Cytochrome P450 Gene Expression Research Articles

Selective role for tumor necrosis factor-α, but not interleukin-1 or Kupffer cells, in down-regulation of CYP3A11 and CYP3A25 in livers of mice infected with a noninvasive intestinal pathogen

Hepatic cytochrome P450 (P450) gene and protein expression are modulated during inflammation and infection. Oral infection of C57BL/6 mice with Citrobacter rodentium produces mild clinical symptoms while selectively regulating hepatic P450 expression and elevating levels of proinflammatory cytokines. Here, we explored the role of cytokines in the regulation of hepatic P450 expression by orally infecting tumor necrosis factor-α (TNFα) receptor 1 null mice (TNFR1−/−), interleukin-1 (IL1) receptor null mice (IL1R1−/−), and Kupffer cell depleted mice with C. rodentium. CYP4A mRNA and protein levels and flavin monooxygenase (FMO)3 mRNA expression levels were down-regulated, while CYP2D9 and CYP4F18 mRNAs remained elevated during infection in wild-type, receptor knockout, and Kupffer cell depleted mice. CYPs 3A11 and 3A25 mRNA levels were down-regulated during infection in wild-type mice but not in TNFR1−/− mice. Consistent with this observation, CYPs 3A11 and 3A25 were potently down-regulated in mouse hepatocytes treated with TNFα. Oral infection of IL1R1−/− mice and studies with mouse hepatocytes indicated that IL1 does not directly regulate CYP3A11 or CYP3A25 expression. Uninfected mice injected with clodronate liposomes had a significantly reduced number of Kupffer cells in their livers. Infection increased the Kupffer cell count, which was attenuated by clodronate treatment. The P450 mRNA and cytokine levels in infected Kupffer cell depleted mice were comparable to those in infected mice receiving no clodronate. The results indicate that TNFα is involved in the regulation of CYPs 3A11 and 3A25, but IL1β and Kupffer cells may not be relevant to hepatic P450 regulation in oral C. rodentium infection.

Evaluation of hepatotoxicity potential of cinnabar-containing An-Gong-Niu-Huang Wan, a patent traditional Chinese medicine

An-Gong-Niu-Huang Wan (AGNH) is a patent traditional Chinese medicine for brain disorders. It contains 10% cinnabar (HgS). Hg is known to produce toxicity to the kidney, brain and liver. Is AGNH safe? Liver is a major organ for drug metabolism, whether the long-term use of AGNH would affect hepatic P450 enzymes is unknown. To address these concerns, mice were given orally cinnabar (300 mg/kg), cinnabar-containing AGNH daily for 44 days, and liver toxicity was examined and compared with that of methylmercury (MeHg, 2.6 mg/kg) and mercuric chloride (HgCl 2, 32 mg/kg). Serum aminotransferases were increased by MeHg and HgCl 2 only. Histopathology showed more severe liver damage in MeHg- and HgCl 2-treated mice than in the cinnabar and AGNH groups. Accumulation of Hg in MeHg- and HgCl 2-treated mice was 96- and 71-fold higher than controls, respectively, but was only 2-fold after cinnabar and AGNH administration. Expressions of metallothionein-1 and heme oxygenase-1, biomarkers for Hg toxicity, were increased by MeHg and HgCl 2, but were not altered in cinnabar- and AGNH-treated mice. Expression of hepatic cytochrome P450 genes, such as Cyp1a1, Cyp1b1 and Cyp4a10 was increased only after MeHg and HgCl 2, and the expressions of Cyp3a11and Cyp3a25 were increased by all treatments, indicating the potential Hg-drug interactions after long-term use of cinnabar-containing traditional medicines. Taken together, the results demonstrate that AGNH is much less hepatotoxic than common mercurials, and that the use of total Hg content to evaluate the toxicity of cinnabar-containing traditional Chinese medicines appears to be inappropriate.

