CYP2C9 mRNA Research Articles

Involvement of the CYP1A Subfamily in Stereoselective Metabolism of Carvedilol in .BETA.-Naphthoflavone-Treated Caco-2 Cells

We have previously reported that the metabolism of S-carvedilol in beta-naphthoflavone (beta-NF)-treated Caco-2 cells is faster than that of R-carvedilol. The aim of the present study was to identify the enzyme responsible for the stereoselective metabolism of carvedilol in the cells. The expression of cytochrome P450 (CYP) 1A1 and CYP1A2 mRNA, but not CYP2D6, CYP3A4, and CYP2C9 mRNA, was increased in beta-NF-treated Caco-2 cells, as compared with non-treated cells. Furafylline, an inhibitor of the CYP1A subfamily, decreased the metabolism of S-carvedilol in Caco-2 cells cultured on plastic dishes. In addition, the glucuronidation of carvedilol was not significant in microsomes of beta-NF-treated Caco-2 cells. On the other hand, the oxidation of S-carvedilol in microsomes of beta-NF-treated Caco-2 cells was faster than that of R-carvedilol, and furafylline decreased the oxidative activity of S-carvedilol. These findings suggested that the CYP1A subfamily was responsible for the stereoselective metabolism of carvedilol in beta-NF-treated Caco-2 cells.

Epoxyeicosatrienoic acids are part of the VEGF-activated signaling cascade leading to angiogenesis

Cytochrome P-450 (CYP) epoxygenases metabolize arachidonic acid to epoxyeicosatrienoic acid (EET) regioisomers, which activate several signaling pathways to promote endothelial cell proliferation, migration, and angiogenesis. Since vascular endothelial growth factor (VEGF) plays a key role in angiogenesis, we assessed a possible role of EETs in the VEGF-activated signal transduction cascade. Stimulation with VEGF increased CYP2C promoter activity in endothelial cells and enhanced CYP2C8 mRNA and protein expression resulting in increased intracellular EET levels. VEGF-induced endothelial cell tube formation was inhibited by the EET antagonist 14,15-epoxyeicosa-5(Z)-enoicacid (14,15-EEZE), which did not affect the VEGF-induced phosphorylation of its receptor or basic fibroblast growth factor (bFGF)-stimulated tube formation. Moreover, VEGF-stimulated endothelial cell sprouting in a modified spheroid assay was reduced by CYP2C antisense oligonucleotides. Mechanistically, VEGF stimulated the phosphorylation of the AMP-activated protein kinase (AMPK), which has also been linked to CYP induction, and the overexpression of a constitutively active AMPK mutant increased CYP2C expression. On the other hand, a dominant-negative AMPK mutant prevented the VEGF-induced increase in CYP2C RNA and protein expression in human endothelial cells. In vivo (Matrigel plug assay) in mice, endothelial cells were recruited into VEGF-impregnated plugs; an effect that was sensitive to 14,15-EEZE and the inclusion of small interfering RNA directed against the AMPK. The EET antagonist did not affect responses observed in plugs containing bFGF. Taken together, our data indicate that CYP2C-derived EETs participate as second messengers in the angiogenic response initiated by VEGF and that preventing the increase in CYP expression curtails the angiogenic response to VEGF.

Growth hormone (GH) secretion, GH-dependent gene expression, and sexually dimorphic body growth in young rats with chronic renal failure

Chronic renal disease results in growth failure in children. This study sought to determine the influences of early renal failure on body growth, growth hormone (GH) secretion, and GH-dependent hepatic gene expression. Neonatal animals were subjected to five-sixth nephrectomy (Nephr) and monitored during growth. Sham-operated male (Sham) and female (Fem) rats served as controls. Whereas Nephr of adult animals causes renal insufficiency, neonatal nephrectomy leads to frank renal failure. In male Nephr compared with Sham animals, GH half-life and GH pulse frequency increased by 1.55- and 1.33-fold, respectively, and GH secretory-burst size decreased by 80%. Approximate entropy analysis quantified more disorderly patterns of GH secretion in Nephr animals, which differed from Sham males, but not from Fem rats. Expression of liver P450 CYP2C11 mRNA, which is dependent upon the male GH pattern, became undetectable, whereas expression of liver P450 CYP2C12 mRNA, which is dependent upon the female GH pattern, increased multifold. Renal failure in young rats abrogates the male pattern of GH pulsatility, abolishes the sexual dimorphism of body weight gain, and induces a female pattern of hepatic gene expression. These data raise the possibility that disruption of pulsatile GH secretion contributes to the growth failure of renal disease.

