Methylmercury: Formation, Sources and Health Effects : Formation, Sources and Health Effects [1 ed.] 9781611222173, 9781617618383

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Copyright © 2011. Nova Science Publishers, Incorporated. All rights reserved. Methylmercury: Formation, Sources and Health Effects : Formation, Sources and Health Effects, Nova Science Publishers,

Copyright © 2011. Nova Science Publishers, Incorporated. All rights reserved. Methylmercury: Formation, Sources and Health Effects : Formation, Sources and Health Effects, Nova Science Publishers,

ENVIRONMENTAL SCIENCE, ENGINEERING AND TECHNOLOGY

METHYLMERCURY

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FORMATION, SOURCES AND HEALTH EFFECTS

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ENVIRONMENTAL HEALTH - PHYSICAL, CHEMICAL AND BIOLOGICAL FACTORS

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ENVIRONMENTAL SCIENCE, ENGINEERING AND TECHNOLOGY

METHYLMERCURY FORMATION, SOURCES AND HEALTH EFFECTS

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ANDREW P. CLAMPET EDITOR

Nova Science Publishers, Inc. New York

Methylmercury: Formation, Sources and Health Effects : Formation, Sources and Health Effects, Nova Science Publishers,

Copyright © 2012 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers’ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works.

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Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. Additional color graphics may be available in the e-book version of this book. LIBRARY OF CONGRESS CATALOGING-IN-PUBLICATION DATA Methylmercury : formation, sources, and health effects / editors, Andrew P. Clampet. p. cm. Includes bibliographical references and index. ,6%1H%RRN 1. Methylmercury--Environmental aspects. 2. Methylmercury--Toxicology. I. Clampet, Andrew P. II. Title. QD412.H6M38 2010 615.9'25663--dc22 2010034015

Published by Nova Science Publishers, Inc. © New York

Methylmercury: Formation, Sources and Health Effects : Formation, Sources and Health Effects, Nova Science Publishers,

CONTENTS i 

Preface Chapter 1

Chapter 2

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Chapter 3

Chapter 4

Chapter 5

The Role of Selenium in Mitigating Mercury Toxicity Lucia A. Seale, Nicholas V. Ralston and Marla J. Berry 

1 

Fish as a Dietary Source of Mercury and Methylmercury, Risks and Benefits Afnan Freije and Maysoon Awadh 

35 

Inexpensive Low-Cost Mercury Speciation by Hydride Generation Atomic Absorption Spectrometry after Ion Exchange Separation in a Fia System (Fia-Ie-Hg-Aas) A. Gredilla, J. Larreta, I. Martinez-Arkarazo, S. Fdez-Ortiz de Vallejuelo, G. Arana, J. C. Raposo, A. de Diego and J. M. Madariaga 

61 

Cathepsin-Dependent Neuronal Death Pathways Induced by Methylmercury Hiroshi Nakanishi and Zhou Wu 

81 

Not Only Concentrations Matter:Some Practical Considerations of In Vitro MeHg Toxicity Studies Jason Y. Chang  

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vi Chapter 6

Chapter 7

Chapter 8

Contents Methylmercury and Total Mercury Contamination in an Aquatic Ecosystem of Hg-Mining River in Guizhou, China Qingyang Liu  Mercury Methylation Versus Demethylation: Main Processes Involved Susana Río Segade, Teresa Dias and Elsa Ramalhosa  MeHg-Exposure through Seafood Consumption: Cholinergic Muscarinic Receptor as a Target of Toxicity and Potential Biomarker of CNS Effects Teresa Coccini, Elisa Roda, Anna F. Castoldi and Luigi Manzo 

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Index

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123 

167 

191 

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PREFACE Methylmercury (MeHg) is considered a major environmental pollutant that bioaccumulates in fish tissues in direct relation to their age and predatory status. Growing evidence indicates that MeHg toxicity induces a selenium deficiency disease since most of the hallmarks of severe selenium deficiency are also present in mercury toxicity cases. The main source of MeHg exposure in humans is through seafood ingestion. This book presents current research from across the globe in the study of methymercury, including the role of selenium in mitigating mercury toxicity; fish as a dietary source of mercury and methylmercury; as well as in vitro MeHg toxicity studies. Chapter 1 - Mercury is a naturally occurring element present throughout the environment that is used in industrial processes and commercial products. Mercury vapors released from coal-fueled and other heavy industries distribute globally, and deposition from the atmosphere is the dominant source of influx in the environment. Inorganic mercury is converted to the bioavailable and potentially toxic methylmercury by anaerobic organisms in aquatic systems including lakes, rivers, and the ocean. Methylmercury (MeHg) is considered a major environmental pollutant that bioaccumulates in fish tissues in direct relation to their age and predatory status. Accumulating evidence suggests that the MeHg sequesters the trace element selenium in the body, thus preventing it from performing its essential physiological roles. Supplemental dietary selenium counteracts the toxic effects of MeHg by replacing the selenium that has been sequestered. Selenium deficiency can lead to neurological, reproductive, and thyroid dysfunction. Growing evidence indicates MeHg toxicity induces a selenium deficiency disease since most of the hallmarks of severe selenium deficiency are also present in mercury toxicity cases. The main source of MeHg exposure in humans is through seafood ingestion. Top marine and freshwater predators (mostly fish and toothed cetaceans) tend to

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Andrew P. Clampet

accumulate large amounts of MeHg in their body and the ratio of mercury to selenium should be considered when evaluating toxicity. Ocean fish species usually exhibit rich selenium to mercury molar ratios, and differ greatly in their tissue accumulation of MeHg. Ongoing studies are investigating the mechanisms of MeHg toxicity and mechanisms of selenium-dependent protection. The mitigating effects of dietary selenium may result in revised seafood consumption recommendations. This review describes reasons why the risks of adverse effects of MeHg exposure are outweighed by the benefits of one to two servings of fish per week. Since unnecessary avoidance of fish consumption could result in significantly higher coronary disease deaths and suboptimal neural development in children, consideration of mercury-selenium interactions may assist regulatory agencies in their efforts to protect and improve public health. Chapter 2 - Mercury occurs naturally and distributed widely by human activities in the environment. Mercury is a heavy metal, an element of the earth with a molecular weight of 200.59g and an atomic number of 80. It is one of the few elements that are mobile, shiny, silver-white, odorless liquid at room temperatures, therefore it is called quicksilver. Mercury is used to make thermometers, diffusion pumps, barometers, mercury vapour lamps, mercury switches, pesticides, batteries, dental preparations, antifouling paints, pigments, and catalysts (Boening, 2000; Sanzo, 2001; UNEP, 2005). Mercury in the environment exists in three forms, elemental or metallic mercury (Hg0), inorganic and organic mercury compounds. Mercury (Hg) can be bound to other compounds as monovalent Hg(I) or divalent mercury (Hg(II) or Hg²+). Both inorganic and organic chemical compounds of mercury (II) are much more numerous than those of mercury (I) (Qian, 2001; UNEP, 2005; Virtanen et al., 2006). Chapter 3 - Ion exchange is presented as a simple, non-expensive technique for the effective separation of inorganic mercury (Hg2+) and methylmercury (CH3Hg+) in hydrochloric or chloride media. For such chemical conditions, the negatively charged HgCl42- is susceptible to be retained by anionic exchangers, while the non-charged CH3HgCl should pass through the resin with negligible retention. Further elution of the retained Hg2+ makes it possible the analysis of both species in a single injection of sample. Preliminary experiments to select the best retention and elution conditions were carried out in a batch mode by using a high precision syringe pump connected to a Pasteur pipette packed with the anionic resin Dowex M-41. Analysis of total mercury in the eluate was performed in a commercial FIA system by cold vapour atomic absorption spectrometry (FIA-HG-QFAAS).

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Preface

iii

The separation step was then implemented in the FIA system by including an Omnifit column packed with the resin immediately after the injection valve. The system was optimised to get maximum sensitivity in the analysis. The absolute detection limits estimated were 1.9 ng of Hg2+ and 0.8 ng of CH3Hg+. The utility of the method was confirmed by analysis of (a) synthetic mixtures of Hg2+ and CH3Hg+ in hydrochloric acid and (b) natural estuarine waters spiked with Hg2+ and CH3Hg+. Chapter 4 - An intoxication of methylmercury (MeHg), also known as Minamata disease, commonly results from the ingestion of MeHgcontaminated food. MeHg is easily absorbed from the intestine and therefore if transported to the central nervous system (CNS) through the blood-brain barrier. Among CNS neurons, the cerebellar granule neurons are especially vulnerable to MeHg intoxication. Exposure to MeHg during development results in an impaired migration of the cerebellar granule neurons and impaired synaptogenesis, thus leading to a disordering of the cerebellar architecture. On the other hand, exposure to MeHg during adulthood can also result in loss of cerebellar granule neurons from the internal granule cell layer, while Purkinje cells remain intact. MeHg has been also reported to affect functions of astrocytes and microglia. Despite the clinical importance, the understanding of the mechanism underlying MeHg-induced neuronal death is still limited. Cathepsins B and D, two major lysosomal proteases in the CNS, have been implicated in neuronal death.Cathepsins B and D are involved in neuronal death through different two pathways: intracellular proteolysis after their leakage from the lysosome to the cytosol of neurons and extracellular proteolysis after secreted from activated microglia.Here, the author summarize possible neuronal and microglial cathepsin-dependent neuronal death pathways induced by various stimuli including MeHg, and then the potential application of inhibitors for cathepsins on MeHg-induced pathological changes in the CNS. Chapter 5 - Tissue culture is a useful tool to study cellular events caused by a variety of toxic compounds. Results from different research groups, however, are difficult to compare when there are some differences in experimental conditions. By using the neurotoxic compound methylmercury (MeHg) as an example, this study demonstrated that the apparent MeHg cytotoxicity on rat C6 glioma cells could vary greatly even if the same MeHg concentration was used. Specifically, the apparent cytotoxicity was very dependent on cell density and the volume of the toxic agent used. An incubation of cells with 2.5 µM MeHg reduced cell viability to ~60% of control 24 hours later if cells were plated at 20,000 cells/well in a 96-well

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plate with 100 µl/well. Cultures with a lower density were much more sensitive to MeHg toxicity. At a fixed concentration, a larger volume of MeHg caused a significantly higher toxicity toward cells. Results of this study and those from other toxic agents were discussed from a methodological point of view. An experimental protocol using cell cultures to screen pharmacological agents against a cytotoxic agent was proposed based on a "commitment point" concept. Chapter 6 - Xiaxi River, located in Wansan, Guizhou province in China, serves as a recipient river of effluents from a large waste Hg-mining area. In order to evaluate the effects ofdischarging effluent on the mercury contamination of the local aquatic ecosystem, sediment cores, water samples and biota samples from Xiaxi River wereanalyzed for total mercury and methylmercury concentration. High concentrations of total mercury (T-Hg) concentrations were found in sediment cores (20.04-36.02μg g-1 dry weight). Ratio of methylmercury (MeHg) to T-Hg was less than 0.04 % insediments and ranged from 48% to 82% in biota samples. The highest level of T-H and MeHg were found in aquatic fish samples. The trophic levels (TLs) of biota samples were calculated by using stable isotopes methods. The relative contents of MeHg were significantly correlated with TLs (R2 = 0.9245) which further confirmed that MeHg can be biotransfered and biomagnified via food chain in this aquatic ecosystem. Chapter 7 - It is well known that mercury presents high toxicity, causing a great damage to the environment and living organisms; however, its properties depend on the mercury species present. Organomercury compounds, where methylmercury is included, cause more concern. Since 60-70’s, several methylation mechanisms are known. Generally, methylmercury can be formed naturally in the aquatic environment by two general pathways: chemical methylation (abiotic) and microbial (biotic)processes. At the same time, methylmercury can be also decomposed abiotically or by the action of several demethylating microbes, or demethylators, ranging from anaerobes to aerobes. Regarding the biotic methylmercury demethylation, two distinct vias - oxidative and reductive might be used by those microorganisms, differing in the final products obtained. In relation to the reductive processes, two pathways might occur. The first one involves the mercury resistance operon (mer) whereas the second one involves sulfide ions; however, the former is considered to be the most common pathway. Regarding the mer operon, some bacteria only carry on a narrow-spectrum operon (merA), being only able to reduce inorganic mercury (Hg(II)) to elemental mercury (Hg0). On the other hand, others beyond this

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Preface

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operon also carry on a broad-spectrum operon (merB). These microorganisms are able to decompose methylmercury to Hg0. Taking into account all of these processes, in the present work the most referred methylation and demethylation mechanisms found in aquatic environments are discussed, as well as the environmental factors that influence them. Factors related with the inorganic mercury/methylmercury availability and those that affect directly the activity of methylators and demethylators are also referred. Generally, the relationships encountered are complex and sometimes significant shifts on the microbial communities may be observed. These changes can alter the processes involving the mercury species, as well as the final products obtained. In conclusion, the abiotic factors and the type of microorganisms that are present in the environment, including their genetic patrimony, influence significantly the presence and the type of the mercury species. Furthermore, there are environmental factors, such as redox conditions, sulfides and organic matter that also affect the mercury dynamic and the equilibrium existents. Chapter 8 - Methylmercury (MeHg) highly bioaccumulates in marine and freshwater fish, biomagnifying in aquatic food webs. The dietary pathway, through the consumption of contaminated fish is the main source of human exposure. MeHg is efficiently absorbed and distributed throughout the body, including the CNS. Pervasive chronic low level Hg exposure, primarily through the widespread consumption of fish, is of concern because there is evidence that low-levels produce subtle neurodevelopmental disabilities. Cerebral cholinergic muscarinic receptors (MRs) are one of the sensitive biochemical CNS endpoints altered by MeHg-exposure. In vivo and in vitro experimental studies, aim at providing mechanistic insights involved in MeHginduced neurotoxicity and pointing out potential biomarkers of CNS changes, investigated the effects of different doses and timing of MeHg exposure on brain and lymphocytes MRs in mature and immature rats. Cerebral data: (i) 1 mg MeHg/kg/day, gestational day(GD)7-post natal(PD)7, enhanced MRs more in dams (87% and 60% in cerebellum and cerebral cortex, respectively) than in PD21 pups (0-50%) in accordance with the higher Hg levels detected in the adult brain (7-9 μg/g) than in offspring’s brain (1.5-2.8 μg/g); (ii) 0.5 mg/kg/day (GD7-PD21) MeHg affected total MRs and M1, M2, and M3 subtypes of the mature and immature rats, in a brainarea-, gender- and time-dependent fashion, also causing reactive gliosis in the cerebellum. Lymphocyte data: 1 mg MeHg/kg/day (GD7-PD7) enhanced MR density in both mature and immature rats, with a more pronounced effect in mothers (Bmax - [density] increase of 139% over control) than in offspring

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(49-73%) as compared with their respective controls in accordance with the higher Hg levels detected in the adult blood (11.3±2.2 μg/mL) than in pups (1.3±0.4 μg/mL). In summary, the lymphocytes data mirrored those found in brain MRs confirming the author previous results in non-pregnant adult rats that supported the predictive value of these peripheral receptors as surrogate markers of CNS changes. In vitro studies showed that MeHg was an almost equipotent inhibitor of the specific muscarinic antagonist 3H-QNB binding to rat and human lymphocyte MRs (IC50 range: 4.1-5.2 μM). Notably, IC50 values for MeHg to lymphocyte MRs were comparable to the Hg levels reached in blood (5-50 μM) of the PD21 rats exposed to MeHg. These findings show that MRs are a sensitive target of CNS in response to MeHg-exposure. The effect of MeHg in peripheral blood cells is in accordance with the data in brain, supporting the use of this peripheral endpoint as a potential biomarker of MeHg-induced cerebral muscarinic alterations. Furthermore, the similarity of MeHg IC50 binding data in human and animal peripheral tissues suggests the possible application of such biomarker to humans exposed to this environmental toxicant.

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Chapter 1

THE ROLE OF SELENIUM IN MITIGATING MERCURY TOXICITY Lucia A. Seale1, Nicholas V. Ralston2 and Marla J. Berry1 1

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Department of Cell and Molecular Biology, John A. Burns School of Medicine, University of Hawaii, Honolulu HI 96813 2 Energy and Environmental Research Center, University of North Dakota, Grand Forks, ND 58202

ABSTRACT Mercury is a naturally occurring element present throughout the environment that is used in industrial processes and commercial products. Mercury vapors released from coal-fueled and other heavy industries distribute globally, and deposition from the atmosphere is the dominant source of influx in the environment. Inorganic mercury is converted to the bioavailable and potentially toxic methylmercury by anaerobic organisms in aquatic systems including lakes, rivers, and the ocean. Methylmercury (MeHg) is considered a major environmental pollutant that bioaccumulates in fish tissues in direct relation to their age and predatory status. Accumulating evidence suggests that the MeHg sequesters the trace element selenium in the body, thus preventing it from performing its essential physiological roles. Supplemental dietary selenium counteracts the toxic effects of MeHg by replacing the selenium that has been sequestered. Selenium deficiency can lead to neurological, reproductive, and thyroid dysfunction. Growing evidence indicates MeHg toxicity

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2

Lucia A. Seale, Nicholas V. Ralston and Marla J. Berry induces a selenium deficiency disease since most of the hallmarks of severe selenium deficiency are also present in mercury toxicity cases. The main source of MeHg exposure in humans is through seafood ingestion. Top marine and freshwater predators (mostly fish and toothed cetaceans) tend to accumulate large amounts of MeHg in their body and the ratio of mercury to selenium should be considered when evaluating toxicity. Ocean fish species usually exhibit rich selenium to mercury molar ratios, and differ greatly in their tissue accumulation of MeHg. Ongoing studies are investigating the mechanisms of MeHg toxicity and mechanisms of selenium-dependent protection. The mitigating effects of dietary selenium may result in revised seafood consumption recommendations. This review describes reasons why the risks of adverse effects of MeHg exposure are outweighed by the benefits of one to two servings of fish per week. Since unnecessary avoidance of fish consumption could result in significantly higher coronary disease deaths and suboptimal neural development in children, consideration of mercury-selenium interactions may assist regulatory agencies in their efforts to protect and improve public health.

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INTRODUCTION "Mad as a hatter" is a well known English expression popularized by the famous children's book "Alice’s Adventures in Wonderland" by Lewis Carroll. Although Carroll's nonsensical (and fantastic) story contributed to the dissemination of this expression, its historical origin is less glamorous. In fact, it derives from mercury toxicity in the work environment. During the 18th and 19th centuries in Europe, mercuric nitrate was used to remove fur from pelts in the making of felt hats, a process called “carroting”. Chronic, prolonged exposure to and inhalation of mercury vapor resulted in "hatter's shakes", a form of neurological damage producing symptoms such as tremors similar to Parkinson's Disease, as well as mental confusion, insomnia, hallucinations and distorted vision. These symptoms are still known today as "Mad Hatter's Syndrome" (reviewed by Wedeen 1989). However, hatters were not the first working-class exposed to mercury toxicity. According to archeological records, the metal was used by Chinese before 2000 BC, found in tombs in Ancient Egypt, used as part of cosmetic applications in Ancient Greece, used as an ingredient of “elixirs” prepared by alchemists during the Dark and Middle Ages (reviewed by Krebs 2006) and was exploited in large amounts to harvest gold and silver ores, and in the mirror making industry during the Renaissance. Mercury was also believed to

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The Role of Selenium in Mitigating Mercury Toxicity

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cure syphilis, tuberculosis and constipation in the 1800’s. It was ingested in a variety of formulations including "blue mass", a pill that contained more than a third elemental mercury or mercury compounds (detailed in Hirschhorn et al. 2001). The first medical description that this heavy metal would cause neurological problems in humans was published in 1823 when physician Sir William Burnett reported to the Royal Society the massive poisoning that had happened on board the Triumph and Phipps, two British ships that salvaged a wrecked Spanish vessel transporting mercury in 1810 (Burnett 1823). The mercury storage containers were destroyed and mercury filled the ship, mixing with bread and other provisions. Mercury vapor was reported to be in all contained decks. Soon thereafter a third of the crewmembers developed extreme salivation, tremor, breathing difficulties, partial paralysis and tooth loss - there was no report of behavioral problems. All animals aboard died (the historical episode is reviewed in Doherty 2004). In the mid 20th century, incidents in Japan brought mercury toxicity to the headlines. From the 1930’s through the 1960’s, an industrial plant discharged tons of methylmercury (MeHg) waste into the waters of Minamata Bay, contaminating the fish that lived in this small, enclosed water body, and ultimately the humans that consumed them. Although severe toxicity was noted in numerous adults, unborn children were affected far more severely. Significant birth anomalies and neurobehavioral disturbances were verified in more than 2,000 people, some of these cases being lethal, while other patients had developed permanent disabilities. Milder cases of the syndrome were characterized by motor control and sensation disturbances. Patients who died displayed severe brain atrophy, cerebral and cerebellar lesions, and pathological changes in cell architecture (reviewed by Ralston 2008). Unfortunately, some patients did not exhibit neurotoxic symptoms of mercury poisoning until up to 5 years after the exposure occurred. This silent latency period, which is unique to MeHg poisoning (Weiss et al. 2002), made it difficult for local physicians to properly connect the symptoms of their disabled patients with the MeHg contamination event. Following this tragic incident, mercury toxicity was rechristened as "Minamata disease". Mercury thus became a more feared chemical and a number of policies and rules were implemented on its handling, use and discharge in order to decrease human exposure. In the United States, specific federal laws, such as the Mercury-containing and Rechargeable Batteries Management Act of 1996 and the Mercury Export Ban Act of 2008, were created to minimize discharge of mercury in the environment. Besides these, the more general Clean Air Act, the Clean Water Act, the Safe Drinking Water Act and the Resource

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Lucia A. Seale, Nicholas V. Ralston and Marla J. Berry

Conservation and Recovery Act all have chapters ruling about limiting mercury levels in the environment. The Environmental Protection Agency (EPA) is responsible for issuing the regulatory rules to these acts and reinforcing compliance. On the other end, the Food and Drug Administration (FDA) is responsible for regulating and monitoring mercury levels on seafood, cosmetics and pharmaceutical drugs and vaccines. Although mercury compounds are gradually being reduced from general industrial use, they are still a main component in the manufacturing of thermometers, dental amalgam, fluorescent lamps and certain skin whitening products. This chapter attempts to grasp the recent ideas on mercury toxicity and examine evidence that mercury’s effect on another trace element, selenium (Se), has pivotal roles in the molecular mechanism of toxicity as well as in mitigating certain aspects of mercury toxicity.

