methylnitronitrosoguanidine and Colonic-Neoplasms

Experimental chemical carcinogenesis in the digestive tract is reviewed, mainly on the basis of information obtained in the laboratories of the National Cancer Center Research Institute. It is generally accepted that cancer is the outcome of DNA damage, resulting in mutation, loss, amplification and recombination of genes. Gastric cancer is no exception. It was shown very early that cancer of the glandular stomach can be produced in rats by administration of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a widely used mutagen. However, this depends on the genotype. Whereas the ACI rat is susceptible to MNNG, the Buffalo rat is resistant and this is a dominantly inherited trait. Genes responsible for the sensitivity to gastric cancer induction are at present under investigation by linkage analysis of rat genome markers. With regard to cancer in humans, our finding that cooked proteinaceous foods can give rise to a series of heterocyclic amines (HCAs) is of major significance. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), one of the most abundant, causes colon cancers in male rats, whereas in females it induces breast cancers. The colon cancers induced by PhIP feature a deletion of G as represented by 5-GGGA-3-->5-GGA-3 in the Apc gene, resulting in a truncated Apc molecule. Microsatellite mutations have also been found in PhIP-induced colon tumors, as in human hereditary non-polyposis colorectal cancer cases. Similarly to the case of gastric cancer production by MNNG, there is a genetic component and F344 rats are more susceptible to PhIP colon carcinogenesis than the ACI/N strain and the gene responsible is being sought. Since carcinogenesis proceeds with accumulation of genetic alteration, often involving genomic instability, exposure to any kind of carcinogenic substances, either xeno- or autobiotics, needs to be reduced as far as possible, taking account of inconvenience at the individual and socio-economical levels.

Topics: Adenomatous Polyposis Coli; Animals; Carrier Proteins; Colonic Neoplasms; DNA, Neoplasm; Gene Deletion; Genes, APC; Humans; Male; Methylnitronitrosoguanidine; Molecular Chaperones; Nerve Tissue Proteins; Rats; Rats, Inbred ACI; Rats, Inbred BUF; Rats, Inbred F344; Rats, Wistar; Receptors, Aryl Hydrocarbon; Stomach Neoplasms

Dietary factors are now considered to be among the most important environmental risk determinants for cancer. In addition to epidemiological studies, experimental animal studies are an important tool to investigate dietary modulation in carcinogenesis. Results of recent experimental studies on the effect of some nutrients indicate that vitamin A did show an inverse relation with the occurrence of preneoplastic respiratory lesions but not with respiratory tract tumors in benzo[a]pyrene-induced respiratory carcinogenesis. Dietary fat increases respiratory tract tumors and preneoplastic lesions. In colon carcinogenesis, a fat-fiber interrelation was noticed in 1,2-dimethyl-hydrazine- and N-methyl-N'-nitro-N-nitrosoguanidine-induced tumors. Preliminary results in prostate carcinogenesis indicate that dietary fat did not influence the incidence of prostate cancer in a recently developed rat model. Some possible mechanisms in colon and prostate carcinogenesis are discussed.

Topics: 1,2-Dimethylhydrazine; Androgens; Animals; Benzo(a)pyrene; Colonic Neoplasms; Diet; Dietary Fats; Dietary Fiber; Dietary Proteins; Dimethylhydrazines; Disease Models, Animal; Drug Synergism; Epidemiologic Methods; Humans; Lung Neoplasms; Male; Methylnitronitrosoguanidine; Models, Biological; Prostatic Neoplasms; Vitamin A

Topics: 1,2-Dimethylhydrazine; Adult; Animals; Azoxymethane; Cholesterol; Cholesterol, Dietary; Cholestyramine Resin; Cocarcinogenesis; Colonic Neoplasms; Diagnosis-Related Groups; Dietary Fats; Dimethylhydrazines; Disease Models, Animal; Epidemiologic Methods; Humans; Methylnitronitrosoguanidine; Methylnitrosourea

Topics: Animals; Antioxidants; Ascorbic Acid; Butylated Hydroxyanisole; Butylated Hydroxytoluene; Carcinogens; Colonic Neoplasms; Ethoxyquin; Glutathione Transferase; Mammary Neoplasms, Experimental; Methylnitronitrosoguanidine; Neoplasms, Experimental; Phenobarbital; Propyl Gallate; Rats; Rats, Inbred F344; Saccharin; Stomach Neoplasms; Substrate Specificity; Urinary Bladder Neoplasms

We propose that SF derived from normal-appearing biopsies of ACR gene carriers exist in an initiated state as the result of a dominant mutation. Based on our studies with the ACR cell system, we further suggest that, although an initiated state is essential to cancer development, not all initiated cells necessarily develop into cancerous cells. The genetic makeup of an initiated cell has been established through linkage between abnormal phenotypic markers and pedigree profiles and through cell hybridization, including initial analysis of gene products. We believe that it is consistent with an autosomal dominant trait. In contrast, cells from patients who are homozygous for chromosomal breakage syndromes, including those with xeroderma pigmentosum, represent an experiment of nature which presumably underlies factors associated with cancer promotion in humans. We have demonstrated that ACR cells can be differentially transformed by oncogenic viruses, a carcinogen (MNNG), and gamma-ray irradiation, and that they can proliferate in vitro after exposure to a tumor promoter (TPA. This simple experimental model provides a novel system for the study of tumor promotion in vitro. We further suggest that, through the use of TPA, various stages associated with cancer development in humans, i.e., initiation through promotion and progression, can be identified in vitro. Attempts to apply these results in vivo are currently in progress. The apparent susceptibility of ACR cells to further transformation by oncogenic viruses and chemical and physical agents indicates that genetic information residing within these cells, probably in the form of a relatively limited and specific number of DNA sequences associated with the ACR mutation, renders them more sensitive to these three distinct classes of carcinogens. We submit that, through our tests on SF, and ACR gene carriers within recognized ACR clusters can be diagnosed at present with sufficient certainty to warrant immediate action. In addition, it seems that the time has arrived for a major undertaking to screen for persons who are likely to be at increased risk of cancer, perhaps through walk-in clinics. An underlying assumption in these studies is that predisposition to cancer, in general, is associated with an autosomal dominant trait in obligatory heterozygote gene carriers.

Topics: Actins; Adenoma; Antigens, Neoplasm; Carcinogens; Cell Transformation, Neoplastic; Cell Transformation, Viral; Cells, Cultured; Cholesterol; Cocarcinogenesis; Colonic Neoplasms; Cytoskeleton; Disease Susceptibility; Genes, Dominant; Humans; Kirsten murine sarcoma virus; Methylnitronitrosoguanidine; Mitochondria; Models, Biological; Mutation; Plasminogen Activators; Prognosis; Rectal Neoplasms

The chemical class of drugs known as the nitrosoureas are a recently developed group of very active alkylating-agent anticancer drugs which are best represented by BCNU, CCNU, and methyl-CCNU (meCCNU). The nitrosoureas are among the most active, if not the most active, anticancer drugs both quantitatively (log kill of sensitive tumor cells in vivo) and qualitatively (spectrum of mouse, rat, and hamster tumors responding to treatment). Therapeutic anticancer activity of the nitrosoureas has been consistently observed with oral as well as parenteral administration. The nitrosoureas are clearly the most active group of anticancer drugs observed against experimental meningeal leukemias and intracerebrally implanted transplantable primary tumors of central nervous system origin (eg, gliomas, ependymoblastomas, and astrocytomas in mice and hamsters). The nitrosoureas have been observed to be less than additive in lethal toxicity for vital normal cells in the mouse in combination with representatives of the other major classes of anticancer agents, eg, purine antagonists, pyrimidine antagonists, inhibitors of DNA polymerase(s) or ribonucleotide reductase(s), mitotic inhibitors, drugs that bind to or intercalate with DNA, and other alkylating agents. Therapeutic synergism against one or more transplantable or spontaneous tumors of mice, rats, or hamsters with one of several nitrosoureas in two-drug combinations with representatives of most of the major classes of anticancer agents listed above has been reported. With a number of advanced-stages mouse tumors, generally considered to be refractory to treatment with most anticancer agents, long-term cures have been obtained with combination-drug or combined-modality (surgery plus chemotherapy) treatment. The demonstrated lack of cross-resistance of several leukemias and solid tumors of mice selected for resistance to BCNU, meCCNU, or other alkylating agents suggests that the widely held opinion that all alkylating agents are very similar in biologic mechanism of action, and therefore resistance to one alkylating agent probably predicts cross-resistance to all alkylating agents, may no longer be tenable. If not, then alkylating-agent drug combinations, either used alone or combined with other treatment modalities (eg, surgery) which have been reported to result in therapeutic improvement in a number of experimental murine tumor systems, may be indicated for serious consideration as surgical adjuvant chemotherapy by s

Topics: Animals; Carmustine; Colonic Neoplasms; Drug Evaluation, Preclinical; Drug Resistance; Drug Therapy, Combination; Leukemia L1210; Lomustine; Lung Neoplasms; Mammary Neoplasms, Experimental; Melanoma; Methylnitronitrosoguanidine; Methylnitrosourea; Mice; Neoplasms, Experimental; Nitrosourea Compounds; Semustine

Physical exercise has been shown to be protective against colon carcinogenesis. Physical exercise, however, covers a wide range of modalities, from which different effects on the human body have been reported. We sought to clarify whether aerobic and resistance trainings would differently affect the development of early carcinogenic events in the colon.. Male BALB/c, C57/BL6, and interleukin 10 knockout (IL-10; on C57/BL6 background) mice were exposed to the carcinogen N-methyl-N'-nitro-N-nitrosoguanidine. BALB/c mice were subjected to either aerobic (swimming) or resistance trainings (climbing a ladder with load attached to the tail). C57/BL6 and IL-10 mice only swam.. In BALB/c carcinogen-exposed mice, aerobic and resistance trainings decreased serum creatine kinase levels (P < 0.001). Although aerobic and resistance trainings reduced the generation of lipid thiobarbituric reactive species (P < 0.01 and P < 0.001), only aerobic exercises enhanced serum glutathione levels aside from carcinogenic exposure (P < 0.05). Carcinogen-exposed and aerobic-trained mice developed 36% less colon preneoplastic lesions than its control group (P < 0.05). Aerobic training reduced colonic subepithelial cyclooxygenase-2 expression in carcinogen-exposed mice (P < 0.001). Interestingly, in this same group, colonic IL-10 expression was upregulated sevenfold (P < 0.001). Current findings were confirmed in C57/BL6 carcinogen-exposed mice, in which aerobic training promoted antipreneoplastic effects (P < 0.05). Knocking IL-10 out of C57/BL6 mice abrogated antipreneoplastic effects of aerobic training on the colon tissue (P > 0.05).. IL-10 is a pivotal element for antipreneoplastic effects of aerobic training on the colon.

