This AOP is licensed under a Creative Commons Attribution 4.0 International License.
Aromatase inhibition leads to male-biased sex ratio via impacts on gonad differentiation
Point of Contact
- Kelvin Santana Rodriguez
- Dan Villeneuve
- Gerald Ankley
- Brendan Ferreri-Hanberry
|Author status||OECD status||OECD project||SAAOP status|
|Under Development: Contributions and Comments Welcome|
This AOP was last modified on July 16, 2022 18:37
|Inhibition, Aromatase||March 14, 2022 08:43|
|Reduction, 17beta-estradiol synthesis by the undifferentiated gonad||July 13, 2022 10:25|
|Increased, Differentiation to Testis||July 13, 2022 10:20|
|Increased, Male Biased Sex Ratio||July 13, 2022 10:15|
|Decrease, Population growth rate||July 08, 2022 07:40|
|Inhibition, Aromatase leads to Increased, Differentiation to Testis||July 11, 2022 15:36|
|Inhibition, Aromatase leads to Reduction, E2 Synthesis by the undifferentiated gonad||July 11, 2022 14:23|
|Inhibition, Aromatase leads to Increased, Male Biased Sex Ratio||July 14, 2022 14:47|
|Reduction, E2 Synthesis by the undifferentiated gonad leads to Increased, Differentiation to Testis||July 11, 2022 15:17|
|Increased, Differentiation to Testis leads to Increased, Male Biased Sex Ratio||July 12, 2022 17:25|
|Increased, Male Biased Sex Ratio leads to Decrease, Population growth rate||July 12, 2022 16:30|
|Fadrozole||November 29, 2016 18:42|
|Letrozole||November 29, 2016 18:42|
|Exemestane||November 12, 2020 01:53|
|Stressor:292 Clotrimazole||November 12, 2020 01:55|
|Prochloraz||November 29, 2016 18:42|
This adverse outcome pathway links inhibition of aromatase activity in teleost fish during gonadogenesis to increased differentiation to testis resulting in a male-biased sex ratio in the population, and ultimately, reduced population sustainability. Most gonochoristic fish species develop either as males or females and do not change sex throughout their life span. However, there is a window of development during gonadal differentiation that can be sensitive to environmental conditions, including exposure to some chemicals. For example, treatment with sex steroids in conjunction with the period of sexual differentiation has been showed to favor ovary or testis development in fish exposed to estrogens or androgens, respectively. Altered synthesis and regulation of endogenous steroids can also affect sexual differentiation in fish. In most vertebrate taxa, aromatase (cytochrome P450 [CYP]19a1) is the rate-limiting enzyme for the conversion of 17β-estradiol (E2) from testosterone (T). Endocrine-active chemicals such as fadrozole, letrozole and exemestane (pharmaceuticals) or prochloraz and propiconazole (fungicides) inhibit aromatase activity. Exposure of some fish species to aromatase inhibitors during sex differentiation can reduce endogenous E2 synthesis, thereby resulting in phenotypic males, the default sex in the absence of estrogen signaling during gonadal differentiation. Given the critical role of female fecundity in determining total numbers of offspring, the resultant male-biased sex ratio can reduce population size, especially if sustained over multiple generations.
AOP Development Strategy
In fish sexual differentiation occurs post hatch and can be influenced by exogenous factors such as chemicals, temperature, pH, population density, social cues and more. As a result, the gonadal sex phenotype in many fish can be altered by environmental conditions experienced during development, particularly in conjunction with sexual differentiation (Scholz and Klüver, 2009). At this stage, the bipotential gonad can differentiate to either testes or ovaries depending both on genetic and environmental factors (Strüssmann and Nakamura, 2002). Sex steroids are among the factors that influence sex differentiation in non-mammalian vertebrates; in many fish species exogenous androgens and estrogens act, respectively, to enhance the development of testes and ovaries in exposed animals (Nakamura 2010). In teleost fish, the relative balance between endogenous estrogens and androgens during sexual differentiation is critical to ensuring normal sex ratios and, ultimately, viable populations. Various homeostatic mechanisms ensure that steroid biosynthesis is appropriately controlled during development. A key biosynthetic enzyme is CYP19a1 (aromatase), which is responsible for the conversion of C19 androgens (e.g., T) to C18 estrogens (e.g., E2) in brain and gonadal tissues of vertebrates (Payne and Hales, 2004; Simpson et al. 1994). In fish, there are two CYP19a1 isoforms, with CYP19a1a mostly expressed in the gonads and CYP19a1b largely expressed in the brain (Callard et al. 2001).
