Activation, PPARα

117-81-7

BJQHLKABXJIVAM-UHFFFAOYNA-N

BJQHLKABXJIVAM-UHFFFAOYSA-N

Di(2-ethylhexyl) phthalate

1,2-Benzenedicarboxylic acid, bis(2-ethylhexyl) ester DEHP 1,2-Benzedicarboxylic acid, bis(2-ethyl-hexyl) ester 1,2-Benzenedicarboxylic acid bis(2-ethylhexyl) ester 1,2-Benzenedicarboxylic acid, 1,2-bis(2-ethylhexyl) ester 1,2-Benzenedicarboxylic acid,bis(2-ethylhexylester) Bis(2-ethylhexyl) 1,2-benzenedicarboxylate Bis(2-ethylhexyl) o-phthalate bis(2-ethylhexyl) phthalate Bis(2-ethylhexyl)phthalat Bis(2-ethylhexyl)phthalate Bisoflex 81 Bisoflex DOP Corflex 400 Di(2-ethylhexyl)phthalate Di(isooctyl) phthalate Di-2-ethylhexlphthalate Di-2-ethylhexyl phthalate DI-2-ETHYLHEXYL-PHTHALATE Diacizer DOP Diethylhexyl phthalate Dioctylphthalate DOF Ergoplast FDO Ergoplast FDO-S ETHYLHEXYL PHTHALATE Eviplast 80 Eviplast 81 Fleximel Flexol DOD Flexol DOP ftlalato de bis(2-etilhexilo) Garbeflex DOP-D 40 Good-rite GP 264 Hatco DOP Jayflex DOP Kodaflex DEHP Kodaflex DOP Monocizer DOP NSC 17069 Palatinol AH Palatinol AH-L Phtalate de Bis (Ethyle-2-Hexyle) Phtalate de bis(2-ethylhexyle) PHTHALATE, BIS(2-ETHYLHEXYL) Phthalic acid di(2-ethylhexyl) ester Phthalic acid, bis(2-ethylhexyl) ester PHTHALIC ACID, BIS(2-ETHYLHEXYL)ESTER PHTHALSAEURE-BIS-(2-AETHYLHEXYL)-ESTER Pittsburgh PX 138 Plasthall DOP Reomol D 79P Sansocizer DOP Sansocizer R 8000 Sconamoll DOP Staflex DOP Truflex DOP Vestinol AH Vinycizer 80 Vinycizer 80K Witcizer 312

DTXSID5020607

637-07-0

KNHUKKLJHYUCFP-UHFFFAOYSA-N

Clofibrate

ethyl-p-chlorophenoxyisobutyrate Propanoic acid, 2-(4-chlorophenoxy)-2-methyl-, ethyl ester 2-(p-Chlorophenoxy)-2-methylpropionic acid ethyl ester Abitrate Amotril Anparton Arteriosan Artevil Ateculon Ateriosan Atheropront Atromid S Atromidin Azionyl Bioscleran Cartagyl Claripex Claripex CPIB Clobren SF Clofibrat clofibrato Clofinit Ethyl (p-chlorophenoxy) isobutyrate Ethyl 2-(4-chlorophenoxy)-2-methylpropionate Ethyl 2-(4-chlorophenoxy)isobutyrate Ethyl 2-(p-chlorophenoxy)-2-methylpropionate Ethyl 2-(p-chlorophenoxy)isobutyrate Ethyl clofibrate Ethyl p-chlorophenoxyisobutyrate Ethyl α-(4-chlorophenoxy)isobutyrate Ethyl α-(4-chlorophenoxy)-α-methylpropionate Ethyl α-(p-chlorophenoxy)isobutyrate Ethyl α-(p-chlorophenoxy)-α-methylpropionate Hyclorate Lipavil Lipavlon Lipomid Liprinal Miscleron Misclerone Neo-Atromid Normolipol NSC 79389 p-Chlorophenoxyisobutyric acid ethyl ester Propionic acid, 2-(p-chlorophenoxy)-2-methyl-, ethyl ester Recolip Regelan Serotinex Sklerepmexe Sklerolip Skleromexe Sklero-Tablinene Ticlobran Xyduril

DTXSID3020336

3771-19-5

XJGBDJOMWKAZJS-UHFFFAOYNA-N

XJGBDJOMWKAZJS-UHFFFAOYSA-N

Nafenopin

DTXSID8020911

52214-84-3

KPSRODZRAIWAKH-UHFFFAOYNA-N

KPSRODZRAIWAKH-UHFFFAOYSA-N

Ciprofibrate

DTXSID8020331

25812-30-0

HEMJJKBWTPKOJG-UHFFFAOYSA-N

Gemfibrozil

Pentanoic acid, 5-(2,5-dimethylphenoxy)-2,2-dimethyl- 2,2-Dimethyl-5-(2,5-xylyloxy)valeric acid 5-(2,5-Dimethylphenoxy)-2,2-dimethylpentanoic acid Decrelip gemfibrozilo Gevilon Lopizid Trialmin 900 Valeric acid, 2,2-dimethyl-5-(2,5-xylyloxy)-

DTXSID0020652

41859-67-0

IIBYAHWJQTYFKB-UHFFFAOYSA-N

Bezafibrate

Propanoic acid, 2-[4-[2-[(4-chlorobenzoyl)amino]ethyl]phenoxy]-2-methyl- Befizal Benzofibrate Bezafibrat bezafibrato Bezalip Bezatol Difaterol

DTXSID3029869

49562-28-9

YMTINGFKWWXKFG-UHFFFAOYSA-N

Fenofibrate

Propanoic acid, 2-[4-(4-chlorobenzoyl)phenoxy]-2-methyl-, 1-methylethyl ester 2-[4-(4-Chlorobenzoyl)phenoxy]-2-methylpropanoic acid 1-methylethyl ester Ankebin Clorofibrate Elasterin Fenobrate Fenofibrat fenofibrato Fenogal Fenotard Isopropyl 2-[p-(p-chlorobenzoyl)phenoxy]-2-methylpropionate Lipanthyl Lipantil Lipicard Lipidil Lipidil Supra Lipirex Lipoclar Lipofene Liposit MeltDose Nolipax NSC 281319 Procetofen Procetofene Procetoken Protolipan Secalip

DTXSID2029874

79902-63-9

RYMZZMVNJRMUDD-HGQWONQESA-N

Simvastatin

Butanoic acid, 2,2-dimethyl-, (1S,3R,7S,8S,8aR)-1,2,3,7,8,8a-hexahydro-3,7-dimethyl-8-[2-[(2R,4R)-tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl]ethyl]-1-naphthalenyl ester (+)-Simvastatin Apo-Simvastatin Bestatin 20 Butanoic acid, 2,2-dimethyl-, 1,2,3,7,8,8a-hexahydro-3,7-dimethyl-8-[2-(tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl)ethyl]-1-naphthalenyl ester, [1S-[1α,3α,7β,8β(2S*,4S*),8aβ]]- Cholestat Co-Simvastatin Kolestevan L 644128-000U Lipinorm Liponorm Lipovas Lodales Modutrol Nor-Vastina Novo-Simvastatin Pms-simvastatin Simastin 20 Simovil Simvastatin lactone Simvotin Sinvacor Sinvascor Sivastin Starstat 20 Synvinolin Valemia Velostatin

DTXSID0023581

PR:000013056

PR peroxisome proliferator-activated receptor alpha

PR:000015715

PR STAR

CHEBI:16113

CHEBI cholesterol

CHEBI:17347

CHEBI testosterone

PR:000016757

PR translocator protein

UBERON:0003135

UBERON male reproductive organ

GO:0035357

GO peroxisome proliferator activated receptor signaling pathway

D005298

MESH fertility

GO:0010467

GO gene expression

GO:0006839

GO mitochondrial transport

GO:0061370

GO testosterone biosynthetic process

WIKI increased

WIKI decreased

WIKI morphological change

Di(2-ethylhexyl) phthalate

2016-11-29T18:42:26

Mono(2-ethylhexyl) phthalate

2016-11-29T18:42:26

Stressor:205 pirinixic acid (WY-14,643)

2020-12-19T09:06:20

Clofibrate

2016-11-29T18:42:27

Nafenopin

2016-11-29T18:42:27

ciprofibrate

2016-11-29T18:42:27

Gemfibrozil Fibrate drug

2016-11-29T18:42:27

2020-03-31T10:24:40

PERFLUOROOCTANOIC ACID

2016-11-29T18:42:27

Bezafibrate

2016-11-29T18:42:27

Fenofibrate

2016-11-29T18:42:27

Simvastatin

2020-05-06T09:41:35

10116

NCBI rat

10090

NCBI mouse

WCS_9606

common toxicological species human

10095

NCBI mice

9606

NCBI Homo sapiens

10090

NCBI Mus musculus

Activation, PPARα

Molecular

Gene expression occurs in a coordinated fashion (Judson et al., 2012). The many observations of altered gene expression following binding of ligand to PPARα led to systematic investigations of the genomic signature that corresponds to PPARα activation (Tamura et al., 2006; Kupershmidt et al., 2010; Rosen et al., 2017; Rooney et al., 2018; Corton et al., 2020; Hill et al., 2020; Lewis et al., 2020). Specific gene with increased expression following PPARα activation include Cyp4a1, Cpt1B, and Lpl. More generally, the pathways activated include: <ul> <li>Genes involved in Metabolism of lipids and lipoproteins</li> <li>Fatty acid metabolism</li> <li>Genes involved in Fatty acid, triacylglycerol, and ketone body metabolism</li> <li>PPAR signaling pathway</li> <li>Peroxisome</li> <li>Genes involved in Cell Cycle</li> </ul> Biological state The Peroxisome Proliferator Activated receptor α (PPARα) belongs to the <a href="/wiki/index.php/Peroxisome_Proliferator_Activated_receptors_(PPARs;_NR1C)" title="Peroxisome Proliferator Activated receptors (PPARs; NR1C)">Peroxisome Proliferator Activated receptors (PPARs; NR1C)</a> steroid/thyroid/retinoid receptor superfamily of transcription factors. Biological compartments PPARα is expressed in high levels in tissues that perform significant catabolism of fatty acids (FAs), such as brown adipose tissue, liver, heart, kidney, and intestine (Michalik et al. 2006). The receptor is present also in skeletal muscle, intestine, pancreas, lung, placenta and testes (Mukherjee et al. 1997), (Schultz et al. 1999). General role in biology PPARs are activated by fatty acids and their derivatives; they are sensors of dietary lipids and are involved in lipid and carbohydrate metabolism, immune response and peroxisome proliferation (Wahli and Desvergne 1999), (Evans, Barish, & Wang, 2004). PAPRα is a also a target of hypothalamic hormone signalling and was found to play a role in embryonic development (Yessoufou and Wahli 2010). Fibrates, activators of PPARα, are commonly used to treat hypertriglyceridemia and other dyslipidemic states as they have been shown to decrease circulating lipid levels (Lefebvre et al. 2006).

Binding of ligands to PPARα is measured using binding assays in vitro and in silico, whereas the information about functional activation is derived from transactivation assays (e.g. transactivation assay with reporter gene) that demonstrate functional activation of a nuclear receptor by a specific compound. Binding of agonists within the ligand-binding site of PPARs causes a conformational change of nuclear receptor that promotes binding to transcriptional co-activators. Conversely, binding of antagonists results in a conformation that favours the binding of co-repressors (Yu and Reddy 2007), (Viswakarma et al. 2010). Transactivation assays are performed using transient or stably transfected cells with the PPARα expression plasmid and a reporter plasmid, respectively. There are also other methods that have been used to measure PPARα activity, such as the Electrophoretic Mobility Shift Assay (EMSA) or commercially available PPARα transcription factor assay kits, see Table 1. The transactivation (stable transfection) assay provides the most applicable OECD Level 2 assay (i.e. In vitro assays providing mechanistic data) aimed at identifying the initiating event leading to an adverse outcome (LeBlanc, Norris, and Kloas 2011). A recent study characterized the PPARα ligand binding domain for the purpose of next-generation metabolic disease drugs (Kamata et al. 2020). The most direct measure of this MIE is microarray profiling from large gene expression databases TG-GATEs and DrugMatrix coupled with t statistical analysis of whole genome expression profiles (Svoboda et al., 2019; Igarashi et al., 2015) From these data, A gene expression signature of 131 PPARα-dependent genes was built using microarray profiles from the livers of wild-type and PPARα-null mice. A quantitative measure of this expression signature is a measure of similarity/correlation between the PPARα signature and positive and negative test sets is provided by the Running Fisher test (Corton et al., 2020; Hill et al., 2020; Kupershmidt et al., 2010; Lewis et al., 2020; Rooney et al., 2018). A gene expression signature of 131 PPARα-dependent genes was built using microarray profiles from the livers of wild-type and PPARα-null mice. A quantitative measure of this expression signature would be a measure of similarity/correlation between the PPARα signature and positive and negative test sets is provided by the Running Fisher test (Kupershmidt et al., 2010; Rooney et al., 2018; Corton et al., 2020). For all substances, MIE activation does not rise monotonically over dose or time. These fluctuations are likely due to variations in cofactor availability or access to the site of transcription (Gaillard et al., 2006; Koppen et al., 2009; Kupershmidt et al., 2010; Ong et al., 2010; Chow et al., 2011; De Vos et al., 2011; Simon et al., 2015). . <table align="left" border="1" cellpadding="1" cellspacing="1" style="height:3px; width:100px"> <caption>Measurements of PPARα Activation</caption> <thead> <tr> <th scope="row">Method/Test</th> <th scope="col">Test Principle</th> <th scope="col">Test Environment</th> <th scope="col">Test Outcome</th> <th scope="col">Assay Type/Domain</th> </tr> </thead> <tbody> <tr> <th scope="row"> molecular modelling; docking simulation </th> <td>Computational simulation of  ligand binding </td> <td>In silico</td> <td>Prediction off binding interaction </td> <td>Quantitative virtual screeings</td> </tr> <tr> <th scope="row">Scintillation proximity binding assay</th> <td>Direct binding of ligand</td> <td>In vitro</td> <td>Identifies compouds that bind to PPARα</td> <td>Qualitative in vitro screening</td> </tr> <tr> <th scope="row">PPARα reporter gene assay</th> <td>Quantify changes in in PPARα activation via a sensitive surrogate </td> <td>In vitro, Ex vivo</td> <td>Measures changes in activity of genes linked to a PPARα receptor element</td> <td>Quantitative in vitro screening</td> </tr> <tr> <th scope="row">Electrophoretic Band Shift</th> <td>determines if a protein or protein mixture will bind to a specific DNA or RNA sequence</td> <td>In vitro</td> <td>Measures cofactor binding by changes in gel mobility</td> <td>Quantitative in vitro screening</td> </tr> <tr> <th scope="row">Microarray profiling</th> <td>Develop MIE-specific sets of gene expression biomarkers</td> <td>In vivo</td> <td>Classification of PPARα biomarker genes with statistical methods</td> <td>Quantitative in vivo screening</td> </tr> </tbody> </table>

PPARα has been identified in frog (Xenopus laevis), mouse, human, rat, fish, hamster and chicken (reviewed in (Wahli and Desvergne 1999)).

