Estrogenic followed by anti-estrogenic effects of PCBs exposure in juvenil fish (Spaurus aurata)

Vitellogenin (Vtg) is a phospho-lipo-glycoprotein produced by oviparous animals in response to estrogen receptor (ER) binding. The presence of Vtg in juvenile and male fish liver and plasma has been used as biomarker to evaluate levels of environmental contaminants as dioxin and PCBs. Interaction of dioxins and PCBs with aryl hydrocarbon receptor (AhR) may affect reproduction by recruitment of estrogen receptor α (ERα). The aim of this study was to investigate the effects of PCB-126, a co-planar PCB prototypical AhR agonist, and of PCB-153, a non-coplanar PCB lacking dioxine-like activity, on Vtg expression in young fish (Spaurus aurata) after a 12 or 24 h exposure to PCBs as well as 48 h following PCBs removal. Vtg expression was evaluated by immunohistochemistry and by Western-blot analysis. Our results showed an increased Vtg expression following PCBs administration, with a maximum level after 12 h of exposure to either PCB-126, PCB-153 or a mixture of both PCBs. Following this estrogenic activity, an anti-estrogenic activity was detected after 24 h of incubation with PCB-126 (alone or mixed with PCB-153), suggested by a decrease in Vtg expression likely through AhR, as a consequence of a hypothetic defence mechanism to endogenous or exogenous ligands.     

Effects of 17alpha-ethynylestradiol on hormonal responses and xenobiotic biotransformation system of Atlantic salmon (Salmo salar)

Pharmaceuticals are ubiquitous pollutants in the aquatic environment where their potential effects on non-target species like fish has only recently become subject of systematic investigations. In the present study, experiments were undertaken to examine the effects of a synthetic pharmaceutical endocrine disruptor, ethynylestradiol (EE2), given in water at 5 or 50 ng/L and sampled at days 0 (control), 3 and 7 after exposure, on hepatic phase I and II biotransformation and hormonal pathways of juvenile salmon using quantitative (real-time) polymerase chain reaction (qPCR), Vtg ELISA and 7-ethoxyresorufin O-deethylase (EROD) catalytic activity. Our data show that EE2 produced time- and concentration-specific modulation of estrogen receptor isoforms (ERalpha, ERbeta) and androgen receptor-beta (ARbeta). EE2 produced a concentration-specific induction of vitellogenin (Vtg) and zona radiata protein (Zr-protein) at day 3 after exposure. At day 7, Vtg and Zr-protein mRNA (and plasma Vtg protein) expression were significantly decreased in the group given 5 ng EE2/L, compared to dimethyl sulfoxide (DMSO) control group. In the xenobiotic biotransformation pathway, EE2 produced a significant increase of aryl hydrocarbon receptor-alpha (AhRalpha) at day 3 in the group given 5 ng EE2/L and AhRbeta was decreased at the same concentration at day 7. While CYP3A was not significantly affected by EE2 exposure, the CYP1A1, AhR nuclear translocator (Arnt) and AhR repressor (AhRR) mRNA showed an apparent EE2 concentration and time-dependent decrease. The expression of uridine diphosphoglucuronosyl transferase (UGT) and glutathione S-transferase class pi-like (GSTpi-like) mRNA were decreased after exposure to 50ng EE2/L at both day 3 and 7 after exposure. The effect of EE2 on the CYP1A1 gene expressions paralleled effect on EROD and AhRR mRNA, suggesting a direct role of EE2 in controlling cellular detoxification machinery. Interestingly, the carrier vehicle, DMSO produced significant time-dependent induction of estrogenic (ERalpha, Vtg and Zr-protein) responses, compared with blank (i.e. without DMSO) controls at day 7 post-exposure. The effect of DMSO totally underscored the observed EE2 effect at day 7 after exposure. In general, these findings support previous reports on the endocrine effects of EE2, in addition to effects on hepatic biotransformation system. In view of the data presented here and our recent studies, the use of DMSO as carrier vehicle in endocrine toxicological experimental studies should be re-evaluated.     

The anti-estrogenicity of Ah receptor agonists in carp (Cyprinus carpio) hepatocytes.

