Fecal mutagens and Bacteroides fragilis levels in the feces of dimethylhydrazine-treated rats: influence of diet

In a study designed to investigate the effects of dietary synergisms on 1,2-dimethylhydrazine (DMH)-induced rat colon carcinogenesis, fecal pellets were examined for the presence of direct-acting fecal mutagens and levels of Bacteroides fragilis group organisms. Intraperitoneal injections of DMH at 10 mg/kg were given for 16 weeks (weeks 3-18) to 160 male F344 rats consuming 4 supplemental dietary factors in all possible combinations. The dietary factors examined were wheat bran (15%), cholesterol (1%), beef tallow (18%) and indole-3-carbinol (IC) (0.1%). Feces were collected 3, 10, 17, 24 and 31 weeks after commencing the dietary treatments and dichloromethane extracts were assayed using the Salmonella typhimurium TA100 without metabolic activation. The numbers of B. fragilis group organisms were enumerated in feces collected at the same time. Most feces samples were negative for mutagens but extracts from weeks 17-31 showed a significant mutagenic response from the IC factor in the diet. The fecal levels of B. fragilis were significantly increased by the inclusion of cholesterol in the diets. The B. fragilis counts and fecal mutagen production were not correlated (r = 0.09), although species of the B. fragilis group have been implicated in the production on human fecal mutagens.     

Pathological features of the colonic tumours induced in rats by the administration of 1,2-dimethylhydrazine.

The parenteral administration of 1,2-dimethylhydrazine to rats caused the development of colonic neoplasms in about 90% of animals by 24--30 weeks of treatment. Usually there were multiple tumours with a mean of 2.7 per rat. The lesions have been classified histologically into adenomata (26% of all tumours) and carcinomata, the latter showing varying degrees of differentiation. No completely anaplastic tumours were seen, and there were none originating in connective tissue. The distributions of the different tumour types along the length of the colon varied. The more benign lesions were situated predominantly in the distal half of the colon, while the poorly differentiated adenocarcinomata were concentrated in the proximal third of the colon. There was good evidence to suggest that adenomata often progressed to frank malignancy in the distal colon. In the proximal part, however, it appeared that tumours frequently developed de novo as poorly differentiated carcinomata. Perhaps regional variations in the kinetic organisation of the normal colonic mucosa somehow influence the nature of the neoplastic change induced by DMH, thus accounting for the differences in tumor distribution. After 24 weeks of DMH treatment there was only a small increase in the mean number of tumours per rat.     

Intervention of Acidophilus-casei dahi and Wheat bran against molecular alteration in colon carcinogenesis

An in vivo trial was conducted on seventy five rats allocated to three groups, first group was DMH control group, second group was Wheat bran-DMH group (WB-DMH) in which wheat bran was given along with DMH (1,2-dimethylhydrazine) injection, third group was Wheat bran-DMH-Ac Dahi group in which both wheat bran and Acidophilus-casei dahi (a probiotic microorganisms fermented dairy product) was given along with DMH injections. Animals received subcutaneous injections of DMH at a dose rate of 20 mg/kg body weight, once weekly for 15 weeks. The rats were dissected at 40th week of experiment and comet assay was done in colonic cells to assess the DNA damage. The c-myc and cox-2 expression was studied in rat tumour. A significant reduction in DNA damage (48.2%) was observed in WB-DMH-Ac Dahi group as compared to DMH control group (87.8%). The c-myc and cox-2 mRNA level was found highest in DMH control group as compared to WB-DMH and WB-DMH-Ac Dahi group. The results of present study show the enhanced protective potential of Acidophilus-casei and wheat bran against DMH induced molecular alteration in colonic cells during carcinogenesis.     

