Direct measurement of aminopyrine N-demethylase and antipyrine hydroxylase activities in a monolayer rat primary isolated hepatocyte system.

This paper describes a simple method for monitoring changes in aminopyrine N-demethylase and antipyrine hydroxylase activities in isolated primary hepatocyte monolayer culture. Aminopyrine N-demethylase activity was determined by monitoring the rate of formation of 14CO2 derived from the N-demethylation of [dimethylamino-14C]aminopyrine (AP). The rate of AP N-demethylation increased linearly with time for 60 min and proportionately with cell concentrations between 4.1 x 10(5) to 1.67 x 10(6) cells/incubation. As expected, non-linear AP N-demethylase kinetics were observed with hepatocytes as well as with microsomal preparations derived from control rats. Hepatocytes prepared from phenobarbital (PB)-pretreated animals exhibited increased AP N-demethylase activity and typical Michaelis-Menten kinetics. In contrast, microsomal preparations from PB-treated animals exhibited non-linear N-demethylase kinetics that differed from the kinetics of preparations derived from control animals. Antipyrine hydroxylase activity was determined by monitoring the rate of formation of non-extractable conjugated 4-hydroxyantipyrine from [N-14C-methyl]antipyrine. Antipyrine hydroxylase activity was found to increase linearly for 120 min and proportionately with cell concentrations. Antipyrine hydroxylation by hepatocytes prepared from control and PB-pretreated animals followed typical Michaelis-Menten kinetics. AP N-demethylase activity immediately after plating was 10 per cent lower than at 4 hr, whereas antipyrine hydroxylase activities were similar. Culturing hepatocytes for 24 hr resulted in a decline to 40 and 60 per cent of control for AP N-demethylase activity and antipyrine hydroxylase activity respectively.