Perinatal Lipopolysaccharide Exposure Downregulates Pregnane X Receptor and Cyp3a11 Expression in Fetal Mouse Liver
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《毒物学科学杂志》
Department of Toxicology
Institute of Clinical Pharmacology, Anhui Medical University, Hefei, 230032, P.R. China
Key Laboratory of Anti-inflammatory and Immunopharmacology of Anhui Province, Hefei, 230032, P.R. China
ABSTRACT
The pregnane X receptor (PXR) is a member of the nuclear receptor superfamily that regulates cytochrome P450 3A (CYP3A) gene transcription in a ligand-dependent manner. Lipopolysaccharide (LPS)-induced downregulation on PXR and cyp3a11 in adult mouse liver has been well characterized. In this study, we investigated the effects of maternal LPS exposure on PXR and cyp3a11 expression in fetal mouse liver. Pregnant ICR mice were injected intraperitoneally with different doses of LPS (0.10.5 mg/kg) on gestational day (GD) 17. PXR and cyp3a11 mRNA levels were determined using RT-PCR. Erythromycin N-demethylase (ERND) activity was used as an indicator of CYP3A expression in this study. Results showed that LPS significantly downregulated PXR and cyp3a11 mRNA levels and ERND activity in fetal liver in a dose-dependent manner. LPS-induced downregulation of PXR and cyp3a11 mRNA expression and ERND activity was attenuated after pregnant mice were pretreated with alpha-phenyl-N-t-butylnitrone (PBN), a free radical spin trapping agent. Additional experiment revealed that LPS significantly increased lipid peroxidation in fetal liver, which was also attenuated by PBN pretreatment. Furthermore, LPS-induced downregulation of PXR and cyp3a11 mRNA expression and ERND activity was prevented by maternal pretreatment with N-acetylcysteine (NAC). Maternal pretreatment with NAC also inhibited LPS-initiated lipid peroxidation and GSH depletion in fetal liver. However, maternal LPS treatment did not affect nitrite plus nitrate concentration in fetal liver. Correspondingly, aminoguanidine, a selective inhibitor of inducible nitric oxide synthase (iNOS), has no effect on LPS-induced downregulation of PXR and cyp3a11 expression and ERND activity in fetal liver. These results indicated that maternal LPS exposure downregulates PXR and cyp3a11 in fetal mouse liver. Reactive oxygen species (ROS) may be involved in LPS-induced downregulation of PXR and cyp3a11 in fetal mouse liver.
Key Words: lipopolysaccharide; fetal liver; pregnane X receptor; cytochrome P450 3A; reactive oxygen species.
INTRODUCTION
The liver is the major organ of amino acid and lipid metabolism, gluconeogenesis, synthesis of serum proteins, and xenobiotic detoxification. The fetal liver functions as the major hematopoietic organ in the mid- to late fetal stage (Dzierzak and Medvinsky, 1995; Hardy and Hayakawa, 2001). With embryonic development, fetal hepatocytes gradually express various types of cytochromes P450 (CYPs) that play a key role in the detoxification of drug or other xenobiotics (de Wildt et al., 1999; Hines and McCarver, 2002; Hulla and Juchau, 1989; Krauer and Dayer, 1991; Rich and Boobis, 1997).
The cytochrome P450 3A (CYP3A) is a member of the cytochrome P-450 monooxygenase superfamily. In humans, CYP3A4 and CYP3A5 gene products account for 30–40% of the total cytochrome P450 in the adult liver, which is responsible for the oxidative metabolism of numerous clinically used drugs and toxicants (Thummel and Wilkinson, 1998). Although CYP3A4 and CYP3A5 in fetal liver are not detectable, fetal hepatocytes express CYP3A7 as early as 50 to 60 days of gestation, with continued significant levels of expression through the perinatal period (Stevens et al., 2003). In mice, cyp3a11 and cyp3a13 are major members of cyp3a subfamily in the adult liver. In the developing mouse embryo, the amount of cyp3a11 and cyp3a13 expression gradually increases with the advancement of embryonic development (Choudhary et al., 2003).
The pregnane X receptor (PXR) is a member of the nuclear receptor superfamily that regulates CYP3A gene transcription in a ligand-dependent manner (Bertilsson et al., 1998; Kliewer et al., 1998; Lehmann et al., 1998). In addition, PXR plays pivotal roles in the regulation of genes contributing to hepatobiliary cholesterol and bile acid homeostasis. Several studies showed that fetal hepatocytes express PXR mRNA (Kamiya et al., 2003; Li et al., 2000; Miki et al., 2005). Furthermore, PXR activity in liver nuclear extracts assayed by gel EMSA exhibit a strict correlation with mRNA levels. Protein levels for PXR also corresponded to the mRNA and functional activity (Balasubramaniyan et al., 2005).
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. 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 (Mathurin et al., 2000). Lipopolysaccharide has been associated with adverse developmental outcome, including intrauterine fetal death (IUFD) and intrauterine growth retardation (IUGR) in animals (Collins et al., 1994; O'Sullivan et al., 1988). On the other hand, numerous studies indicated that inflammation and infection reduce cytochrome P450 levels in various species including human, rat, and mouse (Morgan, 1997, 2000). Lipopolysaccharide-induced downregulation of cyp3a11 has also been demonstrated in a mouse model (Li-Masters and Morgan, 2001; Sewer et al., 1998). Moreover, LPS-induced downregulation of cyp3a is associated with a marked reduction in PXR mRNA and protein levels (Beigneux et al., 2002; Sachdeva et al., 2003). Therefore, the question of whether maternal LPS exposure downregulates PXR and Cyp3a in fetal mouse liver is especially interesting.
In the present study, we investigated the effects of maternal LPS administration on PXR and cyp3a11 expressions in fetal mouse liver. Our results found that LPS downregulates PXR and cyp3a11 in fetal mouse liver. Reactive oxygen species (ROS) may be involved in LPS-induced downregulation of PXR and cyp3a11 in fetal mouse liver. Maternal NAC pretreatment prevents LPS-induced down-regulation of PXR and cyp3a11 in fetal liver by counteracting LPS-induced oxidative stress.
MATERIALS AND METHODS
Chemicals.
Lipopolysaccharide (Escherichia coli LPS, serotype 0127:B8), alpha-phenyl-N-t-butylnitrone (PBN), aminoguanidine (AG), and N-acetylcysteine (NAC) were purchased from Sigma Chemical Co. (St. Louis, MO). All other reagents were from Sigma or as indicated in the specified methods.
Animals and treatments.
The ICR mice (8 10 week-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 1 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 included four separate experiments.
Experiment 1.
To investigate the effects of LPS on PXR and cyp3a11 gene expression in fetal liver, GD 17 ICR mice were injected with different doses of LPS (0.1, 0.2, and 0.5 mg/kg, i.p.). The saline-treated pregnant mice served as controls. Mice were sacrificed at 12 h after LPS treatment for measurement of PXR and cyp3a11 mRNA expression and ERND activity in fetal livers.
Experiment 2.
To investigate the effects of PBN on LPS-induced downregulation of PXR and cyp3a11 gene expression in fetal liver, pregnant mice were divided into four groups randomly. The pregnant mice in the LPS-treated group were injected with 0.2 mg/kg of LPS (i.p.) on GD 17. The pregnant mice in LPS + PBN group were injected with 100 mg/kg of PBN (i.p.) at 30 min before and 3 h after LPS (0.2 mg/kg, i.p.) administration. Saline- and PBN-treated pregnant mice served as controls. Half of the mice were sacrificed at 6 h after LPS treatment for measurement of nitrite plus nitrate and thiobarbituric acid-reactive substance (TBARS) content in fetal livers. The other mice were sacrificed at 12 h after LPS treatment for measurement of PXR and cyp3a11 mRNA expression and ERND activity in fetal livers.
Experiment 3.
To investigate the effects of NAC on LPS-induced downregulation of PXR and cyp3a11 gene expression in fetal liver, pregnant mice were divided into four groups randomly. The pregnant mice in the LPS-treated group were injected with 0.2 mg/kg of LPS (i.p.) on GD 17. Pregnant mice in the LPS + NAC group were injected with NAC (100 mg/kg, i.p.) either at 30 min before or 3 h after LPS (0.2 mg/kg, i.p.) treatment. Saline- and NAC-treated pregnant mice served as controls. Half of the mice were sacrificed at 6 h after LPS treatment for measurements of TBARS and GSH content in fetal livers. The other mice were sacrificed at 12 h after LPS treatment for measurement of PXR and cyp3a11 mRNA expression and ERND activity in fetal livers.
Experiment 4.
