The role of different cytochrome P450 isoforms in in vitro chloroform metabolism

The two CHCl₃ activation pathways have been studied in incubations at different oxygenation conditions with hepatic microsomes from control Sprague Dawley (SD) rats or SD rats treated with different cytochrome P450 inducers (acetone, phenobarbital, pyrazole, dexamethasone, and β-naphthoflavone). The present results provide direct evidence that CHCl₃ concentration is critical in determining the role of different cytochrome P450 isoforms (CYP) and the related effects of metabolic inducers. At 0.1 mM CHCl₃ concentration, the only major contribution to its oxidative biotransformation in liver microsomes from untreated rats was due to CYP2E1, as shown by metabolic inhibition due to 4-methylpyrazole or by anti-CYP2E1 antibodies. Moreover, animal treatments with acetone and pyrazole increased the production of adducts of phosgene to microsomal phospholipid by about 10–15 times. At 5 mM chloroform, in control rat liver microsomes, CYP2B1/2 was the major participant responsible for chloroform activation, while CYP2E1 and CYP2C11 were also significantly involved. Consistently, at this chloroform concentration, the effect of phenobarbital (CYP2B1/2 inducer) was maximal, producing very high levels of adducts. The reductive pathway was expressed at 5 mM CHCl₃ only and was not significantly increased by any of the inducers used. Moreover, it was not inhibited by metyrapone and 4-methylpyrazole or by anti CYP2C11 antibodies. Therefore, it may be concluded that, in the range of chloroform concentrations tested, those CYPs involved in CHCl₃ oxidative bioactivation do not participate in CHCl₃ reduction. Chloroform oxidative metabolism in PB-microsomes could achieve very high absolute rates, much higher than those in C-microsomes; in contrast, the metabolic rates in AC- and PYR-microsomes remained within the activity levels observable in C-microsomes at high chloroform concentration. Therefore, it can be argued that the CYP2B1/2-mediated induction of CHCl₃ activation is the basis for the effect of PB in potentiating chloroform hepatotoxicity. Moreover, processes other than CYP2E1-mediated metabolic induction may be more relevant in the ketones potentiation of chloroform-induced acute toxicity. © 1997 John Wiley & Sons, Inc. J Biochem Toxicol 11: 305–312, 1997.     

Modulation of porcine cytochrome P450 enzyme activities by surgical castration and immunocastration

The present study aimed to evaluate some cytochrome P450 metabolic enzyme activities in hepatic microsomes prepared from entire male pigs (uncastrated pigs), surgically castrated pigs and pigs immunized against gonadotropin-releasing hormone (immunocastrated pigs). The activities of the following enzymes were measured: ethoxyresorufin O-deethylase (EROD, CYP1A1/1A2), methoxyresorufin O-deethylase (MROD, CYP1A2), pentoxyresorufin O-depentylase (PROD, CYP2B), coumarin hydroxylase (COH, CYP2A) and p-nitrophenol hydroxylase (PNPH, CYP2A/2E1). The total cytochrome P450 contents were not affected by either surgical or immunocastration. Hepatic microsomal activities for EROD, PROD, COH and PNPH were lower in entire male pigs compared with surgically castrated and immunocastrated pigs (P < 0.05). Surgically and immunocastrated male pigs were similar with respect to EROD, MROD, PROD and COH activities (P > 0.05), whereas surgically castrated pigs exhibited lower PNPH activity compared with immunocastrated pigs (P = 0.029). The effect of different concentrations of testicular steroids - testosterone, 17β-estradiol, free estrone and androstenone - on enzyme activities was evaluated by in vitro microsomal study. Testosterone at the concentration of 8 pmol/ml inhibited EROD activities and estradiol-17β at the concentration of 1.8 pmol/ml inhibited PROD activities in hepatic microsomes from surgically castrated pigs. The highest concentration of androstenone (7520 pmol/ml) inhibited COH activities, whereas a 42-fold lower concentration of androstenone (180 pmol/ml) stimulated COH activities in surgically castrated pigs. Both free estrone (3.5 pmol/ml) and androstenone (55 pmol/ml) inhibited EROD activities in microsomes from entire male pigs. Stimulation of COH activities by the highest dose of free estrone (18 pmol/ml) was recorded in microsomes from entire male pigs. However, these effects of steroids were not concentration-dependent and the maximum extent did not exceed ±15% variation compared with the controls. There was no inhibition of PNPH activities in the hepatic microsomes from either entire or castrated pigs. In conclusion, we showed that EROD, PROD, COH and PNPH activities were lower in entire male pigs compared with those in surgically and immunocastrated pigs. Direct inhibition by the testicular steroids - testosterone, 17β-estradiol, free estrone and androstenone - was not the primary cause of the reduced enzyme activities.     

