The female reproductive system ages before any other physiological system, making it a sensitive indicator of aging. Early reproductive aging is associated with the early onset of infertility and an increased risk of several diseases. During aging, systemic and reproductive oxidative stress and inflammation levels increase through inflammasome activation, leading to ovarian follicle loss. Other markers of reproductive aging include increased fibrosis and shortening of telomeres in ovarian cells. The factors that accelerate reproductive aging are unclear, but likely involve exposure to endocrine-disrupting chemicals such as phthalates. Di(2-ethylhexyl) phthalate (DEHP) is a widely used phthalate and humans are exposed to it daily. Several studies show that DEHP induces reproductive toxicity by affecting estrous cyclicity, follicle numbers, and hormone levels. However, little is known about the mechanisms underlying DEHP-induced early onset of reproductive aging. Thus, this study tested the hypothesis that dietary exposure to DEHP induces early reproductive aging by affecting inflammation, fibrosis, and the expression of telomere regulators and antioxidant enzymes. Adult CD-1 female mice were exposed to vehicle (corn oil) or DEHP (0.5, 1.5, or 1500 ppm) via the chow for 6 months. Exposure to DEHP increased the expression of antioxidant enzymes and Caspase 3, increased expression of telomere-associated genes, and increased fibrosis levels in the ovary. In addition, DEHP exposure for 6 months altered ovarian and systemic inflammatory status. Collectively, our novel data suggest that 6-month dietary exposure to DEHP may accelerate reproductive aging by affecting several reproductive aging markers 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.

Genomic imprinting in the brain is theorized to provide parental control over offspring social behaviors. Noncanonical genomic imprinting is a form of epigenetic regulation in which one of a gene’s alleles, either that of maternal or paternal inheritance, exhibits a bias towards higher expression of one parental allele compared to the other. This bias can differ depending on tissue type, and the degree of the parental allele expression bias can even vary across anatomical domains within the same tissue. Dopa decarboxylase ( Ddc ) and tyrosine hydroxylase ( Th ) are both noncanonically imprinted genes that preferentially express their maternal alleles in the brain and Ddc also has a paternal allele expression bias in the periphery. These two genes encode catecholamine synthesis enzymes for the production of dopamine (DA), norepinephrine (NE), and epinephrine (E), and Ddc is also in the serotonin (5-HT) synthesis pathway. These four neurotransmitters are critical regulators of social behavior and disruptions to them are implicated in human mental illnesses. Here we investigated the functional effects of noncanonical imprinting of Ddc and Th on social behavior in mice. By using reciprocal heterozygous mutant mice, we tested the impacts of Ddc and/or Th maternally and paternally inherited alleles on aggression, social recognition, dominance, and social preference behaviors. We found that Ddc paternal-null alleles affect aggression and social recognition behavior, Th maternal-null alleles affect sociability preferences, and compound inheritance of Th and Ddc maternal-null alleles influence preferences for social novelty. These results are consistent with Th and Ddc maternal allele biased expression in central monoaminergic systems regulating sociability, and Ddc paternal allele biased expression in peripheral monoaminergic systems regulating aggression and social recognition.### Competing Interest StatementThe authors have declared no competing interest.

Phthalates are endocrine-disrupting chemicals used in consumer products. Although phthalates are obesogens and affect metabolic function, it is unknown if chronic exposure for 6 months to a phthalate mixture alters adipose tissue phenotype in female mice. After vehicle or mixture exposure, white adipose tissue and brown adipose tissue (WAT and BAT) were analyzed for expression of adipogenesis, proliferation, angiogenesis, apoptosis, oxidative stress, inflammation, and collagen deposition markers. The mixture altered WAT morphology, leading to an increase in hyperplasia, blood vessel number, and expression of BAT markers (Adipoq and Fgf2) in WAT. The mixture increased the expression of the inflammatory markers, Il1 beta, Ccl2, and Ccl5, in WAT. The mixture also increased expression of the proapoptotic (Bax and Bcl2) and antiapoptotic (Bcl2l10) factors in WAT. The mixture increased expression of the antioxidant Gpx1 in WAT. The mixture changed BAT morphology by increasing adipocyte diameter, whitening area, and blood vessel number and decreased expression of the thermogenic markers Ucp1, Pgargc1a, and Adrb3. Furthermore, the mixture increased the expression of adipogenic markers Plin1 and Cebpa, increased mast cell number, and increased Il1 beta expression in BAT. The mixture also increased expression of the antioxidant markers Gpx and Nrf2 and the apoptotic marker Casp2 in BAT. Collectively, these data indicate that chronic exposure to a phthalate mixture alters WAT and BAT lipid metabolism phenotypes in female mice, leading to an apparent shift in their normal morphology. Following long-term exposure to a phthalate mixture, WAT presented BAT-like features and BAT presented WAT-like features.

