Fasting serum potassium and long-term mortality in healthy men.

BACKGROUND: Serum potassium levels have been positively associated with cardiovascular mortality, but little is known about the association with cancer mortality and death due to other causes. We examined whether serum levels of potassium were associated with long-term mortality in a healthy cohort. METHODS: Oslo Ischemia Study invited 2341 initially healthy men aged 40-59 years with no use of medication to a comprehensive health survey in 1972. Fasting serum level of potassium (mmol/L) was ascertained at baseline for 1989 men. We have complete follow-up for death throughout 2017. Cox proportional hazard models were used to calculate hazard ratios (HRs) with 95% confidence intervals (CIs) and adjusted for multiple confounders. RESULTS: After a median follow-up of 30 years (interquartile range 21.2-38.7), 1736 deaths were observed, of which 494 were cancer deaths, 688 cardiovascular deaths, and 536 deaths related to other causes. Restricted cubic spline analysis showed that potassium level was linearly and positively associated with long-term cancer mortality; HR per mmol/L 1.8, 95% CI 1.4-2.4. Compared with low levels of potassium (≤ 4.0 mmol/L), men with high levels (≥4.6 mmol/L) showed a significantly 78% higher risk of cancer death. A positive linear association was found for all-cause mortality (HR per mmol/L 1.6, 95% CI 1.4-1.8), and for cardiovascular (HR per mmol/L 1.4, 95% CI 1.1-1.7) and other cause mortality (HR per mmol/L 1.7, 95% CI 1.3-2.2). CONCLUSIONS: These findings suggest that serum potassium level appears to predict long-term mortality in healthy middle-aged men, and it might imply future surveillance strategies for individuals with high serum potassium levels.

Cecal microbiota association with tumor load in a colorectal cancer mouse model.

Background: Colorectal cancer (CRC) is one of the most common cancer types worldwide. The role of the intestinal microbiota in CRC, however, is not well established. In particular, the co-variation between age, tumor progression and microbiota remains largely unknown. Objective and design: We therefore used a recently developed A/J Min/+ mouse model resembling human CRC to investigate how microbial composition in cecum correlates with tumor progression, butyrate and age. Results: We found that the association between the gut microbiota and tumor load was stronger, by far, than the association with both butyrate and age. The strongest direct tumor association was found for mucosal bacteria, with nearly 60% of the significantly correlating operational taxonomic units being correlated with CRC tumor load alone. Conclusion: We favor a systemic association between tumor load and microbiota, since the correlations are associated with tumor load in gut segments other than the cecum (both small and large intestine).

Spontaneous initiation, promotion and progression of colorectal cancer in the novel A/J Min/+ mouse.

The C57BL/6J multiple intestinal neoplasia (Min/+) mouse is a widely used murine model for familial adenomatous polyposis, a hereditary form of human colorectal cancer. However, it is a questionable model partly because the vast majority of tumors arise in the small intestine, and partly because the fraction of tumors that progress to invasive carcinomas is minuscule. A/J mice are typically more susceptible to carcinogen-induced colorectal cancer than C57BL/6J mice. To investigate whether the novel Min/+ mouse on the A/J genetic background could be a better model for colorectal cancer, we examined the spontaneous intestinal tumorigenesis in 81 A/J Min/+ mice ranging in age from 4 to 60 weeks. The A/J Min/+ mouse exhibited a dramatic increase in number of colonic lesions when compared to what has been reported for the conventional Min/+ mouse; however, an increase in small intestinal lesions did not occur. In addition, this novel mouse model displayed a continual development of colonic lesions highlighted by the transition from early lesions (flat ACF) to tumors over time. In mice older than 40 weeks, 13 colonic (95% CI: 8.7-16.3) and 21 small intestinal (95% CI: 18.6-24.3) tumors were recorded. Notably, a considerable proportion of those lesions progressed to carcinomas in both the colon (21%) and small intestine (51%). These findings more closely reflect aspects of human colorectal carcinogenesis. In conclusion, the novel A/J Min/+ mouse may be a relevant model for initiation, promotion and progression of colorectal cancer.

Colorectal Carcinogenesis in the A/J Min/+ Mouse Model is Inhibited by Hemin, Independently of Dietary Fat Content and Fecal Lipid Peroxidation Rate.

