CYP79B1 from Sinapis alba converts tryptophan to indole-3-acetaldoxime

The cytochrome P450 CYP79B1 from Sinapis alba has been heterologously expressed in Escherichia coli and shown to catalyze the conversion of tryptophan to indole-3-acetaldoxime. Three expression constructs were made, one expressing the native protein and two expressing proteins with different N-terminal modifications. The native construct gave the highest yield as estimated by enzymatic activity per liter of culture. Spheroplasts of E. coli expressing CYP79B1 were reconstituted with the Arabidopsis thaliana NADPH:cytochrome P450 reductase ATR1 heterologously expressed in E. coli to obtain enzymatic activity. This indicates that the E. coli electron-donating system, flavodoxin/flavodoxin reductase, does not support CYP79B1 activity. Recombinant CYP79B1 has a K(m) for tryptophan of 29+/-2 microM and a V(max) of 36.5+/-0.7nmolh(-1)(mlculture)(-1). The identity at the amino acid level of CYP79B1 is, respectively, 93 and 84% to CYP79B2 and CYP79B3 from A. thaliana, and 96% to CYP79B5 (Accession No. AF453287) from Brassica napus. The CYP79B subfamily of cytochromes P450 is likely to constitute a group of orthologous genes in the biosynthesis of indole glucosinolates.     

New insight into the biosynthesis and regulation of indole compounds in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

In spite of their silent and sessile life, plants are dynamic organisms that have developed advanced defence strategies in their adaptation to the pressure of herbivores and pathogens. Natural plant products play an important role as chemical weapons in this warfare. Characteristic of cruciferous plants is the synthesis of nitrogen- and sulphur-rich compounds, such as glucosinolates (Mikkelsen et al. 2002) and indole alkaloids (Pedras et al. 2000). Glucosinolates are believed to be largely non-toxic, but upon tissue disruption, they are hydrolyzed by endogenous -thioglucosidases (myrosinases) (Rask et al. 2000) to primarily isothiocyanates and nitriles, which have many biological activities. These include not only important roles as repellents against herbivorous insects and microorganisms, but also as volatile attraction of specialized insects (Wittstock and Halkier 2002). For humans, these compounds serve as cancer-preventive agents, biopesticides, and flavor compounds (Talalay and Fahey 2001). Indole alkaloids are phytoalexins and production of specific alkaloids is usually limited to only a few species. Cruciferous plants include the model plant Arabidopsis, which produces the indole alkaloid camalexin. This review will focus on the central role of indole-3-acetaldoxime (IAOx) in the biosynthesis of indole glucosinolates, camalexin, and the phytohormone IAA.     

Conversion of indole-3-acetaldoxime to indole-3-acetonitrile by plasma membranes from Chinese cabbage

The in vitro conversion of [¹⁴C]-indole-3-acetaldoxime (IAOX) to [¹⁴C]-indole-3-acetonitrile (IAN) by plasma membranes enriched by aqueous two-phase partitioning of Chinese cabbage ( Brassica campestris L. ssp. pekinensis cv. Granat) has been studied. The reaction product was identified by thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC). A reducing agent, e.g. ascorbic acid, was needed as cofactor for the formation of IAN from IAOX. Reduction equivalents and metal ions were not involved in the conversion of IAOX to IAN. The pH optimum for the reaction was at 6.0 and the apparent K_m for IAOX was 6.3 μ M . The enzyme was not inhibited by thiol reagents. The pI of the enzyme was determined to be 7.1 by isoelectric focusing (IEF). Gel permeation chromatography showed one major activity peak of 40 kDa. The reaction is considered as part of a channeling process leading from tryptophan to IAN with IAOX as an intermediate. This process is probably regulated by the indole derivatives IAOX and IAN.     

