Biomedical subjects

T Zacharewski

Publications and source records attributed to T Zacharewski.

At least 19 recordsLinked to original sources

PubMed2002

Distribution of androgen receptor mRNA expression and immunoreactivity in the brain of the green anole lizard.

Male courtship and copulation are androgen dependent in the green anole lizard, and female receptivity can be facilitated by testosterone. However, only a few, and relatively large, regions in the brain have been implicated in the control of these behaviours. In situ hybridization and immunohistochemistry were therefore used to determine in detail where androgens are likely to act in the brains of breeding males and females. A 697-bp fragment of the anole androgen receptor (AR) was cloned from total RNA isolated from the kidney, which contains the highly androgen-sensitive renal sex segment. The cloned fragment spanned part of the C, the entire D, and part of the E domains, and shared a high degree of similarity with the AR of various species. 35S-labelled antisense and sense probes were generated from the 697-bp fragment for use in in situ hybridization, and the AR antibody PG-21 was used for immunohistochemistry. Both sexes consistently had AR mRNA expression and immunoreactivity in areas associated with vertebrate reproductive behaviours and in motor areas of the brainstem. Interestingly, the PG-21 antibody produced labelling in both the nucleus and cytoplasm, including neuronal processes. The distribution of mRNA and immunoreactivity were comparable in males and females, and the amount of labelling was generally similar, although slightly greater in females. The expression pattern of AR in this species supports the idea that distribution is highly conserved among vertebrates, but that it probably does not dictate behavioural differences between the sexes in anoles.

Amino Acid Sequence↗

PubMed2000

Differential estrogen receptor binding of estrogenic substances: a species comparison.

The study investigated the ability of 34 natural and synthetic chemicals to compete with [3H]17beta-estradiol (E2) for binding to bacterially expressed glutathione-S-transferase (GST)-estrogen receptors (ER) fusion proteins from five different species. Fusion proteins consisted of the ER D, E and F domains of human alpha (GST-hERalphadef), mouse alpha (GST-mERalphadef), chicken (GST-cERdef), green anole (GST-aERdef) and rainbow trout ERs (GST-rtERdef). All five fusion proteins displayed high affinity for E2 with dissociation constants (K(d)) ranging from 0.3 to 0.9 nM. Although, the fusion proteins exhibited similar binding preferences and binding affinities for many of the chemicals, several differences were observed. For example, alpha-zearalenol bound with greater affinity to GST-rtERdef than E2, which was in contrast to other GST-ERdef fusion proteins examined. Coumestrol, genistein and naringenin bound with higher affinity to the GST-aERdef, than to the other GST-ERdef fusion proteins. Many of the industrial chemicals examined preferentially bound to GST-rtERdef. Bisphenol A, 4-t-octylphenol and o,p' DDT bound with approximately a ten-fold greater affinity to GST-rtERdef than to other GST-ERdefs. Methoxychlor, p,p'-DDT, o,p'-DDE, p,p'-DDE, alpha-endosulfan and dieldrin weakly bound to the ERs from the human, mouse, chicken and green anole. In contrast, these compounds completely displaced [3H]E2 from GST-rtERdef. These results demonstrate that ERs from different species exhibit differential ligand preferences and relative binding affinities for estrogenic compounds and that these differences may be due to the variability in the amino acid sequence within their respective ER ligand binding domains.

Amino Acid Sequence↗

PubMed2000

Differential binding affinities of PCBs, HO-PCBs, and aroclors with recombinant human, rainbow trout (Onchorhynkiss mykiss), and green anole (Anolis carolinensis) estrogen receptors, using a semi-high throughput competitive binding assay.

