Decrease, androgen receptors (AR) activation

90357-06-5

LKJPYSCBVHEWIU-UHFFFAOYSA-N

Bicalutamide

Casodex CDX Propanamide, N-[4-cyano-3-(trifluoromethyl)phenyl]-3-[(4-fluorophenyl)sulfonyl]-2-hydroxy-2-methyl-

DTXSID2022678

427-51-0

UWFYSQMTEOIJJG-FDTZYFLXSA-N

Cyproterone acetate

3'H-Cyclopropa[1,2]pregna-1,4,6-triene-3,20-dione, 17-(acetyloxy)-6-chloro-1,2-dihydro-, (1β,2β)- 1,2α-Methylene-6-chloro-17α-acetoxy-4,6-pregnadiene-3,20-dione 1,2α-Methylene-6-chloro-pregna-4,6-diene-3,20-dione 17α-acetate 1,2α-Methylene-6-chloro-Δ4,6-pregnadien-17α-ol-3,20-dione acetate 17-acetate de 6-chloro-1-β,2-β-dihydro-17-hydroxy-3'H-cyclopropa[1,2]pregna-1,4,6-triene-3,20-dione 17-acetato de 6-cloro-1-β,2-β-dihidro-17-hidroxi-3'H-ciclopropa[1,2]pregna-1,4,6-trieno-3,20-diona 17α-Acetoxy-6-chloro-1α,2α-methylenepregna-4,6-diene-3,20-dione 3'H-Cyclopropa[1,2]pregna-1,4,6-triene-3,20-dione 3'H-Cyclopropa[1,2]pregna-1,4,6-triene-3,20-dione, 6-chloro-1β,2β-dihydro-17-hydroxy-, acetate 6-Chlor-1-β,2-β-dihydro-17-hydroxy-3'H-cyclopropa[1,2]pregna-1,4,6-trien-3,20-dion-17-acetat 6-Chloro-1,2α-methylene-17α-hydroxy-Δ6-progesterone acetate 6-Chloro-1,2α-methylene-6-dehydro-17α-hydroxyprogesterone acetate 6-Chloro-17-hydroxy-1α,2α-methylenepregna-4,6-diene-3,20-dione acetate 6-chloro-1-β,2-β-dihydro-17-hydroxy-3'H-cyclopropa[1,2]pregna-1,4,6-triene-3,20-dione 17-acetate Androcur Cyprostat Cyproterone 17-O-acetate Cyproterone 17α-acetate Cyproviron NSC 81430 Pregna-4,6-diene-3,20-dione, 6-chloro-17-hydroxy-1α,2α-methylene-, acetate

DTXSID5020366

133855-98-8

ZMYFCFLJBGAQRS-UHFFFAOYNA-N

ZMYFCFLJBGAQRS-UHFFFAOYSA-N

Epoxiconazole

DTXSID1040372

13311-84-7

MKXKFYHWDHIYRV-UHFFFAOYSA-N

Flutamide

Propanamide, 2-methyl-N-[4-nitro-3-(trifluoromethyl)phenyl]- 4-Nitro-3-(trifluoromethyl)isobutyranilide 4'-Nitro-3'-trifluoromethylisobutyranilide Eulexin Flucinom Flutamid flutamida m-Propionotoluidide, α,α,α-trifluoro-2-methyl-4'-nitro- N-(Isopropylcarbonyl)-4-nitro-3-trifluoromethylaniline Niftholide Niftolide NSC 147834 NSC 215876

DTXSID7032004

85509-19-9

FQKUGOMFVDPBIZ-UHFFFAOYSA-N

Flusilazole

NuStar

DTXSID3024235

67747-09-5

TVLSRXXIMLFWEO-UHFFFAOYSA-N

Prochloraz

1H-Imidazole-1-carboxamide, N-propyl-N-[2-(2,4,6-trichlorophenoxy)ethyl]- BTS 40542-7877 N-propil-N-[2-(2,4,6-triclorofenoxi)etil]-1H-imidazol-1-carboxamida N-propyl-N-[2-(2,4,6-trichlorophenoxy)ethyl]-1H-imidazole-1-carboxamide N-Propyl-N-[2-(2,4,6-trichlorophenoxy)ethyl-1H-imidazole-1-carboxamide N-Propyl-N-[2-(2,4,6-trichlorphenoxy)ethyl]-1H-imidazol-1-carboxamid Plocloraz Prelude Sportak Sportake

DTXSID4024270

60207-90-1

STJLVHWMYQXCPB-UHFFFAOYNA-N

STJLVHWMYQXCPB-UHFFFAOYSA-N

Propiconazole

ppz 1H-1,2,4-Triazole, 1-[[2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-yl]methyl]- (.+-.)-1-[2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-yl-methyl]-1H-1,2,4-triazole (.+-.)-1-[2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-ylmethyl]-1H-1,2,4-triazole 1-[[2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-yl]methyl]-1H-1,2,4-triazole 1-[[2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolane-2-yl]methyl]-1H-1,2,4-triazole 1-[[2-(2,4-Dichlorphenyl)-4-propyl-1,3-dioxolan-2-yl]methyl]-1H-1,2,4-triazol 1-[[2-(2,4-diclorofenil)-4-propil-1,3-dioxolan-2-il]metil]-1H-1,2,4-triazol Bamper 25EC Banner Maxx Cane Sett Treatment Fertilome Liquid Systemic Fungicide Microban PZ Microban S 2140 Mycostat P Proconazole PROPICONAZOL Tilt Premium Wocosen Technical Wocosin Wocosin 50TK

