| Context: Triclosan is a synthetic, nonionic and broad spectrum antimicrobial agent that has been used extensively in personal care products. Triclosan has been demonstrated as a potential endocrine disruptor. 11?-hydroxysteroid dehydrogenase type 2(11?-HSD2), a high-affinity dehydrogenase that inactivates biologically active cortisol to inactive cortisone, is highly expressed in human placental syncytiotrophoblasts in order to ensure normal development of fetus in the presence of high levels of maternal cortisol in pregnancy. Therefore 11?-HSD2 is regarded as a placental glucocorticoid barrier.Objective: We investigated the effects of triclosan on 11?-HSD2 and apoptosis and the relationship between these two events in human placental syncytiotrophoblasts.Design: Primary human placental cytotrophoblasts were isolated from term placenta. Previous studies have shown that maximal syncytialization occurs three days after plating. To examine the effects of triclosan on 11b-HSD2 and apoptosis, the cells were treated with triclosan(0, 0.001, 0.01, 0.1, 1 and 10?M) three days after plating in serum-free medium for 24 h. After triclosan treatment, the cells were collected to measure the expression level and antivity of 11b-HSD2. MTT assay, DNA ladder assay, fluorescent dye Hoechst 33342 staining and western blotting were used to measure the changes of cell viability and markers related to apoptosis after triclosan treatment. In order to understand the relationship between the reduction of 11?-HSD2 and apoptosis induced by triclosan in syncytiotrophoblasts, pre-incubation(1 h) of the cells with the apoptosis inhibitor Z-VAD-FMK(30?M) followed by co-incubation(24 h) with triclosan(0.1?M) was carried out.Results: Triclosan inhibited 11?-HSD2 mRNA, protein and activity levels in a concentration-dependent manner from 0.001?M to 10?M. We also found that triclosan decreased the viability of syncytiotrophoblasts in a concentration-dependent manner. The apoptosis induced by triclosan in human placental syncytiotrophoblasts can also be demonstrated by observations of increased nuclear condensation and DNA fragmentation. Staining with Hoechst 33342 showed hyperchromatic fluorescence signal in the nuclei of syncytiotrophoblasts upon exposure to triclosan(0.1?M), which indicates nuclear shrinkage in the apoptotic cells. DNA ladder assay also revealed that exposure to triclosan(0.1?M) increased DNA fragmentation in placental syncytiotrophoblasts. The immunofluorescence staining of cleaved-caspase-3 showed very little staining in the control group, while more intense staining was observed in the triclosan(0.1?M)-treated group. Treatment of placental syncytiotrophoblasts with triclosan for 24 h reciprocally increases pro-apoptotic proteins cleaved-caspase-3 and Bax and decreased anti-apoptotic proteins pro-caspase-3 and Bcl-2 in a concentration-dependent manner. Blocking the apoptosis induced by triclosan in syncytiotrophoblasts using Z-VAD-FMK the inhibition of 11?-HSD2 by triclosan(0.1?M) was also significantly attenuated at m RNA, protein and activity levels.Conclusions: Triclosan may attenuate the expression of placental 11?-HSD2 via the induction of apoptosis of placental syncytiotrophoblasts. This is likely to disrupt the placental glucocorticoid barrier and impair development of fetus. |
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