Epidemiologically relevant phthalate mixture and mono(2-ethyl-5-hydroxyhexyl) phthalate exposure alter cell energy metabolism in primary mouse granulosa cells.

MEHHP exposure (0.22 μM and 22.0 μM), at an epidemiologically relevant level and 100-fold higher, did not alter expression of the antioxidant enzymes Cat, Gsr, Gpx1, or Sod1 at 24 h of exposure ( Fig. 1A ) or 72 h of exposure ( Fig. 1B ) compared to control in primary mouse granulosa cells. EPIDEM phthalate mixture exposure (2.0 μM and 200.0 μM) at an epidemiologically relevant level and 100-fold higher did not alter expression of the antioxidant enzymes Cat, Gsr, Gpx1, or Sod1 at 24 h of exposure ( Fig. 2A ) or 72 h of exposure ( Fig. 2B ) compared to control. MEHHP exposure (0.22 μM and 22.0 μM) did not alter the expression of the glycolytic enzymes Pfkm, Pfkp, Aldoa, Tpi1, Eno1, or Ldha in primary granulosa cells at 24 h of exposure ( Fig. 3A ) or 72 h of exposure ( Fig. 3B ) compared to control. EPIDEM phthalate mixture (2.0 μM and 200.0 μM) did not alter the expression of the glycolytic enzymes Pfkm, Pfkp, Aldoa, Tpi1, Eno1, or Ldha in primary granulosa cells at 24 h of exposure ( Fig. 4A ). Further, EPIDEM phthalate mixture (2.0 μM and 200.0 μM) did not alter the expression of the glycolytic enzymes Pfkm, Pfkp, Aldoa, Tpi1, or Eno1 at 72 h of exposure ( Fig. 4B ) compared to control. However, EPIDEM phthalate mixture (200.0 μM) at 72 h of exposure increased the expression of Ldha ( Fig. 4B ) compared to control. MEHHP (0.22 μM and 22.0 μM) exposure did not alter the expression of Glut 1 or Glut 8 at 24 h of exposure ( Fig. 5A ) or 72 h of exposure ( Fig. 5B ). EPIDEM phthalate mixture exposure (2.0 μM and 200.0 μM) did not alter the expression of Glut 8 at 24 h of exposure ( Fig. 6A ) or 72 h of exposure ( Fig. 6B ). EPIDEM phthalate mixture exposure (2.0 μM) increased the expression of Glut 1 at 24-hour exposure ( Fig. 6A ), but EPIDEM phthalate mixture exposure (2.0 μM and 200.0 μM) did not alter expression of Glut 1 at 72 h of exposure ( Fig. 6B ). MEHHP exposure at 0.22 μM borderline increased total ATP production rate and MEHHP exposure at 22.0 μM significantly increased total ATP production rate at 24 h of exposure ( Fig. 7A ) compared to control. However, MEHHP exposure (0.22 μM and 22.0 μM) did not alter total ATP production rate at 72 h of exposure ( Fig. 7B ) compared to control. MEHHP exposure at 0.22 μM did not alter glycolytic ATP production rate at 24 h of exposure ( Fig. 7A ) or 72 h of exposure ( Fig. 7B ) compared to control. However, MEHHP exposure at 22.0 μM increased glycolytic ATP production rate at 24 h of exposure ( Fig. 7A ) and 72 h of exposure ( Fig. 7B ) compared to control. MEHHP exposure at 0.22 μM did not alter mitochondrial ATP production rate at 24 h of exposure ( Fig. 7A ) or 72 h of exposure ( Fig. 7B ) compared to control. In contrast, MEHHP exposure at 22.0 μM decreased mitochondrial ATP production rate at 72 h of exposure ( Fig. 7B ), but did not alter mitochondrial ATP production rate at 24 h of exposure ( Fig. 7A ) compared to control. EPIDEM phthalate mixture exposure at 2.0 μM significantly increased real-time total ATP production rate, whereas EPIDEM phthalate mixture exposure at 200.0 μM significantly decreased total ATP production rate at 24 h of exposure ( Fig. 8A ) compared to control. EPIDEM phthalate mixture exposure (2.0 μM and 200.0 μM) significantly increased real-time total ATP production rate at 72 h of exposure ( Fig. 8B ) compared to control. EPIDEM phthalate mixture exposure (2.0 μM) significantly increased glycolytic ATP production rate at 24 h of exposure ( Fig. 8A ) and borderline increased glycolytic ATP production rate at 72 h of exposure ( Fig. 8B ) compared to control. EPIDEM phthalate mixture exposure (200.0 μM) significantly increased glycolytic ATP production rate at 24 h of exposure ( Fig. 8A ) compared to control. EPIDEM phthalate mixture exposure at 2.0 μM