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Created at: 2019-01-04 08:31

AOP ID and Title:


AOP 18: PPARα activation in utero leading to impaired fertility in males
Short Title: PPARα activation leading to impaired fertility

Graphical Representation


Authors


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


Status

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

Abstract


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.



Summary of the AOP

Events

Molecular Initiating Events (MIE), Key Events (KE), Adverse Outcomes (AO)

Sequence Type Event ID Title Short name
1 MIE 227 Activation, PPARα Activation, PPARα
2 KE 266 Decrease, Steroidogenic acute regulatory protein (STAR) Decrease, Steroidogenic acute regulatory protein (STAR)
3 KE 447 Reduction, Cholesterol transport in mitochondria Reduction, Cholesterol transport in mitochondria
4 KE 413 Reduction, Testosterone synthesis in Leydig cells Reduction, Testosterone synthesis in Leydig cells
5 KE 446 Reduction, testosterone level Reduction, testosterone level
6 KE 289 Decrease, Translocator protein (TSPO) Decrease, Translocator protein (TSPO)
7 AO 406 impaired, Fertility impaired, Fertility
8 AO 348 Malformation, Male reproductive tract Malformation, Male reproductive tract

Key Event Relationships

Upstream Event Relationship Type Downstream Event Evidence Quantitative Understanding
Decrease, Steroidogenic acute regulatory protein (STAR) adjacent Reduction, Cholesterol transport in mitochondria Moderate
Reduction, Cholesterol transport in mitochondria adjacent Reduction, Testosterone synthesis in Leydig cells Moderate
Reduction, Testosterone synthesis in Leydig cells adjacent Reduction, testosterone level High
Decrease, Translocator protein (TSPO) adjacent Reduction, Cholesterol transport in mitochondria Low
Malformation, Male reproductive tract adjacent impaired, Fertility High
Activation, PPARα non-adjacent Decrease, Steroidogenic acute regulatory protein (STAR) Moderate
Activation, PPARα non-adjacent Decrease, Translocator protein (TSPO) Low
Reduction, testosterone level non-adjacent Malformation, Male reproductive tract High

Overall Assessment of the AOP

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

Life Stage Applicability
Life Stage Evidence
Development High
Taxonomic Applicability
Term Scientific Term Evidence Links
rat Rattus norvegicus Moderate NCBI
human Homo sapiens Low NCBI
mouse Mus musculus Moderate NCBI
Sex Applicability
Sex Evidence
Male High

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

 

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

 

KERs

Biological plausibility

Level of confidence

Empirical Support

Level of confidence

Inconsistencies/Uncertainties

 

     

Dose-response

Temporality

Incidence

   

PPAR alpha, Activation

=>

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

=>

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

=>

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

=>

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

=>

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

=>

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 Consideration

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)


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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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. http://toxsci.oxfordjournals.org/content/43/1/47.short?rss=1&ssource=mfc.

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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. http://www.ncbi.nlm.nih.gov/pubmed/15364543.

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. http://toxsci.oxfordjournals.org/content/64/2/233.abstract?ijkey=b8af27acfe10695847a4e8a9b568882405d071ae&keytype2=tf_ipsecsha.

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. http://www.ncbi.nlm.nih.gov/pubmed/15987825.

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. http://www.ncbi.nlm.nih.gov/pubmed/11889176.

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. http://www.ncbi.nlm.nih.gov/pubmed/12601638.

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. http://www.ncbi.nlm.nih.gov/pubmed/9547862.

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. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2267017&tool=pmcentrez&rendertype=abstract.

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. http://www.ncbi.nlm.nih.gov/pubmed/24496636.


Appendix 1

List of MIEs in this AOP

Event: 227: Activation, PPARα

Short Name: Activation, PPARα

Key Event Component

Process Object Action
peroxisome proliferator activated receptor signaling pathway peroxisome proliferator-activated receptor alpha increased

Stressors

Name
Di(2-ethylhexyl) phthalate
Mono(2-ethylhexyl) phthalate

Biological Context

Level of Biological Organization
Molecular

Cell term

Cell term
eukaryotic cell

Evidence for Perturbation by Stressor


Overview for Molecular Initiating Event

Fibrates are ligands of PPARα (Staels et al. 1998).

Phthalates

MHEP (CAS 4376-20-9) directly binds in vitro to PPARα (Lapinskas et al. 2005) and activates this receptor in transactivation assays PPARα (Lapinskas et al. 2005), (Maloney and Waxman 1999), (Hurst and Waxman 2003), (Bility et al. 2004), (Lampen, Zimnik, and Nau 2003), (Venkata et al. 2006) ]. DEHP (CAS 117-81-7) has not been found to bind and activate PPARα (Lapinskas et al. 2005), (Maloney and Waxman 1999). However, the recent studies shown activation of PPARα (ToxCastTM Data).

Notably, PPARα are responsive to DEHP in vitro as they are translocated to the nucleus (in primary Sertoli cells) (Dufour et al. 2003), (Bhattacharya et al. 2005). Expression of PPARα [mRNA and protein] has been reported to be also modulated by phthtalates: (to be up-regulated in vivo upon DEHP treatment (Xu et al. 2010) and down-regulated by Diisobutyl phthalate (DiBP) (Boberg et al. 2008)).


Perfluorooctanoic Acid (PFOA) is known to activate PPARα (Vanden Heuvel et al. 2006).

Organotin

Tributyltin (TBT) activates all three heterodimers of PPAR with RXR, primarily through its interaction with RXR (le Maire et al. 2009)



Domain of Applicability


Taxonomic Applicability
Term Scientific Term Evidence Links
rat Rattus norvegicus High NCBI
mouse Mus musculus High NCBI
human Homo sapiens High NCBI

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


Key Event Description

Biological state

The Peroxisome Proliferator Activated receptor α (PPARα) belongs to the Peroxisome Proliferator Activated receptors (PPARs; NR1C) steroid/thyroid/retinoid receptor superfamily of transcription factors.

Biological compartments

PPARα is expressed in high levels in tissues that perform significant catabolism of fatty acids (FAs), such as brown adipose tissue, liver, heart, kidney, and intestine (Michalik et al. 2006). The receptor is present also in skeletal muscle, intestine, pancreas, lung, placenta and testes (Mukherjee et al. 1997), (Schultz et al. 1999).

General role in biology

PPARs are activated by fatty acids and their derivatives; they are sensors of dietary lipids and are involved in lipid and carbohydrate metabolism, immune response and peroxisome proliferation (Wahli and Desvergne 1999), (Evans, Barish, & Wang, 2004). PAPRα is a also a target of hypothalamic hormone signalling and was found to play a role in embryonic development (Yessoufou and Wahli 2010).

Fibrates, activators of PPARα, are commonly used to treat hypertriglyceridemia and other dyslipidemic states as they have been shown to decrease circulating lipid levels (Lefebvre et al. 2006).


How it is Measured or Detected

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.


Key event PPARα activation

What is measured?