To investigate the effects of AG on LPS-induced downregulation of PXR and cyp3a11 gene expression in fetal liver, pregnant mice were divided into four groups randomly. The pregnant mice in the LPS-treated group were injected with 0.2 mg/kg of LPS (i.p.) on GD 17. Pregnant mice in the LPS + AG group were injected with AG (100 mg/kg, i.p.) at 30 min before LPS (0.2 mg/kg, i.p.) treatment. Saline- and AG-treated pregnant mice served as controls. Half of the mice were sacrificed at 6 h after LPS treatment for measurement of nitrite plus nitrate concentration in fetal livers. The other mice were sacrificed at 12 h after LPS treatment for measurement of PXR and cyp3a11 mRNA expression and ERND activity in fetal livers.
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.
Isolation of total RNA and reverse transcriptase (RT).
For each sample, 50 mg of fetal liver tissue was collected. Total cellular RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. RNase-free DNase (Promega, Madison, WI) was used to remove genomic DNA. The integrity and concentration of RNA was determined by measuring absorbance at 260 nm followed by electrophoresis on agarose gels. Total RNA was stored at –80°C. For the synthesis of cDNA, 2.0 μg of total RNA from each sample was resuspended in a 20-μl final volume of reaction buffer, which contained 25 mM Tris · HCl, pH 8.3; 37.5 mM KCl; 10 mM dithiothreitol; 1.5 mM MgCl2; 10 mM of each dNTP and 0.5 mg oligo(dT)15 primer (Promega). After the reaction mixture reached 38°C, 400 units of RT (Promega) was added to each tube and the sample was incubated for 60 min at 38°C. Reverse transcription was stopped by denaturing the enzyme at 95°C.
Polymerase chain reaction (PCR) amplification.
The final PCR mixture contained 2 μl of cDNA, 1x PCR buffer, 1.5 mM MgCl2, 200 μM dNTP mixture, 2 U of Taq DNA polymerase, 1 μM sense and antisense primers, and sterile water to 25 μl. The reaction mixture was covered with mineral oil. Polymerase chain reaction for glyceraldehyde-3-phosphate dehydrogenase (gapdh) was performed on each individual sample as an internal positive-control standard. The following primers were synthesized by Shanghai Sangon Biological Engineering Technology and Service Company (Shanghai, China), according to sequence designs previously described by others (Xu et al., 2004). Gapdh, 5'-GAG GGG CCA TCC ACA GTC TTC-3' and 5'-CAT CAC CAT CTT CCA GGA GCG-3'; PXR, 5'-GCG CGG AGA AGA CGG CAG CAT C-3' and 5'-CCC AGG TTC CCG TTT CCG TGT C-3'cyp3a11, 5'-CTC AAT GGT GTG TAT ATC CCC-3' and 5'-CCG ATG TTC TTA GAC ACT GCC-3'. The sizes of amplified PCR products were 340 bp for gapdh, 254 for PXR, 423 bp for cyp3a11, respectively. Both number of cycles and annealing temperature were optimized for each primer pair. For gapdh, amplification was initiated by 3 min of denaturation at 94°C for 1 cycle, followed by 30 cycles at 94°C for 30 s, 56°C for 30 s, and 72°C for 1 min. For PXR, amplification was initiated by 3 min of denaturation at 94°C for 1 cycle, followed by 45 cycles at 94°C for 30 s, 68°C for 30 s, and 72°C for 1 min. After the last cycle of amplification, samples were incubated for 10 min at 72°C. For cyp3a11, amplification was initiated by 3 min of denaturation at 94°C for 1 cycle, followed by 35 cycles at 94°C for 30 s, 56°C for 30 s, and 72°C for 1 min. A final extension of 72°C for 10 min was included. The amplified PCR products were subjected to electrophoresis at 75 V through 1.5% agarose gels (Sigma) for 45 min. The pBR322 DNA digested with Alu I was used for molecular markers (MBI Fermentas). Agarose gels were stained with 0.5 mg/ml ethidium bromide (Sigma,) TBE buffer.
Preparation of liver microsomes.
Microsomes were isolated from livers by differential centrifugation (Haugen and Coon, 1976). All procedures were conducted at 4°C. Tissue was homogenized in four volumes of Tris/chloride buffer, pH 7.4, containing 150 mM potassium chloride and 1 mM EDTA, with a Polytron homogenizer and centrifuged at 10,000 x g for 20 min. The supernatant was collected and centrifuged at 211,000 x g for 40 min. The microsomal pellet was resuspended and washed in sodium pyrophosphate buffer, pH 7.4, containing 1 mM EDTA and centrifuged again at 211,000 x g for 40 min at 4°C. The washed microsomal pellet was resuspended in a trischloride buffer, pH 7.4, containing 20% glycerol, with a ground glass tissue grinder and stored at –80°C. Protein concentrations of microsome samples were measured according to the method of Lowry et al. (1951), using bovine serum albumin as a standard.
CYP3A catalytic activity.
Erythromycin N-demethylase (ERND) was used as an indicator of CYP3A catalytic activity in this study. The ERND was measured according to the method of Werringloer (1978) with a 45-min incubation containing 4 mM erythromycin in the presence of 0.5 mM NADPH and 0.4 mg of microsomal protein in a total assay volume of 1 ml. The rate of formaldehyde formation was determined spectrophotometrically at 412 nm using the Nash reagent. Measurement for ERND catalytic activity was repeated twice for three separately prepared liver microsome samples.
Determination of glutathione content.
The glutathione (GSH) level was determined by the method of Griffith (1980). Proteins of 0.4 ml tissue 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. Then 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, and 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. Glutathione values were expressed as nanomoles per milligram of (nm · mg–1 protein. Protein content was measured according to the method of Lowry et al. (1951).
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 the solution was 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.
Analysis of nitrite plus nitrate concentration in fetal liver.
The stable end products of L-arginine–dependent nitric oxide synthesis, nitrate plus nitrite, were measured in fetal liver using a colorimetric method based on the Griess reaction (Tracey et al., 1995). Briefly, aliquots of homogenates were added to 35% sulfosalicylic acid and vortexed every 5 min for 30 min to deproteinize samples. The samples were then centrifuged at 10, 000 x g at 4°C for 15 min. An aliquot of the supernatant was taken for nitrite and nitrate analysis. Then 20 μl of plasma sample was mixed with 20 μl of 0.31 M phosphate buffer, pH 7.5, 10 μl of 0.1 mM FAD, 10 μl of 1 mM NADPH, 10 ml of nitrate reductase (10 units/ml), and 30 μl of water in a 96-well plate. The reaction was allowed to proceed for 1 h in the dark. The percent conversion of nitrate to nitrite was 98%. To each sample, 1 μl of lactate dehydrogenase (1500 units/ml) and 10 μl of 100 mM pyruvic acid were added and incubated for 15 min at 37°C. The samples were then mixed with an equivalent volume of Griess reagent and incubated for an additional 10 min at room temperature. Nitrite levels were determined colorimetrically at 550 nm with a Universal microplate reader (Bio-Tek Instruments, Inc., Winooski, VT) and a sodium nitrite standard curve.
Statistical analysis.
The PXR and cyp3a11 mRNA levels were normalized to the Gapdh mRNA level in the same samples. The PXR and cyp3a11 mRNA levels of the control were assigned as 100%. Quantified data from analysis of GSH, TBARS and nitrate plus nitrite concentrations, RT-PCR, and ERND assay were expressed as means ± S.E.M. at each point. Analysis of variance (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 LPS on PXR and Cyp3a11 mRNA Expression and ERND Activity in Fetal Mouse Liver
The PXR and cyp3a11 mRNA levels in fetal liver were determined at 12 h after LPS treatment using RT-PCR. The effects of LPS on PXR and cyp3a11 mRNA expression and ERND activity in fetal mouse liver are presented in Figure 1. Results showed that LPS significantly downregulated PXR and cyp3a11 mRNA levels in a dose-dependent manner. Consistent with downregulation of cyp3a11 mRNA, LPS significantly inhibited ERND activity in fetal liver microsomes.
Effects of PBN on LPS-Induced Downregulation of PXR and Cyp3a11 mRNA and ERND Activity in Fetal Liver
The effects of PBN on LPS-induced downregulation of PXR and cyp3a11 mRNA expression and ERND activity are presented in Figure 2. Results showed that PBN pretreatment and posttreatments significantly attenuated LPS-induced downregulation of PXR and cyp3a11 mRNA levels in fetal liver. In addition, PBN pretreatment and posttreatment also significantly attenuated LPS-induced downregulation of ERND activity in fetal liver.
Effects of PBN on LPS-Induced Lipid Peroxidation in Fetal Liver
Lipid peroxidation was quantified by measuring TBARS levels at 6 h after LPS treatment. As shown in Figure 3, LPS significantly increased TBARS level in fetal liver. Furthermore, PBN pretreatment and posttreatment significantly attenuated LPS-induced increases in TBARS level.
