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A recent concept connecting the lipophilicity of organic chemicals with their genotoxicity on a chromosomal level implies that the lipophilic character of organic chemicals determines a certain background of chromosomal genotoxicity that can be addressed as "non-specific". This is opposed to compounds with more "specific" modes of action. Such mechanisms influence the processes of karyokinesis and cytokinesis. A critical partial process for the chromosomal segregation is the dynamics of assembly and disassembly of microtubules. To broaden the present database for such interactions, chemicals were selected based on their lipophilicity (log P between -1.5 and +1.0) and on hints from the literature pointing to possibilities of interaction with the tubulin-microtubule system. Thus, acetamide, acrylamide, methylmethane sulfonate, acetonitrile, acrylonitrile and cyclohexanone were assessed as to their potencies to influence the dynamic processes of microtubule assembly and disassembly in a cell-free system in vitro. These compounds covered a range of log P between -1.5 and 1.0, complementary to compounds investigated earlier. The entire body of data supports the general concept that hydrophobic interactions are connected with non-specific processes, which contribute to a background genotoxicity on a chromosomal level. It also points to the dynamics of microtubule assembly and disassembly as a decisive partial process involved.

A concept relating the lipophilicity of chemicals with their genotoxicity on a chromosomal level had been generated by Schultz and Onfelt (Chem Biol Interact 126:97-123, 2000). It was shown that aneuploidy in Chinese hamster V79 cells was elicited by lipophilic chemicals at concentrations related to their hydrophobicity (log P), whereas toxicants with a specific mode of action acted at concentrations consistently lower than predicted based on log P. We have now combined available data sets on aneuploidy/micronucleus formation with procedures used in QSAR modelling, in order to find new molecular descriptors for modelling non-specific chromosomal genotoxicity, and to optimise combinations thereof. Molecular structures of 26 chemicals, including steroids, were converted into single 3D models using Corina (version 3.20), and 11 descriptors of molecular properties were calculated. The data of 16 compounds assigned to a non-specific mode of action were imported into the QSAR module of the software package Cerius(2) (version 4.10). Applying genetic function approximation (GFA), linear equations were set up relating molecular descriptors with experimental concentrations at which doubling of micronuclei occurred in V79 cells (exp -log C). The number of variables (molecular descriptors) was limited to a maximum of three, and linear and quadratic terms were allowed. Based on the descriptions provided by the GFA procedure, log P was the most suitable single property to describe non-specific genotoxicity [r (2 ) = 0.88], confirming the original concept of Schultz and Onfelt. Using more descriptors (up to three in combination) resulted in an optimization of correlations up to r (2 )= 0.97. Such optimal correlation coefficients were obtained by combinations (a) of the numbers of hydrogen bond acceptors, the polar surface and total surface areas of molecules on one hand, and by (b) the dipole moment, polar surface and total surface descriptors on the other hand. In essence, the relation of polar surface to the total molecular surface appears pivotal to determine the non-specific chromosomal genotoxicity of lipophilic compounds.

The genotyping of N-acetyltransferase 2 (NAT2) by PCR/RFLP methods yields in a considerable percentage ambiguous results. To resolve this methodical problem a statistical approach was applied. PHASE v2.1.1, a statistical program for haplotype reconstruction was used to estimate haplotype pairs from NAT2 genotyping data, obtained by the analysis of seven single nucleotide polymorphisms relevant for Caucasians. In 1,011 out of 2,921 (35%) subjects the haplotype pairs were clearcut by the PCR/RFLP data only. For the majority of the data the applied method resulted in a multiplicity (2-4) of possible haplotype pairs. Haplotype reconstruction using PHASE v2.1.1 cleared this ambiguity in all cases but one, where an alternative haplotype pair was considered with a probability of 0.029. The estimation of the NAT2 haplotype is important because the assignment of the NAT2 alleles *12A, *12B, *12C or *13 to the rapid or slow NAT2 genotype has been discussed controversially. A clear assignment is indispensable in surveys of human bladder cancer caused by aromatic amine exposures. In conclusion, PHASE v2.1.1 software allowed an unambiguous haplotype reconstruction in 2,920 of 2,921 cases (>99.9%).

The synthetic steroid tetrahydrogestrinone is a new "designer drug" and was recently detected to be illegally used in sports. It is chemically closely related to trenbolone that is known as an animal growth promoter. The potencies of trenbolone, tetrahydrogestrinone and testosterone to induce micronuclei in V79 cells in vitro were determined. CREST analysis was employed to differentiate between aneugenic or clastogenic mechanisms. Cytotoxicity and an influence on the cell cycle were assessed in parallel. Incubations with testosterone, at concentrations between 3 and 300 microM, failed to induce micronuclei. By contrast, tetrahydrogestrinone and trenbolone increased the rate of micronuclei significantly, up to a doubling of the micronuclei rate of untreated controls. Tetrahydrogestrinone and trenbolone displayed a bell-shaped dose-response curve, with maximal effects observed at 3 and 30 microM, respectively. The micronuclei induced by tetrahydrogestrinone and trenbolone were predominantly kinetochor (CREST) positive, pointing to an aneugenic mode of action. This may be related to the specific structure of both molecules with a system of activated double bonds. As the genotoxic effect of tetrahydrogestrinone at a chromosomal level appears at a low concentration range, it cannot be ruled out that tetrahydrogestrinone presents a genotoxic hazard on a chromosomal level under conditions of its current misuse in sports.

Polymorphisms within the estrogen metabolic pathway are prime candidates for a possible association with breast cancer risk. We investigated 11 genes encoding key proteins of this pathway for their potential contribution to breast cancer risk. Of these CYP17A1, CYP19A1, EPHX1, HSD17B1, SRD5A2, and PPARG2 participate in biosynthesis, CYP1A1, CYP1B1, COMT, GSTP1, and SOD2 in catabolism and detoxification. We performed a population-based case-control study with 688 incident breast cancer cases and 724 controls from Germany and genotyped 18 polymorphisms by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), PCR based RFLP (restriction fragment length polymorphism), and TaqMan allelic discrimination. Genotype frequencies were compared between cases and controls and odds ratios were calculated by conditional logistic regression. Further statistical analyses were based on cluster analysis, multifactor dimensionality reduction, logic regression, and global testing. Single factor analyses pointed to CYP1B1&#95;1294&#95;GG as a possible breast cancer risk modulator (OR = 2.57; 95% CI: 1.34-4.93) and two way stratification suggested associations between BMI > or = 30 kg/m(2) and COMT&#95;472&#95;GG (P = 0.0076 and P = 0.0026), BMI < 20 kg/m(2) and HSD17B1&#95;937&#95;GG (P = 0.0082) as well as CYP17A1&#95;-34&#95;CC and HRT use > or =10 years (P = 0.0063). Following correction for multiple testing none of these associations remained significant. No significant association between breast cancer risk and genetic polymorphisms was observed in multifactor analyses. The tested polymorphisms of the estrogen metabolic pathway may not play a direct role in breast cancer risk. Therefore, future association studies should be extended to other polymorphisms and other regulatory pathways.

Occupational exposure to aromatic amines is a known bladder cancer risk factor, whereas the impact of exposure to azo dyes, which may release aromatic amines in humans, is at present controversial. Therefore, the impact of occupational exposures to colorants was investigated in 156 bladder cancer cases and 336 controls in the state of North Rhine-Westphalia. All bladder cancer cases and controls (diagnosed with prostate cancer) requested after-care treatment. The subjects were investigated using a questionnaire for all occupations ever performed for more than 6 mo and for exposures to several possible occupational and nonoccupational bladder carcinogens. The relative bladder cancer risk was adjusted for age and smoking. The adjusted odds ratio (OR) for bladder cancer was elevated in 7 painters (OR 1.98, 95% CI 0.64-6.11), 4 hairdressers (OR 4.9, 95% CI 0.85-28.39), and 16 cases who reported a wood processing occupation (OR 1.19, 95% CI 0.58-2.41). Ten of these 16 cases reported chronic exposure to colorants (OR 1.84, 95% CI 0.68-4.95). The results of this epidemiological study confirm the hypothesis that individuals exposed to colorants show an elevated bladder cancer risk.

