Hepatic P450 Research Articles

Effect of Cytochrome P450 Reductase Deficiency on 2-Amino-9H-pyrido[2,3-b]indole Metabolism and DNA Adduct Formation in Liver and Extrahepatic Tissues of Mice.

2-Amino-9H-pyrido[2,3-b]indole (AαC), a carcinogen formed during the combustion of tobacco and cooking of meat, undergoes cytochrome P450 (P450) metabolism to form the DNA adduct N-(deoxyguanosin-8-yl)-2-amino-9H-pyrido[2,3-b]indole (dG-C8-AαC). We evaluated the roles of P450 expressed in the liver and intestine to bioactivate AαC by employing male B6 wild-type (WT) mice, liver-specific P450 reductase (Cpr)-null (LCN) mice, and intestinal epithelium-specific Cpr-null (IECN) mice. Pharmacokinetic parameters were determined for AαC, 2-amino-9H-pyrido[2,3-b]indol-3-yl sulfate (AαC-3-OSO3H), and N(2)-(β-1-glucosidurony1)-2-amino-9H-pyrido[2,3-b]indole (AαC-N(2)-Glu) with animals dosed by gavage with AαC (13.6 mg/kg). The uptake of AαC was rapid with no difference in the plasma half-lives (t1/2) of AαC, AαC-3-OSO3H, and AαC-N(2)-Glu among mouse models. The maximal plasma concentrations (Cmax) and the areas under concentration-time curve (AUC0-24h) of AαC and AαC-N(2)-Glu were 4-24-fold higher in LCN than in WT mice, but they were not different between WT and IECN mice. These findings are consistent with the ablation of hepatic P450 activity in LCN mice. However, the Cmax and AUC0-24h of AαC-3-OSO3H in plasma were not substantially different among the mouse models. Similar pharmacokinetic parameters were obtained with WT and LCN mice treated with a lower AαC dose (1.36 mg kg(-1)). dG-C8-AαC was detected at similar levels in the livers of all three mouse models at the high AαC dose; levels of dG-C8-AαC in colon, bladder, and lung were greater in LCN than in WT mice and were the same in colon of IECN and WT mice. At the low AαC dose, dG-C8-AαC occurred at ∼ 40% lower levels in liver of LCN mouse than in WT mouse liver, but adduct levels remained higher in extrahepatic tissues of LCN mice. Therefore, hepatic P450 plays an important role in detoxication of AαC, but other hepatic or extrahepatic enzymes contribute to the bioactivation of AαC. P450s expressed in the intestine do not appreciably contribute to bioactivation of AαC in mice.

Role of hepatic cytochrome P450 enzymes in the detoxication of aristolochic acid I; effects on DNA adduct, mutation, and tumor formation.

IntroductionHepatic cytochrome P450s (CYPs) play an important role in the metabolism of plant carcinogen, aristolochic acid I (AAI). In the present study, we employed hepatic NADPH-cytochrome P450 reductase null (HRN) gpt delta transgenic mice to investigate the role of hepatic CYPs in the metabolism of AAI. DNA adduct formation, gene mutation, and tumor induction in the liver and kidneys were analyzed. Pharmacokinetic analyses were performed and tissue levels of AAI were determined.ResultsPretreatment with β-naphthoflavone in wild type gpt delta transgenic mice (BNF-WT mice) could increase the rate of clearance of AAI in blood and tissues, and decrease the formation of AAI-DNA adducts in kidney. In contrast, there was reduced clearance of AAI in HRN gpt delta mice, which showed increased concentration of AAI in tissues and increased levels of DNA adducts. The mutant frequencies of gpt gene, induced by AAI, in the kidneys of HRN gpt delta mice were significantly higher than that in WT mice. In the tumor induction assay, after treatment for 2 months with daily doses of 5 mg/kg AAI, mice were kept under observation for 7 months. During this period, papillomatous changes occurred in the forestomach of both WT-AAI mice and HRN gpt delta-AAI mice. Squamous cell carcinomas were found in the forestomach of 2 HRN gpt delta-AAI mice, which had also metastasized to other tissues. In addition, adenomas were found in 2 of 8 HRN gpt delta-AAI mice, in the absence of squamous cell carcinomas.ConclusionThese results indicated that the main role of hepatic CYPs is to aid in the excretion of AAI, and to protect the target organs against AAI induced DNA adduct formation, mutagenesis, and tumorigenesis.Electronic supplementary materialThe online version of this article (doi:10.1186/s41021-015-0010-z) contains supplementary material, which is available to authorized users.

