Langenbeck's Archives of Surgery

, Volume 393, Issue 4, pp 547–555

A cell-based approach to study changes in the pancreas following nicotine exposure in an animal model of injury

Original Article



Cigarette smoking is a recognized risk factor for the induction of pancreatic diseases and is suspected to play a major role in the development of pancreatic cancer in smokers.

Materials and methods

This study was designed to characterize the mechanisms of nicotine-induced injury to the pancreas. AR42Jcells, a stable mutant pancreatic tumor cell line, was chosen for the study because of its stability in culture media and also because of its known secretory capacity, which is like that of a normal pancreatic acinar cell. It is hypothesized that nicotine-induced effects on the pancreas are triggered by oxidative stress induced in pancreatic acinar cell via oxidative stress signaling pathways.


The results from our study showed that, in vitro, nicotine induced generation of oxygen free radicals measured as malondialdehyde, an end product of lipid peroxidation. Treatment of AR42J cells with nicotine induced p-ERK 1/2 activation as confirmed by Western blot and immunofluorescence imaging of cytoplasmic localization of mitogen-activated protein kinase (MAPK) signals. Nicotine enhanced AR42J cell proliferation and cholecystokinin-stimulated amylase release in AR42J cells. These effects of nicotine were confirmed by simultaneous studies conducted on the same cells by hydrogen peroxide, a known oxidative biomarker. Allopurinol, a XOD inhibitor, suppressed these effects induced by nicotine and H2O2 with the exception that cholecystokinin-stimulated amylase release by H2O2 remained unaltered when AR42J cells were preincubated with allopurinol. These results suggest that nicotine-induced effects on pancreatic acinar cells were associated with generation of oxyradical mediated via the XOD pathway. The results have a direct impact on cell proliferation, MAPK signaling, and acinar cell function.


We conclude that nicotine induces oxidative stress in pancreatic acinar cells and that these events trigger pathophysiological changes in the pancreas, leading to increased cell proliferation and injury.


Nicotine AR42J cell Effect on mechanisms Lipid peroxidation MAPK signaling Cell function 


