Microsomal Hydroxylation Research Articles

Effects of Water Miscible Organic Co-solvents on Enzyme Kinetic Parameters of Drug Metabolizing Enzymes in Rat Liver Microsomes

We have investigated the effects of methanol, acetonitrile, dimethyl sulphoxide or dioxane on enzyme kinetic parameters (Km and Vmax) of p-nitrophenol hydroxylation, phenacetin deethylation and metoprolol metabolism in rat liver microsomes, at different solvent concentrations. In the case of p-nitrophenol hydroxylation, methanol (0.1 %-0.75 % v/v) and dimethyl sulphoxide (0.01 %-0.075 % v/v) showed 1.3 and 2.5-fold increase respectively, in the Km with a less marked effect on the Vmax. In contrast, dimethyl sulphoxide (0.1 %-0.75% v/v) showed a 1.96-fold decrease in the Vmax. Unlike, methanol and dimethyl sulphoxide, acetonitrile showed activation of the p-nitrophenol hydroxylation activity as indicated by a 0.7-fold decrease in the Km. In the case of metoprolol metabolism, dioxane resulted in a 1.5 to 3.1-fold increase in the Km of all the three metabolite formation pathways in concentration dependent manner, with less marked effect on Vmax of the reaction. Interestingly, while acetonitrile did not affect the Km for metabolite 1 and 2 formation, the Km of metabolite 3 formation was decreased in a concentration dependent manner again with little effect on all the three Vmax values. Further, methanol had little to no effect on the Vmax of metabolite 1 and 2 formation while metabolite 3 formation was increased by 2-fold. The Km of all three metabolite formations was found to be decreased in presence of methanol. The phenacetin deethylation activity in rat liver microsomes followed atypical kinetics. Methanol and dimethyl sulphoxide did not affect the auto activation kinetics at concentration range studied. Dimethyl sulphoxide and dioxane appeared to be unsuitable for characterizing the Cytochrome450 mediated reactions because they showed a very significant effect on the Vmax/Km ratio, starting at concentrations of 0.025 % v/v and 0.25 % v/v, respectively. Methanol and acetonitrile at concentration <0.5 % v/v appeared to be acceptable solvents for solubilization of substrates metabolized by Cytochrome450s.

Formation of CYP3A-specific metabolites of ibrutinib in vitro is correlated with hepatic CYP3A activity and 4β-hydroxycholesterol/cholesterol ratio.

Ibrutinib is an orally administered Bruton's tyrosine kinase inhibitor approved for the treatment of B-cell malignancies, including chronic lymphocytic leukemia. Ibrutinib is metabolized primarily via oxidation by cytochrome P450 (CYP) 3A4/5 to M37 (the primary active metabolite), M34, and M25. The objectives of this study were to assess the relationship between formation of the major CYP3A-specific ibrutinib metabolites in vitro and hepatic CYP3A activity and protein abundance, and to evaluate the utility of the endogenous CYP3A biomarker, plasma 4β-hydroxycholesterol (4β-HC) to cholesterol ratio, to predict ibrutinib metabolite formation in individual cadaveric donors with matching hepatocytes. Ibrutinib (5μM) was incubated with single-donor human liver microsomes (n=20) and primary human hepatocytes (n=15), and metabolites (M37, M34, and M25) were measured by liquid chromatography-tandem mass spectrometry analysis. CYP3A4/5 protein concentrations were measured by quantitative targeted absolute proteomics, and CYP3A activity was measured by midazolam 1'-hydroxylation. Ibrutinib metabolite formation positively correlated with midazolam 1'-hydroxylation in human liver microsomes and hepatocytes. Plasma 4β-HC and cholesterol concentrations were measured in plasma samples obtained at the time of liver harvest from the same 15 donors with matching hepatocytes. Midazolam 1'-hydroxylation in hepatocytes correlated with plasma 4β-HC/cholesterol ratio. When an infant donor (1 year old) was excluded based on previous ontogeny studies, M37 and M25 formation correlated with plasma 4β-HC/cholesterol ratio in the remaining 14 donors (Spearman correlation coefficients [r] 0.62 and 0.67, respectively). Collectively, these data indicate a positive association among formation of CYP3A-specific ibrutinib metabolites in human hepatocytes, hepatic CYP3A activity, and plasma 4β-HC/cholesterol ratio in the same non-infant donors.