A study of Chinese benzidine workers indicated elevated levels of UDP-glucuronosyltransferase (UGT) 2B7 T/T activity in carriers for development of bladder cancer. The present study was designed to investigate the possible impact of the presence of UGT2B7 genotype on bladder cancer risk in Caucasians. UGT2B7 polymorphism at locus C(802)T (His(268)Tyr) was detected using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)-based procedure. The study group consisted of 211 bladder cancer cases and 210 controls suffering from different urological diseases, but without any history of cancer. Both groups were recruited from a Department of Urology located in a center of former chemical and rubber industries in Germany. Furthermore, 171 bladder cancer cases with a history of occupational exposure to aromatic amines surveyed for compensation due to an occupational disease were investigated. T/T genotype frequencies in bladder cancer cases, urological controls, and exposed patients appeared similar (27 vs. 35 vs. 25%). This study indicated that there were ethnic differences between Caucasian and Chinese general populations with respect to the UGT2B7 genotype. Furthermore, in contrast to an earlier investigation in benzidine-exposed Chinese bladder cancer patients, no relevant differences between bladder cancer patients and urological hospital controls were observed in Germany.

The genotype glutathione S-transferase P1 (GSTP1) influences the risk for bladder cancer among Chinese workers occupationally exposed to benzidine. Studies of Caucasian bladder cancer cases without known occupational exposures showed conflicting results. Research was thus conducted to define the role of GSTP1 genotypes in Caucasian bladder cancer cases with an occupational history of exposure to aromatic amines. DNA from 143 cases reported to the Industrial Professional Associations (Berufsgenossenschaften) in Germany from 1996 to 2004, who had contracted urothelial cancer due to occupational exposure, and 196 patients from one Department of Surgery in Dortmund, without known malignancy in their medical history, were genotyped using real-time polymerase chain reaction (PCR) (LightCycler) in relation to GSTP1 A1578G (Ile105Val) polymorphism. Among the subjects with bladder cancer, 46% presented the AA genotype, 39% the AG genotype, and 15% the GG genotype. In the surgical (noncancer) control group analyzed, 42% presented the AA genotype, 42% the AG genotype, and 16% the GG genotype. A subgroup of bladder cancer cases, represented by 46 painters, showed a distribution of 41% of the AA genotype, 48% of the AG genotype, and 11% of the GG genotype. Data indicated that in Caucasians exposed to aromatic amines the GSTP1 A1578G polymorphism did not appear to play a significant role as a predisposing factor for bladder cancer incidence.

Tobacco smoking, alcohol drinking, and occupational exposures to polycyclic aromatic hydrocarbons are the major proven risk factors for human head and neck squamous-cell cancer (HNSCC). Major research focus on gene-environment interactions concerning HNSCC has been on genes encoding enzymes of metabolism for tobacco smoke constituents and repair enzymes. To investigate the role of genetically determined individual predispositions in enzymes of xenobiotic metabolism and in repair enzymes under the exogenous risk factor tobacco smoke in the carcinogenesis of HNSCC, we conducted a case-control study on 312 cases and 300 noncancer controls. We focused on the impact of 22 sequence variations in CYP1A1, CYP1B1, CYP2E1, ERCC2/XPD, GSTM1, GSTP1, GSTT1, NAT2, NQO1, and XRCC1. To assess relevant main and interactive effects of polymorphic genes on the susceptibility to HNSCC we used statistical models such as logic regression and a Bayesian version of logic regression. In subgroup analysis of nonsmokers, main effects in ERCC2 (Lys751Gln) C/C genotype and combined ERCC2 (Arg156Arg) C/A and A/A genotypes were predominant. When stratifying for smokers, the data revealed main effects on combined CYP1B1 (Leu432Val) C/G and G/G genotypes, followed by CYP1B1 (Leu432Val) G/G genotype and CYP2E1 (-70G>T) G/T genotype. When fitting logistic regression models including relevant main effects and interactions in smokers, we found relevant associations of CYP1B1 (Leu432Val) C/G genotype and CYP2E1 (-70G>T) G/T genotype (OR, 10.84; 95% CI, 1.64-71.53) as well as CYP1B1 (Leu432Val) G/G genotype and GSTM1 null/null genotype (OR, 11.79; 95% CI, 2.18-63.77) with HNSCC. The findings underline the relevance of genotypes of polymorphic CYP1B1 combined with exposures to tobacco smoke.

In 187 bladder cancer cases reported to the employers' liability insurance association in Germany as suspected cases of an occupational disease produced by aromatic amines, N- acetyltransferase-2 (NAT2) activity status, occupational exposure data, period of latency, and clinical parameters were determined. In 83 out of 187 cases surveyed within the period 1991-1999, the NAT2 acetylator status was investigated by determining the molar ratio of an acetylated and a nonacetylated caffeine metabolite in urine (phenotyping) and/or by NAT2 genotyping according to standard polymerase chain reaction (PCR) protocol. The proportion of slow NAT2 acetylators in the surveyed 83 bladder cancer cases was 67%. In the entire group of surveyed 187 cases, mean duration of exposure was 17.6 yr and mean period of latency was 34.7 yr. Occupational exposures to potential bladder carcinogens were observed in 73 occupations, including chemical industry (25%), and occupations as a painter and/or varnisher (23%) were most often encountered. In 12% of the surveyed bladder cancer cases, a second primary malignancy was observed. The NAT2 distribution observed in the 83 cases is comparable to the proportion in 40 occupationally exposed bladder cancer cases in a Department of Urology located close to a former German production site of benzidine-based azo dyes, but higher than in most studies involving NAT2 genetic status in bladder cancer cases.

Tobacco smoke and occupational exposures to chemicals such as polycyclic aromatic hydrocarbons (PAHs) are, aside from alcohol, the major risk factors for development of head and neck squamous-cell cancer (HNSCC). In this study, new statistical methods were applied. We employ new statistical methods to detect genetic interactions perhaps of higher order, that might play a role in developing HNSCC. The underlying study comprises 312 HNSCC cases and 300 controls. Single-nucleotide polymorphisms (SNPs) of PAH metabolizing and repair enzymes, somatic p53 mutations, and tobacco smoke were examined. Key statistical tools for our analysis are methods of unsupervised and supervised learning. In unsupervised learning, one performs cluster analyses based on well-known and new distance measures to find differences in the SNP patterns of cases and controls, and to understand the role of p53. Our main goal in supervised learning was to identify SNPs and SNP interactions that are likely to alter the susceptibility to HNSCC. Logic regression, a classification method well suited for SNPs, was employed as well as a Bayesian generalization that allows for incorporating additional expert knowledge. These methods detected several important interactions, such as an association between CYP1B1, tobacco smokes and p53 mutations and some interactions between CYP1B1 and glutathione S-transferases in smokers, which included a three-way interaction between CYP1B1, CYP2E1-70G>T, and GSTP1 (exon 5).

Exposure to tobacco smoke is an established cause of cancer in humans and cigarette smoking is a risk factor for urinary bladder cancer development. Aromatic amines are believed responsible for the bladder-specific carcinogenic effect, but polycyclic aromatic hydrocarbons (PAHs) are also of potential relevance. Urothelial cells contain a number of xenobiotic-metabolizing enzymes, which enable them to convert pro-carcinogens into reactive intermediates. In a preceding study, it was demonstrated using cultured porcine urinary bladder epithelial cells (PUBEC) that CYP1A1 mRNA is induced in a potent manner by treatment with benzo[a]pyrene (BaP). In the present study, the time dependence of these effects was evaluated and whether PUBEC cultures derived from individual donors respond differently to BaP treatment was determined. CYP1A1 induction was analyzed by quantitative reverse-transcription polymerase chain reaction (RT-PCR), and genotoxic effects were studied using the Comet assay. Incubation of PUBEC with BaP increased CYP1A1 expression and induction of DNA strand breaks in a time-dependent manner. Interindividual differences were found between PUBEC cultures derived from several donor animals with respect to the response to BaP, such that the extent of CYP1A1 induction and magnitude of DNA damage was interrelated. Hence, individual differences in metabolic capacities and responsiveness to xenobiotics of urothelial cells from individual donors may be factors in susceptibility to genotoxic effects induced by PAHs.