BACKGROUND: Intake of red meat is considered a risk factor for colorectal cancer (CRC) development, and heme, the prosthetic group of myoglobin, has been suggested as a potential cause. One of the proposed molecular mechanisms of heme-induced CRC is based on an increase in the rate of lipid peroxidation catalysed by heme. METHODS: In the present work, the novel A/J Min/+ mouse model for Apc-driven colorectal cancer was used to investigate the effect of dietary heme (0.5 µmol/g), combined with high (40 energy %) or low (10 energy %) dietary fat levels, on intestinal carcinogenesis. At the end of the dietary intervention period (week 3-11), spontaneously developed lesions in the colon (flat aberrant crypt foci (flat ACF) and tumors) and small intestine (tumors) were scored and thiobarbituric reactive substances (TBARS), a biomarker for lipid peroxidation was analysed in feces. RESULTS: Dietary hemin significantly reduced colonic carcinogenesis. The inhibitory effect of hemin was not dependent on the dietary fat level, and no association could be established between colonic carcinogenesis and the lipid oxidation rate measured as fecal TBARS. Small intestinal carcinogenesis was not affected by hemin. Fat tended to stimulate intestinal carcinogenesis. CONCLUSIONS: Contradicting the hypothesis, dietary hemin did inhibit colonic carcinogenesis in the present study. The results indicate that fecal TBARS concentration is not directly related to intestinal lesions and is therefore not a suitable biomarker for CRC.

Chronic oral LPS administration does not increase inflammation or induce metabolic dysregulation in mice fed a western-style diet.

Introduction: Lipopolysaccharides (LPS) present in the intestine are suggested to enter the bloodstream after consumption of high-fat diets and cause systemic inflammation and metabolic dysregulation through a process named "metabolic endotoxemia." This study aimed to determine the role of orally administered LPS to mice in the early stage of chronic low-grade inflammation induced by diet. Methods: We supplemented the drinking water with E. coli derived LPS to mice fed either high-fat Western-style diet (WSD) or standard chow (SC) for 7 weeks (n = 16-17). Body weight was recorded weekly. Systemic inflammatory status was assessed by in vivo imaging of NF-κB activity at different time points, and glucose dysregulation was assessed by insulin sensitivity test and glucose tolerance test near the end of the study. Systemic LPS exposure was estimated indirectly via quantification of LPS-binding protein (LBP) and antibodies against LPS in plasma, and directly using an LPS-sensitive cell reporter assay. Results and discussion: Our results demonstrate that weight development and glucose regulation are not affected by LPS. We observed a transient LPS dependent upregulation of NF-κB activity in the liver region in both diet groups, a response that disappeared within the first week of LPS administration and remained low during the rest of the study. However, WSD fed mice had overall a higher NF-κB activity compared to SC fed mice at all time points independent of LPS administration. Our findings indicate that orally administered LPS has limited to no impact on systemic inflammation and metabolic dysregulation in mice fed a high-fat western diet and we question the capability of intestinally derived LPS to initiate systemic inflammation through a healthy and uncompromised intestine, even when exposed to a high-fat diet.

Induction of colorectal carcinogenesis in the C57BL/6J and A/J mouse strains with a reduced DSS dose in the AOM/DSS model.

BACKGROUND: Colorectal cancer (CRC) is one of the most frequently diagnosed cancers worldwide and thus mouse models of CRC are of significant value to study the pathogenesis. The Azoxymethane/Dextran sulfate sodium (AOM/DSS) model is a widely used, robust initiation-promotion model for chemical induction of colitis-associated CRC in rodents. However, the dosage of chemicals, treatment regimens and outcome measures vary greatly among studies employing this model. Thus, the aim of this study was to examine an AOM/DSS model involving a reduced (1%) dose of DSS for induction of carcinogenesis in A/J and C57BL/6J (B6) mice. RESULTS: We show that colonic preneoplastic lesions can be reliably detected in A/J and B6 mice by use of a AOM/DSS model involving a single injection of 10 mg/kg AOM followed by three 7-day cycles of a low-dose (1%) DSS administration. Supporting existing evidence of A/J mice exhibiting higher susceptibility to AOM than B6 mice, our AOM/DSS-treated A/J mice developed the highest number of large colonic lesions. Clinical symptoms in both strains subjected to the AOM/DSS treatment did not persist in-between treatment cycles, demonstrating that the animals tolerated the treatment well. CONCLUSIONS: Our findings suggest that a reduced dose of DSS in the AOM/DSS model can be considered in future studies of early phase colorectal carcinogenesis in the A/J and B6 mouse strains using preneoplastic lesions as an outcome measure, and that such regimen may reduce the risk of early trial terminations to accommodate human endpoints. Overall, our data emphasize the importance of devoting attention towards choice of protocol, outcome measures and mouse strain in studies of CRC in mice according to the study purpose.

