ToxSci Advance Access originally published online on January 16, 2006
Toxicological Sciences 2006 90(2):569-585; doi:10.1093/toxsci/kfj103
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Unique Gene Expression and Hepatocellular Injury in the Lipopolysaccharide-Ranitidine Drug Idiosyncrasy Rat Model: Comparison with Famotidine
* Department of Pharmacology and Toxicology, Center for Integrative Toxicology, National Food Safety and Toxicology Center, Michigan State University, East Lansing, Michigan 48824; Discovery Toxicology, Bristol-Myers Squibb, Princeton, New Jersey; and Drug Safety Evaluation, Bristol-Myers Squibb, Syracuse, New York
1 To whom correspondence should be addressed at 221 National Food Safety and Toxicology Center, Michigan State University, East Lansing, MI 48824. Fax: (517) 432-2310. E-mail: rothr{at}msu.edu.
Received November 8, 2005; accepted January 10, 2006
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
|---|
Rats cotreated with lipopolysaccharide (LPS) and ranitidine (RAN) but not LPS and famotidine (FAM) develop hepatocellular injury in an animal model of idiosyncratic drug reactions. Evaluation of liver gene expression in rats given LPS and/or RAN led to confirmation that the hemostatic system, hypoxia, and neutrophils (PMNs) are critical mediators in LPS/RAN-induced liver injury. We tested the hypothesis that unique gene expression changes distinguish LPS/RAN-treated rats from rats given LPS or RAN alone and from those cotreated with LPS/FAM. Rats were treated with a nonhepatotoxic dose of LPS (44.4 x 106 endotoxin units/kg, iv) or its vehicle. Two hours thereafter they were given RAN (30 mg/kg, iv), FAM (either 6 mg/kg, a pharmacologically equi-efficacious dose, or 28.8 mg/kg, an equimolar dose, iv), or vehicle. They were killed 2 or 6 h after drug treatment for evaluation of hepatotoxicity (2 and 6 h) and liver gene expression (2 h only). At a time before the onset of hepatocellular injury, hierarchical clustering distinguished rats treated with LPS/RAN from those given LPS alone. 205 probesets were expressed differentially to a greater or lesser degree only in LPS/RAN-treated rats compared to LPS/FAM or LPS alone, which did not develop liver injury. These included VEGF, EGLN3, MAPKAPK-2, BNIP3, MIP-2, COX-2, EGR-1, PAI-1, IFN-
, and IL-6. Expression of these genes was confirmed by real-time PCR. Serum concentrations of MIP-2, PAI-1, IFN-, and IL-6 correlated with their respective gene expression patterns. Overall, the expression of several gene products capable of controlling requisite mediators of injury (i.e., hemostasis, hypoxia, PMNs) in this model were enhanced in livers of LPS/RAN-treated rats. Furthermore, enhanced expression of MAPKAPK-2 in RAN-treated rats and its target genes in LPS/RAN-treated rats suggests that p38/MAPKAPK-2 signaling is a regulation point for enhancement of LPS-induced gene expression by RAN. Key Words: inflammation; drug idiosyncrasy; gene array; lipopolysaccharide; hepatotoxicity.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
|---|
Idiosyncratic hepatotoxicity during drug therapy occurs in a small fraction of patients and is not predicted by preclinical animal studies, since these reactions also occur infrequently in animals. Although these reactions are often purported to occur from reactive metabolites and/or a specific immune response originating from metabolite-protein haptens (Ju and Uetrecht, 2002
; Pirmohamed et al., 1996), for the vast majority of drugs causing idiosyncratic drug reactions (IDRs), the underlying mechanism of toxicity is not understood. We and others have hypothesized that an underlying inflammatory stress might precipitate some IDRs (Ganey et al., 2004; Roth et al., 2003; Tafazoli et al., 2005), and animal models have been developed to study the effect of inflammation on sensitivity to drug-induced hepatotoxicity (Buchweitz et al., 2002; Luyendyk et al., 2003).
In one animal model, rats given small nonhepatotoxic doses of bacterial lipopolysaccharide (LPS) and the histamine2 (H2)-receptor antagonist, ranitidine (RAN), developed hepatocellular injury (Luyendyk et al., 2003). Whereas RAN causes rare idiosyncratic hepatotoxicity in patients (Vial et al., 1991), another H2-receptor antagonist, famotidine (FAM), is associated with almost no reports of hepatocellular injury (Luyendyk et al., 2003). Corresponding to the relative propensity of these drugs to cause liver injury in people, LPS/RAN-cotreated rats developed hepatocellular injury, whereas rats given LPS/FAM did not (Luyendyk et al., 2003). Accordingly, for these two drugs, the LPS-cotreatment model could have predicted which one would be associated with an IDR liability in people.
In LPS/RAN-treated rats, midzonal hepatocellular necrosis resembling damage caused by a large, hepatotoxic dose of LPS began to develop within 3 h of drug administration (Luyendyk et al., 2003). In this model, both the hemostatic system and inflammatory cells appear to be critical mediators of injury (Luyendyk et al., 2004a, 2005b). Despite progress in understanding contributing factors to injury in LPS/RAN-treated rats, the properties inherent to RAN but not shared by FAM that allow for RAN's hepatotoxic interaction with LPS are not known. One possibility is that drugs that cause IDRs affect liver homeostasis (e.g., gene expression) in a manner that makes the response to inflammatory stimuli (e.g., LPS) more robust, resulting in hepatocellular injury. For example, RAN might increase the expression of a particular gene product that initiates signaling pathways to hepatocellular injury only when a concurrent inflammatory response is present. Since liver injury does not occur in LPS/FAM-treated rats, FAM would be expected to lack this capacity. Previously, hepatic gene expression was evaluated in rats given LPS and/or RAN, and a unique gene expression pattern occurred in LPS/RAN-treated rats 3 h post-treatment (Luyendyk et al., 2004b). One limitation of this study is that comparison with hepatic gene expression in rats given LPS and FAM was not undertaken. Moreover, differences in hepatic gene expression have not been evaluated in livers of rats given RAN or FAM alone. Direct comparison of hepatic gene expression elicited by these two drugs might help to identify changes critical for LPS/RAN-induced liver injury and possibly RAN idiosyncrasy.
We tested the hypothesis that, at a time near the onset of liver injury (in LPS/RAN-treated rats), global hepatic gene expression could distinguish rats given RAN alone from those given FAM. Furthermore, the hypothesis was tested that LPS/RAN-, but not LPS/FAM-cotreatment triggers not only unique gene expression, but also augments changes in gene expression caused by LPS itself. Genes were grouped based on their expression pattern, and the expression of several was confirmed by real-time PCR and by ELISA at the protein level.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
|---|
Materials.
Unless otherwise noted, all chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). LPS derived from E. coli serotype O55:B5 with an activity of 6.6 x 106 endotoxin units (EU)/mg was used for these studies (Cat. No. L-2880, Lot # 51K4115). This activity was determined using a QCL Chromogenic LAL Endpoint Assay from Cambrex (East Rutherford, NJ).
Animals.
Male, Sprague-Dawley rats (Crl:CD (SD)IGS BR; Charles River, Portage, MI) weighing 250–350 g were used for these experiments. 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 protocols.
