Asbestos: Risks, Environment and Impact 9781612098531

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ASBESTOS: RISKS, ENVIRONMENT AND IMPACT No part of this digital document may be reproduced, stored in a retrieval system or transmitted in any form or by any means. The publisher has taken reasonable care in the preparation of this digital document, 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 herein. This digital document is sold with the clear understanding that the publisher is not engaged in rendering legal, medical or any other professional services.

ASBESTOS: RISKS, ENVIRONMENT AND IMPACT

ANTONIO SOTO AND

GAEL SALAZAR EDITORS

Nova Biomedical Books New York

Copyright © 2009 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. 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. LIBRARY OF CONGRESS CATALOGING-IN-PUBLICATION DATA

ISBN: 978-1-61209-853-1 (eBook)

Published by Nova Science Publishers, Inc.

New York

.

CONTENTS Preface

vii

Chapter 1

Deposition of Man-Made Fibers in the Human Respiratory Airway Wei-Chung Su and Yung Sung Cheng

Chapter 2

Number of Plasmids Transported into Escherichia Coli through the Yoshida Effect and Predicted Structure of the PenetrationIntermediate Naoto Yoshida

1

39

Chapter 3

Prevention and Detection of Asbestos-Related Diseases in Finland Matti S. Huuskonen

59

Chapter 4

Autoantibody Profiles of an Asbestos-Exposed Population Jean C. Pfau, David J. Blake and Marvin J. Fritzler

73

Chapter 5

Epidemiology of Mesothelioma: The Role of Asbestos Massimo Menegozzo, Roberto Pasetto, Simona Menegozzo and Pietro Comba

97

Chapter 6

Asbestos Litigation: Prospects for Legislative Resolution Edward Rappaport

Short Commentary 1 The Efficiency of the CUSCORE Test as Compared to that Applied to SMR in Detection of a Carcinogenic Exposure Rina Chen, Ph.D. Short Commentary 2 Retroperitoneal fibrosis – a new asbestos-related disease Toomas Uibu Short Commentary 3 Endemic Pleural Plaques and Mesotheliomas in Northern Greece Sichletidis Lazaros, MD, FCCP and Chloros Diamantis, MD Short Commentary 4 Immunological Effects of Asbestos Takemi Otsuki, Megumi Maeda, Yoshie Miura, Hiroaki Hayashi, Shuko Murakami, Naoko Kumagai, and Yasumitsu Nishimura

139

155 165

177 193

.

PREFACE Asbestos is a naturally occurring silicate mineral with long, thin fibrous crystals. It is known as the miracle mineral because of its soft and pliant properties, as well as its ability to withstand heat. Asbestos is known to be toxic and the inhalation of asbestos can be potentially lethal. Since the mid 1980s, many uses of asbestos have been banned in several countries. The Environmental Protection Agency also banned the use of asbestos and as a result, man-made fibers have been manufactured and used in many applications to replace asbestos. However, asbestos can still be found naturally in the air outdoors and in some drinkable water, including water from natural sources. As this book discusses, studies conducted in laboratory animals have shown that certain man-made fibers may have biological effects similar to those of asbestos. This book addresses this serious health concern caused by asbestos as well as potential fiber-related lung injury due to the inhalation of manmade fiber aerosols. Chapter 1 The U.S. Environmental Protection Agency banned the use of asbestos in 1989, since that time man-made fibers have been manufactured and used in many applications to replace asbestos. Studies conducted in laboratory animals have shown that certain man-made fibers may have biological effects similar to those of asbestos, implying that potential fiber-related lung injury might be induced in humans due to inhalation of man-made fiber aerosols. Therefore, it is essential to investigate the exposure dosimetry of man-made fibers in the human respiratory airway, which would particularly benefit workers in the man-made fiber industry. A series of intensive fiber deposition experiments using realistic human nasal and orallung airway casts were conducted in our laboratory in order to fully understand the fiber deposition in the human respiratory airway. Man-made carbon, titanium dioxide (TiO2), and glass fibers having various fiber dimensions were employed as the test materials, and these fiber aerosols were delivered into the casts using several inspiratory flow rates representing different human inhalation rates. These experiments provided a large amount of invaluable data, including the dominant deposition mechanism, airway deposition patterns, and regional deposition efficiencies. The deposition results showed that fiber deposition in the nasal and oral airways increases proportionally as the fiber length and fiber inertia increases. The sites of the enhanced deposition were found around the vestibule (nasal airway) and the oropharynx (oral airway). In the tracheobronchial airways, the carina region in each lung

viii

Antonio Soto and Gael Salazar

generation/bifurcation was the preferred deposition site, and the deposition efficiency increased as the fiber length and inertia increased as well. These results imply that the inhaled small and short fibers have a high penetration rate in the human upper airway and cause relatively more fiber deposition in the lower respiratory tract, which could result in potential lung injures. In contrast, the inhaled large and long fibers tend to deposit in the nasal and oral airways and are therefore less harmful for the tracheobronchial airways. It was also interesting to note that the deposition efficiency of fiber was shown to be significantly lower than that of general compact particles, indicating that fibers have a greater chance of entering the human deep lung compared with the compact particles. Based on these results, empirical models were developed for predicting the fiber deposition in the human airway. It is believed that these proposed models can be very useful for occupational health researchers to accurately estimate the fiber deposition in the human respiratory tract for any given fiber exposure scenario. Chapter 2 When a colloidal solution containing nano-sized acicular material, such as chrysotile, and bacterial cells are placed in friction field between hydrogel and a polymer interface, the nanosized acicular material penetrates bacterial cells and forms a complex called a penetrationintermediate. This is known as the Yoshida effect. The hydrogel exposure method is a novel technique that employs the Yoshida effect to transform prokaryotes with a plasmid. The author of the present study has confirmed that two or more of pUC18, pHSG298, and pHSG396 containing the same replication of origin can be simultaneously introduced into Escherichia coli cells using the hydrogel exposure method. Multiple plasmids were maintained stably in E. coli cells, even following cell subculture under the selection pressure by antibiotics. Multiple plasmids were maintained stably in E. coli cells, even after the subculture was repeated. The stability of each plasmid in E. coli cells was as follows: pUC18, pHSG298, and pHSG396. To investigate the number of plasmids introduced into E. coli cells through the Yoshida effect, the author carried transformed E. coli with both pUC18 and pHSG298, which are adsorbed onto chrysotile in a ratio of 1:1. The relative proportion of colonies obtained from a LB plate supplemented with ampicillin, kanamycin, and both, was 6:6:1, respectively. Subsequently, transformation of E. coli via the Yoshida effect was performed using chrysotile adsorbed to pUC18, pHSG298, and pHSG396 in a 1:1:1 ratio. The relative proportion of colonies obtained from a LB plate supplemented with ampicillin, kanamycin, ch1oramphenicol, and all three combined was 15:15:15:1, respectively. It is possible that the penetration-intermediate can only incorporate one plasmid using the Yoshida effect. pUC18- and pHSG298- bound chrysotile were prepared independently. When E. coli cells were exposed to hydrogel using both pUC18- and pHSG298-bound chrysotile, relative proportion of colonies obtained from a LB plate supplemented with ampicillin, and kanamycin was 1:1, respectively. However, no colonies were obtained on a LB plate supplemented with both ampicillin and kanamycin. This result suggests that the structure of the penetration-intermediate with acquired plasmid DNA consists of a bacterial cell penetrated by a single chrysotile fiber. Chapter 3 In 2006, the WHO and ILO decided to recommend a worldwide ban on the use of asbestos products, which is opposed by the countries producing those products. Early diagnosis of asbestos-related diseases, and close monitoring of the health of the patients, aims

Preface

ix

to improve the prognosis of occupational diseases and, at the same time, will secure the patients with the benefits offered by social medicine. About 100,000 people die of asbestos-related diseases throughout the world every year: 60,000 of lung cancer, 30,000 of mesothelioma and 10,000 of asbestosis. The number of asbestos-related cancers is still on the increase in industrial countries and this trend will continue elsewhere in the future. The first occupational diseases caused by asbestos were found more than 100 years ago, and it was known in the 1930s that there was a link between asbestosis and lung cancer. Although a large number of resources have been spent on research into asbestos-related diseases, not a great deal of progress has been made in the medical prevention and treatment of asbestosis, lung cancer or mesothelioma. Radiological follow-up and monitoring the health of workers who have been exposed to asbestos are opposed by those who require proof from randomized mortality follow-ups before the early detection and treatment of lung-cancer in people without symptoms could be considered ethical. There is, however, some good news that encourages action in this area. The new imaging methods used in the diagnosis of lung cancer have proven far superior to conventional chest X-rays and early diagnosis of incipient, small and operable lesions is possible with low-dose spiral tomography. Chapter 4 Asbestos exposure is associated with autoimmune responses including increased serum immunoglobulins, positive autoantibody tests and immune complex deposition. Occupational and environmental asbestos exposures continue to occur world-wide, making this a current human health issue. The premise that asbestos exposure exacerbates autoimmunity is supported by recent studies from an asbestos exposed population in Libby, Montana, USA. Residents of Libby have experienced significant exposures to amphibole asbestos due to the mining of asbestos-contaminated vermiculite near the community over several decades. This community exhibits higher frequencies of positive anti-nuclear antibody (ANA) tests compared to an unexposed control population and what would be expected based on epidemiological data. Systemic autoimmune diseases are characterized by autoantibody profiles that correspond to specific sets of intracellular components. These profiles can be used to help predict disease, establish a diagnosis as well as assess clinical progression. At this time, it is not known whether a discrete clinical or sub-clinical autoimmune entity is associated with amphibole asbestos exposure. However, asbestos exposure leads to clinical manifestations similar to systemic lupus erythematosus (SLE) in asbestos exposed mice. Therefore, we hypothesized that features of certain autoimmune diseases may be associated with asbestos exposure in humans. The purpose of this study was to determine whether a serological signature exists in this asbestos-exposed population, and to compare this profile with other known systemic autoimmune disease autoantibody profiles. Our results indicate that autoantibodies from individuals exposed to amphibole asbestos primarily recognize chromatin, histone and Ro52, similar to patients with systemic lupus erythematosus (SLE). Anti-dsDNA and anti-Ro52 antibodies are also generated in a murine model of asbestos induced autoimmunity, which suggests that asbestos may drive similar immune phenomena in both humans and mice. In addition, we show that an unexpected number of subjects expressed the Scl-topoisomerase I (topo I: also referred to as Scl-70) antibody, typically seen in scleroderma. The subjects with anti-topo I tended to have higher levels of asbestos exposure and more severe lung disease compared to those without topo I,

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whereas there was no association with exposure or lung disease among people with anti-RNP (antibodies to ribonucleoprotein, common in SLE). Finally, we show that some Libby serum samples contained antibodies that bound fibroblasts, a phenomenon that is also seen in scleroderma. By comparison, neither rheumatoid factor (RF) nor anti-cyclic citrullinated peptide (CCP), markers for rheumatoid arthritis (RA), was substantially elevated in the Libby sample set. This information could be extremely valuable for improved screening of exposed populations. In addition, certain autoantibody profiles may serve as diagnostic or prognostic markers relative to asbestos exposure. Chapter 5 Mesothelioma is a rare but lethal disease. This chapter provides an explanation of the disease and descriptive epidemiological data: incidence, mortality, survival, temporal trends and geographical distribution. It also presents etiological data on asbestos and asbestos-type fibers and delves into the effects of the duration and intensity of exposure and the doseresponse relation. It describes the contexts of occupational risk in general terms, details on exposure and the risks associated with specific work sectors. The report then goes on to illustrate multiple scenarios of environmental exposure. Finally, it elaborates on the contribution of the epidemiology of mesothelioma to Public Health initiatives: environmental reclamation, identification of groups at risk and the prevention of asbestos related pathologies in developing countries. Chapter 6 A large volume of litigation has been occasioned by occupational exposure to asbestos, which may ultimately result in payments of $200 billion or more and has already bankrupted numerous companies. This litigation “explosion” has led to a number of innovations in legal process, but some of the more comprehensive settlements were overturned by the Supreme Court, with the Court suggesting that the situation “calls for national legislation.” One approach proposed in the 109th Congress, embodied in H.R. 1957 (Cannon et al.), would conserve the resources of defendant corporations — many of which have been bankrupted by asbestos cases — so that funds could be applied first to workers who are already sick. This would be done by establishing precise standards for proving the presence of asbestos disease for legal purposes, and postponing the cases of those who might show evidences of exposure but are not yet impaired. H.R. 1957 would also apply to the asbestos problem a number of principles known under the more general rubric “tort reform.” The bill receiving the most attention in the 109th Congress, S. 3274 (Specter and Leahy) would also establish standards for proving injury but, instead of taking the tort reform approach, would remove the cases from the court system entirely. In its place would be an administrative system and a special fund to pay claims. S. 3274 would define nine asbestos disease categories, each with a specified level of compensation, ranging from $25,000 to $1.1 million. The fund from which claims would be paid would be financed by assessments on defendant companies and their insurers. Each of the largest firms subject to assessment would be responsible for paying up to $27.5 million per year for up to 30 years, with an overall funding goal of $140 billion. The most debated points of S. 3274 have included the adequacy of the funding scheme, the levels of compensation, medical criteria (especially as regards smoking history), and startup and close-down issues. This report discusses such issues thematically, and will be updated to reflect major legislative actions. A section-by-section analysis of S. 852 (the predecessor version of S.

Preface

xi

3274, as reported by the Judiciary Committee) may be found in CRS Report RS22081, S. 852: The Fairness in Asbestos Injury Resolution Act of 2005, by Henry Cohen. Short Commentary 1 Quite often statistical analyses are carried out in response to a public alert regarding suspected elevated risk of cancer. Usually no control group is available in these ad-hoc instances, and the analysis is based on the SMR, i.e., on the contrast between the observed and the expected number of events. The SMR (Standardized Morbidity (or Mortality) Ratio) is the ratio between the observed and the expected number of events. The expected number is evaluated according to the age and gender specific rates observed in the reference population. Both types of statistical errors are inflated in these analyses [1]. Namely, frequent false alerts coupled with unsatisfactory power of the test are involved in these analyses. This commentary presents the relative efficiency of the CUSCORE test in alleviating the two types of statistical errors. Analyses of colon cancer deaths among asbestos workers in Israel, demonstrate the efficiency of the procedure in detecting clustering and in providing clues indicating that the significant results are not spurious. Some suggestions are presented in order to increase the efficiency in detection and interpretation of the analyses. Short Commentary 2 Retroperitoneal fibrosis (RPF) is an uncommon fibroinflammatory disorder of the retroperitoneal space. The aetiology of RPF has been unknown in most cases. Current opinion suggests that the inflammatory reaction to advanced atherosclerosis and other autoimmune processes are the main causes leading to RPF. Only some reports in the literature have suggested an association between RPF and past asbestos exposure. In our population-based case-control studies we found a strong association between RPF and asbestos exposure. Most of the asbestos-exposed RPF patients had concomitant asbestosrelated pleural fibrosis. Such changes were not found in unexposed RFP patients. Our findings suggest a shared aetiology for RPF and pleural fibrosis and, furthermore, possibly similar pathogenetic mechanisms. Short Commentary 3 Asbestos fibres and other minerals, when present in certain areas of the world, are mainly to blame for the development of endemic pleural plaques, as illustrated in chest x-ray of the inhabitants of those areas1. Mineral fibres when concentrated in traditional materials such as whitewashing were proven to be especially dangerous in Turkey2, in Metsovo in Western Greece3, as well as in the tested area of Almopia, a county of Pella in Northern Greece. In addition to pleural plaques, high frequency of mesothelioma has been found to co-exist4,5. A higher than expected prevalence of pleural plaques compatible with lesions caused by exposure to mineral fibres has been observed in residents of Almopia, in Central Macedonia, Northern Greece, since early 70’s. From information obtained from elderly residents of the village of Megaplatanos, the population of this area used to whitewash their houses with a material made from “white rocks” extracted from a nearby ravine until 1935. Therefore we decided to carry out an epidemiological study of the population of the seven villages located around the aforementioned ravine in order to estimate the prevalence of pleural plaques. We also performed an environmental study to elucidate the source of the pollution, the type of the offending fibres and their concentration in the air. Short Commentary 4

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In: Asbestos: Risks, Environment and Impact Editor: Antonio Soto and Gael Salazar

ISBN 978-1-60692-053-4 © 2009 Nova Science Publishers, Inc.

Chapter 1

DEPOSITION OF MAN-MADE FIBERS IN THE HUMAN RESPIRATORY AIRWAY Wei-Chung Su and Yung Sung Cheng Lovelace Respiratory Research Institute 2425 Ridgecrest Dr. SE, Albuquerque, NM 87108

ABSTRACT The U.S. Environmental Protection Agency banned the use of asbestos in 1989, since that time man-made fibers have been manufactured and used in many applications to replace asbestos. Studies conducted in laboratory animals have shown that certain man-made fibers may have biological effects similar to those of asbestos, implying that potential fiber-related lung injury might be induced in humans due to inhalation of man-made fiber aerosols. Therefore, it is essential to investigate the exposure dosimetry of man-made fibers in the human respiratory airway, which would particularly benefit workers in the man-made fiber industry. A series of intensive fiber deposition experiments using realistic human nasal and orallung airway casts were conducted in our laboratory in order to fully understand the fiber deposition in the human respiratory airway. Man-made carbon, titanium dioxide (TiO2), and glass fibers having various fiber dimensions were employed as the test materials, and these fiber aerosols were delivered into the casts using several inspiratory flow rates representing different human inhalation rates. These experiments provided a large amount of invaluable data, including the dominant deposition mechanism, airway deposition patterns, and regional deposition efficiencies. The deposition results showed that fiber deposition in the nasal and oral airways increases proportionally as the fiber length and fiber inertia increases. The sites of the enhanced deposition were found around the vestibule (nasal airway) and the oropharynx (oral airway). In the tracheobronchial airways, the carina region in each lung generation/bifurcation was the preferred deposition site, and the deposition efficiency increased as the fiber length and inertia increased as well. These results imply that the inhaled small and short fibers have a high penetration rate in the human upper airway and cause relatively more fiber deposition in the lower respiratory tract, which could result in potential lung injures. In contrast, the inhaled large and long fibers tend to deposit in the nasal and oral airways and are therefore less harmful for the tracheobronchial airways. It was also

2

Wei-Chung Su and Yung Sung Cheng

interesting to note that the deposition efficiency of fiber was shown to be significantly lower than that of general compact particles, indicating that fibers have a greater chance of entering the human deep lung compared with the compact particles. Based on these results, empirical models were developed for predicting the fiber deposition in the human airway. It is believed that these proposed models can be very useful for occupational health researchers to accurately estimate the fiber deposition in the human respiratory tract for any given fiber exposure scenario.

1.1. INTRODUCTION Asbestos is a notorious occupational hazard. Selikoff and Lee as well as IARC have documented that exposures to airborne asbestos in the workplace increase the incidence of asbestosis, mesothelioma, and lung cancer for workers exposed to it (Selikoff and Lee 1978; IARC 1987). Since 1989 when the U.S. Environmental Protection Agency banned the use of asbestos, man-made fibers have been manufactured and used in many applications to replace it. However, research conducted in laboratory animals has shown that certain man-made fibers may have biological effects similar to those of asbestos (Miller et al., 1999; Hesterberg and Hart, 2001; IARC, 2002; Kamstrup, et al. 2002). Because of the potential for asbestosrelated lung diseases being induced in humans due to inhalation of man-made fibers (IARC, 2002; Walton, 1982), it is crutial to investigate the exposure dosimetry of man-made fibers in the human respiratory airway in order to provide information for the associated occupational health researchers. Such investigation would particularly benefit those workers in the manmade fiber manufacturing industry. However, due to severe ethical constraints, fiber deposition experiments using human volunteers are strictly not allowed. As a result, only limited information was available on this topic (Myojo, 1987, 1990; Sussman et al., 1991) until now. This lack of experimental data not only kept the nature of fiber deposition in the human respiratory airway from being well known, but also made validating the developed theoretical model difficult. In order to fully understand fiber deposition in the human respiratory airway a series of experiments of fiber deposition in human respiratory airway casts was intensively carried out in the aerosol laboratory of Lovelace Respiratory Research Institute. Realistic replicate casts of the human nasal and oral-lung airways were employed in the deposition studies. These airway replicate casts have been proven to provide reliable experimental data for the deposition studies using compact particles (Cheng et al., 1999, 2001a, b; Zhou and Cheng, 2005; Zwartz and Guilmette, 2001). Three man-made fibers (carbon, titanium dioxide [TiO2], and glass) were used as the test fiber materials and delivered into the airway casts at different constant inspiratory flow rates. The fiber deposition pattern and the regional deposition efficiency in the airway associated with the fiber dimensions and inhalation conditions were investigated in detail. Based on the experimental data acquired, empirical models were proposed for estimating the fiber regional deposition efficiency in the human respiratory airway. From an occupational hygiene viewpoint, the information obtained from this research is invaluable in assessing the fiber exposure dosimetry and can be applied in predicting the fiber deposition in the human respiratory airway for other types of fibers, including asbestos and newly developed man-made fibers.

Deposition of Man-Made Fibers in the Human Respiratory Airway

3

1.2. EXPERIMENTAL METHOD 1.2.1. Human Nasal Airway Cast The major entry into the human respiratory tract is the nasal airway. It is the airway’s first line of defense and acts as a filtration system to prevent hazardous aerosols from entering the lung. The fraction of the inhaled fiber acquired from the nasal airway due to deposition can directly indicate the remaining fraction of the inhaled fiber entering the lower respiratory airway. For the fiber deposition study conducted in this research, a replicate human nasal airway cast was used. This nasal airway cast was made based on the in vivo magnetic resonance imaging (MRI) of a nonsmoking Caucasian male (53 years of age, 73 kg in body mass, and 173 cm tall). The MRI images were taken every 3 mm in the nasal airway (Guilmette and Gagliano, 1994) and the original images obtained from these MRIs were digitized with a GRAF/PEN sonic digitizer (SAC, Southport, CT). The three-dimensional surfaces then were constructed for adjacent perimeter traces using a computer-assisted design software (SmartCAM, Point Control Co., Eugene, OR). The physical nasal airway cast was made by using 1.5-mm-thick acrylic plates and milling them with a computer-controlled micro-milling machine (CAMM 3, Roland DG, Los Angeles, CA). The entire nasal airway cast contains 77 acrylic plates (115.5 mm total length) and consists of complete nasal airway structures, including the anterior region (first 25 plates: 0 – 37.5 mm, with vestibule and nasal valve subregions), turbinate region (middle 32 plates: 37.5 – 85.5 mm, with front and rear turbinate subregions), and posterior region (last 20 plates: 85.5 – 115.5 mm, with entire nasopharynx region). The turbinate region was further divided into superior turbinate (ST, also known as the olfactory area), middle turbinate (MT), and inferior turbinate (IT) sections. The structure and sections of the nasal airway cast used in this research are shown in Figure 1.2.1.

1.2.2. Human Respiratory Airway Casts Cheng and collegues developed production molds of human respiratory airways from in vivo measurements (oral cavity) and cadavers (tracheobronchial airways) (Cheng et al., 1997). These production molds are used to reproduce human respiratory airway casts that have a defined geometric dimension for deposition studies and they have been shown to provide reliable data in experiments of particle deposition in the human respiratory airway (Cheng et al., 1997, 1999, 2001a, 2003; Zhou and Cheng, 2005), making them ideal for advanced fiber deposition research. Two different airway casts (LA and LB) were made from selected production molds. The only information available for these two casts is that the LA cast was made based on a 16year-old male, and the LB cast was made based on a 21-year-old male. The airway casts were made from conductive silicone rubber (KE-4576, Shin-Etsu Chemical Co., Ltd., Tokyo, Japan). Using conductive material is advantageous for the fiber deposition experiments because it can eliminate the unexpected fiber deposition in the airway caused by a possible

4

Wei-Chung Su and Yung Sung Cheng

electrostatic effect. Figure 1.2.2 shows the structure and physical model for the two casts used in this research. Each airway cast consists of an oral cavity, oropharynx, larynx, trachea, and the tracheobronchial airways to the 4th (LA) or 3rd (LB) bifurcation. Table 1.2.1 shows the morphology information for these two casts. Anterior Region

Turbinate Region

Posterior Region

Human Nasal Airway Schematic Diagram and Physical Model ST MT Nasal Valve

Turbinate

Vestibule

IT Nostril

Nasopharynx

25 plates 0-37.5 mm

32 plates 37.5-85.5 mm

20 plates 85.5-115.5 mm

(a)

(b) Su and Cheng, 2005.

Deposition of Man-Made Fibers in the Human Respiratory Airway Figure 1.2.1. Structure and regions of the human nasal airway replica: (a) schematic diagram, (b) physical model.

Lung cast A (LA)

Lung cast B (LB) Su and Cheng, 2006a. Figure 1.2.2. Structure and regions of the human respiratory airway casts.

5

6

Wei-Chung Su and Yung Sung Cheng Table 1.2.1. Airway dimensions of the two human respiratory airway casts Parent (cm)

Cast LA Trachea, D+E1 1st generation, E2+E3 2nd generation, F1 2nd generation, F2 3rd generation, G1 3rd generation, G2 3rd generation, G3 3rd generation, G4 4th generation, H1 4th generation, H2 Cast LB Trachea, D+E1 1st generation, E2+E3+E4 2nd generation, F1 2nd generation, F2 3rd generation, G1 3rd generation, G2 3rd generation, G3 3rd generation, G4

L 7.44 0.35

D 1.58 1.90

0.38 1.26 0.37 0.66 0.24 0.15 0.3 0.84

Daughter (cm) L

B Angle (°)

P Angle (°)

D

L

D

0.32 (to F1)

1.23

1.76 (to F2)

0.71

35

90

1.23 0.65 0.63 0.90 0.77 0.67 0.82 0.6

1.11 (to G1) 0.80 (to G3) 0.50 (to I1) 0.26 (to H1) 0.50 (to I7) 0.50 (to I9) 0.50 (to I3) 0.50 (to I5)

0.64 0.30 0.52 0.82 0.60 0.60 0.59 0.52

1.24 (to G2) 0.63 (to G4) 0.50 (to I2) 0.26 (to H2) 0.50 (to I8) 0.50 (to I10) 0.50 (to I4) 0.50 (to I6)

0.94 0.67 0.54 0.60 0.60 0.55 0.64 0.49

84 35 80 35 50 78 75 92

75 75 65 60 70 50 78 30

5.50 1.80

1.65 1.52

3.00 (to F1)

1.22

0.55 (to F2)

1.36

57

90

0.60 0.85 0.38 0.25 0.85 0.50

1.25 1.34 1.15 0.90 1.00 0.90

0.60 (to G1) 1.50 (to G3) 0.50 (to T1) 0.50 (to T2) 0.50 (to T3) 0.50 (to T4)

1.15 1.11 0.69 0.68 0.71 0.57

0.40 (to G2) 0.35 (to G4) 0.50 (to B1) 0.50 (to B2) 0.50 (to B3) 0.50 (to B4)

0.90 0.90 0.65 0.61 0.64 0.56

74 56 73 53 66 90

81 65 55 14 32 0

Zhou et al., 2007.

1.2.3. Fiber Materials Fiber is defined as elongated particles with an aspect ratio, β (the ratio of the length to the diameter), over 3 (NIOSH, 1994). The key factor affecting affecting fiber deposition in the human airway is the dimension of the fiber. It has been reported that the aerodynamic diameter of a fiber in the air depends primarily on its diameter and only slightly on its length (Stöber et al., 1970; Cheng et al., 1995). The toxicity of a fiber is strongly related to the fiber length. For instance, it has been reported that long and thin fibers have greater toxicity than short and thick fibers (Timbrell, 1982; Lippmann, 1990; Hill et al., 1995; Bernstein et al., 2001). Therefore, a variety of fiber material is needed when conducting fiber deposition studies for investigating the relationship between the fiber dimension and the associated deposition pattern in the human respiratory airway. In this research, three man-made fiber materials were employed in the deposition experiments: carbon, titanium dioxide (TiO2), and glass. All of these fiber materials were uniform in diameter and diverse in length. Using fibers with uniform diameter is a new approach for fiber deposition studies because it provides a simple way to obtain the fiber

Deposition of Man-Made Fibers in the Human Respiratory Airway

7

dimension for the deposited fiber (length measurement only) and a sure way to determine the effect of fiber length or fiber diameter on the deposition pattern. The test carbon fibers were relatively large fiber provided by Hercules, Inc. (Wilmington, DE). These carbon fibers are black in color, conductive, monodisperse in diameter (CMD = 3.66 μm, σg = 1.11), polydisperse in length (CML = 14.83 μm), and have a density measured at 1.83 g/cm3. The test carbon fiber material contains cylindrical fibers and fiber debris. Therefore, in this research, only carbon fibers having lengths longer than 10 μm were counted as contributing to the final deposition data. The TiO2 and glass fibers were short fibers compared with the carbon fiber. The TiO2 fiber used in this research was made at the University of Florida using electro-spinning technology. The TiO2 fiber is monodisperse in diameter (CMD = 0.59 μm, σg = 1.18) and polydisperse in length (CML = 3.20 μm) with a density of 4.23 g/cm3. The glass fiber (JM475/100, Johns Manville Co., Littleton, CO) is nearly monodisperse in diameter (CMD = 0.62, σg = 1.30) and polydisperse in length (CML = 7.67 μm) with a density of 2.56 g/cm3. Similar criteria for fiber measurement were also applied to these two fiber materials so that only TiO2 fibers longer than 2.5 μm and glass fibers longer than 3.5 μm were counted as the deposition data due to the limitation of the visual measurement. Figure 1.2.3 shows images and the statistics of these three man-made fibers.

Figure 1.2.3. The test man-made fiber materials: (a) carbon fiber, (b) TiO2 fiber, and (c) glass fiber.

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Wei-Chung Su and Yung Sung Cheng

The aerodynamic diameter (dae) of the test fiber can be approximately calculated by the equations below (Stöber, 1972):

d ae = d ve

ρ / ρo κ ,

(1)

where dve is the fiber volume equivalent diameter, ρ is the density of fiber, ρo is the density of water, and κ is the dynamic shape factor for a prolate spheroid. For a prolate spheroid flying in the air with its long axis orientating perpendicular to the flow direction, the dynamic shape factor is κ┴.

(

)

8 2 β − 1 β −1 / 3 3 κ⊥ = , 2β 2 −3 2 ln β + β − 1 + β β 2 −1

)

(

(2)

where β is the aspect ratio, and κ// is the dynamic shape factor if the long axis of a prolate spheroid orientates parallel to the flow direction.

(

)

4 2 β − 1 β −1 / 3 3 κ // = 2 β 2 −1 ln β + β 2 − 1 − β 2 β −1

(

)

(3)

If the orientation of a prolate spheroid is randomly in the air, the dynamic shape factor is κr and can be written as (Happel and Brenner, 1973):

1

κr

=

1 3κ //

+

2 3κ ⊥

.

(4)

Based on the equations shown above, for a 20-μm-long carbon fiber in the airflow (diameter = 3.66 μm) with its long axis orientating perpendicular to the flow direction (dynamic shape factor = κ┴), the aerodynamic diameter of the carbon fiber, dae(┴), is 8.4 μm. On the other hand, if the carbon fiber’s long axis is parallel to the flow direction (dynamic shape factor = κ//), the aerodynamic diameter, dae(//), is 9.7 μm. However, if the carbon fiber orientates randomly in the air (dynamic shape factor = κr), the aerodynamic diameter, dae(r), is 8.8 μm. In contrast, a 100-μm-long carbon fiber in the air has a dae(┴), dae(//), and dae(r) of 10.4 μm, 13.0 μm, and 11.3 μm, respectively. Based on the calculation above, Table 1.2.2 summarizes the physical characteristics as well as the estimated dae for these three man-made fibers (all of the measured data were obtained after the fiber materials passed through the aerosol generation devices).

Deposition of Man-Made Fibers in the Human Respiratory Airway

9

1.2.4. Experimental Setup Due to the characteristics of the fiber material, two different setups were used for the fiber deposition experiments. The carbon and glass fiber materials were dry and powder-like, and were aerosolized for the experiments by a small-scale powder disperser (SSPD, Model 3433, TSI Inc., St. Paul, MN). The related apparatus for this experimental setup included a SSPD, a charge neutralizer, the human airway cast, and the backup filter holder. Figure 1.2.4. shows the experimental setup for the fiber deposition study using the SSPD. The dispersed fibers were first delivered to the Kr85 charge neutralizer. Fibers passing through the neutralizer were at Boltzmann equilibrium and then delivered to the airway cast. A filter holder was attached to the nasopharynx of the nasal airway cast (eight or ten filter holders were connected to the ends of the bronchial airways when the lung airway cast is used) for collecting fibers that passed through the entire cast. A typical experiment lasted from 5 to 20 min depending on the inspiratory flow rate selected, and the SSPD rotation plate was set to a speed of 2. All of these operation parameters have been proven to provide a sufficient number of fibers for the deposition study. Table 1.2.2. Comparison of the physical characteristics and aerodynamic diameter for three different man-made fiber materials Fiber Type

Carbon

TiO2

Glass

Density

1.83 g/cm3

4.23 g/cm3

2.56 g/cm3

Diameter

Length

CMD = 3.66 μm

CML = 14.83 μm

σg = 1.11c

σg = 4.00

CMD = 0.59 μm

CML = 3.20 μm

σg = 0.18

σg = 1.52

CMD = 0.62 μm

CML = 7.67 μm

σg = 1.3

σg = 1.57

dae 7.6 – 12.8 μma 8.1 – 14.9 μmb (L = 10 – 300 μm)

2.1 – 2.7 μm 2.2 – 3.0 μm (L = 2.5 – 12.0 μm) 1.8 – 2.3 μm 2.0 – 2.7 μm (L = 3.5 – 20.0 μm)

a

Fiber aerodynamic diameter with random orientation Fiber aerodynamic diameter with parallel orientation c Geometric standard deviation b

The TiO2 fiber aerosol was generated by a medication nebulizer (Up-Mist, Hospitak Inc., Farmingdale, NY) due to its plate-like characteristic of its original material. The apparatus of this experimental setup included a nebulizer, several alumina drying columns, a charge neutralizer, and the filter holder. Figure 1.2.5 shows the experimental setup for the TiO2 fiber deposition study. Before generating the TiO2 fiber aerosol, the TiO2 fiber plates were placed in alcohol and ultrasonicated for 30 s before the aerosol generation process. When generating

10

Wei-Chung Su and Yung Sung Cheng

TiO2 fiber aerosol, the nebulizer generates TiO2 fiber aerosol along with the alcohol aerosol (droplets). The mixed aerosols were transferred to several drying columns and then a Kr85 charge neutralizer. In this way, sufficient time was provided to ensure that the alcohol mist evaporated before entering the cast and only TiO2 fiber aerosol remained and was delivered into the airway cast.

Human nasal airway cast

Kr 85 Filter Assembly

Air + Fiber

Vacuum Small-Scale Powder Disperser

Figure 1.2.4. Schematic diagram of the experimental setup for the fiber deposition study with SmallScale Powder Disperser.

Deposition of Man-Made Fibers in the Human Respiratory Airway

11

Figure 1.2.5. Schematic diagram of the experimental setup for the fiber deposition study with a medical nebulizer.

Silicon oil (550 Fluid, Dow Corning Co., Midland, MI) was applied to the inside surface of the airway casts to simulate the adhesive, wet nature of a real human airway prior to the deposition experiment. The oil coating has another advantage in that it can force fibers to stick to the place where they originally deposited. The deposition experiments were conducted with constant inspiratory flow rates. Four flow rates (7.5, 15, 30, and 43.5 l/min) were used in the nasal airway studies, and three flow rates (15, 43.5, and 60 l/min) were used in the lung airway studies. These inspiratory flow rates ideally cover an adult’s breathing rate during different daily activities from at-rest to moderate work. Three experiments were carried out for each combination of inspiratory flow rate, fiber materials, and airway casts to obtain average deposition values. It is worth noting that in this research, the dimensions of the glottis openings in the respiratory airway casts (LA and LB) were modified based on the inspiratory flow rate conducted to virtually simulate the variation in the laryngeal airways under different respiratory flow rates. The modification of the glottis opening refers to the information reported in Martonen and Lowe (1983).

1.2.5. Sample Preparation, Fiber Counting, and Length Measurement After each run of the deposition experiments the nasal airway cast or the lung airway cast was divided into regions and/or subregions based on the structure of the human nasal or lung airway. Care was taken when the airway cast was separated (nasal airway) or cut into segments (lung airway). Each divided section was flushed by alcohol or brushed several times using an artist’s brush dipped in alcohol. The fiber-alcohol solution from washing each individual airway section was then vacuum-filtered by a 25-mm filtration assembly to allow fibers to uniformly deposit on a 25-mm mixed cellulose ester membrane filter (GSWP,

12

Wei-Chung Su and Yung Sung Cheng

Millipore Co., Bedford, MA). The filters were then dried at room temperature in a dust-free environment and prepared as sample slides for later microscopic counting and measurement. In general, nine sample slides were acquired from each run of the nasal airway deposition experiment, and 22 to 26 sample slides were acquired from each run of the lung airway deposition experiment. Each sample slide represents the fiber deposition in a specific region of the human airway. The sample slides were then examined by an optical microscope (BH-2, Olympus Optical Co., Tokyo, Japan) with a G22 Walton-Beckett graticule (Pyser-SGI Ltd., Kent, UK). Figure 1.2.6 shows an example of deposited fibers in a microscopic viewing area (under 400x magnification). The number of fibers and the length of individual fibers in a viewing area were determined based on National Institute of Occupational Safety and Health (NIOSH) method 7400. In this research, the fiber diameter was not measured in each sample slide due to the fact that the fiber materials used are assumed to be uniform. Each sample slide was counted and measured for 200 fibers or 200 viewing areas, depending on whichever came first. In this way, the averaged number of fiber in certain length categories in a specific human airway region could be obtained. When these deposition data were available for all regions of the airway cast, the deposition pattern for the entire airway cast as well as the deposition efficiency for a specific airway region could then be determined.

Su and Cheng, 2005. Figure 1.2.6. Fiber counting and measurement with G22 Walton-Beckett gratitude under 400x magnification.

1.3. RESULTS AND DISCUSSION 1.3.1. Fiber Deposition in the Human Nasal Airway Figures 1.3.1. and 1.3.2 show the deposition patterns of man-made fibers in the human nasal airway for different length categories and inspiratory flow rates. The nasal airway was divided into three major regions (anterior, turbinate, and posterior) for presenting the

Deposition of Man-Made Fibers in the Human Respiratory Airway

13

deposition pattern in a simple manner. As can be seen, the anterior region is the site of preferred deposition for carbon fibers. The deposition fraction in the anterior region increases as the inspiratory flow rate increases, and it was found that the longer the carbon fiber, the higher the deposition fraction in that region. Besides the anterior region, a considerable number of carbon fibers deposited in the turbinate region, but very few carbon fibers deposited in the posterior region. In contrast, although the total deposition fraction in the nasal airway is small for the TiO2 and glass fibers, relatively more TiO2 and glass fibers deposited in the turbinate region instead of the anterior region, which is significantly different from the results found from carbon fiber. The regional deposition fractions of the TiO2 and glass fibers do not substantially change with different fiber length categories, or with an increase in the inspiratory flow rate.

Su et al., 2008. Figure 1.3.1. Comparison of fiber deposition patterns in the human nasal airway – the relatively short fibers in each fiber material (data shown are the deposition fractions).

14

Wei-Chung Su and Yung Sung Cheng

Su et al., 2008. Figure 1.3.2. Comparison of fiber deposition patterns in the human nasal airway – the relatively long fibers in each fiber material (data shown are the deposition fractions).

