- Email: [email protected]
Genetic susceptibility to environmental toxicants: the interface between human and experimental studies in the development of new toxicological concepts
Toxicology Letters 127 (2002) 321– 327 www.elsevier.com/locate/toxlet
Genetic susceptibility to environmental toxicants: the interface between human and experimental studies in the development of new toxicological concepts Ricarda Thier a,b,*, Klaus Golka a, Thomas Bru¨ning a,c, Yon Ko d, Hermann M. Bolt a b
a Institut fu¨r Arbeitsphysiologie an der Uni6ersita¨t Dortmund (IfADo), Ardeystraße 67, D-44139 Dortmund, Germany Department of Physiology and Pharmacology, School of Biomedical Sciences, Uni6ersity of Queensland, St. Lucia, Queensland 4072, Australia c Berufsgenossenschaftliches Forschungsinstitut fu¨r Arbeitsmedizin, Bu¨rkle-de-la-Camp-Platz 1, D-44789 Bochum, Germany d Department of Internal Medicine (Medizinische Poliklinik), Uni6ersity of Bonn, 53111 Bonn, Germany
Abstract The growing knowledge of the genetic polymorphisms of enzymes metabolising xenobiotics in humans and their connections with individual susceptibility towards toxicants has created new and important interfaces between human epidemiology and experimental toxicology. The results of molecular epidemiological studies may provide new hypotheses and concepts, which call for experimental verification, and experimental concepts may obtain further proof by molecular epidemiological studies. If applied diligently, these possibilities may be combined to lead to new strategies of human-oriented toxicological research. This overview will present some outstanding examples for such strategies taken from the practically very important field of occupational toxicology. The main focus is placed on the effects of enzyme polymorphisms of the xenobiotic metabolism in association with the induction of bladder cancer and renal cell cancer after exposure to occupational chemicals. Also, smoking and induction of head and neck squamous cell cancer are considered. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Genetic susceptibility; Metabolism; Cancer
1. Introduction The growing knowledge on genetic polymorphisms of enzymes metabolising xenobiotics in humans and their connections with individual sus* Corresponding author. Tel.: + 61-7-3365-3222; fax: + 617-3365-1766. E-mail address: [email protected] (R. Thier).
ceptibility towards toxicants has created new and important interfaces between human epidemiology and experimental toxicology. On the one hand, the results of molecular epidemiological studies may provide new hypotheses and concepts, which call for experimental verification (or falsification), and on the other hand, experimental concepts may obtain further proof by molecular epidemiological studies. If applied diligently, these
0378-4274/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 4 2 7 4 ( 0 1 ) 0 0 5 1 5 - X
322
R. Thier et al. / Toxicology Letters 127 (2002) 321–327
possibilities may be combined to lead to new strategies of human-oriented toxicological research. A particularly interesting and very important field of application of such research strategies is occupational toxicology (Bolt, 1996; Thier et al., 2000). A basic and frequent problem of this field is the availability of exposed population cohorts of limited size only. Interplay of human and experimental studies, however, may nevertheless help to arrive at valid conclusions. This overview will present some outstanding examples for such strategies, based on work of our own group.
2. Occupational bladder cancer Since the classical study of Cartwright et al. (1982) the particular susceptibility of ‘slow acetylators’ for the carcinogenic effect of aromatic amines towards the human urothelium has been confirmed in many studies. In Germany, Lewalter and Miksche (1991) described a group of 331 persons having worked between 1951 and 1967 at a benzidine manufacturing plant in Leverkusen, Germany. The phenotypic ratio of slow (160 persons) to fast (171 persons) acetylators within the total cohort was close to 1. At the time of publication, 92 persons of the entire group were diseased with bladder cancer, among them 75 slow and only 17 fast acetylators. Between 1991 and 1993 an investigation of 196 patients with urothelial bladder cancer of the Department of Urology of the local hospital was performed, and the N-acetyltransferase NAT2 status was assessed by phenotyping and genotyping. There was no increased prevalence of slow acetylators in the whole group of patients compared to the general population, but there was a tendency towards higher representation in the patient group of slow acetylators with a specific working history at the local chemical industry (Golka et al., 1996). This suggested that the genetic trait (slow NAT2 acetylator) was of major importance only in a subgroup with specific occupational exposure and that definition of such subgroups is most important.
In a subsequent case-control study in the German Ruhr area, an area of former coal, iron and steel industries, 412 male urothelial bladder cancer patients were compared as to their occupational histories with 414 patients with prostatic hyperplasia as controls. The study resulted in a smoking-adjusted odds ratio of 2.54 (95% C.I.: 1.64–3.93) in former underground hard-coal miners, consistent with results of (the few) existing similar studies in coal mining regions (Golka et al., 1998). A causative reason for bladder cancer specific for coal miners was not known. However, relevant hints could be deduced from the distribution of enzyme polymorphisms in bladder cancer patients of this area, related to their occupation (Golka et al., 1997; Reckwitz et al., 1997): The ratio of slow versus rapid acetylators did not differ between diseased coal miners and diseased patients in general (in contrast to patients specifically exposed to coal tar). However, a tendency was noted towards a higher representation (84%) of the glutathione transferase GSTM1 deficient genotype than found in the general population of the area (54%; Fig. 1). Although this did not point to the involvement of aromatic amines, GSTM1 is involved in the detoxification of carcinogenic dihydrodiol intermediates of polycyclic aromatic hydrocarbons (PAH). This finding induced the working hypothesis that PAH might causally be involved in urothelial cancers, at least under defined occupational circumstances, and led to a series of experimental toxicological studies. First of all, it was of interest whether the urothelium was capable of metabolising precarcinogens to their ultimately effective genotoxic metabolites. For these investigations, cell culture model systems have been developed. As the levels of enzymes metabolising xenobiotics may be markedly altered in under cell culture conditions, only primary cell culture systems proved to be useful for this purpose (Fo¨ llmann et al., 2000). As an experimental system, porcine urinary bladder epithelial cells (PUBEC) had the further advantage that it was based on abattoir materials with unlimited availability. The cells were characterised according to morphologic, physiologic and biochemical parameters. They displayed a number of constitutive phase-I and
R. Thier et al. / Toxicology Letters 127 (2002) 321–327
phase-II enzyme activities, inter alia of CYP1A1 (EROD activity), prostaglandin H synthase, Nacetyltransferase and glutathione transferase (Fo¨ llmann et al., 2000). This was supplemented by genotoxic effects on the cells by carcinogens that require metabolic activation, and demonstrates that the urothelium can no longer be regarded as an inactive target of preformed reactive metabolites of carcinogens in the urine. This gives support to the view that PAH might be a group of carcinogens relevant for human urothelial cancer. The successful primary culturing of human urothelial cells (Do¨ rrenhaus et al., 2000) provides new possibilities to further test this hypothesis. The metabolisms of aromatic amines and aromatic nitro compounds are closely connected, and the same reactive intermediate (nitroso compounds) are involved. Based on findings on experimental carcinogenicity of nitrobenzene (Cattley et al., 1994) and of dinitrotoluene (Leonard et al., 1987), possible human carcinogenicity of the entire group of compounds is being discussed. Dinitrotoluene is of particular importance as soil contaminant in former areas of ammunition production and military facilities, e.g. in the east of Germany. In this context the observation of a cluster of 14 renal cell carcinomas and six urothelial carcinomas of the renal pelvis among a group of 183 formerly dinitrotoluene-exposed miners, in
323
the underground copper mining industry of the former German Democratic Republic, is noteworthy. Investigation of the frequency of some genetic variants revealed that all six persons with this working history and urothelial cancer of the renal pelvis were slow acetylators for NAT2 (Bru¨ ning et al., 1999a). This strengthens a causative role of dinitrotoluene and calls now for further experimental toxicological research in this direction (Bru¨ ning et al., 2001).
