API

Aop: 25

AOP Title

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Aromatase inhibition leading to reproductive dysfunction

Short name:

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Aromatase inhibition leading to reproductive dysfunction

Authors

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


Point of Contact Dan Villeneuve


Contributors

  • Dan Villeneuve

Status

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Author status OECD status OECD project SAAOP status
Open for citation & comment TFHA/WNT Endorsed 1.12 Included in OECD Work Plan


This AOP was last modified on April 18, 2017 14:27

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Revision dates for related pages

Page Revision Date/Time
Decrease, Population trajectory March 20, 2017 17:53
Inhibition, Aromatase December 03, 2016 16:33
Reduction, Plasma 17beta-estradiol concentrations November 29, 2016 19:01
Reduction, Vitellogenin synthesis in liver March 20, 2017 17:50
Reduction, Vitellogenin accumulation into oocytes and oocyte growth/development March 20, 2017 17:51
Reduction, 17beta-estradiol synthesis by ovarian granulosa cells March 20, 2017 17:49
Reduction, Cumulative fecundity and spawning March 20, 2017 17:52
Reduction, Plasma vitellogenin concentrations March 20, 2017 17:51
Inhibition, Aromatase leads to Reduction, 17beta-estradiol synthesis by ovarian granulosa cells November 30, 2016 13:27
Reduction, 17beta-estradiol synthesis by ovarian granulosa cells leads to Reduction, Plasma 17beta-estradiol concentrations March 20, 2017 12:05
Reduction, Plasma 17beta-estradiol concentrations leads to Reduction, Vitellogenin synthesis in liver March 20, 2017 12:28
Reduction, Cumulative fecundity and spawning leads to Decrease, Population trajectory March 20, 2017 13:49
Reduction, Vitellogenin accumulation into oocytes and oocyte growth/development leads to Reduction, Cumulative fecundity and spawning March 20, 2017 13:35
Reduction, Plasma vitellogenin concentrations leads to Reduction, Vitellogenin accumulation into oocytes and oocyte growth/development March 20, 2017 13:21
Reduction, Vitellogenin synthesis in liver leads to Reduction, Plasma vitellogenin concentrations March 20, 2017 12:58
Reduction, Plasma 17beta-estradiol concentrations leads to Reduction, Plasma vitellogenin concentrations March 20, 2017 16:38

Abstract

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This adverse outcome pathway details the linkage between inhibition of gonadal aromatase activity in females and reproductive dysfunction, as measured through the adverse effect of reduced cumulative fecundity and spawning. Initial development of this AOP draws heavily on evidence collected using 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 2012). 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 aromatase, or in silico predictions of the ability to inhibit aromatase, as a means to identify chemicals with known potential to adversely affect fish populations and potentially other oviparous vertebrates.


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
Inhibition, Aromatase Inhibition, Aromatase

Key Events

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Title Short name
Reduction, Plasma 17beta-estradiol concentrations Reduction, Plasma 17beta-estradiol concentrations
Reduction, Vitellogenin synthesis in liver Reduction, Vitellogenin synthesis in liver
Reduction, Vitellogenin accumulation into oocytes and oocyte growth/development Reduction, Vitellogenin accumulation into oocytes and oocyte growth/development
Reduction, 17beta-estradiol synthesis by ovarian granulosa cells Reduction, 17beta-estradiol synthesis by ovarian granulosa cells
Reduction, Cumulative fecundity and spawning Reduction, Cumulative fecundity and spawning
Reduction, Plasma vitellogenin concentrations Reduction, Plasma vitellogenin concentrations

