Aop:7

AOP Title PPARγ activation leading to impaired fertility in adult female rodents Short name: PPAR activation leading to reproductive toxicity Authors

Malgorzata Nepelska, Sharon Munn, Brigitte Landesmann

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: [email protected] Status

Under development: Do not distribute or cite.

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 5/19/2015.

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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 and further to 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

Molecular Initiating Event

1 Aop:7

Molecular Initiating Event PPAR gamma, Activation PPARγ, Activation Short name: PPARγ, Activation

How this Key Event works

Level of Biological Organization Molecular

Biological state

The Peroxisome Proliferator Activated receptor γ (PPARγ) belongs to Peroxisome Proliferator Activated receptors (PPARs; NR1C) steroid/thyroid/retinoid receptor superfamily of transcription factors, which respond to specific ligands by altering gene expression in a cell-specific manner. The PPARγ gene contains three promoters that yield three isoforms, namely, PPAR-γ1, 2 and 3. PPAR-γ1 and γ3 RNA transcripts translate into the identical PPAR-γ1 protein.

Biological compartments

PPARγ is abundantly expressed in adipose tissue, promoting adipocyte differentiation, but is also present in various cells and tissues, for review see (Braissant et al. 1996). PPARγ expression is tissue dependent (L Fajas et al. 1997), (Lluis Fajas, Fruchart, and Auwerx 1998). PPARγ is most highly expressed in white adipose tissue and brown adipose tissue, where it is a master regulator of adipogenesis as well as a potent modulator of whole-body lipid metabolism and insulin sensitivity (Evans, Barish, and Wang 2004), (Tontonoz and Spiegelman 2008). Whereas PPARγ1 is expressed in many tissues, the expression of PPARγ2 is restricted to adipose tissue under physiological conditions but can be induced in other tissues by a high- fat diet (Saraf et al. 2012).

General role in biology

PPARγ is activated after the binding of natural ligands such as polyunsaturated fatty acids and prostaglandin metabolites. It can also be activated by synthetic ligands such as thiazolidinediones (TZDs) (rosiglitazone, pioglitazone or troglitazone) (Lehmann et al., 1995). PPARγ controls many vital processes such as glucose metabolism and inflammation as well as variety of developmental programs(Wahli & Desvergne, 1999), (Rotman et al., 2008), (Wahli & Michalik, 2012). This receptor itself is essential for developmental processes since targeted disruption of this gene results in embryo lethality, due in part to defective placental development, therefore modulation of PPARγ activity may impact endocrine regulated processes during development as well as later in life.

How it is Measured or Detected

Methods that have been previously reviewed and approved by a recognized authority should be included in the Overview section above. All other methods, including those well established in the published literature, should be described here. Consider the following criteria when describing each method: 1. Is the assay fit for purpose? 2. Is the assay directly or indirectly (i.e. a surrogate) related to a key event relevant to the final adverse effect in question? 3. Is the assay repeatable? 4. Is the assay reproducible?

Binding of ligands to PPARγ is measured using binding assays in vitro and in silico, whereas the information about functional activation is derived from the transactivation using e.g. reporter assay with a reporter gene that demonstrates functional activation of a nuclear receptor by a specific compound. Binding of agonists within the ligand-binding site of PPARs causes a conformational change promoting binding to transcriptional

2 Aop:7 coactivators. Conversely, binding of antagonists results in a conformation that favours the binding of corepressors (Yu & Reddy, 2007) (Viswakarma et al., 2010. Transactivation assays are performed using the transient or stably transfected cells with the PPARγ expression plasmid and a reporter plasmid, correspondingly. There are also other methods that have been used to measure PPARγ activity, such as the Electrophoretic Mobility Shift Assay (EMSA) or commercially available PPARγ transcription factor assay kits, see Table 1.

Key event PPARγ activation What is Ligand Binding Transcriptional measured? activity transactivation Method/test binding transcription reporter gene category molecular modelling assay factor assay assay PPARγ Scintillation luciferase Electrophoretic proximity (mouse/rat) Method/test molecular modelling; docking reporter gene Mobility Shift Assay Reporter name binding assay (EMSA) assay Assay Kit

Test In silico In vitro In vitro In vitro, ex environment vivo PPARγ once activated by a Quantifying ligand, the changes in receptor binds luciferase to a promoter direct expression in the element in Computational simulation of binding treated reporter a candidate ligand binding to indicating the gene for Test cells provides a a receptor, Predicts the the mode target gene principle sensitive and activates strength of association or of action surrogate its binding affinity. for PPARα/ measure of the transcription. γ changes in PPAR The bound functional (activated) to activity. DNA PPAR is measured. The changes in A binding interaction Assess the activity of between a small molecule ability of reporter gene ligand and an enzyme compounds levels functionally Protein: DNA protein may result in to bind to linked to a PPAR- Test binding, DNA activation or inhibition of the PPARγ. responsive outcome binding enzyme. If the protein is a Identifies element/promoter activity receptor, ligand binding may the gives information result in agonism or modulators about the activity antagonism of PPARγ. of the PPAR activation. Gene regulation and determining protein: DNA interactions are the detected by the Transcriptional EMSA. EMSA can be activity of used qualitatively to PPARγ can be identify sequence- Predicts the preferred assessed specific DNA-binding orientation of one molecule PPARγ COS-1cell using This assay proteins (such as to a second when bound to transactivation commercially determines transcription factors) each other to form a stable assay (transient (PPRE)3- available kits whether Proprietary rodent in crude lysates and, complex. Knowledge of the transfection with luciferase like e.g. compounds cell line expressing in conjunction with Test preferred orientation in turn human or mouse reporter PPARγ interact the mouse/rat mutagenesis, to background may be used to predict the PPARγ expression construct transcription directly PPARγ identify the strength of association or plasmid and C2C12 factor assay with important binding binding affinity between two pHD(x3)-Luc kit (Abcam, PPARγ. sequences within a molecules using, for reporter plasmid Cambridge, given genes example, scoring functions. USA or upstream regulatory Cayman region. EMSA can Chemical, also be utilized USA). quantitatively to measure thermodynamic and

3 Aop:7 kinetic parameters. Assay type Quantitative Qualitative Quantitative Quantitative Quantitative Quantitative Quantitative In vitro Screening, In vitro Screening Application In vitro Functional functional studies functional activity Functional studies domain Virtual screening screening studies activity (reported (antagonist/agonist) use: agonist) Source Research/commercial Research Research Research commercial commercial Research/commercial (Lapinskas et al., (Feige et al., 2007), (Kaya, 2005), (Maloney & (Feige et Cayman, (Gijsbers Ref Mohr, Waxman, & Vajda, Abcam (Wu, Gao, Waxman, 1999) al., 2007) et al. 2013) 2006) & Wang, 2005)

The transactivation( stable transfection)assay provides the most applicable OECD Level 2 assay aimed at identifying the initiating event leading to adverse outcome (LeBlanc, Norris, & Kloas, 2011). Currently no internationally validated assays are available.

Evidence Supporting Taxonomic Applicability

Name Scientific Name Evidence Links mice Mus musculus Strong NCBI human Homo sapiens Moderate NCBI rat Rattus rattus Moderate NCBI

PPARγ have been identified in frog (Xenopus laevis), mouse, human, rat, fish, hamster and chicken (Wahli & Desvergne, 1999).

Evidence for Chemical Initiation of this Molecular Initiating Event

1.1 Binding and activation of receptor

PPARγ ligands thiazolidinediones (Rosiglitazone, Pioglitazone, Troglitazone) (Lehmann et al. 1995), (Forman et al. 1995), (Willson et al. 2000).

Phthalates

MEHP (CAS 4376-20-9) directly binds to PPARγ (Lapinskas et al. 2005), (ToxCastTM Data)in vitro and in silico (Feige et al. 2007), (Rotman et al. 2008), (Kaya et al. 2006) and activates this receptor in transactivation assays (Maloney & Waxman 1999), (Hurst & Waxman 2003b), (Venkata et al. 2006), (ToxCastTM Data). In summary, there is experimental in vitro evidence for binding and transcriptional activation of PPARγ. DEHP (CAS 117-81-7) was not found to bind and activate PPARγ (Lapinskas et al. 2005), (Maloney & Waxman 1999). However recent studies show activation of PPARγ by DEHP(ToxCastTM Data), (Pereira-Fernandes et al. 2013). DEHP was also found to increase the levels of PPARγ in vitro (Lin et al. 2011). Notably, PPARγ is responsive to DEHP in vitro and is translocated to the nucleus (in primary Sertoli cells) (Dufour et al. 2003), (Bhattacharya et al. 2005).

Parabens

Butylparaben was not found to bind to the PPARγ (ToxCastTM Data), but activated the human PPARγ (ToxCastTM Data), (Pereira-Fernandes et al. 2013) and mPPARγ in reporter gene assay (Taxvig et al., 2012).

Phenols

Bisphenol A was not found to bind to the PPARγ (ToxCastTM Data), but activated the human PPARγ

4 Aop:7 (ToxCastTM Data), (Pereira-Fernandes et al. 2013) but not mouse PPARγ (Taxvig et al., 2012) in reporter gene assay. BPA was also reported to increase PPARγ (mRNA) in ovarian granulosa cell line and human luteinized granulosa cells (Kwintkiewicz, Nishi, Yanase, & Giudice, 2010).

Organotin

Tributyltin (TBT) activates all three heterodimers of PPAR with RXR, primarily through its interaction with RXR (le Maire et al. 2009).

1.2 Activation of target genes

MEHP activation of endogenous PPARγ target genes was evidenced by the stimulation of PPARγ-dependent adipogenesis in the 3T3-L1 cell differentiation model (Hurst & Waxman, 2003).

References

Barak, Y., Nelson, M. C., Ong, E. S., Jones, Y. Z., Ruiz-Lozano, P., Chien, K. R., … Evans, R. M. (1999). PPAR gamma is required for placental, cardiac, and adipose tissue development. Molecular Cell, 4(4), 585– 95.

Braissant, O., Foufelle, F., Scotto, C., Dauça, M., & Wahli, W. (1996). Differential expression of peroxisome proliferator-activated receptors (PPARs): tissue distribution of PPAR-alpha, -beta, and -gamma in the adult rat. Endocrinology, 137(1), 354–66.

Burns, K. A., & Vanden Heuvel, J. P. (2007). Modulation of PPAR activity via phosphorylation. Biochimica et Biophysica Acta, 1771(8), 952–60. doi:10.1016/j.bbalip.2007.04.018

Fajas, L., Auboeuf, D., Raspé, E., Schoonjans, K., Lefebvre, A. M., Saladin, R., … Auwerx, J. (1997). The organization, promoter analysis, and expression of the human PPARgamma gene. The Journal of Biological Chemistry, 272(30), 18779–89.

Fajas, L., Fruchart, J.-C., & Auwerx, J. (1998). PPARγ3 mRNA: a distinct PPARγ mRNA subtype transcribed from an independent promoter. FEBS Letters, 438(1-2), 55–60. doi:10.1016/S0014-5793(98)01273-3

Feige, J. N., Gelman, L., Michalik, L., Desvergne, B., & Wahli, W. (2006). From molecular action to physiological outputs: peroxisome proliferator-activated receptors are nuclear receptors at the crossroads of key cellular functions. Progress in Lipid Research, 45(2), 120–59. doi:10.1016/j.plipres.2005.12.002

Feige, J. N., Gelman, L., Rossi, D., Zoete, V., Métivier, R., Tudor, C., … Desvergne, B. (2007). The endocrine disruptor monoethyl-hexyl-phthalate is a selective peroxisome proliferator-activated receptor gamma modulator that promotes adipogenesis. The Journal of Biological Chemistry, 282(26), 19152–66. doi:10.1074/jbc.M702724200

Gijsbers, Linda, Henriëtte D L M van Eekelen, Laura H J de Haan, Jorik M Swier, Nienke L Heijink, Samantha K Kloet, Hai-Yen Man, et al. 2013. “Induction of Peroxisome Proliferator-Activated Receptor Γ (PPARγ)- Mediated Gene Expression by Tomato (Solanum Lycopersicum L.) Extracts.” Journal of Agricultural and Food Chemistry 61 (14) (April 10): 3419–27. doi:10.1021/jf304790a.

Issemann, I., & Green, S. (1990). Activation of a member of the steroid hormone receptor superfamily by peroxisome proliferators. Nature, 347(6294), 645–650.

Kaya, T., Mohr, S. C., Waxman, D. J., & Vajda, S. (2006). Computational screening of phthalate monoesters for binding to PPARgamma. Chemical Research in Toxicology, 19(8), 999–1009. doi:10.1021/tx050301s

Lapinskas, P. J., Brown, S., Leesnitzer, L. M., Blanchard, S., Swanson, C., Cattley, R. C., & Corton, J. C. (2005). Role of PPARα in mediating the effects of phthalates and metabolites in the liver. Toxicology, 207(1), 149–163.

5 Aop:7 Le Maire, A., Grimaldi, M., Roecklin, D., Dagnino, S., Vivat-Hannah, V., Balaguer, P., & Bourguet, W. (2009). Activation of RXR-PPAR heterodimers by organotin environmental endocrine disruptors. EMBO Reports, 10(4), 367–73. doi:10.1038/embor.2009.8

LeBlanc, G., Norris, D., & Kloas, W. (2011). Detailed Review Paper State of the Science on Novel In Vitro and In Vivo Screening and Testing Methods and Endpoints for Evaluating Endocrine Disruptors, (178).