Cultured hepatocytes of female carp (Cyprinus carpio) were coexposed for 4 days to 200 nM 17beta-estradiol (E2), and concentration ranges of nine known Ah receptor (AhR) agonists: 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3,3'4,4'5-pentachlorobiphenyl (PCB 126), 2,3'4,4'5-pentachlorobiphenyl (PCB 118), beta-naphthoflavone (BNF), benzo(a)pyrene (BaP), benzo(a)anthracene (BaA), diindolylmethane (DIM), 6-methyl-1,3,8-trichlorodibenzofuran (MCDF) and hexachlorobenzene (HCB). TCDD caused a greater than 100-fold induction of cytochrome P4501A (CYP1A) activity, measured as ethoxyresorufin O-deethylase (EROD), with an EC50 of 6 pM. Based on EC50 values, the order of potency as CYP1A inducers was TCDD > PCB 126 > BNF > BaP > BaA > PCB 118. DIM and MCDF caused a lower maximum CYP1A induction (< 9-fold), whereas HCB caused no EROD induction at concentrations up to 6 microM. TCDD, PCB 126, BNF, BaP, and DIM also caused a concentration-dependent suppression of the secretion of the yolk protein vitellogenin (Vtg), relative to E2-treated hepatocytes. Suppression of Vtg secretion was not directly correlated with EROD activity, and the antiestrogenic effects occurred at higher concentrations than the induction of CYP1A. This indicates that the anti-estrogenicity was not caused by increased metabolism of E2 due to induction of CYP1A. Nevertheless, the order of potency of the tested compounds for suppression of Vtg secretion was comparable to the order of potency for CYP1A induction. This concurrence suggests that the anti-estrogenicity of these compounds is AhR-mediated, but does not involve CYP1A. This could be relevant for feral fish populations, as they are frequently exposed to AhR agonists, to an extent that AhR-mediated effects are observed.     

In vivo modulation of nonylphenol-induced zonagenesis and vitellogenesis by the antiestrogen, 3,3'4,4'-tetrachlorobiphenyl (PCB-77) in juvenile fish

Zonagenesis and vitellogenesis (eggshell zona radiata protein (Zrp) and vitellogenin (Vtg) production, respectively), are two estrogen-regulated processes in oviparous vertebrates that are crucial for oocyte maturation. Treatment of juvenile Atlantic salmon (Salmo salar) with nonylphenol (NP; 25 mg kg(-1)) alone or in combination with 3,3',4,4'-tetrachlorobiphenyl (TCB; 0.1 mg kg(-1)) resulted in pronounced elevations of plasma eggshell Zrp and Vtg and their respective liver mRNA levels in two separate experiments. TCB treatment alone caused the elevation of CYP1A mRNA, protein and enzyme levels (7-ethoxyresorufin O-deethylase (EROD)). In experiment 3, which also included the time factor, exposure of juvenile salmon to 10 and 25 mg NP per kg in combination with TCB generally resulted in reduced plasma Zrp and Vtg levels, compared with NP treatments alone. In a fourth experiment, juvenile salmon were exposed to different doses of TCB either 2 days before or 2 days after a single dose (25 mg kg(-1)) of NP. Samples were always collected 5 days after the NP exposure and analyzed for mRNA and protein levels. Generally, TCB doses given 2 days after NP exposure resulted in the elevation of Vtg and Zrp protein and mRNA levels. Vtg and Zrp mRNA levels were also elevated in the groups treated with 0.1 mg TCB 2 days before NP exposure. In all experiments, TCB injection resulted in the induction of liver CYP1A mRNA, CYP1A protein and EROD activity, but no Zrp or Vtg protein/mRNA inductions were observed when given alone. The present study documents for the first time the apparent stimulation of xenoestrogen-induced responses by an antiestrogenic CYP1A-inducer, in fish or any other lower vertebrate. However, the stimulatory or inhibitory effect of TCB on NP-induced responses appear to be dependent on the ratio of NP and TCB doses, and temporal sequence of exposure. Fish hepatic zonagenesis and vitellogenesis continue to provide interesting models for further studies on the mechanisms and possible interactions between endocrine disruptors and CYP1A-inducers, their antiestrogenic and/or estrogen potentiating effects.     

