Abstract

The hepatotoxicity of the analgesic acetaminophen is believed to be mediated by covalent binding to critical proteins. Radiolabeled 3′-hydroxyacetanilide, a regioisomer of acetaminophen, covalently binds to proteins at levels similar to those of acetaminophen, but without toxicity. Covalent binding has recently been detected by Western blot to a 50-kDa microsomal protein that comigrated with CYP2E1 and was accompanied by a loss of the CYP2E1 activity. However, radiolabel studies previously indicated that a significant amount of the radiolabel is lost during electrophoresis. In the present study, 3′-hydroxyacetanilide covalent binding was detected immunohistochemically in liver using an anti-acetaminophen antiserum. 3′-Hydroxyacetanilide (1000 mg/kg, ip) administration to mice resulted in panlobular immunostaining in liver, with the single layer of hepatocytes surrounding the central veins having the greatest intensity of staining. Staining was most intense at 1 hr and somewhat decreased at 3 and 6 hr. In contrast, immunochemical staining indicated that covalent binding of acetaminophen (250 mg/kg, ip) was confined to the centrilobular hepatocytes, the area of the ensuing necrosis. Cobaltous chloride pretreatment decreased the total intensity of the panlobular immunostaining following 3′-hydroxyacetanilide. The CYP2E1 inhibitor diallyl sulfide decreased the intensity of immunostaining in the central vein area only. Western blot analysis indicated diallyl sulfide also eliminated binding to the microsomal 50-kDa protein. These data are consistent with centrilobular binding of 3′-hydroxyacetanilide, mediated in part by CYP2E1, and panlobular binding, mediated by other P450 enzymes.