significantly increased mitochondrial ATP production rate at 24 h of exposure ( Fig. 8A ), but did not alter mitochondrial ATP production rate at 72 h of exposure ( Fig. 8B ) compared to control. EPIDEM phthalate mixture exposure at 200.0 μM significantly decreased mitochondrial ATP production rate at 24 h of exposure ( Fig. 8A ), but increased mitochondrial ATP production rate at 72 h of exposure ( Fig. 8B ) compared to control. MEHHP exposure (0.22 μM and 22.0 μM) significantly increased basal glycolysis at 24 h of exposure ( Fig. 9A ) and 72 h of exposure ( Fig. 9B ). Following the inhibition of mitochondrial respiration with rotenone and antimycin A, MEHHP exposure (0.22 μM and 22.0 μM) significantly increased compensatory glycolysis at 24 h of exposure ( Fig. 9A ) and 72 h of exposure ( Fig. 9B ). EPIDEM phthalate mixture exposure at 2.0 μM significantly increased basal and compensatory glycolysis and at 24 h of exposure ( Fig. 10A ) and 72 h of exposure ( Fig. 10B ) compared to control. However, EPIDEM phthalate mixture exposure at 200.0 μM did not alter basal or compensatory glycolysis at 24 h of exposure ( Fig. 10A ) or 72 h of exposure ( Fig. 10B ) compared to control.

The mixture composition and concentrations of monophthalates in the epidemiologically relevant phthalate mixture (EPIDEM) used in this study were based on urinary phthalates present in women enrolled in the Midlife Women’s Health Study [ 41 – 46 ] and have been used in previous studies on the effects of phthalates on cells from the reproductive organs [ 41 , 47 , 48 ]. The mixture contained 8.1 % mono (2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), 65.6 % monoethyl phthalate (MEP), 1.3 % mono(2-ethylhexyl) phthalate (MEHP), 3.1 % mono(2-ethyl-5-oxohexyl) phthalate (MEOHP), 6.4 % mono (2-ethyl-5-carboxypentyl) phthalate (MECPP), 0.8 % mono (3-carboxypropyl) phthalate (MCPP), 6.7 % monobutyl phthalate (MBP), 5.3 % monoisobutyl phthalate (MiBP), and 2.6 % monobenzyl phthalate (MBzP). Individual phthalates were diluted in dimethyl sulfoxide (DMSO) to make a final mixture stock concentration of 267 mM and 2.67 mM. Prepared stock concentrations were validated by liquid chromatography–tandem mass spectrometry. These stock solutions were further diluted in media at 0.75 μL/mL to obtain working mixture concentrations of 2.0 μM and 200.0 μM, corresponding to the mean level of these phthalates in urine and 100-higher concentration. The vehicle control contained DMSO at the same concentration (0.75 μL/mL) as what was present in the phthalate treatment groups. To test the difference between a phthalate mixture and a single phthalate, cells were also treated with MEHHP (0.22 μM and 22.0 μM) at the levels found in the Midlife Women’s Health Study participants. In previous studies, MEHHP was tested in comparison to multiple phthalate mixtures [ 41 , 47 , 48 ]. Because MEHHP was a common phthalate among the different phthalate mixtures tested, this phthalate was used as a single phthalate exposure comparison. To remain consistent with previous studies, we also selected MEHHP for single phthalate comparison. The concentrations of the single phthalate MEHHP were at the same concentrations of MEHHP present in the phthalate mixture. Chemicals were obtained from the following suppliers: MEHHP catalog number 09171 and dimethyl sulfoxide DMSO catalog number D2650 were purchased from Sigma-Aldrich (St. Louis, MO). MEHP catalog number M542490, MEOHP catalog number M542520, MECPP catalog number M525550, MiBP catalog number M547700, MBzP catalog number M524900, MCPP catalog number M525635, MEP catalog number M542580, and MBP catalog number M525100 were purchased from Toronto Research Chemicals (Toronto, Ontario, Canada). CD-1 mice were obtained from Charles River Laboratories and housed in the College of Veterinary Medicine Animal Facility at the University of Illinois Urbana-Champaign. Animals were maintained in a controlled