Background: Rising morbidity and mortality related to the use of NSAIDs has led to the withdrawal of some of these agents and reconsideration of the adverse effects and usage paradigms of commonly available NSAIDs. Our objective in this study was to assay molecular indicators of acute hepatic injury associated with the administration of indomethacin, a prototypical NSAID, metabolized by the liver that undergoes enterohepatic circulation with associated gastrointestinal adverse effects.
Methods: Analysis of gene expression, using high-throughput, ADME (absorption, distribution, metabolism, excretion)-specific microarrays, was performed on RNA extracted from the livers of control or indomethacin treated rats, in parallel with serum enzyme tests and histological analysis of paraffin-embedded liver specimens. Male Sprague-Dawley rats (n = 45) were administered intraperitoneal injections of indomethacin for 3 days at the recommended normal dose (6.7 mg/kg), indomethacin at a high dose (20 mg/kg) or vehicle alone (controls).
Results: Upon termination of the study on day 4, serum γ-glutamyl transferase activity and alkaline phosphatase/alanine aminotransferase ratios were significantly elevated in both high- and normal-dose cohorts compared with vehicle-treated animals. Diffuse microvascular steatosis was present in hepatic serial sections obtained from all animals subjected to the high-dosage regimen. High-resolution microarray analysis (six replicates/gene/animal) identified 256 genes, after outlier removal, in 17 functional classifications that were significantly altered by the high, but not by the normal dosage. These included depression of 10 of 11 cytochrome P450 genes (2B3, 2C70, 1A2-P2, 4F1, 2E1, 3A1, 2F1, 3AP7, 2C11, phenobarb-inducible P6) and 7 of 9 genes involved in the response to reactive oxygen species (e.g. glutathione reductase, glutathione transferase, and Superoxide dismutase). Of 16 genes associated with toxin removal, nine exhibited significantly decreased transcripts. There was a marked shift away from lipid metabolism (decreased expression of eight genes) towards glucose utilization associated with steatosis. Despite the compromise of detoxification programs and a shift in metabolic substrate utilization, a compensatory remodeling response was activated, including genes for metalloproteases (ADAM10, MMP10, MMP11), integrins (integrin α-1 and α-E1), and extracellular matrix molecules (platelet/endothelial cell adhesion molecule-1 and heparan sulfate proteoglycan, perlecan), as well as transcripts associated with cell proliferation. The expression levels of only five genes were significantly altered among animals receiving the normal indomethacin dosage.
Conclusion: These data confirmed that even brief exposure to indomethacin altered serum enzymatic activities and that high levels significantly altered gene expression in the liver and hepatic histology (by interfering with the clearance of toxins and xenobiotic substrates) and the regulation of basal metabolism.
This is a preview of subscription content, log in to check access.
Buy single article
Instant access to the full article PDF.
Price includes VAT for USA
Davies G, Martin LA, Sacks N, et al. Cyclooxygenase-2 (COX-2), aromatase and breast cancer: a possible role for COX-2 inhibitors in breast cancer chemoprevention. Ann Oncol 2002; 13: 669–78
Mohammed SI, Craig BA, Mutsaers AJ, et al. Effects of the cyclooxygenase inhibitor, piroxicam, in combination with chemotherapy on tumor response, apoptosis, and angiogenesis in a canine model of human invasive urinary bladder cancer. Mol Cancer Ther 2003; 2: 183–8
Wolfe MM, Lichtenstein DR, Singh G. Gastrointestinal toxicity of non-steroidal antiinflammatory drugs. N Engl J Med 1999; 340: 1888–99
Graham DJ. Risk of acute myocardial infarction and sudden death in patients treated with COX-2 selective and non-selective NSAIDs. Memorandum: United States Food and Drug Administration Public Health Advisory: safety of Vioxx [online]. Available from URL: http://www.fda.gov/cder/drug/infopage/vioxx/default.gov [Accessed 2004 Sep 30]
US Food and Drug Administration. FDA News. FDA Announces Series of Changes to the Class of Marketed Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) [online]. Available from URL: http://www.fda.gov/bbs/topics/news/2005/NEW01171 [Accessed 2005 Apr 7]
Falzon M, Whiting PH, Ewen SWB, et al. Comparative effects of indomethacin on hepatic enzymes and histology and on serum indices of liver and kidney function in rat. Br J Exp Pathol 1985; 66: 527–34
Lennon GC. High-throughput gene expression analysis for drug discovery. Drug Discov Today 2000; 5: 59–66
Lockhart DJ. Genomics, gene expression and DNA arrays. Nature 2000; 405: 827–36
Rubenstein K. Commercial aspects of microarray technology: microarrays in cancer: research and applications. Biotechniques 2003 Mar; Suppl.: 52-4
