Dietary n-3 PUFA increases the apoptotic response to 1,2-dimethylhydrazine, reduces mitosis and suppresses the induction of carcinogenesis in the rat colon

The effect of dietary fish oil on colonic crypt cell apoptosis and proliferation was examined in male Wistar rats, 24 and 48 h after administration of 1,2-dimethylhydrazine (DMH), and its influence on the induction of aberrant crypt foci (ACF) in the distal colon was assessed. Rats (125-150 g) fed a high-fat semi-synthetic diet containing corn oil (CO) were given DMH (30 mg/kg body wt) or a sham injection of EDTA/NaCl. Animals were then fed either the CO diet or a diet in which fish oil (EPA 18.7%; DHA 8%) was substituted for corn oil. Subgroups of rats (n = 5) were killed after 24 and 48 h, and crypt cell apoptosis and proliferation were quantified by morphological criteria in isolated intact crypts from the mid and distal colon. Consumption of the fish oil diet (FO) was associated with increased apoptotic cell death (P < 0.001) and suppression of proliferation (P < 0.05) in colonic crypts both 24 and 48 h after DMH. In a second experiment, animals were given three injections of DMH or sham injections of carrier at weekly intervals. For 48 h after each injection animals were fed either the CO or FO diet, but otherwise maintained on the CO throughout. The number and crypt multiplicity of ACF in the distal colon were determined after 18 weeks, and animals given the FO diet for the 48 h period following carcinogen administration were found to have significantly fewer ACF than rats fed the CO diet (P < 0.05). The data demonstrate that the fatty acid composition of the diet is an important determinant in the induction of carcinogenesis by DMH. The proliferative and apoptotic response of the colonic crypt to carcinogen and fish oil, coupled with the reduced incidence of ACF, suggest n-3 PUFA can protect against the carcinogenic effects of DMH by mediating changes in the balance proliferation and cell death.     

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.     

Chemoprevention of 1,2-dimethylhydrazine-induced colonic preneoplastic lesions in Fischer rats by 6-methylsulfinylhexyl isothiocyanate, a wasabi derivative

The preventive effects of dietary exposure to a wasabi derivative 6-methylsulfinylhexyl isothiocyanate (6-MSITC) during the initiation and post-initiation phases on the development of 1,2-dimethylhydrazine (DMH)-induced colonic aberrant crypt foci (ACF), and β-catenin-accumulated crypts (BCAC) were investigated in male F344 rats. To induce ACF and BCAC, rats were given four weekly subcutaneous injections of DMH (40 mg/kg body weight). The rats also received diets containing 200 or 400 ppm 6-MSITC during the initiation or post-initiation phases. The experiment was terminated 12 weeks after the start. DMH exposure produced a substantial number of ACF (323.8±69.7/colon) and BCAC (3.80±1.05/cm(2)) at the end of the study. Dietary administration of 6-MSITC at a dose of 400 ppm during the initiation phase caused a significant reduction in the total number of ACF (52% reduction, P<0.0001), larger ACF (4 or more crypt ACF) (58% reduction, P<0.001) and BCAC (76% reduction, P<0.00001). The dietary exposure to 6-MSITC significantly reduced the size (crypt multiplicity) of BCAC during both initiation and post-initiation treatment when compared to group 1 treated with DMH alone. Immunohistochemically, 6-MSITC administration lowered the proliferating cell nuclear antigen labeling index in ACF and BCAC. In addition, protein levels of hepatic cytochrome P-450 isozymes at 24 h after 6-MSITC exposure were significantly suppressed (P<0.01). The results indicated that 6-MSITC exerted chemopreventive effects in the present short-term colon carcinogenesis bioassay, through alterations in cell proliferation activity and drug metabolizing enzyme levels.     

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.     

