DNA damage in liver,colon, stomach,lung and kidney of BALB/c mice treated with 1,2-dimethylhydrazine

DNA single-strand breaks induced in various organs of BALB/c mice by treatment with a single dose of 1,2-dimethylhydrazine (DMH) were studied by means of the alkaline elution method modified in order to allow the evaluation of DNA damage in vivo with no need of radioactive prelabelling. DNA damage was detected in liver, lung, kidney, stomach and colon mucosa, with the liver showing the greatest amount of damage. Its degree was dependent on the dose and route of administration. A differential effect was evident in colon mucosa from Swiss and C57BL/6 mice which are respectively susceptible and resistant to the induction of bowel tumors by DMH. The higher degree of DNA damage found in liver in comparison with colon mucosa is consistent with the previously reported higher degree of DNA methylation, but does not correlate with the specificity of this carcinogen in inducing tumors of the large intestine in mice given repeated subcutaneous injections.     

DNA alkylation in mice with genetically different susceptibility to 1,2-dimethylhydrazine-induced colon carcinogenesis

The formation and persistence of methylated purines was determined in mice that received a single s.c. injection of 1,2-[14C]dimethylhydrazine (15 mg/kg) and were allowed to survive for 12 or 60 hr. In mice with a low susceptibility to dimethylhydrazine-induced colon carcinogenesis (C57BL/Ha), concentrations of 7-methylguanine and O6-methylguanine in DNA of colon, ileum, and kidney were 40 to 60% less than in mice with a high incidence of colonic tumors (ICR/Ha). In hepatic DNA the extent of methylation was higher in C57BL/Ha than in ICR/Ha mice. The rate of loss of methylated purines from colon DNA was similar in both strains. In all organs investigated the metabolic incorporation of 14C into normal DNA bases was lower in C57BL/Ha than in ICR/Ha mice. It is concluded that the low carcinogenic response of C57BL/Ha mice is due to the smaller extent of initial alkylation of colon DNA, which probably reflects differences in the enzymic metabolism of the parent carcinogen.     

Silk protein, sericin, suppresses colon carcinogenesis induced by 1,2-dimethylhydrazine in mice

Male 5-week old ICR mice were examined for the effect of feeding silk protein, sericin on colon carcinogenesis. In experiment 1, mice were fed the diets supplemented with 1.5% or 3% sericin for five weeks, and given weekly injections of 1,2-dimethylhydrazine (DMH) for the initial three weeks. Supplemental sericin caused a dose-dependent decrease in the development of colonic aberrant crypt foci. In experiment 2, mice were fed the diet supplemented with 3% sericin for 115 days, and given weekly injections of DMH for the initial ten weeks. The incidence and number of colon tumors were suppressed by consumption of sericin. The results suggest a potential usefulness of sericin as a chemopreventive agent for colon carcinogenesis.     

The relationship between 1,2-dimethylhydrazine dose and the induction of colon tumours: tumour development in female SWR mice does not require a K-ras mutational event

In this study we have investigated the relationship between the dose of 1,2-dimethylhydrazine (DMH) and the yield (and location) of tumours in a mouse strain susceptible to colon tumour induction. Female SWR mice were injected with 6.8 mg/kg DMH i.p. once a week for 1, 5, 10 and 20 weeks and the animals were followed for almost 2 years. Administration of increasing doses of DMH resulted in a dose-dependent decrease in survival time. Colon tumours developed in 26, 76 and 87% of mice given a total dose of 34, 68 and 136 mg/kg DMH, respectively: no tumours were detected in animals treated with a total dose of 6.8 mg/kg. Most colon tumours (79%) were located in the distal colon with the remainder being found in the mid colon and none were detected in either the proximal colon or small intestine. As mutations in the K-ras gene are thought to be key events in the pathogenesis of human and rodent colon tumours, we determined the frequency of codon 12 and 13 K-ras mutations in these tumours by restriction site mutation analysis and/or DNA sequencing. A total of 50 colon tumour samples were analysed for codon 12 mutations and of these 29 were also screened for codon 13 mutations. No mutations were detected in either of these codons. The mutational activation of the K-ras gene is not an essential step in the development of DMH-induced colon tumours in female SWR mice and if similar considerations apply to humans, then the aetiological role of alkylating agents may be underestimated from the prevalence of K-ras GC-->AT transitions in human tumours.     

