Effects of carcinogen dosage on experimental colonic carcinogenesis by azoxymethane: an ultrastructural study of grossly normal colonic mucosa

The transmission electron microscopic features of grossly normal colonic mucosa in the azoxymethane [(AOM) CAS: 25843-45-2]-treated rat model of colonic carcinogenesis were studied by the method of concomitant variation. Ten-week-old male F344 rats were given 10 weekly sc injections of AOM at doses of 3, 7, or 14 mg/kg body weight and were killed 1 week or 15 weeks after the last AOM dose. Grossly normal distal left colon was examined using transmission electron microscopy. Three transient dose-dependent features of colonic epithelial cells were identified: a) mitochondrial injury, b) nuclear pleomorphism and loss of polarity, and c) increased numbers of mitotic figures in the lower third of the crypts. Because these dose-dependent features were present only shortly after AOM administration, they appeared to be manifestations of carcinogen toxicity and associated reactive, regenerative epithelial changes. By contrast, four persistent dose-dependent features were identified: a) increased numbers of epithelial cells with enlarged nucleoli, b) increased numbers of mitotic figures in the middle third of crypts, c) reduced numbers of goblet cells, and d) reduced numbers of apical cytoplasmic vacuoles in the upper third of the crypts. Because these features persisted in a dose-dependent manner for many weeks after the last dose of AOM and were present after the latent period to tumor formation, they appear to be morphologic precursors to colonic carcinogenesis in the model.     

Troglitazone, a ligand for peroxisome proliferator-activated receptor gamma, inhibits chemically-induced aberrant crypt foci in rats.

The biological roles of peroxisome proliferator-activated receptors (PPARs) in various diseases, including inflammation and cancer, have been highlighted recently. Although PPARgamma ligand is suspected to play an important role in carcinogenesis, its effects on colon tumorigenesis remain undetermined. The present time-course study was conducted to investigate possible modifying effects of a PPARgamma ligand, troglitazone, on the development and growth of aberrant crypt foci (ACF), putative precursor lesions for colon carcinoma, induced by azoxymethane (AOM) or dextran sodium sulfate (DSS) in male F344 rats. Oral troglitazone (10 or 30 mg / kg body weight (b.w.)) significantly reduced AOM (two weekly subcutaneous injections, 20 mg / kg b.w.)-induced ACF. Treatment with troglitazone increased apoptosis and decreased polyamine content and ornithine decarboxylase (ODC) activity in the colonic mucosa of rats treated with AOM. Gastric gavage of troglitazone also inhibited colitis and ACF induced by DSS (1% in drinking water), in conjunction with increased apoptosis and reduced colonic mucosal polyamine level and ODC activity. Our results suggest that troglitazone, a synthetic PPARgamma ligand, can inhibit the early stage of colon tumorigenesis with or without colitis.     

Troglitazone, a Ligand for Peroxisome Proliferator-activated Receptor γ Inhibits Chemically-induced Aberrant Crypt Foci in Rats

The biological roles of peroxisome proliferator-activated receptors (PPARs) in various diseases, including inflammation and cancer, have been highlighted recently. Although PPARγ ligand is suspected to play an important role in carcinogenesis, its effects on colon tumorigenesis remain undetermined. The present tune-course study was conducted to investigate possible modifying effects of a PPARγ ligand, troglitazone, on the development and growth of aberrant crypt foci (ACF), putative precursor lesions for colon carcinoma, induced by azoxymethane (AOM) or dextran sodium sulfate (DSS) in male F344 rats. Oral troglitazone (10 or 30 mg/kg body weight (b.w.)) significantly reduced AOM (two weekly subcutaneous injections, 20 mg/kg b.w.)-induced ACF. Treatment with troglitazone increased apoptosis and decreased polyamine content and ornithine decarboxylase (ODC) activity in the colonic mucosa of rats treated with AOM. Gastric gavage of troglitazone also inhibited colitis and ACF induced by DSS (1% in drinking water), in conjunction with increased apoptosis and reduced colonic mucosal polyamine level and ODC activity. Our results suggest that troglitazone, a synthetic PPARγ ligand, can inhibit the early stage of colon tumorigenesis with or without colitis.     

