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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.

Although tobacco smoke has been established as a main risk factor in the development of head and neck squamous cell cancer (HNSCC), genetic polymorphisms of xenobiotic metabolizing enzymes are supposed to modulate an individual's susceptibility to smoking-related HNSCC. N-acetyltransferase (NAT) 1 gene is known to be polymorphic and its protein product is implicated in the activation and detoxification of carcinogens, such as aromatic amines, present in tobacco smoke. We developed a rapid and reproducible LightCycler-assisted real-time polymerase chain reaction (PCR) for NAT1 genotyping, which allowed the parallel differentiation of NAT1*3, *4, *10 and *11 alleles and separately of NAT1*14 and *17 alleles within 60 min without the need for further post-PCR processing. In order to investigate the role of the NAT1 gene polymorphism as a risk-modifying factor in HNSCC, we tested for the presence of NAT1*3, *4, *10, *11, *14 and *17 alleles in a case-control study of 291 HNSCC patients and 300 healthy controls of Caucasian origin. Our findings suggest that in Caucasians, the risk of HNSCC is not associated with NAT1 polymorphism. The overall distribution of NAT1 allele frequencies was not significantly different among cases and controls. The presence of the fast acetylator NAT1*10 and NAT1*11 alleles did not significantly increase the risk of HNSCC and no modifying effect of NAT1*10 was observed among smokers. This new approach in NAT1 genotyping substantially increases throughput of sample analysis and, therefore, enhances opportunities to study NAT1 as a risk factor in different cancers in large-scale studies.

Single nucleotide polymorphisms, such as the cytochrome P450 1A1 (CYP1A1) A4889G and T6235C variants, have been reported to be associated with an increased cancer risk. In order to study their role in molecular-epidemiological studies, we developed a single-step procedure for genotyping these two CYP1A1 polymorphisms using real-time polymerase chain reaction (PCR) and subsequent melting curve analysis. Genotypes of 300 unrelated Caucasians, without prior history of cancer, were determined by real-time PCR and compared to genotypes obtained by restriction fragment length polymorphism PCR. In the population examined, the allele frequency of the CYP1A1 G allele at position 4889 was 0·042, and the frequency of the T allele at position 6235 was 0·10. Five percent of samples disagreed between methods. Sequence analysis of discordant samples revealed that the differences are likely attributed to incomplete digestion of amplicons by the RFLP method. These findings confirm the reliability and utility of real-time PCR as a method for large-scale genotyping.

Intratracheal instillation of 51CrCl3 in anaesthetized rabbits resulted in partial absorption. In blood, the absorbed material was entirely confined to the plasma compartment. Only trace amounts were deposited in liver and kidney. By contrast, after similar application of Na, 51CrO4 the bulk of blood radioactivity was present in red blood cells (RBC). Substantial deposition occurred in liver and kidneys. It is concluded that Cr(VI) may enter the body unreduced via the lung and is partially deposited in cells over a prolonged period of time.Since chromium was accumulated in liver after administration of Cr(VI) we investigated the intracellular disposition of Cr(VI) in the isolated perfused liver. No significant sex differences in chromium distribution were observed. At the end of the experiments (1 h), 60% of the applied dose (312μg Cr/liver) was located in the cytosol, whilst 14% was in the mitochondria, 9% in the microsomal pellet and 2% was associated with the nuclei. Gel chromatography of the cytosolic compartment showed that the overwhelming part of chromium was eluted in fractions with an apparent molecular weight of 6,000 dalton. These fractions exhibited absorption maxima at 410nm and 548nm. It is concluded, that cytosolic reduction might be the main intracellular redox pathway for chromates. This view was confirmed by monitoring the reaction of Cr(VI) with GSH in vitro. GSH reduced Cr(VI) without further cofactors under formation of GSH-chromium complexes, which possibly represent major intermediates in the metabolism of Cr(VI).

