Characterization of marmoset CYP2B6: cDNA cloning, protein expression and enzymatic functions.

The common marmoset is a promising species for evaluating the safety of drug candidates. To further understand the capacity for drug metabolism in marmosets, a cDNA encoding a CYP2B enzyme was cloned from the total RNA fraction of marmoset liver by 3'- and 5'-RACE methods. Nucleotide and deduced amino acid sequences showed 90.8 and 86.2% identity, respectively, with human CYP2B6. The marmoset CYP2B6 (marCYP2B6) protein was expressed in insect cells, and its enzymatic properties were compared with those of human (humCYP2B6) and cynomolgus monkey (cynCYP2B6) orthologs in liver and insect cell microsomes. Enzymatic functions were examined for the oxidation of 7-ethoxy-4-(trifluoromethyl)coumarin (7-ETC), bupropion (BUP) and efavirenz (EFV). The kinetic profiles for the oxidation of the three substrates by liver microsomal fractions were similar between humans and cynomolgus monkeys (biphasic for 7-ETC and monophasic for BUP and EFV), but that of marmosets was unique (monophasic for 7-ETC and biphasic for BUP and EFV). Recombinant enzymes, humCYP2B6 and cynCYP2B6, also yielded similar kinetic profiles for the oxidation of the three substrates, whereas marCYP2B6 showed activity only for 7-ETC hydroxylation. In silico docking simulations suggested that two amino acid residues, Val-114 and Leu-367, affect the activity of marCYP2B6. In fact, a marCYP2B6 mutant with substitutions V114I and L367V exhibited BUP hydroxylase activity that was 4-fold higher than that of humCYP2B6, while its EFV 8-hydroxylase activity was only 10% that of the human enzyme. These results indicate that the amino acids at positions 114 and 367 affect the enzymatic capacity of marmoset CYP2B6.

Induction of neurite outgrowth in PC12 cells by artemisinin through activation of ERK and p38 MAPK signaling pathways.

Growth of neurite processes is a critical step in neuronal development, regeneration, differentiation, and response to injury. The discovery of compounds that can stimulate neurite formation would be important for developing new therapeutics against both neurodegenerative disorders and trauma-induced neuronal injuries. Semisynthetic derivatives of artemisinin, an active compound in Artemisia annua, have been effectively used in malaria treatment, but they have been shown to possess neurotoxic potential. In this study, we found unexpectedly that artemisinin and its derivatives induced neurite outgrowth of PC12 cells. Artemisinins containing an endoperoxide bridge such as artemisinin and dihydroartemisinin induced growth of neurite processes at concentrations that were slightly cytotoxic, artemisinin having the most potent maximal effect among them. Deoxyartemisinin, which lacks the endoperoxide bridge, was ineffective. Artemisinin-treated cells expressed increased levels of the neuronal marker ß(III)-tubulin. Artemisinin upregulated phosphorylation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK), critical signaling molecules in neuronal differentiation. Consistent with activation of the two MAPKs, neurite outgrowth induced by artemisinin was inhibited by the MAPK/ERK kinase inhibitor PD98059 and the p38 MAPK inhibitor SB203580. Artemisinin also induced phosphorylation of cyclic AMP response element-binding protein (CREB) that was almost completely attenuated by PD98059 but not by SB203580. Taken together, our results indicate that artemisinin and its derivatives containing the endoperoxide bridge induced differentiation of PC12 cells toward a neuronal phenotype and suggest that both activation of ERK signaling pathway, which leads to CREB phosphorylation, and activation of p38 MAPK signaling pathway are involved in this process.

Effect of aflatoxin B1 on UDP-glucuronosyltransferase mRNA expression in HepG2 cells.

