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

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

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.     

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 DMH 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.     

Azoxymethane-induced colon tumors and aberrant crypt foci in mice of different genetic susceptibility

Azoxymethane (AOM) is an organotropic colon carcinogen that is commonly used to induce colon tumors in rodents. Unlike its parent compound, 1,2-dimethylhydrazine (DMH), a tumor susceptibility phenotype in inbred mice with respect to AOM has not been established. Thus, this study was undertaken to determine whether genetic susceptibility extends to this carcinogen. SWR/J, A/J (both susceptible to DMH carcinogenesis) and AKR/J (resistant) mice were treated with 10 mg/kg AOM i.p. once a week for 8 weeks. Twenty-five weeks after the initial injection, tumor yield was determined. With a single exception, only SWR/J and A/J mice developed tumors, with a distribution that was limited to the distal colon (16.3±1.1 and 36.4±2.4, respectively). The formation of aberrant crypt foci (ACF), putative preneoplastic lesions, was also assessed in whole-mount colons using Methylene Blue staining. Consistent with tumor multiplicity, the total number of ACF was highest in A/J mice, followed by SWR/J mice. In addition, A/J mice had a significantly greater number of large ACF (five or more crypts per foci) than the other strains. Despite the absence of colon tumors, however, AKR/J mice did develop a significant number of ACF. This finding suggests that ACF in resistant mice are persistent but do not progress to tumors.     

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.     

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.     

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.     

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.     

A locus that influences susceptibility to 1, 2-dimethylhydrazine-induced colon tumors maps to the distal end of mouse chromosome 3

While inheritance of mutated alleles of highly penetrant tumor suppressor genes such as retinoblastoma or p53 predisposes individuals to a greatly increased risk of developing cancer, epidemiological data indicate that the majority of sporadic tumors in humans result from interactions of environmental and host genetic factors. The host genetic factors are poorly penetrant tumor susceptibility genes that determine the likelihood that a cancer will arise from carcinogen exposure. The majority of colon tumors in humans are sporadic in nature. 1,2-dimethylhydrazine (DMH)-induced colon tumors in mice provide a useful animal model to identify genes that influence susceptibility to carcinogen-induced colon tumors in humans. A genome-wide scan of genetic crosses of relatively sensitive C57BL/6J with relatively resistant CBA mice treated with DMH revealed a linkage of DMH susceptibility with the distal end of mouse chromosome 3, suggesting that one or more tumor susceptibility genes may map to this region.     

DNA damage and repair in intact animals. Single-stranded regions in rat liver DNA following administration of dimethylnitrosamine.

Rat liver DNA was radioactively labelled by administration of [3H]thymidine following partial hepatectomy. Two weeks later, the rats were treated with the carcinogen, dimethylnitrosamine. DNA was isolated and fractionated by elution from benzoylated DEAE-cellulose with NaCl and caffeine solution. The caffeine-eluted fraction was increased by administration of dimethylnitrosamine. This increase was proportional to the dose of carcinogen injected and persisted for at least 24 h after administration of the carcinogen. These data, together with the results of hydroxyapatite chromatography, suggest that the DNA contains short single-stranded sections associated with much longer regions of native DNA.     

Induction of colon tumors in 1,2-dimethylhydrazine-resistant Lobund Wistar rats by methylazoxymethanol acetate.

Sprague-Dawley and Lobund Wistar rats, which were sensitive and resistant to induction of colon tumors by 1,2-dimethylhydrazine (DMH), respectively, were treated with methylazoxymethanol (MAM), the product of DMH metabolism by the microsomal mixed-function oxidase system. Although the colon tissue in both stocks of rats had similar NAD+-dependent dehydrogenase activities that are considered necessary to activate MAM to an ultimate carcinogen, still a sevenfold greater incidence of colon tumors was found in the Sprague-Dawley rats, and their tumors were more extensive. The results indicated that the difference in susceptibility to colon tumor induction between the rat stocks was partially related to metabolic activation of the DMH and to other, as yet undetermined, endogenous factors.     

Effects of route of administration on tissue distribution of DNA adducts in mice: comparison of 7H-dibenzo(c,g)carbazole, benzo(a)pyrene, and 2-acetylaminofluorene