In a previous study, rats cotreated with normally nonhepatotoxic doses of LPS and RAN developed hepatocellular injury (Luyendyk et al., 2003). These same doses of LPS (44.4 x 106 EU/kg) and RAN (30 mg/kg) were used in the studies presented here. Two doses of FAM were selected for toxicity and gene expression comparisons: (1) a pharmacologically equi-efficacious dose was selected based on relative potencies of RAN and FAM in antagonizing H2-receptors (FAM-EE; 6 mg/kg) (Lin, 1991; Luyendyk et al., 2003; Scarpignato et al., 1987) and (2) a dose equimolar to that of RAN (FAM-EM; 28.8 mg/kg). Rats fasted for 24 h were given LPS or its saline vehicle (Veh) iv, and food was then returned. Two h later they were given RAN, FAM-EE, FAM-EM, or sterile phosphate-buffered saline (PBS), iv. Two or 6 h after drug treatment, rats were anesthetized with sodium pentobarbital (75 mg/kg, ip) for assessment of hepatic gene expression (2 h) and hepatocellular injury (2 and 6 h). To simplify treatment nomenclature for the remainder of the report, the following group designations have been applied: Saline/PBS (Veh/Veh, n = 5), Saline/RAN (Veh/RAN, n = 5), Saline/FAM-EE (Veh/FAM-EE, n = 5), Saline/FAM-EM (Veh/FAM-EM, n = 6), LPS/PBS (LPS/Veh, n = 5), LPS/FAM-EE (n = 5), LPS/FAM-EM (n = 8), and LPS/RAN (n = 9).
Sample collection.
Blood drawn from the dorsal aorta was collected rapidly in BD Vacutainer Plus Plastic Citrate Tubes (Becton-Dickinson, Franklin Lakes, NJ) or allowed to clot at room temperature. Citrated-plasma and serum were collected and aliquots stored at 4°C for clinical chemistry analysis and –80°C for measurement of protein concentrations. Three 100-mg, midlobe pieces of the right medial liver lobe were flash-frozen in liquid nitrogen for RNA isolation. Slices (3–4 mm thick) of the ventral portion of the left lateral lobe were collected and fixed in 10% neutral buffered formalin.
Hepatotoxicity assessment.
Alanine aminotransferase (ALT) activity was evaluated using a Hitachi 917 Chemistry Analyzer (Roche Diagnostics, Inc.). Formalin-fixed sections of liver were routinely embedded in paraffin, sectioned at approximately 5 µm, and stained with hematoxylin and eosin. Three sections of each liver were examined. Acute, multifocal hepatic necrosis was scored according to the system described previously (Luyendyk et al., 2003), in which a score of 0 represents no significant lesion and 5 represents a severe lesion.
RNA isolation and purification.
Total RNA was isolated from a small piece of frozen liver tissue using Trizol reagent (Invitrogen Corporation, Carlsbad, CA) and purified using RNeasy spin columns (Qiagen, Valencia, CA) according to the manufacturers' instructions. Complete removal of DNA was achieved by using Qiagen's RNase-Free DNase Set. The quality of the RNA was evaluated by measuring the 260:280 nm absorbance ratio, and the integrity of 18S and 28S ribosomal RNA bands was assessed by electrophoresis on RNA 6000 Nano labchips (Agilent Technologies, Palo Alto, CA). RNA concentrations were determined from absorbance values at a wavelength of 260 nm using a SpectraMax spectrophotometer (Molecular Devices Corporation, Sunnyvale, CA).
Probe preparation and microarray hybridization.
Sample labeling, hybridization, and staining were carried out according to the Eukaryotic Target Preparation protocol in the Affymetrix Technical Manual for GeneChip Expression Analysis (Version 701028 rev 1). In summary, 5 µg of purified total RNA were used to generate double-stranded cDNA using Superscript reverse transcriptase (Invitrogen Life Technologies) and a T7-oligo (dT) primer. The resulting cDNA was purified using the GeneChip Sample Cleanup Module according to the manufacturer's protocol. The purified cDNA was amplified using BioArray high yield RNA transcription labeling kit (Enzo) according to the manufacturer's instructions to produce biotin-labeled cRNA (complementary RNA) which was then purified using GeneChip Sample Cleanup Module and quantified. Labeled cRNA (20 µg per chip) was fragmented at 94°C for 35 min. The fragmented cRNA (15 µg) was hybridized to the Affymetrix Rat 230 2.0 array for 16 h at 45°C. The hybridized arrays were washed and stained using Streptavidin–Phycoerythrin (Molecular Probes, Carlsbad, CA) and amplified with affinity-purified, biotinylated anti-streptavidin (Vector Laboratories, Inc, Burlingame, CA) using a GeneChip Fluidics Station 450. The arrays were scanned in Affymetrix high-resolution GeneChip scanner 3000 at 570 nm using Genechip Operating software (GCOS, Ver.1.2).
Data analysis.
Raw Affymetrix scan data (CEL files) that met manufacturer's recommended quality criteria were imported into Rosetta Resolver. Downstream analysis was done with the Rosetta Resolver gene expression analysis software (version 5.0, Rosetta Biosoftware, Seattle, WA). Intrachip normalization and background corrections were applied to the hybridizations or profiles, and the replicate profiles were combined in an error-weighted fashion to create expression ratios with each treatment group as the baseline. Interchip scaling was done to normalize intensity brightness, both across multiple microarrays of the same pattern and of different patterns. Error-model-based transformation was then applied to intensity profiles, and the transformed data were corrected for nonlinearity of expression levels. Error-weighted ANOVA was performed on the input data that were partitioned into groups (ratio experiments) to determine whether statistically significant differences existed among the group means. Genes were considered active if p < 0.01 and the fold change for a comparison was at least ± 1.5-fold compared to Veh/Veh-treated rats. Clustering analysis was performed using an agglomerative hierarchical clustering algorithm for which error-weighted Euclidean distance-based measure (emphasizes the magnitude of the fold changes based on the sum of squares of differences in each direction) was used as similarity measurement.
Real-time polymerase chain reaction (PCR) analysis.
Changes in selected transcript levels determined from microarray analyses were also confirmed by real-time PCR. Five µg of RNA were reverse transcribed to cDNA using a High Capacity cDNA Archive kit (Applied Biosystems, Foster City, CA). Real-time PCR was performed with an ABI Prism 7900HT Sequence Detection System (Applied Biosystems) using 2X SYBR Green master mix (Eurogentec, San Diego, CA). Amplification was carried out as follows: 50°C for 2 min (for uracil N-glycosylase incubation); 95°C for 10 min (denaturation); 40 cycles of 95°C for 15 s and 60°C for 30 s (denaturation/amplification). Dissociation curves were created by adding the following steps to the end of the amplification reaction: 95°C for 15 s (denaturation) and 60°C for 15 s, then gradually increasing to 95°C over 20 min, with a final hold at 95°C for 15 s. Primers were designed for selected genes using Primer Express (v2.0, Applied Biosystems) and checked for specificity by BLAST searches. In addition, primers were only used when they gave rise to a single amplicon as revealed by melting curve analysis. Sequences of forward and reverse primers for target genes purchased from Sigma Genosys (The Woodlands, TX) are listed in Table 1. Twenty ng of cDNA samples were amplified in duplicate using 100 nM primers. 18S rRNA was used as an endogenous control to normalize the mRNA target for the differences in the amount of total RNA added to each reaction. Standard curves were constructed for the target mRNA and the endogenous control (18S rRNA) by serial dilution (60, 20, 6.67, 2.22, 0.74 ng cDNA) of the mixture of cDNA samples obtained from the LPS/Veh group. The amount of target gene and endogenous control in samples was determined by linear regression analysis, and the target mRNA abundance was expressed as the ng target gene/ng 18S rRNA ratio.
|
Measurement of blood proteins.
