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. 2021 Jun 27;183(1):117–127. doi: 10.1093/toxsci/kfab085

Mono-n-Butyl Phthalate Distributes to the Mouse Ovary and Liver and Alters the Expression of Phthalate-Metabolizing Enzymes in Both Tissues

Estela J Jauregui 1, Jasmine Lock 2, Lindsay Rasmussen 1, Zelieann R Craig 1,3,✉
PMCID: PMC8502470  PMID: 34175954

Abstract

Humans are exposed to phthalates daily via items such as personal care products and medications. Reproductive toxicity has been documented in mice exposed to di-n-butyl phthalate (DBP); however, quantitative evidence of its metabolite, mono-n-butyl phthalate (MBP), reaching the mouse ovary and its effects on hepatic and ovarian biotransformation enzymes in treated mice is still lacking. Liquid chromatography/tandem mass spectrometry (LC-MS/MS) was employed to quantify MBP levels in liver, serum, and ovary from mice treated with a single or repeated exposure to the parent compound, DBP. Adult CD-1 females were pipet fed once or for 10 days with vehicle (tocopherol-stripped corn oil) or DBP at 1, 10, and 1000 mg/kg/day. Tissues and serum were collected at 2, 6, 12, and 24 h after the single or final dose and subjected to LC-MS/MS. Ovaries and livers were processed for qPCR analysis of selected phthalate-associated biotransformation enzymes. Regardless of duration of exposure (single vs repeated), MBP was detected in the tissues of DBP-treated mice. In single dose mice, MBP levels peaked at ≤6 h and fell close to background levels by 24 h post-exposure. Following the last repeated dose, MBP levels peaked at ≤2 h and fell to background levels by 12 h. Hepatic and ovarian expression of Lpl, Aldh1a1, Adh1, Ugt1a6a, and Cyp1b1 were altered in DBP-treated mice in a time- and dose-specific manner. These findings confirm that MBP reaches the mouse liver and ovary after oral exposure to DBP and influences the expression of hepatic and ovarian phthalate-associated biotransformation enzymes.

Keywords: dibutyl phthalate, ovary, liver, toxicology, phthalate-metabolizing enzymes

The World Health Organization estimates that 48.5 million couples worldwide are infertile; 30% of these cases can be attributed to female factors (Mascarenhas et al., 2012). In the last decade, there has been an increased concern regarding endocrine-disrupting environmental toxicants causing reproductive disorders. Endocrine-disrupting chemicals (EDCs), such as phthalates, alter hormonal homeostasis and cause developmental and reproductive toxicities (Jahnke et al., 2005). In women, phthalates have been associated with an increased incidence of pregnancy loss, decreased oocyte numbers retrieved during in vitro fertilization, and a low rate of live births (Hauser et al., 2016; Messerlian et al., 2016; Toft et al., 2012). Biomonitoring studies have shown that human exposure to phthalates is ubiquitous (Wittassek and Angerer, 2008), with phthalates found in human urine, amniotic fluid, breast milk, and follicular fluid (Calafat et al., 2006; Du et al., 2016; Silva et al., 2004a, 2004b).

In humans and rodents, phthalates are biotransformed in 2 steps. In phase I, the diester phthalate is hydrolyzed to a monoester phthalate by lipases (Lpl). Following phase I hydrolysis, the monoester phthalate diffuses into the systemic circulation and may target tissues, can be excreted in an unchanged form, or further oxidized and/or glucuronidated in additional reactions catalyzed by phase II biotransformation enzymes. These enzymes include aldehyde dehydrogenase (Aldh1a1), alcohol dehydrogenase (Adh1), and UDP-glucuronosyltransferase (Ugt1a6a), followed by excretion via urination. Phthalate diesters are mainly metabolized in the gastrointestinal tract but are also known to be metabolized in other organs such as the liver (Albro and Lavenhar, 1989; Frederiksen et al., 2007; Ito et al., 2005; Silva et al., 2007; Wittassek and Angerer, 2008).

Di-n-butyl phthalate (DBP) is used worldwide as a plasticizer or solvent in many consumer goods such as personal care products and medication coatings. DBP is not covalently bound to plastics; thus, it is easily leached from these products into the environment (Hernández-Díaz et al., 2009; Schettler, 2006). Previous reports have estimated that the general population is exposed to 7–10 μg/kg/day of DBP (Kavlock et al., 2002; IPCS, 1997), patients taking medications are exposed to 1–233 μg/kg/day of DBP (Hernández-Díaz et al., 2009, 2013), and workers in occupational settings are exposed to 0.1–76 μg/kg/day of DBP (Hines et al., 2011). In women undergoing medically assisted reproduction, an average range of 1.58–3.4 ng/ml of the active metabolite of DBP, mono-n-butyl phthalate (MBP), has been detected in follicular fluid (Du et al., 2016, 2019; Wu et al., 2020; Yao et al., 2020; Yuan et al., 2020). It is hypothesized that MBP is the bioactive compound and is responsible for the induced DBP toxicity observed in the reproductive system. Because the majority of DBP in the body is converted to MBP, this study focused on measuring MBP levels after DBP exposure (Fennell et al., 2004; Saillenfait, 1998).

