Combination of curcumin and green tea catechins prevents dimethylhydrazine-induced colon carcinogenesis.

The chemopreventive effects of curcumin and green tea catechins individually and in combination on 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis were studied in male Wister rats following 32 weeks of dietary treatment. The incidence, number and size of colorectal cancer were measured. Colorectal aberrant crypt foci (ACF) were analyzed by methylene blue staining. Proliferation indices and apoptotic indices were determined by PCNA immunostaining and TUNEL assay, respectively. The results showed that dietary curcumin, catechins and combination administration significantly inhibited the total number of ACF per rat. The combination treatment displayed the most potent inhibitory effect, while there was no difference of inhibition between curcumin and catechins-treated groups. The incidence of colorectal cancer in the treated groups was significantly lower than that of positive control group. Compared with the positive control group, the proliferation index was significantly decreased and the apoptotic index was significantly increased in all treatment groups, while the effect of the combination was the greatest among the treated groups. Our findings suggest that the combination of curcumin and catechins may produce a synergistic colon cancer-preventative effect that would be more potent than each of the compounds alone.

Inhibition of dimethylhydrazine-induced aberrant crypt foci and induction of apoptosis in rat colon following oral administration of the glucosinolate sinigrin.

Glucosinolates are sulphur compounds that occur as glycosides in brassica vegetables. In response to tissue disruption they are degraded by thioglucosidase, releasing a range of highly reactive breakdown products, including the isothiocyanates, which we have previously shown to be selectively cytotoxic to undifferentiated colorectal tumour cells (HT29). In the present study we explored the effect of sinigrin on the intestinal mucosa of rats previously treated with dimethylhydrazine (DMH). In the first experiment, a semisynthetic feed containing sinigrin (400 microg/g diet) was provided 6 h after the second of two injections of DMH. The level of apoptosis was measured by morphological assessment of intact microdissected crypts obtained at 18, 24, 38, 48 and 72 h after injection, and compared with control groups given DMH only, or a sham-injection. Higher numbers of apoptotic nuclei were present in colonic tissue from both groups of DMH-treated rats compared with the controls, and the level was significantly higher in DMH-treated rats fed sinigrin compared with those given DMH only (P < 0.02). In a second experiment, rats were given sinigrin (400 microg/g diet) 22 h after the second of two injections of DMH; the level of apoptosis was measured after 48 h and the numbers of aberrant crypt foci (ACF) were measured after 42 days. The level of apoptosis was significantly higher in DMH-treated rats given sinigrin compared with controls (P < 0.05), and the numbers of ACF were significantly lower in sinigrin-treated rats (P < 0.001). There was no statistically significant induction of apoptosis in animals fed sinigrin alone. Sinigrin administered after DMH suppresses induction of ACF. This may be due to increased apoptotic deletion of damaged stem cells in the crypts of animals fed sinigrin.

Melatonin and colon carcinogenesis: I. Inhibitory effect of melatonin on development of intestinal tumors induced by 1,2-dimethylhydrazine in rats.

