Brian Ketterer

Enzymology of Cytosolic Glutathione S- Transferases

Publisher Summary The glutathione S-transferases (GSTs) catalyze a range of reactions of glutathione (GSH) with hydrophobic electrophiles. Their most established role is the glutathione conjugation of electrophiles, which would otherwise cause toxic reactions with macromolecules. These electrophiles frequently arise from the oxidation of xenobiotics by the cytochrome P450 system. This function has been illustrated by a discussion of aflatoxin B, carcinogenesis. The induction in the adult of a fetal GST with substantial activity toward the carcinogenic electrophile AFB1,-exo-oxide can prevent aflatoxin-induced hepatocarcinogenesis in the rat. The distribution of GSTs differs both qualitatively and quantitatively from tissue to tissue with the consequence that GSTs in one tissue may influence the levels of circulating substrates important for an effect in another tissue. With the development of modern technology, it has been particularly interesting to witness the recent determination of the crystal structure of proteins from three of the gene families, which has enabled the mechanism of the binding and activation of glutathione to be determined. From a practical point of view, one of the most interesting areas of recent work concerns the possible use of nontoxic inducers of GSTs in preventing chemical toxicity.

Human glutathione transferase catalysis of the formation of S-nitrosoglutathione from organic nitrites plus glutathione

The kinetics of spontaneous and human glutathione transferase catalysed formation of S‐nitrosoglutathione(GSNO) from glutathione(GSH) and n‐butyl‐ or amyl nitrite have been studied. At physiological pH and temperature, k 2 values of 22.3 and 21.0 M−1 · min−1 were obtained for n‐butyl‐ and amyl nitrites, respectively. Rate enhancements, (kcat /K m × k 2) × 10−4, due to purified human GSH transferases A1−1, A2−2 and Mla−la were, respectively, 7.00, 2.94 and 10.6 for n‐butyl nitrite and 121, 3.92 and 34.5 for amyl nitrite. GSH transferase P1−1 showed no detectable catalysis of the formation of GSNO. The data suggest that the presence of GSTs A1−1, A2−2 or M1−1 contribute substantially to intracellular metabolism of alkyl nitrites to GSNO. The results may be significant with regard to the immunotoxicity of alkyl nitrites.

Metabolism of 1,2-dibromo-3-chloropropane by glutathione S-transferases

The metabolism of 1,2-dibromo-3-chloropropane (DBCP), measured as the formation of water soluble metabolites and metabolites covalently bound to macromolecules, was studied in isolated rat liver, kidney, and testicular cells, in subcellular fractions, and with purified rat and human glutathione S-transferases (GSTs). The rate of formation of water soluble metabolites in the cells were in the order liver > kidney > testis. The rate of covalent macromolecular binding of reactive DBCP metabolites in the different cell types was of the same relative order. Pretreatment of the cells with the GSH depleting agent diethyl maleate (DEM) markedly decreased the rate of covalent binding in all cell types. Both the overall metabolism and the formation of DBCP metabolites that covalently bound to macromolecules, were substantially higher in rat testicular cells compared to hamster testicular cells. Rat liver cytosol and microsomes, and various purified rat and human GSTs extensively metabolized DBCP to water soluble metabolites in the presence of GSH. When compared to isolated cells, substantially lower rates of binding per mg protein could be observed in subcellular fractions. Binding of DBCP was detected in the microsomal and cytosolic fractions in the absence of NADPH, though in microsomes fortified with a NADPH-regenerating system, the generation of reactive DBCP metabolites was approximately doubled. Studies with purified rat GST isozymes showed that the relative overall GSH conjugation activity with DBCP was in the following order: GST form 3-3 > 2-2 approximately 12-12 > 1-1 > 4-4 approximately 8-8 approximately 7-7. Furthermore, human GST forms also readily metabolized DBCP with activities of GST A1-2 > A2-2 approximately A1-1 > M1a-1a > M3-3 approximately P1-1.

Effects of genetic polymorphism and enzyme induction in the glutathione S-transferase family on chemical safety and risk assessment

Glutathione is important in the detoxication of many xenobiotic chemicals and in the bioactivation of some. The glutathione-S-transferases are a super-family of enzymes involved in conjugation of xenobiotics with glutathione. There are wide genetically determined inter-individual differences in the expression of the isoenzymes. These differences may have important implications for the toxicity of compounds such as polycyclic aromatic hydrocarbons and aflatoxins.

Metabolism of Aflatoxin B1 by Human Hepatocytes in Primary Culture

Aflatoxin B1 (AFB1) is a potent liver carcinogen in the rat (Garner et al 1972) and, with hepatitis B infections, a co-carcinogen in human hepatocellular carcinoma (Qian et al 1994). It is metabolized by cytochrome P450 isoenzymes to give a number of products including AFM1, AFP1 and AFQ1 and the exo-and endo-8,9AFB1 oxides (AFBOs), of which exo-AFBO is the ultimate carcinogenic metabolite (Busby & Wogan 1984; Raney et al 1992a).

Stereoselective conjugation of 2-bromocarboxylic acids and their urea derivatives by rat liver glutathione transferase 12-12 and some other isoforms

Glutathione (GSH) conjugation of the separate enantiomers of five 2-bromocarboxylic acids and some of their urea derivatives by rat liver GSH transferases (GSTs) was studied. The liver cytosolic fraction conjugated all compounds, except for (R)-2-bromoisovaleric acid, with a variable degree of stereoselectivity. A GST pool, prepared by S-hexyl-GSH affinity chromatography, conjugated the urea derivatives at a somewhat higher rate but had very little activity towards the carboxylic acids, indicating that much activity towards the latter substrates was due to transferases not bound by the affinity column. Therefore, the activity was studied of some pure GSTs that are bound only slightly by the affinity column towards the separate enantiomers of 2-bromovaleric acid (BV), its urea derivative and 2-bromo-3-phenylpropionic acid (BPP). No activity was detected with transferases 5-5 and 8-8. Transferase 1-1 was active towards all compounds with high activity towards the urea derivatives. Transferase 12-12 showed high, stereospecific activity towards the R enantiomers of BV, its urea derivative and BPP.