Effects of bergamottin on human and monkey drug-metabolizing enzymes in primary cultured hepatocytes.

We investigated the effect of bergamottin, a major furanocoumarin in grapefruit juice, on phase I and phase II drug-metabolizing enzymes using cultured human and monkey hepatocytes. Both cultured systems were compared and evaluated for the direct effects of bergamottin as well as control treatments on liver enzymes. Treatment of hepatocytes with 0.1, 1, 5, and 10 microM bergamottin resulted in a concentration-dependent reduction in CYP3A4 activity (40-100%) in both human and monkey cells, as measured by testosterone 6 beta-hydroxylase activity. Bergamottin was potent at eliciting these inhibitory effects at both basal and induced states of CYP3A. Bergamottin (5 microM) completely inhibited alpha-naphthoflavone-induced ethoxyresorufin O-dealkylase (EROD) and methoxyresorufin O-dealkylase (MROD) activities in human hepatocytes and caused a 100% decrease in EROD activity in monkey hepatocytes. A 48-h exposure of cultured human hepatocytes to bergamottin resulted in increased levels of immunoreactive CYP3A4, CYP1A1, and CYP1A2 proteins, and CYP3A4, CYP1A1, CYP1A2, CYP2B6, and UDP-glucuronosyl transferase mRNAs. There was only a 20 to 30% reduction in glucuronidation and sulfation of 4-methylumbelliferone in human hepatocytes by 10 microM bergamottin and no effect on conjugation in the monkey hepatocytes. These results suggest that bergamottin causes both inhibition of CYP3A and CYP1A1/2 enzymatic activities and induction of correspondent proteins and mRNAs.     

Effect of protein-calorie malnutrition on cytochromes P450 and glutathione S-transferase.

Protein-calorie malnutrition (PCM) can develop both from inadequate food intake and as a consequence of diseases such as cancer and AIDS. Several studies have shown that PCM can alter drug clearance but little information is available on the effect of PCM on individual cytochrome P450 isoforms and phase II conjugation enzymes. The aim of the present study was to begin a systematic evaluation of the effect of PCM on the activity of individual drug metabolizing enzymes in a rat model of PCM. Control and PCM rats received isocaloric diets which contained either 21% or 5% (deficient) protein. After 3 weeks, the animals were sacrificed and microsomal and cytosolic fractions prepared. Ethoxyresorufin O-deethylation (EROD), chlorzoxazone 6-hydroxylation, dextromethorphan N- and O-demethylation and 1-chloro-2,4-dinitrobenzene (CDNB) conjugation were used as measures of CYP1A, CYP2E1, CYP3A2, CYP2D1 and glutathione S-transferase (GST) activity, respectively. Additionally, NADPH-cytochrome P450 reductase activity was measured in the liver microsomes. PCM significantly reduced the maximum velocity (Vmax) of all model reactions studied. However, differential effects were observed with respect to K(m) values of the reactions. The K(m) values for EROD and dextromethorphan N-demethylation were significantly increased in PCM animals, whereas the K(m) values for chlorzoxazone 6-hydroxylation and dextromethorphan O-demethylation were decreased. In contrast, the K(m) value for CDNB conjugation was unchanged. When NADPH-cytochrome P450 reductase activity was compared, a 29% reduction in reductase activity was noted in PCM animals as compared to controls. Thus, it appears that PCM decreases the overall activity of certain phase I and phase II metabolism enzymes in rat liver while exhibiting differential effects on K(m). Furthermore, this reduction in activity may be due in part to diminished activity of cytochrome P450 reductase.     

Evaluation of animal models for intestinal first-pass metabolism of drug candidates to be metabolized by CYP3A enzymes via in vivo and in vitro oxidation of midazolam and triazolam

Abstract

1. To search an appropriate evaluation methodology for the intestinal first-pass metabolism of new drug candidates, grapefruit juice (GFJ)- and vehicle (tap water)-pretreated mice or rats were orally administered midazolam (MDZ) or triazolam (TRZ), and blood levels of the parent compounds and their metabolites were measured by liquid chromatography/MS/MS. A significant effect of GFJ to elevate the blood levels was observed only for TRZ in mice.

2. In vitro experiments using mouse, rat and human intestinal and hepatic microsomal fractions demonstrated that GFJ suppressed the intestinal microsomal oxidation of MDZ and especially TRZ. Substrate inhibition by MDZ caused reduction in 1′-hydroxylation but not 4-hydroxylation in both intestinal and hepatic microsomal fractions. The kinetic profiles of MDZ oxidation and the substrate inhibition in mouse intestinal and hepatic microsomal fractions were very similar to those in human microsomes but were different from those in rat microsomes. Furthermore, MDZ caused mechanism-based inactivation of cytochrome P450 3A-dependent TRZ 1′-hydroxylation in mouse, rat and human intestinal microsomes with similar potencies.

