Nitric Oxide, Superoxide, and Hydrogen Peroxide Production in Brain Mitochondria after Haloperidol Treatment Silvia Lores Arnaiz, 1 Marı ´a Florencia Coronel, and Alberto Boveris Laboratory of Free Radical Biology–Physical Chemistry, School of Pharmacy and Biochemistry, University of Buenos Aires, 1113 Buenos Aires, Argentina Received October 30, 1998, and in revised form March 19, 1999 Inhibition of mitochondrial respiration and free radical induction have been suggested to be in- volved in haloperidol neurotoxicity. In this study, mice were injected i.p. with haloperidol, according to two different treatments: (a) a single injection (1 mg/kg), sacrificed 1 h after the injection (single- dose model); and (b) two injections (1 mg/kg each), sacrificed 24 h after the first dose (double-dose mod- el). Determinations of oxygen consumption and hy- drogen peroxide (H 2 O 2 ) production rate were car- ried out in isolated brain mitochondria. Nitric oxide (NO) and superoxide (O 2  ) production rates were measured in submitochondrial particles (SMP). Single-dose haloperidol treatment produced a 33% inhibition in malate– glutamate-dependent respira- tion, while no significant changes were found after double-dose treatment. NO production was inhib- ited by 39 and 54% in SMP from haloperidol-treated mice (single- and double-dose treatments, respec- tively) (control value: 1.6  0.2 nmol/min mg pro- tein). NO steady-state concentration was estimated at about 16.5 nM and was decreased by 40% by hal- operidol treatment. Increases of 105 and 54% were found in succinate-supported O 2  and H 2 O 2 produc- tion rates, respectively, after haloperidol single- dose treatment. Haloperidol treatment generated a 248% increase in SMP O 2  production rate when measured in the presence of NADH plus rotenone. Our results suggest that haloperidol neurotoxicity would be mediated by a decreased mitochondrial NO production, a decreased intramitochondrial NO steady-state concentration, and by an inhibition of mitochondrial electron transfer with enhancement of O 2  and H 2 O 2 production. This inhibition does not seem to be caused by increased NO or ONOO  formation. © 1999 Academic Press Key Words: nitric oxide; superoxide; hydrogen peroxide; brain mitochondria; haloperidol. The primary production of superoxide radical (O 2 - ) in mitochondrial membranes and the secondary free radical reactions in the mitochondrial membranes and matrix have been indicated as pathogenic causes for several CNS disorders, including Parkin- son and Alzheimer diseases, tardive dyskinesia, Down syndrome, and ischemic stroke (1). Brain derives most of its energy from mitochon- drial oxidative phosphorylation, consuming in the process a disproportionate amount of the body oxy- gen uptake (2). A significant amount of the oxygen consumed by mitochondria is diverted to form O 2 - and hydrogen peroxide (H 2 O 2 ) as byproducts of the respiratory chain biochemical activity. Superoxide is the stoichiometric precursor of H 2 O 2 (3) and it has been recently estimated that O 2 - and H 2 O 2 formation account for about 1 and 0.5% of the O 2 uptake of liver and heart mitochondria under physiological conditions (4). 1 To whom correspondence should be addressed. Fax: (54-11) 4508-3646. E-mail: slarnaiz@ffyb.uba.ar. NITRIC OXIDE: Biology and Chemistry Vol. 3, No. 3, pp. 235–243 (1999) Article ID niox.1999.0229, available online at http://www.idealibrary.com on 235 1089-8603/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.