K-ras mutations in 1,2-dimethylhydrazine-induced colonic tumors: effects of supplemental dietary calcium and vitamin D deficiency

Recent studies from our laboratory have demonstrated that dietary supplemental calcium had no significant effect on the incidence of 1,2-dimethylhydrazine-induced colonic tumors, but did decrease the number of rats with multiple tumors and reduced tumor size. Moreover, concomitant vitamin D deficiency appeared to abolish these protective effects of calcium on colonic tumors in this experimental model. To date, however, the mechanism(s) involved in these phenomena remain unclear. In order to address these important issues, 1,2-dimethylhydrazine-induced colonic tumors from animals on control, Ca(2+)-supplemented, vitamin D-sufficient, and Ca(2+)-supplemented, vitamin D-deficient diets were examined for the presence of ras oncogene mutations. DNA was extracted from each of these tumors. Targeted areas of K-ras and H-ras genes were amplified by the polymerase chain reaction and analyzed for point mutations using allele-specific oligonucleotide hybridization and subsequent DNA sequencing. The results of these studies demonstrated that: (a) approximately one-third of 1,2-dimethylhydrazine-induced colonic carcinomas in the control group had K-ras G to A mutations; (b) no mutations, however, were detected in the cancers of the calcium-supplemented group; (c) concomitant vitamin D deficiency abolished the antimutagenic effect of dietary calcium supplementation (e.g., approximately one-third of cancers in this group again had detectable K-ras mutations); and (d) no H-ras point mutations were detected in colonic tumors from any group. These findings suggest that alterations in K-ras mutations may be one possible mechanism by which calcium and vitamin D status influence colonic carcinogenesis in this experimental model.     

Augmentation of 1,2-dimethylhydrazine-induced colon cancer by experimental colitis in mice: role of dietary vitamin E

Because ulcerative colitis predisposes to colonic cancer, for determination of the effect of colitis on experimental colon carcinogenesis, rectal instillations of peptides that attract and activate neutrophils were used to induce colitis in CD-1 (ICR) BR mice receiving 20 weekly injections of the carcinogen 1,2-dimethylhydrazine [(DMH) CAS: 540-73-8]. From week 4 through week 15 of DMH injections, twice-weekly enemas of formyl-norleucyl-leucyl-phenylalanine were given to DMH-treated mice. The effect of the antioxidant vitamin E in the diet (1,750 IU/kg diet) was studied in another group of mice treated with DMH and having colitis. Four weeks after DMH was discontinued, cancer occurred in 9 of 28 (32%) animals with DMH plus control enemas, in 22 of 29 (76%) animals with DMH plus colitis (P = .001), and in 16 of 28 (57%) animals with DMH plus colitis plus supplemental vitamin E (P = .11 compared with the group with DMH and colitis). Colitis enhances DMH-induced colonic carcinogenesis.     

Supplemental dietary calcium fails to alter the acute effects of 1,2-dimethylhydrazine on O6-methylguanine, O6-alkylguanine-DNA alkyltransferase and cellular proliferation in the rat colon

Prior studies from our laboratory have demonstrated that K-rasG to A mutations were detectable in a high percentage of carcinomaswhich developed in the colons of animals treated with the knowncolonic procarcinogen, 1,2-dimethyl-hydrazine (DMH). Moreover,in this model, the incidence of these mutations was decreasedby a supplemental dietary calcium regimen which concomitantlydecreased the frequency of rats with multiple tumors as wellas tumor size. In an attempt to clarify the possible mechanism(s)involved in this antimutagenic effect of supplemental calcium,two groups of Sprague—Dawley rats were fed semisyntheticdiets containing either 0.87 or 1.80% cakium by weight for 3weeks, s.c. injected with 100 mg/kg of DMH and killed priorto and at various time periods (16-144 h) after injection. Thecolons of animals were analyzed and compared with respect toO⁶-methylguanine content in DNA, O⁶-alkylguanine-DNA alkyltransferaselevels as well as cellular proliferation, as assessed by immunohistochemicalstaining of colonic crypts by bromodeoxyundine. In certain experiments,these parameters were also analyzed in the proximal and distalcolon before and at various times after administration of DMH.The results of these experiments demonstrated that supplementaldietary calcium was not found to influence significantly O⁶-methylguaninelevels, alkyltransferase levels or cellular proliferation inthe entire colon or in either colonic segment before or afterthe acute administration of DMH. DMH did, however, differentiallyalter all three of these biochemical parameters in the colonicsegments (distal > proximal), possibly due to a greater degreeof metabolic activation in the distal colon.     

