Cytochrome P4502C9 (CYP2C9) allele frequencies in Canadian Native Indian and Inuit populations.

CYP2C9 is the major P450 2C enzyme in human liver and contributes to the metabolism of a number of clinically important substrate drugs. This polymorphically expressed enzyme has been studied in Caucasian, Asian, and to some extent in African American populations, but little is known about the genetic variation in Native American populations. We therefore determined the 2C9*2 (Arg144Cys) and 2C9*3 (Ile359Leu) allele frequencies in 153 Native Canadian Indian (CNI) and 151 Inuit subjects by PCR-RFLP techniques. We also present genotyping data for two reference populations, 325 Caucasian (white North American) and 102 Chinese subjects. Genotyping analysis did not reveal any 2C9*4 alleles in the CNI, Inuit, Caucasian, or Chinese individuals. The 2C9*2 allele appears to be absent in Chinese and Inuit populations, but was present in CNI and Caucasian subjects at frequencies of 0.03 and 0.08-0.15, respectively. The 2C9*3 allele was not detected in the Inuit group, but occured in the CNI group (f = 0.06) at a frequency comparable to that of other ethnic groups. This group of Inuit individuals are the first population in which no 2C9*2 or *3 alleles have been detected so far. Therefore, these alleles may be extremely rare or absent, and unless other novel polymorphisms exist in this Inuit group one would not anticipate any CYP2C9 poor metabolizer subjects among this population.

Evidence for the catalysis of dextromethorphan O-demethylation by a CYP2D6-like enzyme in pig liver.

The pig is increasingly being used in pharmacological and toxicological studies, and is the species of choice for future research into xenotransplantation, extracorporeal liver support and hepatocyte-based bioartificial liver. However, relatively little is known about xenobiotic-metabolizing enzymes in this species. In the present study, immunoblotting with polyclonal anti-rat and anti-human cytochrome P450 (CYP) antibodies revealed the presence of proteins in pig liver which cross-reacted with anti-human CYP1A2, CYP2D6 and CYP3A4, and with anti-rat CYP2E1 antibodies. Northern blot analysis demonstrated the presence of mRNA which hybridized to cDNA probes for human CYP2D6, CYP2E1 and CYP3A4, and to an oligonucleotide probe for pig CYP3A29. As there is a lack of a good animal model for CYP2D6, the presence of a CYP2D6-related protein in pig liver was of particular interest. Pig hepatocytes also demonstrated CYP2D6 immunoreactive protein, and mRNA hybridizable to a CYP2D6 cDNA probe. We investigated the ability of pig liver microsomes to catalyse dextromethorphan O-demethylation, a widely-used marker enzyme activity for CYP2D6. This enzyme activity demonstrated biphasic kinetics, with a high affinity apparent K(m1)=6.9+/-3.6 microM and V(max1)=10.5+/-6.1nmol/min/nmol CYP. The reaction was sensitive to inhibition by the CYP2D6-selective inhibitors quinidine, quinine, lobeline and norfluoxetine, whereas chemical inhibitors selective for other CYP isoforms failed to affect the reaction. We conclude that dextromethorphan O-demethylation is catalysed by a CYP2D enzyme which is remarkably similar to human CYP2D6, suggesting potential value of the pig as a model for predicting human metabolism of xenobiotics which undergo CYP2D6-dependent biotransformation.

NAD(P)H:quinone oxidoreductase: polymorphisms and allele frequencies in Caucasian, Chinese and Canadian Native Indian and Inuit populations.