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

Taxonomic Applicability

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Term Scientific Term Evidence Link
medaka Oryzias latipes Moderate NCBI
zebrafish Danio rerio Moderate NCBI
fathead minnow Pimephales promelas Strong 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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This section addresses the relevant domain of applicability (i.e., in terms of taxa, sex, life stage, etc.) and weight of evidence for the overall hypothesised AOP (i.e., including the MIE, KEs and AO) as a basis to consider appropriate regulatory application (e.g., priority setting, testing strategies or risk assessment). It draws upon the evidence assembled for each KER as one of several components which contribute to relative confidence in supporting information for the entire hypothesised pathway. An important component in assessing confidence in supporting information as a basis to consider regulatory application of AOPs beyond that described in Section 6 is the essentiality of each of the key events as a component of the entire pathway. This is normally investigated in specifically-designed stop/reversibility studies or knockout models (i.e., those where a key event can be blocked or prevented). Assessment of the overall AOP also contributes to the identification of KEs for which confidence in the quantitative relationship with the AO is greatest (i.e., to facilitate determining the most sensitive predictor of the AO). Instructions To edit the “Overall Assessment 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 “Overall Assessment 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 “Overall Assessment of the AOP” section on the AOP page.

Domain of Applicability

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  • Sex: The AOP applies to females only. Males have relatively low gonadal aromatase expression and activity and the androgen 11-KT, rather than the estrogen E2 is a stronger driver of reproductive functions in males. That said, at least in fish, there is a potential autocrine and paracrine for estrogens synthesized in the brain in regulating reproductive behaviors. However, those potential effects are addressed through an alternative AOP that shares the MIE of aromatase inhibition.
  • 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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Support for the essentiality of a number of key events in the AOP was provided by several time-course, stop-reversibility, experiments with fathead minnows exposed to aromatase inhibitors.

1. Villeneuve et al. 2009 and 2013 examined a time-course of key event responses to fadrozole as well as the time-course of recovery following cessation of fadrozole delivery. Once fadrozole was removed from the system, ex vivo E2 production increased, followed by increases in plasma E2 concentrations, and then increases in plasma vitellogenin concentrations. Additionally, while exposure to the chemical was on-going, compensatory up-regulation of CYP19a1a gene expression resulted in increases in ex vivo E2 production, followed by increased plasma E2 and plasma VTG. The essentiality of aromatase inhibition relative to impaired E2 production was further supported by the observation of an "overshoot" in E2 production, relative to controls, shortly after cessation of fadrozole delivery.

2. Similar support was provided in a study by Ankley et al. (2009a). Cessation of prochloraz delivery resulted in rapid recovery of ex vivo E2 production and plasma E2 concentrations, with recovery of vitellogenin concentrations lagging slightly behind. Increased expression of cyp19a1a mRNA during the exposure period aligned with increased ex vivo E2 production, and increased plasma E2, compared to the first day of exposure.


Rationale for essentiality calls:

• Aromatase, inhibition: [Strong] There is good evidence from stop/reversibility studies that ceasing delivery of the aromatase inhibitor leads to recovery of the subsequent key events.

• 17beta-estradiol synthesis by ovarian granulosa cells, reduction: [Strong] In both exposure studies and stop/reversibility studies, when ex vivo E2 production (as measure of this KE) recovers either through compensation or due to removal of the stressor, subsequent KEs have been shown to recover after a lag period.

• plasma 17beta-estradiol concentrations, reduction: [Strong] In both exposure studies and stop/reversibility studies, when plasma E2 concentrations recover either through compensation or due to removal of the stressor, subsequent KEs have been shown to recover after a lag period.

• vitellogenin production in liver (transcription, translation), reduction: [Moderate] This endpoint was not specifically examined in stop/reversibility studies with aromatase inhibitors, but biological plausibility provides strong support for the essentiality of this event.

• plasma vitellogenin concentrations, reduction: [Strong] Shown to recover in a predictable fashion consistent with the order of events in the AOP in stop/recovery studies.

• vitellogenin accumulation into oocytes and oocyte growth/development, reduction: [Weak] Some contradictory evidence regarding the essentiality of this event. No stop/reversibility studies have explicitly considered this key event.

• cumulative fecundity and spawning, reductions: [Moderate] By definition, some degree of spawning is required to maintain population.


