人细胞色素P450 3A活性测定

目的:建立测定人细胞色素P4503A酶活性的方法。方法:收集24h尿液,以HPLC法测定尿液中氢化可的松(HC)与6β-羟基氢化可的松(6β-OHC)的含量,以6β-OHC/HC来评估细胞色素P4503A酶的活性。结果:健康志愿者尿中6β-羟基氢化可的松和氢化可的松的比值为(8.5±3.0),肿瘤患者为(6.2±4.5)。结论:细胞色素P4503A酶的活性可以通过测定6β-OHC与HC的比值进行预测。     

Potent inhibition by star fruit of human cytochrome P450 3A (CYP3A) activity.

There has been very limited information on the capacities of tropical fruits to inhibit human cytochrome P450 3A (CYP3A) activity. Thus, the inhibitory effects of tropical fruits on midazolam 1'-hydroxylase activity of CYP3A in human liver microsomes were evaluated. Eight tropical fruits such as common papaw, dragon fruit, kiwi fruit, mango, passion fruit, pomegranate, rambutan, and star fruit were tested. We also examined the inhibition of CYP3A activity by grapefruit (white) and Valencia orange as controls. The juice of star fruit showed the most potent inhibition of CYP3A. The addition of a star fruit juice (5.0%, v/v) resulted in the almost complete inhibition of midazolam 1'-hydroxylase activity (residual activity of 0.1%). In the case of grape-fruit, the residual activity was 14.7%. The inhibition depended on the amount of fruit juice added to the incubation mixture (0.2-6.0%, v/v). The elongation of the preincubation period of a juice from star fruit (1.25 or 2.5%, v/v) with the microsomal fraction did not alter the CYP3A inhibition, suggesting that the star fruit did not contain a mechanism-based inhibitor. Thus, we discovered filtered extracts of star fruit juice to be inhibitors of human CYP3A activity in vitro.     

In vitro inhibition of human cytochrome P450 by cudratricusxanthone A

Cudratricusxanthone A (CTXA) isolated from the roots of Cudrania tricuspidata Bureau (Moraceae) has several biological activities, including hepatoprotective, neuroprotective, anti-inflammatory, monoamine oxidase inhibitory, and antithrombotic activities. In this study, we investigated the potential herb–drug interaction of CTXA and nine cytochrome P450 (CYP) isoforms in pooled human liver microsomes (HLMs) using a cocktail probe assay. CTXA reversibly inhibited the CYP1A2-catalyzed phenacetin O-deethylation, CYP2C8-catalyzed paclitaxel 6-hydroxylation, and CYP2C9-catalyzed diclofenac 4′-hydroxylation with half-maximal inhibitory concentration (IC₅₀) values of 3.9, 4.7, and 2.9 µM, respectively. The IC₅₀ values did not change under different preincubation conditions. CTXA showed marked dose-dependent, but not time-dependent, inhibition of CYP1A2 and 2C9 activities in HLMs. Dixon plots showed typical competitive inhibition of CYP1A2 and CYP2C9 with K_i values of 1.3 and 1.5 µM, respectively. Further, CTXA inhibited CYP2C8 in a non-competitive manner with a K_i value of 2.2 µM. Our results showed that CTXA reversibly inhibits CYP1A2, 2C8, and 2C9.     

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.     

Mechanism of cytochrome P450-3A inhibition by ketoconazole

Objectives Ketoconazole is extensively used as an index inhibitor of cytochrome P450‐3A (CYP3A) activity in vitro and in vivo, but the mechanism of ketoconazole inhibition of CYP3A still is not clearly established. Methods Inhibition of metabolite formation by ketoconazole (seven concentrations from 0.01 to 1.0 µm ) was studied in human liver microsomes (n = 4) at six to seven substrate concentrations for triazolam, midazolam, and testosterone, and at two substrate concentrations for nifedipine. Key findings Analysis of multiple data points per liver sample based on a mixed competitive–noncompetitive model yielded mean inhibition constant K_i values in the range of 0.011 to 0.045 µm . Ketoconazole IC50 increased at higher substrate concentrations, thereby excluding pure noncompetitive inhibition. For triazolam, testosterone, and midazolam α‐hydroxylation, mean values of α (indicating the ‘mix’ of competitive and noncompetitive inhibition) ranged from 2.1 to 6.3. However, inhibition of midazolam 4‐hydroxylation was consistent with a competitive process. Determination of K_i and α based on the relation between 50% inhibitory concentration values and substrate concentration yielded similar values. Pre‐incubation of ketoconazole with microsomes before addition of substrate did not enhance inhibition, whereas inhibition by troleandomycin was significantly enhanced by pre‐incubation. Conclusions Ketoconazole inhibition of triazolam α‐ and 4‐hydroxylation, midazolam α‐hydroxylation, testosterone 6β‐hydroxylation, and nifedipine oxidation appeared to be a mixed competitive–noncompetitive process, with the noncompetitive component being dominant but not exclusive. Quantitative estimates of K_i were in the low nanomolar range for all four substrates.     

