BALB/c mice fed milk or beef protein: differences in response to 1,2-dimethylhydrazine carcinogenesis.

The development of 1,2-dimethylhydrazine (DMH)-induced colon tumors and immune responses were investigated in male BALB/c mice fed six different equicaloric diets. Milk or beef at a low (11%) or high (33%) level supplied the dietary protein, and corn oil (primarily) at a low (5%) or high (30%) level supplied the fat. Eleven weekly injections of DMH (at 20 mg/kg mouse) or saline were administered. At 59 weeks of age, the milk-fed mice had a significantly higher (P less than or equal to .05) colon tumor incidence than the beef-fed mice, 67 and 16%, respectively. Tumor volume and colon weight in the milk-fed mice were also significantly greater. Low natural killer cell activity against [125I]5-iodo-2'-deoxyuridine-labeled colon tumor cells and high serum blocking of antitumor cell activity were observed in the milk-fed-mice. These mice also exhibited higher T-lymphocyte cytotoxicity against colon tumor cells. These results differ from those of our previous studies and those of numerous epidemiologic investigations.

Surgical resection of the rat colon: effects on carcinogenesis by 1,2-dimethylhydrazine.

Following a 3-month treatment with 1,2-dimethylhydrazine (DMH) given weekly (sc), outbred Sprague-Dawley rats were subjected to partial resection of the transverse colon. After surgery, DMH treatment was continued for 6 months, after which the rats were killed. At autopsy, 63% of the tumors were found in the transverse colon. By contrast, only 29% of the tumors found in the "sham-operated" control animals occurred in the transverse colon. Surgical trauma in the colon mucosa subjected to the action of carcinogenic agents appeared to promote the conditions suitable for the development of tumors in that organ.

Effect of dioctyl sodium sulfosuccinate feeding on rat colorectal 1,2-dimethylhydrazine carcinogenesis.

The 1,2-dimethylhydrazine (DMH) rodent model for colorectal carcinogenesis was used to explore the effect of dietary dioctyl sodium sulfosuccinate (DSS) on carcinogenesis. Inbred male F344 rats were divided into two groups of 84 each and fed the following diets: ground chow and 5% corn oil (control group) and ground chow, 5% corn oil, and 1% DSS (experimental group). All rats received high-dose DMH base, 20 mg/kg/week sc for 20 weeks. Twenty rats per group were killed after 3, 4, 5, and 6 months. Duodenum, small intestine, colon, and rectum were dissected out. Each tumor was measured for size and location and evaluated histologically. The percentage of rats bearing tumors in the control and experimental groups did not differ significantly. In each rat there were fewer gastrointestinal tumors in the DSS-fed group of all histologic types combined, at all organ sites, at 5 and 6 months. This difference between the control and DSS-fed rats reached the level of statistical significance for tumors of the duodenum, colon, and rectum and for total gastrointestinal tumors at the 5th month.

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.

Comparative study of the morphologic, histochemical, and proliferative changes induced in the large intestine of ICR/Ha and C57BL/Ha mice by 1,2-dimethylhydrazine.

The mouse model of 1,2-dimethylhydrazine (DMH)-induced colorectal carcinogenesis was studied to determine the susceptibility of different anatomic segments of the large intestine in ICR/Ha (susceptible) and C57BL/Ha (resistant) mice. In ICR/Ha mice numerous exophytic macroscopic neoplasms were found in the distal colon and rectum after 15 weekly injections of DMH (20 mg/kg). The proximal colon was free of any microscopic or macroscopic neoplasms. In contrast, C57BL/Ha mice given the same treatment showed no macroscopic neoplasms. However, foci of dysplastic crypts were observed throughout the large intestine of C57BL/Ha mice with highest incidence in the distal colon and rectum. In some areas dysplastic crypts were clearly invading the muscularis mucosae and were, therefore, microscopic carcinomas (microcarcinomas). Thus C57BL/Ha mice were not totally resistant to the neoplastic stimulus of DMH, and the susceptibility of the large intestine is site-specific in both mouse strains.

Methylated DNA adducts in the large intestine of ICR/Ha and C57BL/Ha mice given 1,2-dimethylhydrazine.