Risk prediction of drug-drug interaction potential of phenytoin and miconazole topical formulations

The drug-drug interaction (DDI) risk of phenytoin with several topical formulations of miconazole is still unclear. The present investigation conducted in vitro-in vivo extrapolation to predict the potential risks. Our data indicated that miconazole potently inhibited phenytoin hydroxylation in both pooled human liver microsomes (HLMs) and recombinant cytochrome P450 2C9 (CYP2C9) with the Ki values of 125 ± 7 nM and 30 ± 2 nM, respectively. Quantitative prediction of DDI risk suggests that, beside intravenous administration or swallowed tablet, combination of phenytoin and miconazole high dose oral gel or buccal tablet may also result in a clinically significant increase of phenytoin AUC (>53%) by the inhibition of miconazole against phenytoin hydroxylation, consequently a higher frequency of adverse events, while the coadministration of miconazole vaginal formulation and phenytoin will be safe.

In vitro characterization of glycyrol metabolites in human liver microsomes using HR-resolution MS spectrometer coupled with tandem mass spectrometry

1. Glycyrol is a coumestan derivative that is isolated from roots of Glycyrrhiza uralensis. Glycyrol exhibits several biological effects, including anti-oxidative and anti-inflammatory effects.2. Herein, we characterized glycyrol metabolism by cytochrome P450 enzymes (CYPs) and UDP-glucuronosyltransferases (UGTs) using human liver microsomes (HLM), human liver cytosol, human intestinal microsomes, or human recombinant cDNA-expressed CYPs and UGTs. The analysis was conducted using high resolution mass spectroscopy (HR-MS) on a Q ExactiveTM HF Hybride Quadrupole-Orbitrap mass spectrometer.3. NADPH-supplemented HLM generated six glycyrol metabolites (M1–M6) via hydroxylation, oxidation, and hydration; both NADPH- and UDPGA-supplemented liver microsomes generated three glucuronides (M7–M9). Reaction phenotyping revealed that CYP1A2 is the primary enzyme responsible for phase I metabolism, with minor involvement of the CYP3A4/5, CYP2D6, and CYP2E1 enzymes. Glucuronidation of glycyrol was primarily mediated by UGT1A1, UGT1A3, UGT1A9, and UGT2B7.4. In conclusion, glycyrol undergoes the efficient metabolic hydroxylation and glucuronidation reactions in human liver microsomes, which are predominantly catalyzed by CYP1A2, UGT1A1/3/9, and UGT2B7.

Improved predictions of time-dependent drug-drug interactions by determination of cytosolic drug concentrations

The clinical impact of drug-drug interactions based on time-dependent inhibition of cytochrome P450 (CYP) 3A4 has often been overpredicted, likely due to use of improper inhibitor concentration estimates at the enzyme. Here, we investigated if use of cytosolic unbound inhibitor concentrations could improve predictions of time-dependent drug-drug interactions. First, we assessed the inhibitory effects of ten time-dependent CYP3A inhibitors on midazolam 1′-hydroxylation in human liver microsomes. Then, using a novel method, we determined the cytosolic bioavailability of the inhibitors in human hepatocytes, and used the obtained values to calculate their concentrations at the active site of the enzyme, i.e. the cytosolic unbound concentrations. Finally, we combined the data in mechanistic static predictions, by considering different combinations of inhibitor concentrations in intestine and liver, including hepatic concentrations corrected for cytosolic bioavailability. The results were then compared to clinical data. Compared to no correction, correction for cytosolic bioavailability resulted in higher accuracy and precision, generally in line with those obtained by more demanding modelling. The best predictions were obtained when the inhibition of hepatic CYP3A was based on unbound maximal inhibitor concentrations corrected for cytosolic bioavailability. Our findings suggest that cytosolic unbound inhibitor concentrations improves predictions of time-dependent drug-drug interactions for CYP3A.

