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.     

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.     

Post-initiation effects of chlorophyllin and indole-3-carbinol in rats given 1,2-dimethylhydrazine or 2-amino-3-methyl- imidazo

Chlorophyllin (CHL) is a water-soluble derivative of chlorophyll, the ubiquitous pigment in green and leafy vegetables, whereas indole-3-carbinol (I3C) is present in cruciferous vegetables such as cabbage, broccoli and cauliflower. In rats initiated with 1,2-dimethylhydrazine (DMH), CHL and I3C reportedly promoted or enhanced the incidence of colon tumors when they were administered after, or during and after the carcinogen exposure, respectively. The same compounds given post-initiation inhibited the formation of colonic aberrant crypts induced by heterocyclic amines, such as 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), but tumor suppression was not examined in the latter studies. In the present investigation, male F344 rats were treated with IQ or DMH during the first 5 weeks of a 1 year study; IQ was given in the diet (0.03%), whereas DMH was administered once a week by s.c. injection (20 mg/kg body wt). Beginning 1 week after the last dose of IQ or DMH until sacrifice, rats received 0.001, 0.01 or 0.1% (w/v) CHL in the drinking water or 0.001, 0.01 or 0.1% I3C in the diet. Compared with controls given carcinogen alone, 0.1% I3C treatment suppressed the multiplicity of IQ-induced colon tumors, and CHL inhibited in a dose-related manner the incidence of IQ-induced liver tumors. However, 0.001% CHL increased significantly the multiplicity of DMH-induced colon tumors while having no effect on the colon tumors induced by IQ. These results indicate that both the choice of carcinogen as well as the dose of the tumor modulator can be important determinants of the events that occur during post-initiation exposure to CHL or I3C. Based on the present findings and data in the literature, it is possible for CHL and I3C to act as tumor promoters or anticarcinogens, depending upon the test species, initiating agent and exposure protocol.     

Presence of well‐differentiated distal,but not poorly differentiated proximal,rat colon carcinomas is correlated with increased cell proliferation in and lengthening of colon crypts

To determine whether colon crypt proliferative parameters were significantly altered by the stage of colon carcinogenesis or the type or location of colon tumors in rats, male Sprague‐Dawley rats received an injection of the carcinogen 1,2‐dimethylhydrazine (12 mg DMH base/kg body weight) or DMH vehicle once a week for 8 weeks, then were killed 24 weeks later. Three hours before sacrifice, rats were injected with 1 mg/kg body weight colchicine to arrest mitotic cells at metaphase. Transverse sections of the colon mucosa were taken 6 cm from the anus and at least 3 cm from any tumor, fixed in formalin, then stained with hematoxylin & eosin (H&E) for analyses of proliferative parameters. Only complete, mid‐axial crypts were scored for mitotic count (MC), crypt proliferative zone (PZ) height and crypt height (CH). Serial tumor sections were stained with H&E for histological evaluation or used in immunohistochemical detection of transforming growth factor α (TGFα). DMH treatment significantly increased MC, PZ and CH regardless of tumor status. The PZ and CH of rats with a carcinoma located in the distal colon were significantly increased compared with DMH‐treated rats without an adenocarcinoma (AC) or with rats which had a tumor located in the proximal colon. Distal colon ACs were found to be well differentiated and to have greater TGFα immunoreactivity than the generally less differentiated proximal colon carcinomas. Distal colon AC production and systemic circulation of a soluble colon crypt stimulating factor such as TGFα may explain the significant increase in PZ and CH in histologically normal colonic mucosa located away from the tumor. Int. J. Cancer 80:68–71, 1999. © 1999 Wiley‐Liss, Inc.     

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.     

