Molecular Pharmacology, Vol 6, 304-314, Copyright © 1970 by the American Society for Pharmacology and Experimental Therapeutics

Fate of Inducer during Induction of Aryl Hydrocarbon Hydroxylase Activity in Mammalian Cell Culture

II. Levels of Intracellular Polycyclic Hydrocarbon during Enzyme Induction and Decay

¹ Section on Developmental Enzymology, Laboratory of Biomedical Sciences, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20014

The induction and degradation of aryl hydrocarbon hydroxylase activity in hamster fetal cell cultures was studied in the presence and absence of actinomycin D and/or cycloheximide; the concentration of polycyclic hydrocarbon in these cells was examined concomitantly. The level of intracellular polycyclic hydrocarbon diminishes after about 30 min of exposure of the cells to the inducer benz[a]anthracene. This decrease is presumably related to the appearance of the induced microsomal oxygenase.

We have estimated the minimal number of molecules of benz[a]anthracene per cell that are sufficient to stimulate the initial rate of hydroxylase induction maximally. In cells exposed to inducer plus cycloheximide and then grown in fresh control medium, no correlation exists between the amount of intracellular polycyclic hydrocarbon physically or covalently bound and the kinetics of hydroxylase induction. Exposure of the cells to benz[a]-anthracene for 20 min or less does not stimulate hydroxylase activity if RNA synthesis is prevented thereafter. Inhibition of RNA synthesis at any time after 30 min of exposure of the cells to inducer does not prevent the initial, maximal rate of microsomal oxidase induction. The amount of intracellular polycyclic hydrocarbon that is sufficient to stimulate hydroxylase activity maximally in cells during the first several hours of exposure to inducer does not stimulate enzyme activity in cells exposed to benz[a]anthracene for 12 hr or more.

In cells previously treated with benz[a]anthracene and then grown in fresh control medium, actinomycin D produces a stimulation of hydroxylase activity for 7-10 hr. There is no correlation between the concentration of intracellular polycyclic hydrocarbon and this stimulatory effect. The rate of disappearance of induced hydroxylase activity is not affected by the concentration of intracellular polycyclic hydrocarbon in the presence of cycloheximide or actinomycin D plus cycloheximide.

Note:
ACKNOWLEDGMENTS The authors appreciate their valuable discussions with Dr. H. V. Gelboin and his encouragement to investigate this aspect of the enzyme induction problem. We also gratefully acknowledge the critical reviews of this manuscript by Drs. G. M. Tomkins, G. Guroff, and J. C. Robinson.

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