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

Abstract

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.

Phthalates are endocrine disrupting chemicals found in a wide variety of consumer products including food packaging, medical bags and tubing, personal care products, children’s toys, and building materials (Cao 2010; Carlos et al. 2021; Gao et al. 2014; Gore et al. 2015; Pagoni et al. 2022; Šimunović et al. 2022; US Agency for Toxic Substances & Disease Registry 2022). Because phthalates leach from these consumer products, humans are exposed to phthalates in a variety of ways including ingestion, inhalation, and dermal absorption (National Research Council 2008; Zhang and Chen 2014); however, ingestion is the most common route of exposure (Wittassek et al. 2011; Wu et al. 2021). Humans are exposed to phthalates daily, and the majority of the U.S. population is continuously exposed to phthalates according to National Health and Nutrition Examination Survey (NHANES) data 2011–2018 (Centers for Disease Control and Prevention (CDC) (2011–2018). This high prevalence of exposure to phthalates leads to many questions regarding how phthalates affect health.

Previous studies have investigated the effects of phthalates on reproduction. Exposure to di(2-ethylhexyl) phthalate (DEHP) or diisononyl phthalate (DiNP) for 10 days led to subfertility, disruption of estrous cyclicity, and alteration of hormone levels in rodents (Chiang and Flaws 2019; Chiang et al. 2020a,b; Hannon et al. 2016). Further, prenatal DEHP exposure adversely affected reproductive outcomes in a transgenerational manner by accelerating the onset of puberty, disrupting estrous cyclicity, and decreasing fertility-related indices in mice (Rattan et al. 2018). Because humans are exposed to multiple phthalates continuously as opposed to a single phthalate at one time, an environmentally relevant mixture of phthalates was developed based on phthalate metabolite levels in the urine of pregnant women in central Illinois (Yazdy et al. 2018). In previous studies, prenatal exposure to this phthalate mixture (Mix), composed of DEHP, DiNP, benzyl butyl phthalate (BzBP), di-n-butyl phthalate (DBP), diisobutyl phthalate (DiBP), and diethyl phthalate (DEP), altered ovarian steroidogenesis, folliculogenesis, estrous cyclicity, and reduced fertility-related indices (Brehm and Flaws 2021; Gill et al. 2021; Zhou et al. 2017b). Further, prenatal exposure to the Mix led to fertility complications in the F2 and F3 generations of female mice (Zhou et al. 2017a).

Prior reproductive toxicology studies have investigated the effects of acute exposure to phthalates, either 10-day adult exposure or prenatal exposure from gestational day 10.5 to birth, on female reproductive outcomes. However, human exposure to phthalates does not typically occur in an acute manner, but rather phthalate exposure occurs daily over a lifetime. Thus, the current study tested the hypothesis that long-term phthalate exposure to DEHP, DiNP, or Mix alters estrous cyclicity and fertility-related indices in female mice.

Materials and methods

 

 

Chemicals

The phthalates were purchased from Sigma-Aldrich (St. Louis, Missouri), had a purity ≥98%, and had the following catalog numbers: DEP (524972), DBP (524980), DiBP (152641), DEHP (D201154), BzBP (308501), and DiNP (376663). Corn oil (Columbus Vegetable Oils, Des Plaines, Illinois) was used as the vehicle control. Monoester phthalate metabolites were purchased from Sigma-Aldrich: mono-2-ethylhexyl phthalate (MEHP, catalog 796832), monomethyl phthalate (MMP, 36926), monobenzyl phthalate (MBzP, 89505), monoethyl phthalate (MEP, SMB00941), and mono-butyl phthalate (MBP, 30751). Some monoester phthalate metabolites were purchased from Toronto Research Chemicals (Toronto, Ontario): mono-(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP, catalog M542510), mono-(2-ethyl-5-carboxypentyl) phthalate (MECPP, catalog M525550), monoisobutyl phthalate (MiBP, M547700), and mono(3-carboxypropyl) phthalate (MCPP, M525635). Mono-(2-ethyl-5-oxohexyl) phthalate (MEOHP, catalog CLM-6640-MT-1.2) was purchased from Cambridge Isotope Laboratories (Tewksbury, Massachusetts).

Animals

Female CD-1 mice at 33 days of age and male CD-1 mice at 7 weeks of age were purchased from Charles River Laboratories (Wilmington, Massachusetts) and housed in the College of Veterinary Medicine vivarium at the University of Illinois Urbana-Champaign (Urbana, Illinois). After shipment, the mice were allowed to acclimate to new surroundings for 2 weeks prior to the beginning of the experiment. Following acclimation, female mice were group housed 3 mice per cage. Mice were housed in polysulfone cages (Allentown, Allentown, New Jersey), with 1/8 corn cob bedding (Shepherd Specialty Papers), environmental enrichment (iso-BLOX, catalog #6060, Envigo), and water purified by reverse osmosis. The University of Illinois Institutional Animal Care and Use Committee approved all animal handling, housing, and procedures.

Study design and dosing

Phthalates were administered to mice ad libitum in the rodent chow, which was formulated by Envigo Teklad Diets (Madison, Wisconsin). The base chow was a modification of the AIN-93G formulation (TD.94045) that replaced soybean oil with corn oil (Columbus Vegetable Oils, Des Plaines, Illinois). Chow with 7% corn oil was used as the vehicle control group. Phthalates were mixed in corn oil and provided to Envigo for chow preparation.

This current study addressed the long-term, chronic effects of phthalates on female reproduction and estrous cyclicity. The phthalate mixture used in this study was composed of DEP (35.2%), DEHP (21.0%), DBP (14.9%), DiBP (8.6%), DiNP (15.1%), and BzBP (5.1%). This environmentally relevant phthalate mixture was developed based on phthalate metabolite concentrations in urine of pregnant women in central Illinois (iKids study) (Yazdy et al. 2018).

