Effect of N-Acetylcysteine on Lipopolysaccharide-Induced Intra-uterine
http://www.100md.com
《毒物学科学杂志》
Department of Toxicology, Anhui Medical University, Hefei, 230032, P. R. China
Key Laboratory of Anti-inflammatory and Immunopharmacology of Anhui Province, Hefei, 230032, P. R. China
ABSTRACT
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 μg/kg, ip) on gestational day (GD) 15–17. 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 significantly alleviated LPS-induced fetal mortality and reversed LPS-induced growth and skeletal development retardation. Correspondingly, pretreatment with NAC significantly attenuated LPS-induced elevation in TNF- concentration in maternal serum and amniotic fluid and lipid peroxidation in maternal and fetal livers. By contrast to pretreatment, posttreatment with NAC had no effect on LPS-induced TNF- 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.
INTRODUCTION
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 developmental outcome, including embryonic resorption, intra-uterine fetal death (IUFD), intra-uterine growth retardation (IUGR), and preterm delivery in rodents (Collins et al., 1994; O'Sullivan 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 maternal serum TNF- production (Bell et al., 2004; Vizi et al., 2001). Maternal LPS exposure resulted in elevation of TNF- levels in amniotic fluid and placenta (Chen et al., 2005; Gayle et al., 2004). TNF- 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- levels contribute to LPS-induced embryo death (Leazer et al., 2002). Furthermore, pentoxifylline, a TNF- 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- production in pregnant females (Kohmura et al., 2000), suggesting that the cause of fetal death cannot be attributed to mother-derived TNF- 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 COX2 expression, followed by a dose-dependent increase in embryo death. COX2 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 significantly increased inducible nitric oxide synthase (iNOS) expression in decidual and myometrial cells and nitric oxide (NO) production in decidual and uterine (Ogando et al., 2003). In addition, aminoguanidine, an inhibitor of iNOS activity, reversed LPS-induced embryonic resorption and abortion (Athanassakis et al., 1999). These results suggest that NO fulfills 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 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 (H2O2), hydroxyl free radicals (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- expression and NF-B 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 significantly reduced fetal mortality and revised fetal growth and skeletal development retardation via counteracting LPS-induced oxidative stress and inhibiting TNF- 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 specified methods.
Animals and Treatments
The ICR mice (8–10 weeks old; male mice: 28–30 g; female mice: 24–26 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 (20–25°C) 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 μg/kg) on GD 15–17. 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 + 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 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 sacrificed 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 five groups randomly. All pregnant mice except controls received an ip injection of LPS (75 μg/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 sacrificed 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 fluid were collected for nitrite plus nitrate analyses.
Experiment 3.
To investigate the effects of NAC on LPS-induced TNF- production, the pregnant mice were divided into four groups randomly. All pregnant mice except controls received an ip injection of LPS (75 μg/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 sacrificed at 1.5 h after LPS treatment. The remaining animals were sacrificed at 6 h after LPS treatment. Maternal serum and amniotic fluid were collected for TNF- 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.
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 ossification of the phalanges, metacarpals, vertebrae, sternatrae, and skull. The size of the anterior fontanel and ossification of the supraoccipital was scored.
Lipid peroxidation assay.
Lipid peroxidation was quantified by measuring TBARS as described previously (Ohkawa et al., 1979). Tissue was homogenized 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% sodium dodecyl 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 95°C 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 centrifugation at 10,000 x 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 Griffith (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 4°C for 5 min. 400 μl of the supernatant was combined with 0.4 ml of 300 mM Na2HPO4, and the absorbance at 412 nm was read against a blank consisting of 0.4 ml supernatant plus 0.4 ml H2O. Then, 100 μl 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- determination.
Mouse TNF- in maternal serum and amniotic fluid were measured by enzyme-linked immunosorbent assay (R&D, Minneapolis, MN), following the manufacturer's 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. Quantified data were expressed as means ± SEM at each point. p < 0.05 was considered statistically significant. ANOVA and the Student-Newmann-Keuls post hoc test were used to determine differences between the treated animals and the control and statistical significance.
