Genotoxicity of Chromium Compounds

GENOTOXICITY OF CHROMIUM

COMPOUNDS

COMPARATIVE STUDY IN LEATHER TANNING AND STAINLESS STEEL WELDING INDUSTRY WORKERS

Margarida Goulart de Medeiros FFUL , 2003

Genotoxicity of Chromium Compounds Comparative Study in Tanning and Welding Industries

Genotoxicidade de Compostos de Crómio Estudo Comparativo em Indústrias de Curtumes e Metalomecânica

Dissertação apresentada à Faculdade de Farmácia da Universidade de Lisboa para a

obtenção do grau de Doutor

Margarida Alexandra Patrício Goulart de Medeiros

2003

Advisor: Prof. Drª Maria Camila Canteiro Batoréu

Co-advisor: Prof. Dr. José Rueff

The experimental work presented in this thesis was performed at the Laboratory of

Toxicology of the Faculty of Pharmacy of the University of Lisbon, at the

Department of Genetics of the Faculty of Medical Sciences of the New University

of Lisbon and at the Department of Pathobiology and Laboratory Medicine of the

Brown University, Providence, RI, USA.

The Portuguese Foundation for Science and Technology (FCT) financially

supported this work, with the PhD grant PRAXIS/BD/18317/98.

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TABLE OF CONTENTS

List of publications xi

List of figures xii

List of tables xiv

List of abbreviations xv

Summary xvii

Resumo xix

1. Introduction 1

1.1. Historical overview 1

1.2. Physical and chemical properties 4

1.3. Essentiality 6

2. Chromium toxicity 7

2.1. Carcinogenic and genotoxic effects 7

2.1.1. Human studies 7

2.1.2. Animal studies 12

2.1.3. Short-term assays 13

2.2. Other toxic effects 14

2.3. Importance of physical and chemical properties in toxicity 15

2.4. Toxicokinetics 16

2.5. Mechanisms of chromium genotoxicity 18

2.6. Elements researched 23

2.6.1. Biomonitoring and molecular epidemiology 23

2.6.2. Oxidative damage in chromium occupational toxicity 28

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2.7. Objectives of the study 29

3. Subjects and their environment 30

3.1. Tanning industry 30

3.2. Welding industry 33

3.3. Control group 34

3.4. Sample collection 36

3.4.1. Blood sampling 36

3.4.2. Urine sampling 36

3.4.3. Leather dust 36

4. Biomarkers of chromium exposure 37

4.1. Overview 37

4.2. Research procedures 39

4.2.1. Total chromium in biological samples 39

4.2.2. Hexavalent chromium in leather dust 43

4.2.3. DNA-protein crosslinks measurement 44

4.3. Results 46

4.3.1. Total chromium in plasma and urine 47


4.3.3. DNA-protein crosslinks 51

4.4. Discussion 53

4.4.1. Chromium in plasma and urine 53


4.4.3. DNA-protein crosslinks 55

5. Biomarkers of chromium effect 57

5.1. Overview 57

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5.2.1. Chromosomal aberrations assay 59

5.2.2. Cytokinesis-blocked micronucleus assay 59

5.3. Results 60

5.3.1. Chromosomal aberrations 60

5.3.2. Micronuclei 65

5.4. Discussion 69

6. Biomarkers of susceptibility to chromium 72

6.1. Overview 72


6.2.1. Genetic susceptibility 75

6.2.2. Challenge assay 75

6.3. Results 75



6.4. Discussion 78



7. Oxidative damage in chromium occupational toxicology 80

7.1. Overview 80


7.2.1. Lipoperoxidation products 80

7.2.2. Thiol antioxidants 81

7.3. Results 82


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7.4. Discussion 86



8. Final discussion and conclusions 88

9. Future perspectives 92

References 93

Appendice 1 111

Appendice 2 115

Acknowledgements 129

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The present work contains techniques and/or experimental data also

presented in the following scientific papers:

Quievryn G, Goulart M, Messer J, Zhitkovich A. Reduction of Cr (VI) by cysteine: significance in human lymphocytes and formation of DNA damage in reactions with variable reduction rates. Mol Cel Biochem, 222:107-118, 2001. M.G. Medeiros, A.S. Rodrigues, M.C. Batoréu, A. Laires, J. Rueff, and A. Zhitkovich. Elevated levels of DNA–protein crosslinks and micronuclei in peripheral lymphocytes of tannery workers exposed to trivalent chromium. Mutagenesis, 18:19-24, 2003. Margarida G. Medeiros, António S. Rodrigues, Maria Camila Batoréu, António Laires, Anatoly Zhitkovich, José Rueff. Biomarkers of chromium exposure and cytogenetic damage in leather tanning and welding industry workers. In: Human Monitoring for Genetic Effects, NATO Science Series, IOS Press. In press. M.G. Medeiros, A.S. Rodrigues, M.C. Batoréu, A. Laires, A. Zhitkovich and J. Rueff. Lipoperoxidation products and thiol antioxidants in chromium exposed workers. Manuscript in preparation. M.G. Medeiros, A.S. Rodrigues, M.C. Batoréu, A. Laires, and J. Rueff. Chromosomal aberrations and response to challenge by bleomycin in lymphocytes of tannery workers and welders. Manuscript in preparation.

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List of Figures Page

1. Van Gogh’s “Sunflowers”, 1888 1 2. Interaction between the different factors of disease 7 3. Toxicokinetic model for chromium 17 4. Uptake-reduction model 18 5. Structures proposed for the phosphate-based Cr(III)-DNA adducts 22 6. The several check points of genotoxicity 26 7. Biomarkers of exposure and effect in the pathway of genotoxic action 26 8. Biomarkers of susceptibility 27 9. General process of industrial leather tanning and possible exposure to

toxic agents 31

10. Preliminary study of the pyrolisis temperature 42 11. Standard chromium curves for plasma before and after correction for

the control sample absorption 42

12. Three replicate injections of 5µg/L Cr standard in urine 42 13. Standard calibration curve for hexavalent chromium determination by

HPLC 43

14. DNA-protein crosslinks measurement by SDS precipitation assay 45 15. Typical calibration curve for PicoGreen® DNA quantification 45 16. Pre and post-shift urinary chromium levels in tannery workers 50 17. Linear relationship between urinary and plasmatic total chromium

concentration in tanners 50

18. Total chromium in urine and plasma of tanners, welders and controls 51 19. DPC values in tanners, welders and controls 52 20. Chromatid and chromosome-type aberrations 58 21. Different types of chromosomal aberrations found in tanners 63 22. Different types of chromosomal aberrations found in welders 63 23. Different types of chromosomal aberrations found in controls 64 24. Frequency of aberrant cells in tanners, welders and controls 64 25. ‰ of micronucleated binucleated lymphocytes in tanners, welders and

controls 67

26. Normal metaphase 68 27. Chromatid break 68 28. Chromosome break 68 29. Chromatid gap 68 30. Dicentric chromosome with fragment 68 31. Tetraradial chromosome 68 32. Normal binucleated lymphocyte 69 33. Binucleated lymphocyte with MN 69 34. Binucleated lymphocyte with MN 69 35. Binucleated lymphocyte with MN 69

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36. Hypothesized process for infidelity of chromosome/DNA repair after exposure to carcinogens

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37. Micronuclei frequencies after challenge assay in tanners, welders and controls

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38. Urinary thiobarbituric acid reactive substances expressed as malondialdehyde (MDA µmol/g creatinine) in tanners, welders and controls

84

39. Glutathione concentration in peripheral blood lymphocytes of tanners, welders and controls

86

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List of Tables Page

1. Standard reduction potentials of chromium compounds 4 2. Solubility of hexavalent chromium compounds at 20-30 ºC 5 3. Genotoxicity studies in human populations exposed to chromium

compounds 11

4. Genotoxicity studies in laboratory animals exposed to chromium compounds

12

5. Demographic data for tannery workers 32 6. Demographic data for welders 33 7. Demographic data for controls (blood) 34 8. Demographic data for controls (urine) 35 9. Comparison of demographic data for the several study groups 35 10. Pre and post-shift urinary chromium in tannery workers 47 11. Chromium concentrations in urine and plasma of tannery workers 48 12. Chromium concentrations in urine and plasma of welders (first sampling) 49 13. Chromium concentrations in urine and plasma of welders 49 14. Hexavalent chromium in leather dust 51 15. DNA-protein crosslinks values in tanners and welders 51 16. Chromosomal aberrations in welders 60 17. Chromosomal aberrations in tanners 61 18. Chromosomal aberrations in controls 62 19. Micronuclei frequency in tannery workers 65 20. Micronuclei frequency in controls 66 21. Micronuclei frequency in welders 67 22. Challenge test in tanners, welders (first sampling) and controls 76 23. Challenge test in tanners, welders (first sampling) and controls 77 24. Thiobarbituric acid reactive substances in urine of tanners, welders and

controls 82

25. Glutathione and cysteine in peripheral blood lymphocytes of tanners, welders and controls

84

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List of Abbreviations

AA amino-acid AAS-ETA atomic absorption spectroscopy with electrothermal atomization BEI biological exposure index BNMN binucleated cell with micronuclei BSA bovine serum albumin CA chromosomal aberration CBPI cytokinesis-blocked proliferative index Cr chromium Cr(III) trivalent chromium Cr(VI) hexavalent chromium CYS cystein DNA deoxyribonucleic acid DPC DNA-protein crosslinks EBV Ebstein-Barr virus EDTA ethilenodiaminotetracetic acid GSH glutathione GST glutathione s-transferase HPLC high performance liquid chromatography HPRT hypoxanthine guanine phosphoribosyltransferase ip intra peritoneal K-SDS potassium-sodium dodecylsulphate salt LD50 lethal dose for 50% kill mBBr monobromobimane MDA malondialdehyde MN micronuclei, micronucleus PBS phosphate buffered saline PCR polymerase chain reaction PMSF phenylmethylsulfonylfluoride po per os, oral intake P-SDS protein-sodium dodecylsulphate complex SCE sister chromatid exchanges SDS sodium dodecylsulphate STEL short-term exposure levels T1/2 half-life TBA thiobarbituric acid TBARS thiobarbituric acid reactive substances TLV threshold limit value TWA time-weighted averages

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xvii

Summary

The toxicity of chromium (Cr) in occupational settings has been essentially

focused on the hexavalent form of the metal, a Group 1 known human carcinogen according to IARC (International Agency for Research on Cancer) classification. Nevertheless, hexavalent chromium has no toxic action until it is reduced inside the cell to lower oxidation states, the most stable being the trivalent form, Cr(III). Therefore, the Cr(III) may be the ultimate intracellular toxicant and could owe its apparent lack of toxicity to a poor permeability in biological membranes. In this case, chronic exposure to moderate or high concentrations of trivalent chromium compounds may be enough to overcome the cellular uptake barrier. In order to further clarify this hypothesis, a comparative biomonitoring in workers exposed to each of the referred oxidation states of chromium was conducted. Two groups of occupationally exposed individuals participated in the study, one of leather tanning industry workers (exposed to trivalent chromium) and one group of stainless steel welders employed in an engine repair workshop (exposed to hexavalent chromium). A control group of non-exposed individuals was also included.

Measurement of chromium concentration in urine and plasma indicated a significantly higher chromium exposure and absorption in both groups of workers when compared to controls, which demonstrates a significantly increased intake of chromium related to occupational exposure in both cases.

Genetic damage was assessed by the determination of micronucleated cells in the cytokinesis blocked micronucleus assay and observation of chromosomal structure aberrations, both in peripheral blood lymphocytes obtained from the workers and controls. There was a significant excess of micronuclei frequency in tannery workers, but the increase was not significant in the welder group, when compared to controls. No statistical significant increase in chromosomal aberrations was registered in either group.

Chromium is a known cross-linking agent, and the determination of DNA-protein crosslinks (DPC) has been proposed and explored as a biomarker for exposure to hexavalent chromium. For the first time, this biomarker was applied to individuals occupationally exposed to Cr(III) compounds. Levels of DPC were found to be significantly increased in both groups of workers, exposed to chromium (III) and (VI). The findings add strength to the initial premise of Cr(III) low cellular intake being overcome by chronic exposure. DNA-protein crosslinks were nevertheless significantly higher in the welder group over control and tanner values.

Oxidative damage was also investigated by the determination of urinary concentrations of thiobarbituric acid reactive substances, which was significantly elevated in both groups of workers. The levels of antioxidant thiols, glutathione and cystein, present in peripheral blood lymphocytes of exposed individuals showed a marked decrease in total glutathione among the welder but not the tanner group,

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which may be related to the response of the cells to the reactive oxygen and radical species resulting from hexavalent chromium reduction.

Peripheral blood lymphocytes collected from tannery workers were challenged in vitro with a single dose of bleomycin, which is a know radiomimetic compound. There was an excess in genetic damage in cells obtained from tannery workers when compared to the control response, although this result was of borderline statistical significance. This may indicate an increased sensitivity of these individuals to a continuous genotoxic aggression.

The genetic polymorphism studied (GSTT1 and GSTM1 genotype) did not seem to influence the metabolism and toxicity of chromium in exposed tannery workers. Due to the number of individuals studied, and the statistical requirements for such an analysis, this specific part of the study must be regarded as a preliminary approach to the influence of genetic susceptibility on the in vivo toxicity of chromium compounds.

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Resumo

O metal crómio (Cr) existe naturalmente na crusta terrestre, apresentando

vários estados de oxidação, dos quais os mais comuns são o trivalente (III), hexavalente (IV) e estado elementar (0). Tal como outros metais de transição, o crómio apresenta um caracter duplo: actua como elemento essencial para a homeostase de vários organismos vivos, e em determinadas condições pode apresentar-se como um agente tóxico para estes mesmos organismos. A toxicidade deste metal tem sido principalmente relacionada com a forma hexavalente, através de estudos epidemiológicos nos quais se observou um aumento de incidência de carcinomas respiratórios em populações ocupacionalmente expostas a compostos de Cr(VI). Estas observações foram reforçadas por estudos toxicológicos em animais de experiência e estudos in vitro que confirmaram o potencial mutagénico e carcinogénico de certos compostos de Cr(VI), levando à sua classificação como carcinogénio humano Grupo I pela International Agency for Research on Cancer (IARC). Comparativamente, a forma trivalente do metal parece apresentar uma toxicidade bastante inferior, o que poderá estar relacionado com a sua menor eficiência de passagem transmembranar e portanto inferior absorção por parte das células. No entanto, o Cr(VI) é incapaz de reagir directamente com o DNA, necessitando redução por constituintes celulares tais como o glutatião, ácido ascórbico e cisteína, entre outros. Em contraste, o Cr(III) reage prontamente com o material genético formando vários tipos de complexos. A redução intracelular de Cr(VI) produz espécies reactivas de oxigénio e de natureza radicalar e formas reduzidas de crómio. Qualquer destes compostos tem a capacidade de lesar o DNA através de mecanismos oxidativos ou da formação de complexos covalentes estáveis, alterando a estrutura do material genético. Assim, os produtos da redução de Cr(VI), incluindo Cr(III), constituem forma tóxica final de crómio dentro da célula. No caso do Cr(III), a toxicidade está aparentemente limitada apenas pela entrada dentro da célula. A exposição crónica a concentrações moderadas ou elevadas de crómio trivalente pode ser suficiente para ultrapassar este factor limitante, tornando necessária uma reavaliação da genotoxicidade de compostos de Cr(III) nestas condições de exposição. Por forma a contribuir para a clarificação desta questão, o presente trabalho experimental foi desenvolvido como um estudo comparativo de biomonitorização em dois grupos de trabalhadores expostos a compostos de crómio no local de trabalho, sendo um dos grupos constituido por 16 soldadores de aço inox pelo método do arco electrico manual, expostos a fumos e poeiras contendo crómio hexavalente, e um outro grupo de 33 trabalhadores de indústria de curtumes, expostos a soluções e poeiras contendo crómio trivalente, utilizado como agente de curtimento das peles animais. Foi igualmente incluido neste estudo um grupo controlo de 30 individuos não expostos a agentes genotóxicos no local de trabalho.

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O nível de exposição a crómio de cada individuo foi avaliado através da determinação da concentração de crómio total presente em fluidos biológicos, nomeadamente plasma e urina, através de espectroscopia de absorção atómica com atomização electrotérmica (câmara de grafite). As colheitas foram realizadas no último dia da semana de trabalho, a meio do turno (imediatamente antes do almoço). Os trabalhadores da industria de curtumes forneceram duas colheitas de urina adicionais, no último dia da semana de trabalho, imediatamente antes e imediatamente após o turno de trabalho de 8 horas. Ambos os grupos expostos apresentaram concentrações de crómio na urina e no plasma significativamente elevadas em relação ao grupo controlo, o que demonstra uma absorção de crómio a partir do ambiente de trabalho em ambos os casos.

A avaliação de uma potencial lesão genética foi realizada recorrendo a ensaios citogenéticos de visualização da frequência de aberrações estruturais em cromossomas em metafase, e da frequência de micronúcleos em células em interfase com citocinese bloqueada. Ambos os ensaios foram realizados a partir de culturas de linfócitos de sangue periférico obtidos de cada individuo participante no estudo, na sequência da colheita de urina. Foi observado um aumento significativo da frequência de micronúcleos em linfócitos de trabalhadores de curtumes, sendo este aumento não significativo no grupo de soldadores. Não se encontraram valores significativamente aumentados no número de aberrações cromossómicas em qualquer dos grupos estudados.

A exposição celular a compostos de crómio resulta na formação de vários tipos de complexos envolvendo Cr e DNA, incluindo ligações cruzadas DNA-Cr-proteínas. A determinação da quantidade destas ligações presente em linfócitos do sangue periférico de individuos expostos foi proposta como um potencial biomarcador de exposição ao crómio hexavalente. Neste estudo este biomarcador foi aplicado pela primeira vez a um grupo de trabalhadores expostos predominantemente a crómio trivalente. O número de ligações cruzadas DNA-proteínas encontrava-se elevado de forma significativa em ambos os grupos de trabalhadores expostos, quer a Cr(III) como a Cr(VI). Apesar dos valores mais elevados terem sido encontrados em soldadores, é de salientar que o facto de se registar um aumento significativo de ligações cruzadas em trabalhadores de curtumes reforça a hipótese inicial da exposição crónica a compostos de Cr(III) poder resultar na chegada de uma relevante dose biologicamente activa de crómio ao interior da célula, apesar da potencial barreira que as membranas celulares representam para esta forma do metal.

O potencial stress oxidativo em células expostas in vivo a compostos de crómio foi também investigado durante este estudo, através da determinação da concentração de produtos de peroxidação lipídica (substâncias reactivas ao ácido tiobarbitúrico) em amostras de urina dos individuos estudados. Esta concentração, foi encontrada elevada de forma significativa em ambos os grupos de trabalhadores em relação ao valor obtido no grupo controlo. Através da determinação dos níveis de antioxidantes do tipo tiol (glutatião e cisteína) em linfócitos de sangue periférico

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dos mesmos individuos foi observada uma marcada e significativa deplecção de glutatião total no grupo dos soldadores, que poderá estar relacionada com uma maior actividade oxidante do crómio hexavalente ao qual estes trabalhadores estão expostos.

Foi ainda avaliada a resposta in vitro de linfócitos de sangue periférico de trabalhadores de curtumes a uma dose de teste de um genotóxico conhecido (bleomicina). A resposta foi avaliada através da frequência de micronúcleos em células com citocinese bloqueada após a exposição a uma baixa concentração de bleomicina, um radiomimético. Linfócitos do grupo controlo foram submetidos ao mesmo tratamento para estabelecimento de um termo de comparação. Foi registado um excesso de lesão genética em linfócitos de trabalhadores de curtumes, cujo valor se encontra no limiar de significância estatistica. Este excesso indica uma potencial sensibilização das células destes individuos para uma agressão genética posterior.

Como uma primeira abordagem ao papel da susceptibilidade genética na toxicologia ocupacional dos compostos de crómio, foi avaliada a influência do genótipo da glutatião S-transferase T e M (GSTT1 e GSTM1) nos resultados dos biomarcadores estudados. Não foi encontrada qualquer influência aparente destes polimorfismos na absorção ou toxicidade do crómio neste ensaio. É importante referir que a análise estatistica da influência de parâmetros genéticos na susceptibilidade de um grupo de individuos necessita um número considerável de casos estudados de modo a obter amostras representativas de cada um dos genótipos em estudo.

Como conclusão, os resultados obtidos durante este estudo permitem reforçar a importância da determinação de ligações cruzadas em linfócitos de sangue periférico como um biomarcador de exposição a compostos de crómio trivalente e hexavalente, fornecendo uma avaliação da dose biologicamente activa do metal a nível celular. Os resultados indicam ainda um potencial risco genotóxico e de stress oxidativo associado à exposição ocupacional dos trabalhadores nas industrias estudadas, que poderá ser devido a alguns ou vários dos compostos presentes no ambiente industrial, incluindo os compostos de crómio.

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1

1. Introduction

1.1. Historical overview

Chromium discovery is linked to the Beresof mines of Siberia, a source of gold, copper, silver, and lead since 1752. The miners were familiar with a red mineral that was found in small amounts accompanying lead ore. Johann Gottlob Lehmann, investigated this mineral, known as crocoite or crocoisite, and in 1766 he found it produced an emerald-green solution when dissolved in muriatic acid (hydrochloric acid). He died the next year when a retort containing arsenic burst upon heating, but in 1797 the French chemist Nicolas-Louis Vauquelin, noting its beauty, scarcity, value equal to gold, and several contradictory chemical analyses, was determined to find the correct composition of crocoite. He boiled pulverized crocoite (PbCrO4) with two parts soda ash obtaining a yellow solution. The solution formed a beautiful red precipitate with a mercury salt, and a yellow precipitate with lead. Adding tin muriatic turned the solution green. In 1798 he precipitated the lead with muriatic acid, dried the green solid, then cooked it for half an hour on a charcoal crucible. Upon cooling he discovered a network or gray metallic needles, weighing one-third of the original. Because of the many colors of its compounds, he gave the new element the name chromium, after the Greek word chroma, meaning color.

The number of products and industrial processes that include chromium compounds is impressive. One of the earliest uses of chromium salts (since the 1800’s) was the production of dyes and pigments. Several chromium compounds possess deep, vivid colors, that became signature tones: for example, Chrome Yellow or lead chromate, used by Van Gogh in his masterpiece series “Sunflowers” (Figure 1), or the award winning Siberian Red, made from crocoite. Some of these pigments are still used today, and may be present in cosmetic products.

Leather tanning was another early use of chromium, developed in the late 19th century in the United States of America, and quickly adopted around the world: today approximately 90% of all leather is produced by chromium tanning. Basic trivalent chromium compounds are used in the leather production as a chelating agent to stabilize collagen fibers in the animal skin, providing it with the known thermal and hydro resistance of leather. Chromium tanning is still the most economically advantageous method to produce good quality leather, and is not likely to be completely replaced by alternative tanning agents in the near future.

Figure 1. Van Gogh's "Sunflowers", 1888

2

In 1893 Henri Moisau used chromium to make the first ferrochromium alloy. Presently, the steel industry, including chrome plating, alloy production, and welding, is the major consumer of the metal. The deposition of a fine layer (0.5-10 µm) of metallic chromium over a structure of another metal or alloy, known as chrome palting, creates a shield against corrosion, given that the oxidation of elemental chromium is extremely slow. This deposition is obtained by electrolysis, with a solution of chromic acid, and using the piece to cover as a cathod. Stainless steel is used as reference to a family of alloys that share an increased resistance to oxidation, chemical inertness, and hardness. A large number of these alloys include chromium at 1-30%. Rust and corrosion inhibition can also be achieved by the use of solutions containing hexavalent chromium, for example in industrial cooling towers.

Other applications of chromium compounds are more related to ludic activities, as in audio recording: because of its paramagnetic behavior, chromic oxide is used in high quality magnetic recording tapes (the spin alignment of unpaired electrons in CrO2 allows magnetic recording and retrieving of sound waves). Also photography pioneers were pleasantly surprised by the chemical change of silver dichromate when exposed to visible light, which allows imprinting of an image onto a solid support.

