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Relationship: 5

Title

A descriptive phrase which clearly defines the two KEs being considered and the sequential relationship between them (i.e., which is upstream, and which is downstream). More help

Reduction, 17beta-estradiol synthesis by ovarian granulosa cells leads to Reduction, Plasma 17beta-estradiol concentrations

Upstream event

The causing Key Event (KE) in a Key Event Relationship (KER). More help

Reduction, 17beta-estradiol synthesis by ovarian granulosa cells

Downstream event

The responding Key Event (KE) in a Key Event Relationship (KER). More help

Reduction, Plasma 17beta-estradiol concentrations

Key Event Relationship Overview

The utility of AOPs for regulatory application is defined, to a large extent, by the confidence and precision with which they facilitate extrapolation of data measured at low levels of biological organisation to predicted outcomes at higher levels of organisation and the extent to which they can link biological effect measurements to their specific causes.Within the AOP framework, the predictive relationships that facilitate extrapolation are represented by the KERs. Consequently, the overall WoE for an AOP is a reflection in part, of the level of confidence in the underlying series of KERs it encompasses. Therefore, describing the KERs in an AOP involves assembling and organising the types of information and evidence that defines the scientific basis for inferring the probable change in, or state of, a downstream KE from the known or measured state of an upstream KE. More help

AOPs Referencing Relationship

AOP Name	Adjacency	Weight of Evidence	Quantitative Understanding	Point of Contact	Author Status	OECD Status
Aromatase (Cyp19a1) reduction leading to impaired fertility in adult female	adjacent	High		Elise Grignard (send email)	Open for citation & comment	Under Review
Aromatase inhibition leading to reproductive dysfunction	adjacent	High	Moderate	Dan Villeneuve (send email)	Open for citation & comment	WPHA/WNT Endorsed
Androgen receptor agonism leading to reproductive dysfunction (in repeat-spawning fish)	adjacent	High	Low	Dan Villeneuve (send email)	Open for citation & comment	WPHA/WNT Endorsed
Prolyl hydroxylase inhibition leading to reproductive dysfunction via increased HIF1 heterodimer formation	adjacent	High	Moderate	Dalma Martinovic-Weigelt (send email)	Under Development: Contributions and Comments Welcome
Unknown MIE leading to reproductive dysfunction via increased HIF-1alpha transcription	adjacent			Dalma Martinovic-Weigelt (send email)	Under Development: Contributions and Comments Welcome
Embryonic Activation of the AHR leading to Reproductive failure, via epigenetic down-regulation of GnRHR	adjacent	High	Moderate	Jon Doering (send email)	Under development: Not open for comment. Do not cite

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) that help to define the biological applicability domain of the KER.In general, this will be dictated by the more restrictive of the two KEs being linked together by the KER. More help

Term	Scientific Term	Evidence	Link
human	Homo sapiens		NCBI
mouse	Mus musculus	Moderate	NCBI
rat	Rattus norvegicus	High	NCBI
fathead minnow	Pimephales promelas	Moderate	NCBI
Fundulus heteroclitus	Fundulus heteroclitus	High	NCBI

Sex Applicability

An indication of the the relevant sex for this KER. More help

Sex	Evidence
Female	High

Life Stage Applicability

An indication of the the relevant life stage(s) for this KER. More help

Term	Evidence
Adult, reproductively mature	High

Key Event Relationship Description

Provides a concise overview of the information given below as well as addressing details that aren’t inherent in the description of the KEs themselves. More help

See plausibility, below.

Evidence Collection Strategy

Include a description of the approach for identification and assembly of the evidence base for the KER. For evidence identification, include, for example, a description of the sources and dates of information consulted including expert knowledge, databases searched and associated search terms/strings. Include also a description of study screening criteria and methodology, study quality assessment considerations, the data extraction strategy and links to any repositories/databases of relevant references.Tabular summaries and links to relevant supporting documentation are encouraged, wherever possible. More help

Evidence Supporting this KER

Addresses the scientific evidence supporting KERs in an AOP setting the stage for overall assessment of the AOP. More help

Updated 03/20/2017.