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MERCURY BIOGEOCHEMISTRY AND HUMAN EXPOSURES Mercury is a naturally occurring heavy metal found in the earth's crust, primarily in the form of mercuric sulfide (called cinnabar) mixed with other minerals in volcanic rocks. Volcanic activities are therefore the original source of virtually all mercury currently active in the biogeochemical cycle. Most coal reserves were formed during periods of volcanic activity as a result of plant and other organic materials deposited in wetlands that eventually petrified. This petrified coal accumulated mercury deposited over millennia, and thus contains significant amounts of cinnabar that was originally MeHg bound to cysteine and other thiol groups in the plants at the time these materials became buried. Today, a major portion of the elemental mercury that is released into the atmosphere and throughout the global environment is a byproduct of coal burning at thermoelectric power plants. Approximately 40% of mercury emitted by the United States from human sources derives from coal-fired power plants. Since the Industrial Revolution, the level of mercury cycling in the atmosphere has risen considerably, and although power plants in most countries have reduced the amount of mercury they release, atmospheric levels have not diminished as much as had been hoped because the number of coal-fired power plants coming on line in developing countries is increasing faster than mitigation efforts in the developed world is decreasing releases. Mercury that had been fossilized in limestone deposits is released in substantial amounts when these deposits are heated to create concrete. A further major source of mercury release involves the elemental mercury used

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The Role of Selenium in Mitigating Mercury Toxicity

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in gold and silver mining. When mining is performed under poorly controlled conditions, substantial amounts of mercury become dispersed in streams close to the sources of extraction. This directly contaminates the local water bodies and contributes to atmospheric mercury loads that are globally dispersed throughout the environment. It is known that aquatic anaerobic bacteria methylate mercury compounds discharged in the environment and convert them into the highly neurotoxic MeHg. Methylmercury moves from the bacteria to the tissues of the invertebrates that consume them, is well absorbed at each level of the food web and results in bioaccummulation in aquatic systems, reaching particularly high levels in large predators like sharks, toothed cetaceans and large or long lived varieties of fish. The concentration levels of MeHg in these animals can reach a million times higher than the water surrounding them. Ingestion of meat from large predatory fish is the primary source of human exposure to MeHg (Wiener and Suchanek 2008). In 2001 the EPA determined the criterion for MeHg limits in fish and shellfish as 0.3 mg/kg tissue wet weight, followed by the FDA in its control guidance document for fish and fisheries products, which limited MeHg to 1 ppm in edible portions of fish and listed as potential safety hazards the following species: bonito (Sarda sarda), halibut (Hippoglossus sp.), Spanish mackerel (Scomberomorus maculatus), king mackerel (Scomberomorus cavalla), marlin (Makaira sp. and Tetrapturus sp.), sharks (superorder Selachimorpha), swordfish (Xiphias gladius) and bluefin tuna (Thunnus maccoyii, Thunnus thynnus and Thunnus orientalis). A recent article suggested the inclusion of bigeye tuna (Thunnus obesus) on mercury advisories as well (Lowenstein et al. 2010). In addition to ingestion, the skin can also readily absorb MeHg. In 1996, there was an outbreak of mercury poisoning in Arizona, California, New Mexico and Texas among MexicanAmerican immigrants due to the use of a skin whitening cream made in Mexico containing “calomel” or mercurious chloride in a concentration of 6 to 10% by weight. Eighty-seven percent of women exposed to mercury in this beauty product showed mercury levels in urine greater than 20 µg/l, which is considered elevated. This incident led to tightening and enforcement of regulations for mercury in cosmetics by the FDA (Centers for Disease Control 1996). The specific mechanisms by which mercury exerts its effects on the human body are still under investigation. The various mercury species are known to elicit different symptoms of toxicity, but these appear to be largely related to distinctions in how these forms distribute in tissues rather than implying differences in molecular mechanisms of intoxication. It has been

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shown that dietary MeHg is almost completely absorbed by the gastrointestinal tract and quickly reaches the bloodstream. Approximately 95% of mercury is taken up by red blood cells and redistributed throughout the body in a few days (Myers and Davidson 1998). The brain is the main target organ, and the developing fetal brain is particularly sensitive. Some mercury compounds, such as MeHg and mercury vapor readily cross the placental barrier (Yoshida 2002). It has been shown that the molecular form present in fish (Harris et al. 2003) is bound to cysteine (Cys). This conjugate (MeHg-Cys) is readily transported from the mother's bloodstream into and across the placenta into the fetal bloodstream. The form present in fetal blood is also expected to be primarily MeHg-Cys and this form rapidly reaches the developing fetal brain, the primary target tissue associated with development of symptoms of MeHg toxicity. MeHg-Cys accumulates in proteins, either in place of methionine (Met) during protein synthesis, or as a result MeHg transfer to Cys moieties of proteins, including fetal hemoglobin. Therefore, because the concentration of hemoglobin is up to 50% higher in fetal blood, it is not surprising that MeHg contents of fetal blood can be 25% higher than in the mother (Amin-Zaki et al. 1976). Once in the fetal bloodstream, MeHg-Cys is taken up by fetal cells via the neutral amino acid carrier system and rapidly reaches the developing central nervous system of the fetus (Kajiwara et al. 1996). Upon crossing the blood-brain barrierin the mammalian brain, MeHg-Cys and elemental mercury (Hg0) are differentially distributed through gray and white matter of the brain, and trigger diverse neurotoxic symptoms. In the specific case of MeHg, early evidence pointed towards this compound causing disruption of intracellular calcium pool regulation and calcium permeability through the plasma membrane. Chronic MeHg exposure results in ultrastructural changes and accumulation of MeHg inside the mitochondria, inhibiting several mitochondrial enzymes and depolarizing mitochondrial membranes, thus decreasing ATP synthesis (Atchison and Hare 1994). Mitochondrial damage from oxidative stress is probably one of the earliest signs of mercury toxicity. Multiple brain regions are affected by MeHg intoxication, including but not limited to cerebellum, parietal cortex and occipital cortex (Sakamoto et al. 1998). Mercury has been shown to interfere with some neurotransmitters in the brain, such as gamma-aminobutyric acid (GABA) and glutamate. It binds the external part of the GABA receptor, leading to an increase in GABA-induced current (Huang and Narahashi 1996). In cell culture models, mercury chloride seems to prevent astrocytes, the first layer of cells found after crossing the

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blood-brain barrier, from clearing glutamate from the synapse, decreasing the activity of glutamate synthetase. However, MeHg is apparently not involved in similar inhibitory processes (Fitsanakis and Aschner 2005), at least in this cell culture model. Taken together, these effects on amino acid metabolism and regulation are responsible for at least some of the neurotoxic effects of mercury. A study performed with a yeast gene library identified L-glutamine-Dfructose-6-phosphate amidotransferase (GFAT) and ubiquitin transferase (Ubc3) as genes conferring protection against MeHg toxicity in yeast cells. The authors suggest activation of the ubiquitination pathway as a protective system to prevent MeHg toxicity through degradation of proteins bound to MeHg (Naganuma et al. 2002). However, this defense mechanism seems to be more involved in clearance of mercury than its action in the cell and presently there are no data in mammals corroborating such role. Since mercury is thermodynamically stable, but kinetically promiscuous, it readily exchanges associations among low molecular weight thiols such as cysteine, homocysteine, and glutathione, acting as transporters and/or distributors of mercury throughout the body. The majority of mercury in the body is transported bound to these thiol compounds or thiol groups of proteins like albumin and metallothionein, and the binding and dissociation rates of these complexes are primarily responsible for the dynamics of mercury transport (Clarkson 2002). The MeHg-Cys adduct biochemically resembles Met. Because of this molecular mimicry, MeHg-Cys is taken up nonspecifically by the L-type large neutral amino acid transporter (SimmonsWillis et al. 2002), the same mediator as for placental mercury transport. At low levels, MeHg-Cys enters the Met cycle without significant pathological effects; but at high levels it does induce toxic effects. This molecular mimicry is certainly involved in MeHg transport between tissue compartments, and may be prominently involved in aspects of a major molecular mechanism of mercury toxicity discussed below. It has been also observed that there is a negative correlation between endogenous concentrations of thiol-containing molecules in the cell and mercury toxicity (Divine et al. 1999), but the effects are not as pronounced as the negative correlations between cellular selenium contents and mercury toxicity (Ralston et al. 2008). Glutathione, one of the major antioxidants involved in protection against strong oxidants and free radicals, is the most abundant thiol-containing molecule in mammalian cells. Binding of MeHg to glutathione decreases glutathione intracellular concentration (Lee et al. (2001); Queiroz et al. (1998) and Zalups (2000) reviewed by Patrick (2002)), thus increasing downstream

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oxidative stress in the system. Lower levels of glutathione peroxidase activity have been reported in cells exposed to MeHg (Kromidas et al. 1990) and glutathione depletion may play an active role in neurological damage, as for the pathogenesis of Parkinson's disease, for example (Martin and Teismann 2009). By direct binding, glutathione also scavenges mercury compounds in the blood thus preventing mercury from entering tissues and cells. Glutathione-MeHg complexes are a major form of mercury transport and elimination through urine and bile in the body (Ballatori and Clarkson 1983). However, because of the slow clearance of this and related forms, it has a halflife on the order of months, clearly indicating why MeHg demonstrates such long-term retention in the body. Glutathione depletion in the blood decreases the available pool for proper antioxidant responses from the body. Because of the strong binding affinity of the sulfur of glutathione for mercury, glutathione has been prescribed clinically for patients with mercury exposure symptoms. However, Aposhian et al. (2003) have challenged this practice, because they found glutathione was not able to reduce mercury concentrations in brain and kidney cells in rats exposed to elemental mercury. The expectation that selenium would first need to diminish the distribution of mercury into vulnerable tissues in order to counteract MeHg toxicity has been a common, but mistaken belief. This belief was based on preliminary assumptions that the molecular mechanism of mercury toxicity involved pseudo-first order reaction kinetics. As will be discussed below, since that preliminary assumption was incorrect, reducing concentrations of mercury in vital tissues is not a precondition for preventing or alleviating its toxicity. However, intracellular glutathione, thioredoxin and other prominent thiomolecules will clearly be molecular targets for mercury binding. Therefore, the intracellular concentrations of these and other thiol containing molecular species are likely to be prominently involved in the molecular mechanism of MeHg intoxication. However, glutathione's contributions in mercury toxicity issues are likely to be indirect rather than proximal.

SELENIUM NUTRITION AND PHYSIOLOGY Selenium is a trace element and an essential nutrient, required for life in a wide range of species, including vertebrates. It was originally considered a toxin, but in the 1950's its vital importance as an essential antioxidant nutrient was first reported (reviewed by Small-Howard and Berry (2005) and Burk and Hill (2005)). Selenium is generally acquired through the diet and is most

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abundan nt in Brazil nuts and seaafood (USDA A National Nutrient N Databbase 2005). The T forms off selenium fouund in the dieet are mainly the amino acids, selenocy ysteine (Sec) and selenom methionine (SeeMet), althouggh other orgaanic and ino organic forms can also be detected in minor m amounnts. All formss of selenium m must be brooken down to the inorganicc form, selenidde, in order too be used in protein syntheesis (see Figurre 2).

Figure 1.The sulfur andd selenium-contaaining amino accids. Although the thio- and seleno-am mino acids are similar in structure, they differr in physiologiccal abundance, reactivity y, and metaboliic roles. Although Met and SeM Met are structurrally and metaboliically analogous, they differ inn having either sulfur s or Se bouund to the γ-carbbon of their side s chains, Meet and SeMet aree incorporated into i proteins annd peptides nonspeciifically in amouunts that reflect their tissue abuundance. Howevver, upon degradattion SeMet is a source of bioloogically essentiaal Se that is needed for de novoo synthesiss of Sec. The molecular m structuures of Cys andd Sec are also annalogues, but Seec is exclussively inserted into i genetically unique and funnctionally elite selenoprotein s families.. (From Ralstonn and Raymond 2010; figure ussed with permisssion).

Durring amino acid synthesis, plants p generally employ selenium and sulfur nonspeccifically in thheir metabolicc processes. Thus, T plants form f methionnine (Met) and a selenomethhionine SeMeet (see Figure 1) in amountts that reflect the relative sulfur and selenium s conccentrations off the soils in which they are i downstream m metabolites are grown. The metabolic processes off SeMet and its t those of Meet in both plannts and animaals (see Figuree 2). generallly analogous to Howeveer, once incorrporated in animal proteins in place of Met, M the seleniium of SeMet is released when the amiino acid is deggraded, and frrom this pointt on a selenium part p company. Organisms frrom the metabolic pathwaays of sulfur and yotes to mann have evolvved a uniquee cotranslatioonal pathway to prokary incorporate selenium into formatioon of the aminno acid Sec. Inn eukaryotes, Sec c whichh typically speecify terminatiion. is insertted via recodiing of UGA codons, The reccoding mechannism is fostereed by the pressence of cis annd trans elemeents in selen noprotein mR RNAs: the Seec Insertion Sequence (SE ECIS) in thee 3'

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untranslated region of mRNAs, a tRNA for Sec, the SECIS-binding protein, SBP2, and the Sec-specific elongation factor, EFsec (Low and Berry 1996).

Figure 2.General scheme of vertebrate selenium metabolism, interactions between MeHg-Cys and Sec, potential distribution of MeHg-Cys and MeHg-Sec into protein biosynthetic cycles and eventual disposition.(From Ralston and Raymond, 2010; figure used with permission).

Selenocysteine is required for the proper physiological and biochemical activity of most of the 25 selenoproteins identified in humans (see Table 1). Although Sec structurally resembles Cys, selenol is largely deprotonated at physiological pH, resulting in higher reactivity than the protonated sulfur on Cys. This difference has been shown to be responsible for increases in enzyme activity mediated by the presence of Sec. Many of the selenoenzymes whose functions have been characterized are involved in oxidative stress defense or detoxification (Rayman 2000). In humans, five selenoprotein isoforms of glutathione peroxidases are responsible for reducing hydroperoxides and lipid and phospholipid hydroperoxides, thus avoiding the propagation of free radicals and reactive oxygen species that could ultimately damage cell viability.

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Table 1. Mammalian selenoprotein/selenoenzyme functions1

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Selenoenzymes with predominantly antioxidant functions: GPx1 Detoxifies peroxides in aqueous compartment of cytosol GPx2 Expressed in cytosol of liver and tissues of the digestive system GPx3 Primarily synthesized in kidney; active in plasma Se transport to other tissues GPx4 Prevents and reverses oxidative damage to lipids in brain, testis and other tissues TrxR1 Interacts with and/or recycles Trx and most other redox regulating metabolic pathways TrxR2 Located in mitochondria, controls and regulates redox state of its local milieu TGR Reduces glutathione disulfide, specific physiological functions undefined SelM Expressed in various tissues with increased levels in the brain, involved in calcium handling SelK May participate in detoxifying mechanisms in the endoplasmic reticulum Sel15 An oxidoreductase that may assist in disulfide formation and protein folding SelN Mutational defects associated with congenital muscular dystrophy and other disorders SelS May participate in detoxifying mechanisms in the endoplasmic reticulum SelW Expressed in a variety of tissues, may regulate redox state of 14-3-3 proteins Selenoenzymes with endocrine functions: DIO1 Activates thyroid hormone, converts T4 (thyroxine) into T3 DIO2 Regulates thyroid hormone statuslocally, activates or inactivates T3 DIO3 Deactivates thyroid hormone in brain, placenta, and pregnant uterus, important in fetus Selenoenzymes with other physiological functions: SPS2 Catalyzes formation of Se-phosphates required for synthesis of Sec to all selenoproteins MsrB1 Repairs oxidatively damaged methionine (R-sulfoxides) SelP Primary Se transporter in plasma (10 Sec/molecule in humans) 1

Information presented in this table was compiled from: Aachmann et al. 2007; Allan et al. 1999; Dikiy et al. 2007; Gao et al. 2007; Gereben et al. 2008; Gladyshev et al. 2004; Gromer, 2005; Labunskyy et al. 2009; Linster and Van Schaftingen, 2007; Lu, et al. 2006; Moghadaszadeh and Beggs 2006; Reeves et al., 2010; Shchedrina et al. 2010.

Glutathione is an essential cofactor in these reactions. The three selenoprotein isoforms of thioredoxin reductases are involved in regeneration

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of many intracellular antioxidants that are required to maintain intracellular redox states, reduce nucleotides that are required for DNA synthesis, and have prominent roles in regulation of gene expression by redox control of transcription factors (Allan et al. (1999) reviewed by Reeves and Hoffmann (2009)). Although their functions are still largely unknown, it has been reported that Selenoprotein K (Lu et al. 2006), Selenoprotein M (Reeves et al. 2010), Selenoprotein N (Arbogast and Ferreiro 2010), Selenoprotein S (Gao et al. 2007), and Selenoprotein 15 (Labunskyy et al. 2009) all participate in detoxifying mechanisms associated with endoplasmic reticulum redox systems (Shchedrina et al. 2010). In addition, three isoforms of iodothyronine selenodeiodinases control thyroid hormone homeostasis via activation of thyroid hormone globally (type 1 deiodinase) and locally (type 2 deiodinase) throughout the body, as well as by inactivating excess circulating thyroid hormone (type 3 deiodinase). Although these are neither antioxidant nor detoxifying reactions, deiodination of thyroid hormones requires thiol cofactors to regenerate the active enzymes (Gereben et al. 2008). Thus, the presence of Sec at the active sites of these enzymes is required to efficiently perform these reactions. Selenium is transported through the body via the circulation bound to plasma proteins and as Sec residues incorporated into the primary sequence of selenoprotein P (SelP), a selenoprotein synthesized in the liver and distributed to the plasma. Selenoprotein P is unique due to its multiple Sec residues per protein molecule: one Sec in the amino terminal region of the protein and a cluster of Sec's in the middle to carboxyterminal region (Burk and Hill 2005). The number of Sec's in SelP sequences varies according to species, ranging from 0 in one Xenopus selenoprotein P isoform to 28 in sea urchins (Lobanov et al. 2008), and according to dietary status, with expression of the full-length protein proportional to dietary selenium intakes. This protein is thought to be a tissue selective transporter of selenium to specific tissues, thus serving as means of homeostatic redistribution of tissue selenium reserves to vital tissues. Neurological dysfunctions and male sterility (Olson et al. 2005) presented by SelP knockout mice (Schomburg et al. (2003) and Hill et al. (2004)) highlight the crucial importance of maintaining selenium homeostasis in the brain and neuroendocrine tissues, which seem to be hierarchically protected against low circulating selenium levels. Dietary selenium deficiency affects the selenium concentrations of these tissues the least; however, when extreme measures such as selective sequestration or genetic knockouts are used to induce unusually severe selenium deficiencies, the brain and neuroendocrine tissues are the most severely affected. The mechanisms of selenium uptake and

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homeostatic redistribution by tissues are under investigation, with recent reports indicating binding of SelP to a protein called megalin in the proximal tubule cells of the kidney (Olson et al. 2008) and to apolipoprotein E receptor2 in the brain (Burk et al. 2007) and in the testis (Olson et al. 2007). Along with selenium delivery, cells have evolved mechanisms to recycle Sec when selenium is limiting, in order to maintain essential redox functions. The recycling system utilizes selenocysteine lyase (Sec lyase), an enzyme that degrades Se into selenide and alanine using pyridoxal phosphate as a cofactor. Selenide can then be reutilized in the de novo Sec biosynthesis pathway immediately prior to its incorporation into newly formed selenoproteins (Lacourciere and Stadtman 2001). Although recycling appears to be an evolutionary adaptation to circumvent or delay onset of the most severe adverse effects that would otherwise occur secondary to limited dietary selenium intakes, it is well established that a low selenium diet can still be harmful to many aspects of health (Akbaraly et al. (2005) and Kupka et al. (2005)). When normal mechanisms of selenium homeostasis are handicapped by genetic knockouts of SelP, limited dietary selenium intakes progressively become accompanied by increasingly severe neurological disturbances including motor and sensory dysfunction and eventually result in death (Burk et al. (2007) and Hill et al. (2004)). Selenium deficiency per se is a pathology of rare occurrence. Keshan Disease, an endemic virally induced cardiomyopathy that can be fatal, is associated with very low levels of dietary selenium intakes and first reported in a selenium poor region of rural China. In this region, local crops are raised in soil that does not contain sufficient amounts of selenium to be absorbed by the plants utilized as food crops. The dietary selenium intakes considered normal vary according to the region and population, with some populations in areas with low soil selenium maintaining normal intakes due to other aspects of diet, such as seafood ingestion (reviewed by Rayman 2008). However, decreased dietary selenium intakes have been noted in recent decades and are a human health concern in certain parts of Europe, including the United Kingdom (Jackson et al. 2004). Sustained selenium deficiency can also impair male reproduction. The testis is hierarchically protected from low selenium levels, and is rather affected only when deprivation becomes chronic. The role of selenium in sperm structural abnormalities and motility has long been known in rats (Wu et al. 1979) and the mechanism behind these impairments has in recent years been under intense investigation. The selenoprotein glutathione peroxidase 4 is highly expressed in the testis and ultimately was identified as the major

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structural component of the mitochondrial capsule (Ursini et al. 1999) and the only major antioxidant enzyme capable of reducing phospholipid hydroperoxides within membrane systems. As part of the sperm structure, glutathione peroxidase 4 cross-links with itself and other proteins, fostering formation of selenodisulfide bridges with glutathione molecules. When selenium levels are adequate, a second glutathione molecule splits the bridge releasing the enzyme as a selenolate form. Upon selenium deprivation, the enzyme is more nonspecific, reacting to different thiols as well which ultimately disrupts the balance in oxidative status of the mature spermatozoa and leads to deleterious effects. The specific mechanism has recently been reviewed in detail (Flohé 2007). More interestingly, the glutathione peroxidase 4 knockout mouse proved to be prenatally lethal, adding robustness to the hypothesis of the fundamental importance of this enzyme not only to reproduction, but ultimately to life (Yant et al. 2003). As the development of SelP knockout mouse showed, the lack of this selenium transporter protein also leads to male infertility. Sperm development is impaired by structural changes, such as flagellar defects and a hairpin-like formation at the mid-piece-piece junction that results in bending of the sperm and ultimately blunts its normal motile function. Although viable, the animal presents sperm abnormalities that mimic the ones presented by wild type animals deprived of selenium on their diets (Olson et al. 2005). Strikingly, dietary selenium supplementation of the SelP knockout mouse does not reverse defect in sperm development, nor does it restore fertility. Besides reproductive and neurological impairment, it is also known that selenium deficiency can worsen certain disease conditions and impair the immune system (Finley (2005) and Hoffmann and Berry (2008)). It is very interesting to note that health damages are magnified if selenium deficiency is accompanied by other nutrient deficiencies (e.g. iodine or vitamin E) or additional metabolic challenges such as mercury exposure.

