Summary of reference chemicals evaluated by the fish short-term reproduction assay, OECD TG229, using Japanese Medaka, Oryzias latipes.

Under the Organisation for Economic Co-operation and Development (OECD), the Ministry of the Environment of Japan (MOE) added Japanese medaka (Oryzias latipes) to the test guideline fish short-term reproduction assay (FSTRA) developed by the United States Environmental Protection Agency (US EPA) using fathead minnow (Pimephales promelas). The FSTRA was designed to detect endocrine disrupting effects of chemicals interacting with the hypothalamic-pituitary-gonadal axis (HPG axis) such as agonists or antagonists on the estrogen receptor (Esr) and/or the androgen receptor (AR) and steroidogenesis inhibitors. We conducted the FSTRA with Japanese medaka, in accordance with OECD test guideline number 229 (TG229), for 16 chemicals including four Esr agonists, two Esr antagonists, three AR agonists, two AR antagonists, two steroidogenesis inhibitors, two progesterone receptor agonists, and a negative substance, and evaluated the usability and the validity of the FSTRA (TG229) protocol. In addition, in vitro reporter gene assays (RGAs) using Esr1 and ARß of Japanese medaka were performed for the 16 chemicals, to support the interpretation of the in vivo effects observed in the FSTRA. In the present study, all the test chemicals, except an antiandrogenic chemical and a weak Esr agonist, significantly reduced the reproductive status of the test fish, that is, fecundity or fertility, at concentrations where no overt toxicity was observed. Moreover, vitellogenin (VTG) induction in males and formation of secondary sex characteristics (SSC), papillary processes on the anal fin, in females was sensitive endpoints to Esr and AR agonistic effects, respectively, and might be indicators of the effect concentrations in long-term exposure. Overall, it is suggested that the in vivo FSTRA supported by in vitro RGA data can adequately detect effects on the test fish, O. latipes, and probably identify the mode of action (MOA) of the chemicals tested.

[Gender-dependent Expression of ERß in AEC â ¡ of Fetal Mice Exposed to Arsenic and Estrogen Receptor Antagonist].

OBJECTIVE: To determine the effects of arsenic and estrogen receptor antagonist (ICI182, 780) on the expression of estrogen receptor beta (ERß) in alveolar Ⅱ epithelial cells (AECⅡ) of female and male mice. METHODS: Nineteen or twenty day fetus mice were obtained through caesarean section of ICR mice. Purified AECⅡ cells were separated from the female and male fetus,respectively,and confirmed using immunofluorescence staining. The cells were exposed to sodium arsenite (NaAsO2) at a low,medium,or high dosage determined by MTT and cultured for 24 h. The NaAsO2 (5 µmol/L) exposed cells were compared with those treated (for 24 h) with dimethyl sulfoxide (DMSO) or ICI182, 780 (1×10-4 mol/L). Apoptosis rates of the cells were measured by flow cytometry. Real-time fluorescence quantitative PCR method and Western blot technique were used to detect the expression ofERßmRNA and protein in AECⅡ. RESULTS: Purity of AECⅡ cells reached (87.0±2.5)%. NaAsO2 exposure was set at a concentration of 0.5 (low),1.25 (medium),and 5 (high) µmol/L. The cells exposed to medium and high dosage of NaAsO2 had higher apoptosis rates than the blank controls (P<0.05),without sex differences. Female cells exposed to medium and high dosage of NaAsO2 had higher levels of expressions ofERßmRNA and protein than the blank controls (P<0.05) and male cells exposed to the same dosage of NaAsO2 (P<0.05). No significant differences were found in the expressions ofERßmRNA and protein between the exposed male cells and the blank controls. ICI182, 780 lowered the expression levels ofERßmRNA and protein in the female exposed cells (P<0.01). CONCLUSION: Arsenic exposure increases expressions of AECⅡ's ERß,more so in female cells than in male cells. This can be blocked by estrogen receptor antagonists.

Estrogenic effects following larval exposure to the putative anti-estrogen, fulvestrant, in the fathead minnow (Pimephales promelas).