A comparative study was undertaken to assess the ability of 44 polychlorinated biphenyls (PCBs), 9 hydroxylated PCBs (HO-PCBs), and 8 aroclors at concentrations ranging from 1 nM to 10 microM to compete with [3H]17beta-estradiol (E2) for binding to bacterially expressed fusion proteins using a semi-high throughput competitive-binding assay. The fusion proteins consisted of the D, E, and F domains of human (alpha), cloned reptilian (Anolis carolinensis) and recloned rainbow trout (Onchorhynkiss mykiss) estrogen receptors (ER) linked to the glutathione S-transferase (GST) protein. GST-hERalphadef (human), GST-aERdef (reptile) and GST-rtERdef (rainbow trout) fusion proteins exhibited high affinity for E2 with dissociation constants (Kd) of 0.4+/-0.1 nM, 0.7+/-0.2 nM, and 0.6+/-0.1 nM, respectively. Of the 44 PCBs examined, only PCBs 104, 184, and 188 effectively competed with [3H]E2 for binding to the GST-rtERdef protein with IC50 values ranging from 0.4-1.3 microM. In contrast, these same congeners only caused a 30% displacement of [3H]E2 in GST-hERalphadef and GST-aERdef proteins. Several additional congeners were found to bind to the GST-rtERdef fusion protein, although the degree of interaction varied among congeners. Among the HO-PCBs, 2',3',4',5'-tetrachloro-4-biphenylol and 2,6,2',6'-tetrachloro-4-biphenylol bound to all three fusion proteins with IC50 values ranging from 0.1-0.3 microM. Dimethyl sulphoxide (DMSO) concentrations of 20% significantly increased the ability of PCBs 104, 184, and 188 to compete with [3H]E2 for binding to the GST-ERdef fusion proteins, whereas at 20% DMSO, a significant reduction in saturable binding was observed. These results demonstrate that ERs from different species exhibit differential ligand preferences and relative binding affinities for PCBs, which can be dramatically affected by DMSO concentration.

Amino Acid Sequence↗

PubMed1998

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.

Animals↗

PubMed1998

13th Meeting of the Scientific Group on Methodologies for the Safety Evaluation of Chemicals (SGOMSEC): alternative testing methodologies for organ toxicity.

In the past decade in vitro tests have been developed that represent a range of anatomic structure from perfused whole organs to subcellular fractions. To assess the use of in vitro tests for toxicity testing, we describe and evaluate the current status of organotypic cultures for the major target organs of toxic agents. This includes liver, kidney, neural tissue, the hematopoietic system, the immune system, reproductive organs, and the endocrine system. The second part of this report reviews the application of in vitro culture systems to organ specific toxicity and evaluates the application of these systems both in industry for safety assessment and in government for regulatory purposes. Members of the working group (WG) felt that access to high-quality human material is essential for better use of in vitro organ and tissue cultures in the risk assessment process. Therefore, research should focus on improving culture techniques that will allow better preservation of human material. The WG felt that it is also important to develop and make available relevant reference compounds for toxicity assessment in each organ system, to organize and make available via the Internet complete in vivo toxicity data, including human data, containing dose, end points, and toxicokinetics. The WG also recommended that research should be supported to identify and to validate biological end points for target organ toxicity to be used in alternative toxicity testing strategies.

Animal Testing Alternatives↗

PubMed1998

Identification and assessment of endocrine disruptors: limitations of in vivo and in vitro assays.

It has been suggested that chemicals and complex mixtures capable of modulating the endocrine system may contribute to adverse health, reproduction, and developmental effects in humans and wildlife. These effects include increased incidence of hormone-dependent cancers, compromised reproductive fitness, and abnormal reproductive system development. In response to public concern, regulatory agencies in North America and Europe are formulating potential strategies to systematically test chemicals and complex mixtures for their endocrine-disrupting activities. Because of the complexity of the endocrine system and the number of potential endocrine disruptor targets, a tiered approach involving a complementary battery of short- and long-term in vivo and in vitro assays that assesses both receptor and nonreceptor-mediated mechanisms of action is being considered. However, the available established assays use a limited number of end points, and significant information gaps exist for other potential targets in the endocrine system. In addition to discussing the merits and limitations of the assays that may be adopted, this paper also highlights potential problems associated with the use of a tiered testing strategy.

