Differential susceptibility of 3 sublines of C57BL6 mice to the induction of colorectal tumors by 1,2-dimethylhydrazine

Eight-week-old mice of 3 sublines of strain $\text{C57BL}\text{6}$ were given s.c. injections of 1,2-dimethylhydrazine (DMH), once weekly for 10 weeks. The highest incidence (85%) of colorectal tumors occurred in $\text{C57BL}\text{6N}$ mice. Colorectal tumors occurred in 43% of $\text{C57BL}\text{6}\text{J}$ mice, while only 3 (10%) $\text{C57BL}\text{6}\text{Ha}$ mice developed these tumors. Possible factors responsible for the differential susceptibility of 3 sublines of $\text{C57BL}\text{6}$ mice to the induction of colorectal tumors by DMH are discussed.     

Colonic neoplasms in mice produced with six injections of 1,2-dimethylhydrazine

A highly effective but reduced injection schedule for the induction of the colon cancer in CF1 female mice is reported using 6 subcutaneous inoculations of 1,2-dimethylhydrazine at a dose level of 20 mg/kg body weight. Between the 20th and 45th week of the experiment, 83% of the animals were tumor-bearing with a frequency of 2.1 colonic neoplasms macroscopically visible per mouse. Tumors were isolated in nature and primarily located in the distal large bowel. Some degree of carpeting of the colonic mucosa and uncountable numbers fo tumors occurred in 30% of mice and these areas of confluent neoplasms also occurred predominantly in the distal colon. This spectrum of distribution of tumors closely parallels that seen in man thus enhancing the value of this time conserving colon tumor model.     

Target tissue DNA damage in inbred mouse strains with different susceptibility to the colon carcinogen 1, 2-dimethylhydrazine

We have compared liver, kidney and colon DNA damage, as singlestrand breaks, in mice with different strain-dependent susceptibilityto the colon-specific carcinogen 1, 2-dimethylhydrazine (DMH).The mouse strains studied were: AKR/J, DBA2 totally resistant;CD1, C57BL/6N moderately susceptible; SWR/J very susceptibleto DMH-induced carcinogenesis. DNA breaks were estimated fromthe elution rate constant (K) according to the alkaline elutiontechnique. At 4 h after carcinogen administration a substantialand comparable DNA damage was found in liver and kidney in allthe strains examined. The DNA fragmentation index, however,reached a maximum value at 2 h after treatment in the liverof the most susceptible strain (SWR/J). About 50% of the liverDNA damage detected in all five strains 4 h after DMH administrationpersisted at 24 h after treatment and was totally repaired at72 h. Kidney DNA damage decreased in 48 h toward the range ofcontrol values. In colon epithelial cells (the carcinogen targettissue) 2 and 4 h after DMH administration the amount of DNAsingle strand breaks was correletable with the strain sensitivityto the carcinogen. In the time interval studied (2–72h after DMH administration) the decrease of colon DNA damagewas linear in the resistant strains. In contrast, in the moresusceptible strain (SWR/J), the amount of DNA breaks remainedhigh up to 24 h after treatment and returned to background levelat 72 h.     

Gastric tumor induction by 1,2-dimethylhydrazine in Wistar rats with intestinal metaplasia caused by X-irradiation.

Five-week-old male Wistar rats were X-irradiated with a total of 20 Gy in 2 equal fractions at a 3-day interval. 1,2-Dimethylhydrazine (DMH) solution was injected i.m. into the back musculature at a dose of 20 mg/kg body weight weekly for 10 weeks, beginning 20 weeks after the final irradiation. Twelve months after the initial carcinogen treatment, tumors in the fundus of the glandular stomach were observed in 5 of 23 animals receiving both X-irradiation and DMH treatment. No tumors of the glandular stomach were observed in the DMH and X-ray alone or nontreatment groups. It is concluded that the presence of intestinal metaplasia may increase sensitivity to the induction of gastric tumors by carcinogens like DMH.     

Metabolism of 1,2-dimethylhydrazine by cultured human colon

The overall metabolism of 1,2-dimethylhydrazine, an organotropiccolon carcinogen in rodents, has been studied using human colonexplant cultures. The binding level of 1,2-dimethylhydrazineto DNA which in this study includes both reaction of metaboliteswith DNA and incorporation of radioactive metabolites into DNA,showed a 100-fold variation among the 120 people studied. Whendifferent anatomical colonic sites were compared, the highestmean binding levels were found in the ascending and sigmoidcolon. No significant difference in the median and mean bindinglevels were observed in nontumorous colon obtained surgicallyfrom patients with colon cancer and colon obtained from immediateautopsy, but decreased mean binding levels were seen in tissuesobtained by surgery from patients with non-cancerous colonicdisorders. Several exogenous chemicals were found to modifythe metabolism. When the colon ex-plants were co-incubated with1,2-dimethylhydrazine and these chemicals, the binding levelof 1,2-dimethylhydrazine to DNA was (a) increased by eitherindole 3-carbinol or phenobarbital, (b) decreased with disulfiram,butylated hydrox-ytoluene, or taurodeoxycholic acid, and (c)unaltered by lithocholic acid.     