Serum MIP-2, IL-6, and IFN-
concentrations were measured using ELISA kits (Biosource International, Camarillo, CA). Serum PAI-1 concentration was measured using an ELISA kit from American Diagnostica (Stamford, CT).
Statistical analysis.
Two-way analysis of variance (ANOVA) with Tukey's test for multiple comparisons was used for comparison of all data with the exception of gene expression filtering and serum concentrations of IL-6 and IFN-. Serum concentrations of these two cytokines were compared by one-way ANOVA within the LPS-treatment groups using Tukey's test to compare group means.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
|---|
Hepatocellular Injury after LPS/RAN-, but Not LPS/FAM-cotreatment
Hepatocellular injury has been shown to be absent to minimal 2 h after LPS/RAN-cotreatment and fully manifested by 6 h (Luyendyk et al., 2003
). In the current study, injury was examined at these same times. Neither LPS or RAN alone, nor Veh/FAM-EE or Veh/FAM-EM treatments caused increases in serum ALT or AST activities at 2 or 6 h (Fig. 1). Transaminase activities increased slightly at 2 h in LPS/RAN-treated rats, and this increase was more pronounced at 6 h (
8-fold). In contrast, serum ALT and AST activities did not increase in rats cotreated with LPS/FAM-EE or LPS/FAM-EM (Fig. 1), confirming previous results (Luyendyk et al., 2003). Histologically, midzonal hepatocellular necrosis was observed in 1 of 9 LPS/RAN-cotreated rats at 2 h, and this lesion became more frequent (5 of 8 rats) and increased in severity at 6 h (Table 2). One LPS/FAM-EM cotreated rat at each time had minimal hepatocellular necrosis.
|
|
Hierarchical Clustering of Hepatic Gene Expression
Hepatic gene expression was evaluated 2 h after drug treatment. Affymetrix 230 2.0 probesets defined as active (see Materials and Methods) were subjected to hierarchical clustering (Fig. 2). Veh- and LPS-treated rats segregated to separate clusters (clusters 1 and 2, respectively). Within cluster 1, Veh/RAN-treated rats formed their own unique cluster (cluster 1a) whereas Veh/FAM-EE-treated and Veh/FAM-EM-treated rats could not be distinguished from Veh/Veh-treated rats (cluster 1b). Within the LPS cluster, with the exception of two LPS/FAM-EM-treated rats (cluster 2e) that clustered with one LPS/RAN-treated rat (2b), the LPS/FAM-treatment groups could not be distinguished from LPS/Veh-treated rats. LPS/RAN-treated rats formed 3 more groups within the LPS-cluster (clusters 2a, 2c, and 2d). Similar segregation of LPS/RAN-treated rats was observed when clustering was performed without the FAM treatment groups (data not shown). Principal component analysis also revealed similar variability in grouping of LPS/RAN-treated rats, and no correlation was identified within this treatment group with clinical chemistry values or histopathology (data not shown).
|
Enhancement of LPS-induced Gene Expression by RAN, but Not FAM
One possible mechanism underlying liver injury caused by LPS/RAN cotreatment is that RAN augments gene expression caused by LPS, culminating in a more robust production of pro-inflammatory mediators and tissue injury. Alternatively, LPS/RAN-cotreatment might alter the expression of a gene not otherwise changed by LPS/Veh. Since liver injury does not occur in LPS/FAM-treated rats, FAM might lack this capacity. To address these possibilities, we identified a set of genes differentially expressed in LPS/RAN-treated rats compared to all other LPS-treatment groups. First, probeset expression in each cotreatment group was compared to expression after treatment with LPS/Veh. Probesets selected for further filtering were those differentially expressed only in LPS/RAN-treated rats compared to LPS/Veh (i.e., probesets that met Criterion A and not Criteria B or C in Table 3). For these probesets, the expression level in LPS/RAN-cotreated rats was then compared to each FAM-cotreatment group (i.e., criteria D and E, Table 3). Probesets meeting both of these criteria were considered to be those differentially expressed in LPS/RAN-treated rats compared to all other treatment groups (Supplemental Table 1). Of the 205 probesets identified, annotation was available for 95. Of these 95 probesets, treatment with LPS alone altered the activity of 52 genes compared to Veh/Veh-treated rats (Criterion F, Table 3). For these 52 genes, RAN cotreatment selectively altered (increased or decreased) the LPS-induced change in gene expression (Table 5). The remaining probesets were not altered by treatment with LPS alone, indicating the gene expression change was unique to LPS/RAN-cotreated rats (Table 4).
|
|
|
Real-time PCR Confirmation
Real-time PCR results confirmed the microarray gene expression data for selected genes that were increased only in LPS/RAN-cotreated rats. Real-time PCR demonstrated that increased expression of mRNAs for the hypoxia-inducible genes EGLN3 (SM-20, prolyl hydroxylase-3) and VEGF (vascular endothelial growth factor) occurred only in LPS/RAN-treated rats (Figs. 3A and 3B, respectively). Among LPS-treatment groups, expression of proapoptotic gene BNIP3 (BCL2/adenovirus E1B 19 kDa-interacting protein 3) increased only in LPS/RAN-treated rats. Treatment with RAN alone but not FAM alone also significantly increased BNIP3 expression (Fig. 3C). Like BNIP3, expression of mRNA encoding the p38 pathway member, MAPKAPK-2 (mitogen activated protein kinase activated protein kinase-2), was increased by RAN alone. Although statistical filtering of gene array results did not identify an increase in MAPKAPK-2 mRNA after treatment with LPS alone, a slight increase was detected by real-time PCR, and the increase in LPS/RAN-treated rats was additive (Fig. 3D).
|
LPS-induced increased RNA expression that was augmented by RAN-cotreatment included the transcription factor egr-1 (early growth response-1), the inflammatory mediators ptgs2 (cyclooxygenase-2, COX-2) and Cxcl2 (macrophage inflammatory protein-2, MIP-2), and the antifibrinolytic factor serpine1 (plasminogen activator inhibitor-1, PAI-1); these results were confirmed by real-time PCR (Fig. 4). Treatment with RAN alone caused a slight increase in COX-2 and MIP-2 expression and a trend towards an increase in egr-1 expression. Veh/FAM-EE and Veh/FAM-EM treatment both led to a slight increase in MIP-2 expression. Confirming gene array results, cotreatment with RAN substantially augmented the LPS-induced expression of each gene, whereas FAM had no effect (Fig. 4).
|
Serum Concentration of MIP-2 and PAI-1
Previous work with the LPS/RAN model implicated PMNs and the hemostatic system as requisite components of pathogenesis of hepatocellular injury (Luyendyk et al., 2004a
, 2005b). In this regard, it is noteworthy that expression of the PMN chemokine MIP-2 and the antifibrinolytic factor PAI-1 was augmented selectively in LPS/RAN-treated rats (Fig. 4). Consistent with the changes in hepatic MIP-2 mRNA, serum MIP-2 protein concentration increased slightly in rats given Veh/RAN or Veh/FAM-EE (Fig. 5A). LPS/Veh-treatment greatly increased serum MIP-2 concentration (410-fold) compared to Veh/Veh-treated rats (40 pg/ml). Neither FAM-EE nor FAM-EM cotreatment increased this further, whereas RAN cotreatment augmented serum MIP-2 concentration by 2.5 times compared to rats given LPS alone (Fig. 5A). Serum PAI-1 concentration also increased in LPS/Veh-treated rats, and this increase was enhanced by RAN but not by FAM (Fig. 5B).