UBERON:0002107

UBERON liver

CL:0000255

CL eukaryotic cell

High High High

Bhattacharya, Nandini, Jannette M Dufour, My-Nuong Vo, Janice Okita, Richard Okita, and Kwan Hee Kim. 2005. “Differential Effects of Phthalates on the Testis and the Liver.” Biology of Reproduction 72 (3) (March): 745–54. doi:10.1095/biolreprod.104.031583. Bility, Moses T, Jerry T Thompson, Richard H McKee, Raymond M David, John H Butala, John P Vanden Heuvel, and Jeffrey M Peters. 2004. “Activation of Mouse and Human Peroxisome Proliferator-Activated Receptors (PPARs) by Phthalate Monoesters.” Toxicological Sciences : An Official Journal of the Society of Toxicology 82 (1) (November): 170–82. doi:10.1093/toxsci/kfh253. Chow, C. C., Ong, K. M., Dougherty, E. J., & Simons, S. S. (2011). Inferring mechanisms from dose-response curves. Methods Enzymol, 487, 465-483. https://doi.org/10.1016/B978-0-12-381270-4.00016-0 Corton, J. C., Hill, T., Sutherland, J. J., Stevens, J. L., & Rooney, J. (2020). A Set of Six Gene Expression Biomarkers Identify Rat Liver Tumorigens in Short-Term Assays. Toxicol Sci. https://doi.org/10.1093/toxsci/kfaa101 De Vos, D., Bruggeman, F. J., Westerhoff, H. V., & Bakker, B. M. (2011). How molecular competition influences fluxes in gene expression networks. PLoS One, 6(12), e28494. https://doi.org/10.1371/journal.pone.0028494 Dufour, Jannette M, My-Nuong Vo, Nandini Bhattacharya, Janice Okita, Richard Okita, and Kwan Hee Kim. 2003. “Peroxisome Proliferators Disrupt Retinoic Acid Receptor Alpha Signaling in the Testis.” Biology of Reproduction 68 (4) (April): 1215–24. doi:10.1095/biolreprod.102.010488. Feige, Jérôme N, Laurent Gelman, Daniel Rossi, Vincent Zoete, Raphaël Métivier, Cicerone Tudor, Silvia I Anghel, et al. 2007. “The Endocrine Disruptor Monoethyl-Hexyl-Phthalate Is a Selective Peroxisome Proliferator-Activated Receptor Gamma Modulator That Promotes Adipogenesis.” The Journal of Biological Chemistry 282 (26) (June 29): 19152–66. doi:10.1074/jbc.M702724200. Gaillard, S., Grasfeder, L. L., Haeffele, C. L., Lobenhofer, E. K., Chu, T.-M., Wolfinger, R., Kazmin, D., Koves, T. R., Muoio, D. M., Chang, C.-y., & McDonnell, D. P. (2006). Receptor-selective coactivators as tools to define the biology of specific receptor-coactivator pairs. Mol Cell, 24(5), 797-803. https://doi.org/10.1016/j.molcel.2006.10.012 Hill, T., Rooney, J., Abedini, J., El-Masri, H., Wood, C. E., & Corton, J. C. (2020). Gene Expression Thresholds Derived From Short-Term Exposures Identify Rat Liver Tumorigens. Toxicol Sci. https://doi.org/10.1093/toxsci/kfaa102 Hurst, Christopher H, and David J Waxman. 2003. “Activation of PPARalpha and PPARgamma by Environmental Phthalate Monoesters.” Toxicological Sciences : An Official Journal of the Society of Toxicology 74 (2) (August): 297–308. doi:10.1093/toxsci/kfg145. Igarashi, Y., Nakatsu, N., Yamashita, T., Ono, A., Ohno, Y., Urushidani, T., & Yamada, H. (2015). Open TG-GATEs: a large-scale toxicogenomics database. Nucleic Acids Res, 43(Database issue), D921-7. https://doi.org/10.1093/nar/gku955 Kamata S, Oyama T, Saito K, Honda A, Yamamoto Y, Suda K, Ishikawa R, Itoh T, Watanabe Y, Shibata T, Uchida K, Suematsu M, Ishii I. PPARα Ligand-Binding Domain Structures with Endogenous Fatty Acids and Fibrates. iScience. 2020;23(11):101727. 10.1016/j.isci.2020.101727 Kaya, Taner, Scott C Mohr, David J Waxman, and Sandor Vajda. 2006. “Computational Screening of Phthalate Monoesters for Binding to PPARgamma.” Chemical Research in Toxicology 19 (8) (August): 999–1009. doi:10.1021/tx050301s. Koppen, A., Houtman, R., Pijnenburg, D., Jeninga, E. H., Ruijtenbeek, R., & Kalkhoven, E. (2009). Nuclear receptor-coregulator interaction profiling identifies TRIP3 as a novel peroxisome proliferator-activated receptor gamma cofactor. Mol Cell Proteomics, 8(10), 2212-2226. https://doi.org/10.1074/mcp.M900209-MCP200 Kupershmidt, I., Su, Q. J., Grewal, A., Sundaresh, S., Halperin, I., Flynn, J., Shekar, M., Wang, H., Park, J., Cui, W., Wall, G. D., Wisotzkey, R., Alag, S., Akhtari, S., & Ronaghi, M. (2010). Ontology-based meta-analysis of global collections of high-throughput public data. PLoS One, 5(9). https://doi.org/10.1371/journal.pone.0013066 Lampen, Alfonso, Susan Zimnik, and Heinz Nau. 2003. “Teratogenic Phthalate Esters and Metabolites Activate the Nuclear Receptors PPARs and Induce Differentiation of F9 Cells.” Toxicology and Applied Pharmacology 188 (1) (April): 14–23. doi:10.1016/S0041-008X(03)00014-0. Lapinskas, Paula J., Sherri Brown, Lisa M. Leesnitzer, Steven Blanchard, Cyndi Swanson, Russell C. Cattley, and J. Christopher Corton. 2005. “Role of PPARα in Mediating the Effects of Phthalates and Metabolites in the Liver.” Toxicology 207 (1): 149–163. Le Maire, Albane, Marina Grimaldi, Dominique Roecklin, Sonia Dagnino, Valérie Vivat-Hannah, Patrick Balaguer, and William Bourguet. 2009. “Activation of RXR-PPAR Heterodimers by Organotin Environmental Endocrine Disruptors.” EMBO Reports 10 (4) (April): 367–73. doi:10.1038/embor.2009.8. LeBlanc, GA, DO Norris, and W Kloas. 2011. “Detailed Review Paper State of the Science on Novel In Vitro and In Vivo Screening and Testing Methods and Endpoints for Evaluating Endocrine Disruptors” (178). Lefebvre, Philippe, Giulia Chinetti, Jean-Charles Fruchart, and Bart Staels. 2006. “Sorting out the Roles of PPAR Alpha in Energy Metabolism and Vascular Homeostasis.” The Journal of Clinical Investigation 116 (3) (March): 571–80. doi:10.1172/JCI27989. Lewis, R. W., Hill, T., & Corton, J. C. (2020). A set of six Gene expression biomarkers and their thresholds identify rat liver tumorigens in short-term assays. Toxicology, 443, 152547. https://doi.org/10.1016/j.tox.2020.152547 Maloney, Erin K., and David J. Waxman. 1999. “Trans-Activation of PPARα and PPARγ by Structurally Diverse Environmental Chemicals.” Toxicology and Applied Pharmacology 161 (2): 209–218. Michalik, Liliane, Johan Auwerx, Joel P Berger, V Krishna Chatterjee, Christopher K Glass, Frank J Gonzalez, Paul A Grimaldi, et al. 2006. “International Union of Pharmacology. LXI. Peroxisome Proliferator-Activated Receptors.” Pharmacological Reviews 58 (4) (December): 726–41. doi:10.1124/pr.58.4.5. Mukherjee, R, L Jow, G E Croston, and J R Paterniti. 1997. “Identification, Characterization, and Tissue Distribution of Human Peroxisome Proliferator-Activated Receptor (PPAR) Isoforms PPARgamma2 versus PPARgamma1 and Activation with Retinoid X Receptor Agonists and Antagonists.” The Journal of Biological Chemistry 272 (12) (March 21): 8071–6. Ong, K. M., Blackford, J. A., Kagan, B. L., Simons, S. S., & Chow, C. C. (2010). A theoretical framework for gene induction and experimental comparisons. Proc Natl Acad Sci U S A, 107(15), 7107-7112. https://doi.org/10.1073/pnas.0911095107 Rooney, J., Hill, T., Qin, C., Sistare, F. D., & Corton, J. C. (2018). Adverse outcome pathway-driven identification of rat liver tumorigens in short-term assays. Toxicol Appl Pharmacol, 356, 99-113. https://doi.org/10.1016/j.taap.2018.07.023 Schultz, R, W Yan, J Toppari, A Völkl, J A Gustafsson, and M Pelto-Huikko. 1999. “Expression of Peroxisome Proliferator-Activated Receptor Alpha Messenger Ribonucleic Acid and Protein in Human and Rat Testis.” Endocrinology 140 (7) (July): 2968–75. doi:10.1210/endo.140.7.6858. Simon, T. W., Budinsky, R. A., & Rowlands, J. C. (2015). A model for aryl hydrocarbon receptor-activated gene expression shows potency and efficacy changes and predicts squelching due to competition for transcription co-activators. PLoS One, 10(6), e0127952. https://doi.org/10.1371/journal.pone.0127952. Staels, B., J. Dallongeville, J. Auwerx, K. Schoonjans, E. Leitersdorf, and J.-C. Fruchart. 1998. “Mechanism of Action of Fibrates on Lipid and Lipoprotein Metabolism.” Circulation 98 (19) (November 10): 2088–2093. doi:10.1161/01.CIR.98.19.2088. Svoboda, D. L., Saddler, T., & Auerbach, S. S. (2019). An Overview of National Toxicology Program’s Toxicogenomic Applications: DrugMatrix and ToxFX.  In Advances in Computational Toxicology (pp. 141-157). Springer. https://link.springer.com/chapter/10.1007/978-3-030-16443-0_8 ToxCastTM Data. “ToxCastTM Data.” US Environmental Protection Agency. <a class="external free" href="http://www.epa.gov/ncct/toxcast/data.html" rel="nofollow" target="_blank">http://www.epa.gov/ncct/toxcast/data.html</a> Vanden Heuvel, John P, Jerry T Thompson, Steven R Frame, and Peter J Gillies. 2006. “Differential Activation of Nuclear Receptors by Perfluorinated Fatty Acid Analogs and Natural Fatty Acids: A Comparison of Human, Mouse, and Rat Peroxisome Proliferator-Activated Receptor-Alpha, -Beta, and -Gamma, Liver X Receptor-Beta, and Retinoid X Rec.” Toxicological Sciences : An Official Journal of the Society of Toxicology 92 (2) (August): 476–89. doi:10.1093/toxsci/kfl014. Venkata, Nagaraj Gopisetty, Jodie a Robinson, Peter J Cabot, Barbara Davis, Greg R Monteith, and Sarah J Roberts-Thomson. 2006. “Mono(2-Ethylhexyl)phthalate and Mono-N-Butyl Phthalate Activation of Peroxisome Proliferator Activated-Receptors Alpha and Gamma in Breast.” Toxicology Letters 163 (3) (June 1): 224–34. doi:10.1016/j.toxlet.2005.11.001. Viswakarma, Navin, Yuzhi Jia, Liang Bai, Aurore Vluggens, Jayme Borensztajn, Jianming Xu, and Janardan K Reddy. 2010. “Coactivators in PPAR-Regulated Gene Expression.” PPAR Research 2010 (January). doi:10.1155/2010/250126. Wahli, Walter, and B Desvergne. 1999. “Peroxisome Proliferator-Activated Receptors: Nuclear Control of Metabolism.” Endocrine Reviews 20 (5) (October): 649–88. Wu, Bin, Jie Gao, and Ming-wei Wang. 2005. “Development of a Complex Scintillation Proximity Assay for High-Throughput Screening of PPARgamma Modulators.” Acta Pharmacologica Sinica 26 (3) (March): 339–44. doi:10.1111/j.1745-7254.2005.00040.x. Xu, Chuan, Ji-An Chen, Zhiqun Qiu, Qing Zhao, Jiaohua Luo, Lan Yang, Hui Zeng, et al. 2010. “Ovotoxicity and PPAR-Mediated Aromatase Downregulation in Female Sprague-Dawley Rats Following Combined Oral Exposure to Benzo[a]pyrene and Di-(2-Ethylhexyl) Phthalate.” Toxicology Letters 199 (3) (December 15): 323–32. doi:10.1016/j.toxlet.2010.09.015. Yessoufou, a, and W Wahli. 2010. “Multifaceted Roles of Peroxisome Proliferator-Activated Receptors (PPARs) at the Cellular and Whole Organism Levels.” Swiss Medical Weekly 140 (September) (January): w13071. doi:10.4414/smw.2010.13071. Yu, Songtao, and Janardan K Reddy. 2007. “Transcription Coactivators for Peroxisome Proliferator-Activated Receptors.” Biochimica et Biophysica Acta 1771 (8) (August): 936–51. doi:10.1016/j.bbalip.2007.01.008. AOPWiki 2016-11-29T18:41:23

2020-12-28T12:48:16

impaired, Fertility

Individual

Biological state capability to produce offspring Biological compartments System General role in biology Fertility is the capacity to conceive or induce conception. Impairment of fertility represents disorders of male or female reproductive functions or capacity.

As a measure, fertility rate, is the number of offspring born per mating pair, individual or population.

High

Adult, reproductively mature

High High High

AOPWiki 2016-11-29T18:41:24

2016-12-02T09:21:49

Decrease, Steroidogenic acute regulatory protein (STAR)

Cellular

Biological state Steroidogenic acute regulatory protein (StAR) functions as a cholesterol transfer protein and acts directly on lipids of the outer mitochondrial membrane to promote cholesterol translocation (Stocco 2001). Reduction of the protein impacts on the amount of substrate available for steroidogenesis. Biological compartments StAR is expressed principally in steroidogenic tissues (Bauer et al. 2000). General role in biology StAR is required for cholesterol shuttling across the mitochondrial membrane and appears to regulate acute steroid production (Clark and Stocco, 1997). Transcriptional or translational inhibition of StAR expression results in a dramatic decrease in steroid biosynthesis, whereas ~10–15% of steroid synthesis appears to be mediated through StAR-independent mechanisms (Manna et al. 2001) (Clark and Stocco, 1997). In contrast, chronically regulated steroid production appears to be largely mediated by increased transcription of steroidogenic enzymes (Hum and Miller 1993).

The StAR expression can be measured by RT-PCR (mRNA) and on the protein level (western blot). The StAR expression as well as other steroidogenic proteins can be measured in vitro cultured Leydig cells. The methods for culturing Leydig cells can be found in the Database Service on Alternative Methods to animal experimentation (DB-ALM): Leydig Cell-enriched Cultures <a rel="nofollow" target="_blank" class="external autonumber" href="http://ecvam-dbalm.jrc.ec.europa.eu/beta/index.cfm/methodsAndProtocols/index?id_met=232">[1]</a> Testicular Organ and Tissue Culture Systems <a rel="nofollow" target="_blank" class="external autonumber" href="http://ecvam-dbalm.jrc.ec.europa.eu/beta/index.cfm/methodsAndProtocols/index?id_met=515">[2]</a>.

StAR has been cloned from many species, and is highly conserved among mammals, birds, amphibians and fish (Bauer et al. 2000).

CL:0000174

CL steroid hormone secreting cell

Moderate Moderate High

Bauer, M P, J T Bridgham, D M Langenau, A L Johnson, and F W Goetz. 2000. “Conservation of Steroidogenic Acute Regulatory (StAR) Protein Structure and Expression in Vertebrates.” Molecular and Cellular Endocrinology 168 (1-2) (October 25): 119–25. Hum, D W, and W L Miller. 1993. “Transcriptional Regulation of Human Genes for Steroidogenic Enzymes.” Clinical Chemistry 39 (2) (February): 333–40. Manna, P R, J Kero, M Tena-Sempere, P Pakarinen, D M Stocco, and I T Huhtaniemi. 2001. “Assessment of Mechanisms of Thyroid Hormone Action in Mouse Leydig Cells: Regulation of the Steroidogenic Acute Regulatory Protein, Steroidogenesis, and Luteinizing Hormone Receptor Function.” Endocrinology 142 (1) (January): 319–31. doi:10.1210/endo.142.1.7900. Stocco, D M. 2001. “StAR Protein and the Regulation of Steroid Hormone Biosynthesis.” Annual Review of Physiology 63 (January): 193–213. doi:10.1146/annurev.physiol.63.1.193. AOPWiki 2016-11-29T18:41:23

2017-09-16T10:14:32

Reduction, Cholesterol transport in mitochondria

Cellular

Biological state Steroidogenesis begins with the transport of cholesterol from intracellular stores into mitochondria. This process involves a series of protein-protein interactions involving cytosolic and mitochondrial proteins located at both the outer and inner mitochondrial membranes. In steroidogenic cells the cholesterol import to the mitochondrial inner membrane is crucial for steroid synthesis (Rone, Fan, and Papadopoulos 2009). This process is facilitated by the Scavenger Receptor Class B, type 1 (SR-B1) [more relevant for rodents, than for humans] that mediates the selective uptake of cholesterol esters from high-density lipoproteins. Steroidogenic acute regulatory protein (STAR) and the translator protein (TSPO) [former peripheral benzodiazepine receptor (PBR)] mediate cholesterol transport from the outer to the inner mitochondrial membrane. The conversion of cholesterol to pregnenolone is done by Cholesterol side-chain cleavage enzyme (P450scc), the start of steroidogenesis [reviewed in (Miller and Auchus 2011)]. Biological compartments In mitochondria of steroidogenic tissues there are two specialized mechanisms related to hormone synthesis: one by which cholesterol is delivered to the mitochondria and the other by which specialized intra-mitochondrial enzymes participate in the synthesis of hormonal steroids. General role in biology Systemic steroid hormones are primarily formed by the gonads, adrenal glands, and during in utero development by the placenta. Some other organs like brain (Baulieu 1998), and heart (Kayes-Wandover and White 2000) have also been identified as steroid-producing tissues, mainly for local needs. The steroid hormones are indispensable for mammalian life. They are made from cholesterol via complex biosynthetic pathways that are initiated by specialized, tissue-specific enzymes in mitochondria. These hormones include glucocorticoids (cortisol, corticosterone) and mineralocorticoids (aldosterone) produced in the adrenal cortex, estrogens (estradiol), progestins (progesterone) and androgens (testosterone, dihydrotestosterone) produced in the gonads, and calciferols (1,25-dihydroxy vitamin D [1,25OH2D]) produced in the kidneys (Miller and Auchus 2011). Cholesterol is the precursor for the synthesis of steroid hormones in mitochondria. Steroidogenesis begins with the metabolism of cholesterol to pregnenolone facilitated by P450scc. The rate of steroid formation depends on the rate of cholesterol transport from intracellular stores to the inner mitochondrial membrane and the loading of P450scc with cholesterol (Miller and Auchus 2011). Interference with one or more of these reactions leads to reduced steroid production.

This KE can be indirectly measured by: 1. Expression of the proteins involved in cholesterol transport by qPCR or Western blot. 3. Cholesterol transport to the mitochondrial inner membrane in intact cells: <ul> <li>Indirectly as pregnenolone formation by cells. The pregnenolone concentration is assayed by commercially available radioimmunoassays and reflects the amount of cholesterol transported to the mitochondrial inner membrane (Charman et al. 2010). </li> <li>Filipin staining is one of the most widely used tools for studying intracellular cholesterol distribution. The fluorescent detergent filipin binds selectively to cholesterol (and not to cholesterol esters) (Schroeder, Holland, and Bieber 1971). Filipin can be only used for the qualitative analysis of cholesterol distribution, since its fluorescence intensity is not necessarily linearly related to cholesterol content. </li> </ul> The cholesterol transport can be measured in vitro cultured Leydig cells. The methods for culturing Leydig cells can be found in the Database Service on Alternative Methods to animal experimentation (DB-ALM): Leydig Cell-enriched Cultures <a rel="nofollow" target="_blank" class="external autonumber" href="http://ecvam-dbalm.jrc.ec.europa.eu/beta/index.cfm/methodsAndProtocols/index?id_met=232">[1]</a> Testicular Organ and Tissue Culture Systems <a rel="nofollow" target="_blank" class="external autonumber" href="http://ecvam-dbalm.jrc.ec.europa.eu/beta/index.cfm/methodsAndProtocols/index?id_met=515">[2]</a>

The enzymes needed for cholesterol transport were found in amphioxus and are present in vertebrates (Albalat et al. 2011).

CL:0000174

CL steroid hormone secreting cell

High High High

Albalat, Ricard, Frédéric Brunet, Vincent Laudet, and Michael Schubert. 2011. “Evolution of Retinoid and Steroid Signaling: Vertebrate Diversification from an Amphioxus Perspective.” Genome Biology and Evolution 3: 985–1005. doi:10.1093/gbe/evr084. Baulieu, E E. 1998. “Neurosteroids: A Novel Function of the Brain.” Psychoneuroendocrinology 23 (8) (November): 963–87. Charman, Mark, Barry E Kennedy, Nolan Osborne, and Barbara Karten. 2010. “MLN64 Mediates Egress of Cholesterol from Endosomes to Mitochondria in the Absence of Functional Niemann-Pick Type C1 Protein.” Journal of Lipid Research 51 (5) (May): 1023–34. doi:10.1194/jlr.M002345. Kayes-Wandover, K M, and P C White. 2000. “Steroidogenic Enzyme Gene Expression in the Human Heart.” The Journal of Clinical Endocrinology and Metabolism 85 (7) (July): 2519–25. doi:10.1210/jcem.85.7.6663. Miller, Walter L, and Richard J Auchus. 2011. “The Molecular Biology, Biochemistry, and Physiology of Human Steroidogenesis and Its Disorders.” Endocrine Reviews 32 (1) (February): 81–151. doi:10.1210/er.2010-0013. Rone, Malena B, Jinjiang Fan, and Vassilios Papadopoulos. 2009. “Cholesterol Transport in Steroid Biosynthesis: Role of Protein-Protein Interactions and Implications in Disease States.” Biochimica et Biophysica Acta 1791 (7) (July): 646–58. doi:10.1016/j.bbalip.2009.03.001. Schroeder, F, J F Holland, and L L Bieber. 1971. “Fluorometric Evidence for the Binding of Cholesterol to the Filipin Complex.” The Journal of Antibiotics 24 (12) (December): 846–9. Steer, C. 1984. “Detection of Membrane Cholesterol by Filipin in Isolated Rat Liver Coated Vesicles Is Dependent upon Removal of the Clathrin Coat.” The Journal of Cell Biology 99 (1) (July 1): 315–319. doi:10.1083/jcb.99.1.315. AOPWiki 2016-11-29T18:41:24

2017-09-16T10:14:33

Reduction, Testosterone synthesis in Leydig cells

Cellular

Biological state Testosterone is a steroid hormone from the androgen group and is found in humans and other vertebrates. Biological compartments In humans and other mammals, testosterone is secreted primarily by the testicles of males and, to a lesser extent, the ovaries of females and other steroidogenic tissues (e.g., brain, adipose). It either acts locally /or is transported to other tissues via blood circulation. Testosterone synthesis takes place within the mitochondria of Leydig cells, the testosterone-producing cells of the testis. It is produced upon stimulation of these cells by Luteinizing hormone (LH) that is secreted in pulses into the peripheral circulation by the pituitary gland in response to Gonadotropin-releasing hormone (GnRH) from the hypothalamus. Testosterone and its aromatized product, estradiol, feed back to the hypothalamus and pituitary gland to suppress transiently LH and thus testosterone production. In response to reduced testosterone levels, GnRH and LH are produced. This negative feedback cycle results in pulsatile secretion of LH followed by pulsatile production of testosterone (Ellis, Desjardins, and Fraser 1983), (Chandrashekar and Bartke 1998). General role in biology Testosterone is the principal male sex hormone and an anabolic steroid. Male sexual differentiation depends on testosterone (T), dihydrotestosterone (DHT), and the expression of androgen receptors by target cells (Manson and Carr 2003). During the development secretion of androgens by Leydig cells is essential for masculinization of the foetus (Nef 2000). The foetal Leydig cells develop in utero. These cells become competent to produce testosterone in rat by gestational day (GD) 15.5, with increasing production thereafter. Peak steroidogenic activity is reached just prior to birth, on GD19 (Chen, Ge, and Zirkin 2009). Testosterone secreted by foetal Leydig cells is required for the differentiation of the male urogenital system late in gestation (Huhtaniemi and Pelliniemi 1992). Foetal Leydig cells also play a role in the scrotal descent of the testis through their synthesis of insulin-like growth factor 3 (Insl3), for review see (Nef 2000). In humans, the first morphological sign of testicular differentiation is the formation of testicular cords, which can be seen between 6 and 7 weeks of gestation. Steroid-secreting Leydig cells can be seen in the testis at 8 weeks of gestation. At this period, the concentration of androgens in the testicular tissue and blood starts to rise, peaking at 14-16 weeks of gestation. This increase comes with an increase in the number of Leydig cells for review see (Rouiller-Fabre et al. 2009). Adult Leydig cells, which are distinct from the foetal Leydig cells, form during puberty and supply the testosterone required for the onset of spermatogenesis, among other functions. Distinct stages of adult Leydig cell development have been identified and characterized. The stem Leydig cells are undifferentiated cells that are capable of indefinite self-renewal but also of differentiation to steroidogenic cells. These cells give rise to progenitor Leydig cells, which proliferate, continue to differentiate, and give rise to the immature Leydig cells. Immature Leydig cells synthesize high levels of testosterone metabolites and develop into terminally differentiated adult Leydig cells, which produce high levels of testosterone. With aging, both serum and testicular testosterone concentrations progressively decline, for review see (Nef 2000). Androgens play a crucial role in the development and maintenance of male reproductive and sexual functions. Low levels of circulating androgens can cause disturbances in male sexual development, resulting in congenital abnormalities of the male reproductive tract. Later in life, this may cause reduced fertility, sexual dysfunction, decreased muscle formation and bone mineralisation, disturbances of fat metabolism, and cognitive dysfunction. Testosterone levels decrease as a process of ageing: signs and symptoms caused by this decline can be considered a normal part of ageing.