As a result of recent advances identifying associations between phthalate burden and adverse reproductive outcomes in women and work modeling the effects of environmentally relevant exposures to phthalates in animal models, it is known that phthalate toxicity occurs at human relevant dose range. Specifically, the CD-1 mouse has been used to model the effects of environmentally relevant exposure to DBP on the reproductive system and identify ovarian folliculogenesis, steroidogenesis (Rasmussen et al., 2017; Sen et al., 2015), cell cycle arrest regulation, and DNA damage repair as pathways disrupted by DBP exposure in vitro and in vivo (Liu and Craig, 2019; Rasmussen et al., 2017). Furthermore, environmentally relevant exposures to a mixture of phthalates have also been shown to result in disrupted steroidogenesis and fertility in mice (Zhou et al., 2017; Zhou and Flaws, 2017). In all these studies, phthalates have been shown to produce low-dose effects that follow a nonmonotonic dose-response; however, to date no pharmacokinetic data regarding phthalate doses that mimic human exposure in mouse models is available. Additionally, no studies have reported on the levels of phthalate metabolites reaching the reproductive tissues of mice exposed to the parent compounds. Therefore, to better understand the disposition of DBP in the female mouse, this study measured the accumulation of MBP in the serum, liver, and ovary of mice treated with a single dose or repeated oral doses of DBP using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Also, phthalate-associated biotransformation enzymes were measured to determine whether their transcription is altered in response to the presence of MBP in the liver and ovary of DBP-treated mice.

MATERIALS AND METHODS

 

 

Chemicals

Dibutyl phthalate (DBP, CAS no. 84-74-2; 99.6% purity) was obtained from Sigma-Aldrich (St Louis, Missouri). Tocopherol-stripped corn oil (catalog no. 401100) was obtained from Dyets Inc. (Bethlehem, Pennsylvania). The isotope-labeled internal standard mono-butyl phthalate-13C4 was purchased from Cambridge Isotope Laboratories, Inc. (Andover, Massachusetts).

Animals and tissue collection

All animal use and experiments were conducted according to the guidelines stated in the United States Public Health Service’s Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, 1996) and approved by the University of Arizona Institutional Animal Care. Adult female CD-1 mice (60 days old) were obtained from Charles River Laboratories (Charles River, California) and housed in single-use BPA-free Innovive Disposable Rodent Caging at the University of Arizona Central Animal Care Facility. Animals were provided with water and food ad libitum. Mice were subjected to 12 L:12 D cycles and maintained at a temperature of 22 ± 1°C. The animals were allowed to acclimate for at least 24 h before handling. Mice were monitored for estrous stage and weight daily during the oral dosing. Animals were euthanized by decapitation under isoflurane anesthesia at 2, 6, 12, and 24 h after the final oral dose. All the major organs, including the liver and ovaries, were dissected and weighed. The liver and ovaries were snap frozen in liquid nitrogen and stored at −80°C for RNA extraction and measurements of MBP levels. One ovary from each animal was used for RNA extraction, and the contralateral ovary was used for MBP measurements. Blood from each mouse was collected, and the serum was separated and stored at −80°C until subsequent LC-MS/MS analysis.

Single and repeated oral dosing with dibutyl phthalate

In the single dose experiment, mice were randomly assigned to be pipet fed with a single dose of tocopherol-stripped corn oil (vehicle) or 1, 10, or 1000 mg/kg of DBP dissolved in vehicle. Mice (n = 5 per treatment/timepoint) were euthanized at 2, 6, 12, and 24 h after the single dose, and their tissues and serum were collected for analysis. For the repeated dosing experiment, a separate group of mice was randomly assigned to be pipet fed for 10 consecutive days with vehicle or 1, 10, 1000 mg/kg/day of DBP dissolved in vehicle. Mice (n = 5 per treatment/timepoint) were euthanized at 2, 6, 12, and 24 h following the last dose, and their tissues and serum were collected for analysis.

Analysis of MBP by liquid chromatography-tandem mass spectrometry

MBP measurements in livers, ovaries, and sera (n = 5 per treatment/timepoint) were performed by the Analytical Chemistry Shared Resource at the University of Arizona Cancer Center. Analysis of MBP levels was performed on a TSQ Quantum Ultra triple quadrupole mass spectrometer with Surveyor HPLC (ThermoFisher Scientific, Waltham, Massachusetts) using the mass transitions 223 → 149 for mono-butyl phthalate and 13C4 227 → 153 for mono-butyl phthalate 13C4. The limit of detection for MBP was 0.005 µg/ml for serum and tissues. The chromatographic technique was performed as described previously (Chen et al., 2012) on a Thermo Betasil phenyl column (50 mm × 2.1 mm, 3 µm particle size, Thermo Electron, Bellefonte, Pennsylvania). The mobile phase was 0.1% acetic acid in water (A) and 0.1% acetic acid in acetonitrile and methanol (84:16) (B) at a flow rate of 220 µl/min. The sample preparation protocol was adapted from Chang et al. (2013). Tissue samples were weighed out (weight recorded) and homogenized in 400 µl acetonitrile with a tissue homogenizer. Samples were then sonicated for 30 s. After centrifugation, 45 µl of the tissue supernatant or serum were used for the LC-MS/MS analysis. Standard curves in samples were prepared by spiking 10 µl of standard solution (diluted in methanol) into 45 µl of serum or tissue supernatant, 10 µl of the internal standard solution was then added, and the proteins of interest precipitated with 150 µl methanol. After centrifugation, 10 µl injection volume of sample was used for analysis. To minimize contamination, solvents were stored in glass containers, and all standard solutions were prepared and stored in glass. All sample processing was carried out using glass tubes, and the autosampler vials contained glass inserts as well. The average extraction recovery for MBP was 82% for serum (Figure 1), 91% for ovary, and 85% for liver.

