Inhibition by dietary curcumin of azoxymethane-induced ornithine decarboxylase, tyrosine protein kinase, arachidonic acid metabolism and aberrant crypt foci formation in the rat colon

The present study was designed to investigate the modulatoryrole of dietary curcumin on (i) azoxymethane (AOM)-induced ornithinedecarboxylase (ODC), tyrosine protein kinase (TPK) and arachidonicadd metabolism in liver and colonic mucosa of male F344 rats,(ii) in vitro arachidonic add metabolism in the liver and colonicmucosa and (iii) AOM-induced aberrant crypt foci (ACF) formationin the colon of F344 rats. At 5 weeks of age groups of animalswere fed one of the experimental diets containing 0 or 2000p.p.m. curcumin. Two weeks later all the animals except thevehicle-treated groups were given s.c. injections of AOM, 15mg/kg body wt, once weekly for 2 weeks. The animals intendedfor biochemical study were killed 5 days later and the colonicmucosa and liver were analyzed for ODC, TPK, lipoxygenase andcyclo-oxygenase metabolites. The animals intended for ACF studywere killed 9 weeks later and analyzed for ACF in the colon.The results indicated that in saline-treated animals dietarycurcumin significantly inhibited the ODC (P<0.001) and TPK(P<0.05) activities in the liver and colonic mucosa. Dietarycurcumin significantly decreased the levels of AOM-induced ODCactivity in the liver and colon (P< 0.0001) and TPK activityin the liver and colon (P<0.01–0.0001) and the formationof 5(S)-, 8(S)-, 12(S)-and 15(S)-hydroxyeicosatetraenoic acids(HETEs) in the liver and colon (P< 0.0001). Also, curcuminsuppressed AOM-induced prostaglandin (PG) and thromboxane (Tx)formation in the liver (PGE₂, PGF₂     

Chemoprevention of azoxymethane-induced intestinal carcinogenesis by a novel synthesized retinoidal butenolide, 5-hydroxy-4-(2-phenyl-(E)-ethenyl)-2(5H)-furanone, in rats

The present study was designed to investigate the modifyingeffects of dietary 5-hydroxy-4-(2-phenyl-(E)-ethenyl)-2(5H)-furanone(KYN-54), a new synthetic retinoidal butenolide, during thepost-initiation phase on azoxymethane (AOM)-induced rat intestinalcarcinogenesis. The number of aberrant crypt foci (ACF) in ratcolon, colonic ornithine decarboxylase (ODC) activity and bromodeoxy-uridine(BrdUrd) labeling index in rat colonic epithelium were alsoassessed. At 7 weeks of age, male F344 rats (except the KYN-54alone and control groups) were given weekly s.c. injectionsof AOM at 15 mg/kg body wt for 3 weeks. Starting 1 week afterthe last injection of AOM, rats (except the control group) werefed a diet containing KYN-54 at concentrations of 100 or 200p.p.m. throughout the experiment All animals were necropsiedat 32 weeks after the start of the experiment. Compared withthe AOM alone group, KYN-54 at both doses reduced the incidenceand multiplicity of tumors in entire intestine (small and largeintestines). In the 200 p.p.m. KYN-54 fed group especially,tumor incidence and multiplicity in the entire intestine werelower compared with the AOM alone group (P < 0.005 and P< 0.05 respectively). Also, the number of ACF/cm² colon inthe groups of rats treated with AOM and KYN-54 at both doseswere significantly lower than that of rats treated with AOMalone (^P < 0.05). Colonic ODC activity and BrdUrd labelingindex in the groups of rats treated with AOM and KYN-54 at bothdoses were slightly lower than those treated with AOM alone.KYN-54 at 200 p.p.m. significantly lowered BrdUrd labeling indexinduced by AOM (P < 0.005). These results suggest that KYN-54might be a promising chemopreventive agent for intestinal neoplasia.     