Effects of NAC on LPS-Induced Downregulation of PXR and Cyp3a11 in Fetal Mouse Liver
To evaluate the effects of NAC on LPS-induced downregulation of PXR and cyp3a11 in fetal liver, PXR and cyp3a11 mRNA expression and ERND activity were determined at 12 h after LPS treatment. Results showed that NAC pretreatment significantly attenuated LPS-induced downregulation of PXR and cyp3a11 mRNA levels in fetal liver (Figs. 4A and 4B). In addition, maternal NAC pretreatment significantly attenuated LPS-induced decrease in ERND activity in fetal liver (Fig. 4C).
Effects of NAC on LPS-Induced GSH Depletion and Lipid Peroxidation in Fetal Liver
The effects of NAC on LPS-induced GSH depletion and lipid peroxidation are shown in Figure 5. As expected, maternal LPS exposure significantly decreased GSH content in fetal liver. On the one hand, NAC pretreatment attenuated LPS-induced GSH depletion in fetal liver. On the other hand, maternal LPS exposure significantly increased TBARS levels in fetal liver. The NAC pretreatment attenuated LPS-induced lipid peroxidation in fetal liver.
DISCUSSION
CYP3A is a member of the cytochrome P-450 monooxygenase superfamily, which is responsible for the oxidative metabolism of numerous clinically used drugs. The pregnan X receptor is the key transactivator of the CYP3A gene. Several studies have demonstrated that LPS downregulates PXR and cyp3a in adult mouse liver (Beigneux et al., 2002; Sachdeva et al., 2003). In this study, we investigated the effects of maternal LPS exposure on PXR and cyp3a11 expression in fetal mouse liver. Results indicated that LPS significantly inhibited the constitutive expression of PXR and cyp3a11 mRNA in fetal liver in a dose-respondent manner. It has been demonstrated that CYP3A is the major enzyme catalyzing erythromycin N-demethylation in mouse liver (Lan et al., 2000). Thus, ERND activity was used as an indicator of CYP3A expression in this study. The present results indicate that maternal LPS exposure significantly represses ERND activity in fetal liver microsomes.
Reactive oxygen species (ROS) are known to influence the expressions of a number of genes and signal transduction pathways (Allen and Tresini, 2000). Lipopolysaccharide, a potent activator for macrophages, stimulates Kupffer cells to generate ROS, such as (Bautista et al., 1990). In addition, several studies found that LPS increased nitrotyrosine, a marker for NO and ONOO– formation in macrophage-rich organs, such as liver (Bian and Murad, 2001; Ottesen et al., 2001). Our earlier studies showed that ROS are involved in LPS-induced downregulation of PXR and cyp3a11 in adult mouse liver (Xu et al., 2004, 2005). To determine the role of ROS on LPS-induced downregulation of PXR and cyp3a11 in fetal liver, alpha-phenyl-N-t-butylnitrone (PBN), a free radical spin trapping agent, was used to decrease LPS-induced ROS production. As expected, maternal PBN pretreatment and posttreatment significantly inhibited LPS-induced lipid peroxidation in fetal liver, suggesting that PBN was effective in the in vivo trapping of free radicals. Interestingly, LPS-induced downregulation of PXR and cyp3a11 mRNA in fetal liver was significantly attenuated in mice pretreated with PBN. In addition, PBN pretreatment also significantly attenuated LPS-induced downregulation on ERND activity in fetal liver. These results indicate that ROS mediate LPS-induced downregulation of PXR and cyp3a11 in fetal liver.
N-acetylcysteine (NAC) is a GSH precursor and direct antioxidant. Recent study has indicated that NAC protected against fetal death and preterm labor induced by maternal inflammation (Buhimschi et al., 2003). The present study showed that NAC prevented LPS-induced GSH depletion and lipid peroxidation in fetal liver. To investigate the effect of NAC on LPS-induced downregulation of PXR and cyp3a11 in fetal liver, pregnant mice were pretreated with NAC at 30 min before LPS administration, followed by additional dose of NAC at 3 h after LPS treatment. Consistent with its antioxidative effect, NAC significantly attenuated LPS-induced downregulation of PXR and cyp3a11 mRNA levels. This antioxidant also prevented the repressive effect of LPS on ERND activity in fetal liver.
Several studies indicated that nitric oxide (NO) mediates LPS-induced inhibition of cytochrome P450 in adult liver (Kitaichi et al., 1999; Muller et al., 1996). A lot of studies have demonstrated that LPS induced NO production in adult liver and maternal serum (Athanassakis et al., 1999; Hida et al., 2003; Zhang et al., 2000). However, the present study showed that there was no difference in nitrate plus nitrite concentration in fetal liver between the LPS-treated group and the control group (data not shown). This result is in agreement with the earlier work by Casado et al. (1997), in which treatment of rats on GD 21 with lipopolysaccharide (LPS) induced inducible nitric oxide synthase (iNOS) expression in maternal liver but completely failed to elicit this response in the corresponding fetal tissue. Furthermore, the present study found that aminoguanidine, a selective inhibitor of iNOS, has no effect on LPS-induced downregulation of PXR and cyp3a11 mRNA expression and ERND activity in fetal liver (data not shown). These results suggest that LPS-induced downregulation of PXRand cyp3a11 in fetal liver is also independent of NO production.
Numerous studies have demonstrated that pro-inflammatory cytokines, such as TNF-, IL-1, and IL-6, are involved in LPS-induced downregulation of cytochrome P450s (Monshouwer et al., 1996; Sewer and Morgan, 1997). Several studies found that LPS induced TNF-, IL-1, and IL-6 in the maternal serum, the amniotic fluid, and the placenta (Bell et al., 2004; Gayle et al., 2004). Therefore, our results do not exclude the involvement of pro-inflammatory cytokines.
In summary, the present results allow us to reach several conclusions. First, maternal LPS exposure downregulates PXR and cyp3a11 in fetal mouse liver; second, ROS may be involved in LPS-induced downregulation of PXR and cyp3a11 in fetal mouse liver; and third, maternal NAC pretreatment prevents LPS-induced downregulation of PXR and cyp3a11 in fetal liver by counteracting LPS-induced oxidative stress in fetal liver.
ACKNOWLEDGMENTS
The Project was supported by National Natural Science Foundation of China (30371667) and Anhui Provincial Natural Science Foundation (050430714). Conflict of interest: none.
REFERENCES
Allen, R. G., and Tresini, M. (2000). Oxidative stress and gene regulation. Free Radic. Biol. Med. 28, 463–499.
Athanassakis, I., Aifantis, I., Ranella, A., Giouremou, K., and Vassiliadis, S. (1999). Inhibition of nitric oxide production rescues LPS-induced fetal abortion in mice. Nitric Oxide 3, 216–224.
Balasubramaniyan, N., Shahid, M., Suchy, F. J., and Ananthanarayanan, M. (2005). Multiple mechanisms of ontogenic regulation of nuclear receptors during rat liver development. Am. J. Physiol. Gastrointest. Liver Physiol. 288, G251–260.
Bautista, A. P., Meszaros, K., Bojta, J., and Spitzer, J. J. (1990). Superoxide anion generation in the liver during the early stage of endotoxemia in rats. J. Leukoc. Biol. 48, 123–128.
Beigneux, A. P., Moser, A. H., Shigenaga, J. K., Grunfeld, C., and Feingold, K. R. (2002). Reduction in cytochrome P-450 enzyme expression is associated with repression of CAR (constitutive androstane receptor) and PXR (pregnane X receptor) in mouse liver during the acute phase response. Biochem. Biophys. Res. Commun. 293, 145–149.
Bell, M. J., Hallenbeck, J. M., and Gallo, V. (2004). Determining the fetal inflammatory response in an experimental model of intrauterine inflammation in rats. Pediatr. Res. 56, 541–546.
Bertilsson, G., Heidrich, J., Svensson, K., Asman, M., Jendeberg, L., Sydow-Backman, M., Ohlsson, R., Postlind, H., Blomquist, P., and Berkenstam, A. (1998). Identification of a human nuclear receptor defines a new signaling pathway for CYP3A induction. Proc. Natl. Acad. Sci. U. S. A. 95, 12208–12213.
Bian, K., and Murad, F. (2001). Diversity of endotoxin-induced nitrotyrosine formation in macrophage-endothelium-rich organs. Free Radic. Biol. Med. 31, 421–429.
Buhimschi, I. A., Buhimschi, C. S., and Weiner, C. P. (2003). Protective effect of N-acetylcysteine against fetal death and preterm labor induced by maternal inflammation. Am. J. Obstet. Gynecol. 188, 203–208.
Casado, M., Velasco, M., Bosca, L., and Martin-Sanz, P. (1997). Absence of NO synthase type II expression in fetal liver from pregnant rats under septic shock conditions. Hepatology 25, 1406–1411.
Choudhary, D., Jansson, I., Schenkman, J. B., Sarfarazi, and M., Stoilov, I. (2003). Comparative expression profiling of 40 mouse cytochrome P450 genes in embryonic and adult tissues. Arch. Biochem. Biophys. 414, 91–100.
