Levels of ammonia and phenols within the cecal and colonic digesta differed signiﬁcantly between dietary treatments (Table 3). W-HAMS, W-HAMSB, and W-HAW had similar effects on both of the protein fermentation products, lowering concentrations or pools signiﬁcantly relative to one or more of the C-LAMS, W-LAMS, and W-LAW treatments. No signiﬁcant effects were found for pools of ammonia in the cecum or pools of phenols in the colon. The number of SSB in colonic DNA (Fig. 1) was signiﬁcantly greater (by 70%) in rats fed W-LAMS than in those fed C-LAMS. However, SSB numbers were signiﬁcantly lower than for the W-LAMS treatment when W-HAMS, W-HAMSB, or W-HAW, but not W-LAW, was consumed. The thickness of the colonic mucus layer did not signiﬁcantly differ between treatment groups (data not shown). Signiﬁcant inverse relationships were found between SSB and the concentrations of SCFA within the hepatic portal vein (acetate: R2 = 0.32, P = 0.04; propionate: R2 = 0.33, P = 0.04; butyrate: R2 = 0.28, P = 0.07; total SCFA: R2 = 0.36, P = 0.02) and cecal concentrations of butyrate (R2 = 0.40, P = 0.009) and total SCFA (R2 = 0.40, P = 0.009) but not acetate (R2 = 0.25) or propionate (R2 = 0.22). There were no signiﬁcant relationships between SSB and cecal SCFA pools or between SSB and colonic concentrations or pools of SCFA. When pools of SCFA in the cecum and colon were combined, an inverse relationship was found between total SCFA and SSB (R2 = 0.46, P = 0.002). A positive relationship was observed between SSB and the concentration of ammonia within the colon (R2 = 0.40, P = 0.014) but not the cecum (R2 = 0.17). There was no signiﬁcant relationship with pools of ammonia within the colon or cecum or with concentrations or pools of phe- nols within the cecum or colon. Different diets were associated with changes in expression of some candidate DNA damage and repair genes. Box plots depicting the impact of diet on the expression of 8 genes associated with DNA damage, repair, and cell growth displaying signiﬁcant alteration by one or more of the diets, as detected by microarray, are shown in Figure 2. The expression of Mdm2 was signiﬁcantly downregulated by W-HAMS and W-HAMSB com- pared to C-LAMS. Mdm2 was also downregulated in rats by W- HAMSB compared to W-LAW and W-HAW. When the effects of W-HAMSB were compared to the effects of W-LAMS, W-LAW, and W-HAW, there was signiﬁcant downregulation of Top1, Parg, and Msh3. Furthermore, Msh3 was also downregulated by W-HAMS compared with W-LAMS, W-LAW, and W-HAW. In contrast, W-HAMSB resulted in the signiﬁcant upregulation of Gmnn, Cebpa, and Ung. Expression of Gmnn was signiﬁcantly higher in rats consuming W-HAMSB than those receiving the C- LAMS, W-HAMS, and W-HAW diets. Cebpa expression was signiﬁcantly higher in the W-HAMSB group than in the W- HAMS group and Ung was signiﬁcantly upregulated in the W- HAMSB group compared with the W-LAMS and W-HAW groups. When the W-HAW treatment was compared to the C- LAMS and W-HAMSB diets, the greatest DNA damage and repair-related transcriptional response was the signiﬁcant down- regulation of Rere. The above microarray differential expression data were conﬁrmed by qRT-PCR (Supplemental Fig. 1), except for Msh3, where differences were reduced and only the elevated expression in the W-LAW group was clearly evident. Fecal microbial population proﬁles were determined using a custom phylogenetic microarray and PCA showed that the proﬁles fell into 3 deﬁned groups: C-LAMS and W-LAMS, W- HAMS and W-HAMSB, and W-LAW and W-HAW (Supplemen- tal Fig. 2). A heat map showing some of the major differences in abundance of selected species and genera in response to diet is presented in Supplemental Figure 3. Lactic acid producing bacteria of clostridial cluster XVI were in signiﬁcantly higher abundance in the W-HAW group compared to the WHAMS and W-HAMSB groups, and Eubacterium ventriosum and Roseburia cecicola, which belong to clostridial cluster XIVa, were signif- icantly higher in both W-HAMS and W-HAMSB groups. Bacteria of clostridial cluster IV, Spirochaeta sp., and the uncultured gram-negative Cytophaga-Flavobacteria-Bacteroi- des group were also signiﬁcantly higher in abundance in rats consuming the RS diets compared with the C-LAMS and W- LAMS diets (data not shown). Discussion The current data conﬁrm our earlier ﬁndings of more colonocyte SSB in rats fed diets high in protein and also that HAMS and HAMSB opposed this damage (6–9,24). These data are consis- tent with ﬁndings from population studies showing increased CRC risk with greater fat and protein consumption but a TABLE 3 Effects of including different sources of starch in a Western diet moderate in protein and fat on ammonia and phenol levels in cecal and colonic digesta of rats1 Variables Cecum ammonia Pool, mmol Concentration, mmol/kg tissue Cecum phenols Pool, mmol Concentration, mmol/kg tissue Colon ammonia Pool, mmol Concentration, mmol/kg tissue Colon phenols Pool, mmol Concentration, mmol/kg tissue C-LAMS W-LAMS W-HAMS W-HAMSB W-LAW W-HAW 24.0 6 3.7 15.6 6 2.7a 10.6 6 1.9a 8.5 6 1.3a 10.0 6 0.9a 7.8 6 0.6a 5.8 6 1.0 5.3 6 1.2a 15.2 6 2.4 13.0 6 1.7a 8.0 6 2.2ab 7.7 6 1.9a 12.1 6 2.2a 9.5 6 0.9a 5.1 6 0.9 4.4 6 0.9a 13.6 6 3.2 4.8 6 0.7b 4.9 6 0.8b 2.0 6 0.2b 3.9 6 1.2b 2.3 6 0.6b 3.1 6 1.2 2.4 6 1.2ab 16.4 6 1.6 6.7 6 0.7b 5.1 6 0.5b 2.0 6 0.2b 6.2 6 1.0b 3.3 6 0.6b 2.4 6 0.7 1.4 6 0.3b 19.3 6 2.9 13.7 6 1.5a 7.8 6 1.6ab 5.7 6 1.1a 9.8 6 0.9a 8.9 6 0.5a 4.3 6 0.7 4.0 6 0.8a 17.5 6 2.0 8.9 6 0.8b 4.2 6 0.7b 2.4 6 0.4b 4.9 6 1.0b 3.4 6 0.7b 3.5 6 1.6 2.5 6 1.1ab 1 Values are mean 6 SEM, n = 4–10. Means in a row with superscripts without a common letter differ, P , 0.05. C-LAMS, control diet containing low amylose maize starch; 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. 836 Conlon et al.