3. Head and neck cancer Biomarker studies are especially useful in the search of tobacco constituents responsible for human cancer manifestations (Thier et al., 2001). This could be exemplified in a recent study on head and neck squamous cell cancer (HNSCC) (Ko et al., 2001). Tobacco use is causally associated HNSCC. In a case-control study effects of genetic variants of the cytochrome P450 enzymes CYP1A1, CYP1B1, glutathione transferases GSTM1, GSTT1, and GSTP1 genes possibly modifying the risk of smoking-related HNSCC were investigated. Allelisms of the CYP1A1, GSTT1, GSTM1, and GSTT1 genes alone were not associated with an increased risk. By contrast, the CYP1B1 codon
Fig. 1. Distribution of NAT2 phenotype (n = 179) and GSTM1 genotype (n = 89) in urinary bladder cancer patients. Data according to Golka et al. (1997).
324
R. Thier et al. / Toxicology Letters 127 (2002) 321–327
432 polymorphism (CYP1B1*1: Val, CYP1B1*2: Leu) was found to be a putative susceptibility factor in smoking-related HNSCC. The frequency of the variant allele CYP1B1*2 was significantly higher (PB0.001) in the group of smoking cases when compared with smoking controls. Likewise, CYP1B1*2 was significantly (P B 0.001) more frequent in smoking cases compared to non-smoking cases. In combination with variants of the GSTT1 gene significant synergistic effects were observed. The impact of the variants of the CYP1B1 gene on HNSCC risk was also reflected by the strong association with the frequency of somatic mutations of the p53 gene. Smokers with the genotype CYP1B1*2/2 were 20 times more likely to show evidence of p53 mutations than were those with CYP1B1*1/1 genotype. Once more, combined genotype analysis of CYP1B1 and GSTM1 or GSTT1 revealed interactive effects on the occurrence of p53 gene mutations. In total, the results of the study (Ko et al., 2001) pointed to polymorphic variants of CYP1B1, relating significantly to the individual susceptibility of smokers to HNSCC. This was paralleled by earlier findings of an association between susceptibility to colorectal cancer and polymorphisms at residue 432 of CYP1B1 (Fritsche et al., 1999). In general, the CYP isoenzyme CYP1B1 plays a role in metabolic activation of a variety of classes of chemical carcinogens, e.g. arylamines, aromatic nitro compounds, and PAH (Shimada et al., 1996). Although the Val432Leu-polymorphism at this site was suggested to have a profound impact on the catalytic activity of the enzyme, this alteration had little effect on classical kinetic parameters of CYP1B1mediated reactions, such as the epoxidation of ( −)-trans-(7R,8R)-benzo(a)pyrene-7,8-dihydrodiol, the O-dealkylation of ethoxyresorufin and the 1%-hydroxylation of bufuranol. However, this specific polymorphism enhanced the Km of the formations of catechol oestrogens (2- and 4-hydroxy-oestradiol) from oestradiol by a factor of 4 (Li et al., 2000). In this situation, further experimental toxicological research is needed to establish the mechanisms by which the Val432Leu-polymorphism of CYP1B1 interferes with tobacco-specific carcino-
genic compounds. Such research should not be restricted to the classical PAH, but must also consider related nitro and amino compounds. This, in turn, will be invaluable for the design of further epidemiological studies on tobacco-induced HNSCC.
4. Renal cell cancer The discussion on renal carcinogenicity of trichloroethylene, in view of human risk assessment, provides a further outstanding example for the interplay of toxicology and epidemiology (Bru¨ ning and Bolt, 2000). Upon repetitive inhalation of trichloroethylene, renal cell tumours were experimentally increased in male rats, and an increased incidence of interstitial cell tumours of the testes was also reported (Maltoni et al., 1988). Studies on the metabolism of trichloroethylene in rodents and in humans have supported the role of bioactivation reactions for the development of tumours following exposure to trichloroethylene. Trichloroethylene is metabolised by two competing pathways, oxidation by cytochromes P450 (mainly CYP2E1) as the major route of metabolism, and conjugation with glutathione by glutathione transferases as a quantitatively minor pathway (Goeptar et al., 1995). Evidence for the existence of the glutathione-dependent pathway in humans has been obtained by the identification of N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine and Nacetyl-S-(2,2-dichlorovinyl)-L-cysteine in human urine after exposure to trichloroethylene (Bru¨ ning et al., 1998). Epidemiological cohort studies addressing the carcinogenicity of trichloroethylene with respect to the renal or urothelial target sites have been evaluated (Weiss, 1996). No clear evidence for an elevated renal or urinary tract cancer risk in trichloroethylene-exposed groups was visible in these exposed populations. However, a cohort study (Henschler et al., 1995) and a casecontrol study (Vamvakas et al., 1998) of workers having been exposed to unusually high levels of trichloroethylene in Germany supported the postulate that trichloroethylene has a nephrocarcinogenic effect in humans. For decades, companies in the study area had used trichloroethylene as main
R. Thier et al. / Toxicology Letters 127 (2002) 321–327
325
Table 1 Frequency of homozygous gene deletions of the human GSTT1 and GSTM1 genes in renal cell carcinoma (RCC) patients and healthy workers Genotype GSTT1 *1/1 or *1/2 *2/2 GSTM1 *1/1 or *1/2 2*/2
RCC patients TRI exposed n =57 (%)
Control group n = 139 (%)
Odds ratio
95% Cl
54 (95) 3 (5)
107 (77) 32 (23)
5.4
1.6–18.4
29 (51) 28 (49)
76 (55) 63 (45)
0.9
0.5–1.6
Both groups had a similar history of high and long-term exposure to trichloroethylene (TRI). Updated data set according to Bru¨ ning (1999).
