CHEBI:1646917beta-estradiolPR:000006100aromataseGO:0006703estrogen biosynthetic processD005298fertilityGO:0042698ovulation cycleGO:0070330aromatase activityGO:0009299mRNA transcription2decreased9disrupted10116ratWCS_9606humanWCS_90988fathead minnow8078Fundulus heteroclitus10090mouse10095mice7955zebra fishReduction, Plasma 17beta-estradiol concentrationsReduction, Plasma 17beta-estradiol concentrationsOrgan<p>Estradiol synthesized by the gonads is transported to other tissues via blood circulation. The gonads are generally considered to be the primary source of estrogens in systemic circulation.</p>
<p>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.</p>
<p>Given that there are numerous genes, like those coding for vertebrate vitellogenins, choriongenins, cyp19a1b, etc. which are known to be regulated by estrogen response elements, targeted qPCR or proteomic analysis of appropriate targets could also be used as an indirect measure of reduced circulating estrogen concentrations. However, further support for the specificity of the individual gene targets for estrogen-dependent regulation should be established in order to support their use.</p>
<p>A line of transgenic zebrafish employing green fluorescence protein under control of estrogen response elements could also be used to provide direct evidence of altered estrogen, with decreased GFP signal in estrogen responsive tissues like liver, ovary, pituitary, and brain indicating a reduction in circulating estrogens (Gorelick and Halpern 2011).</p>
<p>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.</p>
UBERON:0001969blood plasmaNot SpecifiedUnspecificHighAdultHighHighHighHigh<ul>
<li>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.</li>
<li>Baker ME. 2011. Origin and diversification of steroids: co-evolution of enzymes and nuclear receptors. Molecular and cellular endocrinology 334(1-2): 14-20.</li>
<li>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.</li>
<li>Gorelick DA, Halpern ME. Visualization of estrogen receptor transcriptional activation in zebrafish. Endocrinology. 2011 Jul;152(7):2690-703. doi: 10.1210/en.2010-1257. Epub 2011 May 3. PubMed PMID: 21540282</li>
</ul>
2016-11-29T18:41:232017-09-26T11:30:57Reduction, 17beta-estradiol synthesis by ovarian granulosa cellsReduction, 17beta-estradiol synthesis by ovarian granulosa cellsCellular<p>Like all steroids, estradiol is a cholesterol derivative. Estradiol synthesis in ovary is mediated by a number of enzyme catalyzed reactions involving cyp11 (cholesterol side chain cleavage enzyme), cyp 17 (17alpha-hydroxylase/17,20-lyase), 3beta hydroxysteroid dehyrogenase, 17beta hydroxysteroid dehydrogenase, and cyp19 (aromatase). Among those enzyme catalyzed reactions, conversion of testosterone to estradiol, catalyzed by aromatase, is considered to be rate limiting for estradiol synthesis. 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). Reactions involved in synthesis of C-19 androgens are primarily localized in the theca cells and C-19 androgens diffuse from the theca into granulosa cells where aromatase can catalyze their conversion to C-18 estrogens.</p>
<p>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 radioimmunoassay (e.g., Jensen et al. 2001), ELISA, or analytical methods such as LC-MS (e.g., Owen et al. 2014).</p>
<p>OECD TG 456 <a class="external text" href="http://www.oecd-ilibrary.org/environment/test-no-456-h295r-steroidogenesis-assay_9789264122642-en" rel="nofollow" target="_blank">(OECD 2011)</a> is the validated test guideline for an in vitro screen for chemical effects on steroidogenesis, specifically the production of 17ß-estradiol (E2) and testosterone (T).</p>
<p>The synthesis of E2 can be measured in vitro cultured ovarian cells. The methods for culturing mammalian ovarian cells can be found in the Database Service on Alternative Methods to animal experimentation (DB-ALM): Culture of Human Cumulus Granulosa Cells <a class="external text" href="http://ecvam-dbalm.jrc.ec.europa.eu/beta/index.cfm/methodsAndProtocols/index?id_prot=266" rel="nofollow" target="_blank">(EURL ECVAM Protocol No. 92)</a>, Granulosa and Theca Cell Culture Systems <a class="external text" href="http://ecvam-dbalm.jrc.ec.europa.eu/beta/index.cfm/methodsAndProtocols/index?id_met=535" rel="nofollow" target="_blank">(EURL ECVAM Method Summary No. 92)</a>.</p>
<p>Key enzymes needed to synthesize 17β-estradiol first appear in the common ancestor of amphioxus and vertebrates (Markov et al. 2009; Baker 2011). Consequently, it is plausible that this key event is applicable to most vertebrates. This key event is not applicable to invertebrates, which lack the enzymes required to synthesize 17ß-estradiol.</p>
<p> </p>
CL:0000501granulosa cellHighFemaleHighAdult, reproductively matureHighHigh<ul>
<li>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.</li>
<li>Baker ME. 2011. Origin and diversification of steroids: co-evolution of enzymes and nuclear receptors. Molecular and cellular endocrinology 334(1-2): 14-20.</li>
<li>EURL ECVAM Method Summary no 92. Granulosa and Theca Cell Culture Systems - Summary</li>
<li>EURL ECVAM Protocol no 92 Culture of Human Cumulus Granulosa Cells. Primary cell culture method. Contact Person: Dr. Mahadevan Maha M.</li>
<li>Havelock JC, Rainey WE, Carr BR. 2004. Ovarian granulosa cell lines. Molecular and cellular endocrinology 228(1-2): 67-78.</li>
<li>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.</li>
<li>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.</li>
<li>Norris DO. 2007. Vertebrate Endocrinology. Fourth ed. New York: Academic Press.</li>
<li>OECD (2011), Test No. 456: H295R Steroidogenesis Assay, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris.DOI: <a href="http://dx.doi.org/10.1787/9789264122642-en" target="_blank">http://dx.doi.org/10.1787/9789264122642-en</a></li>
<li>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.</li>
<li>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.</li>
<li>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.</li>
<li>Yaron Z. 1995. Endocrine control of gametogenesis and spawning induction in the carp. Aquaculture 129: 49-73.</li>
</ul>
2016-11-29T18:41:222017-09-16T10:14:21impaired, Fertilityimpaired, FertilityIndividual<p><strong>Biological state</strong></p>
<p>capability to produce offspring</p>
<p><strong>Biological compartments</strong></p>
<p>System</p>
<p><strong>General role in biology</strong></p>
<p>Fertility is the capacity to conceive or induce conception. Impairment of fertility represents disorders of male or female reproductive functions or capacity.</p>
<p>As a measure, fertility rate, is the number of offspring born per mating pair, individual or population.</p>
<p><strong>Plausible domain of applicability</strong></p>
<p><strong><em>Taxonomic applicability</em>: </strong>The impaired fertility may also have relevance for fish, mammals, amphibians, reptiles, birds and and invertebrates with sexual reproduction.</p>
<p><strong><em>Life stage applicability</em></strong>: The impaired fertility can be measured at juveniles and adults.</p>
<p><em><strong>Sex applicability</strong></em>: The impaired fertility can be measured in both male and female species. </p>
HighAdult, reproductively matureHighJuvenileHighAdultsHighHighHigh<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">OECD (2001), <em>Test No. 416: Two-Generation Reproduction Toxicity</em>, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris, <a href="https://doi.org/10.1787/9789264070868-en">https://doi.org/10.1787/9789264070868-en</a>.</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">OECD (2018), <em>Test No. 443: Extended One-Generation Reproductive Toxicity Study</em>, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris, <a href="https://doi.org/10.1787/9789264185371-en">https://doi.org/10.1787/9789264185371-en</a>.</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">OECD (2018), <em>Test No. 414: Prenatal Developmental Toxicity Study</em>, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris, <a href="https://doi.org/10.1787/9789264070820-en">https://doi.org/10.1787/9789264070820-en</a>.</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">OECD (2018), "Reproduction/Developmental Toxicity Screening Test (OECD TG 421) and Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (OECD TG 422)", in <em>Revised Guidance Document 150 on Standardised Test Guidelines for Evaluating Chemicals for Endocrine Disruption</em>, OECD Publishing, Paris, <a href="https://doi.org/10.1787/9789264304741-25-en">https://doi.org/10.1787/9789264304741-25-en</a>.</span></span></p>
2016-11-29T18:41:242023-09-14T12:10:12irregularities, ovarian cycleirregularities, ovarian cycleIndividual<p><b>Biological state</b>
</p><p>The female ovarian cycle is the result of a balanced cooperation between several organs and is determined 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
</p><p>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
</p><p>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).
