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Key Event Relationship Overview

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Description of Relationship

Upstream Event Downstream Event/Outcome
17beta-estradiol synthesis by ovarian granulosa cells, Reduction Plasma 17beta-estradiol concentrations, Reduction

AOPs Referencing Relationship

AOP Name Type of Relationship Weight of Evidence Quantitative Understanding
Aromatase (Cyp19a1) reduction leading to impaired fertility in adult female Directly Leads to Strong
Aromatase inhibition leading to reproductive dysfunction Directly Leads to Strong Moderate
Androgen receptor agonism leading to reproductive dysfunction Directly Leads to Strong Weak
Prolyl hydroxylase inhibition leading to reproductive dysfunction via increased HIF1 heterodimer formation Directly Leads to Strong Moderate
Unknown MIE leading to reproductive dysfunction via increased HIF-1alpha transcription Directly Leads to

Taxonomic Applicability

Name Scientific Name Evidence Links
human Homo sapiens NCBI
mouse Mus musculus Moderate NCBI
rat Rattus sp. Strong NCBI

How Does This Key Event Relationship Work

Biological Plausibility

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.

Weight of Evidence

Empirical Support for Linkage

Include consideration of temporal concordance here


  • 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).


  • 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
Study type
E2 production/levels
Phthalates (DEHP)
ex vivo
1500 mg/kg/day
Reduced/increased E2 production in ovary culture
(Laskey & Berman, 1993)
Phthalates (MEHP)
in vitro
From 50 µM
Reduced E2 production (concentration and time dependent in Granulosa cell)
(Davis, Weaver, Gaines, & Heindel, 1994)
Phthalates (MEHP)
in vitro
reduction E2 production (dose dependent)
(Lovekamp & Davis, 2001)
Phthalates (DEHP)
in vivo
300-600 mg/kg/day
reduction E2 levels dose dependent
(Xu et al., 2010),
Phthalates (MEHP)
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)
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 or Inconsistencies

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

Quantitative Understanding of the Linkage

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.

Evidence Supporting Taxonomic Applicability

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.



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