HepaRG Cells as an in Vitro Model for Evaluation of Cytochrome P450 Induction in Humans

HepaRG is a highly differentiated cell line that displays several hepatocyte-like functions, including drug-metabolizing enzymes. In this study, the HepaRG cells were characterized and evaluated as an in vitro model to predict cytochrome P450 (P450) enzyme induction of drugs in humans. Exposure of HepaRG cells to prototypical inducers resulted in induction of CYP1A1, CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, and CYP3A4 mRNA, as well as phenacetin O-dealkylase, bupropion hydroxylase, diclofenac 4'-hydroxylase, and midazolam 1'-hydroxylase activities. The observed induction is consistent with the previously reported expression of the nuclear receptors pregnane X receptor, constitutive androstane receptor, and aryl hydrocarbon receptor, which are necessary for a P450 induction response. To avoid problems with toxicity and solubility, the induction potency of test compounds was evaluated by calculating the concentrations leading to a 2-fold increase of baseline mRNA or enzyme activity levels (F(2) values) instead of EC(50) values from full dose-response curves. For CYP3A4 mRNA, the obtained F(2) values were related to the in vivo exposure [area under the plasma concentration versus time curve (AUC)] of the inducer (AUC/F(2)). This score was then correlated with the decrease in AUC for a CYP3A probe drug, administered before and after treatment with the inducing agent. By using this method an excellent correlation (R(2) = 0.863) was obtained, which implies that the degree of CYP3A induction in vivo can be predicted from CYP3A4 mRNA induction in HepaRG cells. The present study shows that HepaRG cells are a valuable model to be used for prediction of induction of drug-metabolizing P450 enzymes in vivo in humans.

Cytochrome P450 enzyme levels in HepG2 cells and cryopreserved primary human hepatocytes and their induction in HepG2 cells

Early in vitro toxicity screening might improve the success rate of new chemical entities in pharmaceutical development. In previous studies, the advantage of cytotoxicity screening with the HepG2 cell line was shown. Cytotoxicity could be identified for 70% of the compounds in these assays as compared with known toxicity in either in vitro assays in primary hepatocytes, in in vivo assays in rats, or in (pre-)clinical development in humans. The low Phase I and II enzyme levels in HepG2 cells might have been responsible for the fact that 30% of the compounds scored negative. Therefore, we performed two follow-up studies in which Cytochrome P450 (CYP) enzymes and Phase II metabolism were examined. In the present study, the transcript levels of CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 were measured with quantitative PCR. Results showed that transcripts of all CYPs were present in HepG2 cells, however, mRNA levels of most CYPs were dramatically lower than in primary human hepatocytes. These results were confirmed with luminometric assays which were used to measure the enzyme activities of CYP1A1, 1A2, 2C9, and 3A4. Regulation of CYP1A1, 1A2, 2B6, 2C8, 2D6, 2E1, and 3A4 by the aryl hydrocarbon receptor, pregnane X receptor and constitutive androstane receptor was studied in HepG2 cells at the mRNA and/or enzyme level. Regulation of CYP1A1, 1A2, 2B6, and 3A4 mRNA levels was similar to the regulation in primary human hepatocytes. In contrast, CYP2C8 mRNA levels are inducible in primary human hepatocytes, but not in HepG2 cells, after treatment with PXR/CAR activators. Consistent with other studies, CYP2D6 and 2E1 transcript levels were not changed after treatment with AhR, PXR, and CAR activators. Moreover, CYP1A1 and 1A2 enzyme levels could be induced by AhR agonists and CYP3A4 by PXR agonists. As a consequence of the low levels of CYPs in HepG2 cells, cytotoxicity of several compounds might have been missed or underestimated as compared with cytotoxicity in primary human hepatocytes. Inducing HepG2 cells with particular receptor stimulators might lead to higher toxicity for several of the tested compounds. Compared to primary human hepatocytes, HepG2 cells are a relatively easy-to-handle tool to study the up-regulation of CYP1A1, 1A2, 2B6, and 3A4.

