Progressive apoptosis in chorion laeve trophoblast cells of human fetal membrane tissues during in vitro incubation is suppressed by antioxidative reagents Kunio Ohyama 1 , Bo Yuan 1 , Toshio Bessho 2 and Toshio Yamakawa 1 1 Department of Biochemistry, Faculty of Pharmacy, Tokyo University of Pharmacy & Life Science, Tokyo, Japan; 2 Yoneyama Maternity Hospital, Tokyo, Japan Previously, we demonstrated apoptotic cell death in the chorion laeve trophoblast layer of human fetal membrane tissues during the late stages of pregnancy, the progression of apoptosis during incubation in vitro, and its suppression by a low concentration of glucocorticoid hormones. We now report examination of mRNA expression of inflammatory cytokines [interleukin (IL)-1b, IL-6, tumor necrosis factor- a] and antioxidative enzyme genes [heme oxygenase 1, catalase, Mn-superoxide dismutase (SOD), Cu/Zn-SOD, glutathione S-transferase, glutathione reductase and gluta- thione peroxidase] and apoptosis-related genes during in vitro progression of apoptosis with or without glucocorti- coid by a reverse transcription/PCR method. It was shown that the mRNA levels increased in chorion laeve tissue for each cytokine examined and for catalase, heme oxygenase 1 and Mn-SOD in direct correlation with the in vitro incubation period. By Western blotting the existence of Mn-SOD protein, and its slight increase with incubation time, was also shown. The investigation of the influence of antioxidative reagents [pyrrolidine dithiocarbamate (PDTC), N-acetyl- L-cysteine (NAC) and nordihydro- guaiaretic acid (NDGA)] on DNA fragmentation showed that DNA fragmentation in chorion laeve tissues was inhibited by < 50% in the presence of 1 m M PDTC, 30 mM NAC and 1 mM NDGA. These results suggest that apoptotic cell death of the trophoblast layer of chorion tissues may be induced through intracellular oxidative stress at the stage of parturition. Keywords: antioxidant; apoptosis; chorion; fetal membrane; oxidative stress. We have previously reported that (a) a substantial population of trophoblast cells in the chorion laeve tissue of human fetal membrane are induced to undergo apoptosis at the end of pregnancy, (b) that apoptosis progresses rapidly in vitro, and (c) that apoptosis of the trophoblasts in the tissue was suppressed by the presence of a low concentration of glucocorticoids, hydrocortisone and cortisone [1]. Further- more, we found that cultivated trophoblasts prepared primarily from the fetal membrane were induced to undergo apoptosis by tumor necrosis factor (TNF)-a and also that apoptosis was inhibited by low concentrations of gluco- corticoids [1]. From these results we suggested that apoptotic death of trophoblasts plays an important role in spontaneous disruption of the fetal membrane tissues during the final stage of gestation, and that any inflammatory and/or oxidative events may have a central role in the induction of trophoblast apoptosis. Pregnancy is a physiological state accompanied by an increased requirement for tissue oxygen [2]. It was shown by in vitro investigation that the increased oxygen requirement during embryo development increases the rate of reactive oxygen species (ROS) production [3]. It has also been established that ROS damage cell membrane lipids and induce lipid peroxide formation [4,5] as circulating markers of oxidative stress are increased during normal pregnancy. These peroxidized lipids are produced mainly in the placenta [6] and increase in the blood of pregnant women [7]. These observations indicate that there is an increased oxidative stress during pregnancy. It is common knowledge in the field of obstetrics and gynecology that the beginning of spontaneous disruption of fetal membrane may be caused by tissue inflammation. Furthermore, injury caused by oxidative stress may be responsible for developmental retardation and arrest of mammalian preimplantation embryos in vitro [8,9]. However, no detail is known about the mechanism of the disruption and its relationship to the induction of apoptosis. Our hypothesis is