Colonocyte DNA Research Articles

Interactive Effects of Indigestible Carbohydrates, Protein Type, and Protein Level on Biomarkers of Large Intestine Health in Rats.

The effects of indigestible carbohydrates, protein type, and protein level on large intestine health were examined in rats. For 21 days, 12 groups of six 12-week-old male Wistar rats were fed diets with casein (CAS), or potato protein concentrate (PPC), providing 14% (lower protein level; LP), or 20% (higher protein level; HP) protein, and containing cellulose, resistant potato starch, or pectin. Fermentation end-products, pH, and β-glucuronidase levels in cecal digesta, and ammonia levels in colonic digesta were determined. Cecal digesta, tissue weights, cecal and colon morphology, and colonocyte DNA damage were also analyzed. Digesta pH was lower, whereas relative mass of cecal tissue and digesta were higher in rats fed pectin diets than in those fed cellulose. Cecal parameters were greater in rats fed PPC and HP diets than in those fed CAS and LP diets, respectively. Short-chain fatty acid (SCFA) concentrations were unaffected by protein or carbohydrate type. Total SCFA, acetic acid, and propionic acid concentrations were greater in rats fed LP diets than in those fed HP. Cecal pool of isobutyric and isovaleric acids was greater in rats fed PPC than in those fed CAS diets. PPC diets decreased phenol concentration and increased ammonia concentration in cecal and colonic digesta, respectively. Cecal crypt depth was greater in rats fed PPC and HP diets, and was unaffected by carbohydrates; whereas colonic crypt depth was greater in rats fed cellulose. Myenteron thickness in the cecum was unaffected by nutrition, but was greater in the colon of rats fed cellulose. Colonocyte DNA damage was greater in rats fed LP diets than in those fed HP diets, and was unaffected by carbohydrate or protein type. It was found that nutritional factors decreasing cecal digesta weight contribute to greater phenol production, increased DNA damage, and reduced ammonia concentration in the colon.

Effects of Konjac glucomannan, inulin and cellulose on colonocyte DNA Integrity and anti‐genotoxic responses against azoxymethane in mice fed a high‐fat diet

The aim of study was to compare effects of konjac glucomannan (KGM), inulin, and cellulose on the DNA integrity and gene expressions of enzymes in colonocytes of C57BL/6J mice before and within 24 h of a single dose of azoxymethane (AOM). Six‐week‐old mice were fed a control diet with 20% (w/w) corn oil and 1% (w/w) cellulose or that supplemented with KGM, inulin or cellulose (5%, w/w) for 3 weeks. Fresh feces were collected 3 days prior the AOM (10 mg/kg BW, i. p.) injection on day 22. Each mouse was killed before (0 h), or 6, 24 h after the injection. The colonocytes were immediately isolated for further analyses of DNA damage (denoted by tail moment) and gene expressions. The DNA damage of colonocytes was the lowest in the inulin group at 0 h. The increased AOM‐induced DNA damage was ameliorated by KGM and inulin. The initial (0 h) gene expressions of glutathione peroxidase 2 (GPX2) in the colonocytes were lower in the control and cellulose groups, which may lead to AOM‐induced up‐regulation (to ≥ 2 folds of initial level) within 24 h. Addition of KGM or inulin to control diet beneficially promoted the concentration and daily fecal excretion of bifidobacteria and lactobacilli. The present study suggests that KGM and inulin protect the colonocytes from DNA damage induced by AOM, which may be related to their prebiotic effects. This study was supported by the National Science Council Grant NSC‐101–2320‐B‐040–018 ‐MY2, Taiwan.