The site-specific incidence of 1,2-dimethylhydrazine (DMH)-induced neoplastic changes in intestinal segments of ICR/Ha mice correlates with the persistence of O6-methylguanine (O6MGua) after a single carcinogen injection. Six hours after the injection, the amount of O6MGua in four anatomic (proximal to distal) segments was 16.0, 20.8, 37.5, and 52.8 mumol/mol guanine, respectively. Correlation between the incidence of neoplasms and the amount of alkylation was also observed 14, 40, and 96 hours after DMH treatment. Similar levels of O6MGua were found in the corresponding colon segments of C57BL/Ha mice. After repeated treatment (5 wk) with unlabeled DMH, the amount of O6MGua still correlated with the incidence of neoplasms in ICR/Ha mice. However, in each strain the level of O6MGua was significantly lower in pretreated mice than in mice without DMH pretreatment. Furthermore, the amount of adducts in DNA isolated from different crypt depths showed that within a few hours of the DMH injection the amount of adducts was independent of DNA synthetic activity. Although ICR/Ha and C57BL/Ha mice have different susceptibility to DMH-induced colon cancer, this interstrain difference is not reflected in the amounts or persistence of the miscoding base O6MGua.

Nuclear aberrations as a short-term test for genotoxicity to the colon: evaluation of nineteen agents in mice.

The genotoxicity of 16 agents including several hydrazines, nitrosamines, aromatic amines, polycyclic hydrocarbons, and other related compounds and three known inhibitors of carcinogenesis was assessed in the murine colonic nuclear aberration assay. Of the seven agents considered positive for colonic DNA damage, five were large bowel carcinogens. All structural analogues of the intestinal carcinogens that are tumorigenic for other organs, with the exception of benzo[a]pyrene, were negative in the colonic nuclear aberration assay as were all noncarcinogens tested. The metabolic inhibitor disulfiram completely inhibited 1,2-dimethylhydrazine-induced colonic nuclear damage, while inhibition was less marked for the antioxidants butylated hydroxyanisole and caffeic acid. The versatility of the assay as an indicator of colonic genotoxicity resulting from carcinogen exposure is discussed.

Prolonged antitumor effect of indomethacin on autochthonous intestinal tumors in rats.

Intestinal tumors were induced in randomly propagated Lobund Sprague-Dawley rats by dimethylhydrazine dihydrochloride and by methylazoxymethanol acetate. At 14, 63, and 77 days after exposure to a carcinogen, rats were fed, ad libitum, indomethacin in the drinking water (20 mg/liter) for 20 and 40 weeks. The development of intestinal tumors was prevented or retarded significantly compared to that of control animals. Among control rats at 20, 40, and 52 weeks, the numbers of tumors per rat were relatively constant, but the individual tumors were increased in size.

1,2-Dimethylhydrazine-induced nuclear aberrations in A/J and C57BL/6J mouse colonic crypts.

A single exposure to 1,2-dimethylhydrazine [(DMH) CAS: 540-73-8] produces several forms of aberrant nuclei in the crypts of the murine colon. The frequency of nuclear aberrations (NAs) was examined in the distal colonic crypts in DMH-sensitive A/J mice and relatively DMH-resistant C57BL/6J mice before and after a single exposure to DMH. NAs, mitotic figures, and crypt column heights were scored for all animals as a function of time following administration of DMH. In both strains there was a significant increase in the absolute and relative frequency of NAs by 12 hours, with a corresponding drop and subsequent overshoot in the mitotic index by 48 hours after DMH. The temporal changes in crypt column height correlate closely with the temporal changes in frequency of NAs in both strains. The results showed that both inbred strains respond to acute DMH exposure in a similar and parallel fashion over time. It was concluded that the NA index assay is a sensitive method for detecting early DMH exposure. However, this assay does not relate to ultimate outcome after chronic DMH exposure and should not be used as a predictor of eventual neoplastic transformation of colonic mucosa with this carcinogen.

Morphology and distribution of 1,2-dimethylhydrazine dihydrochloride-induced colon tumors and their relationship to gut-associated lymphoid tissue in the rat.

