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Event: 25

Key Event Title

The KE title should describe a discrete biological change that can be measured. It should generally define the biological object or process being measured and whether it is increased, decreased, or otherwise definably altered relative to a control state. For example “enzyme activity, decreased”, “hormone concentration, increased”, or “growth rate, decreased”, where the specific enzyme or hormone being measured is defined. More help

Agonism, Androgen receptor

Short name
The KE short name should be a reasonable abbreviation of the KE title and is used in labelling this object throughout the AOP-Wiki. The short name should be less than 80 characters in length. More help
Agonism, Androgen receptor

Biological Context

Structured terms, selected from a drop-down menu, are used to identify the level of biological organization for each KE. Note, KEs should be defined within a particular level of biological organization. Only KERs should be used to transition from one level of organization to another. Selection of the level of biological organization defines which structured terms will be available to select when defining the Event Components (below). More help

Cell term

Further information on Event Components and Biological Context may be viewed on the attached pdf.The biological context describes the location/biological environment in which the event takes place.  For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable.  For individual/population events, the organ context is not applicable. More help

Organ term

Further information on Event Components and Biological Context may be viewed on the attached pdf.The biological context describes the location/biological environment in which the event takes place.  For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable.  For individual/population events, the organ context is not applicable. More help

Key Event Components

Further information on Event Components and Biological Context may be viewed on the attached pdf.Because one of the aims of the AOP-KB is to facilitate de facto construction of AOP networks through the use of shared KE and KER elements, authors are also asked to define their KEs using a set of structured ontology terms (Event Components). In the absence of structured terms, the same KE can readily be defined using a number of synonymous titles (read by a computer as character strings). In order to make these synonymous KEs more machine-readable, KEs should also be defined by one or more “event components” consisting of a biological process, object, and action with each term originating from one of 22 biological ontologies (Ives, et al., 2017; See List). Biological process describes dynamics of the underlying biological system (e.g., receptor signalling). The biological object is the subject of the perturbation (e.g., a specific biological receptor that is activated or inhibited). Action represents the direction of perturbation of this system (generally increased or decreased; e.g., ‘decreased’ in the case of a receptor that is inhibited to indicate a decrease in the signalling by that receptor).Note that when editing Event Components, clicking an existing Event Component from the Suggestions menu will autopopulate these fields, along with their source ID and description. To clear any fields before submitting the event component, use the 'Clear process,' 'Clear object,' or 'Clear action' buttons. If a desired term does not exist, a new term request may be made via Term Requests. Event components may not be edited; to edit an event component, remove the existing event component and create a new one using the terms that you wish to add. More help
Process Object Action
androgen receptor activity androgen receptor increased

Key Event Overview

AOPs Including This Key Event

All of the AOPs that are linked to this KE will automatically be listed in this subsection. This table can be particularly useful for derivation of AOP networks including the KE. Clicking on the name of the AOP will bring you to the individual page for that AOP. More help
AOP Name Role of event in AOP Point of Contact Author Status OECD Status
Androgen receptor agonism leading to reproductive dysfunction MolecularInitiatingEvent Dan Villeneuve (send email) Open for citation & comment TFHA/WNT Endorsed


This is a structured field used to identify specific agents (generally chemicals) that can trigger the KE. Stressors identified in this field will be linked to the KE in a machine-readable manner, such that, for example, a stressor search would identify this as an event the stressor can trigger. NOTE: intermediate or downstream KEs in one AOP may function as MIEs in other AOPs, meaning that stressor information may be added to the KE description, even if it is a downstream KE in the pathway currently under development.Information concerning the stressors that may trigger an MIE can be defined using a combination of structured and unstructured (free-text) fields. For example, structured fields may be used to indicate specific chemicals for which there is evidence of an interaction relevant to this MIE. By linking the KE description to a structured chemical name, it will be increasingly possible to link the MIE to other sources of chemical data and information, enhancing searchability and inter-operability among different data-sources and knowledgebases. The free-text section “Evidence for perturbation of this MIE by stressor” can be used both to identify the supporting evidence for specific stressors triggering the MIE as well as to define broad chemical categories or other properties that classify the stressors able to trigger the MIE for which specific structured terms may not exist. More help