Weight of Evidence Summary

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Biological plausibility: Biological plausibility refers to the structural or functional relationship between the key events based on our fundamental understanding of "normal biology". In general, the biological plausibility and coherence linking aromatase inhibition through decreases in circulating concentrations of E2 is very solid. The biochemistry of steroidogenesis and the predominant role of the gonad in synthesis of the sex steroids is well established. Similarly, the role of E2 as the major regulator of hepatic vitellogenin production is widely documented in the literature. The direct link between reduced VTG concentrations in the plasma and reduced uptake into oocytes is highly plausible, as the plasma is the primary source of the VTG. However, the direct connection between reduced VTG uptake and impaired spawning/reduced cumulative fecundity is more tentative. It is not clear, for instance whether impaired VTG uptake limits oocyte growth and failure to reach a critical size in turn impairs physical or inter-cellular signaling processes that promote release of the oocyte from the surrounding follicles. In at least one experiment, oocytes with similar size to vitellogenic oocytes, but lacking histological staining characteristic of vitellogenic oocytes was observed (R. Johnson, personal communication). Regulation of oocyte maturation and spawning involves many factors other than vitellogenin accumulation (Clelland and Peng, 2009). At present, the link between reductions in circulating VTG concentrations and reduced cumulative fecundity are best supported by the correlation between those endpoints across multiple experiments, including those that impact VTG via other molecular initiating events (Miller et al. 2007).

 

Concordance of dose-response relationships: Dose response concordance considers the degree to which upstream events are shown to occur at test concentrations equal to or lower than those that cause significant effects on downstream key events, the underlying assumption being that all KEs can be measured with equal precision. There are a limited number of studies in which multiple key events were considered in the same study. These were considered the most useful for evaluating the concordance of dose-response relationships. In general, effects on downstream key events occurred at concentrations equal to or greater than those at which upstream events occurred (Concordance table: [1]). However, there are exceptions. There are cases where no significant effects on estradiol synthesis by ovarian granulosa cells (ovary explants) were observed, but significant effects on plasma E2 or VTG concentrations were observed. Likewise, there are cases where impacts on plasma VTG were observed at concentrations lower than those reported to reduce plasma E2 concentrations. Based on knowledge of the studies in question, the apparent lack of concordance in some cases is driven by two primary factors. First, differences in the sensitivity and dynamic range of the measurements being made. Second, the effects of compensatory responses along the HPG axis. For instance, although ex vivo E2 production is rapidly affected by exposure to fadrozole, it is also a response that is more rapidly corrected through upregulation of aromatase transcripts (see Villeneuve et al. 2009), meaning that it recovers more quickly than plasma concentrations of E2 or plasma VTG concentrations. Thus, at certain time points, one can get an apparent effect on plasma E2 or T without a measurable impact on E2 production by the gonad tissue, because the upstream insult occurred earlier in time and was subsequently offset by a compensatory response, but the compensation has yet to propagate through the pathway. Sensitivity and dynamic range of the measurement methods is also an issue. Vitellogenin concentrations have a highly dynamic range and can change by orders of magnitude. Other endpoints like plasma steroids are regulated in a narrower range, making differences more difficult to distinguish statistically. Therefore, in our assessment, the deviations from concordance do not call the KERs into question.

 

The concentration-dependence of the key event responses with regard to the concentration of aromatase inhibitor has been established in vitro and/or in vivo for nearly all key events in the AOP.

