Aop: 7

Title

Each AOP should be given a descriptive title that takes the form “MIE leading to AO”. For example, “Aromatase inhibition [MIE] leading to reproductive dysfunction [AO]” or “Thyroperoxidase inhibition [MIE] leading to decreased cognitive function [AO]”. In cases where the MIE is unknown or undefined, the earliest known KE in the chain (i.e., furthest upstream) should be used in lieu of the MIE and it should be made clear that the stated event is a KE and not the MIE. More help

Aromatase (Cyp19a1) reduction leading to impaired fertility in adult female

Short name
A short name should also be provided that succinctly summarises the information from the title. This name should not exceed 90 characters. More help
Aromatase (Cyp19a1) reduction leading to reproductive toxicity

Graphical Representation

A graphical summary of the AOP listing all the KEs in sequence, including the MIE (if known) and AO, and the pair-wise relationships (links or KERs) between those KEs should be provided. This is easily achieved using the standard box and arrow AOP diagram (see this page for example). The graphical summary is prepared and uploaded by the user (templates are available) and is often included as part of the proposal when AOP development projects are submitted to the OECD AOP Development Workplan. The graphical representation or AOP diagram provides a useful and concise overview of the KEs that are included in the AOP, and the sequence in which they are linked together. This can aid both the process of development, as well as review and use of the AOP (for more information please see page 19 of the Users' Handbook).If you already have a graphical representation of your AOP in electronic format, simple save it in a standard image format (e.g. jpeg, png) then click ‘Choose File’ under the “Graphical Representation” heading, which is part of the Summary of the AOP section, to select the file that you have just edited. Files must be in jpeg, jpg, gif, png, or bmp format. Click ‘Upload’ to upload the file. You should see the AOP page with the image displayed under the “Graphical Representation” heading. To remove a graphical representation file, click 'Remove' and then click 'OK.'  Your graphic should no longer be displayed on the AOP page. If you do not have a graphical representation of your AOP in electronic format, a template is available to assist you.  Under “Summary of the AOP”, under the “Graphical Representation” heading click on the link “Click to download template for graphical representation.” A Powerpoint template file should download via the default download mechanism for your browser. Click to open this file; it contains a Powerpoint template for an AOP diagram and instructions for editing and saving the diagram. Be sure to save the diagram as jpeg, jpg, gif, png, or bmp format. Once the diagram is edited to its final state, upload the image file as described above. More help

Authors

List the name and affiliation information of the individual(s)/organisation(s) that created/developed the AOP. In the context of the OECD AOP Development Workplan, this would typically be the individuals and organisation that submitted an AOP development proposal to the EAGMST. Significant contributors to the AOP should also be listed. A corresponding author with contact information may be provided here. This author does not need an account on the AOP-KB and can be distinct from the point of contact below. The list of authors will be included in any snapshot made from an AOP. More help

Malgorzata Nepelska, Elise Grignard, Sharon Munn,

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

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

Point of Contact

Indicate the point of contact for the AOP-KB entry itself. This person is responsible for managing the AOP entry in the AOP-KB and controls write access to the page by defining the contributors as described below. Clicking on the name will allow any wiki user to correspond with the point of contact via the email address associated with their user profile in the AOP-KB. This person can be the same as the corresponding author listed in the authors section but isn’t required to be. In cases where the individuals are different, the corresponding author would be the appropriate person to contact for scientific issues whereas the point of contact would be the appropriate person to contact about technical issues with the AOP-KB entry itself. Corresponding authors and the point of contact are encouraged to monitor comments on their AOPs and develop or coordinate responses as appropriate.  More help
Elise Grignard   (email point of contact)

Contributors

List user names of all  authors contributing to or revising pages in the AOP-KB that are linked to the AOP description. This information is mainly used to control write access to the AOP page and is controlled by the Point of Contact.  More help
  • Elise Grignard

Status

The status section is used to provide AOP-KB users with information concerning how actively the AOP page is being developed, what type of use or input the authors feel comfortable with given the current level of development, and whether it is part of the OECD AOP Development Workplan and has been reviewed and/or endorsed. “Author Status” is an author defined field that is designated by selecting one of several options from a drop-down menu (Table 3). The “Author Status” field should be changed by the point of contact, as appropriate, as AOP development proceeds. See page 22 of the User Handbook for definitions of selection options. More help
Author status OECD status OECD project SAAOP status
Open for citation & comment EAGMST Under Review 1.21 Included in OECD Work Plan
This AOP was last modified on June 04, 2021 10:21
The date the AOP was last modified is automatically tracked by the AOP-KB. The date modified field can be used to evaluate how actively the page is under development and how recently the version within the AOP-Wiki has been updated compared to any snapshots that were generated. More help

Revision dates for related pages

Page Revision Date/Time
Reduction, Plasma 17beta-estradiol concentrations September 26, 2017 11:30
Reduction, 17beta-estradiol synthesis by ovarian granulosa cells September 16, 2017 10:14
impaired, Fertility December 02, 2016 09:21
irregularities, ovarian cycle November 29, 2016 19:09
reduction in ovarian granulosa cells, Aromatase (Cyp19a1) May 28, 2021 07:49
Reduction, Plasma 17beta-estradiol concentrations leads to irregularities, ovarian cycle December 03, 2016 16:37
Reduction, 17beta-estradiol synthesis by ovarian granulosa cells leads to Reduction, Plasma 17beta-estradiol concentrations March 20, 2017 12:05
irregularities, ovarian cycle leads to impaired, Fertility December 03, 2016 16:37
reduction in ovarian granulosa cells, Aromatase (Cyp19a1) leads to Reduction, 17beta-estradiol synthesis by ovarian granulosa cells November 29, 2016 20:10

