Estrogenicity of food-associated estrogenic compounds in the fetuses of female transgenic mice upon oral and IP maternal exposure.

The present study investigated to what extent seven food-associated in vitro estrogenic compounds can induce estrogenic effects in the fetuses of pregnant female mice with an estrogen receptor (ER)-mediated luciferase (luc) reporter gene system. The luc-induction was determined either 8h after maternal dosing with a single intraperitoneal (IP) dose or 24h after the last of a series of 8 daily oral dosages. Three known estrogens, 17beta-estradiol (E(2)), 17 alpha-ethynylestradiol (EE) and 17beta-estradiol 3,17-dipropionate (EP) were used as positive controls at 1mg/kgbw and DMSO as solvent control. The food-associated estrogenic compounds tested were: bisphenol A (BPA), nonylphenol (NP) both at 50mg/kgbw, dichlorodiphenyldichloroethylene (p,p'-DDE) at 50mg/kgbw, quercetin at 16.6 mg/kgbw, and di-isoheptyl phthalate (DIHP), di-(2-ethylhexyl) phthalate (DEHP) and di-(2-ethylhexyl) adipate (DEHA) all at 100mg/kgbw. Exposure to E(2), EE and EP resulted in significant luc inductions upon both oral and/or IP dosing in a variety of tissues including liver, tibia and femurs, and upon IP dosing also in fetuses. BPA, NP, DEHA, DEHP, DIHP, DDE and quercetin were unable to significantly induce luc activity in fetuses. However, after maternal oral exposure during gestation to NP, BPA and DIHP placental luc activity was significantly lowered. The results indicate that at the current levels of exposure to food-associated estrogenic compounds, estrogenic effects to the fetus are not expected. The significant luc reduction in the placenta, should be further studied for its significance for fetal development and relevance for the human situation.

Food-associated estrogenic compounds induce estrogen receptor-mediated luciferase gene expression in transgenic male mice.

The present paper aims at clarifying to what extent seven food-associated compounds, shown before to be estrogenic in vitro, can induce estrogenic effects in male mice with an estrogen receptor (ER)-mediated luciferase (luc) reporter gene system. The luc induction was determined in different tissues 8h after dosing the ER-luc male mice intraperitoneally (IP) or 14h after oral dosing. Estradiol-propionate (EP) was used as a positive control at 0.3 and 1mg/kg bodyweight (bw), DMSO as solvent control. The food-associated estrogenic compounds tested at non-toxic doses were bisphenol A (BPA) and nonylphenol (NP) (both at 10 and 50mg/kgbw), dichlorodiphenyldichloroethylene (p,p'-DDE; at 5 and 25mg/kgbw), quercetin (at 1.66 and 16.6mg/kgbw), di-isoheptyl phthalate (DIHP), di-(2-ethylhexyl) phthalate (DEHP) and di-(2-ethylhexyl) adipate (DEHA) all at 30 and 100mg/kgbw. In general IP dosing resulted in higher luc inductions than oral dosing. EP induced luc activity in the liver in a statistically significant dose-related way with the highest induction of all compounds tested which was 20,000 times higher than the induction by the DMSO-control. NP, DDE, DEHA and DIHP did not induce luc activity in any of the tissues tested. BPA induced luc in the liver up to 420 times via both exposure routes. BPA, DEHP and quercetin induced luc activity in the liver after oral exposure. BPA (50mg/kgbw IP) also induced luc activity in the testis, kidneys and tibia. The current study reveals that biomarker-responses in ER-luc male mice occur after a single oral exposure to food-associated estrogenic model compounds at exposure levels 10 to 10(4) times higher than the established TDI's for some of these compounds. Given the facts that (i) the present study did not include chronic exposure and that (ii) simultaneous exposure to multiple estrogenic compounds may be a realistic exposure scenario, it remains to be seen whether this margin is sufficiently high.

Estrogenic potency of food-packaging-associated plasticizers and antioxidants as detected in ERalpha and ERbeta 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.

The stimulation of cell proliferation by quercetin is mediated by the estrogen receptor.

Quercetin causes biphasic modulation of the proliferation of specific colon and mammary cancer cells. In this study, the possible involvement of the estrogen receptor (ER) in the stimulation of cell proliferation by quercetin was investigated. For this purpose, the effect of quercetin on cell proliferation was tested in ER-positive MCF-7 and T47D cells, and in ER-negative HCC-38 and MDA-MB231 cells. Quercetin stimulated proliferation of ER-positive cells only, suggesting this effect to be ER-dependent. In support of these results, quercetin induced ER-ERE-mediated gene expression in a reporter gene assay using U2-OS cells transfected with either ERalpha or ERbeta, with 10(5)-10(6) times lower affinity than 17beta-estradiol (E2) and 10(2)-10(3 )times lower affinity than genistein. Quercetin activated the ERbeta to a 4.5-fold higher level than E2, whereas the maximum induction level of ERalpha by quercetin was only 1.7 fold that of E2. These results point at the relatively high capacity of quercetin to stimulate supposed 'beneficial' ERbeta responses as compared to the stimulation of ERalpha, the receptor possibly involved in adverse cell proliferative effects. Altogether, the results of this study reveal that physiologically relevant concentrations of quercetin can exert phytoestrogen-like activity similar to that observed for the isoflavonoid genistein.