Cabbage and vitamin E: their effect on colon tumor formation in mice

The effects of cabbage and vitamin E on colon carcinogenesis were investigated in Swiss mice treated with 1,2-dimethylhydrazine. Throughout the experiment the mice were fed a laboratory chow diet (46 mg vitamin E per kg) or chow containing 13 g cabbage per 100 g or 180 mg vitamin E per kg. Starting after 31 days of diet treatment the mice received 7 weekly s.c. injections of DMH. They were sacrificed 17 weeks after the first dose of DMH. While diet did not significantly alter colon tumor response, some trends were observed. Female mice given cabbage had a higher incidence (percent of mice with a tumor) and multiplicity (tumors per tumor bearing mouse) of colon tumors. Males were little affected by cabbage apart from a lower incidence of adenocarcinomas. Compared with mice fed the control diet those given vitamin E had a higher colon tumor incidence. This effect, which was stronger in females, was due to an increased incidence of adenomas. Vitamin E had little apparent affect on tumor multiplicity apart from a reduction in adenocarcinomas in females and adenomas in males. The data do not support the view that cabbage and vitamin E are protective against colon cancer.     

Dietary calcium and vitamin D modulate 1,2-dimethylhydrazine-induced colonic carcinogenesis in the rat

To determine whether supplemental dietary calcium and/or vitamin D deficiency are involved in modulating colon cancer induced by 1,2-dimethylhydrazine (DMH), Sprague-Dawley rats were fed diets containing either: (a) a normal content of calcium (0.87%) and phosphorus (0.60%) with 2.2 IU of vitamin D3 per g of feed (group A); (b) the same diet as group A, but with calcium and phosphorus increased to 1.80 and 0.80%, respectively (group B); or (c) a vitamin D-deficient diet with supplemental calcium (1.80%) and phosphorus (0.80%) (group C). After 6 weeks on their respective diets, one-half the animals in each group were given s.c. injections of either vehicle or DMH (20 mg/kg body weight/week) for 26 weeks. Animals were then sacrificed and the incidence of tumors as well as the number of tumors per tumor-bearing rat were determined. Colonic mucosal polyamine levels were measured after 15 weeks of exposure to vehicle or DMH, before development of histologically recognizable neoplasms. The results of these experiments demonstrated that neither calcium supplementation alone nor supplemental calcium in conjunction with vitamin D deficiency altered the incidence of colonic cancer induced by this carcinogen. Supplemental calcium, however, significantly decreased the number of rats with multiple tumors and reduced tumor size. Moreover, vitamin D deficiency abolished these protective effects of calcium on colon cancer in this experimental model. DMH treatment increased polyamine levels in the premalignant colonic mucosa in group A rats. This carcinogen-induced effect was blunted by high dietary calcium. Vitamin D-deficient, calcium-supplemented rats (group C) showed an increase in N1-acetylspermidine, but not the other polyamines, with DMH treatment.     

Induction of beta-glucuronidase activity during dimethylhydrazine carcinogenesis and additive effects of cholic acid and indole

The kinetic change of beta-glucuronidase (beta-G) activity was measured in mouse large intestinal mucosa during dimethylhydrazine (DMH) carcinogenesis with addition of cholic acid and/or indole. The experiment lasted 21 weeks. The enzyme activity began to increase significantly at 5th week after treatment of DMH with cholic acid and/or indole, and at 7th week with DMH alone. Then, increased activity remained the rest of the time. Mouse intestinal cancer induced by DMH injection are also shown to have an increased beta-G activity. The induction of beta-G activity in the early stage of DMH colon carcinogenesis and additive effects of cholic acid and/or indole may imply one mechanism of action of DMH as a carcinogen and cholic acid as a promoter in large intestinal cancer.     

