T. Zacharewski

BISPHENOL A INTERACTS WITH THE ESTROGEN RECEPTOR ALPHA IN A DISTINCT MANNER FROM ESTRADIOL

We investigated the interaction of bisphenol A (BPA, an estrogenic environmental contaminant used in the manufacture of plastics) with the estrogen receptor alpha (ERalpha) transfected into the human HepG2 hepatoma cell line and expanded the study in vivo to examine the effect of BPA on the immature rat uterus. Bisphenol A was 26-fold less potent in activating ER-WT and was a partial agonist with the ERalpha compared to E2. The use of ERalpha mutants in which the AF1 or AF2 regions were inactivated has permitted the classification of ER ligands into mechanistically distinct groups. The pattern of activity of BPA with the ERalpha mutants differed from the activity observed with weak estrogens (estrone and estriol), partial ERalpha agonists (raloxifene or 4-OH-tamoxifen), or a pure antagonist (ICI 182, 780). Intact immature female Sprague-Dawley rats were exposed to BPA alone or with E2 for 3 days. Unlike E2, BPA had no effect on uterine weight; however, like E2, both peroxidase activity and PR levels were elevated, though not to the level induced by E2. Following simultaneous administration, BPA antagonized the E2 stimulatory effects on both peroxidase activity and PR levels but did not inhibit E2-induced increases of uterine weight. These results demonstrate that BPA is not merely a weak estrogen mimic but exhibits a distinct mechanism of action at the ERalpha.

Aroclor 1254 as a 2,3,7,8-tetrachlorodibenzo-p-dioxin antagonist: Effects on enzyme induction and immunotoxicity

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and Aroclor 1254 induced the cytochrome P-450 dependent monooxygenases, aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) in rat hepatoma H-4-II E cells and C57BL/6J mice. It has been proposed that both Aroclor 1254 and 2,3,7,8-TCDD induce these enzymes via a common mechanism which features initial binding to the aryl hydrocarbon (Ah) cytosolic receptor protein. The major difference between these compounds was the relative potency (i.e. 2,3,7,8-TCDD much greater than Aroclor 1254). Cotreatment of rat hepatoma H-4-II E cells or C57BL/6J mice with a dose of 2,3,7,8-TCDD which submaximally induces AHH and EROD and a dose of Aroclor 1254 which exhibited little or no induction activity resulted in significant antagonism of the induction effects of 2,3,7,8-TCDD. For example, cotreatment of C57BL/6J mice with 2,3,7,8-TCDD (15 nmol/kg) and Aroclor 1254 (25, 75 and 150 mumol/kg) resulted in up to 23% antagonism of AHH induction by 2,3,7,8-TCDD. Moreover, cotreatment with a higher dose of the 2,3,7,8-TCDD agonist (30 or 50 nmol/kg) partially reversed some of the antagonism by Aroclor 1254. In vivo antagonism was observed only at Aroclor 1254/2,3,7,8-TCDD molar ratios of 1667:1, 5000:1 and 10,000:1. Administration of 2,3,7,8-TCDD (3.72 nmol/kg) to C57BL/6J mice resulted in a 76% decrease in the splenic plaque forming cell response to sheep red blood cells. This T-cell mediated immunotoxic effect of 2,3,7,8-TCDD segregates with the Ah locus. In contrast, administration of 5, 15, 75 and 150 mumol/kg of Aroclor 1254 resulted in impairment of the immune response only at the highest dose level. However, cotreatment of mice with 2,3,7,8-TCDD (3.72 nmol/kg) and Aroclor 1254 (5, 15 or 75 mumol/kg) resulted in no significant decrease in the plaque forming cell response and complete protection from the immunotoxicity of 2,3,7,8-TCDD. Cotreatment of the mice with Aroclor 1254 (75 mumol/kg) and a higher dose of the 2,3,7,8-TCDD agonist resulted in partial reversal of the protective effects of Aroclor 1254. The in vitro and in vivo data suggest that within specific antagonist/agonist dose ratios, Aroclor 1254 can antagonize at least 2 Ah receptor-mediated effects of 2,3,7,8-TCDD, namely AHH induction and immunotoxicity.