OECD TG 456 <a rel="nofollow" target="_blank" class="external autonumber" href="http://www.oecd-ilibrary.org/environment/test-no-456-h295r-steroidogenesis-assay_9789264122642-en">[1]</a> is the validated test guideline for an in vitro screen for chemical effects on steroidogenesis, specifically the production of 17ß-estradiol (E2) and testosterone (T). The testosterone syntheis can be measured in vitro cultured Leydig cells. The methods for culturing Leydig cells can be found in the Database Service on Alternative Methods to animal experimentation (DB-ALM): Leydig Cell-enriched Cultures <a rel="nofollow" target="_blank" class="external autonumber" href="http://ecvam-dbalm.jrc.ec.europa.eu/beta/index.cfm/methodsAndProtocols/index?id_met=232">[2]</a>, Testicular Organ and Tissue Culture Systems <a rel="nofollow" target="_blank" class="external autonumber" href="http://ecvam-dbalm.jrc.ec.europa.eu/beta/index.cfm/methodsAndProtocols/index?id_met=515">[3]</a>. Testosterone synthesis in vitro cultured cells can be measured indirectly by testosterone radioimmunoassay or analytical methods such as LC-MS.

Key enzymes needed for testosterone production first appear in the common ancestor of amphioxus and vertebrates (Baker 2011). Consequently, this key event is applicable to most vertebrates, including humans.

CL:0000177

CL testosterone secreting cell

High High Low

Chandrashekar, V, and A Bartke. 1998. “The Role of Growth Hormone in the Control of Gonadotropin Secretion in Adult Male Rats.” Endocrinology 139 (3) (March): 1067–74. doi:10.1210/endo.139.3.5816. Ellis, G B, C Desjardins, and H M Fraser. 1983. “Control of Pulsatile LH Release in Male Rats.” Neuroendocrinology 37 (3) (September): 177–83. Huhtaniemi, I, and L J Pelliniemi. 1992. “Fetal Leydig Cells: Cellular Origin, Morphology, Life Span, and Special Functional Features.” Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine (New York, N.Y.) 201 (2) (November): 125–40. Manson, Jeanne M, and Michael C Carr. 2003. “Molecular Epidemiology of Hypospadias: Review of Genetic and Environmental Risk Factors.” Birth Defects Research. Part A, Clinical and Molecular Teratology 67 (10) (October): 825–36. doi:10.1002/bdra.10084. Nef, S. 2000. “Hormones in Male Sexual Development.” Genes & Development 14 (24) (December 15): 3075–3086. doi:10.1101/gad.843800. Rouiller-Fabre, Virginie, Vincent Muczynski, Romain Lambrot, Charlotte Lécureuil, Hervé Coffigny, Catherine Pairault, Delphine Moison, et al. 2009. “Ontogenesis of Testicular Function in Humans.” Folia Histochemica et Cytobiologica / Polish Academy of Sciences, Polish Histochemical and Cytochemical Society 47 (5) (January): S19–24. doi:10.2478/v10042-009-0065-4. AOPWiki 2016-11-29T18:41:24

2017-09-16T10:14:33

Reduction, fetal/adult testosterone

Reduction of testosterone

Tissue

Biological state Testosterone (T) is a steroid hormone from the androgen group. T serves as a substrate for two metabolic pathways that produce antagonistic sex steroids. Biological compartments Testosterone is synthesized by the gonads and other steroidogenic tissues (e.g., brain, adipose), acts locally and/or is transported to other tissues via blood circulation. Leydig cells are the testosterone-producing cells of the testis. General role in biology Androgens, the main male sex steroids, are the critical factors responsible for the development of the male phenotype during embryogenesis and for the achievement of sexual maturation at puberty. In adulthood, androgens remain essential for the maintenance of male reproductive function and behaviour. Apart from their effects on reproduction, androgens affect a wide variety of non-reproductive tissues such as skin, bone, muscle, and brain (Heemers, Verhoeven, & Swinnen, 2006). Androgens, principally T and 5α-dihydrotestosterone (DHT), exert most of their effects by interacting with a specific receptor, the androgen receptor (AR), for review see (Murashima, Kishigami, Thomson, & Yamada, 2015). On the one hand, testosterone can be reduced by 5α-reductase to produce 5α dihydrotestosterone (DHT). On the other hand, testosterone can be aromatized to generate estrogens. Testosterone effects can also be classified by the age of usual occurrence, postnatal effects in both males and females are mostly dependent on the levels and duration of circulating free testosterone.

Testosterone can be measured by immunoassays and by isotope-dilution gas chromatography-mass spectrometry in serum (Taieb et al., 2003), (Paduch et al., 2014). Testosterone levels are measured i.a. in: Fish Lifecycle Toxicity Test (FLCTT) (US EPA OPPTS 850.1500), Male pubertal assay (PP Male Assay) (US EPA OPPTS 890.1500), OECD TG 441: Hershberger Bioassay in Rats (H Assay).

UBERON:0000178

UBERON blood

High High High

Heemers, H. V, Verhoeven, G., & Swinnen, J. V. (2006). Androgen activation of the sterol regulatory element-binding protein pathway: Current insights. Molecular Endocrinology (Baltimore, Md.), 20(10), 2265–77. doi:10.1210/me.2005-0479 Murashima, A., Kishigami, S., Thomson, A., & Yamada, G. (2015). Androgens and mammalian male reproductive tract development. Biochimica et Biophysica Acta, 1849(2), 163–170. doi:10.1016/j.bbagrm.2014.05.020 Paduch, D. A., Brannigan, R. E., Fuchs, E. F., Kim, E. D., Marmar, J. L., & Sandlow, J. I. (2014). The laboratory diagnosis of testosterone deficiency. Urology, 83(5), 980–8. doi:10.1016/j.urology.2013.12.024 Taieb, J., Mathian, B., Millot, F., Patricot, M.-C., Mathieu, E., Queyrel, N., … Boudou, P. (2003). Testosterone measured by 10 immunoassays and by isotope-dilution gas chromatography-mass spectrometry in sera from 116 men, women, and children. Clinical Chemistry, 49(8), 1381–95. AOPWiki 2016-11-29T18:41:24

2023-05-17T08:53:46

Decrease, Translocator protein (TSPO)

Cellular

Biological state Translocator protein (TSPO), previously known as the peripheral benzodiazepine receptor (PBR), is a mitochondrial outer membrane protein implicated in cholesterol import to the inner mitochondrial membrane (Besman et al. 1989). Biological compartments The TSPO is present in virtually all mammalian peripheral tissues (Zisterer and Williams 1997), however highly prominent TSPO protein expression has been identified in steroidogenic tissues (R. R. Anholt et al. 1985), (Wang, Fan, and Papadopoulos 2012). The presence of TSOP has been confirmed in Leydig and Sertoli cells (Morohaku, Phuong, and Selvaraj 2013), granulosa cells (Amsterdam and Suh 1991) and to a lesser extent in thecal cells (Morohaku, Phuong, and Selvaraj 2013). In subcellular fractions, binding sites for the TSOP have been identified to be present in the outer mitochondrial membrane (OMM) (R. R. Anholt et al. 1985), (R. Anholt et al. 1986). Transcriptional regulation of TSPO genes has been examined and recently reviewed (Morohaku, Phuong, and Selvaraj 2013). General role in biology: regulation of lipid transport TSPO mediates the delivery of the substrate cholesterol to the inner mitochondrial side chain cleavage enzyme P450scc (Besman et al. 1989). TSPO ligands stimulate steroidogenesis and induce cholesterol movement from the outer mitochondrial membrane (OMM) to the inner mitochondrial membrane (IMM) (Besman et al. 1989).

TSPO levels can be assayed by standard methods for assessment of gene expression levels like qPCR or direct protein levels by Western blot. The level of TSPO as well as other steroidogenic protein can be measured in vitro cultured Leydig cells. The methods for culturing Leydig cells can be found in the Database Service on Alternative Methods to animal experimentation (DB-ALM): Leydig Cell-enriched Cultures <a class="external autonumber" href="http://ecvam-dbalm.jrc.ec.europa.eu/beta/index.cfm/methodsAndProtocols/index?id_met=232" rel="nofollow" target="_blank">[1]</a>, Testicular Organ and Tissue Culture Systems <a class="external autonumber" href="http://ecvam-dbalm.jrc.ec.europa.eu/beta/index.cfm/methodsAndProtocols/index?id_met=515" rel="nofollow" target="_blank">[2]</a>. <h2>Uncertainties and Inconsistencies</h2> This information needs to be moved to a key event relationship page. TSPO -knockout mice have shown embryonic lethality (Lacapère and Papadopoulos 2003); in contrast recent findings have shown no effect on viability of foetuses (Tu et al. 2014). Aberrant TSPO levels have been linked to multiple diseases, including cancer, endocrine disorders, brain injury, neurodegeneration, ischemia-reperfusion injury and inflammatory diseases (Wang, Fan, and Papadopoulos 2012). However, recent studies have shown opposite results. Peripheral benzodiazepine receptor/translocator protein global knock-out mice are viable and show no effects on steroid hormone biosynthesis (Tu et al. 2014), (Morohaku et al. 2014). As stated in a recent review "At this point in time, a functional designation for TSPO is still actively being sought" (Selvaraj, Stocco, and Tu 2015).

TSPO is a protein that shows high DNA sequence conservation from bacteria to mammals. It is expressed ubiquitously, but most abundant in steroidogenic cells (Yeliseev, Krueger, and Kaplan 1997).

CL:0000174

CL steroid hormone secreting cell

High High Moderate

Amsterdam, A. & Suh, B.S., 1991. An inducible functional peripheral benzodiazepine receptor in mitochondria of steroidogenic granulosa cells. Endocrinology, 129(1), pp.503–10. Anholt, R. et al., 1986. The peripheral-type benzodiazepine receptor. Localization to the mitochondrial outer membrane. J. Biol. Chem., 261(2), pp.576–583. Anholt, R.R. et al., 1985. Peripheral-type benzodiazepine receptors: autoradiographic localization in whole-body sections of neonatal rats. The Journal of pharmacology and experimental therapeutics, 233(2), pp.517–26. Besman, M.J. et al., 1989. Identification of des-(Gly-Ile)-endozepine as an effector of corticotropin-dependent adrenal steroidogenesis: stimulation of cholesterol delivery is mediated by the peripheral benzodiazepine receptor. Proceedings of the National Academy of Sciences of the United States of America, 86(13), pp.4897–901. Lacapère, J.J. & Papadopoulos, V., 2003. Peripheral-type benzodiazepine receptor: structure and function of a cholesterol-binding protein in steroid and bile acid biosynthesis. Steroids, 68(7-8), pp.569–85. Morohaku, K. et al., 2014. Translocator protein/peripheral benzodiazepine receptor is not required for steroid hormone biosynthesis. Endocrinology, 155(1), pp.89–97. Morohaku, K., Phuong, N.S. & Selvaraj, V., 2013. Developmental expression of translocator protein/peripheral benzodiazepine receptor in reproductive tissues. W. Yan, ed. PloS one, 8(9), p.e74509. Papadopoulos, V. et al., 1997. Targeted disruption of the peripheral-type benzodiazepine receptor gene inhibits steroidogenesis in the R2C Leydig tumor cell line. The Journal of biological chemistry, 272(51), pp.32129–35. Tu, L.N. et al., 2014. Peripheral benzodiazepine receptor/translocator protein global knock-out mice are viable with no effects on steroid hormone biosynthesis. The Journal of biological chemistry, 289(40), pp.27444–54. Wang, H.-J., Fan, J. & Papadopoulos, V., 2012. Translocator protein (Tspo) gene promoter-driven green fluorescent protein synthesis in transgenic mice: an in vivo model to study Tspo transcription. Cell and tissue research, 350(2), pp.261–75. Yeliseev, A.A., Krueger, K.E. & Kaplan, S., 1997. A mammalian mitochondrial drug receptor functions as a bacterial “oxygen” sensor. Proceedings of the National Academy of Sciences of the United States of America, 94(10), pp.5101–6. Zisterer, D.M. & Williams, D.C., 1997. Peripheral-type benzodiazepine receptors. General pharmacology, 29(3), pp.305–14. AOPWiki 2016-11-29T18:41:23

2017-09-16T10:14:33

Malformation, Male reproductive tract

Organ

Biological state Male reproductive tract malformations (congenital malformation of male genitalia) comprise any physical abnormality of the male internal or external genitalia present at birth. Some result from excessive or deficient androgen effect, others result from teratogenic effects, or are associated with anomalies of other parts of the body in a recognizable pattern (i.e., a syndrome). The cause of many of these birth defects is unknown. Hypospadias is a defect of the urogenital system, a malformation in which the urethra opens on the underside of the penis instead of the tip. It results from an incomplete closure of the urethral folds, leaving a split on the penis (Kalfa, Philibert, and Sultan 2009). When the urethra opens to the glans or corona of the penis, it is called distal, whereas opening to the shaft or penoscrotal area defines hypospadias as proximal. Androgens regulate the masculinization of external genitalia. Therefore any defects in androgen biosynthesis, metabolism or action during foetal development can cause hypospadias. Gene defects causing disorders of testicular differentiation, conversion of testosterone to dihydrotestosterone or mutations in the androgen receptor can also result in hypospadias (Kalfa et al. 2008). In about 20% of patients with isolated hypospadias there are signs of endocrine abnormalities by the time of diagnosis (Rey et al. 2005). The majority of hypospadias are believed to have a multifactorial etiology, although a small percentage do result from single gene mutations (Baskin, Himes, and Colborn 2001). The only treatment of hypospadias is surgery, thus, prevention is imperative. Biological compartments: reproductive system

Malformations are detected by macroscopically for any structural abnormality or pathological change. The Congenital malformation of the genitalia is a medical term referring to a broad category of conditions that for humans is classified by International Classification of Diseases (ICD) in chapter "Congenital malformations of genital organs" (Q50-Q56) e.g.Q54 Hypospadias, Q53 Undescended testicle. Hypospadias is usually diagnosed during the routine examination after birth. The hypospadias belongs to the category of "Congenital malformation of the genitalia" - a medical term referring to a broad category of conditions as classified in the International Classification of Diseases (ICD) in chapter "Congenital malformations of genital organs" (Q50-Q56) e.g. Q54 Hypospadias. The anogenital distance (AGD) is a sexual dimorphism that results from the sex difference in foetal androgen (DHT) levels (Rhees et al., 1997). The AGD, the distance from the anus to the genitals, is widely used as biomarker of prenatal androgen exposure during a reproductive programming window (Wolf et al. 1999), (McIntyre, Barlow, and Foster 2001), (Macleod et al. 2010). The AGD is a marker of perineal growth and caudal migration of the genital tubercle. It is androgen-dependent in male rodents (Bowman et al. 2003). Measurement of AGD has also been proposed as a quantitative biomarker of foetal endocrine disruptor exposure in humans (Arbuckle et al. 2008), (Dean and Sharpe 2013). A longer (more “masculine”) AGD is typically associated with favourable health outcomes, while a shorter AGD is associated with adverse health outcomes. The AGD in males is approximately double that of females. Less is known about clinical correlates of AGD in females, although one study found that in women a longer AGD was associated with increased odds of multifollicular ovaries (Mendiola et al. 2012). The AGD is reflecting the prenatal hormonal milieu and in addition a biomarker for the risk of reproductive health problems linked to that early hormonal environment (Barrett et al. 2014). In animal studies, AGD measured from the genital tubercle to the anus is a sensitive marker of in utero exposure to androgens and anti-androgens, and is used extensively in animal reproductive toxicology studies (McIntyre, Barlow, and Foster 2001). AGD of each pup should be measured on at least one occasion from pre natal day postnatal day (PND) 0 through PND 4. Pup body weight should be collected on the day the AGD is measured and the AGD should be normalized to a measure of pup size, preferably the cube root of body weight (12). AGD is influenced by the body weight of the animal and therefore, this should be taken into account when evaluating the data (Gallavan et al, 1999). Body weight as a covariable may also be used (Howdeshell et al. 2007). Decreased AGD in male rats is a hallmark of exposure to antiandrogenic substances (Noriega et al, 2009; Christiansen et al, 2010). A statistically significant change in AGD that cannot be explained by the size of the animal indicates an adverse effect of exposure and should be considered in setting the NOAEL (OECD, 2008). The extended one-generation in vivo reproductive toxicity study OECD TG 443 <a class="external autonumber" href="http://www.oecd-ilibrary.org/environment/test-no-443-extended-one-generation-reproductive-toxicity-study_9789264122550-en" rel="nofollow" target="_blank">[1]</a>is used to investigate adverse effects of chemical substances on fertility and developmental toxicity in the rat, in which AGD is measured.

Hypospadias Rodents (Gray et al. 2001) Human (Manson and Carr 2003) Wildlife species (Hayes et al. 2002) AGD Across numerous species, including humans, AGD is longer in males compared to females; for review see (Barrett et al. 2014).