Taurine Is More Effective than Melatonin on Cytochrome P450 2E1 and Some Oxidative Stress Markers in Streptozotocin-Induced Diabetic Rats

Melatonin and taurine have alleviative effects in streptozotocin (STZ)-induced diabetic rats. Male Wistar rats were divided into nondiabetic, diabetic, diabetic melatonin supplemented and diabetic taurine supplemented groups. At the end of the study, both blood and liver were collected for determination of some oxidative stress parameters, and hepatic cytochrome P450 2E1 (CYP2E1) enzyme activity and gene expression. An increased CYP2E1 activity and expression level with a concomitant significant change in oxidative stress parameters were found in STZ-induced diabetic rats. Taurine or melatonin supplementation to the diabetic rats alleviated these experimental parameters with a more significant effect for taurine than that of melatonin. Suppression of β-hydroxybutyrate (β-HB) production by taurine can be one of the mechanisms of a reduction in CYP2E1. Taurine was effective more than melatonin in reducing CYP2E1 activity and expression; therefore antioxidants might prove beneficial in type 1 diabetes associated with manifestations of liver injury.

Evaluation of chlorinated benz[a]anthracene on hepatic toxicity in rats and mutagenic activity in Salmonella typhimurium

Chlorinated benz[a]anthracenes (Cl-BaA) are halogenated aromatic compounds (typified by dioxins) found in the environment at relatively high concentrations. Fischer 344 rats were intragastrically administered 0, 1, or 10 mg of Cl-BaA or its parent compound benz[a]anthracene (BaA) per kg of body weight for 14 consecutive days. Both chemicals at 10 mg/kg/day inhibited the gain in body weight, and consequent increase in relative liver weight. Hepatic gene expression of cytochrome P450 (CYP) 1A1, 1A2, and 1B1 was significantly stimulated by administration of BaA (10 mg/kg/day) compared with the control. After administration of Cl-BaA, only the CYP1A2 gene was significantly induced, even at the lower dosage; CYP1A1 and 1B1 mRNA levels remained unchanged in Cl-BaA-treated rats compared with controls. To elucidate the role of such Cl-BaA exposure and induced CYPs at toxicity onset, we investigated the mutagenicity of BaA and Cl-BaA using Salmonella typhimurium TA98 and TA100. BaA and Cl-BaA at 10 μg/plate produced positive results in both strains in the presence of rat S-9. Incubation of Cl-BaA with recombinant rat CYP1A2 produced a significantly higher number of revertant colonies in TA98 and TA100 than in controls, but no such change was observed for BaA. In conclusion, BaA changes its own physiological and toxicological actions by its chlorination; (1) daily exposure to Cl-BaA selectively induces hepatic CYP1A2 in rats and (2) Cl-BaA induces frameshift mutations in the presence of CYP1A2, although BaA does not exert mutagenicity. This indicates that CYP1A2 may metabolize Cl-BaA to active forms.

Hepatic Differentiation of Human Embryonic Stem Cells Is Promoted by Three-Dimensional Dynamic Perfusion Culture Conditions

The developmental potential of human embryonic stem cells (hESCs) holds great promise to provide a source of human hepatocytes for use in drug discovery, toxicology, hepatitis research, and extracorporeal bioartificial liver support. There are, however, limitations to induce fully functional hepatocytes on conventional two-dimensional (2D) static culture. It had been shown that dynamic three-dimensional (3D) perfusion culture is superior to induce maturation in fetal hepatocytes and prolong hepatic functions of primary adult hepatocytes. We investigated the potential of using a four-compartment 3D perfusion culture to induce hepatic differentiation in hESC. Undifferentiated hESC were inoculated into hollow fiber-based 3D perfusion bioreactors with integral oxygenation. Hepatic differentiation was induced with a multistep growth factor cocktail protocol. Parallel controls were operated under equal perfusion conditions without the growth factor supplementations to allow for spontaneous differentiation, as well as in conventional 2D static conditions using growth factors. Metabolism, hepatocyte-specific gene expression, protein expression, and hepatic function were evaluated after 20 days. Significantly upregulated hepatic gene expression was observed in the hepatic differentiation 3D culture group. Ammonia metabolism activity and albumin production was observed in the 3D directed differentiation culture. Drug-induced cytochrome P450 gene expression was increased with rifampicin induction. Using flow cytometry analysis the mature hepatocyte marker asialoglycoprotein receptor was found on up to 30% of the cells in the 3D system with directed hepatic differentiation. Histological and immunohistochemical analysis revealed structural formation of hepatic and biliary marker-positive cells. In contrast to 2D culture, the 3D perfusion culture induced more functional maturation in hESC-derived hepatic cells. 3D perfusion bioreactor technologies may be useful for further studies on generating hESC-derived hepatic cells.