Naringenin: A weakly estrogenic bioflavonoid that exhibits antiestrogenic activity

Treatment of immature 21-day-old female Sprague-Dawley rats with 17 beta-estradiol (E2) (0.5 microgram/rat) caused a significant increase in uterine wet weight, DNA synthesis, progesterone receptor (PR) binding, and peroxidase activity. At doses as high as 40 mg/rat, the bioflavonoid naringenin did not cause a significant increase in any of these E2-induced responses. However, in rats cotreated with E2 (0.5 microgram/rat) plus naringenin (30 mg/rat); there was a significant decrease in E2-induced uterine wet weight, DNA synthesis, PR binding, and peroxidase activity, indicating that naringenin exhibits antiestrogenic activity in the immature rodent uterus. The binding of uterine nuclear extracts to a 32P-labeled estrogen responsive element (ERE) or progesterone responsive element (PRE) was determined using gel electrophoretic band shift assays. Incubation of [32P]ERE with uterine nuclear extracts from rats treated with naringenin or E2 resulted in the formation of estrogen receptor (ER):ERE complexes; a higher mobility complex was prominent in the extracts from E2-treated rats, whereas a lower mobility complex was observed using nuclear extracts from naringenin-treated animals. There was a significant decrease in the intensity of the E2-induced complex using nuclear extracts from rats treated with E2 plus naringenin. In contrast, transformed cytosol from control rats gave an intense ER:ERE complex, whereas the intensity of the band was decreased markedly using transformed uterine cytosol from treated rats. Formation of a PR:PRE complex was also determined using transformed uterine cytosol. Cytosol from E2-treated rats gave an intense retarded band, whereas only weak bands were observed using cytosols from DMSO- (solvent), naringenin-, or naringenin plus E2-treated cells. The results of in vitro studies showed that 1 nM E2 increased (3- to 4-fold) the growth of MCF-7 human breast cancer cells, whereas 1-1000 nM naringenin had no effect on cell proliferation. In cells cotreated with 1 nM E2 plus 1000 nM naringenin, there was a significant decrease in E2-induced cell growth. In MCF-7 cells transiently transfected with a pS2 promoter-regulated luciferase reporter gene, naringenin exhibited weak estrogenic activity. In cells cotreated with 0.1 or 1.0 microM naringenin plus 1 nM E2, naringenin inhibited E2-induced luciferase activity. The results of these studies confirmed that naringenin is a weak estrogen that also exhibits partial antiestrogenic activity in the female rat uterus and MCF-7 human breast cancer cells.

Polybrominated dibenzo-p-dioxins and related compounds: Quantitative in vivo and in vitro structure-activity relationships

The effects of structure on the in vitro receptor binding affinities, aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) induction potencies in rat hepatoma cells were determined for the following compounds: 2-bromo-, 2,7/2,8-dibromo-, 2,3,7-tribromo-, 2,4,6,8/1,3,7,9-tetrabromo-, 2,3,7,8-tetrabromo-, 1,3,7,8-tetrabromo-, 1,2,3,7,8-pentabromo-, 1,2,4,7,8-pentabromo-, 2,3-dibromo-7,8-dichloro-, 2,8-dibromo-3,7-dichloro- and 2-bromo-3,7,8-trichlorodibenzo-p-dioxin. The structure-activity relationships (SARs) for the polybrominated dibenzo-p-dioxins (PBDDs) were comparable for both in vitro responses: the most active compounds were substituted only in the lateral 2,3,7 and 8 position and the addition of non-lateral or removal of lateral halogen substituents reduced the activity of the resultant compound. The biologic and toxic effects of 2,3,7,8-tetrabromo-, 1,3,7,8-tetrabromo-, 1,2,4,7,8-pentabromo-1,2,3,7,8-pentabromo-, 2-bromo-3,7,8-trichloro- and 2,3-dibromo-7,8-dichlorodibenzo-p-dioxin on several receptor-mediated responses (thymic atrophy, body weight loss, hepatic microsomal AHH and EROD induction) were determined in a dose-response fashion in immature male Wistar rats. A comparison of the ED50 values for the in vivo responses demonstrated that the SARs for the PBDDs and brominated polychlorinated dibenzo-p-dioxins were comparable to those observed for in vitro receptor binding and AHH induction. Moreover, there was an excellent linear correlation between the -log EC50 (in vitro AHH induction) vs. the in vivo -log ED50 (thymic atrophy) and -log ED50 (body wt loss) correlation coefficient, r = 0.97 for all 2 correlations).

Detection of estrogen- and dioxin-like activity in pulp and paper mill black liquor and effluent using in vitro recombinant receptor/reporter gene assays

In vitro recombinant receptor/reporter gene assays were used to examine pulp and paper mill black liquor and effluent for estrogenic, dioxin-like, and antiestrogenic activities. Using MCF-7 cells transiently transfected with a Gal4-estrogen receptor chimeric construct (Gal4-HEGO) and a Gal4-regulated luciferase reporter gene (17m5-G-Luc), it was estimated that black liquor contains 4 ±2 ppb estrogen equivalents, while negligible estrogenic activity was observed in a methanol-extracted pulp and paper mill effluent fraction (MF). A dioxin response element (DRE)-regulated luciferase reporter gene (pGudLuc1.1) transiently transfected into Hepa1c1c7 wild-type cells exhibited a dose-dependent increase in luciferase activity following treatment with 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD), black liquor, and MF. Based on the dose-response curves, black liquor and MF contain 10 ± 4 ppb and 20 ± 6 ppt TCDD equivalents, respectively. Moreover, MF exhibited significant AhR-mediated antiestrogenic activity. These results demonstrate the utility of these bioassays and suggest that the effects observed in fish exposed to pulp and paper mill effluent may be due to unidentified ER and AhR ligands not detected by conventional chemical analysis due to the lack of appropriate standards.