Aspirin suppresses 1,2-dimethylhydrazine-induced alteration of proliferative parameters in rat colonic crypts

Abstract. Human epidemiological reports and rodent experimental research data indicate a possible chemopreventive effect of regular aspirin use for decreasing risk of colon and rectum cancer incidence and mortality. We have previously demonstrated that aspirin can significantly suppress proliferative parameters in normal rat colonic epithelium when examined 24 h following an acute or chronic course of aspirin administration. To investigate whether aspirin would effectively suppress known carcinogen-induced changes in colonic epithelium, rats were given single s.c. injections of either aspirin (50 mg/kg bw) or saline on days 1–3 and either 1,2-dimethylhydrazine (DMH; 12 mg base/kg bw) or DMH vehicle on day 4 of each week for eight consecutive weeks. Rats were sacrificed 4 days after the last aspirin dose and 3 days after the last DMH or DMH vehicle dose. Using the proliferative biomarkers of proliferating cell nuclear antigen positive cells per midaxial crypt section (SCC), crypt proliferative zone height (PZ), crypt differentiated zone height (DZ), and total crypt height (CH), it was found that aspirin does suppress DMH-induced increases in SCC, PZ and CH. The findings demonstrate that aspirin has a long term (i.e. several days) protective effect against early carcinogen-induced proliferative changes in rat colonic crypts which may help account for aspirin's chemopreventive action against colon cancer.     

Urinary and faecal mutagenicity in car mechanics exposed to diesel exhaust and in unexposed office workers

The occurrence of mutagens in the urine and faeces of a group of car mechanics (n = 8) exposed to high concentrations of diesel exhaust in their working place and of a group of office workers (n = 9) not exposed to diesel exhaust during working hours was compared. The aim of the study was to investigate whether the specific diesel exposure and/or other, more lifestyle-related, factors such as diet had any influence on the mutagenicity of excreta. Faeces were collected and pooled for a consecutive period of 48 h, urine was collected in the same period, but in 4 separate portions representing the urine produced during the day and at night on the 2 collection days. Information about food intake was collected by a 2-day dietary record method. Smoking habits and medicinal drug use were recorded as well. Air particulates were collected in and outside the garage during working hours. The mutagenicity of extracts of air particulates (methanol extracts), urine (XAD-2 and XAD-7 extracts) and faeces (acetone, ether and ether-NaOH extracts) was examined in the Ames test. The results did not suggest that exposure to diesel exhaust mutagens enhanced the incidence and/or degree of either faecal or urinary mutagenicity. Urine of 2 mechanics appeared to contain rather high levels of XAD-7 mutagens, but in view of the uneven distribution over the different collection periods any relationship with the exposure to diesel exhaust mutagens seems improbable. Degree and frequency of faecal mutagenicity was higher in office workers than in mechanics. The pattern of faecal mutagenicity was characteristic of that of faecapentaenes. Statistical analysis did not reveal any consistent relationships between urinary and faecal mutagenicity and the various dietary variables.     

Modulatory efficacy of hesperetin (citrus flavanone) on xenobiotic-metabolizing enzymes during 1,2-dimethylhydrazine-induced colon carcinogenesis

Colorectal cancer is the second leading cause of cancer death worldwide with diet playing a prominent role in disease initiation and progression. Diet and nutrition play an important role during this multistep colon carcinogenic process. We have investigated the modulatory efficacy of hesperetin on aberrant crypt foci (ACF) and xenobiotic-metabolizing enzymes on 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis. Male albino Wistar rats were randomly divided into six groups. Group 1 served as control, received modified pellet diet and group 2 rats received 20 mg/kg body weight of hesperetin p.o. every day. Groups 3-6 rats were given subcutaneous injections of 1,2-dimethylhydrazine (20 mg/kg body weight) once a week for 15 weeks to induce ACF in the colon. In addition, rats in group 4 received hesperetin as in group 2 orally for the first 15 weeks (initiation), group 5 rats received hesperetin as in group 2 after the last injection of DMH and continued till the end of the experimental period (post-initiation). Group 6 received hesperetin as in group 2 throughout the entire period of 32 weeks. DMH exposure showed high incidence (90%) of ACF (280 ± 24.5 aberrant crypt/colon) and dysplastic ACF, elevated activities of phase I enzymes and reduced the activities of phase II enzymes in the liver and colonic mucosa of colon cancer bearing rats. Hesperetin supplementation significantly reversed these effects, the effect being more pronounced in group 6 rats (hesperetin supplemented throughout the study period).These findings suggest that hesperetin can significantly reduce the formation of preneoplastic lesions and effectively modulate the xenobiotic-metabolizing enzymes in rats.     

