Ž . Biochimica et Biophysica Acta 1336 1997 403–408 Rat liver ADP-ribose pyrophosphatase-I as an in vitro target of the acetaminophen metabolite N-acetyl-p-benzoquinoneimine Joao Meireles Ribeiro, Antonio Agudo, Marıa Jesus Costas, Jose Carlos Cameselle ) ˜ ´ ´ ´ Unidad de Bioquımica y Biologıa Molecular, Facultad de Medicina, UniÕersidad de Extremadura, Apartado de Correos 108, E-06080 ´ ´ Badajoz, Spain Received 7 March 1997; accepted 3 April 1997 Abstract Ž . N-acetyl-p-benzoquinoneimine NAPQI is the metabolite responsible for acetaminophen hepatotoxicity. ADP-ribose Ž . pyrophosphatase-I ADPRibase-I; EC 3.6.1.13 hydrolyzes protein-glycating ADP-ribose. The results show NAPQI-depen- dent alterations of ADPRibase-I leading to strong inhibition: a fast K increase produced by low concentrations, and a m time-dependent V decrease by higher NAPQI concentrations. Both effects were prevented by thiols, but not reverted by max them, nor by gel filtration of NAPQI-treated enzyme. Liver ADPRibase-I can be a target of NAPQI-dependent arylation. The inhibition or inactivation of the enzyme would contribute to increasing the free ADP-ribose concentration and nonenzymatic ADP-ribosylation, which is coherent with results linking free ADP-ribose-producing pathways to ac- etaminophen toxicity. q 1997 Elsevier Science B.V. Keywords: ADP-ribose, free; ADP-ribose pyrophosphatase; Acetaminophen; Paracetamol; N-Acetyl-p-benzoquinoneimine; Hepatotoxic- ity 1. Introduction Ž Acetaminophen N-acetyl-p-aminophenol or . paracetamol is widely used as analgesic and an- tipyretic because of its low toxicity at therapeutic doses. However, acetaminophen overdosage can be severely hepatotoxic, particularly after heavy alcohol w x drinking 1,2 . The drug is metabolized by hepatic transferases to glucuronide- and sulfo-conjugates, and Abbreviations: ADPRibase-I, ADP-ribose pyrophosphatase-I; DMSO, dimethylsulfoxide; DTT, DL-dithiothreitol; GSH, reduced glutathione; NAPQI, N-acetyl-p-benzoquinoneimine ) Ž . Corresponding author. Fax: q34 24 289468. E-mail: cam- selle@unex.es wx a minor part by cytochrome P-450 to NAPQI 3,a reactive electrophile that, at low concentration, is efficiently detoxified by nonenzymatic reaction with GSH. However, if the sulfation and glucuronidation pathways become saturated, or if cytochrome P-450 is induced, the proportion of drug metabolized to NAPQI increases and, eventually, GSH can be de- pleted. Severe hepatotoxicity and acute liver failure wx can then occur 4 due either to the unrestricted wx reactivity of NAPQI towards proteins 5 , to the oxidative stress related to GSH depletion and redox cycling between quinone and semiquinone forms of w x NAPQI 5,6 , andror to the disruption of intracellular 2q w x Ca homeostasis 7,8 . Concerning the covalent binding of NAPQI, the major type of protein adducts formed involves selective arylation of protein thiols wx 9 . However, although NAPQI binds to many pro- 0304-4165r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. Ž . PII S0304-4165 97 00051-2