Chromium also finds application as a refractory material and other uses in high temperature settings. Chromite ore (FeOCr2O3), the major source of chromium, is used in the manufacture of bricks and lining material for protection of industrial and research facilities against high temperature. Barium chromate is also used in safety matches and pyrotechnics and jointly with calcium chromate in high temperature batteries. Nuclear research applies chromium layers in the radiation shields; in most chemical and life sciences laboratories, the chromo-sulfuric mixture (chromium trioxide in water and concentrated sulfuric acid), used to assure complete chemical cleaning of glass material, was until recently a familiar presence, and the chromate-dichromate equilibrium is used as a pH indicator in analytical chemistry. In medicine, chromium solutions were used as astringents and antiseptics. The artificial radioactive isotope 51Cr was commonly used in medicine to estimate the average life span of red blood cells.

In drilling, organic salts of trivalent chromium (e.g. chromium lignosulfate) are used as adjuvants in perforation for oil and other intents, and hexavalent chromium is present in construction in the composition of cement and derived products. Chromium compounds also have application in wood preserving, textile and automobile industry, and military activities.

Though not intending to be complete, this list may give an idea of the extension of chromium presence in modern human society.

For an element in such high demand, obviously there was an immense effort in extraction and production of chromium compounds, mainly by the mining of ferrochromite ore deposits, followed by the reduction with aluminum, carbon or

3

silicon, to produce metallic chromium, or the roasting of the ore with soda ash, to originate sodium chromate and dichromate.

Such a wide use has led to the exposure of millions of people worldwide to the products and sub-products of extraction, production and industrial use of chromium compounds, and the effects of such exposure were soon visible. The first reports on nasal tumors among Scottish chrome pigment workers date from 1890, and they alerted the scientific community for the problem of chromium occupational toxicology. Several other reports followed those initial findings, and in 1936, lung cancer was accepted in Germany as work-related disease in chromate workers. Since then, a long sequence of epidemiological studies and case reports has accumulated, confirming the earlier findings among the chromate-based industries. Effects of acute and chronic chromium exposure have been reported to the scientific and public community, discussed in the press and even inspired the film industry (see Appendice 1). At the same time, investigations started to point out the importance of the species of chromium present, and from the two most biologically relevant valences, Cr(III) and Cr(VI), hexavalent chromium was the protagonist of toxicity. Other toxic effects of chromium were identified, namely its strong allergen action, nephrotoxicity, non-carcinogenic respiratory effects such as impaired pulmonary function, bronchitis and asthma. But not only deleterious effects were uncovered: in humans and animals deprived of chromium intake, the essential nature of chromium compounds was acknowledged in 1950, and attributed to the trivalent form of the metal. Chromium adds now to the number of micronutrients that have a dual action in the human organism, being essential to its homeostasis but also potentially toxic when concentrations, chemical forms or exposure routes change.

The environmental contamination by chromium waste disposal is also of concern. Chromium is one of the major contaminants in various sites worldwide, including the Superfund sites in the United States (US EPA, 2002). Substantial amounts of chromium enter sewage treatment plants in major cities, and the World Health Organization (WHO, 1988) estimated that waste water from chromium industries ranged from 40 mg/l (tanning industry) to 50 000 mg/l (chrome plating). These levels must be reduced by precipitation before waste water can be discharged, but this process results in the production of chromium-rich muds whose final faith is landfill, incineration or dumping in the ocean. None of these solutions seems ideal. In particular, the use of chromium residues as landfill for construction sites has elicited concern on the safety of surrounding populations.

Finally in 1987, and later in the 1990 reevaluation (IARC, 1987, 1990), the International Agency for Research on Cancer classified hexavalent chromium compounds as a known human carcinogen, Group 1 (sufficient evidence in humans) whereas trivalent and elemental chromium were considered non classifiable as to their carcinogenicity to humans (Group 3).

Nevertheless, chromium remains one of the most widely used industrial metals and is constantly present in our daily life and our household, included in our

4

stainless steel silverware, our photographic film, the toner in our photocopies, the leather in our shoes, and even in cosmetics and vitamins. It seems impossible, in a near future, to completely eliminate occupational and environmental exposure to this metal. Consequently, the need to understand the multiple aspects of chromium toxic action, in order to minimize the risk involved in that exposure, becomes of capital importance. 1.2. Physical and chemical properties

Chromium is a transition metal (Group VIB, atomic weight 52) showing a white lustrous shine when polished, somewhat fragile when pure, but extremely hard when used as an alloy component. The redox biochemistry of chromium is rich, involving oxidation states from -2 to +6, from which by far the most stable are the elemental Cr (0), Cr(III) and Cr(VI) valences (Cohen et al., 1993). Elemental chromium does not occur naturally on Earth. Hexavalent chromium is also mostly man-made, but it exists in the rare mineral crocoite, previously referred. The prevailing common forms of chromium in Nature are the trivalent compounds, present in ferrochromite, the most important source of the metal (Barceloux, 1999). The ground state electron configuration of elemental chromium is 1s2 2s2p6 3s23p63d5 4s2. Divalent chromium compounds are usually blue and have alkaline properties. Cr(III) is a chelating agent, allowing the formation of hexadentate complexes, and its salts are amphoteric and can acquire colors ranging from purple to green. Hexavalent compounds are acidic and usually yellow or orange. Changes in oxidation state are regulated by the reduction potentials presented in Table 1. Solubility of hexavalent chromium compounds seems to be an important factor of toxicity (see Section 2.3) and is summarized in Table 2.

Table 1. Standard reduction potentials of chromium compounds

From Katz and Salem, 1993

5

Table 2. Solubility of hexavalent chromium compounds at 20-30 ºC

From Katz and Salem, 1993

6

1.3. Essentiality

In the 1950’s, experiments conducted by Schwarts and Mertz with rats fed chromium deficient diet demonstrated the importance of this metal in glucose metabolism: without this micronutrient, the animals developed a glucose intolerance that was reversed with chromium supplementation (reviewed in Davis and Vincent, 1997). Human chromium deficiency is linked to protein-calorie malnutrition and also appears in patients receiving total parenteric nutrition devoid of chromium supplementation (Katz and Salem, 1993). In humans and animals, Cr(III) is considered an essential nutrient that plays a role in glucose, fat, and protein metabolism by potentiating action of insulin (ATSDR, 2000). Chromium is believed to be part of the glucose tolerance factor, of uncertain structure, that has been isolated from acid-hydrolyzed porcine kidney powder and from Brewer´s yeast. Deficiency of chromium in humans and other mammals causes symptoms that resemble diabetes (decreased glucose tolerance, increased concentrations of circulating insulin) and related cardiovascular disease (elevated cholesterol and triglyceride levels, reduced high-density lipoprotein cholesterol levels). The U.S. National Research Council recommends a dietary intake of 50–200 µg/day (NRC 1989). Food elements contain chromium in a range of 5-250 mg/kg, with pepper, brewer’s yeast, calf liver, cheese and wheat germ reporting the highest concentrations (Rojas et al., 1999). Natural drinking water usually contains 1-10µg/L of chromium, but this can be higher in regions with substantial geological deposits of the metal.

Chromium is reported to increase lean body mass and to decrease percentage body fat, which in turn may lead to weight loss, although these results are controversial. There are a number of human studies on chromium supplementation in volunteers looking to lose weight or increase their muscle mass, but they seem to show that chromium may have only a small role in these outcomes, when compared with the importance of balanced diet and exercise (Anderson, 1998). Nevertheless, chromium picolinate, a trivalent form of chromium complexed with picolinic acid, is still widely included in human nutritional supplements and has been recommended to use in cattle and swine diets (NRC, 1997).

7

2. Chromium toxicity 2.1. Carcinogenic and genotoxic effects 2.1.1. Human Studies

Epidemiology is defined as the science investigating the effect of different factors and environmental conditions on the incidence and spreading of infectious and non-infectious diseases. Epidemiologists achieve these objectives by collecting data on the natural history and frequency of disease in human populations and analyzing it in correlation with potential causes for those pathological conditions. The long-term aim of epidemiological studies is understanding the role of various factors of disease in the development of a pathological state, and using that information to avoid or limit the extent of the risk (Wieslaw and Maugeri, 2000).

The complexity of the development of a pathological state is understood by modern epidemiology, which assumes a multifactor etiology of a disease, based on the interaction between individual factors, characteristics of the agent and the role of the environment, in a balance that can be represented as follows:

Individual factors make each organism more or less susceptible to the harmful effects of the agent, and are dependent on an array of characteristics including heredity, age, gender, state of nutrition, general health condition, immunity, past diseases or past exposures. Environmental factors create an external background that affects the individual response and also the agent’s properties, and can be divided in three important groups: physicochemical (climate, chemical burden, radioactivity), social (socioeconomic conditions, level of information), and biological parameters (biological pathogens such as bacteria and viruses, their reservoirs and vectors). Many of these factors interact between them and, depending on their combination, their final effect may be reinforced (by addition or by synergism) or weakened (by antagonism).

ENVIRONMENT

INDIVIDUAL

AGENT

Figure 2. Interaction between the various components of disease development (from Wieslaw and Maugeri, 2000)

8

Occupational exposure has been a valuable source of epidemiological research in the etiology of cancer (Siemiatycki, 1995). Out of the list of known human carcinogens, many were discovered in the occupational environment. Typically, an occupational risk factor for cancer is discovered by an occupational clinician observing a cluster of cancer cases originating from the same workplace, or with the same activity. Epidemiologists explore the hypothesis, and it is eventually confirmed or dismissed. Occupational toxicology provides the necessary conditions for such discoveries to take place, namely an exposure to concentrations of the toxic agent higher than for the general population, sufficient to produce an observable excess of effects in the worker group, an obvious link between occupation and frequency of cases, and a confluence of multiple cases into a single medical practice. This has led to a drastic increase in epidemiological cancer research related to occupational exposure in the last 25 years, and a high number of prospective, retrospective and case-control studies available for review.

Many professional or leisure activities may involve human exposure to chromium compounds. The most relevant are the following:

- Stainless steel welding (Cr(VI)) - Chromate production (Cr(VI)) - Chrome plating (Cr(VI)) - Ferrochrome industry (Cr(III) and Cr(VI)) - Chrome pigments (Cr(III) and Cr(VI)) - Leather tanning (mostly Cr(III)) A complete list of such activities is available in the ATSDR “Toxicological

Profile for Chromium”, page 305 (ATSDR, 2000). Several epidemiological studies on the occupational genotoxicity chromium

have been conducted among workers involved in such activities, and those studies have also been reviewed by different authors (Langård, 1990; Lees, 1991). In brief, an indubitable excess of lung cancer cases was found among workers in certain hexavalent chromium-based industries, namely chromate production, chromate pigment production and chrome plating. Increased cancer risk of up to 18-32 times the risk for unexposed populations has been reported (ATSDR, 2000).

Higher number of unexpected lung cancer deaths were found in the earlier studies, showing that informed prevention measures have had an effect in reducing cancer risk, most likely due to a decrease in exposure to the metal in the occupational environment.

Chromate production workers in Germany, Italy, Japan, United Kingdom and the United States of America have consistently shown excess risks for lung cancer (IARC, 1990). Cases of nasal cancer were also reported among these workers, exposed to a variety of forms of chromium, including Cr(VI) and Cr(III) compounds.

9

Pigment production activity is also accepted as involving an increase in lung cancer risk, established in facilities where the insoluble zinc chromate was produced (IARC, 1990). These workers are exposed mainly to Cr(VI) compounds, in the raw products and the final pigments.

Workers in chromium plating were also found to have an excessive frequency of lung cancer, particularly among those with more years of employment at chrome baths (Sorahan et al., 1998). These workers are exposed to Cr(VI) compounds and possibly also to nickel (IARC, 1990).

Stainless steel welders have been reported to have an excess mortality from lung cancer and other types of cancer (Danielsen et al., 1993; Sjögren et al., 1994) but no increased mortality was found by other authors (reviewed in Lees, 1991). Welders are exposed to fumes containing Cr(VI), Cr(III) and possibly also nickel. Ferrochromium industry studies did not provide conclusive information about carcinogenic effects (IARC, 1990).

Other workers potentially exposed to hexavalent chromium, such as masons exposed to cement dust have reported an increased lung cancer risk (Rafnsson et al., 1997).

Tanning industry workers were found to have excessive incidence of lung and upper respiratory tract cancer (Garabrant and Wegman, 1984; Sweeny et al., 1985; Mikoczy et al., 1996) and other types of cancer like soft tissue sarcoma, gastro-intestinal and bladder cancer (Mikoczy et al., 1994; Montanaro et al., 1997) but other studies found no increased risk (Stern et al., 1987). These workers are exposed to Cr(III) compounds, although a Cr(VI) contamination could have been possible in earlier studies, when trivalent chromium was sometimes obtained by reduction from Cr(VI) salts in the tanning industry. This process is no longer in use, and great concern is taken in controlling Cr(VI) contamination, both in the tanning products and the finished leather.

In addition to its confirmed action as a respiratory tract carcinogen, chromium may be implicated in the pathogenesis of other cancers in the gastrointestinal tract, but no conclusive evidence has been present so far.

Occupational exposure to chromium is the most important source of information on its chronic toxicity and carcinogenicity, but is not the only one. Environmental contamination with chromium compounds is also an important source of human exposure, and can occur in the proximity of:

- Landfill sites with chromium-containing wastes - Industrial facilities that manufacture or use chromium and chromium-

containing compounds - Cement-producing plants - Industrial cooling towers that previously used chromium as a rust inhibitor. - Waterways that receive industrial discharges from electroplating, leather

tanning, and textile industries (Jordão et al., 1997)

10

- Busy roadways, because emissions from automobile brake lining and catalytic converters contain chromium, as well as the emissions from fossil fuel combustion (Marletta et al., 1989)

- Tobacco smoke (Antilla et al., 1989) The epidemiological studies focused on environmental exposed populations

have not found conclusive evidence that links environmental exposure to chromium and cancer risk (Rowbotham et al., 2000; Hayes, 1997; Fryzec et al., 2001). Nevertheless, biomonitoring studies have confirmed increased absorption and excretion of chromium among populations who live nearby chromium-contaminated sites (Stern et al., 1992, 1998). Chromium is also present in several nutritional supplements due to its essential role in glucose metabolism. No epidemiological study was found regarding the cancer risk in individuals who ingested these supplements.

The determination of endpoints of genotoxicity among groups of individuals selected by a particular parameter of exposure or individual characteristics may provide us with an estimate on the carcinogenic potential associated with that parameter. The most often studied genotoxicity endpoints are structural and numerical chromosomal aberrations assessed using classical cytogenetic methods or fluorescence in situ hybridisation, micronuclei, DNA damage (adducts, strand breaks, crosslinking, alkali-labile sites) assessed using biochemical/electrophoretic assays or sister chromatid exchanges, protein adducts (if correlated with DNA damage). Mutagenesis among human populations is most commonly assessed by the hypoxanthine-guanine phosphoribosyltransferase (HPRT) mutations.

Some studies are available concerning in vivo potential reproductive toxicity of chromium compounds in occupational exposure settings. Welders have been reported to have reduced sperm quality (Bonde, 1990). A significant association between congenital malformations (oral clefts) and maternal occupation in the leather and shoe manufacturing industry was found by Bianchi et al. (1997).

A summary of data on genotoxicity studies among human populations exposed to chromium is shown in Table 3. Genotoxicity assessments in chromium-exposed populations have been mostly focused on occupational exposed groups, but some data on environmental exposure has been reported.

11

Table 3. Genotoxicity studies in human populations exposed to chromium compounds

Endpoint Exposure Valence Result Reference Chromosomal aberrations

Chromium plating Cr(VI) - Benova et al., 2002

Stainless steel welding

Cr(VI) + Koshi et al., 1994 ; Sarto et al., 1982


Cr(VI) - Husgafvel-Pursiainen et al., 1982 ; Littorin, 1983

Leather Tanning Cr(III) + Sbrana et al., 1991 Chrome alum Cr(III) - Hamamy et al., 1987 Sister chromatid exchanges

Chromium plating Cr(VI) - Nagaya et al., 1986 and 1991 ; Benova et al., 2002

Chromium plating Cr(VI) + Lai et al., 1998 Chromium plating,

stainless steel welding

Cr(VI) + Koshi et al., 1984 ; Lai et al., 1998 ; Sarto et el., 1982 ; Stella et al., 1982


Cr(VI) - Littorin et al., 1983


Cr(VI) + Werfel et al., 1998

Micronuclei Chromium plating Cr(VI) + Benova et al., 2002 Stainless steel

welding Cr(VI) - Littorin et al., 1983

Leather tanning Cr(III) + Goulart et al., 2002 DNA strand breaks Stainless steel

welding Cr(VI) + Werfel et al., 1998

Bichromate production

Cr(VI) - Gao et al., 1994

Crosslinks Chromium plating Cr(VI) - Zhitkovich et al., 1996 Stainless steel

welding Cr(VI) + Costa et al, 1993, 1993ª ;

Popp et al., 1991 Environmental ? + Taioli et al., 1994 ;

Zhitkovich et al., 1996 Acute ingestion of

chromium Cr(VI) and

Cr(III) - Kuykendall et al., 1996

Leather tanning Cr(III) + Goulart et al., 2002 Unscheduled DNA repair

Chromium plating Cr(VI) + Pilliere et al., 1992

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2.1.2. Animal studies

Chronic inhalatory exposure to hexavalent chromium compounds is known to increase cancer frequency in animal studies, as recently reviewed by ATSDR (2000). Mice exposed to 4.3 mg Cr(VI)/m3 had a 2.8-fold greater incidence of lung tumors than controls; lung tumors were observed in 3/19 rats exposed to 0.1 mg Cr(VI)/m 3 as sodium dichromate for 18 months, followed by 12 months of observation. Chronic oral exposure to hexavalent chromium compounds did not show a significant increase in cancer incidence, although rats exposed to 9 mg Na2CrO4/kg/day in drinking water, for 3 generations (880 days) showed a non-significant increase in forestomach carcinoma and papilloma. Chronic and sub-chronic oral bioassays in rats fed diets or drinking water containing up to 2g/kg/day of Cr(III) did not show any increase in cancer frequency (reviewed in US EPA, 1998). Also, no increased tumor frequency was found in chronic and sub-chronic inhalation of Cr(III) compounds, by exposure to dust (7.8 to 13 mg Cr/m3, 0.5 hour/day, 5 days/week for a maximum of 52 weeks), intratracheal instillation (5 to 15 x 0.01 and 0.05 mg, at 2, 4 and 6 weeks intervals), or intrabronchial implantation (2 mg chromium-containing material, 2 years).

Table 4. Genotoxic studies in laboratory animals exposed to chromium compounds Endpoint Animal Valence Dose/duration Result Reference

Chromosomal aberrations

Rats, inhalation

Cr(0), Cr(VI)

1.84 mg/m3, 1 week and 0.55 mg/m3, 2 months

+ Koshi et al., 1987

Mouse, p.o. Cr(VI) 20 mg/kg, gavage + Sarkar et al., 1993

SCE Rat, inhalation

Cr(0), Cr(VI)

1.84 mg/m3, 1 week and 0.55 mg/m3, 2 months

+ Koshi et al., 1987

Micronuclei Mouse, p.o. Cr(VI) 86 mg/kg, gavage - Shindo et al., 1989

Mouse, p.o. Cr(VI) 1-20 ppm in drinking water or 4µg/kg bolus

doses, 2 days

- Mirsalis et al., 1996

DPC Rat, p.o. Cr(VI) 6 mg/kg/day, 3 or 6 weeks

+ Coogan et al., 1991

Rat, i.p. Cr(VI) Bolus dose + Tsapakos et al., 1981

Rat, i.p. Cr(III) Bolus dose - Cupo and Wetterhahn, 1985

UDS Rat, p.o. Cr(VI) 1-20 ppm in drinking water or 4µg/kg bolus

doses, 2 days

- Mirsalis et al., 1996

Lethal dominant assay

Mouse, i.p. Cr(VI) Bolus dose + Paschin et al., 1982

Mutation frequency

Mouse, i.p. Cr(VI) 40 mg/kg, bolus dose 1 or 2 days

+ Itoh and Shima-da, 1997, 1998

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Genotoxic effects of chromium exposure in animals have been studied by several authors (Table 4). Hexavalent chromium has produced relatively consistent positive results in genotoxic endpoints although the majority of experimental protocols are based on exposure routes that do not relate to human exposure conditions. Reports on trivalent chromium short to medium term genotoxicity have been mostly negative.

Decreased fertility was seen in both male and female rats exposed to welding fumes, and the rate of fetal death was increased in exposed pregnant female rats (IARC, 1990). An influence of Cr(III) preconceptional exposure for the development of cancer was found in rats (Yu et al., 1999).

2.1.3. Short-term assays

Hexavalent chromium has consistently proven to be mutagenic in a variety

of living test systems (reviewed in ATSDR, 2000): multicellular (newt larvae, Drosophila melanogaster), prokaryotic (Salmonella typhimurium, Bacillus subtilis, Escherichia coli), eukaryotic (Saccharomyces cerevisiae, Schizosaccharomyces pombe,), and a vast number of in vitro studies in mammal cell cultures (reviewed in Snow, 1992). Exposure of eukaryotic and prokaryotic cells to Cr(VI) can produce oxidative DNA damage, Cr-DNA adducts, DNA-DNA and DNA-protein crosslinks, and mutations (Cohen et al., 1993). Cr(VI) has also been shown to alter gene expression (Hamilton et al., 1998), activate stress-response pathways (Dubrovskaya and Wetterhahn, 1998), trigger apoptosis (Manning et al., 1994) and cause aberrant DNA methylation (Klein et al., 2002). Conversely, only a small portion of trivalent chromium studies has reported positive results. Nevertheless, evidence of mutagenic and genotoxic activity of Cr(III) was found in Salmonella typhimurium, S. cerevisiae (Bronzetti et al., 1986), non-human mammal cells (Elias et al., 1984, Bianchi and Levis, 1988), and human lymphocytes (Friedman et al., 1987; Rajaram et al., 1995).

Despite its widely proven genotoxicity, hexavalent chromium is unreactive towards DNA in acellular systems and in the absence of reducing compounds. Trivalent chromium, on the other hand, is directly reactive towards the genetic material and able to form stable complexes with DNA in acellular systems. These stable adducts have proven to be mutagenic in replicating human cells (Zhitkovich et al., 1995; Tsou et al., 1997). For a review on the mechanism of chromium genotoxicity, see Section 2.5. Trivalent chromium may also have a role in carcinogenesis through an epigenetic mechanism, by affecting the kinetic parameters of DNA synthesis. In the presence of chromium there is an increase in the processivity of DNA-polymerases and a decrease in fidelity, thus increasing the number of possible mutations in the newly formed DNA chain (Snow, 1991, 1994, Singh and Snow, 1998). The nutritional supplement Cr(III) picolinate has also been shown to cleave DNA (Speetjens et al., 1999), but toxicity has been attributed to the organic ligand.

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2.2. Other toxic effects Acute exposure to high doses of chromium compounds is described in a few reports of accidental or suicidal cases. Hexavalent chromium compounds originate ulceration and inflammation of the direct contact tissue (skin, gastro-intestinal organs). When ingested in large amounts, hexavalent chromium produces abdominal pain, vomiting, diarrhea and intestinal bleeding. Death usually derives from renal failure resulting from tubular necrosis, or combined hepatorenal failure. Estimates of LD50 for hexavalent chromium (sodium chromate) range from 50-150 mg/kg body weight, while acute oral toxicity for trivalent compounds (chromium chloride) ranges between 1900-3300 mg/kg body weight. The best therapy for acute oral intoxication seems to be aggressive dialysis, but the administration of ascorbic acid has also been recommended (Bradberry and Vale, 1999) in an attempt to extracellularly reduce Cr(VI) to Cr(III), and slow down the rate of chromium entering the cells (see Section 2.5. for further discussion on the role of ascorbic acid in chromium reduction).

Chronic and sub-chronic exposure to low doses of hexavalent chromium may also induce kidney damage, as reported by increased excretion of enzymes such as n-acetyl-β-glucosaminidase (Liu et al., 1998) and low molecular weight proteins such as β2-microglobulin and retinol-binding protein (Franchini and Mutti, 1988). In vitro exposure of mammal kidney cells to dicromate showed a decrease in viability and increase in cell volume when cells were exposed to 10 µM - 250 µM concentrations of hexavalent chromium (Dartsch et al., 1998).