  1. 1.
    Talamini G, Bassi C, Falconi M, Sartori N, Salvia R, Rigo L, Castagnini A, Di Francesco V, Frulloni L, Bovo P, Vaona B, Angelini G, Vantini I, Cavallini G, Pederzoli P (1999) Alcohol and smoking as risk factors in chronic pancreatitis and pancreatic cancer. Dig Dis Sci 44:1303–1311PubMedCrossRefGoogle Scholar
  2. 2.
    Lin Y, Tamakoshi A, Hayakawa T, Ogawa M, Ohno Y (2000) Cigarette smoking as a risk factor for chronic pancreatitis: a case-control study in Japan. Research Committee on Intractable Pancreatic Diseases. Pancreas 21:109–114PubMedCrossRefGoogle Scholar
  3. 3.
    Maritz GS, Burger B (1992) The influence of maternal nicotine exposure on neonatal lung carbohydrate metabolism. Cell Biol Int Rep 16:1229–1236PubMedCrossRefGoogle Scholar
  4. 4.
    Bose C, Zhang H, Udupa KB, Chowdhury P (2005) Activation of p-ERK1/2 by nicotine in pancreatic tumor cell line AR42J: effects on proliferation and secretion. Am J Physiol Gastrointest Liver Physiol 289:G926–G934PubMedCrossRefGoogle Scholar
  5. 5.
    Chowdhury P, MacLeod S, Udupa KB, Rayford PL (2002) Pathophysiological effects of nicotine on the pancreas: an update. Exp Biol Med (Maywood) 227:445–454Google Scholar
  6. 6.
    Rayford PL, Chowdhury P (2001) Mecamylamine, a nicotinic receptor channel antagonist, affects amylase secretion by isolated pancreatic acinar cells. J Assoc Acad Minor Phys 12:105–108PubMedGoogle Scholar
  7. 7.
    Chowdhury P, Hosotani R, Chang L, Rayford PL (1990) Metabolic and pathologic effects of nicotine on gastrointestinal tract and pancreas of rats. Pancreas 5:222–229PubMedCrossRefGoogle Scholar
  8. 8.
    Chowdhury P (2003) An exploratory study on the development of an animal model of pancreatitis following nicotine exposure. Tob Induced Dis 1(3):213–217CrossRefGoogle Scholar
  9. 9.
    Wetscher GJ, Bagchi M, Bagchi D, Perdikis G, Hinder PR, Glaser K, Hinder RA (1995) Free radical production in nicotine treated pancreatic tissue. Free Radic Biol Med 18:877–882PubMedCrossRefGoogle Scholar
  10. 10.
    Kalpana C, Menon VP (2004) Curcumin ameliorates oxidative stress during nicotine-induced lung toxicity in Wistar rats. Ital J Biochem 53:82–86PubMedGoogle Scholar
  11. 11.
    Piperakis SM, Visvardis EE, Sagnou M, Tassiou AM (1998) Effects of smoking and aging on oxidative DNA damage of human lymphocytes. Carcinogenesis 19:695–698PubMedCrossRefGoogle Scholar
  12. 12.
    Park BK, Chung JB, Lee JH, Suh JH, Park SW, Song SY, Kim H, Kim KH, Kang JK (2003) Role of oxygen free radicals in patients with acute pancreatitis. World J Gastroenterol 9:2266–2269PubMedGoogle Scholar
  13. 13.
    Ganesh PC, Sreejayan, Rao MN (1999) Evidence for oxidant stress in chronic pancreatitis. Indian J Gastroenterol 18:156–157Google Scholar
  14. 14.
    Sanfey H, Bulkley GB, Cameron JL (1985) The pathogenesis of acute pancreatitis. The source and role of oxygen-derived free radicals in three different experimental models. Ann Surg 201:633–639PubMedCrossRefGoogle Scholar
  15. 15.
    Schoenberg MH, Buchler M, Beger HG (1994) Oxygen radicals in experimental acute pancreatitis. Hepatogastroenterology 41:313–319PubMedGoogle Scholar
  16. 16.
    Sweiry JH, Mann GE (1996) Role of oxidative stress in the pathogenesis of acute pancreatitis. Scand J Gastroenterol Suppl 219:10–15PubMedCrossRefGoogle Scholar
  17. 17.
    Czako L, Takacs T, Varga IS, Tiszlavicz L, Hai DQ, Hegyi P, Matkovics B, Lonovics J (1998) Involvement of oxygen-derived free radicals in L-arginine-induced acute pancreatitis. Dig Dis Sci 43:1770–1777PubMedCrossRefGoogle Scholar
  18. 18.
    Klein AS, Joh JW, Rangan U, Wang D, Bulkley GB (1996) Allopurinol: discrimination of antioxidant from enzyme inhibitory activities. Free Radic Biol Med 21:713–717PubMedCrossRefGoogle Scholar
  19. 19.
    Wisner J, Green D, Ferrell L, Renner I (1988) Evidence for a role of oxygen derived free radicals in the pathogenesis of caerulein induced acute pancreatitis in rats. Gut 29:1516–1523PubMedCrossRefGoogle Scholar
  20. 20.
    Das DK, Engelman RM, Clement R, Otani H, Prasad MR, Rao PS (1987) Role of xanthine oxidase inhibitor as free radical scavenger: a novel mechanism of action of allopurinol and oxypurinol in myocardial salvage. Biochem Biophys Res Commun 148:314–319PubMedCrossRefGoogle Scholar