Effects of timing and quantity of chronic dietary ethanol consumption on azoxymethane-induced colonic carcinogenesis and azoxymethane metabolism in Fischer 344 rats

Epidemiological studies have shown an association between consumption of alcoholic beverages and carcinoma of the large bowel, but studies in experimental models of colonic carcinogenesis have yielded conflicting results. We assessed the effects on azoxymethane-induced colonic carcinogenesis of both timing of chronic dietary ethanol consumption relative to carcinogen administration and quantity of ethanol consumption. Ten-week-old male Fischer 344 rats were given 11%, 22%, or 33% of calories as reagent ethanol or no ethanol by pair feeding with Lieber-DeCarli-type liquid diets providing comparable total carbohydrates, proteins, fats, and calories. Ten weekly s.c. injections of the bowel carcinogen azoxymethane (AOM), 7 mg/kg, were given to all rats in weeks 1-10. Three experimental groups were given their respective ethanol diet during acclimatization and AOM administration (preinduction and induction phases) and then were given the no-ethanol diet from week 11 until sacrifice in week 26 (postinduction phase). Three other groups received the no-ethanol diet during acclimatization and AOM administration and then were changed to their respective ethanol diet until sacrifice. The control AOM group received the no-ethanol diet throughout the study. Suppression of colonic tumorigenesis occurred in the groups with high levels of chronic dietary ethanol consumption during acclimatization and AOM administration: in the 33% and 22% diet groups, the prevalence of colonic tumors was 3% and 20% as compared with 50% in control (P less than 0.001 and P less than 0.02, respectively). Tumorigenesis in the left colon was more affected than in the right colon, as tumor prevalence in the left colon was decreased in both the 33% and 22% diet groups (0% in both versus 24% in control, P less than 0.005), whereas prevalence in the right colon was decreased only in the 33% diet group (3% versus 38%, P less than 0.001). By contrast, prevalence of colonic tumors in the 11% diet group was not significantly different from control. Chronic dietary ethanol consumption after AOM administration had no effect on tumor outcome, regardless of quantity of consumption. In an analogous study of [14C]AOM metabolism in rats fed the 33% diet during acclimatization and AOM administration, 14CO2 was exhaled at a slower rate than in rats fed no-ethanol diet (P = 0.05), indicating suppression of AOM metabolism.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of timing of administration and dose of difluoromethylornithine on rat colonic carcinogenesis

During azoxymethane (AOM)-induced colonic carcinogenesis in rats, biphasic induction of ornithine decarboxylase (ODC) activity occurs in the colonic mucosa. The relative contributions of these two phases of ODC induction to the carcinogenesis process were examined by studying the effects of the specific ODC inhibitor, difluoromethylornithine (DFMO), administered during either the initial phase of ODC increase or during both phases continuously. The effects of 1% and 0.25% DFMO administered continuously were also compared. Continuous oral administration of DFMO at 1% (approximately 8 mg/g body wt/wk) and 0.25% (approximately 2 mg/g body wt/wk) produced 93% inhibition of ODC induction by AOM in the right and left colons throughout the study. Despite suppression of ODC activity to near-normal levels, colon tumor incidence at 26 weeks in the right colon was not affected by either initial-phase or continuous administration of DFMO. In contrast, tumor incidence in the left colon was reduced from 35% to 5% by DFMO given continuously at doses of both 1% and 0.25% over the entire 26 weeks (P less than .05). No significant reduction in left colon tumor incidence resulted from the short initial 11-week course of DFMO although the tumor incidence was reduced (15% vs. 35%). Results suggest that the second ("post-initiation") phase of ODC induction may be of particular importance in carcinogenesis.     

Dietary seed oil rich in conjugated linolenic acid from bitter melon inhibits azoxymethane-induced rat colon carcinogenesis through elevation of colonic PPARgamma expression and alteration of lipid composition

