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.

A glutathione S-transferase metabolizing methylene chloride has been isolated from mouse liver using a variety of chromatographic methods. N-terminal and internal amino acid sequences show that the enzyme, designated GST T1-1*, is closely related to the rat Theta-class GST 5-5. The mouse enzyme, molecular mass 25000 Da, has been isolated to homogeneity in active form with an approximate yield of 2% of the cytosolic activity towards methylene chloride. GST T1-1* has a specific activity of about 5.5 micromol/min per mg of protein whereas the rat GST 5-5 is reported to have a specific activity of about 11 micromol/min per mg of protein [Meyer, Coles, Pemble, Gilmore, Fraser and Ketterer (1991) Biochem. J. 274, 409-414], demonstrating that both the rat and mouse enzymes have similar activity with this substrate. Limited evidence was obtained for a second enzyme, with a similar molecular mass (25400 Da), which had an N-terminal sequence identical to that of rat GST 12-12. This protein, which was sequenced from a band on a gel, was extremely labile and could not be isolated to homogeneity. The partially purified enzyme was not active with methylene chloride.

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Method validation, cross-validation between three laboratories and incurred sample re-analysis demonstrated method robustness. The methods were successfully applied to multiple clinical studies.