</p><p><b>General role in biology</b>
</p><p>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.
</p><p><em>
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?
</em>
</p><p><br />
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 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).
</p><p>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).
</p><p>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).
</p><p>Monitoring of oestrus cyclicity is included in OECD test guidelines (Test No. 407: Repeated Dose 28-day Oral Toxicity Study in Rodents, 2008) <a rel="nofollow" target="_blank" class="external autonumber" href="http://www.oecd-ilibrary.org/environment/test-no-407-repeated-dose-28-day-oral-toxicity-study-in-rodents_9789264070684-en">[1]</a>, (Test No. 416: Two-Generation Reproduction Toxicity, 2001)<a rel="nofollow" target="_blank" class="external autonumber" href="http://www.oecd-ilibrary.org/environment/test-no-416-two-generation-reproduction-toxicity_9789264070868-en">[2]</a> and (Test No. 443: Extended One-Generation Reproductive Toxicity Study, 2012) <a rel="nofollow" target="_blank" class="external autonumber" href="http://www.oecd-ilibrary.org/environment/test-no-443-extended-one-generation-reproductive-toxicity-study_9789264122550-en">[3]</a>and in USA EPA OCSPP 890.1450.
</p><p>In vitro testing
</p><p>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).
</p><p>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).
</p>LowModerate<p>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
</p><p>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.
</p><p>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
</p><p>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
</p><p>OECD. (2008). No 43: Guidance document on mammalian reproductive toxicity testing and assessment.
</p><p>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
</p><p>Ryan, K. J. (1982). Biochemistry of aromatase: significance to female reproductive physiology. Cancer Research, 42(8 Suppl), 3342s–3344s.
</p><p>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
</p><p>Test No. 407: Repeated Dose 28-day Oral Toxicity Study in Rodents. (2008). OECD Publishing. doi:10.1787/9789264070684-en
</p><p>Test No. 416: Two-Generation Reproduction Toxicity. (2001). OECD Publishing. doi:10.1787/9789264070868-en
</p><p>Test No. 443: Extended One-Generation Reproductive Toxicity Study. (2012). OECD Publishing. doi:10.1787/9789264185371-en
</p><p>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
</p>2016-11-29T18:41:242016-11-29T19:09:29reduction in ovarian granulosa cells, Aromatase (Cyp19a1)reduction in ovarian granulosa cells, Aromatase (Cyp19a1)Cellular<p><strong>Biological state</strong></p>
<p>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).</p>
<p><strong>Biological compartments</strong></p>
<p>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).</p>
<p><strong>General role in biology</strong></p>
<p>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).</p>
<p>Aromatase levels can be assayed by standard methods for assessment of gene expression levels like: q-PCR or direct protein levels: Western blot or ELISA. The level of aromatase as well as other steroidogenic protein can be measured in vitro cultured ovarian cells. The methods for culturing ovarian cells can be found in the Database Service on Alternative Methods to animal experimentation (DB-ALM): Culture of Human Cumulus Granulosa Cells <a class="external autonumber" href="http://ecvam-dbalm.jrc.ec.europa.eu/beta/index.cfm/methodsAndProtocols/index?id_prot=266" rel="nofollow" target="_blank">[1]</a>, Granulosa and Theca Cell Culture Systems <a class="external autonumber" href="http://ecvam-dbalm.jrc.ec.europa.eu/beta/index.cfm/methodsAndProtocols/index?id_met=535" rel="nofollow" target="_blank">[2]</a>.</p>
<p>However, in early life stage zebrafish embryos are not practical to measure tissue-specific expression. In those cases, whole body measurements can be used as an indicator of this key event.</p>
<p>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).</p>
CL:0000501granulosa cellModerateHighHigh<p>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</p>
<p>Hillier, S. G. (1985). Sex steroid metabolism and follicular development in the ovary. Oxford Reviews of Reproductive Biology, 7, 168–222.</p>
<p>Ryan, K. J. (1982). Biochemistry of aromatase: significance to female reproductive physiology. Cancer Research, 42(8 Suppl), 3342s–3344s.</p>
<p>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</p>
<p>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</p>
<p>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</p>
<p>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</p>
2016-11-29T18:41:242021-05-28T07:49:031758fc80-c77f-4d49-86aa-fc67a889503ad1c7087e-6d2f-499b-86f0-d0d9bff77f2d<p>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.
</p><p>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).
</p><p>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.
