Hemoglobin Adducts and Urinary Metabolites of ... - ACS Publications

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Hemoglobin Adducts and Urinary Metabolites of Arylamines and Nitroarenes Gabriele Sabbioni* Institute of Environmental and Occupational Toxicology, Casella Postale 108, CH-6780 Airolo, Switzerland Alpine Institute of Chemistry and Toxicology, CH-6718 Olivone, Switzerland Walther-Straub-Institut für Pharmakologie und Toxikologie, Ludwig-Maximilians-Universität, D-80336 München, Germany S Supporting Information *

ABSTRACT: Arylamines and nitroarenes are intermediates in the production of pharmaceuticals, dyes, pesticides, and plastics and are important environmental and occupational pollutants. N-Hydroxyarylamines are the toxic common intermediates of arylamines and nitroarenes. N-Hydroxyarylamines and their derivatives can form adducts with hemoglobin (Hb-adducts), albumin, DNA, and tissue proteins in a dose-dependent manner. Most of the arylamine Hb-adducts are labile and undergo hydrolysis in vitro, by mild acid or base, to form the arylamines. According to current knowledge of arylamine adduct-formation, the hydrolyzable fraction is derived from the reaction products of the arylnitroso derivatives that yield arylsulfinamide adducts with cysteine. Hb-adducts are markers for the bioavailability of N-hydroxyarylamines. Hb-adducts of arylamines and nitroarenes have been used for many biomonitoring studies for over 30 years. Hb-adducts reflect the exposure history of the last four months. Biomonitoring of urinary metabolites is a less invasive process than biomonitoring blood protein adducts, and urinary metabolites have served as short-lived biomarkers of exposure to these hazardous chemicals. However, in case of intermittent exposure, urinary metabolites may not be detected, and subjects may be misclassified as nonexposed. Arylamines and nitroarenes and/or their metabolites have been measured in urine, especially to monitor the exposure of workers. This review summarizes the results of human biomonitoring studies involving urinary metabolites and Hb-adducts of arylamines and nitroarenes. In addition, studies about the relationship between Hb-adducts and diseases are summarized.

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CONTENTS

1. Introduction 2. Metabolism of Arylamines and Nitroarenes 3. Biomonitoring 3.1. Use of Biomonitoring Values 4. Biomonitoring of Arylamines and Nitroarenes in Urine 4.1. Introduction 4.2. Experimental Procedures for the Analysis of Urinary Metabolites 4.3. Urinary Metabolites of Arylamines and Nitroarenes in the General Population 4.3.1. Urinary Metabolites of Aniline in the General Population 4.3.2. Urinary Levels of Monocyclic Arylamines in the General Population 4.3.3. Urinary Metabolites of 4-Aminobiphenyl (4ABP) in the General Population 4.3.4. Urinary Metabolites of 1-Nitropyrene (1NP) in the General Population

4.4. Urinary Metabolites of Arylamines and Nitroarenes in Workers 4.4.1. Urinary Metabolites of Aniline, 2-Methylaniline (2MA), and 3MA in Workers 4.4.2. Urinary Metabolites of Benzidine (Bz) Congeners in Workers 4.4.3. Urinary Metabolites of 4,4′-Methylenedianiline (MDA) Congeners in Workers 4.4.4. Urinary Metabolites of Toluenediamines (TDAs) and Phenylenediamines (PDAs) in Workers 4.4.5. Urinary Metabolites of Nitroarenes in Workers 5. Hb-Adducts of Arylamines and Nitroarenes 5.1. Introduction 5.2. Experimental Procedures for the Analysis of Hb-Adducts 5.3. Hb-Adducts of Arylamines in the General Population

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1740 Received: April 28, 2017 Published: September 21, 2017

© 2017 American Chemical Society

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DOI: 10.1021/acs.chemrestox.7b00111 Chem. Res. Toxicol. 2017, 30, 1733−1766

