Redox Status, Estrogen and Progesterone Production by Swine Granulosa Cells Are Impaired by Triclosan

Abstract

Triclosan is a chlorinated biphenolic with a broad spectrum of antiseptic activities used in cosmetics and hygiene products. Continuous exposure can lead to absorption and bioaccumulation of this substance with harmful health effects. In fact, previous studies have shown that Triclosan acts as an endocrine-disrupting chemical on reproductive organs, with consequent negative effects on reproductive physiology. Therefore, to assess potential adverse impacts on fertility, we tested Triclosan on swine granulosa cells, a model of endocrine reproductive cells. We examined its effects on the main features of granulosa cell functions such as cell growth (BrdU incorporation and ATP production) and steroidogenesis (17-β estradiol and progesterone secretion). Moreover, since oxidant−antioxidant balance plays a pivotal role in follicular function, redox status markers (superoxide, hydrogen peroxide and nitric oxide production, enzymatic and non-enzymatic scavenging activity) were studied. Our results show that Triclosan significantly inhibits cell growth (p < 0.001), steroidogenesis (p < 0.001), superoxide and nitric oxide production (p < 0.001), while it increases (p < 0.05) enzymatic defense systems. Collectively, these data suggest a disruption of the main granulosa cell functions, i.e., proliferation and hormone production, as well as an imbalance in redox status. On these bases, we can speculate that Triclosan would impair granulosa cell functions, thus exerting negative effects on reproductive function. Further studies are needed to explore lower Triclosan concentrations and to unravel its mechanisms of action at gene level.

Keywords: cell proliferation; free radicals; ovary; oxidative stress; steroidogenesis.

Figure 1

Result of ATP (A) and 5-bromo-2′deoxyuridine (BrdU) (B) tests carried out on porcine granulosa cells following treatment with Triclosan (1, 10 or 50 µM) for 48 h. The tests evaluate the metabolic activity and proliferation of treated cells, respectively. Data are expressed as counts per second (CPS) (A) and on milli-Abs units (B) and represent the mean ± SD of six replicates/treatment repeated in five experiments (n = 30). Asterisks on points indicate that data show significant differences (p < 0.001).

Figure 2

Results of the production of 17β estradiol (E2) (A) and progesterone (B) by porcine granulosa cells treated for 48 h with Triclosan (1, 10 or 50 µM) detected by ELISA assay. Data are expressed as % vs. control and represent the mean ± SD of six replicates/treatment repeated in five different experiments (n = 30). Asterisks on points indicate that data show significant differences (p < 0.001).

Figure 3

Results of hydrogen peroxide (H₂O₂) (A), superoxide anion (O₂⁻) (B) and nitric oxide (NO) (C) generation using colorimetric assay on porcine granulosa cells treated for 48 h with Triclosan (1, 10 or 50 µM). Data are expressed as milliAbs units (B) and as µM (A,C) and represent the mean ± SD of six replicates/treatment repeated in five different experiments (n = 30). Asterisks on points indicate that data show significant differences (p < 0.05).

Figure 4

Results of non-enzymatic scavenging activity by porcine granulosa cells treated for 48 h with Triclosan (1, 10 or 50 µM) using the FRAP assay ((A); µM), on peroxidase activity ((B); milliAbs), superoxide dismutase activity ((C); U/mL) and on catalase activity ((D); mU/mL). Data represent the mean ± SD of six replicates/treatment repeated in five different experiments (n = 30). Asterisks on points indicate that the data show significant differences (p < 0.05).