Effects of Potato Fiber and Potato‐Resistant Starch on Biomarkers of Colonic Health in Rats Fed Diets Containing Red Meat

The effects of red meat consumption with and without fermentable carbohydrates on indices of large bowel health in rats were examined. Sprague-Dawley rats were fed cellulose, potato fiber, or potato-resistant starch diets containing 12% casein for 2 wk, then similar diets containing 25% cooked beef for 6 wk. After week 8, cecal and colonic microbiota composition, fermentation end-products, colon structure, and colonocyte DNA damage were analyzed. Rats fed potato fiber had lower Bacteroides-Prevotella-Porphyromonas group compared to other diet groups. Colonic Bifidobacterium spp. and/or Lactobacillus spp. were higher in potato fiber and potato-resistant starch diets than in the cellulose diet. Beneficial changes were observed in short-chain fatty acid concentrations (acetic, butyric, and propionic acids) in rats fed potato fiber compared with rats fed cellulose. Phenol and p-cresol concentrations were lower in the cecum and colon of rats fed potato fiber. An increase in goblet cells per crypt and longer crypts were found in the colon of rats fed potato fiber and potato-resistant starch diets. Fermentable carbohydrates had no effect on colonic DNA damage. Dietary combinations of red meat with potato fiber or potato-resistant starch have distinctive effects in the large bowel. Future studies are essential to examine the efficacy of different types of nondigestible carbohydrates in maintaining colonic health during long-term consumption of high-protein diets. Improved understanding of interactions between the food consumed and gut microbiota provides knowledge needed to make healthier food choices for large bowel health. The impact of red meat on large bowel health may be ameliorated by consuming with fermentable dietary fiber, a colonic energy source that produces less harmful by-products than the microbial breakdown of colonic protein for energy. Developing functional red meat products with fermentable dietary fiber could be one way to promote a healthy and balanced macronutrient diet.

Resistant Starches Protect against Colonic DNA Damage and Alter Microbiota and Gene Expression in Rats Fed a Western Diet1–3

Resistant starch (RS), fed as high amylose maize starch (HAMS) or butyrylated HAMS (HAMSB), opposes dietary protein-induced colonocyte DNA damage in rats. In this study, rats were fed Western-type diets moderate in fat (19%) and protein (20%) containing digestible starches [low amylose maize starch (LAMS) or low amylose whole wheat (LAW)] or RS [HAMS, HAMSB, or a whole high amylose wheat (HAW) generated by RNA interference] for 11 wk (n = 10/group). A control diet included 7% fat, 13% protein, and LAMS. Colonocyte DNA single-strand breaks (SSB) were significantly higher (by 70%) in rats fed the Western diet containing LAMS relative to controls. Dietary HAW, HAMS, and HAMSB opposed this effect while raising digesta levels of SCFA and lowering ammonia and phenol levels. SSB correlated inversely with total large bowel SCFA, including colonic butyrate concentration (R2 = 0.40; P = 0.009), and positively with colonic ammonia concentration (R2 = 0.40; P = 0.014). Analysis of gut microbiota populations using a phylogenetic microarray revealed profiles that fell into 3 distinct groups: control and LAMS; HAMS and HAMSB; and LAW and HAW. The expression of colonic genes associated with the maintenance of genomic integrity (notably Mdm2, Top1, Msh3, Ung, Rere, Cebpa, Gmnn, and Parg) was altered and varied with RS source. HAW is as effective as HAMS and HAMSB in opposing diet-induced colonic DNA damage in rats, but their effects on the large bowel microbiota and colonocyte gene expression differ, possibly due to the presence of other fiber components in HAW.

Exfoliated colonocyte DNA levels and clinical features in the diagnosis of colorectal cancer: a cohort study in patients referred for investigation

