Ovarian Metabolism of an Environmentally Relevant Phthalate Mixture

Abstract

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

Keywords: follicles; metabolism; ovary; phthalates.

Figure 1.

Phthalates used in this study. The phthalate mixture is composed of the 6 diester phthalates on the left, arranged top to bottom from smallest to largest by molecular weight. Their primary monoester metabolites are shown on the right side of the arrows. Benzylbutyl phthalate is cleaved primarily to MBzP, but some MBP may also be formed. Note: DiNP is a mixture of isomers. One representative isomer is shown.

Figure 2.

Scheme of the metabolism of phthalates. Parent diester phthalates are easily cleaved to monoester metabolites during phase I metabolism and may also be modified via oxidation, hydroxylation, or hydrolysis. Conjugation occurs during phase II metabolism. In this study, only the primary monoester metabolites were measured.

Figure 3.

Short-chain phthalates detected by LC-MS/MS in neonatal ovary culture media following 4 days of culture in DMSO or 0.1–10 µg/ml phthalate mixture. Graphs represent means ± SEM from 5–6 separate experiments. Asterisks ( * ) indicate significant differences in the treatment (gray bars) from the control (black bars) (p ≤ .05).

Figure 4.

Long-chain phthalates detected by LC-MS/MS in neonatal ovary culture media following 4 days of culture in DMSO or 0.1–10 µg/ml phthalate mixture. Graphs represent means ± SEM from 5–6 separate experiments. Asterisks (*) indicate significant differences in the treatment (gray bars) from the control (black bars) (p ≤ .05). No DiNP or MiNP was detected.

Figure 5.

Short-chain phthalates detected by LC-MS/MS in antral follicle culture media following 4 days of culture in DMSO or 0.1–10 µg/ml phthalate mixture. Graphs represent means ± SEM from 4 separate experiments (n = 12 follicles/treatment/experiment). Asterisks (*) indicate significant differences in the treatment (gray bars) from the control (black bars) (p ≤ .05); ( ^ ) indicates borderline significance compared with control (p ≤ .10).

Figure 6.

Long-chain phthalates detected by LC-MS/MS in antral follicle culture media following 4 days of culture in DMSO or 0.1–10 µg/ml phthalate mixture. Graphs represent means ± SEM from 4 separate experiments (n = 12 follicles/treatment/experiment). Asterisks (*) indicate significant differences in the treatment (gray bars) from the control (black bars) (p ≤ .05). No MiNP was detected.

Figure 7.

Gene expression of metabolizing enzymes in neonatal ovary culture media following 4 days of culture in DMSO or 0.1–10 µg/ml phthalate mixture. Ovaries were collected and subjected to qPRC analysis relative to Rn18s. Graphs represent means ± SEM from 3 separate experiments (n = 3 ovaries/treatment/experiment). Asterisk (*) indicates significant difference in the treatment compared with control (p = .02).

Figure 8.

Gene expression of metabolizing enzymes in antral follicle culture media following 4 days of culture in DMSO or 0.1–10 µg/ml phthalate mixture. Follicles were collected and subjected to qPRC analysis relative to Rn18s. Graphs represent means ± SEM from 4 separate experiments (n = 12 follicles/treatment/experiment). Asterisk (*) indicates significant difference in the treatment compared with control (p = .03).

Figure 9.

Relative gene expression for the 6 investigated metabolizing genes for neonatal ovaries (A) and antral follicles (B) for DMSO vehicle control groups following 4 days of culture. Expression of UGT1a1 is set to 1.

Figure 10.

Protein expression of metabolizing enzymes in culture media following 4 days of culture in DMSO or 1 µg/ml phthalate mixture. Ovaries and follicles were collected and subjected to Western blotting for expression of LPL, ALDH1A1, and GAPDH. No statistical differences were observed between treatment groups. Graph compares relative expression between DMSO treatment groups in both tissues. Graph represent means ± SEM from 3 separate experiments (n = 6 ovaries/treatment/experiment or 50 follicles/treatment/experiment).