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Effect of N-Acetylcysteine on Lipopolysaccharide-Induced Intra-

uterine Fetal Death and Intra-uterine Growth Retardation in Mice


De-Xiang Xu,*
,

,1
Yuan-Hua Chen,* Hua Wang,* Lei Zhao,* Jian-Ping Wang,* and Wei Wei
*Department of Toxicology, Anhui Medical University, Hefei, 230032, P. R. China, and
Key Laboratory of Anti-inammatory and Immunopharmacology of Anhui Province, Hefei, 230032, P. R. China
Received June 30, 2005; accepted August 21, 2005
Lipopolysaccharide (LPS) has been associated with adverse
developmental outcome, including embryonic resorption, intra-
uterine fetal death (IUFD), intra-uterine growth retardation
(IUGR), and preterm delivery. Reactive oxygen species (ROS)
have been associated with LPS-induced developmental toxicity. N-
acetylcysteine (NAC) is a glutathione (GSH) precursor and direct
antioxidant. The present study investigated the effects of NAC on
LPS-induced IUFD and IUGR. All pregnant mice except controls
were injected with LPS (75 mg/kg, ip) on gestational day (GD)
1517. NAC was administered in two different modes. In mode A,
the pregnant mice were pretreated with two doses of NAC (either
50 plus 25 mg/kg or 200 plus 100 mg/kg) before LPS, one (either
50 or 200 mg/kg) at 12 h before LPS and the other (either 25 or
100 mg/kg) at 15 min before LPS. In mode B, the pregnant mice
were administered with two doses of NAC (either 50 plus 25 mg/kg
or 200 plus 100 mg/kg) in 24 h, one (either 50 or 200 mg/kg) injected
immediately after LPS and the other (either 25 or 100 mg/kg)
injected 3 h after LPS. The number of live fetuses, dead fetuses
and resorption sites was counted on GD 18. Live fetuses in each
litter were weighed. Crown-rump and tail lengths were measured
and skeletal development was evaluated. Results showed that
pretreatment with NAC signicantly alleviated LPS-induced fetal
mortality and reversed LPS-induced growth and skeletal de-
velopment retardation. Correspondingly, pretreatment with
NAC signicantly attenuated LPS-induced elevation in TNF-a
concentration in maternal serum and amniotic uid and lipid
peroxidation in maternal and fetal livers. By contrast to pre-
treatment, posttreatment with NAC had no effect on LPS-induced
TNF-a production and lipid peroxidation. When administered
after LPS, NAC did not protect against LPS-induced IUFD and
IUGR and in fact aggravated LPS-induced preterm labor. All
these results indicate that NAC had a dual effect on LPS-induced
IUFD and IUGR. Pretreatment with NAC improves fetal survival
and reverses LPS-induced fetal growth and skeletal development
retardation, whereas posttreatment with NAC aggravates LPS-
induced preterm labor.
Key Words: N-acetylcysteine; lipopolysaccharide; intra-uterine
fetal death; intra-uterine growth retardation.
Lipopolysaccharide (LPS) is a toxic component of cell walls
of gram-negative bacteria and is widely present in the digestive
tracts of humans and animals (Jacob et al., 1997). Humans are
constantly exposed to low levels of LPS through infection.
Gastrointestinal distress and alcohol drinking often increase
permeability of LPS from gastrointestinal tract into blood
(Fukui et al., 1991). LPS has been associated with adverse de-
velopmental outcome, including embryonic resorption, intra-
uterine fetal death (IUFD), intra-uterine growth retardation
(IUGR), and preterm delivery in rodents (Collins et al., 1994;
OSullivan et al., 1988). In humans, gram-negative bacterial
infections are a recognized cause of fetal loss and preterm labor
(Romero et al., 1988).
Many studies have demonstrated that LPS increased mater-
nal serum TNF-a production (Bell et al., 2004; Vizi et al.,
2001). Maternal LPS exposure resulted in elevation of TNF-a
levels in amniotic uid and placenta (Chen et al., 2005; Gayle
et al., 2004). TNF-a has been associated with preterm labor and
delivery caused by gram-negative bacterial infection in humans
(Silver et al., 1994). Animal experiments showed that rapid
increases in the maternal serum TNF-a levels contribute to
LPS-induced embryo death (Leazer et al., 2002). Furthermore,
pentoxifylline, a TNF-a suppressor, reversed LPS-induced
embryonic resorption and abortion (Gendron et al., 1990).
However, a recent study found that LPS-induced IUFD was not
blocked by treatment with anti-TNF antibody that inhibited
LPS-induced TNF-a production in pregnant females (Kohmura
et al., 2000), suggesting that the cause of fetal death cannot be
attributed to mother-derived TNF-a alone.
Eicosanoids have been demonstrated to be important
mediators of LPS-induced adverse developmental outcome.
Silver et al. (1995) reported that pregnant C3H/HeN mice
injected with LPS showed an increase in decidual eicosanoid
production and COX
2
expression, followed by a dose-dependent
increase in embryo death. COX
2
suppressors decreased LPS-
induced fetal mortality and protected against LPS-induced
preterm delivery (Gross et al., 2000; Sakai et al., 2001). Several
studies showed that maternal LPS exposure signicantly
increased inducible nitric oxide synthase (iNOS) expression
in decidual and myometrial cells and nitric oxide (NO)
1
To whom correspondence should be addressed at Department of Toxicol-
ogy, Anhui Medical University, Hefei 230032, PR China. Fax: 86 551
5161179. E-mail: xudex@mail.hf.ah.cn.
Published by Oxford University Press 2005.
TOXICOLOGICAL SCIENCES 88(2), 525533 (2005)
doi:10.1093/toxsci/k300
Advance Access publication September 14, 2005

