<p>Systems Toxicology Unit, Institute for Health and Consumer Protection, Joint Research Centre, European Commission, Via E. Fermi 2749, I-21027 Ispra, Varese, Italy</p>
<!-- Abstract Section, text as generated by author -->
<div id="abstract">
<h2>Abstract</h2>
<p>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.</p>
<h2>Abstract</h2>
<hr>
<p>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.</p>
<br>
</div>
<!-- Background Section, text as generated by author -->
<div id="background">
<br>
</div>
<!-- AOP summary, includes summary of each of the events associated with this aop -->
<!-- Overall assessment section, *** what is included here? *** -->
<div id="overall_assessment">
<h2>Overall Assessment of the AOP</h2>
<p><strong>Biological plausibility, coherence, and consistency of the experimental evidence</strong></p>
<p><strong>Biological plausibility, coherence, and consistency of the experimental evidence</strong></p>
<p>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.</p>
<p><strong>Concordance of dose-response relationships</strong></p>
<p>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.</p>
<p><strong>Temporal concordance among the key events and the adverse outcome</strong></p>
<p>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.</p>
<p><br />
<strong>Strength, consistency, and specificity of association of adverse effect and initiating event</strong></p>
<p>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.</p>
<p><strong>Alternative mechanism(s) or MIE(s) described which may contribute/synergise the postulated AOP</strong></p>
<p>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).</p>
<p>PPARγ activation</p>
<p>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).</p>
<p><br />
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.</p>
<p> </p>
<p><strong>Uncertainties, inconsistencies and data gaps</strong></p>
<p>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).</p>
<p>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).</p>
<p>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).</p>
<p>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.</p>
<p>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.</p>
<p><strong>Life Stage Applicability</strong></p>
<p>This AOP is relevant for developing (prenatal) male.</p>
<p><strong>Taxonomic Applicability</strong></p>
<p>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)</p>
<p>PPAR alpha activation was found to indirectly alter the expression of genes involved in cholesterol transport in mitochondria</p>
</td>
<td>
<p>very weak</p>
</td>
</tr>
<tr>
<td>
<p><em><strong>TSPO; StAR decrease</strong></em></p>
</td>
<td>
<p>Alterations in the amount of cholesterol transport proteins in mitochondria impact on the levels of substrate for steroid hormones production.</p>
</td>
<td>
<p>weak</p>
</td>
</tr>
<tr>
<td>
<p><em><strong>cholesterol transport in mitochondria, reduction</strong></em></p>
</td>
<td>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>Androgens regulate masculinization of the external genitalia. Therefore any defects in androgen biosynthesis, metabolism or action during foetal development can reproductive tract malformation.</p>
<p>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.</p>
</td>
<td>
<p>Moderate</p>
</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>
<p>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.</p>
</td>
<td>
<p>Strong</p>
</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>
<p>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</p>
</td>
<td>
<p>Moderate</p>
</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>
<p>Support for solid temporal relationship is lacking.</p>
</td>
<td>
<p> </p>
</td>
<td>
<p>Moderate</p>
</td>
<td>
<p>No conflicting data</p>
</td>
</tr>
</tbody>
</table>
<p> </p>
<p>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</p>
<h3>Quantitative Consideration</h3>
<p>This AOP is qualitatively described; however it contains also data that may be used for further development of quantitative description.</p>
<h3>Quantitative Consideration</h3>
<p>This AOP is qualitatively described; however it contains also data that may be used for further development of quantitative description.</p>
</div>
<!-- potential consierations, text as entered by author -->
<h2>Considerations for Potential Applications of the AOP (optional)</h2>
<hr>
<p>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).</p>
<h2>Considerations for Potential Applications of the AOP (optional)</h2>
<p>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).</p>
<p>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).</p>
<p>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.</p>
<p>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.</p>
</div>
<!-- reference section, text as of right now but should be changed to be handled as table -->
<div id="references">
<h2>References</h2>
<hr>
<p>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>.</p>
<p>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>.</p>
<p>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>.</p>
<p>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>.</p>
<p>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>.</p>
<p>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>.</p>
<p>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>.</p>
<p>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>.</p>
<p>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>.</p>
<p>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>.</p>
<p>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>.</p>
<p>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>.</p>
<p>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>.</p>
<p>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>.</p>
<p>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>.</p>
<p>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>.</p>
<p>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>.</p>
<p>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>.</p>
<p>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>.</p>
<p>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>.</p>
<p>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>.</p>
<p>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>.</p>
<p>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>.</p>
<p>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>.</p>
<p>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>.</p>
<p>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>.</p>
<p>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>.</p>
<p>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>.</p>
<p>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>.</p>
<p>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>.</p>
<p>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>.</p>
<p>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>.</p>
<p>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>.</p>
<p>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>.</p>
<p>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>.</p>
<p>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>.</p>
<p>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>.</p>
<!-- Evidence for Perturbation of This Event by Stressors -->
<h3>Evidence for Perturbation by Stressor</h3>
<hr>
<h4>Overview for Molecular Initiating Event</h4>
<p>Fibrates are ligands of PPARα (Staels et al. 1998).
</p><p>Phthalates
</p><p>MHEP (CAS 4376-20-9) directly binds <i>in vitro</i> 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).
</p><p>Notably, PPARα are responsive to DEHP <i>in vitro</i> 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 <i>in vivo</i> upon DEHP treatment (Xu et al. 2010) and down-regulated by Diisobutyl phthalate (DiBP) (Boberg et al. 2008)).
</p><p><br />
Perfluorooctanoic Acid (PFOA) is known to activate PPARα (Vanden Heuvel et al. 2006).
</p><p>Organotin
</p><p>Tributyltin (TBT) activates all three heterodimers of PPAR with RXR, primarily through its interaction with RXR (le Maire et al. 2009)
</p>
<br>
<br>
<!-- end Evidence for Perturbation of This Event by Stressors -->
<h4>Domain of Applicability</h4>
<br>
<!-- loop to find taxonomic applicability under event -->
<p>PPARα has been identified in frog (Xenopus laevis), mouse, human, rat, fish, hamster and chicken (reviewed in (Wahli and Desvergne 1999)).</p>
</div>
<!-- end loop for taxons -->
<!-- life stages -->
<div>
</div>
<!-- end life stages -->
<!-- sex terms -->
<div>
</div>
<!-- end sex terms -->
<div>
<p>PPARα has been identified in frog (Xenopus laevis), mouse, human, rat, fish, hamster and chicken (reviewed in (Wahli and Desvergne 1999)).
</p>
<br>
</div>
<!-- event text -->
<h4>Key Event Description</h4>
<p><b>Biological state</b>
</p><p>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.
</p><p><b>Biological compartments</b>
</p><p>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).
</p><p><b>General role in biology</b>
</p><p>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).
</p><p>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).
</p>
<br>
<h4>How it is Measured or Detected</h4>
<p>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). Currently no internationally validated assays for regulatory purposes are available.
<p>Computational simulation of a candidate ligand binding to a receptor, Predicts the strength of association or binding affinity.
</p>
</td>
<td>
<p>Direct binding indicating the mode of action for PPARα
</p>
</td>
<td colspan="3">
<p>Quantifying changes in luciferase expression in the treated reporter cells provides a sensitive surrogate measure of the changes in PPAR functional activity.
</p>
</td>
<td colspan="2">
<p>PPARα once activated by a ligand, the receptor binds to a promoter element in the gene for target gene and activates its transcription. The DNA-bound (activated) PPAR is measured.
</p>
</td></tr>
<tr>
<td>
<p>Test outcome
</p>
</td>
<td>
<p>A binding interaction between a small molecule ligand and an enzyme protein may result in activation or inhibition of the enzyme. If the protein is a receptor, ligand binding may result in agonism or antagonism of the normal activity of the receptor.
</p>
</td>
<td>
<p>Assesses the ability of compounds to bind to PPARα. Identifies the modulators of PPARα.
</p>
</td>
<td colspan="3">
<p>The changes in activity of reporter gene levels functionally linked to a PPAR-responsive element/promoter gives information about the nature of the PPAR activation.
</p>
</td>
<td colspan="2">
<p>Protein: DNA binding, DNA binding activity  
</p>
</td></tr>
<tr>
<td>
<p>Test background
</p>
</td>
<td>
<p>Predicts the preferred orientation of one molecule to a second when bound to each other to form a stable complex. Knowledge of the preferred orientation in turn may be used to predict the strength of association or binding affinity between two molecules using, for example, scoring functions.
</p>
</td>
<td>
<p>This assay determines whether compounds interact directly with PPARs. The type of beads that are involved in the SPA are microscopic in size and within the beads, there is a scintillant which emits light when it is stimulated. Stimulation occurs when radio-labelled molecules interact and bind to the surface of the bead and trigger the bead to emit light.
</p>
</td>
<td>
<p>PPARα/γ COS-1cell transactivation assay (transient transfection with human or mouse PPARα/γ expression plasmid and pHD(x3)-Luc reporter plasmid
</p>
</td>
<td>
<p>(PPRE)3- luciferase reporter construct C2C12
</p>
</td>
<td>
<p>Proprietary rodent cell line expressing the mouse/rat PPARα
</p>
</td>
<td>
<p>Transcriptional activity of PPARα can be assessed using commercially available kits like e.g. PPAR-α transcription factor assay kit.
</p>
</td>
<td>
<p>Gene regulation and determining protein: DNA interactions are detected by the EMSA. EMSA can be used qualitatively to identify sequence-specific DNA-binding proteins (such as transcription factors) in crude lysates and, in conjunction with mutagenesis, to identify the important binding sequences within a given gene upstream regulatory region. EMSA can also be utilized quantitatively to measure thermodynamic and kinetic parameters.
<p>Table 1 Summary of the chosen methods to measure the PPARα activation.
</p>
<br>
<h4>Key Event Description</h4>
<p>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:</p>
<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>
<p><strong>Biological state</strong></p>
<p>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.</p>
<p><strong>Biological compartments</strong></p>
<p>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).</p>
<p><strong>General role in biology</strong></p>
<p>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).</p>
<p>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).</p>
<h4>How it is Measured or Detected</h4>
<p>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).</p>
<p>The most direct measure of this MIE is microarray profiling from<span style="font-size:medium"><span style="font-family:Cambria,serif"><span style="color:#000000"><span style="font-family:Calibri,sans-serif"><span style="color:#191c1f"> 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).</span></span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:"Times New Roman",serif"><span style="color:#000000"><span style="font-family:Arial,sans-serif">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 </span><span style="font-family:Arial,sans-serif">(Kupershmidt et al., 2010; Rooney et al., 2018; Corton et al., 2020)</span><span style="font-size:10pt"><span style="font-family:Times">.</span></span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:"Times New Roman",serif"><span style="color:#000000"><span style="font-family:Arial,sans-serif">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 </span><span style="font-family:Arial,sans-serif">(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)</span><span style="font-family:Arial,sans-serif">.</span></span></span></span></p>
<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>
<h4>References</h4>
<p>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.
</p><p>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.
</p><p>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.
</p><p>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.
</p><p>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.
</p><p>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.
</p><p>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.
</p><p>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.
</p><p>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.
</p><p>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).
</p><p>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.
</p><p>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.
</p><p>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.
</p><p>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.
</p><p>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.
</p><p>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.
</p><p>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.
</p><p>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.
</p><p>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.
</p><p>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.
</p><p>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.
</p><p>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.
</p><p>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.
</p>
<br>
<!-- end event text -->
</div>
<h4>References</h4>
<p>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.</p>
<p>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.</p>
<p>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</p>
<p>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</p>
<p>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</p>
<p>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.</p>
<p>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.</p>
<p>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</p>
<p>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</p>
<p>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.</p>
<p>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</p>
<p>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</p>
<p>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.</p>
<p>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</p>
<p>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</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>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).</p>
<p>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.</p>
<p>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</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>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</p>
<p>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</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>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</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<h3>List of Key Events in the AOP</h3>
<div>
<div>
<h4><a href="/events/266">Event: 266: Decrease, Steroidogenic acute regulatory protein (STAR)</a><br></h4>
<h5>Short Name: Decrease, Steroidogenic acute regulatory protein (STAR)</h5>
<p>StAR has been cloned from many species, and is highly conserved among mammals, birds, amphibians and fish (Bauer et al. 2000).
<p>StAR has been cloned from many species, and is highly conserved among mammals, birds, amphibians and fish (Bauer et al. 2000).
</p>
<br>
</div>
<!-- event text -->
<h4>Key Event Description</h4>
<p><b>Biological state</b>
<h4>Key Event Description</h4>
<p><b>Biological state</b>
</p><p>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.
</p><p><br />
<b>Biological compartments</b>
</p><p>StAR is expressed principally in steroidogenic tissues (Bauer et al. 2000).
</p><p><b>General role in biology</b>
</p><p>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).
</p>
<br>
<h4>How it is Measured or Detected</h4>
<p>The StAR expression can be measured by RT-PCR (mRNA) and on the protein level (western blot).
<h4>How it is Measured or Detected</h4>
<p>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):
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>.
</p>
<br>
<h4>References</h4>
<p>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.
<h4>References</h4>
<p>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.
</p><p>Hum, D W, and W L Miller. 1993. “Transcriptional Regulation of Human Genes for Steroidogenic Enzymes.” Clinical Chemistry 39 (2) (February): 333–40.
</p><p>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.
</p><p>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.
</p>
<br>
<!-- end event text -->
</div>
<div>
<div>
<h4><a href="/events/447">Event: 447: Reduction, Cholesterol transport in mitochondria</a><br></h4>
<h5>Short Name: Reduction, Cholesterol transport in mitochondria</h5>
<p>The enzymes needed for cholesterol transport were found in amphioxus and are present in vertebrates (Albalat et al. 2011).
<p>The enzymes needed for cholesterol transport were found in amphioxus and are present in vertebrates (Albalat et al. 2011).
</p>
<br>
</div>
<!-- event text -->
<h4>Key Event Description</h4>
<p><b>Biological state</b>
<h4>Key Event Description</h4>
<p><b>Biological state</b>
</p><p>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)].
</p><p><br />
<b>Biological compartments</b>
</p><p>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.
</p><p><br />
</p><p><b>General role in biology</b>
</p><p>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.
</p>
<br>
<h4>How it is Measured or Detected</h4>
<p>This KE can be indirectly measured by:
<h4>How it is Measured or Detected</h4>
<p>This KE can be indirectly measured by:
</p><p>1. Expression of the proteins involved in cholesterol transport by qPCR or Western blot.
