Algorithm for the Automated Evaluation of NAT2 Genotypes.

N-Acetyltransferase 2 (NAT2) genotyping by PCR and RFLP-based methods provides information on seven single nucleotide polymorphisms (SNPs) without deriving the chromosomal phase (haplotype). So genotyping results must be processed to get all possible NAT2 haplotype (or allele) combinations. Here we describe the procedure for genotyping and present a program based on Microsoft® Access® which automatically generates all possible haplotype pairs for a given unphased NAT2 genotype. NAT2 haplotypes are important to predict the NAT2 phenotype.

Removal of micropollutants through a biological wastewater treatment plant in a subtropical climate, Queensland-Australia.

BACKGROUND: Municipal wastewaters contain a multitude of organic compounds derived from domestic and industrial sources including active components of pharmaceutical and personal care products and compounds used in agriculture, such as pesticides, or food processing such as artificial sweeteners often referred to as micropollutants. Some of these compounds or their degradation products may have detrimental effects on the environment, wildlife and humans. Acesuflame is one of the most popular artificial sweeteners to date used in foodstuffs. The main objectives of this descriptive study were to evaluate the presence of micropollutants in both the influent and effluent of a large-scale conventional biological wastewater treatment plant (WWTP) in South-East Queensland receiving wastewater from households, hospitals and various industries. METHODS: Based on USEPA Method 1694: Filtered samples were spiked with mass-labelled chemical standards and then analysed for the micropollutants using liquid chromatography coupled with tandem mass spectrometry. RESULTS: The presence of thirty-eight compounds were detected in the wastewater influent to the treatment plant while nine of the compounds in the categories of analgesic, anti-inflammatory, alkaloid and lipid/cholesterol lowering drugs were undetectable (100 % removed) in the effluent. They were: Analgesic: Paracetamol, Salicylic acid, Oxycodone; Anti-inflammatory: Naproxen + ve, Atorvastatin, Indomethacin, Naproxen; Alkaloid: Caffeine; Lipid/cholesterol lowering: Gemfibrozol. CONCLUSIONS: The study results revealed that the micropollutants removal through this biological treatment process was similar to previous research reported from other countries including Europe the Americas and Asia, except for acesulfame, a highly persistent artificial sweetener. Surprisingly, acesulfame was diminished to a much greater extent (>90 %) than previously reported research for this type of WWTPs (45-65 %) that only include physical removal of objects and solids and a biodegradation step.

Head and neck squamous-cell cancer and its association with polymorphic enzymes of xenobiotic metabolism and repair.

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.

Combining passive sampling and toxicity testing for evaluation of mixtures of polar organic chemicals in sewage treatment plant effluent.

Effluent from sewage treatment plants has been associated with a range of pollutant effects. Depending on the influent composition and treatment processes the effluent may contain a myriad of different chemicals which makes monitoring very complex. In this study we aimed to monitor relatively polar organic pollutant mixtures using a combination of passive sampling techniques and a set of biochemistry based assays covering acute bacterial toxicity (Microtox), phytotoxicity (Max-I-PAM assay) and genotoxicity (umuC assay). The study showed that all of the assays were able to detect effects in the samples and allowed a comparison of the two plants as well as a comparison between the two sampling periods. Distinct improvements in water quality were observed in one of the plants as result of an upgrade to a UV disinfection system, which improved from 24x sample enrichment required to induce a 50% response in the Microtox assay to 84x, from 30x sample enrichment to induce a 50% reduction in photosynthetic yield to 125x, and the genotoxicity observed in the first sampling period was eliminated. Thus we propose that biochemical assay techniques in combination with time integrated passive sampling can substantially contribute to the monitoring of polar organic toxicants in STP effluents.

STAR: predicting recombination sites from amino acid sequence.

BACKGROUND: Designing novel proteins with site-directed recombination has enormous prospects. By locating effective recombination sites for swapping sequence parts, the probability that hybrid sequences have the desired properties is increased dramatically. The prohibitive requirements for applying current tools led us to investigate machine learning to assist in finding useful recombination sites from amino acid sequence alone. RESULTS: We present STAR, Site Targeted Amino acid Recombination predictor, which produces a score indicating the structural disruption caused by recombination, for each position in an amino acid sequence. Example predictions contrasted with those of alternative tools, illustrate STAR'S utility to assist in determining useful recombination sites. Overall, the correlation coefficient between the output of the experimentally validated protein design algorithm SCHEMA and the prediction of STAR is very high (0.89). CONCLUSION: STAR allows the user to explore useful recombination sites in amino acid sequences with unknown structure and unknown evolutionary origin. The predictor service is available from http://pprowler.itee.uq.edu.au/star.