Aflatoxin B1 (AFB1) is a potent mycotoxin that induces hepatocellular carcinoma in many animal species, including humans. In this study, we examined the effects of AFB1 on UDP-glucuronosyltransferase (UGT) mRNA expression in HepG2 cells (human hepatocellular carcinoma cell line). The cells were treated with AFB1 for 48 h at a concentration of 10 µM, and their viability (87%) was not significantly different from that of control cells. Reverse transcription polymerase chain reaction (RT-PCR) analysis demonstrated that the mRNAs of four UGT1As (UGT1A1, UGT1A3, UGT1A4 and UGT1A9) and seven UGT2Bs (UGT2B4, UGT2B7, UGT2B10, UGT2B11, UGT2B15, UGT2B17 and UGT2B28) are expressed in HepG2 cells. The mRNAs of aryl hydrocarbon receptor (AhR), pregnane X receptor (PXR), retinoid X receptor (RXR) and glucocorticoid receptor (GR) as transcriptional regulators were also detected. AFB1 significantly increased mRNA levels of UGT1A3, UGT2B10, UGT2B15 and UGT2B17 in HepG2 cells to 2.5-, 2.0-, 1.9- and 1.5-fold, respectively, whereas the mRNA levels of transcriptional regulators were hardly affected by AFB1. These findings suggest that AFB1 induces UGT2B isoforms rather than UGT1A isoforms in HepG2 cells, and that the change may closely contribute to the toxicity of AFB1.

Stereoselective glucuronidation of carvedilol in human liver and intestinal microsomes.

BACKGROUND AND PURPOSE: Carvedilol is used clinically as a ß-adrenoceptor antagonist for the treatment of chronic heart failure and is primarily metabolized into glucuronides by UDP-glucuronosyltransferase (UGT). In this study, the stereoselective glucuronidation of carvedilol by the human liver and intestinal microsomes was examined using racemate and enantiomers. METHODS: Carvedilol glucuronidation activities at substrate concentrations of 1-1,000 µmol/l in human liver and intestinal microsomes were determined by high-performance liquid chromatography with fluorescence detection, and the kinetic parameters were estimated. RESULTS: The activities of S-glucuronidation toward racemic and enantiomeric carvedilol in liver microsomes were higher than those of R-glucuronidation at all substrate concentrations examined. In intestinal microsomes, the activities of S-glucuronidation from racemic and enantiomeric carvedilol at ≤100 µmol/l substrates were higher than those of R-glucuronidation, whereas the glucuronidation activities at ≥200 µmol/l substrates exhibited the opposite stereoselectivity (R > S) compared with those at ≤100 µmol/l substrates. The activities of R- and S-calvedilol glucuronidation from racemate and enantiomers in the liver and intestinal microsomes were decreased at substrate concentrations of ≥100 or 200 µmol/l, and the kinetics at substrate concentrations of 1-100 and 1-1,000 µmol/l fitted with Michaelis-Menten and substrate inhibition models, respectively. The stereoselectivities of CL(int) values for carvedilol glucuronidation followed by Michaelis-Menten and substrate inhibition models were R < S for liver microsomes and R ≈ S for intestinal microsomes. CONCLUSION: These findings demonstrate that the stereoselectivity of carvedilol glucuronidation was different between human liver and intestinal microsomes, and suggest that the difference is due to the tissue-specific expression of UGT isoforms involved in the glucuronidation of carvedilol.

Expression and inducibility of UDP-glucuronosyltransferase 1As in MCF-7 human breast carcinoma cells.

UDP-glucuronosyltransferases (UGTs) are conjugation enzymes, which are regulated in a tissue-specific manner by endogenous and environmental factors. In this study, we focused on UGT1A isoforms broadly expressed in hepatic and extrahepatic tissues and examined the expression and inducibility of UGT1As (UGT1A1 and UGT1A3-1A10) in MCF-7 cells (human breast carcinoma cell line). Reverse transcription polymerase chain reaction (RT-PCR) analysis demonstrated that UGT1A1, UGT1A6 and UGT1A9 mRNAs as well as the mRNAs of transcriptional regulators (AhR, aryl hydrocarbon receptor; Arnt, AhR nuclear translocator; ERα, oestrogen receptor α; ERß, oestrogen receptor ß; and GR, glucocorticoid receptor) are expressed in MCF-7 cells. UGT1A6 mRNA level in MCF-7 cells was significantly increased to 1.9 times by ß-naphthoflavone (BNF), whereas UGT1A1 and UGT1A9 mRNA levels were not affected by BNF. There were no significant changes in the mRNAs of UGT1A1, UGT1A6 and UGT1A9 in MCF-7 cells by treatment with phenobarbital (PB) and dexamethasone (DEX) in MCF-7 cells. The kinetics of 7-ethyl-10-hydroxycamptothecin (SN-38), 5-hydroxytryptamine (5-HT) and 4-methylumbelliferone (4-MU) glucuronidation by microsomes from control and BNF-treated MCF-7 cells fitted with the Michaelis-Menten model, and the V(max) and CL(int) values were significantly increased to 7.5-8.5 times and 5.9-10.4 times by BNF treatment, respectively. These findings suggest that BNF induces UGT1A6 in MCF-7 cells and that the increase may be mediated by AhR but not pregnane X receptor (PXR)/constitutive androstane receptor (CAR). The information gained in this study should help predict and assess the toxicity of environmental chemicals.

