Acetaminophen hepatotoxicity: correspondence of selective protein arylation in human and mouse liver in vitro, in culture, and in vivo.
Human and mouse liver were exposed to an APAP-activating system, in vitro. Subsequent immunochemical analysis of electrophoretically separated proteins with an affinity-purified anti-APAP antibody indicated that when a cytosolic fraction from human liver was incubated with APAP, an NADPH-regenerating system, and mouse microsomes selective APAP binding occurred predominantly to proteins of approximately 38, 58, and 130 kDa. To evaluate whether similar proteins are targeted in situ, primary cultures of human hepatocytes were treated with 10 mM APAP for 4 hr prior to immunochemical analysis. APAP binding was again detected in protein bands of approximately 38, 58, and 130 kDa. In addition, selective binding was also noted to other cytosolic protein bands, e.g., approximately 52 and 62 kDa. For mouse liver, the majority of the binding, in vitro or in culture, was to proteins of approximately 44 and 58 kDa with lesser binding to proteins of approximately 33 and 130 kDa among others. By contrast, at the times monitored, little covalent binding was detected in the 44-kDa region in the human liver experiments. Most noteworthy was the finding that when the protein arylation patterns on liver samples from a human APAP fatality were compared to those from a mouse given a hepatotoxic dose of APAP, the binding patterns were similar to those detected after the in vitro and the culture experiments with mouse and human livers. Furthermore, an immunohistochemical analysis revealed that as with the mouse, APAP covalent binding in the human liver exhibited a distinct zonal pattern consistent with centrilobular binding. That APAP arylation of the 58- and 130-kDa proteins was observed in livers from both mice and humans suggests that the mouse provides a valid model for studying the mechanistic importance of covalent binding. Elucidation of the identities and functions of the common targeted proteins may clarify their toxicological significance.
Birge
R B
RB
SRI International, Menlo Park, California 94025.
Bartolone
J B
JB
Hart
S G
SG
Nishanian
E V
EV
Tyson
C A
CA
Khairallah
E A
EA
Cohen
S D
SD
D000082:Acetaminophen / Q000633:toxicity*; D000293:Adolescent; D000818:Animals; D002478:Cells, Cultured; D002675:Child, Preschool; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D008099:Liver / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D008297:Male; D051379:Mice; D008862:Microsomes, Liver / Q000378:metabolism; D008875:Middle Aged; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism
D011506:Proteins; D000082:Acetaminophen