Lehmann, J. M., Moore, L. B., Smith-Oliver, T. A., Wilkison, W. O., Willson, T. M., & Kliewer, S. A. (1995). An Antidiabetic Thiazolidinedione Is a High Affinity Ligand for Peroxisome Proliferator-activated Receptor (PPAR ). Journal of Biological Chemistry, 270(22), 12953–12956. doi:10.1074/jbc.270.22.12953

Maloney, E. K., & Waxman, D. J. (1999). trans-Activation of PPARα and PPARγ by Structurally Diverse Environmental Chemicals. Toxicology and Applied Pharmacology, 161(2), 209–218.

Michalik, L., Zoete, V., Krey, G., Grosdidier, A., Gelman, L., Chodanowski, P., … Michielin, O. (2007). Combined simulation and mutagenesis analyses reveal the involvement of key residues for peroxisome proliferator-activated receptor alpha helix 12 dynamic behavior. The Journal of Biological Chemistry, 282(13), 9666–77. doi:10.1074/jbc.M610523200

Morán-Salvador, E., López-Parra, M., García-Alonso, V., Titos, E., Martínez-Clemente, M., González-Périz, A., … Clària, J. (2011). Role for PPARγ in obesity-induced hepatic steatosis as determined by hepatocyte- and macrophage-specific conditional knockouts. FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology, 25(8), 2538–50. doi:10.1096/fj.10-173716

Pereira-Fernandes, A., Demaegdt, H., Vandermeiren, K., Hectors, T. L. M., Jorens, P. G., Blust, R., & Vanparys, C. (2013). Evaluation of a screening system for obesogenic compounds: screening of endocrine disrupting compounds and evaluation of the PPAR dependency of the effect. PloS One, 8(10), e77481. doi:10.1371/journal.pone.0077481

ToxCastTM Data, US Environmental Protection Agency. http://www.epa.gov/ncct/toxcast/data.html.

Vanden Heuvel, J. P. (1999). Peroxisome proliferator-activated receptors (PPARS) and carcinogenesis. Toxicological Sciences : An Official Journal of the Society of Toxicology, 47(1), 1–8.

Viswakarma, N., Jia, Y., Bai, L., Vluggens, A., Borensztajn, J., Xu, J., & Reddy, J. K. (2010). Coactivators in PPAR-Regulated Gene Expression. PPAR Research, 2010. doi:10.1155/2010/250126

Wahli, W., & Desvergne, B. (1999). Peroxisome proliferator-activated receptors: nuclear control of metabolism. Endocrine Reviews, 20(5), 649–88.

Wu, B., Gao, J., & Wang, M. (2005). Development of a complex scintillation proximity assay for high- throughput screening of PPARgamma modulators. Acta Pharmacologica Sinica, 26(3), 339–44. doi:10.1111/j.1745-7254.2005.00040.x

Yu, S., & Reddy, J. K. (2007). Transcription coactivators for peroxisome proliferator-activated receptors. Biochimica et Biophysica Acta, 1771(8), 936–51. doi:10.1016/j.bbalip.2007.01.008

Retrieved from https://aopkb.org/aopwiki/index.php/?oldid=26737

Key Events

Event 17beta-estradiol synthesis by ovarian granulosa cells, Reduction Aromatase (Cyp19a1), reduction in ovarian granulosa cells

6 Aop:7 Plasma 17beta-estradiol concentrations, Reduction 17beta-estradiol synthesis by ovarian granulosa cells, Reduction Short name: 17beta-estradiol synthesis by ovarian granulosa cells, Reduction

How this Key Event works

Level of Biological Organization

Within the ovary, aromatase expression and activity is primarily localized in the granulosa cells (reviewed in (Norris 2007; Yaron 1995; Havelock et al. 2004) and others). C-19 androgens diffuse from the theca cells into granulosa cells where aromatase can catalyze their conversion to C-18 estrogens.

How it is Measured or Detected

Due to the importance of both theca and granulosa cells in ovarian steroidogenesis, it is generally impractical to measure E2 production by isolated granulosa cells (Havelock et al. 2004). However, this key event can be evaluated by examining E2 production by intact ovarian tissue explants either exposed to chemicals in vitro (e.g., (Villeneuve et al. 2007; McMaster ME 1995) or in vivo (i.e., via ex vivo steroidogenesis assay; e.g., (Ankley et al. 2007)). Estradiol released by ovarian tissue explants into media can be quantified by RIA (e.g., Jensen et al. 2001), ELISA, or analytical methods such as LC-MS (e.g., Owen et al. 2014).

Evidence Supporting Taxonomic Applicability

Name Scientific Name Evidence Links

Key enzymes needed to synthesize 17β-estradiol first appear in the common ancestor of amphioxus and vertebrates (Baker 2011). Consequently, this key event is applicable to most vertebrates.

References

Norris DO. 2007. Vertebrate Endocrinology. Fourth ed. New York: Academic Press. Havelock JC, Rainey WE, Carr BR. 2004. Ovarian granulosa cell lines. Molecular and cellular endocrinology 228(1-2): 67-78. Yaron Z. 1995. Endocrine control of gametogenesis and spawning induction in the carp. Aquaculture 129: 49-73. Villeneuve DL, Ankley GT, Makynen EA, Blake LS, Greene KJ, Higley EB, et al. 2007. Comparison of fathead minnow ovary explant and H295R cell-based steroidogenesis assays for identifying endocrine- active chemicals. Ecotoxicol Environ Saf 68(1): 20-32. McMaster ME MK, Jardine JJ, Robinson RD, Van Der Kraak GJ. 1995. Protocol for measuring in vitro steroid production by fish gonadal tissue. Canadian Technical Report of Fisheries and Aquatic Sciences 1961 1961: 1-78. Ankley GT, Jensen KM, Kahl MD, Makynen EA, Blake LS, Greene KJ, et al. 2007. Ketoconazole in the fathead minnow (Pimephales promelas): reproductive toxicity and biological compensation. Environ Toxicol Chem 26(6): 1214-1223. Villeneuve DL, Mueller ND, Martinovic D, Makynen EA, Kahl MD, Jensen KM, et al. 2009. Direct effects, compensation, and recovery in female fathead minnows exposed to a model aromatase inhibitor. Environ Health Perspect 117(4): 624-631. Baker ME. 2011. Origin and diversification of steroids: co-evolution of enzymes and nuclear receptors. Molecular and cellular endocrinology 334(1-2): 14-20.

7 Aop:7 Jensen K, Korte J, Kahl M, Pasha M, Ankley G. 2001. Aspects of basic reproductive biology and endocrinology in the fathead minnow (Pimephales promelas). Comparative Biochemistry and Physiology Part C 128: 127-141. Owen LJ, Wu FC, Keevil BG. 2014. A rapid direct assay for the routine measurement of oestradiol and oestrone by liquid chromatography tandem mass spectrometry. Ann. Clin. Biochem. 51(pt 3):360-367.

Retrieved from https://aopkb.org/aopwiki/index.php/?oldid=21775 Aromatase (Cyp19a1), reduction in ovarian granulosa cells Aromatase (Cyp19a1), reduction in ovarian granulosa cells

How this Key Event works

Level of Biological Organization Cellular

Biological state

Aromatase (Cyp19a1, estrogen synthetase, estrogen synthase) is an enzyme responsible for a key step in the biosynthesis of estrogens, in particular it is responsible for conversion of C-19 androgens into C-18 estrogens (E R Simpson et al., 1994), (Ryan, 1982). It is a member of the cytochrome P450 superfamily (Ryan, 1982). The aromatase gene uses multiple promoters in a tissue-specific manner, resulting in a tissue-specific regulation of aromatase activity (Evan R Simpson, 2004). The cAMP/PKA/CREB pathway is considered to be the primary signalling cascade through which the gonadal Cyp19 promoter is regulated (Stocco, 2008).

Biological compartments

Aromatase in the specialized cells of the ovary, hypothalamus, and placenta has a crucial role in reproduction for mammalian and other vertebrates by converting androgens to estrogens. This enzyme is also present in various other tissues, such as skin, fat, bone marrow, liver, adrenals, and testes (Ryan, 1982).

General role in biology

The ovarian aromatase produces systemic and locally acting estrogens for general reproductive functions. The systemic estrogen produced by ovarian aromatase modulates the central nervous system and pituitary functions for the ovarian cycle and in spontaneously ovulating mammals it triggers the release of the ovulatory surge of luteinizing hormone (Ryan, 1982), (Hillier, 1985). Because only a single gene (CYP19) encodes aromatase in humans, targeted disruption of this gene or inhibition of its product effectively eliminates estrogen biosynthesis (Evan R Simpson et al., 2002). Much attention has been given to the regulation of the aromatase gene and its implication in the development and progression of human estrogen-dependent diseases, including breast cancer, endometrial cancer, and endometriosis, see review (Bulun et al., 2005).

How it is Measured or Detected

Methods that have been previously reviewed and approved by a recognized authority should be included in the Overview section above. All other methods, including those well established in the published literature, should be described here. Consider the following criteria when describing each method: 1. Is the assay fit for purpose? 2. Is the assay directly or indirectly (i.e. a surrogate) related to a key event relevant to the final adverse effect in question? 3. Is the assay repeatable? 4. Is the assay reproducible?

Aromatase levels can be assayed by standard methods for assessment of gene expression levels like: q-PCR

8 Aop:7 or direct protein levels: Western blot or ELISA.

Evidence Supporting Taxonomic Applicability

Name Scientific Name Evidence Links rat Rattus sp. Moderate NCBI human Homo sapiens Strong NCBI

Aromatase (CYP19) orthologs are known to be present in most of the vertebrates [see review (E R Simpson et al., 1994)]. In humans, CYP19 transcript is extensively distributed in tissues including ovaries, placenta, adipose, and brain (E R Simpson et al., 1994). In rodents, aromatase is restricted to the gonads and the brain (Stocco, 2008).

References

Bulun, S. E., Lin, Z., Imir, G., Amin, S., Demura, M., Yilmaz, B., … Deb, S. (2005). Regulation of aromatase expression in estrogen-responsive breast and uterine disease: from bench to treatment. Pharmacological Reviews, 57(3), 359–83. doi:10.1124/pr.57.3.6

Hillier, S. G. (1985). Sex steroid metabolism and follicular development in the ovary. Oxford Reviews of Reproductive Biology, 7, 168–222.

Ryan, K. J. (1982). Biochemistry of aromatase: significance to female reproductive physiology. Cancer Research, 42(8 Suppl), 3342s–3344s.

Simpson, E. R. (2004). Aromatase: biologic relevance of tissue-specific expression. Seminars in Reproductive Medicine, 22(1), 11–23. doi:10.1055/s-2004-823023

Simpson, E. R., Clyne, C., Rubin, G., Boon, W. C., Robertson, K., Britt, K., … Jones, M. (2002). Aromatase-- a brief overview. Annual Review of Physiology, 64, 93–127. doi:10.1146/annurev.physiol.64.081601.142703

Simpson, E. R., Mahendroo, M. S., Means, G. D., Kilgore, M. W., Hinshelwood, M. M., Graham-Lorence, S., … Michael, M. D. (1994). Aromatase cytochrome P450, the enzyme responsible for estrogen biosynthesis. Endocrine Reviews, 15(3), 342–55. doi:10.1210/edrv-15-3-342

Stocco, C. (2008). Aromatase expression in the ovary: hormonal and molecular regulation. Steroids, 73(5), 473–87. doi:10.1016/j.steroids.2008.01.017

Retrieved from https://aopkb.org/aopwiki/index.php/?oldid=23712 Plasma 17beta-estradiol concentrations, Reduction Short name: Plasma 17beta-estradiol concentrations, Reduction

How this Key Event works

Level of Biological Organization Individual

Estradiol synthesized by the gonads and other steroidogenic tissues (e.g., brain, adipose) is transported to other tissues via blood circulation.

How it is Measured or Detected

9 Aop:7 Total concentrations of 17β-estradiol in plasma can be measured by radioimmunoassay (e.g., (Jensen et al. 2001)), enzyme-linked immunosorbent assay (available through many commercial vendors), or by analytical chemistry (e.g., LC/MS; Owen et al. 2014). Total steroid hormones are typically extracted from plasma or serum via liquid-liquid or solid phase extraction prior to analysis.

Evidence Supporting Taxonomic Applicability

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

Key enzymes needed to synthesize 17β-estradiol first appear in the common ancestor of amphioxus and vertebrates (Baker 2011). Consequently, this key event is applicable to most vertebrates.

References

Jensen K, Korte J, Kahl M, Pasha M, Ankley G. 2001. Aspects of basic reproductive biology and endocrinology in the fathead minnow (Pimephales promelas). Comparative Biochemistry and Physiology Part C 128: 127-141. Baker ME. 2011. Origin and diversification of steroids: co-evolution of enzymes and nuclear receptors. Molecular and cellular endocrinology 334(1-2): 14-20. Owen LJ, Wu FC, Keevil BG. 2014. A rapid direct assay for the routine measurement of oestradiol and oestrone by liquid chromatography tandem mass spectrometry. Ann. Clin. Biochem. 51(pt 3):360-367.

Retrieved from https://aopkb.org/aopwiki/index.php/?oldid=26590

Adverse Outcome Fertility, impaired ovarian cycle, irregularities Fertility, impaired

How this Key Event works

Level of Biological Organization Individual

Biological state capability to produce offspring

Biological compartments

System

General role in biology

Fertility is the capacity to conceive or induce conception. Impairment of fertility represents disorders of male or female reproductive functions or capacity.