Inhibition of 1, 2-Dimethylhydrazine-Induced Mucin-Depleted Foci and O6-Methylguanine DNA Adducts in the Rat Colorectum by Boiled Garlic Powder

The scavenging capacity of reactive oxygen species, such as hydroxyl radicals, is reported not to decreasein boiled garlic (an odorless garlic preparation). We therefore examined the modifying effect of boiled garlicpowder (BGP) on 1,2-dimethylhydrazine-induced mucin-depleted foci (MDF) and aberrant crypt foci (ACF),preneoplastic lesions, in the rat colorectum. Male F344 rats (5 weeks old) were fed a basal diet, or experimentaldiets containing 5% or 1% BGP for 5 weeks. One week later, all rats were injected s.c. with DMH (40 mg/kg, onceweekly for 2 weeks). At 10 weeks of age, all the rats were sacrificed, and the colorectum was evaluated for MDFand ACF. In rats given DMH and the 5% or 1% BGP diets (Groups 2 and 3), the numbers of MDF decreasedsignificantly in a dose-dependent manner, compared with the DMH and basal diet value (Group 1) (p<0.01). Thenumbers of ACF in Group 2, but not Group 3, showed a non-significant tendency to decrease. Next, the effectsof BGP on the formation of DMH-induced O6-methylguanine (O6-MeG) DNA adducts in rats were studied. MaleF344 rats (5 weeks old) were fed the basal diet, or 10% BGP diet for 5 weeks. All rats were injected i.p. once with40 mg/kg DMH at the end of week 5. The animals were sacrificed 6 hours after DMH injection to analyze theO6-MeG DNA adducts in the colorectal mucosa. Dietary administration of BGP significantly inhibited the O6-MeG DNA adduct levels in the colorectal mucosa, compared with the controls (p<0.01). These results suggestedthat BGP may exert chemopreventive effects against colon carcinogenesis at least in the initiation stage.     

Growth features of aberrant crypt foci that resist modulation by cholic acid

Previously, we demonstrated that feeding rats a diet containing0.2% chotic acid (CHA-diet) resulted in the elimination or remodellingof a number of aberrant crypt foci (ACF) in their primal stages(1–3 crypts/focus). The Present investigation was conductedto determine if ACF with advanced growth features will responddifferently than their primal counterparts to the CHA-diet.Sprague-Dawley male rats were injected with azoxymethane (20mg/kg)and were maintained on a control diet for 21 weeks. At week21, three rats were killed and their colons were assessed forACF. The remaining animals were randomly devided into two groups,which were fed a control diet or a CHA-diet respectively, After3 weeks of feeding, the rats (n = 5) were killed and their colonswere assessed for the number, size (area occupied by each focus)and crypt multiplicity (number of crypts/focus). The CHA-dietresulted in a significant (P     

Inhibition of dimethylhydrazine-induced aberrant crypt foci and induction of apoptosis in rat colon following oral administration of the glucosinolate sinigrin

Glucosinolates are sulphur compounds that occur as glycosides in brassica vegetables. In response to tissue disruption they are degraded by thioglucosidase, releasing a range of highly reactive breakdown products, including the isothiocyanates, which we have previously shown to be selectively cytotoxic to undifferentiated colorectal tumour cells (HT29). In the present study we explored the effect of sinigrin on the intestinal mucosa of rats previously treated with dimethylhydrazine (DMH). In the first experiment, a semisynthetic feed containing sinigrin (400 microg/g diet) was provided 6 h after the second of two injections of DMH. The level of apoptosis was measured by morphological assessment of intact microdissected crypts obtained at 18, 24, 38, 48 and 72 h after injection, and compared with control groups given DMH only, or a sham-injection. Higher numbers of apoptotic nuclei were present in colonic tissue from both groups of DMH-treated rats compared with the controls, and the level was significantly higher in DMH- treated rats fed sinigrin compared with those given DMH only (P < 0.02). In a second experiment, rats were given sinigrin (400 microg/g diet) 22 h after the second of two injections of DMH; the level of apoptosis was measured after 48 h and the numbers of aberrant crypt foci (ACF) were measured after 42 days. The level of apoptosis was significantly higher in DMH-treated rats given sinigrin compared with controls (P < 0.05), and the numbers of ACF were significantly lower in sinigrin-treated rats (P < 0.001). There was no statistically significant induction of apoptosis in animals fed sinigrin alone. Sinigrin administered after DMH suppresses induction of ACF. This may be due to increased apoptotic deletion of damaged stem cells in the crypts of animals fed sinigrin.      