DTXSID8024280

131983-72-7

PPDBOQMNKNNODG-UHFFFAOYNA-N

PPDBOQMNKNNODG-UHFFFAOYSA-N

Triticonazole

5-[(4-Chlorophenyl)methylene]-2,2-dimethyl-1-(1H-1,2,4-triazol-1-ylmethyl)cyclopentanol

DTXSID0032655

107534-96-3

PXMNMQRDXWABCY-UHFFFAOYNA-N

PXMNMQRDXWABCY-UHFFFAOYSA-N

Tebuconazole

1H-1,2,4-Triazole-1-ethanol, .alpha.-(2-(4-chlorophenyl)ethyl)-.alpha. +- 1H-1,2,4-Triazole-1-ethanol, α-[2-(4-chlorophenyl)ethyl]-α-(1,1-dimethylethyl)- (.+-.)-Tebuconazole 1-(4-Chlorophenyl)-4,4-dimethyl-3-(1,2,4-triazol-1-ylmethyl)pentan-3-ol 1H-1,2,4-Triazole-1-ethanol, α-[2-(4-chlorophenyl)ethyl]-α-(1,1-dimethylethyl)-, (.+-.)- 1H-1,2,4-Triazole-1-ethanol,α-[2-(4-chlorophenyl) ethyl]-α-(1,1-dimethylethyl)-, (.+-.)- BAY-HWG 1608 ETHANOL, α-[2-(4-CHLOROPHENYL)ETHYL]-α- (1,1-DIMETHYLETHYL)-1H-1,2,4-TRIAZOLE Ethyltrianol Etiltrianol Fenetrazole Folicur Microban S 2142 Microban TZ Preventol A 8 TEBUCONAZOL Tebuconazole Resp. HWG 1608 Terbutrazole α-[2-(4-Chlorophenyl)-ethyl]-α-(1,1-dimethylethyl)-1H-1,2,4-triazole-1-ethanol α-[2-(4-chlorophenyl)ethyl]-α-(1,1-dimethylethyl)-1H-1,2,4-triazole-1-ethanol α-tert-Butyl-α-(p-chlorophenethyl)-1H-1,2,4-triazole-1-ethanol

DTXSID9032113

50471-44-8

FSCWZHGZWWDELK-UHFFFAOYNA-N

FSCWZHGZWWDELK-UHFFFAOYSA-N

Vinclozolin

2,4-Oxazolidinedione, 3-(3,5-dichlorophenyl)-5-ethenyl-5-methyl- (.+-.)-Vinclozolin BAS 352-04F N-3,5-Dichlorophenyl-5-methyl-5-vinyl-1,3-oxazolidine-2,4-dione N-3,5-Dichlorophenyl-5-methyl-5-vinyloxazolidine-2,4-dione N-3,5-Dichlorphenyl-5-methyl-5-vinyl-1,3-oxazolidin-2,4-dion N-3,5-diclorofenil-5-metil-5-vinil-1,3-oxazolidina-2,4-diona Ornalin Ranilan Ronilan Ronilan 50WP

DTXSID4022361

PR:000004191

PR androgen receptor

GO:0010468

GO regulation of gene expression

WIKI decreased

Bicalutamide

2020-08-07T06:55:53

Cyproterone acetate

2020-05-17T10:13:28

Epoxiconazole

2020-05-16T11:35:44

Flutamide

2016-11-29T18:42:27

Flusilazole

2020-05-16T11:15:34

Prochloraz

2016-11-29T18:42:22

Propiconazole

2017-05-17T13:18:07

Stressor:286 Tebuconazole

2020-08-07T07:00:53

Triticonazole

2020-05-16T11:02:07

2020-05-16T11:09:42

Vinclozalin

2016-11-29T18:42:27

Mercaptobenzole

2016-11-29T18:42:26

Tebuconazole

2017-05-17T13:17:14

Vinclozolin

2020-05-14T11:28:31

WCS_9606

common toxicological species human

10116

NCBI rat

10090

NCBI mouse

WikiUser_25

Wikiuser: Cyauk human and other cells in culture

10116

NCBI rats

Decrease, androgen receptors (AR) activation

Decrease, AR activation

Cellular

Androgen receptor activation is regulated by the binding of androgens. AR activity can be decreased by either a lack of steroidal ligands (testosterone, DHT) or the presence of antagonist compounds. 12

Significance of AR signaling in fetal development can be studied through a conditional deletion of the androgen receptor using a Cre/loxP approach. The recommended animal model for reproductive study is the mouse.3 Also, epidemiological case-studies following mouse or humans expressing a complete androgen insensitivity allow to directly assess the effects of a lack of AR activation on the development.4 Enzyme immunoassay (ELISA) kits for in vitro quantitative measurement of AR activity are available. Androgen receptors activity can be measured using bioassay such as the (Anti-)Androgen Receptor CALUX reporter gene assay.5