A protective role of 1,25-dihydroxyvitamin D3 in chemically induced rat colon carcinogenesis.

The intriguing observation has been made that 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] receptors are present in tissues not involved in calcium homeostasis and that 1,25(OH)2D3 exerts an antiproliferative, differentiation-promoting action in a variety of cancer cell lines, including cells of the large intestine. It was therefore deemed of interest to study 1,25(OH)2D3 expression and biological activity in a murine model of colon carcinogenesis. Colon carcinogenesis was induced in male rats by the sequential administration of 1,2-dimethylhydrazine dihydrochloride (DMH). Levels and binding characteristics of 1,25(OH)2D3 receptors were assessed in control and DMH-treated rat colonic mucosal high-speed supernatants. In concurrent studies, 1,25(OH)2D3 was administered (s.c., 400 ng/rat) prior to, together with and after DMH challenge and the activity of ornithine decarboxylase (ODC), a growth-related DMH-induced enzyme, was determined in colonic cytosols. Serum Ca2+ levels were measured concurrently. Rats submitted to identical treatment schedules were killed 10 weeks after termination of DMH administration and the whole colon was opened and examined for tumors. The results show that (i) rat colonic mucosa possesses a single class of high-affinity 1,25(OH)2D3 receptors; (ii) DMH administration provokes a marked reduction (50%) in 1,25(OH)2D3 binding sites without affecting Kd values; (iii) DMH administered concurrently with 1,25(OH)2D3 suppressed the vitamin D-induced hypercalcemia and restored serum Ca2+ concentrations to basal levels; and (iv) 1,25(OH)2D3 delivered prior to DMH challenge obliterated the typical DMH-induced early colonic ODC activity peak and markedly reduced (50%) the number of colon adenocarcinomas. The present findings indicate that a colon-specific potent carcinogen interferes with the biological expression of 1,25(OH)2D3 and that vitamin D administered prior to a carcinogenic insult is able to reduce significantly the incidence of colon tumors, presumably acting as an antiproliferative or differentiation-promoting agent.     

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.     

The effects of chronic ethanol administration on polyamine content during dimethylhydrazine-induced colorectal carcinogenesis in the rat

Chronic ethanol (EtOH) consumption has been implicated as aco-carcinogen, selectively promoting rectal tumor formation.We studied the effects of EtOH consumption on tumor formationand polyamine content (putrescine, spermidine and spermine)in proximal and distal colon and rectum of Sprague-Dawley ratstreated with the procarcinogen 1,2-dimethylhydrazine (DMH).Sixty-four adult male rats were pair fed nutritionally completeliquid diets with 36% of calories supplied as EtOH or isocaloriccarbohydrates. Both groups received 4 weeks of the liquid dietfollowed by 4 weeks of standard laboratory chow during which50% of the rats in each group received DMH (30 mg/kg) or vehicles.c. weekly. This cycle was repeated four times (32 weeks).Animals were sacrificed at the end of each 8 week cycle andnormal appearing and available tumor bearing tissue from proximaland distal colon and rectum was obtained for polyamine contentand histology. Five animals, unexposed to DMH or EtOH servedas baseline controls. There were no consistent regional differencesin putrescine, spermidine or spermine of baseline controls.A progressive decrease in tissue putrescine was seen in allthree regions of the control group and was significant at 24and 32 weeks versus baseline controls. In all three regions,chronic EtOH consumption prevented the decrease in tissue putrescine.Spermidine content was also significantly increased in the distalcolon of EtOH-treated animals compared to baseline values. Consistentchanges in spermine content were seen in any treatment groupor region. A significant increase in putrescine content of normalappearing and tumor-bearing tissue of DMH treated animals at32 weeks was noted. Chronic EtOH administration did not augmentrectal or colonic polyamine content in DMH-treated animals.Likewise, chronic EtOH consumption did not alter the number,size or distribution of large bowel tumors of DMH treated animals.     