Inhibitory Effects of High Temperature- and Pressure-Treated Garlic on Formation of 1,2-Dimethylhydrazine-Induced Mucin-Depleted Foci and O6-Methylguanine DNA Adducts in the Rat Colorectum

High temperature- and pressure-treated garlic (HTPG) has been reported to have enhanced antioxidativeand cytotoxic activities. However, there have been no reports on chemopreventive effects using animal cancermodels. This study first examined the modifying effects of HTPG on 1,2-dimethylhydrazine (DMH)-inducedmucin-depleted foci (MDF) and aberrant crypt foci (ACF), preneoplastic lesions in the rat colorectum. MaleF344 rats (5 weeks old) were fed basal diet, or experimental diets containing 1% or 3% HTPG for 5 weeks. Oneweek later, all rats were injected s.c. with DMH (40 mg/kg, once weekly for 2 weeks). At 10 weeks of age, all therats were sacrificed, and the colorectum was evaluated for MDF and ACF. In rats given DMH and 3% HTPG,the numbers of MDF were decreased significantly as compared with those of rats given DMH alone (p<0.01),and the numbers of ACF showed a tendency to decrease, although not significantly. Next, the effects of HTPGon the formation of DMH-induced O6-methylguanine (O6-MeG) DNA adducts in rats were studied. Male F344rats (5 weeks old) were fed the basal diet or 10% HTPG diet for 5 weeks. All rats were injected i.p. once with 40mg/kg DMH at the end of week 5. The animals were sacrificed 6 hours after DMH injection to analyze the O6-MeG DNA adducts in the colorectal mucosa and liver. Dietary administration of HTPG significantly reducedthe adduct levels in the colorectal mucosa and liver, compared with the controls (both p<0.01). The activities ofsome detoxification enzymes in the liver of DMH-treated rats were also measured. HTPG significantly reducedthe activity of cytochrome P450 (CYP) 2E1, known to be responsible for activation of DMH in rat liver (p<0.05).In contrast, HTPG significantly enhanced the activities of phase 2 enzymes, quinone reductase (QR) andglutathione S-transferase (GST), in rat liver (both p<0.05). These results suggested that HTPG might havechemopreventive effects against colon carcinogenesis, at least in the initiation stage.     

Dose response and proliferative characteristics of aberrant crypt foci: putative preneoplastic lesions in rat colon

Foci of aberrant crypts (ACF) have been identified in the unsectioned methylene blue stained rodent colons and hypothesized to represent precursor lesions of colon cancer. In the present study, induction and growth characteristics of ACF were investigated in response to a single injection of varying dosages of 1,2-dimethylhydrazine-2HCl (DMH), a colon carcinogen. Female Sprague-Dawley rats were given a single injection of DMH (5-150 mg/kg). Two and 19 weeks after the injection, animals were killed and their distal 10 cm of colons were enumerated for the number and crypt multiplicity of ACF. Number of ACF increased with increasing dosages of DMH plateauing at 100 mg/kg. However, percentage of ACF exhibiting different crypt multiplicity (1 to greater than 4) were similar among different dose groups. Aberrant crypts and normal crypts were enumerated for total number of cells and number and distribution of S-phase cells along the crypt height 19 weeks after DMH injection after autoradiography. The labeling index (LI) (percentage of S-phase cells) and LI along the crypt height were determined. Compared to the surrounding normal crypts, aberrant crypts exhibited significantly higher (P less than 0.05) number of cells (1122 +/- 81 versus 411 +/- 28) and higher (P less than 0.05) LI (21 +/- 1 versus 12 +/- 1). For the eight ACF analysed in the present study, the distribution of S-phase cells in the aberrant crypts were similar to that of normal crypts in that S-phase cells were restricted to the lower two-thirds of the crypts rather than distributed throughout the height of the crypts as reported for adenomatous epithelium.     