that oxidative stress contributes to ROS formation in the placenta and increases gradually during pregnancy. The stress may serve as a signal to initiate and propagate the inflammatory process and result in apoptosis of placental tissues. Accordingly, we investigated the correlation between the progression of trophoblast apoptosis and the induction of inflammatory cytokine and/ or cellular oxidative stress occurrence. In this paper, we describe trophoblast cell apoptosis during in vitro incubation of human fetal membrane tissue, transcription of apoptosis-related genes, inhibition of apoptosis by Correspondence to K. Ohyama, Department of Biochemistry, School of Pharmacy, Tokyo University of Pharmacy & Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-03, Japan. Fax: 181 426 76 5736, Tel.:181 426 76 5792, E-mail: ohyamak@ps.toyaku.ac.jp (Received 20 June 2001, revised 2 October 2001, accepted 4 October 2001) Abbreviations: TNF, tumor necrosis factor; ROS, reactive oxygen species; PDTC, pyrrolidine dithiocarbamate; NAC, N-acetyl- L-cysteine; NDGA, nordihydroguaiaretic acid; IL, interleukin; NFkB, nuclear factor kb; TNFR, TNF receptor; SOD, superoxide dismutase. Eur. J. Biochem. 268, 6182–6189 (2001) q FEBS 2001 antioxidative reagents, and antioxidative enzyme induction in the tissue. MATERIALS AND METHODS Materials Pyrrolidine dithiocarbamate (PDTC) and N-acetyl- L-cysteine (NAC) were from Dojindo Laboratories and Wako Pure chemical Ind. Ltd, respectively. Nordihydro- guaiaretic acid (NDGA), glucocorticoids, cortisone and hydrocortisone were from Sigma Chemical Co. Fetal bovine serum was provided by Bio-Whittaker. The PCR primers for the p53, c-Jun, nuclear factor-kb (NF-kB), TNFa, interleukin (IL)-1b and IL-6 genes were from Clontech Laboratory Inc. The primers for b-actin, Fas antigen, heme oxygenase 1, catalase, glutathione peroxidase 1 and glutathione S-transferase, TNF receptor (TNFR)1, TNFR2, interferon (IFN)-a, IFN-b, IFN-g, glutathione reductase, Mn-superoxide dismutase (SOD) and Cu/Zn-SOD, bcl-2, ICE and FasL genes were from Amersham Pharmacia Biotech. Human fetal membrane Fetal membranes were prepared aseptically from placenta obtained by Cesarean section at the month of normal parturition. Immediately after the Cesarean section, the chorion laeve tissue was separated from the remainder. Fresh specimens were incubated in cultivation medium in the presence of fetal bovine serum before DNA preparation according the method described previously [1]. Primary culture cells were also prepared from these tissues [1]. Preparation and agarose gel electrophoresis of DNA from chorion laeve tissues Preparation and agarose gel electrophoresis of DNA were carried out according to the method reported previously [1]. Between 0.3 and 0.5 g wet weight of tissue was used for the DNA preparation. The DNA sample (20 mgin20mL) was loaded onto 2% agarose (Agarose X, Nippon gene, Tokyo, Japan) gels and separated by electrophoresis. Gels were stained with ethidium bromide (Sigma Chemical Co.) and viewed under UV light. For the fragmentation analysis, photographs of the gel were digitized with a scanner (ScanJet 4c, Hewlett Packard) and software ( DESKSCAN II, ver. 2.3, Hewlett Packard) on a personal computer. Relative amount of DNA was calculated from the density of the gray level on the digitized image using NIH Image 1.60. DNA . 2072 bp was designated high molecular mass DNA and that in the size range 100–2072 bp was designated fragmented DNA. The DNA fragmentation rate was calculated as the relative amount of fragmented DNA in the digitized image. RT-PCR Total RNA was extracted from chorion laeve tissues (0.5 g wet weight) using an RNA extraction kit, ISOGEN (Wako Pure Chemical Industries, Osaka, Japan). Complementary DNA was synthesized from 1 mg of RNA using 100 pmol random hexamers and 100 U Moloney murine leukemia virus reverse transcriptase (GibcoBRL) in a total volume of 20 mL, according to the manufacturer’s instructions. PCR was performed according to the method described previously [10]. Five microliters