Effect of dietary vitamin D and calcium on the growth of androgen-insensitive human prostate tumor in a murine model

Vitamin D deficiency has been associated with increased risk of prostate cancer (PC) in epidemiologic and prospective studies. An association has also been made between high dietary calcium and increased PC risk. In this study, we evaluated the effect of dietary vitamin D and calcium on the growth of human androgen-insensitive prostate tumor in an athymic mouse model. We observed highest tumor growth in the normal calcium - vitamin D-deficient group, while tumor growth between the normal calcium - vitamin D-sufficient, high calcium - vitamin D-sufficient and high calcium - vitamin D-deficient diet-groups did not significantly differ but was significantly lower than that in the normal calcium - vitamin D-deficient group. Our results suggest an important role of dietary vitamin D as a preventive agent in androgen-insensitive PC.     

Consumption of vitamin B6 reduces colonic damage and protein expression of HSP70 and HO-1, the anti-tumor targets, in rats exposed to 1,2-dimethylhydrazine

Mounting evidence indicates that vitamin B6 is a protective factor for colon cancer. Elevations in colonic damage, cell proliferation and heat shock proteins (HSPs, molecular chaperones) have been suggested to be associated with colon carcinogenesis. This study was performed to examine the effect of dietary levels of vitamin B6 (1, 7 or 35 mg pyridoxine HCl/kg diet) for 22 weeks on colon damage, epithelial cell proliferation and expression of HSPs in rats exposed to 1,2-dimethylhydrazine (DMH). Supplemental vitamin B6 with a low vitamin B6 diet (1 mg pyridoxine HCl/kg diet) significantly reduced fecal activity of intestinal alkaline phosphatase (an index of intestinal damage) and the colonic epithelium PCNA labeling index (a marker of cell proliferation). Analysis using ELISA indicated that supplemental vitamin B6 significantly lowered protein levels of colonic HSP70 and heme oxygenase-1, HSP32 (HO-1). However, real-time RT-PCR analysis revealed that the mRNA levels of these HSPs were not decreased by supplemental vitamin B6, suggesting that the lowering effect of vitamin B6 on the colon protein expression of the HSPs is mediated by mechanisms not involving altered gene expression. This study provided evidence that dietary supplemental vitamin B6 suppresses colon damage, epithelial cell proliferation and protein expression of HSP70 and HO-1, the targets for anti-tumor agents, in rats exposed to DMH.     

Inhibition of ornithine decarboxylase with 2-difluoromethylornithine: reduced incidence of dimethylhydrazine-induced colon tumors in mice

2-Difluoromethylornithine (DFMO) was administered to 1,2-dimethylhydrazine (DMH)-treated mice to reduce colonic polyamine levels and mucosal hyperplasia. Mice received 1% DFMO in drinking water throughout the experiment and were given injections of DMH (20 mg/kg) weekly for 28 weeks. DFMO inactivated 93% of colonic ornithine decarboxylase activity. Although DMH treatment did not induce colonic ornithine decarboxylase activity by Week 28, the putrescine content was increased 31% in DMH-treated mice (p less than 0.01). Concurrent treatment with DFMO depressed putrescine content (42 to 63%) and spermidine content (27 to 38%), but it increased spermine content (18 to 22%). At Week 28 of treatment with DMH alone, RNA content was increased 8.6% (p less than 0.01), DNA content 10% (p less than 0.01), DNA specific activity 24% (p less than 0.01), and crypt depth 20% (p less than 0.01), but not in mice receiving DMH and DFMO. At 28 weeks, 13 of 17 mice (76%) treated with DMH alone had histologically confirmed colon cancers; of mice treated with DMH and DFMO, two of 18 (11%) had colonic tumors. Throughout the experiment, 50 colon cancers developed in 16 DMH-treated mice (mean, 3.12 tumors/mouse); three mice treated with DMH and DFMO developed three colon cancers total (p less than 0.001). Reduction of colonic polyamine levels after DFMO treatment prevents proliferative changes induced by DMH and reduces the incidence of tumors.     