  1. Concentration-dependent aromatase inhibition: (Villeneuve et al. 2006; Ankley et al. 2005; M et al. 2004; AM et al. 2000; Shilling et al. 1999)
  2. Concentration-dependent decreases in E2 production in vitro, ex vivo: (Ankley et al. 2002; Villeneuve et al. 2007; Villeneuve et al. 2009; Ankley et al. 2005; a Marca Pereira et al. 2011; Lee et al. 2006).
  3. Concentration-dependent decreases in circulating E2 concentrations: (Ankley et al. 2002; Villeneuve et al. 2009; Ankley et al. 2005; Ankley et al. 2009a; GT et al. 2001)
  4. Concentration-dependent decreases in vitellogenin mRNA expression: (Sun et al. 2010; Sun et al. 2011; Zhang et al. 2008)
  5. Concentration-dependent decreases in circulating vitellogenin concentrations: (Ankley et al. 2002; Villeneuve et al. 2009; Ankley et al. 2005; Ankley et al. 2009a; Sun et al. 2007; GT et al. 2001; Ralston-Hooper et al. 2013)
  6. Concentration-dependent reductions in VTG uptake into oocytes or impaired oocyte development: Concentration-dependence of these effects has not been well demonstrated. The effects, when seen, have typically been documented at the greatest exposure concentration tested, but concentration-dependence of the severity or frequency of the impact was not documented (e.g., (Ankley et al. 2002; Ankley et al. 2005; Sun et al. 2007)
  7. Concentration-dependent reductions in cumulative fecundity: (Ankley et al. 2002; Ankley et al. 2005; Sun et al. 2007; Zhang et al. 2008)
  8. Declining population trajectory: Modeled population trajectories show a concentration-dependent reduction in projected population size, however, those results are driven by the concentration-dependence of cumulative fecundity. Population-level effects have not been measured directly.


Temporal concordance: Temporal concordance refers to the degree to which the data support the hypothesized sequence of the key events; i.e., the effect on KE1 is observed before the effect on KE2, which is observed before the effect on KE3 and so on. Temporal concordance of the AOP from aromatase inhibition to decreased E2 production, decreased circulating E2, and decreased plasma VTG concentrations has been established (e.g., (Villeneuve et al. 2009; Ankley et al. 2009a; Skolness et al. 2011). Temporal concordance has not been established beyond that key event, in large part due to disconnect in the time-scales over which the events can be measured. For example, most small fish used in reproductive toxicity testing will can spawn anywhere from once daily to several days per week. Given the variability in daily spawning rates, it is neither practical nor effective to evaluate cumulative fecundity at a time scale shorter than roughly a week. Since the impacts at lower levels of biological organization can be detected within hours of exposure, lack of impact on cumulative fecundity before the other key events are impacted cannot be effectively measured. Overall, among those key events whose temporal concordance can reasonably be evaluated, the temporal profile observed is consistent with the AOP.

Consistency: We are aware of no cases where the pattern of key events described was observed without also observing a significant impact on cumulative fecundity. The final adverse outcome is not specific to this AOP. Many of the key events included in this AOP overlap with AOPs linking other molecular initiating events to reproductive dysfunction in small fish.

Uncertainties, inconsistencies, and data gaps: The current major uncertainty in this AOP is whether there is a direct biological linkage 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.


Quantitative Considerations

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Assessment of quantitative understanding of the AOP:

At present, quantitative understanding of the AOP is approaching the point where an in vitro measurement of aromatase inhibition could be used as an input parameter into a series of coupled computational models that could generate quantitative predictions across multiple key events (e.g., circulating E2 concentrations, circulating VTG concentrations, predicted impacts on cumulative fecundity, and effects on population trajectories). A sequence of supporting models has been coupled together and predictions have been made for novel aromatase inhibitors (identified through high throughput in vitro screening), but those predictions have not yet been validated experimentally. The present models are also unable to account for pharmacokinetic considerations (e.g., adsorption, distribution, metabolism/biotransformation, and elimination) and have demonstrated only partial success in simulating compensatory/feedback responses to aromatase inhibition (e.g., (Breen et al. 2013).


Considerations for Potential Applications of the AOP (optional)

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  • The present AOP can provide potential support for the use of alternatives to the fish short term reproduction assay as a screen for aromatase inhibitors.
  • The present AOP can serve as a foundation for tiered testing strategies and IATA related to risk assessments on chemicals identified as aromatase inhibitors.
  • The present AOP can be used to guide endpoint selection for effects-based monitoring studies at sites where aromatase inhibition has been identified as a relevant biological activity of interest (e.g., through bioeffects prediction or bioeffects surveillance approaches; see Schroeder et al. 2016).