CYP1 and AhR expression in 7,12-dimethylbenz[a]anthracene-induced mammary carcinoma of rats prenatally exposed to 3,3',4,4',5-pentachlorobiphenyl

We previously reported the finding that prenatal exposure to a relatively low dose of 3,3',4,4',5-pentachlorobiphenyl (PCB126) acted as an enhancing agent for 17-beta-estradiol (E2)-dependent 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat mammary carcinoma, while a high dose decreased it. E2 is a known risk factor for mammary carcinoma, and CYP1A1 and 1B1 (CYP1) are the major enzymes catalyzing 2- and 4-hydroxylation of E2, respectively. We investigated the induction of CYP1 and aryl hydrocarbon receptor (AhR) in DMBA-induced mammary carcinoma using female Sprague-Dawley rats whose dams had been treated (i.g.) with 2.5 ng, 250 ng, 7.5 microg of PCB126/kg or the vehicle on days 13-19 post-conception. Immunohistochemical analysis revealed that the mammary carcinoma of the 250 ng group showed a significantly higher number of nuclei expressing estrogen receptor alpha (ER) and proliferating cell nuclear antigen (PCNA) compared to those of the other groups. Quantitative real-time RT-PCR analysis revealed that the 7.5 microg group showed a significantly higher level of CYP1A1 mRNA, and that the 250 ng group showed significantly higher levels of CYP1B1 mRNA. The level of AhR mRNA was significantly higher in both the 7.5 microg and 250 ng groups. Western blotting analysis was consistent with mRNA changes. It has been revealed that CYP1B1 catalyzes a step in the formation of 4-hydroxylated E2 metabolites, which show quite high mammary carcinogenicity. This study indicates that the enhancement of DMBA-induced mammary carcinogenicity in a relatively low PCB126 dose group might partially involve the higher expression of CYP1B1 and AhR in these carcinomas.     

Polychlorinated biphenyls alter the expression of endothelial nitric oxide synthase mRNA in human umbilical vein endothelial cells

Polychlorinated biphenyls (PCBs) are a group of persistent pollutants that are detected in maternal serum and umbilical cord, suggesting that fetal exposure also needs to be considered. The effects of dioxin-like PCB congeners 3,3',4,4'-tetrachlorobiphenyl (PCB77) and 3,3',4,4',5-pentachlorobiphenyl (PCB126) and a non-dioxin-like compound 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153) on the expression of endothelial nitric oxide synthase (eNOS), known to maintain blood flow to the fetus, in human umbilical vein endothelial cells (HUVECs) were investigated. The mRNA levels of eNOS, aryl hydrocarbon receptor (AhR) and cytochrome P450 (CYP) 1A1 in cells treated with 5 microM PCBs for 24 hours were analysed by real-time RT-PCR. Cells were also treated with alpha-naphthoflavone (alpha NF), an AhR antagonist or ICI 182780, an estrogen receptor (ER) antagonist, one hour prior to PCB exposure, to observe the effects of these receptors on eNOS modulation. Each PCB increased the eNOS mRNA level by 4.5-fold that was markedly inhibited by alphaNF. ERs were also suspected of altering eNOS levels because ICI 182780 treatment resulted in a decrease in the eNOS level. These results suggest that the eNOS mRNA expression increases due to the action of PCBs related to both AhR and ERs in HUVECs, and that maternal PCB exposure could influence fetal circulation.     

Antagonism of aryl hydrocarbon receptor-dependent induction of CYP1A1 and inhibition of IgM expression by di-ortho-substituted polychlorinated biphenyls

Halogenated aromatic hydrocarbons (HAHs) are ubiquitous environment contaminants that produce many of their toxic effects by binding to the aryl hydrocarbon receptor (AhR). However, several investigations have demonstrated that certain polychlorinated biphenyl (PCB) congeners, principally di-ortho-chlorinated PCB congeners, or mixtures containing multiple di-ortho-chlorinated PCBs, inhibit AhR-mediated responses induced by other toxic HAHs. Most relevant to the present study are past reports demonstrating antagonism by these uniquely acting PCB congeners on AhR agonist-mediated inhibition of humoral immune responses. The mechanism responsible for antagonism of AhR agonists by certain PCBs is presently unknown. The present study evaluated the antagonist activity of several di-ortho-substituted PCB congeners [PCB47 (2,2',4,4'), PCB52 (2,2',5,5'), PCB128 (2,2',3,3',4,4'), and PCB153 (2,2',4,4',5,5')] when present in combination with AhR agonists [TCDD (2,3,7,8,-tetrachlorodibenzo-p-dioxin), PCB126 (3,3',4,4',5), and PCB77 (3,3',4,4')] on CYP1A1 induction and inhibition of lipopolysaccharide (LPS)-induced immunoglobulin production in the CH12.LX B cell line. In contrast to non-ortho-substituted PCB (PCB77), which showed additive effects on CYP1A1 induction in combination with TCDD, all of the di-ortho-substituted PCBs examined produced antagonism. Di-ortho-substituted PCB (PCB52) also antagonized TCDD- or PCB126- mediated inhibition of IgM secretion and immunoglobulin heavy chain mRNA expression in the LPS-activated B cells. In addition, PCB52 inhibited TCDD-induced AhR DNA binding to a dioxin-responsive element. Collectively, these results suggest that the mechanism responsible for antagonism by di-ortho-substituted PCB congeners of AhR agonist-mediated CYP1A1 induction and inhibition of antibody responses in B cells occurs through interference with agonist activation of the cytosolic AhR complex.     

