GLUTATHIONE CONJUGATION OF AFLATOXIN-B1 EXO-EPOXIDES AND ENDO-EPOXIDES BY RAT AND HUMAN GLUTATHIONE S-TRANSFERASES

Much evidence supports the view that the rate of conjugation of glutathione (GSH) with aflatoxin B1 (AFB1) exo-epoxide is an important factor in determining the species variation in risk to aflatoxins and that induction of GSH S-transferases can yield a significant protective effect. An assay has been developed in which the enzymatic formation of the conjugates of GSH and AFB1 exo-epoxide and the recently described AFB1 endo-epoxide is measured directly. H-1 NMR spectra are reported for both the AFB1 exo- and endo-epoxide-GSH conjugates. Structural assignments were made by comparison with AFB1 exo- and endo-epoxide-ethanethiol conjugates, for which nuclear Overhauser effects were measured to establish relative configurations. The endo-epoxide was found to be a good substrate for GSH conjugate formation in rat liver cytosol while mouse liver cytosol conjugated the exo-epoxide almost exclusively. Human liver cytosol conjugated both epoxide isomers to much lower extents than did cytosols prepared from rats or mice. Purified rat GSH S-transferases catalyzed the formation of the AFB1 exo-epoxide-GSH conjugate in the order 1-1 approximately 4-4 approximately 3-3 > 2-2 > 4-6 (7-7 and 8-8 did not form the exo-epoxide-GSH conjugate at levels above the nonenzymatic rate). The only rat GSH S-transferases that conjugated the endo-epoxide were 4-4 and 4-6, with 4-4 being the more active. Purified human GSH S-transferases catalyzed the conjugation of the exo-epoxide in the order M1a-1a > A1-1 > A2-2 (P1-1 and M3-3 did not have detectable activity), while only GSH S-transferase M1a-1a conjugated the endo-epoxide. These results are important in the extrapolation of work with animal models to human risk assessment. Further studies on the biological activity of AFB1 endo-epoxide will be required to evaluate its significance; however, the GSH and mercapturic acid derivatives of the endo- as well as the exo-epoxide should be considered in animal and human studies.