Polychlorinated biphenyls (PCBs) were used in industrial applications until they were banned in the 1970s, but they still persist in the environment. Little is known about the long-term effects of exposure to PCB mixtures on the rat ovary during critical developmental periods. Thus, this study tested whether prenatal and postnatal exposures to PCBs affect follicle numbers and gene expression in the ovaries of F1 offspring. Sprague-Dawley rats were treated with vehicle or Aroclor 1221 (A1221) at 1 mg/kg/day during embryonic days 8-18 and/or postnatal days (PND) 1-21. Ovaries from F1 rats were collected for assessment of follicle numbers and differential expression of estrogen receptor 1 (Esr1), estrogen receptor 2 (Esr2), androgen receptor (Ar), progesterone receptor (Pgr), and Ki-67 (Ki67) at PNDs 8, 32, and 60. Sera were collected for measurement of estradiol concentrations. Prenatal exposure to A1221 significantly decreased the number of primordial follicles and the total number of follicles at PND 32 compared to control. Postnatal PCB exposure borderline increased Ki67 gene expression and significantly increased Ki67 protein levels (PND 60) compared to control. Combined prenatal and postnatal PCB exposure borderline decreased Ar expression (PND 8) compared to control. However, PCB exposure did not significantly affect the expression of Pgr, Esr1, and Esr2 or serum estradiol concentrations compared to control at any time point. In conclusion, these data suggest that PCB exposure affects follicle numbers and levels of the proliferation marker Ki67, but it does not affect expression of some sex steroid hormone receptors in the rat ovary.

Phthalates are chemicals ubiquitously used in industry. Individual phthalates have been found to adversely affect female reproduction; however, humans are exposed to a mixture of phthalates daily, primarily through ingestion. Previous studies show that exposure to an environmentally relevant mixture of phthalates (Mix) can affect female reproduction. Little research, however, has been conducted on the effects of short-term (1 month) and long-term (6 months) exposure to Mix on ovarian functions. Thus, this study tested the hypothesis that short-term and longterm exposure to Mix alters ovarian folliculogenesis, serum hormone concentrations, pituitary gene expression, and ovarian expression of genes involved in steroidogenesis, apoptosis, cell cycle regulation, and oxidative stress. Adult CD-1 female mice were exposed to vehicle control (corn oil) or Mix (0.15-1500 ppm) in the chow for 1 or 6 months. Exposure to Mix for 1 month increased the number of atretic follicles (0.15 ppm), altered ovarian gene expression (0.15 ppm, 1500 ppm), and decreased serum testosterone (1.5 ppm) compared to control. Exposure to Mix for 6 months increased serum follicle-stimulating hormone (FSH) (0.15 ppm), decreased serum luteinizing hormone (LH) (0.15 ppm, 1.5 ppm, and 1500 ppm), decreased serum estradiol (1500 ppm), altered pituitary gene expression (1500 ppm), increased the number (1500 ppm) and percentage (1.5 ppm and 1500 ppm) of primordial follicles, and decreased the percentage of preantral (1500 ppm) and antral (1.5 ppm and 1500 ppm) follicles compared to control. These data indicate that exposure to Mix can alter folliculogenesis, steroidogenesis, and gene expression in female mice.