Polychlorinated biphenyl exposure and CYP19 gene regulation in testicular and adrenocortical cell lines.

Exposure to polychlorinated biphenyls (PCBs) disturbs many estrogen-mediated biochemical processes. PCBs may cause these abnormalities by altering expression of the aromatase gene CYP19. This study demonstrated that high concentrations of PCB126 increased basal CYP19 mRNA abundance in mouse testicular Leydig I-10 cells and human adrenocortical H295R cells. Stimulating the cells with chorionic gonadotropin or 8-Br-cAMP concealed the estrogenic effect of PCB126. PCB126 is a powerful ligand for nuclear receptor AhR. Antagonizing the AhR activity of H295R by an inhibitor abolished PCB126-elicited CYP19 induction. However, PCB126 elevated basal CYP19 expression and aromatase activity in a slow progressive manner contrary to the sharp induction of the classic AhR target gene CYP1A1. Exposure of H295R to PCBs with different AhR activation abilities also varied CYP19 and CYP1A1 expression in dissimilar patterns, although the CYP19 mRNA levels were in line with the AhR activation abilities of the congeners. In contrast to PCB126, PCB39, which could not activate AhR and lacked effect on CYP1A1, significantly reduced CYP19 mRNA expression. AhR apparently played an important role in CYP19 gene regulation, but it might regulate CYP19 differently from CYP1A1 in the adrenocortical cells. Regardless of the action mechanism, PCB exposure increases risk for CYP19 dysregulation.     

Fecundity, 17beta-estradiol concentrations and expression of vitellogenin and estrogen receptor genes throughout the ovarian cycle in female Eastern mosquitofish from three lakes in Florida

Previous studies of Eastern mosquitofish in contaminated Lake Apopka, Florida, have documented reduced sperm count and sexual behaviour in males but increased fecundity and liver weight in females, compared to nearby reference lakes. Liver weight can be an indicator of vitellogenin (Vtg) synthesis in fish, such as the mosquitofish. It was therefore hypothesized that estrogenic organochlorine pesticides, present at elevated concentrations in animals from Lake Apopka, could cause the reproductive disorders in males, as well as increase female fecundity. We initiated a test of this hypothesis by examining the relationship between 17beta-estradiol (E2) tissue concentrations, hepatic estrogen receptor alpha (ERalpha) and Vtg A, B and C gene expression and fecundity in sexually mature female Eastern mosquitofish from Lake Apopka and two reference lakes, Lake Woodruff and Lake Orange. We observed that female Eastern mosquitofish from one site in contaminated Lake Apopka produced fewer but bigger embryos than females from the other Lake Apopka site and two reference sites. However, female E2 concentrations and hepatic ERalpha and Vtg A, B and C gene expression showed no overall differences among the four sites, and it is therefore unlikely that the differences in fecundity were caused by estrogenic EDCs. In addition, we observed no induction of any of the three Vtg genes in male Eastern mosquitofish at the two Lake Apopka sites. Based on the well-documented high sensitivity of Vtg induction as a biomarker of xenoestrogen exposure, the evidence from the present study does not support the hypothesis that estrogenic EDCs are affecting reproduction in Eastern mosquitofish living in Lake Apopka. Our experimental design tested specifically for effects mediated via the ER, and e.g. antiandrogenic DDT metabolites might still be of importance for mosquitofish reproduction in Lake Apopka.     

Effects of natural and synthetic estrogens and various environmental contaminants on vitellogenesis in fish primary hepatocytes: comparison of bream (Abramis brama) and carp (Cyprinus carpio).