Biological Plausibility

Addresses the biological rationale for a connection between KEupstream and KEdownstream. This field can also incorporate additional mechanistic details that help inform the relationship between KEs, this is useful when it is not practical/pragmatic to represent these details as separate KEs due to the difficulty or relative infrequency with which it is likely to be measured. More help

While brain, interrenal, adipose, and breast tissue (in mammals) are capable of synthesizing estradiol, the gonads are generally considered the major source of circulating estrogens in vertebrates, including fish (Norris 2007). Consequently, if estradiol synthesis by ovarian granulosa cells is reduced, plasma E2 concentrations would be expected to decrease unless there are concurrent reductions in the rate of E2 catabolism. Synthesis in other tissues generally plays a paracrine role only, thus the contribution of other tissues to plasma E2 concentrations can generally be considered negligible.

Empirical Evidence

Provides specific (citable) evidence that supports the idea of a change in the upstream KE (KEupstream) leading to, or being associated with, a subsequent change in the downstream KE (KEdownstream), assuming the perturbation of KEupstream is sufficient. More help

Include consideration of temporal concordance here

Fish

In multiple studies with aromatase inhibitors (e.g., fadrozole, prochloraz), significant reductions in ex vivo E2 production have been linked to, and shown to precede, reductions in circulating E2 concentrations (Villeneuve et al. 2009; Skolness et al. 2011). It is also notable that compensatory responses at the level of ex vivo steroid production (i.e., rate of E2 synthesis per unit mass of tissue) tend to precede recovery of plasma E2 concentrations following an initial insult (Villeneuve et al. 2009; Ankley et al. 2009a; Villeneuve et al. 2013).
Ex vivo E2 production by ovary tissue collected from female fish exposed to 30 or 300 μg ketoconazole/L showed significant decreases prior to significant effects on plasma estradiol being observed (Ankley et al. 2012).
Ekman et al. (2011) reported significant reductions in ex vivo E2 production and plasma E2 concentrations in female fathead minnows exposed to 0.05 ug/L 17ß-trenbolone. The effect on plasma E2 was observed at an earlier time point (24 h, versus 48 h for E2 production.
Rutherford et al. (2015) reported significant reductions in both E2 production and circulating E2 concentrations in female Fundulus heteroclitus exposed to 5alpha-dihydrotestosterone or 17alpha-methyltestosterone for 14 d. The effects were equipotent in the case of 17alpha-methyltestosterone, but in the case of 5alpha-dihyrotestosterone, the effect on plasma E2 could be detected at a lower dose (10 ug/L) than that at which a significant effect on E2 production was detected (100 ug/L).
In female Fundulus heteroclitus exposed to 17alpha-methyltestosterone for 7 or 14 d, both E2 production and plasma E2 were impacted at the same exposure concentrations (Sharpe et al. 2004).

Mammals

MEHP /DEHP, mice, ex vivo DEHP (10 -100 μg/ml); MEHP (0.1 and 10 μg/ml) dose dependent reduction E2 production (Gupta et al., 2010)
DEHP, rat, in vivo 300-600 mg/kg/day, dose dependent reduction of E2 plasma levels (Xu et al., 2010)

Evidence for rodent and human models is summarized in Table 1.

Compound class	Species	Study type	Dose	E2 production/levels	Reference
Phthalates (DEHP)	rat	ex vivo	1500 mg/kg/day	Reduced/increased E2 production in ovary culture	(Laskey & Berman, 1993)
Phthalates (MEHP)	rat	in vitro	From 50 µM	Reduced E2 production (concentration and time dependent in Granulosa cell)	(Davis, Weaver, Gaines, & Heindel, 1994)
Phthalates (MEHP)	rat	in vitro	100-200µM	reduction E2 production (dose dependent)	(Lovekamp & Davis, 2001)
Phthalates (DEHP)	rat	in vivo	300-600 mg/kg/day	reduction E2 levels dose dependent	(Xu et al., 2010),
Phthalates (MEHP)	human	in vitro	IC(50)= 49- 138 µM (dependent on the stimulant)	reduction E2 production (dose dependent)	(Reinsberg, Wegener-Toper, van der Ven, van der Ven, & Klingmueller, 2009)
Phthalates (MEHP/DEHP)	mice	ex vivo	DEHP (10 -100 μg/ml); MEHP (0.1 and 10 μg/ml)	reduction E2 production (dose dependent)	(Gupta et al., 2010)

Table 1. Summary of the experimental data for decrease E2 production and decreased E2 levels. IC50- half maximal inhibitory concentration values reported if available, otherwise the concentration at which the effect was observed.