Chemoprevention of colonic aberrant crypt foci in Fischer rats by sulforaphane and phenethyl isothiocyanate

Epidemiological studies have linked consumption of broccolito a reduced risk of colon cancer in individuals with the glutathioneS-transferase M1 (GSTM1) null genotype. GSTs are involved inexcretion and elimination of isothiocyanates (ITCs), which aremajor constituents of broccoli and other cruciferous vegetablesand have cancer chemopreventive potential, so it is speculatedthat ITCs may play a role in protection against human coloncancer. However, there is a lack of data from animal studiesto support this. We carried out a bioassay to examine whethersulforaphane (SFN) and phenethyl isothiocyanate (PEITC), majorITCs in broccoli and watercress, respectively, and their correspondingN-acetylcysteine (NAC) conjugates, show any chemopreventiveactivity towards azoxymethane (AOM)-induced colonic aberrantcrypt foci (ACF) in F344 rats. Groups of six male F344 ratswere treated with AOM subcutaneously (15 mg/kg body wt) onceweekly for 2 weeks. SFN and PEITC and their NAC conjugates wereadministered by gavage either three times weekly for 8 weeks(5 and 20 µmol, respectively) after AOM dosing (post-initiationstage) or once daily for 3 days (20 and 50 µmol, respectively)before AOM treatment (initiation stage). The bioassay was terminatedon week 10 after the second AOM dosing and ACF were quantified.SFN, SFN-NAC, PEITC and PEITC-NAC all significantly reducedthe formation of total ACF from 153 to 100–116 (P <0.01) and multicrypt foci from 52 to 27–38 (more thanfour crypts/focus; P < 0.05) during the post-initiation treatment.However, only SFN and PEITC were effective during the initiationphase, reducing the total ACF from 153 to 109–115 (P <0.01) and multicrypt foci from 52 to 35 (more than four crypts/focus;P < 0.05). The NAC conjugates were inactive as anti-initiatorsagainst AOM-induced ACF. These findings provide important laboratoryevidence for a potential role of SFN and PEITC in the protectionagainst colon cancer.     

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.     

Increased induction of aberrant crypt foci by 1,2-dimethylhydrazine in rats fed diets containing purified genistein or genistein-rich soya protein

The isoflavonoid genistein inhibits mitosis and increases apoptosisin a variety of tumour cell lines in vitro, and may exert anticarcinogeniceffects in vivo. To assess its effects on the colon, rats werefed a semi-synthetic control diet, or similar diets enrichedwith genistein (0.25 g/kg), either as the pure isoflavone oras part of a soya protein isolate, for 7 days before receivingsubcutaneous injections of saline or 1,2-dimethylhydrazine (DMH).After 48 h, rats given saline were killed and samples of theirsmall and large intestinal mucosa were obtained for assessmentof crypt cell mitosis and apoptosis by visual analysis of isolatedintact crypts. Rats given DMH were fed control diet and killedafter 48 h for assessment of crypt cytokinetics or maintainedfor 42 days then killed and their colonic mucosa analysed foraberrant crypt foci (ACF). Two further groups were given controldiet before DMH, followed by the genistein or soya-based dietfor 42 days before assessment of ACF. Neither genistein norsoya protein isolate had a significant effect on crypt cellmitosis or apoptosis in untreated rats, or on the proliferativeresponse to treatment with DMH. However, consumption of puregenistein or the soya protein isolate before treatment withDMH was associated with a 3-fold (P < 0.001) or 2-fold (P< 0.05) increase, respectively, in ACF in the distal colon.There was no significant effect of genistein or soya proteinisolate given after DMH treatment. We conclude that genisteinhas no detectable effect on colonic crypt mitosis or apoptosisin the rat in vivo, but that it promotes induction of ACF byan as yet undefined mechanism when fed immediately before treatmentwith DMH.     

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.     

Sequential analysis of K-ras mutations in aberrant crypt foci and colonic tumors induced by azoxymethane in Fischer-344 rats on high-risk diet