The deposition patterns shown in Figures 1.3.1. and 1.3.2 suggest that the anterior region works as an impactor for the human nasal airway. The anterior region blocks out those high momentum fibers inhaled to prevent them from entering the main nasal passage. The air flow distortion in the anterior region (90-degree turn from vertical to horizontal) is the key factor that leads to the impaction of those high momentum fibers in this area, because high momentum fibers have difficulty in turning with the air stream when an abrupt flow direction change encountered. As result, most high momentum fibers, such as carbon fiber, impacted on the inside surface of the anterior region due to the inertia force (Figure 1.3.3a). Conversely, small fibers have low inertia; therefore, they can follow the air stream closely and easily turn with the air flow and then pass through the anterior region. Therefore, fewer TiO2 and glass fibers deposited in this anterior region (Figure 1.3.3b).

Deposition of Man-Made Fibers in the Human Respiratory Airway

15

Q = 30 l/min

(a) Q = 7.5 l/min

(b) Su and Cheng, 2005. Figure 1.3.3. Fiber deposition in the vestibule area for an inspiratory flow rate of (a) Q = 30 l/min, and (b) Q = 7.5 l/min (bottom view of the nostril entrance).

The turbinate region is a unique part of the human nasal airway. Its uniqueness is mainly due to its complicated structure and slot-like airway passage. There are three meatuses in the turbinate region: superior turbinate, middle turbinate, and inferior turbinate. The whole turbinate region has a remarkably large surface area (Proctor, 1982; Mygind, 1985). The air inhaled through the nasal airway is primarily getting humidified and warmed in this region. However, certain nasal diseases might be closely related to particles that deposit in this region. Therefore, it is worth investigating the details of how fiber deposits in the turbinate region. The airflow profile in the turbinate region is laminar when the inspiratory flow rate is small; however, it could be turbulent when there is a medium to high inspiratory flow rate (Swift and Proctor, 1977; Cole, 1982; Zamankhan, et al., 2006). Turbulence in the turbinate region can cause turbulent deposition for inhaled fibers. In order to study fiber deposition in the turbinate region, it is necessary to show the regional fiber deposition efficiency in the

16

Wei-Chung Su and Yung Sung Cheng

turbinate region. Here, the fiber deposition efficiency in the nasal turbinate is defined as the ratio of fibers deposited in the turbinate region to those that entered it. For the purpose of comparison, Table 1.3.1 summarizes the calculated regional deposition efficiencies in the nasal airway for the three fiber materials together with related deposition fractions. Table 1.3.1. Comparison of the regional deposition fractions and the regional deposition efficiencies for different man-made fiber materials in the human nasal airway ANTERIOR Dep. Dep. fraction efficiency (%) Carbon Fiber length: 10–40 μm 15 l/min 35.3 0.35 30 l/min 77.4 0.77 43.5 l/min 82.6 0.83 TiO2 Fiber length: 2–4 μm 15 l/min 2.3 0.02 30 l/min 2.1 0.02 43.5 l/min 2.7 0.03 Glass Fiber length: 4–6 μm 15 l/min 30 l/min 43.5 l/min

1.0 1.3 2.9

0.01 0.01 0.03

Carbon Fiber length: 70–100 μm 15 l/min 47.7 0.51 30 l/min 87.7 0.88 43.5 l/min 82.5 0.82 TiO2 Fiber length: 6–8 μm

Turbinate Dep. Dep. fraction efficiency (%)

Posterior Dep. Dep. fraction efficiency (%)

30.6 19.6 17.2

0.47 0.87 0.99

1.0 0.5 0.1

0.03 0.15 0.51

4.9 3.5 6.3

0.05 0.04 0.07

0.0 1.4 1.0

0.00 0.01 0.01

2.4 1.3 5.1

0.02 0.01 0.05

0.1 0.3 0.2

0.00 0.00 0.00

33.8 11.8 17.4

0.66 0.96 0.99

0.9 0.2 0.1

0.05 0.36 0.72

15 l/min 1.8 30 l/min 7.0 43.5 l/min 1.8 Glass Fiber length: 8–10 μm

0.02 0.07 0.02

3.3 9.8 2.0

0.03 0.11 0.02

0.0 3.8 0.6

0.00 0.05 0.01

15 l/min 30 l/min 43.5 l/min

0.00 0.02 0.05

4.3 2.5 5.0

0.04 0.03 0.05

0.0 0.2 0.1

0.00 0.00 0.00

0.0 1.8 4.8

Su et al., 2008.

As can be seen for carbon fibers, the turbinate deposition fractions were generally smaller than the anterior deposition fractions. However, the turbinate deposition efficiencies were shown to be significantly higher than the anterior deposition efficiency. For instance, the anterior deposition fraction of the long carbon fibers (70 – 100 μm) with a 30 l/min

Deposition of Man-Made Fibers in the Human Respiratory Airway

17

inspiratory flow rate was 87.7%, which is much larger than the turbinate deposition fraction of 11.8%; but the corresponding anterior deposition efficiency was 0.88, which is less than the turbinate deposition efficiency 0.96 under the same condition. This difference between the regional deposition fraction and regional deposition efficiency, however, was not noticeable as shown for the results of TiO2 and the glass fibers. A possible reason might be that there were only a few TiO2 and glass fibers deposited in the nasal airway. Thus, the comparison between the regional deposition fraction and regional deposition efficiency in the anterior and turbinate regions was not pronounced. Nevertheless, the deposition fraction and deposition efficiency in the turbinate region were shown to be larger than those in the anterior region for both for TiO2 and glass fibers. Therefore, it can be concluded that the turbinate deposition efficiencies were generally larger than the anterior deposition efficiencies for all fiber materials in the nasal airway. These results also confirm that the turbinate region is capable of high efficiency deposition by turbulent deposition for fibers or other inhaled particles, especially those with high momentum. This capability is very advantageous for nasal airway drug delivery, such as delivering drug-containing particles to the turbinate region via nasal spray. However, some pollutants or hazardous particles could also have high deposition efficiency in the turbinate region, might thereby causing possible adverse health effects in the human nasal airway.

1.3.2. Fiber Deposition Efficiency in the Human Nasal Airway Figure 1.3.4 shows the deposition efficiency as a function of the impaction parameter (fiber momentum) for man-made fibers in the nasal airway replica. The fiber deposition efficiency is determined by the fraction of the fiber entering the nasal airway that deposited within it. The dae used for calculating the impaction parameter dae2Q is the fiber aerodynamic diameter in random orientation. As can be seen in Figure 1.3.4, the fiber deposition efficiency in the nasal airway tends to be a smooth S-like shape. The lower end of the carbon fiber deposition efficiency data points is ideally connected to the higher end of the TiO2 fiber deposition efficiency data points, which plainly shows the overall continuity between the data sets acquired. Figure 1.3.4 shows that the deposition efficiency of the carbon fiber increased proportionally with the impaction parameter, indicating that impaction is the main deposition mechanism for the carbon fiber used in this research. The deposition efficiency of the carbon fiber can reach 1.0 when the impaction parameter is greater than 50,000 μm2 cm3/s. To the contrary, the impaction parameters of TiO2 and glass fibers were both smaller than 6,000 μm2cm3/s, and partially overlapped. The deposition efficiencies of TiO2 and glass fibers in the nasal airway were shown to be similar, and both were less than 0.2. The deposition efficiencies increased only slightly with an increase in the impaction parameter. There was no significant relationship found between the impaction parameter and the deposition efficiency for the TiO2 and the glass fibers. The discrepancy of the deposition efficiencies between the three fiber materials shown in Figure 1.3.4 might be due to the fact that the diameter and length of the carbon fiber are considerably large compared with those of the TiO2 and glass fibers. This large physical dimension gives carbon fibers a significant dae value and inertia. As a result, the impaction parameter (fiber momentum) as well as the associated deposition efficiency increases substantially when the fiber dae is increased. As shown in Figure 1.3.4, most of the high

18

Wei-Chung Su and Yung Sung Cheng

momentum fibers have a deposition efficiency of 1.0, implying that high momentum fibers do not pass through the nasal airway. Thus, the human nose appears to function well for filtering out large fibers, such as the carbon fibers, to prevent them from entering the lower respiratory tract. On the other hand, the dimensions of the TiO2 and the glass fibers are relatively small compared with the carbon fiber. Therefore, the fiber dae, the impaction parameter, and the associated fiber deposition efficiency are all consequently small. It is interesting to note that the diameters of the TiO2 and the glass fibers are comparable and that the length distribution of the TiO2 fiber is shorter than that of the glass fiber. However, the calculated dae and the related impaction parameters of the TiO2 fiber are, in general, larger than those of the glass fibers. This result is due to the fact that the density of the TiO2 fiber is greater than that of the glass fiber. Moreover, as previously mentioned, the dae of a fiber depends primarily on its diameter and only slightly on its length (Stöber et al., 1970; Timbrell, 1982; Cheng, 1995). Therefore, the TiO2 fiber shows a slightly higher momentum in the nasal airway compared with the glass fiber. Nevertheless, the overall deposition efficiencies of the TiO2 fiber agree well with those of the glass fiber where the values of the impaction parameters for these two fibers overlap. As shown in Figure 1.3.4, the deposition efficiencies of the TiO2 and the glass fibers all ranged from 0.02 – 0.2, which implies that small fibers have a high penetration rate ( ≥0.8) through the nasal airway. Therefore, small fibers may present a hazard to the human lower respiratory tract.

Deposition Efficiency

1.00 0.90

Carbon fiber

0.80

TiO2 fiber

0.70

Glass fiber

0.60 0.50 0.40 0.30 0.20 0.10 0.00 100

1000

10000

100000

d ae Q ( μ m cm /sec) 2

2

3

Su et al., 2008. Figure 1.3.4 Deposition efficiency as a function of the impaction parameter for different man-made fiber materials in the human nasal airway.

Deposition of Man-Made Fibers in the Human Respiratory Airway

19

1.3.3. Comparison of Nasal Deposition between Fibers and Compact Particles Aerosol deposition experiments in the nasal airway have been conducted intensively with compact particles in the inertia regime (Swift, 1991; Cheng et al., 1999, 2001a, b; Zwartz and Guilmette, 2001; Kelly et al., 2004). IMost of the experimental data acquired from those studies agree well with each other and the data are all located within a narrow band when the deposition efficiency is plotted against the impaction parameter. In this research, deposition experiments also were carried out using compact particles. The compact particles had comparable aerodynamic diameters to those of the carbon, TiO2, and glass fibers in order to compare the coincident deposition efficiencies obtained from the fibers. The test particles were fluorescent polymer microspheres (Duke Scientific Co., Palo Alto, CA) with sizes ranging from 2.1 to 10.0 μm (dae = 2.2 to 10.2 μm). Deposition experiments were conducted using a similar experimental method as for the fiber study, and the deposition result was acquired by measuring the florescence intensity in the washed-out solution for each nasal airway region. The deposition pattern and deposition efficiency of the compact particles were obtained using the same method as employed in the fiber study. Figure 1.3.5 shows the deposition efficiency and corresponding deposition patterns for fibers and compact particles in the nasal airway. As can be seen for fibers and compact particles with a large impaction parameter (≥15,000 μm2cm3/s), the deposition efficiencies are at least 0.5 and the fiber deposition efficiencies are smaller than the compact particle deposition efficiencies. The anterior region is the site of preferred deposition in this impaction parameter regime.

1.0

Deposition Efficiency

0.9

Carbon fiber - Su and Cheng (2005) TiO2 fiber

0.8

Glass fiber

0.7

Spherical particles

Spherical particle dae = 10.2 μm Q = 15 l/min dae2Q = 26250

0.6

Spherical particle dae = 5.1 μm Q = 15 l/min dae2Q = 6563

0.5 0.4 0.3

Glass fiber Length 4-6 μm dae = 1.9 μm Q = 15 l/min dae2Q = 902

Carbon fiber Length 90-100 μm dae = 11.3 μm Q = 15 l/min dae2Q = 31738

Carbon fiber Length 10-20 μm dae = 8.3 μm Q = 15 l/min dae2Q = 17295

Spherical particle dae = 2.2 μm Q = 15 l/min dae2Q = 1158

TiO2 fiber Length 6-10 μm dae = 2.5 μm Q = 15 l/min dae2Q = 1596

0.2 0.1 0.0 100

1000

10000

100000

d ae Q ( μ m cm /sec) 2

2

3

Su et al., 2008. Figure 1.3.5 Comparison of the deposition efficiencies and related deposition patterns between fibers and spherical particles.

20

Wei-Chung Su and Yung Sung Cheng

On the other hand, the deposition efficiencies are generally less than 0.2 for those TiO2 fibers, glass fibers, and compact particles having a small impaction parameter (≤5,000 μm2cm3/s). Relatively higher deposition was found in the turbinate region. However, no significant difference was found regarding the deposition efficiency between the fiber aerosol and the compact particles in this small impaction parameter regime. The fiber deposition efficiencies were shown to be fairly close to those of compact particles. These results shown above imply that, in the inertia regime, fibers with low momentum would have a deposition behavior in the human nasal airway similar to compact particles having comparable aerodynamic diameters, while fibers with high momentum would behave differently from compact particles having an equal aerodynamic diameter. The main reason accounting for this difference might be the fiber orientation in the air flow. As stated in Su and Cheng (2005 and 2006b), some theoretical calculations (Asgharian, 1988; Chen and Yu, 1991; Asgharian et al., 1997) and experimental observation (Myojo, 1987) have reported that fibers tend to align themselves with the flow direction when they fly in the air. Having fibers oriented parallel to the air stream can reduce the fiber deposition in the human airway to a certain extent because with this orientation the fiber can well follow the air stream and pass through the nasal airway relatively easier. As a result, the number of fibers deposited in the arway is therefore less than that of compact particles. This might explain the difference of the deposition efficiencies found between the fibers and compact particles.

1.3.4. Empirical Model for Fiber Deposition in the Nasal Airway An attempt was made to find an empirical model that would be practical for estimating the fiber deposition efficiency in the human nasal airway based on all the fiber deposition data described thus far. Kelly and colleagues stated that nasal deposition can be expressed by the following expression (Kelly et al. 2004): E = 1 – exp [- (α dae2 Q)γ],

(5)

where α and γ are constants needed to be determined. In this research, equation (5) was adopted for searching the best-fit equations for fiber and available compact particle data. A nonlinear fitting procedure in SigmaPlot (SPSS Inc., Chicago, IL) was used as the fitting tool. Published compact particle data from Kelly et al. (2004) and that from the current study were used in the fitting process. It is worth noting that the nasal airway cast used by Kelly and colleagues was made from the same MRI scans that were used in this reasearch. The best-fit curve obtained for the fiber deposition efficiency is α = 4.262 x 10-5, γ = 2.46 with R2 = 0.98. The best-fit curve found for compact particle is α = 6.426 x 10-5, γ = 1.89, and R2 = 0.90. Figure 1.3.6 shows the best-fit curves for the deposition of fiber and compact particles in the nasal airway plotted along with the related experimental data. It is clearly shown that given the same impaction parameter, fewer fibers deposit in the human nasal airway compared with compact particles.

21

Deposition of Man-Made Fibers in the Human Respiratory Airway

1.0 Spherical Particle (SLA) - Kelly et al. (2004) Spherical Particle (Viper) - Kelly et al. (2004 γ 2 1-exp[-(α dae Q) ] (Viper) - Kelly et al. (2004)

Deposition Efficiency

0.8

Spherical Particle - Su and Cheng (2005) Spherical Particle γ 2 1-exp[-(α dae Q) ] (spheres) Carbon Fiber - Su and Cheng (2005) TiO2 Fiber

0.6

Glass Fiber γ 2 1-exp[-(α dae Q) ] (fibers)

0.4

0.2

0.0 102

103

104

105

dae2Q (μm2 cm3/sec) Su et al., 2008. Figure 1.3.5 The best-fit equations of the nasal deposition for fibers and spherical particles.

1.3.5. Fiber Deposition in the Human Oral Airway The oral airway is the major air entry for some workers because of their need of a large respiratory flow rate while performing moderate to heavy work. However, due to the smaller inhalation air velocity caused by the larger dimension of the oral airway, aerosol deposition in the oral airway is less efficient compared with that in the nasal airway. Therefore, a considerable portion of the inhaled particles and fibers are able to penetrate the oral airway then enter the tracheobronchial airways when breathing by mouth. The fiber deposition efficiency found in the oral airway can be used as an index of the oral penetration rate for estimating the number of fibers that enters the lower respiratory airway. Figure 1.3.6 shows the fiber deposition efficiency as a function of the impaction parameter dae2Q (dae is the fiber aerodynamic diameter calculated by parallel orientation, and Q is the inspiratory flow rate) in the oral airways for the two casts.

22

Wei-Chung Su and Yung Sung Cheng

1.0 Cast A - Carbon Fiber Cast A - TiO2 Fiber

Deposition efficiency

0.8

Cast A - Glass Fiber Cast B - Carbon Fiber Cast B - TiO2 Fiber Cast B - Glass Fiber

0.6

Particle - Cast A (Cheng et al. 1999) Particle - Cast B

2

Fiber: 1- exp (-0.0000055 d ae Q ) 2

Particle: 1- exp (-0.000276 d ae Q ) (Cheng et al. 1999)

0.4

0.2

0.0 100

1000

10000 2

2

100000

1000000

3

d ae Q (μm cm /sec)

Figure 1.3.6. Deposition efficiency as a function of the impaction parameter for fibers and compact particles in the human oral airway.

In this research, the oral airway here was defined as the regions from the oral cavity to the larynx in the airway casts. The fiber deposition efficiency in the oral airway was determined by the fraction of the fiber entering the oral airway that deposited within it. Also shown in Figure 1.3.6 are the deposition data of compact particles from our previous work for cast LA (Cheng et al., 1999) together with the new results for cast LB from this study. Fluorescent polymer microspheres (Duke Scientific Co., Palo Alto, CA) with an aerodynamic diameter from 0.5 to 16.4 μm were used as the test particles. The deposition studies of the compact particles were carried out using the same experimental method as described in the nasal airway deposition study. As can be seen in Figure 1.3.6, the magnitudes and the configurations of the fiber deposition efficiencies in the oral airways were comparable in both casts, indicating that the intersubject variability was insignificant in this research. There was no significant relationship shown between the impaction parameter and the deposition efficiency for the TiO2 and glass fibers. The deposition efficiencies increased very slightly as the impaction parameter increased. The magnitudes of the deposition efficiencies were all similar and less than 0.1. This result indicates that thin man-made fibers could have a high penetration rate (≥0.9) in the human oral airway. On the other hand, the deposition efficiency for carbon fiber increased proportionally with the impaction parameter and reached 0.6. This result implies that impaction is also the major deposition mechanism for thick man-made fibers in the oral airway. Fibers with large momentum would have difficulty penetrating the oral airway. In general, as shown by the data in Figure 1.3.4 for the nasal airway, Figure 1.3.6 also reveals an overall continuous match between data obtained from different fiber materials in both oral airway casts, and a relationship between the deposition efficiency and the impaction

Deposition of Man-Made Fibers in the Human Respiratory Airway

23

parameter is clearly shown. Based on these data, an empirical model is proposed according to the equation format suggested by Cheng et al. (1999) to express this relationship. DE(oral) = 1 – exp (-(q dae2Q)),

(6)

where DE(oral) is the fiber deposition efficiency in the oral airway, q is a constant needed to be determined, and dae2Q is the fiber impaction parameter. The nonlinear regression program used in finding the nasal airway empirical model was employed again as the fitting tool. All fiber data shown in Figure 1.3.6 were used in the fitting process. The best-fitted parameter was q = 5.47 x 10-6 with R2 = 0.96, and the result was plotted beside the experimental data shown in Figure 1.3.6. As can be seen, this proposed empirical model can well predict the fiber deposition efficiency in the oral airway, and it is believed that this model is useful in any given fiber exposure scenario (within similar experimental conditions) for estimating the fiber deposition in the human oral airway. Figure 1.3.6 also shows that the deposition efficiencies of compact particles are comparable in the two casts in terms of magnitude and configuration, which is similar to the results found for fiber. Cheng et al. (1999) suggested an empirical model for the deposition of compact particles in the oral airway based on the available in vivo data. The predicted values using the suggested model are also shown in Figure 1.3.6. It was interesting to note that the empirical model suggested by Cheng et al. (1999) agreed quite well with the experimental data acquired in this research for both airway casts. This result validated the practicability of the empirical model proposed by Cheng et al. (1999). When the deposition efficiency of compact particles was compared with that of fibers in both airway casts, the data showed that the deposition efficiencies of compact particles were generally higher than those of fibers. As was discussed above in the nasal fiber deposition, this result is presumed to be attributed to the fiber alignment in the inhaled airflow. Here, this deposition phenomenon was shown again in the oral fiber deposition which supports the fact that fibers tend to fly parallel to the air stream in the human respiratory airway. With a prevailing parallel orientation for fiber flying in the human airway, it can be assumed that “interception”, therefore, is not a critical deposition mechanism for fiber deposition in the oral airway (oral cavity, pharynx, and larynx). The major fiber deposition mechanism in the oral airway is solely inertial impaction. The interception could be an important deposition mechanism when the airway’s dimension is similar in scale to the fiber length (e.g., as in the lower tracheobronchial airways).

1.3.6. Fiber Deposition Pattern in the Human Respiratory Airway The aerosol fractional deposition shown in the human airway suggests the initial dose of the contaminant to the respiratory epithelium and how it influences the subsequent particle clearance and redistribution (Schlesinger et al., 1977). Therefore, it is important to study the fiber fractional deposition pattern in the human respiratory airway because it could provide key information for determining the pathogenesis of fiber-related lung diseases. Figures 1.3.7 and 1.3.8 show the selected deposition patterns of the three fiber materials in the two human respiratory airway casts. Panel (a) represents the deposition patterns of relatively short fibers in each fiber material under the small inspiratory flow rate, panel (b) gives the medium-sized

24

Wei-Chung Su and Yung Sung Cheng

fibers in each fiber material under median the flow rate, and panel (c) shows the deposition patterns for relatively long fibers in each fiber material under the large inspiratory flow rate. As can be seen, very few TiO2 and glass fibers deposited in the airway casts. Most of the TiO2 and glass fibers passed through the entire airway cast and collected on the backup filters, independent of the fiber length and inspiratory flow rate. The regional deposition fractions of the TiO2 and glass fibers shown in the two airway casts were rarely larger than 3%. No site of enhanced deposition could be identified. In contrast, a considerable number of carbon fibers were found deposited in both airway casts. The fractional deposition of carbon fiber in a specific airway region was shown to correlate with the fiber length and the inspiratory flow rate. The deposition of the carbon fibers increased as the fiber length or the inspiratory flow rate increased. At the large inspiratory flow rate, the oropharynx in cast LA and the larynx in cast LB were the sites of enhanced deposition for long carbon fibers. The deposition discrepancy between the carbon fiber and the TiO2 and glass fibers might be attributed to the fiber momentum (inertia) that the fiber possessed when flying in the human airway. As mentioned above, the aerodynamic diameter of the carbon fiber is significantly larger than those of the TiO2 and glass fibers. Therefore, the inhaled carbon fibers have substantially larger momentum in the airway than the TiO2 and glass fibers do. Owing to the inertial force, the heavy carbon fibers easily impacted on the airway surface when an air distortion was encountered (oropharynx and larynx). However, the light TiO2 and glass fibers are able to follow the air stream closely and make turns easily within the air distortion due to the small fiber momentum. As a result, a very limited number of TiO2 and glass fibers impacted on the airway surface –– most of them were able to pass through the entire airway cast. Based on the fiber deposition data shown above, it is implied that small fibers such as TiO2 and glass have no significant deposition mechanism in the upper human respiratory airway (down to the 3rd lung generation in this research). Those inhaled fibers having small fiber momentum, such as TiO2 and glass fibers, have a high penetration rate in the human upper airway that could lead to relatively more deposition in the lower respiratory airway, causing potential lung injures. On the other hand, most fibers that have large fiber momentum would deposit in the oral airway, soon to be removed by swallowing and eliminated in the gastrointestinal tract, thereby causing limited harm to the lower respiratory tract.

Deposition of Man-Made Fibers in the Human Respiratory Airway

25

Figure 1.3.7. The fiber deposition patterns (fractional deposition) in the human respiratory airway cast LA.

26

Wei-Chung Su and Yung Sung Cheng

Figure 1.3.8. The fiber deposition patterns (fractional deposition) in the human respiratory airway cast LB.

Deposition of Man-Made Fibers in the Human Respiratory Airway

27

Similar to the result found for fiber deposition in the human respiratory airway, the fiber deposition hotspots were visually observed in both airway casts. Figure 1.3.9 shows the sites of enhanced deposition found in the two airway casts for carbon fiber with an inspiratory flow rate of 43.5 l/min. Panel a of Figure 1.3.9 indicates the region from the oropharynx to the larynx is the enhanced deposition site in the upper airway for both airway casts. In the lower respiratory airway (panels b, c, and d of Figure 1.3.9) the carina in each tracheobronchial bifurcation was found to be the site of preferred deposition. Fibers were easily observed to deposit and accumulate along the carina ridge. This site of enhanced deposition agrees well with what was observed by Myojo (1987) and Sussman et al. (1991) in their experiments.

28

Wei-Chung Su and Yung Sung Cheng

Su and Cheng, 2006a. Figure 1.3.9. The sites of preferred deposition in the two replicas: (a) oral airway, (b) 1st bifurcation, (c) 2nd bifurcation, and (d) 3rd bifurcation.

1.3.7. Fiber Deposition in the Tracheobronchial Airways Many studies have mentioned that particle deposition in the tracheobronchial airways may result in the initiation of chronic respiratory disease and possible bronchial cancer (Chan and Lippmann, 1980; Ermala and Holsti, 1955; Gurman et al., 1984; Schlesinger and Lippmann, 1972). Fibers are elongated particles. From the inhalation toxicology point of view, fibers are more hazardous for the tracheobronchial airway compared with compact particles. Therefore, it is essential to inspect the fiber deposition in the tracheobronchial airways to provide information for etiological research. Figures 1.3.10 to 1.3.13 show the deposition efficiency as a function of Stokes number for fiber deposition in the tracheobronchial airways. Figures 1.3.10 and 1.3.11 demonstrate the fiber deposition efficiencies in the trachea and 1st lung generation. The data shown in Figures 1.3.12 and 1.3.13 are for the 2nd and 3rd lung generations, respectively. A conventional way to present

Deposition of Man-Made Fibers in the Human Respiratory Airway

29

particle deposition in human lung generations is to use the Stokes number as a parameter of the deposition efficiency because it allows data comparison from different deposition conditions and has been widely used in many bifurcating tube deposition studies of compact particles (Johnston et al., 1977; Kim and Garcia, 1991; Kim and Iglesias, 1989) and fibers (Myojo, 1987, 1990). The Stokes number (Stk) for a specific lung generation/bifurcation can be estimated by

ρ o d ae 2U s , Stk = 18ηD

(7)

where ρ0 is the density of water, dae is the aerodynamic diameter of the fiber with parallel orientation, Us is the mean air velocity in the trachea or in the parent tube of a bifurcation, η is the air viscosity, and D is the mean diameter of the trachea or the parent tube of the bifurcation. The deposition efficiency in a specific airway was computed as the fraction of fiber entering the airway that deposited there. The results shown in Figures 1.3.12 and 1.3.13 were composed by pooling all bifurcation data within the same generation. Therefore, each data point shown in Figures 1.3.12 and 1.3.13 represents the fiber deposition efficiency in a certain lung bifurcation within a specific lung generation. As can be seen in Figures 1.3.10 and 1.3.11, the characteristics of the fiber deposition efficiency in the tracheal airway and the 1st lung generation resemble those found in the oral airway (Figure 1.3.6). The TiO2 and glass fibers showed low deposition efficiencies, but the deposition efficiency of the carbon fiber increased with an increase of the fiber inertia. Besides, the tendency and magnitude of the deposition efficiencies were comparable between the two casts. These results might be due to the fact that the airway geometry of the trachea and the 1st lung generation are relatively simple and that the airway dimensions between the two airway casts were comparable. Therefore, the fiber deposition efficiency would have limited intersubject variability under the same flow rate. Empirical models are proposed for fiber deposition in the human tracheal airway and the 1st lung generation/bifurcation based on these experimental data from our research. To develop an empirical model of the tracheal airway, the suggested equation in Chan et al. (1980) for predicting the deposition efficiency of compact particles in the trachea was adopted as the format for determining fiber deposition in the tracheal airway.

30

Wei-Chung Su and Yung Sung Cheng

1.0 0.9

Deposition Efficiency

0.8 0.7

Cast A - Carbon Fiber Cast A - TiO2 Fiber Cast A - Glass Fiber Cast B - Carbon Fiber

0.6

Cast B - TiO2 Fiber

0.5

Cast B - Glass Fiber

0.4 0.3

DE = 2.54 Stk1.23

Empirical model - Fiber

DE = 0.52 Stk0.87

Empirical model - Particle Chan and Schreck (1980)

0.2 0.1 0.0 0.001

0.01

0.1

1

10

Stk Su and Cheng, 2009. Figure 1.3.10. Fiber deposition in the tracheal airway.

DE(trachea) = a Stk b,

(8)

where a and b are constants needed to be determined. The obtained best-fit curve for fiber (a = 0.52, b = 0.87, and R2 = 0.88) was plotted and is shown in Figure 1.3.10. The line for compact particles reported by Chan et al. (1980) is also shown in the same figure. For the empirical model of fiber deposition efficiency in the 1st lung generation, the equation proposed by Kim and Fisher (1999) was used. DE(1st) = 1 –

1 . cStk d + 1

(9)

This equation was originally used for compact particles as well, and c and d are also constants needed to be determined. The suggested curve found for fiber (c = 7.9, d = 2.0 with R2 = 0.90) was plotted and is shown in Figure 1.3.11 together with the curve of the compact particles. As can be seen, the two empirical models proposed well represent the fiber deposition efficiency in the human trachea and the 1st lung generation (Figures 1.3.10 and 1.3.11), and again it was evident that the estimated fiber deposition efficiencies are smaller than the deposition efficiencies of compact particles.

31

Deposition of Man-Made Fibers in the Human Respiratory Airway

1.0 0.9

Cast A - Carbon Fiber Cast A - TiO2 Fiber

Deposition Efficiency

0.8 0.7

Cast A - Glass Fiber Cast B - Carbon Fiber

0.6

Cast B - TiO2 Fiber

0.5

Cast B - Glass Fiber

0.4 0.3

DE = 1 - 1/(7.9 Stk 2.0 +1)

Empirical model - Fiber Empirical model - Particle Kim and Fisher (1999)

0.2

DE = 1 - 1/(9.6 Stk 1.7 +1)

0.1 0.0 0.001

0.01

0.1

1

10

Stk Su and Cheng, 2009. Figure 1.3.11. Fiber deposition in the 1st lung generation airway.

Figures 1.3.12 and 1.3.13 show the fiber deposition efficiency in the 2nd and 3rd lung generations. It is interesting to note that a noticeable discrepancy exists between the airway casts. Cast LB generally had higher fiber deposition efficiencies than cast LA at Stk > 0.1. Additionally, the data sets exhibited wider scattering and larger standard deviation. When taking a closer look at Figures 1.3.12 and 1.3.13, it can be seen that the discrepancy is mainly in the large Stokes number regime caused by the carbon fiber. The deposition efficiencies generated by the TiO2 and glass fibers were still on average low and comparable between the two casts. Kim and his colleagues conducted a series of tests for particle deposition in bifurcating airway models. They concluded that for a single bifurcation tube (Kim and Iglesias, 1989; Kim et al., 1994), the Stokes number is the governing factor for the particle deposition in the lung bifurcation, and the effects of the branching angle, branching asymmetry, flow distribution pattern, and the tube geometry on the particle deposition efficiency were not significant. Moreover, Kim and Fisher (1999) also found that the Stokes number is the main factor affecting the particle deposition in sequential bifurcation tubes, but the deposition efficiency in the sequent bifurcation (the secondary bifurcation) could be influenced by the bifurcation plane angle and the flow asymmetry. Theoretically, given the same Stokes number for fiber transferring in the lung bifurcations, the fiber deposition efficiency in the bifurcation tubes should be similar based on Kim and Fisher’s conclusion. The reason(s) why a discrepancy exists between the casts in this research as shown in Figures 1.3.12 and 1.3.13 is not clear immediately. A possible explanation might be the intricate nature of the human lower respiratory airway.

32

Wei-Chung Su and Yung Sung Cheng

1.0 0.9

Deposition Efficiency

0.8 0.7 0.6 0.5

Cast A - Carbon Fiber Cast A - TiO2 Fiber Cast A - Glass Fiber Cast B - Carbon Fiber Cast B - TiO2 Fiber Cast B - Glass Fiber

0.4 0.3 0.2 0.1 0.0 0.001

0.01

0.1

1

10

Stk Su and Cheng, 2009. Figure 1.3.12. Fiber deposition in the 2nd lung generation airway.

1.0 0.9

Deposition Efficiency

0.8 0.7 0.6 0.5

Cast A - Carbon Fiber Cast A - TiO2 Fiber Cast A - Glass Fiber Cast B - Carbon Fiber Cast B - TiO2 Fiber Cast B - Glass Fiber

0.4 0.3 0.2 0.1 0.0 0.001

0.01

0.1

Stk Su and Cheng, 2009. Figure 1.3.13. Fiber deposition in the 3rd lung generation airway.

1

10

Deposition of Man-Made Fibers in the Human Respiratory Airway

33

It is well-known that the airway system becomes more and more complex in the deeper regions of the tracheobronchial airway. The variety of the bifurcation plane angles and the related flow distribution as well as the effects of the fiber deposition in the adjacent upper bifurcations vary from subject to subject and would, to a certain extent, affect the fiber deposition efficiency in the lung bifurcations. Thus, unlike the trachea airway and the 1st lung generation that have uncomplicated geometry, the deposition efficiencies in the 2nd and 3rd lung generations showed an apparent intersubject variability between casts LA and LB, and it is very difficult at this point to explicitly explain the discrepancy shown in Figures 1.3.12 and 1.3.13 without a detailed investigation. In order to thoroughly understand the results displayed, further study is needed to systematically investigate the integrated effects on the fiber deposition in realistic lung bifurcations.

1.3.8. Comparison of Fiber Deposition in the Human Airways As mentioned above, there have been limited experiments conducted for fiber deposition in the human airway. Figure 1.3.14 and Table 1.3.2 compare the data of the fiber deposition in the tracheobronchial airways acquired in this research with available experimental data reported in Sussman et al. (1991) and Myojo (1987), respectively. Data shown were selected to have comparable experimental conditions, especially for fiber dimension. With this requirement, the experimental data from carbon fiber were ruled out due to their considerable large fiber dimension. The data of Sussman et al. (1991) was obtained by delivering asbestos fiber into a realistic human tracheobronchial airway cast (oral airway was not included). As can be seen in Figure 1.3.14, the regional deposition efficiency shown in the airway casts from different experiments are basically similar except for the glass fiber data of cast LB from this study. The larynx had the highest deposition efficiency in the airway cast, but the deposition efficiency dropped significantly in the trachea. The deposition efficiency for each fiber material was generally below 0.01 in the lung generations in all airway casts. Although the data fluctuated between lung generations, the magnitude of deposition efficiencies was comparable in general. Myojo’s data was acquired by delivering glass fiber into a model bifurcation tube (Myojo 1987). Table 1.3.2 compares the data obtained from this research (from one of the bifurcations in the 4th generation) with Myojo’s data under similar deposition conditions. As shown in the table, a slightly higher deposition efficiency was shown in our study. This difference might be due to the fact that the airway used in Myojo’s study was an artificial single bifurcation tube. As stated in Myojo (1987), the inside surface of the artificial lung bifurcation tube was relatively smooth and regular compared with the realistic lung bifurcation. Therefore, the air flow fields in the realistic lung bifurcation might be more complicated than those in the single artificial lung bifurcation. As a consequence, there may be more fiber deposited in the bifurcation airway of the realistic cast. Nevertheless, both the data sets shown in Table 1.3.2 confirmed that fibers having small momentum rarely deposit in the upper tracheobronchial airways (at least down to the 4th generation). Therefore, a considerable portion of the small fiber inhaled is subject to enter the lower tracheobronchial airways, raising the burden of fiber deposition in the lung.

34

Wei-Chung Su and Yung Sung Cheng 0.02 Asbestos - Sussman 1991 (2-5 μm)

Deposition Efficiency

TiO2 - Cast A (2-6 μm) Glass - Cast A (4-6 μm) TiO2 - Cast B (2-6 μm) Glass - Cast B (4-6 μm)

0.01

0.00 Larynx

Trachea 1st 2nd Human Respiratory Airway

3rd

Su and Cheng, 2009. Figure 1.3.14. Comparison of fiber deposition efficiencies in the human respiratory airway for a 15 l/min inspiratory flow rate.

Table 1.3.2. Comparison of fiber deposition efficiency in lung bifurcations Myojo 1987

This research

Length (cm) Branching angle Flow rate (l/min)

Brass model bifurcation Parent: 0.6 Daughter: 0.4 Parent: 15 45o 1.87 (Steady)

The 4th generationa Parent: 0.6 Daughter: 0.52 and 0.49 Parent: 0.84 46o 1.86 (Steady)

Fiber material Diameter (μm) Length (μm)

Glass fiber 0.53 10–20

Glass fiber 0.62 10–20

Deposition efficiency

0.7 x 10-3

1.3 x 10-3

Bifurcation Diameter (cm)

a

One of the bifurcations in the 4th generation of airway cast LA. Su and Cheng, 2009.

1.4. CONCLUSION This research reports the results obtained from a series of experiments carried out by delivering man-made fibers (carbon, TiO2, and glass fibers) with various dimensions into

Deposition of Man-Made Fibers in the Human Respiratory Airway

35

human respiratory airway casts. The deposition results showed that impaction is the dominant deposition mechanism for fibers having large momentum (e.g., carbon fiber) in the human airway. The regional deposition efficiency of large momentum fiber increases as the fiber momentum increases. The sites of enhanced deposition for the large momentum fibers are located where the air distortion is significant in the airway, such as the anterior region of the nasal airway, the oropharynx-larynx region in the oral airway, and the carina ridge in the lung bifurcation. In contrast, no significant deposition mechanism was found for fibers with small momentum (e.g., TiO2, and glass fibers). Most of the low momentum fibers passed easily through the entire nasal or oral airway and then entered the lower human respiratory tract. These results imply that thin fibers could easily penetrate the upper respiratory airway and have a pathogenic potential in the lower respiratory airway, while thick fibers deposit just beyond the upper tracheobronchial airways and most of them are unable to reach the lower respiratory tract. When comparing the deposition efficiency between fibers and compact particles, it is interesting to note that the deposition efficiencies of large momentum fibers are considerably lower than those of compact particles. However, the discrepancy is not significant for small momentum fibers. This result indicates that fibers might behave differently from compact particles in the air flow for the large momentum regime, while small fibers and compact particles have similar deposition behavior in the small momentum regime. This result is believed to be subject to the fiber alignment in the air stream. Based on all of the experimental data acquired in this research, empirical models were proposed to express the relationships found between the fiber deposition efficiency and the fiber momentum (impaction parameter or Stokes number) for specific regions in the human respiratory airway. The deposition information acquired from these models can be applied in predicting fiber deposition in the human airway and assessing the exposure dosimetry for other fibers, including asbestos and newly developed man-made fibers.

