This study was conducted to investigate the effects of treatment with the prototypical inducers rifampicin (Rif), dexamethasone (Dex), and omeprazole (Ome) on the mRNA levels of drug-metabolizing enzymes in primary cultures of cryopreserved human, cynomolgus monkey, and rat hepatocytes. Analysis was performed by quantitative real-time RT-PCR using primers and TaqMan probes. Treatment with Ome substantially increased the mRNA levels of both CYP1A1 and CYP1A2 in human hepatocytes, but increased only the mRNA level of CYP1A1 in monkey hepatocytes, whereas it had no marked effect on the mRNA levels of CYP1A1 or CYP1A2 in rat hepatocytes. Treatment with Rif or Dex did not markedly affect the mRNA level of CYP1A in any of the hepatocyte cultures under the conditions used. All three inducers increased the mRNA level of CYP3A8 in monkey hepatocytes (in the order Rif>Dex>or=Ome), and a similar profile was observed for the mRNA level of CYP3A4 in human hepatocytes, but the potency of induction was markedly attenuated. In contrast, only Dex substantially increased the mRNA level of CYP3A1 in rat hepatocytes, with Rif and Ome showing no effects. These results indicate that the molecular mechanisms responsible for the regulation of CYP1A2 genes differ between humans and cynomolgus monkeys, although the regulatory mechanisms for CYP1A1 and CYP3A genes are similar.

The mRNA induction of various transporters by rifampicin (Rif), dexamethasone (Dex) and omeprazole (Ome) was investigated in primary cultures of cryopreserved human and rat hepatocytes. Analysis was performed by quantitative real-time RT-PCR using primers and TaqMan probes. In primary cultures of human hepatocytes, mRNA levels of MDR and MRP1 were increased by about 1.5 fold and 1.3 fold, respectively, by exposure to Rif at 2 to 50 microM as compared with 0.1% DMSO-treated controls. MRP2 mRNA levels in the same human hepatocytes were significantly increased by 1.2 to 1.8 fold by exposure to Rif at 50 microM as compared with controls. In primary cultures of rat hepatocytes, Mdr1a and Mdr1b mRNA levels were not increased or only slightly increased at 24 hr by exposure to any of the inducers at 2, 10 or 50 microM. Mrp2 mRNA levels in the same rat hepatocytes were significantly increased by 7 to 45 fold by exposure to Dex at 2 microM as compared with controls. Based on the species differences observed in the present study, primary cultures of cryopreserved hepatocytes from both the human and rat should be useful in preclinical drug development for evaluating candidate drugs for transporter induction.

A method for the analysis of N-acetylcysteine conjugates of catechol estrogens [catechol estrogen mercapturates (CE SRs)], which are likely to be urinary markers of estrogen-induced tumors, was established in this study. The characteristics of the method that was established were (1) cleanup of urine using the immunoaffinity column of CE SRs, (2) detection of catechol estrogens (CEs) and CE SRs by electrochemical detection, which provided the high specificity, and (3) stability of CE SRs through the cleanup. Using this method, the simultaneous quantitation of 2-hydroxy-17beta-estradiol (2-OHE(2)), 4-hydroxy-17beta-estradiol (4-OHE(2)), 2-hydroxyestrone (2-OHE(1)), 4-hydroxyestrone (4-OHE(1)), 2-hydroxyestrone 1-N-acetylcysteine thioether (2-OHE(1) 1SR), 2-hydroxyestrone 4-N-acetylcysteine thioether (2-OHE(1) 4SR), and 4-hydroxyestrone 2-N-acetylcysteine thioether (4-OHE(1) 2SR) in the range of 1-15 ng was performed. We first demonstrated the presence of CE SRs, 2-OHE(1) 1SR and 2-OHE(1) 4SR, in urine from rats treated intraperitoneally with 17beta-estradiol (E(2)) at a dose of 5 mg/kg. In female rats, the amount of 2-OHE(1) 1SR was several-fold greater than that of 2-OHE(1) 4SR, while the presence of 4-OHE(1) 2SR was not confirmed. The level of CEs and CE SRs in male rats was approximately (1)/(2)-(1)/(20) of that in female rats. The excretion rate following administration of 2-OHE(1) at 2 mg/kg and that following the administration of 4-OHE(1) at 2 mg/kg were different in female rats. In addition, 4-OHE(1) 2SR was present in the urine of male Syrian hamsters treated intraperitoneally with E(2), whereas it was absent in rats.

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The effects of ten endocrine disrupting chemicals, i.e., bisphenol A (BPA), p-nonylphenol (NP), p-octylphenol (OP), p-pentylphenol (PP), butyl benzyl phthalate (BBP), dicyclohexyl phthalate (DCHP), di-n-butyl phthalate (DBP), tetrabutyltin (TBT), tri-n-butyltin chloride (TBC), and di-n-butyltin dichloride (DBD), as well as 17 beta-estradiol (E(2)) as a positive control on the microtubule network in Chinese hamster V79 cells in culture were examined by the indirect immunofluorescence method using anti-beta-tubulin antibody. In the whole-animal system, the effects of BPA, NP, OP, BBP, DBD, and E(2) as well as vinblastine sulfate (VB) as a positive control on microtubules in the cytoplasm of Sertoli cells in rats were examined by electron microscopy. In Chinese hamster V79 cells, TBC and DBD showed higher microtubule-disruptive activity than E(2), while other chemicals had less activity than E(2). The ranking for efficiency on microtubule disruption was (TBC falling dots DBD) > (E(2) = TBT) > (BPA = alkylphenols, NP and OP) >> (phthalate esters, BBP, DHP, and DBP). In rats as a whole-animal system, no disrupting effects on the microtubule network in the cytoplasm of Sertoli cells were observed under any environmental chemicals tested, whereas exposure to VB resulted in marked disruption of the microtubule network. The results of this study suggested that some endocrine disrupting chemicals have disrupting effects on the microtubule network in vitro, but no such effects in vivo.