Infections in humans, and inflammatory stimuli in experimental animals, impair hepatic drug metabolism and cause decreases in the activities and expression of many forms of cytochrome P450 (P450). In humans, this can result in elevated plasma levels of therapeutic agents, and subsequent drug-related toxicity. Recent studies in other laboratories have indicated that nitric oxide (NO) produced by hepatic inducible NO synthase (iNOS) may be involved in both catalytic inhibition of cytochrome P450 and in down-regulation of P450 gene expression evoked by bacterial endotoxin. However, these studies have not addressed inflammation of different etiology (that have been reported not to induce hepatic iNOS), and have been performed only in the presence of chemical inducers of P450. Since NO is an important mediator of inflammation, and inhibition of NO synthase is a potential therapeutic modality for inflammatory conditions, it is important to understand the contribution of NO to changes in P450 levels that occur in inflammation. The goals of this project are to define similarities and differences in the suppression of inducible and constitutive P450 activities and gene expression in three different models- of inflammation: a) bacterial endotoxemia; b) a remote localized inflammation; c) injection of an interferon inducer, and to evaluate the contribution of NO to the inhibition of P450 catalytic activity and downregulation of P450 gene expression that occurs in these in vivo models. In these experiments, the role of NO will be evaluated in both phenobarbital-treated and uninduced rats by examining the effects of a NOS inhibitor on the responses of the P450 system to the three different inflammatory stimuli. Plasma levels of nitrogen oxides will be measured to ensure that the inhibitor indeed reduces NO production. Levels of inflammatory cytokines in the plasma will be measured to assess the effects of the inhibitor on the primary systemic response to inflammation. Hepatic levels of protein and mRNAs of representatives of the major P450 subfamilies expressed in liver will be measured by RNA filter hybridization and Western blotting analyses, respectively. Inhibition of the catalytic activities of these forms will be assessed by comparing the levels of expression of the proteins with diagnostic drug and steroid metabolizing activities associated with specific forms of P450. The specific role of hepatic iNOS in P450 inhibition and expression will be tested in primary rat hepatocytes cultured on Matrigel, using NOS inhibitors and inhibitors of tetrahydrobiopterin synthesis to block the activity of induced iNOS. Constitutive as well as phenobarbital-induced P450 expression will be studied in this model.