The histopathology and relationship of sym-dimethylhydrazine dihydrochloride [(DMH) CAS: 306-37-6; 1,2-dimethylhydrazine dihydrochloride]-induced colon tumors to colonic lymphoid aggregates were examined in outbred male Sprague-Dawley rats treated with saline or DMH and sacrificed at three intervals after treatment. The ratio of polypoid:sessile tumors was 71:29 four months after DMH treatment and 62:38 when tumors were fully developed. Colonic lymphoid aggregates were found 3-5 cm from the cecal-colonic junction, near the flexure of the ascending and transverse colon, and 3-5 cm from the rectum. There were no significant differences between saline-treated and DMH-treated rats regarding the size, cellularity, and number of lymphoid aggregates per rat. A significant association (P less than .001) was seen between tumor development and the presence of a lymphoid aggregate in a given segment of the colon. Sessile adenocarcinomas, but not polypoid tumors, were significantly associated (P less than .001) with lymphoid aggregates and usually presented as mucinous tumors adjacent to or intermixed with the lymphoid tissue.

Changes in crypt cell populations of mouse colon during recovery from treatment with 1,2-dimethylhydrazine.

The induction of adenocarcinomas in distal colons of noninbred female Swiss Webster white mice by 1,2-dimethylhydrazine (DMH) is preceded by the development of hyperplasia of mucosal glands, which includes absolute increases in both the total number of cells and labeled cells per crypt. This experiment was designed to determine if these changes are reversible. Groups of noninbred female Swiss Webster white mice were given weekly injections of DMH, 20 mg/kg body weight, for 1--20 weeks and killed 1--52 weeks after treatment. Animals were pulsed with tritiated thymidine ([3H]dTHd) for 1 hour and autoradiographs were made of sections of distal colon. The total number of cells per crypt and number of [3H]dThd-labeled cells per crypt were significantly elevated in animals killed 1 week after termination of treatment. With increasingly longer periods of recovery, base-line values were reached rapidly in animals given one or two treatments; however, reduction in cells was slower and less complete in animals given eight or more treatments. The latency before tumors developed was inversely related to the number of treatments. Animals that received 20 treatments developed more tumors per tumor-bearing animal than did animals given fewer treatments but allowed by survive for long periods. The findings indicate that once induced changes in the mucosa progress to a threshold level, mucosal glands are irreversibly enlarged and the development of tumors is irrevocable.

Effects of sublethal irradiation on changes in crypts of the mouse colon during treatment with 1,2-dimethylhydrazine.

The effects of low-dose X-irradiation on the progression of early neoplastic changes induced in the colon by 1,2-dimethylhydrazine (DMH) were assessed. Outbred female Swiss-Webster mice were treated with DMH only, DMH-irradiation, irradiation only, or 0.001 M EDTA. Animals were pulsed with [3H]thymidine for 1 hour, and the distal colon was radioautographed. The number of labeled cells per crypt in animals tested with DMH-irradiation was greater (P less than 0.01) than the number in "DMH-only" animals, but a corresponding increase in the length of the crypts did not occur. After 16 weeks of treatment, the percentage of animals with focal atypia was similar in the DMH-irradiation group (87%), and the DMH-only group (100%), but there were more focal atypias per animal in the DMH-only group. After 20 weeks, 14% of the DMH-irradiation group had visible tumors compared to 63% of the DMH-only group. The results suggest that DMH increased the number of cells at risk for irradiation; thus the surge in proliferation subsequent to cell depletion was amplified. However, the development of precancerous lesions was not promoted.

Early histopathologic events to evolution of colon cancer in C57BL/6 and CF1 mice treated with 1,2-dimethylhydrazine.

