FIGURE 1 Effects of including different sources of starch in Western diets moderate in protein and fat on DNA SSB (comet assay tail moments) in colonocytes of rats. Values are mean 6 SEM, n = 9– 10. Values without a common letter differ, P , 0.05. C-LAMS, control diet containing low amylose maize starch; SSB, single-strand break; W-HAMS, Western diet containing high amylose maize starch; W- HAMSB, Western diet containing butyrylated high amylose maize starch; W-HAW, Western diet containing high amylose wheat flour; W-LAMS, Western diet containing low amylose maize starch; W- LAW, Western diet containing low amylose wheat flour. lowering of risk by dietary ﬁber. The present study adds new knowledge in showing that W-HAW, but not W-LAW, also signiﬁcantly lowered SSB compared to W-LAMS, although the W-HAW and W-LAW groups were not signiﬁcantly different from each other. Previous animal studies on diet and CRC have generally used genotoxic agents to induce damage (e.g., azoxy- methane). In keeping with the current data, they have shown that aberrant crypt foci or adenoma formation was increased by high dietary protein and that this was opposed by RS (13–16). The mechanisms behind increased SSB and other genetic damage probably involve increased exposure to cytotoxic agents gener- ated by bacteria. One possibility for proteins containing heme iron (such as red meat) is the formation of DNA adducts through bacterial generation of damaging reactive species (34). However, casein (fed in this study) induces damage but does not contain heme, suggesting involvement of other genotoxic protein pro- ducts such as ammonia and phenols (6,24). Higher dietary protein can raise large bowel digesta ammonia in rats (35) and increase CRC risk through enhancing colonocyte proliferation (36). Ammonia is absorbed from the colon in its unprotonated form (i.e., at more alkaline pH). Our earlier studies showed that cecal ammonia level was inversely related to pH, consistent with diminished absorption. In the present study, SSB correlated positively with colonic digesta ammonia concentration. The apparent anomaly between current and previous ﬁndings can be reconciled by the fact that the levels throughout the large bowel may be inﬂuenced by the type of dietary ﬁber (35). There was no signiﬁcant relationship between SSB and large bowel phenol levels, although levels of ammonia and phenols were signiﬁcantly lowered by the RS treatments. Recently, we showed dose-dependent changes in circulating concentrations of several CRC-related biomarkers in rats fed HAMS and red or white (chicken) meat (18). The present data, demonstrating colonic transcriptional changes in genes associ- ated with DNA damage and repair, indicate that these previous ﬁndings may reﬂect altered gene expression. The expression of genes involved in DNA damage recovery were downregulated in rats fed W-HAMSB. These were Mdm2, which encodes a key negative regulator of the p53 tumor suppressor protein that is overexpressed in neoplasia (37); Parg, which is stimulated by DNA strand breaks (38), markers of topological stress, e.g., Top1 (39); and Gmnn, which can kill cancer cells without harming normal ones (40). In contrast, expression of the base-excision repair pathway gene Ung and the cell cycle damage checkpoint gene Cepba were higher in rats fed HAMSB. The changes suggest that protective mechanisms were operating through different reg- ulatory pathways for W-HAW and W-HAMS and /or W-HAMSB with differential expression of Mdm2, Top1, Parg, Gmnn, and Ung. The HAW diet had the most signiﬁcant impact on the chromatin remodeling gene, Rere. These results suggest there are fewer interruptions to DNA integrity by the moderate fat/protein base with the inclusion of HAMS, HAMSB, or HAW to the diet. For W-HAMSB, the underlying protective mechanism probably involves the ﬁne maintenance of genomic homeostasis while pre- venting replication of damaged cells, whereas for W-HAW, chro- matin restructuring seems to be a key feature, suggesting that protection could occur at a different stage of the cell cycle process. This study was carried out over 3 mo and conﬁrms that dietary effects on large bowel SCFA and DNA damage were sustained in the longer term. As expected, SCFA levels were highest in rats fed RS and lowest in those fed LAMS, consistent with its high digestibility. Large bowel SCFA were intermediate in rats fed H-LAW, supporting the suggestion that RS is higher in whole grain foods compared with reﬁned ones (41). This may contribute to the protective effect of whole grains in lowering the risk of diet-related disease through the generation of SCFA (42). However, for maximal effect, a high amylose content may be necessary (43). Digesta total SCFA and butyrate were highest in rats fed W-HAMSB, consistent with acylated starches supplying SCFA both as the esteriﬁed acid and through fermentation of undigested starch. There were substantial differences between W-HAW and the other RS types. Changes in SCFA and pH were distributed more evenly through the large bowel in rats fed W- HAW. This may be of some value, because most chronic large bowel disease (including CRC) is localized in the distal colon and rectum, where SCFA supply is lowest (44). It has been sug- gested that a combination of RS plus nonstarch polysaccharide (NSP) is optimal in ensuring the supply of SCFA to these viscera (45). Our data support this, because HAW contained both ﬁber polysaccharides integrally, whereas the other diets (apart from W-LAW) required the addition of ﬁber. This may also explain a difference in SCFA between W-HAW and other RS treatments – the signiﬁcantly higher propionate levels. The fermentation of NSP mixtures gives different SCFA patterns relative to when they are fed separately (46) and HAW had an altered NSP content as well as more amylose. In contrast to earlier studies, where higher dietary protein induced a dose-dependent thinning of the mucus layer (7), there was no effect of diet on mucus barrier thickness. This may reﬂect the higher level of indigestible matter (as NSP) in this study, because indigestible styrofoam particles increase small intestinal mucin secretion in rats (47). The cereal NSP in the present study might have similar effects. Feeding of RS alters the composition of the large bowel microbiota (48–51) and HAMS can function as a prebiotic in animals (48). In humans, a diet high in RS and NSP raises fecal levels of a range of bacteria, including Ruminococcus bromii, thought to be important for starch degradation and SCFA production (49). In this study, phylogenetic array analysis followed by PCA showed that the gut microbiota proﬁles fell into 3 distinct groupings. One consisted of the C-LAMS and W- Colonic effects of resistant starches 837