The effect of pineal indole hormone melatonin on colon carcinogenesis was firstly studied in rats. Two-month-old outbred female LIO rats were weekly exposed to 15 (experiment 1, groups 1 and 2) or to five (experiment 2, groups 1 and 2) s.c. injections of 1,2-dimethylhydrazine (DMH) at a single dose of 21 mg/kg of body weight. From the day of the first injection of the carcinogen DMH, the rats from groups 2 (experiments 1 and 2) were given melatonin five days a week during the night-time (from 18:00 h to 8:00 h), dissolved in tap water at 20 mg/l. The experiment was finalized in 6 months after the first injection of DMH. In both experiments the majority of tumors were localized in the descending colon. Tumors of the small intestines developed only in rats from experiment 1. Total incidence of colon tumors as well as tumors in different parts of the colon and the mean number of tumors per rat were much higher in rats from both groups in experiment 1 than that in rats from experiment 2. In experiment 1 melatonin failed to influence the total incidence of colon tumors. However, incidence of carcinomas in the ascending colon was significantly reduced (P < 0.01). The multiplicity of total colon tumors per rat, as well as the mean number of tumors, ascending and descending colon per rat, was also decreased under the influence of melatonin (group 2 vs group 1, P < 0.01). In the same experiment, melatonin slightly decreased the depth of tumor invasion and increased number of highly differentiated colon carcinomas induced by DMH. The percentage of small tumours in the descending colon among rats from group 2 was higher than that of group 1. Treatment with melatonin was also followed by a decrease in the multiplicity of DMH-induced tumors of the duodenum (group 2 vs group 1, P < 0.05) and by a decrease in the incidence of jejunum and ileum tumors (group 2 vs group 1, P < 0.05). In experiment 2, the inhibitory effect of melatonin on DMH-induced colon carcinogenesis was much more expressed than that in experiment 1. Thus, in group 1 the incidence of total colon tumors, ascending and descending colon tumors, was significantly decreased in comparison with group 2; also melatonin reduced the number of tumors per rat in the ascending and descending colon. The number of colon tumors that invaded only mucosa was significantly higher in group 2 than in group 1, P < 0.05. The ratio of highly differentiated tumors was increased (P < 0.05) and the ratio of low-differentiated tumors was decreased (P < 0.05) in rats exposed to melatonin (group 4) as compared with group 3. The number of large size tumors in the ascending and descending colon was decreased whereas the number of small size tumors (<10 mm2) was increased in those parts of the colon that were under the influence of melatonin in experiment 2. Thus, our results demonstrate the inhibitory effect of melatonin on intestinal carcinogenesis induced by DMH in rats.

[The effect of prenatal diethylstilbestrol treatment on 1,2-dimethylhydrazine-induced carcinogenesis in C3HA female mice]. / Vliianie prenatal'nogo vozdeistviia diétilstilbéstrola na kantserogenez, indutsiruemyi 1,2-dimetilgidrazinom u myshei-samok linii C3HA.

C3HA female mice received 0.1-0.3 mg/g body weight diethylstilbestrol (DES) on day 17 of gestation. Their descendants received a total of 20 injections of 1.2-dimethylhydrazine (DMH), subcutaneously, weekly, starting from month 2. Prenatal DES significantly inhibited the development of DMH-induced clitoral gland tumors, particularly, hemorrhagic ovarian cysts which were the frequent cause of death due to their rupture into the abdominal cavity. A significant decrease in cyst incidence resulted in higher survival in mice receiving combined therapy. The results were attributed to DES-induced hyperestrogenization which was confirmed by vaginal smears examination.

Inhibition of 1,2-dimethylhydrazine-induced oxidative DNA damage by green tea extract in rat.

Following subcutaneous injection of 1,2-dimethylhydrazine (DMH), which is carcinogenic to rat colon and liver, to Sprague-Dawley rats, a significant increase of 8-hydroxydeoxyguanosine (8-OHdG) was observed in the DNA of colonic mucosa and liver. The 8-OHdG formation reached the maximal level at about 24 h after the DMII injection. On the other hand, no increase of 8-OHdG was observed in the DNA of the kidney. Drinking green tea extract (GTE) for ten days prior to the DMH injection significantly inhibited the formation of 8-OHdG in the colon. These findings demonstrate that DMH causes oxidative damage to the DNA of its target organ, and that GTE protects colonic mucosa from this oxidative damage.

Inositol and inositol hexaphosphate suppress cell proliferation and tumor formation in CD-1 mice.

In previous studies, we have shown that inositol hexaphosphate (InsP6), a constituent of cereal diet, inhibited azoxymethane-induced experimental large intestinal cancer (LIC) in Fischer 344 rats. We now report a similar antineoplastic action of InsP6 in CD-1 mice injected with 1,2-dimethylhydrazine (DMH). We had hypothesized that InsP6 may bring about this effect by undergoing dephosphorylation to lower phosphorylated forms; the ready availability of Ins, to react with phosphates, may increase the total amount of the lower phosphorylated Ins and potentiate the action of InsP6. LIC induced by DMH (15 mg/kg/week x 13) in mice given a mixture of 1% InsP6 + 1% Ins show a significant reduction (P less than 0.005) in LIC prevalence over InsP6 treatment. Surprisingly, Ins, an in vitro growth promoting agent also caused a significant (P less than 0.001) suppression of LIC. InsP6 +/- Ins also showed a concomitant reduction in the mitotic rate in the non-neoplastic epithelium. Body weight data did not suggest any overt toxic effect of long-term administration of InsP6, Ins or InsP6 + Ins. Since InsP6 is antineoplastic in two species of experimental animals, it should, in combination with Ins, be considered in our strategies for prevention of large intestinal cancer.

