Metabolic activation and immunochemical localization of liver protein adducts of the nonsteroidal anti-inflammatory drug diclofenac.
Diclofenac is a nonsteroidal anti-inflammatory agent that is reported to cause serious hepatic injury in some patients. To investigate the possibility that protein adducts derived from reactive intermediates of diclofenac might be responsible for the hepatotoxicity produced by this drug, we recently developed polyclonal antisera that recognized protein adducts of diclofenac. In the present study, we have characterized further the diclofenac adducts in rat liver. Immunoblotting studies showed that diclofenac-labeled hepatic proteins were formed in a dose- and time-dependent manner in rats given diclofenac. Subcellular fractionation of liver homogenates from diclofenac-treated rats showed that a 50-kDa microsomal protein and 110-, 140-, and 200-kDa plasma membrane proteins were labeled preferentially. Immunofluorescence studies of isolated hepatocytes and immunohistochemical analysis of liver slices from diclofenac-treated mice and rats confirmed that plasma membrane proteins were labeled by diclofenac metabolites and showed that the bile canalicular domain of the plasma membrane was a major site of diclofenac adduct formation. Additionally, we found that cytochrome P-450 and UDP-glucuronosyltransferase, but not acyl-CoA synthase, catalyzed the formation of reactive intermediates of diclofenac that were bound covalently to proteins in vitro. The metabolites catalyzed by cytochrome P-450 in vitro were bound exclusively to a 50-kDa microsomal protein, even in the presence of albumin. In contrast, the 110-, 140-, and 200-kDa plasma membrane proteins as well as others appeared to be labeled when diclofenac was activated by UDP-glucuronosyltransferase.(ABSTRACT TRUNCATED AT 250 WORDS)
Hargus
S J
SJ
Laboratory of Chemical Pharmacology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
Amouzedeh
H R
HR
Pumford
N R
NR
Myers
T G
TG
McCoy
S C
SC
Pohl
L R
LR
D000818:Animals; D001711:Biotransformation; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004008:Diclofenac / Q000493:pharmacokinetics / Q000633:toxicity*; D004305:Dose-Response Relationship, Drug; D005455:Fluorescent Antibody Technique; D014453:Glucuronosyltransferase / Q000378:metabolism; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley
D011506:Proteins; D004008:Diclofenac; D003577:Cytochrome P-450 Enzyme System; D014453:Glucuronosyltransferase