Estrogen receptor antagonism leading to reproductive dysfunction

Daniel L. Villeneuve, US EPA Mid-Continent Ecology Division (villeneuve.dan@epa.gov)

This adverse outcome pathway details the linkage between antagonism of estrogen receptor in females and the adverse effect of reduced cumulative fecundity in repeat-spawning fish species. Cumulative fecundity is the most apical endpoint considered in the OECD 229 Fish Short Term Reproduction Assay. The OECD 229 assay serves as screening assay for endocrine disruption and associated reproductive impairment (OECD 2012a). Cumulative fecundity is one of several variables known to be of demographic significance in forecasting fish population trends. Therefore, this AOP has utility in supporting the application of measures of ER antagonism, or in silico predictions of the ability to antagonize ER as a means to identify chemicals with known potential to adversely affect fish populations.

• Overall Assessment of the AOP

• Concordance of dose-response relationships:

• In a 42 d static renewal exposure to tamoxifen, significant, concentration dependent reduction in the number of clutches and cumulative fecundity were observed for zebrafish (Wester et al. 2003).
• A concentration-dependent reduction in circulating vitellogenin concentrations was detected in female medaka exposed to tamoxifen for 21 d (Sun et al. 2007b). Vitellogenin reductions occurred at a lower concentration (i.e., ≥ 25 μg tamoxifen/L) than reductions in fecundity (i.e., 625 μg tamoxifen/L).

• Temporal concordance among the key events and adverse effect: To date, there are no time-course studies that allow for robust evaluation of the temporal concordance of the entire AOP. However, the temporal concordance of some of the key event relationships has been established. Specifically, reductions in transcription of vitellogenin mRNAs have been shown to precede changes in circulating vitellogenin concentrations.
• Consistency:

• In zebrafish exposed to tamoxifen, reductions in the number of clutches and cumulative egg production were predicted to result in population reductions, although this was in conjunction with altered sex ratios as a concurrent effect in a partial life-cycle test (Wester et al. 2003).
• In medaka co-exposed to 17β-estradiol (E2; 200 ng/L) and 10, 50, or 250 μg tamoxifen/L, exposure to 250 μg tamoxifen significantly reduced fecundity compared to both controls and fish exposed to E2 alone (Sun et al. 2009).
• Fecundity was significantly reduced in medaka exposed to 625 μg tamoxifen/L (Sun et al. 2007b).
• Increases in atretic oocytes and oviducts filled with degenerated eggs were observed in female zebrafish exposed to tamoxifen (Wester et al. 2003). Reduced vitellogenin immuno staining was observed in tamoxifen-exposed zebrafish, based on blind semi-quantitative scoring (van der Ven et al. 2007; Wester et al. 2003). The results are therefore consistent with the AOP.
• In Japanese medaka co-exposed to E2 and tamoxifen for 21 d, both plasma vitellogenin and fecundity were reduced in a tamoxifen concentration-dependent manner (Sun et al. 2009). Although from a co-exposure, the results are broadly consistent with the AOP.
• In Japanese medaka exposed to tamoxifen for 21 d, plasma vitellogenin in females was reduced in a concentration-dependent manner and cumulative fecundity was reduced at the maximum concentration tested (Sun et al. 2007b). The results are consistent with the AOP.
• Dietary exposure to tamoxifen was also shown to reduce circulating vitellogenin concentrations in female medaka (Chikae et al. 2004). The results are consistent with the AOP.
• In tilapia co-injected with E2 or o,p-DDT, tamoxifen inhibited the stimulatory effects of E2 and o,p-DDT on plasma vitellogenin (measured as alkaline labile phosphorous). Alkaline labile phosphorous was not reduced following injection with tamoxifen alone (Leanos-Castaneda et al. 2002). These results are neither entirely consistent nor inconsistent with the AOP.

• Uncertainties, inconsistencies, and data gaps:

• In a 42 d in vivo, flow through, exposures to tamoxifen citrate, no significant reductions in circulating vitellogenin or cumulative fecundity were detected (Williams et al. 2007). The results are therefore inconsistent with the AOP.
• Some uncertainty remains regarding which ER subtype(s) regulates vitellogenin gene expression in the liver of fish. In general, the literature suggests a close interplay between several ER subtypes in the regulation of vitellogenesis. Consequently, at present, the AOP is generalized to impacts on all ER subtypes, even though it remains possible that impacts on a particular sub-type may drive the adverse response.