Chemical Research in Toxicology 5.3.1. Hb-Adducts of Arylamines Metabolically Released from Lidocaine and Prilocaine 5.4. Hb-Adducts of Arylamines, Nitroarenes, and Nitrotoluenes in Workers 5.4.1. Hb-Adducts of Monocyclic Arylamines in Workers 5.4.2. Hb-Adducts of 2,4-Difluoroaniline (24DFA) in Workers 5.4.3. Hb-Adducts of Polycyclic Nitroarenes in Workers 5.4.4. Hb-Adducts of 2,4,6-Trinitrotoluene (TNT) in Workers 6. Biomonitoring of Urinary Metabolites and of HbAdducts in the Same Subjects 6.1. Urinary Metabolites and Hb-Adducts of Musk Xylene in the General Population 6.2. Workers Exposed to 4-Chloroaniline (4CA) and Aniline 6.3. Workers Exposed to 2-Methylaniline (2MA) and Aniline 6.4. Workers Exposed to 3-Chloro-4-fluoroaniline (CFA) 6.5. Workers Exposed to Benzidine (Bz) and Azodyes 6.6. Workers Exposed to 3,3′-Dichlorobenzidine 6.7. Workers Exposed to Methylenedianiline (MDA) Congeners 6.8. Workers Exposed to Nitrotoluenes 6.9. Workers Exposed to 2,4,6-Trinitrotoluene (TNT) 6.10. Workers Exposed to Chloronitrobenzenes (CNBs) 7. Estimation of External Dose from Hb-Adduct Levels and Cancer Risk Assessment 8. Correlation of Hb-Adducts and Disease 8.1. Cancer Cases and 4ABP-Hb Adducts 8.1.1. Lung Cancer and 4ABP-Hb Adducts 8.1.2. Bladder Cancer and 4ABP-Hb Adducts 8.2. Bladder Cancer and Hb-Adducts of Monocyclic Arylamines 8.3. Biological Effects and Hb-Adducts in Workers Exposed to Nitroarenes 8.3.1. Nitrotoluenes (NTs) 8.3.2. 2,4,6-Trinitrotolene (TNT) 8.3.3. Chloronitrobenzenes (CNBs) 9. Conclusions Associated Content Supporting Information Author Information Corresponding Author ORCID Funding Notes Biography Acknowledgments Abbreviations References

Review

and in hair dyes.5−8 The bicyclic diamino compounds, such as benzidine (Bz), 3,3′-dichlorobenzidine (DCBz), 3,3′-dimethylbenzidine, and 3,3′-dimethoxybenzidine, have been used for the production of azo dyes.9−11 Arylamines, such as 2,3-, 2,4-, 2,5-, and 2,6-dimethylaniline (26DMA), 2-, 3-, and 4-ethylaniline (4EA), 2-, 3-, and 4-methylaniline (4MA), 1- and 2-naphthylamine, (2NA), 2-, 3-, and 4-aminobiphenyl (4ABP), are present in cigarette smoke.12,13 Arylamines such as 2NA and 4ABP have been found in fumes of cooking oils.14 Several arylamines have been found in indoor and outdoor air.15 Aniline was present at high levels in comparison to the other arylamines.15 Other sources of exposure to arylamines are food dyes,16 tattoo colorants,17 dyes for textiles,18 and hair dyes,7,19,20 arylamines leaching from food packages,21−23 or pesticides synthesized from arylamines, such as propham, chlorpropham, chlordimeform.24 Several arylamines were found in hair dyes25 including N-methylaniline, aniline, diphenylamine, 2MA, 3MA, 4MA, 2-methoxyaniline, 4-chloroaniline, 2-aminophenol, 1- and 2-naphthylamine, and 4-nitroaniline, 1,2-, 1,3-, and 1,4-phenylenediamine (14PDA), 3-aminophenol, 4-aminophenol, 2,4-, 2,6-, and 3,4-toluenediamine (34TDA), 4ABP,26 4-chloro-1,2-phenylenediamine, and 2-nitro-1,4-phenylenediamine. Arylamines are moderately to extremely toxic.10,27,28 The main health hazards linked to arylamine exposure are methemoglobinemia (metHb)29 and cancer.30 These toxic effects are caused by N-oxidized metabolites of arylamines.31−37 Nitrotoluenes,38,39 nitrobenzenes,40,41 dinitrotoluenes,42 and chloronitrobenzenes43,44 are important industrial intermediates used in the production of explosives, plastics, rubber chemicals, polyurethanes, dyes, and pesticides.45 Nitrobenzene, nitrotoluene isomers, and chloronitrobenzenes are present in the environment.46,47 Polyaromatic nitro compounds are air pollutants derived from fuel combustion.48−51 2-Nitrofluorene,52 2-nitronaphthalene,53 1-nitropyrene,54 1,3-dinitropyrene,55 1,6-dinitropyrene,56 1,8-dinitropyrene,57 and 6-nitrochrysene58 are the compounds of major toxicological concern. Cigarette smoke is a major source of human exposure to nitrobenzene.59 Nitroarenes, such as musk xylene, are important fragrances and perfumes.60 Musk xylene has been found in the environment,61−63 human breast milk,64,65 blood,66 and adipose tissue.64 In most cases, the same acute toxic effects are observed from nitroarene exposure as for arylamine exposure because the same reactive intermediates, N-hydroxyarylamine and nitrosoarene, are produced in vivo.67−69 The present review focuses on urinary metabolites and Hb adducts of arylamines and nitroarenes. This review does not include detailed information about all aspects of biomonitoring of arylamines and nitroarenes that was presented 15 years ago.70 DNA adducts of arylamines and protein adducts of heterocyclic arylamines were reviewed recently by Turesky and colleagues.71−75 Therefore, heterocyclic arylamines will not be part of this review. Serum albumin adducts of arylamines and nitroarenes were reviewed recently.76