Problem: Individual differences in susceptibility to trichloroethylene-induced nephrocarcinogenicity may be conferred by genetic polymorphisms of glutathione S-transferases (GST), because enzymes of this group are pivotal for the metabolic activation of trichloroethylene. Because of a potential involvement of N-acetylation in the detoxication of reactive trichloroethylene metabolite(s) to N-acetyl-cysteine derivatives, polymorphisms of the NAT2 gene may also be relevant., Methods: The primary collective used for a re-investigation of these questions was that of a hospital-based case-control study by Brüning et al. (Am J Ind Med 43:274-285, 2003) of 134 renal cell cancer cases (20 cases exposed to trichloroethylene) and 401 matched controls. Genetic polymorphisms of GSTT1, GSTM1, GSTP1 and NAT2 were studied. Additional control collectives of non-diseased persons were used for comparison of allele frequencies., Results: No genetic influences on the development of renal cancer due to trichloroethylene were apparent, related to the deletion polymorphisms of GSTT1 and GSTM1, as well as to the NAT2 rapid/slow acetylator states. However, renal cell cancer cases displayed a somewhat higher proportion of the homozygous GSTP1 313A wild type (GSTP1*A), although this was not statistically significant (chi(2) test: P=0.1071, when using only the original controls of Brüning et al. (2003); P=0.0781 with inclusion of the additional controls)., Conclusion: The re-investigation does not confirm the working hypothesis of an influence of the deletion polymorphisms of the glutathione S-transferases GSTT1 and GSTM1 on renal cell cancer development due to high occupational exposures to trichloroethylene.

Human diet contains weakly estrogenic compounds such as daidzein (DAI) and genistein (GEN), phytoestrogens present in soy and many vegetables as well as bisphenol A (BPA), a contaminant from packing materials and plastic containers for foods and beverages. In light of concerns about hormonally active agents, biomonitoring methods are needed to assess human exposure to such compounds. A method for simultaneous determination of DAI, its metabolite equol (EQ), GEN, and BPA by GC-MS analysis was established, validated and applied to measure concentrations in human urine. Sample preparation involves enzymatic conjugate cleavage, SPE and derivatization by silylation. For GC/MS analysis, deuterated DAI and GEN and( 13)C-BPA are used as internal standards. LOD are 4, 4, 5 and 3 ng/mL urine for DAI, EQ, GEN and BPA, respectively. Interassay variations were 9% for DAI, 15% for EQ, 18% for GEN and 10% for BPA. Simple workup and accuracy of the method are suited for biomonitoring. An analysis of urine samples from 15 adults consuming typical German food revealed dietary exposure to phytoestrogens in all samples: GEN concentrations ranged between 13 and 238 ng/mL, those for DAI ranged from 12 to 356 ng/mL. More than half of the individuals excreted also the more estrogenic metabolite EQ, at levels of 8-128 ng/mL. Higher concentrations (GEN: 820, DAI: 960 and EQ: 1740 ng/mL) were measured in a 24 h urine sample upon ingestion of soy protein (50 g with 12.9 mg DAI and 25.2 mg GEN). Only urine collected after some days on strict phytoestrogen-free diet had undetectable isoflavone levels. BPA was detected in 9 of 15 urine samples ranging from 3 to 11 ng/mL, and at 55 ng/mL in one sample. In conclusion, a reliable method to determine BPA and isoflavones in urine was established and applied in a pilot study: Biomonitoring results show much higher dietary exposure to phytoestrogens than to BPA in German adults.

Tests for chromosomal damage are indispensable in the genotoxicity testing battery. Thus, positive results of clastogenicity or aneugenicity tests are of key relevance in safety assessment and product development. Schultz and Onfelt [N. Schultz, A. Onfelt, Sensitivity of cytokinesis to hydrophobic interactions. Chemical induction of bi- and multi-nucleated cells, Chem. Biol. Interact. 126 (2000) 97-123.] have studied the chemical induction of bi- and multi-nucleated cells in Chinese hamster V79 cells and compared non-specific agents with inducers acting through a known specific mechanism. They separated compounds with a specific action from those with a non-specific action based on lipophilicity, following a theory of hydrophobic interactions with processes of cytokinesis. It appeared possible to broaden the original database of this concept to include aneugenic as well as clastogenic compounds studied in the micronucleus (MN) test. The datasets used for this purpose were (A) the original dataset of Schultz and Onfelt [N. Schultz, A. Onfelt, Sensitivity of cytokinesis to hydrophobic interactions. Chemical induction of bi- and multi-nucleated cells, Chem. Biol. Interact. 126 (2000) 97-123.], and two sets (B, C) of our own data from studies in V79 cells in vitro. As the particular endpoints used were different (A: counts of bi- and multi-nucleated cells, B/C: micronucleus counts) the coherence of the experimental data sets was validated by including compounds belonging to both collections. Data set B included compounds with a specific effect on the mitotic spindle (nitrobenzene and benzonitrile) and data set C included the phytoestrogens genistein and daidzein, as well as a number of hormonal steroids with unknown mode of action. Taking all three data sets (A, B, C) together, the 33 compounds investigated covered a total lipophilicity range of logP between -0.51 (diamide) and 5.65 (17alpha-propylmesterolone). In order to separate statistical outliers (with a specific mode of action to be likely) from the large cluster of compounds with non-specific genotoxicity related to hydrophobic interactions, the method of robust regression was applied. It appeared that all compounds with a specific mode of action were in fact outliers of the lipophilicity rule. Genistein, a weak clastogen causing chromosomal aberrations and being discussed to induce topoisomerase-2 mediated DNA breaks, came close to the statistical borderline between compounds with specific and non-specific chromosomal genotoxicity. A general procedure is proposed, applicable in chemical product development, to screen specific and non-specific modes of action.

We monitored the exposure of hairdressers to oxidative hair dyes for 6 working days under controlled conditions. Eighteen professional hairdressers (3/day) coloured hairdresser's training heads bearing natural human hair (hair length: approximately 30 cm) for 6 h/working day with a dark-shade oxidative hair dye containing 2% [14C]-para-phenylenediamine (PPD). Three separate phases of hair dyeing were monitored: (A) dye preparation/hair dyeing, (B) rinsing/shampooing/conditioning and (C) cutting/drying/styling. Ambient air and personal monitoring samples (vapours and particles), nasal and hand rinses were collected during all study phases. Urine (pre-exposure, quantitative samples for the 0-12, 12-24, 24-48 h periods after start of exposure) and blood samples (blank, 4, 8 or 24 h) were collected from all exposed subjects. Radioactivity was determined in all biological samples and study materials, tools and washing liquids, and a [14C]-mass balance was performed daily. No adverse events were noted during the study. Waste, equipment, gloves and coveralls contained 0.41+/-0.16%, dye mixing bowls 2.88+/-0.54%, hair wash 45.47+/-2.95%, hair+scalp 53.46+/-4.06% of the applied radioactivity, respectively. Plasma levels were below the limit of quantification (10 ng PPDeq/mL). Total urinary 0-48 h excretion of [14C] levels ranged from a total of <2-18 microg PPDeq and was similar in subjects exposed during the different phases of hair dyeing. Minimal air levels at or slightly above the limit of quantification were found in a few personal air monitoring samples during the phases of hair dyeing and hair cutting, but not during the rinsing phase. Air area monitoring samples or nasal rinses contained no measurable radioactivity. Hand residues ranged from 0.006 to 0.15 microg PPDeq/cm2, and were found predominantly after the cutting/drying phase. The mean mass balance of [14C] across the six study days was 102.50+/-2.20%. Overall, the mean, total systemic exposure of hairdressers to oxidative hair dyes during a working day including 6 hair dyeing processes was estimated to be <0.36 microg PPDeq/kg body weight/working day. Our results suggest that (a) current safety precautions for the handling of hair dyes offer sufficient protection against local and systemic exposure and (b) professional exposure to oxidative hair dyes does not pose a risk to human health.