environment (22 ± 1°C, 12 h light:12 h dark cycles) and were provided food (Tekland 2918) and reverse osmosis filtered water ad libitum. All procedures involving animal care, euthanasia, and tissue collection were approved by the Institutional Animal Use and Care Committee at the University of Illinois Urbana-Champaign (protocols 20034 and 23010). Cycling CD-1 female mice aged 32–42 days were euthanized, and the ovaries from 5 to 10 mice were aseptically removed and placed in supplemented α-minimal essential media (α-MEM, catalog number 12561, Gibco Life Technologies, Grand Island, NY) pre-warmed to 37 °C. The protocol for granulosa cell isolation has been published previously [ 41 , 49 ]. Briefly, granulosa cells were released from antral follicles by rupturing the follicles with a watchmaker’s forceps. Media were supplemented with the following: ITS catalog number I3146 (10 μg/mL insulin, 5.5 μg/mL transferrin, 5 ng/mL sodium selenite, Sigma-Aldrich, St. Louis, MO), penicillin-streptomycin (100 U/mL penicillin, 100 μg/mL streptomycin, Gibco Life Technologies, catalog 15140148), 10 % fetal bovine serum (Gibco Life Technologies, catalog 10082–147), and 5 IU/mL recombinant follicle-stimulating hormone (Scripps catalog number F0617 part number 90632). Cells were passed through a 23 G needle 3–4 times and then through a 40 μM cell strainer. Cells were plated in 100 mm culture dishes and expanded 3–4 days in an incubator at 37°C with 5 % CO2 and controlled humidity. Then, cells were detached with 0.05 % trypsin-ethylenediaminetetraacetic acid (EDTA) (Thermo Fisher Scientific, Cat. No. 25200056), replated, and grown for 24 h prior to phthalate exposure. Following exposure with vehicle or phthalates, cells were grown continuously for either 24 or 72 h. Cell density for RNA isolation was 2 × 10 5 cells/well of a 6-well plate. For RNA expression experiments, cells were allowed to attach for 24 h prior to phthalate treatments for 24 or 72 h. Cells were collected and RNA was isolated with Qiagen Micro RNeasy kits. RNA was reverse transcribed to cDNA with Bio-Rad Iscript. DNA transcripts were replicated using Bio-Rad Evagreen supermix and quantified with Bio-Rad CFX96 Real-Time PCR machines. All gene expression data were normalized to the housekeeping gene beta-actin ( ActB ), and primers for each gene are found in Table 1 . The relative fold changes were calculated and compared with the control group using the Pfaffl method [ 50 ]. The Agilent Seahorse XFe96 was used to measure cellular metabolic function. For the Real Time ATP Rate Assay, cells were plated at a density of 1 × 10 4 cells/well in a 96 well Agilent Seahorse plate and allowed to attach for 24 h prior to phthalate treatments. For the Seahorse XF Glycolytic Rate Assay, cells were plated at a density of 2 × 10 4 cells/well in a 96 well Agilent Seahorse plate and allowed to attach for 24 h prior to phthalate treatments. For both assays, manufacturer’s protocols were followed. Statistical analyses were completed using GraphPad Prism software (version 10.4.0 for Windows, GraphPad Software, San Diego, California USA, www.graphpad.com ). Data were represented as mean ± SEM when appropriate. Statistical significance was assigned at p ≤ 0.05 and indicated with an asterisk. Data were considered trending towards significance when p values were above 0.05 and less than 0.1. Statistical differences between treatment groups were determined using one-way ANOVA with Dunnett’s multiple comparisons post hoc test to compare each treatment to the vehicle control. Shapiro-Wilk normality tests were used to determine if data were normally distributed, and Brown-Forsythe tests were used to determine if treatment groups had equal variances. If data were not normally distributed or variances were not equal among treatment groups, then the Kruskal-Wallis nonparametric test was used followed by a Mann-Whitney U test to compare each treatment to the vehicle control.