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2. METABOLISM OF ARYLAMINES AND NITROARENES Arylamines27 are absorbed through the respiratory system, skin, and gastrointestinal tract.77,78 The metabolism of arylamines has been investigated for over 100 years (Figure 1).33,79−84 Ring oxidation, N-oxidation, N-glucuronidation, and N-acetylation are the main metabolic pathways of arylamines in mammals.85,86 N-Oxidation is the crucial step in the production of toxic products from arylamines. Different enzymes are involved in the N-oxidation to produce N-hydroxarylamines and/or reactive

1. INTRODUCTION Arylamines are intermediates in the production of agricultural and pharmaceuticals chemicals, polyurethanes, dyes, and pigments.1−4 Arylamines are used in shoe creams, in inks for pens, 1734


Chemical Research in Toxicology

Review

Figure 1. Short summary of the metabolism of arylamines, with an aniline as a model compound.33,83,144,194 N-Hydroxylation by cytochrome P450 (P4501A2) for planar arylamines and by P4503A4 for bulky arylamines is the predominant mode of activation of arylamines.87,88,99 Other P450s might be involved in N-oxidation as well.90 C-Oxidation yielding phenolic metabolites is generally involved in the detoxification process. However, aminophenols can be metabolized to reactive iminoquinones.262 Acetylases and deacetylases (DAC) have an important role in the metabolism of arylamines. Arylamines are detoxified by polymorphic N-acetyltransferase 2 (NAT2) and excreted in urine. NAT1 appears to function as an O-acetyltransferase (OAT) and as an N,O-acetyltransferase (N,O-AT) when using acetyl coenzyme A or hydroxamic acids, respectively, as acetyl donors. NAT2 appears to act preferentially as an OAT and NAT. Direct N-sulfation to yield sulfamates is a minor pathway of metabolism for most arylamines.86,203,408,409 The arylamines are oxidized to the potentially genotoxic N-hydroxyarylamines. The arylamines or the N-hydroxyarylamines can be glucuronidated by UDP- glucuronosyltransferases (UGT). The N-hydroxyarylamines and the N-glucuronide are transported through the blood in the bladder. The acidic pH of the urine catalyzes the formation of the nitrenium ion (= very short-lived intermediate)410,411 that gives rise to DNA adducts.143,412 In animal experiments, DNA adducts of arylamines and nitroarenes have been found also in other organs.33,413,414 In blood, N-hydroxyarylamines are taken up in the erythrocytes and oxidized to the nitrosoarenes with the formation of metHb. This process is known as the Kiese Redox-cycle.29 Abbreviations: Ac = acetyl; gluc = β,D-glucuronyl.

