants at ERCs. Additionally, both TBH-1 and CAT-2 also acted downstream of JNK-1/JNK MAPK to regulate the response to toxicants at ERCs. These observations suggested that at least the functions of some neurotransmission-related molecular signals in regulating the response to toxicants at ERCs were under the control of JNK MAPK signaling.

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Chapter 9

Response of G Protein-Coupled Receptors and Ion Channels to Toxicants at Environmentally Relevant Concentrations

Abstract Once exposed to environmental toxicants, the bioavailable toxicants will bind and affect certain G protein-coupled receptors (GPCRs) or ion channel on the surface of cytoplasmic membrane to exert their effects on the body of animals. We first introduced and discussed the involvement of GPCRs and downstream G proteins in regulating the response to toxicants at environmentally relevant concentrations (ERCs) and the underlying mechanisms. Moreover, the involvement of ion channels in regulating the response to toxicants at ERCs and the underlying mechanisms were further introduced and discussed. Keywords Environmentally relevant concentrations · G protein-coupled receptors · G proteins · Ion channels · Environmental exposure · Caenorhabditis elegans

9.1

Introduction

The nematode Caenorhabditis elegans can provide a powerful in vivo model for assessing toxicity of various toxicants or stresses [1–7]. The studies on molecular toxicology and target organ toxicology at the whole animal level can be further performed for different toxicants or stresses in nematodes [2, 3, 8–17]. After the exposure, the toxicants potentially attack or bind certain G proteincoupled receptors (GPCRs) or ion channel on the surface of cytoplasmic membrane of different biological barriers, especially the primary targeted biological barriers. After that, the G proteins will further transduce the signals from affected GPCRs to different downstream cytoplasmic signaling pathways. In Chapter 10 of “Molecular Toxicology in Caenorhabditis elegans,” we have introduced and discussed the functions of GPCRs and ion channels and the downstream cytoplasmic signals in the regulation of toxicity of environmental toxicants or stresses in nematodes [2]. In this chapter, we first introduced and discussed the involvement of GPCRs and downstream G proteins in different tissues in regulating the response to toxicants at environmentally relevant concentrations (ERCs) and the underlying mechanisms in nematodes. Moreover, we further introduced and

© The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 D. Wang, Toxicology at Environmentally Relevant Concentrations in Caenorhabditis elegans, https://doi.org/10.1007/978-981-16-6746-6_9

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discussed the involvement of ion channels in different tissues in regulating the response to toxicants at ERCs and the underlying mechanisms in nematodes.

9.2 9.2.1

Response of G Protein-Coupled Receptors to Toxicants at ERCs Response of Intestinal G Protein-Coupled Receptors to Toxicants at ERCs

C. elegans has been frequently used as an animal model to assess the multiple aspects of toxicity induced by nanopolystyrene at ERCs [18–29]. After exposure from L1-larvae to adult Day-3, the nanopolystyrene at concentrations 1 μg/L could cause the significant induction of intestinal ROS production and decrease in locomotion behavior in nematodes [30]. Moreover, after the exposure, the nanopolystyrene could be translocated and accumulated mainly in the intestine in nematodes [31], which implied the crucial role of intestinal GPCRs in response to nanoplastic exposure. Certain number of GPCRs has been identified in different tissues to control stress response to toxicants [2, 3]. For example, in the insulin signaling pathway, the intestinal GPCR of DAF-2 regulated the nanoplastic toxicity by activating the downstream signaling cascade of AGE-1-AKT-1-DAF-16 [30]. To identify other intestinal GPCRs in response to nanopolystyrene exposure, the nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [32]. Previous studies have suggested that some genes encoding GPCRs were potentially involved in the control of response to toxicants or stresses [33–55]. After the exposure, 1–1000 μg/L nanopolystyrene did not significantly affect the expressions of seb-3, avr-14, avr-15, glc-1, dop-3, ser-7, mgl-1, mgl-2, glp-1, unc-63, srh-220, npr-2, frpr-4, unc-68, lin-12, and tkr-1 in wild-type nematodes (Fig. 9.1) [32]. In contrast, exposure to 1–1000 μg/L nanopolystyrene could significantly increase the expressions of paqr-2, npr-1, ced-1, fshr-1, dcar-1, ser-4, and daf-37 and decrease the expressions of dop-2, npr-9, npr-12, npr-4, gtr-1, and npr-8 in wild-type nematodes (Fig. 9.1) [32]. In 1–1000 μg/L nanopolystyrene-exposed nematodes, the increase in expressions of paqr-2, npr-1, ced-1, fshr-1, dcar-1, ser-4, and daf-37 and the decrease in expressions of dop-2, npr-9, npr-12, npr-4, gtr-1, and npr-8 were concentration dependent (Fig. 9.1) [32]. The 1 μg/L is a predicted environmental concentration for nanopolystyrene. These observations indicated the concentrationdependent response of these 13 GPCRs to exposure to nanopolystyrene at ERCs in nematodes. Using reactive oxygen species (ROS) production and locomotion behavior as the endpoints, it was further observed that RNAi knockdown of paqr-2, npr-1, ced-1, fshr-1, dcar-1, ser-4, or daf-37 caused the susceptibility to the nanopolystyrene

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209

Fig. 9.1 Identification of GPCRs involved in the control of response to nanopolystyrene in wildtype nematodes (reprinted with permission from [32]). (a) Effect of nanopolystyrene exposure on expression of genes encoding GPCRs. (b) Effect of RNAi knockdown of genes encoding GPCRs on toxicity of nanopolystyrene in inducing ROS production. L4440, empty vector. The nanopolystyrene exposure concentration was 1 μg/L. (c) Effect of RNAi knockdown of genes encoding GPCRs on toxicity of nanopolystyrene in decreasing locomotion behavior. L4440, empty vector. The nanopolystyrene exposure concentration was 1 μg/L. The nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated)

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9 Response of G Protein-Coupled Receptors and Ion Channels to Toxicants at. . .

Fig. 9.2 Identification of intestinal GPCRs involved in the control of response to nanopolystyrene in nematodes (reprinted with permission from [32]). (a) Candidate GPCR genes expressed in the intestine. (b) Effect of intestinal RNAi knockdown of paqr-2, ced-1, fshr-1, or npr-4 on toxicity of nanopolystyrene in inducing ROS production. L4440, empty vector. The nanopolystyrene exposure concentration was 1 μg/L. The nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated)

toxicity in wild-type nematodes (Fig. 9.1) [32]. Different from this, RNAi knockdown of dop-2, npr-9, npr-12, npr-4, gtr-1, or npr-8 resulted in the resistance to the nanopolystyrene toxicity in wild-type nematodes (Fig. 9.1) [32]. Therefore, in nematodes exposed to nanopolystyrene (1–1000 μg/L), the alteration in expressions of these identified 13 GPCRs mediated a protective response. Considering the fact that the screen was based on the clues from the previous publications, some other potential GPCRs required for the control of response to nanoplastic exposure are still needed to be further examined. In nematodes, the candidate GPCR genes of paqr-2, ced-1, fshr-1, and npr-4 can be expressed in the intestine (Fig. 9.2, Table 9.1) [32]. VP303 is a genetic tool for intestine-specific RNAi knockdown of gene(s). Considering that the VP303 strain has the deficit in locomotion behavior, ROS production was employed as an endpoint to determine the effect of intestinal RNAi knockdown of paqr-2, ced-1, fshr-1, and npr-4 on nanopolystyrene toxicity. After the exposure, intestinal RNAi knockdown of ced-1 or npr-4 did not obviously affect the toxicity of nanopolystyrene in inducing ROS production (Fig. 9.2) [32]. In contrast, after the nanopolystyrene exposure, intestinal RNAi knockdown of paqr-2 or fshr-1 caused

9.2 Response of G Protein-Coupled Receptors to Toxicants at ERCs Table 9.1 Expression patterns of some genes encoding GPCRs (reprinted with permission from [32])

Gene pqar-2 npr-1 ced-1 fshr-1 dcar-1 ser-4 daf-37 dop-2 npr-9 npr-12 npr-4 gtr-1 npr-8

Intestine + + +

+

Neurons + + + + + + + + + + + + +

211 Germline + +

the more severe ROS production compared with that in VP303 nematodes (Fig. 9.2) [32], suggesting the susceptibility of nematodes with intestinal RNAi knockdown of paqr-2 or fshr-1 to nanopolystyrene toxicity. Therefore, GPCRs of PAQR-2 and FSHR-1 could function in the intestine to regulate the response to toxicants (such as nanopolystyrene) at ERCs in nematodes. Therefore, in nanopolystyrene (1–1000 μg/ L)-exposed nematodes, certain number of intestinal GPCRs might exist to regulate the response to nanoplastic exposure. This observation further implies that, in the environment, the nanoplastic exposure may induce the toxic effects on organisms by binding to and affecting functions of GPCRs on the intestinal barrier. In nematodes, some important signaling pathways, including insulin, p38 MAPK, Wnt, and ELT-2 signaling pathways, have been identified to act in the intestine to regulate the response to nanopolystyrene [30, 56–58]. In the insulin signaling pathway, AGE-1 is a kinase acting downstream of insulin receptor DAF-2, DAF-16 is a FOXO transcriptional factor, and SOD-3 is a mitochondrial Mn-SOD acting as a downstream target of DAF-16. In nanopolystyrene-exposed VP303 nematodes, intestinal RNAi knockdown of paqr-2 or fshr-1 could significantly increase the age-1 expression and decrease the expressions of daf-16 and sod-3 (Fig. 9.3) [32]. In the Wnt signaling pathway, BAR-1 is a β-catenin transcriptional factor [57]. In nanopolystyrene-exposed VP303 nematodes, intestinal RNAi knockdown of paqr-2 or fshr-1 could significantly decrease the bar-1 expression (Fig. 9.3) [32]. In the p38 MAPK signaling pathway, PMK-1 is a p38 MAPK. During the control of response to nanopolystyrene, four transcriptional factors (SKN-1, ATF-7, MDT-15, and SBP-1) and one nuclear hormone receptor (NHR-8) acted as the downstream targets of intestinal PMK-1 [56, 59, 60]. In nanopolystyrene-exposed VP303 nematodes, intestinal RNAi knockdown of fshr-1 did not obviously affect the expressions of pmk-1, skn-1, atf-7, mdt-15, sbp-1, and nhr-8, whereas intestinal RNAi knockdown of paqr-2 could significantly decrease the expressions of pmk-1, skn-1, atf-7, mdt-15, sbp-1, and nhr-8 (Fig. 9.3) [32].

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Fig. 9.3 Identification of downstream targets of intestinal PAQR-2 in regulating the response to nanopolystyrene (reprinted with permission from [32]). (a) Effect of intestinal RNAi knockdown of paqr-2 or fshr-1 on gene expressions in nanopolystyrene-exposed nematodes. L4440, empty vector. Bars represent means  SD. **p < 0.01 vs. VP303. (b) Genetic interaction between PAQR-2 and DAF-16, BAR-1, PMK-1, ELT-2, or ATFS-1 in the intestine to regulate the toxicity of nanopolystyrene in inducing ROS production. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). (c) Genetic interaction between PAQR-2 and DAF-16, BAR-1, PMK-1, ELT-2, or ATFS-1 in the intestine to regulate the toxicity of nanopolystyrene in decreasing locomotion behavior. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). The nanopolystyrene exposure concentration was 1 μg/L. The nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3

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ELT-2 is a GATA transcriptional factor required for the control of both functional state of intestinal barrier and response to nanopolystyrene [58]. In nanopolystyreneexposed VP303 nematodes, although intestinal RNAi knockdown of fshr-1 did not obviously affect the elt-2 expression, intestinal RNAi knockdown of paqr-2 could significantly decrease the elt-2 expression (Fig. 9.3) [32]. Autophagy signaling and mitochondrial unfolded protein response (mt UPR) signaling were also involved in the control of response to nanopolystyrene [10, 25]. LGG-1 is a marker of autophagy activation, and transcriptional factor HLH-30 governs the activation of autophagy [2]. However, in nanopolystyreneexposed VP303 nematodes, intestinal RNAi knockdown of paqr-2 or fshr-1 did not significantly affect the expressions of hlh-30 and lgg-1 (Fig. 9.3) [32]. Therefore, the upstream intestinal GPCRs activating or inhibiting the autophagy signaling are still unknown in nanopolystyrene-exposed nematodes. HSP-6 is a marker of mt UPR activation, and transcriptional factor ATFS-1 governs the activation of mt UPR [2]. In nanopolystyrene-exposed VP303 nematodes, although intestinal RNAi knockdown of fshr-1 did not obviously affect the expressions of atfs-1 and hsp-6, intestinal RNAi knockdown of paqr-2 could significantly decrease the expressions of atfs-1 and hsp-6 (Fig. 9.3) [32]. HSP-4 is a marker of endoplasmic reticulum UPR (ER UPR) activation [2, 3]. The activation of ER UPR in nanopolystyrene-exposed nematodes was under the control of p38 MAPK signaling [56]. In nanopolystyrene-exposed VP303 nematodes, intestinal RNAi knockdown of paqr-2 could further significantly decrease the hsp-4 expression (Fig. 9.3) [32]. In nematodes, intestinal overexpression of PAQR-2 induced a resistance to the toxicity of nanopolystyrene in inducing ROS production and in decreasing locomotion behavior (Fig. 9.3) [32]. RNAi knockdown of daf-16 could suppress the resistance of nematodes overexpressing intestinal paqr-2 (Is(Pges-1-paqr-2)) to the toxicity of nanopolystyrene (Fig. 9.3) [32], which suggested that DAF-16 in the insulin signaling pathway acted downstream of intestinal PAQR-2 to regulate the response to nanopolystyrene at ERCs in nematodes. Intestinal overexpression of FSHR-1 resulted in a resistance to the toxicity of nanopolystyrene in inducing ROS production and in decreasing locomotion behavior (Fig. 9.4) [32]. RNAi knockdown of daf-16 suppressed the resistance of nematodes overexpressing intestinal fshr-1 (Is (Pges-1-fshr-1)) to the toxicity of nanopolystyrene (Fig. 9.4) [32], suggesting that the DAF-16 in the insulin signaling pathway acted downstream of intestinal FSHR-1 to regulate the response to nanopolystyrene at ERCs in nematodes. That is, DAF-16 could act downstream of both the PAQR-2 and the FSHR-1 in the intestine to regulate the response to nanoplastic exposure (Fig. 9.4). Therefore, besides the insulin receptor DAF-2, some other intestinal GPCRs (such as PAQR-2 and the FSHR-1) also potentially activated the insulin signaling pathways under certain stress conditions. RNAi knockdown of bar-1 could suppress the resistance of Is(Pges-1-paqr-2) nematodes to the toxicity of nanopolystyrene (Fig. 9.3) [32], suggesting that the BAR-1 in the Wnt signaling pathway acted downstream of intestinal PAQR-2 to regulate the response to nanopolystyrene at ERCs in nematodes. The resistance of Is

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Fig. 9.4 Genetic interaction between FSHR-1 and DAF-16 or BAR-1 in the intestine to regulate the toxicity of nanopolystyrene (reprinted with permission from [32]). (a) Genetic interaction between FSHR-1 and DAF-16 or BAR-1 in the intestine to regulate the toxicity of nanopolystyrene in inducing ROS production. The nanopolystyrene exposure concentration was 1 μg/L. The nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). (b) Genetic interaction between FSHR-1 and DAF-16 or BAR-1 in the intestine to regulate the toxicity of nanopolystyrene in decreasing locomotion behavior. The nanopolystyrene exposure concentration was 1 μg/L. The nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). (c) A diagram showing the molecular basis of intestinal GPCRs (PAQR-2 and FSHR-1) in response to nanoplastic exposure in nematodes

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(Pges-1-fshr-1) nematodes to the toxicity of nanopolystyrene could be inhibited by RNAi knockdown of bar-1 (Fig. 9.4) [32], which suggested that the BAR-1 in the Wnt signaling pathway also acted downstream of intestinal FSHR-1 to regulate the response to nanopolystyrene at ERCs in nematodes. Therefore, BAR-1/β-catenin further functioned downstream of intestinal GPCRs of PAQR-2 and FSHR-1 to regulate the response to nanoplastic exposure. That is, besides the known Wnt receptors, intestinal GPCRs of PAQR-2 and FSHR-1 could potentially activate the Wnt signaling pathway under the nanoplastic exposure conditions. RNAi knockdown of pmk-1 further inhibited the resistance of Is(Pges-1-paqr-2) nematodes to the toxicity of nanopolystyrene (Fig. 9.3) [32], indicating the role of intestinal PAQR-2 as the upstream regulator of p38 MAPK signaling pathway to regulate the response to nanopolystyrene at ERCs in nematodes. These data suggested that at least two upstream signals exist to activate the PMK-1/p38 MAPK in the p38 MAPK signaling pathway. One is the signaling cascade of NSY-1-SEK-1, and the other is the PAQR-2 signaling. The GATA transcriptional factor ELT-2 was required for the response to nanoplastic exposure by activating ERM-1 or CLEC-63/CLEC-85 [58]. In contrast, the upstream regulators for ELT-2 in controlling nanoplastic toxicity remain unknown. Moreover, the resistance of Is(Pges-1-paqr-2) nematodes to the toxicity of nanopolystyrene could be inhibited by RNAi knockdown of elt-2 (Fig. 9.3) [32], suggesting that the intestinal PAQR-2 functioned upstream of ELT-2 to regulate the response to nanopolystyrene at ERCs in nematodes. The ELT-2 is involved in the control of intestinal permeability [58]. Nevertheless, based on the erioglaucine disodium (a blue dye) staining, intestine-specific RNAi knockdown of paqr-2 did not obviously affect the intestinal permeability of nematodes, since the blue dye was mainly distributed in the intestinal lumen like observed in VP303 nematodes [32]. Additionally, RNAi knockdown of atfs-1 suppressed the resistance of Is(Pges-1paqr-2) nematodes to the toxicity of nanopolystyrene (Fig. 9.3) [32], demonstrating that the ATFS-1 further acted downstream of intestinal PAQR-2 to regulate the response to nanopolystyrene at ERCs in nematodes. This provides a novel upstream regulator for the intestinal induction of mt UPR in nematodes.

9.2.2

Response of Neuronal G Protein-Coupled Receptors to Toxicants at ERCs

The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [61]. Feeding is the main source for bioavailability of nanopolystyrene for nematodes [28]. In C. elegans, a large amount of dendrite endings in neuronal cells are located at the mouth [62], which also suggests the potential of direct sense of nanopolystyrene by neuronal cells.

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Using TU3401 strain as a tool for RNAi knockdown in neurons, the effect of neuronal RNAi knockdown of genes encoding neuronal GPCRs on nanopolystyrene toxicity was determined. After the exposure, the susceptibility to nanopolystyrene toxicity in inducing ROS production and in decreasing locomotion behavior was detected in daf-37(RNAi), ser-4(RNAi), dcar-1(RNAi), npr-1(RNAi), and tkr-1 (RNAi) worms (Fig. 9.5) [61]. Meanwhile, the resistance to nanopolystyrene toxicity in inducing ROS production and in decreasing locomotion behavior was observed in gtr-1(RNAi), dop-2(RNAi), npr-12(RNAi), npr-9(RNAi), npr-8(RNAi), npr-4(RNAi), seb-3(RNAi), avr-15(RNAi), mgl-2(RNAi), and frpr-4(RNAi) worms (Fig. 9.5) [61]. In contrast, the toxicity of nanopolystyrene in inducing ROS production and in decreasing locomotion behavior in avr-14(RNAi), glc-1(RNAi), dop-3(RNAi), ser7(RNAi), mgl-1(RNAi), and npr-2(RNAi) worms was similar to that in TU3401 worms (Fig. 9.5) [61]. Meanwhile, the effect of exposure to nanopolystyrene on expression of candidate GPCR genes was investigated. Among the candidate GPCR genes, exposure to nanopolystyrene (1 μg/L) increased the expressions of daf-37, ser-4, dcar-1, and npr-1 and decreased the expressions of gtr-1, dop-2, npr-12, npr-9, npr-8, and npr-4 (Fig. 9.6) [61]. Based on the analysis of both phenotypes and expression levels, the alteration in expressions of these neuronal GPCR genes mediated a protective response to nanopolystyrene. Thus, after exposure to nanopolystyrene (1 μg/L), the nanopolystyrene may possibly induce a protective response in nematodes. In contrast, exposure to nanopolystyrene (1 μg/L) did not affect the expressions of seb3, avr-15, mgl-2, frpr-4, and tkr-1 (Fig. 9.6) [61]. However, the possibility of other five neuronal GPCR genes in regulating the toxicity of nanopolystyrene could not be excluded. That is, a longer exposure duration or a smaller size of nanopolystyrene exposure might also potentially alter the expressions of these neuronal GPCR genes. In C. elegans, JNK MAPK/JNK-1 signaling acted in the neurons to control the PS-NP toxicity by modulating neurotransmissions of octopamine and dopamine between neurons and intestine [63]. In nanopolystyrene-exposed worms, neuronal RNAi knockdown of npr-9, npr-12, or gtr-1 increased the jnk-1 expression, and neuronal RNAi knockdown of dcar-1 decreased the jnk-1 expression [61]. Neuronal RNAi knockdown of jnk-1 resulted in the susceptibility to nanopolystyrene toxicity in inducing ROS production and in decreasing locomotion behavior (Fig. 9.7) [61]. Moreover, neuronal RNAi knockdown of jnk-1 suppressed the resistance of npr-9(RNAi), npr-12(RNAi), or gtr-1(RNAi) worms to the nanopolystyrene toxicity in inducing ROS production and in decreasing locomotion behavior (Fig. 9.7) [61]. Therefore, in the neuronal cells, GPCRs of NPR-9, NPR-12, and GTR-1 functioned upstream of JNK-1 to control the toxicity of toxicants, such as nanopolystyrene. To determine the genetic interaction between DCAR-1 and JNK-1, transgenic strain overexpressing neuronal DCAR-1 (Is(Punc-14-dcar-1)) was generated, and this strain showed the resistance to nanopolystyrene toxicity in inducing ROS production and in decreasing locomotion behavior (Fig. 9.7) [61]. Moreover, jnk-1 RNAi knockdown inhibited the resistance of Is(Punc-14dcar-1) worms to the nanopolystyrene toxicity in inducing ROS production and in decreasing locomotion behavior (Fig. 9.7) [61], suggesting that the neuronal

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Fig. 9.5 Identification of neuronal GPCRs involved in the control of response to PS-NPs (reprinted with permission from [61]). (a) Effect of RNAi knockdown of GPCR genes in neurons on toxicity of PS-NPs in inducing production of ROS. (b) Effect of RNAi knockdown of GPCR genes in neurons on toxicity of PS-NPs in decreasing locomotion behavior. PS-NPs, polystyrene nanoparticles. L4440, empty vector. The PS-NP exposure concentration was 1 μg/L. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated)

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Fig. 9.6 Effect of PS-NP exposure on expressions of GPCR genes in TU3401 worms (reprinted with permission from [61]). PS-NPs, polystyrene nanoparticles. The PS-NP exposure concentration was 1 μg/L. The nematodes were exposed to PS-NPs from L1-larvae to adult Day-3. Bars represent means  SD. **p < 0.01 vs. control

DCAR-1 further functioned upstream of JNK-1 to control the toxicity of toxicants (such as nanopolystyrene) at ERCs in nematodes. That is, the neuronal GPCRs of NPR-9, NPR-12, DCAR-1, and GTR-1 controlled the toxicity of toxicants (such as nanopolystyrene) by positively or negatively modulating the activity of JNK MAPK signaling. Considering that the neuronal JNK MAPK/JNK-1 controlled nanopolystyrene toxicity by regulating neurotransmission of octopamine and dopamine from neuronal cells to intestinal cells [63], these four neuronal GPCRs had the potential to modulate the neurotransmission of octopamine and dopamine in nanopolystyrene-exposed nematodes. In C. elegans, ERK MAPK/MPK-1 signaling acted in neurons to control the nanopolystyrene toxicity by modulating the release of insulin peptides from neurons to intestine [64]. In nanopolystyrene-exposed worms, neuronal RNAi knockdown of npr-8, npr-9, or dop-2 increased the mpk-1 expression, and neuronal RNAi knockdown of dcar-1 or daf-37 decreased the mpk-1 expression [61]. Neuronal RNAi knockdown of mpk-1 induced a susceptibility to the nanopolystyrene toxicity in inducing ROS production and in decreasing locomotion behavior (Fig. 9.8) [61]. Moreover, the resistance of npr-8(RNAi), npr-9(RNAi), or dop-2(RNAi) worms to the nanopolystyrene toxicity in inducing ROS production and in decreasing locomotion behavior was suppressed by neuronal RNAi knockdown of mpk-1 (Fig. 9.8) [61], suggesting that the neuronal NPR-8, NPR-9, and DOP-2 functioned upstream of MPK-1 to control the toxicity to toxicants (such as nanopolystyrene) at ERCs in nematodes. To examine the genetic interaction between MPK-1 and DCAR-1 or DAF-37, transgenic strains overexpressing the neuronal DCAR-1 (Is (Punc-14-dcar-1)) and the neuronal DAF-37 (Is(Punc-14-daf-37)) were generated, and these two transgenic strains exhibited the resistance to the nanopolystyrene toxicity in inducing ROS production and in decreasing locomotion behavior

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Fig. 9.7 Genetic interaction between JNK-1 and neuronal GPCRs in controlling PS-NP toxicity (reprinted with permission from [61]). (a) Genetic interaction between JNK-1 and NPR-9, NPR-12, or GTR-1 in the neurons to control PS-NP toxicity in inducing ROS production. (b) Genetic interaction between JNK-1 and NPR-9, NPR-12, or GTR-1 in the neurons to control PS-NP toxicity in decreasing locomotion behavior. (c) Genetic interaction between JNK-1 and DCAR-1 to control PS-NP toxicity in inducing ROS production. (d) Genetic interaction between JNK-1 and DCAR-1 to control PS-NP toxicity in decreasing locomotion behavior. PS-NPs, polystyrene nanoparticles. L4440, empty vector. The PS-NP exposure concentration was 1 μg/L. Bars represent means  SD. ** p < 0.01 vs. control (if not specially indicated)

(Fig. 9.8) [61]. Moreover, the resistance of Is(Punc-14-dcar-1) or Is(Punc-14-daf37) transgenic worms to the nanopolystyrene toxicity in inducing ROS production and in decreasing locomotion behavior could be inhibited by RNAi knockdown of mpk-1 (Fig. 9.8) [61], suggesting that the neuronal DCAR-1 and DAF-37 further

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Fig. 9.8 Genetic interaction between MPK-1 and neuronal GPCRs in controlling PS-NP toxicity (reprinted with permission from [61]). (a) Genetic interaction between MPK-1 and NPR-8, NPR-9, or DOP-2 in the neurons to control PS-NP toxicity in inducing ROS production. (b) Genetic interaction between MPK-1 and NPR-8, NPR-9, or DOP-2 in the neurons to control PS-NP toxicity in decreasing locomotion behavior. (c) Genetic interaction between MPK-1 and DCAR-1 or DAF-37 to control PS-NP toxicity in inducing ROS production. (d) Genetic interaction between MPK-1 and DCAR-1 or DAF-37 to control PS-NP toxicity in decreasing locomotion behavior. PS-NPs, polystyrene nanoparticles. L4440, empty vector. The PS-NP exposure concentration was 1 μg/L. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated)

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acted upstream of MPK-1 to control the toxicity of toxicants, such as nanopolystyrene, at ERCs in nematodes. Thus, the neuronal GPCRs of NPR-8, NPR-9, DCAR-1, DOP-2, and DAF-37 regulated the toxicity of toxicants (such as nanopolystyrene) by positively or negatively affecting activity of ERK MAPK signaling. More importantly, in nanopolystyrene-exposed worms, both the JNK MAPK signaling and the ERK MAPK signaling were under the control of neuronal GPCRs of NPR-9 and DCAR-1 [61], which implied the multiple functions of neuronal NPR-9 and DCAR-1 in regulating the stress response. Considering that the neuronal ERK MAPK/MPK-1 controlled the nanopolystyrene toxicity by affecting the release of insulin peptides from neuronal cells to intestinal cells [64], these five neuronal GPCRs potentially affected the release of insulin peptides between neurons and intestine in nanopolystyrene-exposed nematodes. In C. elegans, DBL-1/TGF-β signaling acted in the neurons to control the nanopolystyrene toxicity by modulating the activity of TGF-β receptor of SMA-6 in intestinal cells [65]. In nanopolystyrene-exposed worms, neuronal RNAi knockdown of npr-4, npr-8, npr-9, npr-12, gtr-1, or dop-2 could increase the dbl-1 expression, whereas neuronal RNAi knockdown of daf-37 could decrease the dbl1 expression [61]. The single neuronal RNAi knockdown of dbl-1 resulted in a susceptibility to nanopolystyrene toxicity in inducing ROS production and in decreasing locomotion behavior (Fig. 9.9) [61]. The resistance of npr-4(RNAi), npr-8(RNAi), npr-9(RNAi), npr-12(RNAi), gtr-1(RNAi), or dop-2(RNAi) worms to the nanopolystyrene toxicity in inducing ROS production and in decreasing locomotion behavior could be further inhibited by neuronal RNAi knockdown of dbl-1 (Fig. 9.9) [61], suggesting that the neuronal NPR-4, NPR-8, NPR-9, NPR-12, GTR-1, and DOP-2 controlled the toxicity of toxicants (such as nanopolystyrene) at ERCs by inhibiting the activity of DBL-1 in nematodes. The resistance to the nanopolystyrene toxicity in inducing ROS production and in decreasing locomotion behavior observed in Is(Punc-14-daf-37) worms could be further suppressed by RNAi knockdown of dbl-1 (Fig. 9.9) [61], suggesting that the neuronal DAF-37 controlled the toxicity of toxicants (such as nanopolystyrene) at ERCs by activating the activity of DBL-1 in nematodes. That is, these seven neuronal GPCRs differentially modulated activity and release of DBL-1/TGF-β from neuronal cells to other targeted tissues in nanopolystyrene-exposed worms. In C. elegans, neuronal DBL-1/ TGF-β controlled nanopolystyrene toxicity by modulating activity of TGF-β receptor of SMA-6 in intestinal cells [65], which implied that the release of DBL-1/TGF-β between neuronal cells and intestinal cells might be altered in nanopolystyreneexposed worms, and this process was under the control of certain neuronal GPCRs. In C. elegans, DAF-7/TGF-β signaling acted in the neurons to regulate the nanopolystyrene toxicity by modulating the activity of TGF-β receptor of DAF-1 in intestinal cells [15]. In nanopolystyrene-exposed worms, neuronal RNAi knockdown of npr-4, npr-12, or gtr-1 could increase the daf-7 expression; however, neuronal RNAi knockdown of npr-1 could decrease the daf-7 expression [61]. Similarly, single neuronal RNAi knockdown of daf-7 also induced a susceptibility to the nanopolystyrene toxicity in inducing ROS production and in decreasing locomotion behavior (Fig. 9.10) [61]. The resistance of npr-4(RNAi), npr-12(RNAi), or gtr-1

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Fig. 9.9 Genetic interaction between DBL-1 and neuronal GPCRs in controlling PS-NP toxicity (reprinted with permission from [61]). (a) Genetic interaction between DBL-1 and NPR-4, NPR-8, NPR-9, NPR-12, GTR-1, or DOP-2 in the neurons to control PS-NP toxicity in inducing ROS production. (b) Genetic interaction between DBL-1 and NPR-4, NPR-8, NPR-9, NPR-12, GTR-1,

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(RNAi) nematodes to the nanopolystyrene toxicity in inducing ROS production and in decreasing locomotion behavior could be suppressed by neuronal RNAi knockdown of daf-7 (Fig. 9.10) [61], suggesting that the neuronal NPR-4, NPR-12, and GTR-1 controlled the toxicity of toxicants (such as nanopolystyrene) at ERCs by inhibiting the activity of DAF-7 in nematodes. In C. elegans, neuronal overexpression of NPR-1 (Is(Punc-14-npr-1)) caused a resistance to the PS-NP toxicity in inducing ROS production and in decreasing locomotion behavior (Fig. 9.10) [61]. Furthermore, the resistance of Is(Punc-14-npr-1) worms to the nanopolystyrene toxicity in inducing ROS production and in decreasing locomotion behavior could be inhibited by RNAi knockdown of daf-7 (Fig. 9.10) [61], suggesting that the neuronal NPR-1 further controlled the toxicity of toxicants (such as nanopolystyrene) at ERCs by activating the activity of DAF-7 in nematodes. Therefore, in neuronal cells, the GPCRs of NPR-1, NPR-4, NPR-12, and GTR-1 controlled the toxicity of toxicants (such as nanopolystyrene) at ERCs by activating or suppressing activity of DAF-7/TGF-β in nematodes. In C. elegans, neuronal DAF-7/TGF-β controlled nanopolystyrene toxicity by modulating the activity of TGF-β receptor of DAF-1 in intestinal cells [15], which implied that the release of DAF-7/TGF-β between neuronal cells and intestinal cells might also be changed in nanopolystyrene-exposed worms, and this process was also under the control of certain number of neuronal GPCRs.

