An acetate-yielding diet imprints an immune and anti-microbial programme against enteric infection.

OBJECTIVES: During gastrointestinal infection, dysbiosis can result in decreased production of microbially derived short-chain fatty acids (SCFAs). In response to the presence of intestinal pathogens, we examined whether an engineered acetate- or butyrate-releasing diet can rectify the deficiency of SCFAs and lead to the resolution of enteric infection. METHODS: We tested whether a high acetate- or butyrate-producing diet (HAMSA or HAMSB, respectively) condition Citrobacter rodentium infection in mice and assess its impact on host-microbiota interactions. We analysed the adaptive and innate immune responses, changes in gut microbiome function, epithelial barrier function and the molecular mechanism via metabolite sensing G protein-coupled receptor 43 (GPR43) and IL-22 expression. RESULTS: HAMSA diet rectified the deficiency in acetate production and protected against enteric infection. Increased SCFAs affect the expression of pathogen virulence genes. HAMSA diet promoted compositional and functional changes in the gut microbiota during infection similar to healthy microbiota from non-infected mice. Bacterial changes were evidenced by the production of proteins involved in acetate utilisation, starch and sugar degradation, amino acid biosynthesis, carbohydrate transport and metabolism. HAMSA diet also induced changes in host proteins critical in glycolysis, wound healing such as GPX1 and epithelial architecture such as EZR1 and PFN1. Dietary acetate assisted in rapid epithelial repair, as shown by increased colonic Muc-2, Il-22, and anti-microbial peptides. We found that acetate increased numbers of colonic IL-22 producing TCRαß+CD8αß+ and TCRγδ+CD8αα+ intraepithelial lymphocytes expressing GPR43. CONCLUSION: HAMSA diet may be an effective therapeutic approach for fighting inflammation and enteric infections and offer a safe alternative that may impact on human health.

Medically actionable pathogenic variants in a population of 13,131 healthy elderly individuals.

PURPOSE: To measure the prevalence of medically actionable pathogenic variants (PVs) among a population of healthy elderly individuals. METHODS: We used targeted sequencing to detect pathogenic or likely pathogenic variants in 55 genes associated with autosomal dominant medically actionable conditions, among a population of 13,131 individuals aged 70 or older (mean age 75 years) enrolled in the ASPirin in Reducing Events in the Elderly (ASPREE) trial. Participants had no previous diagnosis or current symptoms of cardiovascular disease, physical disability or dementia, and no current diagnosis of life-threatening cancer. Variant curation followed American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) standards. RESULTS: One in 75 (1.3%) healthy elderly individuals carried a PV. This was lower than rates reported from population-based studies, which have ranged from 1.8% to 3.4%. We detected 20 PV carriers for Lynch syndrome (MSH6/MLH1/MSH2/PMS2) and 13 for familial hypercholesterolemia (LDLR/APOB/PCSK9). Among 7056 female participants, we detected 15 BRCA1/BRCA2 PV carriers (1 in 470 females). We detected 86 carriers of PVs in lower-penetrance genes associated with inherited cardiac disorders. CONCLUSION: Medically actionable PVs are carried in a healthy elderly population. Our findings raise questions about the actionability of lower-penetrance genes, especially when PVs are detected in the absence of symptoms and/or family history of disease.

Erratum: Gut microbial metabolites limit the frequency of autoimmune T cells and protect against type 1 diabetes.

This corrects the article DOI: 10.1038/ni.3713.

Gut microbial metabolites limit the frequency of autoimmune T cells and protect against type 1 diabetes.

Gut dysbiosis might underlie the pathogenesis of type 1 diabetes. In mice of the non-obese diabetic (NOD) strain, we found that key features of disease correlated inversely with blood and fecal concentrations of the microbial metabolites acetate and butyrate. We therefore fed NOD mice specialized diets designed to release large amounts of acetate or butyrate after bacterial fermentation in the colon. Each diet provided a high degree of protection from diabetes, even when administered after breakdown of immunotolerance. Feeding mice a combined acetate- and butyrate-yielding diet provided complete protection, which suggested that acetate and butyrate might operate through distinct mechanisms. Acetate markedly decreased the frequency of autoreactive T cells in lymphoid tissues, through effects on B cells and their ability to expand populations of autoreactive T cells. A diet containing butyrate boosted the number and function of regulatory T cells, whereas acetate- and butyrate-yielding diets enhanced gut integrity and decreased serum concentration of diabetogenic cytokines such as IL-21. Medicinal foods or metabolites might represent an effective and natural approach for countering the numerous immunological defects that contribute to T cell-dependent autoimmune diseases.