Characterization of inhibitory effects of perfluorooctane sulfonate on human hepatic cytochrome P450 isoenzymes: focusing on CYP2A6.

Perfluorooctane sulfonate (PFOS) is a chemically stable compound extensively used as oil and water repellent, surface active agents in our daily life. Accumulative research evidence gradually appears the toxicity of PFOS against mammals, but the whole figure remains to be elucidated. The present study was conducted to know the effects of PFOS on human hepatic drug metabolizing-type cytochrome P450 (CYP) isoenzymes such as CYP1A2 (7-ethoxyresorufin as a substrate), CYP2A6 (coumarin), CYP2B6 (7-ethoxy-4-trifluoromethylcoumarin), CYP2C8 (paclitaxel), CYP2C9 (diclofenac), CYP2C19 (S-mephenytoin), CYP2D6 (bufuralol), CYP2E1 (chlorzoxazone) and CYP3A4 (testosterone) in human livers employing their typical substrates. Although all of the oxidation reactions tested were more or less inhibited by PFOS, diclofenac 4'-hydroxylation mediated mainly by CYP2C9 was most strongly inhibited (K(i) value of 40 nM), followed by paclitaxel 6α-hydroxylation mediated mainly by CYP2C8 (K(i) value of 4 µM). The substrate oxidation reactions catalyzed by CYP2A6, CYP2B6, CYP2C19 and CYP3A4 were moderately (K(i) values of 35 to 45 µM), and those by CYP1A2, CYP2D6 and CYP2E1 were weakly inhibited by PFOS (K(i) values of 190-300 µM). The inhibition by PFOS for coumarin 7-hydroxylation mainly catalyzed by human liver microsomal CYP2A6 as well as by the recombinant enzyme was found to be enhanced by the preincubation of PFOS with human liver microsomes and NADPH as compared to the case without preincubation. The inhibition of the human liver microsomal cumarin 7-hydroxylation was PFOS concentration-dependent, and exhibited pseudo-first-order kinetics with respect to preincubation time, yielding K(inact) and K(I) values of 0.06 min(-1) and 23 µM, respectively. These results suggest that the metabolism of medicines which are substrates for CYP2C9 may be altered by PFOS in human bodies, and that PFOS is a mechanism-based inhibitor of CYP2A6.

Functional characterization of human and cynomolgus monkey UDP-glucuronosyltransferase 1A1 enzymes.

