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<h2>AOP ID and Title:</h2>
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AOP 117: Androgen receptor activation leading to hepatocellular adenomas and carcinomas (in mouse and rat)
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<div class="title">AOP 117: Androgen receptor activation leading to hepatocellular adenomas and carcinomas (in mouse and rat)</div>
<strong>Short Title: AR- HCC</strong>
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<h2>Authors</h2>
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<p>Cancer AOP Workgroup. National Health and Environmental Effects Research Laboratory, Office of Research and Development, Integrated Systems Toxicology Division, US Environmental Protection Agency, Research Triangle Park, NC. Corresponding author for wiki entry (wood.charles@epa.gov)
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<p>Cancer AOP Workgroup. National Health and Environmental Effects Research Laboratory, Office of Research and Development, Integrated Systems Toxicology Division, US Environmental Protection Agency, Research Triangle Park, NC. Corresponding author for wiki entry (wood.charles@epa.gov)</p>
<td>Under development: Not open for comment. Do not cite</td>
<td>Open for adoption</td>
<td>Under Development</td>
<td>1.26</td>
<td>Included in OECD Work Plan</td>
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<h2>Abstract</h2>
<p>This putative adverse outcome pathway (AOP) outlines potential key events leading to a tumor outcome in standard carcinogenicity models. This information is based largely on modes of action described previously in cited literature sources and is intended as a resource template for AOP development and data organization. Presentation in this Wiki does not indicate EPA acceptance of a particular pathway for a given reference agent, only that the information has been proposed in some manner. In addition, this putative AOP relates to the model species indicated and does not directly address issues of human relevance.</p>
<h2>Abstract</h2>
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<p>This putative adverse outcome pathway (AOP) outlines potential key events leading to a tumor outcome in standard carcinogenicity models. This information is based largely on modes of action described previously in cited literature sources and is intended as a resource template for AOP development and data organization. Presentation in this Wiki does not indicate EPA acceptance of a particular pathway for a given reference agent, only that the information has been proposed in some manner. In addition, this putative AOP relates to the model species indicated and does not directly address issues of human relevance.
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<p><strong>Taxonomic applicability</strong>: 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.</p>
<p style="text-align:start"><strong><span style="font-size:medium"><span style="font-family:Aptos,sans-serif"><span style="color:#000000"><span style="font-family:Calibri,sans-serif"><span style="color:black">Life stage applicability: </span></span></span></span></span></strong><span style="font-size:medium"><span style="font-family:Aptos,sans-serif"><span style="color:#000000"><span style="font-family:Calibri,sans-serif"><span style="color:black">Androgen receptor is expressed from the fetal period throughout adult life and activation of the androgen receptor controls sexual development during the fetal period and reproductive function as well as effects in other organs during puberty and adulthood </span></span>(Dalton et al., 2010; Luetjens et al., 2012; Naamneh Elzenaty et al., 2022; Sutinen et al., 2017). (added Nov 2024)</span></span></span></p>
<p style="text-align:start"><strong><span style="font-size:medium"><span style="font-family:Aptos,sans-serif"><span style="color:#000000"><span style="font-family:Calibri,sans-serif"><span style="color:black">Sex applicability: </span></span></span></span></span></strong><span style="font-size:medium"><span style="font-family:Aptos,sans-serif"><span style="color:#000000"><span style="font-family:Calibri,sans-serif"><span style="color:black">Androgen receptor is expressed in both males and females and have important roles for sexual development and reproduction as well as effects in other organs in both sexes (</span></span>Naamneh Elzenaty et al., 2022; Sutinen et al., 2017). (added Nov 2024)</span></span></span></p>
<h4>Key Event Description</h4>
<p><strong>Site of action</strong>: 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.</p>
<p><strong>Responses at the macromolecular level</strong>: 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).</p>
<p><strong>AR paralogs</strong>:</p>
<ul>
<li>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).</li>
<li>AR-B has been lost in Cypriniformes, Siluriformes, Characiformes, and Salmoniformes (Douard et al. 2008).</li>
<li>In Percomorphs, AR-B has accumulated significant substitutions in the both ligand binding and DNA binding domains (Douard et al. 2008).</li>
<li>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. </li>
</ul>
<h4>How it is Measured or Detected</h4>
<p><strong>Measurement/detection</strong>:</p>
<ul>
<li><strong>In vitro methods:</strong>
<ul>
<li>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 (<a href="http://www.oecd.org/env/test-no-458-stably-transfected-human-androgen-receptor-transcriptional-activation-assay-for-detection-of-androgenic-agonist-9789264264366-en.htm">OECD 2016</a>).</li>
<li>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).</li>
<li>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).</li>
<li>Expression of androgen responsive proteins like spiggin in primary cell cultures has also been used to detect AR agonist activity (Jolly et al. 2006).</li>
<li><span style="color:black">US EPA Androgen receptor pathway model. The model includes 11 in vitro assays measuring receptor binding, coregulator recruitment, nuclear translocation, transactivation or cell proliferation. A “</span>reduced” model that include fewer assays also exists. <span style="color:black">The output of the AR pathway model provides an AUC value for the potential of a chemical to cause AR agonism and/or AR antagonism. (EPA, 2022; Judson et al., 2020; Kleinstreuer et al., 2017). (added Nov 2024)</span></li>
<li><span style="color:black">CoMPARA: Collaborative Modeling Project for Androgen Receptor Activity. The US EPA lead consortium has developed consensus computational models that can be used to predict androgen receptor binding, agonist or antagonist activity (Mansouri et al., 2020). (added Nov 2024)</span></li>
</ul>
</li>
<li><strong>In vivo methods</strong>:
<ul>
<li>In fish, phenotypic masculinization of females has frequently been used as an indirect measurement of in vivo androgen receptor agonism.
