Compounds Bisphenol Research Articles

Rapid Method for the Separation and Recovery of Endocrine-Disrupting Compound Bisphenol AP from Wastewater

The removal of bisphenol AP (BPAP) by a multiwalled carbon nanotube (MWCNT) was investigated. BPAP, representing typical scenarios for the BPAP-MWCNT interactions, was employed as a probe molecule. It was found that BPAP exhibited great adsorptive affinity to MWCNT, and the adsorption kinetics equilibrium was arrived within 4.0 min following the pseudo-second-order model. The overall rate process was mainly controlled by the external mass transfer. The hydrogen bond, hydrophobic, and π-π stacking interactions were dominant factors for the strong adsorption of BPAP, instead of the pH ionic strength and other ionic species in contaminated water. The MWCNT has higher stability within 8 removal-regeneration recycles, and up to 95% of recovery could be obtained by eluting the adsorbed BPAP on MWCNT adsorbent using ethanol/sodium hydrate solution. The results of the experiment on real samples verified the effectiveness for the recovery and removal of BPAP from wastewater samples.

Stir bar sorptive extraction with EG-Silicone coating for bisphenols determination in personal care products by GC–MS

An easy to perform analytical method for the determination of three bisphenol compounds (BPs) in commonly used personal care products (PCPs) is presented. Ethylene glycol-silicone (EG-Silicone) coated stir bars, which have recently become commercially available, are evaluated in this study for the simultaneous determination of bisphenol A (BPA), bisphenol F (BPF) and bisphenol Z (BPZ) by stir bar sorptive extraction (SBSE) in combination with thermal desorption–gas chromatography–mass spectrometry (TD–GC–MS). This new sorptive extraction phase allows the analysis of these compounds without any previous derivatization procedure. Different parameters affecting both SBSE extraction and thermal desorption were carefully optimized, using experimental designs based on the Taguchi orthogonal arrays. The procedure was applied to analyzing easily bought PCPs, providing detection limits of about 8ngg−1, with precisions lower than 11% in terms of relative standard deviation. Recovery studies performed at two different concentration levels provided satisfactory values for all the compounds. The analyzed personal care samples contained BPA at concentration levels ranging from 30.9 to 88.3ngg−1.

Bisphenol A Inhibits Voltage-Activated Ca2+ Channels in Vitro: Mechanisms and Structural Requirements

Bisphenol A (BPA), a high volume production chemical compound attracts growing attention as a health-relevant xenobiotic in humans. It can directly bind to hormone receptors, enzymes, and ion channels to become biologically active. In this study we show that BPA acts as a potent blocker of voltage-activated Ca(2+) channels. We determined the mechanisms of block and the structural elements of BPA essential for its action. Macroscopic Ba(2+) / Ca(2+) currents through native L-, N-, P/Q-, T-type Ca(2+) channels in rat endocrine GH(3) cells, mouse dorsal root ganglion neurons or cardiac myocytes, and recombinant human R-type Ca(2+) channels expressed in human embryonic kidney (HEK) 293 cells were rapidly and reversibly inhibited by BPA with similar potency (EC(50) values: 26-35 μM). Pharmacological and biophysical analysis of R-type Ca(2+) channels revealed that BPA interacts with the extracellular part of the channel protein. Its action does not require intracellular signaling pathways, is neither voltage- nor use-dependent, and does not affect channel gating. This indicates that BPA interacts with the channel in its resting state by directly binding to an external site outside the pore-forming region. Structure-effect analyses of various phenolic and bisphenolic compounds revealed that 1) a double-alkylated (R-C(CH(3))(2)-R, R-C(CH(3))(CH(2)CH(3))-R), or double-trifluoromethylated sp(3)-hybridized carbon atom between the two aromatic rings and 2) the two aromatic moieties in angulated orientation are optimal for BPA's effectiveness. Since BPA highly pollutes the environment and is incorporated into the human organism, our data may provide a basis for future studies relevant for human health and development.