CYP79F1 and CYP79F2 have distinct functions in the biosynthesis of aliphatic glucosinolates in Arabidopsis

Cytochromes P450 of the CYP79 family catalyze the conversion of amino acids to oximes in the biosynthesis of glucosinolates, a group of natural plant products known to be involved in plant defense and as a source of flavor compounds, cancer-preventing agents and bioherbicides. We report a detailed biochemical analysis of the substrate specificity and kinetics of CYP79F1 and CYP79F2, two cytochromes P450 involved in the biosynthesis of aliphatic glucosinolates in Arabidopsis thaliana. Using recombinant CYP79F1 and CYP79F2 expressed in Escherichia coli and Saccharomyces cerevisiae, respectively, we show that CYP79F1 metabolizes mono- to hexahomomethionine, resulting in both short- and long-chain aliphatic glucosinolates. In contrast, CYP79F2 exclusively metabolizes long-chain elongated penta- and hexahomomethionines. CYP79F1 and CYP79F2 are spatially and developmentally regulated, with different gene expression patterns. CYP79F2 is highly expressed in hypocotyl and roots, whereas CYP79F1 is strongly expressed in cotyledons, rosette leaves, stems, and siliques. A transposon-tagged CYP79F1 knockout mutant completely lacks short-chain aliphatic glucosinolates, but has an increased level of long-chain aliphatic glucosinolates, especially in leaves and seeds. The level of long-chain aliphatic glucosinolates in a transposon-tagged CYP79F2 knockout mutant is substantially reduced, whereas the level of short-chain aliphatic glucosinolates is not affected. Biochemical characterization of CYP79F1 and CYP79F2, and gene expression analysis, combined with glucosinolate profiling of knockout mutants demonstrate the functional role of these enzymes. This provides valuable insights into the metabolic network leading to the biosynthesis of aliphatic glucosinolates, and into metabolic engineering of altered aliphatic glucosinolate profiles to improve nutritional value and pest resistance.     

Comparison of genomic and cDNA sequences of guinea pig CYP2B18 and rat CYP2B2: absence of a phenobarbital-responsive enhancer module in the upstream region of the CYP2B18 gene

Potential mechanisms were investigated whereby CYP2B18, a cytochrome P450 gene exhibiting high constitutive expression but only low levels of phenobarbital-inducibility in the guinea pig liver, may be differentially regulated versus the highly inducible rat CYP2B2 gene. To comparatively assess potential regulatory sequences associated with CYP2B18, a guinea pig genomic library was screened enabling isolation of the CYP2B18 gene. The genomic screening process resulted in the identification of at least four closely-related CYP2B18 genes, designated here as CYP2B18A-D. Of these isolates, CYP2B18A exhibited sequence identical to that of the CYP2B18 cDNA. Further, the deduced amino acid sequence of the CYP2B18 cDNA was identical to that of N-terminal and internally-derived peptide sequences obtained in this investigation from CYP2B18 protein isolated from guinea pig liver. Genomic structural sequences were derived for CYP2B18A, together with the respective 5'-upstream and intronic regions of the gene. Comparison of the CYP2B18A and CYP2B2 gene sequences revealed the lack of repetitive LINE gene sequences in CYP2B18A, putative silencing elements that effect neighboring genes, although these sequences were present in both 5'-upstream and 3'-downstream regions of CYP2B2. We determined that the phenobarbital-responsive enhancer module was absent from the 5'-upstream region as well as the intronic regions of CYP2B18A gene. We hypothesize that the compromised phenobarbital inducibility of CYP2B18A stems from its lack of a functional phenobarbital responsive enhancer module.     