intermediates: (a) P450,87−90 (b) flavine adenine dinucleotide containing monooxygenases,91 (c) prostaglandin H synthase,92−94 (d) myeloperoxidase in breast and lung tissue,94 and (e) lactoperoxidase secreted from the mammary glands.95,96 The main metabolic steps for arylamines are presented in Figure 1. N-Hydroxyarylamines can be detoxified by a microsomal NADH-dependent reductase97 or P450 2S198 that rapidly converts the N-hydroxyarylamines back to the parent amines. N-Hydroxyarylamines can be further metabolized to reactive intermediates such as O-acetyl-N-phenylhydroxylamine or O-sulfonyl-N-phenylhydroxylamine (Figures 1 and 2)83 that react with DNA99 and proteins in organs, and eventually lead to cytotoxic and genotoxic effects. Several important polymorphisms of enzymes involved in the metabolism of arylamines have been discovered: N-acetyltransferases (NAT),100−102 P450s,103−105 glutathione S-transferases (GST),103 UDP-glucuronosyltransferases (UGT),106 and

sulfotransferases (SULT).107,108 At high exposure levels, polymorphisms might not be important. This was shown in smokers.109 Fast acetylators had higher 4ABP-Hb adduct levels than slow acetylators for weak (ratio: < 1.5 μmol nicotine + cotinine/mmol creatinine) and intermediate (ratio: 1.5−2.4) smokers but not for strong smokers (ratio: > 2.5). In studies of humans with high exposures to chloronitrobenzenes110 or nitrotoluenes,111,112 the Hb-adduct levels did not differ significantly between fast and slow acetylators. It appears that at high exposure levels, NAT2 is saturated, which leads to less N-acetylated products but more N-oxidation products catalyzed by P450s. Excellent reviews were written by Rickert et al.67,69,113 about the metabolism of nitroarenes in animals. Nitroarenes are absorbed through the respiratory system, gastrointestinal tract, or skin.40,42,43 The first metabolic step for nitroarenes is the oxidation in the aromatic ring or the reduction of the nitro 1735



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Figure 2. Nomenclature used in the present publication, using aniline as an example, for metabolites of arylamines. Abbreviations: Ac = acetyl; gluc = β,D-glucuronyl.

group.69,114 The same metabolites as those after arylamine exposures are found after nitro reduction. Nitroarenes are reduced by several enzymes in the liver and by the microflora in the gut to the amine, nitro radical anions, N-hydroxyarylamines, and nitrosoarenes.67,115 Nitrobenzene (NB) given orally to male Fisher-344 rats produced the urinary metabolites 3-nitrophenol, 4-nitrophenol, and 4-hydroxyacetanilide.114 These metabolites were excreted as sulfate ester conjugates. The metabolism and the urinary metabolites of 4-nitrotoluene (4NT), 3NT, and 2NT were investigated in male Fischer 344 rats.116 Benzoic acids, nitrobenzoylglycines, and acetamidobenzoic acids were the major metabolites. Several reviews have been written about nitrated polyaromatic hydrocarbons (nitro-PAH).117,118 After intragastric or intraperitoneal administration or inhalation of 1-nitropyrene (1NP), the majority, 50−60% of the given dose, was eliminated in the feces and 15−20% of the dose in the urine. 1NP is metabolized through two routes: P450-mediated C-oxidation and nitroreduction, which are followed by a conjugation reaction catalyzed by UGT, SULT, and NAT. Urinary or fecal metabolites that were observed after enzymatic or chemical pretreatment were hydroxy-1-nitropyrenes (3-, 6-, and 8-OHNP) (chemical structures in Figure S1), hydroxy-N-acetyl-1-aminopyrenes (3-, 6-, and 8-OHNAAP), trans-4,5-dihydro-4,5-dihydroxy-1NP, 1-aminopyrene (1AP), and N-acetyl-1AP (NAAP). The main metabolite identified in the urine of rats administered 1NP was 6-OHNAAP.119 2-Nitrofluorene, often used as a marker for nitro-PAHs, is metabolized in vivo by two different routes.120 After inhalation, hydroxylated nitrofluorenes, which are potent mutagenic metabolites, are formed. After oral administration, 2-nitrofluorene is reduced to 2-aminofluorene and then N-acetylated to 2-acetylaminofluorene, which is a potent carcinogen.121 Ringhydroxylation of 2-acetylaminofluorene leads to detoxification and excretion.