Schroeder, A. L., Ankley, G. T., Houck, K. A. and Villeneuve, D. L. (2016), Environmental surveillance and monitoring—The next frontiers for high-throughput toxicology. Environ Toxicol Chem, 35: 513–525. doi:10.1002/etc.3309

  • A series of computational models aligned with this AOP (i.e., a quantitative AOP construct) can be applied to estimate in vivo bench-mark doses based on in vitro screening results. Case studies evaluating this application are under way.

References

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1. OECD. 2012. Test No. 229: Fish Short Term Reproduction Assay. Paris, France:Organization for Economic Cooperation and Development.

2. Petkov PI, Temelkov S, Villeneuve DL, Ankley GT, Mekenyan OG. 2009. Mechanism-based categorization of aromatase inhibitors: a potential discovery and screening tool. SAR QSAR Environ Res 20(7-8): 657-678.

3. Lephart ED, Simpson ER. 1991. Assay of aromatase activity. Methods Enzymol 206: 477-483.

4. Letcher RJ, van Holsteijn I, Drenth H-J, Norstrom RJ, Bergman A, Safe S, et al. 1999. Cytotoxicity and aromatase (CYP19) activity modulation by organochlorines in human placental JEG-3 and JAR choriocarcinoma cells. Toxicology and applied pharmacology 160: 10-20.

5. Sanderson J, Seinen W, Giesy J, van den Berg M. 2000. 2-chloro-triazine herbicides induce aromatase (CYP19) activity in H295R human adrenocortical carcinoma cells: a novel mechanism for estrogenicity. Toxicological Sciences 54: 121-127.

6. Villeneuve DL, Knoebl I, Kahl MD, Jensen KM, Hammermeister DE, Greene KJ, et al. 2006. Relationship between brain and ovary aromatase activity and isoform-specific aromatase mRNA expression in the fathead minnow (Pimephales promelas). Aquat Toxicol 76(3-4): 353-368.

7. Ankley GT, Kahl MD, Jensen KM, Hornung MW, Korte JJ, Makynen EA, et al. 2002. Evaluation of the aromatase inhibitor fadrozole in a short-term reproduction assay with the fathead minnow (Pimephales promelas). Toxicological Sciences 67: 121-130.

8. Castro LF, Santos MM, Reis-Henriques MA. 2005. The genomic environment around the Aromatase gene: evolutionary insights. BMC evolutionary biology 5: 43.

9. Norris DO. 2007. Vertebrate Endocrinology. Fourth ed. New York: Academic Press.

10. Yaron Z. 1995. Endocrine control of gametogenesis and spawning induction in the carp. Aquaculture 129: 49-73.

11. Havelock JC, Rainey WE, Carr BR. 2004. Ovarian granulosa cell lines. Molecular and cellular endocrinology 228(1-2): 67-78.

12. Villeneuve DL, Ankley GT, Makynen EA, Blake LS, Greene KJ, Higley EB, et al. 2007. Comparison of fathead minnow ovary explant and H295R cell-based steroidogenesis assays for identifying endocrine-active chemicals. Ecotoxicol Environ Saf 68(1): 20-32.

13. McMaster ME MK, Jardine JJ, Robinson RD, Van Der Kraak GJ. 1995. Protocol for measuring in vitro steroid production by fish gonadal tissue. Canadian Technical Report of Fisheries and Aquatic Sciences 1961 1961: 1-78.

14. Ankley GT, Jensen KM, Kahl MD, Makynen EA, Blake LS, Greene KJ, et al. 2007. Ketoconazole in the fathead minnow (Pimephales promelas): reproductive toxicity and biological compensation. Environ Toxicol Chem 26(6): 1214-1223.

15. Villeneuve DL, Mueller ND, Martinovic D, Makynen EA, Kahl MD, Jensen KM, et al. 2009. Direct effects, compensation, and recovery in female fathead minnows exposed to a model aromatase inhibitor. Environ Health Perspect 117(4): 624-631.

