A rapid and sensitive reporter gene that uses green fluorescent protein expression to detect chemicals with estrogenic activity.

A reporter gene sequence was constructed within a eukaryotic expression vector. The altered plasmid contained 2 sequential estrogen response elements (ERE) coupled to a human phosphoglycerate kinase (PGK) promoter inserted upstream from a cDNA sequence encoding enhanced green fluorescent protein (GFP) with a 3'-polyadenylation signal. The plasmid was linearized and transfected into MCF-7 cells, a human breast cancer-derived line that expresses the estrogen receptor (ER). No selectable marker was present in the plasmid, requiring stably transfected cells to be selected by fluorescence-activated cell sorting based on GFP expression after the cells were treated with 10(-9) M 17beta-estradiol (E2). Stably transfected MCF-7 cells (MCF7-ERE) exhibited 2000-3000 times more fluorescence at 488 nm excitation and 512 nm emission than non-transfected cells. MCF7-ERE cells exhibited a linear increase in GFP expression induced over a range of 10(-12) M E2, a concentration giving 2 times the background expression, to maximal expression at 3 x 0(-10) M E2. From the maximal level, GFP expression plateaued, and then declined when E2 was increased to the highest concentration tested, 10(-7) M. 4-Hydroxytamoxifen (TFN-OH) treatment of cells produced a dose-dependent inhibition of E2-induced GFP expression, indicating the interaction of ER in the regulation of GFP gene expression. A series of estrogenic chemicals were evaluated for their capacity to induce GFP expression in MCF7-ERE cells, showing induced expression of GFP at concentrations 2-4 log units higher than the E2 concentration giving maximal GFP expression. The ERE-PGK-GFP reporter gene system is capable of rapid GFP expression in the presence of low concentrations of E2, and of quantifying estrogenicity of chemicals compared with a standard curve of the natural ligand, 17beta-estradiol.     

Estrogen and environmental estrogenic chemicals exert developmental effects on rat hypothalamic neurons and glias.

We investigated effects of 17beta-estradiol (E(2)) and endocrine disrupters, nonylphenol (NP) and bisphenol-A (BPA), focusing on the neuronal development in cultures of fetal rat hypothalamic cells. We applied different concentrations of E(2), NP or BPA to the cultured hypothalamic cells and observed their effects on dendritic and synaptic development by immunocytochemistry using anti-microtubule associated protein-2 (MAP2) and anti-synapsin I antibodies, respectively. Administration of E(2) for 7 days affected MAP2-positive area as well as synapsin I-positive area. NP and BPA also influenced neuronal developments. The significant increase both in MAP2- and synapsin I-positive areas was observed at 10 and/or 100 nM of them, while 1 microM of them reduced the positive areas. Synaptic densities calculated from synapsin I-positive area/MAP2-positive area were not constant among different doses of three chemicals, but increased at 10 and/or 100 nM and decreased at 1 microM. Furthermore, immunostaining of NP-treated cells with the antibody against glial fibrillary acidic protein (GFAP) revealed that glial development was similarly influenced by NP. Therefore, the present results demonstrated that not only E(2) but also the environmental estrogenic chemicals, NP and BPA, affect development of fetal rat hypothalamic cells in vitro.     

Rapid detection assays for multidrug resistance

In attempts to develop simple and rapid assays for detection of multidrug resistance (MDR) doxorubicin-resistant S180 (S180DOX), colchicine-resistant CHO (CHOCOL) and cytarabine (cytosine-arabinoside)-resistant L1210 cell lines (L1210AraC) were investigated. S180DOX and CHOCOL cells express the MDR-phenotype while L1210AraC does not. Using a previously described short-term assay firstly, inhibition of incorporation of radioactive nucleic acid precursors into tumour cells after addition of doxorubicin was measured. Secondly, the accumulation of the fluorescent dye rhodamine 123 (R123) in the different cell lines were analyzed. It could be observed that the resistant S180DOX and CHOCOL cells needed significantly more time to accumulate R123 than their sensitive parental cell lines or L1210AraC cells. Therefore, both assays are adequate tools for the rapid detection of MDR.     

Screening for androgen receptor activities in 253 industrial chemicals by in vitro reporter gene assays using AR-EcoScreen cells.

