The utility of cold-preserved human hepatocytes in studies on cytochrome P450 induction and hepatic drug transport

Abstract

1. Human hepatocytes that had been cold-preserved in SureTran^TM matrix (Abcellute Ltd, Cardiff, UK) were used for studies on cell viability, cytochrome P450 (CYP) 3A4, 2B6 and 1A2 induction and hepatic drug transporters. It has recently been shown that basal CYP activities are maintained in cold-preserved hepatocytes (Palmgren et al., 2012).

2. After 5?d of cold preservation, the viability was still more than 70%, and after 8?d it was around 60%. In hepatocytes that had been cold-preserved for 3?d, the activity of CYP3A4 was induced around 15-fold upon treatment with 8?µM rifampicin for 72?h. For CYP2B6, the activity was induced 4- to 16-fold in hepatocytes that had been cold-preserved for 3?d and thereafter treated with 1?mM phenobarbital for 72?h. The activity of CYP1A2 was low and close to the limit of detection in non-treated cells that had been cold-preserved for up to 3?d, while the activity increased in cells treated with 0.3–25?µM β-naphthoflavone for 72?h. CYP3A4, 2B6 and 1A2 mRNA levels were only determined with hepatocytes from one donor and increased upon treatment with the inducers.

3. Hepatic uptakes of estrone-3-sulfate, taurocholate, ipratropium and rosuvastatin were stable in human hepatocytes that had been cold-preserved for up to 2?d.

4. In summary, cold-preserved human hepatocytes demonstrate retained viability and can advantageously be used for in vitro induction studies and for studies of hepatic uptake transporters.     

Differential inhibition of naringenin on human and rat cytochrome P450 2E1 activity

Cytochrome P450 2E1 (CYP2E1) has been proposed as a molecular target in oxidative stress-associated metabolic diseases. Rats are chosen as model organisms in most experiments studying CYP2E1-related toxicity; however, the human relevance of these results remains unclear. To describe differences in catalysis and inhibition between human and rat CYP2E1, recombinant human and rat CYP2E1 enzymes were treated with different concentrations of naringenin (NAR, 10 nM – 1 mM), and inhibition parameters were calculated. Interspecies differences in the catalytic efficiency for O-demethylation of 7-methoxy-4-(trifluoromethyl)coumarin were revealed (45-fold higher in human CYP2E1 than in the rat enzyme). Additionally, differences in the potency of inhibition of NAR were found (absolute half inhibitory concentration, IC₅₀ = 204 ± 28 and 69 ± 4 μM; inhibition constant, Ki = 9 ± 2 and 161 ± 20 μM in human and rat CYP2E1, respectively). Although NAR exhibited a noncompetitive mechanism of inhibition of both CYP2E1 enzymes, this compound is an irreversible inhibitor of rat CYP2E1 and a reversible inhibitor of the human enzyme. Molecular docking suggested that differences in the potency of inhibition and time dependence between species could be attributable to the differential interactions of NAR with access channels to the CYP2E1 catalytic site. These results highlight the importance of finding the appropriate model to improve the predictability of animal-based assays for human risk assessment.     

Determination of cytochrome P450 1A2 and cytochrome P450 3A4 induction in cryopreserved human hepatocytes