Although phthalates are known as reproductive and developmental toxicants, little is known about the effects of phthalate exposure on a major cell type in the ovary, the granulosa cell [ 51 , 52 ]. To help fill this gap in research, our previous study identified biomarkers of phthalate exposure in ovarian primary cells and ovarian cell lines of human and mouse origin [ 41 ]. Biomarkers of phthalate exposure were identified by sequencing mRNAs of ovarian cells exposed to phthalates. Analyses of differentially expressed genes indicated that although the exposures did not largely affect the expression of individual genes, phthalate exposures led to significant alterations in several biologic processes associated with many gene ontology terms, including ATP generation and cellular response to ATP, regulation of glycolytic process, and regulation of glucose transmembrane transport [ 41 ]. This current study expanded on these findings and tested the hypothesis that exposures to an epidemiologically relevant phthalate mixture and a single phthalate, MEHHP, aberrantly affect cellular metabolism and alter biomarkers of oxidative stress in primary granulosa cells. In addition, we assessed real-time production of ATP in response to phthalate exposure. Our data show that phthalate exposure does not alter antioxidant enzyme gene expression in granulosa cells, but phthalate exposure does affect ATP production rates. Further, our data show that cells exposed to MEHHP and 2.0 μM phthalate mixture rely on glycolysis as a means of energy production in the absence of mitochondrial respiration, whereas cells exposed to 200.0 μM phthalate mixture lack this compensatory ability to fulfill cell energy demands. Oxidative stress is one mechanism that leads to ovarian defects and pathologies [ 53 ]. Alterations in the expression of antioxidant enzymes such as Cat, Gsr, Gpx1 and Sod1 are indicative of oxidative stress and are a cellular mechanism to mediate reactive oxygen species. In the current study, phthalate mixture or MEHHP exposure did not alter the expression of antioxidant stress genes in granulosa cells. In a previous in vitro study, a monophthalate mixture and MEHP exposure led to changes in antioxidant gene expression [ 54 , 55 ]. However, previous in vitro studies utilized whole follicles, composed of granulosa cells, theca cells, and oocytes, exposed to phthalates for 96 h as opposed to only granulosa cells exposed to phthalates for 24 and 72 h in the current study. In previous studies, the phthalate mixture was based on phthalate metabolites in the urine of pregnant women, and was composed of MEHP, MEP, MiBP, MBP, MBzP, and monoisononyl phthalate (MiNP) [ 56 ]. Our study utilized a phthalate mixture based on phthalates in non-pregnant women aged 45–54 years of age, did not contain MiNP, and instead, contained MEHHP, MEOPH, MECPP, and MCPP [ 57 ]. Thus, the results from current and previous studies could differ due to different phthalates and phthalate mixtures and/or different exposure times used in the studies. In addition, the results from current and previous studies could be due to differences in culture methods; the current study used a two-dimensional culture system, whereas some previous studies used three-dimensional structures (whole follicles). It is possible that culturing cells in a two-dimensional versus three-dimensional system alters cell function. The two-dimensional system was selected for the current study, in part, based on the limitations of the Seahorse assay, which has been developed for monolayered cultured cells. The standard placement of the Seahorse analyzer detection probes are positioned 200 μm above the culture plate bottom to yield robust, real-time cellular metabolism data of both oxygen consumption rate and extra cellular acidification rate. Phthalate exposure has been associated with defects in glucose metabolism. Several epidemiology studies have found phthalate exposure to be positively correlated with increased risk of gestational metabolic disorders [ 58 – 62 ]. At the cellular level, DEHP exposure has been shown to impair glucose metabolism in muscle satellite cells, and MEHP exposure has been shown to increase glucose uptake in adipocytes [ 63 , 64 ]. In the current study, MEHHP exposure did not change the expression of Glut1 or Glut8 , but the EPIDEM mixture exposure increased expression of Glut1 at 24 h compared to controls. Because the EPIDEM mixture contains MEHHP as well as eight additional phthalates, it is likely that the EPIDEM mixture-induced increase in Glut1 is in response to a phthalate or combination of phthalates in the EPIDEM mixture other than MEHHP. In previous experimental studies, exposure to DEHP and MEHP altered expression of Glut4 in myotubes, and DEHP altered Glut4 expression in adipocytes [ 65 – 67 ]. Further, DBP exposure altered Glut3 expression and other glucose metabolism factors