Molecular and Cellular Biochemistry

, Volume 204, Issue 1–2, pp 119–126 | Cite as

Pirfenidone inhibits NADPH-dependent microsomal lipid peroxidation and scavenges hydroxyl radicals

  • Hara P. Misra
  • Christine Rabideau


Pirfenidone (Pf), a new broad-spectrum anti-fibrotic agent, is known to offer protection against lung fibrosis in vivo in laboratory animals, and against mitogenesis and collagen formation by human lung fibroblasts in vitro. Because reactive oxygen species are thought to be involved in these events, we investigated the mechanism(s) by which Pf ameliorates oxidative stress and its effects on NADPH-dependent lipid peroxidation. Pf has been shown to cause inhibit NADPH-dependent lipid peroxidation in sheep liver microsomes in a dose-dependent manner. The concentration of Pf required to cause 50% inhibition of lipid peroxidation was ~ 6 mM. Pf was found to be ineffective as a superoxide radical scavenger. Pf was also ineffective in decomposing H2O2 and chelating iron. In deoxyribose degradation assays, Pf was a potent scavenger of hydroxyl radicals with a rate constant of 5.4 × 109 M-1 sec-1. EPR spectroscopy in combination with spin trapping techniques, using a Fenton type reaction and DMPO as a spin-trapping agent, Pf scavenged hydroxyl radicals in a dose-dependent manner. The concentration of Pf required to inhibit 50% signal height was ~ 2.5 mM. Because iron was used in the Fenton reaction, the ability of Pf in chelating iron was verified in a fluorescent competitive assay using calcein as the fluorescent probe. Pf up to 10 mM concentration was ineffective in chelating either Fe2+ or Fe3+ in this system. We propose that Pf exerts its beneficial effects, at least in part, through its ability to scavenge toxic hydroxyl radicals.