Effect of Jiawei Foshou San and its compatibility on hepatic P450 enzyme activity and hepatocyte morphology in rats

To investigate the effect of Jiawei Foshou San and its various combined administration on hepatic P450 enzyme activity and hepatocyte morphology in rats. Rats were orally administered with drugs for four weeks and then sacrificed to prepare liver microsomes. The liver microsomes were incubated with the cocktail method; The metabolites were determined with the rapid liquid chromatography with tandem mass spectrometry (LC-MS/MS) to investigate the hepatocyte P450 enzyme activity. In addition, the hepatic pathological changes were observed by using the hematoxylin and eosin (HE) staining. Compared with the control group, the enzyme activity of CYP1A2 and CYP3A4 in the Jiawei Foshou san group showed a significant rise (P < 0.05); the enzyme activity of CYP1A2, CYP2C9, CYP2D6, CYP2E1 and CYP3A4 in the ferulic acid + ligustrazine group and the ligustrazine + tetrahydropalmatine group showed a significant rise (P < 0.05) ; the enzyme activity of CYP1A2, CYP2D6 and CYP2E1 in the ligustrazine group showed a significant rise (P < 0.05); the enzyme activity of CYP3A4 in the ferulic acid group showed a significant reduction (P < 0.05). After the administration with various drugs, the hepatocyte morphologies in the ferulic acid group and the ligustrazine group were normal. The pathological changes were observed in the tetrahydropalmatine group, such as unclear boundary of hepatic lobules, disordered hepatic cell arrangement, blurred edge, anisokaryosis and infiltration of inflammatory cells. The ferulic acid + tetrahydropalmatine group, the ligustrazine + tetrahydropalmatine group and the Jiawei Foshou San group also showed inflammatory infiltration, but with less pathological changes, particularly the Jiawei Foshou San group. The study result shows that Jiawei Foshou San can induce the enzyme activity of CYP1A2 and CYP3A4, and ligustrazine may be the effective substance for inducing CYP1A2. Its combination with ferulic acid and ligustrazine can significantly reduce the liver toxicity of tetrahydropalmatine.

Hepatic effects of repeated oral administration of diclofenac to hepatic cytochrome P450 reductase null (HRN™) and wild-type mice.

Hepatic NADPH-cytochrome P450 oxidoreductase null (HRN™) mice exhibit normal hepatic and extrahepatic biotransformation enzyme activities when compared to wild-type (WT) mice, but express no functional hepatic cytochrome P450 activities. When incubated in vitro with [(14)C]-diclofenac, liver microsomes from WT mice exhibited extensive biotransformation to oxidative and glucuronide metabolites and covalent binding to proteins was also observed. In contrast, whereas glucuronide conjugates and a quinone-imine metabolite were formed when [(14)C]-diclofenac was incubated with HRN™ mouse liver, only small quantities of P450-derived oxidative metabolites were produced in these samples and covalent binding to proteins was not observed. Livers from vehicle-treated HRN™ mice exhibited enhanced lipid accumulation, bile duct proliferation, hepatocellular degeneration and necrosis and inflammatory cell infiltration, which were not present in livers from WT mice. Elevated liver-derived alanine aminotransferase, glutamate dehydrogenase and alkaline phosphatase activities were also observed in plasma from HRN™ mice. When treated orally with diclofenac for 7 days, at 30 mg/kg/day, the severities of the abnormal liver histopathology and plasma liver enzyme findings in HRN™ mice were reduced markedly. Oral diclofenac administration did not alter the liver histopathology or elevate plasma enzyme activities of WT mice. These findings indicate that HRN™ mice are valuable for exploration of the role played by hepatic P450s in drug biotransformation, but poorly suited to investigations of drug-induced liver toxicity. Nevertheless, studies in HRN™ mice could provide novel insights into the role played by inflammation in liver injury and may aid the evaluation of new strategies for its treatment.

Functional Coupling of ATP-binding Cassette Transporter Abcb6 to Cytochrome P450 Expression and Activity in Liver

Although endogenous mechanisms that negatively regulate cytochrome P450 (P450) monooxygenases in response to physiological and pathophysiological signals are not well understood, they are thought to result from alterations in the level of endogenous metabolites, involved in maintaining homeostasis. Here we show that homeostatic changes in hepatic metabolite profile in Abcb6 (mitochondrial ATP-binding cassette transporter B6) deficiency results in suppression of a specific subset of hepatic P450 activity. Abcb6 null mice are more susceptible to pentobarbital-induced sleep and zoxazolamine-induced paralysis, secondary to decreased expression and activity of Cyp3a11 and Cyp2b10. The knock-out mice also show decrease in both basal and xeno-inducible expression and activity of a subset of hepatic P450s that appear to be related to changes in hepatic metabolite profile. These data, together with the observation that liver extracts from Abcb6-deficient mice suppress P450 expression in human primary hepatocytes, suggest that this mouse model may provide an opportunity to understand the physiological signals and the mechanisms involved in negative regulation of P450s.