degreasing agent without any precautionary measures. The results of both studies were discussed in the literature with reserve. Therefore, further investigations on patients with renal cell carcinoma and with histories of high trichloroethylene exposures were of particular relevance. Excretion of marker proteins in the urine pointed to the existence of toxic damage to the proximal renal tubules by trichloroethylene (Bru¨ ning et al., 1996). In order to investigate persistent nephrotoxic effects of high trichloroethylene exposures independent of the origination of renal cell cancer, a retrospective study was carried out on 39 workers exposed to high levels of trichloroethylene from 1956 to 1975. The control group comprised 46 persons from the same company who were categorised by their age. They were employed in the company’s administrative departments and had not been exposed to trichloroethylene or to other chemicals. The nephrotoxic effect of high trichloroethylene exposures was confirmed, as in the previous studies, using SDS-PAGE, with respect to an increased occurrence of tubular kidney damage among those exposed to trichloroethylene. Those exposed exhibited an increased excretion of the glutathione transferase isozyme GST alpha that is a specific marker for damage of the proximal tubules of the kidney. In contrast, excretions of the glutathione transferase enzyme GST pi, a marker for distaltubular damage, were in the same range for the exposed and the control group (Bru¨ ning et al., 1999b). The results of these biomarker investiga-
tions in diseased humans were consistent with the toxicological thesis of Goeptar et al. (1995) which had been derived on the basis of mechanistic considerations. It postulates that renal cell tumours are only induced by trichloroethylene if the substance causes toxic renal cell damage. Furthermore, 42 patients with renal cell carcinoma and an anamnesis of several years of exposure to trichloroethylene were examined with respect to the presence of the two polymorphic glutathione transferases GSTM1 and GSTT1. Forty-eight persons served as controls, who had similarly been exposed to trichloroethylene, but who to date had no kidney tumour (Bru¨ ning et al., 1997). A shift was observed among those having renal carcinomas after exposure to trichloroethylene in the direction of the presence of either gene GSTM1 but in particular GSTT1 (Table 1). The molecular epidemiological findings, in general, corresponded with the hypothesis that the occurrence of trichloroethylene-induced renal cell carcinomas involves metabolism of trichloroethylene via the glutathione transferase metabolic pathway. It also indicates that the population exhibits considerable differences in susceptibility to the nephrocarcinogenic effect of trichloroethylene on the basis of a genetically determined enzyme supply. In this respect, it is interesting that GSTT1 is expressed in the human kidney (Thier et al., 1997, 1998; Delbanco et al., 2001), but not the cytochrome P450 isozyme CYP2E1, which is connected with the oxidative metabolism of trichloroethylene (Amet et al., 1997).
326
R. Thier et al. / Toxicology Letters 127 (2002) 321–327
Hence, the compilation and synopsis of toxicological, epidemiological and molecular epidemiological studies on trichloroethylene, in total, provide a new type of integrated argumentation for the nephrotoxicity and nephrocarcinogenicity of this particular solvent (Bru¨ ning and Bolt, 2000).
5. Conclusions Current processes leading to a new convergence of disciplines are evident in toxicology. Traditionally, the central and most integral part of toxicology is scientific establishment of toxic effects by animal experimentation, development of toxicological concepts on the basis of physiological and molecular mechanisms, and extrapolation of risks from the animal experiment to humans with the help of knowledge in both toxicodynamics and toxicokinetics. Now, entirely new links appear between epidemiological research in humans and experimental research in toxicology. Thereby, new and more efficient research strategies connect epidemiological and experimental research. Being aware of the growing realisation of the decisive importance of the genetic background of exposed subjects and identification of increasing numbers of respective genetic traits, the term of a new era of toxicology has been coined (Thier and Bolt, 2001). This not only describes new research fields, but also new ways to broaden our knowledge!
Acknowledgements The authors thank the Deutsche Forschungsgemeinschaft, the Deutscher Akademischer Austauschdienst, and the U.S. Environmental Protection Agency for valuable financial support of the studies underlying to this review.
References Amet, Y., Berthon, F., Fournier, G., Dre´ ano, Y., Bardou, L., Cle´ des, J., Me´ nez, J.F., 1997. Cytochrome P450 4A and
2E1 expression in human kidney microsomes. Biochem. Pharmacol. 53, 765 – 771. Bolt, H.M., 1996. Genetic predisposition in occupational toxicology. Arch. Toxicol. (Suppl. 18), 362 – 366. Bru¨ ning, T., 1999. Zur Kanzerogenita¨ t des Lo¨ semittels Trichlorethylen: Neue Ansa¨ tze zur arbeitsmedizinischen Pra¨ vention. Habilitationsschrift. Medical Faculty, University of Bonn. Bru¨ ning, T., Bolt, H.M., 2000. Renal toxicity and carcinogenicity of trichloroethylene: key results, mechanisms, and controversies. Crit. Rev. Toxicol. 30, 253 – 285. Bru¨ ning, T., Golka, K., Makropoulos, V., Bolt, H.M., 1996. Pre-existence of chronic tubular damage in cases of renal cell cancer after long and high exposure to trichloroethylene. Arch. Toxicol. 70, 259 – 260. Bru¨ ning, T., Lammert, M., Kempkes, M., Thier, R., Golka, K., Bolt, H.M., 1997. Influence of polymorphisms of GSTM1 and GSTT1 for risk renal cell cancer in workers with a long term and high occupational exposure to trichloroethylene. Arch. Toxicol. 71, 596 – 599. Bru¨ ning, T., Vamvakas, S., Makropoulos, V., Birner, G., 1998. Acute intoxication with trichloroethylene: clinical symptoms, toxicokinetics, metabolism and development of biochemical parameters for renal damage. Toxicol. Sci. 41, 157 – 165. Bru¨ ning, T., Chronz, C., Thier, R., Havelka, J., Ko, Y., Bolt, H.M., 1999a. Occurrence of urinary tract tumors in miners exposed to dinitrotoluene. J. Occup. Environ. Med. 41, 144 – 149. Bru¨ ning, T., Sundberg, A.G.M., Birner, G., Lammert, M., Bolt, H.M., Appelkvist, E.E., Nilsson, R., Dallner, G., 1999b. Glutathione transferase alpha as a marker for tubular damage after trichloroethylene exposure. Arch. Toxicol. 73, 246 – 254. Bru¨ ning, T., Thier, R., Mann, H., Melzer, H., Bro¨ de, P., Dallner, G., Bolt, H.M., 2001. Pathological excretion patterns of urinary proteins in miners highly exposed to dinitrotoluene. J. Occup. Environ. Med. 43, 610 – 615. Cartwright, R., Ahmad, R.A., Barham-Hall, D., Glashan, R.W., Rogers, H.J., Higgins, E., 1982. Role of N-acetyltransferase phenotypes in bladder carcinogenesis: a pharmacogenetic epidemiological approach to bladder cancer. Lancet 2, 842 – 845. Cattley, R.C., Everitt, J.I., Gross, E.A., Moss, O.R., Hamm, T.E., Popp, J.A., 1994. Carcinogenicity and toxicity of inhaled nitrobenzene in B6C3F1 mice and CD rats. Fundam. Appl. Toxicol. 22, 328 – 340. Delbanco, E.H., Bolt, H.M., Huber, W.W., Beken, S., Geller, F., Philippou, S., Brands, F.H., Bru¨ ning, T., Thier, R., 2001. Glutathione transferase activities in renal carcinomas and adjacent normal renal tissues: factors influencing renal carcinogenesis induced by xenobiotics. Arch. Toxicol. 74, 688 – 694. Do¨ rrenhaus, A., Mu¨ ller, J.I.F., Golka, K., Jedrusik, P., Schulze, H., Fo¨ llmann, W., 2000. Cultures of exfoliated epithelial cells from different locations of the human urinary tract and the renal tubular system. Arch. Toxicol. 74, 618 – 626.