Because there are high levels of phthalates found in food contact materials, such as plastic wrap and plastic containers, ingestion is a major route of exposure for humans (Wu et al. 2021). To mimic human exposure, phthalate exposure was administered via the rodent chow. We used 3 levels of exposure for DEHP, DiNP, and Mix: 0.15 ppm, 1.5 ppm, and 1500 ppm. Each treatment group consisted of 12–14 mice. Based on previous studies that exposed mice to phthalates via the chow, target doses were selected based on the assumption that a 25 g mouse eats approximately 5 g of food/day (Neier et al. 2019a,b, 2020). With these assumptions, the 0.15 ppm dose is roughly equivalent to 24 µg phthalate/kg body weight/day and the 1.5 ppm dose is roughly equivalent to 240 µg phthalate/kg body weight/day. The 0.15 and 1.5 ppm doses fall within the ranges of daily human exposure, infant exposure, and occupational exposure (Kavlock et al. 2002; Koch and Calafat 2009). Previous studies completed in our laboratory have shown that short-term exposures to 20 $\mu$g phthalate/kg body weight/day or 200 $\mu$g phthalate/kg body weight/day have long-term detrimental effects on female reproductive health in mice (Brehm and Flaws 2021; Brehm et al. 2020; Chiang and Flaws 2019; Chiang et al. 2020a,b; Gill et al. 2021; Hannon et al. 2016; Zhou et al. 2017a,b). The highest dose of 1500 ppm, roughly equivalent to 240 mg phthalate/kg body weight/day, was used as a comparison to the 200 mg phthalate/kg body weight/day dose used in previous studies and exceeds typical human exposure, (Agency for Toxic Substances and Disease Registry (ATSDR) 2022; Brehm and Flaws 2021; Brehm et al. 2020; Chiang and Flaws 2019; Chiang et al. 2020a,b; Gill et al. 2021; Zhou et al. 2017a,b). The highest human exposure to DEHP is a result of medical procedures and has an upper limit of 8.5 mg/kg body weight/day (Agency for Toxic Substances and Disease Registry (ATSDR) 2022). The 1500 ppm DEHP exposure is roughly equivalent to 28 times the estimated human exposure to DEHP. The phthalate mixture is composed 6 different individual phthalates, and DEHP accounts for 21% of the phthalate concentration in this mixture. Thus, exposure to DEHP in the 1500 ppm Mix is approximately 6 times higher than the estimated human exposure to DEHP.

Beginning at 6 weeks of age, the mice were administered treatments in the chow ad libitum, continuously for 11 months. Daily continuous exposure to phthalates strongly mimics human exposure to phthalates. Of the 16 urinary phthalates monitored in the NHAHES 2017–2018 study, 10 urinary phthalates were present at or above the limit of detection in 95% of the 2762 participants aged 6 years and above, indicating that the majority of the human population is exposed to phthalates daily (Centers for Disease Control and Prevention (CDC) 2017–2018).

Body weights and food consumption weights

Before the beginning of the experiment, mice were weighed and randomly assigned to treatment groups. For the duration of the experiment, mice were weighed once a week and weight was recorded to the nearest tenth of a gram. Cage food hoppers were filled once a week with treatment chow in excess. Mice were permitted to eat the rodent chow ad libitum. To the nearest tenth of a gram, food added each week was weighed, and food left from the previous week was weighed to calculate how much food had been consumed each week per cage. Because mice were group housed (3 per cage), the food consumed per cage each week was divided by the number of mice in the cage to estimate the amount of food consumed per mouse per week. Lab personnel monitoring body weight and food consumption were also responsible for feeding mice weekly. Because exposure to phthalates was given in the rodent chow and to ensure mice were fed the correct chow, investigators were unblinded to mouse identification and treatment group.

Estrous cyclicity

Mice were vaginally lavaged with phosphate-buffered saline daily for 14 days after 1, 3, 5, 7, and 11 months of phthalate exposure (n = 12–14 females per treatment group). Using previously defined criteria, vaginal cytology determined the stage of estrus (Goldman et al. 2007). Metestrus and diestrus were combined because of the similar vaginal cytology and hormone profiles of these stages. Time spent in each cycle was determined by dividing the number of days the mouse was in that cycle by the total number of days vaginal cytology was read for that time point of phthalate exposure. A cytologist blinded to the mouse identification and treatment group assessed the stage of estrous cyclicity.

Breeding trials and fertility indices

Female mice (n = 6–9 females per treatment group) were exposed to treatment groups for 11 consecutive months and then paired individually with male mice that had not been dosed with phthalates. Methods for breeding trials and fertility indices were completed as previously described (Brehm and Flaws 2021; Chiang and Flaws 2019; Chiang et al. 2020b; Rattan et al. 2018; Ziv-Gal et al. 2015). For fertility trials, investigators were unblinded to mouse identification and treatment group. Females were checked twice daily for the presence of a copulatory plug. If the copulatory plug was present, the male was removed from the cage and the female was housed individually. Females were weighed daily from the start of breeding trials until the presence of a copulatory plug. Females were separated from the male if a copulatory plug was not present within 14 days of breeding. Females were weighed a minimum of twice weekly for the duration of the breeding trial and were considered pregnant if they gained at least 4 g and/or gave birth to pups. Pregnant mice were monitored twice a day for birth and difficulties during labor and delivery. Mating index, gestational index, pregnancy rate, birth rate, dystocia rate, and fertility index were calculated at the conclusion of the breeding trial. Mating index was calculated by dividing the number of females who had a copulatory plug divided by the number of females in that treatment group. Gestational index was calculated by dividing the number of dams that gave birth to live pups by the number of pregnant dams. Pregnancy rate was calculated by dividing the number of dams that were pregnant by the number of females in that treatment group. Birth rate was calculated by dividing number of dams that gave birth to live pups by the number of females in that treatment group. Dystocia rate was calculated by dividing the number of dams with dystocia by the number of dams that were pregnant. Fertility index was calculated by dividing the number of pregnant females by the number of females with a copulatory plug.