</p><p>3. Cholesterol transport to the mitochondrial inner membrane in intact cells:
</p>
<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>
<p>The cholesterol transport can be measured <i>in vitro</i> cultured Leydig cells. The methods for culturing Leydig cells can be found in the Database Service on Alternative Methods to animal experimentation (DB-ALM):
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>
</p>
<br>
<h4>References</h4>
<p>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.
<h4>References</h4>
<p>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.
</p><p>Baulieu, E E. 1998. “Neurosteroids: A Novel Function of the Brain.” Psychoneuroendocrinology 23 (8) (November): 963–87.
</p><p>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.
</p><p>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.
</p><p>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.
</p><p>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.
</p><p>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.
</p><p>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.
</p>
<br>
<!-- end event text -->
</div>
<div>
<div>
<h4><a href="/events/413">Event: 413: Reduction, Testosterone synthesis in Leydig cells</a><br></h4>
<h5>Short Name: Reduction, Testosterone synthesis in Leydig cells</h5>
<p>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.
<p>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.
</p>
<br>
</div>
<!-- event text -->
<h4>Key Event Description</h4>
<p><b>Biological state</b>
<h4>Key Event Description</h4>
<p><b>Biological state</b>
</p><p>Testosterone is a steroid hormone from the androgen group and is found in humans and other vertebrates.
</p><p><b>Biological compartments</b>
</p><p>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).
</p><p><b>General role in biology</b>
</p><p>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).
</p><p>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).
</p><p>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).
</p><p>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.
</p>
<br>
<h4>How it is Measured or Detected</h4>
<p>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).
<h4>How it is Measured or Detected</h4>
<p>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):
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>.
</p><p>Testosterone synthesis in vitro cultured cells can be measured indirectly by testosterone radioimmunoassay or analytical methods such as LC-MS.
</p>
<br>
<h4>References</h4>
<p>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.
<h4>References</h4>
<p>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.
</p><p>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.
</p><p>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.
</p><p>Nef, S. 2000. “Hormones in Male Sexual Development.” Genes & Development 14 (24) (December 15): 3075–3086. doi:10.1101/gad.843800.
</p><p>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.
<td><a href="/aops/307">Aop:307 - Decreased testosterone synthesis leading to short anogenital distance (AGD) in male (mammalian) offspring</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/526">Aop:526 - Decreased, Chicken Ovalbumin Upstream Promoter Transcription Factor II (COUP-TFII) leads to Impaired, Spermatogenesis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/124">Aop:124 - HMG-CoA reductase inhibition leading to decreased fertility</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/18">Aop:18 - PPARα activation in utero leading to impaired fertility in males</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/51">Aop:51 - PPARα activation leading to impaired fertility in adult male rodents </a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/496">Aop:496 - Androgen receptor agonism leading to reproduction dysfunction （in zebrafish）</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/64">Aop:64 - Glucocorticoid Receptor (GR) Mediated Adult Leydig Cell Dysfunction Leading to Decreased Male Fertility</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/120">Aop:120 - Inhibition of 5α-reductase leading to Leydig cell tumors (in rat)</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/288">Aop:288 - Inhibition of 17α-hydrolase/C 10,20-lyase (Cyp17A1) activity leads to birth reproductive defects (cryptorchidism) in male (mammals)</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/570">Aop:570 - Decreased testosterone synthesis leading to hypospadias in male (mammalian) offspring</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/575">Aop:575 - Decreased testosterone synthesis leading to increased nipple retention (NR) in male (rodent) offspring</a></td>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">This key event (KE) is applicable to all mammals, as the synthesis and role of testosterone are evolutionarily conserved (Vitousek et al., 2018). Both sexes produce and require testosterone, which plays critical roles throughout life, from development to adulthood; albeit there are differences in lief stages when testosterone exert specific effects and function (Luetjens & Weinbauer, 2012; Naamneh Elzenaty et al., 2022). Accordingly, this KE applies to both males and females across all life stages, but life stage should be considered when embedding in AOPs. </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Notably, the key enzymes involved in testosterone production first appeared in the common ancestor of amphioxus and vertebrates (Baker, 2011). This suggests that the KE has a broader domain of applicability, encompassing non-mammalian vertebrates. AOP developers are encouraged to integrate additional knowledge to expand its relevance beyond mammals to other vertebrates.</span></span></p>
</div>
<!-- end loop for taxons -->
<!-- life stages -->
<div>
</div>
<!-- end life stages -->
<!-- sex terms -->
<div>
</div>
<!-- end sex terms -->
<div>
<p>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.</p>
<br>
</div>
<h4>Key Event Description</h4>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="background-color:white"><span style="color:black">Testosterone is an endogenous steroid hormone that acts by binding the androgen receptor (AR) in androgen-responsive tissues (Murashima et al., 2015). As with all steroid hormones, testosterone is produced through steroidogenesis, an enzymatic pathway converting cholesterol into all the downstream steroid hormones. Briefly, androstenedione or androstenediol is converted to testosterone by the enzymes 17β-hydroxysteroid dehydrogenase (HSD) or 3β-HSD, respectively. Testosterone can then be converted to the more potent androgen, dihydrotestosterone (DHT) by 5α-reductase, or aromatized by CYP19A1 (Aromatase) into estrogens. Testosterone secreted in blood circulation can be found free or bound to SHBG or albumin (Trost & Mulhall, 2016). </span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="background-color:white"><span style="color:black">Testosterone is produced mainly by the testes (in males), ovaries (in females) and to a lesser degree in the adrenal glands. The output of testosterone from different tissues varies with life stages. During fetal development testosterone is crucial for the differentiation of male reproductive tissues and the overall male phenotype. In adulthood, testosterone synthesis is controlled by the Hypothalamus-Pituitary-Gonadal (HPG) axis. GnRH is released from the hypothalamus inducing LH pulses secreted by the anterior pituitary. This LH surge leads to increased testosterone production, both in testes (males) and ovaries (females). If testosterone reaches low levels, this axis is once again stimulated to increase testosterone synthesis. This feedback loop is essential for maintenance of appropriate testosterone levels (Chandrashekar & Bartke, 1998; Ellis et al., 1983; Rey, 2021).</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="background-color:white"><span style="color:black">By disrupting e.g. steroidogenesis or the HPG-axis, testosterone synthesis or homeostasis may be disrupted and can lead to less testosterone being synthesized and released into circulation. </span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><u><span style="background-color:white"><span style="color:black">General role in biology</span></span></u></span></span></p>
<p style="text-align:justify"><span style="font-size:11.0pt"><span style="background-color:white"><span style="font-family:"Calibri",sans-serif"><span style="color:black">Androgens are essential hormones responsible for the development of the male phenotype during fetal life and for sexual maturation at puberty. In adulthood, androgens remain essential for the maintenance of male reproductive function and behavior but is also essential for female fertility. Apart from their effects on reproduction, androgens affect a wide variety of non-reproductive tissues such as skin, bone, muscle, and brain (Heemers et al 2006). Androgens, principally testosterone and DHT, exert most of their effects by interacting with the AR (Murashima et al 2015). </span></span></span></span></p>
<h4>How it is Measured or Detected</h4>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:10.5pt"><span style="background-color:white"><span style="color:black">Testosterone levels can be quantified in serum (in vivo), cell culture medium (in vitro), or tissue (ex vivo, in vitro). Methods include traditional immunoassays such as ELISA and RIA, advanced techniques like LC-MS/MS, and liquid scintillation spectrometry following radiolabeling (Shiraishi et al., 2008).</span></span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:10.5pt"><span style="background-color:white"><span style="color:black">The H295R Steroidogenesis Assay (OECD TG 456) is (currently; anno 2025) primarily used to measure estradiol and testosterone production. This validated OECD test guideline uses adrenal H295R cells, with hormone levels measured in the cell culture medium (OECD, 2011). H295R adrenocortical carcinoma cells express the key enzymes and hormones of the steroidogenic pathway, enabling broad analysis of steroidogenesis disruption by quantifying hormones in the medium using LC-MS/MS. Initially designed to assess testosterone and estradiol levels, the assay now extends to additional steroid hormones, such as progesterone and pregnenolone. The U.S. EPA’s ToxCast program further advanced this method, enabling high-throughput measurement of 11 steroidogenesis-related hormones (Haggard et al., 2018). While the H295R assay indirectly reflects disruptions in overall steroidogenesis (e.g., changes in testosterone levels), it does not provide mechanistic insights.</span></span></span></span></span></p>
<p><span style="font-size:10.5pt"><span style="background-color:white"><span style="font-family:"Calibri",sans-serif"><span style="color:black">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 may also be measured by: 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).</span></span></span></span></p>
<!-- event text -->
<h4>Key Event Description</h4>
<p><strong>Biological state</strong></p>
<h4>References</h4>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Baker, M.E. (2011). Insights from the structure of estrogen receptor into the evolution of estrogens: implications for endocrine disruption. Biochem Pharmacol, 82(1), 1-8. <a href="https://doi.org/10.1016/j.bcp.2011.03.008" style="color:blue; text-decoration:underline">https://doi.org/10.1016/j.bcp.2011.03.008</a> </span></span></p>
<p>Testosterone (T) is a steroid hormone from the androgen group. T serves as a substrate for two metabolic pathways that produce antagonistic sex steroids.</p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Chandrashekar, V., & Bartke, A. (1998). The Role of Growth Hormone in the Control of Gonadotropin Secretion in Adult Male Rats*. Endocrinology, 139(3), 1067–1074. <a href="https://doi.org/10.1210/endo.139.3.5816" style="color:blue; text-decoration:underline">https://doi.org/10.1210/endo.139.3.5816</a> </span></span></p>
<p><strong>Biological compartments</strong></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Ellis, G. B., Desjardins, C., & Fraser, H. M. (1983). Control of Pulsatile LH Release in Male Rats. Neuroendocrinology, 37(3), 177–183. <a href="https://doi.org/10.1159/000123540" style="color:blue; text-decoration:underline">https://doi.org/10.1159/000123540</a> </span></span></p>
<p>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.</p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Haggard, D. E., Karmaus, A. L., Martin, M. T., Judson, R. S., Setzer, R. W., & Paul Friedman, K. (2018). High-Throughput H295R Steroidogenesis Assay: Utility as an Alternative and a Statistical Approach to Characterize Effects on Steroidogenesis. Toxicological Sciences, 162(2), 509–534. <a href="https://doi.org/10.1093/toxsci/kfx274" style="color:blue; text-decoration:underline">https://doi.org/10.1093/toxsci/kfx274</a> </span></span></p>
<p><strong>General role in biology</strong></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">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 </span></span></p>
<p>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.</p>
<br>
<h4>How it is Measured or Detected</h4>
<p>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).</p>
<br>
<h4>References</h4>
<p>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</p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Luetjens, C. M., & Weinbauer, G. F. (2012). Testosterone: biosynthesis, transport, metabolism and (non-genomic) actions. In Testosterone (pp. 15–32). Cambridge University Press. <a href="https://doi.org/10.1017/CBO9781139003353.003" style="color:blue; text-decoration:underline">https://doi.org/10.1017/CBO9781139003353.003</a> </span></span></p>
