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
|Author status||OECD status||OECD project||SAAOP status|
|Under Development: Contributions and Comments Welcome|
This AOP was last modified on August 03, 2021 17:30
|Inhibition, Aromatase||September 16, 2017 10:14|
|Reduction, 17beta-estradiol synthesis by the undifferentiated gonad||September 15, 2020 13:49|
|Increased, Differentiation to Testis||September 15, 2020 14:01|
|Increased, Male Biased Sex Ratio||August 07, 2020 18:17|
|Decrease, Population trajectory||September 26, 2017 11:33|
|Inhibition, Aromatase leads to Increased, Differentiation to Testis||September 30, 2020 09:34|
|Inhibition, Aromatase leads to Reduction, E2 Synthesis by the undifferentiated gonad||August 04, 2021 16:42|
|Inhibition, Aromatase leads to Increased, Male Biased Sex Ratio||April 13, 2021 11:20|
|Reduction, E2 Synthesis by the undifferentiated gonad leads to Increased, Differentiation to Testis||August 04, 2021 17:48|
|Increased, Differentiation to Testis leads to Increased, Male Biased Sex Ratio||September 17, 2020 09:03|
|Increased, Male Biased Sex Ratio leads to Decrease, Population trajectory||September 17, 2020 06:34|
|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 exposed during gonadogenesis to male-biased sexual differentiation, 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 or chemical pollutants. For example, treatment with exogenous steroid hormone prior to/during 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. For most vertebrate taxa, aromatase (Cyp19a1) is the rate-limiting enzyme for the biosynthesis of 17β - estradiol (E2) from testosterone. Many endocrine disrupting chemicals such as fadrozole, letrozole and exemestane (pharmaceuticals) or prochloraz and propiconazole (fungicides) are known to inhibit aromatase activity. Exposure of some fish species to these types of aromatase inhibitiors, during the critical period of sex differentiation, can reduce endogenous E2 synthesis, thereby producing phenotypic males, the default sex in the absenced of estrogen signaling during the differentiation period. Given the strong role of female fecundity in dictacting population size, a male-biased sex ratio can result in reduced population levels if sustained over multiple generations.
In fish, sexual differentiation occurs post hatching which makes the process potentially susceptible to the action of exogenous factors such as chemicals, temperature, pH, population density, social cues and more. As a result, the 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 be destined to take a testis or an ovary differentiation pathway reliant on both 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 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 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 cytochrome P450 aromatase (CYP19), which is responsible for the conversion of C19 androgens to C18 estrogens in brain and gonadal tissues of vertebrates (Payne and Hales, 2004; Simpson et al. 1994). In fish, there are two CYP19 isoforms due to the teleost-specific whole-genome duplication. CYP19a1a is mostly expressed in the gonads and CYP19a1b is largely expressed in the brain (Callard et al. 2001).
For over 3 decades 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 disrupting sex steroid synthesizing enzymes such as aromatase. Disruption of CYP19 expression or activity can alter the production of estrogens in developing gonads, causing an imbalance in androgen-to-estrogen ratios important to the determination and differentiation of the ovary. In many fish species the “default” gonad type is testis, 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 can lead to population changes.
Summary of the AOP
Molecular Initiating Events (MIE)
Key Events (KE)
Adverse Outcomes (AO)
|Sequence||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 trajectory||Decrease, Population trajectory|
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
Domain of Applicability
The life stage applicable to this AOP is developing embryos and juveniles prior to- or during gonadal differentiation. Since the sexually dimorphic expression of aromatase plays a crucial role in the differentiation to either testis or ovaries in the undifferentiated bipotential gonad (Guiguen et al. 2010), this AOP can be applicable to the stage of development at which aromatase influences gonadal differentiation. This AOP is not applicable to sexually differentiated adults and the precide timing of the sensitive period relevant to this AOP varies by species.
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 zebrafish destined to become females, coiciding 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, J. et al. 2001). Additionally, it has been shown that the period of 7-14 dpf is the most sensitive to chemical inhibition of CYP19 activity, and a continuous exposure of 2-3 weeks is sufficient for the masculinization of the majority of genetic female tilapia (Kwon, J. et al. 2000). This indicates that redirection of sexual differentiation from ovary to testis, can result from a decrease in aromatase expression or activity during early development, prior to or during sex differentiation (OECD 2011).
The molecular initiating event for this AOP occurs prior to or during gonad differentiation. Therefore, the AOP is only applicable to sexually undifferentiated individuals.
The taxonomic applicability of this AOP includes species in the class Osteichthyes. Aromatase itself is well conserved among vertebrates (Wilson JY et al., 2005). However, the degree to which aromatase and subsequent production of endogenous estrogens are involved in sex determination or sexual differentiation varies with species. Fish, amphibians, and reptiles have environmental sex determination and regulation of aromatase expression and sex steroids is closely tied to sex-determining environmental factors (Angelopoulou et al. 2012). In contrast birds and mammals have genetic sex determination and may 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 whether driven by sex determining genes or environmental factors, the role of aromatase expression and activity in sexual differentiation appears universal among vertebrates (Angelopoulou et al. 2012; Sarre et al. 2004; Uller and Helantera, 2011; Ramsey and Crews, 2009). Thus, in principle, the present AOP is likely applicable, to some extent in all vertebrates. However, the quantitative sensitivity relative to the concentration, timing, and route of exposure to aromatase inhibitors is expected to vary widely among species. Given the diversity sex determination and differentiation methods in fish, reptiles, etc. (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 is hard to generalize, although there is reason to believe it is applicable to many species of fish, reptiles, and amphibians, and perhaps less so to birds and mammals.
