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Diclofenac toxicity in human intestine ex vivo is not related to the formation of intestinal metabolites

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Abstract

The use of diclofenac (DCF), a nonsteroidal anti-inflammatory drug, is associated with a high prevalence of gastrointestinal side effects. In vivo studies in rodents suggested that reactive metabolites of DCF produced by the liver or the intestine might be responsible for this toxicity. In the present study, precision-cut intestinal slices (PCIS) prepared from the jejunum of 18 human donors were used as an ex vivo model to investigate whether DCF intestinal metabolites are responsible for its intestinal toxicity in man. PCIS were incubated with a concentration range of DCF (0–600 µM) up to 24 h. DCF (≥400 µM) caused direct toxicity to the intestine as demonstrated by ATP depletion, morphological damage, caspase 3 activation, and lactate dehydrogenase leakage. Three main metabolites produced by PCIS (4′-hydroxy DCF, 5-hydroxy DCF, and DCF acyl glucuronide) were detected by HPLC. Protein adducts were detected by immunohistochemical staining and showed correlation with the intestinal metabolites. DCF induced similar toxicity to each of the samples regardless of the variation in metabolism among them. Less metabolites were produced by slices incubated with 400 µM DCF than with 100 µM DCF. The addition of the metabolic inhibitors such as ketoconazole, cimetidine, or borneol decreased the metabolite formation but increased the toxicity. The results suggest that DCF can induce intestinal toxicity in human PCIS directly at therapeutically relevant concentrations, independent of the reactive metabolites 4′-OH DCF, 5-OH DCF, or diclofenac acylglucuronide produced by the liver or formed in the intestine.

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References

  • Allison MC, Howatson AG, Torrance CJ, Lee FD, Russell RI (1992) Gastrointestinal damage associated with the use of nonsteroidal antiinflammatory drugs. N Engl J Med 327:749–754

    Article  CAS  PubMed  Google Scholar 

  • Atchison CR, West AB, Balakumaran A, Hargus SJ, Pohl LR, Daiker DH, Aronson JF, Hoffmann WE, Shipp BK, Treinen-Moslen M (2000) Drug enterocyte adducts: possible causal factor for diclofenac enteropathy in rats. Gastroenterology 119:1537–1547

    Article  CAS  PubMed  Google Scholar 

  • Bansal R, Prakash J, de Ruijter M, Beljaars L, Poelstra K (2011) Peptide-modified albumin carrier explored as a novel strategy for a cell-specific delivery of interferon gamma to treat liver fibrosis. Mol Pharm 8:1899–1909

    Article  CAS  PubMed  Google Scholar 

  • Bjarnason I, Hayllar J, MacPherson AJ, Russell AS (1993) Side effects of nonsteroidal anti-inflammatory drugs on the small and large intestine in humans. Gastroenterology 104:1832–1847

    CAS  PubMed  Google Scholar 

  • Bjornsson E, Westgaard G, Bjarnason I (2008) Severe injury to the small bowel associated with a short course of diclofenac. Scand J Gastroenterol 43:759–760

    Article  PubMed  Google Scholar 

  • Boelsterli UA, Ramirez-Alcantara V (2011) NSAID acyl glucuronides and enteropathy. Curr Drug Metab 12:245–252

    Article  CAS  PubMed  Google Scholar 

  • Boelsterli UA, Redinbo MR, Saitta KS (2013) Multiple NSAID-induced hits injure the small intestine: underlying mechanisms and novel strategies. Toxicol Sci 131:654–667

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Bort R, Ponsoda X, Jover R, Gomez-Lechon MJ, Castell JV (1999) Diclofenac toxicity to hepatocytes: a role for drug metabolism in cell toxicity. J Pharmacol Exp Ther 288:65–72

    CAS  PubMed  Google Scholar 

  • Boushey RP, Yusta B, Drucker DJ (1999) Glucagon-like peptide 2 decreases mortality and reduces the severity of indomethacin-induced murine enteritis. Am J Physiol 277:E937–E947