Fast uptake kinetics of 51Cr(VI) into red blood cells (RBC) were studied. The uptake was determined as a process composed of two different first order processes with half lives of 23 sec and 10.4 min. Whereas the uptake of Cr(VI) in resuspended RBC (0.9% saline) was almost complete, in blood a portion of approximately 15% of the applied dose remained in plasma and did not penetrate into RBC, even after longer time periods. Over the concentration range studied (10 μM-50mM), the fast initial uptake followed Michaelis-Menten-kinetics. The maximum capacity of this Cr(VI) transport into RBC of man was 3.1 × 108 ions × cell-1 × min-1.It is likely that Cr(VI) is transported under participation of “band-3-protein”;, a physiological anion carrier system of the RBC membrane. Intracellularly, chromium is almost quantitatively bound to hemoglobin, and thereby trapped for the cell's lifetime.

Rats were exposed, in a closed desiccator jar chamber, to concentrations of various halogenated C1 and C2 compounds at which the metabolizing capacities were saturated (Vmax conditions). Within the exposure period of 50 h concentrations of the xenobiotic and of exhaled acetone were monitored in the gas phase of the system. The quantitative extent of acetone exhalation was dependent on the individual compound examined. Acetone exhalation was stimulated in presence of vinyl chloride, vinyl bromide, vinyl fluoride, vinylidene fluoride, cis-and trans-1,2-di-chloroethylene, trichloroethylene, perchloroethylene, methylene chloride, chloroform, carbon tetrachloride and 1,1,2-trichloroethane. No stimulation of acetone exhalation occured with 1,1,1-trichloroethane and with the reference hydrocarbon n-hexane. Also, acetone exhalation was evoked by infusions of either fluoroacetate or chloroacetate, two anticipated or proven metabolites of some haloethylenes; the infusion rates of which were based on calculations of the metabolic rates of vinylidene fluoride and of vinyl chloride, respectively.

Fast transport kinetics of 51Cr (VI) into red blood cells (RBCs) in vitro were studied. No significant species differences were found between RBCs of man and rat. The uptake of 51Cr (VI) by RBCs in whole blood was composed of two different first order processes of different velocities (apparent t1/2 of 22.7 s and 10.4 min for man and 6.9 s and 10.1 min for rat, respectively). However, even after longer time periods a fixed portion of approximately 15% of the administered dose remained in the plasma and did not penetrate into RBCs Over the entire concentration range studied (10 µM–50 mM), the fast initial uptake followed Michaelis-Menten kinetics. The maximal capacity of this Cr(VI) transport into RBCs of man and rat was 3.1×108 CrO42- ions × cell-1 × min-1 and 2.5×108 CrO4-2 ions × cell-1 × min-1, respectively. It is likely that Cr(VI) is transported into RBCs via a physiological anion carrier (“band-3-protein”).

Rats were exposed to [1,2-14C] vinyl chloride. Liver RNA was isolated, hydrolyzed, and the nucleosides separated on Aminex-A-6. Besides the physiological bases and 1,N6-ethenoadenosine, radioactivity was also incorporated into 3,N4-ethenocytidine. Radioactive 3,N4-ethenocytidine moieties were also formed on incubation of polycytidylic acid with rat liver microsomes, NADPH and [14C] vinyl chloride. These alkylation mechanisms are consistent with the mutagenic and cancerogenic properties of vinyl chloride.

Rats were exposed to [1,2-14C] vinyl chloride in a closed system at initial concentrations below 100 ppm. When the system was occupied by 3 rats, a half-life of vinyl chloride in the system's atmosphere of 1.13 ± 0.12 h was observed. The volume of the system was 10.3 l. Calculation of the clearance of vinyl chloride from the system revealed that about 40% of inspired vinyl chloride is absorbed by lung. Therefore, changes in respiration did not influence uptake of vinyl chloride.