9.2.3

Response of Germline G Protein-Coupled Receptors to Toxicants at ERCs

After the exposure, the nanoplastic particles could not only be accumulated in the intestine, but also obviously be accumulated in the reproductive organs, such as gonad [28, 31]. This observation implied that the nanoplastic particles could directly activate or inhibit certain GPCRs in germline cells of nematodes. Previous studies have suggested that some genes encoding germline GPCRs (PAQR-2, GLC-1, SER-7, GLP-1, and CED-1) were potentially required for or associated with stress response [37, 40, 42, 52, 66]. The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [67]. After the exposure, nanopolystyrene (1–1000 μg/L) did not influence glc-1, ser-7, and glp-1 expressions in the germline (Fig. 9.11) [67]. However, exposure to nanopolystyrene (1–1000 μg/  ⁄ Fig. 9.9 (continued) or DOP-2 in the neurons to control PS-NP toxicity in decreasing locomotion behavior. (c) Genetic interaction between DBL-1 and DAF-37 to control PS-NP toxicity in inducing ROS production. (d) Genetic interaction between DBL-1 and DAF-37 to control PS-NP toxicity in decreasing locomotion behavior. PS-NPs, polystyrene nanoparticles. L4440, empty vector. The PS-NP exposure concentration was 1 μg/L. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated)

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Fig. 9.10 Genetic interaction between DAF-7 and neuronal GPCRs in controlling PS-NP toxicity (reprinted with permission from [61]). (a) Genetic interaction between DAF-7 and NPR-4, NPR-12, or GTR-1 in the neurons to control PS-NP toxicity in inducing ROS production. (b) Genetic interaction between DAF-7 and NPR-4, NPR-12, or GTR-1 in the neurons to control PS-NP toxicity in decreasing locomotion behavior. (c) Genetic interaction between DAF-7 and NPR-1 to control PS-NP toxicity in inducing ROS production. (d) Genetic interaction between DAF-7 and NPR-1 to control PS-NP toxicity in decreasing locomotion behavior. PS-NPs, polystyrene nanoparticles. L4440, empty vector. The PS-NP exposure concentration was 1 μg/L. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated)

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Fig. 9.11 Identification of germline GPCRs in response to NPS exposure in wild-type nematodes (reprinted with permission from [67]). (a) TEM image of NPS in K medium before the sonication. (b) Effect of NPS exposure on expressions of genes encoding germline GPCRs in wild-type nematodes. Approximately 40 intact gonads were isolated for the extraction of total RNAs. NPS, nanopolystyrene. Bars represent means  SD. **p < 0.01 vs. control. Statistical significance of differences between treatments was examined using one-way ANOVA

L) increased expressions of paqr-2 and ced-1 in the germline (Fig. 9.11) [67]. In nanopolystyrene (1–1000 μg/L)-exposed C. elegans, the increase in expression levels of paqr-2 and ced-1 in the germline was concentration dependent (Fig. 9.11) [67]. That is, certain number of GPCRs can be activated in the germline by exposure to nanopolystyrene at ERCs in nematodes. This provides the evidence for the existence of direct molecular response in the germline to nanopolystyrene exposure in C. elegans. Using DCL569 strain as a genetic tool for germline RNAi knockdown of gene(s), germline RNAi knockdown of paqr-2 or ced-1 could not result in obvious production of ROS, affect locomotion behavior, and influence brood size under normal conditions (Fig. 9.12) [67]. Germline RNAi knockdown of paqr-2 did not influence the nanopolystyrene toxicity in inducing ROS production, in inhibiting locomotion behavior, and in reducing brood size (Fig. 9.12) [67]. Different from this, the toxicity of nanopolystyrene exposure in inducing ROS production, in inhibiting locomotion behavior, and in reducing brood size was enhanced by germline RNAi knockdown of ced-1 (Fig. 9.12) [67]. Thus, the GPCR CED-1 functioned in germline to control the toxicity of toxicants (such as nanopolystyrene) at ERCs in nematodes. That is, the GPCR CED-1 in the germline can control the stress response by mediating a protective response to certain toxicants, such as the nanopolystyrene, at ERCs in nematodes. The potential targeted genes of CED-1 have been raised during the control of different processes, and some of them can be expressed in the germline [68– 73]. Among 16 potential targeted genes for germline ced-1, germline RNAi knockdown of ced-1 decreased expressions of ced-6, ced-10, dyn-1, vps-34, lst-4, snx-1, snx-6, rab-7, rab-14, and epn-1 in nanopolystyrene-exposed DCL569 nematodes (Fig. 9.13) [67]. Among these ten candidate genes, exposure to nanopolystyrene (1 μg/L) further increased expression levels of ced-10, vps-34, snx-1, rab-7, and rab14 in wild-type nematodes (Fig. 9.13) [67]. In nanopolystyrene-exposed DCL569

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Fig. 9.12 Effect of germline RNAi knockdown of paqr-2 or ced-1 on toxicity of NPS (reprinted with permission from [67]). (a) Effect of germline RNAi knockdown of paqr-2 or ced-1 on toxicity of NPS in inducing ROS production. (b) Effect of germline RNAi knockdown of paqr-2 or ced-1 on toxicity of NPS in decreasing locomotion behavior. NPS, nanopolystyrene. The NPS exposure concentration was 1 μg/L. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). If not specially indicated, the statistical significance of differences between treatments was examined using one-way ANOVA

worms, germline RNAi knockdown of ced-10, vps-34, snx-1, rab-7, or rab-14 caused more severe production of ROS, suppression in locomotion behavior, and reduction in brood size (Fig. 9.13) [67], indicating the susceptibility of ced-10 (RNAi), vps-34(RNAi), snx-1(RNAi), rab-7(RNAi), and rab-14(RNAi) animals to nanopolystyrene toxicity. These findings implied the potential function of CED-10, VPS-34, SNX-1, RAB-7, and RAB-14 as targets for germline CED-1 in controlling response to nanopolystyrene at ERCs in nematodes. In nematodes, ced10 encodes a small GTPase, vps-34 encodes a VPS protein, snx-1 encodes a BAR domain-containing sorting nexin, rab-7 encode a GTPase, and rab-14 also encode a GTPase. In nematodes, the smaller nanoparticles could induce the more severe toxic effects than those having large sizes [1–4]. Therefore, besides the identified possible targets of CED-1 as indicated above, the CED-6, DYN-1, LST-4, SNX-6, and EPN-1 may also potentially function downstream of germline GPCR CED-1 to control the toxicity of nanopolystyrene with the smaller size and at ERCs. To confirm the role of CED-10, VPS-34, SNX-1, RAB-7, or RAB-14 as targets of germline CED-1 in controlling nanopolystyrene toxicity, we examined genetic interaction between CED-1 and CED-10, VPS-34, SNX-1, RAB-7, or RAB-14 in the germline. Germline overexpression of CED-1 significantly suppressed production of ROS, enhanced locomotion behavior, and increased brood size in

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Fig. 9.13 Identification of downstream targets of intestinal CED-1 in regulating the response to NPS exposure (reprinted with permission from [67]). (a) Effect of germline RNAi knockdown of ced-1 on gene expressions in NPS-exposed nematodes. L4440, empty vector. Bars represent means  SD. **p < 0.01 vs. DCL569. Statistical significance of differences between treatments was examined using one-way ANOVA. (b) Effect of NPS exposure on expressions of ced-6, ced10, dyn-1, vps-34, lst-4, snx-1, snx-6, rab-7, rab-14, and epn-1 in wild-type nematodes. Bars represent means  SD. **p < 0.01 vs. control. Statistical significance of differences between treatments was examined using one-way ANOVA. (c) Effect of germline RNAi knockdown of ced-10, vps-34, snx-1, rab-7, or rab-14 on NPS toxicity in inducing ROS production. L4440, empty vector. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). If not specially indicated, the statistical significance of differences between treatments was examined using one-way ANOVA. (d) Effect of germline RNAi knockdown of ced-10, vps-34, snx-1, rab-7, or rab-14 on NPS toxicity in decreasing locomotion behavior. L4440, empty vector. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). If not specially indicated, the statistical significance of differences between treatments was examined using one-way ANOVA. NPS, nanopolystyrene. The NPS exposure concentration was 1 μg/L

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Fig. 9.14 Genetic interaction between CED-1 and CED-10, VPS-34, SNX-1, RAB-7, or RAB-14 in the germline to regulate the response to NPS exposure (reprinted with permission from [67]). (a) Genetic interaction between CED-1 and CED-10, VPS-34, SNX-1, RAB-7, or RAB-14 in the germline to regulate the NPS toxicity in inducing ROS production. (b) Genetic interaction between CED-1 and CED-10, VPS-34, SNX-1, RAB-7, or RAB-14 in the germline to regulate the NPS toxicity in decreasing locomotion behavior. NPS, nanopolystyrene. Exposure concentration of NPS was 1 μg/L. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). If not specially indicated, the statistical significance of differences between treatments was examined using one-way ANOVA

nanopolystyrene-exposed worms (Fig. 9.14) [67], indicating the resistance of animals overexpressing germline CED-1 (Is(Pmex-5-ced-1)) to the nanopolystyrene toxicity. Moreover, RNAi knockdown of ced-10, vps-34, snx-1, rab-7, or rab-14 could cause obvious production of ROS, inhibition in locomotion behavior, and reduction in brood size in nanopolystyrene exposed Is(Pmex-5-ced-1) worms (Fig. 9.14) [67], demonstrating that CED-10, VPS-34, SNX-1, RAB-7, and RAB-14 acted downstream of germline CED-1 to control toxicity of toxicants (such as nanopolystyrene) at ERCs in nematodes. Therefore, the signaling cascade of CED-1-CED-10/VPS-34/SNX-1/RAB-7/RAB-14 in the germline to control

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nanopolystyrene toxicity was raised, which strengthens the understanding of molecular basis for germline in response to exposure to toxicants, such as nanopolystyrene, at ERCs in nematodes. Nevertheless, the future work on the elucidation of molecular signals mediated by germline CED-10, VPS-34, SNX-1, RAB-7, and RAB-14 in controlling the nanopolystyrene toxicity is still needed. In nematodes, the insulin, Wnt, p38 MAPK, and ELT-2 signaling pathways functioned in intestinal cells to control nanopolystyrene toxicity [30, 56–58]. In insulin signaling pathway, DAF-16 is a FOXO transcriptional factor; in Wnt signaling pathway, BAR-1 is a β-catenin transcriptional factor; and in p38 MAPK signaling pathway, PMK-1 is a p38 MAPK. In the insulin, Wnt, and p38 MAPK signaling pathway, only PMK-1/p38 MAPK and signaling cascades of DAF-2-AGE-1-AKT1-DAF-16 and GSK-3-BAR-1 were found to be in response to nanopolystyrene (1 μg/L) exposure [30, 56, 57]. After nanopolystyrene exposure, germline RNAi knockdown of ced-10, vps-34, snx-1, rab-7, or rab-14 could not influence expression levels of bar-1 and elt-2 (Fig. 9.15) [67]. In contrast, in nanopolystyreneexposed DCL569 worms, germline RNAi knockdown of ced-10, rab-7, or rab-14 decreased daf-16 expression, and germline RNAi knockdown of vps-34 or snx-1 decreased pmk-1 expression (Fig. 9.15) [67]. Meanwhile, germline RNAi knockdown of ced-1 could further decrease expressions of daf-16 and pmk-1 in nanopolystyrene-exposed DCL569 worms [67]. Nevertheless, after the nanopolystyrene exposure, germline RNAi knockdown of ced-1 could not affect the expressions of bar-1 and elt-2 [67]. Therefore, CED-1-activated signaling cascade in the germline could control the toxicity of nanopolystyrene at ERCs via affecting activities of p38 MAPK and insulin signaling pathways in intestinal cells in nematodes.

9.3

Response of G Proteins to Toxicants at ERCs

G proteins are composed of α, β, and γ subunits. Nevertheless, the signals from GPCRs are normally transduced by Gα subunits to the downstream cytoplasmic signaling pathways.

9.3.1

Response of Intestinal Gα Subunits to Toxicants at ERCs

The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [74]. Among the 21 genes encoding Gα subunits, 8 genes (egl-30, goa1, gpa-6, gpa-7, gpa-10, gpa-12, gpa-17, and gsa-1) are expressed in the intestine (Fig. 9.16 and Table 9.2) [74]. After the exposure from L1-larvae to adult Day-3,

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9 Response of G Protein-Coupled Receptors and Ion Channels to Toxicants at. . .

Fig. 9.15 Germline RNAi knockdown of ced-10, vps-34, snx-1, rab-7, or rab-14 affected expressions of daf-16 and pmk-1 in NPS exposed DCL569 nematodes (reprinted with permission from [67]). (a) Effect of ced-10, vps-34, snx-1, rab-7, or rab-14 on expressions of daf-16, pmk-1, bar-1, and elt-2 in NPS-exposed DCL569 nematodes. L4440, empty vector. NPS, nanopolystyrene. The NPS exposure concentration was 1 μg/L. Bars represent means  SD. **p < 0.01 vs. DCL569. Statistical significance of differences between treatments was examined using one-way ANOVA. (b) A diagram showing the molecular basis for germline CED-1 in response to NPS exposure in nematodes

0.1 μg/L nanopolystyrene (100 nm) did not affect the expression of any of these eight intestinal genes, and 1–100 μg/L nanopolystyrene did not significantly alter the expressions of gpa-6, gpa-7, gpa-12, gpa-17, and gsa-1 (Fig. 9.16) [74]. Different from these, exposure to nanopolystyrene at the concentration of 1 μg/L could significantly increase the expressions of egl-30 and goa-1 and decrease the gpa-10 expression (Fig. 9.16) [74]. Moreover, the alteration in expressions of egl-30, goa-1, and gpa-10 was concentration dependent in wild-type nematodes exposed to 1–100 μg/L nanopolystyrene (Fig. 9.16) [74]. These observations suggested that

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Fig. 9.16 Genes encoding intestinal Gα subunits dysregulated by nanopolystyrene (reprinted with permission from [23]). (a) Searching for genes encoding Gα subunits expressed in the intestine. (b) Effect of nanopolystyrene exposure on expressions of genes encoding intestinal Gα subunits. Bars represent means  SD. **p < 0.01 vs. control Table 9.2 Expression patterns of genes encoding Gα subunits (reprinted with permission from [74])

Genes egl-30 goa-1 gpa-1 gpa-2 gpa-3 gpa-4 gpa-5 gpa-6 gpa-7 gpa-8 gpa-9 gpa-10 gpa-11 gpa-12 gpa-13 gpa-14 gpa-15 gpa-16 gpa-17 gsa-1 odr-3

Intestine + +

+ +

+ +

Neurons + + + + + + +

Germline

Muscle

+

+ + + + + + + +

+

+

+ + + +

+ +

+

exposure to nanopolystyrene at ERCs could alter the expressions of egl-30, goa-1, and gpa-10 in nematodes. Using ROS production and brood size as the endpoints, the effect of intestinespecific RNAi knockdown of egl-30, goa-1, or gpa-10 on toxicity in

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Fig. 9.17 Nanopolystyrene toxicity in nematodes with intestine-specific RNAi knockdown of egl30, goa-1, or gpa-10 (reprinted with permission from [74]). (a) Effect of intestine-specific RNAi knockdown of egl-30, goa-1, or gpa-10 on nanopolystyrene toxicity in inducing ROS production. (b) Effect of intestine-specific RNAi knockdown of egl-30, goa-1, or gpa-10 on nanopolystyrene toxicity in reducing brood size. L4440, empty vector. Exposure concentration of nanopolystyrene was 1 μg/L. Exposure was performed from L1-larvae to adult Day-3. Bars represent means  SD. ** p < 0.01 vs. control (if not specially indicated)

nanopolystyrene-exposed nematodes was examined. In VP303 nematodes, exposure to nanopolystyrene (1 μg/L) caused the significant induction of ROS production and reduction in brood size (Fig. 9.17) [74]. Intestine-specific RNAi knockdown of goa1 did not obviously affect the toxicity of nanopolystyrene (Fig. 9.17) [74]. In contrast, intestine-specific RNAi knockdown of egl-30 enhanced the toxicity of nanopolystyrene, whereas intestine-specific RNAi knockdown of gpa-10 suppressed the toxicity of nanopolystyrene (Fig. 9.17) [74]. Therefore, Gα subunits of EGL-30 and GPA-10 acted in the intestine to regulate the response to nanopolystyrene at ERCs in nematodes. Moreover, it was observed that exposure to 100 mg/L could significantly decrease the egl-30 expression and increase the gpa-1 expression [74]. That is, at high concentrations (such as in the range of mg/L), the alteration in expressions of intestinal EGL-30 and GPA-10 might mediate the toxicity induction of nanopolystyrene on nematodes. The genetic interaction between EGL-30 and GPA-10 in the intestine to regulate the response to nanopolystyrene was further determined. After the exposure, intestine-specific RNAi knockdown of gpa-10 could significantly decrease the ROS production and increase the brood size in nanopolystyrene-exposed egl-30 (RNAi) nematodes (Fig. 9.18) [74]. That is, the susceptibility of egl-30(RNAi)

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Fig. 9.18 Genetic interaction between EGL-30 and GPA-10 in the intestine to regulate the response to nanopolystyrene (reprinted with permission from [74]). (a) Genetic interaction between EGL-30 and GPA-10 in the intestine to regulate the toxicity of nanopolystyrene in inducing ROS production. (b) Genetic interaction between EGL-30 and GPA-10 in the intestine to regulate the toxicity of nanopolystyrene in reducing brood size. L4440, empty vector. Exposure concentration of nanopolystyrene was 1 μg/L. Exposure was performed from L1-larvae to adult Day-3. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated)

nematodes to nanopolystyrene toxicity could be suppressed by intestine-specific RNAi knockdown of gpa-10. This observation suggested that exposure to nanopolystyrene at ERCs induced two antagonistic Gα subunits (EGL-30 and GPA-10) in the intestine of nematodes. Moreover, this observation implied that, among these two Gα subunits, EGL-30 might play a prominent role in regulating the response to nanopolystyrene, since EGL-30 could genetically act upstream of GPA-10 to regulate the response to nanopolystyrene at ERCs in nematodes. EGL-30 is an ortholog of vertebrate Gαq, and Gαq normally regulate the biological processes by activating PLCβ isoforms (PLCβ) in organisms. egl-8 encodes the PLCβ. After the exposure, RNAi knockdown of egl-18 caused the more severe induction of ROS production and reduction in brood size in nanopolystyreneexposed nematodes compared with those in nanopolystyrene-exposed wild-type nematodes (Fig. 9.19) [74], suggesting the susceptibility of egl-8(RNAi) nematodes to the toxicity of nanopolystyrene. Intestinal overexpression of EGL-30 resulted in the decrease in ROS production and increase in brood size in nanopolystyreneexposed nematodes (Fig. 9.19) [74], suggesting the resistance of nematodes overexpressing intestinal EGL-30 to the toxicity of nanopolystyrene. Moreover, RNAi knockdown of egl-8 could obviously suppressed the resistance of nematodes

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Fig. 9.19 Genetic interaction between EGL-30 and EGL-8 to regulate the response to nanopolystyrene (reprinted with permission from [74]). (a) Genetic interaction between EGL-30 and EGL-8 to regulate the toxicity of nanopolystyrene in inducing ROS production. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). (b) Genetic interaction between EGL-30 and EGL-8 to regulate the toxicity of nanopolystyrene in reducing brood size. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). (c) Effect of intestinespecific RNAi knockdown of egl-30 on expression of egl-8 in nanopolystyrene-exposed VP303 nematodes. Bars represent means  SD. **p < 0.01 vs. VP303. L4440, empty vector. Exposure was performed from L1-larvae to adult Day-3. Exposure concentration of nanopolystyrene was 1 μg/L

overexpressing intestinal EGL-30 to the toxicity of nanopolystyrene in inducing ROS production and in reducing brood size (Fig. 9.19) [74]. Additionally, intestine-specific RNAi knockdown of egl-30 could further significantly decrease the egl-8 expression in nanopolystyrene-exposed nematodes (Fig. 9.19) [74]. These observations demonstrated that the signaling cascade of EGL-30-EGL-8 was activated in nematodes exposed to nanopolystyrene at ERCs in nematodes. During the control of response to nanopolystyrene at ERCs, DAF-16 is a FOXO transcriptional factor, and AGE-1 is a kinase acting downstream of insulin receptor DAF-2 in the insulin signaling pathway, BAR-1 is a β-catenin transcriptional factor in the Wnt signaling pathway, PMK-1 is a p38 MAPK in the p38 MAPK signaling pathway, and MDT-15 and SBP-1, two lipid metabolic sensors, acted downstream of PMK-1 to regulate the response to nanopolystyrene at ERCs in nematodes [30, 56– 60]. In nanopolystyrene-exposed VP303 nematodes, intestine-specific RNAi knockdown of egl-30 or egl-8 could significantly increase the expression of age-1 and

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Fig. 9.20 Intestinal EGL-30 and GPA-10 acted upstream of insulin, p38 MAPK, and/or Wnt signaling pathways to regulate the response to nanopolystyrene (reprinted with permission from [74]). (a) Effect of intestine-specific RNAi knockdown of egl-30, egl-8, or gpa-10 on expressions of age-1, daf-16, pmk-1, mdt-15, sbp-1, and bar-1. Exposure was performed from L1-larvae to adult Day-3. L4440, empty vector. Exposure concentration of nanopolystyrene was 1 μg/L. Bars represent means  SD. **p < 0.01 vs. VP303. (b) A diagram showing the molecular basis of intestinal EGL-30 and GPA-10 in regulating the response to nanopolystyrene in nematodes

decrease the expressions of daf-16, pmk-1, mdt-15, sbp-1, and bar-1 (Fig. 9.20) [74]. Meanwhile, in nanopolystyrene-exposed VP303 nematodes, RNAi knockdown of gpa-10 could significantly increase the expressions of pmk-1, mdt-15, and sbp-1 (Fig. 9.20) [74]. In nanopolystyrene-exposed VP303 nematodes, intestine-specific RNAi knockdown of gpa-10 did not significantly affect the expressions of age-1, daf-16, and bar-1 (Fig. 9.20) [74]. These observations suggested that, besides the insulin receptor DAF-2, some other unidentified GPCR(s) may also exist to modulate the signaling cascade of AGE-1-DAF-16 during the regulation of response to nanopolystyrene at ERCs in nematodes. Considering that EGL-30 could act upstream of GPA-10 to regulate the response to nanopolystyrene, the EGL-30 and the GPA-10 constituted an effect amplification mechanism to regulate the function of intestinal signaling cascade of PMK-1-MDT-15/SBP-1 in regulating the response to nanopolystyrene at ERCs in nematodes. Therefore, in the intestine of nanopolystyrene-exposed nematodes, the signaling cascade of EGL-30-EGL-8 can potentially activate multiple downstream signaling pathways, at least including the insulin, p38 MAPK, and/or Wnt signaling pathways. For the identified intestinal GPCRs in response to nanopolystyrene at ERCs, the genetic interaction between PAQR-2 and FSHR-1 (two GPCRs) and their possible downstream G proteins in the intestine in regulating the toxicity of nanopolystyrene were further determined. Using ROS production and brood size as the endpoints, intestinal RNAi knockdown of gpa-10 could suppress the susceptibility to the toxicity of nanopolystyrene in inducing ROS production and in reducing brood size in pagr-2(RNAi) or fshr-1(RNAi) nematodes (Fig. 9.21) (Y.-H. Yang and D.Y. Wang, unpublished data). Moreover, RNAi knockdown of egl-30 could further suppress the resistance to the toxicity of nanopolystyrene in inducing ROS production and in reducing brood size in nematodes overexpressing intestinal PAQR-2 or

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9 Response of G Protein-Coupled Receptors and Ion Channels to Toxicants at. . .

Fig. 9.21 Genetic interaction between PAQR-2 and FSHR-1 and their downstream G proteins in regulating the toxicity of nanopolystyrene (Y.-H. Yang and D.-Y. Wang, unpublished data). (a) Genetic interaction between PAQR-2 and FSHR-1 and their downstream G proteins in regulating the toxicity of nanopolystyrene in inducing ROS production. (b) Genetic interaction between PAQR-2 and FSHR-1 and their downstream G proteins in regulating the toxicity of nanopolystyrene in decreasing locomotion behavior. L4440, empty vector. Exposure concentration of nanopolystyrene was 1 μg/L. Exposure was performed from L1-larvae to adult Day-3. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated)

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FSHR-1 (Fig. 9.21) (Y.-H. Yang and D.-Y. Wang, unpublished data). Therefore, in the intestine, PAQR-2 and FSHR-1 acted upstream of two Gα subunits of EGL-30 and GPA-10 to regulate the toxicity of nanopolystyrene at ERCs in nematodes.

9.3.2

Response of Neuronal Gα Subunits to Toxicants at ERCs

The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [21]. Among 21 genes encoding Gα subunits, most of them (gsa-1, egl30, goa-1, gpa-1, gpa-2, gpa-3, gpa-4, gpa-5, gpa-6, gpa-7, gpa-8, gpa-9, gpa-10, gpa-11, gpa-12, gpa-13, gpa-14, gpa-15, gpa-16, and odr-3) can be expressed in the neurons. Among the neuronal Gα genes, nanopolystyrene exposure at concentrations 1 μg/L significantly increased egl-30, goa-1, gpa-5, gpa-11, and gpa-15 expressions and decreased gsa-1 and gpa-10 expressions (Fig. 9.22) (Y.-H. Yang and D.-Y. Wang, unpublished data). In 1–100 μg/L nanopolystyrene-exposed wild-type nematodes, the expressional alterations of egl-30, goa-1, gpa-5, gpa-11, gpa-15, gsa-1, and gpa-10 were concentration dependent (Fig. 9.22) (Y.-H. Yang and D.-Y. Wang, unpublished data), which suggested the response of these 7 Gα proteins to nanopolystyrene at ERCs in nematodes. Using TU3401 as a neuronal RNAi tool, it was found that neuronal RNAi knockdown of egl-30, gpa-5, gpa-11, or gpa-15 did not affect the nanopolystyrene toxicity in causing ROS production (Fig. 9.23) (Y.-H. Yang and D.-Y. Wang, unpublished data). In contrast, neuronal RNAi knockdown of goa-1 induced the more severe ROS production and inhibition of locomotion behavior in nanopolystyrene-exposed nematodes, and neuronal RNAi knockdown of gsa-1 or gpa-10 suppressed the nanopolystyrene toxicity in causing ROS production and in inhibiting locomotion behavior (Fig. 9.23) (Y.-H. Yang and D.-Y. Wang, unpublished data). Therefore, the neuronal Gα proteins of GOA-1, GSA-1, and GPA-10 were involved in the control of toxicity of nanopolystyrene at ERCs in nematodes. Some genes encoding neuronal GPCRs involved in the control of nanopolystyrene toxicity have been recently identified in nematodes [61]. Among these GPCR genes, neuronal RNAi knockdown of npr-8, npr-9, npr-12, gtr-1, or dop-2 increased the goa-1 expression in nanopolystyrene-exposed nematodes (Fig. 9.24) (Y.-H. Yang and D.-Y. Wang, unpublished data). Moreover, neuronal RNAi knockdown of goa-1 suppressed the resistance to nanopolystyrene toxicity in causing ROS production and in inhibiting locomotion behavior in npr-8(RNAi), npr9(RNAi), npr-12(RNAi), gtr-1(RNAi), or dop-2(RNAi) nematodes (Fig. 9.24) (Y.-H. Yang and D.-Y. Wang, unpublished data). Therefore, the neuronal GPCRs of NPR-8, NPR-9, NPR-12, GTR-1, and DOP-2 functioned upstream of Gα/GOA-1 to control the toxicity of nanopolystyrene at ERCs in nematodes.

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9 Response of G Protein-Coupled Receptors and Ion Channels to Toxicants at. . .

