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.
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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.
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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