Evaluation of Methylation Biomarkers for Detection of Circulating Tumor DNA and Application to Colorectal Cancer.

Solid tumors shed DNA into circulation, and there is growing evidence that the detection of circulating tumor DNA (ctDNA) has broad clinical utility, including monitoring of disease, prognosis, response to chemotherapy and tracking tumor heterogeneity. The appearance of ctDNA in the circulating cell-free DNA (ccfDNA) isolated from plasma or serum is commonly detected by identifying tumor-specific features such as insertions, deletions, mutations and/or aberrant methylation. Methylation is a normal cell regulatory event, and since the majority of ccfDNA is derived from white blood cells (WBC), it is important that tumour-specific DNA methylation markers show rare to no methylation events in WBC DNA. We have used a novel approach for assessment of low levels of DNA methylation in WBC DNA. DNA methylation in 29 previously identified regions (residing in 17 genes) was analyzed in WBC DNA and eight differentially-methylated regions (DMRs) were taken through to testing in clinical samples using methylation specific PCR assays. DMRs residing in four genes, BCAT1, GRASP, IKZF1 and IRF4, exhibited low positivity, 3.5% to 7%, in the plasma of colonoscopy-confirmed healthy subjects, with the sensitivity for detection of ctDNA in colonoscopy-confirmed patients with colorectal cancer being 65%, 54.5%, 67.6% and 59% respectively.

A prediction model for colon cancer surveillance data.

Dynamic prediction models make use of patient-specific longitudinal data to update individualized survival probability predictions based on current and past information. Colonoscopy (COL) and fecal occult blood test (FOBT) results were collected from two Australian surveillance studies on individuals characterized as high-risk based on a personal or family history of colorectal cancer. Motivated by a Poisson process, this paper proposes a generalized nonlinear model with a complementary log-log link as a dynamic prediction tool that produces individualized probabilities for the risk of developing advanced adenoma or colorectal cancer (AAC). This model allows predicted risk to depend on a patient's baseline characteristics and time-dependent covariates. Information on the dates and results of COLs and FOBTs were incorporated using time-dependent covariates that contributed to patient risk of AAC for a specified period following the test result. These covariates serve to update a person's risk as additional COL, and FOBT test information becomes available. Model selection was conducted systematically through the comparison of Akaike information criterion. Goodness-of-fit was assessed with the use of calibration plots to compare the predicted probability of event occurrence with the proportion of events observed. Abnormal COL results were found to significantly increase risk of AAC for 1 year following the test. Positive FOBTs were found to significantly increase the risk of AAC for 3 months following the result. The covariates that incorporated the updated test results were of greater significance and had a larger effect on risk than the baseline variables.

Bioequivalence of n-3 fatty acids from microencapsulated fish oil formulations in human subjects.

Fish oil n-3 fatty acids (FA) have known health benefits. Microencapsulation stabilises and protects fish oil from oxidation, enabling its incorporation into foods. The aim of the present study was to compare the bioavailability of n-3 FA delivered as two microencapsulated fish oil-formulated powders or fish oil gel capsules (FOGC) taken with a flavoured milk in healthy participants. Formulation 1 (F1) composed of a heated mixture of milk protein-sugar as an encapsulant, and formulation 2 (F2) comprised a heated mixture of milk protein-sugar-resistant starch as an encapsulant. Participants consumed 4 g fish oil (approximately 1·0 g EPA and DHA equivalent per dose). Bioavailability was assessed acutely after ingestion of a single dose by measuring total plasma FA composition over a period of 48 h (n 14) using a randomised cross-over design, and over the short term for a period of 4 weeks using an unblinded parallel design (after daily supplementation) by measuring total plasma and erythrocyte FA composition at baseline and at 2 and 4 weeks (n 47). In the acute study, F1 greatly increased (% Δ) plasma EPA and total n-3 FA levels at 2 and 4 h and DHA levels at 4 h compared with FOGC. The time to reach maximal plasma values (T(max)) was shorter for F1 than for FOGC or F2. In the short-term study, increases in plasma and erythrocyte n-3 FA values were similar for all treatments and achieved an omega-3 index in the range of 5·8-6·3 % after 4 weeks. Overall, the results demonstrated human bioequivalence for microencapsulated fish oil powder compared with FOGC.