AIMS: UDP-glucuronosyltransferase 1A1 (UGT1A1) plays important roles in the glucuronidation of various drugs and endogenous substances. Cynomolgus monkeys are regarded as experimental animals closer to humans in studies on safety evaluation and biotransformation for drug development. In this study, the similarities and differences in the enzymatic properties of UGT1A1 between humans and cynomolgus monkeys were precisely identified. MAIN METHODS: Human and cynomolgus monkey UGT1A1s (humUGT1A1 and monUGT1A1, respectively) were cloned, and the corresponding proteins were heterologously expressed in insect cells. The enzymatic properties of UGT1A1 proteins were characterized by kinetic analysis of 7-hydroxy-4-trifluoromethylcoumarin (7-HFC), estradiol at 3-hydroxy position (E-3OH) and 7-ethyl-10-hydroxycamptothecin (SN-38) glucuronidation. KEY FINDINGS: There were no significant differences in the levels of kinetic parameters for 7-HFC, E-3OH and SN-38 glucuronidation between humans and cynomolgus monkeys in both enzyme sources of liver microsomes and recombinant UGT1A1s. 7-HFC and E-3OH glucuronidation by human liver microsomes exhibited biphasic and sigmoidal kinetics, respectively, whereas the kinetics by cynomolgus monkey liver microsomes fitted the typical Michaelis-Menten model. SN-38 glucuronidation by human and cynomolgus monkey liver microsomes exhibited autoactivation kinetics. In recombinant UGT1A1 enzymes expressed in insect cells, the kinetics of 7-HFC, E-3OH and SN-38 glucuronidation fitted the substrate inhibition (7-HFC glucuronidation) or Hill equation (E-3OH and SN-38 glucuronidation), and each glucuronidation showed the same kinetic profile between humans and cynomolgus monkeys. SIGNIFICANCE: These findings suggest that the enzymatic properties of human and cynomolgus monkey UGT1A1 enzymes are very similar.

Functional characterization of CYP2C8.13 and CYP2C8.14: catalytic activities toward paclitaxel.

Cytochrome P450 2C8 (CYP2C8) plays important roles in the metabolism of various drugs, including the anti-cancer drug, paclitaxel. We recently identified two novel CYP2C8 alleles (CYP2C8*13 and CYP2C8*14; wild-type, CYP2C8*1A) with non-synonymous single nucleotide polymorphisms in a Japanese population. To precisely investigate the effect of amino acid substitutions (CYP2C8*13, Ile223Met; CYP2C8*14, Ala238Pro) on CYP2C8 function, CYP2C8 proteins of the wild-type (CYP2C8.1) and variants (CYP2C8.13 and CYP2C8.14) were heterologously expressed in yeast cells, and their paclitaxel 6alpha-hydroxylation activities were determined. The K(m), V(max) and CL(int) values for paclitaxel 6alpha-hydroxylation of CYP2C8.1 were 2.3 microM, 4.1 pmol/min./pmol CYP and 1.7 microl/min./pmol CYP, respectively. The K(m) value of CYP2C8.14 was significantly higher (2.9-fold) than that of CYP2C8.1. The V(max) value of CYP2C8.14 was comparable to that of CYP2C8.1 and the CL(int) value was reduced to 46% of CYP2C8.1. In contrast, the K(m), V(max) and CL(int) values of CYP2C8.13 were similar to those of CYP2C8.1. These results suggest that Ala238Pro substitution in CYP2C8.14 decreases the affinity toward paclitaxel of the CYP2C8 enzyme, and that the genetic polymorphism of the CYP2C8*14 allele may influence the clinical response to drugs metabolized mainly by CYP2C8.

Functional characterization of human cytochrome P4502E1 allelic variants: in vitro metabolism of benzene and toluene by recombinant enzymes expressed in yeast cells.

Benzene and toluene are common organic solvents currently in worldwide industrial usage, which are metabolized mainly by hepatic cytochrome P450 2E1 (CYP2E1) in humans. Genetic polymorphism of CYP2E1 in 5'-flanking and coding regions has been found previously in Caucasian and Chinese populations. In this study, the effects of CYP2E1 alleles causing amino acid substitutions (CYP2E1*2, CYP2E1*3 and CYP2E1*4; wild-type, CYP2E1.1A) on benzene hydroxylation and toluene methylhydroxylation were studied using recombinant CYP2E1 enzymes of wild-type (CYP2E1.1) and variants (CYP2E1.2 having Arg76His, CYP2E1.3 having Val389Ile and CYP2E1.4 having Val179Ile) expressed in yeast cells. The K (m), V (max) and CL (int) values of CYP2E1.1 were 10.1 mM, 9.38 pmol/min/pmol CYP and 0.99 nL/min/pmol CYP for benzene hydroxylation, and 3.97 mM, 19.9 pmol/min/pmol CYP and 5.26 nL/min/pmol CYP for toluene methylhydroxylation, respectively. The K (m), V (max) and CL (int) values for benzene and toluene metabolism of CYP2E1.2, CYP2E1.3 and CYP2E1.4 were comparable to those of wild-type CYP2E1. These findings may mean that the polymorphic alleles of CYP2E1 causing amino acid substitutions are not directly associated with the metabolic activation of benzene and toluene. The information gained in this study should help to identify the variations in the toxicity of environmental pollutants.

