Mono-(2-ethyl-5-hydroxyhexyl) phthalate promotes uterine leiomyoma cell survival through tryptophan-kynurenine-AHR pathway activation.

Uterine leiomyoma is the most common tumor in women and causes severe morbidity in 15 to 30% of reproductive-age women. Epidemiological studies consistently indicate a correlation between leiomyoma development and exposure to endocrine-disrupting chemical phthalates, especially di-(2-ethylhexyl) phthalate (DEHP); however, the underlying mechanisms are unknown. Here, among the most commonly encountered phthalate metabolites, we found the strongest association between the urine levels of mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), the principal DEHP metabolite, and the risk of uterine leiomyoma diagnosis (n = 712 patients). The treatment of primary leiomyoma and smooth muscle cells (n = 29) with various mixtures of phthalate metabolites, at concentrations equivalent to those detected in urine samples, significantly increased cell viability and decreased apoptosis. MEHHP had the strongest effects on both cell viability and apoptosis. MEHHP increased cellular tryptophan and kynurenine levels strikingly and induced the expression of the tryptophan transporters SLC7A5 and SLC7A8, as well as, tryptophan 2,3-dioxygenase (TDO2), the key enzyme catalyzing the conversion of tryptophan to kynurenine that is the endogenous ligand of aryl hydrocarbon receptor (AHR). MEHHP stimulated nuclear localization of AHR and up-regulated the expression of CYP1A1 and CYP1B1, two prototype targets of AHR. siRNA knockdown or pharmacological inhibition of SLC7A5/SLC7A8, TDO2, or AHR abolished MEHHP-mediated effects on leiomyoma cell survival. These findings indicate that MEHHP promotes leiomyoma cell survival by activating the tryptophan-kynurenine-AHR pathway. This study pinpoints MEHHP exposure as a high-risk factor for leiomyoma growth, uncovers a mechanism by which exposure to environmental phthalate impacts leiomyoma pathogenesis, and may lead to the development of novel druggable targets.

Neonicotinoids differentially modulate nicotinic acetylcholine receptors in immature and antral follicles in the mouse ovary1.

Neonicotinoids are the most widely used insecticides in the world. They are synthetic nicotine derivatives that act as nicotinic acetylcholine receptor (nAChR) agonists. Although parent neonicotinoids have low affinity for the mammalian nAChR, they can be activated in the environment and the body to positively charged metabolites with high affinity for the mammalian nAChR. Imidacloprid (IMI), the most popular neonicotinoid, and its bioactive metabolite desnitro-imidacloprid (DNI) differentially interfere with ovarian antral follicle physiology in vitro, but their effects on ovarian nAChR subunit expression are unknown. Furthermore, ovarian nAChR subtypes have yet to be characterized in the ovary. Thus, this work tested the hypothesis that ovarian follicles express nAChRs and their expression is differentially modulated by IMI and DNI in vitro. We used PCR, RNA in situ hybridization, and immunohistochemistry to identify and localize nAChR subunits (α2, 4, 5, 6, 7 and ß1, 2, 4) expressed in neonatal ovaries and antral follicles. Chrnb1 was expressed equally in neonatal ovaries and antral follicles. Chrna2 and Chrnb2 expression was higher in antral follicles compared to neonatal ovaries and Chrna4, Chrna5, Chrna6, Chrna7 and Chrnb4 expression was higher in neonatal ovaries compared to antral follicles. The α subunits were detected throughout the ovary, especially in oocytes and granulosa cells. IMI and DNI dysregulated expression of multiple nAChR subunits in neonatal ovaries, but only dysregulated one subunit in antral follicles. These data indicate that mammalian ovaries contain nAChRs, and their susceptibility to IMI and DNI exposure varies with the stage of follicle maturity.

The role of the aryl hydrocarbon receptor in mediating the effects of mono(2-ethylhexyl) phthalate in mouse ovarian antral follicles†.

Di(2-ethylhexyl) phthalate (DEHP) is a pervasive environmental toxicant used in the manufacturing of numerous consumer products, medical supplies, and building materials. DEHP is metabolized to mono(2-ethylhexyl) phthalate (MEHP). MEHP is an endocrine disruptor that adversely affects folliculogenesis and steroidogenesis in the ovary, but its mechanism of action is not fully understood. Thus, we tested the hypothesis that the aryl hydrocarbon receptor (AHR) plays a functional role in MEHP-mediated disruption of folliculogenesis and steroidogenesis. CD-1 mouse antral follicles were isolated and cultured with MEHP (0-400 µM) in the presence or absence of the AHR antagonist CH223191 (1 µM). MEHP treatment reduced follicle growth over a 96-h period, and this effect was partially rescued by co-culture with CH223191. MEHP exposure alone increased expression of known AHR targets, cytochrome P450 (CYP) enzymes Cyp1a1 and Cyp1b1, and this induction was blocked by CH223191. MEHP reduced media concentrations of estrone and estradiol compared to control. This effect was mitigated by co-culture with CH223191. Moreover, MEHP reduced the expression of the estrogen-sensitive genes progesterone receptor (Pgr) and luteinizing hormone/choriogonadotropin receptor (Lhcgr) and co-treatment with CH223191 blocked this effect. Collectively, these data indicate that MEHP activates the AHR to impair follicle growth and reduce estrogen production and signaling in ovarian antral follicles.