Ideal colonoscopic surveillance intervals to reduce incidence of advanced adenoma and colorectal cancer.

BACKGROUND AND AIMS: There is limited information about the interplay between multiple risk factors contributing to the risk of advanced neoplasia. We determined the actual risk for advanced neoplasia in relation to lapsed time between colonoscopies in people enrolled in a structured surveillance program. This risk information can be used to guide the selection of optimal surveillance intervals. METHODS: Patients were recruited into programs at two major tertiary hospitals, with a personal or family history of advanced neoplasia. Five thousand one hundred forty-one patients had an index and one or more surveillance colonoscopies. Fifty-one percent had a family history of colorectal neoplasia while the remainder had a personal history. RESULTS: Patients with an immediately prior colonoscopy result (prior result) of advanced adenoma had a risk for advanced neoplasia 7.1 times greater than those with a normal prior result. Cancer as a prior result did not confer a greater risk than either a hyperplastic polyp or a nonadvanced adenoma. Being female reduced risk, age increased risk. Only a family history of a first-degree relative diagnosed under 55, or definite or suspected hereditary nonpolyposis colorectal cancer (HNPCC) conferred an increased risk over a personal history of advanced neoplasia. CONCLUSIONS: Most family history categories did not confer excess risk above personal history of advanced neoplasia. A prior cancer poses less of a risk than a prior advanced adenoma. Based on our models, a person with an advanced adenoma should be scheduled for colonoscopy at 3 years, corresponding to a 15% risk of advanced neoplasia for a male aged under 56. Guidelines should be updated that uses a 15% risk as a benchmark for calculating surveillance intervals.

Resistant starch alters colonic contractility and expression of related genes in rats fed a Western diet.

BACKGROUND AND AIM: Dietary fiber shortens gut transit time, but data on the effects of fiber components (including resistant starch, RS) on intestinal contractility are limited. We have examined RS effects in male Sprague-Dawley rats fed either a high-amylose maize starch (HAMS) or a wholemeal made from high-amylose wheat (HAW) on ileal and colonic contractility ex vivo and expression of genes associated with smooth muscle contractility. METHODS: Rats were fed diets containing 19 % fat, 20 % protein, and either low-amylose maize starch (LAMS), HAMS, wholemeal low-amylose wheat (LAW) or HAW for 11 week. Isolated ileal and proximal colonic sections were induced to contract electrically, or by receptor-independent (KCl) or receptor-dependent agents. Colonic gene expression was assessed using an Affymetrix microarray. RESULTS: Ileal contractility was unaffected by treatment. Maximal proximal colonic contractility induced electrically or by angiotensin II or carbachol was lower for rats fed HAMS and LAW relative to those fed LAMS (P < 0.05). The colonic expression of genes, including cholinergic receptors (Chrm2, Chrm3), serotonin receptors (Htr5a, Htr7), a protease-activated receptor (F2r), a prokineticin receptor (Prokr1), prokineticin (Prok1), and nitric oxide synthase 2 (Nos2), was altered by dietary HAMS relative to LAMS (P < 0.05). HAW did not significantly affect these genes or colonic contractility relative to effects of LAMS. CONCLUSIONS: RS and other fiber components could influence colorectal health through modulation of stool transit time via effects on muscular contractility.

Butyrylated starch affects colorectal cancer markers beneficially and dose-dependently in genotoxin-treated rats.

