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Relationship: 2072

Title

The title of the KER should clearly define the two KEs being considered and the sequential relationship between them (i.e., which is upstream and which is downstream). Consequently all KER titles take the form “upstream KE leads to downstream KE”.  More help

Decreased, cholesterol leads to Decreased, 11KT

Upstream event
Upstream event in the Key Event Relationship. On the KER page, clicking on the Event name under Upstream Relationship will bring the user to that individual KE page. More help
Downstream event
Downstream event in the Key Event Relationship. On the KER page, clicking on the Event name under Upstream Relationship will bring the user to that individual KE page. More help

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

This table is automatically generated upon addition of a KER to an AOP. All of the AOPs that are linked to this KER will automatically be listed in this subsection. Clicking on the name of the AOP in the table will bring you to the individual page for that AOP. More help
AOP Name Adjacency Weight of Evidence Quantitative Understanding Point of Contact Author Status OECD Status
PPARalpha Agonism Impairs Fish Reproduction adjacent High Not Specified Ashley Kittelson (send email) Open for citation & comment

Taxonomic Applicability

Select one or more structured terms 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. Authors can indicate the relevant taxa for this KER in this subsection. The process is similar to what is described for KEs (see pages 30-31 and 37-38 of User Handbook) More help
Term Scientific Term Evidence Link
teleost fish teleost fish High NCBI
Vertebrates Vertebrates Low NCBI

Sex Applicability

Authors can indicate the relevant sex for this KER in this subsection. The process is similar to what is described for KEs (see pages 31-32 of the User Handbook). More help
Sex Evidence
Male High
Female Low

Life Stage Applicability

Authors can indicate the relevant life stage for this KER in this subsection. The process is similar to what is described for KEs (see pages 31-32 of User Handbook). More help

Key Event Relationship Description

Provide a brief, descriptive summation of the KER. While the title itself is fairly descriptive, this section can provide details that aren’t inherent in the description of the KEs themselves (see page 39 of the User Handbook). This description section can be viewed as providing the increased specificity in the nature of upstream perturbation (KEupstream) that leads to a particular downstream perturbation (KEdownstream), while allowing the KE descriptions to remain generalised so they can be linked to different AOPs. The description is also intended to provide a concise overview for readers who may want a brief summation, without needing to read through the detailed support for the relationship (covered below). Careful attention should be taken to avoid reference to other KEs that are not part of this KER, other KERs or other AOPs. This will ensure that the KER is modular and can be used by other AOPs. More help

The cholesterol molecule is the precursor for all steroid hormone synthesis. Cholesterol is obtained from de novo synthesis within cells or uptake of extracellular cholesterol (Eacker et al., 2008), however the dependence on either source varies by species (Klinefelter et al., 2014). Cholesterol is then transported into the inner mitochondrial membrane via the steroidogenic acute regulatory protein (StAR). Cholesterol is then converted to pregnenolone via the enzyme cytochrome P450 side-chain cleavage (cyp11a1). This is the rate-limiting step of steroidogenesis (Arukwe, 2008). Pregnenolone is then used to produce all other steroid hormones. 11-KT is synthesized from testosterone primarily using the enzymes CYP11β1 and HSD11β2 (Yazawa et al., 2008).

Evidence Supporting this KER

Assembly and description of the scientific evidence supporting KERs in an AOP is an important step in the AOP development process that sets the stage for overall assessment of the AOP (see pages 49-56 of the User Handbook). To do this, biological plausibility, empirical support, and the current quantitative understanding of the KER are evaluated with regard to the predictive relationships/associations between defined pairs of KEs as a basis for considering WoE (page 55 of User Handbook). In addition, uncertainties and inconsistencies are considered. More help

Time

Dose

Decreased Cholesterol?

Decreased

11-KT?

Citation

Species

48 hours

1.7, 33, & 70 mg/g Bezafibrate

No

No

Velasco-Santamaría et al. 2011

Danio Rerio

7 days

33 & 70 mg/g Bezafibrate

Yes

No

21 days

1.7 & 33 mg/g Bezafibrate

Yes

No

21 days

70 mg/g Bezafibrate

Yes

Yes

67 days

10 ug/L Gemfibrozil

Decreased ex vivo 11-KT production unless supplemented with 25OH-cholesterol

Fraz et al. 2018

Danio Rerio

21 days

0.04 mg/L Gemfibrozil

Yes

No

Lee et al. 2019

Oryzias latipes

21 days

0.4 & 3.7 mg/L Gemfibrozil

Yes

Yes

Biological Plausibility
Define, in free text, the biological rationale for a connection between KEupstream and KEdownstream. What are the structural or functional relationships between the KEs? For example, there is a functional relationship between an enzyme’s activity and the product of a reaction it catalyses. Supporting references should be included. However, it is recognised that there may be cases where the biological relationship between two KEs is very well established, to the extent that it is widely accepted and consistently supported by so much literature that it is unnecessary and impractical to cite the relevant primary literature. Citation of review articles or other secondary sources, like text books, may be reasonable in such cases. The primary intent is to provide scientifically credible support for the structural and/or functional relationship between the pair of KEs if one is known. The description of biological plausibility 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 (see page 40 of the User Handbook for further information).   More help