Detection of Human CYP2C8, CYP2C9, and CYP2J2 in Cardiovascular Tissues

The cytochrome P450 (P450) enzymes CYP2C8, CYP2C9, and CYP2J2 metabolize arachidonic acid to epoxyeicosatrienoic acids, which are known to be vital in regulation of vascular tone and cardiovascular homeostasis. Because there is limited information regarding the relative expression of these P450 enzymes in cardiovascular tissues, this study examined the expression of CYP2C8, CYP2C9, and CYP2J2 mRNA and protein in human heart, aorta, and coronary artery samples by real-time polymerase chain reaction, immunoblotting, and immunohistochemistry. CYP2J2 and CYP2C9 mRNA levels were highly variable in human hearts, whereas CYP2C8 mRNA was present in lower abundance. CYP2J2 mRNA was approximately 10(3) times higher than CYP2C9 or CYP2C8 in human heart. However, CYP2C9 mRNA was more abundant than CYP2J2 or CYP2C8 in one ischemic heart. In human aorta, mean CYP2C9 mRNA levels were approximately 50 times higher than that of CYP2J2 and 5-fold higher than that of CYP2C8. In human coronary artery, mean values for CYP2C9 mRNA were approximately 2-fold higher than that of CYP2J2 mRNA and 6-fold higher than that of CYP2C8 mRNA. Immunoblotting results show relatively high levels of CYP2J2 and CYP2C8 protein in human hearts, which was confirmed by immunohistochemistry. CYP2C9 protein was also detected at high levels in one ischemic heart by immunoblotting. CYP2C9 was present at higher levels than CYPJ2 in aorta and coronary artery, whereas CYP2C8 protein was below the limits of detection. The expression of CYP2J2 and CYP2C8 in human heart, and CYPC9 and CYP2J2 in aorta and coronary artery is consistent with a physiological role for these enzymes in these tissues.

Broad but Distinct Role of Pregnane X Receptor on the Expression of Individual Cytochrome P450s in Human Hepatocytes

In the present study, we have utilized a target selective human pregnane X receptor-siRNA (hPXR-siRNA)-adenovirus expression system to examine the contribution of hPXR on the gene regulation of drug-metabolizing P450s in human hepatocytes. Introduction of the hPXR-siRNA adenoviral vector reduced the level of PXR mRNA. After infection with Ad hPXR-siRNA, the basal and ligand-activated CYP2A6, CYP2C8, CYP3A4 and CYP3A5 mRNA levels were decreased significantly in dose-dependent manners, whereas CYP2B6, CYP2C9 and CYP2C19 mRNA levels were moderately influenced after infection with Ad hPXR-siRNA. These data suggest the distinct PXR influences on the regulation of these genes. The expression of CYP1A2 and CYP2D6 mRNA were not affected by the introduction of hPXR-siRNA, suggesting that PXR plays no functional role in the expression of either of these genes. This is the first report to compare simultaneously the relative contribution of hPXR on the expression of nine forms of P450 in primary cultured human hepatocytes. Mutual sharing among nuclear receptors of their binding cis-elements becomes clear now. Thus, the present method using the combination of adenovirus-mediated hPXR-siRNA expression and human hepatocytes may offer clear information on the relative role of nuclear receptors such as hPXR on the expression of drug metabolizing genes.

Role of Human Hepatocyte Nuclear Factor 4α in the Expression of Drug-Metabolizing Enzymes and Transporters in Human Hepatocytes Assessed by Use of Small Interfering RNA