Prenatal exposure to Di(2-ethylhexyl) phthalate and high-fat diet synergistically disrupts gonadal function in male mice†.

Prenatal exposure to Di (2-ethylhexyl) phthalate (DEHP) impairs the reproductive system and causes fertility defects in male offspring. Additionally, high-fat (HF) diet is a risk factor for reproductive disorders in males. In this study, we tested the hypothesis that prenatal exposure to a physiologically relevant dose of DEHP in conjunction with HF diet synergistically impacts reproductive function and fertility in male offspring. Female mice were fed a control or HF diet 7 days prior to mating and until their litters were weaned on postnatal day 21. Pregnant dams were exposed to DEHP or vehicle from gestational day 10.5 until birth. The male offspring's gross phenotype, sperm quality, serum hormonal levels, testicular histopathology, and testicular gene expression pattern were analyzed. Male mice born to dams exposed to DEHP + HF had smaller testes, epididymides, and shorter anogenital distance compared with those exposed to HF or DEHP alone. DEHP + HF mice had lower sperm concentration and motility compared with DEHP mice. Moreover, DEHP + HF mice had more apoptotic germ cells, fewer Leydig cells, and lower serum testosterone levels than DEHP mice. Furthermore, testicular mRNA expression of Dnmt1 and Dnmt3a was two to eight-fold higher than in DEHP mice by qPCR, suggesting that maternal HF diet and prenatal DEHP exposure additively impact gonadal function by altering the degree of DNA methylation in the testis. These results suggest that the combined exposure to DEHP and high-fat synergistically impairs reproductive function in male offspring, greater than exposure to DEHP or HF diet alone.

The effects of short-term and long-term phthalate exposures on ovarian follicle growth dynamics and hormone levels in female mice†.

Di(2-ethylhexyl) phthalate and diisononyl phthalate are widely used as plasticizers in polyvinyl chloride products. Short-term exposures to phthalates affect hormone levels, ovarian follicle populations, and ovarian gene expression. However, limited data exist regarding the effects of long-term exposure to phthalates on reproductive functions. Thus, this study tested the hypothesis that short-term and long-term exposure to di(2-ethylhexyl) phthalate or diisononyl phthalate disrupts follicle dynamics, ovarian and pituitary gene expression, and hormone levels in female mice. Adult CD-1 female mice were exposed to vehicle, di(2-ethylhexyl) phthalate, or diisononyl phthalate (0.15 ppm, 1.5 ppm, or 1500 ppm) via the chow for 1 or 6 months. Short-term exposure to di(2-ethylhexyl) phthalate (0.15 ppm) and diisononyl phthalate (1.5 ppm) decreased serum follicle-stimulating hormone levels compared to control. Long-term exposure to di(2-ethylhexyl) phthalate and diisononyl phthalate (1500 ppm) increased the percentage of primordial follicles and decreased the percentages of preantral and antral follicles compared to control. Both phthalates increased follicle-stimulating hormone levels (di(2-ethylhexyl) phthalate at 1500 ppm; diisononyl phthalate at 1.5 ppm) and decreased luteinizing hormone levels (di(2-ethylhexyl) phthalate at 0.15 and 1.5 ppm; diisononyl phthalate at 1.5 ppm and 1500 ppm) compared to control. Furthermore, both phthalates altered the expression of pituitary gonadotropin subunit genes (Cga, Fshb, and Lhb) and a transcription factor (Nr5a1) that regulates gonadotropin synthesis. These data indicate that long-term exposure to di(2-ethylhexyl) phthalate and diisononyl phthalate alters follicle growth dynamics in the ovary and the expression of gonadotropin subunit genes in the pituitary and consequently luteinizing hormone and follicle-stimulating hormone synthesis.

Esr1 but Not CYP19A1 Overexpression in Mammary Epithelial Cells during Reproductive Senescence Induces Pregnancy-Like Proliferative Mammary Disease Responsive to Anti-Hormonals.