Urinary excretion of N1-acetylspermidine and other acetylated and free polyamines in the 1,2-dimethylhydrazine model of experimental rat colon cancer

1,2-Dimethylhydrazine (DMH) is a potent procarcinogen with selectivity for the colon. Recently, it has been demonstrated that levels of N1-acetylspermidine were elevated 2-3-fold in colonic tumors induced by this agent compared to control tissues. To determine whether alterations in the urinary levels of this acetylated polyamine or other polyamines were useful biochemical markers for colon cancer in this experimental model, rats were given s.c. injections of DMH (20 mg/kg body weight/week) or diluent for 26 weeks. One week after the last injection, control and DMH-treated animals were placed in separate metabolic cages and their urine was collected for 24 h. The urinary levels (expressed as nmol/mg creatinine) of putrescine, spermidine, spermine, N1-acetylspermidine, and N8-acetylspermidine were then analyzed by high-performance liquid chromatography. Animals from each group were then sacrificed and their colons were examined for tumors. The results of these studies demonstrated that the urinary level of N1-acetylspermidine was an excellent biochemical marker for colonic tumors induced by DMH. At 18.3 nmol/mg creatinine, N1-acetylspermidine was 100% sensitive and specific for colon cancer. Moreover, urinary levels of N1-acetylspermidine were better for this purpose than the N1-acetylspermidine/N8-acetylspermidine molar ratio, a marker previously suggested to be more specific for certain cancers than free polyamines.     

Calcium inhibits colon carcinogenesis in an experimental model in the rat

Different dietary factors can affect colorectal cancer incidence. However, the effect of increased levels of dietary calcium on neoplasms is unclear. The present study was designed to examine the effect of a low calcium supplement on experimental colon carcinogenesis induced by parenteral administration of dimethylhydrazine (DMH). One hundred and twenty 10-week-old Sprague-Dawley rats were divided into five groups of equal sex distribution. The 10 rats in group A (control group) received no treatment; the 30 rats in group B (DMH group) were injected subcutaneously with 18 weekly doses of 21 mg/kg DMH; the 20 rats in group C (EDTA control group) received EDTA solution only; the 30 rats in group D (calcium group) received calcium at 3.2 g/l by adding calcium lactate to the drinking water from the start until the conclusion of the experiment; and the 30 rats in group E (DMH + calcium group) received oral calcium supplements at the same dose as the rats in group D (calcium group) and the same DMH injections as the rats in group B (DMH group). The rats were sacrificed at 25-34 weeks. In group E, we observed a significant diminution in the number of tumours (P = 0.01); an increase in the number of tumour-free animals (P = 0.006); a change in tumour location towards the distal colon (P < 0.025); more adenomas (P = 0.02); and a diminution of adenocarcinomas and mucinous carcinomas, although this was not significant. We conclude that a low dietary calcium supplement in rats inhibits colon cancer carcinogenesis induced by DMH, and changes tumour location towards the distal colon.     