Immediate early inflammatory gene responses of human umbilical vein endothelial cells to hemorrhagic venom

This report describes a focused immediate early gene response by human umbilical vein endothelial cells (HUVEC) to Echis carinatus snake venom. Primary cultures of HUVEC were used to assess acute inflammatory gene responses. Crude E. carinatus venom (2.5µg/ml) was used to stimulate HUVEC. HUVEC stimulated for 3h with E. carinatus venom showed a focused response to the venom, with significant increases in metallothionein (e.g., MT1H, MT2A, MT1X) and cytochrome P450 (e.g., CYP1A1, CYP1B1) gene expressions compared to non-stimulated controls. Several other genes involved in cell growth and matrix attachment were repressed [e.g., thrombospondin 1 (THBS1), connective tissue growth factor (CTGF)]. These data suggest that acute vascular injury induced by hemorrhagic snake venom initiates an anti-oxidant response primarily involving metallothioneins.

Gene Expression and Mutation Assessment Provide Clues of Genetic and Epigenetic Mechanisms in Liver Tumors of Oxazepam-Exposed Mice

Liver tumors from a previous National Toxicology Program study were examined using global gene expression and mutation analysis to define the mechanisms of carcinogenesis in mice exposed to oxazepam. Five hepatocellular adenomas and 5 hepatocellular carcinomas from male B6C3F1 mice exposed to 5000 ppm oxazepam and 6 histologically normal liver samples from control animals were examined. One of the major findings in the study was upregulation of the Wnt/β-catenin signaling pathway. Genes that activate β-catenin, such as Sox4, were upregulated, whereas genes that inhibit Wnt signaling, such as APC and Crebbp, were downregulated. In addition, liver tumors from oxazepam-exposed mice displayed β-catenin mutations and increased protein expression of glutamine synthetase, a downstream target in the Wnt signaling pathway. Another important finding in this study was the altered expression of oxidative stress-related genes, specifically increased expression of cytochrome p450 genes, including Cyp1a2 and Cyp2b10, and decreased expression of genes that protect against oxidative stress, such as Sod2 and Cat. Increased oxidative stress was confirmed by measuring isoprostane expression using mass spectrometry. Furthermore, global gene expression identified altered expression of genes that are associated with epigenetic mechanisms of cancer. There was decreased expression of genes that are hypermethylated in human liver cancer, including tumor suppressors APC and Pten. Oxazepam-induced tumors also exhibited decreased expression of genes involved in DNA methylation (Crebbp, Dnmt3b) and histone modification (Sirt1). These data suggest that formation of hepatocellular adenomas and carcinomas in oxazepam-exposed mice involves alteration of the Wnt signaling pathway, oxidative stress, and potential epigenetic alterations.

Roles of Mitogen-Activated Protein Kinases in the Regulation of CYP Genes

Cells undergo phenotypic changes after exposure to a wide range of exogenous stimuli that include growth factors, proinflammatory cytokines and environmental chemicals. Such stimuli may arise as components of disease pathogenesis and cellular injury, or as a result of exposure to environmental chemicals and radiation. These stimuli modulate the proliferation and differentiation of cells by altering the regulation of genes that control homeostasis. A generalized response appears to be a decline in the expression and function of many cytochrome P450 (CYP) genes in liver and other tissues. Thus, individuals who have been exposed to such exogenous stimuli often exhibit a decreased capacity for drug clearance, which has important consequences for concurrent drug therapy. Several signaling pathways transduce exogenous stimuli within cells, with the mitogen-activated protein kinases (MAPKs) being one of the most important. Evidence is increasing that MAPKs may impair the expression of multiple CYP genes by modulating the activity of transcription factors, including nuclear receptors, the aryl hydrocarbon receptor, and the activator protein-1 complex. MAPKs catalyze the phosphorylation of transcription complexes that incorporate these factors, which modulates their capacity to transactivate target genes, including CYPs. An understanding of the mechanisms that account for the regulatory impact of MAPKs on the transcriptional factors that regulate CYP genes will provide critical insight into the consequences from exposure to injurious stresses that impact cellular function.