in the Sertoli cells of mice [ 68 ]. In the current study, Glut3 and Glut4 were not detected in granulosa cells via qPCR. However, EPIDEM exposure increased the expression of Glut1 at 24 h, but not at 72 h compared to control, indicating that Glut1 may be an early biomarker of phthalate exposure. It is possible that phthalate exposure altered expression of glucose transporters that were below the limit of detection for qPCR. Many other genes besides glucose transporters are associated with glucose transport, including adiponectin receptors and genes of the solute carrier family. Expression of these other factors associated with glucose transport were not assessed in this study. Glycolysis is a process by which cells convert glucose to ATP. Unlike oxidative phosphorylation in the mitochondria, glycolysis does not require oxygen and takes place in the cytoplasm of the cell. In addition to changes in glucose transporters, defects in glycolysis may indicate phthalate effects on glucose metabolism. Evaluation of glycolytic enzymes indicated that MEHHP did not change expression of these enzymes compared to controls. However, the EPIDEM mixture increased the expression of Ldha , an enzyme required for glycolysis and ATP production. Given that the EPIDEM mixture, but not MEHHP alone increased the expression of Ldha , these data suggest that a phthalate or combination of phthalates in the EPIDEM mixture other than MEHHP is responsible for the phthalate-induced increase in Ldha expression. Little is known regarding phthalate exposure and its effects on cellular metabolism, but phthalates have been shown to cause alterations in the metabolic profile of cardiac cells and fat adipocytes [ 69 , 70 ]. Additionally, the association between phthalates and childhood asthma was found to be mediated by glycolysis [ 71 ]. Effects on glycolysis and ATP synthesis indicate a cellular stress response, often a toxicologic response. MEHHP (22.0 μM) and the phthalate mixture increased glycolytic ATP production rate. In addition to increased basal glycolysis, MEHHP and 2.0 μM phthalate mixture exposure increased compensatory glycolysis when mitochondrial respiration was blocked. To our knowledge, this is the first report of phthalate exposure increasing ATP production during glycolysis. Glycolysis is a less efficient means of ATP production compared to mitochondrial respiration, but in absence of oxygen, glycolysis can be a primary source of cell energy to drive cell proliferation [ 72 ]. In aerobic conditions, glycolysis has been shown to be the main source of ATP in many cancer cells, a phenomenon termed the Warburg Effect. Our results are consistent with those from studies of other chemicals. In some studies, arsenic exposure induced glycolysis in primary human urothelial cells, human bronchial epithelial cells, human prostate epithelial cells, and human dermal fibroblasts, and the arsenic-induced glycolysis was found to be mediated by the HIF-1alpha/GLUT1 pathway [ 73 , 74 ]. Similarly, nickel refining fumes also caused an increase in aerobic glycolysis in vitro and in vivo [ 75 , 76 ]. Additionally, exposure to persistent organic pollutants, such as the dichloro-diphenyltrichloroethane metabolite, dichlorodiphenyldichloroethylene, and perfluorooctane sulfonic acid, has been shown to dysregulate ATP production in human ovarian tissue in vitro by altering the expression of the glycolytic enzymes LDHA and ENO1 [ 77 ]. A recent study reported that increased levels of DEHP metabolites in human ovarian follicles are associated with downregulation of genes in the respiratory electron transport chain and gluconeogenesis pathways in the associated granulosa cells [ 17 ]. Follicle growth was also inhibited in women with higher DEHP levels compared to women with low DEHP levels, suggesting that phthalates may inhibit follicle maturation by disrupting energy homeostasis in women undergoing infertility treatments [ 17 ]. In conclusion, this study found that MEHHP exposure and an epidemiologically relevant phthalate mixture led to aberrations in cell energy metabolism in granulosa cells. In absence of mitochondrial ATP production, cells exposed to MEHHP alone compensated for cell energy demand by increasing glycolytic ATP production. In contrast, cells exposed to the phthalate mixture lack compensatory mechanisms for energy demands. Our data demonstrate that functional endpoints may be more sensitive measures of phthalate exposure effects compared to gene expression assessment. Finally, phthalates are considered as EDCs that act via endocrine modes of action. Our results encourage the exploration of additional mechanisms beyond canonical endocrine pathways. Effects on cellular energy metabolism could have significant impacts on cell and organ function, and further studies should investigate the role of energy metabolism changes in phthalate-linked reproductive toxicity.