Selection of patients for investigation of suspected colorectal cancer is difficult. One possible improvement may be to measure DNA isolated from exfoliated cells collected from the rectum. This was a cohort study in a surgical clinic. Participants were aged ≥40 years and referred for investigation of suspected colorectal cancer. Exclusion criteria were inflammatory bowel disease, previous gastrointestinal malignancy, or recent investigation. A sample of the mucocellular layer of the rectum was taken with an adapted proctoscope (the Colonix system). Haemoglobin, mean cell volume, ferritin, carcino-embryonic antigen and faecal occult bloods were tested. Analysis was by logistic regression. Participation was offered to 828 patients, of whom 717 completed the investigations. Three were lost to follow up. Seventy-two (10%) had colorectal cancer. Exfoliated cell DNA was higher (P<0.001) in cancer (median 5.4 μg/ml [inter-quartile range 1.8,12]) compared with those without cancer (2.0 μg/ml [IQR 0.78,5.5]). Seven variables were independently associated with cancer, including age (odds ratio [OR], 1.05; 95% confidence interval [CI], 1.02,1.08; P<0.001) DNA (OR, 1.05; CI, 1.01,3.6; P=0.01), mean cell volume (OR, 0.93; CI, 0.89,0.97; P=0.001), carcino-embryonic antigen 1.02 per μg/l (CI, 1.00,1.04; P=0.02), male sex (OR, 2.0; CI, 1.1,3.6; P=0.02), rectal bleeding (OR, 2.4; CI, 1.3,4.5; P=0.007) and positive faecal occult blood (OR, 6.7; CI, 3.4, 13; P<0.001). The area under the receiver-operating characteristic curve for the DNA score was 0.65 (0.58-0.72) and for the seven variable model 0.88 (CI, 0.84-0.92). Quantification of exfoliated DNA from rectal cellular material has promise in the diagnosis of colorectal cancer, but this requires confirmation in a larger study.

An arabinoxylan-rich fraction from wheat enhances caecal fermentation and protects colonocyte DNA against diet-induced damage in pigs

Population studies show that greater red and processed meat consumption increases colorectal cancer risk, whereas dietary fibre is protective. In rats, resistant starches (a dietary fibre component) oppose colonocyte DNA strand breaks induced by high red meat diets, consistent with epidemiological data. Protection appears to be through SCFA, particularly butyrate, produced by large bowel carbohydrate fermentation. Arabinoxylans are important wheat fibre components and stimulate large bowel carbohydrate SCFA production. The present study aimed to determine whether an arabinoxylan-rich fraction (AXRF) from wheat protected colonocytes from DNA damage and changed colonic microbial composition in pigs fed with a diet high (30 %) in cooked red meat for 4 weeks. AXRF was primarily fermented in the caecum, as indicated by higher tissue and digesta weights and higher caecal (but not colonic) acetate, propionate and total SCFA concentrations. Protein fermentation product concentrations (caecal p-cresol and mid- and distal colonic phenol) were lower in pigs fed with AXRF. Colonocyte DNA damage was lower in pigs fed with AXRF. The microbial profiles of mid-colonic mucosa and adjacent digesta showed that bacteria affiliating with Prevotella spp. and Clostridial cluster IV were more abundant in both the mucosa and digesta fractions of pigs fed with AXRF. These data suggest that, although AXRF was primarily fermented in the caecum, DNA damage was reduced in the large bowel, occurring in conjunction with lower phenol concentrations and altered microbial populations. Further studies to determine the relationships between these changes and the lowering of colonocyte DNA damage are warranted.

Screening for colorectal cancer: established and emerging modalities

It has been estimated that >95% of cases of colorectal cancer (CRC) would benefit from curative surgery if diagnosis was made at an early or premalignant polyp stage of disease. Over the past 10 years, most developed nation states have implemented mass population screening programs, which are typically targeted at the older (at-risk) age group (>50-60 years old). Conventional screening largely relies on periodic patient-centric investigation, particularly involving colonoscopy and flexible sigmoidoscopy, or else on the fecal occult blood test. These methods are compromised by either low cost-effectiveness or limited diagnostic accuracy. Advances in the development of diagnostic molecular markers for CRC have yielded an expanding list of potential new screening modalities based on investigations of patient stool (for colonocyte DNA mutations, epigenetic changes or microRNA expression) or blood specimens (for plasma DNA mutations, epigenetic changes, heteroplasmic mitochondrial DNA mutations, leukocyte transcriptome profile, plasma microRNA expression or protein and autoantibody expression). In this Review, we present a critical evaluation of the performance data and relative merits of these various new potential methods. None of these molecular diagnostic methods have yet been evaluated beyond the proof-of-principle and pilot-scale study stage and it could be some years before they replace existing methods for population screening in CRC.