Forty Fischer-344 male rats were given a high-risk diet (HRD)that was high in fat, low in fiber and low in calcium. After4 weeks, the rats were given two weekly s.c. injections of azoxymethane(AOM, 15 mg/kg body wt), and remained on the same diet tilldeath. Eight rats were killed at 12 weeks and again at 20 weeksin order to microdissect aberrant crypt foci (ACF containingfour or more crypts/focus from their colons. The remaining 24rats were killed at 30 weeks to harvest colonic tumors. Thepolymerase chain reaction (PCR) was used to amplify specificDNA segments in the K-ras gene from ACF and colonic tumors.The PCR-amplified DNAs were sequenced to identify the pointmutations in codons 12 and 13. All the mutations detected inthe ACF and colonic tumors were G to A transitions in the secondposition of codon 12. These mutations were present in the ACFof 2/8 (25%) and 3/8 (37%) rats at 12 and 20 weeks respectively.The mutations were present in colonic tumors of 7/24(29%) rats.These results provide important evidence for the significanceof K-ras mutations in ACF (>4 crypts/focus) as early markersof malignant potential in the colons of F344 rats exposed toAOM while receiving a high-risk western style diet.     

Initiation and promotion of colonic aberrant crypt foci in rats by 5-hydroxymethy1-2-furaldehyde in thermolyzed sucrose

We have previously shown that thermolyzed sucrose in the dietpromotes the growth of aberrant crypt foci (ACF) in the rat.HPLC analysis of the light caramel colored product showed thatit contained 1% 5-hydroxymethy1-2-furaldehyde (HMF), confirmedby mass and NMR spectroscopy. To determine whether HMF was responsiblefor the promotion of ACF by thermolyzed sucrose, 45 F344 femalerats were initiated with the colon carcinogen azoxymethane (AOM),and a week later were randomized to four groups receiving AIN-76diets containing untreated sucrose, 20% thermolyzed sucrose,20% butanol extracted thermolyzed sucrose (HMF free) or 1% HMF.Thermolyzed sucrose in the diet led to larger ACF as previouslyobserved. Thermolyzed sucrose extracted to remove HMF, did notaffect ACF size, but 1% HMF added to the diet led to a largerACF both with relation to average size and number of ACF oflarger sizes (P < 0.05). To determine whether HMF had initiatingeffects, 172 female F344 rats were given water, HMF (at dosesto 300 mg/kg) or AOM (5 mg/kg) by gavage twice and the totalnumber of ACF was scored 30 days later. The results demonstratedthat HMF induces ACF in a dose-dependent manner (P < 0.02),though the effect was much weaker than that of AOM. We concludethat sugar heated under household cooking conditions may actas both an initiator and a promoter of colon cancer becauseof the presence of HMF.     

Transgenic expression of human MGMT protects against azoxymethane-induced aberrant crypt foci and G to A mutations in the K-ras oncogene of mouse colon

Transgenic mice over-expressing MGMT, which codes for the humanprotein O⁶-alkylguanine-DNA alkyltransferase, are protectedfrom methylating agent-induced thymic lymphomas. In this studywe evaluated the ability of transgenic overexpression of MGMTin the colon to protect mice from the development of azoxymethane(AOM)-inducedaberrant crypt foci (ACF) and mutations in K-ras. Colonic alkyltransferasein MGMT+ transgenic mice was > 5-fold higher than in nontransgenics:10.5 ± 1.1 vs 2.2 ± 1.1 fmol/µg DNA, P => 0.0001. Mice received 20 mg AOM/kg i.p. at 6 weeks or 15mg AOM/kg at 6 and 7 weeks of age, and 8 wks later colons wereexamined for ACF. A significant protective effect of MGMT wasseen in mice given single dose of 20 mg AOM/kg. The incidenceof ACF/colon was lower in MGMT+ mice (2.0 ± 1.2) thanin nontransgenic mice (3.9 ± 1.8, P = 0.02). G to A mutationsin codon 12 of K-ras were detected by PCR-RFLP in ACF and inrandom samples of normal appearing mucosa. The incidence ofACF with mutant K-ras in MGMT transgenic mice (0.6 ±0.7/colon) was significantly reduced compared to nontransgenicmice (2.3 ± 1.7/colon, P = 0.02). We propose that AOMinduces at least two overlapping but not identical premalignantlesions (aberrant crypt foci and K-ras mutations) which canbe prevented by over-expression of MGMT. Thus, MGMT may protectcolonic mucosa from carcinogenesis involving methylating agentssuch as AOM.     