Population studies suggest that greater dietary fiber intake may lower colorectal cancer (CRC) risk, possibly through the colonic bacterial fermentative production of butyrate. Butyrylated starch delivers butyrate to the colon of humans with potential to reduce CRC risk but high doses may exacerbate risk through promoting epithelial proliferation. Here we report the effects of increasing dietary butyrylated high amylose maize starch (HAMSB) on azoxymethane (AOM) induced distal colonic DNA damage, cell proliferation, mucus layer thickness and apoptosis in rats. Five groups of 15 rats were fed AIN-93G based diets containing 0-40% HAMSB for 4 weeks then injected with (AOM) and killed 6 hours later. Large bowel total SCFA, acetate and butyrate pools and hepatic portal venous plasma total SCFA, acetate and butyrate concentrations were higher with greater HAMSB intake. Distal colonic epithelial apoptotic index and colonic mucus thickness increased, while DNA single strand breaks decreased dose-dependently with greater HAMSB intake. Colonocyte proliferation rates were unaffected by diet. These data suggest that increasing large bowel butyrate may reduce the risk of CRC in a dose dependent manner by enhancing apoptotic surveillance in the colonic epithelium for damaged cells without promoting the risk of tumorigenesis through increased cell proliferation.

Little evidence for association between the TGFBR1*6A variant and colorectal cancer: a family-based association study on non-syndromic family members from Australia and Spain.

BACKGROUND: Genome-wide linkage studies have identified the 9q22 chromosomal region as linked with colorectal cancer (CRC) predisposition. A candidate gene in this region is transforming growth factor ß receptor 1 (TGFBR1). Investigation of TGFBR1 has focused on the common genetic variant rs11466445, a short exonic deletion of nine base pairs which results in truncation of a stretch of nine alanine residues to six alanine residues in the gene product. While the six alanine (*6A) allele has been reported to be associated with increased risk of CRC in some population based study groups this association remains the subject of robust debate. To date, reports have been limited to population-based case-control association studies, or case-control studies of CRC families selecting one affected individual per family. No study has yet taken advantage of all the genetic information provided by multiplex CRC families. METHODS: We have tested for an association between rs11466445 and risk of CRC using several family-based statistical tests in a new study group comprising members of non-syndromic high risk CRC families sourced from three familial cancer centres, two in Australia and one in Spain. RESULTS: We report a finding of a nominally significant result using the pedigree-based association test approach (PBAT; p = 0.028), while other family-based tests were non-significant, but with a p-value <; 0.10 in each instance. These other tests included the Generalised Disequilibrium Test (GDT; p = 0.085), parent of origin GDT Generalised Disequilibrium Test (GDT-PO; p = 0.081) and empirical Family-Based Association Test (FBAT; p = 0.096, additive model). Related-person case-control testing using the "More Powerful" Quasi-Likelihood Score Test did not provide any evidence for association (MQLS; p = 0.41). CONCLUSIONS: After conservatively taking into account considerations for multiple hypothesis testing, we find little evidence for an association between the TGFBR1*6A allele and CRC risk in these families. The weak support for an increase in risk in CRC predisposed families is in agreement with recent meta-analyses of case-control studies, which estimate only a modest increase in sporadic CRC risk among 6*A allele carriers.

A panel of genes methylated with high frequency in colorectal cancer.