<table> <tbody> <tr> <td colspan="1" rowspan="1">   </td> <td colspan="1" rowspan="1"> 1 Davey R.A and Grossmann M. (2016) Androgen Receptor Structure, Function and Biology: From Bench to Bedside. Clinical Biochemist Reviews, 37(1): 3-15. PCM4810760 2 Gao W., Bohl C.E. and Dalton J.T. (2005) Chemistry and Structural Biology of Androgen Receptor. Chemical Reviews 105(9): 3352-3370<a href="https://www.google.com/url?q=https://doi.org/10.1021/cr020456u&sa=D&ust=1554891396627000">https://doi.org/10.1021/cr020456u</a>  3 Kaftanovskaya E.M., Huang Z., Barbara A.M., De Gendt K., Verhoeven G., Ivan P. Gorlov, and Agoulnik A.I. (2012) Cryptorchidism in Mice with an Androgen Receptor Ablation in Gubernaculum Testis. Molecular Endocrinology, 26(4): 598-607.<a href="https://www.google.com/url?q=https://doi.org/10.1210/me.2011-1283&sa=D&ust=1554891396628000">https://doi.org/10.1210/me.2011-1283</a>  4 Hutson J.M. (1985) A biphasic model for the hormonal control of testicular descent. Lancet, 24;2(8452): 419-21<a href="https://www.google.com/url?q=http://dx.doi.org/10.1016/S0140-6736(85)92739-4&sa=D&ust=1554891396629000">http://dx.doi.org/10.1016/S0140-6736(85)92739-4</a>  5 van der Burg B., Winter R., Man HY., Vangenechten C., Berckmans P., Weimer M., Witters M. and van der Linden S. (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 <a href="https://www.google.com/url?q=https://doi.org/0.1016/j.reprotox.2010.04.012&sa=D&ust=1554891396630000">https://doi.org/0.1016/j.reprotox.2010.04.012</a>  </td> </tr> </tbody> </table> AOPWiki 2019-04-10T05:04:18

2019-04-10T05:24:20

Altered, Transcription of genes by AR

Cellular

The Androgen Receptor and its function Androgens act by binding to the Androgen receptor (AR) in androgen-responsive tissues (Davey and Grossmann 2016). Human AR mutations and mouse knockout models have established the fundamental role of AR in masculinization and spermatogenesis (Maclean et al.; Walters et al. 2010; Rana et al. 2014). The AR is also expressed in many other tissues such as bone, muscles, ovaries and within the immune system (Rana et al. 2014).   Altered transcription of genes by the AR as a Key Event The AR belongs to the steroid hormone nuclear receptor family. It is a ligand-activated transcription factor with three domains; the N-terminal domain, the DNA-binding domain, and the ligand-binding domain with the latter being the most evolutionary conserved (Davey and Grossmann 2016). Upon activation by ligand-binding, the AR translocate from the cytoplasm to the cell nucleus, dimerizes, binds to androgen response elements in the DNA to modulate gene transcription (Davey and Grossmann 2016). The transcriptional targets varies between different cells and tissues, as well as with developmental stages and is, for instance, dependent on available co-regulators (Bevan and Parker 1999; Heemers and Tindall 2007). Several known and proposed target genes of AR canonical signaling have been identified by analysis of gene expression following treatments with AR agonists (Bolton et al. 2007; Ngan et al. 2009) and can for instance be found in the Androgen-Responsive Gene Database (Jiang et al. 2009).

In vitro Decreased transcription of genes by the AR can be measured by measuring the transcription level of known downstream target genes by RT-qPCR or other transcription analyses approaches, eg transcriptomics. Indirect approaches include the use of transient or stable transactivation assays including the validated OECD test guideline assay, Test No. 458: Stably Transfected Human Androgen Receptor Transcriptional Activation Assay for Detection of Androgenic Agonist and Antagonist Activity of Chemicals (OECD 2016). The stably transfected AR-EcoScreenTM cell line is freely available for the Japanese Collection of Research Bioresources (JCRB) Cell Bank under reference number JCRB1328. These cell-based transcriptional activation assays are typically used to detect AR agonists and antagonists. However, these types of assays are well suited to measure this KE as what they measure is exactly AR transcriptional activity. Other assays along this line include the AR-CALUX reporter gene assay that is derived from human U2-OS cells stably transfected with the human AR and an AR responsive reporter gene (van der Burg et al. 2010). In vivo Known downstream target gene transcription level can be measured in tissues by RT-qPCR or other gene expression analyses approaches.

Both the DNA-binding and ligand-binding domains of the AR are highly evolutionary conserved, whereas the transactivation domain show more divergence, which may affect AR-mediated gene regulation across species (Davey and Grossmann 2016).  Despite certain inter-species differences, AR function mediated through gene expression is highly conserved, with mutation studies from both humans and rodents showing strong correlation for AR-dependent development and function (Walters et al. 2010). This KE is applicable for both sexes, across developmental stages into adulthood, in numerous cells and tissues and across taxa.