</p>
<table border="1" style="border-collapse:collapse;font-size:75%">
<tr>
<td>
<p><b>Compound class</b>
</p>
</td>
<td>
<p><b>species</b>
</p>
</td>
<td>
<p><b>KE: Reduced E2</b>
</p><p><br />
</p>
</td>
<td>
<p><b>KE: Ovarian cycle irregularities</b>
</p>
</td>
<td>
<p><b>Reference</b>
</p>
</td></tr>
<tr>
<td>
<p>Phthalates (DEHP)
</p><p><br />
</p>
</td>
<td>
<p>rat
</p>
</td>
<td>
<p>Reduced serum E2
</p>
</td>
<td>
<p>prolonged the estrous cycle, anovulation (3000 mg/kg/day)
</p>
</td>
<td>
<p>(Davis et al., 1994)
</p>
</td></tr>
<tr>
<td>
<p>Phthalates (DEHP)
</p>
</td>
<td>
<p>rat
</p>
</td>
<td>
<p>Reduced serum E2; FSH, pitutiary FSH, LH
</p>
</td>
<td>
<p>irregular estrous cycles, prolongation of the cycle
</p>
</td>
<td>
<p>(Takai et al., 2009)
</p>
</td></tr>
<tr>
<td>
<p>Phthalates (DEHP)
</p>
</td>
<td>
<p>rat
</p>
</td>
<td>
<p>decreased E2 levels in estrus increased levels in diestrus
</p>
</td>
<td>
<p>alters the estrous cycle
</p>
</td>
<td>
<p>(Laskey & Berman, 1993)
</p>
</td></tr>
<tr>
<td>
<p>Phthalates (DEHP)
</p>
</td>
<td>
<p>rat
</p>
</td>
<td>
<p>Reduced serum E2; FSH, pituitary FSH and LH
</p>
</td>
<td>
<p>continuous diestrus stage
</p>
</td>
<td>
<p>(Hirosawa et al., 2006)
</p>
</td></tr>
<tr>
<td>
<p>Chlorotriazines (trazine)
</p>
</td>
<td>
<p>rat
</p>
</td>
<td>
<p>reduced plasma E2 by 63% and 88% at does 100 or 300 mg/kg ,respectively)
</p>
</td>
<td>
<p>cycle lengthening, and increase in days spent in estrus and decrease in proestrus (100 or 300 mg/kg)
</p>
</td>
<td>
<p>(Eldridge et al., 1994)
</p>
</td></tr>
<tr>
<td>
<p>Phenols (4-tert-octylphenol)
</p>
</td>
<td>
<p>rat
</p>
</td>
<td>
<p>Progesterone elevated
</p>
</td>
<td>
<p>Decreased number of cycles, diestrus was extended
</p>
</td>
<td>
<p>(Laws, 2000)
</p>
</td></tr>
<tr>
<td>
<p>Phthalates (DEHP)
</p>
</td>
<td>
<p>sheep
</p>
</td>
<td>
<p>Progesterone elevated
</p>
</td>
<td>
<p>Increased mean cycle length, Short cycles (dose-dependent)
</p>
</td>
<td>
<p>(Herreros et al., 2013)
</p>
</td></tr>
<tr>
<td>
<p>Phthalates (DEHP)
</p>
</td>
<td>
<p>rat
</p>
</td>
<td>
<p><br />
</p>
</td>
<td>
<p>Deficit in growing follicles and corpora lutea
</p>
</td>
<td>
<p>(Schilling, K., Deckardt. K., Gembardt, Chr., and Hildebrand, 1999)
</p>
</td></tr>
<tr>
<td>
<p>dioxins
</p>
</td>
<td>
<p>rat
</p>
</td>
<td>
<p><br />
</p>
</td>
<td>
<p>Prolonged diestrus
</p>
</td>
<td>
<p>(Li, Johnson, & Rozman, 1995);
</p>
</td></tr></table>
<p><br />
</p><p><br />
</p><p>Table 1 Summary of the empirical evidence supporting KER. Estradiol (E2), luteinising hormone (LH) and follicle stimulating hormone (FSH).
</p><p>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.
</p><p>See Table 1.
</p><p>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.
</p><p>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.
</p><p>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. doi:10.1016/j.repbio.2013.01.177.
</p><p>Hillier, S G. 1985. “Sex Steroid Metabolism and Follicular Development in the Ovary.” Oxford Reviews of Reproductive Biology 7 (January): 168–222.
</p><p>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.
</p><p>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.
</p><p>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.
</p><p>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.”
</p><p>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.
</p><p>Simpson, Evan R. 2004. “Models of Aromatase Insufficiency.” Seminars in Reproductive Medicine 22 (1) (February): 25–30. doi:10.1055/s-2004-823024.
</p><p>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.
</p>2016-11-29T18:41:342016-12-03T16:37:566dd9c937-ac80-416a-a42e-82d27d9267f31758fc80-c77f-4d49-86aa-fc67a889503a<p>See plausibility, below.</p>
<p>Updated 03/20/2017.</p>
<p>While brain, interrenal, adipose, and breast tissue (in mammals) 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. Synthesis in other tissues generally plays a paracrine role only, thus the contribution of other tissues to plasma E2 concentrations can generally be considered negligible.</p>
<h2> </h2>
<p><em>Include consideration of temporal concordance here </em></p>
<p><strong>Fish</strong></p>
<ul>
<li>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).</li>
<li>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).</li>
<li>Ekman et al. (2011) reported significant reductions in ex vivo E2 production and plasma E2 concentrations in female fathead minnows exposed to 0.05 ug/L 17ß-trenbolone. The effect on plasma E2 was observed at an earlier time point (24 h, versus 48 h for E2 production.</li>
<li>Rutherford et al. (2015) reported significant reductions in both E2 production and circulating E2 concentrations in female Fundulus heteroclitus exposed to 5alpha-dihydrotestosterone or 17alpha-methyltestosterone for 14 d. The effects were equipotent in the case of 17alpha-methyltestosterone, but in the case of 5alpha-dihyrotestosterone, the effect on plasma E2 could be detected at a lower dose (10 ug/L) than that at which a significant effect on E2 production was detected (100 ug/L).</li>
<li>In female Fundulus heteroclitus exposed to 17alpha-methyltestosterone for 7 or 14 d, both E2 production and plasma E2 were impacted at the same exposure concentrations (Sharpe et al. 2004). </li>
</ul>
<p><strong>Mammals</strong></p>
<ul>
<li>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)</li>
<li>DEHP, rat, in vivo 300-600 mg/kg/day, dose dependent reduction of E2 plasma levels (Xu et al., 2010)</li>
</ul>
<p>Evidence for rodent and human models is summarized in Table 1.</p>
<p> </p>
<table border="1" style="border-collapse:collapse; font-size:75%">
<tbody>
<tr>
<td>
<p>Compound class</p>
</td>
<td>
<p>Species</p>
</td>
<td>
<p>Study type</p>
</td>
<td>
<p>Dose</p>
</td>
<td>
<p>E2 production/levels</p>
</td>
<td>
<p>Reference</p>
</td>
</tr>
<tr>
<td>
<p>Phthalates (DEHP)</p>
</td>
<td>
<p>rat</p>
</td>
<td>
<p>ex vivo</p>
</td>
<td>
<p>1500 mg/kg/day</p>
</td>
<td>
<p>Reduced/increased E2 production in ovary culture</p>
</td>
<td>
<p>(Laskey & Berman, 1993)</p>
</td>
</tr>
<tr>
<td>
<p>Phthalates (MEHP)</p>
</td>
<td>
<p>rat</p>
</td>
<td>
<p>in vitro</p>
</td>
<td>
<p>From 50 µM</p>
</td>
<td>
<p>Reduced E2 production (concentration and time dependent in Granulosa cell)</p>
</td>
<td>
<p>(Davis, Weaver, Gaines, & Heindel, 1994)</p>
</td>
</tr>
<tr>
<td>
<p>Phthalates (MEHP)</p>
</td>
<td>
<p>rat</p>
</td>
<td>
<p>in vitro</p>
</td>
<td>
<p>100-200µM</p>
</td>
<td>
<p>reduction E2 production (dose dependent)</p>