Effect of combining in vitro estrogenicity data with kinetic characteristics of estrogenic compounds on the in vivo predictive value

With the ultimate aim of increasing the utility of in vitro assays for toxicological risk assessment, a method was developed to calculate in vivo estrogenic potencies from in vitro estrogenic potencies of compounds by taking into account systemic availability. In vitro estrogenic potencies of three model compounds (bisphenol A, genistein, and 4-nonylphenol) relative to ethinylestradiol (EE2), determined with the estrogen receptor alpha (ERα) transcriptional activation assay using hER-HeLa-9903 cells, were taken from literature and used to calculate the EE2 equivalent (EE2EQ) effect doses in the predominantly ERα-dependent rat uterotrophic assay. Compound-specific differences in hepatic clearance relative to the reference compound EE2 were determined in vitro to examine whether in vivo estrogenic potencies reported in literature could be more accurately estimated. The EE2EQ doses allowed to predict in vivo uterotrophic responses within a factor of 6-25 and the inclusion of the hepatic clearance further improved the prediction with a factor 1.6-2.1 for especially genistein and bisphenol A. Yet, the model compounds still were less potent in vivo than predicted based on their EE2 equivalent estrogenic potency and hepatic clearance. For further improvement of the in vitro to in vivo predictive value of in vitro assays, the relevance of other kinetic characteristics should be studied, including binding to carrier proteins, oral bioavailability and the formation of estrogenic metabolites.

Toxicity assessment and vitellogenin expression in zebrafish (Danio rerio) embryos and larvae acutely exposed to bisphenol A, endosulfan, heptachlor, methoxychlor and tetrabromobisphenol A

Organochlorine pesticides and brominated flame retardants, such as tetrabromobisphenol A and polybrominated diphenyl ethers, pose an environmental hazard owing to their persistence, low solubility and estrogenic effects, and concerns have been raised regarding their effects on aquatic biota. In the present study, zebrafish embryos and larvae were used as a model to investigate the sublethal and lethal effects of three different organochlorine pesticides, namely methoxychlor, endosulfan and heptachlor, as well as the flame retardant tetrabromobisphenol A, and its precursor compound bisphenol A. Preliminary data for chemical exposure tests were obtained by determining the 96 h median effective concentration EC50 (hatching rate) and 96 h median lethal concentration LC50 . Quantitative polymerase chain reaction was used to investigate the gene expression levels of the biomarker vitellogenin (vtg1) after 96 h exposures to 10, 25, 50 and 75% of the 96 h EC50 value for embryos and 96 h LC50 value for larvae. The use of vtg1 mRNA induction in zebrafish embryos and larvae was found to be a sensitive biomarker of exposure to these organic compounds, and was helpful in elucidating their adverse effects and setting water quality guidelines.

Simultaneous Determination of Bisphenol Compounds in Canned Food by Matrix Solid-Phase Dispersion and Liquid Chromatography-Tandem Quadrupole Linear Ion Trap Mass Spectrometry

A method based on matrix solid-phase dispersion and liquid chromatography-quadrupole/linear ion trap-mass spectrometry(MSPD-LC/QqLIT) for qualitative and quantitative determination of bisphenol compounds in canned food was presented.Bisphenol residues were extracted with acetonitrile solution containing 0.1% formic acid,followed by a further clean-up procedure using QuEChERS method.Bisphenol compounds were separated upon a C18 column and detected by liquid chromatographic-tandem mass spectrometry.Information dependent acquisition scan function(IDA) combined with enhanced product ion scan(EPI) library was used to confirm the bisphenol compounds,while quantification analysis was operated by MRM mode using external standard method simulta-neously.The recoveries ranged from 49.3%-128.4%(n=6) with RSD from 3.2% to 14.4%(n=6) in 4 kinds of canned food matrices respectively.The method is proved to be quick and effective for simultaneosly qualitative and quatitative determination of bisphenol residues in canned foodstuffs.

Mobilization of endocrine‐disrupting chemicals and estrogenic activity in simulated rainfall runoff from land‐applied biosolids

Municipal biosolids are commonly applied to land as soil amendment or fertilizer as a form of beneficial reuse of what could otherwise be viewed as waste. Balanced against this benefit are potential risks to groundwater and surface water quality from constituents that may be mobilized during storm events. The objective of the present study was to characterize the mobilization of selected endocrine-disrupting compounds, heavy metals, and total estrogenic activity in rainfall runoff from land-applied biosolids. Rainfall simulations were conducted on soil plots amended with biosolids. Surface runoff and leachate was collected and analyzed for the endocrine-disrupting compounds bisphenol A, 17α-ethynylestradiol, triclocarban, triclosan, octylphenol, and nonylphenol; a suite of 16 metals; and estrogenic activity via the estrogen receptor-mediated chemical activated luciferase gene expression (ER-CALUX) bioassay. Triclocarban (2.3-17.3 ng/L), triclosan (<51-309 ng/L), and octylphenol (<4.9-203 ng/L) were commonly detected. Chromium (2.0-22 µg/L), Co (2.5-10 µg/L), Ni (28-235 µg/L), Cu (14-110 µg/L), As (1.2-2.7 µg/L), and Se (0.29-12 µg/L) were quantifiable over background levels. Triclosan, Ni, and Cu were detected at levels that might pose some risk to aquatic life, though levels of metals in the biosolids were well below the maximum allowable regulatory limits. The ER-CALUX results were mostly explained by background bisphenol A contamination and octylphenol in runoff, although unknown contributors or matrix effects were also found.