Figure 1.

Figure 1.

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(Methods). Extracted serum standard curve for MBP. The average extraction recovery for MBP was 82% for serum.

RNA extraction and cDNA synthesis

Total RNA from livers and ovaries of DBP-treated and control mice was extracted using Qiagen RNeasy Micro Kits (Qiagen, Valencia, California) followed by a 15-min Qiagen DNase treatment. RNA concentration for each sample was determined by measuring the absorbance at 260 nm on a Synergy H1m microplate reader using a Take3 microvolume plate (Biotek, Winooski, Vermont). The extracted RNA (0.5 µg) was used to generate cDNA utilizing the iScript cDNA synthesis kit (Bio-Rad, Hercules, California). The cDNA was then used for real-time PCR analysis.

Real-Time PCR

Real-time PCR was performed in triplicate (n = 3–5 per treatment/timepoint) utilizing 1 µl of cDNA (0.5 µg/µl), 2 µl of each primer (20 µM), 10 µl of Ssofast EvaGreen Supermix (Bio-Rad, Hercules, California), and 5 µl of nuclease-free water. Specific primers for Lpl (forward: 5′-TGGCTGACACTGGACAAACA-3′ and reverse: 5′-CCCACTTTCAAACACCCAAACA-3′), Aldh1a1 (forward: 5′- GGTGAGGAGGACTAGTTGTGAC-3′ and reverse: 5′-TCACAACACCTGGGGAACAG-3′), Adh1 (forward: 5-AAGTTTCCGTTGGACCCGTT-3′ and reverse: 5′-CGGTACGGATGCTCTTTCCA-3′), Cyp1b1 (forward: 5′-TCCTCTTTACCAGATACCCGGA-3′ and reverse: 5′-GACATATGGCAGGTTGGGCT-3′), Ugt1a6a (forward: 5′-TCAGATGCTGGCTGATGGTG-3′ and reverse: 5′-AGAAGGCAAGCCATCCTGTC-3′), Gapdh (forward: 5′-ACAACTTTGGCATTGTGGAA-3′ and reverse: 5′-GATGCAGGGATGATGTTCTG-3′), and Rps2 (forward: 5′-CTGACTCCCGACCTCTGGAAA-3′ and reverse: 5′-GAGCCTGGGTCCTCTGAACA-3′) were designed or made as published previously and verified by PrimerBLAST software (Warner et al., 2019). Gene expression data were analyzed using the ΔΔCt model for relative quantification, normalized to housekeeping genes Rps2 and Gapdh, and are represented as mean normalized relative expression. The negative controls (a no template control, a no primer control, and a no reverse transcriptase control) included in the real-time PCR reactions showed no amplification.

Statistical analysis

Data were analyzed using GraphPad Prism 8 software (GraphPad, San Diego, California). Before statistical analysis, all data were subjected to normality and homogeneity of variance tests. Statistical analysis of MBP levels was done by 2-way ANOVA, followed by Dunnett’s post hoc test. Real-time PCR data were analyzed by 1-way ANOVA followed by Dunnett’s post hoc test when investigating treatment or time effects. Additionally, ROUT testing (1%) was performed when extreme values were observed to identify outliers before removal. All data were represented as mean ± standard error of the mean (SEM). Statistical significance was assigned based on p values with a p ≤ .05 considered as significant. Each n represents an individual animal. A priori power analysis (G*Power; F test family; effect size: 0.4; power: 0.8) revealed that a total of 111 mice would be needed for this study. We used a total of 160 mice which provided a power of approximately 0.95.

RESULTS

 

