Induction of CYP2C genes in human hepatocytes in primary culture.

The expression and inducibility of four CYP2C genes, including CYP2C8, -2C9, -2C18, and -2C19, was investigated in primary cultures of human hepatocytes. By the use of RNase protection assay and specific antibodies, each CYP2C mRNA and protein were quantified unequivocally. The four CYP2C mRNAs were expressed in human livers and cultured primary hepatocytes, but only the CYP2C18 protein was not detected. Compounds known to activate the pregnane X receptor (PXR) such as rifampicin, or the constitutively activated receptor (CAR) such as phenobarbital, induced CYP2C8, CYP2C9, and to a lesser extent CYP2C19 mRNAs and proteins. CYP2C18 mRNA was expressed but not inducible. The concentration dependence of CYP2C8 and CYP2C9 mRNAs in response to rifampicin and phenobarbital paralleled that of CYP3A4 and CYP2B6, the maximum accumulation being reached with 10 microM rifampicin and 100 microM phenobarbital. In contrast, dexamethasone produced maximum induction of CYP2C8 and CYP2C9 mRNAs at 0.1 microM while in these conditions neither CYP3A4 nor CYP2B6 was significantly induced. Moreover, the concentration dependence of CYP2C8 and CYP2C9 mRNAs in response to dexamethasone paralleled that of tyrosine aminotransferase. Furthermore, dexamethasone, which has been recently shown to up-regulate PXR and CAR expression through the glucocorticoid receptor, potentiated CYP2C8 and CYP2C9 mRNA induction in response to rifampicin and phenobarbital. Collectively, these results suggest the possible implication of at least three receptors in the regulation of CYP2C8 and CYP2C9 expression, i.e., glucocorticoid receptor, PXR, and/or CAR.

Interleukin-6 negatively regulates the expression of pregnane X receptor and constitutively activated receptor in primary human hepatocytes.

The marked impairment of hepatic drug metabolism during inflammation and infections has been known for many years and shown to result from down-regulation of cytochrome P450s (CYP) by cytokines. However, the mechanism of this repression is unknown. Using primary cultures of human hepatocytes, we show here that interleukin-6 (IL-6) rapidly and markedly decreases the expression of PXR (pregnane X receptor) and CAR (constitutively activated receptor) mRNAs, but does not affect the levels of dioxin receptor and glucocorticoid receptor mRNA. In parallel, IL-6 decreases both rifampicin- and phenobarbital-mediated induction of CYP2B6, CYP2C8, CYP2C9, and CYP3A4. As the transcriptional activity of PXR and CAR is not affected by IL-6 in cell-based reporter assays, our data suggest that the loss of CYP2 and CYP3 inducibility results from the negative regulation of PXR and CAR gene expression by this cytokine.

Regulation of dioxin receptor function by omeprazole.

The intracellular dioxin (aryl hydrocarbon) receptor mediates signal transduction by dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin) and related environmental pollutants and functions as a ligand-activated transcription factor. In this study we have examined the effects on dioxin receptor function of a potentially novel ligand, omeprazole, which is widely clinically used as a gastric anti-ulcer drug. In primary human hepatocytes omeprazole potently induced cytochrome P4501A1 mRNA expression, whereas this effect was not detected in mouse primary hepatocytes. In human hepatoma cells omeprazole was found to induce transcription of reporter genes via the xenobiotic response element that is recognized by the ligand-activated dioxin receptor. In contrast, the human dioxin receptor was not activated by omeprazole upon expression in a receptor-deficient mouse hepatoma cell line. In a reconstituted yeast (Saccharomyces cerevisiae) model system, however, both the mouse and human dioxin receptors were potently activated by omeprazole. Although omeprazole failed to displace dioxin in in vitro ligand binding assays, a residue within the ligand binding domain that is critical for dioxin binding in vitro was also critical for omeprazole responsiveness in vivo. Consistent with this observation, both omeprazole and dioxin responsiveness of the dioxin receptor was inhibited in mutant yeast cells expressing low levels of the molecular chaperone hsp90 that is critical for ligand binding activity. The sulfoxide group that is essential for formation of a planar conversion product of omeprazole was found to be critical for dioxin receptor activation. Taken together, these data suggest that omeprazole represents a precursor for a novel class of dioxin receptor agonists that are bona fide dioxin receptor ligands but generated in a strictly species-specific manner.