1,2-Dimethylhydrazine-induced alterations in N1-acetylspermidine levels in rat distal colonic mucosa: effects of 2-difluoromethylornithine.

Recently, our laboratory has demonstrated that N1-acetylspermidine levels were increased in the distal colonic mucosa of rats administered 1,2-dimethylhydrazine for 15 and 26 weeks. In order to further explore the possible role of this acetylated polyamine in the malignant transformation process induced by this carcinogen, groups of rats were subcutaneously injected weekly with dimethylhydrazine (20 mg/kg body wt.) or diluent for 5, 10, 15 and 26 weeks +/- 1% 2-difluoromethylornithine in the drinking water. The latter agent, an irreversible inhibitor of ornithine decarboxylase, has previously been shown to inhibit colonic tumor formation in this experimental model. At each of these time periods, rats from each group were killed, their proximal and distal colonic mucosa harvested and examined, and compared with respect to polyamine levels, including N1-acetylspermidine, as well as the activities of ornithine decarboxylase, S-adenosylmethionine decarboxylase, spermidine N1-acetyltransferase and polyamine oxidase. The results of these experiments demonstrated that: (1) N1-acetylspermidine levels in the proximal colonic segment of all animals were similar at each time point; (2) N1-acetylspermidine levels were also similar in the distal colons of all animals at 5 and 10 weeks. At 15 weeks, however, the level of N1-acetylspermidine was increased in the dimethylhydrazine-treated distal colonic segment secondary to increases in the activity of spermidine N1-acetyltransferase; and (3) at 26 weeks, the level of this acetylated polyamine remained higher in dimethylhydrazine-treated distal 'uninvolved' colonic mucosa and was markedly elevated in colonic tumors; (4) co-administration of difluoromethylornithine decreased the elevated levels of N1-acetylspermidine to control values in the distal colons of animals treated with carcinogen for 15 and 26 weeks; and (5) difluoromethylornithine markedly reduced the number of tumors induced by dimethylhydrazine in the distal but not proximal colonic mucosa at 26 weeks.

Chloroform inhibition of 1,2-dimethylhydrazine-induced gastrointestinal tract tumors in the Fisher 344 rat.

The effect of chloroform (CHCl3), administered at 0, 900, and 1800 mg/liter in the drinking water, on the carcinogenic potency of 1,2-dimethylhydrazine (DMH) was investigated. Groups of 40 male Fisher 344 rats were given one of the three drinking water solutions for 39 weeks following the subcutaneous injection of 200 mg/kg DMH, a known gastrointestinal (GI) tract carcinogen in this animal strain. When tumors from the GI tract were pooled there was a highly significant (p less than 0.001) decrease in total number of tumors per group with increasing concentration of drinking water CHCl3. In the control group (0 mg/liter CHCl3), 14/39 (36%) of the animals developed tumors of the GI tract, including the duodenum, jejunum, stomach, cecum, and colon. In contrast, the incidence of tumors in the two groups of rats given CHCl3 in the drinking water was significantly lower (p less than 0.001; 900 mg/liter CHCl3, 12.8%; 1800 mg/liter CHCl3, 12.5%). A similar relationship was obtained when colon tumors were analyzed independently (p = 0.01). The incidence of total colon tumors obtained in the control group of this study (10/39, 26%) agrees well with the previous study by B.S. Reddy, K. Watanabe, and J.H. Weisburger (1977, Cancer Res. 37, 4156-4159) conducted in the same rat strain (7/30, 23%). These results demonstrate that CHCl3 in the drinking water inhibits carcinogenesis in the rat GI tract.

Use of polar solvents in chemoprevention of 1,2-dimethylhydrazine-induced colon cancer.