Colonocyte telomere shortening is greater with dietary red meat than white meat and is attenuated by resistant starch

Population studies indicate that greater red meat consumption increases colorectal cancer risk while dietary fibre is protective. Previous work in rats showed that diets high in protein, including red meat, increase colonocyte DNA strand breaks and that this effect is attenuated by resistant starches (RS). Telomeres are long hexamer repeats that protect against spontaneous DNA damage which would lead to chromosomal instability. Telomere shortening is associated with greater risk of colorectal cancer. The current study aimed to determine the effects of cooked red and white meat intake on colonocyte telomere length in rats and whether dietary RS modified their effects. After four weeks of feeding cooked beef or chicken at 15, 25 and 35% of diet with or without RS, colonocyte telomere length was measured. Telomere length decreased in proportion to red meat content of the diet. A similar trend was observed in the white meat group. Colonocyte telomere shortening due to increased dietary meat was attenuated by the inclusion of RS. These data support previous findings of increased colonocyte DNA damage with greater red and white meat intake and also the protective effect of dietary fibre.

OC-078 The diagnostic accuracy of colonocyte dna quantification in colorectal cancer diagnosis using a novel device

<h3>Introduction</h3> Survival from colorectal carcinoma (CRC) has not recently improved in the UK despite various initiatives including rapid investigation clinics and the national screening programme. It can be a difficult disease to diagnose. A new diagnostic device (Colonix®) that obtains an imprint of the rectal mucosa has been developed. Early laboratory and pilot studies suggest that the amount of colonocyte DNA correlates well with colorectal pathology. The aim of this study was to assess the diagnostic accuracy of the Colonix® device in identifying CRC in patients with moderate to high risk of colorectal cancer. <h3>Methods</h3> Patients were referred by their primary care physicians for further investigation according to the NICE guidelines. Participants completed a symptom questionnaire, had routine blood tests, faecal occult blood test (FOBT) and had a Colonix® test in clinic. The main outcome of the study was the presence of CRC. This was determined histologically by conventional investigation. The quantity of DNA in the sample was measured by investigators blinded to the outcome. <h3>Results</h3> 828 patients entered the study: 717 received a Colonix® test. Three were lost to follow-up, leaving 714 patients for analysis of whom 72 (10.1%) had a CRC. The mean (SD) age of those with cancer was 74 years (9.7), and those without 70 (12.4) years. 42 (58%) of the cancers were in males whereas 277 (43%) of those without cancer were male. The mean (SD) DNA score in cancer cases was 7.5 (7.3) ng/ml, and in those without cancer 4.4 (5.6) ng/ml, a significant difference (p&lt;0.001). In univariable logistic regression the odds-ratio of each ng/ml of the DNA score was 1.07 (95% CI 1.04 to 1.11). Using the DNA score alone, the area under the ROC curve was 0.65. The area under the ROC curve improved to 0.87 in multivariable analysis using DNA score, age, mean cell volume (MCV), carcino-embryonic antigen (CEA), positive faecal occult blood test and rectal bleeding. <h3>Conclusion</h3> Colonocyte DNA scores obtained using a Colonix® test, have considerable ability to identify colorectal cancer in the referred population when combined with the results of other simple investigations.

Butyrylated starch protects colonocyte DNA against dietary protein-induced damage in rats

