Minipig cytochrome P450 2E1: comparison with human enzyme.

Cytochrome P450 2E1 was isolated from minipig liver microsomes. The protein has been cloned and the respective cDNA sequenced (GenBank Accession Number AY581116). Minipig CYP2E1 is two residues shorter than its human ortholog. The only difference between pig and minipig sequence is the presence of aspartic acid residue in position 346 contrary to valine in the pig enzyme. Minipig CYP2E1 was shown to be able to convert two prototypical substrates of human CYP2E1, chlorzoxazone and p-nitrophenol, to the respective metabolites. The experiments performed with both the liver microsomal fraction and reconstituted systems with human or minipig CYP2E1 confirmed the similarity of both enzymes. Inhibition with diethyldithiocarbamate gave comparable Ki values for minipig as well as for the human CYP2E1. The results indicate that the systems containing minipig CYP2E1 may be used to model the respective CYP2E1-catalyzed reactions of drug metabolism in humans.     

Antinociceptive and antiedematogenic properties and acute toxicity of Tabebuia avellanedae Lor. ex Griseb. inner bark aqueous extract

Background

The search for an optimal experimental model in pharmacology is recently focused on (mini)pigs as they seem not only to be an alternative source of cells and tissues for xenotherapy but also an alternative species for studies on drug metabolism in man due to similarities between (mini) pig and human drug metabolizing systems. The purpose of this work is to characterize minipig liver microsomal cytochromes P450 (CYPs) by comparing their N-terminal sequences with corresponding human orthologs.

Results

The microsomal CYPs exhibit similar activities to their human orthologous enzymes (CYP3A4, nifedipine oxidation; 2A6, coumarin 7-hydroxylation; 2D6, bufuralol 1'-hydroxylation; 2E1, p-nitrophenol hydroxylation; and 2C9, tolbutamide hydroxylation). Specific minipig CYP (2A, 2C and 3A) enzymes were partially purified and proteins identified by immunostaining (using antibodies against the respective human CYPs) were used for N-terminal amino acid sequencing. From comparisons, it can be concluded that the sequence of the first 20 amino acids at the N-terminus of minipig CYP2A is highly similar to human CYP2A6 (70% identity). The N-terminal sequence of CYP2C shared about 50% similarity with human 2C9. The results on the minipig liver microsomal CYP3A yielded identical data with those obtained for amino acid sequences of the pig CYP3A29 showing 60% identity with human CYP3A4.

Conclusions

Thus, our results further support the view that minipigs may serve as model animals in pharmacological/toxicological studies with substrates of human CYP enzymes, namely, of the CYP3A and CYP2A forms.     

Different in vitro metabolism of paclitaxel and docetaxel in humans, rats, pigs, and minipigs.

We investigated cytochrome P450 (P450)-catalyzed metabolism of the important cancer drugs paclitaxel and docetaxel in rat, pig, minipig, and human liver microsomes and cDNA-expressed P450 enzymes. In rat microsomes, paclitaxel was metabolized mainly to C3'-hydroxypaclitaxel (C3'-OHP) and to a lesser extent to C2-hydroxypaclitaxel (C2-OHP), di-hydroxypaclitaxel (di-OHP), and another unknown monohydroxylated paclitaxel. In pig and minipig microsomes, this unknown hydroxypaclitaxel was the main metabolite, whereas C3'-OHP was a minor product. In minipigs, C2-OHP was the next minor product. In human liver microsomes, 6 alpha-hydroxypaclitaxel (6 alpha-OHP) was the main metabolite, followed by C3'-OHP and C2-OHP. Among different cDNA-expressed human P450 enzymes (CYP1A2, 1B1, 2A6, 2C9, 2E1, and 3A4), only CYP3A4 enzyme formed C3'-OHP and C2-OHP. Docetaxel was metabolized in pig, minipig, rat, and human liver microsomes mainly to hydroxydocetaxel (OHDTX), whereas CYP3A-induced rat microsomes produced primarily diastereomeric hydroxyoxazolidinones. Human liver microsomes from 10 different individuals formed OHDTX at different rates correlated with CYP3A4 content. Troleandomycin as a selective inhibitor of CYP3A inhibited the formation of C3'-OHP, C2-OHP, and di-OHP, as well as the unknown OHP produced in rat, minipig, and pig microsomes. In human liver microsomes, troleandomycin inhibited C3'-OHP and C2-OHP formation, and a suitable inhibitor of human CYP2C8, fisetin, strongly inhibited the formation of 6 alpha-OHP, known to be catalyzed by human CYP2C8. In conclusion, the metabolism of docetaxel is the same in all four species, but metabolism of paclitaxel is different, and 6 alpha-OHP remains a uniquely human metabolite. Pigs and minipigs compared with each other formed the same metabolites of paclitaxel.     