3. These results are useful information in the analysis of data obtained in mouse and rat for the evaluation of first-pass effects of drug candidates to be metabolized by CYP3A enzymes.     

补骨脂素和异补骨脂素对体外细胞色素P450酶活性的抑制和诱导作用

目的探讨补骨脂素(PSO)和异补骨脂素(IPSO)对细胞色素P450(CYP)活性的抑制和诱导作用。方法将人肝微粒体或鼠肝微粒体与PSO或IPSO以及CYP特异性探针底物共孵育30 min,分别以非那西丁O-脱乙基、甲苯磺丁脲4-羟基化、美芬妥因-4-羟基化、右美沙芬O-脱甲基化和咪达唑仑1'-羟基化为同工酶CYP1A2,CYP2C9,CYP2C19,CYP2D6(大鼠2D2)和CYP3A4(大鼠3A1/2)代谢活性的标志,用HPLC-MS/MS法检测相应代谢产物的生成量并计算相应的IC50值,评价PSO和IPSO对5种CYP同工酶的潜在抑制作用。将PSO和IPSO或阳性诱导剂与"三明治"培养大鼠肝原代细胞共孵育72 h后,再加入CYP探针底物孵育1 h,检测相应代谢产物的生成量,与阳性诱导剂组比较,评价二者对CYP1A和CYP3A的诱导作用。结果 PSO和IPSO对人肝微粒体和鼠肝微粒体的CYP1A2均有较强的抑制作用,在人肝微粒体中的IC50值分别为0.17和0.13μmol·L-1;在鼠肝微粒体中的IC50值分别为0.47和0.36μmol·L-1。二者对人肝微粒体的CYP2D6也有中等强度的抑制作用,IC50值分别为3.59和9.51μmol·L-1。PSO和IPSO 100μmol·L-1可分别将大鼠肝细胞的CYP3A活性提高1.18和0.96倍,有一定的诱导作用。结论 PSO和IPSO能显著抑制CYP1A2酶活性,对CYP3A有一定的诱导作用。     

In vivo and in vitro induction of cytochrome P450 enzymes in beagle dogs.

The aim of this study was to determine the in vitro and in vivo effects of several prototypical inducers, namely beta-naphthoflavone, 3-methylcholanthrene, phenobarbital, isoniazid, rifampin, and clofibric acid, on the expression of cytochrome P450 (P450) enzymes in beagle dogs. For the in vitro induction study, primary cultures of dog hepatocytes were treated with enzyme inducers for 3 days, after which microsomes were prepared and analyzed for P450 activities. For the in vivo induction study, male and female beagle dogs were treated with enzyme inducers for 4 days (with the exception of phenobarbital, which was given for 14 days), after which the livers were removed and microsomal P450 activities were determined ex vivo. Treatment of male beagle dog hepatocyte cultures (n = 3) with beta-naphthoflavone or 3-methlychloranthrene resulted in up to a 75-fold increase in microsomal 7-ethoxyresorufin O-dealkylase (CYP1A1/2) activity, whereas in vivo treatment of male and female beagle dogs with beta-naphthoflavone followed by ex vivo analysis resulted in up to a 24-fold increase. Phenobarbital caused a 13-fold increase in 7-benzyloxyresorufin O-dealkylase (CYP2B11) activity in vitro and up to a 9.9-fold increase in vivo. Isoniazid had little or no effect on 4-nitrophenol hydroxylase activity in vitro. Rifampin caused a 13-fold induction of testosterone 6beta-hydroxylase (CYP3A12) activity in vitro and up to a 4.5-fold increase in vivo. Treatment of dogs in vivo or dog hepatocytes in vitro with clofibric acid appeared to have no effect on CYP4A activity as determined by the 12-hydroxylation of lauric acid. In general, the absolute rates (picomoles per minute per milligram of microsomal protein) of P450 reactions catalyzed by microsomes from cultured hepatocytes (i.e., in vitro rates) were considerably lower than those catalyzed by microsomes from dog liver (i.e., ex vivo rates). These results suggest that beagle dogs have CYP1A, CYP2B, CYP2E, and CYP3A enzymes and that the induction profile resembles the profile observed in humans more than in rats.     

