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. Both compounds provoked a dose- and time-dependent increase in CYP1A1 (EROD activity, protein and mRNA levels), but OME was less effective at all the concentrations and times tested. The mechanism of benzimidazole-mediated induction of CYP1A1 was investigated by comparison with 3-MC, a prototypical AhR ligand. As expected, OME and TBZ were unable to displace [(3)H]-TCDD from its binding sites to the AhR in competitive binding studies. Moreover, classic tyrosine kinase inhibitor herbimycin A (HA) inhibited the two benzimidazoles-mediated CYP1A1 inductions, but only partially inhibited the 3-MC-mediated one. Another two tyrosine kinase inhibitors, Lavendustin A (LA) and genistein (GEN), had no effect on CYP1A1 induction by benzimidazoles and 3-MC. These results are consistent with the implication of a tyrosine kinase, most probably the Src tyrosine kinase, in the mechanism of CYP1A1 induction in rat hepatocytes.

Induction of cytochrome P450 1A1 gene expression, oxidative stress, and genotoxicity by carbaryl and thiabendazole in transfected human HepG2 and lymphoblastoid cells.

Carbaryl and thiabendazole, two widely used pesticides, have been shown to induce cytochrome P450 1A1 (CYP1A1) expression, but neither compound is capable of displacing [3H] 2,3,7,8-tetrachlorodibenzo-P-dioxin from its aryl hydrocarbon receptor binding site. In the present study, we investigated the transcriptional regulation of CYP1A1 as well as other genes in various human hepatoma HepG2 cell lines stably transfected with the chloramphenicol acetyl transferase (CAT) reporter gene and cloned under the control of each of 14 promoters or response elements from relevant stress genes. Carbaryl and thiabendazole were found to activate CYP1A1 at the level of transcription, as demonstrated by the dose-dependent increase in reporter CAT and CYP1A1 mRNAs. Moreover, this effect appeared to be mediated via the xenobiotic responsive element (XRE), because both pesticides specifically activated various fusion constructs containing XRE sequences (CYP1A, glutathione S-transferase, and XRE). Carbaryl and to a lesser extent thiabendazole also activated other stress genes such as c-fos and NF-kappaBRE, HSP70 and GRP78, and GADD153 at a transcriptional level. These data suggest that these molecules induce early alert genes, including those known to be sensitive to oxidative stress. This led us to examine the genotoxic effect of carbaryl and thiabendazole by an in vitro DNA repair solid-phase assay. Both compounds provoked a strong DNA-damaging activity in the human lymphoblastoid cell line that constitutively expresses human CYP1A1 cDNA, but not in the parental line, indicating that CYP1A1 is chiefly implicated in carbaryl and thiabendazole genotoxicity. This effect was confirmed on HepG2 cells. These observations support the notion that intracellular signals leading to CYP1A1 induction, oxidative stress, and genotoxicity are intimately related.

Effects of macrolide antibiotics on CYP3A expression in human and rat hepatocytes: interspecies differences in response to troleandomycin.

The effects of various macrolide antibiotics [triacetyloleandomycin (TAO), clarithromycin, azithromycin, roxithromycin, erythromycin base] and the new ketolide HMR3004 on CYP3A expression were evaluated in human and rat hepatocytes. Cells were treated for 3 days with nontoxic concentrations of the drugs, and CYP3A induction was assessed through midazolam hydroxylase activity and Western and Northern blot analyses. In rat hepatocytes, no induction of CYP3A1 expression was observed following exposure to macrolides, even to erythromycin base and TAO (well known in vivo CYP3A1 inducers), whereas dexamethasone and phenobarbital were confirmed to induce this enzyme. In contrast, treatment of fresh and thawed human hepatocytes with TAO, produced an increase of midazolam hydroxylation (4-fold over control). This result was in agreement with the high amount of CYP3A4 protein and mRNA revealed by Western and Northern blot analyses. Other tested macrolides had no induction effect on CYP3A expression. These results confirmed the interspecies variability of CYP3A regulation in hepatocytes and raised the question of its mechanism of induction by macrolides in human liver.

