Mechanisms of Cell Death of Thymocytes Induced by Polyunsaturated, Monounsaturated and Trans-Fatty Acids Mari A ˚ hs, 1 Aparna Prasad, 2 Zafar Aminov, 2 and David O. Carpenter 1,2 * 1 Institute for Health and the Environment, University at Albany, Rensselaer, NY 12144 2 Department of Environmental Health Sciences, School of Public Health, University at Albany, Rensselaer, NY 12144 ABSTRACT Polyunsaturated fatty acids (PUFAs) are rapidly cytotoxic to isolated murine thymocytes, and the degree of cell death has been correlated with changes in membrane fluidity, elevation of intracellular calcium concentration and generation of reactive oxygen species. We have compared the degree of cell death and increase in membrane fluidity of C-20 and C-22 omega-3 and 6 PUFAs to those induced by monounsaturated and trans-fatty acids, and find that concentrations which induce comparable increases in membrane fluidity do not cause comparable cell death. The C-18 omega-6 causes a decrease in membrane fluidity, yet is the most potent in causing cell death. Omega-6 PUFAs are more cytotoxic than omega-3 PUFAs, while monounsaturated and trans-fats show little cytotoxicity and only at much higher concentrations. Cell death is preceded by reductions of both plasma and mitochondrial membrane potential, and occurs via apoptosis. These results indicate that cell death is due to mechanisms other than changes in membrane fluidity. J. Cell. Biochem. 112: 3863–3871, 2011. ß 2011 Wiley Periodicals, Inc. KEY WORDS: OMEGA-3 FATTY ACIDS; APOPTOSIS; MITOCHONDRIA; MEMBRANE FLUIDITY; CALCIUM O mega-3 polyunsaturated fatty acids (PUFAs) are essential to humans and have a variety of biochemical, nutritional and epigenetic properties [Benatti et al., 2004]. They are major components on neuronal membranes [Horrocks and Farooqui, 2004], and deficiency results in impaired learning and visual function [Bourre et al., 1993]. They are also thought to be important for cardiovascular health [Hooper et al., 2006] and are known to reduce risk of sudden cardiac death following a heart attack [Albert et al., 1998]. The mechanism is believed to be that they become incorporated into cardiac muscle membranes, increasing membrane fluidity and as a result reducing entry of sodium and calcium into cells [Leaf et al., 2005]. In contrast, saturated and trans-fats increase risk of cardiovascular disease [Oh et al., 2005; Woodside and Kromhout, 2005]. Omega-6 PUFAs are also essential, but these PUFAs are rarely lacking in the current human diet, leading some to propose that the omega-6 to omega-3 PUFA ratio has important implications for human health [Harbige, 2003; Wijendran and Hayes, 2004; Simopoulos, 2008]. It has been known for many years that exogenous lipids can alter membrane fluidity. Saturated fats [Calder et al., 1994] and cholesterol [Lau and Das, 1995] cause a decrease in fluidity, while omega-3 fatty acids cause an increased fluidity in various cells [Hashimoto et al., 1999, 2001] and membranes including mitochondrial membranes [Stillwell et al., 1997]. Arachidonic acid (C-20, omega-6) (ARA) has also been reported to increase fluidity of endothelial cells [Villacara et al., 1989] and to help Journal of Cellular Biochemistry ARTICLE Journal of Cellular Biochemistry 112:3863–3871 (2011) 3863 Abbreviations: ARA; 20:5n-6, arachidonic acid; ALA; 18:3n-3, a linolenic acid; BAPTA; 1,2-bis(o-aminophenox- y)ethane-N,N,N’,N’-tetraacetic acid; C; carbon; CCCP; carbonyl cyanide 3-chlorophenylhydrazone; DCF; dichloro- fluorescein; DiBAC 4 (3); bis-(1; 3-dibutylbarbituric acid)trimethine oxonol; DPH; 1,6-diphenyl-1,3,5-hexatriene; DHA; 22:6n-3; docosahexaenoic acid; DilC1(5); 1,1 ´ ,3,3,3 ´ -hexamethylindodicarbocyanine idodide; DTA; 22:4n-6; docosatetraenoic acid; EA; t18:1; elaidic acid; EPA; 20:5n-3; eicosapentaenoic acid; HEPES; 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid; LNA; 18:2n-6; linoleic acid; MMP; mitochondrial membrane potential; MUFA; monounsaturated fatty acids; OLA; oleic acid; P; fluorescence polarization; PI; propidium iodide; PMP; plasma membrane potential; PUFAs; polyunsaturated fatty acids; ROS; reactive oxygen species; SEM; standard error of the mean. Mari A ˚ hs and Aparna Prasad contributed equally to this study. Grant sponsor: Linkoping University Medical School; Grant sponsor: Institute for Health and the Environment, University at Albany. Mari A ˚ hs’s present address is Linko ¨ ping University Medical School, Linko ¨ ping, Sweden. *Correspondence to: David O. Carpenter, Institute for Health and the Environment, University at Albany, 5 University Place, A 217, Rensselaer, NY 12144, USA. E-mail: carpent@uamail.albany.edu Received 4 August 2011; Accepted 8 August 2011  DOI 10.1002/jcb.23319  ß 2011 Wiley Periodicals, Inc. Published online 15 August 2011 in Wiley Online Library (wileyonlinelibrary.com).