Topics: Animals; Carcinogens; Colonic Neoplasms; Disease Models, Animal; Humans; Interleukin-10; Male; Methylnitronitrosoguanidine; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Physical Conditioning, Animal; Precancerous Conditions; Resistance Training; Swimming

The alkylating agent N-methyl-N'-nitro-N'-nitrosoguanidine (MNNG) can cause excess DNA strand breaks that lead to poly(ADP-ribose)polymerase-1 (PARP-1) overactivation and cell death (parthanatos). However, the detail mechanism of MNNG-induced parthanatos was not well-investigated. In this study, we used MNNG-treated mouse embryonic fibroblasts (MEFs) to elucidate the signaling pathways of MNNG-induced parthanatos. We found that MNNG-induced cell death accompanied by rapid PARP-1 activation, c-Jun N-terminal kinase (JNK) activation, biphasic reactive oxygen species (ROS) production and intracellular calcium increase. The early ROS production occurring at 1 min and peaking at 5-15 min after MNNG treatment partially resulted from NADPH oxidase. In contrast, the late phase of ROS production occurring at 30 min and time-dependently increasing up to 6h after MNNG treatment was generated by mitochondria. The antioxidant, NAC can abrogate all phenomena caused by MNNG. Results indicate that the calcium rise was downstream of early ROS production, and was involved in PARP-1 and JNK activation. Moreover, the PARP inhibitor was able to reduce MNNG-induced late-phase ROS production, calcium elevation, and cell death. Results further indicated the involvement of RIP1 in sustained ROS production and calcium increase. We characterized the interactive roles of ROS, calcium, JNK, and RIP1 in MNNG-induced cell death. We found that in addition to the alkylating property previously demonstrated, ROS production triggered by MNNG results in enhanced DNA damage and PARP-1 activation. Moreover, intracellular calcium elevation and ROS production have mutual amplification effects and thus contribute to PARP-1-mediated parthanatos.

Topics: Alkylating Agents; Animals; Calcium; Cell Death; Colonic Neoplasms; DNA Damage; Female; Fibroblasts; HCT116 Cells; HeLa Cells; Humans; MAP Kinase Kinase 4; Methylnitronitrosoguanidine; Mice; Oxidative Stress; Poly(ADP-ribose) Polymerases; Reactive Oxygen Species; Signal Transduction; Uterine Cervical Neoplasms

The aberrant crypt foci (ACF), cyclooxygenase 2 (COX-2), and the proliferating cell nuclear antigen (PCNA) are putative biomarkers for colon cancer. To study the association between light (1 g of ethanol/kg bw) and moderate (3 g of ethanol/kg bw) doses of ethanol with the chemical carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), Wistar rats were divided into 6 groups. The colon fragments were collected for histochemical and immunohistochemical analyses, and the liver samples were collected for oxidative stress analysis, with products of lipid peroxidation (malondialdehyde), antioxidant enzymes (glutathione), and vitamin E. The association of light and moderate doses of ethanol with MNNG did not present differences in the oxidative parameters. However, a reduction in vitamin E levels in the carcinogen groups was observed. The association induced a reduction of the COX-2 and PCNA expression. The number of ACF in the group that received a light dose of ethanol had lower rates, while the group that received a moderate dose had the highest rates compared to the control MNNG, demonstrating that the light dose of ethanol could have a protective effect, while the moderate dose could represent a risk during chemical carcinogenesis in rats.

Topics: Aberrant Crypt Foci; Animals; Antioxidants; Biomarkers; Carcinogens; Colon; Colonic Neoplasms; Cyclooxygenase 2; Dose-Response Relationship, Drug; Ethanol; Glutathione; Immunohistochemistry; Lipid Peroxidation; Liver; Male; Malondialdehyde; Methylnitronitrosoguanidine; Oxidative Stress; Proliferating Cell Nuclear Antigen; Rats; Rats, Wistar; Vitamin E

Current published data suggest that DNA mismatch repair (MMR) triggers prolonged G(2) cell cycle checkpoint arrest after alkylation damage from N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) by activating ATR (ataxia telangiectasia-Rad3-related kinase). However, analyses of isogenic MMR-proficient and MMR-deficient human RKO colon cancer cells revealed that although ATR/Chk1 signaling controlled G(2) arrest in MMR-deficient cells, ATR/Chk1 activation was not involved in MMR-dependent G(2) arrest. Instead, we discovered that disrupting c-Abl activity using STI571 (Gleevec, a c-Abl inhibitor) or stable c-Abl knockdown abolished MMR-dependent p73alpha stabilization, induction of GADD45alpha protein expression, and G(2) arrest. In addition, inhibition of c-Abl also increased the survival of MNNG-exposed MMR-proficient cells to a level comparable with MMR-deficient cells. Furthermore, knocking down GADD45alpha (but not p73alpha) protein levels affected MMR-dependent G(2) arrest responses. Thus, MMR-dependent G(2) arrest responses triggered by MNNG are dependent on a human MLH1/c-Abl/GADD45alpha signaling pathway and activity. Furthermore, our data suggest that caution should be taken with therapies targeting c-Abl kinase because increased survival of mutator phenotypes may be an unwanted consequence.

Topics: Antineoplastic Agents; Base Pair Mismatch; Benzamides; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Colonic Neoplasms; DNA Repair; Dose-Response Relationship, Drug; G2 Phase; Humans; Imatinib Mesylate; Methylnitronitrosoguanidine; Models, Biological; Nuclear Proteins; Piperazines; Proto-Oncogene Proteins c-abl; Pyrimidines; Signal Transduction

Aberrant crypt foci (ACF) are microscopic lesions which have been postulated to precede the development of adenomas, precursors of colon cancer. The gastric M1/MUC5AC mucin has also been described as an early marker of colon carcinogenesis in the human and in the rat. To study changes in mucin expression associated with the genesis of tumors, Wistar rats were treated by intrarectal instillations of MNNG, twice a week for 2 weeks, and were sacrificed 10 (n = 20), 14 (n = 20), 22 (n = 20), 30 (n = 10) and 66 (n = 16) weeks after the beginning of the treatment. In the treated rats, the MUC5AC mucin was mainly expressed in ACF compared with the histologically normal mucosae, which showed few isolated MUC5AC-positive normal crypts. During carcinogenesis, the percentage of large ACF [> or =10 aberrant crypts] increased and the number of MUC5AC-positive (NCs) decreased. At Week 30, small tumors were observed arising from large ACF, both types of lesions expressing MUC5AC. At Week 66, large tumors showed remnants of MUC5AC-positive ACF in their adjacent mucosae. This observation suggests that the expression of MUC5AC is associated with the ACF/adenoma sequence and supports the notion of large ACF as precursors of adenomas/adenocarcinomas. Moreover, the expression of MUC5AC in the transitional mucosa adjacent to both rat and human colon tumors suggests that some human tumors could arise from large ACF, and reinforces the concept of the premalignant potential of these lesions.

Topics: Adenocarcinoma; Adenoma; Animals; Antibodies, Monoclonal; Colonic Neoplasms; Disease Progression; Immunohistochemistry; Intestinal Mucosa; Male; Methylnitronitrosoguanidine; Mucin 5AC; Mucins; Precancerous Conditions; Rats; Rats, Wistar; Time Factors

We have evaluated cell survival, apoptosis, and cell cycle responses in a panel of DNA mismatch repair (MMR)-deficient colon and prostate cancer cell lines after alkylation and UV-C damage. We show that although these MMR-deficient cells tolerate alkylation damage, they are as sensitive to UV-C-induced damage as are the MMR-proficient cells. MMR-proficient cells arrest in the S-G2 phase of the cell cycle and initiate apoptosis following alkylation damage, whereas MMR-deficient cells continue proliferation. However, two prostate cancer cell lines that are MMR-deficient surprisingly arrest transiently in S-G2 after alkylation damage. Progression through G1 phase initially depends on the expression of one or more of the D-type cyclins (D1, D2, and/or D3). Analysis of cyclin D1 expression shows an initial MMR-independent decrease in the protein level after alkylation as well as UV-C damage. At later time points, however, only DNA damage-arrested cells showed decreased cyclin D1 levels irrespective of MMR status, indicating that reduced cyclin D1 could be a result of a smaller fraction of cells being in G1 phase rather than a result of an intact MMR system. Finally, we show that cyclin D1 is degraded by the proteasome in response to alkylation damage.