Chronic or sub-chronic human exposure to hexavalent chromium compounds by inhalation increases the risk for respiratory cancer, but also other non-carcinogenic respiratory tract alterations. Symptoms of impaired pulmonary function, assessed by respiratory flow and volume measurements, along with chronic bronchitis, were reported in welders, and the prevalence of effects correlated with both cigarette smoking and the measure of lifetime exposure to welding fumes (Bradshaw et al., 1998). Health surveys among tannery workers reported a high number of complaints on asthma and chronic bronchitis (Őry et al., 1997), with a higher incidence of sickness absence occurring in small tanneries. The lung toxicity of inhaled trivalent chromium compounds has also been evaluated by animal based studies, that showed a clear effect on the alveolar macrophages, namely enlarged lysosomes and increased number of surfactant-like inclusions (Camner and Johansson, 1992; Johansson et al., 1992). In summary, gross and histopathological changes to the respiratory tract resulted from inhalation of Cr(VI) compounds or a combination of Cr(VI) and Cr(III) compounds, but both Cr(VI) and Cr(III) altered macrophage function.

Another known toxic effect of hexavalent chromium is its ability to cause ulceration of the nasal mucosa and skin in inhalatory and dermal exposure, due to its strong oxidizing and corrosive properties. In workers exposed to high

15

concentrations of chromium compounds there is also a more serious risk of nasal septum perforation. The nasal tissue lesions may be due to airborne chromium mist and fumes or to the direct contact of nasal mucosa with contaminated hands. The increase in industrial hygiene measures and the decrease in chromium concentrations in the occupational environment have been successful in diminishing the frequency of such lesions.

Hexavalent chromium is also a strong allergen, second only to nickel, and is a known causal agent of allergic contact dermatitis, with a high number of reported cases among construction workers exposed to cement (Wong et al., 1998). Chromium induces a type IV (delayed) hypersensitivity reaction that involves the production of lymphokines by sensitized T-cells and induces a serious inflammatory condition. Certain sensitized individuals can be so severally affected by their chromium allergy that they have been named “chromium cripples” (Fisher, 1976), as they are practically disabled by a continuous or recurrent dermatitis. Allergic reactions to chromium were also evaluated in exposure to environmental relevant concentrations, but these low levels of exposure seemed insuficient to elicit a response (Fowler et al., 1999). Trivalent chromium may have significantly milder allergenic abilities but positive responses have been obtained in studies with chromate sensitized individual challenged with trivalent chromium solutions (reviewed in Katz and Salem, 1993) or patches of tanned leather (Nygren and Wahlberg, 1998). A surprising example of chromium sensitization is the case of allergenic reactions among black jack card-game players, caused by the chromium-based green pigment of the game tables (Fisher, 1976). The concentration of chromium (total and water) and other metals has also been investigated in beauty products, and high levels of chromium among various commercial products, including some high-profile brands, may pose an allergic threat to sensitized individuals (Sainio et al., 2000).

Cr(VI) also interacts with red blood cells, causing an alteration in size, shape and physiological properties. Hemoglobin is effectively oxidized by Cr(VI) in vitro (Alpoim et al., 1995).

2.3. Importance of physical and chemical properties in chromium toxicity

It is well accepted that the toxicity of metals relies not only on their elemental characteristics but also on the physical-chemical properties of their various compounds. Several metals are known that have significantly different toxic actions according to the characteristics of the specific metal form present, as in the cases of mercury and arsenic (Goyer, 1996). Not all cases have been so well clarified, and chromium toxic action is also a good example of this complexity. The hexavalent form is regarded as the primary toxic threat, due to its easy passage through biological membranes (see uptake-reduction model, Section 2.5.), in contrast with the trivalent form, considered quite less toxic due to less efficient membrane passage. Nevertheless, trivalent chromium absorption has been

16

demonstrated in workers exposed to this valence state, which indicates that the rate of uptake of Cr(III) by the cells may be slower, but effective in chronic occupational exposure settings.

The nature of the salt of chromium present is also an important factor of absorption and toxicity. Organic complexes of trivalent chromium are absorbed to a greater extent than inorganic compounds, due to a better solubility in biological membranes (Kiilunen et al., 1983). The use of organic Cr(III) salts (chromium picolinate) as chromium supplements is based on this feature.

The water solubility of the chromium salts may also condition the toxicity of the metal. Moderately soluble and insoluble or slightly soluble hexavalent chromium compounds appear to be the most potent carcinogens in experimental animals (Katz and Salem, 1993), and the insoluble zinc chromate and slightly soluble (5.8 µg/L at 25ºC) lead chromate seem to be the most potent carcinogens in occupational exposure (Langård, 1990). Soluble chromium is more rapidly cleared from the lung into the systemic circulation, and therefore inhalation of less soluble chromium compounds may result in increased accumulation in the respiratory system, the only confirmed target site for carcinogenesis (De Flora, 2000). This may be the explanation for the inverse correlation between solubility and toxicity. 2.4. Toxicokinetics

Hexavalent and trivalent chromium compounds can be absorbed by inhalation and ingestion. Dermal contact to high chromium concentrations induces systemic toxicity, particularly when a pre-existing condition, such as necrosis of the skin, enhances chromium penetration. Hexavalent chromium is absorbed more efficiently than trivalent chromium, probably due to the easy trans-membrane passage of Cr(VI), as discussed in Section 2.5, but Cr(III) exposed workers also have an increase of chromium concentration in plasma and urine after the work shift.

Lung is the only tissue in the organism for which chromium concentration increases steadily with age in the general population, not occupationally exposed to the metal (WHO, 1988). Lung tissue from workers exposed to chromium has concentrations of the metal that may be several orders of magnitude higher than non-exposed individuals (WHO, 1988). This may reflect an accumulation of chromium that is inhaled from the environment.

17

The model for chromium absortion, distribution and excretion, as proposed by O’Flaherty (1996) is represented in Figure 3. A two-compartment model is accepted for both Cr(III) and Cr(VI), with a slightly shorter first rate half-life for hexavalent chromium, but in both cases there is rapid phase of chromium clearance

mainly due to the metal’s rapid urinary excretion) T½ 7 to24 hours followed by a slow phase, T½ = 15-30 days. Inhaled chromium can be divided into two pools: the larger diameter particles are cleared from the upper respiratory system by mucociliary action, are swallowed and move on to the gastro-intestinal system, while the smaller particles are deposited in the lungs, where chromium may be absorbed or become retained in the pulmonary tissue, depending on the solubility of the compound present, ability to cross cell membranes, reaction with tissue proteins and/or with biological reducers (Perrault et al., 1995). Hexavalent chromium may undergo reduction to Cr(III) in the blood compartment, which may limit the amount of chromium available to enter the cells since membranes are less permeable to the latter. (De

URINE EXCRETION

EXCRETION HAIR, NAILS

DERMAL EXPOSURE

KIDNEY Cr(VI) Cr(III)

LIVER Cr(VI) Cr(III)

BONE Cr(VI) Cr(III)

R

PLASMATIC REDUCTION

Cr(VI) Cr(III)

PLASMA PL

ASM

A R

ED C

ELLS C

r(VI)C

r(III)

GI TRACT Cr(VI) Cr(III)

FECAL EXCRETION

POOL A Cr(VI) Cr(III)

ORAL EXPOSURE

INHALATION EXPOSURE

R

LUNG

POOL B

R

Figure 3. Toxicokinetic model for chromium (adapted from O’Flaherty, 1996).

R

R

Particle diameter >5 µm

18

Flora et al., 1997). Within the tissues, a reduction from Cr(VI) to Cr(III) may increase retention (R) and accumulation of intracellular chromium. Absorbed chromium exists in plasma in the form of stable aqua-complexes and bound to plasmatic proteins (O’Flaherty, 1995). 2.5. Mechanisms of chromium genotoxicity

The mutagenic and genotoxic activity of hexavalent chromium is not yet fully understood. There are several possible mechanistic models describing the genotoxicity of hexavalent chromium, which may complement each other (Cieślak-Golonka, 1995). The most widely accepted is the uptake-reduction model, first proposed by Jennette (1979, 1981) and represented schematically in Figure 4 (adapted from Dartsch et al., 1998).

Figure 4. Uptake-reduction model (adapted from Dartsch et al., 1998)

The uptake of hexavalent chromium by the cell is very efficient: the

chromate ion, which is the predominant form of Cr(VI) in aqueous solution at physiological pH (Cieślak-Golonka, 1995), benefits from a tetrahedral structure similar to essential anions such as sulfate or phosphate. That explains its easy trans-membrane passage, through a general anion transport system, which is blocked to the octahedral structure of the Cr(III) aqua-complexes.

Slow uptake by passive difusion and endocytosis

Extracellular space

Cytoplasm

SO42- ,PO4

3-

Chromate Anion (CrVI) Octahedral

Cr(III) Complex

Anion transport

Anion transport

Cell membrane

CrVI reduced to CrIII by Glutathione, cysteine, ascorbate, cytochrome P450...

Reactive intermediates Radicals and ROS

Chronic toxicity: DNA –protein crosslinks, strand breaks, DNA-chromium adducts.

Acute toxic effects: necrosis and cell death

19

Upon entering the cell, hexavalent Cr reacts rapidly with biological reducers and becomes trapped in lower valence forms. The complex intracellular reduction chemistry of chromium seems to have an important part in the DNA damage. The most common cellular non-enzymatic cellular reducers of chromium are ascorbate, glutathione and cysteine, and these can act as antioxidants as well as active reagents in the destruction of reactive oxygen and radical species (Cieślak-Golonka, 1995). The reduction mechanisms as well as the spectrum of intermediate species formed in the reactions driven by each reducer appear to be different (Kitagawa et al., 1988; Stearns, 1994; Shi, 1994). All three cellular reducers interact with hexavalent chromium under physiological conditions, and a synergistic effect between ascorbic acid and glutathione was reported by Suzuki (1990), making the reduction more efficient (≈30% increase) when these reducers are in 1:10 proportion.

Ascorbic acid in not biologically synthesized by humans but is found naturally among other animals and plants, which provide the necessary intake in human diet. In vivo the most important characteristic of ascorbic acid is its role as a reducer. This reaction can undergo a single- or a two-electron pathway. It has been shown that the interaction of Cr(VI) with ascorbic acid at pH 7.4 originates the intermediate Cr(V) and/or Cr(IV) formation and finally in Cr(III)-dehydroascorbate complex, which is compatible with the two electron reduction pathway. It was also found that the reduction of hexavalent chromium by ascorbate is faster than by glutathione (Suzuki and Fukuda, 1990).

The most important SH-containing compound is glutathione (GSH), or γ-glutamyl-cysteinyl-glycine), and it is also the most abundant low-molecular thiol. GSH occurs in the cell in two forms: reduced (GSH) and oxidized (GSSG), interchangeable as follows:

2GSH ↔ GSSG + 2H+ + 2e- This balance creates an important redox system, which is catalyzed by metal

ions. GSH is responsible to a great extent for the cytoplasmic reduction of the chromate ion, during which several oxygen species are formed, namely superoxide radical (O2

-), hydroxyl ( OH) radical and singlet oxygen. The accepted mechanism for this reduction in neutral pH is as follows (O’Brien and Ozolins, 1989):

Cr(VI) + GSH→ Cr(VI)SG + H+

Cr(VI)SG + GSH → Cr(IV) + GSSG + H+ 2Cr(IV) → Cr(V) + Cr(III)

But in acid solution, the reaction is able to generate reactive species of

radicalar nature from the glutathione molecule (see next page):

20

CrO42- + GSH ↔ O3CrSG- + OH-

Cr(V) + GS GSSG + Cr(IV) Cr(V) Cr(III) This pathway has also been demonstrated to occur under physiological

conditions (O´Brien and Wang, 1992), and the presence of Cr(V) was detected in chick embryo liver and red blood cells, and in whole living animals treated with hexavalent chromium (Liu et al., 1994). The thiyl radical can further proceed the reaction and generate H2O2 in this way:

GS + GSH → GSS G- + H+ GSS G- + O2 → GSSG + O2

- 2 O2

- + 2 H+ → H2O2 + O2 The production of H2O2 during the reduction of Cr(VI) by GSH has been

supported by EPR studies that show that an aqueous solution of Cr(IV)-GSH complex consumes molecular oxygen (Liu et al., 1997).

The intermediate Cr(V) and Cr(IV) or other metals may be involved in a Fenton reaction to generate hydroxyl radicals from the hydrogen peroxide (Shi et al., 1999):

Cr(IV) + H2O2 → Cr(V) + OH- + OH Cr(V) + H2O2 → Cr(VI) + OH- + OH Fe(II) + H2O2 → Fe(III) + OH- + OH

There is much less published work, and knowledge, about cysteine Cr(VI) reduction, but it has also been shown to have an initial higher reduction rate than does glutathione (Connet and Wetterhahn, 1983).

Trivalent chromium does not undergo reduction by the cell, but evidence of oxidative damage was reported by in vitro studies with this valence (Shi et al., 1993; Tsou et al., 1996; Lloyd et al., 1998). Hydroxyl ion radical generation was shown to result from the reaction of Cr(III) with H2O2 and lipid hydroperoxides, although highest values of OH are reached only at alkaline pH (pH 10). The proposed mechanism for the Cr(III)-dependent OH generation is the Haber-Weiss reaction (Shi et al., 1998), as follows:

O2- + Cr(III) → Cr(II) + O2

Cr(II) + H2O2 → Cr(III) + OH+OH-

A possibility of Cr(II) generation when Cr(VI) is reduced by ascorbic acid according to the two-electron transfer module has also been suggested.

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The production of oxygen and radical reactive species during the intracellular metabolism of chromium compounds, described above, is one of the possible mechanisms through which chromium exerts its toxicity. Nevertheless, it has been proven that chromate-exposed cells contain a considerable amount of Cr(III)-DNA adducts, and that the most abundant form (up to 50%) of DNA-damage in these cells are ternary Cr-DNA adducts involving amino-acid ligands (cysteine, histidine and glutamic acid) or glutathione (Voitkun et al., 1998; Zhitkovich et al., 1995). Recently, ascorbic acid as also been shown to be involved in these complexes (Quievryn et al., 2002).

The ternary complexes, or chromium-mediated DNA-protein crosslinks, seem to be formed by the initial complexation of Cr(III) with the amino-acid (AA), and subsequent reaction with DNA (Zhitkovich et al., 1996a). No difference was found between Cr(III)-AA complexes isolated from Cr(VI) in vitro reducing mixtures and those formed by the direct reactions of Cr(III) with AA ligands, therefore the reduction step does not seem to be determinant for this pathway of toxicity. Ternary Cr(III)-DNA adducts with AA or GSH have been found to be mutagenic in human cells (Zhitkovich et al., 1998), the mutagenic activity increasing with the volume of the complex (Zhitkovich,1995). Binary Cr(III)-DNA complexes caused minimal mutagenicity.

Experiments conducted with initial binding of AA to Cr(III) suggest that the initial binary Cr-AA complex probably contains only one AA, since multidentate ligands of Cr(III) are quite stable (Zhitkovich et al., 1996a). The lack of interaction of Cr(III)-AA complexes with nucleosides, as well as the prompt reaction with mononucleotides, points to the phosphate group as the primary binding site. Cr(III) has also been shown to bind preferably to the phosphate group when in aqueous solutions (Fiol et al., 1984). The proposed structures of phosphate-based Cr(III)-DNA adducts are represented by Figure 5 (from Zhitkovich et al., 2001). Numbers I and II represent the S and R isomers of DNA alkylphosphotriesters. Number III shows a possible configuration of the Cr(III) – SDNA adduct; numbers IV, V and VI represent the Cr(III)-RDNA adduct, stabilized by further coordination or hydrogen bonding with other atoms of the purine base, whereas the adduct structure represented on VII does not allow additional stabilization. Numbers IV and V are, respectively, the mutagenic forms of binary Cr(III)-DNA adducts and ternary AA-Cr(III)-DNA adducts proposed by Zhitkovich et al. (2001).

The covalent binding of proteins to DNA can have serious genetic consequences, by disrupting the natural process of protein interaction with DNA, affecting critical events such as gene activating and silencing or the replication of DNA during phase S of the cell cycle (Costa et al., 1993). Indeed, alterations of the cell cycle have been reported by exposure to chromate ions (Xu et al., 1996), and Cr(III) was shown to affect the processivity of DNA polymerase (Snow, 1991, 1994). The previously mentioned mutagenicity of DNA-protein crosslinks can be derived from the difficult access to DNA bases “buried” under those complexes (Briggs and Briggs, 1988).

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I. II.

III. IV.

V. VI.

VII.

Figure 5. Structures proposed for the phosphate-based Cr(III)-DNA adducts (from Zhitkovich et al., 2001). See text for discussion.

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The extra cellular reduction of hexavalent chromium is regarded as a

process of detoxification, transforming chromate to the less permeable form of trivalent chromium (Petrilli and De Flora, 1988). The intracellular reduction may on the other hand, pose a higher threat due the possibility of activating either of the proposed toxic pathways of chromium genotoxicity: the generation of reactive oxygen or radical species and of lower valence forms of chromium. Also, the reduced forms of chromium, particularly Cr(III) compounds have shown to be directly reactive towards DNA (Arakawa et al., 2000) and the only limitation seems to be the slow uptake of this form of chromium by the cell. Nevertheless, there is no known process for the release of Cr(III) from the intracellular medium, and thus there is an accumulation throughout the life span of the cell (Jennette, 1981). This may be specially important in chronic exposure to moderate or high concentrations of Cr(III), as it is the case in occupational settings. As previously mentioned, ascorbic acid was proposed as therapy for hexavalent chromium intoxication, and masks with a filling containing ascorbic acid have been used to reduce the inhalation of Cr(VI) fumes in occupational exposure (Connett and Wetterhahn, 1983). Measures such as these should be evaluated, since the chronic toxicity of lower valence forms of chromium must be carefully examined. 2.6. Elements researched 2.6.1. Biomonitoring and molecular epidemiology

Biological monitoring, or biomonitoring, is defined as the “systematic determination of indicators in human biological fluids such as blood and urine to evaluate the uptake and health effects of chemicals” (Christensen, 1995). Occupational or environmental exposure to chemical and physical toxic agents may pose short and long-term health risks, and consequently several regulations have to be implemented in order to reduce that exposure. Biological monitoring is used for the risk assessment involved in establishing the permissible values for each toxic agent in occupational and environmental exposure, and to verify compliance with existing regulations. The determination of airborne pollutants (environment monitoring), when possible, is another important way to quantify exposure; nevertheless biological monitoring provides an internal dose assessment, reflects all sources and routes of exposure, may give a better estimate of the dose reaching the target site, and is useful to identify subgroups of individuals subject to higher levels of exposure (Christensen, 1995).

The protocol of a biomonitoring study can determinately affect the outcome and value of the research. The strategy for conducting a population monitoring study comprises several phases (compiled from Au, 2000, Albertini et al., 2000 and Zober and Will, 1996):

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A. Decision to perform biomonitoring This can be a straight-forward decision, if there is a continuing program of biomonitoring in a specific population (e.g. blood lead surveillance among lead workers) or it may arise from new toxicological data that triggers a targeted risk assessment directed to a potentially exposed group. In this phase, information on the morbidity and mortality patterns of a population may provide an insight to increased risk. B. Gather of information on the toxic agent Evaluating present knowledge on characteristics such as metabolism, mechanism of action and toxicokinetics of the toxic agent in question is of critical importance, as well as reviewing previous biomonitoring or experimental studies. This should allow the formulation of a hypothesis, and the choice of the appropriate endpoints to be applied. C. Population characteristics In this step, there is the need to consider the size of the population necessary to provide statistical power to the findings of the study. The three important determinants of statistical power are (i) the magnitude of the expected (assumed) effect of exposure to a genotoxic agent, (ii) the variability of the biomarker and (iii) the size of the study population. A preliminary study may be useful in determining the first two parameters; to attain the desired statistical power, the number of subjects should then be defined accordingly (see Albertini et al., 2000, for a list of user-friendly software that allows that calculation). Matched non-exposed controls should also be carefully chosen. Also in this phase other factors must be analyzed, such as the motivation of the population to participate in the study, and ethical considerations regarding, among other factors, the volunteer nature of the study, need for informed consent, assurance of absence of health risk derived from the investigation, non-discrimination of participating and non-participating individuals, privacy of personal information and individual results, information on the global results of the study and access of each individual to his/hers personal results accompanied by a competent interpretation. D. Data to be collected The specific endpoints selected during the second phase of protocol planning must be applied accordingly. Questions such as sample stability in transport and storage must not be neglected. Relevant demographic data among the selected populations must also be collected. E. Interpretation of data The appropriate statistical methods must be selected and applied to analyze and compile the raw data obtained in the previous phase. Conclusions should take in account existing limit values for the toxic agent, risk assessment studies previously undertaken, and potential confounders. For unregulated substances, there may be the need to establish an internal action

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level, otherwise, there is no limit against which to validate results and assess consequences. This internal action level may be derived from (i) other reference values, such as the highest no observed adverse effect level (NOAEL), the no observed effect level (NOEL), or the lowest low observed adverse effect level (LOAEL) (ii) the upper reference concentrations for unexposed populations (background levels) and (iii) the detection limit of the analytical methodology. F. Publishing and archiving of data The results of the study should be communicated to the population involved. If a potential health hazard was found, preventive action should be discussed. The study should also be peer-reviewed and published in the open literature. Raw data should be archived in retrievable format, that allows posterior meta-analysis if required. The main objective of biomonitoring is to identify individuals or groups at

risk of developing negative health effects due to their exposure to a toxic agent, and intervene with the appropriate prevention measures. As previously discussed (Section 2.1.1.) the classical epidemiology approach of identifying associations between exposure to hazardous substances and the development of diseases such as cancer, has provided valuable information on the risk involved in such exposure. Specifically regarding neoplasic conditions, the endpoints for such investigation are commonly cancer mortality and morbility. However, this approach is limited by factors such as the long latency of the disease, the difficulty in estimating past exposure, and the insensitivity to modest increases in common cancers (Perera, 1993). The more stringent regulations on environmental and occupational exposure further contributed for the difficulty of conducting meaningful classical epidemiology investigations, by reducing the number of workers exposed to genotoxic agents and the levels of exposure of those workers (Bonassi and Au, 2002).

In order to improve the epidemiological protocol, Perera and Weinstein (1982) proposed the incorporation of laboratory analytical measurements, and since then a multitude of studies have been conducted to provide useful analytical data regarding exposure and biological effects by the determination of biological indicators or biomarkers. Presently, there is a large number of biomarkers available to evaluate genetic and cancer risk in human populations, representing endpoints for assessing practically the entire spectrum of genotoxic action. Figures 6, 7 and 8 are commonly used representations of the relevance of each type of biomarkers in the several phases of cancer development (from Albertini et al., 1996).

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Figure 6. The several checkpoints of the carcinogenic pathway (from Albertinin et al., 1996).

Figure 7. Biomarkers of exposure and effect in the carcinogenic pathway (from Albertinin et al.,

1996).

Biomarkers of exposure

Biomarkers of effect

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In the context of cancer molecular epidemiology, the biomarkers of exposure can detect genotoxic agents at various levels within the body, including the target molecule, DNA. They are important in determining the internal dose and furthermore, the biologically effective dose to which the individual is subject (Figure 7). Biomarkers of effect measure early genetic damage and may be divided into two groups: reporter type, with potential predictive nature, that detect the increase in genetic damage that may translate into cancer, and disease type, that relate to changes that already indicate an on-going cancer process (Figure 7).

Biomarkers of susceptibility assess inter-individual variability relevant to disease outcome, and may be related to any phase of the genotoxic action, from absorption, metabolism and toxicokinetics, early biological effects and cancer progression (Figure 8).

Figure 8. Biomarkers of susceptibility (from Albertinin et al., 1996). Although this classification has been widely used, it may generate some

doubts in the separation between biomarkers of exposure and genetic effects, since the later are sometimes also used to quantify exposure, and some of the first may be linked to the pathway of genotoxic effect. A more mechanistic terminology has been proposed (Albertini et al., 1996) that refers to a reversible (transient) genotoxic response, related to a measure of exposure, and irreversible (permanent) genotoxic outcome, related to the final effect. Nevertheless, there still are aspects of exposure that are better assessed by the use of irreversible endpoints, that may provide an estimate of cumulative response due to past exposure (as it is the case, for example, of the evaluation of cumulative dose in radiation exposure, that can be quantified by the observation of chromosomal aberrations, namely dicentric structures, in lymphocytes from the exposed subject).

In this study, the classical classification of biomarkers of exposure, effect and susceptibility will be applied.