Tissue-specific mRNA expression profiles of drug-metabolizing enzymes and transporters in the cynomolgus monkey.

Pairs of forward and reverse primers and TaqMan probes specific to each of 19 drug-metabolizing enzymes (cytochrome P450s, UDP-glucuronosyltransferases, glutathione S-transferases, and sulfotransferases) and 5 transporters (ABC and SLC transporters) in the cynomolgus monkey were prepared. The expression level of each target mRNA was analyzed in total RNA obtained from three specimens of various cynomolgus monkey tissues (adrenal gland, brain, heart, kidney, large intestine, liver, lung, pancreas, prostate, small intestine, spleen, testis, and thymus) by real-time reverse transcription PCR using an Applied Biosystems 7500 Fast Real-Time PCR System. The data obtained in the present study provide useful information on tissue-specific profiles of the expression of these target mRNAs in the cynomolgus monkey, and the results are expected to be valuable in establishing drug metabolism- and transporter-mediated screening systems using the cynomolgus monkey for the evaluation of new chemical entities in new drug development.

Stereoselective glucuronidation of propranolol in human and cynomolgus monkey liver microsomes: role of human hepatic UDP-glucuronosyltransferase isoforms, UGT1A9, UGT2B4 and UGT2B7.

The stereoselective glucuronidation of propranolol (PL) in human and cynomolgus monkey liver microsomes, and the roles of human hepatic UDP-glucuronosyltransferase (UGT) isoforms involved in the enantiomeric glucuronidation of PL using recombinant UGT enzymes were investigated. In Michaelis-Menten plots, R- and S-PL glucuronidation by human liver microsomes showed sigmoidal kinetics whereas the kinetics of enantiomeric PL glucuronidation by cynomolgus monkey liver microsomes was monophasic. The Km, Vmax and CLint values of cynomolgus monkey liver microsomes were generally higher than the S50, Vmax and CLmax values of human liver microsomes in R- and S-PL glucuronidation. The glucuronidation of R- and S-PL was catalyzed by at least 3 UGT isoforms: UGT1A9, UGT2B4 and UGT2B7. Michaelis-Menten plots for R- and S-PL glucuronidation by UGT1A9 were monophasic, whereas the kinetics of UGT2B7 showed sigmoidal curves. Enantiomeric R-PL glucuronidation by UGT2B4 showed sigmoidal kinetics, whereas S-PL glucuronidation displayed monophasic kinetics. UGT1A9 showed remarkable stereoselectivity in Vmax and CLint values of R-PL < S-PL. These findings demonstrate that the profiles of enantiomeric PL glucuronidation in human and cynomolgus monkey liver microsomes are largely different and suggest that the human hepatic UGT isoforms UGT1A9, UGT2B4 and UGT2B7 play distinctive roles in enantiomeric PL glucuronidation.

Effect of psychotropic drugs on the 21-hydroxylation of neurosteroids, progesterone and allopregnanolone, catalyzed by rat CYP2D4 and human CYP2D6 in the brain.

We determined the effects of psychotropic drugs on the cytochrome P450 2D (CYP2D)-mediated 21-hydroxylation of progesterone (PROG) and allopregnanolone (ALLO) with the goal of clarifying whether neurosteroid levels are affected by psychotropic drugs in the brain. PROG or ALLO was incubated with rat CYP2D4 or human CYP2D6 in the presence of typical psychotropic drugs, fluoxetine, imipramine, desipramine, mazindol, and GBR12909, and the 21-hydroxylated metabolites of PROG and ALLO were determined by high performance liquid chromatography and liquid chromatography-tandem mass spectrometry, respectively. Fluoxetine competitively inhibited CYP2D4-mediated PROG 21-hydroxylation and increased both Km and Vmax values of CYP2D6-mediated PROG 21-hydroxylation. In addition, fluoxetine competitively inhibited ALLO 21-hydroxylation mediated by CYP2D4 and CYP2D6. Imipramine, desipramine, mazindol, and GBR12909 competitively inhibited PROG 21-hydroxylation mediated by CYP2D4 and/or CYP2D6, and all psychotropic drugs inhibited ALLO 21-hydroxylation mediated by CYP2D4 and/or CYP2D6. The inhibition constants (Ki values) of imipramine, desipramine, and mazindol against the 21-hydroxylation of PROG and ALLO by CYP2D6 were lower than those by CYP2D4. These results indicate that psychotropic drugs including fluoxetine affected the metabolism of neurosteroids, such as PROG and ALLO in the brain, suggesting that the regulation of the neurosteroid levels is modified by central nervous system-active drugs that inhibit brain CYP2D isoforms.