16. Baker ME. 2011. Origin and diversification of steroids: co-evolution of enzymes and nuclear receptors. Molecular and cellular endocrinology 334(1-2): 14-20.

17. Jensen K, Korte J, Kahl M, Pasha M, Ankley G. 2001. Aspects of basic reproductive biology and endocrinology in the fathead minnow (Pimephales promelas). Comparative Biochemistry and Physiology Part C 128: 127-141.

18. Biales AD, Bencic DC, Lazorchak JL, Lattier DL. 2007. A quantitative real-time polymerase chain reaction method for the analysis of vitellogenin transcripts in model and nonmodel fish species. Environ Toxicol Chem 26(12): 2679-2686.

19. Schmieder P, Tapper M, Linnum A, Denny J, Kolanczyk R, Johnson R. 2000. Optimization of a precision-cut trout liver tissue slice assay as a screen for vitellogenin induction: comparison of slice incubation techniques. Aquat Toxicol 49(4): 251-268.

20. Navas JM, Segner H. 2006. Vitellogenin synthesis in primary cultures of fish liver cells as endpoint for in vitro screening of the (anti)estrogenic activity of chemical substances. Aquat Toxicol 80(1): 1-22.

21. Korte JJ, Kahl MD, Jensen KM, Mumtaz SP, Parks LG, LeBlanc GA, et al. 2000. Fathead minnow vitellogenin: complementary DNA sequence and messenger RNA and protein expression after 17B-estradiol treatment. Environmental Toxicology and Chemistry 19(4): 972-981.

22. Tyler C, van der Eerden B, Jobling S, Panter G, Sumpter J. 1996. Measurement of vitellogenin, a biomarker for exposure to oestrogenic chemicals, in a wide variety of cyprinid fish. Journal of Comparative Physiology and Biology 166: 418-426.

23. Tyler C, Sumpter J. 1996. Oocyte growth and development in teleosts. Reviews in Fish Biology and Fisheries 6: 287-318.

24. Leino R, Jensen K, Ankley G. 2005. Gonadal histology and characteristic histopathology associated with endocrine disruption in the adult fathead minnow. Environmental Toxicology and Pharmacology 19: 85-98.

25. Wolf JC, Dietrich DR, Friederich U, Caunter J, Brown AR. 2004. Qualitative and quantitative histomorphologic assessment of fathead minnow Pimephales promelas gonads as an endpoint for evaluating endocrine-active compounds: a pilot methodology study. Toxicol Pathol 32(5): 600-612.

26. Miller DH, Ankley GT. 2004. Modeling impacts on populations: fathead minnow (Pimephales promelas) exposure to the endocrine disruptor 17b-trenbolone as a case study. Ecotoxicology and Environmental Safety 59: 1-9.

27. Ankley GT, Jensen KM, Durhan EJ, Makynen EA, Butterworth BC, Kahl MD, et al. 2005. Effects of two fungicides with multiple modes of action on reproductive endocrine function in the fathead minnow (Pimephales promelas). Toxicol Sci 86(2): 300-308.

28. Ankley GT, Bencic D, Cavallin JE, Jensen KM, Kahl MD, Makynen EA, et al. 2009a. Dynamic nature of alterations in the endocrine system of fathead minnows exposed to the fungicide prochloraz. Toxicol Sci 112(2): 344-353.

29. Skolness SY, Durhan EJ, Garcia-Reyero N, Jensen KM, Kahl MD, Makynen EA, et al. 2011. Effects of a short-term exposure to the fungicide prochloraz on endocrine function and gene expression in female fathead minnows (Pimephales promelas). Aquat Toxicol 103(3-4): 170-178.

30. Breen M, Villeneuve DL, Ankley GT, Bencic DC, Breen MS, Watanabe KH, et al. 2013. Developing Predictive Approaches to Characterize Adaptive Responses of the Reproductive Endocrine Axis to Aromatase Inhibition: II. Computational Modeling. Toxicological sciences : an official journal of the Society of Toxicology.