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production in decidual and uterine (Ogando et al., 2003). In
addition, aminoguanidine, an inhibitor of iNOS activity, re-
versed LPS-induced embryonic resorption and abortion
(Athanassakis et al., 1999). These results suggest that NO
fullls a functional role in LPS-induced embryonic resorption
and abortion.
LPS stimulates macrophages to generate reactive oxygen
species (ROS) and increases nitrotyrosine, a marker for O
d
2
;
NO, and ONOO

formation, in macrophage-rich organs


(Bautista et al., 1990). Excess ROS formation has been
implicated in the teratologic mechanism of several chemicals,
including phenytoin, ethanol, and thalidomide (Kotch et al.,
1995; Parman et al., 1999; Wells et al., 1996; Winn et al.,
1997). The role of ROS on teratogenesis in diabetic pregnancy
has also been demonstrated (Viana et al., 2000). A recent study
showed that ascorbic acid inhibits ROS production, NF-kappa
B activation, and prevents ethanol-induced growth retardation
and microencephaly (Peng et al., 2005). Our earlier study
found that alpha-phenyl-N-t-butylnitrone, a free radical spin-
trapping agent, protected against LPS-induced IUFD and
reversed LPS-induced growth and skeletal developmental
retardation (data not published), indicating that ROS mediated,
at least partially, LPS-induced IUFD and IUGR.
N-acetylcysteine (NAC) is a GSH precursor and direct
antioxidant. As a potent antioxidant, NAC directly scavenges
hydrogen peroxide (H
2
O
2
), hydroxyl free radicals
d
OH; and
hypochloric acid (HOCl) in vitro (Aruoma et al., 1989). NAC
also decreases free radical levels by increasing GSH synthesis
(Neuschwander-Tetri et al., 1996; Song et al., 2004). Several
studies showed that NAC inhibited LPS-induced iNOS and
TNF-a expression and NF-jB activity (Pahan et al., 1998;
Verhasselt et al., 1999). Clinically, NAC has been successfully
used in adult respiratory distress syndrome (Victor et al., 2003).
A recent study showed that pretreatment with NAC protected
against LPS-induced preterm labor (Buhimschi et al., 2003).
However, the effects of NAC on LPS-induced IUFD and IUGR
have not been well characterized.
In this study, we investigated the effects of NAC on LPS-
induced IUFD and IUGR in ICR mice. Our results found that
NAC has a dual effect on LPS-induced IUFD and IUGR.
Pretreatment with NAC signicantly reduced fetal mortality
and revised fetal growth and skeletal development retardation
via counteracting LPS-induced oxidative stress and inhibiting
TNF-a production. However, when administered after LPS
treatment, NAC had no effect on LPS-induced IUFD and IUGR
and in fact worsened LPS-evoked preterm labor.
MATERIALS AND METHODS
Chemicals. Lipopolysaccharide (Escherichia coli LPS, serotype
0127:B8), N-acetylcysteine (NAC) and DL-buthionine-(SR)-sulfoximine
(BSO) were purchased from Sigma Chemical Co. (St. Louis, MO). All the
other reagents were from Sigma or as indicated in the specied methods.
Animals and Treatments
The ICR mice (810 weeks old; male mice: 2830 g; female mice: 2426 g)
were purchased from Beijing Vital River whose foundation colonies were all
introduced from Charles River Laboratories, Inc. The animals were allowed
free access to food and water at all times and were maintained on a 12-h light/
dark cycle in a controlled temperature (2025C) and humidity (50 5%)
environment for a period of one week before use. For mating purposes, four
females were housed overnight with two males starting at 9:00 P.M. Females
were checked by 7:00 A.M. the next morning, and the presence of a vaginal
plug was designated as gestational day (GD) 0. The present study consisted of
three separate experiments.