MOLECULAR MECHANISMS OF MERCURY-SELENIUM INTERACTIONS As stated above, most bioavailable forms of selenium can be metabolized in the body and incorporated into selenoproteins as the amino acid Sec. Mercury has been shown since the 1970's to bind selenium (reviewed by Burk and Hill 2005). In fact, mercury's affinity for selenide is a million times greater

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than its affinity for sulfide (Dyrssen and Wedborg 1991). Since selenide is produced during each cycle of Sec synthesis (Figure 2), while no similar pathway exists for intracellular sulfide, the vulnerability of selenium is markedly greater than would otherwise be expected. A major reason for this difference is the pKa of the side chains of Cys and Sec. The side chain selenol of Sec has a pKa of ~5.5 (Tan et al. 1988) whereas the pKa of the cysteine thiol is 8.30 (Danehy et al. 1968). Therefore, the selenol side chain is fully ionized in the normal physiological pH range and fully active as a nucleophile, but the thiol of cysteine is predominantly protonated and thus uncharged. The biochemical consequences of this difference follow the expected trends since analogues of natural selenoproteins have significantly reduced catalytic activity (2 to 3 orders of magnitude) when Sec is substituted with Cys (Köhrle et al. 2000). With the much greater quantities of Cys and thiols compared to Sec and other selenol forms found in the body, it is true that Cys is the main carrier of MeHg. However, the difference in binding affinity between Cys and Sec favors an equilibrium distribution with the selenium of Sec. Whenever the mercury of MeHg-Cys encounters an available selenium of Sec, the MeHg will preferentially exchange its association to bind with Sec (see Figure 2).

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Silencing of Selenium (SOS-1) By biochemical definition, an irreversible inhibitor is any interfering molecule that forms a covalent bond with the catalytic moiety of an enzyme active site. Since the mercury of MeHg-Cys exchanges from the sulfur of Cys to form a covalent bond with the selenium of Sec, it is by definition an irreversible inhibitor. Without the active selenium of the Sec present, the activity of that enzyme molecule is terminated. Although in many cases, the term irreversible is not necessarily intended to mean a permanent bond has formed, in the case of MeHg binding, the formation of the Hg-Se linkage is so stable that it truly is permanent on biologic, and even geologic, time scales. The "silencing" of the selenium of Sec will not only terminate essential enzyme activities. It will also change the charged sites on selenium transport molecules such as selenoprotein P and glutathione peroxidases. Since charge distributions are important aspects of molecular recognition of protein by selective uptake receptors, replacement of charged Sec with uncharged MeHgSec would inevitably diminish recognition and uptake of selenium transport molecules. Therefore, high MeHg exposures decrease selenoenzyme activities and simultaneously, disrupt selenium homeostasis. Remarkably adept

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homeostatic regulatory mechanisms, normally direct delivery of selenium to preferentially supplied tissues such as placenta, brain, and neuroendocrine tissues that have specific receptors for selective uptake of selenoprotein P. However, when binding by MeHg silences the charged sites on its molecular surface, selenoprotein P will no longer be recognized and delivered to the tissues that require selenium. Among the selenoproteins, the glutathione peroxidases (Chen et al. 2006), selenoprotein P (Chen et al. (2006) and Falnoga et al. (2002)) and selenoprotein W (Kim et al. 2005) were pinpointed as those most probably involved in the mercury detoxification and/or neuronal protection mechanisms against mercury in mammals. Interestingly, these selenoproteins appear to have enhanced binding affinities for mercury compounds in the presence of glutathione. Selenoprotein W mRNA was downregulated by treatment with MeHg in SH-SY5Y neuronal cells, and this regulation was dependent on depletion of intracellular glutathione (Kim et al. 2005). In addition, selenoprotein W was shown to be S-glutathionylated on a cysteine residue in mammals (Whanger 2009). Glutathione peroxidase isoforms are all dependent on glutathione as a cofactor for the peroxidation reaction to occur. The activity of these enzymes is significantly inhibited in the presence of MeHg in SHSY5Y neuronal cells and in brains of mice exposed to MeHg via contamination of drinking water (Franco et al. 2009). The inhibition of glutathione peroxidases renders cells susceptible to oxidative stress, a wellknown mercury toxicity target mechanism. Selenoprotein P, the plasma protein with multiple selenocysteine residues, can bind selectively in an equimolar ratio to mercury-selenium complexes in the presence of glutathione (Yoneda and Suzuki (1997) and Suzuki et al. (1998)). It has been suggested that, because of the specific characteristics required for mercury binding, selenoprotein P is the preferential carrier of mercury-selenium compounds in low, chronic mercury exposure situations, such as are found in marine mammals and mercury mine workers. Enhanced vulnerability of the Sec of selenoenzyme active sites would be expected to occur because of the extensive interactions between thiomolecules and selenoenzymes. When highly exposed to MeHg, the cysteine's of glutathione, thioredoxin, and other thiomolecules become increasingly loaded with MeHg. Since many of the characterized selenoenzymes are actively involved with reducing oxidized thiomolecules such as glutathione, thioredoxin, they will naturally bring the Cys portion of these molecules into close proximity with the enzymes active site Sec. However, when the thiomolecule, e.g., thioredoxin, enters the active site with a MeHg bound to its

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Cys, it potentiates the transfer of MeHg from the Cys of thioredoxin to the Sec of the enzyme. Unfortunately, this natural feature of selenoenzyme activities would greatly enhance silencing of Sec at their active sites.

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Sequestration of Selenium (SOS-2) The selenium that becomes bound to mercury when MeHg transfers from Cys to Sec is permanently retired from participation in future cycles of Sec synthesis as shown in Figure 2. If the selenium resources of the host, and therefore those of the affected cells and their tissues are plentiful, additional selenium is rapidly made available to replace the quantities lost to mercury binding. However, when selenium status is low, either because of poor dietary resources or because of prior depletion from consuming foods with high MeHg-Cys contents, further MeHg-Cys exposures can overwhelm the cells ability to replace the selenium lost to MeHg binding. The MeHg-Sec that is formed may become involved in protein synthesis cycles, and undergo multiple passages of protein synthesis and degradation. However, this would not release the selenium that is bound to the mercury of MeHg. Eventually, MeHg-Sec is degraded into inorganic HgSe, a form that is known to accumulate in cellular lysosomes of tissues of marine mammals (Huggins et al. 2009). Clearly, if all of the cell's selenium resources were sequestered in association with mercury, either as MeHg-Sec cycling in tissue proteins, or as inorganic HgSe particulates localized in cellular lysosomes, none would be available to supply the needs of de novo Sec synthesis in accompaniment with selenoprotein formation. This would naturally result in loss of all these essential selenoenzyme activities in tissues that cannot live without them. It has always been understood that MeHg caused harm in tissues by reacting with biologically important molecules. Therefore, it has been correctly assumed that the basic reaction mechanism was bimolecular. Because of the high binding affinities between sulfur and mercury, it seemed likely that thiomolecules were involved in some way. The toxic tissue concentrations of mercury are ~1 µM, and highly toxic levels are on the order of ~2.5 µM, but the molar concentrations of sulfur in tissues are on the order of ~100,000 µM. Because it was assumed the concentration of intracellular sulfur molecule targets were essentially constant in comparison to that of mercury, knowing the intracellular concentration of the just the mercury would still supply adequate indications of the risk of toxicity associated with that

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level of mercury exposure. Therefore, prior researchers have assumed that the reaction rate equation of the molecular mechanism of mercury toxicity was adequately described by pseudo-first order reaction equations. The significance of normal intracellular tissue concentrations of selenium (~1 µM) in relation to mercury exposures was overlooked because the importance of selenium in physiology was generally unknown among toxicologists. However, since mercury concentrations approaching equimolar were increasingly associated with toxicity, and those with mercury in molar excess of selenium were severely toxic, it is clear that mercury exposures in relation to tissue selenium contents are an important aspect of the equation. Because tissue selenium contents tend to vary in relation to dietary selenium intakes, the accuracy of risk assessments employing pseudo-first order rate reactions based on mercury alone are unlikely to be as reliable as bimolecular equations solved using the concentrations of both elements.

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SOS-1 and SOS-2 Induced Generation of Apoptosis Initiators Futile cycles of formation of selenoenzymes without Sec result in loss of the essential functions listed in Table 2, but an unexpected vulnerability exists when thioredoxin reductase 1 (TrxR1) is produced without Sec. Recent studies indicate that TrxR1 promotes apoptosis when it is made without Sec, and the product has been identified as GRIM-12 (gene associated with retinoid interferon-induced mortality 12) (Anestål and Arnér 2003). Transient transfection with GRIM-12/TrxR1 directly induces cell death. Therefore, conditions of severe selenium deprivation from high MeHg exposures will not only abolish intracellular TRxR1 activities, a loss that may in itself be sufficient to induce cell death in vulnerable brain and neuroendocrine tissues. The disabled protein is itself a potent apoptosis initiator that rapidly induces cell death. The GRIM-12 programmed response may have evolved to limit nutritionally unsupported growth during selenium deprivation. Apoptosis of cells would release selenium that could be used to support growth of neighboring cells and maintain their health. Therefore, under conditions of moderate nutritional selenium deprivation this would ensure that all cells had at least minimally healthy amounts of selenium for supporting essential selenoenzyme activities. However, and along with the consequences of SOS1/SOS-2, GRIM-12 may be responsible for the severe brain damage noted among fetally exposed children of mothers that ate MeHg contaminated foods during catastrophic exposure incidents.

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PROTECTIVE ROLE OF DIETARY SELENIUM AGAINST MERCURY TOXICITY The protective effects of selenium against mercury toxicity in animals have been known for decades (Parizek and Ostadalova 1967), and are known to occur throughout the animal kingdom, including in fish (Yang et al. 2010), birds (Sell 1977) and mammals (Hansen et al. (1981) and Beyrouty and Chan (2006), reviewed by Khan and Wang 2009). Selenium supplementation or selenium-rich diets can mitigate the effects of mercury and mercury compounds (Kosta et al. (1975); Chen et al. (2006); Yang et al. (2010) and Berry and Ralston 2008). In addition, it is intriguing that indigenous human populations living in areas with significant elevated mercury exposure from natural and anthropogenic sources and selenium-rich soil such as the Brazilian Amazon, thus consuming a selenium- and mercury-rich diet may present very low effects (or virtually none) of mercury toxicity, when compared to the levels of mercury to which they were exposed (Lemire et al. (2006) and Berzas-Nevado et al. (2010)). This may be because of the combination of selenium-rich soil and diets of this region. The same protective phenomenon is reported in rats fed varying levels of selenium and MeHg in their diets (Ralston et al. 2008). The rats presented growth retardation upon exposure to MeHg in the diets, but the effects were highly dependent upon dietary selenium intakes. Weanling Long Evans rats fed low selenium diets, 0.1 µmol Se/kg prepared to contain 50 µmol MeHg/kg (~10 ppm) failed to thrive and showed hind limb crossing and had completely stopped gaining weight after 11 weeks. After 18 weeks on this diet, rats were half the size their counterparts that ate diets without added mercury. Among rats exposed to these same MeHg doses, but with either normal or seleniumrich (1.0 or 10.0 µmol Se/kg, respectively) diets, growth and protection against developing neurotoxic effects was proportional to their dietary selenium intakes. Therefore, exposures are not the sole determinant of MeHg toxicity since animals with the same dietary intakes can have either lethal, notable, or no discernable effects. However, it is crucial to note that the effects of MeHg exposures are entirely proportional to dietary Hg:Se molar ratios. Among rats fed diets with 50 µmol MeHg: 10 µmol Se/kg (molar ratio 5:1), there were no observable effects on neurological health or growth. Rats fed diets with 50 µmol MeHg, 1 µmol Se/kg (molar ratio 50:1), demonstrated no observable effects on neurological health, but growth was significantly less after 3 weeks and

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became increasingly impaired as time progressed, although not as severely as among rats fed low selenium diets. Rats fed diets with 50 µmol MeHg and 0.1 µmol Se/kg (molar ratio 500:1), showed depressed weight gain after 3 weeks, and stopped gaining weight after 10 weeks, and started losing weight after 11 weeks. At that time hind limb crossing and impaired walking abilities were first noted, and these symptoms rapidly grew more severe until lethality and declining health and/or lethality made it necessary to terminate the study. A study of dietary selenium's effectiveness in therapeutic treatment of MeHg poisoning found that weight gain was rapidly restored to animals that were rescued with selenium-rich diets, while those that were not rescued continued to progressively worsen (Ralston, manuscript in preparation). The development of worsening neurological signs (hind limb crossing, walking impairments) continued to progress for the first two weeks on the seleniumrich rescue diets, but then recovered to approximately the level they had been at when treatment was initiated. Although the study was continued for some months, none of the MeHg exposed animals made a complete recovery. The surprising aspect of the therapy study was that weight gain recoveries of selenium-rescued animals whose diets no longer contained MeHg were statistically equivalent to those that were fed diets that still contained 50 µmol MeHg/kg. The presence of MeHg in their diets did not affect their weight gain recoveries, but the presence of additional selenium from dietary sources made the difference between life and death. The mitigation of mercury toxicity by selenium may decrease the burden on the antioxidant systems caused by mercury presence. This mitigation mechanism remains under intense debate, but it has been suggested that, due to increased binding affinity, mercury sequesters selenium from its normal usage in the body. Thus, mercury toxicity is manifested primarily through consequent selenium deficiency, and by comparison with the organs they target (mainly in the nervous and reproductive systems), mercury toxicity’s pathological characteristics strongly resemble aggravated cases of selenium deficiency, supporting the concept that mercury toxicity is actually a selenium deficiency disease. Human blood selenium concentrations tend to range between 0.5 to 2.5 μM (~30-200 ppb). Thus, normal blood concentrations of MeHg (less than ~0.01 µM; ~2 ppb), would not induce any complexation-dependent decreases in selenium availability, and no impacts on physiological maintenance of selenoenzyme activities would be expected to occur. However, in the cases of MeHg-contaminated populations (blood concentrations as high as 30 µM MeHg), SOS-1/SOS-2 diversion of selenium from selenoprotein synthesis to

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formation of mercury-selenium complexes could cause an intracellular andsystemic selenium deficiency. Such exposures would decrease the availability of selenium and diminish selenoenzyme activities in vitally important, but highly vulnerable tissues. Khan and Wang (2009) calculated that selenium could only bind a small fraction of the total MeHg in highly exposed individuals, and indicated that selenium-dependent protection against MeHg toxicity did not occur as a result of MeHg sequestration. Their finding underlines our contention that mercury-selenium interaction studies need to focus on MeHg-dependent sequestration of selenium rather than seleniumdependent sequestration of MeHg. Since selenium deficiency is the primary cause for many endocrine, reproductive and neurological pathologies (Bellinger et al. 2009), it is interesting to note that these same systems are also primary targets of mercury toxicity (Castoldi et al. (2001) and Tan et al. (2009)) and present pathologies that reflect those that accompany severe selenium deficiencies. As previously stated, for humans the main source of MeHg exposure is through fish consumption. Meanwhile dietary selenium may be acquired through a variety of foods, although as a class, seafoods are particularly rich in selenium. Although MeHg tends to bioaccumulate throughout the food web, selenium bioaccumulation is only meaningful at the bottom and top of the food web. Selenium is homeostatically controlled. Therefore, in selenium poor ecosystems bioaccumulation and preferential retention of selenium assures adequate amounts are present to support required processes in vulnerable tissues. These tissue concentrations are preserved throughout the normal range of dietary selenium intakes and only deflect slightly as intakes increase. Coaccumulation of mercury and selenium in the form of HgSe occurs in tissues of top predators. In the case of selenium, an in silico search for the Selenocysteine Insertion Sequence (SECIS) element, the specific 3’UTR hairpin cis element that characterizes a selenoprotein mRNAs, revealed the presence of two selenoproteins restricted to fishes, Fep15 (Novoselov et al. 2006) and selenoprotein J (Castellano et al. 2005), which were further characterized in vivo. Fep15 has no known function, but its mammalian homologue selenoprotein 15 was shown to be an ER resident protein involved in disulfide bond formation (Labunskyy et al. 2007). Selenoprotein J was proposed to be a structural protein in the zebrafish eye lens working as a crystallin. The functions of these two proteins in fish remain to be further elucidated. However, a third selenoprotein, called selenoprotein L (Sel L) was also identified in silico in 2007 in zebrafish (Danio rerio) and it was revealed to be

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present only in aquatic organisms (Shchedrina et al. 2007). Selenoprotein L has a unique diselenide Sec-XX-Sec motif, resembling the Cys-XX-Cys motif present in thioredoxin proteins. This suggests selenoprotein L may have a redox function, but the fact that it is restricted to aquatic organisms opens up exciting possibilities for insights into new mechanisms for detoxification of heavy metal compounds in water animals only that could incorporate the fact that MeHg accumulation in aquatic top predators does not seem to affect them pathologically as in land creatures. In addition, an intriguing question regards the fact that selenoprotein W, which is dependent on glutathione binding for function, is not glutathionylated on cysteine residue 37 in fish, a residue found to be glutathionylated in mammals. This could be an important site for mercury binding and the lack of such post-translational modification in fishes may explain also the lack of major physiological disruption even upon the elevated mercury concentrations these animals sometimes present (Whanger 2009).

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FISH CONSUMPTION ADVISORIES Current governmental policies and regulations regarding MeHg exposure from fish used for human consumption do not incorporate selenium levels as a practical indicator of MeHg-related risk. Instead, the 2004 FDA/EPA advisory recommends that children, pregnant women, women who might become pregnant and nursing mothers avoid shark, swordfish, king mackerel, and tilefish consumption and limit other fish consumption up to 2 meals per week. This advisory is intended to maintain MeHg exposures below 1/10 of the Lowest Observable Adverse Effect Levels (LOAEL) established in the Faroe Islands Study (Grandjean et al. 1997). In that study, it was found that high MeHg exposures (~95% originating from eating pilot whale meats, ~5% originating from eating codfish) among the mothers may have caused as much as 0.1 and 0.25 IQ points decreases at the highest levels of exposure. However, based on the findings of the Faroe Islands Study as well as a number of subsequent studies (Ralston 2008), it appears that mercury-selenium molar ratios need to be considered in the seafood safety issue. There is increasing cause for concern regarding the basis for, and potentially adverse effects of the current maternal seafood consumption advisory statement. First, the report on which the advisory was based lists seafood as a source of mercury, but has a very broad definition of "seafood". It includes not only crustaceans and ocean fish that are typically consumed, but also predatory

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whales and large sharks that are not consumed in the United States. This broad definition of seafood is important because as the report notes, the only adverse effects were noted in studies performed in locations where predatory whales or sharks were the major source of MeHg exposure, not fish. As top predators of the marine food chain, toothed cetaceans and sharks are known to accumulate significantly higher levels of MeHg in their bodies compared to bony fish. As a result, the muscle meats of toothed whales and most predatory varieties of shark are unique in being the only foods human consume that are known to contain mercury in substantial molar excess of selenium (Kaneko and Ralston 2007). Although the effects of maternal MeHg exposure on child IQ's were very subtle (0.1-0.25 IQ points) a reanalysis of the Faroe Islands Study found potent protective effects associated with increasing maternal fish consumption (Budtz-Jørgensen et al. 2007), and although the adverse effects of MeHg exposure were purportedly not related to Hg:Se molar ratios (Choi et al. 2008), it is difficult to understand how this could be the case since blood Hg levels were highly correlated with Hg:Se molar ratios (R2>0.97 and 0.98 for cohorts 1 and 2 respectively, p< 0.001 for both cohorts). Therefore, the results of that study clearly indicate that blood MeHg contents were correlated with blood Hg:Se molar ratios. The blood Hg:Se molar ratios in these children are higher than in any other study, approaching and exceeding the 1:1 molar ratios. The pilot whale meat consumed in the Faroe Islands contains 4-5 times more mercury than selenium (Julshamn et al. 1987), making it uniquely different from typical varieties of ocean that usually contain many times more selenium than mercury. Therefore, the current U.S. FDA warnings regarding ocean fish consumption are based on the results of a study that does not properly represent the varieties of fish that are actually consumed. Because of the pronounced differences in Hg:Se molar ratios, the risks of pilot whale meat consumption are unlikely to provide much in the way of useful information regarding the safety of eating ocean fish. Second, no study ever showed detrimental neurological development in children exposed to MeHg derived from marine fish in the diet, the best known in this case being the Seychelles Child Development Study, performed in a community with a heavy consumption of ocean fish. The Seychelles Study showed no significant difference on neurodevelopmental risk for children born from mothers whose intake of mercury on fish were significant high (Myers et al. (2003), recently reviewed with additional data by Myers et al. (2009)). Instead, the most recent, largest, and best designed studies indicate the opposite is the case. A study performed in the United Kingdom showed significant benefits to IQ scores in children whose mothers had more than 340

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Lucia A. Seale, Nicholas V. Ralston and Marla J. Berry

grams of fish/week in their diets (Hibbeln et al. 2007). This result is expected to be due to the improved nutritional status of mothers that eat ocean fish with abundant selenium and omega-3 fatty acid contents. Omega-3 fatty acids boost neural development in fetuses and infants and it is increasingly believed that the beneficial effects of seafood consumption should be highlighted and balanced with the potential risks associated with MeHg exposure. Otherwise, the nutritional benefits of seafood consumption that foster healthy pregnancies and improved child development outcomes will be lost by mothers that avoid seafood out of concern regarding MeHg (Ralston 2008). Third, recent results from a study performed in Hawai'i have been very illuminating regarding potential dietary exposure to MeHg from fish consumption (Kaneko and Ralston 2007). In the referred study, 420 samples of representative sizes of 15 commonly consumed pelagic fish species were collected from the Pacific Ocean and analyzed for their selenium and MeHg contents. All but one pelagic fish species were found to contain selenium in excess of MeHg; Se:Hg molar ratio of 1:1 or more) in their edible meats. Of all the fish studied, only mako shark contained mercury in excess of selenium, (Se:Hg molar ratio 1:2). Fourth, a recent study examined the bioavailability of selenium from fish meats (yellowfin tuna, swordfish, and mako shark) and found that the selenium from all three sources was highly bioavailable, and equally effective as inorganic forms of selenium that have previously been used to test selenium-dependent protection against MeHg toxicity (Ralston, manuscript in preparation). In this study, weanling rats were deprived of dietary selenium for five weeks before being switched to diets containing 0.1, 1.0, or 10 µmol Se/kg, or selenium from delipidated protein derived from ocean fish, added as 10% by mass to the diets to replace an equivalent amount of low selenium protein from torula yeast. After five weeks on these diets the selenium from ocean fish protein was bioaccumulated in liver at levels proportional to the same concentrations of inorganic dietary selenium. Even more importantly, MeHg from ocean fish proteins did not add to the toxicity of 50 µmol MeHg/kg in the diets, but the selenium from the ocean fish potently counteracted MeHg toxicity, completely preventing both observable neurotoxicity (hind limb crossing) and growth inhibition. The results described above support the concept that meats from ocean fish possess sufficient selenium to counterbalance the adverse effects of the MeHg they also contain, and can be consumed without major health concerns. Since maternal avoidance of ocean fish consumption appears to be associated with loss of 5 (Hibbeln et al. 2007) to almost 10 IQ points (Lederman et al.