CL:0000255

CL eukaryotic cell

High Mixed

High

Foetal

High

Adult, reproductively mature

High High High

Bevan C, Parker M (1999) The role of coactivators in steroid hormone action. Exp. Cell Res. 253:349–356 Bolton EC, So AY, Chaivorapol C, et al (2007) Cell- and gene-specific regulation of primary target genes by the androgen receptor. Genes Dev 21:2005–2017. doi: 10.1101/gad.1564207 Davey RA, Grossmann M (2016) Androgen Receptor Structure, Function and Biology: From Bench to Bedside. Clin Biochem Rev 37:3–15 Draskau MK, Boberg J, Taxvig C, et al (2019) In vitro and in vivo endocrine disrupting effects of the azole fungicides triticonazole and flusilazole. Environ Pollut 255:113309. doi: 10.1016/j.envpol.2019.113309 Estrada M, Espinosa A, Müller M, Jaimovich E (2003) Testosterone Stimulates Intracellular Calcium Release and Mitogen-Activated Protein Kinases Via a G Protein-Coupled Receptor in Skeletal Muscle Cells. Endocrinology 144:3586–3597. doi: 10.1210/en.2002-0164 Heemers H V., Tindall DJ (2007) Androgen receptor (AR) coregulators: A diversity of functions converging on and regulating the AR transcriptional complex. Endocr. Rev. 28:778–808 Jiang M, Ma Y, Chen C, et al (2009) Androgen-Responsive Gene Database: Integrated Knowledge on Androgen-Responsive Genes. Mol Endocrinol 23:1927–1933. doi: 10.1210/me.2009-0103 Kjærstad MB, Taxvig C, Nellemann C, et al (2010) Endocrine disrupting effects in vitro of conazole antifungals used as pesticides and pharmaceuticals. Reprod Toxicol 30:573–582. doi: 10.1016/J.REPROTOX.2010.07.009 Laier P, Metzdorff SB, Borch J, et al (2006) Mechanisms of action underlying the antiandrogenic effects of the fungicide prochloraz. Toxicol Appl Pharmacol 213:160–71. doi: 10.1016/j.taap.2005.10.013 Maclean HE, Chu S, Warne GL, Zajact JD Related Individuals with Different Androgen Receptor Gene Deletions MacLeod DJ, Sharpe RM, Welsh M, et al (2010) Androgen action in the masculinization programming window and development of male reproductive organs. In: International Journal of Andrology. Blackwell Publishing Ltd, pp 279–287 Ngan S, Stronach EA, Photiou A, et al (2009) Microarray coupled to quantitative RT&ndash;PCR analysis of androgen-regulated genes in human LNCaP prostate cancer cells. Oncogene 28:2051–2063. doi: 10.1038/onc.2009.68 OECD (2016) Test No. 458: Stably Transfected Human Androgen Receptor Transcriptional Activation Assay for Detection of Androgenic Agonist and Antagonist Activity of Chemicals, OECD Guide. OECD Publishing Rana K, Davey RA, Zajac JD (2014) Human androgen deficiency: Insights gained from androgen receptor knockout mouse models. Asian J. Androl. 16:169–177 Sonneveld E, Jansen HJ, Riteco JAC, et al (2005) Development of Androgen-and Estrogen-Responsive Bioassays, Members of a Panel of Human Cell Line-Based Highly Selective Steroid-Responsive Bioassays. Toxicol Sci 83:136–148. doi: 10.1093/toxsci/kfi005 van der Burg B, Winter R, Man H yen, et al (2010) Optimization and prevalidation of the in vitro AR CALUX method to test androgenic and antiandrogenic activity of compounds. Reprod Toxicol 30:18–24. doi: 10.1016/j.reprotox.2010.04.012 Walters KA, Simanainen U, Handelsman DJ (2010) Molecular insights into androgen actions in male and female reproductive function from androgen receptor knockout models. Hum Reprod Update 16:543–558. doi: 10.1093/humupd/dmq003 AOPWiki 2016-11-29T18:41:23

2020-11-04T11:11:01

Antagonism, Androgen receptor

Molecular

The androgen receptor (AR) and its function Development of the male reproductive system and secondary male characteristics is dependent on androgens (foremost testosterone (T) and dihydrotestosterone (DHT). T and the more biologically active DHT act by binding to the AR (<a href="#_ENREF_4" title="MacLean, 1993 #251">MacLean et al, 1993</a>; <a href="#_ENREF_5" title="MacLeod, 2010 #27">MacLeod et al, 2010</a>; <a href="#_ENREF_8" title="Schwartz, 2019 #252">Schwartz et al, 2019</a>), with human AR mutations and mouse knock-out models having established its pivotal role in masculinization and spermatogenesis (<a href="#_ENREF_9" title="Walters, 2010 #254">Walters et al, 2010</a>). The AR is a ligand-activated transcription factor belonging to the steroid hormone nuclear receptor family (<a href="#_ENREF_1" title="Davey, 2016 #250">Davey & Grossmann, 2016</a>). The AR has three domains; the N-terminal domain, the DNA-binding domain and the ligand-binding domain, with the latter being most evolutionary conserved. Apart from the essential role AR plays for male reproductive development and function (<a href="#_ENREF_9" title="Walters, 2010 #254">Walters et al, 2010</a>), the AR is also expressed in many other tissues and organs such as bone, muscles, ovaries and the immune system (<a href="#_ENREF_7" title="Rana, 2014 #253">Rana et al, 2014</a>).  AR antagonism as Key Event The main function of the AR is to activate gene transcription in cells. Canonical signaling occurs by ligands (androgens) binding to AR in the cytoplasm which results in translocation to the cell nucleus, receptor dimerization and binding to specific regulatory DNA sequences (<a href="#_ENREF_2" title="Heemers, 2007 #255">Heemers & Tindall, 2007</a>). The gene targets regulated by AR activation depends on cell/tissue type and what stage of development activation occur, and is, for instance, dependent on available co-factors. Apart from the canonical signaling pathway, AR can also function through non-genomic modalities, for instance rapid change in cell function by ion transport changes (<a href="#_ENREF_3" title="Heinlein, 2002 #256">Heinlein & Chang, 2002</a>). However, with regard to this specific KE the canonical signaling pathway is what is referred to.

AR antagonism can be measured in vitro by transient or stable transactivation assays to evaluate nuclear receptor activation. There is already a validated assay for AR (ant)agonism adopted by the OECD, Test No. 458: Stably Transfected Human Androgen Receptor Transcriptional Activation Assay for Detection of Androgenic Agonist and Antagonist Activity of Chemicals (<a href="#_ENREF_13" title="OECD, 2016 #257">OECD, 2016</a>). The stably transfected AR-EcoScreenTM cells (<a href="#_ENREF_15" title="Satoh, 2004 #280">Satoh et al, 2004</a>) should be used for the assay and is freely available for the Japanese Collection of Research Bioresources (JCRB) Cell Bank under reference number JCRB1328. Other assays include the AR-CALUX reporter gene assay that is derived from human U2-OS cells stably transfected with the human AR and an AR responsive reporter gene (<a href="#_ENREF_18" title="van der Burg, 2010 #261">van der Burg et al, 2010</a>), various transiently transfected reporter cell lines (<a href="#_ENREF_10" title="Körner, 2004 #282">Körner et al, 2004</a>), and more. Recently developed AR dimerization assay may soon be included in TGs for its improved ability to measure potential stressor-mediated dimerization/activation (<a href="#_ENREF_11" title="Lee, 2021 #288">Lee et al, 2021</a>).