pirfenidone free radicals lipid peroxidation EPR spin trapping antioxidant fibrosis 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Iyer SN, Margolin SB, Hyde DM, Giri SN: Lung fibrosis is ameliorated by pirfenidone fed in diet after the second dose in a three-dose bleomycin-hamster model. Exp Lung Res 24: 119-132, 1998Google Scholar
  2. 2.
    Iyer SN, Wild JS, Schiedt MJ, Margolin SB, Giri SN: Dietary intake of pirfenidone ameliorates bleomycin-induced lung fibrosis in hamsters. J Lab Clin Med 125: 779-785, 1995Google Scholar
  3. 3.
    Kehrer JP, Margolin SB: Pirfenidone diminishes cyclophosphamideinduced lung fibrosis in mice. Toxicol Lett 90: 125-132, 1997Google Scholar
  4. 4.
    Suga H, Teraoka S, Ota K, Komemsushi S, Furutani S, Yamauchi S, Margolin S: Preventive effect of pirfenidone against experimental sclerosing peritonitis in rats. Exp Toxicol Pathol 47: 287-291, 1995Google Scholar
  5. 5.
    Margolin SB, Margolin B, Margolin D: Removal of interstitial pulmonary fibrosis (asbestos-induced) by oral chemotherapy with pirfenidone. Fed Proc 41: 1150, 1982Google Scholar
  6. 6.
    Cain WC, Stuart RW, Lefkowitz DL, Starnes JD, Margolin S, Lefkowitz SS: Inhibition of tumor necrosis factor and subsequent endotoxin shock by pirfenidone. Int J Immunopharm 20: 685-695, 1998Google Scholar
  7. 7.
    Margolin SB, Lefkowitz S: Pirfenidone: A novel pharmacologic agent for prevention and resolution (removal) of lung fibrosis. FASEB J 8: A382, 1994Google Scholar
  8. 8.
    Kaneko M, Inoue H, Nakazawa R, Azuma N, Suziki M, Yamauchi S, Margolin SB, Tsubota K, Saito I: Pirfenidone induces intercellular adhesion molecule-1 (ICAM-1) down regulation on cultured human synovial fibroblasts. Clin Exp Immunol 113: 72-76, 1998Google Scholar
  9. 9.
    Zhang S, Shiels IA, Ambler JS, Taylor SM: Pirfenidone reduces fibronectin synthesis by cultured human retinal pigment epithelial cells. Aust N Z J Ophthalmol 26: S74-S76, 1998Google Scholar
  10. 10.
    Lee BS, Margolin SB, Nowak RA: Pirfenidone: A novel pharmacological agent that inhibits leiomyoma cell proliferation and collagen production. J Clin Endocrinol Metab 83: 219-223, 1998Google Scholar
  11. 11.
    Hay J, Shahzeidi S, Laurent G: Mechanism of bleomycin-induced lung damage. Arch Toxicol 65: 81-94, 1991Google Scholar
  12. 12.
    Parizada B, Werber MM, Nimrod A: Protective effects of human recombinant MnSOD in adjuvant arthritis and bleomycin-induced lung fibrosis. Free Rad Res Commun 15: 297-301, 1991Google Scholar
  13. 13.
    Giri, SN, Misra HP, Chandler DB, Chen Z: Increase in lung prolyl hydroxylase and superoxide dismutase activities during bleomycininduced lung fibrosis in hamsters. Exp Mol Pathol 39: 317-326, 1983Google Scholar
  14. 14.
    Windhager R, Nemethova M, Mutsaers S, Lang S, Kotz R, Kitzmueller E, Lubec G: Evidence for the involvement of the hydroxyl radical in the pathogenesis of excessive connective tissue proliferation in patients with tumor-endoprostheses. Life Sci 62: 1261-1269, 1998Google Scholar
  15. 15.
    Arisawa S, Arisawa T, Ohashi M, Nitta Y, Ikeya T, Asai J: Effect of the hydroxyl radical on fibroblast-mediated collagen remodelling in vitro. Clin Exp Pharmacol Physiol 23: 222-228, 1996Google Scholar
  16. 16.
    De Mattia G, Bravi MC, Laurenti O, Cassone-Faldetta M, Proietti A, De Luca O, Armiento A, Ferri C: Reduction of oxidative stress by oral N-acetyl-L-cysteine treatment decreases plasma soluble vascular cell adhesion molecule-1 concentrations in non-obese, non-dyslipidaemic, normotensive, patients with non-insulin-dependent diabetes. Diabetologia 41: 1392-1396, 1998Google Scholar
  17. 17.
    Steinhauser ML, Kunkel SL, Hogaboam CM, Evanoff H, Strieter RM, Lukacs NW: Macrophage/fibroblast co-culture induces macrophage inflammatory protein-1 alpha production mediated by intercellular adhesion molecule-1 and oxygen radicals. Leuk Biol 64: 636-641, 1998Google Scholar