Identification of the cytochrome P450 isoenzymes involved in the metabolism of diazinon in the rat liver

The metabolism of diazinon, an organophosphorothionate pesticide, to diazoxon and pyrimidinol has been studied in incubations with hepatic microsomes from control Sprague–Dawley (SD) rats or SD rats treated with different P450‐specific inducers (phenobarbital, dexamethasone, β‐napthoflavone, and pyrazole). Results obtained indicate an involvement of CYP2C11, CYP3A2, and CYP2B1/2, whereas CYP2E1 and CYP1A1 do not contribute to the pesticide oxidative metabolism. Indeed, diazinon was metabolized by microsomes from control rats; among the inducers, phenobarbital and dexamethasone only increased the production of either metabolites, although to different extents. The production of the two metabolites is self‐limiting, due to P450 inactivation; therefore, the inhibition of CYP‐specific monooxygenase activities after diazinon preincubation has been used to selectively identify the competent CYPs in diazinon metabolism. Results indicate that, after diazinon preincubation, CYP3A2‐catalyzed reactions (2β‐ and 6β‐testosterone hydroxylation) are very efficiently inhibited; CYP2C11‐ and CYP2B1/2‐catalyzed reactions (2α‐ and 16β‐testosterone hydroxylation, respectively) are weakly inhibited, while CYP2E1‐, CYP2A1/2‐, and CYP1A1/2‐related activities were unaffected. Results obtained by using chemical inhibitors or antibodies selectively active against specific CYPs provide a direct evidence for the involvement of CYP2C11, CYP3A2, and CYP2B1/2, indicating that each of them contributed about 40–50% of the diazinon metabolism, in hepatic microsomes from untreated, phenobarbital‐, and dexamethasone‐treated rats, respectively. The higher diazoxon/pyrimidinol ratio observed after phenobarbital‐treatment together with the significantly more effective inhibition toward diazoxon production exerted by metyrapone in microsomes from phenobarbital‐treated rats supports the conclusion that CYP2B1/2 catalyze preferentially the production of diazoxon. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 13: 53–61, 1999     

Coexpression in yeast of Taxus cytochrome P450 reductase with cytochrome P450 oxygenases involved in Taxol biosynthesis

To maximize redox coupling efficiency with recombinant cytochrome P450 hydroxylases from yew (Taxus) species installed in yeast for the production of the anticancer drug Taxol, a cDNA encoding NADPH:cytochrome P450 reductase from T. cuspidata was isolated. This single-copy gene (2,154 bp encoding a protein of 717 amino acids) resembles more closely other reductases from gymnosperms (approximately 90% similarity) than those from angiosperms (<80% similarity). The recombinant reductase was characterized and compared to other reductases by heterologous expression in insect cells and was shown to support reconstituted taxoid 10beta-hydroxylase activity with an efficiency comparable to that of other plant-derived reductases. Coexpression in yeast of the reductase along with T. cuspidata taxoid 10beta-hydroxylase, which catalyzes an early step of taxoid biosynthesis, demonstrated significant enhancement of hydroxylase activity compared to that supported by the endogenous yeast reductase alone. Functional transgenic coupling of the Taxus reductase with a homologous cytochrome P450 taxoid hydroxylase represents an important initial step in reconstructing Taxol biosynthesis in a microbial host.     