Development and distribution of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced aberrant crypt foci in the rat large intestine.

Aberrant crypt foci (ACF) are generally considered to be preneoplastic lesions for colon cancer. To assess their induction by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a colon carcinogen, we performed a sequential study of ACF morphology and localization. F344 male rats were given PhIP, and methylene blue-stained colon epithelium and isolated crypts were analyzed at weeks 12, 25, 50, and 75. Each crypt was classified into 2 groups, "single" with round bottoms and "bifurcating" displaying V-shaped clefts (indicating proliferation). In combination with the number of crypts in an ACF, this classification was a good indicator for the generation of ACF in line with the fission mechanism of growth. Increasing numbers of crypts in ACF through weeks 12 to 75 and decreased percentages of ACF with bifurcating crypts at the late time points indicated that proliferation of crypts occurs predominantly during the early stages. The distribution pattern showed a significant shift (P < 0.000005) from the distal to the proximal part of the large intestine between weeks 25 and 50. Adenocarcinomas were first found to develop at week 50 in the ascending colon and cecum where bifurcating crypts were generally lacking at weeks 12 and 25. These data suggest the existence of (1) proliferating ACF which contains bifurcating crypt(s) and (2) quiescent or senescent ACF which consists of only single crypts.     

Effect of carcinogen dose fractionation, diet and source of F344 rat on the induction of colonic aberrant crypts by 2-amino-3-methylimidazo[4,5-f]quinoline

Carcinogen dose fractionation, diet and source of laboratory animal were examined as variables in the induction of colonic aberrant crypt foci (ACF) by the heterocyclic amine 2-amino-3-methylimidazo [4, 5-f]quinoline (IQ). In the first experiment, male F344 rats from the National Cancer Institute (NCI rats) were fed AIN-93G diet and, starting in the third week, IQ was given by gavage on alternating days, the total carcinogen dose of 105 mg being fractionated proportionally over 2, 4, 8 or 14 weeks. Only the high dose (2 week) treatment with IQ was effective for the induction of ACF at 16 weeks, producing on average 3.8 ACF/colon versus 0.5 ACF/colon in all other groups (P < 0.05). The 2 week IQ dosing protocol was used in a second experiment in which male F344 rats from Simonsen Laboratories (SN) or NCI were fed AIN-93G, AIN-76A or chow diet. On average, SN rats on chow diet had twice the number of aberrant crypts compared with NCI rats given the same diet and three to four times as many aberrant crypts as NCI rats fed AIN diets. Hepatic cytochrome P4501A1 (CYP1A1) levels were essentially unaffected by diet, but methoxyresorufin O-demethylase activities and CYP1A2 protein levels were increased 2- to 3-fold in animals fed chow versus AIN diets. During the 2 week period of carcinogen administration, IQ markedly induced CYP1A proteins and negated the differences among groups related to diet. No consistent diet-related changes were detected in the activities of aryl sulfotransferase or N-acetyltransferase, but UDP-glucuronosyltransferase activities were elevated 2- to 3-fold in rats given chow versus AIN diets. In summary, high dose treatment with IQ was required for the induction of ACF, rats on the chow diet had more aberrant crypts than those given AIN diets and male F344 rats purchased from different vendors and fed chow diet differed with respect to their sensitivity to induction of ACF.     

Development and Distribution of 2-Amino-1-methyl-6-phenylimidazo[4,5-b]-pyridine (PhIP)-induced Aberrant Crypt Foci in the Rat Large Intestine

