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Aop: 30

AOP Title

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Estrogen receptor antagonism leading to reproductive dysfunction

Short name:

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Estrogen receptor antagonism leading to reproductive dysfunction

Authors

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

Point of Contact

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Dan Villeneuve

Contributors

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  • Dan Villeneuve

Status

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Author status OECD status OECD project SAAOP status
Open for citation & comment EAGMST Under Review 1.12 Included in OECD Work Plan


This AOP was last modified on November 30, 2016 12:22

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Revision dates for related pages

Page Revision Date/Time
Decrease, Population trajectory March 20, 2017 17:53
Antagonism, Estrogen receptor September 16, 2017 10:14
Reduction, Vitellogenin synthesis in liver September 16, 2017 10:16
Reduction, Plasma vitellogenin concentrations September 16, 2017 10:14
Reduction, Vitellogenin accumulation into oocytes and oocyte growth/development September 16, 2017 10:14
Reduction, Cumulative fecundity and spawning March 20, 2017 17:52
Antagonism, Estrogen receptor leads to Reduction, Vitellogenin synthesis in liver November 30, 2016 12:10
Reduction, Vitellogenin synthesis in liver leads to Reduction, Plasma vitellogenin concentrations March 20, 2017 12:58
Reduction, Plasma vitellogenin concentrations leads to Reduction, Vitellogenin accumulation into oocytes and oocyte growth/development March 20, 2017 13:21
Reduction, Vitellogenin accumulation into oocytes and oocyte growth/development leads to Reduction, Cumulative fecundity and spawning March 20, 2017 13:35
Reduction, Cumulative fecundity and spawning leads to Decrease, Population trajectory March 20, 2017 13:49

Abstract

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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.


Background (optional)

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This optional section should be used to provide background information for AOP reviewers and users that is considered helpful in understanding the biology underlying the AOP and the motivation for its development. The background should NOT provide an overview of the AOP, its KEs or KERs, which are captured in more detail below.

Instructions

To add background information, click Edit in the upper right hand menu on the AOP page. Under the “Background (optional)” field, a text editable form provides ability to edit the Background.  Clicking ‘Update AOP’ will update these fields.


Summary of the AOP

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Stressors

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Describes stressors known to trigger the MIE and provides evidence supporting that initiation. This will often be a list of prototypical compounds demonstrated to interact with the target molecule in the manner detailed in the MIE description to initiate a given pathway (e.g., 2,3,7,8-TCDD as a prototypical AhR agonist; 17α-ethynyl estradiol as a prototypical ER agonist). However, depending on the information available, this could also refer to chemical categories (i.e., groups of chemicals with defined structural features known to trigger the MIE). It can also include non-chemical stressors such as genetic or environmental factors. The evidence supporting the stressor will typically consist of a brief description and citation of literature showing that particular stressors can trigger the MIE.

Instructions

To add a stressor associated with an AOP, under “Summary of the AOP” click ‘Add Stressor’ will bring user to the “New Aop Stressor” page. In the Name field, user can search for stressor by name. Choosing a stressor from the resulting drop down populates the field. Selection of an Evidence level from the drop down menu and add any supporting evidence in the text box. Click ‘Add stressor’ to add the stressor to the AOP page.


Molecular Initiating Event

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Title Short name
Antagonism, Estrogen receptor Antagonism, Estrogen receptor

Key Events

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Title Short name
Reduction, Vitellogenin synthesis in liver Reduction, Vitellogenin synthesis in liver
Reduction, Plasma vitellogenin concentrations Reduction, Plasma vitellogenin concentrations
Reduction, Vitellogenin accumulation into oocytes and oocyte growth/development Reduction, Vitellogenin accumulation into oocytes and oocyte growth/development
Reduction, Cumulative fecundity and spawning Reduction, Cumulative fecundity and spawning

Adverse Outcome

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Title Short name
Decrease, Population trajectory Decrease, Population trajectory

Relationships Between Two Key Events (Including MIEs and AOs)

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Network View

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Life Stage Applicability

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Life stage Evidence
Adult, reproductively mature Strong

Taxonomic Applicability

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Term Scientific Term Evidence Link
zebra danio Danio rerio NCBI
fathead minnow Pimephales promelas NCBI
medaka Oryzias latipes NCBI

Sex Applicability

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Sex Evidence
Female Strong

Graphical Representation

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Click to download graphical representation template

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Overall Assessment of the AOP

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  • 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.

 

Domain of Applicability

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Life Stage: This AOP applies to sexually mature animals. Sex: This AOP applies to females. Taxonomic Applicability: Based on the taxonomic applicability of the component key events, this AOP could potentially apply to most oviparous chordates.

Domain(s) of Applicability

  • Sex: The AOP applies to females only
  • Life stages: The relevant life stages for this AOP are reproductively mature adults. This AOP does not apply to adult stages that lack a sexually mature ovary, for example as a result of seasonal or environmentally-induced gonadal senescence (i.e., through control of temperature, photo-period, etc. in a laboratory setting).
  • Taxonomic: At present, the assumed taxonomic applicability domain of this AOP is class Osteichthyes. In all likelihood, the AOP will also prove applicable to all classes of fish (e.g., Agnatha and Chondrithyes as well). Additionally, all the key events described should be conserved among all oviparous vertebrates, suggesting that the AOP may also have relevance for amphibians, reptiles, and birds. However, species-specific differences in reproductive strategies/life histories, ADME (adsorption, distribution, metabolism, and elimination), compensatory reproductive endocrine responses may influence the outcomes, particularly from a quantitative standpoint.