</td>
<td>
<p>(Lovekamp & Davis, 2001)</p>
</td>
</tr>
<tr>
<td>
<p>Phthalates (DEHP)</p>
</td>
<td>
<p>rat</p>
</td>
<td>
<p>in vivo</p>
</td>
<td>
<p>300-600 mg/kg/day</p>
</td>
<td>
<p>reduction E2 levels dose dependent</p>
</td>
<td>
<p>(Xu et al., 2010),</p>
</td>
</tr>
<tr>
<td>
<p>Phthalates (MEHP)</p>
</td>
<td>
<p>human</p>
</td>
<td>
<p>in vitro</p>
</td>
<td>
<p>IC(50)= 49- 138 µM (dependent on the stimulant)</p>
</td>
<td>
<p>reduction E2 production (dose dependent)</p>
</td>
<td>
<p>(Reinsberg, Wegener-Toper, van der Ven, van der Ven, & Klingmueller, 2009)</p>
</td>
</tr>
<tr>
<td>
<p>Phthalates (MEHP/DEHP)</p>
</td>
<td>
<p>mice</p>
</td>
<td>
<p>ex vivo</p>
</td>
<td>
<p>DEHP (10 -100 μg/ml); MEHP (0.1 and 10 μg/ml)</p>
</td>
<td>
<p>reduction E2 production (dose dependent)</p>
</td>
<td>
<p>(Gupta et al., 2010)</p>
</td>
</tr>
</tbody>
</table>
<p><br />
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.</p>
<p>Based on the limited set of studies available to date, there are no known inconsistencies.</p>
<p>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.</p>
<p>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.</p>
<p>• 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.</p>
<p>• 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.</p>
<p>• 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.</p>
HighFemaleHighAdult, reproductively matureNot SpecifiedModerateHighModerateHigh<p>Key enzymes needed to synthesize 17β-estradiol first appear in the common ancestor of amphioxus and vertebrates (Baker 2011). While some E2 synthesis can occur in other tissues, the ovary is recognized as the major source of 17β-estradiol synthesis in female vertebrates. Endocrine actions of ovarian E2 are facilitated through transport via the plasma. Consequently, this key event relationship is applicable to most female vertebrates.</p>
<ul>
<li>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.</li>
<li>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.</li>
<li>Baker ME. 2011. Origin and diversification of steroids: co-evolution of enzymes and nuclear receptors. Molecular and cellular endocrinology 334(1-2): 14-20.</li>
</ul>
<ul>
<li>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.</li>
</ul>
<ul>
<li>Ekman DR, Villeneuve DL, Teng Q, Ralston-Hooper KJ, Martinović-Weigelt D, Kahl MD, Jensen KM, Durhan EJ, Makynen EA, Ankley GT, Collette TW. Use of gene expression, biochemical and metabolite profiles to enhance exposure and effects assessment of the model androgen 17β-trenbolone in fish. Environ Toxicol Chem. 2011 Feb;30(2):319-29. doi: 10.1002/etc.406.</li>
</ul>
<ul>
<li>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.</li>
</ul>
<ul>
<li>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.</li>
</ul>
<ul>
<li>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.</li>
</ul>
<ul>
<li>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.</li>
</ul>
<ul>
<li>Norris DO. 2007. Vertebrate Endocrinology. Fourth ed. New York: Academic Press.</li>
</ul>
<ul>
<li>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.</li>
</ul>
<ul>
<li>Rutherford R, Lister A, Hewitt LM, MacLatchy D. Effects of model aromatizable (17α-methyltestosterone) and non-aromatizable (5α-dihydrotestosterone) androgens on the adult mummichog (Fundulus heteroclitus) in a short-term reproductive endocrine bioassay. Comp Biochem Physiol C Toxicol Pharmacol. 2015 Apr;170:8-18. doi: 10.1016/j.cbpc.2015.01.004.</li>
<li>Sharpe RL, MacLatchy DL, Courtenay SC, Van Der Kraak GJ. Effects of a model androgen (methyl testosterone) and a model anti-androgen (cyproterone acetate) on reproductive endocrine endpoints in a short-term adult mummichog (Fundulus heteroclitus) bioassay. Aquat Toxicol. 2004 Apr 28;67(3):203-15.</li>
<li>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>
<li>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.</li>
<li>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.</li>
<li>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.</li>
</ul>
<ul>
<li>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.</li>
</ul>
2016-11-29T18:41:332017-03-20T12:05:21d1c7087e-6d2f-499b-86f0-d0d9bff77f2d6e6f90f2-ff1e-4819-9897-4aeb3647fa75<p>The ovarian cycle irregularities impact on reproductive capacity of the females that may result in impaired fertility:
</p><p>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).
</p><p>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 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).
</p><p>3. Significant delays in ovulation can result in increased embryonic abnormalities and pregnancy loss (Fugo and Butcher, 1966; Cooper et al., 1994).
</p><p>4. Persistent diestrus indicates temporary or permanent cessation of follicular development and
ovulation, and thus at least temporary infertility.
</p><p>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.
</p><p>6. Lengthening of the cycle may be a result of increased duration of either estrus or diestrus.
</p><p>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.
However, there are other mechanisms that might result in impaired fertility (e.g cellular maturation in ovary).
</p><p>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).
</p><p><br />
</p>
<table border="1" style="border-collapse:collapse;font-size:75%">
<tr>
<td>
<p>Compound class
</p>
</td>
<td>
<p><b>Species</b>
</p>
</td>
<td>
<p><b>AO:ovarian cycle irregularities</b>
</p>
</td>
<td>
<p><b>AO:Impaired fertility</b>
</p>
</td>
<td>
<p>reference
</p>
</td></tr>
<tr>
<td>
<p>Phthalates (DEHP)
</p>
</td>
<td>
<p>rat
</p>
</td>
<td>
<p>5-400 mg/kg/day females differed from the control in the relative amount of time spent in oestrous stages
</p>
</td>
<td>
<p>number of live pups (P0) reduced (400 mg/kg/day)
</p>
</td>
<td>
<p>(Blystone et al., 2010)
</p>
</td></tr>
<tr>
<td>
<p>Phthalates (DEHP)
</p>
</td>
<td>
<p>rat
</p>
</td>
<td>
<p>irregular estrous cycles (3,000 mg/kg/day)
</p>
</td>
<td>
<p>slight decline in pregnancy rate (3,000 mg/kg/day)
</p>
</td>
<td>
<p>(Takai et al., 2009)
</p>
</td></tr>
<tr>
<td>
<p>Phthalates (DEHP)
</p>
</td>
<td>
<p>mice
</p>
</td>
<td>
<p><br />
</p>
</td>
<td>
<p>dose-dependent decreases in fertility
</p>
</td>
<td>
<p>(Lamb, Chapin, Teague, Lawton, & Reel, 1987)
</p>
</td></tr>
<tr>
<td>
<p>Phthalates (DEHP)
</p>
</td>
<td>
<p>mice
</p>
</td>
<td>
<p>No change
</p>
</td>
<td>
<p>abortion rate of 100% in F0 dams (500-mg/kg/day)
</p>
</td>
<td>
<p>(Schmidt, Schaedlich, Fiandanese, Pocar, & Fischer, 2012).