Freshly prepared human hepatocytes are considered as the 'gold standard' for in vitro testing of drug candidates. However, several disadvantages are associated with the use of this model system. The availability of hepatocytes is often low and consequently the planning of the experiments rendered difficult. In addition, the quality of the available cells is in some cases poor. As an alternative, cryopreserved human hepatocytes were validated as a model to study cytochrome P450 1A2 (CYP1A2) and cytochrome P450 3A4 (CYP3A4) induction. In a single blinded experiment, hepatocytes from three separate lots were incubated with three concentrations of different compounds, and compared to non-treated cells and cells incubated with omeprazole or rifampicin. CYP1A2 and CYP3A4 induction was determined by measuring 7-ethoxyresorufin-O-deethylation activity and 6beta-hydroxytestosterone formation, respectively. CYP1A2 and CYP3A4 mRNA and protein expression were analyzed by TaqMan QRT-PCR and immunodetection. Cells responded well to the prototypical inducers with a mean 38.8- and 6.2-fold induction of CYP1A2 and CYP3A4 activity, respectively. Similar as with fresh human hepatocytes, high batch-to-batch variation of CYP1A2 and CYP3A4 induction was observed. Except for 1 and 10 microM rosiglitazone, the glitazones did not significantly affect CYP1A2. A similar result was observed for CYP3A4 activity although CYP3A4 mRNA and protein expression were dose-dependently upregulated. In conclusion, cryopreserved human hepatocytes may be a good alternative to fresh hepatocytes to study CYP1A and 3A induction.     

Effect of zolpidem on human cytochrome P450 activity,and on transport mediated by P-glycoprotein

The influence of high concentrations of zolpidem (100 microM, corresponding to approximately 200 times maximum therapeutic concentrations) on the activity of six human Cytochrome P450 (CYP) enzymes was evaluated in a model system using human liver microsomes. Zolpidem produced negligible or weak inhibition of human CYP1A2, 2B6, 2C9, 2C19, 2D6, and 3A. Transport of rhodamine 123, presumed to be mediated mainly by the energy-dependent efflux transport protein P-glycoprotein, was studied in a cell culture system using a human intestinal cell line. High concentrations of zolpidem (100 microM), exceeding the usual therapeutic range by more than 100-fold, produced only modest impairment of rhodamine 123 transport. The findings indicate that zolpidem is very unlikely to cause clinical drug interactions attributable to impairment of CYP activity or P-gp mediated transport.     

Effect of Pyrethrins on cytochrome P450 forms in cultured rat and human hepatocytes

High doses of Pyrethrins produce liver and thyroid gland tumours in rats by modes of action involving the induction of hepatic xenobiotic metabolising enzymes. The aim of this study was to compare the effects of Pyrethrins with those of the rat liver and thyroid tumour promoter sodium Phenobarbital on some cytochrome P450 (CYP) forms in cultured rat and human hepatocytes. The treatment of female Sprague-Dawley rat and human (both male and female) hepatocytes for 72 h with 0-1000 microM Pyrethrins and 0-1000 microM Phenobarbital did not result in any marked cytotoxicity. In rat hepatocytes both Pyrethrins and Phenobarbital produced an induction of 7-benzyloxy-4-trifluoromethylcoumarin O-debenzylase activity (a CYP1A/2B form marker) and CYP2B1 and CYP2B1/2 mRNA levels. Pyrethrins and Phenobarbital also induced CYP3A-dependent testosterone 6beta-hydroxylase activity in rat hepatocytes. In human hepatocytes Pyrethrins and Phenobarbital induced both testosterone 6beta-hydroxylase activity and CYP3A4 mRNA levels and also increased CYP2B6 mRNA levels. The effects of Pyrethrins and Phenobarbital were concentration-dependent and exhibited a threshold. These results demonstrate that the effects of Pyrethrins on CYP forms in cultured rat and human hepatocytes are qualitatively similar to those of Phenobarbital. Pyrethrins induce CYP2B and CYP3A forms in cultured rat hepatocytes and can induce CYP3A and CYP2B forms in human hepatocytes. While CYP form induction by Pyrethrins, Phenobarbital and related compounds can be associated with liver and thyroid gland tumour formation in rodents, epidemiological data for Phenobarbital suggests that such effects do not occur in humans.     