Mechanism-based inhibition of recombinant human cytochrome P450 3A4 by tomato juice extract

This study investigates whether tomato juice can inhibit cytochrome P450 (CYP) 3A4-mediated drug metabolism. Three commercially available, additive-free tomato juices, along with homogenized fresh tomato, were analyzed for their ability to inhibit testosterone 6β-hydroxylation activity using human recombinant CYP3A4. Results were compared to that of grapefruit juice. Ethyl acetate extracts of the tomato juices moderately reduced residual activity of CYP3A4 testosterone 6β-hydroxylation activity by 19.3-26.2% with 0-min preincubation. Residual activity was strongly reduced by 69.9-83.5% at 20-min preincubation, a reduction similar to that of grapefruit juice extract, known to contain constituents of mechanism-based inhibitors. One juice extract (tomato juice C) showed irreversible dose- and preincubation time-dependent and partial nicotinamide adenine dinucleotide phosphate (NADPH)-dependent inhibition of CYP3A4 activity. Furthermore, we examined whether the CYP3A4 inhibitory effect of tomato juice was substrate dependent by examining midazolam 1'-hydroxylation activity and nifedipine oxidation activity, in addition to testosterone 6β-hydroxylation activity. Tomato juice showed a potent inhibitory effect on nifedipine oxidation activity, which was comparable to that on testosterone 6β-hydroxylation activity; however, it showed a weak inhibitory effect on midazolam 1'-hydroxylation activity. We conclude that tomato juice contains one or more mechanism-based and competitive inhibitor(s) of CYP3A4. Additionally, significant CYP3A4 inhibitory activity did not result from lycopene, a major compound in tomato. Although the active compound was uncertain, a strong CYP3A4 inhibitory activity was observed in other solanaceous plants, i.e., potato, eggplant, sweet pepper, and capsicum. Therefore, responsible compounds in tomato are likely commonly shared among solanaceous vegetables.     

Effect of high-dose vitamin C on hepatic cytochrome P450 3A4 activity

STUDY OBJECTIVES: To investigate the effect of high-dose vitamin C on cytochrome P450 (CYP) 3A4 activity, and to evaluate possible sex-specific effects on CYP3A4 activity. DESIGN: Single-center longitudinal study. SETTING: Tertiary- and specialty-care teaching hospital. SUBJECTS: Fourteen healthy Caucasian adult volunteers (seven men, seven women). INTERVENTION: Subjects self-administered vitamin C 500 mg twice/day for 14 days. MEASUREMENTS AND MAIN RESULTS: Hepatic CYP3A4 activity was measured by using the erythromycin breath test on days 1 (baseline) and 15. Overall, no significant effect of vitamin C on CYP3A4 activity was observed. Sex and baseline results were significant predictors of changes in CYP3A4 activity. In men, mean activity increased by 21.9% (95% confidence interval -3.88-47.6%). The effect in women was not consistent. CONCLUSION: Sex and baseline CYP3A4 activity appeared to influence the effect of vitamin C on CYP3A4 activity.     