|
Effect of Treatment with RAN and FAM Alone on Hepatic Gene Expression
In addition to evaluating the effect(s) of RAN and FAM cotreatment on gene expression in rats given LPS, this study compared the effect on hepatic gene expression of each drug given alone. To this end, probesets determined to be active relative to Veh/Veh-treated rats after administration of each drug alone (i.e., Veh/RAN, Veh/FAM-EE, Veh/FAM-EM treatments) were identified by meeting criteria G, H, or I in Table 3. The number of probesets determined to be active after each treatment is represented in a Venn Diagram (Fig. 6), and specific lists are available in Supplemental Tables 2–8. Approximately 2400 probesets changed in Veh/RAN-treated rats, whereas only approximately 300 changed after treatment with Veh/FAM-EE and even fewer after Veh/FAM-EM. Of the relatively large number of genes altered by RAN, 94% were changed selectively (i.e., not changed by FAM).
|
) or decreased (
) relative to Veh/Veh-treated rats in a given treatment set is shown. Probesets with activities altered by more than one treatment are indicated by an intersection symbol (
). R: probesets changed only after treatment with Veh/RAN (Supplemental Table 2); F-EE: probesets changed only after treatment with Veh/F-EE (Supplemental Table 3); F-EM: probesets changed only after treatment with F-EM (Supplemental Table 4); RSince hepatocellular injury did not occur in rats given RAN alone, these changes in gene expression are likely not sufficient to cause injury. However, the RAN-induced expression of one or more critical gene products might interact with an LPS-induced inflammatory stress to cause liver injury. Accordingly, gene expression changes in LPS/RAN-treated rats that occurred as a consequence of RAN treatment only were identified as those that met criteria I and J, but not criteria G, H, F, and K (Table 3). Two hundred thirty-two probesets met these criteria (Supplemental Table 9) and annotation was currently available for 58 (Table 6). Of particular interest, 8 genes were differentially expressed in LPS/RAN-treated rats compared to all other LPS-treated groups. These included BCL2/adenovirus E1B 19 kDa-interacting protein 3 (BNIP3), cathepsin L (ctsl), glutamate oxaloacetate transaminase 1 (got1), interferon-related developmental regulator 1 (ifrd1), inhibin beta E (inhbe), MAP kinase-activated protein kinase 2 (MAPKAPK-2), and heat shock 70kD protein 1A (Hspa1a) (Table 6, bold font).
|
Hepatic Gene Expression and Serum Concentrations of IFN-
and IL-6As confirmed by real-time PCR above, expression of MAPKAPK-2 mRNA was increased by treatment with RAN alone, but unaffected by treatment with either FAM-EE or FAM-EM (Fig. 3D). MAPKAPK-2 is a downstream kinase in the p38 signaling pathway, which is important for stabilization (i.e., inhibiting mRNA degradation) of numerous inflammatory mediator mRNAs, some of which were expressed to a greater degree in LPS/RAN-treated rats (i.e., COX-2, MIP-2; Table 4) (Rousseau et al., 2002
). In addition to stabilizing mRNAs for MIP-2 and PAI-1, the p38 signaling pathway can stabilize IL-6 and IFN-gamma mRNA (Mavropoulos et al., 2005; Winzen et al., 1999). Probesets for these genes were close to, but did not meet, the statistical criterion for enhanced expression in LPS/RAN-treated rats. Nonetheless, since the data suggested p38 signaling as a pathway to LPS/RAN-induced gene expression, real-time PCR was performed. IL-6 mRNA was significantly increased in livers of all LPS treatment groups (Fig. 7A), and although the increase appeared more than 2-fold larger in LPS/RAN-treated rats compared to rats given LPS alone, the difference was not statistically significant (p = 0.06). Serum IL-6 protein concentration was below the limit of detection (10 pg/ml) in rats given Veh/Veh and was not increased by either RAN or FAM alone. LPS given alone increased serum IL-6 concentration in LPS-treated rats (16 ng/ml), and this was not enhanced by FAM cotreatment at either dose (Fig. 7C). However, RAN cotreatment augmented serum IL-6 concentration in LPS-pretreated rats (68 ng/ml). Like IL-6 mRNA, IFN- mRNA was significantly increased in livers of each LPS-treatment group. Expression in LPS/RAN-treated rats was significantly greater (2-fold) than in rats given LPS/Veh (Fig. 7B). IFN- was not detected in serum of rats given Veh/Veh and was unchanged by either drug given alone (Fig. 7D). LPS caused an increase in serum IFN- concentration (19 ng/ml), and this was enhanced by RAN (47 ng/ml), but not FAM (Fig. 7D).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
|---|
Cotreatment with a small, nonhepatotoxic dose of LPS renders numerous xenobiotics, including some drugs, hepatotoxic in rats (Buchweitz et al., 2002
; Ganey and Roth, 2001; Labib et al., 2002; Lind et al., 1984; Luyendyk et al., 2003). This increase in susceptibility to liver injury has been proposed to be a component of idiosyncratic reactions in people (Roth et al., 2003), and the LPS-pretreatment model in rats might be able to distinguish drugs with idiosyncratic liability preclinically. This study confirmed previous results (Luyendyk et al., 2003) showing that LPS-pretreatment rendered RAN, but not FAM hepatotoxic in rats, consistent with the potential of these H2 antagonists to cause liver injury in people. In addition to hepatotoxicity outcome, global hepatic gene expression and specific genes also distinguished RAN from FAM.
Hepatic gene expression was evaluated at the onset of hepatocellular injury in LPS/RAN-treated rats (i.e., 2 h; a timepoint at which only 1 of 9 rats had minimal necrosis, Table 2) so that changes in gene expression could be associated with the initial pathogenesis. First, hierarchical clustering was used to evaluate global differences among treatment groups. Rats given LPS/FAM-EE or LPS/FAM-EM could not be distinguished from rats given LPS/Veh, suggesting that FAM cotreatment did not greatly alter LPS-induced gene expression. Similar to previous findings (Luyendyk et al., 2003), several groups of LPS/RAN-treated rats were observed within the LPS cluster, and these clustered at various distances from rats given LPS/FAM (Fig. 2). One LPS/RAN-treated rat clustered within the LPS group but separately from other LPS/RAN-treated rats, and a second rat clustered closely to 2 LPS/FAM-EM-treated rats (Fig. 2). Accordingly, like LPS/RAN-induced liver injury, in which serum ALT activity at 6 h varied from 64 to 845 U/L (Fig. 1), LPS/RAN-induced changes in hepatic gene expression were also variable at a time before peak injury. In this study, it was not possible to correlate changes in gene expression with hepatocellular injury for individual animals because livers were taken before injury developed. Nonetheless, gene expression patterns for LPS/FAM-treated rats clustered with rats treated with LPS alone, whereas this was not true for LPS/RAN-treated rats.
