Eicosapentaenoic acid inhibits the growth of liver preneoplastic lesions and alters membrane phospholipid composition and peroxisomal beta-oxidation.

The purpose of this study was to determine whether individual administration of highly purified eicosapentaenoic acid (EPA), one of the main components of the n-3 polyunsaturated fatty acid family, would alter the growth of focal lesions during hepatocarcinogenesis. The protocol used to induce chemical carcinogenesis in liver was the Solt-Farber model (diethylnitrosamine as initiator and 2-acetylaminofluorene and carbon tetrachloride associated with partial hepatectomy as promoters). Proliferative lesions were quantified with the histochemical marker gamma-glutamyltranspeptidase at partial hepatectomy and at sacrifice. The number and size of the gamma-glutamyltranspeptidase-positive foci observed were significantly lower in rats supplemented with EPA. Fatty acid treatment increased EPA and docosapentaenoic acid content in membrane total phospholipids, in phosphatidylethanolamine, and in phosphatidylcholine. The content of arachidonic acid decreased significantly only in total phospholipids and in phosphatidylethanolamine. Fatty acid content of phosphatidylinositol was not modified. Moreover, we observed an increase in the activity of palmitoyl-CoA oxidase, the limiting enzyme of peroxisomal beta-oxidation, the preferential metabolic pathway of n-3 polyunsaturated fatty acid. Conversely, unmodified levels of alpha-tocopherol and unchanged production of lipid peroxidation products (malondialdehyde) were observed. These results suggest that the EPA inhibitory effect on preneoplastic foci development may be related to alteration of fatty acid composition in phospholipid classes and to enhancement of peroxisomal beta-oxidation and H2O2 production.

Cell proliferation, differentiation, and apoptosis are modified by n-3 polyunsaturated fatty acids in normal colonic mucosa.

Supplementation with low doses of eicosapentaenoic (EPA) or docosahexaenoic (DHA) acid was used here to investigate changes in epithelial proliferation, differentiation, and apoptosis in normal rat colonic mucosa. ACI/T rats received by oral administration low doses of purified EPA or DHA ethyl esters (1 g/kg body weight) and colonic mucosa was analyzed for cell proliferation, differentiation, and apoptosis. n-3 Polyunsaturated fatty acid incorporation into membrane phospholipids was investigated as reflections of fatty acid metabolism. Both EPA and DHA suppressed colonocyte proliferation and increased the numbers of differentiating and apoptotic cells without modification of the crypt morphology and the number of cells per crypt columns. A significant incorporation of the supplemented fatty acids into total phospholipids was observed. This enrichment was accompanied by a decreased content in arachidonic acid. The observation that EPA and DHA do not alter crypt morphology although they modify cell turnover in normal colonic mucosa suggests a possible use of these fatty acids as dietary chemopreventive agents.

Dietary supplementation with eicosapentaenoic and docosahexaenoic acid inhibits growth of Morris hepatocarcinoma 3924A in rats: effects on proliferation and apoptosis.

The effect of individual administration of low doses of highly purified eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (1 g/kg body weight) on the growth of Morris hepatocarcinoma 3924A transplanted in ACI/T rats was investigated. Both EPA and DHA inhibited growth of the hepatocarcinoma (50% reduction of tumor weight or volume at the 19th day after transplantation for both of the n-3 PUFA groups). EPA treatment reduced the percentage of proliferating tumor cells labeled with BUdR (10-fold), whereas DHA did not. Conversely, DHA supplementation induced a doubling of the number of cells undergoing apoptosis (labeled by TUNEL), whereas EPA treatment was much less effective. Analysis of changes in phospholipid fatty acids in tumor-cell membranes after both treatments with EPA and DHA showed a significant reduction in arachidonic-acid levels. EPA and docosapentaenoic acid (DPA), its elongation product, were increased in the phospholipids from EPA-treated animals. DHA and EPA, but not DPA, were increased in the DHA-treated group. It is concluded from the results of the present study that the anti-tumoral effect of EPA is related mainly to its inhibition of cell proliferation, whereas that of DHA corresponds with its induction of apoptosis. The alterations in fatty-acid composition induced by EPA or DHA appear to be factors underlying their differential actions on cell proliferation and apoptosis.

Low-dose eicosapentaenoic or docosahexaenoic acid administration modifies fatty acid composition and does not affect susceptibility to oxidative stress in rat erythrocytes and tissues.