UBERON:0000079

UBERON male reproductive system

Moderate High

Arbuckle, Tye E, Russ Hauser, Shanna H Swan, Catherine S Mao, Matthew P Longnecker, Katharina M Main, Robin M Whyatt, et al. 2008. “Meeting Report: Measuring Endocrine-Sensitive Endpoints within the First Years of Life.” Environmental Health Perspectives 116 (7) (July): 948–51. doi:10.1289/ehp.11226. Barrett, Emily S, Lauren E Parlett, J Bruce Redmon, and Shanna H Swan. 2014. “Evidence for Sexually Dimorphic Associations between Maternal Characteristics and Anogenital Distance, a Marker of Reproductive Development.” American Journal of Epidemiology 179 (1) (January 1): 57–66. doi:10.1093/aje/kwt220. Baskin, L S, K Himes, and T Colborn. 2001. “Hypospadias and Endocrine Disruption: Is There a Connection?” Environmental Health Perspectives 109 (11) (November): 1175–83. Bowman, Christopher J, Norman J Barlow, Katie J Turner, Duncan G Wallace, and Paul M D Foster. 2003. “Effects of in Utero Exposure to Finasteride on Androgen-Dependent Reproductive Development in the Male Rat.” Toxicological Sciences : An Official Journal of the Society of Toxicology 74 (2) (August): 393–406. doi:10.1093/toxsci/kfg128. Dean, Afshan, and Richard M Sharpe. 2013. “Clinical Review: Anogenital Distance or Digit Length Ratio as Measures of Fetal Androgen Exposure: Relationship to Male Reproductive Development and Its Disorders.” The Journal of Clinical Endocrinology and Metabolism 98 (6) (June): 2230–8. doi:10.1210/jc.2012-4057. Gray, L E, J Ostby, J Furr, C J Wolf, C Lambright, L Parks, D N Veeramachaneni, et al. 2001. “Effects of Environmental Antiandrogens on Reproductive Development in Experimental Animals.” Human Reproduction Update 7 (3): 248–64. Hayes, Tyrone B, Atif Collins, Melissa Lee, Magdelena Mendoza, Nigel Noriega, A Ali Stuart, and Aaron Vonk. 2002. “Hermaphroditic, Demasculinized Frogs after Exposure to the Herbicide Atrazine at Low Ecologically Relevant Doses.” Proceedings of the National Academy of Sciences of the United States of America 99 (8) (April 16): 5476–80. doi:10.1073/pnas.082121499. Howdeshell, Kembra L, Johnathan Furr, Christy R Lambright, Cynthia V Rider, Vickie S Wilson, and L Earl Gray. 2007. “Cumulative Effects of Dibutyl Phthalate and Diethylhexyl Phthalate on Male Rat Reproductive Tract Development: Altered Fetal Steroid Hormones and Genes.” Toxicological Sciences : An Official Journal of the Society of Toxicology 99 (1) (September): 190–202. doi:10.1093/toxsci/kfm069. Kalfa, Nicolas, Benchun Liu, Ophir Klein, Ming-Hsieh Wang, Mei Cao, and Laurence S Baskin. 2008. “Genomic Variants of ATF3 in Patients with Hypospadias.” The Journal of Urology 180 (5) (November): 2183–8; discussion 2188. doi:10.1016/j.juro.2008.07.066. Kalfa, Nicolas, Pascal Philibert, and Charles Sultan. 2009. “Is Hypospadias a Genetic, Endocrine or Environmental Disease, or Still an Unexplained Malformation?” International Journal of Andrology 32 (3) (June): 187–97. doi:10.1111/j.1365-2605.2008.00899.x. Macleod, D J, R M Sharpe, M Welsh, M Fisken, H M Scott, G R Hutchison, A J Drake, and S van den Driesche. 2010. “Androgen Action in the Masculinization Programming Window and Development of Male Reproductive Organs.” International Journal of Andrology 33 (2) (April): 279–87. doi:10.1111/j.1365-2605.2009.01005.x. Manson, Jeanne M, and Michael C Carr. 2003. “Molecular Epidemiology of Hypospadias: Review of Genetic and Environmental Risk Factors.” Birth Defects Research. Part A, Clinical and Molecular Teratology 67 (10) (October): 825–36. doi:10.1002/bdra.10084. McIntyre, B S, N J Barlow, and P M Foster. 2001. “Androgen-Mediated Development in Male Rat Offspring Exposed to Flutamide in Utero: Permanence and Correlation of Early Postnatal Changes in Anogenital Distance and Nipple Retention with Malformations in Androgen-Dependent Tissues.” Toxicological Sciences : An Official Journal of the Society of Toxicology 62 (2) (August): 236–49. Mendiola, Jaime, Manuela Roca, Lidia Mínguez-Alarcón, Maria-Pilar Mira-Escolano, José J López-Espín, Emily S Barrett, Shanna H Swan, and Alberto M Torres-Cantero. 2012. “Anogenital Distance Is Related to Ovarian Follicular Number in Young Spanish Women: A Cross-Sectional Study.” Environmental Health : A Global Access Science Source 11 (January): 90. doi:10.1186/1476-069X-11-90. OECD. 2012. Test No. 443: Extended One-Generation Reproductive Toxicity Study. OECD Guidelines for the Testing of Chemicals, Section 4. OECD Publishing. doi:10.1787/9789264185371-en. Rey, Rodolfo A, Ethel Codner, Germán Iñíguez, Patricia Bedecarrás, Romina Trigo, Cecilia Okuma, Silvia Gottlieb, Ignacio Bergadá, Stella M Campo, and Fernando G Cassorla. 2005. “Low Risk of Impaired Testicular Sertoli and Leydig Cell Functions in Boys with Isolated Hypospadias.” The Journal of Clinical Endocrinology and Metabolism 90 (11) (November): 6035–40. doi:10.1210/jc.2005-1306. Wolf, C., C. Lambright, P. Mann, M. Price, R. L. Cooper, J. Ostby, and L. E. Gray. 1999. “Administration of Potentially Antiandrogenic Pesticides (procymidone, Linuron, Iprodione, Chlozolinate, P,p’-DDE, and Ketoconazole) and Toxic Substances (dibutyl- and Diethylhexyl Phthalate, PCB 169, and Ethane Dimethane Sulphonate) during Sexual Differen.” Toxicology and Industrial Health 15 (1-2) (February 1): 94–118. doi:10.1177/074823379901500109. AOPWiki 2016-11-29T18:41:24

2017-09-16T10:14:33

<upstream-id>5340a4b8-21db-4ede-ac81-ae345eef82be</upstream-id> <downstream-id>cb49cb80-1037-4b86-aaca-d960e767f7c5</downstream-id> Steroidogenic acute regulatory protein (StAR) mediates the cholesterol transport from the outer to the inner mitochondrial membrane, where it undergoes side chain cleavage by cytochrome P-450 enzyme (P450scc) that yields the steroid precursor, pregnenolone (Besman et al. 1989). The cholesterol transfer within the mitochondria is the rate-limiting step in the production of steroid hormones. Therefore reduced amount/activity of the StAR impairs the cholesterol delivery that is necessary for the hormone biosynthesis.

The first step in steroidogenesis takes place within mitochondria. StAR facilitates the movement of cholesterol from the outer mitochondrial membrane (OMM) to the inner mitochondrial membrane (IMM) for steroidogenesis [reviewed in (Miller and Auchus 2011)]. It is primarily present in steroid-producing cells, including theca cells and luteal cells in the ovary, Leydig cells in the testis and cells in the adrenal cortex.

Down-regulation of StAR and impaired steroidogenesis was reported upon exposure to phthalates i.a. (Barlow et al. 2003), (Borch et al. 2006), for details see Table 1. <table class="wikitable" id="Event436"> <tbody> <tr> <td> </td> <td> </td> <td> </td> <td> KE: StAR, decrease </td> <td> KE: Cholesterol transport, decrease </td> <td> </td> <td> </td> </tr> <tr> <td> Compound </td> <td> Species </td> <td> Effect level </td> <td> </td> <td> </td> <td> Details </td> <td> References </td> </tr> <tr> <td> Phthalate (DBP) </td> <td> rat </td> <td> LOEL=500 mg/kg/day </td> <td> mRNA StAR decrease (by ~34%) </td> <td> reduced Leydig cell lipid content </td> <td> </td> <td> (Barlow et al. 2003) </td> </tr> <tr> <td> Phthalate (DBP) </td> <td> rat </td> <td> LOEL=500 mg/kg/day (GD12-19) </td> <td> </td> <td> decrease uptake of cholesterol Leydig cell mitochondria </td> <td> decreased testosterone, decreased expression of scavenger receptor B1, P450(SCC), steroidogenic acute regulatory protein, and cytochrome p450c17 </td> <td> (Thompson, Ross, and Gaido 2004) </td> </tr> <tr> <td> Phthalate (DBP) </td> <td> rat </td> <td> LOEL=500 mg/kg </td> <td> mRNA and protein StAR decrease </td> <td> </td> <td> 1 dose, Time course analysis (0,5,1,2,3,6,12,18, 24h killed at GD), decreased testosterone in foetal testis </td> <td> (Thompson et al. 2005) </td> </tr> <tr> <td> Phthalate (DEHP) </td> <td> rat </td> <td> LOEL=300 mg/ kg/day   </td> <td> mRNA StAR decrease </td> <td>   </td> <td> dose-dependently reduced StAR, TSOP mRNA (GD 21 testes), also on protein levels in Leydig cells   </td> <td> (Borch et al. 2006) </td> </tr> <tr> <td> Phthalate (DBP) </td> <td> rat </td> <td> LOEL=500 mg/kg/day, (GD12 to 21) </td> <td> mRNA StAR decrease </td> <td>   </td> <td> Testes examined GD 16, 19, and 21, cytochrome P450 side chain cleavage, cytochrome P450c17, decrease. Testicular testosterone and androstenedione decreased (GD 19 and 21) </td> <td> (Shultz 2001) </td> </tr> <tr> <td> Phthalate (MEHP) </td> <td> rat </td> <td> LOEC=250 μM </td> <td> protein StAR decrease (immature and adult Leydig cells) </td> <td> cholesterol transport, decrease (into the mitochondria of immature and adult Leydig cells </td> <td> decreased testosterone by approximately 60%, in vitro ( immature and adult Leydig cells) </td> <td> (Svechnikov, Svechnikova, and Söder 2008) </td> </tr> <tr> <td> Phthalate (DBP) </td> <td> rat </td> <td> LOEL=500 mg/kg/day </td> <td> mRNA StAR decrease </td> <td>   </td> <td> GD 12 -20, examinations on GD20 </td> <td> (Johnson et al. 2011) </td> </tr> </tbody> </table> Table 1 Summary table of empirical support for this KER. LOEC-lowest effect concentration, LOEL- lowest observed effect level, Dibutyl phthalate (DBP), Di-2-ethylhexyl phthalate (DEHP), mono(2-ethylhexyl) phthalate (MEHP).

Some steroidogenesis is independent of StAR; when nonsteroidogenic cells are transfected with the P450scc system, they convert cholesterol to pregnenolone at about 14% of the StAR-induced rate (Lin et al. 1995). The mechanism of StAR-independent steroidogenesis is unclear (Miller and Auchus 2011). Johnson et al proposed the involvment of sterol regulatory element–binding protein (SREBP) in phthalate mediated disruption of steroidogenesis. Their study showed lipid metabolism pathways transcriptionally regulated by SREBP were inhibited in the rat but induced in the mouse, and this differential species response corresponded with repression of the steroidogenic pathway. In rats exposed to 100 or 500 mg/kg DBP from gestational days (GD) 16 to 20, a correlation was observed between GD20 testis steroidogenic inhibition and reductions of testis cholesterol synthesis endpoints including testis total cholesterol levels (Johnson et al. 2011).

Rat see Table 1.

#<Reference::ActiveRecord_Associations_CollectionProxy:0x00005eb8a27cb488> AOPWiki 2016-11-29T18:41:34

2016-12-02T09:28:42

<upstream-id>cb49cb80-1037-4b86-aaca-d960e767f7c5</upstream-id> <downstream-id>55f446b3-d41c-476c-a1b7-8109fa6f8f4f</downstream-id> Production of steroid hormones depends on the availability of cholesterol in the mitochondrial matrix. A decreased amount of cholesterol inside the mitochondria (e. g by decreased expression of enzymes that transport cholesterol like StAR or TSOP) means diminished substrate for hormone (testosterone) production in testes.

Steroid hormones play a critical role in sexual development, homeostasis, stress-responses, carbohydrate metabolism, tumor growth, and reproduction. These hormones are primarily produced in specialized steroidogenic tissues and are synthesized from a common precursor, cholesterol. Mitochondria are a key control point for the regulation of steroid hormone biosynthesis. The first and rate-limiting step in steroidogenesis is the transfer of cholesterol from the outer mitochondrial membrane to the inner mitochondrial membrane, a process dependent on the action of StAR (Stocco, 2001) and the subsequent transport across the inner mitochondrial space into the steroidogenic pathway, which is executed by TSPO (Hauet et al., 2005). Testosterone production by Leydig cells is primarily under the control of luteinizing hormone (LH). Stimulation of the Leydig cells results in the activation of StAR transcription and translation, which facilitates the transfer of cholesterol into the mitochondrial matrix to cholesterol side-chain cleavage cytochrome P450 (P450scc, CYP11A), which converts cholesterol to pregnenolone. Pregnenolone diffuses to the smooth endoplasmic reticulum where it is further metabolized to testosterone via the actions of 3β-hydroxysteroid dehydrogenase Δ5-Δ4-isomerase (3β-HSD), 17α-hydroxylase/C17-20 lyase (P450c17, CYP17), and 17β-hydroxysteroid dehydrogenase type III (17HSD3). For review see (Payne & Hales, 2013). Decreased expression of genes that are responsible for cholesterol transport and steroidogenic enzyme activities in the Leydig cell leads to decreased testosterone production.

There is evidence from experimental work that demonstrates a coordinated reduction in the expression of key genes and proteins that are involved in cholesterol transport and steroidogenesis, together with a corresponding reduction in testosterone in testes. For details see Table 1. Foetal Leydig cells exhibit a high rate of lipid metabolism, which is required for both synthesizing and importing the testosterone precursor cholesterol. Upon exposure to some chemicals mRNA expression of genes in these pathways are profoundly reduced e.g. following 500mg/kg phthalate (DBP) exposure (Johnson, McDowell, Viereck, & Xia, 2011), (Thompson et al., 2005). Additionally, after phthalate exposure testis cholesterol and cholesterol-containing lipid droplets in foetal Leydig cells are also reduced (Barlow et al., 2003), (Johnson et al., 2011), (Lehmann, Phillips, Sar, Foster, & Gaido, 2004).   <table class="wikitable" id="Event438"> <tbody> <tr> <td> </td> <td> </td> <td> </td> <td> KE: Cholesterol transport, reduction </td> <td> KE: Testosterone production/levels, reduction </td> </tr> <tr> <td> Compound </td> <td> Species </td> <td> Effect level </td> <td> Translator protein (TSPO), decrease; Steroidogenic acute regulatory protein (StAR) decrease </td> <td> </td> </tr> <tr> <td> Phthalate (DBP) </td> <td> rat </td> <td> LOEL=500 mg/kg/day </td> <td> mRNA StAR decrease (by ~34%)(Barlow et al., 2003) </td> <td>   </td> </tr> <tr> <td> Phthalate (BBP, DPeP, DEHP, DHP, DiHeP, DCHP, DINP DHeP) </td> <td> rat </td> <td> </td> <td> </td> <td>   </td> </tr> <tr> <td> Phthalate (DBP, DEHP, BBP) </td> <td> Rat </td> <td> LOEL=750 mg/kg/day (GD14-18) </td> <td> </td> <td> testosterone production, reduction ex vivo fetal testes examined on GD18 (Wilson et al., 2004)   </td> </tr> <tr> <td> </td> <td> </td> <td> </td> <td> </td> <td>   </td> </tr> <tr> <td> Phthalate (DBP) </td> <td> rat </td> <td> LOEL=500 mg/kg/day </td> <td> reduced Leydig cell lipid content(Barlow et al., 2003) </td> <td>   </td> </tr> <tr> <td> Phthalate (DBP) </td> <td> rat </td> <td> LOEL=500 mg/kg/day GD 12 -20, examinations on GD20 </td> <td> total cholesterol levels, reduction </td> <td> intratesticular testosterone levels, reduction (by nearly 90%)(Johnson et al., 2011) </td> </tr> <tr> <td> Phthalate (DBP) </td> <td> rat </td> <td> LOEL=500 mg/kg/day (GD12-19) </td> <td> decrease uptake of cholesterol Leydig cell mitochondria gd 19 </td> <td> <pre> testosterone production, reduction ex vivo (Thompson, Ross, & Gaido, 2004) </pre> </td> </tr> <tr> <td> Phthalate (DEHP) </td> <td> mouse </td> <td> LOEL=1 g/kg/day </td> <td> reduced TSPO mRNA </td> <td> testosterone levels, reduction (Gazouli, 2002) </td> </tr> <tr> <td> Phthalate (DEHP) </td> <td> rat </td> <td> LOEL=300 mg/ kg/day   </td> <td> dose-dependently reduced StAR, TSOP mRNA (GD 21 testes), also on protein levels in Leydig cells (Borch, Metzdorff, Vinggaard, Brokken, & Dalgaard, 2006)   </td> <td>   </td> </tr> <tr> <td> Phthalate (DEHP) </td> <td> rat </td> <td> LOEL=300 mg/kg/day </td> <td>   </td> <td> testosterone production, reduction (ex vivo) testosterone levels, reduction (Borch et al., 2006), (Borch, Ladefoged, Hass, & Vinggaard, 2004) </td> </tr> <tr> <td> Phthalate (MEHP) </td> <td> mouse </td> <td> LOEC=100 μM </td> <td> <ul> <li> reduced TSPO mRNA levels by 50%, </li> <li> binding sites decreased by 50% </li> <li> no effect on receptor affinity </li> <li> inhibited the transfer or loading of cholesterol to the inner mitochondrial membrane P450scc. (Gazouli, 2002) </li> </ul> </td> <td>   </td> </tr> <tr> <td> Phthalate (MEHP) </td> <td> rat </td> <td> IC50 =100 μM </td> <td> <ul> <li> inhibited formation of progesterone (Gazouli, 2002) </li> </ul> </td> <td>   </td> </tr> <tr> <td> Phthalate (MEHP) </td> <td> rat </td> <td> LOEC=250 μM </td> <td> cholesterol transport, decrease (into the mitochondria of immature and adult Leydig cells) </td> <td> Testosterone, reduction by approximately 60%, in vitro ( immature and adult Leydig cells) (Svechnikov, Svechnikova, & Söder, 2008) </td> </tr> <tr> <td> Phthalate (DEHP) </td> <td> rat </td> <td> LOEL=750 mg/kg/day </td> <td>   </td> <td> testosterone production reduction, testosterone levels, reduction (testicular and whole-body T levels in fetal and neonatal male rats from GD 17 to PND 2. (Parks, 2000) </td> </tr> <tr> <td> Phthalate (MEHP) </td> <td> rat </td> <td> LOEC=1 μM </td> <td>   </td> <td> testosterone production, reduction dose-dependent (Chauvigné et al., 2011) </td> </tr> <tr> <td> Perfluorooctanoic acid (PFOA) </td> <td> mouse </td> <td> LOEL=5mg/kg/day </td> <td> </td> <td> plasma testosterone, reduction (by 37%)(Li et al., 2011) </td> </tr> <tr> <td> WY-14,643 </td> <td> mouse </td> <td> LOEC=50 mg/kg/day </td> <td> reduced TSPO mRNA </td> <td> Serum testosterone levels, reduction (Gazouli, 2002) </td> </tr> <tr> <td> WY-14,643 </td> <td> rat </td> <td> </td> <td>   </td> <td> No decrease of testosterone ( ex vivo), (Furr, Lambright, Wilson, Foster, & Gray, 2014) </td> </tr> <tr> <td> WY-14,643 </td> <td> mouse </td> <td> LOEC=100 μM </td> <td> Inhibited progesterone synthesis (Gazouli, 2002) </td> <td>   </td> </tr> <tr> <td> Bezafibrate </td> <td> mouse </td> <td> IC50=100 μM </td> <td> <ul> <li> a dose-dependent 10–95% inhibition of the progesterone synthesis at 24 or 72 h </li> <li> inhibited the transfer or loading of cholesterol to the inner mitochondrial membrane P450scc. At 100 μM </li> <li> binding sites of TSPO decreased IC50 of approximately 100 μM </li> <li> decrease TSPO levels by 60% at 100 μM (Gazouli, 2002) </li> </ul> </td> <td>   </td> </tr> <tr> <td> Bezafibrate </td> <td> rat </td> <td> IC50 = 30 μM </td> <td> inhibited formation of progesterone (Gazouli, 2002) </td> <td>   </td> </tr> <tr> <td> Bezafibrate </td> <td> rat </td> <td> IC50 ~10−4 μM </td> <td> </td> <td> testosterone production, reduction (Gazouli, 2002) </td> </tr> <tr> <td> Phthalate (DiBP) </td> <td> rat </td> <td> GD 19 -21 </td> <td> reduced StAR, (Boberg et al., 2008) </td> <td> testicular testosterone production and testicular testosterone levels, (Boberg et al., 2008) </td> </tr> </tbody> </table> Table 1. Summary table of empirical support for this KER. IC50 half maximal inhibitory concentration, LOEC-lowest effect concentration, LOEL- lowest observed effect level, Dibutyl phthalate (DBP), diisobutyl phthalate (DiBP), Bis(2-ethylhexyl) phthalate (DEHP), Dibutyl phthalate (DBP), Bezafibrate and WY-14,643 are PPARα ligands, n.a - not available

Thompson et al investigated time course effects of phthalate on steroidogenesis gene expression and testosterone concentration. The study showed diminished concentration testosterone concentration in the foetal testis by 50% within 1h of treatment with phthalate (DBP). Surprisingly, the diminution in testosterone concentration preceded any alteration in expression of genes in the steroidogenesis pathway. Star mRNA was significantly diminished 2 h after DBP exposure, but Cyp11a1, Cyp17a1, and Scarb1 did not show a significant decrease in expression until 6 h after DBP exposure (Thompson et al., 2005). In utero exposure of rats to PFOA 20 mg/kg did not cause any effect on fetal testosterone (Boberg et.al. 2008) although in mice (adult) the decrease level of testosterone was observed. Testosterone production may also be diminished due to reduction/inhibition of other genes involved in steroidogenesis (e.g. P450scc, Cyp17a1) (Thompson et al., 2004), (Boberg et al., 2008), (Chauvigné et al., 2009), (Chauvigné et al., 2011).