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) can be selected from an ontology. In many cases, individual species identified in these structured fields will be those for which the strongest evidence used in constructing the AOP was available in relation to this KE. More help
Term Scientific Term Evidence Link
fathead minnow Pimephales promelas High NCBI
medaka Oryzias latipes High NCBI

Life Stages

The structured ontology terms for life-stage are more comprehensive than those for taxa, but may still require further description/development and explanation in the free text section. More help
Life stage Evidence
Adult, reproductively mature High

Sex Applicability

No help message More help
Term Evidence
Female High

Key Event Description

A description of the biological state being observed or measured, the biological compartment in which it is measured, and its general role in the biology should be provided. For example, the biological state being measured could be the activity of an enzyme, the expression of a gene or abundance of an mRNA transcript, the concentration of a hormone or protein, neuronal activity, heart rate, etc. The biological compartment may be a particular cell type, tissue, organ, fluid (e.g., plasma, cerebrospinal fluid), etc. The role in the biology could describe the reaction that an enzyme catalyses and the role of that reaction within a given metabolic pathway; the protein that a gene or mRNA transcript codes for and the function of that protein; the function of a hormone in a given target tissue, physiological function of an organ, etc. Careful attention should be taken to avoid reference to other KEs, KERs or AOPs. Only describe this KE as a single isolated measurable event/state. This will ensure that the KE is modular and can be used by other AOPs, thereby facilitating construction of AOP networks. More help

Site of action: The molecular site of action is the ligand binding domain of the AR. This particular key event specifically refers to interaction with nuclear AR.  Downstream KE responses to activation of membrane ARs may be different. The cellular site of action for the molecular initiating event is undefined.

Responses at the macromolecular level: Binding of a ligand, including xenobiotics that act as AR agonists, to the cytosolic AR mediates a conformational shift that facilitates dissociation from accompanying heat shock proteins and dimerization with another AR (Prescott and Coetzee 2006; Claessens et al. 2008; Centenera et al. 2008). Homodimerization unveils a nuclear localization sequence, allowing the AR-ligand complex to translocate to the nucleus and bind to androgen-response elements (AREs) (Claessens et al. 2008; Cutress et al. 2008). This elicits recruitment of additional transcription factors and transcriptional activation of androgen-responsive genes (Heemers and Tindall 2007).

AR paralogs:

  • Most vertebrates have a single gene coding for nuclear AR. However, most fish have two AR genes (AR-A, AR-B) as a result of a whole genome duplication event after the split of Acipenseriformes from teleosts but before the divergence of Osteoglossiformes (Douard et al. 2008).
  • AR-B has been lost in Cypriniformes, Siluriformes, Characiformes, and Salmoniformes (Douard et al. 2008).
  • In Percomorphs, AR-B has accumulated significant substitutions in the both ligand binding and DNA binding domains (Douard et al. 2008).
  • Differential ligand selectivity and subcellular localization has been reported for AR paralogs in some fish species (e.g., Bain et al. 2015), but the difference is not easily generalized based on available data in the literature. 

How It Is Measured or Detected

One of the primary considerations in evaluating AOPs is the relevance and reliability of the methods with which the KEs can be measured. The aim of this section of the KE description is not to provide detailed protocols, but rather to capture, in a sentence or two, per method, the type(s) of measurements that can be employed to evaluate the KE and the relative level of scientific confidence in those measurements. Methods that can be used to detect or measure the biological state represented in the KE should be briefly described and/or cited. These can range from citation of specific validated test guidelines, citation of specific methods published in the peer reviewed literature, or outlines of a general protocol or approach (e.g., a protein may be measured by ELISA).Key considerations regarding scientific confidence in the measurement approach include whether the assay is fit for purpose, whether it provides a direct or indirect measure of the biological state in question, whether it is repeatable and reproducible, and the extent to which it is accepted in the scientific and/or regulatory community. Information can be obtained from the OECD Test Guidelines website and the EURL ECVAM Database Service on Alternative Methods to Animal Experimentation (DB-ALM). ?