Interaction of dietary fat and route of carcinogen administration on 1,2-dimethylhydrazine-induced colon tumorigenesis in rats

Since the results of an earlier study indicating no effect ofdietary fat on dimethylhydrazine (DMH)-induced colon cancerin rats differed from those of other investigators, the presentstudy was initiated to determine if the modulating effect offat intake on colon tumorigenesis was dependent on the routeof DMH administration. Male weanling Sprague-Dawley rats (160)were fed one of two nutritionally balanced diets containing5% or 24% corn oil (CO). Following 3 weeks adaptation to theirrespective diets, 40 rats from each diet group were treatedwith five doses of DMH (30 mg/kg) by intragastric i.g.) gavageor subcutaneous (s.c.) injection, over a 3 week period. Ratswere sacrificed when they showed clinical signs of colon tumorand surviving animals were killed 51 weeks after the initialDMH treatment. The cumulative probability of death with coloncarcinoma did not differ between the dietary or treatment groups.There was no effect of route of administration or dietary faton total intestinal tumor incidence. The number of rats withcolon carcinoma was: 5%CO.IG=25; 24%CO.IG=27; 5%CO.SC=23; 24%CO.SC=19.Polypoid tumor incidence was significantly higher in the 24%CO.SCgroup (12/40) compared to the 5%CO.SC group (3/40) (Chi-squared= 5.25; p <0.03) while sessile tumor incidence was the inverse.Marginally significant differences in tumor morphology werenoted between the IG groups.     

Relationship between dose, time, and tumor yield in mouse dimethylhydrazine-induced colon tumorigenesis

Colorectal tumor yield and volume data were obtained using 355 CF1 mice serially sacrificed up to 84 weeks following various dose levels of the carcinogen 1,2-dimethylhydrazine dichloride (DMH). Several conclusions were reached: (a) With increasing doses of DMH, there was an increased tumor yield and decreased latency period. (b) With repeated doses, there was a rapidly cumulative tumor yield. (c) New tumors continued to accumulate in the colon and rectum even at long intervals after the DMH treatments. This was substantiated by a positive correlation between the number of tumors per colon and the delay after DMH. In addition, when several tumors were present in the same mouse, their sizes were graded rather than uniform. These observations are consistent with a 2- or multi-step carcinogenesis mechanism. The latter implies that DMH induces a permanent transmissible alteration within some cells which thereafter will be at risk for further alterations capable of initiating cancer growth.     

Aberrant crypts correlate with tumor incidence in F344 rats treated with azoxymethane and phytate.

Aberrant crypts are putative preneoplastic lesions that have been proposed as intermediate biomarkers for colon cancer. The goals of these studies were to determine (i) if the colon cancer chemopreventive agent, sodium phytate, when started 1 week after a single dose of carcinogen, has any effect on the development of aberrant crypt foci (ACF) in treated rats; and (ii) if ACF at an early time period under these conditions correlate with the later formation of tumors in similarly treated animals. The number of ACF with four or more crypts was greater (P = 0.02, Mann-Whitney test) in rats with tumors compared with rats without tumors killed at 36 weeks after the injection of azoxymethane (AOM); the total number of ACF was not significantly different in these two groups. The incidence of tumors in F344 rats treated with AOM without phytate was 83% (10/12) compared to 25% (3/12) in rats treated with AOM plus phytate (P = 0.0045, two-tail Fisher's exact test). The finding of more (P = 0.005, Mann-Whitney test) ACF with four or more crypts in rats without phytate than in rats with phytate at 12 weeks after the injection of AOM is consistent with the hypothesis that the development of larger ACF (with four or more crypts) is predictive of the tumor incidence. These results validate the use of this parameter, i.e. ACF with four or more crypts, as an intermediate biomarker for tumor incidence in this system.     

Non-steroidol anti-inflammatory drug effect on crypt cell proliferation and apoptosis during initiation of rat colon carcinogenesis.