Fig. 9.22 Genes encoding neuronal Gα subunits dysregulated by PS-NPs in wild-type nematodes (Y.-H. Yang and D.-Y. Wang, unpublished data). (a) Searching for genes encoding Gα subunits expressed in the neurons. (b) Effect of PS-NP exposure on expressions of genes encoding neuronal Gα subunits. PS-NPs, polystyrene nanoparticles. Bars represent means  SD. **p < 0.01 vs. control

Neuronal RNAi knockdown of dcar-1 or daf-37 also increased gsa-1 and gpa-10 expressions in nanopolystyrene-exposed nematodes (Fig. 9.24) (Y.-H. Yang and D.Y. Wang, unpublished data). Meanwhile, neuronal RNAi knockdown of gsa-1 or gpa-10 inhibited the susceptibility to nanopolystyrene toxicity in causing ROS production and in inhibiting locomotion behavior in dcar-1(RNAi) and daf-37 (RNAi) nematodes (Fig. 9.24) (Y.-H. Yang and D.-Y. Wang, unpublished data). Therefore, the neuronal GPCRs of DCAR-1 and DAF-37 functioned upstream of Gα/GSA-1 or Gα/GPA-10 to control the toxicity of nanopolystyrene at ERCs in nematodes. In nanopolystyrene-exposed nematodes, neuronal RNAi knockdown of dcar-1 or daf-37 decreased the goa-1 expression (Fig. 9.25) (Y.-H. Yang and D.-Y. Wang, unpublished data). Additionally, RNAi knockdown of goa-1 could suppress the resistance of nematodes overexpressing neuronal dcar-1 (Is(Punc-14-dcar-1)) or daf-37 (Is(Punc-14-daf-37)) to nanopolystyrene toxicity in causing ROS production and in inhibiting locomotion behavior (Fig. 9.25) (Y.-H. Yang and D.-Y. Wang, unpublished data). That is, the neuronal GPCRs of DCAR-1 and DAF-37 functioned

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Fig. 9.23 PS-NP toxicity in nematodes with neuronal RNAi knockdown of egl-30, goa-1, gpa-5, gpa-11, gpa-15, gsa-1, or gpa-10 (Y.-H. Yang and D.-Y. Wang, unpublished data). (a) Effect of neuronal RNAi knockdown of egl-30, goa-1, gpa-5, gpa-11, gpa-15, gsa-1, or gpa-10 on PS-NP toxicity in inducing ROS production. (b) Effect of neuronal RNAi knockdown of gsa-1, goa-1, or gpa-10 on PS-NP toxicity in decreasing locomotion behavior. PS-NPs, polystyrene nanoparticles. L4440, empty vector. Exposure concentration of PS-NPs was 1 μg/L. Bars represent means  SD. ** p < 0.01 vs. control (if not specially indicated)

upstream of Gα/GOA-1 to control the toxicity of nanopolystyrene at ERCs in nematodes. In nanopolystyrene-exposed nematodes, the gsa-1 expression was decreased by neuronal RNAi knockdown of npr-8, npr-9, or dop-2 (Fig. 9.25) (Y.-H. Yang and D.-Y. Wang, unpublished data). Moreover, RNAi knockdown of gsa-1 inhibited the susceptibility of nematodes overexpressing neuronal npr-8 (Is(Punc-14-npr-8)), npr9 (Is(Punc-14-npr-9)), or dop-2 (Is(Punc-14-dop-2)) to nanopolystyrene toxicity in causing ROS production and in inhibiting locomotion behavior (Fig. 9.25) (Y.-H. Yang and D.-Y. Wang, unpublished data). Therefore, the neuronal GPCRs of NPR-8, NPR-9, and DOP-2 functioned upstream of Gα/GSA-1 to control the toxicity of nanopolystyrene at ERCs in nematodes.

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9 Response of G Protein-Coupled Receptors and Ion Channels to Toxicants at. . .

Fig. 9.24 Effect of neuronal RNAi knockdown of goa-1, gsa-1, or gpa-10 on PS-NP toxicity in npr-8(RNAi), npr-9(RNAi), npr-12(RNAi), gtr-1(RNAi), dop-2(RNAi), dcar-1(RNAi), or daf-37 (RNAi) nematodes (Y.-H. Yang and D.-Y. Wang, unpublished data). (a) Effect of neuronal RNAi knockdown of npr-8, npr-9, npr-12, gtr-1, dop-2, dcar-1, or daf-37 on expressions of goa-1, gsa-1, or gpa-10 in PS-NP-exposed TU3401 nematodes. Bars represent means  SD. ** p < 0.01 vs. TU3401. (b) Effect of neuronal RNAi knockdown of goa-1, gsa-1, or gpa-10 on PS-NP toxicity in inducing ROS production in npr-8(RNAi), npr-9(RNAi), npr-12(RNAi), gtr-1 (RNAi), dop-2(RNAi), dcar-1(RNAi), or daf-37(RNAi) nematodes. Bars represent means  SD. ** p < 0.01 vs. control (if not specially indicated). (c) Effect of neuronal RNAi knockdown of goa-1, gsa-1, or gpa-10 on PS-NP toxicity in decreasing locomotion behavior in npr-8(RNAi), npr-9 (RNAi), npr-12(RNAi), gtr-1(RNAi), dop-2(RNAi), dcar-1(RNAi), or daf-37(RNAi) nematodes. Bars

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In nanopolystyrene-exposed nematodes, the gpa-10 expression could be further decreased by neuronal RNAi knockdown of npr-8 or dop-2 (Fig. 9.25) (Y.-H. Yang and D.-Y. Wang, unpublished data). Meanwhile, RNAi knockdown of gpa-10 suppressed the susceptibility of Is(Punc-14-npr-8) or Is(Punc-14-dop-2) nematodes to nanopolystyrene toxicity in causing ROS production and in inhibiting locomotion behavior (Fig. 9.25) (Y.-H. Yang and D.-Y. Wang, unpublished data). That is, the neuronal GPCRs of NPR-8 and DOP-2 further functioned upstream of Gα/GPA-10 to control the toxicity of nanopolystyrene at ERCs in nematodes. In nematodes, daf-7 encoding a TGF-β, dbl-1 encoding a TGF-β, mpk-1 encoding a ERK MAPK, jnk-1 encoding a JNK MAPK, and glb-10 encoding a globin protein acted in the neurons to control nanopolystyrene toxicity [15, 64, 65, 75]. In nanopolystyrene-exposed nematodes, neuronal RNAi knockdown of gsa-1 increased jnk-1, mpk-1, and dbl-1 expressions (Fig. 9.26) (Y.-H. Yang and D.-Y. Wang, unpublished data). Meanwhile, neuronal RNAi knockdown of jnk-1, mpk-1, or dbl-1 suppressed the resistance to nanopolystyrene toxicity in causing ROS production and in inhibiting locomotion behavior in gsa-1(RNAi) nematodes (Fig. 9.26) (Y.-H. Yang and D.-Y. Wang, unpublished data), which suggested that the neuronal Gα/GSA-1 functioned upstream of JNK-1/JNK MAPK, MPK-1/ERK MAPK, and DBL-1/TGF-β signals to control the toxicity of nanopolystyrene at ERCs in nematodes. In nanopolystyrene-exposed nematodes, neuronal RNAi knockdown of gpa-10 increased glb-10 and dbl-1 expressions (Fig. 9.26) (Y.-H. Yang and D.-Y. Wang, unpublished data). Moreover, neuronal RNAi knockdown of glb-10 or dbl-1 inhibited the resistance to nanopolystyrene toxicity in causing ROS production and in inhibiting locomotion behavior in gpa-10(RNAi) nematodes (Fig. 9.26) (Y.H. Yang and D.-Y. Wang, unpublished data), which suggested that the neuronal Gα/ GPA-10 functioned upstream of GLB-1/Globin and DBL-1/TGF-β signals to control the toxicity of nanopolystyrene at ERCs in nematodes. In nanopolystyrene-exposed nematodes, neuronal RNAi knockdown of goa-1 decreased jnk-1, mpk-1, daf-7, and dbl-1 expressions (Fig. 9.27) (Y.-H. Yang and D.-Y. Wang, unpublished data). To determine the genetic interactions between neuronal GOA-1 and its targets, the strain overexpressing neuronal goa-1 (Is (Punc-14-goa-1)) was generated. RNAi knockdown of jnk-1, mpk-1, daf-7, or dbl1 could suppress the resistance to nanopolystyrene toxicity in causing ROS production and in inhibiting locomotion behavior in Is(Punc-14-goa-1) nematodes (Fig. 9.27) (Y.-H. Yang and D.-Y. Wang, unpublished data), which suggested that the neuronal Gα/GOA-1 functioned upstream of JNK-1/JNK MAPK, MPK-1/ERK MAPK, DAF-7/TGF-β, and DBL-1/TGF-β signals to control the toxicity of nanopolystyrene at ERCs in nematodes.

 ⁄ Fig. 9.24 (continued) represent means  SD. **p < 0.01 vs. control (if not specially indicated). L4440, empty vector. PS-NPs, polystyrene nanoparticles. Exposure concentration of PS-NPs was 1 μg/L

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9 Response of G Protein-Coupled Receptors and Ion Channels to Toxicants at. . .

Fig. 9.25 Effect of RNAi knockdown of goa-1, gsa-1, or gpa-10 on PS-NP toxicity in nematodes overexpressing neuronal DCAR-1, DAF-37, NPR-8, NPR-9, or DOP-2 (Y.-H. Yang and D.-Y. Wang, unpublished data). (a) Effect of neuronal RNAi knockdown of dcar-1, daf-37, npr-8, npr-9, or dop-2 on expressions of goa-1, gsa-1, or gpa-10 in PS-NP-exposed TU3401 nematodes. L4440, empty vector. Bars represent means  SD. **p < 0.01 vs. TU3401. (b) Effect of RNAi knockdown of goa-1, gsa-1, or gpa-10 on PS-NP toxicity in inducing ROS production in nematodes overexpressing neuronal DCAR-1, DAF-37, NPR-8, NPR-9, or DOP-2. Bars represent means  SD. ** p < 0.01 vs. control (if not specially indicated). (c) Effect of RNAi knockdown of goa-1, gsa-1, or gpa-10 on PS-NP toxicity in decreasing locomotion behavior in nematodes overexpressing neuronal DCAR-1, DAF-37, NPR-8, NPR-9, or DOP-2. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). PS-NPs, polystyrene nanoparticles. Exposure concentration of PS-NPs was 1 μg/L

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Fig. 9.26 Genetic interactions between GSA-1 or GPA-10 and its targets in the neurons to regulate the PS-NP toxicity (Y.-H. Yang and D.-Y. Wang, unpublished data). (a) Effect of neuronal RNAi knockdown of gsa-1 or gpa-10 on expression of genes in PS-NP-exposed TU3401 nematodes. Bars represent means  SD. **p < 0.01 vs. TU3401. (b) Genetic interactions between GSA-1 or GPA-10 and its targets in the neurons to regulate the PS-NP toxicity in inducing ROS production. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). (c) Genetic interactions between GSA-1 or GPA-10 and its targets in the neurons to regulate the PS-NP toxicity in decreasing locomotion behavior. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). L4440, empty vector. PS-NPs, polystyrene nanoparticles. Exposure concentration of PS-NPs was 1 μg/L

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Fig. 9.27 Genetic interactions between GOA-1 and its targets in the neurons to regulate the PS-NP toxicity (Y.-H. Yang and D.-Y. Wang, unpublished data). (a) Effect of neuronal RNAi knockdown of goa-1 on expression of genes in PS-NP-exposed TU3401 nematodes. Bars represent means  SD. ** p < 0.01 vs. TU3401. (b) Genetic interactions between GOA-1 and its targets in the neurons to regulate the PS-NP toxicity in inducing ROS production. Bars represent means  SD. ** p < 0.01 vs. control (if not specially indicated). (c) Genetic interactions between GOA-1 and its targets in the neurons to regulate the PS-NP toxicity in decreasing locomotion behavior. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). L4440, empty vector. PS-NPs, polystyrene nanoparticles. Exposure concentration of PS-NPs was 1 μg/L

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9.3.3

245

Response of Germline Gα Subunits to Toxicants at ERCs

The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [21]. Among the 21 genes encoding Gα subunits, 2 genes (gpa-15 and gpa-16) are expressed in the germline (Fig. 9.28) (Y.-H. Yang and D.-Y. Wang, unpublished data). After the exposure, nanopolystyrene at concentrations ≧ 1 μg/L could significantly increase the expressions of gpa-15 and gpa-16 (Fig. 9.28) (Y.-H. Yang and D.-Y. Wang, unpublished data). Moreover, the alteration in expressions of gpa-15 and gpa-16 was concentration dependent in wild-type nematodes exposed to 1–1000 μg/L nanopolystyrene (Fig. 9.28) (Y.-H. Yang and D.-Y. Wang, unpublished data), suggesting the response of GPA-15 and GPA-16 in the germline to toxicants at ERCs in nematodes. Using ROS production and brood size as the endpoints, the effect of germline RNAi knockdown of gpa-15 or gpa-16 on the toxicity of nanopolystyrene was investigated. Exposure to nanopolystyrene (1 μg/L) could result in the significant induction of ROS production and decrease in locomotion behavior in DCL569 nematodes (Fig. 9.29) (Y.-H. Yang and D.-Y. Wang, unpublished data). After the nanopolystyrene exposure, germline RNAi knockdown of gpa-15 or gpa-16 caused the more severe induction of ROS production and decrease in locomotion behavior compared with those in DCL569 nematodes (Fig. 9.29) (Y.-H. Yang and D.-Y. Wang, unpublished data), suggesting the susceptibility of gpa-15(RNAi) or gpa-16 (RNAi) nematodes to the toxicity of nanopolystyrene. Therefore, the germline Gα proteins of GPA-15 and GPA-16 were involved in the control of toxicity of nanopolystyrene at ERCs in nematodes.

Fig. 9.28 Genes encoding germline Gα subunits dysregulated by nanopolystyrene in wild-type nematodes (Y.-H. Yang and D.-Y. Wang, unpublished data). (a) Searching for genes encoding Gα subunits expressed in the germline. (b) Effect of nanopolystyrene exposure on expressions of genes encoding germline Gα subunits. Bars represent means  SD. **p < 0.01 vs. control

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9 Response of G Protein-Coupled Receptors and Ion Channels to Toxicants at. . .

Fig. 9.29 Genetic interaction between GPA-15 and GPA-16 in the germline to regulate the response to nanopolystyrene (Y.-H. Yang and D.-Y. Wang, unpublished data). (a) Genetic interaction between GPA-15 and GPA-16 in the germline to regulate the toxicity of nanopolystyrene in inducing ROS production. (b) Genetic interaction between GPA-15 and GPA-16 in the germline to regulate the toxicity of nanopolystyrene in decreasing locomotion behavior. L4440, empty vector. Exposure concentration of nanopolystyrene was 1 μg/L. Exposure was performed from L1-larvae to adult Day-3. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated)

Using ROS production and locomotion behavior as the endpoints, the genetic interaction between GPA-15 and GPA-16 in the germline to regulate the response to nanopolystyrene was further examined. After the nanopolystyrene exposure, germline RNAi knockdown of both gpa-15 and gpa-16 resulted in the more severe induction of ROS production and decrease in locomotion behavior compared with those in gpa-15(RNAi) or gpa-16(RNAi) nematodes (Fig. 9.29) (Y.-H. Yang and D.Y. Wang, unpublished data). These observations implied that, in the germline, GPA-15 and GPA-16 acted in different pathways to regulate the toxicity of nanopolystyrene at ERCs in nematodes. As described above, the GPCR CED-1 acted in the germline to regulate the response to nanopolystyrene [67]. In nanopolystyrene-exposed DCL569 nematodes, germline RNAi knockdown of ced-1 did not affect the gpa-15 expression; however, the gpa-16 expression was significantly decreased by germline RNAi knockdown of

9.3 Response of G Proteins to Toxicants at ERCs

247

Fig. 9.30 Genetic interaction between CED-1 and GPA-16 in the germline to regulate the response to nanopolystyrene (Y.-H. Yang and D.-Y. Wang, unpublished data). (a) Effect of germline RNAi knockdown of ced-1 on expressions of gpa-15 and gpa-16 in nanopolystyrene-exposed nematodes. L4440, empty vector. Bars represent means  SD. **p < 0.01 vs. DCL569. (b) Genetic interaction between CED-1 and GPA-16 in the germline to regulate the toxicity of nanopolystyrene in inducing ROS production. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). (c) Genetic interaction between CED-1 and GPA-16 in the germline to regulate the toxicity of nanopolystyrene in decreasing locomotion behavior. Bars represent means  SD. ** p < 0.01 vs. control (if not specially indicated). Exposure concentration of nanopolystyrene was 1 μg/L. Exposure was performed from L1-larvae to adult Day-3

ced-1 (Fig. 9.30) (Y.-H. Yang and D.-Y. Wang, unpublished data). In the germline, the genetic interaction between CED-1 and GPA-16 in regulating the response to nanopolystyrene was further determined. Germline overexpression of CED-1 significantly inhibited the induction of ROS production and increased the locomotion behavior in nanopolystyrene-exposed nematodes (Fig. 9.30) (Y.-H. Yang and D.-Y. Wang, unpublished data), which suggested the resistance of nematodes overexpressing germline ced-1 (Is(Pmex-5-ced-1)) to the toxicity of nanopolystyrene. Moreover, RNAi knockdown of gpa-16 could cause the significant induction of ROS production and decrease in locomotion behavior in nanopolystyrene-exposed Is(Pmex-5-ced-1) nematodes (Fig. 9.30) (Y.-H. Yang

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Fig. 9.31 Identification of downstream targets of germline GPA-15 and GPA-16 in regulating the response to nanopolystyrene (Y.-H. Yang and D.-Y. Wang, unpublished data). (a) Effect of germline-specific RNAi knockdown of gpa-15 or gpa-16 on gene expressions in nanopolystyrene-exposed DCL569 nematodes. L4440, empty vector. Exposure was performed from L1-larvae to adult Day-3. Exposure concentration of nanopolystyrene was 1 μg/L. Bars represent means  SD. **p < 0.01 vs. DCL569. (b) A diagram showing the molecular basis of germline GPA-15 and GPA-16 in regulating the response to nanopolystyrene

and D.-Y. Wang, unpublished data). Therefore, in the germline, GPA-16 acted downstream of GPCR CED-1 to regulate the toxicity of nanopolystyrene at ERCs in nematodes. Previous studies have suggested the involvement of CED-10, VPS-34, SNX-1, RAB-7, RAB-14, NDK-1, NHL-2, WRT-3, PAT-12, and LIN-23 proteins in the germline to regulate the response to nanopolystyrene at ERCs [16, 67]. In nanopolystyrene-exposed nematodes, germline-specific RNAi knockdown of gpa15 significantly decreased the expressions of ced-10, vps-34, and snx-1 and increased the expressions of ndk-1, nhl-2, pat-12, and lin-23 (Fig. 9.31) (Y.-H. Yang and D.-Y. Wang, unpublished data). In nanopolystyrene-exposed nematodes, germline-specific RNAi knockdown of gpa-16 further significantly decreased the expressions of ced-10, vps-34, snx-1, rab-7, and rab-14 and increased the expressions of ndk-1, wrt-3, pat-12, and lin-23 (Fig. 9.31) (Y.-H. Yang and D.-Y. Wang, unpublished data). Therefore, the germline GPA-15 acted upstream of NHL-2, NDK-1, PAT-12, LIN-23, CED-10, VPS-34, and SNX-1 to regulate the toxicity of nanopolystyrene at ERCs in nematodes. In addition, the germline GPA-16 acted upstream of NDK-1, PAT-12, LIN-23, WRT-3, CED-10, VPS-34, SNX-1, RAB-7, and RAB-14 to regulate the toxicity of nanopolystyrene at ERCs in nematodes.

9.4

Response of Ion Channels to Toxicants at ERCs

Besides the GPCRs, the ion channels on cytoplasmic membrane can also function to sense various environmental stimuli in organisms.

9.4 Response of Ion Channels to Toxicants at ERCs

9.4.1

249

Response of Ion Channels to Toxicants at ERCs

Previous studies have raised some genes encoding ion channels required for the control of stress response in nematodes [76–86]. The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [87]. After the exposure, nanopolystyrene (1–1000 μg/L) did not significantly affect expressions of mec-4, mec-2, osm-9, trp-1, tax-2, slo-1, osm-12, unc-103, cup-4, and exc-1 in wild-type worms (Fig. 9.32) [87]. In contrast, exposure to nanopolystyrene (1–1000 μg/L) could increase expressions of egl-19, mec-10, trp-4, trp-2, tax-4, cca-1, unc-2, and unc-93 and decrease the expressions of cng-3, mec-6, ocr-2, deg-1, exc-4, kvs-1, and

Fig. 9.32 Effect of nanopolystyrene exposure on expression of genes encoding ion channels in wild-type nematodes (reprinted with permission from [87]). (a) Raman spectroscopy of nanopolystyrene particles. The Raman spectroscopy analysis indicated that the nanopolystyrene showed the peaks at 1001.61 cm 1 (breathing vibration of benzene ring), at 1031.92 cm 1 (symmetric extension vibration of carbon atoms in benzene ring), at 1201.65 cm 1 (stretching vibration of carbon atoms between benzene ring and polyethylene group), at 1451.12 cm 1 (asymmetric bending vibration of hydrogen atoms), and at 1602.39 cm 1 (asymmetric stretching vibration of benzene ring carbon atoms). (b) TEM image of nanopolystyrene particles in K medium before the sonication. (c) Expression of genes encoding ion channels in nanopolystyrene-exposed nematodes. The nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3. Bars represent means  SD. **p < 0.01 vs. control

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9 Response of G Protein-Coupled Receptors and Ion Channels to Toxicants at. . .

eat-2 in wild-type worms (Fig. 9.32) [87]. After the nanopolystyrene exposure, the increase in expressions of egl-19, mec-10, trp-4, trp-2, tax-4, cca-1, unc-2, and unc93 and the decrease in expressions of cng-3, mec-6, ocr-2, deg-1, exc-4, kvs-1, and eat-2 were concentration dependent (Fig. 9.32) [87]. These observations suggested the response of these 15 ion channel genes to toxicants (such as nanopolystyrene) at ERCs in nematodes.

9.4.2

Ion Channels Involved in Controlling Toxicity of Toxicants at ERCs

The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [87]. Locomotion behavior and production of ROS were used as assessment endpoints to assess the toxicity of nanopolystyrene. Among the dysregulated genes induced by nanopolystyrene exposure, RNAi knockdown of mec-6, mec-10, trp-4, trp-2, ocr-2, deg-1, unc-2, exc-4, or kvs-1 did not affect the toxicity of nanopolystyrene in inducing production of ROS (Fig. 9.33) [87]. Different from this, a decrease in production of ROS and an increase in locomotion behavior were observed in nanopolystyrene-exposed cng-3(RNAi) and eat-2(RNAi) nematodes compared with those in nanopolystyrene-exposed wild-type nematodes (Fig. 9.33) [87], suggesting that the cng-3(RNAi) and eat-2(RNAi) nematodes showed a resistance to the toxicity of nanopolystyrene. Moreover, exposure to nanopolystyrene caused the more severe toxicity in inducing production of ROS and in decreasing locomotion behavior in egl-19(RNAi), tax-4(RNAi), cca-1(RNAi), and unc-93(RNAi) nematodes compared with those in wild-type nematodes (Fig. 9.33) [87], which suggested that the egl-19(RNAi), tax-4(RNAi), cca-1 (RNAi), and unc-93(RNAi) nematodes exhibited a susceptibility to the toxicity of nanopolystyrene. Therefore, CNG-3, EAT-2, EGL-19, CCA-1, TAX-4, and UNC-93 were required in regulating the toxicity of toxicants (such as nanopolystyrene) at ERCs in nematodes.

9.4.3

Intestinal Ion Channels Involved in Controlling Toxicity of Toxicants at ERCs

The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [87]. EGL-19 and CCA-1 can be expressed in the intestine (Table 9.3). VP303 was used as a genetic tool to carry out the intestinal RNAi knockdown of gene(s). After the nanopolystyrene exposure, the production of ROS was enhanced by intestinal RNAi knockdown of egl-19 or cca-1 (Fig. 9.34) [87], which suggested

9.4 Response of Ion Channels to Toxicants at ERCs

251

Fig. 9.33 Identification of ion channels involved in the control of nanopolystyrene toxicity (reprinted with permission from [87]). (a) Identification of ion channels involved in the control of nanopolystyrene toxicity in inducing ROS production. (b) Identification of ion channels involved in the control of nanopolystyrene toxicity in decreasing locomotion behavior. L4440, empty vector. The nanopolystyrene exposure concentration was 1 μg/L. The nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated) Table 9.3 Expression patterns of candidate genes encoding ion channels (reprinted with permission from [87])

Gene cng-3 egl-19 tax-4 cca-1 unc-93 eat-2

Intestine + +

Neurons + + + + + +

Germline

+

252

9 Response of G Protein-Coupled Receptors and Ion Channels to Toxicants at. . .

Fig. 9.34 Identification of intestinal ion channels involved in the control of nanopolystyrene toxicity in inducing ROS production (reprinted with permission from [87]). L4440, empty vector. The nanopolystyrene exposure concentration was 1 μg/L. The nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated)

the susceptibility to nanopolystyrene toxicity in egl-19(RNAi) or cca-1(RNAi) nematodes. That is, the intestinal ion channels of EGL-19 and CCA-1 were required for the toxicity control of toxicants (such as nanopolystyrene) at ERCs in nematodes.

9.4.4

Neuronal Ion Channels Involved in Controlling Toxicity of Toxicants at ERCs

The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [87]. In nematodes, CNG-3, EAT-2, EGL-19, CCA-1, TAX-4, and UNC-93 can be expressed in the neurons (Table 9.3) [87]. Production of ROS and locomotion behavior were employed as toxicity assessment endpoints. Using TU3401 as a genetic tool for neuronal RNAi knockdown of gene(s), neuronal RNAi knockdown of cca-1 could not influence the toxicity of nanopolystyrene in inducing production of ROS (Fig. 9.35) [87]. Different from this, neuronal RNAi knockdown of cng-3 or eat-2 could suppress the production of ROS and increased the locomotion behavior in nanopolystyrene-exposed TU3401 nematodes (Fig. 9.35) [87], suggesting that the nematodes with neuronal RNAi knockdown of cng-3 or eat-2 had the resistance to nanopolystyrene toxicity. Exposure to nanopolystyrene further resulted in the more severe toxicity in inducing production of ROS and in decreasing locomotion behavior in egl-19(RNAi), tax-4(RNAi), and unc-93(RNAi) nematodes compared with those in nanopolystyrene-exposed TU3401 nematodes (Fig. 9.35) [87], suggesting the susceptibility of nematodes with neuronal RNAi knockdown of egl-19, tax-4, or unc-93 to the toxicity of nanopolystyrene. Therefore, the neuronal ion channels of CNG-3, EAT-2, EGL-19, TAX-4, and

9.4 Response of Ion Channels to Toxicants at ERCs

253

Fig. 9.35 Identification of neuronal ion channels involved in the control of nanopolystyrene toxicity (reprinted with permission from [87]). (a) Identification of neuronal ion channels involved in the control of nanopolystyrene toxicity in inducing ROS production. (b) Identification of neuronal ion channels involved in the control of nanopolystyrene toxicity in decreasing locomotion behavior. L4440, empty vector. The nanopolystyrene exposure concentration was 1 μg/L. The nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated)

UNC-93 were required for the toxicity control of toxicants (such as nanopolystyrene) at ERCs in nematodes. Among the ion channels involved in response to nanopolystyrene, only CCA-1 can be expressed in the germline (Table 9.3) [87]. In DCL569 worms, germline RNAi knockdown of cca-1 did not show any obvious alteration in production of ROS and locomotion behavior under the nanopolystyrene exposure condition [87]. Thus, CCA-1 could not function in germline to control the toxicity of nanopolystyrene.

254

9.4.5

9 Response of G Protein-Coupled Receptors and Ion Channels to Toxicants at. . .

Targets for Intestinal and Neuronal Ion Channels in Controlling Toxicity of Toxicants at ERCs

In the intestine, insulin signaling pathway mediated by daf-16, p38 MAPK signaling pathway mediated by pmk-1, Wnt signaling pathway mediated by bar-1, and ELT-2 signaling pathway have been identified to be required for the control of response to nanopolystyrene [30, 56–58]. DAF-16 is a transcriptional factor in the insulin signaling pathway, PMK-1 is a p38 MAPK in the MAPK signaling pathway, and BAR-1 is a β-catenin transcriptional factor in the Wnt signaling pathway. The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [87]. In nanopolystyrene-exposed VP303 nematodes, intestinal RNAi knockdown of cca-1 did not significantly alter expressions of pmk-1 and bar-1 (Fig. 9.36) [87]. In contrast, in nanopolystyrene-exposed VP303 nematodes, intestinal RNAi

Fig. 9.36 Identification of downstream targets of intestinal and neuronal ion channels in regulating the response to nanopolystyrene (reprinted with permission from [87]). (a) Identification of downstream targets of intestinal ion channels in regulating the response to nanopolystyrene. L4440, empty vector. The nanopolystyrene exposure concentration was 1 μg/L. The nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3. Bars represent means  SD. ** p < 0.01 vs. VP303. (b) Identification of downstream targets of neuronal ion channels in regulating the response to nanopolystyrene. L4440, empty vector. The nanopolystyrene exposure concentration was 1 μg/L. The nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3. Bars represent means  SD. **p < 0.01 vs. TU3401. (c) A diagram showing the molecular basis for intestinal and neuronal ion channels in regulating the response to nanopolystyrene in nematodes

9.5 Perspectives

255

knockdown of cca-1 resulted in a significant decrease in elt-2 expression (Fig. 9.36) [87]. Additionally, in nanopolystyrene-exposed VP303 nematodes, intestinal RNAi knockdown of egl-19 significantly inhibited the expressions of daf-16 and elt-2 (Fig. 9.36) [87]. Therefore, in intestinal cells, two signaling cascades (CCA-1ELT-2 and EGL-19-ELT-2/DAF-16) have been raised, and they form a molecular network to control the toxicity of nanopolystyrene at ERCs in nematodes (Fig. 9.36) [87]. During the control of nanopolystyrene toxicity, the activity of DAF-16 was inhibited by insulin receptor DAF-2 [30]. Therefore, at least in insulin signaling pathway, both GPCR(s) and ion channel(s) modulate the related signaling cascade. In the neurons, the JNK MAPK, ERK MAPK, and TGF-β signaling pathway are involved in the control of PS-NP toxicity [15, 63–65]. JNK-1 is a JNK MAPK, MPK-1 is an ERK MAPK, and DBL-1 and DAF-7 are two TGF-β ligands. The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [87]. In nanopolystyrene-exposed TU3401 nematodes, the expressions of jnk-1, mpk-1, and dbl-1 were significantly increased by neuronal RNAi knockdown of eat-2, the expressions of mpk-1 and daf-7 were significantly decreased by neuronal RNAi knockdown of unc-93, and the expressions of jnk-1 and daf-7 were significantly decreased by neuronal RNAi knockdown of tax-4 (Fig. 9.36) [87]. Different from this, neuronal RNAi knockdown of cng-3 or egl-19 did not affect the expressions of jnk-1, mpk-1, dbl-1, and daf-7 in nanopolystyrene-exposed TU3401 nematodes (Fig. 9.36) [87]. Therefore, in neuronal cells, three signaling cascades (EAT-2-DBL-1/JNK-1/MPK-1, TAX-4-JNK-1/DAF-7, and UNC-93-MPK-1/ DAF-7) have been raised, and they organize a protective molecular network to regulate the toxicity of nanopolystyrene at ERCs in nematodes (Fig. 9.36) [87]. Nevertheless, the downstream target(s) for neuronal EGL-19 and CNG-3 in controlling nanopolystyrene toxicity still remain unclear. This finding implied that, besides DBL-1/TGF-β, DAF-7/TGF-β, JNK MAPK, and ERK MAPK signaling pathways, some other unknown signaling pathways in neuronal cells also participate in controlling response to nanopolystyrene exposure.