The environmental pollutant 7H-dibenzo(c,g)carbazole (DBC) has been shown to be a potent carcinogen in various mouse tissues, but displays an unusual degree of hepatocarcinogenicity. We have previously reported that in accord with this activity, mouse liver is the target organ for DBC-DNA binding, with total levels being up to 2700 times greater than in extrahepatic tissues after s.c. administration. To elaborate on this finding, we have directly compared the tissue distribution of DNA damage by three diverse aromatic carcinogens, DBC, benzo(a)pyrene (BP), and 2-acetylaminofluorene (AAF). Following a single topical, p.o., or s.c. administration of 80 mumol/kg of test compound to male BALB/c mice, a 32P-postlabeling assay showed the total number of DBC adducts in liver DNA to be 11-138 times that in kidney, lung, or skin DNA. The degree of hepatic adduction varied as a function of the route of administration, with the highest occurring after topical application and the lowest after s.c. injection. The tissue preference for AAF and BP adducts varied with the route of administration and was much less than for DBC adducts, except that topical application of BP gave DNA adduct levels in skin that were 91-218 times greater than in other tissues. For a given tissue and route of administration, DNA adduction by DBC was 1.7- to 950-fold greater than that by BP and AAF, except in skin where the level of DNA adducts from BP was 3 to 4 times that from DBC. We conclude that (a) DBC exhibits an exceptional and unique preference for liver DNA adduction after different routes of administration; (b) DBC is more potent overall than BP or AAF in causing tissue DNA damage; and (c) for each of the three carcinogens, the route of exposure is a much less important factor than the nature of the carcinogen in determining the tissue distribution of covalent DNA damage.     

DNA fragmentation in some organs of rats and mice treated with cycasin.

Cycasin (methylazoxymethanol-beta-D-glucoside) is carcinogenic in several animal species. It produces a variety of malignant tumours, mainly in the liver of mice, and in the liver, kidney and large intestine in rats. It does not appear to be mutagenic in the Ames test, even in the presence of liver microsome fraction, and it is among those carcinogens (less than 10%) ranked as "false negatives" in this test. The ability of cycasin to damage in vivo liver, kidney, lung and colonic DNA of Wistar rats and C57BL/L mice was investigated by means of alkaline elution technique. Oral single-dose administration of cycasin, in the range of 50-400 mg/kg body weight, produced in the rat a clearly evident dose-dependent DNA fragmentation in the liver, and less marked damage to DNA from kidney and colon mucosa. In mice, the same treatment produced dose-dependent DNA damage only in the liver. DNA repair up to 18 h appeared to be incomplete both in mice and rats. Methylazoxymethanol acetate is considered to be an active form of cycasin. While in vivo methylazoxymethanol acetate caused DNA damage, in vitro it appeared inactive and required metabolic activation, possibly consisting in its hydrolysis by esterase activity, to be able to cause DNA fragmentation.     

Dietary n-3 PUFA increases the apoptotic response to 1,2-dimethylhydrazine, reduces mitosis and suppresses the induction of carcinogenesis in the rat colon

The effect of dietary fish oil on colonic crypt cell apoptosis and proliferation was examined in male Wistar rats, 24 and 48 h after administration of 1,2-dimethylhydrazine (DMH), and its influence on the induction of aberrant crypt foci (ACF) in the distal colon was assessed. Rats (125-150 g) fed a high-fat semi-synthetic diet containing corn oil (CO) were given DMH (30 mg/kg body wt) or a sham injection of EDTA/NaCl. Animals were then fed either the CO diet or a diet in which fish oil (EPA 18.7%; DHA 8%) was substituted for corn oil. Subgroups of rats (n = 5) were killed after 24 and 48 h, and crypt cell apoptosis and proliferation were quantified by morphological criteria in isolated intact crypts from the mid and distal colon. Consumption of the fish oil diet (FO) was associated with increased apoptotic cell death (P < 0.001) and suppression of proliferation (P < 0.05) in colonic crypts both 24 and 48 h after DMH. In a second experiment, animals were given three injections of DMH or sham injections of carrier at weekly intervals. For 48 h after each injection animals were fed either the CO or FO diet, but otherwise maintained on the CO throughout. The number and crypt multiplicity of ACF in the distal colon were determined after 18 weeks, and animals given the FO diet for the 48 h period following carcinogen administration were found to have significantly fewer ACF than rats fed the CO diet (P < 0.05). The data demonstrate that the fatty acid composition of the diet is an important determinant in the induction of carcinogenesis by DMH. The proliferative and apoptotic response of the colonic crypt to carcinogen and fish oil, coupled with the reduced incidence of ACF, suggest n-3 PUFA can protect against the carcinogenic effects of DMH by mediating changes in the balance proliferation and cell death.     

Induction of colon mucosal β-glucuronidase production as a mechanism for 1,2-dimethylhydrazine colon carcinogenesis

Although early studies in germ-free rats showed almost complete dependence on dimethylhydrazine (DMH) colon carcinogenesis upon the presence of colon bacteria, no adequate explanation was given for the 20% tumor incidence observed in germ-free animals. Bacterial activation of liver microsomal products releasing active proximate carcinogens has been the accepted reason for the exquisite specificity DMH has for the colon. Recent work, including the present study, show the colon mucosa is capable of metabolizing carcinogens and activating conjugating forms metabolized in the liver independent of the intestinal microflora. Mucosal β-glucuronidase production was assayed in coded, scraped mucosa samples from the duodenum/jejunum, ileum, right colon, and left colon of normal and DMH-treated rats. Normal mucosal β-glucuronidase production was highest in the left colon followed by the right colon, duodenum, and ileum, respectively. Enzyme production in the left colon was significantly increased 24 hours after injection of 25 mg/kg body weight DMH. No elevation was seen in other mucosal samples. Metabolism of DMH to oxidated forms conjugated to glucuronic acid is well established. Thus, this study offers a possible role for carcinogen, induction of a metabolic enzyme in its target tissue.     