Chemopreventive effect of trans-resveratrol--a phytoalexin against colonic aberrant crypt foci and cell proliferation in 1,2-dimethylhydrazine induced colon carcinogenesis

Prevention of cancer remains a primary need and new chemopreventive agents must be developed for this purpose. Towards this goal, a chemoprevention study was conducted to evaluate the activity of resveratrol (Res), a phytoalexin, as an inhibitor of colon carcinogenesis. Wistar male rats were divided into six groups, group 1 were control rats, group 2 were control rats that received Res (8 mg/kg body wt p.o. everyday), rats in groups 3-6 were treated weekly with 1,2-dimethylhydrazine (DMH, 20 mg/kg body wt, s.c. x 15 times). In addition, groups 4, 5 and 6 received Res as in group 2. Modifying effects were assessed using aberrant crypt foci (ACF) and the extent of histopathological lesions as end point markers. At the end of 30 weeks, Res markedly reduced tumor incidence, the degree of histological lesions and also the size of tumors significantly (P < 0.05) as compared with the rats treated with unsupplemented DMH. The number of ACF consisting of more than six aberrant crypts per rat was observed in group 6 (6.2 +/- 1.4), group 5 (7.7 +/- 1.0) and group 4 (8.2 +/- 1.4) which were significantly lower than that of group 3 (22.3 +/- 2.4) (P < 0.05). The most pronounced inhibition of ACF development was noted in rats fed Res for the entire period and also during the post-initiation period. Also, Res administration lowered the number of argyrophilic nucleolar organizing region-associated proteins (AgNORs) per nucleus in non-lesional colonic crypts, which reflects the cell proliferation activity. Oxidative imbalance in DMH-treatment was significantly (P < 0.01) modulated on Res supplementation as indicated by optimal concentration of thiobarbituric acid reactive substances (TBARS), superoxide dismutase (SOD), catalase (CAT) and reduced glutathione (GSH). The results of our study suggest Res to be an effective chemopreventive agent, which suppresses DMH-induced colon carcinogenesis at various stages.     

The kinetics of DNA alkylation, repair and replication in hepatocytes, Kupffer cells, and sinusoidal endothelial cells in rat liver during continuous exposure to 1,2-dimethylhydrazine

We have previously shown that when rats are continually exposedto 1,2-dimethylhydrazine (SDMH) in a manner that induces malignanthemangioendotheliomas of the liver there was both accumulationof promutagenic DNA damage, as measured by O⁶-methylguanine(O6MG) and a selective increase in cell replication in the targetcell population, the non-parenchymal cells (NPC). Hepatocytesrapidly repaired O6MG and had only modest increases in cellreplication. In this study, we have further separated the NPCinto Kupffer cells (KC) and sinusoidal endothelial cells (SEC)and have determined the kinetics of O6MG and 7-methylguanine(7MG) formation and repair, and cell replication in each individualcell type. O6MG concentrations were highest over the entireexposure period in the SEC, lowest in the hepatocytes, withintermediate concentrations occurring in KC. 7MG concentrationswere lower in SEC than in KC or hepatocytes resulting in greatlyincreased O6/7MG ratios in SEC suggesting a lower capacity forSDMH metabolism and O6MG repair. Moreover, the concentrationsof 7MG and the pattern of accumulation were sufficiently similarin KC and hepatocytes to suggest similar capacities of thesetwo cell types to activate SDMH. Rates of cell replication weregreatly elevated in both KC and SEC by SDMH, while hepatocytereplication was barely affected. These data suggest that SECand to a lesser extent KC have greater potential for initiationdue to mispairing of O6MG than do hepatocytes. The data furthersuggest that alkylation in SEC may arise from metabolites generatedby neighboring hepatocytes or KC.     