Human adult hepatocytes in primary monolayer culture. Maintenance of mixed function oxidase and conjugation pathways of drug metabolism

The stabilities of several drug oxidation and conjugation pathways in human adult hepatocytes have been investigated during 72 hr culture. Cytochrome P-450-dependent mixed function oxidase was measured by the O-dealkylations of ethoxyresorufin (EROD), pentoxyresorufin (PROD) and benzyloxyresorufin (BROD), which are probes for different isozymes of cytochrome P-450 in the rat. EROD declined to 64% of initial fresh cell values after 72 hr in culture, whereas PROD increased to 162% and BROD remained relatively constant. Addition of phenobarbitone to the culture medium selectively increased PROD to a greater extent than EROD and did not affect BROD. NADPH-cytochrome c reductase and NADH-cytochrome b5 reductase were markedly labile during culture, declining to 32% and 22% of fresh cell values respectively. Epoxide hydrolase (EH) showed a large transient increase (2-5-fold) in enzyme activity 24 hr after culture, declining to fresh cell values by 48 hr. UDP-glucuronyltransferase (GT) activity towards phenolphthalein and 1-naphthol also increased (2-3-fold) during the 72 hr of culture, the greater and more rapid increase being observed with phenolphthalein glucuronidation. Sulphotransferase activity declined rapidly within 24 hr of culture, whereas reduced glutathione (GSH) levels and GSH conjugation were maintained at fresh cell values for 72 hr.     

The inhibitory effect of tannic acid on cytochrome P450 enzymes and NADPH-CYP reductase in rat and human liver microsomes.

Tannic acid has been shown to decrease mutagenicity and/or carcinogenicity of several amine derivatives and polycyclic aromatic hydrocarbons in rodents. The purpose of this study was to evaluate the effect of tannic acid on cytochrome P450 (CYP)-catalyzed oxidations using rat liver microsomes (RLM) and human liver microsomes (HLM) as the enzyme sources. In RLM, tannic acid showed a non-selective inhibitory effect on 7-methoxyresorufin O-demethylation (MROD), 7-ethoxyresorufin O-deethylation (EROD), tolbutamide hydroxylation, p-nitrophenol hydroxylation and testosterone 6beta-hydroxylation activities with IC(50) values ranged from 14.9 to 27.4 microM. In HLM, tannic acid inhibited EROD, MROD and phenacetin O-deethylation activities with IC(50) values ranged from 5.1 to 7.5 microM, and diclofenac 4-hydroxylation, dextromethorphan O-demethylation, chlorzoxazone 6-hydroxylation and testosterone 6beta-hydroxylation with IC(50) values ranged from 20 to 77 microM. In baculovirus-insect cell-expressed human CYP 1A1 and 1A2, the IC(50) values of tannic acid for CYP 1A1- and 1A2-catalyzed EROD activities were 23.1 and 2.3 microM, respectively, indicating that tannic acid preferably inhibited the activity of CYP1A2. Tannic acid inhibited human CYP1A2 non-competitively with a Ki value of 4.8 microM. Tannic acid was also found to inhibit NADPH-CYP reductase in RLM and HLM with IC(50) values of 11.8 and 17.4 microM, respectively. These results suggested that the inhibition of CYP enzyme activities by tannic acid may be partially attributed to its inhibition of NADPH-CYP reductase activity.     

Cryopreserved rat, dog and monkey hepatocytes: measurement of drug metabolizing enzymes in suspensions and cultures

Metabolism in fresh and cryopreserved (CP) rat, dog and monkey hepatocyte suspensions and cultures was measured using midazolam (CYP3A), tolbutamide (CYP2C), dextromethorphan (CYP2D) and p-nitrophenol (glucuronosyl S-transferases (UGT), sulphotransferases (ST)). CYP3A, CYP2C9, CYP2D6, UGT and ST enzyme functions in fresh and CP rat, dog and monkey hepatocyte suspensions were retained - CP rat hepatocytes lost some CYP2C activity but this was restored by adding NADPH or by placing the cells in culture, suggesting that the enzyme was still functional. Phase 2 activities were equivalent in fresh and CP hepatocyte suspensions. In some cases, incubation conditions increased the rate of metabolism, possibly reflecting de novo cofactor synthesis. However, this effect was substrate and species dependent and was not always the same in fresh and CP cells. CYP3A, CYP2C, CYP2D, UGT and ST activities at 24 hours of culture of rat and monkey hepatocytes were not compromised by cryopreservation. CYP3A, CYP2D but not CYP2C were lower in 24-hour cultures of CP dog hepatocytes than in fresh cells. Despite being lower than fresh cells, UGT activity in dog CP hepatocytes did not decrease from 0 to 24 hours of culture. Species-specific metabolism of p-nitrophenol could be demonstrated in both CP cell suspensions and cultures. In conclusion, these data suggest that the enzyme characteristics of fresh and CP hepatocytes from each species and under specific incubation conditions should be considered when carrying out metabolism studies of new compounds.     

