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Relationship: 3517

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 Decreased, Pregnenolone levels

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

Decreased, Pregnenolone levels

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

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
mammals	mammals	High	NCBI

Sex Applicability

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

Sex	Evidence
Mixed	High

Life Stage Applicability

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

Term	Evidence
All life stages	High

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

Cholesterol gets transported from the outer (OMM) to the inner mitochondrial membrane (IMM), where it undergoes cleavage of the aliphatic side chain by cytochrome P-450 enzyme (P450scc or CYP11A1) that yields the steroid precursor, pregnenolone (Besman et al., 1989). Impaired cholesterol transport, affects the substrate availability, leading to reduced levels of pregnenolone.

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

The KER describes a generally recognized and understood process, i.e. canonical knowledge. The aim of the literature search was therefore to identify review articles and book chapters that summarise the canonical knowledge. PubMed was searched using key words related to steroidogenesis. The search was restricted to reviews from the last 10 years.

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

In stimulated steroidogenic cells, cholesterol is transported from OMM to the IMM most commonly through the transduceosome protein complex (Aghazadeh et al., 2015). Components of the complex shuttle cholesterol to CYP11A1, which turns it to pregnenolone (Besman et al., 1989).

It is hypothesized that cholesterol is directed to CYP11A1 with the help of the metabolon complex, which shares proteins voltage-dependent anion channel 1 (VDAC1) and translocator protein (TSPO) with the transduceosome complex. The metabolome also contains the ATPase family AAA domain-containing protein 3 (ATAD3). Other than the non-vesicular transport through protein carriers, cholesterol can also be transported through vesicles which requires ATP and accounts for less than 30% of cholesterol transfer (Aghazadeh et al., 2015; Miller, 2017). Availability of the substrate through functioning transfer of cholesterol, along with CYP11A1 lead to production of pregnenolone and therefore initiation of all steroidogenesis.

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

Empirical evidence to demonstrate that decreased cholesterol in mitochondria leads to decreased pregnenolone levels can come from several studies, which indicate that disruption in cholesterol transport leads to steroidogenesis disruption. These studies do not include direct measurements of pregnenolone, but since pregnenolone is the precursor of all the downstream steroids, it can be deduced that its levels are affected. However, disruption in downstream processes cannot be excluded.

- Studies with decreased overall cholesterol, which would also reflect mitochondrial cholesterol levels; Individuals prescribed statins to decrease their cholesterol levels, demonstrate decreased levels of pregnenolone (Mostovaya et al., 2022).

- Studies disrupting proteins of the transduceosome complex (VDAC1, TSPO), would lead to decreased cholesterol transport. Knocking down VDAC1 in fibroblast-like cells resulted in inhibition of pregnenolone synthesis (Bose et al., 2008b, 2008a). Using the TSPO inhibitor 19-Atriol on rodent Leydig cells in vitro, a reduced production of testosterone was observed (Chung et al., 2013). Similarly, TSPO knock-out mice exhibited disrupted steroidogenesis (Barron et al., 2018; Fan et al., 2020; Farhan et al., 2021). (Chan et al., 2008; Hu et al., 2002; Huang et al., 2012; Tajima et al., 2001)

- Lastly, there is evidence from rodent exposure studies that have observed decreased cholesterol uptake in mitochondria along with decreased steroid hormones, presented in Table 1.

Table 1 Summary table of empirical support from rodent exposure studies for this KER. LOEC-lowest effect concentration, LOEL- lowest observed effect level, Dibutyl phthalate (DBP), mono(2-ethylhexyl) phthalate (MEHP).

Compound	Species	Effect level	KEupstream	KEdownstream	References
Phthalate (DBP)	rat	LOEL=500 mg/kg/day (GD12-19)	decrease uptake of cholesterol Leydig cell mitochondria	decreased testosterone, decreased expression of P450scc	(Thompson et al., 2004)
Phthalate (MEHP)	rat	LOEL=500 mg/kg/day (GD12-19)	cholesterol transport, decrease (into the mitochondria of immature and adult Leydig cells	decreased testosterone by approximately 60%, in vitro (immature and adult Leydig cells)	(Svechnikov et al., 2008)

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

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

Mutations of CYP11A1 can affect the activity of the enzyme and therefore the rate at which cholesterol is converted to pregnenolone. In reported cases, CYP11A1 mutations cause 80-90% loss of P450scc activity (Hauffa and Hiort, 2011; Miller and Auchus, 2011). In human adrenal cells, the expression of CYP11A1 is controlled by the adrenocorticotropic hormone (ACTH), therefore ACTH is also a modulating factor (Xing et al., 2010).