Molecular-level analyses of breast carcinogenesis benefit from vivo disease models. Estrogen receptor 1 (Esr1) and cytochrome P450 family 19 subfamily A member 1 (CYP19A1) overexpression targeted to mammary epithelial cells in genetically engineered mouse models induces largely similar rates of proliferative mammary disease in prereproductive senescent mice. Herein, with natural reproductive senescence, Esr1 overexpression compared with CYP19A1 overexpression resulted in significantly higher rates of preneoplasia and cancer. Before reproductive senescence, Esr1, but not CYP19A1, overexpressing mice are tamoxifen resistant. However, during reproductive senescence, Esr1 mice exhibited responsiveness. Both Esr1 and CYP19A1 are responsive to letrozole before and after reproductive senescence. Gene Set Enrichment Analyses of RNA-sequencing data sets showed that higher disease rates in Esr1 mice were accompanied by significantly higher expression of cell proliferation genes, including members of prognostic platforms for women with early-stage hormone receptor-positive disease. Tamoxifen and letrozole exposure induced down-regulation of these genes and resolved differences between the two models. Both Esr1 and CYP19A1 overexpression induced abnormal developmental patterns of pregnancy-like gene expression. This resolved with progression through reproductive senescence in CYP19A1 mice, but was more persistent in Esr1 mice, resolving only with tamoxifen and letrozole exposure. In summary, genetically engineered mouse models of Esr1 and CYP19A1 overexpression revealed a diversion of disease processes resulting from the two distinct molecular pathophysiological mammary gland-targeted intrusions into estrogen signaling during reproductive senescence.

Overexpression of Estrogen Receptor α in Mammary Glands of Aging Mice Is Associated with a Proliferative Risk Signature and Generation of Estrogen Receptor α-Positive Mammary Adenocarcinomas.

Age is a risk factor for human estrogen receptor-positive breast cancer, with highest prevalence following menopause. While transcriptome risk profiling is available for human breast cancers, it is not yet developed for prognostication for primary or secondary breast cancer development utilizing at-risk breast tissue. Both estrogen receptor α (ER) and aromatase overexpression have been linked to human breast cancer. Herein, conditional genetically engineered mouse models of estrogen receptor 1 (Esr1) and cytochrome P450 family 19 subfamily A member 1 (CYP19A1) were used to show that induction of Esr1 overexpression just before or with reproductive senescence and maintained through age 30 months resulted in significantly higher prevalence of estrogen receptor-positive adenocarcinomas than CYP19A1 overexpression. All adenocarcinomas tested showed high percentages of ER+ cells. Mammary cancer development was preceded by a persistent proliferative transcriptome risk signature initiated within 1 week of transgene induction that showed parallels to the Prosigna/Prediction Analysis of Microarray 50 human prognostic signature for early-stage human ER+ breast cancer. CYP19A1 mice also developed ER+ mammary cancers, but histology was more divided between adenocarcinoma and adenosquamous, with one ER- adenocarcinoma. Results demonstrate that, like humans, generation of ER+ adenocarcinoma in mice was facilitated by aging mice past the age of reproductive senescence. Esr1 overexpression was associated with a proliferative estrogen pathway-linked signature that preceded appearance of ER+ mammary adenocarcinomas.

Urinary phthalate metabolite concentrations and hot flash outcomes: Longitudinal associations in the Midlife Women's Health Study.

Midlife in women is an understudied time for environmental chemical exposures and menopausal outcomes. Recent cross-sectional research links phthalates with hot flashes, but little is known regarding such associations over time. Our objective was to estimate longitudinal associations between repeated measures of urinary phthalate metabolite concentrations and hot flash outcomes in midlife women. Using data from the Midlife Women's Health Study (MWHS), a prospective longitudinal study, we fit generalized linear mixed-effects models (GLMMs) and Cox proportional hazards regression models to repeated measures over a 4-year period. Recruitment occurred in Baltimore and surrounding counties, Maryland, USA between 2006 and 2015. Participants were premenopausal/perimenopausal women (n = 744) aged 45-54 years, who were not pregnant, not taking menopausal symptom medication or oral contraceptives, did not have hysterectomy/oophorectomy, and irrespective of hot flash experience. Baseline mean (SD) age was 48.4 (2.45), and 65% were premenopausal. Main outcome measures included adjusted odds ratios (ORs) for 4 self-reported hot flash outcomes (ever experienced, past 30 days experience, weekly/daily, and moderate/severe), and hazard ratios (HRs) for incident hot flashes. We observed mostly increased odds of certain hot flash outcomes with higher concentrations of metabolites of di (2-ethylhexyl) phthalate (DEHP), monoisobutyl phthalate (MiBP), and a molar summary measure of plasticizer phthalate metabolites (DEHP metabolites, mono-(3-carboxypropyl) phthalate (MCPP), monobenzyl phthalate (MBzP)). Some associations between exposures and outcomes indicated decreased odds. In conclusion, phthalate metabolites were associated with certain hot flash outcomes in midlife women. Midlife may be a sensitive period for higher phthalate metabolite concentrations with respect to menopausal symptoms.