Essentiality of the Key Events
Direct support for the essentiality of several of the key events in the AOP has been provided mainly by gene knockout studies of the cyp19a1 gene in zebrafish and Nile tilapia. Studies have demonstrated that cyp19a1b mutant lines of develop as females while cypa19a mutants develop as males suggesting that gonadal aromatase inhibition is crucial step for the subsequent key events to occur.
- 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-/- developed as males. Histological examination (at 120 dpf) of the cyp1a1a-/- mutant showed that all exhibited normal spermatogenesis in the testis with no observable difference between the wild type (+/+) and heterozygous (+/-) males. However, to prove the necessity of E2 synthesis for ovarian differentiation, they performed an experiment to "rescue" the phenotype of cyp19a1a mutant by E2 treatment (0.05, 0.50 and 5.00 nM) over the time of gonadal differentiation (15–30 days port-fertilization). Exposure to E2 resulted in normal functioning ovaries with fully developed perinucleolar oocytes and small amount of stromal tissue, and with the rescue effect observed in some individuals even at the lowest 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 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 leading to a male biased sex ratio population. Additionally, a rescue experiment was performed using E2 on all male mutant cyp1a1a-/- and the results suggested that E2 treatment could rescue the sex ratio defect (9 females among 14 fish).
- Similar support using Nile tilapia was provided in a study by Zhang et al. (2017). Using 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 morphology. The cyp19a1a-/- XX tilapia shifted to the male pathway at as early as 5 days post hatch (dph), as reflected by the gonadal type and were 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 is the key enzyme in the conversion of C19 androgens to C18 estrogens and the biological plausibility linking aromatase inhibition to E2 reduction is very solid. Additionally, the role of E2 as a major regulator for downstream estrogen-responsive genes necessary for proper female gonad development is well documented in literature (Gorelick et al. 2011; Guiguen et al. 2010). The link between E2 reduction for the undifferentiated gonad leading to an increased differentiation to testis is highly plausible. As the levels of estradiol are reduced, ER responsive genes required for proper ovarian differentiation will be downregulated in the bipotential gonad and instead allowing gene expression that leads to the morphological development of the testes due to an imbalance in the androgen to estrogen ratio (Shi et al., 2018; Yin et al. 2017; Zhang et al. 2017). Therefore, it is plausible that estradiol reduction in the undifferentiated gonad at the onset of sexual differentiation promotes testis differentiating in a concentration dependent manner (Baumann et al., 2015; Morthorst et al., 2010). 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 favored 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 more male offspring will be produced (D'Cotta et al., 2001, Kwon et al., 2000; Luzio et al. 2016). Therefore, persistence of such conditions for repeated or prolong periods of times within the habitat of given species, can result in a male-biased population. Empirical evidence supporting the direct link between male biased population and a reduced population sustainability in fish species is lacking. However, increasing biased sex ratios can definitely have significant effects in sustainable fish populations (Marty et al. 2017). A male-biased sex ratio already suggests that the number of breeding females is reduced. If the male-biased sex ratio persists and/or increases over time, the offspring production for such population could eventually decrease and consequently, population productivity would be reduced (Brown et al. 2015; Grayson et al. 2014).
Concordance of Dose Response Relationship
Concentration dependence of the key events in response to the concentration of the aromatase inhibitor has been established for major key events in several teleost fish species using in vivo studies. The best supporting evidence would be studies that considered multiple key events in an in vivo study. However, there were exceptions. The differential sensitivity to inhibition of Cytochrome P450 Aromatase (CYP19) is best measured in vitro (Doering et al. 2019) 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 lead 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 (Danio rerio) exposed to farozole 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), Zebrafish (Danio rerio) exposed to different concentrations of aromatase inhibitors (Exemestane, Fadrozole, Prochloraz) lead to increased number of males in a dose-dependent way (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.
We are aware of no cases where the pattern of key events described was observed without also observing a significant impact on male sex ratios. The 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 during the period of development (ie. androgen receptor agonism, AOP 376) 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.
Considerations for Potential Applications of the AOP (optional)
Sex ratios can be a useful endpoint in risk and hazard assessment of chemicals. In July 2011, the Fish Sexual Development Test (FSDT) has officially been adopted as OECD test guideline no. 234 for the detection of EDCs within the OECD conceptual framework at level 4 (OECD, 2011b). The Fish Sexual Development Test(FSDT; OECD TG 234, OECD, 2011) covers endocrine disruption during the developmental period of sexual differentiation of particularly zebrafish and uses gonadal differentiation and sex ratio as endocrine disruption-associated endpoints for indicating EAS (estrogen, androgen and steroidogenesis) activity (Dang & Kienzler 2019). Therefore, this AOP can provide additional support to the use of alternative measurements in this type of tests by screening for aromatase inhibitors.
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