Any Polymorphisms of CYP2C9 Affects the Biochemical Profile of Diabetic Patients Receiving Glibenclamide

Introduction: Glibenclamide, a hypoglycemic agent, is a member of the sulfonylurea family. It is metabolized by the hepatic P450 CYP2C9. Despite thorough pharmacokinetic characterization of glibenclamide, the evidence for a pharmacogenetic influence on the pharmacodynamics of this medication is scarce. The current study aims to evaluate a possible association between different CYP2C9 alleles and the pharmacodynamic effect of glibenclamide, including hypoglycemic events and required daily glibenclamide dosage. Methods: The patients included were aged ≥ 18 years, diagnosed with type II diabetes mellitus, and treated with metformin and glibenclamide for at least 3 months. Exclusion criteria were treatment with other hypoglycemic agents or with medications that affect CYP2C9 function. Patients underwent a medical interview, including detailed anti-glycemic treatment dosage and duration. Blood was drawn for CYP2C9 allele genotyping, glycosilated hemoglobin A1C, chemistry and a complete blood count. Patients were re-interviewed 1 month after inclusion for hypoglycemic event incidence. The patient’s genotype was classified as wild-type (WT) for *1/*1 alleles, or CYP2C9 polymorphism for any other alleles. Results: Fifty eight patients were recruited. Characteristics were similar in the two groups. Forty patients had WT genotype (69.0%), twelve patients had *1/*2 genotype (20.7%), five (8.6%) had *1/*3 genotype and one (1.7%) had *2/*3 genotype. Patients with CYP2C9 polymorphism had similar A1C levels (7.5 ± 0.99 vs. 7.6 ± 1.3, NS) but suffered more hypoglycemic episodes (≥ 2 episodes in 3 months; 5% vs. 22.2%, p=0.04) under similar glibenclamide dosage (6.5+4.2 vs. 5.3 ± 3.9, NS). Conclusion: Our study suggests that diabetic patients with any CYP2C9 polymorphism have higher tendency to develop hypoglycemia when treated with glibenclamide, compared to those with wild type alleles. Further studies are needed to evaluate the clinical benefit from genotyping CYP2C9 before treatment initiation with glibenclamide or other drugs metabolized by CYP2C9 enzyme. Study points • Most of the sulfonylurea drug family members are metabolized by the hepatic P450 CYP2C9 enzyme to their inactive form. • Polymorphism in CYP2C9 may encode a lower activity CYP2C9 enzyme, with different alleles encoding enzymes with varied activity degree. • Our study shows that any polymorphism in CYP2C9 gene expose patients treated with glibenclamide to higher risk of hypoglycemia compared with the wild type alleles.

Effect of erythropoietin on hepatic cytochrome P450 expression and function in an adenine-fed rat model of chronic kidney disease.

Erythropoietin (EPO) is used to treat anaemia associated with chronic kidney disease (CKD). Hypoxia is associated with anaemia and is known to cause a decrease in cytochrome P450 (P450) expression. As EPO production is regulated by hypoxia, we investigated the role of EPO on P450 expression and function. Male Wistar rats were subjected to a 0.7% adenine diet for 4 weeks to induce CKD. The diet continued for an additional 2 weeks while rats received EPO by i.p. injection every other day. Following euthanasia, hepatic P450 mRNA and protein expression were determined. Hepatic enzyme activity of selected P450s was determined and chromatin immunoprecipitation was used to characterize binding of nuclear receptors involved in the transcriptional regulation of CYP2C and CYP3A. EPO administration decreased hepatic mRNA and protein expression of CYP3A2 (P < 0.05), but not CYP2C11. Similarly, EPO administration decreased CYP3A2 protein expression by 81% (P < 0.001). A 32% decrease (P < 0.05) in hepatic CYP3A enzymatic activity (Vmax ) was observed for the formation of 6βOH-testosterone in the EPO-treated group. Decreases in RNA pol II recruitment (P < 0.01), hepatocyte nuclear factor 4α binding (P < 0.05) and pregnane X receptor binding (P < 0.01) to the promoter region of CYP3A were also observed in EPO-treated rats. Our data show that EPO decreases the expression and function of CYP3A, but not CYP2C in rat liver.