Inhibition of human carbonic anhydrase isozymes I, II and VI with a series of bisphenol, methoxy and bromophenol compounds

Carbonic anhydrase inhibitors (CAI) are valuable molecules as they have several therapeutic applications, including anti-glaucoma activity. In this study, inhibition of three human carbonic anhydrase (hCA, EC 4.2.1.1) isozymes I, II and VI with a series of bisphenol and bromophenol derivatives was investigated. Molecular docking studies of a set of such inhibitors within CA I and II were also performed. KI values of the molecules 2–9 were in the range of 10.025–892.109 μM for hCA I, 1.437–59.107 μM for hCA II and 11.143–919.182 μM for hCA VI, respectively. Reported inhibitory activities of molecules 2–9 will assist in better understanding of structure-activity relationship studies of CAI.

In vitro exposure of Nile tilapia (Oreochromis niloticus) testis to estrogenic endocrine disrupting chemicals: mRNA expression of genes encoding steroidogenic enzymes

Many environmental pollutants can exert adverse effects on exposed organisms, including fish, leading to disruption of the endocrine system. Enzymes involved in the sex steroid biosynthesis are potential targets for the toxic action of pollutants. In this context, we investigated the hypothesis that selected estrogenic chemicals—the pharmaceutical estrogen ethinylestradiol (EE2), the phytoestrogen genistein (GEN), and the industrial compound bisphenol A (BPA)—may cause endocrine disruption by directly disturbing steps of fish steroidogenic pathways. We studied the mRNA expression of eight selected genes encoding steroidogenic enzymes (11β-HSD2, 20β-HSD, 3β-HSD1, 17β-HSD1, 17β-HSD8, 17β-HSD12, CYP19a, CYP19b) by quantitative real-time PCR. Testis slices from adult specimens of the model fish Nile tilapia were exposed in vitro for 3 and 8 h either to individual or to mixture solutions of EE2 (100 ng/L), GEN (200 ng/L), and BPA (10 µg/L); all at the peak concentrations observed in the Douro River estuary (Portugal). Our data revealed that only the mixture of the tested chemicals directly induced the expression of 11β-HSD2, 17β-HSD1, and 17β-HSD12, after 8 h, whereas no effect was seen for chemicals tested individually. The gene expression pattern agrees with the concept of dose addition for environmental mixtures, and for the first time an interference of estrogenic EDCs is reported for 17β-HSD1 and 17β-HSD12.

Bacterial biosensors for evaluating potential impacts of estrogenic endocrine disrupting compounds in multiple species

To study the effects and possible mechanisms of suspected endocrine disrupting compounds (EDCs), a wide variety of assays have been developed. In this work, we generated engineered Escherichia coli biosensor strains that incorporate the ligand-binding domains (LBDs) of the β-subtype estrogen receptors (ERβ) from Solea solea (sole), and Sus scrofa (pig). These strains indicate the presence of ligands for these receptors by changes in growth phenotype, and can differentiate agonist from antagonist and give a rough indication of binding affinity via dose-response curves. The resulting strains were compared with our previously reported Homo sapiens ERβ biosensor strain. In initial tests, all three of the strains correctly identified estrogenic test compounds with a high degree of certainly (Z' typically greater than 0.5), including the weakly binding test compound bisphenol A (BPA) (Z' ≈ 0.1-0.3). The modular design of the sensing element in this strain allows quick development of new species-based biosensors by simple LBD swapping, suggesting its use in initial comparative analysis of EDC impacts across multiple species. Interestingly, the growth phenotypes of the biosensor strains indicate similar binding for highly estrogenic control compounds, but suggest differences in ligand binding for more weakly binding EDCs.