9.5

Perspectives

An increasing evidence has proven the important value of C. elegans for the study of molecular toxicology [2, 3, 88, 89]. After the uptake, the environmental toxicants may cause the toxicity on organisms by activating or inhibiting certain GPCRs. With the nanopolystyrene as the example of environmental toxicants, limited number of intestinal, neuronal, and germline GPCRs have been identified to be involved in the control of toxicity of toxicants at ERCs in nematodes. Therefore, the limited number of GPCRs would be activated or inhibited to mediate the response of nematodes to toxicants at ERCs. In addition, the identified GPCRs in the intestine, neurons, and germline just reflect the binding potential by certain toxicants or response to certain toxicants in different tissues of nematodes.

256

9 Response of G Protein-Coupled Receptors and Ion Channels to Toxicants at. . .

Moreover, further with nanopolystyrene as the example of environmental toxicants, the limited number of intestinal, neuronal, and germline Gα subunits was identified to be required for the control of toxicity of toxicants at ERCs in nematodes. This observation further supports the role of GPCRs in different tissues in response to toxicants at ERCs. More importantly, it was found that only very limited number of Gα subunits exists to transduce the signals of intestinal, neuronal, and germline GPCRs to downstream different signaling pathways in nematodes exposed to certain toxicants at ERCs. Besides the GPCRs, certain number of ion channels were further identified at least in the intestine and neurons to be involved in the control of toxicity of toxicants (such as nanopolystyrene) at ERCs in nematodes. Nevertheless, the possible existence of germline ion channels with the function to control toxicity of toxicants at ERCs still cannot be excluded. Therefore, so far, the obtained evidence supports the roles of both GPCRs and ion channels in different tissues to be required for the control of toxicity of toxicants at ERCs in nematodes.

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Chapter 10

Epigenetic Control of Response to Toxicants at Environmentally Relevant Concentrations

Abstract Epigenetic control is an important molecular mechanism for nematodes in regulating the response to various toxicants or stresses. We first introduced and discussed the involvement of microRNAs (miRNAs), long noncoding RNAs (lncRNAs), and circular RNAs (circRNAs) in regulating the response to toxicants at environmentally relevant concentrations (ERCs). Moreover, we introduced and discussed the underlying mechanisms of certain miRNAs, lncRNAs, and circRNAs in regulating the response to toxicants at ERCs. Furthermore, we introduced and discussed the methylation regulation and histone acetylation regulation-related signals involved in the control of response to toxicants at ERCs and the underlying mechanisms. Keywords Environmentally relevant concentrations · Epigenetic control · Response · Caenorhabditis elegans

10.1

Introduction

The nematode Caenorhabditis elegans has been shown to be useful to detect the toxicity of different environmental toxicants or stresses [1–14]. More importantly, due to the well-described molecular and genetic backgrounds, C. elegans is a powerful animal model for the study of molecular toxicology [2, 3, 15–25]. In nematodes, two groups of epigenetic mechanisms exist to regulate the toxicity of environmental toxicants or stresses. One group of epigenetic mechanisms involved in the regulation of toxicity of environmental toxicants or stresses are mediated by methylation regulation and histone acetylation regulation. Another group of epigenetic mechanisms involved in the regulation of toxicity of environmental toxicants or stresses are at least mediated by microRNAs (miRNAs), long noncoding RNAs (lncRNAs), and circular RNAs (circRNAs). In Chapter 12 of “Molecular Toxicology in Caenorhabditis elegans,” we have introduced the epigenetic regulation of toxicity of environmental toxicants or stresses in nematodes [2]. In this chapter, we focused on the introduction and the discussion of epigenetic mechanisms involved in

© The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 D. Wang, Toxicology at Environmentally Relevant Concentrations in Caenorhabditis elegans, https://doi.org/10.1007/978-981-16-6746-6_10

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the control of response to toxicants at environmentally relevant concentrations (ERCs) in nematodes.

10.2

miRNAs Control of Response to Toxicants at ERCs

In nematodes, the miRNAs can regulate the toxicity of different environmental toxicants, such as carbon-based nanomaterials and fine particulate matter (PM2.5) [26–36]. The miRNAs were also involved in the control of toxicity induction of environmental stresses (such as pathogen infection) in nematodes [37–39].

10.2.1 Response of miRNAs to Toxicants at ERCs In the recent years, C. elegans has been frequently employed to assess the potential toxicity of nanopolystyrene at ERCs [40–47]. Using intestinal reactive oxygen species (ROS) production and locomotion behavior reflected by head thrash and body bend as the endpoints, exposure (L1-larvae to adult Day-3) to nanopolystyrene at concentrations
1 μg/L could cause the significant induction of intestinal ROS production and decrease in locomotion behavior in nematodes [48]. The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [49]. Using the SOLiD sequencing technique, seven dysregulated miRNAs were identified in nanopolystyrene (1 μg/L, a predicted environmental concentration)exposed nematodes (Fig. 10.1) [49]. Among these seven miRNAs, three upregulated miRNAs and four downregulated miRNAs were identified (Fig. 10.1) [49]. The upregulated miRNAs contained mir-35, mir-38, and mir-354, and the downregulated miRNAs contained mir-39, mir-76, mir-794, and mir-1830 (Fig. 10.1) [49]. The dysregulation of miRNAs in nanopolystyrene-exposed nematodes was further confirmed by the qRT-PCR analysis [49]. The qRT-PCR assay confirmed the decrease in expressions of mir-39, mir-76, mir-794, and mir-1830 and the increase in expressions of mir-35, mir-38, and mir-354 in nanopolystyrene (1 μg/L)-exposed nematodes (Fig. 10.2) [49]. Moreover, the expressions of all these examined seven miRNAs were dose-dependent in nematodes exposed to nanopolystyrene (1–100 μg/ L) (Fig. 10.2) [49]. Meanwhile, no obvious alteration in expressions of these examined seven miRNAs was detected in 0.1 μg/L nanopolystyrene-exposed nematodes (Fig. 10.2) [49]. These observations suggested the response of some miRNAs to nanopolystyrene at ERCs in nematodes.

10.2

miRNAs Control of Response to Toxicants at ERCs

265

Fig. 10.1 Dysregulation of microRNAs by nanopolystyrene (1 μg/L) in wild-type nematodes (reprinted with permission from [49]). (a) TEM image of nanopolystyrene particles in K medium. (b) Raman spectroscopy of nanopolystyrene particles. (c) Heat map of identified dysregulated microRNAs in wild-type N2 nematodes after exposure to nanopolystyrene (1 μg/L). (d) Downregulated and upregulated microRNAs in wild-type N2 nematodes after exposure to nanopolystyrene (1 μg/L). Exposure was performed from L1-larvae to adult Day-3

10.2.2 Functional Analysis of miRNAs in Regulating the Response to Toxicants at ERCs The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [49]. Intestinal ROS production and locomotion behavior reflected by thrash and body bend were used as the endpoints [49]. To determine the potential functions of candidate miRNAs in regulating the response to nanopolystyrene, the

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Fig. 10.2 qRT-PCR analysis of microRNA expression in nanopolystyrene-exposed wild-type nematodes (reprinted with permission from [49]). Exposure was performed from L1-larvae to adult Day-3. Bars represent means  SD. **p < 0.01 vs. control

transgenic nematodes overexpressing these miRNAs were generated [49]. Under the normal conditions, nematodes overexpressing mir-35, mir-38, mir-39, mir-76, mir354, mir-794, or mir-1830 did not show the obvious intestinal ROS induction and the alteration in locomotion behavior (Fig. 10.3) [49]. It was found that overexpression of mir-39 or mir-1830 did not obviously affect the toxicity of nanopolystyrene in inducing intestinal ROS production and in decreasing locomotion behavior (Fig. 10.3) [49]. In contrast, the more severe induction of intestinal ROS production and decrease in locomotion behavior were observed in nanopolystyrene-exposed nematodes overexpressing mir-76 or mir-794 compared with nanopolystyrene-exposed wild-type nematodes (Fig. 10.3) [49]. Additionally, overexpression of mir-35, mir-38, or mir-354 suppressed the induction of intestinal ROS production and the decrease in locomotion behavior in nematodes exposed to nanopolystyrene (Fig. 10.3) [49]. These results suggested the involvement of mir-35, mir-38, mir-76, mir-354, and mir-794 in regulating the response to nanopolystyrene at ERCs in nematodes. Among the five candidate miRNAs, the genetic mutants for mir-35 and mir-76 are available. To confirm the role of mir-35 and mir-76 in regulating the response to nanopolystyrene, the effects of mir-35 or mir-76 mutation on nanopolystyrene toxicity were also investigated [49]. After the nanopolystyrene exposure, the mir35 mutant nematodes showed enhanced intestinal ROS production and more severe

10.2

miRNAs Control of Response to Toxicants at ERCs

267

Fig. 10.3 Effect of overexpression of mir-35, mir-38, mir-39, mir-76, mir-354, mir-794, or mir1830 on nanopolystyrene toxicity in nematodes (reprinted with permission from [49]). (a) Effect of overexpression of mir-35, mir-38, mir-39, mir-76, mir-354, mir-794, or mir-1830 on nanopolystyrene toxicity in inducing intestinal ROS production. (b) Effect of overexpression of mir-35, mir-38, mir-39, mir-76, mir-354, mir-794, or mir-1830 on nanopolystyrene toxicity in decreasing locomotion behavior. Nanopolystyrene exposure concentration was 1 μg/L. Exposure was performed from L1-larvae to adult Day-3. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated)

decrease in locomotion behavior compared with wild-type nematodes; however, the mir-76 mutant nematodes exhibited the suppression in intestinal ROS production and the inhibition in the decrease in locomotion behavior (Fig. 10.4) [49].

10.2.3 Molecular Basis for mir-35 in Regulating the Response to Toxicants at ERCs The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [49]. Expression of mir-35 in neurons, muscle, germline, or epidermis

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Fig. 10.4 Effect of mir-35 or mir-76 mutation on nanopolystyrene toxicity in nematodes (reprinted with permission from [49]). (a) Effect of mir-35 or mir-76 mutation on nanopolystyrene toxicity in inducing intestinal ROS production. (b) Effect of mir-35 or mir-76 mutation on nanopolystyrene toxicity in decreasing locomotion behavior. Nanopolystyrene exposure concentration was 1 μg/L. Exposure was performed from L1-larvae to adult Day-3. Bars represent means  SD. ** p < 0.01 vs. control (if not specially indicated)

did not affect the toxicity of nanopolystyrene in inducing ROS production in mir-35 mutant nematodes (Fig. 10.5) [50]. In contrast, expression of intestinal mir-35 could significantly suppress the nanopolystyrene toxicity in inducing ROS production and in decreasing locomotion behavior in mir-35 mutant nematodes (Fig. 10.5) [50], suggesting that mir-35 acted in the intestine to regulate the response to nanopolystyrene at ERCs in nematodes. Based on the search of conserved sites that match the seed region of mir-35, the potential targeted genes of mir-35 were predicted using TargetScan (version 6.2, http://www.targetscan.org/worm_52/). Among the predicted targeted genes of mir35, 11 genes (ndk-1, dab-1, ell-1, alr-1, egl-3, ced-1, cex-2, cyd-1, tsn-1, mab-3, and C30F12.2) are expressed in the intestine (https://www.wormbase.org) (Fig. 10.6) [50]. Among these 11 genes, exposure to nanopolystyrene (1 μg/L) could further significantly decrease the expressions of ndk-1 and cex-2 and increase the expressions of dab-1, alr-1, cyd-1, and C30F12.2 in wild-type nematodes (Fig. 10.6) [50]. Moreover, in nanopolystyrene-exposed wild-type nematodes, mutation of mir-35 could only significantly increase the expressions of ndk-1 and cex-2 (Fig. 10.6) [50]. In nematodes, ndk-1 encodes a homolog of NM23-H1, and cex-2 encodes a calexcitin. It was further observed that intestine-specific RNAi knockdown of ndk-1 or cex-2 resulted in the suppression in ROS production in

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Fig. 10.5 Tissue-specific activity of mir-35 in regulating the response to nanopolystyrene in wildtype nematodes (reprinted with permission from [50]). (a) Tissue-specific activity of mir-35 in regulating the toxicity of nanopolystyrene in inducing ROS production. (b) Intestine-specific activity of mir-35 in regulating the toxicity of nanopolystyrene in decreasing locomotion behavior. Pges-1, Punc-14, Pmyo-3, and Pmlt-7 are intestine-specific, neuron-specific, muscle-specific, and epidermis-specific promoters, respectively. Exposure was preformed from L1-larvae to adult Day-3. Exposure concentration of nanopolystyrene was 1 μg/L. Bars represent means  SD. ** p < 0.01 vs. control (if not specially indicated)

nanopolystyrene-exposed VP303 nematodes (Fig. 10.6) [50], suggesting that the ndk-1(RNAi) and the cex-2(RNAi) nematodes were resistant to the toxicity of nanopolystyrene. These observations suggested the possible role of NDK-1 and CEX-2 as the downstream targets of intestinal mir-35 in regulating the response to nanopolystyrene at ERCs in nematodes. To determine the targeted genes of mir-35 in regulating the response to nanopolystyrene, the genetic interaction between mir-35 and NDK-1 or CEX-2 was examined. It was assumed that mutation or RNAi knockdown of targeted gene can suppress the phenotype of mir-35 mutant nematodes after nanopolystyrene exposure. After the exposure, RNAi knockdown of ndk-1 could suppress the susceptibility of mir-35 mutant nematodes to nanopolystyrene toxicity (Fig. 10.7) [50]. Different from this, RNAi knockdown of cex-2 did not obviously affect the susceptibility of mir-35 mutant nematodes to nanopolystyrene toxicity (Fig. 10.7)

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Fig. 10.6 Identification of potential targeted genes of intestinal mir-35 in regulating the response to nanopolystyrene (reprinted with permission from [50]). (a) Searching for the potential intestinal targeted genes of mir-35 based on TargetScan prediction. (b) Effect of nanopolystyrene exposure on gene expressions in wild-type nematodes. Bars represent means  SD. **p < 0.01 vs. control. (c) Effect of mir-35 mutation on gene expressions in nanopolystyrene-exposed wild-type nematodes. Exposure concentration of nanopolystyrene was 1 μg/L. Bars represent means  SD. ** p < 0.01 vs. wild type. (d) Effect of intestine-specific RNAi knockdown of ndk-1 or cex-2 on toxicity of nanopolystyrene in inducing ROS production in VP303 nematodes. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). Exposure was preformed from L1-larvae to adult Day-3. Exposure concentration of nanopolystyrene was 1 μg/L

[50]. Thus, the NDK-1, not the CEX-2, acted as the target of mir-35 to regulate the response to nanopolystyrene at ERCs in nematodes. During the control of biological processes, NDK-1 normally acts upstream of MPK-1 and kinase suppressors of Ras (KSR-1 and KSR-2). In nematodes, MPK-1, an ERK MAPK, usually acted in the neurons to regulate the response to toxicants, such as graphene oxide [2, 3]. Intestine-specific RNAi knockdown of ndk-1 caused the suppression in induction of ROS production and reduction in brood size, and

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Fig. 10.7 Genetic interaction between mir-35 and NDK-1 or CEX-2 in regulating the response to nanopolystyrene in wild-type nematodes (reprinted with permission from [50]). (a) Genetic interaction between mir-35 and NDK-1 or CEX-2 in regulating the toxicity of nanopolystyrene in inducing ROS production. (b) Genetic interaction between mir-35 and NDK-1 or CEX-2 in regulating the toxicity of nanopolystyrene in decreasing locomotion behavior. Exposure was preformed from L1-larvae to adult Day-3. Exposure concentration of nanopolystyrene was 1 μg/ L. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated)

intestine-specific RNAi knockdown of ksr-1 or ksr-2 resulted in the enhancement in induction of ROS production and reduction of brood size in nanopolystyreneexposed VP303 nematodes (Fig. 10.8) [50], suggesting the resistance of ndk-1 (RNAi) nematodes and the susceptibility of ksr-1(RNAi) or ksr-2(RNAi) nematodes to nanopolystyrene toxicity. Moreover, RNAi knockdown of ksr-1 or ksr-2 could inhibit the resistance of ndk-1(RNAi) nematodes to nanopolystyrene toxicity in inducing ROS production and in reducing brood size (Fig. 10.8) [50]. In nanopolystyrene-exposed VP303 nematodes, intestine-specific RNAi knockdown of ndk-1 could further significantly increase the expressions of ksr-1 and ksr-2 [50]. Therefore, KSR-1 and KSR-2 acted downstream of intestinal NDK-1 to regulate the response to nanopolystyrene at ERCs in nematodes. The intestine is an important response organ to nanopolystyrene, and insulin, p38 MAPK, and Wnt signaling pathways acted in the intestine to respond the nanopolystyrene in nematodes [18, 19, 48]. DAF-16 is a FOXO transcriptional

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Fig. 10.8 Genetic interaction between NDK-1 and KSR-1 or KSR-2 in the intestine to regulate the toxicity of nanopolystyrene in inducing ROS production in VP303 nematodes (reprinted with permission from [50]). Exposure was preformed from L1-larvae to adult Day-3. Exposure concentration of nanopolystyrene was 1 μg/L. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated)

factor in insulin signaling pathway, PMK-1 is a p38 MAPK in p38 MAPK signaling pathway, and BAR-1 is a β-catenin transcriptional factor in Wnt signaling pathway. Intestine-specific RNAi knockdown of daf-16, pmk-1, or bar-1 caused the enhancement in toxicity of nanopolystyrene [18, 19, 48], suggesting the susceptibility of daf16(RNAi), pmk-1(RNAi), and bar-1(RNAi) nematodes to nanopolystyrene toxicity. After the exposure, intestine-specific RNAi knockdown of pmk-1 or bar-1 did not affect the resistance of ndk-1(RNAi) nematodes to nanopolystyrene toxicity (Fig. 10.9) [50]. In contrast, intestine-specific RNAi knockdown of daf-16 could obviously suppress the resistance of ndk-1(RNAi) nematodes to nanopolystyrene toxicity (Fig. 10.9) [50]. In nanopolystyrene-exposed VP303 nematodes, intestinespecific RNAi knockdown of ndk-1 could further significantly increase the expressions of daf-16 and sod-3, a targeted gene of daf-16 [50]. Therefore, NDK-1 could further act upstream of DAF-16 in insulin signaling pathway to regulate the response to nanopolystyrene. That is, the activity of DAF-16 in regulating the response to nanopolystyrene is under the control of two molecular mechanisms. One is the control by the upstream signaling cascade of DAF-2-AGE-1-AKT-1 in the insulin signaling pathway. Another is the epigenetic control by signaling cascade of mir-35NDK-1. The epigenetic control by signaling cascade of mir-35-NDK-1 may play a role of effect amplification for the activity of DAF-16 in regulating the response to nanopolystyrene at ERCs in nematodes. After the exposure, the more severe induction of ROS production and reduction in brood size were detected in nanopolystyrene-exposed daf-16(RNAi);ksr-1(RNAi) nematodes compared with those in nanopolystyrene-exposed daf-16(RNAi) or ksr-1 (RNAi) nematodes (Fig. 10.10) [50]. Similarly, the more severe induction of ROS production and reduction in locomotion behavior were detected in nanopolystyreneexposed daf-16(RNAi)ksr-2(RNAi) nematodes compared with those in

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Fig. 10.9 Genetic interaction between NDK-1 and DAF-16, PMK-1, or BAR-1 in the intestine to regulate the toxicity of nanopolystyrene in inducing ROS production in VP303 nematodes (reprinted with permission from [50]). Exposure was preformed from L1-larvae to adult Day-3. Exposure concentration of nanopolystyrene was 1 μg/L. Bars represent means  SD. ** p < 0.01 vs. control (if not specially indicated)

nanopolystyrene-exposed daf-16(RNAi) or ksr-2(RNAi) nematodes (Fig. 10.10) [50]. Additionally, in nanopolystyrene-exposed VP303 nematodes, RNAi knockdown of ksr-1 or ksr-2 did not affect the daf-16 expression (Fig. 10.10) [50]. These observations suggested that DAF-16 and KSR-1/2 acted in different pathways to regulate the response to nanopolystyrene at ERCs in nematodes.

10.2.4 Molecular Basis for mir-794 in Regulating the Response to Toxicants at ERCs The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [49]. To determine the tissue-specific activity of mir-794 in regulating the response to nanopolystyrene, the transgenic strain overexpressing muscle mir794 and transgenic strain overexpressing intestinal mir-794 were generated. Overexpression of muscle mir-794 did not obviously affect the toxicity of nanopolystyrene in inducing ROS production and in reducing brood size (Fig. 10.11) [51]. In contrast, intestinal overexpression of mir-794 caused the more severe induction of ROS production and reduction in brood size in nanopolystyreneexposed nematodes compared with those in nanopolystyrene-exposed wild-type

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Fig. 10.10 Genetic interaction between DAF-16 and KSR-1 or KSR-2 in the intestine to regulate the response to nanopolystyrene (reprinted with permission from [50]). (a) Genetic interaction between DAF-16 and KSR-1 or KSR-2 in the intestine to regulate the toxicity of nanopolystyrene in inducing ROS production in VP303 nematodes. Exposure was preformed from L1-larvae to adult Day-3. Exposure concentration of nanopolystyrene was 1 μg/L. Bars represent means  SD. ** p < 0.01 vs. control (if not specially indicated). (b) Effect of intestine-specific RNAi knockdown of ksr-1 or ksr-2 on daf-16 expression in nanopolystyrene-exposed VP303 nematodes. Exposure was preformed from L1-larvae to adult Day-3. Exposure concentration of nanopolystyrene was 1 μg/L. Bars represent means  SD. (c) A diagram showing the molecular basis for intestinal mir-35 in regulating the response to nanopolystyrene in nematodes

nematodes (Fig. 10.11) [51]. Therefore, mir-794 acted in the intestine to regulate the response to nanopolystyrene at ERCs in nematodes. Based on the search of conserved sites that match the seed region of mir-794, the potential targeted genes of mir-794 were predicted using TargetScan (version 6.2, http://www.targetscan.org/worm_52/). Among the predicted targeted genes of mir794, 39 genes are expressed in the intestine (https://www.wormbase.org) (Fig. 10.12) [51]. Among these 39 intestinal genes, exposure to nanopolystyrene (1 μg/L) could significantly decrease the expressions of daf-12, daf-5, dct-1, fkh-7, fos-1, nhr-66, octr-1, psa-3, sulp-1, tbc-10, uba-1, and mab-31 and increase the expressions of daf-16, nhr-154, nhr-25, nhr-4, nhr-41, nhr-43, nhr-84, pan-1, rnt-1,

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Fig. 10.11 Tissue-specific activity of mir-794 in regulating the response to nanopolystyrene (reprinted with permission from [51]). (a) Tissue-specific activity of mir-794 in regulating the toxicity of nanopolystyrene in inducing ROS production. (b) Tissue-specific activity of mir-794 in regulating the toxicity of nanopolystyrene in reducing brood size. Exposure concentration of nanopolystyrene was 1 μg/L. Exposure was performed from L1-larvae to adult Day-3. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated)

skn-1, and mdt-15 (Fig. 10.12) [51]. Considering that the expression of mir-794 was decreased by exposure to nanopolystyrene (1 μg/L) [49], it was assumed that the expressions of targeted genes of mir-794 should be increased in nematodes exposed to nanopolystyrene (1 μg/L). The expressions of daf-16, nhr-154, nhr-25, nhr-4, nhr-41, nhr-43, nhr-84, pan-1, rnt-1, skn-1, and mdt-15 in nanopolystyrene-exposed nematodes overexpressing intestinal mir-794 were further examined. After the nanopolystyrene exposure, intestinal overexpression of mir-794 could significantly decrease the expressions of daf-16, nhr-25, skn-1, and mdt-15 (Fig. 10.12) [51]. These observations implied the role of DAF-16, NHR-25, SKN-1, and MDT-15 as the possible targets of intestine mir-794 in regulating the response to nanopolystyrene at ERCs in nematodes. DAF-16 is a FOXO transcriptional factor in the insulin signaling pathway, NHR-25 is a nuclear hormone receptor, SKN-1 is Nrf transcriptional factor in the p38 MAPK signaling pathway, and MDT-15 is a lipid metabolic sensor. Previous studies have demonstrated that DAF-16, SKN-1, and MDT-15 could act in the intestine to regulate the response to nanopolystyrene [15, 18, 48]. Intestinespecific RNAi knockdown of nhr-25 did not obviously affect the toxicity of nanopolystyrene in inducing ROS production and in reducing brood size [51]. Thus, NHR-25 did not act in the intestine to regulate the response to nanopolystyrene.

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Fig. 10.12 Identification of potential targeted genes of intestinal mir-794 in regulating the response to nanopolystyrene (reprinted with permission from [51]). (a) Searching for the potential intestinal targeted genes of mir-794 based on TargetScan prediction. (b) Effect of nanopolystyrene exposure on gene expressions in wild-type nematodes. Bars represent means  SD. **p < 0.01 vs. control. (c) Effect of intestinal mir-794 overexpression on gene expressions in nanopolystyrene-exposed wildtype nematodes. Exposure concentration of nanopolystyrene was 1 μg/L. Bars represent means  SD. **p < 0.01 vs. wild type. Exposure was preformed from L1-larvae to adult Day-3. Exposure concentration of nanopolystyrene was 1 μg/L

To confirm the role of DAF-16, SKN-1, and MDT-15 as the molecular targets of intestinal mir-794 in regulating the response to nanopolystyrene, the intestinal daf16, skn-1, or mdt-15 containing 30 UTR (Is(Pges-1-daf-16 + 30 UTR), Is(Pges-1-skn1 + 30 UTR), or Is(Pges-1-mdt-15 + 30 UTR)) was introduced into the transgenic strain overexpressing intestinal mir-794 (Is(Pges-1-mir-794)). Overexpression of intestinal daf-16, skn-1, or mdt-15 containing 30 UTR caused the suppression in ROS

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Fig. 10.13 Effects of intestinal overexpression of mir-794 on the response to nanopolystyrene in nematodes overexpressing intestinal daf-16, skn-1, or mdt-15 containing 30 UTR (reprinted with permission from [51]). (a) Effects of intestinal overexpression of mir-794 on the toxicity of nanopolystyrene in inducing ROS production in nematodes overexpressing intestinal daf-16, skn1, or mdt-15 containing 30 UTR. (b) Effects of intestinal overexpression of mir-794 on the toxicity of nanopolystyrene in reducing brood size in nematodes overexpressing intestinal daf-16, skn-1, or mdt-15 containing 30 UTR. Exposure concentration of nanopolystyrene was 1 μg/L. Exposure was performed from L1-larvae to adult Day-3. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated)

production and increase in brood size in nanopolystyrene-exposed nematodes (Fig. 10.13) [51], suggesting the resistance of nematodes overexpressing intestinal daf-16, skn-1, or mdt-15 containing 30 UTR to the toxicity of nanopolystyrene. Moreover, overexpression of intestinal mir-794 suppressed the resistance of nematodes overexpressing daf-16, skn-1, or mdt-15 containing 30 UTR to the toxicity of nanopolystyrene (Fig. 10.13) [51]. Therefore, the intestinal mir-794 potentially regulated the response to nanopolystyrene by affecting the activities of daf-16, skn-1, and mdt-15 via binding to their 30 UTRs. These observations further suggested the important function of intestinal mir-794 in mediating an epigenetic regulation

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mechanism by linking insulin and p38 MAPK signaling pathways to regulate the response to nanopolystyrene at ERCs in nematodes.

10.2.5 Molecular Basis for mir-354 in Regulating the Response to Toxicants at ERCs The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [49]. To determine the tissue-specific activity of mir-354 in regulating the response to nanopolystyrene, transgenic animals overexpressing neuronal (Is (Punc-14-mir-354)), intestinal (Is(Pges-1-mir-354)), muscle (Is(Pmyo-3-mir-354)), epidermal (Is(Pmlt-7-mir-354)), and germline mir-354 (Is(Pmex-5-mir-354)) were generated. Using ROS production and locomotion behavior as endpoints, neuronal, muscle, epidermal, or germline overexpression of mir-354 did not obviously affect the toxicity of nanopolystyrene (Fig. 10.14) [52]. In contrast, intestinal overexpression of mir-354 significantly inhibited the toxicity of nanopolystyrene in inducing ROS production and in decreasing locomotion behavior (Fig. 10.14) [52]. These observations suggested that the mir-354 acted in the intestine to regulate the toxicity of nanopolystyrene at ERCs in nematodes. Based on the search of conserved sites that match seed region of mir-354, the potential targeted genes of mir-354 were predicted using TargetScan (http://www. targetscan.org/worm_52/). Among the predicted targeted genes of mir-354, 48 genes can be expressed in the intestine (https://www.wormbase.org) (Fig. 10.15) [52]. Among these 48 intestinal genes, exposure to nanopolystyrene (1 μg/L) could significantly increase the expressions of hlh-30, ant-1.1, shc-1, ags-3, lir-1, aha-1, nhr-43, btbd-10, abts-4, lgg-2, shn-1, mca-3, and ftt-2 and decrease the daf-3 expression (Fig. 10.15) [52]. Considering the fact that the mir-354 expression was increased by nanopolystyrene exposure [49], it can be assumed that the expression of its targeted genes should be decreased by nanopolystyrene exposure. After the nanopolystyrene exposure, intestinal overexpression of mir-354 could further significantly decrease the daf-3 expression (Fig. 10.15) [52]. Moreover, RNAi knockdown of daf-3 suppressed the induction of ROS production and the decrease in locomotion behavior in nanopolystyrene-exposed nematodes (Fig. 10.15) [52], suggesting the resistance of daf-3(RNAi) nematodes to the toxicity of nanopolystyrene. Thus, DAF-3, a transcriptional factor of DAF-7-mediated TGF-β signaling pathway, might act as the downstream target of intestinal mir-354 to regulate the toxicity of nanopolystyrene at ERCs. To confirm the role of DAF-3 as the target of intestinal mir-354 in regulating the response to nanopolystyrene, the genetic interaction between mir-354 and DAF-3 in the intestine to regulate the response to nanopolystyrene was further examined. After the nanopolystyrene exposure, the nematodes overexpressing intestinal daf-3 containing 30 UTR showed the more severe induction of ROS production and

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Fig. 10.14 Tissue-specific activity of mir-354 in regulating the response to nanopolystyrene (reprinted with permission from [52]). (a) Tissue-specific activity of mir-354 in regulating the toxicity of nanopolystyrene in inducing ROS production. (b) Tissue-specific activity of mir-354 in regulating the toxicity of nanopolystyrene in decreasing locomotion behavior. The nanopolystyrene exposure concentration was 1 μg/L. The nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated)

decrease in locomotion behavior compared with wild type (Fig. 10.16) [52], suggesting the susceptibility of Is(Pges-1-daf-3 + 30 UTR) nematodes to the toxicity of nanopolystyrene. Moreover, intestinal overexpression of intestinal mir-354 could suppress the susceptibility of Is(Pges-1-daf-3 + 30 UTR) nematodes to the toxicity of nanopolystyrene in inducing ROS production and in decreasing locomotion behavior (Fig. 10.16) [52]. These observations confirmed the function of DAF-3 as the downstream target of intestinal mir-354 in regulating the toxicity of nanopolystyrene at ERCs.