Abstract

In the abstract section, authors should provide a concise and informative summation of the AOP under development that can stand-alone from the AOP page. Abstracts should typically be 200-400 words in length (similar to an abstract for a journal article). Suggested content for the abstract includes the following: The background/purpose for initiation of the AOP’s development (if there was a specific intent) A brief description of the MIE, AO, and/or major KEs that define the pathway A short summation of the overall WoE supporting the AOP and identification of major knowledge gaps (if any) If a brief statement about how the AOP may be applied (optional). The aim is to capture the highlights of the AOP and its potential scientific and regulatory relevance More help

This AOP links activation of the Peroxisome Proliferator Activated Receptorγ (PPARγ) to reproductive toxicity in adult female. The development of this AOP relies on evidence collected from rodent models and incorporates human mechanistic and epidemiological data. The PPARγ is a ligand-activated transcription factor that belongs to the nuclear receptor family, which also includes the steroid and thyroid hormone receptors. Interest in PPARγ action as a mechanistic basis for effects on the reproductive system arises from the demonstrated relationships between activation of this receptor and impairment of the steroidogenesis leading to reproductive toxicity in rodents. 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 PPARγ, followed by the disruption of the hormonal balance which leads to irregularities of the ovarian cycle that may further be cause of impaired fertility. The PPARγ-initiated AOP to rodent female reproductive toxicity is a first step for structuring current knowledge about a mode of action which is neither ER-mediated nor via direct aromatase inhibition. In the current form the pathway lays a strong basis for linking an endocrine mode of action with an apical endpoint, prerequisite requirement for the identification of endocrine disrupting chemicals. This AOP is complemented with a structured data collection which will serve as the basis for further quantitative development of the pathway.

Background (optional)

This optional subsection should be used to provide background information for AOP reviewers and users that is considered helpful in understanding the biology underlying the AOP and the motivation for its development. The background should NOT provide an overview of the AOP, its KEs or KERs, which are captured in more detail below. Examples of potential uses of the optional background section are listed on pages 24-25 of the User Handbook. More help

Summary of the AOP

This section is for information that describes the overall AOP. The information described in section 1 is entered on the upper portion of an AOP page within the AOP-Wiki. This is where some background information may be provided, the structure of the AOP is described, and the KEs and KERs are listed. More help

Events:

Molecular Initiating Events (MIE)
An MIE is a specialised KE that represents the beginning (point of interaction between a stressor and the biological system) of an AOP. More help
Key Events (KE)
This table summarises all of the KEs of the AOP. This table is populated in the AOP-Wiki as KEs are added to the AOP. Each table entry acts as a link to the individual KE description page.  More help
Adverse Outcomes (AO)
An AO is a specialised KE that represents the end (an adverse outcome of regulatory significance) of an AOP.  More help
Sequence Type Event ID Title Short name
1 MIE 408 reduction in ovarian granulosa cells, Aromatase (Cyp19a1) reduction in ovarian granulosa cells, Aromatase (Cyp19a1)
2 KE 219 Reduction, Plasma 17beta-estradiol concentrations Reduction, Plasma 17beta-estradiol concentrations
3 KE 3 Reduction, 17beta-estradiol synthesis by ovarian granulosa cells Reduction, 17beta-estradiol synthesis by ovarian granulosa cells
4 AO 406 impaired, Fertility impaired, Fertility
5 AO 405 irregularities, ovarian cycle irregularities, ovarian cycle

Relationships Between Two Key Events (Including MIEs and AOs)

This table summarises all of the KERs of the AOP and is populated in the AOP-Wiki as KERs are added to the AOP. Each table entry acts as a link to the individual KER description page.To add a key event relationship click on either Add relationship: events adjacent in sequence or Add relationship: events non-adjacent in sequence.For example, if the intended sequence of KEs for the AOP is [KE1 > KE2 > KE3 > KE4]; relationships between KE1 and KE2; KE2 and KE3; and KE3 and KE4 would be defined using the add relationship: events adjacent in sequence button.  Relationships between KE1 and KE3; KE2 and KE4; or KE1 and KE4, for example, should be created using the add relationship: events non-adjacent button. This helps to both organize the table with regard to which KERs define the main sequence of KEs and those that provide additional supporting evidence and aids computational analysis of AOP networks, where non-adjacent KERs can result in artifacts (see Villeneuve et al. 2018; DOI: 10.1002/etc.4124).After clicking either option, the user will be brought to a new page entitled ‘Add Relationship to AOP.’ To create a new relationship, select an upstream event and a downstream event from the drop down menus. The KER will automatically be designated as either adjacent or non-adjacent depending on the button selected. The fields “Evidence” and “Quantitative understanding” can be selected from the drop-down options at the time of creation of the relationship, or can be added later. See the Users Handbook, page 52 (Assess Evidence Supporting All KERs for guiding questions, etc.).  Click ‘Create [adjacent/non-adjacent] relationship.’  The new relationship should be listed on the AOP page under the heading “Relationships Between Two Key Events (Including MIEs and AOs)”. To edit a key event relationship, click ‘Edit’ next to the name of the relationship you wish to edit. The user will be directed to an Editing Relationship page where they can edit the Evidence, and Quantitative Understanding fields using the drop down menus. Once finished editing, click ‘Update [adjacent/non-adjacent] relationship’ to update these fields and return to the AOP page.To remove a key event relationship to an AOP page, under Summary of the AOP, next to “Relationships Between Two Key Events (Including MIEs and AOs)” click ‘Remove’ The relationship should no longer be listed on the AOP page under the heading “Relationships Between Two Key Events (Including MIEs and AOs)”. More help