Analyses of CYP2C in porcine microsomes

The cytochrome P450 2C (CYP2C) subfamily in human beings includes four different isoenzymes that metabolize different substrates although with some cross reactivity. Some of these substrates (e.g. diclofenac and tolbutamide), have been investigated in porcine microsomes, but without identifying the specific CYP catalysing the reactions. The objective of this study was therefore to test some CYP2C substrates and identify the porcine CYP2C responsible for the reaction. Three substrates, paclitaxel, tolbutamide and omeprazole, were chosen, as they are metabolized by three different CYP2C isoenzymes in human beings. Microsomes, isolated from 20 different pigs, 12 conventional, and 8 minipigs, were incubated with these compounds, and correlations between the metabolism rates of these three substrates were found indicating that the reactions are catalysed by the same enzyme. Male minipigs tend to have higher average activity than female minipigs, which is in contrast to the gender-dependent expression seen for other CYP isoenzymes. The metabolic activities correlated with the protein level determined in Western blotting, using anti-human CYP2C9, indicating that this enzyme is responsible for the reaction. The expression of the CYP2C enzymes was analysed by real-time polymerase chain reaction, using a primer set that could amplify CYP2C8, CYP2C9 and CYP2C19. The melting curves (peaks) revealed that all three genes were present, showing very different expression levels in the various types of pigs. The area of one of the peaks, however, correlated with the CYP2C9-like enzyme concentration, suggesting that this peak represents CYP2C9. Among paclitaxel, tolbutamide and omeprazole, omeprazole is the best probe of CYP2C9-like enzyme in the pig.     

Porcine cytochrome 2A19 and 2E1

Cytochrome P450 (CYP) is a major group of enzymes, which conduct Phase I metabolism. Among commonly used animal models, the pig has been suggested as the most suitable model for investigating drug metabolism in human beings. Moreover, porcine CYP2A19 and CYP2E1 are responsible for the biotransformation of both endogenous and exogenous compounds such as 3‐methylindole (skatole), sex hormones and food compounds. However, little is known about the regulation of porcine CYP2A19 and CYP2E1. In this MiniReview, we summarise the current knowledge about the regulation of porcine CYP2A19 and CYP2E1 by environmental, biological and dietary factors. Finally, we reflect on the need for further research, to clarify the interaction between active feed components and the porcine CYP system.     

Role of gene knockout and transgenic mice in the study of xenobiotic metabolism

The role of P450s in xenobiotic metabolism, toxicity, and carcinogenicity has been studied for many years by using in vitro approaches and limited in vivo investigations. Genetic analysis to study the effects of xenobiotics in intact animals has only recently been carried out by use of gene knockout mice. Mice lacking expression of these enzymes have no or only modest phenotypes, indicating that their xenobiotic-metabolizing enzymes are not critical for mammalian development or physiological homeostasis. The null mice have been used to study the roll of xenobiotic-metabolizing enzymes in chemical toxicity and carcinogenicity. There are marked species differences in the expression and catalytic activities of P450s that metabolize xenobiotics, and this complicates the extrapolation of data obtained in rodents for use in drug development and human risk assessment. This is especially notable between mice and rats, commonly used experimental models, and humans. To begin to develop more predictive models, P450 humanized mice were produced and characterized by using genomic clones containing the complete coding and regulatory regions of genes, as transgenes. Humanized lines expressing CYP2D6 and CYP3A4 human P450 were characterized and found to accurately express human P450 proteins and catalytic activities at levels comparable to or higher than the corresponding activities found in human tissues. These novel mouse lines offer the opportunity to predict human drug and carcinogen metabolism and disposition and to search for endogenous substrates for human P450s.     