Ligand Binding
Transcriptional activity

Method/test category

molecular modelling
binding assay
transactivation reporter gene assay
transcription factor assay

Method/test

name

molecular modelling; docking

Scintillation proximity binding assay

luciferase reporter gene assay

PPARα (mouse/rat) Reporter Assay Kit

Electrophoretic Mobility Shift Assay (EMSA)

Test environment

In silico
In vitro
In vitro
In vitro, ex vivo

Test principle

Computational simulation of a candidate ligand binding to a receptor, Predicts the strength of association or binding affinity.

Direct binding indicating the mode of action for PPARα

Quantifying changes in luciferase expression in the treated reporter cells provides a sensitive surrogate measure of the changes in PPAR functional activity.

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.

Test outcome

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.

Assesses the ability of compounds to bind to PPARα. Identifies the modulators of PPARα.

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.

Protein: DNA binding, DNA binding activity  

Test background

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.

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.

PPARα/γ COS-1cell transactivation assay (transient transfection with human or mouse PPARα/γ expression plasmid and pHD(x3)-Luc reporter plasmid

(PPRE)3- luciferase reporter construct C2C12

Proprietary rodent cell line expressing the mouse/rat PPARα

Transcriptional activity of PPARα can be assessed using commercially available kits like e.g. PPAR-α transcription factor assay kit.

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.

Assay type

Quantitative
Qualitative
Quantitative
Quantitative
Quantitative

Quantitative

Quantitative

Application domain

Virtual screening

In vitro screening

In vitro Screening, functional studies activity (reported use: agonist)

 

In vitro Screening functional activity (antagonist/agonist)

 

 

Ref

(Feige et al. 2007), (Kaya et al. 2006)
(Lapinskas et al. 2005), (Wu, Gao, and Wang 2005)
(Maloney and Waxman 1999)
(Feige et al. 2007)
Indigobiosciences
Abcam

Table 1 Summary of the chosen methods to measure the PPARα activation.


References

Bhattacharya, Nandini, Jannette M Dufour, My-Nuong Vo, Janice Okita, Richard Okita, and Kwan Hee Kim. 2005. “Differential Effects of Phthalates on the Testis and the Liver.” Biology of Reproduction 72 (3) (March): 745–54. doi:10.1095/biolreprod.104.031583.

Bility, Moses T, Jerry T Thompson, Richard H McKee, Raymond M David, John H Butala, John P Vanden Heuvel, and Jeffrey M Peters. 2004. “Activation of Mouse and Human Peroxisome Proliferator-Activated Receptors (PPARs) by Phthalate Monoesters.” Toxicological Sciences : An Official Journal of the Society of Toxicology 82 (1) (November): 170–82. doi:10.1093/toxsci/kfh253.

Dufour, Jannette M, My-Nuong Vo, Nandini Bhattacharya, Janice Okita, Richard Okita, and Kwan Hee Kim. 2003. “Peroxisome Proliferators Disrupt Retinoic Acid Receptor Alpha Signaling in the Testis.” Biology of Reproduction 68 (4) (April): 1215–24. doi:10.1095/biolreprod.102.010488.

Feige, Jérôme N, Laurent Gelman, Daniel Rossi, Vincent Zoete, Raphaël Métivier, Cicerone Tudor, Silvia I Anghel, et al. 2007. “The Endocrine Disruptor Monoethyl-Hexyl-Phthalate Is a Selective Peroxisome Proliferator-Activated Receptor Gamma Modulator That Promotes Adipogenesis.” The Journal of Biological Chemistry 282 (26) (June 29): 19152–66. doi:10.1074/jbc.M702724200.

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.

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.

Lampen, Alfonso, Susan Zimnik, and Heinz Nau. 2003. “Teratogenic Phthalate Esters and Metabolites Activate the Nuclear Receptors PPARs and Induce Differentiation of F9 Cells.” Toxicology and Applied Pharmacology 188 (1) (April): 14–23. doi:10.1016/S0041-008X(03)00014-0.

Lapinskas, Paula J., Sherri Brown, Lisa M. Leesnitzer, Steven Blanchard, Cyndi Swanson, Russell C. Cattley, and J. Christopher Corton. 2005. “Role of PPARα in Mediating the Effects of Phthalates and Metabolites in the Liver.” Toxicology 207 (1): 149–163.

Le Maire, Albane, Marina Grimaldi, Dominique Roecklin, Sonia Dagnino, Valérie Vivat-Hannah, Patrick Balaguer, and William Bourguet. 2009. “Activation of RXR-PPAR Heterodimers by Organotin Environmental Endocrine Disruptors.” EMBO Reports 10 (4) (April): 367–73. doi:10.1038/embor.2009.8.

LeBlanc, GA, DO Norris, and W Kloas. 2011. “Detailed Review Paper State of the Science on Novel In Vitro and In Vivo Screening and Testing Methods and Endpoints for Evaluating Endocrine Disruptors” (178).

Lefebvre, Philippe, Giulia Chinetti, Jean-Charles Fruchart, and Bart Staels. 2006. “Sorting out the Roles of PPAR Alpha in Energy Metabolism and Vascular Homeostasis.” The Journal of Clinical Investigation 116 (3) (March): 571–80. doi:10.1172/JCI27989.

Maloney, Erin K., and David J. Waxman. 1999. “Trans-Activation of PPARα and PPARγ by Structurally Diverse Environmental Chemicals.” Toxicology and Applied Pharmacology 161 (2): 209–218.

Michalik, Liliane, Johan Auwerx, Joel P Berger, V Krishna Chatterjee, Christopher K Glass, Frank J Gonzalez, Paul A Grimaldi, et al. 2006. “International Union of Pharmacology. LXI. Peroxisome Proliferator-Activated Receptors.” Pharmacological Reviews 58 (4) (December): 726–41. doi:10.1124/pr.58.4.5.

Mukherjee, R, L Jow, G E Croston, and J R Paterniti. 1997. “Identification, Characterization, and Tissue Distribution of Human Peroxisome Proliferator-Activated Receptor (PPAR) Isoforms PPARgamma2 versus PPARgamma1 and Activation with Retinoid X Receptor Agonists and Antagonists.” The Journal of Biological Chemistry 272 (12) (March 21): 8071–6.

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.

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.

ToxCastTM Data. “ToxCastTM Data.” US Environmental Protection Agency. http://www.epa.gov/ncct/toxcast/data.html

Vanden Heuvel, John P, Jerry T Thompson, Steven R Frame, and Peter J Gillies. 2006. “Differential Activation of Nuclear Receptors by Perfluorinated Fatty Acid Analogs and Natural Fatty Acids: A Comparison of Human, Mouse, and Rat Peroxisome Proliferator-Activated Receptor-Alpha, -Beta, and -Gamma, Liver X Receptor-Beta, and Retinoid X Rec.” Toxicological Sciences : An Official Journal of the Society of Toxicology 92 (2) (August): 476–89. doi:10.1093/toxsci/kfl014.