Network View

The AOP-Wiki automatically generates a network view of the AOP. This network graphic is based on the information provided in the MIE, KEs, AO, KERs and WoE summary tables. The width of the edges representing the KERs is determined by its WoE confidence level, with thicker lines representing higher degrees of confidence. This network view also shows which KEs are shared with other AOPs. More help

Stressors

The stressor field is a structured data field that can be used to annotate an AOP with standardised terms identifying stressors known to trigger the MIE/AOP. Most often these are chemical names selected from established chemical ontologies. However, depending on the information available, this could also refer to chemical categories (i.e., groups of chemicals with defined structural features known to trigger the MIE). It can also include non-chemical stressors such as genetic or environmental factors. Although AOPs themselves are not chemical or stressor-specific, linking to stressor terms known to be relevant to different AOPs can aid users in searching for AOPs that may be relevant to a given stressor. More help

Life Stage Applicability

Identify the life stage for which the KE is known to be applicable. More help
Life stage Evidence
Adult, reproductively mature High

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) can be selected. In many cases, individual species identified in these structured fields will be those for which the strongest evidence used in constructing the AOP was available in relation to this KE. More help
Term Scientific Term Evidence Link
rat Rattus norvegicus High NCBI
mouse Mus musculus Low NCBI
human Homo sapiens Low NCBI

Sex Applicability

The authors must select from one of the following: Male, female, mixed, asexual, third gender, hermaphrodite, or unspecific. More help
Sex Evidence
Female High

Overall Assessment of the AOP

This section addresses the relevant biological domain of applicability (i.e., in terms of taxa, sex, life stage, etc.) and WoE for the overall AOP as a basis to consider appropriate regulatory application (e.g., priority setting, testing strategies or risk assessment). The goal of the overall assessment is to provide a high level synthesis and overview of the relative confidence in the AOP and where the significant gaps or weaknesses are (if they exist). Users or readers can drill down into the finer details captured in the KE and KER descriptions, and/or associated summary tables, as appropriate to their needs.Assessment of the AOP is organised into a number of steps. Guidance on pages 59-62 of the User Handbook is available to facilitate assignment of categories of high, moderate, or low confidence for each consideration. While it is not necessary to repeat lengthy text that appears elsewhere in the AOP description (or related KE and KER descriptions), a brief explanation or rationale for the selection of high, moderate, or low confidence should be made. More help

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. However, the experimental support is derived from a limited number of studies. The PPARγ activators have been shown to alter steroidogenesis, ovarian cycle and impair reproduction [see reviews (Kay, Chambers, and Foster 2013), (Froment et al. 2006), (Peraza et al. 2006), (Latini et al. 2008), (Martino-Andrade and Chahoud 2010), (Lyche et al. 2009), (Lovekamp-Swan and Davis 2003)]. The biochemistry of steroidogenesis and the predominant role of the ovaries in synthesis of the sex steroids are well established. During the reproductive years the ovary is the central organ providing hormones necessary for the communication between the reproductive tract and the central nervous system, assuring normal reproductive function. Hormonal imbalance may lead to irregularities of the ovarian cycle that could be one of many possible events resulting in decrease fertility.

Concordance of dose-response relationships

This is a qualitative description of the pathway; the currently available studies provide little quantitative information on dose-response relationships between key events (KEs). The experimental data for selected compounds (phthalates, phenols and parabens) reveals concordance between one KE to the next in the sequence, i.e. that each KE occur at first and on lower dose than the following KE. To establish more reliable and quantitative linkages tailored experiments are required.

Temporal concordance among the key events and the adverse outcome

Most of the gathered evidence relies on the measurement of the effects at the same time point (detailed information captured in KER), thus studies providing evidence for complete temporal concordance are missing.

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

PPARγ-null mutation is embryonically lethal due to a defect in placental development ( PPARγ is necessary for angiogenesis)(Barak et al. 1999). Organ (ovary) targeted knock-out studies are needed to more precisely inform on the mechanistic involvement of the PPAR family in the proposed AOP.

The pathway's weak point lies in the linkages between the initial events in the pathway. However, there is evidence supporting both chemical dependent and independent involvement of PPARγ in the female reproductive function:

Chemical independent studies:

1. disruption of PPARγ in ovary using cre/loxP technology led to ovarian dysfunction and female subfertility (30% of animals infertile, reminders had delayed conception and reduced litter size) (Cui et al. 2002)

2. granulosa cell specific deletion of PPARγ in mice results in marked impairment of ovulation due to defective follicular rupture (Kim et al. 2008)

Chemical dependent studies:

3. Antagonist of PPARγ recovered the decrease of aromatase after treatment with MEHP (PPARγ agonist) (Lovekamp-Swan, Jetten, and Davis 2003)

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

Alternative mechanisms relating to the pathway are described in greater detail in the descriptions of KERs.