Quantification of phthalates in urine

Phthalates were quantified in mouse urine and normalized to creatinine to account for differences in urine concentrations. At approximately 2 h after lights were on in the vivarium, spontaneous urine samples were collected from the female mice after 6 months (n = 5–8) and 11 months (n = 4) of vehicle control or phthalate exposure by placing each mouse on a Petri dish and letting it urinate in the dish. Urine samples were collected on 2 consecutive days and pooled for each mouse. Each urine sample (5 µl) was diluted 1:20 and used to determine creatinine concentration by enzyme-linked immunoassay (ELISA) (Creatinine Kit Arbor Assays K002-H1). Samples were prepared according to the manufacturer’s protocol, and absorbances were read on a Bio-Tek Synergy LX multi-mode plate reader. The remainder of the sample volume was submitted to the University of Illinois Urbana-Champaign Roy K. Carver Biotechnology Metabolomics Center for analysis of urinary phthalate metabolite levels. Isotope dilution high-performance liquid chromatography negative-ion electrospray ionization-tandem mass spectrometry (HPLC–MS/MS) and methods adapted from the Centers for Disease Control and Prevention (Silva et al. 2007; Warner et al. 2019) were used to detect phthalate metabolites (MEHP, MEHHP, MECPP, MEOHP, MCPP, MBzP, MEP, MBP, and MiBP). Investigators who quantified urinary phthalates were unblinded to mouse identification and treatment group.

Statistical analyses

Statistical analyses were completed using either GraphPad Prism software (version 9.4.1 for Windows, GraphPad Software, San Diego, California, www.graphpad.com) or SPSS software (IBM Corp. IBM SPSS Statistics for Windows, Version 28.0. Armonk, New York: IBM Corp.). Data were represented as mean ± SEM when appropriate. Statistical significance was assigned at p ≤ .05 and indicated with an asterisk. Data were considered trending towards significance when p-values were above .05 and less than .1. Changes in mouse body weights and weekly food consumption from the start of the experiment were recorded for each indicated time of phthalate exposure, and statistical differences between treatment groups over time were determined using 2-way ANOVA with repeated measures when data were complete. If the data set had missing data points, such as when a mouse died prior to the end of the experiment, the data set was analyzed by fitting a mixed model. Dunnett’s multiple comparisons post hoc tests were used to compare each treatment to the vehicle control. Nominal data were analyzed with a one-sided Fisher’s exact test comparing each treatment to the vehicle control. If data were not normally distributed, the Kruskal-Wallis nonparametric test was used followed by a Mann-Whitney U test to compare each treatment to the vehicle control.

RESULTS

 

Body weight, phthalate exposure, and food consumption

During the 11 months of the study, all mice gained body weight regardless of treatment (Figure 1; n = 12–14; p ≤ .05). DEHP exposure at 0.15 ppm, 1.5 ppm, or 1500 ppm did not significantly change body weight compared to control at any time-point (Figure 1A;n = 12–14 females per treatment group). Similarly, DiNP and Mix exposure (0.15 ppm, 1.5 ppm, or 1500 ppm) did not significantly change body weight compared to control at any time point (Figs. 1C and E; n = 12–14 females per treatment group). Further, DEHP, DiNP, and Mix exposure (0.15 ppm, 1.5 ppm, or 1500 ppm) did not alter food consumption compared to control (Figs. 1B, D, and F).

Figure 1.

Average change in mouse body weight and weekly food consumption over time. Change in body weight (A) and weekly food consumption (B) of mice exposed to vehicle control, 0.15 ppm DEHP, 1.5 ppm DEHP, or 1500 ppm DEHP via the rodent chow for 11 consecutive months. Changes in body weights (C) and weekly food consumption (D) of mice exposed to vehicle control, 0.15 ppm DiNP, 1.5 ppm DiNP, or 1500 ppm DiNP via the rodent chow for 11 consecutive months. Change in body weight (E) and weekly food consumption (F) of mice exposed to vehicle control, 0.15 ppm Mix, 1.5 ppm Mix, or 1500 ppm Mix via the rodent chow for 11 consecutive months. Each treatment group had n = 12–14 female mice. Error bars indicate SEM for each treatment group at each month of exposure.

Urinary phthalate metabolite levels

To verify and monitor phthalate exposure, urinary phthalate metabolites (MEHP, MEOHP, MEHHP, MECPP, MBP, MiBP, MMP, MBzP, MCPP, and MEP) were measured following 6 months of phthalate exposure (Figs. 2A, C, and E) and 11 months of phthalate exposure (Figs. 2B, D, and F). The data indicate that the known metabolites of DEHP exposure, MEHP, MEOHP, and MEHHP, were present in mouse urine after 6 and 11 months of DEHP exposure (Figs. 2A and B). The data also indicate that very low levels of urinary metabolites were present in controls, and that the levels of metabolites in controls were substantially lower than in DEHP treatment groups (Figs. 2A and B). Urinary concentrations of MEHP after 1500 ppm DEHP exposure (542–17 117 ng/ml, 6 months exposure; 675–37 470 ng/ml, 1 year exposure) were higher than urinary concentrations of MEHP after exposure to vehicle control (0.1–0.6 ng/ml, 6 months exposure; 0 ng/ml, 1 year exposure, p < .001). Urinary concentrations of MEOHP after 1500 ppm DEHP exposure (2080–9570 ng/ml, 6 months exposure; 11 887–20 209 ng/ml, 1 year exposure) were higher than urinary concentrations of MEOHP after exposure to vehicle control (0.1–0.9 ng/ml, 6 months exposure; 0–0.2 ng/ml, 1 year exposure, p < .001). Urinary concentrations of MEHHP after 1500 ppm DEHP exposure (4287–23 135 ng/ml, 6 months exposure; 26 434–35 500 ng/ml, 1 year exposure) were higher than the urinary levels of MEHHP after exposure to vehicle control (0–1.5 ng/ml, 6 months exposure; 0–0.2 ng/ml, 1 year exposure, p < .001). No statistical differences were found between control and 0.15 ppm DEHP or between control and 1.5 ppm DEHP groups for urinary concentrations of MEHP, MEOHP, MEHHP, MECPP, MBP, MiBP, MMP, MBzP, MCPP, and MEP.

