Edward T. Morgan

Regulation of Cytochromes P450 During Inflammation and Infection

Hepatic P450 activities are profoundly affected by various infectious and inflammatory stimuli, and this has clinical and toxicological consequences. Whereas the expression of most P450s in the liver is suppressed, some are induced. Many of the effects observed in vivo can be mimicked by pro-inflammatory cytokines and IFNs, and P450s are differentially regulated by these agents. Therefore, different cytokine profiles and concentrations in the vicinity of the hepatocyte in different models of inflammation may result in qualitatively and quantitatively different effects on populations of P450s. In addition to cytokines, glucocorticoids may have an important role in P450 regulation in stress conditions, including that caused by inflammatory stimuli. Although in many cases the decreases in activity are due primarily to a down-regulation of P450 gene transcription, it is likely that modulation of RNA and protein turnover, as well as enzyme inhibition, contributes to some of the observed effects. The mechanisms whereby these effects are produced may also vary with both the P450 under study and the time course of the effect. The complexity of the P450 response to inflammation and infection means that all of the above factors must be considered when trying to predict the effect of a given infectious or inflammatory condition on the clinical or toxic response of humans or animals to an administered drug or toxin. The question of whether the down-regulation of the hepatic P450 system to inflammation or infection is a homeostatic or pathological response cannot be answered at present. It is difficult to discern the physiological benefit of reducing hepatic P450 activities, unless it is to prevent the generation of reactive oxygen species generated by uncoupled catalytic turnover of the enzymes. On the other hand, as we proposed some years ago [64], the suppression of P450 may be due to the livers need to utilize its transcriptional machinery and energy for the synthesis of APPs involved in the inflammatory response. In that case, one could ask why the organism has gone to the trouble of employing differential mechanisms for suppression of P450. One answer could be that the response evolved after the divergence of many of the P450 genes, necessitating the evolution of multiple redundant mechanisms for P450 suppression. In contrast to the down-regulation of P450s in the liver, the induction of several forms in this and other tissues suggests a more specific homeostatic role of these effects, e.g., in generation or catabolism of bioactive metabolites.

Regulation of Drug-Metabolizing Enzymes and Transporters in Infection, Inflammation, and Cancer

This article is a report on a symposium sponsored by the American Society for Pharmacology and Experimental Therapeutics and held at the Experimental Biology 07 meeting in Washington, DC. The presentations discussed the phenomenology, clinical consequences, and underlying mechanisms of cytochrome P450 and drug transporter regulation by inflammatory and infectious stimuli. Although considerable insights into the links between inflammatory mediators and altered hepatic drug clearance pathways have been gained from previous studies with acute inflammatory stimuli, this symposium highlighted recent advances in understanding how these processes operate in other organs and chronic inflammatory states relevant to human diseases. The development of mouse models of live bacterial infection provides excellent opportunities to explore the impact of infection on drug metabolism beyond the well characterized effects of bacterial endotoxin. Altered levels of cytochromes P450 and especially drug transporters due to inflammation in brain, intestine, and placenta have significant implications for the use of many drugs in diverse clinical settings. The consequences of inflammatory cytokine production by tumors for drug safety and efficacy in cancer patients were outlined. Repression of drug clearance pathways by tumor-derived cytokines may result in extreme toxicity to chemotherapy, compromising treatment of many cancers. It is fitting that, in honoring the career contributions and achievements of Dr. Kenneth W. Renton, this symposium reinforced the clinical relevance of this field.

Gene-Specific Effects of Inflammatory Cytokines on Cytochrome P450 2C, 2B6 and 3A4 mRNA Levels in Human Hepatocytes

Cytochromes P450 (P450s) are down-regulated in hepatocytes in response to inflammation and infection. This effect has been extensively studied in animal models, but significantly less is known about responses in humans and even less about responses in the absence of inducing agents. This article focuses on the effects of bacterial lipopolysaccaride (LPS), interleukin-6 (IL-6), tumor necrosis factor-α (TNF), interferon γ (IFN), transforming growth factor-β (TGF) and interleukin-1β (IL-1) on expression of CYP2B6 and the CYP2C mRNAs in human hepatocytes. These effects were compared with responses of the better studied and more abundant CYP3A4. CYP3A4 and CYP2C8 were down-regulated by all cytokine treatments. CYP2C18, which is expressed at very low levels in liver, was unaffected by cytokine treatments. The other CYP2Cs and CYP2B6 showed cytokine-specific effects. CYP2C9 and CYP2C19 showed almost identical response patterns, being down-regulated by IL-6 and TGF but not significantly affected by LPS, TNF, IFN, or IL-1. CYP2B6 mRNA responded only to IL-6 and IFN. IL-6 down-regulated the mRNAs of all P450s studied. Western blot analysis of P450 protein expression supported the mRNA data to a large extent, although some inconsistencies were observed. Our results show that human CYP2C8, 2C9, 2C18, 2C19, 2B6, and 3A4 responses to inflammation are independently regulated and indicate that this fine control may have a critical effect on human drug responses in disease states.