Panda S, Sata TK, Hampton GM, et al. An array of insights: application of DNA chip technology in the study of cell biology. Trends Cell Biol 2003; 13: 151–6
Tan PK, Downey TJ, Spitznagel EL, et al. Evaluation of gene expression measurements from commercial microarray platforms. Nucleic Acids Res 2003; 31: 5676–84
Quakenbush J. Computational analysis of microarray data. Nat Rev Genet 2001; 2: 418–27
Wallenstein MC, Mauss EA. Effect of prostaglandin synthetase inhibitors on experimentally induced convulsions in rats. Pharmacology 1984; 29: 85–93
LaFramboise WA, Bombach KL, Dhir RJ, et al. Molecular dynamics of the compensatory response to myocardial infarct. J Mol Cell Cardiol 2005; 38: 103–17
Hampel FR, Ronchetti EM, Rousseeuw PJ, et al. Robust statistics: the approach based on influence functions. New York: John Wiley & Sons, 1986
Huber PJ. Robust statistics. New York: John Wiley & Sons, 1981
Analytical Methods Committee. Robust statistics: a method of coping with outliers. AMC Technical Brief. London: © Royal Society of Chemistry; 2001 No.6
. Medical College of Wisconsin. Rat genome database [online]. Available from URL: http://rgd.mcw.edu/ [Accessed 2006 May 1]
Dennis G, Sherman BT, Hosack DA, et al. DAVID: Database for Annotation, Visualization, and Integrated Discovery. Genome Biol 2003; 4(5): 3
Université René Descartes, Paris. GENATLAS [online]. Available from URL: http://www.dsi.univ-paris5.fr/genatlas [Accessed 2006 May 1]
Rebhan M, Chalifa-Caspi V, Prilusky J, et al. GeneCards: encyclopedia for genes, proteins and diseases [online]. Weizmann Institute of Science, Bioinformatics Unit and Genome Center. Available from URL: http://bioinformatics.weizmann.ac.il/cards [Accessed 2006 May 1]
LaFramboise WA, Bombach KL, Pogozelski AR, et al. Microarray data source: hepatic gene expression response to acute indomethacin exposure [online]. Available from URL: http://bioinformatics.upmc.edu/laframboise/papers/indo/indo.htm [Accessed 2006 May 1]
Patel S, Lyons-Weiler J. caGEDA: a web application for the integrated analysis of global gene expression patterns in cancer [online]. Appl Bioinformatics 2004; 3 (1): 49-62. Available from URL: http://bioinformatics.upmc.edu [Accessed 2006 May 1]
Tusher VG, Tibshirani R, Chu G. Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci U S A 2001; 98(9): 5116–21
Schumacher HR, Boice JA, Daikh DI, et al. Randomised double blind trial of etoricoxib and indomethacin in treatment of acute gouty arthritis. BMJ 2002; 324: 1488–92
Rubin BR, Burton R, Navarra S, et al. Efficacy and safety profile of treatment with etoricoxib 120mg once daily compared with indomethacin 50mg three times daily in acute gout. Arthritis Rheum 2004; 50: 598–606
Fosslien E. Mitochondrial medicine-molecular pathology of defective oxidative phosphorylation. Ann Clin Lab Sci 2001; 31(1): 25–67
Treem WR, Sokol RJ. Disorders of the mitochondria. Semin Liver Dis 1998; 18: 237–53
Waring JF, Jolly RA, Ciurlionis R, et al. Clustering of hepatotoxins based on mechanism of toxicity using gene expression profiles. Toxicol Appl Pharmacol 2001; 175: 28–42
Omogbai EK, Ozoluna RI, Idaesor PE, et al. Some studies on the rodenticidal action of indomethacin. Drug Chem Toxicol 1999; 22: 629–42
McCarthy DM. Comparative toxicity of nonsteroidal anti-inflammatory drugs. Am J Med 1999; 107: 37S–46S
Yamada T, Hoshino M, Hayakawa T, et al. Bile secretion in rats with indomethacin-induced intestinal inflammation. Am J Physiol 1996; 270: G804–12
Lee M, Kou LT, Whitmore GA, et al. Importance of replication in microarray gene expression studies: statistical methods and evidence from repetitive cDNA hybridizations. Proc Natl Acad Sci U S A 2000; 97: 9834–9
Lyng H, Badiee A, Svendsrud DH, et al. Profound influence of microarray scanner characteristics on gene expression ratios: analysis and procedure for correction. BMC Genomics 2004; 5: 10
Financial support for this study was provided by the Allegheny Heart Institute (#399109), 320 East North Avenue, Pittsburgh, Pennsylvania, PA, USA. The authors express their sincere thanks to Dr Scott Magnuson of GenUs Biosystems (Northbrook, IL, USA) for his support and advice in the early design and performance of this study, Jim George and Mike Pazdon of GE Healthcare Life Sciences (Piscataway, NJ, USA) for their technical support and expertise in establishing the CodeLink™ system in our laboratory at Allegheny General Hospital, and to Ms Mary Wisniewski for the help and services provided by the UPMC clinical chemistry laboratory at Shadyside Hospital. The authors have no conflict of interest pertaining to the conduct or contents of the study and this manuscript.
About this article
Cite this article
LaFramboise, W.A., Bombach, K.L., Pogozelski, A.R. et al. Hepatic Gene Expression Response to Acute Indomethacin Exposure. Mol Diag Ther 10, 187–196 (2006). https://doi.org/10.1007/BF03256457
- Outlier Removal
- Triacyl Glycerol Lipase
- Microvesicular Steatosis
- Indomethacin Administration