Zinc mediated normalization of histoarchitecture and antioxidant status offers protection against initiation of experimental carcinogenesis

The present study evaluated the inhibitory effects of zinc on colonic antioxidant defense system and histoarchitecture during 1,2 dimethylhydrazine (DMH) induced colon carcinogenesis in male Sparque Dawley rats. The rats were segregated into four groups viz., normal control, DMH treated, zinc treated, DMH + zinc treated. Colon carcinogenesis was induced through weekly subcutaneous injections of DMH (30 mg/kg body weight) for 8 weeks. Zinc (in the form of zinc sulphate) was supplemented to rats at a dose level of 227 mg/l in drinking water, ad libitum for the entire duration of the study. Increased lipid peroxidation was accompanied by a decrease in reduced glutathione (GSH), glutathione reductase (GR), glutathione-s-transferase (GST), superoxide dismutase (SOD), and catalase. Administration of zinc to DMH treated rats significantly decreased the lipid peroxidation levels with simultaneous enhancement of GSH, GR, GST, SOD, and Catalase. Histopathological studies from DMH treated rats revealed disorganization of colonic histoarchitecture. However, zinc treatment to DMH treated rats greatly restored normalcy in the colonic histoarchitecture, with no apparent signs of abnormality. Energy Dispersive X-Ray Fluorescence (EDXRF) studies revealed a significant decrease in tissue concentrations of zinc in the colon following DMH treatment, which upon zinc supplementation were recovered to near normal levels. In conclusion, the results of this study suggest that zinc has a beneficial effect during the initiation of key events leading to the development of experimentally induced carcinogenesis.     

Modifying action of neonatal androgenization on 1,2-dimethylhydrazine-induced carcinogenesis in male CBA-strain mice

Newborn male CBA mice received a single treatment with 0.5 mg testosterone propionate. Weekly subcutaneous injections of 1,2-dimethylhydrazine (DMH) were given to 2-month-old mice. The incidence of pararenal angiosarcomas and colonic tumors in neonatally androgenized mice reached 78.5 and 71.0%, respectively by the 35th week after the DMH treatment was commenced. In DMH-treated control mice, the incidence of the above tumors amounted to 25 and 32%, respectively.     

Mutagenic action of a series of epoxides

The mutagenicity of a series of 13 epoxide compounds was studied using a bacterial plate assay system. The histidine-dependent tester strains TA98 (for frameshift mutagens) and TA100 (for base-pair substitution mutagens) of Salmonella typhimurium were used. Mutagenicity was evaluated both with and without the addition of rat liver microsomal extract. Dieldrin, diglycidyl ether of bis phenol A and 3 of its homologues were not mutagenic. Allyl glycidyl ether, n-butyl glycidyl ether, vinyl cyclohexene diepoxide, glycidol, glycidaldehyde, diglycidyl ether, diepoxybutane and diglycidyl ether of substituted glycerine were mutagenic in the TA100 strain, causing reversion of the bacteria to histidine independence. Dose—response curves of the mutagenicity of the latter 4 compounds were obtained. On a molar basis, glycidaldehyde was about 20–50 times more potent in producing mutation that were the other 3 epoxides in the dose—response test. In general, the mutagenicity of the epoxides was not enhanced or diminished by the addition of microsomal extract.     

Chemopreventive efficacy of green tea drinking against 1,2‐dimethyl hydrazine‐induced rat colon carcinogenesis

Colorectal cancer is one of the leading causes of tumour‐related deaths. In the present study, the chemopreventive effect of green tea on 1,2‐dimethylhydrazine (DMH)‐induced colon carcinogenesis was studied in male Wistar rats. The DMH group received subcutaneous injections of DMH (30 mg kg^?1 body weight) once a week for 30 weeks, the normal group received the vehicle of DMH, and the DMH + green tea group received DMH simultaneously with 1% green tea as their sole source of drinking fluid throughout the experimental period. In the DMH group treated with green tea, significant reductions in gene overexpressions of colonic nuclear factor κB (NF‐κB), tumour necrosis factor α, inducible nitric oxide synthase and cyclooxygenase 2, and NF‐κB immunostaining indicates the anti‐inflammatory effect of green tea in attenuating colon cancer. Moreover, the anti‐angiogenic and anti‐invasiveness effects of green tea were revealed as reductions of both vascular endothelial growth factor and matrix metalloproteinase‐7 mRNA expression levels. These effects were confirmed by the significant reduction of serum tumour necrosis factor α, C‐reactive protein levels, inhibition of tumour incidence, and nearly normal survival rate and colonic architecture. It can be concluded that green tea exerts a potent chemopreventive effect on colon carcinogenesis possibly due to the inhibition of NF‐κB. Copyright © 2012 John Wiley & Sons, Ltd.     