Topics: Alkylating Agents; Alkylation; Apoptosis; Base Pair Mismatch; Cell Cycle; Cell Line, Tumor; Cell Survival; Colonic Neoplasms; Cyclin D1; Cysteine Endopeptidases; DNA Damage; DNA Methylation; DNA Repair; DNA, Neoplasm; Humans; Male; Methylnitronitrosoguanidine; Multienzyme Complexes; Prostatic Neoplasms; Proteasome Endopeptidase Complex; Time Factors; Ultraviolet Rays

A rat colonic adenocarcinoma was implanted subcutaneously (s.c.) in nude mice. After 7 days, the animals were divided into different groups. Two groups received subcutaneous injections twice daily with 3 or 6 micro g/kg body weight octreotide, galanin and serotonin. Three groups were respectively treated with 20, 30, and 40 micro g/kg body weight of the previously mentioned bioactive substances. Control group received only saline solution in the same fashion as treated animals. The treatment lasted for 5 days. The tumour volume and weight, the relative density of blood vessels, of tumour necrotic tissue, of apoptotic nuclei and of proliferating nuclei were measured. Apoptosis was detected by in situ labelling of nuclear DNA fragmentation according to TUNEL method, and proliferation by immunocytochemistry. Morphometry was done with the classical stereological point-counting method. Food consumption, animal weight, faeces weight and its water content were measured for 3 days before and after treatment. Triple therapy with 3 and 6 micro g/kg body weight had no effect on any of the parameters measured, except in reducing the relative volume density of tumour blood vessels. Treatment with 20, 30 and 40 micro g/kg body weight of the previously mentioned bioactive substances reduced the tumour volume, the relative volume density of blood vessels and increased the relative volume density of necrotic tissue and of apoptotic nuclei (in the 20 micro g group). However, there was no difference between treated mice and controls regarding the relative volume density of proliferating nuclei. There was no statistical difference between treated animals regarding food consumption, body weight, faeces weight and its water content before and during treatment. The present study confirms that triple therapy with octreotide, galanin and serotonin causes regression of a rat colon carcinoma. It further showed that optimum treatment dose is 20 micro g/kg body weight of each bioactive substance. Moreover, this therapy regime does not show apparent side effects in the experiments carried out on mice.

Topics: Adenocarcinoma; Animals; Apoptosis; Blood Vessels; Carcinogens; Colonic Neoplasms; Drug Therapy, Combination; Galanin; Methylnitronitrosoguanidine; Mice; Mice, Nude; Necrosis; Octreotide; Rats; Serotonin; Transplantation, Heterologous

The 39-kDa DNA polymerase beta (pol beta) is an essential enzyme in short-patch base excision repair pathway. A wild-type and a truncated forms of pol beta proteins are expressed in primary colorectal and breast adenocarcinomas and in a primary culture of renal cell carcinoma. To test whether pol beta has a contributory role in tumorigenicity of human tumor cell lines, we have undertaken a study to determine expression of pol beta in colon, breast, and prostate tumor cell lines. Unlike primary colon tumor cells, three types of pol beta mRNA have been identified in HCT116, LoVo, and DLD1, colon tumor cell lines. A 111-bp-deleted pol beta transcript was expressed in MCF7, a breast tumor cell line, but not in primary breast tumor cells. An expression of a smaller pol beta transcript has been revealed in DU145, a prostate tumor cell line, whereas, a single base (T) deletion in mRNA at codon 191 was found in prostate cancer tissue. Interestingly, a wild-type pol beta transcript was also expressed in all tumor cell lines similar to primary tumor cells. Furthermore, the cell extract of LoVo exhibited highest gap-filling synthesis function of pol beta when the extract of DU145 showed lowest activity. MNNG, a DNA alkylating agent, enhanced the gap-filling synthesis activity in extracts of LoVo cell line. Furthermore, the cellular viability of LoVo and HCT116 cells is sensitive to MNNG when DU145 cells are resistant. These results demonstrate heterogeneity in pol beta mRNA expression, which may be a risk factor related to tumorigenic activities of tumor cell lines.

Topics: Breast Neoplasms; Cell Survival; Colonic Neoplasms; DNA Polymerase beta; DNA Repair; DNA, Neoplasm; Female; Gene Expression; Humans; Male; Methylnitronitrosoguanidine; Neoplasms; Prostatic Neoplasms; RNA, Messenger; RNA, Neoplasm; Tumor Cells, Cultured; Tumor Stem Cell Assay

To clarify the carcinogenic factors--whether it is the kind of carcinogen or their length of exposure--that determine whether colorectal cancer develops from an adenoma or develops de novo in the absence of an adenoma, we histopathologically analyzed a total of 229 rat colon tumors induced by administration of 1,2-dimethyl-hydrazine (DMH) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) for three or 15 weeks. In the three-week-exposure groups, 71% of DMH-induced carcinomas and 82% of MNNG-induced carcinomas coexisted with low-grade dysplasia (adenomatous remnant). However, in the 15-week-exposure groups, lowgrade dysplasia was observed in only 10% of DMH-induced and 27% of MNNG-induced carcinomas. Even in the tumors smaller than 20 mm3, it was observed in only 10% of DMH-induced and 32% of MNNG-induced carcinomas. Furthermore, carcinomas without low-grade dysplasia predominated from the initial period of tumor occurrence. Next, we investigated association of K-ras and APC gene mutations with these carcinogenesis patterns in 80 tumors. K-ras mutations were not detected in any tumors induced by three weeks of exposure. However, in the 15-week-exposure groups, this mutation was observed in 57% of DMH-induced tumors and 13% of MNNG-induced tumors. APC mutations in the region homologous to the human mutation cluster region were observed in only 6% of tumors. Thus, our results suggest that the carcinogenesis patterns in rat colon are dependent on the length of exposure to carcinogen and that K-ras mutations were partly involved in a subset of them.

Topics: 1,2-Dimethylhydrazine; Animals; Carcinogens; Carcinoma; Colonic Neoplasms; Genes, APC; Genes, ras; Methylnitronitrosoguanidine; Mutation; Rats; Rats, Inbred F344; Time Factors

Treatment of colon cancer cells with MNNG causes DNA damage with reduced telomeric signals in a p53-dependent manner, but increased cell cycle arrest in S-G(2)/M by both p53-dependent and independent mechanisms. Results also indicate that cellular levels of TRF2 may play a critical role in MNNG-induced cell cycle arrest and apoptosis of colon cancer cells.

Topics: Apoptosis; Blotting, Western; Cell Cycle; Colonic Neoplasms; DNA; DNA Damage; DNA-Binding Proteins; Flow Cytometry; Humans; In Situ Hybridization, Fluorescence; Methylnitronitrosoguanidine; Polymerase Chain Reaction; Telomeric Repeat Binding Protein 2; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Mismatch recognition in human cells is mediated primarily by a heterodimer of hMSH2 and hMSH6. Cells mutated in both alleles of the hMSH6 gene are deficient in the correction of base/base mispairs and insertion/deletion loops of one nucleotide and thus exhibit a strong mutator phenotype, evidenced by elevated mutation rates and microsatellite instability, as well as by tolerance to methylating agents. The decrease in replication fidelity associated with a loss of mismatch correction implies that with each division, these cells are likely to acquire new mutations throughout their genomes. Should such secondary mutations occur in genes linked to replication fidelity or involved in the maintenance of genomic stability, they might contribute to the observed mutator phenotype. The human colon tumour line HCT15 represents one such case. Although it carries inactivating mutations in both hMSH6 alleles, it has also been shown to contain a missense mutation in the coding sequence of the proofreading domain of the polymerase-delta gene. In an attempt to find out whether the phenotype of HCT15 cells was indeed brought about solely by the lack of hMSH6, we stably transfected them with a vector carrying the wild-type hMSH6 cDNA. Our results show that although the levels of transgenic hMSH6 were low, expression of the wild-type protein resulted in a substantial restoration of mismatch binding, mismatch repair capacity and the stability of mononucleotide repeats, as well as in the reduction of mutation rates. Although methylation tolerance of the hMSH6-expressing cells was not markedly affected, the G2 cell cycle checkpoint, absent in N-methyl-N'-nitro-N-nitrosoguanidine-treated control cells, was restored.

Topics: Base Pair Mismatch; Base Sequence; Colonic Neoplasms; DNA Primers; DNA Repair; DNA-Binding Proteins; DNA, Complementary; G2 Phase; Guanine; Humans; Methylnitronitrosoguanidine; Methylnitrosourea; Microsatellite Repeats; Mutation; Phenotype; Transfection; Tumor Cells, Cultured

We have developed a "comparative growth assay" that complements current assays of drug effects based on cytotoxicity. A co-culture of two cell lines, one of which is fluorescently labeled, is exposed to a cytotoxic agent and the proportion of fluorescent cells is compared with that of a baseline unexposed co-culture. For demonstration purposes, two HCT116 cell lines (an hMLH1 homozygous and an hMLH1 heterozygous mutant), altered by insertion of vector alone or the same vector carrying an insert for the expression of enhanced green fluorescent protein (EGFP), were exposed to numerous "anti-cancer" agents. The assay was further validated in a system of two cell lines differing only in the expression of the breast cancer resistance protein (BRCP). The assay allowed the estimation of the duration of action of a particular agent. Assessment of the agent's differential activity over a given time in culture could be expressed as a selection rate, which we chose to describe on an "average selection per day" basis. We conclude that this assay: 1) provides insight into the differential dynamic effects of chemotherapeutic agents or radiation; and 2) allows, through the use of matched cell lines, the investigation of critical physiologic features that govern cell sensitivity.