The effect of the cytochrome P-450 suicide inactivator, 1-aminobenzotriazole, on the in vivo metabolism and pharmacologic activity of flurazepam

Flurazepam (F) is an extensively prescribed hypnotic (Dalmane) whose in vivo activity has been suggested to be due to its primary metabolites, hydroxyethyl flurazepam (HEF) and N-desalkylflurazepam (DAF). In order to determine the intrinsic pharmacologic activity of F, mice were administered various doses of the cytochrome P-450 suicide inactivator, 1-aminobenzotriazole (ABT), 1 hr before the ip administration of 1 mg/kg 14C-F. One hr after 14C-F, 70 mg/kg pentylenetetrazole was administered iv and the mice were observed for convulsions. F alone offered no protection from convulsion (mean brain concentrations were 3.9, 32, and 11 ng/g for F, DAF, and HEF, respectively). F with 25 mg/kg ABT also offered no protection despite a 6-fold increase in brain concentrations of F. F with 100 mg/kg ABT offered a 57% protection from convulsions (mean brain concentrations were 99, 62, and 41 ng/g for F, DAF, and HEF, respectively). One mg/kg F with 400 mg/kg ABT offered 100% protection from convulsions (brain concentrations were 190, 47, and 18 ng/g for F, DAF, and HEF, respectively). These data indicate that F has intrinsic pharmacologic activity which must be considered when evaluating the pharmacodynamics of F.     

Effects of propofol on human hepatic microsomal cytochrome P450 activities

1. The potential of propofol to inhibit the activity of major human cytochrome P450 enzymes has been examined in vitro using human liver microsomes. Propofol produced inhibition of CYP1A2 (phenacetin O -deethylation), CYP2C9 (tolbutamide 4-hydroxylation), CYP2D6 (dextromethorphan O -demethylation) and CYP3A4 (testosterone 6 beta hydroxylation) activities with IC = 40, 49, 213 and 32 mu M respectively. K for propofol against all of these enzymes with the exception of CYP2D6, where propofolishowed little inhibitory activity, was 30, 30 and 19 mu M respectively for CYPs 1A2, 2C9 and 3A4. 2. Furafylline, sulphaphenazole, quinidine and ketoconazole, known selective inhibitors of CYPs 1A2, 2C9, 2D6 and 3A4 respectively, were much more potent than propofol having IC = 0.8, 0.5, 0.2 and 0.1 mu M; furafylline and sulphaphenazole yielded K = 0.6 and 0.7 mu M respectively. i 3. The therapeutic blood concentration of propofol (20 mu M; 3-4 mu?g ml) together with the in vitro K estimates for each of the major human P450 enzymes have been used to i estimate the extent of cytochrome P450 inhibition, which may be produced in vivo by propofol. This in vitro - in vivo extrapolation indicates that the degree of inhibition of CYP1A2, 2C9 and 3A4 activity which could theoretically be produced in vivo by propofol is relatively low (40-51%); this is considered unlikely to have any pronounced clinical significance. 4. Although propofol has now been used in 190 million people since its launch in 1986,thereare onlysinglereportsofpossible druginteractions between propofoland either alfentanil or warfarin. Consequently, it is difficult to conclude from either the published literature or the ZENECA safety database whether there is any evidence to indicate that propofol produces clinically significant drug interactions through inhibition of cytochrome P450-related drug metabolism.     

Clotrimazole-induced modulation of hepatic cytochrome P450 enzymes in Syrian and Chinese hamsters.

Clotrimazole, an imidazole antifungal drug, is known to induce cytochrome P450 isozymes of the P450IIIA and P450IIB subfamilies in rats. This agent modulated hepatic cytochrome P450 enzymes differently in golden Syrian and Chinese hamsters and also in hamsters and rats. Clotrimazole at a daily intraperitoneal dose of 100 mg/kg for three days increased the amount of cytochrome P450 in the livers of the two hamster strains. In Syrian hamsters, clotrimazole significantly induced the activities of 7-pentoxyresorufin O-dealkylase, coumarin 7-hydroxylase, benzphetamine N-demethylase and testosterone 15 alpha- and 16 alpha-hydroxylases, but reduced those of testosterone 15 beta-, 7 alpha-, 6 beta-, 2 alpha- and 2 beta-hydroxylases. In Chinese hamsters, clotrimazole markedly stimulated the activities of coumarin 7-hydroxylase and testosterone 15 alpha, 16 alpha- and 2 alpha-hydroxylases as well as the formation of androstenedione. Western blot analysis revealed that clotrimazole treatment induced mainly cytochrome P450 isozymes immunorelated to the P450IIB and P450IIA subfamilies in Syrian hamsters and isozymes immunorelated to the P450IIA subfamily in Chinese hamsters. In contrast, in both hamster strains, clotrimazole did not induce the isozymes corresponding to the P450IIIA subfamily.     