Lipid-mediated transfection of normal adult human hepatocytes in primary culture.

The aim of this work was to develop a procedure for the lipid-mediated transfection of DNA into normal adult human hepatocytes in culture. Cells were plated in a serum-free culture medium at various cell densities, on plastic or collagen-coated dishes, both in the absence and in the presence of epidermal growth factor (EGF). The cells were incubated for various periods of time with mixtures of DNA-lipofectin or DNA-3 beta[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-chol) liposomes, and the efficiency of transfection was assessed by measuring the activity of reporter genes, beta-galactosidase or chloramphenicol acetyl-transferase (CAT). For comparison, similar experiments were carried out with human cell lines including HepG2, Caco-2, and WRL68. The efficiency of transfection (in percentage of cells) was not significantly different after transfection with lipofectin or DC-chol and comprised between 0.04 and 1.7% (extreme values) for different cultures. The efficiency of transfection decreased as the age or density of the culture increased and increased in cultures treated with EGF. Direct measurement of the rate of DNA synthesis suggested that the efficiency of transfection was related to the number of cells entering the S phase. Under the same conditions, the efficiency of transfection was one to two orders of magnitude greater in the three cell lines. A plasmid harboring 660 bp of the 5'-flanking region of CYP1A1 (containing two xenobiotic enhancer elements) fused upstream of the promoter of thymidine kinase and the CAT reporter gene was constructed. When this plasmid was transfected in human hepatocytes, CAT activity was induced as expected. We conclude that normal adult human hepatocytes can be transfected with exogenous DNA and that the transfected construct is regulated in the manner expected from in vivo studies.

Induction of CYP1A1 gene by benzimidazole derivatives during Caco-2 cell differentiation. Evidence for an aryl-hydrocarbon receptor-mediated mechanism.

The Caco-2 cell line, derived from a human colon adenocarcinoma, is unique in its property of spontaneously differentiating into a mature enterocyte cell type during its growth in culture. In this work, we compared the response of the CYP1A1 gene with the benzimidazole derivatives omeprazole and lansoprazole, and with the classical inducer beta-naphthoflavone in the Caco-2 cells at various culture stages. In addition, we characterized the Caco-2 aryl-hydrocarbon receptor. The protein-synthesis inhibitor cycloheximide led to a derepression of the CYP1A1 gene transcription, and to a superinduction when combined with either beta-naphthoflavone or benzimidazoles. Taking advantage of the spontaneous differentiation of Caco-2 cells in long-term cultures, we observed a difference in behavior between the classical inducer beta-naphthoflavone and the atypical inducer omeprazole. In the poorly differentiated cells, both compounds elicited comparable dose/response and rate of induction of CYP1A1 gene expression. In the fully differentiated cells, in contrast, the induction by omeprazole was only transient, whereas the response to beta-naphthoflavone was long lasting. The Caco-2 aryl-hydrocarbon receptor exhibited binding characteristics similar to those determined for human liver and other tissues. The induction of CYP1A1 transcription by benzimidazole derivatives in Caco-2 cells occurred with no direct binding of benzimidazole derivatives to the aryl-hydrocarbon receptor, as in human hepatocytes. However, transient transfection experiments clearly showed that the xenobiotic-responsive element enhancer, with which the activated aryl-hydrocarbon receptor interacts, could drive the induction of a heterologous promoter in the presence of benzimidazoles. Finally the presence of the activated aryl-hydrocarbon receptor in the nuclei of the Caco-2 cells exposed to these molecules was clearly demonstrated by gel-retardation experiments. These results question about the mechanism of ligand-independent activation of the aryl-hydrocarbon receptor and intracellular signaling, initiated by benzimidazole derivatives.

