Stimulation of de novo synthesis of cytochrome P-450 by phenobarbital in primary nonproliferating cultures of adult rat hepatocytes.

Primary monolayer cultures of nonproliferating parenchymal cells prepared from adult rat liver and maintained in serum-free medium responded to additions of phenobarbital with concentration-dependent increases in synthesis and accumulation of a cytochrome P-450 protein immunochemically and catalytically indistinguishable from that found in the livers of adult rats treated with phenobarbital. Maximal stimulation of the rate of synthesis of this cytochrome protein by phenobarbital, as much as 20-fold higher than in control cultures (1.01% of the rate of synthesis of total cellular protein), could be achieved when the drug was first added to cultures no older than 24 hr and then was maintained in the medium for 96 hr. In addition to phenobarbital, chemicals classified as "phenobarbital-like" inducers in vivo (mephenytoin, mirex, 2,2',4,4',5,5'-hexabromobiphenyl) induced synthesis in culture of this same immunoreactive protein. Supplementation of the medium with 0.1 microM H2SeO3 plus phenobarbital produced an average 2-fold enhancement in the rate of synthesis of this inducible cytochrome protein as compared to that in cultures receiving phenobarbital alone. Inasmuch as there was a decline in selenium content and in the activity of the seleno-enzyme glutathione peroxidase in hepatocyte cultures maintained in standard culture medium for more than 24 hr, the added selenium appears to correct a spontaneously acquired cellular deficiency in selenium. Contrary to the concept that liver cells placed in culture promptly dedifferentiate with general loss of specialized functions such as cytochrome P-450, our data demonstrate that expression of the phenobarbital-inducible form of cytochrome P-450 is not extinguished in culture, but rather it is masked transiently and is attenuated as the cells adapt to the imperfect conditions of the culture environment.