Conflicting alterations in hepatic expression of CYP3A and enzyme kinetics in rats exposed to 5-fluorouracil: relevance to pharmacokinetics of midazolam

Abstract

1. 5-Fluorouracil (5-FU) is a pyrimidine derivative widely used for the treatment of cancer. In this study, we investigated the effects of 5-FU on the protein expression of hepatic CYP3A and their enzyme activity for metabolizing midazolam (MDZ), a typical substrate of CYP3A, in rat liver microsomes. We also examined the pharmacokinetic behavior of intravenously administered MDZ in rats treated with 5-FU (120?mg/kg, ip).

2. 5-FU was shown to induce hepatic CYP3A2 protein 2?days after administration without changing the expression of CYP3A1/3A23. However, affinity of 5-FU-inducible CYP3A protein to MDZ for its 4- and 1′-hydroxylation was decreased. Furthermore, the susceptibility of MDZ hydroxylation activity to a CYP3A inhibitor differed between the control and 5-FU groups.

3. Pharmacokinetic analysis of the MDZ disposition demonstrated no significant differences in the total clearance (CL_tot) and elimination rate constant (k_e) between the control and 5-FU-treated rats. Lack of alteration in the metabolic clearance of MDZ may be attributable to the induction of CYP3A protein with reduced affinity for the substrate of CYP3A enzymes.

4. Our findings provide novel information regarding the manifestation of inductive and interfering actions of 5-FU toward hepatic CYP3A to help in assessing the pharmacokinetics of CYP3A substrate drugs.     

Human cytochrome P450 3A (CYP3A) mediated midazolam metabolism: the effect of assay conditions and regioselective stimulation by alpha-naphthoflavone, terfenadine and testosterone

The effect of ionic strength, assay constituents, alpha-naphthoflavone (aNF), terfenadine and testosterone on human CYP3A mediated midazolam (MDZ) 1'-hydroxylation (MDZ 1'-OH) and 4-hydroxylation (MDZ 4-OH) in vitro was examined. Increasing concentration of Tris-HCl (Tris) and sodium phosphate (PO4) buffers differentially affected MDZ 1'-OH and MDZ 4-OH formation rates and had a different effect on MDZ metabolism mediated by microsomes containing CYP3A4 versus CYP3A4 and CYP3A5. MDZ metabolism was not affected by PO4 buffer concentration when cumene hydroperoxide (CUOOH) was used as the source of reactive oxygen. Interestingly, the ammonium ion present in the solution of glucose 6-phosphate dehydrogenase was found to inhibit MDZ metabolism. The addition of MgCl2 up to 50 mM and CaCl2 (5-30 mM) had no affect or inhibited MDZ metabolism, respectively. Formation of MDZ 1'-OH by microsomes from adult and fetal liver and expressed CYP3A4 was regioselectively stimulated by aNF (10 microM). In human hepatocytes, aNF stimulated MDZ 1'-OH formation (up to 100%). Terfenadine (20 microM) regioselectively stimulated MDZ 1'-OH formation in Tris (1-200 mM) and PO4 (1-10 mM) buffers by up to 159%. Surprisingly, with expressed CYP3A4, terfenadine (20 microM) inhibited MDZ 1'-OH formation. Terfenadine (20 microM) had little effect on MDZ 1'-OH formation by fetal liver microsomes. Testosterone (10 and 100 microM) regioselectively stimulated (up to 269%) MDZ 4-OH formation by adult liver microsomes and expressed CYP3A4. Testosterone (100 microM) inhibited (> 40%) MDZ 1'-OH and MDZ 4-OH formation by fetal liver microsomes. With adult liver microsomes, aNF and terfenadine had little effect on the Km for MDZ 1'-OH formation. However, the Km for MDZ 4-OH formation was decreased (up to 94%) by 100 microM testosterone. In the presence of CUOOH, no stimulation of MDZ metabolism was observed by aNF, terfenadine or testosterone in adult liver microsomes. These studies indicate that because assay conditions can substantially alter the catalytic activity of CYP3A, caution should be exerted when extrapolating results between in vitro and in vivo, and when results from different laboratories are compared. Further, these results suggest that the stimulation of CYP3A4 may also occur in vivo and, consequently, may have clinical importance.     

Roles of cytochrome P450 3A enzymes in the 2-hydroxylation of 1,4-cineole,a monoterpene cyclic ether,by rat and human liver microsomes