Phthalates are found in plastic food containers, medical plastics, and personal care products. However, the effects of long-term phthalate exposure on female reproduction are unknown. Thus, this study investigated the effects of long-term, dietary phthalate exposure on estrous cyclicity and fertility in female mice. Adult female CD-1 mice were fed chow containing vehicle control (corn oil) or 0.15-1500 ppm of di(2-ethylhexyl) phthalate (DEHP), diisononyl phthalate (DiNP), or a mixture of phthalates (Mix) containing DEHP, DiNP, benzyl butyl phthalate, di-n-butyl phthalate, diisobutyl phthalate, and diethyl phthalate. Measurements of urinary phthalate metabolites confirmed effective delivery of phthalates. Phthalate consumption for 11 months did not affect body weight compared to control. DEHP exposure at 0.15 ppm for 3 and 5 months increased the time that the mice spent in estrus and decreased the time the mice spent in metestrus/diestrus compared to control. DiNP exposure (0.15-1500 ppm) did not significantly affect time in estrus or metestrus/diestrus compared to control. Mix exposure at 0.15 and 1500 ppm for 3 months decreased the time the mice spent in metestrus/diestrus and increased the time the mice spent in estrus compared to control. DEHP (0.15-1500 ppm) or Mix (0.15-1500 ppm) exposure did not affect fertility-related indices compared to control. However, long-term DiNP exposure at 1500 ppm significantly reduced gestational index and birth rate compared to control. These data indicate that chronic dietary exposure to phthalates alters estrous cyclicity, and long-term exposure to DiNP reduces gestational index and birth rate in mice.

Perfluorooctanoic acid (PFOA) is a synthetic fluorosurfactant used in the manufacturing of fluorotelomers. Although PFOA is no longer produced in the United States, it is environmentally persistent and found in imported food packaging, cookware, and textiles. Previous studies have identified developmental toxicity of PFOA, but little is known about the effects of PFOA on the adult ovary. Thus, this study examined the effects of PFOA on hormone levels, ovarian steroidogenic gene expression, and folliculogenesis in mice in vitro and in vivo. For the in vitro studies, antral follicles from adult female mice were cultured with vehicle control or 1, 10, or 100 mu g/ml PFOA for 96 h. For the in vivo studies, adult CD-1 female mice were orally dosed with vehicle control or 1, 5, 10, or 20 mg/kg/day PFOA for 10 days. Gene expression of steroidogenic enzymes, levels of sex steroid hormones, and follicle counts were analyzed. In vitro, PFOA (100 mu g/ml) significantly decreased follicle growth, estradiol and estrone levels, and gene expression of StaR, Cyp11a1, and Hsd3b1 compared with controls. In vivo, exposure to PFOA significantly decreased progesterone and pregnenolone levels (5 mg/kg), increased testosterone levels (1 mg/kg), and increased gene expression of Cyp19a1 (1 mg/kg) compared with controls. Exposure to PFOA also significantly altered follicle counts by decreasing primordial follicles and increasing preantral and antral follicles (5 and 10 mg/kg) compared with controls. Collectively, these data show that PFOA disrupts adult ovarian function in a nonmonotonic matter and may pose a risk for premature ovarian failure.

Widespread use of phthalates as plasticizers in everyday products has led to substantial increase in human exposure. Phthalate metabolites have been shown to have detrimental reproductive health effects on both men and women, with the exact mechanism of toxicity remaining elusive. In this study, we tested the hypothesis that the phthalate metabolites act through peroxisome proliferator activated receptor gamma (PPARγ) using a dual pronged in silico-in vitro approach. Incorporating a Gaussian accelerated molecular dynamics (GaMD) string method, we explored the conformational landscape of PPAR. The resultant trajectory data was then used as a framework for ensemble molecular docking of physiologically relevant phthalate metabolites: monononyl phthalate (MNP), monoisononyl phthalate (MiNP), mono(2-ethylhexyl) phthalate (MEHP), monobenzyl phthalate (MBzP), monobutyl phthalate (MBP), monoisobutyl phthalate (MiBP), and monoethyl phthalate (MEP). High-scoring docked poses on PPARγ from Autodock Vina, MOE, and GLIDE, revealed that a salt bridge between the phthalate carboxylate ester, produced post metabolism, with R288 stabilizes the phthalate metabolites, occupying the ligand binding domain of PPARγ. Productive phthalate poses obtained post clustering were assessed for stability by additional molecular dynamics simulations, with their attenuation effects on protein dynamics gauged via a dynamical network and fluctuation analyses. Additionally, given the high structural similarity of phthalates, ligand relative binding free energies were then quantified using alchemical methods. Experimentally, we show that treatment of granulosa cell cultures with the phthalates led to significant changes in expression of the PPARγ target Fabp4 and to significant increases in activity of luciferase under the control of a PPAR response element. Collectively, these data suggest that certain phthalate metabolites may act through PPARγ as competitive inhibitors for endogenous fatty acids stabilized through R288.