<ul>
<li>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; <a href="http://www.oecd-ilibrary.org/environment/test-no-229-fish-short-term-reproduction-assay_9789264185265-en">OECD 2012</a>).</li>
<li>Anal fin elongation in female western mosquitofish (<em>Gambusia affinis</em>) has similarly been viewed as evidence of AR activation (Raut et al. 2011; Sone et al. 2005).</li>
<li>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 (<a href="http://www.oecd-ilibrary.org/environment/test-no-229-fish-short-term-reproduction-assay_9789264185265-en">OECD 2012</a>).</li>
<li>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.</li>
</ul>
</li>
</ul>
</li>
<li><strong>High Throughput Screening</strong>
<ul>
<li>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:
<ul>
<li>ATG_AR_TRANS A cell based assay that can differentiate agonism from antagonism</li>
<li>NVS_NR_hAR A cell free assay using recombinant human AR. Can detect binding, but cannot distinguish agonism from antagonism.</li>
<li>NVS_NR_rAR A cell free assay using recombinant rat AR. Can detect binding, but cannot distinguish agonism from antagonism.</li>
<li>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.</li>
<li>Tox21_AR_BLA_Agonist_ratio A cell based assay with an inducible reporter. Can distinguish agonists from antagonists.</li>
<li>Tox21_AR_LUC_MDAKB2_agonist A cell based assay with an inducible reporter. Can distinguish agonists from antagonists.</li>
<li>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.</li>
<li>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.</li>
<li>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.</li>
<li>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.</li>
<li>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.</li>
<li>Baker ME. 1997. Steroid receptor phylogeny and vertebrate origins. Molecular and cellular endocrinology 135(2): 101-107.</li>
<li>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.</li>
<li>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.</li>
<li>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.</li>
<li>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.</li>
<li><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Dalton JT, Gao W Androgen Receptor. 2010. In C. M. Bunce & M. J. Campbell (Eds.), Nuclear Receptors (pp. 143–182). Springer Netherlands. https://doi.org/10.1007/978-90-481-3303-1_6</span></span></li>
<li>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</li>
<li>Eick GN, Thornton JW. 2011. Evolution of steroid receptors from an estrogen-sensitive ancestral receptor. Molecular and cellular endocrinology 334(1-2): 31-38.</li>
<li><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">EPA. Summary of the Performance Metrics for the Androgen Receptor (AR) and Estrogen Receptor (ER) Pathway Models and Associated Individual In Vitro Assays. 2002. https://www.epa.gov/chemical-research/exploring-toxcast-data</span></span></li>
<li>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</li>
<li><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Heemers HV, Tindall DJ. Androgen receptor (AR) coregulators: a diversity of functions converging on and regulating the AR transcriptional complex. Endocr Rev. 2007 Dec;28(7):778-808. https://doi.org/10.1210/er.2007-0019</span></span></li>
<li>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.</li>
<li>Jakobsson, S., Borg, B., Haux, C. et al. Fish Physiology and Biochemistry (1999) 20: 79. doi:10.1023/A:1007776016610</li>
<li>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.</li>
<li>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.</li>
<li><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Judson R, Houck K, Paul Friedman K, Brown J, Browne P, Johnston PA, Close DA, Mansouri K, Kleinstreuer N. Selecting a minimal set of androgen receptor assays for screening chemicals. 2002. Regulatory Toxicology and Pharmacology, 117, 104764. https://doi.org/10.1016/j.yrtph.2020.104764</span></span></li>
<li>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.</li>
<li><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Kleinstreuer NC, Ceger P, Watt ED, Martin M, Houck K, Browne P, Thomas RS, Casey WM, Dix DJ, Allen D, Sakamuru S, Xia M, Huang R, Judson R. Development and Validation of a Computational Model for Androgen Receptor Activity. 2017. Chem Res Toxicol. 17;30(4):946-964. https://doi.org/10.1021/acs.chemrestox.6b00347</span></span></li>
<li>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.</li>
<li><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Luetjens C M, Weinbauer GF. Testosterone: biosynthesis, transport, metabolism and (non-genomic) actions. 2012. In Testosterone (pp. 15–32). Cambridge University Press. https://doi.org/10.1017/CBO9781139003353.003</span></span></li>
<li>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.</li>
<li><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Mansouri K, Kleinstreuer N, Abdelaziz AM, Alberga D, Alves VM, Andersson PL, Andrade CH, Bai F, Balabin I, Ballabio D, Benfenati E, Bhhatarai B, Boyer S, Chen J, Consonni V, Farag S, Fourches D, García-Sosa AT, Gramatica P, … Judson RS. CoMPARA: Collaborative Modeling Project for Androgen Receptor Activity. 2020. Environmental Health Perspectives, 128(2), 027002. https://doi.org/10.1289/EHP5580</span></span></li>
<li>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.</li>
<li><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Naamneh Elzenaty R, du Toit T, Flück CE. Basics of androgen synthesis and action. 2022. Best Practice & Research Clinical Endocrinology & Metabolism, 36(4), 101665. https://doi.org/10.1016/j.beem.2022.101665</span></span></li>
<li>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.</li>
<li>OECD (2012), <em>Test No. 229: Fish Short Term Reproduction Assay</em>, OECD Publishing, Paris.<br />
<li>OECD (2016), <em>Test No. 458: Stably Transfected Human Androgen Receptor Transcriptional Activation Assay for Detection of Androgenic Agonist and Antagonist Activity of Chemicals</em>, OECD Publishing, Paris.<br />
<li>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.</li>
<li>Prescott J, Coetzee GA. 2006. Molecular chaperones throughout the life cycle of the androgen receptor. Cancer letters 231(1): 12-19.</li>
<li><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Raut SA, Howell WM, Angus RA. Endocrine-disrupting effects of spironolactone in female western mosquitofish, Gambusia affinis. Environ Toxicol Chem. 2011 Jun;30(6):1376-82. https://doi.org/10.1002/etc.504</span></span></li>
<li><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Sébillot A, Damdimopoulou P, Ogino Y, Spirhanzlova P, Miyagawa S, Du Pasquier D, Mouatassim N, Iguchi T, Lemkine GF, Demeneix BA, Tindall AJ. Rapid fluorescent detection of (anti)androgens with spiggin-gfp medaka. Environ Sci Technol. 2014 Sep 16;48(18):10919-28. https://doi.org/10.1021/es5030977</span></span></li>
<li>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.</li>
<li>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.</li>
<li>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.</li>