Atipamezole is a promising non-discriminative inhibitor against pan-CYP450 including diclofenac 4′-hydroxylation: A comparison with ABT for drug ADME optimization and mechanism study

1-aminobenzotriazole (ABT) is a known P450 enzyme non-selective inactivator that serves as a tool for assessing P450-mediated metabolism. However, many findings demonstrated that ABT was ineffective with human CYP2C9. A profound pan-CYP450 inhibitor is desired avoid the risk of incomplete inhibition of P450, especially CYP2C9. Atipamezole is commonly used to recover animals from sedation-anesthesia induced by α2−adrenoceptor agonists. The purpose of this study is to evaluate atipamezole as a non-selective inhibitor of P450 enzymes and compare it with ABT. Inhibition toward seven major human CYP450 isoform was determined for atipamezole and ABT in human, rat, and dog liver microsomes for the direct and time-dependent inhibition potentials. IC50 values toward human and animal CYPs without preincubation are 0.02–7.93 μM and 20.9–1798 μM for atipamezole and ABT, respectively. The IC50 values of ABT after preincubation shift to 4.06–460 μM. Atipamezole has more effective inhibition to CYP2C9 mediated diclofenac hydroxylation in human and animal liver microsomes with IC50 values of 1.50–5.20 μM than that of ABT at 74.7-460 μM. No IC50 shift was observed for atipamezole to CYP isoforms. In vivo utility of atipamezole was assessed by co-dosing with diclofenac in rats. At 30 mg/kg via oral, atipamezole enhanced the AUC of diclofenac by 13.1-fold and the Cmax by 5.6-fold. Similar enhancement also achieved for ABT (100 mg/kg) with AUC and Cmax increased 9.5 and 4.8-fold. As a reversible pan-CYP inhibitor, atipamezole showed less species difference than ABT. It provides a better and easier to use alternative to ABT for ADME optimization and elucidating mechanistic drug metabolism or toxicity studies.

Sorafenib N-Oxide Is an Inhibitor of Human Hepatic CYP3A4.

The multi-kinase inhibitor sorafenib (SOR) is clinically important in the treatment of hepatocellular and renal cancers and undergoes CYP3A4-dependent oxidation in liver to the pharmacologically active N-oxide metabolite (SNO). There have been reports that kinase inhibitors such as SOR may precipitate pharmacokinetic interactions with coadministered drugs that compete for CYP3A4-mediated biotransformation, but these occur non-uniformly in patients. Clinical evidence also indicates that SNO accumulates in serum of some patients during prolonged SOR therapy. In this study undertaken in hepatic microsomes from individual donors, we assessed the possibility that SNO might contribute to pharmacokinetic interactions mediated by SOR. Enzyme kinetics of CYP3A4-mediated midazolam 1'-hydroxylation in individual human hepatic microsomes were analyzed by non-linear regression and appropriate replots. Thus, SNO and SOR were linear-mixed inhibitors of microsomal CYP3A4 activity (Kis 15 ± 4 and 33 ± 14μM, respectively). To assess these findings, further molecular docking studies of SOR and SNO with the 1TQN crystal structure of CYP3A4 were undertaken. SNO elicited a larger number of interactions with key amino acid residues located in substrate recognition sequences of the enzyme. In the optimal docking pose, the N-oxide moiety of SNO was also found to interact directly with the heme moiety of CYP3A4. These findings suggest that SNO could contribute to pharmacokinetic interactions involving SOR, perhaps in individuals who produce high circulating concentrations of the metabolite.

The activity of brain and liver cytochrome P450 2D (CYP2D) is differently affected by antidepressants in the chronic mild stress (CMS) model of depression in the rat

The effect of two second-generation antidepressants escitalopram and venlafaxine on the activity of brain and liver cytochrome P450 2D (CYP2D) involved in the metabolism of psychotropics and neurotransmitters was determined in the chronic mild stress (CMS) model of depression. Escitalopram or venlafaxine (10 mg/kg ip/day each) were administered to control and CMS rats for 5 weeks. The activity of CYP2D was studied by measurement of the rate of bufuralol 1′-hydroxylation in microsomes derived from the liver or different brain structures. The obtained results indicate that CMS and the studied antidepressants had different effects on the CYP2D activity depending on the location of the enzyme. In the brain, CMS produced an increase in the CYP2D activity in the hippocampus. Chronic escitalopram or venlafaxine had no effect on the CYP2D activity in the brain of nonstressed rats, however, the antidepressants increased the enzyme activity in the frontal cortex, hypothalamus and cerebellum of stressed animals. In the liver, CMS did not affect the CYP2D activity, while chronic escitalopram or venlafaxine significantly decreased the CYP2D activity and protein level in nonstressed and stressed rats.We conclude that: 1) CMS stimulates the CYP2D activity in the hippocampus and triggers the stimulatory effect of antidepressants on CYP2D in other brain structures; 2) the local brain metabolism of CYP2D substrates (neurosteroids, neurotransmitters, psychotropics) may be enhanced by CMS and/or antidepressants; 3) in contrast to the brain, the liver metabolism of CYP2D substrates may be slower during long-term treatment with escitalopram or venlafaxine.