of the reaction solution and Ready-Load TM 100 bp DNA Ladder (GibcoBRL) as DNA size markers were separated by electrophoresis through 2.0% agarose using Tris/borate/EDTA buffer, and the PCR product was viewed on an UV transilluminator after ethidium bromide staining. Western blotting For Western blotting analysis of Mn-SOD and Cu/Zn-SOD, tissue samples (wet weight of 0.2 g) were homogenized with a prechilled Potter-type Teflon homogenizer in 0.4 mL lysis buffer (10 m M Tris/HCl pH 7.4, containing 1% SDS, 1.0 m M sodium ortho-vanadate, 2 mg : mL 21 leupeptin, 2 mg : mL 21 aprotinin, 1 mg : mL 21 pepstatin and 1 mM phenylmethanesulfonyl fluoride) and 0.4 mL of Laemmli buffer [11] containing 100 m M dithiothreitol. Dissolved tissue homogenate was boiled in a water bath for 10 min, and then centrifuged at 13 000 g for 15 min at 4 8C. Protein concentration of the supernatant was determined according to Bradford’s method using the protein assay dye reagent (Bio-Rad Laboratories Ltd) and BSA as the standard [12]. SDS/PAGE was performed with a 12.5% polyacrylamide gel using a Mini-protean II Cell apparatus (Bio-Rad), including prestained molecular mass standards (Gibco-BRL). Protein bands on the gel was transferred to nitrocellulose by using Semi-dry transfer cells (Bio-Rad) according to the Bio-Rad instruction manual. Blotting of the nitrocellulose and use of anti-Mn-SOD or anti-Cu/Zn-SOD antibody (StressGen Biotechnologies Corp.) were carried out according to the supplier’s recommendations. Alkaline phosphatase-labeled goat anti-rabbit IgG and 5-bromo-4-chloro-3-indolyl- phosphate/nitroblue tetrazolium (both from Kirkegaard Perry Laboratories) were used as a secondary antibody and alkaline phosphate substrates, respectively. RESULTS We had established previously that trophoblast cell apoptosis takes place in the chorion laeve tissue during the late stage of pregnancy and that apoptosis progresses during in vitro incubation in culture medium. In the present study we investigated mRNA expression of 23 apoptosis-related genes in chorion laeve tissue prepared from human fetal membranes obtained by cesarean section at 36–37 weeks of pregnancy. mRNA was measured during in vitro incubation in culture medium using reverse transcription (RT)/PCR. The results are shown in Fig. 1. Under conditions in which a house-keeping gene (b-actin) mRNA was constitutively expressed and not altered quantitatively during the incubation period, mRNAs of bcl-2, Fas antigen, P53, TNFa-receptor-1 and -2, ICE (caspase-1), glutathione S-transferase, glutathione peroxidase and glutathione reductase were also expressed constitutively and showed no quantitative alteration. We provisionally named this group of genes group-1. Expression of mRNA for c-Jun, IFN-a, IFN-b, NF-kB, TNFa, IL-1b, IL-6, Cu/Zn-SOD, Mn-SOD, catalase and hemoxygenase-1 increased in comparison with those of the control tissue (no incubation sample) during the incubation period (group-2). FasL and q FEBS 2001 Antioxidants suppress apoptosis in trophoblasts (Eur. J. Biochem. 268) 6183 IFN-g mRNAs were not expressed in the tissue nor during incubation (group-3). During the in vitro incubation period, the influence of the existence of a low concentration of glucocorticoid (10 mg : mL 21 hydrocortisone or cortisone) on various gene mRNA expressions of chorion laeve tissue was investigated, as shown in Fig. 1 also. With the exception of ICE mRNA, group-1 gene mRNA expression was not influenced by the Fig. 1. RT-PCR analysis of mRNA production in chorion laeve tissue during in vitro incubation with or without glucocorticoid. Values at the right-hand side of the electrophoretic pattern show the predicted length of the PCR product. Lane St, 100-bp DNA Ladder (Gibco BRL). Messenger RNA expression in the tissue during in vitro incubation is shown in the upper panels and that in the presence of glucocorticoid (10 mg : mL 21 ) is shown in the lower panels. 6184 K. Ohyama et al. (Eur. J. Biochem. 