MBP Levels in Serum and Tissues After a Single Oral Dose of DBP

Biomonitoring data that assess phthalate doses mimicking human environmental exposure in the mouse model are lacking. Thus, to investigate this, a concentration-time study in CD-1 female mice after a single dose of DBP was performed. MBP was measured in the sera, livers, and ovaries of mice at 2, 6, 12, and 24 h after either a single oral dose of oil (vehicle), 1 mg/kg of DBP (1DBP), 10 mg/kg of DBP (10DBP), or 1000 mg/kg of DBP (1000DBP). In mice treated with oil, background MBP levels were detected in the liver and serum but not in the ovary. MBP was detected in the sera and tissues of all DBP-treated mice. The average background levels at all timepoints detected in vehicle-treated mice were 0.5 ± 0.164 µg/g for liver and 0.009 ± 0.003 µg/ml for serum. In DBP-treated mice, serum MBP levels were highest at 6 h in the 1DBP group (0.018 ± 0.005 µg/ml), whereas levels in the 10DBP (0.290 ± 0.056 µg/ml) and 1000DBP (15.742 ± 2.46 µg/ml) dose groups were significantly highest at 2 and 6 h compared with 12 and 24 h (p ≤ .05). MBP levels in all DBP-treated mice decreased with time after the 6 h time point (Figure 2A). Similarly, the highest concentrations of MBP in the liver of mice treated with 1DBP (2.215 ± 0.319 µg/g) was significantly higher at 6 h, whereas 10DBP (19.358 ± 6.158 µg/g), and 1000DBP (235.098 ± 67.735 µg/g) were detected at 6 h and significantly decreased (p ≤ .05) at 24 h in comparison to the 2 h time point (Figure 2B). In the ovary, MBP levels were highest at 2 h in the 1DBP (2.641 ± 0.889 µg/g) and 10DBP (10.001 ± 1.184 µg/g) dose groups, and at 6 h for the 1000DBP (166.232 ± 46.321 µg/g) dose group. At the 12 h timepoint, MBP levels significantly decreased in the 10DBP and undetectable in the 1000DBP dose group (p ≤ .05) (Figure 2C).

Figure 2.

Figure 2.

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MBP levels in liver, serum, and ovary after 1 oral dose of DBP. Adult female CD-1 mice were pipet fed with 1 dose of vehicle or DBP at 1 mg/kg (1DBP), 10 mg/kg (10DBP), or 1000 mg/kg (1000DBP) and euthanized 2, 6, 12, or 24 h after the dose. Tissues were collected and MBP levels were measured in serum (A), liver (B), and ovary (C) of vehicle and DBP-treated mice (n = 5 per treatment/timepoint) by LC-MS/MS and compared with the 2 h timepoint in the same treatment group. Graphs represent MBP levels as mean ± SEM. Asterisks (*) indicate statistically significant (*p ≤ .05 or **p ≤ .005).

MBP Levels in Serum and Tissues After Repeated DBP Dosing

Given that MBP was detected in the ovary after a single DBP dose, whether MBP accumulates in the ovaries of mice after short-term repeated exposure to DBP like that reported by Sen et al. (2015) was investigated. To determine this, CD-1 mice were treated for 10 consecutive days with oil or DBP at the same levels used in the single dose study (1, 10, and 1000 mg/kg/day), and their MBP levels measured at 2, 6, 12, and 24 h after their last dose. In mice treated with oil, background MBP levels were detected only in the liver (0.092 ± 0.045 µg/g; 60% detection in controls; Figure 3). In DBP-treated mice, MBP was detected in all sera and tissue samples except for the tissues from mice in the 1DBP group. In serum, MBP levels were highest at 2 h in 10DBP (0.762 µg/ml ± 0.096) and 1000DBP (75.604 µg/ml ± 10.736) and significantly decreased (p ≤ .05) with time (Figure 3A). A similar time response was observed in the liver, MBP levels were highest in 10DBP (36.942 ± 3.490 µg/g) and 1000DBP (1255.946 ± 215.288 µg/g) at 2 h and significantly decreased (p ≤ .05) at 6, 12, and 24 h (Figure 3B). Interestingly, MBP levels were only detected in the ovary of mice treated with the 2 highest dosages. In these treatment groups, the highest concentrations of MBP were detected at 2 h for the 10DBP (3.82 ± 1.371 µg/g; 80% detection) and 1000DBP (1186.761 ± 201.256 µg/g; 100% detection) prior to dropping to undetectable levels at 24 h (p ≤ .05; Figure 3C).

Figure 3.

Figure 3.

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MBP levels in liver, serum, and ovary after 10 daily doses of DBP. Adult female CD-1 mice were pipet fed with 10 daily doses of vehicle or DBP at 1 mg/kg/day (1DBP), 10 mg/kg/day (10DBP), or 1000 mg/kg/day (1000DBP) and euthanized 2, 6, 12, or 24 h after the final dose. Tissues were collected and MBP levels were measured in serum (A), liver (B), and ovary (C) of vehicle and DBP-treated mice (n = 5 per treatment/timepoint) by LC-MS/MS and compared with the 2 h timepoint in the same treatment group. Graphs represent MBP levels as mean ± SEM. Asterisks (*) indicate statistically significant (*p ≤ .05, **p ≤ .005, or ***p ≤ .0005) and pound (#) indicates a trend (#p ≤ .10).

Expression of Phthalate-Associated Biotransformation Enzymes in Mouse Liver and Ovary

