Activity and kinetic properties of basal aryl hydrocarbon hydroxylase during proliferation in the transformable C3H 10T1/2 CL8 cell line.

ABSTRACT

Basal aryl hydrocarbon hydroxylase (AHH) activity and its kinetic properties were studied as a function of proliferation in C3H mouse embryo 10T1/2 CL8 cells. Activity was low in freshly plated cells, increased during exponential growth, peaked at confluency, and then declined. The apparent Km-values for benzo[a]pyrene (BP) and NADPH were less in proliferating (approx. 0.37 microM BP, 3.3 nM NADPH) than in confluent cells (0.74-1.39 microM BP, 33.4-53.4 nM NADPH). Cells at different growth states responded differently to benz[a]anthracene (BA) and aminophylline, an inhibitor of cyclic nucleotide phosphodiesterases. When cells were harvested at the mid log phase of growth, 12 h of exposure to aminophylline caused maximum induction, while 24 h of BA treatment were required. In contrast, at early confluence, 12 h of BA treatment gave the greatest levels of activity, while exposure to aminophylline did not induce AHH. In fact, decreases in activity were observed. These differences are indicative of different regulatory mechanisms for BA and aminophylline induction. They also suggest the regulation of basal AHH by cyclic nucleotides changes during growth. The exposure times giving maximum activity were used to determine the kinetic properties of BA-induced activity. As with basal AHH, the Km-value for BP was less in log phase (0.2-0.4 microM BP) than in confluent cells (0.64-1.05 microM BP). Moreover, the Km-values for BP and NADPH in control cultures at confluency (0.10-0.14 microM BP, 15.4-23.2 nM NADPH) were less than those for BA-treated cells (0.64 microM BP, 37.9-54.8 nM NADPH) under the same nutritional conditions. The finding that the Km-values for BP is lower in rapidly dividing cells than in confluent cells may help to explain why proliferating cells are more susceptible to transforming agents.