The objective of the present study was to investigate the consequences of early-life exposure to fulvestrant on estrogenic gene expression in fathead minnow larvae. To address this objective, fathead minnow larvae were exposed to fulvestrant (ICI 182,780) during the window of sexual differentiation between 0 to 30 days post-hatch (dph). The four treatment groups in this study included: filtered water controls (never exposed), solvent controls (ethanol 0.01%), and nominally low (0.10µg/L) and high (10.0µg/L) doses of fulvestrant. Following 30 d exposure to their respective treatment, larvae were transferred to filtered water aquaria and assessed for alterations in endocrine-responsive gene expression (i.e., RT-qPCR), body size and survival. The remaining fish depurated in filtered water until reaching sexual maturity (180dph) for assessment of persistent effects on sex characteristics, reproductive performance and sex ratio. Following the 30-d early life exposure, larvae showed upregulations of the endocrine-responsive genes ar, erß and vtg in response to both low and high doses of fulvestrant, but showed no differences in survival or body mass. Upon reaching sexual maturity under depuration conditions, male minnows previously exposed to fulvestrant as larvae showed reductions in gonad mass along with the feminization of secondary sex characteristics with no observed effects in females. Exposure to fulvestrant had no effects on gonadal histology, reproductive performance or final sex ratio as adults. Results from this study demonstrate that aqueous exposure to fulvestrant is estrogenic in fathead minnow larvae and is capable of feminizing male fish as adults following early life exposure.

The role of estrogen receptor subtypes for induction of delayed effects on the estrous cycle and female reproductive organs in rats.

It has been reported that neonatal exposure to estrogens at relatively low doses can induce early onset anovulation as a delayed effect in female rats. Dysfunction of kisspeptin neurons in the anteroventral periventricular nucleus (AVPV) was proposed to be a trigger for this effect. To determine the roles of estrogen receptor (ER) subtypes in the induction of delayed effects, we conducted a series of experiments using Donryu rats to examine whether neonatal injection of an ERα agonist (PPT), an ERß agonist (DPN) or an ERα antagonist (ICI) could induce delayed effects. Also, involvement of the kisspeptin neurons in the AVPV for induction of delayed effect by PPT and DPN was investigated. We observed that neonatal exposure to PPT, DPN and ICI induced the early onset of abnormal estrous cyclicity after sexual maturation, suggesting that the compounds capable of inducing delayed effects are not limited to ERα agonists. On the other hand, the data suggested the possibility that DPN and ICI functioned partially as ERα agonists in the neonatal brain. Regardless of the agents used, there is a possibility that dysfunction of kisspeptin neurons in the AVPV might contribute to induction of early onset anovulation.

Estrogenic and anti-estrogenic influences in cultured brown trout hepatocytes: Focus on the expression of some estrogen and peroxisomal related genes and linked phenotypic anchors.

Estrogens, estrogenic mimics and anti-estrogenic compounds are known to target estrogen receptors (ER) that can modulate other nuclear receptor signaling pathways, such as those controlled by the peroxisome proliferator-activated receptor (PPAR), and alter organelle (inc. peroxisome) morphodynamics. By using primary isolated brown trout (Salmo trutta f. fario) hepatocytes after 72 and 96h of exposure we evaluated some effects in selected molecular targets and in peroxisomal morphological features caused by: (1) an ER agonist (ethinylestradiol-EE2) at 1, 10 and 50µM; (2) an ER antagonist (ICI 182,780) at 10 and 50µM; and (3) mixtures of both (Mix I-10µM EE2 and 50µM ICI; Mix II-1µM EE2 and 10µM ICI and Mix III-1µM EE2 and 50µM ICI). The mRNA levels of the estrogenic targets (ERα, ERß-1 and vitellogenin A-VtgA) and the peroxisome structure/function related genes (catalase, urate oxidase-Uox, 17ß-hydroxysteroid dehydrogenase 4-17ß-HSD4, peroxin 11α-Pex11α and PPARα) were analyzed by real-time polymerase chain reaction (RT-PCR). Stereology combined with catalase immunofluorescence revealed a significant reduction in peroxisome volume densities at 50µM of EE2 exposure. Concomitantly, at the same concentration, electron microscopy showed smaller peroxisome profiles, exacerbated proliferation of rough endoplasmic reticulum, and a generalized cytoplasmic vacuolization of hepatocytes. Catalase and Uox mRNA levels decreased in all estrogenic stimuli conditions. VtgA and ERα mRNA increased after all EE2 treatments, while ERß-1 had an inverse pattern. The EE2 action was reversed by ICI 182,780 in a concentration-dependent manner, for VtgA, ERα and Uox. Overall, our data show the great value of primary brown trout hepatocytes to study the effects of estrogenic/anti-estrogenic inputs in peroxisome kinetics and in ER and PPARα signaling, backing the still open hypothesis of crosstalk interactions between these pathways and calling for more mechanistic experiments.