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Fig. 10.15 Identification of potential target of intestinal mir-354 in regulating the response to nanopolystyrene (reprinted with permission from [52]). (a) Searching for the potential intestinal targeted genes of mir-354 based on TargetScan prediction. (b) Effect of nanopolystyrene exposure

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10.2.6 Molecular Basis for mir-38 in Regulating the Response to Toxicants at ERCs The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [49]. Based on the search of conserved sites that match seed region of mir-38, the potential targeted genes of mir-38 were predicted using TargetScan (version 6.2, http://www.targetscan.org/worm_52/). Among the predicted targeted genes of mir-38, 17 genes (sup-26, pat-12, nhl-2, ndk-1, dab-1, rig-6, wrt-3, ced-1, cyd-1, tnt-2, tsn-1, doxa-1, C30F12.2, unc-52, egl-1, lin-23, and gld-1) are expressed in the germline (https://www.wormbase.org) (Fig. 10.17) [53]. Among these 17 germline genes, exposure to nanopolystyrene (1 μg/L) from L1-larvae for approximately 6.5-day significantly decreased the expressions of pat-12, nhr-2, ndk-1, wrt-3, tsn-1, and lin-23 and increased the expressions of dab-1, rig-6, cyd1, tnt-2, doxa-1, C30F12.2, unc-52, egl-1, and gld-1 (Fig. 10.17) [53]. Considering the fact that mir-38 expression was increased by exposure to nanopolystyrene [49], it was assumed that the expression of its targeted genes should be decreased in nanopolystyrene-exposed nematodes. Moreover, after nanopolystyrene exposure, overexpression of germline of mir-38 significantly decreased the expressions of pat-12, nhl-2, ndk-1, wrt-3, and lin-23, but did not affect the tsn-1 expression (Fig. 10.17) [53]. Therefore, PAT-12, NHL-2, NDK-1, WRT-3, and LIN-23 might act as the downstream targets of germline mir-38 to regulate the response to toxicants (such as nanopolystyrene) at ERCs. DCL569 was used as a tool for germline-specific RNAi knockdown. Exposure to nanopolystyrene (1 μg/L) caused the significant induction of ROS production and decrease in locomotion behavior in DC569 nematodes (Fig. 10.18) [53]. Moreover, germline-specific RNAi knockdown of pat-12, nhl-2, ndk-1, wrt-3, or lin-23 inhibited the induction of ROS production, as well as the decrease in locomotion behavior, in nanopolystyrene-exposed DCL569 nematodes (Fig. 10.18) [53]. That is, the pat-12(RNAi), nhl-2(RNAi), ndk-1(RNAi), wrt-3(RNAi), and lin-23(RNAi) nematodes showed the resistance to nanopolystyrene toxicity. Therefore, PAT-12, NHL-2, NDK-1, WRT-3, and LIN-23 could function in the germline to regulate the response to toxicants (such as nanopolystyrene) at ERCs.

 ⁄ Fig. 10.15 (continued) on gene expressions in wild-type nematodes. Bars represent means  SD. ** p < 0.01 vs. control. (c) Effect of intestinal mir-354 overexpression on expression of daf-3 in nanopolystyrene-exposed nematodes. Bars represent means  SD. **p < 0.01 vs. wild type. (d) Effect of RNAi knockdown of daf-3 on toxicity of nanopolystyrene in inducing ROS production. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). (e) Effect of RNAi knockdown of daf-3 on toxicity of nanopolystyrene in decreasing locomotion behavior. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). The nanopolystyrene exposure concentration was 1 μg/L. The nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3

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Fig. 10.16 Genetic interaction between mir-354 and DAF-3 in the intestine to regulate the response to nanopolystyrene (reprinted with permission from [52]). (a) Genetic interaction between mir-354 and DAF-3 in the intestine to regulate the toxicity of nanopolystyrene in inducing ROS production. (b) Genetic interaction between mir-354 and DAF-3 in the intestine to regulate the toxicity of nanopolystyrene in decreasing locomotion behavior. The nanopolystyrene exposure concentration was 1 μg/L. The nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated)

To confirm whether PAT-12, NHL-2, NDK-1, WRT-3, and LIN-23 acted as the molecular targets of germline mir-38 in regulating the response to nanopolystyrene, the germline pat-12, nhl-2, ndk-1, wrt-3, or lin-23 containing 30 UTR (Is(Pmex-5pat-12 + 30 UTR), Is(Pmex-5-nhl-2 + 30 UTR), Is(Pmex-5-ndk-1 + 30 UTR), Is(Pmex-5wrt-3 + 30 UTR), or Is(Pmex-5-lin-23 + 30 UTR)) was introduced into the transgenic strain overexpressing germline mir-38 (Is(Pmex-5-mir-38)). Overexpression of germline pat-12, nhl-2, ndk-1, wrt-3, or lin-23 containing 30 UTR caused the enhancement in induction of ROS production and decrease in locomotion behavior in nanopolystyrene-exposed nematodes (Fig. 10.19) [53], suggesting the susceptibility of these strains to the toxicity of nanopolystyrene. Overexpression of germline mir-38 did not affect the ROS production and the locomotion behavior in

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Fig. 10.17 Identification of potential targeted genes of germline mir-38 in regulating the response to nanopolystyrene in nematodes (reprinted with permission from [53]). (a) Searching for the potential germline targeted genes of mir-38 based on TargetScan prediction. (b) Effect of nanopolystyrene exposure on gene expressions in wild-type nematodes. Bars represent means  SD. ** p < 0.01 vs. control. (c) Effect of mir-38 overexpression in germline on gene expressions in nanopolystyrene-exposed wild-type nematodes. Bars represent means  SD. **p < 0.01 vs. wild type. Exposure concentration of nanopolystyrene was 1 μg/L. Exposure was preformed from L1-larvae to adult Day-3

nanopolystyrene-exposed nematodes overexpressing germline pat-12 or lin-23 containing 30 UTR (Fig. 10.19) [53]. Different from this, overexpression of germline mir-38 suppressed the susceptibility of nematodes overexpressing germline nhl-2, ndk-1, or wrt-3 containing 30 UTR to the toxicity of nanopolystyrene in inducing ROS production and in decreasing locomotion behavior (Fig. 10.19) [53]. These observations suggested that NHL-2, NDK-1, and WRT-3 acted as the downstream targets of germline mir-35 during the control of response to nanopolystyrene at ERCs in nematodes. In nematodes, nhl-2 encodes a miRISC cofactor, ndk-1 encodes a homolog of NM23-H1, and wrt-3 encodes a homolog of mammalian peptidylprolyl cis-trans isomerase-like 2 (PPIL2). During the control of biological processes, HER-1, AIN-1, and EKL-1 acted as the potential downstream targets of germline NHL-2 [54–56]. Among these three

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Fig. 10.18 Effect of germline-specific RNAi knockdown of pat-12, nhl-2, ndk-1, wrt-3, or lin-23 on toxicity of nanopolystyrene in nematodes (reprinted with permission from [53]). (a) Effect of germline-specific RNAi knockdown of pat-12, nhl-2, ndk-1, wrt-3, or lin-23 on toxicity of nanopolystyrene in inducing ROS production. (b) Effect of germline-specific RNAi knockdown of pat-12, nhl-2, ndk-1, wrt-3, or lin-23 on toxicity of nanopolystyrene in decreasing locomotion behavior. L4440, empty vector. Exposure was preformed from L1-larvae to adult Day-3. Exposure concentration of nanopolystyrene was 1 μg/L. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated)

genes, germline-specific RNAi knockdown of her-1 or ain-1 did not affect the ROS production and the locomotion behavior in nanopolystyrene-exposed nematodes (Fig. 10.20) [53]. In contrast, germline-specific RNAi knockdown of ekl-1 caused the enhancement in induction of ROS production and decrease in locomotion behavior induced by nanopolystyrene exposure (Fig. 10.20) [53], suggesting the susceptibility of ekl-1(RNAi) nematodes to nanopolystyrene toxicity. Moreover, in nanopolystyrene-exposed nematodes, germline-specific RNAi knockdown of nhl-2 significantly reduced the ekl-1 expression (Fig. 10.20) [53]. These observations suggested the role of EKL-1 as the downstream target of germline NHL-2 in regulating the response to toxicants (such as nanopolystyrene) at ERCs in nematodes. In nematodes, ekl-1 encodes a Tudor domain protein. During the control of biological processes, germline NDK-1 can act upstream of kinase suppressors of Ras (KSR-1 and KSR-2) [57, 58]. Using locomotion behavior and ROS production as the endpoints, the nematodes with germline-specific RNAi

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Fig. 10.19 Effects of germline overexpression of mir-38 on the response to nanopolystyrene in nematodes overexpressing germline pat-12, nhl-2, ndk-1, wrt-3, or lin-23 containing 30 UTR (reprinted with permission from [53]). (a) Effects of germline overexpression of mir-38 on the toxicity of nanopolystyrene in inducing ROS production in nematodes overexpressing germline pat-12, nhl-2, ndk-1, wrt-3, or lin-23 containing 30 UTR. (b) Effects of germline overexpression of mir-38 on the toxicity of nanopolystyrene in decreasing locomotion behavior in nematodes overexpressing germline pat-12, nhl-2, ndk-1, wrt-3, or lin-23 containing 30 UTR. Exposure concentration of nanopolystyrene was 1 μg/L. Exposure was performed from L1-larvae to adult Day-3. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated)

knockdown of ksr-1 or ksr-2 showed the susceptibility to nanopolystyrene toxicity (Fig. 10.21) [53]. More importantly, germline-specific RNAi knockdown of ksr-1 or ksr-2 inhibited the resistance of ndk-1(RNAi) nematodes to the nanopolystyrene toxicity (Fig. 10.21) [53], demonstrating the potential role of KSR-1 and KSR-2 as

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Fig. 10.20 Identification of downstream targets of germline NHL-2 in regulating the response to nanopolystyrene in nematodes (reprinted with permission from [53]). (a) Effect of germline-specific RNAi knockdown of her-1, ain-1, or ehl-1 on ROS production in nanopolystyrene-exposed nematodes. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). (b) Effect of germline-specific RNAi knockdown of her-1, ain-1, or ehl-1 on locomotion behavior in nanopolystyrene-exposed nematodes. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). (c) Effect of germline-specific RNAi knockdown of nhl-2 on ekl-1 expression. Bars represent means  SD. **p < 0.01 vs. DCL569. L4440, empty vector. Exposure concentration of nanopolystyrene was 1 μg/L. Exposure was performed from L1-larvae to adult Day-3

the downstream targets of germline NDK-1 during the control of response to toxicants (such as nanopolystyrene) at ERCs in nematodes. In mammals, PPIL-2 usually regulates the biological processes by affect the activity of BACE1 [59]. The homolog of BACE1 in C. elegans is ASP-2. Exposure to nanopolystyrene (1 μg/L) significantly increased the asp-2 expression (Fig. 10.22) [53]. Meanwhile, germline RNAi knockdown of asp-2 caused the susceptibility to nanopolystyrene toxicity based on the assessment on endpoints of locomotion behavior and ROS production (Fig. 10.22) [53]. Moreover, germline RNAi

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Fig. 10.21 Genetic interaction between NDK-1 and KSR-1 or KSR-2 in the germline to regulate the response to nanopolystyrene in nematodes (reprinted with permission from [53]). (a) Genetic interaction between NDK-1 and KSR-1 or KSR-2 in the germline to regulate the toxicity of nanopolystyrene in inducing ROS production. (b) Genetic interaction between NDK-1 and KSR-1 or KSR-2 in the germline to regulate the toxicity of nanopolystyrene in decreasing locomotion behavior. L4440, empty vector. Exposure concentration of nanopolystyrene was 1 μg/L. Exposure was performed from L1-larvae to adult Day-3. Bars represent means  SD. ** p < 0.01 vs. control (if not specially indicated)

knockdown of asp-2 could inhibit the resistance of wrt-3(RNAi) nematodes to the nanopolystyrene toxicity (Fig. 10.22) [53], demonstrating the potential role of ASP-2 as the downstream target of germline WRT-3 in regulating the response to toxicants (such as nanopolystyrene) at ERCs. Therefore, three proteins (NHL-2, NDK-1, and WRT-3) were identified as the targets of germline mir-38 in regulating the response to nanopolystyrene. During the control of response to nanopolystyrene, these three downstream targets further mediated three signaling cascades (NHL-2EKL-1, NDK-1-KSR-1/2, and WRT-3-ASP-2), which provided an important basis for understanding the mechanism of germline in response to nanoplastic exposure in environmental organisms.

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Fig. 10.22 Identification of downstream target of germline WRT-3 in regulating the response to nanopolystyrene in nematodes (reprinted with permission from [53]). (a) Effect of nanopolystyrene exposure on asp-2 expression. Exposure concentration of nanopolystyrene was 1 μg/L. Exposure was performed from L1-larvae to adult Day-3. Bars represent means  SD. **p < 0.01 vs. control. (b) Genetic interaction between WRT-3 and ASP-2 in the germline to regulate the toxicity of nanopolystyrene in inducing ROS production. L4440, empty vector. Exposure concentration of nanopolystyrene was 1 μg/L. Exposure was performed from L1-larvae to adult Day-3. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). (c) Genetic interaction between WRT-3 and ASP-2 in the germline to regulate the toxicity of nanopolystyrene in decreasing locomotion behavior. L4440, empty vector. Exposure concentration of nanopolystyrene was 1 μg/L. Exposure was performed from L1-larvae to adult Day-3. Bars represent means  SD.

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10.2.7 Molecular Basis for mir-76 in Regulating the Response to Toxicants at ERCs The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [60]. Using TargetScan as a tool, the potential targets for mir-76 were predicted. In nematodes, the mir-76 is expressed in the neurons. Among the predicted targeted genes, anat-1, deg-1, gsa-1, glb-10, ldb-1, nfki-1, pfn-1, set-4, glb-25, and tag-178 can be expressed in the neurons (https://www.wormbase.org) (Fig. 10.23) [60]. In wild-type nematodes, exposure to 1–100 μg/L nanopolystyrene increased the expressions of glb-10 and nfki-1 and decreased the gsa-1 expression (Fig. 10.23) [60]. It was assumed that the expression levels of mir-76’s targets would be increased by exposure to nanopolystyrene. Moreover, mir-76 mutation increased the expressions of glb-10 and nfki-1 in nanopolystyrene-exposed nematodes (Fig. 10.23) [60]. That is, the expressions of glb-10 and nfki-1 were opposite to the expression of mir-76 in nanopolystyrene-exposed nematodes. Therefore, GLB-10 and NFKI-1 might act as the targets of mir-76 to control the toxicity of toxicants (such as nanopolystyrene) at ERCs in nematodes. Using TU3401 as a genetic tool for neuronal RNAi knockdown of gene(s), neuronal RNAi knockdown of glb-10 induced the susceptibility to the nanopolystyrene toxicity in inducing ROS production and in decreasing locomotion behavior (Fig. 10.24) [60]. Therefore, GLB-10 functioned in neuronal cells to control the toxicity of toxicants (such as nanopolystyrene) at ERCs in nematodes. In nematodes, glb-10 encodes a globin protein. Different from this, although mutation of mir-76 increased the expression of nfki-1 in nanopolystyrene-exposed worms, neuronal RNAi knockdown of nfki-1 could not alter the nanopolystyrene toxicity in inducing ROS production and in decreasing locomotion behavior (Fig. 10.24) [60]. Thus, NFKI-1 did not function downstream of neuronal mir-76 in controlling the nanopolystyrene toxicity. Genetic interaction analysis indicated that the RNAi knockdown of glb-10 could notably cause the ROS production and the decrease in locomotion behavior in nanopolystyrene-exposed mir-76 mutant nematodes (Fig. 10.25) [60]. That is, the resistance of mir-76 mutant to nanopolystyrene toxicity was inhibited by RNAi knockdown of glb-10. Moreover, the neuronal glb-10 containing 30 UTR (Punc-14-glb-10 + 30 UTR) was introduced in nematodes overexpressing neuronal mir-76 (Is(Punc-14-mir-76)). Overexpression of neuronal glb-10 containing 30 UTR suppressed the toxicity of nanopolystyrene exposure in inducing ROS production and in decreasing locomotion behavior (Fig. 10.26) [60], indicating the resistance of Is(Punc-14-glb10 + 30 UTR) nematodes to the nanopolystyrene toxicity. Neuronal overexpressing  ⁄ Fig. 10.22 (continued) **p < 0.01 vs. control (if not specially indicated). (d) A diagram showing the molecular basis of germline mir-38 in regulating the response to nanopolystyrene in nematodes

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Fig. 10.23 Identification of potential targeted genes of mir-76 in regulating the response to nanopolystyrene in nematodes (reprinted with permission from [60]). (a) Searching for the potential neuronal targeted genes of mir-76 based on TargetScan prediction. (b) Effect of nanopolystyrene exposure on gene expressions in wild-type nematodes. Bars represent means  SD. ** p < 0.01 vs. control. (c) Effect of mir-76 mutation on expressions of glb-10 and nfki-1 in nanopolystyrene-exposed nematodes. Exposure concentration of nanopolystyrene was 1 μg/L. Bars represent means  SD. **p < 0.01 vs. wild type. Exposure was preformed from L1-larvae to adult Day-3

mir-76 enhanced the toxicity of nanopolystyrene exposure in inducing ROS production and in decreasing locomotion behavior (Fig. 10.26) [60], indicating the susceptibility of Is(Punc-14-mir-76) nematodes to the nanopolystyrene toxicity. Furthermore, overexpression of neuronal mir-76 inhibited the resistance of Is (Punc-14-glb-10 + 30 UTR) nematodes to the nanopolystyrene toxicity in inducing ROS production and in decreasing locomotion behavior (Fig. 10.26) [60]. These results confirmed that GLB-10 functioned as the direct target of neuronal mir-76 in controlling the toxicity of toxicants (such as nanopolystyrene) at ERCs in nematodes.

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Fig. 10.24 Effect of neuronal RNAi knockdown of glb-10 or nfki-1 on response to nanopolystyrene (reprinted with permission from [60]). (a) Effect of neuronal RNAi knockdown of glb-10 or nfki-1 on toxicity of nanopolystyrene in inducing ROS production. (b) Effect of neuronal RNAi knockdown of glb-10 or nfki-1 on toxicity of nanopolystyrene in decreasing locomotion behavior. L4440, empty vector. Exposure was preformed from L1-larvae to adult Day-3. Exposure concentration of nanopolystyrene was 1 μg/L. Bars represent means  SD. ** p < 0.01 vs. control (if not specially indicated)

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In nematodes, the lncRNAs can also regulate the toxicity of different environmental toxicants or stresses, such as graphene oxide (GO) or simulated microgravity [25, 61].

10.3.1 Response of lncRNAs to Toxicants at ERCs The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [62]. Using the Illumina sequencing technique, a number of dysregulated lncRNAs were detected in 1 μg/L nanopolystyrene-exposed nematodes (Fig. 10.27) [62]. The Illumina sequencing yielded 2189 lncRNA transcript isoforms, and 37 lncRNAs were dysregulated by exposure to nanopolystyrene (1 μg/L) (Fig. 10.27) [62]. Among these 37 dysregulated lncRNAs, 22 lncRNAs including 3 known lncRNAs (linc-7, linc-50, and linc-169) were downregulated, and 15 lncRNAs including 5 known lncRNAs (linc-2, linc-9, linc-18, linc-32, and

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Fig. 10.25 Genetic interaction between mir-76 and GLB-10 in regulating the response to nanopolystyrene (reprinted with permission from [60]). (a) Genetic interaction between mir-76 and GLB-10 in regulating the toxicity of nanopolystyrene in inducing ROS production. (b) Genetic interaction between mir-76 and GLB-10 in regulating the toxicity of nanopolystyrene in decreasing locomotion behavior. Exposure was preformed from L1-larvae to adult Day-3. Exposure concentration of nanopolystyrene was 1 μg/L. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated)

linc-61) were upregulated (Fig. 10.27) [62]. All these dysregulated lncRNAs induced by nanopolystyrene exposure were intergenic lncRNAs in nematodes [62]. Considering that the molecular information for most of the novel lncRNAs is absent, the known dysregulated lncRNAs were further focused to examine their dynamic expression in nanopolystyrene-exposed nematodes [62]. First of all, the qRT-PCR assay confirmed the decrease in expressions of linc-7, linc-50, and linc169 and the increase in expressions of linc-2, linc-9, linc-18, linc-32, and linc-61 in nanopolystyrene (1 μg/L)-exposed nematodes (Fig. 10.28) [62]. Except XLOC_013858, the qRT-PCR assay confirmed the dysregulation of other novel lncRNAs in nanopolystyrene (1 μg/L)-exposed nematodes [62]. Moreover, both the decrease in expression of linc-7, linc-50, or linc-169 and the increase in expression of linc-2, linc-9, linc-18, linc-32, or linc-61 were dose-dependent in nematodes exposed to nanopolystyrene (1–100 μg/L) (Fig. 10.28) [62]. Meanwhile, no obvious alteration in expressions of these examined lncRNAs were detected in nanopolystyrene (0.1 μg/L)-exposed nematodes (Fig. 10.28) [62]. These observations suggested the response of some lncRNAs to nanopolystyrene at ERCs in nematodes.

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Fig. 10.26 Effects of neuronal overexpression of mir-76 on the response to nanopolystyrene in nematodes overexpressing neuronal glb-10 containing 30 UTR (reprinted with permission from [60]). (a) Effects of neuronal overexpression of mir-76 on the toxicity of nanopolystyrene in inducing ROS production in nematodes overexpressing neuronal glb-10 containing 30 UTR. (b) Effects of neuronal overexpression of mir-76 on the toxicity of nanopolystyrene in decreasing locomotion behavior in nematodes overexpressing neuronal glb-10 containing 30 UTR. Exposure concentration of nanopolystyrene was 1 μg/L. Exposure was performed from L1-larvae to adult Day-3. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated)

10.3.2 Functional Analysis of lncRNAs in Regulating the Response to Toxicants at ERCs The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [62]. Intestinal ROS production and locomotion behavior reflected by head thrash and body bend were used as the endpoints [62]. The effects of RNAi

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Fig. 10.27 Genome-wide identification of lncRNAs in response to nanopolystyrene (1 μg/L) in nematodes (reprinted with permission from [62]). (a) TEM image of nanopolystyrene. (b) Raman spectroscopy of nanopolystyrene. (c) Heat map of identified dysregulated lncRNAs induced by exposure to nanopolystyrene (1 μg/L) in nematodes. (d) Downregulated and upregulated lncRNAs in nematodes exposed to nanopolystyrene (1 μg/L). Exposure to nanopolystyrene was performed from L1-larvae to adult Day-3

knockdown of linc-2, linc-7, linc-9, linc-18, linc-32, linc-50, linc-61, or linc-169 on toxicity induction of nanopolystyrene were examined in nematodes [62]. RNAi knockdown of linc-7, linc-32, or linc-169 did not obviously affect the nanopolystyrene toxicity in inducing intestinal ROS production and in decreasing

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Fig. 10.28 Confirmation of lncRNAs expression in nanopolystyrene-exposed nematodes by qRT-PCR (reprinted with permission from [62]). Exposure to nanopolystyrene was performed from L1-larvae to adult Day-3. Bars represent means  SD. **p < 0.01 vs. control

locomotion behavior (Fig. 10.29) [62]. In contrast, the more severe intestinal ROS production and locomotion behavior decrease were observed in nanopolystyreneexposed linc-2(RNAi), linc-9(RNAi), or linc-61(RNAi) nematodes compared with nanopolystyrene-exposed wild-type nematodes (Fig. 10.29) [62]. Meanwhile, the suppression in intestinal ROS production and the increase in locomotion behavior were detected in nanopolystyrene-exposed linc-18(RNAi) or linc-50(RNAi) nematodes compared with nanopolystyrene-exposed wild-type nematodes (Fig. 10.29) [62]. These observations suggested two different responses to nanopolystyrene at ERCs mediated by these 5 lncRNAs (linc-2, linc-9, linc-61, linc-18, and linc-50). On the one hand, the alteration in linc-18 mediated the toxicity induction of nanopolystyrene. On the other hand, the alteration in linc-2, linc-9, linc-50, and linc-61 expressions mediated a protective response for nematodes against the nanopolystyrene toxicity.

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Fig. 10.29 Effect of RNAi knockdown of lncRNAs on toxicity of nanopolystyrene in nematodes (reprinted with permission from [62]). (a) Effect of RNAi knockdown of lncRNAs on toxicity of nanopolystyrene in inducing intestinal ROS production. (b) Effect of RNAi knockdown of lncRNAs on toxicity of nanopolystyrene in decreasing locomotion behavior. Exposure concentration of nanopolystyrene was 1 μg/L. Exposure to nanopolystyrene was performed from L1-larvae to adult Day-3. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated)

10.3.3 Intestinal lncRNAs Required for the Control of Response to Toxicants at ERCs The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [63]. In nematodes, linc-61, linc-50, linc-9, and linc-2 can be expressed in the intestine. In the isolated intact intestines, exposure to nanopolystyrene (1–100 μg/L) increased the expressions of linc-61, linc-9, and linc-2 and decreased the linc-50 expression (Fig. 10.30) [63]. The increase in expression levels of linc-61, linc-9, and linc-2 and the decrease in expression level of linc-50 were concentration dependent in nanopolystyrene (1–100 μg/L)-exposed wild-type worms (Fig. 10.30) [63]. Meanwhile, it was found that the intestinal RNAi knockdown of linc-61, linc-9, or linc-2 led to the more severe toxicity of nanopolystyrene in reducing brood size

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Fig. 10.30 The linc-2, linc-9, linc-50, and linc-61 acted in the intestine to regulate the response to PS-NPs (reprinted with permission from [63]). (a) TEM image of nanopolystyrene particles in K medium before the sonication. (b) Effect of PS-NP exposure on expressions of linc-2, linc-9, linc50, and linc-61 in the intestine. For each treatment, 50 intact intestines were isolated and used. Bars represent means  SD. **p < 0.01 vs. control. (c) Effect of intestinal RNAi knockdown of linc-2, linc-9, linc-50, or linc-61 on PS-NP toxicity in inducing ROS production. Exposure concentration of PS-NPs was 1 μg/L. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). (d) Effect of intestinal RNAi knockdown of linc-2, linc-9, linc-50, or linc-61 on PS-NP toxicity in reducing brood size. Exposure concentration of PS-NPs was 1 μg/L. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). PS-NPs, polystyrene nanoparticles

and in inducing ROS production (Fig. 10.30) [63], suggesting the susceptibility of linc-61(RNAi), linc-9(RNAi), and linc-2(RNAi) worms to the nanopolystyrene toxicity. In contrast, intestinal RNAi knockdown of linc-50 suppressed the toxicity of nanopolystyrene in reducing brood size and in inducing ROS production (Fig. 10.30) [63], indicating the resistance of linc-50(RNAi) worms to the

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nanopolystyrene toxicity. These observations suggested that the intestinal linc-61, linc-50, linc-9, and linc-2 were required for the control of response to toxicants (such as nanopolystyrene) at ERCs in nematodes.