Venkata, Nagaraj Gopisetty, Jodie a Robinson, Peter J Cabot, Barbara Davis, Greg R Monteith, and Sarah J Roberts-Thomson. 2006. “Mono(2-Ethylhexyl)phthalate and Mono-N-Butyl Phthalate Activation of Peroxisome Proliferator Activated-Receptors Alpha and Gamma in Breast.” Toxicology Letters 163 (3) (June 1): 224–34. doi:10.1016/j.toxlet.2005.11.001.

Viswakarma, Navin, Yuzhi Jia, Liang Bai, Aurore Vluggens, Jayme Borensztajn, Jianming Xu, and Janardan K Reddy. 2010. “Coactivators in PPAR-Regulated Gene Expression.” PPAR Research 2010 (January). doi:10.1155/2010/250126.

Wahli, Walter, and B Desvergne. 1999. “Peroxisome Proliferator-Activated Receptors: Nuclear Control of Metabolism.” Endocrine Reviews 20 (5) (October): 649–88. Wu, Bin, Jie Gao, and Ming-wei Wang. 2005. “Development of a Complex Scintillation Proximity Assay for High-Throughput Screening of PPARgamma Modulators.” Acta Pharmacologica Sinica 26 (3) (March): 339–44. doi:10.1111/j.1745-7254.2005.00040.x.

Xu, Chuan, Ji-An Chen, Zhiqun Qiu, Qing Zhao, Jiaohua Luo, Lan Yang, Hui Zeng, et al. 2010. “Ovotoxicity and PPAR-Mediated Aromatase Downregulation in Female Sprague-Dawley Rats Following Combined Oral Exposure to Benzo[a]pyrene and Di-(2-Ethylhexyl) Phthalate.” Toxicology Letters 199 (3) (December 15): 323–32. doi:10.1016/j.toxlet.2010.09.015.

Yessoufou, a, and W Wahli. 2010. “Multifaceted Roles of Peroxisome Proliferator-Activated Receptors (PPARs) at the Cellular and Whole Organism Levels.” Swiss Medical Weekly 140 (September) (January): w13071. doi:10.4414/smw.2010.13071.

Yu, Songtao, and Janardan K Reddy. 2007. “Transcription Coactivators for Peroxisome Proliferator-Activated Receptors.” Biochimica et Biophysica Acta 1771 (8) (August): 936–51. doi:10.1016/j.bbalip.2007.01.008.


List of Key Events in the AOP

Event: 266: Decrease, Steroidogenic acute regulatory protein (STAR)

Short Name: Decrease, Steroidogenic acute regulatory protein (STAR)

Key Event Component

Process Object Action
gene expression STAR decreased

AOPs Including This Key Event


Biological Context

Level of Biological Organization
Cellular

Cell term

Cell term
steroid hormone secreting cell

Domain of Applicability


Taxonomic Applicability
Term Scientific Term Evidence Links
mouse Mus musculus Moderate NCBI
human Homo sapiens Moderate NCBI
rat Rattus norvegicus High NCBI

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


Key Event Description

Biological state

Steroidogenic acute regulatory protein (StAR) functions as a cholesterol transfer protein and acts directly on lipids of the outer mitochondrial membrane to promote cholesterol translocation (Stocco 2001). Reduction of the protein impacts on the amount of substrate available for steroidogenesis.


Biological compartments

StAR is expressed principally in steroidogenic tissues (Bauer et al. 2000).

General role in biology

StAR is required for cholesterol shuttling across the mitochondrial membrane and appears to regulate acute steroid production (Clark and Stocco, 1997). Transcriptional or translational inhibition of StAR expression results in a dramatic decrease in steroid biosynthesis, whereas ~10–15% of steroid synthesis appears to be mediated through StAR-independent mechanisms (Manna et al. 2001) (Clark and Stocco, 1997). In contrast, chronically regulated steroid production appears to be largely mediated by increased transcription of steroidogenic enzymes (Hum and Miller 1993).


How it is Measured or Detected

The StAR expression can be measured by RT-PCR (mRNA) and on the protein level (western blot). The StAR expression as well as other steroidogenic proteins can be measured in vitro cultured Leydig cells. The methods for culturing Leydig cells can be found in the Database Service on Alternative Methods to animal experimentation (DB-ALM): Leydig Cell-enriched Cultures [1] Testicular Organ and Tissue Culture Systems [2].


References

Bauer, M P, J T Bridgham, D M Langenau, A L Johnson, and F W Goetz. 2000. “Conservation of Steroidogenic Acute Regulatory (StAR) Protein Structure and Expression in Vertebrates.” Molecular and Cellular Endocrinology 168 (1-2) (October 25): 119–25.

Hum, D W, and W L Miller. 1993. “Transcriptional Regulation of Human Genes for Steroidogenic Enzymes.” Clinical Chemistry 39 (2) (February): 333–40.

Manna, P R, J Kero, M Tena-Sempere, P Pakarinen, D M Stocco, and I T Huhtaniemi. 2001. “Assessment of Mechanisms of Thyroid Hormone Action in Mouse Leydig Cells: Regulation of the Steroidogenic Acute Regulatory Protein, Steroidogenesis, and Luteinizing Hormone Receptor Function.” Endocrinology 142 (1) (January): 319–31. doi:10.1210/endo.142.1.7900.

Stocco, D M. 2001. “StAR Protein and the Regulation of Steroid Hormone Biosynthesis.” Annual Review of Physiology 63 (January): 193–213. doi:10.1146/annurev.physiol.63.1.193.


Event: 447: Reduction, Cholesterol transport in mitochondria

Short Name: Reduction, Cholesterol transport in mitochondria

Key Event Component

Process Object Action
mitochondrial transport cholesterol decreased

Biological Context

Level of Biological Organization
Cellular

Cell term

Cell term
steroid hormone secreting cell

Domain of Applicability


Taxonomic Applicability
Term Scientific Term Evidence Links
mouse Mus musculus High NCBI
human Homo sapiens High NCBI
rat Rattus norvegicus High NCBI

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


Key Event Description

Biological state

Steroidogenesis begins with the transport of cholesterol from intracellular stores into mitochondria. This process involves a series of protein-protein interactions involving cytosolic and mitochondrial proteins located at both the outer and inner mitochondrial membranes. In steroidogenic cells the cholesterol import to the mitochondrial inner membrane is crucial for steroid synthesis (Rone, Fan, and Papadopoulos 2009). This process is facilitated by the Scavenger Receptor Class B, type 1 (SR-B1) [more relevant for rodents, than for humans] that mediates the selective uptake of cholesterol esters from high-density lipoproteins. Steroidogenic acute regulatory protein (STAR) and the translator protein (TSPO) [former peripheral benzodiazepine receptor (PBR)] mediate cholesterol transport from the outer to the inner mitochondrial membrane. The conversion of cholesterol to pregnenolone is done by Cholesterol side-chain cleavage enzyme (P450scc), the start of steroidogenesis [reviewed in (Miller and Auchus 2011)].


Biological compartments

In mitochondria of steroidogenic tissues there are two specialized mechanisms related to hormone synthesis: one by which cholesterol is delivered to the mitochondria and the other by which specialized intra-mitochondrial enzymes participate in the synthesis of hormonal steroids.