Figure 2.

Urinary phthalate metabolite concentrations. Concentrations of urinary metabolites from female mice exposed to vehicle control, 0.15 ppm DEHP, 1.5 ppm DEHP, or 1500 ppm DEHP via the rodent chow for 6 consecutive months (A) and 11 consecutive months (B). Concentrations of urinary metabolites from female mice exposed to vehicle control, 0.15 ppm DiNP, 1.5 ppm DiNP, or 1500 ppm DiNP via the rodent chow for 6 consecutive months (C) and 11 consecutive months (D). Concentrations of urinary metabolites from female mice exposed to vehicle control, 0.15 ppm Mix, 1.5 ppm Mix, or 1500 ppm Mix via the rodent chow for 6 consecutive months (E) and 11 consecutive months (F). Values of MEP urinary concentrations from 2 mice treated with Mix for 11 months fall outside the range of graph F, 84 553 ng/ml and 116 926 ng/ml. Graphs represent metabolite concentration means of each treatment group with SEM and individual values of each mouse indicated. Each treatment group had n = 4–8 females.

The highest levels of urinary metabolites measured with dietary DiNP exposure were MEOHP, MEHHP, MECPP, and MCPP. Dietary exposure to 0.15 ppm DiNP for 6 months led to higher urinary MEOHP concentrations (3–2029 ng/ml, p < .05) and higher urinary MEHHP concentrations (4–4914 ng/ml, p < .0001) than those urinary concentrations after vehicle control exposure (MEOHP, 0.1–0.9 ng/ml; MEHPP, 0–0.6 ng/ml). Dietary exposure to 1500 ppm DiNP for 6 months led to higher MECPP urinary concentrations (372–3003 ng/ml; p < .0001) than vehicle control (0–0.2 ng/ml). Following 1 year of 1500 ppm DiNP exposure, urinary concentrations of MECPP (1079–2335 ng/ml, p < .0001) and MCPP (1582–4381 ng/ml, p < .0001) were higher than vehicle control (MECPP 0.2–0.4 ng/ml; MCPP, 1.6–3.6 ng/ml). No statistical differences were found between control and 1.5 ppm DiNP groups for urinary concentrations of MEHP, MEOHP, MEHHP, MECPP, MBP, MiBP, MMP, MBzP, MCPP, and MEP.

Dietary Mix exposure resulted in higher urinary MEHHP, MBP, MiBP, and MEP concentrations compared to the urinary levels of MEHHP, MBP, MiBP, and MEP after exposure to vehicle control. Dietary exposure to 1500 ppm Mix for 6 months led to higher urinary MEHHP (73–8137 ng/ml, p < .01), MBP (179–18 781 ng/ml, p < .0001), MiBP (232–22 716, p < .0001), and MEP (836–27 140 ng/ml, p < .0001) concentrations compared to vehicle control urinary concentrations of MEHHP (0–1.5 ng/ml), MBP (0–3.6 ng/ml), MiBP (0.6–27.3 ng/ml), and MEP (0–49 ng/ml). Phthalate mixture exposure at 1500 ppm for 1 year resulted in higher urinary MBP (434–21 745, p < .0001), MiBP (246–13 686 ng/ml, p < .01), and MEP (511–84 553, p < .0001) concentrations compared to vehicle control urinary concentrations of MBP (1.6–3.6 ng/ml), MiBP (0–0.5 ng/ml), and MEP (7.1–23.2 ng/ml). No statistical differences were found between 0.15 Mix and control or 1.5 ppm Mix and control urinary concentrations of MEHP, MEOHP, MEHHP, MECPP, MBP, MiBP, MMP, MBzP, MCPP, and MEP.

Effect of phthalate exposure on estrous cyclicity

DEHP exposure (0.15 ppm, 1.5 ppm, or 1500 ppm) for 1 month did not significantly affect the time that the mice spent in estrus or metestrus/diestrus compared to control (Figure 3A;n = 12–14 females per treatment group). Similarly, DEHP exposure at 1.5 and 1500 ppm for 3 months did not significantly affect the time that the mice spent in estrus or metestrus/diestrus compared to control (Figure 3B;n = 12–14 females per treatment group). Further, DEHP exposure at 1.5 and 1500 ppm for 5 months and DEHP exposure at 0.15, 1.5, or 1500 ppm for 7 and 11 months did not affect the time that the mice spent in estrus or metestrus/diestrus compared to control (Figs. 3C–E). In contrast, DEHP exposure at 0.15 ppm for 3 months tended to increase the time that the mice spent in estrus and significantly decreased the time that the mice spent in metestrus/diestrus compared to control (Figure 3B;n = 12–14 females per treatment group; p ≤ .05). Further, DEHP exposure at 0.15 ppm for 5 months increased the time mice spent in estrus and decreased the time that mice spent in metestrus/diestrus (Figure 5C;n = 12–14 females per treatment group; p ≤ .05).

