Endocrine disrupting chemicals, 4-nonylphenol, bisphenol A and butyl benzyl phthalate, impair metabolism of estradiol in male and female rats as assessed by levels of 15α-hydroxyestrogens and catechol estrogens in urine.

Bisphenol A (BPA), 4-nonylphenol (NP) and butyl benzyl phthalate (BBP), termed endocrine-disrupting chemicals, are known to mimic estrogen activity. The effects of these chemicals on 17ß-estradiol (E2 ) metabolism in vivo in rats were examined. Male and female rats were given NP (250 mg kg-1  day-1 ), BPA (250 µg kg-1  day-1 ) or BBP (500 mg kg-1  day-1 ) by gavage for 14 days, followed by a single intraperitoneal injection of E2 (5 mg kg-1 ) on the final day. The urinary excretion over 72 hours of 2-hydroxyestrone 1-N-acetylcysteine thioether, 2-hydroxyestrone 4-N-acetylcysteine thioether, 4-hydroxyestrone 2-N-acetylcysteine thioether, 2-hydroxy-17ß-estradiol (2-OHE2 ), 2-hydroxyestrone (2-OHE1 ), 4-hydroxy-17ß-estradiol, 4-hydroxyestrone, 15α-hydroxyestriol (E4 ), 15α-hydroxy-17ß-estradiol and 15α-hydroxyestrone was measured. Increases in urinary excretion of 2-OHE1 and decreases in E4 were observed in males treated with NP or BBP. Decreases in urinary excretion of 2-OHE2 and E4 were observed in males treated with BPA. Decreases in urinary excretion of 2-OHE1 and 2-OHE2 were observed in females treated with BBP. Normalized liver and weights were increased in both sexes treated with NP or BBP. Histologic observations revealed marked changes in the distal tubules and collecting ducts in the kidneys of rats exposed to NP and BBP, and hypertrophy in the hepatocytes of the centrilobular zone of the liver. No BPA-related effects on organ weight and on liver or kidney histopathology were found. These results suggest that the 14 day oral dosing of NP and BBP disrupted E2 metabolism, resulting from marked morphological and functional alterations in the liver and kidneys. In addition, BPA could induce metabolic and endocrine disruption.

Determination of urinary 15α-hydroxyestrogen levels via immunoaffinity extraction.

15α-Hydroxyestrogens (15α-OHEs) are metabolites of the female hormone estradiol. In this study, to discover physiological markers that can be utilized for monitoring fetal conditions and estrogen-induced cancers, we established a method for quantifying 15α-OHEs in rat urine via immunoaffinity column extraction and HPLC-electrochemical detection, and detected 15α-OHEs in urine obtained male rats treated with estradiol. Notably, the standard curves for quantification obtained using the column were linear over a range of 0.5-50ng 15α-OHEs. The accuracy of the analytical method with cleanup was 97-109% for the three kinds of 15α-OHEs examined, and the intra-assay precision of the measured values had a coefficient of variation of ≤20.6%. Therefore, the theoretical limit of quantification was 0.5ng. However, the actual measured values obtained from the urine of male rats indicated that the detection limits were 0.425, 0.103, and 0.047ng for estetrol, 15α-hydroxyestradiol, and 15α-hydroxyestrone, respectively. Our newly established method for measuring 15α-OHE concentrations in urine could facilitate characterization of the in vivo metabolic profile of 15α-OHEs in mammals under various physiological conditions, which could comprise clinical markers for monitoring human fetal health conditions in mammals.

Comparison of inducibility of CYP1A and CYP3A mRNAs by prototypical inducers in primary cultures of human, cynomolgus monkey, and rat hepatocytes.

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.

Regulation of mRNA expression of MDR1, MRP1, MRP2 and MRP3 by prototypical microsomal enzyme inducers in primary cultures of human and rat hepatocytes.

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.

Effects of prototypical drug-metabolizing enzyme inducers on mRNA expression of housekeeping genes in primary cultures of human and rat hepatocytes.

Quantitative real-time RT-PCR was used to investigate the effects of prototypical drug-metabolizing enzyme inducers rifampicin (Rif), dexamethasone (Dex), and omeprazole (Ome) on mRNA expression levels of the housekeeping genes beta-actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-glucuronidase (GUSB), hypoxanthine phosphoribosyltransferase 1 (HPRT1), peptidylprolylisomerase A (PPIA), TATA box binding protein (TBP), and transferrin receptor (TFRC) in primary cultures of cryopreserved human and rat hepatocytes. The mRNA levels of ACTB, GAPDH, GUSB, PPIA, TBP, and TFRC relative to HPRT1 in human hepatocytes were constant at all concentrations of inducers. However, the mRNA level of GAPDH relative to HPRT1 in rat hepatocytes was markedly increased by Rif. The mRNA levels of GAPDH, GUSB, PPIA, TBP, and TFRC relative to HPRT1 in rat hepatocytes were significantly increased by Dex. ACTB and HPRT1 are suitable internal controls for evaluating mRNA expression levels in primary cultures of human and rat hepatocytes after Rif, Dex, or Ome exposure.