This AOP is licensed under a Creative Commons Attribution 4.0 International License.
Androgen receptor agonism leading to male-biased sex ratio
Point of Contact
- Dan Villeneuve
- Kelvin Santana Rodriguez
|Author status||OECD status||OECD project||SAAOP status|
|Under development: Not open for comment. Do not cite|
This AOP was last modified on July 12, 2021 09:50
|Agonism, Androgen receptor||March 20, 2017 17:44|
|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|
|Agonism, Androgen receptor leads to Increased, Differentiation to Testis||April 12, 2021 10:24|
|Agonism, Androgen receptor leads to Increased, Male Biased Sex Ratio||April 12, 2021 09:31|
|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|
|17beta-Trenbolone||November 29, 2016 18:42|
|Chemical:33664 17-Methyltestosterone||March 23, 2021 13:34|
|5alpha-Dihydrotestosterone||March 14, 2017 12:44|
|Methyldihydrotestosterone||May 07, 2021 15:36|
|11-Keto-testosterone||May 07, 2021 15:36|
This adverse outcome pathway links androgen receptor agonism in teleost fish during gonadogenesis to male-biased sexual differentiation and successively, reduced population sustainability. Sex determination in teleost fishes is highly plastic; it can be genetically or environmentally driven or both depending on species. Species with environmentally-based sex determination in particular can be very sensitive to environmental pollutants during the period of differentiation. Exogenous hormones are of ecological concern because they have the potential to alter gonad development and sex differentiation. Androgens play a crucial role in sex differentiation, sexual maturation, and spermatogenesis in vertebrates and their modes of action are mediated via androgen receptors (ARs). Like many steroid hormones, androgens acts by entering the cell and forming a complex with a its hormone receptor allowing it to enter the nucleus where it can bind to specific short DNA sequences (Androgen Responsive Elements) and serve as transcription factors of androgen mediated genes involved in the male differentiation pathway. Many studies have shown that administration of androgens during early development in some teleost species can strongly bias sexual differentiation to testes. This can result in male biased sex ratios which, if sustained over multiple generations, can impact population fitness and long term viability.
Summary of the AOP
Molecular Initiating Events (MIE)
Key Events (KE)
Adverse Outcomes (AO)
|Sequence||Type||Event ID||Title||Short name|
|1||MIE||25||Agonism, Androgen receptor||Agonism, Androgen receptor|
|2||KE||1790||Increased, Differentiation to Testis||Increased, Differentiation to Testis|
|3||KE||1791||Increased, Male Biased Sex Ratio||Increased, Male Biased Sex Ratio|
|4||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
See details below.
Domain of Applicability
The life stage applicable to this AOP is developing embryos and juveniles prior to- or during the gonadal developmental stage. This AOP is not applicable to sexually differentiated adults.
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 has shown that the ar genes appear to be specific to jawed vertebrates (Gnathostomata), since no ar gene has been reported from Agnatha (Thornton 2001; Hossain et al 2008). 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 might 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 in combination of in vivo and in vitro studies of androgen receptor agonist and antagonist exposures during the critical period of sexual differentiation.
Golan & Levavi-Sivian 2014 exposed genetically female Nile tilapia (Oreochromis niloticus) to 17-a-methyltestosterone (MT) and dihydrotestosterone (DHT), two well-known androgen receptor agonisms that induces the male differentiation pathway and a dose dependent male biased sex ratio (downstream key events). However, when these are combined with androgen antagonist Flutamide, the sex inversion potency of androgen is reduced in a dose-dependent manner. The decrease in sex inversion efficiency caused by flutamide is due to the direct blocking of the androgen binding to its androgen receptor. Therefore, this suggest that androgen receptor agonism ligand binding is required for the subsequent key events to occur.
Crowder et al., 2018 generated zebrafish with a mutation in the ar gene (aruab105/105) and the resulting mutants developed ovaries and displayed female secondary sexual characteristics. The small percentage of mutants that developed as males displayed female secondary sexual characteristics with structurally disorganized testes, and were unable to produce normal levels of sperm. This demonstrates that the AR is required for proper testis development and fertility. This supports the essentiality for the androgen receptor agonism for the testis differentiation pathway to proceed.
In a similar study with zebrafish, Yu et al. 2018 generate ar gene mutant line using CRISPR/Cas9 technology. The resulting showed that the number of female offspring was increased and the resulting ar-null males had female secondary sex characteristics and were infertile due to defective spermatogenesis. This study supports the essentiality for the ar agonism for the development of testis and subsequently a male biased sex ratio.
There is good evidence from a sex inversion treatment via the direct blocking of AR using androgen antagonist that support the specificity of androgen receptor agonism 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.
The biological plausibility linking androgen receptor agonism through the increased differentiation to testis is very solid. Actions of androgens are mediated by the androgen receptor. The Androgen Receptor (AR) is part of the nuclear receptor superfamily. ARs are ligand-dependent transcription factors and contain a highly conserved DNA-binding domain (DBD) and a moderately well conserved ligand-binding domain (LBD) (Hossain et al., 2008). Steroid hormones such as androgens acts by entering the cell and forming a complex with a its hormone receptor. After ligand binding, AR monomers undergo conformational change, dissociate from chaperone proteins, dimerize, and bind coactivator proteins (Bohen et al., 1995; Pratt and Toft, 1997). After this, the complex is translocated to the nucleus where it can bind to specific short DNA sequences (Androgen Responsive Elements) and serve as transcription factors of androgen mediated genes (Harbott et al., 2009). During sexual development, endogenous androgen can therefore induce the upregulation of many genes involved in the male developmental pathway.