Naturalizing laboratory mice by housing in a farmyard-type habitat confers protection against colorectal carcinogenesis.

Living in a farm environment in proximity to animals is associated with reduced risk of developing allergies and asthma, and has been suggested to protect against other diseases, such as inflammatory bowel disease and cancer. Despite epidemiological evidence, experimental disease models that recapitulate such environments are needed to understand the underlying mechanisms. In this study, we show that feralizing conventional inbred mice by continuous exposure to a livestock farmyard-type environment conferred protection toward colorectal carcinogenesis. Two independent experimental approaches for colorectal cancer induction were used; spontaneous (Apc Min/+ mice on an A/J background) or chemical (AOM/DSS). In contrast to conventionally reared laboratory mice, the feralized mouse gut microbiota structure remained stable and resistant to mutagen- and colitis-induced neoplasia. Moreover, the feralized mice exhibited signs of a more mature immunophenotype, indicated by increased expression of NK and T-cell maturation markers, and a more potent IFN-γ response to stimuli. In our study, hygienically born and raised mice subsequently feralized post-weaning were protected to a similar level as life-long exposed mice, although the greatest effect was seen upon neonatal exposure. Collectively, we show protective implications of a farmyard-type environment on colorectal cancer development and demonstrate the utility of a novel animal modeling approach that recapitulates realistic disease responses in a naturalized mammal.

5-Hydroxymethylfurfural and 5-sulfooxymethylfurfural increase adenoma and flat ACF number in the intestine of Min/+ mice.

BACKGROUND: 5-Hydroxymethylfurfural (HMF) is produced in large quantities during the processing of food containing carbohydrates and can be metabolised to 5-sulfooxymethylfurfural (SMF), a reactive intermediate that can bind to DNA and cause mutagenic effects. MATERIALS AND METHODS: Three to six days after birth, multiple intestinal neoplasia (Min/+) mice were given a single subcutaneous injection of either 500 mg/kg body weight (bw) HMF, 25 mg/kg bw SMF or vehicle (0.9 % NaCl), and were euthanised at 12 weeks of age. The number and size of adenomas and flat aberrant crypt foci (ACF) were counted in the intestine. RESULTS: HMF increased the number of small intestinal adenomas (p=0.033), whereas SMF increased the flat ACF number in the large intestine (p=0.025). Treatment with HMF and SMF had no effect on the size of the adenomas. CONCLUSION: These results show that both HMF and SMF are weak intestinal carcinogens in Min/+ mice.

Correspondence between flat aberrant crypt foci and mucin-depleted foci in rodent colon carcinogenesis.

BACKGROUND: Flat aberrant crypt foci (flat ACF) and mucin-depleted foci (MDF) are preneoplastic lesions identified in the colon of carcinogen-treated rodents stained with methylene blue (MB) and high iron diamine-alcian blue (HID-AB), respectively. The correspondence between flat ACF and MDF in the same colon of Min mice treated with azoxymethane (AOM) and of F344 rats treated with 1,2-dimethylhydrazine (DMH) was explored. MATERIALS AND METHODS: The position of each flat ACF was recorded on a digitally constructed photographic map of the MB-stained colon. The same colons were then stained with HID-AB and the position of each MDF was compared with that of flat ACF. RESULTS: The fraction of coincident lesions, identified as both flat ACF and MDF with the two staining methods, was 57% and 42%, in the Min mice and F344 rats, respectively. Flat ACF or MDF not coincident with the two staining methods were either undetectable or ACF with one of the two methods. CONCLUSION: Flat ACF and MDF show considerable, but not total, overlap.

Effects of perinatal exposure to acrylamide and glycidamide on intestinal tumorigenesis in Min/+ mice and their wild-type litter mates.