Scc-1, a novel colon cancer susceptibility gene in the mouse: linkage to CD44 (Ly-24, Pgp-1) on chromosome 2.

Mutations of proto-oncogenes and tumor-suppressor genes lead to neoplastic development. Some germline mutations of these genes increase the tumor susceptibility of their carriers, but the relationship between genes controlling tumor susceptibility and the known oncogenes and tumor-suppressor genes remains unelucidated. Moreover, as tumor susceptibility in mouse is controlled by multiple genes, their identification has been virtually impossible. We therefore developed a new system, the recombinant congenic strains (RCS), which separates individual susceptibility genes into different RC strains, thus facilitating their analysis. To map genes controlling the development of colon cancer, we used the Balb/c-c-STS (CcS/Dem) RC strains. Owing to several unidentified genes, Balb/cHeA mice are relatively resistant and STS/A mice highly susceptible to 1,2-dimethylhydrazine-(DMH)-induced colon adenocarcinomas. Each CcS/Dem strain carries a different subset of about 12.5% of genes of the STS strain on the Balb/c background, and individual STS susceptibility genes became segregated into different RC strains. Using CcS-19, one of the highly susceptible RC strains, we mapped a novel colon tumor susceptibility gene, Scc-1, different from the oncogenes and tumor-suppressor genes known to be involved in colon tumorigenesis, in the vicinity of CD44 (Ly-24, Pgp-1) on chromosome 2. The mapping of the Scc-1 gene indicates that the RCS system can be used to map and study the presently unknown genes which control cancer development.     

大肠粘膜细胞色素P450在致癌剂活化中的作用及其诱导和抑制

本文研究了大肠粘膜细胞和肝细胞微粒体酶系对专一性致大肠癌化合物—二甲基肼(DMH)的代谢活性。实验证明,大肠粘膜细胞如同肝细胞一样,存在依赖细胞色素P450的混合功能氧化酶,能催化DMH的羟化反应.使之转变成具直接致癌活性的终致癌物,其羟化活性大约相当于肝的79.5％。大肠粘膜细胞对DMH的羟化活性及细胞色素P450含量,能被苯巴比妥钠所诱导,如给动物以维生素E,则两者同时降低。文中强调了大肠粘膜细胞色素P450在DMH代谢转变中的重要作用,并讨论了维生素E在预防大肠化学致癌过程中的意义。     

Carcinogen binding to various types of dietary fiber

The percent of the carcinogen 1,2-dimethylhydrazine (DMH) bound to a variety of fibers, such as wheat bran, corn bran, citrus pulp, citrus pectin, and alfalfa, was examined at pH values ranging from 1 to 12. The percent of DMH bound to wheat bran increased from 4% at PH 1 to 55% at pH 2 to 77% at pH 12. A sharp rise in carcinogen binding to corn bran occurred between pH 5% of the DMH was bound and pH 8 where 51% of the DMH was bound. The percent of DMH bound to dehydrated citrus pulp also increased as the pH increased with 10% binding observed at pH 1 and with 57% binding observed at pH 12. Between pH 2 and pH 7, the percent of DMH bound to pectin decreased from 60 to 11%. As the pH became more basic, the percent of DMH bound to pectin increased to 42% at pH 12. The sharpest rise in the percent of DMH bound to alfalfa meal occurred between pH 10.5 and pH 12.0. Results from this experiment showed that the affinity to various types of dietary fibers for the colon carcinogen DMH was differentially affected by pH. These results suggested that the protective effect of certain types of dietary fiber against chemically induced colon cancer my in part be attributed to enhanced carcinogen binding by dietary fiber in the colon.     

Organ-specific effects of 1,2-dimethylhydrazine in hamster

Uptake and metabolism of the carcinogen 1,2-dimethylhydrazine (DMH) were compared in isolated epithelial cells from the colon and the small intestine. A new method was developed to separate colonic epithelial cells into surface columnar cells and crypt cells without the use of any proteolytic enzymes. Colonic columnar cell-enriched fraction exhibited DMH metabolism two to three times higher than that of crypt cells. The carcinogen binding was much lower in the small intestine as compared to the colon. In the small intestine, the crypt cell-enriched fraction showed higher carcinogen binding as compared to villus cells. Pyrazole was found to inhibit DMH binding by isolated small intestinal and colonic epithelial cells. The extent of inhibition was maximum in cells showing the greatest ability to incorporate DMH.