Animals↗

PubMed1998

Toxic equivalency factors (TEFs) for PCBs, PCDDs, PCDFs for humans and wildlife.

An expert meeting was organized by the World Health Organization (WHO) and held in Stockholm on 15-18 June 1997. The objective of this meeting was to derive consensus toxic equivalency factors (TEFs) for polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) and dioxinlike polychlorinated biphenyls (PCBs) for both human, fish, and wildlife risk assessment. Based on existing literature data, TEFs were (re)evaluated and either revised (mammals) or established (fish and birds). A few mammalian WHO-TEFs were revised, including 1,2,3,7,8-pentachlorinated DD, octachlorinated DD, octachlorinated DF, and PCB 77. These mammalian TEFs are also considered applicable for humans and wild mammalian species. Furthermore, it was concluded that there was insufficient in vivo evidence to continue the use of TEFs for some di-ortho PCBs, as suggested earlier by Ahlborg et al. [Chemosphere 28:1049-1067 (1994)]. In addition, TEFs for fish and birds were determined. The WHO working group attempted to harmonize TEFs across different taxa to the extent possible. However, total synchronization of TEFs was not feasible, as there were orders of a magnitude difference in TEFs between taxa for some compounds. In this respect, the absent or very low response of fish to mono-ortho PCBs is most noticeable compared to mammals and birds. Uncertainties that could compromise the TEF concept were also reviewed, including nonadditive interactions, differences in shape of the dose-response curve, and species responsiveness. In spite of these uncertainties, it was concluded that the TEF concept is still the most plausible and feasible approach for risk assessment of halogenated aromatic hydrocarbons with dioxinlike properties.

Animals↗

PubMed1998

Assessment of polystyrene extract for estrogenic activity in the rat uterotrophic model and an in vitro recombinant receptor reporter gene assay.

The purpose of the study was to determine whether polystyrene used in food-contact applications would elicit an estrogenic response when extracts simulating exaggerated conditions of use were subjected to in vivo and in vitro tests. A sample of polystyrene was subjected to extraction conditions that simulate, or exaggerate, the actual food-contact uses of polystyrene to maximize the amount of low molecular weight polystyrene extractables. The food-simulating solvent and the time and temperature conditions recommended by the Food and Drug Administration (FDA) were selected to maximize the level of extractable components from polystyrene. The extract was examined for its estrogenic response in vivo using the immature rat uterotrophic assay and in vitro using an estrogen receptor (ER)-mediated recombinant receptor reporter gene assay. In vivo, the uterine weights of juvenile female Sprague Dawley rats (10 rats/group) were determined after oral gavage exposure to the extract (two dosage levels: one represents the maximum potential daily human exposure to polystyrene extractables and the other represents one-tenth of the maximum exposure level), vehicle control (sesame oil), or positive control [diethylstilbestrol (DES), at 200 micrograms/kg body weight]. In addition, five treatment groups were dosed by subcutaneous injection of either estradiol (1, 50, and 500 micrograms/kg body weight) or DES (2 and 200 micrograms/kg body weight). Dosing began on postnatal day (pnd) 21 and continued daily through pnd 23. Body weights were collected at study initiation (pnd 21) and at necropsy (pnd 24). Body weights were not different statistically between treatment groups at study initiation or at necropsy. Uterine wet weights and uterine weights relative to body weights were significantly increased (p < 0.05) for estradiol at 50 and 500 micrograms/kg, DES at 2 and 200 micrograms/kg, and DES at 200 micrograms/kg (oral) over vehicle control. The polystyrene extract had no effect on uterine wet weight or uterine weights relative to body weights at either level tested. An in vitro recombinant estrogen receptor/reporter gene assay that involved transiently transfecting MCF-7 human breast cancer cells with the chimeric human ER, Ga14-HEGO, consisting of the yeast Ga14 DNA binding domain linked to the ligand binding domain of the human ER and a Ga14 response element (17mer)-regulated reporter gene (17m5-G-Luc) was employed. Dose-dependent induction of the reporter gene, 17m5-G-Luc, was observed with the positive control, 17 beta-estradiol (E2). Induction of greater than 100-fold was obtained following incubation of transfected MCF-7 cells with 10 nM E2 for 24 hours. No induction of reporter gene activity was observed with the polystyrene extracts dissolved in dimethylsulfoxide (0.01, 0.1 or 0.01 mg/ml) using the same assay conditions. These results indicate that polystyrene extract does not elicit ER-mediated activity using the Ga14-HEGO/17m5-G-Luc recombinant receptor/reporter gene assay. In conclusion, extracts from polystyrene produced no estrogenic response in either the rat uterotrophic assay or the MCF-7 cell assay for estrogen receptor-mediated activity.