Productive and non-productive complexes in cytochrome P450-containing systems

The equilibrium dissociation constants K_D, the complex association / dissociation rate constants (k _on /k _off) and lifetimes of the complexes of redox partners were measured for three cytochrome P450-containing monooxygenase systems (P450cam, P450scc, and P450 2B4) under hydroxylation conditions. The Q parameter representing the ratio of protein-protein complex lifetime (τ _lT ) to time required for a single hydroxylation cycle (τ_turnover) was introduced for estimation of productivity of complexes formed within the systems studied. The Q parameter was insignificantly changed upon transition from the oxidation to hydroxylation conditions. Lifetimes (τ _lT ) for the binary complexes formed within the P450cam and the P450scc systems obligatory requiring an intermediate electron transfer protein between the reductase and cytochrome P450 could not realize hydroxylation reactions for substrates with known τ_turnover and so they were non-productive while the binary complexes formed within the P450 2B4 system, not requiring such intermediate electron-transfer protein, appeared to be productive. Formation of ternary complexes was demonstrated under hydroxylation conditions in all three systems. Analysis of Q values led to the conclusion that the ternary complexes formed within the P450cam and the P450scc systems were productive. In the case of the P450 2B4 system, more than half (about 60%) ternary complexes were also found to be productive.     

Establishment of a yeast system that stably expresses human cytochrome P450 reductase: application for the study of drug metabolism of cytochrome P450s in vitro

Cytochrome P450s (CYPs) hold a balance in studying pharmacokinetics, toxico-kinetics, drug metabolism, and drug-drug interactions, which require association with cytochrome P450 reductase (CPR) to achieve optimal activity. A novel system of Saccharomyces cerevisiae useful for expression studies of mammalian microsomal CYPs was established. Human CPR (hCPR) was co-expressed with human CYP3A4 (hCYP3A4) in this system, and two expression plasmids pTpLC and pYeplac195-3A4 containing the cDNA of hCPR and hCYP3A4 were constructed, respectively. The two plasmids were applied first and controlled by phosphoglycerate kinase (PGK) promoter. S. cerevisiae BWG1-7alpha transformed with the expression plasmids produced the respective proteins in the expected molecular sizes reactive with both anti-hCYP3A4 immunoglobulin (Ig) and anti-hCPR Ig. The activity of hCPR in yeast BWG-CPR was 443.2 nmol reduced cytochrome c/min/mg, which was about three times the CPR activity of the microsome prepared from the parental yeast. The protein amount of hCYP3A4 in BWG-CPR/3A4 was 35.53 pmol/mg, and the 6beta-hydroxylation testosterone formation activity of hCYP3A4 expressed was 7.5 nmol/min/nmol CYP, 30 times higher than the activity of hCYP3A4 expressed in the parental yeast, and almost two times the activity of hCYP3A4 from homologous human liver microsome. Meanwhile, BWG-CPR/3A4 retained 100 generations under nonselective culture conditions, indicating this yeast was a mitotically stable transformant. BWG-CPR was further tested daily by the PCR amplification of hCPR of yeast genome, Western blot analysis, and the activity assay of hCPR of yeast microsome. This special expression host for CYPs was validated to be stable and efficient for the expression of CYPs, applying as an effective selection model for the drug metabolism in vitro.     

Cytochromes P450 and experimental models of drug metabolism

For the development of new drugs, evaluation of drug-drug interactions with already known compounds, as well as for better understanding of metabolism pathways of various toxicants and pollutants, we studied the drug metabolism mediated by cytochromes P450. The experimental approach is based on animal drug-metabolising systems. From the ethical as well as rational reasons, the selection of an appropriate system is crucial. Here, it is necessary to decide on the basis of expected CYP system involved. For CYP1A-mediated pathways, all the commonly used experimental models are appropriate except probably the dog. On the contrary, the dog seems to be suitable for modelling of processes depending on the CYP2D. With CYP2C, which is possibly the most large and complicated subfamily, the systems based on monkey ( Maccacus rhesus ) may be a good representative. The CYP3A seems to be well modelled by pig or minipig CYP3A29. Detailed studies on activities with individual isolated CYP forms are needed to understand in full all aspects of inter-species differences and variations.     