Role of Cell Death in Toxicology: Does It Matter How Cells Die?

My research activity started with studies on drug metabolism in rat liver microsomes in the early 1960s. The CO-binding pigment (cytochrome P450) had been discovered a few years earlier and was subsequently found to be involved in steroid hydroxylation in adrenal cortex microsomes. Our early studies suggested that it also participated in the oxidative demethylation of drugs catalyzed by liver microsomes, and that prior treatment of the animals with phenobarbital caused increased levels of the hemoprotein in the liver, and similarly enhanced rates of drug metabolism. Subsequent studies of cytochrome P450-mediated metabolism of toxic drugs in freshly isolated rat hepatocytes characterized critical cellular defense systems and identified mechanisms by which accumulating toxic metabolites could damage and kill the cells. These studies revealed that multiple types of cell death could result from the toxic injury, and that it is important to know which type of cell death results from the toxic injury.

In vivo and in vitro diclofenac 5-hydroxylation mediated primarily by cytochrome P450 3A enzymes in common marmoset livers genotyped for P450 2C19 variants

Common marmosets (Callithrix jacchus) are potentially useful nonhuman primate models for preclinical studies. An anti-inflammatory drug, diclofenac is reportedly metabolized mainly by human cytochrome P450 (P450) 2C9 to 4′-hydroxydiclofenac and minorly by P450 3A4 to 5-hydroxydiclofenac that leads to reactive intermediates. In this study, in vivo pharmacokinetics in six marmosets and in vitro metabolism in nine marmoset liver microsomes were analyzed using diclofenac to evaluate marmosets as preclinical drug metabolism models. In wild-type marmosets genotyped for P450 2C19 p.[(Phe7Leu; Ser254Leu; Ile469Thr)], plasma levels of 5-hydroxydiclofenac and 4′-hydroxydiclofenac were roughly similar, but the homozygotes showed approximately three-times higher plasma levels of 5-hydroxydiclofenac than those of 4′-hydroxydiclofenac after oral administrations of diclofenac (50 mg/kg). Nine marmoset liver microsomes catalyzed mainly diclofenac 5-hydroxylation with no significant effects of the the P450 2C19 genotype, and partly diclofenac 4′-hydroxylation that depended on the P450 2C19 genotype, at substrate concentrations of 10 µM and 100 µM. Both Michaels-Menten constant Km values for diclofenac 4′-hydroxylation in human and marmoset liver microsomes were ∼30 μM and those for diclofenac 5-hydroxylation were ∼120 μM. Ketoconazole significantly suppressed only diclofenac 5-hydroxylation in marmoset or human liver microsomes at 0.030 μM, indicating main contribution of P450 3A enzymes, which were found to be P450 3A5/90 using recombinant marmoset P450 3A enzymes. These results suggest that marmosets would be a functional animal model for in vivo and in vitro metabolites likely generated via arene oxide intermediates of diclofenac by P450 3A enzymes in humans, primarily because marmosets lack the ortholog of human P450 2C9.

Inhibitory Effects of Dimethyllirioresinol, Epimagnolin A, Eudesmin, Fargesin, and Magnolin on Cytochrome P450 Enzyme Activities in Human Liver Microsomes.