The effects of inhaled pollutants on reproduction in marginalized communities: a contemporary review.

Important differences in health that are closely linked with social disadvantage exist within and between countries. According to the World Health Organization, life expectancy and good health continue to increase in many parts of the world, but fail to improve in other parts of the world, indicating that differences in life expectancy and health arise due to the circumstances in which people grow, live, work, and age, and the systems put in place to deal with illness. Marginalized communities experience higher rates of certain diseases and more deaths compared to the general population, indicating a profound disparity in health status. Although several factors place marginalized communities at high risk for poor health outcomes, one important factor is exposure to air pollutants. Marginalized communities and minorities are exposed to higher levels of air pollutants than the majority population. Interestingly, a link exists between air pollutant exposure and adverse reproductive outcomes, suggesting that marginalized communities may have increased reproductive disorders due to increased exposure to air pollutants compared to the general population. This review summarizes different studies showing that marginalized communities have higher exposure to air pollutants, the types of air pollutants present in our environment, and the associations between air pollution and adverse reproductive outcomes, focusing on marginalized communities.

Effects of prenatal and lactational exposure to iodoacetic acid on the F1 generation of mice†.

Water disinfection can generate water disinfection byproducts (DBPs). Iodoacetic acid (IAA) is one DBP, and it has been shown to be an ovarian toxicant in vitro and in vivo. However, it is unknown if prenatal and lactational exposure to IAA affects reproductive outcomes in female offspring. This study tested the hypothesis that prenatal and lactational exposure to IAA adversely affects reproductive parameters in F1 female offspring. Adult female CD-1 mice were dosed with water (control) or IAA (10, 100, and 500 mg/L) in the drinking water for 35 days and then mated with unexposed males. IAA exposure continued throughout gestation. Dams delivered naturally, and pups were continuously exposed to IAA through lactation until postnatal day (PND) 21. Female pups were euthanized on PND 21 and subjected to measurements of anogenital distance, ovarian weight, and vaginal opening. Ovaries were subjected to histological analysis. In addition, sera were collected to measure reproductive hormone levels. IAA exposure decreased vaginal opening rate, increased the absolute weight of the ovaries, increased anogenital index, and decreased the percentage of atretic follicles in female pups compared to control. IAA exposure caused a borderline decrease in the levels of progesterone and follicle-stimulating hormone (FSH) and increased levels of testosterone in female pups compared to control. Collectively, these data show that prenatal and lactational exposure to IAA in drinking water affects vaginal opening, anogenital index, the weight of the ovaries, the percentage of atretic follicles, and hormone levels in the F1 generation in mice.

Associations of maternal anthropometrics with newborn anogenital distance and the 2:4 digit ratio.

