Increased synthesis of glutathione S-transferases in response to anticarcinogenic antioxidants. Cloning and measurement of messenger RNA

Glutathione S-transferase activities in mouse hepatic cytosols are elevated as much as 11-fold following the administration of BHA (2(3)-tert-butyl-4-hydroxyanisole), a widely used antioxidant food additive. Ethoxyquin (1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline) and disulfiram [bis(diethyldithiocarbamyl)disulfide] also enhance the activities of glutathione S-transferases and certain other enzymes. Each of these compounds protects rodents against mutagenic and carcinogenic metabolites. A major (pI 8.7) and a minor (pI 9.3) component of the family of mouse hepatic glutathione S-transferases have been purified to homogeneity. These transferases are immunologically cross-reactive, and have a high degree of NH₂-terminal sequence homology (but are not identical). The enzymes differ in a number of molecular and catalytic properties. The transferases are 12-fold elevated by dietary BHA as demonstrated by immunotitration. The mRNA for the major glutathione S-transferase is increased more than 20-fold in the liver RNA of BHA-fed mice, as determined by translation of total liver mRNA and characterization of the products by immunoprecipitation and sodium dodecyl sulfate-gel electrophoresis or by two-dimensional gel electrophoresis. A cDNA plasmid complementary to glutathione S-transferase mRNA was constructed. Translation of liver mRNA selected by hybridization with this plasmid gave products similar to or identical with glutathione S-transferase polypeptides. The cDNA insert has been partially sequenced and its orientation has been determined. Its sequence corresponds to the NH₂-terminal region (beginning at residue 9) of the amino acid sequence of the glutathione S-transferase with pI 9.3. Hybridization of the ³²P-labeled cDNA plasmid with total liver RNA indicates a 26-fold increase in homologous mRNA in response to the feeding of BHA.