An endogenous colon mitosis inhibitor reduces the increased cell proliferation in colonic epithelium induced by dietary cholic acid and treatment with 1,2-dimethylhydrazine

We have recently purified and identified a tripeptide (pGlu-His-GlyOH)in mouse intestinal extracts which reversibly inhibits normalcolonic epithelial cell renewal in mice. We also found a similarresponse to a single injection of the peptide during bile-acid-inducedhyperproliferation in the colonic epithelium. To investigatethe effect of repeated injections of the same dose of the inhibitorunder various pathological conditions of the colonic mucosawe either fed mice low-calcium cholic acid diet, treated theanimals with a single injection of the colon carcinogen 1,2-dimethyl-hydrazine(DMH), or gave both treatments. The peptide reduced the mitoticrate and the labelling indices in the colonic epithelium duringthe first 5 days of feeding the low-calcium cholic acid diet,and the size of the proliferative compartment was reduced. Proliferatingcells were found significantly closer to the base of the crypisin the peptide-treated animals. The mitotic rate was also reducedby each of repeated peptide injections during the first 72 hin the DMH-treated animals; the labelling indices at 48 and72 h only. By feeding DMH-treated animals low-calcium cholicacid diet, similar results were obtained. However, in all carcinogen-treatedanimals the tripeptide had no effect on the size and localizationof the proliferating cells in the colonic crypts.     

Role of cytochrome P450 in DNA damage produced by treatment of colon cells with 1,2-dimethylhydrazine

Human colon cells (LS174T) were treated with the model colon carcinogen 1,2-dimethylhydrazine (DMH) to determine the production of O(6)-methylguanine DNA adducts. Three known P450 inducers (benzanthracene, pyrazole and phenobarbital) were used to produce different P450 environments in each group of cells prior to treatment with DMH. An increased level of DNA damage of different degrees above uninduced levels was observed in all treated groups. Inhibition of the natural protection systems (glutathione and O(6)-methyltransferase) were also included in the study. Glutathione apparently is not of significant protection against DMH damage in colon cells challenged with DMH. In contrast methyltransferase does exert a protective role in this type of cells by reducing the extent of DNA O(6)-methylguanine adduct formation in colon cells following induction of different panels of cytochrome P450 isoforms.     

Effect of dietary carbohydrates on the growth of dysplastic crypt foci in the colon of rats treated with 1,2-dimethylhydrazine

The effect of dietary starch and sucrose on the growth of foci of dysplastic crypts in the colon (FDC) was studied in female Sprague Dawley rats treated twice p.o. with 25 mg/kg of 1,2-dimethylhydrazine (DMH). After DMH administration, the animals were fed high-fat (23% corn oil, w/w)/low-calcium (0.1%, w/w)/low-cellulose (2%, w/w) diets in which carbohydrates were represented by corn starch (starch diet) or sucrose (sucrose diet) (46%, w/w). The animals were fed for either 30 or 105 days with the experimental diets. The number of FDC was not significantly affected by diet. However, after 30 days the percentage of small FDC (formed by 1-2 dysplastic crypts) was higher in the animals fed the starch diet compared to the animals fed the sucrose diet [90.3 +/- 1.1% (SE) and 82.6 +/- 3.1%, respectively; P less than 0.05]. In contrast, foci formed by 3-4 dysplastic crypts were decreased by the starch diet (P less than 0.05). After 105 days of feeding, the starch diet induced a number of dysplastic crypts/focus lower than that induced by the sucrose diet (2.6 +/- 0.1 and 2.9 +/- 0.1, respectively; P less than 0.05). The percentage of small FDC was also higher in the animals fed the starch diet compared to animals fed the sucrose diet (P less than 0.01). After 30 days of feeding, DMH treatment increased colon proliferative activity in both dietary groups (P less than 0.05). But after 105 days of feeding, proliferation was similar in controls and DMH-treated rats and markedly reduced in animals fed the starch diet (mean labeling index values for both controls and DMH-treated rats were 10.4 +/- 0.8 and 4.4 +/- 0.5 in the sucrose and starch diets, respectively; P less than 0.001). The overall results suggest that starch in high-fat/low-calcium/low-cellulose diets has a protective role against DMH-colon carcinogensis in the rat.     

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.     