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Su, W. C. and Y. S. Cheng (2005). Deposition of Fiber in the Human Nasal Airway. Aerosol Sci. Technol. 39: 888-901. Su, W. C. and Y. S. Cheng: Fiber (2006a). Deposition Pattern in Two Human Respiratory Tract Replicas. Inhalation Toxicology. 18: 749-760. Su, W. C. and Y. S. Cheng (2006b). Deposition of Fiber in a Human Airway Replica. J. Aerosol Sci. 37: 1429-1441. Su, W. C., J. Wu and Y. S. Cheng (2008). Deposition of Man-made Fiber in a Human Nasal Airway. Aerosol Sci. Technol. 42: 173-181. Su, W. C. and Y. S. Cheng (2009). Deposition of Man-made Fibers in Human Respiratory Airway Casts. J. Aerosol Sci. 40: 270-284. Sussman, R. G., Cohen, B. S., and Lippmann, M. (1991). Asbestos fiber deposition in human tracheobronchial cast. I. experimental. Inhalation Toxicology, 3, 145-160. Swift, D. L. (1991). Inspiratory inertial deposition of aerosols in human nasal airway replicate casts: implication for the proposed NCRP lung model, Radiation Protection Dosimetry, 38(1/3), v29-34. Swift, D. L., and Proctor D. F. (1977). Access of air to the respiratory tract. In: Respiratory Defense Mechanisms: Part I, J. D. Brian , D. F. Proctor, and L. M. Reid eds., Dekker, New York, pp. 63-93. Timbrell, V. (1982). Deposition and retention of fibers in human lung. The Annals of Occupational Hygiene, 26, 347-369. Walton, W. H. (1982). The nature, hazards, and assessment of occupational exposure to airborne asbestos dusts: a review, Annals of Occupational Hygiene, 20, 19-23. Zamankhan, P., Ahmadi, G., Wang, Z., Hopke, P. K., Cheng, Y. S., and Su, W.C. (2006). Airflow and deposition of nano-particles in a human nasal cavity. Aerosol Science and Technology, 40, 463-476. Zhou, Y., and Cheng, Y. C. (2005). Particle deposition in a cast of human tracheobronchial airways. Aerosol Science and Technology, 39, 492-500. Zhou, Y., W.-C. Su and Y. S. Cheng (2007). Fiber Deposition in the Tracheobronchial Region: Experimental Measurements. Inhal. Toxicol. 19(13): 1071-1078. Zwartz, G. J., and Guilmette, R. A. (2001). Effect of flow rate on particle deposition in a replica of a human nasal airway. Inhalation Toxicology, 13, 109-127.

In: Asbestos: Risks, Environment and Impact Editor: Antonio Soto and Gael Salazar

ISBN 978-1-60692-053-4 © 2009 Nova Science Publishers, Inc.

Chapter 2

NUMBER OF PLASMIDS TRANSPORTED INTO ESCHERICHIA COLI THROUGH THE YOSHIDA EFFECT AND PREDICTED STRUCTURE OF THE PENETRATION-INTERMEDIATE Naoto Yoshida* Department of Biochemistry and Applied Biosciences, University of Miyazaki, 1-1Gakuen Kibanadai-Nishi, Miyazaki 889-2192, Japan

ABSTRACT When a colloidal solution containing nano-sized acicular material, such as chrysotile, and bacterial cells are placed in friction field between hydrogel and a polymer interface, the nanosized acicular material penetrates bacterial cells and forms a complex called a penetrationintermediate. This is known as the Yoshida effect. The hydrogel exposure method is a novel technique that employs the Yoshida effect to transform prokaryotes with a plasmid. The author of the present study has confirmed that two or more of pUC18, pHSG298, and pHSG396 containing the same replication of origin can be simultaneously introduced into Escherichia coli cells using the hydrogel exposure method. Multiple plasmids were maintained stably in E. coli cells, even following cell subculture under the selection pressure by antibiotics. Multiple plasmids were maintained stably in E. coli cells, even after the subculture was repeated. The stability of each plasmid in E. coli cells was as follows: pUC18, pHSG298, and pHSG396. To investigate the number of plasmids introduced into E. coli cells through the Yoshida effect, the author carried transformed E. coli with both pUC18 and pHSG298, which are adsorbed onto chrysotile in a ratio of 1:1. The relative proportion of colonies obtained from a LB plate supplemented with ampicillin, kanamycin, and both, was 6:6:1, respectively. Subsequently, transformation of E. coli via the Yoshida effect was performed using chrysotile adsorbed to pUC18, pHSG298, and pHSG396 in a 1:1:1 ratio. The * E-mail: [email protected] Tel.: +81-985-58-7218

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relative proportion of colonies obtained from a LB plate supplemented with ampicillin, kanamycin, ch1oramphenicol, and all three combined was 15:15:15:1, respectively. It is possible that the penetration-intermediate can only incorporate one plasmid using the Yoshida effect. pUC18- and pHSG298- bound chrysotile were prepared independently. When E. coli cells were exposed to hydrogel using both pUC18- and pHSG298-bound chrysotile, relative proportion of colonies obtained from a LB plate supplemented with ampicillin, and kanamycin was 1:1, respectively. However, no colonies were obtained on a LB plate supplemented with both ampicillin and kanamycin. This result suggests that the structure of the penetration-intermediate with acquired plasmid DNA consists of a bacterial cell penetrated by a single chrysotile fiber.

INTRODUCTION When a colloidal solution containing nano-sized acicular material [1] and bacterial cells is stimulated by sliding friction at the interface between hydrogel and an interface-forming material, the frictional coefficient increases rapidly and the nano-sized acicular material and bacterial cells form a chestnut bur-shaped complex. This complex grows larger and penetrates bacterial cells, thereby forming a penetration-intermediate, due to the driving force derived from the sliding friction [2]. This effect is called the Yoshida effect, in honor of its discoverer [3]. A hydrogel shear stress greater than or equal to 2.1 N is essential for the Yoshida effect to occur [4], and has been observed with agarose, gellan gum [5], and kappa-carrageenan [6]. In addition, polymers such as polystyrene, polyethylene, acrylonitrile-butadiene rubber and latex rubber, as well as silicate minerals such as quartz and jadeite, are all suitable interfaceforming materials. With regard to nano-sized acicular materials, the Yoshida effect has also been confirmed with multi-walled carbon nanotubes [7], maghemite (gamma-Fe2O3) [8], chrysotile [9, 10] and alpha-sepiolite [11] having diameters of 10-50 nm. Discovering efficient means by which to introduce DNA into bacteria is of great practical importance in genetic engineering and molecular biology [12, 13]. The introduction of exogenous DNA into Escherichia coli was first demonstrated by Mandel and Higa [14], who observed that incubation of a suspension of E. coli cells and bacteriophage lambda DNA in a solution of CaCl2 at 0˚C resulted in transduction. They further showed that exposure to a heat pulse, in which a mixture of cells and DNA was briefly incubated at 42˚C, chilled on ice, and then diluted by the addition of growth medium, improved the frequency of transfection. Hanahan's protocol is one of the best chemical transformation methods presently available, resulting from a series of attempts to attain maximal transformation efficiency [15, 16] after the first demonstration of Ca2+-dependent DNA transfer into E. coli by Cohen [17, 18]. The transformation efficiency of E. coli by plasmid DNA is 105 -109 cells/µg plasmid DNA using the Hanahan method. The Yoshida effect [3] is a phenomenon in which a penetration-intermediate is formed. The author of the present study has previously attempted to introduce genes into bacterial cells by the nano-sized acicular material adsorbed to plasmid DNA through the Yoshida effect. E. coli as a plasmid recipient cell was dispersed in a nano-sized acicular material colloidal solution containing chrysotile adsorbed to plasmid DNA (chrysotile-plasmid-cell mixture). Following this, the chrysotile-plasmid-cell mixture was dropped onto the surface of

Number of Plasmids Transported into Escherichia Coli through the Yoshida Effect.. 41 a hydrogel, such as agarose, and treated physically by sliding a polystyrene streak bar over the hydrogel to create friction (hydrogel exposure). Plasmid DNA was easily incorporated into E. coli, and antibiotic resistance was conferred by transformation [19]. To obtain greater transformation efficiency, the author then attempted to determine optimal transformation conditions. The following conditions resulted in the greatest transformation efficiency: the recipient cell concentration within the chrysotile-plasmid-cell mixture had an optical density greater than or equal to 2 at 550 nm, the vertical reaction force applied to the streak bar was greater than or equal to 40 g, and the rotation speed of the hydrogel was greater than or equal to 34 rpm. The transformation efficiency of E. coli cultured in solid medium was greater than that of E. coli cultured in broth. Under these conditions, the author observed a transformation efficiency of 107 per µg pUC18 DNA. This transformation method is known as the hydrogel exposure method [20]. The advantage of achieving bacterial transformation is that a competent cell preparation is not required. The complications associated with conventional methods are reduced because gene introduction and screening are carried out on the hydrogel simultaneously. In addition to E. coli, other Gram-negative and Gram-positive bacteria are able to acquire plasmid DNA through the Yoshida effect. The hydrogel exposure method has been embraced as superior to conventional methods. With prolonged hydrogel exposure, during which surface moisture may diffuse into the hydrogel, greater concentrations of chrysotile are observed on the hydrogel surface. In addition, chrysotile aggregates exceeding 50 µm resembling chestnut burs are observed on the hydrogel surface [21]. The chrysotile aggregates penetrate the cell membranes of adherent E. coli cells during hydrogel exposure due to sliding friction forces. We have previously demonstrated that plasmids adsorbed to chrysotile aggregates are incorporated into recipient E. coli cells (penetration-intermediate). In penetration-intermediate, plasmid adsorbed to chrysotile was released from chrysotile by approximate 300 bp of sRNA [22, 23], replicated by DNA polymerase, and then transferred to daughter cell[24]. It is generally accepted that plasmids containing the same origin of replication are incompatible [25, 26, 27, 28] and cannot stably co-exist in a cell together. However, several groups have carried out experiments in which plasmids containing the same origin of replication and different antibiotic resistance genes have been introduced simultaneously into bacteria. Two Col E1-derivative plasmids have co-replicated in the same E. coli clone for many cell doublings, irrespective of the rec genotype of host E. coli [29]. Phagemids containing the same origin of replication can co-exist without the need to apply any selection pressure for relatively long periods [30]. These results appear to contradict the widely accepted doctrine of plasmid incompatibility, which is defined as the failure of two coresident plasmids to be stably inherited in the absence of external selection [25]. With regard to the hydrogel exposure method, the author found that multiple plasmids categorized as incompatible containing the same origin of replication were incorporated simultaneously into E. coli cells, and persisted in the present study. The number of plasmids incorporated into each penetration-intermediate through the Yoshida effect and the structure of each penetration-intermediate can be predicted by based on the characteristics of compatible plasmids. In this chapter, the author outlines the number of plasmids capable of being incorporated into a single penetration-intermediate, and discusses the structure of penetrationintermediates.

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MATERIALS AND METHODS E. COLI STRAIN AND PLASMIDS Escherichia coli JM109 (el4-, recA1, endA1, gyrA96, thi-1, hsdR17, supE44, relA1, ∆(lac-proAB), and [F’, traD36 proAB, lacIq Z∆M15]) was used as the recipient cell line. pUC18 [31], pHSG396 [32] and pHSG298 [32] plasmids containing the same origin of replication derived from pMB1 were used as donor plasmids. Various characteristics of these plasmids are shown in Table 1. pUC18, pHSG396 and pHSG299 possess ampicillin chloramphenicol, and kanamycin resistance, as selective markers, respectively. pHSG298 and pHSG396 contain the same consensus multi-cloning site as pUC18. Table 1. Plasmids used in this study Plasmid pUC18 pHSG396 pHSG298

Replicon pMB1 pMB1 pMB1

Size (bp) 2686 2238 2676

Selection marker Ampicillin Chloramphenicol Kanamycin

Accession No. L09136 M19415 M19087

PREPARATION OF THE CHRYSOTILE COLLOIDAL SOLUTION Chrysotile whisker, isolated using a sedimentation procedure [33], was purchased from Waco Chemical Industries, Ltd. (Osaka, Japan). A 0.4 g whisker of chrysotile suspended in 40 ml of deionized distilled water was vigorously shaken for 5 min (at 1500 rpm), and then centrifuged at 2,000 g for 2 min. After centrifugation, the upper phase was discarded to remove excess floating matter. The chrysotile pellet was then re-suspended in 40 ml of deionized distilled water, and vigorously shaken for 5 min (at 1500 rpm). The upper phase obtained by centrifugation at 2,000 g for 2 min was then filtered using a 30 µm mesh size nylon filter (Millipore, USA), autoclaved at 121˚C for 15 min, and then used as the chrysotile colloidal solution (CCS). Of the fibers remaining in the CCS, 95% had diameters of 50-70 nm and lengths of 0.34-2.30 µm, as determined by scanning electron microscopy. When chrysotile density was determined spectrophotometrically, an OD550 between 0.05 and 0.10 was observed, and the concentration of chrysotile fibers in CCS was estimated at 40-50 µg/ml.

STANDARD PROTOCOL OF HYDROGEL EXPOSURE E. coli JM109 colonies grown at 37˚C for 18 hr on LB agar plates were retrieved and suspended in 475 µl of chrysotile colloidal solution containing 10-50 ng of pUC18 DNA, and subsequently added to 25 µl of 4 M NaCl solution as an osmotic stabilizer. This was used as the chrysotile-plasmid-cell colloidal mixture (CPCM). The CPCM density was adjusted spectrophotometrically to 2.0 at 550 nm. Hydrogel plates (8.5 cm in diameter) containing 2% agar (Nakalai tesque), LB nutrients [34] and 50 µg/ml ampicillin, were rapidly dried in a

Number of Plasmids Transported into Escherichia Coli through the Yoshida Effect.. 43 clean room to remove all visible water from the surface before spreading CPCM onto the hydrogel surface. A 50 µl aliquot of CPCM was spread onto each hydrogel plate, after which the hydrogel surface was rubbed for 60 seconds using a polystyrene stir stick (SARSTEDT, Germany) and an automatic turning table (90 rpm), without damaging the agar. The vertical reaction force on the stir stick was maintained at 40 gf/cm2 using a specially designed apparatus known as the Tribos Provider [35]. This is the is the hydrogel exposure method. The Tribos Provider apparatus was purchased as a Gene Injector from Takasaki Scientific Instruments Co. (Kawaguchi, Japan) After incubation for 18-20 h at 37˚C, the number of cell colonies demonstrating antibiotic resistance were counted.

INTRODUCTION OF MULTIPLE PLASMIDS INTO E. COLI CELLS USING THE HYDROGEL EXPOSURE METHOD E. coli JM109 cultured on LB agar plate at 37˚C for 18-20 h were used as plasmid recipient cells. 500 µl of chrysotile colloidal solution was added to a mixture of pUC18 and pHSG298 (0.5 µg each), after which the chrysotile carrying plasmids was washed with distilled water by centrifugation to remove unbound plasmid, and finally dissolved in 500 µl of 200 mM NaCl. The density of the chrysotile carrying plasmids colloidal solution was adjusted spectrophotometrically to 2.0 at 550 nm by the direct addition of recipient E. coli JM109 cells. This was used as the chryostile-plasmid (pUC18 and pHSG298)-cell mixture (CPCM). The same method was used to make the chrysotile-plasmids (pUC18 and pHSG369, pHSG298 and pHSG396, and all three plasmids)-cell mixture. Each CPCMs were exposed to hydrogel on LB-containing 2% agar. Subsequently, the penetration-intermediate(s) formed on the surface of each hydrogel was recovered with 1 ml of LB broth, and then transferred to a test tube and shaken at 37˚C for 60 min. 50 µl of fluid from the test tube was spread onto a LB plate containing multiple antibiotics (ampicillin and kanamycin, ampicillin and chloramphenicol, kanamycin and chloramphenicol, or all three antibiotics). The concentration of each antibiotic on a given LB plate was 50 µg/ml. In order to confirm growth under the selection pressure of multiple antibiotics, single colony demonstrating antibiotics resistance was inoculated onto a new LB agar plate containing multiple antibiotic combinations. Plasmids were then extracted from transformed E. coli demonstrating resistance to multiple antibiotics using a commercial kit (Plasmid Miniprep Kit, BIORAD), after which plasmid size was examined by agarose gel electrophoresis.

PLASMID STABILITY E. coli with acquired resistance to ampicillin and chloramphenicol was cultured in BL broth supplemented with ampicillin, chloramphenicol, and both ampicillin and chloramphenicol, as well as in the absence of antibiotics at 37˚C for 24 h (first culture). Following this, the cells were subcultured into fresh medium supplemented with each of these antibiotics every 24 h over the course of 9 days (2ed to 10th culture). E. coli with acquired resistance to ampicillin and kanamycin were cultured in BL broth supplemented with ampicillin, kanamycin, and both ampicillin and kanamycin, as well as in the absence of

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antibiotics at 37˚C for 24 h (first culture). After this, the cells were subcultured into fresh medium supplemented with each of these antibiotics every 24 h over the course 9 days (2ed to 10th culture). E. coli with acquired resistance to chloramphenicol and kanamycin were cultured in BL broth supplemented with chloramphenicol, kanamycin, and both chloramphenicol and kanamycin, as well as in the absence of antibiotics at 37˚C for 24 h (first culture), followed by subculture into fresh medium supplemented with each of these antibiotics every 24 h over the course of 9 days (2ed to 10th culture). The concentration of each antibiotic within the medium was 50 µg/ml. Plasmids were extracted from E. coli following the first, 5th, and 10th culture using a commercial kit (Plasmid Miniprep Kit, BioRad), after which plasmid size was confirmed by agarose gel electrophoresis.

NUMBER OF PLASMIDS INTRODUCED INTO EACH PENETRATIONINTERMEDIATE USING THE HYDROGEL EXPOSURE METHOD Escherichia coli JM109 cultured on LB agar plate at 37˚C for 18-20 h were used as plasmid recipient cells. 500 µl of chrysotile colloidal solution was added to a mixture of pUC18 and pHSG298 (0.5 µg each), chrysotile carrying plasmids was washed with distilled water following centrifugation to remove unbound plasmid and dissolved in 500 µl of 200 mM NaCl. The density of the chrysotile carrying plasmid colloidal solution was adjusted spectrophotometrically to 2.0 at 550 nm by the direct addition of recipient E. coli JM109 cells. This mixture exposed to hydrogel on LB-containing 2% agar. Subsequently, the penetration-intermediate(s) formed on the surface of the hydrogel was recovered with 1 ml of LB broth, and then transferred to a test tube and shaken at 37˚C for 60 min. 50 µl of fluid from the test tube was spread on LB plates containing ampicillin, kanamycin, and both ampicillin and kanamycin. The number of colonies demonstrating antibiotic resistance, presumably as a result of transformation, was examined for each LB plate after incubation at 37˚C for 18-20 h. 500 µl of chrysotile colloidal solution was added to a mixture of pUC18 , pHSG298, and pHSG396 (0.5 µg each), after which the chrysotile were washed with distilled water following centrifugation to remove unbound plasmid and dissolved in 500 µl of 200 mM NaCl. The density of the chrysotile carrying plasmids colloidal solution was adjusted spectrophotometrically to 2.0 at 550 nm by direct addition of recipient E. coli JM109 cells. This mixture was then exposed to hydrogel on LB-containing 2% agar. Subsequently, the penetration-intermediate(s) formed on the surface of the hydrogel was recovered with 1 ml of LB broth, and then transferred to a test tube and shaken at 37˚C for 60 min. 50 µl of fluid from the test tube was spread onto LB plates containing ampicillin, kanamycin, chloramphenicol and all three antibiotics. The number of colonies demonstrating antibiotic resistance, presumably as a result of transformation, was examined for each LB plate after incubation at 37˚C for 18-20 h.

Number of Plasmids Transported into Escherichia Coli through the Yoshida Effect.. 45

PREDICTED STRUCTURE OF A PENETRATION-INTERMEDIATE Escherichia coli JM109 cells cultured in LB broth at 37˚C for 18-20 h were used as plasmid recipient cells. 500 µl of chrysotile colloidal solution was added to 0.5 µg of pUC18, after which chrysotile carrying pUC18 was washed with distilled water following centrifugation to remove unbound pUC18 and dissolved in 500 µl of 200 mM NaCl (chrysotile carrying pUC18 colloidal solution). A chrysotile carrying pHSG298 colloidal solution was prepared in the same manner described above. Equal volumes (250 µl) of chrysotile carrying pUC18 and carrying pHSG298 colloidal solution were mixed, and the density of mixture adjusted spectrophotometrically to 2.0 at 550 nm by direct addition of recipient E. coli JM109 cells. This mixture was then exposed to hydrogel on LB-containing 2% agar. Subsequently, the penetration-intermediate(s) formed on the surface of the hydrogel was recovered with 1 ml of LB broth, and then transferred to a test tube and shaken at 37˚C for 60 min. 50 µl of fluid from the test tube was spread onto LB plates containing ampicillin, kanamycin, and both ampicillin and kanamycin. The number of colonies demonstrating antibiotic resistance, presumably as a result of transformation, was examined for each LB plate after incubation at 37˚C for 18-20 h.

RESULTS INTRODUCTION OF MULTIPLE PLASMIDS INTO E. COLI USING THE HYDROGELEXPOSURE METHOD E. coli sensitive to a range of antibiotics are often used in transformation experiments in the field of biotechnology. However E. coli transformed by pUC18, pHSG298 and pHSG396, demonstrate resistance to ampicillin, kanamycin, and chloramphenicol, respectively. In a previous study, we found that applying the hydrogel exposure method to a mixture of chrysotile, E. coli JM109 recipient cells and pUC18, resulted in transformation of E. coli JM109 cells with pUC18, thereby conferring ampicillin resistance [36]. A mixture containing chrysotile carrying pUC18 and pHSG396 with E. coli JM109 was subjected to the hydrogel exposure method, after which transformed colonies of E. coli JM109 resistant to both ampicillin and chloramphenicol were obtained. As shown in Figure 1AC, a single colony of transformed E. coli JM109 resistant to ampicillin and chloramphenicol using the hydrogel exposure method was inoculated on a new LB agar plate containing both ampicillin and chloramphenicol, after which further growth was observed. E. coli JM109 harboring either pUC18 or pHSG396 alone did not grow on LB agar containing both ampicillin and chloramphenicol. E coli JM109 transformed to possess both ampicillin and chloramphenicol resistance, E. coli JM109 (pUC18), and E. coli (pHSG396) were cultured in LB broth under selective antibiotic pressure, after which plasmid was extracted from each cultured cell line. Linearized pUC18 and pHSG396 following Hind III digestion appears as single DNA bands corresponding to 2686 bp and 2238 bp, respectively, on agarose gel electrophoresis. As shown in Figure 2A, the plasmids extracted from E. coli JM109 (pUC18) and E. coli JM109 (pHSG396) appeared at the 2686 and 2238 bp positions following Hind III digestion, respectively. Plasmid extracted from E. coli JM109 transformed

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to possess both ampicillin and chloramphenicol resistance produced two DNA bands corresponding to 2686 and 2238 bp following Hind III digestion. This result indicated that pUC18 and pHSG396 co-existed in a single E. coli cell following hydrogel exposure.

Figure 1. Growth of E. coli JM109 harboring multiple plasmids on LB agar plates containing various antibiotics as selection markers. AC: E. coli (pUC18), E. coli (pHSG396), and E. coli transformed to possess both ampicillin and chloramphenicol resistance, were streaked onto LB plates containing both ampicillin and chloramphenicol, AK: E. coli transformed to possess both ampicillin and kanamycin resistance was streaked onto LB plates containing both ampicillin and kanamycin, CK: E. coli transformed to possess both chloramphenicol and kanamycin resistance was streaked onto LB plates containing both chloramphenicol and kanamycin, ACK: E. coli transformed to possess resistance to all three antibiotics was streaked onto LB plates containing ampicillin, chloramphenicol, and kanamycin combined.

A mixture containing chrysotile carrying pUC18 and pHSG298 with E. coli JM109 was subjected to the hydrogel exposure method, resulting in transformed colonies resistant to both ampicillin and kanamycin. As shown in Figure 1AK, a single colony of transformed E. coli JM109 resistant to ampicillin and kanamycin using the hydrogel exposure method was inoculated onto a new LB agar plate containing both ampicillin and kanamycin, after which further growth of E. coli JM109 was observed. E. coli JM109 harboring either pUC18 or

Number of Plasmids Transported into Escherichia Coli through the Yoshida Effect.. 47 pHSG298 alone did not grow under these circumstances. E coli JM109 transformed to possess both ampicillin and kanamycin resistance, E. coli JM109 (pUC18), and E. coli JM109 (pHSG298), was cultured in LB broth under selective antibiotic pressure, after which plasmid was extracted from each cultured cell line. pUC18 and pHSG298 digested by Sma I produces a single DNA band (2686 bp) and two DNA bands (764, 1912 bp), respectively, on agarose gel electrophoresis. As shown in Figure 2B, The plasmid extracted from E. coli JM109 (pUC18) appeared as a single DNA band at 2686 bp following Sma I digestion. The plasmid extracted from E. coli JM109 (pHSG298) appeared as two DNA bands at 764 and 1912 bp following Sma I digestion. Plasmid extracted from E. coli JM109 transformed to possess both ampicillin and kanamycin resistance appeared as three DNA bands corresponding to 764, 1912, and 2686 bp. This result indicated that pUC18 and pHSG298 co-existed in single E. coli JM109 cell following hydrogel exposure.

Figure 2. The plasmid profile of E. coli JM109 showing the presence of multiple plasmids. Lane M indicates a DNA size marker of lambda DNA double digested with HindIII and EcoRI. Panel A: plasmid extracted from E. coli digested with HindIII and subjected to 1% agarose gel electrophoresis. Lanes 1 and 3: pUC18 and pHSG396, respectively. Lane 2: plasmid extracted from E. coli transformed to possess both ampicillin and chloramphenicol resistance. Panel B: plasmid extracted from E. coli digested with SmaI and subjected to 1% agarose gel electrophoresis. Lanes 1 and 3: pUC18 and pHSG298, respectively. Lane 2: plasmid extracted from E. coli transformed to possess both ampicillin and kanamycin resistance. Panel C: plasmid extracted from E. coli digested with EcoRI and subjected to 1% agarose gel electrophoresis. Lanes 1 and 3: pHSG396 and pHSG298, respectively. Lane 2: plasmid extracted from E. coli transformed to possess both chloramphenicol and kanamycin resistance. Panel D: plasmid extracted from E. coli double digested with SmaI and XhoI and subjected to 1% agarose gel electrophoresis. Lanes 1, 2 and 3: pUC18, pHSG396 and pHSG298, respectively. Lane 4: plasmids extracted from E. coli transformed to possess ampicillin, chloramphenicol and kanamycin resistance.

A mixture containing chrysotile carrying pHSG298 and pHSG396 with E. coli JM109 was subjected to the hydrogel exposure method, after which transformed colonies resistant to both kanamycin and chloramphenicol were obtained. As shown in Figure 1CK, a single colony of transformed E. coli JM109 resistant to kanamycin and chloramphenicol using the hydrogel exposure method was inoculated onto a new LB agar plate containing both kanamycin and chloramphenicol, after which further growth of E. coli JM109 was observed. E. coli JM109 harboring either pHSG298 or pHSG396 alone did not grow under these circumstances. E coli JM109 transformed to possess both kanamycin and chloramphenicol

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resistance, E. coli JM109 (pHSG298) and E. coli (pHSG396), was cultured in LB broth under selective antibiotic pressure, after which plasmid was extracted from each cultured cell line. Linearized pHSG298 and pHSG396 by EcoRI digestion produces single DNA bands corresponding 2276 bp and 2238 bp, respectively, on agarose gel electrophoresis. As shown in Figure 2C, the plasmid extracted from E. coli (pHSG298) and E. coli (pHSG396) appeared at the 2276 and 2238 bp positions following EcoRI digestion, respectively. The plasmid extracted from E. coli JM109 transformed to possess both kanamycin and chloramphenicol resistance appeared as two DNA bands corresponding to 2276 and 2238 bp following EcoRI digestion. This result indicated that pHSG298 and pHSG396 co-existed in a single E. coli JM109 cell following hydrogel exposure. A mixture containing chrysotile carrying pUC18, pHSG298 and pHSG396, with E. coli JM109 cells was subjected to hydrogel exposure method, after which transformed colonies resistant to ampicillin, kanamycin and chloramphenicol were obtained. As shown in Figure 1ACK, a single colony of transformed E. coli JM109 resistant to ampicillin, kanamycin and chloramphenicol using the hydrogel exposure method was inoculated onto a new LB agar plate containing ampicillin, kanamycin and chloramphenicol, after which further growth of E. coli JM109 was observed. E. coli JM109 harboring either pUC18, pHSG298 or pHSG396 alone did not grow under these circumstances. E coli JM109 transformed to possess all three antibiotics resistance, E. coli (pUC18), E. coli (pHSG298) and E. coli (pHSG396) was cultured in LB broth under selective antibiotic pressure, after which plasmid was extracted from each cultured cell line. Linearized pUC18 and pHSG396 by both SmaI and XhoI digestion appeares as single DNA bands corresponding 2686 bp and 2238 bp, respectively on agarose gel electrophoresis. pHSG298 digested by both Sma I and XhoI produces two DNA bands (764 and 1912 bp) on agarose gel electrophoresis. As shown in Figure 2D, plasmid extracted from E. coli JM109 (pUC18) appeared as a single DNA band at 2686 bp following both SmaI and XhoI digestion. The plasmid extracted from E. coli JM109 (pHSG396) appeared as single DNA band at 2238 bp following both SmaI and XhoI digestion. The plasmid extracted from E. coli JM109 (pHSG298) produced two DNA bands at 764 and 1912 bp following both SmaI and XhoI digestion. Plasmid extracted from transformed E. coli JM109 resistant to ampicillin, chloramphenicol and kanamycin produced four DNA bands corresponding to 764, 1912, 2238 and 2686 bp following both SmaI and XhoI digestion. This result indicated that pUC18, pHSG396 and pHSG298 co-existed in a single E. coli cell after hydrogel exposure.

PLASMID STABILITY When E. coli JM109 harboring both pUC18 and pHSG396 was subcultured in LB broth containing both ampicillin and chloramphenicol, stable co-existence of the plasmids was observed over 10 subcultures, equivalent to approximately 100 generations (Figure 3). However, the copy number of pHSG396 was less than that of pUC18. When E. coli harboring both pUC18 and pHSG396 was subcultured in LB containing ampicillin alone, persistence of pUC18 was observed over 10 subcultures, while a reduction in the relative copy number of pHSG396 was observed, with disappearance after 5 subcultures. When E. coli JM109 harboring both pUC18 and pHSG396 was subcultured in LB containing chloramphenicol,

Number of Plasmids Transported into Escherichia Coli through the Yoshida Effect.. 49 both plasmids persisted over 10 subcultures. When E. coli JM109 harboring both pUC18 and pHSG396 was subcultured in LB in the absence of antibiotics, persistence of pUC18 was observed over 10 subcultures, while a reduction in the relative copy number of pHSG396 was observed, with disappearance after 5 subcultures (Figure 3).

Figure 3. Stability and compatibility of pUC18 and pHSG396. E. coli JM109 (pUC18, pHSG396) was grown in LB broth with repeated sub-culturing in the presence of ampicillin (Lane Am), chloramphenicol (Lane Cm), and both ampicillin and chloramphenicol (Lane Am Cm), as well as in the absence of antibiotics (Lane Non). Plasmid extracted from each sub-culture of E. coli was digested with HindIII and subjected to 1% agarose gel electrophoresis. Lane M indicates a DNA size marker of lambda DNA double digested with HindIII and EcoRI. Panels A, B, and C show plasmid profiles following the 1st, 5th, and 10th sub-culture of E. coli (pUC18, pHSG396), respectively.

When E. coli JM109 harboring both pUC18 and pHSG298 was subcultured in LB broth containing ampicillin and kanamycin or kanamycin alone, stable co-existence of the plasmids was observed over 10 subcultures, equivalent to approximately 100 generations (Figure 4). When E. coli JM109 harboring both pUC18 and pHSG298 was subcultured in LB broth containing ampicillin alone, persistence of pUC18 was observed over 10 subcultures, while a reduction in the relative copy number of pHSG298 was observed at 10 subcultures. When E. coli JM109 harboring both pUC18 and pHSG298 was subcultured in LB in the absence of antibiotics, persistence of pUC18 was observed over 10 subcultures, while a reduction in the relative copy number of pHSG298 was observed at 10 subcultures (Figure 4).

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Figure 4. Stability and compatibility of pUC18 and pHSG298. E. coli JM109 (pUC18, pHSG298) was grown in LB broth with repeated sub-culturing in the presence ampicillin (Lane Am), kanamycin (Lane km), both ampicillin and kanamycin (Lane Am Km), and in the absence of antibiotics (Lane Non). Extracted plasmid from each sub-culture of E. coli was digested with SmaI and subjected to 1% agarose gel electrophoresis. Panels A, B, and C show plasmid profiles following the 1st, 5th, and 10th subculture of E. coli (pUC18, pHSG298), respectively.

When E. coli JM109 harboring both pHSG298 and pHSG396 was subcultured in LB broth containing kanamycin and chloramphenicol or chloramphenicol alone, stable coexistence of the plasmids was observed over 10 subcultures, equivalent to approximately 100 generations. Prior to subculture, the copy number of pHSG396 was less than that of pUC18. When E. coli JM109 harboring both pHSG298 and pHSG396 was subcultured in LB containing kanamycin alone or in the absence of antibiotics, persistence of pHSG298 was observed over 10 subcultures, while disappearance of pHSG396 was observed at 5 subcultures (Figure 5). These results indicate that plasmid stability in terms of persistence in E. coli is as follows: pUC18 > pHSG298 > pHSG396.

Number of Plasmids Transported into Escherichia Coli through the Yoshida Effect.. 51

Figure 5. Stability and compatibility of pHSG396 and pHSG298. E. coli JM109 (pHSG396, pHSG298) was grown in LB broth with repeated sub-culturing in the presence chloramphenicol (Lane Cm), kanamycin (Lane km), both chloramphenicol and kanamycin (Lane Cm Km), and in the absence of antibiotics (Lane Non). Extracted plasmid from each sub-culture of E. coli was digested with EcoRI and subjected to 1% agarose gel electrophoresis. Panels A, B, and C show plasmid profiles following the 1st, 5th, and 10th sub-culture of E. coli (pHSG396, pHSG298), respectively.

NUMBER OF PLASMIDS INTRODUCED INTO THE PENETRATIONINTERMEDIATE USING THE HYDROGEL EXPOSURE METHOD Colonies demonstrating ampicillin and kanamycin resistance were reliably obtained following exposure of a mixture of chrysotile, pUC18, pHSG298 and E. coli JM109 recipient cells, to the hydrogel. The author of the present study then examined the number of colonies obtained on LB agar plates containing ampicillin, kanamycin, or ampicillin and kanamycin combined. As shown in Table 2, 257 and 273 colonies (mean values from five independent experiments) were observed on LB agar plates containing ampicillin and kanamycin, respectively. Forty four colonies (mean value of five independent experiments) were observed on the LB agar plate containing both ampicillin and kanamycin. Table 2. Number of colony forming units of E. coli JM109 transformed to possess double antibiotic resistance through the Yoshia effect No. of transformants/plate Selection marker

Trial 1

2

3

4

5

Average

Ratio

Ampicillin (Am)

166

70 355 382 310 257

6

Kanamycin (Km)

166 200 352 359 288 273

6

Am & Km

14

17

75

50

64

44

1

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Naoto Yoshida

Colonies demonstrating ampicillin, chloramphenicol, and kanamycin resistance were reliably obtained following exposure of a mixture of chrysotile, pUC18, pHSG298 and pHSG396 with E. coli JM109 recipient cells, to the hydrogel. The author of the present study examined the number of colonies obtained on LB agar plates containing ampicillin, chloramphenicol, kanamycin, or all three antibiotics combined. As shown in Table 3, 1115, 1462 and 1223 colonies (mean values from four independent experiments) were observed on LB agar plates containing ampicillin, chloramphenicol, and kanamycin, respectively. On the other hand, 85 colonies (mean value of four independent experiments) were observed on the LB agar plate containing all three antibiotics. Interference among different patterns of resistance was not observed since E. coli JM109 resistant to all three antibiotics were observed on LB agar plates in the presence or absence of the three antibiotics, and the same number of colonies were observed regardless of the presence of antibiotic. The penetrationintermediate most commonly introduces only one plasmid using the hydrogel exposure method. Table 3. Number of colony forming units of E. coli JM109 transformed to possess multiple antibiotic resistance through the Yoshia effect

Selection marker Ampicillin (Am) Kanamycin (Km) Chloramphenicol (Cm) Am & Km & Cm

1 711 861 823 35

2 677 695 893 43

No. of transformants Trial Average 3 4 1392 1680 1115 1748 1588 1223 2040 2092 1462 142 119 85

Ratio 15 15 15 1

PREDICTED STRUCTURE OF THE PENETRATION-INTERMEDIATE Colonies demonstrating ampicillin and kanamycin resistance were reliably obtained following exposure of a mixture of chrysotile, pUC18, pHSG298 and E. coli recipient cells, to the hydrogel exposure method. While the penetration-intermediate most commonly incorporates only one plasmid with the Yoshida effect, some penetration-intermediates can incorporate multiple plasmids (pUC18 and pHSG298) simultaneously. pUC18-bound and pHSG298-bound chrysotile were prepared independently (Figure 8, inset). It is well known that nucleic acid adsorbs very tightly to clay mineral [37, 38, 39, 40], thus plasmid is not easily released from chrysotile that is composed of clay mineral [41]. Hydrogel exposure was performed to investigate whether E. coli recipient cells could be transformed to demonstrate multiple antibiotic resistance using a newly prepared chrysotile colloidal solution containing equal amounts of pUC18-bound and pHSG298-bound chrysotile. Transformation was evaluated by the number of colonies formed on LB agar plates containing either ampicillin or kanamycin, as well as the number of colonies formed on plates containing both ampicillin and kanamycin, indicating penetration by both pUC18-bound and pHSG298-bound chrysotile. Thousands of colonies formed on LB agar plates containing

Number of Plasmids Transported into Escherichia Coli through the Yoshida Effect.. 53 either ampicillin or kanamycin. However, no colonies formed on LB agar plates containing both ampicillin and kanamycin (Figure 8). This result suggests that single penetrationintermediate which acquire exogenous plasmid accepts single penetration by chrysotile. It is possibly that the structure of penetration-intermediate is the simple state of which single chrysotile penetrates single E. coli cell.

CONCLUSION Velappan et al. have carried out experiments in which plasmids containing the same origin of replication and different antibiotic resistance genes were introduced simultaneously into bacteria. By selecting for resistance to one antibiotic, and subsequently testing these clones for resistance to other antibiotics, they were able to determine the percentage of bacteria containing more than one plasmid and found that plasmid persistence is far more common than generally realized [30]. The plasmids pUC18, pHSG396, and pHSG298 contain the same origin of replication (pMB1) [42, 43] and different antibiotic resistance genes. Our results indicate that these plasmids can be introduced simultaneously into a single E. coli cell through the Yoshida effect, and can be stably maintained in E. coli for periods that are experimentally significant (Figure3-5). Multiple plasmids containing the same replication of origin persisted in E. coli in the presence of external pressure. This contradicts the widely accepted doctrine of plasmid incompatibility and suggests that more than one plasmid may be introduced into the penetration-intermediate(s) formed through the Yoshida effect. Figure 6 is a diagrammatic representation of the introduction of two plasmids into E. coli JM109 cells. The penetration-intermediate must uptake both pUC18 and pHSG298 to acquire ampicillin and kanamycin resistance. The relative proportion of plasmids containing pUC18, PHSG298 or both in the present study was 6:6:1 (Table 2, Figure6). The frequency of acquisition of both pUC18 and pHSG298 by the penetration-intermediate in the present study was 15% as determined by patterns of antibiotic resistance. The relative proportion of plasmids containing pUC18, PHSG298 or both, would be closer to 2:2:1 if every penetration-intermediate was capable of acquiring two plasmids. Figure 7 is a diagrammatic representation of the introduction of three plasmids into E. coli JM109 cells. The penetration-intermediate must uptake all three pUC18, pHSG396, and pHSG298 plasmids to acquire ampicillin, chloramphenicol, and kanamycin resistance. The relative proportion of plasmids containing pUC18, pHSG396, PHSG298, or all three in the present study was 15:15:15:1 (Table 3, Figure7). The frequency of acquisition of all three by the penetration-intermediate was 6% as determined by patterns of antibiotic resistance. A maximum of three plasmids can be introduced into a single E. coli cell through the Yoshida effect. Uptake of a single plasmid by the penetration-intermediate is most common with the Yoshida effect.