Characterization of cytochrome P450 isoenzymes in primary cultures of pig hepatocytes

Despite the fact that pigs are increasingly used in pharmacological and toxicological studies, knowledge on the enzymes which metabolize xenobiotics, in particular cytochrome P450 (CYP) enzymes, in pigs is still very limited. Primary cultures of pig hepatocytes were used to characterize CYP enzymes. The characterization was performed at the level of enzymatic activities, apoprotein and mRNA analyses. Enzyme inducers investigated were β-naphthoflavone (BNF), phenobarbital (PB), dexamethasone (DEX) and rifampicin (RIF). After 48 hr of BNF treatment, CYP1A protein and mRNA levels were increased, and ethoxyresorufin O-deethylation and caffeine 3-demethylation were strongly induced. PB and RIF increased the levels of CYP3A apoprotein and mRNA, whereas BNF down-regulated CYP3A and related activities. PB and RIF treatment resulted in increased ethylmorphine N-demethylation and testosterone hydroxylation, which appears to be the result of CYP3A induction. Hybridization of pig RNA with a human CYP2C9 cDNA probe showed a PB and RIF inducible CYP, which was down-regulated by BNF. Similar inducing effects were observed for tolbutamide, a marker substrate for CYP2C. DEX was not a potent inducer, although some induction of CYP3A mRNA was observed. The present results indicate the absence of CYP2B and probably CYP2D enzymes and activities in pig liver. Despite some dissimilarities, the results indicate that pigs, apart from their very human-like physiology, might represent a more appropriate model species for oxidative drug metabolism in humans than rats.     

Substrates of human cytochromes P450 from families CYP1 and CYP2: analysis of enzyme selectivity and metabolism

A compilation of information relating to substrate metabolism via human cytochromes P450 (CYP) from the CYP1 and CYP2 families is reported. The data presented include details of preferred sites of metabolism and Km values (usually for the expressed enzymes) for each reaction for selected substrates of CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP2E1. Although other P450 databases are available, they do not provide such information as is collated here, and which can prove useful for comparing P450 substrate characteristics. This information can be employed in analysing the structural requirements for human P450 enzyme selectivity and for establishing various rules regarding preferred site of metabolism for selective P450 substrates. For example, in most cases it would appear that there is a set number of intervening 'heavy' atoms (atoms other than hydrogen) between sites of metabolism and key hydrogen bond acceptors (or donors) for human P450 substrates, with the number of intervening atoms being dependent upon the type of P450 involved.     

Structure-activity relationship for human cytochrome P450 substrates and inhibitors

Criteria governing the avidity of substrate binding to human hepatic cytochromes P450 (CYP) associated with Phase 1 metabolism of drugs are described. The results of extensive quantitative structure-activity relationship (QSAR) analyses are reported for substrates of human P450s: CYPIA2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4, representing the enzymes exhibiting major involvement in the metabolism of drug substrates in Homo sapiens. In particular, it is shown that hydrogen bond properties in each class of enzyme-substrate complex are especially important factors in determining substrate binding affinity towards those human P450s which are involved in drug metabolism.     