NAD(P)H:quinone oxidoreductase (NQO1) catalyses the two-electron reduction of quinone compounds. NQO1 is involved in the reductive bioactivation of cytotoxic antitumour quinones such as mitomycin C, but also plays a protective role against the carcinogenicity and mutagenicity of quinones, their precursors and metabolites. Three alleles have been identified in the human population: the functional Arg139/Pro187 allele (which we have termed NQO1*1); the nonfunctional allele Arg139/Ser187 (NQO1*2) and the Trp139/Pro187 allele (NQO1*3), which is associated with a diminished activity. We applied polymerase chain reaction-based genotyping assays to characterize interethnic variability in the frequency of NQO1 alleles in Caucasian (n = 575), Canadian Native Indian (n = 110), Canadian Inuit (n = 83) and Chinese (n = 86) populations. The NQO1*2 allele was found at significantly higher frequencies in Chinese (0.49) and Native North American populations (Inuit 0.46; Canadian Native Indians 0.40) compared with Caucasians (0.16). The NQO1*3 allele was not observed in Inuit individuals, and occurred at a lower frequency than the NQO*2 allele in Caucasians (0.05), Chinese (0.04) and Canadian Native Indians (0.01). Our results predict that a greater proportion of Orientals and related ethnic groups lack, or have reduced, NQO activity relative to Caucasians. Affected individuals may not only exhibit resistance to quinone-based cancer therapy because of a decreased production of cytotoxic drug metabolites, but may also be more susceptible to toxicities associated with toxicants.

Terfenadine-antidepressant interactions: an in vitro inhibition study using human liver microsomes.

AIMS: Inhibition of the metabolism of terfenadine has been associated with torsades de pointes ventricular arrhythmias. The aim of this study was to assess in vitro the potency of the antidepressants nefazodone, sertraline and fluoxetine in inhibiting terfenadine biotransformation. METHODS: Human liver microsomes were incubated with terfenadine and the antidepressants at various concentrations. Formation of the two major metabolites of terfenadine was determined by h.p.l.c. RESULTS: The apparent Km for microsomes from four human livers was 11+/-5 and 18+/-3 microM (mean +/-s.e.mean) for the N-dealkylation and C-hydroxylation pathways, respectively. Nefazodone, sertraline and fluoxetine inhibited terfenadine N-dealkylation with K(i) values of 10+/-4, 10+/-3 and 68+/-15 microM respectively. Inhibition of the C-hydroxylation pathway yielded noncompetitive K(i) values of 41+/-4, 67+/-13 and 310+/-40 microM respectively. CONCLUSIONS: Nefazodone and sertraline were moderately weak in vitro inhibitors of terfenadine metabolism while fluoxetine was a very weak inhibitor. Clinically significant interaction of terfenadine is more likely with nefazodone than sertraline or fluoxetine since therapeutic plasma levels of nefazodone are comparatively higher.

Preclinical evaluation of drug-drug interaction potential: present status of the application of primary human hepatocytes in the evaluation of cytochrome P450 induction.

Cytochrome P450 (CYP) inhibition and induction are the key mechanisms in drug-drug interactions. Aside from clinical studies, primary human hepatocytes may represent the most appropriate experimental system for the evaluation of CYP induction in humans. A consensus of an international panel on the present status and future research directions in the application of primary human hepatocytes in the evaluation of CYP-induction is presented here. The following observations are concluded to be generally true: (1) Human hepatocytes isolated from both biopsy samples and transplantable livers are suitable for induction studies. (2) Hormonally-defined media can be used for the evaluation of CYP induction. (3) Isozyme-selective induction of CYP1A and 3A by known inducers are observed. (4) Reproducibility of induction could be improved by using hepatocytes plated as confluent cultures. (5) Induction could be observed for hepatocytes treated at 1-3 days after culturing. (6) Treatment duration of 2 days in general leads to near maximal induction. (7) In general, there is a good qualitative correlation between human hepatocyte results in vitro and clinical observations in vivo. (8) When the same inducers were evaluated in independent laboratories, similar data were generally observed. We conclude that primary human hepatocytes represent an appropriate model for mechanistic evaluation of CYP induction and as a screening tool for CYP induction potential of xenobiotics. A set of data acceptance criteria are proposed: (1) Positive response should be observed with concurrent positive control chemicals; (2) reproducible observation should be observed with multiple human donors; (3) for negative response, the doses used should not be cytotoxic; and (4) replicate treatment and/or multiple dose treatment should be performed to allow statistical analysis. Future studies should include the further development of on: (1) The inducibility of CYP isozymes other than CYP1A and 3A, and phase II enzymes; (2) further development of culturing condition to allow optimal gene expression; (3) evaluation of the involvement of nonparenchymal cells on CYP induction of parenchymal cells; (4) the and validation of quantitative approaches to extrapolate in vitro data to in vivo data; (5) evaluation of possible individual variations and potential genetic polymorphism in inducibility; (6) further definition of species differences in CYP induction; (7) development of a 'normal' human hepatocyte cell line for CYP induction studies; (8) improvement of cryopreservation procedure of human hepatocytes; (9) definition of the molecular mechanisms of CYP induction; and (10) evaluation of the induction of phase II metabolic pathways.

