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Dan Villeneuve, US EPA Mid-Continent Ecology Division (firstname.lastname@example.org)
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OECD Project 1.12: The Adverse outcome pathways linking aromatase inhibition, androgen receptor agonism, estrogen receptor antagonism, or steroidogenesis inhibition, to impaired reproduction in small repeat-spawning fish species
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This adverse outcome pathway details the linkage between binding and activation of androgen receptor as a nuclear transcription factor in females and reproductive dysfunction as evidenced through reductions cumulative fecundity and spawning in repeat-spawning fish species. Cumulative fecundity is the most apical endpoint considered in the OECD 229 Fish Short Term Reproduction Assay. The OECD 229 assay serves as screening assay for endocrine disruption and associated reproductive impairment (OECD 2012). Cumulative fecundity is one of several variables known to be of demographic significance in forecasting fish population trends. Therefore, this AOP has utility in supporting the application of measures of androgen receptor binding and activation as a nuclear transcription factor as a means to identify chemicals with known potential to adversely affect fish populations.
Summary of the AOP
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Molecular Initiating Event
|Molecular Initiating Event||Support for Essentiality|
|Androgen receptor, Agonism||Strong|
|Population trajectory, Decrease|
Relationships Among Key Events and the Adverse Outcome
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Life Stage Applicability
|Adult, reproductively mature|
|Pimephales promelas||Pimephales promelas||NCBI|
Overall Assessment of the AOP
Weight of Evidence Summary
Provide an overall summary of the weight of evidence based on the evaluations of the individual linkages from the Key Event Relationship pages.
- Biological Plausibility
- The biochemistry of steroidogenesis and the predominant role of the gonad in synthesis of the sex steroids are well established.
- Similarly, the role of E2 as the major regulator of hepatic vitellogenin production is widely documented in the literature.
- The direct link between reduced VTG concentrations in the plasma and reduced uptake into oocytes is highly plausible, as the plasma is the primary source of the VTG.
- The direct connection between reduced VTG uptake and impaired spawning/reduced cumulative fecundity is more tentative. It is not clear, for instance whether impaired VTG uptake limits oocyte growth and failure to reach a critical size in turn impairs physical or inter-cellular signaling processes that promote release of the oocyte from the surrounding follicles. In at least one experiment, oocytes with similar size to vitellogenic oocytes, but lacking histological staining characteristic of vitellogenic oocytes was observed (R. Johnson, personal communication). At present, the link between reductions in circulating VTG concentrations and reduced cumulative fecundity are best supported by the correlation between those endpoints across multiple experiments, including those that impact VTG via other molecular initiating events (Miller et al. 2007).
- At present, negative feedback is the most biologically plausible explanation for the reductions in ex vivo T and E2 production following exposure to 17β-trenbolone. In vitro exposure of fathead minnow ovary tissue to 17β-trenbolone or spironolactone does not cause reductions in T or E2 synthesis at concentrations comparable to those that produce significant responses in vivo (i.e., at non-cytotoxic concentrations; D.L. Villeneuve, unpublished data; C.A. LaLone unpublished data), nor are there any known reports of 17β-trenbolone directly inhibiting steroid biosynthesis. When tested in an in vitro steroidogenesis assay using H295R adrenal carcinoma cells, trenbolone caused a concentration-dependent increase in estradiol production, as opposed to any reductions in steroid hormone concentrations, an effect that was concurrent with increased transcription of CYP19 (aromatase) in the cell line (Gracia et al. 2007). Given the lack of any established direct effect on steroidogenic enzyme activity, negative feedback is currently the most likely explanation for the consistent effects observed in vivo. That said, many uncertainties regarding the exact mechanisms through which an exogenous, non-aromatizable, AR agonist elicits negative feedback remain.
- Biological Plausibility
Concordance of dose-response relationships: There are a limited number of studies in which multiple key events were considered in the same study following exposure to known, non-aromatizable, AR agonists. These were considered the most useful for evaluating the concordance of dose-response relationships. In general, effects on downstream key events occurred at concentrations equal to or greater than those at which upstream events occurred Concordance Table. For exposures to 17b-trenbolone, key events related to steroid production and circulating estradiol and vitellogenin concentrations were impacted at the same dose at which effects on cumulative fecundity were observed. Effects on vitellogenin transcription were only observed at greater concentrations, but data for comparable species and dose ranges were unavailable at present. For two other AR agonists tested in fish, available studies examined a single time-point only. Consequently, it was unclear whether lower effect concentrations for certain downstream KEs, relative to upstream were due to a lack of dose-response concordance, or due to decreased sensitivity of the upstream later in the exposure time-course.