General role in biology

Systemic steroid hormones are primarily formed by the gonads, adrenal glands, and during in utero development by the placenta. Some other organs like brain (Baulieu 1998), and heart (Kayes-Wandover and White 2000) have also been identified as steroid-producing tissues, mainly for local needs. The steroid hormones are indispensable for mammalian life. They are made from cholesterol via complex biosynthetic pathways that are initiated by specialized, tissue-specific enzymes in mitochondria. These hormones include glucocorticoids (cortisol, corticosterone) and mineralocorticoids (aldosterone) produced in the adrenal cortex, estrogens (estradiol), progestins (progesterone) and androgens (testosterone, dihydrotestosterone) produced in the gonads, and calciferols (1,25-dihydroxy vitamin D [1,25OH2D]) produced in the kidneys (Miller and Auchus 2011). Cholesterol is the precursor for the synthesis of steroid hormones in mitochondria. Steroidogenesis begins with the metabolism of cholesterol to pregnenolone facilitated by P450scc. The rate of steroid formation depends on the rate of cholesterol transport from intracellular stores to the inner mitochondrial membrane and the loading of P450scc with cholesterol (Miller and Auchus 2011). Interference with one or more of these reactions leads to reduced steroid production.


How it is Measured or Detected

This KE can be indirectly measured by:

1. Expression of the proteins involved in cholesterol transport by qPCR or Western blot.

3. Cholesterol transport to the mitochondrial inner membrane in intact cells:

  • 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).
  • 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.

The cholesterol transport can be measured in vitro cultured Leydig cells. The methods for culturing Leydig cells can be found in the Database Service on Alternative Methods to animal experimentation (DB-ALM): Leydig Cell-enriched Cultures [1] Testicular Organ and Tissue Culture Systems [2]


References

Albalat, Ricard, Frédéric Brunet, Vincent Laudet, and Michael Schubert. 2011. “Evolution of Retinoid and Steroid Signaling: Vertebrate Diversification from an Amphioxus Perspective.” Genome Biology and Evolution 3: 985–1005. doi:10.1093/gbe/evr084.

Baulieu, E E. 1998. “Neurosteroids: A Novel Function of the Brain.” Psychoneuroendocrinology 23 (8) (November): 963–87.

Charman, Mark, Barry E Kennedy, Nolan Osborne, and Barbara Karten. 2010. “MLN64 Mediates Egress of Cholesterol from Endosomes to Mitochondria in the Absence of Functional Niemann-Pick Type C1 Protein.” Journal of Lipid Research 51 (5) (May): 1023–34. doi:10.1194/jlr.M002345.

Kayes-Wandover, K M, and P C White. 2000. “Steroidogenic Enzyme Gene Expression in the Human Heart.” The Journal of Clinical Endocrinology and Metabolism 85 (7) (July): 2519–25. doi:10.1210/jcem.85.7.6663.

Miller, Walter L, and Richard J Auchus. 2011. “The Molecular Biology, Biochemistry, and Physiology of Human Steroidogenesis and Its Disorders.” Endocrine Reviews 32 (1) (February): 81–151. doi:10.1210/er.2010-0013.

Rone, Malena B, Jinjiang Fan, and Vassilios Papadopoulos. 2009. “Cholesterol Transport in Steroid Biosynthesis: Role of Protein-Protein Interactions and Implications in Disease States.” Biochimica et Biophysica Acta 1791 (7) (July): 646–58. doi:10.1016/j.bbalip.2009.03.001.

Schroeder, F, J F Holland, and L L Bieber. 1971. “Fluorometric Evidence for the Binding of Cholesterol to the Filipin Complex.” The Journal of Antibiotics 24 (12) (December): 846–9.

Steer, C. 1984. “Detection of Membrane Cholesterol by Filipin in Isolated Rat Liver Coated Vesicles Is Dependent upon Removal of the Clathrin Coat.” The Journal of Cell Biology 99 (1) (July 1): 315–319. doi:10.1083/jcb.99.1.315.


Event: 413: Reduction, Testosterone synthesis in Leydig cells

Short Name: Reduction, Testosterone synthesis in Leydig cells

Key Event Component

Process Object Action
testosterone biosynthetic process testosterone decreased

Biological Context

Level of Biological Organization
Cellular

Cell term

Cell term
testosterone secreting cell

Domain of Applicability


Taxonomic Applicability
Term Scientific Term Evidence Links
rat Rattus norvegicus High NCBI
human Homo sapiens High NCBI
mice Mus sp. Low NCBI

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.


Key Event Description

Biological state

Testosterone is a steroid hormone from the androgen group and is found in humans and other vertebrates.

Biological compartments

In humans and other mammals, testosterone is secreted primarily by the testicles of males and, to a lesser extent, the ovaries of females and other steroidogenic tissues (e.g., brain, adipose). It either acts locally /or is transported to other tissues via blood circulation. Testosterone synthesis takes place within the mitochondria of Leydig cells, the testosterone-producing cells of the testis. It is produced upon stimulation of these cells by Luteinizing hormone (LH) that is secreted in pulses into the peripheral circulation by the pituitary gland in response to Gonadotropin-releasing hormone (GnRH) from the hypothalamus. Testosterone and its aromatized product, estradiol, feed back to the hypothalamus and pituitary gland to suppress transiently LH and thus testosterone production. In response to reduced testosterone levels, GnRH and LH are produced. This negative feedback cycle results in pulsatile secretion of LH followed by pulsatile production of testosterone (Ellis, Desjardins, and Fraser 1983), (Chandrashekar and Bartke 1998).

General role in biology

Testosterone is the principal male sex hormone and an anabolic steroid. Male sexual differentiation depends on testosterone (T), dihydrotestosterone (DHT), and the expression of androgen receptors by target cells (Manson and Carr 2003). During the development secretion of androgens by Leydig cells is essential for masculinization of the foetus (Nef 2000). The foetal Leydig cells develop in utero. These cells become competent to produce testosterone in rat by gestational day (GD) 15.5, with increasing production thereafter. Peak steroidogenic activity is reached just prior to birth, on GD19 (Chen, Ge, and Zirkin 2009). Testosterone secreted by foetal Leydig cells is required for the differentiation of the male urogenital system late in gestation (Huhtaniemi and Pelliniemi 1992). Foetal Leydig cells also play a role in the scrotal descent of the testis through their synthesis of insulin-like growth factor 3 (Insl3), for review see (Nef 2000).

In humans, the first morphological sign of testicular differentiation is the formation of testicular cords, which can be seen between 6 and 7 weeks of gestation. Steroid-secreting Leydig cells can be seen in the testis at 8 weeks of gestation. At this period, the concentration of androgens in the testicular tissue and blood starts to rise, peaking at 14-16 weeks of gestation. This increase comes with an increase in the number of Leydig cells for review see (Rouiller-Fabre et al. 2009).