Magnolin, epimagnolin A, dimethyllirioresinol, eudesmin, and fargesin are pharmacologically active tetrahydrofurofuranoid lignans found in Flos Magnoliae. The inhibitory potentials of dimethyllirioresinol, epimagnolin A, eudesmin, fargesin, and magnolin on eight major human cytochrome P450 (CYP) enzyme activities in human liver microsomes were evaluated using liquid chromatography-tandem mass spectrometry to determine the inhibition mechanisms and inhibition potency. Fargesin inhibited CYP2C9-catalyzed diclofenac 4′-hydroxylation with a Ki value of 16.3 μM, and it exhibited mechanism-based inhibition of CYP2C19-catalyzed [S]-mephenytoin 4′-hydroxylation (Ki, 3.7 μM; kinact, 0.102 min−1), CYP2C8-catalyzed amodiaquine N-deethylation (Ki, 10.7 μM; kinact, 0.082 min−1), and CYP3A4-catalyzed midazolam 1′-hydroxylation (Ki, 23.0 μM; kinact, 0.050 min−1) in human liver microsomes. Fargesin negligibly inhibited CYP1A2-catalyzed phenacetin O-deethylation, CYP2A6-catalyzed coumarin 7-hydroxylation, CYP2B6-catalyzed bupropion hydroxylation, and CYP2D6-catalyzed bufuralol 1′-hydroxylation at 100 μM in human liver microsomes. Dimethyllirioresinol weakly inhibited CYP2C19 and CYP2C8 with IC50 values of 55.1 and 85.0 μM, respectively, without inhibition of CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2D6, and CYP3A4 activities at 100 μM. Epimagnolin A, eudesmin, and magnolin showed no the reversible and time-dependent inhibition of eight major CYP activities at 100 μM in human liver microsomes. These in vitro results suggest that it is necessary to investigate the potentials of in vivo fargesin-drug interaction with CYP2C8, CYP2C9, CYP2C19, and CYP3A4 substrates.

CYP2C8-mediated interaction between repaglinide and steviol acyl glucuronide: In vitro investigations using rat and human matrices and in vivo pharmacokinetic evaluation in rats

CYP2C8 is involved in the metabolic clearance of several important drugs and recent reports have shown that acyl glucuronides of gemfibrozil and clopidogrel are potent time-dependent inhibitors of CYP2C8 activity. In the present study, the inhibitory effect of steviol acyl glucuronide (SVAG), a circulating metabolite formed after the ingestion of rebaudioside A, was investigated using in vitro and in vivo systems. Results indicated that SVAG was a reversible but not a time-dependent inhibitor of CYP2C8-mediated paclitaxel 6α-hydroxylation. SVAG was also capable of inhibiting CYP2C8-mediated repaglinide 3′-hydroxylation in human liver microsomes and recombinant human CYP2C8, with Ki values of 15.8 μM and 11.6 μM, respectively. In contrast, SVAG did not exhibit inhibitory effect on CYP2C8 activity in rat liver microsomes. In addition, co-administration of rebaudioside A with repaglinide in rats did not lead to AUC and Cmax changes of repaglinide. Although mathematic prediction using a simplified mechanistic model revealed a moderate interaction potential between repaglinide and SVAG, cautions should be given to patients with hypoglycemia if repaglinide and rebaudioside A are used in combination for the blood sugar control.

Rapid and accurate liquid chromatography and tandem mass spectrometry method for the simultaneous quantification of ten metabolic reactions catalyzed by hepatic cytochrome P450 enzymes.

The hepatic cytochrome P450 enzymes play a central role in the biotransformation of endogenous and exogenous substances. A sensitive high-throughput liquid chromatography with tandem mass spectrometry assay was developed and validated for the simultaneous quantification of the products of ten metabolic reactions catalyzed by hepatic cytochrome P450 enzymes. After the substrates were incubated separately, the samples were pooled and analyzed by liquid chromatography with tandem mass spectrometry using an electrospray ionization source in the positive and negative ion modes. The method exhibited linearity over a broad concentration range, insensitivity to matrix effects, and high accuracy, precision, and stability. The novel method was successfully applied to study the kinetics of phenacetin-O deethylation, coumarin-7 hydroxylation, bupropion hydroxylation, taxol-6 hydroxylation, omeprazole-5 hydroxylation, dextromethorphan-O demethylation, tolbutamide-4 hydroxylation, chlorzoxazone-6 hydroxylation, testosterone-6β hydroxylation, and midazolam-1 hydroxylation in rat liver microsomes.

Hydroxylation and N-dechloroethylation of Ifosfamide and deuterated Ifosfamide by the human cytochrome p450s and their commonly occurring polymorphisms.