Collins, J. G., Smith, M. A., Arnold, R. R., and Offenbacher, S. (1994). Effects of Escherichia coli and Porphyromonas gingivalis lipopolysaccharide on pregnancy outcome in the golden hamster. Infect. Immun. 62, 4652–4655.
de Wildt, S. N., Kearns, G. L., Leeder, J. S., and van den Anker, J. N. (1999). Cytochrome P450 3A: Ontogeny and drug disposition. Clin. Pharmacokinet. 37, 485–505.
Dzierzak, E., and Medvinsky, A. (1995). Mouse embryonic hematopoiesis. Trends Genet. 11, 359–366.
Gayle, D. A., Beloosesky, R., Desai, M., Amidi, F., Nunez, S. E., and Ross, M. G. (2004). Maternal LPS induces cytokines in the amniotic fluid and corticotropin releasing hormone in the fetal rat brain. Am. J. Physiol. Regul. Integr. Comp. Physiol. 286, R1024–1029.
Griffith, O. W. (1980). Determination of glutathione and glutathione disulfide using glutathione reductase and 2-vinylpyridine. Anal. Biochem. 106, 207–212.
Hardy, R. R., and Hayakawa, K. (2001). B cell development pathways. Annu. Rev. Immunol. 19, 595–621.
Haugen, D. A., and Coon, M. J. (1976). Properties of electrophoretically homogeneous phenobarbital-inducible and beta-naphthoflavone-inducible forms of liver microsomal cytochrome P-450. J. Biol. Chem. 251, 7929–7939.
Hida, A. I., Kawabata, T., Minamiyama, Y., Mizote, A., and Okada, S. (2003). Saccharated colloidal iron enhances lipopolysaccharide-induced nitric oxide production in vivo. Free Radic. Biol. Med. 34, 1426–1434.
Hines, R. N., and McCarver, D. G. (2002). The ontogeny of human drug-metabolizing enzymes: phase I oxidative enzymes. J. Pharmacol. Exp. Ther. 300, 355–360.
Hulla, J. E., and Juchau, M. R. (1989). Developmental aspects of P450IIIA: Prenatal activity and inducibility. Drug Metab. Rev. 20, 765–779.
Kamiya, A., Inoue, Y., and Gonzalez, F. J. (2003). Role of the hepatocyte nuclear factor 4alpha in control of the pregnane X receptor during fetal liver development. Hepatology 37, 1375–1384.
Kitaichi, K., Wang, L., Takagi, K., Iwase, M., Shibata, E., Nadai, M., and Hasegawa, T. (1999). Decreased antipyrine clearance following endotoxin administration: In vivo evidence of the role of nitric oxide. Antimicrob Agents Chemother. 43, 2697–2701.
Kliewer, S. A., Moore, J. T., Wade, L., Staudinger, J. L., Watson, M. A., Jones, S. A., McKee, D. D., Oliver, B. B., Willson, T. M., Zetterstrom, R. H., et al. (1998). An orphan nuclear receptor activated by pregnanes defines a novel steroid signaling pathway. Cell 92, 73–82.
Krauer, B., and Dayer, P. (1991). Fetal drug metabolism and its possible clinical implications. Clin. Pharmacokinet. 21, 70–80.
Lan, L. B., Dalton, J. T., and Schuetz, E. G. (2000). Mdr1 limits CYP3A metabolism in vivo. Mol. Pharmacol. 58, 863–869.
Lehmann, J. M., McKee, D. D., Watson, M. A., Willson, T. M., Moore, J. T., and Kliewer, S. A. (1998). The human orphan nuclear receptor PXR is activated by compounds that regulate CYP3A4 gene expression and cause drug interactions. J. Clin. Invest. 102, 1016–1023.
Li, J., Ning, G., and Duncan, S. A. (2000). Mammalian hepatocyte differentiation requires the transcription factor HNF-4alpha. Genes Dev. 14, 464–474.
Li-Masters, T. and Morgan, E. T. (2001). Effects of bacterial lipopolysaccharide on phenobarbital-induced CYP2B expression in mice. Drug Metab. Dispos. 29, 252–257.
Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275.
Mathurin, P., Deng, Q. G., Keshavarzian, A., Choudhary, S., Holmes, E. W., and Tsukamoto, H. (2000). Exacerbation of alcoholic liver injury by enteral endotoxin in rats. Hepatology 32, 1008–1017.
Miki, Y., Suzuki, T., Tazawa, C., Blumberg, B., and Sasano, H. (2005). Steroid and xenobiotic receptor (SXR), cytochrome P450 3A4 and multidrug resistance gene 1 in human adult and fetal tissues. Mol. Cell. Endocrinol. 231, 75–85.
Monshouwer, M., McLellan, R. A., Delaporte, E., Witkamp, R. F., van Miert, A. S., and Renton, K. W. (1996). Differential effect of pentoxifylline on lipopolysaccharide-induced downregulation of cytochrome P450. Biochem. Pharmacol. 52, 1195–1200.
Morgan, E. T. (1997). Regulation of cytochromes P450 during inflammation and infection. Drug Metab. Rev. 29, 1129–1188.
Morgan, E. T. (2000). Regulation of cytochrome p450 by inflammatory mediators: Why and how Drug Metab. Dispos. 29, 207–212.
Muller, C. M., Scierka, A., Stiller, R. L., Kim, Y. M., Cook, D. R., Lancaster, J. R., Jr., Buffington, C. W., and Watkins, W. D. (1996). Nitric oxide mediates hepatic cytochrome P450 dysfunction induced by endotoxin. Anesthesiology 84, 1435–1442.
Ohkawa, H., Ohishi, N., and Yagi, K. (1979). Assay for lipid peroxidation in animal tissues by thiobarbituric acid reaction. Anal. Biochem. 44, 276–278.
O'Sullivan, A. M., Dore, C. J., Boyle, S., Coid, C. R., and Johnson, A. P. (1988). The effect of campylobacter lipopolysaccharide on fetal development in the mouse. J. Med. Microbiol. 26, 101–105.
Ottesen, L. H., Harry, D., Frost, M., Davies, S., Khan, K., Halliwell, B., and Moore, K. (2001). Increased formation of S-nitrothiols and nitrotyrosine in cirrhotic rats during endotoxemia. Free Radic. Biol. Med. 31, 790–798.
Rich, K. J., and Boobis, A. R. (1997). Expression and inducibility of P450 enzymes during liver ontogeny. Microsc. Res. Tech. 39, 424–435.
Sachdeva, K., Yan, B., and Chichester, C. O. (2003). Lipopolysaccharide and cecal ligation/puncture differentially affect the subcellular distribution of the pregnane X receptor but consistently cause suppression of its target genes CYP3A. Shock 19, 469–474.
Sewer, M. B., Barclay, T. B., and Morgan, E. T. (1998). Down-regulation of cytochrome P450 mRNAs and proteins in mice lacking a functional NOS2 gene. Mol. Pharmacol. 54, 273–279.
Sewer, M. B., and Morgan, E. T. (1997). Nitric oxide-independent suppression of P450 2C11 expression by interleukin-1beta and endotoxin in primary rat hepatocytes. Biochem. Pharmacol. 54, 729–737.
Stevens, J. C., Hines, R. N., Gu, C., Koukouritaki, S. B., Manro, J. R., Tandler, P. J., and Zaya, M. J. (2003). Developmental expression of the major human hepatic CYP3A enzymes. J. Pharmacol. Exp. Ther. 307, 573–582.
Thummel, K. E, and Wilkinson, G. R. (1998). In vitro and in vivo drug interactions involving human CYP3A. Annu. Rev. Pharmacol. Toxicol. 38, 389–430.
Tracey, W. R., Tse, J., and Carter, G. (1995). Lipopolysaccharide-induced changes in plasma nitrite and nitrate concentrations in rats and mice: Pharmacological evaluation of nitric oxide synthase inhibitors. J. Pharmacol. Exp. Ther. 272, 1011–1015.
Werringloer, J. (1978). Assay of fomaldehyde generated during microsomal oxidation reactions. Methods Enzymol. 52, 297–302.
Xu, D. X., Wei, W., Sun, M. F., Wei, L. Z., and Wang, J. P. (2005). Melatonin attenuates lipopolysaccharide-induced down-regulation of pregnane X receptor and its target gene CYP3A in mouse liver. J. Pineal Res. 38, 27–34.
Xu, D. X., Wei, W., Sun, M. F., Wu, C. Y., Wang, J. P., Wei, L. Z., and Zhou, C. F. (2004). Kupffer cells and ROS partially mediate LPS-induced down-regulation of nuclear receptor pregnane X receptor and its target gene CYP3A in mouse liver. Free Radic. Biol. Med. 37, 10–22.