Impact of Infectious and Inflammatory Disease on Cytochrome P450–Mediated Drug Metabolism and Pharmacokinetics

Infection and inflammation are associated with downregulation of hepatic and extrahepatic cytochrome P450s as well as other drug‐metabolizing enzymes and transporters. We review the genesis of inflammatory reactions and discuss the relevance of the inflammation‐related regulation of cytochrome P450 enzymes to clinical drug–disease and drug–drug interactions. An understanding of the enzyme specificity and mechanisms of regulation will allow us to make better decisions about adjusting drug dosage regimens when a patients inflammatory status changes.

Regulation of Drug Metabolizing Enzymes and Transporters in Infection, Inflammation, and Cancer

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. Keywords: inflammation; infection; cytokines; toll-like receptors; biologics

Bimodal Regulation of Ceramidase by Interleukin-1β IMPLICATIONS FOR THE REGULATION OF CYTOCHROME P450 2C11 (CYP2C11)

Interleukin 1β (IL-1β) induces the hydrolysis of sphingomyelin (SM) to ceramide (Cer) in primary cultures of rat hepatocytes, and Cer has been proposed to play a role in the down-regulation of cytochrome P450 2C11 (CYP2C11) and induction of α1-acid glycoprotein (AGP) by this cytokine (Chen, J., Nikolova-Karakashian, M., Merrill, A. H. & Morgan, E. T. (1995)J. Biol. Chem. 270, 25233–25238). Nonetheless, some of the features of the down-regulation of CYP2C11 do not fit a simple model of Cer as a second messenger as follows:N-acetylsphinganine (C2-DHCer) is as potent asN-acetylsphingosine (C2-Cer) in suppression of CYP2C11; the IL-1β concentration dependence for SM turnover is different from that for the increase in Cer; and the increase in Cer mass is not equivalent to the amount of SM hydrolyzed nor the time course of SM hydrolysis. In this article, we report that these discrepancies are due to activation of ceramidase by the low concentrations of IL-1β (∼2.5 ng/ml) that maximally down-regulate CYP2C11 expression, whereas higher IL-1β concentrations (that induce AGP) do not activate ceramidase and allow Cer accumulation. This bimodal concentration dependence is demonstrated both by in vitro ceramidase assays and in intact hepatocytes using a fluorescence Cer analog, 6-((N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-Cer (NBD-Cer), and following release of the NBD-fatty acid. IL-1β increases both acid and neutral ceramidase activities, which appear to be regulated by tyrosine phosphorylation because pretreatment of hepatocytes with sodium vanadate increases (and 25 μmgenistein reduces) the basal and IL-1β-stimulated ceramidase activities. Since these findings suggested that sphingosine (and, possibly, subsequent metabolites) is the primary mediator of the down-regulation of CYP2C11 by IL-1β, the effects of exogenous sphingosine and C2-Cer on expression of this gene were compared. Sphingosine was more potent than C2-Cer in down-regulation of CYP2C11 when added alone or with fumonisin B1 to block acylation of the exogenous sphingosine. Furthermore, the suppression of CYP2C11 by C2-Cer (and C2-DHCer) is probably mediated by free sphingoid bases, rather than the short chain Cer directly, because both are hydrolyzed by hepatocytes and increase cellular levels of sphingosine and sphinganine. From these observations we conclude that sphingosine, possibly via sphingosine 1-phosphate, is a mediator of the regulation of CYP2C11 by IL-1β in rat hepatocytes and that ceramidase activation provides a “switch” that determines which sphingolipids are elevated by this cytokine to produce multiple intracellular responses.

Metabolomics Reveals that Hepatic Stearoyl-CoA Desaturase 1 Downregulation Exacerbates Inflammation and Acute Colitis

To investigate the pathogenic mechanism of ulcerative colitis, a dextran sulfate sodium (DSS)-induced acute colitis model was examined by serum metabolomic analysis. Higher levels of stearoyl lysophosphatidylcholine and lower levels of oleoyl lysophosphatidylcholine in DSS-treated mice compared to controls led to the identification of DSS-elicited inhibition of stearoyl-CoA desaturase 1 (SCD1) expression in liver. This decrease occurred prior to the symptoms of acute colitis and was well correlated with elevated expression of proinflammatory cytokines. Furthermore, Citrobacter rodentium-induced colitis and lipopolysaccharide treatment also suppressed SCD1 expression in liver. Scd1 null mice were more susceptible to DSS treatment than wild-type mice, while oleic acid feeding and in vivo SCD1 rescue with SCD1 adenovirus alleviated the DSS-induced phenotype. This study reveals that inhibition of SCD1-mediated oleic acid biogenesis exacerbates proinflammatory responses to exogenous challenges, suggesting that SCD1 and its related lipid species may serve as potential targets for intervention or treatment of inflammatory diseases.