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Figure 6. Diagrammatic representation of an experiment designed to predict the number of plasmids introduced into a penetration-intermediate. Inset: with prolonged hydrogel exposure, greater concentrations of chrysotile were observed on the hydrogel surface. In addition, chrysotile aggregates exceeding 50µm developed on the hydrogel surface. They were shaped like a chestnut bur. The chrysotile aggregates adsorbed plasmid (pUC18, pHSG298) and penetrated E. coli JM109 cells during hydrogel exposure as a result of the sliding friction force generated at the interface of the hydrogel using a stirring stick. The relative ratio of colony forming units seen on LB plates containing ampicillin (Amr), kanamycin (Kmr), and both ampicillin and kanamycin (Amr, Kmr ), was 6:6:1, respectively.

Figure 7. Diagrammatic representation of an experiment designed to predict the number of plasmids introduced into a penetration-intermediate. Inset: the chestnut bur-shaped chrysotile aggregates adsorbed plasmid (pUC18, pHSG396, pHSG298) and penetrated E. coli JM109 cells during hydrogel exposure as a result of the sliding friction force generated at the interface of the hydrogel using a stirring stick. The relative ratio of colony forming units seen on LB plates containing ampicillin (Amr) , kanamycin (Kmr), chloramphenicol (Cmr), and ampicillin, kanamycin and chloramphenicol combined (Amr, Kmr, Cmr), was 15:15:15:1, respectively.

Number of Plasmids Transported into Escherichia Coli through the Yoshida Effect.. 55

Figure 8. Diagrammatic representation of an experiment designed to predict the structure of the penetration-intermediate. The relative ratio of colony forming units seen on LB plates containing either ampicillin (Amr) or kanamycin (Kmr), was 1:1, respectively. E. coli JM109 transformed to possess both ampicillin and kanamycin resistance was not obtained (Amr, Kmr).

With the hydrogel exposure method, plasmid adsorbed to nano-sized acicular material is transported into E. coli cells via the Yoshida effect. The author of the present study demonstrated the introduction of multiple plasmids containing the same origin of replication, thereby conferring resistance to different antibiotics, into E. coli JM109 cells. pUC18-bound and pHSG298-bound chrysotile colloidal solutions were prepared independently, mixed, and then subjected to hydrogel exposure. The pUC18-bound and pHSG298-bound chrysotile complexes must have each penetrated the E. coli JM109 cell to confer ampicillin and kanamycin resistance. Figure 8 is a diagrammatic representation of the introduction of pUC18 and pHSG298 into E. coli JM109 cells. E. coli JM109 were observed to uptake either pUC18 or pHSH298, thereby demonstrating ampicillin or kanamycin resistance, but not both pUC18 and pHSG298 simultaneously. This result suggests that the structure of the penetrationintermediate which acquire exogenous plasmid DNA show penetrated state of E. coli cell with single chrysotile fiber. The author proposed that multiply penetrated E. coli with chrysotile fibers lead cell bursting or lost the function of cell division.

REFERENCES [1] [2]

[3]

Dong, L. and Johnson, D.T. Adsorption of acicular particles at liquid-fluid interfaces and the influence of the line tension. Langmuir. 2005; 21(9): 3838-49. Kurokawa, T., Tominaga, T., Katsuyama, Y., Kuwabara, R., Furukawa, H., Osada, Y., and Gong, J.P. Elastic-hydrodynamic transition of gel friction. Langmuir. 2005; 21(19): 8643-8. Yoshida, N. Discovery and Application of the Yoshida Effect: Nano-Sized Acicular Materials Enable Penetration of Bacterial Cells by Sliding Friction Force. Recent Pat Biotechnol. 2007; 1(3):194-201.

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Naoto Yoshida Stammen, J.A., Williams, S., Ku, D.N., and Guldberg, R.E. Mechanical properties of a novel PVA hydrogel in shear and unconfined compression. Biomaterials, 2001; 22(8): 799-806. Smith, A.M., Shelton, R.M., Perrie, Y., and Harris, J.J. An initial evaluation of gellan gum as a material for tissue engineering applications. J Biomater Appl. 2007; 22(3): 241-54. Hosseinzadeh, H., Pourjavavdi, A. and Zohuriaan-Mehr, M.J. Modified carrageenan. 2. Hydrolyzed crosslinked kappa-carrageenan-g-PAAm as a novel smart superabsorbent hydrogel with low salt sensitivity. J Biomater Sci Polym Ed. 2004; 15(12): 1499-511. Rojas-Chapana, J., Troszczynska, J., Firkowska, I., Morsczeck, C., and Giersig, M. Multi-walled carbon nanotubes for plasmid delivery into Escherichia coli cells. Lab Chip, 2005; 5(5): 536-9. Kang, S.C., Jo, Y.J., Bak, J.P., Kim, K.C., and Kim, Y.S. Evaluation for protein binding affinity of maghemite and magnetite nanoparticles. J Nanosci Nanotechnol. 2007; 7(11):3706-8. Balan, E., Mauri, F., Lemaire, C., Brouder, C., Guyot, F., Saitta, A.M., and Devouard, B. Multiple ionic-plasmon resonances in naturally occurring multiwall nanotubes: infrared spectra of chrysotile asbestos. Phys Rev Lett. 2002; 89(17): 177401. Falini, G., Foresti, E., Gazzano, M., Gualtieri, A.F., Leoni, M., Lesci, I.G., and Roveri, N. Tubular-shaped stoichiometric chrysotile nanocrystals. Chemistry, 2004; 10(12): 3043-9. Kara, M., Yuzer, H., Sabah, E., and Celik, M.S. Adsorption of cobalt from aqueous solutions onto sepiolite. Water Res. 2003; 37(1): 224-32. Listner, K., Bentley, L.K. and Chartrain, M. A simple method for the production of plasmid DNA in bioreactors. Methods Mol Med. 2006; 127: 295-309. Grabherr, R. and Bayer, K. Impact of targeted vector design on Co/E1 plasmid replication. Trends Biotechnol. 2002; 20(6): 257-60. Mandel, M. and Higa, A. Calcium-dependent bacteriophage DNA infection. J Mol Biol. 1970; 53(1): 159-62. Hanahan, D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983; 166(4): 557-80. Inoue, H., Nojima, H. and Okayama, H. High efficiency transformation of Escherichia coli with plasmids. Gene, 1990; 96(1): 23-8. Cohen, G. and Zimmer, Z. Transfection of Escherichia coli by bacteriophage P1 DNA. Mol Gen Genet. 1974; 128(2): 183-6. Cohen, S.N., Chang, A.C. and Hsu, L. Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc Natl Acad Sci U S A. 1972; 69(8): 2110-4. Yoshida, N., Ikeda, T., Yoshida, T., Sengoku, T., and Ogawa, K. Chrysotile asbestos fibers mediate transformation of Escherichia coli by exogenous plasmid DNA. FEMS Microbiol Lett. 2001; 195(2): 133-7. Yoshida, N., Nakajima-Kambe, T., Matsuki, K., and Shigeno, T (2007) Novel plasmid transformation method mediated by chrysotile, sliding friction, and elastic body exposure. Anal Chem Insights, 2007; 2: 9-15.

Number of Plasmids Transported into Escherichia Coli through the Yoshida Effect.. 57 [21] Yoshida, N. and Saeki, Y. Chestnut bur-shaped aggregates of chrysotile particles enable inoculation of Escherichia coli cells with plasmid DNA. Appl Microbiol Biotechnol. 2004; 65(5): 566-75. [22] Vogel, J. and Wagner, E.G. Target identification of small noncoding RNAs in bacteria. Curr Opin Microbiol. 2007; 10(3): 262-70. [23] Levine, E., Zhang, Z., Kuhlman, T., and Hwa, T. Quantitative characteristics of gene regulation by small RNA. PLoS Biol. 2007; 5(9): e229. [24] Yoshida, N. and Ide, K. Appl Microbiol Biotechnol. Plasmid DNA is released from nano-sized acicular material surface by low molecular-weight oligonucleotides: Exogenous plasmid acquisition mechanism for penetration-intermediates based on the Yoshida effect, In press. [25] Novick, R.P. Plasmid incompatibility. Microbiol Rev. 1987; 51(4): 381-95. [26] Nordström, K. and Austin, S.J. Mechanisms that contribute to the stable segregation of plasmids. Annu Rev Genet. 1989; 23: 37-69. [27] Austin, S. and Nordström, K. Partition-mediated incompatibility of bacterial plasmids. Cell, 1990; 60(3):351-4. [28] Bouet, J.Y., Nordström, K. and Lane, D. Plasmid partition and incompatibility--the focus shifts. Mol Microbiol. 2007; 65(6): 1405-14. [29] Hashimoto-Gotoh, T. and Timmis, K.N. Incompatibility properties of Col E1 and pMB1 derivative plasmids: random replication of multicopy replicons. Cell, 1981; 23(1): 229-38. [30] Velappan, N., Sblattero, D., Chasteen, L., Pavlik, P., and Bradbury, A.R. Plasmid incompatibility: more compatible than previously thought? Protein Eng Des Sel. 2007; 20(7): 309-13. [31] Yanisch-Perron, C., Vieira, J. and Messing, J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene, 1985; 33 (1), 103-19. [32] Takeshita, S., Sato, M., Toba, M., Masahashi, W. and, Hashimoto-Gotoh, T. Highcopy-number and low-copy-number plasmid vectors for lacZ alpha-complementation and chloramphenicol- or kanamycin-resistance selection. Gene, 1987; 61(1): 63-74. [33] Pelé, J.P. and Calvert, R.A. Comparative study on the hemolytic action of short asbestos fibers on human, rat, and sheep erythrocytes. Environ Res. 1983; 31(1): 16475. [34] Sambrook, J., Fritsch, E.F. and Maniatis, T. Molecular cloning: a laboratory manual, 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y.: 1986. [35] Yoshida, N. and Saeki, Y. Chrysotile fibers penetrate Escherichia coli cell membrane and cause cell bursting by sliding friction force on agar plates. J Biosci Bioeng. 2004; 97(3): 162-8. [36] Yoshida, N., Kodama, K., Nakata, K., Yamashita, M., and Miwa, T. Escherichia coli cells penetrated by chrysotile fibers are transformed to antibiotic resistance by incorporation of exogenous plasmid DNA. Appl Microbiol Biotechnol. 2002; 60(4): 461-8. [37] Lorenz, M.G., Aardema, B.W. and Wackernagel, W. Highly efficient genetic transformation of Bacillus subtilis attached to sand grains. J Gen Microbiol. 1988; 134(1): 107-12.

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[38] Lorenz, M.G. and Wackernagel, W. Natural genetic transformation of Pseudomonas stutzeri by sand-adsorbed DNA. Arch Microbiol. 1990; 154(4): 380-5. [39] Demanehe, S., Jocteur-Monrozier, L., Quiquampoix, H., and Simonet, P. Evaluation of biological and physical protection against nuclease degradation of clay-bound plasmid DNA. Appl Environ Microbiol. 2001; 67(1): 293-9. [40] Gallori, E., Bazzicalupo, M., Dal Canto L., Fani, R., Nannipieri, P., Vettori, C., and Stotzky, G. Transformation of Bacillus subtilis by DNA bound on clay in non-sterile soil. FEMS Microbiol Ecol. 1994; 15(1-2), 119-26. [41] Mossman, B.T., Bignon, J., Corn, M., Seaton, A., and Gee, J.B. Asbestos: scientific developments and implications for public policy. Science, 1990; 247(4940): 294-301. [42] Cesareni, G., Cornelissen, M., Lacatena, R.M., and Castagnoli, L. Control of pMB1 replication: inhibition of primer formation by Rop requires RNA1. EMBO J. 1984; 3(6): 1365-9. [43] Russell, D.W. and Horiuchi, K. The mutH gene regulates the replication and methylation of the pMB1 origin. J Bacteriol. 1991; 173(10): 3209-14.

In: Asbestos: Risks, Environment and Impact Editor: Antonio Soto and Gael Salazar

ISBN 978-1-60692-053-4 © 2009 Nova Science Publishers, Inc.

Chapter 3

PREVENTION AND DETECTION OF ASBESTOSRELATED DISEASES IN FINLAND Matti S. Huuskonen ABSTRACT In 2006, the WHO and ILO decided to recommend a worldwide ban on the use of asbestos products, which is opposed by the countries producing those products. Early diagnosis of asbestos-related diseases, and close monitoring of the health of the patients, aims to improve the prognosis of occupational diseases and, at the same time, will secure the patients with the benefits offered by social medicine. About 100,000 people die of asbestos-related diseases throughout the world every year: 60,000 of lung cancer, 30,000 of mesothelioma and 10,000 of asbestosis. The number of asbestos-related cancers is still on the increase in industrial countries and this trend will continue elsewhere in the future. The first occupational diseases caused by asbestos were found more than 100 years ago, and it was known in the 1930s that there was a link between asbestosis and lung cancer. Although a large number of resources have been spent on research into asbestos-related diseases, not a great deal of progress has been made in the medical prevention and treatment of asbestosis, lung cancer or mesothelioma. Radiological follow-up and monitoring the health of workers who have been exposed to asbestos are opposed by those who require proof from randomized mortality follow-ups before the early detection and treatment of lung-cancer in people without symptoms could be considered ethical. There is, however, some good news that encourages action in this area. The new imaging methods used in the diagnosis of lung cancer have proven far superior to conventional chest X-rays and early diagnosis of incipient, small and operable lesions is possible with low-dose spiral tomography.

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Matti S. Huuskonen

USE OF ASBESTOS AND EXPOSURE TO ASBESTOS There has been a ban on the use of asbestos in Finland since 1994, and almost no asbestos is used for new building purposes any more. The occupational hygiene limit for asbestos was reduced from 2 fibres/cm3 in 1986 to 0.1 fibres/cm3 in 2005. More than 300,000 tons of asbestos (175 000 tons chrysotile, 120 000 tons anthophyllite and altogether 5 000 tons chrocidolite and amosite) has been used in Finland. An anthophyllite quarry was in operation at Paakkila between 1918 and 1975. Asbestos products were manufactured at various localities between 1923 and 1988. The use of asbestos was at its peak in the 1960s and 1970s. The following jobs have involved exposure to asbestos: asbestos spray-coating, asbestos quarry work, the manufacture of asbestos products, maintenance and repair work, shipbuilding, installation of thermal boilers, lining and dismantling ovens, pipe insulation and other insulation work, the production of building materials, housing construction, property maintenance, and brake and clutch work. In Finland 2/3 of all the asbestos used is in the existing building stock, and more than 3,000 workers may be exposed to dust that contains asbestos in repair and maintenance work if the protection regulations are not observed or the technology is defective. That is why stringent safety regulations apply to the dismantling of building materials that contain asbestos; the work is carried out using special methods and may only be carried out by licensed companies and workers with the relevant training. The waste from dismantling asbestos is transported to landfill sites which are subject to regulations concerning the treatment of asbestos waste. The safety of asbestos-dismantling work is regulated by the Government decision on asbestos work and the National Board of Labour Protection’s decision on acceptable methods and equipment, Table 1 shows the asbestos situation in Finland in 1986, 1992 and 2005 ((Asbestos Committee 1990; Huuskonen et al. 1995; Huuskonen & Rantanen 2006; Laakkonen et al. 2007).

Prevention and Detection of Asbestos-Related Diseases in Finland Table 1. The results achieved during the FIOH Asbestos Program 1987-1992 and its follow-up since 1993

New use of asbestos Prohibition

Import of raw asbestos and its production (tons/year) Total consumption of asbestos (tons/year) Asbestos abatement Regulation

Situation 1986

Situation 1992

Situation 2005

Crocidolite asbestos since1977

All asbestos varieties since 2005 (EU Directive)

2,200

All asbestos varieties with some exceptions since 1993 0

0

2,500

300

10

Unlicensed and poorly controlled

Licensed to special companies and controlled

Licensed to special companies and controlled

Training of abatement No Yes Yes workers Number of trained abatement Below 10 4,000 2,000 workers Below 1 25 Below 20 Revenue of inspection and abatement companies (EUR/year) 770 7,000 5,000 Asbestos samples from inspection and abatement work (number/year) Occupational exposure limit 2.0 fibers/cm2 0.5 fibers/cm3 0.1 fibers/cm3 Diagnosis of occupational diseases. Asbestosis (number/year) 30 133 80 Pleural changes 0 1,390 510 Cancers 5 83 141 Quality of the diagnoses related to occupational diseases and asbestos

61

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Matti S. Huuskonen

Compensation practice of occupational diseases

Mostly asbestosis recognized and compensated

New compensation guidelines for asbestos, cancer and new registration practice of bilateral pleural changes

Mesothelioma panel of pathologists Expert groups for the diagnosis of pneumoconioses (number of group in universities) Diagnostic fiber analysis of lung tissue taken during operation or at autopsy (number/year) Use of (high resolution) computed tomography in the diagnosis of asbestos-related diseases

No

Yes

Compensation for asbestos cancers according to the accepted exposure criteria and registration of asbestosrelated diseases. Asbestos lung cancers are compensated according to the same criteria in Australia, France, Belgium and Germany. Yes

1

5

5

Below 10

around 100

300 (about 15% of lung cancers)

No

HRCT with asbestosis identification diagnosis

For asbestosis, diagnostic HRCT criteria in use, CT spreading in clinical monitoring of occupational disease patients. Debate on screening for lung cancer.

All asbestos diseases under surveillance and follow-up

All asbestos diseases under surveillance and follow-up

Current, retired and previous asbestos workers under surveillance and follow-up More than 20,000

Current, retired and previous asbestos workers under surveillance and follow-up

Surveillance of workers exposed to asbestos Asbestos-related Only occupational diseases asbestosis under surveillance and followup Asbestos-exposed workers Only current workers under surveillance and followup Number of persons under Few hundred surveillance and follow-up of asbestosis cases followed by the FIOH

More than 5,000

Prevention and Detection of Asbestos-Related Diseases in Finland

63

Monitoring the Health Situation of those Who Have Been Exposed to Asbestos The number of people who have worked with asbestos in Finland has been estimated at 200,000, of whom 50,000 to 60,000 are still alive, their average age being over 70. In many instances, asbestos-related diseases do not appear until decades after exposure, so the medical condition of those exposed to asbestos must be monitored even when the exposure has come to an end. An initial check-up is carried out on workers who will be exposed to asbestos before their work starts. Periodic check-ups are then performed at three-year intervals. The occupational health care service monitors the medical condition of those who have been working and exposed to asbestos, and correspondingly the rest of the primary health care service looks after those who do not participate in working life or work independently. The occupational health care services guide those who are finishing their work, including those who will be retiring or rendered unemployed, to apply for check-ups at a place considered suitable. When the occupational health care service or other health care service discovers a person with a finding indicative of an asbestos-related disease or a suspicion of it, he/she is sent for further diagnostic tests at a lung hospital or the outpatient department of occupational medicine services at a university hospital, which organizes regular monitoring of the sick person’s medical condition (Huuskonen 2005; Finnish Institute of Occupational Health and Ministry of Social Affairs and Health, Terveystarkastukset työterveyshuollossa [Health checks in occupational health care] 2006). Between 1990 and 1992, the Institute of Occupational Health carried out broad-based screening of asbestos-related diseases which showed that 22% of those who had worked in housing construction and shipbuilding had pulmonary fibrosis and changes in a pleura (Huuskonen et al. 1995; Koskinen et al. 1998). The Mini-Suomi survey showed that, as a result of work-related exposure to asbestos, 6.8% of men and 2% of women in the population over the age of 30 had bilateral pleural plaques. Radiological findings in the lung caused by asbestos are detectable in more than 100,000 Finns. This incidence, which is extremely high by international standards, has been caused by the general use of anthophyllite asbestos in building materials, which began in the 1920s (Zitting et al. 1995). An HRCT test is better than a lung X-ray for diagnosing diffuse interstitial pulmonary fibrosis, emphysema and changes in a pleura and, correspondingly, low dose spiral computer tomography is better for diagnosing lung cancer. For well founded suspicions of an occupational disease, insurance companies will compensate for the costs of clinical tests in accordance with the Employment Insurance Accidents Act. Insurance companies will pay for the follow-up tests of patients with an occupational disease, and units responsible for the follow-up of asbestos patients will ensure that the insurance system receives information sufficiently often about changes in the clinical condition of a patient in order to determine the disablement (Tiitola et al. 2002; Huuskonen et al. 2001; Kusaka et al. 2005; Nordman et al. 2006; Vierikko et al. 2007).

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DISEASES CAUSED BY ASBESTOS AND MORTALITY FROM ASBESTOSRELATED DISEASES Asbestos causes lung cancer, mesothelioma (pleural cancer and cancer of the peritoneum), asbestosis (pneumoconiosis) and pleural disorders such as parietal pleural fibrosis (plaques), diffuse visceral pleural fibrosis and exudative pleural inflammation (pleuritis). Rounded atelectasis can be created in a fibrotic pleura and the atelectatic lung fiber beneath it. Asbestos also increases the danger of developing carcinoma of the larynx and retroperitoneal fibrosis. The time gap between the first exposure to asbestos and the onset of asbestos-related diseases is generally more than 10 years, and with cancers often 20 to 40 years or more. The greater the exposure to asbestos, the greater the risk of becoming ill. It is not possible to identify a level of exposure below which the risk of asbestos cancers would not have increased (Consensus report; WHO 1998; Uibu et al. 2004). An extremely clear link between the use of asbestos and mortality from asbestos-related diseases is seen when a comparison is made between the connection between the volume of use of asbestos in the 1960s and the mortality caused by asbestos-related diseases in 33 countries at the start of the 2000s. Some of these countries have used asbestos more than Finland in proportion to the population and some have used less. In Western Europe, the Nordic countries, North America and Australia, the use of asbestos was at its peak in the 1960s and 1970s (Lin et al. 2007). The incidence of mesothelioma, which is considered an indicator of exposure to asbestos, has remained at about 70 cases a year in Finland, according to the Finnish Cancer Registry. This is average for the industrial countries. Between 1955 and 2004, a total of about 1,500 people died of mesothelioma. During this time, there were about 4,500 cases of asbestosrelated cancer, assuming that the number of lung cancers caused by asbestos is estimated at double the number of cases of mesothelioma. In the next few years, the number of asbestosrelated cancers is expected to rise from the present level (Tossavainen 2004; Huuskonen et al. 2006; Laakkonen et al. 2007).

Lung Cancer Asbestos can cause all kinds of lung cancers in terms of type of tissue. They can be located in any part of the lung, but occur more frequently in the lower lobes than in cases of lung cancer in the portion of the population without asbestos exposure. Epidemiological studies show that up to between 13% and 35% of all lung cancers have an occupational cause, with asbestos being the biggest single one. When combined, asbestos and smoking multiply the incidence of lung cancer several times over: while about 10% of the male population who smoke die of lung cancer, the figure for insulation workers with high asbestos exposure who also smoke is about 25%. About half of those who have been highly exposed and fallen ill with asbestosis have died of lung cancer. The morbidity from lung cancer starts to increase 10 years from the start of exposure to asbestos and is at its highest after 40 years (Meurman et al. 1994; Karjalainen 1994; Oksa 1998; Henderson et al. 1998; Henderson et al. 2004).

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Mesothelioma The incidence of mesothelioma gives a good picture of the course of the entire epidemic of asbestos-related diseases. In the United States, the incidence of mesothelioma is thought to have peaked in 1997. In the United Kingdom, the incidence of mesothelioma is expected to increase until 2015. In Finland, the annual incidence of mesothelioma has been about 70 cases since the mid-1990s. Besides asbestos fibers and an erionite fiber in Turkey, it has not been proven that there are other causes of mesothelioma in human beings. The number of mesotheliomas in the population and worker groups reflects how common the exposure to asbestos has been. Exposure at work does not need to be strong or long lasting. Crocidolite is the most potent carcinogen of all types of asbestos, but anthophyllite has also been proven to cause mesothelioma. Smoking does not increase the risk. Mesothelioma appears on average 30-50 years and at the shortest 15–20 years from the start of exposure. The younger a person is when asbestos accumulates in the lungs, the more dangerous it is because the fibers have more time to cause diseases (Tuomi 1991; Karjalainen et al. 1994; Takahashi et al. 1999; Tossavainen 2004; Hodgson et al. 2005).

Asbestosis Asbestosis is a pneumoconiosis disease in which the asbestos dust accumulated in the lungs causes fibrosis in the lung tissue (diffuse interstitial fibrosis). Asbestosis is often associated with severe or moderate occupational exposure that has lasted 10-20 years, but short, unusually severe exposure can cause asbestosis in a sufficiently long follow-up. Diffuse interstitial fibrosis starts in the lower lobes, and as it progresses it contracts the lungs. When asbestosis has advanced to a sufficient extent, a typical symptom is emphysema. Asbestosis causes restrictive lung function and a reduction in the overall diffusion capacity and in the specific diffusion capacity. Because of the long latency period (20-40 years) cases of asbestosis are still being found in Finland even though severe exposure had become rare by the 1980s (Oksa et al. 1994; Huuskonen et al. 2001; Nordman el al 2006).

Pleural Diseases Exposure to asbestos can cause parietal pleural fibrosis (pleural plaques), liquid pleural inflammation (pleuritis), diffuse visceral pleural fibrosis and other adhesions and rounded atelectasis (pseudotumor), which consists of a fibrotic pleura and the atelectatic pulmonary fiber under it, which become voluted and form a parenchymal nodus. Bilateral pleural plaques which do not cause symptoms or interfere with the functioning of the lungs if there are not many of them or if they are not related to incipient asbestosis are a reliable sign of exposure to asbestos. Plaques begin to be found 15 years after the exposure starts. In many instances, plaques are found first on one side and become bilateral and calcify during the follow-up. On the other hand, plaques are not found in all asbestos spray-coaters who have been severely exposed. Diffuse visceral pleural fibrosis may cause a reduction in the vital capacity and diffusion capacity in functioning tests. Liquid pleural inflammations may appear during the first 10 years of exposure (Tiitola et al. 2002 (b); Nordman et al. 2006).

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THE ASBESTOS EPIDEMIC - IS IT OVER? The health consequences of the worldwide use of asbestos are starting to be increasingly visible in countries that have sufficiently efficient systems for diagnosis, notification, registration and compensation. The growth rates have created concern about possible overdiagnosis, while concern has also been expressed by trade unions worried about both underdiagnosis and under-reporting. It is difficult to believe that so many research groups in so many countries would exaggerate the problem similarly. From a purely methodological perspective, we also know that the bias is more likely to be an underestimation instead of overestimation of the risk. There are several reasons for this; first, even in the best registered countries such as the Nordic ones under-reporting is substantial due to insufficient coverage of exposure assessment, the high probability that occupational etiology is ignored by the clinicians who do the major part of cancer diagnosis and high pressure from the industry's and insurances' side to deny asbestos-related morbidity. On the other hand, there is very little pressure for over-diagnosis or overestimation of the risk (Rantanen & Henderson 1998). The basic principle of the Finnish Institute of Occupational Health Asbestos Program was simultaneous use of primary, secondary and tertiary preventive strategies. Measures were directed at the working environment, groups of employees or individuals. The Program received unanimous support from society. It will take decades to see how well the primary goal of the Program, a reduction in asbestos-related morbidity, has been reached. Besides the concrete and practical measures, current attitudes in society were of crucial importance in minimizing exposure. The most important measures after the Program include those to ensure the safety of dismantling work and the introduction of new safe substitute materials. The follow-up of exposed and diseased individuals' health has been organized systematically. There has been some pessimism about action on identifying asbestos-related cancers just because the scope for making any effective health interventions is minimal except in the case of smoking control. There is, however, some good news in this area that encourages action. From the ethical point of view it is also the imperative for the medical profession to contribute both to prevention, but also to the detection, early-as- possible diagnosis, treatment and compensation of asbestos-related diseases (Rantanen 1988; Huuskonen et al. 1995; Consensus report 1997; Consensus report 2000; Huuskonen & Rantanen 2006).

Early Lung Cancer Diagnostics It has not been proven that X-ray screening of the lungs reduces lung cancer mortality. About 10% of lung cancer patients are alive 5 years from detection of the disease, and the situation has remained the same during the past decade. One reason for the poor prognosis is that only 20% of lung cancers are found early enough for surgery. In cases where a lung cancer tumor can be surgically removed in its entirety, more than half of the patients live longer than 5 years. The Best Treatment recommendation in 2001 states that there is no clear evidence of the cost effects of screening lung cancer (Finnish Respiratory Society and Finnish Oncology Association 2001). There is now evidence that low dose spiral computer tomography is better than X-ray screening of the lungs for finding incipient lung cancers, which can be removed by radical

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surgery. An ELCAOP study (www.ielcap.org) screened lung cancer every year from 19932005 using spiral computer tomography. The subjects of the study were 31,567 smokers or people who had been exposed to a cancer hazard at their work, but there was no control group. The follow-up found 484 people with lung cancer, 412 of whom (86%) were in stage 1. The prognosis was that 80% of all the patients in the study and 86% of those whose cancer was found early would live for ten years. The best prognosis (92%) was for those whose lung cancer was found at stage 1 and on whom an operation was performed within a month of finding the cancer. There were 8 people in the study in whom cancer was found at stage 1 but who were not given treatment. All of them died within 5 years of lung cancer being diagnosed (The International ELCAP Investigators 2006). The National Lung Cancer Trial (http://nci.hih.gov/nist) is examining whether lung cancer mortality can be reduced through spiral computer tomography screening. Asbestos workers who have smoked are a lung cancer risk group for whom early lung-cancer diagnostics can be considered well founded. Because radiological tests do not reveal changes in cancer that start in the hilus region of the bronchus, sputum cytology has been recommended as a supplementary method for early diagnostics. Early lung cancer biomarkers are being studied widely these days. At the moment, tests that can be applied to diagnostics do not exist (except for a cytological test), but in the future it might be possible to determine from sputum or blood tests whether a patient has lung cancer biomarkers. (Consensus Report 2000; Teppo et al. 2001;.Tiitola et al. 2002(a); Salomaa et al. 2005; Jett 2005; Nymark et al. 2006; The International ELCAP Investigators 2006; Bach et al. 2007; Vierikko et al. 2007).

Mesothelioma Panel A national mesothelioma panel was set up at the Institute of Occupational Health in Finland in 1989. It can be consulted in cases where there are difficulties in terms of differential diagnosis. The cases that have come for consultation are practical cases sent by pathologists or specialists in forensic medicine or cases sent in order to give systematic confirmation of diagnoses for scientific research. There are some 70 cases of mesothelioma every year, 20%-30% of which are confirmed by the panel. Generally speaking, diagnostics are managed well in Finland these days, and only the most difficult cases are sent to the panel for confirmation. In Finland there is no legal obligation to send all mesothelioma cases to the panel for final diagnosis, although such an obligation exists in certain other European countries (Huuskonen et al. 1995; Huuskonen et al. 2006; Nordman et al. 2006).

Expert Groups in Pneumoconiosis Diseases All the university hospitals in Finland have expert groups in pneumoconiosis diseases that can be consulted in problematical cases. The group in Helsinki operates at the Institute of Occupational Health, meeting 10-12 times a year and handling 70-100 cases annually. Most the cases have been sent by non-life insurance companies or appeal bodies in the insurance system. The most usual areas for questions have concerned the differential diagnostics of asbestosis (50%), work-related causes of lung cancer (25%), work-related causes of visceral pleura disease and the extent to which an asbestos-related disease is the cause of death (7%).

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Other infrequent areas for questions have been cases concerning silicosis and cancers other than those caused by asbestos. In other regional expert groups, the areas for questions have been similar to those in Helsinki, but most of the cases are referred by hospitals (Nordman et al. 2006). Every year, the asbestos fiber contents in the lung tissue are examined in about 300 samples (320 in 2005) for occupational disease diagnostics purposes. There are big regional differences in demand, which shows that an asbestosis test has not yet established itself as part of the exposure surveys in those cases where a patient is operated on because the suspicion of an occupational disease. However, in forensic medicine studies of cause of death carried out because there is a suspicion of an occupational disease, tests for asbestos in the lung tissue are generally requested, and regional differences are negligible (Huuskonen et al. 2006).

Registered Asbestos-Related Diseases In Finland, more than 10,000 asbestos-related diseases were recorded in the register for work-related diseases in 1964-2002, 80% of them being pleural diseases. A total of 1,088 asbestos-related lung cancers and 410 mesotheliomas have been registered during this time, 329 being pleural cancers and 18 cancers of the peritoneum (Table 2) (Huuskonen & Rantanen 2006). Table 2. Asbestos-related diseases recorded in the register for work-related diseases Diagnosis

1964–86

1987–91

1992–96

1997–01

2002

2005

Lung cancer Mesothelioma Asbestosis Pleural diseases

14 17 488 not known not known

130 78 400 1,521

468 138 846 3,841

391 146 613 1,756

84 46 88 346

92 42 80 510

8

39

89

24

83

519

2,137

5,332

2,995

588

807

Others and not defined more precisely Total

Today, more than 500 asbestos-related diseases and almost 200 work-related deaths, 100150 of which have been asbestos-related cancers, are registered in Finland every year. The average age of those who have developed asbestos-related cancer was 64 in 2001. The peak for morbidity from asbestos-related cancer is expected to be reached in 2005-2015. After this, the number is estimated to decline, but because of the long latency period, we must expect that new asbestos-related cancer diseases will continue to diagnosed for decades to come (Huuskonen et al. 2006; Nordman et al. 2006). In 2005, a total of 807 cases of diseases caused by asbestos dust were recorded in Finland, accounting for 12% of all occupational diseases and suspicions of occupational disease. The most common disease caused by asbestos was pleural adhesions and thickenings,

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which accounted for 510 (63%) of all asbestos-related diseases. The growth in this figure compared with 2002 is greater than for other asbestos-related diseases. Asbestosis was registered 84 times. The number of asbestosis cases has shown a slight decline in the 2000s. Cancers suspected of being caused by asbestos were recorded in 141 instances, cancers of the respiratory organs in 92 and mesotheliomas in 42. Cancers other than those suspected of being caused by asbestos were recorded in 7 instances. There are no major changes in the number of asbestos-related cancers compared with earlier years (Laakkonen et al. 2007).

INTERNATIONAL COLLABORATION The health consequences of the world-wide use of asbestos are starting to be increasingly visible in countries which have sufficiently efficient systems for diagnosis, notification, registration and compensation. The basic principle of the Asbestos Program was simultaneous use of primary, secondary and tertiary preventive strategies. Measures were directed at the working environment, groups of employees or individuals (Rantanen, 1988; Huuskonen & Rantanen 2006). In 1997, an international group of experts convened to discuss and formulate criteria for the diagnosis of asbestos-related diseases and their attribution to asbestos. The Helsinki Criteria have met widespread acceptance and thereby improved compensation practices in Finland and several other countries. (Consensus report 1997). In 1998 the WHO published the environmental health criteria 203 “Chrysotile Asbestos” and used information from the Helsinki criteria during the preparation of this document (Henderson el al. 1997). After Helsinki a multidisciplinary review of the relationship asbestos exposure and lung cancer, with emphasis on studies published 1997-2004 has been published recently (Henderson et al. 2004). Another international expert meeting on new advances in the radiology and screening of asbestos-related diseases was organised in 2000 [Consensus report 2000]. The international expert group recently published the International Classification of HRCT for Occupational and Environmental Respiratory Diseases (Kusaka et al, 2005). In 2001, the World Trade Organization ruled on the ongoing dispute concerning the banning of asbestos, finding that a national decision to prohibit the substance was justified in the interests of preventing serious health problems. The Finnish Institute of Occupational health contributed to these processes by providing extensive research evidence, reports and policy advice.

CONCLUSION An estimated 50,000 to 60,000 people who have been exposed to asbestos in their work are alive in Finland today. Only some of the asbestos-related diseases are found and registered as occupational diseases because of the time elapsed before falling ill. High-quality and careful monitoring of people who have been under considerable exposure to asbestos at work and of asbestos patients is endeavoring to improve the early diagnostics of occupational diseases and the predictability of illnesses and at the same time to pay attention to the sociomedical benefits of patients with occupational diseases (ILO: general agreements no.

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139 1974 and no. 162 1986). The occupational health care service must guide those who are finishing their work, including those who will be retiring or become unemployed, to apply for check-ups at a place considered suitable (Occupational Health Care Act 1383/2001 and Government Decree on medical examinations in work that presents a special risk of illness). If an asbestos worker is discovered to have a finding indicative of an asbestos-related disease or there is a suspicion of it, clinical tests should be carried at a lung hospital or at the outpatient department of the occupational medicine services at university hospitals. Asbestos workers who are also smokers are a risk group for which early lung-cancer diagnostics and treatment can be considered well-founded (Vierikko et al. 2007).

BIBLIOGRAPHY Asbestos Committee, Ministry of Labor. 1990. Final Report 66/1989, Helsinki, 76 p + appendixes (in Finnish) Bach, P.B., Jett, J.R., Pastorino, U.,. Tockman, M.S., Swensen, S.J., and Begg, C.B. Computed Tomography. Screening and Lung Cancer. JAMA, 2007;229:953-61. Consensus report. 1997. Asbestos, asbestosis, and cancer: the Helsinki criteria for diagnosis and attribution. Scand J Work Environ Health, 23(4):311-316. Consensus report. 2000. International expert meeting on new advances in the radiology and screening of asbestos-related diseases. Scand J Work Environ Health, 26(5):449-454. Henderson, D.W., de Klerk, N.H., Hammar, S.P., Hillerdal, G., Huuskonen, M.S., Leigh, J., Pott, F., Roggli, V.L., Shilkin, K.B., and Tossavainen, A. 1997. Asbestos and lung cancer: is it attributable to asbestosis or to asbestos fiber burden? In: Corrin B, editor. Pathology of lung tumors. New York, Edinburgh, London, Madrid, Melbourne, San Fransisco, Tokyo, Churchill Livingstone, 83-118. Henderson, D.W., Rodelsperger, K., Woitowitz, H.J., and Leigh, J. 2004. After Helsinki: a multidisciplinary review of the relationship between asbestos exposure and lung cancer, with emphasis on studies published during 1997-2004. Pathology, 36(6), 517-550. Hodgson, J.T., McElvenny, D.M., Darnton, A.J., Price, M.J., and Peto, J. The expected burden of mesothelioma mortality in Great Britain from 2002–2050. British Journal of Cancer, 2005;92:587–92. Huuskonen, O., Kivisaari, L., Zitting, A., Taskinen, K., Tossavainen, A., and Vehmas, T. High-resolution computed tomography classification of lung fibrosis for patients with asbestos-related disease. Scand J Work Environ Health, 2001;27(2):106–12. Huuskonen, M.S. 2005. Asbestos-related diseases. In: Evidence-Based Medicine Guidelines. Kunnamo I, editor in chief. Helsinki, The Finnish Medical Sociciety DUODECIM Medical Publications Ltd and John Wiley & Sons, Ltd. 226-228. Huuskonen, M.S., Koskinen, K., Tossavainen, A. ym. Finnish Institute of Occupational Health Asbestos Program 1987-92. Am J Ind Med. 1995;28:123-42. Huuskonen, M.S., Oksa, P., Vehmas, T., Anttila, S., and Tossavainen, A. Asbestisairaudet Suomessa. Suomen Lääkäril 2006;39: 3961- 66. Huuskonen, M.S. and Rantanen, J. Finnish Institute of Occupational Health (FIOH): Prevention and Detection of Asbestos-Related Diseases 1987–2005. Am J Ind Med. 2006;49 (3):215–20.