Recently, there has been great concern about the potential of industrial chemicals to act as endocrine disrupters. In this report, we conducted a pilot study to validate the use of AR-EcoScreen cells for tier 1 screening of androgen receptor (AR) agonist and antagonist activities. From 253 test compounds, we identified two AR agonists and nine antagonists. The two agonists, 2-tert-butylanthraquinone and benzoanthrone, were relatively weak (10% maximal activation of the positive control, 5alpha-dihydrotestosterone, at 2.54x10(-7) and 4.46x10(-6) M, respectively). The most potent antagonist was 3,3'-dichlorobenzidine dihydrochloride (IC50 = 2.28x10(-7) M). The order of the anti-androgenic activities was 3,3'-dichlorobenzidine dihydrochloride>4-diethylaminobenzaldehyde>4,4'-[1-[4-[1-(4-hydroxyphenyl)-1-methylethyl]phenyl]ethylidene]bis[phenol]>2,4,6-trichlorophenylhydrazine = 4-(phenylpropyl)pyridine>2-hydroxy-4-methoxybenzophenone>2,2-bis(4-cyanophenyl)propane>4-methoxy-2-methyldiphenylamine = 2,4-diphenyl-4-methylpentene-1. These results suggest that AR-EcoScreen cell line has the potential to be used as a tool for the large scale tier 1 screening of chemicals for androgen receptor agonist and antagonist activity.     

Possible estrogenic and/or antiandrogenic effects of methoxychlor on prolactin release in male rats

Methoxychlor (MTX) is a pesticide currently used as a substitute for dichloro-diphenyl-trichloroethane (DDT). This organochloride insecticide has some estrogenic properties, and may modify the feedback mechanisms of steroids on the hypothalamus and pituitary. This work was undertaken to explore the possible effects of MTX on the episodic prolactin release and to analyze whether these effects are mediated by dopamine (DA), luteinizing hormone (LH), and/or testosterone. Adult male Sprague-Dawley rats were administered 25 mg/kg/day of MTX in sesame oil for 30 days. Control animals received vehicle only. The episodic prolactin release and plasma testosterone levels were measured as well as the dopamine (DA) content in the median eminence (ME) and in the anterior (AH), mediobasal (MBH), and posterior (PH) hypothalamus. The mean serum prolactin levels and absolute pulse amplitude of the hormone increased after the xenobiotic administration, whereas its relative pulse amplitude diminished. The frequency and duration of prolactin peaks and its half-life were not modified by the treatment with the pesticide. On the other hand, methoxychlor decreased the DA content in ME, increased it in AH, and did not change it in MBH or PH. MTX decreased plasma levels of LH and testosterone compared with controls. These data suggest estrogenic and antiandrogenic effects of MTX on the episodic prolactin secretion; the changes observed in prolactin release could be explained, at least in part, by some of the changes of DA at the hypothalamus and of LH at the pituitary, but not by changes of testosterone at the testicular level.     

Endocrine disruptive effects of chemicals eluted from nitrile-butadiene rubber gloves using reporter gene assay systems

Disposable gloves made of nitrile-butadiene rubber (NBR) are used for contact with foodstuffs rather than polyvinyl chloride gloves containing di(2-ethylhexyl)phthalate (DEHP), because endocrine-disruptive effects are suspected for phthalate diesters including DEHP. However, 4,4'-butylidenebis(6-t-butyl-m-cresol) (BBBC), 2,4-di-t-butylphenol, and 2,2,4-trimetyl-1,3-pentanediol diisobutyrate can be eluted from NBR gloves, and possibly also detected in food. In this study, we examined the endocrine-disrupting effects of these chemicals via androgen receptor (AR) and estrogen receptor (ER)-mediated pathways using stably transfected reporter gene cell lines expressing AR (AR-EcoScreen system) and ER (MVLN cells), respectively. We also examined the binding activities of these chemicals to AR and ER. The IC50 value of BBBC for antagonistic androgen was in the range of 10(-6)M. The strength of inhibition was about 5 times that of a known androgen antagonist, 1,1'-(2,2-dichloroethylidene)bis[4-chlorobenzene] (p,p'-DDE), and similar to that of bisphenol A. The IC50 value of BBBC for antagonistic estrogen was in the range of 10(-6)M. These results suggest that BBBC and its structural homologue, 4,4'-thiobis(6-t-butyl-m-cresol) are androgen and estrogen antagonists. It is therefore necessary to study these chemicals in vivo, and clarify their effect on the endocrine system.     

Validation and application of a reporter gene assay for the determination of estrogenic endocrine disruptor activity in milk

Endocrine disruptors (EDs) are compounds known to interfere with the endocrine system by disturbing the action or pathways of natural hormones which may lead to infertility or cancer.Our diet is considered to be one of the main exposure routes to EDs. Since milk and dairy products are major components of our diet they should be monitored for ED contamination. Most assays developed to date utilise targeted, chromatography based methods which lack information on the biological activity and mixture effects of the monitored compounds.A biological reporter gene assay (RGA) was developed to assess the total estrogen hormonal load in milk. It has been validated according to EU decision 2002/657/EC. Analytes were extracted by liquid–liquid extraction with acetonitrile followed by clean up on a HLB column which yielded good recovery and small matrix effects. The method has been shown to be estrogen specific, repeatable and reproducible, with covariance values below 20%. In conclusion, this method enables the detection of low levels of estrogen hormonal activity in milk with a detection capability of 36 pg g⁻¹ EEQ and has been successfully applied in testing a range of milk samples.     