Adult Leydig cells, which are distinct from the foetal Leydig cells, form during puberty and supply the testosterone required for the onset of spermatogenesis, among other functions. Distinct stages of adult Leydig cell development have been identified and characterized. The stem Leydig cells are undifferentiated cells that are capable of indefinite self-renewal but also of differentiation to steroidogenic cells. These cells give rise to progenitor Leydig cells, which proliferate, continue to differentiate, and give rise to the immature Leydig cells. Immature Leydig cells synthesize high levels of testosterone metabolites and develop into terminally differentiated adult Leydig cells, which produce high levels of testosterone. With aging, both serum and testicular testosterone concentrations progressively decline, for review see (Nef 2000).

Androgens play a crucial role in the development and maintenance of male reproductive and sexual functions. Low levels of circulating androgens can cause disturbances in male sexual development, resulting in congenital abnormalities of the male reproductive tract. Later in life, this may cause reduced fertility, sexual dysfunction, decreased muscle formation and bone mineralisation, disturbances of fat metabolism, and cognitive dysfunction. Testosterone levels decrease as a process of ageing: signs and symptoms caused by this decline can be considered a normal part of ageing.


How it is Measured or Detected

OECD TG 456 [1] is the validated test guideline for an in vitro screen for chemical effects on steroidogenesis, specifically the production of 17ß-estradiol (E2) and testosterone (T). The testosterone syntheis can be measured in vitro cultured Leydig cells. The methods for culturing Leydig cells can be found in the Database Service on Alternative Methods to animal experimentation (DB-ALM): Leydig Cell-enriched Cultures [2], Testicular Organ and Tissue Culture Systems [3].

Testosterone synthesis in vitro cultured cells can be measured indirectly by testosterone radioimmunoassay or analytical methods such as LC-MS.


References

Chandrashekar, V, and A Bartke. 1998. “The Role of Growth Hormone in the Control of Gonadotropin Secretion in Adult Male Rats.” Endocrinology 139 (3) (March): 1067–74. doi:10.1210/endo.139.3.5816.

Ellis, G B, C Desjardins, and H M Fraser. 1983. “Control of Pulsatile LH Release in Male Rats.” Neuroendocrinology 37 (3) (September): 177–83. Huhtaniemi, I, and L J Pelliniemi. 1992. “Fetal Leydig Cells: Cellular Origin, Morphology, Life Span, and Special Functional Features.” Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine (New York, N.Y.) 201 (2) (November): 125–40.

Manson, Jeanne M, and Michael C Carr. 2003. “Molecular Epidemiology of Hypospadias: Review of Genetic and Environmental Risk Factors.” Birth Defects Research. Part A, Clinical and Molecular Teratology 67 (10) (October): 825–36. doi:10.1002/bdra.10084.

Nef, S. 2000. “Hormones in Male Sexual Development.” Genes & Development 14 (24) (December 15): 3075–3086. doi:10.1101/gad.843800.

Rouiller-Fabre, Virginie, Vincent Muczynski, Romain Lambrot, Charlotte Lécureuil, Hervé Coffigny, Catherine Pairault, Delphine Moison, et al. 2009. “Ontogenesis of Testicular Function in Humans.” Folia Histochemica et Cytobiologica / Polish Academy of Sciences, Polish Histochemical and Cytochemical Society 47 (5) (January): S19–24. doi:10.2478/v10042-009-0065-4.


Event: 446: Reduction, testosterone level

Short Name: Reduction, testosterone level

Key Event Component

Process Object Action
testosterone decreased

Biological Context

Level of Biological Organization
Tissue

Organ term

Organ term
blood

Domain of Applicability


Taxonomic Applicability
Term Scientific Term Evidence Links
human Homo sapiens High NCBI
rat Rattus norvegicus High NCBI
mouse Mus musculus High NCBI

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.


Key Event Description

Biological state

Testosterone (T) is a steroid hormone from the androgen group. T serves as a substrate for two metabolic pathways that produce antagonistic sex steroids.

Biological compartments

Testosterone is synthesized by the gonads and other steroidogenic tissues (e.g., brain, adipose), acts locally and/or is transported to other tissues via blood circulation. Leydig cells are the testosterone-producing cells of the testis.

General role in biology

Androgens, the main male sex steroids, are the critical factors responsible for the development of the male phenotype during embryogenesis and for the achievement of sexual maturation at puberty. In adulthood, androgens remain essential for the maintenance of male reproductive function and behaviour. Apart from their effects on reproduction, androgens affect a wide variety of non-reproductive tissues such as skin, bone, muscle, and brain (Heemers, Verhoeven, & Swinnen, 2006). Androgens, principally T and 5α-dihydrotestosterone (DHT), exert most of their effects by interacting with a specific receptor, the androgen receptor (AR), for review see (Murashima, Kishigami, Thomson, & Yamada, 2015). On the one hand, testosterone can be reduced by 5α-reductase to produce 5α dihydrotestosterone (DHT). On the other hand, testosterone can be aromatized to generate estrogens. Testosterone effects can also be classified by the age of usual occurrence, postnatal effects in both males and females are mostly dependent on the levels and duration of circulating free testosterone.


How it is Measured or Detected

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).


References

Heemers, H. V, Verhoeven, G., & Swinnen, J. V. (2006). Androgen activation of the sterol regulatory element-binding protein pathway: Current insights. Molecular Endocrinology (Baltimore, Md.), 20(10), 2265–77. doi:10.1210/me.2005-0479

Murashima, A., Kishigami, S., Thomson, A., & Yamada, G. (2015). Androgens and mammalian male reproductive tract development. Biochimica et Biophysica Acta, 1849(2), 163–170. doi:10.1016/j.bbagrm.2014.05.020

Paduch, D. A., Brannigan, R. E., Fuchs, E. F., Kim, E. D., Marmar, J. L., & Sandlow, J. I. (2014). The laboratory diagnosis of testosterone deficiency. Urology, 83(5), 980–8. doi:10.1016/j.urology.2013.12.024

Taieb, J., Mathian, B., Millot, F., Patricot, M.-C., Mathieu, E., Queyrel, N., … Boudou, P. (2003). Testosterone measured by 10 immunoassays and by isotope-dilution gas chromatography-mass spectrometry in sera from 116 men, women, and children. Clinical Chemistry, 49(8), 1381–95.


Event: 289: Decrease, Translocator protein (TSPO)

Short Name: Decrease, Translocator protein (TSPO)

Key Event Component

Process Object Action
gene expression translocator protein decreased

AOPs Including This Key Event


Biological Context

Level of Biological Organization
Cellular

Cell term

Cell term
steroid hormone secreting cell

Domain of Applicability


Taxonomic Applicability
Term Scientific Term Evidence Links
Homo sapiens Homo sapiens High NCBI
rat Rattus norvegicus High NCBI
Mus musculus Mus musculus Moderate NCBI

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).


Key Event Description

Biological state

Translocator protein (TSPO), previously known as the peripheral benzodiazepine receptor (PBR), is a mitochondrial outer membrane protein implicated in cholesterol import to the inner mitochondrial membrane (Besman et al. 1989).