Since the mid-90s, there has been concern about the potential impacts of endocrine disrupting chemicals (EDCs) in fish and wildlife. Many EDCs can exert effects in early life stages that can lead to potential impacts at the population level. For example, some chemicals have been shown to alter the sexual phenotype of fish by affecting steroidogenic enzymes such as aromatase. Inhibition of CYP19a1 expression or activity can alter the production of estrogens in developing gonads, causing alterations in in androgen-to-estrogen ratios and affecting processes such as gonadal differentiation. In many fish species the “default” gonad type is testes, so when estrogen signaling is reduced there is a a resultant bias toward male-biased sex ratios (Guiguen et al. 2010). When male biased sex ratios occur, the number of breeding females can decrease over time and have negative impacts on population growth and sustainability. The present AOP provides the evidence framework of the negative impacts of aromatase inhibition at early developmental stages of teleost fish during the critical period of sexual differentiation and how this could lead to population-level effects.
Summary of the AOP
Molecular Initiating Events (MIE)
Key Events (KE)
Adverse Outcomes (AO)
|Type||Event ID||Title||Short name|
|MIE||36||Inhibition, Aromatase||Inhibition, Aromatase|
|KE||1789||Reduction, 17beta-estradiol synthesis by the undifferentiated gonad||Reduction, E2 Synthesis by the undifferentiated gonad|
|KE||1790||Increased, Differentiation to Testis||Increased, Differentiation to Testis|
|KE||1791||Increased, Male Biased Sex Ratio||Increased, Male Biased Sex Ratio|
|AO||360||Decrease, Population growth rate||Decrease, Population growth rate|
Relationships Between Two Key Events (Including MIEs and AOs)
|Inhibition, Aromatase leads to Increased, Differentiation to Testis||non-adjacent||High|
|Inhibition, Aromatase leads to Increased, Male Biased Sex Ratio||non-adjacent||Moderate|
Life Stage Applicability
Overall Assessment of the AOP
See details below.
Domain of Applicability
The life stage to which this AOP applies is developing embryos/juveniles during gonadal differentiation. Since the sexually dimorphic expression of aromatase has been shown to play a crucial role in the differentiation to testis vs ovary of the undifferentiated bipotential gonad (Guiguen et al. 2010), the AOP is applicable to the stage of development during which aromatase might influence this process. The precise timing of the sensitive period relevant to this AOP will vary by species, but the AOP is not applicable to differentiated juveniles or to adults.
Studies with zebrafish (Danio rerio) have shown that both brain and gonadal aromatase expression can be observed at 20 days post-fertilization (dpf) with an increase in expression at 25 dpf in fish destined to become females, coinciding with the onset of gonadal differentiation period (Lau et al. 2016). In Nile tilapia (Oreochromis niloticus), aromatase expression can be observed as early as 3-4 dpf with an increase in expression starting at 11 dpf in genetic females (Kwon et al. 2001). Additionally, it has been shown that the period of 7-14 dpf is the most sensitive to chemical inhibition of CYP19a1 activity, and a continuous exposure of 2-3 weeks is sufficient for the masculinization of the majority of genetic female tilapia (Kwon et al. 2000). This clearly indicates alteration of differentiation from ovary to testis results during sex differentiation (OECD 2011).
The molecular initiating event for this AOP occurs during gonad differentiation. Therefore, the AOP is only applicable to sexually undifferentiated individuals.
