Departments of * Pharmacology and Toxicology and Biochemistry and Molecular Biology, National Food Safety and Toxicology Center, Center for Integrative Toxicology, Michigan State University, East Lansing, Michigan 48824; and
Investigative Toxicology, Pharmacia Corporation, Kalamazoo, Michigan
Received February 26, 2004; accepted April 2, 2004
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ABSTRACT |
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Key Words: inflammation; ranitidine; liver; gene array; plasminogen activator inhibitor-1; hypoxia.
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INTRODUCTION |
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Bacterial lipopolysaccharide (endotoxin, LPS) is a potent inflammagen, and exposure to small, noninjurious amounts of LPS renders rats susceptible to liver injury from normally nontoxic doses of hepatotoxic xenobiotics, including some drugs (Roth et al., 2003). For example, rats cotreated with a small, normally nonhepatotoxic dose of LPS and either a small nonhepatotoxic dose of RAN or the phenothiazine antipsychotic drug chlorpromazine develop hepatotoxicity similar to idiosyncratic reactions observed in people treated with either drug (Buchweitz et al., 2002
; Luyendyk et al., 2003
). Halothane, another agent associated with human idiosyncratic hepatotoxicity, causes liver injury in hypoxic rats when they are coexposed to LPS (Lind et al., 1984
). Thus, these studies support the hypothesis that concurrent inflammation might precipitate some idiosyncratic drug reactions. However, mechanisms of toxicity in these animal models are not understood.
LPS recognition by toll-like receptors on Kupffer cells and other inflammatory cells activates signal transduction pathways, leading to cell activation and elaboration of inflammatory mediators (Beutler, 2002). An important component of LPS activation of inflammatory cells such as macrophages is transcriptional activation of numerous genes (Gao et al., 2002
). Many of these gene products such as tumor necrosis factor
(TNF-
) can further activate transcription of other cytokines, adhesion molecules, and neutrophil (PMN) chemokines in other cell types such as endothelial cells (Zhao et al., 2003
). Increased TNF-
mRNA can be detected in livers of rats treated with LPS, and serum TNF-
concentration is markedly increased after LPS exposure (Barton et al., 2001
; Hewett et al., 1993
). Interestingly, TNF-
is important for liver injury from large doses of LPS in a mechanism dependent on PMN activation (Hewett et al., 1993
). Thus, enhanced expression of certain genes after LPS exposure is important for liver injury, and understanding these changes could further elucidate mechanisms of inflammatory tissue injury.
Investigation of gene expression patterns might also identify mechanisms of pathogenesis in models in which modest, noninjurious inflammation potentiates xenobiotic-induced liver injury. For example, cotreatment of rats with a nonhepatotoxic dose of LPS potentiates allyl alcohol (AA)-induced liver injury and results in greater expression of hepatic cyclooxygenase-2 (COX-2) compared to treatment with either agent alone (Ganey et al., 2001). In this model, COX-2 inhibition affords partial protection from liver injury, suggesting that augmented COX-2 gene expression is important for AA/LPS-induced liver injury (Ganey et al., 2001
). In rats cotreated with nonhepatotoxic doses of aflatoxin B1 (AFB1) and LPS, TNF-
mRNA is increased in liver to a level similar to rats treated with LPS alone. However, the serum concentration of TNF-
is significantly greater in AFB1/LPS-treated rats, and this cytokine is causally involved in the potentiation of hepatocellular injury (Barton et al., 2001
). Thus, in other models of LPS-potentiation, a difference in magnitude of gene expression in LPS and LPS/xenobiotic-treated rats may or may not be sufficient to cause liver injury, and post-transcriptional increases in protein or interaction between two genes expressed at otherwise noninjurious levels might contribute to liver injury. The use of gene array technology can facilitate investigation of such interactions. For example, in rats treated with galactosamine and LPS, gene arrays were used to identify changes in gene expression related to inflammation and oxidative stress (Li et al., 2003
).