  • In vitro methods:
    • OECD Test No. 458: Stably transfected human androgen receptor transcriptional activation assay for detection of androgen agonists and antagonists has been reviewed and validated by OECD and is well suited for detection of this key event (OECD 2016).
    • Binding to the androgen receptor can be directly measured in cell free systems based on displacement of a radio-labeled standard (generally testosterone or DHT) in a competitive binding assay (e.g., (Olsson et al. 2005; Sperry and Thomas 1999; Wilson et al. 2007; Tilley et al. 1989; Kim et al. 2010).
    • Cell based transcriptional activation assays are typically required to differentiate agonists from antagonists, in vitro. A number of reporter gene assays have been developed and used to screen chemicals for AR agonist and/or antagonist activity (e.g., (Wilson et al. 2002; van der Burg et al. 2010; Mak et al. 1999; Araki et al. 2005).
    • Expression of androgen responsive proteins like spiggin in primary cell cultures has also been used to detect AR agonist activity (Jolly et al. 2006).
  • In vivo methods
    • In fish, phenotypic masculinization of females has frequently been used as an indirect measurement of in vivo androgen receptor agonism.
      • Development of nuptial tubercles, a dorsal fatpad, and a characteristic banding pattern has been observed in female fathead minnows exposed to androgen agonists (Ankley et al. 2003; Jensen et al. 2006; Ankley et al. 2010; LaLone et al. 2013; OECD 2012).
      • Anal fin elongation in female western mosquitofish (Gambusia affinis) has similarly been viewed as evidence of AR activation (Raut et al. 2011; Sone et al. 2005).
      • In medaka, development of papillary processes, which normally only appear on the second to seventh or eighth fin aray of the anal fin, has also been used as an indirect measure of androgen receptor agonism (OECD 2012).
      • Production of the nest building glue, spiggin, in three female 3-spined sticklebacks (Gasterosteus aculeatus) has also been well documented as an indicator of androgen receptor agonism (Jakobsson et al. 1999; Hahlbeck et al. 2004). Quantification of the spiggin protein in exposed female 3-spined stickleback or green fluorescence protein expression in a transgenic spg1-gfp medaka line (Sébillot et al. 2014) can be used to detect androgen receptor agonism.
  • High Throughput Screening
    • ​Measures of AR agonism have been included in high throughput screening programs, such as US EPA's Toxcast program. Toxcast assays relevant for screening chemicals for their ability to bind and/or activate the AR include:
      • ATG_AR_TRANS A cell based assay that can differentiate agonism from antagonism
      • NVS_NR_hAR A cell free assay using recombinant human AR. Can detect binding, but cannot distinguish agonism from antagonism.
      • NVS_NR_rAR A cell free assay using recombinant rat AR. Can detect binding, but cannot distinguish agonism from antagonism.
      • OT_AR_ARELUC_AG_1440 A cell based assay that measures expression of a reporter gene under control of androgen-responsive elements. Can distinguish agonism from antagonism.
      • Tox21_AR_BLA_Agonist_ratio A cell based assay with an inducible reporter. Can distinguish agonists from antagonists.
      • Tox21_AR_LUC_MDAKB2_agonist A cell based assay with an inducible reporter. Can distinguish agonists from antagonists.
    • Assay descriptions

Domain of Applicability

This free text section should be used to elaborate on the scientific basis for the indicated domains of applicability and the WoE calls (if provided). While structured terms may be selected to define the taxonomic, life stage and sex applicability (see structured applicability terms, above) of the KE, the structured terms may not adequately reflect or capture the overall biological applicability domain (particularly with regard to taxa). Likewise, the structured terms do not provide an explanation or rationale for the selection. The free-text section on evidence for taxonomic, life stage, and sex applicability can be used to elaborate on why the specific structured terms were selected, and provide supporting references and background information.  More help

Taxonomic applicability: Androgen receptor orthologs are primarily limited to vertebrates (Baker 1997; Thornton 2001; Eick and Thornton 2011; Markov and Laudet 2011). Therefore, this MIE would generally be viewed as relevant to vertebrates, but not invertebrates.