Experiment 1. To investigate the effects of NAC on LPS-induced IUFD
and IUGR, the pregnant mice were divided into seven groups randomly. All
pregnant mice except controls received an ip injection of LPS (75 lg/kg) on GD
1517. NAC was administered in two different modes. In mode A, the pregnant
mice were pretreated with two doses of NAC (either 50 plus 25 mg/kg or 200
plus 100 mg/kg) before LPS, one (either 50 or 200 mg/kg) at 12 h before LPS
and the other (either 25 or 100 mg/kg) at 15 min before LPS. DL- buthionine-
(SR)- sulfoximine (BSO) is an inhibitor of glutathione (GSH) synthesis. To
investigate the role of GSH on NAC-mediated protection against LPS-induced
IUFD, the pregnant mice in the NAC BSOLPS group were pretreated with
two doses of BSO (100 mg/kg at 12 h before LPS and 100 mg/kg at 2 h before
LPS) and two doses of NAC (200 mg/kg at 12 h before LPS and 100 mg/kg at
15 min before LPS). In mode B, the pregnant mice were administered two doses
of NAC (either 50 plus 25 mg/kg or 200 plus 100 mg/kg) in 24 h, one (either
50 or 200 mg/kg) injected immediately after LPS and the other (either 25 or
100 mg/kg) injected 3 h after LPS. All dams were sacriced on GD 18 and
gravid uterine weights were recorded. For each litter, the number of live fetuses,
dead fetuses, and resorption sites were counted. Live fetuses in each litter were
weighed. Crown-rump and tail lengths were measured. All fetuses were then
stored in ethanol a minimum of two weeks for subsequent skeletal evaluation.
Experiment 2. To investigate the effects of NAC on LPS-induced lipid
peroxidation, GSH depletion, and NO production, the pregnant mice were
divided into ve groups randomly. All pregnant mice except controls received
an ip injection of LPS (75 lg/kg) on GD 15. In NAC pretreatment group, the
pregnant mice were pretreated with two doses of NAC (200 plus 100 mg/kg)
before LPS, one (200 mg/kg) at 12 h before LPS and the other (100 mg/kg) at
15 min before LPS. To investigate the effects of BSO on GSH synthesis, the
pregnant mice in NAC BSO LPS group were pretreated with two doses of
BSO (100 mg/kg at 12 h before LPS and 100 mg/kg at 2 h before LPS) and two
doses of NAC (200 mg/kg at 12 h before LPS and 100 mg/kg at 15 min before
LPS). In the NAC post-treatment group, the pregnant mice received two doses
of NAC (200 plus 100 mg/kg), one (200 mg/kg, ip) injected immediately after
LPS and the other (100 mg/kg, ip) injected 3 h after LPS. All dams were
sacriced at 6 h after LPS treatment. Maternal liver, placenta, and fetal liver
were dissected for GSH and thiobarbituric acid-reactive substance (TBARS)
measurements. Maternal serum and amniotic uid were collected for nitrite
plus nitrate analyses.
Experiment 3. To investigate the effects of NAC on LPS-induced TNF-a
production, the pregnant mice were divided into four groups randomly. All
pregnant mice except controls received an ip injection of LPS (75 lg/kg) on
GD 15. In the NAC pretreatment group, the pregnant mice were pretreated with
two doses of NAC (200 plus 100 mg/kg) before LPS, one (200 mg/kg) at 12 h
before LPS and the other (100 mg/kg) at 15 min before LPS. In the NAC post-
treatment group, the pregnant mice received two doses of NAC (200 plus
100 mg/kg), one (200 mg/kg, ip) injected immediately after LPS and the other
(100 mg/kg, ip) injected 3 h after LPS. Half of the dams were sacriced at 1.5 h
after LPS treatment. The remaining animals were sacriced at 6 h after LPS
treatment. Maternal serum and amniotic uid were collected for TNF-a
analyses.
All procedures on animals followed the guidelines for humane treatment set
by the Association of Laboratory Animal Sciences and the Center for
Laboratory Animal Sciences at Anhui Medical University.
526 XU ET AL.