Oxidative (permanent) hair dyes contain one or several "primary intermediates" (e.g., p-phenylenediamines, p-aminophenols) and "couplers" (e.g., m-aminophenols, m-hydroxyphenols). In the presence of peroxide, the primary intermediate(s) and the coupler(s) undergo a chemical reaction to form colored oligomers. In the 1970s a number of aromatic amines used in oxidative hair dyes were identified as mutagenic and/or carcinogenic in rodents after lifetime oral administration. In response, regulatory action was taken, and some hair dye ingredients were banned in the European Union. Although recent results suggest that the main "primary intermediate" of oxidative hair dyes, p-phenylenediamine, has a weak genotoxic potential in vitro, it was not mutagenic in a mixture with nonmutagenic couplers, if tested under conditions comparable to those of practical use. Under conditions of use of permanent hair dyes, between 0.1 and 0.5% of the applied p-phenylenediamine may be absorbed through the skin. Acetylation in the skin is a key metabolic step for the primary intermediates p-phenylenediamine and p-aminophenol. Because of the involvement of aromatic amines, the discussion on the carcinogenicity of hair dyes in humans has been focused on urothelial cancer. Numerous epidemiological studies have investigated the risk of bladder cancer associated with the profession as a hairdresser, as well as the risk to consumers of hair dyes. Although some earlier studies suggested an overrepresentation of bladder cancer in male hairdressers, the majority of modern studies do not show an increase in relevant bladder cancer risk for professional or personal use of oxidative hair dyes. Today, there seems to be no relevant bladder cancer risk from the use of oxidative hair dyes. Such a conclusion can be derived from new toxicokinetic and metabolism investigations and is in general accordance with current epidemiological data. Human urothelial cancers, chemically induced by aromatic amines, have typical latency times often longer than 20 years. Since earlier exposures could have an impact decades later, the possibility of bladder cancer in hairdressers having intensively worked with permanent hair dyes during earlier decades (prior to the 1980s) should be taken into account.

N-phenyl-2-naphthylamine (PBNA) represents an example of a suspected carcinogen that is found negative in mutagenicity and clastogenicity testing as well as in long-term animal carcinogenicity bioassays in several species, but for which a carcinogenic risk cannot be excluded because of its metabolic conversion to the known human carcinogen 2-naphthylamine. Also, epidemiologic studies failed to indicate an elevated bladder cancer risk in humans occupationally exposed to PBNA. The amounts of 2-naphthylamine found in the urine of different species including humans after exposure to PBNA indicate unequivocally that PBNA is dephenylated to some extent. These are not explained by the 2-naphthylamine impurities in technical-grade PBNA. To explain the metabolic dephenylation process, it has been suggested that PBNA is metabolized by cytochrome P-450 (CYP) enzymes to the phenolic derivative 4'-hydroxy-N-phenyl-2-naphthylamine, followed by its further oxidation to the quinone imine, which subsequently hydrolyses to form the dephenylation product 2-naphthylamine. Phenolic metabolites from the initial CYP-mediated activation step are rapidly conjugated. Quantitatively, dephenylation of PBNA to 2-naphthylamine is a minor pathway. The dog represents an animal model that appears to approximate the human metabolism and biological activation of PBNA. Based on published data, a worst-case scenario indicates that about 1% of total PBNA taken up is transferred into 2-naphthylamine. However, in vitro as well as in vivo findings with PBNA may point to a significantly smaller conversion rate, as metabolites anticipated from the metabolism of 2-naphthylamine were not detected so far. The assumption, which may well be an overestimation, is compatible with findings in animal experiments, and explains the lack of direct evidence of carcinogenicity of PBNA in both experimental and epidemiological studies.

Acrylamide (AA) is carcinogenic in animals and classified by the International Agency for Research on Cancer as probably carcinogenic in humans. Regarding the AA contents of food the diet significantly contributes to the overall AA burden of the general population. However, it is unclear to which degree the diet, apart from smoking, contributes to the internal AA exposure. Therefore the influence of an AA-free diet on the excretion of urinary mercapturic acid metabolites derived from AA in three healthy volunteers fasting for 48 h was examined. Urinary AA mercapturic acid metabolites were considerably reduced after 48 h of fasting. The levels were even well below the median level in non-smokers. This confirms that the diet is the main source of environmental AA exposure in humans, apart from smoking. Other possible AA sources could be of minor quantitative importance only.

Environmental contamination with 2,4,6-TNT (trinitrotoluene) represents a worldwide problem. Concern for carcinogenicity can be derived from chemically related compounds, especially the dinitrotoluenes. In the metabolism of TNT, the reductive routes are preponderant. The main urinary metabolites of TNT are 4-amino-2,6-dinitrotoluene and 2-amino-4,6-dinitrotoluene. In humans exposed to TNT, the formation of hemoglobin adducts of the amino-dinitrotoluenes is in general concordance with the ratio of urinary excretion. The variations in quantities of excreted metabolites among the different occupational cohorts studied are likely explained by the different routes of exposure to TNT, including dermal uptake. Most studies show that urinary excretion of the amino-dinitrotoluenes (4-amino-dinitrotoluene plus 2-amino-dinitrotoluene) in a range of 1 to 10 mg L(-1) (5-50 microM) are not uncommon--for instance in persons employed with the disposal of military waste. Trinitotoluene is mutagenic in Salmonella typhimurium strains TA98 and TA100, with and without exogenous metabolic activation. Mutagenic activity has been found in urine from workers who were occupationally exposed to TNT. An unpublished 2-year study was reported in 1984 by the IIT Research Institute, Chicago, IL. Fischer 344 rats were fed diets containing 0.4, 2.0, 10, or 50 mg/kg TNT per day. In the urinary bladder, hyperplasia (12 of 47 animals p < .01) and carcinoma (11 of 47 animals, p < .05) were observed at significant levels in high-dose (50 mg kg(-1)) females and in one or two females, respectively, at 10 mg kg(-1). Taking all the available evidence together, the appropriate precautions should be taken.

Although cytosolic glutathione S-transferase (GST) enzymes occupy a key position in biological detoxification processes, two of the most relevant human isoenzymes, GSTT1-1 and GSTM1-1, are genetically deleted (non-functional alleles GSTT1*0 and GSTM1*0) in a high percentage of the human population, with major ethnic differences. The structures of the GSTT and GSTM gene areas explain the underlying genetic processes. GSTT1-1 is highly conserved during evolution and plays a major role in phase-II biotransformation of a number of drugs and industrial chemicals, e.g. cytostatic drugs, hydrocarbons and halogenated hydrocarbons. GSTM1-1 is particularly relevant in the deactivation of carcinogenic intermediates of polycyclic aromatic hydrocarbons. Several lines of evidence suggest that hGSTT1-1 and/or hGSTM1-1 play a role in the deactivation of reactive oxygen species that are likely to be involved in cellular processes of inflammation, ageing and degenerative diseases. There is cumulating evidence that combinations of the GSTM1*0 state with other genetic traits affecting the metabolism of carcinogens (CYP1A1, GSTP1) may predispose the aero-digestive tract and lung, especially in smokers, to a higher risk of cancer. The GSTM1*0 status appears also associated with a modest increase in the risk of bladder cancer, consistent with a GSTM1 interaction with carcinogenic tobacco smoke constituents. Both human GST deletions, although largely counterbalanced by overlapping substrate affinities within the GST superfamily, have consequences when the organism comes into contact with distinct man-made chemicals. This appears relevant in industrial toxicology and in drug metabolism.

Occupational standards concerning allowable concentrations of chemical compounds in the ambient air of workplaces have been established in several countries worldwide. With the integration of the European Union (EU), there has been a need of establishing harmonised Occupational Exposure Limits (OEL). The European Commission Directive 95/320/EC of 12 July 1995 has given the tasks to a Scientific Committee for Occupational Exposure Limits (SCOEL) to propose, based on scientific data and where appropriate, occupational limit values which may include the 8-h time-weighted average (TWA), short-term limits/excursion limits (STEL) and Biological Limit Values (BLVs). In 2000, the European Union issued a list of 62 chemical substances with Occupational Exposure Limits. Of these, 25 substances received a "skin" notation, indicating that toxicologically significant amounts may be taken up via the skin. For such substances, monitoring of concentrations in ambient air may not be sufficient, and biological monitoring strategies appear of potential importance in the medical surveillance of exposed workers. Recent progress has been made with respect to formulation of a strategy related to health-based BLVs.