Both the DNA-binding and ligand-binding domains of the AR are highly evolutionary conserved, whereas the transactivation domain show more divergence which may affect AR-mediated gene regulation across species (<a href="#_ENREF_1" title="Davey, 2016 #250">Davey & Grossmann, 2016</a>). Despite certain inter-species differences, AR function mediated through gene expression is highly conserved, with mutations studies from both humans and rodents showing strong correlation for AR-dependent development and function (<a href="#_ENREF_9" title="Walters, 2010 #254">Walters et al, 2010</a>). This KE is applicable for both sexes, across developmental stages into adulthood, in numerous cells and tissues and across taxa

CL:0000255

CL eukaryotic cell

High Mixed

High

Foetal

Moderate

Embryo

High

During development and at adulthood

High High High High <a name="_ENREF_1">Alapi EM, Fischer J (2006) Table of Selected Analogue Classes. In Analogue-based Drug Discovery, Fischer J, Ganellin CR (eds), p 515. Weinheim: Wiley-VCH Verlag GmbH & Co</a> <a name="_ENREF_2">Davey RA, Grossmann M (2016) Androgen Receptor Structure, Function and Biology: From Bench to Bedside. Clin Biochem Rev 37: 3-15</a> <a name="_ENREF_3">Draskau MK, Boberg J, Taxvig C, Pedersen M, Frandsen HL, Christiansen S, Svingen T (2019) In vitro and in vivo endocrine disrupting effects of the azole fungicides triticonazole and flusilazole. Environ Pollut 255: 113309</a> <a name="_ENREF_4">Foster PM, Harris MW (2005) Changes in androgen-mediated reproductive development in male rat offspring following exposure to a single oral dose of flutamide at different gestational ages. Toxicol Sci 85: 1024-1032</a> <a name="_ENREF_5">Hass U, Scholze M, Christiansen S, Dalgaard M, Vinggaard AM, Axelstad M, Metzdorff SB, Kortenkamp A (2007) Combined exposure to anti-androgens exacerbates disruption of sexual differentiation in the rat. Environ Health Perspect 115 Suppl. 1: 122-128</a> <a name="_ENREF_6">Heemers HV, Tindall DJ (2007) Androgen receptor (AR) coregulators: a diversity of functions converging on and regulating the AR transcriptional complex. Endocr Rev 28: 778-808</a> <a name="_ENREF_7">Heinlein CA, Chang C (2002) The roles of androgen receptors and androgen-binding proteins in nongenomic androgen actions. Mol Endocrinol 16: 2181-2187</a> <a name="_ENREF_8">Kita DH, Meyer KB, Venturelli AC, Adams R, Machado DL, Morais RN, Swan SH, Gennings C, Martino-Andrade AJ (2016) Manipulation of pre and postnatal androgen environments and anogenital distance in rats. Toxicology 368-369: 152-161</a> <a name="_ENREF_9">Kjærstad MB, Taxvig C, Nellemann C, Vinggaard AM, Andersen HR (2010) Endocrine disrupting effects in vitro of conazole antifungals used as pesticides and pharmaceuticals. Reprod Toxicol 30: 573-582</a> <a name="_ENREF_10">Körner W, Vinggaard AM, Térouanne B, Ma R, Wieloch C, Schlumpf M, Sultan C, Soto AM (2004) Interlaboratory comparison of four in vitro assays for assessing androgenic and antiandrogenic activity of environmental chemicals. Environ Health Perspect 112: 695-702</a> <a name="_ENREF_11">Lee SH, Hong KY, Seo H, Lee HS, Park Y (2021) Mechanistic insight into human androgen receptor-mediated endocrine-disrupting potentials by a stable bioluminescence resonance energy transfer-based dimerization assay. Chem Biol Interact 349: 109655</a> <a name="_ENREF_12">MacLean HE, Chu S, Warne GL, Zajac JD (1993) Related individuals with different androgen receptor gene deletions. J Clin Invest 91: 1123-1128</a> <a name="_ENREF_13">MacLeod DJ, Sharpe RM, Welsh M, Fisken M, Scott HM, Hutchison GR, Drake AJ, van den Driesche S (2010) Androgen action in the masculinization programming window and development of male reproductive organs. Int J Androl 33: 279-287</a> <a name="_ENREF_14">OECD. (2016) Test No. 458: Stably Transfected Human Androgen Receptor Transcriptional Activation Assay for Detection of Androgenic Agonist and Antagonist Activity of Chemicals. OECD Guidelines for the Testing of Chemicals, Section 4, Paris.</a> <a name="_ENREF_15">Rana K, davey RA, Zajac JD (2014) Human androgen deficiency: insights gained from androgen receptor knockout mouse models. Asian J Androl 16: 169-177</a> <a name="_ENREF_16">Satoh K, Ohyama K, Aoki N, Iida M, Nagai F (2004) Study on anti-androgenic effects of bisphenol a diglycidyl ether (BADGE), bisphenol F diglycidyl ether (BFDGE) and their derivatives using cells stably transfected with human androgen receptor, AR-EcoScreen. Food Chem Toxicol 42: 983-993</a> <a name="_ENREF_17">Schwartz CL, Christiansen S, Vinggaard AM, Axelstad M, Hass U, Svingen T (2019) Anogenital distance as a toxicological or clinical marker for fetal androgen action and risk for reproductive disorders. Arch Toxicol 93: 253-272</a> <a name="_ENREF_18">Sonneveld E, Jansen HJ, Riteco JA, Brouwer A, van der Burg B (2005) Development of androgen- and estrogen-responsive bioassays, members of a panel of human cell line-based highly selective steroid-responsive bioassays. Toxicol Sci 83: 136-148</a>   <a name="_ENREF_19">van der Burg B, Winter R, Man HY, Vangenechten C, Berckmans P, Weimer M, Witters H, van der Linden S (2010) Optimization and prevalidation of the in vitro AR CALUX method to test androgenic and antiandrogenic activity of compounds. Reprod Toxicol 30: 18-24</a> <a name="_ENREF_20">Vinggaard AM, Niemelä J, Wedebye EB, Jensen GE (2008) Screening of 397 chemicals and development of a quantitative structure--activity relationship model for androgen receptor antagonism. Chem Res Toxicol 21: 813-823</a> <a name="_ENREF_21">Walters KA, Simanainen U, Handelsman DJ (2010) Molecular insights into androgen actions in male and female reproductive function from androgen receptor knockout models. Hum Reprod Update 16: 543-558</a> AOPWiki 2016-11-29T18:41:22