<li>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.</li>
<li><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Sutinen P, Malinen M, Palvimo JJ. Androgen Receptor. 2017. In M. Simoni & I. T. Huhtaniemi (Eds.), Endocrinology of the Testis and Male Reproduction (pp. 395–416). Springer International Publishing. https://doi.org/10.1007/978-3-319-44441-3_12</span></span></li>
<li>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.</li>
<li>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.</li>
<li>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.</li>
<li>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.</li>
<li>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.</li>
<li>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.</li>
<li>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.</li>
<li>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.</li>
<td><a href="/aops/107">Aop:107 - Constitutive androstane receptor activation leading to hepatocellular adenomas and carcinomas in the mouse and the rat</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/117">Aop:117 - Androgen receptor activation leading to hepatocellular adenomas and carcinomas (in mouse and rat)</a></td>
<td><a href="/aops/107">Aop:107 - Constitutive androstane receptor activation leading to hepatocellular adenomas and carcinomas in the mouse and the rat</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/117">Aop:117 - Androgen receptor activation leading to hepatocellular adenomas and carcinomas (in mouse and rat)</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/37">Aop:37 - PPARα activation leading to hepatocellular adenomas and carcinomas in rodents</a></td>
<!-- Evidence for Perturbation of This Event by Stressors -->
<h3>Evidence for Perturbation by Stressor</h3>
<hr>
<br>
<h4>Phenobarbital</h4>
<p><p>Phenobarbital</p>
<p>1. NaPB treatment has been shown to increase replicative DNA synthesis in cultured mouse (Haines et al., 2018c) and rat hepatocytes (Haines et al., 2018c; Hirose et al., 2009).</p>
<p>2. NaPB treatment (1 week 500-2500 ppm in the diet) was shown to significantly increase the BrdU labeling index in the livers of male CD-1 mice and male Wistar rats compared to their respective vehicle-treated controls (Yamada et al., 2014).</p>
<p>3. An increase in replicative DNA synthesis was observed in male and female mice administered 1000 ppm NaPB in the diet for 1 month (Jones et al., 2009).</p>
<p>4. PB at 0, 10, 50, 100 and 500 mg/kg (ppm) in the diet was administered to 8 week old male rats and male mice for 90 days. A significant induction of hepatic replicative DNA synthesis (as determined by [3H]-thymidine incorporation) was observed in the rat liver at 7 days, but had returned to control levels by 14 days. In mice, there was a significant increase in hepatic replicative DNA synthesis throughout treatment (Kolaja et al., 1996a). In both species, the most pronounced effect was observed in the centrilobular region.</p>
</p>
<br>
<h4>Epidermal growth factor</h4>
<p><p>Epidermal growth factor</p>
<p>1. Human epidermal growth factor (hEGF) treatment was shown to significantly increase replicative DNA synthesis, and Ki-67 mRNA levels in human hepatocytes of chimeric mice with humanized livers (human hepatocyte chimeric livers) (Yamada et al., 2014).</p>
<p>2. EGF has been shown to increase the proliferation of mouse (Bowen et al., 2014; Haines et al., 2018c), rat (Bowen et al., 2014; Haines et al., 2018c; Hodges et al., 2000), and human (Haines et al., 2018c; Parzefall et al., 1991) hepatocyte cultures as determined by increase in replicative DNA synthesis compared to appropriate controls.</p>
</p>
<br>
<h4>pirinixic acid</h4>
<p><p>WY-14,643 (pirinixic acid)</p>
<ol>
<li>WY-14,643 (pirinixic acid) is a potent PPARα activator, and its ability to stimulate cell proliferation has been reviewed in Corton et al. (2018).</li>
<p>Epidermal growth factor (EGF) is one of several extracellular ligands of the epidermal growth factor receptor (EGFR). The EGFR signaling pathway is conserved in most animals, in which it controls processes such as cell proliferation, differentiation, adhesion, and migration (Barberan and Cebria, 2018).</p>
<p>Epidermal growth factor (EGF) is one of several extracellular ligands of the epidermal growth factor receptor (EGFR). The EGFR signaling pathway is conserved in most animals, in which it controls processes such as cell proliferation, differentiation, adhesion, and migration (Barberan and Cebria, 2018).</p>
<p>EGFR is a transmembrane protein that is classified as a tyrosine kinase receptor. EGFR has several structural domains: 1) an N-terminal extracellular domain that binds ligands such as EGF, 2) a transmembrane domain, 3) an intracellular domain containing tyrosine kinase activity, and 4) a C-terminal region that contains tyrosine residues that are the sites of autophosphorylation. Ligand binding results in a cascade of events that include EGFR homo-or heterodimerization, activation of the tyrosine kinase domain, tyrosine autophosphorylation, and ultimately the activation of downstream signaling cascades that control various processes in the liver such as proliferation, survival, differentiation, response to injury, and repair (Berasain and Avila, 2014; Komposch and Sibilia, 2015).</p>
<p>EGF has been used as an agent to stimulate proliferation of rat, mouse, and human hepatic cells in culture (Bowen et al., 2014; Haines et al., 2018c; Hodges et al., 2000; Parzefall et al., 1991).</p>
<p>Other mitogenic agents produce a cell proliferation response in rats and mice, but not other mammalian species such as humans, hamsters or dogs. These agents include phenobarbital (a model CAR activator) (Haines et al., 2018c; Hirose et al., 2009; Parzefall et al., 1991), WY-14,643 (pirinixic acid) (a model PPARalpha activator) (Corton et al., 2018) and TCDD (a model AhR activator) (Becker et al., 2015; Budinsky et al., 2014).</p>
<br>
</div>
<!-- event text -->
<h4>Key Event Description</h4>
<p><u>Key Event Description:</u></p>
<h4>Key Event Description</h4>
<p><u>Key Event Description:</u></p>
<p>One of the mechanisms known to induce cell proliferation in the livers of rats and mice occurs through exposure to a mitogen. Mitogenic cell proliferation is characterized by liver enlargement without evidence of necrosis, as opposed to regenerative/compensatory proliferation, in which the liver parenchyma is restored after loss due to necrosis or hepatectomy.</p>
<p>Cell proliferation in the livers of rats and mice occurs through exposure to a mitogen and is characterized by liver enlargement without evidence of necrosis. In contrast, regenerative/compensatory proliferation occurs following loss of liver parenchymal cells from necrosis or hepatectomy.</p>
<p>In mammals that have been administered a mitogenic xenobiotic, several factors impact the nature of the hepatocyte proliferative response. These include the identity of the mitogen, the time course and dose of administration, and the species and strain of the test system. The effects on the liver may be confined to certain lobes, or may be observed throughout the organ (<a href="#_ENREF_8" title="Columbano, 1996 #152">Columbano and Shinozuka, 1996</a>).</p>
