teers administered interferon, decreases in the metabolism Administration of interferons of both the gamma and of antipyrine, 16 theophylline, [18] erythromycin, 20 and hexoalfa/beta classes down-regulates hepatic cytochrome barbital 18 have been observed. Cytokines, including inter-P450 (CYP) genes when administered to humans or rats.
feron gamma (IFN-g), have also been shown to down-regulate In male rats, interferons decrease expression of CYP3A2 the expression and catalytic activities of major CYP enzymes at a pretranslational level, but because interferons also in cultured human hepatocytes. 13 release other cytokines in vivo, it is unclear whether this
In experimental animals, several interferons (including rat is a direct effect on hepatocytes. We therefore examined recombinant IFN-g and natural rat interferon alfa/beta, but the effects of rat recombinant interferon gamma (IFN-g) not heterologous interferons) [21] appeared to decrease exon CYP3A2, other 3A genes, and 2C11 in stable primary pression of CYP genes. The changes in individual CYPs procultures of male rat hepatocytes. Hepatocytes were culduced by interferon-inducing agents and other cytokines aptured on matrigel in Williams' E, and messenger RNAs pear to be relatively nonspecific, with many enzymes being (mRNAs) for 3A2, 3A1-like CYPs, and 2C11 mRNA were suppressed to a similar extent. [3][4][8][10] In earlier work in rats, 22,23 determined by RNase protection assays. CYP3A and we showed that homologous (but not heterologous) interfer-2C11 proteins were immunoquantified, and their cataons administered in vivo produced a relatively specific reduclytic activities were estimated by testosterone hydroxyltion in the important constitutive male-specific enzyme, ation pathways. In control cells, 3A2 mRNA decreased CYP3A2, and this change was exerted at the pretranslational initially but then recovered, and stable levels (15% of level. The decrease in CYP3A protein seemed to account freshly isolated cells) were attained between days 3 and for most of the reduction of total hepatic CYP content in 7. Phenobarbital increased 3A2 mRNA to 60-120% values interferon-treated rats, but changes in other 3A genes have of freshly isolated cells, and mRNA for 3A1-like CYPs not been studied at the molecular level. were increased 20-fold. In both control and phenobarbi-It has been established that the interferons release other tal-treated hepatocytes, rat recombinant IFN-g (33 U/ cytokines in vivo. 25 It is not clear, therefore, whether the mL) reduced mRNA for 3A2 and 3A1-like CYPs, as well effects observed in intact rats are caused by interferons actas 3A protein and testosterone 6b-hydroxylase activity.
ing directly on the hepatocyte or to other cytokines released Interferon had no effect on CYP2C11 at mRNA or protein by the interaction of interferon with other cell types, such levels in untreated cells, although a reduction in 2C11
as Kupffer cells and lymphocytes. Indeed, in earlier work, protein was evident in phenobarbital-treated cultures.
interferon often failed to suppress CYP levels in hepatocyte It is concluded that interferon directly alters expression suspensions or in culture. 4, We therefore conducted experiof constitutive and inducible CYP3A genes in well-differments to ascertain whether interferon acts directly on hepaentiated male rat hepatocytes in culture, but has no eftocytes to decrease expression and activity of CYP 3A2, and fect on constitutive expression of CYP2C11. (HEPATOLto clarify the specificity of this effect. Because CYP3A2 ex-OGY 1996;24:367-373.)
pression initially declines in culture, the first objective of the study was to establish optimal experimental conditions Several viral and bacterial infections, 1 endotoxin, 2,3 immuto maintain 3A2. Having achieved this, it was then possible nization procedures, 1 and interferon-inducing agents 4-6 have to examine down-regulation of CYP genes at steady-state been associated with impaired drug metabolism mediated by levels of messenger RNA (mRNA) expression, and to compare cytochrome P450 (CYP) enzymes. This has been attributed, the results with CYP protein expression and enzymatic activat least in part, to release of cytokines. Thus, tumor necrosis ity. factor a, 2,7,8 the interleukins, (IL), IL-1a and IL-1b, 3,9-14 IL-2, 15 IL-4, 13 and IL-6, 10, and the interferons (see below)
MATERIALS AND METHODS
have all been shown to lower the activity and protein content
Materials. [4-14 C]Testosterone (specific activity 56 mCi/mmol) was of hepatic CYP enzymes. In patients and in healthy volunpurchased from Amersham Australia (Sydney, Australia). Collagenase (type IV), dexamethasone, ethylenediaminetetraacetic acid (EDTA), polyoxyethylene-sorbitan monolaurate (Tween 20), peroxidase-labeled goat anti-rabbit immunoglobulin G, cell culture medium Abbreviations: CYP, cytochrome P450; IL, interleukin; IFN-g, interferon gamma; EDTA, components, steroid standards, and other biochemicals were from ethylenediaminetetraacetic acid; GH, growth hormone; tNA, total nucleic acid; mRNA, mes-Sigma Chemical Co. (St. Louis, MO). Other steroid standards were senger RNA.