Effects of eicosapentaenoic and docosahexaenoic acids dietary supplementation on cell proliferation and apoptosis in rat colonic mucosa

n-3 Polyunsaturated fatty acids (PUFA) have been shown to inhibit chemical-induced carcinogenesis of colon in rats (Reddy, B.S., and Maruyama, H., Cancer Res. 46:3367-3370, 1986) and to normalize altered proliferative patterns of the colonic mucosa in human subjects at high risk for colon cancer (Anti, M., Gastroenterology 103, 883-891, 1992 and Gastroenterology 107, 1709-1715, 1994). To determine whether n-3 PUFA also influence the normal cellular turnover, we investigated the effects of dietary supplementation with eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), the two major n-3 PUFA, on cell proliferation, apoptosis, and crypt morphology in normal colonic mucosa. Moreover we analyzed some metabolic pathways such as fatty acid incorporation into membrane phospholipids and peroxisomal I]-oxidation involved in the effects of n-3 PUFA. Sixty male inbred ACI/T rats were randomly divided into three groups of 20 each, receiving oleic acid (control), EPA, or DHA (1 g/kg body wt/d) as ethyl esters by gastric gavage for 10 d. Cell proliferation and apoptosis were examined by bromodeoxyuridine and by in situ nick end labeling staining, respectively; fatty acid composition of phospholipid (PL) classes by thin-layer chromatography and gas-liquid chromatography and the activity of palmitoyl-CoA oxidase, the limiting enzyme of peroxisomal t-oxidation, were measured fluorimetrically by a coupled peroxidase reaction in mucosal homogenate. Both EPA and DHA inhibited cell proliferation and induced apoptosis in normal colonic mucosa of rats, but they did not modify crypt morphology and the total number of cells per crypt. EPA and DHA treatment decreased arachidonic acid content in phosphatidylethanolamine (PE) and increased EPA in all PL classes. After DHA treatment, DHA itself increased in phosphatidylcholine (PC) and PE but not in phosphatidylinositot (PI). As a consequence of the modifications in fatty acid composition, n-6/n-3 PUFA ratio decreased in total PL, in PC and PE fractions of both EPA and DHA groups. On the contrary, n-6/n-3 PUFA ratio did not change in the PI fraction. Unsaturation index was not modified in total PL as well as in PL classes. A significant increase in peroxisomal ~-oxidation (140% in EPA and 170% in DHA rats) was observed after both EPA and DHA supplementation. The increase in this metabolic pathway resulted in an overproduction of hydrogen peroxide. The changes in cell proliferation and apoptosis were related to changes in the composition of membrane PL and in fatty acid peroxisomal metabolism. The observation that arachidonic acid level is not affected in PC and PI suggests that eicosanoids are not involved in n-3 PUFA effects. Moreover, the induction of peroxisomal ~-oxidation with a consequent increase in hydrogen peroxide production suggests that a mild increase in reactive oxygen species could be implicated in the inhibition of cell proliferation and in the induction of apoptosis by n-3 PUFA. Our finding that EPA and DHA did not modify crypt morphology and the total number of cells per crypt, although they altered cell proliferation and apoptosis in normal colonic mucosa, suggests a possible use of these fatty acids as dietary chemopreventive agents.