The purpose of this study was to test the hypothesis that hepatic gene expression changes could distinguish rats treated with LPS, RAN, or LPS/RAN before the onset of significant liver injury in LPS/RAN-treated rats. Genes were segregated based on their patterns of expression and classified based on the nature of their respective gene products. Real-time PCR and ELISA were used to confirm enhanced expression of plasminogen activator inhibitor-1 (PAI-1), and hepatic fibrin deposition was evaluated to determine if the enhanced PAI-1 expression was associated with tissue fibrin deposition as a functional consequence.
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MATERIALS AND METHODS |
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Animals. Male Sprague-Dawley rats (Crl:CD (SD)IGS BR; Charles River, Portage, MI) weighing 250350 g were used for this study. Animals were fed standard chow (Rodent chow/Tek 8640, Harlan Teklad, Madison, WI) and allowed access to water ad libitum. They were allowed to acclimate for 1 week in a 12-h light/dark cycle prior to use.
Experimental protocol. Rats fasted for 24 h were given 44.4 x 106 EU/kg LPS or its saline vehicle, iv. Two h later, 30 mg/kg RAN or sterile phosphate-buffered saline (PBS) vehicle was administered iv. RAN solution was administered at 2 ml/kg body weight at a rate of approximately 0.15 ml/min. Accordingly, rats were treated with either saline and PBS (Veh group), LPS and PBS (LPS group), saline and RAN (RAN group), or LPS and RAN (LPS/RAN group). Three h later, rats were anesthetized with sodium pentobarbital (50 mg/kg, ip) and killed by exsanguination. Blood was allowed to clot at room temperature, and serum was collected and stored at 80°C until use. Slices (34 mm) of the left lateral liver lobe were collected and fixed in 10% neutral buffered formalin. Three 100-mg midlobe pieces of the right medial lobe were flash frozen in liquid nitrogen for RNA isolation. Groups treated with either vehicle, LPS, or RAN contained 3 rats, whereas 4 rats comprised the LPS/RAN group.
Hepatotoxicity assessment. Hepatic parenchymal cell injury was estimated as an increase in serum alanine aminotransferase (ALT) activity. ALT activity was determined spectrophotometrically using Infinity-ALT reagent from Sigma Chemical Co. (St. Louis, MO). Sinusoidal endothelial cell function was estimated using a commercially available, enzyme-linked immunosorbent assay (ELISA) for hyaluronic acid (Corgenix Medical Corporation, Westminster, CO). Formalin-fixed liver samples (34 samples/rat) were embedded in paraffin, sectioned at 5 µm, stained with hematoxylin and eosin (H&E), and examined by light microscopy.
RNA isolation. Total RNA was isolated from snap-frozen liver samples (approximately 100 mg) in accordance with protocols recommended by Affymetrix, Inc. (Santa Clara, CA) for GeneChip experiments. Total RNA was isolated with Trizol reagent (Invitrogen Corp, Carlsbad, CA) according to manufacturer's instructions. Samples were subsequently passed over a Qiagen RNeasy column (Qiagen Corp., Valencia, CA) for further purification. RNA quality and concentration were assessed by absorbance at 280 and 260 nm and by analysis with a 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA).
Affymetrix GeneChip analysis. RNA isolated from each rat was processed and analyzed as described below using separate Affymetrix RG_U34A arrays. Synthesis of double-stranded cDNA from total RNA, synthesis of biotin-labeled cRNA, fragmentation of the cRNA for target preparation, eukaryotic target hybridization, washing, staining, and scanning of the RG_U34A arrays were carried out according to the Affymetrix GeneChip Expression Analysis Technical Manual (701021, rev. 1). Scan analysis was carried out with both the scaling and normalization factors set to 1. For data normalization, the array was treated as an XYZ-dimensional vector, and normalized by dividing each data point by the Cartesian length (magnitude) of the vector, then multiplied by the average of the magnitudes of the XX arrays in the data set. XYZ is the number of data points on the array and XX is the number of arrays. Normalized signal data were imported into the JMP (Release 5.0.1.2, SAS Institute, Inc.) software for principal component analysis.
