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
|Author status||OECD status||OECD project||SAAOP status|
|Under Development: Contributions and Comments Welcome|
This AOP was last modified on April 22, 2021 10:04
|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 Reduction, E2 Synthesis by the undifferentiated gonad||September 30, 2020 09:42|
|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||September 30, 2020 10:15|
|Inhibition, Aromatase leads to Increased, Differentiation to Testis||September 30, 2020 09:34|
|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 during gonadogenesis leading to a male-biased sex determination and successively, reduced population sustainability. Most gonochoristic fish species, develop either as males or females, and do not change sex throughout their entire life spans. However, there’s a developmental window in which their sex determination can be sensitive to environmental conditions or chemical pollutants. Treatment with steroid hormones prior to sexual differentiation has shown to induce ovary or testis development according to the type of steroid that is administered. For most vertebrate taxa, aromatase (Cyp19a1) is the rate-limiting enzyme for the biosynthesis of 17β - estradiol from testosterone. Many endocrine disrupting chemicals such as fadrozole, letrozole and exemestane are well known chemicals that inhibit the activity aromatase. Exposure during the critical period of sex differentiation in gonochoristic teleost fish with an aromatase inhibitor that blocks estrogen biosynthesis can induce phenotypic males. Given that females carry the major reproductive production of the population, a male-biased sex ratio can result in a reduced population fitness, particularly for those species present in ecosystems that are heavily impacted by human activities.
In fish, sexual differentiation occurs post hatching which makes them susceptible to the action of exogenous factors including hormones, temperature, pH, population density, social cues and more. As a result, the sex phenotype in most fish can be altered depending on the environmental conditions in which they are exposed during development, particularly during the critical period of sexual differentiation. At this stage, the bipotential gonad can be destined to take a testis or an ovary differentiation pathway that is reliant on both the genetic and environmental factors.
Sex steroid hormones are considered the natural inducers of sex differentiation for non-mammalian vertebrates where androgens and estrogens act, respectively, as testis and ovary inducers. In teleost fish, the hormonal balance between estrogens and androgens is essential during the sexual differentiation period and this balance is in turn dependent on the availability and activity of steroid synthesizing enzymes such as aromatase60.
Cytochrome P450 aromatase (CYP19) is the enzyme responsible for the conversion of C19 androgens to C18 estrogens in brain and gonadal tissues of vertebrates52,70. Therefore it a crucial enzyme for the female developmental pathway for many vertebrates. In fish, there are two isoforms of aromatase due to the teleost-specific whole-genome duplication. Cyp19a1a that is mostly expressed in the gonads and cyp19a1b that is expressed in the brain.
In recent years, there has been growing concern about the potential impacts of endocrine disrupting chemicals in the wildlife. Particularly of important concern, is the effects it can exert in early life stages that can lead to major impacts at the population level. Many EDC’s are known to alter the sexual phenotype of fish by disrupting sex steroid synthesizing enzymes. Cyp1a1 can be a potential target for endocrine disrupting chemicals as it catalyzes the final step of estrogen biosynthesis which control crucial developmental and physiological processes.
Disruption of aromatase expression will alter the production rate of estrogens in the developing gonads, increasing an imbalance in the androgen-to-estrogen ratio leading to the disruption of estrogen related biological processes that lead to the determination and differentiation of the ovary. Therefore, as aromatase inhibitors block the synthesis of estrogens (by inhibiting the conversion of androgens to estrogens), the level of androgens in the developing organism increases, inducing testis differentiation and male maturation instead of a female developing pathway7 . When the conditions that favor a male differentiation pathway persists, male biased sex ratios can occur. As a result, the number of breeding females can decrease over time and the population productivity can be affected. Therefore, altered sex ratios can 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 stage of teleost fish particularly during the critical period of sexual differentiation and how can this ongoing exposure on population can lead to a population dysfunction.
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)
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 the gonadal developmental stage. Since the sexually dimorphic expression of aromatase plays a crucial role in the differentiation to either testis or ovaries in the undifferentiated bipotential gonad, this key event relationship can be applicable to the exact stage of development at which the aromatase enzyme works to influence gonadal differentiation. This AOP is not applicable to sexually differentiated adults.