Most evidence for the taxonomic applicability of this AOP comes from species in the class Osteichthyes. Aromatase itself is well conserved among vertebrates (e.g., Wilson et al. 2005). However, the degree to which aromatase and subsequent production of endogenous estrogens such as E2 are involved in sex determination or sexual differentiation varies with species. Many fish, amphibian, and reptile species have environmental sex determination, and regulation of aromatase expression and sex steroids profiles are closely tied to sex-determining environmental factors (Angelopoulou et al. 2012). Alternatively, vertebrates that largely rely on genetic sex determination (birds, mammals) would be anticipated to be less vulnerable to effects of aromatase inhibitors during gonad differentiation, although there remains compelling evidence for an important role of steroid signaling during the process (Angelopoulou et al. 2012). Overall, regardless of differeing roles for aromatase in sexual differentiation, expression appears universal among vertebrates during this life stage (Angelopoulou et al. 2012; Sarre et al. 2004; Uller and Helantera, 2011; Ramsey and Crews, 2009). Thus, in principle, components of the present AOP may have some degree of applicability to all vertebrates. Given the substantial diversity of sex determination and differentiation strategies in fish, amphibians and reptiles (including those from closely related phylogenetic groups; Sarre et al. 2004; Angelopoulou et al. 2012), quantiative sensitivity, and taxonomic domain of appicability of the present AOP are hard to generalize, although there is reason to believe it should have broad applicability in bony fishes.
Essentiality of the Key Events
Direct support for the essentiality of several of the key events in the AOP has been provided by modification/knockout studies of the cyp19a1 gene in zebrafish and Nile tilapia. Specifically:
- Lau et al. (2016) generated insertion/deletion mutations in the zebrafish cyp19a1a gene using TALEN (transcription activator-like effector nuclease) and CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 approaches. All mutant cyp19a1a-/- fish developed as males. Histological examination (at 120 dpf) of the cyp1a1a-/- mutants showed that they exhibited normal spermatogenesis in the testis with no observable difference between the wild type (+/+) and heterozygous (+/-) males. To confirm the necessity of E2 synthesis for ovarian differentiation, they performed an experiment to "rescue" the phenotype of cyp19a1a mutants by E2 treatment (0.05, 0.50 and 5.00 nM) encampassing the period of gonadal differentiation (15–30 days pdf). Treatment with the estrogen resulted in normal functioning ovaries with fully developed perinucleolar oocytes and small amount of stromal tissue, even in some individuals at the lowest E2 concentration (0.05 nM). This supports the essentiality of aromatase inhibition relative to E2 synthesis reduction as a critical step for testis differentiation.
- In a similar study also with zebrafish, Muth-Köhne et al. (2016) generated cyp19a1a and cyp19a1b gene mutant lines and a cyp19a1a;cyp19a1b double-knockout line using TALENs. All cyp19a1a mutants and cyp19a1a;cyp19a1b double mutants developed as males, whereas cyp1a1b double mutant (-/-) had a 1:1 sex ratio similar to the wild type controls. This again supports the essentiality of gonadal aromatase inhibition for testis differentiation that would lead to a male biased sex ratio. Additionally, a small rescue experiment performed using E2 on all male mutant cyp1a1a-/- indicated that E2 treatment could could restore a near normal sex ratio defect (9 females among 14 fish).
- Studies in Nile tilapia similar to those conducted in zebrafish were described by Zhang et al. (2017), who worked with genetic female mutants for cypa19a and cyp19a1b. Results showed that all cyp19a1a+/- XX and cyp19a1a+/+ XX fish developed as females, whereas all cyp19a1a-/- XX and cyp19a1a-/- XY fish developed as males, based on gonad differentiation. The cyp19a1a-/- XX tilapia shifted to the male pathway as early as 5 dph and ultimatelywere fertile. This again provides strong support for the critical role of gonadal aromatase relative to ovarian development.
There is good evidence from gene knockout experiments of the two different isoforms of aromatase that support the specificity of gonadal aromatase inhibition for the subsequent key events to occur.