Zhang, C., Walker, L. M., Hinson, J. A., and Mayeux, P. R. (2000). Oxidant stress in rat liver after lipopolysaccharide administration: Effect of inducible nitric-oxide synthase inhibition. J. Pharmacol. Exp. Ther. 293, 968–972.(De-Xiang Xu, Yuan-Hua Che)
Institute of Clinical Pharmacology, Anhui Medical University, Hefei, 230032, P.R. China
Key Laboratory of Anti-inflammatory and Immunopharmacology of Anhui Province, Hefei, 230032, P.R. China
ABSTRACT
The pregnane X receptor (PXR) is a member of the nuclear receptor superfamily that regulates cytochrome P450 3A (CYP3A) gene transcription in a ligand-dependent manner. Lipopolysaccharide (LPS)-induced downregulation on PXR and cyp3a11 in adult mouse liver has been well characterized. In this study, we investigated the effects of maternal LPS exposure on PXR and cyp3a11 expression in fetal mouse liver. Pregnant ICR mice were injected intraperitoneally with different doses of LPS (0.10.5 mg/kg) on gestational day (GD) 17. PXR and cyp3a11 mRNA levels were determined using RT-PCR. Erythromycin N-demethylase (ERND) activity was used as an indicator of CYP3A expression in this study. Results showed that LPS significantly downregulated PXR and cyp3a11 mRNA levels and ERND activity in fetal liver in a dose-dependent manner. LPS-induced downregulation of PXR and cyp3a11 mRNA expression and ERND activity was attenuated after pregnant mice were pretreated with alpha-phenyl-N-t-butylnitrone (PBN), a free radical spin trapping agent. Additional experiment revealed that LPS significantly increased lipid peroxidation in fetal liver, which was also attenuated by PBN pretreatment. Furthermore, LPS-induced downregulation of PXR and cyp3a11 mRNA expression and ERND activity was prevented by maternal pretreatment with N-acetylcysteine (NAC). Maternal pretreatment with NAC also inhibited LPS-initiated lipid peroxidation and GSH depletion in fetal liver. However, maternal LPS treatment did not affect nitrite plus nitrate concentration in fetal liver. Correspondingly, aminoguanidine, a selective inhibitor of inducible nitric oxide synthase (iNOS), has no effect on LPS-induced downregulation of PXR and cyp3a11 expression and ERND activity in fetal liver. These results indicated that maternal LPS exposure downregulates PXR and cyp3a11 in fetal mouse liver. Reactive oxygen species (ROS) may be involved in LPS-induced downregulation of PXR and cyp3a11 in fetal mouse liver.
Key Words: lipopolysaccharide; fetal liver; pregnane X receptor; cytochrome P450 3A; reactive oxygen species.
INTRODUCTION
The liver is the major organ of amino acid and lipid metabolism, gluconeogenesis, synthesis of serum proteins, and xenobiotic detoxification. The fetal liver functions as the major hematopoietic organ in the mid- to late fetal stage (Dzierzak and Medvinsky, 1995; Hardy and Hayakawa, 2001). With embryonic development, fetal hepatocytes gradually express various types of cytochromes P450 (CYPs) that play a key role in the detoxification of drug or other xenobiotics (de Wildt et al., 1999; Hines and McCarver, 2002; Hulla and Juchau, 1989; Krauer and Dayer, 1991; Rich and Boobis, 1997).
The cytochrome P450 3A (CYP3A) is a member of the cytochrome P-450 monooxygenase superfamily. In humans, CYP3A4 and CYP3A5 gene products account for 30–40% of the total cytochrome P450 in the adult liver, which is responsible for the oxidative metabolism of numerous clinically used drugs and toxicants (Thummel and Wilkinson, 1998). Although CYP3A4 and CYP3A5 in fetal liver are not detectable, fetal hepatocytes express CYP3A7 as early as 50 to 60 days of gestation, with continued significant levels of expression through the perinatal period (Stevens et al., 2003). In mice, cyp3a11 and cyp3a13 are major members of cyp3a subfamily in the adult liver. In the developing mouse embryo, the amount of cyp3a11 and cyp3a13 expression gradually increases with the advancement of embryonic development (Choudhary et al., 2003).
The pregnane X receptor (PXR) is a member of the nuclear receptor superfamily that regulates CYP3A gene transcription in a ligand-dependent manner (Bertilsson et al., 1998; Kliewer et al., 1998; Lehmann et al., 1998). In addition, PXR plays pivotal roles in the regulation of genes contributing to hepatobiliary cholesterol and bile acid homeostasis. Several studies showed that fetal hepatocytes express PXR mRNA (Kamiya et al., 2003; Li et al., 2000; Miki et al., 2005). Furthermore, PXR activity in liver nuclear extracts assayed by gel EMSA exhibit a strict correlation with mRNA levels. Protein levels for PXR also corresponded to the mRNA and functional activity (Balasubramaniyan et al., 2005).
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. 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 (Mathurin et al., 2000). Lipopolysaccharide has been associated with adverse developmental outcome, including intrauterine fetal death (IUFD) and intrauterine growth retardation (IUGR) in animals (Collins et al., 1994; O'Sullivan et al., 1988). On the other hand, numerous studies indicated that inflammation and infection reduce cytochrome P450 levels in various species including human, rat, and mouse (Morgan, 1997, 2000). Lipopolysaccharide-induced downregulation of cyp3a11 has also been demonstrated in a mouse model (Li-Masters and Morgan, 2001; Sewer et al., 1998). Moreover, LPS-induced downregulation of cyp3a is associated with a marked reduction in PXR mRNA and protein levels (Beigneux et al., 2002; Sachdeva et al., 2003). Therefore, the question of whether maternal LPS exposure downregulates PXR and Cyp3a in fetal mouse liver is especially interesting.
In the present study, we investigated the effects of maternal LPS administration on PXR and cyp3a11 expressions in fetal mouse liver. Our results found that LPS downregulates PXR and cyp3a11 in fetal mouse liver. Reactive oxygen species (ROS) may be involved in LPS-induced downregulation of PXR and cyp3a11 in fetal mouse liver. Maternal NAC pretreatment prevents LPS-induced down-regulation of PXR and cyp3a11 in fetal liver by counteracting LPS-induced oxidative stress.
MATERIALS AND METHODS
Chemicals.
Lipopolysaccharide (Escherichia coli LPS, serotype 0127:B8), alpha-phenyl-N-t-butylnitrone (PBN), aminoguanidine (AG), and N-acetylcysteine (NAC) were purchased from Sigma Chemical Co. (St. Louis, MO). All other reagents were from Sigma or as indicated in the specified methods.
Animals and treatments.
The ICR mice (8 10 week-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 1 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 included four separate experiments.
Experiment 1.
To investigate the effects of LPS on PXR and cyp3a11 gene expression in fetal liver, GD 17 ICR mice were injected with different doses of LPS (0.1, 0.2, and 0.5 mg/kg, i.p.). The saline-treated pregnant mice served as controls. Mice were sacrificed at 12 h after LPS treatment for measurement of PXR and cyp3a11 mRNA expression and ERND activity in fetal livers.
Experiment 2.
To investigate the effects of PBN on LPS-induced downregulation of PXR and cyp3a11 gene expression in fetal liver, pregnant mice were divided into four groups randomly. The pregnant mice in the LPS-treated group were injected with 0.2 mg/kg of LPS (i.p.) on GD 17. The pregnant mice in LPS + PBN group were injected with 100 mg/kg of PBN (i.p.) at 30 min before and 3 h after LPS (0.2 mg/kg, i.p.) administration. Saline- and PBN-treated pregnant mice served as controls. Half of the mice were sacrificed at 6 h after LPS treatment for measurement of nitrite plus nitrate and thiobarbituric acid-reactive substance (TBARS) content in fetal livers. The other mice were sacrificed at 12 h after LPS treatment for measurement of PXR and cyp3a11 mRNA expression and ERND activity in fetal livers.
Experiment 3.
To investigate the effects of NAC on LPS-induced downregulation of PXR and cyp3a11 gene expression in fetal liver, pregnant mice were divided into four groups randomly. The pregnant mice in the LPS-treated group were injected with 0.2 mg/kg of LPS (i.p.) on GD 17. Pregnant mice in the LPS + NAC group were injected with NAC (100 mg/kg, i.p.) either at 30 min before or 3 h after LPS (0.2 mg/kg, i.p.) treatment. Saline- and NAC-treated pregnant mice served as controls. Half of the mice were sacrificed at 6 h after LPS treatment for measurements of TBARS and GSH content in fetal livers. The other mice were sacrificed at 12 h after LPS treatment for measurement of PXR and cyp3a11 mRNA expression and ERND activity in fetal livers.
Experiment 4.
To investigate the effects of AG on LPS-induced downregulation of PXR and cyp3a11 gene expression in fetal liver, pregnant mice were divided into four groups randomly. The pregnant mice in the LPS-treated group were injected with 0.2 mg/kg of LPS (i.p.) on GD 17. Pregnant mice in the LPS + AG group were injected with AG (100 mg/kg, i.p.) at 30 min before LPS (0.2 mg/kg, i.p.) treatment. Saline- and AG-treated pregnant mice served as controls. Half of the mice were sacrificed at 6 h after LPS treatment for measurement of nitrite plus nitrate concentration in fetal livers. The other mice were sacrificed at 12 h after LPS treatment for measurement of PXR and cyp3a11 mRNA expression and ERND activity in fetal livers.
