Relationship between the nature of mucus and crypt multiplicity in aberrant crypt foci in the rat colon

Aberrant crypt foci (ACF) induced in the distal colon of F344 male rats, 4, 8, 12 and 35 weeks after the first administration of 1, 2-dimethylhydrazine-2HCl (DMH) were examined to determine whether a correlation exists between the nature of goblet cell mucin and the number of crypts (crypt multiplicity) comprising the ACF. According to the ACF score calculated from the results of the qualitative observation of sulfomucins (SuMs) and sialomucins (SiMs), the ACF in the 4th week showed a weak correlation between the nature of the mucus and crypt multiplicity, and the ACF of each class showed similar mucous profiles. From the 8th week, a significant difference (P < 0.01) was recognized between the ACF consisting of 3 crypts or less and those consisting of 4 crypts or more. The proportion of crypts with SiM predominance showed a decrease in the 8th week in the ACF consisting of 1 crypt and in the 12th week in the ACF consisting of 2 or 3 crypts, implying a recovery tendency. The ACF consisting of more than 4 crypts showed little change over time, retaining the tendency of SiM predominance. Ulex europaeus agglutinin-I (UEA-I) lectin-positive crypts appeared in the ACF. This finding was significantly more prominent (P < 0.001) in the ACF with SiM predominance than in the ACF with SuM predominance at each experimental period, and in the 12th week after the first administration of DMH, the incidence of ACF with UEA-I-reactive mucin was decreased in the ACF groups consisting of 3 crypts or less, compared with the ACF groups consisting of 4 or more crypts. These results suggest that the biological quality of mucus in ACF consisting of 4 or more crypts is different from that in ACF consisting of 3 crypts or less. This difference should be considered when ACF are used as an intermediate biomarker of colon cancer.     

Bifidobacterium longum and lactulose suppress azoxymethane-induced colonic aberrant crypt foci in rats

Bifidobacterium longum has been shown to afford protection against colon tumorigenesis. Lactulose, a keto analog of lactose, serves as a substrate for preferential growth of Bifidobacterium. It is not known whether feeding lactulose along with B. longum will have any advantage over feeding of B. longum alone. To test this combination effect, 61 male Fisher 344 weanling rats were divided into four groups of 15 rats each (16 in the control group) and assigned to one of the following four diets for 13 weeks: (i) AIN76A (control, C); (ii) C + 0.5% B. longum (C+Bl, containing 1 x 10(8) viable cells/g feed); (iii) C + 2.5% lactulose (C+L); (iv) C + 0.5% B. longum + 2.5% lactulose (C+Bl+L). All animals received a s.c. injection of azoxymethane at 16 mg/kg body wt at 7 and 8 weeks of age. Colons of 10 rats from each dietary group were analyzed for aberrant crypt foci (ACF), which are preneoplastic markers. Colonic mucosa and livers from five rats were analyzed for glutathione S-transferase (GST, a Phase II enzyme marker). Results indicate that feeding of lactulose and B. longum singly and in combination reduces the number of ACF (P = 0.0001) and the total number of aberrant crypts significantly (P = 0.0005). The total number of ACF in diets C, C+Bl, C+L and C+Bl+L were 187 +/- 9, 143 +/- 9, 145 +/- 11 and 97 +/- 11 respectively. There was no significant difference in weight gain among treatments. Colonic mucosal GST levels were significantly (P = 0.05) higher in the Bl and L groups compared with group C. Initially there was a mild diarrhea in lactulose-fed rats. There was a positive correlation between higher cecal pH and number of ACF. Results of the study indicate that Bifidobacterium and lactulose exert an additive antitumorigenic effect in rat colon.     