• Griffin et al. reported that morpholino knock-downs of either esr1 (ERα) or esr2b (ERβb) prevented estradiol-mediated induction of vitellogenin expression in zebrafish (Griffin et al. 2013).
• Using selective agonists agonists and antagonists for ERα and ERβ, it was concluded that ERβ was primarily responsible for inducing vitellogenin production in rainbow trout and that compounds exhibiting ERα selectivity would not be detected using a vitellogenin bioassay (Leanos-Castaneda and Van Der Kraak 2007). However, a subsequent study conducted in tilapia concluded that agonistic and antagonistic characteristics of mammalian, isoform-specific ER agonists and antagonists, cannot be reliably extrapolated to piscine ERs (Davis et al. 2010).
• Expression of both ERα1 and ERβ1 were strongly correlated with plasma vitellogenin concentrations over the reproductive cycle of rainbow trout (Nagler et al. 2012).
• Based on RNA interference knock-down experiments Nelson and Habibi proposed a model in which all ER subtypes are involved in E2-mediated vitellogenesis, with ERβ isoforms stimulating expression of both vitellogenin and ERα gene expression, and ERα helping to drive vitellogenesis, particularly as it becomes more abundant following sensitization (Nelson and Habibi 2010).

• There remains uncertainty as to whether there is a direct biological linkage, as opposed to correlation only, between impaired VTG uptake into oocytes and impaired spawning/reduced cumulative fecundity. Plausible biological connections have been hypothesized but have not yet been tested experimentally.

• OECD. 2012a. Test no. 229: Fish short term reproduction assay. Paris, France:Organization for Economic Cooperation and Development.
• Wester P, van den Brandhof E, Vos J, van der Ven L. 2003. Identification of endocrine disruptive effects in the aquatic environment - a partial life cycle assay in zebrafish. (RIVM Report). Bilthoven, the Netherlands:Joint Dutch Environment Ministry
• Sun L, Zha J, Spear PA, Wang Z. 2007b. Tamoxifen effects on the early life stages and reproduction of japanese medaka (oryzias latipes). Environmental toxicology and pharmacology 24:23-29.
• Sun L, Zha J, Wang Z. 2009. Effects of binary mixtures of estrogen and antiestrogens on japanese medaka (oryzias latipes). Aquatic toxicology 93:83-89.
• Williams TD, Caunter JE, Lillicrap AD, Hutchinson TH, Gillings EG, Duffell S. 2007. Evaluation of the reproductive effects of tamoxifen citrate in partial and full life-cycle studies using fathead minnows (pimephales promelas). Environmental toxicology and chemistry / SETAC 26:695-707.
• van der Ven LT, van den Brandhof EJ, Vos JH, Wester PW. 2007. Effects of the estrogen agonist 17beta-estradiol and antagonist tamoxifen in a partial life-cycle assay with zebrafish (danio rerio). Environmental toxicology and chemistry / SETAC 26:92-99.
• Chikae M, Ikeda R, Hasan Q, Morita Y, Tamiya E. 2004. Effects of tamoxifen, 17alpha-ethynylestradiol, flutamide, and methyltestosterone on plasma vitellogenin levels of male and female japanese medaka (oryzias latipes). Environmental toxicology and pharmacology 17:29-33.
• Leanos-Castaneda O, Van Der Kraak G. 2007. Functional characterization of estrogen receptor subtypes, eralpha and erbeta, mediating vitellogenin production in the liver of rainbow trout. Toxicology and applied pharmacology 224:116-125.
• Griffin LB, January KE, Ho KW, Cotter KA, Callard GV. 2013. Morpholino mediated knockdown of eralpha, erbetaa and erbetab mrnas in zebrafish (danio rerio) embryos reveals differential regulation of estrogen-inducible genes. Endocrinology.
• Davis LK, Katsu Y, Iguchi T, Lerner DT, Hirano T, Grau EG. 2010. Transcriptional activity and biological effects of mammalian estrogen receptor ligands on three hepatic estrogen receptors in mozambique tilapia. The Journal of steroid biochemistry and molecular biology 122:272-278.
• Nagler JJ, Cavileer TD, Verducci JS, Schultz IR, Hook SE, Hayton WL. 2012. Estrogen receptor mrna expression patterns in the liver and ovary of female rainbow trout over a complete reproductive cycle. General and comparative endocrinology 178:556-561.
• Nelson ER, Habibi HR. 2010. Functional significance of nuclear estrogen receptor subtypes in the liver of goldfish. Endocrinology 151:1668-1676.
• Miller DH, Jensen KM, Villeneuve DL, Kahl MD, Makynen EA, Durhan EJ, Ankley GT. 2007. Linkage of biochemical responses to population-level effects: a case study with vitellogenin in the fathead minnow (Pimephales promelas). Environ. Toxicol. Chem. 26: 521-527.