The interleukin-2 receptor down-regulates the expression of cytochrome P450 in cultured rat hepatocytes.

BACKGROUND & AIMS: Interleukin (IL) 2 is used in advanced cancers, but its effects on cytochrome P450 remain unknown. Other cytokines down-regulate hepatic cytochrome P450, but it is not known whether this involves cytokine receptors. The aim of this study was to determine whether the IL-2 receptor is expressed on hepatocytes and whether its activation by IL-2 depresses cytochrome P450 in cultured rat hepatocytes. METHODS: A monoclonal antibody specific for the rat IL-2 receptor alpha chain was used to label the receptor, whereas effects on cytochrome P450 were determined after 24 hours of culture with human recombinant IL-2 (5000 U/mL). RESULTS: The presence of the IL-2 receptor in hepatocytes was shown by immunoblots, flow cytometry, and scanning confocal microscopy. IL-2 caused a 46% decrease in total cytochrome P450; a 35%, 35%, 36%, 26%, and 56% decrease in immunoreactive cytochrome P4501A1, 2B, 2C11, 2D1, and 3A, respectively; and a marked decrease in cytochrome P4503A2 and 2C11 messenger RNAs. Addition to the culture medium of the anti-receptor antibody or the tyrosine kinase inhibitor genistein prevented the IL-2-mediated decrease in cytochrome P450. CONCLUSIONS: IL-2 down-regulates the expression of cytochrome P450 genes in cultured rat hepatocytes by interacting with its receptor expressed on hepatocytes.

Evidence for the ligand-independent activation of the AH receptor.

Benzimidazole derivatives are potent inducers of CYP1A1 in rabbit and human hepatocytes, but apparently do not bind the AH receptor. To resolve this paradoxical behaviour, studies have been concerned with the question of whether an alternative ligand-independent mechanism could explain the activation of the AH receptor. From experiments in cultured rabbit hepatocytes we show that benzimidazoles bind early and transiently to an unknown protein. Moreover, they are able to deplete the AHR in a time- and dose-dependent manner. In contrast, benzimidazoles are unable to induce CYP1A1 mRNA in mouse hepa-1 cells and to deplete the high-affinity AHR form from these cells. Taken together these data suggest that a signal transduction pathway, similar to that involved in the ligand-independent activation of steroid receptors, could only activate the low-affinity forms of AHR as those existing in rabbit and human cells.

Electrotransfer of microsomal cytochrome P450 after isoelectric focusing (IEF blots): resolution of human 2A and 3A isozymes.

In this article a procedure is described which allows us to achieve electroblots of membrane-associated proteins after isoelectric focusing. Proteins are resolved in vertical acrylamide slab gels containing ampholines, urea, and nonionic detergents (dodecyl maltoside and Tergitol NP10). After migration the focusing gel is reinforced by a carrier gel (10% acrylamide) polymerized on only one face of the slab. Electroelution of the proteins toward a hydrophobic membrane (Immobilon P) is obtained by adding denaturing agents to the carrier gel (sodium dodecyl sulfate and mercaptoethanol) and by increasing the methanol concentration (60%) in the transfer buffer. This method is applied to analyzing P450 2A and 3A cytochromes from human liver microsomes.

Omeprazole and lansoprazole are mixed inducers of CYP1A and CYP3A in human hepatocytes in primary culture.

The ability of several gastric antiulcer drugs including lansoprazole, cimetidine and ranitidine to affect the expression of human liver microsomal cytochromes P450 comparatively to omeprazole, reported previously to be a CYP1A inducer, was evaluated in primary cultures of human hepatocytes. Poly (A)+ RNA and microsomes extracted from the cells were analyzed in Northern and Western blots with specific cDNA probes and antibodies, and assayed for form-specific monoxygenase activities. Lansoprazole induced both CYP1A1 and CYP1A2 as omeprazole and did not apparently bind to the aryl hydrocarbon receptor with high affinity. Omeprazole sulfone was not an inducer of CYP1A. Omeprazole, omeprazole sulfone and lansoprazole induced CYP3A in approximately 50% of tested cultures, whereas 100% of tested cultures responded to omeprazole and to rifampicin in terms of CYP1A and CYP3A induction, respectively. Finally, cimetidine and ranitidine were not inducers. We conclude that omeprazole and lansoprazole constitute a new class of mixed inducers of CYP1A and CYP3A in human hepatocytes in primary culture and that the induction of CYP3A in response to these molecules could be polymorphic in humans.