Analysis of bisphenols in soft drinks by on-line solid phase extraction fast liquid chromatography–tandem mass spectrometry

In this study, an automated on-line solid-phase extraction coupled to fast liquid chromatography–tandem mass spectrometry (on-line SPE fast LC–MS/MS) method was developed for the simultaneous analysis of bisphenol A (BPA), bisphenol F (BPF), bisphenol E (BPE), bisphenol B (BPB) and bisphenol S (BPS) in canned soft drinks without any previous sample treatment. A C18 (12 μm particle size) loading column was used for the SPE on-line preconcentration before the liquid chromatography baseline separation of bisphenol compounds using a C18 Fused-Core™ (50 mm × 2.1 mm i.d.) column, which took less than 3 min. Gradient elution and heated electrospray were used to reduce matrix effect and improve ionization efficiency. To select the most intense and selective transitions, fragmentation studies were performed by multiple-stage mass spectrometry in an ion trap mass analyzer and tandem mass spectrometry in a triple quadrupole instrument, this latter instrument being used for quantitation in SRM mode. Quality parameters of the method were established and we obtained a simple, fast, reproducible (RSD values lower than 10%) and accurate (precision higher than 93%) method for the analysis of bisphenols in canned soft drinks at the ng L −1 level using matrix-matched calibration.

Toxicity to early life stages on medaka (Oryzias latipes) and in vitro estrogen intensity of bisphenol compounds

Toxicity to early life stages on medaka (Oryzias latipes) and in vitro estrogen intensity of bisphenol compounds

Effect of mixed liquor pH on the removal of trace organic contaminants in a membrane bioreactor

Experiments were conducted over approximately 7 months to investigate the effects of mixed liquor pH (between pH 5 and 9) on the removal of trace organics by a submerged MBR system. Removal efficiencies of ionisable trace organics (sulfamethoxazole, ibuprofen, ketoprofen, and diclofenac) were strongly pH dependent. However, the underlying removal mechanisms are different for ionisable and non-ionisable compounds. High removal efficiencies of these ionisable trace organics at pH 5 could possibly be attributed to their speciation behaviour. At this pH, these compounds exist predominantly in their hydrophobic form. Consequently, they could readily adsorb to the activated sludge, resulting in higher removal efficiency in comparison to under less acidic conditions in the reactor. Removal efficiencies of the two non-ionisable compounds bisphenol A and carbamazepine were relatively independent of the mixed liquor pH. Results reported here suggest an apparent connection between physicochemical properties of the compounds and their removal efficiencies by MBRs.

Measuring Ca 2+-signalling at fertilization in the sea urchin Psammechinus miliaris: Alterations of this Ca 2+-signal by copper and 2,4,6-tribromophenol

During fertilization, eggs undergo a temporary rise in the intracellular concentration of free Ca 2+ ions. Using the membrane permeable acetoxymethylester of the fluorescent calcium indicator dye Fura-2, Fura-2 AM, the Ca 2+-signal at fertilization was not detectable in eggs of the sea urchin Psammechinus miliaris. However, after treatment of the eggs with Fura-2 AM in combination with MK571, an inhibitor for multidrug resistance associated proteins, clear Ca 2+-signals at fertilization could be measured without microinjection of the dye. We used this methodology to detect possible alterations of Ca 2+-signalling at fertilization by exposure of eggs to environmental pollutants. For this purpose, the heavy metal copper, the bromophenol 2,4,6-tribromophenol, the organic compound bisphenol A and the polycyclic aromatic hydrocarbon phenanthrene were tested for their potential to inhibit fertilization success of P. miliaris. Copper and 2,4,6-tribromophenol showed a dose-dependent effect on fertilization rates of P. miliaris and significantly inhibited fertilization at 6.3 µM Cu 2+ and 1 µM 2,4,6-tribromphenol. Bisphenol A significantly inhibited fertilization success at 438 µM while phenanthrene had no effect up to 56 µM. 6.3 µM copper and 100 µM 2,4,6-tribromophenol significantly increased the Ca 2+-signal at fertilization. This alteration may contribute to the reduced fertilization rates of P. miliaris after exposure to copper and 2,4,6-tribromophenol.