Inducibility of UDP-glucuronosyltransferase 1As by beta-naphthoflavone in HepG2 cells.

UDP-glucuronosyltransferases (UGTs) are conjugation enzymes, which are regulated in a tissue-specific manner by endogenous and environmental factors. In this study, we focused on UGT1A isoforms (UGT1A1, UGT1A6 and UGT1A9), mainly expressed in the human liver, and examined the inducibility of UGT1As by beta-naphthoflavone (BNF) in human hepatoma HepG2 cells. The cells were pretreated for 72 h with BNF at concentrations of 25, 50 and 100 microM. 7-Ethyl-10-hydroxycamptothecin (SN-38) glucuronidation, used as a probe for UGT1A1, showed sigmoidal kinetics with a Hill coefficient (n) of 1.2-1.3 in control and BNF-pretreated HepG2 cells. The Vmax values were significantly increased 3.6- to 4.3-fold by BNF, whereas there was no significant change in the S50 values by BNF at any concentration examined. On the other hand, 4-methylumbelliferone (4-MU) glucuronidation as a probe for UGT1A6 and UGT1A9 in the control and BNF-pretreated HepG2 cells exhibited a biphasic kinetic pattern. Although Km1 values for the low-Km phase were similar between the control and BNF-pretreated HepG2 cells, Km2 values for the high-Km phase of BNF-pretreated HepG2 cells were reduced to 54-69% of control HepG2 cells. The values of Vmax1 and Vmax2 for the low- and high-Km phases, respectively, were significantly increased 1.9- to 2.6-fold by BNF at 25 and/or 50 microM but not 100 microM. With respect to Vmax (Vmax1 and Vmax2) and Vmax/Km (Vmax1/Km1 and Vmax2/Km2), the values were significantly increased 2.0- to 3.2-fold by BNF at all concentrations examined. Furthermore, real-time reverse transcription polymerase chain reaction using TaqMan probes demonstrated that BNF concentration-dependently induced mRNA levels of UGT1A1 but not UGT1A6 or UGT1A9 in HepG2 cells (1.3- to 6.0-fold). These results suggest that the inducibility of UGT1A isoforms in HepG2 cells by BNF is different from other aryl hydrocarbon receptor agonists previously reported, and should provide useful information for the prediction of drug-drug interactions and toxicological assessment of environmental chemicals.

The effect of dimethyl sulfoxide on the function of cytochrome P450 2D6 in HepG2 cells upon the co-expression with NADPH-cytochrome P450 reductase.

HepG2 cells, a human hepatoma cell line, stably expressing NADPH-cytochrome P450 reductase (OR) and/or cytochrome P450 2D6 wild-type (CYP2D6-WT) or its variants (Pro34Ser, Gly42Arg, Arg296Cys and Ser486Thr) were established in the present study. The cultivation of HepG2 cells expressing CYP2D6-WT in the culture medium containing dimethyl sulfoxide (DMSO, 0.1% of final concentration) markedly increased the bufuralol (BF) 1''-hydroxylase activity compared with that of control cells when cultivated without DMSO. A similar effect was also observed in HepG2 cells stably expressing CYP2D6 and OR. The addition of hemin in place of DMSO to the culture medium resulted in no increase in the enzyme activity. Western blot analysis revealed that the levels of CYP2D6 protein were similar between DMSO-treated and non-treated HepG2 cells regardless of OR expression. Spectrophotometric analysis of reduced carbon monoxide-difference spectra of HepG2 cells expressing CYP2D6-WT and/or OR demonstrated that the addition of DMSO increased the peak height of functional CYP2D6 at 450 nm. These results suggest that the increase in CYP2D6 activity is attributable to the radical-scavenging effect of DMSO. The HepG2 cell lines stably expressing OR and CYP2D6 or its variants in combination with DMSO treatment may be useful for screening the cytotoxicity of chemical compounds which undergo oxidation by CYP2D6.