1. Oxidation of 1,4-cineole, a monoterpene cyclic ether, was studied in rat and human liver microsomes and recombinant cytochrome P450 (P450 or CYP) enzymes expressed in insect cells in which human P450 and NADPH-P450 reductase cDNAs have been introduced. On analysis with gas chromatography/mass spectrometry, 2- exo -hydroxy-1,4-cineole was identified as a principal oxidation product of 1,4-cineole catalysed by rat and human P450 enzymes. 2. CYP3A4 was a major enzyme involved in the 2-hydroxylation of 1,4-cineole by human liver microsomes, based on the following lines of evidence. First, 1,4-cineole 2-hydroxylation activities catalysed by human liver microsomes were inhibited by ketoconazole, a potent inhibitor of CYP3A activities, and an anti-CYP3A4 antibody. Second, there was a good correlation between CYP3A4 contents and 1,4-cineole 2-hydroxylation activities in liver microsomes of eighteen human samples examined. Finally, of 10 recombinant human P450 enzymes examined, CYP3A4 had the highest activity for 1,4-cineole 2-hydroxylation. 3. Liver microsomal 1,4-cineole 2-hydroxylation activities were induced in rat by pregnenolone 16 α-carbonitrile and dexamethasone and extensively inhibited by ketoconazole, indicative of the possible roles of CYP3A enzymes in this reaction. 4. Kinetic analysis showed that V max / K m for 1,4-cineole 2-hydroxylation catalysed by liver microsomes was higher in a human sample HL-104 (4.6 μM -1?min -1) than those of rat treated with pregnenolone 16 α-carbonitrile (0.49 μM -1?min -1) and dexamethasone (0.36 μM -1?min -1). 5. 1,8-Cineole, a structurally related monoterpene previously shown to be catalysed by CYP3A enzymes, inhibited 1,4-cineole 2-hydroxylation catalysed by human liver microsomes, whereas 1,4-cineole did not inhibit 1,8-cineole 2-hydroxylation activities. Both compounds caused inhibition of testosterone 6 β -hydroxylation by human liver microsomes, the former compound being more inhibitory than the latter. 6. These results suggest that 1,4-cineole and 1,8-cineole, two plant essential oils present in Citrus medica L. var. acida and Eucalyptus polybractea, respectively, are converted to 2-hydroxylated products by CYP3A enzymes in rat and human liver microsomes. It is unknown at present whether the 2-hydroxylation products of these compounds are more active biologically than the parent compound.     

Roles of cytochrome P450 3A enzymes in the 2-hydroxylation of 1,4-cineole, a monoterpene cyclic ether, by rat and human liver microsomes.

1. Oxidation of 1,4-cineole, a monoterpene cyclic ether, was studied in rat and human liver microsomes and recombinant cytochrome P450 (P450 or CYP) enzymes expressed in insect cells in which human P450 and NADPH-P450 reductase cDNAs have been introduced. On analysis with gas chromatography/mass spectrometry, 2-exo-hydroxy-1,4-cineole was identified as a principal oxidation product of 1,4-cineole catalysed by rat and human P450 enzymes. 2. CYP3A4 was a major enzyme involved in the 2-hydroxylation of 1,4-cineole by human liver microsomes, based on the following lines of evidence. First, 1,4-cineole 2-hydroxylation activities catalysed by human liver microsomes were inhibited by ketoconazole, a potent inhibitor of CYP3A activities, and an anti-CYP3A4 antibody. Second, there was a good correlation beteeen CYP3A4 contents and 1,4-cineole 2-hydroxylation activities in liver microsomes of eighteen human samples examined. Finally, of 10 recombinant human P450 enzymes examined, CYP3A4 had the highest activity for 1,4-cineole 2-hydroxylation. 3. Liver microsomal 1,4-cineole 2-hydroxylation activities were induced in rat by pregnenolone 16alpha-carbonitrile and dexamethasone and extensively inhibited by ketoconazole, indicative of the possible roles of CYP3A enzymes in this reaction. 4. Kinetic analysis showed that Vmax/Km for 1,4-cineole 2-hydroxylation catalysed by liver microsomes was higher in a human sample HL-104 (4.6 microM(-1) min(-1)) than those of rat treated with pregnenolone 16alpha-carbonitrile (0.49 microM(-1) min(-1)) and dexamethasone (0.36 microM(-1) min(-1)). 5. 1,8-Cineole, a structurally related monoterpene previously shown to be catalysed by CYP3A enzymes, inhibited 1,4-cineole 2-hydroxylation catalysed by human liver microsomes, whereas 1,4-cineole did not inhibit 1,8-cineole 2-hydroxylation activities. Both compounds caused inhibition of testosterone 6beta-hydroxylation by human liver microsomes, the former compound being more inhibitory than the latter. 6. These results suggest that 1,4-cineole and 1,8-cineole, two plant essential oils present in Citrus medica L. var. acida and Eucalyptus polybractea, respectively, are converted to 2-hydroxylated products by CYP3A enzymes in rat and human liver microsomes. It is unknown at present whether the 2-hydroxylation products of these compounds are more active biologically than the parent compound.     

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.     