Expression changes of CYP2A and CYP3A in microsomes from pig liver and cultured hepatocytes

The P450 enzymes of the liver are responsible for the metabolism of a wide range of chemical compounds, and hepatocytes are used in pharmacological and toxicological in vitro tests. Thus, it is important to know how stable these enzymes are in culture. We measured the activity of CYP2A and CYP3A in microsomes isolated from both pig liver and primary pig hepatocyte cultures, together with the apoprotein concentration using Western blotting. The CYP2A activity and apoprotein concentration decreased rapidly; only about 5 percent remained after 48 hr incubation, whereas the CYP3A activity and apoprotein concentration was constant. CYP3A was induced 3 times after exposure to rifampicin, whereas neither rifampicin nor pyrazole could induce CYP2A. The hepatocytes were also incubated with varying concentration of FCS and autologous serum, however without effect on the stability of CYP2A, nor did different concentrations of growth hormone and testosterone added to the cultures have any effect.     

Oxidation of 1,8-cineole, the monoterpene cyclic ether originated from eucalyptus polybractea, by cytochrome P450 3A enzymes in rat and human liver microsomes.

1,8-Cineole, the monoterpene cyclic ether known as eucalyptol, is one of the components in essential oils from Eucalyptus polybractea. We investigated the metabolism of 1,8-cineole by liver microsomes of rats and humans and by recombinant cytochrome P450 (P450 or CYP) enzymes in insect cells in which human P450 and NADPH-P450 reductase cDNAs had been introduced. 1,8-Cineole was found to be oxidized at high rates to 2-exo-hydroxy-1,8-cineole by rat and human liver microsomal P450 enzymes. In rats, pregenolone-16alpha-carbonitrile (PCN) and phenobarbital induced the 1,8-cineole 2-hydroxylation activities by liver microsomes. Several lines of evidence suggested that CYP3A4 is a major enzyme involved in the oxidation of 1,8-cineole by human liver microsomes: (1), 1,8-cineole 2-hydroxylation activities by liver microsomes were inhibited very significantly by ketoconazole, a CYP3A inhibitor, and anti-CYP3A4 immunoglobulin G; (2), there was a good correlation between CYP3A4 contents and 1,8-cineole 2-hydroxylation activities in liver microsomes of eighteen human samples; and (3), of various recombinant human P450 enzymes examined, CYP3A4 had the highest activities for 1,8-cineole 2-hydroxylation; the rate catalyzed by CYP3A5 was about one-fourth of that catalyzed by CYP3A4. Kinetic analysis showed that K(m) and V(max) values for the oxidation of 1,8-cineole by liver microsomes of human sample HL-104 and rats treated with PCN were 50 microM and 91 nmol/min/nmol P450 and 20 microM and 12 nmol/min/nmol P450, respectively. The rates observed using human liver microsomes and recombinant CYP3A4 were very high among other CYP3A4 substrates reported so far. These results suggest that 1,8-cineole, a monoterpenoid present in nature, is one of the effective substrates for CYP3A enzymes in rat and human liver microsomes.     

Cloning CYP2D21 and CYP3A22 cDNAs from liver of miniature pigs.

To compare the identity of the primary structure of drug-metabolizing cytochrome P450 between miniature pigs and humans, two cDNA clones, coding for miniature pig CYP2D21 and CYP3A22, were isolated. The deduced amino acid sequences of CYP2D21 and CYP3A22 were 78.3 and 75.0% identical to human CYP2D6 and CYP3A4, respectively. These values were nearly the same as those of bovine, dog, and some rodent isoforms, and 12.2 to 18.4% lower than those of nonhuman primates such as cynomolgus monkeys, Japanese monkey, and marmosets. These data indicate that miniature pig P450s are genetically not so close as monkey P450s to human P450s as previously expected. The recombinant CYP2D21 enzyme, however, showed bufuralol 1'-hydroxylase activity, suggesting that miniature pig CYP2D21 is capable of metabolizing some of the same substrates associated with human CYP2D6 despite its low identity to human counterparts.     

Characterization of diuron N-demethylation by mammalian hepatic microsomes and cDNA-expressed human cytochrome P450 enzymes.