Role of cytochrome P450-mediated metabolism and identification of novel thiol-conjugated metabolites in mice with phenytoin-induced liver injury

Phenytoin, 5,5-diphenylhydantoin (DPH), is widely used as an anticonvulsant agent. Severe hepatic injury rarely occurs in patients who received DPH. The development of liver injury is thought to be caused by reactive metabolites; however, the metabolites suggested to contribute to hepatotoxicity have not yet been detected in vivo and their effect on developing the liver injury is largely unknown. We recently demonstrated that DPH treatment decreased hepatic glutathione (GSH) contents, and GSH-depleted condition exacerbated DPH-induced liver injury in mice. The aim of the present study was to identify the reactive metabolite and to investigate the role of P450-mediated metabolisms in DPH-induced liver injury. We identified a novel GSH-conjugated (GS)-DPH, a conjugate of putative electrophilic arene oxide intermediate with GSH, in the bile of mice with DPH-induced liver injury. In plasma, cysteine- or N-acetylcysteine-conjugated DPH was detected, and these thiol conjugates levels were correlated with the plasma alanine aminotransferase (ALT) levels. These changes were significantly reduced by pretreatment with P450 inhibitor. Furthermore, the increases of hepatic P450 activities were in parallel with elevation of plasma thiol conjugates levels. These findings suggest that the arene oxide intermediate, which can be converted to thiol conjugates, is involved in DPH-induced liver injury.

SiRNA-Mediated Knockdown of P450 Oxidoreductase in Rats: A Tool to Reduce Metabolism by CYPs and Increase Exposure of High Clearance Compounds

To develop a tool based on siRNA-mediated knockdown of hepatic P450 oxidoreductase (POR) to decrease the CYP-mediated metabolism of small molecule drugs that suffer from rapid metabolism in vivo, with the aim of improving plasma exposure of these drugs. siRNA against the POR gene was delivered using lipid nanoparticles (LNPs) into rats. The time course of POR mRNA knockdown, POR protein knockdown, and loss of POR enzyme activity was monitored. The rat livers were harvested to produce microsomes to determine the impact of POR knockdown on the metabolism of several probe substrates. Midazolam (a CYP3A substrate with high intrinsic clearance) was administered into LNP-treated rats to determine the impact of POR knockdown on midazolam pharmacokinetics. Hepatic POR mRNA and protein levels were significantly reduced by administering siRNA and the maximum POR enzyme activity reduction (~85%) occurred 2weeks post-dose. In vitro analysis showed significant reductions in metabolism of probe substrates due to POR knockdown in liver, and in vivo POR knockdown resulted in greater than 10-fold increases in midazolam plasma concentrations following oral dosing. Anti-POR siRNA can be used to significantly reduce hepatic metabolism by various CYPs as well as greatly increase the bioavailability of high clearance compounds following an oral dose, thus enabling it to be used as a tool to increase drug exposure in vivo.

Genetic variation in aldo‐keto reductase 1D1 (AKR1D1) affects expression and activity of multiple cytochrome P450s (292.1)

Human liver gene regulatory network analysis identified a cytochrome P450 subnetwork with AKR1D1 as its key regulatory driver. This study assessed the biologic importance of AKR1D1 [key enzyme in synthesis of bile acids, ligand activators of FXR, PXR and CAR, known P450 regulators] to hepatic P450 expression. Overexpression of AKR1D1 in primary human hepatocytes led to increased expression of CYP3A4, 2C8, 2C9, 2C19, and 2B6. Conversely, AKR1D1 knockdown decreased expression of these P450s. Resequencing AKR1D1 from donor livers identified a 3'‐UTR (rs1872930) SNP that was significantly associated with higher AKR1D1 mRNA. AKR1D1 3'‐UTR‐luciferase reporter studies showed that variant allele resulted in higher luciferase activity, suggesting that the SNP increases AKR1D1 mRNA stability and/or translation efficiency. AKR1D1 3'‐UTR SNP was significantly associated with increased hepatic mRNA of multiple P450s (CYP3A4, 2C8, 2C9, 2C19, and 2B6) and CYP3A4, 2C8, 2C19, and 2B6 activities. After adjusting for multiple testing, association remained significant for AKR1D1, CYP2C9, and CYP2C8 mRNA and CYP2C8 activity. These results provide new insights into the variation in expression and activity of P450s that can account for interindividual differences in drug metabolism/efficacy. In conclusion, we provide the first experimental evidence supporting a role for AKR1D1 as a key genetic regulator of P450 network.