Uncertainties and Inconsistencies

Addresses inconsistencies or uncertainties in the relationship including the identification of experimental details that may explain apparent deviations from the expected patterns of concordance. More help

Based on the limited set of studies available to date, there are no known inconsistencies.

Known modulating factors

This table captures specific information on the MF, its properties, how it affects the KER and respective references.1.) What is the modulating factor? Name the factor for which solid evidence exists that it influences this KER. Examples: age, sex, genotype, diet 2.) Details of this modulating factor. Specify which features of this MF are relevant for this KER. Examples: a specific age range or a specific biological age (defined by...); a specific gene mutation or variant, a specific nutrient (deficit or surplus); a sex-specific homone; a certain threshold value (e.g. serum levels of a chemical above...) 3.) Description of how this modulating factor affects this KER. Describe the provable modification of the KER (also quantitatively, if known). Examples: increase or decrease of the magnitude of effect (by a factor of...); change of the time-course of the effect (onset delay by...); alteration of the probability of the effect; increase or decrease of the sensitivity of the downstream effect (by a factor of...) 4.) Provision of supporting scientific evidence for an effect of this MF on this KER. Give a list of references. More help

Quantitative Understanding of the Linkage

Captures information that helps to define how much change in the upstream KE, and/or for how long, is needed to elicit a detectable and defined change in the downstream KE. More help

At present we are unaware of any well established quantitative relationships between ex vivo E2 production (as an indirect measure of granulosa cell E2 synthesis) and plasma E2 concentrations.

There are considerable data available which might support the development of such a relationship. Additionally, there are a number of existing mathematical/computational models of ovarian steroidogenesis (Breen et al. 2013; Shoemaker et al. 2010) and/or physiologically-based pharmacokinetic models of the hypothalamic-pituitary-gonadal axis (e.g., (Li et al. 2011a) that may be adaptable to support a quantitative understanding of this linkage.

• The Breen et al. 2013 model was developed based on in vivo time-course data for fathead minnow and incorporates prediction of compensatory responses resulting from feedback mechanisms operating as part of the hypothalamic-pituitary-gonadal axis.

• The Shoemaker et al. 2010 model is chimeric and includes signaling pathways and aspects of transcriptional regulation based on a mixture of fish-specific and mammalian sources.

• The Li et al. 2011 model is a PBPK-based model that was calibrated from data from fathead minnows, including controls and fish exposed to either 17alpha ethynylestradiol or 17beta trenbolone.

Response-response Relationship

Provides sources of data that define the response-response relationships between the KEs. More help

Time-scale

Information regarding the approximate time-scale of the changes in KEdownstream relative to changes in KEupstream (i.e., do effects on KEdownstream lag those on KEupstream by seconds, minutes, hours, or days?). More help

Known Feedforward/Feedback loops influencing this KER

Define whether there are known positive or negative feedback mechanisms involved and what is understood about their time-course and homeostatic limits. More help

Domain of Applicability

A free-text section of the KER description that the developers can use to explain their rationale for the taxonomic, life stage, or sex applicability structured terms. More help

Key enzymes needed to synthesize 17β-estradiol first appear in the common ancestor of amphioxus and vertebrates (Baker 2011). While some E2 synthesis can occur in other tissues, the ovary is recognized as the major source of 17β-estradiol synthesis in female vertebrates. Endocrine actions of ovarian E2 are facilitated through transport via the plasma. Consequently, this key event relationship is applicable to most female vertebrates.