Aberrant crypt foci (ACF) are generally considered to be preneoplastic lesions for colon cancer. To assess their induction by 2-amino-1-methyl-6-phenylimidazo[4,5- b ]pyridine (PhIP), a colon carcinogen, we performed a sequential study of ACF morphology and localization. F344 male rats were given PhIP, and methylene blue-stained colon epithelium and isolated crypts were analyzed at weeks 12, 25, 50, and 75. Each crypt was classified into 2 groups, "single" with round bottoms and "bifurcating" displaying V-shaped clefts (indicating proliferation). In combination with the number of crypts in an ACF, this classification was a good indicator for the generation of ACF in line with the fission mechanism of growth. Increasing numbers of crypts in ACF through weeks 12 to 75 and decreased percentages of ACF with bifurcating crypts at the late time points indicated that proliferation of crypts occurs predominantly during the early stages. The distribution pattern showed a significant shift ( P < 0.000005) from the distal to the proximal part of the large intestine between weeks 25 and 50. Adenocarcinomas were first found to develop at week 50 in the ascending colon and cecum where bifurcating crypts were generally lacking at weeks 12 and 25. These data suggest the existence of (1) proliferating ACF which contains bifurcating crypt(s) and (2) quiescent or senescent ACF which consists of only single crypts.     

Chemopreventive effects of carotenoids and curcumins on mouse colon carcinogenesis after 1,2-dimethylhydrazine initiation

The present study was carried out to examine the chemopreventive effects of carotenoids such as fucoxanthin, lycopene and lutein as well as curcumin and its derivative, tetrahydrocurcumin (THC), on development of putative preneoplastic aberrant crypt foci (ACF) in colons of mice initiated with 1,2-dimethylhydrazine dihydrochloride (DMH). Influence on proliferation of colonic crypt epithelial cells was also assessed in terms of 5-bromo-2'-deoxyuridine (BrdU) incorporation. Five-week-old B6C3F1 male mice were divided into three groups, groups 1 and 2 being given DMH (20 mg/kg body wt, s.c.) twice a week for 3 weeks. Animals of group 1 were then treated with one of the test compounds, lycopene (0.005% and 0.0025%) or fucoxanthin (0.01%) in the drinking water and lutein (0.05%), curcumin (0.5%) or THC (0.5% and 0.2%) in the diet from weeks 5-12. Group 2 served as a carcinogen alone control and group 3 mice were given test compounds alone. All animals were killed at week 12. Numbers of ACF/mouse in the group 1 treated with fucoxanthin (47.1 +/- 13.7), lutein (42.6 +/- 19.6) or 0.5% THC (46.6 +/- 17.7) were significantly decreased as compared to the control group 2 value (63.3 +/- 19.4) (P < 0.01). Numbers of aberrant crypts (ACs)/mouse were also significantly lower after treatment with lutein (79.9 +/- 34.7) or 0.5% THC (81.8 +/- 32.5) than in the control group (115.1 +/- 37.1) (P < 0.01). BrdU labeling indices (LI) in mice treated with lutein and 0.5% THC were significantly decreased in both upper and lower half compartments of colonic crypts as compared to the controls (P < 0.05 and 0.01, respectively), especially the upper half data corresponding to reduction of ACs/mouse. The results thus suggest that fucoxanthin, lutein, and THC may have potential as chemopreventive agents against colon carcinogenesis.      

Effect of dietary carbohydrates on the growth of dysplastic crypt foci in the colon of rats treated with 1,2-dimethylhydrazine

The effect of dietary starch and sucrose on the growth of foci of dysplastic crypts in the colon (FDC) was studied in female Sprague Dawley rats treated twice p.o. with 25 mg/kg of 1,2-dimethylhydrazine (DMH). After DMH administration, the animals were fed high-fat (23% corn oil, w/w)/low-calcium (0.1%, w/w)/low-cellulose (2%, w/w) diets in which carbohydrates were represented by corn starch (starch diet) or sucrose (sucrose diet) (46%, w/w). The animals were fed for either 30 or 105 days with the experimental diets. The number of FDC was not significantly affected by diet. However, after 30 days the percentage of small FDC (formed by 1-2 dysplastic crypts) was higher in the animals fed the starch diet compared to the animals fed the sucrose diet [90.3 +/- 1.1% (SE) and 82.6 +/- 3.1%, respectively; P less than 0.05]. In contrast, foci formed by 3-4 dysplastic crypts were decreased by the starch diet (P less than 0.05). After 105 days of feeding, the starch diet induced a number of dysplastic crypts/focus lower than that induced by the sucrose diet (2.6 +/- 0.1 and 2.9 +/- 0.1, respectively; P less than 0.05). The percentage of small FDC was also higher in the animals fed the starch diet compared to animals fed the sucrose diet (P less than 0.01). After 30 days of feeding, DMH treatment increased colon proliferative activity in both dietary groups (P less than 0.05). But after 105 days of feeding, proliferation was similar in controls and DMH-treated rats and markedly reduced in animals fed the starch diet (mean labeling index values for both controls and DMH-treated rats were 10.4 +/- 0.8 and 4.4 +/- 0.5 in the sucrose and starch diets, respectively; P less than 0.001). The overall results suggest that starch in high-fat/low-calcium/low-cellulose diets has a protective role against DMH-colon carcinogensis in the rat.     

