The effects of glutathione (GSH) and of purified human and rat GSH S-transferases (GSTs) on the covalent DNA binding of 3 putative ultimate food-borne carcinogens, the N-acetoxy derivatives of 2-amino-i-methyl

The effects of glutathione (GSH) and of purified human and rat GSH S-transferases (GSTs) on the covalent DNA binding of 3 putative ultimate food-borne carcinogens, the N-acetoxy derivatives of 2-amino-i-methyl 6-phenylimidazo(4,5-b)pyridine (PhIP), 2-amino-3-methylimldazo(4,5-f) quinoline (IQ), and 2-amlno-3,8-dlmethylimidazo(4,5-f)quinoxaline (MeIQx), were studied in vitro.GSH (5 mM)alone slightly inhibited (10%) the DNA binding of N-acetoxy-PhIP (100 pM)at pH 7.5, but the binding could be strongly inhibited in the presence ofboth GSH and GSTs. Among human GSTs, the isozyme Al-i (a-class) was most effective (90% Inhibi tion) foHowed by Ai-2 (40% inhibition); the effect of adding A2-2 was negligible, suggesting that the activity exists in subunIt Al. In addition, human GST P1-i (it-class) also had some inhibitory effect (30%). Among the rat GSTs tested, GST 1-2 and GST 12-12 (0-clam), which are the equivalent of human Ai-2 and T2-2, respectively, were able to inhibit DNA binding of N-acetoxy-PhIP (75 and 40%, respectively). This activity toward N-acetoxy-PhIP was dependent on enzyme concentration and was subject to inactivation by triethyltin bromide, a known GST inhibitor. In contrast, the binding of N-acetoxy-IQ or N-acetoxy-MeIQx to DNA was unaffected by addition of the human or rat GSTs; however, GSH alone significantly inhibited (40%) their binding to DNA. High-performance liquid chromatographic analyses of incubation mixtures containing Nacetoxy-PhIP, GSH, and GST Al-i failed to detect GSH conjugates of Phil'. Only oxidized glutathione and the parent amine, PhIP, were do tected as reaction products, suggesting a redox mechanism. GST activity in human hepatic and colon mucosal cytosols was subse quently examined using the synthetic or O-acetyltransferaso-generated N-acetoxy derivatives of PhIP, IQ, and MeIQx as substrates. GST activity toward N-acetoxy-PhIP was expressed in all 8 livers but not in 6 colons. No activity toward N-acetoxy-IQ or N-acetoxy-MeIQx was detected In human liver cytosols. This study indicates that a GST-dependent detoxification pathway may be an Importantdeterminant for the organ specificity of the heterocyclic amine carcinogens. Moreover, the high specificity of the reaction for GST Al-i, which is known to be inducible by cruciferous and yeliow-green vegetable consumption, is consistent with the protective effects of such diets against human colorectal cancer.