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Skeletal examination and evaluation. The fetuses stored in ethanol were
cleared of skin, viscera, and adipose tissue. Fetuses were then incubated in
acetone overnight and subsequently macerated and stained with alizarin red S
for 2 days. After an overnight incubation in 70% ethanol/glycerol/benzyl
alcohol, the fetuses were stored in glycerol until examination. Skeletal
evaluation included determination of the degree of ossication of the
phalanges, metacarpals, vertebrae, sternatrae, and skull. The size of the anterior
fontanel and ossication of the supraoccipital was scored.
Lipid peroxidation assay. Lipid peroxidation was quantied by measuring
TBARS as described previously (Ohkawa et al., 1979). Tissue was homoge-
nized in 9 volumes of 50 mmol/l Tris-HCl buffer (pH 7.4) containing 180
mmol/l KCl, 10 mmol/l EDTA, and 0.02% butylated hydroxytoluene. To 0.2 ml
of the tissue homogenate, 0.2 ml of 8.1% sodiumdodecyl sulfate, 1.5 ml of 20%
acetic acid, 1.5 ml of 0.9% thiobarbituric acid, and 0.6 ml of distilled water
were added and vortexed. The reaction mixture was placed in a water bath at
95C for 1 h. After cooling on ice, 1.0 ml of distilled water and 5.0 ml of
butanol/pyridine mixture (15:1, v/v) were added and vortexed. After centrifu-
gation at 10,000 3 g for 10 min, absorbance of the resulting lower phase was
determined at 532 nm. The TBARS concentration was calculated using 1,1,5,5-
tetraethoxypropane as standard.
Determination of glutathione content. The glutathione (GSH) was
determined by the method of Grifth (1980). Proteins of 0.4 ml liver
homogenates were precipitated by the addition of 0.4 ml of a metaphosphoric
acid solution. After 40 min, the protein precipitate was separated from the
remaining solution by centrifugation at 5000 rpm at 4C for 5 min. 400 ll of
the supernatant was combined with 0.4 ml of 300 mM Na
2
HPO
4
, and the
absorbance at 412 nm was read against a blank consisting of 0.4 ml supernatant
plus 0.4 ml H
2
O. Then, 100 ll DTNB (0.02%, w/v; 20 mg DTNB in 100 ml of
1% sodium citrate) was added to the blank and sample. Absorbance of the
sample was read against the blank at 412 nm. The GSH content was determined
using a calibration curve prepared with an authentic sample. GSH values were
expressed as nmol mg
1
protein. Protein content was measured according to the
method of Lowry et al. (1951).
TNF-a determination. Mouse TNF-a in maternal serum and amniotic
uid were measured by enzyme-linked immunosorbent assay (R&D,
Minneapolis, MN), following the manufacturers instructions.
Statistical analysis. The litter was considered the unit for statistical
comparison among different groups. Fetal mortality was calculated per litter
and then averaged per group. For fetal weight, crown-rump and tail lengths, and
skeletal evaluation, the means were calculated per litter and then averaged per
group. Quantied data were expressed as means SEM at each point. p < 0.05
was considered statistically signicant. ANOVA and the Student-Newmann-
Keuls post hoc test were used to determine differences between the treated
animals and the control and statistical signicance.
RESULTS
Effects of NAC on LPS-Induced IUFD
The number of litters, implantation sites per litter, resorp-
tions per litter, live fetuses per litter, and dead fetuses per litter,
and the incidence of preterm labor are presented in Table 1.
LPS and NAC exhibited no obvious maternal side effects (data
not shown). There were no differences in the number of
implantation sites and resorptions per litter among different
groups. In the control group, there were no pregnant mice
delivered before GD 18. Administration of LPS (75 lg/kg) on
GD 1517 resulted in 8.3% (1/12) preterm delivery. Pre-
treatment with NAC did not inuence the incidence of pre-
term delivery. Surprisingly, posttreatment with NAC (50 plus
25 mg/kg in 24 h) signicantly increased LPS-induced preterm
labor ( p < 0.01).
The effects of NAC on LPS-induced IUFD are presented in
Figure 1. Results showed that maternal LPS administration on
GD 1517 resulted in 63.2% fetal death. The effects of NAC on
LPS-induced IUFD depended on the schedule of treatments.
Pretreatment with NAC signicantly decreased fetal mortality
to 13.7%in the lowdose (50 plus 25 mg/kg) group and 24.7%in
the high dose (200 plus 100 mg/kg) group. However, post-
treatment with NAC had no effect on LPS-induced IUFD. To
investigate the role of GSHon NAC-mediated protection against
LPS-induced IUFD, the pregnant mice were pretreated with
BSO to inhibit GSH synthesis. Interestingly, NAC-mediated
protection was not counteracted by pretreatment with BSO.
Effects of NAC on LPS-Induced IUGR
The effects of NAC on LPS-induced IUGR are presented in
Figures 2A, 2B and 2C. Maternal LPS exposure markedly
decreased fetal weight and crown-rump and tail lengths.
Pretreatment and posttreatment with NAC had little effect on
LPS-induced decrease in fetal weight. Furthermore, pretreat-
ment with NAC signicantly attenuated LPS-induced decrease
in crown-rump and tail lengths, whereas posttreatment had no
effect on LPS-induced decrease in crown-rump and tail lengths.
TABLE 1
Comparison of Fetal Outcomes among Different Groups
Treatments Litters
Implantation sites
per litter x S
Resorptions
per litter x S
Live fetuses
per litter x S
Dead fetuses
per litter x S
Preterm
delivery (%)
Control 19 12.8 2.12 0.58 1.09 12.2 2.10 0.05 1.11 0
LPS (75 lg/kg) 12 13.4 1.98 0.50 0.86 4.75 2.25

