Inflammation and infection have long been known to downregulate the activity and expression of cytochrome P450 (CYP) enzymes involved in hepatic drug clearance. This can result in elevated plasma drug levels and increased adverse effects. Recent information on regulation of human CYP enzymes is presented, as are new developments in our understanding of the mechanisms of regulation. Experiments to study the effects of modulating CYP activities on the inflammatory response have yielded possible insights into the physiological consequences, if not the purpose, of the downregulation. Regulation of hepatic flavin monooxygenases, UDP-glucuronosyltransferases, sulfotransferases, glutathione S-transferases, as well as of hepatic transporters during the inflammatory response, exhibits similarities and differences with regulation of CYPs.

ABSTRACT: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-␣ (TNF), interferon ␥ (IFN), transforming growth factor-␤ (TGF) and interleukin-1␤ (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 downregulated 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.The liver responds to inflammation and infection by increasing levels of acute-phase proteins and concurrently down-regulates liverspecific proteins involved in drug metabolism (Morgan, 1997;Renton, 2004;Aitken et al., 2006). Cytochromes P450 (P450s) are the major drug-metabolizing enzymes in the liver and are significantly down-regulated by infection and inflammatory stimuli (Morgan, 1997;Aitken et al., 2006). The rapid down-regulation of P450 mRNAs (Morgan, 1997) has led to a focus on transcription as the primary mechanism of these effects, and this view is supported by the size and speed of transcriptional down-regulation as demonstrated in rats in vivo (Cheng et al., 2003). In humans, this down-regulation is associated with decreased drug clearance (Kraemer et al., 1982;Rivory et al., 2002;Carcillo et al., 2003;Renton, 2004) and can result in increased incidence of drug toxicity (Kraemer et al., 1982).Three lines of evidence support a major role of inflammatory cytokines as the mediators of P450 regulation in the liver during inflammation. First, cytokines can down-regulate P450 expression in cultures of rodent and human hepatocytes (Abdel-Razzak et al., 1993;Morgan, 1997;Sunman et al., 2004;Aitken et al., 2006). Second, mice with null mutations in cytokine or cytokine receptor genes display diminished P450 down-regulation in response to some inflammatory stimuli (Siewert et al., 2000;Ashino et al., 2004). Third, P450-dependent drug clearance has been correlated inversely with plasma interleukin-6 (IL-6) in patients with tumors (Rivory et al...

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.

A multifamily sequence alignment of the rabbit CYP4A members with the known structure of CYP102 indicates amino acid differences falling within the so-called substrate recognition site(s) (SRS). Chimeric proteins constructed between CYP4A4 and CYP4A7 indicate that laurate activity is affected by the residues within SRS1 and prostaglandin activity is influenced by SRS2-3. Site-directed mutant proteins of CYP4A7 found laurate and arachidonate activity markedly diminished in the R90W mutant (SRS1) and somewhat decreased in W93S. While PGE(1) activity was only slightly increased, the mutant proteins H206Y and S255F (SRS2-3), on the other hand, exhibited remarkable increases in laurate and arachidonate metabolism (3-fold) above wild-type substrate metabolism. Mutant proteins H206Y, S255F, and H206Y/S255F but not R90W/W93S, wild-type CYP4A4, or CYP4A7 metabolized arachidonic acid in the absence of cytochrome b(5). Stopped-flow kinetic experiments were performed in a CO-saturated environment performed to estimate interaction rates of the monooxygenase reaction components. The mutant protein H206Y, which exhibits 3-fold higher than wild-type substrate activity, interacts with CPR at a rate at least 10 times faster than that of wild-type CYP4A7. These experimental results provide insight regarding the residues responsible for modulation of substrate specificity, affinity, and kinetics, as well as possible localization within the enzyme structure based on comparisons with homologous, known cytochrome P450 structures.

Two forms of beta-thymosins, designated thymosin beta 11 and thymosin beta 12, were isolated from trout (Salmo gairdneri) spleen. This suggests that the presence of two beta-thymosins, previously thought to be a property of mammalian tissues only, is a more general phenomenon in vertebrate species. Both trout beta-thymosins were found to be N-terminally blocked by a group identified as acetyl by m.s. Automated protein sequencing of tryptic, thermolytic and Staphylococcus aureus in 41-residue V8 proteinase fragments revealed that one of the two beta-thymosins corresponds to the previously reported 41-residue-long sequence of thymosin beta 11 with two substitutions at positions 5 and 7, i.e. Asn instead of Asp, and Glu instead of Gln, whereas the other beta-thymosin, designated thymosin beta 12, was found to be a 42-residue polypeptide closely similar in sequence to thymosin beta 11, with five substitutions (i.e. at positions 5, 7, 10, 11 and 41, with Asp, Ala, Ser, Asn and Thr instead of Asn, Glu, Ala, Ser and Ser respectively) and one addition at position 42 (Ala). Comparison of the known six sequences of beta-thymosins together with the sequences reported here showed that the sequence similarity of the two beta-thymosins in trout (86%) is greater than that of the two beta-thymosins in mammalian species (74%) and that residues at 28 positions are identical in all beta-thymosins, the longer conserved segments located at positions 16-26 and 31-38.