The alkylation of nucleic acids of rat and mouse in vivo by the carcinogen 1,2-dimethylhydrazine

1,2-Dimethylhydrazine, in contrast to 1-methylhydrazine, is a potent carcinogen for the colon in rats and mice. 1,2-[¹⁴C]Dimethylhydrazine was administered to rats and mice in doses which are carcinogenic following a single dose in the former species, or carcinogenic on repeated administration in the latter species, and the rate of ¹⁴CO₂ exhalation was measured. Exhalation of ¹⁴CO₂ was also studied after administration of single doses of 1-[¹⁴C]methylhydrazine to mice. Incorporation of radioactivity into the nucleic acids of a variety of organs was found at a time after injection (about 6 h) when ¹⁴CO₂ production from both compounds was virtually complete. Methylation of nucleic acids of liver and colon, as indicated by the formation of 7-methylguanine, was observed after treatment with 1,2-dimethylhydrazine and to a smaller extent by a factor of about 10 after treatment with 1-methylhydrazine. Less than 1% of a single dose of 1,2-[¹⁴C]dimethylhydrazine was excreted in the bile of rats as determined by chemical and radioactivity assays. The similarities of the biological and biochemical actions of 1,2-dimethylhydrazine with those of some nitroso compounds and of cycasin (methylazoxymethanol glucoside) are emphasized.     

Rapid induction of colorectal tumors in rats initiated with 1,2-dimethylhydrazine followed by dextran sodium sulfate treatment

To establish a rapid bioassay system with neoplastic end-points for detection of colorectal carcinogenesis modifiers, we evaluated the effects of dextran sodium sulfate (DSS) treatment on the different stages of carcinogenesis in rats initiated with 1,2-dimethylhydrazine (DMH). F344 male rats were given three subcutaneous injections of DMH (40 mg/kg body weight) in a week, and were administered drinking water containing 1.0% DSS ad libitum either during or after the initiation period for a week, or both during and after initiation periods for 2 weeks. At the 10th week of the experiment, although the numbers of aberrant crypt foci were significantly decreased in all groups treated with DSS and given DMH-initiation as compared with DMH alone, dysplastic foci/adenomas/adenocarcinomas were increased. The incidences and multiplicities of these lesions were highest in rats treated with DSS after DMH-initiation period. At the 26th week, the incidences of adenocarcinomas (100 vs. 20% in DMH alone) and their multiplicities (6.6 +/- 0.8/rat vs. 0.2 +/- 0.4/rat in DMH alone) were also highest in this group. These results indicate that short-term DSS-treatment in the post-initiation period significantly accelerates DMH-induced colorectal tumor development in rats, so that this protocol may effective for establishment of a rapid bioassay system with neoplastic end-points.     

The induction of cancer of the large intestine in Macaca fascicularis monkeys with 1,2-dimethylhydrazine

Continuous subcutaneous administration of 16 mg/kg body weight 1,2-dimethylhydrazine three times a month (total dose--1080-3696 mg) to Macaca fascicularis monkeys induced cancer invariably confined to the colon within 34-47 weeks. Biologic, clinical, histologic features and natural course of the tumor proved similar to those of its human counterpart.     

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.      

Beta-Catenin mutations in a mouse model of inflammation-related colon carcinogenesis induced by 1,2-dimethylhydrazine and dextran sodium sulfate