Selective suppression of cytochrome P-450 gene expression by interleukins 1 and 6 in rat liver

Inflammatory stimuli suppress constitutive hepatic expression of the CYP2C11 and CYP2C12 genes in male and female rat livers, respectively. We have shown previously that injection of interleukin-1 (IL1), but not interleukin-6 (IL6), to female rats also suppresses CYP2C12. In the present study, we examined the effects of these cytokines on CYP2C12 expression in rat hepatocyte cultures, and their in vivo effects on expression of multiple cytochrome P-450 (P450) gene products in male rat livers. IL1 suppressed the expression of CYP2C12 mRNA and protein in hepatocytes cultured on Matrigel in the presence of growth hormone. No consistent effect of IL6 was observed. Maximal suppression of CYP2C12 mRNA after 24 h of IL1 treatment reached 12 and 32% of control levels in two separate experiments. The approximate ED50 for IL1 was 5 ng/ml. CYP2C12 protein was suppressed to 28% of control levels as early as 12 h after IL1 treatment. Injection of IL1, low doses of dexamethasone, or both, in male rats produced decreases in total P450, and in CYP3A2 and CYP2C11 mRNA and protein expression similar to effects previously seen for CYP2C12 expression in females. CYP2E1 mRNA and protein was significantly suppressed only by the combination of IL1 and dexamethasone. IL6 treatment of male rats down-regulated the CYP2C11 and CYP2E1 mRNAs at a dose of 4.5 micrograms/kg, which was lower than that required to induce haptoglobin mRNA, a prototype acute phase gene product. CYP2C11 protein content of the microsomes was also decreased by IL6 treatment, with a slower time-course than for suppression of its mRNA. No significant effects of IL6 treatment were seen on CYP3A2 mRNA or CYP3A2/1 proteins. These results demonstrate that IL1 and IL6 treatments in vivo differentially affect subsets of P450 gene products in rat liver.

Mechanisms of cytochrome P450 regulation by inflammatory mediators

Hepatic levels of cytochrome P450 enzymes and their mRNAs are reduced in models of inflammation or infection. The contributions of transcriptional versus post-transcriptional mechanisms to this decline are poorly understood. The transcription of CYP2C11 is rapidly suppressed by administration of bacterial endotoxin (lipopolysaccharide, LPS) to rats, consistent with the finding that the CYP2C11 promoter contains a negative NF-kappa B response element that confers down-regulation of a linked reporter gene by cytokines. Nitric oxide has been proposed to be a mediator of inflammatory suppression of P450 expression, but reports from different laboratories have disagreed on this subject. Recently, we found that LPS suppresses the expression of CYP2B1 by both pre-translational and post-translational mechanisms in rat hepatocytes, the latter being NO-dependent and occurring only at high concentrations of LPS. Studies were conducted in control and NOS2-null mice to determine the contributions of these different mechanisms to CYP2B suppression in vivo.

Nitric oxide-independent suppression of P450 2C11 expression by interleukin-1β and endotoxin in primary rat hepatocytes☆☆☆

Hepatic expression of multiple cytochrome P450 genes is suppressed in the livers of rats undergoing an inflammatory response. Nitric oxide (NO) released during inflammation has been implicated in the decreased activities and expression of several cytochrome P450 isozymes. We examined the role of cytokine-mediated NO release on cytochrome P450 2C11 expression in rat hepatocytes cultured on Matrigel. Lipopolysaccharide (LPS), interleukin-1beta (IL-1beta), IL-6, and tumor necrosis factor-alpha (TNF-alpha), but not interferon-gamma (IFN-gamma), suppressed the expression of P450 2C11 mRNA. Neither IL-6 nor IFN-gamma caused NO release into the medium or induction of inducible nitric oxide synthase (iNOS) mRNA. IL-1beta and LPS were the most effective in causing NO release and iNOS induction, and in down-regulating P450 2C11 mRNA expression. Combinations of the cytokines, IFN-gamma, and LPS produced an additive release of NO but did not synergize to further suppress P450 2C11 mRNA. To investigate the role of NO in the IL-1beta- or LPS-mediated suppression of P450 2C11, N-monomethyl-L-arginine (NMA) was administered at concentrations ranging from 30 to 300 microM. Three hundred micromolar NMA returned NO release back to control levels, but did not affect the IL-1beta- or LPS-mediated down-regulation of P450 2C11 mRNA or protein expression. Our results suggest that NO is not required for IL-1beta- or LPS-mediated down-regulation of P450 2C11 expression in cultured hepatocytes.