1679 Environmental Toxicology and Chemistry, Vol. 28, No. 8, pp. 1679–1686, 2009  2009 SETAC Printed in the USA 0730-7268/09 $12.00 + .00 EFFECTS OF DIETARY N-ACETYLCYSTEINE ON THE OXIDATIVE STRESS INDUCED IN TILAPIA (OREOCHROMIS NILOTICUS) EXPOSED TO A MICROCYSTIN-PRODUCING CYANOBACTERIAL WATER BLOOM MARI ´ A PUERTO,† ANA I. PRIETO,† SILVIA PICHARDO,† ISABEL MORENO,† A ´ NGELES JOS,† ROSARIO MOYANO,‡ and ANA M. CAMEA ´ N*† †Area of Toxicology, Faculty of Pharmacy, University of Seville, Profesor Garcı ´a Gonza ´lez Street Number 2, Seville 41012, Spain ‡Department of Pharmacology, Toxicology and Legal and Forensic Medicine, University of Cordoba, Campus de Rabanales Carretera Madrid-Ca ´diz s/n, Co ´rdoba 14071, Spain ( Received 16 October 2008; Accepted 1 February 2009) Abstract—Fish can be exposed to toxic cyanobacterial cells in natural waters and fish farms and suffer from oxidative damage. The present study investigates the effects of N-acetylcysteine (NAC), a glutathione (GSH) precursor, on the oxidative stress induced by Microcystis cyanobacterial cells containing microcystins (MCs) in tilapia fish (Oreochromis niloticus). Variation in lipid per- oxidation (LPO) levels, carbonyl group content, reduced glutathione to oxidized glutathione ratio (GSH:GSSG), and catalase (Enzyme Commission [EC] 1.11.1.6), superoxide dismutase (SOD; EC 1.15.1.1), glutathione reductase (GR; EC 1.8.1.7), glutathione per- oxidase (GPx; EC 1.11.1.9), and glutathione S-transferase (EC 2.5.1.18) activities in liver and kidney of tilapia exposed to a single oral dose of 120 g MC-LR (with leucine [L] and arginine [R])/fish and killed in 24 h were investigated in the absence and presence of 20.0, 44.0, and 96.8 mg NAC/fish/d. Results showed a protective role of NAC, depending on the dose and the biomarker considered. The increase in LPO (1.9- and 1.4-fold in liver and kidney, respectively) and the decreased protein content and GSH: GSSG in the liver induced by MCs were recovered mainly by the lower doses of NAC employed. Antioxidant enzyme activities increased (range, 1.4- to 1.7-fold) by MCs also were ameliorated by NAC, although the highest level used induced significant alteration of some enzymatic activities, such as SOD, GPx, and GR. Thus, NAC can be considered to be a useful chemoprotectant that reduces hepatic and renal oxidative stress in the prophylaxis and treatment of MC-related intoxications in fish when careful attention is given to its application dose because of its own pro-oxidant activity, as shown in the present study at 96.8 mg NAC/ fish/d. Keywords—Cyanobacteria Microcystin Oreochromis niloticus Oxidative stress N-acetylcysteine INTRODUCTION Microcystins (MCs), the predominant toxins of cyanobac- terial blooms, are cyclic heptapeptide molecules containing both L- and D-amino acids and an unusual hydrophobic C20 D-amino acid commonly termed ADDA (3-amino-9-methoxy- 2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid). More than 70 different MCs are known, and these differ mainly in the two L-amino acids at positions 2 and 4. The most common, and also the most extensively studied, are MC-LR (with leu- cine [L] and arginine [R]), MC-RR (with two arginines), and MC-YR (with tyrosine [Y] and arginine) [1]. Fish are fre- quently exposed to MCs both directly and passively, which consequently causes toxic effects and fish kills [1,2]. Microcystins have been characterized as potent inhibitors of serine and threonine phosphatases, affecting intracellular signalling, cell growth, and differentiation processes [3]. Ox- idative stress induced by exposure to MCs is considered to be involved in the development of MC toxicity in different ex- perimental models [4,5], including fish after oral exposure to Microcystis sp. [6–8]. Alterations of the major antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), and the antioxidant glutathione, in liver of fish exposed to MCs have been well documented, and these biochemical * To whom correspondence may be addressed (camean@us.es). Published on the Web 3/16/2009. responses could be responsible for the difference in sensitivity found among fish belonging to different trophic levels [9]. When reactive oxygen species (ROS) overwhelm the cel- lular defenses, essential biomolecules, such as proteins, lipids, and DNA, are damaged. Previous studies have demonstrated that MC-LR and MC-RR cause oxidative stress and lipid per- oxidation (LPO) in tilapia (Oreochromis niloticus) [10], and cyanobacterial water blooms induce protein oxidation in tench (Tinca tinca) [11]. Changes in cellular glutathione levels in different fish models caused by Microcystis cyanobacteria ex- tracts also have been reported [12,13]. Reduced glutathione (GSH) has been associated with a number of critical cellular regulatory functions, including xenobiotic conjugation of elec- trophiles by means of glutathione S-transferases (GSTs). In fact, the detoxification of MC-LR in the liver occurs by GSH conjugation via the action of GST [14], and the intracellular GSH plays an important role in MC-induced cytotoxicity and cytoskeletal changes in rat hepatocytes [15]. Reduced gluta- thione also behaves as a free radical scavenger, helps in re- generating other antioxidants, and acts as an electron donor in the reduction of peroxides governed by GPx, leading to the formation of glutathione disulfide. Although research in fish has demonstrated that mammalian and piscine systems exhibit similar toxicological and adaptive responses to oxidative stress [16], studies that document the protective effect of some antioxidants against the toxic action of MCs in aquatic organisms are very scarce [17]. Use of vitamin E has been proposed to restore tissue redox status in