How it is Measured or Detected

10 Aop:7 Methods that have been previously reviewed and approved by a recognized authority should be included in the Overview section above. All other methods, including those well established in the published literature, should be described here. Consider the following criteria when describing each method: 1. Is the assay fit for purpose? 2. Is the assay directly or indirectly (i.e. a surrogate) related to a key event relevant to the final adverse effect in question? 3. Is the assay repeatable? 4. Is the assay reproducible?

As a measure, fertility rate is the number of offspring born per mating pair, individual or population.

Evidence Supporting Taxonomic Applicability

Name Scientific Name Evidence Links rat Rattus rattus Strong NCBI mouse Mus musculus Strong NCBI human Homo sapiens Strong NCBI

Regulatory Examples Using This Adverse Outcome

Under REACH, information on reproductive toxicity is required for chemicals with an annual production/importation volume of 10 metric tonnes or more. Standard information requirements include a screening study on reproduction toxicity (OECD TG 421/422) at Annex VIII (10-100 t.p.a), a prenatal developmental toxicity study (OECD 414) on a first species at Annex IX (100-1000 t.p.a), and from March 2015 the OECD 443(Extended One-Generation Reproductive Toxicity Study) is reproductive toxicity requirement instead of the two generation reproductive toxicity study (OECD TG 416). If not conducted already at Annex IX, a prenatal developmental toxicity study on a second species at Annex X (≥ 1000 t.p.a.).

Under the Biocidal Products Regulation (BPR), information is also required on reproductive toxicity for active substances as part of core data set and additional data set (EU 2012, ECHA 2013). As a core data set, prenatal developmental toxicity study (EU TM B.31) in rabbits as a first species and a two-generation reproduction toxicity study (EU TM B.31) are required. OECD TG 443 (Extended One-Generation Reproductive Toxicity Study) shall be considered as an alternative approach to the multi-generation study.) According to the Classification, Labelling and Packaging (CLP) regulation (EC, 200; Annex I: 3.7.1.1): a) “reproductive toxicity” includes adverse effects on sexual function and fertility in adult males and females, as well as developmental toxicity in the offspring; b) “effects on fertility” includes adverse effects on sexual function and fertility; and c) “developmental toxicity” includes adverse effects on development of the offspring.

References

Retrieved from https://aopkb.org/aopwiki/index.php/?oldid=27278 ovarian cycle, irregularities

How this Key Event works

Level of Biological Organization Individual

Biological state

The female ovarian cycle is the result of a balanced cooperation between several organs and is determined

11 Aop:7 by a complex interaction of hormones. Ovarian cycle irregularities include disturbances in the ovarian cycle (e.g. longer cycle, persistent estrus) and/or ovulation problems (deferred ovulation or anovulation). The estrous cycle (also oestrous cycle) comprises the recurring physiologic changes that are induced by reproductive hormones in females. Estrous cycles start after sexual maturity in females and are interrupted by anestrous phases or pregnancies. During this cycle numerous well defined and sequential alterations in reproductive tract histology, physiology and cytology occur, initiated and regulated by the hypothalamic- pituitary-ovarian (HPO) axis. The central feature of the mammalian estrous cycle is the periodic maturation of eggs that will be released at ovulation and luteinisation of the follicles after ovulation to form corpora lutea. Adapted from www.oecd.org/chemicalsafety/testing/43754807.pdf Biological compartments

The cyclic changes that occur in the female reproductive tract are initiated and regulated by the hypothalamic-pituitary-ovarian (HPO) axis. Although folliculogenesis occurs independently of hormonal stimulation up until the formation of early tertiary follicles, the gonadotrophins luteinising hormone (LH) and follicle stimulating hormone (FSH) are essential for the completion of follicular maturation and development of mature preovulatory (Graafian) follicles. The oestrous cycle consists of four stages: prooestrus, oestrus, metoestrus (or dioestrus 1) and dioestrus (or dioestrus 2) orchestrated by hormones. Levels of LH and FSH begin to increase just after dioestrus. Both hormones are secreted by the same secretory cells (gonadotrophs) in the pars distalis of the anterior pituitary (adenohypophysis). FSH stimulates the development of the zona granulosa and triggers expression of LH receptors by granulosa cells. LH initiates the synthesis and secretion of androstenedione and, to a lesser extent, testosterone by the theca interna; these androgens are utilised by granulosa cells as substrates in the synthesis of estrogen. Pituitary release of gonadotrophins thus drives follicular maturation and secretion of estrogen during prooestrus. Gonadotrophin secretion by the anterior pituitary is regulated by luteinising hormone-releasing hormone (LHRH), produced by the hypothalamus. LHRH is transported along the axons of hypothalamic neurones to the median eminence where it is secreted into the hypothalamic-hypophyseal portal system and transported to the anterior pituitary. The hypothalamus secretes LHRH in rhythmic pulses; this pulsatility is essential for the normal activation of gonadotrophs and subsequent release of LH and FSH. Adapted from www.oecd.org/chemicalsafety/testing/43754807.pdf

Follicles that produce estrogens have sequestered pituitary FSH which in turn stimulates the aromatase reaction. Such follicles can undergo normal development and ovulation and contain eggs that readily resume meiosis when released. In the absence of an active local aromatase (i.e., no follicle-stimulating hormone), the follicles and oocytes become atretic and regress without ovulating. If aromatase is present, the estrogen and follicle stimulating hormone can further develop the follicular cells for normal luteal function after ovulation takes place (Ryan, 1982).

General role in biology

A sequential progression of interrelated physiological and behavioural cycles underlines the female's successful production of young. In many but not all species the first and most basic of these is estrous cycle, which is itself a combination of cycles.

How it is Measured or Detected

Methods that have been previously reviewed and approved by a recognized authority should be included in the Overview section above. All other methods, including those well established in the published literature, should be described here. Consider the following criteria when describing each method: 1. Is the assay fit for purpose? 2. Is the assay directly or indirectly (i.e. a surrogate) related to a key event relevant to the final adverse effect in question? 3. Is the assay repeatable? 4. Is the assay reproducible?

The pattern of events in the estrous cycle may provide a useful indicator of the normality of reproductive neuroendocrine and ovarian function in the nonpregnant female. It also provides a means to interpret hormonal, histologic, and morphologic measurements relative to stage of the cycle, and can be useful to monitor the status of mated females. Regular cyclicity is one of the key parameters in assessment of female reproductive function in rodents. Parameters assessed for cyclicity: - Number of cycling females - Number of females with regular cycles - Number of cycles - Estrous cycle length - Percentage of time spent in the

12 Aop:7 various estrous cycle stages Estrous cyclicity provides a method for evaluating the endocrine disrupting activity of each test chemical under physiologic conditions where endogenous concentrations of estrogen vary. Abnormal cycles were defined as one or more estrous cycles in the 21-day period with prolonged estrus (≥3 days) and/or prolonged metestrus or diestrus (≥4 days) within a given cycle (Goldman, Murr, & Cooper, 2007).

Estrous cycle normality can be monitored in the rat and mouse by observing the changes in the vaginal smear cytology. Visual observation of the vagina is the quickest method, requires no special equipment, and is best used when only proestrus or estrus stages need to be identified. For details see: (Westwood, 2008), (Byers, Wiles, Dunn, & Taft, 2012) and OECD guidelines (www.oecd.org).

The observation that animals do not ovulate while exhibiting estrous cycles indicates that estrous cyclicity alone may not be a sufficient surrogate of healthy function of ovaries; the measurements of serum hormones and particularly FSH can contribute to more sensitivity indicators of healthy function of ovaries (Davis, Maronpot, & Heindel, 1994).

In vitro testing

The follicle culture models were developed for the in-vitro production of mature oocytes and used to study the process of folliculogenesis and oogenesis in vitro (Cortvrindt & Smitz, 2002). These in vitro cultures demonstrate near-identical effects to those found in vivo, therefore might be able to acquire a place in fertility testing, replacing some in-vivo studies for ovarian function and female gamete quality testing (Stefansdottir, Fowler, Powles-Glover, Anderson, & Spears, 2014).

Evidence Supporting Taxonomic Applicability

Name Scientific Name Evidence Links mice Mus musculus Weak NCBI rat Rattus sp. Moderate NCBI

The estrous cycle comprises the recurring physiologic changes that are induced by reproductive hormones in most mammalian females. Many of the mechanisms involved in the regulation of the reproductive axis are similar across species (particularly those mediated through the estrogen receptor), assessments of rodent estrous cyclicity can offer insight into potential adverse effects in humans (Goldman, Murr, & Cooper, 2007). While evaluations of vaginal cytology in the laboratory rodent can provide a valuable reflection of the integrity of the hypothalamic-pituitary-ovarian axis, other indices are more useful in humans to determine the functional status of the reproductive system (e.g. menses, basal body temperature, alterations in vaginal pH, cervical mucous viscosity, and blood hormone levels). Nevertheless, since many of the mechanisms involved in the regulation of the reproductive axis are similar across species (particularly those mediated through the estrogen receptor), assessments of rodent estrous cyclicity can offer insight into potential adverse effects in humans (Rasier, Toppari, Parent, & Bourguignon, 2006).

Regulatory Examples Using This Adverse Outcome

Chemicals may be found to interfere with reproductive function in the female rat. This interference is commonly expressed as a change in normal morphology of the reproductive tract or a disturbance in the duration of particular phases of the estrous cycle. This key event lies within the scope of testing for endocrine disrupting activity of chemicals and therefore for testing of female reproductive and developmental toxicity. Monitoring of oestrus cyclicity is included in OECD test guidelines (Test No. 407: Repeated Dose 28-day Oral Toxicity Study in Rodents, 2008), (Test No. 416: Two-Generation Reproduction Toxicity, 2001) and (Test No. 443: Extended One-Generation Reproductive Toxicity Study, 2012) and in USA EPA OCSPP 890.1450. While an evaluation of the estrous cycle in laboratory rodents can be a useful measure of the integrity of the hypothalamic-pituitary-ovarian reproductive axis, it can also serve as a way of insuring that animals exhibiting abnormal cycling patterns are excluded from a study prior to exposure to a test compound. When incorporated as an adjunct to other endpoint measures, a determination of a

13 Aop:7 female's cycling status can contribute important information about the nature of a toxicant insult to the reproductive system. In doing so, it can help to integrate the data into a more comprehensive mechanistic portrait of the effect, and in terms of risk assessment, may provide some indication of a toxicant's impact on human reproductive physiology. Significant evidence that the estrous cycle (or menstrual cycle in primates) has been disrupted should be considered an adverse effect (OECD, 2008). Included should be evidence of abnormal cycle length or pattern, ovulation failure, or abnormal menstruation.

References

Byers, S. L., Wiles, M. V, Dunn, S. L., & Taft, R. A. (2012). Mouse estrous cycle identification tool and images. PloS One, 7(4), e35538. doi:10.1371/journal.pone.0035538

Cortvrindt, R. G., & Smitz, J. E. J. (2002). Follicle culture in reproductive toxicology: a tool for in-vitro testing of ovarian function? Human Reproduction Update, 8(3), 243–54.

Davis, B. J., Maronpot, R. R., & Heindel, J. J. (1994). Di-(2-ethylhexyl) phthalate suppresses estradiol and ovulation in cycling rats. Toxicology and Applied Pharmacology, 128(2), 216–23. doi:10.1006/taap.1994.1200

Goldman, J. M., Murr, A. S., & Cooper, R. L. (2007). The rodent estrous cycle: characterization of vaginal cytology and its utility in toxicological studies. Birth Defects Research. Part B, Developmental and Reproductive Toxicology, 80(2), 84–97. doi:10.1002/bdrb.20106

OECD. (2008). No 43: Guidance document on mammalian reproductive toxicity testing and assessment.

Rasier, G., Toppari, J., Parent, A.-S., & Bourguignon, J.-P. (2006). Female sexual maturation and reproduction after prepubertal exposure to estrogens and endocrine disrupting chemicals: a review of rodent and human data. Molecular and Cellular Endocrinology, 254-255, 187–201. doi:10.1016/j.mce.2006.04.002

Ryan, K. J. (1982). Biochemistry of aromatase: significance to female reproductive physiology. Cancer Research, 42(8 Suppl), 3342s–3344s.

Stefansdottir, A., Fowler, P. A., Powles-Glover, N., Anderson, R. A., & Spears, N. (2014). Use of ovary culture techniques in reproductive toxicology. Reproductive Toxicology (Elmsford, N.Y.), 49C, 117–135. doi:10.1016/j.reprotox.2014.08.001

Test No. 407: Repeated Dose 28-day Oral Toxicity Study in Rodents. (2008). OECD Publishing. doi:10.1787/9789264070684-en

Test No. 416: Two-Generation Reproduction Toxicity. (2001). OECD Publishing. doi:10.1787/9789264070868-en

Test No. 443: Extended One-Generation Reproductive Toxicity Study. (2012). OECD Publishing. doi:10.1787/9789264185371-en

Westwood, F. R. (2008). The female rat reproductive cycle: a practical histological guide to staging. Toxicologic Pathology, 36(3), 375–84. doi:10.1177/0192623308315665

Retrieved from https://aopkb.org/aopwiki/index.php/?oldid=23718

Scientific evidence supporting the linkages in the AOP

14 Aop:7 Event Description Triggers PPAR gamma, Activation Indirectly Aromatase (Cyp19a1), reduction in ovarian Leads to granulosa cells Aromatase (Cyp19a1), reduction in ovarian Directly 17beta-estradiol synthesis by ovarian granulosa cells Leads to granulosa cells, Reduction 17beta-estradiol synthesis by ovarian Directly Plasma 17beta-estradiol concentrations, granulosa cells, Reduction Leads to Reduction Plasma 17beta-estradiol concentrations, Directly ovarian cycle, irregularities Reduction Leads to ovarian cycle, irregularities Indirectly Fertility, impaired Leads to PPAR gamma, Activation Indirectly Leads to Aromatase (Cyp19a1), reduction in ovarian granulosa cells

How Does This Key Event Relationship Work

This KER establishes the link between PPARγ activation and reduced levels of aromatase in ovarian granulosa cells. Aromatase is a key enzyme in steroidogenesis, catalysing the conversion of androgens to estrogens.

