Differential interaction of the methoxychlor metabolite 2,2-bis-(p-hydroxyphenyl)-1,1,1-trichloroethane with estrogen receptors alpha and beta.

Concern that some chemicals in our environment may affect human health by disrupting normal endocrine function has prompted research on interactions of environmental contaminants with steroid hormone receptors. We compared the activity of 2,2-bis-(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE), an estrogenic metabolite of the organochlorine pesticide methoxychlor, at estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta). Human hepatoma cells (HepG2) were transiently transfected with either human or rat ERalpha or ERbeta plus an estrogen-responsive, complement 3-luciferase construct containing a complement 3 gene promoter sequence linked to a luciferase reporter gene. After transfection, cells were treated with various concentrations of HPTE in the presence (for detecting antagonism) or absence (for detecting agonism) of 17beta-estradiol. HPTE was a potent ERalpha agonist in HepG2 cells, with EC50 values of approximately 5 x 10(-8) and 10(-8) M for human and rat ERalpha, respectively. In contrast, HPTE had minimal agonist activity with either human or rat ERbeta and almost completely abolished 17beta-estradiol-induced ERbeta-mediated activity. Moreover, HPTE behaved as an ERalpha agonist and an ERbeta antagonist with other estrogen-responsive promoters (ERE-MMTV and vtERE) in HepG2 and HeLa cells. This study demonstrates the complexity involved in determining the mechanism of action of endocrine-active chemicals that may act as agonists or antagonists through one or more hormone receptors.

Estrogenic activity of a dieldrin/toxaphene mixture in the mouse uterus, MCF-7 human breast cancer cells, and yeast-based estrogen receptor assays: no apparent synergism.

The estrogenic activity of dieldrin, toxaphene, and an equimolar mixture of both compounds (dieldrin/toxaphene) was investigated in the 21-day-old B6C3F1 mouse uterus, MCF-7 human breast cancer cells, and in yeast-based reporter gene assays. Treatment of the animals with 17beta-estradiol (E2) (0.0053 kg/day x3) resulted in a 3.1-, 4.8-, and 7.8-fold increase in uterine wet weight, peroxidase activity, and progesterone receptor binding, respectively. In contrast, treatment with 2.5, 15 and 60 micromol/kg (x3) doses of toxaphene, dieldrin, or dieldrin/toxaphene (equimolar) did not significantly induce a dose-dependent increase in any of the E2-induced responses. The organochlorine pesticides alone and the binary mixture did not bind to the mouse uterine estrogen receptor (ER) in a competitive binding assay using [3H]E2 as the radioligand. In parallel studies, estrogenic activities were determined in MCF-7 cells by using a cell proliferation assay and by determining induction of chloramphenicol acetyl transferase (CAT) activity in MCF-7 cells transiently transfected with plasmids containing estrogen-responsive 5'-promoter regions from the rat creatine kinase B and human cathepsin D genes. E2 caused a 24-fold increase in CAT activity in MCF-7 cells transiently transfected with creatine kinase B and a 3.8-fold increase in cells transiently transfected with the human cathepsin D construct. Treatment of MCF-7 cells with dieldrin, toxaphene, or an equimolar mixture of dieldrin plus toxaphene (10(-8)-10(-5) M) did not significantly induce cell proliferation or CAT activity in the transient transfection experiment with both plasmids. The relative competitive binding of the organochlorine pesticides was determined by incubating MCF-7 cells with 10(-9) M [3H]E2 in the presence or absence of 2 x 10(-7) M unlabeled E2 (to determine nonspecific binding), toxaphene (10(-5) M), dieldrin (10(-5) M), and equimolar concentrations of the dieldrin plus toxaphene mixture (10(-5) M). The binding observed for [3H]E2 in the whole cell extracts was displaced by unlabeled E2, whereas the organochlorine pesticides and binary mixture exhibited minimal to nondetectable competitive binding activity. E2 caused a 5000-fold induction of beta-galactosidase (beta-gal) activity in yeast transformed with the human ER and a double estrogen responsive element upstream of the beta-gal reporter gene. Treatment with 10(-6)-10(-4) M chlordane, dieldrin, toxaphene, or an equimolar mixture of dieldrin/toxaphene did not induce activity, whereas 10(-4) M endosulfan caused a 2000-fold increase in beta-gal activity. Diethylstilbestrol caused a 20-fold increase in activity in yeast transformed with the mouse ER and a single estrogen responsive element upstream of the beta-gal reporter gene. Dieldrin, chlordane, toxaphene, and endosulfan induced a 1.5- to 4-fold increase in activity at a concentration of 2.5 x 10(-5) M. Synergistic transactivation was not observed for any equimolar binary mixture of the pesticides at concentrations of either 2.5 x 10(-5) M or 2.5 x 10(-4) M. The results of this study demonstrate that for several estrogen-responsive assays in the mouse uterus, MCF-7 human breast cancer cells, and yeast-based reporter gene assays, the activities of both dieldrin and toxaphene were minimal, and no synergistic interactions were observed with a binary mixture of the two compounds.