While not directly addressing dose-response concordance, the dependence of the key events on the concentration of the androgen agonist has been established for all key events starting at and down-stream of reduced T synthesis. However, to date we are not aware of any studies that have established a concentration-response relationship between exposure to non-endogenous AR agonists (e.g., xenobiotics, pharmaceuticals) and circulating gonadotropin concentrations in fish or other vertebrates.
- Exposure of female fathead minnows to the AR agonist 17β-trenbolone for 21 d caused concentration-dependent reductions in circulating T, E2, and VTG concentrations over a range from 0.005 to 0.5 μg/L. The concentration response for all three variables had a “U”-shaped concentration response curve which may indicate concentration-dependent differences in the feedback response and/or compensatory processes. Histological evidence of reduced VTG uptake and reduced gonad stage were evident, although the concentration-response of histological effects was not determined. Despite the “U”-shaped concentration-response at the biochemical level, concentration-dependent reductions in cumulative fecundity were observed (Ankley et al. 2003). Effective concentrations were consistent with those causing phenotypic masculinization in female fish.
- Jensen et al. (2006) also demonstrated concentration-dependent reductions in circulating T, E2, and VTG following 21 d of in vivo exposure to 17α-trenbolone (Jensen et al. 2006).
- In a time-course experiment in which female fathead minnows were exposed to to 33 or 472 ng 17β-trenbolone/L ex vivo T, ex vivo E2, plasma E2, and plasma VTG all showed concentration-dependent reductions that were consistent with the AOP (Ekman et al. 2011).
- Exposure of female fathead minnows to spironolactone, a pharmaceutical that binds the fathead minnow AR, for 21 d caused concentration-dependent reductions in cumulative fecundity, plasma VTG and VTG mRNA expression, and plasma E2 concentrations. The frequency and severity of females with decreased yolk accumulation, and increased oocyte atresia was concentration-dependent. The chemical also induced phenotypic masculinization in female fish. (Lalone et al. 2013).
- Exposure of female medaka to spironolactone caused concentration-dependent reductions in cumulative fecundity and VTG mRNA expression (impacts on steroid hormone concentrations were not measured). Spironolactone also caused phenotypic masculinization of female medaka (Lalone et al. 2013).
- Temporal concordance among the key events and adverse effect: Temporal concordance between activation of the AR as a nuclear transcription factor and onset of a negative feedback response resulting in decreased gonadotropin secretion has not been established. Temporal concordance of the key events starting with reduced T biosynthesis and proceeding through reductions in plasma vitellogenin has been established Concordance table. Temporal concordance beyond the key event of reductions in plasma vitellogenin has not been established, in large part due to disconnect in the time-scales over which the events can be measured. For example, most small fish used in reproductive toxicity testing can spawn anywhere from once daily to several days per week. Given the variability in daily spawning rates, it is neither practical nor effective to evaluate cumulative fecundity at a time scale shorter than roughly a week. Since the impacts at lower levels of biological organization can be detected within hours of exposure, lack of impact on cumulative fecundity before the other key events are impacted cannot be effectively measured. Overall, among those key events whose temporal concordance can reasonably be evaluated based on currently available data, the temporal profile observed is consistent with the AOP.
- Consistency: We are aware of no cases where the pattern of key events described was observed without also observing a significant impact on cumulative fecundity. Due to variability in the cumulative fecundity endpoint and potential compensatory responses ((Villeneuve et al. 2009; Villeneuve et al. 2013; Ankley et al. 2009b; Zhang et al. 2008; Ekman et al. 2012), the cumulative fecundity endpoint can be less sensitive than key events measured at lower levels of biological organization. Nonetheless, the occurrence of the final adverse outcome when the other key events are observed is very consistent. The final adverse effect is not specific to this AOP. Many of the key events included in this AOP overlap with AOPs linking other molecular initiating events to reproductive dysfunction in small fish.
- In general, there is a consistent body of evidence linking exposure to an AR agonist to decreased T synthesis, E2 synthesis, circulating E2 and VTG concentrations, and cumulative fecundity in female fish. For example, the association between 17β-trenbolone exposure and reduced vitellogenin concentrations in females has been replicated in over a dozen independent experiments (Ekman et al. 2011; Ankley et al. 2003; Jensen et al. 2006; Ankley et al. 2010; Hemmer et al. 2008; Seki et al. 2006; Brockmeier et al. 2013). However, relatively few exogenous, non-aromatizable, AR agonists have been tested. Other than recent work with spironolactone (Lalone et al. 2013), we are not aware of the profile of responses being demonstrated for other AR agonists.