Effect of genetic polymorphism on the metabolism of endogenous neuroactive substances, progesterone and p-tyramine, catalyzed by CYP2D6.

Metabolic activities toward endogenous substrates in the brain, progesterone and p-tyramine, by cytochrome P450 2D6.2 (CYP2D6.2), CYP2D6.10A, CYP2D6.10C, and P34S, G42R, R296C, and S486T mutants expressed in recombinant Saccharomyces cerevisiae were compared with those by CYP2D6.1 (wild-type) in order to clarify the effects of genetic polymorphism of CYP2D6 on the metabolism of neuroactive steroids and amines in the brain. For the 6beta-hydroxylation of progesterone, the V(max) values for CYP2D6.2, CYP2D6.10A, and the P34S and G42R mutants, were less than half of those for CYP2D6.1, and CYP2D6.10C had a higher K(m) and a lower V(max) than the wild-type. The V(max)/K(m) values for CYP2D6.10A, CYP2D6.10C, and the P34S and G42R mutants were 12-31% of that for CYP2D6. The 16alpha-hydroxylation and 21-hydroxylation of progesterone by CYP2D6.10A, CYP2D6.10C, and the P34S and G42R mutants were not detected, and the R296C mutant had a higher K(m) for the 16alpha-hydroxylation and a lower V(max) for the 21-hydroxylation than those for CYP2D6.1. For dopamine formation from p-tyramine, the K(m) values for CYP2D6.2 and the R296C mutant were higher than those for CYP2D6.1, CYP2D6.10A, and CYP2D6.10C had a higher K(m) and a lower V(max) than the wild-type. The V(max)/K(m) values for CYP2D6.2, CYP2D6.10A, CYP2D6.10C and the P34S, G42R and R296C mutants were less than 45% of those for the wild-type. These results suggest the possibility that the polymorphism of CYP2D6, including CYP2D6*2, CYP2D6*10 and CYP2D6*12, might affect an individual behavior and the central nervous system through endogenous compounds, such as neuroactive steroids and tyramine, in the brain.

An iron-regulated gene required for utilization of aerobactin as an exogenous siderophore in Vibrio parahaemolyticus.

A previous investigation using the Fur titration assay system showed that Vibrio parahaemolyticus possesses a gene encoding a protein homologous to IutA, the outer-membrane receptor for ferric aerobactin in Escherichia coli. In this study, a 5.6 kb DNA region from the V. parahaemolyticus WP1 genome was cloned and two entire genes, iutA and alcD homologues, were identified which are absent from Vibrio cholerae genomic sequences. The V. parahaemolyticus IutA and AlcD proteins share 43 % identity with the Escherichia coli IutA protein and 24 % identity with the Bordetella bronchiseptica AlcD protein of unknown function, respectively. Primer extension analysis revealed that the iutA gene is transcribed in response to low-iron availability from a putative promoter overlapped with a sequence resembling a consensus E. coli Fur-binding sequence. In agreement with the above finding, V. parahaemolyticus effectively utilized exogenously supplied aerobactin for growth under iron-limiting conditions. Moreover, insertional inactivation of iutA impaired growth in the presence of aerobactin and incapacitated the outer-membrane fraction from iron-deficient cells for binding (55)Fe-labelled aerobactin. These results indicate that the V. parahaemolyticus iutA homologue encodes an outer-membrane protein which functions as the receptor for ferric aerobactin. Southern blot analysis revealed that the iutA homologues are widely distributed in clinical and environmental isolates of V. parahaemolyticus. However, additional genes required for ferric aerobactin transport across the inner membrane remain to be clarified.