Omeprazole, an inducer of human CYP1A1 and 1A2, is not a ligand for the Ah receptor.

Omeprazole is a benzimidazole derivative which induces both P450 1A1 and 1A2 in human liver in vitro and in vivo. Northern blot analysis of polyA RNA prepared from primary cultures of human hepatocytes indicates that both 1A1 and 1A2 messages are induced by beta-naphthoflavone and omeprazole. Co-treatment of cells with these inducers and with actinomycin D or cycloheximide results in no accumulation of both mRNA or superinduction of 1A1 mRNA, respectively. 9S enriched fraction of cytosol was prepared either from human hepatocytes in culture or from human liver tissue and analyzed by sucrose density gradient sedimentation for its capacity to bind 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), omeprazole or omeprazole sulfone (a metabolite of omeprazole in man). Whereas 2 microM TCDD displaced almost totally [3H]TCDD from the Ah receptor, both omeprazole and omeprazole sulfone did not, even at 5000-fold molar excess. In addition, when [14C] omeprazole was incubated with 9S enriched fraction of human liver or hepatocyte cytosol, no interaction could be detected in sucrose density gradient. These experiments suggest that omeprazole is not a ligand for the human liver Ah receptor.

Effect of corticosteroids on the expression of cytochromes P450 and on cyclosporin A oxidase activity in primary cultures of human hepatocytes.

Prednisone, prednisolone, and methylprednisolone are currently administered in association with cyclosporin A in the postoperative treatment of transplant patients. The aim of this work was to evaluate the effects of these corticosteroids on the expression of several forms of cytochromes P450 (P450), including P450 1A2, 2D6, 2E1, and 3A, and on cyclosporin A oxidase activity in human liver. For this purpose, human hepatocytes prepared from lobectomies were maintained in culture in a serum-free medium, in collagen-coated dishes, for 96-144 hr, in the absence or presence of 50-100 microM corticosteroids, rifampicin, or dexamethasone. To mimic more closely the current clinical protocol, hepatocyte cultures were also co-treated with corticosteroids and cyclosporin A or ketoconazole (a selective inhibitor of P450 3A). Cyclosporin A oxidase activity, intracellular retention of cyclosporin A oxidized metabolites within hepatocytes, accumulation of P450 proteins and corresponding messages, and de novo synthesis and half-lives of these P450 were measured in parallel in these cultures. Our results, obtained from seven different hepatocyte cultures, showed that 1) dexamethasone and prednisone, but not prednisolone or methylprednisolone, were inducers of P450 3A, at the level of protein and mRNA accumulation, as well as of cyclosporin A oxidase activity, known to be predominantly catalyzed by these P450; 2) although corticosteroids are known to be metabolized in human liver, notably by P450 3A, partial or total inhibition of this P450 by cyclosporin or ketoconazole, respectively, did not affect the inducing efficiency of these molecules; 3) corticosteroids did not affect the half-life of P450 3A or the accumulation of other forms of P450, including 1A2, 2D6, and 2E1; 4) chronic treatment of cells with cyclosporin did not affect P450 3A accumulation; 5) corticosteroids were all competitive inhibitors of cyclosporin A oxidase in human liver microsomes, with Ki values of 61 +/- 12, 125 +/- 25, 190 +/- 38, and 210 +/- 42 microM for dexamethasone, prednisolone, prednisone, and methylprednisolone, respectively; and 6) chronic treatment of cells with corticosteroids did not influence the excretion of oxidized metabolites of cyclosporin from the cells. These results support most of clinical reports dealing with mutual interactions between cyclosporin A and corticosteroids.