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2009) it appears that government advisories need to be updated to reflect current scientific understanding of the fish MeHg issue. Based on the growing evidence of selenium mitigation, Kaneko and Ralston (2007) proposed the Selenium-Health Benefit Value be used as seafood safety criteria. Since this index correlates with both the expected benefits of enhanced selenium status as an outcome of seafood consumption as well as potential risks of MeHg exposures, these criteria appear to offer a balanced indication of anticipated quality of seafoods for maternal consumption. It is anticipated that this new index will be particularly important when applied to freshwater fish consumption issues since their selenium status is highly dependent upon environmental selenium availability. Since a number of factors influence selenium availability as well as MeHg bioaccumulation in freshwater fish, this index will be particularly important for establishing safety or risks associated with their consumption. Efforts are currently underway to assess Hg:Se molar ratios in fish from lakes across the United States. Incorporating selenium into MeHg risk assessments will better inform regulatory and policy decisions intended to improve and protect child health.

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CONCLUSION Further investigation of mercury-selenium interaction mechanisms and downstream genomic responses will be required to improve understanding of details regarding the SOS-1, SOS-2, and GRIM-12 mechanisms of mercury toxicity. However, it is clear that an improved dietary selenium intake increases the intracellular availability of selenium to replace amounts lost to MeHg-Sec binding. Therefore, instead of ocean fish consumption causing health risks through increasing MeHg exposures, consumption of selenium-rich seafoods appear likely to protect against mercury toxicity. Insights into the molecular mechanisms of mercury toxicity will be useful in developing clinical strategies using selenium supplementation to avoid pathological consequences in individuals accidentally exposed to high levels of mercury. Understanding of mercury-selenium interactions will make it possible for regulatory agencies to formulate better-informed maternal seafood consumption advisories that will protect and improve child health.

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ACKNOWLEDGMENTS Dr. Berry's research is supported by grant R01-DK47320 from the National Institute of Diabetes and Digestive and Kidney Diseases. Support for Dr. Ralston's research and preparation of this article was supported by grant NA08NMF4520492 from the National Oceanic and Atmospheric Administration to the University of North Dakota Energy and Environmental Research Center. This article has not been subjected to review by the funding agencies and therefore does not necessarily reflect the views of these entities and no official endorsements should be inferred.

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selenium in beluga whale tissues Environmental Bioindicators 4(4): 291302. Jackson, M. J., Dillon, S. A., Broome, C. S., McArdle, A., Hart, C. A., McArdle, F. (2004). Are there functional consequences of a reduction in selenium intake in UK subjects? Proc. Nutr. Soc. 63(4): 513-7. Julshamn, K., Anderson, A., Ringdal, O., and Mørkøre, J. (1987). Trace elements intake in the Faroe Islands, I. Element levels in edible parts of pilot whales (Globicephalus meleanus).Sci. Total Environ. 65: 53–62. Kajiwara, Y., Yasutake, A., Adachi, T., Hirayama, K. (1996).Methylmercury transport across the placenta via neutral amino acid carrier.Arch. Toxicol. 70(5): 310-4. Kaneko, J. J. and Ralston, N. V. (2007).Selenium and mercury in pelagic fish in the central north pacific near Hawaii.Biol. Trace Elem. Res. 119(3): 242-54. Khan, M. A. and Wang, F. (2009). Mercury-selenium compounds and their toxicological significance: toward a molecular understanding of the mercury-selenium antagonism. Environ. Toxicol. Chem. 28(8): 1567-77. Kim, Y. J., Chai, Y. G., Ryu, J. C. (2005). Selenoprotein W as molecular target of methylmercury in human neuronal cells is down-regulated by GSH depletion. Biochem.Biophys. Res. Commun. 330(4): 1095-102. Köhrle, J., Brigelius-Flohé, R., Böck, A., Gärtner, R., Meyer, O., Flohé, L. (2000). Selenium in biology: facts and medical perspectives. Biol. Chem. 381;849-864. Kosta, L., Byrne, A. R., Zelenko, V. (1975). Correlation between selenium and mercury in man following exposure to inorganic mercury.Nature 254(5497): 238-9. Krebs, R. E. (2006). The history and use of our earth's chemical elements: a reference guide (2nd edition). Westport CT: Greenwood Press. Kromidas, L., Trombetta, L. D., Jamall, I. S. (1990). The protective effects of glutathione against methylmercury cytotoxicity.Toxicol.Lett. 51(1): 67-80. Kupka, R., Garland, M., Msamanga, G., Spiegelman, D., Hunter, D., Fawzi, W. (2005). Selenium status, pregnancy outcomes, and mother-to-child transmission of HIV-1.J. Acquir. Immune Defic.Syndr. 39(2): 203-10. Labunskyy, V. M., Hatfield, D. L., Gladyshev, V. N. (2007). The Sep15 protein family: roles in disulfide bond formation and quality control in the endoplasmic reticulum. IUBMB Life 59(1): 1-5. Labunskyy, V. M., Yoo, M. H., Hatfield, D. L., Gladyshev, V. N. (2009). Sep15, a thioredoxin-like selenoprotein, is involved in the unfolded

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protein response and differentially regulated by adaptive and acute ER stresses. Biochemistry 48(35): 8458-65. Lacourciere, G. M. and Stadtman, T. C. (2001).Utilization of selenocysteine as a source of selenium for selenophosphate biosynthesis.Biofactors 14(1-4): 69-74. Lederman, S. A., Jones, R. L., Caldwell, K. L., Rauh, V., Sheets, S. E., Tang, D., Viswanathan, S., Becker, M., Stein, J. L., Wang, R. Y., Perera, F. P. (2008). Relation between cord blood mercury levels and early child development in a World Trade Center cohort.Environ. Health Perspect 116(8): 1085-91 Lee, Y. W., Ha, M. S., Kim, Y. K. (2001). Role of reactive oxygen species and glutathione in inorganic mercury-induced injury in human glioma cells.Neurochem. Res. 26(11): 1187-93. Lemire, M., Mergler, D., Fillion, M., Passos, C. J., Guimarães, J. R., Davidson, R., Lucotte, M. (2006). Elevated blood selenium levels in the Brazilian Amazon. Sci. Total Environ. 366(1): 101-11. Linster, C. L. and Van Schaftingen, E. (2007). Vitamin C. Biosynthesis, recycling and degradation in mammals. FEBS J. 274(1): 1-22. Lobanov, A. V., Hatfield, D. L., Gladyshev, V. N. (2008). Reduced reliance on the trace element selenium during evolution of mammals.Genome Biol 9(3): R62. Low, S. C. and Berry, M. J. (1996).Knowing when not to stop: selenocysteine incorporation in eukaryotes.Trends Biochem. Sci. 21(6): 203-8. Lowenstein, J. H., Burger, J., Jeitner, C. W., Amato, G., Kolokotronis, S. O., Gochfeld, M. (2010). DNA barcodes reveal species-specific mercury levels in tuna sushi that pose a health risk to consumers. Biol. Lett .[epub ahead of print]. Lu, C., Qiu, F. , Zhou, H., Peng, Y., Hao, W., Xu, J., Yuan, J., Wang, S., Qiang, B., Xu, C., Peng, X. (2006). Identification and characterization of selenoprotein K: an antioxidant in cardiomyocytes. FEBS Lett. 580(22): 5189-97. Martin, H. L. and Teismann, P. (2009). Glutathione--a review on its role and significance in Parkinson's disease. FASEB J. 23(10): 3263-72. Moghadaszadeh, B. and Beggs, A. H. (2006).Selenoproteins and their impact on human health through diverse physiological pathways.Physiology (Bethesda) 21: 307-15. Myers, G. J. and Davidson, P. W. (1998). Prenatal methylmercury exposure and children: neurologic, developmental, and behavioral research. Environ. Health Perspect 106 Suppl 3: 841-7.

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Myers, G. J., Davidson, P. W., Cox, C., Shamlaye, C. F., Palumbo, D., Cernichiari, E., Sloane-Reeves, J., Wilding, G. E., Kost, J., Huang, L. S., Clarkson, T. W. (2003) Prenatal methylmercury exposure from ocean fish consumption in the Seychelles child development study. Lancet 361(9370): 1686-92. Myers, G. J., Thurston, S. W., Pearson, A. T., Davidson, P. W., Cox, C., Shamlaye, C. F., Cernichiari, E., Clarkson, T. W. (2009). Postnatal exposure to methyl mercury from fish consumption: a review and new data from the Seychelles Child Development Study. Neurotoxicology 30(3): 338-49. Naganuma, A., Furuchi, T., Miura, N., Hwang, G. W., Kuge, S. (2002). Investigation of intracellular factors involved in methylmercury toxicity. Tohoku J. Exp. Med. 196(2): 65-70. Novoselov, S. V., Hua, D., Lobanov, A. V., Gladyshev, V. N. (2006). Identification and characterization of Fep15, a new selenocysteinecontaining member of the Sep15 protein family.Biochem. J. 394(Pt 3): 575-9. Olson, G. E., Winfrey, V. P., Hill, K. E., Burk, R. F. (2008). Megalin mediates selenoprotein P uptake by kidney proximal tubule epithelial cells. J. Biol. Chem. 283(11): 6854-60. Olson, G. E., Winfrey, V. P., Nagdas, S. K., Hill, K. E., Burk, R. F. (2005). Selenoprotein P is required for mouse sperm development. Biol. Reprod. 73(1): 201-11. Olson, G. E., Winfrey, V. P., Nagdas, S. K., Hill, K. E., Burk, R. F. (2007). Apolipoprotein E receptor-2 (ApoER2) mediates selenium uptake from selenoprotein P by the mouse testis. J. Biol. Chem. 282(16): 12290-7. Parizek, J. and Ostadalova, I. (1967).The protective effect of small amounts of selenite in sublimate intoxication.Experientia 23(2): 142-3. Patrick, L. (2002). Mercury toxicity and antioxidants: Part 1: role of glutathione and alpha-lipoic acid in treatment of mercury toxicity. Altern. Med. Rev. 7(6): 456-71. Queiroz, M. L., Pena, S. C. Salles, T. S., de Capitani, E. M., Saad, S. T. (1998). Abnormal antioxidant system in erythrocytes of mercury-exposed workers.Hum. Exp. Toxicol. 17(4): 225-30. Ralston, N. V. (2008). Selenium health benefit values as seafood safety criteria. Ecohealth 5(4): 442-55. Ralston, N. V., Ralston, C. R., Blackwell, J. L., Raymond, L. J. (2008). Dietary and tissue selenium in relation to methylmercury toxicity.Neurotoxicology 29(5): 802-11.

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Tan, K. S., Arnold, A.P., Rabenstein, D.L. (1988). Selenium-77 nuclear magnetic resonance studies of selenols, diselenides, and selenyl sulfides. Can. J. Chem. 66: 54-60. Tan, S. W., Meiller, J. C., Mahaffey, K. R. (2009) The endocrine effects of mercury in humans and wildlife. Crit. Rev. Toxicol. 39(3): 228-69. U.S. Department of Agriculture National Nutrient Database for Standard Reference, Release 17. Selenium, Se (µg) Content of Selected Foods.Accessed 2005 July. Available from: URL: http://www.nal.usda. gov/fnic/foodcomp/. Ursini, F., Heim, S., Kiess, M., Maiorino, M., Roveri, A., Wissing, J., Flohé, L. (1999).Dual function of the selenoprotein PHGPx during sperm maturation.Science 285(5432): 1393-6. Wedeen, R. P. (1989). Were the hatters of New Jersey "mad"? Am. J. Ind. Med. 16(2): 225-233. Weiss, B., Clarkson, T. W., Simon, W. (2002). Silent latency periods in methylmercury poisoning and in neurodegenerative disease.Environ. Health Perspect 110(5):851-4. Whanger, P. D. (2009).Selenoprotein expression and function-selenoprotein W. Biochim.Biophys.Acta. 1790(11): 1448-52. Wiener, J. G. and Suchanek, T. H. (2008). The basis for ecotoxicological concern in aquatic ecosystems contaminated by historical mercury mining.Ecol. Appl. 18(8 Suppl): A3-11. Wu, A. S., Oldfield, J. E., Shull, L. R., Cheeke, P. R. (1979). Specific effect of selenium deficiency on rat sperm.Biol. Reprod. 20(4): 793-8. Yang, D. Y., Ye, X. Chen, Y. W., Belzile, N. (2010). Inverse relationships between selenium and mercury in tissues of young walleye (Stizosedion vitreum) from Canadian boreal lakes.Sci. Total Environ. 408(7): 1676-83. Yant, L. J., Ran, Q., Rao, L., Van Remmen, H., Shibatani, T., Belter, J. G., Motta, L., Richardson, A., Prolla, T. A. (2003). The selenoprotein GPX4 is essential for mouse development and protects from radiation and oxidative damage insults. Free Radic. Biol. Med. 34(4): 496-502. Yoneda, S. and Suzuki, K. T. (1997). Equimolar Hg-Se complex binds to selenoprotein P. Biochem. Biophys. Res. Commun. 231(1): 7-11. Yoshida, M. (2002).Placental to fetal transfer of mercury and fetotoxicity.Tohoku J. Exp. Med. 196(2): 79-88. Zalups, R. K. (2000). Molecular interactions with mercury in the kidney.Pharmacol. Rev. 52(1): 113-43.

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Chapter 2

FISH AS A DIETARY SOURCE OF MERCURY AND METHYLMERCURY, RISKS AND BENEFITS Afnan Freije and Maysoon Awadh

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Department of Biology, College of Science, University of Bahrain, Kingdom of Bahrain

CHEMICAL AND PHYSICAL PROPERTIES OF MERCURY Mercury occurs naturally and distributed widely by human activities in the environment. Mercury is a heavy metal, an element of the earth with a molecular weight of 200.59g and an atomic number of 80. It is one of the few elements that are mobile, shiny, silver-white, odorless liquid at room temperatures, therefore it is called quicksilver. Mercury is used to make thermometers, diffusion pumps, barometers, mercury vapour lamps, mercury switches, pesticides, batteries, dental preparations, antifouling paints, pigments, and catalysts (Boening, 2000; Sanzo, 2001; UNEP, 2005). Mercury in the environment exists in three forms, elemental or metallic mercury (Hg0), inorganic and organic mercury compounds. Mercury (Hg) can be bound to other compounds as monovalent Hg(I) or divalent mercury (Hg(II) or Hg²+). Both inorganic and organic chemical compounds of mercury (II) are much more numerous than those of mercury (I) (Qian, 2001; UNEP, 2005; Virtanen et al., 2006).

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In addition to inorganic mercury salts, including mercurous chloride, mercuric chloride, mercuric acetate, and mercuric sulfide, mercury (II) forms an important class of organometallic compounds. The most toxic organometallic compounds is the subclass of short-chain alkyl mercurials in which mercury is attached to the carbon atom of a methyl, ethyl, or propyl group (WHO, 1989; Clarkson, 1998; WHO, 2003; Dorea et al., 2003). The specific gravity of metallic mercury at 20°C is 13.534 g/cm and the vapour pressure is 0.16 Pa (0.0012 mmHg). However, a saturated atmosphere at 20°C contains approximately 15 mg/m³, which is 300 times greater than the recommended health-based occupational exposure limit of 0.05 mg/m³.The boiling point of mercury is 356.73°C (675°F) and it is a poor conductor of heat as compared with other metals but is a good conductor of electricity (WHO, 1990; Wikipedia, 2006). Mercury compounds differ greatly in their solubility values in water in the order: elemental mercury < mercurous chloride < methylmercury chloride < mercuric chloride (WHO, 1990). Due to the high solubility of the methylmercury cation in water, the solubility of methylmercury chloride in water is higher than that of mercurous chloride by about three orders of magnitude (Clarkson, 1988). On the other hand elemental mercury and the halide compounds of alkylmercurials are soluble in non-polar solvents (Clarkson, 1988). Hursh (1985) showed that mercury vapour is more soluble in plasma, whole blood, and haemoglobin than in distilled water or isotonic saline. Mercury readily forms alloys with other metals, such as gold, silver, and tin. These alloys are called amalgams. Therefore, mercury is amalgamated with gold to facilitate its recovery from its ores. Mercury and its compounds are highly poisonous (Levy et al., 2003; UNEP, 2005).

INORGANIC FORMS OF MERCURY Mercury is present in many chemical forms; it occurs naturally as non toxic mercuric sulphide (HgS) cinnabar. Inorganic mercuric compounds (mercury salts) include mercuric oxide (HgO), mercuric chloride HgCl2 (corrosive sublimate - a violent poison), mercurous chloride Hg2Cl2 (calomel, occasionally still used in medicine), mercury fulminate Hg(ONC)2 (a detonator used in explosives), and mercuric sulphide (vermillion, a high-grade paint pigment) (WHO, 1991; WHO, 2003; UNEP, 2005).

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Most inorganic mercury salts are white powders or crystals, while mercuric sulphide which is the most common form of mercury, is a red insoluble salt which turns black after exposure to light. Some mercury salts (such as HgCl2) are volatile to exist as an atmospheric gas. However, the water solubility and chemical reactivity of these inorganic (ionic) mercury gases lead to more rapid deposition from the atmosphere than for metallic mercury. This results in shorter atmospheric lifetimes for these divalent inorganic mercury gases than for the elemental mercury gas (Qian, 2001; WHO, 2003).

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ORGANIC FORMS OF MERCURY When mercury combines with carbon the compounds formed are called “organic” mercury compounds or organomercurials. There are a large number of organic mercury compounds (such as dimethylmercury, phenylmercury, ethylmercury and methylmercury); however, the most common organic mercury compound in the environment is methylmercury. Methylmercury and phenylmercury exist as white crystalline salts (for example, methylmercuric chloride or phenylmercuric acetate). However, dimethylmercury is a colourless liquid (WHO, 1990; UNEP, 2004). Methylmercury (CH3Hg) is the most common organic mercury compound that is generated by natural processes. Methylmercury can build up (bioaccumulate and biomagnify) in fish and marine mammals to levels that are many thousands of times greater than levels in the surrounding water (USGS, 2000; USEPA, 2001; Mahaffey, 2004; Mora et al., 2004; Voegborlo and Akagi, 2007). Methylmercury can be formed in the environment mainly by microbial metabolism (biotic processes) and by chemical processes that do not involve living organisms (abiotic processes). Direct anthropogenic (or humangenerated) sources of methylmercury are currently not known. However, indirectly anthropogenic releases contribute to the methylmercury levels found in nature because of the transformation of other forms (USEPA, 2001; UNEP, 2004).

SOURCES OF MERCURY IN THE ENVIRONMENT Mercury occurs naturally and is distributed throughout the environment by both natural processes and human activities (Qian, 2001; FVSRC, 2005). The

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sources of mercury releases to biosphere as grouped by the United Nations Environment Programme (UNEP, 2004; UNEP, 2005) in four categories were:

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NATURAL SOURCES Natural sources of mercury releases by: volcanic activity, weathering of rocks, evaporation from water and soil surfaces, forest fires and degradation of minerals (FVSRC, 2005; UNEP, 2005). The natural mercury emissions are beyond control, considered as a part of the local and global living environment. Regions with high concentrations in surface rocks are characterized by high mercury emissions to the atmosphere. Emissions of mercury from water and soil surfaces today, are composed of both natural sources and re-emission of previous mercury deposition from both natural sources and anthropogenic. This makes it very difficult to determine the actual natural mercury emissions (UNEP, 2005). There are indications that anthropogenic emissions of mercury around the globe have resulted in deposition rates today which are 1.5 to 3 times higher than those during pre-industrial times. However, in and around industrial areas the deposition rates have increased by 2 to 10 times during the last 200 years (Lindquist et al., 1984; Bergan et al., 1999). The global natural emission was estimated by Mason et al. (1994) at about 1650 metric tons/year. This estimate was updated by Lamborg et al. (2002) to around 1400 metric tons/year. However, Bergan and Rohde (2001) had estimated global natural emission of about 2400 metric tons, in which 1100 was emitted from oceans and 1320 from land sources.

ANTHROPOGENIC RELEASES FROM THE MOBILIZATION OF MERCURY IMPURITIES IN MATERIALS Naturally, mercury is present in coal and other fossil fuels. On the other hand, mercury present in minerals like lime for cement production and soils (such as agricultural soils subject to acidification management) and metal ores including for example zinc, copper and gold ore. Today production of coalfired power is deemed the single largest global source of atmospheric mercury emissions (Pacyna and Pacyna, 2000).

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In primary and recycled materials mercury impurities constitute major contributions to the total global mercury burden. Processing of secondary raw materials, like iron and steel for example, can also be a significant source of mercury releases, and emission control technologies are often necessary. In this case the origin of the mercury may be both natural impurities and as a result of intentional use of mercury in products/components in iron/steel scrap (switches, air-bag activators etc.) (USGS, 2000; Qian, 2001; FVSRC, 2005). The majority of atmospheric anthropogenic emissions are released as gaseous elemental mercury (Hgº). This is capable of being transported over very long distances (USEPA, 1997). The remaining part of air emissions are in the form of gaseous divalent compounds (such as HgCl2) or bound to particles present in the emission gas (UNEP, 2005). These species have a shorter atmospheric lifetime than elemental vapor and will deposit via wet or dry processes within roughly 100 to 1000 kilometers. However, significant conversion between mercury species may occur during atmospheric transport, which will affect the transport distance (UNEP, 2005). The atmospheric residence time of elemental mercury is in the range of months to roughly one year. Atmospheric residence time here designates the time span from a given mercury molecule is emitted to the atmosphere till it is deposited on land or in water. It does not include subsequent re-emission to the atmosphere. This makes transport on a hemispherical scale possible and emissions in any continent can thus contribute to the deposition in other continents. For example, contributions of external sources to anthropogenic mercury depositions to Europe and Asia were estimated to be about 20 percent and 15 percent, respectively. Similarly up to 50 percent of anthropogenic mercury deposited to North America is from external sources (Travnikov and Ryaboshapko, 2002). Furthermore, mercury is also capable of re-emissions from water and soil surfaces. This process greatly enhances the overall residence time of mercury in the environment. Recent findings indicate re-emission rates of approximately 20 percent over a two-year period, based on stable mercury isotope measurements in north-western Ontario, Canada (Lindberg et al., 2001).