Our previous short-term experiment demonstrated that seed oil from bitter melon (Momordica charantia) (BMO), which is rich in cis(c)9, trans(t)11, t13-conjugated linolenic acid (CLN), inhibited the development of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF). In our study, the possible inhibitory effect of dietary administration of BMO on the development of colonic neoplasms was investigated using an animal colon carcinogenesis model initiated with a colon carcinogen AOM. Male F344 rats were given subcutaneous injections of AOM (20 mg/kg body weight) once a week for 2 weeks to induce colon neoplasms. They also received diets containing 0.01%, 0.1% or 1% BMO for 32 weeks, starting 1 week before the first dosing of AOM. At the termination of the study (32 weeks), AOM induced 83% incidence (15/18 rats) of colonic adenocarcinoma. Dietary supplementation with 0.01% and 0.1% BMO caused significant reduction in the incidence (47% inhibition by 0.01% BMO, p<0.02; 40% inhibition by 0.1% BMO, p<0.05; and 17% inhibition by 1% BMO) and the multiplicity (64% inhibition by 0.01% BMO, p<0.005; 58% inhibition by 0.1% BMO, p<0.02; and 48% inhibition by 1% BMO, p<0.05) of colonic adenocarcinoma, though a clear dose response was not observed. Such inhibition was associated with the increased content of CLA (c9,t11-18:2) in the lipid composition in colonic mucosa and liver. Also, BMO administration in diet enhanced expression of peroxisome proliferator-activated receptor (PPAR) gamma protein in the nonlesional colonic mucosa. These findings suggest that BMO rich in CLN can suppress AOM-induced colon carcinogenesis and the inhibition might be caused, in part, by modification of lipid composition in the colon and liver and/or increased expression of PPARgamma protein level in the colon mucosa.     

Suppression of Azoxymethane-induced Rat Colon Aberrant Crypt Foci by Dietary Protocatechuic Acid

The modifying effect of dietary exposure to protocatechuic acid (PCA) on the development of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) was investigated in male F344 rats. The effects of PCA feeding on the silver-stained nucleolar organizer regions protein (AgNORs) count in the colonic epithelial cells and on the ornithine decarboxylase (ODC) activity in the colonic mucosa were also estimated. Animals were given weekly s.c. injections of AOM (15 mg/kg body weight) for 3 weeks to induce ACF. These rats were fed diet containing 1000 or 2000 ppm PCA for 5 weeks, starting one week before the first dosing of AOM. All rats were killed 2 weeks after the last AOM injection, to measure the number of ACF, ODC activity, and AgNORs count per nucleus in the colon. In rats given AOM and PCA, the frequency of ACF/colon was significantly decreased compared with that in rats given AOM alone ( P < 0.005 at 1000 and P < 0.05 at 2000 ppm). ODC activity in the colon of rats given AOM and PCA at both doses was also significantly lower than that of rats treated with AOM alone ( P < 0.05). Similarly, the mean AgNORs count in rats fed PCA was significantly smaller than that of rats treated with AOM alone ( P < 0.0001). Treatment with PCA alone did not affect these three biomarkers. These results provide further evidence that PCA could be a chemopreventive agent against rat colon carcinogenesis.     

Catalpa seed oil rich in 9t,11t,13c-conjugated linolenic acid suppresses the development of colonic aberrant crypt foci induced by azoxymethane in rats

Catalpa (Catalpa ovata) seed oil (CPO) is a unique oil that contains a high amount of 9trans,11trans,13cis-conjugated linolenic acid. In the present study, we investigated whether dietary administration with CPO affects the development of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) in male F344 rats to elucidate its possible cancer chemopreventive efficiency. Also, the effect of CPO on the fatty acid composition of liver tissue and colonic mucosa, the serum levels of total cholesterol and triglyceride, and the mRNA expression of cyclooxygenase (COX)-2 in the colonic mucosa were measured. In addition, the cell proliferation activity and apoptotic index in the colonic mucosa were estimated immunohistochemically. Animals were given two weekly subcutaneous injections of AOM (20 mg/kg body weight). They also received the experimental diet containing 0.01%, 0.1% or 1% CPO for 4 weeks, starting one week before the first dosing of AOM. AOM exposure produced a substantial number of ACF (99+/-28) at the end of the study (week 4). Dietary administration of CPO reduced the number of ACF (AOM + 0.01% CPO, 32+/-11, P<0.001; AOM + 0.1% CPO, 35+/-18, P<0.001; AOM + 1% CPO, 18+/-10, P<0.001). 9t,11t-conjugated linoleic acid was detected in the liver tissue and colonic mucosa of rats fed the CPO-containing diet. Additionally, dietary administration with CPO decreased the serum triglyceride level and the expression of COX-2 mRNA in the colonic mucosa. The indices of cell proliferation and apoptosis in the colonic mucosa of rats treated with AOM and 1% CPO have significant differences when compared with the AOM alone group. These findings suggest the possible chemopreventive activity of CPO in the early phase of colon carcinogenesis.     