Widespread use of phthalates as solvents and plasticizers leads to everyday human exposure. The mechanisms by which phthalate metabolites act as ovarian toxicants are not fully understood. Thus, this study tested the hypothesis that the phthalate metabolites monononyl phthalate (MNP), monoisononyl phthalate (MiNP), mono(2ethylhexyl) phthalate (MEHP), monobenzyl phthalate (MBzP), monobutyl phthalate (MBP), monoisobutyl phthalate (MiBP), and monoethyl phthalate (MEP) act through peroxisome proliferator-activated receptors (PPARs) in mouse granulosa cells. Primary granulosa cells were isolated from CD-1 mice and cultured with vehicle control (dimethyl sulfoxide) or MNP, MiNP, MEHP, MBzP, MBP, MiBP, or MEP (0.4-400 mu M) for 24 h. Following culture, qPCR was performed for known PPAR targets, Fabp4 and Cd36. Treatment with the phthalate metabolites led to significant changes in Fabp4 and Cd36 expression relative to control in dose-dependent or nonmonotonic fashion. Primary granulosa cell cultures were also transfected with a DNA plasmid containing luciferase expressed under the control of a consensus PPAR response element. MNP, MiNP, MEHP, and MBzP caused dose-dependent changes in expression of luciferase, indicating the presence of functional endogenous PPAR receptors in the granulosa cells that respond to phthalate metabolites. The effects of phthalate metabolites on PPAR target genes were inhibited in most of the cultures by co-treatment with the PPAR-gamma inhibitor, T0070907, or with the PPAR-alpha inhibitor, GW6471. Collectively, these data suggest that some phthalate metabolites may act through endogenous PPAR nuclear receptors in the ovary and that the differing structures of the phthalates result in different levels of activity.

Phthalates are a family of chemicals that can be found in plastic and personal care products used by consumers every day and they are known endocrine disrupting chemicals that can disrupt female reproduction. In previous studies, an environmentally relevant phthalate mixture was shown to affect female reproduction in a trans generational manner. However, limited information was available on the effect of phthalate mixtures on ovarian steroidogenesis and folliculogenesis. Ovarian steroidogenesis is important for producing hormones needed for reproduction and ovarian regulation, and folliculogenesis is essential for the development of ovarian follicles and successful fertility. Thus, this study tested the hypothesis that prenatal exposure to an environmentally relevant phthalate mixture adversely affects ovarian steroidogenesis and folliculogenesis in the F1 generation of adult female mice. Pregnant dams (F0 generation) were orally dosed with vehicle control or a phthalate mixture (20 mu g/kg/day-500 mg/kg/day) daily from gestational day 10 to birth, and the adult F1 females were the offspring of the dosed dams. The ovaries of the F1 generation were collected at postnatal day 60. One ovary was used for histological examination of the numbers and percent of different follicle types. The other ovary was used to measure expression of theca and granulosa cell enzymes. Additionally, sera were collected for measuring hormone levels. The results indicate that prenatal exposure to the phthalate mixture decreases hormone levels and gene expression, alters the transitioning of follicle types, and leads to a higher incidence of atresia in the F1 generation offspring.