10.3.4 Downstream Targets for Intestinal lncRNAs in Controlling the Response to Toxicants at ERCs The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [63]. In nematodes, the binding site regions in transcriptional factors by lncRNAs have been examined by ChIP-SEQ, and the antibodies corresponding to the transcriptional factors were further applied for immunoprecipitating nucleic acids [64, 65]. Among the genes encoding transcriptional factors with binding potential to linc-2, exposure to nanopolystyrene increased the expression levels of daf-16, hlh30, fkh-2, and dve-1 and decreased the expression levels of ham-1 and nhr-77 [63]. For the genes encoding transcriptional factors with binding potential to linc9, exposure to nanopolystyrene increased the expression levels of daf-16, skn-1, and dve-1 and decreased the expression level of nhr-77 [63]. Among the genes encoding transcriptional factors with binding potential to linc-50, exposure to nanopolystyrene increased the expression levels of daf-16, hlh-30, skn-1, and dve-1 and decreased the expression level of ham-1 [63]. For the genes encoding transcriptional factors with binding potential to linc-61, exposure to nanopolystyrene increased the expression levels of daf-16, skn-1, hlh-30, fkh-2, and dve-1 and decreased the expression levels of ham-1 and nhr-77 [63]. For the dysregulated genes encoding intestinal transcriptional factors with binding potential to linc-61, linc-50, linc-9, or linc-2 in nanopolystyrene-exposed wildtype nematodes, the effects of intestinal RNAi knockdown of linc-61, linc-50, linc-9, or linc-2 on expression levels of these genes were determined in nanopolystyreneexposed VP303 nematodes. After the nanopolystyrene exposure, intestinal RNAi knockdown of linc-2 inhibited the expression level of daf-16 and increased the expression level of ham-1, intestinal RNAi knockdown of linc-9 increased the nhr-77 expression, intestinal RNAi knockdown of linc-50 increased the expression levels of daf-16 and dve-1 and suppressed the ham-1 expression, and intestinal RNAi knockdown of linc-61 inhibited the expression levels of daf-16, fkh-2, and dve-1 (Fig. 10.31) [63]. DAF-16 is FOXO transcriptional factor, HAM-1 is STOX transcriptional factor, NHR-77 is nuclear hormone receptor, DVE-1 is transcriptional factor required for control of mitochondrial unfolded protein response (mt UPR), and FKH-2 is forkhead transcriptional factor. Among the candidate targets of intestinal linc-61, linc-50, linc-9, and linc-2, the nanopolystyrene toxicity in inducing ROS production was not affected by intestinal RNAi knockdown of ham-1 (Fig. 10.32) [63]. In contrast, intestinal RNAi knockdown of daf-16, dve-1, or fkh-2 induced the more severe nanopolystyrene toxicity in

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Fig. 10.31 Effect of intestinal RNAi knockdown of linc-2, linc-9, linc-50, or linc-61 on gene expressions in PS-NP-exposed VP303 nematodes (reprinted with permission from [63]). PS-NPs, polystyrene nanoparticles. L4440, empty vector. Exposure concentration of PS-NPs was 1 μg/L. Bars represent means  SD. **p < 0.01 vs. VP303

inducing the ROS production and in reducing the brood size (Fig. 10.32) [63], demonstrating the susceptibility of daf-16(RNAi), dve-1(RNAi), and fkh-2(RNAi) worms to the nanopolystyrene toxicity. Additionally, intestinal RNAi knockdown of nhr-77 inhibited the nanopolystyrene toxicity in inducing the ROS production and in reducing the brood size (Fig. 10.32) [63], indicating the resistance of nhr-77 (RNAi) worms to the nanopolystyrene toxicity. Therefore, DAF-16, DVE-1, FKH-2, and NHR-77 possibly functioned as downstream targets of intestinal linc-61, linc50, linc-9, and/or linc-2 to regulate the toxicity of toxicants (such as nanopolystyrene) at ERCs in nematodes. To confirm the downstream targets of intestinal linc-9 and linc-50, the genetic interactions between linc-9 and linc-50 and their possible target(s) in the intestine to regulate the nanopolystyrene toxicity were determined. Intestinal RNAi knockdown of nhr-77 suppressed the susceptibility of linc-9(RNAi) nematodes to the nanopolystyrene toxicity in inducing ROS production and in reducing brood size (Fig. 10.33) [63], demonstrating the function of NHR-77 as downstream target of intestinal linc-9 to control the toxicity of toxicants (such as nanopolystyrene) at ERCs in nematodes. Insulin, p38 MAPK, Wnt, and ELT-2 signaling pathways functioned in intestinal cells to be involved in the control of nanopolystyrene toxicity [18–20, 48]. PMK-1 is a p38 MAPK, and BAR-1 is a β-catenin. Moreover, after the nanopolystyrene exposure, although the intestinal RNAi knockdown of nhr-77 did not obviously influence expression levels of pmk-1, bar-1, and elt-2, intestinal RNAi knockdown of nhr-77 increased the daf-16 expression [63]. Additionally, RNAi knockdown of daf-16 suppressed the resistance of nhr-77(RNAi) nematodes to the

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Fig. 10.32 Effect of intestinal RNAi knockdown of daf-16, ham-1, nhr-77, dve-1, or fkh-2 on toxicity of PS-NPs (reprinted with permission from [63]). (a) Effect of intestinal RNAi knockdown of daf-16, ham-1, nhr-77, dve-1, or fkh-2 on toxicity of PS-NPs in inducing ROS production. (b) Effect of intestinal RNAi knockdown of daf-16, nhr-77, dve-1, or fkh-2 on toxicity of PS-NPs in reducing brood size. PS-NPs, polystyrene nanoparticles. L4440, empty vector. Exposure concentration of PS-NPs was 1 μg/L. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated)

toxicity of nanopolystyrene in inducing ROS production and in reducing brood size in [63]. Therefore, NHR-77 functioned upstream of intestinal DAF-16 to control the toxicity of toxicants (such as nanopolystyrene) at ERCs in nematodes. In addition, although the RNAi knockdown of dve-1 did not obviously affect the resistance of linc-50(RNAi) nematodes to nanopolystyrene toxicity, intestinal RNAi knockdown of daf-16 inhibited the resistance of linc-50(RNAi) nematodes to the nanopolystyrene toxicity in inducing ROS production and in reducing brood size (Fig. 10.33) [63]. Thus, the DAF-16, but not the DVE-1, functioned downstream of intestinal linc-50 to control the toxicity of toxicants (such as nanopolystyrene) at ERCs in nematodes. To confirm the targets of intestinal linc-2 and linc-61, the genetic interactions between intestinal linc-2 and linc-61 and their possible target(s) to control

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Fig. 10.33 Genetic interactions between linc-9 and linc-50 and their target(s) in the intestine to regulate the PS-NP toxicity (reprinted with permission from [63]). (a) Genetic interactions between linc-9 and linc-50 and their target(s) in the intestine to regulate the PS-NP toxicity in inducing ROS production. (b) Genetic interactions between linc-9 and linc-50 and their target(s) in the intestine to regulate the PS-NP toxicity in reducing brood size. L4440, empty vector. Exposure concentration of PS-NPs was 1 μg/L. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated)

nanopolystyrene toxicity were determined. The transgenic strains with the overexpression of intestinal linc-2 (Is(Pges-1-linc-2)) and intestinal linc-61 (Is (Pges-1-linc-61)) exhibited the resistance to the toxicity of nanopolystyrene in inducing ROS production and in reducing brood size (Fig. 10.34) [63]. Moreover, the RNAi knockdown of daf-16 inhibited the resistance of Is(Pges-1-linc-2) worms to the nanopolystyrene toxicity in inducing ROS production and in reducing brood size (Fig. 10.34) [63], suggesting that DAF-16 functioned downstream of intestinal linc-2 to control the toxicity of toxicants (such as nanopolystyrene) at ERCs in nematodes. Additionally, RNAi knockdown of daf-16, dve-1, or fkh-2 further inhibited the resistance of Is(Pges-1-linc-61) worms to the nanopolystyrene toxicity in inducing ROS production and in reducing brood size (Fig. 10.34) [63], indicating

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Fig. 10.34 Genetic interactions between linc-2 and linc-61 and their target(s) in the intestine to regulate the PS-NP toxicity (reprinted with permission from [63]). (a) Genetic interactions between linc-2 and linc-61 and their target(s) in the intestine to regulate the PS-NP toxicity in inducing ROS production. Exposure concentration of PS-NPs was 1 μg/L. Bars represent means  SD. ** p < 0.01 vs. control (if not specially indicated). (b) Genetic interactions between linc-2 and linc-61 and their target(s) in the intestine to regulate the PS-NP toxicity in reducing brood size. Exposure concentration of PS-NPs was 1 μg/L. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). (c) A diagram showing the molecular basis of intestinal lncRNAs in mediating induction of protective response to PS-NPs in worms

that DAF-16, DVE-1, and FKH-2 functioned downstream of intestinal linc-61 to control the toxicity of toxicants (such as nanopolystyrene) at ERCs in nematodes. After the nanopolystyrene exposure, although intestinal RNAi knockdown of fkh2 did not influence the expression levels of bar-1, pmk-1, and elt-2, RNAi knockdown of fkh-2 could suppress the daf-16 expression [63]. The transgenic worm with overexpression of intestinal fkh-2 (Is(Pges-1-fkh-2)) showed the resistance to nanopolystyrene toxicity in inducing ROS production and in reducing brood size [63]. It was further found that the resistance of Is(Pges-1-fkh-2) worms to the nanopolystyrene toxicity in inducing ROS production and in reducing brood size could be inhibited by daf-16 RNAi knockdown [63], suggesting that DAF-16 also

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functioned downstream of intestinal FKH-2 to control the toxicity of toxicants (such as nanopolystyrene) at ERCs in nematodes.

10.4

circRNAs Control of Response to Toxicants at ERCs

10.4.1 Response of circRNAs to Toxicants at ERCs In nematodes, exposure to graphene oxide (GO) could cause various aspects of toxicity [33, 66–75]. The GO was selected as the example of environmental toxicants, and the nematodes were exposed to GO from L1-larvae to adult Day-1 [76]. Using the Illumina HiSeq2500 sequencing technique, 43 dysregulated circRNAs were identified in GO (1 mg/L)-exposed nematodes [76]. The 33 known circRNAs were further focused to confirm their expressions in GO-exposed nematodes using qRT-PCR technique [76]. These 33 known circRNAs are exonshuffling-derived circRNAs, and 28 dysregulated circRNAs were validated in GO (1 mg/L)-exposed wild-type nematodes by qRT-PCR assay (Fig. 10.35) [76]. Moreover, among these 28 dysregulated circRNAs induced by GO (1 mg/L) exposure, the expressions of 5 circRNAs were further significantly altered by exposure to GO (100 μg/L) (Fig. 10.35) [76]. These five dysregulated circRNAs included four downregulated circRNAs (circ_0000115, circ_0000201, circ_0000247, and circ_0000665) and one upregulated circRNA (circ_0000308) in nematodes (Fig. 10.35) [76].

10.4.2 Functional Analysis of circRNAs in Regulating the Response to Toxicants at ERCs The GO was selected as the example of environmental toxicants, and the nematodes were exposed to GO from L1-larvae to adult Day-1 [76]. To determine the function of the five candidate circRNAs (circ_0000115, circ_0000201, circ_0000247, circ_0000308, and circ_0000665) in regulating the GO toxicity, RNAi knockdown was performed in nematodes [76]. RNAi knockdown of corresponding host genes for these five circRNAs did not affect the expressions of these five circRNAs [76]. Intestinal ROS production and locomotion behavior reflected by head thrash and body bend were employed as the toxicity assessment endpoints [76]. After the exposure, it was found that RNAi knockdown of circ_0000115, circ_0000247, circ_0000308, or circ_0000665 caused the resistance of nematodes to GO toxicity in inducing intestinal ROS production and in decreasing locomotion behavior (Fig. 10.36) [76]. In contrast, RNAi knockdown of circ_0000201 resulted in the susceptibility of nematodes to GO toxicity in inducing intestinal ROS production

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Fig. 10.35 Validation of circRNAs expression in GO wild-type exposed nematode via qRT-PCR analysis (reprinted with permission from [76]). (a) GO exposure concentration was 1 mg/L. (b) GO exposure concentration was 100 μg/L GO. Prolonged exposure to GO was performed from L1-lavae to adult Day-1. Bars represent means  SD. **p < 0.01 vs. control

and in decreasing locomotion behavior (Fig. 10.36) [76]. Therefore, all these five circRNAs were involved in the regulation of response to GO.

10.4.3 Functional Analysis of circ_0000115 in Regulating the Response to Toxicants at ERCs The GO was selected as the example of environmental toxicants, and the nematodes were exposed to GO from L1-larvae to adult Day-1 [76]. The circ_0000115 is the most downregulated circRNA in nematodes exposed to GO [76]. Intestinal ROS production was used as an endpoint [76]. Using the RNAi knockdown genetic tools (VP303 used for RNAi knockdown in intestine, NR222 used for RNAi knockdown in epidermis, TU3401 used for RNAi knockdown in neurons, and WM118 used for

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Fig. 10.36 Functional analysis of candidate circRNAs in regulating ROS production (a) and locomotion behavior (b) (reprinted with permission from [76]). GO concentration is 100 μg/L. Prolonged exposure to GO was performed from L1-lavae to adult Day-1. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated)

RNAi knockdown in muscle), it was found that RNAi knockdown of circ_0000115 in epidermis or muscle did not affect the GO toxicity in inducing intestinal ROS production (Fig. 10.37) [76]. Different from this, RNAi knockdown of circ_0000115 in the intestine or neurons could induce a resistance to GO toxicity in inducing intestinal ROS production (Fig. 10.37) [76], suggesting that the circ_0000115 acted in the intestine and the neurons to regulate the response to GO in nematodes. To identify the potential targets of circ_0000115 during the control of GO toxicity, a RNA pull-down experiment was performed to pull down proteins with or without the biotinylated probe of circ_0000115 [76]. According to the results of circ_0000115 pull-down assay for the sample collected from nematodes exposure to GO (100 μg/L), most of the pulled down proteins were in the supernatant [76]. A visible protein band between the molecular weights of 40–55 kDa appeared in the sample with the biotinylated probe of circ_0000115, and this band was not present in the control without the probe (Fig. 10.23) [76]. Using mass spectrometry technique, the proteins in this band were examined [76]. According to the score of circ_0000115-protein binding capacity using the RPISeq website, IFC-2 protein ranked first as a possible target protein of circ_0000115 [76]. The potential nucleotide binding sites in IFC-2 amino acid sequence and potential amino acid binding sites in circ_0000115 sequence were analyzed by PRIdictor (Fig. 10.38) [76]. The

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Fig. 10.37 Tissue-specific activity of circ_0000115 in regulating GO toxicity in inducing ROS production (reprinted with permission from [76]). GO concentration is 100 μg/L. Prolonged exposure to GO was performed from L1-lavae to adult Day-1. Bars represent means  SD. ** p < 0.01 vs. control (if not specially indicated)

IFC-2 protein contains eight possible nucleotide binding sites located at amino acids 276, 363, 389, 422, 436, 448, 497, and 578, respectively (Fig. 10.38) [76]. Meanwhile, the circ_0000115 contains multiple sites that may bind amino acids (Fig. 10.38) [76], suggesting the molecular interaction between circ_0000115 and IFC-2 protein. In nematodes, IFC-2 is a protein present in the cytoplasm of intestinal cells and plays an important role in maintaining the intestinal morphological structure [76]. The intestinal lumen of ifc-2 (RNAi) nematodes was considerably widened [76]. Exposure to GO (100 μg/L) could significantly decrease the ifc-2 expression [76]. Meanwhile, after the GO exposure, intestine-specific RNAi knockdown of circ_0000115 further noticeably increased the ifc-2 expression [76]. Moreover, after the GO exposure, intestinal RNAi knockdown of ifc-2 induced a more significant change of irregularly widened intestinal lumen [76]. Additionally, intestinal RNAi knockdown of ifc-2 caused the more significant induction of intestinal ROS production in GO-exposed nematodes compared with that in GO-exposed VP303 nematodes [76], which suggested the role of IFC-2 as target of intestinal circ-0000115 in regulating the GO toxicity.

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Fig. 10.38 circRNA pull-down assay combined with mass spectrometry analysis (reprinted with permission from [76]). (a) Diagram showing the preparation of circ_0000115 pull-down biotin probe. B biotinylation. (b) The result of circ_0000115 pull-down in SDS-PAGE electrophoresis. Arrowhead indicates the position for IFC-2. (c) PRIdictor prediction result of the possible nucleotide binding sites in IFC-2. (d) PRIdictor prediction result of the possible amino acid binding sites in circ_0000115

10.5

Epigenetic Control of Response to Toxicants at ERCs by Histone Methylation-Related Signals

10.5.1 Response of Histone Methylation-Related Signals to Toxicants at ERCs The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [77]. During the control of stress response, some genes associated with methylation regulation play important functions [78–82]. Among these genes, exposure to nanopolystyrene (1–100 μg/L) did not affect expressions of set-25, jmjd-1.2, jmjd-3.1, his-24, wdr-5.1, rbr-2, set-2, and ash-2 (Fig. 10.39) [77]. Additionally, exposure to 1–10 μg/L nanopolystyrene also did not alter set-16 expression (Fig. 10.39) [77]. Different from these, exposure to nanopolystyrene (1–100 μg/L) decreased met-2 expression, and exposure to 100 μg/L nanopolystyrene increased set-16 expression (Fig. 10.39) [77].

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Fig. 10.39 MET-2 regulated the toxicity of PS-NPs (reprinted with permission from [77]). (a) Effect of PS-NP exposure on expressions of genes in wild-type nematodes. Bars represent means  SD. **p < 0.01 vs. control. (b) Effect of RNAi knockdown of met-2 on PS-NP toxicity in inducing ROS production in wild-type nematodes. Exposure concentration of PS-NPs was 1 μg/ L. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). (c) Effect of RNAi knockdown of met-2 on PS-NP toxicity in decreasing locomotion behavior in wild-type nematodes. Exposure concentration of PS-NPs was 1 μg/L. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). PS-NPs, polystyrene nanoparticles

10.5.2 Functional Analysis of MET-2 in Regulating the Response to Toxicants at ERCs Although the exact environmental concentrations of nanoplastics are still unclear, the predicted environmental concentrations of nanoplastics have been raised 1 μg/ L. Production of ROS and locomotion behavior were applied as endpoints to assess the nanopolystyrene toxicity. Both production of ROS and decrease in locomotion behavior were suppressed in nanopolystyrene-exposed met-2(RNAi) worms compared with those in nanopolystyrene-exposed wild-type worms (Fig. 10.39) [77]. Therefore, RNAi knockdown of met-2 induced a resistance to toxicity of toxicants at ERCs in nematodes.

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10.5.3 Tissue-Specific Activity of MET-2 in Regulating the Toxicity of Toxicants at ERCs The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [77]. Based on information in WormBase (https://www.wormbase.org), MET-2 can be expressed in neuron, intestine, muscle, epidermis, and germline. Using TU3401, VP303, WM118, NR222, or DCL569 as genetic tools, RNAi knockdown of met-2 in neuron, intestine, muscle, epidermis, or germline was performed. Based on observation on ROS production, RNAi knockdown of met-2 in neuron, muscle, or epidermis did not alter nanopolystyrene toxicity (Fig. 10.40) [77]. Different from this, RNAi knockdown of met-2 in the intestine or germline reduced the toxicity of nanopolystyrene in causing production of ROS and in decreasing locomotion behavior (Fig. 10.40) [77]. These observations indicated

Fig. 10.40 Tissue-specific activity of MET-2 in regulating PS-NP toxicity (reprinted with permission from [77]). (a) Tissue-specific activity of MET-2 in regulating PS-NP toxicity in inducing ROS production. (b) Germline-specific activity of MET-2 in regulating PS-NP toxicity in decreasing locomotion behavior. PS-NPs, polystyrene nanoparticles. L4440, empty vector. Exposure concentration of PS-NPs was 1 μg/L. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated)

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the role of intestinal and germline MET-2 in regulating the toxicity of toxicants (such as nanopolystyrene) at ERCs in nematodes.

10.5.4 Targets for Intestinal MET-2 in Regulating the Toxicity of Toxicants at ERCs The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [77]. During the control of PS-NP toxicity, DAF-16-mediated insulin signaling pathway, PMK-1-mediated p38 MAPK signaling pathway, BAR-1medaited Wnt signaling pathway, and ELT-2 signaling pathway played their important functions in intestinal cells [18–20, 48]. In nanopolystyrene-exposed VP303 worms, although pmk-1 expression could not be altered by met-2 RNAi knockdown, expressions of daf-16, bar-1, and elt-2 were increased by met-2 RNAi knockdown (Fig. 10.41) [77]. Moreover, daf-16 RNAi knockdown suppressed the resistance to nanopolystyrene toxicity in causing production of ROS in met-2(RNAi) worms (Fig. 10.41) [77]. Similarly, bar-1 RNAi knockdown inhibited the resistance to PS-NP toxicity in inducing production of ROS in met-2(RNAi) worms (Fig.10.41) [77]. Additionally, the resistance of met-2(RNAi) worms to nanopolystyrene toxicity in inducing production of ROS was suppressed by elt-2 RNAi knockdown (Fig. 10.41) [77]. These data suggested the roles of DAF-16/FOXO transcriptional factor, BAR-1/β-catenin, and ELT-2 as downstream targets of MET-2 in the intestine to control the toxicity of toxicants (such as nanopolystyrene) at ERCs in nematodes.

10.5.5 Targets for Germline MET-2 in Regulating the Toxicity of Toxicants at ERCs The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [77]. During the control of PS-NP toxicity, some regulatory proteins (such as LIN-23, PAT-12, WRT-3, NHL-2, and NDK-1) have been identified in germline cells [53]. In nanopolystyrene-exposed DCL569 worms, although the lin23, nhl-2, and ndk-1 expressions were not affected by met-2 RNAi knockdown, the pat-12 and wrt-3 expressions were significantly decreased by met-2 RNAi knockdown (Fig. 10.42) [77]. To determine the interaction between MET-2 and PAT-12 or WRT-3 in germline cells to control PS-NP toxicity, transgenic strain overexpressing germline MET-2 (Is (Pmex-5-met-2)) was generated. In Is(Pmex-5-met-2) worms, the nanopolystyrene

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Fig. 10.41 Target identification for intestinal MET-2 in regulating PS-NP toxicity (reprinted with permission from [77]). (a) Effect of intestinal RNAi knockdown of met-2 on expressions of daf-16, pmk-1, bar-1, and elt-2 in PS-NP-exposed VP303 worms. Bars represent means  SD. ** p < 0.01 vs. VP303. (b) Genetic interaction between MET-2 and DAF-16 in the intestine to regulate PS-NP toxicity in inducing ROS production. Bars represent means  SD. ** p < 0.01 vs. control (if not specially indicated). (c) Genetic interaction between MET-2 and BAR-1 in the intestine to regulate PS-NP toxicity in inducing ROS production. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). (d) Genetic interaction between MET-2 and ELT-2 in the intestine to regulate PS-NP toxicity in inducing ROS production. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). L4440, empty vector. Exposure concentration of PS-NPs was 1 μg/L. PS-NPs, polystyrene nanoparticles

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Fig. 10.42 Target identification for germline MET-2 in regulating PS-NP toxicity (reprinted with permission from [77]). (a) Effect of germline RNAi knockdown of met-2 on expressions of ndk-1, nhl-2, wrt-3, pat-12, and lin-23 in PS-NP-exposed DCL569 worms. L4440, empty vector. Exposure concentration of PS-NPs was 1 μg/L. Bars represent means  SD. **p < 0.01 vs. DCL569. (b) Genetic interaction between MET-2 and WRT-3 or PAT-12 in the germline to regulate PS-NP toxicity in inducing ROS production. L4440, empty vector. Exposure concentration of PS-NPs was 1 μg/L. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). (c) Genetic

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toxicity in inducing ROS production and in decreasing locomotion behavior was further enhanced (Fig. 10.42) [77]. Moreover, the susceptibility of Is(Pmex-5-met-2) worms to nanopolystyrene toxicity could be suppressed by RNAi knockdown of pat12 or wrt-3 (Fig. 10.42) [77]. Therefore, PAT-12 and WRT-3 functioned as downstream targets of MET-2 in germline cells to control toxicity of toxicants (such as nanopolystyrene) at ERCs in nematodes.

10.6

Epigenetic Control of Response to Toxicants at ERCs by Acetylation-Related Signals

10.6.1 Response of Acetylation-Related Signals to Toxicants at ERCs The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [24]. During the control of stress response, some genes associated with histone acetylation regulation play important roles [83–85]. Among these histone acetylation-associated genes, exposure to nanopolystyrene (1–100 μg/L) did not cause any alteration in expressions of mys-1, trr-1, natc-1, and sir-2.4 (Fig. 10.43) [24]. Different from this, exposure to nanopolystyrene (1–100 μg/L) increased the cbp-1 expression (Fig. 10.43) [24], suggesting the response of CBP-1 to nanopolystyrene at ERCs in nematodes. In nematodes, CBP-1 is an acetyltransferase.

10.6.2 Functional Analysis of Acetylation-Related Signals in Regulating the Response to Toxicants at ERCs The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [24]. Production of ROS and locomotion behavior were used as endpoints to assess nanopolystyrene toxicity. Compared to wild-type worms, exposure to nanopolystyrene resulted in the more severe production of ROS and decrease in locomotion behavior in cbp-1(RNAi) worms (Fig. 10.43) [24]. Thus, in cbp-1  ⁄ Fig. 10.42 (continued) interaction between MET-2 and WRT-3 or PAT-12 in the germline to regulate PS-NP toxicity in decreasing locomotion behavior. L4440, empty vector. Exposure concentration of PS-NPs was 1 μg/L. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). (d) A diagram showing the molecular basis of MET-2 in intestine and germline to control PS-NP toxicity in nematodes. PS-NPs, polystyrene nanoparticles

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Fig. 10.43 CBP-1 regulated the toxicity of PS-NPs (reprinted with permission from [24]). (a) Effect of PS-NP exposure on expressions of genes in wild-type nematodes. Bars represent means  SD. **p < 0.01 vs. control. (b) Effect of RNAi knockdown of cbp-1 on PS-NP toxicity in inducing ROS production in wild-type nematodes. Exposure concentration of PS-NPs was 1 μg/ L. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). (c) Effect of RNAi knockdown of cbp-1 on PS-NP toxicity in decreasing locomotion behavior in wild-type nematodes. Exposure concentration of PS-NPs was 1 μg/L. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). PS-NPs, polystyrene nanoparticles

(RNAi) worms, the susceptibility to toxicity of toxicants (such as nanopolystyrene) can be detected, which suggested the involvement of CBP-1 in regulating the toxicity of nanopolystyrene at ERCs in nematodes.

10.6.3 Tissue-Specific Activity of CBP-1 in Regulating Toxicity of Toxicants at ERCs The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [24]. Based on the information in WormBase (https://www.wormbase. org), CBP-1 can be expressed in neurons, intestine, muscle, epidermis, and germline. Using TU3401, VP303, WM118, NR222, or DCL569 as genetic tools, RNAi knockdown of cbp1-1 in neuron, intestine, muscle, epidermis, or germline was

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Fig. 10.44 Tissue-specific activity of CBP-1 in regulating PS-NP toxicity (reprinted with permission from [24]). (a) Tissue-specific activity of CBP-1 in regulating PS-NP toxicity in inducing ROS production. (b) Neuronal- and germline-specific activities of CBP-1 in regulating PS-NP toxicity in decreasing locomotion behavior. PS-NPs, polystyrene nanoparticles. L4440, empty vector. Exposure concentration of PS-NPs was 1 μg/L. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated)

carried out. Based on observation on ROS production, RNAi knockdown of cbp-1 in muscle or epidermis could not affect nanopolystyrene toxicity (Fig. 10.44) [24]. In contrast, RNAi knockdown of cbp-1 in neuronal cells, intestinal cells, or germline cells caused formation of more severe production of ROS in nanopolystyreneexposed TU3401, VP303, or DCL569 worms (Fig. 10.44) [24]. Moreover, RNAi knockdown of cbp-1 in neuronal cells or germline cells also resulted in formation of more severe inhibition in locomotion behavior in nanopolystyrene-exposed TU3401 or DCL569 worms (Fig. 10.44) [24]. Therefore, these data demonstrated the tissue activities of CBP-1 in neurons, intestine, and germline to control the toxicity of toxicants (such as nanopolystyrene) at ERCs in nematodes.

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10.6.4 Targets of Intestinal CBP-1 in Regulating Toxicity of Toxicants at ERCs The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [24]. During the control of PS-NP toxicity, ELT-2 signaling pathway, BAR-1-mediated Wnt signaling pathway, PMK-1-mediated p38 MAPK signaling pathway, and DAF-16-mediated insulin signaling pathway were identified in intestinal cells [18–20, 48]. In nanopolystyrene-exposed VP303 worms, cbp-1 RNAi knockdown caused a remarkable decrease in the expressions of pmk-1 and daf-16 rather than the expressions of elt-2 and bar-1 (Fig. 10.45) [24]. To determine genetic interaction between CBP-1 and PMK-1 or DAF-16, transgenic worm overexpressing intestinal CBP-1 (Is(Pges-1-cbp-1)) was generated. Using production of ROS and locomotion behavior as endpoints, the resistance to nanopolystyrene was observed in Is(Pges-1-cbp-1) worms (Fig. 10.45) [24]. Moreover, pmk-1 or daf-16 RNAi knockdown could suppress the resistance to nanopolystyrene toxicity observed in Is(Pges-1-cbp-1) worms (Fig. 10.45) [24]. Therefore, in the intestine, PMK-1 and DAF-16 functioned as downstream targets of CBP-1 to control the toxicity of toxicants (such as nanopolystyrene) at ERCs in nematodes.

10.6.5 Targets of Neuronal CBP-1 in Regulating Toxicity of Toxicants at ERCs The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to adult Day-3 [24]. During the control of PS-NP toxicity, DBL-1 and DAF-7-mediated TGF-β signaling pathways, MPK-1-mediated ERK MAPK signaling pathway, and JNK-1-mediated JNK MAPK signaling pathway were identified in neuronal cells [46, 52, 86, 87]. In nanopolystyrene-exposed TU3401 worms, although dbl-1 and mpk-1 expressions were not altered by cbp-1 RNAi knockdown, the daf-7 and jnk-1 expressions were decreased by cbp-1 RNAi knockdown (Fig. 10.46) [24]. To detect genetic interaction between CBP-1 and DAF-7 or JNK-1, transgenic worm overexpressing neuronal CBP-1 (Is(Punc-14-cbp-1)) was generated. Using production of ROS and locomotion behavior as endpoints, the resistance to toxicity of nanopolystyrene was also detected in Is(Punc-14-cbp-1) worms (Fig. 10.46) [24]. Furthermore, daf-7 or jnk-1 RNAi knockdown inhibited the resistance to nanopolystyrene toxicity in Is(Punc-14-cbp-1) worms (Fig. 10.46) [24]. Therefore, in neuronal cells, DAF-7 and JNK-1 acted as downstream targets of CBP-1 to control the toxicity of toxicants (such as nanopolystyrene) at ERCs in nematodes.