Effects of chronic dietary beer and ethanol consumption on experimental colonic carcinogenesis by azoxymethane in rats

Epidemiological studies have shown an association between consumption of alcoholic beverages, particularly beer, and carcinoma of the large bowel, especially the rectum. We studied the effects of chronic dietary beer and ethanol consumption on experimental colonic carcinogenesis, fecal bile acid and neutral sterol levels, fecal bacterial flora, and colonic epithelial DNA synthesis. Ten-week-old male Fischer 344 rats were pair fed throughout the study with Lieber-DeCarli-type liquid diets providing comparable total carbohydrates, proteins, fats, and calories. The diets provided 23 or 12% of calories as alcohol in beer (Hi-Beer and Lo-Beer groups), 18 or 9% of calories as reagent ethanol (Hi-EtOH and Lo-EtOH groups), or no alcohol (control group). After 3 weeks of dietary acclimatization, 10 weekly s.c. injections of the bowel carcinogen azoxymethane, 7 mg/kg, were given (weeks 1-10). At necropsy in week 26, the high alcohol groups (Hi-Beer and Hi-EtOH) showed a significantly reduced incidence of tumors in the right colon (42 and 46% versus 81% in control, P less than 0.01 and P = 0.02) but no effect on left colonic tumorigenesis. By contrast, the low alcohol groups (Lo-Beer and Lo-EtOH) showed a trend toward increased incidence and proportion of tumors in the left colon (incidence of 42 and 35% versus 15% in control, P = 0.06 for Lo-Beer; 28 and 30% of tumors in left colon versus 11%, P = 0.08 and P = 0.07) but no effect on right colonic tumorigenesis. Numbers of right colonic tumors were inversely correlated with alcohol consumption of all rats (r = -0.350, P less than 0.001), but left colonic tumors were not correlated. Fecal bile acid and neutral sterol levels, fecal bacterial counts, and colonic epithelial DNA synthesis did not correlate with the effects of alcohol consumption on colonic tumorigenesis. Our findings suggest that: modulation of experimental colonic tumorigenesis by chronic dietary beer and ethanol consumption was due to alcohol rather than other beverage constituents; tumorigenesis in the right and left colon was affected differentially by the levels of alcohol consumption, reflecting complex interactions among the potential mechanisms for alcohol effects in the model used.     

Chemoprevention of colonic aberrant crypt foci by an inducible nitric oxide synthase-selective inhibitor

Inducible nitric oxide synthase (iNOS) is overexpressed in colonic tumors of humans and also in rats treated with a colon carcinogen. iNOS appear to regulate cyclooxygenase-2 (COX-2) expression and production of proinflammatory prostaglandins, which are known to play a key role in colon tumor development. Experiments were designed to study the inhibitory effects of S,S'-1,4-phenylene-bis(1,2-ethanediyl)bis-isothiourea (PBIT) a selective iNOS-specific inhibitor, measured against formation of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF). Beginning at 5 weeks of age, male F344 rats were fed experimental diets containing 0 or 50 p.p.m. of PBIT, or 2000 p.p.m. of curcumin (non-specific iNOS inhibitor). One week later, rats were injected s.c. with AOM (15 mg/kg body wt, once weekly for 2 weeks). At 17 weeks of age, all rats were killed, colons were evaluated for ACF formation and colonic mucosa was assayed for isoforms of COX and NOS activities. Both COX and iNOS activities in colonic mucosa of the AOM-treated rats were significantly induced. Importantly, 50 p.p.m. PBIT suppressed AOM-induced colonic ACF formation to 58% (P < 0.0001) and crypt multiplicity containing four or more crypts per focus to 78% (P < 0.0001); it also suppressed AOM-induced iNOS activity. Curcumin inhibited colonic ACF formation by 45% (P < 0.001). These observations suggest that iNOS may play a key regulatory role in colon carcinogenesis. Developing iNOS-specific inhibitors may provide a selective and safe chemopreventive strategy for colon cancer treatment.     

