The role of protein tyrosine kinases in CYP1A1 induction by omeprazole and thiabendazole in rat hepatocytes.

Benzimidazoles compounds like omeprazole (OME) and thiabendazole (TBZ) mediate CYP1A1 induction differently from classical aryl hydrocarbon receptor (AhR) ligands, 3-methylcholanthrene (3-MC) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). To clarify the involvement of an intracellular signal pathway in CYP1A1 induction by OME and TBZ, the TBZ, OME and 3-MC signal-transducing pathways were compared by using specific protein tyrosine kinase inhibitors in primary culture of rat hepatocytes. The effect of OME and TBZ (75-250 microM) on cytochrome P450 1A1 (CYP1A1) expression was therefore studied in primary cultures of rat hepatocytes after 24 h, 48 h and 72 h of exposure.

Diflubenzuron, a benzoyl-urea insecticide, is a potent inhibitor of TCDD-induced CYP1A1 expression in HepG2 cells.

Diflubenzuron (DFB) belongs to a group of compounds called benzoyphenyl ureas acting as chitin synthesis inhibitors, which also inhibit growth of B16 murine melanomas. The present study was designed to investigate the effect of this insecticide, on CYP1A1 expression and induction in human hepatoma cells HepG2. Treatment of HepG2 cells over 72 h with noncytotoxic concentrations of DFB resulted in a strong dose-dependent decrease in constitutive ethoxyresorufin-O-deethylase activity.

Molecular characteristics of carbaryl, a CYP1A1 gene inducer.

Carbaryl belongs to a series of compounds that activate the CYP1A1 gene. This study demonstrates the inability of carbaryl to compete with 2,3,7,8-tetrachlorodibenzo-p-dioxin for binding to the rat aryl hydrocarbon (dioxin) receptor. Structural and physicochemical properties of this insecticide, in relation to the requirements for binding to the aryl hydrocarbon receptor, are described.

Hepatic Ah receptor binding affinity for 2,3,7,8-tetrachlorodibenzo-p-dioxin: similarity between beagle dog and cynomolgus monkey.

Hepatic AhR binding affinity for [3H]-2,3,7,8-tetrachlorodibenzo-p-dioxin ([3H]TCDD) was compared between two species widely used as laboratory animals: beagle dog and cynomolgus monkey (Macaca fascicularis). The enriched 9S fractions from both species were obtained by sucrose gradient sedimentation. After incubation with [3H]TCDD, dextran-coat charcoal treatment (10 mg/ml) revealed that dog and monkey possess an AhR with a low binding affinity for [3H]TCDD.

Knockout animals in toxicology: assessment of toxin bioactivation pathways using mice deficient in xenobiotic metabolizing enzymes.

Genetically engineered animal models represent a substantial improvement in in vivo assessment of toxic pathways. Several transgenic mouse lines have been designed to detect specific toxic markers in response to xenobiotic exposure. They are suitable for in vivo large scale screening of potentially toxic effects of drugs and other xenobiotics, and are used as bioassay models for carcinogenicity testing.

Cytochrome 1A1 induction by primaquine in human hepatocytes and HepG2 cells: absence of binding to the aryl hydrocarbon receptor.

Malaria remains the most prevalent infectious disease of tropical and subtropical areas of the world. It represents a crucial problem in public health care, affecting 750 million people annually, of whom at least two million die. Various antimalarials currently used were studied for their capability to induce expression of the cytochrome P450 1A1 (CYP1A1) gene, an enzyme that plays an important role in the activation of xenobiotics to genotoxic derivatives.

Effects of carbaryl and naphthalene on rat hepatic CYP1A1/2: potential binding to Ah receptor and 4S benzo(a)pyrene-binding protein.

Activation of the CYP1A1 gene has been described to be mediated by the cytosolic Ah receptor (AhR) and a possible cooperative role of the 4S benzo(a)pyrene-binding protein (4S protein). Carbaryl (CAR) has been shown to induce human CYP1A1 gene expression without binding to the human AhR. In this study, Sprague-Dawley rats received a single i.