Interaction of environmental estrogens with the estrogen receptor (ER) has been shown in various fish species. Our objective was to compare the sensitivity of bream (Abramis brama) to (xeno-)estrogens with that of the carp (Cyprinus carpio), by measuring the effects of 17beta-estradiol (E2), estrone (E1), ethynylestradiol (EE2), bisphenol A (BPA), nonylphenol (NP), methoxychlor (MXCL), and halogenated aromatic hydrocarbons (HAHs) such as polychlorinated biphenyls (PCB126, PCB118), 2,3,7,8-tetrachlorodibenzo-dioxin (TCDD), and 2,3,4,7,8-pentachlorodibenzofuran (PCDF) on vitellogenesis in primary hepatocytes. Comparing the EC50 values in bream hepatocytes: EE2 (0.1-0.2 microM) < E1 (0.6-0.2 microM) < E2 (1.9 microM) with those of carp hepatocytes EE2 (0.03-0.06 microM) < E2 (0.3 microM) approximately E1 (0.2-0.3 microM) we found differences in sensitivity and ranking of the estrogenic potency of E2 and E1, indicating interspecies differences. Exposure to BPA, NP, MXCL, and HAHs did not or only weakly induce vitellogenesis. Bream hepatocytes coexposed to E2 and TCDD, PCB126 or PCDF showed a concentration-dependent inhibition of E2-induced vitellogenesis. IC50 (concentration of a compound that elicits 50% inhibition of E2-induced vitellogenesis) values determined in bream were: TCDD (0.02-0.09 nM) < PCB126 (0.35-0.1 nM) < PCDF (2.0-0.1) and in carp were: TCDD (0.01 nM) < PCB126 (0.4 nM). PCB118 showed no (anti-)estrogenic response. IC50 values and benchmark-concentration for TCDD and PCB126 in bream and carp hepatocytes were in the same range, indicating similar sensitivity to these compounds. Due to their anti-estrogenic capacity with benchmark-concentrations in the pM range TCDD, PCDF, and PCB126 may form a potential hazard for the reproductive success of fish species by inhibition of vitellogenesis.     

Brain cytochrome P450 aromatase gene isoforms and activity levels in atlantic salmon after waterborne exposure to nominal environmental concentrations of the pharmaceutical ethynylestradiol and antifoulant tributyltin.

In this study, the effects of two environmental endocrine disruptors, the synthetic pharmaceutical estrogen (ethynylestradiol, EE2) and antifoulant (tributyltin, TBT) representing two different modes of action on the endocrine system, were studied on brain steroidogenic pathway of juvenile Atlantic salmon (Salmo salar). Neurosteroidogenesis was studied using brain aromatase gene isoforms and enzyme activity, in parallel with typical xenoestrogen responses, such as brain estrogen receptor (ERalpha) and plasma vitellogenin (Vtg) levels. Fish were exposed to nominal waterborne EE2 (5 and 50 ng/l) and TBT (50 and 250 ng/l) concentrations dissolved in dimethyl sulfoxide (DMSO), singly and in combination. Gene expressions were quantified using real-time PCR with gene-specific primers, aromatase activity was analyzed using the tritiated water-release assay, and plasma Vtg was analyzed using competitive ELISA. Our data show that EE2 induced a concentration-specific modulation of P450aromA, P450aromB, and aromatase activity in addition to ERalpha and plasma Vtg levels in juvenile salmon at day 3 postexposure. TBT exposure caused both the elevation and inhibition of P450aromA, P450aromB, and aromatase activity levels, depending on concentration, at day 7 postexposure. TBT elevated and inhibited ERalpha and plasma Vtg and also antagonized EE2-induced expression of the studied variables at day 7 postexposure. Interestingly, the carrier vehicle DMSO modulated the receptor-mediated and non-receptor-mediated estrogenic responses at day 7 postexposure, compared to day 3. In general, these findings suggest that the exposed animals are experiencing impaired steroidogenesis and modulations of receptor-mediated endocrine responses. Given the integral role of neurosteroids in homeostatic process, growth, metabolism, reproduction, and development of central nervous system and function, these effects may have serious impact on this endocrine pathway and potentially affect organismal reproductive performance and health. In conclusion, the regulation of steroidogenesis is a fundamental mechanism involved in the biosynthesis of important biological compounds, irrespective of organ; therefore, the search for the molecular targets of xenoestrogens, given singly and also in combination, in these pathways will increase our understanding of organismal endocrine disruption and potential consequences.     