Evidence for Perturbation by Stressor

Overview for Molecular Initiating Event

When a specific MIE can be defined (i.e., the molecular target and nature of interaction is known), in addition to describing the biological state associated with the MIE, how it can be measured, and its taxonomic, life stage, and sex applicability, it is useful to list stressors known to trigger the MIE and provide evidence supporting that initiation. This will often be a list of prototypical compounds demonstrated to interact with the target molecule in the manner detailed in the MIE description to initiate a given pathway (e.g., 2,3,7,8-TCDD as a prototypical AhR agonist; 17α-ethynyl estradiol as a prototypical ER agonist). Depending on the information available, this could also refer to chemical categories (i.e., groups of chemicals with defined structural features known to trigger the MIE). Known stressors should be included in the MIE description, but it is not expected to include a comprehensive list. Rather initially, stressors identified will be exemplary and the stressor list will be expanded over time. For more information on MIE, please see pages 32-33 in the User Handbook.

Characterization of chemical properties: Androgen receptor binding chemicals can be grouped into two broad structural domains, steroidal and non-steroidal (Yin et al. 2003). Steroidal androgens consist primarily of testosterone and its derivatives (Yin et al. 2003). Many of the non-steroidal AR binding chemicals studied are derivatives of well known non-steroidal AR antagonists like bicalutamide, hydroxyflutamide, and nilutamide (Yin et al. 2003). Nonetheless, a number of QSARs and SARs that consider AR binding of both these pharmaceutical agents as well as environmental chemicals have been developed (Waller et al. 1996; Serafimova et al. 2002; Todorov et al. 2011; Hong et al. 2003; Bohl et al. 2004). However, it has been shown that very minor structural differences can dramatically impact function as either an agonist or antagonist (Yin et al. 2003; Bohl et al. 2004; Norris et al. 2009), making it difficult at present to predict agonist versus antagonist activity based on chemical structure alone.

In vivo considerations: A variety of steroidal androgens can be converted to estrogens in vitro through the action of cytochrome P450 19 (aromatase). Structures subject to aromatization may behave in vivo as estrogens despite exhibiting potent androgen receptor agonism in vitro.


Chemical is a non-aromatizable androgen.