Effect of a calcium-enriched diet on the colonic epithelial hyperproliferation induced by N-methyl-N'-nitro-N-nitrosoguanidine in rats on a low calcium and fat diet

We examined whether hyperproliferation of colonic crypt epithelium during cancer induction by N-methyl-N-nitro-N-nitrosoguanidine (MNNG), in rats on a low fat and calcium diet could be reduced by added calcium p.o. From the age of 4 weeks, 104 male Sprague-Dawley rats received a low fat (3.5%), low calcium (0.05% calcium ion), and low vitamin D (0.4 IU/g) diet. Sixty-four also had calcium salts, derived from either calcium lactate or solubilized calcium carbonate, added to their drinking water; therefore their total calcium intake was about 1% of daily diet. At age 12 weeks the rats were divided into 4 treatment groups: 8 rats, not receiving added calcium, had rectal saline instillations weekly (saline control group) and were sacrificed after a further 28 weeks; 3 groups of 32 rats each received intrarectal MNNG (1.5 mg) weekly. One group, not receiving added calcium, was the MNNG control group; while the second group also received added calcium lactate, and the third group received calcium carbonate. Groups of 24 were sacrificed periodically until 28 weeks of treatment. Rats were sacrificed and epithelial proliferation was estimated, 1 week after the last intrarectal instillation, by in vivo labeling with tritiated thymidine and measuring the ratio of labeled to total colonic crypt epithelial cells. The mean labeling index of the MNNG treated and added calcium groups were significantly higher (8.7-9.5%) than that of the saline controls (2.8%) only at week 28; however, it was then still significantly less than that of the MNNG controls not having added calcium (17.9%). Hyperproliferation, during induction of colonic cancer by MNNG in rats on a low calcium diet, can be reduced by a calcium enriched diet even in the presence of a low fat intake.     

Protective Role of Aspirin,Vitamin C,and Zinc and their Effects on Zinc Status in the DMH-Induced Colon Carcinoma Model

Chemoprotection refers to the use of specific natural or synthetic chemical agents to suppress or prevent theprogression to cancer. The purpose of this study is to assess the protective effect of aspirin, vitamin C or zinc ina dimethyl hydrazine (DMH) colon carcinoma model in rats and to investigate the effect of these supplementson changes associated with colonic zinc status. Rats were randomly divided into three groups, group 1 (aspirin),group 2 (vitamin C) and group 3 (zinc), each being subdivided into two groups and given subcutaneous injectionof DMH (30 mg/kg body wt) twice a week for 3 months and sacrificed at 4 months (A-precancer model) and6 months (B-cancer model). Groups 1, 2, 3 were simultaneously given aspirin, vitamin C, or zinc supplementrespectively from the beginning till the end of the study. It was observed that 87.5% of rats co-treated with aspirinor vitamin C showed normal colonic histology, along with a significant decrease in colonic tissue zinc at bothtime points. Rats co-treated with zinc showed 100% reduction in tumor incidence with no significant change incolonic tissue zinc. Plasma zinc, colonic CuZnSOD (copper-zinc superoxide dismutase) and alkaline phosphataseactivity showed no significant changes in all 3 cotreated groups. These results suggest that aspirin, vitamin Cor zinc given separately, exert a chemoprotective effect against chemically induced DMH colonic preneoplasticprogression and colonic carcinogenesis in rats. The inhibitory effects are associated with maintaining the colonictissue zinc levels and zinc enzymes at near normal without significant changes.     

Effects of dietary fish oil and corn oil on protein kinase C distribution in the rat colon with and without 1,2 dimethylhydrazine treatment and in rat colonic adenocarcinoma

The activity and subcellular distribution of Protein Kinase C (PKC) was determined in the colons of Sprague-Dawley rats that were fed either a low fat rat chow or rat chow supplemented with 17% corn oil (40% ingested calories as fat). Rats given the high fat diets were either given no carcinogen or treated prior to or subsequent to the initiation of the test diets with 1,2 dimethylhydrazine (DMH). Rats were sacrificed and PKC activity determined in the soluble and particulate fractions of the colonic tissue 13 weeks after the initiation of the diets or DMH treatment, which was before tumor induction. In addition several rats were maintained on their diets until colon tumor formation occurred and PKC activity determined in the colonic tumor and compared to age matched control colonic tissue. In the absence of DMH, fish oil and corn oil equally augmented PKC activity and decreased the ratio of soluble/particulate PKC. With DMH treatment, corn oil augmented PKC as above, but fish oil supplementation resulted in a pattern of PKC activity and distribution more typical of a low fat diet, particularly when fish oil supplementation preceded DMH treatment. PKC activity in DMH induced colonic carcinomas was markedly depressed regardless of the fat source in the diet, when compared to colonic tissue from a non-DMH treated age matched low fat control.     