Lpl has been implicated in the phase I biotransformation of phthalates in humans and rodents; whereas Aldh1a1, Adh1, and Ugt1a6a have been described as carrying out subsequent oxidation and/or glucuronidation reactions (Albro and Lavenhar, 1989; Frederiksen et al., 2007; Ito et al., 2005; Silva et al., 2007). Detection of MBP in the ovary of DBP-treated mice suggests that if the ovary has the appropriate enzymatic machinery, it may metabolize phthalates locally. Therefore, utilizing qPCR, the expression of transcripts for these 5 biotransformation enzymes and Cyp1b1, a cytochrome P450 biotransformation enzyme known to be induced by phthalates ( Chen et al., 2012), were measured in the ovary and liver of the control groups at 2 h. As expected, in the liver, Aldh1a1 and Adh1 were significantly enriched by a 13- and 15-fold change, respectively, when compared with the housekeeping gene Gapdh (p ≤ .005; Figure 4A). Additionally, the Lpl and Ugt1a6a, and an enzyme known to be induced by phthalates, Cyp1b1, were also detected in the liver (Figure 4A). In the ovary, Lpl, Aldh1a1, Adh1, Ugt1a6a, and Cyp1b1 were also expressed, with Aldh1a1 being highly enriched by 1.75-fold change (p ≤ .005) when compared with the reference gene, Gapdh (Figure 4B). Even though Lpl, Adh1, Ugt1a6a, and Cyp1b1 were also detected in the ovary, their expressions were significantly lower (p ≤ .005) than Gapdh (Figure 4B).

Figure 4.

Figure 4.

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Expression of DBP-metabolizing enzymes in liver and ovary. Expression of Lpl, Aldh1a1, Adh1, Ugt1a6a, and Cyp1b1 were analyzed in the liver (A) and ovary (B) of vehicle treated mice at the 2 h timepoint by qPCR (n = 3–5). Normalized relative expression of DBP-metabolizing enzymes were compared with normalized relative expression of Gapdh. Gene expression data are presented as mean normalized relative expression ± SEM. Asterisks (*) indicate statistically significant when compared with Gapdh (*p ≤ .05, **p ≤ .005, and ***p ≤ .0005).

Expression of Phthalate-Associated Biotransformation Enzymes After a Single Oral Dose of DBP

Using qPCR, this study measured the expression of Lpl, Aldh1a1, Adh1, Ugt1a6a, and Cyp1b1 in liver and ovary samples collected at 2, 6, 12, and 24 h after a single oral dose of DBP. In the liver, there were no significant differences in the expression of Lpl in DBP-treated mice versus vehicle controls (Figure 5A). Aldh1a1 expression was increased when compared with controls at 2 h in the 1DBP (p ≤ .10) and 10DBP (p ≤ .005) groups (Figure 5B). The expression of Adh1 was also significantly increased (p ≤ .05) in the 1DBP group at 12 and 24 h, and in the 10DBP (p ≤ .05) dose group at 12 h when compared with controls (Figure 5C). In contrast, Ugt1a6a transcript levels were significantly decreased (p ≤ .05) at 6 h in the 1DBP group and at 2, 6, and 12 h in the 10DBP group (Figure 5D). Finally, hepatic Cyp1b1 gene expression was significantly increased (p ≤ .05) at 24 h in the 10DBP group (Figure 5E). Additionally, to explore time-dependent changes in the phthalate metabolism genes, the expression of each gene was compared within treatment groups to their corresponding 2 h time point. The expression of Lpl significantly decreased (p ≤ .05) in the control group and in the 1000DBP group at 12 h in comparison to the 2 h time point; however, all the other genes did not vary with time in the oil groups. Interestingly, Adh1 significantly increased at the 6 h and then decreased at the 12 and 24 h time points. Ugt1a6a also significantly increased in the 1DBP (p ≤ .05) and 10DBP (p ≤ .005) groups at 24 h when compared with their corresponding 2 h timepoint. Additionally, there was an increasing trend (p ≤ .10) observed in the expression of Cyp1b1 in the 1DBP and 10DBP groups at 24 h.

Figure 5.

Figure 5.

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Gene expression of DBP-metabolizing enzymes in liver after 1 oral dose of vehicle or DBP. Adult female CD-1 mice were pipet fed with on dose of vehicle or DBP at 1 mg/kg (1DBP), 10 mg/kg (10DBP), or 1000 mg/kg (1000DBP) and euthanized 2, 6, 12, or 24 h after the dose. Expression of Lpl (A), Aldh1a1 (B), Adh1 (C), Ugt1a6a (D), and Cyp1b1 (E) in the liver were analyzed by qPCR, normalized to housekeeping genes, and compared with controls at each timepoint (n = 3–5 per treatment/timepoint). Gene expression data are presented as mean normalized relative expression ± SEM. Asterisks (*) indicate statistically significant (*p ≤ .05, or **p ≤ .005) and pound (#) indicates a trend (#p ≤ .10).

Knowing that phthalate-metabolizing enzymes are also expressed in the ovary, whether their expression was also altered following a single oral dose of DBP was investigated next. The expression of Lpl transcript was significantly increased (p ≤ .005) in the 1DBP and 10DBP dose groups at the 12 and 24 h when compared with controls at the same timepoints in the ovary (Figure 6A). Interestingly, Aldh1a1 expression was also significantly increased (p ≤ .0005) in the 10DBP group at 6 and 24 h compared with controls (Figure 6B). Similarly, Adh1 was significantly increased in the 1DBP group at 12 h, whereas in the 10DBP group, it was significantly increased (p ≤ .05) at 6 and 12 h versus controls (Figure 6C). There was no change in the expression of Ugt1a6a in any of the treatment levels and timepoints (Figure 6D). Finally, Cyp1b1 expression levels were significantly increased (p ≤ .05) at 2 and 12 h in the 1DBP and 10DBP groups compared with their respective controls (Figure 6E). As with hepatic expression of these genes, time-dependent changes were investigated by comparing each time point to 2 h within each treatment group. It was observed that Lpl and Aldh1a1 significantly decreased (p ≤ .05) at 6, 12, and 24 h in the oil group when compared with 2 h. Additionally, Ugt1a6a was significantly increased (p ≤ .005) in the 1000DBP at 24 h, whereas Cyp1b1 was significantly decreased (p ≤ .05) in the 10DBP group at 12 h.