The contributing MIE in the pathway proposed is activation of PPARα supported by experimental evidence of dual activation of PPARα/γ by MEHP leading to decreased expression and activity of aromatase in granulosa cells (Lovekamp-Swan, Jetten, and Davis 2003) and inhibition of aromatase expression upon activation of PPARα by the ligand, fenofibrate, in the ovary of mouse (Toda et al. 2003).

The relation of PPARγ activation to other enzymes in steroidogenesis and reduced estradiol production PPARγ ligands were shown to modulate other enzymes involved in steroidogenesis

  • upstream of aromatase:

• Steroidogenic acute regulatory protein (StAR)

StAR was up regulated by PPARγ ligands (rosiglitazone and pioglitazone) in human granulosa cells in vitro (Seto-Young et al. 2007) and by MEHP in rat granulosa cells (Svechnikova, Svechnikova, and Söder 2011). StAR facilitates that rapid mobilization of cholesterol for initial catalysis to pregnenolone by the P450-side chain cleavage enzyme located within the mitochondria ( see review (Payne and Hales 2013)).

• 3β-hydroxysteroid dehydrogenase (3β-HSD)

Contradictory results were found on the effect of PPARγ ligands on 3β-HSD enzyme. Work on porcine granulosa cells has found that troglitazone competitively inhibits 3β-HSD enzyme activity (Gasic et al. 1998). Opposite results were obtained with another agonist of PPARγ (rosiglitazone) that stimulated 3βHSD protein expression and activity in porcine ovarian follicles (Rak-Mardyła and Karpeta 2014). 3β-HSD catalyses the conversion of pregnenolone to progesterone see review (Payne and Hales 2013)

• 17-alpha-hydroxylase (P450c17, CYP 17) Conflicting reports have arisen regarding the effect of PPARγ agonists on the expression and activity of this enzyme, mRNA production was unchanged following porcine thecal cell exposure to PPARγ ligand (Schoppee 2002), whilst other reports indicate CYP17 expression inhibition by PPARγ (rosiglitazone) agonist in ovarian follicles (Rak-Mardyła and Karpeta 2014). P450c17converts progesterone to androgen see review (Payne and Hales 2013)

  • downstream of aromatase:

Reduced production of estradiol may result from alteration of the enzymes upstream of aromatase (described above) or by increasing estradiol catabolism (altering Cyp1b1 and 17-βHSD IV, which are involved in estradiol conversion to catechol estrogens and estrone respectively).

• 17β-Hydroxysteroid dehydrogenase (17β-HSD)

Agonist of PPARγ (rosiglitazone) was found to inhibit 17β-HSD protein expression in ovarian follicles (Rak-Mardyła and Karpeta 2014), whereas increase in enzyme expression was noted upon treatment of granulosa cells by phthalate (MEHP) (Lovekamp-Swan, Jetten, and Davis 2003). 17β-Hydroxysteroid dehydrogenase (17β-HSD) metabolises estradiol to estrone see review (Payne and Hales 2013). For example, in vitro studies with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) investigating steroid production in human luteinizing granulosa cells (hLGC) showed estradiol decreased without changing either aromatase protein or its enzyme activity (Morán et al. 2000). Studies by the same laboratory identified P450c17 as a molecular target for endocrine disruption of hLGC specifically decreasing the supply of androgens for E2 synthesis (Morán et al. 2003). Reduced levels of estradiol production may result from increased inactivation of E2 via conversion to estrone as shown in isolated mouse small preantral follicles upon phthalate (MEHP) treatment (Lenie and Smitz 2009) and granulosa cells (Lovekamp-Swan, Jetten, and Davis 2003). Taken together, these findings provide strong evidence for the direct effect of PPARγ agonists on ovarian synthesis and secretion of hormones.

Reduced levels of estradiol and irregularities of ovarian cycle

The impact on ovarian cycle may result from a defect in hypothalamic-pituitary-gonadal (HPG) axis signalling, other than by alteration of estradiol level. MEHP inhibited follicle-simulating hormone (FSH) mediated stimulation of adenylate cyclase and progesterone synthesis in primary cultures of rat granulosa cells (Treinen, Dodson, and Heindel 1990).

Uncertainties, inconsistencies and data gaps

The current major uncertainty in this AOP is the basis of the functional relationship between the PPARγ, activation leading to Aromatase (Cyp19a1), reduction in ovarian granulosa cells. The possible mechanisms have been proposed and investigated, however there is lack of dose response and temporal data supporting the relationship (Lovekamp-Swan, Jetten, and Davis 2003), (Fan et al. 2005), (Mu et al. 2001). The pattern of the PPARγ expression in ovarian follicles is not steady, unlike expression of PPARα and δ. This fact adds to the complexity to the interpretation of mechanisms involved in the pathway. The PPARγ is down-regulated in response to the LH surge (C M Komar et al. 2001), but only in follicles that have responded to the LH surge (Carolyn M Komar and Curry 2003). Because PPARγ is primarily expressed in granulosa cells, it may influence development of these cells and their ability to support normal oocyte maturation. PPARγ could also potentially affect somatic cell/oocyte communication not only by impacting granulosa cell development, but by direct effects on the oocyte. Modulation of the PPARγ activity/expression in the ovary therefore, could potentially affect oocyte developmental competence. There is high strength, as well as specificity starting from the association between the reductions of E2 production leading to fertility impairment in females. Consistency of key events in the AOP is supported by several lines of evidence deriving from in vitro and in vivo studies that support PPARγ activation as an important actor in reproductive toxicity in rodents [(Kay, Chambers, and Foster 2013), (Froment et al. 2006), (Peraza et al. 2006), (Latini et al. 2008), (Martino-Andrade and Chahoud 2010), (Lyche et al. 2009), (Lovekamp-Swan and Davis 2003)].