Sustained use of non-steroidal anti-inflammatory drugs (NSAIDs) may prevent colorectal cancer. However, the optimal drug, period of efficacy and mechanism(s) of action are unknown. Experiments were undertaken to determine which of several NSAIDs would modulate colon crypt cell proliferation or apoptosis when given during the initiation phase of 1,2-dimethylhydrazine (DMH)-induced rat colon cancer. Colon crypts located both away from and over an aggregate of lymphoid nodules (ALN) were examined. Rats were injected with aspirin, indomethacin, nabumetone, sodium salicylate, 16,16-dimethyl prostaglandin E2 or saline for 3 days and DMH or DMH vehicle on day 4 of each week for 8 weeks, then killed 3 days after the last DMH injection. At the time of killing, DMH had significantly increased crypt cell proliferation but not apoptosis. There was significantly more cell proliferation and apoptosis in crypts over the ALN than away from the ALN. Aspirin and salicylate increased proliferation and apoptosis in crypts over the ALN. Finally, the distributional peaks of cell proliferation and apoptosis were shifted significantly closer together after DMH. Thus, DMH increases proliferation and alters the distribution of proliferating and apoptotic cells in colon crypts early in carcinogenesis. Aspirin may suppress tumour incidence via salicylate by enhancing apoptosis in carcinogen-initiated cells.     

Modification by five antioxidants of 1,2-dimethylhydrazine-initiated colon carcinogenesis in F344 rats

The effects of antioxidants given in the post initiation phaseof colon tumor development were investigated in male F344 ratstreated with 1 ,2-dimethylhydrazine (DMH). Animals (20/group)were given s.c. injections of DMH at a dose of 20 mg/kg oncea week for four consecutive weeks. One week after the last injection,rats were fed diet containing 5% sodium L-asorbate (SA), 0.5%butylated hydroxyanisole (BHA), 0.8% ethoxyquin (EQ), 1.0% propylgallate or 0.5% butylated hydroxytoluene (BHT) for 36 weeks.A control group was fed the basal diet not containing antioxidants.The experiment was terminated 40 weeks after the first injectionof DMH and all intestinal tumors were confirmed histologically.SA significantly increased the incidence of adenomas and thenumber of tumors per rat of the colon (especially of the distalcolon). Although EQ and BHT did not affect the number of ratswith colon tumors, the number of tumors per rat occurring inthe distal colon was significantly increased by EQ while beingdecreased by BHT. No modification of tumor development was observedwith BHA or PG. Thus, modification of tumor development by SA,EQ and BHT was apparent, mainly in the distal colon.     

Evaluation of butylated hydroxyanisole, myo-inositol, curcumin, esculetin, resveratrol and lycopene as inhibitors of benzo[a]pyrene plus 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced lung tumorigenesis in A/J mice.

The potential activities of butylated hydroxyanisole (BHA), myo-inositol, curcumin, esculetin, resveratrol and lycopene-enriched tomato oleoresin (LTO) as chemopreventive agents against lung tumor induction in A/J mice by the tobacco smoke carcinogens benzo[a]pyrene (BaP) and 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were evaluated. Groups of 20 A/J mice were treated weekly by gavage with a mixture of BaP and NNK (3 micromol each) for 8 weeks, then sacrificed 26 weeks after the first carcinogen treatment. Mice treated with BHA (20 or 40 micromol) by gavage 2 h before each dose of BaP and NNK had significantly reduced lung tumor multiplicity. Treatment with BHA (20 or 40 micromol) by gavage weekly or with dietary BHA (2000 ppm), curcumin (2000 ppm) or resveratrol (500 ppm) from 1 week after carcinogen treatment until termination had no effect on lung tumor multiplicity. Treatment with dietary myo-inositol (30,000 ppm) or esculetin (2000 ppm) from 1 week after carcinogen treatment until termination significantly reduced lung tumor multiplicity, with the effect of myo-inositol being significantly greater than that of esculetin. Treatment with dietary LTO (167, 1667 or 8333 ppm) from 1 week before carcinogen treatment until termination had no effect on lung tumor multiplicity. The results of this study demonstrate that BHA is an effective inhibitor of BaP plus NNK-induced lung tumorigenesis in A/J mice when administered during the period of carcinogen treatment and that, among the compounds tested, myo-inositol is most effective after carcinogen treatment.     

