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Thimerosal is a widely used preservative in health care products, especially in vaccines. Due to possible adverse health effects, investigations on its metabolism and toxicity are urgently needed. An in vivo study on chronic toxicity of thimerosal in rats was inconclusive and reports on genotoxic effects in various in vitro systems were contradictory. Therefore, we reinvestigated thimerosal in the cytochalasin B block micronucleus test. Glutathione S-transferases were proposed to be involved in the detoxification of thimerosal or its decomposition products. Since the outcome of genotoxicity studies can be dependent on the metabolic competence of the cells used, we were additionally interested whether polymorphisms of glutathione S-transferases (GSTM1, GSTT1, or GSTP1) may influence the results of the micronucleus test with primary human lymphocytes. Blood samples of six healthy donors of different glutathione S-transferase genotypes were included in the study. At least two independent experiments were performed for each blood donor. Significant induction of micronuclei was seen at concentrations between 0.05-0.5 micro g/ml in 14 out of 16 experiments. Thus, genotoxic effects were seen even at concentrations which can occur at the injection site. Toxicity and toxicity-related elevation of micronuclei was seen at and above 0.6 micro g/ml thimerosal. Marked individual and intraindividual variations in the in vitro response to thimerosal among the different blood donors occurred. However, there was no association observed with any of the glutathione S-transferase polymorphism investigated. In conclusion, thimerosal is genotoxic in the cytochalasin B block micronucleus test with human lymphocytes. These data raise some concern on the widespread use of thimerosal.

In general, the biological activation of nephrocarcinogenic chlorinated hydrocarbons proceeds via conjugation with glutathione. It has mostly been assumed that the main site of initial conjugation is the liver, followed by a mandatory transfer of intermediates to the kidney. It was therefore of interest to study the enzyme activities of subgroups of glutathione transferases (GSTs) in renal cancers and the surrounding normal renal tissues of the same individuals (n = 21). For genotyping the individuals with respect to known polymorphic GST isozymes the following substrates with differential specificity were used: 1-chloro-2,4-dinitrobenzene for overall GST activity (except GST theta); 7-chloro-4-nitrobenzo-2-oxa- 1,3-diazole for GST alpha; 1,2-dichloro-4-nitro-benzene for GST mu; ethacrynic acid and 4-vinylpyridine for GST pi; and methyl chloride for GST theta. In general, the normal tissues were able to metabolize the test substrates. A general decrease in individual GST enzyme activities was apparent in the course of cancerization, and in some (exceptional) cases individual activities, expressed in the normal renal tissue, were lost in the tumour tissue. The GST enzyme activities in tumours were independent of tumour stage, or the age and gender of the patients. There was little influence of known polymorphisms of GSTM1, GSTM3 and GSTP1 upon the activities towards the test substrates, whereas the influence of GSTT1 polymorphism on the activity towads methyl chloride was straightforward. In general, the present findings support the concept that the initial GST-dependent bioactivation step of nephrocarcinogenic chlorinated hydrocarbons may take place in the kidney itself. This should be a consideration in toxicokinetic modelling.

Fifty-nine persons with industrial handling of low levels of acrylonitrile (AN) were studied. As part of a medical surveillance programme an extended haemoglobin adduct monitoring [N-(cyanoethyl)valine, CEV; N-(methyl)valine. MV: N-(hydroxyethyl)valine, HEV] was performed. Moreover, the genetic states of the polymorphic glutathione transferases GSTM1 and GSTT1 were assayed by polymerase chain reaction (PCR). Repetitive analyses of CEV and MV in subsequent years resulted in comparable values (means, 59.8 and 70.3 microg CEV/1 blood; 6.7 and 6.7 microg MV/1 blood). Hence, the industrial AN exposures were well below current official standards. Monitoring the haemoglobin adduct CEV appears as a suitable means of biomonitoring and medical surveillance under such exposure conditions. There was also no apparent correlation between the CEV and HEV or CEV and MV adduct levels. The MV and HEV values observed represented background levels, which apparently are not related to any occupational chemical exposure. There was no consistent effect of the genetic GSTM1 or GSTT1 state on CEV adduct levels induced by acrylonitrile exposure. Therefore, neither GSTM1 nor GSTT1 appears as a major AN metabolizing isoenzyme in humans. The low and physiological background levels of MV were also not influenced by the genetic GSTM1 state, but the MV adduct levels tended to be higher in GSTT1- individuals compared to GSTT1 + persons. With respect to the background levels of HEV adducts observed, there was no major influence of the GSTM1 state, but GST- individuals displayed adduct levels that were about 1/3 higher than those of GSTT1 + individuals. The coincidence with known differences in rates of background sister chromatid exchange between GSTT1- and GSTT1 + persons suggests that the lower ethylene oxide (EO) detoxification rate in GSTT1- persons, indicated by elevated blood protein hydroxyethyl adduct levels, leads to an increased genotoxic effect of the physiological EO background.

