API

Aop: 18

AOP Title

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PPARα activation in utero leading to impaired fertility in males

Short name:

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PPARα activation leading to impaired fertility

Authors

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Malgorzata Nepelska, Elise Grignard, Sharon Munn,

Systems Toxicology Unit, Institute for Health and Consumer Protection, Joint Research Centre, European Commission, Via E. Fermi 2749, I-21027 Ispra, Varese, Italy

Corresponding author: sharon.munn@ec.europa.eu; elise.grignard@ec.europa.eu

Point of Contact

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Elise Grignard

Contributors

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  • Elise Grignard

Status

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Author status OECD status OECD project SAAOP status
Open for citation & comment EAGMST Under Review 1.21 Included in OECD Work Plan


This AOP was last modified on December 02, 2016 10:31

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Revision dates for related pages

Page Revision Date/Time
Activation, PPARα September 16, 2017 10:14
impaired, Fertility December 02, 2016 09:21
Decrease, Steroidogenic acute regulatory protein (STAR) September 16, 2017 10:14
Reduction, Cholesterol transport in mitochondria September 16, 2017 10:14
Reduction, Testosterone synthesis in Leydig cells September 16, 2017 10:14
Reduction, testosterone level September 16, 2017 10:14
Decrease, Translocator protein (TSPO) September 16, 2017 10:14
Malformation, Male reproductive tract September 16, 2017 10:14
Decrease, Steroidogenic acute regulatory protein (STAR) leads to Reduction, Cholesterol transport in mitochondria December 02, 2016 09:28
Reduction, Cholesterol transport in mitochondria leads to Reduction, Testosterone synthesis in Leydig cells December 02, 2016 10:16
Reduction, Testosterone synthesis in Leydig cells leads to Reduction, testosterone level December 02, 2016 10:18
Activation, PPARα leads to Decrease, Steroidogenic acute regulatory protein (STAR) December 02, 2016 10:12
Decrease, Translocator protein (TSPO) leads to Reduction, Cholesterol transport in mitochondria December 02, 2016 10:14
Activation, PPARα leads to Decrease, Translocator protein (TSPO) December 02, 2016 10:13
Malformation, Male reproductive tract leads to impaired, Fertility December 02, 2016 10:21
Reduction, testosterone level leads to Malformation, Male reproductive tract December 02, 2016 10:20

Abstract

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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.


Background (optional)

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This optional section should be used to provide background information for AOP reviewers and users that is considered helpful in understanding the biology underlying the AOP and the motivation for its development. The background should NOT provide an overview of the AOP, its KEs or KERs, which are captured in more detail below.

Instructions

To add background information, click Edit in the upper right hand menu on the AOP page. Under the “Background (optional)” field, a text editable form provides ability to edit the Background.  Clicking ‘Update AOP’ will update these fields.


Summary of the AOP

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Stressors

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Describes stressors known to trigger the MIE and provides evidence supporting that initiation. This will often be a list of prototypical compounds demonstrated to interact with the target molecule in the manner detailed in the MIE description to initiate a given pathway (e.g., 2,3,7,8-TCDD as a prototypical AhR agonist; 17α-ethynyl estradiol as a prototypical ER agonist). However, depending on the information available, this could also refer to chemical categories (i.e., groups of chemicals with defined structural features known to trigger the MIE). It can also include non-chemical stressors such as genetic or environmental factors. The evidence supporting the stressor will typically consist of a brief description and citation of literature showing that particular stressors can trigger the MIE.

Instructions

To add a stressor associated with an AOP, under “Summary of the AOP” click ‘Add Stressor’ will bring user to the “New Aop Stressor” page. In the Name field, user can search for stressor by name. Choosing a stressor from the resulting drop down populates the field. Selection of an Evidence level from the drop down menu and add any supporting evidence in the text box. Click ‘Add stressor’ to add the stressor to the AOP page.


Molecular Initiating Event

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Title Short name
Activation, PPARα Activation, PPARα

Key Events

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Title Short name
Decrease, Steroidogenic acute regulatory protein (STAR) Decrease, Steroidogenic acute regulatory protein (STAR)
Reduction, Cholesterol transport in mitochondria Reduction, Cholesterol transport in mitochondria
Reduction, Testosterone synthesis in Leydig cells Reduction, Testosterone synthesis in Leydig cells
Reduction, testosterone level Reduction, testosterone level
Decrease, Translocator protein (TSPO) Decrease, Translocator protein (TSPO)

Adverse Outcome

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Title Short name
impaired, Fertility impaired, Fertility
Malformation, Male reproductive tract Malformation, Male reproductive tract

Relationships Between Two Key Events (Including MIEs and AOs)