Identification and developmental expression of the full complement of Cytochrome P450 genes in Zebrafish

BackgroundIncreasing use of zebrafish in drug discovery and mechanistic toxicology demands knowledge of cytochrome P450 (CYP) gene regulation and function. CYP enzymes catalyze oxidative transformation leading to activation or inactivation of many endogenous and exogenous chemicals, with consequences for normal physiology and disease processes. Many CYPs potentially have roles in developmental specification, and many chemicals that cause developmental abnormalities are substrates for CYPs. Here we identify and annotate the full suite of CYP genes in zebrafish, compare these to the human CYP gene complement, and determine the expression of CYP genes during normal development.ResultsZebrafish have a total of 94 CYP genes, distributed among 18 gene families found also in mammals. There are 32 genes in CYP families 5 to 51, most of which are direct orthologs of human CYPs that are involved in endogenous functions including synthesis or inactivation of regulatory molecules. The high degree of sequence similarity suggests conservation of enzyme activities for these CYPs, confirmed in reports for some steroidogenic enzymes (e.g. CYP19, aromatase; CYP11A, P450scc; CYP17, steroid 17a-hydroxylase), and the CYP26 retinoic acid hydroxylases. Complexity is much greater in gene families 1, 2, and 3, which include CYPs prominent in metabolism of drugs and pollutants, as well as of endogenous substrates. There are orthologous relationships for some CYP1 s and some CYP3 s between zebrafish and human. In contrast, zebrafish have 47 CYP2 genes, compared to 16 in human, with only two (CYP2R1 and CYP2U1) recognized as orthologous based on sequence. Analysis of shared synteny identified CYP2 gene clusters evolutionarily related to mammalian CYP2 s, as well as unique clusters.ConclusionsTranscript profiling by microarray and quantitative PCR revealed that the majority of zebrafish CYP genes are expressed in embryos, with waves of expression of different sets of genes over the course of development. Transcripts of some CYP occur also in oocytes. The results provide a foundation for the use of zebrafish as a model in toxicological, pharmacological and chemical disease research.

Investigation of breed and sex effects on cytochrome P450 gene expression in cattle liver

Many cytochrome P450 enzymes are involved in xenobiotic metabolism and elimination. In humans, genetic variation in some of these enzymes contributes to inter-individual drug responses, sometimes having significant clinical effects. Transcript levels of eight P450 genes were evaluated in liver to investigate potential differences in breed and sex in cattle. In Angus calves, heifers appeared to have higher gene expression than steers for two of the eight genes. In Angus and Simmental pregnant cows, Angus appeared to have higher gene expression for three of the eight genes. Transcript evaluation is just the first step toward determining if differences exist between breeds and sexes in enzyme catalytic activity. However, others ( Giantin et al., 2008) have shown correlations between transcript levels and catalytic activity in other cattle breeds. Therefore breed and/or sex of an animal may need to be considered before administering a dose of a xenobiotic due to the potential for harmful drug residues in foodstuffs as well as improper treatment of disease conditions.

Three-Dimensional Perfusion Bioreactor Culture Supports Differentiation of Human Fetal Liver Cells

The ability of human fetal liver cells to survive, expand, and form functional tissue in vitro is of high interest for the development of bioartificial extracorporeal liver support systems, liver cell transplantation therapies, and pharmacologic models. Conventional static two-dimensional culture models seem to be inadequate tools. We focus on dynamic three-dimensional perfusion technologies and developed a scaled-down bioreactor, providing decentralized mass exchange with integral oxygenation. Human fetal liver cells were embedded in a hyaluronan hydrogel within the capillary system to mimic an in vivo matrix and perfusion environment. Metabolic performance was monitored daily, including glucose consumption, lactate dehydrogenase activity, and secretion of alpha-fetoprotein and albumin. At culture termination cells were analyzed for proliferation and liver-specific lineage-dependent cytochrome P450 (CYP3A4/3A7) gene expression. Occurrence of hepatic differentiation in bioreactor cultures was demonstrated by a strong increase in CYP3A4/3A7 gene expression ratio, lower alpha-fetoprotein, and higher albumin secretion than in conventional Petri dish controls. Cells in bioreactors formed three-dimensional structures. Viability of cells was higher in bioreactors than in control cultures. In conclusion, the culture model implementing three-dimensionality, constant perfusion, and integral oxygenation in combination with a hyaluronan hydrogel provides superior conditions for liver cell survival and differentiation compared to conventional culture.