At the level of specific gene expression changes, several differences might make LPS/RAN-treated rats uniquely susceptible. For example, LPS/RAN-treatment might uniquely alter the expression of a gene product required for liver injury. Several genes encoding products associated with cell death were increased to a greater degree in LPS/RAN-treated rats compared to other LPS-treated groups. These included the hypoxia-inducible, pro-apoptotic factors BNIP3 (Bruick, 2000), EGLN3 (Freeman et al., 2003; Straub et al., 2003), Nr4a1 (Lin et al., 2004; Yoo et al., 2004), and RTP801 (Shoshani et al., 2002). The increased expression of these and other hypoxia-inducible genes (e.g., VEGF; Fig. 3) is consistent with the observation that hypoxia occurred only in livers of LPS/RAN-treated rats (Luyendyk et al., 2004a). Interestingly, an agent that attenuated hypoxia also reduced injury in this model (Luyendyk et al., 2005b), suggesting a role for hypoxia in the pathogenesis. Taken together, the results indicate that LPS/RAN-treatment results in uniquely enhanced expression of several hypoxia-inducible cell death factors.
Another gene that increased only in LPS/RAN-treated rats (Fig. 3) was vascular endothelial growth factor (VEGF), suggesting that the mechanisms required for its upregulation are in place only in LPS/RAN-cotreated rats. Interestingly, although treatment with RAN alone was without effect on VEGF expression (Fig. 3), expression of the VEGF receptor Flt-1 (FLT1) was increased by RAN to a value similar to that seen in LPS/RAN-treated rats (Table 6). This combination of increases in both ligand and receptor in LPS/RAN-treated rats suggests a possible role for VEGF signaling in LPS/RAN-induced liver injury. Among other stimuli, inflammatory cytokines and hypoxia increase VEGF expression (Xiong et al., 1998). Inhibition of coagulation system activation reduced hypoxia in LPS/RAN-treated rats (Luyendyk et al., 2005b) but was without effect on VEGF expression (data not shown), suggesting that in this model VEGF is induced by a coagulation/hypoxia-independent pathway.
One possibility is that VEGF causes coagulation system activation by upregulation of procoagulant factors in LPS/RAN-treated rats. For example, VEGF can upregulate procoagulant tissue factor on monocytes and endothelial cells (Clauss et al., 1996; Mechtcheriakova et al., 1999). Tissue factor is an important regulator of LPS-induced coagulation system activation in endotoxemia (Pawlinski et al., 2004). One transcription factor important for the upregulation of tissue factor is early growth response-1 (egr-1; Pawlinski et al., 2003). VEGF treatment increases egr-1 mRNA expression in endothelial cells (Liu et al., 2000) and nuclear translocation and binding of egr-1 to the tissue factor promoter (Mechtcheriakova et al., 1999). The observation that hepatic expression of egr-1 mRNA was also enhanced by LPS/RAN-cotreatment (Fig. 4) raises the possibility that VEGF participates in this upregulation. Interestingly, the amount of tissue factor mRNA in LPS/RAN-treated rats was greater than all other LPS-treated groups (Table 5). Thus, greater expression in LPS/RAN-treated rats of VEGF, the VEGF receptor FLT1, the downstream transcription factor egr-1, and a product of a gene regulated by egr-1 (i.e., tissue factor), collectively represents one possible pathway to enhanced hepatic fibrin deposition that could contribute to liver damage. Similar to the expression pattern of egr-1, mRNA as well as serum protein concentration for the antifibrinolytic factor plasminogen activator inhibitor-1 (PAI-1) were increased in LPS/RAN-treated rats (Figs. 4 and 5). Interestingly, PAI-1 expression was diminished in egr-1 knockout mice in ischemia-reperfusion and transplantation models of tissue injury (Okada et al., 2001; Yan et al., 2000). Inasmuch as fibrin deposition is important in LPS/RAN-induced liver injury, enhanced egr-1 expression might contribute both to expression of procoagulant (e.g., tissue factor) and antifibrinolytic (e.g., PAI-1) factors.
In addition to egr-1 and PAI-1, several genes were changed by LPS and to a greater degree in LPS/RAN-treated rats. For example, expression of the proinflammatory genes MIP-2 and COX-2 was increased in LPS/RAN-treated rats. In one model of LPS-potentiation of hepatotoxicity, COX-2 expression was augmented by cotreatment and was important for hepatocellular injury (Ganey et al., 2001). We have reported that COX-2 is not required for LPS/RAN-induced hepatocellular injury (Luyendyk et al., 2005a). The expression of MIP-2 mRNA was associated with a large increase in serum concentration of this chemokine (Fig. 5). This is interesting in light of the critical importance of PMNs for LPS/RAN-induced liver injury (Luyendyk et al., 2005b). Moreover, rdc1, a chemokine receptor recently renamed CXCR7 (Balabanian et al., 2005), was expressed to a greater degree in livers of LPS/RAN-treated rats compared to all other LPS-treated groups (Table 5). The role of CXCR7 expression in the LPS/RAN model is not clear, although its ligand, CXCL12/stromal cell-derived factor-1alpha (SDF-1), is important for maintenance of blood PMNs and regulation of PMN chemotaxis (Struyf et al., 2005; Suratt et al., 2004). PMN chemokines are important in some (Bertini et al., 2004), but not other models of inflammatory liver injury (Dorman et al., 2005). In addition to the pathogenic implication for enhanced expression of these gene products, the similarity in pattern of expression of COX-2, MIP-2, and PAI-1 suggests that the increases in their mRNAs occur by a similar mechanism. In this respect, it is interesting that the hypoxia-induced expression of PAI-1 and LPS-induced expression of COX-2 and MIP-2 require p38 signaling (Kietzmann et al., 2003; Lasa et al., 2000; Rousseau et al., 2002).
Increased expression of COX-2, MIP-2, and other inflammatory mediators in response to LPS is mediated in part by AU-rich element-binding proteins and mRNA stabilization that requires the downstream p38 pathway member, MAPKAPK-2 (Dean et al., 2004; Rousseau et al., 2002). LPS-induced stabilization of mRNAs encoding inflammatory mediators (e.g., IL-6, COX-2) and translation of some inflammatory cytokines (e.g., tumor necrosis factor-
[TNF-]) is reduced in MAPKAPK-2 deficient cells or mice and blunted by inhibitors of p38 (Kotlyarov and Gaestel, 2002; Lasa et al., 2000; Neininger et al., 2002; Rousseau et al., 2002). Treatment with either RAN or LPS alone, but not FAM, caused upregulation of MAPKAPK-2 mRNA in liver, and an additive increase was observed in LPS/RAN-cotreated rats (Fig. 3). Since treatment with RAN alone did not result in liver damage, the upregulation of MAPKAPK-2 is insufficient to cause toxicity. However, it is possible that in the face of p38 activation by LPS pretreatment, RAN-induced MAPKAPK-2 expression stabilizes mRNAs for cytokines initially expressed after LPS alone (e.g., MIP-2; Fig. 4). In addition, consistent with its regulation by MAPKAPK-2 at the translational level, expression of TNF- mRNA was not enhanced in LPS/RAN-treated rats, although its concentration in the plasma was significantly greater (unpublished results). This prolonged and/or enhanced production of inflammatory mediators might result in tissue injury in LPS/RAN-treated rats. Indeed, induction of inflammatory cytokines and hepatocellular injury caused by galactosamine/LPS-cotreatment is reduced in MAPKAPK-2 deficient mice, suggesting a role for p38/MAPKAPK-2 signal transduction in the pathogenesis of this liver injury (Kotlyarov et al., 1999).