268) q FEBS 2001 addition of glucocorticoid to the incubation medium. ICE mRNA was expressed constitutively but decreased on addition of glucocorticoid. In group-2 genes, mRNA levels of c-Jun, IFN-a and IFN-b decreased significantly with the addition of glucocorticoid, like that of the ICE gene, whereas NF-kB mRNA expression was almost the same with and without glucocorticoid, and TNFa, IL-1b and IL-6 increased more than in the absence of glucocorticoid. In group-2, mRNAs of catalase, heme oxygenase 1, Cu/Zn- SOD and Mn-SOD increased at an earlier time than in the absence of glucocorticoid. Next, we investigated the existence and quantitative alteration of Mn-SOD and Cu/Zn-SOD proteins in chorion laeve tissue during in vitro incubation using Western blotting analysis. With specific antibodies against Mn- and Cu/Zn-SODs, a single band was shown for each SOD (Fig. 2). From a calculation curve prepared from mobility of standard proteins, the molecular masses of Mn- and Cu/Zn- SODs were estimated as 25 kDa and 23 kDa, respectively. Whereas Mn-SOD showed a distinct band and the band increased slightly with the elapse of in vitro incubation time, that of Cu/Zn-SOD was barely detectable under the experimental conditions used. It was not possible to obtain a clearer band for Cu/Zn-SOD by increasing only the amount of specific antibody used; to demonstrate a clear band for Cu/Zn-SOD (of the same intensity as that of the Mn-SOD band) more than three times the amount of protein used in the experiment illustrated in Fig. 2 were required (data not shown). To investigate the role of any oxidative factors in the induction of apoptotsis in trophoblasts we studied the influence of the antioxidant reagents PDTC, NAC, NDGA and alloprinol on DNA fragmentation in chorion laeve tissue during in vitro incubation. The concentration of each antioxidant reagent was determined by preliminary examination using cultured chorion laeve cells. With these concentrations, cultured chorion laeve cells showed no indication of undergoing either apoptosis or necrosis (data not shown). The upper and lower panels of Fig. 3A–D show the electrophoretic mobility of extracted DNA using agarose gel electrophoresis and DNA fragmentation rates, respect- ively. As shown in Fig. 3A 1 m M PDTC inhibited an increase in DNA laddering during incubation: inhibition was . 45% compared with nontreated incubation at 4 h. With 30 m M NAC, inhibition of DNA fragmentation was started at 3 h incubation, and inhibition was . 50% for a 5-h period (Fig 3B). The addition of 1 m M NDGA to the culture medium inhibited DNA fragmentation from 2–3 h (Fig. 3C) and this inhibition increased gradually during incubation; at 4 h incubation inhibition was 52%. However, 1m M alloprinol did not influence DNA fragmentation (Fig. 3D). DISCUSSION In this report, we describe the quantification of changes in mRNA levels of 23 apoptosis-related genes in chorion laeve tissue during in vitro incubation of human fetal membranes obtained at term. Based on the extent of quantitative alteration, we have divided these genes into three groups. bcl-2, Fas, P53, TNFR-1, TNFR-2, ICE, glutathione S-transferase, glutathione peroxidase and glutathione reductase genes were included in a group showing constitutive expression during the incubation period (group-1); this group, except for the ICE, showed no alteration of expression after the addition of glucocorticoid. In group-2 genes, which increased during incubation, mRNA levels for c-Jun, IFN-a and IFN-b decreased in the presence of glucocorticoid, those of NF-kB and TNFa were at the same level as in the absence of glucocorticoid, those of IL-1b and IL-6 were higher than in the absence of glucocorticoid, and those of Cu/Zn-SOD, Mn-SOD, catalase and hemeoxygenase-1 increased at an earlier time than in the absence of glucocorticoid. From an indication that FasL and INF-g mRNA expression could not be detected (group- 3), a direct contribution of the Fas/Fas L system to apoptosis in the tissue is not suggested. In these alterations of gene expression, it is noticeable that the expression of ICE and c-Jun mRNA decreased, and that the presence of