Aryl Hydrocarbon Hydroxylase Induction in Mammalian Liver Cell Culture

Abstract

Abstract Aryl hydrocarbon hydroxylase activity is inducible in rat fetal hepatocytes in culture by phenobarbital, the insecticide, 2,2-bis(p-chlorophenyl)-1,1,1-trichloroethane (p,p'-DDT), benz[a]anthracene, or 3-methylcholanthrene. Between the 2nd and 4th day of age of the primary cultures, the maximal net rate at which the hydroxylase activity accumulates is about the same when phenobarbital, benz[a]anthracene, or 3-methylcholanthrene is in the growth medium at optimal concentrations. The optimal inducing concentrations for each compound are 2.0 mm phenobarbital, 100 µm p,p'-DDT, 13 µm benz[a]anthracene, and 0.75 µm 3-methylcholanthrene. The kinetic data of the oxygenase induction and degradation are the same in liver cell cultures exposed to either phenobarbital or benz[a]anthracene; the time required for doubling the hydroxylase activity is about 3.0 hours, and the initial half-life of the induced enzyme system is about 10½ hours. An additive effect is obtained when either phenobarbital or p,p'-DDT is present with a polycyclic hydrocarbon in the medium, but not when the hepatocytes are treated with phenobarbital plus p,p'-DDT, or with the combination of benz[a]anthracene plus 3-methylcholanthrene. In fetal liver cells exposed to phenobarbital for 3 days, total hepatocellular RNA synthesis does not change significantly or may increase slightly, whereas in hepatocytes treated with benz[a]anthracene for 1 to 3 days, gross RNA synthesis decreases about 30%. There is a 15 to 20% decline in total protein synthesis in liver cell cultures treated with either phenobarbital or benz[a]anthracene for 1 to 3 days. The apparent Michaelis constants for the control hydroxylase and the enzyme system inducible by either phenobarbital or benz[a]anthracene are similar, ranging between 2 and 4 µm in the presence of the substrate, benzo[a]pyrene.