Differential effects of CYP2E1 status on the metabolic activation of the colon carcinogens azoxymethane and methylazoxymethanol.

Methylazoxymethanol (MAM) and its chemical and metabolic precursor, azoxymethane (AOM), both strong colon carcinogens in rodents, can be metabolically activated by CYP2E1 in vitro. Using CYP2E1-null mice, we found that CYP2E1 deficiency differentially affects the activation of AOM and MAM, as reflected in DNA guanine alkylation in the colon and in the formation of colonic aberrant crypt foci (ACF). Male and female inbred 129/SV wild-type (WT) and CYP2E1-null (null) mice were treated with 189 micromol/kg of either AOM or methylazoxymethyl acetate (MAMAc), and 7-methylguanine (7-MeG) and O(6)-methylguanine (O(6)-MeG) were measured in the DNAs of various organs. The levels of O(6)-MeG (as pmol/nmol guanine) in the liver, colon, kidney, and lung of male null mice treated with AOM were 87, 48, 70, and 43% lower, respectively, than in AOM-treated WT mice. In null mice treated with MAMAc, the DNA O(6)-MeG levels were lower by 38% in the liver but were higher by 368, 146, and 194% in the colon, kidney, and lung, respectively, compared with the same organs of WT mice treated in the same way. Determination of ACF revealed that although AOM-induced ACF formation was significantly lower in the null group than in the WT group, MAMAc-induced ACF formation was significantly higher in the null group than in the WT group. These results demonstrate an important role for CYP2E1 in the in vivo activation of AOM and MAM and suggest that agents that modify CYP2E1 activity at the tumor initiation stage might either enhance or inhibit colon carcinogenesis, depending on whether AOM or MAMAc is used as the carcinogen. The mechanism of this effect is discussed.     

Purple Rice Extract Supplemented Diet Reduces DMHInduced Aberrant Crypt Foci in the Rat Colon by Inhibition of Bacterial β-Glucuronidase

Background: Purple rice has become a natural product of interest which is widely used for health promotion.This study investigated the preventive effect of purple rice extract (PRE) mixed diet on DMH initiation of coloncarcinogenesis. Materials and Methods: Rats were fed with PRE mixed diet one week before injection of DMH(40 mg/kg of body weight once a week for 2 weeks). They were killed 12 hrs after a second DMH injection tomeasure the level of O6-methylguanine and xenobiotic metabolizing enzyme activities. Results: In rats thatreceived PRE, guanine methylation was reduced in the colonic mucosa, but not in the liver, whereas PRE did notaffect xenobiotic conjugation, with reference to glutathione-S-transferase or UDP-glucuronyl transferase. After5 weeks, rats that received PRE with DMH injection had fewer ACF in the colon than those treated with DMHalone. Interestingly, a PRE mixed diet inhibited the activity of bacterial β-glucuronidase in rat feces, a criticalenzyme for free methylazoxymethanol (MAM) release in the rat colon. These results indicated that purple riceextract inhibited β-glucuronidase activity in the colonic lumen, causing a reduction of MAM-induced colonicmucosa DNA methylation, leaded to decelerated formation of aberrant crypt foci in the rat colon. Conclusions:The supplemented purple rice extract might thus prevent colon carcinogenesis by the alteration of the colonicenvironment, and thus could be further developed for neutraceutical products for colon cancer prevention.     

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.     

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.     

Inhibition of Azoxymethane-induced DNA Adduct Formation by Aloe arborescens var. natalensis

To clarify the possible mechanisms of inhibition of azoxymethane (AOM)-induced aberrant crypt foci (ACF) in ‍the rat colorectum by freeze-dried whole leaves of Aloe arborescens var. natalensis (Kidachi aloe) (hereinafter referred ‍to as ALOE) and commercial crude aloin (Sigma A-0451; from Curacao aloe) (hereinafter ALOIN), we studied the ‍effects of ALOE and ALOIN on the formation of AOM-induced DNA adducts (O6-methylguanine; O6-MeG) in rats. ‍Male F344 rats (4 weeks old) were fed a basal diet, or experimental diets containing 5%ALOE or 0.25%ALOIN for ‍5 weeks. All rats were injected s.c. twice with 15 mg/kg AOM, once at the end of week 1, and once at the end of week ‍2. The animals were sacrificed 6 hours after the second injection to analyze DNA adducts (O6-MeG) in the colorectum. ‍Dietary administration of ALOE significantly inhibited the O6-MeG levels (50% reduction) compared with controls, ‍whereas the O6-MeG levels in the ALOIN-fed rats showed a tendency to decrease (by 30%), although not significantly. ‍In this study, we also measured the enzyme activity and mRNA level of cytochrome (CYP) 2E1, known to be responsible ‍for the activation of AOM, in rat liver. ALOE-fed rats showed significantly reduced CYP2E1 enzymatic activity ‍(27% reduction) compared with controls. On the other hand, the activity in ALOIN-fed rats tended to decrease by ‍11%, although not significantly. The CYP2E1 mRNA levels in ALOE- and ALOIN-fed rats were slightly reduced ‍(9.7% and 5.2%, respectively). These results may explain, at least in part, the previously observed inhibitory effects ‍of ALOE and ALOIN, especially ALOE on AOM-induced ACF formation in the rat colorectum.     

