Expression of biotransformation enzymes in human fetal olfactory mucosa: potential roles in developmental toxicity

High levels of cytochrome P450 are present in the olfactory mucosa (OM) in mammalian animals and contribute to the known tissue-selective toxicity of numerous chemical compounds. Olfactory toxicity in the perinatal period may have a greater impact on behavior, growth, and development than in adults. To establish a molecular basis for determining the risk of developmental toxicity in OM, the expression of several cytochrome P450 enzymes, as well as NADPH-cytochrome P450 reductase and microsomal epoxide hydrolase, was examined in hepatic and nasal microsomes prepared from human fetal tissues at gestational day 91-125. The relative microsomal concentrations of these biotransformation enzymes were determined on immunoblots. Expression of CYP2A, CYP2J2, the reductase, and epoxide hydrolase was detected in both OM and liver. The microsomal levels of these enzymes were generally lower in OM than in liver of the same fetuses, except for the CYP2A-related proteins, which were expressed in OM at much higher levels. OM expression of CYP2A6, CYP2A13, CYP2B6, and CYP2J2 mRNAs was detected using RNA-PCR. These results document, for the first time, prenatal expression of xenobiotic-bioactivating cytochrome P450 enzymes in human OM and suggest that the human fetal OM may be a preferred target tissue for the toxicity of maternally derived chemical compounds that are activated by the CYP2A enzymes.     

Expression and function of cytochrome p450-dependent enzymes in human skin cells

Scientific interest in defining the human body's ability to limit the effects of administered drugs and xenobiotics dates from the mid-19th century when developing knowledge and techniques in the field of organic chemistry first made such studies possible. The first experimental evidence documenting the existence of cytochrome p450 (CYP) dates to the year 1955, when an enzyme system capable of oxidizing xenobiotic compounds was identified in the endoplasmic reticulum of liver homogenates. From these days on several studies analyzed the expression and function of metabolizing phase I enzymes in liver cells. Due to the unique structural features of human skin, little was known about the expression and function of CYP enzymes in this tissue and their role in uptake, metabolism and elimination of xenobiotics as well as endogenous substrates. Lasting recent years it has become clear that human skin cells express various CYP enzymes, including CYP26AI which is responsible for the metabolism of retinoic acid in skin cells. It has been also shown that CYP enzyme expression patterns are cell type and tissue specific and that in skin cells this differs significantly from its expression in other environmental interfaces such as the liver, lung and gastrointestinal tract. Therefore knowledge of skin-specific CYP expression and function is a prerequisite for pharmacological studies of the skin.     

细胞色素P450 3A在胎肾上腺和胎肝中的表达

采用酶学和逆转录聚合酶链式反应 ( RT-PCR)技术 ,研究与药物代谢密切相关的细胞色素P450 3A( CYP3A)在胎肾上腺和胎肝中的表达 .结果表明 ,胎肾上腺微粒体中苄非他明 ,氨基比林 ,红霉素 N-脱甲基酶和睾酮 6β-羟化酶活性分别为胎肝微粒体的 2 1 % ,2 60 % ,1 0 5%和 33% .CYP诱导剂苯巴比妥能显著增加体外培养胎肾上腺细胞中苄非他明和氨基比林 N-脱甲基酶活性 ,诱导剂地塞米松也能明显诱导红霉素 N-脱甲基酶活性 .RT- PCR表明胎肾上腺和胎肝中存在 CYP3A7m RNA表达 .说明胎肾上腺和胎肝中存在 CYP3A亚族 .上述结果提示处于发育时期的肾上腺具有与胎肝能力相当的药物代谢功能 .     

Cytochrome P-450 in human fetal liver. Properties of P-450 HFLa,a form of cytochrome P-450 in human fetal livers

