In vitro metabolism of aflatoxin B1 by normal and tumorous liver tissue from Thailand

Liver tissues were obtained hom 20 liver cancer patients from Thailand, an area where the incidence of this tumour is high and where exposure to aflatoxin occurs. The expression of hepatic cytochrome P450s (P450) and glutathione S-transferase (GST) was examined and this expression was compared to the in vitro metabolism of aflatoxin B-1 (AFB(1)). There was a >10-fold inter-individual variation in expression of the various P450s including CYP3A4 (57-fold), CYP2B6 (56-fold) and CYP2A6 (120-fold). Microsomal metabolism of AFB(1) to AFB(1) 8,9-epoxide (as measured by AFB(1) tris-diol formation) and aflatoxin Q(1) (AFQ(1)), the major metabolite produced, was significantly correlated with CYP3A3/4 expression (P < 0.001) and, to a lesser extent, with CYP2B6 expression (P < 0.01). There was a significantly reduced expression of major P450 proteins in microsomes from liver tumours compared to microsomes from the paired normal liver when analysed by Western immunoblot analysis. The production of AFQ(1) and AFB(1) tris-diol was almost uniformly reduced in tumours, but interestingly, the production of AFP(1) was significantly increased. The immunoreactive expression of the major human classes of cytosolic GSTs (alpha, mu and pi) was also analyzed in normal and tumorous liver tissue. The expression of GSTA (alpha) and GSTM (mu) class proteins was markedly decreased and GSTP (pi) increased in the majority of tumour cytosols compared to normal liver. The cytosolic GST activity (1-chloro-2,4-dinitrobenzene conjugation) was significantly lower in liver tumours compared to normal liver (193 +/- 149 versus 875 +/- 299 nmol/min/mg, P < 0.0001), as was glutathione peroxidase (GPx) activity (cumene hydroperoxide) (26 +/- 23 versus 70 +/- 26 mnol/min/mg respectively, P < 0.0005). Ten out of 14 individuals (71%) were homozygous mull when genotyped for GSTM1. There was no detectable conjugation of AFB(1) 8,9-epoxide to glutathione by cytosol either from tumorous or normal liver. Thus, capacity of human cytosols to conjugate reactive AFB(1) metabolites to GSH resembled AFB(1)-sensitive species such as rat, trout and duck rather than resistant species such as mouse and hamster. These data indicate a strong capacity of multiple forms of human hepatic P450s to metabolize AFB(1) to both the reactive intermediate AFB(1) 8,9-epoxide and the detoxification product AFQ(1). These results suggest that in view of the lack of significant GST-mediated protection against AFB(1) in human liver, variations in expression of hepatic P450, due either to genetic polymorphisms or to modulation by environmental factors, may be important determinants in the risk of liver cancer development in AFB(1)-exposed populations.