Exposure to Mono-n-Butyl Phthalate in Women with Endometriosis and Its Association with the Biological Effects on Human Granulosa Cells

Abstract

To study the association between urinary phthalate metabolite levels, endometriosis, and their effects on human granulosa cells, we recruited patients who underwent laparoscopy to confirm endometriosis (n = 123) and control patients (n = 78). Liquid chromatography-tandem mass spectrometry was used to measure the following five urinary phthalate metabolites: mono-n-butyl phthalate (MnBP), mono(2-ethylhexyl) phthalate, monobenzyl phthalate, mono(2-ethyl-5-oxo-hexyl) phthalate, and mono(2-ethyl-5-hydroxyhexyl) phthalate. Urinary MnBP levels were higher in patients with endometriosis than in controls after multivariable logistic regression including the number of deliveries, body mass index, and use of medicine as covariables. MnBP correlates with other phthalate metabolites. Previous studies found that endometriosis was a detrimental condition for granulosa cells. In our study, we observed whether MnBP affected granulosa cells. MnBP treatment altered the gene expression of BIRC5, BUB1B, CDC20, cyclin B1, IL-1β, TNF-α, inhibin-B, StAR, and P450ssc and attenuated the ratio of the mitochondrial membrane potential in human granulosa cells. Moreover, MnBP decreased the expression of the anti-Mullerian hormone. These findings suggest that MnBP concentration is associated with endometriosis and may affect the health and steroidogenesis of human granulosa cells.

Keywords: anti-Mullerian hormone; granulosa cells; mitochondrial membrane potential; mono-n-butyl phthalate (MnBP), endometriosis; phthalate.

Figure 1

Correlations between urinary MnBP concentration and the concentrations of (A) MEHP, (B) MBzP, (C) MEOHP, and (D) MEHHP. The total sample size was 205. The coefficient of determination (r²) was (A) 0.321, (B) 0.354, (C) 0.4688, and (D) 0.3822. All correlations had p values < 0.0001. Mono-n-butyl phthalate (MnBP), mono(2-ethylhexyl) phthalate (MEHP), monobenzyl phthalate (MBzP), mono(2-ethyl-5-oxo-hexyl) phthalate (MEOHP), and mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP).

Figure 2

Effects of MnBP treatment on the expression of (A) BIRC5, (B) BUB1B, (C) CDC20, and (D) cyclin B1 in human granulosa cells. The expression levels of mRNA for the baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5), mitotic checkpoint serine/threonine kinase beta (BUB1B), cell division cycle 20 (CDC20), and cyclin B1 were measured using the reverse transcription quantitative polymerase chain reaction. Human granulosa cells were seeded overnight and treated with solvent control (ACN) and 67.5, 250, or 500 µg/mL of MnBP for 24 h. The expression levels of BIRC5, BUB1B, CDC20, and cyclin B1 were normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data are shown as mean ± SD compared to those of the solvent control. * p < 0.05. ** p < 0.01.

Figure 3

Effects of MnBP treatment on the expression of (A) IL-1β and (B) TNF-α. The expression levels of mRNA for the interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) were measured using the reverse transcription quantitative polymerase chain reaction. Human granulosa cells were seeded overnight and treated with solvent control (ACN) and 67.5, 250, or 500 µg/mL of MnBP for 24 h. The expression levels of IL-1β and TNF-α were normalized to that of GAPDH. Data are shown as mean ± SD compared to those of the solvent control. * p < 0.05.

Figure 4

Effects of MnBP on the ratio of mitochondrial membrane potential in human granulosa cells. Human granulosa cells were seeded overnight in 96-well plates and treated with solvent control (ACN) and 67.5, 250, or 500 µg/mL of MnBP for 72 h. The treated cells were stained with JC-1 solution (a cationic dye) and incubated at 37 °C in 5% CO₂ for 30 min. The ratio of the fluorescence intensity of J-aggregates (excitation and emission at 550 and 600 nm, respectively) to that of J-monomer (excitation and emission at 485 and 535 nm, respectively) was used as the indicator of cell health. * p < 0.05.

Figure 5

Effects of MnBP on the expression of (A) AMH and (B) inhibin B in human granulosa cells. The expression levels of mRNA for the AMH and inhibin B was measured using the reverse transcription quantitative polymerase chain reaction. Human granulosa cells were seeded overnight and treated with solvent control (ACN) and 67.5, 250, or 500 µg/mL of MnBP for 24 h. The expression levels of AMH and inhibin B were normalized to that of GAPDH. Data are shown as mean ± SD compared to those of the solvent control. * p < 0.05. ** p < 0.01.

Figure 6

Effects of MnBP on the expression of (A) StAR and (B) P450ssc in human granulosa cells. The expression levels of mRNA for the steroidogenic acute regulatory protein (StAR) and cytochrome cholesterol side-chain cleavage enzyme (P450scc) were measured using the reverse transcription quantitative polymerase chain reaction. Human granulosa cells were seeded overnight and treated with solvent control (ACN) and 67.5, 250, or 500 µg/mL of MnBP for 24 h. The expression levels of StAR and P450ssc were normalized to that of GAPDH. Data are shown as mean ± SD compared to those of the solvent control. * p < 0.05. ** p < 0.01.