R. Thier et al. / Toxicology Letters 127 (2002) 321–327 Fo¨ llmann, W., Guhe, C., Weber, S., Birkner, S., Ma¨ hler, S., 2000. Cultured porcine urinary bladder epithelial cells as a screening model for genotoxic effects of aromatic amines: characterisation and application of the cell culture model. ATLA 28, 833 – 854. Fritsche, E., Bru¨ ning, T., Jonkmanns, C., Ko, Y., Bolt, H.M., Abel, J., 1999. Detection of cytochrome P450 1B1 Bfr I polymorphism: genotype distribution in healthy German individuals and in patients with colorectal carcinoma. Pharmacogenetics 9, 405 –408. Goeptar, A.R., Commandeur, J.N.M., van Ommen, B., van Bladeren, P.J., Vermeulen, N.P.E., 1995. Metabolism and kinetics of trichloroethylene in relation to carcinogenicity. Relevance of the mercapturic acid pathway. Chem. Res. Toxicol. 8, 3 – 21. Golka, K., Prior, V., Blaskewicz, M., Cascorbi, M., Scho¨ ps, W., Kierfeld, G., Roots, I., Bolt, H.M., 1996. Occupational history and genetic N-acetyltransferase polymorphism in urothelial cancer patients of Leverkusen, Germany. Scand. J. Work Environ. Health 22, 332 – 338. Golka, K., Reckwitz, T., Kempkes, M., Cascorbi, I., Blaszkewicz, M., Reich, S., Roots, I., Soekeland, J., Schulze, H., Bolt, H.M., 1997. N-Acetyltransferase 2 (NAT-2) and glutathione S-transferase m (GSTM1) in bladder cancer patients in a highly industrialized area. Int. J. Occup. Environ. Health 3, 105 –110. Golka, K., Bandel, T., Schlaefke, S., Reich, S.E., Reckwitz, T., Urfer, W., Bremicker, K.D., Neugebauer, W., Soekeland, J., Bolt, H.M., 1998. Urothelial cancer of the bladder in an area of former coal, iron, and steel industries in Germany: a case-control study. Int. J. Occup. Environ. Health 4, 79 – 84. Henschler, D., Vamvakas, S., Lammert, M., Dekant, W., Kraus, B., Bru¨ ning, T., Ulm, K., 1995. Increased incidence of renal cell tumors in a cohort of cardbord workers exposed to trichloroethylene. Arch. Toxicol. 69, 291 –299. Ko, Y., Abel, J., Harth, V., Bro¨ de, P., Antony, C., Donat, S., Fischer, H.P., Ortiz-Pallardo, M.E., Thier, R., Sachinidis, A., Vetter, H., Bolt, H.M., Herberhold, C., Bru¨ ning, T., 2001. Association of CYP1B1 codon 432 mutant allele in head and neck squamous cell cancer is reflected by somatic mutations of p53 in tumor tissue. Cancer Res. 61, 4398 – 4404. Leonard, T.B., Graichen, M.E., Popp, J.A., 1987. Dinitrotoluene isomer-specific hepatocarcinogenesis in F344 rats. J. Natl. Cancer Inst. 79, 1313 –1319.
327
Lewalter, J., Miksche, L., 1991. Empfehlungen zur arbeitsmedizinischen Pra¨ vention depositions- und dispositionsbedingter Arbeitsstoff-Beanspruchungen. 31. Jahrestagung der Deutschen Gesellschaft fu¨ r Arbeitsmedizin. Gentner, Stuttgart, pp. 135 – 139. Li, D.N., Seidel, A., Pritchard, M.P., Wolf, C.R., Friedberg, T., 2000. Polymorphisms in P450 CYP1B1 affect the conversion of estradiol to the potentially carcinogenic metabolite 4-hydroxyestradiol. Pharmacogenetics 10, 343 – 353. Maltoni, C., Lefemine, G., Cotti, G., Perino, G., 1988. Longterm carcinogenicity bioassays on trichloroethylene administered by inhalation to Sprague – Dawley rats and Swiss and B6C3F1 mice. Ann. N. Y. Acad. Sci. 534, 316 – 342. Reckwitz, T., Golka, K., Kempkes, M., Blaszkewicz, M., Bolt, H.M., Soekeland, H., Schulze, H., 1997. Berufliche und außerberufliche Risikofaktoren fu¨ r Harnblaenkarzinogene in einer hochindustrialisierten Region. Aktuel. Urol. 28, 105 – 108. Shimada, T., Hayes, C.L., Yamazaki, H., Amin, S., Hecht, S.S., Guengerich, F.P., Sutter, T.R., 1996. Activation of chemically diverse procarcinogens by human cytochrome P-450 1B1. Cancer Res. 56, 2979 – 2984. Thier, R., Bolt, H.M., 2001. The new era of toxicology. Trends Pharmacol. Sci., 11, 549 – 550. Thier, R., Wiebel, F.A., Schulz, T.G., Hinkel, A., Bru¨ ning, T., Bolt, H.M., 1997. Comparison of GST theta activity in liver and kidney of four species. Arch. Toxicol. (Suppl. 20), 471 – 474. Thier, R., Delbanco, E.H., Wiebel, F.A., Hallier, E., Bolt, H.M., 1998. Determination of glutathione transferase (GSTT1-1) activities in different tissues based on formation of radioactive metabolites using 35S-glutathione. Arch. Toxicol. 72, 811 – 815. Thier, R., Lewalter, J., Bolt, H.M., 2000. Species differences in acrylonitrile metabolism and toxicity between experimental animals and humans based on observations in human accidental poisonings. Arch. Toxicol. 74, 184 – 189. Thier, R., Lewalter, J., Selinski, S., Bolt, H.M., 2001. Re-evaluation of the effect of smoking on the methylation of N-terminal valine in haemoglobin. Arch. Toxicol. 75, 270 – 273. Vamvakas, S., Bru¨ ning, T., Thomasson, B., Lammert, M., Baumu¨ ller, A., Bolt, H.M., Dekant, W., Birner, G., Henschler, D., Ulm, K., 1998. Renal cell cancer correlated with occupational exposure to trichloroethylene. J. Cancer Res. Clin. Oncol. 124, 374 – 382. Weiss, N.L., 1996. Cancer in relation to occupational exposure to trichloroethylene. Occup. Environ. Med. 53, 1 – 5.