Mechanism-based inhibition of cytochrome P450 3A4 by therapeutic drugs

Consistent with its highest abundance in humans, cytochrome P450 (CYP) 3A is responsible for the metabolism of about 60% of currently known drugs. However, this unusual low substrate specificity also makes CYP3A4 susceptible to reversible or irreversible inhibition by a variety of drugs. Mechanism-based inhibition of CYP3A4 is characterised by nicotinamide adenine dinucleotide phosphate hydrogen (NADPH)-, time- and concentration-dependent enzyme inactivation, occurring when some drugs are converted by CYP isoenzymes to reactive metabolites capable of irreversibly binding covalently to CYP3A4. Approaches using in vitro, in silico and in vivo models can be used to study CYP3A4 inactivation by drugs. Human liver microsomes are always used to estimate inactivation kinetic parameters including the concentration required for half-maximal inactivation (K(I)) and the maximal rate of inactivation at saturation (k(inact)).Clinically important mechanism-based CYP3A4 inhibitors include antibacterials (e.g. clarithromycin, erythromycin and isoniazid), anticancer agents (e.g. tamoxifen and irinotecan), anti-HIV agents (e.g. ritonavir and delavirdine), antihypertensives (e.g. dihydralazine, verapamil and diltiazem), sex steroids and their receptor modulators (e.g. gestodene and raloxifene), and several herbal constituents (e.g. bergamottin and glabridin). Drugs inactivating CYP3A4 often possess several common moieties such as a tertiary amine function, furan ring, and acetylene function. It appears that the chemical properties of a drug critical to CYP3A4 inactivation include formation of reactive metabolites by CYP isoenzymes, preponderance of CYP inducers and P-glycoprotein (P-gp) substrate, and occurrence of clinically significant pharmacokinetic interactions with coadministered drugs.Compared with reversible inhibition of CYP3A4, mechanism-based inhibition of CYP3A4 more frequently cause pharmacokinetic-pharmacodynamic drug-drug interactions, as the inactivated CYP3A4 has to be replaced by newly synthesised CYP3A4 protein. The resultant drug interactions may lead to adverse drug effects, including some fatal events. For example, when aforementioned CYP3A4 inhibitors are coadministered with terfenadine, cisapride or astemizole (all CYP3A4 substrates), torsades de pointes (a life-threatening ventricular arrhythmia associated with QT prolongation) may occur.However, predicting drug-drug interactions involving CYP3A4 inactivation is difficult, since the clinical outcomes depend on a number of factors that are associated with drugs and patients. The apparent pharmacokinetic effect of a mechanism-based inhibitor of CYP3A4 would be a function of its K(I), k(inact) and partition ratio and the zero-order synthesis rate of new or replacement enzyme. The inactivators for CYP3A4 can be inducers and P-gp substrates/inhibitors, confounding in vitro-in vivo extrapolation. The clinical significance of CYP3A inhibition for drug safety and efficacy warrants closer understanding of the mechanisms for each inhibitor. Furthermore, such inactivation may be exploited for therapeutic gain in certain circumstances.     

Non-specific inhibition of human cytochrome P450-catalyzed reactions by hemin

Hemin, a stable form of heme, is known to have an antimutagenic effect. Inhibitory effects of hemin on the cytochrome P450 (CYP)-catalyzed reactions of human liver microsomes and reconstituted systems containing purified CYP and NADPH-cytochrome P450 reductase (NPR) were seen. Hemin non-specifically inhibited all of the microsomal CYP activities examined. Hemin also inhibited 7-ethoxyresorufin O-deethylation, 3-[2-(N,N-diethyl-N-methylammonium)ethyl]-7-methoxy-4-methylcoumarin O-demethylation, and testosterone 6beta-hydroxylation catalyzed by purified CYPs 1A2, 2D6, and 3A4, with IC50 values of 27, 19, and 2.4 microM, respectively. Hemin also inhibited reduction of cytochrome c and ferricyanide by NPR, as much as 47%. Spectrally detectable CYP was destroyed in human liver microsomes and in a reconstituted system in the presence of hemin and an NADPH-generating system. We propose that the antimutagenic effect of hemin might be due to inhibition of CYP and NPR enzymes involved in the bioactivation of mutagens.     

Effects of N-terminal modification of recombinant human cytochrome P450 1A2 on catalytic activity

• The high-level expression of mammalian cytochrome P450 in bacteria usually requires modification of the amino-terminal region of the enzyme. The effect of altering amino acids in the N-terminus of human recombinant CYP1A2 on its catalytic activity was investigated herein.

• Rates of 7-ethoxyresorufin O-deethylation by CYP1A2a (a form made by altering the amino acids LLL of CYP1A2 to RER at positions 3–5) in reconstituted systems were significantly low compared with those of other CYP1A2?N-terminal variants at a low ratio of cytochrome P450 to NADPH-cytochrome P450 reductase, but not at higher reductase concentrations.

• CYP1A2a-dependent ethoxyresorufin O-deethylase activity in a cumene hydroperoxide-supported system was approximately 2-fold higher than other CYP1A2?N-terminal variants.

• Our results suggest that modification of three N-terminal amino acids in CYP1A2 alters the interaction between CYP1A2 and the reductase in reconstituted phospholipid vesicles and in the bicistronic membranes.

