H. M. Bolt

Relevance of the deletion polymorphisms of the glutathione S-transferases GSTT1 and GSTM1 in pharmacology and toxicology

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.

The reduction of chromium (VI) to chromium (III) by glutathione: An intracellular redox pathway in the metabolism of the carcinogen chromate

The capacity of glutathione (GSH) to reduce Cr(VI) to Cr(III) in vitro was investigated. The reaction was determined spectrophotometrically by following the absorption of Cr(VI) at 370 nm. At stoichiometric conditions (molar ratio Cr(VI)/GSH of 1:3) the reduction was strongly dependent on the solutions pH. It was much slower at pH 7.4 than at pH values below 5. An excess of GSH (100- or 1000-fold) accelerated the reaction. In any case, 3 GSH molecules were required to reduce 1 molecule of chromate. Incubation of human red blood cells (RBC) with an excess of Na2CrO4 (10 mM) decreased the GSH content of the cells to 10% of the original amount. This depletion of GSH was similar to that obtained when RBC were incubated with 62 mM diethylmaleate (DEM), a well known GSH depleting agent. Sephadex G-100 chromatography of lysates from human RBC incubated with radioactive chromate (51Cr(VI] showed a strong affinity of 51Cr for hemoglobin: 97% of the applied dose was bound to hemoglobin whilst only minor amounts of 51Cr were found in the low-molecular fractions. However, incubations of prepared lysates (as opposed to intact cells) with 10 mM Na2 51CrO4 markedly raised the chromium content of low-molecular fractions (probably GSH-Cr-complexes), probably indicative of a role of GSH in the intra-cellular reduction of Cr(VI) to Cr(III), the latter being regarded as the ultimately toxic species of this metal.

Glutathione S-transferase GSTM1 and GSTT1 null genotypes as potential risk factors for urothelial cancer of the bladder.

Abstract Onehundred-and-thirteen patients with cancer of the urinary bladder (cases) were examined with respect to the frequency of null genotypes of the polymorphic glutathione S-transferases GSTM1 and GSTT1. The allelic background in the German population of the area was evaluated by analysing 170 newborns (controls). The frequency of GSTM1 and GSTT1 null genotypes in this population, using methods based upon internal standard controlled polymerase chain reaction (PCR), was 0.54 and 0.18 respectively. An elevated relative bladder cancer risk of GSTM1 null genotype carriers was indicated by comparison of this background with the data of the bladder cancer cases (OR = 1.81; 95% CI [1.10, 2.98]; p = 0.019). The frequencies of the GSTT1 null genotype in the total group of bladder cancer cases versus controls did not differ statistically. However, a significantly higher relative risk of bladder cancer for the GSTT1 null genotype was detected in the cases-subgroup of non-smokers (OR = 3.84; 95% CI [1.21, 12.23]; p = 0.023). Thus, the GSTT1 null genotype might represent a minor risk factor for human bladder cancer which should be further investigated.

Interaction of metal salts with cytoskeletal motor protein systems.

Interactions of chemicals with the microtubular network of cells may lead to genotoxicity. Micronuclei (MN) might be caused by interaction of metals with tubulin and/or kinesin. The genotoxic effects of inorganic lead and mercury salts were studied using the MN assay and the CREST analysis in V79 Chinese hamster fibroblasts. Effects on the functional activity of motor protein systems were examined by measurement of tubulin assembly and kinesin-driven motility. Lead and mercury salts induced MN dose-dependently. The no-effect-concentration for MN induction was 1.1 microM PbCl(2), 0.05 microM Pb(OAc)(2) and 0.01 microM HgCl(2). The in vitro results obtained for PbCl(2) correspond to reported MN induction in workers occupationally exposed to lead, starting at 1.2 microM Hg(II) (Vaglenov et al., 2001, Environ. Health Perspect. 109, 295-298). The CREST Analysis indicate aneugenic effects of Pb(II) and aneugenic and additionally clastogenic effects of Hg(II). Lead (chloride, acetate, and nitrate) and mercury (chloride and nitrate) interfered dose-dependently with tubulin assembly in vitro. The no-effect-concentration for lead salts in this assay was 10 microM. Inhibition of tubulin assembly by mercury started at 2 microM. The gliding velocity of microtubules along immobilised kinesin molecules was affected by 25 microM Pb(NO(3))(2) and 0.1 microM HgCl(2) in a dose-dependent manner. Our data support the hypothesis that lead and mercury genotoxicity may result, at least in part, via disturbance of chromosome segregation via interaction with cytoskeletal proteins.