Genetic variation in aldo‐keto reductase 1D1 (AKR1D1) affects expression and activity of multiple cytochrome P450s (LB595)

Human liver gene regulatory network analysis identified a cytochrome P450 subnetwork with AKR1D1 as its key regulatory driver. This study assessed the biologic importance of AKR1D1 [key enzyme in synthesis of bile acids, ligand activators of FXR, PXR and CAR, known P450 regulators] to hepatic P450 expression. Overexpression of AKR1D1 in primary human hepatocytes led to increased expression of CYP3A4, 2C8, 2C9, 2C19, and 2B6. Conversely, AKR1D1 knockdown decreased expression of these P450s. Resequencing AKR1D1 from donor livers identified a 3'‐UTR (rs1872930) SNP that was significantly associated with higher AKR1D1 mRNA. AKR1D1 3'‐UTR‐luciferase reporter studies showed that variant allele resulted in higher luciferase activity, suggesting that the SNP increases AKR1D1 mRNA stability and/or translation efficiency. AKR1D1 3'‐UTR SNP was significantly associated with increased hepatic mRNA of multiple P450s (CYP3A4, 2C8, 2C9, 2C19, and 2B6) and CYP3A4, 2C8, 2C19, and 2B6 activities. After adjusting for multiple testing, association remained significant for AKR1D1, CYP2C9, and CYP2C8 mRNA and CYP2C8 activity. These results provide new insights into the variation in expression and activity of P450s that can account for interindividual differences in drug metabolism/efficacy. In conclusion, we provide the first experimental evidence supporting a role for AKR1D1 as a key genetic regulator of P450 network.

Deletion of 30 Murine Cytochrome P450 Genes Results In Viable Mice With Compromised Drug Metabolism

In humans, 75% of all drugs are metabolized by the cytochrome P450-dependent monooxygenase system. Enzymes encoded by the CYP2C, CYP2D, and CYP3A gene clusters account for ∼80% of this activity. There are profound species differences in the multiplicity of cytochrome P450 enzymes, and the use of mouse models to predict pathways of drug metabolism is further complicated by overlapping substrate specificity between enzymes from different gene families. To establish the role of the hepatic and extrahepatic P450 system in drug and foreign chemical disposition, drug efficacy, and toxicity, we created a unique mouse model in which 30 cytochrome P450 genes from the Cyp2c, Cyp2d, and Cyp3a gene clusters have been deleted. Remarkably, despite a wide range of putative important endogenous functions, Cyp2c/2d/3a KO mice were viable and fertile, demonstrating that these genes have evolved primarily as detoxification enzymes. Although there was no overt phenotype, detailed examination showed Cyp2c/2d/3a KO mice had a smaller body size (15%) and larger livers (20%). Changes in hepatic morphology and a decreased blood glucose (30%) were also noted. A five-drug cocktail of cytochrome P450 isozyme probe substrates were used to evaluate changes in drug pharmacokinetics; marked changes were observed in either the pharmacokinetics or metabolites formed from Cyp2c, Cyp2d, and Cyp3a substrates, whereas the metabolism of the Cyp1a substrate caffeine was unchanged. Thus, Cyp2c/2d/3a KO mice provide a powerful model to study the in vivo role of the P450 system in drug metabolism and efficacy, as well as in chemical toxicity.

Role of Hepatic and Intestinal P450 Enzymes in the Metabolic Activation of the Colon Carcinogen Azoxymethane in Mice

P450-mediated bioactivation of azoxymethane (AOM), a colon carcinogen, leads to the formation of DNA adducts, of which O6-methylguanine (O6-mG) is the most mutagenic and contributes to colon tumorigenesis. To determine whether P450 enzymes of the liver and intestine both contribute to AOM bioactivation in vivo, we compared tissue levels of AOM-induced DNA adducts, microsomal AOM metabolic activities, and incidences of colonic aberrant crypt foci (ACF) among wild-type (WT), liver-specific P450 reductase (Cpr)-null (LCN), and intestinal epithelium-specific Cpr-null (IECN) mice. At 6 h following AOM treatment (at 14 mg/kg, s.c.), O6-mG and N7-mG levels were highest in the liver, followed by the colon, and then small intestine in WT mice. As expected, hepatic adduct levels were significantly lower (by >60%) in LCN mice but unchanged in IECN mice, whereas small-intestinal adduct levels were unchanged or increased in LCN mice but lower (by >50%) in IECN mice compared to that in WT mice. However, colonic adduct levels were unchanged in IECN mice compared to that in WT mice and increased in LCN mice (by 1.5–2.9-fold). The tissue-specific impact of the CPR loss in IECN and LCN mice on microsomal AOM metabolic activity was confirmed by rates of formation of formaldehyde and N7-mG in vitro. Furthermore, the incidence of ACF, a lesion preceding colon cancer, was similar in the three mouse strains. Thus, AOM-induced colonic DNA damage and ACF formation is not solely dependent on either hepatic or intestinal microsomal P450 enzymes. P450 enzymes in both the liver and intestine likely contribute to AOM-induced colon carcinogenesis.