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ANTHROPOGENIC RELEASES FROM MERCURY USED IN PRODUCTS AND PROCESSES Mercury is used in many products and industrial processes. Due to releases during manufacturing, leaks, disposal or incineration of spent products or other releases (UNEP, 2004; UNEP, 2005).

EMISSION OF MERCURY TO THE AIR The anthropogenic sources of mercury that releases to the air include mining and smelting, industrial processes involving the use of mercury such as chlor-alkali production facilities and production of cement (UNEP, 2004; UNEP, 2005; McCrary et al., 2006).

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EMISSION OF MERCURY TO THE WATER To the surface waters mercury is released from naturally occurring mercury in soils and rocks and from industrial activities, including, leather tanning, paper and pulp mills, electroplating, and chemical manufacturing. On the other hand, wastewater treatment facilities may also release mercury to water. Mercury in the air is an indirect source of mercury to surface waters; it is deposited from rain and other processes to water surfaces and to soils. Mercury may also be mobilized from sediments if disturbed by flooding and/or dredging (Qian, 2001; UNEP, 2004; UNEP, 2005).

EMISSION OF MERCURY TO THE SOIL In soil the sources of mercury include direct application of fertilizers and fungicides and disposal of solid waste, including batteries and thermometers, to landfills. Municipal incinerator ash disposal in landfills and the application of sewage sludge to crop land result in increased levels of mercury in soil (UNEP, 2004; FVSRC, 2005; UNEP, 2005).

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RE-MOBILIZATION OF HISTORIC ANTHROPOGENIC MERCURY Mercury released previously can be deposited in soils, sediments, water bodies landfills and waste/tailings piles (Boening, 2000; UNEP, 2004; UNEP, 2005).

GLOBAL CYCLING, TRANSPORT AND FATE OF MERCURY IN THE ENVIRONMENT

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Global cycling of mercury is a complex process. When mercury evaporates from surface waters and soils to the atmosphere, is re-deposited on land and surface water, and then is absorbed by soil or sediments. After redeposition on land and water, mercury is commonly volatilized back to the atmosphere as a gas, or as adherents to particulates, or be methylated. Emitted mercury vapour is converted to soluble forms (e.g. Hg II) and deposited by rain onto soil and water (Boening, 2000; USGS, 2000; USEPA, 2001; Booth and Zeller, 2005).

MERCURY SPECIES AND TRANSFORMATION IN AQUATIC ENVIRONMENT In water methylmercury is the most common form of mercury, quickly enters the aquatic food chain. In most adult fish, 90% to 100% of the mercury is methylmercury (Bloom, 1992; Niencheski et al., 2001; Mahaffey, 2004). Marine aquatic organisms at all levels accumulate mercury in their tissues, where organic mercury is retained for long periods. A number of factors that affect the susceptibility of aquatic organisms to mercury, include the life-cycle stage (the larval stage being particularly sensitive), temperature, acidity, dissolved organic matter (DOC) and salinity of surrounding water (Downs et al., 1998; Morel et al., 1998; Ikingura and Akagi, 2003; Harding et al., 2006; Murphy et al., 2007). The ratio of the methylmercury concentration in fish tissue to the concentration of inorganic mercury in water is usually between 10,000 and 100,000 to one (WHO, 1989). The change of mercury from inorganic form to methylated forms is the first step in the aquatic bioaccumulation process. This can occur by microbial

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metabolism (biotic processes) such as by certain bacteria and by chemical processes that do not involve living organisms (abiotic processes). Bacteria that process sulfate (SO4)-2 take up mercury in its inorganic form and convert it to methylmercury, through metabolic processes. The conversion of inorganic mercury to methylmercury is important because its toxicity is greater and organisms require longer time to eliminate methylmercury (Morel et al. 1998; USGS, 2000). These methylmercury containing bacteria may be consumed by the next higher level in the food chain, or excrete the methylmercury to the water where it adsorb to plankton which are also consumed by the next level in the food chain. Once methylmercury is released, it enters the food chain by rapid diffusion and tight binding to proteins. Animals consume higher concentrations of mercury at each successive level of the food chain and accumulate methylmercury faster than they eliminate it. Thus small environmental concentration of methylmercury can readily accumulate to potentially harmful concentrations in fish, fish eating wildlife and people (Nakagawa et al., 1997; Morel et al., 1998; USGS, 2000, Sehee et al., 2010). The formation of methylmercury in aquatic systems is influenced by a wide variety of environmental factors. The efficiency of microbial mercury methylation generally depends on factors such as microbial activity and the concentration of bioavailable mercury (rather than the total mercury pool), which in turn are influenced by parameters such as temperature, pH, redox potential and the presence of inorganic and organic complexing agents. (Ullrich et al., 2001; Seiders, 2006) Certain bacteria also demethylate mercury, thereby forming some natural constraints on the build-up of methylmercury (Marvin-Dipasquale et al., 2000, Bailey et al., 2001). Since both methylation and demethylation processes occur, environmental methylmercury concentrations reflect net methylation rather than actual rates of methylmercury synthesis. Numerous bacterial strains capable of demethylating and methylmercury are known, including both aerobic and anaerobic species, but demethylation appears to be predominantly accomplished by aerobic organisms. Bacterial demethylation has been demonstrated both in sediments and in the water column of freshwater lakes. Degradation of methyl and phenyl mercury by fresh water algae has also been described (Ullrich et al., 2001). Purely chemical methylation of mercury is also possible if suitable methyl donors are present. The relative importance of abiotic versus biotic methylation mechanisms in the natural aquatic environment has not yet been

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established, but it is generally believed that mercury methylation is predominantly a microbially mediated process (Ullrich et al., 2001). Mason and Fitzgerald (1996; 1997) have reviewed aspects of the cycle of mercury in oceans and other waters. From open ocean studies, it is apparent that elemental mercury, dimethylmercury and, to a lesser extent, methylmercury are common constituents of the dissolved mercury pool in deep ocean waters. In open ocean surface waters dimethylmercury is lacking, maybe as a result of decomposition in the presence of light and an additional potential loss via evaporation from the water surface. Recent results suggest that low oxygen conditions are not necessary for the formation of dimethylmercury in the open oceans. This contrasts with temperate lake waters where methylmercury is more commonly occurring than dimethylmercury. Studies in freshwater and estuarine environments have shown that methylation of mercury is primarily taking place under low oxygen conditions and mainly by sulphate-reducing bacteria.

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POTENTIAL SOURCES OF EXPOSURE AND OCCURRENCE OF MERCURY IN HUMAN TISSUES Humans are exposed to mercury from different sources. Dental amalgam is the most important source for elemental mercury vapour exposure. On the other hand, using skin-lightening creams and soaps that contain mercury and using mercury for cultural/ritualistic purposes or in traditional medicine, can also result in substantial exposure to inorganic or elemental mercury (Levy et al., 2003; UNEP, 2004). Dietary intake is the dominant source of exposure to mercury for the general population. Fish and seafood products are the main source of methylmercury in the diet; studies have shown that methylmercury concentrations in fish and shellfish are approximately 1,000 to 10,000 times greater than in other foods (Rice et al., 2000; Dorea, 2009). Once released into the environment, inorganic mercury is converted to organic mercury (methylmercury) which is the primary form that accumulates in fish and shellfish. Methylmercury biomagnifies up the food chain as it is passed from a lower food chain level to a subsequently higher food chain level through consumption of prey organisms or predators. Fish at the top of the aquatic food chain, bioaccumulate methylmercury approximately 1 to 10

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million times greater than dissolved methylmercury concentrations found in surrounding waters (Lawrence and Mason, 2001; USEPA, 2001).

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RISKS AND BENEFITS OF EATING FISH Fish and shellfish are an important part of a healthy diet. Fish and shellfish contain high quality protein and other essential nutrients. They are also low in saturated fat, and contain omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (Daviglus et al., 2002; Patterson, 2002; Burger and Gochfeld, 2006). A variety of potential health benefits are associated with omega-3 fatty acids, including prevention of cardiovascular disease (CVD), cardiac autonomic nervous effect, hypertension, autoimmune diseases and cancers (Connor, 2000; Kris, 2002; Burger and Gochfeld, 2004; Burger and Gochfeld, 2006, Thurston et al., 2007; Yaginum-Sakurai et al., 2009; Yourifuji et al., 2010). In developing children, omega-3 fatty acids play a role in brain development and visual acuity. A well-balanced diet that includes a variety of fish and shellfish can contribute to heart health and children's proper growth and development. So, women and young children in particular should include fish or shellfish in their diets due to the many nutritional benefits (Burger and Gochfeld, 2004; Oken et al., 2005; Kuntz et al., 2009). The special requirements for the developing nervous system in late gestation and early childhood make it important for pregnant and lactating women to have sufficient dietary intake of omega-3 fatty acids, which is most readily achieved by eating fish. By including moderate amounts of a variety of fish in a balanced diet, and choosing fish that are low in methylmercury, women can provide these essential nutrients to their babies both in uterus and through breast milk, while at the same time minimizing their exposure to environmental pollutants (Mahaffey, 2004; Virtanen et al., 2006). Nutritional benefits may be compromised by the health risks of toxic contaminants in many fish. Potentially, some fish are sufficiently contaminated with environmental pollutants, such as methylmercury (Clarkson, 2002). Therefore, balancing the benefits and harm remains a risk communication challenge (Knuth et al., 2003; Gochfeld, 2004; Huang et al., 2005; Willett, 2005; Verger et al., 2007; Mahaffey et al., 2008).

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FISH CONSUMPTION LIMITS Levels of methylmercury, measured in parts per million (ppm) or (mg/Kg) wet weight, vary greatly, based upon the species, size and age of the fish (Kureishy, 1993; Al-Majed and Perston, 2000 a,b; Sadiq et al., 2002; Kehrig et al, 2007; Magalhaes et al., 2007). In general, methylmercury levels for most fish range from less than 0.01 to 0.5 mg/kg, according to the Food and Drug Administration (FDA). The average concentration for commercially important species is less than 0.3 mg/kg. However, in large predator fish, methylmercury levels can reach up to 1 mg/kg, which is the limit allowed by the FDA in fish intended for human consumption (Lacerda et al., 2000; USEPA, 2001; Chen et al., 2004; Voegborlo and Akagi, 2007; USFDA, 2006). The risks from mercury in fish and shellfish depend on the amount of fish and shellfish eaten and the level of mercury in the fish and shellfish. Therefore, the FDA and the Environmental Protection Agency (EPA) are advising women who may become pregnant, pregnant women, nursing mothers, and young children to avoid some types of fish and eat fish and shellfish that are lower in mercury (Mahaffey, 2004; USFDA, 2006).

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TOXICITY OF MERCURY AND METHYLMERCURY Pharmacokinetics Effects Methylmercury carries the largest risk due to its high mobility in the body. Approximately 95% of methylmercury ingested is absorbed through the gastrointestinal tract, and is distributed to all tissues in about 30 hours. Methylmercury is somewhat lipophilic, allowing it to pass through lipid membranes of cells and facilitating its distribution to all tissues, where it binds readily to proteins fish muscle tissue. Skinning, trimming and cooking the fish do not significantly reduce the mercury concentration in the fillet. Since moisture is lost during cooking, the concentration of mercury after cooking is actually higher than it is in the fresh uncooked fish (USEPA, 2001; Clarkson, 2002; Mahaffey, 2004; Virtanen et al., 2006). On the other hand, Methylmercury is present in the body as water-soluble complexes mainly, if not exclusively, attached to the sulphur atom of thiol ligands. It enters the endothelial cells of the blood brain barrier as a complex

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with L-cysteine. The process is so specific that the complex with the optical isomer D-cysteine is not transported. Structurally, the L-complex is similar to the large neutral amino acid L-methionine and is carried across the cell membrane on the large neutral amino acid carrier (Clarkson, 2002). Following ingestion and distribution, about 5% of the methylmercury is found in blood and 10% in the brain. The methylmercury concentration in red blood cells is approximately 20 times that in the plasma (Clarkson, 2002). On the other hand, methylmercury can accumulate in hair and toenails and can be used as indicators for long term mercury exposure. In hair, mercury levels about 250 times larger than blood levels (WHO, 1990). Methylercury is also distributed to the fur and feathers of wild life. The highest mercury levels in humans are generally found in the kidneys (Levy et al., 2003). Methylmercury crosses the placental barrier with cord blood levels similar or slightly higher than levels in maternal blood. Fetal brain mercury levels are approximately 5-7 times higher than in maternal blood. The excretion process for methylmercury involves its demethylation, converting it (half life in body is about 70-80 days) to inorganic form, then elimination from the body in the feces (UNEP, 2005).

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ACUTE TOXICITY The estimated lethal dose of methylmercury is 10 to 60 mg/kg. Acute high-level exposures to methylmercury may result in impaired central nervous system function, damage of kidney and failure, gastrointestinal damage, cardiovascular collapse, shock, and death (Harda, 1995; USEPA, 2001).

CHRONIC TOXICITY Both elemental mercury and methylmercury produce a variety of health effects at relatively high exposures. While recent studies indicate that lower dose exposure can have effects on the cardiovascular and immune systems, neurotoxicity is the effect of greatest concern. This is true whether exposure occurs to the developing embryo or fetus during pregnancy or to adults and children (Grandjean et al., 1997; Gulson et al., 1998; Hites et al., 2004). Two major episodes of long-term methylmercury poisoning through fish consumption has been recorded in Japan. The first occurred in the early 1950s among people, fish consuming domestic animals such as cats, and wildlife

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living near Minamata City on the shores of Minamata Bay, Kyushu, Japan. The source of the methylmercury contamination was effluent from a chemical factory that used mercury as a catalyst and discharged wastes into the bay where it accumulated in fish and shellfish that were a dietary staple of this population (Qian, 2001, UNEP, 2005). In 1965, another methylmercury poisoning incident occurred in the area of Niigata, Japan. The signs and symptoms of the disease in Niigata were similar to those of methylmercury poisoning in Minamata (JECFA, 2003; UNEP, 2005). Methylmercury poisoning also occurred in Iraq following consumption of seed grain that had been treated with a fungicide containing methylmercury. The first outbreak occurred prior to 1960; the second occurred in the early 1970s (USEPA, 2001; UNEP, 2005). In this case, imported mercury-treated seed grains that arrived after the planting season were ground into flour and baked into bread. Unlike the longterm exposures in Japan, the epidemic of methylmercury poisoning in Iraq was short in duration lasting approximately 6 months. The signs and symptoms of methylmercury poisining disease in Iraq were predominantly in the nervous system, including difficulty with peripheral vision or blindness, sensory disturbances, incoordination, impairment of walking, and slurred speech. Both children and adults were affected (Shao et al., 2006).

DEVELOPMENTAL TOXICITY Data are available on developmental effects in animals. Also, convincing data from a number of human studies (i.e., Minamata and Iraq) indicate that methylmercury causes subtle to severe neurological effects depending on dose and individual susceptibility. EPA considers methylmercury to have sufficient human and animal data to be classified as a developmental toxicant. Methylmercury accumulates in body tissue; consequently, maternal exposure occurring prior to pregnancy can contribute to the overall maternal body burden and result in exposure to the developing fetus. In addition, infants may be exposed to methylmercury through breast milk (Ubillus et al., 2000; USEPA, 2001; Renee et al., 2006; Davidson et al., 2008; Fonseca et al., 2008; Sakamoto et al., 2008, Rand et al. 2009). Some infants born to mothers who had consumed methylmercury contaminated grain (particularly during the second trimester of pregnancy)

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showed nervous system damage even though the mother was only slightly affected or asymptomatic (Carrington, 2004).

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MUTAGENICITY, NEUROTOXICITY AND CYTOTOXICITY Methylmercury appears to be clastogenic but not to be a point mutagen; that is, mercury causes chromosome damage but not small heritable changes in DNA. EPA has classified methylmercury as being of high concern for potential human germ cell mutagenicity (USEPA, 2001). Chronic exposure to Methylmercury may lead to induced-neurotoxicity that can be identified by three major mechanisms resulting in neural cell death as well as cytoarchitectural alterations in the nervous system. The major three mechanisms include calcium homeostasis disruption, overproduction of reactive protein species inducing oxidative stress or reduction of antioxidative defenses, and interactions with sulfhydryl groups (Rice, 2008; Ceccatelli et al., 2010). Morphological and cytotoxic effects has also being associated with feeding on fish contaminated with Methylmercury. The liver of exposed individuals showed infiltration of leukocytes, increased number of melanomacrophage centers, lesions and necrotic areas in Disse’s space, chaos and disorder in cytoskeleton organization suggesting toxic effect in hepatocytes. Head kidney showed increased necrotic areas, increased centers of melanomacrophages, increased phagocytic areas suggesting too slow mechanisms of defense to Methylmercury (Mela et al., 2007).

DISTRIBUTION OF MERCURY IN FISH SAMPLES Several factors are associated with the variation in Hg concentrations in fish including trophic level, age, and size of fish (Kureishy, 1993; Al-Majed and Preston, 2000a; Billard and Lecointre, 2001). Higher concentrations of Hg are usually detected in larger fish than smaller fish especially within a species. Fish that are high in the food chain biomagnify contaminant such as Hg and accumulate them in higher concentrations (Nakagawa et al., 1997; Al-Majed and Preston, 2000a; Mora et al., 2004; Freije and Awadh; 2009). Carnivore’s fish can sometimes be an exception, having higher concentrations due to their

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trophic position. Those carnivores’ fish can be used as a good indicator of mercury pollution monitoring (Vigh et al., 1996). Several studies on mercury accumulation in fish have shown a significant positive correlation between T-Hg and MeHg suggesting that more organic mercury (MeHg) accumulate in fish muscles than inorganic mercury (Hg) (Akagi et al., 1995; Al-Majed and Preston, 2000a; Mora et al., 2004; Freije and Awadh, 2009). MeHg is one of the most toxic forms of Hg in aquatic ecosystems which is bioavailable and biomagnify in the food chain resulting in relatively high levels in large fish. The proportion of MeHg relative to Hg in fish species studied ranged from 87.5 to 97.6% (Chvojka et al., 1990; Anderson and Depledge, 1997; Kannan et al., 1998; Al-Majed and Preston, 2000a; Campbell et al., 2003; Freije and Awadh, 2009). The T-Hg and MeHg concentrations of fish muscles also correlate positively with fish weight and forklength which also represent the age and hence exposure (Allen-Gil et al., 1997; Phillips et al., 1997; Stafford, 1997; Romeo et al., 1999; Al-Majed and Preston, 2000a; Billard and Lecointre, 2001; Agusa et al., 2004; Freije and Awadh, 2009). Generally, diet and living environment influence mercury and methylmercury content in muscle tissues of fish (Vigh et al., 1996; Svobodoval et al., 2004; Sarica et al., 2005; Sehee et al., 2010). It is well established that nearly all of mercury in fish muscles (≥95%) occur as methylmercury and is mostly transferred to fish through their food. Therefore, fish species that feed on other fish have higher mercury concentrations which increases in these predators as they age (Gray, 2002).In general, muscles of predatory fish contain higher concentrations of T-Hg and MeHg, than nonpredatory fishes. The lowest concentrations of T-Hg and MeHg recorded were in the muscles of fish which primarily feed on plankton, whereas the highest concentrations were detected in long-lived top predator fish (AlArrayed et al., 1999; Fishbase, 2006; Freije and Awadh, 2009). The trophic positions of the different species should also be taken in consideration when comparing T-Hg and MeHg in fish. Fish that are at the top of the food chain as well as being an epiplagic and a demersal fish feeding respectively on Small fish show a relatively higher T-Hg and MeHg contents in comparison with other fish species. On the other hand, fish that are predominantly planktonic surface feeder have the lowest concentrations of THg and MeHg. (Carpenter et al., 1997; Fisher and Bianchi, 1984; Anderson and Depledge, 1997; Al-Majed and Preston, 2000a; Surette et al., 2006; Freije and Awadh; 2009). Anderson and Depledge (1997) reported an obvious

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difference between the contaminant distribution in epipelagic, mesopelagic and demersal fish. They have suggested that the epipelagic fish are more exposed to atmospheric inputs and photochemical production of MeHg, wheres demersal fish are more influenced by sediment conditions (Surette et al., 2006).

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HUMAN HEALTH RISK ASSESSMENT The benefits of fish consumption are well documented in the literature. Fish is an excellent source of omega-3 fatty acids that reduce cholesterol levels and the incidence of stroke, heart disease and pre-term delivery (Daviglus et al., 2002; Patterson, 2002; Conner, 2000; Whelton et al., 2004; Gochfeld and Burger, 2005; Fernandes and Venkatraman, 2006). Despite these benefits, fish consumption is the main pathway for human exposure for MeHg (Rice et al., 2000). MeHg can cross the placenta posing a direct effect on the developing fetus. Chronic exposure of fetus to MeHg can cause infants to be born with mental retardation and to exhibit cerebral palsy like symptoms (Gulson et. al., 1998). The ingestion of mercury contaminated fish for long term induce numbness in extremities, tremors, spasms, personality and behavioral changes, difficulty in walking, defenses, blindness, and finally lead to death (IOM, 1991). The action level of T-Hg and MeHg in fish (0.5-1.0 mg/kg wet weight) is considered as the upper tolerance limit for human consumption by several authorities including the United States Food and Drug Administration (USFDA), the Australian National Health and Medical Research Council (NHMRC), the Common-wealth and Victorian Statutory, and the USEPA (AlHashimi and Al-Zorba, 1991, USFDA, 2006). However the WHO permissible limits is 0.5 mg/kg wet weight for T-Hg and 0.3 mg/kg wet weight for Me-Hg (WHO, 1990), whereas Burger and Gochfeld (2006) considered this limit as a regulatory level rather than risk level.