In a previous study, we developed a novel mouse model for colitis-related carcinogenesis, utilizing a single dose of azoxymethane (AOM) followed by dextran sodium sulfate (DSS) in drinking water. In the present study, we investigated whether colonic neoplasms can be developed in mice initiated with a single injection of another genotoxic colonic carcinogen 1,2-dimethylhydrazine (DMH), instead of AOM and followed by exposure of DSS in drinking water. Male crj: CD-1 (ICR) mice were given a single intraperitoneal administration (10, 20 or 40 mg/kg body weight) of DMH and 1-week oral exposure (2% in drinking water) of a non-genotoxic carcinogen, DSS. All animals were killed at week 20, histological alterations and immunohistochemical expression of beta-catenin, cyclooxygenase (COX-2) and inducible nitric oxide synthase (iNOS) were examined in induced colonic epithelial lesions (colonic dysplasias and neoplasms). Also, the beta-catenin gene mutations in paraffin-embedded colonic adenocarcinomas were analyzed by the single strand conformation polymorphism method, restriction enzyme fragment length polymorphism and direct sequencing. The incidences of colonic neoplasms with dysplastic lesions developed were 100% with 2.29+/-0.95 multiplicity, and 100% with 10.38+/-4.00 multiplicity in mice given DMH at doses of 10 mg/kg or 20 mg/kg and 2%DSS, respectively. Although approximately half of the mice given DMH at a dose of 40 mg/kg bodyweight were dead after 2-3 days after the injection, mice who received DMH 40 mg/kg and 2%DSS had 100% incidence of colonic neoplasms with 9.75+/-6.29 multiplicity. Immunohistochemical investigation revealed that adnocarcinomas, induced by DMH at all doses and 2%DSS, showed positive reactivities against beta-catenin, COX-2 and iNOS. In DMH/DSS-induced adenocarcinomas, 10 of 11 (90.9%) adenocacrcinomas had beta-catenin gene mutations. Half of the mutations were detected at codon 37 or 41, encoding serine and threonine that are direct targets for phosphorylation by glycogen synthase kinase-3beta. The present results suggests that, as in the previously reported model (AOM/DSS) our experimental protocol, DMH initiation followed by DSS, may provide a novel and useful mouse model for investigating inflammation-related colon carcinogenesis and for identifying xenobiotics with modifying effects.     

Tumours of the anal region induced in mice by 1,2-dimethylhydrazine

CBA female mice treated with 1,2-dimethylhydrazine developed a high incidence of benign and malignant tumours in the anal region. Many of these tumours originated from the perianal glands rather than the epidermis.     

Apoptosis (cell death) induced in mouse bowel by 1,2-dimethylhydrazine, methylazoxymethanol acetate, and gamma-rays

Apoptosis is a pattern of cell death involving nuclear pyknosis, cytoplasmic condensation, and karyorrhexis. The frequency of apoptosis after treatment with two colon carcinogens and radiation was studied in the crypts of five different portions of mouse bowel. When 1,2-dimethylhydrazine (DMH) was injected s.c., the earliest rise in apoptotic incidence after a high dose (200 mg/kg) was noted at 3 h in small intestine and at 6 h in large bowel. After i.p. administration of methylazoxymethanol (MAM) acetate, apoptotic cells were seen in large bowel after 3 h. When the plateau values attained after high doses of DMH were compared, many apoptotic cells were found in the lower part of the large bowel, whereas few such cells were observed in the small intestine and the upper part of the large bowel. This finding was reversed in the case of radiation-induced apoptosis. In the descending colon, a definite circadian rhythm in the apoptotic incidence was observed 6 h after injection of DMH or MAM acetate. Apoptosis showed a high incidence when these drugs were given between 2400 h and 0900 h, but a low incidence after administration between 1200 h and 2100 h. In the small intestine a rhythm was also noted for MAM acetate, but not significantly for DMH.     

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

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

Inhibitory effects of freeze-dried milk fermented by selected Lactobacillus bulgaricus strains on carcinogenesis induced by 1,2-dimethylhydrazine in rats and by diethylnitrosamine in hamsters

Fermented milk products might be used for cancer chemoprevention due to their putative anticarcinogenic and antitumor activities. The diet was supplemented with freeze-dried milk fermented by Lactobacillus bulgaricus strain LBB.B 144 (product FFM.B 144) added throughout the experiment at doses of 1.3 g and 2.5 g per rat, 5 times a week starting 3 weeks before the first carcinogen injection. This treatment significantly inhibited, by 26.2-28.6% and by 34.2%, the total intestinal carcinogenesis induced by 1,2-dimethylhydrazine (DMH, 21 mg/kg, s.c., once per week for 20 weeks) in male and female BD6 rats, respectively. FFM.B144 decreased the tumor incidence and multiplicity in large bowel, caecum, and duodenum. Protective effects were better expressed in female animals, with exception of that observed in duodenum. Supplementation of diet with freeze-dried milk fermented by Lactobacillus bulgaricus strain LBB.B5 (product FFM.B5) inhibited DMH-induced carcinogenesis only in the large bowel, but had no significant protective effect when all intestinal tumors were taken into account. However, both freeze-dried products favorably shifted the differentiation of large bowel tumors by increasing the proportion of benign and highly differentiated malignant tumors and decreasing in parallel the number of poorly differentiated carcinomas without influencing the tumor size. A lower number of cases with visible mesenterial metastasis was also observed in FFM-treated rats. In addition, both FFM.B 144 and FFM.B5 significantly inhibited, by 26-33%, the induction in the same rats of ear-duct tumors. FFM.B144 but not FFM.B5 was also effective in inhibiting the tracheal carcinogenesis induced in Syrian golden hamsters by diethylnitrosamine (DEN, 100 mg/kg, two s.c. injections), the protective effect being better expressed in female animals. The anticarcinogenic potential of some fermented milk products might be exploited in chemoprevention of cancer in humans.     

