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Relationship: 438

Title

A descriptive phrase which clearly defines the two KEs being considered and the sequential relationship between them (i.e., which is upstream, and which is downstream). More help

Reduction, Cholesterol transport in mitochondria leads to Reduction, Testosterone synthesis in Leydig cells

Upstream event

The causing Key Event (KE) in a Key Event Relationship (KER). More help

Reduction, Cholesterol transport in mitochondria

Downstream event

The responding Key Event (KE) in a Key Event Relationship (KER). More help

Reduction, Testosterone synthesis in Leydig cells

Key Event Relationship Overview

The utility of AOPs for regulatory application is defined, to a large extent, by the confidence and precision with which they facilitate extrapolation of data measured at low levels of biological organisation to predicted outcomes at higher levels of organisation and the extent to which they can link biological effect measurements to their specific causes.Within the AOP framework, the predictive relationships that facilitate extrapolation are represented by the KERs. Consequently, the overall WoE for an AOP is a reflection in part, of the level of confidence in the underlying series of KERs it encompasses. Therefore, describing the KERs in an AOP involves assembling and organising the types of information and evidence that defines the scientific basis for inferring the probable change in, or state of, a downstream KE from the known or measured state of an upstream KE. More help

AOPs Referencing Relationship

AOP Name	Adjacency	Weight of Evidence	Quantitative Understanding	Point of Contact	Author Status	OECD Status
PPARα activation in utero leading to impaired fertility in males	adjacent	Moderate		Arthur Author (send email)	Open for citation & comment	Under Review
PPARα activation leading to impaired fertility in adult male rodents	adjacent	Moderate		Evgeniia Kazymova (send email)	Not under active development	Under Development

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) that help to define the biological applicability domain of the KER.In general, this will be dictated by the more restrictive of the two KEs being linked together by the KER. More help

Term	Scientific Term	Evidence	Link
mice	Mus sp.	Moderate	NCBI
rat	Rattus norvegicus	High	NCBI
human	Homo sapiens	Low	NCBI

Sex Applicability

An indication of the the relevant sex for this KER. More help

Life Stage Applicability

An indication of the the relevant life stage(s) for this KER. More help

Key Event Relationship Description

Provides a concise overview of the information given below as well as addressing details that aren’t inherent in the description of the KEs themselves. More help

Production of steroid hormones depends on the availability of cholesterol in the mitochondrial matrix. A decreased amount of cholesterol inside the mitochondria (e. g by decreased expression of enzymes that transport cholesterol like StAR or TSOP) means diminished substrate for hormone (testosterone) production in testes.

Evidence Collection Strategy

Include a description of the approach for identification and assembly of the evidence base for the KER. For evidence identification, include, for example, a description of the sources and dates of information consulted including expert knowledge, databases searched and associated search terms/strings. Include also a description of study screening criteria and methodology, study quality assessment considerations, the data extraction strategy and links to any repositories/databases of relevant references.Tabular summaries and links to relevant supporting documentation are encouraged, wherever possible. More help

Evidence Supporting this KER

Addresses the scientific evidence supporting KERs in an AOP setting the stage for overall assessment of the AOP. More help

Biological Plausibility

Addresses the biological rationale for a connection between KEupstream and KEdownstream. This field can also incorporate additional mechanistic details that help inform the relationship between KEs, this is useful when it is not practical/pragmatic to represent these details as separate KEs due to the difficulty or relative infrequency with which it is likely to be measured. More help

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

Empirical Evidence

Provides specific (citable) evidence that supports the idea of a change in the upstream KE (KEupstream) leading to, or being associated with, a subsequent change in the downstream KE (KEdownstream), assuming the perturbation of KEupstream is sufficient. More help

There is evidence from experimental work that demonstrates a coordinated reduction in the expression of key genes and proteins that are involved in cholesterol transport and steroidogenesis, together with a corresponding reduction in testosterone in testes. For details see Table 1. Foetal Leydig cells exhibit a high rate of lipid metabolism, which is required for both synthesizing and importing the testosterone precursor cholesterol. Upon exposure to some chemicals mRNA expression of genes in these pathways are profoundly reduced e.g. following 500mg/kg phthalate (DBP) exposure (Johnson, McDowell, Viereck, & Xia, 2011), (Thompson et al., 2005). Additionally, after phthalate exposure testis cholesterol and cholesterol-containing lipid droplets in foetal Leydig cells are also reduced (Barlow et al., 2003), (Johnson et al., 2011), (Lehmann, Phillips, Sar, Foster, & Gaido, 2004).