Inconsistencies

Agonists of PPARγ were found to impact on steroidogenesis; however contradictory data show their effect on different stages of the process as well the direction of the effect(see above). Some in vivo studies also reported two-way effect on the estradiol production by PPARγ agonists. This effect may be attributed to the different measurements during different stages of estrous cycle. The phase of the estrous cycle, in which hormones are measured, may influence the readout of compound effect. In rats treated with DEHP increase in estradiol production was observed in ovarian cells (ex vivo) extracted during diestrus phase, however there was decrease in estradiol when the cells were extracted during estrus stage (Laskey and Berman 1993). In alignment with this result increased levels of estradiol were found in sheep proceeding the estrus phase (Herreros et al. 2013).

Data Gaps: There is a limited number of studies investigating the effect of PPARγ and its role in female reproductive function, in order to establish a more quantitative and temporal coherent linkage of the MIE to the subsequent key events studies are required. For example: the plausible mechanism of activation of a PPARγ, RXR and involvement of NFkappaB and their role in transcriptional repression of the aromatase gene could be investigated in modified transactivation assays to measure NFkappaB repression, rather than transactivation. Similar assays have been already generated, for estrogen receptor-mediated transrepression (Quaedackers et al. 2001).

Domain of Applicability

The relevant biological domain(s) of applicability in terms of sex, life-stage, taxa, and other aspects of biological context are defined in this section. Biological domain of applicability is informed by the “Description” and “Biological Domain of Applicability” sections of each KE and KER description (see sections 2G and 3E for details). In essence the taxa/life-stage/sex applicability is defined based on the groups of organisms for which the measurements represented by the KEs can feasibly be measured and the functional and regulatory relationships represented by the KERs are operative.The relevant biological domain of applicability of the AOP as a whole will nearly always be defined based on the most narrowly restricted of its KEs and KERs. For example, if most of the KEs apply to either sex, but one is relevant to females only, the biological domain of applicability of the AOP as a whole would be limited to females. While much of the detail defining the domain of applicability may be found in the individual KE and KER descriptions, the rationale for defining the relevant biological domain of applicability of the overall AOP should be briefly summarised on the AOP page. More help

This AOP is relevant for mature females for details see reviews [(Kay, Chambers, and Foster 2013), (Froment et al. 2006), (Peraza et al. 2006), (Latini et al. 2008), (Martino-Andrade and Chahoud 2010), (Lyche et al. 2009), (Lovekamp-Swan and Davis 2003)].

The experimental support for the pathway is based on rodent models and other mammals (pig, sheep) including human mechanistic and epidemiological data. The experimental animal data are assumed relevant for consideration of human risk.

This AOP applies to females only for details see reviews [(Kay, Chambers, and Foster 2013), (Froment et al. 2006), (Peraza et al. 2006), (Latini et al. 2008), (Martino-Andrade and Chahoud 2010), (Lyche et al. 2009), (Lovekamp-Swan and Davis 2003)].

Essentiality of the Key Events

An important aspect of assessing an AOP is evaluating the essentiality of its KEs. The essentiality of KEs can only be assessed relative to the impact of manipulation of a given KE (e.g., experimentally blocking or exacerbating the event) on the downstream sequence of KEs defined for the AOP. Consequently evidence supporting essentiality is assembled on the AOP page, rather than on the independent KE pages that are meant to stand-alone as modular units without reference to other KEs in the sequence.The nature of experimental evidence that is relevant to assessing essentiality relates to the impact on downstream KEs and the AO if upstream KEs are prevented or modified. This includes: Direct evidence: directly measured experimental support that blocking or preventing a KE prevents or impacts downstream KEs in the pathway in the expected fashion. Indirect evidence: evidence that modulation or attenuation in the magnitude of impact on a specific KE (increased effect or decreased effect) is associated with corresponding changes (increases or decreases) in the magnitude or frequency of one or more downstream KEs.When assembling the support for essentiality of the KEs, authors should organise relevant data in a tabular format. The objective is to summarise briefly the nature and numbers of investigations in which the essentiality of KEs has been experimentally explored either directly or indirectly. See pages 50-51 in the User Handbook for further definitions and clarifications.  More help

KRs WoE

Essentiality - KEs

level of confidence

     

PPAR gamma, Activation

PPARγ activation was found to indirectly alter the expression of aromatase

weak

Aromatase (Cyp19a1), reduction in ovarian granulosa cells

Aromatase is the cytochrome P450 enzyme complex responsible for the conversion of androgens to estrogens during steroidogenesis which is a key reaction in the sex differentiation in vertebrates. Alterations in the amount of aromatase present or in the catalytic activity of the enzyme will alter the levels of estrogens in tissues and dramatically disrupt estrogen hormone action.

moderate

17beta-estradiol synthesis by ovarian granulosa cells

While both brain and adrenal tissue are capable of synthesizing estradiol, the gonads are generally considered the major source of circulating estrogens in vertebrates, including fish (Norris 2007). Consequently, if estradiol synthesis by ovarian granulosa cells is reduced, plasma E2 concentrations would be expected to decrease unless there are concurrent reductions in the rate of E2 catabolism.