Biomarkers of susceptibility

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2.6.2. Oxidative damage in chromium occupational toxicology

As described in Section 2.5, chromium mechanism of genotoxicity may be

described by a bifurcated pathway, with genetic damage resulting from either oxidative damage (that may be responsible for effects such as DNA double-strand breaks, abasic sites, and base mutations) or a metal-binding pathway (that would account for the Cr-DNA adducts and the cross-linking activity). The relative importance of each pathway of toxicity is controversial. Oxidative damage was extensively demonstrated in chromium-exposed cells and experimental animals, (Witmer et al., 1994; Bagchi et al., 1995, 2002; Stohs et al., 2001; Sugden and Martin, 2002). Nevertheless, recent studies have pointed to the preponderance of Cr-DNA complex formation in the final mutagenic outcome (Zhitkovich et al., 2001). The reduction of chromium seems to be a starting point for both pathways, particularly in the case of hexavalent chromium exposure. Trivalent chromium is also able to participate in reduction processes that generate oxidative damage in vitro (Shi et al., 1998).

As previously referred, several experimental studies have provided substantial evidence for oxidative damage after exposure to various chromium compounds, both in the trivalent and hexavalent forms. Considerably less researchers have addressed the role of in vivo oxidative damage in human exposure to chromium compounds, although the available previous reports point to an increase in oxidative stress in both Cr(VI) and Cr(III) occupational exposure (Gromadzin’ska et al., 1996; Huang et al., 1999), assessed by the concentration of lipoperoxidation products in urine or plasma.

Several biological components may be responsible for the intracellular reduction of chromium. The final products, as well as the efficiency of that reaction have been shown to vary substantially when different reducers are involved (Kitagawa et al., 1988; Stearns, 1994; Shi, 1994). It seems likely that the levels of those reducers are affected by the interaction with a continuous entry of chromium in the cell, particularly in chronic occupational exposure. In this view, a variation in the available intracellular concentrations of important reducers may affect the possible reaction pathways and result in a modulation of the genotoxic outcome associated with chromium exposure. Unbalancing the homeostasis of cellular antioxidants and reducer molecules may also affect cellular response to other compounds. The intracellular levels of biological reducers that are directly involved in chromium reduction may provide an insight on the potential effect of chromium in the protection mechanisms of the cell.

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2.7. Objectives of the study Genotoxicity studies have mainly been focused on the hexavalent chromium forms, a IARC group I known human carcinogen, but still there are many questions to be answered on the in vivo mechanisms that rule chromium toxicity. Specifically, the relative importance of oxidative processes and DNA adduct formation and the role of other valence states in the carcinogenic action have not been completely clarified. Reduced forms of chromium, particularly trivalent chromium, the predominant form of the metal in biological systems and the environment, has an unclear toxic potential. Its reactivity towards biological macromolecules, including DNA, and its possible epigenetic role in carcinogenesis, seem to be limited only by a poor membranar passage, possibly overcome to a certain extent in chronic occupational exposure.

This study is aimed to compare the profile of several exposure, effect and susceptibility biomarkers in two groups of workers exposed to the two main oxidation states of chromium, hexavalent and trivalent. The analysis of the biological response to those endpoints will allow us to explore the following questions:

1) How do the internal dose and biological effective dose relate in Cr(VI) and Cr(III) exposure?

2) Do Cr(III) occupationally exposed individuals incur in genotoxic risk? 3) What is the importance of selected genetic characteristics in the toxic

outcome of chromium exposure? 4) Is there an acquired resistance or susceptibility of the cells to further

genotoxic damage, after exposure to chromium? 5) Is there a role for oxidative damage in occupational chromium toxicity? 6) What protocol of biomonitoring for genetic risk should be implemented in

chromium-exposed workers?

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3. Subjects and their environment 3.1. Tanning industry

Leather tanning is the process of converting raw hides or skins into leather in order to prevent them from decaying, make them resistant to moisture and keep them supple and durable. The surface of the hides and skins presents the hair and oil glands and is known as the grain side; the flesh side is much smoother and softer and makes the best quality leather. The main three types of hides used in the leather industry are from cattle, sheep and pigs, although more exotic products from other sources are also in the market (for example, tanned skin of camel, reptiles, and even fish). Tanning is essentially the reactions of collagen fibres in the skin with tannins, including trivalent chromium compounds, aluminum, and vegetable tannins (extracted from tree bark), among others. The general process for industrial leather tanning is shown in Figure 9.

Briefly, hides are first trimmed and washed to remove salts and other solids and to restore moisture, then fleshed and devoid of hair, usually with calcium hydroxide (lime). Bating and pickling remove the unwanted calcium hydroxide after hair removal and prepare the hide for the tanning action. Pickling usually leaves the skin in an acid pH (pH 3 or lower). The basic chromium sulfate is added and the pH is raised moderately. Following tanning, the chrome-tanned leather is piled down, wrung, and graded for thickness and quality, split into flesh and grain layers, and polished to the desired thickness. When necessary, there may be a re-tanning step, and in almost all cases, dying and fat-liquoring (adding oil to the skin to replace the oil lost in beam-house and tanyard processes). The final stage of finishing gives the leather its final look and may involve further buffing, imprinting of patterns or coating with varnish.

The potential exposure to toxic compounds is noted on the right side of Figure 9. Exposure to chromium compounds may occur in the warehouse, where chromium compounds are stored and weighed, and the solutions are made, in the tanyard area, where the leather is tanned and in the subsequent processing steps that involve production of particulate matter from the leather, since at that stage the tanned hide is already highly rich in chromium. Workers from the warehouse (Wh), tanning (T), splitting and polishing (common facilities - S) and buffing and dying (common facilities - D) areas of a bovine leather producing industry in Alcanena, Portugal, participated in this study. The industry’s occupational health service was responsible for initial contacts with the workers in order to obtain informed consent for their participation. Information on the tannery workers is summarized in Table 5.

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Setting out

Drying

Conditioning

Staking, dry milling

Buffing

Finishing and plating

Receiving and storing hides

Trimming

Soaking and washing

Fleshing

Unhairing

Bathing

Pickling

Tanning

Wringing/Siding

Splitting

Polishing

Retanning

Bleaching and coloring

Fat liquoring

BEAMHOUSE

TANYARD

RETAN, COLOR, FAT LIQUOR

FINISHING

Sulfides, NH3

Chromium

PM

Possible VOC

Possible VOC

Possible PM, VOC, NH3

PM

VOC

Figure 9. General process of industrial leather tanning and possible exposure to toxic agents. PM – particulate matter, VOC – volatile organic compounds

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D, dying and buffing; S, Splitting and shaving; T, tanning; Wh, warehouse; m, male; f, female

Table 5. Demographic data for tannery workers # Type of work Smoker (yes/no) Gender Age (years) Years employed 1 D y M 41 18 2 S n M 35 15 3 T n M 58 20 4 S n M 65 37 5 Wh n M 54 18 6 Wh n M 25 6 7 S y M 42 18 9 D y M 51 16

10 S n M 33 16 11 D n M 37 17 12 D n M 31 13 13 D n M 46 29 14 Wh n M 32 14 15 D n M 42 27 16 S n F 38 8 18 D n M 57 27 19 S n M 51 31 20 T n M 41 12 21 S n M 33 13 22 S y M 39 22 24 D n M 26 2 25 D y M 28 10 28 S y M 39 5 30 D y M 21 1 31 Wh n M 46 30 32 S n M 49 28 33 S y M 40 13 34 D n M 48 18 35 S n M 27 10 36 D n M 42 5 37 Wh n M 32 14 38 D y M 55 14 39 D n M 45 28

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3.2. Welding industry

Stainless steel welding is widely used in the production and repair of a large variety of metal equipment and machinery. There are at least 10 major types of stainless steel welding, including manual metal arc welding, which is one of the activities that involve the highest potential inhalation exposure to hexavalent chromium (Hewitt, 2001).

Manual metal arc consists of using a stainless steel rod (electrode) to establish a current between an AC/DC source of power and the piece to weld. Stainless steel has a high resistance to electrical current, therefore applying high amperage to the system generates high temperature that melts both the base material and the electrode. The electrode is gradually consumed during the process, and needs to be continuously fed. The melted metal pool generated in the process fuses as it cools down to produce the final welded piece. To maintain the constant flow of high amperage current to the work zone, the stainless steel electrode is covered with a coating of current-carrying ions that help transpose the current to the metal. The process generates abundant amounts of smoke and fumes containing relevant quantities of hexavalent chromium (50-90% of the total chromium present in the fumes), oxidized from the metallic chromium in the rod and the welded piece by the high temperature in the presence pf atmospheric oxygen.

Workers from a motor repairing workshop in Santarém, Portugal, involved in the welding process full time or as a frequent activity, participated in the study. The industry’s occupational health service was responsible for initial contacts with the workers in order to obtain informed consent for their participation. Table 6 summarizes demographic information on the welders. (W, welding; m, male) Table 6. Demographic data for welders

# Activity Smoker (yes/no) Gender Age (years) Years employed 45 W n m 40 17 46 W y m 43 29 47 W y m 38 22 48 W n m 48 22 49 W n m 44 29 50 W n m 42 15 51 W n m 44 31 53 W n m 40 24 54 W y m 43 18 55 W y m 41 11 56 W n m 41 4 57 W y m 24 4 58 W y m 25 4 59 W y m 52 3 60 W n m 63 3 61 W y m 27 1

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3.3. Control group

The control group was gathered from blood bank donors selected for not being occupationally exposed to potential genotoxic agents in their present or former professional activity. Control urine samples were obtained from a group of non-exposed workers from several activities. The use of an industry control group, originating for example from the administrative services, was not possible due to the very low number of available control individuals in the industries studied. Tables 7 and 8 summarize demographic information on controls. Table 7. Demographic data for controls (blood)

# Activity Smoker (yes/no) Gender Age (years) 1 R n F 27 2 R n f 40 3 Te y f 42 4 P n m 38 5 S n f 25 6 R y m 40 7 S y m 22 8 Se y m 28 9 D n m 64

10 C n m 53 11 Cl n m 42 12 Cl n m 23 13 T n m 45 14 R n m 46 15 Se n m 70 16 D y m 44 17 T y m 31 18 Cl y m 35 19 Ta n m 56 20 S y m 48 21 M n m 28 22 T n m 49 23 Te y m 40 24 M n m 64 25 C n m 48 26 Me y m 56 27 S y m 21 28 Se n m 55 29 Cl y m 57 30 T y m 53 31 D n m 56

C, carpenter; Cl, clerical; D, driver; M, machine operator; Me, mechanic; R, researcher; S, student; Se, security; T, teacher; Ta, tailor; Te, technician; m, male; f, female.

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Table 8. Demographic data for controls (urine)

# Activity Smoker (yes/no) Gender Age (years) 1 C y m 38 2 Cl n m 50 3 Cl n f 46 4 Cl y m 45 5 T n f 59 6 T y m 47 7 M n m 60 8 Ce y m 43 9 T n m 31

10 Cl n m 49 11 T n m 38 12 R y m 40 13 Me y m 42 14 C y m 45 15 C n m 47 16 D n m 65 17 R n m 27 18 R n f 28 19 R y f 30 20 R n f 24

C, carpenter; Ce, civil engineer; Cl, clerical; D, driver; M, machine operator; Me, mechanic; R, researcher; Se, security; T, teacher; Te, technician Table 9 summarizes the information presented in the previous tables in the form of mean ± standard deviation, in order to facilitate comparison between groups.

Table 9. Comparison of demographic data for the several study groups Age (±stdev) Years employed (±stdev) % Smokers % Males

Tanners 41±11 17±9 25 97 Welders 41±10 15±11 20 100

Controls (blood) 43±13 42 87 Controls (urine) 43±11 40 75

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3.4. Sample collection 3.4.1. Blood sampling

Venous blood (25ml) was collected from each worker and control subject in

the last day of the workweek, immediately before the lunch break, and transferred aseptically into disposable polypropylene tubes containing 15 UI of lithium heparin (BBraun, Melsungen, Germany). Ordinary metal needles were used in venipuncture, since they have been shown not to add measurable amounts of chromium to samples (Finley et al., 1997). The samples were transported to the laboratory at low temperature and minimal vibration, and arrived there within 2 hours.

Lymphocytes were immediately isolated from 8 ml of blood by a standard Ficol/sodium diatrizoate protocol using Histopaque-1077 (Sigma, St. Louis, MO, USA). Plasma was obtained by centrifugation of 5 ml of whole blood, 1000 rpm for 10 minutes at room temperature. All samples were kept at –70ºC until analysis.

A selected group of 5 welders (numbers 45, 49, 51, 54, and 56) involved in full-time manual metal arc welding was sampled twice within a one-year interval. 3.4.2. Urine sampling

A spot urine sample was obtained from all subjects (worker and control

groups) on the last day of the workweek before the lunch break. Additionally, spot urine samples were obtained from 25 tannery workers on

the last day of the workweek (Friday), in the morning (pre-shift) and at the end of the working day (post-shift) to evaluate the absorption during work shift. Urine samples were collected in sterile polyethylene containers, aliquotized, and frozen at –70ºC until analysis were performed. Creatinine was quantified in all urines by a standard picric acid assay (Kit 555-A, Sigma Diagnostics, St. Louis, MO, USA).

A group of 5 welders (numbers 45, 49, 51, 54, and 56) involved in full-time manual metal arc welding was sampled twice within a one-year interval. 3.4.3. Leather dust

Leather dust samples were collected from the residues produced by the splitting and polishing machinery in the tanning industry studied.

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4. Biomarkers of chromium exposure 4.1. Overview

If the existence of dangerous substances in the workplace cannot be excluded, it must be assured that the levels to which the workers are exposed do not pose a health risk. The necessary risk assessment relies on epidemiological and experimental studies to regulate a number of limits that should be implemented and inspected. The limits can be expressed in terms of allowable atmospheric concentration or permissible biological concentrations for the chemical or its metabolites. The first type of regulated limits includes the American Conference of Governmental Industrial Hygienists (ACGIH) published TLV or Threshold Limit Values. Other regulatory agencies have imposed limits that are equivalent in concept to the TLVs, varying in name, for example, the Occupational Safety and Health Administration’s (OSHA) PEL (permissible exposure levels). TLVs are concentration limits under which nearly all workers may be exposed day after day without adverse effect. TLV include 3 categories: TWA, time-weighted averages, a value for normal 8h work shift and 40-hour workweek, STEL, short term exposure limit, a value for allowable concentrations for a short period of time, usually 15 minutes, and TLV-C, or TLV-ceiling, a value that can not be exceeded, even briefly. Concentrations of the toxic agent or its metabolites in body fluids are regulated as biological TLV, or BEI, biological exposure indices (Lauwerys, 1996). A list of regulated limits and guidelines applied to chromium exposure is available in ATSDR (2000). The TLVs for Cr(III) and Cr(VI) in the Portuguese regulation are 0.5 and 0.05 mg/m3, respectively. Zinc chromate has a lower TLV of 0.01 mg/m3 (IPQ, 1990).

Traditionally, exposure assessment was based on questionnaires and historical information such as employment records and air sampling data. More recently, biological monitoring is considered a useful and more precise instrument to determine the level of exposure to a toxic agent. The most direct protocol of monitoring human exposure to a toxic agent is the measure of that same substance, or its metabolites, in body fluids of exposed individuals. This is highly specific and (depending on the techniques available) very sensitive to the potential toxic. The results obtained can be used to calculate the internal exposure doses and determine the dose-response relationship (Bonassi and Au, 2002).

Several factors can impair the reliability of body fluid concentrations to estimate the level of metal exposure, and they should be considered during the planning of the biomonitoring protocol and interpretation of results (Christensen, 1995). These include toxicokinetics (the timing of sampling should be decided according to the biological half life of the toxic); demographic and life-style factors such as age and sex or smoking and diet habits that can be responsible for altered levels of metals in body fluids; past exposure conditions: inter-individual

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differences in absorption, distribution, biotransformation and excretion (the number of subjects studied should be enough to average out these variations); intra-individual variation (it is advisable to obtain 2-3 samples from each subject and register the mean value of those determinations); sensitivity that must continue to be improved; and contamination, given the ubiquous nature of certain elements in the environment and the low levels in biological samples (as it is the case for cobalt, nickel and chromium, among others).

Different types of biological samples have been used to estimate exposure to toxic metals, but the most common include readily available material such as urine, whole blood, plasma or serum, red blood cells, lymphocytes, hair, and nails.

Analysis of blood and derived products is a common technique for monitoring exposure, but because of its invasive nature, it is not the preferred method for routine biological monitoring. Urine sampling is widely used and is regarded as the simplest and most practical technique of evaluating chromium absorption. Nevertheless, the use of urine chromium concentrations as a biomarker of exposure has some limitations. The half-life of the metal in the blood stream is short followed by rapid urinary excretion or storage in body tissues such as bone, kidney or liver, so urinary levels, as well as plasma determinations, may not provide an indication of low chronic exposure to chromium (Anderson et al., 1993; Bukowski et al., 1991; Finley et al., 1996). Furthermore, because all urinary excreted chromium is in the trivalent form, it provides no information on the valence state absorbed (Minoia and Cavalleri, 1988).

Red blood cell analysis is considered a biomarker of exposure to hexavalent chromium, on the basis of the easy membrane passage of Cr(VI), intracellular reduction and binding with cellular components, in opposition to the relative membrane impermeability of trivalent chromium which tends to bind to plasma proteins such as transferrin, without entering the erythrocyte (Wiegand et al., 1988). Chromium is thought to stay trapped for the life-time of the cell, approximately 120 days (for this reason 51Cr was used to estimate the average life-span of red blood cells, as previously stated). This is somewhat controversial, since it has also been reported that the half-life of chromium in the red blood cells may be shorter than the cell life span (O’Flaherty, 1995). This technique is invasive and involves a difficult analytic procedure, in addition to producing conflicting results in low dose exposures.

In addition to the classical use of biological indicators described above, other techniques have been implemented to assess exposure and, furthermore, estimate the biological effective dose in exposed individuals. The quantification of adducts to macromolecules (proteins and DNA), determination of DNA strand breaks (COMET assay), and of sister chromatid exchanges in peripheral lymphocytes of exposed individuals are representative examples of those biomarkers of exposure. Among these, DNA adducts are the most relevant to the disease process in prospective studies (Shuker and Farmer, 1992), and are preferred to protein adduct determination.

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As previously discussed, it has been extensively proven that exposure of cells to hexavalent chromium results in the formation of several types of lesions (DNA strand breaks, DNA-DNA and DNA-protein crosslinks, and modified nucleotides, including oxidized bases), but the most important are complexes containing chromium and DNA. These complexes can have either binary or ternary structure, the latter being described as crosslinks between DNA and amino-acids or peptide compounds.

Protein-DNA interactions have important roles various aspects of gene expression and inheritance, and if the normal course of such interaction is altered, serious consequences may come up. Certain proteins have a role in the expression of specific DNA sequences, resulting in the silencing or further transcription of specific genes. Others are covalently anchored to DNA and have a great importance in stabilizing chromatin structure. The chromium-mediated cross-linking of proteins or amino-acids to DNA (DPC) has the potential to disrupt that fine tuning and result in impaired cell function and toxicity (Manning et al., 1994).

The measurement of these crosslinks has been proposed and explored as a biomarker of chromium exposure and elevated DNA-protein crosslinks have been reported in welders (Costa et al., 1993, 1993a, 1996; Zhitkovich et al., 1998), chrome platers (Costa et al., 1996; Zhitkovich et al., 1996) and residents of a chromium contaminated area (Taioli et al., 1995). Until now, no study has been directed to evaluating DPC in Cr(III) exposed workers. In view of the in vitro properties of trivalent chromium compounds and the evidence that, despite the difficult cellular uptake of this form, there is relevant absorption of Cr(III) in occupational settings, this study proposes to investigate the in vivo formation of DPC in Cr(VI) as well as Cr(III) exposure. To our knowledge, no previous evaluation of DPC among trivalent chromium exposed workers has been conducted. 4.2. Research procedures 4.2.1. Total chromium in biological samples

The accurate and precise determination of very low concentrations of chromium in biological samples (in the range of parts per billion, or µg/L) is a challenging analytical procedure. At these low levels, very few techniques have sufficient sensitivity for the analysis, namely neutron activation, inductively coupled plasma - mass spectrometry and electro-thermal atomization atomic absorption spectrometry (ETA-AAS). The most common and readily available tool for the determination of chromium in biological material is the last referred technique (Harzdorf and Lewalter, 1997). Briefly, ETA-AAS is a measurement of the quantity of atoms of a certain element assessed by the absorption of radiation at a specific wavelength. ETA-AAS uses a graphite cylinder (“furnace”) to receive a

40

small amount of sample (5-50 µl) that is heated to very high temperatures by passing electrical current through the graphite tube, which becomes incandescent. The sample is successively dryed, pyrolised (to decompose organic matter) and atomized, under an inert atmosphere of argon to prevent damage of the graphite tube and reaction with the elements under analysis.

The graphite tube is in the path of a radiation beam emited by a lamp specific for the element under analysis. The most commonly used and better performance lamps are the hollow cathod type. The cathod in these lamps is made of or filled with the element to be analised under an inert atmosphere of Argon. The system is subject to a voltage of aproximately 300 V. At this voltage, some atoms of the metal present in the cathod rise to excited state and and fall back into basal state by emitting radiation in a wavelenth spectrum that is characteristic to the element. The radiation emmited is directed to pass through the cloud of atoms inside the graphite cylinder, that works as the cuvette in the traditional spectrophotometry.

The ETA-AAS method is a variation of the classical atomic absorption spectrophotometry, where the sample is nebulized and injected into a flame fed by oxidant and fuel gases, where it is atomized. The advantages of ETA-AAS are increased sensitivity (due to a longer length of time of the atoms in the optical path and a higher temperature reached, that assures complete atomization), simplified sample preparation, and low volume of sample required.

The performance of ETA-AAS can be improved by the application of several technical developments, namely:

- L´vov platform. The incorporation of a flat or curved platform in the graphite tube, where the sample is deposited. Because the temperature of the sample in the platform lags behind the rising temperature of the walls of the furnace, the analyte does not vaporize until the tube reaches a constant temperature, improving reproducibility.

- Pyrolitically coated graphite tubes. These present a more uniform surface and a better heat transference to the sample.

- Background correction. Three types of background correction are available in ETA-AAS: tungsten/halogen, deuterium and Zeeman effect.

- Matrix modifiers. The most commonly used are Mg(NO3)2 and Pd(NO3)2, which are added to the sample in order to convert the analyte present into a non-volatile form, to minimize losses during pyrolisis.

- Alternative gas during the pyrolisis. In order to improve organic matter decomposition, and avoid generation of fumes during atomization that would raise the background interference, an alternative gas (oxygen or mixtures of air) may be used during the pyrolisis phase, followed by a purge by argon prior to atomization.

41

- Automated sample injector. The manual injection of samples is a non-advisable procedure in ETA-AAS, and the use of an automated sample injector significantly improves reproducibility. In any technical settings, one common concern in chromium analysis is

avoiding contamination of the samples that can significantly alter the results obtained. Samples should be collected in disposable, “metal free” polypropylene or polytetrafluoroethylene (PTFE) flask and tubes, use of glass material should be minimized and the glass used should be first soaked overnight in HNO3 15%, then rinsed in deionized water and dried dust free. Ultra-pure deionized water and low metal content solvents should be used to prepare all solutions.

Chromium determinations in urine and plasma were conducted according to the protocol described in Granadillo et al. (1994), with some modifications to adapt the technique to the instrumentation used, a Perkin Elmer 2380 Atomic Absorption Spectrophotometer.

Chromium is a refractory element (melting point ~1857 ± 20ºC, boiling point 2672ºC), therefore the use of matrix modifiers to prevent its volatilization seems unnecessary, and the use of a relatively high pyrolisis temperature seems permitted. That was also the conclusion of the preliminary study of maximum temperatures for pyrolisis, as shown in Figure 10, that finally set pyrolisis temperature at 1350ºC. This temperature was chosen according to the observed decrease in absorvance and the increase in variation of the measure at higher temperatures This preliminary study was conducted with a inorganic chromium standard of 5 µg/L, and an atomization temperature set at 2500ºC.

Urine and plasma total chromium was quantified by the use of a standard calibration curve obtained by adding known amounts of chromium to a control sample. Plasma samples were diluted with Triton X-100 solution (1:4), to avoid flocculation and to assure homogeneity. Urine samples were diluted 1:1 with Triton X-100 solution acidified with 0.01 mol/L nitric acid for further stabilization. The complete protocol is described in Appendice 2, Protocol 1. A standard calibration curve in plasma is shown in Figure 11, along with correction for the control sample absorption. Three replicate injections of 5 µg/L Cr standard are represented in Figure 12. Limit of detection for this method was 0,37 µg/L, based on an average of 10 blank replicates and 3 times the standard deviation of those results (Westgard, 2002).