Azoxymethane-induced beta-catenin-accumulated crypts in colonic mucosa of rodents as an intermediate biomarker for colon carcinogenesis

It is now well established that bile acids act as colon tumor promoters. However, a previous study provided conflicting data showing that dietary exposure of cholic acid (CHA), a primary bile acid, inhibits the carcinogen-induced formation of aberrant crypt foci (ACF), possible preneoplastic lesions, in colonic mucosa of rodents. Recently we found beta-catenin-accumulated crypts (BCAC) in colonic mucosa of rats initiated with azoxymethane (AOM) and provided evidence that BCAC might be preneoplastic lesions independent from ACF. In the present study, we investigated the modifying effects of dietary CHA on the formation of BCAC as well as ACF in male F344 rats after exposure to AOM to determine if the differences in the effect of CHA on these lesions could account for this discrepancy. The results indicate that administration of CHA (0.5%) in the diet during the post-initiation phase significantly reduced the total number, multiplicity and size of ACF (P < 0.00001) in AOM-exposed colonic mucosa as reported previously. The number of ACF even with >4 aberrant crypts/focus was also decreased significantly (P < 0.0002), suggesting that the large ACF are little resistant to continuous feeding of 0.5% CHA diet. Interestingly, the dietary CHA significantly enhanced both the multiplicity (P < 0.002) and size (P < 0.00001), but not the incidence, of AOM-induced BCAC when compared with the control diet group. Importantly, the number of large BCAC with >6 crypts/lesion was increased significantly by the dietary CHA (P < 0.003). Our results support the concept that BCAC are precursors of colon tumors and indicate the usefulness of BCAC as intermediate biomarkers for colon carcinogenesis, although the methodology for their detection requires further improvement.     

The chemopreventive role of ursodeoxycholic acid in azoxymethane-treated rats: suppressive effects on enhanced group II phospholipase A2 expression in colonic tissue

Great interest has been focused on the chemoprevention of colonic carcinogenesis by oral administration of ursodeoxycholic acid (UDCA) because its administration reportedly reduces the incidence of colon cancer in animal experiments. To elucidate the precise role of UDCA in the chemoprevention of azoxymethane-induced colon carcinogenesis, we examined the expression levels of group II phospholipase A2 in the colonic tissue of UDCA-treated and untreated rats and correlated the levels with the findings of aberrant crypt foci, putative preneoplastic lesions. Twelve weeks after azoxymethane exposure, the total number of aberrant crypt foci in 0.4% UDCA-fed rats and 1% UDCA-fed rats was significantly decreased compared to the untreated animals. The mucosal concentrations of PGE2 and 6-keto PGF1alpha were significantly lower in the UDCA-treated rats than in untreated rats. In correlation with lowering, the enhanced activity, protein mass and mRNA levels of group II phospholipase A2 were significantly attenuated in the UDCA-treated animals. The chemopreventive role of UDCA in colon carcinogenesis may lie in its modulation of the arachidonate metabolism in colonic mucosa.     

Preventive effects of chrysin on the development of azoxymethane-induced colonic aberrant crypt foci in rats

The modifying effects of dietary feeding with chrysin (5,7-dihydroxyflavone) on the development of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) were investigated in male F344 rats. We also assessed the effect of chrysin on mitosis and apoptosis in 'normal appearing' crypts. To induce ACF, rats were given two weekly subcutaneous injections of AOM (20 mg/kg body weight). They also received an experimental diet containing chrysin (0.001 or 0.01%) for 4 weeks, starting 1 week before the first dose of AOM. AOM exposure produced a substantial number of ACF (73+/-13/rat) at the end of the study (week 4). Dietary administration of chrysin caused significant reduction in the frequency of ACF: 0.001% chrysin, 37+/-17/rat (49% reduction, P<0.001); and 0.01% chrysin, 40+/-10/rat (45% reduction, P<0.001). In addition, chrysin administration significantly reduced the mitotic index and significantly increased the apoptotic index in 'normal appearing' crypts. These findings might suggest a possible chemopreventive activity of chrysin in the early step of colon tumorigenesis through modulation of cryptal cell proliferation activity and apoptosis.     

Chemoprevention of Azoxymethane-induced Rat Colon Carcinogenesis by a Xanthine Oxidase Inhibitor, 1'-Acetoxychavicol Acetate