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The International Early Lung Cancer Action Program Investigators. Survival of Patients with Stage I Lung Cancer Detected on CT Screening. The NEW ENGLAND

JOURNAL of MEDICINE, 2006;355:1763-71. Jett, J.R. Update on CT Screening and nodule evaluation. Pulmonary Perspectives, 2005;22(3):1–3. Karjalainen, A. Occupational asbestos exposure, pulmonary fiber burden and lung cancer in the Finnish population. Helsinki: Helsingin yliopisto, Työterveyslaitos 1994. Karjalainen, A., Meurman, L., and Pukkala, E. Four cases of mesothelioma in miners. Occup Environ Med. 1994;51:212–5. Koskinen, K., Zitting, A., Tossavainen, A., Rinne, J.P., Roto, P., Kivekäs, J., Reijula, K., and Huuskonen, M.S. 1998. Radiographic abnormalities among Finnish construction, shipyard and asbestos industry workers. Scand J Work Environ Health, 24(2):109-117. Kusaka, Y., Hering, K.G., Parker, J.E., toim. International Classification of HRCT for Occupational and Environmental Respiratory Diseases. Tokyo: Springer-Verlag 2005. Laakkonen, R., Palo, L., Saalo, A., Jolanki, R., Mäkinen, I., and Kauppinen, T. Ammattitaudit ja ammattitautiepäilyt 2005. Työterveyslaitos, Helsinki 2007. Lin, R.T., Takahashi, K., Karjalainen, A., Hoshuyama, T., Wilson, D., Kameda, T., Chan C.C., Wen, C.P., Furuya, S., Higashi, T., Chien, L.C., and Ohtaki, M. Ecological association between asbestos-related diseases and historical asbestos consumption: an international analysis. Lancet, 2007;369:844 -9. Meurman, L., Pukkala, E. and Hakama, M.1994. Incidence of cancer among anthofyllite asbestos miners in Finland. Occup. Environ. Med. 51:42-45. Nordman, H., Oksa, P., Karjalainen, A., and Koskinen, H. Asbestisairauksien diagnostiikka ja seuranta. Työ ja ihminen Tutkimusraportti 28. Helsinki, Työterveyslaitos 2006. Nymark, P., Wikman, H., Ruosaari, S., Hollmen, J., Vanhala, E., Karjalainen, A., Anttila, S., and Knuutila, S. Identification of Spesific Gene Copy Number Changes in AsbestosRelated Lung Cancer, Cancer Res. 2006;66:5737-42. Oksa, P. Asbestosis, its detection and predictors of progression and cancer. People and Work Research Reports 17. Helsinki: Finnish Institute of Occupational Health 1998. Oksa, P., Suoranta, H., Koskinen, H., Zitting, A., and Nordman, H. High resolution computed tomography in the early detection of asbestosis. Occup Environ Health, 1994;65:299– 304. Rantanen, J. 1988. Preventive strategies for occupational cancer in Finland. In: Primary Prevention of Cancer edited by W. J. Eylenbosch, N. Can Larebeke and A. M. Depoerter. Raven Press, Ltd. New York, 313-35. Rantanen, J. and Henderson, D.W. 1998. The global asbestos epidemic - need for diagnostic criteria for outcomes. In: Advances in the prevention of Occupational Respiratory Diseases. Proceedings of the 9th International Conference on Occupational Respiratory Diseases, Kyoto, Japan 13-16 October, 1997. Chiyotani K, Hosoda Y, Aizawa Y, editors. Amsterdam, Lausanne, New York, Oxford, Shannon, Singapore, Tokyo. Elsevier, Excerpta Medica International Congress Series 1153. 259-265. Salomaa, E.R., Sallinen, S., Hiekkanen, H., and Liippo, K. Delays in the Diagnosis and Treatment of Lung Cancer. Chest, 2005;128:4:2282-8. Suomen keuhkolääkäriyhdistys ry ja Suomen onkologiayhdistys ry. Keuhkosyövän hoitosuositus. Käypä hoito -suositus. Duodecim 2001;117:894–908.

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Takahashi K, Huuskonen MS, Tossavainen A, Higashi T, Okubo T, Rantanen J. Ecological relationship between mesothelioma incidence/mortality and asbestos consumption in ten western countries and Japan. Journal of Occupational Health 1999;41:8–11. Teppo, L., Anttila, A., Auvinen, A., and Hakama, M. Syöpätautien seulonta edelleen tarpeellista ja tehokasta. Duodecim. 2000;116:893–901. Terveystarkastukset työterveyshuollossa. Asbesti ja muut silikaatit, mm. keraamiset kuidut. Työterveyslaitos ja Sosiaali- ja terveysministeriö. Helsinki, Vammalan kirjapaino 2005. s. 350-9. Tiitola, M., Kivisaari, L., Huuskonen, M.S., Mattson, K., Koskinen, H., Lehtola, H., Zitting, A., and Vehmas, T. 2002 (a). Computed tomography screening for lung cancer in asbestos-exposed workers. Lung Cancer, 35(1):17-22.. Tiitola, M., Kivisaari, L., Zitting, A., Kaleva, S., Tossavainen, A., Huuskonen, M.S., and Vehmas, T. (b). Computed tomography of asbestos-related pleural abnormalities. Int Arch Occup Environ Health, 2002;75:224-228. Tossavainen, A. Global use of asbestos and the incidence of mesothelioma. Int J Occup Environ Health, 2004;10:22–5. Tuomi, T. Asbestos exposure of Finnish mesothelioma patients. Electron microscopic analysis of mineral fibres in the lung tissue and bronchoalveolar lavage fluid.Helsinki: Kuopion yliopisto, Työterveyslaitos 1991. Uibu, T., Oksa, P., Auvinen, A., Honkanen, E., Saha, H., Uitti, J., and Roto, P. Asbestos exposure as a risk factor for retroperitoneal fibrosis. Lancet, 2004;363:1422–6. Vierikko, T., Järvenpää, R., Autti, T., Oksa, P., Huuskonen, M.S., Kaleva, S., Laurikka, J., Kajander S, Paakkola, K., Saarelainen, S., Salomaa, E.R., Tukiainen, P., Uitti, J., Vanhanen, M., and Vehmas, T. Chest CT screening of workers with asbestos exposure:lung tumors, benign nodules and incidental findings. European Respiratory Journal, 2007:29:78-84. World Health Organization. 1998. "Chrysotile Asbestos". Geneva; Environmental Health Criteria 203. International Programme on Chemical Safety, 197 p. World Trade Organization. 12 March 2001. European Communities - Measures Affecting Asbestos and Asbestos Containing Products. WT/DS135/AB/R. Zitting, A., Karjalainen, A., Impivaara, O., Tossavainen, A., Kuusela, T., Mäki, J., and Huuskonen, M.S. Radiographic small lung opacities and pleural abnormalities as a consequence of asbestos exposure in an adult population. Scand J Work Environ Health, 1995;21:470-77.

In: Asbestos: Risks, Environment and Impact Editor: Antonio Soto and Gael Salazar

ISBN 978-1-60692-053-4 © 2009 Nova Science Publishers, Inc.

Chapter 4

AUTOANTIBODY PROFILES OF AN ASBESTOSEXPOSED POPULATION Jean C. Pfau∗1, David J. Blake2 and Marvin J. Fritzler3 1

Department of Biomedical and Pharmaceutical Sciences, University of Montana, Missoula MT 2 Johns Hopkins University, Baltimore MD 3 Department of Medicine, University of Calgary, Calgary, Alberta

ABSTRACT Asbestos exposure is associated with autoimmune responses including increased serum immunoglobulins, positive autoantibody tests and immune complex deposition. Occupational and environmental asbestos exposures continue to occur world-wide, making this a current human health issue. The premise that asbestos exposure exacerbates autoimmunity is supported by recent studies from an asbestos exposed population in Libby, Montana, USA. Residents of Libby have experienced significant exposures to amphibole asbestos due to the mining of asbestos-contaminated vermiculite near the community over several decades. This community exhibits higher frequencies of positive anti-nuclear antibody (ANA) tests compared to an unexposed control population and what would be expected based on epidemiological data. Systemic autoimmune diseases are characterized by autoantibody profiles that correspond to specific sets of intracellular components. These profiles can be used to help predict disease, establish a diagnosis as well as assess clinical progression. At this time, it is not known whether a discrete clinical or sub-clinical autoimmune entity is associated with amphibole asbestos exposure. However, asbestos exposure leads to clinical manifestations similar to systemic lupus erythematosus (SLE) in asbestos exposed mice. Therefore, we hypothesized that features of certain autoimmune diseases may be associated with asbestos exposure in humans. The purpose of this study was to determine whether a serological signature exists in this asbestos-exposed population, and to compare this profile with other known systemic autoimmune disease autoantibody profiles. Our results indicate that autoantibodies from individuals exposed to amphibole asbestos primarily recognize chromatin, histone and Ro52, similar to patients with systemic lupus erythematosus (SLE). Anti-dsDNA and anti-Ro52 antibodies are also generated in a murine model of asbestos induced autoimmunity, which suggests that ∗

Contact Information: Jean C. Pfau, Ph.D. Skaggs 283, Department of Biomedical and Pharmaceutical Sciences, University of Montana, Missoula MT 59812, (406) 243-4529. [email protected]

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Jean C. Pfau, David J. Blake and Marvin J. Fritzler asbestos may drive similar immune phenomena in both humans and mice. In addition, we show that an unexpected number of subjects expressed the Scl-topoisomerase I (topo I: also referred to as Scl-70) antibody, typically seen in scleroderma. The subjects with antitopo I tended to have higher levels of asbestos exposure and more severe lung disease compared to those without topo I, whereas there was no association with exposure or lung disease among people with anti-RNP (antibodies to ribonucleoprotein, common in SLE). Finally, we show that some Libby serum samples contained antibodies that bound fibroblasts, a phenomenon that is also seen in scleroderma. By comparison, neither rheumatoid factor (RF) nor anti-cyclic citrullinated peptide (CCP), markers for rheumatoid arthritis (RA), was substantially elevated in the Libby sample set. This information could be extremely valuable for improved screening of exposed populations. In addition, certain autoantibody profiles may serve as diagnostic or prognostic markers relative to asbestos exposure.

INTRODUCTION Occupational and environmental exposures to biologically active forms of silicon oxides such as crystalline silica and amphibole fibers (asbestos and asbestiform minerals) continue to occur and pose serious health risks because of their abundance in the earth’s crust and their use in many materials in developed and developing nations. Studies regarding health effects from these exposures are critical at many levels, from epidemiological data to exploring cellular and molecular mechanisms, so that the information can be used in setting regulatory exposure standards, minimizing exposure risks, screening exposed populations, anticipating healthcare costs in industry, and exploring preventive and therapeutic modalities. While acknowledging the impact and severity of the cancers and fibrotic disorders associated with silica and asbestos, this chapter focuses on the complex, enigmatic, and often devastating systemic autoimmune disorders. As early as 1914, an association between occupational exposures of inhaled crystalline silica1 and autoimmunity was suggested when Bramwell reported increased frequencies of diffuse systemic sclerosis (SSc) in stone masons [1]. However, documented reports of the increased prevalence of SLE in workers occupationally exposed to silica first appeared in the 1990’s [2, 3]. Case reports and population-based studies have continued to enrich and strengthen the data supporting the association between crystalline silica and SLE. The historical literature linking silica exposure with autoimmune diseases is summarized in recent reviews [4-6]. While the background literature regarding silica is fairly extensive, studies regarding asbestos exposures and autoimmunity have been more limited. Studies of the humoral responses following asbestos exposure appear in the literature beginning around 1965 when the presence of rheumatoid factor (RF) and antinuclear antibodies (ANA) were reported in asbestos workers [7, 8]. Interestingly, despite clear associations between silica exposure and systemic autoimmune diseases (SAID) such as systemic lupus erythematosus (SLE), SSc and rheumatoid arthritis (RA), asbestos exposure has not yet been linked with any particular autoimmune or connective tissue disorder. There are several possibilities for this knowledge gap, including a lack of statistical power due to relatively small or diffuse exposure cohorts, difficulties in exposure assessment, mild or sub-clinical entities that remain

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undetected or masked by other pathology, or alternatively, an absence of any associated autoimmune pathology despite the presence of the autoantibodies. In addition, because of the latency and complexity of diseases such as SLE [9], establishing an association between exposure and actual clinical disease is difficult. Asbestos exposure in humans is associated with the development of malignant and nonmalignant diseases including lung cancer, pulmonary fibrosis (asbestosis) as well as an increased frequency of autoantibody production. Although the commercial use of asbestos has decreased in the United States since the 1970s, asbestos exposure remains a major environmental health concern. More than 30 million tons of asbestos have been mined in the US alone [10] and more than 27 million people were exposed to asbestos between 1940 and 1979 [11]. As late as 1989, the use of asbestos products in the U.S. exceeded 55,000 tons per year. Developed countries have restricted the use of asbestos because of its harmful public health effects. However, asbestos is now a major concern in developing countries due to increased marketing and commercial use [12, 13]. Additionally, in 2002 the vermiculite mine and community of Libby, MT were designated by the Environmental Protection Agency (EPA) as Superfund sites due to the amphibole asbestos contamination of vermiculite, which led to exposure throughout the mine site and surrounding area. Significant health issues have become evident in Libby and many other sites where the vermiculite was shipped for processing [14]. Hence, asbestos exposure remains an immediate environmental health concern not only in Libby but throughout the United States and in developing countries.

AMPHIBOLE ASBESTOS CONTAMINATION IN LIBBY, MT From 1924 to 1990, Zonolite mountain near Libby, Montana was the site of a vermiculite mining operation. In 1963, W.R. Grace bought the Zonolite mining operations, which subsequently provided approximately 80% of the world’s supply of vermiculite [15]. Unfortunately the vermiculite extracted from the Libby mine was contaminated with naturally-occurring asbestos fibers. The mine closed in 1990 after almost 60 years of operation. The first emergency response team from the EPA was sent to Libby in November 1999. The EPA began collecting air, soil and insulation samples. In October of 2002 the vermiculite mine and surrounding community of Libby were designated as EPA Superfund sites due to the asbestos contamination of vermiculite. The various mining, transportation, and processing activities as well as the personal and commercial use of vermiculite in the community led to widespread environmental exposures in the Libby area, which resulted in significant asbestos-related diseases even in residents with short exposure times of less than a year [16]. The extensive asbestos exposure has led to considerable health problems in the community including reduced pulmonary function, enhanced autoimmune responses and increased mortality from lung cancer, malignant mesothelioma and fibrosis [17, 18]. The asbestos-related disease (ARD) observed in patients from Libby include interstitial fibrosis, pleural plaques, and mesothelioma, with a subset of patients developing rapid progression of disease. 1

A distinction is made between crystalline silica and either amorphous silica or silicone, as the immunological effects of the latter two have not been established and remain controversial.

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The asbestos contamination of Libby vermiculite has been characterized as both regulated asbestos fibers (e.g., tremolite and other amphibole forms) and unregulated fibers (e.g., winchite and richterite) [19]. The mixture of amphibole fibers in the Libby amphibole sample differs in terms of fiber length and metallic cations adhered to the fiber surface. Moreover, these amphibole fibers are thought to be more pathogenic than serpentine asbestos fibers because the longer amphibole fibers cannot be readily cleared from the lung [20]. The premise that asbestos exposure exacerbates autoimmune responses in humans is supported by two recent studies of the asbestos exposed population in Libby. According to data from these studies, exposure to Libby amphibole asbestos increases the risk for SAID by more than 50% [21]. Moreover, residents of Libby have higher frequencies of positive ANA tests compared to those from an unexposed control population [22]. Anecdotal concerns have been raised by the residents regarding SLE or SLE-like connective tissue disease, and at a recent conference in Denver, Colorado addressing the epidemiology of asbestos-related diseases related to the Libby vermiculite2, physicians present agreed that autoimmune/connective tissue diseases needed to be included in a thorough evaluation of asbestos-related pathologies. This is particularly important due to reports that those individuals who manifest positive ANA tests and/or rheumatic symptoms may experience more severe pulmonary complications ([22], and personal communication, B. Black, M.D., Libby Center for Asbestos Related Disease). An analysis of the specific targets of autoantibodies (AA) from residents in Libby, MT can provide insight and help define the clinical diagnoses in this exposed population. The spectrum of AA related to amphibole asbestos exposure may have implications in elucidating the immunologic response to asbestos and provide a biomarker for clinical screening. The objective of this study was to characterize the specific AA found in serum samples from the Libby cohort and to determine if the presence of AA correlated with a particular clinical pattern.

MATERIALS AND METHODS Human samples. All samples were acquired according to approved University of Montana Institutional Review Board protocols intending to protect the well-being and confidentiality of all subjects. The recruitment and collection of samples described in this chapter have been reported previously [21, 22], and include subjects from both Libby and Missoula MT, with Missoula as the unexposed reference population. Wherever data is compared between Libby and Missoula, sample sets were matched by age and sex. Missoula is similar to Libby in being located in a mountain valley, subject to similar climatic conditions, including winter inversions, dry summers, and exposure to smoke from forest fires and wood burning. Because prevailing winds in Missoula are from the west and Libby is well to the north, there is no detectable transfer of asbestos from the Libby airshed to the Missoula airshed. Therefore, on a relative basis, while one cannot exclude the possibility 2

July 23-24, 2007. Sponsored by the Environmental Protection Agency, with participation from Centers for Disease Control, Agency for Toxic Substances and Disease Registry, NIOSH, several university researchers, the Montana Department of Environmental Quality, and the community of Libby MT. For more information: www.epa.gov/region8/toxics_pesticides/asbestos/

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that there could have been some minimal asbestos exposure by the Missoula population, it is an acceptable reference population for the Libby subjects who were definitely exposed to asbestos. Subjects in both communities are fairly homogeneous in terms of ethnicity; the majority of residents are of northern European descent, with 94.7% Caucasian in Lincoln County and 93.6% Caucasian in Missoula County according to Montana census data. The presence of physician-diagnosed autoimmune disease among Libby subjects was not assessed in the screening. None of the Missoula subjects recruited had diagnosed SAID, although this was not a criterion for exclusion. Subjects were excluded when data indicated that they were currently using medications that are known to be associated with positive ANA tests and drug-induced lupus (DIL), specifically chlorpromazine, hydralazine, isoniazid, methyldopa, minocycline, procainamide, quinidine. However, many other drugs may be associated with DIL but were not among the exclusion criteria used for this study.

Sample and Data Collection The blood samples were collected, and serum samples were obtained and frozen by standardized clinical methods to prevent differences due to handling. The samples were blinded with only sex and age noted, and stored at -80°C until assayed. Coded information regarding disease status and exposure was obtained from a questionnaire and screening data from the Agency for Toxic Substances and Disease Registry (ATSDR), which performed a screening in Libby during 2000-2001 [23]. ARD and asbestos exposure rankings. The lung diseases reported in Libby have been previously described [24, 25], and include primarily pleural and some interstitial abnormalities. In our Libby sample set, Asbestos-Related Disease (ARD) status, based on data recorded in the database primarily as a result of the ATSDR screenings, was ranked on a scale of 0 – 3 as previously described [22]. The rankings combine pleural and interstitial abnormalities, and were intentionally simplified, based on radiographic evidence of single versus extensive plaques or interstitial abnormalities, as well as spirometric evidence of functional deficits. For example, a subject with single-lobe pleural plaques and no functional deficit would be scored at 1, subjects with more extensive plaques or fibrosis but without functional deficits received a score of 2, whereas a subject with bilateral plaques and/or fibrosis with effects on spirometry was scored at 3. To further stratify the sample sets by disease types would have made the subsets too small for statistical analysis. Exposure status was ranked on a scale of 0 – 4, where zero means no known exposure, 1 = 1-2 routes of exposure for less than 5 years, 2 = 3 or more routes of exposure for less than 5 years, 3 = 1-2 routes of exposure for ≥ 5 years, and 4 = 3 or more routes of exposure for ≥ 5 years [22].

Detection of Serum Autoantibodies A clinical test for autoantibodies to nuclear antigens (ANA assay) was performed at a screening dilution of the sera. All serum samples were diluted 1:40 in phosphate-buffered saline (PBS) and tested by indirect immunofluorescence (IIF) on a commercially prepared and fixed HEp-2 cell substrate (ImmunoConcepts Inc., Sacramento, CA), according to manufacturer’s instructions. The staining pattern and relative fluorescence intensity were compared with known positive and negative controls using a Zeiss fluorescence microscope

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with 40x objective and recorded as positive (1+ to 4+) or negative (0). The staining pattern was also noted and recorded. The same microscope and settings were used for all samples, and the slides were read by two independent readers.

Addressable Laser Bead Immunosorbent Assay (ALBIA) Analyses of antibodies to eight extractable nuclear antigens (ENAs) commonly seen in SAID (Sm, RNP, Ro52, SS-A60, SS-B, topo I, Jo-1, Ribosomal P, chromatin) were performed using an ALBIA kit (QUANTA Plex™ SLE Profile 8; INOVA Diagnostics) according to the manufacturer’s instructions, on a Luminex multiplex system (MiraiBio, Alameda, CA). The values were compared using Starstation 2.0 software (Applied Cytometry Systems, Sacramento, CA) to negative and graduated positive control reagents provided with the kit, and determined to be low or high positive, or negative.

Quantification of IgM Rheumatoid factor (RF), Anti-Cyclic Citrullinated Peptide (CCP), Topo I, and Histone by ELISA IgM RF, anti-CCP, anti-topo I, and anti-histone antibodies in the serum samples were measured by ELISA according to the manufacturer’s protocol (Quanta Lite™, INOVA Diagnostics). The plates were read on the SpectraMax plate reader (Molecular Devices, Sunnyvale, CA). Optical density (OD) values were compared with known controls provided with the kit and rated as negative or positive (low or high).

Cell-Based ELISA for Anti-Fibroblast Antibodies MRC-5 human lung fibroblast cells (American Type Culture Collection, ATCC) were plated to 96-well plates at 10,000 cells per well in DMEM media containing 10% fetal calf serum and antibiotics, and allowed to adhere and grow for 48 hr at 37oC in a 5% CO2 incubator. The cells were washed with warm PBS and then blocked in PBS containing 5% goat serum with 0.5% Tween-20 for 1 hr at 37oC. Serum samples were applied as the primary antibody at 1:100 dilution and allowed to stain the cells for 1 hr at 37oC with occasional gentle rocking of the plate by hand. After gentle washing with blocking buffer, the secondary antibody (HRP-Goat anti-human IgG) was applied at 1:5000 dilution in blocking buffer, and again allowed to stain for an hour as above. After gentle washing 2x with blocking buffer, HRP-TMB substrate (Sigma) was added to the wells and allowed to develop color. The plates were read on a microtiter plate reader at 450 nm.

Statistical Analysis Serum samples that recognized one of the nine autoantigens as described above were classified as positive and treated as ordinal data. Alternatively, serum samples that were

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unreactive to the autoantigens were classified as negative. Extent of lung disease or exposure was classified as ordinal data as 4 or 5 point levels, respectively. Data was analyzed nonparametrically through two approaches. Data was analyzed using contingency tables using subject scores through the chi-square test. Data was also analyzed using Mann-Whitney analysis using subject scores. Where appropriate, a one-way ANOVA analysis was used to observe trends among multiple groups of data. Statistical significance was established as a two-tailed probability of type I error occurring at less than 5%. Analysis was performed on Prism 4.0 software (GraphPad, San Diego, CA).

RESULTS Frequency of Antinuclear Antibodies Seventy randomly chosen serum samples from Libby, MT were tested by IIF for ANA and were determined to be positive or negative based on known controls. Fifty of the 70 serum samples (70%) were ANA positive, which is consistent with previously published results for this population (16). The intensity and the staining patterns visible on the ANA tests were also recorded (Table I). The staining patterns that were the most prevalent were homogeneous (indicative of antibodies to chromatin components), nucleolar and speckled staining patterns (Figure 1). The most common ANA pattern from Missoula was the speckled pattern, consistent with data from normal populations, in which approximately 30-32% screen positive for ANA at a 1:40 dilution of the serum [26]. In addition, nine Libby samples showed a filamentous cytoplasmic pattern indicative of cytoskeletal staining (indicated as “cytosk” in Table I), and one had a cytoplasmic speckled staining pattern (indicated as “cytosp” in Table I, subject # 70).

Autoantibody Characterization The ALBIA, which detects reactivity to nine autoantigens commonly targeted in SAID, was used to characterize the specific target antigens of the autoantibodies found in the Libby cohort. Sixteen of the 70 total Libby serum samples (23%) had positive ALBIA tests and most were reactive to a single target antigen (Table I). The most common autoantibodies detected in the Libby serum samples were autoantibodies to chromatin (44% of the positive samples) and autoantibodies to Ro52 (19% of the positive samples). Serum samples that had antibodies to chromatin were further analyzed on a histone ELISA. Of the seven samples that were positive against chromatin, five samples tested positive against histones (71%). No serum samples reacted with Jo-1, Sm or Ribo P. Figure 2 shows the distribution of positive tests against the target antigens.

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Percent Positive

16 14 12 10 8 6 4 2

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om

er

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uc le ol a

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Figure 1. Percentages of ANA IIF patterns for Libby samples. A total compilation of the ANA staining patterns at screening dilution 1:40 of 189 Libby serum samples is shown as a percent of the total. Mean age = 57 ± 39.5 years. MND= Multiple Nuclear Dot. ALBIA Positive

8

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SSA/60

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Figure 2. Frequencies of specific autoantibodies in Libby serum samples by ALBIA. The specific targets recognized by AAs from Libby serum samples were characterized with an addressable laser bead immunoassay as described in Materials and Methods. Samples that tested positive for chromatin were further analyzed for reactivity against H2/H3/H4 histones by ELISA.

Topo-I antibodies were also analyzed by ELISA, which detected not only the two samples positive by ALBIA, but also an additional 6 samples. The ELISA data had a better correlation with the ANA positive status of the samples, especially nucleolar and homogeneous staining patterns. The total positive for Scl-70 was 8 of 70 (11%).

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IgM RF and Anti-CCP Frequency RF is an autoantibody directed at the Fc region of IgG and is usually of IgM isotype. RF is associated with a number of inflammatory diseases and is not specific for RA (10). The presence of these autoantibodies may contribute to disease by the formation of immune complexes in tissues. In a separate set of 45 randomly selected Libby samples, the presence of anti-CCP was also measured, due to the sensitivity and specificity of this test for RA [27]. Both antibodies were measured semi-quantitatively by ELISA. Twenty-four of the seventy total Libby serum samples (34%) were positive for RF IgM (Table I), which is consistent with previously published results [22]. None of the samples tested higher than 25 RF units in this assay, suggesting a low titer. Three of 45 samples (6.6%) were positive for anti-CCP, with two in the low positive range and one in the high positive range for this assay (Data not shown).

Correlation of Autoantibodies with Extent of Exposure and ARD To determine whether an association existed between the presence of a positive ALBIA test (Figure 2) and the extent of exposure (ranked by years of exposure as well as number of pathways), the presence or absence of AA to one of the nine autoantigens was classified as ordinal data and designated as positive or negative, respectively. The data were analyzed using a chi-square test and a non-parametric Mann-Whitney U-test. No significant association was observed between the presence of these autoantibodies and the extent of exposure. Subjects with positive ALBIA tests had a slightly higher exposure status, but this difference was not statistically significant (Figure 3a) (P = 0.1312). The presence of individual autoantibody specificities was also generally not correlated with extent of exposure, represented here by samples positive for anti-RNP (Figure 3b). Because pulmonary abnormalities are common in SAID, it was important to determine whether the presence of particular autoantibodies was associated with the severity of asbestos-related lung disease (ARD). The only autoantibody specificity that showed a significant association with ARD score was topo-I as measured by ELISA (Figure 3c). The association of anti-topo-I with extent of exposure and with ARD was only found to be significant when all eight samples were used in the analysis, to include those positive by ELISA, as described above. This raises the possibility that the lack of association for the other autoantibodies could be attributed to the limited size of the groups analyzed.

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4

A

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Figure 3. Asbestos exposure and lung disease scores for subjects with and without antibodies to ENA. Using a scoring system applied to exposure and lung disease data for each of the Libby subjects, the mean scores for subjects with and without these antibodies are shown. There was no significant difference in exposure or disease scores for subjects positive for any ENA Ab (A), nor for anti-RNP alone (B). C. DNA Topo 1 positive subjects had a significantly higher score for both parameters, suggesting that these antibodies are associated with higher exposure and more severe lung disease. (* = p< 0.05 by Mann-Whitney non-parametric U test).

Presence of Antibodies to Fibroblasts Patients with SAID also have been shown to produce autoantibodies against non-nuclear antigens, such as cell surface proteins of fibroblasts. The presence of anti-fibroblast antibodies (AFA) was assessed using a cell-based ELISA, measuring the binding of serum antibodies to MRC-5 human fibroblasts. Figure 4a shows that several of the Libby serum samples contain antibodies that bind fibroblasts, increasing the mean optical density (OD) of the Libby samples to a value significantly higher than the mean OD of the Missoula samples. The majority of the high binding samples appear to occur among those who were positive for anti-topo-I by ELISA (Figure 4b).

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1.400

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Libby Topo I+

Figure 4. Antifibroblast antibodies in Libby serum samples. Serum samples from Missoula and Libby were analyzed by cell-based ELISA for anti-fibroblast antibodies (AFA). A. At least a subset of the Libby samples had quite high binding, making the mean OD significantly higher than for Missoula. B. High AFA binding occurred in the Scl-70+ group. * = p < 0.05 by unpaired t test.

DISCUSSION The ubiquitous nature of silicon oxides as a major elemental component of our planet poses tremendous challenges for identifying and tracking exposures. High exposures primarily occur occupationally during mining and sandblasting as well as the manufacture of pottery, insulation, ceramics, glass, absorbents, dessicants, filler, and abrasive cleaners. In many of these cases, the biologically active component is crystalline silica, as a result of charge interactions with silicon oxide radicals that form during fracturing processes [28]. Additionally, fibrous silicates such as amphibole asbestos fibers remain significant health

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hazards. Asbestos is a general term for hydrated silicate fibers that are strong and heatresistant. Because of its high tensile strength and ability to absorb heat, asbestos has been used extensively as building materials in roofing, siding, fire protection material, heating and electrical wire insulation, appliance components, sheet flooring, ceiling and floor tiles, and drywall. Asbestos fibers are categorized into two separate families based on morphology. Amphibole fibers are straight rod-like fibers that include fibers such as amosite, tremolite, crocidolite and Libby amphibole asbestos (Figure 5). In contrast, serpentine fibers are curved pliable fibers. The serpentine fiber family includes a single fiber type, chrysotile, which accounts for much of the world’s asbestos consumption. Asbestos fibers can remain airborne in contaminated areas and eventually settle into soil, sediment and tree bark [29, 30]. Therefore, the toxic potential of asbestos fibers remains for long periods in contaminated areas once they are introduced into the environment. Although there appear to be some similarities between silica and asbestos in terms of induction of autoantibodies and immune complexes, the pathologies associated with crystalline versus fibrous silicates appear to be somewhat unique, necessitating the separate study of these two silicate families. In particular, the pulmonary manifestations of silicosis versus asbestosis are readily distinguishable and classified separately [31, 32]. Although several studies show increased frequencies and titers of autoantibodies with asbestos exposure, published studies indicate that asbestos is more commonly associated with cancer rather than SAID, possibly due to differential effects on the activation of T cells, though the mechanism of this difference remains unclear [33, 34].

Figure 5. Light micrograph of Libby amphibole mixture. This material, collected from six sites around the Libby vermiculite mine has been labeled as “Libby Six-Mix” by U.S. Geological Survey who kindly provided the sample [19].

This is further complicated by the fact that there are many forms of asbestos that vary in terms of surface charge and adsorbed ions, leading to different biological effects [35, 36].

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Nevertheless, since both mineral silicates cause apoptosis and oxidative stress in various cell types and both are considered to be immune “adjuvants”, continued investigation of their association with SAID is warranted because the similarities and differences between these groups will guide our understanding of environmental induction of autoimmunity. Residents of Libby, MT have higher frequencies and titers of positive ANA tests, which correlated to the extent of exposure and the severity of disease [22]. AA characterized from the Libby cohort were initially described as recognizing more than one ENA [22]. However, the present characterization indicates that, in most cases, AA from the asbestos exposed cohort recognize a single antigen. The sensitivity and specificity of techniques for detecting specific AA are constantly being improved; therefore, the study reported in this chapter used some of the more advanced techniques and diagnostic platforms in order to better enable a more specific assessment of the serological profile. The initial detection of AA has classically used indirect immunofluorescence for ANA on HEp-2 cell substrates. This screening test offers the appropriate level of sensitivity, however there are now more specific confirmatory tests for the detection and semi-quantification of ENA antibodies in human sera [37]. With the exception of antibodies to cytoplasmic antigens, such as Jo-1, ribo-P and Ro52, it is rare to have a positive anti-ENA antibody test in the absence of a positive ANA test. In order to correctly screen a patient for the presence of AA, it has been suggested that a two stage testing is performed that includes an initial ANA screening test followed by a confirmatory anti-ENA test [38]. Cytoplasmic staining, seen in 14% of the Libby samples, may become more clinically relevant as the specific target antigens are identified. The cytoplasmic pattern seen with the Libby samples was primarily filamentous, suggesting antibodies to cytoskeletal components, without evidence of antibodies to mitochondria, Golgi complex, endosomes, GW bodies or lysosomes [39]. All samples that were positive for cytoskeletal staining were also positive for ANA. Interestingly, autoreactivity to cytoskeletal proteins have been noted at high frequencies in RA patients. In a study comparing patients with RA, progressive SSc, or SLE with normal volunteers, the frequencies of fibrous or filamentous cytoplasmic staining were 76%, 30%, 6% and 4%, respectively [40]. The most common ALBIA ANA from the Libby cohort were reactive to chromatin, histones and Ro52. This AA profile is similar to those found in patients with SLE [41]. The homogeneous staining pattern, which was predominant in this population, is usually associated with antibodies to DNA, histone, or the macromolecular DNA/histone complex referred to as nucleosomes or chromatin. Previous data indicated that less than half of the ANA positive Libby subjects with homogeneous staining had positive tests to dsDNA, as measured by Crithidia luciliae testing [22]. The current study indicated that the antibodies targeted chromatin with some reactivity to histones. Antibodies to chromatin may occur earlier in a disease course, with antibodies to dsDNA and histone following due to epitope spreading. Interestingly, anti-histone AA have been reported in SSc patients exhibiting pulmonary fibrosis [42]. Anti-histone antibodies are also found in approximately 40% of patients with SLE, and are associated with symptomatic drug-induced lupus. Antibodies against certain components of histone, such as H2A-H2B dimer, are thought to be characteristic of drug-induced lupus while reactivity to the H1 and H2B subunits of histones is common in idiopathic SLE [43, 44]. Since the assay used to determine anti-histone reactivity was not specific for any one of the histones (except that H1 has been removed), the anti-histone antibodies observed could be against the H2A, H2B, H3 or H4 core subunits of

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the nucleosome. Therefore, further characterization may be needed to determine the specific core histones and their respective epitopes targeted by these antibodies. DIL generally resolves upon removal of the implicated drug, but since asbestos remains in the lung, the induced lupus would not likely resolve. Because patients with DIL often do not fulfill the criteria for idiopathic SLE, presenting with vague symptoms such as arthralgias, myalgia, malaise and fever, this could be an interesting explanation for the difficulty in identifying a discrete clinical autoimmune entity associated with asbestos exposure. In addition to anti-chromatin antibodies, anti-Ro52 was the next most common autoantibody found in the Libby samples. These AAs have also been identified in patients with idiopathic SLE and other connective tissue diseases [45, 46]. A murine model of asbestos-induced autoimmunity was recently established to investigate the mechanisms by which asbestos triggers systemic autoimmunity [47]. Asbestos exposed mice developed positive antinuclear antibody tests and mild glomerulonephritis suggestive of a systemic lupus erythematosus (SLE)-like disease. Interestingly, the murine SLE-like disease was characterized by the production of AAs that recognize dsDNA and Ro52. These data support the premise that amphibole asbestos generates a serological SLE-like disease in both humans and in mice. Moreover, because humans and mice exposed to amphibole asbestos generate similar AA profiles, the alterations of the immune response by amphibole asbestos may be comparable. Therefore, the murine model of asbestos induced autoimmunity is both relevant and useful to study the immunological effects of amphibole asbestos. Further, the presence of antibodies to Ro52 raises the interesting question of its role in autoimmunity that was heightened when it was identified as an E3 ubiquitin ligase [48]. The possibility that autoantigens become antigenic due to proteolytic degradation has been postulated [49, 50]. It is possible that exposure to environmental toxins may cause protein misfolding leading to aberrant ubiquitination of targets by Ro52 (including self-ubiquitination) thus resulting in ineffective proteosomal degradation of target proteins, that may then lead to an autoimmune response. In addition, Ro52 has been shown to accumulate in apoptotic blebs during programmed cell death induced by a variety of oxidant challenges including asbestos [51, 52]. The data suggest that asbestos impairs proteolytic cleavage of Ro52 in murine macrophages, and the protein is then recognized by mouse autoantibodies as well as a commercial monoclonal antibodies raised against amino acids 141-280 of the 52 kD polypeptide by both immunocytochemistry and Western blotting of asbestos-treated cells undergoing apoptosis [51]. Because SAID patients often present with a specific pattern or selection of AA specificities, and also that some AA appear early in the course of the disease, it is possible and very intriguing that these AA might occur in distinct patterns following specific exposures. An association was shown between silica exposure and the production of antiDNA and anti-Sm autoantibodies, both commonly found in lupus patients [53]. Remarkably, the autoantibody profiles in our asbestos-exposed cohort did not overlap reported profiles for silica-exposures except for topo-I. However, due to the wide range of antibodies, the presence of autoantibodies in controls, and variability between individuals, it would take larger number of samples from various exposures and compared to unexposed controls to see if a consistent pattern developed [54]. Silica exposure has been associated with the presence of RF and an increased risk of RA [55, 56]. In addition, there was some evidence of a higher rate of RA in Libby than might be expected [21]. Low titer IgM RF was present in 34% of the Libby subjects, compared to

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approximately 36% in Missoula samples [22]. Anti-CCP antibodies were seen in 6.6% of the Libby samples, where a frequency of 1-2% might be expected in a normal population. In order to determine the significance of this result, it would be important to follow up with additional samples compared to a gender and age matched set of samples from Missoula. The frequency of anti-CCP may be elevated in Libby particularly since this autoantibody appears to be predictive of incipient RA, which would be consistent with the epidemiological study. This is also interesting in view of the presence of AA to cytoskeletal components, which may also be a marker for RA [40]. However, a caveat is that the cases for RA in the study by Noonan, et al. [57], were self-reported so that although the surveys indicated that their RA was physician diagnosed, the reported cases may have included some non-RA patients, such as osteoarthritis. Finally, arthritis is an early symptom of SLE as well, often occurring prior to diagnosis suggesting that some of the RA cases could be evolving to that condition. One of the anecdotal concerns of the Libby population has been the incidence of diseases not formally classified as rheumatoid, such as fibromyalgia and chronic fatigue syndrome (CFS). Although these diseases remain poorly defined serologically, there is some evidence of increased frequency of positive ANA tests in CFS patients [58, 59]. The diagnostic challenges associated with these diseases, especially in the presence of other chronic diseases, will make assessment of the true prevalence of these diseases in Libby a difficult task. Nevertheless, the presence of a high frequency of positive ANA that were not also positive by other tests for antibodies to ENA suggests that there may be unidentified antibodies in these subjects. Antibodies to nuclear envelope antigens were reported in CFS patients [59], giving a staining pattern that could be mistaken for a homogeneous pattern, which was the most common pattern seen in the Libby samples. However, other factors can cause positive ANA tests without reactivity to the ENAs tested in this study, including smoking or thyroid disease. There is no evidence of unusually high rates of thyroid disease or smoking in Libby, however, compared to other communities in Montana. Approximately 20-22% of adults in both Libby and Missoula smoke tobacco, according to the Montana Department of Public Health and Human Services, so smoking is unlikely to be the cause of ANA differences between these populations. There is some evidence that AA appear prior to the onset of clinically overt disease [60]. A thorough analysis of an 80-patient cohort as well as a literature review in 1970, led by British researchers Turner-Warwick and Parkes, concluded that there was growing evidence not only that asbestos drives autoimmune responses, but also that there was an intriguing and potentially critical association of these asbestos-induced AA with the severity and progression of the pulmonary disease seen in the asbestos exposed subjects [8]. Interestingly, exposure to asbestos and silica lead to patterns of illness that are very similar to SAID including both pulmonary fibrosis and serum ANA [22, 61, 62]. Although the mechanisms leading to the progression of these conditions have not been fully explained, there is evidence that some of the lung pathologies seen with both asbestos and silica exposures are immunologically mediated [63-65]. Although many of the cells and mediators involved in the initiation of silicate-associated pulmonary fibrosis have been studied, the reasons for the chronic and progressive nature of silicosis and asbestosis remain unclear. Interestingly, analysis of the data from the earlier study of AA in Libby subjects [22] showed a significant association of positive ANA tests with higher lung disease scores. In addition, when the CDC/ATSDR (Agency for Toxic Substances and Disease Registry) performed its screening in Libby during 2000-2001, of 7307 screening participants, 494