Figure 6.

Figure 6.

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Gene expression of DBP-metabolizing enzymes in ovaries after 1 oral dose of vehicle or DBP. Adult female CD-1 mice were pipet fed with 1 dose of vehicle or DBP at 1 mg/kg (1DBP), 10 mg/kg (10DBP), or 1000 mg/kg (1000DBP) and euthanized 2, 6, 12, or 24 h after the dose. Expression of Lpl (A), Aldh1a1 (B), Adh1 (C), Ugt1a6a (D), and Cyp1b1 (E) in the ovary were analyzed by qPCR, normalized to housekeeping genes, and compared with controls at each timepoint (n = 3–5 per treatment/timepoint). Gene expression data are presented as mean normalized relative expression ± SEM. Asterisks (*) indicate statistically significant (*p ≤ .05, **p ≤ .005, ***p ≤ .0005, or ****p ≤ .00005) and pound (#) indicates a trend (#p ≤ .10).

Expression of Phthalate-Metabolizing Enzymes After Repeated Oral Dosing With DBP

Because a single dose of DBP altered the gene expression of some of the phthalate-metabolizing enzymes and Cyp1b1 in the ovary and liver, a subsequent experiment investigated whether a similar pattern would be observed in tissues of mice treated with 10 daily doses of DBP. In the liver of mice treated with DBP for 10 days, Lpl mRNA was significantly decreased (p ≤ .05) in mice treated with 1DBP and 10DBP at 2 h when compared with controls at 2 h (Figure 7A). There was no difference in the expression of Aldh1a1 but a noticeable variation in its expression at 24 h (Figure 7B). An increasing trend in Adh1 expression was observed in the 10DBP group at 24 h when compared with control (Figure 7C). The Ugt1a6a gene expression was significantly increased (p ≤ .05) or showed an increasing trend in the 1DBP, 10DBP, and 1000DBP groups at 2 h. Interestingly, at the 6 h timepoint, Ugt1a6a was significantly increased (p ≤ .05) in the 1DBP and the 10DBP groups in comparison to control (Figure 7D). The expression of Cyp1b1 was also significantly increased (p ≤ .05) by at least 9-fold in 1DBP, 10DBP, and 1000DBP at 2 h when compared with control (Figure 7E). Time-dependent changes were observed in the expression of Aldh1a1, Adh1, Ugt1a6a, and Cyp1b1 when compared within treatment groups to their corresponding 2 h time point. The expression of Aldh1a1 and Adh1 was significantly increased (p ≤ .05) at 12 h in the 1000DBP group compared with 2 h. Ugt1a6a decreased in 1DBP at 24 h (p ≤ .005), in 10DBP at 12 h (p ≤ .10), and in the 1000DBP group at 6, 12, and 24 h (p ≤ .0005). Additionally, there was a decreasing trend in the expression of Cyp1b1 in the 1DBP at 12 h.

Figure 7.

Figure 7.

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Gene expression of DBP-metabolizing enzymes in livers after 10 oral doses of vehicle or DBP. Adult female CD-1 mice were pipet fed with 10 daily dosages of vehicle or DBP at 1 mg/kg/day (1DBP), 10 mg/kg/day (10DBP), or 1000 mg/kg/day (1000DBP) and euthanized 2, 6, 12, or 24 h after the final dose. Expression of Lpl (A), Aldh1a1 (B), Adh1 (C), Ugt1a6a (D), and Cyp1b1 (E) in the liver were analyzed by qPCR, normalized to housekeeping genes, and compared with controls at each timepoint (n = 5 per treatment/timepoint). Gene expression data are presented as mean normalized relative expression ± SEM. Asterisks (*) indicate statistically significant (*p ≤ .05, **p ≤ .005, or ***p ≤ .0005) and pound (#) indicates a trend (#p ≤ .10).

In the ovary, qPCR analysis showed significantly increased (p ≤ .05) expression of Lpl in the 10DBP dose group and an increasing trend (p ≤ .10) in the 1000DBP dose group at 6 h when compared with controls. In all 3 dose groups, Lpl expression levels significantly decreased (p ≤ .05) or showed a decreasing trend at 12 h in comparison to control (Figure 8A). The expression of Aldh1a1 was significantly increased (p ≤ .05) in all 3 dose groups at 24 h when compared with their respective control (Figure 8B). The expression of Adh1 was significantly increased in the 10DBP group at 2 h and in the 1DBP group at 6 h when compared with control at the same timepoints (Figure 8C). In the ovary, the expression of Ugt1a6a or Cyp1b1 did not change (Figs. 8D and 8E). When time-dependent changes of these same genes were evaluated, Aldh1a1 significantly decreased (p ≤ .05) in the oil at 6 and 24 h when compared with 2 h. The expression of Lpl significantly decreased (p ≤ .05) in the 1000DBP group at 12 h in comparison to 2 h. Interestingly, Aldh1a1 significantly increased (p ≤ .05) in the 1DBP at 6, 12, and 24 h compared with the 2 h time point. Additionally, Adh1 significantly increased (p ≤ .05) in the 1DBP at 12 h when compared with its expression at 2 h. In the 1DBP group, there was also an increasing trend of Ugt1a6a and a decreasing trend of Cyp1b1 at 6 h in comparison to their corresponding 2 h time point.