In our studies to find natural compounds with chemopreventive efficacy in foods, using azoxymethane (AOM)-induced colonic aberrant crypt foci and colonic mucosal cell proliferation as biomarkers, a x an thine oxidase inhibitor, 1′-acetoxychavicol acetate (ACA), present in the edible plant Languas galanga from Thailand was found to be effective. This study was conducted to test the ability of ACA to inhibit AOM-induced colon tumorigenesis when it was fed to rats during the initiation or post-initiation phase. Male F344 rats were given three weekly s.c. injections of AOM (15mg/kg body weight) to induce colonic neoplasms. They were fed diet containing 100 or 500 ppm ACA for 4 weeks, starting one week before the first dosing of AOM (the initiation feeding). The other groups were fed the ACA diet for 34 weeks, starting one week after the last AOM injection (the post-initiation feeding). At the termination of the study (week 38), AOM had induced 71% incidence of colonic adenocarcinoma (12/17 rats). The initiation feeding with ACA caused significant reduction in the incidence of colon carcinoma (54% inhibition by 100 ppm ACA feeding and 77% inhibition by 500 ppm ACA feeding, P=0.03 and P=0.001, respectively). The post-initiation feeding with ACA also suppressed the incidence of colonic carcinoma (45% inhibition by 100 ppm ACA feeding and 93% inhibition by 500 ppm ACA feeding, P=0.06 and P=0.00003, respectively). Such inhibition was dose-dependent and was associated with suppression of proliferation biomarkers, such as ornithine decarboxylase activity in the colonic mucosa, and blood and colonic mucosal polyamine contents. ACA also elevated the activities of phase II enzymes, glutathione S-transferase (GST) and quinone reductase (QR), in the liver and colon. These results indicate that ACA could inhibit the development of AOM-induced colon tumorigenesis through its suppression of cell proliferation in the colonic mucosa and its induction of GST and QR. The results confirm our previous finding that ACA feeding effectively suppressed the development of colonic aberrant crypt foci. These findings suggest possible chemopreventive ability of ACA against colon tumorigenesis.     

Intake of beer inhibits azoxymethane-induced colonic carcinogenesis in male Fischer 344 rats

Modulatory effects of beer consumption on azoxymethane (AOM)-induced rat colonic carcinogenesis in male Fischer 344 rats were investigated. Single cell gel electrophoresis assay indicated that DNA damage of colonocytes, induced by a single AOM injection (15 mg/kg body weight), was significantly reduced in rats fed beer or malt extract for 2 weeks. Examination of aberrant crypt foci (ACF) formation in colonic mucosa, induced by AOM (15 mg/kg body weight; twice weekly), revealed that feeding of beer during the whole experimental period of 5 weeks significantly reduced the number of ACF by 35%. In the post-initiation protocol, a reduction in ACF formation by 26% was not significant. The efficacy in inhibition of ACF formation varied with the brand of beer. ACF formation was significantly reduced in rats treated with freeze-dried beer (FD Beer), but not with ethanol, suggesting that nonvolatile components of beer are responsible for the reduction. Significant suppression of ACF formation was observed in groups treated with hot water extract of malt, especially with extracts of colored malts, although no reduction was observed by feeding with hops extract. A long-term experiment of 42 weeks indicated that intake of beer decreased tumor incidence by 22% and decreased the number of neoplastic lesions, including adenocarcinomas and adenomas, by 44%. These results suggest that components of beer have chemopreventive effects on colonic carcinogenesis induced by AOM and that intake of beer may contribute to a reduction in the risk of cancer susceptibility.     

Citrus auraptene inhibits chemically induced colonic aberrant crypt foci in male F344 rats

The modifying effect of dietary administration of auraptene isolated from the peel of citrus fruit (Citrus natsudaidai Hayata) on the development of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) was investigated in rats. Male F344 rats were given s.c. injections of AOM (15 mg/kg body wt) once a week for 3 weeks to induce ACF. They also received diets containing 100 or 500 p.p.m. auraptene for 5 weeks, starting 1 week before the first dose of AOM. At termination of the study (week 5) dietary administration of auraptene caused a significant reduction in the frequency of ACF in a dose- dependent manner (P < 0.05). Feeding of auraptene suppressed expression of cell proliferation biomarkers (5-bromo-2'-deoxyuridine labeling- index, ornithine decarboxylase activity, polyamine content and number of silver stained nucleolar organizer region protein particles) in the colonic mucosa and the occurrence of micronuclei caused by AOM. Also, auraptene increased the activities of phase II enzymes (glutathione S- transferase and quinone reductase) in the liver and colon. These findings might suggest that inhibition of AOM-induced ACF may be associated, in part, with increased activity of phase II enzymes in the liver and colon and suppression of cell proliferation in the colonic mucosa.      