Biological monitoring and Biological Limit Values (BLV): the strategy of the European Union.

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.

Genotoxicity of inorganic lead salts and disturbance of microtubule function.

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.

Disturbed microtubule function and induction of micronuclei by chelate complexes of mercury(II).

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.

Genotoxicity of inorganic mercury salts based on disturbed microtubule function.

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.

Chromosomal genotoxicity of nitrobenzene and benzonitrile.

In order to investigate the chromosomal genotoxicity of nitrobenzene and benzonitrile, we studied the induction of micronuclei (MN) by these test compounds in V79 cells, as well as effects on the formation and stability of microtubules and on motor protein functions. No cytotoxicity was seen in V79 cell cultures in terms of Neutral red uptake after 18 h treatment with up to 1 mM nitrobenzene or 1 mM benzonitrile. Subsequently, a concentration range up to 100 micro M was used in the experiments on induction of MN. Both test compounds exhibit a weak, but definitely positive test result compared to the solvent (DMSO) control. Minimal effect concentrations of nitrobenzene and benzonitrile appeared as low as 0.01 micro M, and no-effect-concentrations were between 0.001 and 0.005 micro M. Clearly enhanced MN rates were found at 0.1 micro M and higher. Both, nitrobenzene and benzonitrile, induced mostly kinetochor (CREST)-positive micronuclei, thus characterising the chromosomal effects as aneugenic. In cell-free assays, a slight effect on tubulin assembly was observed at 1 mM nitrobenzene without addition of DMSO. Higher concentrations (5 mM) led to secondary effects. In presence of 1% DMSO, nitrobenzene exerted no detectable effect on tubulin assembly up to the solubility limit in water of about 15 mM. For benzonitrile in presence of DMSO, a clear dose-response of inhibition of tubulin assembly at 37 degrees C was seen above the no-effect-concentration of 2 mM, with an IC(50) of 13 mM and protein denaturation starting above a level of about 20 mM. The nature of the effects of nitrobenzene and benzonitrile on the association of tubulin to form microtubules was confirmed by electron microscopy. Treatment by either 5 mM nitrobenzene or 13 mM benzonitrile plus 1% DMSO left the microtubular structure intact whereas 5 mM nitrobenzene, in absence of DMSO, led to irregular cluster formations. The experiments demonstrate that both nitrobenzene and benzonitrile, in millimolar concentration ranges, may lead to interference with tubulin assembly in a cell-free system. The functionality of the tubulin-kinesin motor protein system was assessed using the microtubule gliding assay. Nitrobenzene affected the gliding velocity in a concentration-dependent manner, starting at about 7.5 micro M and reaching complete inhibition of motility at 30 micro M, whereas benzonitrile up to 200 micro M did not affect the kinesin-driven gliding velocity. The micronucleus assay data demonstrate a chromosomal endpoint of genotoxicity of nitrobenzene and benzonitrile. Aneugenic effects of both compounds occur at remarkably low concentrations, with lowest-effect-concentrations being 0.1 micro M. This points to the relevance of interactions with the cellular spindle apparatus.

Markers of genetic susceptibility in human environmental hygiene and toxicology: the role of selected CYP, NAT and GST genes.