Mitotic responsiveness of cultured adult human hepatocytes to epidermal growth factor, transforming growth factor alpha, and human serum.

The present study was undertaken to evaluate the ability of human hepatocytes to respond in culture to various mitotic agents including epidermal growth factor (EGF), transforming growth factor alpha (TGF-alpha), or serum from patients with fulminant hepatitis. Human hepatocytes were maintained in culture on collagen-coated plates in a chemically and hormonally defined serum-free medium at low cell density. Twelve hours after plating, cultures were treated with increasing amounts of EGF (1-100 ng/mL), TGF-alpha (1-100 ng/mL), or human serum (1%-10%) for 0-96 hours. Proliferative response was assessed by determining against time the rate of DNA synthesis by [3H]thymidine incorporation in DNA, the labeling index, the expression of cyclin A, the amount of DNA, and the number of cells. The rate of DNA synthesis reached a maximum after 48 hours of treatment with 20 ng/mL EGF, 40 ng/mL TGF-alpha, or 5%-10% of human serum (fulminant hepatitis); the average increase with respect to untreated cells was 4.35 times with EGF, 5.4 times with TGF-alpha, and 4-6 times with serum from patients with fulminant hepatitis. The maximum expression of cyclin A coincided with the maximum of DNA synthesis. After 72 hours of treatment with EGF or human serum (fulminant hepatitis), the amount of DNA increased by 75%-100% (P less than 0.001) and the number of cells by 50% (P less than 0.001). These results show that adult human hepatocytes respond to mitogens, as expected from previous studies on animal hepatocytes, and provide experimental basis for future investigations in the field of human liver regeneration.

Effects of imidazole derivatives on cytochromes P450 from human hepatocytes in primary culture.

The expression of several forms of cytochrome P450 including P450 1A2, 2D6, 2E1, and 3A was investigated in human hepatocytes maintained in primary culture for 96 h in the absence or presence of 50 microM of various imidazole derivatives. These included ketoconazole, clotrimazole, miconazole, fluconazole, secnidazole and metronidazole. In addition, the typical inducers rifampicin and beta-naphthoflavone were used for comparison. Western and Northern blot analysis of microsomes and RNA prepared from these cultures as well as de novo synthesis experiments revealed that, among the imidazole derivatives tested, only clotrimazole was a strong rifampicin-like inducer of P450 3A. The expression of the other forms of P450 tested was not affected by the treatments. Analysis of the inhibition of 13 monoxygenase activities, including ethoxyresorufin and phenacetin O-deethylases, coumarin 7 alpha-, lauric acid 11- and 12-, mephenytoin 4-, debrisoquin 4-, and aniline hydroxylases, benzphetamine, aminopyrine, mephenytoin and erythromycin demethylases, and cyclosporin oxidase (representative of 10 different forms of P450 in human liver microsomes) revealed that ketoconazole was a strong and selective in vitro inhibitor of P450 3A (cyclosporin oxidase) with a Ki less than 1 microM. Clotrimazole and miconazole were also strong inhibitors of P450 3A-mediated activities in contrast to the other imidazole derivatives.

Induction, regulation and messenger half-life of cytochromes P450 IA1, IA2 and IIIA6 in primary cultures of rabbit hepatocytes. CYP 1A1, 1A2 and 3A6 chromosome location in the rabbit and evidence that post-transcriptional control of gene IA2 does not involve mRNA stabilization.