  18. 18.
    Kamp DW, Graceffa P, Pryor WA, Weitzman SA: The role of free radicals in asbestos-induced diseases. Free Rad Biol Med 12: 293-315, 1992Google Scholar
  19. 19.
    Kim S, Dehnez F, Kim KY, Holt JT, Sporn MB, Roberts AB: Activation of the second promoter of the transforming growth factor-β1 gene by transforming growth factor-β1 and phorbol ester occurs through the same target sequences. J Biol Chem 264: 19373-19378, 1989Google Scholar
  20. 20.
    Poli G, Kinter A, Justement JS, Kehrl JH, Bressler P, Stanley S, Fauci AS: Tumor necrosis factor-α functions in an autocrine manner in the induction of human immunodeficiency virus expression. Proc Natl Acad Sci USA 87: 782-785, 1990Google Scholar
  21. 21.
    Maher JJ, Tzagarakis C, Gimenez A: Malondialdehyde stimulates collagen production by hepatic lipocytes only upon activation in primary culture. Alcohol & Alcoholism 29: 605-610, 1994Google Scholar
  22. 22.
    Darr D, Combs S, Pinnell S: Ascorbic acid and collagen synthesis: Rethinking a role for lipid peroxidation. Arch Biochem Biophys 307: 331-335, 1993Google Scholar
  23. 23.
    Rust P, Eichler I, Renner S, Elmadfa I: Effects of long-term oral betacarotene supplementation on lipid peroxidation in patients with cystic fibrosis. Int J Vit Nutr Res 68: 83-87, 1998Google Scholar
  24. 24.
    Poli G, Parola M: Oxidative damage and fibrogenesis. Free Rad Biol Med 22: 287-305, 1997Google Scholar
  25. 25.
    Das KC, Misra HP: Antiarrhythmic agents: Scavengers of hydroxyl radicals and inhibitors of NADPH-dependent lipid peroxidation in bovine lung microsomes. J Biol Chem 267: 19172-19178, 1992Google Scholar
  26. 26.
    Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254, 1976Google Scholar
  27. 27.
    Bernheim F, Bernheim MLC, Wilbur KM: The reactions between thiobarbituric acid and the oxidation products of certain lipids. J Biol Chem 174: 257-264, 1948Google Scholar
  28. 28.
    McCord JM, Fridovich I: Superoxide dismutase: An enzymic function for erythrocuprein (hemocuprein). J Biol Chem 244: 6049-6055, 1969Google Scholar
  29. 29.
    Misra HP: Adrenochrome assay for superoxide dismutase. In: R.A. Greenwald (ed). CRC Handbook of Methods for Oxygen Radical Research. CRC Press, Florida, 1985, pp 237-241Google Scholar
  30. 30.
    Misra HP, Fridovich I: The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J Biol Chem 247: 3170-3175, 1972Google Scholar
  31. 31.
    Halliwell B, Gutteridge JMC, Aruoma OI: The deoxyribose method: A simple test tube assay for determination of rate constants for reactions of hydroxyl radicals. Anal Biochem 165: 215-219, 1987Google Scholar
  32. 32.
    Breuer W, Epsztejn S, Milligram P, Cabantchik IZ: Transport of iron and other transition metals into cells as revealed by a fluorescent probe. Am J Physiol 268: C1354-C1361, 1995Google Scholar
  33. 33.
    McCord JM, Fridovich I: The reduction of cytochrome c by milk xanthine oxidase. J Biol Chem 243: 5753-5760, 1968Google Scholar
  34. 34.
    Thurman RG, Ley HG, Scholz R: Hepatic microsomal ethanol oxidation, hydrogen peroxide formation and the role of catalase. Eur J Biochem 25: 420-430, 1972Google Scholar
  35. 35.
    Turner MG II, Rosen GM: Spin trapping of superoxide and hydroxyl radicals with substituted pyrroline 1-oxide. J Med Chem 29: 2439-2444, 1986Google Scholar
  36. 36.
    Zang LY, Misra HP: EPR kinetic studies of superoxide radicals generated during the autoxidation of 1-methyl-4-phenyl-2,3-dihydropyridinium, a bioactivated intermediate of Parkinsonianinduced neurotoxin 1-methyl-4-phenyl-1.2.3,6-tetrahydropyridine. J Biol Chem 267: 23601-23608, 1992Google Scholar
  37. 37.
    Chance B, Maehly AC: Assay of catalase and peroxidases. Meth Enzymol 2: 764-775, 1955Google Scholar
  38. 38.