<p>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</p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Murashima, A., Kishigami, S., Thomson, A., & Yamada, G. (2015). Androgens and mammalian male reproductive tract development. Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms, 1849(2), 163–170. <a href="https://doi.org/10.1016/j.bbagrm.2014.05.020" style="color:blue; text-decoration:underline">https://doi.org/10.1016/j.bbagrm.2014.05.020</a> </span></span></p>
<p>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</p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Naamneh Elzenaty, R., du Toit, T., & Flück, C. E. (2022). Basics of androgen synthesis and action. Best Practice & Research Clinical Endocrinology & Metabolism, 36(4), 101665. <a href="https://doi.org/10.1016/j.beem.2022.101665" style="color:blue; text-decoration:underline">https://doi.org/10.1016/j.beem.2022.101665</a> </span></span></p>
<p>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.</p>
<br>
<!-- end event text -->
</div>
<div>
<div>
<h4><a href="/events/289">Event: 289: Decrease, Translocator protein (TSPO)</a><br></h4>
<h5>Short Name: Decrease, Translocator protein (TSPO)</h5>
</div>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">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. <a href="https://doi.org/10.1016/j.urology.2013.12.024" style="color:blue; text-decoration:underline">https://doi.org/10.1016/j.urology.2013.12.024</a> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Rey, R. A. (2021). The Role of Androgen Signaling in Male Sexual Development at Puberty. Endocrinology, 162(2). <a href="https://doi.org/10.1210/endocr/bqaa215" style="color:blue; text-decoration:underline">https://doi.org/10.1210/endocr/bqaa215</a> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Shiraishi, S., Lee, P. W. N., Leung, A., Goh, V. H. H., Swerdloff, R. S., & Wang, C. (2008). Simultaneous Measurement of Serum Testosterone and Dihydrotestosterone by Liquid Chromatography–Tandem Mass Spectrometry. Clinical Chemistry, 54(11), 1855–1863. <a href="https://doi.org/10.1373/clinchem.2008.103846" style="color:blue; text-decoration:underline">https://doi.org/10.1373/clinchem.2008.103846</a> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">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.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Trost, L. W., & Mulhall, J. P. (2016). Challenges in Testosterone Measurement, Data Interpretation, and Methodological Appraisal of Interventional Trials. The Journal of Sexual Medicine, 13(7), 1029–1046. <a href="https://doi.org/10.1016/j.jsxm.2016.04.068" style="color:blue; text-decoration:underline">https://doi.org/10.1016/j.jsxm.2016.04.068</a> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Vitousek, M. N., Johnson, M. A., Donald, J. W., Francis, C. D., Fuxjager, M. J., Goymann, W., Hau, M., Husak, J. F., Kircher, B. K., Knapp, R., Martin, L. B., Miller, E. T., Schoenle, L. A., Uehling, J. J., & Williams, T. D. (2018). HormoneBase, a population-level database of steroid hormone levels across vertebrates. Scientific Data, 5(1), 180097. <a href="https://doi.org/10.1038/sdata.2018.97" style="color:blue; text-decoration:underline">https://doi.org/10.1038/sdata.2018.97</a> </span></span></p>
<p>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).</p>
</div>
<!-- end loop for taxons -->
<!-- life stages -->
<div>
</div>
<!-- end life stages -->
<!-- sex terms -->
<div>
</div>
<!-- end sex terms -->
<div>
<p>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).</p>
<br>
</div>
<!-- event text -->
<h4>Key Event Description</h4>
<p><strong>Biological state</strong></p>
<h4>Key Event Description</h4>
<p><strong>Biological state</strong></p>
<p>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).</p>
<p><strong>Biological compartments</strong></p>
<p>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).</p>
<p><strong>General role in biology: regulation of lipid transport</strong></p>
<p>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).</p>
<br>
<h4>How it is Measured or Detected</h4>
<p>TSPO levels can be assayed by standard methods for assessment of gene expression levels like qPCR or direct protein levels by Western blot.</p>
<h4>How it is Measured or Detected</h4>
<p>TSPO levels can be assayed by standard methods for assessment of gene expression levels like qPCR or direct protein levels by Western blot.</p>
<p>The level of TSPO as well as other steroidogenic protein can be measured <em>in vitro</em> 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>.</p>
<h2>Uncertainties and Inconsistencies</h2>
<p>This information needs to be moved to a key event relationship page.</p>
<p>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).</p>
<br>
<h4>References</h4>
<p>Amsterdam, A. & Suh, B.S., 1991. An inducible functional peripheral benzodiazepine receptor in mitochondria of steroidogenic granulosa cells. Endocrinology, 129(1), pp.503–10.</p>
<h4>References</h4>
<p>Amsterdam, A. & Suh, B.S., 1991. An inducible functional peripheral benzodiazepine receptor in mitochondria of steroidogenic granulosa cells. Endocrinology, 129(1), pp.503–10.</p>
<p>Anholt, R. et al., 1986. The peripheral-type benzodiazepine receptor. Localization to the mitochondrial outer membrane. J. Biol. Chem., 261(2), pp.576–583.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<td><a href="/aops/7">Aop:7 - Aromatase (Cyp19a1) reduction leading to impaired fertility in adult female</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/51">Aop:51 - PPARα activation leading to impaired fertility in adult male rodents </a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/18">Aop:18 - PPARα activation in utero leading to impaired fertility in males</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/64">Aop:64 - Glucocorticoid Receptor (GR) Mediated Adult Leydig Cell Dysfunction Leading to Decreased Male Fertility</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/348">Aop:348 - Inhibition of 11β-Hydroxysteroid Dehydrogenase leading to decreased population trajectory </a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/349">Aop:349 - Inhibition of 11β-hydroxylase leading to decresed population trajectory </a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/396">Aop:396 - Deposition of ionizing energy leads to population decline via impaired meiosis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/398">Aop:398 - Decreased ALDH1A (RALDH) activity leading to decreased fertility via disrupted meiotic initiation of fetal oogonia </a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/492">Aop:492 - Glutathione conjugation leading to reproductive dysfunction via oxidative stress</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/345">Aop:345 - Androgen receptor (AR) antagonism leading to decreased fertility in females</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/592">Aop:592 - DBEPE-induced DNA strand breaks and LDH activity inhibition leading to population growth rate decline via energy metabolism disrupt and apoptosis</a></td>
<p><strong>Plausible domain of applicability</strong></p>
<p><strong><em>Taxonomic applicability</em>: </strong>The impaired fertility may also have relevance for fish, mammals, amphibians, reptiles, birds and and invertebrates with sexual reproduction.</p>
<p><strong><em>Life stage applicability</em></strong>: The impaired fertility can be measured at juveniles and adults.</p>
<p><em><strong>Sex applicability</strong></em>: The impaired fertility can be measured in both male and female species. </p>
</div>
<!-- end loop for taxons -->
<!-- life stages -->
<div>
</div>
<!-- end life stages -->
<!-- sex terms -->
<div>
</div>
<!-- end sex terms -->
<div>
</div>
<!-- event text -->
<h4>Key Event Description</h4>
<p><strong>Biological state</strong></p>
<h4>Key Event Description</h4>
<p><strong>Biological state</strong></p>
<p>capability to produce offspring</p>
<p><strong>Biological compartments</strong></p>
<p>System</p>
<p><strong>General role in biology</strong></p>
<p>Fertility is the capacity to conceive or induce conception. Impairment of fertility represents disorders of male or female reproductive functions or capacity.</p>
<br>
<h4>How it is Measured or Detected</h4>
<p>As a measure, fertility rate, is the number of offspring born per mating pair, individual or population.</p>
<h4>How it is Measured or Detected</h4>
<p>As a measure, fertility rate, is the number of offspring born per mating pair, individual or population.</p>
<br>
<h4>Regulatory Significance of the AO</h4>
<p>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.).</p>
<h4>Regulatory Significance of the AO</h4>
<p>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.).</p>
<p>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.</p>
<br>
<!-- end event text -->
</div>
<div>
<div>
<h4><a href="/events/348">Event: 348: Malformation, Male reproductive tract</a><br></h4>
<h5>Short Name: Malformation, Male reproductive tract</h5>
</div>
<h4>References</h4>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">OECD (2001), <em>Test No. 416: Two-Generation Reproduction Toxicity</em>, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris, <a href="https://doi.org/10.1787/9789264070868-en">https://doi.org/10.1787/9789264070868-en</a>.</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">OECD (2018), <em>Test No. 443: Extended One-Generation Reproductive Toxicity Study</em>, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris, <a href="https://doi.org/10.1787/9789264185371-en">https://doi.org/10.1787/9789264185371-en</a>.</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">OECD (2018), <em>Test No. 414: Prenatal Developmental Toxicity Study</em>, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris, <a href="https://doi.org/10.1787/9789264070820-en">https://doi.org/10.1787/9789264070820-en</a>.</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">OECD (2018), "Reproduction/Developmental Toxicity Screening Test (OECD TG 421) and Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (OECD TG 422)", in <em>Revised Guidance Document 150 on Standardised Test Guidelines for Evaluating Chemicals for Endocrine Disruption</em>, OECD Publishing, Paris, <a href="https://doi.org/10.1787/9789264304741-25-en">https://doi.org/10.1787/9789264304741-25-en</a>.</span></span></p>
<p>Rodents (Gray et al. 2001) Human (Manson and Carr 2003) Wildlife species (Hayes et al. 2002)</p>
<p>AGD Across numerous species, including humans, AGD is longer in males compared to females; for review see (Barrett et al. 2014).</p>
<br>
</div>
<!-- event text -->
<h4>Key Event Description</h4>
<p><strong>Biological state</strong></p>
<h4>Key Event Description</h4>
<p><strong>Biological state</strong></p>
<p><strong>Male reproductive tract malformations</strong> (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.</p>
<p><strong>Hypospadias</strong> 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.</p>
<p>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.</p>
<h4>How it is Measured or Detected</h4>
<p>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.</p>
<p>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).</p>
<p><br />
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.</p>
<br>
<h4>Regulatory Significance of the AO</h4>
<p>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.</p>
<h4>Regulatory Significance of the AO</h4>
<p>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.</p>
<p>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.).</p>
<p>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.</p>
<p>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.</p>
<p>AGD is a reproductive endpoint, assessment of AGD is mandatory in OECD TG 443, 415/416 (OECD 2012).</p>
<br>
<h4>References</h4>
<p><br />
<h4>References</h4>
<p><br />
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.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<br>
<!-- end event text -->
</div>
<h2>Appendix 2</h2>
<h2>List of Key Event Relationships in the AOP</h2>
<!-- Evidence for relationship links section, this lists the relationships and then supports them -->
<div id="evidence_supporting_links">
<hr>
<h3>List of Adjacent Key Event Relationships</h3>
<div>
<div id="evidence_supporting_links">
<h3>List of Adjacent Key Event Relationships</h3>
<div>
<h4><a href="/relationships/436">Relationship: 436: Decrease, Steroidogenic acute regulatory protein (STAR) leads to Reduction, Cholesterol transport in mitochondria</a></h4>
<td><a href="/aops/18">PPARα activation in utero leading to impaired fertility in males</a></td>
<td>adjacent</td>
<td>Moderate</td>
<td></td>
</tr>
</thead>
<tbody>
<tr>
<th><a href="/aops/18">PPARα activation in utero leading to impaired fertility in males</a></th>
<th>adjacent</th>
<th>Moderate </th>
<th></th>
</tr>
</tbody>
</table>
</div>
<h4>Evidence Supporting Applicability of this Relationship</h4>
<br>
<!-- loop to find taxonomic applicability under relationship -->
<div>
</div>
<!-- end loop for taxons -->
<!-- loop to find life stages under relationship -->
<div>
</div>
<!-- end loop for life stages -->
<!-- sex terms -->
<div>
</div>
<!-- end sex terms -->
<p>Rat see Table 1.</p>
<h4>Key Event Relationship Description</h4>
<p>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.</p>
<!-- if nothing shows up in any of these fields, then evidence supporting this KER will not be displayed -->
<h4>Evidence Supporting this KER</h4>
</tbody>
</table>
</div>
<h4>Evidence Supporting Applicability of this Relationship</h4>
<p style="text-align:justify">The steroidogenic acute regulatory protein (StAR or STARD1) belongs to the larger family of START proteins, which all include a steroidogenic acute regulatory domain (STARD) (Tugaeva & Sluchanko, 2019). This domain can be found in genomes from plants, bacteria, protists, and animals, but not in archaea or yeast (Tugaeva & Sluchanko, 2019). However, the STARD1 subfamily is found in vertebrates. </p>
<p style="text-align:justify">This KER is applicable to both sexes as the role of StAR is essential for both (Lin et al., 1995; Stocco 2002; Miller, 2011).</p>