Vanadate supplements and 1,2-dimethylhydrazine induced colon cancer in mice: increased thymidine incorporation without enhanced carcinogenesis

Because vanadate ion is a potent mitogen and accumulates in the gut of rodents fed vanadate supplements, effects of ammonium metavanadate in drinking water (10 ppm or 20 ppm) were studied on the development of large bowel neoplasms in mice treated with 1,2-dimethylhydrazine (DMH) (20 mg kg-1 weekly for 20 weeks). In the colon at 30 weeks DMH treatment caused a 14% increase in RNA content, an 18% increase in DNA content, and 33% deeper crypts. Vanadate at either 10 ppm or 20 ppm decreased RNA content by approximately 11%. Although vanadate increased thymidine incorporation 210% to 550% compared with controls, it had no influence on the attack rate, incidence, or histological type of tumours induced by DMH.     

Site specific reduction of colon cancer incidence, without a concomitant reduction in cryptal cell proliferation, in 1,2-dimethylhydrazine treated rats by diets containing 10% pectin with 5% or 20% corn oil

The effects of specific dietary interventions on incidence ofcarcinogen-induced cancer and on cryptal cell proliferationin areas of the colon located either over aggregates of lymphoidnodules (ALN) or away from ALN was investigated. Groups of dimethylhydrazine(DMH) treated rats or non-DMH-treated rats were fed a basalAIN-76 diet less fiber of any type, or the basal fiber freediet supplemented with 10% pectin and with 5%, 10%, or 20% cornoil. The adenocarcinoma (AC) incidence was determined in regionsof the colon, i.e. ascending, descending, descending over theALN and descending away from the ALN. The results indicate that:(i) factors associated with ALN promote AC formation, (ii) dietarymodifications (addition of pectin and of 20% corn oil to thediet) each cause significant site specific suppression of ACincidence, (iii) DMH-treatment rendered crypts non-responsiveto the suppression of cryptal cell proliferation which occurredin the rats not treated with DMH (suggestive of a DMH-inducedloss in the regulation of cell proliferation) and (iv) reductionof AC incidence was not always accompanied by reduction in cryptcell proliferation. Studies of intervention procedures designedto prevent colon cancer should take into account the colon sitespecific tumorigenic response to the preventive agent and shouldnot rely on a single biomarker to predict the efficacy of theintervention.     

Inhibition of DNA synthesis by 1,2-dimethylhydrazine and methylazoxymethanol acetate in rabbit colon mucosa in organ culture.

When maintained in organ culture, colon mucosa from male New Zealand White rabbits showed a near-normal mucosal morphology and linear incorporation of [3H]thymidine into mucosal DNA up to 36 hours of incubation. Explants from the descending colon had a higher DNA synthetic activity than did other segments of the large bowel. Inhibition of DNA synthesis in colon explants by 1,2-dimethylhydrazine (DMH) and methylazoxymethanol (MAM) acetate was dose-dependent. When DNA synthesis was determined after an 18-hour incubation, MAM acetate inhibited DNA synthesis at concentrations of 50, 100, 150, and 200 microgram/ml. With the same concentration of DMH, little or no inhibition was observed. At the concentration of 200 microgram/ml, both carcinogens significantly inhibited DNA synthesis after 3 and 6 hours of incubation. With longer incubation, the inhibitory effect of DMH appeared to be reversible, whereas DNA synthesis was continuously inhibited by MAM acetate up to 18 hours of incubation. No altered uptake of [3H]thymidine by colon explants incubated in the presence of DMH or MAM acetate for 18 hours was observed. No morphologic changes were seen in colon explants treated with 200 microgram MAM acetate/ml for 18 hours. Physostigmine sulfate had no influence on MAM acetate-induced inhibition of DNA synthesis in colon explants. These in vitro observations reflected a direct action of DMH and MAM acetate on the colon mucosa and supported the possibilility that colon epithelial cells contain enzymes capable of activating DMH and MAM acetate to their alkylating carcinogens.     

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.     

Inhibitors for the mutagenicities of colon carcinogens, 1,2-dimethylhydrazine and azoxymethane, in the host-mediated assay.

Inhibitory effects of several chemicals on the mutagenicities of 1,2-dimethylhydrazine (DMH) and azoxymethane (AOM) for Salmonella typhimurium G46 in the host-mediated assay were investigated. They were carbon disulfide (CS2), tetraethylthiuram disulfide (disulfiram, DSF), sodium diethyldithiocarbamate (SDDC), ethylene-bis(dithiocarbamato) manganese (Maneb), pyrazole (PZ), aminoacetonitrile hydrogen sulfate (AAN), and sodium selenite (SE). All the compounds, except for SE, inhibited the mutagenicities of DMH and AOM.     