8.17 1.95

8.3
LPS NAC (pretreatment, 50 plus 25 mg/kg) 11 15.5 1.97 1.36 1.96 12.27 2.46* 1.91 2.09* 0
LPS NAC (pretreatment, 200 plus 100 mg/kg) 14 12.9 3.53 0.75 1.06 9.14 2.25* 3.07 1.64* 14.3
LPS NAC (pretreatment, 200 plus 100 mg/kg)BSO 10 14.3 2.11 0.80 0.92 12.6 1.70* 0.9 1.11* 0
LPS NAC (posttreatment, 50 plus 25 mg/kg) 17 13.8 2.22 1.22 1.99 6.35 2.18 6.76 1.52 47.1*
LPS NAC (posttreatment, 200 plus 100 mg/kg) 20 13.3 2.87 0.28 0.57 6.00 2.84 7.05 2.44 20.0

Signicantly different from control, p < 0.05.


*Signicantly different from LPS group, p < 0.05.
LPS-INDUCED FETAL GROWTH RETARDATION 527

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To investigate the role of GSH on NAC-mediated protection
against LPS-induced IUGR, the pregnant mice were pretreated
with BSO to inhibit GSH synthesis. Results showed that BSO
pretreatment did not inuence NAC-mediated protection
against LPS-induced IUGR.
Effects of NAC on LPS-Induced Skeletal Developmental
Retardation
The effects of NAC on LPS-induced skeletal development
retardation are presented in Table 2. Results showed that fetal
skeleton in LPS-treated mice exhibited fewer ossication
centers in caudal vertebrae, anterior and posterior phalanges
as compared with the control. In addition, maternally admin-
istered LPS also retarded fetal supraoccipital ossication. Both
pretreatment and posttreatment with NAC signicantly atten-
uated LPS-induced skeletal development retardation.
Effects of NAC on LPS-Induced Lipid Peroxidation
Lipid peroxidation was quantied by measuring TBARS.
The effects of NAC on LPS-induced lipid peroxidation are
presented in Figures 3A, 3B, and 3C. Results showed that LPS
signicantly increased TBARS level in maternal liver, pla-
centa, and fetal liver. Pretreatment with NAC signicantly
attenuated LPS-induced lipid peroxidation in maternal liver
and fetal liver. However, post-treatment with NAC had no
effect on LPS-induced lipid peroxidation in maternal liver,
placenta, and fetal liver.
FIG. 1. The effects of NAC on LPS-induced IUFD. The pregnant mice
were administered with LPS and NAC as described in Materials and Methods.
The pregnant mice were sacriced on GD 18. The number of live fetuses and
dead fetuses were counted. Numbers of litters in each group were presented in
parentheses. Pre-NAC(75) LPS, pretreatment with NAC (50 plus 25 mg/kg)
before LPS (lg/kg); Pre-NAC(300) LPS, pretreatment with NAC (200 plus
100 mg/kg) before LPS (lg/kg); Pre-NAC(300) BSO LPS, pretreatment
with NAC (200 plus 100 mg/kg) plus BSO (100 plus 100 mg/kg) before LPS
(lg/kg); LPS Post-NAC(75), posttreatment with NAC (50 plus 25 mg/kg)
after LPS (lg/kg); LPS Post-NAC(300), posttreatment with NAC (200 plus
100 mg/kg) after LPS (lg/kg). p < 0.05 as compared with control group. *p <
0.05 as compared with LPS-treated group.
FIG. 2. The effects of NAC on LPS-induced IUGR. The pregnant mice
were administered with LPS and NAC as described in Materials and Methods.
Pregnant mice were sacriced on GD 18. Fetal weight and crown-rump and tail
lengths were measured. Numbers of litters in each group were presented in
parentheses. Pre-NAC(75) LPS, pretreatment with NAC (50 plus 25 mg/kg)
before LPS (lg/kg); Pre-NAC(300) LPS, pretreatment with NAC (200 plus
100 mg/kg) before LPS (lg/kg); Pre-NAC(300) BSO LPS, pretreatment
with NAC (200 plus 100 mg/kg) plus BSO (100 plus 100 mg/kg) before LPS
(lg/kg); LPS Post-NAC(75), posttreatment with NAC (50 plus 25 mg/kg)
after LPS (lg/kg); LPS Post-NAC(300), posttreatment with NAC (200 plus
100 mg/kg) after LPS (lg/kg). (A) The effects of NAC on LPS-induced
decrease in fetal weight. (B) The effects of NAC on LPS-induced decrease in