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.
Isolation of total RNA and reverse transcriptase (RT).
For each sample, 50 mg of fetal liver tissue was collected. Total cellular RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. RNase-free DNase (Promega, Madison, WI) was used to remove genomic DNA. The integrity and concentration of RNA was determined by measuring absorbance at 260 nm followed by electrophoresis on agarose gels. Total RNA was stored at –80°C. For the synthesis of cDNA, 2.0 μg of total RNA from each sample was resuspended in a 20-μl final volume of reaction buffer, which contained 25 mM Tris · HCl, pH 8.3; 37.5 mM KCl; 10 mM dithiothreitol; 1.5 mM MgCl2; 10 mM of each dNTP and 0.5 mg oligo(dT)15 primer (Promega). After the reaction mixture reached 38°C, 400 units of RT (Promega) was added to each tube and the sample was incubated for 60 min at 38°C. Reverse transcription was stopped by denaturing the enzyme at 95°C.
Polymerase chain reaction (PCR) amplification.
The final PCR mixture contained 2 μl of cDNA, 1x PCR buffer, 1.5 mM MgCl2, 200 μM dNTP mixture, 2 U of Taq DNA polymerase, 1 μM sense and antisense primers, and sterile water to 25 μl. The reaction mixture was covered with mineral oil. Polymerase chain reaction for glyceraldehyde-3-phosphate dehydrogenase (gapdh) was performed on each individual sample as an internal positive-control standard. The following primers were synthesized by Shanghai Sangon Biological Engineering Technology and Service Company (Shanghai, China), according to sequence designs previously described by others (Xu et al., 2004). Gapdh, 5'-GAG GGG CCA TCC ACA GTC TTC-3' and 5'-CAT CAC CAT CTT CCA GGA GCG-3'; PXR, 5'-GCG CGG AGA AGA CGG CAG CAT C-3' and 5'-CCC AGG TTC CCG TTT CCG TGT C-3'cyp3a11, 5'-CTC AAT GGT GTG TAT ATC CCC-3' and 5'-CCG ATG TTC TTA GAC ACT GCC-3'. The sizes of amplified PCR products were 340 bp for gapdh, 254 for PXR, 423 bp for cyp3a11, respectively. Both number of cycles and annealing temperature were optimized for each primer pair. For gapdh, amplification was initiated by 3 min of denaturation at 94°C for 1 cycle, followed by 30 cycles at 94°C for 30 s, 56°C for 30 s, and 72°C for 1 min. For PXR, amplification was initiated by 3 min of denaturation at 94°C for 1 cycle, followed by 45 cycles at 94°C for 30 s, 68°C for 30 s, and 72°C for 1 min. After the last cycle of amplification, samples were incubated for 10 min at 72°C. For cyp3a11, amplification was initiated by 3 min of denaturation at 94°C for 1 cycle, followed by 35 cycles at 94°C for 30 s, 56°C for 30 s, and 72°C for 1 min. A final extension of 72°C for 10 min was included. The amplified PCR products were subjected to electrophoresis at 75 V through 1.5% agarose gels (Sigma) for 45 min. The pBR322 DNA digested with Alu I was used for molecular markers (MBI Fermentas). Agarose gels were stained with 0.5 mg/ml ethidium bromide (Sigma,) TBE buffer.
Preparation of liver microsomes.
Microsomes were isolated from livers by differential centrifugation (Haugen and Coon, 1976). All procedures were conducted at 4°C. Tissue was homogenized in four volumes of Tris/chloride buffer, pH 7.4, containing 150 mM potassium chloride and 1 mM EDTA, with a Polytron homogenizer and centrifuged at 10,000 x g for 20 min. The supernatant was collected and centrifuged at 211,000 x g for 40 min. The microsomal pellet was resuspended and washed in sodium pyrophosphate buffer, pH 7.4, containing 1 mM EDTA and centrifuged again at 211,000 x g for 40 min at 4°C. The washed microsomal pellet was resuspended in a trischloride buffer, pH 7.4, containing 20% glycerol, with a ground glass tissue grinder and stored at –80°C. Protein concentrations of microsome samples were measured according to the method of Lowry et al. (1951), using bovine serum albumin as a standard.
CYP3A catalytic activity.
Erythromycin N-demethylase (ERND) was used as an indicator of CYP3A catalytic activity in this study. The ERND was measured according to the method of Werringloer (1978) with a 45-min incubation containing 4 mM erythromycin in the presence of 0.5 mM NADPH and 0.4 mg of microsomal protein in a total assay volume of 1 ml. The rate of formaldehyde formation was determined spectrophotometrically at 412 nm using the Nash reagent. Measurement for ERND catalytic activity was repeated twice for three separately prepared liver microsome samples.
Determination of glutathione content.
The glutathione (GSH) level was determined by the method of Griffith (1980). Proteins of 0.4 ml tissue 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. Then 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, and 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. Glutathione values were expressed as nanomoles per milligram of (nm · mg–1 protein. Protein content was measured according to the method of Lowry et al. (1951).
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 the solution was 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.
Analysis of nitrite plus nitrate concentration in fetal liver.
The stable end products of L-arginine–dependent nitric oxide synthesis, nitrate plus nitrite, were measured in fetal liver using a colorimetric method based on the Griess reaction (Tracey et al., 1995). Briefly, aliquots of homogenates were added to 35% sulfosalicylic acid and vortexed every 5 min for 30 min to deproteinize samples. The samples were then centrifuged at 10, 000 x g at 4°C for 15 min. An aliquot of the supernatant was taken for nitrite and nitrate analysis. Then 20 μl of plasma sample was mixed with 20 μl of 0.31 M phosphate buffer, pH 7.5, 10 μl of 0.1 mM FAD, 10 μl of 1 mM NADPH, 10 ml of nitrate reductase (10 units/ml), and 30 μl of water in a 96-well plate. The reaction was allowed to proceed for 1 h in the dark. The percent conversion of nitrate to nitrite was 98%. To each sample, 1 μl of lactate dehydrogenase (1500 units/ml) and 10 μl of 100 mM pyruvic acid were added and incubated for 15 min at 37°C. The samples were then mixed with an equivalent volume of Griess reagent and incubated for an additional 10 min at room temperature. Nitrite levels were determined colorimetrically at 550 nm with a Universal microplate reader (Bio-Tek Instruments, Inc., Winooski, VT) and a sodium nitrite standard curve.
Statistical analysis.
The PXR and cyp3a11 mRNA levels were normalized to the Gapdh mRNA level in the same samples. The PXR and cyp3a11 mRNA levels of the control were assigned as 100%. Quantified data from analysis of GSH, TBARS and nitrate plus nitrite concentrations, RT-PCR, and ERND assay were expressed as means ± S.E.M. at each point. Analysis of variance (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 LPS on PXR and Cyp3a11 mRNA Expression and ERND Activity in Fetal Mouse Liver
The PXR and cyp3a11 mRNA levels in fetal liver were determined at 12 h after LPS treatment using RT-PCR. The effects of LPS on PXR and cyp3a11 mRNA expression and ERND activity in fetal mouse liver are presented in Figure 1. Results showed that LPS significantly downregulated PXR and cyp3a11 mRNA levels in a dose-dependent manner. Consistent with downregulation of cyp3a11 mRNA, LPS significantly inhibited ERND activity in fetal liver microsomes.
Effects of PBN on LPS-Induced Downregulation of PXR and Cyp3a11 mRNA and ERND Activity in Fetal Liver
The effects of PBN on LPS-induced downregulation of PXR and cyp3a11 mRNA expression and ERND activity are presented in Figure 2. Results showed that PBN pretreatment and posttreatments significantly attenuated LPS-induced downregulation of PXR and cyp3a11 mRNA levels in fetal liver. In addition, PBN pretreatment and posttreatment also significantly attenuated LPS-induced downregulation of ERND activity in fetal liver.
Effects of PBN on LPS-Induced Lipid Peroxidation in Fetal Liver
Lipid peroxidation was quantified by measuring TBARS levels at 6 h after LPS treatment. As shown in Figure 3, LPS significantly increased TBARS level in fetal liver. Furthermore, PBN pretreatment and posttreatment significantly attenuated LPS-induced increases in TBARS level.
Effects of NAC on LPS-Induced Downregulation of PXR and Cyp3a11 in Fetal Mouse Liver
To evaluate the effects of NAC on LPS-induced downregulation of PXR and cyp3a11 in fetal liver, PXR and cyp3a11 mRNA expression and ERND activity were determined at 12 h after LPS treatment. Results showed that NAC pretreatment significantly attenuated LPS-induced downregulation of PXR and cyp3a11 mRNA levels in fetal liver (Figs. 4A and 4B). In addition, maternal NAC pretreatment significantly attenuated LPS-induced decrease in ERND activity in fetal liver (Fig. 4C).