Rat adult hepatocytes in primary pure and mixed monolayer culture. Comparison of the maintenance of mixed function oxidase and conjugation pathways of drug metabolism

The stabilities of several drug oxidation and conjugation pathways in adult rat hepatocytes were investigated in two systems: a primary pure culture lasting 3 days and a primary mixed culture (hepatocytes co-cultured with epithelial cells) lasting 10 days. The cytochrome P450 content in hepatocytes drastically declined within 48 hr in both culture systems. Cytochrome P450-dependent mixed function oxidase was measured by the O-dealkylation of ethoxyresorufin (EROD) and of pentoxyresorufin (PROD). UPD-glucuronosyl transferase (UDP-GT) activity was measured using 1-naphthol and morphine as substrates. In both culture systems, the activities of enzymes belonging to the 3-methylcholanthrene-inducible family, namely EROD and 1-naphthol UDP-GT, were much better maintained than those of PROD and morphine UDP-GT, which belong to the phenobarbitone-inducible family: in pure cultures, EROD and 1-naphthol UDP-GT activities declined to 60% of initial values within 3 days; in mixed cultures, EROD activity was stable throughout the 10 day culture period, whereas that of 1-naphthol UDP-GT was stable until day 4 but had declined to 70% of the initial value by day 8. In contrast, PROD and morphine UDP-GT activities declined to approx. 30% of the initial values within 2 days in both culture systems, and had dropped to approx. 10% of the initial value within 8 days in mixed culture. Reduced glutathione (GSH) levels fluctuated, but remained high throughout culture. GSH conjugation declined to 40% of initial values within 3 days in pure culture, whereas it remained relatively constant in mixed culture. Comparison of these two culture systems therefore showed that although the inclusion of epithelial cells did prolong hepatocyte viability, there was a change in relative enzyme activities in both systems, suggesting a shift towards a more de-differentiated drug metabolism pattern.     

Quantification of CYP1A1 and 2B1/2 in rat hepatocytes cultured in microwells by immunological methods

The quantification of P-450 enzymes (CYP proteins) is of great relevance for drug metabolism and toxicity studies. Their measurement in cultured cells requires a sufficient amount of cellular protein to isolate a minimal amount of functional microsomes. The difficulties in obtaining microsomes from a small number of cultured hepatocytes make it necessary to use more sensitive methods. Immunological methods can overcome such drawbacks. However, their use is prevented by the fact that P-450 enzymes are membrane proteins that require the use of detergents for adequate solubilization which, in turn, can interfere with the antigen-antibody recognition. A sensitive immunoassay (competitive indirect ELISA) was developed which allowed quantification of CYP1A1 and CYP2B1/2 in small culture plates with a reduced number of cells. The main advantages of this ELISA were: (1) precise measurement of both proteins over a wide range of antigen concentration (10–10,000 fmol CYP proteins); (2) minimal sample handling allowing reproducible titrations in small number of cells; (3) minimization of interference by detergents; (4) an excellent correlation (r = 0.96–0.98) between the immunologically determined CYP1A1 and CYP2B1/2, and the activities of ethoxyresorufin-O-deethylase (EROD) and pentoxyresorufin-O-depentylase (PROD), respectively. This assay allowed a precise monitoring of the expression of CYP proteins in cultured cells. Upon incubation of hepatocytes with 3-methylcholanthrene the levels of immunoreactive CYP1A1 and CYP2B1/2 were paralleled by an increase of the enzymatic activities of EROD and PROD, respectively. In cultured rat hepatocytes treated with phenobarbital, significant EROD activity was detected in the absence of immunoreactive CYP1A1 induction. Analogously, there was a dissimilarity between enzymatic activity and protein levels in ageing cultures: the decrease of PROD activity preceded by several hours that of the immunoreactive CYP2B1/2, which emphasizes the need to determine individual CYPs, in addition to their enzymatic activities.     

3-甲基胆蒽对大鼠肝细胞细胞色素P450 1A的诱导作用

目的　研究 3 甲基胆蒽 (3 methylcholanthrene,3 MC)对原代培养大鼠肝细胞细胞色素P450 1A(CYP 1A)酶活力的诱导作用 ,并探讨其时间 效应和剂量 效应关系。方法　原位 2步胶原酶灌流法分离大鼠肝细胞 ,无血清条件下培养细胞 ,并用不同剂量的 3 MC诱导肝细胞 2 4h或 48h。乙氧基试卤灵 (ethoxyresorufin,EOR)为CYP 1A酶活力探针药 ,高效液相色谱法 (HPLC)测定试卤灵 (resorufin,RSF)浓度。结果　实验条件下对照组和 3 MC诱导组的RSF均可被检测 ,与对照细胞相比 ,除 0 0 0 5μmol L的 3 MC对原代培养大鼠肝细胞EROD无显著诱导作用外 ,其余剂量 (0 0 5 ,0 5 ,5μmol L)的 3 MC均明显诱导酶活力 ,酶活力分别被上调至对照组的 5 0 8,2 3 3 ,33 6倍 (P <0 0 1 ) ,3 MC的诱导作用在 2 4h达最强 ,而其在 48h的诱导作用与 2 4h相似。3 MC对原代培养大鼠肝细胞CYP 1A酶活力的诱导作用呈明显的剂量依赖性。结论　 3 MC对大鼠原代培养肝细胞CYP 1A酶活力有明显的诱导作用     