strong

Plasma 17beta-estradiol concentrations, Reduction

Estrogens are crucial for female fertility, as proved by the severe reproductive defects observed when their synthesis is blocked.

strong

ovarian cycle irregularities

A sequential progression of interrelated physiological and behavioural cycles underlines the female reproductive function.

strong

Fertility, impaired

Impaired Fertility is the endpoint of reproductive toxicity

strong

Evidence Assessment

The biological plausibility, empirical support, and quantitative understanding from each KER in an AOP are assessed together.  Biological plausibility of each of the KERs in the AOP is the most influential consideration in assessing WoE or degree of confidence in an overall hypothesised AOP for potential regulatory application (Meek et al., 2014; 2014a). Empirical support entails consideration of experimental data in terms of the associations between KEs – namely dose-response concordance and temporal relationships between and across multiple KEs. It is examined most often in studies of dose-response/incidence and temporal relationships for stressors that impact the pathway. While less influential than biological plausibility of the KERs and essentiality of the KEs, empirical support can increase confidence in the relationships included in an AOP. For clarification on how to rate the given empirical support for a KER, as well as examples, see pages 53- 55 of the User Handbook.  More help

KERs

Biological plausibility

level of confidence

Empirical Support

level of confidence

Inconsistencies/Uncertainties

     

Dose-response

Temporality

Incidence

   

PPARγ, Activation =>

Aromatase (Cyp19a1), reduction in ovarian granulosa cells

There is functional relationship between PPARγ activation and reduction in aromatase levels. Several mechanisms have been investigated; however there is no established consensus.

Moderate

  • KEup occurs at lower dose than KEdown(dose response concordance)
  • occurrence of the key events at similar dose and time point
  • Support for solid temporal relationship is lacking

Weak

Limited data, for details see KER pages

Aromatase (Cyp19a1), reduction in ovarian granulosa cells =>

17beta-estradiol synthesis by ovarian granulosa cells

Within the ovary, aromatase expression and activity is primarily localized in the granulosa cells. Therefore, changes in ovarian aromatase can generally be assumed to directly impact E2 synthesis by the granulosa cells.

Moderate

  • KEup occurs at lower dose than KEdown(dose response concordance)
  • occurrence of the key events at similar dose and time point
  • Support for solid temporal relationship is lacking

moderate

Limited data

17beta-estradiol synthesis by ovarian granulosa cells, Reduction =>

Plasma 17beta-estradiol concentrations

The gonads are generally considered the major source of circulating estrogens, consequently, if estradiol synthesis by ovarian granulosa cells is reduced, plasma E2 concentrations would be expected to decrease.

Strong

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

moderate

Limited data

Plasma 17beta-estradiol concentrations, Reduction =>

ovarian cycle irregularities

Alterations in relationships among the hypothalamic, pituitary, and ovarian components of the reproductive axis can have marked effects on cyclicity. A toxicological insult to any one of these sites can disrupt the cycle and block ovulation.

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

moderate

 

ovarian cycle irregularities =>

Fertility, impaired

A sequential progression of interrelated physiological and behavioural cycles underlines the female's fertility and successful production of offspring.

Strong

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

moderate

 

Table 1 Weight of Evidence Summary. The underlying questions for the content of table: Dose-response: Does the empirical evidence support that a change in KEup leads to an appropriate change in KEdown?; Temporality: 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?

Quantitative Understanding

Some proof of concept examples to address the WoE considerations for AOPs quantitatively have recently been developed, based on the rank ordering of the relevant Bradford Hill considerations (i.e., biological plausibility, essentiality and empirical support) (Becker et al., 2017; Becker et al, 2015; Collier et al., 2016). Suggested quantitation of the various elements is expert derived, without collective consideration currently of appropriate reporting templates or formal expert engagement. Though not essential, developers may wish to assign comparative quantitative values to the extent of the supporting data based on the three critical Bradford Hill considerations for AOPs, as a basis to contribute to collective experience.Specific attention is also given to how precisely and accurately one can potentially predict an impact on KEdownstream based on some measurement of KEupstream. This is captured in the form of quantitative understanding calls for each KER. See pages 55-56 of the User Handbook for a review of quantitative understanding for KER's. More help

Considerations for Potential Applications of the AOP (optional)

At their discretion, the developer may include in this section discussion of the potential applications of an AOP to support regulatory decision-making. This may include, for example, possible utility for test guideline development or refinement, development of integrated testing and assessment approaches, development of (Q)SARs / or chemical profilers to facilitate the grouping of chemicals for subsequent read-across, screening level hazard assessments or even risk assessment. While it is challenging to foresee all potential regulatory application of AOPs and any application will ultimately lie within the purview of regulatory agencies, potential applications may be apparent as the AOP is being developed, particularly if it was initiated with a particular application in mind. This optional section is intended to provide the developer with an opportunity to suggest potential regulatory applications and describe his or her rationale.To edit the “Considerations for Potential Applications of the AOP” section, on an AOP page, in the upper right hand menu, click ‘Edit.’ This brings you to a page entitled, “Editing AOP.” Scroll down to the “Considerations for Potential Applications of the AOP” section, where a text entry box allows you to submit text. In the upper right hand menu, click ‘Update AOP’ to save your changes and return to the AOP page or 'Update and continue' to continue editing AOP text sections.  The new text should appear under the “Considerations for Potential Applications of the AOP” section on the AOP page. More help

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 PPARγ 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 to sex steroids disruption and effects on reproductive tract and fertility. This AOP forms the starting point on an AOP network mapping 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

List the bibliographic references to original papers, books or other documents used to support the AOP. More help

Cui, Yongzhi, Keiko Miyoshi, Estefania Claudio, Ulrich K Siebenlist, Frank J Gonzalez, Jodi Flaws, Kay-Uwe Wagner, and Lothar Hennighausen. 2002. “Loss of the Peroxisome Proliferation-Activated Receptor Gamma (PPARgamma ) Does Not Affect Mammary Development and Propensity for Tumor Formation but Leads to Reduced Fertility.” The Journal of Biological Chemistry 277 (20) (May 17): 17830–5. doi:10.1074/jbc.M200186200.

