The cDNA encoding human glutathione S-transferase (GST) T1 has been expressed as two recombinant forms in Escherichia coli that could be purified by affinity chromatography on either IgGSepharose or nickel-agarose ; one form of the transferase was synthesized from the pALP 1 expression vector as a Staphylococcus aureus protein A fusion, whereas the other form was synthesized from the pET-20b expression vector as a C-terminal polyhistidine-tagged recombinant. The yields of the two purified recombinant proteins from E. coli cultures were approx. 15 mg\l for the protein A fusion and 25 mg\l for the C-terminal polyhistidine-tagged GST T1-1. The purified recombinant proteins were catalytically active, although the protein A fusion was typically only 5-30 % as active as the histidine-tagged GST. Both recombinant forms could catalyse the conjugation of glutathione with the model substrates 1,2-epoxy-3-(4h-

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Two murine Theta-class glutathione S-transferases (GSTs), mGSTT1 and mGSTT2, have been cloned and sequenced. The murine cDNAs, together with the published sequences of the rat and human enzymes, were used to design oligonucleotide probes in order to determine the distribution of mRNA for these enzymes in the liver and lung of rat, mouse and human. The mRNA distribution was compared with that of enzyme protein determined with an antibody to rat GSTT2-2. Both the antibody and the oligonucleotide probes gave the same distribution patterns. Both enzymes were present at significantly higher concentrations in mouse tissues than in rat or human tissues. In mouse liver, both enzymes were localized in specific cell types and in nuclei. Although the distribution of GSTT2-2 in rat liver was similar to that seen in the mouse, GSTT1-1 was not localized in a specific cell type or in the nuclei of either rat or human liver. In the lungs, very high concentrations of the Theta enzymes were present in mouse-lung Clara cells and ciliated cells, with much lower levels in the Clara cells only of rat lung. Low levels of human transferase GSTT1-1 were detected in a small number of Clara cells and ciliated cells at the alveolar/ bronchiolar junction. The relative activities between species, and the cellular and sub-cellular distribution within the liver and lungs of each species, provides an explanation for the species-specificity of methylene chloride, a mouse-specific carcinogen activated by glutathione S-transferase GSTT1-1.

Thiamethoxam is a neonicotinoid insecticide that is not a mutagen, but it did cause a significant increase in liver cancer in mice, but not rats, in chronic dietary feeding studies. Previous studies in mice have characterized a carcinogenicity mode of action that involved depletion of plasma cholesterol, cell death, both as single cell necrosis and as apoptosis, and sustained increases in cell replication rates. In a study reported in this article, female rats have been exposed to thiamethoxam in their diet at concentrations of 0, 1000, and 3000 ppm for 50 weeks, a study design directly comparable to the mouse study in which the mode of action changes were characterized. In rats, thiamethoxam had no adverse effects on either the biochemistry or histopathology of the liver at any time point during the study. Cell replication rates were not increased, in fact they were significantly decreased at several time points. The lack of effect on the rat liver is entirely consistent with the lack of liver tumor formation in the two-year cancer bioassay. Comparisons of the metabolism of thiamethoxam in rats and mice have shown that concentrations of the parent chemical were either similar or higher in rat blood than in mouse blood in both single dose and the dietary studies strongly indicating that thiamethoxam itself is unlikely to play a role in the development of liver tumors. In contrast, the concentrations of the two metabolites, CGA265307 and CGA330050, shown to play a role in the development of liver damage in the mouse, were 140- (CGA265307) and 15- (CGA330050) fold lower in rats than in mice following either a single oral dose, or dietary administration of thiamethoxam for up to 50 weeks. Comparisons of the major metabolic pathways of thiamethoxam in vitro using mouse, rat, and human liver fractions have shown that metabolic rates in humans are lower than those in the rat suggesting that thiamethoxam is unlikely to pose a hazard to humans exposed to this chemical at the low concentrations found in the environment or during its use as an insecticide.

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