Di(2-ethylhexyl) phthalate (DEHP) is a chemical commonly used as a plasticizer to render polyvinyl chloride products more durable and flexible. Although exposure to DEHP has raised many health concerns due to the identification of DEHP as an endocrine disruptor, it is still used in consumer products, including polyvinyl chloride plastics, medical tubing, car interiors, and children's toys. To investigate the impact of early life exposure to DEHP on the ovary and testes, newborn piglets were orally dosed with DEHP (20 or 200 mg/kg/day) or vehicle control (tocopherol-stripped corn oil) for 21 days. Following treatment, ovaries, testes, and sera were harvested for histological assessment and measurement of steroid hormone levels. In male piglets, progesterone and pregnenolone levels were significantly lower in both treatment groups compared to control, whereas in female piglets, progesterone was significantly higher in the 20 mg group compared to control, indicating sex-specific effects in a non-monotonic manner. Follicle numbers and gene expression of steroidogenic enzymes and apoptotic factors were not altered in treated ovaries compared to controls. In DEHP-treated testes, germ cell migration was impaired and germ cell death was significantly increased compared to controls. Overall, the results of this study suggest that neonatal exposure to DEHP in pigs leads to sex-specific disruption of the reproductive system.

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: ƩPhthalates; DEHP: ƩDEHP), exposure sources (plastics: ƩPlastic; personal care products: ƩPCP), and modes of action (anti-androgenic: ƩAA). 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 ƩPlastic 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.

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 Bcl2110, 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.

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 mu g/mL), or phthalate metabolite mixture (0.1-100 mu g/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 mu g/mL) compared to controls. The metabolite mixture also significantly increased the expression of cell cycle inhibitors (100 mu g/mL) and the antiapoptotic factor Bcl2l10 (10 mu g/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 mu g/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. Summary sentence Environmentally relevant mixtures of phthalates and phthalate metabolites disrupt the cell cycle and increase apoptosis in neonatal mouse ovaries.

Background: Phthalate exposure is associated with altered reproductive function, but little is known about associations between phthalate and hormone levels in midlife women. Methods: This cross-sectional analysis includes 45-54-year-old pre- and perimenopausal women from Baltimore, MD and its surrounding counties enrolled in the Midlife Women's Health Study (n = 718). Serum and urine samples were collected from participants once a week for four consecutive weeks to span the menstrual cycle. Serum samples were assayed for estradiol, testosterone, progesterone, sex hormone binding globulin (SHBG), follicle-stimulating hormone (FSH), and anti-Mullerian hormone (AMH), and geometric means were calculated for each hormone across all four weeks. Urine samples were analyzed for nine phthalate metabolites from pools of one-to-four urine samples. Phthalate metabolite concentrations were specific gravity-adjusted and assessed as individual metabolites or as molar sums of metabolites from common parents (di(2-ethylhexyl) phthalate metabolites, FDEHP), exposure sources (plastic, FPlastics; personal care products, FPCP), biological activity (anti-androgenic, FAA), and sum of all metabolites (FPhthalates). We used linear regression models to assess overall associations of phthalate metabolites with hormones, controlling for important demographic, lifestyle, and health factors. We also explored whether associations differed by menopause status, body mass index (BMI), and race/ethnicity. Results: Most participants were non-Hispanic white (67%) or black (29%), college-educated (65%), employed (80%), and had somewhat higher mean urinary phthalate metabolite concentrations than other U.S. women. Overall, the following positive associations were observed between phthalate metabolites and hormones: FDEHP (%Delta: 4.9; 95%CI: 0.5, 9.6), FPlastics (%Delta: 5.1; 95%CI: 0.3, 10.0), and FAA (%Delta: 7.8; 95%CI: 2.3, 13.6) with estradiol; MiBP (%Delta: 6.6; 95%CI: 1.5, 12.1) with testosterone; FDEHP (%Delta: 8.3; 95%CI: 1.5, 15.6), FPlastics (%Delta: 9.8; 95%CI: 2.4, 17.7), MEP (%Delta: 4.6; 95%CI: 0.1, 9.2), FPCP (%Delta: 6.0; 95%CI: 0.2, 12.2), FPhthalates (%Delta: 9.0; 95%CI: 2.1, 16.5), and FAA (%Delta: 12.9; 95%CI: 4.4, 22.1) with progesterone; and MBP (%Delta: 8.5; 95%CI: 1.2, 16.3) and FAA (%Delta: 9.0; 95%CI: 1.3, 17.4) with AMH. Associations of phthalate metabolites with hormones differed by menopause status (strongest in premenopausal women for estradiol, progesterone, and FSH), BMI (strongest in obese women for progesterone), and race/ethnicity (strongest in nonHispanic white women for estradiol and AMH).