Fan, WuQiang, Toshihiko Yanase, Hidetaka Morinaga, Yi-Ming Mu, Masatoshi Nomura, Taijiro Okabe, Kiminobu Goto, Nobuhiro Harada, and Hajime Nawata. 2005. “Activation of Peroxisome Proliferator-Activated Receptor-Gamma and Retinoid X Receptor Inhibits Aromatase Transcription via Nuclear Factor-kappaB.” Endocrinology 146 (1) (January): 85–92. doi:10.1210/en.2004-1046.

Froment, P, F Gizard, D Defever, B Staels, J Dupont, and P Monget. 2006. “Peroxisome Proliferator-Activated Receptors in Reproductive Tissues: From Gametogenesis to Parturition.” The Journal of Endocrinology 189 (2) (May): 199–209. doi:10.1677/joe.1.06667.

Gasic, S, Y Bodenburg, M Nagamani, A Green, and R J Urban. 1998. “Troglitazone Inhibits Progesterone Production in Porcine Granulosa Cells.” Endocrinology 139 (12) (December): 4962–6. doi:10.1210/endo.139.12.6385.

Herreros, Maria A, Teresa Encinas, Laura Torres-Rovira, Rosa A Garcia-Fernandez, Juana M Flores, Jose M Ros, and Antonio Gonzalez-Bulnes. 2013. “Exposure to the Endocrine Disruptor di(2-Ethylhexyl)phthalate Affects Female Reproductive Features by Altering Pulsatile LH Secretion.” Environmental Toxicology and Pharmacology 36 (3) (November): 1141–9. doi:10.1016/j.etap.2013.09.020.

Kay, Vanessa R, Christina Chambers, and Warren G Foster. 2013. “Reproductive and Developmental Effects of Phthalate Diesters in Females.” Critical Reviews in Toxicology 43 (3) (March): 200–19. doi:10.3109/10408444.2013.766149.

Kim, Jaeyeon, Marcey Sato, Quanxi Li, John P Lydon, Francesco J Demayo, Indrani C Bagchi, and Milan K Bagchi. 2008. “Peroxisome Proliferator-Activated Receptor Gamma Is a Target of Progesterone Regulation in the Preovulatory Follicles and Controls Ovulation in Mice.” Molecular and Cellular Biology 28 (5) (March): 1770–82. doi:10.1128/MCB.01556-07.

Komar, C M, O Braissant, W Wahli, and T E Curry. 2001. “Expression and Localization of PPARs in the Rat Ovary during Follicular Development and the Periovulatory Period.” Endocrinology 142 (11) (November): 4831–8. doi:10.1210/endo.142.11.8429.

Komar, Carolyn M, and Thomas E Curry. 2003. “Inverse Relationship between the Expression of Messenger Ribonucleic Acid for Peroxisome Proliferator-Activated Receptor Gamma and P450 Side Chain Cleavage in the Rat Ovary.” Biology of Reproduction 69 (2) (August): 549–55. doi:10.1095/biolreprod.102.012831.

Laskey, J.W., and E. Berman. 1993. “Steroidogenic Assessment Using Ovary Culture in Cycling Rats: Effects of Bis (2-Diethylhexyl) Phthalate on Ovarian Steroid Production.” Reproductive Toxicology 7 (1) (January): 25–33. doi:10.1016/0890-6238(93)90006-S.

Latini, Giuseppe, Egeria Scoditti, Alberto Verrotti, Claudio De Felice, and Marika Massaro. 2008. “Peroxisome Proliferator-Activated Receptors as Mediators of Phthalate-Induced Effects in the Male and Female Reproductive Tract: Epidemiological and Experimental Evidence.” PPAR Research 2008 (January): 359267. doi:10.1155/2008/359267.

Lenie, Sandy, and Johan Smitz. 2009. “Steroidogenesis-Disrupting Compounds Can Be Effectively Studied for Major Fertility-Related Endpoints Using in Vitro Cultured Mouse Follicles.” Toxicology Letters 185 (3) (March 28): 143–52. doi:10.1016/j.toxlet.2008.12.015.

Lovekamp-Swan, Tara, and Barbara J. Davis. 2003. “Mechanisms of Phthalate Ester Toxicity in the Female Reproductive System.” Environmental Health Perspectives 111 (2) (October 28): 139–145. doi:10.1289/ehp.5658.

Lovekamp-Swan, Tara, Anton M Jetten, and Barbara J Davis. 2003. “Dual Activation of PPARalpha and PPARgamma by Mono-(2-Ethylhexyl) Phthalate in Rat Ovarian Granulosa Cells.” Molecular and Cellular Endocrinology 201 (1-2) (March 28): 133–41.

