Tài liệu Báo cáo Y học: Progressive apoptosis in chorion laeve trophoblast cells of human fetal membrane tissues during in vitro incubation is suppressed by antioxidative reagents pptx

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:
(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]. We postulate
that any oxidative conditions (resulting in an occurrence of
oxidative stress) may be created in chorion laeve tissues of
human fetal membranes at the term of gestation through
mitochondrial mechanisms, and that these conditions may

cause spontaneous fetal membrane disruption prior to
parturition.
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