Weight of Evidence

Biological Plausibility

Peroxisome proliferator-activated receptor γ (PPARγ) is master switch of lipid metabolism and cell differentiation, their role has also been acknowledged in regulation of reproductive function and development [reviewed by (P Froment et al., 2006), (Minge, Robker, & Norman, 2008)]. The PPARs are implicated in regulation of steroidogenesis from in vitro data [reviewed by (Carolyn M Komar, 2005)].

PPARγ involvement in aromatase regulation in granulosa cells

The PPARγ is activated upon the ligand binding in granulosa cells, and then indirectly alters the expression of aromatase, the rate-limiting enzyme in conversion of androgens to estrogens (Kwintkiewicz, Nishi, Yanase, & Giudice, 2010), (Lovekamp-Swan, Jetten, & Davis, 2003), (Mu et al., 2000). The ligands of PPARγ were also shown to regulate other enzymes involved in steroidogenesis (Dupont, Chabrolle, Ramé, Tosca, & Coyral-Castel, 2008). All PPAR isoforms have been detected in both human and rodent ovary [reviewed by (Carolyn M Komar, 2005)]. In female rats the PPARγ have been detected in granulosa cells.

• PPARγ is primarily expressed in the granulosa cells and pre-ovulatory follicles, less strongly in the theca cells and corpus luteum where its expression increases after ovulation and falls after the LH surge, (C M Komar, Braissant, Wahli, & Curry, 2001). In the absence of fertilization or embryo implantation, PPARγ expression decreases as a result of corpus luteum regression (Viergutz, Loehrke, Poehland, Becker, & Kanitz, 2000).

• PPARγ is directly involved in oocyte maturation and ovulation [reviewed by (P Froment et al., 2006)]

Additional studies have shown that PPARγ is active in the ovary (P Froment et al. 2003).

The precise molecular mechanism by which PPARγ regulates aromatase is unclear given the fact that the proximal promoter regulating aromatase expression in the rat ovary does not contain an obvious peroxisome

15 Aop:7 proliferator response element (PPRE) (Young and McPhaul 1997). There are plausible ways by which the PPARγ (as transcriptionally active PPAR:RXR heterodimer) could modify the transcription of aromatase including activation of RXR competition for binding sites on DNA and competition for limiting co-activators required for gene transcription. A new insight in the mechanism of regulation of the aromatase gene and activation of PPAR gamma and RXR was brought by Fan et al proposing disruption of NF-κB interaction with the aromatase promoter (Fan et al. 2005). The authors showed that activation of PPARγ and RXR impaired the interaction between NF-κB and aromatase promoter II and the p65 based transcription in both ovarian and fibroblast cells in a PPARγ-dependent manner (Fan et al. 2005). Studies supporting that hypothesis show that both PPARγ ligand (Troglitazone) and RXR ligand (LG100268) suppress aromatase activity in human granulosa cells (Mu et al. 2000), (Mu et al. 2001) and together causing a greater reduction than either compound alone (Mu et al. 2000).

Another possibility is that PPARγ is able to modify protein–protein interactions involved in the transcription of aromatase. Activation of PPARγ may recruit cofactors away from aromatase to inhibit normal transcription. Further studies are necessary to determine how PPARγ transcriptionally repress aromatase.

Empirical Support for Linkage

Agonists of PPARγ were shown to also decrease aromatase in human and rodent ovarian cells: Bisphenol A, in vitro, human: (Kwintkiewicz et al., 2010) showing significant PPARγ activation and reduction of aromatase from the same dose (40μM).

MEHP, in vitro , rat: at 50 μM 45% decrease mRNA aromatase (Lovekamp-Swan et al., 2003) DEHP, in vivo , rat: dose dependent increase of PPARγ and reduction of aromatase (300-600mg/kg/day), measured at the same time point (Xu et al., 2010).

The treatment of the granulosa cells with PPARγ ligand (Troglitazone) results in an inhibition of the aromatase protein levels and/or activity in a dose-dependent manner (Mu et al., 2000), (Mu et al., 2001). Table 1 provides the experimental support for the implication of PPARγ in aromatase regulation (limited quantitative and taxonomical aspects are included).

Inhibition studies

The involvement of each PPARγ subtype in the suppression of aromatase by MEHP was shown by Lovekamp-Swan et al. MEHP alone suppressed aromatase by 48% and the PPARγ (GW 347845) agonists alone decreased aromatase by 30 % . As expected, the PPARγ antagonist (GR 259662) completely blocked the suppression of aromatase by the PPARγ-selective agonist (Lovekamp-Swan et al., 2003).

Table 1 summaries the studies and compounds activating PPARγ and causing decrease level of aromatase (in granulosa cells) including limited quantitative and taxonomical aspects. Data for activation of PPARα are also included as it is hypothesised contributing/synergising pathway.

KE:PPAR activation KE:Aromatase Species Reference decrease Compound PPARα PPARγ PPARα PPARγ binding binding activation activation <4- <0.1-60 µM > ToxCast, (Venkata et al., 2006), (Hurst & <12-30µM > 100µM > EC50 =6.2 At 167µM decrease Waxman, 2003)(Lapinskas et al., 2005), MEHP Ki=15µM Ki=12 µM, human EC50 µM/AC50=55.1 mRNA (Reinsberg, Wegener-Toper, van der Ven, van AC50=18.3µM =3.2 µM µM der Ven, & Klingmueller, 2009) <0.5- <2-200µM> At 50 μM 45% Mouse, (Hurst & Waxman, 2003)(Lovekamp-Swan et MEHP n.f. n.f. 100µM> EC50 =10.1 decrease mRNA rat al., 2003) EC50 μM =0.6 μM

1 μM 50% activity, at 100 μM 90% (Willson, Brown, Sternbach, & Henke, 2000), Troglitazone inactive n.i. inactive EC50 = 0.55 human μM activity100 μM (Mu et al., 2001) decreased mRNA

16 Aop:7 Mouse, (Willson et al., 2000), (Lovekamp-Swan et al., Troglitazone inactive n.i. inactive EC50 = 0.78 1 μM, 45%-48 μM decrease mRNA rat 2003)

Table 1 Table summarising the quantitative relationship between activation of PPAR α&γ and decreased levels of aromatase across the species. AC50- and EC50 - half maximal effective concentration values, reported if available, Ki -inhibition constant, n.f.= not found, n.i.= not investigated.

Uncertainties or Inconsistencies

There is substantial evidence in literature supporting the KER, however the underlying mechanism are to be investigated, together with other pathways involved in aromatase down regulation. The pattern of the PPARγ expression in ovarian follicles is not steady, unlike expression of PPARs α 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, Braissant, Wahli, & Curry, 2001), but only in follicles that have responded to the LH surge (Carolyn M Komar & 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. Some evidence implies that the regulatory role of PPARγ might be connected to the other events in estradiol synthesis like the impairment of cholesterol transport to mitochondria (Cui et al., 2002).

PPARα The experimental data supports the parallel involvement of another member of PPAR superfamily of nuclear receptors, PPARα. PPARα was shown to be implicated in the down regulation of aromatase in rat: in vitro (Lovekamp-Swan et al., 2003); in vivo (Xu et al., 2010) and in mice in vivo (Toda, Okada, Miyaura, & Saibara, 2003). The ovarian aromatase promoter contains one half of a PPRE (peroxisome proliferator response element), which is the binding site for steroidogenic factor 1 (SF-1) (Young & McPhaul, 1997). While it is unknown whether PPARα can compete for binding on an incomplete response element, disruption of SF-1 binding to this half site would disrupt normal aromatase transcription. Studies by S. Plummer et al showed that PPARα and SF1 share a common coactivator (S. Plummer, Sharpe, Hallmark, Mahood, & Elcombe, 2007), (S. M. Plummer et al., 2013), CREB-binding protein (CBP), which is present in limiting concentrations (McCampbell, 2000). Binding of CBP to PPARα could therefore starve SF1 a cofactor essential for its transactivation functions. Surprisingly, aromatase levels were increased in ovaries of PPARα-null mice upon treatment with PPARα ligand (Toda et al., 2003).

PPARα was also reported to regulate other enzymes involved in steroidogenesis like: 17 beta- hydroxysteroid dehydrogenase type IV (HSD IV) (Corton et al., 1996), 3 beta-hydroxysteroid dehydrogenase (Wong, Ye, Muhlenkamp, & Gill, 2002) or 11beta-hydroxysteroid dehydrogenase type (Hermanowski- Vosatka et al., 2000). While PPARα/γ activators (like MEHP ) suppress aromatase, they showed no effect on Cholesterol side-chain cleavage enzyme (P450scc) in granulosa cells, demonstrating a more specific effect on steroidogenesis (Lovekamp-Swan et al., 2003). Experiments with PPARα-null mice indicate involvement of the receptor in reproductive toxicity, however cannot be entirely explained by the activation of PPARα mediated pathway as PPARα-null mice remain sensitive to DEHP-mediated reproductive toxicity (Ward et al. 1998), which implies other players including PPARγ. The above evidence supports the involvement of PPARα in regulation of steroidogenesis on its different steps. As PPARα is found primarily in the theca and stroma and the expression of PPARα in granulosa cells is very low (Carolyn M Komar, 2005) therefore it might be involved in steps in steroidogenesis upstream of aromatase.

Retinoid X Receptor (RXR)

Chemicals are able to activate RXR–PPARγ through RXR because this heterodimer interacts poorly with co- repressors in vivo and belongs to the group of so-called ‘permissive’ heterodimers, which can be stimulated by RXR ligands on their own (Germain, Iyer, Zechel, & Gronemeyer, 2002). Studies demonstrated that a PPARγ ligand and/or a RXR ligand decreased the aromatase activity in both cultured human ovarian granulosa cells (Mu et al., 2000), (Mu et al., 2001) and human granulosa-like tumor KGN cells (Kwintkiewicz

17 Aop:7 et al., 2010) and combined treatment causes a greater reduction than either compound alone (Mu et al., 2000), (Mu et al., 2001).

Inconsistencies

No effect on aromatase protein expression was observed after PPARγ ligand (rosiglitazone) treatment in porcine ovarian follicles (Rak-Mardyła & Karpeta, 2014).

Quantitative Understanding of the Linkage

Is it known how much change in the first event is needed to impact the second? Are there known modulators of the response-response relationships? Are there models or extrapolation approaches that help describe those relationships?

Evidence Supporting Taxonomic Applicability

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

See the Table 1.

References

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Bhattacharya, N., Dufour, J. M., Vo, M.-N., Okita, J., Okita, R., & Kim, K. H. (2005). Differential effects of phthalates on the testis and the liver. Biology of Reproduction, 72(3), 745–54. doi:10.1095/biolreprod.104.031583

Corton, J., Bocos, C., Moreno, E., Merritt, A., Marsman, D., Sausen, P., … Gustafsson, J. (1996). Rat 17 beta-hydroxysteroid dehydrogenase type IV is a novel peroxisome proliferator-inducible gene. Mol. Pharmacol., 50(5), 1157–1166.

Cui, Y., Miyoshi, K., Claudio, E., Siebenlist, U. K., Gonzalez, F. J., Flaws, J., … Hennighausen, L. (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), 17830–5. doi:10.1074/jbc.M200186200

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Plummer, S., Sharpe, R. M., Hallmark, N., Mahood, I. K., & Elcombe, C. (2007). Time-dependent and compartment-specific effects of in utero exposure to Di(n-butyl) phthalate on gene/protein expression in the fetal rat testis as revealed by transcription profiling and laser capture microdissection. Toxicological Sciences : An Official Journal of the Society of Toxicology, 97(2), 520–32. doi:10.1093/toxsci/kfm062

Rak-Mardyła, A., & Karpeta, A. (2014). Rosiglitazone stimulates peroxisome proliferator-activated receptor gamma expression and directly affects in vitro steroidogenesis in porcine ovarian follicles. Theriogenology, 82(1), 1–9. doi:10.1016/j.theriogenology.2014.02.016

Reinsberg, J., Wegener-Toper, P., van der Ven, K., van der Ven, H., & Klingmueller, D. (2009). Effect of mono-(2-ethylhexyl) phthalate on steroid production of human granulosa cells. Toxicology and Applied Pharmacology, 239(1), 116–23. doi:10.1016/j.taap.2009.05.022

Rotman, N., Haftek-Terreau, Z., Lücke, S., Feige, J., Gelman, L., Desvergne, B., & Wahli, W. (2008). PPAR Disruption: Cellular Mechanisms and Physiological Consequences. CHIMIA International Journal for Chemistry, 62(5), 340–344. doi:10.2533/chimia.2008.340

Toda, K., Okada, T., Miyaura, C., & Saibara, T. (2003). Fenofibrate, a ligand for PPARalpha, inhibits aromatase cytochrome P450 expression in the ovary of mouse. Journal of Lipid Research, 44(2), 265–70. doi:10.1194/jlr.M200327-JLR200

ToxCastTM Data. “ToxCastTM Data.” US Environmental Protection Agency. http://www.epa.gov/ncct/toxcast/data.html.