Studies with zebrafish have shown that both brain and gonadal aromatase expression can be observed at 20 days post-fertilization with and increase in expression at 25 days post-fertilization in zebrafish destined to become females which also coincided with onset of gonadal differentiation period (Lau et al. 2016). In tilapia, aromatase expression can be observed as early as 3-4 days post fertilization with and increase in expression starting at 11 days post-fertilization in genetic females (Kwon, J. et al. 2001). Additionally, it has been shown that the period of 7-14 days post-fertilization is the most sensitive towards an aromatase inhibitor and that a consecutive exposure of 2-3 weeks is sufficient for the masculinization of the majority of genetic female tilapia fish (Kwon, J. et al. 2000). This suggest that to redirect the sexual differentiation pathway from ovary to testis, an alteration of aromatase expression will be most effective during the early developmental stage prior and during the critical sex differentiation period.
The molecular initiation event for this AOP occurs prior to gonad differentiation. Therefore, this AOP is only applicable to sexually undifferentiated individuals.
The taxonomic applicability of this AOP is the class Osteichthyes. However, phylogenetic analysis among mammalian, amphibian, reptile, bird, and fish has shown that aromatase is well conserved among all vertebrates (Wilson JY et al., 2005). Additionally, CYP19 was detected in the amphioxus suggesting that it has possible origin in primitive chordates. Therefore, because all key events in the present AOP can be applicable to most non-mammalian vertebrates, it is probable that this AOP could be relevant to amphibians, reptiles and birds as well. Though, the outcomes mind differ due to species-specific differences.
Essentiality of the Key Events
Support for the essentiality of several of the Key Events in the AOP was provided mainly by gene knockout of the cyp1a1 gene in zebrafish and tilapia. Teleost fish have two genes encoding for aromatase; cyp1a1a that is mainly expressed in the gonads and cyp1a1b expressed in the brain. Studies have demonstrated that mutant lines of cyp1a1b develop as females while cypa1a mutants develop as males suggesting that gonadal aromatase inhibition is crucial step for the subsequent key events to occur.
- Lau et al. 201613 generated indel mutations in zebrafish cyp19a1a gene using TALEN and CRISPR/Cas9 approaches. All mutant cyp19a1a-/- developed as males. Histological examination (at 120 days post-fertilization) of the cyp1a1a-/- mutant showed that all exhibited normal spermatogenesis in the testis with no observable difference to the wild type (+/+) and heterozygous (+/-) males. However, to prove the role 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). The result showed that exposure to E2 caused normal ovarian formation with fully developed perinucleolar oocytes and little amount of stromal tissues, and the effect could be 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.
- On a similar study with zebrafish, Muth-Köhne et al. 20168 generated cyp19a1a and cyp19a1b gene mutant lines and a cyp19a1a;cyp19a1b double-knockout line in zebrafish using transcription activator-like effector nucleases (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 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 17 β-estradiol on all male mutant cyp1a1a-/- and the results suggested that treatment could rescue the sex ratio defect (9 females among 14 fish).
- Similar support using Nile tilapia (Oreochromis niloticus) was provided in a study by Zhang et al. 201712. Using genetic female mutant for cypa1a and cyp1a1b. Results showed that all cyp19a1a+/- XX and cyp19a1a+/+ XX fish developed as females, whereas all cyp19a1a-/- XX and cyp19a1a-/- XY fish developed as males. The cyp19a1a-/- XX tilapia shifted to the male pathway at as early as 5 days after hatch (dah), as reflected by the gonadal expression and were fertile. This supports the essentiality of gonadal aromatase inhibition during early development for a testis differentiation pathway to be induced.
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
Biological plausibility provides strong support for the essentiality of this event for the subsequent key events to occur.
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.
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 or permanent 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, in most of them 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 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 most studies based on the 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 leading to 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 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 specific 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 at the developmental stage.
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 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. 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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