If p38/MAPKAPK-2 signaling is operative in livers of LPS/RAN-treated rats, we would expect enhanced expression of not only MIP-2 and COX-2 mRNAs, but also other cytokines, including IL-6 and IFN- (Kotlyarov and Gaestel, 2002). Although the gene expression pattern for these cytokines in the array study did not quite meet our statistical criteria, we evaluated IL-6 and IFN by real-time PCR. Although the increase in IL-6 mRNA did not reach statistical significance, expression of mRNA for IFN- was augmented in liver, and the serum protein concentration of both cytokines was markedly and selectively augmented in serum of LPS/RAN-treated rats (Fig. 7). Accordingly, identification of genes expressed to a greater degree in LPS/RAN-treated rats suggested the possibility that p38/MAPKAPK-2 signaling was important for elevated levels of certain cytokine mRNAs.
The roles of IFN- in liver injury are increasingly understood. For example, acetaminophen (APAP)-induced liver injury is reduced in IFN- deficient mice (Ishida et al., 2002) and natural killer (NK) cells are thought to be an important source of IFN- in livers of APAP-treated mice (Liu et al., 2004). IFN--receptor deficiency in mice provided protection against LPS/Gal-induced hepatocellular injury (Car et al., 1994). In addition, normally noncytotoxic concentrations of IFN- and another cytokine produced after LPS-treatment, TNF-, synergistically kill hepatocytes in culture at concentrations that are not toxic when each cytokine is given alone (Adamson and Billings, 1993). Thus, augmented expression of IFN- by RAN cotreatment might render a nontoxic dose of LPS hepatotoxic. The role of IFN- in LPS/RAN-induced liver injury is not fully characterized, although future studies will characterize the role of NK cells and the effect of antibody neutralization of IFN-.
Although the focus of this discussion is on genes increased in expression by LPS/RAN-treatment, the expression level of several genes was also selectively decreased (Tables 4 and 5). These changes might also be important for liver injury. Notably, LPS/RAN-treatment caused a reduction in the expression of hepatic nuclear factor 4 (HNF4). Several studies have indicated a role for HNF in regulation of hepatocyte gene expression and homeostasis (Watt et al., 2003). Although HNF4 deficiency causes embryonic lethality, hepatocyte-specific deletion of HNF4 caused altered lipid and bile acid homeostasis (Hayhurst et al., 2001; Watt et al., 2003). Moreover, LPS treatment reduced hepatic nuclear HNF4 levels and DNA binding activity in rats (Cheng et al., 2003). Thus, a reduction in expression of HNF4 or other cytoprotective or anti-inflammatory genes by LPS/RAN-cotreatment might alter hepatocellular function and contribute to the pathogenesis of liver injury.
In summary, RAN was rendered hepatotoxic in rats undergoing a normally noninjurious inflammatory response precipitated by LPS, whereas hepatotoxicity was not observed in rats cotreated with LPS/FAM. Importantly, this result correlates with the occurrence of idiosyncratic hepatotoxicity for these two drugs in people, supporting the predictive potential of this model. Although clinical chemistry and histopathology did not distinguish RAN from FAM when given alone, marked changes in global hepatic gene expression occurred after treatment with RAN but not FAM, suggesting altered liver homeostasis. Hierarchical clustering also distinguished the effects of RAN alone on hepatic gene expression as well as those of LPS/RAN-treated rats from those given LPS alone. Thus, gene expression analysis revealed potentially deleterious changes caused by RAN alone in the absence of hepatocellular injury and, at a time before liver injury, distinguished LPS/RAN-treated rats destined to develop injury from those given LPS alone. Ideally, it would be interesting to compare the expression profile elicited by LPS/RAN-treatment with that of a hepatotoxic dose of RAN. However, increasing doses of RAN resulted in animal deaths without causing hepatocellular injury (Luyendyk et al., 2003). Accordingly, though RAN did not cause hepatocellular injury alone, LPS pretreatment revealed both the hepatotoxic potential of RAN and gene expression changes likely to be important in LPS/RAN-treated rats. Importantly, neither FAM alone nor LPS/FAM could be distinguished from respective controls either by gene expression or clinical chemistry/histopathology.
Statistical filtering of gene expression data revealed genes expressed only, or to a greater extent, in livers of LPS/RAN-treated rats. Several of these were confirmed by real-time PCR, and mRNA changes for some were associated with elevated serum protein concentration. Gene expression changes caused by RAN alone, including increased expression of the VEGF receptor and MAPKAPK-2, suggested potential mechanisms through which enhancement of LPS-induced gene expression could occur. This observation led to confirmation of the hypothesis that the expression of two other potentially important cytokines not identified by gene expression filtering, IL-6 and IFN-, was enhanced in LPS/RAN-treated rats. Overall, the results indicate that LPS/RAN-cotreatment enhances the expression of genes encoding products involved in processes known to be critical to the pathogenesis of LPS/RAN-induced hepatocellular injury, including enhancement of coagulation, inhibition of fibrinolysis, enhanced PMN chemotaxis, and hypoxia-inducible cell death. These gene expression results not only suggested potential mechanisms of LPS/RAN pathogenesis, but could aid in the identification of biomarkers to predict idiosyncratic toxicity of drugs.
|
SUPPLEMENTARY DATA |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
|---|
Supplementary data are available online at http://toxsci.oxfordjournals.org/. Genes differentially expressed in LPS/RAN-treated rats compared to other treatment groups are shown in Supplemental Table 1. Supplemental Tables 2–8 contain genes displayed in Venn diagram format in Figure 6. Supplemental Table 9 contains genes differentially expressed in LPS/RAN-treated rats as a consequence of a RAN effect. Statistical filters applied to generate these lists are described in the Results section.
|
ACKNOWLEDGMENTS |
|---|
This work was supported by a grant from the National Institutes of Health (DK061315) and by Bristol-Myers Squibb. J.P.L. was partially supported by training grant number 5 T32 ES07255 from the National Institute of Environmental Health Sciences (NIEHS), and The Society of Toxicology's Novartis Graduate Fellowship. The authors wish to thank Sandra Newport and Colin North for assistance with the in vivo portion of these studies.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
|---|
Adamson, G. M., and Billings, R. E. (1993). Cytokine toxicity and induction of NO synthase activity in cultured mouse hepatocytes. Toxicol Appl. Pharmacol. 119, 100–107.[CrossRef][Web of Science][Medline]
Balabanian, K., Lagane, B., Infantino, S., Chow, K. Y., Harriague, J., Moepps, B., Arenzana-Seisdedos, F., Thelen, M., and Bachelerie, F. (2005). The chemokine SDF-1/CXCL12 binds to and signals through the orphan receptor RDC1 in T lymphocytes. J. Biol. Chem. 280, 35760–35766.
Bertini, R., Allegretti, M., Bizzarri, C., Moriconi, A., Locati, M., Zampella, G., Cervellera, M. N., Di, C. V., Cesta, M. C., Galliera, et al. (2004). Noncompetitive allosteric inhibitors of the inflammatory chemokine receptors CXCR1 and CXCR2: Prevention of reperfusion injury. Proc. Natl. Acad. Sci. U.S.A. 101, 11791–11796.
Bruick, R. K. (2000). Expression of the gene encoding the proapoptotic Nip3 protein is induced by hypoxia. Proc. Natl. Acad. Sci. U.S.A. 97, 9082–9087.
Buchweitz, J. P., Ganey, P. E., Bursian, S. J., and Roth, R. A. (2002). Underlying endotoxemia augments toxic responses to chlorpromazine: Is there a relationship to drug idiosyncrasy? J. Pharmacol. Exp. Ther. 300, 460–467.