Venkata, N. G., Robinson, J. a, Cabot, P. J., Davis, B., Monteith, G. R., & Roberts-Thomson, S. J. (2006). Mono(2-ethylhexyl)phthalate and mono-n-butyl phthalate activation of peroxisome proliferator activated- receptors alpha and gamma in breast. Toxicology Letters, 163(3), 224–34. doi:10.1016/j.toxlet.2005.11.001

Viergutz, T., Loehrke, B., Poehland, R., Becker, F., & Kanitz, W. (2000). Relationship between different stages of the corpus luteum and the expression of the peroxisome proliferator-activated receptor gamma protein in bovine large lutein cells. Journal of Reproduction and Fertility, 118(1), 153–61.

Willson, T. M., Brown, P. J., Sternbach, D. D., & Henke, B. R. (2000). The PPARs: from orphan receptors to drug discovery. Journal of Medicinal Chemistry, 43(4), 527–50.

Wong, J. S., Ye, X., Muhlenkamp, C. R., & Gill, S. S. (2002). Effect of a peroxisome proliferator on 3 beta- hydroxysteroid dehydrogenase. Biochemical and Biophysical Research Communications, 293(1), 549–53. doi:10.1016/S0006-291X(02)00235-8

Xu, C., Chen, J.-A., Qiu, Z., Zhao, Q., Luo, J., Yang, L., … Shu, W. (2010). Ovotoxicity and PPAR-mediated aromatase downregulation in female Sprague-Dawley rats following combined oral exposure to

20 Aop:7 benzo[a]pyrene and di-(2-ethylhexyl) phthalate. Toxicology Letters, 199(3), 323–32. doi:10.1016/j.toxlet.2010.09.015

Young, M., & McPhaul, M. J. (1997). Definition of the elements required for the activity of the rat aromatase promoter in steroidogenic cell lines. The Journal of Steroid Biochemistry and Molecular Biology, 61(3-6), 341–8.

Synthetic ligand, rosiglitazone stimulates AMP-activated protein kinase (AMPK) and enhances the meiotic resumption of mouse oocytes (Dupont, Reverchon, Cloix, Froment, & Ramé, 2012). Retrieved from https://aopkb.org/aopwiki/index.php/?oldid=26970 Aromatase (Cyp19a1), reduction in ovarian granulosa cells Directly Leads to 17beta-estradiol synthesis by ovarian granulosa cells, Reduction

How Does This Key Event Relationship Work

Aromatase is the cytochrome P450 enzyme complex responsible for the conversion of androgens to estrogens during steroidogenesis [reviewed by (Simpson et al., 1994)], which is a key reaction in the sex differentiation in vertebrates. Reduction in level of aromatase or in the catalytic activity of the aromatase itself will reduce the levels of estrogens in tissues and dramatically disrupt estrogen (E2) hormone action.

Weight of Evidence

Biological Plausibility

Aromatase in the specialized cells of the ovary, hypothalamus, and placenta clearly serves crucial role in reproduction for mammalian and other vertebrates by converting the androgens to estrogens. Therefore, the coordinated and cell-specific expression of the aromatase (Cyp19a1) gene in the ovary plays a key role in the 17beta-estradiol (E2) synthesis. Within the ovary, aromatase expression and activity is primarily localized in the granulosa cells (reviewed in (Havelock, Rainey, & Carr, 2004). C-19 androgens diffuse from the theca cells into granulosa cells where aromatase can catalyze their conversion to C-18 estrogens. Therefore, inhibition, decrease of level or activity of ovarian aromatase can generally be assumed to directly impact E2 synthesis by the granulosa cells.

Empirical Support for Linkage

Environmental agents, toxicants, and various natural products can impact on aromatase activity and/or alteration in protein levels to result in reduced levels of estrogen.

Studies providing evidence for the linkage of aromatase decrease and decreased E2 production include: Bisphenol A: in vitro, human: significant reduction of aromatase from 40μM and decreased E2 production from 80 μM (Kwintkiewicz, Nishi, Yanase, & Giudice, 2010) at the same time point. MEHP, in vitro:

• human, from 10μM decreased aromatase activity (dose dependent), at 167 μM decrease in mRNA levels of aromatase and from 10μM decrease of estradiol production (dose dependent), measured at the same time point (48h) (Reinsberg, Wegener-Toper, van der Ven, van der Ven, & Klingmueller, 2009)

• rat, dose response decrease in aromatase levels from 50μM, dose dependent decrease of E2 production from 100μM, at the same (48 h) (Lovekamp & Davis, 2001).

• rat, decrease in aromatase levels at 100μg/ml DEHP, 10μg/ml MEHP and dose dependent decrease of E2

21 Aop:7 production from 10μg/ml DEHP, 0.1μg/ml MEHP and at the same time point (96 h) (Gupta et al., 2010).

Table 1 summarises available empirical evidence.

Study Compound Species type comments Aromatase decrease levels/activity Reference (Davis, Weaver, in decrease activity of aromatase and dose and time decrease activity of aromatase 100 Gaines, & Heindel, MEHP rat vitro dependent decrease of E2 production µM E2 production 50-100 µM 1994), ex decrease aromatase level and dose decrease of E2 aromatase level 50µM (Lovekamp & Davis, MEHP rat vivo production E2 production 100-200 µM 2001)

in Does dependent reduction of E2 levels, and Does DEHP rat vivo dependent reduction decrease aromatase expression 300-600mg/kg/day (Xu et al., 2010),

in Dose dependent reduction E2 production and reduction E2 levels IC(50)= 49- 138 (Reinsberg et al., MEHP human vitro reduction aromatase of expression µM, at 167µM decrease aromatase 2009)

E2 production at DEHP (10 - MEHP/DEHP ex dose dependent E2 production, and reduction of 100 μg/ml);MEHP (0.1 and 10 μg/ml) mice (Gupta et al., 2010) vivo aromatase levels Aromatase levels DEHP (100 μg/ml); MEHP 0.1 μg/ml

This KE describes decreased levels and/or availability of aromatase different from aromatase inhibition.

Uncertainties or Inconsistencies

Availability or reduced aromatase levels

Studies by Davis et al showed that MEHP impacts on availability (degradation) of aromatase as the decrease in E2 production is evident after the treatment with transcription and translation blockers (actinomycin D or cycloheximide). MEHP was further decreased E2 production independently of the presence of inhibitors pointing out at mechanisms of degradation rather than aromatase synthesis (Davis et al., 1994). MEHP can indirectly impact on aromatase rates by decreasing necessary cofactors (availability) or activation of aromatase inhibitors. Treinin et al showed in vitro dose dependent inhibition of progesterone production by MEHP in granulosa cells and reduced FSH-stimulated cAMP accumulation in granulosa cells implicating a direct or indirect effect of MEHP on FSH receptor (Treinen, Dodson, & Heindel, 1990). Similar effects of cAMP accumulation were seen in Sertoli cells (Lloyd & Foster, 1988), (Heindel & Chapin, 1989), (Heindel & Powell, 1992). Since granulosa and Sertoli cells share several structural and functional characteristics this mechanism is plausible. Study by Ma et al showed that inhaled DEHP (5 and 25 mg/m3) increased levels of LH and E2 in serum of prepubertal rats, and it increased ovarian Cyp19a1 expression (Ma et al., 2006), which is in disagreement with the key event relationship. This difference might be due to measurements of hormones during different phases of the estrous cycle, alterations in the experimental approaches used (in vivo versus in vitro) as well as exposure routes and doses given.

Quantitative Understanding of the Linkage

Is it known how much change in the first event is needed to impact the second? Are there known modulators of the response-response relationships? Are there models or extrapolation approaches that help describe those relationships?

Several mechanistically-based models of ovarian steroidogenesis have been developed (Breen et al. 2013; Breen et al. 2007; Shoemaker et al. 2010; Quignot and Bois 2013). These may be adaptable to predict in vitro E2 production and/or plasma E2 concentrations from in vitro or in vivo measurements of changes of

22 Aop:7 aromatase expression/availability.

Evidence Supporting Taxonomic Applicability

Name Scientific Name Evidence Links

See table 1.

References

Davis, B. J., Weaver, R., Gaines, L. J., & Heindel, J. J. (1994). Mono-(2-ethylhexyl) phthalate suppresses estradiol production independent of FSH-cAMP stimulation in rat granulosa cells. Toxicology and Applied Pharmacology, 128(2), 224–8. doi:10.1006/taap.1994.1201

Gupta, R. K., Singh, J. M., Leslie, T. C., Meachum, S., Flaws, J. a, & Yao, H. H.-C. (2010). Di-(2-ethylhexyl) phthalate and mono-(2-ethylhexyl) phthalate inhibit growth and reduce estradiol levels of antral follicles in vitro. Toxicology and Applied Pharmacology, 242(2), 224–30. doi:10.1016/j.taap.2009.10.011

Havelock, J. C., Rainey, W. E., & Carr, B. R. (2004). Ovarian granulosa cell lines. Molecular and Cellular Endocrinology, 228(1-2), 67–78. doi:10.1016/j.mce.2004.04.018

Heindel, J. J., & Chapin, R. E. (1989). Inhibition of FSH-stimulated cAMP accumulation by mono(2- ethylhexyl) phthalate in primary rat Sertoli cell cultures. Toxicology and Applied Pharmacology, 97(2), 377– 85.

Heindel, J. J., & Powell, C. J. (1992). Phthalate ester effects on rat Sertoli cell function in vitro: effects of phthalate side chain and age of animal. Toxicology and Applied Pharmacology, 115(1), 116–23.

Kwintkiewicz, J., Nishi, Y., Yanase, T., & Giudice, L. C. (2010). Peroxisome proliferator-activated receptor- gamma mediates bisphenol A inhibition of FSH-stimulated IGF-1, aromatase, and estradiol in human granulosa cells. Environmental Health Perspectives, 118(3), 400–6. doi:10.1289/ehp.0901161

Lloyd, S. C., & Foster, P. M. (1988). Effect of mono-(2-ethylhexyl)phthalate on follicle-stimulating hormone responsiveness of cultured rat Sertoli cells. Toxicology and Applied Pharmacology, 95(3), 484–9.

Lovekamp, T. N., & Davis, B. J. (2001). Mono-(2-ethylhexyl) phthalate suppresses aromatase transcript levels and estradiol production in cultured rat granulosa cells. Toxicology and Applied Pharmacology, 172(3), 217–24. doi:10.1006/taap.2001.9156

Ma, M., Kondo, T., Ban, S., Umemura, T., Kurahashi, N., Takeda, M., & Kishi, R. (2006). Exposure of prepubertal female rats to inhaled di(2-ethylhexyl)phthalate affects the onset of puberty and postpubertal reproductive functions. Toxicological Sciences : An Official Journal of the Society of Toxicology, 93(1), 164– 71. doi:10.1093/toxsci/kfl036

Reinsberg, J., Wegener-Toper, P., van der Ven, K., van der Ven, H., & Klingmueller, D. (2009). Effect of mono-(2-ethylhexyl) phthalate on steroid production of human granulosa cells. Toxicology and Applied Pharmacology, 239(1), 116–23. doi:10.1016/j.taap.2009.05.022

Simpson, E. R., Mahendroo, M. S., Means, G. D., Kilgore, M. W., Hinshelwood, M. M., Graham-Lorence, S., … Michael, M. D. (1994). Aromatase cytochrome P450, the enzyme responsible for estrogen biosynthesis. Endocrine Reviews, 15(3), 342–55. doi:10.1210/edrv-15-3-342

Treinen, K. A., Dodson, W. C., & Heindel, J. J. (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), 334–40.

23 Aop:7 Xu, C., Chen, J.-A., Qiu, Z., Zhao, Q., Luo, J., Yang, L., … Shu, W. (2010). Ovotoxicity and PPAR-mediated aromatase downregulation in female Sprague-Dawley rats following combined oral exposure to benzo[a]pyrene and di-(2-ethylhexyl) phthalate. Toxicology Letters, 199(3), 323–32. doi:10.1016/j.toxlet.2010.09.015

Retrieved from https://aopkb.org/aopwiki/index.php/?oldid=26976 17beta-estradiol synthesis by ovarian granulosa cells, Reduction Directly Leads to Plasma 17beta-estradiol concentrations, Reduction

How Does This Key Event Relationship Work

Weight of Evidence

Biological Plausibility

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.

Empirical Support for Linkage

Include consideration of temporal concordance here

Fish

In multiple studies with aromatase inhibitors (e.g., fadrozole, prochloraz), significant reductions in ex vivo E2 production have been linked to, and shown to precede, reductions in circulating E2 concentrations (Villeneuve et al. 2009; Skolness et al. 2011). It is also notable that compensatory responses at the level of ex vivo steroid production (i.e., rate of E2 synthesis per unit mass of tissue) tend to precede recovery of plasma E2 concentrations following an initial insult (Villeneuve et al. 2009; Ankley et al. 2009a; Villeneuve et al. 2013). Ex vivo E2 production by ovary tissue collected from female fish exposed to 30 or 300 μg ketoconazole/L showed significant decreases prior to significant effects on plasma estradiol being observed (Ankley et al. 2012).

Mammals

MEHP /DEHP, mice, ex vivo DEHP (10 -100 μg/ml); MEHP (0.1 and 10 μg/ml) dose dependent reduction E2 production (Gupta et al., 2010) DEHP, rat, in vivo 300-600 mg/kg/day, dose dependent reduction of E2 plasma levels (Xu et al., 2010)

Evidence for rodent and human models is summarized in Table 1.