2,2-Bis(4-Chlorophenyl)-1,1-Dichloroethylene Stimulates Androgen Independence in Prostate Cancer Cells through Combinatorial Activation of Mutant Androgen Receptor and Mitogen-Activated Protein Kinase Pathways

Therapy resistance represents a major clinical challenge in disseminated prostate cancer for which only palliative treatment is available. One phenotype of therapy-resistant tumors is the expression of somatic, gain-of-function mutations of the androgen receptor (AR). Such mutant receptors can use noncanonical endogenous ligands (e.g., estrogen) as agonists, thereby promoting recurrent tumor formation. Additionally, selected AR mutants are sensitized to the estrogenic endocrine-disrupting compound (EDC) bisphenol A, present in the environment. Herein, screening of additional EDCs revealed that multiple tumor-derived AR mutants (including T877A, H874Y, L701H, and V715M) are sensitized to activation by the pesticide 2,2-bis(4-chlorophenyl)-1,1-dichloroethylene (DDE), thus indicating that this agent may impinge on AR signaling in cancer cells. Further investigation showed that DDE induced mutant AR recruitment to the prostate-specific antigen regulatory region, concomitant with an enhancement of target gene expression, and androgen-independent proliferation. By contrast, neither AR activation nor altered cellular proliferation was observed in cells expressing wild-type AR. Activation of signal transduction pathways was also observed based on rapid phosphorylation of mitogen-activated protein kinase (MAPK) and vasodilator-stimulated phosphoprotein, although only MAPK activation was associated with DDE-induced cellular proliferation. Functional analyses showed that both mutant AR and MAPK pathways contribute to the proliferative action of DDE, as evidenced through selective abrogation of each pathway. Together, these data show that exposure to environmentally relevant doses of EDCs can promote androgen-independent cellular proliferation in tumor cells expressing mutant AR and that DDE uses both mutant AR and MAPK pathways to exert its mitogenic activity.

Cetyltrimethylammonium Bromide-Coated Magnetic Nanoparticles for the Preconcentration of Phenolic Compounds from Environmental Water Samples

The research presented in this paper investigates the adsorption of cation surfactants--cetyltrimethylammonium bromide (CTAB) and cetylpyridinium chloride (CPC)--onto magnetic nanoparticles and the application of this mixed hemimicelles solid-phase extraction (SPE) method for the preconcentration of several typical phenolic compounds-bisphenol A (BPA), 4-tertoctylphenol (4-OP), and 4-n-nonylphenol (4-NP)--from environmental water samples. In this novel SPE method, the charged surfactants CTAB and CPC form mixed hemimicelles on Fe3O4 nanoparticles (Fe3O4 NPs), which causes retention of analytes by strong hydrophobic and electrostatic interactions. The SPE method combines the advantages of mixed hemimicelles and magnetic nanoparticles. In order to provide guidelines forthe mixed hemimicelles SPE method development, surfactant adsorption isotherms and zeta-potential isotherms were also investigated. The main factors affecting the adsolubilization of analytes, such as the amount of Fe3O4 NPs and surfactants, the type of surfactants, the solution pH,the sample loading volume, and the desorption conditions, were investigated and optimized. A concentration factor of 800 was achieved by the extraction of 800 mL of several environmental water samples using this SPE method. Under the selected conditions, detection limits obtained for BPA, 4-OP, and 4-NP were 12, 29, 34 ng/L, respectively. The accuracy of the method was evaluated by recovery measurements on spiked samples, and good recoveries (68-104%) with low relative standard deviations from 2 to 7% were achieved. The advantages of this new SPE method include high extraction yields, high breakthrough volumes, short analysis times, and easy preparation of sorbents. To the best of our knowledge, this is the first time that a mixed hemimicelles SPE method based on magnetic separation and nanoparticles has been used for the pretreatment of environmental water samples.

Structural Analysis of Bisphenol-A and its Methylene, Sulfur, and Oxygen Bridged Bisphenol Analogs

The molecular structures of bisphenol-A, C(CH3)2(p-C6H4OH)2 (monoclinic, P21/n, a = 11.1940(6) A, b = 18.738(1) A, c = 17.8623(9) A, β = 100.571(1)°), its methylene (CH2) and heteroatom (S, O) bridged analogs, CH2(p-C6H4OH)2 (monoclinic, P21/n, a = 5.4351(1) A, b = 20.7895(3) A, c = 8.8432(1) A, β = 93.419(1)°), S(p-C6H4OH)2 (monoclinic, P21/n, a = 5.5115(1) A, b = 21.0666(2) A, c = 8.6917(1) A, β = 92.962(1)°) and O(p-C6H4OH)2 (orthorhombic, Pbcn, a = 5.2745(5) A, b = 8.2724(8) A, c = 22.085(2) A), have been determined by X-ray diffraction at 115 K, revealing structural differences in the series of compounds. The results show that the Caryl–Xbridge–Caryl angles (Xbridge = C(CH3)2, CH2, S, O) span a range greater than 14°. The dihedral angle between the planes of the hydroxyphenyl groups also varies according to the identity of the bridging group, varying from 67.24(2)° in S(p-C6H4OH)2 to 85.82(4)° in C(CH3)2(p-C6H4OH)2. In addition, the pitch angle, which more accurately describes the propeller-like nature of these bisphenolic compounds, varies from 39.88(3)° in S(p-C6H4OH)2 to 59.62(7)° in C(CH3)2(p-C6H4OH)2. Ab initio electronic structure calculations predict very similar bond lengths and angles to those observed crystallographically; however, the predicted dihedral angles and pitch angles are quite different, suggesting that these features are greatly influenced by crystal packing.