Figure 3.

The effects of DEHP exposure on estrous cyclicity after 1 month (A), 3 months (B), 5 months (C), 7 months (D), and 11 months (E) of exposure. Estrous cyclicity was monitored daily for a 2-week period at each exposure time point. Treatment groups included vehicle control, 0.15 ppm DEHP, 1.5 ppm DEHP, or 1500 ppm DEHP in the rodent chow fed ad libitum. Bar graphs represent mean percentages of each treatment group with SEM and individual values of each mouse indicated. Each treatment group had n = 12–14 female mice. Asterisks (*) indicate significant differences compared to the vehicle control group (p ≤ .05).

Figure 5.

The effects of phthalate mixture exposure on estrous cyclicity after 1 month (A), 3 months (B), 5 months (C), 7 months (D), and 11 months (E) of exposure. Estrous cyclicity was monitored daily for a 2-week period at each exposure time point. Treatment groups included vehicle control, 0.15 ppm Mix, 1.5 ppm Mix, or 1500 ppm Mix in the rodent chow fed ad libitum. Bar graphs represent mean percentages of each treatment group with SEM and individual values of each mouse indicated. Each treatment group had n = 12–14 female mice. Asterisks (*) indicate significant differences compared to the vehicle control group (p ≤ .05), and carets (  ^ ) indicate borderline significant differences compared to the vehicle control group (p < .10).

DiNP exposure at 0.15 ppm, 1.5 ppm, or 1500 ppm for 1 month did not significantly change time that the mice spent in estrus or metestrus/diestrus compared to control (Figure 4A;n = 12–14 females per treatment group). Similarly, DiNP exposure at 0.15, 1.5, or 1500 ppm for 5 or 11 months did not significantly affect the time that the mice spent in estrus or metestrus/diestrus compared to control (Figs. 4C and E; n = 12–14 females per treatment group). In contrast, DiNP exposure at 1.5 ppm for 3 months tended to increase (p = .06) the time that the mice spent in estrus and tended to decrease (p = .07) the time that mice spent in metestrus/diestrus compared to control (Figure 4B;n = 12–14 females per treatment group). Further, DiNP exposure at 0.15 ppm for 7 months tended to decrease (p = .06) the time that the mice spent in estrus and tended to increase (p = 0.07) the time that the mice spent in metestrus/diestrus compared to control (Figure 4D;n = 12–14 females per treatment group).

Figure 4.

The effects of DiNP exposure on estrous cyclicity after 1 month (A), 3 months (B), 5 months (C), 7 months (D), and 11 months (E) of exposure. Estrous cyclicity was monitored daily for a 2-week period at each exposure time point. Treatment groups included vehicle control, 0.15 ppm DiNP, 1.5 ppm DiNP, or 1500 ppm DiNP in the rodent chow fed ad libitum. Bar graphs represent mean percentages of each treatment group with SEM and individual values of each mouse indicated. Each treatment group had n = 12–14 female mice. Carets (  ^ ) indicate borderline significant differences compared to the vehicle control group (p < .10).

Mix exposure at 1500 ppm for 1 month tended to decrease the time the mice spent in metestrus/diestrus compared to control (Figure 5A;n = 12–14 females per treatment group; p = .09). Further, Mix exposure at 0.15 ppm and 1500 ppm for 3 months significantly increased the time the mice spent in estrus and decreased the time the mice spent in metestrus/diestrus compared to control (Figure 5B;n = 12–14 females per treatment group; p ≤ .05). However, Mix exposure did not significantly affect estrous cyclicity at any other doses or time-points compared to control (Figure 5; n = 12–14 females per treatment group).

Effect of phthalate exposure on fertility indices

DEHP exposure (0.15 ppm, 1.5 ppm, or 1500 ppm) for 12 months did not affect mating index, gestation index, pregnancy rate, fertility index, birth rate, or dystocia rate compared to control (Figure 6; n = 7–9 per treatment group). Similarly, Mix exposure at 0.15 ppm, 1.5 ppm, or 1500 ppm did not affect mating index, gestation index, pregnancy rate, fertility index, birth rate, or dystocia rate compared to control (Figure 7; n = 6–9 per treatment group). In contrast, DiNP exposure at 1.5 ppm tended to decrease the gestational index (p = .09) compared to control, and DiNP exposure at 1500 ppm significantly decreased the gestational index (p = .01) and birth rate (p = .04) compared to control (Figure 8; n = 7–9 per treatment group).

Figure 6.

The effects of DEHP exposure on female fertility indices following 12 months of exposure to vehicle control, 0.15 ppm DEHP, 1.5 ppm DEHP, or 1500 ppm DEHP in the rodent chow fed ad libitum. Fertility was monitored by recording mating index (A), gestational index (B), pregnancy rate (C), fertility index (D), birth rate (E), and dystocia rate (F) following the first pregnancy of each dam. Graphs represent mean percentages. Each treatment group was composed of n = 7–9 female mice per treatment group.

Figure 7.

The effects of Mix exposure on female fertility indices following 12 months of exposure to vehicle control, 0.15 ppm Mix, 1.5 ppm Mix, or 1500 ppm Mix in the rodent chow fed ad libitum. Fertility was monitored by recording mating index (A), gestational index (B), pregnancy rate (C), fertility index (D), birth rate (E), and dystocia rate (F) following the first pregnancy of each dam. Graphs represent mean percentages. Each treatment group was composed of n = 6–9 female mice per treatment group.

Figure 8.