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Network View

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Life Stage Applicability

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Life stage Evidence
Development Strong

Taxonomic Applicability

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Term Scientific Term Evidence Link
rat Rattus norvegicus Moderate NCBI
human Homo sapiens Weak NCBI
mouse Mus musculus Moderate NCBI

Sex Applicability

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Sex Evidence
Male Strong

Graphical Representation

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Click to download graphical representation template

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Overall Assessment of the AOP

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Biological plausibility, coherence, and consistency of the experimental evidence

In the presented AOP it is hypothesized that the key events occur in a biologically plausible order prior to the development of adverse outcomes. The PPARα activators have been shown to alter steroidogenesis and impair reproduction [see reviews (Corton and Lapinskas 2004), (Latini et al. 2008), (David 2006)]. However, there are some conflicting reports on the involvement of PPARα as MIE of the proposed AOP (Johnson, Heger, and Boekelheide 2012), (David 2006). The biochemistry of steroidogenesis and the predominant role of the gonad in synthesis of the sex steroids are well established. Steroidogenesis is a complex process that is dependent on the availability of cholesterol in mitochondria. Perturbation of genes responsible for cholesterol transport and steroidogenic enzyme activities in the Leydig cell will lead to a decrease in testicular testosterone (T) production. As a consequence, androgen-dependent tissue differentiation/development is adversely affected. The physical manifestation of this event may be reproductive tract malformation and possibly leads to impaired fertility.

Concordance of dose-response relationships

This is a qualitative description of the pathway; the currently available studies provide quantitative information on dose-response relationships only partially. Experimental data are based on exposure to phthalates and indicate that key events of this pathway occur at similar dose levels. The effects of altered gene expression levels that are responsible for the cholesterol transport into the Leydig cells were shown at >50 mg/kg/bw, a dose at which foetal T was decreased and anatomical malformations (hypospadias) were produced (Mylchreest, Cattley, and Foster 1998), (Mylchreest 2000), (Akingbemi 2001), (Lehmann et al. 2004). Tailored experiments are required for the exploration of quantitative linkages.

Temporal concordance among the key events and the adverse outcome

This AOP bridges two life stages: the AOs are results of the chemical exposure during a critical prenatal period for male development, the masculinization programming window (MPW), within which androgens must act to ensure the correct development of the male reproductive tract (Welsh et al. 2008). Therefore, the AOP focuses on the exposures within the MPW (15.5–18.5 GD days in rats). The temporal relationship of exposure to gestation day has been investigated using phthalates and it has been demonstrated that the gestational timing of exposure is important for the production of the adverse effects on the male reproductive tract (reviewed in (Ema 2002)). Moreover, the temporal relationship between alterations of gene expression and changes in testosterone production has been investigated for phthalates (DBP) (Lehmann et al. 2004), (Thompson et al. 2005). Initial increases in gene expression are followed by decreases in the expression of genes which are associated with steroidogenesis. The observed decreased steroidogenesis and subsequent decrease in testosterone levels is well established as precursors to anatomical changes in the developing male reproductive tract. Thus, those key events of gene expression are temporally consistent with subsequent events, however complete temporal concordance studies are missing.


Strength, consistency, and specificity of association of adverse effect and initiating event

The strength of the chosen chemical initiators as PPARα activators was shown to partially correlate with their ability to act as a male reproductive toxicant (Corton and Lapinskas 2004). The presented key events leading to a decrease in steroidogenesis are plausible and consistent with the observed effects. There is coherence between decreased testosterone synthesis and malformations.

Alternative mechanism(s) or MIE(s) described which may contribute/synergise the postulated AOP

The inhibitory effect of PPARα activation seems to be attributable to an impairment of the multistep process of cholesterol mobilization, transport into mitochondria, and steroidogenesis leading to impaired androgens production. Therefore, it is plausible that several other mechanisms may contribute to/synergise with this AOP. For example, activation of other isoforms of PPARs (PPARβ/δ or/and γ) is hypothesised to be relevant for the pathway (Lapinskas et al. 2005), (Shipley and Waxman 2004).

PPARγ activation

Opposing effects of PPARγ ligands (thiazolidinediones, TZD) on androgen levels and/or production in male humans (Dunaif et al. 1996), (Bloomgarden, Futterweit, and Poretsky 2001), (Vierhapper, Nowotny, and Waldhäusl 2003) and animal models have been described (Kempná et al. 2007), (Gasic et al. 1998), (Mu et al. 2000), (Arlt, Auchus, and Miller 2001), (Minge, Robker, and Norman 2008), (Gasic et al. 2001), (Veldhuis, Zhang, and Garmey 2002). In rats no effects of PPARγ ligand (rosiglitazone) on production or total circulating testosterone levels were seen (Boberg et al. 2008), however a decrease in basal or induced testosterone production occurred in the Leydig cells of rosiglitazone-treated rats (Couto et al. 2010).