Animals↗

PubMed1997

Antiestrogenic activity of hydroxylated polychlorinated biphenyl congeners identified in human serum.

Several hydroxylated polychlorinated biphenyls (PCBs) identified in human serum have been synthesized and these include 2,2',3,4',5,5'-hexachloro-4-biphenylol; 2,3,3',4',5-pentachloro-4-biphenylol; 2',3,3',4',5-pentachloro-4-biphenylol; 2,2',3,3',4',5-hexachloro-4-biphenylol; 2,2',3,3',4',5,5'-heptachloro-4-biphenylol; 2,2',3,4',5,5',6-heptachloro-4-biphenylol; and 2,2',3',4,4',5,5'-heptachloro-3-biphenylol. The hydroxy-PCBs exhibited minimal binding to the rat uterine cytosolic estrogen receptor (ER) and did not induce proliferation of estrogen-responsive MCF-7 human breast cancer cells at concentrations ranging from 10(-5) to 10(-8) M. The estrogenic activity of these compounds was further investigated utilizing two estrogen-responsive in vitro bioassays, namely, (i) HeLa cells stably transfected with a Gal4:human ER chimera and a 17-mer-regulated luciferase reporter gene, and (ii) MCF-7 cells transiently transfected with a full-length human ER expression plasmid and a plasmid containing an estrogen-responsive vitellogenin A2 promoter linked to a chloramphenicol acetyl transferase (CAT) reporter gene. None of the hydroxy-PCBs significantly induced luciferase activity in the stably transfected HeLa cells or CAT activity in MCF-7 cells at concentrations as high as 10(-5) M. The antiestrogenic effects of the hydroxy-PCBs were also investigated using the same bioassays in which the cells were cotreated with 17beta-estradiol plus the hydroxy-PCBs. All of the hydroxy-PCB congeners inhibited one or more estrogenic response, and one congener, 2,2',3,4',5,5',6-heptachloro-4-biphenylol, inhibited 17beta-estradiol-induced cell proliferation and CAT activity in MCF-7 cells and luciferase activity in HeLa cells.

Adenocarcinoma↗

PubMed1997

Hydroxylated polychlorinated biphenyls (PCBs) as estrogens and antiestrogens: structure-activity relationships.