			KE: Cholesterol transport, reduction	KE: Testosterone production/levels, reduction
Compound	Species	Effect level	Translator protein (TSPO), decrease; Steroidogenic acute regulatory protein (StAR) decrease
Phthalate (DBP)	rat	LOEL=500 mg/kg/day	mRNA StAR decrease (by ~34%)(Barlow et al., 2003)
Phthalate (BBP, DPeP, DEHP, DHP, DiHeP, DCHP, DINP DHeP)	rat
Phthalate (DBP, DEHP, BBP)	Rat	LOEL=750 mg/kg/day (GD14-18)		testosterone production, reduction ex vivo fetal testes examined on GD18 (Wilson et al., 2004)

Phthalate (DBP)	rat	LOEL=500 mg/kg/day	reduced Leydig cell lipid content(Barlow et al., 2003)
Phthalate (DBP)	rat	LOEL=500 mg/kg/day GD 12 -20, examinations on GD20	total cholesterol levels, reduction	intratesticular testosterone levels, reduction (by nearly 90%)(Johnson et al., 2011)
Phthalate (DBP)	rat	LOEL=500 mg/kg/day (GD12-19)	decrease uptake of cholesterol Leydig cell mitochondria gd 19	testosterone production, reduction ex vivo (Thompson, Ross, & Gaido, 2004)
Phthalate (DEHP)	mouse	LOEL=1 g/kg/day	reduced TSPO mRNA	testosterone levels, reduction (Gazouli, 2002)
Phthalate (DEHP)	rat	LOEL=300 mg/ kg/day	dose-dependently reduced StAR, TSOP mRNA (GD 21 testes), also on protein levels in Leydig cells (Borch, Metzdorff, Vinggaard, Brokken, & Dalgaard, 2006)
Phthalate (DEHP)	rat	LOEL=300 mg/kg/day		testosterone production, reduction (ex vivo) testosterone levels, reduction (Borch et al., 2006), (Borch, Ladefoged, Hass, & Vinggaard, 2004)
Phthalate (MEHP)	mouse	LOEC=100 μM	reduced TSPO mRNA levels by 50%, binding sites decreased by 50% no effect on receptor affinity inhibited the transfer or loading of cholesterol to the inner mitochondrial membrane P450scc. (Gazouli, 2002)
Phthalate (MEHP)	rat	IC50 =100 μM	inhibited formation of progesterone (Gazouli, 2002)
Phthalate (MEHP)	rat	LOEC=250 μM	cholesterol transport, decrease (into the mitochondria of immature and adult Leydig cells)	Testosterone, reduction by approximately 60%, in vitro ( immature and adult Leydig cells) (Svechnikov, Svechnikova, & Söder, 2008)
Phthalate (DEHP)	rat	LOEL=750 mg/kg/day		testosterone production reduction, testosterone levels, reduction (testicular and whole-body T levels in fetal and neonatal male rats from GD 17 to PND 2. (Parks, 2000)
Phthalate (MEHP)	rat	LOEC=1 μM		testosterone production, reduction dose-dependent (Chauvigné et al., 2011)
Perfluorooctanoic acid (PFOA)	mouse	LOEL=5mg/kg/day		plasma testosterone, reduction (by 37%)(Li et al., 2011)
WY-14,643	mouse	LOEC=50 mg/kg/day	reduced TSPO mRNA	Serum testosterone levels, reduction (Gazouli, 2002)
WY-14,643	rat			No decrease of testosterone ( ex vivo), (Furr, Lambright, Wilson, Foster, & Gray, 2014)
WY-14,643	mouse	LOEC=100 μM	Inhibited progesterone synthesis (Gazouli, 2002)
Bezafibrate	mouse	IC50=100 μM	a dose-dependent 10–95% inhibition of the progesterone synthesis at 24 or 72 h inhibited the transfer or loading of cholesterol to the inner mitochondrial membrane P450scc. At 100 μM binding sites of TSPO decreased IC50 of approximately 100 μM decrease TSPO levels by 60% at 100 μM (Gazouli, 2002)
Bezafibrate	rat	IC50 = 30 μM	inhibited formation of progesterone (Gazouli, 2002)
Bezafibrate	rat	IC50 ~10−4 μM		testosterone production, reduction (Gazouli, 2002)
Phthalate (DiBP)	rat	GD 19 -21	reduced StAR, (Boberg et al., 2008)	testicular testosterone production and testicular testosterone levels, (Boberg et al., 2008)