Induction of colon tumors in 1,2-dimethylhydrazine-resistant Lobund Wistar rats by methylazoxymethanol acetate.

Sprague-Dawley and Lobund Wistar rats, which were sensitive and resistant to induction of colon tumors by 1,2-dimethylhydrazine (DMH), respectively, were treated with methylazoxymethanol (MAM), the product of DMH metabolism by the microsomal mixed-function oxidase system. Although the colon tissue in both stocks of rats had similar NAD+-dependent dehydrogenase activities that are considered necessary to activate MAM to an ultimate carcinogen, still a sevenfold greater incidence of colon tumors was found in the Sprague-Dawley rats, and their tumors were more extensive. The results indicated that the difference in susceptibility to colon tumor induction between the rat stocks was partially related to metabolic activation of the DMH and to other, as yet undetermined, endogenous factors.     

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 sodium ascorbate on 1,2-dimethylhydrazine- or methylnitrosourea-induced colon carcinogenesis in rats

The effect of dietary sodium ascorbate (SA) on colon carcinogenesisevoked by 1,2-dimethylhydrazine (DMH) or N-methyl-N-nitrosourea(MNU) was studied in female F344 rats. Animals were fed dietscontaining 0, 0.25 and 1% of SA and given s.c. a single doseof 150 mg DMH/kg body wt., 10 weekly s.c. injections of 20 mgDMH/kg body wt. or intrarectal administration of 2 mg MNU, twicea week for 2 weeks. The incidence of colon and kidney tumorswas lower in rats fed the 0.25 or 1% SA and treated with a singledose of DMH than in the animals fed the diet without SA; however,the tumor incidences did not differ between the SA- and controldiet-fed animals and treated with multiple doses of DMH or MNU.     

Effect of polyamine oxidase inhibition on the colonic malignant transformation process induced by 1,2-dimethylhydrazine

Recent studies of colon adenocarcinomas in humans and experimentally induced colonic tumors in rodents have demonstrated selective elevations in the level of N1-acetylspermidine in these malignant tissues. The exact relationship of these alterations in acetylated polyamine levels to the malignant transformation process, however, remains unclear. In order to clarify this issue, rats were given s.c. injections of 1,2-dimethylhydrazine (DMH; 20 mg/kg body wt/week) or diluent for up to 26 weeks. After 10 weeks of carcinogen treatment, one-half of the animals in each group were also concomitantly given i.p. injections of MDL 72527 (20 mg/kg body wt/week), a specific inhibitor of polyamine oxidase, until they were killed. Animals were killed after 15 weeks of DMH treatment and polyamine levels as well as the activities of polyamine oxidase, ornithine decarboxylase and spermidine-N1-acetyltransferase were measured and compared in rat proximal and distal colonic mucosa of each group. Polyamine levels were also assessed in each of these groups after 26 weeks of treatment with this carcinogen +/- MDL 72527. In addition, in view of recent studies that have indicated that polyamines may influence certain oncogenes in human colonic carcinoma cells, tumors from DMH +/- MDL 72527 were analyzed for K-ras mutations. The results of these experiments demonstrated for the first time that: (i) MDL 72527 was a specific inhibitor of polyamine oxidase in normal and malignant colonic tissue; (ii) concomitant administration of this agent with DMH enhanced the elevation of colonic N1-acetylspermidine and significantly reduced the mean colonic tumor burden, as assessed by total tumor area per rat, produced by this carcinogen alone; (iii) analysis of K-ras mutations revealed a similar incidence (62-69%) in adenocarcinomas for both groups (+/- MDL 72527); (iv) however, analysis of the K-ras-mutated and non-mutated tumors revealed that in both carcinogen-treated groups (+/- MDL 72527), tumors with such mutations were smaller than their counterparts without such genetic alterations. Moreover, MDL 72527 reduced the average size of tumors, with and without such mutations, to a similar extent.