The objective of the research is to elucidate the role and mechanism of action of cytokines mediators of the acute phase response in regulation of cytochrome P450 (P450), a family of hemoproteins responsible for biotransformation of many drugs, other chemicals, and certain endogenous substrates. The cytokines to be studied are interleukin 6 (IL-6), interleukin 1 (IL-1), interferon (INF), and tumor necrosis factor (TNF). Cytokine effects on P450 may explain altered biotransformation occurring during infection and inflammation. In addition, effects on P450 by cytokines when used in cancer immunotherapy or other immune disorders may result in drug-drug interaction, influencing the disposition and toxicity of concurrently used drugs. Although effects on P450 have been shown during the acute phase response, or after the administration of cytokines, because of the complex interplay among the cytokines to affect each others synthesis, release, and biological activity it is unclear from in vivo studies which cytokines directly regulate P450. The project will use cultured rat hepatocytes to determine which cytokines directly regulate P450, and if P450 isoforms are differentially affected by cytokines. Rat hepatocytes will be cultured on Matrigel matrix in Williams E medium containing insulin and dexamethasone. Recently, such rat hepatocyte cultures been shown to be inducible by phenobarbital (PB) for P450IIB 1/2 to levels obtained in vivo. Preliminary results with hepatocyte culture demonstrated a direct effect of IL-6 to inhibit the PB induction of P450IIB 1/2. Hepatocyte cultures will be used to further elucidate the concentration response and temporal relationship for IL-6 to alter PB induction of benzyloxyresorufin O-deethylase activity, a measure of P450IIB1/2 enzymatic activity. Western blot analysis using antibodies to P450IIB1/2 will be used to study effects of IL-6 on the level and turnover of P45011B 1/2 protein. Solution hybridization on total RNA of hepatocytes will be performed using a labelled cDNA oligonucleotide probe for P450IIB1 and P450IIB2 mRNA to evaluate effects of IL-6 on mRNA level and turnover. Transcription rate will be determined using nuclear run-off assay. Similar experiments will be used to evaluate the ability of IL-1, TNF and INF to inhibit P45011B 1/2 induction. Thirdly, the project will determine if IL-6, IL- 1, TNF, and INF affect the induction of other P450 isoforms in rat hepatocyte cultures. Effects of cytokines on 3- methylcholanthrene induction of P450IA1/2 and ethanol induction of P450IIE1 will be examined. Ethoxyresorufin O-deethylase activity, and p-nitrophenol hydroxylase activity will be assayed as indices of P450IA1/2, and P450IIE enzymatic activity, respectively. Specific antibodies and labelled cDNA oligonucleotide probes for P450IA1/2, and P450IIE1 will be used in Western blot analysis and solution hybridization assays, respectively, to assess cytokine effects on level and turnover of protein and mRNA. Knowledge of the effects of cytokines on P450 is of utmost importance for understanding alterations in pharmacokinetics during infection or inflammation, as well as during cancer immunotherapy.