Genetic susceptibility to environmental toxicants: the interface between human and experimental studies in the development of new toxicological concepts Ricarda Thier a,b,*, Klaus Golka a, Thomas Bru¨ning a,c, Yon Ko d, Hermann M. Bolt a b
a Institut fu¨r Arbeitsphysiologie an der Uni6ersita¨t Dortmund (IfADo), Ardeystraße 67, D-44139 Dortmund, Germany Department of Physiology and Pharmacology, School of Biomedical Sciences, Uni6ersity of Queensland, St. Lucia, Queensland 4072, Australia c Berufsgenossenschaftliches Forschungsinstitut fu¨r Arbeitsmedizin, Bu¨rkle-de-la-Camp-Platz 1, D-44789 Bochum, Germany d Department of Internal Medicine (Medizinische Poliklinik), Uni6ersity of Bonn, 53111 Bonn, Germany
Abstract The growing knowledge of the genetic polymorphisms of enzymes metabolising xenobiotics in humans and their connections with individual susceptibility towards toxicants has created new and important interfaces between human epidemiology and experimental toxicology. The results of molecular epidemiological studies may provide new hypotheses and concepts, which call for experimental verification, and experimental concepts may obtain further proof by molecular epidemiological studies. If applied diligently, these possibilities may be combined to lead to new strategies of human-oriented toxicological research. This overview will present some outstanding examples for such strategies taken from the practically very important field of occupational toxicology. The main focus is placed on the effects of enzyme polymorphisms of the xenobiotic metabolism in association with the induction of bladder cancer and renal cell cancer after exposure to occupational chemicals. Also, smoking and induction of head and neck squamous cell cancer are considered. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Genetic susceptibility; Metabolism; Cancer
1. Introduction The growing knowledge on genetic polymorphisms of enzymes metabolising xenobiotics in humans and their connections with individual sus* Corresponding author. Tel.: + 61-7-3365-3222; fax: + 617-3365-1766. E-mail address: [email protected] (R. Thier).
ceptibility towards toxicants has created new and important interfaces between human epidemiology and experimental toxicology. On the one hand, the results of molecular epidemiological studies may provide new hypotheses and concepts, which call for experimental verification (or falsification), and on the other hand, experimental concepts may obtain further proof by molecular epidemiological studies. If applied diligently, these
0378-4274/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 4 2 7 4 ( 0 1 ) 0 0 5 1 5 - X
322
R. Thier et al. / Toxicology Letters 127 (2002) 321–327
possibilities may be combined to lead to new strategies of human-oriented toxicological research. A particularly interesting and very important field of application of such research strategies is occupational toxicology (Bolt, 1996; Thier et al., 2000). A basic and frequent problem of this field is the availability of exposed population cohorts of limited size only. Interplay of human and experimental studies, however, may nevertheless help to arrive at valid conclusions. This overview will present some outstanding examples for such strategies, based on work of our own group.
2. Occupational bladder cancer Since the classical study of Cartwright et al. (1982) the particular susceptibility of ‘slow acetylators’ for the carcinogenic effect of aromatic amines towards the human urothelium has been confirmed in many studies. In Germany, Lewalter and Miksche (1991) described a group of 331 persons having worked between 1951 and 1967 at a benzidine manufacturing plant in Leverkusen, Germany. The phenotypic ratio of slow (160 persons) to fast (171 persons) acetylators within the total cohort was close to 1. At the time of publication, 92 persons of the entire group were diseased with bladder cancer, among them 75 slow and only 17 fast acetylators. Between 1991 and 1993 an investigation of 196 patients with urothelial bladder cancer of the Department of Urology of the local hospital was performed, and the N-acetyltransferase NAT2 status was assessed by phenotyping and genotyping. There was no increased prevalence of slow acetylators in the whole group of patients compared to the general population, but there was a tendency towards higher representation in the patient group of slow acetylators with a specific working history at the local chemical industry (Golka et al., 1996). This suggested that the genetic trait (slow NAT2 acetylator) was of major importance only in a subgroup with specific occupational exposure and that definition of such subgroups is most important.
In a subsequent case-control study in the German Ruhr area, an area of former coal, iron and steel industries, 412 male urothelial bladder cancer patients were compared as to their occupational histories with 414 patients with prostatic hyperplasia as controls. The study resulted in a smoking-adjusted odds ratio of 2.54 (95% C.I.: 1.64–3.93) in former underground hard-coal miners, consistent with results of (the few) existing similar studies in coal mining regions (Golka et al., 1998). A causative reason for bladder cancer specific for coal miners was not known. However, relevant hints could be deduced from the distribution of enzyme polymorphisms in bladder cancer patients of this area, related to their occupation (Golka et al., 1997; Reckwitz et al., 1997): The ratio of slow versus rapid acetylators did not differ between diseased coal miners and diseased patients in general (in contrast to patients specifically exposed to coal tar). However, a tendency was noted towards a higher representation (84%) of the glutathione transferase GSTM1 deficient genotype than found in the general population of the area (54%; Fig. 1). Although this did not point to the involvement of aromatic amines, GSTM1 is involved in the detoxification of carcinogenic dihydrodiol intermediates of polycyclic aromatic hydrocarbons (PAH). This finding induced the working hypothesis that PAH might causally be involved in urothelial cancers, at least under defined occupational circumstances, and led to a series of experimental toxicological studies. First of all, it was of interest whether the urothelium was capable of metabolising precarcinogens to their ultimately effective genotoxic metabolites. For these investigations, cell culture model systems have been developed. As the levels of enzymes metabolising xenobiotics may be markedly altered in under cell culture conditions, only primary cell culture systems proved to be useful for this purpose (Fo¨ llmann et al., 2000). As an experimental system, porcine urinary bladder epithelial cells (PUBEC) had the further advantage that it was based on abattoir materials with unlimited availability. The cells were characterised according to morphologic, physiologic and biochemical parameters. They displayed a number of constitutive phase-I and
R. Thier et al. / Toxicology Letters 127 (2002) 321–327
phase-II enzyme activities, inter alia of CYP1A1 (EROD activity), prostaglandin H synthase, Nacetyltransferase and glutathione transferase (Fo¨ llmann et al., 2000). This was supplemented by genotoxic effects on the cells by carcinogens that require metabolic activation, and demonstrates that the urothelium can no longer be regarded as an inactive target of preformed reactive metabolites of carcinogens in the urine. This gives support to the view that PAH might be a group of carcinogens relevant for human urothelial cancer. The successful primary culturing of human urothelial cells (Do¨ rrenhaus et al., 2000) provides new possibilities to further test this hypothesis. The metabolisms of aromatic amines and aromatic nitro compounds are closely connected, and the same reactive intermediate (nitroso compounds) are involved. Based on findings on experimental carcinogenicity of nitrobenzene (Cattley et al., 1994) and of dinitrotoluene (Leonard et al., 1987), possible human carcinogenicity of the entire group of compounds is being discussed. Dinitrotoluene is of particular importance as soil contaminant in former areas of ammunition production and military facilities, e.g. in the east of Germany. In this context the observation of a cluster of 14 renal cell carcinomas and six urothelial carcinomas of the renal pelvis among a group of 183 formerly dinitrotoluene-exposed miners, in
323
the underground copper mining industry of the former German Democratic Republic, is noteworthy. Investigation of the frequency of some genetic variants revealed that all six persons with this working history and urothelial cancer of the renal pelvis were slow acetylators for NAT2 (Bru¨ ning et al., 1999a). This strengthens a causative role of dinitrotoluene and calls now for further experimental toxicological research in this direction (Bru¨ ning et al., 2001).