An intervention study was performed on 28 workers exposed by inhalation to styrene in the reinforced plastics industry and 20 controls not occupationally exposed to the compound. The workers involved were 14 laminators exposed to a time-weighted average of approximately 40 ppm styrene and 14 formers exposed to an average of about 10 ppm styrene. Ambient air monitoring data and the concentration of mandelic acid in the urine were used for the assessment of exposure. From each subject, peripheral blood lymphocytes were analysed for sister chromatid exchanges (SCEs). In the laminators, the mean SCE frequency was significantly higher than in the controls in both the group of smokers (9.59 ± 0.77 SCEs/cell vs 7.23 ± 1.00 SCEs/cell) and the group of non-smokers (10.25 ± 1.08 SCEs/cell vs 5.98 ± 0.60 SCEs/cell). The mean SCE frequency of the formers (7.42 ± 128 SCEs/cell in smokers) did not differ statistically from the controls (7.23 ± 1.00 SCEs/cell in smokers). No evaluation was made for non-smoking formers since all but one worker in this group were smokers. In order to comply with a lowering of the occupational exposure limit (MAK value) for occupational exposure to styrene in the Federal Republic of Germany from 100 ppm to 20 ppm, considerable technical and hygienic improvements were made at the work site of the laminators. This intervention led to a reduction of average exposure of these workers by inhalation from 40 ppm to approximately 20 ppm. One year after these improvements were made, a second investigation was performed. In all but one of the laminators, the concentration of mandelic acid in urine had dropped considerably. The SCE frequency in blood lymphocytes of the laminators had likewise dropped significantly to 7.74 ± 0.59 SCEs/cell in the non-smokers. In the smokers, it was also lower than on the first occasion (9.02 ± 1.19), yet statistical evaluation was not possible due to insufficient numbers. Overall, the results of the intervention study show that the lowering of the occupational exposure limit for styrene to 20 ppm in Germany was justified and that a reduction of occupational exposure to the chemical has led to a prevention of adverse cytogenetic effects.

[6,7-3H] Ethinyloestradiol (50 μg) was administered intravenously to volunteers and the free extractable ethinyloestradiol in the plasma was measured. The compound showed a biphasic plasma decline. The half-life of the second phase was 7.5 ± 1.7 (sd) hours. Administration of rifampicin (600 mg for 6 days) shifted the half-life of ethinyloestradiol to 3.3 ± 0.9 h while the apparent volume of distribution for the second phase of climination was not changed.When [2,4,6,7-3H] ethinyloestradiol (100 μg) was administered orally, some of the tritium was released by oxidative metabolism from the steroid and transformed to tritiated water (HTO) which equilibrated with whole body water. This portion, normally 7.17 ± 1.66 % of the tritium dose, was increased by previous administration of rifampicin to 10.62 ± 2.27 %. The initial rate of oxidation of [2,4,6,7-3H]ethinyloestradiol was increased more than twofold by rifampicin treatment. The results are consistent with previous findings that rifampicin induces the oestrogen-2-hydroxylase in the endoplasmic reticulum of human liver, and explain the reduced effectiveness of ethinyloestradiol in oral contraceptives, if the patients are treated with rifampicin.

Summary Microsomal uptake irreversible protein binding of labelled trichloroethylene was measured following incubation with rat liver microsomes in an all-glass vacuum system.

Summary Rats have been pretieatet with 6-nitro-1.2.3-benzothiadiazole which completely blocks metabolism of vinyl chloride. If the animals are exposed to atmospheric vinyl chloride, formation of an equilibrium between the compound in the gas phase and in the animal's orgnism is observed. Unmetabolized vinyl chloride is concentrated in adipose tissue. The distribution pattern of vinyl chloride in different organs of the rat is constant over the concentration range of 25–10,000 ppm of vinyl chloride in the exposure atmosphere. Distribution of metabolites of vinyl chloride contrasts to that of the original compound; metabolites primarily are concentrated in liver and in kidneys.

The present concept is that mestranol must be demethylated to ethinyl-oestradiol in order to have oestrogenic activity. This activation of mestranol has been studied in vivoby means of radiospirometry and in vitroincubations with rat liver microsomes.During the microsomal incubation of doubly labelled [4-14C] mestranol and [3H-methoxy] mestranol with an NADPH-regenerating system 14C-labelled ethinyloestradiol was formed as the main metabolite, which was identified by crystallization to constant specific activity. In addition, a minor metabolite was found, which had the unaltered methoxy group. The enzymatic reaction was complete after 20 min. Pre-treatment of the rats with phenobarbital caused a threefold increase in ethinyloestradiol production.The in vivoresults after application of mestranol tritiated in the methoxy group show an increase of the body water specific activity up to a maximum on the 2nd day. In the control rats 34.5% and in phenobarbital pre-treated rats 47.7% of the tritium was metabolized to body water. DDT-pre-treatment caused a HTO production of 60%. These values represent a minimal demethylation rate for the in vivoconditions.