One of the major forms of cytochrome P-450, named P-450 HFLa, of human fetal livers was purified and characterized. The cytochrome P-450 preparation had an apparent molecular weight of 51,500 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. N-Terminal amino acid sequence of P-450 HFLa was similar but not identical to that of P-450NF involved in nifedipine oxidation in adult human livers. P-450 HFLa catalyzed 16 alpha-hydroxylation of dehydroepiandrosterone 3-sulfate (DHEA-sulfate) in a reconstituted system. The concentration of P-450 HFLa in liver homogenates from human fetuses highly correlated with the activity of DHEA-sulfate 16 alpha-hydroxylase. Furthermore, anti-P-450 HFLa antibodies inhibited the 16 alpha-hydroxylation. P-450 HFLa was also found to catalyze the mutagenic activation of aflatoxin B1 (AFB1) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). The antibodies to P-450 HFLa inhibited efficiently the mutagen-producing activities from AFB1 and IQ in human fetal livers. The nucleotide and the deduced amino acid sequences of lambda HFL33 containing the entire coding region for a form of cytochrome P-450 related to P-450 HFLa, were highly similar to but clearly distinct from those of NF25 and HLp complementary deoxyribonucleic acids. The oligonucleotide probes specific to the coding and 3'-noncoding region of lambda HFL 33 (oli-HFL and oli-HFL3', respectively) gave hybridizable bands with ribonucleic acid (RNA) from fetal but not adult livers. In contrast, an oligonucleotide probe specific to the coding region of NF 25 and HLp (oli-NF) gave hybridizable bands with RNA from only adult but not fetal livers.     

Advances in human cytochrome p450 and personalized medicine

Among all the drug metabolic enzymes, cytochrome P450 (CYP450) superfamily acts as an important role responsible for the oxidation of almost 90% currently used drugs. As variations of Single Nucleotide Polymorphism (SNPs) in human CYP450 genes will cause different drug effects and even adverse effects, studies on SNPs of human CYP450 genes can be used for indicating the most possible genes associated with human diseases and relevant therapeutic targets, predicting the drug efficacy and adverse drug response, investigating individual gene specific properties and then providing personalized and optimal clinic therapies. Recently, some new bioinformatics methods are introduced in SNPs researches, which significantly facilitate the development of drug and medicine. The review will focus on a brief introduction of the SNPs of human drug metabolic enzymes and their relationships with personalized medicine. Besides, common bioinformatics analysis methods and some latest progresses and applications in this area will also be discussed.     

Expression and function of cytochrome p450 in brain drug metabolism

Cytochrome P450 (CYP, P450) is the collective term for a superfamily of heme-containing membrane proteins responsible for the metabolism of approximately 70 - 80 % of clinically used drugs. Besides the liver and other peripheral organs, P450 isoforms are expressed in glial cells and neurons of the brain. To enlighten their function and significance is a topic of high interest, as most of the neuroactive drugs used in therapy today are not only substrates, but also inducers of brain P450s with far reaching consequences. First of all, brain P450s are regulated differentially from those in liver. The availability of the prosthetic heme group appears to be essential for correct membrane insertion and enzymatic functionality of brain P450s. Furthermore, although not contributing to body's overall drug metabolism, brain P450s fulfil particular functions within specific cell types of the brain. In astrocytes of brain's border lines P450 isoforms are expressed at very high level. They form a metabolic barrier regulating drugs' influx, modulate blood-flow regulation, and act as signalling enzymes in inflammation. In neurons, however, P450s apparently have different function. In specified brain regions such as hypothalamus, hippocampus and striatum they provide signalling molecules like steroids and fatty acids necessary for neuronal outgrowth and maintenance. Induction of these P450s by neuroactive drugs can alter steroid hormone signalling directly in drug target cells, which may cause clinically relevant side effects like reproductive disorders and sexual or mental dysfunction. The understanding of brain P450 function appears to be of major interest in long-term drug mediated therapy of neurological diseases.     

Effect of starvation and chlormethiazole on cytochrome P450s of rat nasal mucosa

Cytochrome P450 (CYP) enzymes of nasal tissue are relatively resistant to induction by classical inducers. In the present study, the effects of starvation on the expression of CYP1A, 2A, 2B, 2C, 2E, 2G, and 3A subfamilies in the nasal mucosa of rat were studied. Fasting for 72 hr caused an increase in 2E1-dependent p-nitrophenol hydroxylase and 1A-dependent ethoxy- (or methoxy) resorufin dealkylase activities, but did not affect either 2A-linked coumarin hydroxylase or the testosterone hydroxylase activity, the latter reaction being a marker of several CYPs including 2G1. Whereas increases in 2E1- and 1A- associated catalytic activities were accompanied by a concomitant increase in the corresponding apoproteins as determined by immunoblotting, immunoactive protein bands reactive with antibodies raised against rat 1A1, 2B1, 2C11, 3A1 or rabbit nasal 2A10/11 and 2G1 were not altered. Fasting also increased CYP2E1 and CYP1A2 on the mRNA level, but did not alter CYP1A1 mRNA as determined by hybridization with cDNA probes selective for these cytochromes. A reiterative administration of chlormethiazole, a specific inhibitor of 2E1 in liver, strongly inhibited many CYPs, including 2E1, 1A2, 2G1, and 2A in the nasal mucosa, but did not influence expression of 2B or 3A as determined by immunoblotting or catalytic activities. The chlormethiazole-mediated inhibition of 1A1 and 2E1 was demonstrated to be at the mRNA level. These results suggest that fasting induces the gene expression of 2E1 and 1A2 and that the mechanisms involved in the regulation of CYPs in the nasal mucosa are tissue-specific. The inducibility of the above-mentioned isoforms may have a significant role in the clearance of drugs and bioactivation of inhaled compounds.     