Essentiality of the Key Events

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The essentiality of various of the KEs is influential in considering confidence in an overall hypothesised AOP for potential regulatory application being secondary only to biological plausibility of KERs (Meek et al., 2014; 2014a). The defining question for determining essentiality (included in Annex 1) relates to whether or not downstream KEs and/or the AO is prevented if an upstream event is experimentally blocked. It is assessed, generally, then, on the basis of direct experimental evidence of the absence/reduction of downstream KEs when an upstream KE is blocked or diminished (e.g., in null animal models or reversibility studies). Weight of evidence for essentiality of KEs would be considered high if there is direct evidence from specifically designed experimental studies illustrating essentiality for at least one of the important key events [e.g., stop/reversibility studies, antagonism, knock out models, etc.) moderate if there is indirect 25 evidence that experimentally induced change of an expected modulating factor attenuates or augments a key event (e.g., augmentation of proliferative response (KEupstream) leading to increase in tumour formation (KEdownstream or AO)) and weak if there is no or contradictory experimental evidence of the essentiality of any of the KEs (Annex 1).

Instructions

To edit the “Essentiality of the Key Events” section, on an AOP page, in the upper right hand menu, click ‘Edit.’ This brings you to a page entitled, “Editing AOP.” Scroll down to the “Essentiality of the Key Events” section, where a text entry box allows you to submit text. In the upper right hand menu, click ‘Update AOP’ to save your changes and return to the AOP page.  The new text should appear under the “Essentiality of the Key Events” section on the AOP page.


Weight of Evidence Summary

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The weight of evidence for each of the KERs comprising the AOP are ranked moderate to strong. Biological plausibility at the molecular and cellular level of the early key events is very strong. Some uncertainties regarding the mechanistic details of the connection between reduced vtg availability and uptake limit the strength of evidence to some degree. However, there are considerable evidence to support the idea that ER antagonism can ultimately lead to reproductive failure. Overall weight of evidence is moderate.


Quantitative Considerations

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A quantitative relationship between ER antagonism (the MIE) and reductions in vitellogenin transcription and translation have not been well established. However, a correlative relationship between plasma vitellogenin concentrations and cumulative fecundity has been reported (Miller et al. 2007) and applied for quantitative modeling (Ankley et al.


Considerations for Potential Applications of the AOP (optional)

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At their discretion, the developer may include in this section discussion of the potential applications of an AOP to support regulatory decision-making. This may include, for example, possible utility for test guideline development or refinement, development of integrated testing and assessment approaches, development of (Q)SARs / or chemical profilers to facilitate the grouping of chemicals for subsequent read-across, screening level hazard assessments or even risk assessment. While it is challenging to foresee all potential regulatory application of AOPs and any application will ultimately lie within the purview of regulatory agencies, potential applications may be apparent as the AOP is being developed, particularly if it was initiated with a particular application in mind. This optional section is intended to provide the developer with an opportunity to suggest potential regulatory applications and describe his or her rationale. Detailing such considerations can aid the process of transforming narrative descriptions of AOPs into practical tools. In this context, it is necessarily beneficial to involve members of the regulatory risk assessment community on the development and assessment team. The Network view which is generated based on assessment of weight of evidence/degree of confidence in the hypothesized AOP taking into account the elements described in Section 7 provides a useful summary of relevant information as a basis to consider appropriate application in a regulatory context. Consideration of application needs then, to take into consideration the following rank ordered qualitative elements: Confidence in biological plausibility for each of the KERs Confidence in essentiality of the KEs Empirical support for each of the KERs and overall AOP The extent of weight of evidence/confidence in both these qualitative elements and that of the quantitative understanding for each of the KERs (e.g., is the MIE known, is quantitative understanding restricted to early or late key events) is also critical in determining appropriate application. For example, if the confidence and quantitative understanding of each KER in a hypothesised AOP are low and or low/moderate and the evidence for essentiality of KEs weak (Section 7), it might be considered as appropriate only for applications with less potential for impact (e.g., prioritisation, category formation for testing) versus those that have immediate implications potentially for risk management (e.g., in depth assessment). If confidence in quantitative understanding of late key events is high, this might be sufficient for an in depth assessment. The analysis supporting the Network view is also essential in identifying critical data gaps based on envisaged regulatory application.

Instructions

To edit the “Considerations for Potential Applications of the AOP” section, on an AOP page, in the upper right hand menu, click ‘Edit.’ This brings you to a page entitled, “Editing AOP.” Scroll down to the “Considerations for Potential Applications of the AOP” section, where a text entry box allows you to submit text. In the upper right hand menu, click ‘Update AOP’ to save your changes and return to the AOP page.  The new text should appear under the “Considerations for Potential Applications of the AOP” section on the AOP page.


References

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  • 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.