Histological and immunohistochemical observations of mucin-depleted foci (MDF) stained with Alcian blue, in rat colon carcinogenesis induced with 1,2-dimethylhydrazine dihydrochloride

The usefulness of mucin-depleted foci (MDF), which have recently been proposed as a new preneoplastic biomarker in rat colon carcinogenesis, was histologically investigated in rat colonic tissues treated with 1,2-dimethylhydrazine dihydrochloride (DMH). The relationship among aberrant crypt foci (ACF), MDF and beta-catenin accumulated crypts (BCAC) was examined by comparing the corresponding computer-captured images. Twelve male F344 rats were given DMH s.c. at a dose of 40 mg/kg body weight, once a week for 2 weeks, and randomly divided into two groups. Rats in group 1 were given normal drinking water, while those in group 2 were given drinking water containing indomethacin (IND) at 16 ppm for 6 weeks. All animals were sacrificed 8 weeks after the first DMH treatment. The resected colons were fixed in 10% formalin, and stained with Alcian blue for observation of ACF and MDF. Histological and immunohistochemical analysis revealed that the numbers of ACF, MDF and overlapping lesions in group 2 (treated with IND) were significantly decreased, compared with those in group 1. The number of BCAC in group 2 was also significantly lower than that in group 1. The reduction (61.5%) of MDF by IND was much greater than that (29.3%) of ACF. Analyses of the computer-captured images indicated that MDF had more frequent dysplastic changes and overexpression of beta-catenin than did ACF. MDF having over 4 crypts or MDF with the appearance of ACF corresponded well to BCAC. These results suggest that MDF may be useful as an early biomarker in colon carcinogenesis.     

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

Azoxymethane (AOM) is an organotropic colon carcinogen that is commonly used to induce colon tumors in rodents. Unlike its parent compound, 1,2-dimethylhydrazine (DMH), a tumor susceptibility phenotype in inbred mice with respect to AOM has not been established. Thus, this study was undertaken to determine whether genetic susceptibility extends to this carcinogen. SWR/J, A/J (both susceptible to DMH carcinogenesis) and AKR/J (resistant) mice were treated with 10 mg/kg AOM i.p. once a week for 8 weeks. Twenty-five weeks after the initial injection, tumor yield was determined. With a single exception, only SWR/J and A/J mice developed tumors, with a distribution that was limited to the distal colon (16.3±1.1 and 36.4±2.4, respectively). The formation of aberrant crypt foci (ACF), putative preneoplastic lesions, was also assessed in whole-mount colons using Methylene Blue staining. Consistent with tumor multiplicity, the total number of ACF was highest in A/J mice, followed by SWR/J mice. In addition, A/J mice had a significantly greater number of large ACF (five or more crypts per foci) than the other strains. Despite the absence of colon tumors, however, AKR/J mice did develop a significant number of ACF. This finding suggests that ACF in resistant mice are persistent but do not progress to tumors.     

