Aflatoxins are mycotoxins commonly found in nature. Aflatoxin B1 (AFB1) has a wide range of biological activities affecting animals, including acute toxicity, teratogenicity, mutagenicity and carcinogenicity. It is often found as a contaminant in foodstuffs such as corn, peanuts and other grains. Cytochrome p450s (CYP) are involved in the metabolism of AFB1. Conjugation of AFB-8,9-oxide (AFBO) to glutathione, mediated by glutathione-s-transferases, is regarded as an important detoxification step. The key human GSTM1 is deleted in half the population, but its role is in protection is unknown since it has weaker activity for AFBO conjugation than the most active rodent GSTs. This study investigated the role of hCYP1A2 in activating AFB1 to AFBO, and the subsequent detoxification by conjugation with specific glutathione-s-transferases. To study the interaction of these phase i and phase ii enzymes, we utilized V79MZ (V79) hamster lung fibroblasts stably transfected with human cytochrome p450 1a2, then super-transfected with either human gst mu-1 (V79h1A2/hGSTM1, clone 9) or mouse GST alpha-3 (V79h1A2/mGSTA3, clone 42). A cell line transfected with empty vector Δpcep4Δ (V79h1A2 Δ10) was also tested as a control. Mice are resistant to the genotoxic effects of afb1 due to their high expression of gst alpha-3, therefore, V79h1A2/mGSTA3 was predicted to show protection. The cells expressing hCYP1A2 activated the AFB1 as evidenced by the >20-fold enhancement of cytotoxicity compared to non-expressing V79cells (IC50 = 5.4 nM vs. >100 nM in the parent cell line). Protection against cytotoxicity in cells co-expressing hGSTM1 was 1.4-fold (IC50 = 7.4 nM; not significant) but cells expressing mGSTA3 showed a 4.4-fold protection (IC50 = 24 nM). Protection by the GSTs against mutagenicity of AFB1 at the hprt locus was stronger overall than the protection against cytotoxicity, and was most evident at the highest concentration of 10 nM. Fold protection against AFB1 mutagenicity at the highest dose was 2.4-fold in cells expressing hGSTM1 and 3.6-fold in cells expressing mGSTA3. When DNA alkylation was investigated, the protection of both GSTs closely mirrored their protection against mutagenicity, with cells expressing hGSTM1 and mGSTA3 showing 2-fold and 3.3-fold reduction in AFB1-DNA adducts, respectively, at 48 hrs of exposure.
[Proc Amer Assoc Cancer Res, Volume 46, 2005]