The effects of structure on the estrogenicity and antiestrogenicity of hydroxylated polychlorinated biphenyls were investigated using the following estrogen-sensitive assays: competitive binding to the rat and mouse cytosolic estrogen receptor (ER); immature rat and mouse uterine wet weight, peroxidase and progesterone receptor (PR) levels; induction of luciferase activity in HeLa cells stably transfected with a Gal4:human ER chimera and a 17mer-regulated luciferase reporter gene; proliferation of MCF-7 human breast cancer cells; induction of chloramphenicol acetyl transferase (CAT) activity in MCF-7 cells transiently transfected with a full-length human ER expression plasmid and a plasmid containing an estrogen-responsive vitellogenin A2 promoter linked to a CAT reporter gene. The chemicals synthesized for this study contained a 4-hydroxy group in one ring, a 2- or 3-chloro substituent meta or ortho to the hydroxyl group, and variable substitution (2',3',4',5'-, 2',3',4',6'-, 2',3',5',6'-tetrachloro and 2',4',6'-trichloro) in the chlorophenyl ring. The compounds included: 2,2',3',4',5'- (A), 2,2',3',4',6'- (B), and 2,2',3',5',6'-pentachloro- (C); 2,2',4',6'-tetrachloro-4-biphenylol (D); 2',3,3',4',5'- (E), 2',3,3',4',6'- (F), and 2',3,3',5',6'-pentachloro (G); and 2',3,4',6'-tetrachloro-4-biphenylol (H). With the exception of 2',3,4',6'-tetrachloro-4-biphenylol (H), all of the compounds competitively bound to the mouse and rat ER with relative binding affinities [compared to 17beta-estradiol (E2)] varying from 1.4 x 10(-3) to 5.3 x 10(-5). The structure-ER binding relationships for the hydroxy-PCB congeners were different in the rat and mouse, and no dose-dependent estrogenic activities were observed in the mouse or rat uterus. Several hydroxy-PCB congeners exhibited antiestrogenic activity (primarily in the mouse uterus) and two compounds, 2,2',3',5',6- and 2,2',3',4',6'-pentachloro-4-biphenylol, inhibited E2-induced uterine wet weight, PR binding, and peroxidase activity in the mouse uterus. 2,2',3',4',5'- and 2,2',3',4',6'-Pentachloro-4-biphenylol induced CAT activity in MCF-7 cells transiently transfected with the Vit-CAT plasmid; the remaining congeners did not induce CAT activity but exhibited antiestrogenic activity in MCF-7 cells cotreated with 10(-9) E2 plus 10(-5) M hydroxy-PCBs. Complementary structure-estrogenicity relationships were observed utilizing the HeLa cell luciferase induction and MCF-7 cell proliferation assays. The placement of the 2- or 3-chloro groups in the phenolic ring had minimal effects on estrogenic activity, whereas 2,4,6-trichloro- and 2,3,4,6-tetrachloro substitution in the chlorophenyl ring (B, D, F, and H) were required for this response. Substitution in the phenolic ring was also not important for structure-antiestrogenicity relationships, and the most active compounds (A, C, E, and G) contained 2',3',4',5'- and 2',3',5',6'-tetrachlorophenyl groups. Thus, structure-estrogenicity/antiestrogenicity relationships for this series of hydroxy-PCBs were complex and response-specific.

Animals↗

PubMed1997

Organochlorine exposure and risk for breast cancer.

Organochlorine industrial compounds, combustion products and pesticides have been widely identified in the environment and residues have been detected in extracts prepared from fish, wildlife, human tissues as well as human milk and serum. Many of these compounds possess sex steroid activities and therefore have the potential to disrupt endocrine-regulated homeostasis. Organochlorines which exhibit hormonal activity include: (i) polychlorinated biphenyls (PCBs), hydroxylated PCBs, o,p'-DDT, and other organochlorine insecticides which exhibit estrogen receptor (ER) agonist activities; (ii) p,p'-DDE, a ligand for the androgen receptor which exhibits antiandrogen activity; (iii) PCBs, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and related aromatic hydrocarbons which bind the aryl hydrocarbon (Ah) receptor and exhibit tissue-specific antiestrogenic activity; and (iv) hydroxylated aromatics which bind transthyretin, a thyroid hormone binding protein. Although, it has been suggested that the estrogenic activity of PCBs and DDE may be a contributing factor for development of breast cancer in women, levels of these compounds are not consistently elevated in breast cancer patients and there is no evidence that women occupationally-exposed to relatively high levels of PCBs or DDE exhibit an increased incidence of breast cancer. In contrast, epidemiology studies suggest that women exposed to high levels of TCDD during an industrial accident in Seveso, Italy, have a decreased incidence of both breast and endometrial cancer. Based on the dietary intake of hormone or antihormone mimics derived from natural compounds in food, the estrogenic contribution of organochlorine compounds is small and their role in development of breast cancer is questionable.