</p>
</td></tr>
<tr>
<td>
<p>Phthalates (DEHP)
</p>
</td>
<td>
<p>sheep
</p>
</td>
<td>
<p>dose-dependent effect on the duration of the estrous cycles shortening of the ovulatory cycles due mainly to a reduction in the size and lifespan of CL
</p>
</td>
<td>
<p><br />
</p>
</td>
<td>
<p>(Herreros, Gonzalez-Bulnes, et al., 2013)
</p>
</td></tr>
<tr>
<td>
<p>Phthalates (DEHP)
</p>
</td>
<td>
<p>sheep
</p>
</td>
<td>
<p>No effect on ovulatory efficiency
</p>
</td>
<td>
<p><br />
</p>
</td>
<td>
<p>(Herreros, Encinas, et al., 2013)
</p>
</td></tr>
<tr>
<td>
<p>Phthalates (DEHP)
</p>
</td>
<td>
<p>rat
</p>
</td>
<td>
<p>No changes in F0, increase of cycle by 0.4 day in F1 at 10,000ppm
</p>
</td>
<td>
<p>18% and 21% decrease in live pups/litter F0 at 7500ppm and 10,000ppm respectively, no viable litters (F1 10,000 ppm ~643.95mg/kg/day)
</p>
</td>
<td>
<p>(NTP, 2005)
</p>
</td></tr>
<tr>
<td>
<p>Phthalates (DEHP)
</p>
</td>
<td>
<p>rat
</p>
</td>
<td>
<p>Deficit in growing follicles and corpora lutea
</p>
</td>
<td>
<p>4-fold increase in females with stillborn pups in F0 at 9000ppm 2.1-fold Postimplantation loss in F0 at 9000ppm
</p>
</td>
<td>
<p>(Schilling, K., Deckardt. K., Gembardt, Chr., and Hildebrand, 1999)
</p>
</td></tr>
<tr>
<td>
<p>Phthalates (DEHP)
</p>
</td>
<td>
<p>rat
</p>
</td>
<td>
<p>prolong the estrous cycle, anovulation
</p>
</td>
<td>
<p><br />
</p>
</td>
<td>
<p>(Davis, Maronpot, & Heindel, 1994)
</p>
</td></tr>
<tr>
<td>
<p>Phthalates
</p>
</td>
<td>
<p><br />
</p>
</td>
<td>
<p><br />
</p>
</td>
<td>
<p>Reduced fertility and fecundity
</p>
</td>
<td>
<p>(Wolf et al., 1999)
</p>
</td></tr>
<tr>
<td>
<p>Organochlorine (methoxychlor)
</p>
</td>
<td>
<p>rat
</p>
</td>
<td>
<p>Decreased number of cycles, extended diestrus and estrus
</p>
</td>
<td>
<p><br />
</p>
</td>
<td>
<p>(Laws, 2000)
</p>
</td></tr>
<tr>
<td>
<p>Organotins tributyltin chloride (TBTCl)
</p>
</td>
<td>
<p>rat
</p>
</td>
<td>
<p>At 125 ppm vaginal opening and impaired estrous cyclicity
</p>
</td>
<td>
<p><br />
</p>
</td>
<td>
<p>(Ogata et al., 2001)
</p>
</td></tr></table>
<p><br />
Table 1 Summary the empirical evidence supporting the KER.
</p><p><br />
</p><p>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).
</p><p>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.
</p><p><b>Inconsistencies</b>
</p><p>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).
</p><p>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).
</p><p>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.
</p><p><br />
</p><p><b>Mice</b>
</p>
<ul>
<li>environmental air pollution (Mohallem et al., 2005)
</li>
<li>phthalates (DEHP)
</li>
<li>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).
</li>
<li>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 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).
</li>
</ul>
<p><b>Rat</b>
phthalates (DEHP)
</p>
<ul>
<li>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.
</li>
</ul>
<ul>
<li>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).
</li>
</ul>
<p><b>Human</b>
</p><p>Studies showing a correlation between decreased fertility and;
</p>
<ul>
<li>professional activity (Olsen, 1994)
</li>
<li>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).
</li>
<li>smoking (Hull, North, Taylor, Farrow, & Ford, 2000)
</li>
<li>the use of certain drugs or radiation exposure (Dobson & Felton, 1983)
</li>
</ul>
<p>For the taxonomic applicability see also the Table 1.
</p><p>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.
</p><p>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
</p><p>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.
</p><p>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
</p><p>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
</p><p>Chao, H.-R., Wang, S.-L., Lin, L.-Y., Lee, W.-J., & Päpke, O. (2007). Placental transfer of polychlorinated 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
</p><p>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.
</p><p>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
</p><p>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.
</p><p>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
</p><p>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
</p><p>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.
</p><p>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.
</p><p>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
</p><p>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.
</p><p>Massaro, E. J. (Ed.). (1997). Handbook of Human Toxicology, Volume 236. Taylor & Francis.
</p><p>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
</p><p>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
</p><p>NTP. (2005). Multigenerational Reproductive Assessment by Continuous Breeding when Diethylhexylphthalate (CAS 117-81-7).
</p><p>OECD. (2008). No 43: Guidance document on mammalian reproductive toxicity testing and assessment.
</p><p>Ogata, R., Omura, M., Shimasaki, Y., Kubo, K., Oshima, Y., Aou, S., & Inoue, N. (2001). Two-generation reproductive toxicity study of tributyltin chloride in female rats. Journal of Toxicology and Environmental Health. Part A, 63(2), 127–44. doi:10.1080/15287390151126469
</p><p>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.
</p><p>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.
</p><p>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
</p><p>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.
</p><p>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
</p><p>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
</p>2016-11-29T18:41:342016-12-03T16:37:5620361076-cf32-4c5d-9f55-739180df6fe66dd9c937-ac80-416a-a42e-82d27d9267f3<p>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.
</p><p>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.
</p><p>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.
</p><p><br />
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:
</p><p>• 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)
</p><p>• 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).
</p><p>• rat, decrease in aromatase levels at 100μg/ml DEHP, 10μg/ml MEHP and dose dependent decrease of E2 production from 10μg/ml DEHP, 0.1μg/ml MEHP and at the same time point (96 h) (Gupta et al., 2010).
</p><p>Table 1 summarises available empirical evidence.