After administration of the intestinal carcinogen 1,2-dimethylhydrazine (DMH), C57BL/6J and CF1 mice were observed for early precursor lesions to large bowel cancer. Among the initial events seen following DMH treatment, an abrupt reduction in colonic DNA synthesis was the earliest lesion detectable. The frequency of aberrant colonic nuclei rose shortly after DMH treatment, reaching a maximum value 24 hours later and remaining elevated for 3 days following the exposure. Mucin changes, detected histochemically, and cell kinetic alterations in crypt proliferation rates were observed much later and were a constant feature for both strains following 4 weekly treatments with DMH, while carcinomas appeared in all animals 32 weeks after the start of DMH treatment. The quantitative comparison of these histopathologic observations for the early detection of colon cancer suggests that the induction of colonic nuclear aberrations in the mucosa of the large bowel might provide a sensitive and rapid indication of genotoxicity to this organ and thus might provide the basis for a screening methodology for colon carcinogens.

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.

Carcinogenicity of deuterium-labeled 1,2-dimethylhydrazine in mice.

To study the effect of deuterium substitution on the carcinogenicity of 1,2-dimethylhydrazine (DMH) in mice, two comparisons were made between DMH and its fully methyl-deuterated analogue, [2H6]DMH. In a lifetime study with the CBA strain, groups of 19-30 animals of each sex were dosed s.c. weekly with 8 mg/kg of either DMH or [2H6]DMH for 8, 16, or 32 weeks. In the second study, female CF-1 mice were given DMH or [2H6]DMH in 10 weekly s.c. doses of 12 mg/kg each (13.2 mg/kg for [2H6]DMH) and examined for colon tumors 36 weeks after the first dose. Deuteration significantly decreased tumor incidence in the colon of males (P less than 0.01) and the anal tissue of both sexes (P less than 0.05) but increased that of hepatomas and lung tumors in males (P less than 0.01). Substrate deuteration did not significantly affect overall incidence of any other tumor type, however, including hemangioendotheliomas and kidney tumors in both sexes, as well as colon, uterine, ovarian, liver, and lung tumors in females. The results indicate that C--H bond breakage is kinetically important in the activation of DMH to its ultimately carcinogenic form in organs such as the male colon (relative risk in DMH-versus [2H6]DMH-treated animals approximately equal to 6), and that inhibition of this process by substrate deuteration allows a diversionary mechanism having a smaller isotope effect to become relatively more extensive. The qualitatively different effect in other organs (e.g., kidney, relative risk approximately equal to 1) supports recent suggestions that the net mechanism of activation can differ from one target tissue to another, possibly by striking a different balance between parallel metabolic pathways. The lack of a significant isotope effect on overall colon tumor incidence in females of either strain suggests that differences in relative importance among competing enzymes may also be responsible for sexual dimorphism in tumor induction by DMH.

Effect of two kinds of pectin and guar gum on 1,2-dimethylhydrazine initiation of colon tumors and on fecal beta-glucuronidase activity in the rat.

The effect of 5% low-methoxylated pectin, high-methoxylated pectin, and guar gum on 1,2-dimethylhydrazine initiation of colon cancer was investigated using groups of 30 rats. The growth of the rats in the different groups was very similar to that of control group fed a fiber-free diet. Both kinds of pectin increased the multiplicity of color tumors, whereas guar gum did not significantly influence carcinogenesis. Bacterial beta-glucuronidase activity in feces and colonic content was the same in pectin-fed rats and controls but significantly lower in the guar gum group. Thus, it was not related to the number of tumors in each group.

Selenium inhibition of 1,2-dimethylhydrazine-induced colon carcinogenesis.

The inhibitory effect of selenium (Na2SeO3) on 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis in male Sprague-Dawley rats is presented. A 4-ppm selenium supplement to the drinking water was provided concurrently with DMH treatment and continued until death or sacrifice. Rats were administered 10 weekly injections of 10 mg DMH per kg body weight. Thirtyone weeks following the tenth DMH injection, all surviving animals were sacrificed. At sacrifice, the colon tumor incidence in DMH-only controls was 8 of 28 (29%). Selenium supplementation significantly (p less than 0.01) reduced the colon tumor incidence to 1 of 37 (3%). The cumulative colon tumor incidence for all animals found dead or sacrificed was also significantly (p less than 0.05) reduced from 11 of 40 in DMH controls to 3 of 40 in DMH-selenium-supplemented rats. The total number of colon tumors was reduced from 13 to 3, and the average number of tumors per rat from 1.2 to 1.0 by supplemental selenium. The majority (greater than 65%) of all tumors were located in the distal colon. The serum glutamic oxaloacetic transaminase, alkaline phosphatase, and complete blood count were normal and equivalent for the DMH only, DMH-selenium, and untreated control groups in this study. The glutathione S-transferase activity in liver cytosol preparations was increased from 39.6 +/- 7.3 (S.D.) microM product/min/mg (DMH only) to 67.6 +/- 5.8 microM product/min/mg by selenium only and to 54.3 +/- 10.6 microM product/min/mg in selenium-DMH-treated rats. Protection by selenium may in part be attributed to enhanced detoxification of carcinogenic electrophiles.