E2 Synthesis by the undifferentiated gonad
There is evidence from a stop (by cyp19a1knockout) and recovery (through compensation) experiment where E2 can rescue the sex ratio altered due to the gonadal aromatase gene knockout suggesting that E2 depletion is necessary for the subsequent key events to occur.
Differentiation to Testis
By definition, differentiation to testis is required for a male reproductive phenotype.
Male Biased Sex Ratio
Breeding females (and both sexes) are necessary for population sustainability. A male biased sex population suggests a reduced offspring production and consequentially reduced population sustainability.
This is the terminal key event in the AOP. Its essentiality for progression to downstream events in the sequence cannot be evaluated.
Aromatase catalyzes the conversion of T to E2, so the biological plausibility of aromatase inhibition leading to reductions in available E2 is clear. Additionally, the role of E2 as a major regulator of normal female gonad development is well documented (Gorelick et al. 2011; Guiguen et al. 2010). The link between E2 reductions leading to increased differentiation of the bipotential gonad to testis is highly plausible. As E2 signaling is reduced, ER responsive genes required for ovarian differentiation will be downregulated in the bipotential gonad resulting in a default development of testes (Yin et al. 2017; Zhang et al. 2017). Therefore, it is plausible that E2 reduction in the undifferentiated gonad at the onset of sexual differentiation would promote testis formation possibly in a concentration dependent manner. The direct link between increased differentiation to testis leading to a male biased sex ratio is also well supported by biological plausibility. If the conditions that favor a male producing phenotype (in this case, the aromatase inhibitor) overlap with the critical period of sex differentiation in a given population, it is reasonable that relatively more male offspring will be produced (D'Cotta et al., 2001, Kwon et al., 2000; Luzio et al. 2016). Therefore, exposure of sensitive species to aromatase inhibition for an extended period of time during reproducitve development plausibly would result in a male-biased population. Empirical evidence supporting the direct link between male biased cohorts and a reduced population sustainability in fish species is limited. However, biased sex ratios can definitely impact fish populations (Marty et al. 2017). For example, a male-biased sex ratio would logically lead to a reduction in the number of breeding females such that over time decreases in offspring would result in population declines (Brown et al. 2015; Grayson et al. 2014). Miller et al. (2022) recently developed a model specifically designed to capture the effects of male-biased sex ratios on population trajectories in fathead minnows (Pimephales promelas).
Concordance of Dose Response Relationship
There have been a number of in vitro and in vivo studies, primarily in fish, that have examined the effects of known aromatase inhibitors on different KEs in the AOP. Some of these studies measured only one KE in the AOP and/or employed just a single dose of a given stressor, so cannot be directly used to explore dose-response concordance.
The differential sensitivity to inhibition of CYP191 likely is best measured in vitro (Doering et al. 2019b) but most studies that support this AOP are performed in vivo. There are cases in which the significant effect of reduced E2 was either not measured, measured at a time period outside the critical differentiation period, only one concentration of an aromatase inhibitor was used (Ruksana et al. 2010) or they were gene knockout studies (Yin et al. 2017; Zhang et al. 2017). Therefore these could not be considered for the dose-response relationship. Additionally, increased differentiation to testes is observed via histological examinations in which most studies using aromatase inhibitors only determined the general presence of male or female first and secondary characteristics but a degree of differentiation or differentiation stage of the gonads was not measured nor reported in some studies based on the exposure doses. The most observable dose response relationship for this AOP was for the non-adjacent relationship between aromatase inhibition and an increased male biased sex ratio in which several studies using multiple concentrations of an aromatase inhibitor led to an increased number of males in a dose-dependent way.