Chemopreventive effect of 2-(allylthio)pyrazine (2-AP) on rat colon carcinogenesis induced by azoxymethane (AOM)

An investigation was conducted to assess the chemopreventive effects of 2-(allylthio)pyrazine (2-AP), synthesized for potential use as a chemopreventive agent, after administration during the pre-initiation and post-initiation stages in a rat colon carcinogenesis model with azoxymethane (AOM). One hundred, 5-week-old, male F344 rats were randomly divided into two experiments (n = 50 each). Experiment 1 rats were randomly divided into three groups: Group 1 rats were pre-treated with 2-AP (25 or 50 mg/kg body weight, 3 consecutive days through the route of intragastric intubations) before AOM (20 mg/kg body weight, single subcutaneous (s.c.) injection) initiation. Group 2 rats were treated with AOM alone. Group 3 rats were given 2-AP alone without AOM initiation. The animals were killed at the end of each experiment (week 5) and the aberrant crypt foci (ACF) of the colonic mucosa were assessed after staining with methylene blue. Experiment 2 rats were randomly divided into three groups: Group 1 rats were given 2-AP (10, 25 or 50 mg/kg body weight, five-times intragastric intubations per week for 5 weeks from week 3) after AOM (15 mg/kg body weight, three s.c. injections) initiation for 2 weeks. Group 2 rats were treated with AOM alone. Group 3 rats were given 2-AP alone without AOM initiation. The animals were killed at the end of the experiment (week 8) and the ACF of the colonic mucosa were quantified. Total numbers of ACF/colon in Group 1 rats (pre-treated with 2-AP) tended to decrease (2-AP, 50 mg/kg body weight) or increase (2-AP, 100 mg/kg body weight) depending on the dose level. Total numbers of ACF/colon in Group 1 rats (treated with AOM followed by 2-AP, all subgroups; 160.8 +/- 38.0; 161.8 +/- 38.1; 137.1 +/- 48.4) were decreased significantly compared with the values in Group 2 rats (AOM alone; 214.8 +/- 48.1) (P < 0.05 or 0.01). The highest dose group (2-AP, 50 mg/kg body weight) had the lowest levels of total numbers of ACF/colon among the three subgroups. Total numbers of aberrant crypts (AC)/colon of the highest dose group (340.1+/- 117.9) decreased significantly compared with the value for Group 2 rats (AOM alone; 545.1 +/- 38.3). These results thus suggest that 2-AP may have potential as a chemopreventive agent against rat colon carcinogenesis after administration of AOM during the post-initiation stage.     

Dietary‐feeding of grape seed extract prevents azoxymethane‐induced colonic aberrant crypt foci formation in fischer 344 rats

Chemoprevention by dietary agents/supplements has emerged as a novel approach to control various malignancies, including colorectal cancer (CRC). This study assessed dietary grape seed extract (GSE) effectiveness in preventing azoxymethane (AOM)‐induced aberrant crypt foci (ACF) formation and associated mechanisms in Fischer 344 rats. Six‐week‐old rats were injected with AOM, and fed control diet or the one supplemented with 0.25% or 0.5% (w/w) GSE in pre‐ and post‐AOM or only post‐AOM experimental protocols. At 16 wk of age, rats were sacrificed and colons were evaluated for ACF formation followed by cell proliferation, apoptosis, and molecular analyses by immunohistochemistry. GSE‐feeding caused strong chemopreventive efficacy against AOM‐induced ACF formation in terms of up to 60% (P < 0.001) reduction in number of ACF and 66% (P < 0.001) reduction in crypt multiplicity. Mechanistic studies showed that GSE‐feeding inhibited AOM‐induced cell proliferation but enhanced apoptosis in colon including ACF, together with a strong decrease in cyclin D1, COX‐2, iNOS, and survivin levels. Additional studies showed that GSE‐feeding also decreased AOM‐caused increase in β‐catenin and NF‐κB levels in colon tissues. Compared to control animals, GSE alone treatment did not show any considerable change in these biological and molecular events in colon, and was nontoxic. Together, these findings show the chemopreventive efficacy of GSE against the early steps of colon carcinogenesis in rats via likely targeting of β‐catenin and NF‐κB signaling, and suggest its potential usefulness for the prevention of human CRC. © 2010 Wiley‐Liss, Inc.     