The effects of DiNP exposure on female fertility indices following 12 months of exposure to vehicle control, 0.15 ppm DiNP, 1.5 ppm DiNP, or 1500 ppm DiNP in the rodent chow fed ad libitum. Fertility was monitored by recording mating index (A), gestational index (B), pregnancy rate (C), fertility index (D), birth rate (E), and dystocia rate (F) following the first pregnancy of each dam. Graphs represent mean percentages. Each treatment group was composed of n = 7–9 female mice per treatment group. Asterisks (*) indicate significant differences compared to the vehicle control (p ≤ .05), and carets (  ^ ) indicate borderline significant differences compared to the vehicle control (p < .10).

DISCUSSION

This study tested the hypothesis that chronic daily exposure to DEHP, DiNP, or a mixture of phthalates alters estrous cyclicity and fertility-related parameters in CD-1 mice. Prior studies showed that adult exposure to DEHP or DiNP for 10 days alters estrous cyclicity for up to 9 months post-dosing in mice (Chiang and Flaws 2019). This study builds on those previous findings by investigating the effects of chronic phthalate exposure (DEHP, DiNP, or a mixture of phthalates) on estrous cyclicity, following 1 month, 3 months, 5 months, 7 months, and 11 months of exposure. Additionally, in previous studies, DEHP adult exposure or DiNP adult exposure for 10 days reduced the fertility index in mice (Chiang and Flaws 2019). The current study expands on these findings by determining the effect of chronic DEHP, DiNP, or phthalate mixture exposure on fertility indices following 12 months of phthalate exposure. Collectively, the data from this study show that chronic exposure to phthalates via the diet alters estrous cyclicity and long-term exposure to DiNP reduces gestational index and birth rate in mice.

In this study, mice were exposed to phthalates via the food chow. This exposure route is relevant to human phthalate exposure, which also occurs largely by ingestion of foods covered in food packaging containing phthalates (Wittassek et al. 2011; Wu et al. 2021). All the mice in this study ate similar amounts of food, indicating that the observed significant effects of phthalates on estrous cyclicity or fertility-related indices were not due to mice consuming more or less food than the control. Increases in urinary phthalate metabolites compared to control also indicate effective delivery of long-term phthalate exposure via the chow.

The selected urinary metabolites were measured because they represent a panel of metabolites used in several human and rodent studies and serve as a “gold standard” for monitoring phthalate exposure (Meling et al. 2020; Warner et al. 2019; Warner et al. 2021a,b; Ziv-Gal et al. 2016). Our data indicate that long-term exposure to phthalates via the chow results in detectable levels of these urinary phthalates and that the mice in the study were continuously consuming the chow containing phthalates. Measures were taken to eliminate exogenous phthalate exposure, such as the use of reverse osmosis drinking water and cages designed to not leach phthalates. Many of the urinary phthalate metabolites in the vehicle control group were undetectable, such as MEHP, or fell within a low concentration range, such as MEHHP (0–0.2 ng/ml). However, a few of the urinary metabolites, such as MBP (1.6–3.6 ng/ml), were detected in low levels in the vehicle control group, indicating that it is difficult to completely eliminate phthalate exposure. Phthalates are present in drinking water, and DEHP concentrations are detected at 0.16–170 µg/l of drinking water in the United States (Shelby 2006). Treating drinking water by reverse osmosis will remove 90–99% of environmental contaminants, but it is possible that the low levels of phthalates detected in the urine from control animals came from the drinking water. Furthermore, urine samples are exposed to plastic tubing when phthalates are measured by HPLC–MS/MS. Because phthalates are noncovalently bound to plastics, phthalates can leach from plastics (Rowdhwal and Chen 2018). Thus, samples exposed to laboratory plastics are a possible source of the urinary phthalates detected in control mice.

The 0.15 ppm and 1.5 ppm phthalate doses were chosen as doses of phthalates relevant to human exposure. Cumulative urinary metabolite concentrations (MEHP, MEOPH, MEHHP, MECPP, MBP, MiBP, MBzP, MCPP, and MEP) from adult women in the Midlife Women’s Health Study (MWHS) fall in the range of 124–411 ng/ml and adult women in NHANES had urinary metabolite concentrations of 42–311 ng/ml (Warner et al. 2021b). Cumulative urinary phthalate metabolites in the 1.5 ppm DEHP (37–92 ng/ml) and 1.5 ppm Mix (70–149 ng/ml) groups fall within the range of urinary metabolite concentrations of adult women. The cumulative urinary concentration of 0.15 ppm DEHP (12–30 ng/ml) and 0.15 ppm Mix (18–35 ng/ml) was below the range of urinary metabolite concentrations in adult women from the MWHS and NHANES studies. Although levels of total urinary phthalate metabolites in mice were comparable to those in adult women, differences were observed in the levels of individual urinary phthalate metabolites. For example, urinary concentrations of MEHP were undetectable in mice exposed to 0.15 ppm DEHP and 0.15 ppm Mix, whereas urinary MEHP concentrations were detectable in MWHS adult women (2.7–9.3 ng/ml) and NHANES adult women (0.6–3.1 ng/ml). Similarly, urinary MBzP concentrations in mice exposed to 0.15 DEHP (0–0.1 ng/ml) and 0.15 Mix (0.2–0.5 µg/ml) were lower than urinary MBzP concentrations in MWHS adult women (5.4–16.1 ng/ml) and NHANES adult women (1.8–19.4 ng/ml). These differences in individual urinary phthalate metabolite concentrations between mice and humans may stem from species differences in metabolism (Ito et al. 2014).