3. Head and neck cancer Biomarker studies are especially useful in the search of tobacco constituents responsible for human cancer manifestations (Thier et al., 2001). This could be exemplified in a recent study on head and neck squamous cell cancer (HNSCC) (Ko et al., 2001). Tobacco use is causally associated HNSCC. In a case-control study effects of genetic variants of the cytochrome P450 enzymes CYP1A1, CYP1B1, glutathione transferases GSTM1, GSTT1, and GSTP1 genes possibly modifying the risk of smoking-related HNSCC were investigated. Allelisms of the CYP1A1, GSTT1, GSTM1, and GSTT1 genes alone were not associated with an increased risk. By contrast, the CYP1B1 codon
Fig. 1. Distribution of NAT2 phenotype (n = 179) and GSTM1 genotype (n = 89) in urinary bladder cancer patients. Data according to Golka et al. (1997).
324
R. Thier et al. / Toxicology Letters 127 (2002) 321–327
432 polymorphism (CYP1B1*1: Val, CYP1B1*2: Leu) was found to be a putative susceptibility factor in smoking-related HNSCC. The frequency of the variant allele CYP1B1*2 was significantly higher (PB0.001) in the group of smoking cases when compared with smoking controls. Likewise, CYP1B1*2 was significantly (P B 0.001) more frequent in smoking cases compared to non-smoking cases. In combination with variants of the GSTT1 gene significant synergistic effects were observed. The impact of the variants of the CYP1B1 gene on HNSCC risk was also reflected by the strong association with the frequency of somatic mutations of the p53 gene. Smokers with the genotype CYP1B1*2/2 were 20 times more likely to show evidence of p53 mutations than were those with CYP1B1*1/1 genotype. Once more, combined genotype analysis of CYP1B1 and GSTM1 or GSTT1 revealed interactive effects on the occurrence of p53 gene mutations. In total, the results of the study (Ko et al., 2001) pointed to polymorphic variants of CYP1B1, relating significantly to the individual susceptibility of smokers to HNSCC. This was paralleled by earlier findings of an association between susceptibility to colorectal cancer and polymorphisms at residue 432 of CYP1B1 (Fritsche et al., 1999). In general, the CYP isoenzyme CYP1B1 plays a role in metabolic activation of a variety of classes of chemical carcinogens, e.g. arylamines, aromatic nitro compounds, and PAH (Shimada et al., 1996). Although the Val432Leu-polymorphism at this site was suggested to have a profound impact on the catalytic activity of the enzyme, this alteration had little effect on classical kinetic parameters of CYP1B1mediated reactions, such as the epoxidation of ( −)-trans-(7R,8R)-benzo(a)pyrene-7,8-dihydrodiol, the O-dealkylation of ethoxyresorufin and the 1%-hydroxylation of bufuranol. However, this specific polymorphism enhanced the Km of the formations of catechol oestrogens (2- and 4-hydroxy-oestradiol) from oestradiol by a factor of 4 (Li et al., 2000). In this situation, further experimental toxicological research is needed to establish the mechanisms by which the Val432Leu-polymorphism of CYP1B1 interferes with tobacco-specific carcino-
genic compounds. Such research should not be restricted to the classical PAH, but must also consider related nitro and amino compounds. This, in turn, will be invaluable for the design of further epidemiological studies on tobacco-induced HNSCC.
4. Renal cell cancer The discussion on renal carcinogenicity of trichloroethylene, in view of human risk assessment, provides a further outstanding example for the interplay of toxicology and epidemiology (Bru¨ ning and Bolt, 2000). Upon repetitive inhalation of trichloroethylene, renal cell tumours were experimentally increased in male rats, and an increased incidence of interstitial cell tumours of the testes was also reported (Maltoni et al., 1988). Studies on the metabolism of trichloroethylene in rodents and in humans have supported the role of bioactivation reactions for the development of tumours following exposure to trichloroethylene. Trichloroethylene is metabolised by two competing pathways, oxidation by cytochromes P450 (mainly CYP2E1) as the major route of metabolism, and conjugation with glutathione by glutathione transferases as a quantitatively minor pathway (Goeptar et al., 1995). Evidence for the existence of the glutathione-dependent pathway in humans has been obtained by the identification of N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine and Nacetyl-S-(2,2-dichlorovinyl)-L-cysteine in human urine after exposure to trichloroethylene (Bru¨ ning et al., 1998). Epidemiological cohort studies addressing the carcinogenicity of trichloroethylene with respect to the renal or urothelial target sites have been evaluated (Weiss, 1996). No clear evidence for an elevated renal or urinary tract cancer risk in trichloroethylene-exposed groups was visible in these exposed populations. However, a cohort study (Henschler et al., 1995) and a casecontrol study (Vamvakas et al., 1998) of workers having been exposed to unusually high levels of trichloroethylene in Germany supported the postulate that trichloroethylene has a nephrocarcinogenic effect in humans. For decades, companies in the study area had used trichloroethylene as main
R. Thier et al. / Toxicology Letters 127 (2002) 321–327
325
Table 1 Frequency of homozygous gene deletions of the human GSTT1 and GSTM1 genes in renal cell carcinoma (RCC) patients and healthy workers Genotype GSTT1 *1/1 or *1/2 *2/2 GSTM1 *1/1 or *1/2 2*/2
RCC patients TRI exposed n =57 (%)
Control group n = 139 (%)
Odds ratio
95% Cl
54 (95) 3 (5)
107 (77) 32 (23)
5.4
1.6–18.4
29 (51) 28 (49)
76 (55) 63 (45)
0.9
0.5–1.6
Both groups had a similar history of high and long-term exposure to trichloroethylene (TRI). Updated data set according to Bru¨ ning (1999).