Biological compartments

The TSPO is present in virtually all mammalian peripheral tissues (Zisterer and Williams 1997), however highly prominent TSPO protein expression has been identified in steroidogenic tissues (R. R. Anholt et al. 1985), (Wang, Fan, and Papadopoulos 2012). The presence of TSOP has been confirmed in Leydig and Sertoli cells (Morohaku, Phuong, and Selvaraj 2013), granulosa cells (Amsterdam and Suh 1991) and to a lesser extent in thecal cells (Morohaku, Phuong, and Selvaraj 2013). In subcellular fractions, binding sites for the TSOP have been identified to be present in the outer mitochondrial membrane (OMM) (R. R. Anholt et al. 1985), (R. Anholt et al. 1986). Transcriptional regulation of TSPO genes has been examined and recently reviewed (Morohaku, Phuong, and Selvaraj 2013).

General role in biology: regulation of lipid transport

TSPO mediates the delivery of the substrate cholesterol to the inner mitochondrial side chain cleavage enzyme P450scc (Besman et al. 1989). TSPO ligands stimulate steroidogenesis and induce cholesterol movement from the outer mitochondrial membrane (OMM) to the inner mitochondrial membrane (IMM) (Besman et al. 1989).


How it is Measured or Detected

TSPO levels can be assayed by standard methods for assessment of gene expression levels like qPCR or direct protein levels by Western blot.

The level of TSPO as well as other steroidogenic protein can be measured in vitro cultured Leydig cells. The methods for culturing Leydig cells can be found in the Database Service on Alternative Methods to animal experimentation (DB-ALM): Leydig Cell-enriched Cultures [1], Testicular Organ and Tissue Culture Systems [2].

Uncertainties and Inconsistencies

This information needs to be moved to a key event relationship page.

TSPO -knockout mice have shown embryonic lethality (Lacapère and Papadopoulos 2003); in contrast recent findings have shown no effect on viability of foetuses (Tu et al. 2014). Aberrant TSPO levels have been linked to multiple diseases, including cancer, endocrine disorders, brain injury, neurodegeneration, ischemia-reperfusion injury and inflammatory diseases (Wang, Fan, and Papadopoulos 2012). However, recent studies have shown opposite results. Peripheral benzodiazepine receptor/translocator protein global knock-out mice are viable and show no effects on steroid hormone biosynthesis (Tu et al. 2014), (Morohaku et al. 2014). As stated in a recent review "At this point in time, a functional designation for TSPO is still actively being sought" (Selvaraj, Stocco, and Tu 2015).


References

Amsterdam, A. & Suh, B.S., 1991. An inducible functional peripheral benzodiazepine receptor in mitochondria of steroidogenic granulosa cells. Endocrinology, 129(1), pp.503–10.

Anholt, R. et al., 1986. The peripheral-type benzodiazepine receptor. Localization to the mitochondrial outer membrane. J. Biol. Chem., 261(2), pp.576–583.

Anholt, R.R. et al., 1985. Peripheral-type benzodiazepine receptors: autoradiographic localization in whole-body sections of neonatal rats. The Journal of pharmacology and experimental therapeutics, 233(2), pp.517–26.

Besman, M.J. et al., 1989. Identification of des-(Gly-Ile)-endozepine as an effector of corticotropin-dependent adrenal steroidogenesis: stimulation of cholesterol delivery is mediated by the peripheral benzodiazepine receptor. Proceedings of the National Academy of Sciences of the United States of America, 86(13), pp.4897–901.

Lacapère, J.J. & Papadopoulos, V., 2003. Peripheral-type benzodiazepine receptor: structure and function of a cholesterol-binding protein in steroid and bile acid biosynthesis. Steroids, 68(7-8), pp.569–85.

Morohaku, K. et al., 2014. Translocator protein/peripheral benzodiazepine receptor is not required for steroid hormone biosynthesis. Endocrinology, 155(1), pp.89–97. Morohaku, K., Phuong, N.S. & Selvaraj, V., 2013. Developmental expression of translocator protein/peripheral benzodiazepine receptor in reproductive tissues. W. Yan, ed. PloS one, 8(9), p.e74509.

Papadopoulos, V. et al., 1997. Targeted disruption of the peripheral-type benzodiazepine receptor gene inhibits steroidogenesis in the R2C Leydig tumor cell line. The Journal of biological chemistry, 272(51), pp.32129–35.

Tu, L.N. et al., 2014. Peripheral benzodiazepine receptor/translocator protein global knock-out mice are viable with no effects on steroid hormone biosynthesis. The Journal of biological chemistry, 289(40), pp.27444–54.

Wang, H.-J., Fan, J. & Papadopoulos, V., 2012. Translocator protein (Tspo) gene promoter-driven green fluorescent protein synthesis in transgenic mice: an in vivo model to study Tspo transcription. Cell and tissue research, 350(2), pp.261–75.

Yeliseev, A.A., Krueger, K.E. & Kaplan, S., 1997. A mammalian mitochondrial drug receptor functions as a bacterial “oxygen” sensor. Proceedings of the National Academy of Sciences of the United States of America, 94(10), pp.5101–6. Zisterer, D.M. & Williams, D.C., 1997. Peripheral-type benzodiazepine receptors. General pharmacology, 29(3), pp.305–14.


List of Adverse Outcomes in this AOP

Event: 406: impaired, Fertility

Short Name: impaired, Fertility

Key Event Component

Process Object Action
fertility decreased

Biological Context

Level of Biological Organization
Individual

Domain of Applicability


Taxonomic Applicability
Term Scientific Term Evidence Links
rat Rattus norvegicus High NCBI
mouse Mus musculus High NCBI
human Homo sapiens High NCBI

Key Event Description

Biological state

capability to produce offspring

Biological compartments

System

General role in biology

Fertility is the capacity to conceive or induce conception. Impairment of fertility represents disorders of male or female reproductive functions or capacity.


How it is Measured or Detected

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


Regulatory Significance of the AO

Under REACH, information on reproductive toxicity is required for chemicals with an annual production/importation volume of 10 metric tonnes or more. Standard information requirements include a screening study on reproduction toxicity (OECD TG 421/422) at Annex VIII (10-100 t.p.a), a prenatal developmental toxicity study (OECD 414) on a first species at Annex IX (100-1000 t.p.a), and from March 2015 the OECD 443(Extended One-Generation Reproductive Toxicity Study) is reproductive toxicity requirement instead of the two generation reproductive toxicity study (OECD TG 416). If not conducted already at Annex IX, a prenatal developmental toxicity study on a second species at Annex X (≥ 1000 t.p.a.).

Under the Biocidal Products Regulation (BPR), information is also required on reproductive toxicity for active substances as part of core data set and additional data set (EU 2012, ECHA 2013). As a core data set, prenatal developmental toxicity study (EU TM B.31) in rabbits as a first species and a two-generation reproduction toxicity study (EU TM B.31) are required. OECD TG 443 (Extended One-Generation Reproductive Toxicity Study) shall be considered as an alternative approach to the multi-generation study.) According to the Classification, Labelling and Packaging (CLP) regulation (EC, 200; Annex I: 3.7.1.1): a) “reproductive toxicity” includes adverse effects on sexual function and fertility in adult males and females, as well as developmental toxicity in the offspring; b) “effects on fertility” includes adverse effects on sexual function and fertility; and c) “developmental toxicity” includes adverse effects on development of the offspring.


