Original article Arachidonic acid cytotoxicity in leukocytes: implications of oxidative stress and eicosanoid synthesis Celine Pompeia a, *, Jofre J.S. Freitas b , Jung S. Kim c , Szulim B. Zyngier c , Rui Curi d a National Institute of Deafness and other Communication Disorders, National Institutes of Health, Room 4249, Building 50, 50 South Drive, Bethesda, MD 20892, USA b Department of Physiological and Morphological Sciences, Center of Biological Sciences, University of Para State, Brazil c Department of Pharmacology, Institute of Biomedical Sciences I, University of São Paulo, Brazil d Department of Physiology and Biophysics, University of São Paulo, Brazil Received 26 June 2001; accepted 15 May 2002 Abstract Arachidonic acid (AA)-induced cytotoxicity was evaluated in leukocytes: the human leukemia cell lines HL-60, Jurkat and Raji and in rat lymphocytes. Such cytotoxicity was dose- and time-dependent. At concentrations below 5 μM, AA was not toxic; at 10–400 μM, AA induced apoptosis and at concentrations beyond 400 μM, necrosis. The minimum exposure time to trigger cell death was of around 1 h, but the effect was increased by longer exposure times until 6–24 h. Apoptosis was morphologically characterized by a decrease in cell and nuclear volume, chromatin condensation and DNA fragmentation and the presence of lipid bodies, without changes in organelle integrity. Biochemically, AA-induced apoptosis was associated with internucleosomal fragmentation and caspase activation, evaluated by PARP cleavage and the use of a caspase inhibitor. Necrosis was characterized by increased cell volume, presence of loose chromatin, appearance of vacuoles, loss of membrane integrity and of the definition of organelles. The apoptotic effect of AA was studied as to oxidative–reductive imbalance and the participation of eicosanoids. Apoptotic AA treatment was accompanied by an increase in the quantity of thiobarbituric acid reactive substances (TBARS), low-level chemiluminescence and in the glutathione disulfide/reduced glutathione ratio, indicating oxidative stress. The addition of tocopherol, ascorbate, prostaglandin E 2 and lipoxygenase inhibitors delayed cell death, whereas the inhibition of cyclooxygenase promoted AA-induced cell death. Cell treatment with AA was accompanied by increased cellular production of LTB 4 . AA, therefore, is cytotoxic at physiological and supraphysiological concentrations, causing apoptosis and necrosis. Cell treatment with apoptotic concentrations of AA involves oxidative stress and changes in eicosanoid biosynthesis. © 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Arachidonic acid; Leukocytes; Oxidative stress; Apoptosis; Necrosis; Eicosanoids 1. Introduction Arachidonic acid (AA) is an essential polyunsaturated fatty acid (PUFA) that participates directly or after its conversion to eicosanoids in the modulation of the function of various organs and systems, such as the digestive, reproductive, renal and immune systems. Moreover, AA is an important mediator of inflammation, as can be confirmed by the fact that among the most used pharmaceutical substances are the inhibitors of AA metabolism or release that act as anti-inflammatory and analgesic drugs. AA is metabolized into eicosanoids via specific enzymes, such as cyclooxygenases (COX), which generate prostaglandins (PG) and thromboxanes (TX); lipoxygenase, which leads to the production of leukotrienes (LT), lipoxins (LX), hydrox- yeicosatetraenoic acids (HETEs) and hydroperoxyeicosatet- raenoic acids (HPETEs); and cytochromes P-450, which participate in the generation of PGs, TXs, HETEs, HPETEs, epoxyeicosatrienoic (EETEs) acids, among others. Abbreviations: AA, arachidonic acid; ASA, acetylsalicylic acid; Asc, ascorbate; COX, cyclooxygenase; EtOH, ethanol; FSC, forward scattering; GSSG, glutathione disulfide; LD50, lethal dose 50%; LT, leukotriene; Nd, not determined; NDGA, nordihydroguaiaretic acid; NSAID, non-steroidal anti-inflammatory drug; PARP, poly(ADP)-ribose polymerase; PBS, phosphate-buffered saline; PG, prostaglandin; PUFA, polyunsaturated fatty acids; S.D., standard deviation; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; SSC, side scattering; TBARS, thiobar- bituric acid reactive substances; toco, tocopherol * Corresponding author. Tel.: +1-301-402-1601; fax: +1-301-402-1765. E-mail address: cpompeia@nidcd.nih.gov (C. Pompeia). Biology of the Cell 94 (2002) 251–265 www.elsevier.com/locate/biocell © 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. PII: S 0 2 4 8 - 4 9 0 0 ( 0 2 ) 0 1 2 0 0 - 5