fetal crown-rump length. (C) The effects of NAC on LPS-induced decrease in
tail lengths. p < 0.05 as compared with control group. *p < 0.05 as compared
with LPS-treated group.
528 XU ET AL.

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Effects of NAC on GSH Content
The effects of NAC on GSH content are presented in
Figures 4A, 4B, and 4C. Results showed that a single dose of
LPS signicantly decreased GSH content in maternal liver,
placenta, and fetal liver. However, NAC did not elevate GSH
levels in maternal liver, placenta, and fetal liver of LPS-treated
pregnant mice. As expected, BSO pretreatment aggravated
LPS-induced GSH depletion in maternal liver, placenta, and
fetal liver. Interestingly, posttreatment with NAC worsened
LPS-induced GSH depletion in maternal liver and placenta.
Effects of NAC on LPS-Induced TNF-a Expression
The effects of LPS on LPS-induced TNF-a production are
presented in Figure 5. Results showed that TNF-a concen-
trations in maternal serum and amniotic uid were signicantly
increased at 1.5 h after LPS treatment. TNF-a concentrations in
maternal serum returned to control level at 6 h after LPS
treatment. However, TNF-a concentrations in amniotic uid
remained a high level in LPS-treated group. Pretreatment with
NAC signicantly attenuated LPS-induced TNF-a concentra-
tion in maternal serum and amniotic uid, whereas posttreat-
ment with NAC had no effect on LPS-induced TNF-a mRNA
production.
DISCUSSION
The present study investigated the effect of pretreatment
with NAC on LPS-induced IUFD and IUGR. The pregnant
mice were pretreated with two doses of NAC (either 50 plus
25 mg/kg or 200 plus 100 mg/kg) before LPS, one (either 50 or
200 mg/kg) at 12 h before LPS and the other (either 25 or 100
mg/kg) at 15 min before LPS. Our results indicated that
pretreatment with NAC markedly improved fetal survival,
increased fetal weight and crown-rump and tail lengths, and
revised LPS-induced skeletal development retardation. Fur-
thermore, pretreatment with NAC signicantly attenuated LPS-
induced increase in TBARS level in maternal and fetal livers,
suggesting that NAC-mediated protection against LPS-induced
IUFD and IUGR is, at least partially, associated with decreased
lipid peroxidation.
The antioxidant activity of NAC primarily involves two
mechanisms (Aruoma et al., 1989): (1) NAC acts as a free
radical scavenger, directly scavenges H
2
O
2
, HOCl, and
d
OH;
and (2) NAC acts as a precursor of GSH to facilitate
intracellular GSH synthesis. To investigate the role of GSH
on NAC-mediated protection against LPS-induced IUFD and
IUGR, we measured the reduced GSH content in maternal
liver, placenta, and fetal liver. As expected, maternal LPS
exposure signicantly decreased GSH level in maternal liver,
placenta, and fetal liver. However, pretreatment with NAC had
no effect on LPS-induced GSH depletion. Furthermore, DL-
buthionine- (SR)- sulfoximine (BSO), an inhibitor of GSH
synthesis, did not inuence NAC-mediated protection against
LPS-induced IUFD and IUGR, although LPS-induced hepatic
GSH depletion was aggravated by BSO in NAC-pretreated
pregnant mice. These results suggest that NAC-mediated
protection against LPS-evoked IUFD and IUGR is not
attributed to increased GSH synthesis but most likely due to
its strong ROS-scavenging effect.
Numerous studies demonstrated that TNF-a plays an
important role in LPS-induced developmental toxicity (Silver
TABLE 2
The Effects of NAC on LPS-Induced Fetal Skeletal Development Retardation
Control
(n 19)
LPS
(n 12)
NAC(75) LPS
(n 11)
NAC(300) LPS
(n 14)
NAC(300) BSO LPS
(n 10)
LPS NAC(75)
(n 17)
LPS NAC(300)
(n 20)
No. examined
Fetuses 231 57 135 120 126 76 112
Scores
Supraoccipital bone
a
1.02 0.01 2.15 0.25