<br>
<p>In mammals, the nature of the hepatocyte proliferative response is shaped by the identity of the mitogen, the time course and dose of administration, and the species and strain of the test animal (<a href="#_ENREF_8" title="Columbano, 1996 #152">Columbano and Shinozuka, 1996</a>).</p>
<h4>How it is Measured or Detected</h4>
<p>There are several well-characterized and well-accepted techniques that have been used to detect mitogenic proliferation in vitro and in vivo (Peffer et al., 2018b). These include the detection of labeled nucleosides or nucleoside analogs that have been incorporated into newly synthesized DNA, or the detection of endogenous markers of proliferation such as antigen Ki-67 or proliferating cell nuclear antigen (PCNA) (Kee et al., 2002; Muskhelishvili et al., 2003; Wood et al., 2015). Several of these techniques may involve immunohistochemical techniques to detect proliferating cells, thus allowing for the detection of proliferation within specific tissue sections. For each of these methods, a labeling index (fraction of labeled cell population/total number of cells in population) is calculated, and this index can be statistically compared between different groups (Wood et al., 2015).</p>
<h4>How it is Measured or Detected</h4>
<p>Mitogenic proliferation in vitro and in vivo is measured by the incorporation of labeled nucleosides or nucleoside analogs into newly synthesized DNA (Peffer et al., 2018b), the detection of endogenous markers of proliferation such as antigen Ki-67 or proliferating cell nuclear antigen (PCNA) (Kee et al., 2002; Muskhelishvili et al., 2003; Wood et al., 2015), and other immunohistochemical techniques to detect proliferating cells. For each of these methods, a labeling index (fraction of labeled cell population/total number of cells in population) is calculated, and this index can be statistically compared between different groups (Wood et al., 2015).</p>
<p><u>Nucleoside and nucleoside analog labeling</u>. Actively proliferating cells undergo DNA synthesis in a highly regulated process during the S (synthesis) phase of the cell cycle. Once the DNA of a cell is replicated during S phase, the cell undergoes mitosis. This results in two cells, each of which has a complete copy of the genome. The DNA replication that occurs in S phase may be detected by the incorporation radiolabeled (e.g., 3H-thymidine) into the newly synthesized DNA, which can be detected from isolated livers using standard autoradiographic techniques. Nucleoside analogs may also be incorporated into the newly-synthesized DNA, including 5-bromo-2-deoxyuridine (BrdU) or 5-ethyl-2’-deoxy uridine (EdU), which may be detected using standard immunohistochemical and biolabeling techniques, respectively (Cavanagh et al., 2011). Drawbacks of the use of nucleoside analogs include concerns regarding the proper administration (dose, route of administration and length of exposure) to animals that allow for adequate labeling without inducing considerable toxicity (Cavanagh et al., 2011; Cohen, 2010). In addition, nucleoside/nucleoside analog incorporation techniques are not specific for the detection of proliferation but may also identify cells that are undergoing DNA synthesis during apoptosis or DNA repair.</p>
<p><u>Endogenous markers of proliferation.</u> Ki-67 and PCNA are endogenous proteins expressed by mammalian cells that are in active phases of the cell cycle (G1, S, G2, M) and are not expressed in quiescent (G0) cells (Dietrich, 1993; Eldrige et al., 1993; Scholzen and Gerdes, 2000). They are detected in hepatocytes using standard immunohistochemical techniques. The advantage of using endogenous markers is that they do not require administration of exogenous markers for labeling, and they can be used for both prospective and retrospective cell proliferation analysis. A direct comparison of BrdU, Ki67 and PCNA labeling in various proliferating tissues of male Sprague-Dawley rats (Muskhelishvili et al., 2003) has indicated that Ki67 and BrdU immunohistochemistry methods gave similar labelling index results, whereas PCNA immunohistochemistry was not concordant with these methods and gave highly variable results. These authors suggested that PCNA is less accurate as a measure of cell proliferation because it has a long half-life and can be retained in cells that are not dividing, and is more involved in DNA repair mechanisms than Ki67. As a result, Ki67 has emerged as a more preferred endogenous marker for assessing cell proliferation in hepatocytes in recent years compared to PCNA.</p>
<br>
<h4>References</h4>
<p><a name="_ENREF_1">Barberan, S. and Cebria, F. (2018), The role of the EGFR signaling pathway in stem cell differentiation during planarian regeneration and homeostasis. <em>Semin Cell Dev Biol</em>, 10.1016/j.semcdb.2018.05.011. </a></p>
<p> </p>
<h4>References</h4>
<p><a name="_ENREF_1">Barberan, S. and Cebria, F. (2018), The role of the EGFR signaling pathway in stem cell differentiation during planarian regeneration and homeostasis. <em>Semin Cell Dev Biol</em>, 10.1016/j.semcdb.2018.05.011. </a></p>
<p><a name="_ENREF_2">Becker, R. A., Patlewicz, G., Simon, T. W., Rowlands, J. C. and Budinsky, R. A. (2015), The adverse outcome pathway for rodent liver tumor promotion by sustained activation of the aryl hydrocarbon receptor. <em>Regul Toxicol Pharmacol</em> <strong>73</strong>, 172-90, 10.1016/j.yrtph.2015.06.015. </a></p>
<p> </p>
<p><a name="_ENREF_3">Berasain, C. and Avila, M. A. (2014), The EGFR signalling system in the liver: from hepatoprotection to hepatocarcinogenesis. <em>J Gastroenterol</em> <strong>49</strong>, 9-23, 10.1007/s00535-013-0907-x. </a></p>
<p> </p>
<p><a name="_ENREF_4">Bowen, W. C., Michalopoulos, A. W., Orr, A., Ding, M. Q., Stolz, D. B. and Michalopoulos, G. K. (2014), Development of a chemically defined medium and discovery of new mitogenic growth factors for mouse hepatocytes: mitogenic effects of FGF1/2 and PDGF. <em>PLoS One</em> <strong>9</strong>, e95487, 10.1371/journal.pone.0095487. </a></p>
<p> </p>
<p><a name="_ENREF_5">Budinsky, R. A., Schrenk, D., Simon, T., Van den Berg, M., Reichard, J. F., Silkworth, J. B., Aylward, L. L., Brix, A., Gasiewicz, T., Kaminski, N., Perdew, G., Starr, T. B., Walker, N. J. and Rowlands, J. C. (2014), Mode of action and dose-response framework analysis for receptor-mediated toxicity: The aryl hydrocarbon receptor as a case study. <em>Crit Rev Toxicol</em> <strong>44</strong>, 83-119, 10.3109/10408444.2013.835787. </a></p>
<p> </p>
<p><a name="_ENREF_6">Cavanagh, B. L., Walker, T., Norazit, A. and Meedeniya, A. C. (2011), Thymidine analogues for tracking DNA synthesis. <em>Molecules</em> <strong>16</strong>, 7980-93, 10.3390/molecules16097980. </a></p>
<p> </p>
<p><a name="_ENREF_7">Cohen, S. M. (2010), Evaluation of possible carcinogenic risk to humans based on liver tumors in rodent assays: the two-year bioassay is no longer necessary. <em>Toxicol Pathol</em> <strong>38</strong>, 487-501, 10.1177/0192623310363813. </a></p>