Induction of tumors in mice with the herbicide succinic acid 2,2-dimethylhydrazide.

A solution of 2% succinic acid 2,2-dimethylhydrazide was given continuously in the drinking water of 6-week-old randomly bred albino mice for the remainder of their lives. The treatment gave rise to tumors of blood vessels, lungs, and kidneys. The tumor incidences in these tissues in the controls were 6, 18, and 0%, whereas in the treated groups the corresponding tumor incidences were 73, 73, and 5%. Light microscopic examination revealed typical angiomas and angiosarcomas of blood vessels, adenomas and adenocarcinomas of lungs, and adenomas of kidneys. The study thus demonstrates the tumorigenicity of the herbicide, succinic acid 2,2-dimethylhydrazide. Since the residues of this chemical occur in fruit, the human population is exposed to it. The environmental implication of this finding and the fact that the hydrazines as a class have tumorigenic properties are discussed.

Site-specific induction of nuclear anomalies (apoptotic bodies and micronuclei) by carcinogens in mice.

The usefulness of nuclear anomalies (NA) as a short-term test for indication of carcinogens in the mouse colon has been suggested previously by experiments in which colon-specific carcinogens induced NA in the colon, whereas non-colon carcinogens were, in general, impotent in that organ. We have extended this work to other sites in the digestive tract of female C57BL/6 mice treated with gamma-rays, 1,2-dimethylhydrazine dihydrochloride, or N-methylnitrosourea. Each agent induced NA at all of the sites examined. The frequency of NA at different times after treatment depended upon both the agent used and the site examined. 1,2-Dimethylhydrazine dihydrochloride (which is known to induce tumors predominantly in the colon) induces NA with the highest efficiency (relative to gamma-rays) in the descending colon. N-Methylnitrosourea (which induces tumors mainly in the forestomach) induces NA with the highest efficiency in the forestomach. These results further support the usefulness of the assay in that the frequency of NA produced at the various sites by 1,2-dimethylhydrazine dihydrochloride and N-methylnitrosourea correlates with that found in the carcinogenicity studies.

Effect of alterations in the quality and quantity of dietary fat on 1,2-dimethylhydrazine-induced colon tumorigenesis in rats.

The effect of alterations in the quality and quantity of dietary fat on 1,2-dimethylhydrazine-induced colon cancer in rats was studied. Weanling Sprague-Dawley rats were fed semipurified diets containing 24% beef fat, 24% corn oil, 24% Crisco, or the three fats in equal parts to make a total of 5% fat with other macronutrients and micronutrients adjusted to balance the ratios of nutrient to calorie. After 4 weeks of dietary treatment, all rats, except vehicle-treated animals, received 1,2-dimethylhydrazine (15 mg/kg) by gavage, once a week for 5 weeks. The animals were fed the experimental diets until intestinal tumors developed, and surviving animals were sacrificed at 60 weeks. There was no effect of any of the high-fat diets tested on intestinal tumor incidence, latency, size, or frequency. All groups contained the same proportion of adenomas (less than 3%) as well as adenocarcinomas classified as mucinous. In the group fed 24% Crisco, tumors occurred with greater frequency in the proximal section of the colon than in lower segments, but the distribution was approximately uniform in the other groups. Cumulative probability of death with colon carcinoma was lowest in the 24% Crisco group, but the other high-fat groups did not differ significantly from the 5% mixed fat group nor from one another.