Prevention by retinoids of azoxymethane-induced tumors and aberrant crypt foci and their modulation of cell proliferation in the colon of rats

Retinoids are proposed chemopreventive agents that inhibit cell proliferation and induce differentiation. Their ability to prevent azoxymethane (AOM)-induced aberrant crypt foci (ACF) and tumors and to modulate cell proliferation was investigated in the colon of male F344 rats. Thirteen retinoids were evaluated for prevention of ACF and two of them, 9-cis-retinoic acid (RA) and 4-(hydroxyphenyl)retinamide (4-HPR), were also evaluated for prevention of colon cancer. The retinoids were administered continuously in the diet starting 1 week prior to the first of two weekly 15 mg/kg i.p. injections of AOM and for a total of either 5 or 36 weeks in order to evaluate their effect on colonic ACF and tumors. At a concentration of 1 mmol/kg diet, 2-(carboxyphenyl)retinamide caused the greatest reduction (57.7%) in the yield of ACF. 9-cis-RA was toxic at 1 mmol/kg so that it was evaluated at 0.1 mmol/kg, resulting in a 41.6% reduction in ACF. The ability of the retinoids to reduce the proliferating cell nuclear antigen (PCNA) labeling index in ACF and in non-involved crypts correlated with their ability to prevent ACF. Both 9-cis-RA (0.1 and 0.2 mmol/kg diet) and 4-HPR (1 and 2 mmol/kg diet) were highly effective in decreasing the yield of AOM-induced colon tumors. In summary, retinoids were demonstrated to reduce cell proliferation and to prevent ACF and tumors in the colon, suggesting promise as preventive agents for colon cancer.     

Azoxymethane-induced colon tumors and aberrant crypt foci in mice of different genetic susceptibility

INTRODUCTION: Virtual endoscopy is a new 3D technique which permits to depict the inner surface of anatomic cavities. We report our experience in the study of the nasal cavity and paranasal sinuses. MATERIAL AND METHODS: CT examinations of the maxillofacial region were obtained using 1.5 or 3.0 mm slices, 1.5 or 3.0 mm table feed, 120 kV, 140 mA, 2 s scan time, standard and high resolution algorithms for bony structures and a field of view of 14-16 cm. The images were transferred on a workstation and processed with the Navigator virtual endoscopy software (General Electric). A threshold value ranging -300 to -550 UH was applied. RESULTS: We report a series of virtual images of the nasal cavity which includes rhinopharynx vault, torius tubarius, choanae, turbinates, tubal orifice and osteomeatal complex. Moreover, images of frontal sinus and tear duct, of polyps and fracture of maxillary sinus are also presented. 3D virtual endoscopy provides a clear visualization of the anatomic structures of the nasal cavity and sinuses. The images are similar to those of conventional endoscopy. Moreover virtual endoscopy visualizes the paranasal sinuses, which are not accessible at conventional endoscopy. The main limitations of this new technique are the arbitrary choice of the threshold value and the homogenization of different tissue densities, which reduces the contrast resolution. CONCLUSIONS: Virtual endoscopy can presently be considered a complementary technique of the standard axial and coronal CT examination. It provides an effective demonstration of the anatomy of these structures and shows areas which are difficult to visualize with conventional endoscopy. This technique could be of help in didactical activity; its clinical application has to be verified.     

Inhibition by wheat bran cereals of the development of aberrant crypt foci and colon tumours

BACKGROUND: Double inactivation by solvent/detergent treatment plus heating at 100 degrees C for 30 minutes after lyophilization has been adopted to improve viral safety of factor VIII and factor IX concentrates, particularly with respect to non-lipid-enveloped viruses. The aim of this study was to evaluate the safety of concentrates exposed to these virucidal methods. STUDY DESIGN AND METHODS: Twenty-six previously untreated hemophiliacs, 19 with factor VIII deficiency and 7 with factor IX deficiency, were investigated in a prospective multicenter study over a 12-month follow-up period by the use of serologic and virologic markers for lipid- and non-lipid-enveloped viruses (human immunodeficiency virus types 1 and 2; hepatitis A, B, and C viruses; B19 parvovirus antibodies; and B19 DNA). Overall, 270,000 U of factor VIII and 102,000 U of factor IX concentrate were administered during the study period. RESULTS: None of the 26 patients seroconverted for human immunodeficiency virus or hepatitis C virus. Hepatitis B virus markers remained negative in the 10 unvaccinated hemophiliacs. No hepatitis A virus seroconversion occurred among 17 susceptible patients. B19 seroconversion (IgM) and B19 viremia were observed within 2 weeks of the first concentrate infusion in 8 of 15 susceptible patients, 5 of 11 treated with factor VIII and 3 of 4 with factor IX concentrate. CONCLUSION: This prospective study indicates that very high temperatures applied to lyophilized concentrates appear to prevent the transmission of hepatitis A virus to hemophiliacs. However, B19 parvovirus still contaminates concentrates despite the use of this robust virucidal method.     