The kinetic properties of bacterial and rat liver glutathione S-transferases (GST) active with dichloromethane (DCM) were compared. The theta class glutathione S-transferase (rGSTTI-1) from rat liver had an affinity for dihalomethanes lower by three orders of magnitude (K(app) > 50 mM) than the bacterial DCM dehalogenase/GST from Methylophilus sp. DM11. Unlike the bacterial DCM dehalogenase, the rat enzyme was unable to support growth of the dehalogenase minus Methylobacterium sp. DM4-2cr mutant with DCM. Moreover, the presence of DCM inhibited growth with methanol of the DM4-2cr transconjugant expressing the rat liver GSTT1-1. In Salmonella typhimurium TA1535, expression of rat and bacterial DCM-active GST from a plasmid in the presence of DCM yielded up to 5.3 times more reversions to histidine prototrophy in the transconjugant expressing the rat enzyme. Under the same conditions, however, GST-mediated conversion of DCM to formaldehyde was lower in cell-free extracts of the transconjugant expressing the rat GSTT1 than in the corresponding strain expressing the bacterial DCM dehalogenase. This provided new evidence that formaldehyde was not the main toxicant associated with GST-mediated DCM conversion, and indicated that an intermediate in the transformation of DCM by GST, presumably S-chloromethylglutathione, was responsible for the observed effects. The marked differences in substrate affinity of rat and bacterial DCM-active GST, as well as in the toxicity and genotoxicity associated with expression of these enzymes in bacteria, suggest that bacterial DCM dehalogenases/GST have evolved to minimise the toxic effects associated with glutathione-mediated catalysis of DCM conversion.

Glutathione transferase (GST) GSTT1-1 is involved in the biotransformation of several chemicals widely used in industry, such as butadiene and dichloro methane DCM. The polymorphic hGSTT1-1 may well play a role in the development of kidney tumours after high and long-term occupational exposure against trichloroethylene. Although several studies have investigated the association of this polymorphism with malignant diseases little is known about its enzyme activity in potential extrahepatic target tissues. The known theta-specific substrates methyl chloride (MC) dichloromethane and 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) were used to assay GSTT1-1 activity in liver and kidney of rats, mice, hamsters and humans differentiating the three phenotypes (non-conjugators, low conjugators, high conjugators) seen in humans. In addition GSTT1-1 activity towards MC and DCM was determined in human erythrocytes. No GSTT1-1 activity was found in any tissue of non-conjugators (NC). In all organs high conjugators (HC) showed twofold higher activity towards MC and DCM than low conjugators (LC). The activity in human samples towards EPNP was too close to the detection limit to differentiate between the three conjugator phenotypes. GSTT1-1 activity towards MC was two to seven-times higher in liver cytosol than in kidney cytosol. The relation for MC between species was identical in both organs: mouse > HC > rat > LC > hamster > NC. In rats, mice and hamsters GSTT1-1 activity in liver cytosol towards DCM was also two to seven-times higher than in the kidney cytosol. In humans this activity was twice as high in kidney cytosol than in liver cytosol. The relation between species was mouse > rat > HC > LC > hamster > NC for liver, but mouse > HC > LC/rat > hamster/NC for kidney cytosol. The importance to heed the specific environment at potential target sites in risk assessment is emphasized by these results.

A new polymorphic form of glutathione S-transferase (GST), metabolising monohalogenated methanes, ethylene oxide and dichloromethane, has been purified from human erythrocytes and characterized. Several characteristics, such as similar elution patterns on different chromatographic matrices, KM-values and activity towards antibodies, confirm a previous assumption that this novel GST is a class theta enzyme. Although the presence or absence of the enzyme activity in human red blood cells is parallel with the polymorphism of the human GST T1 gene, the new GST theta in red blood cells may differ from the known GST T1-1 enzyme from other tissues in terms of substrate specificity, since established GST T1-1 substrates [1,2-epoxy-3-(p-nitro-phenoxy)propane and p-nitro-benzyl chloride] are not metabolized. The substrate specificity of the new enzyme in erythrocytes resembles more closely that of GST T2-2, most likely due to a common N-terminal modification which modifies substrate binding. The new polymorphic GST-isoform in human red blood cells therefore may be considered to represent an N-terminally modified isoform of GST T1-1.