Phthalates are environmental toxicants found in frequently used plastics such as food wrapping, medical bags and tubing, personal care products, children’s toys, and building materials [ 1 – 8 ]. Because phthalates leach from plastics, humans are continuously exposed to these toxicants. According to National Health and Nutrition Examination Survey, the majority of the US population is exposed to phthalates [ 9 ]. Phthalates are endocrine disrupting chemicals (EDCs) with deleterious effects on the endocrine systems of both males and females [ 4 , 5 ]. In women, phthalates have been linked with reduced fertility, reduced ovarian reserve, diabetes, and other endocrine disorders such as polycystic ovarian syndrome and endometriosis [ 5 , 10 – 13 ]. Among women seeking infertility treatment, increased urinary metabolites of di (2-ethylhexyl) phthalate (DEHP) were associated with decreased antral follicle counts and increased pregnancy loss [ 14 , 15 ]. Additionally, DEHP metabolites in ovarian follicles were associated with reduced follicle growth in response to gonadotropin stimulation, disrupted cholesterol and steroid metabolism, as well as increased inflammation [ 16 , 17 ]. Phthalate and phthalate alternative (DINCH) mixtures have been positively associated with follicle-stimulating hormone levels, which is an indicator of diminished ovarian reserve [ 18 , 19 ]. Decreased ovarian reserve could explain why women with high levels of MEHHP and mono-ethyl-oxohexyl phthalates (MEOHP) had mean ages of menopause 1.86–3.8 years earlier than women with lower levels of exposure [ 20 , 21 ]. These findings lead to concerns regarding phthalate exposure and women’s health. Animal studies corroborate epidemiological evidence associating phthalates with reduced fertility, endocrine disruption, and accelerated reproductive aging in females. In experimental studies, diisononyl phthalate (DiNP) decreased time to mating, gestation index, fertility index, and birth rate in female mice [ 22 , 23 ]. The negative effects of phthalates on fertility are likely attributed, at least in part, to effects on the ovary. In previous experimental studies, DEHP exposure decreased primordial and total follicle numbers in mice, and a phthalate mixture increased atretic follicles and decreased preantral and antral follicles compared to control [ 24 – 28 ]. Dibutyl phthalate (DBP) exposure also increased the number of atretic follicles and decreased antral follicles compared to control [ 29 , 30 ]. DEHP exposure induced early reproductive aging by affecting inflammation, fibrosis, and the expression of telomere regulators and antioxidant enzymes in the ovary [ 31 ]. The toxic effects of phthalate exposure on the ovary are correlated with aberrant effects on steroidogenesis. In vivo, DEHP and DiNP exposure decreased serum estradiol and testosterone levels, and a phthalate mixture decreased testosterone levels in female mice [ 25 , 32 ]. The negative effects of phthalates on steroidogenesis in females has been recapitulated in vitro. In cultured human mural granulosa cells, DBP exposure decreased estradiol and progesterone production, and mono (2-ethylhexyl) phthalate (MEHP) decreased estradiol levels [ 33 , 34 ]. DEHP and its metabolite, MEHP, inhibited growth of antral follicles and reduced estradiol levels in vitro, and a mixture of phthalates decreased growth of antral follicles and decreased androstenedione, testosterone, estrone, and estradiol levels compared to control in cultured mouse follicles [ 35 , 36 ]. In rat granulosa cells, DEHP exposure decreased estradiol and progesterone levels and MEHP exposure decreased estradiol and progesterone levels [ 37 – 39 ]. In cultured mouse granulosa cells, DEHP exposure decreased progesterone levels, decreased cell proliferation, and increased cell apoptosis [ 40 ]. Collectively, these studies demonstrate that phthalates affect follicle numbers/health and sex steroid hormone synthesis. As the primary source of steroid hormones and the organ essential for successful ovulation, the ovary is required for sexual, reproductive, and overall women’s health. Although several studies indicate that phthalates have toxic effects on the ovary, few studies have identified the pathways by which phthalate exposure affects the ovary. In a previous study, granulosa cells, a primary ovarian cell required for steroid production, were exposed to epidemiologically relevant phthalates and subjected to RNA sequencing [ 41 ]. Analyses of transcriptomic data across multiple mouse and human ovarian cell models revealed shared Gene Ontology (GO) term clusters associated with cell energy metabolism. For example, in mouse granulosa cells, MEHHP and epidemiological phthalate mixtures affected terms such as “negative regulation of ATP biosynthetic process,” “mitochondrial FAD transmembrane transport,” and “glycogen biosynthetic process,” suggesting that phthalates may disrupt cell energy metabolism. Because of our previous RNA sequencing results, the current study was designed to identify functional alterations in cell energy mechanisms affected by phthalate exposure. Thus, the current study tested the hypothesis that exposure to an epidemiological phthalate mixture or MEHHP affects cell energy metabolism in granulosa cells.