42

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rban

ce o

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Figure 10. Preliminary study of the pyrolisis temperature

y = 0,0162x + 0,0504R2 = 0,9923

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Figure 11. Standard chromium curves for plasma before (left) and after correction for the control sample absorption.

Figure 12. Three replicate injections of 5 µg/L Cr standard in urine. Each square represents 0,1 units

of absorvance.

43

4.2.2. Hexavalent chromium in leather dust

In the tanning industry, hexavalent chromium is controlled in both the tannin (basic Cr(III) sulfate) and the final product, due to chemical safety regulations for leather, imposed by regulatory agencies or required for the affixation of quality labels such as the European Eco-Label (Font et al., 1998). The intermediate product is not controlled, because it would be unlikely that it would contain any relevant amounts of Cr(VI) if this is not detected by the initial and final analysis. Still, the present study included a determination of hexavalent chromium in the leather dust obtained during the intermediate phases of the process (splitting and polishing).

After an extraction phase with 3 different pH buffers, the extracts were combined with 1,5-diphenylcarbazide in acid pH, and the resulting chromium-diphenylcarbazide complex was quantified by HPLC (Appendice 2, Protocol 2) with UV/Visible spectrophotometry detection. Typical standard calibration curve is presented in Figure 13. Limit of detection for this method was 0.78 ng/ml of Cr(VI), based on a signal to noise ratio of 3:1 (Snyder, 1997).

Figure 13. Standard calibration curve for hexavalent chromium determination by HPLC

y = 2046,7xR2 = 0,9994

0

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0 10 20 30 40 50 60

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44

4.2.3. DNA-protein crosslinks measurement

Several methods have been developped with the purpose of quantifying DNA-protein crosslinks (DPC), including alkaline elution, filter retention, immunochemical, blotting and restriction enzime pattern methods (Zhitkovich and Costa, 1992). Most of these methods require specialized instrumentation and relatively high amounts of starting material. The classical approach to the measurement of DPC is alkaline elution (Kohn and Ewing, 1979), that measures the speed with which DNA fragments pass through a filter, given that protein-linked DNA will be slower than free DNA. This is a sensitive technique, but it poses technical difficulties in application to large-scale biomonitoring, being a time consuming procedure allowing the processing of only a limited number of samples in a few days. Also, it does not allow the isolation of DPC for further characterization, and may not be absolutely specific towards covalentely bound proteins, since in less stringent protocols proteins that are reversibly attached to DNA may be equivocally quantified as DPC.

A new protocol of determining DPC in cells was proposed by Zhitkovich and Costa (1992) and was applied in this study (Figure 14). It is based on a method to quantify topoisomerases (Muller, 1983), starting with the lysis of the cells by sodium dodecylsulfate (SDS), shearing of DNA by passing through a 25G needle, and precipitation of the proteins by the addition of a potassium salt. The anionic detergent SDS is known to bind to proteins but not to DNA, and the addition of potassium chloride results in an K-SDS precipitate that drags down proteins with it. Harsh treatment is applied to dissociate non-covalentely bound DNA (2% SDS, 3 heat cycles at 50ºC). The precipitate is repeatedly washed to remove unbound DNA and finally treated with proteinase K. Cross-linked DNA is released and quantified in the suppernatant by fluorescence, using bisbenzimide (Hoechst® 33258) or PicoGreen® fluorescent DNA dye. DPCs are quantified as the percentage of SDS-precipitable DNA versus total DNA in the sample. The complete protocol is present in Appendice 2 (Protocol 3). A typical standard curve for DNA quantification by PicoGreen® is shown in Figure 15. Limit of detection for DNA fluorimetric determination is 0.73 ng/ml, based on an average of 10 blank replicates and 3 times the standard deviation of those results (Westgard, 2000).

The average size of DNA fragments obtained from the shearing step should be controlled to assure that it does not vary substantially between samples, since it may affect the final DPC quantification (Appendice 2, Protocol 4).

45

Figure 14. DNA-protein crosslinks measurement by SDS precipitation assay (adapted from Costa et al., 1993)

y = 243.88x + 259.19R2 = 0.998

0

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Figure 15. Typical calibration curve for PicoGreen ® DNA quantification.

Cellular Lysis in SDS

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46

4.3. Results All results presented in the following tables were analyzed for normal distribution using the Kolmogorov-Smirnov test. When no significant difference from normality was found, Student’s t-test was applied for comparison between group means, and group values are presented as mean ± standard deviation. Non-parametric tests were applied when data distribution significantly deviated from normality, and are referred to when result is presented. Where no test is specified, Student’s t-test and parametric analysis was applied. All calculations were performed using Microsoft® Excel 2000, with Analyse-It™ Version 1.66.

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4.3.1. Chromium concentration in plasma and urine

Table 10. Pre and post-shift urinary chromium in tannery workers (µg chromium/g creatinine)

# Morning Afternoon 1 0.59 0.95 2 0.73 5.43 3 3.39 9.05 4 0.54 1.37 5 6.90 31.07 6 4.68 3.34 7 0.94 0.91 9 1.07 1.74

10 0.45 1.30 11 0.34 1.51 12 0.20 0.89 13 a) 1.72 14 3.96 a) 15 1.07 a) 16 0.88 9.25 18 1.34 2.74 19 1.80 2.49 20 25.09 6.53 21 0.54 0.84 22 1.40 a) 24 1.65 2.04 25 0.78 1.85 28 1.39 3.79 30 a) 23.46 31 a) a) 32 2.44 3.64 33 1.04 2.12 34 4.34 6.19 35 2.11 2.20 36 a) a) 37 14.68 30.95 38 1.42 1.27 39 a) 1.69

a) contaminated urine (discarded)

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Table 11. Chromium concentrations in urine and plasma of tannery workers

# UCr (µg/L) Creatinine(g/L) UC Cr (µg/g creat.) PCr (µg/L) 1 4.55 1.03 4.42 1.14 2 4.13 0.91 4.55 1.06 3 1.93 0.63 3.04 2.73 4 4.39 1.07 4.10 2.73 5 9.22 1.12 8.26 11.67 6 7.68 1.60 4.81 5.76 7 2.02 1.67 1.21 1.21 9 4.01 1.60 2.50 1.29

10 4.85 2.49 1.95 0.68 11 a) 1.46 12 2.59 0.82 3.16 4.39 13 1.70 1.17 1.45 0.19 14 2.31 1.51 1.53 3.79 15 1.79 1.20 1.49 2.65 16 4.81 1.98 2.43 0.53 18 1.78 0.84 2.11 1.74 19 3.92 1.16 3.37 1.21 20 3.37 2.11 1.60 3.26 21 2.14 1.56 1.38 0.91 22 5.18 0.93 5.56 4.47 24 2.41 1.27 1.90 3.71 25 1.13 1.09 1.04 3.02 28 a) 1.59 30 2.59 0.87 2.98 2.42 31 4.88 2.86 1.70 1.52 32 1.08 1.07 1.02 1.75 33 1.32 0.85 1.56 1.36 34 2.62 1.79 1.46 2.65 35 3.16 1.68 1.88 1.36 36 1.78 0.59 3.02 1.82 37 46.87 2.43 19.27 26.75 38 2.83 1.80 1.57 2.20 39 1.60 0.94 1.71 1.52

a) contaminated urine (discarded); creat., creatinine; P, plasmatic; U, urinary; UC, urinary corrected.

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Table 12. Chromium concentrations in urine and plasma of welders (first sampling)

# UCr (µg/L) Creatinine (g/L) UCCr (µg/g creat.) PCr (ppb) 45 2.02 1.25 1.62 1.90 49 2.04 0.85 2.41 1.19 51 1.54 1.01 1.52 2.02 54 1.28 0.60 2.13 0.55 56 2.17 1.20 1.80 2.10

U, urinary; UC, urinary corrected; P, plasmatic; creat., creatinine. Table 13. Chromium concentrations in urine and plasma of welders

# UCr (µg/L)) Creatinine (g/L) UCCr (ug/g creat.) PCr (ppb) 45 0.55 0.93 0.59 2.12

46 0.23 2.12 0.11 0.00

47 3.33 1.63 2.04 3.13

48 1.25 1.69 0.74 0.00

49 0.85 0.95 0.89 6.39

50 2.15 1.37 1.57 7.15

51 4.13 2.20 1.88 3.34

53 5.63 2.72 2.07 2.58

54 8.61 4.31 2.00 2.42

55 0.45 0.65 0.69 8.01

56 4.40 2.53 1.74 5.11

57 3.32 1.98 1.68 1.87

58 2.11 0.69 3.08 3.09

59 1.67 0.74 2.27 1.63

60 2.12 0.63 3.39 2.18

61 3.37 1.53 2.20 3.31 U, urinary; UC, urinary corrected; P, plasmatic; creat., creatinine.

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Figure 16. Pre and post-shift urinary chromium levels in tannery workers

Figure 17. Linear relationship between urinary and plasmatic total chromium concentration in tanners.

y = 1,34x - 0,95Pearson's r = 0,93

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51

Figure 18. Total chromium in urine and plasma of tanners, welders and controls. UCr, corrected urinary chromium; PCr, plasmatic chromium. * p<0.005; ** p<0.0005, Mann-Whitney test.

4.3.2. Hexavalent chromium concentration in leather dust Table 14. Hexavalent chromium in leather dust Extraction pH Cr(VI) µg/g of leather

Acid 2.50±1.56 Neutral nd Basic 19.52±13.48

Values presented are mean ± stdev; nd, non detected 4.3.3. DNA-protein crosslinks Table 15. DNA-protein cross-link values in tanners, welders and controls (as % of crosslinked DNA)

Tanners Welders (1st sampling) Welders Controls # DPC % # DPC % # DPC % # DPC % 4 0.97 45 2.67 45 1,56 4 0,69 5 1.07 46 1,76 5 0,61 6 1.25 47 0,49 6 0,51

14 0.84 48 1,33 7 0,4 18 1.08 49 1.89 49 0,56 8 0,61 20 0.81 50 0,99 9 0,38

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UCr ug/g creatininePCr ug/L

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***

52

Table 15. DNA-protein cross-link values in tanners, welders and controls (continued) Tanners Welders (1st sampling) Welders Controls

# DPC % # DPC % # DPC % # DPC % 21 0.61 51 0.47 51 1.58 10 0.7 22 0.79 53 1.46 11 0.66 24 0.92 54 3.46 54 2.48 12 0.68 34 0.6 55 1.20 13 1.12 36 1.01 56 2.60 56 1.74 14 0.36 37 0.68 57 1.53 15 0.7 38 0.66 58 0.25 16 0.48 39 0.86 59 0.37 17 0.18

60 1.02 18 0.46 61 0.76 19 0.33 20 0.38 21 0.49 22 0.49 23 0.94 24 0.64 25 0.74 26 0.48

Figure 19. DPC values in tanners, welders and controls. * p=0.006;**p=0.013.

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4.4. Discussion 4.4.1. Chromium determinations in plasma and urine

Biological monitoring of exposed individuals is a powerful tool to assess increased body burden of potentially toxic compounds. In the case of chromium, several authors have reported an increase in chromium concentrations in biological samples of workers following exposure to chromium in various activities, including stainless steel welding (Edmé et al., 1997), chromate production (Minoia and Cavalieri, 1988), chrome plating (Lukanova et al., 1996) and leather tanning (Randal and Gibson, 1987; Minoia and Cavalieri, 1988).

In the present study, and in harmony with the previous findings described above, both exposed groups showed a significant increase in plasmatic and urinary chromium when compared to non-exposed control subjects (Figure 18). Trivalent and hexavalent chromium exposure seem to be similarly efficient in raising chromium levels in body fluids of exposed workers. Again it is confirmed that in trivalent chromium based industries there is substantial absorption of the metal due to continuous exposure to a chromium-rich environment.

Spot samples of urine collected midday, at the end of the workweek showed an excess in chromium concentration in tanners of about 3.7-fold in comparison to control mean value, whereas welders had a 2.4-fold increase. Plasma concentrations displayed a similar profile. There is no significant difference in chromium concentrations between welders and tanners, although the tanner group reports higher mean values, which is somewhat surprising, given the documented higher efficiency in Cr(VI) uptake. The levels may reflect a better cellular uptake of hexavalent chromium, and consequent clearance from the plasma into the tissues, which may be related to its higher toxic potential. No correlation was found between smoking habits and the levels of urinary or plasmatic chromium. The small amounts of chromium present in tobacco (0.24–14.6 mg/kg, ATSDR, 2000), do not seem relevant when compared to the levels of occupational exposure. Also, no correlation is reported between years of employment and chromium concentrations, possibly because a steady state of chromium absorption and excretion is rapidly achieved soon after the activity is initiated.

Although no significant difference in total chromium concentrations was found between different activities in tanners, a higher mean value is reported for workers from the warehouse facilities, who are responsible for weighting the basic chromium sulfate used in the tanning process.

It is possible, in the case of chromium biomonitoring, to make an assessment of the increase in metal absorption during the work shift by collecting a sample before and after the shift. Given that chromium has a first compartment half-life of 7-24 hours, the values measured will reflect the exposure during an

54

average of 12 hours prior to the sample collection. The main excretion pathway is urine, and urinary levels are expected to be increased after the work shift, with a relative “wash out” effect during the overnight break period. There should be some accumulation of chromium within the body, and a gradual increase in pre-shift and post-shift values should happen throughout the workweek.

An evaluation of pre and post-shift chromium concentration in the tanner group was performed, by collecting urine samples in the morning, immediately before starting work, when possible. There was a definite elevation of the post-shift values when compared to the beginning of the shift in almost every worker studied (Figure 16), with an median 2- fold increase for the overall group (95% confidence interval 1.4 - 2.7, Hodges-Lehman method). These findings indicate a substantial chromium intake, absorption and excretion of Cr(III) during the daily eight hours of work.

The comparison of urinary and plasmatic chromium concentrations in tanners found a positive correlation between results (Pearson’s r = 0.93, Figure 17). Apparently, and despite the relatively short half-life of chromium in the blood compartment, in chronic occupational exposure there are relevant amounts of chromium being absorbed, distributed throughout the organism and being excreted in urine. Therefore, chromium levels in either plasma or urine seem to be a valid measure of exposure in routine biomonitoring. 4.4.2. Hexavalent chromium in leather dust Total chromium content in tanned leather is very high, reaching 3% (w/w). A large fraction of the metal is covalently bound to the organic macromolecules of the original skin, namely collagen, but a relevant amount of chromium is leachable and can be extracted by treatment of leather with water or buffered solutions. As mentioned previously, there are a number of imposed limits to the amount of hexavalent chromium that may be present in the finished leather. In order to imposed these limits, it is necessary to accurately quantify Cr(VI) in the leather, but the methods accepted for that determination vary between regulatory agencies. This is critical because it is presently well accepted that extracting soluble Cr(VI) from leather without altering the oxidation state of the chromium present is very difficult to achieve (Font et al., 1998). Oxidation of chromium in the leather sample has been proven to occur in model tests using alkaline buffers for the extraction step, particularly when the extraction is made in the presence of air (Harzdorf and Lewalter, 1997). For that reason, most of the standard methods (such as the IUC 18, the official IULTCS standard method for Cr(VI) determination, and the DIN 53314) propose an extraction with only a neutral pH buffer (pH 7.5), under inert atmosphere of nitrogen or argon. The results obtained in the present study are in agreement with these guidelines, showing that only when the pH deviates from neutrality there is a detectable amount of Cr(VI) that is then likely to be derived from artifacts such as

55

oxidation of the chromium in the sample. Furthermore, the tanned leather produced in the participating industry is continuously monitored for hexavalent chromium, and no Cr(VI) has been detected in those routine controls. 4.4.3. DNA-protein crosslinks

DNA-protein crosslinks (DPC) are a promising biomarker of exposure to assess the biologically efective dose in chromium exposure. DPC levels have been previously quantified in several populations that were either occupationally or environmentally exposed to hexavalent chromium compounds. An increase in DPC was found in workers exposed to hexavalent chromium compounds, namely chrome platers (Zhitkovich et al., 1996) and welders (Costa et al., 1993; Zhitkovich et al., 1998). Environmentally exposed populations have also been investigated for this biomarker, showing positive results when compared to control subjects (Costa et al., 1996; Taioli et al., 1995). DPC values were correlated with red blood cell chromium or chromium exposure estimates. No study was located in the available literature concerning DPC formation in trivalent chromium exposed workers.

DPC formation deriving from oral exposure to Cr(VI) and Cr(III) has been evaluated by Kuykendall et al. (1996), who conducted a measurement of DPC in lymphocytes of human volunteers who ingested hexavalent chromium solutions as a bolus dose of 5 mg of Cr(VI) or Cr(VI) reduced by dilution in orange juice. No elevated DPC were found in these subjects, but the study conditions may have contributed to the absence of findings. DPC were determined only in the same day of dosing, which may not allow the length of time necessary for DPC formation in lymphocytes of subjects. The study did comprise an evaluation of the time course of DPC in cultured lymphocytes (EBV-transformed Burkitt’s lymphoma cells), and reported a period of 4 hours as enough to obtain significant elevation of DPC values, but this cell line was exposed to concentrations much higher than the reported higher mean concentrations of chromium in plasma of study volunteers: the lower concentration used was about 200-fold higher than the highest chromium values in plasma of the human volunteers.

The results obtained in the present study showed a significant increase in DPC values in tannery workers when compared to the control group (0.88% vs 0.57%, p=0.006, Figure 19). Furthermore, there was a positive correlation between chromium concentrations and DPC, in both urine (r=0.54, p=0.005, Pearson's test) and plasma (r=0.45, p=0.001, Pearson's test) of leather tanning workers. This is the first report of increased DPC values in a trivalent chromium exposed group, and may be an indication of potential CrIII toxicity in occupational exposure.

The welder group presented an even higher value of DPC, also significantly different from control values (2.22% vs 0.57%, p=0.013). Both hexavalent and trivalent chromium-exposed groups had a significant excess of chromium in biological samples, reflecting an increased body burden of the metal, and showed higher levels of DPC when compared to control individuals. The obvious

56

difference between the welder and tanner group (more than 2.5 fold) may reflect the different cellular uptake of the main valence states to which these groups are exposed.

A small group of welders was sampled twice within a one year interval. The group is involved full-time in manual metal arc stainless steel welding. There is a weak but positive correlation between the two sets of values (Pearson’s r = 0.49).

These results support the causal relationship between chromium exposure and increased lymphocyte DPC levels.

DPC determinations were not affected by bias factors such as age or smoking. This is in agreement with previous reports (Costa et al., 1993) and characterizes one of the advantages of DPC as an exposure biomarker.

Some previous studies have suggested the existence of a plateau on DPC values, that would reach saturation levels and not accompany the increase in internal doses of chromium above 7-8 ppb in red blood cells (Costa et al., 1996). No such effect is reported here.

DPC are not absolutely specific of chromium, given that other compounds may act as crosslinking agents: trans and cis-platine (Banjar et al.., 1984), nickel (Costa et al., 1993), formaldehyde (Speit et al., 2000) and lead (Wu et al., 2002) are some examples. In this case, an evaluation of the possible concomitant exposure to other compounds in the industrial settings under study is extremely important is characterizing the role of chromium in the measured DPC levels. In the tanning process, the only identified candidate to a potential cross-linking action is formaldehyde, used in some industries as a leather preserver. The industrial engineers at the participating tannery denied using the chemical in their tanning processes. Stainless steel is an alloy of iron and chromium that may contain nickel, among other elements. The composition of the electrodes used in manual metal arc welding at the participating industry was obtained, and it was confirmed that, although nickel was one of the components of the alloy, it was in considerable lower amount in proportion to chromium (1.24% chromium versus 0.02% nickel, 620 fold difference). Therefore the results obtained may be considered most likely due to chromium action than to any other potential confounder. The positive correlation between DPC and chromium concentration in both plasma and urine adds strenght to that evidence.

Oxidative damage products, and namely lipoperoxidation products, may also act as crosslinking agents (Voitkun and Zhitkovich, 1999). Considering the possible bifurcated pathway of chromium toxicity, involving both oxidative damage and formation of Cr-DNA adducts (see Section 2.5), this biomarker may benefit from added sensitivity by being a potential endpoint for both pathways of genotoxicity.

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5. Biomarkers of chromium effect 5.1. Overview

Biomarkers of genotoxic effect are related to irreversible genotoxic outcome and can be measured at the chromosome level or at the gene level. Punctual alterations in the genetic material, involving only a few thousand DNA bases, can be measured by mutation assays, the most commonly used being the hypoxanthine guanine phosphoribosyltransferase (HPRT) mutations in lymphocytes T (identified as purine analogue resistant cells) and the glycophorin A (GPA) mutation in red blood cells. Other gene mutations that are specific of various types of cancer are also potential and highly relevant biomarkers of effect, and may be identifiable by polymerase chain reaction amplification-based technologies.

The protocols available to measure genotoxic effects at the chromosome level include classical cytogenetic protocols such as chromosomal aberrations, micronuclei and sister chromatid exchanges observation (Wolf, 1998). The single cell electrophoresis assay, or Comet assay may also be considered an effect biomarker; this assay measures double strand DNA breaks by the length of migration of the DNA in each cell when submitted to electrophoresis.

Chromosomal aberration (CA) assay in peripheral blood lymphocytes stimulated by a mitogen and blocked in metaphase by a spindle poison (colchicine or colcemid) is the most extensively used and best validated biomarker of genotoxic effect. Exposure to toxic agents usually induces changes in chromosome structure due to breaks and rearranges, of chromatid or chromosome type; most frequently, these result either from direct DNA breakage or replication of a damaged DNA template. Few genotoxic agents induce direct DNA breakage, the classical example being ionizing radiation or radiomimetic agents such as bleomycin. More commonly, a compound induces alterations in DNA (such as adducts) that require that the cells undergo S-phase of DNA replication before a structural aberration is detected (these are called S-phase-dependent genotoxic agents). Types of CA are represented in Figure 20, next page. Not all figures represented can be visualized by ordinary optic microscopy.

The frequency of CAs has been proven to be positively correlated with the incidence of cancer, which may give it a predictive value (Hagmar et al., 1998). The majority of cancer cells show chromosomal alterations such as deletions and translocations, which means that CA formation may be part of the development of cancer. Another point in favor of this assay is the persistence of detectable effects, since cells with small deletions and gain or loss of certain chromosomes, can survive. A major disadvantage of CA analysis is that it is time-consuming, and requires well-trained technicians to insure proper scoring of slides. Recently, molecular techniques have been proposed to improve the output of this assay, namely by the use of fluorescence in situ hybridization (FISH). A number of

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chromosome and region-specific fluorescent probes have been developed to detect, respectively, chromosome rearrangements (by chromosome painting) or chromosome-region breaks (tandem-probe assay).

Another cytogenetic protocol extensively used as a biomarker of genotoxic

effect is the determination of micronuclei in cultured cytokinesis-blocked peripheral blood lymphocytes. Micronuclei are fragments of genetic material that contain either acentric fragments (resulting from DNA breaks), whole chromosomes, or complex rearrangements that are unable to properly attach and be pulled to the poles by the mitotic spindle. This generates chromosomal material that is not included in any of the final nuclei, and remains in the cytoplasm of the cell, involved by its own nuclear envelope. Instead of detecting the chromosomal aberration in the cell metaphase, we detect the loss of portions of genetic material from the nucleus after the cell completes nuclear division. The advantage of this assay is that it simplifies the scoring, and is able to detect clastogenic (loss of portions of chromosomes) and aneuploidic (whole chromosome loss) effects. Inclusively, the origin of the fragment visualized as a MN can be determined by the

Figure 20. Chromatid and chromosome-type aberrations (from Savage, 2000)

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use of anti-kinetochore marked antibodies or fluorescent probe for centromeric DNA sequences: both identify MN that are likely to be the result of a whole chromosome loss. A disadvantage of the MN assay is that it may have lower sensitivity than the CA method, because it requires the completion of the cell cycle; severely damaged cells may not be able to do so and are not scored. In compensation, a larger number of cells can be scored in this assay in comparison to CA.

Both chromosomal aberrations and micronuclei in cultured peripheral blood lymphocytes were applied in this study as biomarkers of effect. 5.2. Research procedures 5.2.1. Chromosomal aberration assay

Chromosomal aberration (CA) assay is based on the observation of cells arrested in the metaphase step of mitosis, when the chromosomes attain sufficient condensation to allow visualization of their structure.