of early biological effects, (e) markers of altered function, and (f) markers of clinical disease. This review describes the advances made in the analysis of urinary metabolites (internal dose) and Hb-adducts (biologically effective dose) of arylamines and nitroarenes. 3.1. Use of Biomonitoring Values. In the USA, biomonitoring studies are performed on thousands of people for a long period (https://www.cdc.gov/exposurereport/). The results are published regularly on the homepage of National Report on Human Exposure to Environmental Chemicals. The environmental pollutants and/or their metabolites are monitored in urine and serum. Only in the case of acrylamide and glycidamide were Hb-adducts measured. No arylamines were included in the studies. In Canada,130 Korea,131 Japan,132 Germany,133,134 and other European countries,135−138 biomonitoring programs were implemented. The major problem of such studies is the toxicological interpretation of the values found. In most animal experiments, the toxic effects of chemicals have been related to the dose of the chemical and not to the internal dose, such as blood levels or urinary metabolites of the chemical. Therefore, pathological effects cannot be related to these biomarkers since they were not determined in the experiments. For DNA adducts, such comparisons exist.139−141 The levels of 4ABP-DNA adducts found in bladder cancer cases were compared with the levels of 4ABP-DNA adducts detected in animal carcinogenicity studies.142−144 For populations with low exposure, it is very difficult to relate the biomonitoring results with biological effects. Such biological effects can be seen mainly in highly exposed people.111,145 One approach is to estimate the actual dose from the biomarker levels using pharmacokinetic modeling.146−150 The extrapolated dose can then be compared to the known toxic dose values. The following frameworks were created to establish an order for biomonitoring values. Such efforts have been summarized in a recent review.151 The Human Biomonitoring Commission in Germany uses as reference value the 95th percentile (RV95) of the measured pollutant concentration levels in blood or urine found in the general population.152−154 RV95 values are statistically derived values, which have no health relevance. Such reference values are valuable to individuate uncommon exposures in biomonitoring studies. Populations with levels of toxic compounds above the RV95 level might necessitate interventions to reduce the exposure to such a chemical. RV95 values for arylamines found in urine have been established in Germany155 (see Tables 1−3).156

3. BIOMONITORING Biomarkers were developed to establish the exposure and effects of xenobiotics and susceptibility of individuals.70,122−126 The biomonitoring paradigm was developed 30 years ago.127−129 Biomarkers were classified according to single steps in the progressive nature of disease: (a) markers of susceptibility, (b) markers of the internal dose (parent compound and metabolites in urine), (c) markers of the biologically effective dose (DNA-, protein-, and blood protein adducts), (d) markers 1736



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Table 1. Mean Hb-Adduct Levels of Arylamines (AAs) and Mean Urinary Levels of AAs

a U-AA-tot, 2 M HCl, 80 °C, 1 h. bU-AA-tot, 4.7 M NaOH, 80 °C, 2 h. cU-AA-free, extraction from urine without pretreatment. dU-AA-tot, 3.6 M H2SO4, 80 °C, 2 h. eMouse. fPhenmedipham. gHCl derivative. hHBI = hemoglobin binding index = (mmol AA/mol Hb)/dose (mmol/kg). iCSF oral cancer slope factor (mg/kg-bw)−1. jTD50 value in mg/kg-bw. kChronic RfD-value in mg/kg-day. lNSRL no significant risk level in μg/day, California EPA. Abbreviations: ca = cancer case, co = controls, con = consumer, ex = exposed, hd = hair dresser, ns = nonsmokers, po = postshift, pr = preshift, s = smokers; italic numbers = geometric mean, underlined number = median, 2MeOA = 2-methoxyaniline.