Inhibition of dietary fat-promoted colon carcinogenesis in rats by supplemental calcium or vitamin D3

A 2 x 2 x 2 factorial experimental design was used to investigatethe effects of supplemental calcium (Ca) (0.5% versus 1.0% ofdiet as Ca gluconate) and vitamin D₃ (D) (1000 IU/kg diet versus2000 IU/kg diet) on 1,2-dimethylhydrazine-induced colon carcinogenesisin male F344 rats promoted with a 20% corn oil diet. Animalson the high-fat (HF) diet had an increased tumor incidence comparedto the low-fat (LF) control diet (86% versus 53%, P < 0.05)and supplemental Ca or D reduced this to or below the LF incidence(HF + Ca: 53%, HF + D: 47%). However, supplemental Ca or D hadno inhibitory effect with LF diets (LF + Ca: 67%, LF + D: 60%).The results of this study indicate a possible role for supplementalCa or D in the prevention of colon cancer, effective only inHF diets.     

Colon-specific regulation of vitamin D hydroxylases--a possible approach for tumor prevention

Epidemiological data suggest a protective role of calcium and vitamin D against colorectal tumor pathogenesis. 1,25-dihydroxyvitamin D3 (1,25-D3) is a key determinant of calcium homeostasis, cell proliferation and differentiation. Calcium in the intestinal lumen functions as a growth regulator and may prevent cancer by direct reduction of colonocyte proliferation. While calcium or vitamin D can counteract proliferation by itself, they could also interact if nutritional calcium were to modulate colonic vitamin D synthesis. In this paper we demonstrate that colonic and renal vitamin D hydroxylases are regulated independently. When mice were fed a modified AIN-76 diet containing low dietary calcium (0.1 or 0.04%) fecal calcium content was as low as 5% of that found in mice on a 0.9% calcium containing diet. Low fecal calcium concentration enhanced proliferating cell nuclear antigen expression in the colon mucosa and reduced that of the cyclin dependent kinase inhibitor p21. While low dietary calcium did not affect colonic expression of VDR or 25-hydroxyvitamin D3 1alpha-hydroxylase (CYP27B1) mRNA, it influenced their renal expression in the expected manner by elevating the CYP27B1 expression and reducing VDR and 25-hydroxyvitamin D3 24-hydroxylase (CYP24) expression. In contrast, low calcium diets significantly augmented colonic CYP24 mRNA expression, but only in the ascending colon. This might result in reduced colonic accumulation of 1,25-D3 during hyperproliferation caused by low dietary calcium and might support site-specific tumorigenesis. The important realization that low dietary calcium by itself is a risk factor for colorectal carcinogenesis and that colonic and renal vitamin D hydroxylases indeed are regulated differently from each other will provide novel approaches for colon cancer prevention.     

Multiple dietary factors in the enhancement of dimethylhydrazine carcinogenesis: main effect of indole-3-carbinol

The effects of multiple dietary influences on 1,2-dimethylhydrazine [(DMH) CAS: 540-73-8]-induced colon cancer in rats were studied. A 2(4) factorial experimental design was used to examine the main and interactive effects of 15% wheat bran (WB), 1% cholesterol (CH) with cholic acid, 20% beef tallow (BT), and 0.1% indole-3-carbinol (IC) on 160 male F344 rats treated ip with DMH (10 mg/kg) weekly for 16 weeks. The test diets were fed for 3 weeks before, 16 weeks during, and 12 weeks after DMH administration. At necropsy, total weight gain, liver and spleen weights, serum CH levels, liver aryl hydrocarbon hydroxylase (AHH) activity, and the size, number, incidence, and location of intestinal tumors were analyzed for dietary factor effects. The most significant inducer of tumors was the combination of CH + BT + IC acting in synergism. The single main effect most responsible for tumor morbidity was IC, which appeared to enhance tumorigenesis via its role as an inducer of AHH activity. The WB decreased tumor incidence and burden when added to diets also containing CH, but it otherwise increased tumor burden per tumor-bearing animal and incidence in all other diets. This study demonstrated the need for examining synergistic and antagonistic interactions among dietary initiators and/or promoters of colon carcinogenesis, as well as implicating IC as a significant factor in the development of DMH-induced tumors in rats.     