Whole heparinized peripheral blood obtained from the subjects was cultivated in supplemented Ham’s F-10 culture medium for 48 hours, the last three hours in the presence of a mitotic spindle inhibitor (colchicine or Colcemide®). Addition of phytohaemagglutinin to the culture stimulated the lymphocytes to undergo division. The 48h incubation time has been shown to produce a high yield of first metaphase cells, which is important because the frequency of CA is greatly decreased in second generation cells, due to the dilution of damage among daughter cells and the loss of damaged cells that do not proliferate further.

At the end of incubation, cells were harvested by centrifugation, submitted to a hypotonic shock to eliminate cytoplasm and fixed with methanol/acetic acid solution. The slides may be prepared immediately, or the cells may be stored in the fixative solution at –20ºC (see detailed protocol in Appendice 2, Protocol 5). 100 complete metaphases (46 centromeres), were scored by subject. CAs were classified according to Rueff et al. (1993). Briefly, CAs were classified as chromatid or chromosome gaps, chromatid or chromosome breaks intrachanges and interchanges (see Figure 20). Mitotic index was determined by counting the number of metaphases per 1000 interphase nuclei. 5.2.2. Micronuclei assay

Micronuclei (MN) are fragments of genetic material present in the cytoplasm that may be expressed in dividing cells. Its frequency is increased by exposure to genotoxic agents. Because MN are expressed in cells that have completed a cellular division, the standard technique proposed by Fenech and

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Morley (1985) makes use of cytochalasin-B to inhibit cytokinesis, and allow identification of binuclear cells.

Whole heparinized peripheral blood obtained from the subjects was cultivated in supplemented Ham’s F-10 culture medium for 72 hours; addition of phytohaemagglutinin to the culture stimulated the lymphocytes to undergo division. At 44 hours of incubation, cytochalasin-B is added to the culture medium. At the end of incubation, cells were harvested by centrifugation, submitted to a mild hypotonic shock to enlarge the cytoplasm of the cell and improve scoring. The cells are then either smeared or cytocentrifuged onto clean glass slides, allowed to dry, fixed, and stained by Giemsa. (see complete protocol in Appendice 2, Protocol 6). Cells may also be fixed while still in the tube and stored at –20ºC until slides are prepared. 1000 binucleated cells with preserved cytoplasm are scored by subject, according to criteria described in Kirsch-Volders et al. (2000). The cytokinesis-blocked proliferative index was determined according to Surrallés et al. (1995). 5.3. Results

All results presented in the following tables were analyzed for

normal distribution using the Kolmogorov-Smirnov test. When no significant difference from normality was found, Student’s t-test was applied for comparison between group means, and group values are presented as mean ± standard deviation. Non-parametric tests were applied when data distribution significantly deviated from normality, and are referred to when result is presented. Where no test is specified, Student’s t-test and parametric analysis was applied. All calculations were performed using Microsoft® Excel 2000, with Analyse-It™ Version 1.66. 5.3.1.Chromosomal aberrations Table 16. Chromosomal aberrations in welders

Aberrant Cells # Gaps Breaks Interchanges Intrachanges Incl. gaps Excl. gaps Mitotic Index ‰

45 6 6 22 46 8 1 9 1 18 47 4 1 1 6 2 11 48 5 4 1 10 5 38 49 3 1 4 4 28 50 5 2 1 8 3 14 51 4 2 6 2 17 53 5 4 9 4 11 54 7 3 1 11 4 39 55 5 3 8 3 31 56 6 1 7 1 13

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Table 16. Chromosomal aberrations in welders (continued) Aberrant Cells

# Gaps Breaks Interchanges Intrachanges Incl. gaps Excl. gaps Mitotic Index ‰ 57 3 3 10 58 8 8 1 17 9 7 59 6 2 8 2 14 60 1 2 3 2 11 61 3 2 5 2 9 Table 17. Chromosomal aberrations in tanners

Aberrant Cells # Gaps Breaks Interchanges Intrachanges Incl. gaps Excl. gaps Mitotic Index ‰ 1 2 2 4 2 6 2 1 1 2 1 7 3 4 1 5 1 4 4 6 2 8 2 6 5 4 2 6 2 9 6 8 1 9 1 39 7 9 9 1 1 11 2 6

10 8 1 2 11 3 5 11 3 1 4 1 20 12 8 2 10 2 16 13 3 3 40 14 8 3 11 3 5 15 5 3 8 3 5 16 3 3 6 3 6 18 4 1 1 6 2 2 19 5 4 9 4 9 20 7 5 1 13 6 6 21 2 1 3 1 11 22 6 2 8 2 4 24 4 1 5 1 10 25 7 4 11 4 14 28 4 2 6 2 4 30 7 1 8 1 6 32 10 10 1 33 5 2 7 2 9 34 6 6 8 35 9 5 14 5 4 36 8 1 9 1 7 37 3 3 16 38 4 4 3 39 11 2 13 2 6

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Table 18. Chromosomal aberrations in controls

Aberrant Cells # Gaps Breaks Interchange Intrachange Incl. gaps Excl. gaps Mitotic Index ‰ 1 3 3 6 3 35 2 1 1 7 3 1 1 2 1 8 4 3 2 5 2 16 5 4 1 5 1 15 6 5 2 7 2 8 7 8 8 1 1 13 9 8 4 12 4 10

10 3 2 5 2 12 11 3 2 5 2 6 12 1 1 7 13 2 4 6 4 9 14 2 2 4 2 5 15 4 4 8 4 12 16 1 1 5 17 1 1 22 18 1 3 1 5 4 3 19 3 2 5 2 14 20 3 1 4 1 5 21 4 1 5 1 5 22 2 3 5 3 9 23 5 5 12 24 4 3 7 3 5 25 5 2 7 2 18 26 4 5 9 5 22 27 2 1 3 1 9 28 2 3 5 3 4 29 2 1 3 1 12 30 5 4 9 4 11 31 3 1 4 1 14

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Figure 21. Different types of chromosomal aberrations found in tanners.

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Figure 22. Different types of chromosomal aberrations found in welders.

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Figure 23. Different types of chromosomal aberrations found in controls.

Figure 24. Frequency of aberrant cells in tanners, welders and controls. Total values including and excluding gaps are presented. * p<0.05.

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5.3.2 Micronuclei Table 19. Micronuclei frequency in tannery workers

Subject %BN ‰BNMN CBPI 1 35.8 6 1.41 2 37.0 8 1.49 3 33.2 9 1.36 4 32.3 7 1.37 5 38.4 12 1.42 6 45.2 5 1.61 7 22.9 4 1.27 8 32.0 6 1.38 9 23.9 6 1.28

10 48.3 5 1.53 11 44.6 16 1.49 12 62.0 9 1.74 13 27.6 4 1.31 14 59.9 7 1.68 15 47.7 7 1.54 16 36.9 10 1.39 17 34.5 5 1.40 18 47.9 6 1.57 19 50.4 5 1.58 20 19.6 8 1.23 21 56.1 2 1.68 22 45.0 5 1.52 23 23.2 4 1.26 24 43.1 5 1.49 25 Culture failed 26 32.1 3 1.34 27 42.0 4 1.49 28 44.6 4 1.59 29 38.8 4 1.47 30 32.4 9 1.43 31 54.5 3 1.70 32 24.7 9 1.27 33 Culture failed

BN, binucleated cells; BNMN, binucleated cells with micronuclei; CBPI, cytokinesis blocked proliferation index.

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Table 20. Micronuclei frequency in controls

# %BN ‰BNMN CBPI 1 47.7 3 1.55 2 45.8 4 1.57 3 46.0 3 1.56 4 60.4 4 1.78 5 33.6 2 1.41 6 36.8 7 1.40 7 33.2 3 1.39 8 44.0 5 1.45 9 36.6 5 1.39

10 38.3 3 1.52 11 42.8 3 1.49 12 37.5 4 1.47 13 53.5 3 1.72 14 47.1 4 1.52 15 30.4 2 1.39 16 37.8 3 1.41 17 Culture failed 18 32.8 3 1.38 19 29.3 4 1.36 20 35.0 8 1.38 21 35.8 7 1.36 22 35.1 6 1.46 23 Culture failed 24 35.1 2 1.36 25 33.0 5 1.35 26 28.4 11 1.30 27 29.3 1 1.36 28 Culture failed 29 38.8 3 1.43 30 32.9 1 1.37

BN, binucleated cells; BNMN, binucleated cells with micronuclei; CBPI, cytokinesis blocked proliferation index.

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Table 21. Micronuclei frequency in welders

# %BN ‰BNMN CBPI 45 52.7 3 1.70 46 40.2 3 1.50 47 53.5 7 1.69 48 52.6 10 1.64 49 46.9 11 1.55 50 66.6 5 1.77 51 66.0 2 1.75 53 60.0 3 1.73 54 46.0 12 1.54 55 56.8 2 1.73 56 54.6 8 1.65 57 57.7 2 1.62 58 52.3 6 1.59 59 53.0 5 1.71 60 49.8 10 1.61 61 58.1 4 1.69

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Figure 25. ‰ of micronucleated binucleated lymphocytes in tanners, welders and controls. *p<0.01.

*

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Examples of chromosomal aberration preparations obtained in the present study

Figure 26.Normal metaphase. Figure 27. Chromatid break.

Figure 28. Chromosome break. Figure 29. Chromatid gap.

Figure 30. Dicentric chromosome with fragment. Figure 31. Tetraradial chromosome.

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Examples of cytokinesis-blocked micronucleus assay preparations obtained in the present study

Figure 32. Normal binucleated lymphocyte. Figure 33. Binucleated lymphocyte with MN.

Figure 34. Binucleated lymphocyte with MN. Figure 35. Binucleated lymphocyte with MN. 5.4. Discussion

Previous cytogenetic damage studies conducted in populations exposed to chromium compounds have provided somewhat inconsistent results, as previously mentioned in Section 2.1.1.

Cytogenetic damage among leather tannery workers has not deserved much attention in biomonitoring studies, nevertheless at least two previous reports were found in the available literature (Sbrana et al., 1991; Cid et al., 1991). Both studies showed small increases in cytogenetic damage (measured by chromosomal aberrations in peripheral blood lymphocytes and micronuclei in exfoliated urotelial

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cells), that were statistically significant in individuals directly involved in the tanning process (Sbrana et al., 1991).

The present cytogenetic analysis of micronuclei in the lymphocytes of workers in the tanner group showed mean values significantly higher than in the controls (6.35±2.94 ‰ versus 3.58±1.69 ‰, p<0.01). Although the non-specific nature of the endpoint does not allow further clarification of the agent (or agents) responsible for the genetic damage, the results indicate an increase in genotoxic risk related to occupational exposure in the leather tanning industry. Workers employed in the final polishing department had a significantly higher frequency of micronuclei in binucleated lymphocytes than the combined other groups (11±3.37 ‰ vs 5.7±2.20 ‰, p=0.046). In this step of the leather production process, the leather is close to the end of the production process, and has already received not only the tannin (basic chromium sulfate) and the dyes. Therefore, the dust produced may contain other potentially genotoxic agents (IARC, 1981), which may explain the higher frequency in micronuclei among workers of this department.

In the chromosomal aberration assay, there is a non-significant increase chromosomal aberration frequencies (excluding gaps) in tanners. If chromosome and chromatid gaps are included, the difference to controls becomes significant. Although certain authors have sustained the inclusion of chromatid and chromosome gaps in the total aberration results (Paz-y-Miño et al., 2002), this study follows a more conservative approach that excludes these structures. Despite that fact, these findings still suggest a higher genotoxic risk in the tanning industry workers.

Previous studies have reported the in vitro induction of micronuclei by trivalent chromium (chromium chloride) in human diploid fibroblasts, showing a predominance of aneugenic over clastogenic effects (Seoane and Dulout, 2001). This may explain the apparent non correlation between the two cytogenetic endpoints, given that the chromosomal aberration assay only includes cells that present complete metaphases (46 chromosomes). In the particular case of chromium, a better estimate of genetic damage may be attained by the micronuclei assay, able to detect both clastogens and aneuploidic agents.

Several studies conducted with welder populations have failed to report increased cytogenetic damage, even when biological indicators of exposure are elevated (Husgafvel-Pursiainen et al., 1982; Littorin et al., 1983; Nagaya et al., 1989, 1991; Jelmert et al., 1995), while in other studies significant increases have been reported (Popp et al., 1991; Knudsen et al., 1992; Jelmert et al., 1994; Lai et al., 1998). Indeed, welding fumes are classified by IARC as possibly carcinogenic to humans (Group 2B), partly due to the inconsistency of findings in human and animal genotoxicity studies (IARC, 1990).

Possible explanations for the absence of positive results in genotoxicity studies that focus on welders have been presented by other authors, namely the lower exposure of the lymphocytes to chromium that enters the body by inhalation and may be retained in the lung, which is after all the primary target organ for chromium genotoxicity (Littorin et al., 1983), the selective elimination of

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genetically damaged cells from the blood stream, and even a possible supressor effect of other compounds that welders are exposed to, namely nickel (Katifsis et al., 1996; Katifsis et al., 1998). The suggestion that blood lymphocytes may not be the most appropriate cell test system for biological monitoring to Cr(VI) has been supported by some authors (Benova et al., 2002; Surrales et al., 1995) but a recent study on the frequency of micronuclei in nasal cells of stainless steel welders produced no positive results, indicating that the absence of findings is not directly dependent on the cells used (Huvinen et al., 2002).

In the present study, a similar inconclusive pattern of response to genotoxic effect biomarkers is reported in welders. The welder group registered an increase in genetic damage measured by chromosomal aberrations and micronuclei, although this increase is not statistically significant when compared to control values. However, it must be noted that the mean micronuclei levels for welders are above the mean plus standard deviation of the control group, therefore the results can be interpreted as pointing to a potential increased risk for genetic damage in welders.

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6. Biomarkers of susceptibility to chromium 6.1. Overview

In the context of cancer molecular epidemiology, biomarkers of

susceptibility refer to the determination of inter-individual differences that affect the response to genotoxic aggressions. These differences can be genetically determined or acquired due to previous exposures to chemical or physical agents. Assessment of individual susceptibility may be accomplished by genetic screening for relevant polymorphic genes (related to genotype differences), or by evaluation of the response of pre-exposed cells to a challenge dose of a known genotoxic agent (assessment of phenotype differences).

Humans have considerable genetic heterogeneity. Genetic variation is the basis of evolutionary processes, that then leads to the fixation of one or another allele at a locus, enhancing adaptive integrity of the genome. The importance of genetic variability in susceptibility to disease development has been identified long ago, namely as a tool to prevent occupational health risks by shifting susceptible individuals to other job functions were they would not be placed at risk (reviewed in Gochfeld, 1997).

Genetic screening, or genotyping, is the most important group of biomarkers of susceptibility. Genotyping may increase our ability to identify small groups of individuals at high risk of developing cancer due to a genotoxic aggression, and avoid interpretation problems due to the dilution of the effects in the general population. Sub-areas of genetic susceptibility screening include the screening of inheritable variations in carcinogen metabolizing enzymes, inherited differences in DNA repair mechanisms, and germ-line mutations in tumor-associated genes (Ishibe and Kelsey, 1997). The last one consists on determining inherited cancer-predisposing genes that are associated with much higher risk and show strong familial incidence patterns. These are not further discussed here (for a review, see Li, 1995).

The genes studied in the genetic screening protocols are genes considered polymorphic, that, by definition, show variations in sequence with a frequency greater than one in hundred subjects. Although many polymorphisms are not functional, some may code for critical proteins and have an impact on cell functions, namely on enzymatic activity. The polymorphic genes are associated with relatively low, but relevant, increase in cancer risk, and may be strongly dependent on exposure to produce significant differences in response (Rothman and Hayes, 1995).

A great number of studies have been focused on the identification and study of differences in the metabolism of carcinogens, because those variations have been associated with an increased susceptibility to cancer development. Several enzymes

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that are affected by genetic polymorphisms have been identified, including N-acetyltransferases (NAT), Glutathione-S-transferases (GST) and cytochrome P450 enzymes.

Glutathione-S-transferases (GST) are an important family of conjugation enzymes known to catalyze the conjugation of glutathione with different species of electrophilic compounds. They play a major part in the cellular detoxification system and probably evolved to protect the cell against reactive oxygen metabolites (Landi, 2000). GSTs are involved in the detoxification of reactive electrophilic metabolites, such as products of lipoperoxidation, for example 4-hydroxynonenal, or epoxides derived from the biotransformation of polyciclic aromatic hydrocarbons (Ketterer, 1998).

Biotransformation, involving metabolic activation and detoxification, has an important role in determining the ultimate effects of exposure to chemical carcinogens. Depending on the enzyme, there may be one or several mutant alleles that produce enzyme variants with reduced or increased efficiency in comparison with the wild-type form. In some cases, such as the null glutathione S-transferases M1 (GSTM1) and T1 (GSTT1), the functional enzyme is completely missing. Case control studies have reported a high frequency of null-genotypes in lung and bladder cancer patients (Rothman and Hayes, 1995; Ryberg et al., 1997). The different biological response to a genotoxic agent due to genetic polymorphisms can also be evaluated by cytogenetic methods, and the association of certain genotypes with increased risk of genetic damage has been found both in vitro as in vivo (reviewed by Norppa, 1997).

The present study investigated the role of GSTM1 and GSTT1 genotype in the endpoints determined among the study subjects, to evaluate possible genetic susceptibility to their occupational exposure. It should be pointed out that the study of genetic polymorphisms normally demands a considerable number of individuals to produce statistically significant results, considering the low frequency of some of the genotypes present. In this view, this study should be considered a preliminary approach to the investigation of genetic susceptibility in chromium occupational toxicity.

The individual susceptibility to genotoxic damage can also be estimated by the response of cultured cells to a challenge dose of a known genotoxic agent, using a cytogenetic endpoint. Two altered responses may arise from this challenge, when we compare cells that were previously exposed to genotoxic agents, in vivo or in vitro, to non-exposed cells: either the previously exposed cells present an excessive genetic damage, showing an increased susceptibility to the aggression, or they have acquired a defense mechanism, by a process named adaptive response, that allows them to limit the extent of the damage, and have a smaller increase in the genetic damage than non-exposed cells.

Increased response of in vivo genotoxic-exposed cells to a subsequent in vitro exposure may be related to an impaired DNA repair process, due to alterations in the DNA structure or the repair enzymes induced by the previous exposure.

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These alterations difficult correct repair of the damage induced by the challenge agent and may indicate that the exposed individuals have increased health risk (Au et al., 1996). According to this hypothesis, chemical agents that react with DNA or proteins can, even at low doses, interfere with DNA repair, causing an increased response to posterior lesion by another agent, as represented in Figure 36. In this example the challenge agent is gamma radiation. The adducts formed during exposure to the first genotoxic are still present when cells are exposed to radiation, and there is a more difficult repair of damage due to their presence. Finally, the observation of chromosomal aberrations allows the scoring of the typical radiation-induced alterations: chromosome and chromatid breaks and rearrangement of chromosomes to form dicentric structures. In vivo or in vitro exposure to compounds such as 1,3-butadiene and nickel acetate has been shown to reduce DNA repair efficiency in exposed cells, measured by the challenge assay outlined above (reviewed in Au, 2001).

Another possible response to the challenge dose is exactly the opposite: the cells may be somewhat protected against the genotoxic aggression, and turn out a lower extent of genetic damage, when compared to equally challenged cells that had no previous genotoxic exposure. This cellular defense may be due to the activation of multiple repair systems, increased protein synthesis or antioxidant activity (reviewed in Oliveira et al., 1998). Nevertheless, this effect has been questioned, as it may derive from a selective elimination of extensively injured

Figure 36. Hypothesized process for infidelity of chromosome/DNA repair after exposure to carcinogens. The formation of chromosome aberrations identified as dicentrics and

breaks is shown (from Au, 1993)

(4 different chromosomes)

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cells (by apoptosis or necrosis), and a false scoring of the less damaged cells as the global population (Wojcik et al., 1996). 6.2. Research procedures 6.2.1. Genetic susceptibility

Glutathione S-transferases (GSTs) are a superfamily of polymorphic enzymes involved in conjugation of reactive intermediates to soluble glutathione forms, playing an important role in the detoxification of endogenous and exogenous toxicants (Landi, 2000). The polymorphic genes GSTM1, GSTT1 and GSTP1 are involved in the detoxification of a variety of potential carcinogenic compounds such as hydrocarbon diol-epoxides, steroids and genotoxic lipoperoxidation products. DNA was extracted from whole blood samples of all workers and controls were collected into 10 ml heparinized tubes and stored at –20 °C until use. Genotyping for GSTM1 and GSTT1 gene deletions was carried out by a polymerase chain reaction (PCR) in multiplex process, and detected by visualization of amplification products in an agarose gel, stained with ethidium bromide (see Appendice 2, Protocol 7). 6.2.2. Challenge assay

Whole heparinized peripheral blood obtained from the subjects was cultivated in supplemented Ham’s F-10 culture medium with addition of phytohaemagglutinin to the culture stimulate the lymphocytes to undergo division (same procedure as in Protocols 5 and 6, Appendice 2). At 24 hours of incubation, bleomycin (Delta Laboratories, Queluz, Portugal) was added to the culture medium to a final concentration of 3 µg/ml. After adding bleomycin to the culture, the same protocol as described for the cytokinesis-blocked micronuclei assay was followed (see Appendice 2, Protocol 6), as this was the cytogenetic endpoint chosen to evaluate genetic damage after exposure to the challenge compound.

Bleomycin is a radiomimetic chemical that has a direct action on the genetic material, generating the same clastogenic effects as radiation. This chemical was chosen to avoid the variability due to metabolizing enzymes in the case of xenobiotics that require activation. 6.3. Results

All results presented in the following tables were analyzed for

normal distribution using the Kolmogorov-Smirnov test. When no significant

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difference from normality was found, Student’s t-test was applied for comparison between group means, and group values are presented as mean ± standard deviation. Non-parametric tests were applied when data distribution significantly deviated from normality, and are referred to when result is presented. Where no test is specified, Student’s t-test and parametric analysis was applied. All calculations were performed using Microsoft® Excel 2000, with Analyse-It™ Version 1.66. 6.3.1. Genetic susceptibility

Table 22. Challenge test in tanners, welders (first sampling) and controls Tanners Welders Controls

# GSTM1 GSTT1 # GSTM1 GSTT1 # GSTM1 GSTT1 2 - - 45 - + 1 - + 3 - + 49 - - 2 - + 4 + - 51 + - 3 - - 5 - + 54 + - 4 + + 6 - + 56 + + 5 + - 7 - + + - 9 + + 7 - +

10 - - 8 + - 11 + + 9 - - 12 - - 10 + - 13 - + 11 + + 14 - - 12 + - 15 + + 13 + - 16 + + 16 + + 20 - + 18 + - 21 - + 19 + + 22 + + 20 + + 24 - - 21 + + 25 - - 23 + - 28 + + 24 - + 30 + + 25 + - 33 + + 26 - - 34 + + 27 - - 35 - + 29 - - 36 - - 31 + - 37 - + 32 + + 38 + +

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6.3.2. Challenge assay Table 23. Challenge test in tanners, welders (first sampling) and controls

Tanners Welders Controls # BNMN # BNMN # BNMN 2 35.0 45 27.0 1 32.0 3 49.0 49 20.0 2 30.0 4 30.0 51 41.0 3 32.0 5 72.0 54 17.0 4 12.0 6 27.0 56 30.0 5 48.0 7 77.0 7 36.0 9 18.0 8 13.0

10 21.0 9 40.0 11 56.0 10 34.0 12 19.0 11 42.0 13 43.0 12 20.0 14 48.0 13 31.0 15 19.0 16 22.0 16 61.0 18 42.0 20 55.0 19 26.0 21 50.0 20 34.0 22 11.0 21 30.0 24 43.0 23 20.0 25 60.0 24 58.0 28 29.0 25 16.0 30 44.0 26 38.0 33 42.0 27 24.0 34 32.0 29 46.0 35 24.0 31 30.0 36 20.0 32 54.0 37 58.0 38 36.0

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Figure 37. Micronuclei frequencies after challenge assay in tanners, welders and controls. BNMN,

binucleated micronucleated lymphocytes. 6.4. Discussion 6.4.1. Genetic susceptibility

No significant pattern of influence of the genetic polymorphisms of GSTM1 and GSTT1 enzymes in the results of biomarkers of exposure or effect was found in the groups studied, although an increase in the urinary lipoperoxidation products was found in the double null genotypes (for results and discussion, see Chapter 7).

