Modulation of colonocyte proliferation and apoptosis by dietary fish oil in experimental colorectal carcinogenesis.

Abstract

Accumulating epidemiological and experimental evidence demonstrates an inverse relationship between consumption of marine fish oils rich in eicosapentaenoic acid (205, n-3, EPA) and development of colorectal cancer. Clinical studies on patients at high risk of developing colorectal cancer have shown that low level fish oil dietary supplementation can normalise aberrant proliferative patterns associated with the early stages of neoplasia[ 11. In chemically induced rodent models of colorectal neoplasia, diets rich in n-3 fatty acids such as EPA significantly reduce the incidence and multiplicity of colorectal adenocarcinomas relative to diets rich in n-6 fatty acids[2]. Further studies on a number of human tumour cell lines have clearly demonstrated a selective low dose tumoricidal capacity for EPA, proving cytotoxic or at least cytostatic to tumour cells but not to normal cells[3]. A number of mechanisms have been proposed to account for these anticarcinogenic effects including augmentation of free-radical generation leading to loss of cell viability in sensitive cell populations[4], inhibition of cyclooxygenase and lipoxygenase eicosanoid synthesising enzyme systems[ll, modulation of cell signalling pathway@] and gene expression[6]. Ultimately fish oil exerts its effects through control of cellular proliferation[7,8] and recent evidence suggests EPA may induce apoptosis both in vivo[9] and in vitro[lO]. The objectives of this study are firstly to determine the effects of short-term fish oil dietary supplementation on colonocyte proliferation and spontaneous apoptosis in the distal colon of normal healthy animals. Secondly, to provide insight into the influence of fish oil on crypt cell kinetics immediately following carcinogenic insult with 1,2 dimethylhydrazine (DMH), since modifications of the initiating stages of chemical carcinogenesis are likely to effect neoplastic outcome within the colon. Forty male Wistar rats fed a semi-synthetic diet containing starch (280g/kg), sucrose (380g/kg), casein (200g/kg), corn oil (CO) (80g/kg) and a balanced mineral and vitamin mix were divided randomly into four groups (Con-CO, Con-FO, DMHCO, and DMH-FO) prior to the start of the experiment. Accordingly, half the animals were injected subcutaneously with DMH (30mg/kg body weight), the remaining controls with an equal volume of saline. Immediately, the Con-FO and DMH-FO assigned groups were fed a freshly prepared diet where corn oil was substituted by fish oil ( EPA 18.7%:DHA 8.0%). The ConCO and DMH-CO groups remained on the standard corn oil diet. Five animals from each group were killed at 24 hours and at 48 hours and their colons removed and flushed with saline. Tissue samples were collected from the distal end of the colon, fixed in ethano1:acetic acid (75:25), and stained with Feulgen's reagent. Crypts were then micro-dissected from the tissue, lightly compressed under a cover-slip and morphologically identifiable mitosis and apoptosis counted in twenty crypts per animal (100 cryptdgroup) under a light microscope. Data was collated and significance determined by two-way analysis of variance. As expected, injection of DMH significantly reduced proliferation and increased apoptosis in the distal colon both 24 hours and 48 hours following administration. The effects of fish oil dietary supplementation were most marked after 48 hours (see Table 1). Proliferation was lower in the distal colon in FO compared to CO fed rats both in the presence and absence of DMH, though the effect was not statistically significant at 24 hours. Apoptosis was significantly raised by fish oil at both 24 and 48 hours in the presence and absence of DMH. Table 1. hffect of diet and DMH on proliferation and apoptosis in the distal colon after 48 hours. Each value represents the mean f S.E.M for 100 crypts. Mitosis Apoptosis DMH Corn oil Fish oil :::: :::; 2: O.O f b