It is the ultimate goal of the intended REACH process (Registration, Evaluation and Authorization of Chemicals) of the European Union to identify substances of hazardous properties and to evaluate the risks of human and environmental exposure. During the last few months there has been a controversial discussion as to what extent in vitro studies and consideration of structure activity relationship provide sufficient information to waive repeated exposure studies. Industry as well as certain regulatory agencies or NGOs support this approach and propose that repeated dose studies may only be required beyond 100 t/a. From a toxicological point of view it has to be stressed that this discussion primarily considers the cost reduction and protection of animals, whereas protection of human health and the environment are secondary. In vitro studies only allow identification of specific hazardous properties which can be detected by the specific test system. Moreover, appropriate information on the dose response of adverse effects, identification of thresholds and NOELs that are essential for risk characterization cannot be obtained from these studies. Consequently, identification of all relevant hazardous properties and endpoints of adverse effects can only be determined in the intact animal by repeated dose studies such as 28-day or 90-day studies. In the absence of such information the hazard identification is incomplete and there is no basis for appropriate risk assessment of human exposure. Thus, any waiving of repeated dose studies in animals bears the probability of unforeseen effects in case of acute or continuous human exposure. From this the undersigning European Toxicologists conclude: 1. The intention of REACH is to identify hazardous properties in order that a reliable risk assessment can be made and measures taken to deal with chemicals posing a significant risk. 2. The recent debate has centered on ways in which the well established in vivo methods for risk assessment can be bypassed. 3. The evidence that the available alternatives would support such replacement is weak. Progress to improve their value for risk assessment purposes is bound to be slow because the issues are very complex. As a group of European Toxicologists we strongly support the need for more research support in these areas, but we believe that over claims for progress is damaging their development. 4. Under the circumstances only two options are available: to reduce very substantially the estimation of hazard and risk with inevitable adverse consequences for human health and environmental protection, or to continue the existing methods until properly validated new methods are available.

We investigated the human metabolism of AA to the mercapturic acids N-acetyl-S-(2-carbamoylethyl)-L-cysteine (AAMA) and N-(R/S)-acetyl-S-(2-carbamoyl-2-hydroxyethyl)-L: -cysteine (GAMA) which are derived from AA itself and from its oxidative genotoxic metabolite glycidamide (GA), respectively. A healthy male volunteer received a single dose of about 1 mg deuterium-labelled acrylamide (d(3)-AA), representing 13 microg/kg body weight, in drinking water. Urine samples before dosing and within 46 h after the dose were analysed for d(3)-AAMA and d(3)-GAMA by LC-ESI-MS/MS. A first phase of increase in urinary concentration was found to last 18 h with a broad plateau between 8 and 18 h for AAMA, and 22 h for GAMA. Elimination half-lives of both AAMA and GAMA were estimated to be approximately 3.5 h for the first phase and more than 10 h up to few days for the second phase. Total recovery in urine after 24 h was about 51% as the sum of AAMA and GAMA and hereby well in accordance with former studies in rats. After 2 days AAMA, accounting for altogether 52% of the total AA dose, was the major metabolite of AA in humans. GAMA, accounting for 5%, appeared as a minor metabolite of AA. In humans we found a urinary ratio of 0.1 for GAMA/AAMA compared to previously reported values of 0.2 for rats and 0.5 for mice. Therefore, the metabolic fate of AA in humans was more similar to that in rats than in mice as already demonstrated in terms of the haemoglobin adducts. Consequently a genotoxic potency of AA mediated by GA could be supposed to be comparable in rats and humans.

Automobile drivers are exposed to several organic hydrocarbons. Concentrations measured in passenger compartments have been reported to range between 13 and 560 microg/m(3) for benzene, 33-258 microg/m(3) for toluene, 20-250 microg/m(3) for xylene (mixed isomers) and 3-23 microg/m(3) for trimethylbenzene (mixed isomers). These aromatic hydrocarbons are emitted from gasoline and from materials inside a car. In the present study we evaluated, whether these exposures pose a potential risk to the health of drivers. Therefore, we derived maximum exposure levels inside cars for chronic (ELIA(chronic)) and short-term (STELIA) exposure. The lowest ELIA's(chronic) for benzene, toluene, xylene and trimethylbenzene were 0.083, 1.2, 8.8 and 0.31 mg/m(3), respectively. The respective STELIA's were 16, 30, 29 and 25 mg/m(3). Obviously concentrations of toluene, xylene and trimethylbenzene inside cars do not exceed their individual STELIA's. In contrast, benzene seems to be problematic, since concentrations inside cars amount up to 0.56 mg/m(3), which exceeds the ELIA(chronic) derived for benzene. This should not be underestimated, since benzene is a genotoxic carcinogen that probably acts by non-threshold mechanisms. In conclusion, concentrations of toluene, xylene and trimethylbenzene usually observed inside cars are unlikely to pose a risk to the health of drivers. A systematic toxicological evaluation of the risk associated with benzene exposure in cars seems to be necessary.

Expression of cytochromes P450 CYP1A1, CYP1B1, CYP2E1 and CYP4B1 was analysed on the transcript level in human urothelial cells obtained by various methods. As a source of urothelial cells, exfoliated cells in urine samples were used. Their expression profiles were determined either immediately after centrifugal enrichment (n=4) or after their cultivation and propagation (n=8). Another source of urothelial cells were ureter specimens from surgical subjects (n=4). Generally, expression was most prominent for CYP1B1 and CYP4B1 among the CYP transcripts analysed. CYP1B1 mRNA was detected in all samples investigated except for one ureter specimen. CYP4B1 mRNA was present in cell cultures from three out of eight healthy subjects, in three out of four directly investigated urinary sediments and in the cells of all five ureter specimens of four donors investigated after resection and subsequent cell culture. In most cases, CYP2E1 transcript levels were lower than those of CYP1B1 and CYP4B1. CYP2E1 mRNA was detected in cell cultures of six out of eight healthy subjects, in one out of four urinary sediments and in three out of five ureter specimens. CYP1A1 mRNA was clearly observed only in cells from resected ureters. In cell cultures the relative mRNA expression levels varied with subjects interindividually, intraindividually and also during the time of cell culture. The study demonstrates constitutive mRNA expressions of xenobiotic metabolising CYP enzymes in human urothelial cells obtained by different methods. In particular, transcripts of CYP1B1 and CYP4B1 are present, coding for enzymes which are active in the metabolism of polycyclic aromatic hydrocarbons and arylamines, respectively.

In this study we investigated human metabolism and excretion of DEHP after intravenous exposure. For this purpose we determined the five major DEHP metabolites in urine samples of a volunteer before and after a platelet donation (dual-needle technique). Plateletpheresis procedures are known to cause a significant DEHP exposure. We observed a sharp increase in urinary DEHP metabolite concentrations after the procedure. Maximum concentrations of 5OH-MEHP, 5oxo-MEHP, 5cx-MEPP and MEHP observed 4 h after the procedure were 822, 729, 577 and 388 microg/l respectively. 2cx-MMHP was excreted at highest concentrations after 8 h (201 microg/l). Due to longer elimination half-times, 5cx-MEPP and 2cx-MMHP were the major metabolites excreted in urine 24 h after the exposure. The 24-h-cumulative excretion of 363 microg 5cx-MEPP, 353 microg 5OH-MEHP, 309 microg 5oxo-MEHP, 178 microg MEHP and 133 microg 2cx-MMHP indicates an absolute exposure of our volunteer of about 2.6 mg DEHP. Related to the body weight this equals a dose of 31.6 microg/kg body weight/day. This indicates that current risk or preventive limit values for DEHP such as the RfD of the US EPA (20 microg/kg/day) and the TDI of the European Union (20-48 microg/kg/day) can be exceeded on the day of the plateletpheresis. The amount of the dose excreted in urine, distribution of the metabolites in urine and all other elimination characteristics after intravenous DEHP exposure are comparable to oral exposure. There are no indications that toxicokinetic behaviour and the toxicity of DEHP are fundamentally different after the two routes of exposure. Therefore, toxicological endpoints observed for DEHP after oral application should also be considered relevant for medical procedures causing intravenous DEHP exposure, like apheresis procedures. Especially women in their reproductive age need to be protected from DEHP exposures exceeding the above mentioned preventive limit values.