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(6.7%) indicated that they had been diagnosed with either SLE, SSc, or RA [21]. Of those 494 participants from the Libby cohort with suspected SAID, 171 (35%) have had pleural and/or interstitial abnormalities indicated on chest radiographs that were confirmed by two Breaders. These data suggest that the proportion of radiographic abnormalities among those with suspected autoimmune disease (35%) was almost double the proportion of radiographic abnormalities among the entire Libby cohort (approximately 18%) [24]. These data are supported by a study by Tamura, et al, in which positive ANAs were more frequent in asbestos-exposed subjects who had pulmonary lesions [66]. A 3-year follow-up study of those patients demonstrated that positive ANAs were correlated with the progression of asbestosis [67]. Although it could be argued that AA are simply a result of the chronic lung inflammation in asbestos-exposed lungs, the alternate hypothesis is that there is an autoimmune component to these pulmonary pathologies. This chapter reports that the only specificity that showed a significant association with scored pulmonary disease (ARD) was topo-I. Anti-topo-I was detected by two techniques: ALBIA (ENA8, INOVA Diagnostics) and ELISA (INOVA) (Table 1). The latter assay appeared more sensitive and revealed positive tests on eight subjects, compared to two by ALBIA. The results by ELISA were more consistent with ANA data of nucleolar staining. Therefore, all eight samples were used for subsequent analyses. An association between antitopo-I and pulmonary disease has been reported [68], although that association is much stronger with subjects testing positive for anti-topo-I by immunodiffusion rather than by ELISA [69]. Two hypotheses have been proposed to explain this relationship. One is the possibility that the AA actually enter the cell where anti-DNA topoisomerase I is able to inhibit enzyme activity [70], resulting in cell death and therefore tissue damage. Second, antitopo I antibodies from SSc patients have been shown to bind to fibroblasts and initiate proinflammatory/profibrotic cascades [71, 72]. These studies suggest that antibodies targeting various cell types can modulate cell function and therefore contribute to pathology. The possible role of AA to fibroblasts, endothelial, and epithelial cells in vascular and fibrotic disorders is receiving considerable attention as the evidence of their pathogenicity expands. Antibodies to endothelial cells have been implicated in vasculitis [73], SSc [74], and SLE [75]. Anti-epithelial cell antibodies are being studied in cryptogenic fibrosing alveolitis [76] and non-allergic asthma [77]. Anti-fibroblast antibodies (AFA) have primarily been studied as a possible factor in the fibrosis of patients with SSc [78, 79]. However, despite the growing concern about an autoimmune process in these diseases, very few of the target antigens have been clearly identified, and the mechanism of lost tolerance to those antigens is unknown. Early techniques did not allow the sensitivity of detection of certain autoantibody specificities that is now available, and previous studies did not specifically link certain autoantibodies with these complex pulmonary/autoimmune combinations. Table I. Data from 70 randomly selected Libby samples as described. Blank = no data

89

Autoantibody Profiles of an Asbestos-Exposed Population Patient

Age

Gender

Disease

1 2 3 4 5 7 8 9 11 14 17 18 19 36 38 39 40 41 44 47 49 50 53 58 60 65 66 68 70 73 74 77 79 81 83 86 89 92 98 99 100 102 118 119 120 121 122 123 126 127 129 135 136 142 164 167 176 177 180 185 190 196 198 207 224 225 356 360 366 371

72 59 65 48 54 47 48 65 48 48 43 36 62 57 61 69 78 43 51 66 49 63 58 60 53 73 52 45 69 41 44 55 42 51 32 59 56 66 52 71 66 64 79 70 79 69 39 36 75 60 50 62 44 62 39 33 99 79 62 49 44 66 35 69 49 41

M F F M M F F M M M M M F M M F M M M M F F F M M F F F F M M F F F F M M M M M M M F F M F F M F F M M F M F M F F F F F M M M F M

3 3 3 3 2 0 1 2 3 2 2 2 2 3 3 3 3 3 2 3 1 2 1 3 1 3 1 1 2 1 0 2 0 2 1 2 2 3

53

M

2

71

F

3

3 2 2 2 3 3 0 0 2 2 0 0 3 2 1 3 2 0 3 0 1 1 3 0 0

Exposure ANA score and pattern 4 0 3 2 spkl 3 3 hom/spkl 1 0 4 1 spkl/cytsk 3 3 nuc 3 0 4 3 hom 4 3 hom/cytsk 2 0 2 3 spkl 4 1 nuc 3 2 nuc/cytsk 3 2 nuc 2 3 hom 3 2 nuc/cytsk 4 2 nuc 4 2 cent 1 0 3 4 hom 3 0 3 3 hom 3 2 nuc/cytsk 1 0 4 4 hom/cytsp 3 2 hom 1 0 1 0 4 3 hom, cytsp 3 4 hom 3 1 nuc 3 3 hom/nuc 3 1 spkl 3 4 cent 1 0 3 1 spkl 3 2 hom/spkl 3 3 atyp 2 0 4 2 hom 3 1 hom/cytsk 3 1 spkl/cytsk 3 4 hom/cytsk 3 3 hom/nuc 2 2 hom 3 4 nuc 1 0 2 1 nuc 3 1 hom 3 2 spkl 4 3 spkl 3 3 nuc 1 4 spkl 0 0 3 3 spkl 4 0 3 0 1 2spkl 3 2 spkl 4 1 spkl 3 0 4 0 3 0 3 0 1 2 spkl 1 2 hom 2 hom/nuc 4 2 hom/nuc 1 hom/nuc 1 1 hom

SLE 9 Profile ALBIA 0 0 SSB 0 Ro52 Chromatin, Histone RNP 0 Topo-1 0 0 0 0 0 Chromatin Chromatin, Histone 0 0 0 0 0 SSA-60 0 RNP Chromatin, Histone 0 0 0 Chromatin 0 Topo-1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Chromatin, Histone 0 0 0 0 0 0 0 0 0 0 Ro52 0 0 0 0 0 0 0 0 0 Chromatin, Histone Ro52 0 0

Topo-1 by ELISA

Topo-1

Topo-1 Topo-1

Topo-1

Topo-1

Topo-1 Topo-1

Topo-1

RF IgM 0 0 0 0 0 0 0 1 0 1 0 0 0 1 1 1 1 0 0 0 0 1 1 1 1 0 0 0 1 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1 1 0 0 0 1 0 0 0 1 1 1 1 0 1 0 0 1 0 0 0 0 1 1 0 1 0

New techniques allow exquisitely sensitive detection of large numbers of specificities, easily screened in large numbers of subjects. Modern radiographic techniques have also dramatically improved the ability to classify pulmonary outcomes of exposures compared to connective tissue diseases [80]. This chapter simply reports the presence of AFA in some asbestos-exposed subjects from Libby, illustrating the importance of follow up on these

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subjects to determine whether these antibodies might be markers for rapid progression or exacerbation of pleural or interstitial disease. An association of these antibodies with clinical progression might lead to excellent targets for therapeutic intervention.

CONCLUSION The purpose of this study was to detect a serological signature in the asbestos exposed population of Libby, MT, and compare this profile with other known SAID autoantibody profiles, including those seen with silica exposure. While the variability in the samples makes comparisons difficult, autoantibody profiles of this asbestos-exposed population show similarities to those of patients with idiopathic SLE, drug-induced SLE, and scleroderma, due to the apparently high frequency of anti-topo-I. The presence of AFA may indicate a direct role for asbestos-induced AA in accelerating fibrotic manifestations of the exposure. This information may improve screening of asbestos-exposed populations and could serve to point toward diagnostic or prognostic markers relative to asbestos exposure. A marker that appears earlier after exposure and that is correlated with eventual autoimmune disease would be a great benefit. Future studies include the exploration of the specific autoantigen targets for the AFA in mouse and human models, and prospective study of this population to determine the relationship between ANA, AFA and clinical progression of both autoimmune and pulmonary pathologies. Finally, collaboration with medical doctors that treat and advise patients from Libby, MT would provide necessary clinical diagnostic data for future studies. Joining clinical observations and the serological characterization presented here has an excellent chance to help accurately characterize clinical autoimmune manifestations of asbestos exposure, and therefore to screen, diagnose and treat asbestos exposed individuals.

ACKNOWLEDGEMENTS The authors gratefully acknowledge the Libby Center for Asbestos Related Diseases (Brad Black, M.D., Director) for assistance with sample acquisition, and Roger Diegel, M.D., Rheumatology, for his review of this Chapter.

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[79] Chizzolini, C., Raschi, E., Rezzonico, R., Testoni, C., Mallone, R., Gabrielli, A., Facchini, A., Del Papa, N., Borghi, M. O., Dayer, J. M., Meroni, P. L. (2002). Autoantibodies to fibroblasts induce a proadhesive and proinflammatory fibroblast phenotype in patients with systemic sclerosis. Arthritis Rheum., 46, 1602-13. [80] Muravov, O. I., Kaye, W. E., Lewin, M., Berkowitz, Z., Lybarger, J. A., Campolucci, S. S., Parker, J. E. (2005). The usefulness of computed tomography in detecting asbestosrelated pleural abnormalities in people who had indeterminate chest radiographs: the Libby, MT, experience. Int. J. Hyg. Environ. Health, 208, 87-99.

Funding: CDC 822092 NIH ES04804 NIH ES012596 NCRR P20 017670

In: Mesothelioma from Bench Side to Clinic Editor: Alfonso Baldi, pp. 25-66

ISBN: 978-1-60021-789-0 © 2008 Nova Science Publishers, Inc.

Chapter 5

EPIDEMIOLOGY OF MESOTHELIOMA: THE ROLE OF ASBESTOS Massimo Menegozzo1, Roberto Pasetto2, Simona Menegozzo1 and Pietro Comba2 1

Campania Regional Operating Center (COR) of the National Mesothelioma Registry (ReNaM), Experimental Medicine Department, Second University of Naples, Naples, Italy 2 Department of Environment and Primary Prevention, Istituto Superiore di Sanità, Rome, Italy

ABSTRACT Mesothelioma is a rare but lethal disease. This chapter provides an explanation of the disease and descriptive epidemiological data: incidence, mortality, survival, temporal trends and geographical distribution. It also presents etiological data on asbestos and asbestos-type fibers and delves into the effects of the duration and intensity of exposure and the dose-response relation. It describes the contexts of occupational risk in general terms, details on exposure and the risks associated with specific work sectors. The report then goes on to illustrate multiple scenarios of environmental exposure. Finally, it elaborates on the contribution of the epidemiology of mesothelioma to Public Health initiatives: environmental reclamation, identification of groups at risk and the prevention of asbestos related pathologies in developing countries.

DEFINITION Malignant mesothelioma is a rare and very severe form of cancer of the serous membranes of mesothelial origin, with an unfortunate prognosis and a highly limited survival time [1, 2, 3]. Malignant mesothelioma attacks the serous membranes originating from the embryonal mesodermal folium and has the following clinical manifestations: • pleural mesothelioma • peritoneal mesothelioma • pericardial mesothelioma • mesothelioma of the tunica vaginalis testis

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Massimo Menegozzo, Roberto Pasetto, Simona Menegozzo et al. From a histological perspective, it appears in three primary forms: • • •

epithelial sarcomatoid biphasic or mixed

This classification is approximate as the availability of wide ranging samples of mesothelioma determines a significant variability of the histological picture. Nevertheless, classification in the three istotypes is sufficiently correlated both with the prognosis and with the evaluation criteria for surgery: epitheliomorphic mesotheliomas have a better prognosis and are susceptible to a therapeutic approach with an improved probability of survival. Diagnosis of a malignant pleural mesothelioma involves numerous difficulties relating to possible superimposition of such clinical contexts as carcinoma of the lung in a juxtapleural location, pleural metastasis of the extra-pulmonary primary carcinomas, reactive pleuritis. Peritoneal mesothelioma is also associated with problems of differential diagnostics relative to peritoneal metastasis of primary carcinomas, with peritoneal localization of TBC. Mesotheliomas of the pericardium and of the tunica vaginalis testis are rare. The principal etiological agent of malignant mesothelioma is asbestos [4,5,6], although other agents cannot be excluded for a limited percentage of cases, such as environmental exposure to other types of complex silicates found in nature [7,8] and previous exposure to ionizing radiation. Diagnosis of mesothelioma is currently based on several basic parameters: (a) patient’s clinical history: ( work anamnesis with special regard to evaluation of a previous exposure to asbestos, clinical outcome, course of the illness, temporal evolution up to exitus ); (b) imunohistochemical assessment: based on the availability of a set of markers that allow for a differential diagnosis between pleural mesothelioma and pulmonary adenocarcinoma [9]. The best set of biomarkers consists of: positive biomarkers per MM: calretinina and cytokeratine 5/6 and WT 1; negative biomarkers per MM: CEA and MOC-31

• •

(c) image diagnostics using standard chest x-rays ( I.L.O. ‘ 80 ) [10] and new imaging techniques: • • •

TC [11] PET [12] Magnetic Resonance [13]

(d) serum markers capable of acting as precocious biomarkers for the onset of mesothelioma: • •

SMRP ( Serum Mesothelin-Related Protein ) [14] Auto antibodies P53 [15]

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N-ERC/mesothelin [16] Osteopontina [17]

(e) profile of gene expression: [18,19,20] Numerous studies have been conducted since 1999 to assess the presence of any specific profiles of gene expression (micro-array analysis) to define the criteria for differential diagnostics between pleural mesothelioma and lung cancer. A set of genes has consequently been identified that accompany the transformation of the mesothelial cell into a cancerous cell by modifying its activity by means of gene over-expression for some and of under-expression for others. The study of gene expression has proven useful not only for the differential diagnosis of pleural mesothelioma, but also for an evaluation of the prognosis.

DESCRIPTIVE EPIDEMIOLOGY OF MESOTHELIOMA Incidence Mesothelioma may be considered as a rare disease. The incidence of “natural” pleural mesothelioma, of background type (no exposure to asbestos), is estimated to be less than 1 case per million per year [7], but the number of cases is much higher in populations that are exposed to asbestos. An assessment of the distribution of cases of mesothelioma conducted using a database with indices of the proportion of workers exposed to carcinogenic agents and their level of exposure by region, throughout the world, has led to the following distribution of the 43,000 deaths estimated for the year 2000: Eastern Mediterranean 2,300, Africa 3,100, Americas 3,200, Europe 7,000, Southeast Asia 11,100, Western Pacific 16,300 [21]. During the 20th century asbestos was part of the industrialization process. According to the data provided by the U.S. Geological Survey between 1900 and 2003, approximately 182 million tons of asbestos were extracted [22], reaching a production peak of 5 million tons in the 70s. In 2003, 2.15 million tons of chrysotile (white asbestos) were extracted, with the principal producers being, in decreasing order: Russia (878,000 tons), Kazakhstan, China, Canada, Brazil (195,000 tons). The causal relationship between exposure to asbestos and mesothelioma is a consolidated one and in many countries a relation has been observed between the pattern of mortality/incidence by mesothelioma and the consumption curves of asbestos, with a lag time of approximately 30-40 years [23,24] (table 1). An estimate of the incidence in different countries can be made starting with the data of the International Association of Cancer Registries (IACR). It must first be pointed out that many countries have no cancer registries and so the estimates of incidence based on such registries are not complete. Furthermore, even in countries that may have one or more registries, the surveys are based on local compilations and generally cover a limited section of territory. The data found in the most recent IACR publication of the series “Cancer Incidence in Five Continents”, refer to 186 registries from 57 countries for the years 1993-1997 [25]. The estimated incidence varies between a minimum corresponding to the limit of detection of rates, a number of registered cases of less than 5, equaling an incidence of slightly higher than

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1 case per one million per year, in poorer countries and with an inferior coverage of the population, to a standardized rate (using as reference the age structure of the world population) of 6 cases per 100,000 per year among men in regions of Australia and in Italy. Cases observed in women are an average of 3 to 4 times less than those observed in men. The crude rates, indicating the extent of the disease by country, without considering age incidence, have higher values (between 4 and 12 cases per 100,000 men per year) in the western countries of Europe, in Australia and in the United States.

Mortality Mortality from pleural mesothelioma, according to the International Classification of Diseases (ICD), is considered within the category of malignant tumors of the pleura (ICD IX edition, 163.0-163.9). This category includes, in addition to mesotheliomas of the pleura, other primary tumors located in the pleura, prevalently sarcomas, which are very rare, and erroneously classified secondary tumors, for the most part metastatic carcinomas of primary tumors originating in the breast and lung. Mesotheliomas in other sites are not categorized separately. Furthermore, the percentage of pleural mesotheliomas present in cancer registries is generally greater than 80% of the total number of mesotheliomas and exceeds 90% in more numerous case series [25]. Mortality from pleural mesothelioma is, on the epidemiological level, a reasonable approximation of the incidence of pleural mesothelioma. The quality of the registration of causes of death by pleural mesothelioma has been assessed on a limited number of cases in Australia [26], the United States [27], Italy [28] and France [29]. The percentage of pleural mesotheliomas correctly classified in the category of death from malignant tumor of the pleura varied, in the years preceding 1990, between 23% and 76% considering different periods of study, different stages and different sub-groups analyzed (i.e. by sex). An estimate of 40% of false positives and 18% of false negatives was recently reported on the basis of a regional, Italian, registry of mesothelioma cases [30]. There is a general consensus that the misclassification of individual cases attains a sort of compensation at the level of population, making the mortality data usable for analyses of descriptive epidemiology. Mesothelioma of the pleura is a lethal pathology with a median survival rate of less than one year from diagnosis [2,31,32]. In addition, while the incidence data are based on cancer registries, with consequent limitation of the population covered by the survey, mortality data are often available for large populations and are based on current data flows that are temporally stable and standardized. For these reasons mortality data are used for an indirect estimate of the incidence of pleural mesothelioma. In addition to being the only type of evaluation possible for frequency of pathology in nations without cancer registries, this estimate may be better than the estimate obtained from real incidence data, especially for countries with cancer registries that have insufficient population coverage and geographic heterogeneity for frequency of disease. Mortality data are generally used for geographic type descriptions and for predictions of temporal trends.

Survival

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Mesothelioma is currently an incurable disease. Survival does not appear to have changed substantially over time, reflecting both the gravity of the disease and the inefficacy of therapies used to date. The average survival rate for peritoneal mesothelioma is less than that of pleural mesothelioma, about 6 months from diagnosis, and the two genders differ greatly [32]. This difference, in favor of women, is probably attributable to a misclassification of female tumors of the genital tract [2]. Recognized prognostic factors are age, histological type and diagnostic certainty [2, 33, 34]. There is an inverse relation between age and survival, with survival being less at an advanced age [32]. The risk from fibrous mesotheliomas is approximately three times that of epithelial and biphasic mesotheliomas [32, 33, 34]. Finally, diagnostic certainty also appears to be a predictive factor. Mesotheliomas classified as suspect are attributed a higher risk factor than those classified as certain. This may be linked to the tendency to avoid invasive diagnostic procedures for patients in an advanced stage of the illness and in generally precarious conditions [32].

Temporal Trends The epidemic of mesotheliomas is increasing in countries that have made vast use of asbestos in the past. The epidemic curve, in fact, follows that of consumption over time. It has been estimated that there have been more than 20,000 asbestos-related lung tumors and 10,000 mesotheliomas per year in the period immediately preceding the year 2000 in the populations of Western Europe, Scandinavia, North America, Japan and Australia [35]. The complete ban on the importation, manufacture, sale and use of asbestos or its restriction was applied successively in these countries beginning at the end of the 80s and a complete ban on asbestos was adopted by a progressively increasing number of states worldwide (www.ibas.btinternet.co.uk). On the basis of incidence and mortality trends, we deduce that the epidemic curve of mesothelioma has already reached its peak in the United States, at least among men [36] and the “plateau” is expected to be reached in the period 2010-2020 in Great Britain [37], France [38], Holland [39], Finland [40], Sweden [41], New Zealand [42] and Australia [31]. In Italy, the maximum level of the epidemic may follow a year or so later, using as reference the peak of consumption leading up to the 80s [43]. While the use of asbestos has been prohibited or reduced in some countries, it is increasing in Asia, Latin America and in the Commonwealth of Independent States (Byelorussia, Russian Federation, Ukraine) [44]. The first 5 countries for asbestos consumption in the year 2000 were, in order of priority: Russia (447,000 tons), China, Brazil, India, Thailand (121,000 tons) [45] . These data lead to the reasonable supposition that a decline in preventable mesothelioma related deaths in industrialized countries will be followed by a growth in developing countries.

Geographic Distribution

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The “macro” geographic distribution of mesothelioma has already been described in the paragraph concerning incidence. In respect of individual countries, epidemiological monitoring systems based on specific registries of the pathology or on assessments of mortality data, provide a more refined description and a detailed distribution of mesotheliomas on a national level [46, 47, 48, 49, 50, 51]. These types of analyses indicate that clusters of mesotheliomas are concentrated in areas of the country with major industrial plants in which in the asbestos is extracted (quarries and milling plants), processed as a primary raw material (i.e. production of asbestos cement), used as a component of the production process (i.e. used for insulation in shipyards) and is widely found in the structures of industrial systems (i.e. insulation in heating pipes for chemical plants). Table 1. Cases of mesothelioma in the 90s and consumption of asbestos in the 70s. Countries listed by decreasing order of cases observed (modified by Tossavainen 2004 [45]) Country United States Great Britain Germany Italy France

Cases (year) 2,800 (2000) 1,595 (1999) 1,007 (1997) 930 (1995) 750 (1996)

Australia Netherlands Sweden Finland New Zealand Norway

490 (2000) 377 (1997) 105 (1996) 74 (1999) 50 (1996) 48 (1995)

Asbestos tons (year) 552,000 (1975) 170,000 (1970) 230,000 (1975) 140,000 (1975) 143,000 (1970) 70,000 (1970) 49,000 (1976) 20,000 (1970) 11,000 (1970) 8,000 (1970) 8,000 (1970)

These systems allow us to verify, in space and time, those areas of the country that are most dangerous and thus requiring the implementation of medical surveillance systems for subjects previously exposed and centers of excellence for treatment of asbestos related diseases. Generally, epidemiological monitoring draws attention to situations in which asbestos exposure is already known but, in some cases, it may highlight situations of nonrecognized occupational exposure (i.e. non-asbestos textile factories [52]) or special conditions such, for example, as those resulting from a natural contamination of the soil by asbestos-type fibers (exposure to fluoro-edenite in a town in Sicily [53]).

ETIOLOGICAL ROLE OF ASBESTOS AND OTHER FIBERS Asbestos Asbestos consists of a series of fibrous minerals found in nature and is easy to extract and process. The different types of asbestos found in nature are classified into two large families:

Epidemiology of Mesothelioma: The Role of Asbestos

• •

103

serpentines in which the principal mineral is chrysotile amphyboles, encompassing several different species: • • • • •

crocidolite amosite anthophyllite tremolite actinolite

The fibrous structure of asbestos makes it almost indestructible, non-flammable, resistant to acids and corrosives, highly flexible, resistant to stress, and has excellent properties for absorbing heat and noise. Because of its extraordinary characteristics, it is estimated that asbestos was used in more than 3,000 products and objects during the XX century. Possible negative effects of this mineral on human health began to appear just a few years after it began to be extensively used, especially in the textile industry. As indicated in the book “Late lessons from early warnings” [54], the first observation of possible harm to human health caused by exposure to asbestos may be attributed to Lucy Deane, one of the first Women Inspectors of Factories in the UK. Writing in 1898, Deane included asbestos work as one of the four dusty occupations which came under observation that year. She went on to observe that: ‘the evil effects of asbestos dust have also instigated a microscopic examination of the mineral dust by HM Medical Inspector. Clearly revealed was the sharp glass-like jagged nature of the particles, and where they are allowed to rise and to remain suspended in the air of the room in any quantity, the effects have been found to be injurious as might have been expected.’[55] The pathological consequences of occupational exposure to asbestos are amply recognized in international medical literature ( the first works are all in English medical literature) and bear the following dates: • • • • • •

•

1907: recognition of the first cases of asbestosis in workers of textile plants in Great Britain [56]; 1927: Cooke coins the term asbestosis for the pulmonary fibrosis induced by inhalation of asbestos fibers [57]; 1930: recognition of asbestosis as an occupational disease caused by occupational exposure to asbestos [58]; 1931: first regulation on the use of asbestos in Great Britain through issue of the “Asbestos Industry Regulations “ [59]; 1935: initial studies on the possibility of bronchial carcinomas caused by occupational exposure to asbestos [60]; In 1943 Wedler cites the presence of “pleural carcinomas” in his German statistics on the onset of tumors of the respiratory system in individuals suffering from asbestosis [61]; In 1947 Mallory presents a case of pleural mesothelioma in a worker exposed to asbestos [62];

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•

•

1955: the inquiries carried out by Doll demonstrate in an incontrovertible manner that exposure to asbestos leads to a significant incidence of death by pulmonary cancer [63]; 1960: Wagner’s studies of workers in the asbestos mines of South Africa identify pleural mesothelioma as a typical tumor caused by occupational exposure to asbestos [64]; subsequent studies conducted by Wagner’s group in South Africa and by Selikoff in the U.S. confirm this data, extending the risk of mesothelioma not only to the pleura but to all mucosa of mesothelial origin ( peritoneum [65], pericardium, tunica vaginalis testis);

In spite of the fact that the negative effects on human health were already known in the 60s, it was only at the beginning of the 80s that a decrease in the world production of asbestos begins to be noted, going from 4.7 million tons in 1980 to 1.5 million tons in the year 2000. However, this decrease is not homogeneously distributed world wide. In this sense it is paradoxical that Canada, though prohibiting the use of asbestos within its own territory, continues to extract asbestos from its mines and to export it widely, accompanied by a capillary propaganda campaign on the safe use of chrysotile asbestos.

Other Mineral Fibers Implicated in the Etiopathogenesis of Mesotheliomas In addition to asbestos in its serpentine and amphybole varieties, other minerals with a fibrous structure have been recognized as causal factors of incidence of mesothelioma in the populations exposed. Specifically, we refer to the data provided by literature on two mineral species: • •

erionite: this fibrous mineral is found in the regions of Anatolia and is considered responsible for the epidemic of mesotheliomas registered in those regions [66]. fluoro-edenite: a new mineral species only recently identified in the vicinity of the town of Biancavilla in Sicily, responsible for the epidemic of mesotheliomas among the inhabitants of the town [67].

Both these mineral fibers and related epidemiological findings are dealt with in the section on environmental exposure to asbestos and other mineral fibers.

Duration and Intensity of Exposure to Asbestos and Dose-Response Relation A dose-response relation between the level of exposure to asbestos, estimated according to the work performed, and the inception of mesothelioma, was initially described by Newhouse [68] and by Newhouse and Berry [69] among workers in the textile industry. In 1979 Seidman et al [70] wrote “using the duration of work activity in an amosite plant as a measure of the dose of asbestos, we noted that in general the lower the dose the greater the time required for onset of the adverse experience of mortality, and even when this does appear it is less severe. This fact has important implications in controlling cancer: if it is not

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possible to completely avoid exposure to carcinogenic agents, a reduced exposure can delay the onset of adverse effects and the frequency of their occurrence”. The issue was taken up by other authors such as Armstrong et al. [71] who described the relationship between cumulative exposure to asbestos and mortality by mesothelioma among crocidolite miners in Australia. Tuomi et al. [72] and Rogers et al. [73], respectively in Finland and Australia, also wrote of the significant relation between the risk of mesothelioma and the concentration of asbestos fibers in the pulmonary tissues. Of particular importance is the contribution made by Iwatsubo et al. [74] who conducted a case-control study demonstrating that a dose-response relation already existed at an exposure of 0.5 ff/ml/year (fibers per milliliter per year). And finally it should be noted that the risk associated with continuous exposure is higher than the risk associated with intermittent exposure [64]. In a comment on this work, Siemiatycki and Boffetta [75] examined the study in detail, concluding that the inquiry is valid, even though the quantitative aspects require further replication. In another contribution, Boffetta [76] reviewed the quantitative aspects of asbestos induced cancer, suggesting that the incidence of mesothelioma may vary according to latency period, exposure expressed in fibers/milliliter, to the constant that expresses the specific carcinogenic power per type of fiber and type of industry, to the minimum latency required to reveal an increase in mesothelioma and to the effect of the latency period. The significance of this model lies in the fact that each brief period of exposure leads to an addition to the subsequent risk, proportionate to the concentration of dust at that time, multiplied by the p-th power of the time that has passed since that moment. Berry et al [77] found a significant exposure/response relation for pleural mesothelioma in a cohort of 5,000 subjects exposed to asbestos in the city of London between 1933 and 1980. Hodgson and Darnton [5], demonstrate how pleural mesothelioma increases sublinearly with the cumulative dose, even though a linear relation is compatible with the data, and do not believe they can assume the existence of a threshold. In accordance with the above, it should be noted that there is a correlation between the duration of exposure to asbestos and the quantity of asbestos bodies found in lung tissue and between the duration of exposure and the total quantity of fibers in the lungs [78]. In the monograph by the World Health Organization [79] on the medical effects of chrysotile, observations on mesothelioma indicate that in the mining sector, which has the greatest number of mesotheliomas, there were no cases associated with exposures of less than two years while it appeared that there was a clear dose-response relation, with crude rates (in terms of cases /1000 persons-year) between 0.15 (for lower cumulative exposure) and 0.95 (for cumulative exposures three times greater). Finally, the American Conference of Governmental Industrial Hygienists (ACGIH)[80] recently re-confirmed the existence of a dose-response relation for pleural mesothelioma. Thus prolonged exposure to asbestos is not a mandatory condition for the onset of mesothelioma. There is an appreciable increased risk of mesothelioma also as a result of brief exposure to asbestos, as documented by Seidman et al. [70] and subsequently confirmed by other authors, including Roggli [78], Neumann et al [81] and Leigh et al [31]. The latter two works are of special interest as they refer to two rather extensive case series, respectively the mesothelioma registries for Germany and for Australia. In Germany, Neumann et al examined

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1,605 cases of mesothelioma, identifying an occupational history of exposure to asbestos in 70% of the subjects studied. The average duration of exposure was 17.5 years, with a range of between 1 month and 56 years. In Australia, Leigh et al studied 6,329 cases of mesothelioma diagnosed between 1945 and 2000, for 82% of whom there was documented exposure to asbestos. Three percent (3%) of the cases had been exposed for less than 3 months. To be noted is the fact that there are dose-response relations in connection with occupational exposure and also for non-occupational environmental exposure [82,83]. Hansen’s study in particular, conducted on a community residing near a crocidolite mine, demonstrates that for subjects not occupationally asbestos exposed the incidence of mesothelioma increases in relation to the period of latency, the duration of exposure and the cumulative exposure. Concerning the periods of latency, understood as the time between the beginning of exposure and manifestation of the disease, tumors generally follow normal or log-normal distributions [84]. This means that the probability of discovering very high or very low individual data increases with increased observations. Estimates on the latency of mesothelioma were provided by Prof. Selikoff’s group as part of the extensive study conducted on a cohort of asbestos workers [85]. For the 356 cases of pleural mesothelioma observed, the average latency was 33.8 years, with a standard derivation of 8.9 years. Subsequently, additional data were provided by other authors. Shorter latency periods are normally around 15 years and the longer ones around 60-70 years [86,87]. With reference to asbestos caused cancer, a final observation concerns the role played by the size of the fibers. Some authors have measured the length and diameter of the fibers that, surpassing the lung-pleura barrier, were deposited on the parietal pleura near the sites in which the mesothelioma develops. The majority of these fibers are particularly short (less than 5 μm) and thin (diameter less than 0.25 μm), as observed by various authors [88,89,90,91]. This observation has led some to maintain that only ultrafine fibers play an etiologic role in mesothelioma [92]. In actual fact, the issue is more complex, as there are elements supporting the theory that the pleura-pulmonary barrier may also be exceeded by larger particles and fibers [93,94], and that fibers may reach areas distant from the lung by migration or diffusion [95,96]. The most commonly held opinion is that asbestos fibers of any length can provoke a pathological response [97,98].

Occupational Exposure The enormous quantity of asbestos produced during the course of the past century, in spite of the progressive decrease in production beginning in the 80s, is a strong risk factor for humans on a global scale because since it is non biodegradable, it has an injurious potential for an unlimited period of time. The etiological role of asbestos in determining mesotheliomas is characterized by a correlation between the cumulative dose [74] and the incidence of mesotheliomas in the exposed population, without defining a threshold to exclude the induction of mesothelioma. From the data available in literature on mesothelioma registries, based on an evaluation of exposure to asbestos obtained through the indirect method of interviews, we note that in reference to occupational and non occupational contexts, exposure is present in up to approximately 80% of the cases [99,100,101].

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Thus we can state that, in terms of human health, asbestos as such, or asbestos as part of complex materials, represented and may still represent a significant occupational risk characterized by different methods of action:

a) Direct Occupational •

• •

•

•

•

Miners tasked with direct extraction of the mineral, in serpentine and amphybole form (an increasingly decreasing cohort in western countries, but still present in countries that still carry out mineral extraction ). Miners tasked with extraction of minerals other than asbestos, but exposed to the presence of asbestos minerals contaminated by the rocks extracted.. Workers in the production of materials containing asbestos at different levels of friability, according to the matrix used ( i.e. asbestos cement, asbestos glues, asbestos resins, braking systems ). Workers in the sector of application of materials containing asbestos for the insulation of buildings, means of transportation, etc. ( i.e. ships, trains, buses, trams, airplanes ). Workers in the sector of maintenance and demolition of buildings insulated with asbestos, means of transportation, systems ( i.e. heating plants, insulated pipes ), equipment ( i.e. contactors ), other devices (i.e. vehicle braking systems, cranes) containing material with an asbestos content. Workers tasked with removing insulation from materials containing asbestos (an important activity during the present phase of radical elimination of the asbestos risk).

b) Indirect Occupational •

Workers who do not perform activities requiring the handling or manipulation of materials containing asbestos, but who work alongside other departments that are not adequately separated and in which processes take place using materials containing asbestos. This situation in particular is found inside large industrial hangars with unseparated work units, in which there is no effective form of primary prevention to trap asbestos fibers at the source nor adequate industrial cleaning of the departments.

c) Environmental Occupational •

Workers exposed to inhalation of asbestos fibers not from direct occupational exposure but from contamination by airborne fibers issuing from sprayed application of asbestos, or from roofs of industrial hangars containing asbestos cement and/or other asbestos material found in the work environment;

Obviously there are some work environments in which there may exist more than one type of occupational exposure to asbestos fibers, such as:

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•

insulation of railway carriages applied using a sprayed mixture of crocidolite, water and glue, in an occupational environment where there occur simultaneous activities such as molding of asbestos cartons, in hangars covered with segments of Eternit in an unsatisfactory state of preservation; seamen working in engine rooms insulated with asbestos and who live in roomscabins insulated with materials containing asbestos.

We must not forget that asbestos fibers dispersed in the work environment may lead to forms of extra occupational risk in living environments, in two ways: diffusion to nearby living environments, when the dispersion of asbestos fibers in the work environment is such that it can be transmitted to the nearby ecosystem by air currents. This can lead to significant contamination, especially if the production plant is in a highly urbanized area; • diffusion to domestic living environments and to family members of workers occupationally exposed to the asbestos risk, through contamination of work clothes brought into the home for cleaning; Numerous epidemiological studies have identified significant excessive rates of mortality by mesothelioma in many productive sectors that involve occupational exposure to asbestos [4]. The risk of mesothelioma resulting from occupational risk to asbestos varies according to the different occupational sectors in which asbestos is used. This variability refers to the different types of asbestos used (serpentines and/or amphyboles), intensity of exposure ( estimated both as average concentration of airborne asbestos fibers and as time of exposure), environmental and individual prevention measures implemented. In respect of the intensity of exposure required to produce a significant incidence of mesothelioma in an exposed working population, studies based on the fiber content in lungs of unexposed subjects, subjects who have died from asbestosis and subjects deceased from mesothelioma, have demonstrated that the level of exposure to asbestos fibers for individuals dying of mesothelioma is significantly greater that the level of exposure of subjects who have not been exposed ( background ) but also significantly lower than the average level of exposure for subjects who have died of asbestosis [102]. As to latency (time between the initial exposure to asbestos and the insurgence of mesothelioma) the Consensus Panel for 1997 [103] has determined that a minimum latency of 10 years from initial exposure is required to attribute a nexus between exposure to asbestos and mesothelioma. Recent data relative to the second report of the National Registry of Mesotheliomas in Italy ( ReNaM ) [47], dealing with a total of 5,173 cases during the period 1993 – 2001 and reconstructing exposure by means of interviews, indicate that: •

•

In 77.2% of the cases of mesothelioma, exposure to asbestos was distributed as follows: • 67.4% from occupational exposure ( certain, probable, possible ) • 4.3% from familial exposure • 4.2% from environmental exposure

Epidemiology of Mesothelioma: The Role of Asbestos • •

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1.3% from exposure to an extra occupational leisure activity or hobby

An average latency of 43.6 years between the beginning of the exposure and appearance of the disease

Data indicates a tendentially decreasing risk in the mining sector, while the impact seems to be much greater in the sector of secondary processes. The productive sectors most affected are construction, shipbuilding, the mechanical and metallurgical industry, the sector of construction and repair of railways and the asbestos cement industry. Along with these traditional sectors, the ReNaM study also highlights sectors in which there is no direct processing of materials containing asbestos but whose work environment does contain asbestos as an insulation material (sugar refineries, chemical, oil extraction and refining industries, production of electrical energy). All data is reported in the table contained in the aforementioned ReNAM 2006 Report:

Table 2. Number of exposures per economic sector of exposure (ReNaM recodification) and period of diagnosis for cases of malignant mesothelioma caused by certain, probable or possible occupational exposure (period of incidence 1993 – 2001) Category

1993-1995

%

1996-1998

%

1999-2001

%

Total

%

Metallurgical and mechanical industry

21

5.5

59

7.2

111

7.1

191

6.9

Metallurgical industry

17

4.5

35

4.3

64

4.1

116

4.2

Oil extraction and refining

6

1.6

10

1.2

15

1.0

31

1.1

Extraction of minerals

3

0.8

2

0.2

10

0.6

15

0.5

Manufacturing of metal products

23

6.0

36

4.4

88

5.6

147

5.3

Textile industry

2

0.5

30

3.7

103

6.6

135

4.9

Non metallurgical mineral industry (excluding asbestos cement)

7

1.8

10

1.2

18

1.2

35

1.3

Asbestos cement industry

21

5.5

26

3.2

54

3.5

101

3.7

Railways

21

5.5

45

5.5

63

4.0

129

4.7

Shipyards Production and maintenance of means of transportation; vehicle and motor vehicle repair shops (excluding ship and railway yards)

65

17.0

150

18.4

161

10.3

376

13.6

11

2.9

32

3.9

63

4.0

106

3.8

Food and beverage industry (excluding sugar refineries)

9

2.4

8

1.0

39

2.5

56

2.0

Sugar refineries

10

2.6

9

1.1

22

1.4

41

1.5

Chemical and plastics industry

15

3.9

20

2.5

62

4.0

97

3.5

Rubber industry

5

1.3

8

1.0

23

1.5

36

1.3

Wood and wood products industry

0

0.0

2

0.2

5

0.3

7

0.3

Tobacco industry

0

0.0

0

0.0

1

0.1

1

0.0

Tanning and curing industry, manufacturing of leather and fur products

0

0.0

1

0.1

3

0.2

4

0.1

Clothes manufacturing

1

0.3

0

0.0

10

0.6

11

0.4

Glass and ceramics industry

3

0.8

12

1.5

24

1.5

39

1.4

Paper and paper products (including publishing) Other manufacturing industries (furniture, jewelry, musical instruments, sports items, etc.)