Biphasic response of checkpoint control proteins in hyperoxia: exposure to lower levels of oxygen induces genome maintenance genes in experimental baboon BPD

Objective of the study is to evaluate the modifying potential of p-methoxycinnamic acid (p-MCA), an active rice bran phenolic acid on biotransforming bacterial enzymes and xenobiotic metabolizing enzymes in 1,2-dimethylhydrazine-induced rat colon carcinogenesis. 48 male albino wistar rats were divided into six groups. Group1 (control) received modified pellet diet and 0.1 % carboxymethylcellulose; group2 received modified pellet diet along with p-MCA (80 mg/kg b.wt. p.o.) everyday for 16 weeks; groups 3-6 received 1,2-dimethylhydrazine (DMH) (20 mg/kg b.wt.) subcutaneous injection once a week for the first 4 weeks, while groups 4–6 received p-MCA at three different doses of 20, 40 and 80 mg/kg b.wt. p.o. everyday for 16 weeks. A significant increase in carcinogen-activating enzymes (cytochrome P450, cytochrome b5, cytochrome P4502E1, NADH-cytochrome-b5-reductase and NADPH-cytochrome-P450 reductase) with concomitant decrease in phaseII enzymes, DT-Diaphorase, glutathione S-transferase, UDP-glucuronyl-transferase and gamma glutamyltransferase were observed in group3 compared to control. DMH treatment significantly increased the activities of feacal and colonic bacterial enzymes (β-glucosidase, β-galactosidase, β-glucuronidase, nitroreductase, sulphatase and mucinase). p-MCA supplementation (40 mg/kg b.wt) to carcinogen exposed rats inhibited these enzymes, which were near those of control rats. The formation of dysplastic aberrant crypt foci in the colon and the histopathological observations of the liver also supports our biochemical findings. p-MCA (40 mg/kg b.wt.) offers remarkable modulating efficacy of biotransforming bacterial and xenobiotic metabolizing enzymes in colon carcinogenesis.     

Sulindac enhances cell proliferation in DMH-treated mouse colonic mucosa

In a previous study we reported that the NSAID sulindac had a marked inhibitory effect on the development of colonic tumours in mice treated with the carcinogen 1,2-dimethylhydrazine (DMH). In this study we examined the effects of sulindac in respect of cell-kinetic changes in mouse colonic mucosa as determined by flash labelling with the thymidine analogue bromodeoxyuridine (BrdUrd) at varying intervals during the process of colonic carcinogenesis. We also investigated the possibility that these changes may be modulated by misoprostol a prostaglandin E₁ analogue. Four groups of 36 mice each were treated for 18 weeks with the following drug/s respectively: (1) DMH; (2) DMH and sulindac; (3) DMH, sulindac and misoprostol; and (4) DMH and misoprostol. Three animals from each group were killed each week between the sixth week and the eighteenth week after the start of the experiment. A 1-h flash label technique was employed and paraffin sections of colonic mucosa were examined. For each animal a total of 50 perfect axially cut crypts were chosen and the following parameters determined: crypt length, labelling index and labelling index distribution: the data were analysed using the computer program GLIM. For each of the four groups, crypt lengths increased significantly with the duration of treatment with no significant difference between the groups. In sulindac-treated animals the labelling index for all positions increased with duration of treatment whereas for animals not treated with sulindac there was no significant difference in labelling index with respect to duration of treatment. The administration of misoprostol did not appear to significantly alter the effects of sulindac. It is postulated that the observed increase in cell proliferation could be a compensatory phenomenon occurring secondary to loss of crypt epithelial cells by apoptosis induced by sulindac. Also the finding of an increase in labelling index mediated by a chemopreventive agent indirectly questions the rationale behind the therapeutic manipulation of crypt cell proliferation in order to reduce the risk of colon cancer.     