</p><p><br />
</p>
<table border="1" style="border-collapse:collapse;font-size:75%">
<tr>
<td>
<p>Compound
</p>
</td>
<td>
<center>Species</center>
</td>
<td>
<center>Study type</center>
</td>
<td>
<center>comments</center>
</td>
<td>
<center>Aromatase decrease levels/activity</center>
</td>
<td>
<center>Reference</center>
</td></tr>
<tr>
<td>
<p>MEHP
</p>
</td>
<td>
<center>rat</center>
<center></center>
</td>
<td>
<center>in vitro</center>
</td>
<td>
<center>decrease activity of aromatase and dose and time dependent decrease of E2 production</center>
</td>
<td>
<center>decrease activity of aromatase 100 µM</center>
<center>E2 production 50-100 µM</center>
</td>
<td>
<center>(Davis, Weaver, Gaines, & Heindel, 1994),</center>
</td></tr>
<tr>
<td>
<p>MEHP
</p>
</td>
<td>
<center>rat</center>
<center></center>
</td>
<td>
<center>ex vivo</center>
</td>
<td>
<center>decrease aromatase level and dose decrease of E2 production</center>
</td>
<td>
<center>aromatase level 50µM</center>
<center>E2 production 100-200 µM</center>
</td>
<td>
<center>(Lovekamp & Davis, 2001)</center>
<center></center>
</td></tr>
<tr>
<td>
<p>DEHP
</p>
</td>
<td>
<center>rat</center>
</td>
<td>
<center>in vivo</center>
</td>
<td>
<center>Does dependent reduction of E2 levels, and Does dependent reduction decrease aromatase expression</center>
</td>
<td>
<center>300-600mg/kg/day</center>
</td>
<td>
<center>(Xu et al., 2010),</center>
</td></tr>
<tr>
<td>
<p>MEHP
</p>
</td>
<td>
<center>human</center>
</td>
<td>
<center>in vitro</center>
</td>
<td>
<center>Dose dependent reduction E2 production and reduction aromatase of expression</center>
</td>
<td>
<center>reduction E2 levels IC(50)= 49- 138 µM, at 167µM decrease aromatase </center>
</td>
<td>
<center>(Reinsberg et al., 2009)</center>
</td></tr>
<tr>
<td>
<p>MEHP/DEHP
</p><p><br />
</p>
</td>
<td>
<center>mice</center>
</td>
<td>
<center>ex vivo</center>
</td>
<td>
<center>dose dependent E2 production, and reduction of aromatase levels</center>
</td>
<td>
<center>E2 production at DEHP (10 -100 μg/ml);MEHP (0.1 and 10 μg/ml)</center>
<center>Aromatase levels DEHP (100 μg/ml); MEHP 0.1 μg/ml</center>
</td>
<td>
<center>(Gupta et al., 2010)</center>
</td></tr></table>
<p>This KE describes decreased levels and/or availability of aromatase different from aromatase inhibition.
</p><p><b>Upstream events</b>
An upstream event has been postulated to involve PPARγ activation, however the studies confirming its role in the reduction of aromatase levels are incomplete.
The mechanisms involving Peroxisome Proliferator Activated receptor γ activation leading to aromatase (Cyp19a1) reduction relating to the pathway are described in greater detail in the page <a href="/wiki/index.php/Peroxisome_Proliferator_Activated_receptor_%CE%B3_activation_indirectly_leads_to_aromatase_(Cyp19a1)_reduction" title="Peroxisome Proliferator Activated receptor γ activation indirectly leads to aromatase (Cyp19a1) reduction">Peroxisome Proliferator Activated receptor γ activation indirectly leads to aromatase (Cyp19a1) reduction </a>.
</p><p><b>Availability or reduced aromatase levels</b>
</p><p><br />
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.
</p><p><em>
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?
</em>
</p><p><br />
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 aromatase expression/availability.
</p><p>See table 1.
</p><p>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
</p><p>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
</p><p>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
</p><p>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.
</p><p>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.
</p><p>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
</p><p>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.
</p><p>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
</p><p>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
</p><p>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
</p><p>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
</p><p>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.
</p><p>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
</p>2016-11-29T18:41:342016-11-29T20:10:18Aromatase (Cyp19a1) reduction leading to impaired fertility in adult femaleAromatase (Cyp19a1) reduction leading to reproductive toxicity<p>Malgorzata Nepelska, Elise Grignard, Sharon Munn,</p>
<p>Systems Toxicology Unit, Institute for Health and Consumer Protection, Joint Research Centre, European Commission, Via E. Fermi 2749, I-21027 Ispra, Varese, Italy</p>
<p>Corresponding author: sharon.munn@ec.europa.eu; elise.grignard@ec.europa.eu</p>
Open for citation & commentEAGMST Under ReviewIncluded in OECD Work Plan1.21<p>This AOP links activation of the Peroxisome Proliferator Activated Receptorγ (PPARγ) to reproductive toxicity in adult female. The development of this AOP relies on evidence collected from rodent models and incorporates human mechanistic and epidemiological data. The PPARγ is a ligand-activated transcription factor that belongs to the nuclear receptor family, which also includes the steroid and thyroid hormone receptors. Interest in PPARγ action as a mechanistic basis for effects on the reproductive system arises from the demonstrated relationships between activation of this receptor and impairment of the steroidogenesis leading to reproductive toxicity in rodents. PPARs play important roles in the metabolic regulation of lipids, of which cholesterol, in particular being a precursor of steroid hormones, makes the link between lipid metabolism to effects on reproduction. The key events in the pathway comprise the activation of PPARγ, followed by the disruption of the hormonal balance which leads to irregularities of the ovarian cycle that may further be cause of impaired fertility. The PPARγ-initiated AOP to rodent female reproductive toxicity is a first step for structuring current knowledge about a mode of action which is neither ER-mediated nor via direct aromatase inhibition. In the current form the pathway lays a strong basis for linking an endocrine mode of action with an apical endpoint, prerequisite requirement for the identification of endocrine disrupting chemicals. This AOP is complemented with a structured data collection which will serve as the basis for further quantitative development of the pathway.</p>
<p>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.).</p>
<p>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.</p>
<p>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 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.