Identification of human cytochrome p450 isozymes involved in diphenhydramine N-demethylation.

Diphenhydramine is widely used as an over-the-counter antihistamine. However, the specific human cytochrome P450 (P450) isozymes that mediate the metabolism of diphenhydramine in the range of clinically relevant concentrations (0.14-0.77 microM) remain unclear. Therefore, P450 isozymes involved in N-demethylation, a main metabolic pathway of diphenhydramine, were identified by a liquid chromatography-mass spectrometry method developed in our laboratory. Among 14 recombinant P450 isozymes, CYP2D6 showed the highest activity of diphenhydramine N-demethylation (0.69 pmol/min/pmol P450) at 0.5 microM. CYP2D6 catalyzed diphenhydramine N-demethylation as a high-affinity P450 isozyme, the K(m) value of which was 1.12 +/- 0.21 microM. In addition, CYP1A2, CYP2C9, and CYP2C19 were identified as low-affinity components. In human liver microsomes, involvement of CYP2D6, CYP1A2, CYP2C9, and CYP2C19 in diphenhydramine N-demethylation was confirmed by using P450 isozyme-specific inhibitors. In addition, contributions of these P450 isozymes estimated by the relative activity factor were in good agreement with the results of inhibition studies. Although an inhibitory effect of diphenhydramine on the metabolic activity of CYP2D6 has been reported previously, the results of the present study suggest that it is not only a potent inhibitor but also a high-affinity substrate of CYP2D6. Therefore, it is worth mentioning that the sedative effect of diphenhydramine might be caused by coadministration of CYP2D6 substrate(s)/inhibitor(s). In addition, large differences in the metabolic activities of CYP2D6 and those of CYP1A2, CYP2C9, and CYP2C19 could cause the individual differences in anti-allergic efficacy and the sedative effect of diphenhydramine.     

人胎肾上腺线粒体细胞色素P—450体外脱甲基功能

AIM: To explore the capacity and characteristics of adrenal mitochondria to metabolize xenobiotics in vitro in human fetus. METHODS: Subcellular fractions of fetal adrenal were prepared by differential centrifugation. Mitochondrial P-450 system was proved by spectral analyses and SDS-PAGE. The formaldehyde formation contents were measured with Nash reagent. RESULTS: The erythromycin N-demethylation linearly increased in the protein concentration (1-4 mg)- and incubation time (10-30 min)-dependent manners. A typical concentration-effect relationship appeared with erythromycin 0.067-1 mmol.L-1 and a positive correlation (r = 0.641, P < 0.05) existed between erythromycin N-demethylation and gestation months. The N-demethylation values (nmol.s-1/g protein) of erythromycin (2.7 +/- 0.8), benzfetamine (1.1 +/- 0.5), and aminophenazone (0.9 +/- 0.4) in mitochondria were 89% (P > 0.05), 162% (P < 0.01), and 62% (P < 0.01), respectively, of those in microsomes. There was correlation between mitochondria and microsomes in the N-demethylation of erythromycin (r = 0.708, P < 0.05) and benzfetamine (r = 0.707, P < 0.05). Troleandomycin stimulated erythromycin N-demethylation in adrenal mitochondria as well as in adrenal and liver microsomes in vitro. CONCLUSION: Fetal adrenal mitochondria, with multiple P-450 isoforms and greater capacity of demethylation, play a role in drug-metabolism during fetal development.     