Event: 348: Malformation, Male reproductive tract

Short Name: Malformation, Male reproductive tract

Key Event Component

Process Object Action
male reproductive organ morphological change

AOPs Including This Key Event


Biological Context

Level of Biological Organization
Organ

Organ term

Organ term
male reproductive system

Domain of Applicability


Taxonomic Applicability
Term Scientific Term Evidence Links
human Homo sapiens Moderate NCBI
rat Rattus norvegicus High NCBI

Hypospadias

Rodents (Gray et al. 2001) Human (Manson and Carr 2003) Wildlife species (Hayes et al. 2002)

AGD Across numerous species, including humans, AGD is longer in males compared to females; for review see (Barrett et al. 2014).


Key Event Description

Biological state

Male reproductive tract malformations (congenital malformation of male genitalia) comprise any physical abnormality of the male internal or external genitalia present at birth. Some result from excessive or deficient androgen effect, others result from teratogenic effects, or are associated with anomalies of other parts of the body in a recognizable pattern (i.e., a syndrome). The cause of many of these birth defects is unknown.

Hypospadias is a defect of the urogenital system, a malformation in which the urethra opens on the underside of the penis instead of the tip. It results from an incomplete closure of the urethral folds, leaving a split on the penis (Kalfa, Philibert, and Sultan 2009). When the urethra opens to the glans or corona of the penis, it is called distal, whereas opening to the shaft or penoscrotal area defines hypospadias as proximal. Androgens regulate the masculinization of external genitalia. Therefore any defects in androgen biosynthesis, metabolism or action during foetal development can cause hypospadias. Gene defects causing disorders of testicular differentiation, conversion of testosterone to dihydrotestosterone or mutations in the androgen receptor can also result in hypospadias (Kalfa et al. 2008). In about 20% of patients with isolated hypospadias there are signs of endocrine abnormalities by the time of diagnosis (Rey et al. 2005). The majority of hypospadias are believed to have a multifactorial etiology, although a small percentage do result from single gene mutations (Baskin, Himes, and Colborn 2001). The only treatment of hypospadias is surgery, thus, prevention is imperative.


Biological compartments: reproductive system


How it is Measured or Detected

Malformations are detected by macroscopically for any structural abnormality or pathological change. The Congenital malformation of the genitalia is a medical term referring to a broad category of conditions that for humans is classified by International Classification of Diseases (ICD) in chapter "Congenital malformations of genital organs" (Q50-Q56) e.g.Q54 Hypospadias, Q53 Undescended testicle. Hypospadias is usually diagnosed during the routine examination after birth. The hypospadias belongs to the category of "Congenital malformation of the genitalia" - a medical term referring to a broad category of conditions as classified in the International Classification of Diseases (ICD) in chapter "Congenital malformations of genital organs" (Q50-Q56) e.g. Q54 Hypospadias.

The anogenital distance (AGD) is a sexual dimorphism that results from the sex difference in foetal androgen (DHT) levels (Rhees et al., 1997). The AGD, the distance from the anus to the genitals, is widely used as biomarker of prenatal androgen exposure during a reproductive programming window (Wolf et al. 1999), (McIntyre, Barlow, and Foster 2001), (Macleod et al. 2010). The AGD is a marker of perineal growth and caudal migration of the genital tubercle. It is androgen-dependent in male rodents (Bowman et al. 2003). Measurement of AGD has also been proposed as a quantitative biomarker of foetal endocrine disruptor exposure in humans (Arbuckle et al. 2008), (Dean and Sharpe 2013). A longer (more “masculine”) AGD is typically associated with favourable health outcomes, while a shorter AGD is associated with adverse health outcomes. The AGD in males is approximately double that of females. Less is known about clinical correlates of AGD in females, although one study found that in women a longer AGD was associated with increased odds of multifollicular ovaries (Mendiola et al. 2012). The AGD is reflecting the prenatal hormonal milieu and in addition a biomarker for the risk of reproductive health problems linked to that early hormonal environment (Barrett et al. 2014). In animal studies, AGD measured from the genital tubercle to the anus is a sensitive marker of in utero exposure to androgens and anti-androgens, and is used extensively in animal reproductive toxicology studies (McIntyre, Barlow, and Foster 2001). AGD of each pup should be measured on at least one occasion from pre natal day postnatal day (PND) 0 through PND 4. Pup body weight should be collected on the day the AGD is measured and the AGD should be normalized to a measure of pup size, preferably the cube root of body weight (12). AGD is influenced by the body weight of the animal and therefore, this should be taken into account when evaluating the data (Gallavan et al, 1999). Body weight as a covariable may also be used (Howdeshell et al. 2007). Decreased AGD in male rats is a hallmark of exposure to antiandrogenic substances (Noriega et al, 2009; Christiansen et al, 2010). A statistically significant change in AGD that cannot be explained by the size of the animal indicates an adverse effect of exposure and should be considered in setting the NOAEL (OECD, 2008).


The extended one-generation in vivo reproductive toxicity study OECD TG 443 [1]is used to investigate adverse effects of chemical substances on fertility and developmental toxicity in the rat, in which AGD is measured.


Regulatory Significance of the AO

In regulatory hazard identification and risk assessment of chemicals malformations of male genitalia are considered as a chemically induced adverse outcome that is used for risk assessment and management purposes. The prenatal developmental toxicity study (TG 414) is the method for examining embryo-foetal toxicity as a consequence of exposure during pregnancy. Parental and offspring growth, development and viability are the relevant endpoints in generation studies (OECD TG 415/416/443). These guidelines are implemented in a number of occasions where the reproductive /developmental toxicity have to be assessed in order to comply with relevant EU regulations.

Under REACH, information on reproductive toxicity is required for chemicals with an annual production/importation volume of 10 metric tonnes or more. Standard information requirements include a screening study on reproduction toxicity (OECD TG 421/422) at Annex VIII (10-100 t.p.a), a prenatal developmental toxicity study (OECD 414) on a first species at Annex IX (100-1000 t.p.a), and from March 2015 the OECD 443(Extended One-Generation Reproductive Toxicity Study) is reproductive toxicity requirement instead of the two generation reproductive toxicity study (OECD TG 416). If not conducted already at Annex IX, a prenatal developmental toxicity study on a second species at Annex X (≥ 1000 t.p.a.).

Under the Biocidal Products Regulation (BPR), information is also required on reproductive toxicity for active substances as part of core data set and additional data set (EU 2012, ECHA 2013). As a core data set, prenatal developmental toxicity study (EU TM B.31) in rabbits as a first species and a two-generation reproduction toxicity study (EU TM B.31) are required. OECD TG 443 (Extended One-Generation Reproductive Toxicity Study) shall be considered as an alternative approach to the multi-generation study.