<p> </p>
<p><a name="_ENREF_8">Columbano, A. and Shinozuka, H. (1996), Liver regeneration versus direct hyperplasia. <em>FASEB J</em> <strong>10</strong>, 1118-28. </a></p>
<p> </p>
<p><a name="_ENREF_9">Corton, J. C., Peters, J. M. and Klaunig, J. E. (2018), The PPARalpha-dependent rodent liver tumor response is not relevant to humans: addressing misconceptions. <em>Arch Toxicol</em> <strong>92</strong>, 83-119, 10.1007/s00204-017-2094-7. </a></p>
<p> </p>
<p><a name="_ENREF_10">Dietrich, D. R. (1993), Toxicological and pathological applications of proliferating cell nuclear antigen (PCNA), a novel endogenous marker for cell proliferation. <em>Crit Rev Toxicol</em> <strong>23</strong>, 77-109, 10.3109/10408449309104075. </a></p>
<p> </p>
<p><a name="_ENREF_11">Eldrige, S. R., Butterworth, B. E. and Goldsworthy, T. L. (1993), Proliferating cell nuclear antigen: a marker for hepatocellular proliferation in rodents. <em>Environ Health Perspect</em> <strong>101 Suppl 5</strong>, 211-8, 10.1289/ehp.93101s5211. </a></p>
<p> </p>
<p><a name="_ENREF_12">Haines, C., Elcombe, B. M., Chatham, L. R., Vardy, A., Higgins, L. G., Elcombe, C. R. and Lake, B. G. (2018c), Comparison of the effects of sodium phenobarbital in wild type and humanized constitutive androstane receptor (CAR)/pregnane X receptor (PXR) mice and in cultured mouse, rat and human hepatocytes. <em>Toxicology</em> <strong>396-397</strong>, 23-32, 10.1016/j.tox.2018.02.001. </a></p>
<p> </p>
<p><a name="_ENREF_13">Hirose, Y., Nagahori, H., Yamada, T., Deguchi, Y., Tomigahara, Y., Nishioka, K., Uwagawa, S., Kawamura, S., Isobe, N., Lake, B. G. and Okuno, Y. (2009), Comparison of the effects of the synthetic pyrethroid Metofluthrin and phenobarbital on CYP2B form induction and replicative DNA synthesis in cultured rat and human hepatocytes. <em>Toxicology</em> <strong>258</strong>, 64-9. </a></p>
<p> </p>
<p><a name="_ENREF_14">Hodges, N. J., Orton, T. C., Strain, A. J. and Chipman, J. K. (2000), Potentiation of epidermal growth factor-induced DNA synthesis in rat hepatocytes by phenobarbitone: possible involvement of oxidative stress and kinase activation. <em>Carcinogenesis</em> <strong>21</strong>, 2041-7. </a></p>
<p> </p>
<p><a name="_ENREF_15">Jones, H. B., Orton, T. C. and Lake, B. G. (2009), Effect of chronic phenobarbitone administration on liver tumour formation in the C57BL/10J mouse. <em>Food Chem Toxicol</em> <strong>47</strong>, 1333-40, 10.1016/j.fct.2009.03.014. </a></p>
<p> </p>
<p><a name="_ENREF_16">Kee, N., Sivalingam, S., Boonstra, R. and Wojtowicz, J. M. (2002), The utility of Ki-67 and BrdU as proliferative markers of adult neurogenesis. <em>J Neurosci Methods</em> <strong>115</strong>, 97-105. </a></p>
<p> </p>
<p><a name="_ENREF_17">Kolaja, K. L., Stevenson, D. E., Johnson, J. T., Walborg, E. F., Jr. and Klaunig, J. E. (1996a), Subchronic effects of dieldrin and phenobarbital on hepatic DNA synthesis in mice and rats. <em>Fundam Appl Toxicol</em> <strong>29</strong>, 219-28. </a></p>
<p> </p>
<p><a name="_ENREF_18">Komposch, K. and Sibilia, M. (2015), EGFR Signaling in Liver Diseases. <em>Int J Mol Sci</em> <strong>17</strong>, 10.3390/ijms17010030. </a></p>
<p> </p>
<p><a name="_ENREF_19">Muskhelishvili, L., Latendresse, J. R., Kodell, R. L. and Henderson, E. B. (2003), Evaluation of cell proliferation in rat tissues with BrdU, PCNA, Ki-67(MIB-5) immunohistochemistry and in situ hybridization for histone mRNA. <em>J Histochem Cytochem</em> <strong>51</strong>, 1681-8. </a></p>
<p> </p>
<p><a name="_ENREF_20">Parzefall, W., Erber, E., Sedivy, R. and Schulte-Hermann, R. (1991), Testing for induction of DNA synthesis in human hepatocyte primary cultures by rat liver tumor promoters. <em>Cancer Res</em> <strong>51</strong>, 1143-7. </a></p>
<p> </p>
<p><a name="_ENREF_21">Peffer, R. C., LeBaron, M. J., Battalora, M., Bomann, W. H., Werner, C., Aggarwal, M., Rowe, R. R. and Tinwell, H. (2018b), Minimum datasets to establish a CAR-mediated mode of action for rodent liver tumors. <em>Regul Toxicol Pharmacol</em> <strong>96</strong>, 106-120, 10.1016/j.yrtph.2018.04.001. </a></p>
<p> </p>
<p><a name="_ENREF_22">Scholzen, T. and Gerdes, J. (2000), The Ki-67 protein: from the known and the unknown. <em>J Cell Physiol</em> <strong>182</strong>, 311-22, 10.1002/(sici)1097-4652(200003)182:3<311::aid-jcp1>3.0.co;2-9. </a></p>
<p> </p>
<p><a name="_ENREF_23">Wood, C. E., Hukkanen, R. R., Sura, R., Jacobson-Kram, D., Nolte, T., Odin, M. and Cohen, S. M. (2015), Scientific and Regulatory Policy Committee (SRPC) Review: Interpretation and Use of Cell Proliferation Data in Cancer Risk Assessment. <em>Toxicol Pathol</em> <strong>43</strong>, 760-75, 10.1177/0192623315576005. </a></p>
<p> </p>
<p><a name="_ENREF_24">Yamada, T., Okuda, Y., Kushida, M., Sumida, K., Takeuchi, H., Nagahori, H., Fukuda, T., Lake, B. G., Cohen, S. M. and Kawamura, S. (2014), Human hepatocytes support the hypertrophic but not the hyperplastic response to the murine nongenotoxic hepatocarcinogen sodium phenobarbital in an in vivo study using a chimeric mouse with humanized liver. <em>Toxicol Sci</em> <strong>142</strong>, 137-57, 10.1093/toxsci/kfu173. </a></p>
<td><a href="/aops/107">Aop:107 - Constitutive androstane receptor activation leading to hepatocellular adenomas and carcinomas in the mouse and the rat</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/117">Aop:117 - Androgen receptor activation leading to hepatocellular adenomas and carcinomas (in mouse and rat)</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/118">Aop:118 - Chronic cytotoxicity leading to hepatocellular adenomas and carcinomas (in mouse and rat)</a></td>
<td><a href="/aops/107">Aop:107 - Constitutive androstane receptor activation leading to hepatocellular adenomas and carcinomas in the mouse and the rat</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/117">Aop:117 - Androgen receptor activation leading to hepatocellular adenomas and carcinomas (in mouse and rat)</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/118">Aop:118 - Chronic cytotoxicity leading to hepatocellular adenomas and carcinomas (in mouse and rat)</a></td>
<td><a href="/aops/107">Aop:107 - Constitutive androstane receptor activation leading to hepatocellular adenomas and carcinomas in the mouse and the rat</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/108">Aop:108 - Inhibition of pyruvate dehydrogenase kinase leading to hepatocellular adenomas and carcinomas (in mouse and rat)</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/117">Aop:117 - Androgen receptor activation leading to hepatocellular adenomas and carcinomas (in mouse and rat)</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/118">Aop:118 - Chronic cytotoxicity leading to hepatocellular adenomas and carcinomas (in mouse and rat)</a></td>