Moderate High Low

See Table 1.

#<Reference::ActiveRecord_Associations_CollectionProxy:0x00005eb8a273cf80> AOPWiki 2016-11-29T18:41:34

2016-12-02T10:16:58

<upstream-id>55f446b3-d41c-476c-a1b7-8109fa6f8f4f</upstream-id> <downstream-id>2297c412-0985-40e3-b66b-9c93906abb9d</downstream-id> Impairment of testosterone production in testes directly impacts on testosterone levels.

Within the testes, steroid synthesis takes place within the mitochondria of Leydig cells. Testosterone production by Leydig cells is primarily under the control of LH. LH indirectly stimulates the transfer of cholesterol into the mitochondrial matrix to cholesterol side-chain cleavage cytochrome P450 (P450scc, CYP11A), which converts cholesterol to pregnenolone. Pregnenolone diffuses to the smooth endoplasmic reticulum where it is further metabolized to testosterone via the actions of 3β-hydroxysteroid dehydrogenase Δ5-Δ4-isomerase (3β-HSD), 17α-hydroxylase/C17-20 lyase (P450c17, CYP17), and 17β-hydroxysteroid dehydrogenase type III (17HSD3). For review see (Payne & Hales, 2013). Therefore, inhibition or impairment of the testosterone production directly impacts on the levels of testosterone.

There is evidence from experimental work that demonstrates a coordinated, dose-dependent reduction in the production of testosterone and consecutive reduction of testosterone levels in foetal testes and in serum, see Table 1. <table class="wikitable" id="Event439"> <tbody> <tr> <td>   </td> <td>   </td> <td>   </td> <td> KE: testosterone synthesis, reduction </td> <td> KE: testosterone, reduction </td> <td>   </td> <td>   </td> </tr> <tr> <td> Compound </td> <td> Species </td> <td> Effect level </td> <td>   </td> <td>   </td> <td> Details </td> <td> References </td> </tr> <tr> <td> Phthalates (DEHP) </td> <td> rat </td> <td> LOEL =300 mg/kg/day </td> <td> testicular testosterone production, reduction (ex vivo) </td> <td> testicular testosterone levels, reduction, no change plasma testosterone </td> <td> testosterone levels at GD 21 in male rat fetuses exposed to 0, 10, 30, 100, or 300 mg /kg bw/day from GD 7 to GD 21 testicular testosterone production ex vivo </td> <td> (Borch, Metzdorff, Vinggaard, Brokken, & Dalgaard, 2006) </td> </tr> <tr> <td> Phthalates (DBP) </td> <td> rat </td> <td> LOEL =50 mg/kg/day </td> <td>   </td> <td> testicular testosterone levels, reduction, </td> <td> Testicular testosterone was reduced >50 mg/kg/day </td> <td> (Shultz, 2001) </td> </tr> <tr> <td> Phthalates (DEHP) </td> <td> rat </td> <td> LOEL=300 mg/kg/day </td> <td> fetal testicular testosterone production, reduction </td> <td>   </td> <td>   </td> <td> (Borch, Ladefoged, Hass, & Vinggaard, 2004)   </td> </tr> <tr> <td> Phthalates (DEHP) </td> <td> rat </td> <td> LOEL=300 mg/kg/day </td> <td>   </td> <td> testicular testosterone levels, reduction, </td> <td>   </td> <td> (Borch et al., 2004)   </td> </tr> <tr> <td> Phthalates (DEHP) </td> <td> rat </td> <td> LOEL=300 mg/kg/day </td> <td>   </td> <td> No change plasma testosterone </td> <td>   </td> <td> (Borch et al., 2004)   </td> </tr> <tr> <td> Phthalates (DEHP) </td> <td> rat </td> <td> LOEL=100 mg/kg/day </td> <td>   </td> <td> Serum testosterone levels, reduction, </td> <td>   </td> <td> (Akingbemi, 2001)   </td> </tr> <tr> <td> Phthalates (DEHP) </td> <td> rat </td> <td> LOEL=750 mg /kg /day </td> <td>   </td> <td> testicular testosterone levels, reduction, by 60 – 85% </td> <td>   </td> <td> (Parks, 2000) </td> </tr> <tr> <td> Phthalates (DEHP) </td> <td> rat </td> <td> LOEL=750 mg /kg/day </td> <td>   </td> <td> testosterone levels, reduction, fetuses on GD 17 (71% lower than controls) and 18 (47% lower than controls) </td> <td>   </td> <td> (Parks, 2000)   </td> </tr> <tr> <td> Phthalates (DEHP) </td> <td> rat </td> <td> LOEL=750mg/kg/day   </td> <td> ex vivo testosterone production, reduction by 50% </td> <td>   </td> <td>   </td> <td> (Wilson et al., 2004)   </td> </tr> <tr> <td> Phthalates (DEHP) </td> <td> rat </td> <td> LOEL=234 mg/kg/day   </td> <td>   </td> <td> serum testosterone levels, reduction, </td> <td>   </td> <td> (Culty et al., 2008)   </td> </tr> <tr> <td> Phthalates (DEHP) </td> <td> rat </td> <td> LOEL=1250 mg/kg/day </td> <td> ex vivo foetal testicular production </td> <td>   </td> <td>   </td> <td> (Culty et al., 2008)   </td> </tr> <tr> <td> Phthalates (DEHP) </td> <td> rat </td> <td> ED50=444,2 mg/kg/day </td> <td> ex vivo foetal testicular production, reduction   </td> <td>     </td> <td>   </td> <td> (Hannas et al., 2012)   </td> </tr> <tr> <td> Phthalates (DHP) </td> <td> rat </td> <td> ED50=75.25 mg/kg/day   </td> <td> ex vivo foetal testicular production, reduction </td> <td>   </td> <td>   </td> <td> (Hannas et al., 2012)   </td> </tr> </tbody> </table> Table 1. Summary table for empirical support for this KER. ED50 - half maximal effective concentration, LOEL- lowest observed effect level, Dibutyl phthalate (DBP), Bis(2-ethylhexyl) phthalate (DEHP), Dihexyl Phthalate (DHP).

High High Moderate

Ses Table 1.

#<Reference::ActiveRecord_Associations_CollectionProxy:0x00005eb8a29533f0> AOPWiki 2016-11-29T18:41:34

2016-12-02T10:18:05

<upstream-id>2b5c1d7a-b354-44ec-8807-51af86f827b3</upstream-id> <downstream-id>5340a4b8-21db-4ede-ac81-ae345eef82be</downstream-id> The direct link of PPARα in regulation of the cholesterol transport in mitochondria and hormone synthesis derives from studies demonstrating that PPARα may act as indirect transrepressor of the key steroidogenic factor-1 (SF-1) (S. Plummer et al. 2007), (S. M. Plummer et al. 2013). SF-1 is a transcription factor essential for expression of genes involved in steroidogenesis (including Steroidogenic acute regulatory protein (StAR)).

The PPARα is expressed in foetal rat Leydig cells (Boberg et al. 2008), (S. M. Plummer et al. 2013) and in adult rat Leydig cells (Schultz et al. 1999). Recent studies have shown that foetal testes contained PPARα protein–binding peaks in CYP11a, StAR, and CYP17a regulatory regions (S. M. Plummer et al. 2013). Binding of PPARα to promoter of steroidogenic gene occurs at binding sites different from those of SF-1, indicating that PPARα may be an indirect repressor of SF1 binding. Moreover, it is possible that PPARα could act via sequestration of the shared coactivator CBP (S. M. Plummer et al. 2013). PPARα and SF-1 share a common coactivator, CREB-binding protein (CBP), which is present in limited concentrations (McCampbell 2000). Binding of CBP to PPARα could therefore starve SF-1 from a cofactor essential for its transactivation functions. SF-1 controls transcription of the StAR gene (Sugawara et al. 1996). Steroidogenic acute regulatory (StAR) protein plays a critical role in the movement of cholesterol from the outer to the inner mitochondrial membrane (Stocco 2001). Hence, it seems likely that the ability of PPARα to interfere with SF-1 binding/transactivation caused by exposure to chemicals (e.g. phthalates) could affect the StAR expression and the cholesterol transport in mitochondria.

PPARα agonists can suppress Leydig cell steroidogenesis (Gazouli 2002), and downregulate steroidogenic genes including StAR (Borch et al. 2006), (Lehmann et al. 2004), (Liu et al. 2005), for details see Table 1. Moreover, PPARα agonists, which do not directly transrepress the StAR promoter, have been found to downregulate the expression of this gene in steroidogenic tissues (in mice ovaries) (Toda et al. 2003).   <table class="wikitable" id="KER369"> <tbody> <tr> <td> Compound </td> <td> species </td> <td> KE: PPARα, Activation </td> <td> KE: StAR, Decrease </td> </tr> <tr> <td> Phthalate (MBzP ) </td> <td> human </td> <td> EC50 = 30 μM human (Hurst and Waxman 2003) </td> <td> n.a. </td> </tr> <tr> <td> Phthalate (DBP) </td> <td> rodent </td> <td> EC50 = 21 μM MBzP EC50 = 63 μM (Hurst and Waxman 2003) MBuP </td> <td> LOEC=50 mg/kg/day (Borch et al. 2006) (Lehmann et al. 2004), (Shultz 2001) </td> </tr> <tr> <td> Phthalate (MEHP) </td> <td> rodent </td> <td> LOEC=30 μM (Bility et al. 2004) </td> <td> LOEC=300 mg/kg/day (Borch et al. 2006) </td> </tr> <tr> <td> Phthalate (MEHP) </td> <td> human </td> <td> Ki=15 µM (Lapinskas et al. 2005) EC50 = 3.2 μM (Hurst and Waxman 2003) </td> <td> n.a. </td> </tr> <tr> <td> Phthalate (DiBP) </td> <td> rat </td> <td> LOEC=600 mg/kg/day, decrease of PPARα mRNA, at GD 19 and 21 in testes of foetuses (Boberg et al. 2008) </td> <td> LOEC=600 mg/kg/day, decrease of StAR mRNA at GD 19 and 21 in testes of foetuses (Boberg et al. 2008) </td> </tr> <tr> <td> Econazole </td> <td> rodent </td> <td> AC50=0.0729 μM (ToxCastTM Data) </td> <td> LOEC=25 μM, mouse MA-10 Leydig tumor cell line, Decrease in StAR activity and/or expression (Walsh, Kuratko, and Stocco 2000) </td> </tr> <tr> <td> Perfluorooctanoate (PFOA) </td> <td> mice </td> <td> 3T3-L1 cells transfected with human, mouse and rat PPARα (Vanden Heuvel et al. 2006) </td> <td> LOEC= 5.0 mg/kg/day mRNA StAR decrease in the testis of wild-type mice, LOEC=1.0 mg/kg/day mRNA StAR decrease in testis of PPARα-humanized mice (not in the PPARα –null mice) (Li et al. 2011) </td> </tr> <tr> <td> Fenofibrate </td> <td> mice </td> <td> PPAR agonist Increase PPARα protein (Toda et al. 2003). </td> <td> Decrease of StAR (protein level, in mice ovaries) (Toda et al. 2003) </td> </tr> </tbody> </table> Table 1. Summary table for empirical support of KER. ED50 - half maximal effective concentration, LOEC-lowest observed effect concentration, Bis(2-ethylhexyl) phthalate (DEHP), Dibutyl phthalate (DBP), diisobutyl phthalate (DiBP), mono-sec-butyl phthalate (MBuP), n.a - not available.

Uncertainties PPARα was also shown to regulate Translator protein (TSPO), which is a mitochondrial outer membrane protein implicated in cholesterol import to the inner mitochondrial (for details see <a href="/wiki/index.php/Relationship:370" title="Relationship:370">Relationship:370</a>). Moreover, there is evidence that activated PPARα regulates the expression of enzymes involved in steroid metabolism (17β-hydroxysteroid dehydrogenase IV, 11β-hydroxysteroid dehydrogenase I, and 3β-hydroxysteroid dehydrogenase V (Hermanowski-Vosatka et al. 2000), (Corton et al. 1996), (Wong et al. 2002)). Inconsistencies In utero rat exposure to the PPARα agonist Wy-14,643 did not reduce fetal testis steroidogenic gene expression or testosterone production (Hannas et al. 2012).

High Moderate Low

See Table 1.

#<Reference::ActiveRecord_Associations_CollectionProxy:0x00005eb8a2d0c208> AOPWiki 2016-11-29T18:41:33

2016-12-02T10:12:02

<upstream-id>d61aa1ac-faba-4c5e-a5a8-99006004a82e</upstream-id> <downstream-id>cb49cb80-1037-4b86-aaca-d960e767f7c5</downstream-id> Translocator Protein (TSPO) mediates the first step of cholesterol transport to the inner mitochondrial membrane cytochrome P-450 side chain cleavage enzyme (P450scc) (Besman et al. 1989). TSPO ligands stimulate steroidogenesis and induce cholesterol movement from the outer mitochondrial membrane (OMM) to the inner mitochondrial membrane (IMM) (Besman et al. 1989). Therefore reduced amount/activity of the TSPO impairs the cholesterol delivery that is necessary for the hormone biosynthesis.

The TSPO was first identified as a peripheral tissue diazepam binding site [known as peripheral-type benzodiazepine receptor (PBR)] and since then it has been implicated in many cellular processes. Amongst these are steroid biosynthesis, protein import, heme biosynthesis, immunomodulation, cellular respiration and oxidative processes. The TSPO is present in virtually all mammalian peripheral tissues (Zisterer and Williams 1997), however highly prominent TSPO protein expression has been identified in steroidogenic tissues (R. R. Anholt et al. 1985),(Wang, Fan, and Papadopoulos 2012). The presence of TSPO was confirmed in Leydig and Sertoli cells (Morohaku, Phuong, and Selvaraj 2013), granulosa cells (Amsterdam and Suh 1991) and to lesser extent in thecal cells (Morohaku, Phuong, and Selvaraj 2013). In subcellular fractions, binding sites for the TSPO were identified to be present in the OMM (R. R. Anholt et al. 1985), (R. Anholt et al. 1986).

The decreased TSPO protein levels leads to decreased cholesterol transport into Leydig cells (Gazouli 2002), (Borch et al. 2006). Moreover, Thompson et al., observed decreased uptake of cholesterol in Leydig cell mitochondria upon exposure to phthalates (Thompson, Ross, and Gaido 2004).

Targeted disruption of TSPO in rat Leydig R2C cells reduced steroidogenesis (Papadopoulos et al. 1997). However, recent experiments with TSPO knockdown in steroidogenic cells was not shown to affect steroid hormone biosynthesis (Tu et al. 2014) as well as in a specific deletion of TSPO in steroidogenic Leydig cells did not impair their synthesis of testosterone (Morohaku et al. 2014). As stated in the recent review "At this point in time, a functional designation for TSPO is still actively being sought" (Selvaraj, Stocco, and Tu 2015).

Moderate Low Low

Rat (Papadopoulos et al., 1997)

#<Reference::ActiveRecord_Associations_CollectionProxy:0x00005eb8a2d5cd98> AOPWiki 2016-11-29T18:41:34

2016-12-02T10:14:54

<upstream-id>2b5c1d7a-b354-44ec-8807-51af86f827b3</upstream-id> <downstream-id>d61aa1ac-faba-4c5e-a5a8-99006004a82e</downstream-id> Activation of PPARα leads to decreased expression of cholesterol transport (TSPO) gene in steroidogenic cells (e.g. Leydig cell) and as a consequence the amount of cholesterol transported into mitochondria decreases (impact on steroid production).

PPARs are nuclear receptors that among many other functions regulate genes involved in cholesterol uptake and transport (Xie, Yang, and DePierre 2002), (Gazouli 2002), (Campioli et al. 2011). The indirect link of PPAR receptors in regulation of the cholesterol transport in mitochondria derives from studies demonstrating PPARα dependent control of TSOP (Gazouli 2002), (Campioli et al. 2011). PPARα is present in steroidogenic cells e.g. of the testes during its development as well as in adult testes (Schultz et al. 1999), (Boberg et al. 2008) and modulation of its activity has been shown to impact on TSOP transcriptional activity (Gazouli 2002). The exact mechanisms of this relationship are not known.