gluco- corticoid could not suppress the expression of inflammatory cytokines including TNFa, IL-1b and IL-6. Also, pro- duction of mRNA of the antioxidant enzymes catalase, Mn- SOD, and heme oxygenase 1 is detected at an earlier time when incubated in the presence of glucocorticoid than that in its absence. We have already reported that the presence of a lower concentration of glucocorticoid during incubation suppressed the progression of trophoblast cell apoptosis in the tissue, and that apoptosis of primary cultured human chorion trophoblast cells induced by TNFa was also suppressed by this lower concentration of glucocorticoid [1]. These previous results suggested that the expression of mRNA of inflammatory cytokines and/or inflammation may play a role in the induction and process of apoptotic cell death of trophoblasts in chorion laeve tissues during the late Fig. 2. Expression of Mn- and Cu/Zn-SOD proteins during in vitro incubation of chorion laeve tissue. Western blotting analyses (A) and (B) show Mn- and Cu/Zn-SOD protein expressions, respectively. Values indicated on the right-hand side are the molecular masses of Mn- and Cu/Zn-SODs (25 kDa and 23 kDa). Lane M, molecular mass markers: 110 kDa, phosphorylase B; 78 kDa, BSA; 49 kDa, ovalbumin; 35.6 kDa, carbonic anhydrase; 29 kDa, soybean trypsin inhibitor; 21.1 kDa, lysozyme. q FEBS 2001 Antioxidants suppress apoptosis in trophoblasts (Eur. J. Biochem. 268) 6185 Fig. 3. Effect of antioxidants on DNA fragmentation of chorion laeve tissue during in vitro incubation. Tissue was incubated in medium containing 1 m M PDTC (A), 30 mM NAC (B), 1 mM NDGA (C) or 1 mM alloprinol (D). the upper and lower parts in each figure show electrophoretic patterns of DNA prepared from the tissue on a 2% agarose gel and the DNA fragmentation ratio, respectively. 6186 K. Ohyama et al. (Eur. J. Biochem. 268) q FEBS 2001 stage of pregnancy. However, the results presented here show that expression of inflammatory cytokine gene mRNAs increased during incubation, but was not altered by glucocorticoid. These apparently inconsistent results could be explained by assuming that the role of these cytokines in apoptosis may already be complete at the time the fetal membrane tissues were prepared, or that the role of these cytokines may be connected indirectly with the progression of apoptosis. It has been reported for the anti-inflammatory mechanism of glucocorticoid, that the glucocorticoid receptor protein (which is a transcription regulatory factor) binds to the Jun domain, and that the binding is dependent on the amount of glucocorticoid [13]. In addition, this binding results in exhibition of anti-inflammatory effects through inactivation of the AP-1 function [14,15]. These reports are consistent with our finding that the expression of c-Jun, and also ICE genes, are depressed by the presence of glucocorticoids, and furthermore that glucocorticoid could not inhibit the expression of inflammatory cytokine mRNAs. Our prelimi- nary experiment shows that the presence of 10 25 M of glucocorticoid receptor antagonist, RU-486, during incu- bation inhibited the suppression of trophoblast apoptosis in the tissue by glucocorticoid (data not shown). This suggests that the suppression of trophoblast apoptosis is achieved through a cellular glucocorticoid receptor. Consequently, it can be assumed that apoptosis suppression by glucocorticoid may be connected to the mechanism of its anti-inflammatory function, and that apoptosis suppression by glucocorticoid in the tissue may also be affected by suppression of specific gene functions, such as c-Jun. It is known that there is an increase in fetal plasma glucocorticoid concentration due to maturation and sustained activation of the fetal hypothalamic–pipuitary– adrenal axis towards the end of gestation and at the onset of labor in most mammalian species that have been studied [16,17]. Simultaneously with the increase in fetal gluco- corticoid concentration at the onset of labor there is a progressive increase in plasma, amniotic fluid, and