    CAS  PubMed  Google Scholar 

  • Cheng YT, Wu CH, Ho CY, Yen GC (2012) Catechin protects against ketoprofen-induced oxidative damage of the gastric mucosa by up-regulating Nrf2 in vitro and in vivo. J Nutr Biochem 24:475–483

    Article  PubMed  Google Scholar 

  • Cho S, Wood A, Bowlby MR (2007) Brain slices as models for neurodegenerative disease and screening platforms to identify novel therapeutics. Curr Neuropharmacol 5:19–33

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Davies NM, Saleh JY, Skjodt NM (2000) Detection and prevention of NSAID-induced enteropathy. J Pharm Pharm Sci 3:137–155

    CAS  PubMed  Google Scholar 

  • Fortun PJ, Hawkey CJ (2007) Nonsteroidal antiinflammatory drugs and the small intestine. Curr Opin Gastroenterol 23:134–141

    CAS  PubMed  Google Scholar 

  • Galetin A, Houston JB (2006) Intestinal and hepatic metabolic activity of five cytochrome P450 enzymes: impact on prediction of first-pass metabolism. J Pharmacol Exp Ther 318:1220–1229

    Article  CAS  PubMed  Google Scholar 

  • Graaf IA, Groothuis GM, Olinga P (2007) Precision-cut tissue slices as a tool to predict metabolism of novel drugs. Expert Opin Drug Metab Toxicol 3:879–898

    Article  PubMed  Google Scholar 

  • Groothuis GM, de Graaf IA (2012) Precision-cut intestinal slices as in vitro tool for studies on drug metabolism. Curr Drug Metab 14:112–119

    Article  Google Scholar 

  • Guengerich FP (2005) Principles of covalent binding of reactive metabolites and examples of activation of bis-electrophiles by conjugation. Arch Biochem Biophys 433:369–378

    Article  CAS  PubMed  Google Scholar 

  • Hargus SJ, Amouzedeh HR, Pumford NR, Myers TG, McCoy SC, Pohl LR (1994) Metabolic activation and immunochemical localization of liver protein adducts of the nonsteroidal anti-inflammatory drug diclofenac. Chem Res Toxicol 7:575–582

    Article  CAS  PubMed  Google Scholar 

  • Hui L, Gao S, Liu T, Cao C, Guo J, Hao R, Yi Y, Li C, Zhao Y, Liang A (2011) Evaluation of vitro hepatotoxicity of monocrotaline by precision-cut liver slice technique. Zhongguo Zhong Yao Za Zhi 36:628–632

    PubMed  Google Scholar 

  • Kalgutkar AS, Didiuk MT (2009) Structural alerts, reactive metabolites, and protein covalent binding: how reliable are these attributes as predictors of drug toxicity? Chem Biodivers 6:2115–2137

    Article  CAS  PubMed  Google Scholar 

  • Kararli TT (1995) Comparison of the gastrointestinal anatomy, physiology, and biochemistry of humans and commonly used laboratory animals. Biopharm Drug Dispos 16:351–380

    Article  CAS  PubMed  Google Scholar 

  • Kasper M, Seidel D, Knels L, Morishima N, Neisser A, Bramke S, Koslowski R (2004) Early signs of lung fibrosis after in vitro treatment of rat lung slices with CdCl2 and TGF-beta1. Histochem Cell Biol 121:131–140

    Article  CAS  PubMed  Google Scholar 

  • Kenny JR, Maggs JL, Meng X, Sinnott D, Clarke SE, Park BK, Stachulski AV (2004) Syntheses and characterization of the acyl glucuronide and hydroxy metabolites of diclofenac. J Med Chem 47:2816–2825

    Article  CAS  PubMed  Google Scholar 

  • Khojasteh SC, Oishi S, Nelson SD (2010) Metabolism and toxicity of menthofuran in rat liver slices and in rats. Chem Res Toxicol 23:1824–1832

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Kretz-Rommel A, Boelsterli UA (1993) Diclofenac covalent protein binding is dependent on acyl glucuronide formation and is inversely related to P450-mediated acute cell injury in cultured rat hepatocytes. Toxicol Appl Pharmacol 120:155–161