Effects of bile acids on 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-induced aberrant crypt foci and DNA adduct formation in the rat colon

The effects of deoxycholic acid (DCA) and ursodeoxycholic acid (UDCA) on 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced aberrant crypt foci (ACF) in the rat colon were examined. The effect of these bile acids on DNA adduct formation by PhIP in the colon was then analyzed, since the main action of PhIP is the formation of DNA adducts and subsequent gene mutations. For the ACF study, male F344 rats were administered PhIP-HCl (75 mg/kg, 10 doses) by gavage, and a diet containing bile acid (0.4% DCA or UDCA) was provided from 3 days before the first dose of PhIP for 8 weeks. The mean number of ACF per colon of DCA, UDCA and controls were 9.9, 2.4 and 5.5, respectively. The ACF number was significantly increased by DCA and decreased by UDCA (P<0.001). To examine the effect of bile acids on DNA adduct formation, male F344 rats were fed a diet supplemented with bile acids (0.1 or 0.4% of DCA and UDCA) 7 days prior to the PhIP administration. All rats were administered a single dose of PhIP-HCl (50 mg/kg) by gavage and sacrificed 48 hours later. DNA adduct levels of the 0.1% UDCA, 0.1% DCA and controls were 2.93 (adducts/10(7) nucleotides), 2.65 and 1.10, respectively. Those of 0.4% UDCA, 0.4% DCA and controls were 1.64, 1.30 and 1.00, respectively. The PhIP-DNA adduct level was significantly increased by administration of 0.1% UDCA, 0.1% DCA (P<0.05) and 0.4% UDCA (P<0.01). The increasing effect of both DCA and UDCA on PhIP-induced DNA adduct formation was unexpected, and was not directly associated with ACF formation.     

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.      

DNA adduct formation and assessment of aberrant crypt foci in vivo in the rat colon mucosa after treatment with N-methyl-N-nitrosourea

N-Nitroso-compound DNA adduct formation in vivo and occurrenceof aberrant crypt foci (ACF) were studied in the rat colon mucosaafter a single, local treatment with a carcinogen, N-methyl-N-nitrosourea(MNU), using a simple surgical approach. A segment of F344 ratcolon was ligated to make a pouch and injected with MNU solution.For the study of DNA adduct formation, the solution contained50 µCi of [³H]MNU. The results demonstrated that similarranges of carcinogen dose, i.e. 0.15x10^–2 –1.5x10^–2MMNU, could induce both DNA adduct formation and appearanceof ACF in the rat colon with both parameters showing a nearbylinear dose dependence. HPLC analysis revealed the DNA adductsto include both 7-methylguanine (7-mGua) and O⁶-methylguanine(O⁶-mGua) with the 7-mGua/O⁶-mGua ratio being 8.2–11.3:1in the system used. Assessment of ACF development from 4 to16 weeks after MNU treatment at a dose of 7.5x10^–2 M showedthe numbers to increase up to the 8th week, followed by a decreaseat weeks 12 and 16, when 40% of the ACF counted at the peaktime point were still present. The percentage of large ACF (     

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.     

Tumor suppressor functions for the Cdk inhibitor p21 in the mouse colon

The Cdk inhibitor p21 regulates p53-mediated growth arrest following DNA damage. It is expressed during epithelial differentiation in a variety of organs including colon. We investigated susceptibility of p21-deficient mice to the colon carcinogen azoxymethane (AOM). After AOM injections, rodents develop putative premalignant lesions called aberrant crypt foci (ACF) that are localized to the distal three centimeters of the colon. p21-deficient mice developed significantly higher numbers of ACF than wild-type mice in response to AOM, and these were not restricted to distal colon. After AOM treatment, increased numbers of lymphoid aggregates were detected in p21-deficient colon. Proliferation was similar in wild type and p21-deficient colon before and after AOM injection, but AOM-induced apoptosis was detected only in wild-type crypt epithelial cells, and not in the p21-deficient colon. The proapoptotic function uncovered for p21 was unexpected, because p21 acts as an inhibitor of apoptosis in many systems, and is not required for p53-dependent apoptosis. Enhanced formation of ACF in p21-deficient mice supports a tumor suppressor function for p21 in the colon. Reduced apoptosis of colon epithelial cells with deleterious mutations may be an initiating event in the formation of ACF, with inflammatory cell cytokine expression contributing to their further expansion.     