Hershberger assay for antiandrogenic effects of phthalates

The antiandrogenic effects of seven phthalates, di(2-ethylhexyl) phthalate (DEHP), dibutyl phthalate (DBP), butyl benzyl phthalate (BBP), di-isononyl phthalate (DINP), di-isodecyl phthalate (DIDP), di-n-heptyl phthalate (DnHP), and mono-2-ethyhexyl phthalate (MEHP), were investigated by Hershberger assay in castrated male SD rats. An androgen agonist, testosterone (0.4 mg/kg/d), was administered for 10 consecutive days by subcutaneous (s.c.) injection as a positive control. Additionally, 20, 100, or 500 mg/kg body weight (bw)/d of 6 phthalates (DEHP, DBP, BBP, DINP, DIDP, or DnHP) or 10, 50, or 250 mg/kg bw/d of MEHP, the primary metabolite of DEHP, were also administered orally in combination with testosterone (0.4 mg/kg/d, s.c.) for 10 consecutive days, respectively. In the testosterone-treated groups, glans penis, seminal vesicles, ventral prostate, and levator ani/bulbocavernosus muscles (LABC) weights were found to be significantly increased. Ventral prostate weights were significantly decreased in animals treated with DEHP or DBP at doses of 20 mg/kg bw/d or above, 500 mg/kg bw/d DIDP, and 250 mg/kg bw/d MEHP. Seminal vesicles weights were also significantly decreased by DEHP at > 100 mg/kg bw/d, DINP at > 20 mg/kg bw/d, DIDP at 500 mg/kg bw/d, or MEHP at 50 or 250 mg/kg bw/d, respectively. In addition, LABC weights were decreased by DEHP at 500 mg/kg bw/d, DINP at 500 mg/kg bw/d, and MEHP at 50 or 100 mg/kg bw/d. These data suggest that some phthalates possess antiandrogenic activity, and that multiple cross-talk between androgen, estrogen, and steroid hormone receptors occurs.     

Potential estrogenic effects of bisphenol-A estimated by in vitro and in vivo combination assays

The potential estrogenic activities of bisphenol-A were investigated in vitro (E-screen and estrogen receptor competitive binding bioassays) and in vivo (uterotrophic assay). Uterotrophic responses were evaluated using mature ovariectomized Sprague-Dawley female rats treated subcutaneously with bisphenol A (1, 5, 10, 50, and 100 mg/kg/day), E2 (0.3 microgram/kg), and DES (0.3 microgram/kg) for 3 consecutive days. In a MCF-7 cell proliferation assay, E2 and DES used as positive estrogens induced maximum proliferation of MCF-7 cells at 1.0 nM, whereas BPA slightly induced MCF-7 cell proliferation at a higher level of 0.1 microM and maximum proliferation at 10 microM. In a competitive binding assay, E2 and DES showed inhibition of 17 beta-[3H]estradiol binding to the rat uterus ER with an IC50 of 1.0 nM and 0.5 nM, respectively. However, BPA had an IC50 of 5 microM, which was approximately 5,000 or 10,000-fold greater than the IC50 of E2 and DES. In uterotrophic assays, uterus (wet and blotted) and vagina weights were significantly increased at the dose of BPA 100 mg/kg/day in OVX Sprague-Dawley rats. These studies demonstrate that BPA exhibits weak estrogenic activity in all experimental systems, and thus its migration from epoxy resins or polycarbonate products should be controlled not to exceed a safety levels for humans.     

Assessment of styrene oligomers eluted from polystyrene-made food containers for estrogenic effects in in vitro assays

Recently, several substances from among the huge numbers of chemicals used by mankind have been implicated as instigators of disrupted endocrine function and related human health problems. Polystyrene (PS) is one of the most frequently used resins in the world, and the styrene oligomer dissolved out from PS has been designated as a potential trigger of estrogen-like activity in the Wingspread Declaration and the Japan Environment Agency's SPEED98 [JEA (Japan Environment Agency) Strategic Problem on Environmental Endocrine Disruptors '98 (SPEED) '98), http://www.env.go.jp/en/pol/speed98/sp98.html]. In order to assess the endocrine disrupting effect of styrene oligomers, we tested one styrene monomer (SM), three styrene dimers (SDs) and seven styrene trimers (STs), newly isolated from optical isomers, known to dissolve in small amounts from cup noodle containers made of polystyrene by the estrogen receptor binding assay, luciferase reporter gene assay, and human breast cancer cell MCF-7 proliferation assay. In all three tests, none of the SM, SDs and STs showed any significant activity. Accordingly, we concluded that these substances have no estrogenic activity.     