Car, B. D., Eng, V. M., Schnyder, B., Ozmen, L., Huang, S., Gallay, P., Heumann, D., Aguet, M., and Ryffel, B. (1994). Interferon gamma receptor deficient mice are resistant to endotoxic shock. J. Exp. Med. 179, 1437–1444.
Clauss, M., Weich, H., Breier, G., Knies, U., Rockl, W., Waltenberger, J., and Risau, W. (1996). The vascular endothelial growth factor receptor Flt-1 mediates biological activities. Implications for a functional role of placenta growth factor in monocyte activation and chemotaxis. J. Biol. Chem. 271, 17629–17634.
Cheng, P. Y., Wang, M., and Morgan, E. T. (2003). Rapid transcriptional suppression of rat cytochrome P450 genes by endotoxin treatment and its inhibition by curcumin. J. Pharmacol. Exp. Ther. 307, 1205–1212.
Dean, J. L., Sully, G., Clark, A. R., and Saklatvala, J. (2004). The involvement of AU-rich element-binding proteins in p38 mitogen-activated protein kinase pathway-mediated mRNA stabilisation. Cell Signal. 16, 1113–1121.[CrossRef][Web of Science][Medline]
Dorman, R. B., Gujral, J. S., Bajt, M. L., Farhood, A., and Jaeschke, H. (2005). Generation and functional significance of CXC chemokines for neutrophil-induced liver injury during endotoxemia. Am. J. Physiol. Gastrointest. Liver Physiol. 288, G880–G886.
Freeman, R. S., Hasbani, D. M., Lipscomb, E. A., Straub, J. A., and Xie, L. (2003). SM-20, EGL-9, and the EGLN family of hypoxia-inducible factor prolyl hydroxylases. Mol. Cells 16, 1–12.[Web of Science][Medline]
Ganey, P. E., Barton, Y. W., Kinser, S., Sneed, R. A., Barton, C. C., and Roth, R. A. (2001). Involvement of cyclooxygenase-2 in the potentiation of allyl alcohol-induced liver injury by bacterial lipopolysaccharide. Toxicol. Appl. Pharmacol. 174, 113–121.[CrossRef][Web of Science][Medline]
Ganey, P. E., Luyendyk, J. P., Maddox, J. F., and Roth, R. A. (2004). Adverse hepatic drug reactions: inflammatory episodes as consequence and contributor. Chem. Biol. Interact. 150, 35–51.[CrossRef][Web of Science][Medline]
Ganey, P. E., and Roth, R. A. (2001). Concurrent inflammation as a determinant of susceptibility to toxicity from xenobiotic agents. Toxicology 169, 195–208.[CrossRef][Web of Science][Medline]
Hayhurst, G. P., Lee, Y. H., Lambert, G., Ward, J. M., and Gonzalez, F. J. (2001). Hepatocyte nuclear factor 4alpha (nuclear receptor 2A1) is essential for maintenance of hepatic gene expression and lipid homeostasis. Mol. Cell Biol. 21, 1393–1403.
Ishida, Y., Kondo, T., Ohshima, T., Fujiwara, H., Iwakura, Y., and Mukaida, N. (2002). A pivotal involvement of IFN-gamma in the pathogenesis of acetaminophen-induced acute liver injury. FASEB J. 16, 1227–1236.
Ju, C., and Uetrecht, J. P. (2002). Mechanism of idiosyncratic drug reactions: Reactive metabolite formation, protein binding and the regulation of the immune system. Curr. Drug Metab. 3, 367–377.[CrossRef][Medline]
Kietzmann, T., Jungermann, K., and Gorlach, A. (2003). Regulation of the hypoxia-dependent plasminogen activator inhibitor 1 expression by MAP kinases. Thromb. Haemost. 89, 666–673.[Web of Science][Medline]
Kotlyarov, A., and Gaestel, M. (2002). Is MK2 (mitogen-activated protein kinase-activated protein kinase 2) the key for understanding post-transcriptional regulation of gene expression? Biochem. Soc. Trans. 30, 959–963.[CrossRef][Web of Science][Medline]
Kotlyarov, A., Neininger, A., Schubert, C., Eckert, R., Birchmeier, C., Volk, H. D., and Gaestel, M. (1999). MAPKAP kinase 2 is essential for LPS-induced TNF-alpha biosynthesis. Nat. Cell Biol. 1, 94–97.[CrossRef][Web of Science][Medline]
Labib, R., Turkall, R., and bdel-Rahman, M. S. (2002). Endotoxin potentiates the hepatotoxicity of cocaine in male mice. J. Toxicol. Environ. Health A 65, 977–993.[CrossRef][Web of Science][Medline]
Lasa, M., Mahtani, K. R., Finch, A., Brewer, G., Saklatvala, J., and Clark, A. R. (2000). Regulation of cyclooxygenase 2 mRNA stability by the mitogen-activated protein kinase p38 signaling cascade. Mol. Cell Biol. 20, 4265–4274.
Lin, B., Kolluri, S. K., Lin, F., Liu, W., Han, Y. H., Cao, X., Dawson, M. I., Reed, J. C., and Zhang, X. K. (2004). Conversion of Bcl-2 from protector to killer by interaction with nuclear orphan receptor Nur77/TR3. Cell 116, 527–540.[CrossRef][Web of Science][Medline]
Lin, J. H. (1991). Pharmacokinetic and pharmacodynamic properties of histamine H2-receptor antagonists. Relationship between intrinsic potency and effective plasma concentrations. Clin. Pharmacokinet. 20, 218–236.[Web of Science][Medline]
Lind, R. C., Gandolfi, A. J., Sipes, I. G., and Brown, B. R. J. (1984). The involvement of endotoxin in halothane-associated liver injury. Anesthesiology 61, 544–550.[Web of Science][Medline]
Liu, L., Tsai, J. C., and Aird, W. C. (2000). Egr-1 gene is induced by the systemic administration of the vascular endothelial growth factor and the epidermal growth factor. Blood 96, 1772–1781.
Liu, Z. X., Govindarajan, S., and Kaplowitz, N. (2004). Innate immune system plays a critical role in determining the progression and severity of acetaminophen hepatotoxicity. Gastroenterology 127, 1760–1774.[CrossRef][Web of Science][Medline]
Luyendyk, J. P., Lehman-McKeeman, L. D., Nelson, D. M., Bhaskaran, V., Car, B. D., Cantor, G. H., North, C. M., Newport, S. W., Maddox, J. F., Ganey, P. E., et al. (2005a). Augmentation of lipopolysaccharide-induced gene expression and liver injury by ranitidine, but not famotidine. Toxicol. Sci. 84(S-1), 1930 (Abstract).
Luyendyk, J. P., Maddox, J. F., Green, C. D., Ganey, P. E., and Roth, R. A. (2004a). Role of hepatic fibrin in idiosyncrasy-like liver injury from lipopolysaccharide-ranitidine coexposure in rats. Hepatology 40, 1342–1351.[CrossRef][Web of Science][Medline]
Luyendyk, J. P., Maddox, J. F., Cosma, G. N., Ganey, P. E., Cockerell, G. L., and Roth, R. A. (2003). Ranitidine treatment during a modest inflammatory response precipitates idiosyncrasy-like liver injury in rats. J. Pharmacol. Exp. Ther. 307, 9–16.
Luyendyk, J. P., Mattes, W. B., Burgoon, L. D., Zacharewski, T. R., Maddox, J. F., Cosma, G. N., Ganey, P. E., and Roth, R. A. (2004b). Gene expression analysis points to hemostasis in livers of rats cotreated with lipopolysaccharide and ranitidine. Toxicol. Sci. 80, 203–213.