Applications of primary human hepatocytes in the evaluation of pharmacokinetic drug-drug interactions: evaluation of model drugs terfenadine and rifampin.

The utility of primary human hepatocytes in the evaluation of drug-drug interactions is being investigated in our laboratories. Our initial approach was to investigate whether drug-drug interactions observed in humans in vivo could be reproduced in vitro using human hepatocytes. Two model drugs were studied: terfenadine and rifampin, representing compounds subjected to drug-drug interactions via inhibitory and induction mechanisms, respectively. Terfenadine was found to be metabolized by human hepatocytes to C-oxidation and N-dealkylation products as observed in humans in vivo. Metabolism by human hepatocytes was found to be inhibited by drugs which are known to be inhibitory in vivo. Ki values for the various inhibitors were derived from the in vitro metabolism data, resulting in the following ranking of inhibitory potency: For the inhibition of C-oxidation, ketoconazole > itraconazole > cyclosporin approximately troleandomycin > erythromycin > naringenin. For the inhibition of N-dealkylation, itraconazole > or = ketoconazole > cyclosporin > or = naringenin > or = erythromycin > or = troleandomycin. Rifampin induction of CYP3A, a known effect of rifampin in vivo, was also reproduced in primary human hepatocytes. Induction of CYP3A4, measured as testosterone 6 beta-hydroxylation, was found to be dose-dependent, treatment duration-dependent, and reversible. The induction effect of rifampin was observed in hepatocytes isolated from all 7 human donors studied, with ages ranging from 1.7 to 78 years. To demonstrate that the rifampin-induction of testosterone 6 beta-hydroxylation could be generalized to other CYP3A4 substrates, we evaluated the metabolism of another known substrate of CYP3A4, lidocaine. Dose-dependent induction of lidocaine metabolism by rifampin is observed. Our results suggest that primary human hepatocytes may be a useful experimental system for preclinical evaluation of drug-drug interaction potential during drug development, and as a tool to evaluate the mechanism of clinically observed drug-drug interactions.

cDNA-expressed human cytochrome P450 isozymes. Inactivation by porphyrinogenic xenobiotics.

A number of xenobiotics are known to exert their porphyrinogenic effects in rodents and chick embryos through mechanism-based inactivation of certain cytochrome P450 (P450) isozymes. To facilitate the extrapolation of results from test animals to humans, we have assessed the ability of three prototype porphyrinogenic compounds-namely, 3,5-diethoxycarbonyl-1,4-dihydro-2,6-dimethyl-4-ethylpyridine (DDEP), 3-[2-(2,4,6-trimethylphenyl)thioethyl]-4-methylsydnone (TTMS), and allylisopropylacetamide (AIA)-to cause mechanism-based inactivation of cDNA-expressed human P450s 1A1, 1A2, 2C9-Arg144 (2C9), 2D6-Val374 (2D6), and 3A4 in microsomes from human lymphoblastoid cell lines (Gentest Corp., Woburn, MA). The following catalytic markers of human P450 isozymes were used: ethoxyresorufin O-deethylase (P450s 1A1 and 1A2), diclofenac 4-hydroxylation (P4502C9), dextromethorphan O-demethylase (P4502D6), and testosterone 6 beta-hydroxylation (P4503A4). We found that DDEP and TTMS caused mechanism-based inactivation of cDNA-expressed human P450s 1A1, 1A2, and 3A4, whereas only DDEP was able to cause mechanism-based inactivation of cDNA-expressed human P4502C9; neither xenobiotic caused mechanism-based inactivation of cDNA-expressed human P4502D6. A comparison of the human P450 isozyme data with results previously obtained in rat and chick embryo liver showed a close correspondence between the results obtained with P450s 1A and 3A, but not the P4502C subfamily. Because several rat isozymes (P450s 2A1, 2B1, 2C6, 2C11, and 3A1) undergo inactivation by AIA, it was noteworthy that AIA did not inactivate any of the cDNA-expressed human P450 isozymes. Because mechanism-based inactivation of P450 isozymes is related to the porphyrinogenicity of xenobiotics, our results demonstrate the importance of supplementing studies of mechanism-based inactivation of P450 isozymes in animal models with similar studies on cDNA-expressed human P450 isozymes.