<td><a href="/aops/107">Aop:107 - Constitutive androstane receptor activation leading to hepatocellular adenomas and carcinomas in the mouse and the rat</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/108">Aop:108 - Inhibition of pyruvate dehydrogenase kinase leading to hepatocellular adenomas and carcinomas (in mouse and rat)</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/117">Aop:117 - Androgen receptor activation leading to hepatocellular adenomas and carcinomas (in mouse and rat)</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/118">Aop:118 - Chronic cytotoxicity leading to hepatocellular adenomas and carcinomas (in mouse and rat)</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/37">Aop:37 - PPARα activation leading to hepatocellular adenomas and carcinomas in rodents</a></td>
<td><a href="/aops/107">Constitutive androstane receptor activation leading to hepatocellular adenomas and carcinomas in the mouse and the rat</a></td>
<td>adjacent</td>
<td>High</td>
<td>Moderate</td>
</tr>
</thead>
<tbody>
<tr>
<th><a href="/aops/107">Constitutive androstane receptor activation leading to hepatocellular adenomas and carcinomas in the mouse and the rat</a></th>
<th>adjacent</th>
<th>High </th>
<th>High</th>
</tr>
<tr>
<th><a href="/aops/117">Androgen receptor activation leading to hepatocellular adenomas and carcinomas (in mouse and rat)</a></th>
<th>adjacent</th>
<th> </th>
<th></th>
</tr>
</tbody>
</table>
</div>
<h4>Evidence Supporting Applicability of this Relationship</h4>
<br>
<!-- loop to find taxonomic applicability under relationship -->
<p>Studies in various species, or in isolated hepatocytes from various mammalian species including humans, have demonstrated that CAR activators such as phenobarbital or metofluthrin produce a cell proliferation response that is seen in mice or rats, but not in hamsters, guinea pigs or humans (Hasmall and Roberts, 1999; Hirose et al., 2009; James and Roberts, 1996; Yamada et al., 2014; Yamada et al., 2009). Accordingly, phenobarbital and other CAR activators do not produce liver tumors in long term studies in hamsters (Diwan et al., 1986; Elcombe et al., 2014). Consistent with the lack of effects on proliferation, Diwan et al. (1986) also reported that in Syrian hamsters, phenobarbital treatment at 500 ppm in the drinking water did not produce any increases in preneoplastic foci of cellular alteration compared to groups that received an initiator alone. Therefore, this key event of increased foci in the liver has strong data indicating it is specific to mice and rats, the species which also develop hepatocellular tumors in response to known CAR activators.</p>
</div>
<!-- end loop for taxons -->
<!-- loop to find life stages under relationship -->
<div>
</div>
<!-- end loop for life stages -->
<!-- sex terms -->
<div>
</div>
<!-- end sex terms -->
<p>Studies in various species, or in isolated hepatocytes from various mammalian species including humans, have demonstrated that CAR activators such as phenobarbital or metofluthrin produce a cell proliferation response that is seen in mice or rats, but not in hamsters, guinea pigs or humans (Hasmall and Roberts, 1999; Hirose et al., 2009; James and Roberts, 1996; Yamada et al., 2014; Yamada et al., 2009). Accordingly, phenobarbital and other CAR activators do not produce liver tumors in long term studies in hamsters (Diwan et al., 1986; Elcombe et al., 2014). Consistent with the lack of effects on proliferation, Diwan et al. (1986) also reported that in Syrian hamsters, phenobarbital treatment at 500 ppm in the drinking water did not produce any increases in preneoplastic foci of cellular alteration compared to groups that received an initiator alone. Therefore, this key event of increased foci in the liver has strong data indicating it is specific to mice and rats, the species which also develop hepatocellular tumors in response to known CAR activators.</p>
<h4>Key Event Relationship Description</h4>
<p>Based on altered gene expression under the influence of CAR activation, an increase in cell proliferation of hepatocytes leads to a greater chance of normal, spontaneous errors in DNA replication and thus a higher proportion of altered hepatocytes. The hepatocytes with abnormal DNA can exhibit cell-cell communication differences from normal hepatocytes, and experience greater cell division even in the presence of contact inhibition with other hepatocytes. The islands of more actively dividing hepatocytes can be detected via histology based both on the larger numbers of cells (hyperplasia) and possibly a characteristic staining property of the clonally expanded cells (foci of cellular alteration – either eosinophilic, basophilic or clear cell). Thus, a higher rate of proliferation in the rodent liver leads to greater prevalence of altered hepatocytes, which clonally expand to generate an increase in preneoplastic foci.</p>
<!-- if nothing shows up in any of these fields, then evidence supporting this KER will not be displayed -->
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p>The increased cell replication rate in the liver due to CAR activation (i.e. via a mitogenic signaling) is similar to other well-understood modes of action where an increase in cell proliferation leads to an eventual increase in preneoplastic foci, such as PPARα activating ligands and AhR activating ligands, which also lead to an increase in preneoplastic foci via clonal expansion of transformed hepatocytes. In mice lacking the CAR receptor, including initiation-promotion assays, the upstream events (e.g. CAR activation, altered gene expression, and increased cell proliferation) and the downstream events (e.g. preneoplastic foci) are all blocked, providing strong support for the biological plausibility of this Key Event Relationship (Huang et al., 2005; Tamura et al., 2015; Tamura et al., 2013; Yamamoto et al., 2004).</p>
<strong>Empirical Evidence</strong>
<p>The observed increase in numbers of preneoplastic foci, usually with eosinophilic staining properties, is observed with great regularity in mode of action work of CAR activating xenobiotics where histopathology at later times has been examined. This increase in foci (mixed or eosinophilic) after 2 years was observed at tumorigenic dose levels with metofluthrin in male rats (Deguchi et al., 2009), and at tumorigenic dose levels in mice treated with phenobarbital (Jones et al., 2009). With TCPOBOP in mice, multiple eosinophilic foci were reported to co-occur along with an increased incidence of eosinophilic adenomas and carcinomas after 60 weeks of treatment (Diwan et al., 1992). With well-studied CAR activators such as phenobarbital and TCPOBOP, increased cell proliferation has been detected at similar dose levels where increased altered foci are seen (Geter et al., 2014; Huang et al., 2005; Kolaja et al., 1996a; Kolaja et al., 1996b) (Tables 2 and 3); therefore, there is strong support for the linkage of these earlier key events with CAR activators leading to an increase in pre-neoplastic foci.</p>
<strong>Uncertainties and Inconsistencies</strong>
<p>The incidence of altered foci, and their histological staining properties (e.g. eosinophilic, basophilic, clear cell, mixed) are not always reported in published studies of carcinogenicity with CAR activating compounds. In addition, the timing of interim or final sacrifices and histopathology data may possibly miss a window of time (for certain molecules) where the increase in preneoplastic foci can be quantified. However, the consistent findings with well-known CAR activating compounds and their absence in CAR knockout mouse studies provide a strong basis for their existence in the CAR AOP.</p>