3

0.8

0.2

8

0.5

13

0.5

5

1.3

1.5

26

1.7

43

1.6

2 12

Table 2. (Continued) Category Construction

1993-1995

%

1996-1998

%

1999-2001

%

Total

%

50

13.1

105

12.9

252

16.1

407

14.7

Production of electrical energy and gas

7

1.8

13

1.6

21

1.3

41

1.5

Re-cycling

0

0.0

1

0.1

7

0.4

8

0.3

Agriculture and husbanding industry

1

0.3

8

1.0

13

0.8

22

0.8

Fishing

0

0.0

1

0.1

2

0.1

3

0.1

Hotels, restaurants, bars

0

0.0

3

0.4

4

0.3

7

0.3

Sales (wholesale and retail)

15

3.9

20

2.5

37

2.4

72

2.6

Marine transportation

13

3.4

25

3.1

37

2.4

75

2.7

Ground and air transportation

9

2.4

24

2.9

62

4.0

95

3.4

Maritime cargo movements

13

3.4

27

3.3

34

2.2

74

2.7

Public administration

2

0.5

7

0.9

17

1.1

26

0.9

Education

1

0.3

0

0.0

9

0.6

10

0.4

Military Defense

16

4.2

42

5.1

49

3.1

107

3.9

Banks, insurance, postal system

0

0.0

5

0.6

5

0.3

10

0.4

Health and social services

4

1.0

8

1.0

26

1.7

38

1.4

Other

3

0.8

17

2.1

16

1.0

36

1.3

Non Classified

0

0.0

1

0.1

0

0.0

1

0.0

382

100

816

100

1.564

100

2.762

100

Total

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DESCRIPTION OF TYPES OF EXPOSURE AND RISK IN VARIOUS OCCUPATIONAL SECTORS The following section contains detailed information on exposure to asbestos and its health consequences for individual occupational sectors. They are only a part of the work activities affected by the asbestos risk, but are nevertheless representative of the various types of exposure, according to the categories previously defined.

Production of Asbestos Cement Asbestos cement has surely been one of the most widely used products among those containing asbestos. It is estimated that approximately 70% of asbestos is used for this product. Factories for the production of asbestos cement products have been located throughout the world, since the first was established in Austria (Vocklabruck) in 1895 [104]. Other locations were Casale Monferrato - Italy ( 1907 ), Lund ( 1907 ) Sweden, Salonit Anhovo ( 1921) Slovenia, Split ( 1920 ) Croatia, Aalborg ( 1927 ) Denmark, Chongqin ( 1939 ) China, Osasco ( 1941 ) Brazil, Slemmestad ( 1942 ) Norway, Toronto ( 1948 ) Ontario – Canada, Acco ( 1952 ) Israel, Tyler ( 1954 ) Texas – U.S., Tarnow ( 1959 ) Poland, Pusan ( 1969 ) South Korea, Mina-Abdulla ( 1977 ) Kuwait. Numerous epidemiological studies have been conducted on the injurious effects on humans of exposure to asbestos fibers released by the manufacture of asbestos cement, as reported in a review conducted by Magnani and Colleagues [105]. It is interesting to note that various types of asbestos are used in the asbestos cement mixture, the principal one being chrysotile. Studies of mortality for cohorts exposed to asbestos cement have involved more general aspects relating to the carcinogenic nature of chrysotile, denied by some authors who maintain that cancer from asbestos ( and specifically mesothelioma ) is to be attributed to contamination by amphybole asbestos ( tremolite ) [106]. In contrast to this theory of a potentially safe use of chrysotile, the study by Yano and colleagues [107], conducted on a cohort of asbestos cement workers in Chongquin, China, demonstrates that this cohort, exposed to chrysotile lacking any tremolite contamination, also had a significant incidence of mesotheliomas. It should also be noted that epidemiological studies have frequently highlighted the incidence of cases of mesothelioma caused by extra occupational environmental pollution from contamination of the ecosystem located near the cement/asbestos companies ( paraoccupational exposure ) [108,109,110] or contamination of the domestic environment when washing work clothes at home [111].

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Insulation and Maintenance Activities of Insulated Industrial Plants The insulation of industrial plants and means of marine and rail transportation has often been assigned to companies that performed solely this activity, both for the application of insulation using materials containing asbestos ( to install panels and/or spray applications using mixtures of amphyboles – water – glue ), and subsequent maintenance and removal of deteriorated insulation material. These groups of workers are high risk cohorts because of the intensity of exposure and the prevalence of amphyboles (crocidolite and amosite), considered the most dangerous type of asbestos for the insurgence of mesotheliomas. The first great epidemiological study was conducted by Selikoff who investigated a cohort of approximately 17,800 insulation workers registered with the International Association of Heat and Frost Insulators and Asbestos Workers ( AFL-CIO-CLC ) operating in the U.S. and Canada. This same group was also the subject of a follow-up epidemiological study beginning in 01.01.1967. The results of this study published over various dates, were an extraordinary contribution not only to further knowledge of the incidence of mesothelioma in a cohort of insulation workers, but also because they cleared up a great many issues relating to the effects on human health caused by occupational exposure to asbestos [85,112]. Overall, up to 1992, 457 workers died of malignant mesothelioma, with an uncommon prevalence of deaths by peritoneal mesothelioma in respect of pleural mesothelioma. Additional epidemiological studies of cohorts of insulators have confirmed the high incidence of mesothelioma [113,114], and in particular the high prevalence of peritoneal mesothelioma [115].

Construction, Maintenance, Restoration and Repair of Ships Since the 1940s numerous studies have been published, in different countries, on the neoplastic risks in the shipyard industry. The wealth of data available is proof of the magnitude of asbestos consumption in ship construction since the beginning of the 1900s, as a result of the conversion from sails to the steam engine, and the subsequent introduction of iron and steel hulls. Up to the 1970s there has always been an increasingly greater demand for new ships and an uninterrupted increase and expansion of navigation companies. A significant increase in the number of ships under construction was provided by the Second World War: we saw a forced development of military fleets in Germany, Japan, Great Britain, Italy and an even greater one with the participation of the United States in the conflict and the American decision to rapidly equip a fleet that would allow the U.S. to be present simultaneously in the European and in the Asiatic theatre. It is only in the past few decades that naval construction has retrenched: in the Europe of 15 nations the number of employees was approximately 350,000 in 1975, decreasing to 85,000 in 2002 with the disappearance of almost two thirds of the shipyards (in Italy marine employees were 36,260 in 1975 and 13,438 in 2002). The first indication of pleural mesotheliomas among shipyard workers in Italy was provided by Zanardi and colleagues [116]: it refers to 8 lung tumors and 1 case of mesothelioma observed in Liguria.

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In 1972 Zannini stated that in examining “several thousand workers from all the shipyards of Liguria ” he found “approximately 700 cases of pleura-pulmonary asbestosis, some of which were associated with pulmonary and pleural neoplasia - 6 cases of pulmonary neoplasia and 3 cases of pleural mesothelioma; it is important to specify that 5 of these neoplasias were found in workers in the insulation sector and 4 in subjects performing other work” [117]. The authors state that they found “a greater incidence of pleurapulmonary neoplasia (ship repair workers in the port of Genova) compared to other port workers and the population of the rest of the city ". During this same period the first data was published on the incidence of cases of mesothelioma in shipyard workers of Trieste and Monfalcone [118]. The progressive study of these cases indicates that in the period between 1968 and 2000, the number of mesotheliomas observed increased from 20 to 557, and that the majority of the afflicted had undergone occupational exposure in a shipyard [100]. A study of mortality on 3,890 shipyard workers (construction and repair) of Genova, using the mortality of the general population of Genova for comparison [119], indicates an increased general mortality (SMR – Standardized Mortality Ratio – 108.8) from tumors (SMR 122.6), and an excessive mortality for all the different sites of the respiratory system: SMR of 148.2 – 442 – 138.6 for lung tumors, primary pleural tumors, tumors of the larynx. Also important was a mortality study of 893 employees of the Davidson Company of Genova, which carries out insulation work using materials containing asbestos, including sprayed applications, in shipyards, railway carriage construction companies and in the building construction sector [114]. The assessment of mortality, compared with that of the Italian population, is based on 97 deaths. These exceed the rate of total mortality by 41%, for tumors by 65% and for respiratory tumors by 102%. There is a marked excess of mortality by primary pleural tumors (based on 4 deaths) and by primary peritoneal tumors (based on 2 cases). In recent years we have become aware of a serious public health problem: ships insulated with asbestos and scheduled to be demolished are being sent to the “southern part of the world”, particularly to India [120]. Of the approximately 45,000 ocean-going ships in the world, about 700 are taken out of service every year. In the early 1970s, shipbreaking was a highly mechanized industrial operation carried out in the shipyards of Great Britain, Taiwan, Mexico, Spain, and Brazil. Rigorous environmental and health and safety standards have shifted this activity to poorer Asian nations. It is estimated that over 100,000 workers are employed at shipbreaking yards worldwide. By the end of the 1990s, 70% of ships were being scrapped in India. Studies conducted in the area of Hamburg, Germany, site of the asbestos textile industries and civilian and military shipyards, have identified progressively greater cases of mesothelioma [121], dozens of which are related to shipyards. Cases of mesothelioma in subjects not directly exposed have been noted also in family members of asbestos insulators. In Holland, in 1958, Van der Schoot published a study of three cases of malignant mesothelioma in individuals working in the sector of shipyard insulation [122]. Stumphius [123] writes that he has identified asbestos corpuscles in approximately 60% of the employees of a shipyard on the isle of Walcheren whom he had examined. He presents a case study of 25 cases of mesothelioma, identified in the period 1962-1968, for which he reconstructs the work place and the presence of exposure to asbestos: these are subjects who

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lived near the shipyards and 22 had worked in the naval shipyard of Royal Schelde in some capacity. Concerning Great Britain, an initial survey of cases of mesothelioma in shipyards dates to 1958 [124] and refers to 13 mesotheliomas, observed in the shipyards of Belfast. The author reports of an increase in the problem and in a subsequent article the cases had become 45 [125]. The deceased had asbestos corpuscles in the lungs while the presence of pulmonary asbestosis was infrequent. Examples of other cases of mesotheliomas are provided by McEwen, who reports on 83 cases of mesothelioma observed during the period 1950-1967 in Scotland [126] and the extensive study by Wagner on 622 cases of mesothelioma, with exposure to asbestos occurring prevalently in shipyards [127].

Construction, Maintenance, Restoration and Repair of Railway Carriages The evaluation of cases of mesothelioma in this sector can benefit from previous studies on the use and methods of use of asbestos in the rail sector and the results of several epidemiological studies. [64, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143]. The most extensive description of cases of mesothelioma resulting from the use of asbestos in steam locomotives was conducted by Mancuso and colleagues on railway workers in the United States during the 20s. During the period between the two world wars, the asbestos used in railway carriages in Italy was contained in objects: belts, ropes and cartons containing chrysotile asbestos in steam coupling devices (pipes bringing the steam from the boiler or heater to the wagon), in the pipes used to convey steam located in the underlying steam boxes and, in the interior of the wagons, to the hot air vents (heating vents) located under the passenger seats or to the backrest in some types of wagons. In electrical locomotives, asbestos was present in the contactors, in the rheostats, as sheathing for the cables subject to heat stress and in the heaters located in the conductor’s cabin. This first exposure to asbestos in Italy therefore took place in the sites in which the locomotives and wagons were built. Only later did the railway company use asbestos for repairs and maintenance requiring small, medium and major interventions. The great change in Italy occurred with the decision of the State Railway System in 1956 (FFSS), that would later lead to similar decisions by private railway companies, to use trains with a body insulated with crocidolite mixed with water and vinyl glue, applied by sprayer. It is interesting to note that the use of asbestos in railroads was not limited only to Italy: in fact cases of mesothelioma or epidemiological studies report on its use and describe its effects in several European countries (Switzerland [144], Scandinavia [145], England [51], Germany [81], Holland [146]) and non European countries (Australia [50], South Africa [64]). What is original is that the consequences of the decision to insulate using spray applied crocidolite were not evaluated in Italy until the 80s, an aspect that differentiates Italy from other countries.

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A useful comparison in this respect is found in England. Descriptions of environmental surveys indicate that railways in England began to be insulated using spray applied asbestos from the end of the 30s, a technique that was simultaneously much used in ship construction. Spray applied insulation was patented and immediately implemented upon issue of the first English law protecting from asbestos in 1931. This method of application however was not governed by any regulation. The English Labor Inspectorate (HSE) responsible for monitoring enforcement decided to undertake environmental measurements to assess the intensity of exposure considered to be high and ordered employers to provide safety devices for the respiratory system not only for insulation workers but also for other workers located in their proximity. The extension of the safety measures to “bystanders” worried employers and led to a contrast that would be resolved only with new legislation on asbestos in 1969 [147]. Regarding work with the risk of exposure to asbestos in the field of transportation, railway machinists were among the first subjected to exposure of sprayed insulation easily accessible from the interior of the conductor’s cabin. Among those tasks with the most direct exposure in rail construction and repair are those of boiler operator and pipe-fitters, employees who worked on the steam pipes extending from the boiler wagon to the passenger wagon. Riggers, pipe-fitters and electricians were involved in the systems installation and furnishings of new trains. Panel installers, carpenters, upholsterers, plumbers and electricians were all subject to direct exposure during maintenance and repair operations. As for indirect exposure the list is much longer, as work sites in which rail repair and maintenance operations were conducted had no separations or measures to contain exposure, thus leading to widespread contamination of the entire hangars in which the work took place, a judgment confirmed by the results of some environmental measures.

Personnel Travelling in Means of Transportation Insulated with Asbestos: Ships and Trains The exposure to asbestos suffered by embarked personnel is a consequence of the vast use of asbestos for several decades by the ship building industry undertaking marine construction, repair and restoration. Occupational exposure to asbestos by embarked naval personnel coincides with the beginning of the industrial use of asbestos, at the end of the 1800s. More specifically, the modern era of asbestos use can be dated to an article appearing in the prestigious review The Engineer on 22 June 1883, titled “Asbestos and its applications“ [148]. In this article the writer refers to an initial industrial application of asbestos introduced by John Bell in 1879, by insulating steam engines with material containing asbestos. This application found immediate use in the navies of Great Britain and Germany. The use of asbestos was extensive in ship building since the end of the 1800s. It was especially widespread in the navies because of its thermal insulation and fireproofing properties. Asbestos made it possible to create areas inside ships that could be insulated in case of fire, by using special firewalls; other objectives were also attained, such as thermal and acoustic insulation of engine rooms and related heating systems [149]. During the First World War the use of asbestos greatly increased in the navies and merchant marine worldwide.

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118

It should finally be noted that exposure to asbestos by seamen was prolonged and went beyond simple occupational exposure since work environments were the same as living environments, providing a 24hours exposure. Concerning the installation of asbestos in the hull of the ship, with the consequent risk of occupational exposure, we refer to the Italian document developed by the Coordinamento per la Prevenzione nei luoghi di lavoro delle Regioni e delle Province Autonome di Trento e Bolzano dated 30 August 2000 [150]. Table 3 illustrating the distribution of materials containing asbestos in the structure of the ship shows the vast extent of its use within the entire ship: Specifically, embarked personnel were subjected to the greatest exposure in the following compartments: (a) Lodgings for on board personnel: upon conclusion of work hours embarked naval personnel remained exposed to the risk of asbestos even in the “living environment”. (b) Engine room: particularly significant exposure due to the massive use of materials containing asbestos as insulation for engines and also because of the generally confining and insufficiently ventilated space of the engine room. It should also be borne in mind that the vibrations typical of an engine room is a factor that strongly conditions the dispersion of asbestos fiber from the matrix. (c) Passenger lounges: the activity of steward could also expose to the risk of occupational inhalation of airborne asbestos fibers. The first indications of the risk of asbestos for seamen in Italy are attributed to Puntoni et al [151] as part of a study of port workers in Genova and to Bianchi et al [152]. These writers describe three cases of pleural mesothelioma in embarked subjects on different types of ships, and in discussing the cases point out three aspects: exposure takes place especially during the phases of inspection, maintenance and repair; the fact that exposure is not massive nor continuous does not eliminate the risk; engine room personnel are the most exposed. In the U.S., Jones et al [153] examined the chest x-rays of over 5,000 naval machinists, observing pleural alterations caused by asbestos in 12% of the subjects; the prevalence of these contexts was higher (27%) among those who had been embarked for a greater number of years. Also in the U.S., Selikoff et al [154] studied more than 3,000 seamen and observed pleural or pulmonary alterations caused by asbestos in 35% of the subjects. The prevalence of these alterations was greater in engine room personnel, intermediate in deck workers and lesser among stewards. Confirmation was provided by a smaller study conducted in Greece [155]: in a group of 141 seamen, 41% had at least one radiological sign of damage from asbestos. Table 3. Material with the possible presence of asbestos inside ships [150] Area

GARAGE

Material, equipment, furnishings Ceiling and/or covering of room and perimeter walls Pipes Conduits for electrical lines Doors

Type of material containing asbestos Flocks, Sprayed, Panels Cement mixture, Fabric, Gaskets, Cushions Plaits, Packing, Stucco, Cement mixture Panels, Cement mixture

Epidemiology of Mesothelioma: The Role of Asbestos

CORRIDORS AND STAIRS

LODGING FOR EMBARKED PERSONNEL

PASSENGER LOUNGES

Walls False ceiling Doors Ventilation ducts Pipes Cabin walls False ceiling Doors Ventilation ducts Pipes Conduits for electrical lines Walls Ceilings and false ceilings Doors Ventilation ducts Pipes ( hot water, washing, fire prevention, etc..) Conduits for electrical lines Engine gas exhaust collectors

ENGINE ROOMS AND AUXILIARY MOTORS

Smoke exhaust pipes for boilers and incinerators Pipes (steam, hot water, fire prevention, etc.. ) Conduits for electrical lines Ventilation ducts Reefer cells Kitchen

MISCELLANEOUS Ovens and stoves Ventilation ducts Pipes

119

Sprayed, Panels, Cement mixture Sprayed, Panels, Cement mixture Panels, Cement mixture Gaskets, Plastering Cement mixture, Gaskets Sprayed, Panels, Cement mixture Sprayed, Panels, Cement mixture Panels, Cement mixture Gaskets, Stucco Cement mixture, Fabric, Gaskets Plaits, Packing, Stucco, Cement mixture Sprayed, Panels, Cement mixture Sprayed, Panels, Cement mixture Panels, Cement mixture Gaskets, Stucco Cement mixture, Fabric, Gaskets Plaits, Packing, Stucco, Cement mixture Insulation cushions, Fabric, Mattresses, Cement mixture, Gaskets Insulating cushions, Fabric, Mattresses, Cement mixture, Gaskets Cement mixture, Fabric, Plaits, Gaskets Plaits, Packing, Stucco, Cement mixture Gaskets, Stucco Sprayed, Panels, Mattresses, Cement mixture Sprayed, Panels, Cement mixture Panels, Plaits, Stucco, Slabs of asbestos cement Gaskets, Stucco Cement mixture, Gaskets

The first quantitative sign of the risk of mesothelioma among seamen is based on the seven cases observed in a study of an English cohort of 13,000 officers and seamen of the Navy, versus two expected cases [156]. In Italy a study of a cohort of 984 seamen in Civitavecchia, embarked at least once between 1936 and 1975, displayed a significant increase in mortality by lung tumors, with a trend associated with the duration of employment as seaman; one subject died of mesothelioma of the pleura [157]. On the basis of all the data that emerged during the decade 1980-90, Greenberg [158] affirmed that the massive quantities of materials containing asbestos on ships, also subjected to continuous vibrations and mechanical stress, was the cause of the frequent radiological alterations observed in seamen and of the increased risk of cancer that was beginning to become obvious. The Australian Registry of Mesotheliomas (1945-2000) reports 224 cases of seamen out of a total of 3,008 activities involving exposure to asbestos [31]. In Finland [159], a case-control study of 10 cases of mesothelioma observed in a cohort of 30,000 seamen embarked during the period 1960-80, revealed a significant increase in the

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risk associated with the activity of machinist (OR –Odds Ratio- 7.50, conf. int. 95% 1.53-36.7 with a 10 year latency; OR 9.75, conf. int. 95% 1.88-50.6 with a 20 year latency). In Sweden [160], the Standardized Incidence Ratio (SIR) for pleural mesothelioma for seamen in the period 1961-1998 was 2.83 (conf. int. 95% 1.41-5.09).

Industrial and Civilian Construction Work The use of asbestos in the construction sector came about because of its thermal and acoustic insulation properties and the fact that it was fireproof. This particular use was extensive in North America (Canada and U.S.) and was applied using the crocidolite spray method [161]. The percentage of asbestos used in construction products varied from approximately 20% for floors to approximately 80% for insulation of heating pipes [162]. The considerable quantity of material containing asbestos in the Twin Towers of the WTC in New York caused significant environmental contamination from asbestos fibers after the September 11 attacks. [163] Epidemiological studies have progressively highlighted the risk of mesothelioma in workers in the construction sector [164]: we must note in fact that the significance of this occupational exposure is also in relation to the large number of construction workers, even though the concentration of asbestos fibers does not reach the same levels as in the shipyard and railway sectors. We estimate that in the U.S. construction workers form the largest group of workers occupationally exposed to asbestos. Baker assessed that in 1980 the number of construction workers exposed to asbestos was approximately 7,500,000. [165]. Additional epidemiological studies conducted by Robinson et al [166] on a cohort of 31,068 electricians in the construction sector revealed an excessive level of mortality by mesotheliomas. Similar data was found by Peto in the United Kingdom, who also indicated that this category will have the greatest increase of mesothelioma cases in the near future [167].

Textile Sector An assessment of the risk associated with occupational exposure considered potentially carcinogenic is provided by the International Agency for Research on Cancer (IARC). The IARC included the activity of the textile sector in group 2B [168] on the basis of the limited evidence of carcinogenicity provided by available studies. This assessment is based on the increased incidence of bladder cancer among dyers and millers (azoic dyes) and of carcinoma of the paranasal sinuses among weavers (dust from fibers and fabric). A case review by the Sloan-Kettering Cancer Center in 1982 evidences the appearance of mesotheliomas in textile workers that is not, however, associated with a possible exposure to asbestos [169]; In the past, with the exception of the infrequent production of fireproof fabrics (containing asbestos), the asbestos risk in the textile industry has never been sufficiently studied.

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Eventually, however, some studies began to document a possible risk of occupational exposure to minerals for workers of this sector: −

−

−

−

−

studies conducted in the city of Prato (Tuscany) drew attention to an increased risk of lung cancer among the fabric sorters (exposed in the 50s) and weavers (employed in the 70s), suggesting asbestos and mineral oils, respectively, as carcinogenic factors [170] and to an observable cluster of mesothelioma cases associated with re-use of asbestos bags [171]; some cases of malignant mesothelioma (MM) appearing among silk spinners in Lombardy may have been caused by exposure to asbestos dispersed into the air by the insulation and fittings on pipes [172]; in Piedmont there was a case of mesothelioma in a worker assigned to maintenance of pipes bringing water and steam to the dying and finishing sections in the textile industry [173]; the Mesotheliomas Registry for Tuscany indicated the need to further study possible exposure in the textile department, as subjects who perform different tasks may be affected [174]; some parts of textiles machinery (ferrules, gaskets) may be points of potential dispersion [175].

A recent review of cases was conducted by the Mesothelioma Registry for Lombardy [176]. The ReNaM archives contain 90 cases of malignant mesothelioma from occupational exposure in the textile sector, excluding workers in weaving and spinning of fireproof materials containing asbestos. The anatomic location of the disease that is most involved is the pleural area with 88 cases (97.8%). There are also 2 cases involving the peritoneum (2.2%) and none for the pericardium and the tunica vaginalis testis. The clinical diagnosis according to the ISPESL criteria [177] was certain in 80 cases (88.9%), probable in 7 cases (7.8%) and possible in 3 cases (3.3%). Occupational etiology was certain in 49 cases (54.4%), probable in 15 cases (16.7%) and possible in 26 cases (28.9%).

Installation and Repair of Lifts The braking systems on lifts have lining with an asbestos content. When in operation, asbestos fibers are released and collect on the floor of the elevator booth. There is exposure to asbestos during the installation and maintenance and repair phase of lifts. Cases of mesotheliomas among lift workers were observed by Huncharek [178] and colleagues and by Tonut [179]. Epidemiological studies showing a significant incidence of malignant mesothelioma in lift workers were conducted by Bruno and colleagues in 1999 [180].

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ENVIRONMENTAL EXPOSURE Even since the initial indication [64] of a relationship between asbestos and malignant mesothelioma (MM) the substantial risk of exposure linked to the environment in a wider sense has also emerged. This includes: • •

paraoccupational exposure from matter released into the surrounding environment by mining and processing systems; residential exposure from the presence in the immediate vicinity of the house or inside the house of materials contaminated by asbestos, co-habitation with workers exposed to asbestos, the performance of daily activities, including play by children, using items containing contaminated materials.

In the original series of 33 cases of mesothelioma observed by Wagner and colleagues, circumstances of non-occupational exposure were the only ones present in the anamnesis of 14 cases. Since that time, the epidemic of mesotheliomas in the crocidolite mining areas of South Africa has provided additional proof of the consequences of environmental exposure [181]; furthermore, the mining area of Wittenoom Gorge in Australia, where crocidolite is extracted, was the object of extensive studies to investigate the dose-response relation at different levels of environmental exposure, thanks to the availability of environmental measures [82]. In a completely different context, the inquiry conducted in London a few years after Wagner’s report used a case-control approach to highlight the high risk of MM from exposures defined as “neighboring” [182]. Overall, 9 of the 76 cases for which a complete history was available had as the sole significant condition a relative who had worked in the asbestos industry, and an additional 11 had lived within half a mile from an asbestos industry. The description of the epidemic of MM linked to environmental exposure to erionite, in the villages of Karain in central Anatolia, Turkey [183,184] became a turning point in the perception of the problem of non occupational exposure to asbestiform fibers. In the case of erionite, a mineral without any commercial or industrial use, the cases observed could not be etiologically attributed to occupational conditions. Almost simultaneously another group of cases was described in the population of Tuzkoy, about 50 km from Karain [185], in an area also contaminated by erionite, and another aggregate in the population of Cermik, in southeast Anatolia [186], this time in relation to the use of local materials strongly contaminated by fibrous tremolite, used as plaster. More recently, numerous works [187,188,189,190] have allowed for updating of the incidence of mesothelioma by environmental exposure to asbestos, in the areas of eastern Anatolia. Epidemics with characteristics very similar to the above have been described in Greece [191], Cyprus [192], Corsica [193], New Caledonia [194], China [195], Italy [196], Montana and California [197]. Three systematic reviews [7,198,199] have recently summarized the current state of knowledge, even though the results of the study on the residents of Wittenoom, cited above, are not included. These reviews emphasized the part played by today non occupational exposure in explaining a significant number of the cases of MM observed, and the fact that

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the strong association between these exposures and the risk of MM is not a modest one. It was also pointed out that lacking appropriate countermeasures the exposures could prolong the epidemic of MM in countries in which there is no longer any industrial use of asbestos. The contribution of environmental and familial exposure to the overall incidence of malignant mesothelioma is certainly not a negligible one, especially among women [111]. The risk to which subjects are exposed can reach levels comparable to those of occupational cohorts [200]. Of the 5,173 cases found in the ReNaM Archive [201] there is currently sufficient information to evaluate the exposure for 3,552 cases, approximately 69%. In 302 cases, equal to approximately 9% of cases with defined exposure, a familial or environmental exposure has been determined, in the absence of any occupational exposures. Of a total of 187 cases of environmental exposure attributed to the cases classified as having an environmental etiology, one third (76) are attributable to proximity to plants that produce asbestos cement items. There are also various periods of habitation in the proximity of shipyards and ports (16 periods), iron and steel industries/foundries, chemical/petrochemical plants, electrical plants (15), railway lines (12), asbestos textile industries (9). In general, the production sectors most frequently associated with environmental cases of MM have also used amphyboles (amosite and crocidolite). Nevertheless, the most widely used type of asbestos was certainly chrysotile and we cannot ignore that there is epidemiological evidence of an increased risk of mesotheliomas caused by environmental contamination induced by the extraction and processing of chrysotile [202]. One highly important case was discovered in the town of Biancavilla, Italy, where there was such a high concentration of deaths by malignant tumor of the pleura that an investigation was conducted, confirming many cases as MM and identifying the etiology of the epidemic in the exploitation of materials from a local quarry, contaminated by a fibrous asbestiform mineral not previously known, fluoroedenite [203]. The potential of injury to one’s health caused by exposure to asbestos linked to natural sources in the living environment, when such sources are disturbed by some anthropical activity, was also demonstrated by two inquiries conducted on a small aggregate of cases in two areas distant from each other, one in Piedmont and one in Basilicata, but associated by the presence of serpentinites contaminated by tremolite [202,204]. The latency, measured between the beginning of the first exposure and diagnosis, goes from a minimum of 6 years up to 84 years, with an average of 46.5 (±14.7). The duration of exposure varies from 2 to 84 years, with an average of 32.1 (±19.6). Of 144 cases, 137 (67 men and 70 women) had a pleural localization, 6 (3 men and 3 women) peritoneal, and one pericardium. Cases with a non pleural localization are rare and analytic studies to determine environmental risk have not been conducted, even though there are well documented cases [205]. In many, if not all, areas in which a significant share of environmental etiology has been noted, there have also been cases of domestic origin, caused by one or more family members working in the asbestos industry and bringing fibers into the home, on their work clothes or other items [111]. In the ReNaM Archives their number is equivalent to the number of environmental contaminations. However, there is a clear predominance of women among the familial cases,

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compared with the substantial equilibrium between the two sexes in the environmental cases. Another characteristic of familial exposures is the wider range of work processes to which they are associated: production of asbestos cement (22 work periods), asbestos weaving (9), naval construction and repair (21), railway construction and repair (11). More than half of the work periods involving exposure referred to activities and tasks in which asbestos was not present as a raw material, but was installed on systems, such as in the chemical, rubber and sugar refining industry (10 periods of work), in the production of electrical energy (6), in iron and steel industries and foundries (9), in textiles (8), railways (8), and in material or objects of more or less widespread use, as in construction (18 periods of work), installation and maintenance of electrical or hydraulic machinery and systems (11) and vehicle production (5). Latency, measured from the beginning of the initial exposure to actual diagnosis, extends from a minimum of 19 years to 86 years, with an average of 47.8 (±14.6). The duration of exposure varies from less than one year to 78 years, with an average of 21.6 (±13.5). Of 150 cases, 146 (23 men and 123 women) had pleural localization and 4 (1 man and 3 women) peritoneal. The cases with peritoneal localization all have a histologically confirmed diagnosis, with certain mesothelioma in two cases and probable mesothelioma in another two. The occupation of the relatives exposed were, respectively, a cast iron foundry, forging, construction and glassworks. Latency could be estimated only for two cases, and was very long: 49 and 70 years.

Urban Environment Some authors have debated the existence of an “urban factor” for mesothelioma (Hemminki et al 2003 [206], Polednak 2003 [207]) in light of the extensive use of asbestos in construction over the past decades, the aging of buildings, the increased release of fibers from the cement matrix caused by the deterioration of the structures, and especially the massive release of spray applied fibers (Marconi et al 1987 [208]). In a large city like Rome, there are more cases of mesothelioma without occupational exposure to asbestos than in the surrounding areas, with a greater proportion of women, compared to the rest of the Latium Region (Ascoli et al 2004 [209]). This observation could be explained by the greater number of subjects exposed to asbestos in the homes and offices of a metropolitan area compared with the surrounding territory.

CONTRIBUTION OF THE EPIDEMIOLOGY OF MESOTHELIOMA TO PUBLIC HEALTH INITIATIVES In light of the above, we believe epidemiological studies of mesothelioma serve a dual purpose, in accordance with the evolution of prevention policies in the different countries. The two circumstances to be kept separate are those in countries that have stopped using asbestos and those that still use it. Unquestionably, epidemiological surveillance of mesothelioma as a whole and consequent ascertainment of the medical impact of asbestos are

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more evolved in countries that have banned this agent compared to those that still allow its extraction and use in work places and in daily life. [210]. In the more advanced countries that had made wide use of asbestos in the course of the XX century and banned it in the past few decades, epidemiological studies of mesothelioma determine the priorities to be followed in environmental reclamation and identify the groups at high risk who require greater protection.

Environmental Reclamation and Identification of Groups at Risk Determination of priorities for environmental reclamation must generally consider the foreseeable number of cases, estimated according to the number of subjects exposed, the level of exposure and the course of exposure over time, as well as the different types of exposure, in other words, identification of those sections of the population experiencing the highest level of exposure in the absence of appropriate mechanisms providing legal and social protection or simply, access to information [211]. This establishes a link between identification of groups at risk through epidemiological observation, and the consequent implementation of environmental reclamation procedures. This course of action requires a high level of integration among medical and environmental operators, cooperation between scientists and administrators and the involvement of the communities affected, leading to less arbitrary decision-making processes founded on scientific evidence and, in the end, appreciably more equitable.

Prevention of the Asbestos Pathology in Developing Countries The role of epidemiology in countries that still allow the use of asbestos, mostly located in developing areas of the world, is more complex. Here the medical impact of asbestos is often denied, or underestimated, for two principal reason: the active role played by the asbestos industry in influencing the scientific community, government bodies and workers’ organizations, including through the contrived re-proposition of scientific controversies of little significance for the implementation of appropriate preventive measures [212], and the inadequacy of epidemiological surveillance systems considering the relatively brief time that has passed since the initial widespread diffusion of asbestos and the consequent non attainment of sufficient latency [213]. Nonetheless, there is such a huge amount of epidemiological studies and prognostic models of the epidemic curve, that it is possible to imagine future scenarios even in these regions of the world. If current methods of using asbestos remain in effect and only limited measures of prevention are implemented, the medical impact for workers and for the general population will be dramatic. There are no alternatives to a complete ban on the use of asbestos [214]. This conclusion, advocated by minority, though highly qualified, voices in the past (Collegium Ramazzini 2004) [215] has also recently been reached by the World Health Organization [216], whose strategy to eliminate asbestos pathologies includes banning the use of this agent, encouraging the use of alternative materials, prevention of exposure during reclamation and the improvement of early diagnostic procedures, medical treatment, rehabilitation and social support to the afflicted. To this end, the WHO recommends the implementation of national plans to eliminate diseases caused by asbestos by promoting

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training, cooperation among the institutions involved and, in the final analysis, an increased awareness of the problem.

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In: Mesothelioma from Bench Side to Clinic Editor: Alfonso Baldi, pp. 25-66

ISBN: 978-1-60021-789-0 © 2008 Nova Science Publishers, Inc.

Chapter 6

ASBESTOS LITIGATION: PROSPECTS FOR LEGISLATIVE RESOLUTION *

Edward Rappaport Analyst in Industry Economics and Finance Domestic Social Policy Division

ABSTRACT A large volume of litigation has been occasioned by occupational exposure to asbestos, which may ultimately result in payments of $200 billion or more and has already bankrupted numerous companies. This litigation “explosion” has led to a number of innovations in legal process, but some of the more comprehensive settlements were overturned by the Supreme Court, with the Court suggesting that the situation “calls for national legislation.” One approach proposed in the 109th Congress, embodied in H.R. 1957 (Cannon et al.), would conserve the resources of defendant corporations — many of which have been bankrupted by asbestos cases — so that funds could be applied first to workers who are already sick. This would be done by establishing precise standards for proving the presence of asbestos disease for legal purposes, and postponing the cases of those who might show evidences of exposure but are not yet impaired. H.R. 1957 would also apply to the asbestos problem a number of principles known under the more general rubric “tort reform.” The bill receiving the most attention in the 109th Congress, S. 3274 (Specter and Leahy) would also establish standards for proving injury but, instead of taking the tort reform approach, would remove the cases from the court system entirely. In its place would be an administrative system and a special fund to pay claims. S. 3274 would define nine asbestos disease categories, each with a specified level of compensation, ranging from $25,000 to $1.1 million. The fund from which claims would be paid would be financed by assessments on defendant companies and their insurers. Each of the largest firms subject to assessment would be responsible for paying up to $27.5 million per year for up to 30 years, with an overall funding goal of $140 billion. The most debated points of S. 3274 have included the adequacy of the funding scheme, the levels of compensation, medical criteria (especially as regards smoking history), and start-up and close-down issues. This report discusses such issues thematically, and will be updated to reflect major legislative actions. A section-by-section analysis of S. 852 (the predecessor version of S. 3274, as reported by the Judiciary Committee) may be found in CRS Report RS22081, S. 852: The Fairness in Asbestos Injury Resolution Act of 2005, by Henry Cohen. *

Excerpted from CRS Report RL32286, dated August 23, 2006.

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Asbestos has been widely used as an insulation material, friction product (e.g., in brakes and clutches) and textile reinforcement, due to its unique combination of strength, flexibility and resistance to heat and corrosion. Over the years, scientific studies have increasingly implicated the material as a cause of debilitating, fatal lung diseases. Protective standards have been adopted and progressively tightened, but human exposures continue to occur through ongoing use and from legacy buildings and equipment.[1] Moreover, cases of asbestosis, lung cancer and other diseases will be emerging for years to come because they occur after a long latency. Although most cases of asbestos-related disease have occurred from occupational exposure, few of the affected workers have been able to obtain medical and financial assistance from their employers under state workers compensation law. (This was due to various legal hurdles, such as time limitations, and the difficulty of proving that lung cancer was caused by employment.) However, many have successfully sued the manufacturers of asbestos under claims of products liability, to such an extent that many large firms have been forced into bankruptcy. This litigation “explosion” has led to calls for legislation that would expedite the settlement process through administrative alternatives. The legislation that has advanced the farthest is S. 852, which was marked up by the Judiciary committee on 26 May 2005 and debated in the Senate during 7 to 14 February 2006. A budgetary point of order was raised by Senator Ensign and sustained by a vote of 58 to 41 (60 being needed to proceed further with the debate), sending the bill back to committee. In May 2006, a revised version reflecting the managers’ amendment and other proposals raised in the floor debate was introduced as S. 3274. This report describes how the asbestos litigation process has evolved, and then discusses the legislative “fixes” that have been tried or proposed. The discussion is thematic, highlighting the sub-issues remaining most in dispute. For a section-by-section explanation, see CRS Report RS22081, S. 852: The Fairness in Asbestos Injury Resolution Act of 2005, by Nathan Brooks.

SCOPE OF LITIGATION It is estimated that at least 730 thousand people had brought asbestos-related personal injury suits against more than 8,000 defendants by the end of 2002, and the number of new claims each year appears to be still increasing.[2] Typically, each plaintiff sues dozens of defendants, so the total volume of litigation is quite substantial. The total amount spent on asbestos litigation (awards and expenses) has been on the order of $70 billion, most of this expenditure being financed by defendant companies and their insurers. The total ultimate bill may be on the order of $200 to 250 billion. The amounts awarded in individual cases are difficult to estimate, as most are resolved confidentially by settlements, but among cases that have gone to trial and succeeded,[3] the average award has been about $1.8 million. Negotiated settlements tend to be considerably less, however. Minus the legal expenses of both plaintiffs and defendants, about 42% of total spending has been reaching the claimants as their net recovery.