RESULTS
Effects of NAC on LPS-Induced IUFD
The number of litters, implantation sites per litter, resorptions 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 μg/kg) on GD 15–17 resulted in 8.3% (1/12) preterm delivery. Pretreatment with NAC did not influence the incidence of preterm delivery. Surprisingly, posttreatment with NAC (50 plus 25 mg/kg in 24 h) significantly 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 15–17 resulted in 63.2% fetal death. The effects of NAC on LPS-induced IUFD depended on the schedule of treatments. Pretreatment with NAC significantly decreased fetal mortality to 13.7% in the low dose (50 plus 25 mg/kg) group and 24.7% in the high dose (200 plus 100 mg/kg) group. However, posttreatment with NAC had no effect on LPS-induced IUFD. To investigate the role of GSH on 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, pretreatment with NAC significantly 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. 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 influence 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 ossification centers in caudal vertebrae, anterior and posterior phalanges as compared with the control. In addition, maternally administered LPS also retarded fetal supraoccipital ossification. Both pretreatment and posttreatment with NAC significantly attenuated LPS-induced skeletal development retardation.
Effects of NAC on LPS-Induced Lipid Peroxidation
Lipid peroxidation was quantified by measuring TBARS. The effects of NAC on LPS-induced lipid peroxidation are presented in Figures 3A, 3B, and 3C. Results showed that LPS significantly increased TBARS level in maternal liver, placenta, and fetal liver. Pretreatment with NAC significantly 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.
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 significantly 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- Expression
The effects of LPS on LPS-induced TNF- production are presented in Figure 5. Results showed that TNF- concentrations in maternal serum and amniotic fluid were significantly increased at 1.5 h after LPS treatment. TNF- concentrations in maternal serum returned to control level at 6 h after LPS treatment. However, TNF- concentrations in amniotic fluid remained a high level in LPS-treated group. Pretreatment with NAC significantly attenuated LPS-induced TNF- concentration in maternal serum and amniotic fluid, whereas posttreatment with NAC had no effect on LPS-induced TNF- 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. Furthermore, pretreatment with NAC significantly 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 H2O2, HOCl, and 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 significantly 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 influence 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- plays an important role in LPS-induced developmental toxicity (Silver et al., 1994). NAC is an inhibitor of TNF- 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- production in maternal serum and amniotic fluid, suggesting that NAC-mediated protection against LPS-induced IUFD and IUGR might also be associated with inhibition of TNF- 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 H2O2 and potentiate "auto-oxidize" process of NAC (Oikawa et al., 1999). Having undergone auto-oxidation, thiols no longer act as "antioxidants." Once initiated, these reactions can produce additional ROS including H2O2, and 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 influenced 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 significantly 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 NAC administration. 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 oxidative stress and inhibiting TNF- 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). Conflict of interest: none declared.
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Key Laboratory of Anti-inflammatory and Immunopharmacology of Anhui Province, Hefei, 230032, P. R. China
ABSTRACT
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 μg/kg, ip) on gestational day (GD) 15–17. 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 significantly alleviated LPS-induced fetal mortality and reversed LPS-induced growth and skeletal development retardation. Correspondingly, pretreatment with NAC significantly attenuated LPS-induced elevation in TNF- concentration in maternal serum and amniotic fluid and lipid peroxidation in maternal and fetal livers. By contrast to pretreatment, posttreatment with NAC had no effect on LPS-induced TNF- 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.
INTRODUCTION
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 developmental outcome, including embryonic resorption, intra-uterine fetal death (IUFD), intra-uterine growth retardation (IUGR), and preterm delivery in rodents (Collins et al., 1994; O'Sullivan 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 maternal serum TNF- production (Bell et al., 2004; Vizi et al., 2001). Maternal LPS exposure resulted in elevation of TNF- levels in amniotic fluid and placenta (Chen et al., 2005; Gayle et al., 2004). TNF- 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- levels contribute to LPS-induced embryo death (Leazer et al., 2002). Furthermore, pentoxifylline, a TNF- 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- production in pregnant females (Kohmura et al., 2000), suggesting that the cause of fetal death cannot be attributed to mother-derived TNF- 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 COX2 expression, followed by a dose-dependent increase in embryo death. COX2 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 significantly increased inducible nitric oxide synthase (iNOS) expression in decidual and myometrial cells and nitric oxide (NO) production in decidual and uterine (Ogando et al., 2003). In addition, aminoguanidine, an inhibitor of iNOS activity, reversed LPS-induced embryonic resorption and abortion (Athanassakis et al., 1999). These results suggest that NO fulfills 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 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 (H2O2), hydroxyl free radicals (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- expression and NF-B 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 significantly reduced fetal mortality and revised fetal growth and skeletal development retardation via counteracting LPS-induced oxidative stress and inhibiting TNF- 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 specified methods.