3种肝源永生化细胞药物代谢酶表达差异比较研究（英文）

目的比较3种人源肝永生化细胞LX-2,L02和HepG2药物代谢酶的表达差异,确定适合于特定细胞色素P450(CYP)亚型研究的细胞类型。方法体外培养人源传代肝细胞L02,LX-2和HepG2,以奥美拉唑(Ome)、地塞米松(Dex)、苯巴比妥钠(Phe)、异烟肼(Iso)和利福平(Rif)0,5,10,20和40μmol·L~(-1)进行诱导。CCK-8法检测细胞存活,实时荧光定量PCR(RT-qPCR)法检测LX-2细胞中CYP1A2,CYP2B6,CYP2C9,CYP2C19,CYP2D6,CYP2E1和CYP3A4基因表达水平;Western印迹法检测LX-2,L02和HepG2细胞中以上7种CYP亚型蛋白表达水平;LX-2,L02和HepG2经Rif 5,10,20和40μmol·L~(-1)诱导后,Luciferin-PFBE法检测细胞中CYP3A4酶活性变化。结果 CCK-8结果显示,与相应细胞对照组相比,Ome,Dex,Phe,Iso和Rif(≤40μmol·L~(-1))作用于LX-2,L02和HepG2细胞24 h,细胞存活率均>80%;RT-qPCR法检测结果显示,与LX-2细胞对照组相比,Phe诱导CYP2B6(P<0.05)和CYP2D6(P<0.01)表达上升;Western蛋白印迹结果显示,L02,LX-2和HepG2细胞经Ome,Dex,Phe,Iso和Rif 40μmol·L~(-1)处理后,CYP1A2,CYP2B6,CYP2C9,CYP2C19,CYP2D6,CYP2E1和CYP3A4蛋白表达存在差异。L02细胞中CYP2C9(IA=1.58±0.07),CYP2C19(IA=0.96±0.02)和CYP3A4(IA=1.30±0.01),LX-2细胞中CYP2B6(IA=1.48±0.01)和CYP2D6(IA=1.46±0.02),HepG2细胞中的CYP1A2(IA=1.62±0.19)和CYP2E1(IA=1.49±0.10)分别具有最高的表达丰度。CYP3A4酶活性检测显示,Rif处理L02,LX-2和HepG2细胞24 h后,CYP3A4活性变化无统计学差异。结论 L02,LX-2和HepG2细胞中CYP亚型蛋白表达丰度差异提示,L02细胞适用于CYP2C9,CYP2C19和CYP3A4实验;LX-2细胞适用于CYP2B6和CYP2D6实验;HepG2细胞适用于CYP1A2和CYP2E1实验。     

Inhibition of drug-metabolizing enzyme activity in human hepatic cytochrome P450s by bisphenol A

Effect of bisphenol A on drug-metabolizing enzyme activities by human hepatic cytochrome P450s (CYP) was investigated. We measured aminopyrine N-demethylation by eleven kinds of cDNA-expressed CYPs. CYP2C19 and CYP2B6 catalyzed most efficiently the aminopyrine N-demethylation, followed by CYP2C8 and CYP2D6. Bisphenol A (1 mM) most efficiently inhibited aminopyrine N-demethylation by CYP2C8 and CYP2C19 by 82% and 85%, respectively, whereas inhibition of the activities by CYP 2B6 and 2D6 was less than 40%. Bisphenol A exhibited a noncompetitive-type inhibition of aminopyrine N-demethylase activity by CYP2C8 with Ki value of 97 microM. Additionally, we investigated the inhibitory effect of bisphenol A on CYP2C19-mediated S-mephenytoin 4-hydroxylation. Bisphenol A exhibited a mixed-type inhibition with Ki value of 113 microM. These results suggest that bisphenol A inhibits human hepatic CYP activities, especially CYP2C8 and CYP2C19.     