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The resulting liabilities have forced some 73 companies into bankruptcy in the last 30 years, 37 of them since January 1, 2000. Among the most prominent of these firms are Armstrong World Industries, Babcock and Wilcox, Federal Mogul, Johns-Manville, OwensCorning, U.S. Gypsum and W. R. Grace. Bankruptcy is not a desirable outcome for either the defendant firms or the claimants. Claims can be put on hold for five years or more, and in some cases the trusts established to take care of victims have been able to pay only 5% to 10% of what was expected. A subsidiary question is the extent to which defendants can rely on their insurance companies to cover their liabilities, an issue that is occasioning substantial litigation of its own.[4]

PROCEDURAL IMPROVISATION The unprecedented scale of litigation has induced courts and the parties to develop new structures for resolution of cases. Whereas, at first, defendants vigorously contested such issues as whether a worker was “injured,” whether the cause was asbestos exposure, and which manufacturer’s asbestos was the particular asbestos at fault, by the 1980s new court procedures and decisions were establishing clearer bases for liability. Some judges encouraged consolidation of cases, for example, by selecting a few individual cases to go to trial as representative of the whole. Defendants found that their best opportunity was to negotiate settlements through attorneys representing thousands of claims at a time, with the amounts for each individual to be determined by schedules of factors such as disease type. By the 1990s, the leading law firms representing claimants had standing agreements with the major defendants for settling claims (though that system has since lost much of its viability). The bankruptcy courts have been a notable forum for resolving cases en masse, beginning with the pathbreaking Manville Trust.[5] In 1988, after six years under court supervision, Johns-Manville Corp. emerged from bankruptcy 50% owned by a trust charged with compensating current and future asbestos liability claimants. Administrative procedures were developed to streamline claims handling. The trust’s operating expenditures are only 5% of benefits paid, and lawyers representing claimants cannot charge more than 25%. Thus, claimants receive 70% of what the trust pays out. Unfortunately, though, the amounts paid are quite low, since the assets of the trust have only been adequate to pay 5% to 10% of full value. The system became a model for other, solvent companies. Congress also codified the process for a bankrupt firm to resolve its liability for all pending and future claims via such trusts.[6] In short, some observers believe that through such innovations “asbestos litigation was transformed in fact — although not in form — into a quasi-administrative regime.”[7] Much of the resulting concepts and language feed into the legislative measures discussed below. Most recently, some corporations, including Halliburton, Honeywell and the Europeanbased manufacturer ABB, have presented plans by which claims are to be resolved by the bankruptcies of their subsidiaries rather than the parent corporation, which would then be able to carry on freed of asbestos liabilities. This would make use of the 1994 bankruptcy law amendment, but leave the parent corporation solvent and still in control of its operations (unlike the Manville model, which put control of the whole corporation under the trust).[8]

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Finally, many had expected eventually to come to a final resolution of most cases by “global” settlements. However, the two prominent asbestos settlements that were fully litigated up to the Supreme Court were overturned there.[9] The key features of the Georgine settlement were (1) definitive criteria for proving exposure and illness, in a simplified and expedited process, (2) standardized compensation for actual illness only, (3) preservation of the right to compensation later if disease (or worsened disease) occurs later, (4) a cap on attorney fees, and (5) a limited right to opt out and rely on one’s ordinary right to sue. These settlements were rejected for not meeting the requirements for establishing class actions under Federal Rule of Civil Procedure 23. Georgine was found wanting because various subgroups of claimants (and potential claimants) were in widely varying circumstances, so that common elements did not predominate among their cases. Also, adequate representation was not broadly enough assured, especially for those who might become aware of their injury only in the future. These defects were not adequately overcome by the agreement’s provision allowing potential plaintiffs to opt out. The Ortiz class was established under a different subsection of Rule 23 that did not require meeting such criteria, but the Court said it had not been demonstrated convincingly enough that the settlement qualified for this alternate rule subsection (assets of defendants insufficient to meet liabilities). What was notable about these cases is that members of the Supreme Court expressed discomfort with having to reject settlements with some merit for not meeting the detailed requirements of federal court procedure (which, of course, has its own merits). As stated by Justice Ginsburg in the Georgine case, “Rule 23, which must be ... applied with the interests of absent class members in close view, cannot carry the large load ... heaped upon it.” More pointedly, Justice Souter in Ortiz commented that “this litigation defies customary judicial administration and calls for national legislation.” Thus, each of several hoped-for routes toward resolution — bankruptcy court, class actions, or consolidation of individual cases in one court (which is possible for federal court cases) has run into significant impediments in recent years. At the same time, differences have emerged between claimants who are critically ill and others who may be less sick (or show abnormal x-rays without apparent illness) but who sue immediately, either because of legal deadlines (“statutes of limitations”) or because they fear that funds may not be available later.

POLICY ALTERNATIVES Status Quo Despite warnings that the asbestos problem is reaching “crisis” proportions, it could be argued that the current legal regime has distinct advantages and should be allowed to proceed as it is, or with minor improvements. The current system is providing substantial assistance to large numbers of victims, most of whom do not have to pay lawyers’ fees unless and until compensation is received. From a public policy perspective, the fact that defendant companies are the ones financing the benefits may be considered broadly beneficial. That is, companies in all industries are being put on notice that allowing harm to occur to employees and the public can be fatal to their own financial well-being.

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On the other side of the ledger, the current system is not likely to have adequate resources to fully compensate all claimants. A substantial portion of the resources that are available is used to run the system rather than directly benefit claimants. It is also disorganized, with no oversight to assure that compensation is allocated primarily to those with the most compelling cases.

Changes in Tort Law Some observers see asbestos litigation as part and parcel of broader problems with personal injury litigation that justify more general “tort reform,” especially in cases with thousands of plaintiffs. Many specific measures have been suggested, such as caps on punitive damages, limitations on joint and several liability, and more narrowly specifying the court(s) in which each plaintiff can bring his/her case.[10] A tort innovation peculiar to asbestos is the “pleural registry.” In a number of states, this device enables one to make a tentative filing when one learns of one’s injury (often upon diagnosis of pleural plaques),[11] and thus meet the legal deadline even though no (or minor) impairment has yet occurred. Trial of the claim is delayed until serious symptoms occur. And if a non-malignant case is established, a potential claim for cancer would be put aside until that disease becomes evident (a two disease rule). The idea behind the postponing of cases — many of which will never progress to debilitating disease — is to allow immediate resources to be concentrated on those with the most serious immediate problems.

Medical Criteria Building on the pleural registry concept, H.R. 1957 (Cannon et al.) would hold in abeyance statutes of limitations and other time limits until such time as impairment may occur and be diagnosed. Then, before plaintiffs can proceed further with an asbestos or silica[12] suit, they would first make a prima facie case that they have a physical impairment and that exposure to asbestos has been a substantial contributing factor. The bill specifies detailed medical criteria for establishing these claims. Factors to be considered include employment and smoking history, x-ray evidence of abnormalities such as pleural thickening or opacities, pathological evidence of lung scarring, and impaired breathing shown by measures such as forced vital capacity. H.R. 1957 also incorporates a number of general tort reform features, as well as others to address the techniques of some lawyers and their medical associates who allegedly have generated thousands of questionable cases. Thus, the bill attempts to avoid diagnosis “mills” by requiring that the prima facie evidence be developed by a qualified physician who “has or had a doctor-patient relationship” with the plaintiff. Among the tort reform ideas in H.R. 1957: • •

Venue: Cases may only be brought in federal court for the district where the plaintiff resides or experienced the asbestos exposure. Consolidation of cases: Only with the consent of all parties.

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•

•

Burden of proof: It must be shown that the conduct of each defendant and exposure to each defendant’s asbestos or silica was a substantial contributing factor to the disease. Proportional liability: One implication of this provision is that full damages would not be received if some potential defendants have not been sued or have become bankrupt. Cap on non-economic losses: $250 thousand ($500 thousand in cases of mesothelioma). Also, punitive damages not available.

A similar measure — though differing in particulars — was introduced as a substitute amendment during debate on S. 852 (see infra) but tabled on a vote of 70-27.) The philosophy of “medical criteria” bills is that the court system (i.e., the federal court system) can handle the asbestos issue if cases are dealt with in an orderly, reasonable way. This means, above all, that awards be made if and when actual impairment occurs, on the basis of reliable, relevant evidence. Given these conditions, it is held that there would be no need for new governmental agencies and levies.[13]

Administrative System Two bills in the 108th Congress (H.R. 1114 and S. 2290) would have established administrative systems to settle claims along the lines of the Georgine settlement. Their intent was to circumvent the seemingly insurmountable requirements of the Federal Rules of Civil Procedure — while assuring a reasonable measure of justice for all parties. (New versions of S. 2290 have been introduced in the 109th Congress, and will be discussed extensively in the next section.) Like the other bills just discussed, H.R. 1114 (Kirk) would require all claimants to first establish that they have an eligible asbestos-related medical condition; failing that, their right to future action would be preserved until such time as impairment occurs. The unique feature of H.R. 1114 was its establishment of a new agency within the Justice Department, the Office of Asbestos Compensation (OAC). The OAC would not only determine medical eligibility, but would also take direct part in litigation and settlement. First (upon issuing a claimant a certificate of medical eligibility), the OAC, acting through a Trustee, would receive offers of settlement from both sides. The Trustee would also make offers of its own to claimants. If a claimant accepts the Trustee’s offer, the Trustee would assume the claim and pursue it against the defendants. Claimants could accept or reject any offers they wish, and for any cases not settled, either pursue a regular lawsuit or an administrative proceeding under the auspices of the OAC. A federal fund would be established for the purpose of facilitating the Trustee’s assumption of claims, with the intention of the fund breaking even financially in the long run.

THE TRUST FUND APPROACH In 2003, the Senate Judiciary Committee began an extensive series of hearings on the asbestos issue and facilitated negotiations involving groups of diverse interests. Out of this

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process emerged a series of bills. At the beginning of the 109th Congress, the new chair of the committee, Senator Specter, introduced S. 852 with seven co-sponsors of both parties. It was reported in June 2005 (Senate Report 109-97) and debated in February 2006, failing a test vote at that time. A revised version, S. 3274, was introduced in May. The House has not taken up the asbestos issue since 2000.[14] (For further explanation of specific provisions, see CRS Report RS22081, S. 852: The Fairness in Asbestos Injury Resolution Act of 2005, by Henry Cohen.) S. 3274 would facilitate the resolution of cases, not only, like the other bills, by specifying medical eligibility criteria, but also by establishing a schedule of benefits — ranging to over $1 million for mesothelioma — and a fund financed by assessments on defendant companies and their insurers, through which all claims would be paid. Each of the largest firms subject to assessment would be responsible for paying up to $27.5 million per year for as long as 30 years. The funding target is $140 billion, consisting of $90 billion from defendant companies, $46 billion from insurance companies, and about $4 billion from liquidation of existing bankruptcy trusts. The federal government is expressly excluded from any payment obligation. In what follows, we discuss S. 3274 specifically, highlighting the particular points that have been most difficult and/or still attract opposition to the bill. They fall mostly under the headings of: funding adequacy, acceptability of the compensation schedule, basis for the diagnostic categories, and start-up and close-down issues. (Most of these points, it may be noted, were addressed in the 2003 committee report on S. 1125 (108th Congress), which still constitutes a useful overview despite subsequent changes in the details.[15])

Funding Adequacy A number of unknowns mean the bill’s stated funding capacity of $140 billion, a substantial sum by any measure, may yet not suffice to pay all scheduled benefits. The Congressional Budget Office (CBO) estimated that the ultimate total claims would probably be in the range of $120 to 150 billion.[16] Combined with administrative and financing costs, revenues of at most $140 billion might not be sufficient. Moreover, some private estimates indicated that claims might be as much as double CBO’s figures.[17] The bill features a fixed schedule of benefits, while the adequacy of funding is addressed through a number of contingency measures (e.g., a “guaranteed payment account”). The revenue side of the equation thus becomes a bit complicated. In any case, it should be recognized that the “headline” figure of $140 billion is a goal or estimate rather than a fixed mandate. Actual assessments on defendant companies will be determined by their assignment into tiers and sub-tiers, these being defined by the companies’ historical asbestos payments and recent (2002) sales revenue. Annual assessments (for 30 years) will range from $27.5 million (a company with historical asbestos payments greater than $75 million and falling within the top quintile of these companies by revenue) down to the smallest assessment, $100 thousand (a company with asbestos payments of $1 - $5 million and revenues in the smallest third of these companies). There is a blanket exemption for small businesses (as defined by the Small Business Act, 15 U.S.C. 632) and various “inequity adjustments.” The bill also requires $46 billion from the insurance industry, but leaves the allocation among companies to a special commission (Subtitle II B).[18] On the expenditure side, some flexibility could be

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provided by annual financial reviews, which would lead to reevaluation (and tightening) of diagnostic criteria if the number of claims in any category differs greatly (25% +/-) from CBO projections. The debate in the Senate in February 2006 ultimately turned on the financial question. A point of order was raised and sustained that CBO’s projections indicated at least the possibility of expenditures exceeding revenues in periods after the first 10 years of operation. The bill provides, however, that if funding proves inadequate, the program will be terminated and cases revert to the court system. This question of “sunset” is dealt with in the final section of this report.

Compensation Adequacy The diagnostic categories and their compensable amounts are shown in table 1. (The amounts are in 2005 dollars and are to be adjusted for inflation in future years.) At least three types of consideration have guided the development of these numbers: (1) the pattern of awards given by courts or agreed in settlements, (2) the severity of symptoms and prognosis for each category, and (3) the likelihood that asbestos is the principal cause of disease. For example, non-lung cancers (Level VI) are paid less than one-fifth of what is paid for mesothelioma (Level X). This is not only because mesothelioma is one of the most lethal of cancers (usually resulting in death within 18 months), but also because mesothelioma is almost always caused by asbestos.[19] (Diagnosis categories are discussed in more detail in the next section.) Table 1. Asbestos Disease Categories and Compensable Amounts Level I II III IV V VI VII A VII B VII C VIII A VIII B VIII C IX

Disease or Condition Asbestosis — normal lung function Mixed disease (asbestosis + other) with impairment Asbestosis — TLCa 60-80% Severe asbestosis — TLC 50-60% Disabling asbestosis — TLC < 50% “Other” cancers (non-lung) Lung cancer with pleural disease — smokers — former smokers — non-smokers Lung cancer with asbestosis — smokers — former smokers — non-smokers Mesothelioma

Award Amount Medical monitoring only $25,000 $100,000 $400,000 $850,000 $200,000 $300,000 $725,000 $800,000 $600,000 $975,000 $1,100,000 $1,100,000

Source: S. 852, Sections 121(d), 131. a. TLC means Total Lung Capacity. For full diagnostic descriptions, see bill, subsection 121(d).

Claimants could receive net payments somewhat less than these amounts due to certain contingencies, particularly:

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Attorney fees: While a program of assistance in filing claims (including information about pro bono attorneys) would be established, claimants could use their own fee-based attorneys. However, the bill would limit fees to 5% of the benefit received. Collateral sources: Benefits would be reduced for collateral sources (i.e., other payments received from defendants and insurers). However, there would be no offset for workers compensation or veterans benefits. The case of railroad and maritime workers is complicated because they are covered by the Federal Employers Liability Act (FELA, 45 U.S.C. 51 et seq.) or Jones Act (46 U.S.C. App. 688), both of which have aspects both of workers compensation and of tort liability. S. 3274 (subsection 131(b)(4)) deals with this complication by awarding — in lieu of what they might have gotten by workers compensation — an additional “special adjustment” equal to 110% of the average amount received by asbestos victims (at the claimant’s disease level) in recent years.

Diagnostic Categories Eligibility for benefits would require certain kinds of evidence, including documentation of occupational exposure to asbestos (preceding a minimum 10-year latency period), smoking history, physical examination, pulmonary function test, x-rays (and possibly CAT scans), and pathology report.[20] With this evidence, administrators are to apply the criteria in Subsection 121(d) and determine the highest of the 10 disease levels to which each claimant belongs (if any). The goal is a non-adversarial system that is prompt, efficient, and as accurate as possible in a field where there are substantial scientific uncertainties. While in some respects the benefit of the doubt is given to claimants, on the other hand the system is meant to eliminate screening “mills” that produce thousands of claims upon evidence that is fragmentary at best, if not fraudulent.[21]

Disputed Categories Several of the disease categories have drawn criticism on the ground that they are not credibly linked to asbestos exposure. Among these are as follows: •

•

Simple asbestosis (Level I). It is agreed on all sides that claimants at Level I are not impaired (“ill”), hence do not receive cash compensation, only the right to monitoring. If illness on other levels is subsequently found, compensation can then be claimed. Some dispute the rationale for monitoring, arguing that being at Level I does not imply any higher probability of subsequent illness than for other workers who are not at Level I. On the other side it is argued that, as done with many toxic substances, all exposed workers should get screening regardless of whether they show symptoms.[22] “Other cancers” (Level VI). There is dispute here on whether asbestos causes nonlung cancers (such as colorectal). Proponents of the provision note that the existing bankruptcy trusts compensate for non-lung cancers, but opponents claim that this is due to quirks of bankruptcy bargaining dynamics. At any rate, an independent physician panel must determine in each Level VI case that asbestos was a substantial contributing factor to the cancer. The bill would also mandate a study by the Institute

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Edward Rappaport of Medicine, the conclusions of which will be binding, regarding the issue of causation at Level VI. Lung cancer without evidence of asbestos disease. Previous versions of the bill included a level for lung cancer without evidence of pleural disease or asbestosis. But S. 3274 has no such level, in effect accepting the argument that when asbestos causes lung cancer, there is almost always identifiable asbestos damage. “Mixed dust” / Silicosis. Oftentimes asbestos exposure occurs in combination with silicosis. Concern about potential use of “mixed dust” lawsuits to circumvent the ending of asbestos suits[23] led to a new subsection, 403(b). It requires, in effect, that anyone suing over silica disease must show that asbestos was not a substantial contributing factor to one’s impairment.

The Tobacco Question Underlying most of these disputes is the relevance of smoking history. The committee report stated that “The Fund is not intended to be a compensation system for smokers, which would otherwise overwhelm the Fund, leaving no money for asbestos victims.” Thus the compensation scheme discounts the awards to smokers in two ways. First, the three lung cancer levels (two in the current version of the bill) are distinguished by the degree of pathology or x-ray evidence linking the cancer to asbestos. Implicitly, a higher probability is attributed to other causes (e.g., tobacco or radon) where the link to asbestos causation is less certain (and a third level was deleted altogether). Second, levels are divided explicitly into sub-levels for smokers, former smokers[24] and non-smokers. The resulting scheme has been criticized from both sides. On the one hand, as noted, some claim that asbestos is almost never the cause of cancer without also causing clinical asbestosis, so there should be no Level VII. On the other side, plaintiff advocates note that a high percentage of the blue collar workers most exposed to asbestos were indeed smokers, so that the widely publicized figure of up to $1 million for lung cancer would be received by very few. A key point of disagreement is whether there is synergy between tobacco and asbestos in causing cancer. Many believe that there is such a synergistic effect (i.e., when one is exposed to both asbestos and tobacco), the risk of lung cancer is enhanced greatly beyond the sum of the two factors independently. If this is so, then it could be argued that the awards to smokers should not be reduced very much vis-avis non-smokers. However, differing testimony on the matter was received by the committee and consensus not reached.[25] Diagnostic Quality Control In addition to the foregoing disagreements about defining eligible medical categories, there is the issue of types of evidence to be deemed credible. In the existing tort law system, plaintiffs present evidence favorable to their case and defendants have an opportunity to challenge it. Since S. 3274 would replace tort law with a non-adversarial, administrative system, it explicitly defines what kinds of evidence are necessary and acceptable, and requires auditing of the results. Subsection 121(b) sets general rules for expertise of those developing evidence. Thus, (1) x-ray interpretations must be done by “B-readers,” a certification overseen by the National Institute for Occupational Safety and Health; (2) pulmonary function testing for asbestos

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(Levels III to V) is to be done in accordance with the standards of the American Thoracic Society; and (3) diagnosis of malignancies (Levels VI through IX) must be done by boardcertified pathologists.[26] Furthermore, guidelines are to be developed relating to CT scans in the diagnosis of lung damage, for future use in the program (subsection 121(h)). Section 115 provides for reviews and audits, including the empaneling of independent Breaders to spot check accuracy of submitted readings. Certain B-readers who have previously been paid for testimony are disqualified from the program. The Administrator is also instructed to develop methods for evaluating general medical evidence. If the evidence presented by particular physicians or facilities is found “not consistent with prevailing medical practices or the applicable requirements of this act,” consequences may include disqualification. Finally, Section 401 provides criminal penalties for fraud or false statements.

Transition Issues S. 3274 would pre-empt all cases pending on the date of enactment, and all future cases, in favor of the new system.[27] Possibly hundreds of thousands of cases would be transferred at the outset, so that getting the system established and making what are supposed to be prompt decisions might be an administrative as well as financial challenge. Concern has been expressed about at least three transition issues: claimants potentially caught between systems, status of settlements, and a possible cash “crunch” in the first few years.

Pending Cases Concern has been expressed about claimants who may have their pending cases dismissed but must wait for the new system to begin. Proposals have been made (and included in S. 1125, 108th Congress, as reported) to delay termination of tort proceedings until the administrative system was up and running, but this was not included in subsequent versions of the bill. Rather, S. 852 contains certain deadlines for getting the process going. First, case processing is to begin immediately, even before funding is established. “Exigent health claims” (e.g., cases of terminal illness) are to be given priority, and the administrator is to certify within nine months of enactment that the office is ready to review and pay exigent claims. In cases of mesothelioma, payment of 50% is to be made within 30 days of approval, the rest in six months. On another track, incentives are given (subsection 106(f)(2)) to promptly reach negotiated settlements for mesothelioma cases. For participants in existing bankruptcy settlements, the trusts are to set aside 10-12 % of their assets to continue making payments until the Fund is operational. Finally, the office must be ready to start processing all types of claim within 24 months. If the start-up deadlines are not met, claimants may pursue their cases in court (at least until such time as the program does become operational). Pending Settlements Subsection 403(c) of S. 3274 would terminate “inventory” or “matrix” agreements, which are open-ended, standing arrangements that pay specified amounts to claimants who qualify currently or in the future. One’s view on whether these or other agreements should be terminated will probably correspond with one’s overall evaluation of the fairness of the proposed system vis-avis the current tort system. For example, it is argued that some

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companies that have agreed to settlements will pay much less under the bill’s terms.[28] But, as argued by the committee majority for passage (108th Congress), “The purported unfairness of preempting non-final settlement agreements ... [etc.] ... rests on the faulty premise that the existing system is somehow fair.”[29]

Funding Schedule As noted, a large number of cases may be expected at the outset. Concern has been repeatedly expressed, during the debate on an asbestos resolution fund, about whether sufficient amounts would be available to promptly pay a large expected number of claims in the first years. Final decisions (in uncontested cases) could possibly start coming within 180 days of enactment, and the first (40%) installment on each claim is to be paid within one year of the award decision.[30] In anticipation of this demand, the bill front-loads the collection of funds. The stated goals of the funding program are to: (a) collect $3 billion each year from defendant companies; (b) collect more than $20 billion from insurers over the first five years;[31] and (c) receive the assets of bankruptcy trusts within six months of enactment. This would come to more than $35 billion within five years. Moreover, the compensation fund could borrow, both from the Federal Financing Bank and the private sector. The limits of borrowing authority are not specified numerically, but defined in terms of what could be repaid from anticipated revenues over the subsequent 10 years (Subsection 221(b)(3)).[32] The bill also authorizes surcharges for various contingencies. In order to provide greater assurance to those claimants who are most ill, there is a lockbox-type mechanism (Section 221(c)). Under this provision, the administrator would establish separate accounts for each of the most serious diagnoses (Levels IX, VIII, V and IV) and reserve needed funds to them first. Implicitly, claimants in other levels would not be paid if sufficient funds are not available for the four protected levels.

SUNSET The ultimate guaranty that the program would not add to the federal deficit is that the fund’s fiscal status will be regularly monitored and, if insolvency is foreseeable and unavoidable, the program will be wound up and cases be returned to the court system. Although the nominal life of the program is 30 years, it could be terminated earlier if the fund is found to be inadequate to meet all claims. With each annual report, the administrator is to analyze whether the fund will be able to pay all claims as due at any time within the next five years. If such shortfall is projected, the administer is to make recommendations for reform or termination. Recommendations are referred to a special commission consisting of five Cabinet members, who will hold public hearings and forward its recommendations to the Congress. However, if the administrator, at any time after claim processing has begun, conducts an operational review and determines “that if any additional claims are resolved, the Fund will not have sufficient resources when needed[33] to pay 100% of all resolved claims while also meeting all other obligations...,” then the program is to terminate 180 days thereafter. Thus the annual report, with its five-year financial projections, gives regular opportunities for deliberative mid-course corrections. But if insolvency (inability to meet all

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currently assumed obligations) ever becomes imminent, the administrator is authorized to terminate the program. In that case, unresolved or new asbestos cases can be pursued in court, albeit with certain restrictions. In particular, there would not be as wide a choice of venue as at present. The suits could be filed either in the federal courts or in a state court where the claimant resides or experienced the asbestos exposure. In the debate on the budget point of order, critics of the bill disputed whether such a scenario would ever be followed, arguing that political pressures to “save” the program in the event of a financial shortfall would be irresistible. Thus, they cautioned, the acceptability of a possible shortfall should be considered before going ahead with the program.

REFERENCES The Environmental Protection Agency issued a regulation in 1986 that would have banned virtually all major uses, but most of the rule was overturned by the Fifth Circuit Court of Appeals (Corrosion-Proof Fittings, 947 F.2d 1201). A stand-alone bill in the 108th Congress, S. 1115 (Murray), would have mandated such a ban (with a procedure for EPA to allow exceptions). One of the bills for resolving litigation, S. 3274 (Specter and Leahy), would also include a ban on future usage. S. 668 (Specter) would establish criminal penalties for willful violation of occupational asbestos standards. Current regulatory standards are described in CRS Report RS21042, Asbestos: Federal Regulation of Uses, by Edward Rappaport. Quantitative data cited here are from Stephen Carroll et al., Asbestos Litigation (Santa Monica, CA: RAND Institute for Civil Justice, 2005). Available at [http://www.rand.org/ pubs/monographs/MG162/index.html]. About two-thirds of plaintiffs going to trial win and receive awards. Randy Maniloff, “Asbestos: Insurance Coverage Issues on a Changing Landscape,” Mealey’s Litigation Report: Insurance, July 9, 2002. Homepage: [http://www.mantrust.org]. Section 111 of the Bankruptcy Reform Act of 1994 (P.L. 103-394), also known as Section 524(g). Stephen Carroll et al., Asbestos Litigation Costs and Compensation, An Interim Report (Santa Monica, CA: RAND Institute for Civil Justice, 2002), p. 26. Susan Warren and Alexei Barrionuevo, “Halliburton to Settle Asbestos Claims,” Wall Street Journal (Dec. 19, 2002), pp. A3, A6. A recent ruling has cast doubt on the efficacy of this strategy. See Russell Gold and Goran Mijuk, “ABB Fails to get Court Approval for Asbestos Plan,” Wall Street Journal (Dec. 3, 2004), pp. A3, A8. Amchem Products v. Windsor, 521 U.S. 591 (1997) [also known as the Georgine case] and Ortiz v. Fibreboard, 527 U.S. 815 (1999). See Deborah Hensler, “As Time Goes By: Asbestos Litigation after Amchem and Ortiz,” Texas Law Review, v. 80, no. 7 (June 2002), pp. 1899-1924. Tort reform is discussed more generally in CRS Report 95-797, Federal Tort Reform Legislation: Constitutionality and Selected Statutes, by Henry Cohen.

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Pleural plaques resemble calluses. They are patches of tough sinewy tissue which form on the inside of the chest wall and show up in chest x-rays. They are generally thought of as an indicator of asbestos exposure rather than a disease. H.R. 1957 notes in its findings section that the number of silicosis cases has been rising rapidly and that “it is necessary to address silica legislation to avoid an asbestos-like litigation crisis.” Debate on Cornyn amendment SA 2749 in Congressional Record, February 9, 2006. pp. S882-S885 and S944-S957. H.R. 1283 (Hyde)/S. 758 (Ashcroft) in the 106th Congress (H.Rept. 106-782). See Palmer, Elizabeth. “House Panel Approves Bill to Federalize Asbestos Cases,” CQ Weekly, March 18, 2000, pp. 598-599. U.S. Congress, Senate Committee on the Judiciary, The Fairness in Asbestos Injury Resolution Act of 2003, 108th Cong. 1st sess., S.Rept. 108-118 (Washington: GPO, 2003), 227 p. (Hereafter cited as “Committee report”). CBO cost estimate for S. 852, August 25, 2005. Bates White, LLC. Analysis of S. 852. September 2005. [http://www.bateswhite.com / news/pdf/2005_Bates_FAIR_Act_report.pdf]. Groups of insurance companies (possibly even all companies in one group) can agree to any allocation of their total liability among themselves. Section 404, meanwhile, adjusts the obligations of insurers and reinsurers to each other and to defendant companies. For general information about asbestos-related diseases, see [http://www.nlm.nih.gov/ medlineplus/asbestos.html], and [http://www.health.nih.gov/ result.asp?disease_id=54]. There are special allowances for victims of the World Trade Center attack and Hurricane Katrina, and for residents of Libby, Montana (who received substantial exposure from the dust of a vermiculite mine). The bill mandates a study of whether significant exposure occurred to people living near about 18 sites around the country where vermiculite was processed. On allegations of fraudulent testing, see Sen. Kyl’s statement, Committee report pp. 95-98. Also, Parloff, Roger. “Diagnosing for Dollars,” Fortune, 13 June 2005. p. 97 ff. Compare Committee report pp. 98-99 and pp. 212-213. The bill also allocates $20 to $30 million per year for at least five years for a screening program “for individuals at high risk of asbestos-related disease.” (Sect. 221(c)) The issue gained considerable attention in the wake of an unexpected court ruling in June 2005. (Glater, Jonathan. “The Tort Wars, at a Turning Point,” New York Times, October 9, 2005, Business p. 1,7) The Senate Judiciary Committee held a hearing on the issue on February 2, 2006. Those who quit at least 12 years before diagnosis. Both sides were supported by expert witnesses. The committee majority in favor of the bill relied particularly on testimony of Dr. James Crapo of the University of Colorado. The dissenting minority claimed a “scientific consensus” for synergy as expressed by institutions such as the National Toxicology Program (Department of Health and Human Services) and the International Agency for Research on Cancer. Compare Committee report pp. 64-66 with pp. 200-202. According to the bill text, diagnoses of non-malignant conditions (Levels I through V) can be rendered by any physician. However, the Committee report (at p. 39) expressed the intent that “the documentation would be provided by an appropriately board-certified

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physician in occupational medicine or pulmonary medicine,” while recognizing that access to same “may not be feasible for all claimants due to geographical constraints.” There are a few exceptions. For example, non-consolidated cases may proceed if at the presentation of evidence stage. (Subsection 403(d)(2)) The January 2006 bankruptcy settlement of USG Corp. (the former U.S. Gypsum) explicitly provides that if an asbestos bill is passed in the 109th Congress, then the company will put $900 million into its trust fund for injury claims (roughly what it estimates it would owe to the government fund), but the company will deposit another $3 billion if no such bill is enacted. Johnson, Fawn. USG Corp. Announces Asbestos Settlement that Would Resolve 150,000 Workers’ Claims. Daily Labor Report, 31 January 2006. p. A7, A8. Compare Committee report pp. 69-71 with pp. 206-208. The administrator can extend the claim payment schedule to four years instead of three years “if such action is warranted in order to preserve the overall solvency of the Fund” (Subsection 133(a)(3)). Even so, 50% of each award must be paid within two years. $2.7 billion in years 1 and 2, $5.075 billion in years 3 through 5. Repayment is not a general government obligation, but “is limited solely to amounts available in the ... Fund” (Subsection 221(b)(4)). The phrase “when needed” would seem to allow for the slowing of payments from a threeyear to a four-year schedule as authorized by Subsection 133(a)(3).

In: Mesothelioma from Bench Side to Clinic Editor: Alfonso Baldi, pp. 25-66

ISBN: 978-1-60021-789-0 155 © 2008 Nova Science Publishers, Inc.

Short Commentary 1

THE EFFICIENCY OF THE CUSCORE TEST AS COMPARED TO THAT APPLIED TO SMR IN DETECTION OF A CARCINOGENIC EXPOSURE Rina Chen, Ph.D. BioForum, Applied Knowledge Center, Ness-Ziona, Israel

INTRODUCTION Quite often statistical analyses are carried out in response to a public alert regarding suspected elevated risk of cancer. Usually no control group is available in these ad-hoc instances, and the analysis is based on the SMR, i.e., on the contrast between the observed and the expected number of events. The SMR (Standardized Morbidity (or Mortality) Ratio) is the ratio between the observed and the expected number of events. The expected number is evaluated according to the age and gender specific rates observed in the reference population. Both types of statistical errors are inflated in these analyses [1]. Namely, frequent false alerts coupled with unsatisfactory power of the test are involved in these analyses. This commentary presents the relative efficiency of the CUSCORE test in alleviating the two types of statistical errors. Analyses of colon cancer deaths among asbestos workers in Israel, demonstrate the efficiency of the procedure in detecting clustering and in providing clues indicating that the significant results are not spurious. Some suggestions are presented in order to increase the efficiency in detection and interpretation of the analyses.

BACKGROUND False alerts (type 1 error) are likely to be frequent because of the ad hoc situation. This frequency may indeed be high and of a real concern here, since an incidental alarm may be elicited in any one of indefinite number of communities. The relative inefficiency of the test (type 2 error) in detecting a real cluster is related to the facts that the number of events in the data set is usually scarce, and that only some of the events might be clustered. The possibility

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that the cluster is embedded within the data set is related to one or more of the following reasons: the long latent period, the possibility that the initiation time of exposure is unknown and the possibility that the later events of the data have not been induced by the exposure and therefore are not clustered. The later cases may not be clustered when the study covers a long period of time extending beyond the maximum reasonable latent period. The underlying assumption is that only a small number of individuals are likely to be sensitive to any specific carcinogenic exposure and that at a certain point of time all the sensitive individuals have been diagnosed. Thus for example, suppose that the data to be analyzed is related to exposure in a workplace. All the workers in the data set were recruited 40 years ago. If the maximum reasonable latent period is 30 years, no excessive number of diagnoses should be expected during the last 10 years of the study. In fact, it is even possible that a smaller than expected number of diagnoses appear during the last 10 years. This will be the case if in addition to inducing cancer, the exposure also enhances the carcinogenic process among those who would become cases regardless of the exposure. Hence in this example, contrary to our intuition, the power of a test applied to data of the first 30 years would be larger than that applied to data of 40 years. This situation calls for applying a surveillance type test. Surveillance systems have long been used in monitoring the quality of manufactured products [2]. Malfunction of the production process is declared when the percentage of defected produced items is too large in a consistent manner. The statistical surveillance techniques applied to such data, are based on a score that is being accumulated over successive periods. The accumulated score is either increased or decreased at the end of each period according to whether or not the percentage of defected items indicates deterioration in the production process. The analysis is terminated when the score gets larger than a predetermined value and recycled (returns to 0) when the accumulated data is negative. In the first case the result is significant, in the second case the procedure is recycled because no deterioration in the process until that time has been indicated. Hypothetically, the SMR could be considered as a relevant statistic for a test based on a surveillance approach. In which case the SMR will be evaluated for each consecutive subperiod. Several consecutive large SMRs may indicate clustering. However, this approach is usually not applicable to data associated with public alerts, since each estimates of SMR may include 0 or a very small number of events. A statistic representing an estimate of 1/SMR can efficiently be used when the data are scarce. The statistic termed RI (Relative Interval) was suggested for several procedures associated with detection and investigation of disease clustering. These include procedures for: monitoring [3-5], ad hoc tests of significance [6-8] and post alarm interpretation and confirmation of clustering [1,9,10].

THE TIME INTERVAL BETWEEN EVENTS The RI is a statistic evaluated for each time interval between successive observed events. Thus, the period under study is divided into sub-periods, each defined by the actual observed data, rather than by pre-specified units of calendar time. Hence by definition, each sub-period includes one and only one event.

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The RI is defined as the expected number of events between two consecutive observed events. Accordingly, it is the time interval between consecutive events measured by standardized units. Each time unit is the time expected until the next event. In terms of months, the unit may change from one year to another, and/or from one disease to another, and/or from one community to another. But it is always the expected time for one event. Since by definition, one event is observed in each time interval, and the RI is the expected number of events, the RI can be viewed as the ratio between the expected and the observed number of events (i.e., as RI/1.0), namely as the reciprocal of the SMR. The RI follows the exponential distribution [4]. Under the null hypothesis, the mean of the RI is 1.0; hence the sum of the RIs over the period is practically the expected number of events in the study.

THE CUSCORE TEST As mentioned above, the RI is a time measurement that is expected to be 1.0 under stable conditions, and smaller than 1.0 under elevated risks. A clustering phenomenon will be indicated if several consecutive RIs are small. Based on this idea, the CUSCORE test of significance was derived [1] from the CUSCORE monitoring technique [11]. The CUSCORE test was shown to be more efficient than a test applied to the SMR when only some of the events are clustered [6]. Thus the test may, to some extant, control the statistical error of type 2. As will be shown later, it is useful also in alleviation the type 1 error, by providing clues regarding the possibility of superfluous significance. The CUSCORE test is based on an accumulated score. For that test each observed RI is defined as either “short” or “long”. The time interval between events is defined as short if the RI is shorter than a critical value k and long otherwise. This critical value is evaluated by a Markov Chain approach, assuming that the probability for a short interval follows the exponential distribution [6,10]. The critical value (k) is presented in Table 1 for 5% significance level by the number of events (S). The score of the test is 0 at start (prior to the first event), and either increases or decreases by 1.0 at each event. It increases if the next RI is “short” and decreases if it is “long”. The test is recycled when the score is 0 and a long RI is observed. A cluster is detected when the CUSCORE equals 5. Hence, the accumulated score is between 0 and 5. The following simple fictitious example demonstrates the procedure. Table 2 presents the dates of the 8 events observed among, say 200 workers over the period 1970-2002. The time intervals between consecutive diagnoses are also presented in Table 2. The interval associated with the first case is the time from January 1970 until the date of the first diagnosis. For ease of demonstration, it is assumed that all these workers were recruited in January 2000 and were followed up through out the entire period. It is also assumed that under stable conditions, 0.25 diagnoses are expected at each year of the study period (namely 0.0208 events per month). These two assumptions are of course unrealistic, since neither the composition nor the risks remain constant over the years. Even if all individuals are being followed over the entire period, the expected number of events should be changed because of aging. Table 2 presents the time interval between consecutive diagnoses in months and in RI units, and the score of the test. The RI is obtained as the product: Number of months*(0.25/12). The critical value for 8 events is k=0.546. No clustering is detected in

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these data. In fact the number of events observed is about that expected. The expected number of events is 0.25*33=8.25 (it is also the sum of RI + the expected number between June 13, 2002 and December 31 2002).

THE EFFICIENCY OF THE CUSCORE TEST IN A CONCRETE EXAMPLE The relative efficiency of the CUSCORE test in detection and in providing clues for interpretation of the results can be demonstrated by the published [7] results of analyses applied to colon cancer cases among 2208 asbestos Jewish male workers in a cement factory in Israel during Jan 1987-Dec. 1997. These data include the dates of death of each of the 12 asbestos workers who died with colon cancer during those 11 years. Analyses of these data were carried out by both the SMR and by the CUSCORE test. For both analyses the expected number of events was calculated according to the age specific mortality rates from colon cancer among Jewish males in Israel during 1990-92 [12]. The expected number of deaths during the entire period of the study is 7.94. Accordingly, SMR=1.51 and the 90% confidence limits are 0.87-2.45. Thus using a one-sided test, the SMR is not significant at 5% level (actually P