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.     

Sequential and morphological analyses of aberrant crypt foci formation in mice of differing susceptibility to azoxymethane-induced colon carcinogenesis

Aberrant crypt foci (ACF), putative preneoplastic lesions, are early morphological changes induced by the colon carcinogen azoxymethane (AOM). Although inbred mice differ markedly in their susceptibility to AOM carcinogenesis, we have previously shown that ACF develop in both resistant and sensitive mouse strains after AOM treatment. The purpose of this study was to examine the sequential development and identify the morphological characteristics of ACF induced by AOM in the distal colon of sensitive and resistant mice. A/J (highly susceptible), SWR/J (relatively susceptible) and AKR/J (resistant) mice were treated with 10 mg/kg AOM or saline i.p. once a week for 6 weeks and were killed at 1, 2, 4, 6, 9 and 24 weeks after the last injection. The distal colons were stained with methylene blue and the numbers of ACF and tumors determined. Tumors were present as early as 4 weeks after AOM exposure in SWR/J and A/J mice and increased in frequency throughout the study in both strains. No tumors developed in the AKR/J mice. ACF, however, formed in all strains of mice. The greatest difference between susceptible and resistant strains was in the number of large ACF that developed at later time points. Furthermore, morphometric analysis revealed that A/J mice had the highest percentage of dysplastic ACF, followed by SWR/J mice. These data indicate that the difference in cancer risk from AOM may be due to the lack of progression of smaller ACF in the resistant mice and to the development of dysplasia in a higher percentage of ACF from susceptible strains.     

Estrous cycle modification of rat mammary gland DNA alkylation by N-methyl-N-nitrosourea

Virgin Sprague-Dawley rats exhibiting regular estrous cycles were used as a model system to determine whether the level of circulating estrogen modifies the alkylation pattern of mammary gland DNA by a direct-acting carcinogen, N-methyl-N-nitrosourea (NMU). The concentration of 7-methylguanine and O6-methylguanine were similar in mammary epithelial DNA 0.25, 0.50, and 1.0 h after i.v. injection of 50 mg/kg body weight NMU on different days of the rat estrous cycle. However, O6-methylguanine was significantly higher in mammary gland DNA 8 and 24 h after a single i.v. dose of carcinogen on proestrus or estrus, compared to rats receiving carcinogen on diestrus. There was no difference in the 7-methylguanine levels at 8 h in any group, but this adduct was higher in estrous-treated rats at 24 h. The ratio of O6-methylguanine to 7-methylguanine was significantly lower at 8 h in mammary gland DNA from diestrous-injected rats, and this difference reflected the lower level of O6-methylguanine adducts in this group. In contrast, O6-methylguanine concentrations in DNA extracted from the liver of the same animals were virtually identical at all time periods examined. 7-Methylguanine levels were higher in the liver at 0.5, 1, 8, and 24 h post-NMU in proestrus as compared with diestrous-injected rats. The observed adduct clearance suggests that rat mammary epithelium may contain repair systems capable of removing O6-methylguanine. These results also suggest that the initial removal of the O6-methylguanine lesions in mammary epithelial DNA (rather than the initial rate of alkylation) is affected by the hormonal environment during carcinogen exposure. This effect may be tissue specific since removal of O6-methylguanine from liver DNA is apparently not altered by the stage of the estrous cycle at which NMU is administered.     

Inhibition of O(6)-methylguanine-DNA methyltransferase increases azoxymethane-induced colonic tumors in rats