Effect of dietary oil intake on hepatic cytochrome P450 activity in the rat

The objective of the present study was to investigate whether or not different dietary oils, commonly used as drug vehicles, alter hepatic microsomal drug metabolism in the rat. Male adult Sprague-Dawley rats were administered 1 mL/kg/d of either corn, olive, sesame, or soybean oil via oral gavage for 7 days. An additional rat group was given an equal volume of water each day to serve as a control. We found that the hepatic cytochrome P450 (CYP) 3A2 protein level increased by 16% (p < 0.01) in rats given soybean oil compared with control rats. In contrast, CYP2C11 protein levels decreased by 32% (p < 0.01) in the corn oil group and by 31% (p < 0. 01) in rats given olive oil. The changes in the in vitro production of 6beta- and 2alpha-hydroxytestosterone, markers of CYP3A2 and 2C11 activities, respectively, were consistent with their protein levels, although not statistically different than controls. The results demonstrate that dietary oils may have differential effects on specific hepatic CYP isoforms and may add to the variability in metabolism when xenobiotics are administered using dietary oils as vehicles.     

Effect of lindane on hepatic and brain cytochrome P450s and influence of P450 modulation in lindane induced neurotoxicity.

Oral administration of lindane (2.5, 5, 10 and 15 mg/kg, body weight) for 5 days was found to produce a dose-dependent increase in the activity of P450 dependent 7-ethoxyresorufin-O-deethylase (EROD), 7-pentoxyresorufin-O-dealkylase (PROD) and N-nitrosodimethylamine demethylase (NDMA-d) in rat brain and liver. A significant increase in the hepatic and brain P450 monooxygenases was also observed when the duration of exposure of low dose (2.5 mg/kg) of lindane was increased from 5 days to 15 or 21 days. As observed with different doses, the magnitude of induction in the activity of P450 monooxygenases was several fold higher in liver microsomes when compared with the brain. Western blotting studies have indicated that the increase in the P450 enzymes could be due to the increase in the expression of P450 1A1/1A2, 2B1/2B2 and 2E1 isoenzymes. In vitro studies using organic inhibitors specific for individual P450 isoenzymes and antibody inhibition experiments have further demonstrated that the increase in the activity of PROD, EROD and NDMA-d are due to the increase in the levels of P450 2B1/2B2, 1A1/1A2 and 2E1 isoenzymes, respectively. Induction studies have further shown that while pretreatment of 3-methylcholanthrene (MC), an inducer of P4501A1/1A2, did not produce any significant effect in the incidence of lindane induced convulsions, pretreatment with phenobarbital (PB), an inducer of P450 2B1/2B2 or ethanol, an inducer of P450 2E1 catalysed reactions, significantly increased the incidence of lindane induced convulsions. Similarly, when the P450-mediated metabolism of lindane was blocked by cobalt chloride incidence of convulsions was increased in animals treated with lindane indicating that lindane per se or its metabolites formed by PB or ethanol inducible P450 isoenzymes are involved in its neurobehavioral toxicity.     