Analysis of Substrate Specificity of Pig CYP2B22 and CYP2C49 towards Herbicides by Transgenic Rice Plants

We introduced two novel types of pig (Sus scrofa) cytochrome P450, CYP2B22 and CYP2C49, into rice plants (Oryza sativa L. cv. ‘Nipponbare’) to produce herbicide-tolerant plants and to confirm the metabolic activities of the cytochrome P450 species. In germination tests, both types of transgenic plants showed tolerance to various herbicides with different modes of action. CYP2B22 rice plants showed tolerance towards 12 herbicides including chlortoluron (100 μM), amiprofos-methyl (2.5 μM), pendimethalin (10 μM), metolachlor (2.5 μM), and esprocarb (20 μM). CYP2C49 rice plants showed tolerance towards 13 herbicides, including chlortoluron (100 μM), norflurazon (0.5 μM), amiprofos-methyl (2.5 μM), alachlor (0.8 μM), and isoxaben (1 μM). The herbicide tolerance was considered to reflect the substrate specificity of the introduced P450 species. We used ¹⁴C-labeled metolachlor and norflurazon to confirm the P450 activity in the transgenic rice plants. The herbicides were metabolized more quickly in the transgenic rice plants than in the nontransgenic rice plants. Therefore, CYP2B22 and CYP2C49 rice plants became more tolerant to various herbicides than nontransgenic control plants because of accelerated metabolism of the herbicides by the introduced P450 species. Assuming that public and commercial acceptance is forthcoming, these transgenic rice plants may become useful tools for the breeding of herbicide-tolerant crops.     

Cytochrome P450 CYP79B2 from Arabidopsis catalyzes the conversion of tryptophan to indole-3-acetaldoxime, a precursor of indole glucosinolates and indole-3-acetic acid

Glucosinolates are natural plant products known as flavor compounds, cancer-preventing agents, and biopesticides. We report cloning and characterization of the cytochrome P450 CYP79B2 from Arabidopsis. Heterologous expression of CYP79B2 in Escherichia coli shows that CYP79B2 catalyzes the conversion of tryptophan to indole-3-acetaldoxime. Recombinant CYP79B2 has a K(m) of 21 microm and a V(max) of 7.78 nmol/h/ml culture. Inhibitor studies show that CYP79B2 is different from a previously described enzyme activity that converts tryptophan to indole-3-acetaldoxime (Ludwig-Müller, J. , and Hilgenberg, W. (1990) Phytochemistry, 29, 1397-1400). CYP79B2 is wound-inducible and expressed in leaves, stem, flowers, and roots, with the highest expression in roots. Arabidopsis overexpressing CYP79B2 has increased levels of indole glucosinolates, which strongly indicates that CYP79B2 is involved in indole glucosinolate biosynthesis. Our data show that oxime production by CYP79s is not restricted to those amino acids that are precursors for cyanogenic glucosides. Our data are consistent with the hypothesis that indole glucosinolates have evolved from cyanogenesis. Indole-3-acetaldoxime is a precursor of the plant hormone indole-3-acetic acid, which suggests that CYP79B2 might function in biosynthesis of indole-3-acetic acid. Identification of CYP79B2 provides an important tool for modification of the indole glucosinolate content to improve nutritional value and pest resistance.     

Improvement in the expression of CYP2B6 by co-expression with molecular chaperones GroES/EL in Escherichia coli

Improvement of CYP2B6 expression was examined by co-expression with molecular chaperones GroES/EL. Although a CO-reduced difference spectrum was not detected in Escherichia coli transformed only by the CYP2B6-expressing vector, co-expression of GroES/EL resulted in high-level expression which reached over 2000 nmol P450/L. CYP2B6 was purified from the E. coli membrane with a high yield. Purified CYP2B6 showed 7-ethoxy-4-trifluoromethylcoumarin O-deethylase activity in a reconstitution system. This expression system would be useful for the production of large amounts of active CYP2B6 and for the detailed analysis of the enzyme.     