Spreeta-based biosensor assays for endocrine disruptors

The construction and performance of an automated low-cost Spreeta™-based prototype biosensor system for the detection of endocrine disrupting chemicals (EDCs) is described. The system consists primarily of a Spreeta miniature liquid sensor incorporated into an aluminum flow cell holder, dedicated to support a Biacore chip frame, in combination with a simple pressurized air-driven fluid system. During the optimization, a monoclonal antibody (MAb)-based immunoassay for the estrogenic compound bisphenol A (BPA) was used as a model. After the optimization two thyroxine transport protein inhibition assays for thyroid endocrine disruptors were implemented. The average noise of the system for 1 min of baseline was 1.1 μRIU (refractive index units) and it could be operated in the range of 18–22 °C with a minimum baseline drift (5–10 μRIU/100 min). Optimum signal to noise ratio (S/N R) was obtained using a flow cell height of 100 μm and a flow rate of 180 μl/min. The sensitivity of the Spreeta-based biosensor inhibition assays implemented (50% inhibition concentration (IC 50) of 30.2 nM for BPA using MAb12 and 12.3 and 11.6 nM for thyroxine (T4) using thyroxine-binding globulin (TBG) and recombinant transthyretin (rTTR), respectively) was comparable to the sensitivity previously obtained using a Biacore 3000 (IC 50 of 39.9 nM for BPA and 8.6 and 13.7 nM, respectively, for T4). The results indicate that the alternative prototype system can be used in combination with ready-to-use biosensor chip surfaces and it is potentially a useful tool for the bioeffect-related screening of EDCs.

Study on mutagenic effects of bisphenol A diglycidyl ether (BADGE) and its derivatives in the Escherichia coli tryptophan reverse mutation assay

The di-epoxy compound bisphenol A diglycidyl ether (BADGE), its first and second hydrolysis products (BADGE·H 2O and BADGE·2H 2O, respectively) and its bis-chlorohydrin derivative (BADGE·2HCl) were examined for their mutagenicity in the Escherichia coli tryptophan reverse mutation test with strains WP2, WP2 uvrA and IC3327. The assays were performed in the presence and absence of exogenous metabolic activation (S9 fraction from rat liver). The di-epoxy compound BADGE was able to induce mutagenic effects in strains WP2 uvrA and IC3327 and the epoxy-diol BADGE·H 2O also showed a positive response with these strains, although the latter was less potent than the former. On the other hand, the lack of mutagenic activity of BADGE·2H 2O and BADGE·2HCl was also demonstrated.

Estrogenic Potency of Food-Packaging-Associated Plasticizers and Antioxidants As Detected in ERα and ERβ Reporter Gene Cell Lines

This study presents the estrogenic potency of 21 food-packaging-associated compounds determined for the first time, using two transfected U2-OS (human osteoblasts devoid of endogenous estrogen receptors) estrogen receptor (ER) alpha and beta cell lines. Six plasticizers and three antioxidants were slightly estrogenic in the ERalpha cells. The model compounds bisphenol A and nonylphenol, one plasticizer [tris(2-ethylhexyl)trimellitate (TEHTM)], and two antioxidants (propyl gallate and butylated hydroxyanisole) were estrogenic in both ERalpha and ERbeta cells. Compared to estradiol (E2), these compounds appeared to be relatively more estrogenic in the ERbeta cells than in the ERalpha cells. Three sorbitol-based plasticizers activated neither ERalpha nor ERbeta and may be good replacements of existing plasticizers. All responses were additive with the response of E2. This indicates that they may contribute to the total effects of the pool of estrogenic compounds humans are exposed to. The estrogenic potencies of these compounds, together with the suggested beneficial effect of ERbeta-mediated responses and adverse ERalpha-mediated effects, support the importance of detecting characteristics for ERalpha and ERbeta response separately in independent models, as done in the present study.