Effects of silver nanoparticles on rat hepatic cytochrome P450 enzyme activity

Silver nanoparticles (AgNPs) are increasingly used in various products and consequentially the potential adverse effects associated with exposure to them are of concern. This study investigated the effects of AgNPs on the hepatic drug-metabolizing enzymes of the cytochrome P450 (CYP) families 1, 2 and 3, using both in vitro and in vivo biological assays. AgNPs were orally administered to Sprague-Dawley rats at various concentrations (0-1000 mg/kg body weight/day) for 2 weeks. No effect was found on the plasma levels of ALT, AST and ALP in all treated rat groups, and no significant change in the activities of CYP1A, CYP2C, CYP2D, CYP2E1 and CYP3A was observed for all tested AgNP doses. The results correlated with the observation that no AgNPs were detected in the liver sections of the tested rats. However, the in vitro system using rat liver microsomes demonstrated a strong inhibition of CYP2C (IC(50)?= 28 μg/mL) and CYP2D (IC(50)?= 23 μg/mL) activities, but not of CYP1A, CYP2E1 and CYP3A activities (IC(50) > 100 μg/mL) at concentrations up to 100 μg/mL of AgNPs. The inhibitory effect of AgNPs on these CYPs indicates the possibility of the AgNP-drug interaction when co-administered with some medicines and this may cause adverse effects to patients.     

Effects of silver nanoparticles on rat hepatic cytochrome P450 enzyme activity

Silver nanoparticles (AgNPs) are increasingly used in various products and consequentially the potential adverse effects associated with exposure to them are of concern. This study investigated the effects of AgNPs on the hepatic drug-metabolizing enzymes of the cytochrome P450 (CYP) families 1, 2 and 3, using both in vitro and in vivo biological assays. AgNPs were orally administered to Sprague-Dawley rats at various concentrations (0–1000?mg/kg body weight/day) for 2 weeks. No effect was found on the plasma levels of ALT, AST and ALP in all treated rat groups, and no significant change in the activities of CYP1A, CYP2C, CYP2D, CYP2E1 and CYP3A was observed for all tested AgNP doses. The results correlated with the observation that no AgNPs were detected in the liver sections of the tested rats. However, the in vitro system using rat liver microsomes demonstrated a strong inhibition of CYP2C (IC₅₀?=?28 µg/mL) and CYP2D (IC₅₀?=?23 µg/mL) activities, but not of CYP1A, CYP2E1 and CYP3A activities (IC₅₀ > 100 µg/mL) at concentrations up to 100 µg/mL of AgNPs. The inhibitory effect of AgNPs on these CYPs indicates the possibility of the AgNP-drug interaction when co-administered with some medicines and this may cause adverse effects to patients.     

Inhibitory effect of natural furanocoumarins on human microsomal cytochrome P450 3A activity

To investigate the possible drug interaction with herbal medicine, furanocoumarin derivatives isolated from several Umbelliferous crude drugs were examined for their inhibitory effects on a typical human drug metabolizing enzyme, cytochrome P450 3A (CYP3A). Most furanocoumarins tested at 0.1 mM reduced microsomal testosterone 6beta-hydroxylation as an index of CYP3A activity to less than 50% of the control. In particular, the dimer and trimer derivatives of furanocoumarins showed striking inhibition, whose potencies were similar to that of a typical CYP3A inhibitor, ketoconazole. Preincubation of dimer types of furanocoumarins increased suppression but not most of the monomer derivatives, suggesting that the inhibition on CYP3A activity was caused by at least plural mechanisms. These results raised the possibility that the furanocoumarin containing herbal medicines may alter pharmacokinetics of co-ingested drugs similar to the case with grapefruit juice.     

SM-1对人肝微粒体细胞色素P450亚型酶的抑制作用

目的观察SM-1对人肝细胞色素P450酶系统中7种亚型酶活性的影响。方法在体外将一定浓度的底物或SM-1与人肝微粒体孵育30 min,分为对照组(底物)和实验组(底物和SM-1),以非那西丁、安非他酮、紫杉醇、甲苯磺丁脲、奥美拉唑、右美沙芬、睾酮为CYP探针底物,应用HPLC法检测各探针底物代谢量,计算抑制率,评价SM-1对人肝微粒体CYP1A2、2B6、2C8、2C9、2C19、2D6、3A4酶的抑制活性。结果 SM-1对CYP1A2、2B6、2C8、2C9、2C19、2D6、3A4的抑制率分别是0.05%、3.37%、0.08%、2.07%、4.20%、-0.15%和10.84%。结论 SM-1对CYP3A4可能有抑制作用,临床用药时应注意因CYP酶抑制引起的药物相互作用。     