The hydroxylation and N-dechloroethylation of deuterated ifosfamide (d4IFO) and ifosfamide (IFO) by several human P450s have been determined and compared. d4IFO was synthesized with deuterium at the alpha and alpha' carbons to decrease the rate of N-dechloroethylation and thereby enhance hydroxylation of the drug at the 4' position. The purpose was to decrease the toxic and increase the efficacious metabolites of IFO. For all of the P450s tested, hydroxylation of d4IFO was improved and dechloroethylation was reduced as compared with nondeuterated IFO. Although the differences were not statistically significant, the trend favoring the 4'-hydroxylation pathway was noteworthy. CYP3A5 and CYP2C19 were the most efficient enzymes for catalyzing IFO hydroxylation. The importance of these enzymes in IFO metabolism has not been reported previously and warrants further investigation. The catalytic ability of the common polymorphisms of CYP2B6 and CYP2C9 for both reactions were tested with IFO and d4IFO. It was determined that the commonly expressed polymorphisms CYP2B6*4 and CYP2B6*6 had reduced catalytic ability for IFO compared with CYP2B6*1, whereas CYP2B6*7 and CYP2B6*9 had enhanced catalytic ability. As with the wild-type enzymes, d4IFO was more readily hydroxylated by the polymorphic variants than IFO, and d4IFO was not dechloroethylated by any of the polymorphic forms. We also assessed the use of specific inhibitors of P450 to favor hydroxylation in human liver microsomes. We were unable to separate the pathways with these experiments, suggesting that multiple P450s are responsible for catalyzing both metabolic pathways for IFO, which is not observed with the closely related drug cyclophosphamide.

Evaluation of metabolism dependent inhibition of CYP2B6 mediated bupropion hydroxylation in human liver microsomes by monoamine oxidase inhibitors and prediction of potential as perpetrators of drug interaction

The objective of the study was to evaluate the metabolism dependent inhibition of CYP2B6 catalyzed bupropion hydroxylation in human liver microsomes by monoamine oxidase (MAO) inhibitors and to predict the drug–drug interaction potential of monoamine oxidase inhibitors as perpetrators of drug interaction. Human liver microsomal CYP2B6 activities were investigated using bupropion hydroxylation as probe substrate marker. The results from single point time dependent inhibition and shift assays suggest that clorgyline, pargyline, phenelzine, and selegiline were metabolism based inhibitors of CYP2B6. In IC50 shift assays, clorgyline, pargyline, phenelzine and selegiline are metabolism based inhibitors of CYP2B6 with fold shit of 3.0-, 3.7-, 2.9-, and 11.4-fold respectively. The inactivation of clorgyline was characterized by KI value of 2.5±0.3 and kinact value of 0.045±0.001min−1. Phenelzine inactivated CYP2B6 with KI and kinact values of 44.9±6.9μM and 0.085±0.003min−1 respectively. Inactivation of selegiline was characterized with KI and kinact values of 22.0±3.3 and 0.074±0.002min−1 respectively. The inactivation caused by these inhibitors was not reversed by dialysis indicating irreversible inhibition. Based on the mechanistic static model, selegiline showed an increase in the area under the curve (AUC) of efavirenz and bupropion by 1.01-fold. Phenelzine predicted to cause an increase in the AUC of efavirenz and bupropion by 9.4- and 2.4-fold respectively considering unbound hepatic inlet concentrations of phenelzine. In conclusion, the results from this study demonstrated that MAO inhibitors can inactivate human liver microsomal CYP2B6. The likelihood of drug interaction when selegiline co-administered with CYP2B6 substrates is remote. Caution is required while co-administering phenelzine with substrates that are exclusively metabolized by CYP2B6 enzyme and substrates that have narrow therapeutic index.

Characterization of phase I metabolism of resibufogenin and evaluation of the metabolic effects on its antitumor activity and toxicity.