Heme Exporter FLVCR1a Regulates Heme Synthesis and Degradation and Controls Activity of Cytochromes P450

Background & AimsThe liver has one of the highest rates of heme synthesis of any organ. More than 50% of the heme synthesized in the liver is used for synthesis of P450 enzymes, which metabolize exogenous and endogenous compounds that include natural products, hormones, drugs, and carcinogens. Feline leukemia virus subgroup C cellular receptor 1a (FLVCR1a) is plasma membrane heme exporter that is ubiquitously expressed and controls intracellular heme content in hematopoietic lineages. We investigated the role of Flvcr1a in liver function in mice.MethodsWe created mice with conditional disruption of Mfsd7b, which encodes Flvcr1a, in hepatocytes (Flvcr1afl/fl;alb-cre mice). Mice were analyzed under basal conditions, after phenylhydrazine-induced hemolysis, and after induction of cytochromes P450 synthesis. Livers were collected and analyzed by histologic, quantitative real-time polymerase chain reaction, and immunoblot analyses. Hepatic P450 enzymatic activities were measured.ResultsFlvcr1afl/fl;alb-cre mice accumulated heme and iron in liver despite up-regulation of heme oxygenase 1, ferroportin, and ferritins. Hepatic heme export activity of Flvcr1a was closely associated with heme biosynthesis, which is required to sustain cytochrome induction. Upon cytochromes P450 stimulation, Flvcr1afl/fl;alb-cre mice had reduced cytochrome activity, associated with accumulation of heme in hepatocytes. The expansion of the cytosolic heme pool in these mice was likely responsible for the early inhibition of heme synthesis and increased degradation of heme, which reduced expression and activity of cytochromes P450.ConclusionsIn livers of mice, Flvcr1a maintains a free heme pool that regulates heme synthesis and degradation as well as cytochromes P450 expression and activity. These findings have important implications for drug metabolism.

Cytochrome P450 mRNA Expression in the Rodent Brain: Species-, Sex-, and Region-Dependent Differences

Cytochrome P450 (P450) enzymes play a critical role in the activation and detoxication of many neurotoxic chemicals. Although research has largely focused on P450-mediated metabolism in the liver, emerging evidence suggests that brain P450s influence neurotoxicity by modulating local metabolite levels. As a first step toward better understanding the relative role of brain P450s in determining neurotoxic outcome, we characterized mRNA expression of specific P450 isoforms in the rodent brain. Adult mice (male and female) and rats (male) were treated with vehicle, phenobarbital, or dexamethasone. Transcripts for CYP2B, CYP3A, CYP1A2, and the orphan CYP4X1 and CYP2S1 were quantified in the liver, hippocampus, cortex, and cerebellum by quantitative (real-time) polymerase chain reaction. These P450s were all detected in the liver with the exception of CYP4X1, which was detected in rat but not mouse liver. P450 expression profiles in the brain varied regionally. With the exception of the hippocampus, there were no sex differences in regional brain P450 expression profiles in mice; however, there were marked species differences. In the liver, phenobarbital induced CYP2B expression in both species. Dexamethasone induced hepatic CYP2B and CYP3A in mice but not rats. In contrast, brain P450s did not respond to these classic hepatic P450 inducers. Our findings demonstrate that P450 mRNA expression in the brain varies by region, regional brain P450 profiles vary between species, and their induction varies from that of hepatic P450s. These novel data will be useful for designing mechanistic studies to examine the relative role of P450-mediated brain metabolism in neurotoxicity.

Effect of Siwu decoction and its combined administration on hepatic P450 enzymatic activity and mRNA expression in rats