<h4>Key Event Relationship Description</h4>
<p>Based on altered gene expression under the influence of CAR activation, an increase in cell proliferation of hepatocytes leads to a greater chance of normal, spontaneous errors in DNA replication and thus a higher proportion of altered hepatocytes. The hepatocytes with abnormal DNA can exhibit cell-cell communication differences from normal hepatocytes, and experience greater cell division even in the presence of contact inhibition with other hepatocytes. The islands of more actively dividing hepatocytes can be detected via histology based both on the larger numbers of cells (hyperplasia) and possibly a characteristic staining property of the clonally expanded cells (foci of cellular alteration – either eosinophilic, basophilic or clear cell). Thus, a higher rate of proliferation in the rodent liver leads to greater prevalence of altered hepatocytes, which clonally expand to generate an increase in preneoplastic foci.</p>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p>The increased cell replication rate in the liver due to CAR activation (i.e. via a mitogenic signaling) is similar to other well-understood modes of action where an increase in cell proliferation leads to an eventual increase in preneoplastic foci, such as PPARα activating ligands and AhR activating ligands, which also lead to an increase in preneoplastic foci via clonal expansion of transformed hepatocytes. In mice lacking the CAR receptor, including initiation-promotion assays, the upstream events (e.g. CAR activation, altered gene expression, and increased cell proliferation) and the downstream events (e.g. preneoplastic foci) are all blocked, providing strong support for the biological plausibility of this Key Event Relationship (Huang et al., 2005; Tamura et al., 2015; Tamura et al., 2013; Yamamoto et al., 2004).</p>
<p>The observed increase in numbers of preneoplastic foci, usually with eosinophilic staining properties, is observed with great regularity in mode of action work of CAR activating xenobiotics where histopathology at later times has been examined. This increase in foci (mixed or eosinophilic) after 2 years was observed at tumorigenic dose levels with metofluthrin in male rats (Deguchi et al., 2009), and at tumorigenic dose levels in mice treated with phenobarbital (Jones et al., 2009). With TCPOBOP in mice, multiple eosinophilic foci were reported to co-occur along with an increased incidence of eosinophilic adenomas and carcinomas after 60 weeks of treatment (Diwan et al., 1992). With well-studied CAR activators such as phenobarbital and TCPOBOP, increased cell proliferation has been detected at similar dose levels where increased altered foci are seen (Geter et al., 2014; Huang et al., 2005; Kolaja et al., 1996a; Kolaja et al., 1996b) (Tables 2 and 3); therefore, there is strong support for the linkage of these earlier key events with CAR activators leading to an increase in pre-neoplastic foci.</p>
<h4>References</h4>
<p>[see reference list at end of this AOP; it includes all cited references]</p>
<strong>Uncertainties and Inconsistencies</strong>
<p>The incidence of altered foci, and their histological staining properties (e.g. eosinophilic, basophilic, clear cell, mixed) are not always reported in published studies of carcinogenicity with CAR activating compounds. In addition, the timing of interim or final sacrifices and histopathology data may possibly miss a window of time (for certain molecules) where the increase in preneoplastic foci can be quantified. However, the consistent findings with well-known CAR activating compounds and their absence in CAR knockout mouse studies provide a strong basis for their existence in the CAR AOP.</p>
<h4>References</h4>
<p>[see reference list at end of this AOP; it includes all cited references]</p>
<td><a href="/aops/107">Constitutive androstane receptor activation leading to hepatocellular adenomas and carcinomas in the mouse and the rat</a></td>
<td>adjacent</td>
<td>High</td>
<td>Not Specified</td>
</tr>
<tr>
<td><a href="/aops/117">Androgen receptor activation leading to hepatocellular adenomas and carcinomas (in mouse and rat)</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<th>AOP Name</th>
<th>Adjacency</th>
<th>Weight of Evidence</th>
<th>Quantitative Understanding</th>
<td><a href="/aops/118">Chronic cytotoxicity leading to hepatocellular adenomas and carcinomas (in mouse and rat)</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
</thead>
<tbody>
<tr>
<th><a href="/aops/107">Constitutive androstane receptor activation leading to hepatocellular adenomas and carcinomas in the mouse and the rat</a></th>
<th>adjacent</th>
<th>High </th>
<th>High</th>
</tr>
<tr>
<th><a href="/aops/117">Androgen receptor activation leading to hepatocellular adenomas and carcinomas (in mouse and rat)</a></th>
<th>adjacent</th>
<th> </th>
<th></th>
</tr>
<tr>
<th><a href="/aops/118">Chronic cytotoxicity leading to hepatocellular adenomas and carcinomas (in mouse and rat)</a></th>
<th>adjacent</th>
<th> </th>
<th></th>
</tr>
</tbody>
</table>
</div>
<h4>Evidence Supporting Applicability of this Relationship</h4>
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<p>Phenobarbital and other CAR activators do not produce liver tumors in long term studies in hamsters (Diwan et al., 1986; Elcombe et al., 2014). Consistent with the lack of effects on proliferation and on tumor development, Diwan et al. (1986) also reported that phenobarbital treatment at 500 ppm in the drinking water did not produce any increases in preneoplastic foci of cellular alteration compared to groups that received an initiator alone. Further, treatment of CAR knockout mice lacking the CAR nuclear receptor with phenobarbital or TCPOBOP produced none of the early key events (e.g. altered expression of CAR-responsive cell cycle genes, increased cell proliferation) and no increases in altered foci or tumors (Huang et al., 2005; Yamamoto et al., 2004). Therefore, the development of increased foci in the liver in response to treatment with CAR activators has strong data indicating it is specific to mice and rats, the species which also develop hepatocellular tumors in response to known CAR activators.</p>
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<p>Phenobarbital and other CAR activators do not produce liver tumors in long term studies in hamsters (Diwan et al., 1986; Elcombe et al., 2014). Consistent with the lack of effects on proliferation and on tumor development, Diwan et al. (1986) also reported that phenobarbital treatment at 500 ppm in the drinking water did not produce any increases in preneoplastic foci of cellular alteration compared to groups that received an initiator alone. Further, treatment of CAR knockout mice lacking the CAR nuclear receptor with phenobarbital or TCPOBOP produced none of the early key events (e.g. altered expression of CAR-responsive cell cycle genes, increased cell proliferation) and no increases in altered foci or tumors (Huang et al., 2005; Yamamoto et al., 2004). Therefore, the development of increased foci in the liver in response to treatment with CAR activators has strong data indicating it is specific to mice and rats, the species which also develop hepatocellular tumors in response to known CAR activators.</p>