<strong>Biological Plausibility</strong>
<p>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.</p>
<h4>Key Event Relationship Description</h4>
<p style="text-align:justify">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.</p>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p style="text-align:justify">The first step in steroidogenesis takes place within mitochondria. Cholesterol, the precursor molecule of steroids, is stored in either the plasma membrane or lipid droplets primarily of steroidogenic cells. These include theca cells and luteal cells in the ovary, Leydig cells in the testis and cells in the adrenal cortex, brain and placenta. The cholesterol delivery to the inner mitochondrial membrane (IMM), which contains insignificant cholesterol amounts, is accomplished either by ATP-dependent vesicular transport or through non-vesicular transport through protein carriers (Aghazadeh et al., 2015). </p>
<p style="text-align:justify">The non-vesicular transport of the hydrophobic cholesterol through the aqueous intermembrane space of the mitochondria is regulated by the transduceosome protein complex. The complex is assembled upon hormonal stimulation and consists of cytosolic proteins, including StAR and outer mitochondrial membrane ones like voltage-dependent anion channel 1 (VDAC1) and translocator protein (TSPO) (Aghazadeh et al., 2015). It is hypothesized that kinases, with the most prominent being protein kinase A (PKA), activate transcription factors that trigger StAR transcription and also activate StAR protein (Tugaeva & Sluchanko, 2019). Subsequently, StAR binds cholesterol and in response TSPO and VDAC1 shuttle cholesterol to P450scc. The cholesterol transfer through the tranduceosome accounts for more than 70% of the cholesterol transport to the mitochondria, therefore, any decrease on StAR levels would result in a decrease of cholesterol transport (Miller, 2017). This estimation refers to non-steroidogenic cells, and non-vesicular transport is estimated to be higher in steroidogenic cells, as it is more efficient.</p>
<strong>Empirical Evidence</strong>
<p>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.</p>
<strong>Empirical Evidence</strong>
<p style="text-align:justify">The role of StAR in cholesterol transport becomes evident in individuals with StAR deficiency. These individuals have a condition called congenital lipoid adrenal hyperplasia (LCAH), characterized by an inability to synthesize steroids and accumulated fatty deposits in steroidogenic cells, attributed to the failure of cholesterol transport to mitochondria (Stocco, 2002). These individuals can have underdeveloped genitalia and may require lifelong hormone replacement therapy. Additionally, mouse StAR knock-out studies treated with corticosteroid replacement demonstrate the same phenotype (Caron et al., 1997; Hasegawa et al., 2000).</p>
<p>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.</p>
<p>decrease uptake of cholesterol Leydig cell mitochondria</p>
</td>
<td>
<p>decreased testosterone, decreased expression of scavenger receptor B1, P450(SCC), steroidogenic acute regulatory protein, and cytochrome p450c17</p>
</td>
<td>
<p>(Thompson, Ross, and Gaido 2004)</p>
</td>
</tr>
<tr>
<td>
<p>Phthalate (DBP)</p>
</td>
<td>
<p>rat</p>
</td>
<td>
<p>LOEL=500 mg/kg</p>
</td>
<td>
<p>mRNA and protein StAR decrease</p>
</td>
<td> </td>
<td>
<p>1 dose, Time course analysis (0,5,1,2,3,6,12,18, 24h killed at GD), decreased testosterone in foetal testis</p>
</td>
<td>
<p>(Thompson et al. 2005)</p>
</td>
</tr>
<tr>
<td>
<p>Phthalate (DEHP)</p>
</td>
<td>
<p>rat</p>
</td>
<td>
<p>LOEL=300 mg/ kg/day</p>
<p> </p>
</td>
<td>
<p>mRNA StAR decrease</p>
</td>
<td>
<p> </p>
</td>
<td>
<p>dose-dependently reduced StAR, TSOP mRNA (GD 21 testes), also on protein levels in Leydig cells</p>
<p> </p>
</td>
<td>
<p>(Borch et al. 2006)</p>
</td>
</tr>
<tr>
<td>
<p>Phthalate (DBP)</p>
</td>
<td>
<p>rat</p>
</td>
<td>
<p>LOEL=500 mg/kg/day, (GD12 to 21)</p>
</td>
<td>
<p>mRNA StAR decrease</p>
</td>
<td>
<p> </p>
</td>
<td>
<p>Testes examined GD 16, 19, and 21, cytochrome P450 side chain cleavage, cytochrome P450c17, decrease.</p>
<p>Testicular testosterone and androstenedione decreased (GD 19 and 21)</p>
</td>
<td>
<p>(Shultz 2001)</p>
</td>
</tr>
<tr>
<td>
<p>Phthalate (MEHP)</p>
</td>
<td>
<p>rat</p>
</td>
<td>
<p>LOEC=250 μM</p>
</td>
<td>
<p>protein StAR decrease (immature and adult Leydig cells)</p>
</td>
<td>
<p>cholesterol transport, decrease (into the mitochondria of immature and adult Leydig cells</p>
</td>
<td>
<p>decreased testosterone by approximately 60%, in vitro ( immature and adult Leydig cells)</p>
</td>
<td>
<p>(Svechnikov, Svechnikova, and Söder 2008)</p>
</td>
</tr>
<tr>
<td>
<p>Phthalate (DBP)</p>
</td>
<td>
<p>rat</p>
</td>
<td>
<p>LOEL=500 mg/kg/day</p>
</td>
<td>
<p>mRNA StAR decrease</p>
</td>
<td>
<p> </p>
</td>
<td>
<p>GD 12 -20, examinations on GD20</p>
</td>
<td>
<p>(Johnson et al. 2011)</p>
</td>
</tr>
</tbody>
</table>
<p>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).</p>
<strong>Uncertainties and Inconsistencies</strong>
<p style="text-align:justify">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).</p>
<p style="text-align:justify">Additionally, the non-StAR-dependent transport can occur through vesicular transport or through non-specific lipid transport proteins called sterol carrier protein 2 or x (SCP2/SCPx) (Galano et al., 2022). These transport proteins are hypothesized to be a supplementary mechanism to StAR dependent cholesterol transport.</p>
<strong>Uncertainties and Inconsistencies</strong>
<p>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).</p>
<h4>Quantitative Understanding of the Linkage</h4>
<strong>Time-scale</strong>
<p style="text-align:justify">It is estimated that one molecule of StAR protein can transport 400 molecules of cholesterol per minute (Elustondo et al., 2017).</p>
<p>Mutations affecting S194 and S187 phosphorylation sites of StAR lead to LCAH (Aghazadeh et al., 2015). Phosphorylation of S194 can induce StAR activity by two-fold.</p>
<p>Barlow, Norman J, Suzanne L Phillips, Duncan G Wallace, Madhabananda Sar, Kevin W Gaido, and Paul M D Foster. 2003. “Quantitative Changes in Gene Expression in Fetal Rat Testes Following Exposure to Di(n-Butyl) Phthalate.” Toxicological Sciences : An Official Journal of the Society of Toxicology 73 (2) (June): 431–41. doi:10.1093/toxsci/kfg087.</p>
<h4>References</h4>
<p>Aghazadeh, Y., Zirkin, B. R., & Papadopoulos, V. (2015). Pharmacological Regulation of the Cholesterol Transport Machinery in Steroidogenic Cells of the Testis. In Vitamins and Hormones (Vol. 98, pp. 189–227). Academic Press Inc. https://doi.org/10.1016/bs.vh.2014.12.006 Barlow, Norman J, Suzanne L Phillips, Duncan G Wallace, Madhabananda Sar, Kevin W Gaido, and Paul M D Foster. 2003. “Quantitative Changes in Gene Expression in Fetal Rat Testes Following Exposure to Di(n-Butyl) Phthalate.” Toxicological Sciences : An Official Journal of the Society of Toxicology 73 (2) (June): 431–41. doi:10.1093/toxsci/kfg087. </p>
<p>Besman, M J, K Yanagibashi, T D Lee, M Kawamura, P F Hall, and J E Shively. 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) (July): 4897–901.</p>
<p>Besman, M J, K Yanagibashi, T D Lee, M Kawamura, P F Hall, and J E Shively. 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) (July): 4897–901. </p>
<p>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.</p>
<p>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. </p>
<p>Johnson, Kamin J, Erin N McDowell, Megan P Viereck, and Jessie Q Xia. 2011. “Species-Specific Dibutyl Phthalate Fetal Testis Endocrine Disruption Correlates with Inhibition of SREBP2-Dependent Gene Expression Pathways.” Toxicological Sciences : An Official Journal of the Society of Toxicology 120 (2) (April): 460–74. doi:10.1093/toxsci/kfr020.</p>
<p>Caron, K. M., Soo, S.-C., Wetsel, W. C., Stocco, D. M., Clark, B. J., & Parker, K. L. (1997). Targeted disruption of the mouse gene encoding steroidogenic acute regulatory protein provides insights into congenital lipoid adrenal hyperplasia. In Medical Sciences (Vol. 94). www.pnas.org. </p>
<p>Lin, D, T Sugawara, J F Strauss, B J Clark, D M Stocco, P Saenger, A Rogol, and W L Miller. 1995. “Role of Steroidogenic Acute Regulatory Protein in Adrenal and Gonadal Steroidogenesis.” Science (New York, N.Y.) 267 (5205) (March 24): 1828–31.</p>
<p>Elustondo, P., Martin, L. A., & Karten, B. (2017). Mitochondrial cholesterol import. Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids, 1862(1), 90–101. https://doi.org/10.1016/j.bbalip.2016.08.012 </p>
<p>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.</p>
<p>Galano, M., Venugopal, S., & Papadopoulos, V. (2022). Role of STAR and SCP2/SCPx in the Transport of Cholesterol and Other Lipids. In International Journal of Molecular Sciences (Vol. 23, Issue 20). MDPI. https://doi.org/10.3390/ijms232012115 </p>
<p>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.</p>
<p>Hasegawa, T., Zhao, L., Caron, K. M., Majdic, G., Suzuki, T., Shizawa, S., Sasano, H., & Parker, K. L. (2000). Developmental Roles of the Steroidogenic Acute Regulatory Protein (StAR) as Revealed by StAR Knockout Mice. In Molecular Endocrinology (Vol. 14). https://academic.oup.com/mend/article/14/9/1462/2751100 Johnson, Kamin J, Erin N McDowell, Megan P Viereck, and Jessie Q Xia. 2011. “Species-Specific Dibutyl Phthalate Fetal Testis Endocrine Disruption Correlates with Inhibition of SREBP2-Dependent Gene Expression Pathways.” Toxicological Sciences : An Official Journal of the Society of Toxicology 120 (2) (April): 460–74. doi:10.1093/toxsci/kfr020. </p>
<p>Svechnikov, Konstantin, Irina Svechnikova, and Olle Söder. 2008. “Inhibitory Effects of Mono-Ethylhexyl Phthalate on Steroidogenesis in Immature and Adult Rat Leydig Cells in Vitro.” Reproductive Toxicology (Elmsford, N.Y.) 25 (4) (August): 485–90. doi:10.1016/j.reprotox.2008.05.057.</p>
<p>Lin, D, T Sugawara, J F Strauss, B J Clark, D M Stocco, P Saenger, A Rogol, and W L Miller. 1995. “Role of Steroidogenic Acute Regulatory Protein in Adrenal and Gonadal Steroidogenesis.” Science (New York, N.Y.) 267 (5205) (March 24): 1828–31. </p>
<p>Thompson, Christopher J, Susan M Ross, and Kevin W Gaido. 2004. “Di(n-Butyl) Phthalate Impairs Cholesterol Transport and Steroidogenesis in the Fetal Rat Testis through a Rapid and Reversible Mechanism.” Endocrinology 145 (3) (March): 1227–37. doi:10.1210/en.2003-1475.</p>
<p>Men, Y., Fan, Y., Shen, Y., Lu, L., & Kallen, A. N. (2017). The steroidogenic acute regulatory protein (StAR) is regulated by the H19/let-7 axis. Endocrinology, 158(2), 402–409. https://doi.org/10.1210/en.2016-1340 </p>
<p>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.</p>
<p>Miller, W. L. (2017). Steroidogenesis: Unanswered Questions. In Trends in Endocrinology and Metabolism (Vol. 28, Issue 11, pp. 771–793). Elsevier Inc. https://doi.org/10.1016/j.tem.2017.09.002 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. </p>
<p>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. </p>
<p>Stocco, D. M. (2002). Clinical disorders associated with abnormal cholesterol transport: mutations in the steroidogenic acute regulatory protein. www.elsevier.com/locate/mce Svechnikov, Konstantin, Irina Svechnikova, and Olle Söder. 2008. “Inhibitory Effects of Mono-Ethylhexyl Phthalate on Steroidogenesis in Immature and Adult Rat Leydig Cells in Vitro.” Reproductive Toxicology (Elmsford, N.Y.) 25 (4) (August): 485–90. doi:10.1016/j.reprotox.2008.05.057. </p>
<p>Thompson, Christopher J, Susan M Ross, and Kevin W Gaido. 2004. “Di(n-Butyl) Phthalate Impairs Cholesterol Transport and Steroidogenesis in the Fetal Rat Testis through a Rapid and Reversible Mechanism.” Endocrinology 145 (3) (March): 1227–37. doi:10.1210/en.2003-1475. </p>
<p>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. </p>
<p>Tugaeva, K. V., & Sluchanko, N. N. (2019). Steroidogenic Acute Regulatory Protein: Structure, Functioning, and Regulation. In Biochemistry (Moscow) (Vol. 84, pp. 233–253). Pleiades journals. https://doi.org/10.1134/S0006297919140141</p>
</div>
<br>
<div>
<div>
<h4><a href="/relationships/438">Relationship: 438: Reduction, Cholesterol transport in mitochondria leads to Reduction, Testosterone synthesis in Leydig cells</a></h4>
<!-- loop to find life stages under relationship -->
<div>
</div>
<!-- end loop for life stages -->
<!-- sex terms -->
<div>
</div>
<!-- end sex terms -->
<p>See Table 1.</p>
<h4>Key Event Relationship Description</h4>
<p>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.</p>
<!-- if nothing shows up in any of these fields, then evidence supporting this KER will not be displayed -->
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p>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.</p>
<h4>Key Event Relationship Description</h4>
<p>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.</p>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p>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.</p>
<strong>Empirical Evidence</strong>
<p>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).</p>
<strong>Empirical Evidence</strong>
<p>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).</p>
<p>dose-dependently reduced StAR, TSOP mRNA (GD 21 testes), also on protein levels in Leydig cells (Borch, Metzdorff, Vinggaard, Brokken, & Dalgaard, 2006)</p>
<p> </p>
</td>
<td>
<p> </p>
</td>
</tr>
<tr>
<td>
<p>Phthalate (DEHP)</p>
</td>
<td>
<p>rat</p>
</td>
<td>
<p>LOEL=300 mg/kg/day</p>
</td>
<td>
<p> </p>
</td>
<td>
<p>testosterone production, reduction (ex vivo) testosterone levels, reduction (Borch et al., 2006), (Borch, Ladefoged, Hass, & Vinggaard, 2004)</p>
</td>
</tr>
<tr>
<td>
<p>Phthalate (MEHP)</p>
</td>
<td>
<p>mouse</p>
</td>
<td>
<p>LOEC=100 μM</p>
</td>
<td>
<ul>
<li>
<p>reduced TSPO mRNA levels by 50%,</p>
</li>
<li>