Gazouli et al showed that PPAR activators (Bezafibrate , MEHP ) inhibited the transfer or loading of cholesterol to the inner mitochondrial membrane in MA-10 mouse Leydig tumor cells and decreased levels of TSOP protein. Additionally, in R2C (Leydig tumor cell line) inhibited the formation of progesterone. The levels of the TSPO protein were decreased in testes of adult mice exposed to and DEHP (Gazouli 2002). Moreover, the finding that benzafibrate and phthalates inhibit the hormone-induced and constitutively sustained steroidogenesis with similar IC50 values indicates that these compounds act on a common regulatory component of the steroidogenic pathway (Gazouli 2002). In in vivo studies with rats exposed to DEHP the levels of TSOP (mRNA) were decreased dose-dependently in foetal testes (GD 21 testes), also on protein levels in Leydig cells (Borch et al. 2006). For details see Table1. <table border="1" style="border-collapse:collapse; font-size:75%"> <tbody> <tr> <td> Compound </td> <td> KE: PPARα, Activation </td> <td> KE: TSPO, Decrease </td> <td> species </td> <td> Details </td> <td> References </td> </tr> <tr> <td> Phthalate (DBP) </td> <td> EC50=30μM </td> <td> n.a. </td> <td> human </td> <td> Monobenzyl phthalate (MBzP) metabolite of DBP </td> <td> (Hurst and Waxman 2003) </td> </tr> <tr> <td> Phthalate (DBP) </td> <td> EC50=21μM * EC50=63μM** </td> <td> LOEC=100 μM </td> <td> rodent </td> <td> *MBzP **mono-sec-butyl phthalate (MBuP) metabolite of DBP </td> <td> (Hurst and Waxman 2003),(Gazouli 2002) </td> </tr> <tr> <td> Phthalate (DEHP) </td> <td> LOEC=30μM </td> <td> LOEC=300 mg/kg /d   </td> <td> rodent </td> <td> MEHP metabolite of DEHP </td> <td> (Bility et al. 2004), (Gazouli 2002), (Borch et al. 2006) </td> </tr> <tr> <td> Phthalate (DEHP) </td> <td> Ki=15µM; EC50 = 3.2μM </td> <td> n.a. </td> <td> human </td> <td> MEHP metabolite of DEHP </td> <td> (Lapinskas et al. 2005), (Hurst and Waxman 2003) </td> </tr> <tr> <td> Bezafibrate </td> <td> EC50=55μM </td> <td> LOEC=100 μM </td> <td> rodent </td> <td> TSPO decreased by 80% in MA-10 Leydig cells </td> <td> (Willson et al. 2000), (Gazouli 2002) </td> </tr> <tr> <td> WY-14,643 </td> <td> EC50=0.00027μM </td> <td> LOEC=50 mg/kg/d </td> <td> rodent </td> <td>   </td> <td> (Pinelli et al. 2005), (Gazouli 2002) </td> </tr> </tbody> </table> Table 1. Summary table of empirical support for this KER. ED50 - half maximal effective dose, LOEC-lowest observed effect concentration, Bis(2-ethylhexyl) phthalate (DEHP), Dibutyl phthalate (DBP), WY-14,643 and Bezafibrate ligands of PPARα, n.a.- not available

The exact mechanisms of this relationship are not known.   Treatment of adult mice with PPARα activator (DEHP or WY-14,643) resulted in reduced levls of circulating testosterone and testis TSPO mRNA, consistent with the in vitro effects (Gazouli 2002). In contrast, liver TSPO mRNA levels have been increased, indicating a tissue-specific regulation of TSOP expression by PPARα activator (Gazouli 2002). In the PPARα-null mice, compared with the wild-type controls, circulating testosterone levels were decreased suggesting a positive constitutive role for PPARα in maintaining Leydig cell steroid formation. Surprisingly, treatment of the PPARα-null mice with PPARα activators (DEHP and WY-14,643) restored testosterone formation and TSPO mRNA returned to normal levels, suggesting PPARα-independent pathways might be involved in the regulation of TSPO genes and steroidogenesis (Gazouli 2002). In support of this hypothesis, an other study demonstrated that part of the toxic effect of phthalate (DEHP) on testis was retained in PPARα-null mice (Ward et al. 1998). There is some evidence involving additional PPARs in transcriptional regulation of TSPO: <ul> <li>PPARβ/δ (Campioli et al. 2011);</li> <li>PPARγ isoform was also detected in testes (Boberg et al. 2008) and it was reduced by treatment of DEHP in parallel with the reduction of TSPO regulation (Borch et al. 2006).</li> </ul>   A genomic study does not support the hypothesis that activation of PPARα/γ pathways is involved in the effects of phthalates on sexual differentiation of the male rat, as Wy-14,643 (PPARα activator) has no effect on testosterone production and the PPARγ isoform has not been detected in testes at gestation day 14-18 (Hannas et al. 2012). Differential patterns of TSPO expression in the foetal rat testis have been observed upon phthalate (DBP) treatment, whereas TSPO mRNA up-regulated protein levels were decreased in Leydig cells (Lehmann et al. 2004).

High Low Low

See Table 1.

#<Reference::ActiveRecord_Associations_CollectionProxy:0x00005eb8a2dda1a8> AOPWiki 2016-11-29T18:41:33

2016-12-02T10:13:35

<upstream-id>a7cf349d-e09e-48ba-b1a2-a8c0f53586fe</upstream-id> <downstream-id>4535a791-f011-4a5b-874f-1cbd65c4a100</downstream-id> Impairment in the normal development of the male reproductive tract (e.g. genital abnormality and/or cryptorchidism) can impact on fertility later in life.

Hypospadias next to cryptorchidism belongs to the most common male reproductive disorders that manifest at birth and may have a common origin in foetal life (Skakkebaek, Rajpert-De Meyts, and Main 2001) and are associated with decreased fertility (Thorup et al. 2010).

Human Asklund et al that semen quality was reduced in men with hypospadias and additional genital disorders, predominately cryptorchidism (Asklund et al. 2010). In another study by Bracka, 25% of 41 hypospadias patients including 26 patients also with cryptorchidism had a lower sperm density (Bracka 1989). Men with a history of cryptorchidism have an increased risk of infertility (Thorup et al. 2010). Eisenberg et al. found shorter AGD among infertile men as compared with fertile men (Eisenberg et al. 2011). Rat In rodents in utero exposure to agents known to disrupt androgen mediated pathways corrupts normal male genital development with a decrease in genital length (ie phallus length, AGD) and impaired testosterone and sperm production (Macleod et al. 2010), (Cowin et al. 2010), including exposure to phthalates (NTP 2005).

High High Low

Human and Rat see "Empirical Support for Linkage"

#<Reference::ActiveRecord_Associations_CollectionProxy:0x00005eb8a2e3b9a8> AOPWiki 2016-11-29T18:41:34

2016-12-02T10:21:50

<upstream-id>2297c412-0985-40e3-b66b-9c93906abb9d</upstream-id> <downstream-id>a7cf349d-e09e-48ba-b1a2-a8c0f53586fe</downstream-id> Male sexual differentiation in general depends on testosterone (T), dihydrotestosterone (DHT), and the expression of androgen receptors by target cells (Manson and Carr 2003). Disturbances in the balance of this endocrine system by either endogenous or exogenous factors may lead to male reproductive tract, malformations (e.g. hypospadias, cryptorchidism). Reduction in T levels during foetal development subsequently lower levels of its metabolite DHT lead also to impaired growth of the perineum with reduced anogential distance (AGD) (Bowman et al. 2003).

Hypospadias The role of foetal androgens (T and DHT) is crucial for the development of the male reproductive tract especially during the first trimester of pregnancy. Androgens regulate masculinization of external genitalia. T is necessary for stabilization and differentiation of the Wolffian structures (e.g., the epididymis, vas deferens and seminal vesicles) and also for normal development of the foetal testes; DHT, produced locally from testosterone, is required for normal development of the genital tubercle and urogenital sinus into the external genitalia and prostate (Murashima et al. 2015). Therefore any defects in androgen biosynthesis, metabolism or action during development can cause hypospadias (Rey et al. 2005). The environmental factors with anti-androgenic activity may alter the complex regulation of male sex differentiation during foetal life (Kalfa et al., 2008). Although the cause in most cases is unknown, hypospadias has been associated with aberrant androgen signalling during development (Wolf et al. 1999). The aetiology of this frequent malformation has not been elucidated despite intensive investigation (Kalfa, Philibert, and Sultan 2009). Hypospadias thus appears at the crossroads of genetic, endocrine and environmental mechanisms (Kalfa, Philibert, and Sultan 2009). Anogential distance (AGD) The anogenital distance (AGD) is a sexual dimorphism that results from the sex difference in foetal androgen (DHT) levels (Rhees et al., 1997). The AGD is a marker of perineal growth and caudal migration of the genital tubercle. It is androgen-dependent in male rodents (Bowman et al. 2003). During development, androgens stimulate the growth of the perineal region between the sex papilla and the anus, resulting in an increased AGD in male offspring (Bowman et al. 2003). The AGD, is believed to be a biomarker of prenatal androgen exposure in many species, and in humans it has been associated with several adverse reproductive health outcomes in adults. AGD reflects foetal androgen exposure only within a discrete masculinization programming window (MPW), during which development of male reproductive organs is taking place (Wolf et al. 1999), (Macleod et al. 2010). Cryptorchidism Undescended testis (UDT), also called cryptorchidism, is the most frequent congenital malformation in males, occurring in 2–5% of full-term male births (Hadziselimovic 2002) (Brucker-Davis et al. 2008). Testosterone and insulin-like peptide 3 (INSL3) are two major Leydig cell hormones that regulate physiological testicular descent during foetal development (Virtanen et al. 2007). Most cases of cryptorchidism remain idiopathic but epidemiological and experimental studies have suggested a role of both genetic and environmental factors. Studies e. g.(Gray et al. 2000) have shown that maternal administration of certain chemicals (phthalate esters) during the critical intrauterine period of sexual differentiation alters development of both androgen- and insl3-dependent tissues. Cryptorchidism is shown to be linked with increased risk of hypofertility and testicular cancer (Fénichel et al. 2015).

Hypospadias Reduced T production during the male rat development lead to hypospadias (Mylchreest, Cattley, and Foster 1998), (Mylchreest 2000), (Gray et al. 2000), (Parks 2000), (Wilson et al. 2004); this outcome is associated with Leydig cell function. Anogential distance (AGD) The decreased AGD has been associated with the perturbation of androgen-mediated development of the reproductive tract in rat males which were exposed to anti-androgens in utero (Wolf et al. 1999), (McIntyre et al. 2000), (McIntyre, Barlow, and Foster 2001). Several studies have demonstrated that exposure to phthalates results in decreased anogenital distance in human males (S. H. Swan et al. 2015), (Bornehag et al. 2015), presumably due to lowered testosterone levels (Suzuki et al. 2012), (Jurewicz and Hanke 2011), (Shanna H Swan et al. 2005), for details see Table 1. <table class="wikitable" id="Event608"> <tbody> <tr> <td> </td> <td> </td> <td> </td> <td> KE: testosterone, reduction </td> <td> KE: AGD, decreased </td> <td> </td> <td> </td> </tr> <tr> <td> Compound </td> <td> Species and strain: Doses: duration, [measurement day] </td> <td> Effect level </td> <td> </td> <td> </td> <td> Details </td> <td> References </td> </tr> <tr> <td> Phthalates (DEHP) </td> <td> rat, 0, 10, 30, 100, or 300 mg /kg bw/day : 7- 21 GD, [GD 21] </td> <td> LOEL=-300 mg /kg bw/day </td> <td> Testicular testosterone levels, reduction, no change plasma testosterone </td> <td> </td> <td> </td> <td> (Borch et al. 2006) </td> </tr> <tr> <td> Phthalates (DEHP) </td> <td> rat, GD 3- PND 21 </td> <td> LOEL=750 mg /kg bw/day </td> <td> </td> <td> Anogenital distance decreased </td> <td> Gestational and lactational </td> <td> (Moore et al. 2001) </td> </tr> <tr> <td> Phthalates (DEHP) </td> <td> rat </td> <td> LOEL=750 mg /kg bw/day </td> <td> reduction in T production, and reduced testicular and whole-body T levels in fetal and neonatal male </td> <td> Anogenital distance decreased </td> <td> Exposure from (GD) 14 to postnatal day (PND) 3, AGD reduced by 36% in exposed male </td> <td> (Parks 2000) </td> </tr> <tr> <td> Phthalates (DEHP) </td> <td> rat </td> <td> LOEL=15 mg /kg bw/day </td> <td> Decreased testosterone levels </td> <td> Effects on Sperm production </td> <td> </td> <td> (Andrade et al. 2006) </td> </tr> <tr> <td> Phthalates (DEHP) </td> <td> rat </td> <td> LOEL=5 mg /kg bw/day </td> <td> </td> <td> chryptorchidism </td> <td> </td> <td> (Andrade et al. 2006) </td> </tr> <tr> <td> Phthalates (DEHP) </td> <td> rat </td> <td> LOEL=1.215 mg /kg bw/day </td> <td> Decreased testosterone levels </td> <td> Effects on Sperm production </td> <td> </td> <td> (Andrade et al. 2006) </td> </tr> <tr> <td> </td> <td> </td> <td> </td> <td> </td> <td> </td> <td> </td> <td> </td> </tr> <tr> <td> Phthalates (DnHP) </td> <td> rat </td> <td> LOEL=250 mg /kg bw/day </td> <td> </td> <td> Anogenital distance decreased </td> <td> </td> <td> (Saillenfait, Gallissot, and Sabaté 2009) </td> </tr> <tr> <td> Phthalates (DEHP) </td> <td> rat </td> <td> LOEL=300 mg/kg/day </td> <td> </td> <td> Anogenital distance decreased </td> <td> </td> <td> (Jarfelt et al. 2005) </td> </tr> <tr> <td> Phthalates (DBP) </td> <td> rat </td> <td> LOEL=500 mg/kg/day </td> <td> </td> <td> Anogenital distance decreased </td> <td> throughout pregnancy until postnatal day 20 </td> <td> (Mylchreest, Cattley, and Foster 1998) </td> </tr> <tr> <td> Phthalates dicyclohexyl phthalate (DCHP) </td> <td> rat </td> <td> LOEL=6000 ppm </td> <td> </td> <td> Anogenital distance decreased </td> <td> F1 and F2 6000 ppm, and decrease of AGD and appearance of areola mammae were observed in the F1 male 6000 ppm and F2 male receiving doses of 1200 ppm or 6000 ppm. </td> <td> (Hoshino, Iwai, and Okazaki 2005) </td> </tr> <tr> <td> Phthalate (DBP) </td> <td> rat </td> <td> LOEL=500 mg/kg/day </td> <td> intratesticular testosterone levels, reduction (by nearly 90%) </td> <td> Anogenital distance decreased </td> <td> GD 12 -20, examinations on GD20 </td> <td> (Johnson et al. 2011) </td> </tr> <tr> <td> Phthalates (MEHP) </td> <td> Human </td> <td>   </td> <td> </td> <td> Anogenital index decreased </td> <td> Urine concentration of phthalates metabolites MEHP associated with reduced AGI, suggestive association of sum of DEHP metabolites with reduced AGI </td> <td> (Suzuki et al. 2012), </td> </tr> <tr> <td> Phthalates (MEP), (MBP), (MBzP), (MiBP) </td> <td> human </td> <td>   </td> <td> </td> <td> Urinary concentrations of phthalate metabolites inversely related to AGI </td> <td> 134 boys 2-36 months of age </td> <td> (Shanna H Swan et al. 2005) </td> </tr> <tr> <td> Phthalates (MEHP, MBP) </td> <td> human </td> <td> MBP= 78.4 ng/mL* in urine; 85.2 ng/mL* in amniotic fluid MEHP =24.9 ng/mL * in urine; 22.8 ng/mL* in amniotic fluid </td> <td> </td> <td> In girls, decreased AGD in relation to amniotic fluid levels of MBP and MEHP. No associations found in boys </td> <td> Amniotic fluid and urine concentrations of phthalate metabolites </td> <td> (Huang et al. 2009) </td> </tr> </tbody> </table> Table 1 Summary of experimental evidence for the KER. Lowest-Observed-Effect-Level (LOEL), Dibutyl phthalate (DBP), diisobutyl phthalate (DiBP), di-n-hexyl phthalate (DnHP), monobutyl phthalate (MBP); Bis(2-ethylhexyl) phthalate (DEHP) mono-(2-ethylhexyl) phthalate (MEHP); monoethyl phthalate (MEP), monobenzyl phthalate (MBzP), monoisobutyl phthalate (MiBP); anogenital index (AGI)-weight normalised index of AGD median.

Hypospadias Epidemiological studies have demonstrated an association between foetal estrogen exposure and hypospadias (Klip et al. 2002), (Brouwers et al. 2007). However, the molecular mechanism underlying this association is unknown (Wang and Baskin 2008), (Blaschko, Cunha, and Baskin 2012). Anogential distance (AGD) Study by Huang et al did not found associations with the phthalates metabolites in the male AGD, however in females in relation to amniotic fluid levels of MBP and MEHP (Huang et al. 2009).

High High Low

Hypospadias Maternal exposure to estrogenic and antiandrogenic endocrine disrupting compounds has been implicated in increased risk of cryptorchidism and hypospadias in human male offspring without statistical significance (Morales-Suárez-Varela et al. 2011). AGD Across numerous species, including humans, AGD is longer in males compared to females; for review see (Barrett et al. 2014).

#<Reference::ActiveRecord_Associations_CollectionProxy:0x00005eb8a2ea2400> AOPWiki 2016-11-29T18:41:34

2016-12-02T10:20:16

PPARα activation in utero leading to impaired fertility in males

PPARα activation leading to impaired fertility

Arthur Author

Malgorzata Nepelska, Elise Grignard, Sharon Munn, Systems Toxicology Unit, Institute for Health and Consumer Protection, Joint Research Centre, European Commission, Via E. Fermi 2749, I-21027 Ispra, Varese, Italy Corresponding author: sharon.munn@ec.europa.eu; elise.grignard@ec.europa.eu

BY-SA

EAGMST Under Review

1.21

1.0

This AOP links the activation of Peroxisome Proliferator Activated Receptor α (PPARα) to the developmental/reproductive toxicity in male. The development of this AOP relies on evidence collected from rodent models and incorporates human mechanistic and epidemiological data. The PPARα is a ligand-activated transcription factor that belongs to the nuclear receptor family, which also includes the steroid and thyroid hormone receptors. The hypothesis that PPARα action is the mechanistic basis for effects on the reproductive system arises from limited experimental data indicating relationships between activation of this receptor and impairment of steroidogenesis leading to reproductive toxicity. PPARs play important roles in the metabolic regulation of lipids, of which cholesterol, in particular, being a precursor of steroid hormones, makes the link between lipid metabolism to effects on reproduction. The key events in the pathway comprise the activation of PPARα, followed by the disruption cholesterol transport in mitochondria, impairment of hormonal balance which leads to malformation of the reproductive tract in males which may lead to impaired fertility. The PPARα-initiated AOP to rodent male developmental toxicity is a first step for structuring current knowledge about a mode of action which is neither AR-mediated nor via direct steroidogenesis enzymes inhibition. In the current form the pathway lays a strong basis for linking an endocrine mode of action with an apical endpoint, a prerequisite requirement for the identification of endocrine disrupting chemicals. This AOP is complemented with a structured data collection which will serve as the basis for further quantitative development of the pathway.

Fibrates are ligands of PPARα (Staels et al. 1998). Phthalates MHEP (CAS 4376-20-9) directly binds in vitro to PPARα (Lapinskas et al. 2005) and activates this receptor in transactivation assays PPARα (Lapinskas et al. 2005), (Maloney and Waxman 1999), (Hurst and Waxman 2003), (Bility et al. 2004), (Lampen, Zimnik, and Nau 2003), (Venkata et al. 2006) ]. DEHP (CAS 117-81-7) has not been found to bind and activate PPARα (Lapinskas et al. 2005), (Maloney and Waxman 1999). However, the recent studies shown activation of PPARα (ToxCastTM Data). Notably, PPARα are responsive to DEHP in vitro as they are translocated to the nucleus (in primary Sertoli cells) (Dufour et al. 2003), (Bhattacharya et al. 2005). Expression of PPARα [mRNA and protein] has been reported to be also modulated by phthtalates: (to be up-regulated in vivo upon DEHP treatment (Xu et al. 2010) and down-regulated by Diisobutyl phthalate (DiBP) (Boberg et al. 2008)). Perfluorooctanoic Acid (PFOA) is known to activate PPARα (Vanden Heuvel et al. 2006). Organotin Tributyltin (TBT) activates all three heterodimers of PPAR with RXR, primarily through its interaction with RXR (le Maire et al. 2009)

Under REACH, information on reproductive toxicity is required for chemicals with an annual production/importation volume of 10 metric tonnes or more. Standard information requirements include a screening study on reproduction toxicity (OECD TG 421/422) at Annex VIII (10-100 t.p.a), a prenatal developmental toxicity study (OECD 414) on a first species at Annex IX (100-1000 t.p.a), and from March 2015 the OECD 443(Extended One-Generation Reproductive Toxicity Study) is reproductive toxicity requirement instead of the two generation reproductive toxicity study (OECD TG 416). If not conducted already at Annex IX, a prenatal developmental toxicity study on a second species at Annex X (≥ 1000 t.p.a.). Under the Biocidal Products Regulation (BPR), information is also required on reproductive toxicity for active substances as part of core data set and additional data set (EU 2012, ECHA 2013). As a core data set, prenatal developmental toxicity study (EU TM B.31) in rabbits as a first species and a two-generation reproduction toxicity study (EU TM B.31) are required. OECD TG 443 (Extended One-Generation Reproductive Toxicity Study) shall be considered as an alternative approach to the multi-generation study.) According to the Classification, Labelling and Packaging (CLP) regulation (EC, 200; Annex I: 3.7.1.1): a) “reproductive toxicity” includes adverse effects on sexual function and fertility in adult males and females, as well as developmental toxicity in the offspring; b) “effects on fertility” includes adverse effects on sexual function and fertility; and c) “developmental toxicity” includes adverse effects on development of the offspring.