Table 1. Summary table of empirical support for this KER. IC50 half maximal inhibitory concentration, LOEC-lowest effect concentration, LOEL- lowest observed effect level, Dibutyl phthalate (DBP), diisobutyl phthalate (DiBP), Bis(2-ethylhexyl) phthalate (DEHP), Dibutyl phthalate (DBP), Bezafibrate and WY-14,643 are PPARα ligands, n.a - not available

Uncertainties and Inconsistencies

Addresses inconsistencies or uncertainties in the relationship including the identification of experimental details that may explain apparent deviations from the expected patterns of concordance. More help

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

Known modulating factors

This table captures specific information on the MF, its properties, how it affects the KER and respective references.1.) What is the modulating factor? Name the factor for which solid evidence exists that it influences this KER. Examples: age, sex, genotype, diet 2.) Details of this modulating factor. Specify which features of this MF are relevant for this KER. Examples: a specific age range or a specific biological age (defined by...); a specific gene mutation or variant, a specific nutrient (deficit or surplus); a sex-specific homone; a certain threshold value (e.g. serum levels of a chemical above...) 3.) Description of how this modulating factor affects this KER. Describe the provable modification of the KER (also quantitatively, if known). Examples: increase or decrease of the magnitude of effect (by a factor of...); change of the time-course of the effect (onset delay by...); alteration of the probability of the effect; increase or decrease of the sensitivity of the downstream effect (by a factor of...) 4.) Provision of supporting scientific evidence for an effect of this MF on this KER. Give a list of references. More help

Quantitative Understanding of the Linkage

Captures information that helps to define how much change in the upstream KE, and/or for how long, is needed to elicit a detectable and defined change in the downstream KE. More help

Response-response Relationship

Provides sources of data that define the response-response relationships between the KEs. More help

Time-scale

Information regarding the approximate time-scale of the changes in KEdownstream relative to changes in KEupstream (i.e., do effects on KEdownstream lag those on KEupstream by seconds, minutes, hours, or days?). More help

Known Feedforward/Feedback loops influencing this KER

Define whether there are known positive or negative feedback mechanisms involved and what is understood about their time-course and homeostatic limits. More help

Domain of Applicability

A free-text section of the KER description that the developers can use to explain their rationale for the taxonomic, life stage, or sex applicability structured terms. More help

See Table 1.

References

List of the literature that was cited for this KER description. More help

Barlow, N. J., Phillips, S. L., Wallace, D. G., Sar, M., Gaido, K. W., & Foster, P. M. D. (2003). Quantitative changes in gene expression in fetal rat testes following exposure to di(n-butyl) phthalate. Toxicological Sciences : An Official Journal of the Society of Toxicology, 73(2), 431–41. doi:10.1093/toxsci/kfg087

Boberg, J., Metzdorff, S., Wortziger, R., Axelstad, M., Brokken, L., Vinggaard, A. M., … Nellemann, C. (2008). Impact of diisobutyl phthalate and other PPAR agonists on steroidogenesis and plasma insulin and leptin levels in fetal rats. Toxicology, 250(2-3), 75–81. doi:10.1016/j.tox.2008.05.020

Borch, J., Ladefoged, O., Hass, U., & Vinggaard, A. M. (2004). Steroidogenesis in fetal male rats is reduced by DEHP and DINP, but endocrine effects of DEHP are not modulated by DEHA in fetal, prepubertal and adult male rats. Reproductive Toxicology (Elmsford, N.Y.), 18(1), 53–61. doi:10.1016/j.reprotox.2003.10.011

Borch, J., Metzdorff, S. B., Vinggaard, A. M., Brokken, L., & Dalgaard, M. (2006). Mechanisms underlying the anti-androgenic effects of diethylhexyl phthalate in fetal rat testis. Toxicology, 223(1-2), 144–55. doi:10.1016/j.tox.2006.03.015

Chauvigné, F., Plummer, S., Lesné, L., Cravedi, J.-P., Dejucq-Rainsford, N., Fostier, A., & Jégou, B. (2011). Mono-(2-ethylhexyl) phthalate directly alters the expression of Leydig cell genes and CYP17 lyase activity in cultured rat fetal testis. PloS One, 6(11), e27172. doi:10.1371/journal.pone.0027172