Topics: Anti-Bacterial Agents; Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Breast Neoplasms; Cell Culture Techniques; Cell Division; Coculture Techniques; Colonic Neoplasms; Dose-Response Relationship, Drug; Dose-Response Relationship, Radiation; Drug Resistance, Neoplasm; Flow Cytometry; Fluorescent Dyes; Genetic Vectors; Gentamicins; Green Fluorescent Proteins; Humans; Luminescent Proteins; Methylnitronitrosoguanidine; Mitoxantrone; Mutation; Neoplasm Proteins; Time Factors; Transfection; Tumor Cells, Cultured

Small polydisperse circular DNA (spcDNA) is a heterogeneous population of extrachromosomal circular molecules present in a large variety of eukaryotic cells. Elevated amounts of total spcDNA are related to endogenous and induced genomic instability in rodent and human cells. We suggested spcDNA as a novel marker for genomic instability, and speculated that spcDNA might serve as a mutator. In this study, we examine the presence of telomeric sequences on spcDNA. We report for the first time the appearance of telomeric repeats in spcDNA molecules (tel-spcDNA) in rodent and human cells. Restriction enzyme analysis indicates that tel-spcDNA molecules harbor mostly, if not exclusively, telomeric repeats. In rodent cells, tel-spcDNA levels are higher in transformed than in normal cells and are enhanced by treatment with carcinogen. Tel-spcDNA is also detected in some human tumors and cell lines, but not in others. We suggest, that its levels in human cells may be primarily related to the amount of the chromosomal telomeric sequences. Tel-spcDNA may serve as a unique mutator, through specific mechanisms related to the telomeric repeats, which distinguish it from the total heterogeneous spcDNA population. It may affect telomere dynamics and genomic instability by clastogenic events, alterations of telomere size and sequestration of telomeric proteins.

Topics: Animals; Carcinoma; Cell Line; Chromosomes; Colonic Neoplasms; Cricetinae; Deoxyribonucleases, Type II Site-Specific; DNA Probes; DNA, Circular; DNA, Neoplasm; Electrophoresis, Gel, Two-Dimensional; Embryo, Mammalian; HeLa Cells; Humans; In Situ Hybridization, Fluorescence; MAP Kinase Kinase Kinases; Methylnitronitrosoguanidine; Molecular Weight; Mutagens; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Rats; Repetitive Sequences, Nucleic Acid; Telomere

Multicell spheroids were exposed to DNA-damaging agents with the aim of determining whether prompt DNA damage could be predictive for cell killing and drug resistance. Chinese hamster V79 cells, SiHa human cervical carcinoma cells, and WiDr human colon carcinoma cells were grown as spheroids and exposed to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4-nitroquinoline-1-oxide (4NQO), doxorubicin, etoposide, actinomycin D, 1-(2-nitro-1-imidazolyl)-3-aziridino-2-propanol (RSU 1069), 3-amino-1,2,4-benzotriazine-1,4-dioxide (tirapazamine), and nitrogen mustard. Average DNA damage measured using the alkali comet assay generally correlated with cell killing irrespective of exposure times or drug concentration. However, better predictive power was achieved by using DNA damage levels in individual cells to identify the fraction of cells containing sufficient numbers of DNA strand breaks to cause death. Using this concept of a "threshold" for DNA damage, cell survival could be predicted for exposure to 4NQO, tirapazamine, nitrogen mustard, RSU 1069, and actinomycin D and was largely independent of cell type. The threshold value varied for each drug. For 4NQO, tirapazamine, and RSU 1069, DNA damage equivalent to about 10,000 strand breaks/cell was not toxic to cells of any spheroid type. Conversely, for actinomycin D, any DNA damage above background levels (approximately 100 breaks) was toxic for all three cell types. For some DNA-damaging drugs, the lack of correlation between DNA damage and cell killing was also informative. For etoposide and doxorubicin, no common threshold for cell killing could be determined, consistent with the hypothesis that DNA damage is only one of the actions of these drugs leading to cell death. For MNNG, the tail moment threshold varied significantly for the different spheroid types, probably indicating differences in repair. Overall, for five of the eight drugs, DNA damage measured using the comet assay was an effective and quantitative method of predicting drug cytotoxicity in complex multicelled systems.

Topics: 4-Nitroquinoline-1-oxide; Animals; Carcinogens; Cell Death; Cells, Cultured; Colonic Neoplasms; Cricetinae; Cricetulus; DNA Damage; Drug Screening Assays, Antitumor; Female; Fibroblasts; Humans; Lung; Methylnitronitrosoguanidine; Predictive Value of Tests; Spheroids, Cellular; Uterine Cervical Neoplasms

Sequential endoscopic observation of dog colons was performed during colon carcinogenesis. Two beagle dogs were given suppositories containing N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) every day for five months. In month 3, aberrant crypt foci (ACF), a putative preneoplastic lesion, were found in the colons of both dogs, but not in an untreated dog. The frequency of ACF increased until month 10, and then decreased. In month 9, very small lesions, less than 1 mm in diameter, which were similar to human early flat tumors, were first noticed. One of these lesions grew to about 7 mm in size without a change in its shape for 10 months. There were more than ten flat-type tumors in the two dogs, but such lesions were not found in the untreated dog. By biopsy, two of the lesions were proved to be well-differentiated adenocarcinomas histologically. Four polypoid lesions were found in one of the carcinogen-treated dogs. Thus, flat-type adenocarcinomas were induced in the dog colon by ENNG, and their development was followed by magnifying endoscopy.

Topics: Adenocarcinoma; Animals; Carcinogens; Colon; Colonic Neoplasms; Colonic Polyps; Disease Models, Animal; Dog Diseases; Dogs; Endoscopy; Methylnitronitrosoguanidine; Precancerous Conditions

An interposed colon segment has been clinically reconstructed as a gastric substitute. The purpose of this study is to establish a rat model of colonic interposition and to investigate serial mucosal changes and adaptation of interposed colon mucosa under prolonged exposure to bile and pancreatic juice reflux.. About 80% of the glandular stomach was resected, and a 3-cm segment of the transverse colon interposed isoperistaltically between the remnant stomach and duodenum. Epithelial proliferation, aberrant crypt foci (ACF), and tumors in the interposed colon segment were investigated after 4 months of administration of N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) to rats (ENNG-treated rats).. In the interposed colon, crypt lengths increased significantly, and the number of goblet cells per crypt per 1 mm decreased significantly compared to those in the remnant colon, whether ENNG was administered or not. Both crypt lengths and the number of goblet cells in the interposed colon of controls and ENNG-treated rats showed no significant difference. A proliferating cell nuclear antigen (PCNA) labeling index (LI) of the remnant colon was almost 30% in both controls and ENNG-treated rats. In controls, the PCNA LI in the interposed colon at 4, 8, and 12 months after surgery was 30.8, 31.8, and 47.8%. In ENNG-treated rats, the PCNA LI in the nontumorous mucosa of the interposed colon was 44.9, 55.4, and 61.5% at the above postsurgical intervals. ACF and carcinoma were observed only in the interposed colon of ENNG-treated rats. ACF was observed as early as 4 months after surgery, and its incidence increased serially. Both the incidence of carcinogenesis and the number of tumors had increased 8 months after surgery.. We established a rat model of colonic interposition following gastrectomy. The adaptation of interposed colon mucosa was well conducted. A malignant condition, however, was induced in the interposed colon segment serving as a gastric substitute because of both carcinogen predisposition and prolonged exposure to bile and pancreatic juice reflux.

Topics: Adaptation, Physiological; Animals; Bile; Carcinogens; Colon; Colonic Neoplasms; Epithelium; Gastrectomy; Intestinal Mucosa; Male; Methylnitronitrosoguanidine; Pancreatic Juice; Proliferating Cell Nuclear Antigen; Rats; Rats, Wistar; Time Factors

The phenomenon of alkylation tolerance has been observed in cells that are deficient in some component of the DNA mismatch repair (MMR) system. An alkylation-induced cell cycle arrest had been reported previously in one MMR-proficient cell line, whereas a MMR-defective clone derived from this line escapes from this arrest. We examined human cancer cell lines to determine if the cell cycle arrest were dependent upon the MMR system. Growth characteristics and cell cycle analysis after MNNG treatment were ascertained in seven MMR-deficient and proficient cell lines, with and without confirmed mutations in hMLH1 or hMSH2 by an in vitro transcription/translation assay. MMR-proficient cells underwent growth arrest in the G2 phase of the cell cycle after the first S phase, whereas MMR-deficient cells escaped an initial G2 delay and resumed a normal growth pattern. In the HCT116 line corrected for defective MMR by chromosome 3 transfer, the G2 phase arrest lasted more than five days. In another MMR-proficient colon cancer cell line, SW480, cell death occurred five days after MNNG treatment. A competent MMR system appears to be necessary for G2 arrest or cell death after alkylation damage, and this cell cycle checkpoint may allow the cell to repair damaged DNA, or prevent the replication of mutated DNA by prohibiting clonal expansion.