Dietary resistant starch (RS), as a high amylose maize starch (HAMS), prevents dietary protein-induced colonocyte genetic damage in rats, possibly through the short-chain fatty acid (SCFA) butyrate produced by large bowel bacterial RS fermentation. Increasing butyrate availability may improve colonic health and dietary high amylose maize butyrylated starch (HAMSB) is an effective method of achieving this goal. In this study, rats (n = 8 per group) were fed diets containing high levels (25%) of dietary protein as casein with 10 or 20% dietary HAMSB and HAMS. Colonocyte genetic damage was measured by the comet assay and was 2-fold higher in rats fed 25% protein than those fed 15% protein (P < 0.001). Concurrent feeding of 25% protein and either HAMS or HAMSB lowered genetic damage significantly relative to a low-RS high-protein control diet. The 20% HAMSB diet was twice as effective as 20% HAMS in opposing genetic damage. Large bowel digesta butyrate was significantly increased in rats fed 20% compared with 10% HAMS and in rats fed 20% compared with 10% HAMSB. The levels were significantly higher in the HAMSB groups relative to the HAMS groups. Hepatic portal venous SCFA were higher in rats fed HAMS and highest in those fed HAMSB. Caecal digesta ammonia was increased by HAMSB and correlated negatively with digesta pH. Ammonia is cytotoxic and lower digesta pH could lower its absorption, possibly contributing to lower genetic damage. Delivery of butyrate to the large bowel by HAMSB could reduce colorectal cancer risk by preventing diet-induced colonocyte genetic damage.

Differential effects of dietary whey, casein and soya on colonic DNA damage and large bowel SCFA in rats fed diets low and high in resistant starch

Feeding higher levels of dietary animal protein (as casein or red meat) increases colonic DNA damage and thins the colonic mucus barrier in rats. Feeding resistant starch (RS) reverses these changes and increases large bowel SCFA. The present study examined whether high dietary dairy (casein or whey) or plant (soya) proteins had similar adverse effects and whether dietary RS was protective. Adult male rats were fed diets containing 15 or 25 % casein, whey or soya protein with or without 48 % high amylose starch (as a source of RS) for 4 weeks. DNA damage was measured in isolated colonocytes using the comet assay. Higher dietary casein and soya (but not whey) increased colonocyte DNA damage. DNA damage was highest with soya when fed at 15 or 25 % protein without RS. Dietary RS attenuated protein-induced colonocyte DNA damage in all groups but it remained significantly higher in rats fed 25 % soya compared with those fed 15 % protein. Dietary protein level did not affect colonic mucus thickness overall but the barrier was thinner in rats fed high dietary casein. This effect was reversed by feeding RS. Caecal total SCFA and butyrate pools were higher in rats fed RS compared with digestible starch. Caecal and faecal SCFA were unrelated to genetic damage but correlated with mucus thickness. The present data confirm that higher dietary protein affected colonocyte DNA and colonic mucus thickness adversely but that proteins differ in their effects on these indices of colon health. The data show also that these changes were reversed by RS.

Dose-dependent reduction of dietary protein-induced colonocyte DNA damage by resistant starch in rats correlates more highly with caecal butyrate than with other short chain fatty acids

Previous studies have shown increased levels of colonocyte DNA damage (as measured by the comet assay) and thinning of the colonic mucus layer in rats fed higher dietary protein as casein or red meat with highly digestible starch. Feeding resistant starch (RS) as high amylose maize starch (HAMS) opposed these changes. However, the dietary level of HAMS was relatively high (48% by weight) so this study was conducted to establish whether HAMS had the same effects at lower dietary levels. Adult male rats were fed a diet containing 25% casein with 0%, 10%, 20%, 30% or 40% HAMS for 4 wk. DNA single strand breaks and 8-hydroxyguanosine levels were measured in isolated colonocytes by the comet assay. As expected, comet tail moment was greatest and the mucus barrier thinnest in rats fed 0% HAMS. DNA damage was reduced and the mucus barrier thickened in a logarithmic dose-dependent manner by HAMS. There was no significant difference in 8-hydroxyguanosine between dietary groups. Caecal and faecal short chain fatty acid (SCFA) pools rose with the increased level of dietary HAMS. DNA damage of colonocytes correlated negatively with caecal SCFA but the strongest correlation was with caecal butyrate, which is consistent with the proposed role of this SCFA in promoting a normal cell phenotype. These data show that HAMS prevents protein-induced colonic DNA damage in a dose-dependent manner. Inclusion of 10% HAMS was found to be sufficient to oppose colonocyte DNA damage, and to increase caecal and faecal SCFA pools.