Prevention of colonic aberrant crypt foci and modulation of large bowel microbial activity by dietary coffee fiber, inulin and pectin

The present experiments were aimed at developing novel dietary fibers to aid in reduction of colon cancer risk. We assessed the effects of coffee (non-fiber fraction), coffee fiber (arabino-galactose polymer) and inulin (oligo-fructose) in male F344 rats using formation of azoxymethane (AOM)-induced aberrant crypt foci (ACF) in the colon as the measure of preventive efficacy (or lack of such). At 5 weeks of age, groups of rats were fed the AIN-76A (control) and experimental diets that contained 1% coffee, 10% coffee fiber, 10% inulin, 10% pectin (positive control for fiber) or 200 p.p.m. piroxicam (a known ACF inhibitor). At 7 weeks of age, all animals were s.c injected with AOM (15 mg/kg body wt) once weekly for 2 weeks. All rats were killed 8 weeks after the last AOM injection and ACF were counted. The contents of the cecum were analyzed for bacterial beta-glucuronidase activity and short-chain fatty acids (SCFAs). Dietary administration of coffee fiber significantly suppressed AOM-induction of colonic ACF, in terms of total number, as well as crypt multiplicity and number of ACF/cm2 colon (P < 0.01-0.001). Inulin diet had no significant effect on total ACF, but had reduced the number of ACF/cm2 (P < 0.05). Whereas coffee had no effect on ACF formation, 10% pectin diet and 200 p.p.m. piroxicam significantly suppressed colonic ACF (P < 0.001) as had been expected. A significant reduction of cecal beta-glucuronidase activity was observed in the rats fed coffee, coffee fiber and pectin diets. Further, coffee fiber, inulin and pectin increased cecal SCFA levels 3- to 5-fold. These results suggest that coffee fiber can prevent colon cancer risk. Further studies are warranted to determine the full potential of this fiber in pre-clinical efficacy studies.      

Prevention of colonic preneoplastic lesions by the probiotic Lactobacillus acidophilus NCFMTM in F344 rats

The experiments described here were aimed at developing novel probiotic strains that may aid in the reduction of colon cancer risk. We assessed the potential anticancer properties of Lactobacillus acidophilus NCFMTM in male F344 rats using inhibition of the formation of azoxymethane (AOM)-induced aberrant crypt foci (ACF) in the colon as the measure of preventive efficacy. At 6 weeks of age, groups of rats were fed the experimental diets containing 0, 2% or 4% lyophilized cultures of L. acidophilus NCFMTM. At 7 weeks of age, all animals in each dietary group, except the vehicle-treated rats, were s.c. injected with AOM (15 mg/kg body weight) once weekly for two weeks. The vehicle-treated groups were given s.c. injections of normal saline. All rats were necropsied 10 weeks after the last AOM injection and ACF in formalin-fixed, methylene blue-stained colonic tissues were counted under the light microscope. The contents of the cecum were analyzed for bacterial beta-glucuronidase activity. Diet supplementation with the probiotic strain NCFMTM significantly suppressed AOM-induction of colonic ACF, in terms of total number, as well as crypt multiplicity and number of ACF/cm2 colon (P<0.01 - 0.001). NCFMTM inhibited AOM-induced colonic ACF formation in a dose-dependent manner (P<0.01). A significant dose-dependent reduction of cecal beta-glucuronidase activities was observed in the rats fed 2% (P<0.04) and 4% (P<0.0001) NCFMTM. These results suggest that Lactobacillus acidophilus NCFMTM may potentially prevent colon cancer development. Further studies are warranted to determine the full potential of this probiotic strain in preclinical efficacy studies.     

Modifying effects of Terminalia catappa on azoxymethane-induced colon carcinogenesis in male F344 rats.