The potent colon carcinogen, 1,2-dimethylhydrazine induces mutations primarily in the colon

1,2-Dimethylhydrazine (DMH) is a potent colon carcinogen that is commonly used as an initiator in studies of the effects of diet on colon cancer. Previous studies have shown that although this compound produces multiple tumors in the colons in most individuals of every species tested, it is, at best, marginally mutagenic in the bone marrow (micronuclei) and small intestine (Dlb-1 mutations). Here we report its mutagenicity in the primary target tissue, the colonic epithelium, by means of the Mutatrade markMouse cII assay, an assay for intragenic mutations in a lambda shuttle vector that is integrated into the genome of these mice. Animals were treated with 0, 10, 20, or 30 mg/ml of DMH, either as a single injection or as multiple weekly injections, and mutations were measured in both the small intestine and colon. In the small intestine, there was an increase in mutant frequency following a single injection of DMH, but this was significant only at 30 mg/kg [induced mutant frequency (MF) = 18 x 10(-5) mutants/plaque]. In the colon, following a single treatment of DMH, there was a significant increase in mutant frequency at doses of 20 and 30 mg/kg (induced MF = 17 x 10(-5) and 23 x 10(-5) mutants/plaque, respectively). Following ten injections of 20 mg/kg of DMH, there was a greater than ten-fold increase in mutations in the colon (MF = 275 x 10(-5) mutants/plaque) than the small intestine (MF = 25 x 10(-5) mutants/plaque). These results show that DMH, under the conditions typically used for dietary studies, induces large numbers of mutations in the tissue in which it induces most cancers.     

Apoptotic differences in experimentally induced colorectal rat tumours

The presence of apoptotic bodies and of intraepithelial lymphocytes (IELs) were assessed in colorectal adenomas and adenocarcinomas induced in 158 rats by two different carcinogens: 1,2 dimethylhydrazine (DMH) and glutamic acid pyrolysate (GLU-P-1 and 2). Apoptotic granules were present in 97.5% ( n=40) of the 41 GLU-induced adenomas and adenocarcinomas, but only in 20.5% ( n=24) of the 117 DMH-induced tumours. IELs were found in 95.1% ( n=39) of the 41 GLU-induced tumours but only in 21.4% (n=25) of the 117 DMH-induced neoplasias. The differences were significant ( p< 0.001). The presence of IELs and apoptotic granules in GLU tumours (and their absence in the majority of the DMH tumours) is new evidence that IELs are the cells from which many of the apoptotic granules — seen in colorectal neoplasias — derive. GLU neoplasias were induced following daily treatment, for 24 months (about half the life span of the animals) and DMH neoplasias by weekly doses, for a period of only 2.8-6 months. It would appear that 'slowly growing' colorectal GLU neoplasias often attract IELs and trigger lymphocytic apoptosis whereas 'quickly growing' DMH tumours seldom evoke those reactions.     

Mutagens in human faeces. Are they relevant to cancer of the large bowel?

1. Mutagenic activity has been detected in faecal extracts, prepared by a number of methods, from donors living under widely differing geographical, cultural and dietary circumstances. Faecal extracts cause point mutations in bacteria and chromosomal damage in cultured mammalian cells. 2. The claims that nitroso compounds are present in human faeces have been retracted, and the chemical nature of faecal mutagens is still unknown. Indirect evidence suggests the presence of several classes of mutagen. 3. The use of different methods of mutation assay gives conflicting estimates of the proportion of people who excrete mutagenic faeces. There is wide variation in mutagenic activity between different stool samples from one person, and between different stool samples from different people. There is conflicting evidence for inhibition or enhancement of the mutagenicity of reference mutagens by faecal extracts. The effects of air oxidation on the mutagenicity of faecal extracts have not been investigated in detail. 4. It has been claimed that the proportion of people excreting mutagenic faeces is higher in groups representing populations at high risk of large-bowel cancer than in groups at low risk of large-bowel cancer. For the reasons given in paragraph 3, these claims must be regarded as premature. 5. The part played by faecal mutagens in the aetiology of large-bowel cancer has yet to be determined.     