Breast Neoplasms↗

PubMed1996

Assessing the estrogenic and dioxin-like activities of chemicals and complex mixtures using in vitro recombinant receptor-reporter gene assays.

In vitro recombinant receptor-reporter gene assays have been used to assess and rank the potency of chemicals and complex mixtures suspected of possessing estrogen and (or) aryl hydrocarbon receptor (AhR) mediated activity. The environmental estrogen (E2) bioassay consists of a Gal4-human estrogen receptor chimeric construct (Gal4-HEGO) and a Gal4-regulated luciferase reporter gene (17m5-G-Luc) that have been stably integrated into HeLa cells. The assay exhibits 10-fold induction in luciferase reporter gene activity following treatment with 1 nM 17 beta-estradiol and has a detection limit of approximately 5 pg of 17 beta-estradiol/mL. The AhR bioassay uses Hepa 1c1c7 wild-type cells transiently transfected with a dioxin response element regulated luciferase reporter gene. These assays were used to assess the estrogen and dioxin-like activities of naringenin, atrazine, and simazine and complex mixtures such as pulp and paper mill black liquor and urban air particulates. The activities of these chemicals and complex mixtures are confirmed using the pure antiestrogen ICI 164,384 and in in vitro gel retardation assays. Results of this study demonstrate the utility of in vitro recombinant receptor-reporter gene assays in identifying and assessing the estrogenic and dioxin-like activities of chemicals and complex mixtures.

Air Pollutants↗

PubMed1996

Failure of chloro-S-triazine-derived compounds to induce estrogen receptor-mediated responses in vivo and in vitro.

The potential estrogenic activities of atrazine and simazine were investigated in vivo using the immature female Sprague-Dawley rat uterus and in vitro using the estrogen-responsive MCF-7 human breast cancer cell line and the estrogen-dependent recombinant yeast strain PL3. Animals that were dosed with 50, 150, or 300 mg/kg of atrazine or simazine alone for 3 consecutive days did not exhibit any significant increases in uterine wet weight while decreases in cytosolic progesterone receptor (PR) binding levels and uterine peroxidase activity were observed. 17 beta-estradiol (E2)-induced increases in uterine wet weight were not significantly affected by cotreatment with either chemical; however, some dose-independent decreases in E2-induced cytosolic PR binding and uterine peroxidase activity were observed. In vitro, atrazine and simazine did not affect basal or E2-induced MCF-7 cell proliferation or the formation of nuclear PR-DNA complexes as determined by gel electrophoretic mobility shift assays. In addition, these chloro-S-triazines did not display agonist activity or antagonize E2-induced luciferase activity in MCF-7 cells transiently transfected with a Gal4-human estrogen receptor chimera (Gal4-HEGO) and a Gal4-regulated luciferase reporter gene (17m5-G-Luc). Moreover, the estrogen-dependent PL3 yeast strain was not capable of growth on minimal media supplemented with atrazine or simazine in place of E2. Collectively, these results indicate that the reported estrogenic and antiestrogenic effects elicited by these chemicals are not mediated by the estrogen receptor.

Animals↗

PubMed1995

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.

Animals↗

PubMed1995

Synthesis and characterization of hydroxylated polychlorinated biphenyls (PCBs) identified in human serum.

Hydroxylated polychlorinated biphenyls (PCBs) have been identified in wildlife and human samples. Most of these compounds are highly chlorinated (penta-hepatachloro) and contain a single meta- or para-hydroxyl group. Using the Cadogan coupling procedure, the following hydroxy-PCBs congeners were synthesized: 2,3,3',4',5-pentachloro-4-biphenylol, 2,3',4,4',5-pentachloro-3-biphenylol, 2',3,3',4',5-pentachloro-4-biphenylol, 2,2',3',4,4'-pentachloro-3-biphenylol, 2,2',3,3',4',5-pentachloro-4-biphenylol, 2,2',3',4,4',5-hexachloro-3-biphenylol, 2,2',3,4',5,5'-hexachloro-4-biphenylol, 2,2',3,3',4',5,5'-heptachloro-4-biphenylol, 2,2',3',4,4',5,5'-hepatachloro-3-biphenylol, 2,2',3,4',5,5',6-heptachloro-4-biphenylol. Many of these compounds have been detected as residues in human serum and current studies are investigating their activities as agonists and antagonists for several endocrine-mediated responses.

