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.

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.

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.

The effects of a phthalate metabolite mixture on antral follicle growth and sex steroid synthesis in mice.

Phthalates are used as solvents and plasticizers in a wide variety of consumer products. Most people are exposed to phthalates as parent compounds through ingestion, inhalation, and dermal contact. However, these parent compounds are quickly metabolized to more active compounds in several tissues. Although studies indicate that phthalate metabolites reach the ovary, little is known about whether they are ovarian toxicants. Thus, this study tested the hypothesis that phthalate metabolites influence the expression of genes involved in sex steroid synthesis, cell cycle regulation, cell death, oxidative stress, and key receptors, as well as production of sex steroid hormones by mouse antral follicles. The selected metabolite mixture 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). Antral follicles from adult CD-1 mice were cultured for 96 h with vehicle control (DMSO) or metabolite mixture (0.065-325 µg/mL). Growth of follicles in culture was monitored every 24 h. Total RNA was isolated after 24 and 96 h and used for gene expression analysis. Media were collected and subjected to hormone analysis. Exposure to the phthalate mixture inhibited follicle growth, decreased expression of steroidogenic enzymes, and altered the levels of sex steroids relative to control. The mixture, primarily at the two highest doses, also altered expression of cell cycle regulators, apoptotic factors, oxidative stress genes, and some receptors. Collectively, these data suggest that mixtures of phthalate metabolites can directly impact follicle health.

Incorporation of charged residues in the CYP2J2 F-G loop disrupts CYP2J2-lipid bilayer interactions.

CYP2J2 epoxygenase is an extrahepatic, membrane bound cytochrome P450 (CYP) that is primarily found in the heart and mediates endogenous fatty acid metabolism. CYP2J2 interacts with membranes through an N-terminal anchor and various non-contiguous hydrophobic residues. The molecular details of the motifs that mediate membrane interactions are complex and not fully understood. To gain better insights of these complex protein-lipid interactions, we employed molecular dynamics (MD) simulations using a highly mobile membrane mimetic (HMMM) model that enabled multiple independent spontaneous membrane binding events to be captured. Simulations revealed that CYP2J2 engages with the membrane at the F-G loop through hydrophobic residues Trp-235, Ille-236, and Phe-239. To explore the role of these residues, three F-G loop mutants were modeled from the truncated CYP2J2 construct (Δ34) which included Δ34-I236D, Δ34-F239H and Δ34-I236D/F239H. Using the HMMM coordinates of CYP2J2, the simulations were extended to a full POPC membrane which showed a significant decrease in the depth of insertion for each of the F-G loop mutants. The CYP2J2 F-G loop mutants were expressed in E. coli and were shown to be localized to the cytosolic fraction at a greater percentage relative to construct Δ34. Notably, the functional data demonstrated that the double mutant, Δ34-I236D/F239H, maintained native-like enzymatic activity. The membrane insertion characteristics were examined by monitoring CYP2J2 Trp-quenching fluorescence spectroscopy upon binding nanodiscs containing pyrene phospholipids. Relative to the Δ34 construct, the F-G loop mutants exhibited lower Trp quenching and membrane insertion. Taken together, the results suggest that the mutants exhibit a different membrane topology in agreement with the MD simulations and provide important evidence towards the involvement of key residues in the F-G loop of CYP2J2.

Endocannabinoids anandamide and 2-arachidonoylglycerol are substrates for human CYP2J2 epoxygenase.

The endocannabinoids, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), are arachidonic acid (AA) derivatives that are known to regulate human cardiovascular functions. CYP2J2 is the primary cytochrome P450 in the human heart and is most well known for the metabolism of AA to the biologically active epoxyeicosatrienoic acids. In this study, we demonstrate that both 2-AG and AEA are substrates for metabolism by CYP2J2 epoxygenase in the model membrane bilayers of nanodiscs. Reactions of CYP2J2 with AEA formed four AEA-epoxyeicosatrienoic acids, whereas incubations with 2-AG yielded detectable levels of only two 2-AG epoxides. Notably, 2-AG was shown to undergo enzymatic oxidative cleavage to form AA through a NADPH-dependent reaction with CYP2J2 and cytochrome P450 reductase. The formation of the predominant AEA and 2-AG epoxides was confirmed using microsomes prepared from the left myocardium of porcine and bovine heart tissues. The nuances of the ligand-protein interactions were further characterized using spectral titrations, stopped-flow small-molecule ligand egress, and molecular modeling. The experimental and theoretical data were in agreement, which showed that substitution of the AA carboxylic acid with the 2-AG ester-glycerol changes the binding interaction of these lipids within the CYP2J2 active site, leading to different product distributions. In summary, we present data for the functional metabolomics of AEA and 2-AG by a membrane-bound cardiovascular epoxygenase.