Ah receptor: relevance of mechanistic studies to human risk assessment.

Studies of the toxic actions of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in numerous animal models and in human and animal cells in culture have established that the most characteristic pathologic lesions produced by this compound result from events initiated by the interaction of TCDD with a specific intracellular receptor protein, the Ah receptor. Although most research on the interaction of TCDD with the Ah receptor has focused on establishing involvement of this receptor complex in specific toxic responses, recent application of modern cell and molecular biology techniques is yielding new insights into the mechanism(s) of signal transduction. Elucidation of these mechanisms is essential for understanding the molecular basis of the cell and species specificity which is a hallmark of TCDD toxicity. This knowledge should provide the framework for development of a more toxicologically relevant risk assessment model.

Estrogenicity of bisphenol A in a human endometrial carcinoma cell line.

The ability of bisphenol A (BPA) to affect human estrogen receptor (ER) binding, expression of progesterone receptor (PR) mRNA and protein, and cell proliferation has been measured in the human endometrial cell line, ECC-1. Although less potent than 17beta-estradiol, BPA was able to bind to the human uterine ER. BPA also induced both mRNA and protein to levels similar to E2. BPA-mediated PR mRNA induction was antagonized by ICI, suggesting an ER-mediated pathway. Finally, E2 produced a 2-fold increase in cell number, while BPA showed no difference compared with vehicle control. The increase by E2 was inhibited by treatment with the either ICI 182,780 (ICI) or BPA, suggesting similar binding sites. Although ER binding is similar, E2 affected both proliferation and PR expression, while BPA only affected PR gene expression. The results of this study provide evidence that two ER agonists can act differentially in vitro to affect the expression of genes involved in regulating cellular growth and development, though the human risk potential remains to be determined.

Effects of low concentrations of benzene on mouse hematopoietic cells in vivo: a preliminary report.

Evaluation of benzene-induced hematotoxicity following exposure to low concentration is important for understanding mechanisms of toxicity and determining the dose response at benzene levels close to the current occupational exposure limit (1 ppm). Male B6C3F1 mice were exposed to 0, 1, 10, 100, or 200 ppm benzene by inhalation for 6 hr/day, 5 days/week, for 1, 2, 4, or 8 weeks. At each sampling time, we evaluated primitive and committed progenitor cells, differentiating and maturing lineage-specific cells, and stromal cells in the bone marrow; T and B lymphocytes of the spleen and thymus; micronucleated reticulocytes and erythrocytes; and standard blood parameters. At 100 and 200 ppm benzene, there were rapid and significant reductions in number of reticulocytes in the blood, B lymphocytes in the bone marrow and spleen, and an increased frequency of micronucleated reticulocytes in the bone marrow. At 10 ppm, the only parameter affected was a transient reduction in the number of splenic B lymphocytes. There were no significant effects induced by 1 ppm benzene in this study. The present study suggests numbers of B lymphocytes and maturing erythrocytes, and frequency of micronucleated reticulocytes are sensitive indicators of benzene-induced hematotoxicity and will be useful in further investigation of the hematotoxicity induced by 10 to 100 ppm benzene.