Topics: Alkylating Agents; Carcinoma; Colonic Neoplasms; DNA Damage; DNA Repair; DNA-Binding Proteins; Female; G2 Phase; Humans; Methylnitronitrosoguanidine; Models, Genetic; MutS Homolog 2 Protein; Neoplasms; Ovarian Neoplasms; Proto-Oncogene Proteins; RNA, Messenger; Sequence Deletion; Stem Cells; Tumor Cells, Cultured

Lactic acid bacteria (LAB) are proposed to have several beneficial effects, including the inactivation of carcinogens. We have studied the potential of Lactobacillus acidophilus (from a commercially available yogurt), Lactobacillus gasseri (P79), Lactobacillus confusus (DSM20196), Streptococcus thermophilus (NCIM 50083), Bifidobacterium breve and Bifidobacterium longum (from human infant stool) to prevent the induction of DNA damage by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG, 7.5 mg/kg body wt) in colon cells of the rat. Using the new technique of single cell microgel electrophoresis, all investigated strains were antigenotoxic toward MNNG after a single dose of 10(10) viable cells/kg body wt p.o. eight hours before the carcinogen. One-half and one-tenth of this initial dose resulted in a loss of protective activity. High doses of heat-treated L. acidophilus strains were also not antigenotoxic. One mechanism of the preventive effect could be that bacterial metabolites or components are responsible. Accordingly, selected examples were investigated in vitro in colon cells of the rat. Metabolically active L. acidophilus cells, as well as an acetone extract of the culture, prevented MNNG-induced DNA damage. Different cell fractions from L. acidophilus (cytoplasm, cell wall skeleton, cell wall) were devoid of antigenotoxic activity, whereas the peptidoglycan fraction and whole freeze-dried cells were antigenotoxic. As a second carcinogen, 1,2-dimethylhydrazine (DMH) was used. A dose- and time-response study was first performed to assess the effects of DMH in several segments of the gastrointestinal (GI) tract. Exposure for 16 hours to 15 or 25 mg DMH/kg body wt p.o. induced DNA damage in cells of the distal colon of rats, whereas no cytotoxicity was seen. Pretreatment orally with LAB on four consecutive mornings before DMH gavage (8 hours after the last LAB application) revealed that L. acidophilus, L. confusus, L. gasseri, B. longum, and B. breve inhibited the genotoxic effect of DMH. One of four S. thermophilus and one of three Lactobacillus delbrueckeii ssp. bulgaricus strains were also protective. Heat-treated L. acidophilus did not inhibit DMH-induced genotoxicity. A few aliquots of the colon cells were processed immunohistochemically for the presence of the "proliferation cell nuclear antigen" (PCNA). DMH treatment did not increase PCNA, nor was there any modulation by LAB. The effect of L. acidophilus on foreign compound-metabolizing enzymes (Phase I

Topics: 1,2-Dimethylhydrazine; Animals; Anticarcinogenic Agents; Antimutagenic Agents; Bifidobacterium; Carcinogens; Colon; Colonic Neoplasms; Dimethylhydrazines; DNA Damage; Immunohistochemistry; Lactobacillus; Lactobacillus acidophilus; Male; Methylnitronitrosoguanidine; Proliferating Cell Nuclear Antigen; Rats; Rats, Sprague-Dawley

The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) has been implicated in resistance of human brain tumors to alkylating agents. We observed that 14 human medulloblastoma- and glioma-derived cell lines differ in sensitivity to the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), as shown by their 28-fold range in 10% survival dose (LD10). By using the substrate analogue inhibitor O6-benzylguanine (O6-BG), we showed that the contribution of MGMT to resistance varies widely, as evidenced by 3- to 30-fold reductions in LD10 among the lines, and varies up to 20-fold among subpopulations of individual lines. Importantly, variability in resistance, manifested as a 20-fold range in LD10, persists after measurable MGMT is eliminated, disclosing differential contributions of other resistance mechanisms to survival. Cells exposed to MNNG while suspended in growth medium are more resistant than cells alkylated as subconfluent monolayers, and MGMT accounts for a smaller proportion of their resistance. Notably, the MGMT content of the lines is not statistically correlated with MNNG resistance or with potentiation of killing by O6-BG, even though MGMT is a biochemically demonstrated determinant of resistance. In contrast, the same lines vary less in resistance to the ethylating agent N-ethylnitrosourea (ENU), and MGMT makes only a small contribution to resistance. Our results strongly indicate that resistance to both MNNG and ENU is multifactorial.

Topics: Alkylating Agents; Antineoplastic Agents; Brain Neoplasms; Cell Adhesion; Colonic Neoplasms; Culture Media; Drug Resistance; Drug Screening Assays, Antitumor; Ethylnitrosourea; Glioma; Medulloblastoma; Methylation; Methylnitronitrosoguanidine; Methyltransferases; O(6)-Methylguanine-DNA Methyltransferase; Tumor Cells, Cultured

We studied the increasing expression of the p53 tumor suppressor gene in Sprague-Dawley rats, chemically induced to develop colon cancer. p53 expression was evaluated histochemically at various stages of tumor progression (during a period of 40 weeks) that can be followed by colonic hyperproliferation labeled by 3H-thymidine incorporation. We found that high level nuclear expression of p53 protein correlates with progression of malignancy in carcinogen-induced animals, whereas cytoplasmic staining is related to the onset and early development of malignancy.

Topics: Adenocarcinoma; Animals; Azoxymethane; Cell Nucleus; Colonic Neoplasms; Cytoplasm; Epithelium; Gene Expression; Genes, p53; Immunohistochemistry; Male; Methylnitronitrosoguanidine; Mutation; Rats; Rats, Sprague-Dawley; Tumor Suppressor Protein p53

Curcumin (diferuloylmethane), a yellow pigment that is obtained from the rhizomes of Curcuma longa Linn., is a major component of turmeric and is commonly used as a spice and food-coloring agent. The inhibitory effects of feeding commercial grade curcumin (77% curcumin, 17% demethoxycurcumin, and 3% bisdemethoxycurcumin) in AIN 76A diet on carcinogen-induced tumorigenesis in the forestomach, duodenum, and colon of mice were evaluated. Administration p.o. of commercial grade curcumin in the diet inhibited benzo(a)pyrene-induced forestomach tumorigenesis in A/J mice, N-ethyl-N'-nitro-N-nitrosoguanidine-induced duodenal tumorigenesis in C57BL/6 mice, and azoxymethane (AOM)-induced colon tumorigenesis in CF-1 mice. Dietary commercial grade curcumin was given to mice at: (a) 2 weeks before, during, and for 1 week after carcinogen administration (during the initiation period); (b) 1 week after carcinogen treatment until the end of the experiment (during the postinitiation period); or (c) during both the initiation and postinitiation periods. Feeding 0.5-2.0% commercial grade curcumin in the diet decreased the number of benzo(a)pyrene-induced forestomach tumors per mouse by 51-53% when administered during the initiation period and 47-67% when administered during the postinitiation period. Feeding 0.5-2.0% commercial grade curcumin in the diet decreased the number of N-ethyl-N'-nitro-N-nitrosoguanidine-induced duodenal tumors per mouse by 47-77% when administered during the postinitiation period. Administration of 0.5-4.0% commercial grade curcumin in the diet both during the initiation and postinitation periods decreased the number of AOM-induced colon tumors per mouse by 51-62%. Administration of 2% commercial grade curcumin in the diet inhibited the number of AOM-induced colon tumors per mouse by 66% when fed during the initiation period and 25% when fed during the postinitiation period. The ability of commercial grade curcumin to inhibit AOM-induced colon tumorigenesis is comparable to that of pure curcumin (purity greater than 98%). Administration of pure or commercial grade curcumin in the diet to AOM-treated mice resulted in development of colon tumors which were generally smaller in number and size as compared to the control group of AOM-treated mice. These results indicate that not only did curcumin inhibit the number of tumors per mouse and the percentage of mice with tumors but it also reduced tumor size. Histopathological examination of the tumors sho

Topics: Adenocarcinoma; Adenoma; Adenoma, Villous; Animals; Azoxymethane; Benzo(a)pyrene; Carcinogens; Colonic Neoplasms; Curcumin; Duodenal Neoplasms; Female; Male; Methylnitronitrosoguanidine; Mice; Stomach Neoplasms

The human colon tumor cell line HCT 116 is known to have a homozygous mutation in the mismatch repair gene hMLH1 on human chromosome 3, to exhibit microsatellite instability, and to be defective in mismatch repair. In order to determine whether the introduction of a normal copy of hMLH1 gene restores mismatch repair activity and corrects microsatellite instability, a single human chromosome 3 from normal fibroblasts was transferred to HCT 116 cells via microcell fusion. As a control, human chromosome 2 was also transferred to HCT 116 cells. Two HCT 116 microcell hybrid clones that received a single copy of chromosome 2 (HCT 116 + ch2) and two that received a single copy of chromosome 3 (HCT 116 + ch3) were isolated and characterized. A G-G mismatch in M13-derived heteroduplex DNA was efficiently repaired in cell extracts from HCT 116 + ch3 cells, but not in those of parent HCT 116 cells or HCT 116 + ch2 cells. Microsatellite alterations at the D5S107 locus containing CA repeats were seen in 8 of 80 subclones from HCT 116 cells, and in 13 of 150 subclones from HCT 116 + ch2 cells. In contrast, none of the 225 subclones derived from mismatch repair-proficient HCT 116 + ch3 cells showed alterations in the microsatellite at the same locus. The effect of introducing chromosome 3 on the sensitivity of HCT 116 cells to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was examined, since enhanced tolerance to MNNG is accompanied by loss of mismatch repair activity in several cell lines. Within 3 days after treatment with 5 microM MNNG, HCT 116 + ch3 cells became morphologically flat and stopped growing. Their colony-forming ability, determined 10 days after treatment, was reduced 200-fold when compared to MNNG-treated parental HCT 116 and HCT 116 + ch2 cells. These results support the hypothesis that mutations in both alleles of the hMLH1 gene are necessary for the manifestation of defective mismatch repair and microsatellite instability and for enhanced MNNG tolerance. The results also suggest that the mismatch repair system contributes to the process that causes growth arrest in response to DNA damage by alkylating agents.

