Immunochemical quantitation of 3-(cystein-S-yl)acetaminophen protein adducts in subcellular liver fractions following a hepatotoxic dose of acetaminophen.
The hepatotoxicity of acetaminophen correlates with the formation of 3-(cystein-S-yl)acetaminophen protein adducts. Using a sensitive and specific immunochemical assay, we quantitated the formation of these protein adducts in liver fractions and serum after administration of a hepatotoxic dose of acetaminophen (400 mg/kg) to B6C3F1 mice. Adducts in the cytosolic fraction increased to 3.6 nmol/mg protein at 2 hr and then decreased to 1.1 nmol/mg protein by 8 hr. Concomitant with the decrease in adducts in the cytosol, 3-(cystein-S-yl)acetaminophen protein adducts appeared in serum and their levels paralleled increases in serum alanine aminotransferase. Microsomal protein adducts peaked at 1 hr (0.7 nmol/mg protein) and subsequently decreased to 0.2 nmol/mg at 8 hr. The 4000 g pellet (nuclei, plasma membranes, and cell debris) had the highest level of adducts (3.5 nmol/mg protein), which remained constant from 1 to 8 hr. Evaluation of fractions purified from a 960 g pellet indicated that the highest concentration of 3-(cystein-S-yl)acetaminophen protein adducts was located in plasma membranes and mitochondria; peak levels were 10.3 and 5.1 nmol/mg respectively. 3-(Cystein-S-yl)acetaminophen protein adducts were detected in nuclei only after enzymatic hydrolysis of the proteins. The localization of high levels of 3-(cystein-S-yl)acetaminophen protein adducts in plasma membranes and mitochondria may play a critical role in acetaminophen toxicity.
Division of Biochemical Toxicology, National Center for Toxicological Research, Jefferson, AR 72079-9502.
N R
NR
Pumford
D W
DW
Roberts
R W
RW
Benson
J A
JA
Hinson
D000082:Acetaminophen / Q000032:analysis / Q000097:blood / Q000378:metabolism* / Q000633:toxicity; D000410:Alanine Transaminase / Q000097:blood; D000818:Animals; D016227:Benzoquinones; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D056486:Chemical and Drug Induced Liver Injury; D003545:Cysteine / Q000032:analysis / Q000097:blood / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D007097:Imines / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000648:ultrastructure*; D008297:Male; D051379:Mice; D008862:Microsomes, Liver / Q000378:metabolism; D008930:Mitochondria, Liver / Q000378:metabolism; D011809:Quinones / Q000378:metabolism / Q000494:pharmacology
D016227:Benzoquinones; D007097:Imines; D011809:Quinones; C043967:acetaminophen-cysteine conjugate; D000082:Acetaminophen; D000410:Alanine Transaminase; C028473:N-acetyl-4-benzoquinoneimine; D003545:Cysteine