Inhibition of 1,2-dimethylhydrazine induced colon genotoxicity in rats by the administration of probiotic curd

Epidemiologic and experimental studies suggest that the probiotic organisms are effective in preventing colon carcinogenesis, which is the major cause of mortality and morbidity in western countries. Keeping this in view, a curd (a common Indian fermented milk product) was prepared by the addition of probiotic cultures Lactobacillus acidophilus, Lactobacillus casei and curd culture Lactococcus lactis biovar. diacetylactis. In present study, we have evaluated the anti tumor effect of probiotic curd by monitoring the DNA damage through comet assay. The rats were allocated to four groups, first group was DMH control group, second group was probiotic curd group in which probiotic curd was given along with DMH (1,2-dimethylhydrazine) injection, third group was normal curd group in which normal curd was given along with DMH injection and fourth group was normal control group. Animals received subcutaneous injection of DMH dissolved in normal saline at a dose rate of 20 mg/kg body weight, once weekly for 15 weeks. The rats were dissected at 40th week of experiment and comet assay was done in colonic cells to assess the DNA damage. A significant reduction in DNA damage (54.7%) was observed in probiotic curd group as compared to DMH control group (88.1%). The probiotic curd was effective to significantly reduce the L:W ratio in comparison to DMH control group and normal curd. The results of present study show the protective effects of probiotic curd against DMH induced genotoxicity in colonic cells.     

Vanadium inhibits placental glutathione S-transferase (GST-P) positive foci in 1,2-dimethyl hydrazine induced rat colon carcinogenesis

Vanadium (V) has recently been found to possess potent anti-neoplastic activity in rat colon carcinogenesis. In the present study attempts have been made to investigate the expression of the number and area of aberrant crypt foci positive for placental glutathione S-transferase (GST-P) during 1,2-dimethyl hydrazine (DMH)-induced rat colon carcinogenesis. Male Sprague Dawley rats were randomly divided into four groups. Rats in group A were designed as normal controls. Group B animals received DMH once a week (20 mg/kg body wt.) intraperitoneally for 16 weeks. Group C rats received the same treatment of DMH as in group B, along with 0.5-ppm vanadium as ammonium monovanadate ad libitum in drinking water throughout the experiment. Vanadium alone was given to Group D rats without any DMH injection. The expression of the number and the area of aberrant crypt foci (ACF) positive for GST-P was maximum in DMH-treated group. Vanadium-treated rats significantly reduced (P < 0.001) the expression of GST-P positive ACF cells (by 71.13%) for the entire period of the study. Moreover the histopathological examination also showed that vanadium action could minimize the aberrant crypt foci (P < 0.001). Furthermore, vanadium supplementation also elevated SOD activities in both liver and colon (P < 0.01, P < 0.02 and P < 0.01, P < 0.02 respectively) when compared to their carcinogen counterparts. Our results confirm that vanadium is particularly effective in limiting the action of the carcinogen, thereby establishing its anticarcinogenicity in chemically induced rat colon carcinogenesis.     

Effect of quality and quantity of dietary fat and dimethylhydrazine in colon carcinogenesis in rats.

The effect, quality, and quantity of dietary fat on colon tumor induction by DMH were studied in rats exposed to a given regimen for two generations prior to treatment with DMH. Animals fed a 20% corn oil or 20% lard and treated with DMH had a higher incidence of colonic tumors than did rats fed a 5% corn oil, 5% lard or Purina lab chow and treated similarly. The quality of fat had no major difference on the incidence of colonic tumors.     

Citrus nobiletin inhibits azoxymethane-induced large bowel carcinogenesis in rats

The inhibitory effects of dietary feeding of citrus nobiletin on azoxymethane (AOM)-induced rat colon carcinogenesis using a long-term bioassay were investigated. Five-week old male F344 rats were initiated with two weekly subcutaneous injections of AOM (20 mg/kg bw) to induce colonic tumors. They were also given the diets containing 0.01% or 0.05% nobiletin for 34 weeks, starting one week after the last dosing of AOM. At the end of the study, the incidence of colonic adenocarcinoma were 67% in the AOM alone group, 55% in the AOM-->0.01% nobiletin group, 35% (p<0.05) in the AOM-->0.05% nobiletin group. Also, nobiletin feeding reduced the cell-proliferation activity, increased the apoptotic index, and decreased the prostaglandin E2 content in colonic adenocarcinoma and/or colonic mucosa. These findings might suggest that citrus nobiletin has chemopreventive ability against AOM-induced rat colon carcinogenesis.