    Article  CAS  PubMed  Google Scholar 

  • Kretz-Rommel A, Boelsterli UA (1994) Mechanism of covalent adduct formation of diclofenac to rat hepatic microsomal proteins. Retention of the glucuronic acid moiety in the adduct. Drug Metab Dispos 22:956–961

    CAS  PubMed  Google Scholar 

  • Laine L, Goldkind L, Curtis SP, Connors LG, Yanqiong Z, Cannon CP (2009) How common is diclofenac-associated liver injury? Analysis of 17,289 arthritis patients in a long-term prospective clinical trial. Am J Gastroenterol 104:356–362

    Article  PubMed  Google Scholar 

  • Lim MS, Lim PL, Gupta R, Boelsterli UA (2006) Critical role of free cytosolic calcium, but not uncoupling, in mitochondrial permeability transition and cell death induced by diclofenac oxidative metabolites in immortalized human hepatocytes. Toxicol Appl Pharmacol 217:322–331

    Article  CAS  PubMed  Google Scholar 

  • LoGuidice A, Ramirez-Alcantara V, Proli A, Gavillet B, Boelsterli UA (2010) Pharmacologic targeting or genetic deletion of mitochondrial cyclophilin D protects from NSAID-induced small intestinal ulceration in mice. Toxicol Sci 118:276–285

    Article  CAS  PubMed  Google Scholar 

  • LoGuidice A, Wallace BD, Bendel L, Redinbo MR, Boelsterli UA (2012) Pharmacologic targeting of bacterial beta-glucuronidase alleviates nonsteroidal anti-inflammatory drug-induced enteropathy in mice. J Pharmacol Exp Ther 341:447–454

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Macpherson AB, Jarnason I (1993) NSAIDS and gastropathy. Pharmacoeconomics 3:83–84

    Article  CAS  PubMed  Google Scholar 

  • Maiden L, Thjodleifsson B, Theodors A, Gonzalez J, Bjarnason I (2005) A quantitative analysis of NSAID-induced small bowel pathology by capsule enteroscopy. Gastroenterology 128:1172–1178

    Article  PubMed  Google Scholar 

  • Maiden L, Thjodleifsson B, Seigal A, Bjarnason II, Scott D, Birgisson S, Bjarnason I (2007) Long-term effects of nonsteroidal anti-inflammatory drugs and cyclooxygenase-2 selective agents on the small bowel: a cross-sectional capsule enteroscopy study. Clin Gastroenterol Hepatol 5:1040–1045

    Article  PubMed  Google Scholar 

  • McConnell EL, Fadda HM, Basit AW (2008) Gut instincts: explorations in intestinal physiology and drug delivery. Int J Pharm 364:213–226

    Article  CAS  PubMed  Google Scholar 

  • Miners JO, Birkett DJ (1998) Cytochrome P4502C9: an enzyme of major importance in human drug metabolism. Br J Clin Pharmacol 45:525–538

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Obach RS, Zhang QY, Dunbar D, Kaminsky LS (2001) Metabolic characterization of the major human small intestinal cytochrome p450s. Drug Metab Dispos 29:347–352

    CAS  PubMed  Google Scholar 

  • O’Brien PJ, Irwin W, Diaz D, Howard-Cofield E, Krejsa CM, Slaughter MR, Gao B, Kaludercic N, Angeline A, Bernardi P, Brain P, Hougham C (2006) High concordance of drug-induced human hepatotoxicity with in vitro cytotoxicity measured in a novel cell-based model using high content screening. Arch Toxicol 80:580–604

    Article  PubMed  Google Scholar 

  • Ponsoda X, Bort R, Jover R, Gomez-Lechon MJ, Castell JV (1995) Molecular mechanism of diclofenac hepatotoxicity: association of cell injury with oxidative metabolism and decrease in ATP levels. Toxicol In Vitro 9:439–444