Topics: Base Sequence; Chromosome Banding; Chromosomes, Human, Pair 2; Chromosomes, Human, Pair 3; Colonic Neoplasms; DNA Repair; DNA, Satellite; Drug Tolerance; Humans; Methylnitronitrosoguanidine; Molecular Sequence Data; Point Mutation; Polymerase Chain Reaction; Transfection; Tumor Cells, Cultured; Tumor Stem Cell Assay

Consumption of fermented dairy foods has been linked to reduced incidence of colon cancer in population groups. Recently, biologically active compounds have been isolated from these products. Bacterial proteinases, produced by dairy starter cultures, generate a variety of peptides from casein. Some of these casein-derived peptides are likely to alter intestinal cell kinetics. Effects on colon cell kinetics because of the presence of casein-derived peptides may be a mechanism through which fermented dairy foods reduce the risk of colon cancer. We have used two intestinal cell lines (IEC-6 cells, derived from normal rat intestine, and Caco-2 cells, derived from human colon adenocarcinoma) to identify casein peptides that affect intestinal cell kinetics. Cell culture media containing casein were inoculated with three commercial starter cultures and incubated for 4, 8, or 24 h. The bacteria-conditioned media were then filter-sterilized and incubated with the intestinal cells for 6 or 24 h. Rates of [3H]thymidine incorporation and cell cycle kinetics determined by flow cytometry were affected by the culture-modified media in both cell lines. The IEC-6 cells tended to reduce, and Caco-2 cells to increase, rates of cell division after exposure to the media. Intestinal cell response varied among the starter cultures. The results support the use of intestinal cell cultures to identify casein peptides generated by dairy starter cultures, which affect intestinal cell kinetics.

Topics: Adenocarcinoma; Animals; Bacteria; Caseins; Cell Cycle; Cell Line; Colonic Neoplasms; Culture Media, Conditioned; DNA; Humans; Hydrolysis; Kinetics; Lactobacillus; Methylnitronitrosoguanidine; Peptides; Rats; Streptococcus; Tumor Cells, Cultured

Effects of the dietary antioxidants alpha-tocopherol (alpha-Toc), t-butylhydroquinone (TBHQ), propyl gallate (PG) and butylated hydroxytoluene (BHT) were examined using a multi-organ carcinogenesis model. Groups of 20 F344 male rats were treated with a single intragastric administration of 100 mg/kg body weight N-methyl-N'-nitro-N-nitrosoguanidine, a single intragastric administration of 750 mg/kg body weight N-ethyl-N-hydroxyethylnitrosamine, two subcutaneous injections of 0.5 mg/kg body weight N-methylbenzyl-nitrosamine and four subcutaneous injections of 40 mg/kg body weight 1,2-dimethylhydrazine. At the same time the rats were given 0.1% N-dibutylnitrosamine for 4 weeks and then 0.1% 2,2'-dihydroxy-di-n-propylnitrosamine for 2 weeks in the drinking water, for a total carcinogen exposure period of 6 weeks. Starting 3 days thereafter the rats received 1% alpha-Toc, 1% TBHQ, 1% PG or 0.7% BHT in the diet, or basal diet alone. Further groups of 10-15 animals each were treated with antioxidant alone or basal diet alone as controls. Surviving animals were killed at the end of week 36. Histopathological examination showed that alpha-Toc increased the incidence of glandular stomach atypical foci but reduced the incidence and multiplicity of kidney atypical tubules. TBHQ significantly elevated the incidences of esophageal papillary or nodular (PN) hyperplasias and papillomas, as well as forestomach papillomas, but significantly decreased the multiplicity of colon adenocarcinomas. PG was only effective in reducing the multiplicity of kidney atypical tubules. BHT enhanced the development of thyroid hyperplasias, but strongly reduced the incidence and multiplicity of colon adenocarcinomas. This compound was also associated with lowered incidence and multiplicity of renal cell tumors. None of the agents studied was unequivocal in exerting either positive or negative influence.

Topics: 1,2-Dimethylhydrazine; Animals; Anticarcinogenic Agents; Antioxidants; Butylated Hydroxytoluene; Carcinogens; Colon; Colonic Neoplasms; Dimethylhydrazines; Gastrointestinal Neoplasms; Hydroquinones; Hyperplasia; Kidney; Kidney Neoplasms; Male; Methylnitronitrosoguanidine; Neoplasms, Experimental; Nitrosamines; Organ Size; Precancerous Conditions; Propyl Gallate; Rats; Rats, Inbred F344; Vitamin E

We previously immortalized human oral keratinocytes by transfection with recombinant human papillomavirus type 16 (HPV-16) DNA and established two cell lines. These transfected cells were morphologically different from the normal counterpart, contained intact HPV-16 DNA in an integrated form, and expressed numerous viral genes. These cells contained lower levels of wild-type p53 protein and higher levels of c-myc mRNAs compared to normal cells. However, they proliferated only in keratinocyte growth medium containing a low level of calcium and were not tumorigenic in nude mice. A HPV-16-immortalized cell line was exposed to either 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone or N-methyl-N'-nitro-N-nitrosoguanidine. Four chemically transformed cell colonies were isolated. These cells proliferated well in Dulbecco's minimum essential medium containing a physiological level of calcium. They contained, similar to the immortalized counterpart, integrated HPV-16 sequences and lower levels of both wild-type p53 protein and DCC messages compared to normal cells. Among the chemically transformed cells, two colonies obtained from 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone exposure demonstrated an enhanced proliferation capacity in nude mice and transcribed a substantially higher amount of HPV-16 E6/E7, epidermal growth factor receptors, and c-myc genes compared with the immortalized counterpart. These experiments indicate that malignant transformation of oral keratinocytes can be caused by a sequential combined effect of "high risk" HPV and tobacco-related carcinogens.

Topics: Base Sequence; Carcinogens; Cell Division; Cell Line, Transformed; Cell Transformation, Neoplastic; Cells, Cultured; Colonic Neoplasms; DNA Primers; ErbB Receptors; Exons; Genes, myc; Genes, p53; Genes, ras; Humans; Keratinocytes; Male; Methylnitronitrosoguanidine; Molecular Sequence Data; Mouth Neoplasms; Mutagenesis; Nicotiana; Nitrosamines; Oligonucleotides, Antisense; Papillomaviridae; Plants, Toxic; Polymerase Chain Reaction; Transforming Growth Factor alpha

The aim of this study was to determine whether changes in serum free radicals may be useful for the early detection of precancerous conditions in the rat colon after treatment with a direct carcinogen (N-Methyl-N'-Nitro-N-Nitrosoguanidine, MNNG) and a secondary bile acid (deoxycholic acid, DCA) as a tumor promoter. It was shown that a significant increase in the concentrations of free radicals in sera of rats following their treatment with MNNG and DCA was observed as early as the 18th week after the beginning of the treatment. Since our results have shown that these alterations occurred in parallel with neoplastic transformations in the rat colon, it suggests that the increase in serum free radicals reflected the precancerous situation in the animals and may be useful in the early detection of cancer development. The possible role of free radicals in deoxycholate-induced liver toxicity was discussed.

Topics: Animals; Colonic Neoplasms; Deoxycholic Acid; Free Radicals; Gentisates; Hydroxybenzoates; Male; Methylnitronitrosoguanidine; Precancerous Conditions; Rats; Rats, Sprague-Dawley

Methods of morphological, histochemical and immunohistochemical analyses were used to further characterize differences between tumourous and adjacent grossly normal tissues in chemically-induced colon cancer in rats. Colon tumors were induced by the treatment of rats with 1,2-dimethylhydrazine or with N-methyl-N'-nitro-N-nitrosoguanidine alone or with subsequent treatment with deoxycholic bile acid. Tissues were studied morphologically (for the presence of goblet cells in the colon crypts, and the extent of infiltration of lymphocytes into the crypts and between them), histochemically (for the presence of positive reaction to neutral and acid mucopolysaccharides) and immunohistochemically (for the presence of tissue polypeptide antigen). All data were evaluated quantitatively, and index of tissue damage was calculated for both tumorous and non-tumorous tissues. Significant morphological differences were found between tumorous and adjacent apparently normal tissue. Histochemically and immunohistochemically, both types of tissue reacted very similarly to exposure to the carcinogens. Index of damage was significantly different from normal untreated colon in both kinds of tissue. It was suggested that precancerous state in tissue adjacent-to-tumor could be detected using the combination of these methods.

Topics: 1,2-Dimethylhydrazine; Animals; Carcinogens; Colon; Colonic Neoplasms; Deoxycholic Acid; Dimethylhydrazines; Glycosaminoglycans; Histocytochemistry; Immunohistochemistry; Male; Methylnitronitrosoguanidine; Rats; Rats, Sprague-Dawley; Time Factors

Topics: 1,2-Dimethylhydrazine; Aging; Alkylating Agents; Animals; Carcinogens; Colonic Neoplasms; Dimethylhydrazines; Female; Methyl Methanesulfonate; Methylnitronitrosoguanidine; Methylnitrosourea; Precancerous Conditions; Rats; Rats, Wistar

The development of tumorigenic conditions in the carcinogen-exposed rat colon was studied using selected morphological, histochemical, immunohistochemical and biochemical methods of analysis. Rats were treated with two carcinogens: 1,2-dimethylhydrazine and N-methyl-N'-nitro-N-nitrosoguanidine alone or with deoxycholic acid as a tumor promoter. It was found that 3 months after treatment of animals with the carcinogens the following changes were developed in colonic tissue: infiltration of lymphocytes in the mucous membrane, high increase in mitotic index among epithelial cells, negative reactions of colonic cells for neutral mucopolysaccharides and sulfomucins and positive reactions to carboxyl groups, nonsulfated acid mucosubstances and tissue polypeptide antigens. An increase in the activity of ornithine decarboxylase in colonic tissue was developed within the same time period and has been seen only in those tissues which were characterized by the development of precancerous conditions. Individual variations were observed in the manifestation of the studied parameters in rat neoplastic colonic tissues. It is suggested that these differences reflect an individual sensitivity of animals to carcinogens and the magnitude of the dysplastic processes induced in the colon.