Arabidopsis mutants in the C-S lyase of glucosinolate biosynthesis establish a critical role for indole-3-acetaldoxime in auxin homeostasis

We report characterization of SUPERROOT1 (SUR1) as the C-S lyase in glucosinolate biosynthesis. This is evidenced by selective metabolite profiling of sur1, which is completely devoid of aliphatic and indole glucosinolates. Furthermore, following in vivo feeding with radiolabeled p-hydroxyphenylacetaldoxime to the sur1 mutant, the corresponding C-S lyase substrate accumulated. C-S lyase activity of recombinant SUR1 heterologously expressed in Escherichia coli was demonstrated using the C-S lyase substrate djenkolic acid. The abolishment of glucosinolates in sur1 indicates that the SUR1 function is not redundant and thus SUR1 constitutes a single gene family. This suggests that the "high-auxin" phenotype of sur1 is caused by accumulation of endogenous C-S lyase substrates as well as aldoximes, including indole-3-acetaldoxime (IAOx) that is channeled into the main auxin indole-3-acetic acid (IAA). Thereby, the cause of the "high-auxin" phenotype of sur1 mutant resembles that of two other "high-auxin" mutants, superroot2 (sur2) and yucca1. Our findings provide important insight to the critical role IAOx plays in auxin homeostasis as a key branching point between primary and secondary metabolism, and define a framework for further dissection of auxin biosynthesis.     

The Arabidopsis UDP‐glycosyltransferases UGT79B2 and UGT79B3, contribute to cold,salt and drought stress tolerance via modulating anthocyanin accumulation

The plant family 1 UDP‐glycosyltransferases (UGTs) are the biggest GT family in plants, which are responsible for transferring sugar moieties onto a variety of small molecules, and control many metabolic processes; however, their physiological significance in planta is largely unknown. Here, we revealed that two Arabidopsis glycosyltransferase genes, UGT79B2 and UGT79B3, could be strongly induced by various abiotic stresses, including cold, salt and drought stresses. Overexpression of UGT79B2/B3 significantly enhanced plant tolerance to low temperatures as well as drought and salt stresses, whereas the ugt79b2/b3 double mutants generated by RNAi (RNA interference) and CRISPR‐Cas9 strategies were more susceptible to adverse conditions. Interestingly, the expression of UGT79B2 and UGT79B3 is directly controlled by CBF1 (CRT/DRE‐binding factor 1, also named DREB1B) in response to low temperatures. Furthermore, we identified the enzyme activities of UGT79B2/B3 in adding UDP‐rhamnose to cyanidin and cyanidin 3‐O‐glucoside. Ectopic expression of UGT79B2/B3 significantly increased the anthocyanin accumulation, and enhanced the antioxidant activity in coping with abiotic stresses, whereas the ugt79b2/b3 double mutants showed reduced anthocyanin levels. When overexpressing UGT79B2/B3 in tt18 (transparent testa 18), a mutant that cannot synthesize anthocyanins, both genes fail to improve plant adaptation to stress. Taken together, we demonstrate that UGT79B2 and UGT79B3, identified as anthocyanin rhamnosyltransferases, are regulated by CBF1 and confer abiotic stress tolerance via modulating anthocyanin accumulation.     

Regulation of phenobarbital-induction of CYP2B and CYP3A genes in rat cultured hepatocytes: involvement of several serine/threonine protein kinases and phosphatases

We investigated the involvement of diverse protein kinases and phosphatases in the transduction pathways elicited by phenobarbital (PB), a well-known inducer of some hepatic cytochromes P450 (CYP). Different inhibitors or activators of protein kinases or phosphatases were assessed for their ability to modulate PB-induction of CYP2B and CYP3A mRNA expression. Rat hepatocytes in primary culture were treated with the test compounds one hour prior to, and then continuously, in the absence or presence of 1 mmol/L PB for 24 h. By northern blot analysis of CYP2B1/2 and 3A1/2 gene expression, we first confirmed the negative role of the adenosine 3′:5′ cyclic monophosphate (cAMP)/protein kinase A pathway and the positive role of some serine/threonine protein phosphatases in the mechanism of PB-induction. The present data further suggested that Ca²⁺/calmodulin-dependent protein kinases II (independently of Ca²⁺) and extracellular signal-regulated kinases 1/2 (ERK1/2) might function respectively as positive and negative regulator in the PB-induction of CYP2B and CYP3A. In contrast, protein kinases C and phosphatidylinositol-3-kinase did not appear to be involved, while the role of tyrosine kinases remained unclear. We conclude that a complex network of phosphorylation/dephosphorylation events might be crucial for PB-induction of rat CYP2B and CYP3A. This revised version was published online in July 2006 with corrections to the Cover Date.     