A comparative study on the effects of 2,3,7,8,-tetrachlorodibenzo-p-dioxin polychlorinated biphenyl126 and estrogen in human bronchial epithelial cells

Epidemiological studies on 2,3,7,8,-tetrachlorodibenzo-p-dioxin (TCDD) exposure indicated high incidences of pulmonary dysfunctions and lung cancer. Animal studies also demonstrated lung cancer development in female, but not in male, rats exposed to TCDD. Such effects, however, have not been reported in polychlorinated biphenyls (PCB) exposure. In our present study, we have investigated the effects of TCDD and PCB126, with or without cotreatment with 17 beta-estradiol (E2), on a human bronchial epithelial cell line BEAS-2B. We found that treatment with either TCDD or PCB126 alone reduced cell numbers as well as thymidine incorporation. Cell death, however, was only detected in PCB126-, but not TCDD-, treated cultures. The TCDD-induced cell reduction, therefore, could not be contributed to cell death. Meanwhile, because TCDD- and PCB126-enhanced CYP1A1 and CYP1B1 expressions were significantly reduced by the AhR antagonist and CYP1 inhibitor alpha-naphthoflavone (ANF), this indicated that the effects of TCDD and PCB126 were AhR and cytochrome p450 1 dependent. We also found that while E2 itself did not alter CYP1A1 and CYP1B1 expressions, cotreatment of E2 with TCDD or PCB126 would significantly enhance TCDD-, but not PCB126-, induced toxicity. We further demonstrated that in the presence of E2, 1 nM TCDD increased the production of E2 metabolites, 2-methoxyestradiol (2-MeOE2) and 4-methoxyestradiol (4-MeOE2). PCB126, however, only increased 2-MeOE2 formation without significant induction of 4-MeOE2. We believe that these metabolites, especially 4-MeOE2, interacted with TCDD to further suppress cell growth. Our data provided the first demonstration on the enhancement of TCDD-induced toxicity in human lung cells via interaction with estrogen.     

Effect of in vitro estrogenic pesticides on human oestrogen receptor alpha and beta mRNA levels

Nine widely distributed pesticides were recently demonstrated to possess potential estrogenic properties in oestrogen receptor (ER) transactivation and/or E-screen assays. We tested the effect of these nine pesticides on the human ERalpha and ERbeta mRNA steady state levels in the mamma cancer fibroblast MCF-7BUS cells using on-line RT-PCR. Like 17beta-oestradiol (E2), fenarimol significantly decreased the ERalpha and increased the ERbeta mRNA level. Endosulfan and pirimicarb alone decreased the ERalpha mRNA level weakly. After co-exposure with E2, all the tested pesticides counteracted the E2-induced decrease of the ERalpha mRNA level, but only significantly for prochloraz, dieldrin, and tolchlofos-methyl. Alone no pesticides affected the ERbeta mRNA level significantly, but chlorpyrifos increased the mRNA level weakly. Co-exposure with E2 elicited a significant increased ERbeta mRNA level by prochloraz, fenarimol, endosulfan, dieldrin, and tolchlofos-methyl, whereas no significant effect of the carbamate pesticides on the ERbeta mRNA level was observed. This study demonstrated that organochlor and organophosphorous pesticides possess the ability to interfere with the ERalpha and ERbeta mRNA steady state levels.     

Design, synthesis, and estrogenic activity of a novel estrogen receptor modulator--a hybrid structure of 17beta-estradiol and vitamin E in hippocampal neurons

We recently discovered that ICI 182,780 (1), an antagonist of estrogen receptor (ER)-dependent proliferation in reproductive tissues, functions as an estrogenic agonist in primary neurons. The present study investigated whether the agonist properties of 1 in neurons could be translated into structural analogs. 7alpha-[(4R,8R)-4,8,12-trimethyltridecyl]estra-1,3,5-trien-3,17beta-diol (2), a hybrid structure of 17beta-estradiol and vitamin E, was synthesized and found to bind to both ERalpha and ERbeta. In vitro analyses demonstrated that 2 was neuroprotective and effective in activating molecular mechanisms associated with estrogenic agonist activity in rat primary hippocampal neurons. Collectively, the data support an estrogenic agonist profile of 2 action comparable to 1 in primary neurons, confirming that estrogenic activity of 1 in neurons is not a unique phenomenon. These results provide support for the development of a brain-selective ER modulator, with potential as an efficacious and safe estrogen alternative to prevent Alzheimer's disease and cognitive decline in postmenopausal women.