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Akagi, H., Malm, O., Branches, F.J.P., Kinjo, Y., Kashima, Y., Guimaraes, J.R.D., Haraguchi, K., Pfeiffer, W.C., Takizawa, Y., Kato, H. (1995) Human Exposure to Mercury due to Gold-mining in the Tapajos River Basin, Amazon, Brazil: Speciation of Mercury in Human Hair, Blood, and Urine. Water, Air and Soil Pollution 80, 85-94. Al-Arrayed, F.H., Al-Maskati, H., Abdullah, F.J. (1999) n-3 polyunsaturated fatty acids content of some edible fish from Bahrain waters. Estuarine, coastal and shelf Science49,109-114. Al-Hashimi, A.H., Al-Zorba, M.A. (1991) Mercury in some commercial fish from Kuwait: a pilot study. The Science of the Total Environment106, 7182. Allen-Gil, S.M., Gubala, C.P., Landers, D.H., Lasorsa, B.K., Crecelius, E.A., Curtis, L.R. (1997) Heavy metal accumulation in sediment and freshwater fish in US Arctic lakes. Environmental Toxicology and Chemistry 16, 733741. Al-Majed, N.B., Preston, M.R. (2000a) An assessment of the total and methyl mercury content of zooplankton and fish tissue collected from Kuwait territorial waters. Marine Pollution Bulletin40, 298–307. Al-Majed, N.B., Preston, M.R. (2000b) Factors influencing the total mercury and methyl mercury in the hair of the fishermen of Kuwait. Environmental Pollution 109, 239–250. Anderson, J.L., Depledge, M.H. (1997) A survey of total mercury and methymercury in edible fish and invertebrates from Azorean waters. Marine Environmental Research44(3), 331-350. Bailey, E.A., Gray, J.E. and Hines, M.E. (2001) Mercury transformations in soils near mercury mines in Alaska. Materials and Geoenvironment 48(1), 212-218. Bergan, T., Gallaedo, L., Rohde, H. (1999) Mercury in the global atmospherea three dimensional model study. Atmospheric Environment33, 15751585. Bergan, T., Rohde, H. (2001) Oxidation of elemental mercury in the atmosphere; constraints imposed by global scale modeling. Journal ofAtmospheric Chemistry 40, 191-212. Billard, R., Lecointre, G. (2001) Biology and conservation of sturgeon and paddle fish. Reviews in Fish Biology and Fisheries 10, 355-392. Bloom, N.S. (1992) On the chemical form of mercury in edible fish and marine invertebrate tissue. Canadian Journal of Fisheries and Aquatic Science 49, 1010-1017.

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Davidson, P.W., Strain, J.J., Myers, G.J., Thurston, S.W., Bonham, M.P. et al. (2008) Neurodevelopmental effects of maternal nutitional status and exposure to methylmercury from eating fish during pregnancy. NeuroToxicology 29(5), 767-775. Daviglus, M., Sheeshka, J., Murkin, E. (2002) Health benefits from eating fish. Comments Toxicology(8), 345–74. Dorea, J.G., Barbosa, A.C., Souzade, J., Fadini, P., Jardim, W.F. (2003) Piranhas (Serrasalmus spp.) as markers of mercury bioaccumulation in Amazonian ecosystems. Exotoxicology and Environmetal Safety 59(10), 57-63. Dorea, J.G. (2009) Studies of fish consumption as source of methylmercury should consider fish-meal-fed farmed fish and other animal foods. Environmental Research 109(1), 131-132. Downs, S.G., MacLeod, C.L., Lester, J.N., (1998) Mercury in precipitation and its relation to bioaccumulation in fish: A literature review. Water Air Soil Pollution 108, 149-187. Fernandes, Ph.D.G., Venkatraman, Ph.D.J. (2006) Role of omega-3 fatty acids in health and disease. Nutrition Research, Obesity, DietaryLlipids and Hyperlipidemia13(1), S19-S45. Fischer, W., Bianchi G. (eds.). (1984) FAO species identification sheets for fishery purposes. Western Indian Ocean (Fishing Area 51). Prepared and printed with the support of the Danish International Development Agency (DANIDA). FAO, Rome. Vol. 1-6. Fonseca, M. de F., Dorea, J. G., Bastos, W. R., Marques, R. C., Torres, J. P.M., Malm, O. (2008) Poor psychometric scores of children living in isolated riverine and agrarian communities and fish-methylmercury exposure. NeuroToxicology 29(6), 1008-1015. Freije, A., Awadh, M (2009) Total and methylmercury intake associated with fish consumption in Bahrain. Water and Environment Journal 23(2), 155164. FVSRC, Food and Veterinary Service Research Centre (2005) Determination of mercury and methylmercury contamination level in industrially utilized predatory freshwater fish in Latvia. Research project report(PVD), Riga. Gray, J.S., (2002) Biomagnification in marine systems: the perspective of an ecologist. Marine Pollution Bulletin 45, 46-52. Grandjean, P., Weihe P, White R., Debes, F., Araki, S., Yokoyama, K. (1997) Cognitive deficit in 7-year-old children with prenatal exposure to methylmercury. Neurotoxicology Teratology 20, 1-12.

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Gochfeld, M. (2004) Cases of mercury exposure, bioavailability, and absorption. Ecotoxicology and Environmental Safety 56,174–9. Gochfeld, M., Burger, J. (2005) Good fish/bad fish: a composite benefit-risk by dose curve. NeuroToxicology26, 511-520. Gulson, B.L., Mahaffey, K.R., Jameson, C.W., Mizon, K.J., Korsch, M.J., Cameron, M.A., et al. (1998) Mobilization of lead from the skeleton during the postnatal period is larger than during pregnancy. Journal of Laboratory of Clinical Medicine 131, 324–9. Harada, M., (1995) Minamata disease: methylmercury poisoning in Japan caused by environmental pollution. Critical Reviews in Toxicology 25, 125. Harding, k.M., Gowland, J.A., Dillon, P.J. (2006) Mercury concentration in black flies Simulium spp. (Diptera, Simuliidae) from soft-water streams in Ontario, Canada. Environmental pollution 143, 529-535. Hites, R.A., Foran, J.A., Carpenter, D.O., Hamilton, M.C., Knuth, B.A. and Schwager, S.J. (2004) Global assessment of organic contaminants in farmed salmon. Science303, 226–229. Huang, L-S, Cox, C., Myers, G.J., Davidson, P.W., Cernichiari, E., Shamlaye, C.F., Sloane-Reeves, J., Clarkson, T.W. (2005) Exploring nonlinear association between prenatal methylmercury exposure from fish consumption and child development: evaluation of the Seychelles Child Development Study nine-year data using semiparametric additive models. Environmental Research 97(1), 100-108. Hursh, J.B. (1985) Particle coefficients of mercury (203Hg) vapour between air and biological fluids. Journal of applied Toxicology (5), 327-332. Ikingura, J.R., Akagi, H. (2003) Total mercury and methylmercury levels in fish from hydroelectric reservoirs in Tanzania. The Science of the Total Environment 304(1-3), 355-368. (IOM) Institute of Medicine (1991) Seafood Safety. Washington, DC: National Academy Press. JECFA (2003) Joint Expert Committee on food additives of the Food Agriculture Organization and the World Health Organization. Available: www.sciencedirect.com. Kannan, K., Smith, G., Lee, F., Windom, L., Hertmuller, I., Macauley, M., Summers, K. (1998). Distribution of total mercury and methylmercury in water, sediment, and fish from South Florida estuaries. Archives of Environmental Contamination and Toxicology 34, 109-118.

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Kehrig, H. do A., Howard, B.M., Malm, O. (2008) Methylmercury in a predatory fish (Cichla spp.) inhabiting the Brazilian Amazon. Environmental Pollution 154(1), 68-76. Knuth, B.A., Connelly, A., Sheeshka, N., Patterson, J. (2003) Weighing health benefit and health risk information when consuming sport–caught fish. Risk Analysis 23, 1185–97. Kris, E.P.M., Harris, W.S., Appel, L.J. (2002) Fish consumption, fish oil, omega-3 fatty acids and cardiovascular disease. Nutrition committee 106, 2747-57. Kuntz, S.W., Hill, W.G., Linkenbach, J.W., Lande, G, Larsson, L. (2009) Methylmercury risk and awareness among American Indian women of childbearing age living on an inland northwest reservation. Environmental Research 109(6), 753-759. Kureishy, T.W., (1993) Concentration of heavy metals in marine organisms around Qatar before and after the Gulf War oil spill. Marine Pollution Bulletin 27, 183–186. Lacerda, L.D., Paraquetti, H.H.M., Marins, R.V., Rezedende, C.E., Zalmon, I.R., Gomes, M.P. (2000) Mercury content in shark species from the South –Eastern Brazilian Coast. Reviews in Brazilian Biology 60, 571-576. Lamborg, C.H., Fitzgerald, W.F., O’Donnell, J., Torgerson, T. (2002) A nonsteady-state compartmental model of global-scale mercury biogeo chemistry with interhemispheric atmospheric gradients. Lawrence, A.L., Mason, R.P. (2001) Factors controlling the bioaccumulation of mercury and methylmercury by the estuarine amphipod Leptocheirus plumulosus. Environmental Pollution 111, 217-231. Levy, M., Schwartz, S., Dijak, M., Weber, J.P., Tardif, R., Rouah, F. (2003) Childhood urine mercury exertion: dental amalgam and fish consumption as exposure factors. Environmental Research 94(3), 283-290. Lindberg, S. E., Wallschlager, D., Prestbo, E. M., Bloom, N. S., Price, J. and Reinhart, D. (2001) Methylated mercury species in municipal waste landfill gas sampled in Florida, USA. Atmospheric Environment 35, 40114015. Lindquist, O., Jernelov, A., Johansson, K., Rohde, H. (1984) Mercury in the Swedish Environment. Global and local sources, report 1816. National Swedish Environmental Protection Agency, Stockholm, 121 pp. Mahaffey, K.R. (2004) Fish and shellfish as dietary sources of methylmercury and the omega-3 fatty acids eicosahexaenoic and docasahexaenoic acid: Risks and benefits. Environmental Research95, 414-428.

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Mahaffey, K.R., Clickner, R.P., Jeffries, R. A. (2008) Methylmercury and omega-3 fatty acids: Co-occurrence of dietary sources with emphasis on fish and shellfish. Environmental Research 107(10), 20-29. Magalhaes, M.C., Costa, V., Menezes, G. M., Pinho, M.R., Santos, R.S., Monteiro, L.R. (2007) Intra- and inter-specific variability in total and methylmercury bioaccumulation by eight marine fish species from the Azores. Marine Pollution Bulletin 54(10), 1654-1662. Marvin-Dipasquale, M., Agee, J., McGowan, C., Oremland, R., Thomas, M., Krabbenhoft, D. and Gilmour, C.C. (2000) Methyl-mercury Degradation Pathways: A Comparison among Three Mercury Impacted Ecosystems. Environmental Science and Technology 34, 4908-4916. Mason, R.P., Fitzgerald, W.F., and Morel, M.M. (1994) The biogeochemical cycling of elemental mercury: Anthropogenic influences. Geochimica et Cosmochimica Acta 58(15), 3191-3198. Mason, R.P., Fitzgerald, W.F. (1996) Sources, sinks and biochemical cycling of mercury in the ocean. In: Baeyens, W., Ebinghaus, R. and Valiliev, O. (eds.): Global and regional mercury cycles: Sources, fluxes and mass balances. NATO ASI Series, 2. Environment - Vol. 21. Kluwer Academic Publishers, Dordrecht, The Netherlands. Mason, R.P., Fitzgerald, W.F. (1997) Biogeochemical cycling of mercury in the marine environment. In: Sigel, A. and Sigel, H.: Metal ions in biological systems. Marcel Dekker, Inc. 34, 53-111. McCrary, J.K., Castro, M., McKaye, K.R. (2006) Mercury in fish from two Nicaraguan lakes: A recommendation for increased monitoring of fish for international commerce. Environmental Pollution 141, 513-518. Mela, M., Randi, M.A.F., Ventura, D.F., Carvalho, C.E.V., Pelletier, E., Oliveria Ribeiro, C.A. (2007) Effect of dietry methylmercury on liver and kidney histology in the neotropical fish Hoplias malabaricus. Ecotoxicology and Environmental safety 68(3), 426-435. Mora, S.J., Fowler, S.W., Wyse, E., Azemard, S. (2004) Distribution of heavy metals in marine bivalves, fish and coastal sediments in the Gulf and Gulf of Oman. Marine Pollution Bulletin 49(5-6), 410-24. Morel, F.M.M., Kraepiel, A.M.L., Amyot, M. (1998) The chemical cycle and bioaccumulation of mercury. Annual Reviews of Ecology and Systematic 29,543-566. Murphy, C.A., Rose, K.A., Alvarez, M.del C., Fuiman, L.A. (2008) Modeling larval fish behavior: Scaling the sublethal effects of methylmercury to population-relevant endpoints. Aquatic Toxicology 86(4), 470-484.

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Nakagawa, R., Yumita, Y., Hiromoto, M. (1997) Total mercury intake from fish and shellfish by Japanese people. Chemosphere 35, 2909-2913. Niencheski, L.F., Windom, H.L., Baraj, B., Wells, D., Smith, R. (2001) Mercury in fish from Patos and Mirim Lagoons, Southern Brazil. Marine Pollution Bulletin 42(12), 1403-1406. Oken, E., Wright, R.S., Kleinman, K.P., Bellinger D., Amarasiriwardena, C.J. (2005) Maternal fish consumption, hair mercury, and infant cognition in a US. Health Perspective 113, 1376-1380. Pacyna, J.M., Pacyna, E.G. (2000) Assessment of emissions/discharges of mercury reaching the Arctic environment. The Norwegian Institute for Air Research, NILU Report Or 7/2000, Kjeller, Norway. Patterson, J. (2002) Introduction–comparative dietary risk: balance the risks and benefits of fish consumption. Comments Toxicology 8, 337–344. Phillips, R., Heilprin, J., Hart, A. (1997) Mercury accumulation in barred sand bass (Paralabrax nebulifer) near a large wastewater outfall in the Southern California Bight. Marine Pollution Bulletin34, 96-102. Qian, J. (2001) Mercury species in environmental samples studied by spectroscopic methods (doctoral thesis). Printed by: SLU, Grafiska Enheten, Umea, Sweden. Rand, M.D., Dao, J.C., Clason, T.A. (2009) Methylmercury distribution of embryonic neural development in Drosophila. NeuroToxicology 30(5), 794-802. Renee, L., Sato, M.D., Gaylyn, G.L.M.D., Steve S. Phd. (2006) Antepartum seafood consumption and mercury levels in newborn cord blood. American Journal of Obstetrics and Gynecology 194(6), 1683-1688. Rice, G., Swartout J., Mahaffey K., Schoeny R. (2000) Derivation of U.S. EPS's oral Reference Dose (RfD) for methylmercury. Drug Chemistry Toxicology 23,41–54. Rice, D.C. (2008) Overview of modifiers of methylmercury neurotoxicity: Chemicals, nutrients, and the social environment. NeuroToxicology 29(5), 761-766. Romeo, M., Siau, Y., Sidoumou, Z., GnassiapBarelli, M. (1999) Heavy metal distribution in different fish species from the Mauritania coast. The Science of the Total Environment 232, 169-175. Sadiq, M., Saeed, T., Fowler, S.W. (2002) Seafood contamination. In:Khan, N.Y., Munawar, M., Price, A.R.G., Editors, 2002. The Gulf Ecosystem: Health and Sustainability, Bakhuys Publishers, Leiden, pp. 327-351. Sakamoto, M., Kubota, M., Murata, K., Nakai, K., Sonoda, I., Satoh, H. (2008) Changes in mercury concentrations of segmental maternal hair

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during gestation and their correlations with other biomarkers of fetal exposure to methylmercury in the Japanese population. Environmental Research 106(2), 270-276. Sanzo, J.M., Dorronsoro, M., Amiano, P., Amurrio, A., Aguinagalde, F.X., Azpiri, M.A. and EPIC Group of Spain. (2001) Estimation and validation of mercury intake associated with fish consumption in an EPIC cohort of Spain. Public Health Nutrition 4(5), 981-988. Sarica, J., Amyot, M., Hare, L., Blanchfield, P., Bodaly, R.A., Hintelmann, H., Lucotte, M. (2005) Mercury transfer from fish carcasses to scavengers in boreal lakes: the use of stable isotopes of mercury. Environmental Pollution 134, 13-22. Sehee, O., Moon-Kyung, K., Seung-Muk, Y., Kyung-Duk, Z. (2010) Distribution of total mercury and sediments and fishes in Lake Shihwa, Korea. Science of The Total Environment 408(5), 1059-1068. Seiders, K. (2006) Measuring mercury trends in freshwater fish in Washington State. Quality assurance project plan. Available: www.ecy.wa.gov/ biblio/0603103.html. Shao, L.j., Gab, W.E., Su, Q.D. (2006) Determination of total and inorganic mercury in fish samples with on-line oxidation coupled to atomic fluorescence spectrometry. Analytica Chimica Acta 562, 128-133. Stafford, P. (1997) Mercury concentrations in Maine sport fishes. Transactions of the American Fisheries Society 126, 144-152. Surette, C., Lucotte, M., Tremblay, A. (2006) Influence of intensive fishing on the partitioning of mercury and methylmercury in three lakes of Northen Quebec. Science of the Total environment 368, 248-261. Svobodova, Z., Celechovska, O., Kolarova, J., Randak, T., Zlabek, V., 2004. Assessment of metal contamination in the upper reaches of the Ticha Orlice river. Czech Journal of Animal Sciences 49, 458-464. Thurston, S.W., Bvet, P., Myers, G.J., Davidson, P. W., Georger, L.A., Shamlaye, C., Clarkson, T.W. (2007) Does prenatal methylmercury exposure from fish consumoption affect blood pressure in childhood. NeuroToxicology 28(5), 924-930. Travnikov, O., Ryaboshapko, A. (2002) Modelling of mercury hemispheric transport and depositions. EMEP/MSC-E Technical Report 6/2002, Meteorological Synthesizing Center-East, Moscow, Russia. Ubillus, F., Alergria, A., Barbera, R., Farre, R., Lagarda, M.J. (2000) Methyl mercury and inorganic mercury determination in fish by cold vapour generation atomic absorption spectrometry. Food chemistry 71(4),529533.

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Ullrich, S. M., Tanton, T.W., Abdrashitova, S. A. (2001) Mercury in the Aquatic Environment: A Review of Factors affecting Methylation. Critical Reviews in Environmental Science and Technology 31(3), 241293. UNEP, United Nations Environment Programme (2004) Regional awarenessraising workshop on mercury pollution. A global problem that needs to be addressed, UNEP, Geneva, Switzerland. Available: http//www.chem.unep. ch/mercury/. UNEP, United Nations Environment Programme (2005) Global mercury assessment, UNEP, Geneva, Switzerland. Available:http://www.chem. unep.ch/MERCURY/Report/Final%20Assessment%20report.htm. USEPA, United States Environmental Protection Agency(1997) Mercury study report to congress,Vol.1:Executive summary. Available:www.epa. gov/ttn/caaa/t3/reports/volume1.pdf. USEPA, United States Environmental Protection Agency (2001) Mercury update: Impact on fish advisories. EPA Fact sheet, June 2001. Available: http://www.epa.gov/ost/fish, June 2001. USFDA Food and Drug Administration(2006) Mercury levels in commercial fish and shellfish. Available: www.cfsan.fda.gov/-frf/sea-mehg. USGS,United States Geological Survey (2000) Mercury in the environment. U.S. Geological Survey,Fact sheet 146-0. Available:www.usgs.gov/ themes/factsheet/146-00/index.html. Verger, P., Houdart, S., Marette, S., Roosen, J., Blanchemanche, S. (2007) Impact of a risk-benefit advisory on fish consumotion and dietary exposure to methylmercury in France. Regulatory Toxicology and Pharmacology 48(3), 259-269. Vigh, P., Mastala, Z., Balogh, V. (1996) Comparison of Heavy metal concentration of grass carp in a shallow eutrophic lake and a fish pond (possible effect of food contamination). Chemosphere 32, 691-701. Virtanen, J.K., Rissanen, T.H., Voutilaninen, S., Tuomainen, T.P. (2006) Mercury as a risk factor for cardiovascular diseases. Journal of Nutritional Biochemistry.In Press, Corrected Proof, Available online 16 June 2006. http://www.sciencedirect.com/ Voegborlo, R.B., Akagi, H. (2007) Determination of mercury in fish by cold vapour atomic absorption spectrometry using an automatic mercury analyzer. Food Chemistry 100, 853-858. Whelton, S.P., He J., Whelton, P.K., Muntner, P. (2004) Meta-analysis of observational studies on fish intake and coronary heart disease. American Journal of Cardiology 93(9), 1119-1123.

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WHO/IPCS, World Health Organisation, International Programme on Chemical Safety (1989) Mercury – Environmental aspects. Environmental Health Criteria No 86, Geneva, Switzerland. WHO/IPCS, World Health Organisation, International Programme on Chemical Safety (1990) Methylmercury. Environmental Health Criteria No 101, Geneva, Switzerland. WHO/IPCS, World Health Organisation, International Programme on Chemical Safety (1991) Inorganic mercury. Environmental Health Criteria No 118, Geneva, Switzerland. WHO/IPCS, World Health Organisation, International Programme on Chemical Safety (2003) Elemental mercury and inorganic mercury compounds. Concise International Chemical Assessment Document No 50. Willett, W.C. (2005) Fish balancing health risks and benefits. American Journal of Preventive Medicine 29, 320–74. Yaginuma-Sakurai, K., Murata, K., Shimada, M., Nakai, K., Kurokawa, N., Kameo, S., Satoh, H. (2010) Intervention study on cardiac autonomic nervous effects of methylmercury from seafood. Neurotoxicology and Teratology 32(2), 240-245. Yorifuji, T. Tsuda, T., Kashima, S., Takao, S., Harada, M. (2010) Long-term exposure to methylmercury and its effect on hypertension in Mnamata. Environmental Research 110(1), 40-46.

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In: Methylmercury: Formation, Sources… ISBN: 978-1-61761-838-3 Editor: Andrew P. Clampet © 2012 Nova Science Publishers, Inc.

Chapter 3

INEXPENSIVE LOW-COST MERCURY SPECIATION BY HYDRIDE GENERATION ATOMIC ABSORPTION SPECTROMETRY AFTER ION EXCHANGE SEPARATION IN A FIA SYSTEM (FIA-IE-HG-AAS)

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A. Gredilla, J. Larreta, I. Martinez-Arkarazo, S. Fdez-Ortiz de Vallejuelo, G. Arana, J. C. Raposo, A. de Diego∗ and J. M. Madariaga Kimika Analitikoa Saila, Euskal Herriko Unibertsitatea, 644 P.K., E-48080 Bilbao, Spain

ABSTRACT Ion exchange is presented as a simple, non-expensive technique for the effective separation of inorganic mercury (Hg2+) and methylmercury (CH3Hg+) in hydrochloric or chloride media. For such chemical conditions, the negatively charged HgCl42- is susceptible to be retained by anionic exchangers, while the non-charged CH3HgCl should pass through the resin with negligible retention. Further elution of the retained Hg2+ makes it possible the analysis of both species in a single injection of sample. Preliminary experiments to select the best retention and elution ∗

Corresponding author; e-mail address: [email protected]

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A. Gredilla, J. Larreta, I. Martinez-Arkarazo et al. conditions were carried out in a batch mode by using a high precision syringe pump connected to a Pasteur pipette packed with the anionic resin Dowex M-41. Analysis of total mercury in the eluate was performed in a commercial FIA system by cold vapour atomic absorption spectrometry (FIA-HG-QFAAS). The separation step was then implemented in the FIA system by including an Omnifit column packed with the resin immediately after the injection valve. The system was optimised to get maximum sensitivity in the analysis. The absolute detection limits estimated were 1.9 ng of Hg2+ and 0.8 ng of CH3Hg+. The utility of the method was confirmed by analysis of (a) synthetic mixtures of Hg2+ and CH3Hg+ in hydrochloric acid and (b) natural estuarine waters spiked with Hg2+ and CH3Hg+.