Up-regulation of CYP expression in hepatoma cells stably transfected by chimeric nuclear receptors

Most hepatoma cell lines lack proper expression and induction of cytochrome P450 (CYP) enzymes and this deficiency hampers their use as in vitro models for drug and xenobiotic metabolism. According to previous studies, the poor expression of CYP enzymes may be due to decreases in CYP gene transcription. Two nuclear receptors (NRs), the pregnane X receptor (PXR) and the constitutive androstane receptor (CAR), are known to regulate many genes involved in xenobiotic metabolism and disposition. Here, we studied the expression of different CYP, NR and NR co-regulator genes in hepatoma cell lines. Next, we created “chimeric NR” constructs by cloning the strong activation domain from the p65 subunit of transcription factor NF-κB and appending it to either N- or C-termini of the human CAR or PXR. We established that these chimeric NRs displayed enhanced trans-activation potential as compared to the unmodified NRs, and showed that transient transfection of a single chimeric NR increased the expression of several CYPs simultaneously. Finally, stable cell lines expressing a chimeric NR had elevated levels of CYP3A4, CYP2B6 and CYP2C9 mRNAs and CYP3A4-mediated metabolism when compared to the wild-type hepatoma cells. These findings establish a proof of principle how improved metabolic cell models could be designed.

CYP3A4 Catalytic Activity Is Induced in Confluent Huh7 Hepatoma Cells

Drug-induced hepatotoxicity is an important cause for disapproval, limitations of use, or withdrawal of drugs, and there is a high need for reproducible in vitro systems that can predict such toxicity. In this study, we show that confluent growth of the human hepatoma cell line Huh7 up to 5 weeks results in increased gene expression of several cytochromes P450 (P450s), UDP-glucuronosyltransferases, transporters, transcription factors, and several liver-specific genes, as measured by low-density array. The most striking effect was seen for CYP3A4 expression. Western blot analysis revealed increased amounts of CYP3A4 together with increased levels of NADPH-P450 reductase, cytochrome b(5), and albumin with prolonged time of confluence. By using the CYP3A4-specific substrates luciferin 6' benzyl ether, testosterone, and midazolam, we could confirm that the increased CYP3A4 gene expression also was accompanied by a similar increase in catalytic activity, inhibitable by the CYP3A4-selective inhibitor ketoconazole. The CYP3A4 activity in confluent cells was also inducible by rifampicin. Finally, the cell system could support the CYP3A4-dependent hepatotoxic activation of aflatoxin B(1), which was effectively inhibited by ketoconazole. Our results show that Huh7 cells grown confluent differentiate into a more metabolically competent cell line, especially with regard to CYP3A4.

Transcriptomic analysis of chilling stress in Phaseolus spp.

The wild bean species Phaseolus angustissimus has shown a greater ability to survive chilling temperatures than the cultivated bean Phaseolus vulgaris. Arrays of cDNA clones from cold acclimated crowns of Medicago sativa were used to measure low temperature-induced changes in the expression profiles of both bean species and compare them to each other. Macroarray hybridization results indicate that P. vulgaris failed to sustain homeostatic expression levels of more genes than did P. angustissimus after 3 days of chilling temperature exposure. Expression patterns of selected genes were further characterized with RT-PCR using plants exposed to various durations of chilling. The most notable difference between the two species in these gene expression experiments was that P. angustissimus was able to recover the expression of a cytochrome P450 species under chilling temperature exposure whereas P. vulgaris failed to sustain levels of cytochrome P450 gene expression levels. Results of these experiments suggest that a key to chilling tolerance in Phaseolus is maintenance of homeostatic gene expression levels under chilling conditions.