Oxygen radical formation during cytochrome P450-catalyzed cyclosporine metabolism in rat and human liver microsomes at varying hydrogen ion concentrations

The role of pH in uncoupling the electron-flux between oxidoreductase and cytochrome P450 (P450) or P450 and cyclosporine (CyA) and resulting in the generation of oxygen radicals was investigatedin vitro in rat and human liver microsomal preparations. Since the electron-flux from NADPH to cytochrome c via oxidoreductase showed a fairly constant reduction activity from pH 7.0–9.5, the generation of oxygen radicals at the level of P450-Cyclosporine (instead of oxidoreductase-P450) was investigated. The effects of increasing pH on oxygen radical formation was measured by the thiobarbituric acid assay (TBA) and the adrenochrome reaction. The trends in oxygen radical production were correlated with benzphetamine metabolism (production of formaldehyde) and CyA metabolism (analyzed by high performance liquid chromatography). The TBA assay showed increased MDA-detected lipid peroxidation (unrelated to autooxidation) at pH<8.0 and pH>8.0 (rat and human, respectively) while the adrenochrome reaction showed decreased oxygen radical production. When these results were compared to benzphetamine (a substrate of P450 2B and 3A) metabolism and CyA (a substrate of P450 3A) metabolism, increased metabolism followed the pH-dependent trend of MDA-detected lipid peroxidation. Benzphetamine metabolism with formaldehyde production and depletion of parent compound during CyA metabolism were increased at pH<8.0 in the rat samples and at pH>8.0 in the human samples. This parallel relation suggests that the increased metabolism of CyA at lower pH in rats and higher pH in humans may be the result of favorable interactions of P450 with Cyclosporine that also result in increased oxygen radical-related lipid peroxidation.Abbreviations CCl₄ carbon tetrachloride - CyA cyclosporin A - EDTA ethylenediaminetetraacetic acid - HPLC high performance liquid chromatography - MDA malondialdehyde - MFO mixed function oxidase - MICROS microsomes - NADPH nicotinamide adenine dinucleotide phosphate - TBA thiobarbituric acid This work was supported by Grant No. CA-53191 from the National Cancer Institute DHHW     

Purification of rat liver microsomal cytochrome P-450b without the use of nonionic detergent