Keywords: mercury speciation, ionic exchange, flow injection, hydride generation, atomic absorption spectrometry.

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1. INTRODUCTION Anthropogenic uses of mercury during the last decades have derived in increasingly concentrations of this metal in different environmental compartments [1]. Elemental mercury (Hg0), inorganic mercury (Hg2+) and methylmercury (CH3Hg+) are the most important species in natural water systems [2]. CH3Hg+ is the most toxic species due to its ability to cross the cell wall, and easily incorporates into the food chain [3, 4]. Hg2+ can transform into CH3Hg+ through biotic and abiotic ways, unless the mechanisms still remain unclear [2]. Mercury speciation analysis has been a continuous research topic during the last 20-30 years. Hyphenations between chromatography (gas or liquid) and atomic [5, 6] or mass detectors [7, 8] are the approaches more often used lastly. Combination with hydride generation or related techniques provides improved sensitivity and considerably reduces the matrix effects [6]. Preconcentration techniques (cryogenic trapping, solid phase extraction and microextraction, adsorption onto solid traps) are however still necessary in the analysis of certain samples. The use of sulphydryl cotton fibers has allowed separation of Hg2+ from CH3Hg+ and, simultaneously, preconcentration of the latter [9]. Most usually, the instrumentation involved in mercury speciation analysis is expensive in terms of equipment and maintenance [10]; although some private laboratories have incorporated such instrumentation, speciation

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of mercury has not yet become a routine analytical methodology in environmental laboratories. Ion exchange is a relatively cheap alternative to separate chemical species. It is versatile and usually provides an important clean up of the sample and a remarkable preconcentration of the analyte. It can be easily implemented in flow injection systems, resulting in automated and very reproducible procedures. Despite these characteristics, the potentiality of ion exchange in mercury speciation analysis has been only scarcely investigated by now [11]. More recently, it has been applied to remediation procedures [12, 13]. CH3Hg+ and Hg2+ exhibit different ionic character in a chloride medium. Thermodynamic data of these chemical systems (stability constants collected from [14-16]) shows that, at pH < 10 most of Hg2+ is in the anionic form HgCl42- while CH3Hg+ remains as its neutral chloro complex species, CH3HgCl. At these conditions, an anionic resin should retain the Hg(II) as HgCl42- and let methylmercury (as CH3HgCl) freely pass through it with negligible retention. In this work, we investigate the separation of CH3Hg+ and Hg2+ (in chloride medium) making use of an anionic exchange resin, e. g., Dowex M41. This resin was selected among other similar exchangers in a previous work [17]. First, the best conditions for the i) negligible retention of CH3Hg+, ii) quantitative retention of Hg2+, and iii) further quantitative elution of Hg2+ have been studied in a series of experiments performed in batch mode. Second, and based on the previous results, we propose an inexpensive method for the sequential analysis (single injection of sample) of CH3Hg+ and Hg2+ in an aqueous sample by cold vapour atomic absorption spectrometry in a FIA system, after separation of the analytes in a microcolumn packed with the anionic resin, Dowex M-41. Finally, we show the potentiality of the proposed method to make mercury speciation analysis in natural water samples, by analysing estuarine waters previously spiked with known amounts of CH3Hg+ and Hg2+. The aim of developing such an inexpensivea low-cost, but accurate, method for mercury speciation using standard atomic absorption instrumentation (cold vapour and quartz furnace AAS) is to help private laboratories working in the field of environmental analysis going further with mercury analysis issues apart from its total concentration.

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2. EXPERIMENTAL PROCEDURE 2.1. Safety Considerations Organomercury compounds, and specially methylmercury are extremely toxic. It may cause neurological damage as well as kidney malfunction. Direct contact with the skin may lead to death. Precautions and adequate clothing are absolutely necessary when manipulating the reagent. Disposal of all mercurycontaining waste from the experiment was performed in accordance with facility guidelines.

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2.2. Reagents Stock solutions of inorganic mercury (Hg2+) and methylmercury (CH3Hg+) (1000 μg·mlmL-1) were prepared, respectively, by dissolving the appropriate amount of mercury(II) chloride (HgCl2, Puratrem, Strem Chemicals; Bischeim, France) in several drops of nitric acid (69%, Tracepur, Merck; Darmstadt, Germany) or methylmercury chloride (CH3HgCl, Puratrem, Strem Chemicals; Bischeim, France) in several drops of acetone (p.a., Lab-Scan; Dublin, Ireland) and, in both cases, diluting to the mark with MilliQ quality water (conductivity< 0.05 μS·cm-1) (Millipore; Bedford, USA). These stock solutions were kept in dark at 4ºC for a maximum period of three months. Dilutions of these stocks in 2.0 mol·dm-3 hydrochloric acid (HCl, 36%, Tracepur, Merck; Darmstadt, Germany) were used to produce working solutions (single CH3Hg+, single Hg2+ or mixtures of both) at different concentrations. These diluted solutions were also kept in dark at 4ºC for a maximum of oneweek. Aqueous solutions of L(+)cysteine (C3H8ClNO2S·H2O, p. a., Merck; Darmstadt, Germany), sodium thiosulphate thiosulfate (Na2S2O3·5H2O, p. a., Merck; Darmstadt, Germany), dihydrate disodium ethylenediamine tetraacetate (Na2EDTA·2H2O, p. a., Panreac.; Barcelona, Spain), sodium sulfide (Na2S, p. a., Panreac.; Barcelona, Spain) and potassium thiocyanate (KSCN, puriss. p. a., Fluka; Buchs, Switzerland) were checked as eluents of the anion exchanger in the batch experiments. All of them were prepared by dissolving the adequate amount of the reagent in water. 2 mol·dm-3 hydrochloric solutions of L(+)cysteine were also used in the on-line elution experiments.

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The weakly basic anion exchange resin considered during the experiments, e. g., Dowex M-41 (Dow Chemical; Horgen, Switzerland) was used directly in their original chloride form. It was thoroughly counter current washed up with diluted sodium chloride solution and then with water before use. Solutions of 0.2% (w/v) sodium tetrahydroborate (NaBH4, p. a., Merck; Darmstadt, Germany), freshly prepared by dissolving the reagent in 0.05% (w/v) sodium hydroxide (NaOH, p. a., Merck; Darmstadt, Germany), were used as reducing agent in the derivatization step. Aqueous HCl solutions of different concentrations were used as carrier in the FIA system. The volatilised analytes were transported to the quartz furnace by a constant flow of argon (99.999%, Carburos Metálicos; Barcelona, Spain).

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2.3. Instrumentation Masses of reagents were weighted in a Mettler-AE 200 balance (instrumental precision ±0.0001 g, Mettler Toledo GMBH; Greifensee, Switzerland). Dilutions were prepared using 20, 100, 1000 and 5000 μL micropipettes (Eppendorf; Hamburg, Germany). A Metrohm 775 Dosimat high precision syringe pump was used in the experiments performed in batch mode to deliver precise volumes of solution through a Pasteur pipette (about 8 cm length and 6 mm i. d.) packed with the resin. The concentration of total mercury in the eluates was performed by cold vapour atomic absorption spectrometry. Atomic mercury was generated by on-line chemical reduction in a FIA system (FIAS 400, Perkin Elmer; Wellesley, USA), comprising two multi-channel peristaltic pumps, electronically controlled switching valves, one injection port, a merging zone and one liquid-gas phase separator. A flow of argon gas transported the mercury vapour generated to a hot T-shaped quartz furnace mounted in the light pathway of an atomic absorption spectrometer (4110ZL, Perkin Elmer; Wellesley, USA), equipped with an Hg electrodeless discharge lamp (Perkin-Elmer; Wellesley, USA). The 253.7 nm line was used in all the measurements. The whole system was automatically controlled by the AA Winlab 1.0 software (Perkin Elmer; Wellesley, USA). The measurement system is schematically shown in Figure 1a. The on-line speciation measurements were performed in a system similar to that shown in Figure 1a. In this case, an Omnifit column packed with the resin was inserted immediately after the injection valve (Figure 1b). Five different microcolumns with varying geometry (see Table 1) were checked to look for the best performance of the system.

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Figure 1. Schematic diagram of the measuring system for total mercury, FIA-HGQFAAS (a) and mercury speciation, FIA-IE-HG-QFAAS (b). PP1: sample pump; PP2: carrier pump; IP: injection port; G-L: gas-liquid separator; QFAAS: quartz furnace atomic absorption spectrometer.

Table 1. Identification and geometrical characteristics of the microcolumns checked in the FIA-IE-HG-QFAAS system Identification Length (cm) Diameter (mm) Weight of resin packed (g)

C1 10 5 ∼2

C2 10 6.6 ∼ 2.5

C3 10 10 ∼6

C4 10 3 ∼1

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C5 25 3 ∼2

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2.4. Analytical Procedure

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Retention and Elution Experiments in Batch Mode The Pasteur pipette was packed with about 2 g of wet resin, plugged with quartz wool and the outlet of the syringe was connected to the top of the pipette. In the retention experiments for Hg2+, two fractions of water were first passed through the pipette. Then, six fractions of Hg2+ solution (about 10 μg·L1 ) were propelled through the pipette. Finally, six fractions of water were used to rinse the pipette. A similar procedure was followed in the retention experiments for CH3Hg+, using a CH3Hg+ solution of about 20 μg·L-1. In the elution experiments, two fractions of water were first passed through the resin, and then, successively, two fractions of Hg2+ solution (about 10 μg·L-1), two more fractions of water, six fractions of a solution of the elution agent investigated (0.01 mol·dm-3 L(+)cysteine, 0.1 mol·dm-3 Na2S2O3, 0.1 mol·dm-3 Na2EDTA, 0.1 mol·dm-3 Na2S, 0.1 mol·dm-3 KSCN, 8 mol·dm-3 HCl and 2, 6, 8 and 10 mol·dm-3 HNO3) and, finally, three more fractions of water. The fractions were of 10 mL in all the cases and the flow rate of the syringe was always fixed at 1 mL·min-1. All the fractions were collected in separate flasks and analysed for total mercury following the procedure described below. Analysis of Total Mercury by FIA-HG-QFAAS The optimal experimental conditions found for the analysis of total mercury by FIA-HG-QFAAS using the system schematised in Figure 1a are as follows: 500 μL of sample containing CH3Hg+ or Hg2+ are injected in a flow of carrier solution (3% HCl), which is mixed with a stream of %0.2 NaBH4. Reaction takes place in the merging zone of the FIA system and the volatile mercury compound produced (CH3HgH or Hg0) is driven to the quartz furnace, hold at 900°C (CH3Hg+) or 100°C (Hg2+), by argon at a flow rate of 50 mL·min-1. The absorption is monitored along the process at 253.7 nm using a 0.7 nm slit. The analytical signal used is the height of the peak obtained in each case. The speed of the peristaltic pump propelling the solutions is fixed at 120 rev·min-1. In the optimisation experiments, some of the parameters (temperature of the quartz furnace, speed of the peristaltic pump and concentration of the carrier solution) were varied according to the experimental design. The analysis of each sample was, at least, twice repeated. When quantification was necessary, a calibration graph with, at least, five standard solutions was prepared in advance. A blank was always injected after the

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analysis of each sample to look for possible memory effects, which were never observed. A control solution with a known concentration of mercury was injected sometimes along the working day to follow the overall sensitivity of the system.

Analysis of CH3Hg+ and Hg2+ by FIA-IE-HG-QFAAS The analysis of the CH3Hg+ and Hg2+ in a liquid sample in a unique injection of sample by FIA-IE-HG-QFAAS was performed in two consecutive steps using the system shown in Figure 1b. In the first step (the CH3Hg+ mode), 500 μL of sample (sample pump at 100 rev·min-1) were injected in the flow of carrier (HCl 3%), pumped at 120 rev·min-1. The Hg2+ got retained in the C2 microcolumn (10 cm length, 6.6 mm i. d.), while the CH3Hg+ freely passed through it, was converted into CH3HgH, reached the quartz furnace and its absorbance was monitored and stored. In the second step (the Hg2+ mode), a 2 mol·dm-3 hydrochloric solution of L(+)cysteine (0.1 mol·dm-3) solution took the place of the HCl 3% as carrier and the pumps started working (the carrier pump at 120 rev·min-1 and the sample pump at 100 rev·min-1). The Hg2+ retained in the microcolumn during the previous step was then eluted in the flow of L(+)cysteine, reduced to elemental mercury and its signal monitored and stored. The absorbance was measured in both cases at the 253.7 nm line with a slit of 0.7 nm. The quartz furnace was always kept constant at 900°C. At this temperature, the sensitivity for Hg2+ is lower, but still high enough to determine the analyte in the samples considered in this work. In this way, the analysis time was considerably reduced, because the system needs about 90 minutes to cool down from 900°C to 100°C. In the optimisation experiments, the speed of the carrier pump, the concentration of the L(+)cysteine solution and/or the geometry of the column varied according to the experimental design.

3. RESULTS AND DISCUSSION 3.1. Optimisation of the FIA-HG-QFAAS Measuring System The working conditions recommended by Perkin Elmer for the analysis of total mercury [18] require that all the mercury present in the sample is in the form of Hg2+. The company proposes to heat the quartz furnace at 100°C. At this temperature, if CH3Hg+ is present in the sample, the CH3HgH produced in

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the derivatization reaction will not be transformed into elemental mercury and, consequently, a lower signal will be registered. If CH3Hg+ is the only mercury species present, no signal will be obtained at all. This is, in fact, the most probable situation in our experiments if the resin is working correctly. Taking this into account, we decided to optimise the analysis of total mercury, both in solutions containing only Hg2+ or only CH3Hg+ (10 μg·L-1), using the system shown in Figure 1a. First, a full factorial design with three factors at three different levels with four replicates of the central point was used in each case (Hg2+ or CH3Hg+) to check if the temperature of the quartz furnace (T), the speed of the peristaltic pump (F) pumping the carrier (HCl) and the reagent (NaBH4) and the concentration of the carrier solution (C) statistically influenced the analytical signal. The level of each factor at each experiment is shown in Table 2. The temperature of the quartz tube was not further increased due to instrumental limitations of the system. Pump speed higher than 120 rev·min-1 resulted in formation of bubbles and saturation of the gas-liquid separator.

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Table 2. Levels considered to construct the 2n-1 full factorial design used to check the influence of the temperature of the quartz furnace (T), the speed of the carrier pump (F) and the concentration of the carrier solution (C) on the sensitivity of the analysis of total mercury by FIA-HGQFAAS Factor T (°C) F (rev·min-1) C (% )

Low 100 40 2

Central 500 80 4

High 900 120 6

Analysis of variance (ANOVA) of the results showed that all the factors or any of their combinations significantly influence the sensitivity of the analysis at a 95% confidence level in both cases. In preliminary experiments, it was observed that solutions of NaBH4 more concentrated than 0.2% produced heavy bubbling which collapsed the gas-liquid phase separator, so that the concentration recommended by the manufacturer (0.2%) was used without further optimisation. Second, a central composite design (the same three factors at five different levels, see Table 3) was considered in each case (Hg2+ or CH3Hg+). Non-linear regression analysis of the results by the NLREG program [19] allowed defining polynomials to describe the surface of response in each case (Figure 2).

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Finally, the maximum of each surface showed the optimal conditions for each analysis (T= 900°C, F = 120 rev·min-1, C = 3% for CH3Hg+ and T= 100°C, F = 120 rev·min-1, C = 3% for Hg2+ respectively).

Figure 2. Response surfaces obtained for Hg2+ (a) and CH3Hg+ (b) in the optimisation process as a function of the temperature of the quartz furnace (T) and the speed of the peristaltic pump (F). The concentration of the carrier HCl has been kept constant at 3%.

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Table 3. Levels considered to construct the central composite design used to define the polynomials which best describe the dependence of the responses with the temperature of the quartz furnace (T), the speed of the carrier pump (F) and the concentration of the carrier solution (C) in the analysis of total mercury by FIA-HG-QFAAS Factor T (°C) F (rev·min-1) C (% )

Low 100 40 2

-α 260 55 2.8

Central 500 80 4

+α 740 105 5.2

High 900 120 6

3.2. Experiments in Batch Mode with the Dowex M-41 Resin

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The most appropriate working conditions providing maximum retention of Hg2+ and minimum retention of CH3Hg+ with the Dowex M-41 resin were first selected after several experiments carried out in batch mode following the procedure described in the experimental section. The results are summarised in Figure 3.

Figure 3. Retention of Hg2+ (white bars) and CH3Hg+ (black bars) by the Dowex MP41 resin (see text for experimental details). The horizontal line represents the signal of a CH3Hg+ solution (20 μg·L-1), which has not been passed through the resin.

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No statistical difference between the signal of a blank (about 0.0008 units of absorbance) and that of an Hg2+ fraction was indicative of quantitative retention. On the other hand, the signal of each CH3Hg+ fraction was compared with that of an equivalent CH3Hg+ solution (20 μg·L-1), which has not been passed through the resin. An effective and quantitative retention of Hg2+ was observed. In addition, CH3Hg+ passed through the resin efficiently and quickly. The results obtained in the elution experiments are shown in Figure 4 in the case of HCl 8.0 mol·dm-3, HNO3 8.0 mol·dm-3, HNO3 10.0 mol·dm-3 and L(+)cysteine 0.01 mol·dm-3. Again, the signal of an equivalent Hg2+ solution (20 μg·L-1), which has not been passed through the Pasteur pipette, has been taken as reference. The seventh fraction corresponds to the first injection of elution agent. As it can be observed, only L(+)cysteine 0.01 mol·dm-3 is able to force the quantitative elution of all the Hg2+ previously retained in the Dowex M-41 resin. In addition, all the Hg2+ is recovered in a unique fraction.

Figure 4. Elution of the Hg+2 retained in the Dowex M-41 resin by HCl 8.0 mol·dm-3, HNO3 8.0 mol·dm-3, HNO3 10.0 mol·dm-3 and L(+)cysteine 0.01 mol·dm-3. The horizontal line represents the signal of a Hg2+ solution (20 μg·L-1), which has not been passed through the resin.

The rest of elution compounds checked showed a significantly lower capacity to back-extract Hg2+ from the resin. Solutions of 0.1 mol·dm-3 Na2S2O3, 0.1 mol·dm-3 Na2EDTA, 0.1 mol·dm-3 Na2S, 0.1 mol·dm-3 KSCN

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and 2 and 6 mol·dm-3 HNO3 were also checked as elution agents. Similar profiles were obtained, with recoveries always lower than those observed for L(+)cysteine.

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3.3. Experiments On-Line: Optimization of the FIA-IE-HGQFAAS System for Mercury Speciation Preliminary Experiments In several preliminary experiments, the microcolumn C1 (10 cm length, 5 mm i. d.) packed with the Dowex M-41 resin was used to investigate i) the influence of the speed of the carrier pump on the retention of Hg2+, ii) the change in sensitivity that the implementation of the microcolumn into the FIA system causes in the CH3Hg+ analysis, iii) the influence of the speed of the carrier pump on the sensitivity of the analysis of CH3Hg+ and iv) the influence of increasing amounts of Hg2+ in the sample on the analysis of CH3Hg+. Summarising, at a pump speed over 60 rev·min-1 (pump speeds from 40 to 120 rev·min-1 were checked), the resin packed in the C1 column is not able to quantitatively retain the Hg2+ contained in 500 μL of a 10 μg·L-1 Hg2+ solution. Second, the inclusion of an empty microcolumn into the FIA system reduces the sensitivity of the CH3Hg+ analysis in a factor of about two (the slope of a calibration line obtained with a pump speed of 40 rev·min-1 changes from ∼0.0040 a. u. / (μg·L-1) to ∼0.0018 a. u. / (μg·L-1)). If the column is packed with the resin, the change in the slope is even higher (∼0.0015 a. u. / (μg·L-1)). This is directly related to the fact that wider and lower peaks are obtained as absorbance signal with the microcolumn due to dispersion effects in the FIA system. Third, the sensitivity of the CH3Hg+ analysis (using the column packed with the resin) slowly increases with the speed of the carrier pump (∼0.0015 a. u. / (μg·L-1) at F = 40 rev·min-1 to ∼0.0019 a. u. / (μg·L-1) at F = 80 rev·min-1). Finally, increasing amounts of Hg2+ in the sample only influence the CH3Hg+ analysis if at the current working conditions the resin is not able to retain the Hg2+ quantitatively (this happens with a speed of the carrier pump over 60 rev·min-1 for the C1 microcolumn). In that case, of course, a higher signal than expected is obtained. Selection of the Best Microcolumn Geometry In a first experiment, the retention of Hg2+ on columns of different geometry packed with the Dowex M-41 resin was investigated. 500 μL of a

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solution of 20 μg·L-1 of Hg2+ were injected in the system and the signal obtained working in the CH3Hg+ mode was compared with that obtained in the analysis of a blank (about 0.0008 a. u.). The results are shown in Table 4. The effect of the internal diameter is clearly observed in the first three columns considered (C2, C3 and C4 with the same length, 10 cm, and varying internal diameter). While the narrowest one (3 mm i. d.) is not able to retain all the Hg2+ even at the lowest speed, the widest one (10 mm i. d.) quantitatively retains Hg2+ at all the speeds considered. The C2 microcolumn (6.6 mm i. d.) lets escaping only a low amount of Hg2+ when working at the highest speed checked.

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Table 4. Retention of Hg2+ on columns of different geometry packed with the Dowex M-41 resin at different speeds of the carrier pump. 500 μL of Hg2+ solution (20 μg·L-1) were injected in the system (CH3Hg+ mode). Signal of a blank: 0.0008 ± 0.0002 a. u

Pump speed (rev·min-1) ↓ 40

Absorbance obtained with each microcolumn (a. u.) C2 C3 C4 C5 0.0006 0.0006 0.0105 0.0005

60 80 100 120

0.0008 0.0011 0.0007 0.0016

0.0006 0.0006 0.0006 0.0008

0.0204 0.0294 0.0380 0.0450

0.0007 0.0008 0.0009 0.0011

The results seem to be clearly related to the amount of resin packed in each column and the residence time of the sample inside the column. The experiment was repeated with the C1, C2 and C5 microcolumns using Hg2+ solutions at different concentrations between 5 and 200 μg·L-1. The results, shown in Figure 5., confirm the previous conclusions and fix the maximum amount of Hg2+ susceptible to be retained in each case. After these experiments, the C1 and C4 microcoloumns were eliminated from the list of candidates to be used in the FIA-IE-HG-QFAAS system, due to their poor capacity to retain Hg2+. Another set of experiments was carried out to investigate the loose in sensitivity due to dispersion effects caused by the insertion of each column packed with the Dowex M-41 resin in the FIA system.