Stereoselectivity of human cytochrome p450 in metabolic and inhibitory activities

This review focuses on stereoselectivity of human cytochrome P450 (P450 or CYP) in the area of metabolism and inhibition. A meta-analysis was performed based on the reported values regarding (1) values of the Michaelis-Menten constant (K(m)), maximal velocity (V(max)), and intrinsic clearance (V(max)/K(m)) for 45 metabolic reactions of 19 substrates and (2) inhibition constants (K(i)) for 6 inhibitors. The median (R)/(S)-enantiomer ratios of the K(m), V(max), and V(max)/K(m) values for CYP1A2, CYP2B6, CYP2C19, CYP2D6, and CYP3A4 were in the range of 0.80-1.53, whereas the median ratios of V(max), and V(max)/K(m) values for CYP2C9 were 0.43 and 0.60, respectively. In addition, the parameters for metabolic reactions (25-80%) of (R)-enantiomers of these P450s were comparable to those of (S)-enantiomers (the ratios were between 0.5 and 2), whereas 45-69% of V(max) and V(max)/K(m) values for the (R)-enantiomer in CYP2C9 were less than half of those for the (S)-enantiomer, although the kinetic behavior of the stereoselectivity depended on the metabolic reaction. There is a limited number of reports regarding stereoselective inhibition and induction in vitro. The present information gives insight into the contribution of stereoselectivity to metabolism mediated by P450s and the risk of adverse drug-drug interactions due to stereoselectivity.     

Inhibitory effect of glyburide on human cytochrome p450 isoforms in human liver microsomes.

The inhibitory effect of glyburide [International Nonproprietary Name (INN), glibenclamide] on CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 activities was evaluated using pooled human liver microsomes. Glyburide strongly inhibited CYP2C9-catalyzed S-warfarin and phenytoin metabolism in a competitive manner, with Ki (IC50) values of 2.4 (11.3) microM and 3.1 (9.4) microM, respectively. CYP3A4-catalyzed midazolam 1-hydroxylation was inhibited by glyburide with a Ki (IC50) value of 42.5 (90.0) microM. However, glyburide showed no appreciable inhibitory effect on CYP1A2, CYP2C8, CYP2C19, CYP2E1, or CYP2D6. In summary, glyburide showed potent inhibition on CYP2C9 and weak inhibition on CYP3A4, whereas it had minimal or no inhibitory effect on the other cytochromes p450 examined. It is anticipated that clinically significant drug-drug interactions will ensue when glyburide is coadministered with agents that are cleared primarily by the CYP2C9-mediated pathway and those with narrow therapeutic ranges.     

Partial purification and properties of cytochrome P450 from homogenates of human fetal livers.

Using ω-amino-n-octyl Sepharose 4B and hydroxylapatite columns, cytochrome P450 was purified to approx. 6.7 nmoles per mg of protein from the 105,000 g precipitate of homogenates of human fetal livers. The partially purified preparation of cytochrome P450 was free of detectable amounts of cytochrome b₅, NADPH-cytochrome P450 reductase and NADH-cytochrome b₅ reductase. The absolute spectrum of the preparation exhibited a peak at 417 nm in the Soret region, indicating that this cytochrome P450 is a low spin species. Binding of aniline and SKF 525-A to this partially purified preparation of cytochrome P450 produced a modified type II and a type I difference spectra, respectively. As judged by Ouchterlony double diffusion analysis, the cytochrome P450 preparation did not cross react with antibody against cytochrome P450 isolated from the livers of phenobarbital-pretreated rats. In reconstituted systems, the cytochrome exhibited considerable activity for aniline hydroxylation but only a low ethlymorphine N-demethylation activity compared to cytochrome P450 isolated from the livers of phenobarbital-pretreated rats.     

人胎肾上腺线粒体细胞色素P-450体外脱甲基功能(英文)

目的:了解胎肾上腺线粒体代谢外源性化合物的能力和特征,方法:制备亚细胞组分,酶学检测脱甲基反应代谢产物—甲醛的含量,结果:在光谱分析和SDS-PAGE证实线粒体存在P-450的基础上,进一步证明线粒体P-450具有脱甲基功能,其脱甲基作用呈蛋白浓度(1-4 mg)和反应时间(10-30 min)依赖性增加,与底物浓度间有良好的量效关系,并与胎龄呈正比,线粒体中红霉素、苄非他明和氨基比林的脱甲基反应分别为微粒体中的89％,162％和62％。醋竹桃霉素增强肾上腺的红霉素脱甲基反应。结论:胎肾上腺线粒体有较强的脱甲基功能,提示胎儿肾上腺线粒体兼有药物代谢功能。     

Characterization of human cytochrome p450 enzymes involved in the metabolism of cilostazol.