Mechanisms of ketamine-involved regulation of cytochrome P450 gene expression

Importance of the field: Ketamine, a widely used intravenous anesthetic agent, is biotransformed by cytochrome P450s (CYPs). Considerable attention is devoted to the ketamine-involved regulation of CYP gene expression. Assessing how ketamine regulates CYP gene expression is significant in recognizing the pharmacology of this anesthetic agent and its clinical application.Areas covered in this review: In this review, the authors discuss the effects of ketamine on the regulation of CYP gene expression and its possible mechanisms from the aspects of cytoskeletal remodeling, mitochondrial dysfunction, and alterations of intracellular ATP levels and calcium homeostasis.What the reader will gain: This review may provide readers with more clues for recognizing and avoiding possible ketamine–drug interactions.Take home message: Ketamine may inhibit CYP3A4 expression possibly through reducing calcium mobilization and mitochondrial ATP synthesis and consequently disturbing cytoskeleton remodeling.

Molecular analysis of pyrethroid resistance conferred by target insensitivity and increased metabolic detoxification in Plutella xylostella

The pyrethroid resistance of the diamondback moth Plutella xylostella (L.) is conferred by increased gene expression of cytochrome P450 to detoxify the insecticide and/or through gene mutation of the sodium channel, which makes the individual insensitive to pyrethroids. However, no information is available about the correlation between the increased metabolic detoxification and the target insensitivity in pyrethroid resistance. Frequencies of pyrethroid-resistant alleles (L1014F, T929I and M918I) and two resistance-related mutations (A1101T and P1879S) at the sodium channel and expression levels of the cytochrome P450 gene CYP6BG1 were examined individually using laboratory and field strains of P. xylostella. Real-time quantitative PCR analysis using the laboratory strains revealed that levels of larval expression of the resistant strain, homozygous for the pyrethroid-resistant alleles other than the M918I, are significantly higher than those of the susceptible strain. In the field strains, the expression levels in insects having the same resistant alleles as those of the resistant strains varied greatly among individuals. The expression levels were not significantly higher than those in the heterozygotes. Significant correlation between the target insensitivity and the increased metabolic detoxification in pyrethroid resistance of P. xylostella was observed in the laboratory but not in the field.

Resistance to Fusarium Head Blight and Seedling Blight in Wheat Is Associated with Activation of a Cytochrome P450 Gene

ABSTRACT One plant genotype displays a resistance phenotype at one development stage but a susceptible reaction to the same pathogen at another stage, which is referred to here as resistance inversion. In wheat, Fusarium head blight (FHB)-resistant cv. Sumai3 showed a Fusarium seedling blight (FSB)-susceptible reaction whereas FHB-susceptible cv. Annong8455 exhibited FSB resistance when challenged with a Fusarium asiaticum strain that produces deoxynivalenol (DON). The resistance to FHB and FSB in wheat was closely associated with expression of a plant cytochrome P450 gene in response to FHB pathogens and mycotoxins. Quantitative real-time polymerase chain reaction analyses showed that expression of nine defense-related genes in spikes and seedlings was induced by the fungal infection, in which a massive accumulation of a plant cytochrome P450 gene, CYP709C1, was clearly associated with the resistance reaction in both seedling and spike. The FHB-resistant Sumai3 accumulated 7-fold more P450 transcripts than did the FHB-susceptible Annong8455, while 84-fold more P450 transcripts were accumulated in the FSB-resistant Annong8455 than the FSB-susceptible Sumai3. A Fusarium strain with a disrupted Tri5 gene, which is not able to produce the first enzyme essential for trichothecene mycotoxin biosynthesis, also induced more P450 transcripts in FHB- and FSB-resistant cultivars. The fungal activation of the P450 gene was more profound in the FSB-resistant reaction than the FHB-resistant reaction relative to their susceptible counterparts. DON triggered a differential expression of the P450 gene with comparable patterns in spikes and seedlings in a resistance-dependent manner. These results may provide a basis for dissecting mechanisms underlying FHB and FSB resistance reactions in wheat and revealing functions of the cytochrome P450 in plant detoxification and defense.