Gas Chromatography-Mass Spectrometry↗

PubMed1993

Efficient transactivation by retinoic acid receptors in yeast requires retinoid X receptors.

All-trans and 9-cis retinoic acids are natural derivatives of vitamin A that modulate gene expression as a consequence of binding to nuclear retinoic acid receptors (RARs) and retinoid X receptors (RXRs). RXRs form heterodimers with RARs in vitro, and such complexes display enhanced binding affinities for cognate DNA response elements. As yeast is devoid of endogenous RARs and RXRs, we used this organism to investigate whether transactivation in vivo requires RAR/RXR heterodimers. Using a domain-swapping approach, we demonstrate that chimeric RAR alpha 1 and RXR alpha containing the DNA-binding domain of the estrogen receptor activate transcription of a cognate reporter gene in yeast, independently of each other. These activities result from an inducible transcription activation function located in the ligand-binding domains of RAR alpha 1 and RXR alpha and a constitutive activation function located in the A/B region of RAR alpha 1. The inducible activation function of RXR alpha is induced exclusively by 9-cis-retinoic acid in this system. Transactivation of a reporter gene containing a retinoic acid response element by RAR alpha was considerably increased by RXR alpha, even in the absence of ligand. Optimal induction was achieved with 9-cis-retinoic acid, which stimulates the activity of both receptors. This study illustrates the utility of yeast to investigate signal transduction by retinoids in the absence of endogenous RARs, RXRs, and detectable retinoic acid isomerization.

Animals↗

PubMed1993

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

Animals↗

PubMed1992

Characterization and purification of human retinoic acid receptor-gamma 1 overexpressed in the baculovirus-insect cell system.

The full-length cDNA for the human retinoic acid receptor-gamma 1 (RAR-gamma 1) has been expressed to high levels in Spodoptera frugiferda (Sf9) cells using the baculovirus expression system. Western blot analysis revealed that RAR-gamma 1 expression increased between 32 and 60 h post-infection. The recombinant receptor was expressed primarily as a nuclear protein and displayed a molecular mass of 50 kDa as determined by SDS/PAGE and gel-filtration chromatography, consistent with its cDNA-deduced size. Based on ligand binding, 2 x 10(6) RAR-gamma 1 molecules were expressed per Sf9 cell, a level approx. 2000 times greater than in mammalian cells. The receptor was partially purified 300-fold by sequential anion-exchange, gel-filtration and DNA affinity chromatographies. The overexpressed receptor specifically bound all-trans-retinoic acid (RA) and the synthetic retinoid CD367 with high affinity (Kd 0.15 nM and 0.23 nM respectively). The RA metabolites 4-hydroxy-RA and 4-oxo-RA were poor competitors for [3H]CD367 binding to recombinant RAR-gamma 1 (K(i) > 1 microM), indicating that 4-oxidation of RA greatly reduces its affinity for RAR-gamma 1. Gel-retardation analysis demonstrated that RAR-gamma 1 specifically bound the RA response element of the mouse RAR-beta gene. RAR-gamma 1 species expressed from recombinant baculovirus (in Sf9 cells) and vaccinia virus (in HeLa cells) exhibited similar affinities for RA and CD367 and had comparable DNA-binding properties in gel-retardation experiments. Moreover, a similar requirement for additional DNA-binding stimulatory factor(s) was observed in both cases. These results provide a basis for the use of baculovirus-expressed RAR-gamma 1 in further functional and structural studies.

Amino Acid Sequence↗