degreasing agent without any precautionary measures. The results of both studies were discussed in the literature with reserve. Therefore, further investigations on patients with renal cell carcinoma and with histories of high trichloroethylene exposures were of particular relevance. Excretion of marker proteins in the urine pointed to the existence of toxic damage to the proximal renal tubules by trichloroethylene (Bru¨ ning et al., 1996). In order to investigate persistent nephrotoxic effects of high trichloroethylene exposures independent of the origination of renal cell cancer, a retrospective study was carried out on 39 workers exposed to high levels of trichloroethylene from 1956 to 1975. The control group comprised 46 persons from the same company who were categorised by their age. They were employed in the company’s administrative departments and had not been exposed to trichloroethylene or to other chemicals. The nephrotoxic effect of high trichloroethylene exposures was confirmed, as in the previous studies, using SDS-PAGE, with respect to an increased occurrence of tubular kidney damage among those exposed to trichloroethylene. Those exposed exhibited an increased excretion of the glutathione transferase isozyme GST alpha that is a specific marker for damage of the proximal tubules of the kidney. In contrast, excretions of the glutathione transferase enzyme GST pi, a marker for distaltubular damage, were in the same range for the exposed and the control group (Bru¨ ning et al., 1999b). The results of these biomarker investiga-
tions in diseased humans were consistent with the toxicological thesis of Goeptar et al. (1995) which had been derived on the basis of mechanistic considerations. It postulates that renal cell tumours are only induced by trichloroethylene if the substance causes toxic renal cell damage. Furthermore, 42 patients with renal cell carcinoma and an anamnesis of several years of exposure to trichloroethylene were examined with respect to the presence of the two polymorphic glutathione transferases GSTM1 and GSTT1. Forty-eight persons served as controls, who had similarly been exposed to trichloroethylene, but who to date had no kidney tumour (Bru¨ ning et al., 1997). A shift was observed among those having renal carcinomas after exposure to trichloroethylene in the direction of the presence of either gene GSTM1 but in particular GSTT1 (Table 1). The molecular epidemiological findings, in general, corresponded with the hypothesis that the occurrence of trichloroethylene-induced renal cell carcinomas involves metabolism of trichloroethylene via the glutathione transferase metabolic pathway. It also indicates that the population exhibits considerable differences in susceptibility to the nephrocarcinogenic effect of trichloroethylene on the basis of a genetically determined enzyme supply. In this respect, it is interesting that GSTT1 is expressed in the human kidney (Thier et al., 1997, 1998; Delbanco et al., 2001), but not the cytochrome P450 isozyme CYP2E1, which is connected with the oxidative metabolism of trichloroethylene (Amet et al., 1997).
326
R. Thier et al. / Toxicology Letters 127 (2002) 321–327
Hence, the compilation and synopsis of toxicological, epidemiological and molecular epidemiological studies on trichloroethylene, in total, provide a new type of integrated argumentation for the nephrotoxicity and nephrocarcinogenicity of this particular solvent (Bru¨ ning and Bolt, 2000).
5. Conclusions Current processes leading to a new convergence of disciplines are evident in toxicology. Traditionally, the central and most integral part of toxicology is scientific establishment of toxic effects by animal experimentation, development of toxicological concepts on the basis of physiological and molecular mechanisms, and extrapolation of risks from the animal experiment to humans with the help of knowledge in both toxicodynamics and toxicokinetics. Now, entirely new links appear between epidemiological research in humans and experimental research in toxicology. Thereby, new and more efficient research strategies connect epidemiological and experimental research. Being aware of the growing realisation of the decisive importance of the genetic background of exposed subjects and identification of increasing numbers of respective genetic traits, the term of a new era of toxicology has been coined (Thier and Bolt, 2001). This not only describes new research fields, but also new ways to broaden our knowledge!
Acknowledgements The authors thank the Deutsche Forschungsgemeinschaft, the Deutscher Akademischer Austauschdienst, and the U.S. Environmental Protection Agency for valuable financial support of the studies underlying to this review.
References Amet, Y., Berthon, F., Fournier, G., Dre´ ano, Y., Bardou, L., Cle´ des, J., Me´ nez, J.F., 1997. Cytochrome P450 4A and
2E1 expression in human kidney microsomes. Biochem. Pharmacol. 53, 765 – 771. Bolt, H.M., 1996. Genetic predisposition in occupational toxicology. Arch. Toxicol. (Suppl. 18), 362 – 366. Bru¨ ning, T., 1999. Zur Kanzerogenita¨ t des Lo¨ semittels Trichlorethylen: Neue Ansa¨ tze zur arbeitsmedizinischen Pra¨ vention. Habilitationsschrift. Medical Faculty, University of Bonn. Bru¨ ning, T., Bolt, H.M., 2000. Renal toxicity and carcinogenicity of trichloroethylene: key results, mechanisms, and controversies. Crit. Rev. Toxicol. 30, 253 – 285. Bru¨ ning, T., Golka, K., Makropoulos, V., Bolt, H.M., 1996. Pre-existence of chronic tubular damage in cases of renal cell cancer after long and high exposure to trichloroethylene. Arch. Toxicol. 70, 259 – 260. Bru¨ ning, T., Lammert, M., Kempkes, M., Thier, R., Golka, K., Bolt, H.M., 1997. Influence of polymorphisms of GSTM1 and GSTT1 for risk renal cell cancer in workers with a long term and high occupational exposure to trichloroethylene. Arch. Toxicol. 71, 596 – 599. Bru¨ ning, T., Vamvakas, S., Makropoulos, V., Birner, G., 1998. Acute intoxication with trichloroethylene: clinical symptoms, toxicokinetics, metabolism and development of biochemical parameters for renal damage. Toxicol. Sci. 41, 157 – 165. Bru¨ ning, T., Chronz, C., Thier, R., Havelka, J., Ko, Y., Bolt, H.M., 1999a. Occurrence of urinary tract tumors in miners exposed to dinitrotoluene. J. Occup. Environ. Med. 41, 144 – 149. Bru¨ ning, T., Sundberg, A.G.M., Birner, G., Lammert, M., Bolt, H.M., Appelkvist, E.E., Nilsson, R., Dallner, G., 1999b. Glutathione transferase alpha as a marker for tubular damage after trichloroethylene exposure. Arch. Toxicol. 73, 246 – 254. Bru¨ ning, T., Thier, R., Mann, H., Melzer, H., Bro¨ de, P., Dallner, G., Bolt, H.M., 2001. Pathological excretion patterns of urinary proteins in miners highly exposed to dinitrotoluene. J. Occup. Environ. Med. 43, 610 – 615. Cartwright, R., Ahmad, R.A., Barham-Hall, D., Glashan, R.W., Rogers, H.J., Higgins, E., 1982. Role of N-acetyltransferase phenotypes in bladder carcinogenesis: a pharmacogenetic epidemiological approach to bladder cancer. Lancet 2, 842 – 845. Cattley, R.C., Everitt, J.I., Gross, E.A., Moss, O.R., Hamm, T.E., Popp, J.A., 1994. Carcinogenicity and toxicity of inhaled nitrobenzene in B6C3F1 mice and CD rats. Fundam. Appl. Toxicol. 22, 328 – 340. Delbanco, E.H., Bolt, H.M., Huber, W.W., Beken, S., Geller, F., Philippou, S., Brands, F.H., Bru¨ ning, T., Thier, R., 2001. Glutathione transferase activities in renal carcinomas and adjacent normal renal tissues: factors influencing renal carcinogenesis induced by xenobiotics. Arch. Toxicol. 74, 688 – 694. Do¨ rrenhaus, A., Mu¨ ller, J.I.F., Golka, K., Jedrusik, P., Schulze, H., Fo¨ llmann, W., 2000. Cultures of exfoliated epithelial cells from different locations of the human urinary tract and the renal tubular system. Arch. Toxicol. 74, 618 – 626.