Resistant Starch Attenuates Colonic DNA Damage Induced by Higher Dietary Protein in Rats

: Epidemiologic studies suggest that dietary complex carbohydrates are protective against colorectal cancer but dietary protein may increase risk. However, experimental data to support these relationships are scant. We have shown in rats that consumption of a high-protein (25% casein) diet for 4 wk resulted in a twofold increase in damage to colonocyte DNA compared with a low-protein (15% casein) diet. This was associated with thinning of the colonic mucous barrier and increased levels of fecal p-cresol. Addition of resistant starch as a high-amylose maize starch to the diet increased cecal short-chain fatty acid pools and attenuated DNA damage, suggesting protection against genotoxic agents. In humans, this could translate to altered risk of colonic cancer.

Microsatellite analysis of fecal DNA for colorectal cancer detection.

The advent of noninvasive methods of testing for colorectal cancer that have a high level of specificity and sensitivity is eagerly awaited. Thirty patients with sporadic colorectal cancer and 11 patients with hereditary nonpolyposis colon cancer (HNPCC) enrolled in this study. We analyzed the loss of heterozygosity (LOH) in matched genomic DNA extracted from blood and surgical specimens (tumor and tumor-free colonic mucosa), and the corresponding oral rinse and stool specimens using seven microsatellite loci (APC, p53, DCC, hMLH1, D9S162, D9S171, and IFNA). To reduce the normal colonocyte DNA contamination of the stool samples, we compared three different methods for fecal genomic DNA extraction. As normal controls, we analyzed the LOH using the oral rinse and stool samples from 15 individuals without cancer. The LOH determined from the oral rinse and the stool samples matched those determined from the blood and the neoplastic tissue. All patients with HNPCC had microsatellite alterations at hMLH-1 in tumor DNA and corresponding fecal DNA. There were significant differences in the frequency of p53-LOH and D9S171-LOH between the group with sporadic disease and those with HNSCC (P = 0.0031 and 0.0294, respectively). Two cases with D9S162-LOH were detected in individuals without cancer. For the patients with sporadic disease, using p53 and adenomatous polyposis coli (APC), the sensitivity of the fecal DNA analysis was 96.7% (95% CI, 83-100) with a specificity of 100%. We demonstrate that LOH analysis using oral rinse and stool samples may be a suitable screening tool for colorectal cancer.

Short chain fatty acids differentially modulate cellular phenotype and c-myc protein levels in primary human nonmalignant and malignant colonocytes.

Short chain fatty acids may protect colonic mucosa against neoplastic transformation by modulating colonocyte phenotype, DNA synthesis, and c-myc levels. To test this hypothesis, nonmalignant and malignant human colonocytes were isolated from surgical specimens and treated with 10 mM acetate, propionate, or butyrate. Markers of cellular phenotype, DNA synthesis, and c-myc protein levels were assayed by alkaline phosphatase and dipeptidyl dipeptidase IV activities, [3H]thymidine labeling, and western blotting, respectively. Butyrate, in particular, exerted discordant effects on alkaline phosphatase (P < 0.05), and c-myc levels (P < 0.05, N > or = 6) in nonmalignant and malignant human colonocytes. DPDD was unaffected by any of the short chain fatty acids tested. [3H]Thymidine labeling was differentially stimulated by short chain fatty acids in both cell types and greater DNA synthesis rates were observed in malignant colonocytes (P < 0.005, N = 16). These data suggest that in vitro, butyrate, in particular, may differentially modulate phenotype, DNA synthesis, and c-myc in nonmalignant and malignant human colonocytes.