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.

Differential gene expression in response to methoxychlor and estradiol through ERalpha, ERbeta, and AR in reproductive tissues of female mice.

The reproductive and developmental effects of 17beta-estradiol (E2) and methoxychlor (MXC) observed in treated rodents appear to be linked to some unique but also overlapping patterns of gene expression. The MXC metabolite 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE) was previously shown to have selective agonist activity through estrogen receptor alpha (ERalpha) and antagonist activity through ERbeta and androgen receptor (AR). To discover gene families regulated by HPTE and E2, and to characterize similarities and differences in patterns of gene expression induced by these selective ER ligands, we analyzed tissues from mice treated for 3 days with a combined treatment of E2 and HPTE (E2 + HPTE), or the antiandrogen flutamide (FLU). RNA from uteri and ovaries was analyzed with cDNA microarrays and real-time RT-PCR. Results indicate that HPTE and E2 acted similarly to regulate most gene families in the uterus, which expresses predominantly ERalpha. However, in both the uterus and the ovary, there were a few genes that displayed differential patterns of gene regulation by E2 or HPTE treatment, presumably through ERbeta, AR, or other unidentified pathways. In the uterus, progesterone receptor, ERalpha, AR, insulin-like growth factor 1, insulin-like growth factor binding protein 5, and clusterin mRNAs were significantly reduced with both E2 or HPTE treatments, whereas cathepsin B was induced. Conversely, in the ovary, induction of cathepsin B by E2 was reversed after cotreatment with HPTE, and ERbeta expression was induced similarly by HPTE and FLU but not by E2. In addition, E2 uniquely regulated glutathione peroxidase 3, glutathione S-transferase, and cytochrome P450 17alpha-hydroxylase, with no effect of HPTE or FLU treatments. This analysis demonstrated several gene families that appear to be regulated in a ligand-specific pattern, which may explain the unique but overlapping reproductive tissue pathologies following exposure to E2 and MXC.

Altered gene profiles in fetal rat testes after in utero exposure to di(n-butyl) phthalate.

Di(n-butyl) phthalate (DBP) has antiandrogenic-like effects on the developing reproductive tract in the male rat and produces regions of interstitial cell hyperplasia and gonocyte degeneration in the developing fetal testes at maternal doses of 100-500 mg/kg/day. Neither DBP nor its primary metabolites interact with the androgen receptor in vitro. The present study was performed to examine gene expression in the fetal rat testes following in utero DBP exposure. Pregnant Sprague-Dawley rats received corn oil, DBP (500 mg/kg/day), or flutamide (reference antiandrogen, 50 mg/kg/day) by gavage daily from gestation day (GD) 12 to 21. Dose levels were selected to maximize fetal response with minimal maternal toxicity. Testes were isolated on GD 16, 19, and 21. Global changes in gene expression were determined by microarray analysis. Selected genes were further examined by quantitative RT-PCR. DBP, but not flutamide, reduced expression of the steroidogenic enzymes cytochrome P450 side chain cleavage, cytochrome P450c17, and steroidogenic acute regulatory protein. Testicular testosterone and androstenedione were decreased on GD 19 and 21, while progesterone was increased on GD 19 in DBP-exposed testes. Testosterone-repressed prostate message-2 (TRPM-2) was upregulated, while c-kit (stem cell factor receptor) mRNA was downregulated following DBP exposure. TRPM-2 and bcl-2 protein staining was elevated in GD 21 DBP-exposed Leydig and Sertoli cells. Results of this study have led to the identification of several possible mechanisms by which DBP can induce its antiandrogenic effects on the developing male reproductive tract without direct interaction with the androgen receptor. Our results suggest that the antiandrogenic effects of DBP are due to decreased testosterone synthesis. In addition, enhanced expression of cell survival proteins such as TRPM-2 and bcl-2 may be involved in DBP-induced Leydig cell hyperplasia, whereas, downregulation of c-kit may play a role in gonocyte degeneration. Future studies will explore the link between these identified gene expression alterations and ultimate adverse responses.