Azoxymethane (AOM) causes O(6)-methylguanine adduct formation which leads to G-->A transitions. Their repair is carried out by O(6)-methylguanine-DNA methyltransferase (MGMT). To evaluate the importance of this repair event in AOM-induced carcinogenesis, we examined the effect of O(6)-benzylguanine (BG), a potent inhibitor of MGMT, on colonic tumor development. Rats were treated weekly for 2 weeks at 0 and 24 h with BG (60 mg/kg body wt i.p.) or vehicle (40% polyethylene glycol, PEG-400), followed 2 h after the first dose of BG with AOM (15 mg/kg body wt) or vehicle (saline) i.p. Rats were killed 35 weeks later and tumors harvested and DNA extracted. In the AOM-treated groups, BG caused a significant increase in tumor incidence with tumors in 65.9%, versus 30.8% in the AOM/PEG-treated group (P < 0.05). In the BG/AOM group there was also a significant increase in tumor multiplicity, with 2.3 tumors/tumor-bearing rat, versus 1.6 tumors/tumor- bearing rat in the AOM/PEG group (P < 0.05). Since O(6)-methylguanine adducts can cause activating mutations in the K-ras and beta-catenin genes, we examined the effects of BG on these mutations. In the BG group there were seven mutations in codon 12 or 13 of exon 1 of the K-ras gene in 51 tumors examined, compared with no K-ras mutations in 17 tumors analyzed in the AOM/PEG group (P = 0.12). In the BG/AOM group there were 10 mutations in exon 3 of the beta-catenin gene among 48 tumors evaluated, compared with six mutations in 16 tumors analyzed in the PEG/AOM group (P = 0.16). In summary, MGMT inhibition increases AOM-induced colonic tumor incidence and multiplicity in rats.     

Enhanced growth of colorectal aberrant crypt foci in fasted/refed rats involves changes in TGFbeta1 and p21CIP expressions

We previously demonstrated that fasting/refeeding enhances the initiation phase of liver and colorectal carcinogenesis in rats. The present study was undertaken to establish whether cycles of fasting/refeeding carried out during the promotion phase of carcinogenesis may also affect the formation of aberrant crypt foci (ACF), preneoplastic lesions induced in the colon by azoxymethane (AOM). We were also interested in studying whether this effect might be mediated by changes in the proliferation, apoptosis or expression of TGFbeta1 and p21CIP genes in the colon. 44 male Fisher 344 rats were given a single dose of AOM (20 mg/kg s.c.) and one week later, they were exposed to 5 cycles of 4 days fasting followed by 7-10 days of refeeding (refed rats); controls were regularly fed; the rats were killed 2, 8 or 30 days after the last cycle of fasting. Fasting/refeeding caused a dramatic increase in crypt multiplicity when compared with regularly fed rats (AC/ACF was 4.30 +/- 1.3 in refed and 2.38 +/- 0.4 in regularly fed rats, P < 0.005 means +/- SD), while no significant changes were observed in the number of ACF/colon. In the two experimental groups, cell proliferation was higher in ACF than in the surrounding mucosa, but proliferative indexes were higher and the apoptotic index lower in ACF of refed rats compared with regularly fed rats. TGFbeta1 expression was higher in the ACF of refed rats than in those of fully fed controls while p21CIP was less expressed in refed rats than in controls. These results suggest that fasting/refeeding is a risk factor for colon cancer and must be taken into account for cancer prevention in humans.     

Effect of disulfiram on 1,2-dimethlhydrazine- and azoxymethane- induced aberrant crypt foci

We have previously reported a method for visualizing the mucosalsurface of fixed unsectioned rodent colons at the crypt leveland have identified lesions, termed aberrant crypt foci (ACF),in the colons of carcinogen-treated rodents. We hypothesizedthat ACF represent the precursor lesions (PL) of colon cancer.In the present study, the effect of feeding disulfiram (DSF)added to a semi-synthetic diet (0.5% or 1% by wt) on 1,2-dimethylhydrazine(DMH) and azoxymethane (AOM) induced ACF was investigated. DSFhas been shown to inhibit DMH and AOM-induced colon cancer.Therefore, it was reasoned that if ACF represent PL then theirinduction and growth should also be inhibited by DSF. CF1 femalemice were randomly divided into three groups of 30 each. Group1 was fed a diet containing 1% DSF for 9 days prior to and 1day after receiving a single i.p. injection of either DMH, AOMor saline. Group 2 was fed a diet containing 1% DSF for 9 daysprior to and 14 days after receiving a single i.p. injectionof DMH, AOM or saline, whereas group 3 received control dietthroughout the experimental duration. All animals were killed5 weeks after receiving the injections. It was observed thatfeeding DSF, for 9 days prior to and for either 1 day or 14days after the administration of a single injection of DMH,resulted in a complete inhibition of ACF. DSF feeding for 9days prior to and 1 day after AOM injection resulted in a significantlygreater number of ACF compared to the control group (12 ? 2.3vs 7.2 ? 1.2); whereas DSF feeding for a longer duration (i.e.9 days prior to and 14 days after AOM treatment) was associatedwith a significantly lower number of ACF compared to those fedDSF for only one day after AOM treatment (4.1 ? 0.6 vs 12.4? 2.3) and a lower number compared to the control group (4.1? 0.6 vs 7.2 ? 1.2).     