Cytotoxic effects and induction of cytochromes P450 1A1/2 by insecticides, in hepatic or epidermal cells: binding capability to the Ah receptor.

Insecticides deserve particular attention since the general population is potentially exposed to such chemicals through many routes. We therefore tested the comparative acute and chronic toxicity of chemicals belonging to the major insecticides families (DDT, malathion and tetrachlorvinphos, carbaryl, cypermethrin, diflubenzuron), in hepatocytes, HepG2 and HaCaT cell lines. Two kinds of end-points were used: cytotoxicity parameters and CYP1A1 induction.

The 4S benzo(a)pyrene-binding protein is not a transcriptional activator of Cyp1a1 gene in Ah receptor-deficient (AHR -/-) transgenic mice.

In an effort to better understand the role of the 4S benzo(a)pyrene-binding protein in the induction of CYP1A1 by PAHs, we used a genetically engineered mouse line deficient in Ah receptor (AHR -/-). First, we demonstrated through binding experiments analyzed by sucrose gradient sedimentation and gel permeation chromatography that AHR -/- mice have no detectable AHR protein. In contrast, this AHR-deficient line expressed a 4S protein which efficiently binds BP as it does in hepatic cytosol from C57BL/6 mice.

The 8S benzo(a)pyrene-binding protein is an aldehyde dehydrogenase regulated by the Ah receptor.

8S Benzo(a)pyrene-binding proteins from liver cytosol of mouse and rabbit have been partially purified by gel permeation chromatography and affinity chromatography on 1-aminopyrene-Sepharose columns. These proteins, which bind polycyclic aromatic hydrocarbons and daunorubicine, have been identified, by microsequencing, as aldehyde dehydrogenases composed of polypeptides of 54 kDa. Using Ah receptor-deficient (AHR-/-) transgenic mice it has been shown that the amount as well as the binding capabilities of 8S protein was strongly altered in these mice, suggesting that its expression was partially under the control of the Ah receptor.

Ah receptor-dependent CYP1A induction by two carotenoids, canthaxanthin and beta-apo-8'-carotenal, with no affinity for the TCDD binding site.

The assays of several phase I and phase II xenobiotic-metabolizing enzyme activities, as well as CYP1A immunoblot analysis, were performed in liver microsomes and cytosol of male C57BL/6 mice (Ah receptor-responsive), of male DBA/2 mice (Ah receptor-low responsive) and of female Ah receptor gene knockout mice that were fed diets containing 300 mg/kg of a nonprovitamin A carotenoid, canthaxanthin, or a provitamin A carotenoid, beta-apo-8'-carotenal for 14 days, or which were injected i.p. with 3-methylcholanthrene.

Carbaryl induces CYP1A1 gene expression in HepG2 and HaCaT cells but is not a ligand of the human hepatic Ah receptor.

In spite of increasing numbers of insecticides used in agriculture, there are serious concerns regarding the potential risks of exposure to these agents. Carbaryl is one of the most important carbamate insecticides and has been used for about 30 years to control a wide range of pests. The study was designed to investigate if, among various insecticides currently used in world agriculture, this compound could induce human CYP1A1, an enzyme known to play an important role in the chemical activation of xenobiotics to genotoxic derivatives.

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.

Effect of exposure of rabbit hepatocytes to sulfur-containing anthelmintics (oxfendazole and fenbendazole) on cytochrome P4501A1 expression.

The expression of cytochrome P4501A1 and 1A2 was investigated in rabbit hepatocytes maintained in primary cultures for 96 hr in the absence or presence of 100 mum of the benzimidazole anthelmintics oxfendazole or fenbendazole. Total cytochrome P-450, ethoxyresorufin O-deethylase and acetanilide hydroxylase activities were significantly increased in cell cultures receiving benzimidazoles. These increases were more marked after exposure of cultured hepatocytes to oxfendazole (OFZ) than to fenbendazole (FBZ).