The number of subjects involved in this project is not likely to be sufficient to obtain a valid conclusion from this study, given the statistical requirements for a thorough genetic susceptibility analysis. Also, cancer susceptibility due to chemical exposure is likely to be affected by a balance between various metabolizing enzymes, and so the assessment of a very small number of gene polymorphisms may not be enough to in the establishment of the risk profile (Bartsch and Hietanen, 1996). Some authors also suggest that the effect of genotype is more pronounced at low doses and that individual susceptibility may be irrelevant under exceptionally high exposure conditions (Srám and Binková, 2000)

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6.4.2. Challenge assay The challenge assay is based on the notion that a cell that has been exposed to a toxic aggression is likely to have an altered response to further damage. The challenge assay has been applied to the study of a considerable number of toxic agents including radiation, alkylating compounds, solvents, and metals (Oliveira et al., 1998). Chromium in vivo exposure has only recently deserved some attention in this context. One previous study on genotoxicity and radioresistance in lymphocytes obtained from electroplating workers was performed by Vaglenov et al. (1999). The frequency of micronuclei was determined with and without in vitro gamma irradiation in peripheral blood lymphocytes obtained from the workers. Base line frequency of micronucleated cells was significantly increased in chrome platers compared to controls, but after the challenge test micronuclei frequency in the cells obtained from workers was lower than the irradiated control response. The results indicated a possible adaptive response resulting from hexavalent chromium exposure. The challenge assay conducted during the present study focused mainly on the leather-tanning workers. An increased micronuclei frequency in response to the challenge test was found in the lymphocytes of tanners when compared to the control group, both in absolute values and after the frequency of micronuclei obtained without challenge exposure was subtracted. The excess in micronuclei frequency over control response to challenge was statistically significant when the baseline response was subtracted (33.4±18.6‰ versus 22.7±15.2‰, p=0.018). The increase in genotoxic response to the bleomycin challenge in tanners may be related to a less efficient DNA repair when the cell is burdened with stable complexes between chromium and DNA, such as DNA-protein crosslinks, which are increased in the tanner group. Another possible reason is a direct impairement of DNA repair systems, that involve a balance of multiple enzymes and are therefore an important target for possible toxic action, at the DNA or protein level (Au et al., 1996). The proposed epigenetic mechanism of toxic action of trivalent chromium may also have a role in the amplification of genotoxic effects induced by other compounds. Cr(III) has been shown to increase the processivity and decrease the fidelity of DNA replication by DNA polymerases, at physiological relevant concentrations (Singh and Snow, 1998). The replication of DNA has been evaluated in vitro in both acellular systems and in E. coli transfected with genetic material previously treated with CrCl3 (0.4-50µM). Both conditions showed a significant increase in misincorporation of bases during replication, possibly to a tighter bond between polymerase and DNA template and a bypass of damaged or altered DNA bases (Snow, 1991). The findings suggest that tannery workers may have acquired an increased susceptibility to occupational or environmental mutagens, which can place them at higher genotoxic risk in subsequent exposures.

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7. Oxidative damage in chromium occupational toxicity

7.1. Overview

As previously discussed, one of the possible mechanisms for chromium

induced cytotoxicity is the by oxidative damage through the generation of reactive oxygen and radicalar species. Although hexavalent chromium is more likely to undergo redox cycling responsible for initiating oxidative stress in exposed cells, trivalent chromium is also able to generate hydroxyl radical (see Section 2.5). The abundant presence of membrane phospholipids at sites where reactive oxygen species are formed makes them easily accessible endogenous targets for lipoperoxidation (De Zwart et al., 1999). Lipoperoxidation occurs as a chain reaction initiated by free radicals, which propagates itself and can result in the formation of many equivalents of lipid peroxides. This process has been implicated in diverse pathological conditions, including atherosclerosis (Holvoet and Collen, 1998), aging (Spiteller, 2001), rheumatoid arthritis (Henrotin et al., 1992) and cancer (Marnett, 2000). It is also involved in the toxicity of pesticides (Bismuth et al., 1990), solvents (Brattin et al., 1985) and metals (Kasprzak, 1995).

Oxidative DNA damage was shown to be accompanied by a significant decrease in intracellular antioxidants (Lenton, et al., 1999). As previously discussed, some of the most important non-enzymatic cellular reducers of chromium are ascorbic acid, glutathione and cysteine. The exposure of cells to metals may alter GSH concentration: for example, exposure to low doses of mercury chloride starts by increasing intracellular GSH, probably by increased synthesis, and higher doses significantly deplete GSH levels. In previous studies intracellular concentration of GSH, but not ascorbate, has been shown to influence the amount of DPC formed in vitro (Capellmann et al., 1995). This provides evidence that this antioxidant may be involved in the mechanism of the cell against chromium-induced toxicity. The evaluation of thiol antioxidants in chromium-exposed populations may be of importance to further clarify the toxicity mechanisms of different valence states, and has been included in this study. 7.2. Research procedures 7.2.1. Lipoperoxidation products

The extension of the oxidative catabolism of lipid membranes can be evaluated by several endpoints, including the measure of exhaled alkanes (mainly ethane and pentane), determination of isoprostanes and of a variety of aldehydes in biological samples (reviewed in DeZwart et al., 1999). The most widely used method is the quantification of malondialdehyde (MDA), one of the stable

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aldehydic products of lipoperoxidation, present in biological samples such as whole blood, plasma or urine (Gutteridge, 1995; DeZwart et al., 1999). Urinary lipid peroxidation products are a good index of lipoperoxidation, as they most probably reflect the global oxidative status of the whole organism and urine is an easily accessible sample (Cadenas et al., 1996). MDA contained in the sample reacts with thiobarbituric acid (TBA) to form a complex that can be quantified by spectrophotometry and fluorimetry. Nevertheless, this reaction is not specific of MDA, since other compounds (other aldehydes, bilirubin) can react with thiobarbituric acid, and other chromophores present in the biological samples may absorb or fluoresce in the same wavelengths as the MDA-TBA complex. In an attempt to overcome this limitation, the TBA-MDA complex is extracted from the reaction mixture with an organic solvent, and the quantification is made using this extract. Even so, the results of the TBA-MDA assay are presented as thiobarbituric acid reactive substances (TBARS), expressed as MDA.

Protocols have been developed to apply HPLC separation prior to quantification of the TBA-MDA complex (Templar et al., 1999), which allows improvement of the specificity of the determination. However, results obtained previously showed no added advantage to the HPLC protocol when groups of exposed individuals are compared, particularly for population screening (Borba and Monteiro, unpublished results). The fluorescent measurements of TBARS eliminates most of the interference occurring in spectrophotometric assays, increases its sensitivity and strongly correlates with the HPLC methods. It adds the advantages of being an economical, straightforward, fast technique, ideal for medium to large-scale population screening.

Quantification of thiobarbituric acid reactive substances was based on the technique described by Dousset et al. (1983). Thiobarbituric acid was added to duplicate aliquots of plasma or urine samples and heated at 100ºC for 10 minutes in order to favor the formation of the complex. Butilhydroxytoluene was added to the reaction medium to avoid oxidation of lipids in the sample during the 100ºC incubation. The complex was extracted from the aqueous phase by a fixed volume of n-butanol, and the fluorescence of the organic solution was measured. A calibration curve was prepared from 1,1,3,3-tetraetoxipropane, which hydrolyses to malondialdehyde in water (see detailed protocol in Appendice 2, Protocol 8). 7.2.3. Thiol antioxidants

Several methods are available to quantify glutathione in biological samples, most commonly by one enzymatic, fluorometric or colorimetric techniques. The HPLC detection of the fluorescent complexes of GSH is considered the most reliable assay method. Direct fluorimetric measurements of the reaction technique was shown to be affected by confounding factors such as interfering compounds and sample size, and therefore may not be a reliable method for biological samples (Floreani et al., 1997).

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The reaction of available –SH groups with the fluorescent dye monobromobimane generates a stable fluorescent complex that can be separated from the reaction mixture by HPLC and quantified by fluorimetry. Lysis of the cells followed by an extraction step prior to the reaction with the dye enables us to obtain the desired cellular components and to simplify the sample matrix. In this study, an extraction of cellular thiols made possible to determine glutathione (GSH) and cysteine (CYS) in isolated peripheral blood lymphocytes performed by the HPLC separation and quantification of their fluorescent derivatives. Because the glutathione complex is formed only with the reduced form of the molecule, the reaction was performed with and without dithiotreitol (found to completely convert oxidized GSSG into its reduced form). CYS and GSH were well resolved from non-specific reaction products and the fluorescence-based determinations offered a very high sensitivity of thiol measurements (see detailed protocol in Appendice 2, Protocol 8). Limit of detection for this method was 0.067 nmol of CYS and 0.091 nmol of GSH, based on a signal to noise ratio of 3:1 (Snyder, 1997). 7.3. Results

All results presented in the following tables were analyzed for normal

distribution using the Kolmogorov-Smirnov test. When no significant difference from normality was found, Student’s t-test was applied for comparison between group means, and group values are presented as mean ± standard deviation. Non-parametric tests were applied when data distribution significantly deviated from normality, and are referred to when result is presented. Where no test is specified, Student’s t-test and parametric analysis was applied. All calculations were performed using Microsoft® Excel 2000, with Analyse-It™ Version 1.66.

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7.3.1. Lipoperoxidation products Table 24. Thiobarbituric acid reactive substances expressed as malondialdehyde (MDA µmol/g creatinine) in urine of tanners, welders and controls

Tanners Welders Controls # MDA # MDA # MDA 1 1.42 45 3.21 1 0.65 2 2.01 46 1.02 2 1.03 3 29.23 47 0.89 3 0.46 4 2.66 48 0.91 4 0.55 5 1.01 49 2.90 5 0.92 6 0.81 50 1.41 6 1.06 7 2.22 51 0.66 7 1.48 9 1.06 53 1.16 8 0.61

10 1.12 54 0.70 9 0.62 12 2.88 55 3.47 10 0.40 13 1.03 56 0.66 11 0.78 14 1.67 57 0.84 12 0.64 15 1.38 58 3.11 13 0.92 16 1.26 59 2.90 14 1.22 18 1.50 60 4.22 15 0.88 19 1.03 61 1.67 16 0.82 20 1.10 17 0.87 21 1.06 18 0.68 22 1.44 19 0.74 24 0.95 20 0.85 25 1.61 30 1.37 31 0.76 32 2.34 33 1.70 34 1.36 35 1.07 36 1.40 37 0.00 38 1.04 39 1.22

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0,00

0,50

1,00

1,50

2,00

2,50

3,00


Urin

ary

MD

A um

ol/g

cre

atin

ine

Figure 38. Urinary thiobarbituric acid reactive substances expressed as malondialdehyde (MDA

µmol/g creatinine) in tanners, welders and controls. * p<0.005; ** p<0.001. 7.3.2. Thiol antioxidants

Table 25. Glutathione (GSH, nmol/g protein) and cysteine (CYS, nmol/g protein) in peripheral blood lymphocytes of tanners and welders.

Tanners Welders # GSH CYS # GSH CYS 4 134.20 12.60 45 31.31 12.10 5 100.00 9.80 46 159.62 14.00 6 148.00 8.40 47 89.15 14.10

14 82.00 6.00 48 27.70 19.40 18 82.30 14.40 49 57.40 15.10 20 148.20 10.49 50 106.09 21.74 21 108.50 14.50 51 70.87 14.52 22 69.60 9.40 53 79.79 16.35 24 133.40 16.70 54 31.04 6.36 34 81.10 7.10 55 69.77 14.30 36 174.50 23.20 56 54.59 11.19 37 124.00 11.50 57 42.07 18.62 38 119.20 18.20 58 112.66 23.09 39 91.30 14.60 59 48.91 10.02

60 28.71 15.88 61 46.79 9.59

*

**

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Table 25. Glutathione (GSH, nmol/g protein) and cysteine (CYS, nmol/g protein) in peripheral blood lymphocytes of controls

# GSH CYS 4 85.80 11.50 5 181.80 32.00 6 68.40 16.90 7 73.20 11.20 8 78.70 10.70 9 122.70 16.50

10 148.50 17.00 11 194.70 20.90 12 73.60 15.10 13 76.50 17.70 14 117.00 16.30 15 95.60 19.70 16 86.90 6.80 17 88.00 4.70 18 126.30 25.30 19 85.50 44.50 20 93.30 9.80 4 85.80 11.50 5 181.80 32.00 6 68.40 16.90 7 73.20 11.20 8 78.70 10.70 9 122.70 16.50

10 148.50 17.00 11 194.70 20.90 12 73.60 15.10 13 76.50 17.70 14 117.00 16.30 15 95.60 19.70 16 86.90 6.80 17 88.00 4.70 18 126.30 25.30 19 85.50 44.50 20 93.30 9.80 21 79.50 11.70 22 23.40 2.40 23 121.80 13.50 24 94.00 14.00 25 62.60 9.90 26 118.20 11.60

86

0.00

20.00

40.00

60.00

80.00

100.00

120.00

140.00

160.00


Glu

tath

ione

nm

ol/m

g pr

otei

n

Figure 39. Glutathione concentration in peripheral blood lymphocytes of tanners, welders and

controls. *p<0.001. 7.4. Discussion 7.4.1. Lipoperoxidation products

Numerous studies have shown increased indices of lipid peroxidation in laboratory animals and biological systems exposed to chromium. Fewer studies have been conducted to evaluate the role of lipid peroxidation in occupational exposure to the metal. In one previous study (Huang et al., 1999), 25 chrome-plating workers were studied for malondialdehyde (MDA) urinary concentrations and antioxidant status (antioxidant enzymes levels – superoxide dismutase, glutathione peroxidase, catalase) and compared to a reference group of 28 controls. Significantly elevated urinary and plasmatic concentrations of chromium and MDA, and positive correlation between them, were found. No significant difference in antioxidant status between exposed and control groups was reported.

In the present study, both occupationally exposed groups show significantly higher concentration of TBARS expressed as MDA, which indicates an increase in oxidative stress related to their occupational exposure. Stainless steel welders, exposed to fumes containing hexavalent chromium, have the highest value of TBARS in the three groups. This may be due to the generation of reactive oxygen species and radicals by the Cr(VI) intracellular metabolism. There is a positive correlation between urinary TBARS and plasmatic total chromium concentrations

*

*

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found, which supports previous findings of a correlation between urinary and plasmatic malondialdehyde and hexavalent chromium exposure.

The tanner group, exposed to trivalent chromium, also registered significantly elevated concentrations of TBARS when compared to control values. As previously mentioned trivalent chromium is able to generate hydroxyl radicals and to induce oxidative DNA damage in vitro, in the presence of hydrogen peroxide. The high values of lipoperoxidation products may reflect the same process in vivo, although no positive correlation was found between TBARS and chromium values in this group. The present data does not allow further clarification of the origin of increased lipoperoxidation in tanners, due to the multi-exposure to which the workers are subject and the non-specificity of this endpoint.

7.4.2. Thiol antioxidants

HPLC measurements of GSH and CYS concentrations in freshly isolated peripheral lymphocytes from unexposed human subjects found that GSH was the major thiol in these cells with almost 8-fold molar excess over CYS. However, GSH, but not CYS, was significantly lower in cells obtained from subjects in the welder group. A possible explanation for the low concentrations of GSH in lymphocytes of Cr(VI) exposed welders is the formation of stable coordinate complexes between the metal and GSH, that would decrease the amount of available glutathione molecule. DNA-protein crosslinks are significantly elevated among these workers, and these complexes have been reported to involve GSH in their formation. Oxidative stress, also found in welders, may contribute to depletion of GSH, due to its role in radical scavenging.

In the tanner group, neither gluthatione nor cysteine are significantly altered when compared to control values, showing that, if they are involved in the intracellular metabolism or toxicity of chromium or other compounds to which these workers are exposed, their turnover is enough to maintain intracellular concentrations within normal values.

The decrease in GSH reported in welders seems more likely to be an outcome of the combined oxidative stress-mediated depletion and the participation of GSH in binding of metals, forming stable complexes that either remain in the cell (for example as DNA-protein crosslinks) or are excreted. This could explain the disparity in glutathione profiles between the tanner and welder group, both showing significantly elevated lipoperoxidation products in urine. In the tanner case, the access of trivalent chromium to the cell is slow, and although it is able to induce formation of DNA-protein crosslinks, these are in lower extent than hexavalent chromium exposure. The higher concentration of intracellular chromium in welders, with a dramatic increase in DNA-Cr-GSH complexes formed, may be responsible for the decrease in available glutathione.

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8. Final discussion and conclusions

Occupational exposure to chromium compounds has long been a cause of concern for the health safety of workers. It has also been a important source of information to expand our understanding of chromium toxicity in order to develop valuable prevention strategies.

The objectives of this project were presented as questions regarding several aspects of the occupational toxicology of chromium. Let us now analyze the possible answers for those questions provided by the results obtained in the present study.

1) How do the internal dose and biological effective dose relate in Cr(VI) and Cr(III) exposure?

Chromium-exposed individuals consistently show increased concentrations of the metal in biological samples, when compared to non-exposed individuals. In the present study, both leather tanning industry workers and welders had significant excess in urinary and plasmatic chromium concentrations in comparison to control values. Considering that leather tanners are exposed to trivalent chromium, since Cr(VI) contamination is absent or unlikely, it is proven that chronic occupational exposure is enough to overcome the limitations in Cr(III) absorption. The difference in cellular uptake between exposure to trivalent or hexavalent chromium does not seem to result in differences in the internal dose of chromium between the two groups of workers. A somewhat diverse pattern is found when a biomarker of exposure related to the biologically effective dose is investigated. DNA-protein crosslinks (DPC) are an index of the action of chromium on the DNA, and give an estimate of the chromium that ultimately reached the cells and is affecting the structure of the target for genotoxic action. DPC are significantly elevated in both exposed groups in comparison to control values. This is the first report on in vivo DPC formation in individuals exposed to Cr(III), and in this way it is shown that the trivalent form of chromium may be able not only to be absorbed into the organism but to reach the intracellular medium in sufficient amounts to produce a detectable amount of DNA-Cr-protein complexes. But, as importantly, the difference between DPC levels in Cr(III) and Cr(VI) exposed groups is quite impressive, showing that for the same internal dose, as determined by the classical indicators of exposure, considerably different amounts of the toxic are able to reach the target molecule. Cr(VI) ability to be easily and efficiently uptaken by cells is widely accepted as a basis for its toxicity. The measure of the biologically effective dose of chromium, making use of molecular epidemiology biomarkers such as DPC seems to be a more important

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assessment of the potential genotoxic risk involved in the exposure, in contrast with the simple evaluation of extracellular concentrations of the metal that may not give a good estimate of the risk of chromium-related DNA effects.

2) Do Cr(III) occupationally exposed individuals incur in genotoxic risk?

Genotoxic effect is the result of combined processes of lesion and repair that are affected by differences in several critical points, such as toxicokinetics, metabolism, and repair systems. The biological endpoints validated for the evaluation of that effect relate basically to the visualization of cytogenetic abnormalities, such as chromosomal aberrations. Unfortunatelly, these endpoints have low specificity to the original genotoxic agent responsible for their formation, and may inclusively show a cumulative result of several aggressions. For that reason, interpretation of results from cytogenetic assays conducted in multi-exposed human populations must always be done with some caution. In the present study, we can identify the global leather tanner group as incurring in possibly increased genotoxic risk derived from their occupational exposure. The excess in cytogenetic lesion reported is probably the result of their multiexposure to several potentially genotoxic agents, including chromium.

3) What is the importance of selected genetic characteristics in the toxic

outcome of chromium exposure? Genetic polymorphisms of metabolizing enzymes usually result in discrete differences in biotransformation efficiency in the resulting individual phenotype. These small variations may be potentiated by the exposure to relevant amounts of a given toxic compound, to the point of becoming detectable as different responses to common biomarkers of exposure or effect in genotoxic studies. Nevertheless, and because of the low frequency of certain polymorphims, the reliable evaluation of the genetic variation in the toxic outcome usually requires reasonably sized populations. In the present case, although no influence of the genetic polymorphims studied was found, the conclusions are hindered by the number of subjects available for the study. Therefore it should be considered a contribution to the study of the influence of glutathione S-transferase GSTM1 and GSTT1 genotypes in chromium toxicity, rather then a fully conclusive investigation.

4) Is there an acquired resistance or susceptibility of the cells to further genotoxic damage, after exposure to trivalent chromium? When lymphocytes obtained from leather tanning workers were challenged in vitro with a radiomimetic agent, the cells showed an excessive response in comparison to control, non previously exposed, cells. The findings suggest an

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increase in the susceptibility to genotoxic damage that may be related to the worker’s exposure to the industrial chemicals, namely Cr(III) compounds. Although the role of chromium in this sensitizing effect cannot be characterized further at this point, again due to the presence of other chemical and physical agents in the industry, the excessive response to the challenge test may imply that exposure to a leather tanning industrial environment may lower cellular protection against genotoxic agents. To be confirmed, the present data increases the concern on the potential genotoxic risk in Cr(III) exposed populations.

5) Is there a role for oxidative damage in occupational chromium toxicity? A great deal of evidence has been accumulated on the in vitro generation of oxidative stress in the presence of mainly hexavalent, but also trivalent chromium. This study presented an opportunity to investigate the in vivo oxidative pathway, comparing two groups exposed to Cr(VI) and Cr(III). There is a significant excess of lipoperoxidation products in both worker groups when compared to non-exposed controls, suggesting an increase in oxidative processes due to occupational exposure to chromium compounds. This may an indication of in vivo Cr(III) generated oxidative stress, and another proof that Cr(VI) is able to generate oxidative damage. Although the manual metal arc activity is regarded as causing some of the highest acute exposures to Cr(VI), it must be mentioned that stainless steel, as a ferrochromium alloy, also contains considerable amounts of iron, that are likely to contribute to the total oxidative stress found. The depletion of intracellular glutathione, only found in welders, reinforces the idea that these workers are subject to oxidative damage, due to exposure to welding fumes. The absence of decreases in glutathione concentration in tanners may be linked to a slower entry of Cr in the cell, allowing a more efficient regeneration of GSH from GSSG, without a detectable variation in the available glutathione.

6) What protocol of biomonitoring for genetic risk should be implemented in chromium-exposed workers? Being this a small scale project to study and biomonitor the genotoxic effects among occupationally exposed populations, it can be considered as a preliminary approach to continuing biomonitoring. In light of that, and because cytogenetic biomarkers are investigated, it is suggested that the background values of cytogenetic damage be determined for each worker at the beginning of the activity, that can be stored and used for later comparison. Tanners should be routinely biomonitored for chromium exposure by the determination of chromium in urine, a non-invasive high output screening method. In individuals presenting higher chromium levels in urine, the biologically active dose should be assessed, namely by the DNA-protein

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crosslinks assay, and a cytogenetic damage measurement conducted, to be compared with background values for the same worker. The biological monitoring of welders by the classical indicators of exposure may not provide the most reliable results, given the lack of correlation with the biologically effective dose, previously discussed. Given the classification of hexavalent chromium as a known human carcinogen, a more rigorous strategy of biomonitoring should be implemented. The results presented here suggest that these workers would benefit from a routine program of DNA protein crosslinks determination, as a biomarker for biologically effective dose, and regular cytogenetics evaluation to follow carefully relevant changes from background values.

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9. Future perspectives

Sometimes a human monitoring project raises as many questions as answers, and the present study does not deviate too much from that scenery. Clearly there are a number of uncertainties and research needs suggested by the results obtained, as it would be expected in a pilot biomonitoring study. The application of the biomonitoring strategy defined in Chapter 8 to a larger population, possibly by integrating several industries, could be useful in further exploring chromium genotoxicity in occupational exposure. Other important points that merit further development are enumerated below:

- The increased genotoxic damage found in both worker groups represents a

concern for the long-term risk of occupational cancer derived from the leather tanning and welding activities. The findings should serve to alert occupational health services, administration parties and the workers to the critical importance of preventive measures and continuous control of health risk through routine monitoring procedures.

- As this is, to our knowledge, the first report on the in vivo ability of trivalent chromium to form DNA-protein crosslinks in exposed individuals, it is expected that other populations may be studied and the value of DPC as a biomarker of biologically effective dose to all forms of chromium can be further established.