Androgen receptor antagonism by the organophosphate insecticide fenitrothion.

Organophosphate insecticides represent one of the most widely used classes of pesticides with high potential for human exposure in both rural and residential environments. We investigated the interaction of the organophosphothioate pesticide fenitrothion (O,O-dimethyl O-(4-nitro-m-tolyl) phosphorothioate) with the human androgen receptor (AR). Fenitrothion blocked dihydrotestosterone-dependent AR activity in a concentration-dependent and competitive manner in HepG2 human hepatoma liver cells transiently transfected with human AR and an AR-dependent luciferase reporter gene. Schild regression analysis yielded an equilibrium dissociation constant value of 2.18 x 10(-8) M. To determine the antiandrogenic potential of fenitrothion in vivo, 7-week-old castrated Sprague-Dawley rats were dosed once a day for 7 days with testosterone propionate (50 microg/day, sc) plus gavage doses of either corn oil vehicle or fenitrothion (15 or 30 mg/kg/day). An additional group of rats was given testosterone propionate and flutamide (50 mg/kg/day). Motor activity and acetylcholinesterase activity in whole blood and brain were also assessed. Both fenitrothion and the reference antiandrogen flutamide caused significant decreases in the ventral prostate, seminal vesicle, and levator ani plus bulbocavernosus muscles tissue weights. In contrast, blood acetylcholinesterase activity, a standard biomarker of organophosphate poisoning, was only inhibited at the higher dose of fenitrothion (30 mg/kg). Our results demonstrate that fenitrothion is a competitive AR antagonist, comparable in potency to the pharmaceutical antiandrogen flutamide and more potent, based on in vitro assays, than the known environmental antiandrogens linuron and p,p'-, 2,2-bis(p-hydroxyphenyl)-1,1-dichloroethylene ( p,p'-DDE).

Methyl tertiary butyl ether-induced endocrine alterations in mice are not mediated through the estrogen receptor.

Chronic exposure to methyl tertiary butyl ether (MTBE) altered the rodent tumor incidence of endocrine-sensitive tissues and decreased the incidence of estrogen-dependent uterine cystic hyperplasia in mice. To test the hypothesis that changes in the incidence of tumors in female B6C3F1 mice after MTBE exposure are secondary to endocrine alterations, we exposed female mice to the carcinogenic dose of MTBE vapor (8000 ppm) for 3 or 21 days or 4 or 8 months under conditions similar to a previous 2-year bioassay. MTBE exposure significantly decreased body weight gain and ovary and pituitary weight at 4 and 8 months and uterine weight at all time points. After 8 months of exposure, MTBE significantly increased the length of the estrous cycle by increasing the mean number of days in both the estrus and the nonestrus stages. Histological evaluation of H&E-stained tissues showed a decrease in the number of uterine glands after subchronic MTBE exposure. DNA synthesis, as measured by the incorporation of 5-bromo-2'-deoxyuridine (BrdU), was decreased in uterine glandular and luminal epithelial cells after MTBE exposure for 3 or 21 days or 4 or 8 months. MTBE exposure decreased the number of epithelial layers in the cervix and vagina at all time points. DNA synthesis was decreased in cervical and vaginal epithelium after 21 days of MTBE. Decreased zona reticularis of adrenal glands was found after 4 and 8 months of MTBE exposure without changes in BrdU incorporation. MTBE did not competitively bind to estrogen receptor. MTBE exposure did not alter serum estrogen levels or alter the location or intensity of estrogen receptor immunoreactivity in the uterus, cervix, and vagina. These data indicate that while MTBE exposure causes multiple endocrine-related tissue and cellular responses, these effects are not mediated through the estrogen receptor.

