Immunoblot analysis of protein containing 3-(cystein-S-yl)acetaminophen adducts in serum and subcellular liver fractions from acetaminophen-treated mice

Toxicol Appl Pharmacol. 1990 Jul;104(3):521-32. doi: 10.1016/0041-008x(90)90174-s.


The hepatotoxicity of acetaminophen is believed to be mediated by the metabolic activation of acetaminophen to N-acetyl-p-benzoquinone imine which covalently binds to cysteinyl residues on proteins as 3-(cystein-S-yl)acetaminophen adducts. The formation of these adducts in hepatic protein correlates with the hepatotoxicity. In this study, the formation of 3-(cystein-S-yl)acetaminophen adducts in specific cellular proteins was investigated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and detected using affinity-purified antisera specific for 3-(cystein-S-yl)acetaminophen adducts on immunoblots. These techniques were used to investigate the liver 10,000g supernatant and serum from B6C3F1 mice that received hepatotoxic doses of acetaminophen. More than 15 proteins containing 3-(cystein-S-yl)acetaminophen adducts were detected in the liver 10,000g supernatant. The most prominent protein containing 3-(cystein-S-yl)acetaminophen adducts in the hepatic 10,000g supernatant had a relative molecular mass of 55 kDa. Serum proteins containing 3-(cystein-S-yl)acetaminophen adducts had molecular masses similar to those found in the liver 10,000g supernatant (55, 87, and approximately 102 kDa). These data, combined with our previous findings describing the temporal relationship between the appearance of 3-(cystein-S-yl)acetaminophen adducts in protein in the serum and the decrease in the levels of 3-(cystein-S-yl)acetaminophen adducts in protein in the liver, suggested that liver adducts were released into the serum following lysis of hepatocytes. The temporal relationship between the formation of specific adducts and hepatotoxicity in mice following a hepatotoxic dose of acetaminophen was examined using immunoblots of mitochondria, microsomes, cytosol, and plasma membranes. Hepatotoxicity indicated by serum alanine aminotransferase levels was increased at 2 and 4 hr after dosing. The cytosolic fraction contained numerous proteins with 3-(cystein-S-yl)acetaminophen adducts, the most intensely stained of which was a 55-kDa protein. 3-(Cystein-S-yl)acetaminophen adducts were detected in the 55-kDa liver protein 30 min after dosing and prior to the development of significant toxicity. Examination of gels suggested that maximal levels of immunochemically detectable adducts in the 55-kDa protein occurred at 1-2 hr, with a decrease in intensity 4 hr after dosing. The presence of 3-(cystein-S-yl)acetaminophen adducts in proteins prior to hepatotoxicity suggests a threshold for adduct formation in the development of toxicity. Protein in microsomes which contained 3-(cystein-S-yl)acetaminophen adducts ranged in molecular weight from 38 to approximately 106 kDa. The major proteins containing 3-(cystein-S-yl)acetaminophen adducts in the mitochondria had molecular masses of 39, 50, 68, and 79 kDa.(ABSTRACT TRUNCATED AT 400 WORDS)

MeSH terms

  • Acetaminophen / metabolism*
  • Alanine Transaminase / analysis
  • Animals
  • Binding, Competitive
  • Blotting, Western
  • Cell Membrane / analysis
  • Cytosol / analysis
  • Electrophoresis, Polyacrylamide Gel
  • Liver / analysis*
  • Male
  • Mice
  • Mice, Inbred Strains
  • Microsomes / analysis
  • Mitochondria / analysis
  • Proteins / analysis


  • Proteins
  • Acetaminophen
  • Alanine Transaminase