Role of reactive oxygen species in organophosphate insecticide phosalone toxicity in erythrocytes in vitro I. Altuntas a, *, N. Delibas a , D.K. Doguc a , S. Ozmen b , F. Gultekin a a Suleyman Demirel University, School of Medicine, Department of Biochemistry and Clinical Biochemistry, Isparta, Turkey b Department of Anesthesiology and Reanimation, Suleyman Demirel University, School of Medicine, Isparta, Turkey Accepted 30 November 2002 Abstract Reactive oxygen species (ROS) caused by organophosphates may be involved in the toxicity of various pesticides. Therefore, in this study we aimed to investigate how an organophosphate insecticide, phosalone, affects lipid peroxidation (LPO) and the anti- oxidant defence system in vitro. For this purpose, the effects of various doses of phosalone on LPO and the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT) in erythrocytes were studied. Each phosalone dose was incubated with a previously prepared erythrocyte sample at +4  C for 0, 60 and 180 min. After incubation, the levels of mal- ondialdehyde (MDA) and the activities of SOD, GSH-Px and CAT were determined. Phosalone caused an increase in MDA for- mation and a decrease in the activities of SOD, GSH-Px and CAT. However, these effects were seen only at extremely high concentrations of phosalone and these concentrations were in the lethal range. Therefore, we suggest that ROS may not involve in the toxic effects of the pesticidal use of phosalone in low concentrations. # 2003 Elsevier Science Ltd. All rights reserved. Keywords: Antioxidant enzymes; Lipid peroxidation; Phosalone 1. Introduction The widespread use of organophosphate insecticides (OPIs) has long been shown to exert deleterious effects on living organisms (Gultekin et al., 2001). In general, OPIs are neurotoxic in nature by acting as inhibitors of neuronal cholinesterase (ChE) activity. However, some studies reported that OPIs caused lipid peroxidation (LPO). In these studies, LPO has been suggested as one of the molecular mechanisms involved in OPI-induced toxicity (Gupta et al., 1992; Yamano and Morita 1992; Bagchi et al., 1995; Bachowski et al., 1997; Gultekin et al. 2000b, 2001). Previous studies from the present laboratory focused on in vitro and in vivo effects only of chlorpyrifos-ethyl, one of the organophosphate insecticides (OPIs), on LPO and antioxidant enzymes status. The results indi- cated that chlorpyrifos-ethyl caused increased LPO in erythrocytes. It was also shown that chlorpyrifos-ethyl significantly altered the activities of the main anti- oxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT) (Gultekin et al., 2000b, 2001). In the region of Isparta, Turkey, the OPIs commonly used in agricultural production were reported to be phosalone, chlorpyrifos-ethyl, methidathion, fenthion, diazinon, parathion methyl, malathion and oxydemeton methyl in recent years (Agricultural Ministry Office of Isparta Province). Therefore, we have now a great interest in evaluating the toxicity of these OPIs by series of in vitro and in vivo studies in our laboratory. Phosalone (O,O-diethyl-S-(6-chloro-2-oxobenzox- azolin-3-yl-methyl)-phosphorodithioate) has attracted our interest for its effect on LPO and activities of some antioxidant enzymes in vitro or in vivo since no experi- mental results have been reported with phosalone on LPO of human or rat erythrocytes. We have proposed that possible application of various doses of phosalone 0887-2333/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0887-2333(02)00133-9 Toxicology in Vitro 17 (2003) 153–157 www.elsevier.com/locate/toxinvit Abbreviations: CAT, catalase; ChE, cholinesterase; GSH-Px, glu- tathione peroxidase; INT, 2-(4-iodophenyl)-3-(4-nitrophenol)-5-phe- nyltetrazolium chloride; LPO, lipid peroxidation; MDA, malondialdehyde; OPIs, organophosphate insecticides; ROS, reactive oxygen species; SOD, superoxide dismutase. TBA, thiobarbituric acid; TCA, trichloroacetic acid. * Corresponding author. Fax: +90-246-2371651. E-mail address: irfanaltuntas@hotmail.com (I. Altuntas).