In regulatory hazard identification and risk assessment of chemicals malformations of male genitalia are considered as a chemically induced adverse outcome that is used for risk assessment and management purposes. The prenatal developmental toxicity study (TG 414) is the method for examining embryo-foetal toxicity as a consequence of exposure during pregnancy. Parental and offspring growth, development and viability are the relevant endpoints in generation studies (OECD TG 415/416/443). These guidelines are implemented in a number of occasions where the reproductive /developmental toxicity have to be assessed in order to comply with relevant EU regulations. Under REACH, information on reproductive toxicity is required for chemicals with an annual production/importation volume of 10 metric tonnes or more. Standard information requirements include a screening study on reproduction toxicity (OECD TG 421/422) at Annex VIII (10-100 t.p.a), a prenatal developmental toxicity study (OECD 414) on a first species at Annex IX (100-1000 t.p.a), and from March 2015 the OECD 443(Extended One-Generation Reproductive Toxicity Study) is reproductive toxicity requirement instead of the two generation reproductive toxicity study (OECD TG 416). If not conducted already at Annex IX, a prenatal developmental toxicity study on a second species at Annex X (≥ 1000 t.p.a.). Under the Biocidal Products Regulation (BPR), information is also required on reproductive toxicity for active substances as part of core data set and additional data set (EU 2012, ECHA 2013). As a core data set, prenatal developmental toxicity study (EU TM B.31) in rabbits as a first species and a two-generation reproduction toxicity study (EU TM B.31) are required. OECD TG 443 (Extended One-Generation Reproductive Toxicity Study) shall be considered as an alternative approach to the multi-generation study. According to the Classification, Labelling and Packaging (CLP) regulation (EC, 200; Annex I: 3.7.1.1): a) “reproductive toxicity” includes adverse effects on sexual function and fertility in adult males and females, as well as developmental toxicity in the offspring; b) “effects on fertility” includes adverse effects on sexual function and fertility; and c) “developmental toxicity” includes adverse effects on development of the offspring. AGD is a reproductive endpoint, assessment of AGD is mandatory in OECD TG 443, 415/416 (OECD 2012).

adjacent

Not Specified

Moderate adjacent

Not Specified

Moderate adjacent

Not Specified

High adjacent

Not Specified

Low adjacent

Not Specified

High non-adjacent

Not Specified

Moderate non-adjacent

Not Specified

Low non-adjacent

Not Specified

High

High Male

High

Development

Moderate Low Moderate

Biological plausibility, coherence, and consistency of the experimental evidence In the presented AOP it is hypothesized that the key events occur in a biologically plausible order prior to the development of adverse outcomes. The PPARα activators have been shown to alter steroidogenesis and impair reproduction [see reviews (Corton and Lapinskas 2004), (Latini et al. 2008), (David 2006)]. However, there are some conflicting reports on the involvement of PPARα as MIE of the proposed AOP (Johnson, Heger, and Boekelheide 2012), (David 2006). The biochemistry of steroidogenesis and the predominant role of the gonad in synthesis of the sex steroids are well established. Steroidogenesis is a complex process that is dependent on the availability of cholesterol in mitochondria. Perturbation of genes responsible for cholesterol transport and steroidogenic enzyme activities in the Leydig cell will lead to a decrease in testicular testosterone (T) production. As a consequence, androgen-dependent tissue differentiation/development is adversely affected. The physical manifestation of this event may be reproductive tract malformation and possibly leads to impaired fertility. Concordance of dose-response relationships This is a qualitative description of the pathway; the currently available studies provide quantitative information on dose-response relationships only partially. Experimental data are based on exposure to phthalates and indicate that key events of this pathway occur at similar dose levels. The effects of altered gene expression levels that are responsible for the cholesterol transport into the Leydig cells were shown at >50 mg/kg/bw, a dose at which foetal T was decreased and anatomical malformations (hypospadias) were produced (Mylchreest, Cattley, and Foster 1998), (Mylchreest 2000), (Akingbemi 2001), (Lehmann et al. 2004). Tailored experiments are required for the exploration of quantitative linkages. Temporal concordance among the key events and the adverse outcome This AOP bridges two life stages: the AOs are results of the chemical exposure during a critical prenatal period for male development, the masculinization programming window (MPW), within which androgens must act to ensure the correct development of the male reproductive tract (Welsh et al. 2008). Therefore, the AOP focuses on the exposures within the MPW (15.5–18.5 GD days in rats). The temporal relationship of exposure to gestation day has been investigated using phthalates and it has been demonstrated that the gestational timing of exposure is important for the production of the adverse effects on the male reproductive tract (reviewed in (Ema 2002)). Moreover, the temporal relationship between alterations of gene expression and changes in testosterone production has been investigated for phthalates (DBP) (Lehmann et al. 2004), (Thompson et al. 2005). Initial increases in gene expression are followed by decreases in the expression of genes which are associated with steroidogenesis. The observed decreased steroidogenesis and subsequent decrease in testosterone levels is well established as precursors to anatomical changes in the developing male reproductive tract. Thus, those key events of gene expression are temporally consistent with subsequent events, however complete temporal concordance studies are missing. Strength, consistency, and specificity of association of adverse effect and initiating event The strength of the chosen chemical initiators as PPARα activators was shown to partially correlate with their ability to act as a male reproductive toxicant (Corton and Lapinskas 2004). The presented key events leading to a decrease in steroidogenesis are plausible and consistent with the observed effects. There is coherence between decreased testosterone synthesis and malformations. Alternative mechanism(s) or MIE(s) described which may contribute/synergise the postulated AOP The inhibitory effect of PPARα activation seems to be attributable to an impairment of the multistep process of cholesterol mobilization, transport into mitochondria, and steroidogenesis leading to impaired androgens production. Therefore, it is plausible that several other mechanisms may contribute to/synergise with this AOP. For example, activation of other isoforms of PPARs (PPARβ/δ or/and γ) is hypothesised to be relevant for the pathway (Lapinskas et al. 2005), (Shipley and Waxman 2004). PPARγ activation Opposing effects of PPARγ ligands (thiazolidinediones, TZD) on androgen levels and/or production in male humans (Dunaif et al. 1996), (Bloomgarden, Futterweit, and Poretsky 2001), (Vierhapper, Nowotny, and Waldhäusl 2003) and animal models have been described (Kempná et al. 2007), (Gasic et al. 1998), (Mu et al. 2000), (Arlt, Auchus, and Miller 2001), (Minge, Robker, and Norman 2008), (Gasic et al. 2001), (Veldhuis, Zhang, and Garmey 2002). In rats no effects of PPARγ ligand (rosiglitazone) on production or total circulating testosterone levels were seen (Boberg et al. 2008), however a decrease in basal or induced testosterone production occurred in the Leydig cells of rosiglitazone-treated rats (Couto et al. 2010). Moreover, there are contradicting reports as to the presence of PPARγ in the foetal testes (Hannas et al. 2012). Few others transcription factors involved in regulation of lipid metabolism are hypothesized to mediate effects on fetal Leydig cell gene expression like sterol regulatory element–binding protein (SREBP) (Lehmann et al. 2004), (Shultz 2001), CCAAT/enhancer-binding protein-β (CEBPB) (Kuhl, Ross, and Gaido 2007) or NR5A1 (also known as steroidogenic factor 1; Sf1) (Borch et al. 2006). The downstream effects in the pathway might be due to the constellation of earlier events in fetal Leydig cells leading to decrease testosterone production and connected adverse outcomes. Alternative/synergistic MIEs relating to this pathway are hypothesised in the KER description. At present there are no strong views on the other possible MIEs.   Uncertainties, inconsistencies and data gaps The major uncertainty in this AOP is the functional relationship between (MIE) PPARα activation leading to cholesterol transport reduction; possible mechanisms have been proposed but strong experimental support is missing and some conflicting data are reported. The dose response data to support this relationship are lacking. Studies exploring the role of PPARα using PPARα knockout mice showed that prenatal exposure to phthalates caused developmental malformations in both wild-type and PPARα knockout mice, thus suggesting a PPARα-independent mechanism. However, it is difficult to draw any conclusion on the role of PPARα in phthalate-related reproductive toxicity since the intrauterine administration of phthalate (DEHP) occurred before the critical period of reproductive tract differentiation (Peters et al. 1997). Intrauterine DEHP-treated PPARα-deficient mice, developed delayed testicular, renal and developmental toxicities, but no liver toxicity, compared to wild types, thus confirming the early observation by Lee et al. about the PPARα dependence of liver response and, more importantly, indicating that DEHP may induce reproductive toxicity through both PPARα-dependent and -independent mechanism (Ward et al. 1998). PPARα-independent reproductive toxicity observed by Ward et al. may conceivably be mediated by other PPAR isoforms, such as PPARβ and PPARγ, or by a non-receptor-mediated organ-specific mechanism (Barak et al. 1999). Other studies showed that the administration of DEHP resulted in milder testis lesions and higher testosterone levels in PPARα-null mice than in wild-type mice (Gazouli 2002). A more recent report, investigating the role of PPARα, showed decreased testosterone levels in PPARα(−/−) null control mice, suggesting a positive constitutive role for PPARα in maintaining Leydig cell steroid formation (Borch et al. 2006). Inconsistencies Genomic studies by Hannas et al., demonstrated that PPARα agonist Wy-14,643, did not reduce foetal testicular testosterone production following gestational day 14–18 exposure, suggesting that the antiandrogenic activity of phthalates is not PPARα mediated (Hannas et al. 2012). Similarly, recent report by Furr et al. did not observe testosterone decrease after administration of Wy-14,643 in rat ( ex vivo) (Furr et al. 2014). Data Gaps: Complete/pathway driven studies to investigate the effects of PPARs and their role in male reproductive development are lacking. For establishing a solid quantitative and temporal coherent linkage, mode of action framework analysis for PPAR α mediated developmental toxicity are needed. This approach has been applied for the involvement of PPAR α in liver toxicity (Corton et al. 2014), (Wood et al. 2014).   Empirical information on dose-response relationships between the KEs, are not available, however there are solid empirical data that would inform a computational, predictive model for reproductive toxicity via PPARα activation. Life Stage Applicability This AOP is relevant for developing (prenatal) male. Taxonomic Applicability The experimental support for the pathway is mainly based on the animal (rat studies). Conflicting reports comes from the studies on mouse. Studies in mice report contradictory results. Recently, studies by Furr et al revealed that fetal T production can be inhibited by exposure to a phthalates in utero (CD-1 mice), but at a higher dose level than required in rats and causing systemic effects (Furr et al. 2014). However there are some earlier reports that chronic dietary administration of phthalates produces adverse testicular effects and reduces fertility in CD-1 mice (Heindel et al. 1989) Sex Applicability This AOP applies to males only.

<table border="1" style="border-collapse:collapse; font-size:75%"> <tbody> <tr> <td> KRs </td> <td> Essentiality - KEs </td> <td> level of confidence </td> <td> </td> <td> </td> <td>   </td> </tr> <tr> <td> PPAR alpha, Activation   </td> <td> PPAR alpha activation was found to indirectly alter the expression of genes involved in cholesterol transport in mitochondria </td> <td> very weak </td> </tr> <tr> <td> TSPO; StAR decrease </td> <td> Alterations in the amount of cholesterol transport proteins in mitochondria impact on the levels of substrate for steroid hormones production. </td> <td> weak </td> </tr> <tr> <td> cholesterol transport in mitochondria, reduction </td> <td> Production of steroid hormones depends on the availability of cholesterol to the enzymes in the mitochondrial matrix. Decreasing the amount of cholesterol inside the mitochondria will result in a diminished amount of substrate for hormone (testosterone) synthesis. </td> <td> moderate </td> </tr> <tr> <td> Testosterone synthesis, reduction </td> <td> The gonads are generally considered the major source of circulating androgens. Consequently, if testosterone synthesis by testes is reduced, testosterone concentrations would be expected to decrease unless there are concurrent reductions in the rate of T catabolism. </td> <td> strong </td> </tr> <tr> <td> Testosterone, reduction </td> <td> Male sexual differentiation in general depends on androgens (T, dihydrotestosterone (DHT)), disturbances in the balance of this endocrine system by either endogenous or exogenous factors lead to male reproductive tract malformation. </td> <td> strong </td> </tr> <tr> <td> Male reproductive tract malformations </td> <td> Androgens regulate masculinization of the external genitalia. Therefore any defects in androgen biosynthesis, metabolism or action during foetal development can reproductive tract malformation. </td> <td> strong </td> </tr> <tr> <td> Fertility, impaired </td> <td> Impaired fertility is the endpoint of reproductive toxicity </td> <td> strong </td> </tr> </tbody> </table>

<table border="1" style="border-collapse:collapse; font-size:75%"> <tbody> <tr> <td> KERs </td> <td> Biological plausibility </td> <td> Level of confidence </td> <td colspan="3"> Empirical Support </td> <td> Level of confidence </td> <td> Inconsistencies/Uncertainties   </td> </tr> <tr> <td> </td> <td> </td> <td> </td> <td> Dose-response </td> <td> Temporality </td> <td> Incidence </td> <td> </td> <td> </td> </tr> <tr> <td> PPAR alpha, Activation => Translator protein (TSPO), Decrease   </td> <td> There is functional relationship between PPARα activation and reduction in TSPO levels. </td> <td> Very Weak </td> <td> <ul> <li>KEs occur at similar dose levels</li> </ul> </td> <td> <ul> <li>occurrence of the key events at similar dose and time point</li> <li>Support for solid temporal relationship is lacking</li> </ul> </td> <td>   </td> <td> Very Weak </td> <td> Some conflicting data </td> </tr> <tr> <td> PPAR alpha, Activation => Steroidogenic acute regulatory protein (StAR), decrease </td> <td> There is functional relationship between PPARα activation and reduction in StAR levels. </td> <td> Weak </td> <td> <ul> <li>KEs occur at similar dose levels</li> </ul> </td> <td> <ul> <li>Support for solid temporal relationship is lacking.</li> </ul> </td> <td>   </td> <td> Weak </td> <td> Some conflicting data </td> </tr> <tr> <td> Steroidogenic acute regulatory protein (StAR), decrease and Translator protein (TSPO), Decrease => cholesterol transport in mitochondria, reduction   </td> <td> Changes in cholesterol transport proteins can generally be assumed to directly impact levels of cholesterol transport. </td> <td> Moderate </td> <td> <ul> <li>KEs occur at similar dose levels</li> </ul> </td> <td> <ul> <li>Support for solid temporal relationship is lacking.</li> </ul> </td> <td>   </td> <td> Moderate </td> <td> Some conflicting data </td> </tr> <tr> <td> cholesterol transport in mitochondria, reduction => testosterone synthesis, reduction </td> <td> Decreasing the amount of cholesterol inside the mitochondria (e. g by decreasing the expression of enzymes like StAR or TSOP) will result in a diminished amount of substrate for hormone (testosterone) synthesis. </td> <td> Moderate </td> <td> <ul> <li>KEs occur at similar dose levels</li> </ul> </td> <td> <ul> <li>occurrence of the key events at similar dose and time point</li> <li>Support for solid temporal relationship is lacking.</li> </ul> </td> <td>   </td> <td> Moderate </td> <td> Some conflicting data </td> </tr> <tr> <td> testosterone, reduction => Male reproductive tract malformations </td> <td> Reduction in testosterone (T) levels produced in the Leydig cell subsequently lowers the availability of its metabolite; Dihydrotestosterone (DHT).that regulates masculinization of external genitalia. Therefore any defects in androgen biosynthesis, metabolism or action during development can cause male reproductive tract malformation. </td> <td> Strong </td> <td> <ul> <li>KEs occur at similar dose levels</li> </ul> </td> <td> <ul> <li>occurrence of the key events at similar dose and time point</li> <li>Support for solid temporal relationship is lacking.</li> </ul> </td> <td>   </td> <td> Strong </td> <td> No conflicting data </td> </tr> <tr> <td> Male reproductive tract malformations => Fertility, impaired </td> <td> Male reproductive tract malformations (congenital malformation of male genitalia) comprise any physical abnormality of the male internal or external genitalia present at birth, which may impair on fertility later in life </td> <td> Moderate </td> <td> <ul> <li>KEs occur at similar dose levels</li> </ul> </td> <td> <ul> <li>occurrence of the key events at similar dose and time point</li> </ul> Support for solid temporal relationship is lacking. </td> <td>   </td> <td> Moderate </td> <td> No conflicting data </td> </tr> </tbody> </table>   Table 1 Weight of Evidence Summary Table. The underlying questions for the content of the table: Dose-response Does the empirical evidence support that a change in KEup leads to an appropriate change in KEdown?; Does KEup occur at lower doses and earlier time points than KE down and is the incidence of KEup > than that for KEdown?: Incidence Is there higher incidence of KEup than of KEdown?; Inconsistencies/Uncertainties: Are there inconsistencies in empirical support across taxa, species and stressors that don’t align with expected pattern for hypothesized AOP? n.a not applicable

This AOP is qualitatively described; however it contains also data that may be used for further development of quantitative description.

1. The AOP describes a pathway which allows for the detection of sex steroid--related endocrine disrupting modes of action, with focus on the identification of substances which affect the reproductive system. In the current form the pathway lays a strong basis for linking endocrine mode of action with an apical endpoint, a prerequisite requirement for identification of endocrine disrupting chemicals (EDC). EDCs require specific evaluation under REACH (1907/2006, Registration, Evaluation, Authorisation and Restriction of Chemicals (EU, 2006)), the revised European plant protection product regulation 1107/2009 (EU, 2009) and use of biocidal products 528/2012 EC (EU, 2012).Amongst other agencies the US EPA is also giving particular attention to EDCs (EPA, 1998). 2. This AOP structurally represents current knowledge of the pathway from PPARα activation to impaired fertility that may provide a basis for development (and interpretation) of strategies for Integrated Approaches to Testing Assessment (IATA) to identify similar substances that may operate via the same pathway related tosex steroids disruptionand effects on reproductive tract and fertility. This AOP forms the starting point on an AOP network mapping to modes of action for endocrine disruption. 3. The AOP could inform the development of quantitative structure activity relationships, read-across models, and/or systems biology models to prioritize chemicals for further testing.