  21. 21.
    Grisham MB, Hernandez LA, Granger DN (1986) Xanthine oxidase and neutrophil infiltration in intestinal ischemia. Am J Physiol 251:G567–G574PubMedGoogle Scholar
  22. 22.
    Moorhouse PC, Grootveld M, Halliwell B, Quinlan JG, Gutteridge JM (1987) Allopurinol and oxypurinol are hydroxyl radical scavengers. FEBS Lett 213:23–28PubMedCrossRefGoogle Scholar
  23. 23.
    Fox IH (1976) Inborn errors of purine and pyrimidine metabolism. Clin Perinatol 3:133–140PubMedGoogle Scholar
  24. 24.
    Peterson DA, Kelly B, Gerrard JM (1986) Allopurinol can act as an electron transfer agent. Is this relevant during reperfusion injury? Biochem Biophys Res Commun 137:76–79PubMedCrossRefGoogle Scholar
  25. 25.
    Mikulikova D, Bosmansky K, Bosak V, Ondrasik M (1989) The effect of allopurinol on lysosomal enzyme release. Z Rheumatol 48:26–29PubMedGoogle Scholar
  26. 26.
    Parks DA, Granger DN (1986) Contributions of ischemia and reperfusion to mucosal lesion formation. Am J Physiol 250:G749–G753PubMedGoogle Scholar
  27. 27.
    Sawamura M, Sun SH, Ozaki K, Ishikawa J, Ukeda H (1999) Inhibitory effects of citrus essential oils and their components on the formation of N-nitrosodimethylamine. J Agric Food Chem 47:4868–4872PubMedCrossRefGoogle Scholar
  28. 28.
    Shahraki A, Fukunari A, Stone TW (2004) The mechanism of inhibition by xanthine of adenosine A1-receptor responses in rat hippocampus. Neurosci Lett 365:162–166PubMedCrossRefGoogle Scholar
  29. 29.
    Valencia A, Moran J (2004) Reactive oxygen species induce different cell death mechanisms in cultured neurons. Free Radic Biol Med 36:1112–1125PubMedCrossRefGoogle Scholar
  30. 30.
    Fatokun AA, Stone TW, Smith RA (2007) Hydrogen peroxide mediates damage by xanthine and xanthine oxidase in cerebellar granule neuronal cultures. Neurosci Lett 416:34–38PubMedCrossRefGoogle Scholar
  31. 31.
    Chiarugi P (2003) Reactive oxygen species as mediators of cell adhesion. Ital J Biochem 52:28–32PubMedGoogle Scholar
  32. 32.
    Zienolddiny S, Ryberg D, Haugen A (2000) Induction of microsatellite mutations by oxidative agents in human lung cancer cell lines. Carcinogenesis 21:1521–1526PubMedCrossRefGoogle Scholar
  33. 33.
    Christophe J (1994) Pancreatic tumoral cell line AR42J: an amphicrine model. Am J Physiol 266:G963–G971PubMedGoogle Scholar
  34. 34.
    Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72:248–254PubMedCrossRefGoogle Scholar
  35. 35.
    Jung DH (1980) Preparation and application of Procion Yellow starch for amylase assay. Clin Chim Acta 100:7–11PubMedCrossRefGoogle Scholar
  36. 36.
    Ogawa Y, Kobayashi T, Nishioka A, Kariya S, Ohnishi T, Hamasato S, Seguchi H, Yoshida S (2004) Reactive oxygen species-producing site in radiation-induced apoptosis of human peripheral T cells: involvement of lysosomal membrane destabilization. Int J Mol Med 13:69–73PubMedGoogle Scholar
  37. 37.
    Riley PA (1994) Free radicals in biology: oxidative stress and the effects of ionizing radiation. Int J Radiat Biol 65:27–33PubMedCrossRefGoogle Scholar
  38. 38.
    Iijima R, Takahashi H, Namme R, Ikegami S, Yamazaki M (2004) Novel biological function of sialic acid (N-acetylneuraminic acid) as a hydrogen peroxide scavenger. FEBS Lett 561:163–166PubMedCrossRefGoogle Scholar
  39. 39.
    Guan W, Osanai T, Kamada T, Hanada H, Ishizaka H, Onodera H, Iwasa A, Fujita N, Kudo S, Ohkubo T, Okumura K (2003) Effect of allopurinol pretreatment on free radical generation after primary coronary angioplasty for acute myocardial infarction. J Cardiovasc Pharmacol 41:699–705PubMedCrossRefGoogle Scholar
  40. 40.
    Fridovich I (1986) Biological effects of the superoxide radical. Arch Biochem Biophys 247:1–11PubMedCrossRefGoogle Scholar
  41. 41.
    Fujita T (2002) Formation and removal of reactive oxygen species, lipid peroxides and free radicals, and their biological effects. Yakugaku Zasshi 122:203–218PubMedCrossRefGoogle Scholar
  42. 42.
    Yamamoto Y, Ogino K, Igawa G, Matsuura T, Kaetsu Y, Sugihara S, Matsubara K, Miake J, Hamada T, Yoshida A, Igawa O, Yamamoto T, Shigemasa C, Hisatome I (2006) Allopurinol reduces neointimal hyperplasia in the carotid artery ligation model in spontaneously hypertensive rats. Hypertens Res 29:915–921PubMedCrossRefGoogle Scholar
