Comparative metabolism, covalent binding and toxicity of BHT congeners in rat liver slices

The metabolism, covalent binding and hepatotoxicity of butylated hydroxytol uene (BHT, 4-methyl-2,6-di-t-butylphenol) and two congeners (E-BHT, 4-ethyl -2,6-di-t-butylphenol; I-BHT, 4-isopropyl-2,6-di-t-butylphenol) were compar ed using precision-cut liver slices prepared from phenobarbital (PB)-treate d male Sprague-Dawley rats. At equimolar concentrations (1 mM) BHT was the most toxic of the three compounds, causing an 80% decrease in cell viabilit y over a 6 h incubation period. E-BHT was intermediate in toxicity while th e isopropyl derivative was relatively nontoxic. Intracellular glutathione l evels decreased prior to the onset of cytotoxicity. The cytochrome P450 inh ibitor metyrapone completely inhibited the toxicity of all three Compounds. The rates of metabolism of the three compounds to glutathione conjugates w ere compared in both PB-treated microsomes and PB-induced liver slices. In both models, the rate of formation was greatest for BHT, followed by E-BHT and I-BHT. Synthetic quinone methides (QMs) were prepared from each parent phenol and the rates of reactivity with three nucleophiles (water, methanol and glutathione) were compared. With each nucleophile, BHTQM was the most reactive, while I-BHTQM was the least reactive. Finally, covalent binding t o protein was assessed in two ways. First, alkylation of an isolated model protein (bovine insulin) was measured in a microsomal enzyme activation sys tem by mass spectrometry. Incubations with BHT produced the greatest extent of protein alkylation, followed by E-BHT, while no alkylation was observed with I-BHT. In the second system, covalent binding to cellular protein was assessed in rat liver PB microsomes and tissue slices by Western blotting using an antibody specific for the tert-butylphenol portion of the compound s. Binding was greatest for BHT, intermediate for E-BHT and could not be de tected for I-BHT. The alkylation pattern for E-BHT was strikingly similar t o that of BHT, suggesting that both compounds bound similar proteins. In su mmary, our results suggest that for hindered phenols such as BHT, increasin g the length of the 4-alkyl substituent retards the rate of formation of re active intermediates, significantly reduces the electrophilicity of the rea ctive intermediate, and greatly reduces the amount but not the selectivity of covalent binding to cellular protein, thereby reducing the toxicity of t he parent compound. (C) 2001 Elsevier Science Ireland Ltd. All rights reser ved.