intrauterine tissue concentration of prostaglandin (PG), in particular PGE2 followed by an increase in PGF2a [18]. Recent evidence has suggested that the increase in fetal glucocorticoid production directs the increase in intrauterine PG production and onset of labor [18]. These PGs have been also identified as key mediators of the events of labor including cervical ripening, uterine contractility, membrane rupture, utero-placental blood flow, and fetal adaptation to the process of labor [18]. These finding suggest that glucocorticoids in fetal and maternal plasma have a central role in process of labor through an PG production. Fetal membranes obtained from term human pregnancies may show marked increases in PGE2 output, and in the expression of type-2 cyclo-oxygenase before labor [19]. A number of factors, including the pro-inflammatory cytokines IL-1, IL-6 and TNFa [20– 22], cause similar changes in type-2 cyclo-oxygenase expression and PG output when added to fetal membranes in vitro. These findings demonstrated that PG production by intrauterine tissues may be up-regulated by proinflammatory cytokines. Furthermore, an anti-inflammatory cytokine such as IL-10 inhibited the output of PGE2 from intact fetal membranes under basal and lipopolisaccharide-stimulated conditions [23]. These reports suggest that human labor at all gestational ages may have similarities to a general inflammatory response. Recently, it has been also reported that PG induced apoptosis in human placental tissue trophoblast cells during normal pregnancy [24]. We consider that glucocorticoids may have two functional roles in apoptotic events in fetal membrane tissues: apoptotic inhibition and apoptotic induction through their anti-inflammatory function and PG production, respectively. We also suggest that these competitive functions may be regulated by alteration of their quantitative balance during gestation and at the onset labor, and that the balance also regulates the spontaneous rupture of fetal membrane at the end of gestation. It was shown by Western blotting analysis that Mn-SOD protein existed in the tissue and increased slightly during incubation; however, Cu/Zn-SOD protein was barely detectable under the analytical conditions used. In the experiment reported, both of the SOD-specific antibodies were used according to the supplier’s recommendations; we consider that these were optimal conditions because we were unable to improve the clarity of the Cu/Zn-SOD band on Western blotting by increasing the amounts of antibody used; to improve the clarity a higher amount of protein was required for electrophoresis, indicating that the mRNA of Mn-SOD protein was expressed in the tissue at higher levels than that of Cu/Zn-SOD. Recently, it has become clear that a lower concentration of ROS and/or lower level of oxidative stress may play a role in the mechanism of apoptosis [25], and that oxidative stress and chronic inflammation are related, perhaps being an inseparable phenomenon [26]. It is also well established that the activation of apoptosis is associated with ROS produced by mitochondria [27], that the generation of ROS in mitochondria was activated by TNFa [28] and that apoptosis could be blocked or delayed by antioxidants such as NAC [29]. These observations suggest that ROS formation in tissues and/or cells contributes to the induction of oxidative stress associated with chronic inflammation, leading to apoptotic cell death. Cells are protected from ROS by an antioxidant defense system. The initial enzymes in this system are the SODs, which in eukaryotic cells are characterized by their metal requirement and subcellular localization. Mn-SOD is found in the mitochondria [30,31]. The report that the Mn-SOD mRNA level was induced in response to acute inflammatory mediators, lipopolysacchar- ide, IL-1 and TNFa in pulmonary epithelial cells [32] strongly suggests an important role for Mn-SOD in the acute inflammatory response. It has been shown previously that over-expression of Mn-SOD in human breast tumor MCF-7 cells suppressed