CHANGES IN THE CONCENTRATION OF SEVEN FORMS OF CYTOCHROME-P-450 IN PRIMARY CULTURES OF ADULT-RAT HEPATOCYTES

  • 1 January 1985
    • journal article
    • research article
    • Vol. 27  (1) , 125-132
Abstract
Primary monolayer cultures of adult rat hepatocytes were prepared and the losses of cytochromes P-450 measured with the use of specific antibodies directed against purified forms of hepatic cytochrome P-450 which predominate in untreated rats (P-450UT-A, P-450UT-F) or in rats treated with phenobarbital (P-450PB-B/D, P-450PB-C, P-450PB/PCNE) or with 3-methylcholanthrene (P-450.beta.NF-B, P-450.beta.NF/ISF-G). In hepatocytes prepared from an untreated rat and incubated in control medium, total cytochrome P-450, measured spectrally as CO-binding hemoprotein, declined 68% during the first 72 h in culture. The sum of the immunoreactive cytochromes P-450 declined only 24%, indicating that loss of heme rather than of protein accounts for much of the well-known loss of cytochromes P-450 in hepatocyte cultures. In cultures prepared from untreated rats or from rats treated with phenobarbital or with 3-methylcholanthrene, individual forms of cytochrome P-450 declined at markedly differing rates. Incubation of cultures in 3 different media previously reported to maintain levels of total cytochrome P-450 failed to prevent the decline in total cytochrome P-450 during the first 24-72 h in culture. In cultures incubated in medium containing metyrapone, the level of holocytochrome P-450 was maintained at the initial value during the first 72 h, apparently by preventing the net loss of cytochrome P-450 heme and by increasing the concentrations of immunoreactive P-450PB/PCN-E and P-450.beta.NF-B. Medium containing nicotinamide increased the proportion of P-450.beta.NF-R relative to the other forms of cytochrome P-450, whereas cysteine-free medium increased P-450UT-F. Evidently loss of cytochrome P-450 in cultured hepatocytes involves loss of its heme moiety coupled with changes in the concentrations of the individual forms. Recognition of these changes as influenced by specific components of the culture medium is important when using primary hepatocyte cultures for study of xenobiotic metabolism and toxicity in the liver.