Technical dinitrotoluene (consisting of 2,4- and 2,6-dinitrotoluene isomers) has been widely used as explosives. Both technical isomers are mutagenic in Salmonella typhimurium TA98 strains and carcinogenic in rodents. 2,4-dinitrotoluene shows a dose-dependency of malignant tumors of the kidneys, liver, and mammary glands in rats and mice. In this case report, we discuss a cluster of three cases of urothelial cancer amongst a group of about 60 workers exposed to dinitrotoluenes. The workers were employed in the manufacturing of nitrotoluene explosives in the former German Democratic Republic. The cases occurred within a period of 12 years (1990-2002) leading to a 15.9 fold higher incidence of cancer of the urinary bladder than of the federal state where the chemical factory was located. The observation of the cluster of urothelial cancer in persons highly exposed to nitrotoluenes underlines the putative human carcinogenicity of dinitrotoluenes with the human urothelium as a relevant target tissue.

Molecular epidemiological studies are now a powerful tool to determine differential genetic susceptibilities to cancer-causing agents, and to obtain information on potential mechanisms. Cytochrome P450 (CYP) allelic variants are considered biomarkers of susceptibility to cancer. Such variants have an influence on the bioactivation and thereby on the potency of chemical carcinogens. This is very much straight forward for tobacco smoke-related human cancers. A new aspect is the implication of CYP1B1 in tobacco smoke-related cancers at several organ sites. On this basis, the present review is focused on lung, breast, urinary bladder and head and neck cancer. The CYP profile of the human lung includes CYP1A1, -1B1, -2A6, -2A13, -2B6, -2C18, -2E1, -2F1, -3A5 and -4B1. Polycyclic aromatic hydrocarbons (PAHs) and nitrosamines, as active components of tobacco smoke, appear as primary chemical factors for lung malignancies. For human mammary cancer, the use of hormone replacement therapy (HRT) has been shown to be associated with an increase of breast cancer risk, and there seems to be a link between risks caused by HRT use and modifying polymorphisms of drug/xenobiotic enzymes. Specifically, an association of the CYP1B1*3/*3 genotype with increased breast cancer risks has been postulated. Cigarette smoking is a major cause of human urinary bladder cancer. Arylamines, PAHs and nitrosamines are locally activated within the urothelium. Important CYPs in the bladder epithelium of experimental animals and man are CYP1B1 and -4B1. Alcohol consumption and tobacco smoking are known as the major causes of head and neck cancers. Recently, it appears that a polymorphic variant CYP1B1*3/*3 relates significantly to the individual susceptibility of smokers to head and neck cancer, supporting the view that PAH are metabolically activated through CYP1B1. It appears that CYP1B1 plays a key role for the activation of carcinogens at several organ targets, with a likelihood of complex gene-environment interactions implying Phase II enzymes.

The metabolism of di(2-ethylhexyl)phthalate (DEHP) in humans was studied after three doses of 0.35 mg (4.7 microg/kg), 2.15 mg (28.7 microg/kg) and 48.5 mg (650 microg/kg) of D4-ring-labelled DEHP were administered orally to a male volunteer. Two new metabolites, mono(2-ethyl-5-carboxypentyl)phthalate (5cx-MEPP) and mono[2-(carboxymethyl)hexyl]phthalate (2cx-MMHP) were monitored for 44 h in urine and for 8 h in serum for the high-dose case, in addition to the three metabolites previously analysed: mono(2-ethyl-5-hydroxyhexyl)phthalate (5OH-MEHP), mono(2-ethyl-5-oxohexyl)phthalate (5oxo-MEHP) and mono(2-ethylhexyl)phthalate (MEHP). For the medium- and low-dose cases, 24 h urine samples were analysed. Up to 12 h after the dose, 5OH-MEHP was the major urinary metabolite, after 12 h it was 5cx-MEPP, and after 24 h it was 2cx-MMHP. The elimination half-lives of 5cx-MEHP and 2cx-MMHP were between 15 and 24 h. After 24 h 67.0% (range: 65.8-70.5%) of the DEHP dose was excreted in urine, comprising 5OH-MEHP (23.3%), 5cx-MEPP (18.5%), 5oxo-MEHP (15.0%), MEHP (5.9%) and 2cx-MMHP (4.2%). An additional 3.8% of the DEHP dose was excreted on the second day, comprising 2cx-MMHP (1.6%), 5cx-MEPP (1.2%), 5OH-MEHP (0.6%) and 5oxo-MEHP (0.4%). In total about 75% of the administered DEHP dose was excreted in urine after two days. Therefore, in contrast to previous studies, most of the orally administered DEHP is systemically absorbed and excreted in urine. No dose dependency in metabolism and excretion was observed. The secondary metabolites of DEHP are superior biomonitoring markers compared to any other parameters, such as MEHP in urine or blood. 5OH-MEHP and 5oxo-MEHP in urine reflect short-term and 5cx-MEHP and 2cx-MMHP long-term exposure. All secondary metabolites are unsusceptible to contamination. Furthermore, there are strong hints that the secondary oxidised DEHP metabolites-not DEHP or MEHP-are the ultimate developmental toxicants.

The existing systems of classification of carcinogens should include a distinction between genotoxic and non-genotoxic chemicals. For non-genotoxic chemicals, permissible exposure levels can be derived at which no relevant human cancer risks are anticipated. While genotoxic carcinogens can induce chromosomal effects without mutagenic action, non-DNA-reactive genotoxins affecting topoisomerase or the spindle, or those having an exclusively aneugenic effect can be carcinogenic only at high, toxic doses. Specific mechanisms of clastogenicity and processes of carcinogenesis based on reactive oxygen have practical thresholds. Since reactive oxygen species (ROS) are generally genotoxic, the question is whether chemicals that increase ROS production will add to endogenously produced background levels and lead to nonlinear dose-effect relationships. Taking into account the presence of endogenous carcinogens, it is now becoming evident that carcinogenic risk extrapolation to low doses must be considered according to the mode of action.

Lead compounds are known genotoxicants, principally affecting the integrity of chromosomes. Lead chloride and lead acetate induced concentration-dependent increases in micronucleus frequency in V79 cells, starting at 1.1 microM lead chloride and 0.05 microM lead acetate. The difference between the lead salts, which was expected based on their relative abilities to form complex acetato-cations, was confirmed in an independent experiment. CREST analyses of the micronuclei verified that lead chloride and acetate were predominantly aneugenic (CREST-positive response), which was consistent with the morphology of the micronuclei (larger micronuclei, compared with micronuclei induced by a clastogenic mechanism). The effects of high concentrations of lead salts on the microtubule network of V79 cells were also examined using immunofluorescence staining. The dose effects of these responses were consistent with the cytotoxicity of lead(II), as visualized in the neutral-red uptake assay. In a cell-free system, 20-60 microM lead salts inhibited tubulin assembly dose-dependently. The no-observed-effect concentration of lead(II) in this assay was 10 microM. This inhibitory effect was interpreted as a shift of the assembly/disassembly steady-state toward disassembly, e.g., by reducing the concentration of assembly-competent tubulin dimers. The effects of lead salts on microtubule-associated motor-protein functions were studied using a kinesin-gliding assay that mimics intracellular transport processes in vitro by quantifying the movement of paclitaxel-stabilized microtubules across a kinesin-coated glass surface. There was a dose-dependent effect of lead nitrate on microtubule motility. Lead nitrate affected the gliding velocities of microtubules starting at concentrations above 10 microM and reached half-maximal inhibition of motility at about 50 microM. The processes reported here point to relevant interactions of lead with tubulin and kinesin at low dose levels.

A comparative study of N-acetyltransferase 2 (NAT2) genotyping and phenotyping (caffeine test method) was performed on 211 persons to elucidate apparent discrepancies in the assignment of NAT2*12 and NAT2*13 alleles which occur in the literature. The study used the standard procedures of genotyping (two PCR runs and application of seven restriction enzymes) and phenotyping (determination of the two caffeine metabolites 5-acetylamino-6-formylamino-3-methyluracil (AFMU) and 1-methylxanthine (1X)), as documented in detail and validated by the Deutsche Forschungsgemeinschaft. The data were consistent with an AFMU/1X molar ratio of 0.85 as cut-off point (antimode) between phenotypically slow and rapid acetylators. Under this provision, several R/S allele combinations did not comply, either fully or partly, with their associated phenotypes. In particular, there was a wide phenotypic overlap of the alleged rapid allele combination groups (i) NAT2*12A/*5A; NAT2*12C/*5D; NAT2*4/*5B, (ii) NAT2*13/*6B; NAT2*4/*6A, and (iii) NAT2*13/*7A; NAT2*4/*7B. These groups obviously contained both phenotypically rapid and slow acetylators. If one assumes that the presence of one "wild type" allele NAT2*4 defines a rapid acetylator the assignment of the alleles NAT2*12A, NAT2*12C, and NAT*13 as determinants of a rapid acetylator phenotype must be questioned. This refers in particular to the nucleotide changes A803G (NAT2*12A, NAT2*12C) and C282T (NAT2*13). Based on discussions in the literature and the data presented here, there is accumulating evidence that current assignments of the NAT2*12 and NAT2*13 alleles as determinants of a rapid acetylator state should be reconsidered.