To study the effect of Siwu decoction (SWD) compound and its combined administration on hepatic P450 enzymatic activity and mRNA expression in rats. Rats were orally administered with SWD and water decoction combined with other medicines for two weeks, and then sacrificed. Their livers were perfused with normal saline to prepare liver micrisomes. Mixed probe and liver microsome in vitro incubation method were adopted to detect the effect of SWD on hepatic cytochrome P450. The real-time quantitative polymerase chain reaction (Q-PCR) was used to detect the effect of SWD on the expression of hepatic cytochrome P450. Compared with the control group, the SWD compound group showed higher CYP1A2 enzymatic activity (P < 0.05); Rehmanniae-paeoniae, angelicae-paeoniae, angelicae-rhizome, paeoniae-rhizome groups had lower CYP1A2 and CYP2C19 enzymatic activities (P < 0.05); And the compound group, the single component group and the combination group showed lower CYP2B6 enzymatic activities (P < 0.05). The compound could up-regulated the mRNA expression of CYP2B1 (P < 0.05); And the four single components could down-regulated the mRNA expression of CYP2B1 (P < 0.05). SWD compound had the effect in inducing CYP1A2 enzymatic activity. The rehmanniae-paeoniae group and the angelicae-paeoniae group had identical enzymatic activity with the control group, but significant down-regulation in CYP1A2 enzymatic activity after being combined with paeoniae. The compound and its combined administration showed the inhibitory effect on CYP2B6 enzymatic activity, particularly being combined with angelicae. The compound showed identical effect with the four single components in terms of CYP1A2 mRNA expression and enzymatic activity.

Regulation of Intestinal Cytochrome P450 Expression by Hepatic Cytochrome P450: Possible Involvement of Fibroblast Growth Factor 15 and Impact on Systemic Drug Exposure

Tissue-specific deletion of the gene for NADPH-cytochrome P450 (P450) reductase (CPR), the essential electron donor to all microsomal P450 enzymes, in either liver or intestine, leads to upregulation of many P450 genes in the tissue with the Cpr deletion. Here, by studying the liver-specific Cpr-null (LCN) mouse, we examined whether an interorgan regulatory pathway exists, such that a loss of hepatic CPR would cause compensatory changes in intestinal P450 expression and capacity for first-pass metabolism of oral drugs. We show for the first time that intestinal expression of CYP2B, 2C, and 3A proteins was increased in LCN mice by 2- to 3-fold compared with wild-type (WT) mice, accompanied by significant increases in small intestinal microsomal lovastatin-hydroxylase activity and systemic clearance of oral lovastatin (at 5 mg/kg). Additional studies showed that the hepatic Cpr deletion, which caused large decreases in bile acid (BA) levels in the liver, intestine, plasma, and intestinal content, led to drastic decreases in the mRNA levels of intestinal fibroblast growth factor 15 (FGF15), a target gene of the BA receptor farnesoid X receptor. Furthermore, treatment of mice with FGF19 (the human counterpart of mouse FGF15) abolished the difference between WT and LCN mice in small intestinal (SI) CYP3A levels at 6 hours after the treatment. Our findings reveal a previously unrecognized direct role of intestinal FGF15/19 in the regulation of SI P450 expression and may have profound implications for the prediction of drug exposure in patients with compromised hepatic P450 function.

Marsdenia tenacissima extract inhibits gefitinib metabolism in vitro by interfering with human hepatic CYP3A4 and CYP2D6 enzymes

Ethnopharmacological relevanceThe stem of Marsdenia tenacissima (Roxb.) Wight et Arn. is mainly produced in Yunnan China and has long been used as a medicine to treat cancer in China. Xiao-Ai-Ping injection, the water-soluble part of the stem of Marsdenia tenacissima, is administrated as an anti-cancer agent in clinics for decades. Our previous study showed that Marsdenia tenacissima extract (MTE) restored gefitinib sensitivity in gefitinib-resistant non-small cell lung cancer (NSCLC) cells, but the mechanism involved is unknown. Gefitinib undergoes hepatic metabolism predominantly through human cytochrome P450 (CYP) 3A4 and CYP2D6 enzymes. This study aims to evaluate whether MTE interferes with gefitinib metabolism via human hepatic P450 enzymes. Material and methodsA cocktail-substrate assay was used to test the effect of MTE on major CYP enzyme activities by incubation of pooled human liver microsomes with specific substrate probes of CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 in the absence and presence of MTE. Recombinant human CYP450 enzymes were used to predict in vitro gefitinib metabolic clearance in the absence and presence of MTE. The metabolites of the substrate probes and gefitinib were detected by high-performance liquid chromatographic tandem mass spectrometry (HPLC-MS/MS). Human hepatoma HepG2 cells were used to investigate the effect of gefitinib alone or in combination with MTE on CYP3A4 and CYP2D6 mRNA and protein expression. ResultsThe cocktail-substrate assay showed that MTE inhibited CYP450 activities in human liver microsomes with the inhibition rate of 3A4>2C9>2C19>1A2>2D6. The co-administration of MTE with gefitinib significantly decreased the in vitro intrinsic clearance (Clint) of gefitinib by 2.6 and 4.0-fold for CYP2D6 and CYP3A4, respectively, but did not affect other CYP450s. CYP2D6 and CYP3A4 mRNA and protein expression in human hepatoma HepG2 cells were greatly reduced in the combined gefitinib and MTE treatment. ConclusionWe demonstrate that MTE inhibits gefitinib metabolism by interfering with CYP3A4 and CYP2D6. Meanwhile, MTE combined with gefitinib down-regulates the mRNA and protein expression of CYP3A4 and CYP2D6 in the HepG2 cells. Thus, these data suggest that MTE is a promising herbal medicine to enhance gefitinib efficacy through improving its metabolic stability.

