Under conditions of innate immune system activation (i.e., inflammation), the functions of specific cytochrome P450 enzymes, other drug metabolizing enzymes (DMEs), and drug transporters (DTs) are altered in the liver, small intestine, lung, kidney, and central nervous system (CNS). Many of these effects are primarily manifest at the transcriptional/RNA level, leading to corresponding changes in protein levels and function. This not only leads to altered drug and xenobiotic toxicity and action in diseased humans, but also has importance for disease therapy with biologic drugs that target inflammatory mediators or their receptors. Major roles for proinflammatory cytokines such as interleukin‐6 (IL‐6), IL‐1β, and tumor necrosis factor‐α(TNFα) are inferred from the abilities of these agents to affect DMEs and DTs in cultured cells and in vivo , but the in vivo contributions of cytokines to regulation of these proteins in different inflammatory disease states is still poorly understood.

Exposure to inflammatory agents or cytokines causes the suppression of cytochrome P450 (CYP) enzyme activities and expression in liver and primary hepatocyte cultures. We showed previously that phenobarbital-induced CYP2B protein is downregulated in primary cultures of rat hepatocytes after exposure to bacterial endotoxin (lipopolysaccharide) in a nitric oxide (NO) -dependent manner. In this study, we found that CYP2B proteins in primary rat hepatocyte cultures were suppressed >60% after 6 h of treatment with interleukin-1␤ (IL-1). This effect was NO-dependent, and treatment of cells with the NO donors (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl) aminodiazen-1-ium-1,2-diolate (NOC-18), S-nitrosoglutathione, and S-nitroso-N-acetylpenicillamine also suppressed CYP2B proteins. However, the down-regulation by IL-1 was insensitive to inhibition of cGMP-dependent protein kinases. Cytochrome P450s (CYP) 4 are products of multigene families, and many CYPs are involved in biosynthesis and catabolism of physiologically important active molecules such as steroid hormones, sterol, and fatty acids (1). One-third of them are involved in clearance of xenobiotic substances, most of which are expressed in the liver. Inflammation and infection cause the down-regulation of enzyme activity and expression of many CYPs leading to decreased drug clearance, elevation of plasma drug levels, and drug toxicity (2). Inflammation and infection can decrease the metabolic clearances of CYP substrates by 20 -70% (3).Substantial evidence exists to suggest a role of nitric oxide in the regulation of CYP enzyme activities in inflammation. NO is a free radical molecule with important regulatory roles in vasodilation, neurotransmission, inflammation, and cell signaling (4 -6). Inducible nitric-oxide synthase (NOS2, inducible NOS) is induced during infection and inflammation in vivo and in cell cultures treated with bacterial lipopolysaccharide (LPS) or cytokines (7), resulting in high levels of NO production in the cells. It is known that nitric oxide and NO donors are capable of inhibiting the catalytic activities of hepatic microsomal P450s (8 -14). For example, Chlamydia infection resulted in the reduction of CYP1A-and 2B-related metabolism by 49% in mouse liver (15), which was blocked by NOS inhibitors. Three mechanisms are suggested for inhibition of activity; 1) reversible ligation of NO to (primarily ferrous) P450 heme (8); 2) oxidation of P450 protein thiols (16); 3) nitration of specific tyrosine residues on the enzyme (17). CYP8A1 (prostacyclin synthase) and CYP2B1 have been shown to undergo tyrosine nitration by peroxynitrite in cell culture and in vitro, respectively (13,17).Although it is clear that NO production is not globally responsible for the down-regulation of CYP gene expression that occurs in inflammation and infection (18 -20), several reports suggested that inhibition of NOS can attenuate the down-regulation of some hepatic CYP mRNAs or proteins (21-23) or that NO donors can down-regulate some CYP mRNAs (24,25). This evidenc...

