Substrate-inducible Microsomal Aryl Hydroxylase in Mammalian Cell Culture

TLDR

The aryl hydroxylase enzyme system is inducible in hamster fetal cell cultures. The study uses a mammalian cell culture system to investigate microsomal enzyme induction mechanisms relevant to carcinogenesis and drug and steroid metabolism. The enzyme resides in the microsomal fraction, requires NADPH, O₂, pH 7.5, and divalent cations, and its activity is quantified by a sensitive spectrophotofluorometric assay detecting hydroxylated benzo[a]pyrene products. Exposure to carbon monoxide, EDTA, or trypsin inhibits the enzyme, which has a Km of ~0.6 µM for benzo[a]pyrene and accepts multiple polycyclic aromatic hydrocarbons as substrates.

Abstract

Abstract The aryl hydroxylase enzyme system is inducible in hamster fetus cell cultures. The enzyme system is localized in the 105,000 x g pellet (microsomal fraction), has an absolute requirement for NADPH and molecular O2, a pH optimum of pH 7.5, and a partial requirement for divalent cations. Exposure to carbon monoxide reduces the enzyme activity. Treatment with ethylenediaminetetraacetate or trypsin completely prevents enzymatic activity. The Km for the hydroxylase is approximately 0.6 µm with benzo[a]pyrene as substrate. Benz[a]anthracene, 7,12-dimethylbenz[a]anthracene, 3-methylcholanthrene, dibenz[a,h]anthracene, and dibenz[a,c]anthracene are also substrates for the enzyme system. The spectrophotofluorometric determination of hydroxylated benzo[a]pyrene products is sufficiently sensitive to detect 10-12 mole per ml and, hence, has great utility in measuring the hydroxylase activity of cells grown in culture. This mammalian cell culture system is advantageous for the study of the mechanism of microsomal enzyme induction and the related areas of carcinogenesis and drug and steroid metabolism.