Inhibition of hepatocarcinogenic responses to 1,2-dimethylhydrazine by diallyl sulfide, a component of garlic oil

The mechanisms by which diallyl sulfide (DAS), a component ofgarlic oil, inhibits hepatocarcinogenicity of 1,2-dimethylhydrazine(DMH) were examined in male Fischer 344 rats. Rats were subjectedto partial hepatectomy to stimulate hepatocellular proliferationrequired for initiation by DMH (50–200 mg/kg i.p.) given12 h later. Initiation was assessed by the numbers of foci andnodules of hepatocytes that were positive for -glutamyl transpeptidase(-GT) or glutathione-S-transferase-P (GST-P) after 6 weeks promotionby orotic acid (1% in semi-purified diet). DAS at doses above50 mg/kg (by gavage) administered 1 h before DMH (50 mg/kg)partially reduced the numbers of -GT and GST-P-positive foci.By comparison, all doses of DAS (25–100 mg/kg) completelyprevented liver necrosis by DMH (200 mg/kg). DAS substantiallyreduced macromolecular binding of [¹⁴C]DMH in cultured livercells, but had no effect on their levels of gluta-thione-S-transferase,glutathione reductase or glutathione peroxidase at 18 h. Thesefindings suggest that low dosages of DAS which reduce DMH bindingappear more likely to inhibit hepatocarcinogenicity by reducingthe promoting influences of post-necrotic regeneration thanby preventing initiation.     

Dietary resistant starch type 3 prevents tumor induction by 1,2-dimethylhydrazine and alters proliferation, apoptosis and dedifferentiation in rat colon

Some epidemiological and experimental studies suggest that consumption of resistant starch is preventive against colon cancer. Resistant starch leads to a fermentation-mediated increase in the formation of short-chain fatty acids, with a particularly high butyrate fraction in large bowel. Butyrate is considered to be protective against colon cancer because it causes growth arrest and apoptosis and regulates expression of proteins involved in cellular dedifferentiation in various tumor cell lines in culture. We sought to investigate these processes under conditions of a carcinogenicity experiment in vivo. In the present study, 1,2-dimethylhydrazine-treated Sprague-Dawley rats were fed standard diet (n=12) or diet with 10% hydrothermally modified Novelose 330, a resistant starch type 3 (RS3), replacing digestible starch (n=8). After 20 weeks tumor number, epithelial proliferation, apoptosis, immunoreactivity of carcinogenesis-related proteins [protein kinase C-delta (PKC-delta), heat shock protein 25 (HSP25) and gastrointestinal glutathione peroxidase (GI-GPx)], as well as mucin properties were evaluated in proximal and distal colon in situ. No tumors developed under RS3 diet, compared to a tumor incidence of 0.6+/-0.6 (P<0.05) under the standard diet. RS3 decreased the number of proliferating cells, the length of the proliferation zone and the total length of the crypt in the distal colon, but not proximal colon, and enhanced apoptosis in both colonic segments. It induced PKC-delta and HSP25 expression, but inhibited GI-GPx expression in the epithelium of distal colon. RS3 increased the number of predominantly acidic mucin containing goblet cells in the distal colon, but had no effect on the goblet cell count. We conclude that hydrothermally treated RS3 prevented colon carcinogenesis, and that this effect was mediated by enhanced apoptosis of damaged cells accompanied by changes in parameters of dedifferentiation in colonic mucosa.     

Enhancing effect of vitamin E on murine intestinal tumorigenesis by 1,2-dimethylhydrazine dihydrochloride

The effect of the antioxidant vitamin E on the tumor-inducing ability of 1,2-dimethylhydrazine dihydrochloride (1,2-DMH) was investigated in randomly bred Swiss mice. Three groups of mice that were 6 weeks of age at the beginning of the experiment received the following treatments: a) vitamin E acetate [DL-alpha-tocopheryl acetate (TA)] at a 4% dose level in a powdered diet for life; b) 1,2-DMH, 10 weekly sc injections at 20 micrograms/g body weight; c) combination of a and b treatments. The administration of TA enhanced the tumorigenicity of 1,2-DMH, as evidenced by statistically significant incidences of tumors in the duodenum, cecum, colon, rectum, and anus. The present finding apparently is in contrast with the reported inhibitory effect of TA on colon carcinogenesis by 1,2-DMH.