Original Contribution EFFECT OF KUPFFER CELL INACTIVATION ON ETHANOL-INDUCED PROTEIN ADDUCTS IN THE LIVER ONNI NIEMEL ¨ A,* † SEPPO PARKKILA, †‡ BLAIR BRADFORD, § YUJI IIMURO, § MARKKU PASANEN,  and RONALD G. THURMAN § *EP Central Hospital, Seina ¨joki, Finland; † Department of Clinical Chemistry and ‡ Institute of Medical Technology, University of Tampere, Tampere, Finland; § Laboratory of Hepatobiology and Toxicology, Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; and  Department of Pharmacology, University of Oulu, Oulu, Finland (Received 28 January 2002; Revised 16 April 2002; Accepted 23 April 2002) Abstract—Tissue deposition of protein adducts derived from ethanol metabolism and lipid peroxidation, has been suggested to play a role in the initiation of alcoholic liver disease. The mechanisms modulating adduct formation have, however, remained unclear. We used immunohistochemical methods to examine acetaldehyde (AA) and malondialde- hyde (MDA) adducts and cytochrome P4502E1 and P4503A2 expression in rats after administration of (i) an ethanol-diet (n = 6), (ii) ethanol-diet plus gadolinium chloride (GdCl 3 ), a selective Kupffer cell toxicant (n = 7), or (iii) control diet (n = 6). A 4 week ethanol treatment resulted in liver steatosis, necrosis, and inflammation and deposition of protein adducts with both AA and MDA, which colocalized with areas of fatty change. The intensities (mean  SD) of the immunohistochemical reactions for both AA (2.50  1.23) and MDA (3.00  1.10) adducts were significantly higher in the ethanol-fed animals than in the controls (0.083  0.20) (0.16  0.25) (p  .001). GdCl 3 prevented adduct accumulation, the mean immunohistochemistry scores being 0.86  1.07 for AA and 1.64  0.63 for MDA, the former showing a more striking reduction (p  .01). The hepatic cytochrome enzymes were not different in the ethanol-fed groups with or without GdCl 3 . The data indicates that Kupffer cells are involved in the generation of protein adducts with both acetaldehyde and ethanol-induced lipid peroxidation products in alcoholic liver disease. © 2002 Elsevier Science Inc. Keywords—Ethanol metabolism, Lipid peroxidation, Alcoholic liver disease, Free radicals INTRODUCTION Ethanol metabolism in the liver is associated with enhanced oxidative stress and generation of reactive aldehydic products, which can bind to proteins and cellular constituents [1– 4]. Previous immunohisto- chemical studies have revealed the occurrence of ac- etaldehyde (AA)-, and malondialdehyde (MDA)-de- rived protein adducts in alcoholic livers, which coincide with elevations of serum liver-derived en- zymes and precede histological signs of liver disease [5–10]. Generation of hybrid adducts with malondial- dehyde and acetaldehyde in vivo as a result of ethanol consumption has also been demonstrated [11]. The mechanisms responsible for the formation of various adducts have, however, not been established. Several lines of recent evidence have indicated that Kupffer cells, the resident macrophages of the liver, play a key role in the pathogenesis of ethanol-induced liver disease [12–14]. Kupffer cells are highly responsive to the effects of ethanol and become readily activated by ethanol treatment [15], whereas inactivation of Kupffer cells with gadolinium chloride (GdCl 3 ) may prevent liver pathology [13,16]. The present experiments were set out to examine the association between protein adducts with AA and MDA, cytochromes P4502E1 (CYP2E1) and P4503A2 (CYP3A), and liver histology in ethanol-treated rats with and without Kupffer cell inhibition. The data supports a role for Kupffer cells in modulating adduct formation in alcoholic liver disease. Address correspondence to: Onni Niemela ¨, M.D., Professor of Lab- oratory Medicine, EP Central Hospital Laboratory, FIN-60220 Seina ¨- joki, Finland; Tel: +358 (6) 415-4719; Fax: +358 (6) 415-4924; E-Mail: onni.niemela@epshp.fi. Free Radical Biology & Medicine, Vol. 33, No. 3, pp. 350 –355, 2002 Copyright © 2002 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/02/$–see front matter PII S0891-5849(02)00894-8 350