Toxicokinetics of p -tert-octylphenol in male Wistar rats

Abstract Only weak oestrogenic activity has been reported for p-alkylphenols compared with the physiological hormone 17β-estradiol. Despite the low potency, there is concern that due to bioaccumulation oestrogenically efficient blood levels could be reached in humans exposed to trace levels of p-alkylphenols. To address these concerns, toxicokinetic studies with p-tert-octylphenol [OP; p-(1,1,3,3-tetramethylbutyl)-phenol] as a model compound have been conducted in male Wistar rats. OP blood concentrations were determined by GC-MS in rats receiving either single oral (gavage) applications of 50 or 200mg OP/kg body wt or a single intravenous injection of 5mg/kg body wt. The OP blood concentration was ∼1970ng/ml immediately after a single intravenous application, decreased rapidly within 30 min, and was no longer detectable 6–8h after application. The curve of blood concentration vs time was used to calculate an elimination half-life of 310min. OP was detected in blood as early as 10min after gavage administration, indicating rapid initial uptake from the gastrointestinal tract; maximal blood levels reached 40 and 130ng/ml after applications of 50 and 200mg/kg, respectively. Using the area under the curve (AUC) of blood concentration vs time, low oral bioavailabilities of 2 and 10% were calculated for the 50 and 200mg/kg groups, respectively. OP toxicokinetics after repeated administration was investigated in male Wistar rats receiving daily gavage administrations of 50 or 200mg OP/kg body wt for 14 consecutive days. Profiles of OP blood concentration vs time determined on day 1 and day 14 were similar, indicating that repeated oral gavage administration did not lead to increased blood concentrations. Another group of rats received OP via drinking water saturated with OP (∼8mg/l, corresponding to a mean daily dose of ∼800μg/kg) over a period of up to 28 days. OP was not detected in any blood sample from animals treated via drinking water (detection limit was 1–5ng/ml blood). OP concentrations were also analysed in tissues obtained from the repeated gavage (14 days) and drinking water groups (14 and 28 days). In the 50mg/kg group, low OP concentrations were detected in fat and liver from some animals at average concentrations of 10 and 7ng/g tissue, respectively. OP was not detected in the other tissues analysed from this group. In the 200mg/kg group, OP was found in all tissues analysed except testes (fat, liver, kidney, muscle, brain and lung had average concentrations of 1285, 87, 71, 43, 9 and 7ng/g tissue, respectively). OP was not detected in tissues of animals receiving OP via drinking water for 14 or 28 days, except in muscle and kidney tissue of one single animal receiving OP for 14 days. Using rat liver fractions it was demonstrated that OP was conjugated via glucuronidation and sulphation in vitro. A Vmax of 11.24 nmol/(min * mg microsomal protein) and a Km of 8.77μmol/l were calculated for enzyme-catalysed OP glucuronidation. For enzyme-catalysed sulphation, a Vmax of 2.85nmol/(minT15*mg protein) and a Km of 11.35μmol/l were calculated. The results indicate that OP does not bioaccumulate in rats receiving low oral doses, in agreement with the hypothesis of a rapid first-pass elimination of OP by the liver after oral ingestion, via glucuronidation and sulphation. Only if these detoxification pathways are saturated may excessive doses lead to bioaccumulation.

Experimental toxicology of formaldehyde

SummaryFormaldehyde is a reactive chemical which undergoes spontaneous reactions with various cellular constituents. Mutagenicity data may be interpreted on the background of this behavior. Mice are better able to reduce the irritating effect of formaldehyde than rats and to reduce their ventilation rate when formaldehyde acts on the respiratory tract. Subacute exposure of rats to concentrations higher than 2 ppm inhibits mucociliary clearance of the nasal epithelium and leads to progressive histological and ultrastructural lesions at this site. The occurrence of squamous cell carcinomas of the nasal epithelium of rats after 2 years inhalation of 14.3 ppm formaldehyde (CIIT study) is probably the result of chronic and recurrent local toxicity; this is supported by species differences in susceptibility to the tissue damaging and carcinogenic effect of formaldehyde (rat, mouse, hamster). Data on formaldehyde-DNA interaction further support the argument that a direct risk extrapolation from the formaldehyde effects in rats to those expected for man is not possible.