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.     

Associations between polymorphisms in DNA repair genes and TP53 mutations in non-small cell lung cancer

This study was conducted to identify genetic factors predisposing to TP53 mutations in patients with non-small cell lung cancer (NSCLC). A comprehensive panel of potentially functional single nucleotide polymorphisms (SNPs) in DNA repair genes was evaluated in relation to TP53 mutations. Thirty-seven SNPs in 28 DNA repair genes were genotyped by a sequenome mass spectrometry-based genotyping assay in 173 NSCLCs and the associations with TP53 mutations in the entire coding exons (exons 2-11), including splicing sites of the gene, were analyzed. Four SNPs (XPA rs1800975, OGG1 rs1052133, ADPRT rs1136410, and NBS1 rs1805794) were significantly associated with the prevalence of TP53 mutations in multivariate analysis for each SNP. When the 4 SNPs were combined, the prevalence of TP53 mutations was increased as the number of bad genotypes increased (P_trend = 0.001). Patients with 3 and 4 bad genotypes had a significantly higher frequency of TP53 mutations than those with 0-1 bad genotypes (adjusted odds ratio = 5.18, 95% confidence interval = 1.51-17.81, P = 0.01 and adjusted odds ratio = 18.26, 95% confidence interval = 2.87-116.09, P = 0.002, respectively). These findings suggest that the 4 SNPs may modulate the occurrence of TP53 mutations and contribute to lung carcinogenesis. However, larger studies are required to confirm our findings in other ethnic populations.     

Mutational and LOH analyses of p53 alleles in colon tumors induced by 1,2-dimethylhydrazine in F1 hybrid mice

To elucidate the role of p53 in colon tumorigenesis in mice,we examined allele loss and mutational alteration of the p53gene in colon tumors induced by 1,2-dimethylhydrazine (DMH)in F₁ hybrid mice. Intragenic polymorphism of the p53 gene amongparental strains enabled us to assess allele loss of the p53gene and also to determine parental origin of mutated and/orlost alleles. Allele loss was detected in two of 163 tumorsheterozygous for the p53 gene. Polymerase chain reaction-single-strandconformation polymorphism analysis of p53 exons 5–8 revealed33 mutations in 20 of 182 colon tumors, the incidence beinglower than that in human colon cancers. The majority of thesemutations were of transition type: G: A transitions at non-CpGsites were most prevalent, while those at CpG sites were lesscommon. Distribution of the mutations along p53 amino acid sequencerevealed a difference in the location of ‘hot spots’between mice and humans. Incidence of p53 alterations did notdiffer among alleles of different parental origins, suggestingthat genetic changes in DMH-induced mouse colon tumors had occurredindependently of parental origin and DMH susceptibility. Detailedanalysis of p53 mutations on each allele revealed intratumoralheterogeneity in mouse colon tumors. The low incidence of p53mutations and rare allele loss suggest that p53 alteration playsonly a minor role in colon tumorigenesis in mice.     

Interrelationships between cellular proliferation,DNA alkylation and age as determinants of ethylnitrosourea-induced neoplasia

Thirty-day-old Sprague-Dawley rats were used to study the persistence of DNA lesions (e.g., O⁶-alkylguanine) induced by various doses of ethylnitrosourea (ENU). Cellular proliferation was measured as an increment of DNA content per organ at 7 days post-treatment. We observed that the persistence of O⁶-EtGua was not affected by the various dose levels. Comparing the 3 organs, the persistence of O⁶-EtGua ranked in the order of brain > kidney > liver, while the percent increase in DNA content was measured as liver > kidney > brain. When the target specificity of ENU carcinogenesis in 30-day-old rats was compared to that following transplacental exposure in terms of its relationship to the persistence of DNA lesions and the rate of tartet cellular proliferation, it permitted the conclusion that induction of neoplasia in target cells is not only determined by persistent DNA lesions but also by the rate of proliferation of target cells at the time of exposure.