Tissue and Cell 43 (2011) 384–391 Contents lists available at SciVerse ScienceDirect Tissue and Cell j our na l ho me p age: www.elsevier.com/locate/tice Chronic ethanol consumption induces histopathological changes and increases nitric oxide generation in the rat liver Luis F. Tirapelli a , Marcelo E. Batalhão b , Ana L. Jacob-Ferreira a , Daniela P. Tirapelli a , Evelin C. Carnio b , José E. Tanus-Santos a , Regina H. Queiroz c , Sergio A. Uyemura c , Cláudia M. Padovan d , Carlos R. Tirapelli b,∗ a Faculty of Medicine of Ribeirão Preto, University of São Paulo (USP), Brazil b College of Nursing of Ribeirão Preto, USP, Brazil c Faculty of Pharmaceutical Sciences of Ribeirão Preto, USP, Brazil d Faculty of Philosophy Science and Letters of Ribeirão Preto, USP, Brazil a r t i c l e i n f o Article history: Received 22 March 2011 Received in revised form 11 August 2011 Accepted 17 August 2011 Available online 17 September 2011 Keywords: Ethanol Liver Nitric oxide Metalloproteinase Histopathological changes a b s t r a c t In the present work we evaluated the effect of ethanol consumption in histopathological liver changes and several biochemical biomarkers employed in the detection of hepatic dysfunction. Male Wistar rats were treated with ethanol 20% (vol/vol) for 6 weeks. Histopathological investigation of livers from ethanol-treated animals revealed steatosis. Indices of hepatic function (transaminases) and mitochon- drial respiration were not altered in ethanol-treated rats. Chronic ethanol consumption did not alter malondialdehyde (MDA) levels in the liver. Ethanol consumption induced a significant increase on hepatic nitrite and nitrate levels. Treatment with ethanol increased both mRNA expression and immunostain- ing of iNOS, but not eNOS. Finally, ethanol consumption did not alter hepatic levels of metalloproteinase (MMP)-2 and MMP-9. We conclude that alterations on biochemical biomarkers (nitrite and nitrate levels) and histopathology occurred in ethanol-treated rats, supporting the practice of including both types of evaluation in toxicity studies to detect potential ethanol-related hepatic effects. In our model of ethanol consumption, histopathological liver changes were accompanied by elevation in nitrite and nitrate levels indicating increased nitric oxide (NO) generation. Since iNOS-derived NO contributes to hepatic injury, the increased levels of NO described in our study might contribute to a progressive hepatic damage. Therefore, increases in NO generation may be an early indicator of ethanol-induced liver damage. © 2011 Elsevier Ltd. All rights reserved. 1. Introduction Alcoholic liver disease is one of the major causes of morbidity and mortality among alcoholics in the world (Grant et al., 1988). It is proposed that excessive consumption of ethanol causes liver dis- ease by a combination of several pathologic mechanisms including oxidative stress, inflammation, redox alterations, and mitochon- drial damage (Beier and McClain, 2010). For this reason a wide variety of new biomarkers reflecting liver status are being used to evaluate the hepatic effect of excessive ethanol consumption. The impact of ethanol consumption in hepatic function is traditionally evaluated by histological analysis and measure- ments of serum transaminases (Conigrave et al., 2003). Ethanol ∗ Corresponding author at: Departamento de Enfermagem Psiquiátrica e Ciências Humanas, Laboratório de Farmacologia, Universidade de São Paulo, Escola de Enfer- magem de Ribeirão Preto, Avenida Bandeirantes 3900, 14040-902, Ribeirão Preto, SP, Brazil. Tel.: +55 16 36020532. E-mail address: crtirapelli@eerp.usp.br (C.R. Tirapelli). induces oxidative stress in the liver (Dey and Cederbaum, 2006), which is associated with enhanced production of reactive oxygen species (ROS). The latter damage lipids and proteins, resulting in increased malondialdehyde (MDA) levels, which have been used as a biomarker for ethanol-induced hepatic dysfunction (Baldi et al., 1993). In addition to ROS, nitric oxide (NO) is also important in the development of hepatic injury. NO is produced from l-arginine by the catalic action of NO synthase (NOS) (Moncada and Higgs, 1993), which is an enzyme that occurs in three major isoforms: endothelial (eNOS), neuronal (nNOS) and inducible (iNOS) (Forstermann et al., 1998). iNOS from Kupffer cell play a role in alcoholic liver disease, since ethanol toxicity was significantly attenuated in iNOS knock- out mice and in mice treated with 1400 W, a selective iNOS inhibitor (McKim et al., 2003). In fact, sustained generation of NO by iNOS, may turn on a broad spectrum of sequelae, from lipid peroxidation to DNA damage and pro-apoptotic effects (Davis et al., 2001) impli- cating iNOS to the pathogenesis of ethanol-induced liver injury. On the other hand, NO produced from eNOS may be protective, since inhibition of this enzyme enhanced liver damage (McKim et al., 2003). 0040-8166/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tice.2011.08.003