Topics: 1,2-Dimethylhydrazine; Animals; Biomarkers, Tumor; Carcinogens; Colonic Neoplasms; Deoxycholic Acid; Dimethylhydrazines; Histocytochemistry; Immunohistochemistry; Male; Methylnitronitrosoguanidine; Mitotic Index; Ornithine Decarboxylase; Peptides; Precancerous Conditions; Rats; Rats, Sprague-Dawley; Tissue Polypeptide Antigen

Human cell-free extracts were used to detect activities specifically incising O6-methylguanine (m6G) paired with C or T in DNA. A 45-bp double-stranded DNA containing one m6G across from a T (m6G:T) was the test substrate. Extracts from glioblastoma cell lines A172 and A1235 (lacking the m6G-specific repair protein m6G-DNA methyltransferase, MGMT) and colon carcinoma cell line HT29, containing MGMT, showed incision activities specific for the T strand of m6G:T [and G:T, as reported previously by Wiebauer and Jiricny (1989)] substrates, but did not cleave m6G:C (or G:C) substrates. Competition experiments showed that the activity was similar to, if not identical with, the activity in human cells that incises G:T mismatches. The incision sites were similar to those recognized by human G:T- or G:A-specific mismatch enzymes, i.e., the phosphodiester bonds both 3' and 5' to the poorly matched T, suggesting the glycolytic removal of the poorly matched T followed by backbone incisions by class I or II AP endonucleases. Three experiments in which MGMT was inactivated showed that the m6G:T incision activity was not simply due to a two-step mechanisms in which MGMT would first mediate conversion of the m6G:T substrate to a G:T substrate which would serve as a substrate for G:T incision. Extracts from HT29 contained a DNA-binding factor, possibly DNA sequence-specific, that inhibited incision of the m6G:T (but not the G:T) substrate, that was removed by the addition of synthetic DNA to the reaction.

Topics: Base Composition; Base Sequence; Binding, Competitive; Colonic Neoplasms; Deoxyribonuclease I; DNA; DNA Repair; Glioma; Guanine; Humans; Kinetics; Methylnitronitrosoguanidine; Methyltransferases; Molecular Sequence Data; O(6)-Methylguanine-DNA Methyltransferase; Thymine; Tumor Cells, Cultured

Transmission electron microscopy (TEM) was used to study ultrastructural changes that accompanied the tumorous transformation of the descending rat colon epithelial cells, following short treatment with a direct carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), with subsequent prolonged treatment with secondary bile acids, lithocholic (LCA) and deoxycholic (DCA), which enhanced tumor formation. Colon epithelial cells after long treatment with bile acids alone were characterized by the presence of an irregular nuclear membrane, ring-shaped rough endoplasmic reticulum (RER), collagen-like tonofilaments and membrane-bound mucous vacuoles. Tumor cells which developed following treatment with MNNG alone were characterized by the irregular shape of the nuclear membrane and, sometimes, by polynuclei, accumulation of large amounts of mitochondria, loss of cell-cell contacts and by endocytosis of the cell membrane. After combined treatment with MNNG and LCA, many mitochondria lost their membranous envelope; in the cytoplasm many collagen-like tonofilaments, ring-shaped RER and many free ribosomes were present. After treatment with MNNG and DCA, many polysomes were found in the cytoplasm. It was apparent that treatment with MNNG alone caused the development of adenocarcinoma-like tumors, while additional treatment with secondary bile acids significantly enhanced these changes, which were accompanied by the development of atypia and anaplasia of epithelial cells, with many irregularities in intracellular organization.

Topics: Animals; Bile Acids and Salts; Colon; Colonic Neoplasms; Deoxycholic Acid; Epithelium; Lithocholic Acid; Male; Methylnitronitrosoguanidine; Rats; Rats, Inbred Strains; Rectum

The role of selected histopathological methods was evaluated for the characterization of tumourous tissues resulting from chemically induced carcinogenesis in rats. Colon cancer was induced by treating rats with a direct carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), alone or with subsequent treatments with secondary bile acids. Morphological studies showed that the tumorigenic effect of MNNG resulted in changes in the shape of the crypts, disappearance of mucous (goblet) cells, and marked increases in the mitotic index, the size of nuclei and the number of aberrant nuclei. A significant number of lymphocytes infiltrated between the crypts and into their lumens. A sharp decrease in concentrations of neutral mucopolysaccharides and sulfomucins and an increase in the amount of vic-glycol groups and nonsulfated mucosubstances were demonstrated in tumourous epithelial cells. A moderate positive reaction to tissue polypeptide antibody was observed in these cells. The tumor-promoting effects of secondary bile acids were detected morphologically by an increase in the amount of connective tissue that infiltrated newly formed tumors. The increased number of micronuclei in otherwise histologically unaltered colon tissues adjacent to a tumor suggests that these regions represent a precancerous stage in the development of tumors. The important role of morphological methods in the evaluation of the effects of carcinogen and tumor promoters and in the detection of various phases in experimental carcinogenesis was discussed.

Topics: Animals; Cell Nucleus; Colonic Neoplasms; Glycosaminoglycans; Histocytochemistry; Immunohistochemistry; Male; Methylnitronitrosoguanidine; Mitotic Index; Mucins; Neoplasms, Experimental; Peptides; Rats; Rats, Inbred Strains

Large bowel carcinoma was induced in rats by injecting MNNG intrarectally, and changes in the large bowel mucosa prostaglandin (PG) with time were determined. The PGE2 levels of the colonic mucosa in a control group were 20.9 +/- 9.7 (pg/mg total protein) at 5 weeks, 25.5 +/- 9.7 at 10 weeks, 26.5 +/- 18.1 at 20 weeks, and 34.8 +/- 12.7 at 40 weeks. The PGE2 levels in the MNNG-treated group were 44.7 +/- 6.2 at 5 weeks, 43.1 +/- 14.9 at 10 weeks, 70.1 +/- 23.4 at 20 weeks, and 79.7 +/- 54.1 at 40 weeks. The intrinsic PGE2 levels of the noncancerous mucosa were thus significantly higher for the MNNG group than for the control group at all weeks. At 40 weeks, the PGE2 levels of cancer lesions were significantly high compared with those of the noncancerous area. In the cancerous lesions, 6-keto-PGF1 alpha and PGF2 alpha decreased and TXB2 increased significantly at 40 weeks. The observations demonstrated that PGE2 was implicated as a promoter in the development and proliferation of carcinoma in MNNG-induced large bowel carcinogenesis in rats.

Topics: Animals; Colon; Colonic Neoplasms; Dinoprostone; Intestinal Mucosa; Male; Methylnitronitrosoguanidine; Rats; Rats, Inbred Strains; Time Factors

A 3 x 3 factorial experiment was conducted to examine how dietary fiber (wheat bran) and fat (lard) interactively affect the genesis of N-methyl-N'-nitro-N-nitrosoguanidine-induced colon cancer in rats. Groups of 30 male 4-week-old Wistar rats were fed ad libitum one of nine experimental diets containing either 15 (low), 27.5 (medium), or 40% (high) energy as fat in combination with 0.7 (low), 2.2 (medium), or 3.8 g (high) fiber/100 kcal for a period of 37 weeks. After 4 weeks, each rat received a total of five weekly intrarectal instillations of 6 mg N-methyl-N'-nitro-N-nitrosoguanidine/kg. The highest colon carcinoma incidence and the highest total number of carcinomas of the colon were observed in the animals fed the medium-fat/medium-fiber diet. The highest number of polyps and a relatively high polyp incidence occurred in the animals on the high-fat/low-fiber diet. An enhancing effect of fat on both the tumor incidence and tumor multiplicity was clearly present for the low-fiber diets, whereas fat had no effect when the fiber content of the diet was high. In general, the results showed a nonlinear dose-response relationship for fiber and fat. These results indicate that both dietary fiber and fat affect colon carcinogenesis in a complex, interactive manner.

Topics: Adenocarcinoma; Animals; Body Weight; Colonic Neoplasms; Colonic Polyps; Dietary Fats; Dietary Fiber; Eating; Energy Metabolism; Male; Methylnitronitrosoguanidine; Rats; Rats, Inbred Strains

We examined whether hyperproliferation of colonic crypt epithelium during cancer induction by N-methyl-N-nitro-N-nitrosoguanidine (MNNG), in rats on a low fat and calcium diet could be reduced by added calcium p.o. From the age of 4 weeks, 104 male Sprague-Dawley rats received a low fat (3.5%), low calcium (0.05% calcium ion), and low vitamin D (0.4 IU/g) diet. Sixty-four also had calcium salts, derived from either calcium lactate or solubilized calcium carbonate, added to their drinking water; therefore their total calcium intake was about 1% of daily diet. At age 12 weeks the rats were divided into 4 treatment groups: 8 rats, not receiving added calcium, had rectal saline instillations weekly (saline control group) and were sacrificed after a further 28 weeks; 3 groups of 32 rats each received intrarectal MNNG (1.5 mg) weekly. One group, not receiving added calcium, was the MNNG control group; while the second group also received added calcium lactate, and the third group received calcium carbonate. Groups of 24 were sacrificed periodically until 28 weeks of treatment. Rats were sacrificed and epithelial proliferation was estimated, 1 week after the last intrarectal instillation, by in vivo labeling with tritiated thymidine and measuring the ratio of labeled to total colonic crypt epithelial cells. The mean labeling index of the MNNG treated and added calcium groups were significantly higher (8.7-9.5%) than that of the saline controls (2.8%) only at week 28; however, it was then still significantly less than that of the MNNG controls not having added calcium (17.9%). Hyperproliferation, during induction of colonic cancer by MNNG in rats on a low calcium diet, can be reduced by a calcium enriched diet even in the presence of a low fat intake.