Evidence for the mechanism of action of the 2,3,7,8-tetrachlorodibenzo-p-dioxin-mediated decrease of nuclear estrogen receptor levels in wild-type and mutant mouse hepa 1c1c7 cells

Treatment of wild-type Hepa 1c1c7 cells with 1 nM [3H]-17 beta-estradiol resulted in the rapid accumulation of the nuclear estrogen receptor complex whose levels were maximized within 1 hr. Cotreatment of the cells with 10 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and [3H]-17 beta-estradiol did not affect the nuclear estrogen receptor levels 1 hr after addition of the radioligand; however, pretreatment of the cells for 1, 6, 24 or 42 hr with 10 nM TCDD prior to the addition of the radiolabeled hormone caused a greater than 50% decrease in nuclear estrogen receptor levels (determined by velocity sedimentation analysis) 1 hr after the addition of [3H]-17 beta-estradiol. In parallel experiments in which 10 nM TCDD was added 6 hr prior to the radiolabeled hormone, TCDD caused a 63 and 74% decrease in immunodetectable cytosolic and nuclear estrogen receptor protein levels, respectively, in the wild-type Hepa 1c1c7 cells. The nuclear estrogen receptor was also detected in two Hepa 1c1c7 mutant (class 1 and class 2) cell lines which have been characterized previously as TCDD non-responsive due to either decreased aryl hydrocarbon (Ah) receptor levels or a defect in the accumulation of transcriptionally active nuclear Ah receptor complexes, respectively. Treatment of these mutant cell lines with TCDD and [3H]-17 beta-estradiol (as described above) caused only a minimum (class 1) or non-detectable (class 2) decrease in nuclear estrogen receptor binding activity or immunodetectable protein levels. These results, coupled with the structure-dependent differences in the activities of TCDD (a strong Ah receptor agonist) and 2,8-dichlordibenzo-p-dioxin (a weak Ah receptor agonist) in this assay system, support a role for the Ah receptor in the TCDD-mediated decrease of the nuclear estrogen receptor in mouse Hepa 1c1c7 cells. In addition, actinomycin D and cycloheximide both inhibited the TCDD-mediated decrease of nuclear estrogen receptor levels in the Hepa 1c1c7 wild-type cells, and these results suggest that TCDD may induce specific gene products which are involved in this process.

Comparative potencies of Aroclors 1232, 1242, 1248, 1254, and 1260 in male Wistar rats--assessment of the toxic equivalency factor (TEF) approach for polychlorinated biphenyls (PCBs).

Immature male Wistar rats were treated with several different doses of the commercial polychlorinated biphenyls (PCBs) Aroclors 1232, 1242, 1248, 1254, and 1260 (10, 40, 160, 480, and 2000 mg/kg) and the effects on body weight gain, thymic atrophy, and the induction of hepatic microsomal aryl hydrocarbon hydroxylase (AHH), ethoxyresorufin O-deethylase (EROD), and pentoxyresorufin O-deethylase (PROD) activities were measured 14 days after treatment. A significant inhibition in body weight gain was observed only in rats treated with high doses of Aroclors 1232 and 1248 and thymic atrophy was not observed for any of the Aroclors. All the Aroclors caused a dose-dependent increase in hepatic microsomal AHH, EROD, and PROD activities. The corresponding ED50 values for the induction of AHH-EROD activities varied from 51 to 678 mg/kg. Aroclor 1260 was the least active inducer of the P4501A1-mediated enzyme activities. In contrast, Aroclor 1260 was a potent inducer of PROD activity (ED50 = 37 mg/kg), but Aroclors 1232, 1242, 1248, and 1254 did not induce 50% of the maximal response at the highest dose used in this experiment (2000/kg). Previous studies have quantitated the levels of those PCB congeners which induce AHH or EROD activities in Aroclors 1232, 1242, 1254, and 1260 and their potencies or toxic equivalency factors (TEFs) relative to that of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) have also been estimated or experimentally determined. Using highly conservative TEF values it was demonstrated that the calculated ED50s for the Aroclors as inducers of AHH and EROD activity were significantly lower than the observed ED50 values.2

pS2 (TFF1) levels in human breast cancer tumor samples: correlation with clinical and histological prognostic markers.

