Pyridine effects on P450IIE1, IIB and IVB expression in rabbit liver: characterization of high- and low-affinity pyridine N-oxygenases

Abstract

The effects of pyridine exposure on expression of cytochromes P450IIE1, IIB and IVB in rabbit hepatic microsomes and their respective role in pyridine N-oxide production has been examined. Immunoblot analysis revealed that pyridine administration caused a substantial increase in P450IIE1 levels, failed to affect P450IIB content and marginally increased the expression of P450IVB. In an effort to implicate specific forms of P450 in pyridine N-oxide production, the kinetics of pyridine N-oxide formation in uninduced and induced rabbit hepatic microsomal preparations were obtained. Pyridine-induced microsomes exhibited a single low Km value of 81 microM with a approximately 2.5-fold increase in Vmax (2.44 nmol/min/mg protein) relative to uninduced microsomes. Interestingly, pyridine N-oxide production in phenobarbital-induced microsomes were also monophasic, exhibiting a single, high Km value of 949 microM and a Vmax of 3.3 nmol/min/mg protein, a approximately 10-fold increase over the uninduced preparations. In contrast, uninduced and isosafrole-induced rabbit hepatic microsomes both exhibited biphasic kinetics; uninduced microsomes gave Km values of 85 and 973 microM, whereas isosafrole-induced microsomes yielded Km values of 229 and 1733 microM, respectively, with a Vmax somewhat less than uninduced microsomes. When kinetic data were normalized for P450 content, a pronounced substrate specificity was detected for both pyridine- and phenobarbital-induced microsomes. para-Nitrophenol hydroxylase activity was enhanced approximately 6-fold in pyridine-induced microsomes consistent with elevated levels of P450IIE1. para-Nitrophenol competitively inhibited (Ki = 13 microM) the production of pyridine N-oxide in pyridine-induced microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)