The carcinogenicity of vinyl chloride in humans was recognized in 1974 based on observations of hepatic angiosarcomas in highly exposed workers. A multiplicity of endpoints has been demonstrated. The primary target organ, the liver, displays differential susceptibilities of hepatocytes and sinusoidal cells, which are modified by factors of age and dose. There is consistency in organotropism between experimental animals and humans. Vinyl chloride is a pluripotent carcinogen, predominantly directed toward hepatic endothelial (sinusoidal) cells, and second toward the parenchymal cells of the liver. The similarity of results between experimental animals and humans is a solid basis of an amalgamation of experimental and epidemiological risk estimates. Vinyl chloride requires metabolic activation for carcinogenicity and mutagenicity, and toxicokinetics are a key to interpret the dose response. Practically the entire initial metabolism of vinyl chloride is oxidative. At higher exposure concentrations this is nonlinear, and metabolic saturation of metabolism in rats is reached at about 250 ppm. This is consistent with the plateau of hepatic angiosarcoma incidence in rat bioassays. Physiologically based pharmacokinetic/toxicokinetic (PBPK) models have been developed and successfully applied within the frame of human cancer risk assessments. The major DNA adduct induced by vinyl chloride (approximately 98% of total adducts in rats), 7-(2-oxoethyl)guanine, is almost devoid of promutagenic activity. The clearly promutagenic "etheno" adducts N2,3-ethenoguanine and 3,N4-ethenocytosine each represent approximately 1% of the vinyl chloride DNA adducts in rats, and 1,N6-ethenoadenine is found at even lower concentrations. Etheno adducts appear to have a long persistence and are repaired by glycosylases. Vinyl chloride represents a human carcinogen for which a series of mechanistic events connects exposure with the carcinogenic outcome. These include (1) metabolic activation (to form chloroethylene oxide), (2) DNA binding of the reactive metabolite (to exocyclic etheno adducts), (3) promutagenicity of these adducts, and (4) effects of such mutations on protooncogenes/tumor suppressor genes at the gene and gene product levels. In rat hepatocytes, a further event is a biomarker response. Cancer prestages (enzyme-altered foci), as quantitative biomarkers, provide a tool to study dose response even within low dose ranges where a carcinogenic risk cannot be seen in cancer bioassays directly. Such biomarker responses support a linear nonthreshold extrapolation for low-dose assessment of carcinogenic risks due to vinyl chloride. Published risk estimates based on different sets of data (animal experiments, epidemiological studies) appear basically consistent, and on this basis an angiosarcoma risk of approximately 3 x 10(-4) has been deduced by extrapolation, for exposure to 1 ppm vinyl chloride over an entire human working lifetime. An important point that should be considered in regulatory standard settings is the presence of a physiological background of those etheno DNA adducts, which are also produced by vinyl chloride. Likely reasons for this background are oxidative stress and lipid peroxidation. In essence, fundamentals of the hepatocarcinogenicity of vinyl chloride appear now well established, providing a solid scientific basis for regulatory activities.

Mechanistic data must be incorporated into the assessment of the human risks of chemical carcinogens, for trichloroethylene in particular. The total weight of evidence compiled from data on the genotoxicity of trichloroethylene indicates that systemic mutagenic activity is not expressed in vivo. New epidemiologic data on trichloroethylene generated in Denmark showed a steady decline of occupational exposure to trichloroethylene since 1947, but with occasions of high exposures before 1980. Earlier exposures were related to a slight increase in risk of non-Hodgkin's lymphoma, renal carcinoma, and esophageal carcinoma. Such confounders as coexposure to other industrial solvents (non-Hodgkin's lymphoma) and alcohol drinking (esophageal cancer) must be discussed. In Germany, a new consecutive case-control study on kidney cancer confirmed that the risk of renal cell cancer is significantly elevated in persons reporting long-term (several years) exposure to trichloroethylene. The results of all these studies indicate that low exposure (not higher than the current Occupational Exposure Limits) is not associated with increased risk of malignancy. In the kidney, trichloroethylene can act as a complete carcinogen at the stages of both tumor induction and tumor promotion/progresssion in a in a dose-dependent manner. Different types of kidney cancer can be triggered by different genes. Clear-cell renal carcinoma, preferentially induced by trichloroethylene, is linked with the homozygous inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene. In highly exposed subjects, the local genotoxic effect of trichloroethylene results from bioactivation pathways leading to renal VHL gene damage and renal cell carcinomas. The view that renal cell cancer development after long-term (several years) and high (with prenarcotic episodes) occupational exposure to trichloroethylene is due to chronic damage of the proximal tubule of the kidney as a key step argues for the existence of a practical threshold. Prolonged and unusually high-dose exposure to trichloroethylene is deemed a strong risk factor for the development of renal cell cancer. The findings of experimental, mechanistic, and epidemiologic studies lead to the conclusion that trichloroethylene can be considered a human carcinogen for which a practical threshold appears likely and below which no relevant carcinogenic effect is to be expected.

Consumption of tobacco products is the most relevant risk factor for the development of bladder cancer beside occupational contributions. In order to investigate mechanisms of tobacco smoke components in bladder carcinogenesis we have introduced a primary epithelial cell culture system derived from porcine urinary bladder as a suitable representative for the corresponding human tissue under physiological conditions. Two independent readouts were selected as markers for genotoxic events. Changes in the expression level of several toxicologically relevant genes should serve as indicators for early response, while classical genotoxic endpoints monitored manifested damages. Here, we present the first results of our study with benzo(a)pyrene (BaP) as a member of polycyclic aromatic hydrocarbons (PAHs) found in tobacco smoke. Cells treated with BaP show a dramatic increase in the expression of CYP1A1 that appears to be both indicator of and contributor for BaP toxicity. Genes coding for other proteins relevant in xenobiotic metabolism, signal transduction or tumor suppression show moderate effects or no enhancement of their expression levels. Comet assay and micronucleus test did show a significant, dose-dependent increase in DNA damages or aberrations after cell division. While these effects are conforming to the response at the mRNA expression level, they are less pronounced and require rather higher dosages of the chemical.

Although millions of individuals are exposed to emissions from articles inside cars, relatively little has been published about possible adverse health effects and about exposure levels that can be considered safe or "acceptable". Xylene, formaldehyde and acetaldehyde represent typical examples of relevant volatile organic substances (VOC) released from articles inside cars. Recently, a concept for derivation of maximum exposure levels for volatile organic substances in cars has been published. In the present study we applied this concept to derive maximum exposure levels for xylene, formaldehyde and acetaldehyde and compared the resulting concentrations to exposure levels usually found inside of cars. We derived Short Term Exposure Levels Inside Automotive Vehicles (STELIA) of 29, 0.125 and 15.3 mg/m(3) for xylene, formaldehyde and acetaldehyde, respectively. These STELIAs should not be exceeded during short-term exposures, for instance when starting a car that had been heated up during parking in the sun. Exposure Levels Inside Automotive Vehicles (ELIA, chronic) for chronic exposure to non-genotoxic substances were 8.8, 0.125 and 0.635 mg/m(3) for systemic as well as 17.6, 0.125 and 1.7 mg/m(3) for local exposure to xylene, formaldehyde and acetaldehyde, respectively. Although, it is known that exposure limits for carcinogenic substances should be treated with caution, encouraged by the well documented threshold mechanisms we nevertheless derived ELIAs for Carcinogenic and Mutagenic Substances (ELIA, cm) resulting in 0.125 and 0.635 mg/m(3) for formaldehyde and acetaldehyde. If these ELIAs are matched against average concentrations of xylene, formaldehyde and acetaldehyde found in cars at 23 degrees C (1.22, 0.048 and 0.042 mg/m(3)), there is no reason for concern. With respect to STELIAs and extrapolated concentrations at 65 degrees C (14.7, 1.47 and 1.68 mg/m(3), for xylene, formaldehyde and acetaldehyde, respectively), however, a reduction of the concentration of formaldehyde may be necessary.