1.23 0.50* 1.26 0.51* 1.25 0.55* 1.13 0.34* 1.29 0.61*
Number ossied
Sternum 6.00 0.00 5.87 0.26 6.00 0.00 5.98 0.05 5.99 0.02 5.85 0.33 5.96 0.08
Metacarpus 4.00 0.00 3.88 0.29 4.00 0.00 4.00 0.00 4.00 0.00 4.00 0.00 4.00 0.00
Anterior phalanx 4.00 0.00 3.08 1.42

3.98 0.05* 3.97 0.08* 3.90 0.25 4.00 0.01* 3.97 0.06*
Metatarsus 4.98 0.01 4.56 0.39

4.84 0.16 4.91 0.13* 4.89 0.17* 4.81 0.32 4.77 0.14
Posterior phalanx 4.95 0.02 2.99 2.09

4.56 0.38* 4.51 0.47* 4.42 0.63* 4.76 0.34* 4.58 0.33*
Caudal vertebrae 6.30 0.21 3.67 0.91

5.15 0.79* 4.68 0.48 4.84 1.13 4.37 0.83 4.38 0.73
Note. NAC(75) LPS, pretreatment with NAC (50 plus 25 mg/kg) before LPS (lg/kg); NAC(300) LPS, pretreatment with NAC (200 plus 100 mg/kg)
before LPS (lg/kg); NAC(300) BSO LPS, pretreatment with NAC (200 plus 100 mg/kg) plus BSO (100 plus 100 mg/kg) before LPS (lg/kg); LPS NAC
(75), posttreatment with NAC (50 plus 25 mg/kg) after LPS (lg/kg); LPS NAC(300), posttreatment with NAC (200 plus 100 mg/kg) after LPS (lg/kg);
Numbers of litters in each group were presented in parentheses.
a
Supraoccipital bone scores: 1 well ossied, 4 completely unossied.

Signicantly different from control, p < 0.05.


*Signicantly different from LPS group, p < 0.05.
LPS-INDUCED FETAL GROWTH RETARDATION 529

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FIG. 3. The effects of NAC on LPS-induced lipid peroxidation. The
pregnant mice were administered with LPS and NAC as described in Materials
and Methods. The pregnant mice were sacriced at 6 h after LPS treatment.
Maternal liver and placenta were excised for TBARS measurement. Data were
expressed as means SEMof twelve mice in each point. p <0.05 as compared
with control group. *p < 0.05 vs. LPS-treated control.
FIG. 4. The effects of NAC on GSH content. The pregnant mice were
administered with LPS and NAC as described in Materials and Methods. The
pregnant mice were sacriced at 6 h after LPS treatment. Maternal liver and
placenta were excised for GSH measurement. Data were expressed as means
SEM (n 12). p < 0.05 as compared with control group. *p < 0.05 as
compared with LPS-treated group.
530 XU ET AL.