Acute inflammation and immunoresponses induced by ortho-phthalaldehyde in mice

ortho-Phthalaldehyde (OPA) has been used as a safe alternative disinfectant instead of glutaraldehyde; however, recently some adverse effects of OPA were reported in patients and medical professions. We examined the acute toxicity of OPA in male ICR mice injected with 0.125-0.5% OPA and killed some animals 1 day after a single OPA injection, and others 1 or 13 days after two OPA injections 5 days apart. Hematology, blood cell counts, specific antibody production, organ weights, hepatic enzymes, hepatic histopathology and gene expression of cytochrome P450 (CYP) mRNA in liver were examined. Single OPA injections elevated leukocyte counts, the proportion of neutrophils, alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Two OPA injections dose-dependently increased leukocyte counts, the proportion of neutrophils, ALT and AST, and decreased alkaline phosphatase. Leukocyte counts and proportions of neutrophils normalized 13 days after the second of two injections. However, both ALT and AST remained high in mice given higher OPA doses. Significant increased liver-to-body weight ratio and mild hepatic lesions were observed. Gene expression of CYP1a1 and CYP2e1 revealed a tendency of up-regulation 1 day after two OPA injections. However, expression of these genes was then down-regulated 13 days after OPA injections. OPA induced specific IgE and IgG significantly in the sera, suggesting that OPA acts as a hapten. Overall, OPA caused acute inflammation and acted as a haptenic allergen, although it caused only mild liver injury. Such evidence suggested that careful washing and prevention of exposure were needed after OPA disinfection of medical instruments.

Cytochrome 3A and 2E1 in human liver tissue: Individual variations among normal Japanese subjects

Aims The metabolism of drugs, xenobiotic compounds, and other endogenous/exogenous substrates generally begins with their oxidation through cytochrome P450 (CYP). The results of recent pharmacogenetic analyses have demonstrated CYP's polymorphisms to be related to individual differences in metabolism, but only a limited number of CYP3A4 and CYP2E1 variant alleles influence enzymatic activities. Therefore, CYP gene expression profiling of both normal and pathological human livers should provide critical information for an evaluation of the biological significance of CYPs. Main methods In our present study, we first characterized the individual differences in CYP3A4 and CYP2E1 expression levels among Japanese normal or non-pathological liver tissue obtained from autopsy or surgery using immunohistochemistry and quantitative RT-PCR array of phase I metabolic enzymes with combined laser capture microscopy and qPCR analysis. Key findings Both CYP3A4 and CYP2E1 mRNA and proteins were predominantly detected in hepatocytes surrounding central veins in normal liver, but there were marked individual differences in both CYP3A4 and CYP2E1 mRNA and proteins among the 23 Japanese subjects examined. Individual differences in CYP3A and CYP2E1 subtypes were also detected in the livers obtained from monozygotic neonatal Japanese female twins with different survival periods. CYP3A and CYP2E1-positive cells were decreased in number in non-pathological hepatocytes of diseased livers compared to those in disease-free livers from autopsy. Significance The above results suggest that individual differences in CYP3A4 and CYP2E1 exist among normal human liver tissues and in non-pathological hepatocytes between diseased and normal liver, and these differences may be important in evaluating the pharmacodynamics of various substances.

Alteration of cardiac cytochrome P450-mediated arachidonic acid metabolism in response to lipopolysaccharide-induced acute systemic inflammation

Cytochrome P450 (CYP) generated cardioprotective metabolites, epoxyeicosatrienoic acids (EETs), and cardiotoxic metabolites, hydroxyeicosatetraenoic acids (HETEs) levels are determined by many factors, including the induction or repression of the CYP enzymes, responsible for their formation. Therefore, we examined the effect of acute inflammation on the expression of CYP epoxygenases and CYP omega-hydroxylases in the heart, kidney, and liver and the cardiac CYP-mediated arachidonic acid metabolism. For this purpose, male Sprague-Dawley rats were injected intraperitoneally with LPS (1mg/kg). After 6, 12, or 24h, the tissues were harvested and the expression of CYP genes and protein levels were determined using real time-PCR, and Western blot analyses, respectively. Arachidonic acid metabolites formations were determined by liquid chromatography-electron spray ionization-mass spectrometry LC-ESI-MS. Our results showed that inflammation significantly decreased the CYP epoxygenases expression in the heart, kidney and liver with a concomitant decrease in the EETs produced by these enzymes. In contrast to CYP expoxygenses, inflammation differentially altered CYP omega-hydroxylases expression with a significant increase in 20-HETE formation. The present study demonstrates for the first time that acute inflammation decreases CYP epoxygenases and their associated cardioprotective metabolites, EETs while on the other hand increases CYP omega-hydroxylases and their associated cardiotoxic metabolites, 20-HETE. These changes may be involved in the development and/or progression of cardiovascular diseases by inflammation.