Luyendyk, J. P., Shaw, P. J., Green, C. D., Maddox, J. F., Ganey, P. E., and Roth, R. A. (2005b). Coagulation-mediated hypoxia and neutrophil-dependent hepatic injury in rats given lipopolysaccharide and ranitidine. J. Pharmacol. Exp. Ther. 314, 1023–1031.
Mavropoulos, A., Sully, G., Cope, A. P., and Clark, A. R. (2005). Stabilization of IFN-gamma mRNA by MAPK p38 in IL-12- and IL-18-stimulated human NK cells. Blood 105, 282–288.
Mechtcheriakova, D., Wlachos, A., Holzmuller, H., Binder, B. R., and Hofer, E. (1999). Vascular endothelial cell growth factor-induced tissue factor expression in endothelial cells is mediated by EGR-1. Blood 93, 3811–3823.
Neininger, A., Kontoyiannis, D., Kotlyarov, A., Winzen, R., Eckert, R., Volk, H. D., Holtmann, H., Kollias, G., and Gaestel, M. (2002). MK2 targets AU-rich elements and regulates biosynthesis of tumor necrosis factor and interleukin-6 independently at different post-transcriptional levels. J. Biol. Chem. 277, 3065–3068.
Okada, M., Fujita, T., Sakaguchi, T., Olson, K. E., Collins, T., Stern, D. M., Yan, S. F., and Pinsky, D. J. (2001). Extinguishing Egr-1-dependent inflammatory and thrombotic cascades after lung transplantation. FASEB J. 15, 2757–2759.
Pawlinski, R., Pedersen, B., Kehrle, B., Aird, W. C., Frank, R. D., Guha, M., and Mackman, N. (2003). Regulation of tissue factor and inflammatory mediators by Egr-1 in a mouse endotoxemia model. Blood 101, 3940–3947.
Pawlinski, R., Pedersen, B., Schabbauer, G., Tencati, M., Holscher, T., Boisvert, W., Andrade-Gordon, P., Frank, R. D., and Mackman, N. (2004). Role of tissue factor and protease-activated receptors in a mouse model of endotoxemia. Blood 103, 1342–1347.
Pirmohamed, M., Madden, S., and Park, B. K. (1996). Idiosyncratic drug reactions. Metabolic bioactivation as a pathogenic mechanism. Clin. Pharmacokinet. 31, 215–230.[Web of Science][Medline]
Roth, R. A., Luyendyk, J. P., Maddox, J. F., and Ganey, P. E. (2003). Inflammation and drug idiosyncrasy–is there a connection? J. Pharmacol. Exp. Ther. 307, 1–8.
Rousseau, S., Morrice, N., Peggie, M., Campbell, D. G., Gaestel, M., and Cohen, P. (2002). Inhibition of SAPK2a/p38 prevents hnRNP A0 phosphorylation by MAPKAP-K2 and its interaction with cytokine mRNAs. EMBO J. 21, 6505–6514.[CrossRef][Web of Science][Medline]
Scarpignato, C., Tramacere, R., and Zappia, L. (1987). Antisecretory and antiulcer effect of the H2-receptor antagonist famotidine in the rat: Comparison with ranitidine. Br. J. Pharmacol. 92, 153–159.[Web of Science][Medline]
Shoshani, T., Faerman, A., Mett, I., Zelin, E., Tenne, T., Gorodin, S., Moshel, Y., Elbaz, S., Budanov, A., Chajut, A., et al. (2002). Identification of a novel hypoxia-inducible factor 1-responsive gene, RTP801, involved in apoptosis. Mol. Cell Biol. 22, 2283–2293.
Straub, J. A., Lipscomb, E. A., Yoshida, E. S., and Freeman, R. S. (2003). Induction of SM-20 in PC12 cells leads to increased cytochrome c levels, accumulation of cytochrome c in the cytosol, and caspase-dependent cell death. J. Neurochem. 85, 318–328.[Web of Science][Medline]
Struyf, S., Gouwy, M., Dillen, C., Proost, P., Opdenakker, G., and Van, D. J. (2005). Chemokines synergize in the recruitment of circulating neutrophils into inflamed tissue. Eur. J. Immunol. 35, 1583–1591.[CrossRef][Web of Science][Medline]
Suratt, B. T., Petty, J. M., Young, S. K., Malcolm, K. C., Lieber, J. G., Nick, J. A., Gonzalo, J. A., Henson, P. M., and Worthen, G. S. (2004). Role of the CXCR4/SDF-1 chemokine axis in circulating neutrophil homeostasis. Blood 104, 565–571.
Tafazoli, S., Spehar, D. D., and O'Brien, P. J. (2005). Oxidative stress mediated idiosyncratic drug toxicity. Drug Metab Rev. 37, 311–325.[Web of Science][Medline]
Vial, T., Goubier, C., Bergeret, A., Cabrera, F., Evreux, J. C., and Descotes, J. (1991). Side effects of ranitidine. Drug Saf. 6, 94–117.[Web of Science][Medline]
Watt, A. J., Garrison, W. D., and Duncan, S. A. (2003). HNF4: A central regulator of hepatocyte differentiation and function. Hepatology 37, 1249–1253.[CrossRef][Web of Science][Medline]
Winzen, R., Kracht, M., Ritter, B., Wilhelm, A., Chen, C. Y., Shyu, A. B., Muller, M., Gaestel, M., Resch, K., and Holtmann, H. (1999). The p38 MAP kinase pathway signals for cytokine-induced mRNA stabilization via MAP kinase-activated protein kinase 2 and an AU-rich region-targeted mechanism. EMBO J. 18, 4969–4980.[CrossRef][Web of Science][Medline]
Xiong, M., Elson, G., Legarda, D., and Leibovich, S. J. (1998). Production of vascular endothelial growth factor by murine macrophages: Regulation by hypoxia, lactate, and the inducible nitric oxide synthase pathway. Am. J. Pathol. 153, 587–598.
Yan, S. F., Fujita, T., Lu, J., Okada, K., Shan, Z. Y., Mackman, N., Pinsky, D. J., and Stern, D. M. (2000). Egr-1, a master switch coordinating upregulation of divergent gene families underlying ischemic stress. Nat. Med. 6, 1355–1361.[CrossRef][Web of Science][Medline]
Yoo, Y. G., Yeo, M. G., Kim, D. K., Park, H., and Lee, M. O. (2004). Novel function of orphan nuclear receptor Nur77 in stabilizing hypoxia-inducible factor-1alpha. J. Biol. Chem. 279, 53365–53373.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  X. Deng, J. Lu, L. D. Lehman-McKeeman, E. Malle, D. L. Crandall, P. E. Ganey, and R. A. Roth p38 Mitogen-Activated Protein Kinase-Dependent Tumor Necrosis Factor-{alpha}-Converting Enzyme Is Important for Liver Injury in Hepatotoxic Interaction between Lipopolysaccharide and Ranitidine J. Pharmacol. Exp. Ther., July 1, 2008; 326(1): 144 - 152. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. F. Tukov, J. P. Luyendyk, P. E. Ganey, and R. A. Roth The Role of Tumor Necrosis Factor Alpha in Lipopolysaccharide/Ranitidine-Induced Inflammatory Liver Injury Toxicol. Sci., November 1, 2007; 100(1): 267 - 280. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||