Figure 8.

Figure 8.

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Gene expression of DBP-metabolizing enzymes in ovaries after 10 oral doses of vehicle or DBP. Adult female CD-1 mice were pipet fed with 10 daily dosages of vehicle or DBP at 1 mg/kg/day (1DBP), 10 mg/kg/day (10DBP), or 1000 mg/kg/day (1000DBP) and euthanized 2, 6, 12, or 24 h after the final dose. Expression of Lpl (A), Aldh1a1 (B), Adh1 (C), Ugt1a6a (D), and Cyp1b1 (E) in the ovary were analyzed by qPCR, normalized to housekeeping genes, and compared with controls at each timepoint (n = 5 per treatment/timepoint). Gene expression data are presented as mean normalized relative expression ± SEM. Asterisks (*) indicate statistically significant (*p ≤ .05 or **p ≤ .005) and pound (#) indicates a trend (# p ≤ .10).

DISCUSSION

CD-1 female mice were exposed to a single dose or 10 repeated daily doses of DBP, measured levels of MBP, and determined the expression of phthalate-associated biotransformation enzymes in their tissues. MBP levels were detected in serum, liver, and ovary of DBP-treated mice after a single and repeated oral exposure. These findings demonstrate that MBP reaches the ovary after oral exposure to the parent compound and that MBP levels found in the serum, liver and ovary are commensurate to the level of DBP ingested. MBP levels in the ovary, serum, and liver of single dose and repeated dosing mice peaked early (≤6 h single; ≤2 h repeated) after oral DBP exposure but differed in terms of peak concentration and the timing of their fall back to background levels. Additionally, enzymes involved in the biotransformation of phthalates were detected in the mouse tissues tested, and their transcript levels were found to be altered in the livers and ovaries of DBP-treated mice.

Epidemiologic studies have estimated the general population exposure to DBP at 7–10 µg/g/day with ingestion being proposed as the main exposure route (IPCS, 1997; Kavlock et al., 2002; Silva et al., 2005). In women undergoing medically assisted reproduction, MBP levels in follicular fluid have been reported to range on average 1.58–3.4 ng/ml, thus providing evidence that the ovary receives direct exposure to this phthalate (Du et al., 2016, 2019; Wu et al., 2020; Yao et al., 2020; Yuan et al., 2020). Unfortunately, data on the distribution of MBP to the ovary in mouse models exposed to DBP are lacking. In this study, mice were treated with single or repeated doses of DBP at 1 and 10 mg/kg and a high dose of 1000 mg/kg to mimic low and high exposure levels used in toxicological studies. Using LC-MS/MS, MBP was detected in the serum, liver, and ovary of mice treated with single or repeated doses of DBP. Interestingly, the average concentration of MBP detected in the ovaries of DBP-treated mice in this study was higher than MBP levels found in the ovarian follicular fluid of women. Specifically, this finding provides evidence that animal studies using dosages of DBP ≥1 mg/kg/day are significantly overexposing the ovary to MBP when compared with levels reported in human follicular fluid (Du et al., 2016, 2019; Wu et al., 2020; Yao et al., 2020; Yuan et al., 2020). Based on allometric scaling (US USDA and Nair and Jacob, 2016), dose equivalence between mouse and human can be obtained by using 12.3 as the conversion factor. Present findings, taken together with allometric scaling and species differences in chemical disposition, will help further refine the design of animal studies modeling human relevant exposures.

In the single-dose study, differences in the timing and magnitude of peak concentrations of MBP were observed according to matrix (ie, sera vs tissues) and between different dose groups (ie, low vs high doses in tissues). First, regardless of dose, serum MBP levels were lower than those detected in liver and ovary of mice treated with a single oral dose of DBP. This could be explained by removal of MBP from serum as result of tissue distribution because rapid distribution (distribution half-life of 5.77 ± 1.14 min) has been reported in rats treated with DBP via the IV route (30 mg/kg; Chang et al., 2013). Protein binding could be an additional mechanism via which MBP could be decreased in serum given that binding to human serum albumin has been described for DBP (Yue et al., 2014) and other phthalates (Wang and Zhang, 2015; Xie et al., 2011; Yue et al., 2014). As is typical with most xenobiotics, MBP could be leaving serum via glomerular filtration as this route has been described as a significant route of elimination in humans and rats for phthalates with lower hydrophobicity such as DBP and MBP (Domínguez-Romero and Scheringer, 2019). This is supported by work showing that rats exposed to DBP clear more than 80% of the dose within 24–48 h of exposure (Williams and Blanchfield, 1975) and that the half-life of MBP elimination after a 30-mg/kg intravenous dose is 1.7 h (Kremer et al., 2005). Finally, differences observed in the timing of MBP peak concentrations observed between dose levels in livers and ovaries could be related to biotransformation and elimination. This is supported by the present findings showing dose-specific differential expression of inducible biotransformation enzymes in both tissues and published work showing that dose influences the pattern of phthalate metabolites found in urine (Domínguez-Romero and Scheringer, 2019).