2022-06-15T06:17:59

Nipple retention (NR), increased

nipple retention, increased

Individual

In common laboratory strains of rats and mice, females typically have 6 (rats) or 5 (mice) pairs of nipples along the bilateral milk lines. In contrast, male rats and mice do not have nipples. This is unlike e.g., humans where both sexes have 2 nipples (Schwartz et al., 2021). In laboratory rats, high levels of dihydrotestosterone (DHT) induce regression of the nipples in males (Imperato-McGinley & Gautier, 1986; Kratochwil, 1977; Kratochwil & Schwartz, 1976). Females, in the absence of this DHT surge, retain their nipples. This relationship has also been shown in numerous rat studies with perinatal exposure to anti-androgenic chemicals (Schwartz et al., 2021). Hence, if juvenile male rats and mice possess nipples, it is considered a sign of perturbed androgen action early in life. This KE was first published by Pedersen et al (2022).

Nipple retention (NR) is visually assessed, ideally on postnatal day (PND) 12/13 (OECD, 2018; Schwartz et al., 2021). However, PND 14 is also an accepted stage of examination (OECD, 2013). Depending on animal strain, the time when nipples become visible can vary, but the assessment of NR in males should be conducted when nipples are visible in their female littermates (OECD, 2013). Nipples are detected as dark spots (or shadows) called areolae, which resemble precursors to a nipple rather than a fully developed nipple. The dark area may or may not display a nipple bud (Hass et al., 2007). Areolae typically emerge along the milk lines of the male pups corresponding to where female pups display nipples. Fur growth may challenge detection of areolae after PND 14/15. Therefore, the NR assessment should be conducted prior to excessive fur growth. Ideally, all pups in a study are assessed on the same postnatal day to minimize variation due to maturation level (OECD, 2013). NR is occasionally observed in controls. Hence, accurate assessment of NR in controls is needed to detect substance-induced effects on masculine development (Schwartz et al., 2021). It is recommended by the OECD guidance documents 43 and 151 to record NR as a quantitative number rather than a qualitative measure (present/absent or yes/no response). This allows for more nuanced analysis of results, e.g., high control values may be recognized (OECD, 2013, 2018). Studies reporting quantitative measures of NR are therefore considered stronger in terms of weight of evidence. Reproducibility of NR results is challenged by the measure being a visual assessment prone to a degree of subjectivity. Thus, NR should be assessed and scored blinded to exposure groups and ideally be performed by the same person(s) within the same study.

The applicability domain of NR is limited to male laboratory strains of rats and mice from birth to juvenile age.

High Male

High

Birth to < 1 month

High High Hass, U., Scholze, M., Christiansen, S., Dalgaard, M., Vinggaard, A. M., Axelstad, M., Metzdorff, S. B., & Kortenkamp, A. (2007). Combined exposure to anti-androgens exacerbates disruption of sexual differentiation in the rat. Environmental Health Perspectives, 115(suppl 1), 122–128. Imperato-McGinley, J., Binienda, Z., Gedney, J., & Vaughan, E. D. (1986). Nipple Differentiation in Fetal Male Rats Treated with an Inhibitor of the Enzyme 5α-Reductase: Definition of a Selective Role for Dihydrotestosterone. Endocrinology, 118(1), 132–137. Kratochwil, K. (1977). Development and Loss of Androgen Responsiveness in the Embryonic Rudiment of the Mouse Mammary Gland. DEVELOPMENTAL BIOLOGY, 61, 358–365. OECD. (2008). Guidance document 43 on mammalian reproductive toxicity testing and assessment. Environment, Health and Safety Publications, 16(43). OECD. (2013). Guidance document supporting OECD test guideline 443 on the extended one-generation reproductive toxicity test. Environment, Health and Safety Publications, 10(151). OECD. (2016a). Test Guideline 421: Reproduction/Developmental Toxicity Screening Test. OECD Guidelines for the Testing of Chemicals, 421. <a href="http://www.oecd.org/termsandconditions/" style="color:#0563c1; text-decoration:underline">http://www.oecd.org/termsandconditions/</a> OECD. (2016b). Test Guideline 422: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test. OECD Guidelines for the Testing of Chemicals, 422. <a href="http://www.oecd.org/termsandconditions/" style="color:#0563c1; text-decoration:underline">http://www.oecd.org/termsandconditions/</a> OECD. (2018). Test Guideline 443: Extended one-generation reproductive toxicity study. OECD Guidelines for the Testing of Chemicals, 443. <a href="http://www.oecd.org/termsandconditions/" style="color:#0563c1; text-decoration:underline">http://www.oecd.org/termsandconditions/</a> Pedersen, E. B., Christiansen, S., & Svingen, T. (2022). AOP key event relationship report: Linking androgen receptor antagonism with nipple retention. Current Research in Toxicology, 3, 100085. Schwartz, C. L., Christiansen, S., Hass, U., Ramhøj, L., Axelstad, M., Löbl, N. M., & Svingen, T. (2021). On the Use and Interpretation of Areola/Nipple Retention as a Biomarker for Anti-androgenic Effects in Rat Toxicity Studies. Frontiers in Toxicology, 3, 730752. AOPWiki 2020-05-11T10:07:08

