Effects of human and rat glutathione S-transferases on the covalent DNA binding of the N-acetoxy derivatives of heterocyclic amine carcinogens in vitro: a possible mechanism of organ specificity in their carcinogenesis.

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-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP), 2-amino-3-methylimidazo(4,5-f)quinoline (IQ), and 2-amino-3,8-dimethylimidazo(4,5-f)quinoxaline (MeIQx), were studied in vitro. GSH (5 mM) alone slightly inhibited (10%) the DNA binding of N-acetoxy-PhIP (100 microM) at pH 7.5, but the binding could be strongly inhibited in the presence of both GSH and GSTs. Among human GSTs, the isozyme A1-1 (alpha-class) was most effective (90% inhibition) followed by A1-2 (40% inhibition); the effect of adding A2-2 was negligible, suggesting that the activity exists in subunit A1. In addition, human GST P1-1 (pi-class) also had some inhibitory effect (30%). Among the rat GSTs tested, GST 1-2 and GST 12-12 (theta-class), which are the equivalent of human A1-2 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 N-acetoxy-PhIP, GSH, and GST A1-1 failed to detect GSH conjugates of PhIP. Only oxidized glutathione and the parent amine, PhIP, were detected as reaction products, suggesting a redox mechanism. GST activity in human hepatic and colon mucosal cytosols was subsequently examined using the synthetic or O-acetyltransferase-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 important determinant for the organ specificity of the heterocyclic amine carcinogens. Moreover, the high specificity of the reaction for GST A1-1, which is known to be inducible by cruciferous and yellow-green vegetable consumption, is consistent with the protective effects of such diets against human colorectal cancer.