Substrate-inducible Microsomal Aryl Hydroxylase in Mammalian Cell Culture

Abstract

Aryl hydroxylase, derived from hamster fetus cells grown in culture, is an inducible microsomal enzyme system which requires NADPH and molecular oxygen. The addition of benz[a]anthracene or other polycyclic hydrocarbons to the culture medium causes significant increases in the amount of hydroxylase activity: after a lag period of 35 min, the level of hydroxylase activity increases linearly for 16 to 32 hours, at which time the levels are 15- to 40-fold higher than that of control cells. Under similar conditions, neither corticosteroids nor phenobarbital has any effect on the aryl hydroxylase activity. The ability to induce the hydroxylase activity is greatest in cells entering logarithmic growth phase and in secondary and tertiary cultures. Aryl hydroxylase is also induced in fetal cells from mouse and rat; induction is much lower in chick and guinea pig. The aryl hydroxylase enzyme system is inducible in hamster fetus cells derived from lung, small intestine, liver, limb, and brain, and is greatest in cells from older fetuses. The carbon monoxide-binding microsomal cytochrome from cells exposed to benz[a]anthracene has an absorption peak at 446 mµ, compared to 450 mµ for the microsomal fraction from control cells. This alteration may be related to the increased aryl hydroxylase activity. The addition of actinomycin D, puromycin, or cycloheximide simultaneously with benz[a]anthracene completely prevents the induction of hydroxylase activity. In cells previously induced for 25 hours, either the removal of inducer or the addition of puromycin (with or without inducer) causes a fall in aryl hydroxylase activity after 3 hours. The half-life of the induced enzyme activity is 3.3 ± 1.2 hours. The addition of actinomycin D, alone or with inducer, to previously induced cells causes a greater increase in enzyme activity for 4 hours than does benz[a]anthracene alone. These studies suggest that the induction of aryl hydroxylase activity involves an activation of specific genes and changes in their transcription into messenger RNA.