Only fibres promoting a stable butyrate producing colonic ecosystem decrease the rate of aberrant crypt foci in rats

Abstract

Background: Dietary fibres have been proposed as protective agents against colon cancer but results of both epidemiological and experimental studies are inconclusive.

Aims: Hypothesising that protection against colon cancer may be restricted to butyrate producing fibres, we investigated the factors needed for long term stable butyrate production and its relation to susceptibility to colon cancer.

Methods: A two part randomised blinded study in rats, mimicking a prospective study in humans, was performed using a low fibre control diet (CD) and three high fibre diets: starch free wheat bran (WB), type III resistant starch (RS), and short chain fructo-oligosaccharides (FOS). Using a randomised block design, 96 inbred rats were fed for two, 16, 30, or 44 days to determine the period of adaptation to the diets, fermentation profiles, and effects on the colon, including mucosal proliferation on day 44. Subsequently, 36 rats fed the same diets for 44 days were injected with azoxymethane and checked for aberrant crypt foci 30 days later.

Results: After fermentation had stabilised (44 days), only RS and FOS produced large amounts of butyrate, with a trophic effect in the large intestine. No difference in mucosal proliferation between the diets was noted at this time. In the subsequent experiment one month later, fewer aberrant crypt foci were present in rats fed high butyrate producing diets (RS, p=0.022; FOS, p=0.043).

Conclusion: A stable butyrate producing colonic ecosystem related to selected fibres appears to be less conducive to colon carcinogenesis.

Figure 1

Protocol for the study of experimental diet fermentation. Rats (n=96) were randomised before the start of the experiment into 24 blocks (six blocks for each feeding period), one block representing a group of four rats of the same litter and sex. Staggered inclusion of blocks allowed the processing of only one block per sacrifice day, within a period (about one hour) short enough to ensure that all contents could be considered as at the same stage of fermentation. As a possible "experimenter effect" related to the long study period could not be eliminated, inclusion of the 24 blocks was randomised so that all sacrifice days were determined before the experiment began. Blocks were included (median age 46 days) after sex determination and according to these criteria. The four rats of each block, housed in a single cage, were fed successively A03 breeding diet and A04 maintenance diet (UAR). Two weeks before day 0 (D0), rats were housed one per cage and fed powdered maintenance diet. At D0 (median age 72 days), each animal from a block received one of the four experimental diets (table of permutated randomised blocks) and underwent the feeding period allocated by randomisation. Animals were weighed weekly throughout the experiment, from week 1 (at D2) to the day of sacrifice: D16, D30, or D44. Previous studies showed that short chain fatty acid concentrations increased following consumption of the meal and then stabilised during the 8-12 hour postprandial period. Even fed at libitum, rats had the highest consumption of food at the beginning of the dark period. Rats were thus sacrificed 10 hours later, one block at a time, in the order of their codes.

Figure 2

Changes over time in the fermentation of the four diets (low fibre control diet (CD), starch free wheat bran enriched diet (WB), type III resistant starch enriched diet (RS), and short chain fructo-oligosaccharide enriched diet (FOS)) along the large intestine. Twenty one blocks (84 rats) instead of 24 were used for this part of the study as some samples were lost. On the y axis are the concentrations of short chain fatty acids (acetate, propionate, and butyrate), expressed in µmol/g wet content. Values from the caecum (C), and proximal (P) and distal (D) colon of each rat are linked together, each line thus representing the individual fermentation pattern along the large intestine of one rat. When there were missing values for proximal colon concentrations (low content), points were plotted to mark the concentrations in the caecum and distal colon, but not linked.

Figure 3

Changes over time in the relationships between the major short chain fatty acids (SCFAs) issued from fermentation of the four diets (low fibre control diet (CD), starch free wheat bran enriched diet (WB), type III resistant starch enriched diet (RS), and short chain fructo-oligosaccharide enriched diet (FOS)). The axes indicate individual mean large intestine concentrations (an average of caecum, and proximal and distal colon concentrations, expressed in µmol/g wet content) of acetate, propionate, and butyrate for each rat. These values are linked to form a triangle representing the mean fermentation pattern of the rat. The triangle area is proportional to global SCFA production, and the ratio (relative concentration) of each SCFA can be determined from the shape of the triangle, regardless of its size: the more acute the angle, the higher the ratio. Each dark gray box indicates the mean values of one SCFA concentration for a given time and diet. Colour coding for feeding periods is yellow for D2, blue for D16, dark blue for D30, and red for D44.

Figure 4

Residual starch concentration in the caecum of rats fed the resistant starch enriched diet (RS) for 2, 16, 30, or 44 days. Each point represents one rat.

Figure 5

Time and interindividual effects on the mean butyrate large intestine concentration (µmol/g wet content) in rats from the 21 blocks (four rats of the same litter, sex, and feeding period) in this part of the study. The four rats of one block are on the same vertical. To facilitate interpretation, lines have been drawn linking the butyrate concentrations of animals fed the same diet for a given period. On D44, mean butyrate concentrations were higher for the short chain fructo-oligosaccharide enriched diet (FOS) than for the type III resistant starch enriched diet (RS), but this was only due to higher caecal concentrations.