Data analysis and determination of gene activity. To determine which probesets changed after treatment compared to Veh-treated rats, the following steps were performed. Preliminary filtering was performed using the transcript detection call as described in the Affymetrix Statistical Reference Guide. Probe sets that did not have at least 2 samples in any treatment group with "present" or "marginal" detection calls were eliminated from further analysis. Assessment of gene activity was made by Student's t-test using R software (version 1.7.0, www.r-project.org). Adjustment for multiple comparisons was made using a false discovery rate (FDR) criterion (Benjamini and Hochberg, 1995). The FDR provided an approach capable of decreasing, to a user-determined level, the likelihood of committing a type I error, at the same time as providing a manageable number of probesets for continued analysis. For this study, the 1000 most active probesets compared to the vehicle-treated group were identified for each treatment (i.e., LPS, RAN, or LPS/RAN) by an FDR criterion with
= 0.05. Each probeset that changed relative to its expression in vehicle-treated rats was then assigned to a set defined by the treatment or treatments that produced a change in its activity (i.e., LPS/RAN [LR], L, R, L
R, LR
L, LR
R, LR
L
R, where "
" indicates intersection of sets). The resulting sets were visualized using a Venn diagram (Fig. 3). Student's t-test was used to compare the expression of FDR-active probesets after LPS/RAN treatment, with their expression after treatment with either agent alone. Genes with greater expression in LPS/RAN-treated rats compared with either agent given alone were identified in the LR, LR
L, LR
R and LR
L
R sets (see below). Genes with similar expression in LPS/RAN- and LPS-treated rats, or in LPS/RAN- and RAN-treated rats were identified in the LR
L and LR
L
R or in the LR
R and LR
L
R sets, respectively. Genes expressed to a greater degree in rats treated with LPS alone as compared to rats cotreated with LPS/RAN were identified in the L and LR
L sets.
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Reverse transcription and real-time PCR. RNA quantification was performed on the same samples from the gene array experiment using a Spectramax Microplate Spectrophotometer (Molecular Devices, Sunnyvale, CA). Random priming was performed in a final volume of 12.5 µl using 500 ng of total RNA, 7.5 mM Random Hexamers (Amersham Biosciences, Piscataway, NJ), and 1 mM dNTPs (Invitrogen, Carlsbad, California). RNA was denatured at 65°C for 5 min and chilled on ice. Reverse Transcription (RT) master mix was prepared in a final volume of 12.5 µl with a final concentration of 20 U/ml of Superscript II RNAse H-Reverse Transcriptase, 4 U/ml RNaseOut, 2 mM dithiothreitol, and 1X 1st Strand RT buffer (Invitrogen, Carlsbad, California). Denatured RNA and RT master mix were combined in total volume of 25 µl. The reverse transcriptase reaction was performed at room temperature for 10 min, at 42°C for 60 min, and then at 70°C for 15 min in an MJ Research Thermocycler (MJ Research, Inc., Reno, NV).
The following oligonucleotide primers, designed using Oligo 6 program software (Molecular Biology Insights, Cascade, CO), were used to quantify mRNA levels of the following genes by real-time PCR analyses. Early growth response-1 (egr-1): upper primer-5' TGA ACG CAA GAG GCA TAC CA 3'; lower primer- 5' GAG CCC GGA GAG GAG TAA GAG 3'. B-cell translocation gene-2 (btg-2): upper primer- 5' CCA GCC AGT CAC CCT TAG TG 3'; lower primer- 5' CGG GCA GAG TGT TTG GTA AGT 3'. Plasminogen activator inhibitor-1 (PAI-1): upper primer- 5' AAC CCA GGC CGA CTT CA 3'; lower primer- 5' CAT GCG GGC TGA GAC TAG AAT 3'. Ribosomal protein L19 (Rpl19): upper primer- 5' CTC GAT GCC GGA AGA ACA C 3'; lower primer- 5' CGA GCG TTG GCA GTA CCC 3'. A 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) was used to analyze purities of RNA and PCR products.
