Role of Free Radicals in Primary Nonfunction of Marginal Fatty Grafts from Rats Treated Acutely with Ethanol

  1. Zhi Zhong1,
  2. Henry Connor1,
  3. Robert F. Stachlewitz1,
  4. Moritz von Frankenberg1,
  5. Ronald P. Mason3,
  6. John J. Lemasters2 and
  7. Ronald G. Thurman1
  1. Departments of 1Pharmacology (Z.Z., H.C., R.F.S., M.v.F., R.G.T.) and 2Cell Biology and Anatomy (J.J.L.), University of North Carolina, Chapel Hill, North Carolina 27599-7365, and 3Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709 (R.P.M.)

    Abstract

    Acute treatment with one large dose of ethanol, which mimics binge drinking, causes marginal fatty liver and decreases survival significantly after liver transplantation in rats, yet mechanisms remain unclear. Therefore, we evaluated the possible role of free radicals in primary nonfunction caused by acute ethanol. Female donor rats were administered ethanol (5 g/kg orally) 20 hr before explantation, and grafts were stored in UW cold storage solution for 24–42 hr before implantation. Free radicals were trapped with α-(4-pyridyl 1-oxide)-N-tert-butylnitrone after transplantation, and adducts were detected using electron spin resonance spectrometry. Ethanol increased a carbon-centered radical adduct in bile ∼2-fold and elevated serum lipid hydroperoxides ∼4-fold. Ethanol also increased transaminase release 3.7-fold and decreased bile production by 55%. Catechin, a free radical scavenger, minimized the increase in free radicals, blunted transaminase release, and elevated bile production significantly, indicating that free radical production plays an important role in ethanol-induced fatty graft injury. GdCl3 (20 mg/kg intravenously), a selective Kupffer cell toxicant, largely blocked the increases in free radical and lipid hydroperoxide production caused by ethanol. In addition, ethanol nearly doubled white blood cell adhesion after transplantation, leading to increased superoxide production in fatty grafts. GdCl3 largely blocked leukocyte adhesion as well as superoxide production. Allopurinol, an inhibitor of xanthine oxidase, also diminished free radical production, blunted transaminase release, and improved bile production in fatty grafts significantly. Taken together, we conclude that free radical formation increases in ethanol-induced fatty grafts due mainly to activation of Kupffer cells and increased adhesion of white blood cells. Antioxidants can effectively block free radical formation and minimize injury to marginal fatty grafts caused by binge drinking.

    Footnotes

    • Send reprint requests to: Dr. Ronald G. Thurman, Department of Pharmacology, CB# 7365, Mary Ellen Jones Building, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7365. E-mail:thurman{at}med.unc.edu

    • This work was supported in part by National Institutes of Health Grant AA09156 and a graduate student fellowship from the Institute of Nutrition, University of North Carolina (Z.Z.).

    • Abbreviations:
      UW
      University of Wisconsin
      4-POBN
      α-(4-pyridyl 1-oxide)-N-tert-butylnitrone
      AST
      aspartate aminotransferase
      MCDP
      10-N-methylcarbamoyl-3,7-dimethylamino-10-H-phenothiazine
      HBSS
      Hanks’ balanced salt solution
      SOD
      superoxide dismutase
      ANOVA
      analysis of variance
      PMA
      phorbol-12-myristate-13-acetate
      MDA
      mitochondrial malondialdehyde
      • Received March 28, 1997.
      • Accepted July 21, 1997.
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    1. Molecular Pharmacology November 1, 1997 vol. 52 no. 5 912-919
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