Induction of cytochrome P450 3A by retinoids in rat hepatocyte culture.

1. Rat hepatocytes cultured on a Matrigel matrix were exposed for 48 h to all-trans-retinoic acid, 9-cis-retinoic acid, 13-cis-retinoic acid or fenretinide. 2. Cytochrome P450 3A (CYP3A) RNA levels were increased by approximately eightfold in hepatocytes treated with the retinoids compared to control cultures. 3. CYP1A1 and CYP1A2 RNA levels were only slightly affected or unaffected by the retinoids. 4. The induction of CYP3A by these therapeutically-useful retinoids suggests that they may share a common mechanism for accelerated drug catabolism and acquired clinical resistance.

CYP2D6-related oxidation polymorphism in a Canadian Inuit population.

The xenobiotic oxidation polymorphism associated with cytochrome P450 2D6 (CYP2D6) was investigated in 152 genetically related and unrelated healthy Inuit subjects living in the High Arctic of eastern Canada. Phenotyping was based on HPLC determination of the CYP2D6-related dextromethorphan metabolic ratio in overnight urine samples after oral administration of 30 mg dextromethorphan hydrobromide. The log metabolic ratio was bimodally distributed, with three subjects classified as poor metabolizers (PMs). In subjects unrelated in the first degree, the incidence of the PM phenotype was 3 of 90 or 3.3%. PCR-based analyses of DNA for variants of the CYP2D6 gene demonstrated that the PMs of dextromethorphan had the defective allele CYP2D6*4. The estimated frequency of the CYP2D6*4 allele was 0.067-0.083, which is lower than the frequency in Caucasians but higher than the frequency in Oriental populations. The CYP2D6*3 and the CYP2D6*6 alleles were not detected in the Inuit population. The CYP2D6*10 allele was present in only four unrelated subjects, classified as extensive metabolizers (EMs), resulting in an estimated allele frequency of 0.022, which is much lower than in Oriental populations. This study demonstrated the existence of the CYP2D6 polymorphism in Canadian Inuit, while the frequencies of allelic variants of CYP2D6 point to the uniqueness of this population. Several important therapeutic drugs that are being prescribed in Arctic communities will have altered pharmacokinetics in PMs of CYP2D6.

Cytotoxicity of unsaturated metabolites of valproic acid and protection by vitamins C and E in glutathione-depleted rat hepatocytes.

Valproic acid (VPA) and the unsaturated metabolites, 2-ene VPA and (E)-2,(Z)-3'-diene VPA, demonstrated dose-dependent cytotoxicity in primary cultures of rat hepatocytes, as evaluated by lactate dehydrogenase (LDH) leakage. Cellular glutathione (GSH) was depleted by adding buthionine sulfoximine (BSO) to the culture medium. Induction of cytochrome P450 by pretreatment of rats with phenobarbital or pregnenolone-16 alpha-carbonitrile enhanced the cytotoxicity of parent VPA in BSO-treated hepatocytes. The cytotoxicity of 4-ene VPA was apparent in BSO-treated hepatocytes with detectable loss of cell viability at 1 microM of added 4-ene VPA. Depletion of cellular GSH also increased the cytotoxicities of 2-ene VPA and (E)-2,(Z)-3'-diene VPA. The cytotoxicity of 2-ene VPA was comparable to or higher than that of VPA, producing loss of viability at concentrations > or = 5 mM. Time-course evaluation of hepatocyte response to 4-ene VPA in the GSH-depleted state revealed a delayed cytotoxicity with no effect during the first 12 h of exposure followed by a pronounced toxicity between 12 and 14 h. Two major GSH conjugates of 4-ene VPA metabolites, namely 5-GS-4-hydroxy VPA lactone and 5-GS-3-ene VPA, were detected in 4-ene VPA treated hepatocytes. Consistent with this finding, a 50% decrease in cellular GSH levels was observed following 4-ene VPA treatment. Under similar conditions, neither toxicity nor the GSH conjugated metabolite were detected in cells treated with the alpha-fluorinated 4-ene VPA analogue (alpha-F-4-ene VPA). The antioxidants, vitamin C and vitamin E, demonstrated a cytoprotective effect against 4-ene VPA-induced injury in GSH-depleted hepatocytes. These results are in support of hepatocellular bioactivation of VPA via 4-ene VPA to highly reactive species, which are detoxified by GSH. The susceptibility of hepatocytes to VPA metabolite-mediated cytotoxicity depends on cellular GSH homeostasis.