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Calibration curves for CH3Hg+ were constructed using CH3Hg+ solutions and pump speeds ensuring a quantitative retention of Hg2+ in the case of each column considered (C2: 40 rev·min-1; C3: 120 rev·min-1; C5: 100 rev·min-1). The slope of the calibration obtained in each case (C2: ∼0.0007 a. u. /(μg·L-1); C3: ∼0.0005 a. u. / (μg·L-1); C5: ∼0.0023 a. u. / (μg·L-1)) was compared with that obtained by the FIA-HG-QFAAS system (no column, ∼0.0040 a. u. / (μg·L-1)).

Figure 5. (Continued)

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Figure 5. Retention of Hg2+ on columns of different geometry (C1 (a), C2 (b) and C5 (c)) packed with the Dowex M-41 resin at different speeds of the carrier pump. 500 μL of Hg2+ solution (from 5 to 200 μg·L-1) were injected in the system (CH3Hg+ mode). Signal of a blank: 0.0008 ± 0.0002 a. u.

The results show a loose in sensitivity which can be defined as important in the case of the C2 and C3 columns and moderate in the case of the C5 one. The result of the C2 column, however, greatly improves if a slightly higher pump speed is used to do the analysis. After these experiments, the C3 column was also eliminated from the list of candidates. According to the results obtained in terms of capacity to retain Hg2+ and loose of sensitivity in the CH3Hg+ analysis, the C2 microcolumn was finally selected to be used in the FIA-IE-HG-QFAAS system for mercury speciation. The C5 one was also considered as a good alternative, but the packing of this column with the resin results in a much more tedious and time-consuming process.

Selection of the Best Elution Conditions Finally several experiments were conducted in order to study the influence of the speed of the carrier pump and the concentration of L(+)cysteine on the elution of the retained Hg2+ from the column in the FIA-IE-HG-QFAAS system. First, an injection of 500 μL of a solution containing about 20 μg·L-1 of Hg2+ were injected in the CH3Hg+ mode. Then, the retained Hg2+ was eluted and measured in the Hg2+ mode, using different pump speeds (40, 80 and 120 rev·min-1) and L(+)cysteine concentrations (0.01 and 0.1 mol·dm-3). The

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results indicate that the sensitivity of the analysis increases with the pump speed. Even at the highest speed checked, 0.01 mol·dm-3 L(+)cysteine was not able to liberate all the Hg2+ previously retained in the column. The use of a ten times more concentrated solution as carrier, however, resulted in the quantitative elution of the analyte at these conditions. Accordingly, a 0.1 mol·dm-3 L(+)cysteine solution as carrier (in HCl 3%), pumped at 120 rev·min1 , were the experimental conditions selected for the second step (Hg2+ mode) of the analysis by FIA-IE-HG-QFAAS.

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3.4. Analytical Figures of Merit Good calibration graphs, with correlation coefficients always over 0.99 (up to 50 μg·L-1), were obtained for the analysis of both CH3Hg+ (slope, 0.0007 units of absorbance per ppb of CH3Hg+) and Hg2+ (slope, 0.0027 units of absorbance per ppb of Hg2+) by FIA-IE-HG-QFAAS using the best conditions described in the experimental procedure. Absolute detection limits of 0.8 ng of CH3Hg+ and 1.9 ng of Hg2+ were estimated for a sample volume of 500 μL (based on three times the standard deviation of the blank). The reproducibility of the system (5.4% for CH3Hg+ and 7.4% for Hg2+) was calculated as the relative standard deviation of the slope of seven calibration lines constructed in different and consecutive days. The accuracy was checked analysing synthetic mixtures of CH3Hg+ and Hg2+ of varying composition by the proposed method and by another independent method (Eth-GCMIP/AED). This method comprises the ethylation of the sample, the extraction and preconcentration of the obtained volatile compounds in an hexane phase, and the analysis of the organic phase by capillary gas chromatography with microwave induced plasma-atomic emission detection [20]. The results obtained by both methods did not differ significantly at a 95% confidence level.

3.5. Applicability of the Method: Analysis of Real Samples Doped with the Analytes Estuarine water samples collected in different points of the NerbioiIbaizabal estuary (Bilbao, Basque Country) were filtered (45 μm), acidified with HCl and spiked with CH3Hg+ and Hg2+ to a final concentration of 15

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μg·L-1 each. Before spiking the samples, they were analysed for total mercury by FIA-HG-QFAAS as described in the experimental section. In all the cases the concentration was below the detection limit of the technique. The spiked samples were analysed for CH3Hg+ and Hg2+ following the procedure proposed in this work. The results are shown in Table 5. As it can be observed, the recoveries obtained were always satisfactory, in samples with either high or low salinity. Table 5. Analysis of estuarine water samples collected in the NerbioiIbaizabal estuary (Bilbao, Basque Country) spiked with CH3Hg+ and Hg2+ to a final concentration of 15 μg·L-1 each by FIA-IE-HG-QFAAS using the best conditions found in this work Sample 1 2 3 4

Conductivity (mS·cm-1) 24.2 5.3 0.4 0.3

Concentration (μg·L-1) CH3Hg+ Hg+2 14.39 ± 1.30 14.23 ± 1.05 14.39 ± 1.32 14.13 ± 1.14 14.56 ± 1.24 15.91 ± 0.99 14.48 ± 1.21 15.07 ± 1.00

Recovery (%) CH3Hg+ Hg+2 96 ± 8 95 ± 7 96 ± 8 94 ± 8 97 ± 8 106 ± 7 97 ± 8 101 ± 6

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CONCLUSION Ion exchange is presented as an appropriate, easy-to-use and cheap alternative to other more expensive instrumental separation techniques for mercury speciation. Its easy implementation in a flow injection system has also been demonstrated, which carries a further potential for automation and reproducibility of the technique. The analytical method proposed here has demonstrated its potential utility in the analysis of water samples, for both fresh and saline waters. The detection limit of the method does not allow by now, however, the direct analysis of real samples due to the low concentrations usually found in natural waters. This problem can be solved by the inclusion on-line of any preconcentration method in the FIA system and/or the use of any other more sensitive detection technique. We are currently working in those two directions. Although not checked here, the system as presented is susceptible to be used in the analysis of extracts from solid environmental samples, such as sediments or biota, which usually show considerably higher concentrations of both CH3Hg+ and Hg2+.

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ACKNOWLEDGMENTS This work received financial support from the OKAMET Project, Programme of the UNESCO Chair on Sustainable Development and Environmental Education of the University of the Basque Country (ref. UNESCO 09/23). A. Gredilla acknowledges her pre-doctoral fellowship from the University of the Basque Country.

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Gustin, MS; Kolker, A; Gardfeldt, K. Transport and fate of mercury in the environment. Appl. Geochem., 2008 23, 343-344. Ullrich SM; Tanton TW; Abdrashitova SA. Mercury in the Aquatic Environment: A Review of Factors Affecting Methylation. Crit. Rev. Environ. Sci. Technol., 2001 31, 241-293. World Health Organization (WHO). Hazardous chemicals: main risks to children’s health. Fact Sheet EURO/02/04. 2004. Available from www.euro.who.int/mediacentre/FactSheets/20040405_1. Boudou A; Ribeyre F. Mercury in the food web: accumulation and transfer mechanisms. In: Sigel A, Sigel H editors. Metal Ions in Biological Systems: Mercury and Its Effects on Environment and Biology. New York: Marcel Dekker, inc.; 1997; 289-319. Yin Y; Liu J; He B; Shi J; Jiang G. Mercury speciation by a high performance liquid chromatography-atomic fluorescence spectrometry hyphenated system with photo-induced chemical vapor generation reagent in the mobile phase. Microchim. Acta, 2009 167, 289-295. Sánchez-Uría JE; Sanz-Medel A. Inorganic and methylmercury speciation in environmental samples. Talanta, 1998 47, 509-524. Ray SJ; Andrade F; Gamez G; McClenathan D; Rogers D; Schilling G; Wetzel W; Hieftje GM. Plasma-source mass spectrometry for speciation analysis: state-of-the-art.J. Chromat. A, 2004 1050, 3-34. de Souza SS; Rodrigues JL; de Oliveira Souza VC; Barbosa F Jr. A fast sample preparation procedure for mercury speciation in hair samples by high-performance liquid chromatography coupled to ICP-MS. J. Anal. At. Spectrom., 2010 25, 79-83. Cai Y; Jaffe R; Azaam A; Jones RD. Determination of organomercury compounds in aqueous samples by capillary gas chromatography-atomic

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A. Gredilla, J. Larreta, I. Martinez-Arkarazo et al. fluorescence spectrometry following solid-phase extraction. Anal. Chim. Acta, 1996 334, 251-259. Heumann KG. Hyphenated techniques - the most commonly used method for trace elemental speciation analysis. Anal. Bioanal. Chem., 2002 373, 323-324. May K; Stoeppler M; Reisinger K. Studies in the ratio total mercury/methylmercury in the aquatic food chain. Tox. Environ. Chem., 1987 13, 153-159. Ferreira LM; De Carvalho JMR. Mercury removal from chloro-alkali plant waste waters by ion exchange. Environ. Technol., 1997 18, 433440. Monteagudo JM; Ortiz MJ. Removal of inorganic mercury from mine waste water by ion exchange. J. Chem. Technol. Biotechnol., 2000 75, 767-772. Högfeldt, E. (1982). Stability Constants of Metal-Ion Complexes. Part A: Inorganic Ligands.Oxford, Great Britain: Pergamon Press. Sanz J; Raposo JC; de Diego A; Madariaga JM. Appl. Organometal. Chem., 2002 16, 339-346. Sanz J; Raposo JC; Madariaga JM. Potentiometric study of the hydrolysis of (CH3)Hg+ in NaClO4: construction of a thermodynamic model. Appl. Organometal. Chem., 2000 14, 499-506. Sanz J; Raposo JC; Larreta J; Martinez-Arkarazo I; de Diego A; Madariaga JM. On-line separation for the speciation of mercury in natural waters by flow injection-cold vapor-atomic absorption spectrometry. J. Sep. Sci., 2004 27, 1202-1210. Perkin-Elmer. (1994). Flow Injection Mercury Hydride Analysis: Recommended Analytical Conditions and General Information, Part No. B050-1820, Publication B3505. Norwalk, USA: Perkin-Elmer Corporation. Sherrod PH. NLREG-Nonlinear Regression Analysis Program. 2010. Available from http://www.nlreg.com/NLREG.pdf. Sanz J; de Diego A; Raposo JC; Madariaga JM. Routine analysis of mercury species using commercially available instrumentation: chemometric optimization of the instrumental variables. Anal. Chim. Acta, 2003 486, 255-267.

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Chapter 4

CATHEPSIN-DEPENDENT NEURONAL DEATH PATHWAYS INDUCED BY METHYLMERCURY Hiroshi Nakanishi∗ and Zhou Wu Department of Aging Science and Pharmacology, Faculty of Dental Science, Kyushu University, Fukuoka 812-8582, Japan

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ABSTRACT An intoxication of methylmercury (MeHg), also known as Minamata disease, commonly results from the ingestion of MeHg-contaminated food. MeHg is easily absorbed from the intestine and therefore if transported to the central nervous system (CNS) through the blood-brain barrier. Among CNS neurons, the cerebellar granule neurons are especially vulnerable to MeHg intoxication. Exposure to MeHg during development results in an impaired migration of the cerebellar granule neurons and impaired synaptogenesis, thus leading to a disordering of the cerebellar architecture. On the other hand, exposure to MeHg during adulthood can also result in loss of cerebellar granule neurons from the internal granule cell layer, while Purkinje cells remain intact. MeHg has been also reported to affect functions of astrocytes and microglia. Despite the clinical importance, the understanding of the mechanism underlying MeHg-induced neuronal death is still limited. Cathepsins B and D, two major lysosomal proteases in the CNS, have been implicated in neuronal ∗

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Hiroshi Nakanishi and Zhou Wu death.Cathepsins B and D are involved in neuronal death through different two pathways: intracellular proteolysis after their leakage from the lysosome to the cytosol of neurons and extracellular proteolysis after secreted from activated microglia.Here, we summarize possible neuronal and microglial cathepsin-dependent neuronal death pathways induced by various stimuli including MeHg, and then the potential application of inhibitors for cathepsins on MeHg-induced pathological changes in the CNS.

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INTRODUCTION An intoxication of methylmercury (MeHg), also known as Minamata disease, commonly results from the ingestion of MeHg-contaminated food. MeHg is thought to cause a form of cerebral palsy, which is characterized by abnormal muscle tone, reflexes, motor development and coordination. MeHg is easily absorbed from the intestine and therefore it transported to the central nervous system (CNS) through the blood-brain barrier. In parallel with the clinical symptoms of cerebral palsy, cerebellar granuleneurons are especially vulnerable to MeHg intoxication among the CNS neurons [Castoldi et al., 2001]. Exposure to MeHg during development results in an impaired migration of the cerebellar granule neurons and impaired synaptogenesis, thus leading to a disordering of the cerebellar architecture [Choi et al., 1978]. On the other hand, exposure to MeHg during adulthood can also result in loss of the cerebellar granule neurons from the internal granule cell layer, while Purkinje cells remain intact [Nagashima et al., 1996; Wakabayashi et al., 1995]. MeHg has been also reported to affect functions of astrocytes [Ascher, 1996; Ascher et al., 1996] and to induce apoptosis of microglia [Garg et al., 2006; Nishioku et al., 2000] and neural stem cells [Tamm et al., 2006, 2008]. The degeneration of the cerebellar granule neurons by MeHg involves an apoptotic process based on the ultrastructural features and intranucleosomal DNA fragmentation [Nagashima et al., 1996; Nagashima, 1997]. In agreement with the data in vivo, apoptosis has been also described in rodent cultured cerebellar granule neurons [Castoldi et al., 2000; Dare et al., 2001]. However, MeHg induces both apoptosis and necrosis in a concentration-dependent mannerin the cultured cerebellar granule neurons [Castoldi et al., 2000; Kunimoto, 1994]. A group of proteasesin the endosomal/lysosomal proteolytic system is designated as cathepsin which is derived from the Greek term meaning “to digest” [Nakanishi, 2003c]. Although the primary function of cathepsins has

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been believed to degrade proteins by bulk proteolysis in lysosomes, the recent results from the cathepsin gene knockouts have revealed that cathepsins carry out their specific functions by limited proteolysis of proteins [Saftig et al., 1995; Turk et al., 2000; Reinheckel et al., 2001]. It is likely that limited proteolysys is exerted by cathepsins in less acidic intracellular compartments such as early and late endosomes. There is increasing evidence that disturbance of normal balance and extralysosomal localization of cathepsins lead to neurodegeneration in Alzheimer’s disease, stroke, and lysosomal storage diseases [Cataldo et al., 1990, 1991; Nakanishi et al., 1993, 1994, 1997, 2001; Nakanishi and Wu, 2009; Nixon, 2000; Koike et al., 2000]. Furthermore, it is now widely accepted that apoptosis is often associated with release of cathepsins into the cytosol [Boya and Kroemer, 2008; Chwieralski et al., 2006; Gucciardi et al., 2000, 2004; Johansson et al., 2010; Kirkegaard and Jäättelä, 2009; Turk et al., 2000; Turk and Stoka, 2007; Yamashima, 2000; Yamashima and Oikawa, 2009]. Activated microglia also release some members of cathepsins to induce neuronal death [Gan et al., 2004; Kim et al., 2007; Nakanishi, 2003a, b, c, 2007; Wendt et al., 2009]. This chapter summarizes the current knowledge concerning the roles of neuronal and microglial cathepsins B and Din neuronal death induced by a variety of stimuli including MeHg, because cathepsins B and D are most abundant in the brain.

CATHEPSIN B IN NEURONAL DEATH Apoptosis can be initiated by both extrinsic and intrinsic pathways. The extrinsic pathway begins by binding of appropriate ligands to their surface receptors of cells, which leads to activation of procaspases-8. Caspase-8 can either activate procaspase-3 directly or indirectly via its cleavage of Bid to truncated Bid (tBid). tBid is transported through mitochondrial membrane where it augments ologomerization of Bax and induces intrinsic pathway of apoptosis by promoting the release of cytochrome c. On the other hand, the intrisinc pathway is initiated by dysfunction of mitochondria and the consequent release of cytochrome c, which leads to activation of procaspases9 and in consequent procaspase-3. There is substantial evidence that cathepsin B (EC 3.4.22.1), a typical cysteine lysosomal protease, is involved in the apoptotic process. Cathepsin B has been implicated in the activation of the proinflammatory caspase 11 and 1 [Schotte et al., 1998; Vancomperolle et al., 1998]. Furthermore, a new notion

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that cathepsin B is released from the lysosomes into the cytosol to activate apoptotic pathway has been emerged. Cytosolic cathepsin B can cleave the Bcl-2 family member Bid [Stoka et al., 2001], which may lead to cytochrome c release from the mitochondria, thus subsequently causing caspase activation. The importance of cathepsin B-dependent pathway was also demonstrated in tumor necrosis factor (TNF)-α-induced apoptosis in hepatocytes [Guicciardi et al., 2000]. The release of cathepsin B into the cytosol has been also implicated in neuronal death following ischemia [Yamashima et al., 1998; Yamashima, 2000; Yamashima and Oikawa, 2009]. Activated μ-calpain translocates to lysosomal membranes of hippocampal neurons prior to release of cathepsin B after ischemia in primate. Furthermore, m-calpain as well as caspase-8 can permeabilizes the membrane of isolated lysosomes [Guicciardi et al., 2000]. Theses observations lead to the formation of the “Calpain-Cathepsin hypothesis”, whereby the activation of μ-calpain results in the lysosomal membrane permeability causing release of cathepsins from the lysosomes to the cytosol. In addition to the functions of intracellular proteolysis, there is accumulating evidence that cathepsin B released from microglia may play a crucial pathological role in the CNS [Banati et al., 1993; Nakanishi, 2003a, b]. Ryan et al. [1995] used an immortalized murine microglial cell line, BV-2 cells, to demonstrate that cathepsin B was secreted in a heavy chain form, in addition to the proform, upon stimulation with lipopolysaccharide (LPS). It has recently been demonstrated that cathepsin B secreted from microglia is a major causative factor of microglia-induced neuronal apoptosis [Kingham and Pocock, 2001]. More recently, Gan et al. [2004] conducted functional genomic studies to identify cathepsin B as one of the genes transcriptionally induced by amyloid-β(Aβpeptides in microglial cell line, BV-2 cells. They have also further shown that an inhibition of cathepsin B in BV-2 cells using either small interference RNA-mediated gene silencing or a specific inhibitor for cathepsin B, CA074, leads to a decrease in the neurotoxic effects caused by Aβ-activated BV-2 cells. Furthermore, the culture supernatants of LPS-stimulated microglial cells were toxic to neuronal cells [Wendt et al., 2009]. Experiments with membrane-permeable and membrane impermeable cysteine protease inhibitors indicated that this toxic effects was related to the intracellular roles of cathepsins in microglial activation.

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Cathepsin-Dependent Neuronal Death Pathways…

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CATHEPSIN B IN METHYLMERCURY-INDUCED NEURONAL DEATH As reported previously [Miyamoto et al., 2001], neuronal pyknotic changes were observed in the cerebellar granule cell layer and occipital cortex, but not in the hippocampus or the brain stem after chronic treatment with MeHg in adult rats. We have found that the immunoreactivity for cathepsin B was markedly increased in activated microglia accumulated in the granule cell layer of the cerebellum after chronic treatment with MeHg [Sakamoto et al., 2008]. Cathepsin B was upregulated in activated microglia in advance to the cerebellar granule neuronal death (figure 1A). On the other hand, intense aggregate immunoreactivity for cystatin C, a potent endogenous inhibitor of cysteine proteases including cathepsin B, was found in Purkinje cells. Although theimmunoreactivity for cystatin C in cerebellar granule neurons was decreased, some cells still had relative intense cystatin C immunoreactivity (figure 1B). These cystatin C-immunostained cells in the granule cell layer corresponded well with OX42-immunostained microglia. Immuno-histochemical observations were substantiated by immunoblotting analyses. The soluble fractions of the cerebellum from rats after various treatment schedules with MeHg were subjected to immunoblot analyses using anti-cathepsin B and anti-cystatin C antibodies. Soluble fractions of the cerebellum from vehicle-treated rats exhibited slight immunoreactivities for cathepsin B and cystatin C (figure 1C, D). On the day after the final treatment with MeHg for 12 consecutive days, a significant increase in the immunoreactivities for both cathepsin B and cystatin C was observed. Cathepsin B was increased as the mature form with apparent molecular mass of 29 kDa (single-chain form) and 26 kDa (heavy chain of the double-chain form). The single-chain form was present at much higher levels than the heavy chain. Eight days after the final treatment with MeHg for 12 consecutive days, there was a marked increase in the amount of both cathepsin B and cystatin C. These observations indicate that a marked increase in the cathepsin B immunoreactivity in the granule cell layer after treatment with MeHg is closely associated with an accumulation of activated microglia. On the other hand, the cystatin C immunoreactivity was increased mainly in Purkinje cells and partially in activated microglia in the granule cell layer, while the cerebellar granule neurons showed rather decreased immunoreactivity.

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Figure 1. A, B: Immunofluorescent CLSM images for cathepsin B (A) and cystatin C (B) in the cerebellum. V: vehicle treatment; MeHg (12d): on the day after the final administration of MeHg for 12 consecutive days, MeHg (12+3d): 3 days after the final administration of MeHg for 12 consecutive days; MeHg (12+5d): 5 days after the final administration of MeHg for 12 consecutive days; MeHg (12+8d): 8 days after the final administration of MeHg for 12 consecutive days. CB: cathepsin B, CC: cystatin C. Scale bars = 70μm. C, D: Immunoblots from SDS-polyacrylamide gel electrophoresis of whole cerebellum extracts developed with antibody specific for cathepsin B (C) or cystatin C (D). V: cerebellum extracts of vehicle rats; MeHg (12d): cerebellum extracts of rats prepared on the day after the final administration of MeHg; MeHg (12+3d): cerebellum extracts of rats prepared 3 days after the final administration of MeHg for 12 consecutive days; cerebellum extracts of rats prepared 5 days after the final administration of MeHg for 12 consecutive days; cerebellum extracts of rats prepared 8 days after the final administration of MeHg for 12 consecutive days. Each column and bar represents the mean±S.E. of three rats. Asterisks indicate significant differences from the vehicle-treated value (**P