In vitro inhibitory effect of 1-aminobenzotriazole on drug oxidations catalyzed by human cytochrome P450 enzymes: a comparison with SKF-525A and ketoconazole

1-Aminobenzotriazole (ABT) is widely used as a non-specific inhibitor of animal cytochrome P450 (CYP). In the present study, the inhibitory effect of ABT was investigated on drug oxidations catalyzed by human CYP isoforms. This inhibitory effect was compared with that of SKF-525A, another non-specific inhibitor, and ketoconazole, a potent inhibitor of CYP3A. Bacurovirus-expressed recombinant human CYP isoforms were used as an enzyme source. The specific activities for human CYP isoforms are: phenacetin O-deethylation, for CYP1A2; diclofenac 4'-hydroxylation, for CYP2C9; S-mephenytoin 4'-hydroxylation, for CYP2C19; bufuralol 1'-hydroxylation, for CYP2D6; chlorzoxazone 6-hydroxylation, for CYP2E1; testosterone 6beta-hydroxylation, nifedipine oxidation, and midazolam 1'-hydroxylation, for CYP3A4. ABT inhibited both CYP1A2-dependent activity (Ki=330 microM) and CYP2E1-dependent activity (Ki=8.7 microM). In contrast, SKF-525A weakly inhibited CYP1A2-dependent activities (46% inhibition at 1200 microM) and CYP2E1-dependent activities (65% inhibition at 1000 microM). ABT exhibited the highest Ki value for CYP2C9-dependent diclofenac 4'-hydroxylation among those determined by this assay (Ki=3500 microM). Moreover, SKF-525A showed strong inhibition of CYP2D6-dependent bufuralol 1'-hydroxylation (Ki=0.043 microM). Ketoconazole inhibited all tested drug oxidations, however, its inhibitory effect on CYP1A2-dependent activities was very weak (50% inhibition at 120 microM). ABT, SKF-525A, and ketoconazole showed different selectivity and had a wide range of Ki values for the drug oxidations catalyzed by human CYP enzymes. Therefore, we conclude that inhibitory studies designed to predict the contribution of CYP enzymes to the metabolism of certain compounds should be performed using multiple CYP inhibitors, such as ABT, SKF-525A, and ketoconazole.     

Recombinant enzymes overexpressed in bacteria show broad catalytic specificity of human cytochrome P450 2W1 and limited activity of human cytochrome P450 2S1

Human cytochromes P450 2S1 and 2W1 have received only limited attention with regard to characterization of function. Both cytochromes P450 have been reported to be overexpressed in human tumors, and cytochrome P450 2S1 is induced by carcinogenic polycyclic hydrocarbons. We report methods for high-level expression and purification of both cytochromes P450 from Escherichia coli, with the goal of establishing function. The level of expression of human cytochrome P450 2W1 achieved using codon optimization for E. coli was 1800 nmol of cytochrome P450 per liter of culture, the highest level achieved in this laboratory to date. Assays with a number of the typical cytochrome P450 substrates showed no detectable activity, including some for which qualitative reports have appeared in the literature. Cytochrome P450 2W1 catalyzed benzphetamine N-demethylation (k(cat), 3.8/min) and arachidonic acid oxidation, albeit at a very low rate (approximately 0.05/min). In a umu genotoxicity screen, cytochrome P450 2W1 catalyzed the activation of several procarcinogens, particularly polycyclic hydrocarbon diols, but cytochrome P450 2S1 did not. The bioactivation of procarcinogens by cytochrome P450 2W1 may be of significance in the context of reports of preferential expression of the enzyme in tumors, in that activation of procarcinogens could lead to the accumulation of mutations and enhance the carcinogenic process.