31. Breen MS, Villeneuve DL, Breen M, Ankley GT, Conolly RB. 2007. Mechanistic computational model of ovarian steroidogenesis to predict biochemical responses to endocrine active compounds. Annals of biomedical engineering 35(6): 970-981.

32. Shoemaker JE, Gayen K, Garcia-Reyero N, Perkins EJ, Villeneuve DL, Liu L, et al. 2010. Fathead minnow steroidogenesis: in silico analyses reveals tradeoffs between nominal target efficacy and robustness to cross-talk. BMC systems biology 4: 89.

33. Quignot N, Bois FY. 2013. A computational model to predict rat ovarian steroid secretion from in vitro experiments with endocrine disruptors. PloS one 8(1): e53891.

34. Ankley GT, Bencic DC, Cavallin JE, Jensen KM, Kahl MD, Makynen EA, et al. 2009b. Dynamic nature of alterations in the endocrine system of fathead minnows exposed to the fungicide prochloraz. Toxicological sciences : an official journal of the Society of Toxicology 112(2): 344-353.

35. Villeneuve DL, Breen M, Bencic DC, Cavallin JE, Jensen KM, Makynen EA, et al. 2013. Developing Predictive Approaches to Characterize Adaptive Responses of the Reproductive Endocrine Axis to Aromatase Inhibition: I. Data Generation in a Small Fish Model. Toxicological sciences : an official journal of the Society of Toxicology.

36. Ankley GT, Cavallin JE, Durhan EJ, Jensen KM, Kahl MD, Makynen EA, et al. 2012. A time-course analysis of effects of the steroidogenesis inhibitor ketoconazole on components of the hypothalamic-pituitary-gonadal axis of fathead minnows. Aquatic toxicology 114-115: 88-95.

37. Li Z, Kroll KJ, Jensen KM, Villeneuve DL, Ankley GT, Brian JV, et al. 2011a. A computational model of the hypothalamic: pituitary: gonadal axis in female fathead minnows (Pimephales promelas) exposed to 17alpha-ethynylestradiol and 17beta-trenbolone. BMC systems biology 5: 63.

38. A A, A G. 2003. Eggshell and egg yolk proteins in fish: hepatic proteins for the next generation: oogenetic, population, and evolutionary implications of endocrine disruption. Comparative Hepatology 2(4): 1-21.

39. Sun L, Wen L, Shao X, Qian H, Jin Y, Liu W, et al. 2010. Screening of chemicals with anti-estrogenic activity using in vitro and in vivo vitellogenin induction responses in zebrafish (Danio rerio). Chemosphere 78(7): 793-799.

40. Iguchi T, Irie F, Urushitani H, Tooi O, Kawashima Y, Roberts M, et al. 2006. Availability of in vitro vitellogenin assay for screening of estrogenic and anti-estrogenic activities of environmental chemicals. Environ Sci 13(3): 161-183.

41. Murphy CA, Rose KA, Thomas P. 2005. Modeling vitellogenesis in female fish exposed to environmental stressors: predicting the effects of endocrine disturbance due to exposure to a PCB mixture and cadmium. Reproductive toxicology 19(3): 395-409.

42. Murphy CA, Rose KA, Rahman MS, Thomas P. 2009. Testing and applying a fish vitellogenesis model to evaluate laboratory and field biomarkers of endocrine disruption in Atlantic croaker (Micropogonias undulatus) exposed to hypoxia. Environmental toxicology and chemistry / SETAC 28(6): 1288-1303.

43. Ankley GT, Miller DH, Jensen KM, Villeneuve DL, Martinovic D. 2008. Relationship of plasma sex steroid concentrations in female fathead minnows to reproductive success and population status. Aquatic toxicology 88(1): 69-74.

44. Schmid T, Gonzalez-Valero J, Rufli H, Dietrich DR. 2002. Determination of vitellogenin kinetics in male fathead minnows (Pimephales promelas). Toxicol Lett 131(1-2): 65-74.

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