     

Effects of N-terminal modification of recombinant human cytochrome P450 1A2 on catalytic activity

1. The high-level expression of mammalian cytochrome P450 in bacteria usually requires modification of the amino-terminal region of the enzyme. The effect of altering amino acids in the N-terminus of human recombinant CYP1A2 on its catalytic activity was investigated herein. 2. Rates of 7-ethoxyresorufin O-deethylation by CYP1A2a (a form made by altering the amino acids LLL of CYP1A2 to RER at positions 3-5) in reconstituted systems were significantly low compared with those of other CYP1A2 N-terminal variants at a low ratio of cytochrome P450 to NADPH-cytochrome P450 reductase, but not at higher reductase concentrations. 3. CYP1A2a-dependent ethoxyresorufin O-deethylase activity in a cumene hydroperoxide-supported system was approximately 2-fold higher than other CYP1A2 N-terminal variants. 4. Our results suggest that modification of three N-terminal amino acids in CYP1A2 alters the interaction between CYP1A2 and the reductase in reconstituted phospholipid vesicles and in the bicistronic membranes.     

Inhibition of human cytochrome P450 3A4 activity by zinc(II) ion

Effects of Zn2+ on the activity and conformation of cytochorome P450 3A4 (CYP3A4) were investigated. Zn2+ specifically inhibited the testosterone 6beta-hydroxylation activity of CYP3A4 with an IC50 value of 27 microM. Zn2+ inhibited the CO-binding spectra of CYP3A4 reduced by NADPH-cytochrome P450 reductase (CPR) and NADPH only in the presence of b5. Zn2+-induced conformational changes of CYP3A4 were monitored by CD and intrinsic fluorescence. Zn2+ showed no significant effects on the activity of CYP3A4 supported by tert-butyl hydroperoxide, an oxygen surrogate, and on the reduction of b5 by CPR and NADPH. These results suggest that the inhibitory effects of Zn2+ come from preventing the stimulation of b5 on CYP3A4 activity.     

CYP3A4酶介导的人类药物代谢性别差异

药代动力学的性别差异是临床上药物疗效的重要影响因素。引起药代动力学性别差异的比较重要的分子因素包括药物代谢酶和药物转运体等。CYP3A4是人体含量最丰富的酶,具有广泛的代谢底物。大部分体内、外研究表明CYP3A4底物的代谢具有明显的性别差异,女性体内的代谢快于男性。该文系统综述人肝脏CYP3A4酶介导的药物代谢性别差异的研究进展。     

Potent inhibition of human cytochrome P450 1 enzymes by dimethoxyphenylvinyl thiophene

Cytochrome P450 (P450) 1 enzymes such as P450 1A1, 1A2, and 1B1 are known to be involved in the oxidative metabolism of various procarcinogens and are regarded as important target enzymes for cancer chemoprevention. Previously, several hydroxystilbene compounds were reported to inhibit P450 1 enzymes and were rated as candidate chemopreventive agents. In this study, we investigated the inhibitory effect of 2-[2-(3,5-dimethoxyphenyl)vinyl]-thiophene (DMPVT), produced from the chemical modification of oxyresveratrol, on the activities of P450 1 enzymes. The inhibitory potential by DMPVT on the P450 1 enzyme activity was evaluated with the Escherichia coli membranes of the recombinant human cytochrome P450 1A1, 1A2, or 1B1 coexpressed with human NADPH-P450 reductase. DMPVT significantly inhibited ethoxyresorufin O-deethylation (EROD) activities with IC50 values of 61, 11, and 2 nM for 1A1, 1A2, and 1B1, respectively. The EROD activity in DMBA-treated rat lung microsomes was also significantly inhibited by DMPVT in a dose-dependent manner. The modes of inhibition by DMPVT were non-competitive for all three P450 enzymes. The inhibition of P450 1B1-mediated EROD activity by DMPVT did not show the irreversible mechanism-based effect. The loss of EROD activity in P450 1B1 with DMPVT incubation was not blocked by treatment with the trapping agents such as glutathione, N-acetylcysteine, or dithiothreitol. Taken together, the results suggested DMPVT to be a strong noncompetitive inhibitor of human P450 1 enzymes that should be considered as a good candidate for a cancer chemopreventive agent in humans.     