Hepatocyte nuclear factor 4alpha (HNF4alpha) is an important transcription factor in hepatic gene expression. Here, we have investigated the role of HNF4alpha in the expression of drug-metabolizing enzymes and transporters in human hepatocytes using an adenovirus expressing human HNF4alpha-small interfering RNA (hHNF4alpha-siRNA). The hHNF4alpha-siRNA effectively reduced the mRNA and nuclear protein levels of hHNF4alpha in a concentration-dependent manner. The hHNF4alpha-siRNA also decreased the mRNA levels of CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, UGT1A1, UGT1A9, SULT2A1, ABCB1, ABCB11, ABCC2, OATP1B1 and OCT1, as well as those of PXR and CAR. To discern the role of these nuclear receptors, we co-infected hepatocytes with hHNF4alpha-siRNA and PXR- or CAR-expressing adenovirus. The hHNF4alpha-siRNA-induced reductions of the enzyme and transporter mRNA levels were not restored except CYP2B6 mRNA levels, which were returned to the control level by overexpressing CAR. Furthermore, although hHNF4alpha-siRNA did not significantly affect the fold-induction of CYP2B6, CYP2C8, CYP2C9, or CYP3A4 mRNA levels following treatment with CYP inducers, the levels in hHNF4alpha-suppressed cells fell significantly compared to the control. These results suggest that HNF4alpha plays a dominant role in the expression of drug-metabolizing enzymes and transporters in human hepatocytes, and that HNF4alpha expression levels is a possible determinant for inter-individual variations in the expression of these enzymes and transporters.

Identification and Partial Characterization of a Novel CYP2C9 Splicing Variant Encoding a Protein Lacking Eight Amino Acid Residues

CYP2C9 is known as an enzyme responsible for the metabolism of various clinically important drugs. Recently, we cloned a cDNA corresponding to a CYP2C9 splicing variant (SV), which seemed to have an open reading frame of a protein with 482 amino acid residues. To investigate whether or not the SV can be translated as a functionally active protein, we expressed the CYP2C9SV in insect cells, and spectrophotometric and enzymatic properties were characterized. The CYP2C9SV protein showed a typical reduced CO-difference spectrum, indicating that the translated protein binds a heme moiety. However, CYP2C9SV did not metabolize tolbutamide or diclofenac at all, suggesting that the SV protein appeared to lack the ability to catalyze reactions mediated by CYP2C9. Although the CYP2C9SV mRNA was detected in all human liver samples examined in this study by real-time PCR, the level was generally low, ranging between 0.7 and 9.6% of the normal CYP2C9 mRNA. These results suggest that the CYP2C9SV protein is unlikely to contribute to CYP2C9 activities, although it appears to be expressed in most individuals.

Potential Impact of Steatosis on Cytochrome P450 Enzymes of Human Hepatocytes Isolated from Fatty Liver Grafts

Liver grafts discarded for transplantation because of macrosteatosis can constitute a valuable source of human hepatocytes for in vitro metabolic and pharmacotoxicological studies or for therapeutic applications. A condition for using hepatocyte suspensions for these purposes is the preservation of their metabolic competence and, particularly, drug-metabolizing enzymes. A reduction in microsomal cytochrome P450 (P450) activities was observed in fatty livers (>40% steatosis) with respect to normal tissue. Similarly, decreased levels of 7-ethoxycoumarin O-deethylation and testosterone metabolism were observed in human hepatocyte cultures prepared from steatotic liver tissue. To clarify the potential impact of lipid accumulation on human hepatic P450 enzymes, we have used an in vitro model of "cellular steatosis" by incubation of cultured hepatocytes with increasing concentrations (0.25-3 mM) of long-chain free fatty acids (FFA). A dose-dependent accumulation of lipids in the cytosol is induced by FFA mixture. Hepatocytes exposed to 1 mM FFA for 14 h showed lower activity values of CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2D6, CYP2E1, and CYP3A4 enzymes than nontreated hepatocytes (about 45-65% reduction). This treatment also produced significant decreases in CYP1A2, CYP2A6, CYP2C9, CYP2D6, CYP2E1, and CYP3A4 mRNA to about 55 to 75% of mRNA levels in control cells. Our results suggest that although human hepatocytes isolated from steatotic liver show reduced P450 activities, they are metabolically competent and can be used for drug metabolism studies.

