Effects of fish oil and butyrate on diet-mediated apoptosis at the promotion stage of colon carcinogenesis

Abstract

We have previously shown that dietary fish oil and the fiber pectin protect against colon cancer in rats by increasing apoptosis induced by reactive oxygen species (ROS) at the initiation stage of tumorigenesis. We hypothesized that fish oil would incorporate into the cardiolipin of colonic mitochondrial membranes, creating an environment in which butyrate, a fermentation product of pectin, would also increase ROS and lead to apoptosis, as evidenced by decreased mitochondrial membrane potential (MMP), enhanced caspase-3 activity and cytochrome c translocation from the mitochondria, thus protecting against colon cancer by removing DNA damaged cells at the promotion stage of carcinogenesis. Sixty rats were provided a diet containing 15% corn or fish oil for 11 wk and injected with azoxymethane (AOM) or saline at wk 3 and 4. At wk 11, colonocytes were exposed to +/- butyrate ex vivo for 30 or 60 min. ROS and MMP were measured using fluorescence microscopy, and cytochrome c concentration and caspase-3 activity were measured using ELISA assays. Cardiolipin fatty acid enrichment was measured via TLC and GC. Butyrate increased ROS (p<0.0001) regardless of diet or treatment group. In colonic crypts from fish oilconsuming rats, butyrate reduced MMP (p=0.05). However, butyrate had no effect on MMP if the rats were consuming corn oil. In colonocytes from rats consuming fish oil, butyrate decreased mitochondrial cytochrome c (11%; p=0.02) concomitant with an increase in caspase-3 activity (17%; p=0.04) in the distal colon. In fish oil-fed animals, the n-3 fatty acids DHA and EPA were incorporated into cardiolipin at the expense of n-6 fatty acids. Regression analysis revealed a positive relationship between DHA (R=0.49, p=0.03) and EPA (R=0.59, p=0.02) and cytosolic cytochrome c content. As the percentage of DHA and EPA in the cardiolipin increased, the level of cytochrome c in the cytosol increased. These relationships were not seen in rats consuming corn oil and suggest that these results, induced only by the combination of butyrate with fish oil, may lead to increased apoptosis at the promotion stage of colon carcinogenesis via a mitochondria-mediated mechanism.