Compound Species Study Dose E2 production/levels Reference class type Phthalates rat ex 1500 mg/kg/day Reduced/increased E2 production in ovary (Laskey & Berman, 1993) (DEHP) vivo culture Phthalates rat in From 50 µM Reduced E2 production (concentration (Davis, Weaver, Gaines, & Heindel, 1994) (MEHP) vitro and time dependent in Granulosa cell) Phthalates rat in 100-200µM reduction E2 production (dose dependent) (Lovekamp & Davis, 2001)

24 Aop:7 (MEHP) vitro Phthalates rat in 300-600 mg/kg/day reduction E2 levels dose dependent (Xu et al., 2010), (DEHP) vivo Phthalates in IC(50)= 49- 138 µM (Reinsberg, Wegener-Toper, van der Ven, (MEHP) human vitro (dependent on the reduction E2 production (dose dependent) van der Ven, & Klingmueller, 2009) stimulant) Phthalates mice ex DEHP (10 -100 μg/ml); reduction E2 production (dose dependent) (Gupta et al., 2010) (MEHP/DEHP) vivo MEHP (0.1 and 10 μg/ml)

Table 1. Summary of the experimental data for decrease E2 production and decreased E2 levels. IC50- half maximal inhibitory concentration values reported if available, otherwise the concentration at which the effect was observed.

Uncertainties or Inconsistencies

Quantitative Understanding of the Linkage

At present we are unaware of any well established quantitative relationships between ex vivo E2 production (as an indirect measure of granulosa cell E2 synthesis) and plasma E2 concentrations.

There are considerable data available which might support the development of such a relationship. Additionally, there are a number of existing mathematical/computational models of ovarian steroidogenesis (Breen et al. 2013; Shoemaker et al. 2010) and/or physiologically-based pharmacokinetic models of the hypothalamic-pituitary-gonadal axis (e.g., (Li et al. 2011a) that may be adaptable to support a quantitative understanding of this linkage. • The Breen et al. 2013 model was developed based on in vivo time-course data for fathead minnow and incorporates prediction of compensatory responses resulting from feedback mechanisms operating as part of the hypothalamic-pituitary-gonadal axis. • The Shoemaker et al. 2010 model is chimeric and includes signaling pathways and aspects of transcriptional regulation based on a mixture of fish-specific and mammalian sources. • The Li et al. 2011 model is a PBPK-based model that was calibrated from data from fathead minnows, including controls and fish exposed to either 17alpha ethynylestradiol or 17beta trenbolone.

Evidence Supporting Taxonomic Applicability

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

Fish

Mouse

Rat

Human

For details see Table 1 and description in biological plausibility.

References

Fish

25 Aop:7 Norris DO. 2007. Vertebrate Endocrinology. Fourth ed. New York: Academic Press. Villeneuve DL, Mueller ND, Martinovic D, Makynen EA, Kahl MD, Jensen KM, et al. 2009. Direct effects, compensation, and recovery in female fathead minnows exposed to a model aromatase inhibitor. Environ Health Perspect 117(4): 624-631. Skolness SY, Durhan EJ, Garcia-Reyero N, Jensen KM, Kahl MD, Makynen EA, et al. 2011. Effects of a short-term exposure to the fungicide prochloraz on endocrine function and gene expression in female fathead minnows (Pimephales promelas). Aquat Toxicol 103(3-4): 170-178. Ankley GT, Bencic DC, Cavallin JE, Jensen KM, Kahl MD, Makynen EA, et al. 2009a. Dynamic nature of alterations in the endocrine system of fathead minnows exposed to the fungicide prochloraz. Toxicological sciences : an official journal of the Society of Toxicology 112(2): 344-353. Villeneuve DL, Breen M, Bencic DC, Cavallin JE, Jensen KM, Makynen EA, et al. 2013. Developing Predictive Approaches to Characterize Adaptive Responses of the Reproductive Endocrine Axis to Aromatase Inhibition: I. Data Generation in a Small Fish Model. Toxicological sciences : an official journal of the Society of Toxicology. Ankley GT, Cavallin JE, Durhan EJ, Jensen KM, Kahl MD, Makynen EA, et al. 2012. A time-course analysis of effects of the steroidogenesis inhibitor ketoconazole on components of the hypothalamic- pituitary-gonadal axis of fathead minnows. Aquatic toxicology 114-115: 88-95. Shoemaker JE, Gayen K, Garcia-Reyero N, Perkins EJ, Villeneuve DL, Liu L, et al. 2010. Fathead minnow steroidogenesis: in silico analyses reveals tradeoffs between nominal target efficacy and robustness to cross-talk. BMC systems biology 4: 89. Li Z, Kroll KJ, Jensen KM, Villeneuve DL, Ankley GT, Brian JV, et al. 2011a. A computational model of the hypothalamic: pituitary: gonadal axis in female fathead minnows (Pimephales promelas) exposed to 17alpha-ethynylestradiol and 17beta-trenbolone. BMC systems biology 5: 63.

Mammals

Davis, B J, R Weaver, L J Gaines, and J J Heindel. 1994. “Mono-(2-Ethylhexyl) Phthalate Suppresses Estradiol Production Independent of FSH-cAMP Stimulation in Rat Granulosa Cells.” Toxicology and Applied Pharmacology 128 (2) (October): 224–8. doi:10.1006/taap.1994.1201.

Gupta, Rupesh K, Jeffery M Singh, Tracie C Leslie, Sharon Meachum, Jodi a Flaws, and Humphrey H-C Yao. 2010. “Di-(2-Ethylhexyl) Phthalate and Mono-(2-Ethylhexyl) Phthalate Inhibit Growth and Reduce Estradiol Levels of Antral Follicles in Vitro.” Toxicology and Applied Pharmacology 242 (2) (January 15): 224–30. doi:10.1016/j.taap.2009.10.011.

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.

Lovekamp, T N, and B J Davis. 2001. “Mono-(2-Ethylhexyl) Phthalate Suppresses Aromatase Transcript Levels and Estradiol Production in Cultured Rat Granulosa Cells.” Toxicology and Applied Pharmacology 172 (3) (May 1): 217–24. doi:10.1006/taap.2001.9156.

Reinsberg, Jochen, Petra Wegener-Toper, Katrin van der Ven, Hans van der Ven, and Dietrich Klingmueller. 2009. “Effect of Mono-(2-Ethylhexyl) Phthalate on Steroid Production of Human Granulosa Cells.” Toxicology and Applied Pharmacology 239 (1) (August 15): 116–23. doi:10.1016/j.taap.2009.05.022.

Xu, Chuan, Ji-An Chen, Zhiqun Qiu, Qing Zhao, Jiaohua Luo, Lan Yang, Hui Zeng, et al. 2010. “Ovotoxicity and PPAR-Mediated Aromatase Downregulation in Female Sprague-Dawley Rats Following Combined Oral Exposure to Benzo[a]pyrene and Di-(2-Ethylhexyl) Phthalate.” Toxicology Letters 199 (3) (December 15): 323–32. doi:10.1016/j.toxlet.2010.09.015.

Retrieved from https://aopkb.org/aopwiki/index.php/?oldid=26983 Plasma 17beta-estradiol concentrations, Reduction Directly Leads to ovarian cycle, irregularities

26 Aop:7

How Does This Key Event Relationship Work

The development and the function of the female reproductive tract depends upon hormone concentrations and balance. Changes in this fine-tuned hormonal machinery may result in reproductive system dysfunction (e.g. menstrual cycle irregularities, impaired fertility, endometriosis, polycystic ovarian syndrome). Ovarian estrogen is the major component of negative and positive feedback for pituitary release of gonadotrophic hormones; therefore abnormal alterations in the estradiol levels result in irregularities of the ovarian cycle.

Weight of Evidence

Biological Plausibility

Estrogens are crucial for female and male fertility, as proved by the severe reproductive defects observed when their synthesis (Simpson, 2004), (Schomberg et al., 1999) are blocked. As a secreted hormone, estradiol modulates the structure and function of female reproductive tissues, such as the uterus and oviduct. Estradiol is also one of the principal determinants of pituitary neuron functioning and is critical in enabling these cells to exhibit fluctuating patterns of biosynthetic and secretory activity and to generate the preovulatory surge of luteinising hormone (LH) (Hillier, 1985). Estradiol also contributes to cyclical variations in sexual female behaviour. Suppression of estradiol levels results in increased serum follicle stimulating hormone (FSH) levels and an absence of LH surges necessary for ovulation (Everett, 1961), (Davis, Maronpot, & Heindel, 1994) and changes the length of the cycle (Eldridge et al., 1994).

Empirical Support for Linkage

A disruption of cycling caused by xenobiotic treatment can induce changes of length of the phases or cause an irregular pattern with cycles of extended duration and/or impact on ovulation. Measurements of serum estradiol reflect primarily the activity of the ovaries. As such, they are useful in the detection of baseline estrogen in irregularities of ovarian cycle. Suppressed levels of estradiol were found to impact on ovulation (Davis et al., 1994) and cycle duration (Hirosawa, Yano, Suzuki, & Sakamoto, 2006), (Eldridge et al., 1994), (Davis et al., 1994). Table 1 summarises common classes of chemicals shown to cause cycle irregularities through modulation of the hormonal balance.

KE: Reduced E2 Compound species KE: Ovarian cycle irregularities Reference class

Phthalates (DEHP) prolonged the estrous cycle, anovulation (3000 rat Reduced serum E2 mg/kg/day) (Davis et al., 1994)

irregular estrous cycles, prolongation of the Phthalates rat Reduced serum E2; FSH, pitutiary (Takai et al., 2009) (DEHP) FSH, LH cycle

Phthalates rat decreased E2 levels in estrus alters the estrous cycle (Laskey & Berman, 1993) (DEHP) increased levels in diestrus

Phthalates rat Reduced serum E2; FSH, pituitary continuous diestrus stage (Hirosawa et al., 2006) (DEHP) FSH and LH

reduced plasma E2 by 63% and cycle lengthening, and increase in days spent in Chlorotriazines 88% at does 100 or 300 mg/kg estrus and decrease in proestrus (100 or 300 (Eldridge et al., 1994) (trazine) rat ,respectively) mg/kg)

27 Aop:7 Phenols (4- Decreased number of cycles, diestrus was tert- rat Progesterone elevated extended (Laws, 2000) octylphenol)

Phthalates sheep Progesterone elevated Increased mean cycle length, Short cycles (Herreros et al., 2013) (DEHP) (dose-dependent)

(Schilling, K., Deckardt. K., Phthalates Gembardt, Chr., and (DEHP) rat Deficit in growing follicles and corpora lutea Hildebrand, 1999) dioxins rat Prolonged diestrus (Li, Johnson, & Rozman, 1995);

Table 1 Summary of the empirical evidence supporting KER. Estradiol (E2), luteinising hormone (LH) and follicle stimulating hormone (FSH).

Uncertainties or Inconsistencies

The impact on the ovarian cycle may result from defect in hypothalamic-pituitary-gonadal (HPG) axis signalling, other than by alteration of estradiol level. Table 1 shows some chemicals which impact on other hormones and cause irregularities of ovarian cycle.

Quantitative Understanding of the Linkage

Is it known how much change in the first event is needed to impact the second? Are there known modulators of the response-response relationships? Are there models or extrapolation approaches that help describe those relationships?

Evidence Supporting Taxonomic Applicability

Name Scientific Name Evidence Links

See Table 1.

References

Davis, B J, R R Maronpot, and J J Heindel. 1994. “Di-(2-Ethylhexyl) Phthalate Suppresses Estradiol and Ovulation in Cycling Rats.” Toxicology and Applied Pharmacology 128 (2) (October): 216–23. doi:10.1006/taap.1994.1200.

Eldridge, J C, D G Fleenor-Heyser, P C Extrom, L T Wetzel, C B Breckenridge, J H Gillis, L G Luempert, and J T Stevens. 1994. “Short-Term Effects of Chlorotriazines on Estrus in Female Sprague-Dawley and Fischer 344 Rats.” Journal of Toxicology and Environmental Health 43 (2) (October): 155–67. doi:10.1080/15287399409531912.

Everett, J. W. 1961. “The Mammalian Female Reproductive Cycle and Its Controlling Mechanisms.” Sex and Internal Secretions I. Herreros, Maria a, Antonio Gonzalez-Bulnes, Silvia Iñigo-Nuñez, Ignacio Contreras- Solis, Jose M Ros, and Teresa Encinas. 2013. “Toxicokinetics of di(2-Ethylhexyl) Phthalate (DEHP) and Its Effects on Luteal Function in Sheep.” Reproductive Biology 13 (1) (March): 66–74.

28 Aop:7 doi:10.1016/j.repbio.2013.01.177.

Hillier, S G. 1985. “Sex Steroid Metabolism and Follicular Development in the Ovary.” Oxford Reviews of Reproductive Biology 7 (January): 168–222.

Hirosawa, Narumi, Kazuyuki Yano, Yuko Suzuki, and Yasushi Sakamoto. 2006. “Endocrine Disrupting Effect of Di-(2-Ethylhexyl)phthalate on Female Rats and Proteome Analyses of Their Pituitaries.” Proteomics 6 (3) (February): 958–71. doi:10.1002/pmic.200401344.

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. Laws, S. C. 2000. “Estrogenic Activity of Octylphenol, Nonylphenol, Bisphenol A and Methoxychlor in Rats.” Toxicological Sciences 54 (1) (March 1): 154–167. doi:10.1093/toxsci/54.1.154.

Li, X, D C Johnson, and K K Rozman. 1995. “Effects of 2,3,7,8-Tetrachlorodibenzo-P-Dioxin (TCDD) on Estrous Cyclicity and Ovulation in Female Sprague-Dawley Rats.” Toxicology Letters 78 (3) (August): 219– 22.