The tumorigenic capacity of acrylamide (AA) in the intestine of C57BL/6J Min/+ mice, as well as in their wild-type (wt) litter mates was investigated. In Experiment 1, the mice were s.c. injected with 10 or 50 mg/kg body weight (bw) of AA or glycidamide (GA) at week 1 and 2 after birth. In Experiment 2, the mice were given 50 mg/kg bw/injection of AA or GA 1 week before birth to the dam, alone or in combination with exposure of the pups at week 1 and 2 after birth. Following GA exposure at week 1 and 2, the formation of small intestinal tumors in Min/+ mice increased in a dose-dependent manner (r(s) = 0.32, p = 0.008): a 1.3-fold increase in the number of tumors with 50 mg/kg bw GA compared to the controls (p < 0.05). In the wt litter mates, there was a dose response relationship between the GA exposure and the frequency of animals with one or more intestinal neoplasm (intestinal tumors + aberrant crypt foci) (p = 0.018): at 50 mg/kg bw of GA an 8-fold induction was found compared to the controls (p = 0.017). In Experiment 2, Min/+ mice exposed to GA in utero had fewer small intestinal tumors than the controls (p < 0.05). However, following GA exposure the number of intestinal tumors correlated positively with the number of injections (small intestine: r(s) = 0.32, p = 0.002; colon: r(s) = 0.27, p = 0.01). When exposed early in life, GA is a weak intestinal tumorigen in Min/+ mice and their wt litter mates.

Urinary acrylamide metabolites as biomarkers for short-term dietary exposure to acrylamide.

It has previously been reported that heat-treated carbohydrate rich foods may contain high levels of acrylamide resulting in consumers being inadvertently exposed to acrylamide. Acrylamide is mainly excreted in the urine as mercapturic acid derivatives of acrylamide and glycidamide. In a clinical study comprising of 53 subjects, the urinary excretion of these metabolites was determined using solid-phase extraction and liquid chromatography with positive electrospray MS/MS detection. The median (range) total excretion of acrylamide in urine during 24 h was 16 (7-47) microg acrylamide for non-smokers and 74 (38-106) microg acrylamide for smokers, respectively. It was found that the median intake estimate in the study based on 24 h dietary recall was 21 (13-178) and 26 (12-67) for non-smokers and smokers, respectively. The median dietary exposure to acrylamide was estimated to be 0.47 (range 0.17-1.16) microg/kg body weight per day. In a multiple linear regression analysis, the urinary excretion of acrylamide metabolites correlated statistically significant with intake of aspartic acid, protein, starch and coffee. Consumption of citrus fruits correlated negatively with excretion of acrylamide metabolites.

Flat dysplastic aberrant crypt foci are related to tumorigenesis in the colon of azoxymethane-treated rat.

We evaluated the role of aberrant crypt foci (ACF) as biomarkers of colon cancer by studying the sequential development (6-28 weeks) from early lesion to tumor in the colon of azoxymethane-exposed F344 rats (15 mg/kg bw x 2). Surface examination of unsectioned methylene blue-stained colon preparations, transilluminated in the inverse light microscope, revealed two types of early lesions: classic elevated ACF and small flat lesions, which we denoted flat ACF and which were characterized by bright blue staining, compressed crypt openings, and crypts not elevated above the surrounding mucosa. At a later stage, the crypts surrounding large flat ACF became enlarged, a change that slightly raised the structure; principally, large flat ACF and nascent tumors displayed the same surface morphology. Furthermore, flat ACF with 18.6 +/- 10.6 crypt/focus and tumors showed a uniform picture of severe dysplasia with frequent presence of Paneth cells, compressed crypts, cytoplasmic/nuclear overexpression of beta-catenin, and nuclear overexpression of cyclin D1. In contrast, classic elevated ACF with 5.3 +/- 2.5 crypts/focus did not display such changes: they showed mainly hyperplasia, mild or moderate dysplasia but never severe dysplasia. Along the time course, the number of flat ACF + tumors, including microscopic and macroscopic, was virtually constant, approximately 2.5 lesions/rat. The number of classic elevated ACF was initially approximately 180 lesions/rat and terminally approximately 80 lesions/rat. Flat ACF grew significantly faster than classic elevated ACF. In conclusion, our data indicate a continuous developmental growth from small flat dysplastic ACF to the stage of a tumor. In contrast, classic elevated ACF do not seem to be as closely related to tumorigenesis.

Effects of dietary beef, pork, chicken and salmon on intestinal carcinogenesis in A/J Min/+ mice.