Effects of NAC on LPS-Induced GSH Depletion and Lipid Peroxidation in Fetal Liver
The effects of NAC on LPS-induced GSH depletion and lipid peroxidation are shown in Figure 5. As expected, maternal LPS exposure significantly decreased GSH content in fetal liver. On the one hand, NAC pretreatment attenuated LPS-induced GSH depletion in fetal liver. On the other hand, maternal LPS exposure significantly increased TBARS levels in fetal liver. The NAC pretreatment attenuated LPS-induced lipid peroxidation in fetal liver.
DISCUSSION
CYP3A is a member of the cytochrome P-450 monooxygenase superfamily, which is responsible for the oxidative metabolism of numerous clinically used drugs. The pregnan X receptor is the key transactivator of the CYP3A gene. Several studies have demonstrated that LPS downregulates PXR and cyp3a in adult mouse liver (Beigneux et al., 2002; Sachdeva et al., 2003). In this study, we investigated the effects of maternal LPS exposure on PXR and cyp3a11 expression in fetal mouse liver. Results indicated that LPS significantly inhibited the constitutive expression of PXR and cyp3a11 mRNA in fetal liver in a dose-respondent manner. It has been demonstrated that CYP3A is the major enzyme catalyzing erythromycin N-demethylation in mouse liver (Lan et al., 2000). Thus, ERND activity was used as an indicator of CYP3A expression in this study. The present results indicate that maternal LPS exposure significantly represses ERND activity in fetal liver microsomes.
Reactive oxygen species (ROS) are known to influence the expressions of a number of genes and signal transduction pathways (Allen and Tresini, 2000). Lipopolysaccharide, a potent activator for macrophages, stimulates Kupffer cells to generate ROS, such as (Bautista et al., 1990). In addition, several studies found that LPS increased nitrotyrosine, a marker for NO and ONOO– formation in macrophage-rich organs, such as liver (Bian and Murad, 2001; Ottesen et al., 2001). Our earlier studies showed that ROS are involved in LPS-induced downregulation of PXR and cyp3a11 in adult mouse liver (Xu et al., 2004, 2005). To determine the role of ROS on LPS-induced downregulation of PXR and cyp3a11 in fetal liver, alpha-phenyl-N-t-butylnitrone (PBN), a free radical spin trapping agent, was used to decrease LPS-induced ROS production. As expected, maternal PBN pretreatment and posttreatment significantly inhibited LPS-induced lipid peroxidation in fetal liver, suggesting that PBN was effective in the in vivo trapping of free radicals. Interestingly, LPS-induced downregulation of PXR and cyp3a11 mRNA in fetal liver was significantly attenuated in mice pretreated with PBN. In addition, PBN pretreatment also significantly attenuated LPS-induced downregulation on ERND activity in fetal liver. These results indicate that ROS mediate LPS-induced downregulation of PXR and cyp3a11 in fetal liver.
N-acetylcysteine (NAC) is a GSH precursor and direct antioxidant. Recent study has indicated that NAC protected against fetal death and preterm labor induced by maternal inflammation (Buhimschi et al., 2003). The present study showed that NAC prevented LPS-induced GSH depletion and lipid peroxidation in fetal liver. To investigate the effect of NAC on LPS-induced downregulation of PXR and cyp3a11 in fetal liver, pregnant mice were pretreated with NAC at 30 min before LPS administration, followed by additional dose of NAC at 3 h after LPS treatment. Consistent with its antioxidative effect, NAC significantly attenuated LPS-induced downregulation of PXR and cyp3a11 mRNA levels. This antioxidant also prevented the repressive effect of LPS on ERND activity in fetal liver.
Several studies indicated that nitric oxide (NO) mediates LPS-induced inhibition of cytochrome P450 in adult liver (Kitaichi et al., 1999; Muller et al., 1996). A lot of studies have demonstrated that LPS induced NO production in adult liver and maternal serum (Athanassakis et al., 1999; Hida et al., 2003; Zhang et al., 2000). However, the present study showed that there was no difference in nitrate plus nitrite concentration in fetal liver between the LPS-treated group and the control group (data not shown). This result is in agreement with the earlier work by Casado et al. (1997), in which treatment of rats on GD 21 with lipopolysaccharide (LPS) induced inducible nitric oxide synthase (iNOS) expression in maternal liver but completely failed to elicit this response in the corresponding fetal tissue. Furthermore, the present study found that aminoguanidine, a selective inhibitor of iNOS, has no effect on LPS-induced downregulation of PXR and cyp3a11 mRNA expression and ERND activity in fetal liver (data not shown). These results suggest that LPS-induced downregulation of PXRand cyp3a11 in fetal liver is also independent of NO production.
Numerous studies have demonstrated that pro-inflammatory cytokines, such as TNF-, IL-1, and IL-6, are involved in LPS-induced downregulation of cytochrome P450s (Monshouwer et al., 1996; Sewer and Morgan, 1997). Several studies found that LPS induced TNF-, IL-1, and IL-6 in the maternal serum, the amniotic fluid, and the placenta (Bell et al., 2004; Gayle et al., 2004). Therefore, our results do not exclude the involvement of pro-inflammatory cytokines.
In summary, the present results allow us to reach several conclusions. First, maternal LPS exposure downregulates PXR and cyp3a11 in fetal mouse liver; second, ROS may be involved in LPS-induced downregulation of PXR and cyp3a11 in fetal mouse liver; and third, maternal NAC pretreatment prevents LPS-induced downregulation of PXR and cyp3a11 in fetal liver by counteracting LPS-induced oxidative stress in fetal liver.
ACKNOWLEDGMENTS
The Project was supported by National Natural Science Foundation of China (30371667) and Anhui Provincial Natural Science Foundation (050430714). Conflict of interest: none.
REFERENCES
Allen, R. G., and Tresini, M. (2000). Oxidative stress and gene regulation. Free Radic. Biol. Med. 28, 463–499.
Athanassakis, I., Aifantis, I., Ranella, A., Giouremou, K., and Vassiliadis, S. (1999). Inhibition of nitric oxide production rescues LPS-induced fetal abortion in mice. Nitric Oxide 3, 216–224.
Balasubramaniyan, N., Shahid, M., Suchy, F. J., and Ananthanarayanan, M. (2005). Multiple mechanisms of ontogenic regulation of nuclear receptors during rat liver development. Am. J. Physiol. Gastrointest. Liver Physiol. 288, G251–260.
Bautista, A. P., Meszaros, K., Bojta, J., and Spitzer, J. J. (1990). Superoxide anion generation in the liver during the early stage of endotoxemia in rats. J. Leukoc. Biol. 48, 123–128.
Beigneux, A. P., Moser, A. H., Shigenaga, J. K., Grunfeld, C., and Feingold, K. R. (2002). Reduction in cytochrome P-450 enzyme expression is associated with repression of CAR (constitutive androstane receptor) and PXR (pregnane X receptor) in mouse liver during the acute phase response. Biochem. Biophys. Res. Commun. 293, 145–149.
Bell, M. J., Hallenbeck, J. M., and Gallo, V. (2004). Determining the fetal inflammatory response in an experimental model of intrauterine inflammation in rats. Pediatr. Res. 56, 541–546.
Bertilsson, G., Heidrich, J., Svensson, K., Asman, M., Jendeberg, L., Sydow-Backman, M., Ohlsson, R., Postlind, H., Blomquist, P., and Berkenstam, A. (1998). Identification of a human nuclear receptor defines a new signaling pathway for CYP3A induction. Proc. Natl. Acad. Sci. U. S. A. 95, 12208–12213.
Bian, K., and Murad, F. (2001). Diversity of endotoxin-induced nitrotyrosine formation in macrophage-endothelium-rich organs. Free Radic. Biol. Med. 31, 421–429.
Buhimschi, I. A., Buhimschi, C. S., and Weiner, C. P. (2003). Protective effect of N-acetylcysteine against fetal death and preterm labor induced by maternal inflammation. Am. J. Obstet. Gynecol. 188, 203–208.
Casado, M., Velasco, M., Bosca, L., and Martin-Sanz, P. (1997). Absence of NO synthase type II expression in fetal liver from pregnant rats under septic shock conditions. Hepatology 25, 1406–1411.
Choudhary, D., Jansson, I., Schenkman, J. B., Sarfarazi, and M., Stoilov, I. (2003). Comparative expression profiling of 40 mouse cytochrome P450 genes in embryonic and adult tissues. Arch. Biochem. Biophys. 414, 91–100.