Detection of human CYP2Cs in heart, aorta and coronary tissues

CYP2C and CYP2J enzymes metabolize arachidonic to epoxyeicosatrienoic acids (EETs). Expression of these enzymes in the cardiovascular system may have a direct relationship to hypertension and cardiovascular diseases, such as acute myocardial infarction (AMI). CYP2C polymorphisms have also been reported to result in an increased risk of AMI. Drugs that inhibit CYP2C9 activity are cardioprotective in animal models of reperfusion injury. In this study, normal heart, coronary and aorta tissues were obtained from individuals of various ages, sex and races. We then measured the abundance of CYP2C8, CYP2C9 and CYP2J2, in human heart, aorta and coronary samples by Real-time PCR, immunoblotting and immunohistochemistry. Immunoblotting results show that CYP2J2 and CYP2C8 were detected in human heart, coronary vessels and aorta. The relative abundance of CYP2C9 and CYP2J2 mRNA were highly variable in human heart from different individuals, CYP2C8 was detected in only 1/8 hearts. In contrast, in human aorta, CYP2C9 mRNA levels were ~100-fold higher than either CYP2J2 or CYP2C8. In coronary artery, the levels of CYP2C9, CYP2J2, and CYP2C8 mRNA were more comparable but in general CYP2C9 was slightly higher. The presence of CYP2C9, and CYP2J2 and somewhat lower levels of CYP2C8 in cardiovascular tissues suggests that CYP2C9 and CYP2J2 have physiologically relevant roles in the production of vasoactive eicosanoids which may be important in modulating vascular tone and homeostasis. This research was supported by the intramural division of the NIEHS.

Assessment of Induction of Cytochrome P450 by NO-1886 (Ibrolipim), a Lipoprotein Lipase-Promoting Agent, in Primary Cultures of Human Hepatocytes and in Female Rat Liver

The mRNA levels of human cytochrome P450 (CYP)2Cs and CYP3As in primary cultures of freshly isolated human hepatocytes were assessed after exposure to NO-1886 and rifampicin, a typical inducer of CYP3As. mRNA levels were analyzed by real-time RT-PCR using an ABI PRISM 7700 Sequence Detector system. Exposure to NO-1886 for 24 hr at a concentration of less than 10 microM showed only a tendency to reduce or increase the expression levels of CYP2C8, CYP2C9, CYP2C19, CYP3A4, or CYP3A5 mRNA. A higher concentration (50 microM) of NO-1886 induced an increase in CYP2C8 mRNA and a decrease in CYP2C19 mRNA, and these changes continued after additional culture for 24 hr in fresh medium without NO-1886. The expression level of CYP3A4 mRNA after exposure to NO-1886 for 24 hr at 50 microM was about twice that in controls. Following additional culture for 24 hr in fresh medium without NO-1886, the expression of CYP3A4 mRNA was comparable to that in controls. On the other hand, the expression levels of CYP2C9 and CYP3A5 mRNA showed small and variable changes in each donor even at a high concentration (50 microM) of NO-1886. Furthermore, the pharmacokinetics of NO-1886 during repeated oral administration for 14 days was studied in female rats. The pharmacokinetic parameters of NO-1886 were nearly the same on days 1, 7, and 14 of repeated administration. The hepatic microsomal content of CYP isoforms was not affected by repeated administration for 7 days at a dose of 1 to 30 mg/kg in female rats, although the total CYP content was increased at a dose of 30 mg/kg. The expression levels of CYP1A2, CYP2B2, CYP2C12, and CYP2E1 mRNA in primary cultures of rat hepatocytes were not affected by exposure to NO-1886 at 2, 10, or 50 microM. The expression levels of CYP3A1 mRNA in primary cultures of rat hepatocytes were not affected by exposure to NO-1886 at 2 or 10 microM, but were increased, with large individual variation, by exposure at 50 microM. The mRNA expression levels in rat hepatocytes exposed to concentrations comparable to free plasma levels did not change significantly, which was consistent with the equivalence in the in vivo plasma concentrations observed on days 1 and 14 of repeated administration. These results suggest that repeated administration of NO-1886 at clinical doses does not significantly affect the expression levels of CYP isoforms in human liver, although the mRNA levels of the CYP isoforms involved in the metabolism of NO-1886 were increased by exposure to higher concentrations of NO-1886 in human hepatocytes in vitro.