</p>adjacentNot SpecifiedHighadjacentNot SpecifiedHighadjacentNot SpecifiedModeratenon-adjacentNot SpecifiedModerate<table border="1" style="border-collapse:collapse; font-size:75%">
<tbody>
<tr>
<td>
<p><strong>KRs WoE</strong></p>
</td>
<td>
<p><strong>Essentiality - KEs</strong></p>
</td>
<td>
<p><strong>level of confidence</strong></p>
</td>
</tr>
<tr>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr>
<td>
<p><a href="/wiki/index.php/Event:228" title="Event:228">PPAR gamma, Activation </a></p>
<p> </p>
</td>
<td>
<p>PPARγ activation was found to indirectly alter the expression of aromatase</p>
</td>
<td>
<p>weak</p>
</td>
</tr>
<tr>
<td>
<p><a href="/wiki/index.php/Event:408" title="Event:408">Aromatase (Cyp19a1), reduction in ovarian granulosa cells</a></p>
</td>
<td>
<p>Aromatase is the cytochrome P450 enzyme complex responsible for the conversion of androgens to estrogens during steroidogenesis which is a key reaction in the sex differentiation in vertebrates. Alterations in the amount of aromatase present or in the catalytic activity of the enzyme will alter the levels of estrogens in tissues and dramatically disrupt estrogen hormone action.</p>
</td>
<td>
<p>moderate</p>
</td>
</tr>
<tr>
<td><a href="/wiki/index.php/Event:3" title="Event:3">17beta-estradiol synthesis by ovarian granulosa cells</a></td>
<td>
<p>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.</p>
</td>
<td>
<p>strong</p>
</td>
</tr>
<tr>
<td>
<p><a href="/wiki/index.php/Event:219" title="Event:219">Plasma 17beta-estradiol concentrations, Reduction</a></p>
</td>
<td>
<p>Estrogens are crucial for female fertility, as proved by the severe reproductive defects observed when their synthesis is blocked.</p>
</td>
<td>
<p>strong</p>
</td>
</tr>
<tr>
<td>
<p><a href="/wiki/index.php/Event:405" title="Event:405">ovarian cycle irregularities</a></p>
</td>
<td>
<p>A sequential progression of interrelated physiological and behavioural cycles underlines the female reproductive function.</p>
</td>
<td>
<p>strong</p>
</td>
</tr>
<tr>
<td>
<p><a href="/wiki/index.php/Event:406" title="Event:406">Fertility, impaired</a></p>
</td>
<td>
<p>Impaired Fertility is the endpoint of reproductive toxicity</p>
</td>
<td>
<p>strong</p>
</td>
</tr>
</tbody>
</table>
HighFemaleHighAdult, reproductively matureHighLowLow<p><strong>Biological plausibility, coherence, and consistency of the experimental evidence</strong></p>
<p>In the presented AOP it is hypothesized that the key events occur in a biologically plausible order prior to the development of adverse outcomes. However, the experimental support is derived from a limited number of studies. The PPARγ activators have been shown to alter steroidogenesis, ovarian cycle and impair reproduction [see reviews (Kay, Chambers, and Foster 2013), (Froment et al. 2006), (Peraza et al. 2006), (Latini et al. 2008), (Martino-Andrade and Chahoud 2010), (Lyche et al. 2009), (Lovekamp-Swan and Davis 2003)]. The biochemistry of steroidogenesis and the predominant role of the ovaries in synthesis of the sex steroids are well established. During the reproductive years the ovary is the central organ providing hormones necessary for the communication between the reproductive tract and the central nervous system, assuring normal reproductive function. Hormonal imbalance may lead to irregularities of the ovarian cycle that could be one of many possible events resulting in decrease fertility.</p>
<p><strong>Concordance of dose-response relationships</strong></p>
<p>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.</p>
<p>Temporal concordance among the key events and the adverse outcome</p>
<p>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.</p>
<p><strong>Strength, consistency, and specificity of association of adverse effect and initiating event</strong></p>
<p>PPARγ-null mutation is embryonically lethal due to a defect in placental development ( PPARγ is necessary for angiogenesis)(Barak et al. 1999). Organ (ovary) targeted knock-out studies are needed to more precisely inform on the mechanistic involvement of the PPAR family in the proposed AOP.</p>
<p>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:</p>
<p>Chemical independent studies:</p>
<p>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)</p>
<p>2. granulosa cell specific deletion of PPARγ in mice results in marked impairment of ovulation due to defective follicular rupture (Kim et al. 2008)</p>
<p><br />
Chemical dependent studies:</p>
<p>3. Antagonist of PPARγ recovered the decrease of aromatase after treatment with MEHP (PPARγ agonist) (Lovekamp-Swan, Jetten, and Davis 2003)</p>
<p><br />
Alternative mechanism(s) or MIE(s) described which may contribute/synergise the postulated AOP</p>
<p>Alternative mechanisms relating to the pathway are described in greater detail in the descriptions of KERs.</p>
<p>The contributing MIE in the pathway proposed is activation of PPARα supported by experimental evidence of dual activation of PPARα/γ by MEHP leading to decreased expression and activity of aromatase in granulosa cells (Lovekamp-Swan, Jetten, and Davis 2003) and inhibition of aromatase expression upon activation of PPARα by the ligand, fenofibrate, in the ovary of mouse (Toda et al. 2003).</p>
<p>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</p>
<ul>
<li>upstream of aromatase:</li>
</ul>
<p>• Steroidogenic acute regulatory protein (StAR)</p>
<p>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)).</p>
<p>• 3β-hydroxysteroid dehydrogenase (3β-HSD)</p>
<p>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)</p>
<p>• 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)</p>
<ul>
<li>downstream of aromatase:</li>
</ul>
<p>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).</p>
<p><br />
• 17β-Hydroxysteroid dehydrogenase (17β-HSD)</p>
<p>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.</p>
<p>Reduced levels of estradiol and irregularities of ovarian cycle</p>
<p>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).</p>
<p><br />
<strong>Uncertainties, inconsistencies and data gaps</strong></p>
<p>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)].</p>
<p>Inconsistencies</p>
<p>Agonists of PPARγ were found to impact on steroidogenesis; however contradictory data show their effect on different stages of the process as well the direction of the effect(see above). Some in vivo studies also reported two-way effect on the estradiol production by PPARγ agonists. This effect may be attributed to the different measurements during different stages of estrous cycle. The phase of the estrous cycle, in which hormones are measured, may influence the readout of compound effect. In rats treated with DEHP increase in estradiol production was observed in ovarian cells (ex vivo) extracted during diestrus phase, however there was decrease in estradiol when the cells were extracted during estrus stage (Laskey and Berman 1993). In alignment with this result increased levels of estradiol were found in sheep proceeding the estrus phase (Herreros et al. 2013).</p>
