Modulation of aflatoxin B1 biotransformation by beta-naphthoflavone in isolated rabbit lung cells.

Abstract

The cytotoxic and carcinogenic mycotoxin aflatoxin (AF) B1 (AFB1) is biotransformed by the cytochrome P450 monooxygenases (CYP) to a number of relatively nontoxic metabolites, as well as to the ultimate toxic metabolite, AFB1-8,9-epoxide. In a number of tissues and species, AFB1 hydroxylation to the relatively nontoxic metabolite, AFM1, is induced by beta-naphthoflavone (BNF) treatment. Although the liver is the principle target organ for AFB1 toxicity, the mycotoxin is also toxic and carcinogenic to respiratory tissues. To determine if BNF treatment alters the extent of pulmonary AFB1 bioactivation by enhancing detoxification and thereby decreasing epoxidation, the effects of BNF on pulmonary AFB1 metabolism were examined. Rabbit lung cells, isolated by protease digestion and centrifugal elutriation, were incubated with [3H]AFB1. In nonciliated bronchiolar epithelial (Clara) cell-enriched (45-50%) fractions, [3H]AFM1 production (pmol/mg DNA per 2 h) was increased by prior treatment of rabbits with BNF (80 mg/kg per day, 3 and 2 days before cell isolation) as follows: with 1.0 microM [3H]AFB1; control, 10.6 +/- 2.3; BNF, 30.0 +/- 6.4; with 0.10 microM [3H]AFB1; control, 9.4 +/- 4.7; BNF, 20.6 +/- 5.9. With 1.0 microM [3H]AFB1, prior treatment of animals with BNF abolished formation of [3H]aflatoxicol (AFL) but not [3H]AFQ1. The activation (epoxidation) of [3H]AFB1 was measured indirectly as covalent binding to endogenous DNA. With 1.0 microM [3H]AFB1, treatment of rabbits with BNF did not alter DNA binding (pmol/mg DNA per 2 h) in the Clara cell-enriched fraction: control, 103 +/- 41; BNF, 114 +/- 49. However, with 0.10 microM [3H]AFB1, DNA binding in the same fraction was 47% lower in cells from BNF treated animals: control, 17.4 +/- 4.2; BNF, 9.3 +/- 3.9. Formation of 8,9-dihydro-8,9-dihydroxy-AFB1, and the glutathione conjugate of the aflatoxin epoxide (AFB1-GSH) were not detectable at the AFB1 concentration and time point studied, in cells from either BNF-treated or control rabbits. Incubation of isolated, unseparated lung cells from untreated rabbits with 5.0 to 50 microM BNF decreased [3H]AFB1-DNA binding in the presence of 0.1 microM [3H]AFB1 by 35 to 77%, while lower BNF concentrations did not alter DNA binding. In lung cells isolated from BNF treated rabbits, BNF was not detectable (i.e. < 0.5 microM detection limit). Therefore, the amount of BNF present in isolated rabbit lung cells following in vivo treatment with BNF was below that required to directly inhibit AFB1-DNA adduct formation. The decrease in AFB1-DNA binding from rabbits treated with BNF is apparently due to the selective induction of CYP isozymes and related increases in AFM1 formation, and not to direct inhibition of epoxidation or enhanced conjugation of AFB1-8,9-epoxide with glutathione.