To examine the effect of the polar solvents on 1,2-dimethylhydrazine (DMH)-induced colon cancer, 100 male Sprague-Dawley rats were randomly allocated to a control and three treatment groups. Treated animals received N-methylformamide (NMF), dimethylsulfoxide (DMSO), or methylsulfonylmethane (MSM) added to drinking water 1 week before carcinogen injections commenced and for the duration of the experiment. Primary tumors were detected by serial laparotomy under ether anesthesia performed at 2-month intervals and commencing after carcinogen injections had been completed. The average time to tumor onset was significantly delayed in rats receiving NMF and MSM (P = 0.0141 and 0.0398 respectively, Mantel-Haenszel test). In addition, fewer poorly differentiated tumors were noted in treatment groups. No weight loss or toxicity was observed. These findings demonstrate that the polar solvents significantly reduce the latent period to tumor onset in DMH-induced colon cancer and indicate the need to further investigate such compounds as chemopreventive agents.

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 of garlic oil, inhibits hepatocarcinogenicity of 1,2-dimethylhydrazine (DMH) were examined in male Fischer 344 rats. Rats were subjected to partial hepatectomy to stimulate hepatocellular proliferation required for initiation by DMH (50-200 mg/kg i.p.) given 12 h later. Initiation was assessed by the numbers of foci and nodules of hepatocytes that were positive for gamma-glutamyl transpeptidase (gamma-GT) or glutathione-S-transferase-P (GST-P) after 6 weeks promotion by orotic acid (1% in semi-purified diet). DAS at doses above 50 mg/kg (by gavage) administered 1 h before DMH (50 mg/kg) partially reduced the numbers of gamma-GT and GST-P-positive foci. By comparison, all doses of DAS (25-100 mg/kg) completely prevented liver necrosis by DMH (200 mg/kg). DAS substantially reduced macromolecular binding of [14C]DMH in cultured liver cells, but had no effect on their levels of glutathione-S-transferase, glutathione reductase or glutathione peroxidase at 18 h. These findings suggest that low dosages of DAS which reduce DMH binding appear more likely to inhibit hepatocarcinogenicity by reducing the promoting influences of post-necrotic regeneration than by preventing initiation.

[Effect of ascorbic acid on the induction of uterine sarcomas in mice]. / Vliianie askorbinovoi kisloty na induktsiiu sarkom matki u myshei.

Administration of ascorbic acid (0.3, 0.75 or 1.5%) in drinking water started after the treatment of female CBA mice with 1,2-dimethylhydrazine and estradiol-dipropionate inhibited the growth of uterine sarcomas. The inhibitory effect depended upon dosage to some extent. When administered together with estradiol-dipropionate to intact mice, ascorbic acid arrested uterine growth associated with estrogen treatment.

Chemoprevention of colorectal neoplasms. Ascorbic acid and beta-carotene.

The organospecific, 1,2-dimethylhydrazine-induced rat tumor model was used to test tumor formation in groups of animals receiving regular chow, powdered chow with 7%/wt ascorbic acid supplement, pelleted chow with 1%/wt beta-carotene supplement, and pelleted chow with placebo beadlets. Following a 16-week induction period, animals were killed and tumor formation was recorded. Tumor formation in the ascorbic acid supplement group was found to be significantly less than the control group. The beta-carotene group showed no difference in tumor formation compared with the placebo-beadlet control group. Tumor incidence was generally the same between the two control groups, and the ascorbic acid group had significantly fewer tumors than the beta-carotene group. In sum, ascorbic acid supplements in high doses significantly decreased tumor formation, whereas beta-carotene supplements in moderately high doses had no effect on tumor formation in this model.

Inhibition of 1,2-dimethylhydrazine-induced colon tumorigenesis in Balb/c mice by dehydroepiandrosterone.

Long-term administration of dehydroepiandrosterone (DHEA) to Balb/c mice significantly inhibits the rate of appearance of 1,2-dimethylhydrazine (DMH)-induced macroscopic colon and anal tumors and microscopic precursor and malignant lesions. The steroid, which has previously been shown to inhibit spontaneous breast cancer and chemically induced lung tumors in mice, may find application as a chemopreventive in individuals at high risk for developing colon cancer.