Mutations in the ATAD3 family of proteins alter cholesterol metabolism (Desai and Campanella, 2019). TSPO mutations have been shown to affect the rate of steroid synthesis (Owen et al., 2017).

Modulating Factor (MF)	MF Specification	Effect(s) on the KER	Reference(s)

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

Conversion of cholesterol to pregnenolone is considered a slow reaction, with each CYP11A1 catalysing twenty molecules of cholesterol per minute (Auchus and Miller, 2016). As pregnenolone, like other steroids, is not stored but synthesized on demand, effects on the key event upstream will have effects on the levels of pregnenolone within minutes.

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

Taxonomic applicability.

This KER refers to the conversion of cholesterol to pregnenolone through CYP11A1. The CYP11A1 gene is only found in vertebrates and is best characterized in mammals (Slominski et al., 2015).

Life stage applicability

In vivo rodent studies have demonstrated its essentiality as early as fetal life (Huang et al., 2012).

Sex applicability

This KER is applicable to both sexes as cholesterol transport and appropriate pregnenolone levels are essential to females and males (Auchus and Miller, 2016; Miller and Auchus, 2011).

References

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

Aghazadeh, Y., Zirkin, B.R., Papadopoulos, V., 2015. Pharmacological Regulation of the Cholesterol Transport Machinery in Steroidogenic Cells of the Testis, in: Vitamins and Hormones. Academic Press Inc., pp. 189–227. https://doi.org/10.1016/bs.vh.2014.12.006

Auchus, R.J., Miller, W.L., 2016. The Principles, Enzymes, and Pathways of Human Steroidogenesis. Endocrinology: Adult and Pediatric 2–2, 1695-1716.e8. https://doi.org/10.1016/B978-0-323-18907-1.00097-4

Barron, A.M., Ji, B., Kito, S., Suhara, T., Higuchi, M., 2018. Steroidogenic abnormalities in translocator protein knockout mice and significance in the aging male. Biochem J 475, 75–85. https://doi.org/10.1042/BCJ20170645

Besman, M.J., Yanagibashi, K., Lee, T.D., Kawamura, M., Hall, P.F., Shively, J.E., 1989. Identification of des-(Gly-Ile)-endozepine as an effector of corticotropin-dependent adrenal steroidogenesis: stimulation of cholesterol delivery is mediated by the peripheral benzodiazepine receptor. Proc Natl Acad Sci U S A 86, 4897–4901. https://doi.org/10.1073/PNAS.86.13.4897

Bose, M., Whittal, R.M., Gairola, C.G., Bose, H.S., 2008a. Molecular mechanism of reduction in pregnenolone synthesis by cigarette smoke. Toxicol Appl Pharmacol 229, 56–64. https://doi.org/10.1016/J.TAAP.2008.01.007

Bose, M., Whittal, R.M., Miller, W.L., Bose, H.S., 2008b. Steroidogenic Activity of StAR Requires Contact with Mitochondrial VDAC1 and Phosphate Carrier Protein. J Biol Chem 283, 8837. https://doi.org/10.1074/JBC.M709221200

Chan, J.K., Lin, L., Huang, N., Quigley, C.A., AvRuskin, T.W., Achermann, J.C., Miller, W.L., 2008. Severe combined adrenal and gonadal deficiency caused by novel mutations in the cholesterol side chain cleavage enzyme, P450scc. J Clin Endocrinol Metab 93, 696–702. https://doi.org/10.1210/JC.2007-2330

Chung, J.Y., Chen, H., Midzak, A., Burnett, A.L., Papadopoulos, V., Zirkin, B.R., 2013. Drug ligand-induced activation of translocator protein (TSPO) stimulates steroid production by aged brown Norway rat Leydig cells. Endocrinology 154, 2156–2165. https://doi.org/10.1210/EN.2012-2226