Effect of vegetable and carotenoid consumption on aberrant crypt multiplicity, a surrogate end-point marker for colorectal cancer in azoxymethane-induced rats

Epidemiological studies indicate that increased vegetable consumption reduces the risk of colorectal cancer mortality. In the present study we have investigated the effect of consumption of standard diets supplemented with freeze-dried vegetables (peas, spinach, sprouts and broccoli) and carotenoids (all-trans beta-carotene and palm oil carotenoid extract) on surrogate end-point markers for colorectal cancer in an azoxymethane-induced rat model. Mean aberrant crypt multiplicity was reduced (19%) by the pea-supplemented diet only (P < 0.05). The vegetable-induced effect was more apparent in aberrant crypt foci with higher multiplicity. Intervention with diets supplemented with peas, spinach, sprouts and a mix of all vegetables reduced the number of foci with >2 aberrant crypts/focus by 37, 26, 23 and 26%, respectively (P < 0.05). Even more pronounced effects were observed in foci with >3 aberrant crypts/focus, with reductions of approximately 50% in the pea and spinach intervention groups. All-trans beta-carotene and palm oil-derived carotenoids, supplied at similar doses to those expected in the vegetable diets, inhibited ACM only marginally. Aberrant crypt foci formation in groups fed a sprout-supplemented diet prior to or following azoxymethane treatment was similar, indicating that this effect is due to inhibition of promotion rather than initiation of colorectal carcinogenesis. Vegetable and carotenoid consumption did not affect in situ proliferation of colonic crypt cells, as assessed by semi-automated image analysis of bromodeoxyuridine (BrdU)-positive nuclei. BrdU-negative nuclei of colonic crypt cells were reduced slightly in the combined vegetable groups, as compared with the control (P < 0.05). These data: (i) are in line with epidemiological evidence regarding beneficial effects of vegetable consumption on colorectal carcinogenesis; (ii) indicate that consumption of several types of vegetables inhibits early post-initiation events in colorectal carcinogenesis; (iii) suggest that the vegetable-induced effect is more pronounced in advanced lesions; (iv) indicate that the carotenoid content of the vegetables (alpha- and beta-carotene) contributes only marginally to the vegetable-induced effects.     

Inhibitory effect of dietary monoglucosylceramide 1-O-beta-glucosyl-N-2'-hydroxyarachidoyl-4,8-sphingadienine on two different categories of colon preneoplastic lesions induced by 1,2-dimethylhydrazine in F344 rats

Sphingolipids display a wide spectrum of biological activities, including cell growth, differentiation and apoptosis. However, precise mechanisms by which these compounds exert anticancer or cancer-preventive effects are not known. In the present study, we evaluated the preventive efficacy of enriched dietary monoglucosylceramide 1-O-beta-glucosyl-N-2'-hydroxyarachidoyl-4,8-sphingadienine (G(1)CM) on 1,2-dimethylhydrazine (DMH)-induced aberrant crypt foci (ACF) and beta-catenin-accumulated crypt (BCAC) formation in F344 rats during initiation stage. We also examined whether G(1)CM affects cell proliferation and apoptosis in these lesions. Pure G(1)CM was isolated from rice bran. Forty-two rats were divided randomly into five experimental groups. Rats in groups 1-3 were given subcutaneous injections of DMH (40 mg/kg body weight) once a week for 2 weeks. One week before the first injection of DMH, rats in groups 2 and 3 were fed a diet containing 200 and 1,000 p.p.m. G(1)CM, respectively, for 5 weeks. Rats in group 4 were fed a diet containing 1,000 p.p.m. G(1)CM. Rats in group 5 were given the basal diet alone and served as untreated controls. The experiment was terminated 5 weeks after the start. Dietary G(1)CM at both doses (groups 2 and 3) significantly inhibited the induction of ACF and BCAC (P<0.001) when compared to group 1 treated with DMH alone. In groups 2 and 3, the proliferating cell nuclear antigen labeling indices of epithelial cells in ACF and BCAC were also lower than in group 1 (P<0.0001 for ACF, P<0.05 for BCAC). These results, that dietary G(1)CM has possible chemopreventive effects in the present short-term colon carcinogenesis bioassays, suggest that longer exposure may cause suppression of tumor development.     

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.