Functional investigations of thromboxane synthase (CYP5A1) in lipid bilayers of nanodiscs.

CYP5A1 is a membrane-associated cytochrome P450 that metabolizes the cyclooxygenase product prostaglandin (PGH2 ) into thromboxane A2 (TXA2 ), a potent inducer of vasoconstriction and platelet aggregation. Although CYP5A1 is an ER-bound protein, the role of membranes in modulating the thermodynamics and kinetics of substrate binding to this protein has not been elucidated. In this work, we incorporated thromboxane synthase into lipid bilayers of nanodiscs for functional studies. We measured the redox potential of CYP5A1 in nanodiscs and showed that the redox potential is within a similar range of other drug-metabolizing P450 enzymes in membranes. Further, we showed that binding of substrate to CYP5A1 can induce conformational changes in the protein that block small-molecule ligand egress by measuring the kinetics of cyanide binding to CYP5A1 as a function of substrate concentration. Notably, we observed that sensitivity to cyanide binding was different for two substrate analogues, U44069 and U46619, thus indicating that they bind differently to the active site of CYP5A1. We also characterized the effects of the different lipids on CYP5A1 catalytic activity by using nanodiscs to create unary, binary, and ternary lipid systems. CYP5A1 activity increased dramatically in the presence of charged lipids POPS and POPE, as compared to the unary POPC system. These results suggest the importance of lipid composition on modulating the activity of CYP5A1 to increase thromboxane formation.

Hypoxia/reoxygenation impairs memory formation via adenosine-dependent activation of caspase 1.

After hypoxia, a critical adverse outcome is the inability to create new memories. How anterograde amnesia develops or resolves remains elusive, but a link to brain-based IL-1 is suggested due to the vital role of IL-1 in both learning and brain injury. We examined memory formation in mice exposed to acute hypoxia. After reoxygenation, memory recall recovered faster than memory formation, impacting novel object recognition and cued fear conditioning but not spatially cued Y-maze performance. The ability of mice to form new memories after hypoxia/reoxygenation was accelerated in IL-1 receptor 1 knockout (IL-1R1 KO) mice, in mice receiving IL-1 receptor antagonist (IL-1RA), and in mice given the caspase 1 inhibitor Ac-YVAD-CMK. Mechanistically, hypoxia/reoxygenation more than doubled caspase 1 activity in the brain, which was localized to the amygdala compared to the hippocampus. This reoxygenation-dependent activation of caspase 1 was prevented by broad-spectrum adenosine receptor (AR) antagonism with caffeine and by targeted A1/A2A AR antagonism with 8-cyclopentyl-1,3-dipropylxanthine plus 3,7-dimethyl-1-propargylxanthine. Additionally, perfusion of adenosine activated caspase 1 in the brain, while caffeine blocked this action by adenosine. Finally, resolution of anterograde amnesia was improved by both caffeine and by targeted A1/A2A AR antagonism. These findings indicate that amygdala-based anterograde amnesia after hypoxia/reoxygenation is sustained by IL-1ß generated through adenosine-dependent activation of caspase 1 after reoxygenation.

Long-term exposure to di(2-ethylhexyl) phthalate, diisononyl phthalate, and a mixture of phthalates alters estrous cyclicity and/or impairs gestational index and birth rate in 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.

Dietary exposure to an environmentally relevant phthalate mixture alters follicle dynamics, hormone levels, ovarian gene expression, and pituitary gene expression in female mice.

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 long-term 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.

Prenatal and postnatal exposure to polychlorinated biphenyls alter follicle numbers, gene expression, and a proliferation marker in the rat ovary.

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.

The biobehavioral and neuroimmune impact of low-dose ionizing radiation.