Sodium cholate, Emulgen 911, and (3-[(-cholamidopropyl)-dimethyl- ammonio]-1-propanesulfonate) (CHAPS) were selected to examine the effects of ionic, nonionic, and zwitterionic detergents on testosterone hydroxylation catalyzed by four purified isozymes of rat liver microsomal cytochrome P-450, namely P-450a, P-450b, P-450c, and P-450h, in reconstituted systems containing optimal amounts of dilauroylphosphatidylcholine and saturating amounts of NADPH- cytochrome P-450 reductase (reductase). The major phenobarbital-inducible form of rat liver microsomal cytochrome P-450, designated P-450b, was extremely sensitive to the inhibitory effects of Emulgen 911, which is used in several procedures to purify this and other forms of cytochrome P-450. In contrast, sodium cholate and CHAPS had little effect on the catalytic activity of cytochrome P-450b, even at ten times the concentration of Emulgen 911 effecting 50% inhibition (IC-50). By substituting the zwitterionic detergent CHAPS for Emulgen 911, we purified cytochrome P-450b without the use of nonionic detergent. The protein is designated cytochrome P-450b^* to distinguish it from cytochrome P-450b purified with the use of Emulgen 911. NADPH-cytochrome P-450 reductase was also purified both with and without the use of nonionic detergent. The absolute spectra of cytochrome P-450b and P-450b^* were indistinguishable, as were the carbon monoxide (CO)- and metyrapone-difference spectra of the dithionite-reduced hemoproteins. When reconstituted with NADPH-cytochrome P-450 reductase and dilauroylphosphatidylcholine, cytochromes P-450b and P-450b^* catalyzed the N-demethylation of benzphetamine and aminopyrine, the 4-hydroxylation of aniline, the O-dealkylation of 7-ethoxycoumarin, the 3-hydroxylation of hexobarbital, and the 6-hydroxylation of zoxazolamine. Both hemo-proteins catalyzed the 16α- and 16β-hydroxylation of testosterone, as well as the 17-oxidation of testosterone to androstenedione. Both hemoproteins were poor catalysts of erythromycin demethylation and benzo[a]pyrene 3-/9-hydroxylation. The rate of biotransformation catalyzed by cytochrome P-450b^* was up to 50% greater than the rate catalyzed by cytochrome P-450b when reconstituted with either reductase or reductase^*. The activity of cytochrome P-450b and P-450b^* increased up to 50% when reconstituted with reductase^* instead of reductase. In addition to establishing the feasibility of purifying an isozyme of rat liver microsomal cytochrome P-450 without the use of nonionic detergent, these results indicate that the catalytic activity of cytochrome P-450 is not unduly compromised by residual contamination with the nonionic detergent Emulgen 911.     

In vitro metabolism of fipronil by human and rat cytochrome P450 and its interactions with testosterone and diazepam

Fipronil (5-amino-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-[(trifluoromethyl)sulfinyl]-1H-pyrazole-3-carbonitrile) is a highly active, broad spectrum insecticide from the phenyl pyrazole family, which targets the gamma-amino butyric acid (GABA) receptor. Although fipronil is presently widely used as an insecticide and acaricide, little information is available with respect to its metabolic fate and disposition in mammals. This study was designed to investigate the in vitro human metabolism of fipronil and to examine possible metabolic interactions that fipronil may have with other substrates. Fipronil was incubated with human liver microsomes (HLM) and several recombinant cytochrome P450 (CYP) isoforms obtained from BD Biosciences. HPLC was used for metabolite identification and quantification. Fipronil sulfone was the predominant metabolite via CYP oxidation. The K(m) and V(max) values for human liver microsomes are 27.2 microM and 0.11 nmol/mg proteinmin, respectively; for rat liver microsomes (RLM) the K(m) and V(max) are 19.9 microM and 0.39 nmol/mg proteinmin, respectively. CYP3A4 is the major isoform responsible for fipronil oxidation in humans while CYP2C19 is considerably less active. Other human CYP isoforms have minimal or no activity toward fipronil. Co-expression of cytochrome b(5) (b(5)) is essential for CYP3A4 to manifest high activity toward fipronil. Ketoconazole, a specific inhibitor of CYP3A4, inhibits 78% of the HLM activity toward fipronil at a concentration of 2 microM. Oxidative activity toward fipronil in 19 single-donor HLMs correlated well with their ability to oxidize testosterone. The interactions of fipronil and other CYP3A4 substrates, such as testosterone and diazepam, were also investigated. Fipronil metabolism was activated by testosterone in HLM but not in CYP3A4 Supersomes. Testosterone 6beta-hydroxylation in HLM was inhibited by fipronil. Fipronil inhibited diazepam demethylation but had little effect on diazepam hydroxylation. The results suggest that fipronil has the potential to interact with a wide range of xenobiotics or endogenous chemicals that are CYP3A4 substrates and that fipronil may be a useful substrate for the characterization of CYP3A4 in HLM.     