STUDY QUESTION: Are maternal anthropometrics associated with anogenital distance (AGD) and 2:4 digit ratio (2:4D) in newborns? SUMMARY ANSWER: Select maternal anthropometrics indicative of obesity or increased adiposity are associated with elongated AGD in daughters. WHAT IS KNOWN ALREADY: Excessive maternal weight or adiposity before or in early pregnancy may impact child reproductive, and other hormonally mediated, development. AGD and 2:4D are proposed markers of in utero reproductive development. STUDY DESIGN, SIZE, DURATION: This study includes 450 mother/newborn dyads participating in the Illinois Kids Development Study (I-KIDS), a prospective pregnancy cohort from Champaign-Urbana, IL, USA. Participants included in the current study enrolled between 2013 and 2018. PARTICIPANTS/MATERIALS, SETTING, METHODS: Most mothers in this study were college-educated (82%) and non-Hispanic White (80%), and 55% were under- or normal weight before pregnancy. Pregnant women aged 18-40 years reported pre-pregnancy weight and height to calculate pre-pregnancy BMI. At 8-15 weeks gestation, we measured waist and hip circumference, and evaluated weight, % body fat, visceral fat level, % muscle and BMI using bioelectrical impedance analysis. Within 24 h of birth, we measured newborn 2nd and 4th left/right digits to calculate the 2:4D. In daughters, we measured AGDAF (anus to fourchette) and AGDAC (anus to clitoris). In sons, we measured AGDAS (anus to scrotum) and AGDAP (anus to base of the penis). MAIN RESULTS AND THE ROLE OF CHANCE: Select maternal anthropometrics were positively associated with AGD in newborn daughters, but not sons. For example, AGDAC was 0.73 mm (95% CI: 0.15, 1.32) longer for every interquartile range (IQR) increase in pre-pregnancy BMI and 0.88 mm (95% CI: 0.18, 1.58) longer for every IQR increase in hip circumference, whereas AGDAF was 0.51 mm (95% CI: 0.03, 1.00) and 0.56 mm (95% CI: 0.03, 1.09) longer for every IQR increase in hip and waist circumference, respectively. Quartile analyses generally supported linear associations, but additional strong associations emerged in Q4 (versus Q1) of maternal % body fat and visceral fat levels with AGDAC. In quartile analyses, we observed only a few modest associations of maternal anthropometrics with 2:4D, which differed by hand (left versus right) and newborn sex. Although there is always the possibility of spurious findings, the associations for both measures of female AGD were consistent across multiple maternal anthropometric measures, which strengthens our conclusions. LIMITATIONS, REASONS FOR CAUTION: Our study sample was racially and ethnically homogenous, educated and relatively healthy, so our study may not be generalizable to other populations. Additionally, we may not have been powered to identify some sex-specific associations, especially for 2:4D. WIDER IMPLICATIONS OF THE FINDINGS: Increased maternal weight and adiposity before and in early pregnancy may lengthen the female AGD, which warrants further investigation. STUDY FUNDING/COMPETING INTEREST(S): This publication was made possible by the National Institute for Environmental Health Sciences (NIH/NIEHS) grants ES024795 and ES022848, the National Institute of Child Health and Human Development grant R03HD100775, the U.S. Environmental Protection Agency grant RD83543401 and National Institute of Health Office of the Director grant OD023272. Its contents are solely the responsibility of the grantee and do not necessarily represent the official views of the US EPA or NIH. Furthermore, the US EPA does not endorse the purchase of any commercial products or services mentioned in the publication. This project was also supported by the USDA National Institute of Food and Agriculture and Michigan AgBioResearch. The authors declare no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Constitutive expression of Steroidogenic factor-1 (NR5A1) disrupts ovarian functions, fertility, and metabolic homeostasis in female mice.

Steroid hormones regulate various aspects of physiology, from reproductive functions to metabolic homeostasis. Steroidogenic factor-1 (NR5A1) plays a central role in the development of steroidogenic tissues and their ability to produce steroid hormones. Inactivation of Nr5a1 in the mouse results in a complete gonadal and adrenal agenesis, absence of gonadotropes in the pituitary and impaired development of ventromedial hypothalamus, which controls glucose and energy metabolism. In this study, we set out to examine the consequences of NR5A1 overexpression (NR5A1+) in the NR5A1-positive cell populations in female mice. Ovaries of NR5A1+ females presented defects such as multi-oocyte follicles and an accumulation of corpora lutea. These females were hyperandrogenic, had irregular estrous cycles with persistent metestrus and became prematurely infertile. Furthermore, the decline in fertility coincided with weight gain, increased adiposity, hypertriglyceridemia, hyperinsulinemia, and impaired glucose tolerance, indicating defects in metabolic functions. In summary, excess NR5A1 expression causes hyperandrogenism, disruption of ovarian functions, premature infertility, and disorders of metabolic homeostasis. This NR5A1 overexpression mouse provides a novel model for studying not only the molecular actions of NR5A1, but also the crosstalk between endocrine, reproductive, and metabolic systems.

Determinants of urinary phthalate biomarker concentrations in pre- and perimenopausal women with consideration of race.

BACKGROUND/OBJECTIVES: Phthalates are endocrine disruptors in consumer plastics and personal care products. Our objectives were to identify determinants of phthalate biomarkers in women during the hormonally-sensitive midlife period, and to consider differences between non-Hispanic White and Black women. METHODS: We used information from the Midlife Women's Health Study of pre- and peri-menopausal women from Baltimore, Maryland (enrolled 2006-2015). We collected sociodemographic/health information via baseline questionnaires or during clinic visits and measured nine phthalate metabolites in pools of 2-4 urines collected across one menstrual cycle. We calculated molar sums of metabolites to estimate exposure to di(2-ethylhexyl) phthalate (ΣDEHP), personal care product phthalates (ΣPCPs), and phthalates in plastics (ΣPlastics). Accounting for meaningful predictors from bivariable analyses, our multivariable linear regression models evaluated determinants of phthalate biomarkers in all women (n = 689), non-Hispanic White women only (n = 467), or non-Hispanic Black women only (n = 195). RESULTS: In multivariable analyses of all women, those who were perimenopausal, widowed/divorced, non-Hispanic Black, with higher family income, with lower BMI, or who reported more frequent nausea had higher monoethyl phthalate (MEP) and ΣPCP. Non-Hispanic White women who were perimenopausal had lower mono-(3-carboxypropyl) phthalate (MCPP) and monobutyl phthalate (MBP), those who consume alcohol had higher mono-isobutyl phthalate (MiBP), and those with higher BMI had lower MEP and higher MCPP. Alternatively, widowed/divorced Black women had higher ΣDEHP, monobenzyl phthalate (MBzP), and ΣPlastics, whereas Black women with higher income had higher MEP and ΣPCP. Black women who described themselves as having "as much" physical activity as others or who reported a skin condition had lower MBzP and MCPP, respectively. CONCLUSION: We identified important determinants of phthalate biomarkers in midlife women and observed some differences by race. Future studies could consider reasons for these differences when developing interventions to reduce phthalate disparities and related health effects.