- Only limited information regarding the in vivo generation of oxidative stress by trivalent chromium compounds is presented here. These findings should be complemented by the analysis of other oxidative status indicators to confirm and clarify the results obtained. Depletion of glutathione in welding workers is a critical deleterious effect that may have serious consequences in cell function and antioxidant protection, and it also deserves further investigation.

- The definition of a cutoff value to the biologically effective dose of chromium, namely as determined by the DNA-protein crosslinks assay, would be most valuable to identify individuals at higher genotoxic risk. This will probably require the validation of the biomarker of exposure, and establishing the correlation between peripheral lymphocyte and target organ (mainly lung) DPC levels.

- In an expansion phase of Toxicogenomics, the systematic study of the influence of genetic characteristics in the toxicology of metals is should be undertaken in a near future, as our knowledge of the Human genome expands. This preliminary study may have been a modest contribution to that major effort.

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Sugden KD, Martin BD. (2002) Guanine and 7,8-dihydro-8-oxo-guanine-specific oxidation in DNA by chromium(V). Environ Health Perspect, 110(Suppl 5): 725-728.

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Appendice 1

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Erin Brockovich: The Film Universal Pictures, 2000

Erin Brockovich, written by Susannah Grant and Richard LaGravenese stars Julia Roberts in the true story of a twice-divorced mother of three young children, who struggles to be taken seriously. While working as a file clerk in a small law firm, she stumbles upon a cover-up involving contaminated water in a nearby town which is causing devastating illnesses. Through sheer determination, she convinces her boss (Ed Masry) to allow her to investigate, and in the process uncovers proof that the power company Pacific Gas and Electric knowingly misled the small town of Hinkley, California into believing the water they drank was safe, when in actuality, it was tainted with hexavalent chromium. Although the local citizens are initially leery of becoming involved, Erin's brash manner and ability to speak to them clearly-and frankly-earns their trust.

In 1993, PG&E settled with the over 600 plaintiffs for $333 million, the largest settlement ever for health-related damages in a direct-action lawsuit. [trailer of the film Erin Brockovich, Universal Pictures, 2000, is included in the CD

that accompanies the thesis]

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Appendice 2

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1. DETERMINATION OF TOTAL CHROMIUM IN URINE AND PLASMA BY ETA-AAS (adapted from Granadillo et al., 1994) Standards Spectrosol® trivalent chromium standard solution (BHD Laboratory Supplies, Poole, UK): dilute to final concentrations of 0,5 to 10 µg/L in plasma or urine pool (intermediated dilutions made in 0,01M nitric acid). Blank: dH2O + Triton X-100 (1:4) or dH2O + Triton X-100 with 0.01 mol/L nitric acid (1:1). Sample Dilution Plasma 1 + 4 in 0.1% (v/v) Triton X-100 (Sigma, St. Louis, MO, USA). Urine 1 + 1 in 0.1% (v/v) Triton X-100 with 0.01 mol/L nitric acid. Technical parameters

- 20 µl injection volume; - Pyrolitic coated graphite tubes with L’vov platform (Perkin-Elmer,

Norwalk, CT, USA); - Chromium hollow cathod Lumina ® lamp (Perkin Elmer, Norwalk, CT,

USA); - Deuterium lamp background correction; - Wavelength: 357,9 nm; - Monochromator slit: 0,7 nm.

Graphite furnace conditions

Temperature Ramp Hold Argon Flow 1. Dry I 80 1 5 300 ml/min 2. Dry II 210 10 10 300 ml/min 3. Pyrolisis 1350 15 15 300 ml/min 4. Atomization 2500 0 5 0 ml/min 5. Cleanout 2650 2 2 300 ml/min

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2. DETERMINATION OF HEXAVALENT CHROMIUM IN LEATHER DUST BY HPLC (adapted from NIOSH method 7600 and Padarauskas et al., 1998) Standards Sodium chromate tetra hydrated Na2CrO4.4H2O (Aldrich, Milwaukee, WI, USA): dilute in dH2O to final concentrations of 10 to 50 ng/ml. Blank: reaction mixture using dH2O as “sample”. Extraction 1. In three 50 ml tubes, combine 500 mg of leather dust with 8 ml of different pH solutions:

- 0,5 N H2SO4 - PBS Buffer pH 7.0

NaCl 8 g NaH2PO4 0.6 g Na2HPO4 2.72g

+800 ml dH2O, set pH 7.0 with HCl, complete 1L with dH2O - 2% NaOH-3%Na2CO3

2. Let it sit in a rotating platform for 60 minutes at room temperature. 3. Centrifuge for 5 minutes at 4000 rpm. 4. Filter 2 ml of supernatant through 0.2 µm pore filter to remove all leather dust. Reaction with 1,5-dyphenilcarbazide 1. Prepare (daily) the solution of 1,5-dyphenilcarbazide (Aldrich, Milwaukee, WI, USA): 5 mg 1,5-dyphenilcarbazide + 1 ml acetone (dissolve) + 1 ml dH2O 2. Add 300 µl of sample (or standard) to eppendorfs containing 532 µl 0.5N H2SO4, 50 µl dyphenilcarbazide solution and dH2O to complete 1 ml of final reaction volume (alkaline sample reaction needs to be further acidified by adding 20 µl concentrated H2SO4 to optimize reaction). 3. Vortex and allow the reaction to proceed for 15 minutes. The colored complex is stable for 3 hours. Quantification by HPLC Equipment: Shimadzu LC-10Advp Liquid Chromatographer with UV/Visible detector Wavelength: 540 nm Column: Ultrasphere ODS (5 µm, 250 x 4.6 mm) Mobile phase: 20% acetonitrile, 80% acetic acid 0.25% (isocratic) Flow rate: 1.2 ml/min Injection volume: 20 µl

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3. DETERMINATION OF DNA-PROTEIN CROSSLINKS IN PERIPHERAL BLOOD LYMPHOCYTES BY K-SDS PRECIPITATION ASSAY (adapted from Zhitkovich and Costa, 1992) Buffers PBS pH 7.2 (see Protocol 1) DPC lysis buffer (can be prepared in advance and stored at room temperature) 1% SDS 20 mM Tris-HCl 2mM EDTA, pH 8 1 mM phenylmethylsulfonylfluoride, PMSF (Sigma, St. Louis, MO, USA), added when the buffer is used. 0,4 mg/ml bovine serum albumin, BSA (Sigma, St. Louis, MO, USA), added when the buffer is used. DPC wash buffer (can be prepared in advance and stored at 5ºC) 200 mM KCl 20 mM Tris-HCl, pH 7.5 2 mM EDTA, pH 8.0 Lymphocyte isolation and lysis 1. Isolate lymphocytes from 8 ml of heparinized whole blood by standard Ficol/sodium diatrizoate method (Histopaque-1077, Sigma, St. Louis, Mi, USA). Wash three times with sterile PBS pH 7.2. 2. Re-suspend cell pellet in 750 µl PBS pH 7.2; insuring homogeneity, pipette 250 µl of lymphocyte suspension to 3 eppendorfs containing 500 µl DPC lysis buffer to which PMSF and BSA have been recently added (the half life of PMSF in water is 30 minutes at room temperature; the stock solutions of PMSF should be made in absolute ethanol). 3. Prepare 3 blanks by adding 250 µl PBS pH 7.2 to 500 µl DPC lysis buffer. 4. Vortex at highest setting, 5 seconds. 5. Place at -70ºC. DPCs are stable for several month at this storage temperature. DPC assay 1. Thaw samples at 37ºC 2. Shear each sample through a 25 Gauge needle in a 2 ml syringe (aspirate and expel 5 times). Place at –70ºC again for 10 minutes. 3. Thaw samples at 37ºC. Add 500 µl of DPC wash buffer (a precipitate should form). Vortex at highest setting for 5 seconds. 4. Place in water bath at 50ºC for 10 minutes. 5. Place samples on ice for 5 minutes.

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6. Centrifuge samples at 4000 rpm for 5 minutes at 4ºC (swing-out bucket centrifuge). 7. Remove supernatant to a marked tube (all supernatants for each sample should be gathered in a marked tube, as they will be needed to measure non-crosslinked DNA). 8. Add 1 ml of DPC wash buffer to each sample. Vortex at highest setting until pellet is fully dispersed. 9. Place in water bath at 50ºC for 5 minutes. 10. Place samples on ice for 5 minutes. 11. Centrifuge samples at 4000 rpm for 5 minutes at 4ºC. 12. Remove supernatant Repeat steps 8-11 twice (total 3 washes with 1 ml wash buffer) and combine supernatants for each sample in one tube. 13. Add 0.5 mg/ml proteinase K (Sigma, St. Louis, MI, USA) to DPC wash buffer and add 500 µl of this solution to each sample. Vortex to disperse pellet. 14. Leave samples at 50ºC overnight. 15. Add 50 µl of 4 mg/ml BSA solution to each sample. Place samples on ice 30 minutes. 16. Centrifuge samples at 4000 rpm for 20 minutes at 4ºC. DNA quantification 1. Prepare a calibration curve with 0 to 200 ng calf thymus DNA (Gibco BRL Life Technologies, Grand Island, NY, USA) per well for multi-well fluorescence reader, (Tecan SPECTRA Fluor Plus,), or 0 to 400 ng calf thymus DNA for standard fluorescence (Hitachi F-2000 Fluorescence Spectrophotometer). 2. Combine 100 µl of each sample obtained from proteinase K treatment or combined supernatant with 100 µl of a 180x dilution of PicoGreen ® (Molecular Probes, Eugene, OR, USA), for multi-well fluorescence reader, or 350 µl of sample with 350 µl of 180x dilution of PicoGreen ® for standard fluorescence. 3. Allow to react for 15 minutes in the dark. 4. Read fluorescence at 485 excitation, 535 emission. Calculate the percentage of crosslinked DNA from the total DNA in the sample.

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4. DETERMINATION OF THE WEIGHTED AVERAGE LENGTH OF DNA FRAGMENTS DNA sample 1. Digest 1 ml of combined supernatant from the DPC quantification protocol

(Protocol 3) with 0.2 mg/ml proteinase K for 1 hour at 50ºC. 2. Add 0,2M NaCl to 0,6 ml of sample from point 1. 3. Add 2 volumes of 100% ethanol. 4. Leave at –20ºC overnight. 5. Centrifuge at 1400 rpm, 20 minutes at 4ºC. 6. Remove supernatant. 7. Dry the pellet in a Vacufuge for 8 minutes. 8. Dissolve pellet in 50 µl dH2O. Buffers TAE buffer stock solution (50x)

242 g Tris base 57.1 ml glacial acetic acid 100 ml 0.5 M EDTA pH 8 add dH2O to 1000 ml

Loading buffer stock solution (6x)

150 mg Ficol 400 2.5 mg bromophenol blue add dH2O to 1ml

Agarose gel 1. Dissolve 1% agarose in TAE 1x in a heat resistant flask: to prepare a 50 ml gel

add 0.5 g agarose to 49 ml dH2O and 1 ml TAE 50x. 2. Microwave the solution until it looks clear (use care when removing solution

from the microwave, a sudden ebullition may project the hot contents). 3. Cool down slightly, without allowing to reach gel state. 4. Pour into electrophoresis apparatus. 5. Put the “comb” in place before the gel becomes solid. 6. Let it stand for 30 minutes 7. Carefully remove “comb” to leave the well intact. Electrophoresis 1. Mix 1-4 µl of sample with 1x loading buffer diluted with dH2O to a final

volume adequate to the well size (e.g., for a 25 µl well prepare 20 µl sample

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solution by adding 2 µl of sample, 3.3 µl of loading buffer 6x and 14.7 µl of dH2O).

2. Fill the electrophoresis apparatus with TAE 1x until the gel is completely covered.

3. Pipette a DNA fragment size standard (DNA ladder) into one or more wells in the gel, according to the number of samples analyzed. λ-Hind III was used to compared DNA fragments.

4. Pipette the samples into the remaining wells. 5. Turn on power for the electrophoresis. An initial voltage of 10v was used for 5

minutes, followed by 60v for 1 hour. Gel staining 1. Turn off power and carefully remove the gel into a solution of ethidium

bromide (250 ml dH2O plus 25 µl of 10 mg/mg ethidium bromide). 2. Place in a rotating table for 15 min (gentle rotation). 3. Wash twice with dH2O for 10 minutes each time. Visualizing the DNA bands Digitized images of ehtidium bromide stained gels were aquired on a GelDOc 2000 photo-documentation system (BioRad) and the band density information was analysed for weighted average fragment length in Microsoft Excel.

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5. CHROMOSOMAL ABERRATIONS IN PERIPHERAL BLOOD LYMPHOCYTES Cell cultures 1. Add 500 µl of whole blood to 4.5 ml Ham’s F-10 medium supplemented with 24% fetal calf serum (Sigma, St Louis, MO, USA), pennicilin (100 IU/ml), streptomycin (100 µg/ml), 1% L-glutamine, and 1% heparin (50 IU/ml) (BBraun, Melsungen, Germany). 2. Add 2% (v/v) phytohemaglutinine (PHA-M form, Gibco BRL Life Technologies, Grand Island, NY, USA). 3. Close the tubes thightly and incubate at 37ºC for 45 hours. 4. Add colchicine (Fluka, Buchs, Switzerland) to a final concentration of 0.5 µg/ml was added and the culture continued for further 3 hours. Preparation of slides 1. Harvest cells by centrifugation 5 minutes, 314 g, room temperature. 2. Resuspend the cell pellet gently in 8 ml of a hypotonic solution of 0.075 M KCl. Leave in contact at 37ºC for 5 min. 3. Centrifuge cells 5 minutes, 314 g, room temperature, discard supernatant. 4. Loosen the cell pellet. 5. Add 4 ml of cold methanol/acetic acid (3:1), gently swirling the tube. 6. Centrifuge cells 5 minutes, 314 g, room temperature, discard supernatant. 7. Repeat steps 9 and 10 until supernatant is clear. 8. Ressuspend in a small volume of fixative. 9. Drop suspension on water coated slides (3-5 drops per slide) and allow to dry before staining with 4% Giemsa in 0.01 M. phosphate buffer pH 6.8 for 10 minutes. 10. Close the slides with Entellan® (Merck, Darmstadt, Germany) Scoring of slides 1. Code the slides and score 100 complete metaphases, presenting 46 centromeres, per case, at 1,250x magnification. Classification of chromosomal aberrations was made according to criteria described in Rueff et al.(1993). 2. Determine mitotic index by counting the number of metaphases per 1000 nuclei.

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6. MICRONUCLEI IN CYTOKINESIS BLOCKED PERIPHERAL BLOOD LYMPHOCYTES Cell cultures 1. Add 500 µl of whole blood to 4.5 ml Ham’s F-10 medium supplemented with 24% fetal calf serum (Sigma, St Louis, MO, USA), pennicilin (100 IU/ml), streptomycin (100 µg/ml), 1% L-glutamine, and 1% heparin (50 IU/ml) (BBraun, Melsungen, Germany). 2. Add 2% (v/v) phytohemaglutinine (PHA-M form, Gibco BRL Life Technologies, Grand Island, NY). 3. Close the tubes tightly and incubate at 37ºC. 4. At 44h of incubation, add cytochalasin-B (Sigma, St. Louis, MO, USA) to a final concentration of 12.5 µM. (6 µg/ml, stock solution 8.34 mM prepared in DMSO). Preparation of slides 1. After a total of 72 hours of culture, harvest cells by centrifugation 120g, 10 minutes, at room temperature. 2. Wash twice with RPMI 1640 (pH 7.2), supplemented with 2% fetal calf serum. 3. Centrifuge for 7 minutes at 120g, room temperature. 4. Remove supernatant and subject cells to a mild hypotonic treatment, consisting of a mixture (pH 7.2) of RPMI 1640:deionised water 1:4, supplemented with 2% fetal calf serum. 5. Centrifuge for 5 minutes at 120g. 6. Remove supernatant and loosen pellet. 7. Place small drops of cell pellet in clean dry slides, and perform smears. 8. Air dry slides overnight. 9. Fix with freshly prepared ice-cold methanol/acetic acid (3:1) for 20 min. 10. Allow to dry and stain with 4% Giemsa in 0.01 M phosphate buffer pH 6.8 for 8 min. 11. Close the slides with Entellan® (Merck, Darmstadt, Germany). Scoring of slides 1. Code the slides and score 1000 binucleated lymphocytes with well-preserved cytoplasm for micronuclei at (magnification 500x for detection, 1,250x for confirmation), identified according to the criteria described by Kirsch-Volders et al. (2000). 2. Score 1000 lymphocytes for the cytokinesis blocked proliferation index (CBPI), calculated according to Surrallés et al. (1995): CBPI= [MI+2MII+3(MIII+MIV)]/total number of cells, where MI-MIV are the number of cells with one to four nuclei.

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7. GENOTYPING OF HUMAN SUBJECTS DNA extraction Genomic DNA was obtained from 250 µl of whole blood using a commercially available kit according to the manufacturer instructions (QIAamp DNA extraction kit; Qiagen, Hilden, Germany). DNA samples were stored at –20 °C until analysis. Genotyping GSTM1 and GSTT1 genotyping for gene deletions were carried out by a multiplex PCR as follows: DNA samples were amplified with the primers (Perkin Elmer Corp): 5'-GAACTCCCTGAAAAGCTAAAGC-3' (upstream) and 5'-GTTGGGCTCAAATATACGGTGG -3' (downstream) for GSTM1 which produced a 219 bp product, 5'-TCACCGGATCATGGCCAGCA-3' (upstream) and 5'-TTCCTTACTGGTCCTCACATCTC-3' (downstream) for GSTT1 which produced a 459-bp product. The amplification of albumin gene with the primers 5'-GCCCTCTGCTAACAAGTCCTAC-3' (upstream) and 5'-GCCCTAAAAAGAAAATCCCCAATC-3' (downstream) was used as an internal control and produced a 350-bp product. PCR was performed in a final volume of 50 µl, consisting of DNA (0.1 µg) dNTP (0.2 mM each) (Perkin Elmer) MgCl2 (2.5 mM), each primer (1.0, 0.3 and 0.2 µM for GSTM1, GSTT1 and albumin respectively), AmplitaqGold polymerase (1.25 units) (Perkin Elmer), reaction buffer and 2% DMSO. Amplification was performed with an initial denaturation at 95°C for 12 minutes, followed by 35 cycles of amplification performed at 94 °C for 1 minute, 62 °C for 1 min and 72 °C for 1 min, and a final extension at 72 °C for 10 min, using a GeneAmp 9600 thermal cycler (Perkin Elmer Corp). A 1/10 dilution of the amplified products was visualized in an ethidium bromide stained 1.5 % agarose gel. All the genotype determinations were carried out twice in independent experiments and all the inconclusive samples were reanalysed.

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8. DETERMINATION OF LIPOPEROXIDATION PRODUCTS IN PLASMA AND URINE BY THE THIOBARBITURIC ACID ASSAY (adapted from Dousset et al., 1983) Standards 1,1,3,3-tetraetoxipropane (Sigma, St. Louis, MO, USA): dilute in dH2O to final concentrations of 0.5 to 3.5 µM. Blank: reaction mixture using dH2O as “sample”. Thiobarbituric acid reagent 1. Add 200 mg thiobarbituric acid to 9 ml of 2N NaOH. 2. Adjust pH 7.4 with 7% perchloric acid. 3. Complete volume of 25 ml with dH2O (solution A). 4. Add 2 volumes of Solution A to 1 volume of 7% perchloric acid (TBA reagent). Reaction with thiobarbituric acid 1. Pipette duplicate aliquotes of 0.5 ml urine or plasma into screw-cap, heat

resistant glass tubes, containing 25µl of butilhidroxitoluene 0.6% in absolute alcohol.

2. Add 750 µl of freshly prepared TBA reagent. 3. Close the tubes tightly, vortex, and place in a boiling bath for ten minutes. 4. Cool down to room temperature. 5. Add 1.5 ml n-butanol to each tube, close the tube tightly and vortex thoroughly. 6. Centrifuge for 15 minutes at 3000 g. Quantification Carefully remove each organic phase into a different tube, mix and measure fluorescence of the solution at 515 nm excitation, 553 nm emission (Hitachi F-2000 Fluorescence Spectrophotometer).

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9. QUANTIFICATION OF TOTAL THIOL ANTIOXIDANTS IN PERIPHERAL BLOOD LYMPHOCYTES Standards Glutathione and cysteine (both from Sigma, St. Louis, MO, USA): dilute in 40 mM methanesulfonic acid to final concentrations of 1 to 10 nmol/ml. Blank: reaction mixture using 40 mM methanesulfonic acid solution as “sample”. Thiol extraction 1. Wash isolated lymphocytes twice in cold sterile PBS pH 7.2 (see Protocol 1). 2. Suspend cells in 50 µl PBS, 5 mM diethylenetriaminepentacetic acid (DPTA). 3. Add 200 µl of 50 mM methanesulfonic acid. 4. Vortex and perform 2 freeze-thaw cycles at –70ºC. 5. Centrifuge at 12,000 g, 10 min, room temperature (fixed-angle rotor

centrifuge). 6. Collect supernatant and use in the reaction with monobromobimane. Reaction with monobromobimane (mBBr) 1. Solubilize mBBr (Sigma, St.Louis, MO, USA) in acetonitrile (stock solution,

27.12 mg/ml). 2. Add 50 mM of HEPES buffer (pH 8), 5 mM of DPTA, 32 mM NaOH, 5 µl of

mBBr stock solution (2 mM), and dH2O to complete 150 µl of reaction mixture (respect this order). For the samples, make another set of reaction mixture including 2 mM dithiotreitol.

3. Add 100 µl of thiol sample or standard and mix. 4. Allow the reaction to proceed 10 minutes in the dark. 5. After 10 minutes, add 25 mM methanesulfonic acid to stop the reaction. Quantification by HPLC Equipment: Shimadzu LC-10Advp Liquid Chromatographer with fluorescence detector Wavelength: 390 nm excitation, 480 nm emission Column: Ultrasphere ODS (5 µm, 250 x 4.6 mm) Mobile phase methanol, acetic acid 0.25%, gradient (see Figure A1) Flow rate: 1.2 ml/min Injection volume: 20 µl

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15%

100%

25%15%

0%

15%

30%

45%

60%

75%

90%

105%

0 5 10 15 20

Run time (m inutes)

Perc

enta

ge o

f met

hano

l

Figure A1. HPLC mobile phase (methanol/acetic acid 0.25%) gradient for thiol-mBBr complex

separation

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Acknowlegements

Through four years of work, many people that I met along the way left an imprint in my work and in my mind. They all, in different ways, contributed to the final result presented here. The memorable experiences are perhaps the best acknowledgement of all. Even so, I cannot go without mentioning the valuable help and support that I was fortunate to receive from several people in my life:

My husband António for his patience, endless love and support, and my family for the continuous encouragement and pride, and specially my father for giving me the inspiration and help for this work,

My advisor Professor Maria Camila Batoréu, as well as Professor Ana Paula Santos, for believing in me from the start, and my co-advisor Professor José Rueff for welcoming me so openly into the his laboratory,

Professor António Sebastião Rodrigues, for his enthusiasm, knowledge, guidance, sense of humor, energy, and Professor Antonio Laires, for our straight to the point and helpful discussions, and for his always constructive critics,

Everyone at the Genetics Department of the Faculty of Medical Sciences of the New University of Lisbon, for the warm friendship they extended to me, specially Dr. Nuno Oliveira, who opened me a door to this family, Dra. Octavia Gil, who taught me the cytogenetics techniques, Isabel Santos, who helps make this Department a Home, D. Lucrécia and D. Manuela, always making the extra effort to keep everybody working, to Dr. Helena Borba and Dr. Margarida Monteiro, for the help and companionship, Professor Aldina Braz, for sharing helpful insights in the cytogenetics techniques, Dr. Michel Kranendonk, for being an example of scientific competence, Dr. Jorge Gaspar, for his help with the genotyping work, Dr. João Paulo Teixeira, for his encouragement and meeting tips, and all those who “shared” the bench with me, for exceptional companionship and friendship that will endure for the years to come,

Everyone at the Toxicology Laboratory and the Instrumental Methods Section of the Faculty of Pharmacy of the University of Lisbon, for understanding my unusual schedules, and for the technical help that was so important in my first steps in this work,

Dr. Jorge Justo for his interest in this project, Sr. Zito for his efficient connection to the workers and everyone at the participating industries.

To all my deepest appreciation.

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Documents

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