Developmental effects of dietary phytoestrogens in Sprague-Dawley rats and interactions of genistein and daidzein with rat estrogen receptors alpha and beta in vitro.

Estrogenic isoflavones, such as genistein and daidzein, are present in virtually all natural-ingredient rodent diets that use soy as a source of protein. Since these compounds are endocrine-active, it is important to determine whether the amounts present in rodent diets are sufficient to affect sexual development. The present study consisted of in vitro and in vivo parts. In the in vitro portion, human hepatoma cells were transfected with either rat estrogen receptor (ER) alpha or beta plus an estrogen-responsive luciferase reporter gene. Genistein and daidzein were complete agonists at both ERs, genistein being more potent than daidzein, and both compounds were more potent at ER beta than ER alpha. In combined studies with estradiol, genistein exerted additive effects with estradiol in vitro. In the in vivo portion of the study, groups of six pregnant Sprague-Dawley females were fed one of the following four diets, and the pups were maintained on the same diets until puberty: (1) a natural-ingredient, open-formula rodent diet (NIH-07) containing 16 mg genistein and 14 mg daidzein per 100 g of feed; (2) a soy- and alfalfa-free diet (SAFD) in which casein and corn oil were substituted for soy and alfalfa meal and soy oil, respectively, that contained no detectable isoflavones; (3) SAFD containing 0.02% genistein (GE.02); or (4) SAFD containing 0.1% genistein (GE.1). In the GE.1 group, effects of dietary genistein included a decreased rate of body-weight gain, a markedly increased (2.3-fold) uterine/body weight (U/BW) ratio on postnatal day (pnd) 21, a significant acceleration of puberty among females, and a marginal decrease in the ventral prostate weight on postnatal day (pnd) 56. However, developmental differences among the groups fed SAFD, GE.02, or NIH-07 were small and suggested minimal effects of phytoestrogens at normal dietary levels. In particular, on pnd 21, the U/BW ratio of the GE.02 and NIH-07 groups did not differ significantly from that of the SAFD group. Only one statistically significant difference was detected between groups fed SAFD and NIH-07: the anogenital distance (AGD) of female neonates on pnd 1 whose dams were fed NIH-07 was 12% larger than that of neonates whose dams were fed SAFD. The results suggest that normal amounts of phytoestrogens in natural-ingredient rodent diets may affect one developmental parameter, the female AGD, and that higher doses can affect several other parameters in both males and females. Based on these findings, we do not suggest replacing soy- and alfalfa-based rodent diets with phytoestrogen-free diets in most developmental toxicology studies. However, phytoestrogen-free diets are recommended for endocrine toxicology studies at low doses, to determine whether interactive effects may occur between dietary phytoestrogens and man-made chemicals.

Modulation of stromal cell function in DBA/2J and B6C3F1 mice exposed to benzene or phenol.

Inbred B6C3F1 (B6) mice are more resistant to benzene myelotoxicity than are inbred DBA/2J (D2) mice. This difference may be due to increased sensitivity of the hemopoietic microenvironment in D2 mice to benzene or benzene metabolites relative to B6 mice. The objectives of this investigation were to determine whether stromal cells which support hemopoiesis in the marrow were more sensitive to benzene in D2 mice than in B6 mice and to determine whether these strains would continue to express differences in susceptibility to phenol, an oxidative metabolite of benzene. Mice were given benzene (100 mg/kg) or phenol (100 mg/kg) intraperitoneally, twice a day for four consecutive days. On Day 5 marrow cell suspensions were removed from mice given benzene or phenol and from controls, plated in culture and assayed for (1) the relative number of adherent stromal cell (ASC) colonies present, (2) the number of granulocyte/monocyte precursors from benzene or phenol treated mice that could be supported by ASC from normal mice in coculture, and (3) the number of granulocyte/monocyte precursors from normal mice which could be supported by ASC from benzene or phenol treated mice. After benzene administration, only reductions in body weight and marrow cellularity followed the expected pattern and were reduced to a greater extent among D2 mice than B6 mice. Benzene had no significant effect on ASC colonies or on the number of granulocyte/monocyte precursors present. In contrast, the ability of ASC to support hemopoiesis of granulocyte/monocytes from normal donors was reduced to a greater degree among B6 mice than among D2 mice which paradoxically showed an increase in the ability to support hemopoiesis in coculture. Phenol significantly reduced the ability of ASC to support hemopoiesis of granulocyte/monocyte precursors but no preferential effect between strains was evident. These results suggest that benzene, but not phenol, is metabolized differently between the two strains and that bone marrow stromal cells, components of the hemopoietic microenvironment, are sensitive targets for benzene or its oxidative metabolites.