BACKGROUND: The development of colorectal cancer (CRC) is accompanied by extensive epigenetic changes, including frequent regional hypermethylation particularly of gene promoter regions. Specific genes, including SEPT9, VIM1 and TMEFF2 become methylated in a high fraction of cancers and diagnostic assays for detection of cancer-derived methylated DNA sequences in blood and/or fecal samples are being developed. There is considerable potential for the development of new DNA methylation biomarkers or panels to improve the sensitivity and specificity of current cancer detection tests. METHODS: Combined epigenomic methods - activation of gene expression in CRC cell lines following DNA demethylating treatment, and two novel methods of genome-wide methylation assessment - were used to identify candidate genes methylated in a high fraction of CRCs. Multiplexed amplicon sequencing of PCR products from bisulfite-treated DNA of matched CRC and non-neoplastic tissue as well as healthy donor peripheral blood was performed using Roche 454 sequencing. Levels of DNA methylation in colorectal tissues and blood were determined by quantitative methylation specific PCR (qMSP). RESULTS: Combined analyses identified 42 candidate genes for evaluation as DNA methylation biomarkers. DNA methylation profiles of 24 of these genes were characterised by multiplexed bisulfite-sequencing in ten matched tumor/normal tissue samples; differential methylation in CRC was confirmed for 23 of these genes. qMSP assays were developed for 32 genes, including 15 of the sequenced genes, and used to quantify methylation in tumor, adenoma and non-neoplastic colorectal tissue and from healthy donor peripheral blood. 24 of the 32 genes were methylated in >50% of neoplastic samples, including 11 genes that were methylated in 80% or more CRCs and a similar fraction of adenomas. CONCLUSIONS: This study has characterised a panel of 23 genes that show elevated DNA methylation in >50% of CRC tissue relative to non-neoplastic tissue. Six of these genes (SOX21, SLC6A15, NPY, GRASP, ST8SIA1 and ZSCAN18) show very low methylation in non-neoplastic colorectal tissue and are candidate biomarkers for stool-based assays, while 11 genes (BCAT1, COL4A2, DLX5, FGF5, FOXF1, FOXI2, GRASP, IKZF1, IRF4, SDC2 and SOX21) have very low methylation in peripheral blood DNA and are suitable for further evaluation as blood-based diagnostic markers.

Repair and removal of azoxymethane-induced O6-methylguanine in rat colon by O6-methylguanine DNA methyltransferase and apoptosis.

Azoxymethane (AOM) is an alkylating agent that generates mutagenic and carcinogenic O(6)-methylguanine (O(6)meG) adducts in DNA. O(6)meG has been detected in human colonic DNA; hence, understanding the innate cellular events occurring in response to the formation of O(6)meG is important in developing preventive strategies for colorectal cancer. We explored the time-course, dose-response, and kinetics of O(6)meG formation and its removal by the DNA repair protein, O(6)-methylguanine DNA methyltransferase (MGMT), and apoptosis. In rats given AOM (10 mg/kg), the formation of O(6)meG occurs within 2 h of exposure, accompanied by rapid depletion of MGMT activity and followed by the induction of an acute apoptotic response that peaks at 6-8 h. MGMT repair and apoptosis are dependent on AOM dose and O(6)meG load. Apoptosis is initiated only when a high O(6)meG load is present and MGMT activity is fully depleted. AOM, 10 mg/kg, overwhelms MGMT repair for about 96 h and renewed MGMT activity is only observed once O(6)meG is no longer detectable. A threshold for apoptosis is observed at 6 h after 6 mg/kg AOM, when a high O(6)meG persists and MGMT activity is very low. These data suggest that apoptosis is probably triggered by O(6)meG, but only once the capacity of MGMT to repair O(6)meG is exhausted. In the colonic epithelium, apoptosis may be complementary to MGMT, in terms of minimising potentially mutagenic events and maintaining a healthy genome.

Identification of potential pathways involved in induction of apoptosis by butyrate and 4-benzoylbutyrate in HT29 colorectal cancer cells.

Butyrate and its analogues have long been investigated as potential chemotherapeutic agents. Our previous structure-activity relationship studies of butyrate analogues revealed that 4-benzoylbutyrate had comparable in vitro effects to butyrate when used to treat HT29 and HCT116 colorectal cancer cell lines. The aim of this study was to identify potential mechanisms associated with the antitumorigenic effects of 4-benzoylbutyrate. In this study, butyrate, 3-hydroxybutyrate and 4-benzoylbutyrate were also investigated for their effects on histone deacetylase (HDAC) activity and histone H4 acetylation in HT29 and HCT116 cells. The biological effects of these analogues on HT29 cells were further investigated using quantitative proteomics to determine the proteins potentially involved in their apoptotic and antiproliferative effects. Because 3-hydroxybutyrate had minimal to no effect on apoptosis, proliferation or HDAC activity, this analogue was used to identify differentially expressed proteins that were potentially specific to the apoptotic effects of butyrate and/or 4-benzoylbutyrate. Butyrate treatment inhibited HDAC activity and induced H4 acetylation. 4-Benzoylbutyrate inhibited HDAC activity but failed to enhance H4 acetylation. Proteomic analysis revealed 20 proteins whose levels were similarly altered by both butyrate and 4-benzoylbutyrate. Proteins that showed common patterns of differential regulation in the presence of either butyrate or 4-benzoylbutyrate included c-Myc transcriptional targets, proteins involved in ER homeostasis, signal transduction pathways and cell energy metabolism. Although an additional 23 proteins were altered by 4-benzoylbutyrate uniquely, further work is required to understand the mechanisms involved in its apoptotic effects.