Species differences in butadiene metabolism between mice and rats evaluated by inhalation pharmacokinetics

Metabolism of 1,3-butadiene to 1,2-epoxybutene-3 in rats follows saturation kinetics. Comparative investigation of inhalation pharmacokinetics in mice also revealed a saturation pattern. For both species “linear” pharmacokinetics apply at exposure concentrations below 1000 ppm 1,3-butadiene; saturation of butadiene metabolism is observed at atmospheric concentrations of about 2000 ppm.For mice metabolic clearance per kg body weight in the lower concentration range where first order metabolism applies was 7300ml×h−1 (rat: 4500 ml×h−1). Maximal metabolic elimination rate (Vmax) was 400 μmol×h−1 ×kg−1 (rat: 220 μmol ×h−1×kg−1). This shows that 1,3-butadiene is metabolized by mice at higher rates compared to rats.Based on these investigations, the metabolic elimination rates of butadiene in both species were calculated for the exposure concentrations applied in two inhalation bioassays with rats and with mice. The results show that the higher rate of butadiene metabolism in mice when compared to rats may only in part be responsible for the considerable difference in the susceptibility of both species to butadiene-induced carcinogenesis.

Fast uptake kinetics in vitro of 51Cr (VI) by red blood cells of man and rat

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”).

A comparative investigation of the metabolism of methyl bromide and methyl iodide in human erythrocytes

SummaryHuman erythrocyte cytoplasm was incubated in head space vials with either methyl bromide or methyl iodide. The decline in concentration of the two methyl halides was monitored by gas chromatography. Simultaneously, the production of S-methylglutathione was determined by thin layer chromatography. In parallel experiments, boiled erythrocyte cytoplasm was used in order to determine non-enzymatic conjugation. Furthermore, inhibition experiments with sulfobromophthalein were performed. The results were compared with previous findings on the metabolism of methyl chloride. In contrast to methyl chloride, both methyl bromide and methyl iodide showed a significant non-enzymatic conjugation with glutathione. In addition, an enzymatic conjugation could be observed in the erythrocyte cytoplasm of the majority of the population, whereas a minority lacks this enzymatic activity. This is consistent with findings on methyl chloride. Inhibition experiments show that a minor form of the erythrocyte glutathione transferase may be responsible for the enzymatic conjugation. Of the three monchalogenated methanes, methyl bromide is the substrate with the highest affinity for the conjugating enzyme(s). In the case of methyl iodide, non-enzymatic reaction overweighs the enzymatic process. There are possible implications of the results for occupational health and the toxicity of the substances.

Sister chromatid exchange frequency in cultured isolated porcine urinary bladder epithelial cells (PUBEC) treated with ochratoxin A and alpha.

The mycotoxin ochratoxin A (OTA) and its metabolite ochratoxin alpha (OT-alpha) were investigated, to examine their potency to induce sister chromatid exchages (SCE) in cultured poricine urinary baldder epithedial cells (PUBEC) (primary cluture). Serum-free cultured PUBEC were incubated for 5 h with either OTA or OT-alpha, respectively, and subsequently cutured in the presence of 5-bromo-2-deoxyuridine (BrdU). After two cell cycles, mitosis was inhibited by the colchicine derivative Colcemid, cells were fixed and chromosomes were prepared for SCE analysis. For OTA, a dose-dependent increase in SCE frequency was measured in concentrations between 100 pM and 100 nM OTA. At 100nM OTA, SCE frequency increased by about 41%, compared to the base SCE level (7.27 SCEs per chromosome set, solvent control). Higher concentrations of OTA were cytotoxic. The metabolite OT-alpha also increased SCE frequency, but at higher concentrations. At a concentration of 10μM OT-alpha, an increase of about 55% was detected. OT-alpha showed no cytotoxic effect. There results indicate that OTA is genotoxic in this in vitro system, which represents the urinary bladder epithelium, a target organ of OTA in vivo. It could also be shown that OT-alpha, which is said to be non-toxic, is genotoxic in this assay at higher concentrations.