Effects of various enzyme inducers on monooxygenase, glutathione S-transferase and epoxide hydrolase activities in cultured hepatoma cells

Abstract

Compounds which induce microsomal monooxygenase, cytosolic glutathione S-transferase or microsomal epoxide hydrolase, in vivo were added to the medium of cultured Reuber hepatoma cells for about one generation time. Many compounds, including polycyclic hydrocarbons, 7,8-benzoflavone (7,8-BF), 5,6-benzoflavone (5,6-BF), Aroclor 1254 and some heteropolycyclic aromatic compounds led to an about 30-fold increase in monooxygenase activity with 7-ethoxycoumarin as substrate. The concentration required for maximal induction varied considerably among this group of compounds and was less than 0.5 μM for 3-methylcholanthrene, benzo[ a]pyrene, dibenz[ a, h]anthracene, picene and 5,6-BF, about 10 μM for 7,8-BF, 7-methylbenz[ c]acridine, 12-methylbenzo[ a]acridine and Aroclor 1254 and about 150 μM for benzo[ e]pyrene. In contrast, the maximal increase with 2-aminofluorene was only 4-fold, which suggests a different mechanism for the increase in activity. Phenobarbital and pregnenolone-16α-carbonitrile caused only slight increases at very high doses, whereas trans-stilbene oxide and quercetin were completely inactive. In contrast to the potent induction of monooxygenase activity, none of these compounds affected glutathione S-transferase activities and only three compounds (5,6-BF, 7,8-BF and 12-methylbenzo[ a]acridine) affected epoxide hydrolase. These effects, 1.3–2-fold increases in activity, were due to enzyme activation, in that they occurred immediately after addition of the compounds to the cell culture or cell homogenate. In contrast, the increase in monooxygenase activity (studied in detail with 5,6-BF) was only observed several hours after the exposure of the cells to the test compound and was preceded by a period in which the activity was decreased up to 90%. This study shows that when 5,6-BF and 7,8-BF are used to determine whether the toxic effect of a compound is mediated by monooxygenases, effects of these flavones on epoxide hydrolase activity should also be taken into account. The different time course of the flavone-induced effects on monooxygenase and epoxide hydrolase activities and the lack of effect of other monooxygenase inducers on epoxide hydrolase could be used to differentially manipulate the two enzyme activities, establishing experimental conditions whereby the toxicological effects of altering either enzyme could be independently investigated.