Effects of a phthalate metabolite mixture on both normal and tumoral human prostate cells.

Phthalates represent a group of substances used in industry that have antiandrogenic activity and are found in different concentrations in human urine and plasma. More than 8 million tons of phthalates are used each year, predominantly as plasticizers in polyvinyl chloride (PVC) products. Phthalates are widely used in everyday consumer products and improperly discarded into the environment. Furthermore, in vivo studies carried out in our laboratory showed that a mixture of phthalates, equivalent to the mixture used in this study, deregulated the expression of genes and miRNAs associated with prostatic carcinogenic pathways. Thus, this study was designed to establish an in vitro model to assess pathways related to cell survival, proliferation, apoptosis, and biosynthesis of miRNAs, using both normal and tumoral prostatic epithelial cells exposed to an environmentally relevant mixture of phthalate metabolites. Tumor (LNCaP) and normal (PNT-2) prostatic epithelial cell lines were exposed for 24 and 72 h to vehicle control or the phthalate mixture. The selected metabolite mixture (1000 µmol/L) consisted of 36.7% monoethyl phthalate (MEP), 19.4% mono(2-ethylhexyl) phthalate (MEHP), 15.3% monobutyl phthalate (MBP), 10.2% monoisobutyl phthalate (MiBP), 10.2% monoisononyl phthalate (MiNP), and 8.2% monobenzyl phthalate (MBzP). Gene expression was performed by qRT-PCR and cell migratory potential was measured using cell migration assays. Our results showed that the mixture of phthalates increased cell turnover, oxidative stress, biosynthesis, and expression of miRNAs in LNCaP cells; thus, increasing their cellular expansive and migratory potential and modulating tumor behavior, making them possibly more aggressive. However, these effects were less pronounced in benign cells, demonstrating that, in the short term, benign cells are able to develop effective mechanisms or more resistance against the insult.

Iodoacetic acid exposure alters the transcriptome in mouse ovarian antral follicles.

Iodoacetic acid (IAA) is an unregulated water disinfection byproduct that is an ovarian toxicant. However, the mechanisms of action underlying IAA toxicity in ovarian follicles remain unclear. Thus, we determined whether IAA alters gene expression in ovarian follicles in mice. Adult female mice were dosed with water or IAA (10 or 500 mg/L) in the water for 35-40 days. Antral follicles were collected for RNA-sequencing analysis and sera were collected to measure estradiol. RNA-sequencing analysis identified 1063 differentially expressed genes (DEGs) in the 10 and 500 mg/L IAA groups (false discovery rate FDR < 0.1), respectively, compared to controls. Gene Ontology Enrichment analysis showed that DEGs were involved with RNA processing and regulation of angiogenesis (10 mg/L) and the cell cycle and cell division (500 mg/L). Pathway Enrichment analysis showed that DEGs were involved in the phosphatidylinositol 3-kinase and protein kinase B (PI3K-Akt), gonadotropin-releasing hormone (GnRH), estrogen, and insulin signaling pathways (10 mg/L). Pathway Enrichment analysis showed that DEGs were involved in the oocyte meiosis, GnRH, and oxytocin signaling pathways (500 mg/L). RNA-sequencing analysis identified 809 DEGs when comparing the 500 and 10 mg/L IAA groups (FDR < 0.1). DEGs were related to ribosome, translation, mRNA processing, oxidative phosphorylation, chromosome, cell cycle, cell division, protein folding, and the oxytocin signaling pathway. Moreover, IAA exposure significantly decreased estradiol levels (500 mg/L) compared to control. This study identified key candidate genes and pathways involved in IAA toxicity and can help to further understand the molecular mechanisms of IAA toxicity in ovarian follicles.

Iodoacetic acid affects estrous cyclicity, ovarian gene expression, and hormone levels in mice†.