    Parola M, Muraca R, Dianzani I, Barrera G, Leonarduzzi G, Bendinelli P, Piccoletti R, Poli G: Vitamin E dietary supplementation inhibits transforming growth factor β1 gene expression in the rat liver. FEBS Lett 308: 267-270, 1992Google Scholar
  39. 39.
    Mourelle M, Muriel P, Favari L, Franco T: Prevention of CCl4-induced liver cirrhosis by silymarin. Fundam Clin Pharmacol 3: 183-191, 1989Google Scholar
  40. 40.
    Parola M, Leonarduzzi G, Biasi F, Albano E, Biocca ME, Poli G, Dianzani MU: Vitamin E dietary supplementation protects against carvon tetrachloride-induced chronic liver damage and cirrhosis. Hepatology 16: 1014-1021, 1992Google Scholar
  41. 41.
    Kalyanaraman B: Detection of toxic free radicals in biology and medicine. In: E. Hodgson, J.R. Bend, R.M. Philpot (eds). Reviews in Biochemical Toxicology, vol. 4. Elsevier Biochemical, New York, 1982, pp 73-139Google Scholar
  42. 42.
    Yamamoto Y, Kamiya Y: In: A. Sevanian (ed). Lipid Peroxidation in Biological Systems. American Oil Chemists Society, Champaign, IL, 1988, pp 32-50.Google Scholar
  43. 43.
    Svingen AS, O'Neal, FO, Aust SD: The role of superoxide and singlet oxygen in lipid peroxidation. Photochem Photobiol 28: 803-809, 1978Google Scholar
  44. 44.
    Cohen G: The generation of hydroxyl radicals in biologic systems: Toxicological aspects. Photochem Photobiol 28: 669-675, 1978Google Scholar
  45. 45.
    Zweier JL, Flaherty JT, Weisfeldt ML: Direct measurement of free radical generation following reperfusion of ischemic myocardium. Proc Natl Acad Sci USA 84: 1404-1407, 1987Google Scholar
  46. 46.
    Roney PI, Holian A: Possible mechanism of chrysotile asbestosstimulated superoxide anion production in guinea pig alveolar macrophages. Toxicol Appl Pharmacol 100: 132-144, 1989Google Scholar
  47. 47.
    Sugiura Y, Kikuchi T: Formation of superoxide and hydroxyl radicals in iron (II)-bleomycin-oxygen system: Electron spin resonance detection by spin trapping. J Antibiot 31: 1310-1312, 1978Google Scholar
  48. 48.
    Sugiura Y, Suzuki T, Kuwahara J, Tanaka H: On the mechanism of hydrogen peroxide-, superoxide-, and ultraviolet light-induced DNA cleavages in inactive bleomycin-iron (III) complex. Biochem Biophys Res Commun 105: 1511-1518, 1982Google Scholar
  49. 49.
    Hoyt, DG, Lazo JS: Acute pneumocyte injury, poly (ADP-ribose) polymerase activity, and pyridine nucleotide levels after in vitro exposure of murine lung slices to cyclophosphamide. Biochem Pharmacol 48: 1757-1765, 1994Google Scholar
  50. 50.
    Vereckei A, Blazovics A, Gyorgy I, Feher E, Toth M, Szenasi G, Zsinka A, Foldiak G, Feher J: The role of free radicals in the pathogenesis of amiodarone toxicity. J Cardiovasc Electrophysiol 4: 161-177, 1993Google Scholar
  51. 51.
    Kovacs, EJ: Fibrogenic cytokines: The role of immune mediators in the development of scar tissue. Immunol Today 12: 17-23, 1991Google Scholar
  52. 52.
    Zang LY, van Kuijk FJGM, Misra HP: EPR studies of spin-trapped free radicals in paraquat-treated lung microsomes. Biochem Mol Biol Interact 37: 255-262, 1995Google Scholar
  53. 53.
    Fong K, McCay PB, Poyer JL, Keele BB, Misra H: Evidence that peroxidation of lysosomal membranes is initiated by hydroxyl free radicals produced during flavin enzyme activity. J Biol Chem 248: 7792-7797, 1973Google Scholar
  54. 54.
    Hickel, B: Absorption spectra and kinetics of methyl and ethyl radicals in water. J Phys Chem 79: 1054-1059, 1975Google Scholar
  55. 55.
    Uri N: Physio-chemical aspects of autoxidation. In: W.O. Ludenberg (ed). Autoxidation and Antioxidants, (ch. 2). Wiley-Interscience, New York, 1961, pp 55-106Google Scholar

Copyright information

© Kluwer Academic Publishers 2000

Authors and Affiliations

  • Hara P. Misra
    • 1
  • Christine Rabideau
    • 1
  1. 1.Center of Molecular Medicine and Infection Diseases, Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary MedcineVirginia Polytechnic Institute and State UniversityBlacksburgUSA

Personalised recommendations