Chemical defenses of crucifers: elicitation and metabolism of phytoalexins and indole-3-acetonitrile in brown mustard and turnip

The metabolism of the cruciferous phytoalexins brassinin and cyclobrassinin, and the related compounds indole-3-carboxaldehyde, glucobrassicin, and indole-3-acetaldoxime was investigated in various plant tissues of Brassica juncea and B. rapa. Metabolic studies with brassinin showed that stems of B. juncea metabolized radiolabeled brassinin to indole-3-acetic acid, via indole-3-carboxaldehyde, a detoxification pathway similar to that followed by the "blackleg" fungus (Phoma lingam/Leptosphaeria maculans). In addition, it was established that tetradeuterated brassinin was incorporated into the phytoalexin brassilexin in B. juncea and B. rapa. On the other hand, the tetradeuterated indole glucosinolate glucobrassicin was not incorporated into brassinin, although the chemical structures of brassinins and indole glucosinolates suggest an interconnected biogenesis. Importantly, tetradeuterated indole-3-acetaldoxime was an efficient precursor of phytoalexins brassinin, brassilexin, and spirobrassinin. Elicitation experiments in tissues of Brassica juncea and B. rapa showed that indole-3-acetonitrile was an inducible metabolite produced in leaves and stems of B. juncea but not in B. rapa. Indole-3-acetonitrile displayed antifungal activity similar to that of brassilexin, was metabolized by the blackleg fungus at slower rates than brassinin, cyclobrassinin, or brassilexin, and appeared to be involved in defense responses of B. juncea.     

Exploring QSAR for CYP11B2 binding affinity and CYP11B2/CYP11B1 selectivity of diverse functional compounds using GFA and G/PLS techniques

A data set of a series of 132 structurally diverse compounds with cytochrome 11B2 and 11B1 (CYP11B2 and CYP11B1) enzyme inhibitory activities was subjected to molecular shape analysis to explore contributions of shape features as well as electronic, structural, and physicochemical parameters toward enzyme inhibitory activities, in search of appropriate molecular scaffolds with optimum substitutions for highly potent CYP11B2 inhibitors. Genetic function approximation (GFA) and genetic partial least squares (G/PLS) were used as chemometric tools for modeling, and the derived equations were of acceptable statistical quality considering both internal and external validation parameters (Q²: 0.514–0.659, R²_pred: 0.510–0.734). The G/PLS models with spline option for CYP11B2 and CYP11B1 inhibition and selectivity modeling appeared to be the best models based on r_m²_(overall) criterion. The study indicates the importance of the pyridinylnaphthalene and pyridylmethylene-indane scaffolds with less polar and electrophilic substituents for optimum CYP11B2 inhibitory activity and CYP11B2/CYP11B1 selectivity.     

Stereoselective metabolism of methadone N-demethylation by cytochrome P4502B6 and 2C19