Styrene monomer primarily induces CYP2B1 mRNA in rat liver

To determine the cytochrome P450 (CYP) primarily expressed after styrene exposure, seven forms of hepatic CYP mRNA in rats treated with 600 mg kg−1 styrene were examined. CYP1A2, CYP2B1/2, CYP2E1 and CYP3A2 mRNA were observed using real-time LightCycler PCR. The amount of CYP2B1 mRNA was significantly increased, 47-fold compared with controls, suggesting that this CYP is the primary cytochrome P450 in rats exposed to styrene. Significant increases in the amount of CYP2E1, CYP1A2 and CYP2B2 mRNA were also observed after styrene exposure, and their increase levels were 3.1-, 1.7- and 1.7-fold higher than controls, respectively. Western blot analysis also indicated that the protein levels of CYP2B1, CYP2B2, CYP2E1 and CYP1A2 showed clear increases after styrene treatment, corresponding to their mRNA expression. CYP2C11 mRNA decreased significantly in rats after styrene exposure. CYP1A1 was detected at the mRNA level in rat liver, but it was not detected at the protein level. The expression of epoxide hydrolase (EH), involved in Phase I drug metabolism, was also examined. EH mRNA increased 2-fold compared with controls after styrene exposure. Styrene thus appears to be a chemical compound that induces multiple CYPs. The results demonstrate that CYP2B1 is the primarily induced CYP form by styrene treatment to rats at acute toxic level.

Distribution of cytochrome P450 2C, 2E1, 3A4, and 3A5 in human colon mucosa

BackgroundDespite the fact that the alimentary tract is part of the body's first line of defense against orally ingested xenobiotica, little is known about the distribution and expression of cytochrome P450 (CYP) enzymes in human colon. Therefore, expression and protein levels of four representative CYPs (CYP2C(8), CYP2E1, CYP3A4, and CYP3A5) were determined in human colon mucosa biopsies obtained from ascending, descending and sigmoid colon.MethodsExpression of CYP2C, CYP2E1, CYP3A4, and CYP3A5 mRNA in colon mucosa was determined by RT-PCR. Protein concentration of CYPs was determined using Western blot methods.ResultsExtensive interindividual variability was found for the expression of most of the genes. However, expression of CYP2C mRNA levels were significantly higher in the ascending colon than in the sigmoid colon. In contrast, mRNA levels of CYP2E1 and CYP3A5 were significantly lower in the ascending colon in comparison to the descending and sigmoid colon. In sigmoid colon protein levels of CYP2C8 were significantly higher by ~73% than in the descending colon. In contrast, protein concentration of CYP2E1 was significantly lower by ~81% in the sigmoid colon in comparison to the descending colon.ConclusionThe current data suggest that the expression of CYP2C, CYP2E1, and CYP3A5 varies in different parts of the colon.

Down-regulation of astroglial CYP2C, glucocorticoid receptor and constitutive androstane receptor genes in response to cocaine in human U373 MG astrocytoma cells

Psychostimulant drugs abuse is associated with an increased risk of stroke. Cytochromes P450 (CYP), especially the astrocytic members of the CYP2C subfamily may play an important role in the modulation of cerebrovascular functions, by generating vasodilatator metabolites from arachidonic acid (AA). Our study examined the regulation of CYP2C genes in response to cocaine or amphetamine in the human astrocyte-like U373 MG cells, using reverse transcription-polymerase chain reaction (RT-PCR) and western-blot analysis. A treatment for 48 h with increasing concentrations of cocaine caused a significant down-regulation of CYP2C8 and CYP2C9 genes and decreased the protein level. These effects were not observed with amphetamine. One mechanism of the CYP2C mRNA regulation implicates various specific receptors including glucocorticoid receptor (GR) and constitutive androstane receptor (CAR). Effects of cocaine on CYP2C were accompanied by a decrease in the GR and CAR gene expression suggesting that these nuclear receptors could be involved in the CYP2C repression by cocaine in the U373 MG cell line. These findings represent a possible molecular mechanism involved in the cerebrovascular risk associated with cocaine abuse.