Neo-angiogenesis and the premalignant micro-circulatory augmentation of early colon carcinogenesis

Spectroscopic techniques have demonstrated that in the microscopically normal mucosa, there is an increase in mucosal micro-circulation in patients harboring neoplasia elsewhere in the colon (i.e. marker of field carcinogenesis). However, the physiological and molecular basis of this early increase in blood supply (EIBS) has not been elucidated. We, therefore, investigated the microvessel density (MVD) and angiogenic gene expression in the premalignant colonic mucosa from the well-validated azoxymethane (AOM)-treated rat experimental model of colon carcinogenesis. Fisher 344 rats were treated with AOM (15 mg/kg i.p.) or saline and euthanized 14 weeks later (a time-point that precedes carcinoma development). Colon sections were studied for MVD via immunohistochemical assessment for CD31 and location was compared with optical assessment of mucosal hemoglobin with low-coherence enhanced backscattering spectroscopy (LEBS). Finally, we performed a pilot real-time PCR angiogenesis microarray (84 genes) from the microscopically normal colonic mucosa of AOM and age-matched saline treated rats. AOM treatment increased MVD in both the mucosa and submucosa of the rats (125% increase in mucosa; p<0.007, and 96% increase in submucosa; p<0.02) but the increase was most pronounced at the cryptal base consistent with the LEBS data showing maximal hemoglobin augmentation at 200-225 μm depth. Microarray analysis showed striking dysregulation of angiogenic and anti-angiogenic factors. We demonstrate, for the first time, that neo-angiogenesis occurs in the microscopically normal colonic mucosa and was accentuated at the bottom of the crypt. This finding has potential implications as a biomarker for risk-stratification and target for chemoprevention.     

The effect of an oral administration of Lactobacillus casei strain shirota on azoxymethane-induced colonic aberrant crypt foci and colon cancer in the rat

The preventive effect of oral administration of viable Lactobacillus casei strain Shirota (LcS) on azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) and colon cancers in the rat was investigated. The study consisted of two experiments; in a short-term experiment (Exp-I), the inhibitory effect of 8- and 12-week treatments with LcS. Forty rats each received weekly a subcutaneous injection of AOM at a dose of 15 mg/kg of body weight for 5 weeks. Eight and twelve weeks after the start of the carcinogen treatment, each subgroup of rats were sacrificed, and the colon and the mesenteric lymph nodes (MLN) were removed. The number of ACFs and the surface marker of lymphocytes derived from the MLN were investigated. The large ACF (those comprising four or more aberrant crypts per focus) had significantly decreased in the rats which had consumed the LcS diet. And oral administration of viable LcS significantly recovered CD8 positive lymphocytes to the levels in the control group. In a long-term experiment (Exp-II), 30 rats each received weekly a subcutaneous injection of AOM at a dose of 7. 4 mg/kg of body weight for 10 weeks. Twenty-five weeks after the start of the carcinogen treatment, each subgroup of rats were sacrificed, and the colon were removed. The number and incidence of colon cancers were investigated. The number of rats with colon cancers and the number of colon cancers per rat, were significantly decreased in the rats which had consumed the LcS diet. LcS inhibited chemically-induced colon carcinogenesis in the rat. CD8 positive T lymphocytes may play a key role in the preventive effect against colon carcinogenesis.     

Effect of dietary oligofructose and inulin on colonic preneoplastic aberrant crypt foci inhibition

Oligofructose and inulin, naturally-occurring fermentable chicory fructans, have been shown to stimulate the growth of bifidobacteria which are regarded as beneficial strains in the colon and inhibit colon carcinogenesis in the laboratory animal models. The present study was designed to determine the effect of oligofructose and inulin on the azoxymethane (AOM)-induced preneoplastic lesions such as aberrant crypt foci (ACF) formation in the colon of male F344 rats. At 5 weeks of age, groups of animals were fed the AIN-76A (control) and the experimental diets containing 10% oligofructose or inulin. At 7 weeks of age, all animals received s.c. injection of AOM dissolved in normal saline at a dose rate of 15 mg/kg body wt, once weekly for 2 weeks. The animals were necropsied 7 weeks after the last AOM injection, and the ACF were visualized under light microscopy in the formalin-fixed, unsectioned methylene blue-stained colons. They were distinguished by their increased size, more prominent epithelial cells and pericryptal space. The feeding of oligofructose or inulin significantly inhibited the ACF formation and the crypt multiplicity in the colon. The degree of ACF inhibition was more pronounced in animals fed inulin than in those fed oligofructose. The findings suggest that chicory fructan supplements inhibit ACF formation, an early preneoplastic marker of malignant potential in the process of colon carcinogenesis.