Methadone is a clinically used opioid agonist that is oxidatively metabolized by cytochrome P450 (CYP) isoforms to a stable metabolite, EDDP. Methadone is a chiral drug administered as the racemic mixture of (R)-(-)- and (S)-(+)-methadone, but (R)-methadone is the active isomer. The cytochrome P450 (CYP) isoform involved in methadone's metabolism is thought to be CYP3A4, but human drug-drug interaction studies are not consistent with this. The ability of the common human drug-metabolizing CYPs (obtained from baculovirus-infected insect cell supersomes) to generate 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrilidine (EDDP) from racemic methadone was examined and then determined if the CYP isoforms metabolized methadone stereoselectively. Only CYP2B6, 2C19, and 3A4 generated measurable EDDP from 1 microg/ml of racemic methadone. The hierarchy of EDDP generation was CYP2B6 > CYP2C19 >/= CYP3A4. At 10 microg/ml of methadone, CYP2C9 and CYP2D6 also generated EDDP, but in at least 10-fold lower quantities than CYP2B6. Michaelis-Menten kinetic data demonstrated that CYP2B6 had the highest V(max) (44 ng/min/10pmol) and the lowest K(m) (12.6 microg/ml) for EDDP formation of all the CYP isoforms. In human liver microsomes with high and low CYP2B6 expression but equivalent CYP3A4 expression, high CYP2B6 expression microsomes generated twice the amount of EDDP from 10 microg/ml of methadone than low CYP2B6 expression microsomes. When stereoselective metabolism of racemic methadone by CYP2B6, 2C19, and 3A4 was examined using an enantiospecific methadone assay, CYP2B6 preferentially metabolized (S)-methadone, CYP2C19 preferentially metabolized (R)-methadone, and CYP3A4 showed no preference. These data suggest that multiple CYPs metabolized methadone but CYP2B6 had the highest V(max)/K(m). In addition, only CYP2B6 and 2C19 showed stereoselective metabolism. Our data could explain why the plasma concentration ratio of R/S methadone is variable and why drugs that induce CYP2B6 such as nevirapine and efavirenz also induce methadone metabolism, while the CYP3A4 inducer rifabutin has no effect on methadone pharmacokinetics.     

氯吡格雷对大鼠肝药物酶CYP3A4 和CYP1A2 表达的影响

目的：氯吡格雷主要由CYP3A4 催化使其激活,CYP1A2 也参与氯吡格雷活化。关于氯吡格雷对肝微粒体酶的影响国内外 文献报道不多,因此本实验通过检测肝细胞色素氧化酶CYP3A4 和CYP1A2 的表达,探讨氯吡格雷对大鼠肝药物酶的影 响。方法：生理盐水为对照组,氯吡格雷设高、中、低三个剂量组（27,13.5,6.75mg/kg/d）,雄性健康大鼠连续灌胃给药7天,脱臼处 死,取肝组织,通过western blot法检测大鼠肝脏CYP3A4 和CYP1A2 蛋白表达情况。结果：1）、氯吡格雷抑制大鼠CYP3A4 蛋白 表达,氯吡格雷高中低剂量组分别比生理盐水组大鼠CYP3A4 蛋白表达量降低（P<0.05）;氯吡格雷低中高剂量组间进行比较,大 鼠CYP3A4 蛋白表达量呈梯度减少（P<0.05）;2）、氯吡格雷抑制大鼠CYP1A2 蛋白表达,氯吡格雷高中低剂量组分别比生理盐水 组大鼠CYP1A2 蛋白表达量降低（P<0.05）,氯吡格雷低中高剂量组间进行比较,大鼠CYP1A2 蛋白表达量呈梯度减少（P<0.05）。 结论：氯吡格雷使肝细胞色素氧化酶CYP3A4 和CYP1A2 的表达量减少,因此氯吡格雷高、中、低3 个剂量组均不同程度的抑制 大鼠肝脏CYP3A4 和CYP1A2 的表达,提示当氯吡格雷与某些主要经CYP3A4 和CYP1A2 代谢的药物合用时,发生代谢性相关 作用的可能性大。     

Dihydrobenzisoxazole-4-one compounds are novel selective inhibitors of aldosterone synthase (CYP11B2) with in vivo activity