    Article  CAS  PubMed  Google Scholar 

  • Poon GK, Chen Q, Teffera Y, Ngui JS, Griffin PR, Braun MP, Doss GA, Freeden C, Stearns RA, Evans DC, Baillie TA, Tang W (2001) Bioactivation of diclofenac via benzoquinone imine intermediates-identification of urinary mercapturic acid derivatives in rats and humans. Drug Metab Dispos 29:1608–1613

    CAS  PubMed  Google Scholar 

  • Power I, Cumming AD, Pugh GC (1992) Effect of diclofenac on renal function and prostacyclin generation after surgery. Br J Anaesth 69:451–456

    Article  CAS  PubMed  Google Scholar 

  • Pumford NR, Myers TG, Davila JC, Highet RJ, Pohl LR (1993) Immunochemical detection of liver protein adducts of the nonsteroidal antiinflammatory drug diclofenac. Chem Res Toxicol 6:147–150

    Article  CAS  PubMed  Google Scholar 

  • Quinn CM, Bjarnason I, Price AB (1993) Gastritis in patients on non-steroidal anti-inflammatory drugs. Histopathology 23:341–348

    Article  CAS  PubMed  Google Scholar 

  • Roskott AM, Nieuwenhuijs VB, Leuvenink HG, Dijkstra G, Ottens P, de Jager MH, Gonalves Dias Pereira P, Fidler V, Groothuis GM, Ploeg RJ, de Graaf IA (2010) Reduced ischemia-reoxygenation injury in rat intestine after luminal preservation with a tailored solution. Transplantation 90:622–629

    Article  CAS  PubMed  Google Scholar 

  • Sarda S, Page C, Pickup K, Schulz-Utermoehl T, Wilson I (2012) Diclofenac metabolism in the mouse: novel in vivo metabolites identified by high performance liquid chromatography coupled to linear ion trap mass spectrometry. Xenobiotica 42:179–194

    Article  CAS  PubMed  Google Scholar 

  • Seitz S, Boelsterli UA (1998) Diclofenac acyl glucuronide, a major biliary metabolite, is directly involved in small intestinal injury in rats. Gastroenterology 115:1476–1482

    Article  CAS  PubMed  Google Scholar 

  • Seitz S, Kretz-Rommel A, Oude Elferink RP, Boelsterli UA (1998) Selective protein adduct formation of diclofenac glucuronide is critically dependent on the rat canalicular conjugate export pump (Mrp2). Chem Res Toxicol 11:513–519

    Article  CAS  PubMed  Google Scholar 

  • Sibilia J, Deray G, Montalescot G (2006) What do we know about the cardiovascular toxicity of the NSAIDs? Presse Med 35(Suppl 1):11–23

    Article  PubMed  Google Scholar 

  • Somasundaram S, Rafi S, Hayllar J, Sigthorsson G, Jacob M, Price AB, Macpherson A, Mahmod T, Scott D, Wrigglesworth JM, Bjarnason I (1997) Mitochondrial damage: a possible mechanism of the “topical” phase of nsaid induced injury to the rat intestine. Gut 41:344–353

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Somasundaram S, Sigthorsson G, Simpson RJ, Watts J, Jacob M, Tavares IA, Rafi S, Roseth A, Foster R, Price AB, Wrigglesworth JM, Bjarnason I (2000) Uncoupling of intestinal mitochondrial oxidative phosphorylation and inhibition of cyclooxygenase are required for the development of NSAID-enteropathy in the rat. Aliment Pharmacol Ther 14:639–650

    Article  CAS  PubMed  Google Scholar 

  • Strassburg CP, Kneip S, Topp J, Obermayer-Straub P, Barut A, Tukey RH, Manns MP (2000) Polymorphic gene regulation and interindividual variation of UDP-glucuronosyltransferase activity in human small intestine. J Biol Chem 275:36164–36171

    Article  CAS  PubMed  Google Scholar 

  • Tang W (2003) The metabolism of diclofenac-enzymology and toxicology perspectives. Curr Drug Metab 4:319–329