Cilostazol (OPC-13013; 6-[4-(1-cyclohexl-1H-tetrazol-5-yl)butoxy]-3,4-dihydro-2(1H)-quinolinone) is widely used as an antiplatelet vasodilator agent. In vitro, the hydroxylation of the quinone moiety of cilostazol to OPC-13326 [6-[4-(1-cyclohexyl-1H-tetrazol-5-yl)butoxy]-3,4-dihydro-4-hydroxy-2(1H)-quinolinone], is the predominant route, and the hydroxylation of the hexane moiety to OPC-13217 is the second most predominant route. This study was carried out to identify and kinetically characterize the human cytochrome P450 (P450) isozymes responsible for the formation of the two major metabolites of cilostazol, namely, OPC-13326 and OPC-13217 [3,4-dihydro-6-[4-[1-(cis-4-hydroxycyclohexyl)-1H-tetrazol-5-yl)butoxy]-2(1H)-quinolinone)]. In in vitro studies using 14 recombinant human P450 isozymes, CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP2J2, CYP3A4, CYP3A5, and CYP4A11, cilostazol was metabolized to OPC-13326 mainly by CYP3A4 (K(m) = 5.26 muM, intrinsic clearance (CL(int)) = 0.34 microl/pmol P450/min), CYP1B1 (K(m) = 11.2 microM, CL(int) = 0.03 microl/pmol P450/min), and CYP3A5 (K(m) = 2.89 microM, CL(int) = 0.05 microl/pmol P450/min) and to OPC-13217 mainly by CYP3A5 (K(m) = 1.60 microM, CL(int) = 0.57 microl/pmol P450/min), CYP2C19 (K(m) = 5.95 microM, CL(int) = 0.16 microl/pmol P450/min), CYP3A4 (K(m) = 5.35 microM, CL(int) = 0.10 microl/pmol P450/min), and CYP2C8 (K(m) = 33.8 microM, CL(int) = 0.009 microl/pmol P450/min). The present study showed that the two major metabolites of cilostazol in vitro, namely, OPC-13326 and OPC-13217, are mainly catalyzed by CYP3A4 and CYP3A5, respectively.     

Genetic polymorphism of human cytochrome p450 involved in drug metabolism

Recent advances in human gene analysis promoted by the human genome project have brought us a massive amount of information. These data can be seen and analyzed by personal computer through individual Web sites. As a result, the best use of bioinformatic is essential for recent molecular biology research. Genetic polymorphism of drug-metabolizing enzymes influences individual drug efficacy and safety through the alteration of pharmacokinetics and disposition of drugs. Considerable amounts of data have now accumulated as allelic differences of various drug metabolizing enzymes. Current understanding of genotype information on cytochrome P450 is hereby summarized, based on the Web site for their use in individual optimization of drug therapy.     

Bioactivation of tamoxifen by recombinant human cytochrome p450 enzymes

Tamoxifen is a major drug used for adjuvant chemotherapy of breast cancer; however, its use has been associated with a small but significant increase in risk of endometrial cancer. In rats, tamoxifen is a hepatocarcinogen, and DNA adducts have been observed in both rat and human tissues. Tamoxifen has been shown previously to be metabolized to reactive products that have the potential to form protein and DNA adducts. Previous studies have suggested a role for P450 3A4 in protein adduct formation in human liver microsomes, via a catechol intermediate; however, no clear correlation was seen between P450 3A4 content of human liver microsomes and adduct formation. In the present study, we investigated the P450 forms responsible for covalent drug-protein adduct formation and the possibility that covalent adduct formation might occur via alternative pathways to catechol formation. Recombinant P450 3A4 catalyzed adduct formation, and this correlated with the level of uncoupling in the P450 incubation, consistent with a role of reactive oxygen species in potentiating adduct formation after enzymatic formation of the catechol metabolite. Whereas P450s 1A1, 2D6, and 3A5 generated catechol metabolite, no covalent adduct formation was observed with these forms. By contrast, P450 2B6, 2C19, and rat liver microsomes catalyzed drug-protein adduct formation but not catechol formation. Drug protein adducts formed specifically with P450 3A4 in incubations using membranes isolated from bacteria expressing P450 3A4 and reductase, as well as in reconstitutions of purified 3A4, suggesting that the electrophilic species reacted preferentially with the P450 enzymes concerned.