<h4>Key Event Relationship Description</h4>
<p>Clonally expanded cells (foci of cellular alteration – either eosinophilic, basophilic or clear cell) have been shown to be increased at tumorigenic dose levels of CAR activators such as phenobarbital, TCPOBOP and metofluthrin. As discussed for earlier key events, the CAR-mediated events that lead to an increase in altered foci lead to a greater abundance of cells with mutations in their DNA that are less responsive to normal cell-cell signaling and control mechanisms. As a result, these foci are considered preneoplastic lesions, and can progress with time into adenomas and carcinomas. The continued CAR-mediated stimulus for increased cell proliferation within these foci (e.g. as demonstrated in studies by Kolaja et al., 1996b) will also provide an environment where the mutant cells can survive and develop into tumors.</p>
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<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p>The development of liver tumors in rodents, whether spontaneously or induced by a non-genotoxic carcinogen, has consistently included the development of altered foci as a precursor step to hepatocellular adenomas and carcinomas (Goldsworthy and Fransson-Steen, 2002; Tamura et al., 2015). These foci are considered preneoplastic lesions, and their ability to progress to form adenomas and/or carcinomas in rodents has been previously recognized. In the case of CAR activators, an increased incidence of preneoplastic foci has been consistently shown to precede tumor development, and there is a high biological plausibility for this Key Event Relationship (Elcombe et al., 2014; Goldsworthy and Fransson-Steen, 2002; Jones et al., 2009; Lake, 2009).</p>
<h4>Key Event Relationship Description</h4>
<p>Clonally expanded cells (foci of cellular alteration – either eosinophilic, basophilic or clear cell) have been shown to be increased at tumorigenic dose levels of CAR activators such as phenobarbital, TCPOBOP and metofluthrin. As discussed for earlier key events, the CAR-mediated events that lead to an increase in altered foci lead to a greater abundance of cells with mutations in their DNA that are less responsive to normal cell-cell signaling and control mechanisms. As a result, these foci are considered preneoplastic lesions, and can progress with time into adenomas and carcinomas. The continued CAR-mediated stimulus for increased cell proliferation within these foci (e.g. as demonstrated in studies by Kolaja et al., 1996b) will also provide an environment where the mutant cells can survive and develop into tumors.</p>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p>The development of liver tumors in rodents, whether spontaneously or induced by a non-genotoxic carcinogen, has consistently included the development of altered foci as a precursor step to hepatocellular adenomas and carcinomas (Goldsworthy and Fransson-Steen, 2002; Tamura et al., 2015). These foci are considered preneoplastic lesions, and their ability to progress to form adenomas and/or carcinomas in rodents has been previously recognized. In the case of CAR activators, an increased incidence of preneoplastic foci has been consistently shown to precede tumor development, and there is a high biological plausibility for this Key Event Relationship (Elcombe et al., 2014; Goldsworthy and Fransson-Steen, 2002; Jones et al., 2009; Lake, 2009).</p>
<strong>Empirical Evidence</strong>
<p>The observed increase in numbers of preneoplastic foci, usually with eosinophilic staining properties, is observed with great regularity in mode of action work of CAR activating xenobiotics where histopathology at later times has been examined. This increase in foci (mixed or eosinophilic) after 2 years was observed at tumorigenic dose levels with metofluthrin in male rats (Deguchi et al., 2009), and at tumorigenic dose levels in mice treated with phenobarbital (Jones et al., 2009). With TCPOBOP in mice, multiple eosinophilic foci were reported to co-occur along with an increased incidence of eosinophilic adenomas and carcinomas after 60 weeks of treatment (Diwan et al., 1992).</p>
<strong>Empirical Evidence</strong>
<p>The observed increase in numbers of preneoplastic foci, usually with eosinophilic staining properties, is observed with great regularity in mode of action work of CAR activating xenobiotics where histopathology at later times has been examined. This increase in foci (mixed or eosinophilic) after 2 years was observed at tumorigenic dose levels with metofluthrin in male rats (Deguchi et al., 2009), and at tumorigenic dose levels in mice treated with phenobarbital (Jones et al., 2009). With TCPOBOP in mice, multiple eosinophilic foci were reported to co-occur along with an increased incidence of eosinophilic adenomas and carcinomas after 60 weeks of treatment (Diwan et al., 1992).</p>
<p>In addition, experiments where the MIE (CAR activation) is blocked have been performed with these model CAR activators. For phenobarbital and TCPOBOP in mice, the early key events and the progression to increased altered foci and hepatocellular tumors were all blocked in CAR knockout mice (Huang et al., 2005; Yamamoto et al., 2004). Foci of cellular alteration in CAR knockout mice were also prevented in an initiation-promotion model using the CAR activators cyproconazole and fluconazole (Tamura et al., 2015), and the incidence of adenomas and carcinomas was similarly decreased (Tamura et al., 2015). Thus, there is strong support for the involvement of CAR activation in these mechanisms, and that the stated sequence of key events following CAR activation leads to an increase in pre-neoplastic foci and then liver tumors in mice and rats.</p>
<p> </p>
<strong>Uncertainties and Inconsistencies</strong>
<p>The incidence of altered foci, and their staining properties (e.g. eosinophilic, basophilic, clear cell, mixed) are not always reported in published studies of carcinogenicity with CAR activation compounds. However, the consistent findings with well-known CAR activating compounds and their absence in CAR knockout mouse studies provide a strong basis for their existence in the CAR AOP.</p>
<strong>Uncertainties and Inconsistencies</strong>
<p>The incidence of altered foci, and their staining properties (e.g. eosinophilic, basophilic, clear cell, mixed) are not always reported in published studies of carcinogenicity with CAR activation compounds. However, the consistent findings with well-known CAR activating compounds and their absence in CAR knockout mouse studies provide a strong basis for their existence in the CAR AOP.</p>