Lyche, Jan L, Arno C Gutleb, Ake Bergman, Gunnar S Eriksen, AlberTinka J Murk, Erik Ropstad, Margaret Saunders, and Janneche U Skaare. 2009. “Reproductive and Developmental Toxicity of Phthalates.” Journal of Toxicology and Environmental Health. Part B, Critical Reviews 12 (4) (April): 225–49. doi:10.1080/10937400903094091.

Martino-Andrade, Anderson Joel, and Ibrahim Chahoud. 2010. “Reproductive Toxicity of Phthalate Esters.” Molecular Nutrition & Food Research 54 (1) (January): 148–57. doi:10.1002/mnfr.200800312.

Morán, F M, A J Conley, C J Corbin, E Enan, C VandeVoort, J W Overstreet, and B L Lasley. 2000. “2,3,7,8-Tetrachlorodibenzo-P-Dioxin Decreases Estradiol Production without Altering the Enzyme Activity of Cytochrome P450 Aromatase of Human Luteinized Granulosa Cells in Vitro.” Biology of Reproduction 62 (4) (April): 1102–8.

Morán, F M, C A VandeVoort, J W Overstreet, B L Lasley, and A J Conley. 2003. “Molecular Target of Endocrine Disruption in Human Luteinizing Granulosa Cells by 2,3,7,8-Tetrachlorodibenzo-P-Dioxin: Inhibition of Estradiol Secretion due to Decreased 17alpha-hydroxylase/17,20-Lyase Cytochrome P450 Expression.” Endocrinology 144 (2) (March): 467–73. doi:10.1210/en.2002-220813.

Mu, Y M, T Yanase, Y Nishi, R Takayanagi, K Goto, and H Nawata. 2001. “Combined Treatment with Specific Ligands for PPARgamma:RXR Nuclear Receptor System Markedly Inhibits the Expression of Cytochrome P450arom in Human Granulosa Cancer Cells.” Molecular and Cellular Endocrinology 181 (1-2) (July 5): 239–48.

Payne, Anita H., and Dale B. Hales. 2013. “Overview of Steroidogenic Enzymes in the Pathway from Cholesterol to Active Steroid Hormones.” Endocrine Reviews (July 1).

Peraza, Marjorie a, Andrew D Burdick, Holly E Marin, Frank J Gonzalez, and Jeffrey M Peters. 2006. “The Toxicology of Ligands for Peroxisome Proliferator-Activated Receptors (PPAR).” Toxicological Sciences : An Official Journal of the Society of Toxicology 90 (2) (April): 269–95. doi:10.1093/toxsci/kfj062.

Quaedackers, M E, C E Van Den Brink, S Wissink, R H Schreurs, J A Gustafsson, P T Van Der Saag, and B B Van Der Burg. 2001. “4-Hydroxytamoxifen Trans-Represses Nuclear Factor-Kappa B Activity in Human Osteoblastic U2-OS Cells through Estrogen Receptor (ER)alpha, and Not through ER Beta.” Endocrinology 142 (3) (March): 1156–66. doi:10.1210/endo.142.3.8003.

Rak-Mardyła, Agnieszka, and Anna Karpeta. 2014. “Rosiglitazone Stimulates Peroxisome Proliferator-Activated Receptor Gamma Expression and Directly Affects in Vitro Steroidogenesis in Porcine Ovarian Follicles.” Theriogenology 82 (1) (July 1): 1–9. doi:10.1016/j.theriogenology.2014.02.016.

Schoppee, P. D. 2002. “Putative Activation of the Peroxisome Proliferator-Activated Receptor Impairs Androgen and Enhances Progesterone Biosynthesis in Primary Cultures of Porcine Theca Cells.” Biology of Reproduction 66 (1) (January 1): 190–198. doi:10.1095/biolreprod66.1.190.

Seto-Young, Donna, Dimiter Avtanski, Marina Strizhevsky, Grishma Parikh, Parini Patel, Julia Kaplun, Kevin Holcomb, Zev Rosenwaks, and Leonid Poretsky. 2007. “Interactions among Peroxisome Proliferator Activated Receptor-Gamma, Insulin Signaling Pathways, and Steroidogenic Acute Regulatory Protein in Human Ovarian Cells.” The Journal of Clinical Endocrinology and Metabolism 92 (6) (June): 2232–9. doi:10.1210/jc.2006-1935.

Svechnikova, Konstantin, Irina Svechnikova, and Olle Söder. 2011. “Gender-Specific Adverse Effects of Mono-Ethylhexyl Phthalate on Steroidogenesis in Immature Granulosa Cells and Rat Leydig Cell Progenitors in Vitro.” Frontiers in Endocrinology 2 (January): 9. doi:10.3389/fendo.2011.00009.

Toda, Katsumi, Teruhiko Okada, Chisata Miyaura, and Toshiji Saibara. 2003. “Fenofibrate, a Ligand for PPARalpha, Inhibits Aromatase Cytochrome P450 Expression in the Ovary of Mouse.” Journal of Lipid Research 44 (2) (February): 265–70. doi:10.1194/jlr.M200327-JLR200.

Treinen, K A, W C Dodson, and J J Heindel. 1990. “Inhibition of FSH-Stimulated cAMP Accumulation and Progesterone Production by mono(2-Ethylhexyl) Phthalate in Rat Granulosa Cell Cultures.” Toxicology and Applied Pharmacology 106 (2) (November): 334–40.