CYP2C19 genotyping and associated mephenytoin hydroxylation polymorphism in a Canadian Inuit population.

The CYP2C19-associated oxidation polymorphism of mephenytoin was investigated in an Inuit population living in the high Arctic of Canada. Results were obtained for 152 subjects, of whom 90 were unrelated to first degree relatives. Phenotyping was based on the capillary gas chromatographic determination of the S/R enantiomeric ratio in overnight urine collected after a dose of 100 mg (R,S)-mephenytoin. The phenotype was confirmed by determining the S/R enantiomeric ratio after acid treatment of urine samples, and for some subjects, by determining urinary recovery of 4'-hydroxymephenytoin using capillary electrophoresis analysis. DNA was analysed for the m1 and m2 mutations of CYP2C19. Three of 152 subjects (2.0%; 95% confidence limits: 0.0-4.2%) were phenotypically classified as poor metabolizers (PMs). Genotype analysis characterized three individuals as homozygous, and 28 individuals as heterozygous for the m1 mutation, the remaining individuals being homozygous for the wild-type allele. The genotype of the three PMs was concordant with that of the phenotype. DNA fingerprinting confirmed that these three individuals were genetically unrelated. The allele frequency of the CYP2C19m1 mutation, determined in unrelated subjects, was 0.12 (95% confidence limits: 0.07-0.17). CYP2C19m2 was not detected in this population. Thus, the Canadian Inuit resemble Caucasian rather than Asian populations in both the incidence of PM phenotype and the molecular basis of the polymorphism.

Evaluation of drug interactions in intact hepatocytes: Inhibitors of terfenadine metabolism.

Terfenadine has been associated with several adverse drug interactions and it was of interest to develop in vitro systems to explain and predict such interactions. The metabolism of terfenadine was studied using intact hepatocytes from primary human and rat hepatocyte cultures, and the immortalized human hepatoma cell line HepG2. Rates and routes of biotransformation were analysed by HPLC. Terfenadine was extensively metabolized by all three cell culture systems during exposure periods ranging from 4 to 24 hr. Human and rat hepatocytes and HepG2 cells formed products of C-oxidation (an acid metabolite and its precursor alcohol metabolite). Human hepatocytes also formed the N-dealkylation product azacyclonol. Several cytochrome P4503A (CYP3A) substrates and inhibitors were evaluated for their ability to inhibit terfenadine biotransformation. In rat hepatocytes, ketoconazole, erythromycin and troleandomycin failed to inhibit; in HepG2 cells, only ketoconazole potently inhibited terfenadine metabolism. In human hepatocytes, ketoconazole, itraconazole, erythromycin, troleandomycin, cyclosporin and naringenin inhibited terfenadine metabolism. The results suggest that human hepatocytes may be a useful system for screening for inhibitors of terfenadine metabolism.

Induction of CYP3A and associated terfenadine N-dealkylation in rat hepatocytes cocultured with 3T3 cells.

