2(3)-Tert-Butyl-4-Hydroxyanisole Inhibits Oxidative Metabolism of Aflatoxin B 1 in Isolated Rat Hepatocytes

Abstract

Previous studies indicate that dietary administration of phenolic antioxidants, 2(3)-tert-butyl-4-hydroxyanisole (BHA) and 3,5-di-tert-butyl-4-hydroxytoluene, inhibits the carcinogenic effect of a number of chemical carcinogens including aflatoxin B₁ (AFB₁). Induction of hepatic enzymes, such as glutathione S-transferase, UDP-glucuronyltransferase, and epoxide hydrolase, has been shown to be responsible for the reduction of AFB₁ cytotoxic and carcinogenic effects. The effect of BHA on AFB₁ activation was examined in vitro utilizing isolated rat hepatocytes and liver microsomes. In hepatocytes, the total AFB₁ content and bound form of AFB₁ were 3.4 and 1.4 pmol/10⁶ cells, respectively. In the cell-free microsomal activating system, 2.2 pmol were activated per mg of microsomal protein during 60 min of incubation. BHA (0.1–0.5 mM) inhibited AFB₁ activation and binding in both systems in a dose-dependent manner; in hepatocytes, 90% inhibition was observed at 0.5 mM. Analyzing various AFB₁ adducts, BHA (0.25 mM)-treated hepatocytes contained a significantly reduced amount of AFB₁ macromolecular adducts. The antioxidant neither stimulated nor inhibited the cytosolic glutathione S-transferase and microsomal UDP-glucuronyltransferase activities. Analysis of various hydroxylated (aflatoxins M₁ and Q₁ (AFM, and AFQ₁)) and demethylated (aflatoxin P₁ (AFP₁)) metabolites of AFB₁ in both the conjugated and unconjugated form indicated that there was a 30–50% reduction of unconjugated AFP₁, AFQ₁, and AFM₁, whereas AFB₁ was increased 3-fold. There was no significant change of conjugated metabolites. The effect of BHA on AFB₁ activation in hepatocytes was compared with that of other cytochrome P-450 inhibitors; the ED₅₀ values of SKF 525A, BHA, and metyrapone were 9 μM, 40 μM, and 280 μM, respectively. In the cell-free microsomal system, biotransformation of AFB₁ to AFP₁, AFM₁, and AFQ₁ was also inhibited. Kinetic analysis of p-nitroanisole O-demethylase activity of rat liver microsomes demonstrated that BHA inhibited noncompetitively with an apparent K_i of 90 μM. In the absence of enzyme induction, the phenolic antioxidant, BHA, blocks the oxidative biotransformation of AFB₁ in isolated hepatocytes.