The International Agency for Research on Cancer has classified red meat as "probably carcinogenic to humans" (Group 2A). In mechanistic studies exploring the link between intake of red meat and CRC, heme iron, the pigment of red meat, is proposed to play a central role as a catalyzer of luminal lipid peroxidation and cytotoxicity. In the present work, the novel A/J Min/+ mouse was used to investigate the effects of dietary beef, pork, chicken, or salmon (40% muscle food (dry weight) and 60% powder diet) on Apc-driven intestinal carcinogenesis, from week 3-13 of age. Muscle food diets did not differentially affect carcinogenesis in the colon (flat ACF and tumors). In the small intestine, salmon intake resulted in a lower tumor size and load than did meat from terrestrial animals (beef, pork or chicken), while no differences were observed between the effects of white meat (chicken) and red meat (pork and beef). Additional results indicated that intestinal carcinogenesis was not related to dietary n-6 polyunsaturated fatty acids, intestinal formation of lipid peroxidation products (thiobarbituric acid reactive substances, TBARS), or cytotoxic effects of fecal water on Apc-/+ cells. Notably, the amount of heme reaching the colon appeared to be relatively low in this study. The greatest tumor load was induced by the reference diet RM1, underlining the importance of the basic diets in experimental CRC. The present study in A/J Min/+ mice does not support the hypothesis of a role of red meat in intestinal carcinogenesis.

Prevalent location of flat dysplastic aberrant crypt foci near lymphoid follicles in the colon of azoxymethane-treated rats.

In the colon of F344 rats treated with 2 x 15 mg/kg body weight of azoxymethane (AOM), the density (number of lesions/cm2/rat) of flat aberrant crypt foci (ACF) was 13-fold higher (p < 0.05) in the surface area of mucosa immediately adjacent to lymphoid follicles compared with the density of these lesions in the rest of the mucosa. A similar prevalent location near lymphoid follicles was observed for tumours, but not for the classic elevated ACF. The lymphoid follicle-associated flat ACF had the same characteristics as those located in the rest of the mucosa: i.e. severe dysplasia and Wnt pathway stimulation.

Detection and Characterization of Flat Aberrant Crypt Foci (Flat ACF) in the Novel A/J Min/+ Mouse.

BACKGROUND/AIM: Flat aberrant crypt foci (flat ACF) and mucin-depleted foci (MDF) have previously been described as preneoplastic colonic lesions. We used the novel A/J Min/+ mouse model, that demonstrates extensive spontaneous colon carcinogenesis to refine the method of detection of flat ACF and further characterize and define them as early lesions by histological examination and comparison with MDF. MATERIALS AND METHODS: Colons were stained with methylene blue (MB) for flat ACF detection and restained with high-iron diamine-alcian blue (HID-AB) for MDF detection. RESULTS: Optimal flat ACF recognition required at least 24 h of storage post-MB staining and adherence to a set of characteristics. The fraction of flat ACF corresponding with MDF was 93%. Flat ACF/MDF displayed the same picture of severe dysplasia, lack of mucus and goblet cells and accumulation of cytoplasmic ß-catenin. CONCLUSION: The easily detectable flat ACF are reliable surface biomarkers of Apc-driven colon carcinogenesis.

Effect of Dietary Fibers on Cecal Microbiota and Intestinal Tumorigenesis in Azoxymethane Treated A/J Min/+ Mice.

Foods naturally high in dietary fiber are generally considered to protect against development of colorectal cancer (CRC). However, the intrinsic effect of dietary fiber on intestinal carcinogenesis is unclear. We used azoxymethane (AOM) treated A/J Min/+ mice, which developed a significantly higher tumor load in the colon than in the small intestine, to compare the effects of dietary inulin (IN), cellulose (CE) or brewers spent grain (BSG) on intestinal tumorigenesis and cecal microbiota. Each fiber was tested at two dose levels, 5% and 15% (w/w) content of the AIN-93M diet. The microbiota was investigated by next-generation sequencing of the 16S rRNA gene (V4). We found that mice fed IN had approximately 50% lower colonic tumor load than mice fed CE or BSG (p<0.001). Surprisingly, all three types of fiber caused a dose dependent increase of colonic tumor load (p<0.001). The small intestinal tumor load was not affected by the dietary fiber interventions. Mice fed IN had a lower bacterial diversity than mice fed CE or BSG. The Bacteroidetes/Firmicutes ratio was significantly (p = 0.003) different between the three fiber diets with a higher mean value in IN fed mice compared with BSG and CE. We also found a relation between microbiota and the colonic tumor load, where many of the operational taxonomic units (OTUs) related to low tumor load were significantly enriched in mice fed IN. Among the OTUs related to low tumor load were bacteria affiliated with the Bacteroides genus. These results suggest that type of dietary fiber may play a role in the development of CRC, and that the suppressive effect of IN on colonic tumorigenesis is associated with profound changes in the cecal microbiota profile.

Scanning electron microscopy of colonic lesions in 1,2-dimethylhydrazine-treated rats.