Use of a cocktail of probe substrates for drug-metabolizing enzymes for the assessment of the metabolic capacity of hepatocyte preparations

A cocktail of the following probe substrates for human drug-metabolizing enzymes was used to characterize hepatocyte preparations: phenacetin (for CYP1A2), diclofenac (CYP2C9), diazepam (CYP2C19), bufuralol (CYP2D6), midazolam (CYP3A4/5) and 7-hydroxycoumarin (for glucuronidation and sulphation). The cocktail was incubated with cryopreserved human, dog or minipig hepatocytes or with freshly prepared rat hepatocytes. Sample analysis was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in an Open Access environment that allowed less experienced MS operators to login, submit and analyse sample sets using predefined settings without the immediate attendance of an experienced analyst. Intrinsic clearances (CLint) were calculated from the disappearance of the compounds from the incubations. Initially, the cocktail used for human, rat and dog hepatocyte incubations contained 7-ethoxycoumarin instead of 7-hydroxycoumarin. However, 7-ethoxycoumarin had an inhibitory effect on the metabolism of phenacetin. The highest CLint estimated with human and dog hepatocytes was observed for 7-hydroxycoumarin. For rat and minipig hepatocytes, the highest CLint was observed for bufuralol. In incubations with dog and minipig hepatocytes, the lowest CLint was seen with diclofenac, whereas for human and rat hepatocytes, the lowest value was observed with diazepam and phenacetin, respectively. When the cocktail was incubated together with human hepatocytes and 1 microM ketoconazole, the CLint of midazolam was decreased to about 7.5% of the control value, whereas the metabolism of the other cocktail compounds was virtually unaffected by this CYP3A inhibitor. It is suggested that a cocktail of specific human probe substrates for drug-metabolizing enzymes can be used routinely for the determination of the metabolic capacity of hepatocyte preparations in order to ensure the quality and reproducibility of experiments. Moreover, a cocktail of specific probe substrates can also be a useful tool for studies on enzyme inhibition.     

Effect of frying-meat emission particulate on 17beta-estradiol 2- and 4-hydroxylation in human lung adenocarcinoma CL5 cells

The effect of airborne frying-meat emission particulate (FMEP) on metabolism of 17beta-estradiol (E(2)) to potentially toxic catechol estrogens 2- and 4-hydroxyestradiol (2- and 4-OH-E(2)) was determined using human lung adenocarcinoma CL5 cells treated with organic extracts of beef FMEP. E(2) was incubated with microsomes prepared from untreated CL5 cells or cells treated with 200 microg/ml FMEP extract for 6 h. E(2) metabolites formed were analyzed by high-performance liquid chromatography (HPLC). The results revealed that treatment with FMEP produced three-and twofold increases of 2- and 4-hydroxylation of E(2), respectively. Monooxygenase activity and immunoblot analyses showed that FMEP markedly induced microsomal 7-ethoxyresorufin O-deethylase (EROD) activity and cytochrome P-450 (CYP) IAI and CYPIBI protein levels. Similar increases in E(2) hydroxylation, EROD activity, and CYP protein levels were observed with HepG2 human hepatoma and MCF-7 human breast cancer cells treated with FMEP or 1 microM dibenz[a,h]anthracene. Cotreatment of CL5 cells with FMEP extract and 2 microM alpha-naphthoflavone, an arylhydrocarbon receptor antagonist, blocked the inductive effects of FMEP on E(2) hydroxylation and EROD activity. Additions of 0.01, 0.1, or 1 microM alpha-naphthoflavone, a CYP inhibitor, to microsomes produced concentration-dependent decreases in E(2) 2-hydroxylation and EROD activity of CL5 cells induced by dibenz[a,h]anthracene. The present finding demonstrates that FMEP can increase formation of 2-OH-E(2) and 4-OH-E(2) by human lung cells, and induction of CYP1A1 and CYP1B1 is a potential mechanism underlying increased E(2) metabolism. The toxicological significance of FMEP and estrogen interaction warrants further investigation.     

Roles of human CYP2A6 and 2B6 and rat CYP2C11 and 2B1 in the 10-hydroxylation of (-)-verbenone by liver microsomes.