List of the literature that was cited for this KE description. Ideally, the list of references, should conform, to the extent possible, with the OECD Style Guide ( (OECD, 2015). More help
  • Ankley GT, Gray LE. Cross-species conservation of endocrine pathways: a critical analysis of tier 1 fish and rat screening assays with 12 model chemicals. Environ Toxicol Chem. 2013 Apr;32(5):1084-7. doi: 10.1002/etc.2151. Epub 2013 Mar 19. PubMed PMID: 23401061.
  • Ankley GT, Jensen KM, Kahl MD, Durhan EJ, Makynen EA, Cavallin JE, Martinović D, Wehmas LC, Mueller ND, Villeneuve DL. Use of chemical mixtures to differentiate mechanisms of endocrine action in a small fish model. Aquat Toxicol. 2010 Sep 1;99(3):389-96. doi: 10.1016/j.aquatox.2010.05.020. Epub 2010 Jun 4. PubMed PMID: 20573408.
  • Ankley GT, Jensen KM, Makynen EA, Kahl MD, Korte JJ, Hornung MW, Henry TR, Denny JS, Leino RL, Wilson VS, Cardon MC, Hartig PC, Gray LE. Effects of the androgenic growth promoter 17-beta-trenbolone on fecundity and reproductive endocrinology of the fathead minnow. Environ Toxicol Chem. 2003 Jun;22(6):1350-60. PubMed PMID: 12785594.
  • Araki N, Ohno K, Nakai M, Takeyoshi M, Iida M. 2005. Screening for androgen receptor activities in 253 industrial chemicals by in vitro reporter gene assays using AR-EcoScreen cells. Toxicology in vitro : an international journal published in association with BIBRA 19(6): 831-842.
  • Bain PA, Ogino Y, Miyagawa S, Iguchi T, Kumar A. Differential ligand selectivity of androgen receptors α and β from Murray-Darling rainbowfish (Melanotaenia fluviatilis). Gen Comp Endocrinol. 2015 Feb 1;212:84-91. doi: 10.1016/j.ygcen.2015.01.024. PubMed PMID: 25644213.
  • Baker ME. 1997. Steroid receptor phylogeny and vertebrate origins. Molecular and cellular endocrinology 135(2): 101-107.
  • Bohl CE, Chang C, Mohler ML, Chen J, Miller DD, Swaan PW, et al. 2004. A ligand-based approach to identify quantitative structure-activity relationships for the androgen receptor. Journal of medicinal chemistry 47(15): 3765-3776.
  • Centenera MM, Harris JM, Tilley WD, Butler LM. 2008. The contribution of different androgen receptor domains to receptor dimerization and signaling. Molecular endocrinology 22(11): 2373-2382.
  • Claessens F, Denayer S, Van Tilborgh N, Kerkhofs S, Helsen C, Haelens A. 2008. Diverse roles of androgen receptor (AR) domains in AR-mediated signaling. Nuclear receptor signaling 6: e008.
  • Cutress ML, Whitaker HC, Mills IG, Stewart M, Neal DE. 2008. Structural basis for the nuclear import of the human androgen receptor. Journal of cell science 121(Pt 7): 957-968.
  • Douard V, Brunet F, Boussau B, Ahrens-Fath I, Vlaeminck-Guillem V, Haendler B, Laudet V, Guiguen Y. The fate of the duplicated androgen receptor in fishes: a late neofunctionalization event? BMC Evol Biol. 2008 Dec 18;8:336. doi: 10.1186/1471-2148-8-336. PubMed PMID: 19094205
  • Eick GN, Thornton JW. 2011. Evolution of steroid receptors from an estrogen-sensitive ancestral receptor. Molecular and cellular endocrinology 334(1-2): 31-38.
  • Hahlbeck E, Katsiadaki I, Mayer I, Adolfsson-Erici M, James J, Bengtsson BE. The juvenile three-spined stickleback (Gasterosteus aculeatus L.) as a model organism for endocrine disruption II--kidney hypertrophy, vitellogenin and spiggin induction. Aquat Toxicol. 2004 Dec 20;70(4):311-26
  • Hong H, Fang H, Xie Q, Perkins R, Sheehan DM, Tong W. 2003. Comparative molecular field analysis (CoMFA) model using a large diverse set of natural, synthetic and environmental chemicals for binding to the androgen receptor. SAR and QSAR in environmental research 14(5-6): 373-388.
  • Jakobsson, S., Borg, B., Haux, C. et al. Fish Physiology and Biochemistry (1999) 20: 79. doi:10.1023/A:1007776016610
  • Jensen KM, Makynen EA, Kahl MD, Ankley GT. Effects of the feedlot contaminant 17alpha-trenbolone on reproductive endocrinology of the fathead minnow. Environ Sci Technol. 2006 May 1;40(9):3112-7. PubMed PMID: 16719119.
  • Jolly C, Katsiadaki I, Le Belle N, Mayer I, Dufour S. 2006. Development of a stickleback kidney cell culture assay for the screening of androgenic and anti-androgenic endocrine disrupters. Aquatic toxicology 79(2): 158-166.
  • Kim TS, Yoon CY, Jung KK, Kim SS, Kang IH, Baek JH, et al. 2010. In vitro study of Organization for Economic Co-operation and Development (OECD) endocrine disruptor screening and testing methods- establishment of a recombinant rat androgen receptor (rrAR) binding assay. The Journal of toxicological sciences 35(2): 239-243.