Furr, J. R., Lambright, C. S., Wilson, V. S., Foster, P. M., & Gray, L. E. (2014). A short-term in vivo screen using fetal testosterone production, a key event in the phthalate adverse outcome pathway, to predict disruption of sexual differentiation. Toxicological Sciences : An Official Journal of the Society of Toxicology, 140(2), 403–24. doi:10.1093/toxsci/kfu081

Gazouli, M. (2002). Effect of Peroxisome Proliferators on Leydig Cell Peripheral-Type Benzodiazepine Receptor Gene Expression, Hormone-Stimulated Cholesterol Transport, and Steroidogenesis: Role of the Peroxisome Proliferator-Activator Receptor . Endocrinology, 143(7), 2571–2583. doi:10.1210/en.143.7.2571

Hauet, T., Yao, Z.-X., Bose, H. S., Wall, C. T., Han, Z., Li, W., … Papadopoulos, V. (2005). Peripheral-type benzodiazepine receptor-mediated action of steroidogenic acute regulatory protein on cholesterol entry into leydig cell mitochondria. Molecular Endocrinology (Baltimore, Md.), 19(2), 540–54. doi:10.1210/me.2004-0307

Johnson, K. J., McDowell, E. N., Viereck, M. P., & Xia, J. Q. (2011). Species-specific dibutyl phthalate fetal testis endocrine disruption correlates with inhibition of SREBP2-dependent gene expression pathways. Toxicological Sciences : An Official Journal of the Society of Toxicology, 120(2), 460–74. doi:10.1093/toxsci/kfr020

Lehmann, K. P., Phillips, S., Sar, M., Foster, P. M. D., & Gaido, K. W. (2004). Dose-dependent alterations in gene expression and testosterone synthesis in the fetal testes of male rats exposed to di (n-butyl) phthalate. Toxicological Sciences : An Official Journal of the Society of Toxicology, 81(1), 60–8. doi:10.1093/toxsci/kfh169

Li, Y., Ramdhan, D. H., Naito, H., Yamagishi, N., Ito, Y., Hayashi, Y., … Nakajima, T. (2011). Ammonium perfluorooctanoate may cause testosterone reduction by adversely affecting testis in relation to PPARα. Toxicology Letters, 205(3), 265–72. doi:10.1016/j.toxlet.2011.06.015 Miller, W. L. (2007). Steroidogenic acute regulatory protein (StAR), a novel mitochondrial cholesterol transporter. Biochimica et Biophysica Acta, 1771(6), 663–76. doi:10.1016/j.bbalip.2007.02.012

Parks, L. G. (2000). The Plasticizer Diethylhexyl Phthalate Induces Malformations by Decreasing Fetal Testosterone Synthesis during Sexual Differentiation in the Male Rat. Toxicological Sciences, 58(2), 339–349. doi:10.1093/toxsci/58.2.339

Payne, A. H., & Hales, D. B. (2013). Overview of Steroidogenic Enzymes in the Pathway from Cholesterol to Active Steroid Hormones. Endocrine Reviews. Stocco, D. M. (2001). StAR protein and the regulation of steroid hormone biosynthesis. Annual Review of Physiology, 63, 193–213. doi:10.1146/annurev.physiol.63.1.193

Svechnikov, K., Svechnikova, I., & Söder, O. (2008). Inhibitory effects of mono-ethylhexyl phthalate on steroidogenesis in immature and adult rat Leydig cells in vitro. Reproductive Toxicology (Elmsford, N.Y.), 25(4), 485–90. doi:10.1016/j.reprotox.2008.05.057

Thompson, C. J., Ross, S. M., & Gaido, K. W. (2004). Di(n-butyl) phthalate impairs cholesterol transport and steroidogenesis in the fetal rat testis through a rapid and reversible mechanism. Endocrinology, 145(3), 1227–37. doi:10.1210/en.2003-1475

Thompson, C. J., Ross, S. M., Hensley, J., Liu, K., Heinze, S. C., Young, S. S., & Gaido, K. W. (2005). Differential steroidogenic gene expression in the fetal adrenal gland versus the testis and rapid and dynamic response of the fetal testis to di(n-butyl) phthalate. Biology of Reproduction, 73(5), 908–17. doi:10.1095/biolreprod.105.042382

Wilson, V. S., Lambright, C., Furr, J., Ostby, J., Wood, C., Held, G., & Gray, L. E. (2004). Phthalate ester-induced gubernacular lesions are associated with reduced insl3 gene expression in the fetal rat testis. Toxicology Letters, 146(3), 207–15.