Folate Deficiency In Vitro Induces Uracil Misincorporation and DNA Hypomethylation and Inhibits DNA Excision Repair in Immortalized Normal Human Colon Epithelial Cells

Epidemiological studies have indicated that folic acid protects against a variety of cancers, particularly cancer of the colorectum. Folate is essential for efficient DNA synthesis and repair. Moreover, folate can affect cellular S-adenosylmethionine levels, which regulate DNA methylation and control gene expression. We have investigated the mechanisms through which folate affects DNA stability in immortalized normal human colonocytes (HCEC). DNA strand breakage, uracil misincorporation, and DNA repair, in response to oxidative and alkylation damage, were determined in folate-sufficient and folate-deficient colonocytes by single cell gel electrophoresis. In addition, methyl incorporation into genomic DNA was measured using the bacterial enzyme Sss1 methylase. Cultured human colonocyte DNA contained endogenous strand breaks and uracil. Folate deficiency significantly increased strand breakage and uracil misincorporation in these cells. This negative effect on DNA stability was concentration dependent at levels usually found in human plasma (1-10 ng/ml). DNA methylation was decreased in HCEC grown in the absence of folate. Conversely, hypomethylation was not concentration dependent. Folate deficiency impaired the ability of HCEC to repair oxidative and alkylation damage. These results demonstrate that folic acid modulates DNA repair, DNA strand breakage, and uracil misincorporation in immortalized human colonocytes and that folate deficiency substantially decreases DNA stability in these cells.

DNA stability and genomic methylation status in colonocytes isolated from methyl-donor-deficient rats.

Epidemiological studies report an inverse relationship between intake of the B vitamin folic acid and colon cancer. Folate is important for DNA synthesis and repair. Moreover, the production of S-adenosylmethionine (SAM), essential for normal DNA methylation and gene expression, is dependent on folic acid. Folate deficiency may increase the risk of malignant transformation by perturbing these pathways. The principal aim of this study was to determine the effects of folate deficiency on DNA stability and DNA methylation in rat colonocytes in vivo. As the metabolic pathways of folate and other dietary methyl donors are closely linked, the effects of methionine and choline deficiency were also evaluated. Male Hooded-Lister rats were fed a diet deficient in folic acid, or in methionine and choline, or in folate, methionine and choline for 10 weeks. DNA strand breakage and misincorporated uracil were determined in isolated colonocytes using alkaline single cell gel electrophoresis. Global DNA methylation was measured in colonic scrapings. Folate was measured in plasma, erythrocyte and liver samples. Methyl donor deficiency induced DNA strand breakage in colonocytes isolated from all experimental groups. Uracil levels in colonocyte DNA remained unchanged compared with controls. DNA methylation was unaffected either by folate and/or methionine and choline depletion. Rats fed a folate-deficient diet had less folate in plasma, red blood cells and liver than controls. Folate and methyl deficiency in vivo primarily affects DNA stability in isolated colonocytes of rats, without affecting overall DNA methylation.

Dietary flavonoids protect human colonocyte DNA from oxidative attack in vitro.

Epidemiological studies suggest that antioxidant polyphenols in the human diet may protect against diseases such as cancer. In this study we investigated the cytoprotective potential of the flavonoids, quercetin, myricetin, kaempferol and rutin against oxidative DNA damage in human colonocytes in vitro. Caco-2 cells, which display specialised enterocyte/colonocyte cell functions, were used as an in vitro model for human colonocytes. Hydrogen peroxide was employed as the oxidant. DNA damage (strand breakage, oxidised purines and oxidised pyrimidines) was determined using the alkaline single cell gel electrophoresis or comet assay. Cell growth and viability were measured. Hydrogen peroxide caused a dose-dependent increase in DNA strand breakage in human colonocytes, presumably via oxygen free radical generation. Quercetin and myricetin protected Caco-2 cells against oxidative attack. In addition, quercetin decreased hydrogen peroxide-mediated inhibition of growth. Neither rutin nor kaempferol was effective. However, quercetin, while inhibiting DNA strand breakage, did not alter the levels of oxidised bases following peroxide treatment. The antifungal agent ketoconazole, prevented quercetin cytoprotection in Caco-2 cells, indicating that P450-mediated metabolism may alter the efficacy of the flavonoids against oxidative DNA damage. Flavonoids, particularly quercetin, the most abundant flavonoid in the human diet, are likely to be important in defending human colonocytes from oxidative attack.