The process of steroid hormone biosynthesis is well understood, and cholesterol is the precursor for all steroid hormones.

Uncertainties and Inconsistencies
In addition to outlining the evidence supporting a particular linkage, it is also important to identify inconsistencies or uncertainties in the relationship. Additionally, while there are expected patterns of concordance that support a causal linkage between the KEs in the pair, it is also helpful to identify experimental details that may explain apparent deviations from the expected patterns of concordance. Identification of uncertainties and inconsistencies contribute to evaluation of the overall WoE supporting the AOPs that contain a given KER and to the identification of research gaps that warrant investigation (seep pages 41-42 of the User Handbook).Given that AOPs are intended to support regulatory applications, AOP developers should focus on those inconsistencies or gaps that would have a direct bearing or impact on the confidence in the KER and its use as a basis for inference or extrapolation in a regulatory setting. Uncertainties that may be of academic interest but would have little impact on regulatory application don’t need to be described. In general, this section details evidence that may raise questions regarding the overall validity and predictive utility of the KER (including consideration of both biological plausibility and empirical support). It also contributes along with several other elements to the overall evaluation of the WoE for the KER (see Section 4 of the User Handbook).  More help

Although Al-Habsi et al. (2016) show female zebrafish exposed to gemfibrozil and/or atorvastatin have decreased cholesterol and testosterone, decreased testosterone was not seen in males. Although several papers show 11KT is generally correlated with testosterone concentrations (Spanò et al., 2004; Maclatchy & Vanderkraak 1995; Lorenzi et al., 2008), it’s uncertain if 11KT was actually affected.

11KT levels can have high variability between fish. Although Lee et al. (2019) shows a decrease in testosterone and 11KT in a 21-day study, steroid measurements from the 155-day study showed no significant effects. This is possibly due to limited samples size (n=3-5).

Response-response Relationship
This subsection should be used to define sources of data that define the response-response relationships between the KEs. In particular, information regarding the general form of the relationship (e.g., linear, exponential, sigmoidal, threshold, etc.) should be captured if possible. If there are specific mathematical functions or computational models relevant to the KER in question that have been defined, those should also be cited and/or described where possible, along with information concerning the approximate range of certainty with which the state of the KEdownstream can be predicted based on the measured state of the KEupstream (i.e., can it be predicted within a factor of two, or within three orders of magnitude?). For example, a regression equation may reasonably describe the response-response relationship between the two KERs, but that relationship may have only been validated/tested in a single species under steady state exposure conditions. Those types of details would be useful to capture.  More help

Velasco-Santamaría et al. (2011) sampled male zebrafish fed several doses of bezafibrate (1.7, 33, & 70 mg BZF/g food) at several timepoints (48 hours, 7 days, and 21 days). Decreased plasma cholesterol is observed after 7 days to 33 mg/g. However, 11-KT isn’t significantly decreased until 21 days to 70 mg/g. There is a positive linear correlation between cholesterol and 11KT (r=0.291, p=0.0004). These decreases are observed without significant changes to cyp11a1 or StAR.

Male medaka exposed to gemfibrozil for 21 days show decreased cholesterol with doses of 0.03, 0.3, and 3.0 mg/L. However, decreases in 11KT is only significant at doses of 0.3 and 3.0 mg/L (Lee et al. 2019).

Time-scale
This sub-section should be used to provide 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?). This can be useful information both in terms of modelling the KER, as well as for analyzing the critical or dominant paths through an AOP network (e.g., identification of an AO that could kill an organism in a matter of hours will generally be of higher priority than other potential AOs that take weeks or months to develop). Identification of time-scale can also aid the assessment of temporal concordance. For example, for a KER that operates on a time-scale of days, measurement of both KEs after just hours of exposure in a short-term experiment could lead to incorrect conclusions regarding dose-response or temporal concordance if the time-scale of the upstream to downstream transition was not considered. More help

Decreases in cholesterol in Zebrafish due to bezafibrate exposure can be seen after 7 days, however, decreases in plasma 11-KT aren’t significant until 14 days later (Velasco-Santamaría et al. 2011).