2023-01-11T05:53:25

<upstream-id>2d26af16-bf7b-4b83-a044-4dd35c324839</upstream-id> <downstream-id>82caae23-9810-4e8a-bcc8-fc999bc58a17</downstream-id>

#<Reference::ActiveRecord_Associations_CollectionProxy:0x00005eb8a2ffe948> AOPWiki 2020-05-11T07:39:00

2020-05-11T07:39:00

<upstream-id>2d26af16-bf7b-4b83-a044-4dd35c324839</upstream-id> <downstream-id>c5f9206d-8590-4e40-8dee-8b3074c64b07</downstream-id> Several chemicals can antagonize the androgen receptor (AR) in vitro, resulting in decreased AR activation. Decreased AR activation can lead to incomplete reproductive development in males, which can be expressed in several ways. One endpoint affected is areola/nipple retention (NR), which in vivo studies have shown to be linked to suppressed AR activation. NR in rat and mouse toxicity studies is considered an adverse effect (i.e., an AO).

Strategi was described by Pedersen et al (2022): A semi-systematic literature search was conducted during March 2022 in the peer-reviewed databases PubMed and Web of Science, using the search terms “(Nipple) AND (retain* OR retention) AND (androgen)” as well as “(Androgen receptor OR AR) AND (active*) AND (nipple OR areolae) AND (retain* OR retention)”. These searches resulted in 138 papers in total. Upon removal of duplicates, papers were screened according to title, abstract and ultimately full text based on pre-defined inclusion criteria. In vivo studies were included if (i) the study was carried out in mice or rats, (ii) NR in males was investigated as an endpoint, (iii) AR antagonism was the suspected mechanism of action and (iv) anti-androgenic effects of single substance exposures (i.e., not studies on chemical mixtures) were investigated. In vitro studies were included if they contained mechanistic information on AR inhibition by chemical stressors.

The biological plausibility of a link between decreased AR activation and increased NR in male rats is high. The relationship is supported by numerous studies showing that several potent AR antagonists in vitro induce NR in vivo. However, in the literature review conducted for this KER, no studies in mice were found to fulfill the inclusion criteria. The present KER is hence exclusively a description of the situation in rats, although it is believed that the link also exists in mice. The AR is activated through binding of either testosterone or dihydrotestosterone (DHT), the latter having the highest affinity for the AR. Upon binding, the AR translocates to the target cell nucleus where it acts as a transcription factor (Albert, 2018). NT has been shown to be more dependent on DHT-signaling, which suggests that chemicals inducing increased NR also have a higher affinity for the AR than DHT in order to outcompete DHT for AR binding, although supra-high doses of chemicals with lower AR affinity could be speculated to also outcompete T or DHT. The general principle of higher affinity, however, has been confirmed by in vitro studies (Gray et al., 2019; Hass et al., 2012; McIntyre et al., 2000).

Table 2 lists chemical stressors shown to antagonize the AR in vitro as well as causing NR in male rat offspring in vivo. Additional information from the in vivo studies, including the animal species and strain, as well as the doses tested, the dosing period and the time of measurement of NR are specified in this table. The lowest dose yielding a significant increase of retained nipples in male rat pups is defined as the LOAEL. Conversely, the NOAEL represents the highest tested dose yielding no significant increase in NR. Note that the given NOAEL and LOAEL values are highly dependent on study design. Significant values are marked with an asterisk. Table 3 shows a list of stressors shown to have AR antagonistic properties in vitro or in other in vivo studies, but for which the doses tested in vivo did not produce a significant effect on NR. In this list, the lowest tested dose is reported, and the NOAEL presents the highest dose tested which produced no statistically significant effect on NR. Apart from the NOAEL, the information given in Table 3 is identical to Table 2.

A major challenge with NR as a biomarker is the subjectivity of the measure. In juvenile rat pups, nipples are only present as areolae, i.e., dark shadows with or without a nipple bud. This means that the experience of the personnel assessing the presence and number of areolae/nipples can influence the results. Furthermore, the results are likely prone to larger variation if several assessors are used to record NR within the same study. To minimize these sources of uncertainty, assessors must be trained to recognize areolae and not look for fully developed nipples. Moreover, the number of assessors should be limited to one or two, and they should always be blinded to exposure groups. Another factor that may affect NR results is the age of the rat pups at the time of assessment. OECD guidelines have standardized the time for measuring occurrence of NR to be optimal at PD 12 or 13, when they are visible in female littermates (OECD, 2013). However, assessment of permanent NR is not included in any international guidelines. Hence, if NR is measured in older offspring, the time of measurement is not consistent between studies and varies between PD 20 and PD 100. Thus, conclusions on whether NR is permanent or not may differ based on study design. This distinction between a transient and a permanent effect is important from a regulatory perspective, since only a permanent effect will be categorized as a malformation according to OECD guidance document 43 (OECD, 2008).