Iodoacetic acid (IAA) is a water disinfection byproduct that is an ovarian toxicant in vitro. However, information on the effects of IAA on ovarian function in vivo was limited. Thus, we determined whether IAA exposure affects estrous cyclicity, steroidogenesis, and ovarian gene expression in mice. Adult CD-1 mice were dosed with water or IAA (0.5-500 mg/L) in the drinking water for 35-40 days during which estrous cyclicity was monitored for 14 days. Ovaries were analyzed for expression of apoptotic factors, cell cycle regulators, steroidogenic factors, estrogen receptors, oxidative stress markers, and a proliferation marker. Sera were collected to measure pregnenolone, androstenedione, testosterone, estradiol, inhibin B, and follicle-stimulating hormone (FSH) levels. IAA exposure decreased the time that the mice spent in proestrus compared to control. IAA exposure decreased expression of the proapoptotic factor Bok and the cell cycle regulator Ccnd2 compared to control. IAA exposure increased expression of the proapoptotic factors Bax and Aimf1, the antiapoptotic factor Bcl2l10, the cell cycle regulators Ccna2, Ccnb1, Ccne1, and Cdk4, and estrogen receptor Esr1 compared to control. IAA exposure decreased expression of Sod1 and increased expression of Cat, Gpx and Nrf2. IAA exposure did not affect expression of Star, Cyp11a1, Cyp17a1, Hsd17b1, Hsd3b1, Esr2, or Ki67 compared to control. IAA exposure decreased estradiol levels, but did not alter other hormone levels compared to control. In conclusion, IAA exposure alters estrous cyclicity, ovarian gene expression, and estradiol levels in mice.

Environmentally relevant mixtures of phthalates and phthalate metabolites differentially alter the cell cycle and apoptosis in mouse neonatal ovaries†.

Phthalates are a group of chemicals used as additives in various consumer products, medical equipment, and personal care products. Phthalates and their metabolites are consistently detected in humans, indicating widespread and continuous exposure to multiple phthalates. Thus, environmentally relevant mixtures of phthalates and phthalate metabolites were investigated to determine the effects of phthalates on the function of the ovary during the neonatal period of development. Neonatal ovaries from CD-1 mice were cultured with dimethyl sulphoxide (DMSO; vehicle control), phthalate mixture (0.1-100 µg/mL), or phthalate metabolite mixture (0.1-100 µg/mL). The phthalate mixture was composed of 35% diethyl phthalate, 21% di(2-ethylhexyl) phthalate, 15% dibutyl phthalate, 15% diisononyl phthalate, 8% diisobutyl phthalate, and 5% benzylbutyl phthalate. The phthalate metabolite mixture was composed of 37% monoethyl phthalate, 19% mono(2-ethylhexyl) phthalate, 15% monobutyl phthalate, 10% monoisononyl phthalate, 10% monoisobutyl phthalate, and 8% monobenzyl phthalate. After 96 h of culture, ovaries were harvested for histological analysis of folliculogenesis, gene expression analysis of cell cycle and apoptosis regulators, and immune staining for cell proliferation and apoptosis. The metabolite mixture significantly decreased the number and percentage of abnormal follicles (100 µg/mL) compared to controls. The metabolite mixture also significantly increased the expression of cell cycle inhibitors (100 µg/mL) and the antiapoptotic factor Bcl2l10 (10 µg/mL) compared to controls. The phthalate mixture did not significantly alter gene expression or follicle counts, but ovaries exposed to the phthalate mixture (0.1 µg/mL) exhibited marginally significantly increased apoptosis as revealed by DNA fragmentation staining. Overall, these data show that parent phthalates and phthalate metabolites differentially impact ovarian function.

REPRODUCTIVE TOXICOLOGY: Effects of chemical mixtures on the ovary.

The ovaries play a critical role in female reproductive health because they are the site of oocyte maturation and sex steroid hormone production. The unique cellular processes that take place within the ovary make it a susceptible target for chemical mixtures. Herein, we review the available data regarding the effects of chemical mixtures on the ovary, focusing on development, folliculogenesis, and steroidogenesis. The chemical mixtures discussed include those to which women are exposed to environmentally, occupationally, and medically. Following a brief introduction to chemical mixture components, we describe the effects of chemical mixtures on ovarian development, folliculogenesis, and steroidogenesis. Further, we discuss the effects of chemical mixtures on corpora lutea and transgenerational outcomes. Identifying the effects of chemical mixtures on the ovaries is paramount to preventing and treating mixture-inducing toxicity of the ovary that has long-term consequences such as infertility and ovarian disease.

Phthalate exposures and one-year change in body mass index across the menopausal transition.