Long-term culture of hepatocytes has been challenged by the loss of differentiated functions. In particular, there is a rapid decline in cytochrome P450 (CYP). In this study, we cocultured rat hepatocytes with 3T3 fibroblasts for 10 days, and examined hepatocyte viability, morphology, and expression of CYP3A. Terfenadine was incubated with the cultures, and its biotransformation was quantitatively analyzed by HPLC. Terfenadine is metabolized by two major pathways: C-hydroxylation to an alcohol metabolite which is further oxidized to a carboxylic acid, and N-dealkylation to azacyclonol. In rat liver, only the N-dealkylation pathway appears to be mediated by CYP3A since anti-rat CYP3A antibody inhibited azacyclonol but not alcohol metabolite formation in incubations of terfenadine with liver microsomes. Freshly isolated rat hepatocytes were seeded on top of confluent 3T3 cells. Cultures were maintained in Williams' E medium supplemented with 10% fetal bovine serum and either 0.1 mumol/L or 5 mumol/L dexamethasone. In pure hepatocyte cultures, viability, as determined by lactate dehydrogenase (LDH) activity, decreased steadily to less than 30% of initial levels by day 10. In cocultures, LDH activity remained high and was 70% of initial levels on day 10. The half-life of terfenadine disappearance was optimally maintained in cocultures treated with 5 mumol/L dexamethasone, and was associated with the increased formation of azacyclonol. On day 5, nearly 50% of added 5 mumol/L terfenadine was converted to azacyclonol within 6 h, whereas the conversion was only 4% on day 1. Western and RNA-slot blot analyses confirmed that treatment with 5 mumol/L dexamethasone induced CYP3A mRNA expression and CYP3A protein expression. This coculture system could offer a useful approach in the study of drugs and xenobiotics metabolized by CYP3A.

Terfenadine metabolism in human liver. In vitro inhibition by macrolide antibiotics and azole antifungals.

To determine whether the clinical adverse interactions of terfenadine with azole antifungals and macrolide antibiotics may be related to inhibition of terfenadine biotransformation, an in vitro system was developed to follow the metabolism of terfenadine by rat liver S9 or human liver microsomes. When test compounds were coincubated with terfenadine, the metabolites formed and unchanged terfenadine was quantitatively analyzed by HPLC. Five metabolites of terfenadine were formed by rat liver S9: predominantly alcohol metabolite (III), with four minor metabolites--azacyclonol (I), acid metabolite (II), an unidentified metabolite (IV), and a new ketone metabolite (V). By human liver microsomes, two major metabolites were formed: azacyclonol (I) and alcohol metabolite (III). Ketoconazole, fluconazole, itraconazole, erythromycin, clarithromycin, and troleandomycin potently inhibited terfenadine metabolism by human liver (IC50 = 4-10 microM), but inhibition by rat liver was weaker (IC50 = 87-218 microM) and 18% maximally for troleandomycin. Other CYP3A substrates (cyclosporin A, naringenin, and midazolam) also demonstrated potent inhibition of terfenadine biotransformation in human liver microsomes (IC50 = 17-24 microM). Substrates of other P450 families [sparteine (CYP2D6), caffeine (CYP1A), and diclofenac (CYP2C)] only very weakly inhibited terfenadine metabolism. Dixon plot analyses for human liver revealed competitive/reversible inhibition by the azole antifungals and macrolide antibiotics of azacyclonol and alcohol metabolite formations.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative cytotoxicity of non-steroidal anti-inflammatory drugs in primary cultures of rat hepatocytes.

The cytotoxicities of 12 non-steroidal anti-inflammatory drugs (NSAIDs) in primary monolayer cultures of rat hepatocytes were compared. Toxicity was determined by measuring the release of lactate dehydrogenase into the culture medium after 20 hr of exposure. Diflunisal was the most cytotoxic, followed, in order, by mefenamic acid, diclofenac, indomethacin, flurbiprofen, piroxicam, sulindac and ibuprofen. Ketoprofen, naproxen, tolmetin and acetylsalicylic acid (ASA) were the least cytotoxic. Phenobarbital pretreatment in vivo potentiated the in vitro toxicity of diclofenac, ketoprofen and piroxicam, and SKF525-A addition to the medium reduced their toxicity. These results indicate that the cytocidal hepatotoxicity of diclofenac, ketoprofen and piroxicam may be mediated, at least partially, by cytochrome P-450 metabolism. The cytotoxicity of the other nine NSAIDs appears not to be significantly influenced by cytochrome P450 modulation.