Potent inhibition of human cytochrome P450 1B1 by tetramethoxystilbene

Human cytochrome P450 1B1 (CYP1B1) is found mainly in extrahepatic tissues and is overexpressed in a variety of human tumors. Metabolic activation of 17β-estradiol (E2) to 4-hydroxy E2 by CYP1B1 has been postulated to be an important factor in mammary carcinogenesis. The inhibition of recombinant human CYP1B1 by 2,2′,4,6′-tetramethoxystilbene (TMS) was investigated using either the Escherichia coli membranes of recombinant human CYP1B1 coexpressed with human NADPH-P450 reductase or using purified enzyme. 2,2′,4,6′-TMS showed potent and selective inhibition of ethoxyresorufin O-deethylation (EROD) activity of CYP1B1 with IC₅₀ values of 2 nM. 2,2′,4,6′-TMS exhibited 175-fold selectivity for CYP1B1 over CYP1A1 (IC₅₀, 350 nM) and 85-fold selectivity for CYP1B1 over CYP1A2 (IC₅₀, 170 nM). However, inhibition of human NADPH-P450 reductase activity by 2,2′,4,6′-TMS was negligible. The modes of inhibition by 2,2′,4,6′-TMS were noncompetitive for CYP1A1 and CYP1B1. Moreover, 2,2′,4,6′-TMS significantly suppressed EROD activity and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced CYP1A1 or CYP1B1 gene expression in human tumor cells such as HepG2 and MCF-10A. Taken together, our results indicate that 2,2′,4,6′-TMS is a potently selective inhibitor of human CYP1B1 as well as a suppressor of CYP1B1 expression and may be a valuable tool for determining enzyme properties of human CYP1B1.     

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.     

Substrate-dependent modulation of the catalytic activity of CYP3A by erlotinib

Aim:

To ascertain the effects of erlotinib on CYP3A, to investigate the amplitude and kinetics of erlotinib-mediated inhibition of seven major CYP isoforms in human liver microsomes (HLMs) for evaluating the magnitude of erlotinib in drug-drug interaction in vivo.

Methods:

The activities of 7 major CYP isoforms (CYP1A2, CYP2A6, CYP3A, CYP2C9, CYP2D6, CYP2C8, and CYP2E1) were assessed in HLMs using HPLC or UFLC analysis. A two-step incubation method was used to examine the time-dependent inhibition of erlotinib on CYP3A.

Results:

The activity of CYP2C8 was inhibited with an IC₅₀ value of 6.17±2.0 μmol/L. Erlotinib stimulated the midazolam 1′-hydroxy reaction, but inhibited the formation of 6β-hydroxytestosterone and oxidized nifedipine. Inhibition of CYP3A by erlotinib was substrate-dependent: the IC₅₀ values for inhibiting testosterone 6β-hydroxylation and nifedipine metabolism were 31.3±8.0 and 20.5±5.3 μmol/L, respectively. Erlotinib also exhibited the time-dependent inhibition on CYP3A, regardless of the probe substrate used: the value of K_I and k_inact were 6.3 μmol/L and 0.035 min⁻¹ for midazolam; 9.0 μmol/L and 0.045 min⁻¹ for testosterone; and 10.1 μmol/L and 0.058 min⁻¹ for nifedipine.

Conclusion:

The inhibition of CYP3A by erlotinib was substrate-dependent, while its time-dependent inhibition on CYP3A was substrate-independent. The time-dependent inhibition of CYP3A may be a possible cause of drug-drug interaction, suggesting that attention should be paid to the evaluation of erlotinib''s safety, especially in the context of combination therapy.     

豆腐果苷对人肝微粒体CYP450酶体外抑制作用研究

目的:通过评价豆腐果苷在体外对人肝微粒体CYP450酶的7种亚型酶活性的影响,预测服用豆腐果苷可能出现的食物-药物及药物-药物代谢的影响。方法:将豆腐果苷与CYP450酶7种亚型的特异性探针底物咖啡因(CYP1A2)、右美沙芬(CYP2D6)、甲苯磺丁脲(CYP2C9)、S-美芬妥因(CYP2C19)、氯唑沙宗(CYP2E1)、香豆素(CYP2A6)及咪达唑仑(CYP3A4)与人肝微粒体进行孵育反应,采用HPLC和LC-MS/MS法测定对应的7种代谢产物(1,7-二甲基黄嘌呤、去甲右美沙芬、4-羟基甲苯磺丁脲、4-羟基美芬妥因、6-羟基氯唑沙宗、7-羟基香豆素和1-羟基咪达唑仑)的浓度,与对照组比较,确定豆腐果苷对以上7种亚酶活性的影响。结果:豆腐果苷在1～100μmol.L-1时对7种酶的抑制作用均无明显统计学意义(P>0.05)。结论:豆腐果苷可能不会引起有临床意义的CYP450酶抑制现象的发生。     

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.