Collective urinary metabolite concentrations of 0.15 ppm DiNP (7.7–18.8 ng/ml) and 1.5 ppm DiNP (4.9–15.7 ng/ml) in mice fell below the range of urinary metabolite concentrations in adult women. Further, the range of urinary metabolite concentrations in the 1.5 ppm DiNP group was unexpectedly less than the range in the 0.15 ppm DiNP group. Unlike other phthalates such as DEHP, DiNP is a mixture of structurally similar chemical compounds (Saravanabhavan and Murray 2012). Standards of the primary DiNP metabolites for HPLC–MS/MS are not readily available (Saravanabhavan and Murray 2012). Thus, it is possible that the panel of urinary metabolites selected for this study does not capture the primary DiNP urinary metabolites of the mouse.

The 1500 ppm doses of DEHP, DiNP, and Mix, were chosen as toxicological doses of phthalates and for comparison with similar doses used in previous studies. The cumulative urinary phthalate concentrations of mice exposed to 1500 ppm DEHP (40–97 µg/ml) and 1500 ppm Mix (105–178 µg/ml) were about 1000-fold higher than the cumulative urinary phthalate concentration of adult women. The cumulative urinary phthalate concentration of 1500 ppm DiNP (3–10 µg/ml) was less than cumulative urinary phthalate concentration of 1500 ppm DEHP or 1500 ppm Mix.

Although DEHP and DiNP are both present in the Mix treatment groups, the amounts of DEHP and DiNP in the Mix treatment groups are less than in the single phthalate groups. The mixture of phthalates is composed of 21% DEHP and 15.1% DiNP, resulting in a lower exposure to each of these individual phthalates in the Mix groups. For example, urinary MEHHP in the 1500 ppm Mix group (6–9.9 µg/ml) is far less than urinary MEHHP in the 1500 ppm DEHP group (26–35 µg/ml). Further, urinary metabolites in the Mix groups are cumulative concentrations from 6 individual phthalates, and multiple phthalates can be metabolized into similar metabolites excreted in the urine.

Phthalates did not affect mouse body weight compared to control, indicating that the observed phthalate-induced effects on estrous cyclicity, gestational index, and birth rate are due to phthalate exposure and not differences in food consumption between treatment groups. A recent study found that dietary DEHP exposure at 0.05 and 5 mg/kg body weight for 3.5 months increased body weight compared to control in C57BL/6 J mice (Su et al. 2022). Our study, however, was with an outbred strain of mice, CD-1, whereas the previous study used an inbred strain of mice, C57BL/6J. Further, the previous study dosed mice via dough pill, whereas the current study had phthalates added to the normal chow. These differences in mice strains and method of dietary exposure could explain the difference in weight gain between these studies.

DEHP exposure at 0.15 ppm for 3 months and 5 months increased the time spent in estrus and decreased the time spent in metestrus/diestrus compared to control. Previous studies that investigated the effects of short-term exposure to phthalates on cyclicity corroborate our findings. Specifically, a study showed that 10 days of DEHP adult exposure increased the time spent in estrus compared to control and another study showed that 6 weeks of DEHP exposure increased the time spent in estrus to control in rodents (Adam et al. 2021; Hannon et al. 2014). In contrast, one study showed that 10 days of DEHP adult exposure at 20 $\mu$g/kg body weight, a comparable dose to 0.15 ppm DEHP, did not alter estrous cyclicity at 3 and 9 months post-dosing in mice (Chiang and Flaws 2019). Similarly, the current study showed that DEHP exposure for 7 months or 11 months did not alter estrous cyclicity. In this study, DEHP exposure affected estrous cyclicity at 3 and 5 months, but not at 7 and 11 months of exposure. Mammals are born with a finite number of oocytes, and reduced oocyte number, reduced oocyte quality, and reduction in hormone levels characterize female reproductive aging (Coxworth and Hawkes 2010; Kushnir et al. 2012; Shirasuna and Iwata 2017). Because of reduced ovarian function over time, phthalates may elicit more severe effects on estrous cyclicity during the mouse’s reproductive prime as opposed to later in the reproductive aging process. Collectively, these data suggest that the effects of DEHP exposure on estrous cyclicity are dependent upon dose, the age of the mice during exposure, and exposure duration.

Mix exposure at 0.15 ppm and 1500 ppm, but not at 1.5 ppm, decreased the time that the mice spent in estrus following 3 months exposure compared to control. These data indicate that the phthalate mixture has a nonmonotonic dose-response effect on estrous cyclicity. Endocrine disrupting chemicals are known to cause nonmonotonic dose responses, specifically for receptor-mediated effects (Vandenberg 2014). Interestingly, in a previous study, Mix exposure during gestation altered estradiol levels in adult mice in a nonmonotonic dose-response manner (Gill et al. 2021). Steroid hormones, such as estrogens and progestins, and peptides, such as inhibins and activins, regulate the estrous cycle via the concerted action of positive and negative feedback loops on the hypothalamus-pituitary-ovarian (HPG) axis. The current findings together with previous results indicate that the phthalate mixture affects estrogen signaling in a nonlinear manner to affect estrous cyclicity. However, it is possible that the phthalate mixture may also affect other steroid or peptide signaling pathways, causing effects on estrous cyclicity.

Chronic exposure to DiNP, but not DEHP or Mix at 1500 ppm significantly reduced gestational index, and chronic exposure to DiNP at 1.5 ppm tended to reduce gestational index. In contrast, the mating and fertility indices did not differ in any treatment group. These data indicate that DiNP exposure does not affect the ability of the female to mate or become pregnant, but DiNP exposure does reduce the ability of the female to give birth to live pups. Interestingly, a recent pooled study of 16 cohorts of women concluded that phthalate exposure is a risk factor associated with pregnancy complications such as preterm delivery (Welch et al. 2022). A number of mechanisms have been proposed to mediate the action of phthalates on preterm delivery, including an increase in oxidative stress, poor trophoblast differentiation, preterm growth restriction, and negative effects on the thyroid hormone system, which lead to pregnancy complications (Adibi et al. 2010; Aung et al. 2020; Bereketoglu and Pradhan 2022; Busgang et al. 2022; Ferguson et al. 2017). Several rodent studies have reported that phthalate exposure in the range of 50–500 mg/kg body weight/day leads to negative impacts on the placenta (Seymore et al. 2022). Further, in a study that exposed mice to dietary phthalates for 14 weeks, DBP, DEHP, and di-n-hexyl phthalate reduced fertility and decreased the number of live pups (Lamb et al. 1987). Although phthalate exposure in adult mice did not result in preterm birth in this study, DiNP decreased gestational index and birth rate, and trended towards increasing dystocia. Collectively, these data indicate that DiNP exposure leads to pregnancy complications.