<p>binding sites decreased by 50%</p>
</li>
<li>
<p>no effect on receptor affinity</p>
</li>
<li>
<p>inhibited the transfer or loading of cholesterol to the inner mitochondrial membrane P450scc. (Gazouli, 2002)</p>
</li>
</ul>
</td>
<td>
<p> </p>
</td>
</tr>
<tr>
<td>
<p>Phthalate (MEHP)</p>
</td>
<td>
<p>rat</p>
</td>
<td>
<p>IC50 =100 μM</p>
</td>
<td>
<ul>
<li>
<p>inhibited formation of progesterone (Gazouli, 2002)</p>
</li>
</ul>
</td>
<td>
<p> </p>
</td>
</tr>
<tr>
<td>
<p>Phthalate (MEHP)</p>
</td>
<td>
<p>rat</p>
</td>
<td>
<p>LOEC=250 μM</p>
</td>
<td>
<p>cholesterol transport, decrease (into the mitochondria of immature and adult Leydig cells)</p>
</td>
<td>
<p>Testosterone, reduction by approximately 60%, in vitro ( immature and adult Leydig cells) (Svechnikov, Svechnikova, & Söder, 2008)</p>
</td>
</tr>
<tr>
<td>
<p>Phthalate (DEHP)</p>
</td>
<td>
<p>rat</p>
</td>
<td>
<p>LOEL=750 mg/kg/day</p>
</td>
<td>
<p> </p>
</td>
<td>
<p>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)</p>
</td>
</tr>
<tr>
<td>
<p>Phthalate (MEHP)</p>
</td>
<td>
<p>rat</p>
</td>
<td>
<p>LOEC=1 μM</p>
</td>
<td>
<p> </p>
</td>
<td>
<p>testosterone production, reduction dose-dependent (Chauvigné et al., 2011)</p>
</td>
</tr>
<tr>
<td>
<p>Perfluorooctanoic acid (PFOA)</p>
</td>
<td>
<p>mouse</p>
</td>
<td>
<p>LOEL=5mg/kg/day</p>
</td>
<td> </td>
<td>
<p>plasma testosterone, reduction (by 37%)(Li et al., 2011)</p>
<p>testicular testosterone production and testicular testosterone levels, (Boberg et al., 2008)</p>
</td>
</tr>
</tbody>
</table>
<p><br />
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</p>
<strong>Uncertainties and Inconsistencies</strong>
<p>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).</p>
<strong>Uncertainties and Inconsistencies</strong>
<p>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).</p>
<p>Barlow, N. J., Phillips, S. L., Wallace, D. G., Sar, M., Gaido, K. W., & Foster, P. M. D. (2003). Quantitative changes in gene expression in fetal rat testes following exposure to di(n-butyl) phthalate. Toxicological Sciences : An Official Journal of the Society of Toxicology, 73(2), 431–41. doi:10.1093/toxsci/kfg087</p>
<h4>References</h4>
<p>Barlow, N. J., Phillips, S. L., Wallace, D. G., Sar, M., Gaido, K. W., & Foster, P. M. D. (2003). Quantitative changes in gene expression in fetal rat testes following exposure to di(n-butyl) phthalate. Toxicological Sciences : An Official Journal of the Society of Toxicology, 73(2), 431–41. doi:10.1093/toxsci/kfg087</p>
<p>Boberg, J., Metzdorff, S., Wortziger, R., Axelstad, M., Brokken, L., Vinggaard, A. M., … Nellemann, C. (2008). Impact of diisobutyl phthalate and other PPAR agonists on steroidogenesis and plasma insulin and leptin levels in fetal rats. Toxicology, 250(2-3), 75–81. doi:10.1016/j.tox.2008.05.020</p>
<p>Borch, J., Ladefoged, O., Hass, U., & Vinggaard, A. M. (2004). Steroidogenesis in fetal male rats is reduced by DEHP and DINP, but endocrine effects of DEHP are not modulated by DEHA in fetal, prepubertal and adult male rats. Reproductive Toxicology (Elmsford, N.Y.), 18(1), 53–61. doi:10.1016/j.reprotox.2003.10.011</p>
<p>Borch, J., Metzdorff, S. B., Vinggaard, A. M., Brokken, L., & Dalgaard, M. (2006). Mechanisms underlying the anti-androgenic effects of diethylhexyl phthalate in fetal rat testis. Toxicology, 223(1-2), 144–55. doi:10.1016/j.tox.2006.03.015</p>
<p>Chauvigné, F., Plummer, S., Lesné, L., Cravedi, J.-P., Dejucq-Rainsford, N., Fostier, A., & Jégou, B. (2011). Mono-(2-ethylhexyl) phthalate directly alters the expression of Leydig cell genes and CYP17 lyase activity in cultured rat fetal testis. PloS One, 6(11), e27172. doi:10.1371/journal.pone.0027172</p>
<p>Furr, J. R., Lambright, C. S., Wilson, V. S., Foster, P. M., & Gray, L. E. (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), 403–24. doi:10.1093/toxsci/kfu081</p>
<p>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), 2571–2583. doi:10.1210/en.143.7.2571</p>
<p>Hauet, T., Yao, Z.-X., Bose, H. S., Wall, C. T., Han, Z., Li, W., … Papadopoulos, V. (2005). Peripheral-type benzodiazepine receptor-mediated action of steroidogenic acute regulatory protein on cholesterol entry into leydig cell mitochondria. Molecular Endocrinology (Baltimore, Md.), 19(2), 540–54. doi:10.1210/me.2004-0307</p>
<p>Johnson, K. J., McDowell, E. N., Viereck, M. P., & Xia, J. Q. (2011). Species-specific dibutyl phthalate fetal testis endocrine disruption correlates with inhibition of SREBP2-dependent gene expression pathways. Toxicological Sciences : An Official Journal of the Society of Toxicology, 120(2), 460–74. doi:10.1093/toxsci/kfr020</p>
<p>Lehmann, K. P., Phillips, S., Sar, M., Foster, P. M. D., & Gaido, K. W. (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), 60–8. doi:10.1093/toxsci/kfh169</p>
<p>Li, Y., Ramdhan, D. H., Naito, H., Yamagishi, N., Ito, Y., Hayashi, Y., … Nakajima, T. (2011). Ammonium perfluorooctanoate may cause testosterone reduction by adversely affecting testis in relation to PPARα. Toxicology Letters, 205(3), 265–72. doi:10.1016/j.toxlet.2011.06.015 Miller, W. L. (2007). Steroidogenic acute regulatory protein (StAR), a novel mitochondrial cholesterol transporter. Biochimica et Biophysica Acta, 1771(6), 663–76. doi:10.1016/j.bbalip.2007.02.012</p>
<p>Parks, L. G. (2000). The Plasticizer Diethylhexyl Phthalate Induces Malformations by Decreasing Fetal Testosterone Synthesis during Sexual Differentiation in the Male Rat. Toxicological Sciences, 58(2), 339–349. doi:10.1093/toxsci/58.2.339</p>
<p>Payne, A. H., & Hales, D. B. (2013). Overview of Steroidogenic Enzymes in the Pathway from Cholesterol to Active Steroid Hormones. Endocrine Reviews. Stocco, D. M. (2001). StAR protein and the regulation of steroid hormone biosynthesis. Annual Review of Physiology, 63, 193–213. doi:10.1146/annurev.physiol.63.1.193</p>
<p>Svechnikov, K., Svechnikova, I., & Söder, O. (2008). Inhibitory effects of mono-ethylhexyl phthalate on steroidogenesis in immature and adult rat Leydig cells in vitro. Reproductive Toxicology (Elmsford, N.Y.), 25(4), 485–90. doi:10.1016/j.reprotox.2008.05.057</p>
<p>Thompson, C. J., Ross, S. M., & Gaido, K. W. (2004). Di(n-butyl) phthalate impairs cholesterol transport and steroidogenesis in the fetal rat testis through a rapid and reversible mechanism. Endocrinology, 145(3), 1227–37. doi:10.1210/en.2003-1475</p>
<p>Thompson, C. J., Ross, S. M., Hensley, J., Liu, K., Heinze, S. C., Young, S. S., & Gaido, K. W. (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), 908–17. doi:10.1095/biolreprod.105.042382</p>
<p>Wilson, V. S., Lambright, C., Furr, J., Ostby, J., Wood, C., Held, G., & Gray, L. E. (2004). Phthalate ester-induced gubernacular lesions are associated with reduced insl3 gene expression in the fetal rat testis. Toxicology Letters, 146(3), 207–15.</p>
</div>
<br>
<div>
<h4><a href="/relationships/439">Relationship: 439: Reduction, Testosterone synthesis in Leydig cells leads to Reduction, testosterone level</a></h4>
<div>
<h4><a href="/relationships/2125">Relationship: 2125: Reduction, Testosterone synthesis in Leydig cells leads to Decrease, circulating testosterone levels</a></h4>
<!-- loop to find life stages under relationship -->
<div>
</div>
<!-- end loop for life stages -->
<!-- sex terms -->
<div>
</div>
<!-- end sex terms -->
<p>Ses Table 1.</p>
<h4>Key Event Relationship Description</h4>
<p>Impairment of testosterone production in testes directly impacts on testosterone levels.</p>
<!-- if nothing shows up in any of these fields, then evidence supporting this KER will not be displayed -->
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p>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.</p>
<h4>Key Event Relationship Description</h4>
<div>
<p>Impairment of testosterone production in testes directly impacts on testosterone levels.</p>
</div>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p>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.</p>
<strong>Empirical Evidence</strong>
<p>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.</p>
<strong>Empirical Evidence</strong>
<p>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.</p>
<p>testicular testosterone production, reduction (ex vivo)</p>
</td>
<td>
<p>testicular testosterone levels, reduction, no change plasma testosterone</p>
</td>
<td>
<p>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</p>
</td>
<td>
<p>(Borch, Metzdorff, Vinggaard, Brokken, & Dalgaard, 2006)</p>
</td>
</tr>
<tr>
<td>
<p>Phthalates</p>
<p>(DBP)</p>
</td>
<td>
<p>rat</p>
</td>
<td>
<p>LOEL =50 mg/kg/day</p>
</td>
<td>
<p> </p>
</td>
<td>
<p>testicular testosterone levels, reduction,</p>
</td>
<td>
<p>Testicular testosterone was reduced >50 mg/kg/day</p>
<p>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.</p>
<h4>References</h4>
<p>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.</p>
<p>Borch, Julie, Ole Ladefoged, Ulla Hass, and Anne Marie Vinggaard. 2004. “Steroidogenesis in Fetal Male Rats Is Reduced by DEHP and DINP, but Endocrine Effects of DEHP Are Not Modulated by DEHA in Fetal, Prepubertal and Adult Male Rats.” Reproductive Toxicology (Elmsford, N.Y.) 18 (1): 53–61. doi:10.1016/j.reprotox.2003.10.011.</p>
<p>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.</p>
<p>Culty, Martine, Raphael Thuillier, Wenping Li, Yan Wang, Daniel B Martinez-Arguelles, Carolina Gesteira Benjamin, Kostantinos M Triantafilou, Barry R Zirkin, and Vassilios Papadopoulos. 2008. “In Utero Exposure to Di-(2-Ethylhexyl) Phthalate Exerts Both Short-Term and Long-Lasting Suppressive Effects on Testosterone Production in the Rat.” Biology of Reproduction 78 (6) (June): 1018–28. doi:10.1095/biolreprod.107.065649.</p>
<p>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.</p>
<p>Parks, L. G. 2000. “The Plasticizer Diethylhexyl Phthalate Induces Malformations by Decreasing Fetal Testosterone Synthesis during Sexual Differentiation in the Male Rat.” Toxicological Sciences 58 (2) (December 1): 339–349. doi:10.1093/toxsci/58.2.339.</p>
<p>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.</p>
<p>Wilson, Vickie S., Christy Lambright, Johnathan Furr, Joseph Ostby, Carmen Wood, Gary Held, and L.Earl Gray. 2004. “Phthalate Ester-Induced Gubernacular Lesions Are Associated with Reduced insl3 Gene Expression in the Fetal Rat Testis.” Toxicology Letters 146 (3) (February): 207–215. doi:10.1016/j.toxlet.2003.09.012.</p>
</div>
<br>
<div>
<div>
<h4><a href="/relationships/437">Relationship: 437: Decrease, Translocator protein (TSPO) leads to Reduction, Cholesterol transport in mitochondria</a></h4>
<!-- loop to find life stages under relationship -->
<div>
</div>
<!-- end loop for life stages -->
<!-- sex terms -->
<div>
</div>
<!-- end sex terms -->
<p>Rat (Papadopoulos et al., 1997)</p>
<h4>Key Event Relationship Description</h4>
<p>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.</p>
<!-- if nothing shows up in any of these fields, then evidence supporting this KER will not be displayed -->
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p>The 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).</p>
<strong>Empirical Evidence</strong>
<p>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).</p>
<strong>Uncertainties and Inconsistencies</strong>
<p>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).</p>
<h4>Key Event Relationship Description</h4>
<p>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.</p>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p>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).</p>
<strong>Empirical Evidence</strong>
<p>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).</p>
<strong>Uncertainties and Inconsistencies</strong>
<p>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).</p>
<h4>References</h4>
<p>Amsterdam, A., & Suh, B. S. (1991). An inducible functional peripheral benzodiazepine receptor in mitochondria of steroidogenic granulosa cells. Endocrinology, 129(1), 503–10. doi:10.1210/endo-129-1-503 Anholt, R., Pedersen, P., De Souza, E., & Snyder, S. (1986). The peripheral-type benzodiazepine receptor. Localization to the mitochondrial outer membrane. J. Biol. Chem., 261(2), 576–583. Anholt, R. R., De Souza, E. B., Oster-Granite, M. L., & Snyder, S. H. (1985). Peripheral-type benzodiazepine receptors: autoradiographic localization in whole-body sections of neonatal rats. The Journal of Pharmacology and Experimental Therapeutics, 233(2), 517–26.</p>
<h4>References</h4>
<p>Amsterdam, A., & Suh, B. S. (1991). An inducible functional peripheral benzodiazepine receptor in mitochondria of steroidogenic granulosa cells. Endocrinology, 129(1), 503–10. doi:10.1210/endo-129-1-503 Anholt, R., Pedersen, P., De Souza, E., & Snyder, S. (1986). The peripheral-type benzodiazepine receptor. Localization to the mitochondrial outer membrane. J. Biol. Chem., 261(2), 576–583. Anholt, R. R., De Souza, E. B., Oster-Granite, M. L., & Snyder, S. H. (1985). Peripheral-type benzodiazepine receptors: autoradiographic localization in whole-body sections of neonatal rats. The Journal of Pharmacology and Experimental Therapeutics, 233(2), 517–26.</p>
<p>Besman, M. J., Yanagibashi, K., Lee, T. D., Kawamura, M., Hall, P. F., & Shively, J. E. (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), 4897–901.</p>
<p>Borch, J., Metzdorff, S. B., Vinggaard, A. M., Brokken, L., & Dalgaard, M. (2006). Mechanisms underlying the anti-androgenic effects of diethylhexyl phthalate in fetal rat testis. Toxicology, 223(1-2), 144–55. doi:10.1016/j.tox.2006.03.015</p>
<p>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), 2571–2583. doi:10.1210/en.143.7.2571</p>
<p>Morohaku, K., Pelton, S. H., Daugherty, D. J., Butler, W. R., Deng, W., & Selvaraj, V. (2014). Translocator protein/peripheral benzodiazepine receptor is not required for steroid hormone biosynthesis. Endocrinology, 155(1), 89–97. doi:10.1210/en.2013-1556</p>