Downregulation of Mouse Hepatic CYP3A Protein by 3-Methylcholanthrene Does Not Require Cytochrome P450-Dependent Metabolism

The aryl hydrocarbon receptor (AHR)-dependent induction of cytochromes P450 (P450) such as CYP1A1 by 3-methylcholanthrene (MC) and related polycyclic aromatic hydrocarbons is well characterized. We reported previously that MC treatment triggers a pronounced downregulation, particularly at the protein level, of mouse hepatic Cyp3a11, a counterpart of the key human drug-metabolizing enzyme CYP3A4. To determine whether this effect of MC requires hepatic microsomal P450 activity, we studied liver Cpr-null (LCN) mice with hepatocyte-specific conditional deletion of the NADPH-cytochrome P450 oxidoreductase gene. In vehicle-treated animals, basal levels of CYP3A11 mRNA and CYP3A protein immunoreactivity were elevated by approximately 9-fold in LCN mice compared with wild-type (WT) mice, whereas CYP3A catalytic activity was profoundly compromised in LCN mice. MC treatment caused suppression of CYP3A11 mRNA, CYP3A protein immunoreactivity, and CYP3A catalytic activity in WT mice, and the MC effects at the mRNA and protein levels were maintained in LCN mice. Flavin-containing monooxygenase-3 (Fmo3) induction by MC was suggested previously to occur via an AHR-dependent mechanism requiring conversion of the parent compound to DNA-damaging reactive metabolites; however, hepatic FMO3 mRNA levels were dramatically increased by MC in both WT and LCN mice. MC did not function as a mechanism-based inactivator of CYP3A enzymes in hepatic microsomes prepared from untreated WT mice, under conditions in which 1-aminobenzotriazole caused marked NADPH-dependent loss of total P450 content and CYP3A catalytic activity. These results indicate that MC downregulates mouse hepatic CYP3A protein via a pretranslational mechanism that does not require hepatic microsomal P450-dependent activity.

Genetic variation in aldo-keto reductase 1D1 (AKR1D1) affects the expression and activity of multiple cytochrome P450s.

Human liver gene regulatory (Bayesian) network analysis was previously used to identify a cytochrome P450 (P450) gene subnetwork with Aldo-keto reductase 1D1 (AKR1D1) as a key regulatory driver of this subnetwork. This study assessed the biologic importance of AKR1D1 [a key enzyme in the synthesis of bile acids, ligand activators of farnesoid X receptor (FXR), pregnane X receptor (PXR), and constitutive androstane receptor (CAR), known transcriptional regulators of P450s] to hepatic P450 expression. Overexpression of AKR1D1 in primary human hepatocytes led to increased expression of CYP3A4, CYP2C8, CYP2C9, CYP2C19, and CYP2B6. Conversely, AKR1D1 knockdown decreased expression of these P450s. We resequenced AKR1D1 from 98 donor livers and identified a 3'-untranslated region (UTR) (rs1872930) single nucleotide polymorphism (SNP) significantly associated with higher AKR1D1 mRNA expression. AKR1D1 3'-UTR-luciferase reporter studies showed that the variant allele resulted in higher luciferase activity, suggesting that the SNP increases AKR1D1 mRNA stability and/or translation efficiency. Consistent with AKR1D1's putative role as a driver of the P450 subnetwork, the AKR1D1 3'-UTR SNP was significantly associated with increased hepatic mRNA expression of multiple P450s (CYP3A4, CYP2C8, CYP2C9, CYP2C19, and CYP2B6) and CYP3A4, CYP2C8, CYP2C19, and CYP2B6 activities. After adjusting for multiple testing, the association remained significant for AKR1D1, CYP2C9, and CYP2C8 mRNA expression and CYP2C8 activity. These results provide new insights into the variation in expression and activity of P450s that can account for interindividual differences in drug metabolism/efficacy and adverse drug events. In conclusion, we provide the first experimental evidence supporting a role for AKR1D1 as a key genetic regulator of the P450 network.