<p>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).</p>
<p>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)].</p>
<p>The experimental support for the pathway is based on rodent models and other mammals (pig, sheep) including human mechanistic and epidemiological data. The experimental animal data are assumed relevant for consideration of human risk.</p>
<p>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)].</p>
<table border="1" style="border-collapse:collapse; font-size:75%">
<tbody>
<tr>
<td>
<p><strong>KRs WoE</strong></p>
</td>
<td>
<p><strong>Essentiality - KEs</strong></p>
</td>
<td>
<p><strong>level of confidence</strong></p>
</td>
</tr>
<tr>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr>
<td>
<p><a href="/wiki/index.php/Event:228" title="Event:228">PPAR gamma, Activation </a></p>
<p> </p>
</td>
<td>
<p>PPARγ activation was found to indirectly alter the expression of aromatase</p>
</td>
<td>
<p>weak</p>
</td>
</tr>
<tr>
<td>
<p><a href="/wiki/index.php/Event:408" title="Event:408">Aromatase (Cyp19a1), reduction in ovarian granulosa cells</a></p>
</td>
<td>
<p>Aromatase is the cytochrome P450 enzyme complex responsible for the conversion of androgens to estrogens during steroidogenesis which is a key reaction in the sex differentiation in vertebrates. Alterations in the amount of aromatase present or in the catalytic activity of the enzyme will alter the levels of estrogens in tissues and dramatically disrupt estrogen hormone action.</p>
</td>
<td>
<p>moderate</p>
</td>
</tr>
<tr>
<td><a href="/wiki/index.php/Event:3" title="Event:3">17beta-estradiol synthesis by ovarian granulosa cells</a></td>
<td>
<p>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.</p>
</td>
<td>
<p>strong</p>
</td>
</tr>
<tr>
<td>
<p><a href="/wiki/index.php/Event:219" title="Event:219">Plasma 17beta-estradiol concentrations, Reduction</a></p>
</td>
<td>
<p>Estrogens are crucial for female fertility, as proved by the severe reproductive defects observed when their synthesis is blocked.</p>
</td>
<td>
<p>strong</p>
</td>
</tr>
<tr>
<td>
<p><a href="/wiki/index.php/Event:405" title="Event:405">ovarian cycle irregularities</a></p>
</td>
<td>
<p>A sequential progression of interrelated physiological and behavioural cycles underlines the female reproductive function.</p>
</td>
<td>
<p>strong</p>
</td>
</tr>
<tr>
<td>
<p><a href="/wiki/index.php/Event:406" title="Event:406">Fertility, impaired</a></p>
</td>
<td>
<p>Impaired Fertility is the endpoint of reproductive toxicity</p>
</td>
<td>
<p>strong</p>
</td>
</tr>
</tbody>
</table>
<table border="1" style="border-collapse:collapse; font-size:75%">
<tbody>
<tr>
<td>
<p><strong>KERs </strong></p>
</td>
<td>
<p><strong>Biological plausibility</strong></p>
</td>
<td>
<p><strong>level of confidence</strong></p>
</td>
<td colspan="3">
<p><strong>Empirical Support</strong></p>
</td>
<td>
<p><strong>level of confidence</strong></p>
</td>
<td>
<p><strong>Inconsistencies/Uncertainties</strong></p>
</td>
</tr>
<tr>
<td> </td>
<td> </td>
<td> </td>
<td>
<p>Dose-response</p>
</td>
<td>
<p>Temporality</p>
</td>
<td>
<p>Incidence</p>
</td>
<td> </td>
<td> </td>
</tr>
<tr>
<td>
<p><em><strong>PPARγ, Activation =></strong></em></p>
<p><em><strong>Aromatase (Cyp19a1), reduction in ovarian granulosa cells</strong></em></p>
</td>
<td>
<p>There is functional relationship between PPARγ activation and reduction in aromatase levels. Several mechanisms have been investigated; however there is no established consensus.</p>
</td>
<td>
<p>Moderate</p>
</td>
<td>
<ul>
<li>KEup occurs at lower dose than KEdown(dose response concordance)</li>
</ul>
<p> </p>
</td>
<td>
<ul>
<li>occurrence of the key events at similar dose and time point</li>
<li>Support for solid temporal relationship is lacking</li>
</ul>
</td>
<td>
<p> </p>
</td>
<td>
<p>Weak</p>
</td>
<td>
<p>Limited data, for details see KER pages</p>
</td>
</tr>
<tr>
<td>
<p><em><strong>Aromatase (Cyp19a1), reduction in ovarian granulosa cells =></strong></em></p>
<p><em><strong>17beta-estradiol synthesis by ovarian granulosa cells</strong></em></p>
</td>
<td>
<p>Within the ovary, aromatase expression and activity is primarily localized in the granulosa cells. Therefore, changes in ovarian aromatase can generally be assumed to directly impact E2 synthesis by the granulosa cells.</p>
</td>
<td>
<p>Moderate</p>
</td>
<td>
<ul>
<li>KEup occurs at lower dose than KEdown(dose response concordance)</li>
</ul>
</td>
<td>
<ul>
<li>occurrence of the key events at similar dose and time point</li>
<li>Support for solid temporal relationship is lacking</li>
</ul>
</td>
<td>
<p> </p>
</td>
<td>
<p>moderate</p>
</td>
<td>
<p>Limited data</p>
</td>
</tr>
<tr>
<td>
<p><em><strong>17beta-estradiol synthesis by ovarian granulosa cells, Reduction =></strong></em></p>
<p><em><strong>Plasma 17beta-estradiol concentrations</strong></em></p>
</td>
<td>
<p>The gonads are generally considered the major source of circulating estrogens, consequently, if estradiol synthesis by ovarian granulosa cells is reduced, plasma E2 concentrations would be expected to decrease.</p>
</td>
<td>
<p>Strong</p>
</td>
<td>
<ul>
<li>KEs occur at similar dose levels</li>
</ul>
</td>
<td>
<ul>
<li>occurrence of the key events at similar dose Support for solid temporal relationship is lacking</li>
</ul>
</td>
<td>
<p> </p>
</td>
<td>
<p>moderate</p>
</td>
<td>
<p>Limited data</p>
</td>
</tr>
<tr>
<td>
<p><em><strong>Plasma 17beta-estradiol concentrations, Reduction =></strong></em></p>
<p><em><strong>ovarian cycle irregularities </strong></em></p>
</td>
<td>
<p>Alterations in relationships among the hypothalamic, pituitary, and ovarian components of the reproductive axis can have marked effects on cyclicity. A toxicological insult to any one of these sites can disrupt the cycle and block ovulation.</p>
</td>
<td>
<p>Strong</p>
</td>
<td>
<ul>
<li>KEs occur at similar dose levels</li>
</ul>
</td>
<td>
<ul>
<li>occurrence of the key events at similar dose and time point</li>
<li>Support for solid temporal relationship is lacking</li>
</ul>
</td>
<td>
<p> </p>
</td>
<td>
<p>moderate</p>
</td>
<td> </td>
</tr>
<tr>
<td>
<p><em><strong>ovarian cycle irregularities =></strong></em></p>
<p><em><strong>Fertility, impaired </strong></em></p>
</td>
<td>
<p>A sequential progression of interrelated physiological and behavioural cycles underlines the female's fertility and successful production of offspring.</p>
</td>
<td>
<p>Strong</p>
</td>
<td>
<ul>
<li>KEs occur at similar dose levels</li>
</ul>
<p> </p>
</td>
<td>
<ul>
<li>occurrence of the key events at similar dose and with temporal relationship</li>
<li>Support for solid temporal relationship is lacking.</li>
</ul>
</td>
<td>
<p> </p>
</td>
<td>
<p>moderate</p>
</td>
<td> </td>
</tr>
</tbody>
</table>
<p> </p>
<p>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?</p>
<p>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).</p>
<p>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.</p>
<p>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.</p>
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