ABSTRACT: There is experimental evidence that dietary fish oil, which contains the n-3 fatty acid family, i.e., EPA and DHA, protects against colon tumor development, in part by increas- ing apoptosis. Since mitochondria can act as central execution- ers of apoptosis, we hypothesized that EPA and DHA incorpo- ration into colonocyte mitochondrial membranes, owing to their high degree of unsaturation, would enhance susceptibility to damage by reactive oxygen species (ROS) generated via ox- idative phosphorylation. This, in turn, would compromise mito- chondrial function, thereby initiating apoptosis. To test this hy- pothesis, colonic crypts were isolated from rats fed either fish oil, purified n-3 fatty acid ethyl esters, or corn oil (control). Di- etary lipid source had no effect on colonic mitochondrial phos- pholipid class mole percentages, although incorporation of EPA and DHA was associated with a reduction in n-6 fatty acids known to enhance colon tumor development, i.e., linoleic acid (LNA) and its metabolic product, arachidonic acid (ARA). Se- lect compositional changes in major phospholipid pools were correlated to alterations in mitochondrial function as assessed by confocal microscopy. The mol% sum of LNA plus ARA in cardiolipin was inversely correlated with ROS (P = 0.024). Ethanolamine glycerophospholipid ARA (P = 0.046) and choline glycerophospholipid LNA (P = 0.033) levels were posi- tively correlated to mitochondrial membrane potential. In con- trast, ethanolamine glycerophospholipid EPA (P = 0.042) and DHA (P = 0.024) levels were negatively correlated to mitochon- drial membrane potential. Additionally, EPA and DHA levels in choline glycerophospholipids (P = 0.026) were positively corre- lated with caspase 3 activity. These data provide evidence in vivo indicating that dietary EPA and DHA induce compositional changes in colonic mitochondrial membrane phospholipids that facilitate apoptosis. Paper no. L8900 in Lipids 37, 193–199 (February 2002). Highly polyunsaturated dietary n-3 FA, i.e., EPA (20:5n-3) and DHA (22:6n-3), are protective against colon cancer in epi- demiological, clinical, and experimental studies (1–6). These FA are found in high concentration in fish and marine oils. It is clear from recently published studies (5,7,8) that fish oil is protective against experimentally induced colon cancer in part by up-regulating colonocyte apoptosis. In contrast, dietary lipids rich in n-6 FA, found in a variety of vegetable oils, en- hance the development of colon tumors and suppress apopto- sis (5,6,8). These data are noteworthy because a decrease in apoptosis, rather than an increase in cell proliferation, is a bet- ter predictor of colon tumorigenesis (9). However, the mecha- nism(s) by which EPA and DHA promote apoptosis in the colon remain to be elucidated. In many cell types, mitochondria are considered the cen- tral effectors of apoptosis (10). Evidence suggests that incor- poration of marine lipids containing EPA and DHA into mi- tochondrial membranes increases susceptibility to damage by reactive oxygen species (ROS) (11). In addition, in vitro, DHA is capable of increasing cell oxidant production by ac- cumulating in cardiolipin, where it alters electron transport efficiency (12). These data suggest that alterations in mito- chondrial function can be attributed to changes in the acyl chains of mitochondrial phospholipids, which are altered by the composition of dietary fat. Surprisingly, to date, no at- tempt has been made to quantify the incorporation in vivo of EPA and DHA into colonic mitochondrial phospholipid classes. Therefore, we compared the effect of dietary fish oil containing EPA and DHA vs. highly purified fatty acid ethyl esters (FAEE) containing EPA and DHA or corn oil, lacking EPA and DHA, on the levels of colonocyte mitochondrial phospholipid classes and their FA composition. In addition, in order to better understand the importance of the biochemi- cal effects of n-3 FA on colonocyte biology, we tested the hy- pothesis that dietary EPA and DHA induce compositional changes in select membrane phospholipid pools that promote apoptogenic events in vivo. MATERIALS AND METHODS Materials. Reagents for mitochondrial preparation and en- zyme assays were from Sigma (St. Louis, MO). Corn oil was kindly donated by Traco Labs (Champaign, IL). Vacuum-de- odorized menhaden fish oil was provided by the National In- stitutes of Health Fish Oil Test Material Program, Southeast Center (Charleston, SC). Dietary components were purchased from Bioserv (Frenchtown, NJ). All FAEE were from Copyright © 2002 by AOCS Press 193 Lipids, Vol. 37, no. 2 (2002) *To whom correspondence should be addressed at 442 Kleberg Biotechnol- ogy Center, 2471 TAMU, Texas A&M University, College Station, TX 77843-2471. E-mail: r-chapkin@tamu.edu Abbreviations: ARA, arachidonic acid (20:4n-6); ChoGpl, choline glycerophos- pholipids; CMH 2 -DCFDA, 5 (and 6)-chloromethyl-2′,7′-dichlorodihydro- fluorescein diacetate, acetylester; EtnGpl, ethanolamine glycerophospholipids; FAEE, fatty acid ethyl ester; LNA, linoleic acid (18:2n-6); Ptd 2 Gro, cardiolipin; ROS, reactive oxygen species; ∆Ψ mt , mitochondrial membrane potential. Dietary n-3 PUFA Alter Colonocyte Mitochondrial Membrane Composition and Function Robert S. Chapkin a–c, * Mee Young Hong a , Yang-Yi Fan a , Laurie A. Davidson a,c , Lisa M. Sanders a , Cara E. Henderson a , Rola Barhoumi b,c , Robert C. Burghardt b,c , Nancy D. Turner a,c , and Joanne R. Lupton a–c a Molecular and Cell Biology Section, Faculty of Nutrition, b Department of Veterinary Anatomy and Public Health, and c Center for Environmental and Rural Health, Texas A&M University, College Station, Texas