Kinetics of bromodichloromethane metabolism by cytochrome P450 isoenzymes in human liver microsomes

The kinetic constants for the metabolism of bromodichloromethane (BDCM) by three cytochrome P450 (CYP) isoenzymes have been measured in human liver microsomes. The three CYP isoenzymes, CYP2E1, CYP1A2 and CYP3A4, have been identified previously as important in the metabolism of this compound. To measure the constants for each isoenzyme, enzyme-specific inhibitory antibodies were used to block the activities for two of the three isoenzymes. CYP2E1 was found to have the lowest K(m), 2.9 microM, and the highest catalytic activity, k(cat). The K(m) for the other isoenzymes, CYP1A2 and CYP3A4, were about 60 microM with lower values of k(cat). Apparent kinetic constants obtained from two microsomal samples that were not inhibited were consistent with these results. In addition, 11 human microsome samples characterized for 10 CYP activities were correlated with the metabolism of 9.7 microM BDCM by each sample; statistical analysis showed a correlation with CYP2E1 activity only. This result is consistent with the finding that CYP2E1 is the only isoenzyme with a K(m) lower than the BDCM concentration used. The kinetic constants obtained from the inhibited microsomes were compared to similar results from recombinant human isoenzyme preparations containing only one CYP isoenzyme. The results for CYP2E1 were very similar, while the results for CYP1A2 were somewhat less similar and there was a substantial divergence for CYP3A4 in the two systems. Possible reasons for these differences are differing levels of CYP reductase and/or differing makeup of the membrane lipid environment for the CYPs. Because of the low levels of BDCM exposure from drinking water, it appears likely that CYP2E1 will dominate hepatic CYP-mediated BDCM metabolism in humans.     

昆虫细胞色素P450研究的一些新进展

报道了有关细胞色素P45 0研究的一些新发现。果蝇和冈比亚按蚊基因组测序的完成 ,使人类对昆虫P45 0的多样性有一完整的概念 ,已查明果蝇和冈比亚按蚊基因组中分别含有 90种和 1 1 1种P45 0基因。P45 0介导的果蝇对DDT的抗性被证明是Cyp6g1基因超量表达的结果。昆虫可以窃听植物分子信号 (水杨酸、茉莉酮酸 ) ,通过P45 0的诱导机制增强自身对植物防御物质的反防御能力。从分子水平上鉴定了 2个参与蜕皮素合成的线粒体P45 0基因。细胞色素P45 0在昆虫信息素降解中的作用得到鉴定。     

The cytochrome P450 genesis locus: the origin and evolution of animal cytochrome P450s

The neighbourhoods of cytochrome P450 (CYP) genes in deuterostome genomes, as well as those of the cnidarians Nematostella vectensis and Acropora digitifera and the placozoan Trichoplax adhaerens were examined to find clues concerning the evolution of CYP genes in animals. CYP genes created by the 2R whole genome duplications in chordates have been identified. Both microsynteny and macrosynteny were used to identify genes that coexisted near CYP genes in the animal ancestor. We show that all 11 CYP clans began in a common gene environment. The evidence implies the existence of a single locus, which we term the ‘cytochrome P450 genesis locus’, where one progenitor CYP gene duplicated to create a tandem set of genes that were precursors of the 11 animal CYP clans: CYP Clans 2, 3, 4, 7, 19, 20, 26, 46, 51, 74 and mitochondrial. These early CYP genes existed side by side before the origin of cnidarians, possibly with a few additional genes interspersed. The Hox gene cluster, WNT genes, an NK gene cluster and at least one ARF gene were close neighbours to this original CYP locus. According to this evolutionary scenario, the CYP74 clan originated from animals and not from land plants nor from a common ancestor of plants and animals. The CYP7 and CYP19 families that are chordate-specific belong to CYP clans that seem to have originated in the CYP genesis locus as well, even though this requires many gene losses to explain their current distribution. The approach to uncovering the CYP genesis locus overcomes confounding effects because of gene conversion, sequence divergence, gene birth and death, and opens the way to understanding the biodiversity of CYP genes, families and subfamilies, which in animals has been obscured by more than 600 Myr of evolution.     