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Fig. 10.45 Identification of targets of intestinal CBP-1 in regulating PS-NP toxicity (reprinted with permission from [24]). (a) Effect of intestinal RNAi knockdown of cbp-1 on expressions of genes in PS-NP-exposed VP303 nematodes. Bars represent means  SD. **p < 0.01 vs. VP303. (b) Genetic interaction between CBP-1 and DAF-16 or PMK-1 in the intestine to regulate the toxicity of PS-NPs in inducing ROS production. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). (c) Genetic interaction between CBP-1 and DAF-16 or PMK-1 in the intestine to regulate the toxicity of PS-NPs in decreasing locomotion behavior. Bars represent means  SD. ** p < 0.01 vs. control (if not specially indicated). PS-NPs, polystyrene nanoparticles. L4440, empty vector. Exposure concentration of PS-NPs was 1 μg/L

10.6.6 Targets of Germline CBP-1 in Regulating Toxicity of Toxicants at ERCs The nanopolystyrene (100 nm) was selected as the example of environmental toxicants, and the nematodes were exposed to nanopolystyrene from L1-larvae to

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Fig. 10.46 Identification of targets of neuronal CBP-1 in regulating PS-NP toxicity (reprinted with permission from [24]). (a) Effect of neuronal RNAi knockdown of cbp-1 on expressions of genes in PS-NP-exposed TU3401 nematodes. Bars represent means  SD. **p < 0.01 vs. TU3401. (b) Genetic interaction between CBP-1 and DAF-7 or JNK-1 in neurons to regulate the toxicity of PS-NPs in inducing ROS production. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). (c) Genetic interaction between CBP-1 and DAF-7 or JNK-1 in neurons to regulate the toxicity of PS-NPs in decreasing locomotion behavior. Bars represent means  SD. ** p < 0.01 vs. control (if not specially indicated). PS-NPs, polystyrene nanoparticles. L4440, empty vector. Exposure concentration of PS-NPs was 1 μg/L

adult Day-3 [24]. Several germline genes (pat-12, lin-23, wrt-3, nhl-2, and ndk-1) required for the control of PS-NP toxicity have been identified [53]. After nanopolystyrene exposure, although expressions of pat-12, lin-23, wrt-3, and ndk1 were not affected by cbp-1 RNAi knockdown, the nhl-2 expression could be increased by cbp-1 RNAi knockdown in DCL569 worms (Fig. 10.47) [24]. Using production of ROS and locomotion behavior as assessment endpoints, we further

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Fig. 10.47 Identification of targets of germline CBP-1 in regulating PS-NP toxicity (reprinted with permission from [24]). (a) Effect of germline RNAi knockdown of cbp-1 on expressions of genes in PS-NP-exposed DCL569 nematodes. Bars represent means  SD. **p < 0.01 vs. DCL569. (b) Genetic interaction between CBP-1 and NHL-2 in the germline to regulate the toxicity of PS-NPs in inducing ROS production. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). (c) Genetic interaction between CBP-1 and NHL-2 in the germline to regulate the toxicity of PS-NPs in decreasing locomotion behavior. Bars represent means  SD. ** p < 0.01 vs. control (if not specially indicated). PS-NPs, polystyrene nanoparticles. L4440, empty vector. Exposure concentration of PS-NPs was 1 μg/L

observed that the susceptibility to nanopolystyrene toxicity in cbp-1(RNAi) worms could be inhibited by nhl-2 RNAi knockdown (Fig. 10.47) [24]. That is, in the germline, NHL-2 can function as a target of CBP-1 to control the toxicity of toxicants (such as nanopolystyrene) at ERCs in nematodes. It was found that expressions of some insulin peptides (DAF-28, INS-39, and INS-4) could be decreased by nanopolystyrene exposure [87]. These three insulin peptides can be expressed in the germline (https://www.wormbase.org). Among the genes encoding these three insulin peptides, the daf-28 expression could be decreased by nhl-2 RNAi knockdown in DCL569 worms after nanopolystyrene exposure (Fig. 10.48) [24]. To determine genetic interaction between NHL-2 and DAF-28, transgenic worm overexpressing germline NHL-2 (Is(Pmex-5-nhl-2)) was

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Fig. 10.48 Genetic interaction between NHL-2 and insulin peptides in the germline to regulate PS-NP toxicity (reprinted with permission from [24]). (a) Effect of germline RNAi knockdown of cbp-1 on expressions of ins-4, ins-39, and daf-28. Bars represent means  SD. ** p < 0.01 vs. DCL569. (b) Genetic interaction between NHL-2 and DAF-28 in the germline to regulate the toxicity of PS-NPs in inducing ROS production. Bars represent means  SD. ** p < 0.01 vs. control (if not specially indicated). (c) Genetic interaction between NHL-2 and DAF-28 in the germline to regulate the toxicity of PS-NPs in decreasing locomotion behavior. Bars represent means  SD. **p < 0.01 vs. control (if not specially indicated). PS-NPs, polystyrene nanoparticles. L4440, empty vector. Exposure concentration of PS-NPs was 1 μg/L

generated. The susceptibility to toxicity of nanopolystyrene was found in Is(Pmex-5nhl-2) worms as reflected by the alterations in endpoints of production of ROS and locomotion behavior (Fig. 10.48) [24]. Moreover, the susceptibility to nanopolystyrene toxicity in Is(Pmex-5-nhl-2) worms could be suppressed by daf28 RNAi knockdown (Fig. 10.48) [24]. Therefore, in the germline, DAF-28 could

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further function as a target of NHL-2 to control the toxicity of toxicants (such as nanopolystyrene) at ERCs in nematodes.

10.7

Perspectives

During the control of stress response, C. elegans is not only useful to determine the gene functions but also helpful to elucidate the mechanisms of epigenetic control [2, 3, 88–91]. For the response to toxicants at ERCs, usually only very limited number of miRNAs are involved in the control of this process. For example, based on both the expression pattern and the functional analysis, only five miRNAs (mir35, mir-794, mir-354, mir-38, and mir-76) were identified to be required for the control of response to toxicants at ERCs. Among these five miRNAs, mir-35, mir794, and mir-354 acted in the intestine to regulate the response to toxicants at ERCs, mir-76 acted in the neurons to regulate the response to toxicants at ERCs, and mir-38 acted in the germline to regulate the response to toxicants at ERCs. Nevertheless, even in the intestine, these identified miRNAs could not act as the upregulators of all known molecular signaling pathways involved in the control of toxicants at ERCs. In the intestine, the mir-35 acted upstream of both the signaling cascade of NDK-1KSR-1/KSR-2 and the signaling cascade of NDK-1-DAF-16 to regulate the response to toxicants at ERCs. The intestinal mir-794 acted upstream of DAF-16, SKN-1, and MDT-15 to regulate the response to toxicants at ERCs. The intestinal mir-354 acted upstream of DAF-3 to regulate the response to toxicants at ERCs. That is, in the intestine, these identified miRNAs could even act upstream of the same molecular signals (such as DAF-16) to regulate the response to toxicants at ERCs. These observations implied that at least the already identified miRNA-mediated epigenetic control could not cover all aspects of regulation for the response to toxicants at ERCs. In the neurons, the mir-76 regulated the toxicity of toxicants at ERCs by modulating the heme homeostasis-related molecular signaling. So far, the information on molecular basis for response of germline to toxicants is very limited. The identified downstream targets of germline mir-38 provided an important basis for our understanding the response of germline to toxicants at ERCs in nematodes. For the identification of lncRNAs involved in the control of response to toxicants at ERCs, the nanopolystyrene was further selected as the example. Among the known lncRNAs, only five known lncRNAs (linc-2, linc-9, linc-18, linc-50, and linc-61) were identified to be involved in the control of response to toxicants at ERCs. Among these five known lncRNAs, four lncRNAs (linc-2, linc-9, linc-50, and linc-61) were further identified in the intestinal cells to be required for the control of response to toxicants at ERCs. During the response to toxicants (such as nanopolystyrene) at ERCs, only limited number of transcriptional factors functioned as the downstream targets of these four intestinal lncRNAs. linc-2 acted upstream of DAF-16, linc-9 acted upstream of NHR-77, linc-50 functioned upstream of DAF-16, and linc-61 regulated the functions of DAF-16, DVE-1, and FKH-2 to control the

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nanopolystyrene toxicity. That is, only limited number of lncRNAs in different tissues may be activated or inhibited to be in response to toxicants at ERCs. For the identification of circRNAs involved in the control of response to toxicants at ERCs, the GO was selected as the example. Among the dysregulated circRNAs, only five circRNAs (circ_0000115, circ_0000201, circ_0000247, circ_0000308, and circ_0000665) were identified to be involved in the control of response to toxicants at ERCs. That is, similarly, only very limited number of circRNAs are involved in the control of response to toxicants at ERCs. For the identification of histone methylation regulation-related signals involved in the control of response to toxicants at ERCs, the nanopolystyrene was selected as the example. Among the histone methylation regulation-related signals, MET-2, a H3K9 methyltransferase, was identified for the response to toxicants at ERCs. During the control of toxicity of toxicants at ERCs, MET-2 could function in both intestine and germline. In the intestine, MET-2 functioned upstream of ELT-2, BAR-1, and DAF-16 to control the toxicity of toxicants. In the germline, MET-2 functioned upstream of PAT-12 and WRT-3 to control the toxicity of toxicants. These observations support the important role of MET-2-mediated histone methylation regulation in controlling the response to toxicants at ERCs. Moreover, the obtained data suggested that some other histone methylation regulation-related signals may still exist in different tissues (such as in neurons) to control the toxicity of toxicants at ERCs. For the identification of acetylation regulation-related signals involved in the control of response to toxicants at ERCs, the nanopolystyrene was further selected as the example. Among the acetylation regulation-related signals, CBP-1, an acetyltransferase, was identified for the response to toxicants at ERCs. During the control of toxicity of toxicants at ERCs, CBP-1 could function in the intestine, neurons, and germline. In the intestine, CBP-1 controlled the toxicity of toxicants by modulating functions of insulin and p38 MAPK signaling pathways. In the neurons, CBP-1 controlled the toxicity of toxicants by affecting functions of DAF-7/TGF-β and JNK MAPK signaling pathways. In the germline, CBP-1 controlled the toxicity of toxicants by suppressing NHL-2 activity, and NHL-2 further regulated the toxicity of toxicants by modulating insulin communication between germline and the intestine. These findings confirmed the crucial role of CBP-1-mediated acetylation regulation in controlling the response to toxicants at ERCs. The other acetylation regulation-related signals in controlling the toxicity of toxicants at ERCs still need the further identification.

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88. Liu, H.-L., Qiu, Y.-X., & Wang, D.-Y. (2021). Alteration in expressions of ion channels in Caenorhabditis elegans exposed to polystyrene nanoparticles. Chemosphere, 273, 129686. 89. Yang, Y.-H., Dong, W.-T., Wu, Q.-L., & Wang, D.-Y. (2021). Response of G protein-coupled receptor CED-1 in germline to polystyrene nanoparticles in Caenorhabditis elegans. Nanoscale Advances, 3, 1997–2006. 90. Yang, Y.-H., Dong, W.-T., Wu, Q.-L., & Wang, D.-Y. (2021). Induction of protective response associated with expressional alterations in neuronal G protein-coupled receptors in polystyrene nanoparticle exposed Caenorhabditis elegans. Chemical Research in Toxicology, 34, 1308. https://doi.org/10.1021/acs.chemrestox.0c00501 91. Yang, Y.-H., Wu, Q.-L., & Wang, D.-Y. (2021). Dysregulation of G protein-coupled receptors in the intestine by nanoplastic exposure in Caenorhabditis elegans. Environmental Science. Nano, 8, 1019–1028.

Chapter 11

Molecular Networks in Different Tissues in Response to Toxicants at Environmentally Relevant Concentrations

Abstract Different molecular signaling pathways will organize certain molecular networks to regulate the response to toxicants at environmentally relevant concentrations (ERCs). In this chapter, we first introduced the molecular networks formed in the intestine, neurons, and germline, respectively, in regulating the response to toxicants at ERCs. Moreover, the possible signaling communications between different tissues in regulating the response to toxicants at ERCs were introduced and discussed. In addition, the basic conclusions for the response to toxicants at ERCs in nematodes were further discussed. Keywords Environmentally relevant concentrations · Molecular network · Environmental exposure · Caenorhabditis elegans

11.1

Introduction

An increasing evidence has indicated the high sensitivity of Caenorhabditis elegans to various environmental exposures [1–5]. In Chap. 1 of this book, we introduced the sensitivity of C. elegans in detecting the possible transgenerational toxicity of toxicants at ERCs. Moreover, C. elegans can provide a powerful platform for the studies of both molecular toxicology and target organ toxicology [6–11]. In Chaps. 2–10 of this book, we have introduced the functions of different aspects of signaling pathways in regulating the response to toxicants at ERCs in nematodes. In this chapter, we further summarize the molecular networks in the intestine, the neurons, and the germline in regulating the response to toxicants at ERCs in nematodes. Moreover, we also introduced the already raised signaling communications between different tissues in regulating the response to toxicants at ERCs in nematodes.

© The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 D. Wang, Toxicology at Environmentally Relevant Concentrations in Caenorhabditis elegans, https://doi.org/10.1007/978-981-16-6746-6_11

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Molecular Network in the Intestine in Regulating Response to Toxicants at ERCs

11.2.1 Molecular Signaling Network in the Intestine in Response to Toxicants at ERCs With the nanopolystyrene as the example of environmental toxicants, the molecular signaling network in the intestine to regulate the response to toxicants at ERCs was identified in nematodes [5, 12–24]. In the raised molecular signaling network in the intestine, at least there are seven basic signaling pathways found to be activated or inhibited after exposure to toxicants (such as nanopolystyrene) at ERCs in nematodes (Fig. 11.1). These seven basic signaling pathways acting in intestine to regulate the response to toxicants at ERCs are: 1. p38 MAPK signaling pathway. In the p38 MAPK signaling pathway, only the PMK-1/p38 MAPK could be activated in the intestine, and the activated PMK-1/ p38 MAPK further regulated the response to toxicants at ERCs by at least activating downstream three transcriptional factors, SKN-1, ATF-7, and NHR-8 (Fig. 11.1). 2. DAF-7/TGF-β signaling pathway. In the intestine, the DAF-1/TGF-β receptor could be activated by DAF-7/TGF-β, and the activated DAF-1/TGF-β receptor further regulated the response to toxicants at ERCs by activating two downstream signaling cascades, DAF-8-DAF-5 and DAF-8-DAF-3 (Fig. 11.1). 3. DBL-1/TGF-β signaling pathway. In the intestine, the TGF-β receptor SMA-6 could be activated by DBL-1/TGF-β, and the activated TGF-β receptor SMA-6 further regulated the response to toxicants at ERCs by activating two downstream signaling cascades, SMA-4-SMA-9 and SMA-4-MAB-31 (Fig. 11.1). 4. Heme homeostasis-related signaling pathway. In the heme-related signaling pathway, the intestinal HRG-5 could be suppressed by neuronal GLB-10/globin, and the intestinal HRG-5 further regulated the response to toxicants at ERCs by inhibiting two downstream transcriptional factors, ELT-2 and HIF-1 (Fig. 11.1). 5. Wnt signaling pathway. In the Wnt signaling pathway, the GSK-3, a component of APC complex, was inhibited in the intestine, and the GSK-3 further regulated the response to toxicants at ERCs by suppressing the function of BAR-1-POP-1 complex (Fig. 11.1). 6. Insulin signaling pathway. In the intestine, the insulin receptor DAF-2 was inhibited, and the insulin receptor DAF-2 further suppressed the activity and the function of transcriptional factor DAF-16 via activating downstream kinase cascade of AGE-1-AKT-1 during the control of response to toxicants at ERCs (Fig. 11.1). 7. NDK-1 signaling pathway. In the intestine, the NDK-1, a homolog of NM23H1, was inhibited, and the NDK-1 further regulated the response to toxicants at ERCs by suppressing function of transcriptional factor DAF-16 in the insulin signaling pathway or kinase KSR-1/KSR-2 (Fig. 11.1).

11.2

Molecular Network in the Intestine in Regulating Response to Toxicants at ERCs

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Fig. 11.1 Molecular network in the intestine to regulate the response to toxicants at ERCs in nematodes

In the cells of organisms, the transcriptional factors act with the role of switch for the control of different biological processes. Therefore, besides the seven basic signaling pathways introduced above, some transcriptional factor-mediated signaling pathways have been further identified in the intestine to be required for the control of response to toxicants (such as nanopolystyrene) at ERCs in nematodes (Fig. 11.1). These intestinal transcriptional factors are as follows: 1. SKN-1. SKN-1 is a Nrf transcriptional factor in the p38 MAPK signaling pathway. During the control of response to toxicants at ERCs, the intestinal transcriptional factor SKN-1/Nrf could at least activate three downstream targets, MDT-15-SBP-1 signaling cascade, GST-4/GST-5, and XBP-1 (Fig. 11.1).

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2. ATF-7. ATF-7 is also a transcriptional factor in the p38 MAPK signaling pathway. During the control of response to toxicants at ERCs, the intestinal transcriptional factor ATF-7 acted upstream of another transcriptional factor of XBP-1 (Fig. 11.1). 3. NHR-8. NHR-8 is another transcriptional factor acting downstream of PMK-1/ p38 MAPK. During the control of response to toxicants at ERCs, the intestinal transcriptional factor NHR-8 regulated the activity of another transcriptional factor of DAF-12 (Fig. 11.1). 4. MDT-15 and SBP-1. MDT-15 and SBP-1 are two transcriptional factors required for the control of fat storage, and they organize a signaling cascade of MDT-15-SBP-1 during the control of response to toxicants at ERCs (Fig. 11.1). The intestinal transcriptional factor SBP-1 further regulated the response to toxicants at ERCs by activating downstream signaling cascade of FAT-6-CYP-35A3/CLEC-67/LYS-7 or HSP-4, a marker of mitochondrial unfolded protein response (mt UPR) (Fig. 11.1). 5. XBP-1. XBP-1 is a transcriptional factor governing the activation of mt UPR during the control of response to toxicants at ERCs (Fig. 11.1). 6. DAF-5 and DAF-3. DAF-5 and DAF-3 are two transcriptional factors in the DAF-7/TGF-β signaling pathway. DAF-5 and DAF-3 regulated the response to toxicants at ERCs by functioning upstream of another transcriptional factor DAF-12 (Fig. 11.1). 7. DAF-12. DAF-12 is a transcriptional factor required for the control of several aspects of metabolisms, such as fat metabolism. During the control of response to toxicants at ERCs, the transcriptional factor DAF-12 activated the downstream signaling cascade of FAT-6-UGT-18/PGP-6 (Fig. 11.1). 8. SMA-9. SMA-9 is a transcriptional factor in the DBL-1/TGF-β signaling pathway. The transcriptional factor SMA-9 regulated the response to toxicants at ERCs by activating the downstream ELT-2, another transcriptional factor (Fig. 11.1). 9. MAB-31. MAB-31 is also a transcriptional factor in the DBL-1/TGF-β signaling pathway. The transcriptional factor MAB-31 regulated the response to toxicants at ERCs by activating the downstream DAF-16, a transcriptional factor in the insulin signaling pathway (Fig. 11.1). 10. ELT-2. ELT-2 a transcriptional factor required for the control of functional state of intestinal barrier. Meanwhile, the transcriptional factor ELT-2 could further regulate the response to toxicants at ERCs by activating downstream CLEC-63/ CLEC-85, ERM-1, and ATFS-1. ERM-1 is also required for the control of functional state of intestinal barrier. ATFS-1 is a transcriptional factor governing the activation of endoplasmic reticulum UPR (ER UPR). 11. HIF-1. HIF-1 is a transcriptional factor required for the control hypoxic stress. During the control of toxicants at ERCs, the downstream targets of HIF-1 remain still unclear. 12. ATFS-1 and DVE-1. ATFS-1 and DVE-1 are two transcriptional factors required for the control of ER UPR during the control of response to toxicants at ERCs.

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13. BAR-1 and POP-1. BAR-1 and POP-1 form a complex in the Wnt signaling pathway. During the control of response to toxicants at ERCs, the transcriptional factor POP-1 activated the downstream signaling cascade of PRX-5-KAT-1/ ACOX-1.6 or DAF-16, a transcriptional factor in the insulin signaling pathway (Fig. 11.1). 14. DAF-16. The FOXO transcriptional factor DAF-16 in the insulin signaling pathway regulated the response to toxicants at ERCs by activating downstream signaling cascade of UBL-1-HSP-6 reflecting the ER UPR activation, GPB-2, SOD-3 (a mitochondrial Mn-SOD), MTL-1, or HLH-30, a transcriptional factor governing the activation of autophagy (Fig. 11.1). 15. FKH-2 and NHR-77. FKH-2 and NHR-77 are two transcriptional factors, and they regulated the response to toxicants at ERCs by activating or inhibiting activity of DAF-16, a transcriptional factor in the insulin signaling pathway (Fig. 11.1). 16. HLH-30. HLH-30 is a transcriptional factor required for the control of autophagy activation during the control of response to toxicants at ERCs (Fig. 11.1).

11.2.2 G Protein-Coupled Receptor (GPCR)-Mediated Molecular Signaling Network in the Intestine in Response to Toxicants at ERCs In organisms, the GPCRs on the cytoplasmic membrane contribute to the sense of environmental stimuli, and the GPCR signals are further transduced by downstream Gα proteins. With the nanopolystyrene as the example of environmental toxicants, the GPCR-mediated molecular signaling network in the intestine in response to toxicants at ERCs was identified in nematodes [17, 22, 23, 25–27]. In the intestine, so far, at least eight GPCRs have been identified on the cytoplasmic membrane to be required for the control of response to toxicants (such as nanopolystyrene) at ERCs in nematodes (Fig. 11.2). These GPCRs further contributed to the formation of intestinal molecular network involved in the control of response to toxicants at ERCs. 1. DAF-1. On the one hand, the intestinal receptor DAF-1 can receive the signal from neuronal DAF-7/TGF-β. On the other hand, the intestinal TGF-β receptor DAF-1 could be activated by exposure to toxicants at ERCs. The intestinal TGF-β receptor DAF-1 regulated the response to toxicants at ERCs by activating the downstream DAF-8 (Fig. 11.2). 2. SMA-6. On the one hand, the intestinal receptor SMA-6 received the signal from neuronal DBL-1/TGF-β. On the other hand, the intestinal TGF-β receptor SMA-6 could also be activated by exposure to toxicants at ERCs. The intestinal TGF-β receptor SMA-6 regulated the response to toxicants at ERCs by activating the downstream SMA-4 (Fig. 11.2).

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Fig. 11.2 GPCR-mediated molecular network in the intestine to regulate the response to toxicants at ERCs in nematodes

3. DOP-1. Besides in the neurons, the intestinal receptor DOP-1 can also sense and receive the signal from neurotransmitter of dopamine released from the neurons. In addition, the intestinal dopamine receptor DOP-1 could be activated by exposure to toxicants at ERCs. The intestinal dopamine receptor DOP-1 could act upstream of PMK-1/p38 MAPK signaling to regulate the response to toxicants at ERCs (Fig. 11.2). 4. PAQR-2. The upstream ligand(s) for intestinal GPCR PAQR-2 are still unclear during the control of response to toxicants at ERCs. After the exposure to toxicants at ERCs, the intestinal GPCR PAQR-2 regulated the activity of PMK-1/p38 MAPK, and BAR-1/β-catenin in the Wnt signaling pathway, and DAF-16/FOXO transcriptional factor in the insulin signaling pathway via affecting functions of downstream two Gα proteins, GPA-10 and EGL-30 (Fig. 11.2). Moreover, the intestinal GPCR PAQR-2 could further act upstream of ELT-2 to regulate the response to toxicants at ERCs (Fig. 11.2). 5. FSHR-1. The upstream ligand(s) for intestinal GPCR FSHR-1 are also still unclear during the control of response to toxicants at ERCs. After the exposure to toxicants at ERCs, the intestinal GPCR FSHR-1 affected the activity of PMK-1/p38 MAPK, BAR-1/β-catenin in the Wnt signaling pathway, and DAF-16/FOXO transcriptional factor in the insulin signaling pathway via regulating functions of downstream two Gα proteins, GPA-10 and EGL-30 (Fig. 11.2). 6. DAF-2. DAF-2 is an insulin receptor to receive the signals of insulin peptides released from the neurons or the germline during the control of response to

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Molecular Network in the Intestine in Regulating Response to Toxicants at ERCs

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toxicants at ERCs. The intestinal insulin receptor DAF-2 regulated the response to toxicants at ERCs by suppressing the activity and the function of FOXO transcriptional factor DAF-16 (Fig. 11.2). 7. OCTR-1 and SER-6. On the one hand, the intestinal receptors OCTR-1 and SER-6 received the signal from neurotransmitter of octopamine released from the neurons. On the other hand, these two intestinal octopamine receptors could also be activated or inhibited by exposure to toxicants at ERCs (Fig. 11.2). The downstream targets for intestinal octopamine receptors of OCTR-1 and SER-6 are still unknown during the control of response to toxicants at ERCs.

11.2.3 Ion Channel-Mediated Molecular Signaling Network in the Intestine in Response to Toxicants at ERCs Besides the GPCRs, the ion channels on cytoplasmic membrane also contribute to the sense of environmental stimuli in organisms. With the nanopolystyrene as the example of environmental toxicants, the ion channel-mediated molecular signaling network in the intestine in response to toxicants at ERCs was identified in nematodes [28]. In the intestine, so far, only two ion channels have been identified on the cytoplasmic membrane to be required for the control of response to toxicants (such as nanopolystyrene) at ERCs in nematodes (Fig. 11.3). These two ion channels also

Fig. 11.3 Ion channel-mediated molecular network in the intestine to regulate the response to toxicants at ERCs in nematodes

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contribute to the formation of molecular network in the intestine required for the control of response to toxicants at ERCs. 1. CCA-1. The intestinal ion channel of CCA-1 could be activated by exposure to toxicants at ERCs. The intestinal ion channel of CCA-1 regulated the response to toxicants at ERCs by activating the downstream ELT-2, a transcriptional factor required for the control of functional state of intestinal barrier (Fig. 11.3). 2. EGL-19. The intestinal ion channel of EGL-19 could also be activated by exposure to toxicants at ERCs. The intestinal ion channel of EGL-19 regulated the response to toxicants at ERCs by activating the downstream ELT-2 or DAF-16 in the insulin signaling pathway (Fig. 11.3). That is, the activity of intestinal FOXO transcriptional factor DAF-16 could be affected by both the GPCRs and the ion channels on cytoplasmic membrane during the control of response to toxicants at ERCs (Fig. 11.3).

11.2.4 Epigenetic Control-Mediated Molecular Signaling Network in the Intestine in Response to Toxicants at ERCs In organisms, the gene expressions can be regulated by some epigenetic control mechanisms, such as microRNAs (miRNAs), long noncoding RNAs (lncRNAs), methylation regulation, and acetylation regulation. With the nanopolystyrene as the example of environmental toxicants, the epigenetic control-mediated molecular signaling network in the intestine in response to toxicants at ERCs was identified in nematodes [19–21, 23, 29, 30]. Firstly, three miRNAs have been identified in the intestine to control the response to toxicants at ERCs by inhibiting the activity of their downstream direct targets. These three intestinal miRNAs are mir-35, mir-354, and mir-794. 1. mir-35. In nematodes, the expression of mir-35 was increased by exposure to toxicants (such as nanopolystyrene) at ERCs. During the control of response to toxicants at ERCs, the intestinal mir-35 enhanced the function of FOXO transcriptional factor DAF-16 in the insulin signaling pathway by inhibiting the activity of its target of NDK-1 (Fig. 11.4). 2. mir-354. In nematodes, the expression of mir-354 was increased by exposure to toxicants (such as nanopolystyrene) at ERCs. During the control of response to toxicants at ERCs, the intestinal mir-354 inhibited the activity of its target of DAF-3, a transcriptional factor in the DAF-7/TGF-β signaling pathway (Fig. 11.4). 3. mir-794. In nematodes, the expression of mir-794 was increased by exposure to toxicants (such as nanopolystyrene) at ERCs. During the control of response to toxicants at ERCs, the intestinal mir-794 suppressed the activity of its targets of DAF-16, a FOXO transcriptional factor in the insulin signaling pathway, and two

11.2

Molecular Network in the Intestine in Regulating Response to Toxicants at ERCs

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Fig. 11.4 Epigenetic control-mediated molecular network in the intestine to regulate the response to toxicants at ERCs in nematodes

transcriptional factors (SKN-1 and MDT-15) in the p38 MAPK signaling pathway (Fig. 11.4). Secondly, so far, at least four lncRNAs have been identified in the intestine to be required for the regulation of response to toxicants at ERCs. The identified four intestinal lncRNAs are lin-2, linc-9, lin-50, and linc-61. 1. linc-2. In nematodes, the expression of linc-2 was increased by exposure to toxicants (such as nanopolystyrene) at ERCs. The intestinal linc-2 regulated the response to toxicants at ERCs by directly activating DAF-16, FOXO transcriptional factor in the insulin signaling pathway (Fig. 11.4). 2. linc-9. In nematodes, the expression of linc-9 was increased by exposure to toxicants (such as nanopolystyrene) at ERCs. The intestinal linc-9 could regulate the response to toxicants at ERCs by indirectly increasing the expression of DAF-16 in the insulin signaling pathway via inhibiting its downstream direct target of NHR-77, another transcriptional factor (Fig. 11.4). 3. linc-50. In nematodes, the expression of linc-50 was decreased by exposure to toxicants (such as nanopolystyrene) at ERCs. The intestinal linc-50 regulated the

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response to toxicants at ERCs by directly suppressing DAF-16, FOXO transcriptional factor in the insulin signaling pathway (Fig. 11.4). 4. linc-61. In nematodes, the expression of linc-61 was increased by exposure to toxicants (such as nanopolystyrene) at ERCs. The intestinal linc-61 regulated the response to toxicants at ERCs by directly activating DAF-16, a FOXO transcriptional factor in the insulin signaling pathway, or DVE-1, a transcriptional factor governing the mt UPR activation (Fig. 11.4). Moreover, the intestinal linc-61 could further regulate the response to toxicants at ERCs by indirectly increasing the expression of DAF-16 in the insulin signaling pathway via activating its downstream direct target of FKH-2, another transcriptional factor (Fig. 11.4). MET-2, a H3K9 methyltransferase, mediated a molecular mechanism of histone methylation in nematodes. The expression of MET-2 could be inhibited by exposure to toxicants at ERCs. The intestinal MET-2 regulated the response to toxicants at ERCs by suppressing the functions of ELT-2, BAR-1/β-catenin in the Wnt signaling pathway, and DAF-16/FOXO transcriptional factor in the insulin signaling pathway (Fig. 11.4). CBP-1, a acetyltransferase, mediated a molecular mechanism of histone acetylation in nematodes. The expression of CBP-1 could be increased by exposure to toxicants at ERCs. The intestinal CBP-1 regulated the response to toxicants at ERCs by enhancing the functions of PMK-1/p38 MAPK and DAF-16/FOXO transcriptional factor in the insulin signaling pathway (Fig. 11.4). In nematodes, two important phenotypes have been observed in the epigenetic control-mediated molecular network in the intestine to regulate the response to toxicants at ERCs. First of all, at least so far, most of the epigenetic control in the intestine has been found to target to the DAF-16/FOXO transcriptional factor in the insulin signaling pathway during the control of response to toxicants at ERCs (Fig. 11.4). This further implies the crucial function of insulin signaling pathway in regulating the response to toxicants at ERCs in organisms. Moreover, most of epigenetic control-related molecules (such as mir-794, linc-61, MET-2, and CBP-1) in the intestine had multiple downstream targets during the control of response to toxicants at ERCs.