<p>Morohaku, K., Phuong, N. S., & Selvaraj, V. (2013). Developmental expression of translocator protein/peripheral benzodiazepine receptor in reproductive tissues. PloS One, 8(9), e74509. doi:10.1371/journal.pone.0074509</p>
<p>Papadopoulos, V., Amri, H., Li, H., Boujrad, N., Vidic, B., & Garnier, M. (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), 32129–35.</p>
<p>Selvaraj, V., Stocco, D. M., & Tu, L. N. (2015). TRANSLOCATOR PROTEIN (TSPO) AND STEROIDOGENESIS: A REAPPRAISAL. Molecular Endocrinology (Baltimore, Md.), me20151033. doi:10.1210/me.2015-1033</p>
<p>Thompson, C. J., Ross, S. M., & Gaido, K. W. (2004). Di(n-butyl) phthalate impairs cholesterol transport and steroidogenesis in the fetal rat testis through a rapid and reversible mechanism. Endocrinology, 145(3), 1227–37. doi:10.1210/en.2003-1475</p>
<p>Tu, L. N., Morohaku, K., Manna, P. R., Pelton, S. H., Butler, W. R., Stocco, D. M., & Selvaraj, V. (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), 27444–54. doi:10.1074/jbc.M114.578286</p>
<p>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), 261–75. doi:10.1007/s00441-012-1478-5</p>
<p>Zisterer, D. M., & Williams, D. C. (1997). Peripheral-type benzodiazepine receptors. General Pharmacology, 29(3), 305–14.</p>
</div>
<br>
<div>
<h4><a href="/relationships/405">Relationship: 405: Malformation, Male reproductive tract leads to impaired, Fertility</a></h4>
<div>
<h4><a href="/relationships/405">Relationship: 405: Malformation, Male reproductive tract leads to decreased, Fertility</a></h4>
<p>Human and Rat see "Empirical Support for Linkage"</p>
</div>
<!-- end loop for taxons -->
<!-- loop to find life stages under relationship -->
<div>
</div>
<!-- end loop for life stages -->
<!-- sex terms -->
<div>
</div>
<!-- end sex terms -->
<p>Human and Rat see "Empirical Support for Linkage"</p>
<h4>Key Event Relationship Description</h4>
<p>Impairment in the normal development of the male reproductive tract (e.g. genital abnormality and/or cryptorchidism) can impact on fertility later in life.</p>
<!-- if nothing shows up in any of these fields, then evidence supporting this KER will not be displayed -->
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p>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).</p>
<h4>Key Event Relationship Description</h4>
<p>Impairment in the normal development of the male reproductive tract (e.g. genital abnormality and/or cryptorchidism) can impact on fertility later in life.</p>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p>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).</p>
<strong>Empirical Evidence</strong>
<p><strong>Human</strong></p>
<strong>Empirical Evidence</strong>
<p><strong>Human</strong></p>
<p>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).</p>
<p><strong>Rat</strong></p>
<p>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).</p>
<p>Asklund, C, T K Jensen, K M Main, T Sobotka, N E Skakkebaek, and N Jørgensen. 2010. “Semen Quality, Reproductive Hormones and Fertility of Men Operated for Hypospadias.” International Journal of Andrology 33 (1) (February): 80–7. doi:10.1111/j.1365-2605.2009.00957.x.</p>
<h4>References</h4>
<p>Asklund, C, T K Jensen, K M Main, T Sobotka, N E Skakkebaek, and N Jørgensen. 2010. “Semen Quality, Reproductive Hormones and Fertility of Men Operated for Hypospadias.” International Journal of Andrology 33 (1) (February): 80–7. doi:10.1111/j.1365-2605.2009.00957.x.</p>
<p>Bracka, A. 1989. “A Long-Term View of Hypospadias.” British Journal of Plastic Surgery 42 (3) (May): 251–5.</p>
<p>Cowin, Prue A, Elspeth Gold, Jasna Aleksova, Moira K O’Bryan, Paul M D Foster, Hamish S Scott, and Gail P Risbridger. 2010. “Vinclozolin Exposure in Utero Induces Postpubertal Prostatitis and Reduces Sperm Production via a Reversible Hormone-Regulated Mechanism.” Endocrinology 151 (2) (February): 783–92. doi:10.1210/en.2009-0982.</p>
<p>Eisenberg, Michael L, Michael H Hsieh, Rustin Chanc Walters, Ross Krasnow, and Larry I Lipshultz. 2011. “The Relationship between Anogenital Distance, Fatherhood, and Fertility in Adult Men.” PloS One 6 (5) (January): e18973. doi:10.1371/journal.pone.0018973.</p>
<p>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.</p>
<p>Mendiola, Jaime, Richard W Stahlhut, Niels Jørgensen, Fan Liu, and Shanna H Swan. 2011. “Shorter Anogenital Distance Predicts Poorer Semen Quality in Young Men in Rochester, New York.” Environmental Health Perspectives 119 (7) (July): 958–63. doi:10.1289/ehp.1103421.</p>
<p>NTP. 2005. “Multigenerational Reproductive Assessment by Continuous Breeding When Diethylhexylphthalate (CAS 117-81-7).”</p>
<p>Skakkebaek, N E, E Rajpert-De Meyts, and K M Main. 2001. “Testicular Dysgenesis Syndrome: An Increasingly Common Developmental Disorder with Environmental Aspects.” Human Reproduction (Oxford, England) 16 (5) (May): 972–8.</p>
<p>Thorup, Jorgen, Robert McLachlan, Dina Cortes, Tamara R. Nation, Adam Balic, Bridget R. Southwell, and John M. Hutson. 2010. “What Is New in Cryptorchidism and Hypospadias - A Critical Review on the Testicular Dysgenesis Hypothesis.” Journal of Pediatric Surgery. doi:10.1016/j.jpedsurg.2010.07.030.</p>
</div>
<br>
<h3>List of Non Adjacent Key Event Relationships</h3>
<div>
<h3>List of Non Adjacent Key Event Relationships</h3>
<div>
<h4><a href="/relationships/369">Relationship: 369: Activation, PPARα leads to Decrease, Steroidogenic acute regulatory protein (STAR)</a></h4>
<!-- loop to find life stages under relationship -->
<div>
</div>
<!-- end loop for life stages -->
<!-- sex terms -->
<div>
</div>
<!-- end sex terms -->
<p>See Table 1.</p>
<h4>Key Event Relationship Description</h4>
<p>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)).</p>
<!-- if nothing shows up in any of these fields, then evidence supporting this KER will not be displayed -->
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p>The 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.</p>
<h4>Key Event Relationship Description</h4>
<p>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)).</p>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p>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.</p>
<strong>Empirical Evidence</strong>
<p>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).</p>
<strong>Empirical Evidence</strong>
<p>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).</p>
<p> </p>
<table class="wikitable" id="KER369">
<tbody>
<tr>
<td>
<p>Compound</p>
</td>
<td>
<p>species</p>
</td>
<td>
<p>KE: PPARα, Activation</p>
</td>
<td>
<p>KE: StAR, Decrease</p>
</td>
</tr>
<tr>
<td>
<p>Phthalate (MBzP )</p>
</td>
<td>
<p>human</p>
</td>
<td>
<p>EC50 = 30 μM human (Hurst and Waxman 2003)</p>
</td>
<td>
<p>n.a.</p>
</td>
</tr>
<tr>
<td>
<p>Phthalate (DBP)</p>
</td>
<td>
<p>rodent</p>
</td>
<td>
<p>EC50 = 21 μM MBzP</p>
<p>EC50 = 63 μM (Hurst and Waxman 2003) MBuP</p>
</td>
<td>
<p>LOEC=50 mg/kg/day (Borch et al. 2006) (Lehmann et al. 2004), (Shultz 2001)</p>
</td>
</tr>
<tr>
<td>
<p>Phthalate (MEHP)</p>
</td>
<td>
<p>rodent</p>
</td>
<td>
<p>LOEC=30 μM (Bility et al. 2004)</p>
</td>
<td>
<p>LOEC=300 mg/kg/day (Borch et al. 2006)</p>
</td>
</tr>
<tr>
<td>
<p>Phthalate (MEHP)</p>
</td>
<td>
<p>human</p>
</td>
<td>
<p>Ki=15 µM (Lapinskas et al. 2005)</p>
<p>EC50 = 3.2 μM</p>
<p>(Hurst and Waxman 2003)</p>
</td>
<td>
<p>n.a.</p>
</td>
</tr>
<tr>
<td>
<p>Phthalate (DiBP)</p>
</td>
<td>
<p>rat</p>
</td>
<td>
<p>LOEC=600 mg/kg/day, decrease of PPARα mRNA, at GD 19 and 21 in testes of foetuses (Boberg et al. 2008)</p>
</td>
<td>
<p>LOEC=600 mg/kg/day, decrease of StAR mRNA at GD 19 and 21 in testes of foetuses (Boberg et al. 2008)</p>
</td>
</tr>
<tr>
<td>
<p>Econazole</p>
</td>
<td>
<p>rodent</p>
</td>
<td>
<p>AC50=0.0729 μM (ToxCastTM Data)</p>
</td>
<td>
<p>LOEC=25 μM, mouse MA-10 Leydig tumor cell line, Decrease in StAR activity and/or expression (Walsh, Kuratko, and Stocco 2000)</p>
</td>
</tr>
<tr>
<td>
<p>Perfluorooctanoate (PFOA)</p>
</td>
<td>
<p>mice</p>
</td>
<td>
<p>3T3-L1 cells transfected with human, mouse and rat PPARα (Vanden Heuvel et al. 2006)</p>
</td>
<td>
<p>LOEC= 5.0 mg/kg/day mRNA StAR decrease in the testis of wild-type<em> </em>mice, LOEC=1.0 mg/kg/day mRNA StAR decrease in testis of</p>
<p><em>PPARα-humanized</em> mice (not in the PPAR<em>α</em> –null mice) (Li et al. 2011)</p>
</td>
</tr>
<tr>
<td>
<p>Fenofibrate</p>
</td>
<td>
<p>mice</p>
</td>
<td>
<p>PPAR agonist</p>
<p>Increase PPARα protein (Toda et al. 2003).</p>
</td>
<td>
<p>Decrease of StAR (protein level, in mice ovaries) (Toda et al. 2003)</p>
</td>
</tr>
</tbody>
</table>
<p>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.</p>
<strong>Uncertainties and Inconsistencies</strong>
<p><strong>Uncertainties</strong></p>
<strong>Uncertainties and Inconsistencies</strong>
<p><strong>Uncertainties</strong></p>
<p>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)).</p>
<p><strong>Inconsistencies</strong> 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).</p>
<p>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. <a class="external free" href="http://www.ncbi.nlm.nih.gov/pubmed/15310864" rel="nofollow" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed/15310864</a>.</p>
<h4>References</h4>
<p>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. <a class="external free" href="http://www.ncbi.nlm.nih.gov/pubmed/15310864" rel="nofollow" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed/15310864</a>.</p>
<p>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>.</p>
<p>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>.</p>
<p>Corton, JC, C Bocos, ES Moreno, A Merritt, DS Marsman, PJ Sausen, RC Cattley, and JA Gustafsson. 1996. “Rat 17 Beta-Hydroxysteroid Dehydrogenase Type IV Is a Novel Peroxisome Proliferator-Inducible Gene.” Mol. Pharmacol. 50 (5) (November 1): 1157–1166. <a class="external free" href="http://molpharm.aspetjournals.org/content/50/5/1157.abstract?ijkey=a767a7a5a99dd83cc9fe3e4b601372c7ea4caa62&keytype2=tf_ipsecsha" rel="nofollow" target="_blank">http://molpharm.aspetjournals.org/content/50/5/1157.abstract?ijkey=a767a7a5a99dd83cc9fe3e4b601372c7ea4caa62&keytype2=tf_ipsecsha</a>.</p>
<p>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>.</p>
<p>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>.</p>
<p>Hermanowski-Vosatka, A, D Gerhold, S S Mundt, V A Loving, M Lu, Y Chen, A Elbrecht, et al. 2000. “PPARalpha Agonists Reduce 11beta-Hydroxysteroid Dehydrogenase Type 1 in the Liver.” Biochemical and Biophysical Research Communications 279 (2) (December 20): 330–6. doi:10.1006/bbrc.2000.3966. <a class="external free" href="http://www.ncbi.nlm.nih.gov/pubmed/11118287" rel="nofollow" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed/11118287</a>.</p>
<p>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. <a class="external free" href="http://www.ncbi.nlm.nih.gov/pubmed/12805656" rel="nofollow" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed/12805656</a>.</p>
<p>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>.</p>
<p>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>.</p>
<p>Li, Yufei, Doni Hikmat Ramdhan, Hisao Naito, Nozomi Yamagishi, Yuki Ito, Yumi Hayashi, Yukie Yanagiba, et al. 2011. “Ammonium Perfluorooctanoate May Cause Testosterone Reduction by Adversely Affecting Testis in Relation to PPARα.” Toxicology Letters 205 (3) (September 10): 265–72. doi:10.1016/j.toxlet.2011.06.015. <a class="external free" href="http://www.ncbi.nlm.nih.gov/pubmed/21712084" rel="nofollow" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed/21712084</a>.</p>
<p>Liu, Kejun, Kim P Lehmann, Madhabananda Sar, S Stanley Young, and Kevin W Gaido. 2005. “Gene Expression Profiling Following in Utero Exposure to Phthalate Esters Reveals New Gene Targets in the Etiology of Testicular Dysgenesis.” Biology of Reproduction 73 (1) (July): 180–92. doi:10.1095/biolreprod.104.039404. <a class="external free" href="http://www.ncbi.nlm.nih.gov/pubmed/15728792" rel="nofollow" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed/15728792</a>.</p>
<p>McCampbell, A. 2000. “CREB-Binding Protein Sequestration by Expanded Polyglutamine.” Human Molecular Genetics 9 (14) (September 1): 2197–2202. doi:10.1093/hmg/9.14.2197. <a class="external free" href="http://hmg.oxfordjournals.org/content/9/14/2197.abstract?ijkey=c35580e57df64d1fc98fb242bf4ed19362a4a3ce&keytype2=tf_ipsecsha" rel="nofollow" target="_blank">http://hmg.oxfordjournals.org/content/9/14/2197.abstract?ijkey=c35580e57df64d1fc98fb242bf4ed19362a4a3ce&keytype2=tf_ipsecsha</a>.</p>
<p>Plummer, Simon M, Dhritiman Dan, Joanne Quinney, Nina Hallmark, Richard D Phillips, Michael Millar, Sheila Macpherson, and Clifford R Elcombe. 2013. “Identification of Transcription Factors and Coactivators Affected by Dibutylphthalate Interactions in Fetal Rat Testes.” Toxicological Sciences : An Official Journal of the Society of Toxicology 132 (2) (April): 443–57. doi:10.1093/toxsci/kft016. <a class="external free" href="http://www.ncbi.nlm.nih.gov/pubmed/23358192" rel="nofollow" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed/23358192</a>.</p>
<p>Plummer, Simon, Richard M Sharpe, Nina Hallmark, Isobel Kim Mahood, and Cliff Elcombe. 2007. “Time-Dependent and Compartment-Specific Effects of in Utero Exposure to Di(n-Butyl) Phthalate on Gene/protein Expression in the Fetal Rat Testis as Revealed by Transcription Profiling and Laser Capture Microdissection.” Toxicological Sciences : An Official Journal of the Society of Toxicology 97 (2) (June 1): 520–32. doi:10.1093/toxsci/kfm062. <a class="external free" href="http://www.ncbi.nlm.nih.gov/pubmed/17379624" rel="nofollow" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed/17379624</a>.</p>
<p>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. <a class="external free" href="http://www.ncbi.nlm.nih.gov/pubmed/10385388" rel="nofollow" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed/10385388</a>.</p>