Infrequent somatic mutation of the adenomatous polyposis coli gene in aberrant crypt foci of human colon tissue

BACKGROUND: The authors examined somatic mutations of the adenomatous polyposis coli (APC) gene in 84 human aberrant crypt foci (ACF) to determine whether APC gene mutations were involved in the histologic progression of ACF. METHODS: Mutation cluster regions of the APC gene were subjected to polymerase chain reaction single-strand conformation polymorphism analysis and direct sequencing. RESULTS: Four kinds of deletion were detected in the mutation cluster regions of APC gene in five ACF. APC mutation was detected in 1 of 18 ACF with Stage I abnormalities (6%). Four of 10 adenomatous ACF (40%) harbored the mutation. There were no mutations in 56 hyperplastic ACF. CONCLUSIONS: These results suggest that APC mutations may be involved initially in only a limited number of adenomas in ACF.     

Carrageenan given as a jelly, does not initiate, but promotes the growth of aberrant crypt foci in the rat colon

We report a case of sperm cell seminoma caused by trauma. The data in the literature indicate the frequency is less than 5% of all seminomas. This case was exclusively located in the gonads and was a pure form. Orchidectomy with high ligature of the cord and adjuvant radiotherapy at the dose of 25 Gy centered on the para-aortic and subdiaphragmatic chains is adequate treatment. When inguinoscrotal surgery is performed, this zone must be irradiated with 25 Gy. Prognosis is satisfactory: 5-year survival is 100% with this protocol.     

Aberrant crypt foci of the colon as precursors of adenoma and cancer

BACKGROUND: Aberrant crypt foci of the colon are possible precursors of adenoma and cancer, but these lesions have been studied mainly in surgical specimens from patients who already had colon cancer. METHODS: Using magnifying endoscopy, we studied the prevalence, number, size, and dysplastic features of aberrant crypt foci and their distribution according to age in 171 normal subjects, 131 patients with adenoma, and 48 patients with colorectal cancer. We also prospectively examined the prevalence of aberrant crypt foci in 11 subjects (4 normal subjects, 6 with adenoma, and 1 with cancer) before and after the administration of 100 mg of sulindac three times a day for 8 to 12 months and compared the results with those in 9 untreated subjects (4 normal subjects and 5 with adenoma). All 20 subjects had aberrant crypt foci at base line. RESULTS: We identified 3155 aberrant crypt foci, 161 of which were dysplastic; the prevalence and number increased with age. There were significant (P<0.001) correlations between the number of aberrant crypt foci, the presence of dysplastic foci, the size of the foci, and the number of adenomas. After sulindac therapy, the number of foci decreased, disappearing in 7 of 11 subjects. In the untreated control group, the number of foci was unchanged in eight subjects and slightly increased in one (P<0.001 for the difference between the groups). CONCLUSIONS: Aberrant crypt foci, particularly those that are large and have dysplastic features, may be precursors of adenoma and cancer.     

Effects of miso and NaCl on the development of colonic aberrant crypt foci induced by azoxymethane in F344 rats

This case reports the early diagnosis and treatment of an iatrogenic acute localized periodontitis arising from the incorrect use of orthodontic elastic rubber bands to close a maxillary midline diastema in an eight-year-old girl. The case illustrates the misdiagnosis and mismanagement of a transitional diastema in the "ugly duckling" stage of the mixed dentition. The nine-year follow-up shows that thorough periodontal and orthodontic management has allowed successful retention of both maxillary permanent central incisors, which had initially pathological grade 3 vertical mobility, little remaining alveolar bone, and a hopeless prognosis.     