Inherited genetic traits co-determine the susceptibility of an individual to a toxic chemical. Special emphasis has been put on individual responses to environmental and industrial carcinogens, but other chronic diseases are of increasing interest. Polymorphisms of relevant xenobiotic metabolising enzymes may be used as toxicological susceptibility markers. A growing number of genes encoding enzymes involved in biotransformation of toxicants and in cellular defence against toxicant-induced damage to the cells has been identified and cloned, leading to increased knowledge of allelic variants of genes and genetic defects that may result in a differential susceptibility toward environmental toxicants. "Low penetrating" polymorphisms in metabolism genes tend to be much more common in the population than allelic variants of "high penetrating" cancer genes, and are therefore of considerable importance from a public health point of view. Positive associations between cancer and CYP1A1 alleles, in particular the *2C I462V allele, were found for tissues following the aerodigestive tract. Again, in most cases, the effect of the variant CYP1A1 allele becomes apparent or clearer in connection with the GSTM1 null allele. The CYP1B1 codon 432 polymorphism (CYP1B1*3) has been identified as a susceptibility factor in smoking-related head-and-neck squameous cell cancer. The impact of this polymorphic variant of CYP1B1 on cancer risk was also reflected by an association with the frequency of somatic mutations of the p53 gene. Combined genotype analysis of CYP1B1 and the glutathione transferases GSTM1 or GSTT1 has also pointed to interactive effects. Of particular interest for the industrial and environmental field is the isozyme CYP2E1. Several genotypes of this isozyme have been characterised which seem to be associated with different levels of expression of enzyme activity. The acetylator status for NAT2 can be determined by genotyping or by phenotyping. In the pathogenesis of human bladder cancer due to occupational exposure to "classical" aromatic amines (benzidine, 4-aminodiphenyl, 1-naphthylamine) acetylation by NAT2 is regarded as a detoxication step. Interestingly, the underlying European findings of a higher susceptibility of slow acetylators towards aromatic amines are in contrast to findings in Chinese workers occupationally exposed to aromatic amines which points to different mechanisms of susceptibility between European and Chinese populations. Regarding human bladder cancer, the hypothesis has been put forward that genetic polymorphism of GSTM1 might be linked with the occurrence of this tumour type. This supports the hypothesis that exposure to PAH might causally be involved in urothelial cancers. The human polymorphic GST catalysing conjugation of halomethanes, dihalomethanes, ethylene oxide and a number of other industrial compounds could be characterised as a class theta enzyme (GSTT1) by means of molecular biology. "Conjugator" and "non-conjugator" phenotypes are coincident with the presence and absence of the GSTT1 gene. There are wide variations in the frequencies of GSTT1 deletion (GSTT1*0/0) among different ethnicities. Human phenotyping is facilitated by the GST activity towards methyl bromide or ethylene oxide in erythrocytes which is representative of the metabolic GSTT1 competence of the entire organism. Inter-individual variations in xenobiotic metabolism capacities may be due to polymorphisms of the genes coding for the enzymes themselves or of the genes coding for the receptors or transcription factors which regulate the expression of the enzymes. Also, polymorphisms in several regions of genes may cause altered ligand affinity, transactivation activity or expression levels of the receptor subsequently influencing the expression of the downstream target genes. Studies of individual susceptibility to toxicants and gene-environment interaction are now emerging as an important component of molecular epidemiology.

The cytochrome P-450 isoenzyme CYP2E1 in the biological processing of industrial chemicals: consequences for occupational and environmental medicine.

The importance of the isoform CYP2E1 of the human cytochrome P-450 superfamily of enzymes for occupational and environmental medicine is derived from its unique substrate spectrum that includes a number of highly important high-production chemicals, such as aliphatic and aromatic hydrocarbons, solvents and industrial monomers (i.a. alkanes, alkenes, aromatic and halogenated hydrocarbons). Many polymorphic genes, such as CYP2E1, show considerable differences in allelic distribution between different human populations. The polymorphic nature of the human CYP2E1 gene is significant for inter-individual differences in toxicity of its substrates. Since the substrate spectrum of CYP2E1 includes many compounds of basic relevance to industrial toxicology, a rationale for metabolic interactions of different CYP2E1 substrates is provided. In-depth research into the inter-individual phenotypic differences of human CYP2E1 enzyme activities was enabled by the recognition that the 6-hydroxylation of the drug chlorzoxazone is mediated by CYP2E1. Studies on CYP2E1 phenotyping have pointed to inter-individual variations in enzyme activities. There are consistent ethnic differences in CYP2E1 enzyme expression, mostly demonstrated between European and Japanese populations, which point to a major impact of genetic factors. The most frequently studied genetic polymorphisms are the restriction fragment length polymorphisms PstI/ RsaI (mutant allele: CYP2E1*5B) located in the 5'-flanking region of the gene, as well as the DraI polymorphism (mutant allele: CYP2E1*6) located in intron 6. These polymorphisms are partly related, as they form the common allele designated CYP2E1*5A. Striking inter-ethnic differences between Europeans and Asians appear with respect to the frequencies of the CYP2E1*5A allele (only approximately 5% of Europeans are heterozygous, but 37% of Asians are, whilst 6% of Asians are homozygous). Available studies indicate a wide variation in human CYP2E1 expression, which are very likely based on complex gene-environment interactions. Major inter-ethnic differences are apparent on the genotyping and the phenotyping levels. Selected cases are presented where inter-ethnic variations of CYP2E1 may provide likely explanations for unexplained findings concerning industrial chemicals that are CYP2E1 substrates. Possible consequences of differential inter-individual and inter-ethnic susceptibilities are related to individual expressions of clinical symptoms of chemical toxicity, to results of biological monitoring of exposed workers, and to the interpretation of results of epidemiological or molecular-epidemiological studies.