Health related human biomonitoring values I150 and II151 and biomonitoring equivalents values151,157−161 were derived for many compounds but not for arylamines and nitroarenes. For occupational health purposes, special biomonitoring values were established, such as the Biological Exposure Indices (BEIs) (http://www.acgih.org/tlv-bei-guidelines/biologicalexposure-indices-introduction, American Conference of Governmental Industrial Hygienists), BAT values (Biologische Arbeitsstoff-Toleranzwerte, biological tolerance values), and BLW162 (Biologische Leitwerte, biological guideline values) by the German Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area, and the biological monitoring guidance values (BMGV)163 by the governmental human and safety laboratory in Great Britain (Table 4). The BEI normally specifies the amount of a compound below which

nearly all workers should not encounter adverse health effects. BEIs generally represent the levels of biomarkers in samples obtained from healthy workers who have been exposed to xenobiotics at the threshold limit value. BEI values are established for both noncarcinogenic and carcinogenic substances. The BAT-value is the chemical concentration, its metabolites, or an effect parameter at which the health of a worker is not adversely affected even if the worker is repeatedly exposed during long periods. BAT-values are only established for noncarcinogenic chemicals. BLW are derived for carcinogenic substances and for substances without sufficient toxicological data; these are likewise established as averages.162 The BAR values162 (Biologische Arbeitsstoff-Referenzwerte = biological occupational chemical reference value) are established by the German Commission for the Investigation of Health Hazards of 1737



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U-AA-tot, 2 M HCl, 80 °C, 1 h. bU-AA-tot, 3.6 M H2SO4, 80 °C, 2 h. 2-Amino-4-chloro-5-fluorophenyl sulfate concentration given in μg/g creatinine. dHCl derivative. eHBI = hemoglobin binding index = (mmol AA/mol Hb)/dose (mmol/kg). fCSF oral cancer slope factor (mg/kg-bw)−1. gTD50 value in mg/kg-bw. hRfD-value in mg/kg-day. i NSRL no significant risk level in μg/day, California EPA. Abbreviations: ns = nonsmokers, s = smokers; numbers underlined = median.

Biological Monitoring Database (BMDB) of the human and safety laboratory in Great Britain.163 Data for arylamines are listed in Table 4. Generally, derivation of biomonitoring values from urine and plasma is based upon the assumption that the levels of the measured chemicals are at steady state after continuous exposures. Such an exposure scenario might apply to workers in the industry; however, exposure to chemicals in the environment is not continuous, and levels might vary a lot. Therefore, interpretation of such biomonitoring values should be done with caution. Protein adducts such as Hb-adducts reflect the exposure history of the last 120 days, if the Hb-adducts are stable (section 7.).164,165 In contrast, urinary metabolites or noncovalently bound metabolites in blood generally reflect the exposure of the past 24−48 h. Therefore, levels of stable Hb-adducts vary less than the concentrations of urinary metabolites. Thus, Hb-adduct values have the potential to be a better exposure marker for risk assessment than urinary levels of metabolites. Such an approach has been used only in a few cases. Tannenbaum et al. established a toxicokinetic approach to evaluate the exposure dose from the Hb-adduct levels of 4ABP (section 7.).146 Using this method, the daily dose of benzidine (Bz),166 4-chloroaniline (4CA),167 and 2,4,6-trinitrotoluene (TNT)145 was extrapolated from the measured Hb-adducts to estimate the cancer risk for the workers.168−171

Chemical Compounds in the Work Area. These BAR values are similar to the reference values of the German HBM Commission. The difference between a BAR value and a reference value (RV95) is that a BAR value was derived from persons of working age with the aim to assess occupational exposure, whereas the RV95-values were deduced from the general population with the aim to assess environmental exposures. Great Britain developed such an approach for setting BMGV-values163 based on the 90th percentile value of biological monitoring data from workplaces employing good occupational hygiene practice and which should be applicable across similar workplaces using the same substances in similar processes. The data were stored in the

4. BIOMONITORING OF ARYLAMINES AND NITROARENES IN URINE 4.1. Introduction. Urinary metabolites represent only the exposure of the past 24−48 h. The ratio of arylamines to N-acetylarylamines has been used to estimate the N-acetylator phenotype.172−174 The analysis of arylamines in urine was performed in several studies: Bz,175 3,3-dimethylbenzidine,176 3,3-dimethoxybenzidine,176 2MA,177 MDA,178,179 MOCA,180−182 toluenediamines,183−185 and 3,5-dichloroaniline in people exposed to vinclozoline and iprodione,186 3,4- and 3,5-dichloroaniline,187,188 4ABP in smokers and nonsmokers,189−191 and aniline.192,193 In some studies, DNA-adducts were determined in urinary