References

List of the literature that was cited for this KER description. More help

Ankley GT, Bencic DC, 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. Toxicological sciences : an official journal of the Society of Toxicology 112(2): 344-353.
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.
Baker ME. 2011. Origin and diversification of steroids: co-evolution of enzymes and nuclear receptors. Molecular and cellular endocrinology 334(1-2): 14-20.

Davis, B J, R Weaver, L J Gaines, and J J Heindel. 1994. “Mono-(2-Ethylhexyl) Phthalate Suppresses Estradiol Production Independent of FSH-cAMP Stimulation in Rat Granulosa Cells.” Toxicology and Applied Pharmacology 128 (2) (October): 224–8. doi:10.1006/taap.1994.1201.

Ekman DR, Villeneuve DL, Teng Q, Ralston-Hooper KJ, Martinović-Weigelt D, Kahl MD, Jensen KM, Durhan EJ, Makynen EA, Ankley GT, Collette TW. Use of gene expression, biochemical and metabolite profiles to enhance exposure and effects assessment of the model androgen 17β-trenbolone in fish. Environ Toxicol Chem. 2011 Feb;30(2):319-29. doi: 10.1002/etc.406.

Gupta, Rupesh K, Jeffery M Singh, Tracie C Leslie, Sharon Meachum, Jodi a Flaws, and Humphrey H-C Yao. 2010. “Di-(2-Ethylhexyl) Phthalate and Mono-(2-Ethylhexyl) Phthalate Inhibit Growth and Reduce Estradiol Levels of Antral Follicles in Vitro.” Toxicology and Applied Pharmacology 242 (2) (January 15): 224–30. doi:10.1016/j.taap.2009.10.011.

Laskey, J.W., and E. Berman. 1993. “Steroidogenic Assessment Using Ovary Culture in Cycling Rats: Effects of Bis (2-Diethylhexyl) Phthalate on Ovarian Steroid Production.” Reproductive Toxicology 7 (1) (January): 25–33. doi:10.1016/0890-6238(93)90006-S.

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.

Lovekamp, T N, and B J Davis. 2001. “Mono-(2-Ethylhexyl) Phthalate Suppresses Aromatase Transcript Levels and Estradiol Production in Cultured Rat Granulosa Cells.” Toxicology and Applied Pharmacology 172 (3) (May 1): 217–24. doi:10.1006/taap.2001.9156.

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

Reinsberg, Jochen, Petra Wegener-Toper, Katrin van der Ven, Hans van der Ven, and Dietrich Klingmueller. 2009. “Effect of Mono-(2-Ethylhexyl) Phthalate on Steroid Production of Human Granulosa Cells.” Toxicology and Applied Pharmacology 239 (1) (August 15): 116–23. doi:10.1016/j.taap.2009.05.022.

Rutherford R, Lister A, Hewitt LM, MacLatchy D. Effects of model aromatizable (17α-methyltestosterone) and non-aromatizable (5α-dihydrotestosterone) androgens on the adult mummichog (Fundulus heteroclitus) in a short-term reproductive endocrine bioassay. Comp Biochem Physiol C Toxicol Pharmacol. 2015 Apr;170:8-18. doi: 10.1016/j.cbpc.2015.01.004.
Sharpe RL, MacLatchy DL, Courtenay SC, Van Der Kraak GJ. Effects of a model androgen (methyl testosterone) and a model anti-androgen (cyproterone acetate) on reproductive endocrine endpoints in a short-term adult mummichog (Fundulus heteroclitus) bioassay. Aquat Toxicol. 2004 Apr 28;67(3):203-15.
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.
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.
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.
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.

Xu, Chuan, Ji-An Chen, Zhiqun Qiu, Qing Zhao, Jiaohua Luo, Lan Yang, Hui Zeng, et al. 2010. “Ovotoxicity and PPAR-Mediated Aromatase Downregulation in Female Sprague-Dawley Rats Following Combined Oral Exposure to Benzo[a]pyrene and Di-(2-Ethylhexyl) Phthalate.” Toxicology Letters 199 (3) (December 15): 323–32. doi:10.1016/j.toxlet.2010.09.015.