Expression of the glutathione S-transferase (GST) subclasses alpha, mu and pi was investigated immunohistochemically in 20 normal or hyperplastic mesothelium and in 57 malignant mesothelioma cases. These results were correlated with survival and also with P-170 glycoprotein expression. Nearly all the non-neoplastic mesothelium cases were positive for GST alpha and pi. About half of the non-neoplastic cases were positive for mu. Twenty-nine (51%) malignant mesotheliomas were positive for at least one of the GST species; 21 (37%) showed immunoreactivity for alpha, 18 (31.5%) for mu and 21 (37%) for pi. A total of 54 mesothelioma cases displayed immunoreactivity for the P-170 glycoprotein. For GST pi and GST mu, a statistical significance between expression and increased survival was found (respectively P = 0.012 and 0.024) while for GST alpha no significance was found. The results of this study demonstrate that expression of GST pi correlates positively with increased survival in malignant mesothelioma. It is also concluded that, in mesothelioma, GST and P-170 glycoprotein may contribute to the resistance to cytotoxic drugs frequently observed in these tumours. No correlation between GST and P-170 expression was demonstrated.

Three new glutathione transferase (GST) genes from the housefly Musca domestica are described. These genes, identified as MdGST-2, -3, and -4, were from cDNA clones obtained from a cDNA bank in phage lambda. The bank was prepared using poly(A)+ RNA from a housefly that is highly resistant to organophosphate insecticides because of enhanced expression of multiple members of the glutathione transferase gene family. The DNA sequence of each is reported and has a complete open reading frame that specified an amino acid sequence similar to other dipteran glutathione transferases. Based on phylogenetic analysis, we can conclude that the insect glutathione transferase gene family falls into two groups, each of which evolves at a different rate, presumably due to differences in functional constraints. We show that MdGST-1 (and their homologues from Drosophila and Lucilia) evolve at a significantly slower rate than the other members of the gene family. Each housefly GST cDNA was inserted into a bacterial plasmid expression system and a glutathione transferase activity was expressed in Escherichia coli. The transcription pattern of each of these glutathione transferases was examined in a variety of different housefly strains that are known to differ in their resistance to organophosphate insecticides due to different patterns of glutathione transferase expression. We found that the level of transcription for two of our clones was positively correlated with the level of organophosphate resistance.

The expression of three classes of glutathione S-transferases (GSTs), Alpha, Mu, and Pi was investigated in the nasal mucosae of rats during development using immunohistochemical methods. GST Alpha and Mu were first detected in the supranuclear region of sustentacular cells on embryonic days 16. The Bowman's glands expressed differential patterns of immunoreactivity during development, beginning at postnatal day (P) 2 and P6 for Alpha and Mu classes, respectively and being greatest at P11 for both. The acinar cells of vomeronasal glands in the vomeronasal organ expressed Alpha and Mu classes of GSTs from P11 onwards. In the septal organ of Masera, the supranuclear region of sustentacular cells expressed GSTs from P11 with little or no variation during development. In the respiratory mucosa, Alpha and Mu classes of GSTs were detected at the brush borders of ciliated cells and in the acinar cells of posterior septal glands, but not in anterior septal or respiratory glands located on the turbinates. Compared to olfactory mucosa, the changes in immunoreactivity for GSTs were less pronounced in the respiratory mucosa during development. Specific GST Pi immunoreactivity was not detected in the nasal mucosae at any stage of development studied. The occurrence of GSTs in the nasal mucosa, including olfactory, vomeronasal, septal, and respiratory epithelia, suggests that the GSTs are actively involved in the biotransformation of xenobiotics including odorants and pheromones, and may also participate in perireceptor processes such as odorant clearance. In addition, we have developed a working model describing the cellular localization of certain phase I (e.g., cytochrome P-450s) and phase II (e.g., GSTs, gamma-glutamyl transpeptidase) biotransformation enzymes in the olfactory mucosa and their proposed roles in xenobiotic metabolism.