Nephrotoxicity and nephrocarcinogenicity of dinitrotoluene: new aspects to be considered.

Technical dinitrotoluene (DNT) is a mixture of 2,4- and 2,6-DNT. In humans, industrial or environmental exposure can occur orally, by inhalation, or by skin contact. The classification of DNT as an 'animal carcinogen' is based on the formation of malignant tumors in kidneys, liver, and mammary glands of rats and mice. Clear signs of toxic nephropathy were found in rats dosed with DNT, and the concept was derived of an interrelation between renal toxicity and carcinogenicity. Recent data point to the carcinogenicity of DNT on the urinary tract of exposed humans. Between 1984 and 1997, 6 cases of urothelial cancer and 14 cases of renal cell cancer were diagnosed in a group of 500 underground mining workers in the copper mining industry of the former GDR and having high exposures to explosives containing technical DNT. The incidences of both urothelial and renal cell tumors in this group were 4.5 and 14.3 times higher, respectively, than anticipated on the basis of the cancer registers of the GDR. The genotyping of all identified tumor patients for the polymorphic enzymes NAT2, GSTM1, and GSTT1 identified the urothelial tumor cases as exclusively 'slow acetylates'. A group of 161 miners highly exposed to DNT was investigated for signs of subclinical renal damage. The exposures were categorized semi-quantitatively into 'low', 'medium', 'high', and 'very high'. A straight dose-dependence of the excretion of urinary biomarker proteins with the ranking of exposure was seen. Biomarker excretion (alpha1-microglobulin, glutathione S-transferases alpha and pi) indicated that DNT-induced damage was directed toward the tubular system. New data on DNT-exposed humans appear consistent with the concept of cancer initiation by DNT isomers and the subsequent promotion of renal carcinogenesis by selective damage to the proximal tubule. The differential pathways of metabolic activation of DNT appear to apply to the proximal tubule of the kidney and to the urothelium of the renal pelvis and lower urinary tract as target tissues of carcinogenicity.

Melatonin synthesis: a possible indicator of intolerance to shiftwork.

HYPOTHESIS: Melatonin synthesis, which is directly controlled by the central circadian pacemaker indicates the circadian phase better than rectal temperature. METHODS: Thirty four men (16-32 years, 7 morning, 13 neither, 14 evening types) performed a constant routine (24-26-hr bedrest, < 30 lux, 18-20 degrees C, hourly isocaloric diet). Salivary melatonin level was determined hourly and rectal temperature was continuously recorded. RESULTS: The nadir of rectal temperature occurred 1.5 hr (P = 0.017), the onset of melatonin synthesis 3 hr earlier (P < 0.0001) in morning than in evening types. Morningness was not related to the quantitative but significantly to the temporal parameters, closer to those of melatonin than of rectal temperature. CONCLUSIONS: The melatonin onset is a more reliable indicator of the diurnal type than the nadir of rectal temperature. As morningness has been associated with intolerance to shiftwork, melatonin profiling provides a suitable basis for the establishment of directed preventive measures.

Cytochrome P450 1B1, a new keystone in gene-environment interactions related to human head and neck cancer?

Alcohol consumption and tobacco smoking are major causes of head and neck cancers, and regional differences point to the importance of research into gene-environment interactions. Much interest has been focused on polymorphisms of CYP1A1 and of GSTM1 and GSTT1, but a number of studies have not demonstrated significant effects. This has mostly been ascribed to small sample sizes. In general, the impact of polymorphisms of metabolic enzymes appears inconsistent, with some reports of weak-to-moderate associations, and with others of no elevation of risks. The classical cytochrome P450 isoenzyme considered for metabolic activation of polycyclic aromatic hydrocarbons (PAH) is CYP1A1. A new member of the CYP1 family, CYP1B1, was cloned in 1994, currently representing the only member of the CYP1B subfamily. A number of single nucleotide polymorphisms of the CYP1B1 gene have been reported. The amino acid substitutions Val432Leu ( CYP1B1*3) and Asn453Ser ( CYP1B1*4), located in the heme binding domain of CYP1B1, appear as likely candidates to be linked with biological effects. CYP1B1 activates a wide range of PAH, aromatic and heterocyclic amines. Very recently, the CYP1B1 codon 432 polymorphism ( CYP1B1*3) has been identified as a susceptibility factor in smoking-related head-and-neck squamous cell cancer. The impact of this polymorphic variant of CYP1B1 on cancer risk was also reflected by an association with the frequency of somatic mutations of the p53 gene. Combined genotype analysis of CYP1B1 and the glutathione transferases GSTM1 or GSTT1 has pointed to interactive effects. This provides new molecular evidence that tobacco smoke-specific compounds relevant to head and neck carcinogenesis are metabolically activated through CYP1B1 and is consistent with a major pathogenetic relevance of PAH as ingredients of tobacco smoke.