A six-week exposure to gemfibrozil, a cholesterol-lowering pharmaceutical, is sufficient to lower 11-KT levels in the plasma, testes, and whole-body samples of male Zebrafish (Fraz et al. 2018). A 21-day exposure to gemfibrozil is sufficient to lower plasma cholesterol and 11-KT levels in male Japanese Medaka (Lee et al. 2019).

Known modulating factors
This sub-section presents information regarding modulating factors/variables known to alter the shape of the response-response function that describes the quantitative relationship between the two KEs (for example, an iodine deficient diet causes a significant increase in the slope of the relationship; a particular genotype doubles the sensitivity of KEdownstream to changes in KEupstream). Information on these known modulating factors should be listed in this subsection, along with relevant information regarding the manner in which the modulating factor can be expected to alter the relationship (if known). Note, this section should focus on those modulating factors for which solid evidence supported by relevant data and literature is available. It should NOT list all possible/plausible modulating factors. In this regard, it is useful to bear in mind that many risk assessments conducted through conventional apical guideline testing-based approaches generally consider few if any modulating factors. More help
Known Feedforward/Feedback loops influencing this KER
This subsection should define whether there are known positive or negative feedback mechanisms involved and what is understood about their time-course and homeostatic limits? In some cases where feedback processes are measurable and causally linked to the outcome, they should be represented as KEs. However, in most cases these features are expected to predominantly influence the shape of the response-response, time-course, behaviours between selected KEs. For example, if a feedback loop acts as compensatory mechanism that aims to restore homeostasis following initial perturbation of a KE, the feedback loop will directly shape the response-response relationship between the KERs. Given interest in formally identifying these positive or negative feedback, it is recommended that a graphical annotation (page 44) indicating a positive or negative feedback loop is involved in a particular upstream to downstream KE transition (KER) be added to the graphical representation, and that details be provided in this subsection of the KER description (see pages 44-45 of the User Handbook).  More help

Decreases in plasma cholesterol are correlated with a slight increase in StAR in zebrafish (Velasco-Santamaría et al. 2011). This is a possible compensatory mechanism to increase the amount of cholesterol in the mitochondria.

Domain of Applicability

As for the KEs, there is also a free-text section of the KER description that the developer can use to explain his/her rationale for the structured terms selected with regard to taxonomic, life stage, or sex applicability, or provide a more generalizable or nuanced description of the applicability domain than may be feasible using standardized terms. More help

Taxanomic Applicability: The understanding of steroid hormone biosynthesis is developed from human and rodent studies but is generally conserved among vertebrates. Cyp11a1, which performs the first step of converting cholesterol to steroid hormones, is only found in vertebrates (Slominski et al., 2015). However, the relationship may not be relevant or studied in organisms in which 11KT isn't a primary androgen. 11KT is particularly relevant teleost fish as it is the dominant androgen and involved in testicular development and courtship behavior (Brantley et al., 1993; Barannikova et al., 2004; Gemmell et al., 2019). Evidence supporting this KER comes from a few fish species, including zebrafish and medaka, but is biologically plausible for all teleost fish.

Sex Applicability: Male and female fish use the same biological processes to produce steroids and express the necessary enzymes. In most fish species 11KT is significantly lower in females versus males, however a a few species of the order Perciformes show no sexual dimorphism (Lokman et al. 2002). In species with sexual dimorphism, males could show more significant effects resulting from lowered 11-KT than females. Decreased production of 11-KT in females may not be detectable due to low baseline production, however there are few studies available showing the relationship between cholesterol and 11KT in female fish. 

Life-Stage Applicability:

References

List of the literature that was cited for this KER description using the appropriate format. Ideally, the list of references should conform, to the extent possible, with the OECD Style Guide (OECD, 2015). More help

Al-Habsi, A.A., A. Massarsky, T.W. Moon (2016) “Exposure to gemfibrozil and atorvastatin affects cholesterol metabolism and steroid production in zebrafish (Danio rerio)”, Comparative Biochemistry and Physiology, Part B, Vol. 199, Elsevier, pp. 87-96. http://dx.doi.org/10.1016/j.cbpb.2015.11.009

Arukwe, A. (2008) “Steroidogenic acute regulatory (StAR) protein and cholesterol side-chain cleavage (P450scc)-regulated steroidogenesis as an organ-specific molecular and cellular target for endocrine disrupting chemical in fish”, Cell Biology and Toxicology, Vol. 24, Springer, pp. 527-540. https://doi.org/10.1007/s10565-008-9069-7