Chromate metabolism in liver microsomes

The carcinogenicity and mutagenicity of various chromium compounds have been found to be markedly dependent on the oxidation state of the metal. The carcinogen chromate was reduced to chromium(III) by rat liver microsomes in vitro. Metabolism of chromate by microsomal enzymes occurred only in the presence of either NADPH or NADH as cofactor. The chromium(III) generated upon metabolism formed a complex with the NADP⁺ cofactor. Significant binding of chromium to DNA occurred only when chromate was incubated in the presence of microsomes and NADPH. Specific inhibitors of the mixed function oxidase enzymes, 2′-AMP, metyrapone, and carbon monoxide, inhibited the rate of reduction of chromate by microsomes and NADPH. The possible relationship of metabolism of chromate and its interaction with nucleic acids to its carcinogenicity and mutagenicity is discussed.     

Cytochrome b5 involvement in cytochrome P450 monooxygenase activities in house fly microsomes

The involvement of cytochrome b₅ in different cytochrome P450 monooxygenase and palmitoyl CoA desaturase activities in microsomes from insecticide-resistant (LPR) house flies was determined using a specific polyclonal antiserum developed against house fly cytochrome b₅. Anti-b₅ antiserum inhibited the reduction of cytochrome b₅ by NADH-cytochrome b₅ reductase. The antiserum also inhibited palmitoyl CoA desaturase, methoxycoumarin-O-demethylase (MCOD), ethoxycoumarin-O-deethylase (ECOD), and benzo[a]pyrene hydroxylase (aromatic hydrocarbon hydroxylase, AHH) activities. However, methoxyresorufin-O-demethylase (MROD) and ethoxyresorufin-O-deethy-lase (EROD) activities were not affected by this antiserum. These results demonstrate that cytochrome b₅ is involved in fatty acyl CoA desaturase activities and in certain cytochrome P450 monooxygenase activities (i.e., MCOD, ECOD, and AHH) in LPR house fly microsomes. Other cytochrome P450 monooxygenase activities (i.e., MROD and EROD) may not require cytochrome b₅. The results suggest that cytochrome b₅ involvement with cytochrome P450 monooxygenase activities is dependent upon the cytochrome P450 isoform involved. © 1994 Wiley-Liss, Inc.     

细胞色素P450与植物的次生代谢

细胞色素Ｐ４５０是动植物及微生物体内的一类重要的混合功能的血红素氧化还原酶类，是一个基因超家族。它可催化多种化学反应，在防御生物免受外界侵害方面有重要作用。植物Ｐ４５０广泛参与次生代谢产生有植保素功能的物质和对除草剂、杀虫剂等外毒素的生物转化解毒代谢。许多植物Ｐ４５０已被分离纯化，更多的植物Ｐ４５０的基因被克隆和外源表达。对植物Ｐ４５０的表达调控也取得了一些进展，但在抗虫和抗除草剂的农作物基因工程方面尚在起步阶段。     

Anatomical distribution of NADPH-cytochrome P450 reductase and cytochrome P4502D forms in rat brain: Effects of xenobiotics and sex steroids

Since the brain is not a homogeneous organ, but one dependent upon the well orchestrated interaction of numerous parts, pathology in one nucleus may have a large impact upon its overall function. Hence, the anatomical distribution of the P450 monoxygenase system in brain, as well as the regulation of its expression, is important in elucidating its function in that organ. In order to study these issues, female rats-both ovariectomized and not-were treated with a number of xenobiotic compounds and sex steroids. The brains from these animals were dissected into 8 discrete regions and the presence and relative level of message for P4502D and P450 reductase determined using polymerase chain reaction. Results of this investigation indicate the presence of mRNA for reductase and P4502D isoforms throughout the rat brain. In addition, quantitative PCR was utilized to demonstrate the effects of xenobiotics (phenobarbital, β-naphthoflavone, imipramine) and sex hormones (testosterone, estrogen) on the levels of these messages in the female rat brain. Significant induction of message for P4502D forms was noted with testosterone in the absence of estrogen. The level of mRNA for reductase was not significantly influenced by any of the treatments, however. These results raise the issue of a sexual dimorphism in the rat regarding P4502D expression in brain.     

