Aop:7

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AOP Title

Aromatase (Cyp19a1) reduction leading to impaired fertility in adult female
Short name: Aromatase (Cyp19a1) reduction leading to reproductive toxicity

Authors

Malgorzata Nepelska, Elise Grignard, Sharon Munn,

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

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

Status

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OECD Project 1.21: Three Adverse Outcome Pathways from Peroxisome Proliferator-Activated Receptors (PPARs) Activation Leading to Reproductive Toxicity in Rodents

This AOP was last modified on 12/5/2016.

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Abstract

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.

Summary of the AOP

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Molecular Initiating Event

Molecular Initiating Event Support for Essentiality
Aromatase (Cyp19a1), reduction in ovarian granulosa cells Strong

Key Events

Event Support for Essentiality
Plasma 17beta-estradiol concentrations, Reduction Strong
17beta-estradiol synthesis by ovarian granulosa cells, Reduction Strong

Adverse Outcome

Adverse Outcome
Fertility, impaired
ovarian cycle, irregularities

Relationships Among Key Events and the Adverse Outcome

Event Description Triggers Weight of Evidence Quantitative Understanding
17beta-estradiol synthesis by ovarian granulosa cells, Reduction Directly Leads to Plasma 17beta-estradiol concentrations, Reduction Strong
Plasma 17beta-estradiol concentrations, Reduction Directly Leads to ovarian cycle, irregularities Strong
ovarian cycle, irregularities Indirectly Leads to Fertility, impaired Moderate
Aromatase (Cyp19a1), reduction in ovarian granulosa cells Directly Leads to 17beta-estradiol synthesis by ovarian granulosa cells, Reduction Moderate

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Life Stage Applicability

Life Stage Evidence Links
Adult, reproductively mature Strong

Taxonomic Applicability

Name Scientific Name Evidence Links
rat Rattus sp. Strong NCBI
mouse Mus sp. Weak NCBI
human Homo sapiens Weak NCBI

Sex Applicability

Sex Evidence Links
Female Strong

Graphical Representation

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Overall Assessment of the AOP

Biological plausibility, coherence, and consistency of the experimental evidence

In the presented AOP it is hypothesized that the key events occur in a biologically plausible order prior to the development of adverse outcomes. 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).

Weight of Evidence Summary

Summary Table
Provide an overall summary of the weight of evidence based on the evaluations of the individual linkages from the Key Event Relationship pages.

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?

Essentiality of the Key Events

Molecular Initiating Event Summary, Key Event Summary
Provide an overall assessment of the essentiality for the key events in the AOP. Support calls for individual key events can be included in the molecular initiating event, key event, and adverse outcome tables above.

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

Quantitative Considerations

Summary Table
Provide an overall discussion of the quantitative information available for this AOP. Support calls for the individual relationships can be included in the Key Event Relationship table above.

Applicability of the AOP

Life Stage Applicability

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)].

Taxonomic Applicability

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.

Sex Applicability
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)].

Considerations for Potential Applications of the AOP (optional)

1. The AOP describes a pathway which allows for the detection of sex steroid-related endocrine disrupting modes of action, with focus on the identification of substances which affect the reproductive system. In the current form the pathway lays a strong basis for linking endocrine mode of action with an apical endpoint, a prerequisite requirement for identification of endocrine disrupting chemicals (EDC). EDCs require specific evaluation under REACH (1907/2006, Registration, Evaluation, Authorisation and Restriction of Chemicals (EU, 2006)), the revised European plant protection product regulation 1107/2009 (EU, 2009) and use of biocidal products 528/2012 EC (EU, 2012).Amongst other agencies the US EPA is also giving particular attention to EDCs (EPA, 1998).

2. This AOP structurally represents current knowledge of the pathway from 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

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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.

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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.