A previous study showed that short-term DEHP and DiNP adult exposure at 20 $\mu$g/kg body weight did not reduce gestational index, but short-term exposure to DEHP or DiNP reduced the fertility index at 3 months post-dosing, but not at 9 months post-dosing (Chiang and Flaws 2019). The current study shows that DiNP exposure at 1.5 ppm and 1500 ppm, but not at 0.15 ppm decreased gestational index compared to control. Given that DiNP at 0.15 ppm is similar to a dose of 20 $\mu$g/kg body weight, these data suggest that differences in DiNP outcomes stem from differences in length of exposure. Further, the previous study on short-term effects of phthalates was a fertility study in adult mice at 5 months of age and the current study was a fertility study of adult mice at 13 months of age. The differences in age of the dams may also explain the differences in the effects of phthalate exposure on gestational index and fertility index between these 2 studies.

For many of the time points and exposure doses, phthalate exposure decreased the time mice spent in diestrus/metestrus and increased the time mice spend in estrus compared to control. Circulating progesterone levels are highest in diestrus/metestrus and lowest in estrus (Walmer et al. 1992). It is possible that mice exposed to phthalates that spend less time in diestrus/metestrus also have lower circulating levels of progesterone than controls. Furthermore, for many of the same time points and exposure doses, phthalate exposure decreased gestational index and did not alter mating index, which indicates a defect in pregnancy after successful mating has occurred. Progesterone levels are required for the maintenance of pregnancy (Milligan and Finn 1997). Conceivably, less time spent in diestrus/metestus and decreased gestation index with phthalate exposure could be an indication of lower progesterone levels with phthalate exposure compared to control. Future studies should focus on measurement of progesterone in mice exposed to the same doses of phthalates used in this study.

Although DEHP and DiNP are both included in the phthalate mixture, not all reproductive effects are shared between the mixture groups and the single phthalate exposure groups. After 3 months of Mix exposure, time spent in estrus after 0.15 ppm and 1500 ppm exposure increased compared to control. Furthermore, increased time spent in estrus was also present with 0.15 ppm DEHP, but not with 1500 ppm DEHP after 3 months and 5 months of exposure compared to control. The DiNP exposures resulted in the least severe estrous cyclicity phenotypes, with a borderline increase in estrus at 1.5 ppm DiNP after 3 months exposure and a borderline decrease in estrus at 0.15 ppm DiNP after 7 months of exposure. DEHP accounts for 21% of the phthalate exposure in Mix, and DiNP makes up 15.1% of phthalate exposure in Mix, resulting in the 0.15 ppm Mix containing 0.03 ppm DEHP and 0.02 ppm DiNP. The 1500 ppm Mix exposure contains 315 ppm DEHP and 227 ppm DiNP. The exposure to other phthalates in the mixture, DEP, DBP, DiBP, and BzBP, likely leads to the increase in time spent in estrus with Mix exposure compared to control. Additionally, DEHP and DiNP could act synergistically with the other phthalates to induce this phenotype.

Little is known about the molecular targets and cellular mechanisms of phthalate and phthalate metabolite action. Among the DEHP, DiNP, and Mix treatment groups, differences in effects on estrous cyclicity and fertility indices could be a result of metabolites differentially targeting cellular mechanisms and signaling pathways. Each phthalate diester is metabolized into a unique set of monoester metabolites (Warner et al. 2019, 2021a). Our laboratory has previously shown that DEHP and DiNP have differing effects on mouse fertility and estrous cyclicity (Chiang and Flaws 2019). Further, we have previously shown that monoester phthalate metabolites have differing abilities to bind the peroxisome proliferator-activated receptors (PPARs) (Meling et al. 2022). Because PPARs are known to affect steroidogenesis and ovarian biology (Froment et al. 2006; Komar 2005), one possible mechanism by which the different phthalates elicit different effects on ovarian cyclicity and fertility may be through PPAR pathways.

In conclusion, this study found that long-term exposure to DiNP, DEHP, or a mixture of phthalates alters estrous cyclicity up to 7 months of exposure. Additionally, the breeding study data indicate that DiNP exposure decreases fertility-related indices. Specifically, DiNP exposure reduced gestational index and reduced birth rate. Future studies should examine the mechanisms by which chronic exposure to phthalates alters estrous cyclicity and chronic exposure to DiNP reduces gestational index and birth rate.

FUNDING

National Institutes of Health (NIH) (R01 ES028661 to J.F.; R01 ES034112-01A1 and T32 ES007326).

DECLARATION OF CONFLICTING INTERESTS

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

AUTHOR CONTRIBUTIONS

Mary J. Laws: Conceptualization; Project administration; Supervision; Validation; Writing—review & editing. Daryl D. Meling: Conceptualization; Project administration; Supervision; Validation; Writing—review & editing Ashley R. K. Deviney: Data collection, Writing-review & editing. Ramsés Santacruz-Márquez: Conceptualization; Project administration; Supervision; Validation; Writing—review & editing. Jodi A. Flaws: Conceptualization; Funding acquisition; Project administration; Resources; Supervision; Validation; Writing—review & editing.