R. Thier et al. / Toxicology Letters 127 (2002) 321–327 Fo¨ llmann, W., Guhe, C., Weber, S., Birkner, S., Ma¨ hler, S., 2000. Cultured porcine urinary bladder epithelial cells as a screening model for genotoxic effects of aromatic amines: characterisation and application of the cell culture model. ATLA 28, 833 – 854. Fritsche, E., Bru¨ ning, T., Jonkmanns, C., Ko, Y., Bolt, H.M., Abel, J., 1999. Detection of cytochrome P450 1B1 Bfr I polymorphism: genotype distribution in healthy German individuals and in patients with colorectal carcinoma. Pharmacogenetics 9, 405 –408. Goeptar, A.R., Commandeur, J.N.M., van Ommen, B., van Bladeren, P.J., Vermeulen, N.P.E., 1995. Metabolism and kinetics of trichloroethylene in relation to carcinogenicity. Relevance of the mercapturic acid pathway. Chem. Res. Toxicol. 8, 3 – 21. Golka, K., Prior, V., Blaskewicz, M., Cascorbi, M., Scho¨ ps, W., Kierfeld, G., Roots, I., Bolt, H.M., 1996. Occupational history and genetic N-acetyltransferase polymorphism in urothelial cancer patients of Leverkusen, Germany. Scand. J. Work Environ. Health 22, 332 – 338. Golka, K., Reckwitz, T., Kempkes, M., Cascorbi, I., Blaszkewicz, M., Reich, S., Roots, I., Soekeland, J., Schulze, H., Bolt, H.M., 1997. N-Acetyltransferase 2 (NAT-2) and glutathione S-transferase m (GSTM1) in bladder cancer patients in a highly industrialized area. Int. J. Occup. Environ. Health 3, 105 –110. Golka, K., Bandel, T., Schlaefke, S., Reich, S.E., Reckwitz, T., Urfer, W., Bremicker, K.D., Neugebauer, W., Soekeland, J., Bolt, H.M., 1998. Urothelial cancer of the bladder in an area of former coal, iron, and steel industries in Germany: a case-control study. Int. J. Occup. Environ. Health 4, 79 – 84. Henschler, D., Vamvakas, S., Lammert, M., Dekant, W., Kraus, B., Bru¨ ning, T., Ulm, K., 1995. Increased incidence of renal cell tumors in a cohort of cardbord workers exposed to trichloroethylene. Arch. Toxicol. 69, 291 –299. Ko, Y., Abel, J., Harth, V., Bro¨ de, P., Antony, C., Donat, S., Fischer, H.P., Ortiz-Pallardo, M.E., Thier, R., Sachinidis, A., Vetter, H., Bolt, H.M., Herberhold, C., Bru¨ ning, T., 2001. Association of CYP1B1 codon 432 mutant allele in head and neck squamous cell cancer is reflected by somatic mutations of p53 in tumor tissue. Cancer Res. 61, 4398 – 4404. Leonard, T.B., Graichen, M.E., Popp, J.A., 1987. Dinitrotoluene isomer-specific hepatocarcinogenesis in F344 rats. J. Natl. Cancer Inst. 79, 1313 –1319.
327
Lewalter, J., Miksche, L., 1991. Empfehlungen zur arbeitsmedizinischen Pra¨ vention depositions- und dispositionsbedingter Arbeitsstoff-Beanspruchungen. 31. Jahrestagung der Deutschen Gesellschaft fu¨ r Arbeitsmedizin. Gentner, Stuttgart, pp. 135 – 139. Li, D.N., Seidel, A., Pritchard, M.P., Wolf, C.R., Friedberg, T., 2000. Polymorphisms in P450 CYP1B1 affect the conversion of estradiol to the potentially carcinogenic metabolite 4-hydroxyestradiol. Pharmacogenetics 10, 343 – 353. Maltoni, C., Lefemine, G., Cotti, G., Perino, G., 1988. Longterm carcinogenicity bioassays on trichloroethylene administered by inhalation to Sprague – Dawley rats and Swiss and B6C3F1 mice. Ann. N. Y. Acad. Sci. 534, 316 – 342. Reckwitz, T., Golka, K., Kempkes, M., Blaszkewicz, M., Bolt, H.M., Soekeland, H., Schulze, H., 1997. Berufliche und außerberufliche Risikofaktoren fu¨ r Harnblaenkarzinogene in einer hochindustrialisierten Region. Aktuel. Urol. 28, 105 – 108. Shimada, T., Hayes, C.L., Yamazaki, H., Amin, S., Hecht, S.S., Guengerich, F.P., Sutter, T.R., 1996. Activation of chemically diverse procarcinogens by human cytochrome P-450 1B1. Cancer Res. 56, 2979 – 2984. Thier, R., Bolt, H.M., 2001. The new era of toxicology. Trends Pharmacol. Sci., 11, 549 – 550. Thier, R., Wiebel, F.A., Schulz, T.G., Hinkel, A., Bru¨ ning, T., Bolt, H.M., 1997. Comparison of GST theta activity in liver and kidney of four species. Arch. Toxicol. (Suppl. 20), 471 – 474. Thier, R., Delbanco, E.H., Wiebel, F.A., Hallier, E., Bolt, H.M., 1998. Determination of glutathione transferase (GSTT1-1) activities in different tissues based on formation of radioactive metabolites using 35S-glutathione. Arch. Toxicol. 72, 811 – 815. Thier, R., Lewalter, J., Bolt, H.M., 2000. Species differences in acrylonitrile metabolism and toxicity between experimental animals and humans based on observations in human accidental poisonings. Arch. Toxicol. 74, 184 – 189. Thier, R., Lewalter, J., Selinski, S., Bolt, H.M., 2001. Re-evaluation of the effect of smoking on the methylation of N-terminal valine in haemoglobin. Arch. Toxicol. 75, 270 – 273. Vamvakas, S., Bru¨ ning, T., Thomasson, B., Lammert, M., Baumu¨ ller, A., Bolt, H.M., Dekant, W., Birner, G., Henschler, D., Ulm, K., 1998. Renal cell cancer correlated with occupational exposure to trichloroethylene. J. Cancer Res. Clin. Oncol. 124, 374 – 382. Weiss, N.L., 1996. Cancer in relation to occupational exposure to trichloroethylene. Occup. Environ. Med. 53, 1 – 5.