Genetic susceptibility to environmental toxicants: the interface between human and experimental studies in the development of new toxicological concepts.

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.

Enhancement of the chemoprotective enzymes glucuronosyl transferase and glutathione transferase in specific organs of the rat by the coffee components kahweol and cafestol.

The coffee components kahweol and cafestol (K/C) have been reported to protect the colon and other organs of the rat against the formation of DNA adducts by 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP) and aflatoxin B1. PhIP is a cooked-food mutagen to which significant human exposure and a role in colon cancer etiology are attributed, and, interestingly, such cancers appear to develop at a lower rate in consumers of coffees with high amounts of K/C. Earlier studies in rodent liver have shown that a key role in the chemopreventive effect of K/C is likely to be due to the potential of these compounds to induce the detoxification of xenobiotics by glutathione transferase (GST) and to enhance the synthesis of the corresponding co-factor glutathione. However, mutagens like PhIP may also be detoxified by UDP-glucuronosyl transferase (UDPGT) for which data are lacking regarding a potential effect of K/C. Therefore, in the present study, we investigated the effect of K/C on UDPGT and, concomitantly, we studied overall GST and the pattern of individual GST classes, particularly GST-theta;, which was not included in earlier experiments. In addition, we analyzed the organ-dependence of these potentially chemopreventive effects. K/C was fed to male F344 rats at 0.122% in the chow for 10 days. Enzyme activities in liver, kidney, lung, colon, salivary gland, pancreas, testis, heart and spleen were quantified using five characteristic substrates and the hepatic protein pattern of GST classes alpha, mu, and pi was studied with affinity chromatography/HPLC. Our study showed that K/C is not only capable of increasing overall GST and GST classes alpha, mu, and pi but also of enhancing UDGPT and GST-theta. All investigated K/C effects were strongest in liver and kidney, and some response was seen in lung and colon but none in the other organs. In summary, our results show that K/C treatment leads to a wide spectrum of increases in phase II detoxification enzymes. Notably, these effects occurred preferentially in the well perfused organs liver and kidney, which may thus not only contribute to local protection but also to anti-carcinogenesis in distant, less stimulated organs such as the colon.

Possible impact of human CYP2E1 polymorphisms on the metabolism of acrylonitrile.

Case reports of human accidental poisonings point to significant individual differences in human acrylonitrile metabolism and toxicity. A cohort of 59 persons with industrial handling of low levels of acrylonitrile has repetitively been studied from 1994 through 1999 as part of a medical surveillance programme. The analyses included adduct determinations of N-terminal N-(cyanoethyl)valine in haemoglobin and genotypings of the following cytochrome P-450 2E1 (CYP2E1) polymorphisms: G-1259C and C-1019T (two subjects heterozygous), A-316G (three subjects heterozygous), T-297A (15 subjects heterozygous), G-35T (eight subjects heterozygous), G4804A (two subjects heterozygous), T7668A (six subjects heterozygous). N-(Cyanoethyl)valine adduct levels were, if any, only slightly influenced by smoking and mainly determined by the external acrylonitrile exposures. The individual means and medians of N-(cyanoethyl)valine levels over the entire observation period were compared with the CYP2E1 variants (Wilcoxon rank sum test). No influences of the investigated CYP2E1 polymorphisms on the N-(cyanoethyl)valine levels appeared at the 5% level. However, there was a trend, at a level of P approximately 0.1, pointing to higher acrylonitrile-specific adduct levels in persons with the A-316G mutation. Higher adduct levels would be compatible with a slower CYP2E1-mediated metabolism of acrylonitrile and with lower extents of toxification to cyanide.