A study on the regulation and induction of expression of cytochromes P450-IA1, IA2 and IIIA6 genes has been undertaken using primary cultures of adult rabbit hepatocytes grown in a serum-free chemically and hormonally defined medium. In 72-h-old cultures, 50 microM beta-naphthoflavone induced both IA1 and IA2 mRNA, the maximal level being reached after 4 h and 12 h, respectively. This was shown to result from an increase in the rate of transcription of gene IA1. In contrast, gene IA2 was constitutively transcribed in untreated cells, but mRNA only accumulated in the presence of beta-naphthoflavone which, however, did not affect the rate of transcription. Actinomycin D fully blocked induction of both IA1 and IA2 mRNA in response to their inducer. In untreated cells the presence of cycloheximide allowed a 'constitutive' expression of gene IA1, while in beta-naphthoflavone-treated cells, it produced a super-induction of IA1 but no modification of IA2 gene expression. Rifampicin (50 microM) strongly increased the IA1 mRNA level and rate of transcription only in cycloheximide-treated cells. Rifampicin and dexamethasone, two prototypical inducers of P450-IIIAs, induced both large and small IIIA6 mRNAs in a time-dependent fashion, the maximum level being reached after 24 h. This was related to a large increase in the rate of transcription of the gene. Cycloheximide significantly decreased the accumulation of both IIIA6 mRNAs in response to rifampicin, while actinomycin D fully blocked induction. The half-lives of IA1, IA2 and IIIA6 mRNAs were determined by two different methods, namely actinomycin D and [3H]uridine-chase experiments. In untreated cells, the half-lives for IA1, IA2 and IIIA6 mRNAs were 14 h, 16 h and 19 h, respectively when determined by the uridine chase and 18 h, 25 h and 22 h when determined by the actinomycin-D chase. These values were not modified significantly in cells treated with beta-naphthoflavone or rifampicin, indicating that neither of these inducers affected the stability of IA1 and IA2 or IIIA6 messages, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Developmental expression of rabbit cytochrome P450 CYP1A1, CYP1A2 and CYP3A6 genes. Effect of weaning and rifampicin.

Developmental expression of CYP1A1, CYP1A2 and CYP3A6 in the rabbit have been studied. Cytochromes P450IA1, P450IA2 and P450IIIA6 exhibited comparable patterns of developmental expression. Present at low level (less than 0.05 nmol/mg) in the new born animal up to week 3, these proteins sharply accumulated between weeks 3 and 4 to reach a maximum by week 4 (P450IA1, 0.2 nmol/mg; P450IA2, 0.8 nmol/mg; P450IIIA6, 0.12 nmol/mg) and decreased in the adult (P450IA1, 0.2 nmol/mg; P450IA2, 0.4 nmol/mg; P450IIIA6, 0.09 nmol/mg). Cytochromes P450IA1 and P450IA2 were not expressed in the untreated fetus. Onset of CYP3A6 gene expression occurred at day 30 of gestation and both transcription and mRNA accumulation were transplacentally inducible by rifampicin only shortly before birth, i.e. after treatment of the females between days 28 and 30 of gestation. Both long (1.85 kb) and short (1.7 kb) mRNA transcripts were expressed in untreated or rifampicin-treated fetuses. CYP3A6 gene expression was also induced by rifampicin in 1-week-old and 2-week-old animals. Developmental expression of CYP1A1 and CYP1A2 genes was shown to be closely related to the diet change accompanying weaning which occurs at weeks 3-4. In animals subjected to either delayed (week 6) or early (week 2) weaning, sharp accumulation of messages, proteins and related activities were delayed or anticipated accordingly with respect to normal weaning. Artificially scheduled weaning gave similar results when repeated with biological-grade lucern (grown in the absence of chemical fertilizers, pesticides, etc.), the main constituent of commercial rabbit chow. While CYP3A6 gene expression could be brought forward by early weaning at week 2, both message and protein did not exhibit increased accumulation after delayed weaning at week 6, and remained at the low level of the new born animal. Treatment of 1-week-old and 2-week-old animals with triiodothyronine or of 3-week-old animals with propylthiouracil, an antithyroid factor, did not modify the normal pattern of developmental expression of genes CYP1A1, CYP1A2 and CYP3A6. It is concluded that (a) the onset of CYP3A6 gene expression in the fetus occurs at day 30 of gestation, (b) expression of this gene may be induced transplacentally by rifampicin, (c) CYP1A1, CYP1A2 and CYP3A6 gene expression is sharply activated at weaning, and (d) thyroid hormones appear not to be responsible for the pattern of developmental expression of these genes in the rabbit.