Post-transcriptional stabilization of urokinase plasminogen activator mRNA by 2,3,7,8-tetrachlorodibenzo-p-dioxin in a human keratinocyte cell line.

The actions of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a potent rodent carcinogen and suspected human carcinogen, are mediated by the Ah receptor, a ligand-activated transcription factor. Genes altered by TCDD at the transcriptional level in the transformed human keratinocyte cell line SCC-12F include cytochrome P4501A1 (CYP1A1), CYP1B1, transforming growth factor-beta 2, and plasminogen activator inhibitor-2 (PAI-2). Plasminogen activators are serine proteases involved in a number of cell processes, including migration, proliferation, growth factor activation, and tumorigenesis. In this study we investigated the effect of TCDD on other members of the plasminogen activator family. We report that in addition to the transcriptional induction of PAI-2, treatment of SCC-12F cells with 10 nM TCDD also resulted in an increase in urokinase-plasminogen activator (u-PA) mRNA. Induction of u-PA mRNA was maximal by 12 hr and remained approximately twofold above control levels for the 48-hr assay period. Transcription of u-PA was not altered by TCDD as determined by nuclear runoff analysis. Instead, induction of u-PA occurred as a result of a stabilization of the u-PA mRNA following TCDD treatment. Tissue-plasminogen activator and PAI-1 expression were not altered by TCDD. Thus, TCDD acts through different mechanisms in SCC-12F cells to induce both a plasminogen activator and a specific inhibitor of plasminogen activation. These results, together with our earlier results showing an induction of TGF-alpha by TCDD as a result of a stabilization of the TGF-alpha mRNA, demonstrate the importance of both transcriptional and post-transcriptional events in the regulation of gene expression by TCDD.

Regulation of gene expression and acceleration of differentiation in human keratinocytes by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a suspected human carcinogen, is believed to produce its toxic and carcinogenic effects by altering expression of growth-regulatory factors. TCDD alters the expression of a number of specific genes in the transformed human keratinocyte cell line, SCC-12F, including transforming growth factor-alpha (TGF-alpha), TGF-beta 2, plasminogen activator inhibitor-2 (PAI-2), and interleukin-1 beta (IL-1 beta). To determine whether nontransformed human keratinocytes (NHK) respond similarly to TCDD, we studied the effect of TCDD on NHK growth and differentiation, and gene expression. NHK were treated prior to reaching confluence with 10 nM TCDD and evaluated at 1, 2, 3, and 5 days following treatment for the effect of TCDD on cell number, morphology, involucrin levels, mRNA expression, and protein concentrations. TCDD altered both the mRNA and protein concentrations of TGF-alpha, TGF-beta 2, PAI-2, and IL-1 beta. The mRNA level for u-PA, a plasminogen activator that is inhibited by PAI-2, was not altered following TCDD treatment. However, u-PA protein levels were significantly induced, indicating an effect of TCDD on u-PA synthesis, secretion, or turnover. TCDD enhanced NHK differentiation, as determined by an increase in involucrin expression. TCDD did not alter cell number or colony-forming efficiency, suggesting that TCDD was enhancing the differentiation of cells already committed to terminal differentiation. These results demonstrate that treatment of NHK with TCDD results in the simultaneous modulation of expression of a number of growth-regulatory proteins and suggest that the growth and differentiation response of human keratinocytes to TCDD is due to a complex interaction of these diverse proteins.