Concentration dependent aromatase inhibition:
- Immunohistochemical analyses revealed that fish at 35 dah treated with higher concentrations of EM (500, 1000 and 2000 μg/g feed) had no reaction against P450arom but cells with strongly immunopositive responses against P450arom were evident in the lowest dose of EM (100 μg/g feed) similar to the differentiating ovaries of the control fish; these cells occurred as clusters in the vicinity of blood vessels (Ruksana et al. 2010)
Concentration dependent increased differentiation to testes:
- Studies with zebrafish exposed to fadrozole resulted in masculinization at different biological effect levels in a concentration-dependent manner as evidenced from a significantly increased maturity of testes (Muth-Köhne et al. 2016)
Concentration dependent increased male biased sex ratio:
- Nile tilapia (Oreochromis niloticus), fathead minnow (Pimephales promelas), and zebrafish exposed to different concentrations of known aromatase inhibitors (exemestane, fadrozole, prochloraz) lead to increased number of males in a dose-dependent manner (Kwon et al., 2000; Uchida et al., 2004; Ruksana et al. 2010; Thorpe et al., 2011, Holbech et al., 2012).
Concentration dependent decline in population trajectory:
- Modeled population trajectories for male skews of zebrafish exposed to clotrimazole show a concentration-dependent reduction in projected population growth and viability (Brown et al. 2015). Population-level effects have not been measured directly.
Temporal concordance of the AOP from aromatase inhibition to decreased E2 production, increased differentiation to testes and increased male -biased sex ratio (e.g., (Ruksana et al., 2010; Yin et al. 2017; Zhang et al. 2017) has been established. However, beyond that key event, temporal concordance has not yet been established possibly due limiting capability to test and/or document particular population viability in situ. From the evidence gathered for this particular AOP, the best way to determine population viability is via multifactorial population viability analyses that generate the distribution of likely fates for a population exposed to endocrine disrupting chemicals that affect aromatase activity.
There have been a number of in vitro and in vivo studies, primarily in fish, that have examined the effects of known aromatase inhibitors on different KEs in the AOP. Some of these studies measured only one KE in the AOP and/or employed just a single dose of a given stressor, so cannot be directly used to explore dose-response concordance. However, even with these limitations, they can still demonstrate that the overall AOP is consistent with expectations in a variety of species exposed to known inhibitors of aromatase. For example... (concordance table)
Male-biased sex ratios are not specific to this AOP. Many of the KEs included overlap with another AOP (#376) linking activation of the androgen receptor to male biased sex ratios.
Uncertainties, inconsistencies, and data gaps
Currently the major uncertainty in this AOP is the biological linkage between E2 synthesis reduction by the undifferentiated gonad leading to an increased, differentiation to testis. Biological plausibility connections have been established, but experimental measurements of E2 during the particular period of differentiation is lacking.
Known Modulating Factors
There is not yet a sufficient quantitative understanding of this overall AOP to predict the degree to which aromatase inhibition would result in population-level impacts. That said, there are models available suitable for the quantitative prediction of changes in E2 levels caused by degree of aromatase inhibition in some small fish species (Conolly et al. 2018; Doering et al. 2019a), as well as the effects of different (male-biased) sex ratios on fathead minnow population size (Miller et al. 2022). However, there currently are no quantitative data/models relating reductions in E2 to the degree of (increased) differentiation to male gonads and/or male-biased cohorts of fish.
Considerations for Potential Applications of the AOP (optional)
Altered sex ratios in fish can be a useful diagnostic endpoint for identifying EDCs both in field and lab settings. For example, the Fish Sexual Development Test (FSDT) has formally been adopted by the Organisation of Economic Cooperation and Development (OECD) as a test guideline (No. 234) for the detecting EDCs (OECD, 2011b). The FDST is conducted in zebrafish during early development, including sexual differentiation, and uses gonadal differentiation and skewed sex ratios to detect estrogen, androgen and steroidogenesis activity of test chemicals (Dang & Kienzler 2019). This AOP directly supports the mechanistic basis for assays such as the FDST. The AOP also supports the use of in vitro assays that measure aromatase inhibition by test chemicals as a basis for predicting apical impacts on fish (e.g., Conolly et al. 2018; Doering et al. 2019a; 2019b).
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