Calpain released from dying hepatocytes mediates progression of acute liver injury induced by model hepatotoxicants

Liver injury is known to progress even after the hepatotoxicant is long gone and the mechanisms of progressive injury are not understood. We tested the hypothesis that hydrolytic enzymes such as calpain, released from dying hepatocytes, destroy the surrounding cells causing progression of injury. Calpain inhibitor, N-CBZ-VAL-PHE-methyl ester (CBZ), administered I h after a toxic but nonlethal dose of CCl4 (2 ml/kg, ip) to male Sprague Dawley rats substantially mitigated the progression of liver injury (6 to 48 h) and also led to 75% protection against CCl4-induced lethality following a lethal dose (LD75) of CCl4 (3 ml/kg). Calpain leakage in plasma and in the perinecrotic areas increased until 48 h and decreased from 72 It onward paralleling progression and regression of liver injury, respectively, after CCl4 treatment. Mitigation of progressive injury was accompanied by substantially low calpain in perinecrotic areas and in plasma after CBZ treatment. Normal hepatocytes incubated with the plasma collected from CCl4-treated rats (collected at 12 It when most of the CCl4 is eliminated) resulted in extensive cell death prevented by CBZ. Cell-impermeable calpain inhibitor E64 also protected against progression of CCl4-induced liver injury, thereby confirming the role of released calpain in progression of liver injury. Following CCl4 treatment, calpain-specific breakdown of a-fodrin increased, while it was negligible in rats receiving CBZ after CCl4. Hepatocyte cell death in incubations containing calpain was completely prevented by CBZ. Eighty percent of Swiss Webster mice receiving a lethal dose (LD80) of acetarainophen (600 mg/kg, ip) survived if CBZ was administered I h after acetaminophen, suggesting that calpain-mediated progression of liver injury is neither species nor chemical specific. These findings suggest the role of calpain in progression of liver injury. (C) 2003 Elsevier Inc. All rights reserved.