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. Moreover, DFB significantly decreased CYP1A1 induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) after 24 h exposure, as demonstrated by ethoxyresorufin-O-deethylase (EROD) activity and Northern blot analysis. Additional studies were performed both on parental HepG2 cells and HepG2-241c.1, which were stably transfected with the chloramphenicol acetyltransferase (CAT) reporter gene, cloned under the control of the human CYP1A1 promoter (-1140 to +59). Ribonuclease protection assays (RPA) analysis clearly demonstrated an inhibition of CYP1A1 transcription in both cell lines. Surprisingly, in corresponding experiments using 3-methylcholanthrene (3-MC) as a CYP1A1 inducer, DFB was less effective. Finally, in competitive binding studies using a 9S-enriched fraction of HepG2 cytosol, DFB was capable of displacing [(3)H]-2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) from its Ah receptor binding site. Taken together, these results support the involvement of a transcriptional mechanism in the inhibition of CYP1A1 expression in HepG2 cells by DFB, possibly via an Ah receptor antagonism.

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. Except for cypermethrin and diflubenzuron, all these chemicals exerted a cytotoxic effect in hepatocytes and HaCaT, but not in HepG2 cells. However, the induction of the EROD activity appeared more sensitive since a response was detected at lower concentrations. Significant differences were observed between the cell types and the insecticides. Furthermore, these chemicals were unable to displace [3H]TCDD from its binding sites, suggesting that they would not be a ligand of the Ah receptor. The experimental approach used herein may be a good means for predicting the acute and chronic toxicity of pesticides.

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. Studies on HepG2 and HaCaT cell lines showed that carbaryl is capable of increasing, in a dose-dependent manner, both the ethoxyresorufin rufin-O-dec, O-deethylase activity and the steady-state concentrations of CYP1A1 mRNA, suggesting a transcriptional activation of this gene. When alpha-naphthoflavone, a partial Ah receptor (AhR) antagonist, and 8-methoxypsoralen, which interferes with the binding of activated AhR to the xenobiotic responsive element (XRE), were added to the cultures, CYP1A1 induction was suppressed. However, competitive binding studies using the 9S enriched fraction of human cytosol indicated that carbaryl did not displace [3H]TCDD from AhR. These data, together with the activation of a XRE-directed CAT reporter gene by carbaryl, suggest that induction of CYP1A1 involves the participation of the AhR and the XRE, but is not mediated by a direct carbaryl-receptor interaction. An alternative ligand-independent mechanism should be considered. Therefore, although carbaryl concentration in food is very low, care should be taken because of its possible adverse effects in human health through liver and skin, given the well established toxicological importance of CYP1A1 induction.

Comparative study of CYP1A1 induction by 3-methylcholanthrene in various human hepatic and epidermal cell types.

Hepatocytes and keratinocytes are among the most widely used cells in pharmaco-toxicology, but a limitation of these models is the provision of human tissues on a regular basis. The suitability of HepG2, HaCaT and HESV cell lines as an acceptable substitute for primary cultures was examined. In these cell types, the effects of 3-methylcholanthrene (3-MC) were analysed on CYP1A1 gene expression, a crucial CYP subfamily in the activation of chemical carcinogens. Ethoxyresorufin O-deethylase (EROD) activity was never detected in HESV cells, but in other cell types it was stimulated in a concentration-dependent manner (maximal induction, 1-2.5 mum). Above this peak induction the effect fell rapidly. Northern blot analysis of CYP1A1 mRNA agreed with the trends obtained for EROD values. However, the decrease of the EROD activity observed at the highest 3-MC concentrations was not correlated with CYP1A1 mRNA reduction. This study also demonstrated that 3-MC is capable of significantly inducing CYP1A1 in HaCaT cells (17-fold over control), as in human hepatocytes (six- to 18-fold) and HepG2 (fourfold). Therefore, in contrast to SV40-immortalized keratinocytes (HESV), spontaneously immortalized keratinocytes (HaCaT) may constitute a valuable tool for studying epidermal CYP1A1 gene regulation by xenobiotics.