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.

Thiabendazole is an inducer of cytochrome P4501A1 in cultured rabbit hepatocytes.

The effect of TBZ (30-100 microM) was investigated on cytochromes P450 of cultured rabbit hepatocytes considered 72 h after plating. At the highest concentrations and without apparent cellular toxicity, the drug provokes a dose-dependent increase in total microsomal cytochrome P450 and a rise in EROD activity which was correlated to a specific increase in P4501A1 level. Northern blot analysis of RNA reveals an increased level of mRNA specific to P4501A1.

The pig as a model for studying AH receptor and other PAH-binding proteins in man.

In this report we compare the three major binding proteins existing in hepatic cytosols of pig and human. Many data suggest that for the AH receptor, which mediates the biological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin, a structural as well as a functional variability exists across species. Similar conclusions can be drawn from the interspecies characterization of benzo(a)pyrene binding proteins, namely the 4S protein and the 8S protein.

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.

Modulating effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on skin carcinogenesis initiated by the weak inducer 7,12-dimethylbenz(a)anthracene.

The effects of topical pretreatment of CF1-Swiss mice with TCDD on the carcinogenesis induced by DMBA were studied. We also determined the intrinsic features of DMBA as an aryl hydrocarbon hydroxylase (AHH) inducer through either its binding ability to Ah receptor or its inducing effects on benzo(a)pyrene (BP) hydroxylase or DMBA hydroxylase. DMBA is a poor ligand of the Ah receptor (26-fold and 4.

Detection and characterization of a novel hepatic 8 S binding protein for benzo[a]pyrene distinct from the Ah receptor.

Using fractionation procedures such as sucrose gradient sedimentation and gel permeation chromatography, a novel cytosolic binding protein for benzo[a]pyrene has been detected in liver of nonmammalian and mammalian species including human. This protein, called 8 S protein, cosediments with the Ah receptor after centrifugation in sucrose density gradient but can be separated from the Ah receptor and 4 S protein by gel permeation chromatography. Owing to its binding characteristics, the 8 S protein is clearly distinct from the Ah receptor.

Characterization of the 4 S polycyclic aromatic hydrocarbon-binding protein in human liver and cells.

The 4 S polycyclic aromatic hydrocarbon (PAH)-binding protein (PBP) is a soluble protein that binds PAHs with high affinity in mouse, rat, and rabbit. Until now, this protein had not been detected in human placenta or human cells in culture by cytosol labeling and gradient centrifugation assay. Thanks to a preliminary fractionation of cytosol by sedimentation on sucrose gradient or/and gel permeation chromatography, we found that PBP was present in liver, MCF-7 cell line, and hepatocytes of human.

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.

Mechanism of antimutagenicity of wheat sprout extracts.

In this paper we have demonstrated that wheat sprout extract, which has been shown to be antimutagenic towards benzo[a]pyrene (BP), reduced formation of BP metabolites by hepatic microsomes of either benzo[a]pyrene- or phenobarbital-treated rats as analyzed in high-pressure liquid chromatography (HPLC). Comparing the time dependence of profiles and values of BP metabolites, formed in experiments in which the same dose of wheat sprout extract was added to the incubation medium, it has been observed that the later this extract was added the higher the percent of BP that was metabolized. In a bacterial test (cytochrome P450 induction assay) high inhibition of mutagenic activity of cyclophosphamide and ethidium bromide, in the presence of wheat sprout extract, reflected decreased levels of cytochromes P4502B1 and P4501A1 respectively.

Induction and characterization of cytochromes P450IA and -IIB in the newt, Pleurodeles waltl.

The newt (Pleurodeles waltl) is an amphibian species used in a mutagenicity test (micronucleus). This study was carried out to establish if the inducibility of hepatic cytochromes P450 of this species is similar to that of the rat. Our results showed that the newt is characterized by a lower level of hepatic cytochrome P450-dependent activities than the rat.