Cytochrome P450 mRNA expression in peripheral blood lymphocytes as a predictor of enzyme induction

Previous reports have supported the concept that messenger ribonucleic acid (mRNA) concentrations for cytochrome P450 (CYP) enzymes in peripheral blood mononuclear cells may be predictive of systemic enzyme activity. We investigated whether changes in mRNA expression for CYP1A2,CYP2C19, CYP2D6 and CYP3A4 in peripheral blood lymphocytes (PBLs) may serve as surrogate markers for changes in CYP enzyme activity following the administration of rifampin. On day 1 and day 9 of the study, 12 healthy volunteers were administered caffeine 100 mg, debrisoquine 10 mg and omeprazole 40 mg orally, along with midazolam 0.025 mg/kg intravenously. Blood samples and urine were collected for 8 h after drug administration. The subjects took rifampin 300 mg (n = 6) or 600 mg (n = 6) daily on days 2-8. Total RNA was isolated from PBLs on day 1 and day 9, and mRNA expression for the CYP enzymes and hGAPDH were determined by means of quantitative, real-time, reverse-transcriptase polymerase chain reaction. CYP1A2 activity was estimated by calculating the plasma paraxanthine to caffeine AUC ratio (caffeine metabolic ratio; CMR), CYP2C19 activity by the 2-h omeprazole hydroxylation index (HI), CYP2D6 activity by the urinary debrisoquine recovery ratio (DBRR) and CYP3A4 activity by midazolam clearance. Median midazolam clearance (0.362 to 0.740 l/kg/h), omeprazole HI (0.752 to 0.214), CMR (0.365 to 0.450) and DBRR (0.406 to 0.479) all changed significantly following rifampin, consistent with the expected enzyme induction. CYP1A2,CYP2D6 and CYP3A4 mRNA content were measurable in all samples. CYP2C19 mRNA was inconsistently detectable. There were no significant correlations between changes in enzyme activity and mRNA expression by Spearman's rank order correlation. The results do not support the use of mRNA expression assays for CYP1A2, CYP2C19, CYP2D6 and CYP3A4 enzymes in PBLs as surrogates for quantifying changes in systemic enzyme activity in the setting of enzyme induction.

Cytochrome P450-2C11 mRNA Is Not Expressed in Endothelial Cells Dissected from Rat Renal Arterioles

Background: Cytochrome P450 (CYP) isoenzymes (CYP2C and CYP2J) are involved in the production of epoxyeicosatrienoic acids, which are postulated as endothelium-derived hyperpolarizing factors (EDHFs). We hypothesized that if CYP2C11 is involved in the EDHF-mediated responses, its mRNA should be expressed in endothelial cells. We, therefore, examined the mRNA expression of CYP2C11 in endothelial cells of renal arterioles. Methods: Laser microdissection was applied to isolate endothelial cells from the renal arterioles of 4 male and 4 female Wistar rats. As a positive control of CYP2C11 expression, hepatocytes were also dissected from these rats. RNA was isolated and real-time quantitative polymerase chain reaction (Q-PCR) analysis was applied. Results: Q-PCR analysis showed that CYP2C11 mRNA was not expressed in laser microdissected endothelial cells of renal arterioles of male and female rats. CYP2C11 mRNA expression was highly abundant in hepatocytes dissected from male livers, but in female livers hardly any CYP2C11 mRNA was detected. Conclusion: We have shown that endothelial cells can be dissected from small renal arterioles by laser microdissection to study the mRNA expression of specific genes by Q-PCR. Using this novel tool, we demonstrated that the CYP2C11 mRNA was not expressed in the endothelial cells of renal arterioles. Therefore, we speculate that CYP2C11 does not contribute to the EDHF-mediated responses in renal arterioles.

AhR and PPARa: antagonistic effects on CYP2B and CYP3A, and additive inhibitory effects on CYP2C11

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates a spectrum of toxic and biological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds. The peroxisome proliferator-activated receptor alpha (PPARα) is a member of the nuclear receptor super-family of ligand-activated transcription factors and it functions as an obligate heterodimer with retinoid X-receptor alpha RXRα. The aim was to investigate whether the negative cross-talk recently proposed by the present authors between AhR and PPARα on CYP4A and CYP1A has any impact on other cytochrome P450 enzymes. Treatment of male Wistar rats with a PPARα ligand clofibric acid (CA) induced CYP2B1/2 and CYP3A proteins, activities, and the mRNA expression of CYP2B1, CYP2B2, CYP3A1 and CYP3A2, and suppressed CYP2C11 protein, activities and mRNA expression. AhR ligand Sudan III (S.III) treatment decreased basal and CA-induced CYP2B, CYP3A and CYP2C11 protein, activities and mRNA expression. To the best of the authors’ knowledge, this is the first study showing the presence of mutual effects of AhR and PPARα on CYP2B and CYP3A and an additive inhibitory effect on CYP2C11 in the livers of male rats.