  43. 43.
    Inkster ME, Cotter MA, Cameron NE (2007) Treatment with the xanthine oxidase inhibitor, allopurinol, improves nerve and vascular function in diabetic rats. Eur J Pharmacol 561:63–71PubMedCrossRefGoogle Scholar
  44. 44.
    Spahr L, Bresson-Hadni S, Amann P, Kern I, Golaz O, Frossard JL, Hadengue A (2007) Allopurinol, oxidative stress and intestinal permeability in patients with cirrhosis: an open-label pilot study. Liver Int 27:54–60PubMedCrossRefGoogle Scholar
  45. 45.
    Lee WY, Lee SM (2006) Synergistic protective effect of ischemic preconditioning and allopurinol on ischemia/reperfusion injury in rat liver. Biochem Biophys Res Commun 349:1087–1093PubMedCrossRefGoogle Scholar
  46. 46.
    Vincent AM, Stevens MJ, Backus C, McLean LL, Feldman EL (2005) Cell culture modeling to test therapies against hyperglycemia-mediated oxidative stress and injury. Antioxid Redox Signal 7:1494–1506PubMedCrossRefGoogle Scholar
  47. 47.
    Heunks LM, Vina J, van Herwaarden CL, Folgering HT, Gimeno A, Dekhuijzen PN (1999) Xanthine oxidase is involved in exercise-induced oxidative stress in chronic obstructive pulmonary disease. Am J Physiol 277:R1697–R1704PubMedGoogle Scholar
  48. 48.
    Baud O, Greene AE, Li J, Wang H, Volpe JJ, Rosenberg PA (2004) Glutathione peroxidase-catalase cooperativity is required for resistance to hydrogen peroxide by mature rat oligodendrocytes. J Neurosci 24:1531–1540PubMedCrossRefGoogle Scholar
  49. 49.
    Meiners S, Ludwig A, Lorenz M, Dreger H, Baumann G, Stangl V, Stangl K (2006) Nontoxic proteasome inhibition activates a protective antioxidant defense response in endothelial cells. Free Radic Biol Med 40:2232–2241PubMedCrossRefGoogle Scholar
  50. 50.
    Martindale JL, Holbrook NJ (2002) Cellular response to oxidative stress: signaling for suicide and survival. J Cell Physiol 192:1–15PubMedCrossRefGoogle Scholar
  51. 51.
    Yang B, Oo TN, Rizzo V (2006) Lipid rafts mediate H2O2 prosurvival effects in cultured endothelial cells. FASEB J 20:1501–1503PubMedCrossRefGoogle Scholar
  52. 52.
    Zhou Y, Wang Q, Evers BM, Chung DH (2005) Signal transduction pathways involved in oxidative stress-induced intestinal epithelial cell apoptosis. Pediatr Res 58:1192–1197PubMedCrossRefGoogle Scholar
  53. 53.
    Watanabe N, Zmijewski JW, Takabe W, Umezu-Goto M, Le Goffe C, Sekine A, Landar A, Watanabe A, Aoki J, Arai H, Kodama T, Murphy MP, Kalyanaraman R, Darley-Usmar VM, Noguchi N (2006) Activation of mitogen-activated protein kinases by lysophosphatidylcholine-induced mitochondrial reactive oxygen species generation in endothelial cells. Am J Pathol 168:1737–1748PubMedCrossRefGoogle Scholar
  54. 54.
    Abdulnour RE, Peng X, Finigan JH, Han EJ, Hasan EJ, Birukov KG, Reddy SP, Watkins JE, III, Kayyali US, Garcia JG, Tuder RM, Hassoun PM (2006) Mechanical stress activates xanthine oxidoreductase through MAP kinase-dependent pathways. Am J Physiol Lung Cell Mol Physiol 291:L345–L353PubMedCrossRefGoogle Scholar
  55. 55.
    Vorbach C, Harrison R, Capecchi MR (2003) Xanthine oxidoreductase is central to the evolution and function of the innate immune system. Trends Immunol 24:512–517PubMedCrossRefGoogle Scholar
  56. 56.
    Zhang J, Jin N, Liu Y, Rhoades RA (1998) Hydrogen peroxide stimulates extracellular signal-regulated protein kinases in pulmonary arterial smooth muscle cells. Am J Respir Cell Mol Biol 19:324–332PubMedGoogle Scholar
  57. 57.
    Song HJ, Lee TS, Jeong JH, Min YS, Shin CY, Sohn UD (2005) Hydrogen peroxide-induced extracellular signal-regulated kinase activation in cultured feline ileal smooth muscle cells. J Pharmacol Exp Ther 312:391–398PubMedCrossRefGoogle Scholar
  58. 58.
    Granados MP, Salido GM, Pariente JA, Gonzalez A (2005) Effect of H2O2 on CCK-8 evoked changes in mitochondrial activity in isolated mouse pancreatic acinar cells. Biol Cell 97:847–856PubMedCrossRefGoogle Scholar
  59. 59.
    Weitberg AB, Corvese D (1993) Oxygen radicals potentiate the genetic toxicity of tobacco-specific nitrosamines. Clin Genet 43:88–91PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  1. 1.Department of Physiology & Biophysics, Slot # 505University of Arkansas for Medical SciencesLittle RockUSA

Personalised recommendations