Desai, R., Campanella, M., 2019. Exploring mitochondrial cholesterol signalling for therapeutic intervention in neurological conditions. Br J Pharmacol 176, 4284. https://doi.org/10.1111/BPH.14697

Fan, J., Campioli, E., Sottas, C., Zirkin, B., Papadopoulos, V., 2020. Amhr2-Cre-Mediated Global Tspo Knockout. J Endocr Soc 4. https://doi.org/10.1210/JENDSO/BVAA001

Farhan, F., Almarhoun, M., Wong, A., Findlay, A.S., Bartholomew, C., Williams, M.T.S., Hurd, T.W., Shu, X., 2021. Deletion of tspo causes dysregulation of cholesterol metabolism in mouse retina. Cells 10. https://doi.org/10.3390/CELLS10113066/S1

Hauffa, B., Hiort, O., 2011. P450 side-chain cleavage deficiency--a rare cause of congenital adrenal hyperplasia. Endocr Dev 20, 54–62. https://doi.org/10.1159/000321215

Hu, M.C., Hsu, N.C., Hadj, N.B. El, Pai, C.I., Chu, H.P., Wang, C.K.L., Chung, B.C., 2002. Steroid deficiency syndromes in mice with targeted disruption of Cyp11a1. Mol Endocrinol 16, 1943–1950. https://doi.org/10.1210/ME.2002-0055

Huang, C.C.J., Shih, M.C.M., Hsu, N.C., Chien, Y., Chung, B.C., 2012. Fetal Glucocorticoid Synthesis Is Required for Development of Fetal Adrenal Medulla and Hypothalamus Feedback Suppression. Endocrinology 153, 4749–4756. https://doi.org/10.1210/EN.2012-1258

Miller, W.L., 2017. Steroidogenesis: Unanswered Questions. Trends in Endocrinology and Metabolism. https://doi.org/10.1016/j.tem.2017.09.002

Miller, W.L., Auchus, R.J., 2011. The Molecular Biology, Biochemistry, and Physiology of Human Steroidogenesis and Its Disorders. Endocr Rev 32, 81. https://doi.org/10.1210/ER.2010-0013

Mostovaya, M., Kalinchenko, S., Vorslov, L., 2022. PSUN66 Statin Intake as a Cause of Decreased Steroid Hormone Secretion. J Endocr Soc 6, A729. https://doi.org/10.1210/JENDSO/BVAC150.1504

Owen, D.R., Fan, J., Campioli, E., Venugopal, S., Midzak, A., Daly, E., Harlay, A., Issop, L., Libri, V., Kalogiannopoulou, D., Oliver, E., Gallego-Colon, E., Colasanti, A., Huson, L., Rabiner, E.A., Suppiah, P., Essagian, C., Matthews, P.M., Papadopoulos, V., 2017. TSPO mutations in rats and a human polymorphism impair the rate of steroid synthesis. Biochem J 474, 3985–3999. https://doi.org/10.1042/BCJ20170648

Slominski, A.T., Li, W., Kim, T.K., Semak, I., Wang, J., Zjawiony, J.K., Tuckey, R.C., 2015. Novel activities of CYP11A1 and their potential physiological significance. J Steroid Biochem Mol Biol 151, 25. https://doi.org/10.1016/J.JSBMB.2014.11.010

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 25, 485–490. https://doi.org/10.1016/j.reprotox.2008.05.057

Tajima, T., Fujieda, K., Kouda, N., Nakae, J., Miller, W.L., 2001. Heterozygous mutation in the cholesterol side chain cleavage enzyme (p450scc) gene in a patient with 46,XY sex reversal and adrenal insufficiency. J Clin Endocrinol Metab 86, 3820–3825. https://doi.org/10.1210/JCEM.86.8.7748

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, 1227–1237. https://doi.org/10.1210/en.2003-1475

Xing, Y., Parker, C.R., Edwards, M., Rainey, W.E., 2010. ACTH is a potent regulator of gene expression in human adrenal cells. J Mol Endocrinol 45, 59–68. https://doi.org/10.1677/JME-10-0006