Real-time PCR reactions using SYBR Green dye methodology were prepared in a final volume of 25 µl per reaction, with 1 ng of cDNA and 1X SYBR Green PCR Master Mix (PE Applied Biosystems, Foster City, CA). Primer mixture was prepared in 5 µl per reaction with a final concentration of 0.3 µM per primer. SYBR Green real-time PCR was performed using an ABI 7900 (PE Applied Biosystems, Foster City, CA). Relative amounts of target were calculated using the comparative CT method with ribosomal protein L19 (RPL19, RefSeq Accession # NM_031103) as an endogenous reference and a calibrator consisting of RNA pooled from all livers of vehicle-treated rats.
Immunohistochemistry. Liver samples evaluated for fibrin immunohistochemistry were from the 3-h post-treatment time point in a previous study (Luyendyk et al., 2003). A 1 cm3 block of liver cut from the left medial lobe was frozen for 8 min in liquid nitrogen-chilled isopentane, then stored at 80°C until processing. Eight-mm-thick sections of frozen liver were fixed in 10% buffered formalin containing 2% acetic acid for 30 min at room temperature. This fixation protocol solubilizes all fibrinogen and fibrin except for cross-linked fibrin; therefore, only cross-linked fibrin remains in sections of liver (Schnitt et al., 1993
). Sections were blocked with PBS containing 10% horse serum (i.e., blocking solution; Vector Laboratories) for 30 min, and this was followed by incubation overnight at 4°C with goat anti-rat fibrinogen antibody diluted (1:1000, ICN Pharmaceuticals, Aurora, OH) in blocking solution. Next, sections were incubated for three h with donkey anti-goat secondary antibody conjugated to Alexa 594 (1:1000, Molecular Probes, Eugene, OR) in blocking solution for 3 h. Sections were washed three times, 5 min each, with PBS and visualized using a fluorescent microscope. No staining was observed in controls for which the primary or secondary antibody was eliminated from the staining protocol. Liver sections from all treatment groups that were compared morphometrically were stained at the same time.
Quantification of fibrin staining. Fluorescent staining in sections of liver was visualized with an Olympus AX-80T microscope (Olympus, Lake Success, NY). Ten randomly chosen digital images (100X magnification) were captured using a SPOT II camera and SPOT advanced software (Diagnostic Instruments, Sterling Heights, MI). Samples were coded such that the evaluator was not aware of treatment. Each digital image encompassed a total area of 1.4 mm2 and contained several centrilobular and periportal regions. Quantification of immunostaining was performed with Scion Image Beta 4.0.2 (Scion Corporation, Frederick, MD) using the method described by Copple et al. (2002). Ten random fields analyzed for each liver section were averaged and counted as a replicate, i.e., each replicate represents a different rat.
Evaluation of serum PAI-1 concentration. Serum total PAI-1 concentration (i.e., inactive, active, and bound to plasminogen activator) was evaluated using a commercially available ELISA purchased from American Diagnostica, Inc. (Greenwich, CT.).
Statistical analysis. Two-way analysis of variance with Tukey's test for multiple comparisons was used for analysis of clinical chemistry, immunohistochemistry, and ELISA. The criterion for significance was p < 0.05.
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RESULTS |
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Genes with specific treatment-related expression patterns were identified in the sets described above and further categorized into groups using a secondary statistical filter as described in Materials and Methods. Genes with greater expression in LPS/RAN-treated rats compared with either agent given alone were identified in the LR, LRL, LR
R, and LR
L
R sets (Table 1). Overexpression of one or more genes in this group might be a determinant of liver injury in rats treated with LPS/RAN. In addition, groups of genes with similar expression in LPS/RAN- and LPS-treated rats (from LR
L and LR
L
R sets) or in LPS/RAN- and RAN-treated rats (from LR
R and LR
L
R sets) were identified. Since liver injury does not occur after treatment of rats with LPS or RAN alone, expression of these genes might be important if an interaction occurs between two or more gene products. Lastly, a group of genes expressed to a greater degree in rats treated with LPS alone as compared to rats cotreated with LPS/RAN was identified in the L and LR
L sets. The importance of this group lies in the possibility that RAN might interfere with the upregulation by LPS of a gene that protects against liver injury.