11.3

Molecular Network in the Neurons in Regulating Response to Toxicants at ERCs

11.3.1 Molecular Signaling Network in the Neurons in Response to Toxicants at ERCs With the nanopolystyrene as the example of environmental toxicants, the molecular signaling network in the neurons to regulate the response to toxicants at ERCs was identified in nematodes [22–24, 27, 31]. In the raised molecular signaling network in the neurons, at least there are five basic signaling pathways were identified to be

11.3

Molecular Network in the Neurons in Regulating Response to Toxicants at ERCs

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Fig. 11.5 Molecular network in the neurons to regulate the response to toxicants at ERCs in nematodes

activated after exposure to toxicants (such as nanopolystyrene) at ERCs in nematodes (Fig. 11.5). These five basic signaling pathways acting in the neurons to regulate the response to toxicants at ERCs are: 1. JNK MAPK signaling pathway. In the neurons, the glutamate receptor of GLR-4 could be activated by exposure to toxicants at ERCs, and the GLR-4 further acted upstream of JNK-1/JNK MAPK to regulate the response to toxicants at ERCs (Fig. 11.5). During the control of response to toxicants at ERCs, the neuronal JNK-1/JNK MAPK further functioned upstream of SNB-1, which controlled the neurotransmission of dopamine and octopamine in nematodes (Fig. 11.5). 2. ERK MAPK signaling pathway. In the neurons, the tyramine receptor of TYRA-2 could be activated by exposure to toxicants at ERCs, and the TYRA-2 further acted upstream of MPK-1/ERK MAPK to regulate the response to toxicants at ERCs (Fig. 11.5). During the control of response to toxicants at ERCs, the neuronal MPK-1/ERK MAPK further regulated the release of three insulin peptides, INS-4, INS-39, and DAF-28 (Fig. 11.5). 3. DAF-7/TGF-β signaling pathway. In the neurons, the DAF-7/TGF-β could be activated by exposure to toxicants at ERCs (Fig. 11.5). The detailed upstream

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regulators, including the GPCRs, for the neuronal DAF-7/TGF-β in regulating the response to toxicants at ERCs remain still unclear. 4. DBL-1/TGF-β signaling pathway. In the neurons, the DBL-1/TGF-β could be activated by exposure to toxicants at ERCs, and the function of neuronal DBL-1/ TGF-β in regulating the response to toxicants at ERCs was under the control of two signaling cascades, SMOC-1-ZAG-1 and SMOC-1-ADT-2 (Fig. 11.5). In addition, the glutamate receptor further regulated the function of neuronal DBL-1/TGF-β in controlling the response to toxicants at ERCs by suppressing its activity (Fig. 11.5). 5. Heme homeostasis-related signaling pathway. In the neurons, the GLB-10/ globin could be activated by exposure to toxicants at ERCs, and the neuronal GLB-10/globin regulated the response to toxicants at ERCs by activating the downstream HRG-7 (Fig. 11.5).

11.3.2 GPCR-Mediated Molecular Signaling Network in the Neurons in Response to Toxicants at ERCs With the nanopolystyrene as the example of environmental toxicants, the GPCRmediated molecular signaling network in the neurons in response to toxicants at ERCs was identified in nematodes [32]. In the neurons, so far, at least 12 GPCRs have been identified on the cytoplasmic membrane to be required for the control of response to toxicants (such as nanopolystyrene) at ERCs in nematodes (Fig. 11.6). During the control of response to toxicants at ERCs, these 12 neuronal GPCRs usually affected the functions of DBL-1/TGF-β signaling, MPK-1/ERK MAPK signaling, JNK-1/JNK MAPK signaling, and/or DAF-7/TGF-β signaling (Fig. 11.6). In contrast, the upstream GPCRs involved in regulating the GLB-10mediated heme homeostasis signaling remain still largely unclear in nematodes. During the control of response to toxicants at ERCs, the functions of neuronal GPCRs in affecting DBL-1/TGF-β signaling, MPK-1/ERK MAPK signaling, JNK-1/JNK MAPK signaling, and/or DAF-7/TGF-β signaling were normally transduced by three Gα proteins, GOA-1, GSA-1, and GPA-10, and we have introduced and discussed this in Chap. 9 in this book. The 12 neuronal GPCRs are GLR-8, NPR-1, NPR-4, NPR-8, TYRA-2, NPR-9, NPR-12, GLR-4, DCAR-1, GTR-1, DOP-2, and DAF-37 (Fig. 11.6). 1. GLR-8. The expression of GLR-8 could be inhibited by exposure to toxicants at ERCs (Fig. 11.6). The neuronal GPCR GLR-8 regulated the response to toxicants at ERCs by suppressing the function of DBL-1/TGF-β signaling (Fig. 11.6). 2. NPR-1. The expression of NPR-1 could be increased by exposure to toxicants at ERCs (Fig. 11.6). The neuronal GPCR NPR-1 regulated the response to toxicants at ERCs by activating the function of DAF-7/TGF-β signaling (Fig. 11.6).

11.3

Molecular Network in the Neurons in Regulating Response to Toxicants at ERCs

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Fig. 11.6 GPCR-mediated molecular network in the neurons to regulate the response to toxicants at ERCs in nematodes

3. NPR-4. The expression of NPR-4 could be inhibited by exposure to toxicants at ERCs (Fig. 11.6). The neuronal GPCR NPR-4 regulated the response to toxicants at ERCs by suppressing the functions of DBL-1/TGF-β signaling and DAF-7/TGF-β signaling (Fig. 11.6). 4. NPR-8. The expression of NPR-8 could be inhibited by exposure to toxicants at ERCs (Fig. 11.6). The neuronal GPCR NPR-8 regulated the response to toxicants at ERCs by suppressing the functions of DBL-1/TGF-β signaling and MPK-1/ERK MAPK signaling (Fig. 11.6). 5. TYRA-2. The expression of TYRA-2 could be increased by exposure to toxicants at ERCs (Fig. 11.6). The neuronal tyramine GPCR TYRA-2 regulated the response to toxicants at ERCs by activating the function of MPK-1/ERK MAPK signaling (Fig. 11.6). 6. NPR-9. The expression of NPR-9 could be inhibited by exposure to toxicants at ERCs (Fig. 11.6). The neuronal GPCR NPR-9 regulated the response to toxicants at ERCs by suppressing the functions of DBL-1/TGF-β signaling, MPK-1/ ERK MAPK signaling, and JNK-1/JNK MAPK signaling (Fig. 11.6). 7. NPR-12. The expression of NPR-12 could be inhibited by exposure to toxicants at ERCs (Fig. 11.6). The neuronal GPCR NPR-12 regulated the response to

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toxicants at ERCs by suppressing the functions of DBL-1/TGF-β signaling, JNK-1/JNK MAPK signaling, and DAF-7/TGF-β signaling (Fig. 11.6). GLR-4. The expression of GLR-4 could be increased by exposure to toxicants at ERCs (Fig. 11.6). The neuronal glutamate GPCR GLR-4 regulated the response to toxicants at ERCs by activating the function of JNK-1/JNK MAPK signaling (Fig. 11.6). DCAR-1. The expression of DCAR-1 could be increased by exposure to toxicants at ERCs (Fig. 11.6). The neuronal GPCR DCAR-1 regulated the response to toxicants at ERCs by activating the functions of MPK-1/ERK MAPK signaling and JNK-1/JNK MAPK signaling (Fig. 11.6). GTR-1. The expression of GTR-1 could be inhibited by exposure to toxicants at ERCs (Fig. 11.6). The neuronal GPCR GTR-1 regulated the response to toxicants at ERCs by suppressing the functions of DBL-1/TGF-β signaling, JNK-1/ JNK MAPK signaling, and DAF-7/TGF-β signaling (Fig. 11.6). DOP-2. The expression of DOP-2 could be inhibited by exposure to toxicants at ERCs (Fig. 11.6). The neuronal dopamine GPCR DOP-2 regulated the response to toxicants at ERCs by suppressing the functions of DBL-1/TGF-β signaling and MPK-1/ERK MAPK signaling (Fig. 11.6). DAF-37. The expression of DAF-37 could be increased by exposure to toxicants at ERCs (Fig. 11.6). The neuronal GPCR DAF-37 regulated the response to toxicants at ERCs by activating the functions of DBL-1/TGF-β signaling and MPK-1/ERK MAPK signaling (Fig. 11.6).

11.3.3 Ion Channel-Mediated Molecular Signaling Network in the Neurons in Response to Toxicants at ERCs With the nanopolystyrene as the example of environmental toxicants, the ion channel-mediated molecular signaling network in the neurons in response to toxicants at ERCs was identified in nematodes [28]. In the neurons, so far, at least five ion channels have been identified on the cytoplasmic membrane to be required for the control of response to toxicants (such as nanopolystyrene) at ERCs in nematodes (Fig. 11.7). These five neuronal ion channels are CNG-3, EAT-2, TAX-4, UNC-93, and EGL-19 (Fig. 11.7). 1. CNG-3. The expression of CNG-3 could be inhibited by exposure to toxicants at ERCs (Fig. 11.7). The downstream targets for neuronal ion channel CNG-3 in regulating the response to toxicants at ERCs remain still unclear (Fig. 11.7). 2. EAT-2. The expression of EAT-2 could be inhibited by exposure to toxicants at ERCs (Fig. 11.7). The neuronal ion channel EAT-2 regulated the response to toxicants at ERCs by suppressing the functions of DBL-1/TGF-β signaling, MPK-1/ERK MAPK signaling, and JNK-1/JNK MAPK signaling (Fig. 11.7).

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Molecular Network in the Neurons in Regulating Response to Toxicants at ERCs

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Fig. 11.7 Ion channel-mediated molecular network in the neurons to regulate the response to toxicants at ERCs in nematodes

3. TAX-4. The expression of TAX-4 could be increased by exposure to toxicants at ERCs (Fig. 11.7). The neuronal ion channel TAX-4 regulated the response to toxicants at ERCs by activating the functions of JNK-1/JNK MAPK signaling and DAF-7/TGF-β signaling (Fig. 11.7). 4. UNC-93. The expression of UNC-93 could be increased by exposure to toxicants at ERCs (Fig. 11.7). The neuronal ion channel UNC-93 regulated the response to toxicants at ERCs by activating the functions of MPK-1/ERK MAPK signaling and DAF-7/TGF-β signaling (Fig. 11.7). 5. EGL-19. The expression of EGL-19 could be increased by exposure to toxicants at ERCs (Fig. 11.7). The downstream targets for neuronal ion channel EGL-19 in regulating the response to toxicants at ERCs remain still unclear (Fig. 11.7).

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11.3.4 Epigenetic Control-Mediated Molecular Signaling Network in the Neurons in Response to Toxicants at ERCs With the nanopolystyrene as the example of environmental toxicants, the epigenetic control-mediated molecular signaling network in the neurons in response to toxicants at ERCs was identified in nematodes [24, 30]. Firstly, the miRNA of mir-76 has been identified in the neurons to regulate the response to toxicants at ERCs. In nematodes, the expression of mir-76 was decreased by exposure to toxicants (such as nanopolystyrene) at ERCs. During the control of response to toxicants at ERCs, the neuronal mir-76 regulated the function of GLB-10/globin signaling by inhibiting the activity of GLB-10/globin in the heme homeostasis-related signaling pathway (Fig. 11.8). Moreover, the expression of CBP-1, an acetyltransferase, was increased by exposure to toxicants at ERCs, and the neuronal CBP-1 regulated the response to toxicants at ERCs by activating the functions of JNK-1/JNK MAPK signaling and DAF-7/TGF-β signaling (Fig. 11.8).

Fig. 11.8 Epigenetic control-mediated molecular network in the neurons to regulate the response to toxicants at ERCs in nematodes

11.4

11.4

Molecular Network in the Germline in Regulating Response to Toxicants at ERCs

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Molecular Network in the Germline in Regulating Response to Toxicants at ERCs

11.4.1 Molecular Signaling Network in the Germline in Response to Toxicants at ERCs With the nanopolystyrene as the example of environmental toxicants, the molecular signaling network in the germline to regulate the response to toxicants at ERCs was identified in nematodes [33–35]. In the raised molecular signaling network in the germline, at least there are seven basic signaling pathways identified to be activated after exposure to toxicants (such as nanopolystyrene) at ERCs in nematodes (Fig. 11.9). These seven basic signaling pathways acting in the germline to regulate the response to toxicants at ERCs are: 1. Apoptosis and DNA damage-related signaling pathway. In the germline, the apoptosis and DNA damage-related signaling pathway could be activated by exposure to toxicants at ERCs (Fig. 11.9). During the control of response to toxicants at ERCs, HUS-1, CLK-2, CED-1, and EGL-1 constituted the signaling cascade for DNA damage-related signaling pathway, and CED-3, CED-4, and CED-9 constituted the signaling cascade of apoptosis-related signaling pathway (Fig. 11.9).

Fig. 11.9 Molecular network in the germline to regulate the response to toxicants at ERCs in nematodes

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2. NHL-2 signaling. In the germline, the NHL-2 expression could be inhibited by exposure to toxicants at ERCs (Fig. 11.9). The germline NHL-2 acted upstream of EKL-1 to regulate the response to toxicants at ERCs by increasing its activity (Fig. 11.9). 3. NDK-1 signaling. In the germline, the NDK-1 expression could be inhibited by exposure to toxicants at ERCs (Fig. 11.9). The germline NDK-1 acted upstream of KSR-1 and KSR-2 to regulate the response to toxicants at ERCs by suppressing their activity (Fig. 11.9). 4. PAT-12 signaling. In the germline, the PAT-12 expression could be inhibited by exposure to toxicants at ERCs (Fig. 11.9). The detailed downstream targets for germline PAT-12 to regulate the response to toxicants at ERCs remain still unclear (Fig. 11.9). 5. LIN-23 signaling. In the germline, the LIN-23 expression could be inhibited by exposure to toxicants at ERCs (Fig. 11.9). The detailed downstream targets for germline LIN-23 to regulate the response to toxicants at ERCs remain still unclear (Fig. 11.9). 6. WRT-3 signaling. In the germline, the WRT-3 expression could be inhibited by exposure to toxicants at ERCs (Fig. 11.9). The germline WRT-3 acted upstream of ASP-2 to regulate the response to toxicants at ERCs by suppressing its activity (Fig. 11.9). 7. CED-10/VPS-34/SNX-1/RAB-7/RAB-14 signaling. In the germline, the expressions of CED-10, VPS-34, SNX-1, RAB-4, and RAB-14 could be increased by exposure to toxicants at ERCs (Fig. 11.9). During the control of response to toxicants at ERCs, the signaling order of CED-10, VPS-34, SNX-1, RAB-4, and RAB-14 needs the further examination.

11.4.2 GPCR-Mediated Molecular Signaling Network in the Germline in Response to Toxicants at ERCs With the nanopolystyrene as the example of environmental toxicants, the GPCRmediated molecular signaling network in the germline in response to toxicants at ERCs was identified in nematodes [34]. In the germline, the GPCR of CED-1 has been identified on the cytoplasmic membrane to be required for the control of response to toxicants (such as nanopolystyrene) at ERCs in nematodes (Fig. 11.10). During the control of response to toxicants at ERCs, the germline GPCR CED-1 regulated the functions of NDK-1, PAT-12, LIN-23, WRT-3, CED-10, VPS-34, SNX-1, RAB-7, and RAB-14 by activating the GPA-16, a Gα protein (Fig. 11.10). Meanwhile, the activated another Gα protein of GPA-15 could regulate the response to toxicants at ERCs by affecting the functions of NHL-2, NDK-1, PAT-12, LIN-23, CED-10, VPS-34, and SNX-1 (Fig. 11.10). Nevertheless, the upstream germline GPCR(s) activating or inhibiting the Gα protein of GPA-15 remain still unclear during the control of response to toxicants at ERCs (Fig. 11.10).

11.4

Molecular Network in the Germline in Regulating Response to Toxicants at ERCs

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Fig. 11.10 GPCR-mediated molecular network in the germline to regulate the response to toxicants at ERCs in nematodes

11.4.3 Epigenetic Control-Mediated Molecular Signaling Network in the Germline in Response to Toxicants at ERCs With the nanopolystyrene as the example of environmental toxicants, the epigenetic control-mediated molecular signaling network in the germline in response to toxicants at ERCs was identified in nematodes [29, 30, 33]. Firstly, the miRNA of mir-38 has been identified in the germline to regulate the response to toxicants at ERCs. In nematodes, the expression of germline mir-38 was increased by exposure to toxicants (such as nanopolystyrene) at ERCs. The germline mir-38 regulated the response to toxicants at ERCs by inhibiting the activity of downstream targets of NHL-2, NDK-1, and WRT-3 (Fig. 11.11). Moreover, the expression of germline MET-2, a H3K9 methyltransferase, was inhibited by exposure to toxicants at ERCs. The germline MET-2 could regulate the response to toxicants at ERCs by activating the functions of downstream targets of WRT-3 and PAT-12 (Fig. 11.11). In addition, the expression of germline CBP-1, an acetyltransferase, was increased by exposure to toxicants at ERCs. The germline CBP-1 could regulate the response to toxicants at ERCs by suppressing the function of downstream target of NHL-2, which in turns further activated the EKL-1 and DAF-28, an insulin peptide in the germline (Fig. 11.11).

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Fig. 11.11 Epigenetic control-mediated molecular network in the germline to regulate the response to toxicants at ERCs in nematodes

11.5

Signaling Communications Between Different Tissues in Regulating the Response to Toxicants at ERCs

11.5.1 Signaling Communications Between Neurons and Intestine in Regulating the Response to Toxicants at ERCs So far, most of the identified signaling communications are on those between neurons and intestine during the regulation of response to toxicants at ERCs. With the nanopolystyrene as the example of environmental toxicants, six signaling cascades reflecting the signaling communications between neurons and intestine have been identified during the control of response to toxicants at ERCs in nematodes [22–24, 27, 31]. The identified six signaling pathways are insulin, dopamine neurotransmission-related, octopamine neurotransmission-related, DBL-1/TGF-β, DAF-7/TGF-β, and heme homeostasis-related signaling pathways. 1. Insulin signaling pathway. During the control of response to toxicants at ERCs, three insulin peptides of INS-4, INS-39, and DAF-28 could be inhibited in the neurons (Fig. 11.12). The neuronal insulin peptides of INS-4, INS-39, and DAF-28 further regulated the response to toxicants at ERCs by affecting the function of intestinal insulin receptor after the release from the neurons

11.5

Signaling Communications Between Different Tissues in Regulating the. . .

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Fig. 11.12 Signaling communications between neurons and intestine in regulating the response to toxicants at ERCs in nematodes

(Fig. 11.12). In the neurons, the expressions of three insulin peptides of INS-4, INS-39, and DAF-28 were suppressed by the activated MPK-1/ERK MAPK in nematodes exposed to toxicants at ERCs (Fig. 11.12). In the intestine of nematodes exposed to toxicants at ERCs, the insulin receptor DAF-2 suppressed the

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expression and the function of FOXO transcriptional factor DAF-16 by activating the downstream kinase cascade of AGE-1-AKT-1 (Fig. 11.12). Dopamine neurotransmission-related signaling pathway. During the control of response to toxicants at ERCs, the expression of CAT-2 required for the dopamine synthesis could be inhibited in the neurons (Fig. 11.12), implying the suppression in dopamine synthesis. The released dopamine from the neurons further regulated the response to toxicants at ERCs by affecting the function of its receptor DOP-1 in the intestine (Fig. 11.12). In the neurons, the expression of CAT-2 was suppressed by the activated JNK-1/JNK MAPK in nematodes exposed to toxicants at ERCs (Fig. 11.12). In the intestine, the dopamine receptor DOP-1 regulated the response to toxicants at ERCs by activating the downstream PMK-1/p38 MAPK (Fig. 11.12). Octopamine neurotransmission-related signaling pathway. During the control of response to toxicants at ERCs, the expression of TBH-1 required for the octopamine synthesis could be activated in the neurons (Fig. 11.12), implying the increase in octopamine synthesis. The released octopamine from the neurons further regulated the response to toxicants at ERCs by affecting the function of its receptors of OCTR-1 and SRE-6 in the intestine (Fig. 11.12). In the neurons, the expression of TBH-1 was increased by the activated JNK-1/JNK MAPK in nematodes exposed to toxicants at ERCs (Fig. 11.12). DBL-1/TGF-β signaling pathway. During the control of response to toxicants at ERCs, the DBL-1/TGF-β could be activated in the neurons (Fig. 11.12). The neuronal DBL-1/TGF-β further regulated the response to toxicants at ERCs by affecting the function of its receptor of SMA-6 in the intestine after the release from the neurons (Fig. 11.12). In the intestine of nematodes exposed to toxicants at ERCs, the DBL-1/TGF-β receptor SMA-6 enhanced the expression and the function of two transcriptional factors (SMA-9 and MAB-31) by activating the downstream SMA-4 (Fig. 11.12). DAF-7/TGF-β signaling pathway. During the control of response to toxicants at ERCs, the DAF-7/TGF-β could be activated in the neurons (Fig. 11.12). The neuronal DAF-7/TGF-β further regulated the response to toxicants at ERCs by affecting the function of its receptor of DAF-1 in the intestine after the release from the neurons (Fig. 11.12). In the intestine of nematodes exposed to toxicants at ERCs, the DAF-7/TGF-β receptor DAF-1 suppressed the expression and the function of two transcriptional factors (DAF-3 and DAF-5) by activating the downstream DAF-8 (Fig. 11.12). Heme homeostasis-related signaling pathway. During the control of response to toxicants at ERCs, the GLB-10/globin could be activated in the neurons, and the neuronal GLB-10/globin acted upstream of HRG-7 to regulate the response to toxicants at ERCs (Fig. 11.12). The neuronal HRG-7 further regulated the response to toxicants at ERCs by affecting the function of intestinal HRG-5 (Fig. 11.12). In the intestine, the HRG-5 regulated the response to toxicants at ERCs by suppressing the functions of two transcriptional factors (ELT-2 and HIF-1) (Fig. 11.12).

11.5

Signaling Communications Between Different Tissues in Regulating the. . .

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11.5.2 Signaling Communications Between Germline and Intestine in Regulating the Response to Toxicants at ERCs With the nanopolystyrene as the example of environmental toxicants, the signaling cascades reflecting the signaling communications between germline and intestine have been identified during the control of response to toxicants at ERCs in nematodes [34]. In the germline, the VPS-34/SNX-1 signaling and the CED-10/RAB-7/ RAB-14 signaling could be activated by exposure to toxicants at ERCs (Fog. 11.13). The germline VPS-34/SNX-1 signaling regulated the response to toxicants at ERCs by affecting the function of intestinal PMK-1/p38 MAPK (Fig. 11.13). Meanwhile, the germline CED-10/RAB-7/RAB-14 signaling regulated the response to toxicants at ERCs by affecting the function of intestinal DAF-16, a FOXO transcriptional factor in the insulin signaling pathway (Fig. 11.13). In the germline, both the VPS-34/SNX-1 signaling and the CED-10/RAB-7/RAB-14 signaling were activated by the GPCR CED-1 during the control of response to toxicants at ERCs (Fig. 11.13).

11.5.3 Signaling Communications Between Different Neurons in Regulating the Response to Toxicants at ERCs With the nanopolystyrene as the example of environmental toxicants, the signaling cascades reflecting the signaling communications between different neurons have been further identified during the control of response to toxicants at ERCs in nematodes. In the neurons, the increase in TDC-1 expression and the decrease in EAT-4 expression implied the alteration in tyramine synthesis and glutamate synthesis after the exposure to toxicants at ERCs (Fig. 11.14). After the release from one neuron, the neurotransmitter of tyramine regulated the response to toxicants at ERCs by affecting the function of its receptor of TYRA-2 in another neuron (Fig. 11.14). In the neurons, the tyramine receptor TYRA-2 regulated the response to toxicants at ERCs by activating the downstream MPK-1/ERK MAPK signaling (Fig. 11.14). After the release from one neuron, the neurotransmitter of glutamate regulated the response to toxicants at ERCs by affecting the function of its receptors of GLR-4 and GLR-8 in another neuron (Fig. 11.14). In the neurons, the glutamate receptor GLR-4 regulated the response to toxicants at ERCs by activating the downstream JNK-1/ JNK MAPK signaling, and the glutamate receptor GLR-8 regulated the response to toxicants at ERCs by suppressing the downstream DBL-1/TGF-β signaling (Fig. 11.14).

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Fig. 11.13 Signaling communications between germline and intestine in regulating the response to toxicants at ERCs in nematodes

11.6

Basic Conclusions

1. The alterations in enhancing sensitivity or bioavailability were helpful for detecting the toxicity of toxicants at ERCs. The toxicity of toxicants at ERCs could be detected in nematodes with enhanced sensitivity caused by certain

11.6

Basic Conclusions

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Fig. 11.14 Signaling communications between different neurons in regulating the response to toxicants at ERCs in nematodes

treatments or genetic mutations. In addition, the toxicity of toxicants at ERCs could also be detected in nematodes with enhanced permeability of intestinal barrier caused by certain physical injury or genetic mutations. 2. A relatively simple molecular network was formed to be required for the control of response to toxicants at ERCs. Compared with the gene number in

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the nematode genome, definitely, the identified molecular network required for the control of response to toxicants at ERCs is very simple, and this molecular network is organized with limited number of genes in nematodes. That is, in the nematode genome, only very limited genes are involved in the control of response to toxicants at ERCs, and most of the genes do not participate in or possibly only exert very limited effects on the control of response to toxicants at ERCs. These limited number of genes may be the most important and conserved genes during the control of response to toxicants at ERCs. The molecular network in response to toxicants at ERCs was normally formed in potentially bioavailable organs or tissues. With the nanopolystyrene as the example of toxicants, the molecular network in regulating the response to toxicants at ERCs could be found in the intestine, the neurons, and the germline. Cross talks among signaling pathways contributed to the formation of molecular networks required for the control of response to toxicants at ERCs. In the raised molecular network required for the control of response to toxicants at ERCs, the cross talks among different signaling pathways have been frequently detected. In addition, we still cannot exclude the possibility for the existence of cross talks among the other signaling pathways. Certain molecular regulators contributed to the formation of molecular networks required for the control of response to toxicants at ERCs. The limited number of GPCRs and ion channels has been found to activate or inhibit multiple downstream signaling pathways during the control of response to toxicants at ERCs. In addition, some epigenetic control-related molecules (such as miRNAs, lncRNAs, H3K9 methyltransferase MET-2, and acetyltransferase CBP-1) were also involved in the control of response to toxicants at ERCs. Certain signaling communications between different tissues could be formed during the control of response to toxicants at ERCs. So far, during the control of response to toxicants at ERCs, the identified signaling communications contained the signaling communication from neurons to the intestine, the signaling communication from germline to the intestine, and the signaling communication between different neurons. New signaling cascades could be formed in some signaling pathways during the control of response to toxicants at ERCs. At least in Wnt and p38 MAPK signaling pathways, the novel signaling cascades could be identified to be required for the control of response to toxicants at ERCs, which are very different from the known signaling pathways under normal conditions.

11.7

Perspectives

C. elegans is a wonderful model to determine the molecular response of organisms to exposure to toxicants at ERCs [1, 2, 6, 7]. With the nanopolystyrene as the example of environmental toxicants, we here introduced and discussed the intestinal molecular network, the neuronal molecular network, and the germline molecular network

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Perspectives

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required for the control of response to toxicants at ERCs. So far, the obtained molecular network involved in the control of response to toxicants at ERCs was relatively simple and contained only limited number of genes and signaling pathways. In the intestine, the neurons, and the germline, the molecular networks could be formed by cross talks among different signaling pathways, induced by certain number of GPCRs or ion channels, and modulated by certain forms of epigenetic controls during the regulation of response to toxicants at ERCs. In nematodes, the obtained molecular network required for the control of response to toxicants at ERCs so far only contained very limited number of genes. Then, a question has been raised that whether these genes involved in the organization of molecular network can cover all aspects of nematodes in response to toxicants at ERCs at the whole animal level. If they cannot, what molecules further govern the other aspects of nematodes in response to toxicants at ERCs? For example, besides the nematodes genes, whether the microbe genes also participate in the control of nematodes in response to toxicants at ERCs? With the miRNAs as the example of epigenetic regulations, so far the identified miRNAs could not cover all or most of the aspects for molecular network-mediated response to toxicants at ERCs. One possibility is that the functions of epigenetic regulation may be limited during the control of response to toxicants at ERCs. Another possibility is that some other forms of epigenetic regulations are also required, and different forms of epigenetic regulations function together to regulate the molecular network organized by genes during the control of response to toxicants at ERCs. So far, the identified molecular signals and signaling pathways are mainly found in the intestine, the neurons, and the germline during the control of response to toxicants at ERCs. This may be largely due to the selected toxicant of nanopolystyrene, which is a form of nanoparticles. The intestine, the neurons, and the germline were the potential bioavailable tissues for nanopolystyrene in nematodes. Thus, this implies such a possibility that the molecular signals and signaling pathways may be further possibly identified in other tissues in nematodes after exposure to soluble toxicants with severe toxicity at ERCs. Different forms of signaling communications have been found during the control of response to toxicants at ERCs in nematodes. The observed signaling communications involved in the control of response to toxicants at ERCs are mainly formed from neurons to the intestine or from germline to the intestine. These findings imply that the orientations for signaling communications from neurons to the intestine and from germline to intestine are the most important forms during the control of response to toxicants at ERCs. In contrast, the orientations for signaling communications from the intestine to neurons and from the intestine to germline may be the important forms during the control of response to toxicants at high concentrations. Therefore, the biological events in the intestine may be very crucial for the induction of toxicity in nematodes exposed to toxicants at ERCs. In the obtained molecular network required for the control of response to toxicants at ERCs, some important signaling pathways definitely have not been identified. Besides this, most of the downstream targets for the identified transcriptional

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factors still remain largely unclear during the control of response to toxicants at ERCs. Therefore, more efforts are still required for the more thoroughly understanding the molecular response to different forms of toxicants at ERCs in nematodes. In nematodes, the obtained molecular network organized by limited number of genes implied that these limited number of genes may play pivotal roles in regulating the toxicity of toxicants. This may also provide useful clue for identifying the pivotal regulatory genes for the occurrence of certain diseases, especially those required for the early occurrence of certain diseases in humans. In addition, we still need to further ask whether certain and relatively simple biochemical network, physiological network, and structural alterations are also induced in organisms after exposure to toxicants at ERCs.

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Index

C Caenorhabditis elegans, 1, 2, 4, 23, 33, 34, 42, 47, 48, 57, 63, 64, 82, 89, 91, 127, 133, 134, 153, 159, 160, 178, 185, 186, 190, 201, 207, 208, 215, 216, 218, 221, 223, 225, 255, 263, 264, 286, 321, 329, 354

D Development-related signaling pathways, 89–128

E Environmental exposures, 1, 89, 127, 178, 329 Environmentally relevant concentrations (ERCs), 1–24, 33, 34, 36, 38–42, 47–59, 64–84, 89–128, 133–154, 159–180, 185–202, 207–256, 263–322, 329–356 Epigenetic controls, 263–322, 336, 338, 355 ERK MAPK signaling pathway, 57, 77–79

G G protein-coupled receptors (GPCRs), 82–84, 127, 207–256, 333–336, 340–342, 346, 351, 354, 355 G proteins, 207, 229–248

I Insulin signaling pathway, 47–59, 82–84, 128, 208 Ion channels, 207–256, 335, 336, 342, 343, 354, 355

J JNK MAPK signaling pathway, 72–76, 202

M Metabolism-related signaling pathways, 133–154 Molecular networks, 255, 329–356

N Neurotransmission-related molecular signals, 185–202

O Oxidative stress-related molecular signals, 33–42

P P38 MAPK signaling pathway, 64–66, 82–84 Protective response-related signaling pathways, 159–180

© The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 D. Wang, Toxicology at Environmentally Relevant Concentrations in Caenorhabditis elegans, https://doi.org/10.1007/978-981-16-6746-6

359

360 R Responses, 12, 21–24, 33–42, 47–59, 63–84, 89–128, 133–154, 159–180, 185–202, 207–256, 263–322, 329–356

Index T Toxicity assessment, 1, 3–5, 7–9, 11, 12, 33, 34, 40, 71, 162, 171, 252, 303