Collins, J. G., Smith, M. A., Arnold, R. R., and Offenbacher, S. (1994). Effects of Escherichia coli and Porphyromonas gingivalis lipopolysaccharide on pregnancy outcome in the golden hamster. Infect. Immun. 62, 4652–4655.
de Wildt, S. N., Kearns, G. L., Leeder, J. S., and van den Anker, J. N. (1999). Cytochrome P450 3A: Ontogeny and drug disposition. Clin. Pharmacokinet. 37, 485–505.
Dzierzak, E., and Medvinsky, A. (1995). Mouse embryonic hematopoiesis. Trends Genet. 11, 359–366.
Gayle, D. A., Beloosesky, R., Desai, M., Amidi, F., Nunez, S. E., and Ross, M. G. (2004). Maternal LPS induces cytokines in the amniotic fluid and corticotropin releasing hormone in the fetal rat brain. Am. J. Physiol. Regul. Integr. Comp. Physiol. 286, R1024–1029.
Griffith, O. W. (1980). Determination of glutathione and glutathione disulfide using glutathione reductase and 2-vinylpyridine. Anal. Biochem. 106, 207–212.
Hardy, R. R., and Hayakawa, K. (2001). B cell development pathways. Annu. Rev. Immunol. 19, 595–621.
Haugen, D. A., and Coon, M. J. (1976). Properties of electrophoretically homogeneous phenobarbital-inducible and beta-naphthoflavone-inducible forms of liver microsomal cytochrome P-450. J. Biol. Chem. 251, 7929–7939.
Hida, A. I., Kawabata, T., Minamiyama, Y., Mizote, A., and Okada, S. (2003). Saccharated colloidal iron enhances lipopolysaccharide-induced nitric oxide production in vivo. Free Radic. Biol. Med. 34, 1426–1434.
Hines, R. N., and McCarver, D. G. (2002). The ontogeny of human drug-metabolizing enzymes: phase I oxidative enzymes. J. Pharmacol. Exp. Ther. 300, 355–360.
Hulla, J. E., and Juchau, M. R. (1989). Developmental aspects of P450IIIA: Prenatal activity and inducibility. Drug Metab. Rev. 20, 765–779.
Kamiya, A., Inoue, Y., and Gonzalez, F. J. (2003). Role of the hepatocyte nuclear factor 4alpha in control of the pregnane X receptor during fetal liver development. Hepatology 37, 1375–1384.
Kitaichi, K., Wang, L., Takagi, K., Iwase, M., Shibata, E., Nadai, M., and Hasegawa, T. (1999). Decreased antipyrine clearance following endotoxin administration: In vivo evidence of the role of nitric oxide. Antimicrob Agents Chemother. 43, 2697–2701.
Kliewer, S. A., Moore, J. T., Wade, L., Staudinger, J. L., Watson, M. A., Jones, S. A., McKee, D. D., Oliver, B. B., Willson, T. M., Zetterstrom, R. H., et al. (1998). An orphan nuclear receptor activated by pregnanes defines a novel steroid signaling pathway. Cell 92, 73–82.
Krauer, B., and Dayer, P. (1991). Fetal drug metabolism and its possible clinical implications. Clin. Pharmacokinet. 21, 70–80.
Lan, L. B., Dalton, J. T., and Schuetz, E. G. (2000). Mdr1 limits CYP3A metabolism in vivo. Mol. Pharmacol. 58, 863–869.
Lehmann, J. M., McKee, D. D., Watson, M. A., Willson, T. M., Moore, J. T., and Kliewer, S. A. (1998). The human orphan nuclear receptor PXR is activated by compounds that regulate CYP3A4 gene expression and cause drug interactions. J. Clin. Invest. 102, 1016–1023.
Li, J., Ning, G., and Duncan, S. A. (2000). Mammalian hepatocyte differentiation requires the transcription factor HNF-4alpha. Genes Dev. 14, 464–474.
Li-Masters, T. and Morgan, E. T. (2001). Effects of bacterial lipopolysaccharide on phenobarbital-induced CYP2B expression in mice. Drug Metab. Dispos. 29, 252–257.
Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275.
Mathurin, P., Deng, Q. G., Keshavarzian, A., Choudhary, S., Holmes, E. W., and Tsukamoto, H. (2000). Exacerbation of alcoholic liver injury by enteral endotoxin in rats. Hepatology 32, 1008–1017.
Miki, Y., Suzuki, T., Tazawa, C., Blumberg, B., and Sasano, H. (2005). Steroid and xenobiotic receptor (SXR), cytochrome P450 3A4 and multidrug resistance gene 1 in human adult and fetal tissues. Mol. Cell. Endocrinol. 231, 75–85.
Monshouwer, M., McLellan, R. A., Delaporte, E., Witkamp, R. F., van Miert, A. S., and Renton, K. W. (1996). Differential effect of pentoxifylline on lipopolysaccharide-induced downregulation of cytochrome P450. Biochem. Pharmacol. 52, 1195–1200.
Morgan, E. T. (1997). Regulation of cytochromes P450 during inflammation and infection. Drug Metab. Rev. 29, 1129–1188.
Morgan, E. T. (2000). Regulation of cytochrome p450 by inflammatory mediators: Why and how Drug Metab. Dispos. 29, 207–212.
Muller, C. M., Scierka, A., Stiller, R. L., Kim, Y. M., Cook, D. R., Lancaster, J. R., Jr., Buffington, C. W., and Watkins, W. D. (1996). Nitric oxide mediates hepatic cytochrome P450 dysfunction induced by endotoxin. Anesthesiology 84, 1435–1442.
Ohkawa, H., Ohishi, N., and Yagi, K. (1979). Assay for lipid peroxidation in animal tissues by thiobarbituric acid reaction. Anal. Biochem. 44, 276–278.
O'Sullivan, A. M., Dore, C. J., Boyle, S., Coid, C. R., and Johnson, A. P. (1988). The effect of campylobacter lipopolysaccharide on fetal development in the mouse. J. Med. Microbiol. 26, 101–105.
Ottesen, L. H., Harry, D., Frost, M., Davies, S., Khan, K., Halliwell, B., and Moore, K. (2001). Increased formation of S-nitrothiols and nitrotyrosine in cirrhotic rats during endotoxemia. Free Radic. Biol. Med. 31, 790–798.
Rich, K. J., and Boobis, A. R. (1997). Expression and inducibility of P450 enzymes during liver ontogeny. Microsc. Res. Tech. 39, 424–435.
Sachdeva, K., Yan, B., and Chichester, C. O. (2003). Lipopolysaccharide and cecal ligation/puncture differentially affect the subcellular distribution of the pregnane X receptor but consistently cause suppression of its target genes CYP3A. Shock 19, 469–474.
Sewer, M. B., Barclay, T. B., and Morgan, E. T. (1998). Down-regulation of cytochrome P450 mRNAs and proteins in mice lacking a functional NOS2 gene. Mol. Pharmacol. 54, 273–279.
Sewer, M. B., and Morgan, E. T. (1997). Nitric oxide-independent suppression of P450 2C11 expression by interleukin-1beta and endotoxin in primary rat hepatocytes. Biochem. Pharmacol. 54, 729–737.
Stevens, J. C., Hines, R. N., Gu, C., Koukouritaki, S. B., Manro, J. R., Tandler, P. J., and Zaya, M. J. (2003). Developmental expression of the major human hepatic CYP3A enzymes. J. Pharmacol. Exp. Ther. 307, 573–582.
Thummel, K. E, and Wilkinson, G. R. (1998). In vitro and in vivo drug interactions involving human CYP3A. Annu. Rev. Pharmacol. Toxicol. 38, 389–430.
Tracey, W. R., Tse, J., and Carter, G. (1995). Lipopolysaccharide-induced changes in plasma nitrite and nitrate concentrations in rats and mice: Pharmacological evaluation of nitric oxide synthase inhibitors. J. Pharmacol. Exp. Ther. 272, 1011–1015.
Werringloer, J. (1978). Assay of fomaldehyde generated during microsomal oxidation reactions. Methods Enzymol. 52, 297–302.
Xu, D. X., Wei, W., Sun, M. F., Wei, L. Z., and Wang, J. P. (2005). Melatonin attenuates lipopolysaccharide-induced down-regulation of pregnane X receptor and its target gene CYP3A in mouse liver. J. Pineal Res. 38, 27–34.
Xu, D. X., Wei, W., Sun, M. F., Wu, C. Y., Wang, J. P., Wei, L. Z., and Zhou, C. F. (2004). Kupffer cells and ROS partially mediate LPS-induced down-regulation of nuclear receptor pregnane X receptor and its target gene CYP3A in mouse liver. Free Radic. Biol. Med. 37, 10–22.
Zhang, C., Walker, L. M., Hinson, J. A., and Mayeux, P. R. (2000). Oxidant stress in rat liver after lipopolysaccharide administration: Effect of inducible nitric-oxide synthase inhibition. J. Pharmacol. Exp. Ther. 293, 968–972.(De-Xiang Xu, Yuan-Hua Che)