Acetaminophen is a mild analgesic and antipyretic agent known to cause centrilobular hepatic necrosis at toxic doses. Although this may be due to a direct interaction of reactive acetaminophen metabolites with hepatocyte proteins, recent studies have suggested that cytotoxic mediators produced by parenchymal and nonparenchymal cells also contribute to the pathophysiological process. Nitric oxide is a highly reactive oxidant produced in the liver in response to inflammatory mediators. In the present studies we evaluated the role of nitric oxide in the pathophysiology of acetaminophen-induced liver injury. Treatment of male Long Evans Hooded rats with acetaminophen (1 g/kg) resulted in damage to centrilobular regions of the liver and increases in serum transaminase levels, which were evident within 6 hours of treatment of the animals and reached a maximum at 24 hours. This was correlated with expression of inducible nitric oxide synthase (iNOS) protein in these regions. Hepatocytes isolated from both control and acetaminophen-treated rats were found to readily synthesize nitric oxide in response to inflammatory stimuli. Cells isolated from acetaminophen-treated rats produced more nitric oxide than cells from control animals. Production of nitric oxide by cells from both control and acetaminophen-treated rats was blocked by aminoguanidine, a relatively specific inhibitor of iNOS. Arginine uptake and metabolism studies revealed that the inhibitory effects of aminoguanidine were due predominantly to inhibition of iNOS enzyme activity. Pretreatment of rats with aminoguanidine was found to prevent acetaminophen-induced hepatic necrosis and increases in serum transaminase levels. This was associated with reduced nitric oxide production by hepatocytes. Inhibition of toxicity was not due to alterations in acetaminophen metabolism since aminoguanidine had no effect on hepatocyte cytochrome P4502E1 protein expression or N-acetyl-pbenzoquinone-imine formation. Taken together, these data demonstrate that nitric oxide is an important mediator of acetaminophen-induced hepatotoxicity. (HEPATOLOGY 1998; 26:748-754.)
Nitric oxide is a highly reactive oxidant produced by liver parenchymal and nonparenchymal cells from L-arginine via an inducible form of nitric oxide synthase (iNOS). Overproduction of nitric oxide in the liver has been implicated as an important event in endotoxin shock and in other models of hepatic inflammation and injury. The mechanism underlying the actions of nitric oxide in the liver is unknown. Nitric oxide has been reported to down-regulate cytochrome P450, to suppress liver protein and DNA synthesis, 2,18-20 and to induce apoptosis and necrosis, 2,21 and these activities may contribute to hepatotoxicity. Nitric oxide also inhibits catalase activity, suggesting that it may alter the detoxification of cytotoxic oxygen radicals. In this regard, nitric oxide is known to react with superoxide anion, forming peroxynitrite, an even more potent oxidizing agent. Peroxynitrite can react directly with sulfhydryl residues in cell membranes leading to lipid peroxidation, as well as with DNA, resulting in cytotoxicity. Acetaminophen is a mild analgesic and antipyretic agent that causes acute liver failure accompanied by centrilobular hepatic necrosis in cases of overdose. Recent studies have suggested that tissue injury is mediated in part by soluble products produced by hepatocytes and/or resident and inflammatory macrophages including tumor necrosis factor β£, interleukin 1, and reactive oxygen intermediates. The present studies were designed to examine the potential role of nitric oxide in the pathophysiology of acetaminopheninduced hepatotoxicity. We found that pretreatment of rats with aminoguanidine, a relatively selective inhibitor of iNOS, blocked acetaminophen-induced hepatotoxicity. These data suggest that reactive nitrogen intermediates contribute to tissue injury induced by this hepatotoxicant.
MATERIALS AND METHODS
Animals and Treatments.
Male, specific pathogen-free Long Evans Hooded rats weighing approximately 100 g each were obtained from Charles River (Wilmington, MA). Animals were housed in microisolator cages and were provided with food and water ad libitum. All animals received humane care in compliance with the institution' s guidelines criteria for humane care, as outlined in Guide for the Care and Use of Laboratory Animals, prepared by the National Academy of Sciences and published by the National Institutes of Health. Rats were pretreated with aminoguanidine (100 mg/kg/d, subcutaneously) or saline control for 3 days. Thirty minutes after the last injection, animals were treated with acetaminophen (1 g/kg, intraperitoneally) or vehicle control (20% Tween-80 in normal saline).