In view of the promising future for use of n-3 polyunsaturated fatty acids (PUFA) in the prevention of cancer and cardiovascular diseases, it is necessary to ensure that their consumption does not result in detrimental oxidative effects. The aim of the present work was to test a hypothesis that low doses of eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) do not induce harmful modifications of oxidative cell metabolism, as modifications of membrane fatty acid composition occur. Wistar rats received by gavage oleic acid, EPA, or DHA (360 mg/kg body weight/day) for a period of 1 or 4 wk. Fatty acid composition and alpha-tocopherol content were determined for plasma, red blood cell (RBC) membranes, and liver, kidney, lung, and heart microsomal membranes. Susceptibility to oxidative stress induced by tert-butylhydroperoxide was measured in RBC. EPA treatment increased EPA and docosapentaenoic acid (DPA) content in plasma and in all the membranes studied. DHA treatment mainly increased DHA content. Both treatments decreased arachidonic acid content and n-6/n-3 PUFA ratio in the membranes, without modifying the Unsaturation Index. No changes in tissue alpha-tocopherol content and in RBC susceptibility to oxidative stress were induced by either EPA or DHA treatment. The data suggest that EPA and DHA treatments can substantially modify membrane fatty acids, without increasing susceptibility to oxidative stress, when administered at low doses. This opens the possibility for use of low doses of n-3 PUFA for chemoprevention without risk of detrimental secondary effects.

n-3 fatty acids induce oxidative modifications in human erythrocytes depending on dose and duration of dietary supplementation.

The present work was performed to study an optimal dose and duration of dietary n-3 polyunsaturated fatty acid (PUFA) supplementation that would not result in harmful modifications of oxidative cell metabolism. Forty healthy subjects were divided into four groups that received 2.5 g/d eicosapentaenoic acid (EPA) + docosahexaenoic acid (DHA), 5.1 g EPA + DHA/d, 7.7 g EPA + DHA/d, or placebo. Fatty acid composition, tocopherol status, and susceptibility to lipid peroxidation induced in vitro by 2,2'-azobis-(2-amidinopropane) (AAPH) were evaluated in human red blood cell (RBC) membranes on days 30 and 180. n-3 PUFA treatment increased EPA and DHA concentrations in RBC membranes in a time-dependent manner in all of the n-3 PUFA groups. These modifications occurred with concomitant dose- and time-dependent increases in the membrane unsaturation index. After 30 d of treatment with n-3 PUFAs, alpha-to-copherol significantly increased in RBC membranes of the intermediate- and high-dose groups. Because of the higher concentration of this antioxidant in these groups, the susceptibility of RBC membranes to peroxidation was decreased. However, after 180 d of treatment, alpha-tocopherol decreased to baseline values and AAPH-induced lipid peroxidation increased in a dose-dependent manner. These results show that high doses of dietary n-3 PUFAs, as well as long-time treatments, affect human RBC susceptibility to lipid peroxidation by changes in fatty acid composition and tocopherol content.

Dietary fish oil inhibits human erythrocyte Mg,NaK-ATPase.

The effects of long-term treatment with a high dose (7.7 g/day) of n-3 polyunsaturated fatty acids (PUFA) were studied for human red blood cells (RBCs). RBCs isolated from healthy subjects treated for 30 and 180 days with n-3 PUFA showed the following modifications: (1) a time dependent modification of membrane fatty acid composition with a concomitant increase in membrane lipid unsaturation; (2) an increase in lipid peroxidation, expressed as malondialdehyde release, induced in vitro by t-butyl hydroperoxide (t-BOOH); (3) a time-dependent decrease in susceptibility to hemolysis, expressed as K+ leakage, induced in vitro by t-BOOH; (4) a time-dependent decrease in total and ouabain-insensitive Mg,NaK-ATPase activity. These results suggest that long term dietary supplementation with high doses of n-3 PUFA significantly modifies RBC structure and function that might lead to harmful side effects.

n-3 PUFA and alpha-tocopherol control of tumor cell proliferation.

Subjects at high risk for colon cancer received different doses of fish oil on a 30-day randomized double-blind trial to evaluate the chemopreventive effect of n-3 fatty acids against colorectal cancer. Using rectal mucosal proliferation, assessed with 3H-thymidine autoradiography, fish oil induced in the treated groups but not in the placebo group a change in the proliferative pattern, which resulted similar to that observed in low risk population; in the same groups rectal mucosal n-3 fatty acid content increased, where arachidonic acid level decreased. Moreover, n-3 PUFA treatment induced modifications of Vitamin E status. The results suggest that n-3 PUFA could protect high-risk subjects from colon cancer by a mechanism involving a modulation of Vitamin E.