Prevention of the development of preneoplastic lesions, aberrant crypt foci, by a water-soluble extract from cultured medium of Ganoderma lucidum (Rei-shi) mycelia in male F344 rats

The modifying effects of a dietary water-soluble extract from cultured medium of Ganoderma lucidum (Rei-shi or Mannentake) mycelia (MAK) on the development of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) were investigated in male F344 rats. Rats were given subcutaneous injections of AOM (20 mg/kg body weight) once a week for three weeks to induce ACF and fed on diets containing 0, 1.25, 2.5 and 5.0% MAK for five weeks, starting one week before the first dose of carcinogen. MAK significantly and dose-dependently prevented the development of ACF, decreasing the total number of AC and inhibiting cyst formation. MAK (2.5 and 5.0%) also significantly reduced the longitudinal-cross section areas of colon epithelium. MAK in all doses significantly reduced the PCNA positive index, area of the germinal region and number of cells per half crypt. In an additional in vitro experiment, MAK inhibited anchorage-independent growth of several colon carcinoma cell lines. The present results thus indicate that dietary MAK could act as a preventive agent for colon carcinogenesis.     

Extract of vinegar "Kurosu" from unpolished rice inhibits the development of colonic aberrant crypt foci induced by azoxymethane

The modifying effects of administrating an ethyl acetate extract of "Kurosu" (EK), a vinegar made from unpolished rice, on development of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) were investigated in male F344 rats. We also assessed the effects of EK on proliferating cell nuclear antigen (PCNA) index in ACF, prostaglandin (PG) E2 expression in the colonic mucosa and activities of detoxifying enzymes of glutathione S-transferase (GST) and quinone reductase (QR) in the liver. To induce ACF, rats were given two weekly subcutaneous injections of AOM (20 mg/kg body wt). They also received drinking water containing 0, 0.05, 0.1 or 0.2% EK for 4 weeks, starting 1 week before the first dosing of AOM. AOM exposure produced 140 +/- 23 ACF/rat at the end of the study (week 4). EK administration dose-dependently inhibited ACF formation and inhibition by 0.2% EK was statistically significant (P < 0.002). Treatment with EK significantly lowered PCNA index in ACF and reduced PGE2 content in the colonic mucosa, while EK elevated liver GST and QR activities. These findings suggest that EK may be effective for inhibiting colonic ACF, through induction of liver GST and QR and possibly alteration of PGE2 production.     

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     

Prevention by aspirin and its combination with alpha-difluoromethylornithine of azoxymethane-induced tumors, aberrant crypt foci and prostaglandin E2 levels in rat colon

The dose-response relationship in male F344 rats was determined for the ability of aspirin administered in the diet to prevent azoxymethane (AOM)-induced colon cancer and aberrant crypt foci (ACF) and to reduce prostaglandin E2 (PGE2) levels. Starting at either 7 or 22 weeks of age, the rats received aspirin. All rats received two doses of AOM (15 mg/kg each on days 7 and 14) and were killed on day 36. The lowest concentrations of aspirin to prevent ACF or reduce PGE2 levels were 600 and 400 mg/kg, respectively. To evaluate the prevention of tumors, rats received either 0 or 400 mg/kg aspirin for a total of 39 weeks with AOM (30 mg/kg) administered 7 days after the start of treatment. Aspirin had no effect on the yield of colon tumors. In a second experiment, rats started to receive 0, 200, 600 or 1800 mg/kg aspirin or 1000 mg/kg alpha-difluoromethylornithine (DFMO) +/- aspirin. Eight and 15 days later, all the rats received 15 mg/kg AOM. Eleven weeks later, animals that were receiving the control diet started to receive 0, 200, 600 or 1800 mg/kg aspirin; 1000 or 3000 mg/kg DFMO; or 1000 mg/kg DFMO + 200 or 600 mg/kg aspirin. The animals were killed 32 weeks later. DFMO effectively reduced the yield of colon tumors when administered starting either before or after AOM while aspirin was much weaker. The combination of aspirin + DFMO administered after AOM was synergistic. Both aspirin and DFMO decreased the Mitotic Index, while apoptosis was increased only by DFMO. Our results demonstrated that aspirin and DFMO could prevent colon cancer when administered after AOM. Furthermore, aspirin reduced ACF, PGE2 levels and mitosis at concentrations that did not prevent cancer. In contrast, the ability to enhance apoptosis did correlate with the prevention of cancer.