Resistant starches protect against colonic DNA damage and alter microbiota and gene expression in rats fed a Western diet.

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 (R(2) = 0.40; P = 0.009), and positively with colonic ammonia concentration (R(2) = 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.

Butyrate delivered by butyrylated starch increases distal colonic epithelial apoptosis in carcinogen-treated rats.

Animal studies show that increasing large bowel butyrate concentration through ingestion of butyrylated or resistant starches opposes carcinogen-induced tumorigenesis, which is consistent with population data linking greater fiber consumption with lowered colorectal cancer (CRC) risk. Butyrate has been shown to regulate the apoptotic response to DNA damage. This study examined the impact of increasing large bowel butyrate concentration by dietary butyrylated starch on the colonic epithelium of rats treated with the genotoxic carcinogen azoxymethane (AOM). Four groups of 10 male rats were fed AIN-93G based-diets containing either low amylose maize starch (LAMS), LAMS with 3% tributyrin, 10% high amylose maize starch (HAMS) or 10% butyrylated HAMS (HAMSB). HAMS and HAMSB starches were cooked by heating in water. After 4 weeks, rats were injected once with AOM and killed 6 h later. Rates of apoptosis and proliferation were measured in colonic epithelium. Short-chain fatty acid concentrations in large bowel digesta and hepatic portal venous plasma were higher in HAMSB than all other groups. Apoptotic rates in the distal colon were increased by HAMSB and correlated with luminal butyrate concentrations but cellular proliferation rates were unaffected by diet. The increase in apoptosis was most marked in the base and proliferative zone of the crypt. Regulation of luminal butyrate using HAMSB increases the rates of apoptotic deletion of DNA-damaged colonocytes. We propose this pro-apoptotic function of butyrate plays a major role reducing tumour formation in the AOM-treated rat and that these data support a potential protective role of butyrate in CRC.

Is the tissue persistence of O(6)-methyl-2'-deoxyguanosine an indicator of tumour formation in the gastrointestinal tract?

Azoxymethane (AOM) is a methylating agent capable of inducing mutations in DNA by forming adducts with DNA bases. It has been used to understand the mechanisms involved in colon carcinogenesis. Of the adducts formed in response to AOM, O(6)-methyl-2'-deoxy-guanosine (O(6)-mdGua) is the most mutagenic. Based on studies in rodents of the abundance and persistence of DNA adducts in various tissues after treatment with alkylating agents, previous results suggest, as a generalization, that the longer O(6)-mdGua adducts remain unrepaired in the cells of a tissue, the greater the risk for tumorigenesis. To test this hypothesis, we have built on these studies, expanding the number of tissues in which O(6)-mdGua abundance and persistence were examined and correlating these data with tumour distribution and abundance in rats maintained for 26 weeks after the treatment with AOM. Our study revealed firstly the existence of groups of tissues that developed relatively large amounts (proximal and distal colon, proximal small intestine (SI), liver and kidney) and relatively low levels (stomach, distal SI, bladder, spleen, blood and lung) of O(6)-mdGua after AOM exposure. Secondly, while all tissues showed an increase in adduct levels at 6h after mutagen treatment and most showed a significant drop in adduct levels between 6h and 48h (stomach, proximal and distal SI, liver, spleen, blood and lung), one group of tissues displayed O(6)-mdGua levels that did not decrease at 48h (proximal and distal colon, kidney and bladder). Predictably, the colon displayed tumours 26 weeks after treatment. Interestingly, however, the proximal SI also displayed significant tumour formation at that time. Our findings demonstrate (1) a direct association between exposure to O(6)-mdGua and tumours of the distal colon and (2) a dissociation of the relationship between adduct clearance and tumorigenesis in the SI. This diversity of response in the gastrointestinal tract warrants further analysis.