Barannikova, I.A., L.V. Bayunova, T.B. Semenkova (2004) “Serum levels of testosterone, 11-ketotestosterone and oestradiol-17β in three species of sturgeon during gonadal development and final maturation induced by hormonal treatment”, Journal of Fish Biology, Vol. 64(5), Wiley-Blackwell, pp. 1330-1338. https://doi.org/10.1111/j.0022-1112.2004.00395.x

Brantley, R.K., J.C. Wingfield, A.H. Bass (1993) “Sex steroid levels in Porichthys notatus, a fish with alternative reproductive tactics, and a review of the hormonal bases for male dimorphism among teleost fishes”, Hormones and Behavior, Vol. 27(3), Elsevier, pp. 332-347. https://doi.org/10.1006/hbeh.1993.1025

Eacker, S. M. et al. (2008) “Hormonal regulation of testicular steroid and cholesterol homeostasis”, Molecular Endocrinology, Vol. 22(3), pp. 623-635. https://doi.org/10.1210/me.2006-0534

Fraz, S., A.H. Lee, J.Y. Wilson (2018) “Gemfibrozil and carbamazepine decrease steroid production in zebrafish testes (Danio rerio)”, Aquatic Toxicology, Vol. 198, Elsevier, pp. 1-9. https://doi.org/10.1016/j.aquatox.2018.02.006

Gemmell, N.J. et al. (2019) “Natural sex change in fish”, in Sex Determination in Vertebrates, Vol. 134, Academic Press, pp. 71-117. doi: 10.1016/bs.ctdb.2018.12.014.

Klinefelter, G.R., J.W. Laskey, R.P. Amann (2014) “Statin drugs markedly inhibit testosterone production by rat Leydig cells in vitro: Implications for men”, Reproductive Toxicology, Vol. 45, Elsevier, pp. 52-58. https://doi.org/10.1016/j.reprotox.2013.12.010

Lee, G. et al. (2019) “Effects of gemfibrozil on sex hormones and reproduction related performances of Oryzias latipes following long-term (155 d) and short-term (21 d) exposure”, Ecotoxicology and Environmental Safety, Vol. 173, Elsevier, pp. 174-181. https://doi.org/10.1016/j.ecoenv.2019.02.015

Lokman, P.M. et al. (2002) “11-Oxygenated androgens in female teleosts: prevalence, abundance, and life history implications”, General and Comparative Endocrinology, Vol. 129, Academic Press, pp. 1-12. doi: 10.1016/s0016-6480(02)00562-2

Lorenzi, V. et al. (2008) “Diurnal patterns and sex differences in cortisol, 11-ketotestosterone, testosterone, and 17β-estradiol in the bluebanded goby (Lythrypnus dalli)”, General and Comparative Endocrinology, Vol. 155(2)., Elsevier, pp. 438-446. https://doi.org/10.1016/j.ygcen.2007.07.010

MacLatchy, D.L., G.J. Vanderkraak (1995) “The phytoestrogen β-sitosterol alters the reproductive endocrine status of goldfish”, Toxicology and Applied Pharmacology, Vol. 134(2), Elsevier, pp. 305-312. https://doi.org/10.1006/taap.1995.1196

Slominski, A.T. et al. (2015) “Novel activities of CYP11A1 and their potential physiological significance”, The Journal of Steroid Biochemistry and Molecular Biology, Vol. 151, Elsevier, pp. 25-37. https://doi.org/10.1016/j.jsbmb.2014.11.010

Spanó, L. et al. (2004) “Effects of atrazine on sex steroid dynamics, plasma vitellogenin concentration and gonad development in adult goldfish (Carassius auratus)”, Aquatic Toxicology, Vol. 66(4), Elsevier, pp. 369-379. https://doi.org/10.1016/j.aquatox.2003.10.009

Velasco-Santamaría, Y.M. et al. (2011) “Bezafibrate, a lipid-lowering pharmaceutical, as a potential endocrine disruptor in male zebrafish (Danio rerio)”, Aquatic Toxicology, Vol. 105, Elsevier, pp. 107-118. doi:10.1016/j.aquatox.2011.05.018

Yazawa, T. (2008) “Cyp11b1 is induced in the murine gonad by luteinizing hormone/human chorionic gonadotropin and involved in the production of 11-ketotestosterone, a major fish androgen: Conservation and evolution of the androgen metabolic pathway”, Endocrinology, Vol. 149(4), Oxford Academy, pp. 1786-1792. https://doi.org/10.1210/en.2007-1015