6,7-Dihydro-5H-2,1-benzisoxazol-4-one analogs are potent inhibitors of aldosterone synthase (CYP11B2) with selectivity over the highly homologous enzyme cortisol synthase (CYP11B1). These compounds are unique among inhibitors of CYP11B2 in their lack of a strong-heme binding group such as a pyridine or imidazole. Poor metabolic stability in hepatocyte incubations was found to proceed via a reduction of the isoxazole ring. While the enzyme responsible for the reductive metabolism remains unknown, the rate of metabolism could be attenuated by the addition of polar functionality. The in vitro CYP11B2 potency and selectivity were confirmed in vivo in a cynomolgus monkey model by the inhibition of ACTH stimulated aldosterone production without impacting plasma cortisol concentrations.     

氯吡格雷对大鼠肝药物酶CYP3A4和CYP1A2表达的影响

目的：氯吡格雷主要由CYP3A4催化使其激活,CYPlA2也参与氯吡格雷活化。关于氯吡格雷对肝微粒体酶的影响国内外文献报道不多,因此本实验通过检测肝细胞色素氧化酶CYP3A4和CYPlA2的表达,探讨氯吡格雷对大鼠肝药物酶的影响。方法：生理盐水为对照组,氯吡格雷设高、中、低三个剂量组（27,13．5,6．75mg／kg／d）,雄性健康大鼠连续灌胃给药7天,脱臼处死,取肝组织,通过westernblot法检测大鼠肝脏CYP3A4和CYPlA2蛋白表达情况。结果：1）、氯吡格雷抑制大鼠CYP3A4蛋白表达,氯吡格雷高中低剂量组分别比生理盐水组大鼠CYP3A4蛋白表达量降低（P〈0．05）;氯吡格雷低中高剂量组间进行比较,大鼠CYP3A4蛋白表达量呈梯度减少（P〈0．05）;2）、氯吡格雷抑制大鼠CYPlA2蛋白表达,氯吡格雷高中低剂量组分别比生理盐水组大鼠CYPlA2蛋白表达量降低（P〈0．05）,氯吡格雷低中高剂量组间进行比较,大鼠CYPlA2蛋白表达量呈梯度减少（P〈0．05）。结论：氯吡格雷使肝细胞色素氧化酶CYP3A4和CYPlA2的表达量减少,因此氯吡格雷高、中、低3个剂量组均不同程度的抑制大鼠肝脏CYP3A4和CYPlA2的表达,提示当氯吡格雷与某些主要经CYP3A4和CYPlA2代谢的药物合用时,发生代谢性相关作用的可能性大。     

Long-term enhancement of cytochrome P450 2B1/2 expression in rat hepatocyte spheroids through adenovirus-mediated gene transfer

Tissue-like structures of cells organized in vitrohave a great potential for a number of clinical and biomedical applications. Cell functions may be modulated with gene delivery, improving the characteristics of these structures. Hepatocytes that self-assemble into spheroids can be transduced through adenovirus-mediated gene transfer. An adenoviral vector (AdGFP) was employed to deliver a gene encoding for green fluorescent protein (GFP) in rat hepatocyte spheroids. GFP fluorescence was detected for at least one month. Furthermore, the rat cytochrome P450 2B1 gene (CYP2B1) was transferred through infection with a recombinant adenovirus (AdCYP2B1) in hepatocyte spheroids cultured in suspension. The CYP2B1/2 mRNA and apoprotein levels were continuously higher for over 23 days compared to phenobarbital-induced and control cultures. P450-catalyzed pentoxyresorufin-O-dealkylation activity was also high in the AdCYP2B1-infected spheroids. In these spheroid cultures, albumin and urea levels were similar to those in uninfected spheroid cultures, indicating that expression of the CYP2B1transgene did not impair these liver-specific functions. Hepatocyte spheroids transduced by recombinant adenoviral vectors can be efficiently used for drug metabolism studies, in implantation, and in bioartificial liver devices. This revised version was published online in August 2006 with corrections to the Cover Date.