REGULATION OF RAT GLUTATHIONE-S-TRANSFERASE A5 BY CANCER CHEMOPREVENTIVE AGENTS - MECHANISMS OF INDUCIBLE RESISTANCE TO AFLATOXIN B-1

The rat can be protected against aflatoxin B-1 (AFB(1)) hepatocarcinog enesis by being fed on a diet containing the synthetic antioxidant eth oxyquin. Evidence suggests that chemoprotection against AFB(1) is due to increased detoxification of the mycotoxin by one or more inducible drug-metaboIising enzymes. The glutathione S-transferase (GST) isoenzy mes in rat liver that contribute to ethoxyquin-induced chemoprotection against AFB(1) have been identified by protein purification. This app roach resulted in the isolation of several heterodimeric class alpha G ST(1) all of which contained the A5 subunit and possessed at least 50- fold greater activity towards AFB(1)-8,9-epoxide than previously studi ed transferases, Molecular cloning and heterologous expression of rat GSTA5-5 has led to the demonstration that it exhibits substantially gr eater activity for AFB(1)-8,9-epoxide than other rat transferases. The 45 homodimer can also catalyse the conjugation of glutathione with ot her epoxides, such as trans-stilbene oxide and 1,2-epoxy-3-(4'-nitroph enoxy)propane, and possesses high catalytic activity for the reactive aldehyde 4-hydroxynonenal. Western blotting has shown that the A5 subu nit is not only induced by ethoxyquin but that it is also induced by o ther cancer chemopreventive agents, such as butylated hydroxyanisole, oltipraz, benzyl isothiocyanate, indole-3-carbinol and coumarin. In ad dition to GSTA5, we have identified a novel aflatoxin-aldehyde reducta se (AFAR) that is similarly induced by ethoxyquin. However, immunoblot ting has shown that GSTA5 and AFAR are not always co-ordinately regula ted by chemoprotectors. In order to gain a better understanding of the mechanisms responsible for the induction of GSTA5 protein, the GSTA5 gene has been cloned. It was isolated on two overlapping bacteriophage lambda clones and found to be approximate to 12 kb in length. The tra nscriptional start site of GSTA5 has been identified 228 bp upstream f rom the ATG translational initiation codon. Computer-assisted analysis of the upstream sequence has indicated the presence of a putative ant ioxidant responsive element (located between - 421 and - 429 bp) which may be responsible for the induction of GSTA5 by chemopreventive agen ts. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.