Comparison of the cytotoxicity of ACE inhibitors and effects of cytochrome P-450 and glutathione modulation in primary rat hepatocyte cultures.

Hepatotoxicity has been reported with angiotensin-converting enzyme (ACE) inhibitors. The mechanism of liver injury is not known. In the present study, primary rat hepatocytes were used to investigate the cytotoxicity of ACE inhibitors. Captopril, enalapril, fosinopril and quinapril were cytotoxic, as measured by lactate dehydrogenase release at 24 hr, with the rank order of toxicity as quinapril (LC (50) = 0.28 m m ) > fosinopril (LC (50) = 0.4 m m ) > enalapril (LC (50) = 2.0 m m ) > captopril (LC (50) = 20 m m ) . Enalaprilat, the de-esterified metabolite of enalapril, and lisinopril, the lysine analogue of enalaprilat, were not cytotoxic. In vivo pretreatment with pregnenolone-16alpha-carbonitrile (cytochrome P-450 (CYP) 3A induction) enhanced the toxicities of enalapril, fosinopril and quinapril but did not affect the response to captopril. Induction with phenobarbital (CYP2B), beta-naphthoflavone (CYP1A) or ethanol (CYP2E1) did not influence the cytotoxicity of any of the ACE inhibitors. The hypothesis of a CYP3A-mediated bioactivation in the cytotoxicity of enalapril, fosinopril and quinapril was further supported by observations of reduced toxicity in the presence of SKF525-A and troleandomycin. The enhancement of cytotoxicity after pretreatment of the cultures with buthionine sulfoximine suggested a role for glutathione in protection, possibly by detoxifying unknown reactive metabolites.

Comparative cytotoxicity of angiotensin-converting enzyme inhibitors in cultured rat hepatocytes.

Captopril and enalapril, angiotensin-converting enzyme inhibitors (ACEIs), have been associated with idiosyncratic hepatotoxicity. Such drug reactions may be caused by the formation of reactive metabolites by cytochrome P450 isozymes, which can then cause direct or immune-mediated toxicity. Previously, we have demonstrated that enalapril cytotoxicity in primary cultures of rat hepatocytes was due, at least in part, to cytochrome P450-dependent metabolism, and that glutathione was involved in the detoxification process. In the present study, we extended our investigations into mechanisms of cytotoxicity, using rat hepatocyte cultures, to captopril and three recently marketed ACEIs: fosinopril, lisinopril and quinapril. After 24 hr of exposure to lisinopril or enalaprilat (the deesterified metabolite of enalapril), hepatocytes did not show any evidence of cytotoxicity, measured by lactate dehydrogenase leakage, even at 10 mM drug concentrations. The other ACEIs were toxic to the liver cells, with the rank order of toxicity as quinapril (LC50 = 0.28 mM) > fosinopril (LC50 = 0.4 mM) > enalapril (LC50 = 2.0 mM) > captopril (LC50 = 20 mM). In vivo pretreatment of rats with pregnenolone-16 alpha-carbonitrile to induce isozymes of the P450 3A subfamily significantly enhanced the cytotoxicities of quinapril, fosinopril and enalapril but did not affect captopril cytotoxicity. Pretreatment with P450 inducers selective for other isozyme subfamilies (ethanol, beta-naphthoflavone and phenobarbital) did not alter the in vitro toxicity of any of the ACEIs. Co-incubation with SKF525-A (15 microM) or troleandomycin (0.1 mM) reduced the hepatocidal toxicities of quinapril, fosinopril and enalapril. Preincubation with buthionine sulfoximine (2 mM) enhanced the cytotoxicities of quinapril, fosinopril, enalapril and captopril. The results of this study indicate that like enalapril, quinapril and fosinopril can also undergo P450 3A-dependent bioactivation and require maintenance of glutathione status for detoxification, and that captopril causes cytotoxicity independent of cytochrome P450 metabolism.