Suppression of bone marrow stromal cell function by benzene and hydroquinone is ameliorated by indomethacin.

Administration of benzene to mice will inhibit bone marrow stromal cell-supported hemopoiesis in culture. Hydroquinone, a major metabolite of benzene, will cause a similar inhibition of stromal cell function in vitro. Stromal cells produce both an inducer (colony-stimulating factor) and an inhibitor (prostaglandin E2; PGE2) of hemopoiesis. This research was conducted to determine if prostaglandin synthesis is involved in the suppression of stromal cell function by benzene and hydroquinone. Male B6C3F1 mice were administered benzene (100 mg/kg), indomethacin (1 mg/kg), or benzene plus indomethacin twice a day for 4 consecutive days. On Day 5 bone marrow cells were removed to determine the effect of treatment. In a second series of experiments mouse bone marrow stromal cells in culture were treated with hydroquinone (10(-7) to 10(-4) M), indomethacin (10(-6) M), or a combination of hydroquinone plus indomethacin. Stromal cell function was based on the ability of the treated stromal cells to support granulocyte/monocyte colony development in coculture. The results demonstrated that preadministration of indomethacin in vivo ameliorated benzene-induced inhibition of bone marrow stromal cell function. In vitro, indomethacin ameliorated hydroquinone toxicity to stromal cell function. Benzene administration in vivo induced elevated PGE2 in bone marrow samples which were prevented by preadministration of indomethacin. However, hydroquinone in vitro did not induce a consistent increase in PGE2 levels. These results suggested that toxicity to stromal cells was not due solely to increased prostaglandin synthetase activity.

Inhibition of androgen receptor-dependent transcriptional activity by DDT isomers and methoxychlor in HepG2 human hepatoma cells.

Recent reports have raised new concerns that chemicals in our environment may disrupt normal reproduction and development through inhibition of androgen receptor function. This heightened concern has also increased our need for methods that allow us to characterize chemical interaction with the androgen receptor. In this report we describe an androgen receptor assay that utilizes the HepG2 human hepatoma cell line transiently transfected with the human androgen receptor and an androgen-responsive reporter. We used this assay to characterize the interaction with the androgen receptor of several steroidal and nonsteroidal chemicals, including isomers of DDT and methoxychlor. Chemicals were tested either in the absence (for determining agonist activity) or presence of 10(-7) M dihydrotestosterone (for determining antagonist activity). Testosterone and dihydrotestosterone were equally potent agonists in this assay. Estradiol and progesterone displayed partial agonist/antagonist activity. Flutamide was a complete agonist, whereas its hydroxylated metabolite, hydroxyflutamide, only partially antagonized and displayed some agonist activity at 10(-6) M and above. o,p'-DDT, o,p'-DDE, o,p'-DDD, p,p'-DDT, p,p'-DDE, and p, p'-DDD all behaved as antagonists at concentrations above 10(-6) M. p,p'-DDE also showed some agonist activity at 10(-5) M. Methoxychlor was only weakly antagonistic while its hydroxylated metabolite, HPTE, was approximately 10-fold more potent. Our results demonstrate that the HepG2 assay is a sensitive and specific method for detecting chemical interaction with the androgen receptor. This reporter gene assay, which we have used to characterize interaction with both the estrogen and androgen receptors, coupled with more extensive in vivo studies, should be useful for determining the role of multiple steroid receptors in the mechanism of action of endocrine active chemicals.