a c


U-AA-tot, 2 M HCl, 80 °C, 1 h. bU-AA-tot, 0.1 M NaOH, 90 °C, 15 h. cHCl derivative. dU-AA-tot, 3.6 M H2SO4, 80 °C, 2 h. eng/g creatinine. HBI = hemoglobin binding index = (mmol AA/mol Hb)/dose (mmol/kg). gCSF oral cancer slope factor (mg/kg-bw)−1. hTD50 value in mg/kg-bw. iRfD-value in mg/kg-day. jNSRL no significant risk level in μg/day, California EPA. kHBI = 725,274 742,273 and 1007.394 Abbreviations: ca = cancer case, cg = cigarettes, co = controls, ex = exposed, m = male, ns = nonsmokers, s = smokers, w = woman; numbers in italic = geometric mean. a f

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Urinary levels were reported with or without creatinine correction such as μg/g creatinine and μg/L, respectively. Barr et al.200 determined a median level of 1.13 g creatinine per liter of urine in non-Hispanic whites (n = 8150), 1.53 g/L in nonHispanic blacks (n = 6664), and 1.23 g/L in Mexican-Americans (n = 6496). The values listed in this review were mostly performed with non-Hispanic whites. The American Conference of Governmental Industrial Hygienists proposes a reference value of 1 g creatinine/L urine, reviewed by Sauvé et al.201 Therefore, the values listed in this review as μg/L or μg/g are comparable. 4.2. Experimental Procedures for the Analysis of Urinary Metabolites. Urine is usually the main path of excretion of arylamines and nitroarenes. The major metabolites are N-acetylated and nonacetylated aminophenols, which are excreted as glucuronides or sulfates.86,202−210 Urine is the main biological matrix used for biomonitoring studies. Pre- and postshift urine samples are used for biomonitoring studies of workers. Ideally, samples should be taken also after the workers return from vacations in order to distinguish between environmental and occupational exposure. For the sample work up, there are different approaches since the chemicals are present as conjugates or free compounds. The conjugates are mostly glucuronides, sulfates, and acetylated compounds (Figures 2 and 3). The partition coefficients for some potential metabolites of aniline have been calculated with the program Marvin Sketch by ChemAxon (www.chemaxon.com) using the ChemAxon module for the logP calculations. This yields to the following logD values at pH 7.4 (Figure 2): aniline (logD = 1.26), acetanilide (1.37), N-acetyl-4-aminophenol (1.09), 4-aminophenol (0.97), phenylsulfamate (−1.91), 4-aminophenyl sulfate (−2.04), N-acetamidophenyl sulfate (−1.93), aniline-N-glucuronide (−4.06), 4-acetamidophenyl-glucuronide (−4.74), and 4-aminophenyl-glucuronide (−4.81). Extraction of urine with organic solvents without pretreatment (e.g., enzymes or acid) at pH > 7.4 yields unconjugated aniline, acetanilide, and aminophenols. The conjugates are too polar logD < −1.9 in order to be extractable in sufficient amounts under such conditions. Arylamines (AA) obtained from untreated urine have been classified as free amines (U-AA-free). Boiling of urine at 80 °C for 45 min cleaves unstable N-glucuronide-AA conjugates211 and increases the amount of extractable arylamines (U-AA-heat).211 Enzymatic

Table 4. Assessment Values in Biological Material from Germany,162 Great Britain,163 Sweden, 236 and France 235

The urinary levels were obtained after acid hydrolysis for 2 h at 80 °C in 3 M H2SO4,162,397 for 1 h at 80 °C in 3 M H2SO4,258 or for 1.5 h at 100 °C in 1.63 M H2SO4.163,398 Urine was heated in base236 at 80 °C in 0.3 M NaOH for 24 h or at 80 °C in 5 M NaOH235 overnight. The conditions used were different in the 4 countries. In Great Britain, the urinary values were listed as μmol compound/mol creatinine (cr). For nonHispanic whites, the median levels of creatinine in 1 L urine were 1.13 g.200 The American Conference of Governmental Industrial Hygienists proposes a reference value of 1 g creatinine/L urine, as reviewed by Sauvé et al.201 Therefore, the urinary levels listed as μg/g creatinine (cr) correspond more or less to the values given as μg arylamine per liter of blood. bThe original published values were 100 μg/L blood for aniline and

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