Induction of aberrant crypt foci and flat-type adenocarcinoma in the colons of dogs by N-ethyl-N''-nitro-nitrosoguanidine and their sequential changes

Dengue virus infections have been well known for many years; still dengue virus is regarded as an 'emerging' pathogen, as the disease profile is changing. Its geographical range and overall incidence, and the incidence of the associated complications, dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS), are on the increase. Modern-day travel and increasing urbanization seem to be the main contributing factors. In order to estimate the risk of infection during long-term stays in dengue-endemic countries, we tested sera obtained from 323 development aid workers and their family members who had spent on average 9.8 years in dengue-endemic regions for the presence of dengue virus antibodies. Dengue virus antibody screening was done by a commercially available immunofluorescence test (IF). Reactive samples were re-tested by an in-house IF and also tested for cross-reactivity to yellow fever virus using yellow fever IF and neutralization test (NT). Evaluation of the results revealed that the screening test has a specificity of at least 63.2%. In 12 of 19 initially positive cases crossreacting antibodies against yellow fever virus could be ruled out. Three cases remained indeterminable, whereas four of the reactive and 10 (out of 12) of the borderline reactive cases showed crossreactivity with yellow fever virus, probably due to previous vaccination. We found seroprevalence rates of 4.3% with no significant differences related to gender or area of upbringing. Seroprevalence rates were evaluated according to region of suspected or confirmed infection. In two cases the dengue infection had taken a classical clinical course; in another three cases an extraordinary febrile illness was reported in the history. None of the other seropositive individuals had a history of an illness possibly attributable to dengue virus infection. Our results show that there definitely is a risk for long-term expatriates to acquire (mostly non- or oligo-symptomatic) dengue infection, which might be important especially in the light of the supposed aetiology of DHF or DSS as a secondary infection with another dengue virus serotype.     

Expression of G protein alpha subunits in normal rat colon and in azoxymethane-induced colonic neoplasms

BACKGROUND & AIMS: Heterotrimeric G proteins are important in growth-regulating signal transduction. The aim of this study was to characterize the relative expression of G protein alpha subunits in rat colonocytes, colonocyte antipodal plasma membranes, and colonic neoplasms. METHODS: Antipodal plasma membranes were prepared from isolated colonocytes. Azoxymethane was administered to rats to induce colonic neoplasms. K-ras mutations in the neoplasms were determined by oligonucleotide hybridization and confirmed by primer mediated-restriction fragment length polymorphism. Colonocyte and tumor homogenates or membranes were probed for Galpha subunits by Western blotting with isoform-specific antibodies. RESULTS: The expressions of Galphai2, alphai3, and alphaq/11 were significantly enriched in the basolateral compared with brush border fraction of colonic antipodal plasma membranes. In neoplasms without K-ras mutations, the expression of Galphai2 increased 4-fold, Galphas(long) increased 2.5-fold, and Galphai3 increased 1.5-2-fold. Expression did not differ among tumor grades. K-ras mutations were associated with lowered expression of G proteins, especially Galphao. CONCLUSIONS: In colonocytes, Galpha subunits are localized primarily in basolateral plasma membranes. The increased expressions of Galphai2 and, to a lesser degree, Galphai3 and Galphas(long) in tumors was independent of tumor grade but was modulated by the presence of K-ras mutations.     

Effects of epidermal growth factor and dimethylhydrazine on crypt size, cell proliferation, and crypt fission in the rat colon. Cell proliferation and crypt fission are controlled independently

Crypt fission is now established as an important mechanism of intestinal growth and regeneration. It has been proposed that increased crypt size is the stimulus for crypt fission, because crypts preparing for fission are generally larger. Consequently, we investigated the effects of epidermal growth factor (EGF) and dimethylhydrazine, which are both known to stimulate crypt cell proliferation, on crypt fission in the rat intestine. We also examined whether the effects of EGF on both proliferation and crypt fission are modified by the pretreatment with dimethylhydrazine for 16 weeks, dimethylhydrazine was then discontinued for 8 weeks, followed by intravenous infusion of EGF for 1 week. There were four groups: vehicle alone, EGF alone, dimethylhydrazine alone, and dimethylhydrazine followed by EGF infusion. The rats were killed at 25 weeks and rates of intestinal crypt cell production, crypt size, and crypt fission were determined. Intravenously infused EGF significantly increased crypt cell production rate, but the magnitude of the effect decreased from the proximal to the distal colon. EGF caused an increase in crypt area, possibly reflecting an increase in crypt size. Importantly dimethylhydrazine had no significant effect on crypt cell production rate nor on crypt area in the distal colon, but it did cause an increase in crypt area in the mid-colon. The crypt fission index was significantly decreased by EGF and increased by dimethylhydrazine. There was no qualitative interaction between EGF and dimethylhydrazine. These results demonstrate the marked proliferative effect of intravenously infused EGF in the colon of orally fed rats, with significant site effects (P = 0.0007); the effect was greatest in the proximal colon and disappeared in the distal colon. The observation that EGF reduced crypt fission indicates that increased cell proliferation, per se, is not a stimulus for crypt fission. This is further supported by the observation that dimethylhydrazine increases crypt fission in crypts of normal size in the distal colon without significantly increasing cell proliferation. These results suggest that increasing crypt cellularity by proliferation is not sufficient to induce crypt fission, and factors other than increased crypt size by proliferation can control crypt fission. It is also probable that cell proliferation and crypt fission are independently regulated. Crypt fission appears to play a considerable role in the intestinal response to carcinogens.     