Omeprazole is an aryl hydrocarbon-like inducer of human hepatic cytochrome P450.

Omeprazole is a new drug used for its high efficiency as an inhibitor of gastric acid secretion. This substituted benzimidazole molecule had been shown to decrease several liver cytochrome P450-mediated monooxygenase activities both in vitro and in vivo. The present work was undertaken to determine whether this drug was an inducer of cytochrome P450 in humans. Primary cultures of human hepatocytes were maintained in a serum-free, chemically defined medium for 0-96 hours in the absence or in the presence of omeprazole (1-100 mumol/L) or of other cytochrome P450 inducers such as 3-methylcholanthrene, beta-naphthoflavone, or rifampicin for comparison. Omeprazole produced a time- and concentration-dependent increase in (a) cytochrome P450IA2 accumulation determined by western blot in microsomes from omeprazole-treated cells, while the level of other cytochrome P450 forms including P450IID6, IIE1, and IIIA was not increased in the same culture; (b) several monoxygenase activities, including phenacetin deethylase and acetanilide hydroxylase (cytochrome P450IA2) and ethoxyresorufin deethylase and benzpyrene hydroxylase (cytochrome P450IA1); (c) cytochrome P450IA2 de novo synthesis, determined by immunoprecipitation of cell lysate from [3H]Leu-labeled cells; (d) cytochromes P450IA1 and IA2 mRNAs, determined by northern blot analysis. An in vivo study was carried out on liver microsomes from five patients for whom hepatic biopsy specimens were available before and after repeated administration of omeprazole (20 mg/day for 4 days). In all cases, several-fold increases in cytochrome P450IA2 and specific cytochrome P450IA subfamily-dependent monooxygenase activities were observed in agreement with the results from cell culture. It was concluded that omeprazole is an aryl hydrocarbon-like inducer of cytochrome P450 secretion in human liver both in vitro and in vivo. This drug is therefore likely to increase the metabolism of any xenobiotic specifically oxidized by a cytochrome P450IA subfamily. This could potentiate the hepatotoxicity of phenacetin or paracetamol and activation of procarcinogens.

Complete sequence of cytochrome P450 3c cDNA and presence of two mRNA species with 3' untranslated regions of different lengths.

Two cDNAs (pLM3c 4.1 and pLM3c 6.1) coding for rabbit cytochrome P450 3c were sequenced. cDNA 4.1 (1768 bp) exhibits an open reading frame from nucleotides 74 to 1576 encoding the 501 amino acid residues of the entire protein. cDNA 6.1 (189 bp) appears to encode the last 24 amino acids. Comparative amino acid sequence analysis indicated that P450 PCN1, PCN2, and HLp from rat and man, were 70, 67, and 73% homologous, respectively, to P450 3c. According to the cytochrome P450 nomenclature, the P450 3c gene is termed P450IIIA4. Comparison of the nucleotide sequences indicated that cDNA 6.1 was 100% homologous to cDNA 4.1. However, whereas a poly(A) tract started 23 nucleotides after the AATAAA consensus sequence in cDNA 6.1, cDNA 4.1 had a 3' untranslated region extending 101 bp beyond the polyadenylation signal, which lacked poly(A). This observation is consistent with the previous finding that both cDNA 4.1 and 6.1 hybridized with two distinct species of poly(A)RNA (1700 and 1850 bases) from rabbit liver. The extreme 3'-end 79-bp fragment of cDNA 4.1 therefore was isolated by subcloning in pUC12 (clone p18-Rsa I) and used to probe Northern blots of poly(A)RNA from control and rifampicin-treated rabbit liver. In contrast to cDNA 4.1 and 6.1, p18-Rsa I cDNA hybridized only with the largest (1850 bases) mRNA species. We conclude that rabbit liver contains two P450 3c mRNA species differing in the length of their 3' untranslated region.