<p>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>. 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. <a class="external free" href="http://www.ncbi.nlm.nih.gov/pubmed/11181954" rel="nofollow" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed/11181954</a>.</p>
<p>Sugawara, T, J A Holt, M Kiriakidou, and J F Strauss. 1996. “Steroidogenic Factor 1-Dependent Promoter Activity of the Human Steroidogenic Acute Regulatory Protein (StAR) Gene.” Biochemistry 35 (28) (July 16): 9052–9. doi:10.1021/bi960057r. <a class="external free" href="http://www.ncbi.nlm.nih.gov/pubmed/8703908" rel="nofollow" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed/8703908</a>.</p>
<p>Toda, Katsumi, Teruhiko Okada, Chisata Miyaura, and Toshiji Saibara. 2003. “Fenofibrate, a Ligand for PPARalpha, Inhibits Aromatase Cytochrome P450 Expression in the Ovary of Mouse.” Journal of Lipid Research 44 (2) (February): 265–70. doi:10.1194/jlr.M200327-JLR200. <a class="external free" href="http://www.ncbi.nlm.nih.gov/pubmed/12576508" rel="nofollow" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed/12576508</a>. 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>.</p>
<p>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. <a class="external free" href="http://www.ncbi.nlm.nih.gov/pubmed/16731579" rel="nofollow" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed/16731579</a>.</p>
<p>Walsh, L P, C N Kuratko, and D M Stocco. 2000. “Econazole and Miconazole Inhibit Steroidogenesis and Disrupt Steroidogenic Acute Regulatory (StAR) Protein Expression Post-Transcriptionally.” The Journal of Steroid Biochemistry and Molecular Biology 75 (4-5) (December 31): 229–36. <a class="external free" href="http://www.ncbi.nlm.nih.gov/pubmed/11282276" rel="nofollow" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed/11282276</a>.</p>
<p>Wong, Jean S, Xiaoqin Ye, Christy R Muhlenkamp, and Sarjeet S Gill. 2002. “Effect of a Peroxisome Proliferator on 3 Beta-Hydroxysteroid Dehydrogenase.” Biochemical and Biophysical Research Communications 293 (1) (April 26): 549–53. doi:10.1016/S0006-291X(02)00235-8. <a class="external free" href="http://www.ncbi.nlm.nih.gov/pubmed/12054636" rel="nofollow" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed/12054636</a>.</p>
</div>
<br>
<div>
<div>
<h4><a href="/relationships/370">Relationship: 370: Activation, PPARα leads to Decrease, Translocator protein (TSPO)</a></h4>
<!-- loop to find life stages under relationship -->
<div>
</div>
<!-- end loop for life stages -->
<!-- sex terms -->
<div>
</div>
<!-- end sex terms -->
<p>See Table 1.</p>
<h4>Key Event Relationship Description</h4>
<p>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).</p>
<!-- if nothing shows up in any of these fields, then evidence supporting this KER will not be displayed -->
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p>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.</p>
<h4>Key Event Relationship Description</h4>
<p>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).</p>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p>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.</p>
<strong>Empirical Evidence</strong>
<p>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.</p>
<strong>Empirical Evidence</strong>
<p>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.</p>
<p>Monobenzyl phthalate (MBzP) metabolite of DBP</p>
</td>
<td>
<p>(Hurst and Waxman 2003)</p>
</td>
</tr>
<tr>
<td>
<p>Phthalate (DBP)</p>
</td>
<td>
<p>EC50=21μM *</p>
<p>EC50=63μM**</p>
</td>
<td>
<p>LOEC=100 μM</p>
</td>
<td>
<p>rodent</p>
</td>
<td>
<p>*MBzP</p>
<p>**mono-<em>sec</em>-butyl phthalate (MBuP) metabolite of DBP</p>
</td>
<td>
<p>(Hurst and Waxman 2003),(Gazouli 2002)</p>
</td>
</tr>
<tr>
<td>
<p>Phthalate (DEHP)</p>
</td>
<td>
<p>LOEC=30μM</p>
</td>
<td>
<p>LOEC=300 mg/kg /d</p>
<p> </p>
</td>
<td>
<p>rodent</p>
</td>
<td>
<p>MEHP metabolite of DEHP</p>
</td>
<td>
<p>(Bility et al. 2004), (Gazouli 2002), (Borch et al. 2006)</p>
</td>
</tr>
<tr>
<td>
<p>Phthalate (DEHP)</p>
</td>
<td>
<p>Ki=15µM;</p>
<p>EC50 = 3.2μM</p>
</td>
<td>
<p>n.a.</p>
</td>
<td>
<p>human</p>
</td>
<td>
<p>MEHP metabolite of DEHP</p>
</td>
<td>
<p>(Lapinskas et al. 2005), (Hurst and Waxman 2003)</p>
</td>
</tr>
<tr>
<td>
<p>Bezafibrate</p>
</td>
<td>
<p>EC50=55μM</p>
</td>
<td>
<p>LOEC=100 μM</p>
</td>
<td>
<p>rodent</p>
</td>
<td>
<p>TSPO decreased by 80% in MA-10 Leydig cells</p>
</td>
<td>
<p>(Willson et al. 2000), (Gazouli 2002)</p>
</td>
</tr>
<tr>
<td>
<p>WY-14,643</p>
</td>
<td>
<p>EC50=0.00027μM</p>
</td>
<td>
<p>LOEC=50 mg/kg/d</p>
</td>
<td>
<p>rodent</p>
</td>
<td>
<p> </p>
</td>
<td>
<p>(Pinelli et al. 2005), (Gazouli 2002)</p>
</td>
</tr>
</tbody>
</table>
<p>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</p>
<strong>Uncertainties and Inconsistencies</strong>
<p>The exact mechanisms of this relationship are not known.</p>
<strong>Uncertainties and Inconsistencies</strong>
<p>The exact mechanisms of this relationship are not known.</p>
<p> </p>
<p>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).</p>
<p>There is some evidence involving additional PPARs in transcriptional regulation of TSPO:</p>
<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>
<p> </p>
<p>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).</p>
<p>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.</p>
<h4>References</h4>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>Campioli, Enrico, Amani Batarseh, Jiehan Li, and Vassilios Papadopoulos. 2011. “The Endocrine Disruptor Mono-(2-Ethylhexyl) Phthalate Affects the Differentiation of Human Liposarcoma Cells (SW 872).” Edited by Vasu D. Appanna. PloS One 6 (12) (January 21): e28750. doi:10.1371/journal.pone.0028750.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>Pinelli, Alessandra, Cristina Godio, Antonio Laghezza, Nico Mitro, Giuseppe Fracchiolla, Vincenzo Tortorella, Antonio Lavecchia, et al. 2005. “Synthesis, Biological Evaluation, and Molecular Modeling Investigation of New Chiral Fibrates with PPARalpha and PPARgamma Agonist Activity.” Journal of Medicinal Chemistry 48 (17) (August 25): 5509–19. doi:10.1021/jm0502844.</p>
<p>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.</p>
<p>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.</p>
<p>Willson, T M, P J Brown, D D Sternbach, and B R Henke. 2000. “The PPARs: From Orphan Receptors to Drug Discovery.” Journal of Medicinal Chemistry 43 (4) (February 24): 527–50.</p>
<p>Xie, Yi, Qian Yang, and Joseph W DePierre. 2002. “The Effects of Peroxisome Proliferators on Global Lipid Homeostasis and the Possible Significance of These Effects to Other Responses to These Xenobiotics: An Hypothesis.” Annals of the New York Academy of Sciences 973 (November): 17–25.</p>
</div>
<br>
<div>
<h4><a href="/relationships/608">Relationship: 608: Reduction, testosterone level leads to Malformation, Male reproductive tract</a></h4>
<div>
<h4><a href="/relationships/3205">Relationship: 3205: Decrease, circulating testosterone levels leads to Malformation, Male reproductive tract</a></h4>
<!-- loop to find life stages under relationship -->
<div>
</div>
<!-- end loop for life stages -->
<!-- sex terms -->
<div>
</div>
<!-- end sex terms -->
<p>Hypospadias</p>
</div>
<div>
</div>
<div>
</div>
<p>Hypospadias</p>
<p>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).</p>
<p>AGD</p>
<p>Across numerous species, including humans, AGD is longer in males compared to females; for review see (Barrett et al. 2014).</p>
<h4>Key Event Relationship Description</h4>
<p>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).</p>
<h4>Key Event Relationship Description</h4>
<p>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).</p>
<!-- if nothing shows up in any of these fields, then evidence supporting this KER will not be displayed -->
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p><strong>Hypospadias</strong></p>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p><strong>Hypospadias</strong></p>
<p>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).</p>
<p><strong>Anogential distance (AGD)</strong></p>
<p>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).</p>
<p><strong>Cryptorchidism</strong></p>
<p>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).</p>
<strong>Empirical Evidence</strong>
<p>Hypospadias</p>
<strong>Empirical Evidence</strong>
<p>Hypospadias</p>
<p>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.</p>
<p>Anogential distance (AGD)</p>
<p>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.</p>
<p>Testicular testosterone levels, reduction, no change plasma testosterone</p>
</td>
<td> </td>
<td> </td>
<td>
<p>(Borch et al. 2006)</p>
</td>
</tr>
<tr>
<td>
<p>Phthalates</p>
<p>(DEHP)</p>
</td>
<td>
<p>rat, GD 3- PND 21</p>
</td>
<td>
<p>LOEL=750 mg /kg bw/day</p>
</td>
<td> </td>
<td>
<p>Anogenital distance decreased</p>
</td>
<td>
<p>Gestational and lactational</p>
</td>
<td>
<p>(Moore et al. 2001)</p>
</td>
</tr>
<tr>
<td>
<p>Phthalates</p>
<p>(DEHP)</p>
</td>
<td>
<p>rat</p>
</td>
<td>
<p>LOEL=750 mg /kg bw/day</p>
</td>
<td>
<p>reduction in T production, and reduced testicular and whole-body T levels in fetal and neonatal male</p>
</td>
<td>
<p>Anogenital distance decreased</p>
</td>
<td>
<p>Exposure from (GD) 14 to postnatal day (PND) 3, AGD reduced by 36% in exposed male</p>
</td>
<td>
<p>(Parks 2000)</p>
</td>
</tr>
<tr>
<td>
<p>Phthalates</p>
<p>(DEHP)</p>
</td>
<td>
<p>rat</p>
</td>
<td>
<p>LOEL=15 mg /kg bw/day</p>
</td>
<td>
<p>Decreased testosterone levels</p>
</td>
<td>
<p>Effects on Sperm production</p>
</td>
<td> </td>
<td>
<p>(Andrade et al. 2006)</p>
</td>
</tr>
<tr>
<td>
<p>Phthalates</p>
<p>(DEHP)</p>
</td>
<td>
<p>rat</p>
</td>
<td>
<p>LOEL=5 mg /kg bw/day</p>
</td>
<td> </td>
<td>
<p>chryptorchidism</p>
</td>
<td> </td>
<td>
<p>(Andrade et al. 2006)</p>
</td>
</tr>
<tr>
<td>
<p>Phthalates</p>
<p>(DEHP)</p>
</td>
<td>
<p>rat</p>
</td>
<td>
<p>LOEL=1.215 mg /kg bw/day</p>
</td>
<td>
<p>Decreased testosterone levels</p>
</td>
<td>
<p>Effects on Sperm production</p>
</td>
<td> </td>
<td>
<p>(Andrade et al. 2006)</p>
</td>
</tr>
<tr>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr>
<td>
<p>Phthalates</p>
<p>(DnHP)</p>
</td>
<td>
<p>rat</p>
</td>
<td>
<p>LOEL=250 mg /kg bw/day</p>
</td>
<td> </td>
<td>
<p>Anogenital distance decreased</p>
</td>
<td> </td>
<td>
<p>(Saillenfait, Gallissot, and Sabaté 2009)</p>
</td>
</tr>
<tr>
<td>
<p>Phthalates</p>
<p>(DEHP)</p>
</td>
<td>
<p>rat</p>
</td>
<td>
<p>LOEL=300 mg/kg/day</p>
</td>
<td> </td>
<td>
<p>Anogenital distance decreased</p>
</td>
<td> </td>
<td>
<p>(Jarfelt et al. 2005)</p>
</td>
</tr>
<tr>
<td>
<p>Phthalates</p>
<p>(DBP)</p>
</td>
<td>
<p>rat</p>
</td>
<td>
<p>LOEL=500 mg/kg/day</p>
</td>
<td> </td>
<td>
<p>Anogenital distance decreased</p>
</td>
<td>
<p>throughout pregnancy until postnatal day 20</p>
</td>
<td>
<p>(Mylchreest, Cattley, and Foster 1998)</p>
</td>
</tr>
<tr>
<td>
<p>Phthalates</p>
<p>dicyclohexyl phthalate (DCHP)</p>
</td>
<td>
<p>rat</p>
</td>
<td>
<p>LOEL=6000 ppm</p>
</td>
<td> </td>
<td>
<p>Anogenital distance decreased</p>
</td>
<td>
<p>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.</p>
<p>Urine concentration of phthalates metabolites MEHP associated with reduced AGI, suggestive association of sum of DEHP metabolites with reduced AGI</p>
</td>
<td>
<p>(Suzuki et al. 2012),</p>
</td>
</tr>
<tr>
<td>
<p>Phthalates (MEP), (MBP), (MBzP), (MiBP)</p>
</td>
<td>
<p>human</p>
</td>
<td>
<p> </p>
</td>
<td> </td>
<td>
<p>Urinary concentrations of phthalate metabolites inversely related to AGI</p>
</td>
<td>
<p>134 boys 2-36 months of age</p>
</td>
<td>
<p>(Shanna H Swan et al. 2005)</p>
</td>
</tr>
<tr>
<td>
<p>Phthalates</p>
<p>(MEHP, MBP)</p>
</td>
<td>
<p>human</p>
</td>
<td>
<p>MBP= 78.4 ng/mL* in urine; 85.2 ng/mL* in amniotic fluid</p>
<p>MEHP =24.9 ng/mL * in urine; 22.8 ng/mL* in amniotic fluid</p>
</td>
<td> </td>
<td>
<p>In girls, decreased AGD in relation to amniotic fluid levels of MBP and MEHP. No associations found in boys</p>
</td>
<td>
<p>Amniotic fluid and urine concentrations of phthalate metabolites</p>
</td>
<td>
<p>(Huang et al. 2009)</p>
</td>
</tr>
</tbody>
</table>
<p>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.</p>
<strong>Uncertainties and Inconsistencies</strong>
<p>Hypospadias</p>
<strong>Uncertainties and Inconsistencies</strong>
<p>Hypospadias</p>
<p>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).</p>
<p><br />
Anogential distance (AGD)</p>
<p>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).</p>
<p>Andrade, Anderson J M, Simone W Grande, Chris E Talsness, Konstanze Grote, and Ibrahim Chahoud. 2006. “A Dose-Response Study Following in Utero and Lactational Exposure to Di-(2-Ethylhexyl)-Phthalate (DEHP): Non-Monotonic Dose-Response and Low Dose Effects on Rat Brain Aromatase Activity.” Toxicology 227 (3) (October 29): 185–92. doi:10.1016/j.tox.2006.07.022. <a class="external free" href="http://www.ncbi.nlm.nih.gov/pubmed/16949715" rel="nofollow" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed/16949715</a>.</p>
<h4>References</h4>
<p>Andrade, Anderson J M, Simone W Grande, Chris E Talsness, Konstanze Grote, and Ibrahim Chahoud. 2006. “A Dose-Response Study Following in Utero and Lactational Exposure to Di-(2-Ethylhexyl)-Phthalate (DEHP): Non-Monotonic Dose-Response and Low Dose Effects on Rat Brain Aromatase Activity.” Toxicology 227 (3) (October 29): 185–92. doi:10.1016/j.tox.2006.07.022. <a class="external free" href="http://www.ncbi.nlm.nih.gov/pubmed/16949715" rel="nofollow" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed/16949715</a>.</p>
<p>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. <a class="external free" href="http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3864710&tool=pmcentrez&rendertype=abstract" rel="nofollow" target="_blank">http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3864710&tool=pmcentrez&rendertype=abstract</a>.</p>