The actions of 2,3,7,8-tetrachlorodibenzo-p-dioxin on transforming growth factor-beta2 promoter activity are localized to the TATA box binding region and controlled through a tyrosine kinase-dependent pathway.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a widespread environmental contaminant and suspected human carcinogen, is believed to act by altering expression of a number of genes involved in cell growth control. In a previous study, we demonstrated the transcriptional down regulation of transforming growth factor-beta2 (TGF-beta2) by TCDD. To identify the region of the TGF-beta2 promoter necessary for the observed down-regulation by TCDD, we studied the effect of TCDD on a series of TGF-beta2 gene promoter deletions ranging from 1391 to 64 base pairs upstream of the transcription start site. We demonstrate that the effect of TCDD on TGF-beta2 promoter activity is localized to the TATA box sequence. The effect of TCDD on TGF-beta2 transcription is dose-dependent, exhibiting saturation kinetics maximal by 10 nM. Time course experiments show that the maximum decrease (30-50%) in promoter activity by a 10 nM dose of TCDD is complete by 24 hr. DNAase I footprinting and gel shift experiments indicate a single shifted protein complex in this region that we conclude is the transcription initiation complex. TCDD does not appear to significantly alter this complex suggesting that gross alterations in the proteins associated with this sequence do not occur. Treatment of the cells with various protein kinase inhibitors had no significant effect on the TCDD-induced decrease in promoter activity with the exception of genistein, a tyrosine kinase inhibitor. Genistein reverses the effect of TCDD on TGF-beta2 promoter activity back to control levels. Thus, TCDD can modulate gene transcription by acting at the transcription initiation complex via a tyrosine kinase-dependent pathway.

2,3,7,8-Tetrachlorodibenzo-p-dioxin-dependent regulation of transforming growth factors-alpha and -beta 2 expression in a human keratinocyte cell line involves both transcriptional and post-transcriptional control.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a potent modulator of epithelial cell growth and differentiation, has been shown to induce transforming growth factor (TGF)-alpha in cultures of human keratinocytes and in the human keratinocyte cell line, SCC-12F. In this report, we investigated the mechanisms by which TCDD alters TGF-alpha expression. In addition, we studied the actions of TCDD on TGF-beta 1 and TGF-beta 2 expression. Treatment of SCC-12F cells with TCDD resulted in an increase in TGF-alpha and a reduction in TGF-beta 2 mRNA levels while mRNA levels for TGF-beta 1 were unchanged. Changes in TGF-alpha and TGF-beta 2 expression were maximal by 24 h. No change in the rate of transcription of TGF-alpha was detected following treatment with TCDD as determined by nuclear run-off analysis. TCDD treatment resulted in a stabilization of TGF-alpha mRNA as judged by an approximately 2-fold higher level of TGF-alpha mRNA in treated versus control cells in the presence of actinomycin D. In contrast to TGF-alpha, the rate of transcription of TGF-beta 2 was significantly reduced following TCDD treatment. These findings demonstrate that the induction of TGF-alpha expression in SCC-12F cells by TCDD occurs post-transcriptionally, primarily by mRNA stabilization, while TGF-beta 2 expression is reduced due to a decrease in the rate of TGF-beta 2 gene transcription.

Effects of phthalate esters on the developing reproductive tract of male rats.

Phthalate esters are a large group of chemical agents used predominantly as plasticizers and solvents. Certain members of this chemical class have been shown to cause reproductive and developmental toxicity. Recent attention has focused on the potential of these agents to interfere with male reproductive development through a postulated antiandrogenic mechanism. Observations have focused on di-n-butyl phthalate (DBP), di-(2-ethylhexyl) phthalate (DEHP) and butyl benzylphthalate, with most information relating to dose-response relationships obtained for DBP. Neither DBP, DEHP nor their major metabolites interacted with human or rodent androgen receptors (AR) in transcriptional activation assays. DBP was administered during the critical window of development of the male reproductive system, after which the resulting offspring were examined until adulthood. DBP elicited marked effects on the developing male reproductive tract, including malformations of the epididymis and vas deferens, and hypospadias. Retention of thoracic nipples/areolae and reductions in anogenital distance were also noted. Surprisingly, Leydig cell adenomas were induced in some male offspring at 100 days of age. All these events occurred in the absence of any toxicity in the pregnant dam. Examination of testes from fetal rats indicated markedly reduced testosterone levels and increased Leydig cell numbers after DBP administration to the dams. Leydig cells were positive for AR and 3-betahydroxysteroid dehydrogenase.