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, exposure to androgens) overlap with the critical period of sex differentiation in a given population, it is reasonable that more phenotypic males will be produced (Orn et al., 2003; Seki et al., 2004; Bogers et al., 2006; Morthorst et al., 2010; Baumann et al., 2014; Golan & Levavi-Sivian 2014). Therefore, persistence of androgen exposure for repeated or prolong periods of times within the habitat of given fish 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
The concentration-dependence of the key event responses with regard to the concentration of androgens has been established in vivo for some key events in the AOP. In general, effects on downstream key events occurred at concentrations equal to or greater than those at which upstream events occurred. Few studies that looked at multiple key events on this AOP were considered to be stronger support when evaluating the dose response relationship between key events. However, binding to the androgen receptor (the MIE) was not directly measured in any of the in vivo studies as this is often measured with in vitro studies. In fish, phenotypic masculinization of females has been used as an indirect measurement of in vivo androgen receptor agonism. However, in this case/aop, androgen receptor agonism is occurring prior to sexual differentiation and the resulting “phenotypic measurement” for the in vivo study is already considered as a separate downstream key event. Therefore, because support for the upstream key event is done in a different system (in vitro) than for the downstream key events (in vivo) , support for the dose concordance of the androgen receptor agonism (upstream) is done via in vitro studies that targeted the specific steroids used on the in vivo studies of the downstream key event.
- Concentration depended androgen receptor agonism (in vitro)
- COS Whole Cell Binding Assay with fathead minnow AR (fhAR) were used in competitive binding experiments testing several natural and synthetic steroids, some of which are environmental contaminants, such as R188, 17B-trenbolone , and 17a-trenbolone. All showed a concentration dependent displacement of [3H]R1881 binding proving to be high affinity ligands for the fmAR. (Wilson et al., 2004).
- The synthetic steroids, R1881 and methytestosterone, had the highest affinities of all the chemicals tested with IC50 values of about 1.6 nM, followed by the synthetic steroids 17α- and 17β-trenbolone with IC50 values of about 8 and 16 nM, respectively.
- Of the natural steroids, dihydrotestosterone was the strongest competitor with an IC50 of about 20 nM. The IC50 for the fish specific androgen, 11- ketotestosterone, was approximately 40 nM, followed by both testosterone and androstenedione at about 100 nM
- Important to note that all of the above steroids tested were used in the in vivo studies that were selected to support this AOP demonstrating that all bound to the fhAR with a higher affinity than 11- ketotestosterone.
- Concentration dependent increased differentiation to testes:
- Studies with Zebrafish (Danio rerio) exposed to 17β-trenbolone and Japanese medaka (Oryzias latipes) resulted in masculinization at different biological effect levels in a concentration-dependent manner as evidenced from a significantly increased maturity of testes (Orn et al., 2006; Morthorst et al., 2010; Baumann et al., 2014)) for some studies, this was determined ether by the abundance of spermatozoa and/or by the area of the testis.
- Concentration depended increased male biased sex ratio:
- Zebrafish (Danio rerio) exposed to different concentrations of 17β-trenbolone and Dihydrotestosterone lead to increased number of males in a dose-dependent way (Orn et al., 2003; Morthorst et al., 2010; Baumann et al., 2013; Baumann et al., 2014)
- Concentration depended decline in population trajectory:
- Modeled population trajectories show a concentration-dependent reduction in projected population size (Brown et al 2015, Miller et al. 2021?) based strongly on the ratio of male to female. Population-level effects have not been measured directly based on androgen receptor agonism.
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 in teleost fish species. There are cases however, in which exposure to aromatizable androgens such as Methyltestosterone may lead to feminization of fish. This is due in part by excess aromatizable androgens available that can be converted to E2 and favor a female developmental pathway.
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. Aromatase inhibition, AOP 346) to male biased sex ratios.
Uncertainties, inconsistencies, and data gaps
It’s important to note that the use of aromatizable instead of non-aromatizable androgens for sex reversals exposures can lead to excess aromatizable androgens being converted to E2 and inducing a female developmental pathway. Chinook salmon (Oncorhynchus tshawytscha) exposed to Methyltestosterone showed a concentration dependent increase in percentage of males where 100% males were obtained at doses 400 ug/L. but at higher concentrations, the percentage of males decreased and at 10,000ug/L. the percentage of males reduced to 79.4%. (Peferrer & Donaldson, 1993).
However, there have been similar cases to the latter but with the use of non-aromatizable androgens such as Dihydrotestosterone in which the primary cause of this feminization is not clear yet. Bogers et al. 2006 exposed Fathead minnow (Pimephales promelas) to Dihydrotestosterone where fish exposed to 0.1 μg/L all had developed testes with one fish showing mixed sex (testis–ova). Exposure to 0.32 μg/L resulted in 80% males and 20% mixed gonads. But at 1.0ug/L the percentage of males reduced to 40% (40% female, 10% undifferentiated and 10% mixed gonad). Additionally, it has been reported that oral administration of non aromatizable androgen dihydrotestosterone to sexually undifferentiated Channel Catfish (Ictalurus punctatus) resulted in all female populations (Davis et al. 1992).
Based on the evidence reviewed during development of this AOP, quantitative understanding of the KERs is limited at this time.
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 when screening for sex steroid hormones or any other type of EDC affecting androgen receptors.
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