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et al., 1994). NAC is an inhibitor of TNF-a production
(Neuschwander-Tetri et al., 1996; Peristeris et al., 1992; Victor
and De la Fuente, 2002). The present study showed that
pretreatment with NAC dramatically attenuated LPS-induced
TNF-a production in maternal serum and amniotic uid,
suggesting that NAC-mediated protection against LPS-induced
IUFD and IUGR might also be associated with inhibition of
TNF-a production.
The present study investigated the effects of posttreatment
with NAC on LPS-induced IUFD and IUGR. By contrast to
pretreatment, posttreatment with NAC had no effect on LPS-
induced IUFD and IUGR. Furthermore, the present study found
that posttreatment with NAC aggravated LPS-evoked preterm
delivery.
Usually, NAC is mentioned as an antioxidant. However,
NAC and other thiol chemicals have been demonstrated to be
also pro-oxidants (Sagrista et al., 2002; Shen et al., 2000). The
interaction of thiols with reactive radicals could generate thiyl
radicals, which, in turn, may impart a pro-oxidant function.
Actually, the presence of metals, such as Cu (II), and the
presence of ROS, such as H
2
O
2
and O
d
2
; potentiate auto-
oxidize process of NAC (Oikawa et al., 1999). Having
undergone auto-oxidation, thiols no longer act as antiox-
idants. Once initiated, these reactions can produce additional
ROS including O
d
2
; H
2
O
2
, and
d
OH: According to the report
by Sprong et al. (1998), NAC behaves either as anti- or pro-
oxidants depending on the dose administered. A low dose
(275 mg/kg in 24 h) of NAC protected rats against LPS-
mediated oxidative stress, whereas a high dose NAC(900 mg/kg
in 24 h) increased LPS-induced lung injury and mortality. Chan
et al. (2001) found that the local redox environment inuenced
the effect of NAC. In a serum-depleted environment (0.1% fetal
bovine serum), NAC inhibited LPS-induced activation of the
mitogen-activated protein kinases, namely extracellular signal-
regulated kinase, p38mapk, and c-Jun NH2-terminal kinase
(JNK). By contrast, NAC enhanced LPS induction of p38mapk
and JNK phosphorylation in the presence of 10% serum. The
present study showed that pretreatment with NAC signicantly
attenuated LPS-induced lipid peroxidation in maternal and fetal
liver. By contrast, posttreatment with NAC had no effect on
LPS-induced lipid peroxidation in maternal liver, placenta, and
fetal liver, and in fact aggravated LPS-induced GSH depletion
in maternal liver and placenta. These results suggest that NAC
behaves either as an antioxidant or a prooxidant depending on
the schedule of NACadministration. When administered before
LPS, NAC behaved as an antioxidant and protected against
LPS-induced IUFD and IUGR. However, when administered
after LPS, NAC behaved as a pro-oxidant and worsened LPS-
induced preterm labor.
In summary, the present study indicates that NAC has a dual
role on LPS-induced developmental toxicity. The effects of
NAC on LPS-induced IUFD and IUGR depend on the schedule
of NAC administration. Pretreatment with NAC improves fetal
survival and reverses LPS-induced fetal growth and skeletal
development retardation via counteracting LPS-induced oxi-
dative stress and inhibiting TNF-a production. However,
posttreatment with NAC has little effect on LPS-induced IUFD
and IUGR. Actually, when administered after LPS, NAC may
behave as a prooxidant and worsen LPS-induced preterm labor.
ACKNOWLEDGMENTS
The project was supported by National Natural Science Foundation of China
(30371667) and Anhui Provincial Natural Science Foundation (050430714).
Conict of interest: none declared.
FIG. 5. The effects of NAC on LPS-induced TNF-a production. The
pregnant mice were administered with LPS and NAC as described in Materials
and Methods. The pregnant mice were sacriced either at 1.5 h or at 6 h after
LPS. Maternal serum and amniotic uid were collected for measurement of
TNF-a concentration using ELISA. (A) The effects of NAC on TNF-a con-
centration in maternal serum. (B) The effects of NAC on TNF-a concentration
in amniotic uid. Data were expressed as means SEM (n 8). p < 0.05
as compared with control group. *p < 0.05 as compared with LPS-treated
group.
LPS-INDUCED FETAL GROWTH RETARDATION 531

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