(-)-Verbenone, a monoterpene bicyclic ketone, is a component of the essential oil from rosemary species such as Rosmarinus officinalis L., Verbena triphylla, and Eucalyptus globulus and is used for an herb tea, a spice, and a perfume. In this study, (-)-verbenone was found to be converted to 10-hydroxyverbenone by rat and human liver microsomal cytochrome p450 (p450) enzymes. The product formation was determined by high-performance liquid chromatography with UV detection at 251 nm. There was a good correlation between activities of coumarin 7-hydroxylation and (-)-verbenone 10-hydroxylation catalyzed by liver microsomes of 16 human samples, indicating that CYP2A6 is a principal enzyme in (-)-verbenone 10-hydroxylation in humans. Human recombinant CYP2A6 and CYP2B6 catalyzed (-)verbenone 10-hydroxylation at Vmax values of 15 and 21 nmol/min/nmol p450 with apparent Km values of 16 and 91 microM, respectively. In contrast, rat CYP2A1 and 2A2 did not catalyze (-)-verbenone 10-hydroxylation at all, suggesting that there were species-related differences in the catalytic properties of human and rat CYP2A enzymes in the metabolism of (-)-verbenone. In the rat, recombinant CYP2C11, CYP2B1, and CYP3A2 catalyzed (-)-verbenone 10-hydroxylation with Vmax and Km ratios (ml/min/nmol p450) of 0.73, 0.20, and 0.03, respectively. Male-specific CYP2C11 was a major enzyme in (-)-verbenone 10-hydroxylation by untreated rat livers, and CYP2B1 catalyzed this reaction in liver microsomes of phenobarbital-treated rats. Rat CYP2C12, a female-specific enzyme, did not catalyze (-)verbenone 10-hydroxylation. These results suggest that human CYP2A6 and rat CYP2C11 are the major catalysts in the metabolism of (-)-verbenone by liver microsomes and that there are species-related differences in human and rat CYP2A enzymes and sex-related differences in male and female rats in the metabolism of (-)-verbenone.     

Equally potent inhibitors of cholesterol synthesis in human hepatocytes have distinguishable effects on different cytochrome P450 enzymes

Six 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (the present cholesterol-lowering drugs known as statins), lovastatin (L), simvastatin (S), pravastatin (P), fluvastatin (F), atorvastatin (A) and cerivastatin (C) are shown to be potent inhibitors of cholesterol synthesis in human hepatocytes, the target tissue for these drugs in man. All six inhibited in the nM range (IC(50) values: 0.2-8.0 nM). As daily used cholesterol-lowering drugs they are likely coadministered with other drugs. While several cytochrome P450 (CYP) enzymes are involved in drug metabolism in the liver and thus play an important role in drug-drug interaction it was investigated which of these enzymes are influenced by the active forms of the six statins. These enzyme activities were studied in human liver microsomal preparations, and in simian and human hepatocytes in primary culture. The following CYP reactions were used: nifedipine aromatization (CYP3A4), testosterone 6beta-hydroxylation (CYP3A4), tolbutamide methylhydroxylation (CYP2C9), S-mephenytoin 4-hydroxylation (CYP2C19), bufuralol 1'-hydroxylation (CYP2D6), aniline 4-hydroxylation (CYP2E1), coumarin 7-hydroxylation (CYP2A6) and 7-ethoxyresorufin O-dealkylation (CYP1A1/2). In the human liver microsomes the statins (concentrations up to 400 microM) did not influence the CYP1A1/2 activity and hardly the CYP2A6 and CYP2E1 activities. Except P, the other five statins were stronger inhibitors of the CYP2C19 activity with IC(50) values around 200 microM and the same holds for the effect of A, C and F on the CYP2D6 activity. L and S were weaker inhibitors of the latter enzyme activity, whereas P did not influence both activities. About the same was observed for the statin effect on CYP2C9 activity, except that F was a strong inhibitor of this activity (IC(50) value: 4 microM). Using the assay of testosterone 6beta-hydroxylation the CYP3A4 activity was decreased by L, S and F with IC(50) values of about 200 microM and a little more by C and A (IC(50) around 100 microM). P had hardly an effect on this activity. To a somewhat less extent the same trend was seen when CYP3A4 activity was measured using nifedipine as substrate. The inhibitory effects observed in microsomes were verified in suspension culture of freshly isolated hepatocytes from Cynomolgus monkey (as a readily available model) and of human hepatocytes. In general the same trends were seen as in the human microsomes, except that in some cases the inhibition of the CYP activity was less, possibly by the induction of the particular CYP enzyme by incubation of the cells with a particular statin. F remained a strong inhibitor of CYP2C9 activity in human and monkey hepatocytes. A induced the CYP2C9 in monkey hepatocytes but was an inhibitor of the CYP2C9 in human hepatocytes. A, S, L and C were moderate inhibitors in both cellular systems of CYP3A4. P was not affecting any of the CYP activities in the three systems studied. It is concluded that different CYP enzymes interact with different statins and therefore differences in between these drugs are to be expected when drug-drug interaction is considered.