Resibufogenin (RB), one of the major active compounds of the traditional Chinese medicine Chansu, has displayed great potential as a chemotherapeutic agent in oncology. However, it is a digoxin-like compound that also exhibits extremely cardiotoxic effects. The present study aimed to characterize the metabolic behaviors of RB in humans as well as to evaluate the metabolic effects on its bioactivity and toxicity. The phase I metabolic profile in human liver microsomes was characterized systemically, and the major metabolite was identified as marinobufagenin (5β-hydroxylresibufogenin, 5-HRB) by liquid chromatography-mass spectrometry and nuclear magnetic imaging techniques. Both cytochrome P450 (P450) reaction phenotyping and inhibition assays using P450-selective chemical inhibitors demonstrated that CYP3A4 was mainly involved in RB 5β-hydroxylation with much higher selectivity than CYP3A5. Kinetic characterization demonstrated that RB 5β-hydroxylation in both human liver microsomes and human recombinant CYP3A4 obeyed biphasic kinetics and displayed similar apparent kinetic parameters. Furthermore, 5-HRB could significantly induce cell growth inhibition and apoptosis in A549 and H1299 by facilitating apoptosome assembly and caspase activation. Meanwhile, 5-HRB displayed very weak cytotoxicity of human embryonic lung fibroblasts, and in mice there was a greater tolerance to acute toxicity. In summary, CYP3A4 dominantly mediated 5β-hydroxylation and was found to be a major metabolic pathway of RB in the human liver, whereas its major metabolite (5-HRB) displayed better druglikeness than its parent compound RB. Our findings lay a solid foundation for RB metabolism studies in humans and encourage further research on the bioactive metabolite of RB.

Hepatic bile acid metabolism and expression of cytochrome P450 and related enzymes are altered in Bsep −/− mice

The bile salt export pump (BSEP/Bsep; gene symbol ABCB11/Abcb11) translocates bile salts across the hepatocyte canalicular membrane into bile in humans and mice. In humans, mutations in the ABCB11 gene cause a severe childhood liver disease known as progressive familial intrahepatic cholestasis type 2. Targeted inactivation of mouse Bsep produces milder persistent cholestasis due to detoxification of bile acids through hydroxylation and alternative transport pathways. The purpose of the present study was to determine whether functional expression of hepatic cytochrome P450 (CYP) and microsomal epoxide hydrolase (mEH) is altered by Bsep inactivation in mice and whether bile acids regulate CYP and mEH expression in Bsep (-/-) mice. CYP expression was determined by measuring protein levels of Cyp2b, Cyp2c and Cyp3a enzymes and CYP-mediated activities including lithocholic acid hydroxylation, testosterone hydroxylation and alkoxyresorufin O-dealkylation in hepatic microsomes prepared from female and male Bsep (-/-) mice fed a normal or cholic acid (CA)-enriched diet. The results indicated that hepatic lithocholic acid hydroxylation was catalyzed by Cyp3a/Cyp3a11 enzymes in Bsep (-/-) mice and that 3-ketocholanoic acid and murideoxycholic acid were major metabolites. CA feeding of Bsep (-/-) mice increased hepatic Cyp3a11 protein levels and Cyp3a11-mediated testosterone 2β-, 6β-, and 15β-hydroxylation activities, increased Cyp2b10 protein levels and Cyp2b10-mediated benzyloxyresorufin O-debenzylation activity, and elevated Cyp2c29 and mEH protein levels. We propose that bile acids upregulate expression of hepatic Cyp3a11, Cyp2b10, Cyp2c29 and mEH in Bsep (-/-) mice and that Cyp3a11 and multidrug resistance-1 P-glycoproteins (Mdr1a/1b) are vital components of two distinct pathways utilized by mouse hepatocytes to expel bile acids.

Gene Expression Modulation of Two Biosynthesis Pathways via Signal Transduction in &amp;lt;i&amp;gt;Cochliobolus heterostrophus&amp;lt;/i&amp;gt;

G-protein-linked pathways have evolved to allow responses to extracellular agonists (hormones, neurotransmitters, odors, chemoattractants, light and nutrients) in eukaryotic cells, ranging from simpler systems, including yeasts, filamentous fungi and slime molds, to more complex organisms, such as mammals. Although the role of G-protein and mitogen-activated protein kinase (MAPK) in filamentous fungi has been studied for over a decade, downstream elements are less known, and the study of target genes has evolved mainly in recent years. Here, we examined the involvement of G-protein subunits and MAPK in controlling the expression of two distinct target genes. These genes were selected from an array database according to their unique expression profile and the role of closely related genes found in other Ascomycetes. One of these genes is BPH, which encodes the enzyme responsible for cytochrome P450-dependent benzoate hydroxylation in microsomes. The other gene is CIPA, which encodes isoflavone reductase (IfR), an enzyme involved in the synthesis of phytoalexin, which catalyzes an intermediate step in pisatin biosynthesis. The expression profile of these two genes was determined in a series of signaling deficiency mutants that were grown on different media using a DNA microarray. Comparison of the expression profile in the two wild type strains and mutants deficient in the G-protein α or β subunits or in MAPK, revealed a unique control mechanism for the BPH and CIPA genes. The two genes are highly expressed during the infection of the host plant leaves and may associate with the fungal response to the host. Signaling via G-protein or MAPK was shown to be related to cascades that altered the expression of these genes in response to the growth condition. This work demonstrates that signal transduction pathways are controlling genes that, although sharing an environmental dependent response, participate in distinct biosynthesis pathways. Moreover, the transcriptional profile may point to distinct and shared roles of the signaling components.

