Elsevier

Life Sciences

Volume 82, Issues 11–12, 12 March 2008, Pages 623-630
Life Sciences

Imbalance between ROS production and elimination results in apoptosis induction in primary smooth chorion trophoblast cells prepared from human fetal membrane tissues

https://doi.org/10.1016/j.lfs.2007.12.016Get rights and content

Abstract

We have previously demonstrated that induction of apoptosis was observed in the smooth chorion trophoblast cells of human fetal membranes prepared at term, and that apoptosis progressed rapidly during in vitro incubation of the tissues. Furthermore, we identified the contribution of ROS production system (e.g., oxidant enzymes, such as iNOS and Cox-2) to the apoptosis induction in the chorion cells, suggesting an important role of the two inducible enzymes in the induction process. In this study, we examined the role of ROS elimination system (e.g., antioxidant enzymes, such as glutathione peroxidase (GPx) and catalase) in the apoptosis induction of the chorion cells, since the apoptosis induction by oxidative stress is a result of imbalance between production and elimination of ROS. Treatment of chorion and amnion cells with mercaptosuccinic acid (MS, GPx inhibitor) and 3-amino-1,2,4-triazole (ATZ, catalase inhibitor) resulted in an inhibition of GPx and catalase activity, respectively. Furthermore, incubation with MS alone induced apoptosis in the chorion cells and apoptosis level was enhanced by the addition of ATZ, while ATZ alone hardly induced apoptosis in the chorion cells. However, none of these reagents induced apoptosis in the amnion cells. Moreover, an increase of the level of hemeoxygenase-1 gene expression was observed only in the amnion cells when both antioxidant enzyme activities were suppressed. Therefore, we concluded that GPx played a more critical role than catalase in the control of the apoptosis induction of the chorion cells, suggesting that the threshold levels of stress tolerance in the chorion cells are much lower than those in the amnion cells.

Introduction

The rupture of fetal membrane (FM) is an essential part of the delivery process, accompanied by the onset of uterine contractions (Challis, 2000). Although it has been hypothesized that some biochemical events, including degradation of the connective tissue (Stegemann and Stalder, 1967), reduced elasticity (Artal et al., 1976) and decreased thickness (Helmig et al., 1991) of FM, which contribute to the rupture at the normal delivery, the mechanisms involved in the FM rupture during the birth process are little known. In our previous studies, we proposed that apoptosis induction in the chorion tissues is one of the mechanisms of FM rupture associated with labor (Ohyama et al., 1998, Ohyama et al., 2001, Yuan et al., 2006). The induction of apoptosis was observed in the smooth chorion trophoblast layer of human FM prepared at the term. The extent of apoptosis induction in the trophoblast layer progressed rapidly during in vitro incubation of the tissues (Ohyama et al., 1998). We also demonstrated that the progression of apoptosis was suppressed by the presence of antioxidative reagents, suggesting that an intracellular oxidative stress played critical roles in the induction of apoptosis observed in the trophoblast layer at the stage of parturition (Ohyama et al., 2001). Furthermore, we demonstrated that the expression levels of Bax, Bak and Bad mRNAs increased, while those of Bcl-2 mRNA decreased; the expression levels of pro-caspase-3 and -9, proform-poly (ADP-ribose) polymerase decreased; and a leakage of cytochrome c from mitochondria in the chorion tissues was observed during the in vitro incubation. These results suggested that the apoptosis induction observed in the chorion trophoblasts was induced by oxidative stress resulting in mitochondria damage (Ohyama et al., 2001, Yuan et al., 2006).

Moreover, we demonstrated that an inducible nitric oxide synthase (iNOS) inhibitor, a general and a selective cyclooxygenase (Cox)-2 inhibitors suppressed in vitro progression of the apoptosis, and that an NO donor reagent induced apoptosis in primary cultured chorion cells. These results suggested for the first time that iNOS and Cox-2, both of which are known to play a central role in reactive oxygen species (ROS) production (Payne et al., 1995), participated in the induction of chorion cells apoptosis through production of ROS (Yuan et al., 2006). Oxidative stress, as a result of an imbalance between the production and elimination of ROS within the cells (Margaret and Amanda, 1996), is known to induce apoptosis in various cell types (Payne et al., 1995). Therefore, we have been investigating on the contribution of ROS production system (e.g., oxidant enzymes like iNOS and Cox-2) to the trophoblasts apoptosis in the chorion tissues. However, the study concerning a possible contribution of ROS elimination system (e.g., antioxidant enzymes like glutathione peroxidase (GPx), catalase) to the apoptosis induction in the chorion trophoblasts has not yet performed at cellular level.

In this study, in order to reveal the relationship between ROS elimination system and the chorion trophoblasts apoptosis induction, we investigated the contribution of antioxidant enzymes, such as GPx and catalase, to the apoptosis induction in the primary cultured chorion cells, using known inhibitors for GPx, such as mercaptosuccinic acid (MS) and for catalase, such as 3-amino-1,2,4-triazole (ATZ) (Shiba and Shimamoto, 1999, Ishihara et al., 2005).

Section snippets

Materials

MS and ATZ were purchased from Sigma Ltd. (St. Louis, MO, USA). Agarose X for DNA fragmentation analysis was purchased from Nippon Gene (Tokyo, Japan). GPx assay kit and catalase assay kit were purchased from Cayman Chemical (Ann Arbor, MI, USA).

Preparation of primary cultured cells from human fetal membrane

Fetal membranes were prepared aseptically from placenta by Cesarean section at the month of normal parturition as described (Ohyama et al., 1998). Primary culture chorion and amnion cells were prepared from freshly prepared fetal membrane tissues

Effects of MS (GPx inhibitor) and ATZ (catalase inhibitor) on intracellular GPx and catalase activity in primary culture chorion and amnion cells

Primary culture chorion and amnion cells were treated with MS and ATZ at final concentrations of 14 mM and 40 mM, respectively, either for 24 or 48 h. GPx activity observed in untreated chorion cells (ca. 1200 units/mg of protein) was similar to that in untreated amnion cells (Fig. 1A and B). In the chorion cells, GPx activity after 24-h incubation with MS alone or in combination with ATZ decreased to 700 units/mg of protein, and the activity was not changed after 48-h incubation (Fig. 1A). In

Discussion

We have previously demonstrated that the apoptosis induction of trophoblasts in smooth chorion tissues of human fetal membranes (FM) was mediated through the intracellular oxidative stress, resulting in mitochondria damage at the term (Ohyama et al., 1998, Ohyama et al., 2001, Yuan et al., 2006). We have also demonstrated that an inducible nitric oxide synthase (iNOS) inhibitor, a general and a selective cyclooxygenase (Cox)-2 inhibitors suppressed in vitro progression of the trophoblasts

Conclusions

We proposed that inability of ROS elimination system as a result of reducing antioxidant enzyme activities induced apoptosis only in the chorion trophoblast layer when exposed to oxidative stress. Our results suggest that the threshold levels of stress tolerance in the amnion cells are much higher than those in the chorion cells, which results in more vulnerable to apoptosis induction in the chorion cells by oxidative stress.

Acknowledgments

This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology and by the Promotion and Mutual Aid Corporation for Private Schools of Japan.

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