Toxic effects of environmental chemicals on the immune system

The main focus of the discipline of immunotoxicology is the study of alterations in immunological mechanisms caused by chemicals and drugs. These alterations may result in increased incidence of infections or the development of tumors. The major interest of immunotoxicology lies in the evaluation of the results of toxicity testing in rodents for the purpose of risk assessment for humans. As J. G. Vos and colleagues explain, such immunotoxicity testing is preferably carried out as a tiered system consisting of screening followed by functional studies of selected chemicals. This approach has been successful in identifying notorious environmental pollutants as immunotoxic chemicals. Evidence that the non-mammalian immune system can also be damaged by toxic environmental pollutants emphasizes the importance of ecotoxicology.     

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.     

Novel mammalian cell lines expressing reporter genes for the detection of environmental chemicals activating endogenous aryl hydrocarbon receptors (ArhR) or estrogen receptors (ER)

We have constructed two vector systems (pDMS5, pSAB2) containing the promoter regions of the human CYP1A1 gene including xenobiotic response elements or the promoter region of the Xenopus laevis vitellogenin A2 gene including estrogen response elements, respectively, and the genes for green fluorescent protein and firefly luciferase. These vectors were transfected into CHO-K1 cells. Transiently transfected cells consistently responded to 1 nmol/l TCDD (dioxin) or 10 nmol/l 17ss-estradiol, respectively, with a 3-5 fold increase in luciferase activity. Permanent cell lines were selected by culturing transiently transfected cells under continued presence of antibiotics and dilution cloning. Cells which had stably integrated the vector-DNA into the genomic DNA were selected. SiF6 cells responded to treatment with TCDD, PCB126, benzo(a)pyrene or indirubin-3'-monoxime in the concentration range between 0 and 1 micromol/l. SiG12 cells responded to treatment with bisphenol A, 4-MBC and DDT in the concentration range between 0 and 10 micromol/l. Compared with the controls, luciferase mRNA-abundance (semi-quantitative RT-PCR) and luciferase activity (luminescence assay) were elevated up to 3-fold. Resveratrol or tamoxifen, respectively, worked as full antagonists. Luciferase expression was increased upon treatment of cells with extracts of spiked soil samples indicating that our systems are suited for screening of environmental samples.     

Hormones and testis development and the possible adverse effects of environmental chemicals

Development of a fetus into a phenotypic male depends, first, on testis formation and second, on hormone production by the fetal testis. Disorders of testicular hormone production or action can lead in severe cases to phenotypic abnormalities or can predispose towards impaired reproductive health. Evidence for deteriorating human male reproductive health, especially an increase in testicular cancer, points to disturbed (hormonal) development of the fetal testis. By comparison of testicular dysgenesis in humans and exposure to certain phthalates in fetal rats, the similarities in outcomes and testicular cell–cell disruption are highlighted as are the pathways via which oestrogenic and (especially) anti-androgenic environmental chemicals might act to induce such changes. The susceptibility of sperm production in adulthood to ‘hormonal’ disruption in fetal and neonatal life is also discussed. Though it is concluded that no direct evidence links human exposure to environmental chemicals and male reproductive disorders that stem from disturbed testis development, this is based mainly on lack of information. Using the example of phthalates, for which new data have emerged, it is argued that until the appropriate in vivo studies are undertaken, the safety of hormonally active environmental chemicals, especially in mixtures, will continue to give cause for concern as far as testicular development is concerned.     

Comparison of the reporter gene assay for ER-alpha antagonists with the immature rat uterotrophic assay of 10 chemicals

We performed a reporter gene assay for estrogen receptor (ER)-alpha agonists and antagonists of 10 chemicals that showed both estrogen agonistic and reduced the estrogenic effect of ethinyl estradiol in a rat uterotrophic assay. The chemicals tested by the immature uterotrophic assay were p-(tert-pentyl)phenol, 4,4'-thiobis-phenol, 4,4'-(hexafluoroisopropylidene)diphenol, 2,2-bis(4-hydroxyphenyl)-4-methyl-n-pentane, 4,4'-(octahydro-4,7-methano-5H-inden-5-ylidene)bisphenol, 4-(phenylmethyl)phenol, 4,4'-dihydroxybenzophenone, 2,2',4,4'-tetrahydroxybenzophenone, 4-hydroxybenzophenone and 2,4,4'-trihydroxybenzophenone. Although all chemicals examined in this study were positive in the reporter gene assay for ER-alpha agonists, 4,4'-(octahydro-4,7-methano-5H-inden-5-ylidene)bisphenol was only positive in the reporter gene assay for ER-alpha antagonists. These findings demonstrate that results of the reporter gene assay for ER-alpha agonists correlated well with those of the uterotrophic assay, but antagonistic change of 9 of 10 chemicals in the uterotrophic assay was not detected by the reporter gene assay for ER-alpha antagonists.