- Uncertainties, inconsistencies, and data gaps: There are three major areas of uncertainty and data gaps in the current AOP. *First, there remains considerable uncertainty as to the specific mechanism(s) through which AR agonism elicits a negative feedback response at the level of the hypothalamus and/or pituitary. There is also a substantial data gap relative to establishing that exposure to an AR agonist like 17β-trenbolone causes concentration-dependent reductions in circulating gonadotropins.
- The second major uncertainty in this AOP relates to whether there is a direct biological linkage between impaired VTG uptake into oocytes and impaired spawning/reduced cumulative fecundity. Plausible biological connections have been hypothesized, but have not yet been tested experimentally.
- A third uncertainty pertains to the chemical domain of applicability. In vivo, a number of chemicals that are detected as androgens in in vitro screening assays such as receptor binding assays or ligand-activated transcriptional assay can be aromatized to functional estrogens. Thus, in vivo such compounds may produce a profile of effects more consistent with estrogen receptor activation than AR activation or may produced mixed effects characteristic of either estrogen or androgen exposures. Examples of such aromatizable androgens include, testosterone, methyltestosterone, and androstenedione. Consequently, caution is warranted in applying this AOP based on in vitro screening data alone, without consideration for possible conversion to estrogens.
Essentiality of the Key Events
- In general, few studies have directly addressed the essentiality of the proposed sequence of key events.
- Ekman et al. 2011 provide evidence that in fathead minnow, cessation of trenbolone exposure resulted in recovery of plasma E2 and VTG concentrations which were depressed by continuous exposure to 17beta trenbolone. This provides some support for the essentiality of these two key events.
- Essentiality of the proposed negative feedback key event is supported by experimental work that evaluated the ability of AR agonists to reduce T or E2 production in vitro. In vitro exposure of fathead minnow ovary tissue to 17β-trenbolone or spironolactone does not cause reductions in T or E2 synthesis at concentrations comparable to those that produce significant responses in vivo (i.e., at non-cytotoxic concentrations; D.L. Villeneuve, unpublished data; C.A. LaLone unpublished data), nor are there any known reports of 17β-trenbolone directly inhibiting steroid biosynthesis. When tested in an in vitro steroidogenesis assay using H295R adrenal carcinoma cells, trenbolone caused a concentration-dependent increase in estradiol production, as opposed to any reductions in steroid hormone concentrations, an effect that was concurrent with increased transcription of CYP19 (aromatase) in the cell line (Gracia et al. 2007).
- Assessment of quantitative understanding of the AOP: At present, the quantitative understanding of the AOP is insufficient to directly link a measure of chemical potency as an AR agonist (e.g., as measured in a transcriptional activation assay) to a predicted effect concentration at the level of cumulative fecundity. However, a number of mechanistic and statistical models are sufficiently developed to facilitate predictions of cumulative outcomes based on intermediate key event measurements such as circulating vitellogenin concentrations. Because the current models were developed based on a fairly limited range of model compounds and species, the general applicability and degree of accuracy and precision in the model-derived predictions remains uncertain.
Applicability of the AOP
Domain(s) of Applicability
- Chemical: This AOP applies to non-aromatizable androgens. Compounds which can bind the AR in vitro, but are converted to high potency estrogens in vivo through aromatization do not produce the profile of effects described in the present AOP (e.g., methyltestosterone [Ankley et al. 2001; Pawlowski et al. 2004]; androstenedione [OECD 2007]).
- Sex: The AOP applies to females only.
- Life stages: The relevant life stages for this AOP are reproductively mature adults. This AOP does not apply to adult stages that lack a sexually mature ovary, for example as a result of seasonal or environmentally-induced gonadal senescence (i.e., through control of temperature, photo-period, etc. in a laboratory setting).
- Taxonomic: At present, the assumed taxonomic applicability domain of this AOP is iteroparous teleost fish species. However, to date the majority of toxicological data on which this AOP is based has been limited to two small fish species, fathead minnow (Pimephales promelas) and Japanese medaka (Oryzias latipes). Given that species dependent differences in endocrine feedback responses, likely associated with different reproductive strategies, have been reported the applicability domain may prove more restricted than currently assumed.
- Reductions in plasma VTG concentrations and/or hepatic VTG mRNA abundance in females following exposure to 17β-trenbolone has been observed in Pimephales promelas, Oryzias latipes, Danio rerio, (Seki et al. 2006), Cyprinodon variegatus (Hemmer et al. 2008), Gambusia holbrooki and Gambusia affinis (Brockmeier et al. 2013)
- European eel may be an exception to the generalizability of the negative feedback response to a non-aromatizable xenoandrogen (Huang et al. 1997).
Considerations for Potential Applications of the AOP (optional)
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