    Article  CAS  PubMed  Google Scholar 

  • Tang W, Stearns RA, Bandiera SM, Zhang Y, Raab C, Braun MP, Dean DC, Pang J, Leung KH, Doss GA, Strauss JR, Kwei GY, Rushmore TH, Chiu SH, Baillie TA (1999) Studies on cytochrome P-450-mediated bioactivation of diclofenac in rats and in human hepatocytes: identification of glutathione conjugated metabolites. Drug Metab Dispos 27:365–372

    CAS  PubMed  Google Scholar 

  • van de Kerkhof EG, de Graaf IA, de Jager MH, Meijer DK, Groothuis GM (2005) Characterization of rat small intestinal and colon precision-cut slices as an in vitro system for drug metabolism and induction studies. Drug Metab Dispos 33:1613–1620

    Article  PubMed  Google Scholar 

  • van de Kerkhof EG, Ungell AL, Sjoberg AK, de Jager MH, Hilgendorf C, de Graaf IA, Groothuis GM (2006) Innovative methods to study human intestinal drug metabolism in vitro: precision-cut slices compared with using chamber preparations. Drug Metab Dispos 34:1893–1902

    Article  PubMed  Google Scholar 

  • van de Kerkhof EG, de Graaf IA, Groothuis GM (2007) In vitro methods to study intestinal drug metabolism. Curr Drug Metab 8:658–675

    Article  PubMed  Google Scholar 

  • Vickers AE, Rose K, Fisher R, Saulnier M, Sahota P, Bentley P (2004) Kidney slices of human and rat to characterize cisplatin-induced injury on cellular pathways and morphology. Toxicol Pathol 32:577–590

    Article  CAS  PubMed  Google Scholar 

  • Wang AG, Xia T, Yuan J, Yu RA, Yang KD, Chen XM, Qu W, Waalkes MP (2004) Effects of phenobarbital on metabolism and toxicity of diclofenac sodium in rat hepatocytes in vitro. Food Chem Toxicol 42:1647–1653

    Article  CAS  PubMed  Google Scholar 

  • Watkins JB, Klaassen CD (1983) Chemically-induced alteration of UDP-glucuronic acid concentration in rat liver. Drug Metab Dispos 11:37–40

    CAS  PubMed  Google Scholar 

  • Wolfe MM, Lichtenstein DR, Singh G (1999) Gastrointestinal toxicity of nonsteroidal antiinflammatory drugs. N Engl J Med 340:1888–1899

    Article  CAS  PubMed  Google Scholar 

  • Zhu Y, Zhang QY (2012) Role of intestinal cytochrome p450 enzymes in diclofenac-induced toxicity in the small intestine. J Pharmacol Exp Ther 343:362–370

    Article  CAS  PubMed Central  PubMed  Google Scholar 

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Acknowledgments

The authors thank Dr. J. H. Proost and Dr. A. Taneja (University of Groningen) for their advice on the statistical analysis. Dr. Leonie Beljaars (University of Groningen) is acknowledged for her advice on the staining intensity measurement.

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The authors declare no conflict of interest.

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Authors and Affiliations

  1. Division of Pharmacokinetics, Toxicology and Targeting, Department of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV, Groningen, The Netherlands

    Xiaoyu Niu, Inge A. M. de Graaf, Miriam Langelaar-Makkinje & Geny M. M. Groothuis

  2. Division of Analytical Biochemistry, Department of Pharmacy, University of Groningen, Groningen, The Netherlands

    Peter Horvatovich

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  1. Xiaoyu Niu
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  2. Inge A. M. de Graaf
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Correspondence to Geny M. M. Groothuis.

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204_2014_1242_MOESM1_ESM.tif

Supplementary table 1 Inhibition of metabolites formation raw values. Compared with DCF200 µM alone, the addition of borneol (B), ketoconazole (K), cimetidine (C) inhibited the formation of DAG, 4′-OH DCF, 5-OH DCF, respectively. UD: under detection level. NA: not analyzed. (TIFF 5608 kb)

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Niu, X., de Graaf, I.A.M., Langelaar-Makkinje, M. et al. Diclofenac toxicity in human intestine ex vivo is not related to the formation of intestinal metabolites. Arch Toxicol 89, 107–119 (2015). https://doi.org/10.1007/s00204-014-1242-6

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