Follow-up of tumor development in the colons of living rats and implications for chemoprevention trials: assessment of aspirin-difluoromethylornithine combination

We aimed to establish a reliable procedure allowing the follow-up of tumor development by computed tomographic (CT) colonography in an animal model of colon carcinogenesis in order to assess the chemopreventive efficacy of aspirin and difluoromethylornithine (DFMO) given in combination. Fischer rats received an intraperitoneal injection (25 mg/kg) of dimethylhydrazine (DMH) once a week for two weeks in order to initiate colon carcinogenesis. Five months after the last injection of DMH, a first CT colonography was performed and rats were then randomly separated into two groups (control and experimental). The experimental group received a 0.1% mixture of aspirin and DFMO in drinking water. CT colonography was performed at 6, 7 and 8 months. Data showed a precise correlation between location and size of tumors found at autopsy and those detected by CT colonography at 8 months. All tumors were also detected on the CT views obtained previously. Animals of the aspirin/DFMO group exhibited an inactivation of ornithine decarboxylase, a key enzyme in polyamine biosynthesis, and a two-fold reduction in the prostaglandin E2 content of the colonic mucosa (p<0.01). In rats with tumors at the start of the aspirin/DFMO treatment, a significant slow-down of tumor development was observed. In contrast, in rats where no tumors were detected at the start of the treatment, tumor formation was inhibited. Our data show that CT colonography represents a reliable method to assess in a living animal the efficacy of chemopreventive agents.     

Chemopreventive Effect of Amorphophallus campanulatus (Roxb.) Blume Tuber Against Aberrant Crypt Foci and Cell Proliferation in 1, 2-Dimethylhydrazine Induced Colon Carcinogenesis

Colorectal cancer is one of the leading causes of cancer death, both in men and women. This study investigatedthe effects of Amorphophallus campanulatus tuber methanolic extract (ACME) on aberrant crypt foci (ACF)formation, colonic cell proliferation, lipid peroxidative damage and the antioxidant status in a long term preclinicalmodel of 1, 2-dimethylhydrazine (DMH) induced colon carcinogenesis in rats. Male Wistar rats were dividedinto six groups, viz., group I rats served as controls; group II rats treated as drug controls receiving 250 mg/kg body weight of ACME orally; group III rats received DMH (20 mg/kg body weight) subcutaneously once aweek for the first 15 weeks; groups IV, V and VI rats received ACME along with DMH during the initiation,post- initiation stages and the entire period of the study, respectively. All the rats were sacrificed at the end of 30weeks and the intestinal and colonic tissues from different groups were subjected to biochemical and histologicalstudies. Administration of DMH resulted in significant (p≤0.05) intestinal and colonic lipid peroxidation (MDA)and reduction of antioxidants such as catalase, glutathione peroxidase, glutathione reductase, glutathione-Stransferaseand reduced glutathione. Whereas the supplementation of ACME significantly (p≤0.05) improvedthe intestinal and colonic MDA and reduced glutathione levels and the activities of antioxidant enzymes inDMH intoxicated rats. ACME administration also significantly suppressed the formation and multiplicity ofACF. In addition, the DMH administered rats showed amplified expression of PCNA in the colon and decreasedexpression of this proliferative marker was clearly noted with initiation, post-initiation and entire period ofACME treatment regimens. These results indicate that ACME could exert a significant chemopreventive effecton colon carcinogenesis induced by DMH.