IMMUNOCHEMICAL COMPARISON OF 3'-HYDROXYACETANILIDE AND ACETAMINOPHEN BINDING IN MOUSE-LIVER

The hepatotoxicity of the analgesic acetaminophen is believed to be me diated by covalent binding to critical proteins. Radiolabeled 3'-hydro xyacetanilide, a regioisomer of acetaminophen, covalently binds to pro teins at levels similar to those of acetaminophen, but without toxicit y. Covalent binding has recently been detected by Western blot to a 50 -kDa microsomal protein that comigrated with CYP2E1 and was accompanie d by a loss of the CYP2E1 activity. However, radiolabel studies previo usly indicated that a significant amount of the radiolabel is lost dur ing electrophoresis. In the present study, 3'-hydroxyacetanilide coval ent binding was detected immunohistochemically in liver using an anti- acetaminophen antiserum. 3'-Hydroxyacetanilide (1000 mg/kg, ip) admini stration to mice resulted in panlobular immunostaining in liver, with the single layer of hepatocytes surrounding the central veins having t he greatest intensity of staining. Staining was most intense at 1 hr a nd somewhat decreased at 3 and 6 hr. In contrast, immunochemical stain ing indicated that covalent binding of acetaminophen (250 mg/kg, ip) w as confined to the centrilobular hepatocytes, the area of the ensuing necrosis. Cobaltous chloride pretreatment decreased the total intensit y of the panlobular immunostaining following 8'-hydroxyacetanilide. Th e CYP2E1 inhibitor diallyl sulfide decreased the intensity of immunost aining in the central vein area only. Western blot analysis indicated diallyl sulfide also eliminated binding to the microsomal 50-kDa prote in, These data are consistent with centrilobular binding of 3'-hydroxy acetanilide, mediated in part by CYP2E1, and panlobular binding, media ted by other P450 enzymes.