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Real-Time PCR
Genes were selected for real-time PCR analysis based on the FDR filter results in addition to treatment comparisons using Spotfire Decision Site for Genomics. Real-time PCR was performed for three of the genes with increased expression in LPS/RAN-treated rats: PAI-1, egr-1, and btg-2. PAI-1 expression was significantly increased in LPS-treated and RAN-treated rats by 102- and 10-fold, respectively, but by nearly 700-fold in LPS/RAN-treated rats (bars, Fig. 4A). This pattern of expression was consistent with signal intensities for the corresponding Affymetrix probeset (circles, Fig. 4A) identified as active by the FDR filter. For egr-1 (Fig. 4B), a significant increase in mRNA was observed in livers from LPS/RAN-cotreated rats, but not in livers of rats treated with LPS or RAN alone. This was consistent with signal intensities for the associated Affymetrix probeset (circles) identified by the FDR as active only in LPS/RAN-cotreated rats (Fig. 4B). Btg-2 expression was significantly increased in rats given LPS or RAN alone by 25- and 4-fold, respectively, but a much greater increase (67-fold) occurred in rats cotreated with LPS/RAN (Fig. 4C). Although a single probeset for btg-2 (M60921_at) is shown for comparison (circles, Fig. 4C), all 3 probesets for this gene determined to be active by the FDR filter had a similar expression pattern (M60921_at, M60921_g_at, rc_AA944156_s_at).
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DISCUSSION |
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A less obvious possibility is that genes expressed to a similar degree after LPS or LPS/RAN treatment or to a similar degree after RAN or LPS/RAN treatment are important for liver injury in LPS/RAN-treated rats. Since liver injury does not develop in rats treated with LPS or RAN alone, induction of such a gene is probably not sufficient to cause liver injury by itself but may be involved in the pathogenesis of liver injury if it interacts with one or more gene products. Several genes were identified with similar expression in livers after treatment with LPS compared to treatment with LPS/RAN (see LPS/RAN LPS in Table 1, Supplemental Table 9). Genes in this group were related to inflammation or could be identified as LPS-inducible (Supplemental Table 9). For example, genes encoding inflammatory cytokines, including TNF-
and interleukin-1ß as well as inducible nitric oxide synthase, showed similar expression after LPS treatment or LPS/RAN cotreatment. Furthermore, cell surface molecules including the adhesion molecules lectin-like, oxidized low-density lipoprotein receptor-1 (LOX-1), CD14 and CD38, the transcription factors CCAAT/enhancer binding protein (C/EBP)-
and ß, and products involved in signal transduction such as Janus kinase 2 (Jak2) and phosphodiesterase 4B (PDE4B) fit this pattern of expression.
PDE4B is an important regulator of inflammatory responses, including expression of cytokines and activation of inflammatory cells such as PMNs (Jin and Conti, 2003; Essayan, 1999
). In this regard, it is of interest that hepatic lesions that develop in LPS/RAN-treated rats are laden with PMNs (Luyendyk et al., 2003
). Regulation of inflammation by PDE4B or other gene products may be essential for LPS/RAN-induced liver injury but not sufficient to produce liver injury in the absence of RAN cotreatment. Indeed, RAN can sensitize hepatocytes to the cytotoxic effects of PMN-derived factors (Luyendyk et al., 2003
). It therefore seems possible that RAN sensitizes hepatocytes to become injured from otherwise noninjurious upregulation of pro-inflammatory genes, leading to idiosyncratic hepatotoxicity.