Schilling, K., Deckardt. K., Gembardt, Chr., and Hildebrand, B. 1999. “Di-2-Ethylhexyl Phthalate – Two- Generation Reproduction Toxicity Range-Finding Study in Wistar Rats. Continuos Dietary Administration.”

Schomberg, D W, J F Couse, A Mukherjee, D B Lubahn, M Sar, K E Mayo, and K S Korach. 1999. “Targeted Disruption of the Estrogen Receptor-Alpha Gene in Female Mice: Characterization of Ovarian Responses and Phenotype in the Adult.” Endocrinology 140 (6) (June): 2733–44. doi:10.1210/endo.140.6.6823.

Simpson, Evan R. 2004. “Models of Aromatase Insufficiency.” Seminars in Reproductive Medicine 22 (1) (February): 25–30. doi:10.1055/s-2004-823024.

Takai, Ryo, Shuji Hayashi, Junpei Kiyokawa, Yoshika Iwata, Saori Matsuo, Masami Suzuki, Keiji Mizoguchi, Shuichi Chiba, and Toshiaki Deki. 2009. “Collaborative Work on Evaluation of Ovarian Toxicity. 10) Two- or Four-Week Repeated Dose Studies and Fertility Study of Di-(2-Ethylhexyl) Phthalate (DEHP) in Female Rats.” The Journal of Toxicological Sciences 34 Suppl 1 (I) (January): SP111–9.

Retrieved from https://aopkb.org/aopwiki/index.php/?oldid=27006 ovarian cycle, irregularities Indirectly Leads to Fertility, impaired

How Does This Key Event Relationship Work

The ovarian cycle irregularities impact on reproductive capacity of the females that may result in impaired fertility:

1. Irregular cycles may reflect impaired ovulation. Extended vaginal estrus usually indicates that the female cannot spontaneously achieve the ovulatory surge of LH (Huang and Meites, 1975). The persistence of regular vaginal cycles after treatment does not necessarily indicate that ovulation occurred, because luteal tissue may form in follicles that have not ruptured. However, that effect should be reflected in reduced fertility. Conversely, subtle alterations of cyclicity can occur at doses below those that alter fertility (Gray et al., 1989).

2. Persistent or constant vaginal cornification (or vaginal estrus) may result from one or several effects. Typically, in the adult, if the vaginal epithelium becomes cornified and remains so in response to toxicant exposure, it is the result of the agent’s estrogenic properties (i.e., DES or methoxychlor), or the ability of the agent to block ovulation. In the latter case, the follicle persists and endogenous estrogen levels bring about the persistent vaginal cornification. Histologically, the ovaries in persistent estrus will be atrophied

29 Aop:7 following exposure to estrogenic substances. In contrast, the ovaries of females in which ovulation has been blocked because of altered gonadotropin secretion will contain several large follicles and no corpora lutea. Females in constant estrus may be sexually receptive regardless of the mechanism responsible for this altered ovarian condition. However, if ovulation has been blocked by the treatment, an LH surge may be induced by mating (Brown-Grant et al., 1973; Smith, E.R. and Davidson, 1974) and a pregnancy or pseudopregnancy may ensue. The fertility of such matings is reduced (Cooper et al., 1994).

3. Significant delays in ovulation can result in increased embryonic abnormalities and pregnancy loss (Fugo and Butcher, 1966; Cooper et al., 1994).

4. Persistent diestrus indicates temporary or permanent cessation of follicular development and ovulation, and thus at least temporary infertility.

5. Prolonged vaginal diestrus, or anestrus, may be indicative of agents (e.g., polyaromatic hydrocarbons) that interfere with follicular development or deplete the pool of primordial follicles (Mattison and Nightingale, 1980) or agents such as atrazine that interrupt gonadotropin support of the ovary (Cooper et al., 1996). Pseudopregnancy is another altered endocrine state reflected by persistent diestrus. The ovaries of anestrous females are atrophic, with few primary follicles and an unstimulated uterus (Huang and Meites, 1975). Serum estradiol and progesterone are abnormally low.

6. Lengthening of the cycle may be a result of increased duration of either estrus or diestrus.

Weight of Evidence

Biological Plausibility

In females, normal reproductive function involves the appropriate interaction of central nervous system, anterior pituitary, oviducts, uterus, cervix and ovaries. During the reproductive years the ovary is the central organ in this axis. The functional unit within the ovary is the follicle which is composed of theca; granulosa cells and the oocyte. The somatic compartment synthesizes and secrets hormones (steroids and growth factors) necessary for the orchestration of the inter-relationship between the other parts of the reproductive tract and the central nervous system. Oestrus cycle is under strict hormonal control, therefore perturbations of hormonal balance lead to perturbations of normal cyclicity (change in number of cycles or duration of each phase) and/or ovulation problems leading to impaired female reproductive function.

Empirical Support for Linkage

Many chemicals are found to interfere with reproductive function in the female. This interference is commonly expressed as a change in normal morphology of the reproductive tract or in ovarian cycle irregularities (disturbance in the duration of particular phases of the estrous cycle and/or ovulation problems). Monitoring estrous cyclicity provides a means to identify alterations in reproductive functions which are mediated through nonestrogenic as well as estrogenic mechanisms (Blasberg, Langan, & Clark, 1997), (Clark, Blasberg, & Brandling-Bennett, 1998). Adverse alteration in the nonpregnant female reproductive system have been observed at dose levels below those that result in reduced fertility or produce other overt effects on pregnancy or pregnancy outcomes. A disruption of cycling caused by xenobiotic treatment can induce a persistent estrus, a persistent diestrus, an irregular pattern with cycles of extended duration and ovulation problems. Common classes of chemicals have been shown to cause cycle irregularities in rats, humans, and non-human primates. Examples include the polychlorinated biphenyls (PCBs) and dioxins, which are associated with such irregularities in rats and humans (e.g (Li, Johnson, & Rozman, 1995) (Meerts et al., 2004), (Chao, Wang, Lin, Lee, & Päpke, 2007) and various agricultural pesticides, including herbicides, fungicides, and fumigants for review see (Bhattacharya & Keating, 2012),(Bretveld, Thomas, Scheepers, Zielhuis, & Roeleveld, 2006).

30 Aop:7 Compound Species AO:ovarian cycle irregularities AO:Impaired fertility reference class

5-400 mg/kg/day females differed from the Phthalates number of live pups (P0) reduced (400 (Blystone et al., control in the relative amount of time spent in (DEHP) rat mg/kg/day) 2010) oestrous stages

Phthalates rat irregular estrous cycles (3,000 mg/kg/day) slight decline in pregnancy rate (3,000 (Takai et al., 2009) (DEHP) mg/kg/day)

(Lamb, Chapin, Phthalates Teague, Lawton, & (DEHP) mice dose-dependent decreases in fertility Reel, 1987)

(Schmidt, Schaedlich, Phthalates abortion rate of 100% in F0 dams (500- Fiandanese, Pocar, & (DEHP) mice No change mg/kg/day) Fischer, 2012).

dose-dependent effect on the duration of the estrous cycles shortening of the ovulatory cycles (Herreros, Gonzalez- Phthalates sheep (DEHP) due mainly to a reduction in the size and Bulnes, et al., 2013) lifespan of CL

Phthalates sheep No effect on ovulatory efficiency (Herreros, Encinas, et (DEHP) al., 2013)

18% and 21% decrease in live pups/litter F0 at 7500ppm and 10,000ppm respectively, no Phthalates rat No changes in F0, increase of cycle by 0.4 day (NTP, 2005) (DEHP) in F1 at 10,000ppm viable litters (F1 10,000 ppm ~643.95mg/kg/day)

(Schilling, K., 4-fold increase in females with stillborn pups Deckardt. K., Phthalates rat Deficit in growing follicles and corpora lutea in F0 at 9000ppm 2.1-fold Postimplantation Gembardt, Chr., and (DEHP) loss in F0 at 9000ppm Hildebrand, 1999)

Phthalates rat prolong the estrous cycle, anovulation (Davis, Maronpot, & (DEHP) Heindel, 1994)

Phthalates Reduced fertility and fecundity (Wolf et al., 1999)

organochlorine rat Decreased number of cycles, extended diestrus (Laws, 2000) (methoxychlor) and estrus

Table 1 Summary the empirical evidence supporting the KER.

It is known that exposure to 17-β-estradiol can disrupt the normal 4- to 5-day estrous cycle in adult female rats by inducing an extended period of diestrus consistent with pseudopregnancy within 5–7 days after the exposure (Gilmore & McDonald, 1969). This is due to the estrogen-dependent increase in prolactin that rescues ovarian corpora lutea and the subsequent synthesis and release of progesterone (Cooper, R. L., and Goldman, 1999). Significant evidence that the estrous cycle (or menstrual cycle in primates) has been disrupted should be considered an adverse effect (OECD, 2008).

Uncertainties or Inconsistencies

31 Aop:7 Chemicals may be found to interfere with reproductive function in the female. This interference is commonly expressed as a change in normal morphology of the reproductive tract or a disturbance in the duration of particular phases of the estrous cycle. However, menstrual cyclicity is affected by many parameters such as age, nutritional status, stress, exercise level, certain drugs, and the use of contraceptive measures that alter endocrine feedback. In nonpregnant females, repetitive occurrence of the four stages of the estrous cycle at regular, normal intervals suggests that neuroendocrine control of the cycle and ovarian responses to that control are normal. Even normal, control animals can show irregular cycles. However, a significant alteration compared with controls in the interval between occurrence of estrus for a treatment group is cause for concern. Generally, the cycle will be lengthened or the animals will become acyclic. Therefore changes in cyclicity should be interpreted with caution and not judged adverse without a comprehensive consideration of additional relevant endpoints in a weight-of-evidence approach.

Inconsistencies

Two generation studies by Tyl et al with Butyl benzyl phthalate (BBP) did not observe effects in F0 females on any parameters of estrous cycling, mating, or gestation. However, F1 females carrying F2 litters at and reduced number of total and live pups/litter at birth, with no effects on pre- or postnatal survival (Tyl et al., 2004).

Quantitative Understanding of the Linkage

Is it known how much change in the first event is needed to impact the second? Are there known modulators of the response-response relationships? Are there models or extrapolation approaches that help describe those relationships?

Evidence Supporting Taxonomic Applicability

Name Scientific Name Evidence Links

In many instances, human female reproductive toxicity of an agent is suspected based on studies performed in experimental animals. The neuroendocrinology, steroid biochemistry, and other physiologic events in the females of most small experimental species often used (mouse, rat, hamster) are similar in their susceptibility to disruption by toxicants (Massaro, 1997).

Although the assessment of the human ovarian cycle may have a variety of biomarkers distinct from those in rats, many of the underlying endocrine mechanisms associated with successful follicular development, ovulation, pregnancy, and parturition are homologous between the two (for review see (Bretveld et al., 2006). For this reason, a toxicant-induced perturbation of ovarian cycles in female rats suggest that a compound may function as a reproductive toxicant in human females.

Mice

environmental air pollution (Mohallem et al., 2005) phthalates (DEHP) abortion rate of 100% in F0 dams in the 500-mg/kg/day was observed, in F1 females found that the total number of F2 embryos (exposed to DEHP only as germ cells) was not impaired. However, in the 0.05- and 5-mg DEHP groups, 28% and 29%, respectively, of the blastocysts were degenerated, compared with 8% of controls (Schmidt et al., 2012). Lamb et al. studied fertility effects of DEHP in mice (both sexes) and found that DEHP caused dose- dependent decreases in fertility. DBP exposure resulted in a reduction in the numbers of litters per pair and of live pups per litter and in the proportion of pups born alive at the 1.0% amount, but not at lower dose levels. A crossover mating trial demonstrated that female mice, but not males, were affected by DBP, as shown by significant decreases in the percentage of fertile pairs, the number of live pups per litter, the proportion of pups born alive, and live pup weight. DHP in the diet resulted in dose-related

32 Aop:7 adverse effects on the numbers of litters per pair and of live pups per litter and proportion of pups born alive at 0.3, 0.6, and 1.2% DHP in the diet. A crossover mating study demonstrated that both sexes were affected. DEHP (at 0.1 and 0.3%) caused dose-dependent decreases in fertility and in the number and the proportion of pups born alive. A crossover mating trial showed that both sexes were affected by exposure to DEHP. These data demonstrate the ability of the continuous breeding protocol to discriminate the qualitative and quantitative reproductive effects of the more and less active congeners as well as the large differences in reproductive toxicity attributable to subtle changes in the alkyl substitution of phthalate esters (Lamb et al., 1987).

Rat phthalates (DEHP)

female rats exposed to a high dose of DEHP (3,000 mg/kg/day) had irregular estrous cycles and a slight decline in pregnancy rate (Takai et al., 2009). At 1,000 mg/kg bw/day over a period of 4 weeks did not disturb female fertility or early embryo development. There was significant evidence that 5, 15, 50, and 400 mg /kg/day females differed from the control females in the relative amount of time spent in oestrous stages, however no changes were revealed in the number of females with regular cycles, cycle length, number of cycles, and in number of cycling females across the dose groups as compared to the control females The litter size (number of live pups) produced by the P0 generation was significantly reduced in the 400 mg/kg/day dose group (Blystone et al., 2010).

Human

Studies showing a correlation between decreased fertility and;

professional activity (Olsen, 1994) phthalates (DEHP) In occupationally exposed women to high concentration of phthalates exhibit hypoestrogenic anovulary cycles and was associated with decreased pregnancy rate and higher miscarriage rates (Aldyreva,M.V.,Klimove,T.S.,Iziumova,A.S.,Timofeevskaia,L.A., 1975). smoking (Hull, North, Taylor, Farrow, & Ford, 2000) the use of certain drugs or radiation exposure (Dobson & Felton, 1983)

For the taxonomic applicability see also the Table 1.