The surface morphology of late colonic lesions in F344 rats treated with 1,2-dimethylhydrazine was studied by scanning electron microscopy. At week 31 after carcinogen treatment, the surface epithelial characteristics of different types of lesions observed in the colonic mucosa were compared, namely classic elevated aberrant crypt foci (ACF), flat lesion and gross tumour. Classic elevated ACF were easily observed as structures with enlarged crypts elevated from the background mucosa. When the various ACF were compared, or when the ACF were compared with the background mucosa, no ultrastructural differences, or differences in the density of goblet cells were found. The flat lesion showed an epithelium without goblet cells and crypts with small openings harbouring a large number of loose, undefined, dysplastic epithelial cells. These changes appeared to be linked to the malignant development since they were also characteristic of the examined tumour.

Increased levels of PPARbeta/delta and cyclin D1 in flat dysplastic ACF and adenomas in Apc(Min/+) mice.

BACKGROUND: In Apc(Min/+) (Min; multiple intestinal neoplasia) mice two separate populations of aberrant crypt foci (ACF) develop in the colon after azoxymethane (AOM) exposure. ACF(Min), with a flat appearance, severe dysplasia and increased beta-catenin expression, are related to adenoma development, whereas classic ACF, with elevated structure, hyperplasia and normal beta-catenin level, are probably not. MATERIALS AND METHODS: The expressions of peroxisome proliferator-activated receptors (PPARs) beta/delta, cyclin D1 and beta-catenin in ACF, adenoma and normal tissue from AOM-treated Apc(Min/+) mice and a familial adenomatous polyposis (FAP) patient colon tumour were assessed by immunohistochemistry and immunoblotting. RESULTS: The flat ACF (ACF(Min)) displayed increased cytoplasmic levels of beta-catenin, and increased levels of cyclin D1 and PPARbeta/delta. In contrast, the expression in classic ACF resembled normal mucosa. Adenomas from Apc(Min/+) mice, as well as a FAP patient colon tumour, displayed increased nuclear and cytoplasmic levels of beta-catenin, and the same expression patterns of cyclin D1 and PPARbeta/delta as those found in flat ACF. CONCLUSION: In addition to activation of the Wnt signalling pathway in both flat ACF and in adenomas in Apc(Min/+) mice, the increased expression of PPARbeta/delta in these lesions could be a target for pro-inflammatory signals important for growth and reduced apoptosis.

Effects of hemin and nitrite on intestinal tumorigenesis in the A/J Min/+ mouse model.

Red and processed meats are considered risk factors for colorectal cancer (CRC); however, the underlying mechanisms are still unclear. One cause for the potential link between CRC and meat is the heme iron in red meat. Two pathways by which heme and CRC promotion may be linked have been suggested: fat peroxidation and N-nitrosation. In the present work we have used the novel A/J Min/+ mouse model to test the effects of dietary hemin (a model of red meat), and hemin in combination with nitrite (a model of processed meat) on intestinal tumorigenesis. Mice were fed a low Ca2+ and vitamin D semi-synthetic diet with added hemin and/or nitrite for 8 weeks post weaning, before termination followed by excision and examination of the intestinal tract. Our results indicate that dietary hemin decreased the number of colonic lesions in the A/J Min/+ mouse. However, our results also showed that the opposite occurred in the small intestine, where dietary hemin appeared to stimulate tumor growth. Furthermore, we find that nitrite, which did not have an effect in the colon, appeared to have a suppressive effect on tumor growth in the small intestine.

Loss of heterozygosity and nonsense mutation in Apc in azoxymethane-induced colonic tumours in min mice.

C57BL/6J Min/+ mice, which carry a nonsense mutation in Apc, were injected twice neonatally with 5 mg azoxymethane (AOM) /kg body weight. AOM treatment in comparison with untreated Min mice increased the incidence and number of colonic tumours from 6/14 to 22/24 (incidence) and 0.64+/-0.9 to 4.0+/-3.5 tumours per mice, respectively. Colonic tumours were analysed for loss of heterozygosity (LOH) in Apc, and 32 of the samples showed LOH whereas 14 did not. In untreated Min mice, all 8 tumours had LOH in Apc. All tumour samples from the AOM-treated Min mice were analysed for nonsense mutations between codons 686 and 1217 in the Apc gene, and one sample had a G-->A transition mutation in codon 1047. No beta-catenin mutations in the region coding for phosphorylation sites important for degradation were found. In conclusion, the main mechanism for colonic tumour induction in AOM-induced Min mice is LOH in Apc, but Apc nonsense mutations may also occur.