The quantitative understanding of the relationship between decreased AR activity and NR is challenged by the fact that the potency of AR antagonism in vitro is not proportional to the magnitude of NR observed in vivo (Gray et al., 2019). Hence, predicting in vivo effects based on in vitro data is not possible. However, in vitro studies can give indications of which chemicals might exhibit anti-androgenic effects in vivo and should be subject to further testing (Hass et al., 2012). Development of more representative in vitro models is necessary if in vivo tests are to be phased out entirely.

No response-response relationship has been identified.

NR manifest in juvenile male rat pup offspring in response to reduced androgen signaling, e.g. resulting from exposure to an anti-androgenic chemical stressor during fetal development. Developmental sensitivity during fetal development is highest during the so-called male masculinization programming window (MPW) which in rats is between gestational day (GD) 15 and 19 (Welsh et al., 2008). A study in which pregnant rat dams were exposed to the AR antagonist vinclozolin for two-day periods during gestation showed that GD 16–17 was the most sensitive period for increased NR in male offspring (Wolf et al., 2000). A similar study using di-n-butyl phthalate (reduces testosterone levels) also showed that GD 16–17 was the most sensitive period for increased NR in male rats (Carruthers & Foster, 2005). However, to determine if other chemical stressors also have the highest antagonistic potential towards the AR during GD 16-17, further studies with a similar design would be informative. NR can only be recorded when pups are old enough to display them, yet before excessive fur has developed. Hence, the most accurate results can be obtained from assessing the number of nipples on PD 12–14 depending on rat strain and the time of female littermates displaying nipples (OECD, 2013).

No feedback loops that could influence the KER have been identified.

High Male

High

Development

High High

#<Reference::ActiveRecord_Associations_CollectionProxy:0x00005eb8a30d67a8> AOPWiki 2020-05-11T10:24:31

2023-01-11T06:22:46

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#<Reference::ActiveRecord_Associations_CollectionProxy:0x00005eb8a31299a8> AOPWiki 2020-05-11T06:50:06

2020-05-11T06:50:06

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#<Reference::ActiveRecord_Associations_CollectionProxy:0x00005eb8a31764b0> AOPWiki 2020-05-11T10:24:11

2020-05-11T10:24:11

Androgen receptor (AR) antagonism leading to nipple retention (NR) in male (mammalian) offspring

AR antagonism leading to NR

Evgeniia Kazymova

Sofie Christiansen; National Food Institute, Technical University of Denmark, Kongens Lyngby, 2800 Denmark Louise Ramhøj; National Food Institute, Technical University of Denmark, Kongens Lyngby, 2800 Denmark Terje Svingen; National Food Institute, Technical University of Denmark, Kongens Lyngby, 2800 Denmark

BY-SA

Under Development

1.108

2.0

This AOP links Androgen receptor antagonism during fetal life with nipple retention (NR) in male rat offspring. NR, measured around 2 weeks post partum, is a marker for feminization of male rat fetuses and is associated with general feminization of male offspring. Although NR is not a directly applicable measure in humans (male humans normally retain two nipples), it is nevertheless considered an ‘adverse outcome’ in OECD test guidelines; NR measurements are mandatory in specific tests for developmental and reproductive toxicity in chemical risk assessment (TG 443, TG 421/422, TG 414). The AR is a nuclear receptor involved in the transcriptional regulation of various target genes during development and adulthood across species. Its main ligand is testosterone and dihydrotestosterone (DHT). Under normal physiological conditions, testosterone produced mainly by the testicles, is converted in peripheral tissues by 5α-reductase into DHT, which in turn binds AR and activates downstream target genes. AR signaling is necessary for normal masculinization of the developing fetus, and AR action in male rats signals the nipple anlagen to regress, leaving males with no nipples. <div>The key events in this pathway is antagonism of the AR in target cells of the nipple anlagen, which leads to inactivation of the AR and failure to suppress development of the nipples. In this instance, the local levels of testosterone or DHT may be normal, but prevented from binding the AR.</div>

A large number of drugs and chemicals have been shown to antagonise the AR using various AR reporter gene assays. The AR is specifically targeted in AR-sensitive cancers, for example the use of the anti-androgenic drug flutamide in treating prostate cancer (<a href="#_ENREF_1" title="Alapi, 2006 #262">Alapi & Fischer, 2006</a>). Flutamide has also been used in several rodent in vivo studies showing anti-androgenic effects (feminization of male offspring) evident by e.g. short anogenital distance (AGD) in males (<a href="#_ENREF_4" title="Foster, 2005 #53">Foster & Harris, 2005</a>; <a href="#_ENREF_5" title="Hass, 2007 #76">Hass et al, 2007</a>; <a href="#_ENREF_8" title="Kita, 2016 #34">Kita et al, 2016</a>). QSAR models can predict AR antagonism for a wide range of chemicals, many of which have shown in vitro antagonistic potential (<a href="#_ENREF_17" title="Vinggaard, 2008 #263">Vinggaard et al, 2008</a>).

NR is recognized by the OECD as a relevant measure for anti-androgenic effects and is mandatory in the test guidelines Extended One Generation Reproductive Toxicity Study, TG 443 (OECD, 2018) and the two screening studies for reproductive toxicity, TGs 421/422 (OECD, 2016a, 2016b). The endpoint is also described in the guidance documents 43 (OECD, 2008) and 151 (OECD, 2013). Furthermore, NR data can be used in chemical risk assessment for setting the No Observed Adverse Effect Level (NOAEL) as stated in the OECD guidance document 151 (OECD, 2013): “A statistically significant change in nipple retention should be evaluated similarly to an effect on AGD as both endpoints indicate an adverse effect of exposure and should be considered in setting a NOAEL”.

adjacent

Not Specified

High adjacent

Moderate

Moderate adjacent

Low

Moderate non-adjacent

Low

Moderate

AOPWiki 2020-05-11T10:14:48

2023-09-25T16:27:03