apoptosis induced by ROS-generating agents [33], and that in cultured pheochromocytoma PC6 cells overexpression of Mn-SOD prevented apoptosis induced by Fe 21 , amyloid beta peptide and nitric oxide- generating agents [34]. ROS plays a critical role in activation of NF-kB, AP-1, c-Jun kinase and apoptosis induced by TNFa in MCF-7 cells, and these phenomena were blocked by Mn-SOD through inhibition of NF-kB, AP-1, c-Jun kinase activation and the resulting caspase-3 activation [35]. Consequently, our results showing the earlier expression of antioxidant enzyme mRNAs and the blotting analysis of the SODs suggest that an oxidative stress originating in the mitochondria may play an important role in the occurrence of trophoblast cell apoptosis. q FEBS 2001 Antioxidants suppress apoptosis in trophoblasts (Eur. J. Biochem. 268) 6187 The glutathione redox cycle also represents one of the most important defenses against oxidative stress, which is an enzyme-coupled system containing glutathione, glutathione peroxidase and glutathione reductase [36]. mRNAs for the redox state regulating enzymes glutathione, glutathione S-transferase, glutathione peroxidase and glutathione reductase were expressed constitutively and the levels during incubation with glucocorticoid were almost the same as those without. These results suggest that the redox state in trophoblasts in the tissue may not be related directory to the regulation of apoptosis. Furthermore, we examined the influences of antioxidant reagents, PDTC, NAC and NDGA on DNA fragmentation in chorion laeve tissue during incubation. As lower concentrations of ROS, and also nitrogen intermediates, can induce apoptosis in various cells [25], if these oxidants can induce apoptosis, then antioxidants should be able to inhibit apoptosis. Dithiocarbamates, including PDTC, exert antioxidant effects in cells by eliminating hydrogen peroxide, scavenging the superoxide radical [37,38], peroxinitrite and the hydroxyl radical [39], and blocking NF-kB activation [40]. It has also been demon- strated recently that PDTC acts as an oxidizing agent in cells, rather than as an antioxidant, through the inhibition of NF-kB activation [41]. NAC is a thiol-containing anti- oxidant and acts as a nucleophile and a precursor of reduced glutathione [42]. It has also been reported that NAC protects the cells from TNFa-induced U937 cell apoptosis by maintaining mitochondrial integrity and function [43], and ricin-induced apoptosis of U937 cells by maintaining an intracellular reducing condition by acting as a thiol supplier [44]. As several antioxidant reagents other than NDGA failed to block CD95-ligand-mediated human malignant glioma cell apoptosis [45] and NDGA protection of TNFa- mediated L929 cell apoptosis did not appear to involve removal of cytotoxic H 2 O 2 or O 2 – radical [46], it is suggested that the protection effect of NDGA in apoptosis of these cells may be due to its lipoxygenase inhibition. From our results that three antioxidants depressed the progression of apoptosis (but not completely), it is suggested that the apoptosis depressing effects may be implicated by their antioxidative effects, and that the mechanism of these effects may be caused by their radical scavenging activity. However, these antioxidants could have functions other than an antioxidant effect, as described above. Accordingly, it should be considered that the mechanism of the trophoblast apoptosis may be not simple. We conclude that the stress conditions increase during incubation, and that the expression of antioxidant enzyme mRNAs containing Mn-SOD increase to counterbalance these phenomena. The induction and increase of oxidative stress during pregnancy has been indicated by increasing lipid peroxidation in pregnant women, including lipid hydroperoxides and malondialdehyde as circulating markers of oxidative stress [5]. The increase of lipid peroxisides indicates the increase of ROS, because lipid peroxides are formed when lipids interact with ROS [47]. 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