Repeated dosing is commonly utilized in risk assessment as it mimics daily exposure of varying lengths in humans. Therefore, this study investigated the fate of MBP in serum, liver, and ovary and its effects on biotransformation enzyme expression after a subacute (10-day) oral exposure to DBP. Compared with the single-dose study, mice in the repeated dosing regimen had higher peak serum and tissue MBP levels. Alhhough it is tempting to conclude that this is the result of accumulation over the repeated dosing, this idea is not supported by data showing an earlier drop to background MBP levels in repeated versus single dosed mice in this study and various published reports (Fennell et al., 2004; Foster et al., 1983; Saillenfait et al., 1998; Tanaka et al., 1978; Williams and Blanchfield, 1975). It is more likely that these differences between single and repeated dosing regimens are the result of adaptation in the disposition of DBP, particularly absorption and biotransformation. This idea is supported by data showing a more diverse change in hepatic and ovarian biotransformation enzyme transcript in single versus repeated dosing groups in this study. Indeed, altered biotransformation activity in the liver and ovary could result in more detection of MBP versus other metabolites or conjugates. Moreover, by altering esterase expression and gastrointestinal uptake, repeated dosing may cause a state in which more absorbed DBP and MBP are available to be distributed to these tissues. Unfortunately, these processes have not yet been evaluated in mice and, thus, will require future studies.

Transcript levels of selected biotransformation enzymes were evaluated first in untreated ovaries to detect endogenous expression after single and repeated exposure to oral DBP. This study’s findings that Lpl, Aldh1a1, Adh1, Ugt1a6a, and Cyp1b1 transcripts were detected in the adult mouse ovary, with Aldh1a1 showing the highest expression relative to the reference gene demonstrate that the ovary has the capacity to biotransform phthalates locally. Indeed, this study’s data in whole animals are consistent with findings from Warner et al., who showed expression of these enzyme transcripts and conversion from parent to monoester phthalates in vitro using isolated antral follicles and cultured neonatal ovaries from mice (Warner et al., 2019). When evaluating the liver and ovaries from single versus repeatedly dosed mice, a greater number of differentially expressed transcripts were observed in the single dose livers and ovaries. Early increases in hepatic and ovarian Aldh1a1 and Adh1 transcripts, when translated into increased enzyme activity, are likely to result in increased formation of oxidative metabolites and could explain the loss of hepatic and ovarian MBP over time. Among the differences in toxicogenomic response to single versus repeated exposure, the effects of dosing on Ugt1a6a and Lpl were noteworthy. Specifically, it was noted that downregulation of hepatic Ugt1a6a at various timepoints in single dose mice contrasted with upregulation in repeatedly dosed mice. Similarly, ovarian Lpl was upregulated in single dose mice but downregulated at various timepoints in repeatedly exposed mice. Interestingly, the expression of Cyp1b1, a cytochrome P450 enzyme known to be regulated by the aryl hydrocarbon receptor, and to metabolize estradiol to several hydroxylation products (Hayes et al., 1996), was upregulated in the liver and ovary of some of the DBP-treated groups. This result was consistent with other reports indicating that DEHP and DBP upregulate Cyp1b1 transcript (Chen et al., 2012; Ernst et al., 2014; Sen et al., 2015; Zou et al., 2020). Consequently, if these gene expression changes translate to significant differences in MBP oxidation, conjugation, and estradiol breakdown, this could explain discrepancies in the timing of peak and fall to background MBP levels between the 2 dosing regimens and some endocrine disrupting effects of DBP, respectively. Finally, the highest dose used in this study did not result in significant changes in hepatic and ovarian biotransformation gene expression, an observation that evidences the existence of a nonmonotonic dose-response and further supports the mechanism of action of DBP as an EDC.

In conclusion, this study demonstrates that MBP reaches the adult mouse ovary after oral administration of DBP, that the ovary expresses the biotransformation machinery involved in metabolizing phthalates, and highlights differences in DBP disposition between single and repeated dosing schemes. Although additional studies will be needed to fully characterize the mechanisms underlying the differences in disposition observed here, these findings are useful in the risk assessment of DBP as they can facilitate predicting internal doses achieved in animal models and inform how they can be adjusted to better mimic human internal exposures.

ACKNOWLEDGEMENTS

The authors wish to acknowledge members of the Craig Lab for their technical help during tissue collections. The authors also thank Dr. Sherry Chow and Wade Chew for their technical advice and analysis of MBP levels by LC-MS/MS.

FUNDING

National Institute on Environmental Health Sciences (NIEHS) (R01 ES026998 to Z.R.C., P30 ES000669 to Southwest Environmental Health Sciences Center); the National Cancer Institute (P30 CA23074 to Analytical Chemistry Shared Resource, University of Arizona Cancer Center).

DECLARATION OF CONFLICTING INTERESTS

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

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