Contaminated soils represent a potential health risk for the human population. Risk assessment for humans requires specific methods, which must reflect the peculiarities of human behaviour, physiology and biochemistry with respect to contaminant uptake and processing. Biomarkers of effect or exposure have become an appropriate tool. Organic pollutants influence the expression profile of cytochromes P450 (CYP), and CYP1A1 has been shown to be a suitable biomarker for polycyclic aromatic hydrocarbons (PAH). The latter are widely distributed in soils and constitute an important soil contamination. Upon intake of PAH-contaminated soils, CYP1A1 is induced in various organs of rats and minipigs. Increased CYP1A1-levels in lung, kidney and spleen, after oral soil intake, indicate that contaminants escape the primary duodenal and hepatic metabolism and reach further organs. Dose-response relationships reveal that induction effects are to be expected in children based on known exposure conditions. Generally, CYP1A1-induction does not correlate with results of toxicity tests with lower organisms, performed with the same soils. The organic carbon content is largely responsible for this discrepancy. It severely affects the toxicity of soil bound PAH for microorganisms, but obviously affects the mobilization efficiency for PAH in the gastro-intestinal tract of mammals to a minor extent. Soil remediation by different methods may result in a significant reduction of the PAH content and of toxicity. Ingestion of remediated soils by rats shows, however, that the induction potential for CYP1A1 is only slightly decreased after remediation. This means that the major inducing components resist biological remediation or soil washing and remain in the soil. Because data obtained with experimental animals form the guiding principle for in vitro tests to be developed, the suitability of the animal model used for extrapolations to humans has to be proven. Upon soil ingestion, minipigs show a tissue-specific response pattern, which substantially differs from that of rats, which are widely used as animal models. It is not known which response pattern resembles that of man. In summary, cytochromes P450, in particular CYP1A1, are suitable biomarkers to assess the bioavailability of soil bound contaminants and their effects on mammalian species. There are, however, a number of questions to be answered in order to develop an in vitro test for human risk assessment. This concerns, for example, the identification of the suitable animal model, the identification of biomarkers for other contaminants and concepts to transpose the in vivo data to in vitro technologies or to mathematical modelling.

Interactions of mercury(II) with the microtubule network of cells may lead to genotoxicity. Complexation of mercury(II) with EDTA is currently being discussed for its employment in detoxification processes of polluted sites. This prompted us to re-evaluate the effects of such complexing agents on certain aspects of mercury toxicity, by examining the influences of mercury(II) complexes on tubulin assembly and kinesin-driven motility of microtubules. The genotoxic effects were studied using the micronucleus assay in V79 Chinese hamster fibroblasts. Mercury(II) complexes with EDTA and related chelators interfered dose-dependently with tubulin assembly and microtubule motility in vitro. The no-effect-concentration for assembly inhibition was 1 microM of complexed Hg(II), and for inhibition of motility it was 0.05 microM, respectively. These findings are supported on the genotoxicity level by the results of the micronucleus assay, with micronuclei being induced dose-dependently starting at concentrations of about 0.05 microM of complexed Hg(II). Generally, the no-effect-concentrations for complexed mercury(II) found in the cell-free systems and in cellular assays (including the micronucleus test) were identical with or similar to results for mercury tested in the absence of chelators. This indicates that mercury(II) has a much higher affinity to sulfhydryls of cytoskeletal proteins than to this type of complexing agents. Therefore, the suitability of EDTA and related compounds for remediation of environmental mercury contamination or for other detoxification purposes involving mercury has to be questioned.

This study investigated the hypothesis that the chromosomal genotoxicity of inorganic mercury results from interaction(s) with cytoskeletal proteins. Effects of Hg2+ salts on functional activities of tubulin and kinesin were investigated by determining tubulin assembly and kinesin-driven motility in cell-free systems. Hg2+ inhibits microtubule assembly at concentrations above 1 microM, and inhibition is complete at about 10 microM. In this range, the tubulin assembly is fully (up to 6 microM) or partially (~6-10 microM) reversible. The inhibition of tubulin assembly by mercury is independent of the anion, chloride or nitrate. The no-observed-effect-concentration for inhibition of microtubule assembly in vitro was 1 microM Hg2+, the IC50 5.8 microM. Mercury(II) salts at the IC50 concentrations partly inhibiting tubulin assembly did not cause the formation of aberrant microtubule structures. Effects of mercury salts on the functionality of the microtubule motility apparatus were studied with the motor protein kinesin. By using a "gliding assay" mimicking intracellular movement and transport processes in vitro, HgCl2 affected the gliding velocity of paclitaxel-stabilised microtubules in a clear dose-dependent manner. An apparent effect is detected at a concentration of 0.1 microM and a complete inhibition is reached at 1 microM. Cytotoxicity of mercury chloride was studied in V79 cells using neutral red uptake, showing an influence above 17 microM HgCl2. Between 15 and 20 microM HgCl2 there was a steep increase in cell toxicity. Both mercury chloride and mercury nitrate induced micronuclei concentration-dependently, starting at concentrations above 0.01 microM. CREST analyses on micronuclei formation in V79 cells demonstrated both clastogenic (CREST-negative) and aneugenic effects of Hg2+, with some preponderance of aneugenicity. A morphological effect of high Hg2+ concentrations (100 microM HgCl2) on the microtubule cytoskeleton was verified in V79 cells by immuno-fluorescence staining. The overall data are consistent with the concept that the chromosomal genotoxicity could be due to interaction of Hg2+ with the motor protein kinesin mediating cellular transport processes. Interactions of Hg2+ with the tubulin shown by in vitro investigations could also partly influence intracellular microtubule functions leading, together with the effects on the kinesin, to an impaired chromosome distribution as shown by the micronucleus test.

In 1972, Herbert Remmer's group at the University of Tübingen had developed a micro method to assess cytochrome P450 contents and activities of drug metabolising enzymes in needle biopsies from human liver. Upon application of this method to patients receiving different kinds of drug therapy, Herbert Remmer was the first to describe that total human hepatic cytochrome P450 was markedly elevated by the new anti-tuberculosis drug rifampicin. Similar observations were made for the antimycotic clotrimazol. In 1975, Herbert Remmer's group described the unique species difference that induction of cytochrome P450 by rifampicin did not occur in the rat. After the first clinical reports of impaired effectiveness of oral contraception in persons treated with rifampicin, studies at Herbert Remmer's Institute showed a 4-fold increase, after repetitive rifampicin administration to humans, in the ability of hepatic microsomes to ortho-hydroxylate the contraceptive estrogen ethinylestradiol, compared to microsomes from untreated normal subjects. Subsequent pharmacokinetic investigations were compatible with this induction of the estrogen-2-hydroxylase by rifampicin and provided a rational explanation for the classical drug interaction between rifampicin and oral contraceptives. These early studies, in the 1970s in Tübingen, were followed by further developments. It was realized that the cytochrome P450 isoenzyme 3A4 (CYP3A4) is the major CYP isozyme in the human liver metabolizing a variety of xenobiotics and endobiotics, being also responsible for the 2-hydroxylation of ethinylestradiol. The inducibility of CYP3A4 by barbiturates and rifampicin explains the effects of inducers to enhance the clearance of ethynylestradiol and thereby to reduce the effectiveness of oral contraceptives, rifampicin being one of the most potent inducers of human CYP3A4 gene expression. Since 1998, novel "orphan" members of the nuclear hormone receptor superfamily were cloned from mouse, rat, rabbit, and human origin. These so-called pregnane X receptors (PXR), across species, are activated by inducers of CYP3A4 expression. It now appears that PXR is a key mediator of complex induction processes of xenobiotic processing enzymes, which are triggered by rifampicin and other inducers. Studies of the structure and substrate affinities of PXR have provided the rational explanation of the unique species difference of rifampicin induction between humans and rats that was first described by Herbert Remmer.