The purpose of this study was to determine the role of nitric oxide (NO) in the down-regulation of human CYP enzymes and mRNAs by an inflammatory stimulus in cultured human hepatocytes. We focused on CYP2B6, because previous studies showed that rat CYP2B proteins undergo an NOdependent degradation in response to inflammatory stimuli. To ensure high level expression of CYP2B6, the inducer phenytoin was present at all times. Stimulation of cells with a mixture of TNFα, IL-1β and IFNγ (ILmix) down-regulated CYP2B6 mRNA and protein to 9% and 19% of control levels. The NO donor NOC-18 down-regulated CYP2B6 protein to 30% of control, with only a small effect on CYP2B6 mRNA. NOS inhibitors attenuated the down-regulation of CYP2B6 protein, but not mRNA, by ILmix. These findings demonstrate that the post-transcriptional NO dependent down-regulation of CYP2B enzymes, observed previously in rat hepatocytes, is conserved in human CYP2B6. This mechanism is specific for CYP2B6 among the enzymes tested. No evidence was found for regulation of CYP2E1 mRNA or protein by NO. NOC-18 treatment down-regulated CYP3A4 mRNA to 50% of control. However, NOS inhibitors failed to block the effects of ILmix on CYP3A4 expression.

ABSTRACT:Whereas many cytochrome P450 enzymes are transcriptionally suppressed by inflammatory stimuli, down-regulation of CYP2B protein by the inflammatory cytokine interleukin (IL)-1␤ is nitric oxide (NO)-dependent and occurs via polyubiquitination and proteasomal degradation. Here, we used iTRAQ proteomic analysis to search for other proteins that are potentially down-regulated by cellular NO in cultured rat hepatocytes, and we identified CYP3A1 as one such protein. Therefore, we examined whether CYP3A proteins, like CYP2B, undergo NO-and proteasome-dependent degradation in response to cytokine treatment of rat hepatocytes. In cultured rat hepatocytes treated with phenobarbital, IL-1␤ stimulation failed to down-regulate CYP3A1 mRNA within 24 h of treatment, whereas CYP3A protein was down-regulated to 40% of control within 6 h, showing the post-transcriptional down-regulation of CYP3A1 protein.

After infection with Citrobacter rodentium, murine hepatic cytochrome P450 (P450) mRNAs are selectively regulated. Several serum proinflammatory cytokines are elevated, the most abundant being interleukin-6 (IL6). To elucidate the role of cytokines in the regulation of P450s during infection, we orally infected wild-type, IL6(Ϫ/Ϫ), or interferon-␥(Ϫ/Ϫ) [IFN␥(Ϫ/Ϫ)] female C57BL/6J mice with C. rodentium and analyzed hepatic P450 expression 7 days later. The majority of P450 mRNAs were equally affected by infection in each genotype, indicating that IL6 and IFN␥ are not the primary mediators of P450 downregulation in this disease model. The down-regulation of CYP3A11 and CYP3A13 and induction of CYP2D9 mRNAs were attenuated in the IL6(Ϫ/Ϫ) mice, suggesting a role of IL6 in the regulation of only these P450s. Similar evidence implicated IFN␥ in the regulation of CYP2D9, CYP2D22, CYP3A11, CYP3A25, and CYP4F18 mRNAs in C. rodentium infection and CYP2B9, CYP2D22, and CYP2E1 in the bacterial lipopolysaccharide model of inflammation. This is the first indication of an in vivo role for IFN␥ in hepatic P450 regulation in disease states. The deficiency of IL6 or IFN␥ affected serum levels of the other cytokines. Moreover, experiments in cultured hepatocytes demonstrated that tumor necrosis factor ␣ (TNF␣) is the most potent and efficacious of the cytokines tested in the regulation of murine P450 expression. It is therefore possible that part of the IFN␥(Ϫ/Ϫ) and IL6(Ϫ/Ϫ) phenotypes could be attributed to the reduced levels of TNF␣ and part of the IFN␥(Ϫ/Ϫ) phenotype could be caused by reduced levels of IL6.