<p>Blaschko, Sarah D, Gerald R Cunha, and Laurence S Baskin. 2012. “Molecular Mechanisms of External Genitalia Development.” Differentiation; Research in Biological Diversity 84 (3) (October): 261–8. doi:10.1016/j.diff.2012.06.003. <a class="external free" href="http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3443292&tool=pmcentrez&rendertype=abstract" rel="nofollow" target="_blank">http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3443292&tool=pmcentrez&rendertype=abstract</a>.</p>
<p>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>.</p>
<p>Bornehag, Carl-Gustaf, Fredrik Carlstedt, Bo Ag Jönsson, Christian H Lindh, Tina K Jensen, Anna Bodin, Carin Jonsson, Staffan Janson, and Shanna H Swan. 2015. “Prenatal Phthalate Exposures and Anogenital Distance in Swedish Boys.” Environmental Health Perspectives 123 (1) (January): 101–7. doi:10.1289/ehp.1408163. <a class="external free" href="http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=4286276&tool=pmcentrez&rendertype=abstract" rel="nofollow" target="_blank">http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=4286276&tool=pmcentrez&rendertype=abstract</a>.</p>
<p>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. <a class="external free" href="http://www.ncbi.nlm.nih.gov/pubmed/12773767" rel="nofollow" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed/12773767</a>.</p>
<p>Brouwers, Marijn M, Wouter F J Feitz, Luc A J Roelofs, Lambertus A L M Kiemeney, Robert P E de Gier, and Nel Roeleveld. 2007. “Risk Factors for Hypospadias.” European Journal of Pediatrics 166 (7) (July): 671–8. doi:10.1007/s00431-006-0304-z. <a class="external free" href="http://www.ncbi.nlm.nih.gov/pubmed/17103190" rel="nofollow" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed/17103190</a>.</p>
<p>Brucker-Davis, Françoise, Kathy Wagner-Mahler, Isabelle Delattre, Béatrice Ducot, Patricia Ferrari, André Bongain, Jean-Yves Kurzenne, Jean-Christophe Mas, and Patrick Fénichel. 2008. “Cryptorchidism at Birth in Nice Area (France) Is Associated with Higher Prenatal Exposure to PCBs and DDE, as Assessed by Colostrum Concentrations.” Human Reproduction (Oxford, England) 23 (8) (August): 1708–18. doi:10.1093/humrep/den186. <a class="external free" href="http://www.ncbi.nlm.nih.gov/pubmed/18503055" rel="nofollow" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed/18503055</a>.</p>
<p>Eisenberg, Michael L, Tina K Jensen, R Chanc Walters, Niels E Skakkebaek, and Larry I Lipshultz. 2011. “The Relationship between Anogenital Distance and Reproductive Hormone Levels in Adult Men.” The Journal of Urology 187 (2) (February): 594–8. doi:10.1016/j.juro.2011.10.041. <a class="external free" href="http://www.ncbi.nlm.nih.gov/pubmed/22177168" rel="nofollow" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed/22177168</a>.</p>
<p>Fénichel, Patrick, Najiba Lahlou, Patrick Coquillard, Patricia Panaïa-Ferrari, Kathy Wagner-Mahler, and Françoise Brucker-Davis. 2015. “Cord Blood Insulin-like Peptide 3 (INSL3) but Not Testosterone Is Reduced in Idiopathic Cryptorchidism.” Clinical Endocrinology 82 (2) (February): 242–7. doi:10.1111/cen.12500. <a class="external free" href="http://www.ncbi.nlm.nih.gov/pubmed/24826892" rel="nofollow" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed/24826892</a>.</p>
<p>Gray, L E, J Ostby, J Furr, M Price, D N Veeramachaneni, and L Parks. 2000. “Perinatal Exposure to the Phthalates DEHP, BBP, and DINP, but Not DEP, DMP, or DOTP, Alters Sexual Differentiation of the Male Rat.” Toxicological Sciences : An Official Journal of the Society of Toxicology 58 (2) (December): 350–65. <a class="external free" href="http://www.ncbi.nlm.nih.gov/pubmed/11099647" rel="nofollow" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed/11099647</a>.</p>
<p>Hadziselimovic, F. 2002. “Cryptorchidism, Its Impact on Male Fertility.” European Urology 41 (2) (February): 121–3. <a class="external free" href="http://www.ncbi.nlm.nih.gov/pubmed/12074397" rel="nofollow" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed/12074397</a>. Hoshino, Nobuhito, Mayumi Iwai, and Yoshimasa Okazaki. 2005. “A Two-Generation Reproductive Toxicity Study of Dicyclohexyl Phthalate in Rats.” The Journal of Toxicological Sciences 30 Spec No (December): 79–96. <a class="external free" href="http://www.ncbi.nlm.nih.gov/pubmed/16641545" rel="nofollow" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed/16641545</a>.</p>
<p>Huang, Po-Chin, Pao-Lin Kuo, Yen-Yin Chou, Shio-Jean Lin, and Ching-Chang Lee. 2009. “Association between Prenatal Exposure to Phthalates and the Health of Newborns.” Environment International 35 (1) (January): 14–20. doi:10.1016/j.envint.2008.05.012. <a class="external free" href="http://www.ncbi.nlm.nih.gov/pubmed/18640725" rel="nofollow" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed/18640725</a>. Jarfelt, Kirsten, Majken Dalgaard, Ulla Hass, Julie Borch, Helene Jacobsen, and Ole Ladefoged. 2005. “Antiandrogenic Effects in Male Rats Perinatally Exposed to a Mixture of di(2-Ethylhexyl) Phthalate and di(2-Ethylhexyl) Adipate.” Reproductive Toxicology (Elmsford, N.Y.) 19 (4): 505–15. doi:10.1016/j.reprotox.2004.11.005. <a class="external free" href="http://www.ncbi.nlm.nih.gov/pubmed/15749265" rel="nofollow" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed/15749265</a>.</p>
<p>Johnson, Kamin J, Erin N McDowell, Megan P Viereck, and Jessie Q Xia. 2011. “Species-Specific Dibutyl Phthalate Fetal Testis Endocrine Disruption Correlates with Inhibition of SREBP2-Dependent Gene Expression Pathways.” Toxicological Sciences : An Official Journal of the Society of Toxicology 120 (2) (April): 460–74. doi:10.1093/toxsci/kfr020. <a class="external free" href="http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3061485&tool=pmcentrez&rendertype=abstract" rel="nofollow" target="_blank">http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3061485&tool=pmcentrez&rendertype=abstract</a>.</p>
<p>Jurewicz, Joanna, and Wojciech Hanke. 2011. “Exposure to Phthalates: Reproductive Outcome and Children Health. A Review of Epidemiological Studies.” International Journal of Occupational Medicine and Environmental Health 24 (2) (June): 115–41. doi:10.2478/s13382-011-0022-2. <a class="external free" href="http://www.ncbi.nlm.nih.gov/pubmed/21594692" rel="nofollow" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed/21594692</a>. 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. <a class="external free" href="http://www.ncbi.nlm.nih.gov/pubmed/18637150" rel="nofollow" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed/18637150</a>.</p>
<p>Klip, Helen, Janneke Verloop, Jan D van Gool, Marlies E T A Koster, Curt W Burger, and Flora E van Leeuwen. 2002. “Hypospadias in Sons of Women Exposed to Diethylstilbestrol in Utero: A Cohort Study.” Lancet 359 (9312) (March 30): 1102–7. doi:10.1016/S0140-6736(02)08152-7. <a class="external free" href="http://www.ncbi.nlm.nih.gov/pubmed/11943257" rel="nofollow" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed/11943257</a>.</p>
<p>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. <a class="external free" href="http://www.ncbi.nlm.nih.gov/pubmed/20002220" rel="nofollow" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed/20002220</a>. 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. <a class="external free" href="http://www.ncbi.nlm.nih.gov/pubmed/14745936" rel="nofollow" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed/14745936</a>.</p>
<p>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. <a class="external free" href="http://www.ncbi.nlm.nih.gov/pubmed/11452136" rel="nofollow" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed/11452136</a>.</p>
<p>McIntyre, B S, N J Barlow, D G Wallace, S C Maness, K W Gaido, and P M Foster. 2000. “Effects of in Utero Exposure to Linuron on Androgen-Dependent Reproductive Development in the Male Crl:CD(SD)BR Rat.” Toxicology and Applied Pharmacology 167 (2) (September 1): 87–99. doi:10.1006/taap.2000.8998. <a class="external free" href="http://www.ncbi.nlm.nih.gov/pubmed/10964759" rel="nofollow" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed/10964759</a>.</p>
<p>Moore, R W, T A Rudy, T M Lin, K Ko, and R E Peterson. 2001. “Abnormalities of Sexual Development in Male Rats with in Utero and Lactational Exposure to the Antiandrogenic Plasticizer Di(2-Ethylhexyl) Phthalate.” Environmental Health Perspectives 109 (3) (March): 229–37. <a class="external free" href="http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1240240&tool=pmcentrez&rendertype=abstract" rel="nofollow" target="_blank">http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1240240&tool=pmcentrez&rendertype=abstract</a>.</p>
<p>Morales-Suárez-Varela, María M, Gunnar V Toft, Morten S Jensen, Cecilia Ramlau-Hansen, Linda Kaerlev, Ane-Marie Thulstrup, Agustín Llopis-González, Jørn Olsen, and Jens P Bonde. 2011. “Parental Occupational Exposure to Endocrine Disrupting Chemicals and Male Genital Malformations: A Study in the Danish National Birth Cohort Study.” Environmental Health : A Global Access Science Source 10 (1) (January): 3. doi:10.1186/1476-069X-10-3. <a class="external free" href="http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3033238&tool=pmcentrez&rendertype=abstract" rel="nofollow" target="_blank">http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3033238&tool=pmcentrez&rendertype=abstract</a>.</p>
<p>Murashima, Aki, Satoshi Kishigami, Axel Thomson, and Gen Yamada. 2015. “Androgens and Mammalian Male Reproductive Tract Development.” Biochimica et Biophysica Acta 1849 (2) (February): 163–170. doi:10.1016/j.bbagrm.2014.05.020. <a class="external free" href="http://www.sciencedirect.com/science/article/pii/S1874939914001266" rel="nofollow" target="_blank">http://www.sciencedirect.com/science/article/pii/S1874939914001266</a>.</p>
<p>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>.</p>
<p>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>.</p>
<p>Parks, L. G. 2000. “The Plasticizer Diethylhexyl Phthalate Induces Malformations by Decreasing Fetal Testosterone Synthesis during Sexual Differentiation in the Male Rat.” Toxicological Sciences 58 (2) (December 1): 339–349. doi:10.1093/toxsci/58.2.339. <a class="external free" href="http://toxsci.oxfordjournals.org/content/58/2/339.abstract" rel="nofollow" target="_blank">http://toxsci.oxfordjournals.org/content/58/2/339.abstract</a>.</p>
<p>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. <a class="external free" href="http://www.ncbi.nlm.nih.gov/pubmed/16131574" rel="nofollow" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed/16131574</a>.</p>
<p>Saillenfait, Anne-Marie, Frédéric Gallissot, and Jean-Philippe Sabaté. 2009. “Differential Developmental Toxicities of Di-N-Hexyl Phthalate and Dicyclohexyl Phthalate Administered Orally to Rats.” Journal of Applied Toxicology : JAT 29 (6) (August): 510–21. doi:10.1002/jat.1436. <a class="external free" href="http://www.ncbi.nlm.nih.gov/pubmed/19391110" rel="nofollow" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed/19391110</a>.</p>
<p>Suzuki, Y, J Yoshinaga, Y Mizumoto, S Serizawa, and H Shiraishi. 2012. “Foetal Exposure to Phthalate Esters and Anogenital Distance in Male Newborns.” International Journal of Andrology 35 (3) (June): 236–44. doi:10.1111/j.1365-2605.2011.01190.x. <a class="external free" href="http://www.ncbi.nlm.nih.gov/pubmed/21696396" rel="nofollow" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed/21696396</a>.</p>
<p>Swan, S. H., S. Sathyanarayana, E. S. Barrett, S. Janssen, F. Liu, R. H. N. Nguyen, and J. B. Redmon. 2015. “First Trimester Phthalate Exposure and Anogenital Distance in Newborns.” Human Reproduction 30 (4) (February 18): 963–72. doi:10.1093/humrep/deu363. <a class="external free" href="http://www.ncbi.nlm.nih.gov/pubmed/25697839" rel="nofollow" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed/25697839</a>.</p>
<p>Swan, Shanna H, Katharina M Main, Fan Liu, Sara L Stewart, Robin L Kruse, Antonia M Calafat, Catherine S Mao, et al. 2005. “Decrease in Anogenital Distance among Male Infants with Prenatal Phthalate Exposure.” Environmental Health Perspectives 113 (8) (August): 1056–61. <a class="external free" href="http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1280349&tool=pmcentrez&rendertype=abstract" rel="nofollow" target="_blank">http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1280349&tool=pmcentrez&rendertype=abstract</a>.</p>
<p>Virtanen, Helena E, Dina Cortes, Ewa Rajpert-De Meyts, E Martin Ritzén, Agneta Nordenskjöld, Niels E Skakkebaek, and Jorma Toppari. 2007. “Development and Descent of the Testis in Relation to Cryptorchidism.” Acta Paediatrica (Oslo, Norway : 1992) 96 (5) (May): 622–7. doi:10.1111/j.1651-2227.2007.00244.x. <a class="external free" href="http://www.ncbi.nlm.nih.gov/pubmed/17462055" rel="nofollow" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed/17462055</a>.</p>
<p>Wang, Ming-Hsien, and Laurence S Baskin. 2008. “Endocrine Disruptors, Genital Development, and Hypospadias.” Journal of Andrology 29 (5): 499–505. doi:10.2164/jandrol.108.004945. <a class="external free" href="http://www.ncbi.nlm.nih.gov/pubmed/18497336" rel="nofollow" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed/18497336</a>.</p>
<p>Wilson, Vickie S, Christy Lambright, Johnathan Furr, Joseph Ostby, Carmen Wood, Gary Held, and L Earl Gray. 2004. “Phthalate Ester-Induced Gubernacular Lesions Are Associated with Reduced insl3 Gene Expression in the Fetal Rat Testis.” Toxicology Letters 146 (3) (March 2): 207–15. <a class="external free" href="http://www.ncbi.nlm.nih.gov/pubmed/14687758" rel="nofollow" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed/14687758</a>.</p>
<p>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. <a class="external free" href="http://tih.sagepub.com/content/15/1-2/94.abstract?ijkey=9190cbc3a5effe489f5f27911b833ff5e3f1a689&keytype2=tf_ipsecsha" rel="nofollow" target="_blank">http://tih.sagepub.com/content/15/1-2/94.abstract?ijkey=9190cbc3a5effe489f5f27911b833ff5e3f1a689&keytype2=tf_ipsecsha</a>.</p>