According to the Classification, Labelling and Packaging (CLP) regulation (EC, 200; Annex I: 3.7.1.1): a) “reproductive toxicity” includes adverse effects on sexual function and fertility in adult males and females, as well as developmental toxicity in the offspring; b) “effects on fertility” includes adverse effects on sexual function and fertility; and c) “developmental toxicity” includes adverse effects on development of the offspring.

AGD is a reproductive endpoint, assessment of AGD is mandatory in OECD TG 443, 415/416 (OECD 2012).


References


Arbuckle, Tye E, Russ Hauser, Shanna H Swan, Catherine S Mao, Matthew P Longnecker, Katharina M Main, Robin M Whyatt, et al. 2008. “Meeting Report: Measuring Endocrine-Sensitive Endpoints within the First Years of Life.” Environmental Health Perspectives 116 (7) (July): 948–51. doi:10.1289/ehp.11226.

Barrett, Emily S, Lauren E Parlett, J Bruce Redmon, and Shanna H Swan. 2014. “Evidence for Sexually Dimorphic Associations between Maternal Characteristics and Anogenital Distance, a Marker of Reproductive Development.” American Journal of Epidemiology 179 (1) (January 1): 57–66. doi:10.1093/aje/kwt220.

Baskin, L S, K Himes, and T Colborn. 2001. “Hypospadias and Endocrine Disruption: Is There a Connection?” Environmental Health Perspectives 109 (11) (November): 1175–83.

Bowman, Christopher J, Norman J Barlow, Katie J Turner, Duncan G Wallace, and Paul M D Foster. 2003. “Effects of in Utero Exposure to Finasteride on Androgen-Dependent Reproductive Development in the Male Rat.” Toxicological Sciences : An Official Journal of the Society of Toxicology 74 (2) (August): 393–406. doi:10.1093/toxsci/kfg128.

Dean, Afshan, and Richard M Sharpe. 2013. “Clinical Review: Anogenital Distance or Digit Length Ratio as Measures of Fetal Androgen Exposure: Relationship to Male Reproductive Development and Its Disorders.” The Journal of Clinical Endocrinology and Metabolism 98 (6) (June): 2230–8. doi:10.1210/jc.2012-4057.

Gray, L E, J Ostby, J Furr, C J Wolf, C Lambright, L Parks, D N Veeramachaneni, et al. 2001. “Effects of Environmental Antiandrogens on Reproductive Development in Experimental Animals.” Human Reproduction Update 7 (3): 248–64.

Hayes, Tyrone B, Atif Collins, Melissa Lee, Magdelena Mendoza, Nigel Noriega, A Ali Stuart, and Aaron Vonk. 2002. “Hermaphroditic, Demasculinized Frogs after Exposure to the Herbicide Atrazine at Low Ecologically Relevant Doses.” Proceedings of the National Academy of Sciences of the United States of America 99 (8) (April 16): 5476–80. doi:10.1073/pnas.082121499.

Howdeshell, Kembra L, Johnathan Furr, Christy R Lambright, Cynthia V Rider, Vickie S Wilson, and L Earl Gray. 2007. “Cumulative Effects of Dibutyl Phthalate and Diethylhexyl Phthalate on Male Rat Reproductive Tract Development: Altered Fetal Steroid Hormones and Genes.” Toxicological Sciences : An Official Journal of the Society of Toxicology 99 (1) (September): 190–202. doi:10.1093/toxsci/kfm069.

Kalfa, Nicolas, Benchun Liu, Ophir Klein, Ming-Hsieh Wang, Mei Cao, and Laurence S Baskin. 2008. “Genomic Variants of ATF3 in Patients with Hypospadias.” The Journal of Urology 180 (5) (November): 2183–8; discussion 2188. doi:10.1016/j.juro.2008.07.066.

Kalfa, Nicolas, Pascal Philibert, and Charles Sultan. 2009. “Is Hypospadias a Genetic, Endocrine or Environmental Disease, or Still an Unexplained Malformation?” International Journal of Andrology 32 (3) (June): 187–97. doi:10.1111/j.1365-2605.2008.00899.x.

Macleod, D J, R M Sharpe, M Welsh, M Fisken, H M Scott, G R Hutchison, A J Drake, and S van den Driesche. 2010. “Androgen Action in the Masculinization Programming Window and Development of Male Reproductive Organs.” International Journal of Andrology 33 (2) (April): 279–87. doi:10.1111/j.1365-2605.2009.01005.x.

Manson, Jeanne M, and Michael C Carr. 2003. “Molecular Epidemiology of Hypospadias: Review of Genetic and Environmental Risk Factors.” Birth Defects Research. Part A, Clinical and Molecular Teratology 67 (10) (October): 825–36. doi:10.1002/bdra.10084.

McIntyre, B S, N J Barlow, and P M Foster. 2001. “Androgen-Mediated Development in Male Rat Offspring Exposed to Flutamide in Utero: Permanence and Correlation of Early Postnatal Changes in Anogenital Distance and Nipple Retention with Malformations in Androgen-Dependent Tissues.” Toxicological Sciences : An Official Journal of the Society of Toxicology 62 (2) (August): 236–49.

Mendiola, Jaime, Manuela Roca, Lidia Mínguez-Alarcón, Maria-Pilar Mira-Escolano, José J López-Espín, Emily S Barrett, Shanna H Swan, and Alberto M Torres-Cantero. 2012. “Anogenital Distance Is Related to Ovarian Follicular Number in Young Spanish Women: A Cross-Sectional Study.” Environmental Health : A Global Access Science Source 11 (January): 90. doi:10.1186/1476-069X-11-90.

OECD. 2012. Test No. 443: Extended One-Generation Reproductive Toxicity Study. OECD Guidelines for the Testing of Chemicals, Section 4. OECD Publishing. doi:10.1787/9789264185371-en.

Rey, Rodolfo A, Ethel Codner, Germán Iñíguez, Patricia Bedecarrás, Romina Trigo, Cecilia Okuma, Silvia Gottlieb, Ignacio Bergadá, Stella M Campo, and Fernando G Cassorla. 2005. “Low Risk of Impaired Testicular Sertoli and Leydig Cell Functions in Boys with Isolated Hypospadias.” The Journal of Clinical Endocrinology and Metabolism 90 (11) (November): 6035–40. doi:10.1210/jc.2005-1306.

Wolf, C., C. Lambright, P. Mann, M. Price, R. L. Cooper, J. Ostby, and L. E. Gray. 1999. “Administration of Potentially Antiandrogenic Pesticides (procymidone, Linuron, Iprodione, Chlozolinate, P,p’-DDE, and Ketoconazole) and Toxic Substances (dibutyl- and Diethylhexyl Phthalate, PCB 169, and Ethane Dimethane Sulphonate) during Sexual Differen.” Toxicology and Industrial Health 15 (1-2) (February 1): 94–118. doi:10.1177/074823379901500109.


Appendix 2

List of Key Event Relationships in the AOP