Tributyltin (TBT) chloride is an endocrine disrupting chemical associated with reproductive complications. Studies have shown that TBT targets the reproductive tract, impairing ovarian folliculogenesis, and uterine morphophysiology. In this investigation, we assessed whether subchronic and low dose of TBT exposure results in abnormal ovarian follicular reserve and other irregularities in female mice. TBT was administered to female mice (500 ng/kg/day for 12 days via gavage), and reproductive tract morphophysiology was assessed. We further assessed reproductive tract inflammation and oxidative stress. Improper functioning of the reproductive tract in TBT mice was observed. Specifically, irregular estrous cyclicity and abnormal ovarian morphology coupled with reduction in primordial and primary follicle numbers was observed, suggesting ovarian reserve depletion. In addition, improper follicular development and a reduction in antral follicles, corpora lutea, and total healthy ovarian follicles together with an increase in cystic follicles were apparent. Evidence of uterine atrophy, reduction in endometrial gland number, and inflammation and oxidative stress were seen in TBT mice. Further, strong negative correlations were observed between testosterone levels and primordial, primary, and total healthy ovarian follicles. Thus, these data suggest that the subchronic and low dose of TBT exposure impaired ovarian follicular reserve, uterine gland number, and other reproductive features in female mice.

Cytochrome P450 2C8 (CYP2C8) epoxygenase is responsible for the metabolism of over 60 clinically relevant drugs, notably the anticancer drug Taxol (paclitaxel, PAC). Specifically, there are naturally occurring polymorphisms, CYP2C8*2 and CYP2C8*3, that display altered PAC hydroxylation rates despite these mutations not being located in the active site. Herein, we demonstrate that these polymorphisms result in a greater uncoupling of PAC metabolism by increasing the amount of hydrogen peroxide formed per PAC turnover. Anaerobic stopped-flow measurements determined that these polymorphisms have altered first electron transfer kinetics, compared to CYP2C8*1 (wildtype), that suggest electron transfer from cytochrome P450 reductase (CPR) is disfavored. Therefore, these data demonstrate that these polymorphisms affect the catalytic cycle of CYP2C8 and suggest that redox interactions with CPR are disrupted.

Phthalates are synthetic chemicals with widespread human exposure due to their use as additives in consumer products. Phthalate diesters are hydrolyzed in the environment and in the body to monoesters that may be more toxic than the parent compounds. This study tested the hypothesis that adult mouse antral follicles, but not neonatal ovaries, are able to metabolize an environmentally relevant mixture of phthalates. Whole neonatal ovaries and isolated adult antral follicles from CD-1 mice were cultured in media treated with vehicle control or 0.1-10 µg/ml of a mixture composed of 35% diethyl phthalate (DEP), 21% di(2-ethylhexyl) phthalate (DEHP), 15% dibutyl phthalate (DBP), 15% diisononyl phthalate (DiNP), 8% diisobutyl phthalate (DiBP), and 5% benzylbutyl phthalate (BzBP). After 4 days of culture, media were subjected to high-performance liquid chromatography tandem mass spectrometry to measure the amounts of diester phthalates and monoester metabolites. Ovaries and follicles were collected to measure the gene and protein expression of the enzymes required for phthalate metabolism. Monoester metabolites for all phthalates except DiNP were detected in the media for both culture types at most doses. The long-chain phthalates (BzBP, DEHP, and DiNP) were metabolized less than the short-chain phthalates (DEP, DBP, and DiBP) compared with respective controls. Expression of metabolizing enzymes was observed for all treatment groups in both culture types. These data indicate that mouse ovaries are capable of metabolizing low doses of phthalates and suggest that metabolic capacity differs for follicles at different stages of development.