Whereas some genes were induced after LPS- and LPS/RAN-treatment to a similar degree, the expression of others was increased after LPS treatment but showed an attenuated response after cotreatment with RAN (see LPS/RAN < LPS in Table 1, Supplemental Table 11). This pattern is of interest because RAN might prevent expression of gene products that downregulate inflammation or cell death signals, thereby enhancing inflammation to tissue-damaging levels and/or activating cell death pathways. Indeed, within this group, several members of signal transduction pathways were identified, including protein kinase C (PKC)-epsilon. Interestingly, PKC-epsilon and another gene with this expression pattern, oxygen-regulated protein (150 kDa) (ORP150), have been implicated in protection against ischemic stress (Gray et al., 1997; Ozawa et al., 1999
). This suggests the possibility that livers from LPS/RAN-cotreated rats are more sensitive to harmful effects of local ischemia due to loss of protective gene products. In addition, expression of the proteasome subunits LMP2 and LMP7 was observed in LPS-treated rats, but this increase was attenuated after LPS/RAN treatment. Cells lacking LMP2 and LMP7 have defective NF-KB translocation and are sensitive to TNF-
-induced apoptosis (Hayashi and Faustman, 2000
). Overall, these and other genes expressed to a greater degree after LPS treatment (heat shock 70 kDa protein 1A, heme oxygenase-2, dnaJ homolog subfamily ß member 9) compared to LPS/RAN cotreatment may confer some degree of cytoprotection, perhaps by decreasing sensitivity to hypoxia, inflammatory mediators, or oxidative stress.
Genes expressed to a greater degree in LPS/RAN-cotreated rats compared to treatment with either agent alone could represent a population with mechanistic importance if the gene product became expressed at or above the level required to participate in liver injury. Our analysis revealed that genes characterized by this expression pattern were primarily hypoxia-inducible or involved in inflammation (see LPS/RAN>LPS and >RAN in Table 1, Supplemental Table 8, Table 2). Several of the hypoxia-inducible genes in this group can also participate in inflammatory responses. For example, CxCl10 (interferon-inducible cytokine IP-10) is induced under hypoxic conditions and modulates recruitment and retention of inflammatory cells (Neville et al., 1997). The hypoxia-inducible transcription factor egr-1 is involved in cell death signaling (Thiel and Cibelli, 2002
), but it can also influence inflammatory responses by altering cytokine expression (Yan et al., 1999
; Shi et al., 2002
). The observation that numerous hypoxia-inducible genes are expressed in LPS/RAN-cotreated rats suggests the possibility that hypoxia is involved in liver injury in this idiosyncrasy model. However, further studies are required to confirm tissue hypoxia in livers of LPS/RAN-cotreated rats and its role in pathogenesis.
Another gene product expressed to a larger degree in LPS/RAN-cotreated rats compared to rats treated with LPS or RAN alone is PAI-1. PAI-1 is induced by various stimuli, including LPS, inflammatory cytokines, and hypoxia, and is expressed by several cells in the liver, including parenchymal and endothelial cells (Binder et al., 2002; Kietzmann et al., 1999
; Hamaguchi et al., 2003
). Although the cell source and mechanism of enhanced PAI-1 expression in livers of LPS/RAN-cotreated rats is not known, one possibility is that RAN may indirectly augment expression by increasing levels of cytokines known to induce PAI-1, such as TNF-
or IL-1. Interestingly, although hepatocellular liver injury was not observed at this early time after LPS/RAN cotreatment, serum HA concentration was significantly increased, suggesting altered SEC homeostasis. This elevation in serum HA concentration supports SECs as a potential source of PAI-1. Perturbation of SECs by hypoxia (Kietzman et al., 1999
) or altered signal transduction may be responsible for augmented PAI-1 expression in endothelial cells in LPS/RAN-treated rats. P38 mitogen-activated protein kinase is involved in induction of PAI-1 during hypoxia but does not appear to be important in induction of PAI-1 by TNF-
(Hamaguchi et al., 2003
; Kietzmann et al., 2003
). Another possibility is that cotreatment with RAN influences PAI-1 expression at the transcriptional level. Gruber et al. (2003)
demonstrated that the PAI-1 promoter contains a response element for the orphan receptor Nur77 (NGFI-B, TR3). Furthermore, Nur77 overexpression in human umbilical vein endothelial cells activates a luciferase reporter gene controlled by a PAI-1 promoter, and Nur77 is necessary for induction of PAI-1 expression by TNF-
(Gruber et al., 2003
). Since Nur77 is also a hypoxia-inducible gene (Choi et al., 2004
), its expression might be expected to be enhanced in LPS/RAN-treated rats, since numerous other hypoxia-inducible genes, including PAI-1, were identified in this group (Table 2). Although probesets for Nur77 did not emerge as active after LPS/RAN-cotreatment, the expression of these was more than 10-fold greater than vehicle-control in 3 of 4 LPS/RAN-treated rats, whereas less than 2-fold changes occurred in livers of rats given LPS or RAN alone. This result raises the possibility that Nur77 is important for enhanced expression of PAI-1 in LPS/RAN-treated rats. However, additional experiments are necessary to prove such a connection.
