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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
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
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.