Diuron, a widely used herbicide and antifouling biocide, has been shown to persist in the environment and contaminate drinking water. It has been characterized as a "known/likely" human carcinogen. Whereas its environmental transformation and toxicity have been extensively examined, its metabolic characteristics in mammalian livers have not been reported. This study was designed to investigate diuron biotransformation and disposition because metabolic routes, metabolizing enzymes, interactions, interspecies differences, and interindividual variability are important for risk assessment purposes. The only metabolic pathway detected by liquid chromatography/mass spectometry in human liver homogenates and seven types of mammalian liver microsomes including human was demethylation at the terminal nitrogen atom. No other phase I or phase II metabolites were observed. The rank order of N-demethyldiuron formation in liver microsomes based on intrinsic clearance (V(max)/K(m)) was dog > monkey > rabbit > mouse > human > minipig > rat. All tested recombinant human cytochrome P450s (P450s) catalyzed diuron N-demethylation and the highest activities were possessed by CYP1A1, CYP1A2, CYP2C19, and CYP2D6. Relative contributions of human CYP1A2, CYP2C19, and CYP3A4 to hepatic diuron N-demethylation, based on average abundances of P450 enzymes in human liver microsomes, were approximately 60, 14, and 13%, respectively. Diuron inhibited relatively potently only CYP1A1/2 (IC(50) 4 microM). With human-derived and quantitative chemical-specific data, the uncertainty factors for animal to human differences and for human variability in toxicokinetics were within the range of the toxicokinetics default uncertainty/safety factors for chemical risk assessment.     

Pharmacogenetics of P450 oxidoreductase: effect of sequence variants on activities of CYP1A2 and CYP2C19

OBJECTIVES: All microsomal cytochrome P450s enzymes, including those that metabolize the majority of clinically used drugs, require electron transfer through P450 oxidoreductase (POR). Mutations in human POR cause altered steroidogenesis and congenital malformations, but the clinical effects on drug metabolism are unclear. We examined the effects of POR sequence variants on two drug-metabolizing P450 enzymes, CYP1A2 and CYP2C19. METHODS: Our previous sequencing of the human POR gene in POR-deficient patients and in 842 normal individuals identified 35 sequence variants. We expressed these 35 POR sequence variants in bacteria, reconstituted them with the CYP enzymes in vitro, and assayed their activities with human CYP1A2 and CYP2C19. RESULTS: POR variants affected the activities of these enzymes to different extents. Disease-causing POR mutations A287P and R457H diminished catalysis by CYP1A2 and CYP2C19 to barely detectable levels. POR A503V, a polymorphism found in 28% of alleles in the normal population, had 85% of wild-type activity with CYP1A2 and 113% of wild-type activity with CYP2C19. Q153R, a disease-causing mutation that severely impaired steroidogenic activity and cytochrome c reduction, increased the activity of CYP1A2 to 144% and CYP2C19 activity to 284% of control. CONCLUSION: The activity of individual POR mutants may vary greatly depending on the electron recipient used to assay activity. Thus, the activity of a POR mutant to support catalysis by a particular P450 enzyme cannot be predicted by the activity of that POR mutant in an assay with a different P450 or with cytochrome c.     

Heterotropic cooperativity in oxidation mediated by cytochrome p450

Cytochrome P450s (P450 or CYPs) comprise a superfamily of enzymes that catalyze the oxidation of a wide variety of xenobiotic chemicals. Although most of P450 inhibitors decrease the metabolic activities mediated by the corresponding P450 forms, unexpected phenomena, which are called as activation or heterotropic cooperativity, have been often observed. We summarize Michaelis-Menten constants (K(m)), maximal velocities (V(max)), V(max)/K(m) (intrinsic clearance) values, and/or metabolic activities for 22 activators and 24 substrates (30 reactions) mainly mediated by CYP3A4 among human P450 forms. Although an allosteric mechanism has been invoked to explain the cooperativity, the activation patterns or phenomena are dependent on substrates and selected enzyme sources in vitro. Interestingly, recent studies have been shown that human P450 forms other than CYP3A4, such as CYP1A2, CYP2C8, CYP2C9, CYP2D6, and CYP3A7, are also activated by some compounds, whereas there are few reports on CYP3A5. Several models describing interaction among substrates, effectors, and enzymes have been proposed, however, the detailed mechanism for the activation is still generally unknown even though some crystal structures have been shown. A few cases of the cooperativity of CYP3A in experimental animals have been presented, whereas the clinical significance of P450 cooperativity is still unclear. The collective findings provide fundamental and useful information for the activation of P450s by chemicals despite some contradictive kinetic parameters for the same reactions reported. To understand causal factor(s) and mechanism(s) for such different reports summarized here is still one of the hot research topics to be solved in current activation reactions.     