Animals and Treatments
The ICR mice (8–10 weeks old; male mice: 28–30 g; female mice: 24–26 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 (20–25°C) 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 μg/kg) on GD 15–17. 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 + 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 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 sacrificed 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 five groups randomly. All pregnant mice except controls received an ip injection of LPS (75 μg/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 sacrificed 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 fluid were collected for nitrite plus nitrate analyses.
Experiment 3.
To investigate the effects of NAC on LPS-induced TNF- production, the pregnant mice were divided into four groups randomly. All pregnant mice except controls received an ip injection of LPS (75 μg/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 sacrificed at 1.5 h after LPS treatment. The remaining animals were sacrificed at 6 h after LPS treatment. Maternal serum and amniotic fluid were collected for TNF- 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.
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 ossification of the phalanges, metacarpals, vertebrae, sternatrae, and skull. The size of the anterior fontanel and ossification of the supraoccipital was scored.
Lipid peroxidation assay.
Lipid peroxidation was quantified by measuring TBARS as described previously (Ohkawa et al., 1979). Tissue was homogenized 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% sodium dodecyl 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 95°C 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 centrifugation at 10,000 x 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 Griffith (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 4°C for 5 min. 400 μl of the supernatant was combined with 0.4 ml of 300 mM Na2HPO4, and the absorbance at 412 nm was read against a blank consisting of 0.4 ml supernatant plus 0.4 ml H2O. Then, 100 μl 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- determination.
Mouse TNF- in maternal serum and amniotic fluid were measured by enzyme-linked immunosorbent assay (R&D, Minneapolis, MN), following the manufacturer's 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. Quantified data were expressed as means ± SEM at each point. p < 0.05 was considered statistically significant. ANOVA and the Student-Newmann-Keuls post hoc test were used to determine differences between the treated animals and the control and statistical significance.
RESULTS
Effects of NAC on LPS-Induced IUFD
The number of litters, implantation sites per litter, resorptions 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 μg/kg) on GD 15–17 resulted in 8.3% (1/12) preterm delivery. Pretreatment with NAC did not influence the incidence of preterm delivery. Surprisingly, posttreatment with NAC (50 plus 25 mg/kg in 24 h) significantly 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 15–17 resulted in 63.2% fetal death. The effects of NAC on LPS-induced IUFD depended on the schedule of treatments. Pretreatment with NAC significantly decreased fetal mortality to 13.7% in the low dose (50 plus 25 mg/kg) group and 24.7% in the high dose (200 plus 100 mg/kg) group. However, posttreatment with NAC had no effect on LPS-induced IUFD. To investigate the role of GSH on 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, pretreatment with NAC significantly 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. 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 influence 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 ossification centers in caudal vertebrae, anterior and posterior phalanges as compared with the control. In addition, maternally administered LPS also retarded fetal supraoccipital ossification. Both pretreatment and posttreatment with NAC significantly attenuated LPS-induced skeletal development retardation.
Effects of NAC on LPS-Induced Lipid Peroxidation
Lipid peroxidation was quantified by measuring TBARS. The effects of NAC on LPS-induced lipid peroxidation are presented in Figures 3A, 3B, and 3C. Results showed that LPS significantly increased TBARS level in maternal liver, placenta, and fetal liver. Pretreatment with NAC significantly 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.
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 significantly 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- Expression
The effects of LPS on LPS-induced TNF- production are presented in Figure 5. Results showed that TNF- concentrations in maternal serum and amniotic fluid were significantly increased at 1.5 h after LPS treatment. TNF- concentrations in maternal serum returned to control level at 6 h after LPS treatment. However, TNF- concentrations in amniotic fluid remained a high level in LPS-treated group. Pretreatment with NAC significantly attenuated LPS-induced TNF- concentration in maternal serum and amniotic fluid, whereas posttreatment with NAC had no effect on LPS-induced TNF- 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. Furthermore, pretreatment with NAC significantly 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 H2O2, HOCl, and 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 significantly 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 influence 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- plays an important role in LPS-induced developmental toxicity (Silver et al., 1994). NAC is an inhibitor of TNF- 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- production in maternal serum and amniotic fluid, suggesting that NAC-mediated protection against LPS-induced IUFD and IUGR might also be associated with inhibition of TNF- 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 H2O2 and potentiate "auto-oxidize" process of NAC (Oikawa et al., 1999). Having undergone auto-oxidation, thiols no longer act as "antioxidants." Once initiated, these reactions can produce additional ROS including H2O2, and 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 influenced 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 significantly 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 NAC administration. 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 oxidative stress and inhibiting TNF- 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). Conflict of interest: none declared.
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