Metabolism of the carcinogen 1,2-dimethylhydrazine by isolated human colon microsomes and human colon tumor cells in culture.

Human colon microsomes catalyze the metabolism of the model colon carcinogen 1,2-dimethylhydrazine. Activity appears to be distributed in a gradient towards the lower end of the colon. Highest activities were observed for microsomes prepared from the descending segment of the colon with the transverse segment exhibiting lower activities, while the ascending segment showed the lowest rate of metabolism. Dimethylhydrazine metabolism in each segment is inhibited significantly by inhibitors of the cytochrome P-450-dependent mixed function oxidase system. Microsomes prepared from a human colon tumor cell also catalyze the metabolism of 1,2-dimethylhydrazine. Metabolic activity in the cell line can be induced two-fold by treatment of cells with phenobarbital and three-fold by treatment of the cells with phenobarbital plus hydrocortisone. These results show that human colon activates 1,2-dimethylhydrazine and suggest that the human colon may be capable of activating other carcinogens in situ.

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.

Interference with dimethylhydrazine induction of colon tumors in mice by epsilon-aminocaproic acid.

The antifibrinolytic agent epsilon-aminocaproic acid given in the drinking water to Swiss ICR/Ha mice significantly counteracted the appearance of colorectal tumors induced by 21 weekly infections of 1,2-dimethylhydrazine. The drug affected both the number and the location of the tumors and, in some animals, altogether prevented their appearance. The low concentrations of epsilon-aminocaproic acid in the plasma of four control mice given the agent labeled with carbon-14 for 3 days suggest that the effect may depend not on inhibition of plasminogen activator activity, but on interference with the binding of some substance to the strong lysine binding site of plasminogen.

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 carcinogenesis evoked by 1,2-dimethylhydrazine (DMH) or N-methyl-N-nitrosourea (MNU) was studied in female F344 rats. Animals were fed diets containing 0, 0.25 and 1% of SA and given s.c. a single dose of 150 mg DMH/kg body wt., 10 weekly s.c. injections of 20 mg DMH/kg body wt. or intra-rectal administration of 2 mg MNU, twice a week for 2 weeks. The incidence of colon and kidney tumors was lower in rats fed the 0.25 or 1% SA and treated with a single dose of DMH than in the animals fed the diet without SA; however, the tumour incidences did not differ between the SA- and control diet-fed animals and treated with multiple doses of DMH or MNU.

Effect of inhibitors of tumorigenesis on the formation of O6-methylguanine in the colon of 1,2-dimethylhydrazine-treated rats.

The level of O6-methylguanine (O6MeGua) in the colonic DNA of rats treated with 1,2-dimethylhydrazine was determined. The effect of various tumorigenesis inhibitors on the formation of this modified base was also studied. Rats were given a single s.c. injection of 1,2-[14C]dimethylhydrazine. Six hr later, they were killed, and colonic DNA was extracted and analyzed by high-pressure liquid chromatography. The inhibitors tested were disulfiram (DSF), pyrazole, sodium selenite, butylated hydroxyanisole, butylated hydroxytoluene, potassium ascorbate, and 13-cis-retinoic acid. The level of O6MeGua in control rats was 29.9 [(O6MeGua X 10(6)/guanine)]. When rats were fed 0.25% (w/w) DSF, this value was reduced to 10.2, and at 0.5% DSF there was no detectable O6MeGua formed. Injection of pyrazole (40 mg/kg i.p.) 2 hr prior to 1,2-dimethylhydrazine treatment reduced the O6MeGua level to 2.4. All the other tumorigenesis inhibitors had no effect on either O6MeGua levels or the cpm/mg DNA in treated rats. With O6MeGua as a measure of the extent of initiation, these results confirm that DSF and pyrazole inhibit the initiation phase of carcinogenesis. This is to be expected as both have been shown to block the metabolism of azoxymethane, which is a crucial metabolite in the activation of 1,2-dimethylhydrazine. The other substances, all known tumorigenesis inhibitors, may act on the promotional phase of carcinogenesis and are worthy of further study for the role in cancer prevention.