The modifying effects of dietary administration of an herb, Terminalia catappa (TC), were investigated on rat colon carcinogenesis induced by a carcinogen azoxymethane (AOM). The number of aberrant crypt foci (ACF) and beta-catenin accumulated crypts (BCACs) in the colon, and proliferating cell nuclear antigen (PCNA) labelling index in the colonic epithelium were examined in a total of 36 male F344 rats. All animals were randomly divided into five experimental groups (4-10 rats in each group). At 6 weeks of age, rats in groups 1, 2 and 3 were given s.c. injections of AOM once a week for 2 weeks at a concentration of 20 mg/kg body weight. One week before the first injection of AOM, rats in groups 2 and 3 were fed a diet containing 0.02 and 0.1% TC, respectively, throughout the experiment. Rats in group 4 were fed a diet containing 0.1% TC. Rats in group 5 were served as untreated controls. All animals were sacrificed at the experimental week 5 after the start of the experiment. Oral administration of TC at both doses significantly decreased the numbers of both ACF/colon/rat (P<0.05 for 0.02% TC, P<0.005 for 0.1% TC) and BCAC/cm/rat (P<0.05 for both 0.02 and 0.1% TC), when compared with the control group (group 1). Colonic PCNA labelling index in groups 2 and 3 was also significantly lower than that in group 1 (P<0.001 for 0.02% TC, P<0.005 for 0.1% TC). These results suggest that TC has a potent short-term chemopreventive effect on biomarkers of colon carcinogenesis and this effect may be associated with the inhibition of the development of ACF and BCACs.     

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.     

Increased Cell Proliferation of Azoxymethane-induced Aberrant Crypt Foci of Rat Colon

Aberrant crypt foci (ACF) were induced in the colon of F344 rats by s.c. injection of azoxymethane (AOM) twice in a three day-interval and examined after 4 and 12 weeks. The number and crypt multiplicity of ACF in each section of rat colon increased during this period. Histologically, aberrant crypts consisted of proliferating atypical epithelial cells. Cell proliferation of ACF consisting of 4 aberrant crypts [ACF(4)] and 2 aberrant crypts [ACF(2)], and normal crypts in the colon of rats treated with AOM [normal crypts/AOM(+)] or saline [normal crypts/AOM(-)] was investigated by measurement of the mitotic index, proliferating cell nuclear antigen-labeling index (PCNA-LI), and 5-bromo-2'-deoxyuridine-labeling index (BrdU-LI). All three parameters of the cell proliferative activity of ACF(4) were higher than those of normal crypts/AOM(+) and normal crypts/AOM(-). The PCNA-LI and BrdU-LI in ACF(2) were the same as those in ACF(4). These findings suggest that ACF have increased cell proliferative activity. The correlation of these three parameters confirmed that the PCNA-LI is also a useful parameter for evaluating cell proliferative activity in ACF. The presence of many cells stained by PCNA in the upper portion of ACF suggested that ACF have more G₁ phase cells, which readily respond to mitogenic stimulation, than G₀ phase cells, which are predominant in normal crypts.     

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.     

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.     

Modifying effect of tuna orbital oil rich in docosahexaenoic acid and vitamin D3 on azoxymethane-induced colonic aberrant crypt foci in rats

The modifying effect of dietary tuna (Thunnus thynnus orientalis) orbital oil rich in docosahexaenoic acid (DHA) and vitamin D3 (VD3) on the development of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) was investigated in male F344 rats. Animals were given three weekly subcutaneous injections of AOM (15 mg/kg body weight) to induce ACF. The rats were fed the experimental diet containing 5% tuna orbital oil (low fish oil), 23.5% tuna orbital oil (high fish oil), 5% corn oil (low corn oil) or 23.5% corn oil (high corn oil) for 5 weeks, starting 1 week before the first dose of AOM. Animals were sacrificed 2 weeks after the last AOM injection to count colonic ACF and assay the expression of cyclooxygenase (COX)-1 and -2. High corn oil diet significantly increased the development of ACF, when compared with low corn oil diet (P<0.005). High fish oil diet also increased ACF formation compared with low fish oil diet (P<0.01), but the increase was smaller than high corn oil diet. The frequency of ACF was significantly lower in the rats fed high fish oil diet than high corn oil diet (P<0.02). Moreover, frequency of ACF consisted of 4 or more crypts in rats fed the high fish oil diet was significantly lower than that of rats given high corn oil diet. COX-1 and COX-2 expression did not significantly differ among the groups. These results suggest that fish oil derived from tuna, which contains high amounts of DHA and VD3, suppresses the formation and growth of ACF without affecting COX-1 and COX-2 expression, and may have a preventive effect on colon carcinogenesis.