bowel and so contributes to total dietary ﬁber intake. In the large bowel, RS is fermented by the microbiota, releasing SCFA. The major SCFA (acetate, propionate, and butyrate) are thought to generally contribute to optimal colonic function, but butyrate is thought to be particularly important (10–12). It is a preferred metabolic substrate for colonocytes and acts to maintain a normal phenotype in these cells through several complementary mechanisms. In our studies on RS, dietary protein, and DNA damage, the strongest protective relationships were with large bowel butyrate (7), consistent with its proposed role in promoting colonic integrity. This suggestion is supported by nutritional studies with HAMSB. Starches acylated to a high degree of substitution (such as HAMSB) pass into the large bowel where the esteriﬁed SCFA is released by bacterial action. HAMSB consumption produces a sustained rise in large bowel SCFA, speciﬁcally as the esteriﬁed acid, and is as effective as HAMS in opposing diet-induced DNA damage (9). Other laboratory studies include experiments in rodents treated with a carcinogen (azoxy- methane) where RS signiﬁcantly lowers precancerous lesions and tumor burden (13–16). Collectively, these experimental data provide mechanistic support for the dose-dependent reduction in CRC risk with greater ﬁber intake, which was found in the EPIC study (3) and also in an early case-control study that showed attenuation by ﬁber of excess risk with greater protein consump- tion (17). In addition to direct effects of butyrate, there are changes in circulating biomarkers that suggest adaptive changes in gene expression (18). RS may also selectively stimulate the growth of beneﬁcial bacterial that are resident in the colon, thereby contributing to a lower risk of diseases, including CRC (19). RS occurs in foods for a variety of reasons, including the degree of gelatinization of the starch, i.e., the less gelatinized a starch, the lower its ileal digestibility and the higher its RS content. One of the important determinants of gelatinization is the relative content of amylose and amylopectin. The former is a smaller, linear polymer that gelatinizes relatively slowly on heating with water compared with amylopectin. It is also quicker to retrograde on cooling. Both factors contribute to the presence of RS in processed foods made with HAMS (20). Recently, we described a short-term feeding trial in which rats were fed a high amylose wheat ﬂour (HAW) generated by RNA interference and showed that HAW raised large bowel SCFA (21), consistent with greater RS content. HAW is a genetically modiﬁed grain and we deemed it important to determine whether it was as effective as other RS sources in opposing diet-induced colonic DNA damage in rats as a prelude to feeding trials in humans. Changes in large bowel bacterial populations and selected colonocyte genes associated with cellular integrity were also measured. Materials and Methods Animals and diets. Sixty male Sprague-Dawley rats of ~200 g weight were obtained from the Animal Resource Centre, Murdoch University, Perth, Australia. They were housed in wire-bottomed cages in a room with controlled temperature (238C) and lighting (a 12-h-light/-dark cycle) and allowed free access to food and water. The rats were randomly assigned to 1 of 6 groups (n = 10/group) and fed the respective experimental diets for 11 wk. The dietary compositions (Supplemental Table 1) were based on the AIN-93 diet (22). The composition of dietary wheat components (Supplemental Table 2) was determined using standard analytical methods as previously described (21). In the maize starch diets, casein (~80% protein) was the main protein source, with the remainder coming from wheat bran (~20%) (Supplemental Table 2). The control diet contained 7% fat, 13% protein, a highly digestible starch [low amylose maize starch (LAMS); National Starch Food Innovation], and 22% wheat bran as the ﬁber source. The control treatment is designated C-LAMS. The other diets, all moderate in fat (19%) and protein (20%) (deemed a Western diet), differed primarily with respect to the sources and forms of polysaccharides. The polysaccharide sources were LAMS, HAMS (Hi-maize, National Starch Food Innovation), HAMSB (prepared by National Starch Food Innovation with a degree of substi- tution of 0.23), HAW, or a commercial low amylose wheat ﬂour (LAW) [CSIRO Plant Industry (21)]. These treatments with a Western diet background are designated W-LAMS, W-HAMS, W-HAMSB, W-LAW, and W-HAW, respectively. LAMS, HAMSB, and HAMS were obtained as puriﬁed ingredients (i.e., powder), whereas LAW and HAW were added as a ground meal. Diets were balanced for the high levels of ﬁber and protein by changing wheat bran, HAMS, or casein levels in the formulations where appropriate (Supplemental Table 2). The fat used in the diets was a blend of palm and canola oils prepared by Goodman Fielder Limited (Australia) that contained ~39% SFA, 46% MUFA, and 15% PUFA. Group food intakes and individual body weights were monitored daily throughout the study. At the completion of the dietary intervention period, rats were anesthetized with 4% halothane/oxygen to allow collection of gut tissues, digesta, and hepatic portal vein blood at the time animals were killed. The rats were not starved before anesthesia and may be regarded as being in a postabsorptive state as sampling started at 0830 h on each day. Ex- perimental procedures were approved by the Animal Ethics Committee of CSIRO Food and Nutritional Sciences and complied with the Australian Code of Practice for the Care and Use of Animals for Scientiﬁc Purposes. Measurement of DNA strand breaks. A 6-cm segment of colon was removed from rats at a point 3 cm from the distal end for isolation of colonocytes to enable measurement of DNA single-stranded breaks (SSB) using a single-cell gel electrophoresis (comet) assay (23). Comet tail moment (a product of tail length and the fraction of DNA in the tail) was calculated for 50 cells from each of 3 slides per rat using Comet Score v1.5 software (TriTek). Cells with morphology indicative of apoptosis or necrosis were excluded from analyses. Colonic mucus layer thickness measurement. Mucus layer thick- ness was measured as previously described (24). For each animal, 10 measurements were taken at different points along 3 tissue segments, giving 30 thickness measurements in total. Mean thickness was calculated using an image analysis program (24). SCFA, phenols, and ammonia. Frozen fecal and cecal digesta were thawed and then distilled and homogenized with heptanoic acid added as internal standard (to give 5 mmol/kg of feces). The contents were analyzed for SCFA in duplicate by GLC as previously described (25). Portal vein plasma SCFA concentrations were determined by diethyl ether extraction (26). Total SCFA levels were calculated from the sum of acetic, propionic, butyric, isobutyric, caproic, isovaleric, and valeric acid. Phenol levels were determined by a previously described method (27), and ammonia was measured using the indophenol blue procedure (28). Microbiota analysis. A custom phylogenetic microarray developed and validated for gut bacteria was used to analyze the microbiota (29). The cecal contents from the 6 dietary treatment groups (n = 10/group) were collected and pooled randomly to 2 groups for each treatment (n = 5/ group). Each of the 2 cecal pools from each treatment group were processed as follows and run on the microarray. RNA was extracted from the samples as previously described (30). Prokaryote 16S ribosomal RNA genes were ampliﬁed using the primer sets 27F (59-AGAGTTT- GATCMTGGCTCAG-39) and T7/1492R (59-TCTAATACGACT CAC- TATAGGGGGYTACCTTGTTACGACTT-39; the underlined region is modiﬁed to include a T7 promoter sequence) (31). The PCR amplicons were puriﬁed with the MinElute PCR puriﬁcation kit (Qiagen) and then added as a template for in vitro transcription-based synthesis of single- stranded RNA (cRNA) using the MEGAScript T7 In Vitro transcription kit (Ambion). After puriﬁcation with a MEGAclear kit (Ambion), 1 mg of the sample cRNA and 140 ng of standard cRNA were labeled at the same time using Label IT mArray Cy5 reagent (Mirus) for 1 h at 378C while protected from light and then 0.1 volumes of the 103 stop reagent were added (Mirus) to terminate the labeling reaction. The labeled Colonic effects of resistant starches 833