References

Aldyreva,M.V.,Klimove,T.S.,Iziumova,A.S.,Timofeevskaia,L.A. (1975). The effect of phthalate plasticizers on the generative function. Gig.Tr.Prof.Zabol., (19), 25–29.

Bhattacharya, P., & Keating, A. F. (2012). Impact of environmental exposures on ovarian function and role of xenobiotic metabolism during ovotoxicity. Toxicology and Applied Pharmacology, 261(3), 227–35. doi:10.1016/j.taap.2012.04.009

Blasberg, M. E., Langan, C. J., & Clark, A. S. (1997). The effects of 17 alpha-methyltestosterone, methandrostenolone, and nandrolone decanoate on the rat estrous cycle. Physiology & Behavior, 61(2), 265–72.

Blystone, C. R., Kissling, G. E., Bishop, J. B., Chapin, R. E., Wolfe, G. W., & Foster, P. M. D. (2010). Determination of the di-(2-ethylhexyl) phthalate NOAEL for reproductive development in the rat: importance of the retention of extra animals to adulthood. Toxicological Sciences : An Official Journal of the Society of Toxicology, 116(2), 640–6. doi:10.1093/toxsci/kfq147

Bretveld, R. W., Thomas, C. M. G., Scheepers, P. T. J., Zielhuis, G. A., & Roeleveld, N. (2006). Pesticide exposure: the hormonal function of the female reproductive system disrupted? Reproductive Biology and Endocrinology : RB&E, 4(1), 30. doi:10.1186/1477-7827-4-30

Chao, H.-R., Wang, S.-L., Lin, L.-Y., Lee, W.-J., & Päpke, O. (2007). Placental transfer of polychlorinated

33 Aop:7 dibenzo-p-dioxins, dibenzofurans, and biphenyls in Taiwanese mothers in relation to menstrual cycle characteristics. Food and Chemical Toxicology : An International Journal Published for the British Industrial Biological Research Association, 45(2), 259–65. doi:10.1016/j.fct.2006.07.032

Clark, A. S., Blasberg, M. E., & Brandling-Bennett, E. M. (1998). Stanozolol, oxymetholone, and testosterone cypionate effects on the rat estrous cycle. Physiology & Behavior, 63(2), 287–95.

Cooper, R. L., and Goldman, J. M. (1999). Vaginal cytology. In An Evaluation and Interpretation of Reproductive Endpoints for Human Health Risk Assessment. Washington. Davis, B. J., Maronpot, R. R., & Heindel, J. J. (1994). Di-(2-ethylhexyl) phthalate suppresses estradiol and ovulation in cycling rats. Toxicology and Applied Pharmacology, 128(2), 216–23. doi:10.1006/taap.1994.1200

Dobson, R. L., & Felton, J. S. (1983). Female germ cell loss from radiation and chemical exposures. American Journal of Industrial Medicine, 4(1-2), 175–90.

Gilmore, D. P., & McDonald, P. G. (1969). Induction of prolonged diestrus in the rat by a low level of estrogen. Endocrinology, 85(5), 946–8. doi:10.1210/endo-85-5-946 Herreros, M. A., Encinas, T., Torres- Rovira, L., Garcia-Fernandez, R. A., Flores, J. M., Ros, J. M., & Gonzalez-Bulnes, A. (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), 1141–9. doi:10.1016/j.etap.2013.09.020

Herreros, M. A., Gonzalez-Bulnes, A., Iñigo-Nuñez, S., Contreras-Solis, I., Ros, J. M., & Encinas, T. (2013). Toxicokinetics of di(2-ethylhexyl) phthalate (DEHP) and its effects on luteal function in sheep. Reproductive Biology, 13(1), 66–74. doi:10.1016/j.repbio.2013.01.177

Hull, M. G., North, K., Taylor, H., Farrow, A., & Ford, W. C. (2000). Delayed conception and active and passive smoking. The Avon Longitudinal Study of Pregnancy and Childhood Study Team. Fertility and Sterility, 74(4), 725–33.

Lamb, J. C., Chapin, R. E., Teague, J., Lawton, A. D., & Reel, J. R. (1987). Reproductive effects of four phthalic acid esters in the mouse. Toxicology and Applied Pharmacology, 88(2), 255–69.

Laws, S. C. (2000). Estrogenic Activity of Octylphenol, Nonylphenol, Bisphenol A and Methoxychlor in Rats. Toxicological Sciences, 54(1), 154–167. doi:10.1093/toxsci/54.1.154

Li, X., Johnson, D. C., & Rozman, K. K. (1995). Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on estrous cyclicity and ovulation in female Sprague-Dawley rats. Toxicology Letters, 78(3), 219–22.

Massaro, E. J. (Ed.). (1997). Handbook of Human Toxicology, Volume 236. Taylor & Francis.

Meerts, I. A. T. M., Hoving, S., van den Berg, J. H. J., Weijers, B. M., Swarts, H. J., van der Beek, E. M., … Brouwer, A. (2004). Effects of in utero exposure to 4-hydroxy-2,3,3’,4',5-pentachlorobiphenyl (4-OH- CB107) on developmental landmarks, steroid hormone levels, and female estrous cyclicity in rats. Toxicological Sciences : An Official Journal of the Society of Toxicology, 82(1), 259–67. doi:10.1093/toxsci/kfh251

Mohallem, S. V., de Araújo Lobo, D. J., Pesquero, C. R., Assunção, J. V., de Andre, P. A., Saldiva, P. H. N., & Dolhnikoff, M. (2005). Decreased fertility in mice exposed to environmental air pollution in the city of Sao Paulo. Environmental Research, 98(2), 196–202. doi:10.1016/j.envres.2004.08.007

NTP. (2005). Multigenerational Reproductive Assessment by Continuous Breeding when Diethylhexylphthalate (CAS 117-81-7).

OECD. (2008). No 43: Guidance document on mammalian reproductive toxicity testing and assessment.

Olsen, J. (1994). Is human fecundity declining--and does occupational exposures play a role in such a decline if it exists? Scandinavian Journal of Work, Environment & Health, 20 Spec No, 72–7.

34 Aop:7 Schilling, K., Deckardt. K., Gembardt, Chr., and Hildebrand, B. (1999). Di-2-ethylhexyl phthalate – two- generation reproduction toxicity range-finding study in Wistar rats. Continuos dietary administration.

Schmidt, J.-S., Schaedlich, K., Fiandanese, N., Pocar, P., & Fischer, B. (2012). Effects of di(2-ethylhexyl) phthalate (DEHP) on female fertility and adipogenesis in C3H/N mice. Environmental Health Perspectives, 120(8), 1123–9. doi:10.1289/ehp.1104016

Takai, R., Hayashi, S., Kiyokawa, J., Iwata, Y., Matsuo, S., Suzuki, M., … Deki, T. (2009). Collaborative work on evaluation of ovarian toxicity. 10) Two- or four-week repeated dose studies and fertility study of di-(2-ethylhexyl) phthalate (DEHP) in female rats. The Journal of Toxicological Sciences, 34 Suppl 1(I), SP111–9.

Tyl, R. W., Myers, C. B., Marr, M. C., Fail, P. a, Seely, J. C., Brine, D. R., … Butala, J. H. (2004). Reproductive toxicity evaluation of dietary butyl benzyl phthalate (BBP) in rats. Reproductive Toxicology (Elmsford, N.Y.), 18(2), 241–64. doi:10.1016/j.reprotox.2003.10.006

Wolf, C., Lambright, C., Mann, P., Price, M., Cooper, R. L., Ostby, J., & Gray, L. E. (1999). Administration of potentially antiandrogenic pesticides (procymidone, linuron, iprodione, chlozolinate, p,p’-DDE, and ketoconazole) and toxic substances (dibutyl- and diethylhexyl phthalate, PCB 169, and ethane dimethane sulphonate) during sexual differen. Toxicology and Industrial Health, 15(1-2), 94–118. doi:10.1177/074823379901500109

Retrieved from https://aopkb.org/aopwiki/index.php/?oldid=27009

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.

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

35 Aop:7 PPARγ-null mutation is embryonically lethal (Barak et al. 1999). Organ targeted knock-out studies would more precisely inform on the mechanistic involvement of the PPAR family.

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 and 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:

36 Aop:7

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

37 Aop:7 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

Event Description Triggers Weight of Evidence

PPAR gamma, Activation Indirectly Aromatase (Cyp19a1), reduction in Weak Leads to ovarian granulosa cells Aromatase (Cyp19a1), reduction in Directly 17beta-estradiol synthesis by ovarian Moderate ovarian granulosa cells Leads to granulosa cells, Reduction 17beta-estradiol synthesis by ovarian Directly Plasma 17beta-estradiol Strong granulosa cells, Reduction Leads to concentrations, Reduction Plasma 17beta-estradiol concentrations, Directly ovarian cycle, irregularities Strong Reduction Leads to ovarian cycle, irregularities Indirectly Fertility, impaired Moderate Leads to

KERs Biological level of Empirical Support level of Inconsistencies/Uncertainties plausibility confidence confidence Dose-response Temporality Incidence

There is functional occurrence PPARγ, relationship between KEup occurs of the key Activation => PPARγ activation and at lower events at reduction in dose than similar dose Aromatase aromatase levels. KEdown(dose and time Moderate Weak Limited data, for details see KER (Cyp19a1), Several mechanisms response point pages reduction in have been concordance) Support for ovarian investigated; however solid granulosa cells there is no established temporal consensus. relationship is lacking

Aromatase Within the ovary, (Cyp19a1), occurrence aromatase expression of the key reduction in and activity is ovarian events at primarily localized in KEup occurs similar dose granulosa cells the granulosa cells. at lower => and time Therefore, changes in Moderate dose than point moderate Limited data ovarian aromatase can KEdown(dose 17beta- Support for generally be assumed response estradiol solid to directly impact E2 concordance) synthesis by temporal synthesis by the ovarian relationship granulosa cells. granulosa cells is lacking

17beta- The gonads are estradiol generally considered

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synthesis by occurrence the major source of of the key ovarian circulating estrogens, granulosa events at consequently, if KEs occur at similar dose cells, estradiol synthesis by Strong moderate Limited data Reduction => similar dose Support for ovarian granulosa cells levels solid is reduced, plasma E2 Plasma temporal concentrations would 17beta- relationship be expected to estradiol is lacking decrease. concentrations

Alterations in relationships among occurrence Plasma the hypothalamic, of the key 17beta- pituitary, and ovarian events at estradiol components of the similar dose reproductive axis can KEs occur at and time concentrations, Strong moderate Reduction => have marked effects similar dose point on cyclicity. A levels Support for ovarian cycle toxicological insult to solid irregularities any one of these sites temporal can disrupt the cycle relationship and block ovulation. is lacking

occurrence A sequential of the key progression of events at ovarian cycle interrelated KEs occur at similar dose irregularities physiological and similar dose and with => behavioural cycles Strong levels temporal moderate underlines the relationship Fertility, female's fertility and Support for impaired successful production solid of offspring. temporal relationship is lacking.

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 Support for Essentiality PPAR gamma, Activation Weak

Key Event Support for Essentiality 17beta-estradiol synthesis by ovarian granulosa cells, Reduction Strong Aromatase (Cyp19a1), reduction in ovarian granulosa cells Moderate Plasma 17beta-estradiol concentrations, Reduction Strong

KRs WoE Essentiality - KEs level of confidence

PPAR gamma, Activation PPARγ activation was found to indirectly alter the expression of aromatase weak

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Aromatase Aromatase is the cytochrome P450 enzyme complex responsible for the conversion of androgens to estrogens (Cyp19a1), during steroidogenesis which is a key reaction in the sex differentiation in vertebrates. Alterations in the moderate reduction in amount of aromatase present or in the catalytic activity of the enzyme will alter the levels of estrogens in ovarian tissues and dramatically disrupt estrogen hormone action. granulosa cells

17beta-estradiol While both brain and adrenal tissue are capable of synthesizing estradiol, the gonads are generally considered synthesis by the major source of circulating estrogens in vertebrates, including fish (Norris 2007). Consequently, if estradiol strong ovarian synthesis by ovarian granulosa cells is reduced, plasma E2 concentrations would be expected to decrease granulosa cells unless there are concurrent reductions in the rate of E2 catabolism.

Plasma 17beta- estradiol Estrogens are crucial for female fertility, as proved by the severe reproductive defects observed when their strong concentrations, synthesis is blocked. Reduction

ovarian cycle A sequential progression of interrelated physiological and behavioural cycles underlines the female reproductive strong irregularities function.

Fertility, Impaired Fertility is the endpoint of reproductive toxicity strong impaired

Quantitative Considerations

Event Description Triggers Quantitative Understanding

PPAR gamma, Activation Indirectly Aromatase (Cyp19a1), reduction in Leads to ovarian granulosa cells Aromatase (Cyp19a1), reduction in Directly 17beta-estradiol synthesis by ovarian granulosa cells Leads to ovarian granulosa cells, Reduction 17beta-estradiol synthesis by ovarian Directly Plasma 17beta-estradiol granulosa cells, Reduction Leads to concentrations, Reduction Plasma 17beta-estradiol Directly ovarian cycle, irregularities concentrations, Reduction Leads to ovarian cycle, irregularities Indirectly Fertility, impaired Leads to

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 incorporates human mechanistic and epidemiological data. The experimental animal data are assumed relevant for consideration of human risk.

Sex Applicability

40 Aop:7 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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