Akingbemi, B. T. 2001. “Modulation of Rat Leydig Cell Steroidogenic Function by Di(2-Ethylhexyl)Phthalate.” Biology of Reproduction 65 (4) (October 1): 1252–1259. doi:10.1095/biolreprod65.4.1252. <a class="external free" href="http://www.biolreprod.org/content/65/4/1252.long" rel="nofollow" target="_blank">http://www.biolreprod.org/content/65/4/1252.long</a>. Arlt, W, R J Auchus, and W L Miller. 2001. “Thiazolidinediones but Not Metformin Directly Inhibit the Steroidogenic Enzymes P450c17 and 3beta -Hydroxysteroid Dehydrogenase.” The Journal of Biological Chemistry 276 (20) (May 18): 16767–71. doi:10.1074/jbc.M100040200. <a class="external free" href="http://www.ncbi.nlm.nih.gov/pubmed/11278997" rel="nofollow" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed/11278997</a>. Barak, Y, M C Nelson, E S Ong, Y Z Jones, P Ruiz-Lozano, K R Chien, A Koder, and R M Evans. 1999. “PPAR Gamma Is Required for Placental, Cardiac, and Adipose Tissue Development.” Molecular Cell 4 (4) (October): 585–95. <a class="external free" href="http://www.ncbi.nlm.nih.gov/pubmed/10549290" rel="nofollow" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed/10549290</a>. Bloomgarden, Z T, W Futterweit, and L Poretsky. 2001. “Use of Insulin-Sensitizing Agents in Patients with Polycystic Ovary Syndrome.” Endocrine Practice : Official Journal of the American College of Endocrinology and the American Association of Clinical Endocrinologists 7 (4): 279–86. doi:10.4158/EP.7.4.279. <a class="external free" href="http://www.ncbi.nlm.nih.gov/pubmed/11497481" rel="nofollow" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed/11497481</a>. Boberg, Julie, Stine Metzdorff, Rasmus Wortziger, Marta Axelstad, Leon Brokken, Anne Marie Vinggaard, Majken Dalgaard, and Christine Nellemann. 2008. “Impact of Diisobutyl Phthalate and Other PPAR Agonists on Steroidogenesis and Plasma Insulin and Leptin Levels in Fetal Rats.” Toxicology 250 (2-3) (September 4): 75–81. doi:10.1016/j.tox.2008.05.020. <a class="external free" href="http://www.ncbi.nlm.nih.gov/pubmed/18602967" rel="nofollow" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed/18602967</a>. Borch, Julie, Stine Broeng Metzdorff, Anne Marie Vinggaard, Leon Brokken, and Majken Dalgaard. 2006. “Mechanisms Underlying the Anti-Androgenic Effects of Diethylhexyl Phthalate in Fetal Rat Testis.” Toxicology 223 (1-2) (June 1): 144–55. doi:10.1016/j.tox.2006.03.015. <a class="external free" href="http://www.sciencedirect.com/science/article/pii/S0300483X0600165X" rel="nofollow" target="_blank">http://www.sciencedirect.com/science/article/pii/S0300483X0600165X</a>. Corton, J Christopher, Michael L Cunningham, B Timothy Hummer, Christopher Lau, Bette Meek, Jeffrey M Peters, James A Popp, Lorenz Rhomberg, Jennifer Seed, and James E Klaunig. 2014. “Mode of Action Framework Analysis for Receptor-Mediated Toxicity: The Peroxisome Proliferator-Activated Receptor Alpha (PPARα) as a Case Study.” Critical Reviews in Toxicology 44 (1) (January): 1–49. doi:10.3109/10408444.2013.835784. <a class="external free" href="http://www.ncbi.nlm.nih.gov/pubmed/24180432" rel="nofollow" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed/24180432</a>. Corton, J. Christopher, and Paula J Lapinskas. 2004. “Peroxisome Proliferator-Activated Receptors: Mediators of Phthalate Ester-Induced Effects in the Male Reproductive Tract?” Toxicological Sciences 83 (1) (October 13): 4–17. doi:10.1093/toxsci/kfi011. <a class="external free" href="http://www.ncbi.nlm.nih.gov/pubmed/15496498" rel="nofollow" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed/15496498</a>. Couto, Janaína A, Karina L A Saraiva, Cleiton D Barros, Daniel P Udrisar, Christina A Peixoto, Juliany S B César Vieira, Maria C Lima, Suely L Galdino, Ivan R Pitta, and Maria I Wanderley. 2010. “Effect of Chronic Treatment with Rosiglitazone on Leydig Cell Steroidogenesis in Rats: In Vivo and Ex Vivo Studies.” Reproductive Biology and Endocrinology : RB&E 8 (1) (January): 13. doi:10.1186/1477-7827-8-13. <a class="external free" href="http://www.rbej.com/content/8/1/13" rel="nofollow" target="_blank">http://www.rbej.com/content/8/1/13</a>. David, RM. 2006. “Proposed Mode of Action for in Utero Effects of Some Phthalate Esters on the Developing Male Reproductive Tract.” Toxicologic Pathology. doi:10.1080/01926230600642625. <a class="external free" href="http://tpx.sagepub.com/content/34/3/209.short" rel="nofollow" target="_blank">http://tpx.sagepub.com/content/34/3/209.short</a>. Dunaif, A, D Scott, D Finegood, B Quintana, and R Whitcomb. 1996. “The Insulin-Sensitizing Agent Troglitazone Improves Metabolic and Reproductive Abnormalities in the Polycystic Ovary Syndrome.” The Journal of Clinical Endocrinology and Metabolism 81 (9) (September): 3299–306. doi:10.1210/jcem.81.9.8784087. <a class="external free" href="http://www.ncbi.nlm.nih.gov/pubmed/8784087" rel="nofollow" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed/8784087</a>. Ema, Makoto. 2002. “Antiandrogenic Effects of Dibutyl Phthalate and Its Metabolite, Monobutyl Phthalate, in Rats.” Congenital Anomalies 42 (4) (December): 297–308. doi:10.1111/j.1741-4520.2002.tb00896.x. <a class="external free" href="http://doi.wiley.com/10.1111/j.1741-4520.2002.tb00896.x" rel="nofollow" target="_blank">http://doi.wiley.com/10.1111/j.1741-4520.2002.tb00896.x</a>. Furr, Johnathan R, Christy S Lambright, Vickie S Wilson, Paul M Foster, and Leon E Gray. 2014. “A Short-Term in Vivo Screen Using Fetal Testosterone Production, a Key Event in the Phthalate Adverse Outcome Pathway, to Predict Disruption of Sexual Differentiation.” Toxicological Sciences : An Official Journal of the Society of Toxicology 140 (2) (August 1): 403–24. doi:10.1093/toxsci/kfu081. <a class="external free" href="http://toxsci.oxfordjournals.org/content/140/2/403.full" rel="nofollow" target="_blank">http://toxsci.oxfordjournals.org/content/140/2/403.full</a>. Gasic, S, Y Bodenburg, M Nagamani, A Green, and R J Urban. 1998. “Troglitazone Inhibits Progesterone Production in Porcine Granulosa Cells.” Endocrinology 139 (12) (December): 4962–6. doi:10.1210/endo.139.12.6385. <a class="external free" href="http://www.ncbi.nlm.nih.gov/pubmed/9832434" rel="nofollow" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed/9832434</a>. Gasic, S, M Nagamani, A Green, and R J Urban. 2001. “Troglitazone Is a Competitive Inhibitor of 3beta-Hydroxysteroid Dehydrogenase Enzyme in the Ovary.” American Journal of Obstetrics and Gynecology 184 (4) (March): 575–9. doi:10.1067/mob.2001.111242. <a class="external free" href="http://www.sciencedirect.com/science/article/pii/S0002937801774340" rel="nofollow" target="_blank">http://www.sciencedirect.com/science/article/pii/S0002937801774340</a>. Gazouli, M. 2002. “Effect of Peroxisome Proliferators on Leydig Cell Peripheral-Type Benzodiazepine Receptor Gene Expression, Hormone-Stimulated Cholesterol Transport, and Steroidogenesis: Role of the Peroxisome Proliferator-Activator Receptor .” Endocrinology 143 (7) (July 1): 2571–2583. doi:10.1210/en.143.7.2571. <a class="external free" href="http://endo.endojournals.org/content/143/7/2571" rel="nofollow" target="_blank">http://endo.endojournals.org/content/143/7/2571</a>. Hannas, Bethany R, Christy S Lambright, Johnathan Furr, Nicola Evans, Paul M D Foster, Earl L Gray, and Vickie S Wilson. 2012. “Genomic Biomarkers of Phthalate-Induced Male Reproductive Developmental Toxicity: A Targeted RT-PCR Array Approach for Defining Relative Potency.” Toxicological Sciences : An Official Journal of the Society of Toxicology 125 (2) (February): 544–57. doi:10.1093/toxsci/kfr315. <a class="external free" href="http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3262859&tool=pmcentrez&rendertype=abstract" rel="nofollow" target="_blank">http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3262859&tool=pmcentrez&rendertype=abstract</a>. Heindel, J J, D K Gulati, R C Mounce, S R Russell, and J C Lamb. 1989. “Reproductive Toxicity of Three Phthalic Acid Esters in a Continuous Breeding Protocol.” Fundamental and Applied Toxicology : Official Journal of the Society of Toxicology 12 (3) (April): 508–18. <a class="external free" href="http://www.ncbi.nlm.nih.gov/pubmed/2731665" rel="nofollow" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed/2731665</a>. Johnson, Kamin J, Nicholas E Heger, and Kim Boekelheide. 2012. “Of Mice and Men (and Rats): Phthalate-Induced Fetal Testis Endocrine Disruption Is Species-Dependent.” Toxicological Sciences : An Official Journal of the Society of Toxicology 129 (2) (October): 235–48. doi:10.1093/toxsci/kfs206. <a class="external free" href="http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3491958&tool=pmcentrez&rendertype=abstract" rel="nofollow" target="_blank">http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3491958&tool=pmcentrez&rendertype=abstract</a>. Kempná, Petra, Gaby Hofer, Primus E Mullis, and Christa E Flück. 2007. “Pioglitazone Inhibits Androgen Production in NCI-H295R Cells by Regulating Gene Expression of CYP17 and HSD3B2.” Molecular Pharmacology 71 (3) (March): 787–98. doi:10.1124/mol.106.028902. <a class="external free" href="http://www.ncbi.nlm.nih.gov/pubmed/17138841" rel="nofollow" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed/17138841</a>. Kuhl, Adam J, Susan M Ross, and Kevin W Gaido. 2007. “CCAAT/enhancer Binding Protein Beta, but Not Steroidogenic Factor-1, Modulates the Phthalate-Induced Dysregulation of Rat Fetal Testicular Steroidogenesis.” Endocrinology 148 (12) (December): 5851–64. doi:10.1210/en.2007-0930. <a class="external free" href="http://www.ncbi.nlm.nih.gov/pubmed/17884934" rel="nofollow" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed/17884934</a>. Lapinskas, Paula J., Sherri Brown, Lisa M. Leesnitzer, Steven Blanchard, Cyndi Swanson, Russell C. Cattley, and J. Christopher Corton. 2005. “Role of PPARα in Mediating the Effects of Phthalates and Metabolites in the Liver.” Toxicology 207 (1): 149–163. <a class="external free" href="http://www.sciencedirect.com/science/article/pii/S0300483X04005633" rel="nofollow" target="_blank">http://www.sciencedirect.com/science/article/pii/S0300483X04005633</a>. Latini, Giuseppe, Egeria Scoditti, Alberto Verrotti, Claudio De Felice, and Marika Massaro. 2008. “Peroxisome Proliferator-Activated Receptors as Mediators of Phthalate-Induced Effects in the Male and Female Reproductive Tract: Epidemiological and Experimental Evidence.” PPAR Research 2008 (January): 359267. doi:10.1155/2008/359267. <a class="external free" href="http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2225463&tool=pmcentrez&rendertype=abstract" rel="nofollow" target="_blank">http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2225463&tool=pmcentrez&rendertype=abstract</a>. Lehmann, Kim P, Suzanne Phillips, Madhabananda Sar, Paul M D Foster, and Kevin W Gaido. 2004. “Dose-Dependent Alterations in Gene Expression and Testosterone Synthesis in the Fetal Testes of Male Rats Exposed to Di (n-Butyl) Phthalate.” Toxicological Sciences : An Official Journal of the Society of Toxicology 81 (1) (September 1): 60–8. doi:10.1093/toxsci/kfh169. <a class="external free" href="http://toxsci.oxfordjournals.org/content/81/1/60.abstract?ijkey=99364980d6548f969a82406deb6600873a38be36&keytype2=tf_ipsecsha" rel="nofollow" target="_blank">http://toxsci.oxfordjournals.org/content/81/1/60.abstract?ijkey=99364980d6548f969a82406deb6600873a38be36&keytype2=tf_ipsecsha</a>. Minge, Cadence E, Rebecca L Robker, and Robert J Norman. 2008. “PPAR Gamma: Coordinating Metabolic and Immune Contributions to Female Fertility.” PPAR Research 2008 (January): 243791. doi:10.1155/2008/243791. <a class="external free" href="http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2246065&tool=pmcentrez&rendertype=abstract" rel="nofollow" target="_blank">http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2246065&tool=pmcentrez&rendertype=abstract</a>. Mu, Y M, T Yanase, Y Nishi, N Waseda, T Oda, A Tanaka, R Takayanagi, and H Nawata. 2000. “Insulin Sensitizer, Troglitazone, Directly Inhibits Aromatase Activity in Human Ovarian Granulosa Cells.” Biochemical and Biophysical Research Communications 271 (3) (May 19): 710–3. doi:10.1006/bbrc.2000.2701. <a class="external free" href="http://www.ncbi.nlm.nih.gov/pubmed/10814527" rel="nofollow" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed/10814527</a>. Mylchreest, Eve. 2000. “Dose-Dependent Alterations in Androgen-Regulated Male Reproductive Development in Rats Exposed to Di(n-Butyl) Phthalate during Late Gestation.” Toxicological Sciences 55 (1) (May 1): 143–151. doi:10.1093/toxsci/55.1.143. <a class="external free" href="http://www.toxsci.oupjournals.org/cgi/doi/10.1093/toxsci/55.1.143" rel="nofollow" target="_blank">http://www.toxsci.oupjournals.org/cgi/doi/10.1093/toxsci/55.1.143</a>. Mylchreest, Eve, Russell C. Cattley, and Paul M. D. Foster. 1998. “Male Reproductive Tract Malformations in Rats Following Gestational and Lactational Exposure to Di( N -Butyl) Phthalate: An Antiandrogenic Mechanism?” Toxicological Sciences 43 (1) (May 1): 47–60. doi:10.1093/toxsci/43.1.47. <a class="external free" href="http://toxsci.oxfordjournals.org/content/43/1/47.short?rss=1&ssource=mfc" rel="nofollow" target="_blank">http://toxsci.oxfordjournals.org/content/43/1/47.short?rss=1&ssource=mfc</a>. Peters, J M, M W Taubeneck, C L Keen, and F J Gonzalez. 1997. “Di(2-Ethylhexyl) Phthalate Induces a Functional Zinc Deficiency during Pregnancy and Teratogenesis That Is Independent of Peroxisome Proliferator-Activated Receptor-Alpha.” Teratology 56 (5) (November): 311–6. doi:10.1002/(SICI)1096-9926(199711)56:5<311::AID-TERA4>3.0.CO;2-#. <a class="external free" href="http://www.ncbi.nlm.nih.gov/pubmed/9451755" rel="nofollow" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed/9451755</a>. Shipley, Jonathan M, and David J Waxman. 2004. “Simultaneous, Bidirectional Inhibitory Crosstalk between PPAR and STAT5b.” Toxicology and Applied Pharmacology 199 (3) (October 15): 275–84. doi:10.1016/j.taap.2003.12.020. <a class="external free" href="http://www.ncbi.nlm.nih.gov/pubmed/15364543" rel="nofollow" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed/15364543</a>. Shultz, V. D. 2001. “Altered Gene Profiles in Fetal Rat Testes after in Utero Exposure to Di(n-Butyl) Phthalate.” Toxicological Sciences 64 (2) (December 1): 233–242. doi:10.1093/toxsci/64.2.233. <a class="external free" href="http://toxsci.oxfordjournals.org/content/64/2/233.abstract?ijkey=b8af27acfe10695847a4e8a9b568882405d071ae&keytype2=tf_ipsecsha" rel="nofollow" target="_blank">http://toxsci.oxfordjournals.org/content/64/2/233.abstract?ijkey=b8af27acfe10695847a4e8a9b568882405d071ae&keytype2=tf_ipsecsha</a>. Thompson, Christopher J, Susan M Ross, Janan Hensley, Kejun Liu, Susanna C Heinze, S Stanley Young, and Kevin W Gaido. 2005. “Differential Steroidogenic Gene Expression in the Fetal Adrenal Gland versus the Testis and Rapid and Dynamic Response of the Fetal Testis to Di(n-Butyl) Phthalate.” Biology of Reproduction 73 (5) (November): 908–17. doi:10.1095/biolreprod.105.042382. <a class="external free" href="http://www.ncbi.nlm.nih.gov/pubmed/15987825" rel="nofollow" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed/15987825</a>. Veldhuis, Johannes D, George Zhang, and James C Garmey. 2002. “Troglitazone, an Insulin-Sensitizing Thiazolidinedione, Represses Combined Stimulation by LH and Insulin of de Novo Androgen Biosynthesis by Thecal Cells in Vitro.” The Journal of Clinical Endocrinology and Metabolism 87 (3) (March): 1129–33. doi:10.1210/jcem.87.3.8308. <a class="external free" href="http://www.ncbi.nlm.nih.gov/pubmed/11889176" rel="nofollow" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed/11889176</a>. Vierhapper, H, P Nowotny, and W Waldhäusl. 2003. “Reduced Production Rates of Testosterone and Dihydrotestosterone in Healthy Men Treated with Rosiglitazone.” Metabolism: Clinical and Experimental 52 (2) (February): 230–2. doi:10.1053/meta.2003.50028. <a class="external free" href="http://www.ncbi.nlm.nih.gov/pubmed/12601638" rel="nofollow" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed/12601638</a>. Ward, J M, J M Peters, C M Perella, and F J Gonzalez. 1998. “Receptor and Nonreceptor-Mediated Organ-Specific Toxicity of di(2-Ethylhexyl)phthalate (DEHP) in Peroxisome Proliferator-Activated Receptor Alpha-Null Mice.” Toxicologic Pathology 26 (2): 240–6. <a class="external free" href="http://www.ncbi.nlm.nih.gov/pubmed/9547862" rel="nofollow" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed/9547862</a>. Welsh, Michelle, Philippa T K Saunders, Mark Fisken, Hayley M Scott, Gary R Hutchison, Lee B Smith, and Richard M Sharpe. 2008. “Identification in Rats of a Programming Window for Reproductive Tract Masculinization, Disruption of Which Leads to Hypospadias and Cryptorchidism.” The Journal of Clinical Investigation 118 (4) (April): 1479–90. doi:10.1172/JCI34241. <a class="external free" href="http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2267017&tool=pmcentrez&rendertype=abstract" rel="nofollow" target="_blank">http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2267017&tool=pmcentrez&rendertype=abstract</a>. Wood, Charles E, Micheal P Jokinen, Crystal L Johnson, Greg R Olson, Susan Hester, Michael George, Brian N Chorley, et al. 2014. “Comparative Time Course Profiles of Phthalate Stereoisomers in Mice.” Toxicological Sciences : An Official Journal of the Society of Toxicology 139 (1) (May): 21–34. doi:10.1093/toxsci/kfu025. <a class="external free" href="http://www.ncbi.nlm.nih.gov/pubmed/24496636" rel="nofollow" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed/24496636</a>. AOPWiki 2016-11-29T18:41:16

2023-09-25T16:26:44