Inhibition of development of N,N'-dimethylhydrazine-induced rat colonic aberrant crypt foci by pre, post and simultaneous treatments with 24R,25-dihydroxyvitamin D3

It has recently been reported that new vitamin D3 derivatives can exert inhibitory effects on colon carcinogenesis in rats. In the present study the chemopreventive potential of 24R,25-dihydroxyvitamin D3 (24R,25(OH)2vitamin D3) was assessed in a murine model of colon carcinogenesis. In experiment 1, male 6-week-old F344 rats were administered N,N'-dimethylhydrazine (DMH) 20 mg/kg s.c. once a week 4 times. The rats were fed 24R,25(OH)2vitamin D3 at 10 ppm in the diet prior to (pre), together with (simultaneous) or after (post) DMH treatment. Modifying effects were assessed using aberrant crypt foci (ACF), putative preneoplastic lesions, as the end point markers in this model of colon carcinogenesis. After 8 weeks, pre and more markedly simultaneous administration of 24R,25(OH)2vitamin D3 was found to have reduced the total numbers of ACF and significantly inhibited the development of foci. After 16 weeks, numbers of foci with > or = 4 crypts, which are more likely to progress to tumors, were significantly reduced. The most pronounced inhibition of ACF development was noted in rats fed the 24R,25(OH)2vitamin D3 after DMH administration. The reduction was particularly marked in the proximal colon. Blood levels of calcium were not significantly increased over the control levels in groups administered DMH and the vitamin. Immunohistochemical staining showed numbers of proliferating cell nuclear antigen-positive cells to be lower in the colonic epithelia of rats fed the vitamin D3 metabolite than in the controls. In experiment 2, the effect of 24R,25(OH)2vitamin D3 on the alterations in c-fos, c-myc and c-jun oncogene expression in response to DMH administration was examined by northern blot analysis. The early increase in expression of ornithine decarboxylase (ODC) activity was not altered by 24R,25(OH)2vitamin D3. The results suggest that 24R,25(OH)2vitamin D3 is a cancer chemopreventive agent which may suppresses DMH induction of lesions and their subsequent development via an antiproliferative action.     

Effect of soya bean saponins on azoxymethane-induced preneoplastic lesions in the colon of mice

The effect of saponins isolated from soya bean flour on the incidence of aberrant crypt foci (ACF) induced by azoxymethane (AOM) in the colonic wall of CF1 mice was investigated. Four weekly injections of AOM, a known colon carcinogen, were administered to mice. One week after the last injection, mice were placed on an AIN-76 diet supplemented with 3% soya bean saponins or continued on the basal AIN-76 diet. Another group of mice was placed on the saponin diet without AOM initiation to observe the effect of saponins on the growth characteristics of mice. Dietary intake of soya saponins significantly reduced the incidence of ACF at the end of 14 weeks (postinitiation). Noninitiated mice maintained on a similar soya bean saponin-supplemented diet did not show any adverse effects on the growth and overall health of the animals. These findings suggest that soya bean saponins can play an important role in inhibiting the incidence of ACF in the colon of mice.     

