Detoxication of Aflatoxin B1 as a Model for Carcinogen Metabolism

S. Langouët, W. W. Johnson, A. Guillouzo and F.P. Guengerich
Department of Biochemistry and Center in Molecular Toxicology
Vanderbilt University
Nashville, TN
and INSERM U456
University de Rennes I
France

Aflatoxin B1 (AFB1) is a powerful carcinogen which plays an important role in the etiology of human liver cancers. This procarcinogen is activated by cytochrome P450 (CYP) enzymes to give a number of products including the exo-8,9-epoxide which is responsible for its mutagenic and hepatocarcinogenic potential (1,2). Mainly human CYP3A4 and CYP1A2 at a lower extent are involved in the activation to the epoxide and in the formation to less dangerous metabolites. The reaction of exo-8,9-epoxide with DNA involves the intercalation of the epoxide between base pairs and proton field is postulated to provide acid catalysis to the conjugation reaction (3). The detection of several metabolites in primary cultures of human hepatocytes clearly shows that only glutathione (GSH) transferase M1-positive individuals are able to conjugate the epoxide to GSH (4). This observation is in agreement with the variation of enzyme efficiency by estimation of kinetic parameters among individual recombinant GSH transferases which are in the order (rat) 10-10 >>3-3 > (human) M1-1 > T1-1> A1-1 > P1-1 > A2-2 (5). The hydrolysis of the epoxide constitutes another detoxication pathway against AFB1 and is mainly due to spontaneous reaction rather than catalyzed by epoxide hydrolase since rat and human epoxide hydrolases show very little rate acceleration of hydrolysis of AFB1 epoxide (6,7). The knowledge of these data on reaction of the exo-8,9-epoxide is critical to predict the best strategies for chemoprotection. The effects of potential chemoprotective agents such as oltipraz, a synthetic dithiolethione, and sulforaphane, an isothiocyanate, were investigated. For this purpose, we used primary cultures of rat and human hepatocytes which have been proved to be a good model for metabolism studies (8-10). We were able to show that their protection is due to an inhibition of activation of AFB1 in addition to a GSH transferase-dependent inactivation of the carcinogenic exo-epoxide (4). Indeed both CYP1A and CYP3A4 are inhibited by oltipraz and sulforaphane while GSH transferases A1 and A2 are mainly induced compared to GSH transferases M1 (4,11,12).

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