Relationship between Differential Hepatic microRNA Expression and Decreased Hepatic Cytochrome P450 3A Activity in Cirrhosis

Background and AimLiver cirrhosis is associated with decreased hepatic cytochrome P4503A (CYP3A) activity but the pathogenesis of this phenomenon is not well elucidated. In this study, we examined if certain microRNAs (miRNA) are associated with decreased hepatic CYP3A activity in cirrhosis.MethodsHepatic CYP3A activity and miRNA microarray expression profiles were measured in cirrhotic (n=28) and normal (n=12) liver tissue. Hepatic CYP3A activity was measured via midazolam hydroxylation in human liver microsomes. Additionally, hepatic CYP3A4 protein concentration and the expression of CYP3A4 mRNA were measured. Analyses were conducted to identify miRNAs which were differentially expressed between two groups but also were significantly associated with lower hepatic CYP3A activity.ResultsHepatic CYP3A activity in cirrhotic livers was 1.7-fold lower than in the normal livers (0.28 ± 0.06 vs. 0.47 ± 0.07mL* min-1*mg protein-1 (mean ± SEM), P=0.02). Six microRNAs (miR-155, miR-454, miR-582-5p, let-7f-1*, miR-181d, and miR-500) had >1.2-fold increase in cirrhotic livers and also had significant negative correlation with hepatic CYP3A activity (range of r = -0.44 to -0.52, P <0.05). Notably, miR-155, a known regulator of liver inflammation, had the highest fold increase in cirrhotic livers (2.2-fold, P=4.16E-08) and significantly correlated with hepatic CYP3A activity (r=-0.50, P=0.017). The relative expression (2-ΔΔCt mean ± SEM) of hepatic CYP3A4 mRNA was significantly higher in cirrhotic livers (21.76 ± 2.65 vs. 5.91 ± 1.29, P=2.04E-07) but their levels did not significantly correlate with hepatic CYP3A activity (r=-0.43, P=0.08).ConclusionThe strong association between certain miRNAs, notably miR-155, and lower hepatic CYP3A activity suggest that altered miRNA expression may regulate hepatic CYP3A activity.

Metabolism of Dietary Flavonoids in Liver Microsomes

Flavonoids undergo substantial hepatic metabolism and the metabolites might significantly contribute to the effects of these dietary constituents. The metabolites of flavonoids in liver can be summarized as follows: 1) For flavones, the hydroxylation appears to occur at the C-4'-, C-3', C-6 and C-8- position when there is a single or no hydroxy group on the B-ring. The methoxyl groups positioned at the C-7, C-6 or C-4'- position of flavones are demethylated. The glucuronidation occurs at the 6, 7, 4' or 3'- hydroxyl moiety. Flavone glycosides and aglycones appear to undergo similar metabolic pathways. 2) For flavonols, the hydroxylation appears at the 3' and 4'- position and flavonols with a 4'-methoxy group are easily O-demethylated to their corresponding hydroxylated analogs. The glucuronidation takes place at the 7, 3, 3' and 4'- hydroxyl moiety. 3) For isoflavones, the microsomal hydroxylation is observed at the C-3'-, C-6 and C-8- position when there is a single or no hydroxy group on the B-ring. The demethylation takes place at the C-6- or C-4'- position when there is one methoxy group on C-4' or C-6-position, respectively. The glucuronidation occurs at the 4, or 5- hydroxyl moiety. 4) For chalcones, the C-3'-, and C-8- positions undergo hydroxylation. The C-8- position is a very active site for metabolism of chalcones.