Promoting effects of phenobarbital on the enzyme-altered foci induced by intrahepatic gamma-ray-irradiation in the rat liver

Radiation-induced carcinogenesis of the rat liver using iridium-192 seeds as an intrahepatic radioactive source was studied by enzyme histochemical means. Rats were divided into six groups according to various combinations of one or two iridium-192 or stainless steel seeds and whether they were given a diet containing 0.05% phenobarbital (PB) or a basal diet (BD). Each group were sacrificed at 20, 40, and 60 weeks after intrahepatic insertion of the iridium-192 or stainless steel seeds. gamma-Glutamyl transpeptidase (GGT), glucose-6-phosphatase (G6Pase), and adenosine triphosphatase (ATPase) were stained in the liver tissues, and GGT-positive foci were quantified. Liver neoplasm was not evident, but enzyme-altered foci (EAF) were induced by gamma-ray irradiation. At every point (20, 40, and 60 weeks) after the insertion of the seeds, the GGT-positive area was larger in the rats given than those given BD. Moreover, despite the iridium-192 radioactivity decay, EAF developed continuously in the rats given PB, and persisted in those given BD from 40 to 60 weeks after insertion. These results indicated that phenobarbital promotes the development of EAF initiated by irradiation, as it promotes the process of chemical carcinogenesis in the rat liver.     

Cell death and cell proliferation contribute to the enhanced growth of foci by fasting in rat medial colon

We have recently shown that fasting before initiation markedly stimulated the growth of aberrant crypt foci (ACF) induced by azoxymethane (AOM) in the rat medial colon. Here we investigated the mechanisms by which fasting enhanced the growth of ACF. Rats were exposed to 4 day-starvation, then they were given AOM (20 mg/kg) on the first day of refeeding. 4 day-fasting depressed cell proliferation as shown by the decreased mitotic index and enhanced cell death by apoptosis. On the first day of refeeding, apoptotic index remained higher than control values, while mitotic index markedly increased in the colonic epithelium of fasted/ refed rats. The administration of AOM induced an apoptotic wave, that was higher in controls, and a transient drop in the mitotic index that recovered quickly in the fasted/refed group. These data suggest that starvation-induced apoptosis represents the mitogenic stimulus to increase the rates of cell proliferation responsible for the enhanced growth of ACF in fasted/refed rats.     

(E)-2-hexenal-induced DNA damage and formation of cyclic 1,N2-(1,3-propano)-2'-deoxyguanosine adducts in mammalian cells

(E)-2-Hexenal (hexenal), a natural flavor compound, acts as directly genotoxic agent and forms cyclic 1,N2-propano adducts with deoxyguanosine. Formation of this adduct in isolated DNA and in cells was studied with a modified 32P-postlabeling procedure including HPLC separation, nuclease P1 enrichment, two-dimensional TLC of adducted nucleotide bisphosphates on PEI-cellulose, and quantification of adduct spots by liquid scintillation counting. Adduct formation with the more reactive crotonaldehyde was included for comparison. Synthesized adducted dG-3'-phosphates served as external standards for identification and quantification. In calf thymus DNA, hexenal (0.2 mM) shows a time dependent formation of adducts, yielding 1.55 pmol/mumol of DNA at 5 h incubation. With crotonaldehyde (0.2 mM) the adduct rate was about 10-fold higher. Hexenal also generated 1,N2-propano-dG adducts in the human lymphoblastoid Namalva cell line (0.2 mM, 1 h, 86 fmol/mumol of DNA) and in primary rat colon mucosa cells (0.4 mM, 30 min, 50 fmol/mumol of DNA). In primary colon mucosa cells from rats and humans, hexenal and crotonaldehyde (0.4 mM, 30 min) induced DNA damage, detected by single cell microgel electrophoresis (comet assay). In primary rat gastric mucosa cells, hexenal was only weakly active, inducing detectable DNA damage in 20% of cells at 0.8 mM concentration. In contrast, primary mucosa cells from rat esophagus were as sensitive as colon cells. After single oral application of hexenal to rats (up to 320 mg/kg body wt) DNA damage was not detectable in gastrointestinal mucosa. Analysis of hexenal in selected flavored foods revealed concentrations up to 14 ppm (0.14 mM) that are comparable to its natural occurrence in some fruits and vegetables (up to 30 ppm). Thus, the concentration range selected for the toxicological studies described here clearly is relevant: Hexenal, at concentrations found in food, exerts genotoxic effects in cells from rat and human gastrointestinal tract.