Moreover, there are contradicting reports as to the presence of PPARγ in the foetal testes (Hannas et al. 2012). Few others transcription factors involved in regulation of lipid metabolism are hypothesized to mediate effects on fetal Leydig cell gene expression like sterol regulatory element–binding protein (SREBP) (Lehmann et al. 2004), (Shultz 2001), CCAAT/enhancer-binding protein-β (CEBPB) (Kuhl, Ross, and Gaido 2007) or NR5A1 (also known as steroidogenic factor 1; Sf1) (Borch et al. 2006). The downstream effects in the pathway might be due to the constellation of earlier events in fetal Leydig cells leading to decrease testosterone production and connected adverse outcomes. Alternative/synergistic MIEs relating to this pathway are hypothesised in the KER description. At present there are no strong views on the other possible MIEs.

 

Uncertainties, inconsistencies and data gaps

The major uncertainty in this AOP is the functional relationship between (MIE) PPARα activation leading to cholesterol transport reduction; possible mechanisms have been proposed but strong experimental support is missing and some conflicting data are reported. The dose response data to support this relationship are lacking. Studies exploring the role of PPARα using PPARα knockout mice showed that prenatal exposure to phthalates caused developmental malformations in both wild-type and PPARα knockout mice, thus suggesting a PPARα-independent mechanism. However, it is difficult to draw any conclusion on the role of PPARα in phthalate-related reproductive toxicity since the intrauterine administration of phthalate (DEHP) occurred before the critical period of reproductive tract differentiation (Peters et al. 1997). Intrauterine DEHP-treated PPARα-deficient mice, developed delayed testicular, renal and developmental toxicities, but no liver toxicity, compared to wild types, thus confirming the early observation by Lee et al. about the PPARα dependence of liver response and, more importantly, indicating that DEHP may induce reproductive toxicity through both PPARα-dependent and -independent mechanism (Ward et al. 1998). PPARα-independent reproductive toxicity observed by Ward et al. may conceivably be mediated by other PPAR isoforms, such as PPARβ and PPARγ, or by a non-receptor-mediated organ-specific mechanism (Barak et al. 1999). Other studies showed that the administration of DEHP resulted in milder testis lesions and higher testosterone levels in PPARα-null mice than in wild-type mice (Gazouli 2002). A more recent report, investigating the role of PPARα, showed decreased testosterone levels in PPARα(−/−) null control mice, suggesting a positive constitutive role for PPARα in maintaining Leydig cell steroid formation (Borch et al. 2006).

Inconsistencies Genomic studies by Hannas et al., demonstrated that PPARα agonist Wy-14,643, did not reduce foetal testicular testosterone production following gestational day 14–18 exposure, suggesting that the antiandrogenic activity of phthalates is not PPARα mediated (Hannas et al. 2012). Similarly, recent report by Furr et al. did not observe testosterone decrease after administration of Wy-14,643 in rat ( ex vivo) (Furr et al. 2014).

Data Gaps: Complete/pathway driven studies to investigate the effects of PPARs and their role in male reproductive development are lacking. For establishing a solid quantitative and temporal coherent linkage, mode of action framework analysis for PPAR α mediated developmental toxicity are needed. This approach has been applied for the involvement of PPAR α in liver toxicity (Corton et al. 2014), (Wood et al. 2014).

 

Domain of Applicability

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Empirical information on dose-response relationships between the KEs, are not available, however there are solid empirical data that would inform a computational, predictive model for reproductive toxicity via PPARα activation.

Life Stage Applicability

This AOP is relevant for developing (prenatal) male.

Taxonomic Applicability

The experimental support for the pathway is mainly based on the animal (rat studies). Conflicting reports comes from the studies on mouse. Studies in mice report contradictory results. Recently, studies by Furr et al revealed that fetal T production can be inhibited by exposure to a phthalates in utero (CD-1 mice), but at a higher dose level than required in rats and causing systemic effects (Furr et al. 2014). However there are some earlier reports that chronic dietary administration of phthalates produces adverse testicular effects and reduces fertility in CD-1 mice (Heindel et al. 1989)

Sex Applicability

This AOP applies to males only.