The expression of pS2 (TFF1) has been previously shown to identify patients with improved response to anti–hormonal therapy and more favorable outcome. In the current study, 100 human breast carcinoma samples obtained from the Manitoba Breast Tumor Bank were analyzed for pS2 mRNA using a quantitative, competitive reverse transcriptase–polymerase chain reaction (qcRT–PCR) assay. A pS2/ß–actin cut–off criterion of 0.010 was established to classify tumors as either pS2 positive or pS2 negative. pS2 mRNA levels were positively associated with both ER and PR, with the majority of ER+ (59) and PR+ (60) tumors also being positive for pS2. In addition, a significant linear correlation was observed between the amount of pS2 mRNA and ER (p<0.0001) and PR (p<0.0001) protein. pS2 mRNA levels also exhibited an inverse association with tumor size and histological grade, consistent with the observation that pS2 is primarily expressed in small (T <u20092.0u2009cm), but well differentiated tumors (Grades I and II). No associations were observed with tumor cell type, patient age, or lymph node status. The strong correlation displayed between pS2 and a number of currently used breast cancer prognostic markers supports the clinical use of pS2 to further assess tumor status and patient outcome.

Applications of the in vitro aryl hydrocarbon hydroxylase induction assay for determining “2,3,7,8- tetrachlorodibenzo-p-Dioxin equivalents”: Pyrolyzed brominated flame retardants

The pyrolysis of brominated flame retardants FR 300 BA (decabromobiphenyl) ether, FireMaster BP-6 (polybrominated biphenyls), Bromkal 70-5-DE (primarily pentabromodiphenylether), Bromkal 70-DE (primarily penta and tetrabromodiphenylether) and Bromkal G1 (pentabromodiphenylether) resulted in the formation of relatively high levels of polybrominated dibenzofurans (PBDFs) and dibenzo-p-dioxins (PBDDs as determined by gas chromatography-mass spectrometric analysis. The dose response EC50 values for the induction of aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) by the flame retardant pyrolysates was determined in rat hepatoma H-4-II E cells and compared to the relative induction activities of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and the concentrations of 2,3,7,8-TCDD equivalents were calculated. The range of 2,3,7,8-TCDD equivalents levels (micrograms/g or ppm) derived from values obtained from the AHH and EROD bioassays for each of the pyrolyzed flame retardant samples was: 174-194, 480-1400, 2140-4680, 6740-8780 and 3920-5260 ppm for FR 300 BA, FireMaster BP-6, Bromkal 70 DE, Bromkal 70-5 DE and Bromkal G1, respectively. The in vivo dose-response effects of 2 pyrolyzed flame retardants were determined in immature male Wistar rats and compared to the dose-response activities of 2,3,7,8-TCDD. The in vivo responses which were measured included hepatic microsomal AHH and EROD induction, body weight loss and thymic atrophy. For the pyrolyzed FireMaster BP-6 and Bromkal 70-5 DE samples, the range of calculated in vivo 2,3,7,8-TCDD equivalents (ppm in sample) for the 4 in vivo bioassays was 520-1780 ppm and 3860-8960 ppm, respectively. The excellent overlap between the in vivo and in vitro 2,3,7,8-TCDD equivalents for the 2 flame retardant pyrolysate extracts supports the utility of the in vitro induction bioassay for quantitatively determining 2,3,7,8-TCDD equivalents for mixtures containing toxic halogenated aryl hydrocarbons.

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on I-compounds in hepatic DNA of sprague-dawley rats: Sex-specific effects and structure-activity relationships

The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds on the specific patterns of age-dependent I-compound DNA adducts in the liver of male and female Sprague-Dawley rats were determined by the 32P-postlabeling assay. In female rats, TCDD causes a dose-dependent decrease of several individual and total hepatic I-compound levels after administration of 1 and 5 micrograms/kg per week for 4 weeks. In contrast, no such effects were observed in male Sprague-Dawley rats treated with the 5 micrograms/kg dose level of TCDD. The relative effects of TCDD, 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PCDD) and 1,2,4,7,8-PCDD on hepatic I-compound levels in the susceptible female Sprague-Dawley rats were determined using a dose of 5 micrograms/kg per week for 4 weeks. The two compounds which are substituted in all four lateral positions, namely TCDD and 1,2,3,7,8-PCDD, caused a significant decrease in hepatic I-compound levels, whereas 1,2,4,7,8-PCDD which is substituted in only three lateral positions was inactive. The structure-activity relationships observed for the effects of these compounds on hepatic I-compounds correlated with their corresponding structure-Ah receptor binding and structure-toxicity relationships. The results are therefore consistent with a role for the Ah receptor in the TCDD-mediated reduction in hepatic I-compound levels in female Sprague-Dawley rats. These results and data from previous studies demonstrate a correlation between the susceptibility of an organ/species to the carcinogenic effects of TCDD and the reduction of I-compound levels. The significance of this correlation in the development of TCDD-induced carcinogenesis has not been delineated.