ADP-ribose pyrophosphatase-I partially purified from livers of rats overdosed with acetaminophen reveals enzyme inhibition in vivo reverted in vitro by dithiothreitol.
Free ADP-ribose reacts nonenzymatically with proteins and can lead to intracellular damage. The low-Km ADP-ribose pyrophosphatase-I (ADPRibase-I) is well suited to control free ADP-ribose and nonenzymatic ADP-ribosylation. In vitro, the acetaminophen metabolite N-acetyl-p-benzoquinoneimine (NAPQI) decreases ADPRibase-I Vmax and increases Km, effects not reverted by dithiothreitol (DTT) and attributed to enzyme arylation. The present study was conducted to test whether acetaminophen overdose affected ADPRibase-I in vivo. Rats pretreated with 3-methylcholanthrene and L-buthionine-[S,R]-sulfoximine to potentiate acetaminophen toxicity received an intraperitoneal dose of either acetaminophen (800 mg/ kg; n = 5) or vehicle (n = 3). ADPRibase-I partially purified from acetaminophen-overdosed rats showed a decreased Vmax (0.32+/-0.09 versus 0.60+/-0.03 mU/mg of liver protein; p<0.01) not reverted by DTT and an increased Km for ADP-ribose (1.39+/-0.31 versus 0.67+/-0.05 microM; p<0.01) that, contrary to the in vitro NAPQI effect, was reverted by DTT. Incubation of partially purified ADPRibase-I from normal rat liver with oxidized glutathione elicited a time- and dose-dependent, DTT-reverted increase of Km, without change of Vmax. The results indicate that the activity of ADPRibase-I can be regulated by thiol exchange and that the increase of Km, elicited by acetaminophen overdosage was related to the oxidative stress caused by the drug. It remains to be seen whether an increase of free ADP-ribose concomitant to ADPRibase-I inhibition could contribute to the hepatotoxicity of acetaminophen.
Ribeiro
J M
JM
Unidad de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad de Extremadura, Badajoz, Spain.
Costas
M J
MJ
Cameselle
J C
JC
D000082:Acetaminophen / Q000506:poisoning*; D000818:Animals; D004229:Dithiothreitol / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000506:poisoning*; D004793:Enzyme Reactivators / Q000494:pharmacology*; D005260:Female; D008099:Liver / Q000187:drug effects* / Q000201:enzymology; D011755:Pyrophosphatases / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D051381:Rats; D017208:Rats, Wistar
D004791:Enzyme Inhibitors; D004793:Enzyme Reactivators; D000082:Acetaminophen; C058796:ADP-ribose pyrophosphatase I; D011755:Pyrophosphatases; D004229:Dithiothreitol