BACKGROUND: The menopausal transition is a hormonally sensitive period associated with changes in body weight. Phthalates are ubiquitous endocrine disrupting chemicals that could disrupt weight homeostasis, but it is unknown whether this occurs during the menopausal transition. OBJECTIVES: Our objectives were to (1) determine if phthalate exposure in pre- and perimenopausal women was associated with one-year change in body mass index (BMI), and (2) determine if these associations differed across the menopausal transition. METHODS: We addressed our objectives using data from 524 participants enrolled in the Midlife Women's Health Study. We calculated change in BMI from baseline to first follow-up visit approximately one year later. Phthalate exposures were approximated by measuring urinary metabolites in pools of two-to-four spot urine samples collected across a four-week period at baseline. We molar-converted and summed mono-(2-ethylhexyl) phthalate (mEHP), mono (2-ethyl-5-hydroxyhexyl) phthalate (mEHHP), mono (2-ethyl-5-oxohexyl) phthalate (mEOHP), and mono (2-ethyl-5-carboxypentyl) phthalate (mECPP) to approximate exposure to di-(2-ethylhexyl) phthalate (∑DEHP); ∑DEHP, mono (3-carboxypropyl) phthalate (mCPP), and monobenzyl phthalate (mBzP) to approximate exposure to plasticizer phthalates (∑Plastics); and monoethyl phthalate (mEP), monobutyl phthalate (mBP), and monoisobutyl phthalate (miBP) to approximate exposure to phthalates from personal care products (∑PCP). We used multivariable linear regression models to evaluate associations of specific gravity-adjusted ln-transformed phthalate metabolites or sums with one-year BMI change, and also considered whether associations differed depending on each woman's menopausal status change from baseline to first follow-up. RESULTS: At baseline, most women were premenopausal (67.8%), non-Hispanic white (67.9%), and college educated (65.8%). Overall, urinary phthalate metabolites or sums were not associated with one-year BMI change. Stratified analysis identified positive associations between ∑DEHP (and three of its metabolites: MEHP, MEHHP, and MEOHP) and one-year BMI change among women who transitioned from peri-to post-menopause from baseline to first follow-up. For example, in these women, with each doubling of ∑DEHP, BMI increased by 0.65 kg/m2 (95%CI: 0.17, 1.13) from baseline to first follow-up. Personal care product-associated phthalate metabolites (mBP and mEP) were negatively associated with one-year BMI change among women who remained perimenopausal from baseline to first follow-up, while miBP and mEP were positively associated with one-year BMI change among women who transitioned from peri-to post-menopause. CONCLUSION: We found the strongest associations between some phthalates and one-year BMI change in women who transitioned from peri-to post-menopause from baseline to first follow-up. This supports previous evidence that the menopausal transition is a hormonally sensitive period in women's lives. To establish whether phthalate exposure contributes to body weight changes associated with the menopausal transition, substantially more research is needed to corroborate our findings.

Urinary phthalate metabolite concentrations and hot flashes in women from an urban convenience sample of midlife women.

CONTEXT: Phthalate exposure is associated with altered reproductive function, but little is known about associations of phthalate exposure with risk of hot flashes. OBJECTIVE: To investigate associations of urinary phthalate metabolite levels with four hot flash outcomes in midlife women. DESIGN: A cross-sectional study of the first year of a prospective cohort of midlife women, the Midlife Women's Health Study (2006-2015), a convenience sample from an urban setting. PARTICIPANTS: 728 multi-racial/ethnic pre- and perimenopausal women aged 45-54 years. OUTCOME MEASURES: Women completed questionnaires about hot flash experience and provided 1-4 urine samples over four consecutive weeks that were pooled for analysis. Phthalate metabolites were assessed individually and as molar sums representative of common compounds (all phthalates: Æ©Phthalates; DEHP: Æ©DEHP), exposure sources (plastics: Æ©Plastic; personal care products: Æ©PCP), and modes of action (anti-androgenic: Æ©AA). Covariate-adjusted logistic regression models were used to assess associations of continuous natural log-transformed phthalate metabolite concentrations with hot flash outcomes. Analyses were conducted to explore whether associations differed by menopause status, body mass index (BMI), race/ethnicity, and depressive symptoms. RESULTS: Overall, 45% of women reported a history of hot flashes. Compared to women who never experienced hot flashes, every two-fold increase in Æ©Plastic was associated with 18% (OR: 1.18; 95%CI: 0.98, 1.43) and 38% (OR: 1.38; 95%CI: 1.11, 1.70) higher odds of experiencing hot flashes in the past 30 days and experiencing daily/weekly hot flashes, respectively. Some associations of phthalates with certain hot flash outcomes differed by menopause status, BMI, race/ethnicity, and depressive symptoms. CONCLUSIONS: This study suggests that phthalates are associated with hot flash experience and may impact hot flash risk in women who are susceptible to experiencing hot flashes.