The greater expression of the PAI-1 gene in livers of LPS/RAN-treated rats (Fig. 4A) was reflected in enhanced concentration of PAI-1 protein in serum (Fig. 5), suggesting a functional consequence to its induction. PAI-1 has many physiological roles, including inhibition of fibrinolysis and modulation of inflammatory cell migration (Binder et al., 2002; Marshall et al., 2003
). Consistent with its antifibrinolytic activity and pattern of expression, significant fibrin deposits occur only in livers of rats given LPS/RAN. Concurrently elevated serum concentrations of both HA and PAI-1 suggest the possibility that altered SEC homeostasis might favor activation of the hemostatic system and fibrin deposition in liver. Fibrin deposition could cause local ischemia, and resultant hypoxia might contribute to the development of liver injury. This hypothesis is consistent with the observation that numerous hypoxia-inducible genes were expressed in livers of LPS/RAN-cotreated rats (Table 3). Inasmuch as the PAI-1 gene is hypoxia-inducible, the presence of hypoxia might further enhance its expression, triggering a cascade of hemostatic dysregulation. Additionally, hypoxic upregulation of egr-1 could facilitate coagulation system activation by upregulating tissue factor on liver-cell membranes (Pawlinski et al., 2003
). Overall, the data suggest that enhanced PAI-1 expression in LPS/RAN-cotreated rats encourages formation of fibrin clots, possibly resulting in disrupted liver blood flow and hepatocellular hypoxia that could contribute to the development of necrosis. Preliminary studies showing protection from liver injury by fibrinolytic or anticoagulant drugs in LPS/RAN-treated rats support this hypothesis (Luyendyk et al., 2004
).
In summary, rats were treated with either a nonhepatotoxic dose of LPS or its vehicle and with either RAN or its vehicle. Of the four treatments, only LPS/RAN treatment results in liver injury (Luyendyk et al., 2003). At a time before the onset of significant hepatotoxicity in LPS/RAN-treated rats, hierarchical clustering of hepatic gene expression segregated animals by treatment. Hypoxia-inducible genes, including PAI-1 and egr-1, were expressed to a greater degree in livers after LPS/RAN treatment compared to either agent given alone. The enhanced PAI-1 gene expression was reflected in increased PAI-1 protein in serum. Significant fibrin deposits in liver sinusoids were observed only in LPS/RAN-cotreated rats, consistent with the antifibrinolytic activity of PAI-1. Overall, the results suggest that altered expression of genes promoting hemostasis might contribute to liver injury in LPS/RAN-cotreated rats. The studies presented represent a snapshot in time at one dose; examination of gene expression at other times after treatment and other doses of LPS and RAN will illuminate this connection further. The association of hypoxia-inducible gene expression with hepatic fibrin deposition in LPS/RAN-cotreated rats is consistent with the development of tissue ischemia/hypoxia as a contributing factor to liver pathogenesis in this model of RAN idiosyncrasy.
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Supplemental data |
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ACKNOWLEDGMENTS |
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NOTES |
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3 Present address: Bristol Myers Squibb Co., New Brunswick, NJ.
1 To whom correspondence should be addressed at the Department of Pharmacology and Toxicology, National Food Safety and Toxicology Center, Michigan State University, East Lansing, MI 48824. Fax: (517) 432-2310. E-mail: rothr{at}msu.edu.
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