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Induction of Cytochrome P-450 and 5-Aminolevulinate Synthase Activities in Cultured Rat Hepatocytes¹

V. A. Medical Center, White River Junction, Vermont 05001 [P. R. S., W. J. B., S. A. H., J. F. S.]; Department of Biochemistry, Dartmouth Medical School, Hanover, New Hamsphire 03756 [P. R. S., S. A. H., J. F. S.]; and Division of Clinical Toxicology and Environmental Medicine, Department of Medicine, Medical College of Virginia, Richmond, Virginia 23298 [P. S. G.]

Cytochromes P-450IIB1 and P-450IIB2 were recently shown to be inducible in rat hepatocyte cultures maintained on a reconstituted extracellular tumor matrix (Matrigel) as indicated by increases in P-450IIB1 and -IIB2 mRNAs and immunoreactive proteins (J. Cell. Physiol., 134: 309–323, 1988). Here we show that treatment of cultured rat hepatocytes with phenobarbital and other compounds known to induce P-450IIB1/2 in vivo increased spectral cytochrome P-450, immunoreactive proteins, and benzyloxy- and pentoxy-resorufin dealkylases, activites known to be specific for cytochrome P-450IIB1/2. These increases were observed when cells were cultured on either Matrigel or collagen matrix in Williams E medium. Cytochrome P-450III was also increased by phenobarbital and dexamethasone on either matrix. Propoxycoumarin depropylase activity, which has been proposed as a specific activity catalyzed by cytochrome P-450III, was increased 3–4-fold more by treatment with 3-methylcholanthrene than by phenobarbital or dexamethasone. The activity catalyzed by P-450III could be distinguished from that catalyzed by other P-450 forms using the specific inhibitor triacetyloleandomycin. Benzoyloxyresorufin dealkylase was also increased in these cells by treatment with 2,4,5,2',4',5'-hexachlorobiphenyl, glutethimide, or mephenytoin. Treatment with phenobarbital or 2-allyl-2-isopropylacetamide slightly induced 5-aminolevulinate synthase activity. 5-Aminolevulinate synthase activity was slightly increased in cells treated with phenobarbital or 2-allyl-2-isopropylacetamide. Succinyl acetone also induced 5-aminolevulinate synthase activity and, in combination with either of the other two drugs, synergistically increased the enzyme activity regardless of whether cells were cultured on collagen or Matrigel. These results indicate that with simple and economical enzyme assays for bolocytochrome P-450 and 5-aminolevulinate synthase, the rat hepatocyte culture system can be used for studies of the interrelationships between phenobarbital induction of cytochrome P-450 and heme metabolism.

¹ Supported by USPHS Grants CA25012 and AA07146.

² To whom requests for reprints should be addressed, at VA (151), White River Junction, VT 05001.

Received 12/29/89. Revised 5/ 9/90.

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