In the clinical setting, repeated exposures (10-30) to low-doses of ionizing radiation (≤200 cGy), as seen in radiotherapy for cancer, causes fatigue. Almost nothing is known, however, about the fatigue inducing effects of a single exposure to environmental low-dose ionizing radiation that might occur during high-altitude commercial air flight, a nuclear reactor accident or a solar particle event (SPE). To investigate the short-term impact of low-dose ionizing radiation on mouse biobehaviors and neuroimmunity, male CD-1 mice were whole body irradiated with 50 cGy or 200 cGy of gamma or proton radiation. Gamma radiation was found to reduce spontaneous locomotor activity by 35% and 36%, respectively, 6 h post irradiation. In contrast, the motivated behavior of social exploration was un-impacted by gamma radiation. Examination of pro-inflammatory cytokine gene transcripts in the brain demonstrated that gamma radiation increased hippocampal TNF-α expression as early as 4 h post-irradiation. This was coupled to subsequent increases in IL-1RA (8 and 12 h post irradiation) in the cortex and hippocampus and reductions in activity-regulated cytoskeleton-associated protein (Arc) (24 h post irradiation) in the cortex. Finally, restraint stress was a significant modulator of the neuroimmune response to radiation blocking the ability of 200 cGy gamma radiation from impairing locomotor activity and altering the brain-based inflammatory response to irradiation. Taken together, these findings indicate that low-dose ionizing radiation rapidly activates the neuroimmune system potentially causing early onset fatigue-like symptoms in mice.

Perfluorooctanoic Acid Disrupts Ovarian Steroidogenesis and Folliculogenesis in Adult 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 µg/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 µg/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.

Phthalate monoesters act through peroxisome proliferator-activated receptors in the mouse ovary.

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(2-ethylhexyl) 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 µM) 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-γ inhibitor, T0070907, or with the PPAR-α 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.

Prenatal exposure to an environmentally relevant phthalate mixture alters ovarian steroidogenesis and folliculogenesis in the F1 generation of adult female mice.

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 transgenerational 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 µg/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.

Urinary phthalate metabolite concentrations and serum hormone levels in pre- and perimenopausal women from the Midlife Women's Health Study.

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-Müllerian 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, ∑DEHP), exposure sources (plastic, ∑Plastics; personal care products, ∑PCP), biological activity (anti-androgenic, ∑AA), and sum of all metabolites (∑Phthalates). 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: ∑DEHP (%Δ: 4.9; 95%CI: 0.5, 9.6), ∑Plastics (%Δ: 5.1; 95%CI: 0.3, 10.0), and ∑AA (%Δ: 7.8; 95%CI: 2.3, 13.6) with estradiol; MiBP (%Δ: 6.6; 95%CI: 1.5, 12.1) with testosterone; ∑DEHP (%Δ: 8.3; 95%CI: 1.5, 15.6), ∑Plastics (%Δ: 9.8; 95%CI: 2.4, 17.7), MEP (%Δ: 4.6; 95%CI: 0.1, 9.2), ∑PCP (%Δ: 6.0; 95%CI: 0.2, 12.2), ∑Phthalates (%Δ: 9.0; 95%CI: 2.1, 16.5), and ∑AA (%Δ: 12.9; 95%CI: 4.4, 22.1) with progesterone; and MBP (%Δ: 8.5; 95%CI: 1.2, 16.3) and ∑AA (%Δ: 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 non-Hispanic white women for estradiol and AMH). CONCLUSIONS: We found that phthalate metabolites were positively associated with several hormones in midlife women, and that some demographic and lifestyle characteristics modified these associations. Future longitudinal studies are needed to corroborate these findings in more diverse midlife populations.

Early postnatal exposure to di(2-ethylhexyl) phthalate causes sex-specific disruption of gonadal development in pigs.

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.

Iodoacetic acid inhibits follicle growth and alters expression of genes that regulate apoptosis, the cell cycle, estrogen receptors, and ovarian steroidogenesis in mouse ovarian follicles.

The reaction between disinfectants and organic matter or inorganic matter in source water generates disinfection by-products (DBPs) such as iodoacetic acid (IAA). DBPs are associated with health effects such as bladder cancer and adverse reproductive outcomes, but the effects of IAA on the ovary are not well known. This study determined whether IAA exposure affects ovarian follicle growth, steroidogenesis, and expression of apoptotic factors, cell cycle regulators, estrogen receptors, and steroidogenic factors in vitro. IAA exposure significantly decreased follicle growth, expression of cell cycle stimulators, and the proliferation marker Ki67. In contrast, IAA increased expression of the cell cycle inhibitor Cdkn1a. Moreover, IAA exposure increased expression of pro-apoptotic factors, whereas it decreased expression of anti-apoptotic factors. IAA exposure also altered expression of steroidogenic factors and estrogen receptors, disrupting steroidogenesis. These data demonstrate that IAA exposure inhibits follicle growth, decreases cell proliferation, and alters steroidogenesis in mouse ovarian follicles in vitro.

Subchronic and Low Dose of Tributyltin Exposure Leads to Reduced Ovarian Reserve, Reduced Uterine Gland Number, and Other Reproductive Irregularities in Female Mice.

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.