  • LaLone CA, Villeneuve DL, Cavallin JE, Kahl MD, Durhan EJ, Makynen EA, Jensen KM, Stevens KE, Severson MN, Blanksma CA, Flynn KM, Hartig PC, Woodard JS, Berninger JP, Norberg-King TJ, Johnson RD, Ankley GT. Cross-species sensitivity to a novel androgen receptor agonist of potential environmental concern, spironolactone. Environ Toxicol Chem. 2013 Nov;32(11):2528-41. doi: 10.1002/etc.2330. Epub 2013 Sep 6. PubMed PMID: 23881739.
  • Mak P, Cruz FD, Chen S. 1999. A yeast screen system for aromatase inhibitors and ligands for androgen receptor: yeast cells transformed with aromatase and androgen receptor. Environmental health perspectives 107(11): 855-860.
  • Markov GV, Laudet V. 2011. Origin and evolution of the ligand-binding ability of nuclear receptors. Molecular and cellular endocrinology 334(1-2): 21-30.
  • Norris JD, Joseph JD, Sherk AB, Juzumiene D, Turnbull PS, Rafferty SW, et al. 2009. Differential presentation of protein interaction surfaces on the androgen receptor defines the pharmacological actions of bound ligands. Chemistry & biology 16(4): 452-460.
  • OECD (2012), Test No. 229: Fish Short Term Reproduction Assay, OECD Publishing, Paris. DOI:
  • OECD (2016), Test No. 458: Stably Transfected Human Androgen Receptor Transcriptional Activation Assay for Detection of Androgenic Agonist and Antagonist Activity of Chemicals, OECD Publishing, Paris. DOI:
  • Olsson P-E, Berg A, von Hofsten J, Grahn B, Hellqvist A, Larsson A, et al. 2005. Molecular cloning and characterization of a nuclear androgen receptor activated by 11-ketotestosterone. Reproductive Biology and Endocrinology 3: 1-17.
  • Prescott J, Coetzee GA. 2006. Molecular chaperones throughout the life cycle of the androgen receptor. Cancer letters 231(1): 12-19.
  • Serafimova R, Walker J, Mekenyan O. 2002. Androgen receptor binding affinity of pesticide "active" formulation ingredients. QSAR evaluation by COREPA method. SAR and QSAR in environmental research 13(1): 127-134.
  • Sone K, Hinago M, Itamoto M, Katsu Y, Watanabe H, Urushitani H, Tooi O, Guillette LJ Jr, Iguchi T. Effects of an androgenic growth promoter 17beta-trenbolone on masculinization of Mosquitofish (Gambusia affinis affinis). Gen Comp Endocrinol. 2005 Sep 1;143(2):151-60. Epub 2005 Apr 13. PubMed PMID: 16061073.
  • Sperry TS, Thomas P. 1999. Identification of two nuclear androgen receptors in kelp bass (Paralabrax clathratus) and their binding affinities for xenobiotics: comparison with Atlantic croaker (Micropogonias undulatus) androgen receptors. Biology of reproduction 61(4): 1152-1161.
  • Stanko JP, Angus RA. In vivo assessment of the capacity of androstenedione to masculinize female mosquitofish (Gambusia affinis) exposed through dietary and static renewal methods. Environ Toxicol Chem. 2007 May;26(5):920-6. PubMed PMID: 17521138.
  • Thornton JW. 2001. Evolution of vertebrate steroid receptors from an ancestral estrogen receptor by ligand exploitation and serial genome expansions. Proceedings of the National Academy of Sciences of the United States of America 98(10): 5671-5676.
  • Tilley WD, Marcelli M, Wilson JD, McPhaul MJ. 1989. Characterization and expression of a cDNA encoding the human androgen receptor. Proceedings of the National Academy of Sciences of the United States of America 86(1): 327-331.
  • Todorov M, Mombelli E, Ait-Aissa S, Mekenyan O. 2011. Androgen receptor binding affinity: a QSAR evaluation. SAR and QSAR in environmental research 22(3): 265-291.
  • van der Burg B, Winter R, Man HY, Vangenechten C, Berckmans P, Weimer M, et al. 2010. Optimization and prevalidation of the in vitro AR CALUX method to test androgenic and antiandrogenic activity of compounds. Reproductive toxicology 30(1): 18-24.
  • Waller CL, Juma BW, Gray EJ, Kelce WR. 1996. Three-dimensional quantitative structure-activity relationships for androgen receptor ligands. Toxicology and Applied Pharmacolgy 137: 219-227.
  • Wilson VS, Bobseine K, Lambright CR, Gray LE. 2002. A novel cell line, MDA-kb2, that stably expresses an androgen- and glucocorticoid-responsive reporter for the detection of hormone receptor agonists and antagonists. Toxicological Sciences 66: 69-81.
  • Wilson VS, Cardon MC, Gray LE, Jr., Hartig PC. 2007. Competitive binding comparison of endocrine-disrupting compounds to recombinant androgen receptor from fathead minnow, rainbow trout, and human. Environmental toxicology and chemistry / SETAC 26(9): 1793-1802.
  • Yin D, He Y, Perera MA, Hong SS, Marhefka C, Stourman N, et al. 2003. Key structural features of nonsteroidal ligands for binding and activation of the androgen receptor. Molecular pharmacology 63(1): 211-223.