Essentiality of the Key Events

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KRs

Essentiality - KEs

level of confidence

   

 

PPAR alpha, Activation

 

PPAR alpha activation was found to indirectly alter the expression of genes involved in cholesterol transport in mitochondria

very weak

TSPO; StAR decrease

Alterations in the amount of cholesterol transport proteins in mitochondria impact on the levels of substrate for steroid hormones production.

weak

cholesterol transport in mitochondria, reduction

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.

moderate

Testosterone synthesis, reduction

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.

strong

Testosterone, reduction

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.

strong

Male reproductive tract malformations

Androgens regulate masculinization of the external genitalia. Therefore any defects in androgen biosynthesis, metabolism or action during foetal development can reproductive tract malformation.

strong

Fertility, impaired

Impaired fertility is the endpoint of reproductive toxicity

strong


Weight of Evidence Summary

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KERs

Biological plausibility

Level of confidence

Empirical Support

Level of confidence

Inconsistencies/Uncertainties

 

     

Dose-response

Temporality

Incidence

   

PPAR alpha, Activation

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Translator protein (TSPO), Decrease

 

There is functional relationship between PPARα activation and reduction in TSPO levels.

Very Weak

  • KEs occur at similar dose levels
  • occurrence of the key events at similar dose and time point
  • Support for solid temporal relationship is lacking

 

Very Weak

Some conflicting data

PPAR alpha, Activation

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Steroidogenic acute regulatory protein (StAR), decrease

There is functional relationship between PPARα activation and reduction in StAR levels.

Weak

  • KEs occur at similar dose levels
  • Support for solid temporal relationship is lacking.

 

Weak

Some conflicting data

Steroidogenic acute regulatory protein (StAR), decrease and Translator protein (TSPO), Decrease

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cholesterol transport in mitochondria, reduction

 

Changes in cholesterol transport proteins can generally be assumed to directly impact levels of cholesterol transport.

Moderate

  • KEs occur at similar dose levels
  • Support for solid temporal relationship is lacking.

 

Moderate

Some conflicting data

cholesterol transport in mitochondria, reduction

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testosterone synthesis, reduction

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.

Moderate

  • KEs occur at similar dose levels
  • occurrence of the key events at similar dose and time point
  • Support for solid temporal relationship is lacking.

 

Moderate

Some conflicting data

testosterone, reduction

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Male reproductive tract malformations

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.

Strong

  • KEs occur at similar dose levels
  • occurrence of the key events at similar dose and time point
  • Support for solid temporal relationship is lacking.

 

Strong

No conflicting data

Male reproductive tract malformations

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Fertility, impaired

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

Moderate

  • KEs occur at similar dose levels
  • occurrence of the key events at similar dose and time point

Support for solid temporal relationship is lacking.

 

Moderate

No conflicting data

 

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


Quantitative Considerations

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


Considerations for Potential Applications of the AOP (optional)

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1. The AOP describes a pathway which allows for the detection of sex steroid--related endocrine disrupting modes of action, with focus on the identification of substances which affect the reproductive system. In the current form the pathway lays a strong basis for linking endocrine mode of action with an apical endpoint, a prerequisite requirement for identification of endocrine disrupting chemicals (EDC).

EDCs require specific evaluation under REACH (1907/2006, Registration, Evaluation, Authorisation and Restriction of Chemicals (EU, 2006)), the revised European plant protection product regulation 1107/2009 (EU, 2009) and use of biocidal products 528/2012 EC (EU, 2012).Amongst other agencies the US EPA is also giving particular attention to EDCs (EPA, 1998).

2. This AOP structurally represents current knowledge of the pathway from PPARα activation to impaired fertility that may provide a basis for development (and interpretation) of strategies for Integrated Approaches to Testing Assessment (IATA) to identify similar substances that may operate via the same pathway related tosex steroids disruptionand effects on reproductive tract and fertility. This AOP forms the starting point on an AOP network mapping to modes of action for endocrine disruption.

3. The AOP could inform the development of quantitative structure activity relationships, read-across models, and/or systems biology models to prioritize chemicals for further testing.


References

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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. http://www.biolreprod.org/content/65/4/1252.long.

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. http://www.ncbi.nlm.nih.gov/pubmed/11278997.

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. http://www.ncbi.nlm.nih.gov/pubmed/10549290.

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. http://www.ncbi.nlm.nih.gov/pubmed/11497481.

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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. http://www.sciencedirect.com/science/article/pii/S0300483X0600165X.

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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. http://www.ncbi.nlm.nih.gov/pubmed/15496498.

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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. http://toxsci.oxfordjournals.org/content/140/2/403.full.

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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. http://www.sciencedirect.com/science/article/pii/S0002937801774340.

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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. http://www.ncbi.nlm.nih.gov/pubmed/17138841.

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. http://www.ncbi.nlm.nih.gov/pubmed/17884934.

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