Nadph-initiated cytochrome P450-dependent free iron-independent microsomal lipid peroxidation: Specific prevention by ascorbic acid

In this paper we demonstrate that ascorbic acid specifically prevents NADPH-initiated cytochrome P450 (P450)-mediated microsomal lipid peroxidation in the absence of free iron. Lipid peroxidation has been evidenced by the formations of conjugated dienes, lipid hydroperoxide and malondialdehyde. Other scavengers of reactive oxygen species including superoxide dismutase, catalase, glutathione, -tocopherol, uric acid, thiourea, mannitol, histidine, -carotene and probucol are ineffective to prevent the NADPH-initiated P450-mediated free iron-independent microsomal lipid peroxidation. Using a reconstituted system comprised of purified NADPH-P450 reductase, P450 and isolated microsomal lipid or pure L--phosphatidylcholine diarachidoyl, a mechanism has been proposed for the iron-independent microsomal lipid peroxidation and its prevention by ascorbic acid. It is proposed that the perferryl moiety P450 Fe³⁺. O₂ initiates lipid peroxidation by abstracting methylene hydrogen from polyunsaturated lipid to form lipid radical, which then combines with oxygen to produce the chain propagating peroxyl radical for subsequent formation of lipid peroxides. Apparently, ascorbic acid prevents initiation of lipid peroxidation by interacting with P450 Fe³⁺. O₂. (Mol Cell Biochem 166: 35-44, 1997)     

In vitro metabolism and inductive or inhibitive effect of DL111 on rat cytochrome P4501A enzyme

In vitro metabolism and the inductive or inhibitive effect of DL111, a non-hormonal early pregnancy-terminating agent, toward cytochrome P450 (CYP) enzymes in rat liver microsomes were studied. In vitro metabolism of DL111 was performed in different rat liver microsomes (pretreated with phenobarbital (PB), dexamethasone (Dex), beta-naphthoflavone (BNF), DL111, respectively) and the catalytic abilities of these microsomes for DL111 were compared with control group. DL111 was well metabolized in microsomes pretreated with beta-naphthoflavone and itself. The K(m) and V(max) was 41.76 +/- 3.26 microM and 15.34 +/- 1.03 nM min(-1) mg(-1) protein for beta-naphthoflavone group, 48.17 +/- 6.06 microM and 17.54 +/- 1.79 nM min(-1)mg(-1) protein for DL111 group, 77.81 +/- 4.73 microM and 3.087 +/- 0.202 nM min(-1)mg(-1) protein for control group, respectively. The rats were pretreated intraperitoneally with the same daily dose of DL111 for different days. The DL111-pretreated microsomal enzymatic activities were evaluated by measuring the metabolic abilities for specific substrates of various enzymes. The results showed that DL111 had the same inductive function as beta-naphthoflavone (the specific inducer of CYP1A) toward rat liver microsomes. The inhibitive effect of DL111 on CYP1A was investigated by coincubating DL111 with the specific substrates of CYP1A-ethoxyresorufin or phenacetin in the microsome induced by beta-naphthoflavone, and the inhibitive level was compared with fluvoxamine (Flu), the specific inhibitor of CYP1A. DL111 inhibited significantly the metabolism of phenacetin and ethoxyresorufin with the inhibition constant (K(i)) 6.836 +/- 0.10 and 1.222 +/- 0.230 microM, respectively and its inhibition potential on CYP1A was higher than fluvoxamine.     

原核生物细胞色素P450酶系及其应用

细胞色素P450酶系广泛分布于各种生物中,它们通常由一组基因超家族编码并含有血红素,能够催化一系列化学反应,具有多种生物学功能。特别是原核生物P450酶在催化内源性和外源性化合物的反应中具有重要的工业生产应用价值,成为近年来P450酶系研究的热点。本文对近年来原核生物P450酶系的重组表达和生物催化领域的研究进展进行综述。