Utilization of Human Liver Microsomes to Explain Individual Differences in Paclitaxel Metabolism by CYP2C8 and CYP3A4

Paclitaxel is widely used for treatment of malignant tumors. Paclitaxel is metabolized by CYP2C8 and CYP3A4, and these enzymes are known to differ between individuals, although the details have not been clarified. Recent progress in pharmacogenetics has shown that genetic polymorphisms of metabolic enzymes are related to these individual differences. We investigated the effect of the polymorphisms on paclitaxel metabolism by analyzing metabolic activities of CYP2C8 and CYP3A4 and expressions of mRNA and protein. Production of 6α-hydroxypaclitaxel, a metabolite of CYP2C8, was 2.3-fold larger than 3′-p-hydroxypaclitaxel, a metabolite of CYP3A4. Significant inter-individual differences between these two enzyme activities were shown. The expressions of mRNA and protein levels correlated well with the enzyme activities, especially with CYP3A4. Although it was previously reported that CYP2C8*3 showed lower activity than the wild type, two subjects that had the CYP2C8*3 allele did not show lower activities in our study. Inter-individual differences in paclitaxel metabolism may be related to CYP2C8 and CYP3A4 mRNA expression. CYP2C8 is the primary metabolic pathway of paclitaxel, but there is a “shifting phenomenon” in the metabolic pathway of paclitaxel in the liver of some human subjects.

Characterization of recombinant CYP2C11: a vitamin D 25-hydroxylase and 24-hydroxylase

Studies were performed to further characterize the male-specific hepatic recombinant microsomal vitamin D 25-hydroxlase CYP2C11, expressed in baculovirus-infected insect cells, and determine whether it is also a vitamin D 24-hydroxylase. 25- and 24-hydroxylase activities were compared with those of 10 other recombinant hepatic microsomal cytochrome P-450 enzymes expressed in baculovirus-infected insect cells. Each of them 25-hydroxylated vitamin D2, vitamin D3, 1alpha-hydroxyvitamin D2 (1alphaOHD2), and 1alpha-hydroxyvitamin D3 (1alphaOHD3). CYP2C11 had the greatest activity with these substrates, except vitamin D3, which had the same activity as four of the other enzymes. The descending order of 25-hydroxylation by CYP2C11 was 1alphaOHD3 > 1alphaOHD2 > vitamin D2 > vitamin D3. Each of the recombinant cytochrome P-450 enzymes 24-hydroxylated 1alphaOHD2. CYP2C11 had the greatest activity. 24-Hydroxylation of 1alphaOHD3 was very low, and there was none with vitamin D3. Only CYP2C11 24-hydroxylated vitamin D2. Structures of vitamin D metabolites, including 24-hydroxyvitamin D2, 1,24(S)-dihydroxyvitamin D2, and 1,24-dihydroxyvitamin D3, were confirmed by HPLC and gas chromatography retention times and characteristic mass spectrometric fragmentation patterns. In male rats, hypophysectomy significantly reduced body weight, liver weight, hepatic CYP2C11 mRNA expression, and 24- and 25-hydroxylation of 1alphaOHD2. Expression of CYP2J3 and CYP2R1 mRNA did not change. In male rat hepatocytes, CYP2C11 mRNA expression and 24- and 25-hydroxylation were significantly reduced after culture for 24 h compared with uncultured cells. Expression of CYP2J3 and CYP2R1 either increased or did not change. It is concluded that CYP2C11 is a male-specific hepatic microsomal vitamin D 25-hydroxylase that hydroxylates vitamin D2, vitamin D3, 1alphaOHD2, and 1alphaOHD3. CYP2C11 is also a vitamin D 24-hydroxylase.