In vitro characterization of the metabolism of haloperidol using recombinant cytochrome p450 enzymes and human liver microsomes.

A systematic in vitro study was carried out to elucidate the enzymes responsible for the metabolism of haloperidol (HAL) using human liver microsomes and recombinant human cytochrome P450 isoenzymes. In the first series of experiments, recombinant cytochrome P450 (P450) isoenzymes were used to evaluate their catalytic involvement in the metabolic pathways of HAL. Recombinant CYP3A4, CYP3A5, and CYP1A1 were shown to be able to catalyze the metabolism of HAL to its pyridinium analog (HP(+)) and the oxidation of reduced HAL (RH) back to HAL; Recombinant CYP3A4, CYP3A5, CYP1A1, CYP2C19, CYP2C8, CYP2C9, and CYP2D6 were able to catalyze the dealkylation of HAL to 4-(4-chlorophenyl)-4-hydroxypiperidine (CPHP). CYP3A4 was capable of metabolizing HAL to its tetrahydropyridine analog 4-(4-chlorophenyl)-1-[4-(4-fluorophenyl)-4-oxobutyl]-1,2,3,6-tetrahydropyridine and metabolizing to CPHP; CYP3A4 and CYP3A5 were able to metabolize RH to its pyridinium analog (RHP(+)); CYP1A1, CYP1A2, and CYP3A4 were able to catalyze the oxidation of RHP(+) to HP(+). In the second series of experiments, the metabolic activities of human liver microsomes from 12 donors were correlated with catalytic activities of selective substrates of different P450 isoenzymes and immuno-reactivities toward different P450 isoenzymes. CYP3A4 activities were found to correlate to all the seven metabolic pathways of HAL mentioned above. This suggests a prominent role for CYP3A4 in the metabolism of HAL. Interestingly, it was found that recombinant CYP1A1 has the highest activity for oxidizing RHP(+) to HP(+). The activity of recombinant CYP1A1 was 50 times higher than CYP1A2 and 220 times higher than CYP3A4.     

Human cytochrome p450 family 4 enzymes: function, genetic variation and regulation

The microsomal cytochrome P450 (CYP) family 4 monooxygenases are the major fatty acid omega-hydroxylases. These enzymes remove excess free fatty acids to prevent lipotoxicity, catabolize leukotrienes and prostanoids, and also produce bioactive metabolites from arachidonic acid omega-hydroxylation. In addition to endogenous substrates, recent evidence indicates that CYP4 monooxygenases can also metabolize xenobiotics, including therapeutic drugs. This review focuses on human CYP4 enzymes and updates current knowledge concerning catalytic activity profiles, genetic variation and regulation of expression. Comparative differences between the human and rodent CYP4 enzymes regarding catalytic function and conditional expression are also discussed.     

细胞色素P450氧化酶基因多态性对药物代谢影响的研究进展

细胞色素P450氧化酶是人体内的一种重要代谢酶,它能代谢和/或活化许多种药物、前致癌物、前毒物和致变剂。细胞色素P450氧化酶的基因具有遗传多态性,在人类存在等位基因的突变体。本文从基因结构、底物和影响其活性的因素、遗传多态性的分子机制等方面综述细胞色素P450氧化酶的研究进展。