Topics: Animals; Calcium, Dietary; Cell Division; Colon; Colonic Neoplasms; Dietary Fats; Epithelium; Male; Methylnitronitrosoguanidine; Precancerous Conditions; Rats; Rats, Inbred Strains; Reference Values

To clarify the organo-specificity of small intestinal cancer, Wistar strain male rats were operated on as follows: Interposition of ileal loop in distal colon for group I and simple laparotomy for group II. MNNG was given via the rectum at the dose of 2.5 mg/day for 2 weeks from the second postoperative week. After intravenous injection of BrdU 50 mg/kg 40 weeks thereafter, these rats were sacrificed. Tumor incidence was almost the same (77% and 78% for group I and II, respectively), but with an obvious difference in site of occurrence, and the number of tumors per unit area was 0.153 in large intestine and 0.015 in interposed ileum for group I and 0.119 in large intestine for group II. Labeling indices of BrdU in the interposed ileal mucosa and control ileal mucosa were 8.2 and 9.7%, respectively without a great difference there between, but were significantly higher compared with 5.1% in colonic mucosa. The above results suggested possible resistance of the interposed ileal mucosa to the provocation of tumor by MNNG compared with the colonic mucosa because of quick cell turnover of the epithelium in the small intestinal mucosa.

Topics: Animals; Colonic Neoplasms; Ileum; Interphase; Intestinal Mucosa; Male; Methylnitronitrosoguanidine; Rats; Rats, Inbred Strains

We studied a series of 40 rats at various stages of colorectal carcinoma, as induced by N-methyl-N-nitro-Nitrosoguanidine. Lymphokine containing supernatants were obtained simultaneously from splenic and peripheral lymphocytes, after exposure to rat colon cancer antigen in vitro. The lymphokine was found capable of performing Macrophage Migration Inhibition (MIF) when obtained from rats with: carcinoma through serosa, carcinoma of submucosa, carcinoma of the mucosa and carcinoma in situ. All control rats were free of cancer and were MIF negative. The MIF response in this study was evaluated as a marker of chemically induced colorectal carcinoma in rats in order to better understand the lymphocyte response to tumor progression from atypia to adenocarcinoma of the colon.

Topics: Adenocarcinoma; Animals; Antigens, Neoplasm; Colonic Neoplasms; Lymphokines; Macrophage Migration-Inhibitory Factors; Male; Methylnitronitrosoguanidine; Rats; Rats, Inbred Strains

Studies have been initiated to find compounds that can trap direct-acting carcinogens within the lumen of the gastrointestinal tract and thus prevent these carcinogens from attacking tissues of the host. Sodium 4-mercaptobenzene sulfonate (4-MBSNa) is a potent nucleophile and was found to react rapidly in vitro with the direct-acting carcinogen beta-propiolactone (BPL). In further investigations 4-MBSNa was shown to inhibit mutagenesis resulting from exposure of Salmonella typhimurium strain TA-100 to BPL and a second direct-acting carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine. Subsequent experiments were performed to determine if 4-MBSNa would inhibit BPL-induced carcinogenesis in vivo. In the first of these, 4-MBSNa was administered by p.o. intubation to female A/J mice 5 min before p.o. administration of BPL. Under these conditions inhibition of carcinogenesis of the forestomach occurred. In a second experiment, 4-MBSNa was given by rectal intubation 5 min before BPL also administered intrarectally. Administration of BPL intrarectally produced adenomatous polyps of the large intestine. The occurrence of these neoplasms was inhibited by the prior administration of 4-MBSNa. The data presented show that 4-MBSNa has the capacity to trap direct-acting carcinogens and to inhibit the occurrence of BPL-induced neoplasia.

Topics: Animals; Benzenesulfonates; Colonic Neoplasms; Female; Lactones; Male; Methylnitronitrosoguanidine; Mice; Propiolactone; Rats; Rats, Inbred F344; Stomach Neoplasms

This study was conducted to determine the effects of ammonium acetate alone or in combination with sodium cholate upon N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced colon carcinogenesis in rats. Ammonia, acetate, and deconjugated bile acids are produced by microbial enzymes in the gastrointestinal lumen. One hundred twenty male Sprague-Dawley rats, weighing 196 +/- 2 g at 8 wk of age, were given four intrarectal doses of MNNG (2 mg/dose) over 2 wk. They were then randomly assigned among four treatment groups, each containing 30 rats. The groups were arranged in a 2 x 2 factorial design and given intrarectal infusions of the agents under study in 0.3 ml of double-distilled water 3 times weekly for 52 wk beginning 4 wk after the initial MNNG treatment. The experimental treatments were: double-distilled water as control; ammonium acetate (24.8 mg of ammonia); sodium cholate (2 mg of cholic acid); and a combination of ammonium acetate and sodium cholate. Ammonium acetate treatment increased the number of rats with fecal blood 4-fold after 56 wk, and this was associated with a higher incidence of adenocarcinomas with a polypoid morphology. The incidence and total number of carcinomas in situ (high grade dysplasia) increased with ammonium acetate treatment. Ammonium acetate increased the total number of adenocarcinomas. Sodium cholate had no significant main effects on the incidence or morphology of colon lesions. The data support the conclusion that ammonium acetate treatment acted as a promoting agent in MNNG-induced colon carcinogenesis.

Topics: Acetates; Animals; Cholic Acid; Cholic Acids; Colon; Colonic Neoplasms; Male; Methylnitronitrosoguanidine; Rats; Rats, Inbred Strains

The expression of Ha-ras oncogene product in rat gastrointestinal carcinomas induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or 1, 2-dimethylhydrazine (DMH) was studied by Western blotting and immunohistochemistry using anti-Ha-ras p 21 oncoprotein antibody. In Western blotting, high levels of c-Ha-ras p 21 were found in serially transplantable rat duodenal carcinomas induced by MNNG and rat colon carcinomas induced by DMH. Immunohistochemically, c-Ha-ras p 21 immunoreactivity was detected in 3 (16.7%) of 17 MNNG-induced stomach carcinomas and in 21 (63.6%) of 33 DMH-induced colon carcinomas, respectively. In the colon carcinomas, c-Ha-ras p 21 immunoreactivity in deeply invasive tumors was stronger than that in superficially invasive tumors and was expressed in all subserosal tumors. Moreover, all of the metastatic colon carcinomas had c-Ha-ras p 21 immunoreactive tumor cells. These findings suggest that c-Ha-ras p 21 expression plays an important role in tumor proliferation, invasion and metastasis of DMH-induced colon carcinoma.

Topics: Adenocarcinoma; Animals; Colonic Neoplasms; Dimethylhydrazines; Methylnitronitrosoguanidine; Oncogene Protein p21(ras); Oncogene Proteins, Viral; Proto-Oncogenes; Rats; Rats, Inbred Strains; Stomach Neoplasms

2-Chloroethyl (methylsulfonyl)methanesulfonate (ClEtSoSo) was more toxic to the BE (Mer-) cell line than to the HT-29 (Mer+) colon carcinoma. The sensitivity of the BE cells closely paralleled the induction of DNA interstrand cross-links by ClEtSoSo. No DNA interstrand crosslink formation was detected in the HT-29 cells after exposure to ClEtSoSo. Pretreatment of the HT-29 cells with methylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine or streptozotocin increases their sensitivity to ClEtSoSo. Little or no increase in the toxicity of ClEtSoSo was found in BE cells after methylating agent pretreatment. Despite the increase in cell killing, no DNA interstrand cross-links were induced by ClEtSoSo after N-methyl-N'-nitro-N-nitrosoguanidine pretreatment. In contrast, streptozotocin pretreatment allowed ClEtSoSo to form DNA interstrand cross-links in HT-29 cells. The production of DNA strand breaks by ClEtSoSo was observed in HT-29 cells both with and without methylating agent pretreatment. These results suggest that the mechanism of ClEtSoSo may differ from other chloroethylating agents such as the chloroethylnitrosoureas. In addition, there may be a difference in the mechanism by which streptozotocin or N-methyl-N'-nitro-N-nitrosoguanidine pretreatment causes an increased cell killing in a previously resistant human colon carcinoma cell line.

Topics: Alkylating Agents; Antineoplastic Agents; Carcinoma; Cells, Cultured; Colonic Neoplasms; DNA Repair; DNA, Neoplasm; Humans; Mesylates; Methylnitronitrosoguanidine; Streptozocin

The two secondary biliary acids (lithocholic and deoxycholic acids) were co-mutagenic when they were each co-incubated with dimethylhydrazine in the presence of Salmonella typhimurium TA 100. These observations were extended to other toxic chemicals, acting as direct (N-methyl-N'nitro-N-nitrosoguanidine and 2-nitrofluorene) or indirect (2-acetylaminofluorene) mutagens. Lithocholic and deoxycholic acids show a similar behavior towards genotoxic molecules. Nevertheless two differences must be noted. Lithocholic acid was the stronger co-mutagen. When lithocholic acid inhibited mutagenic activity of 2-nitrofluorene, deoxycholic acid did not modify it. Interactions between the two secondary bile acids occurred so that the co-mutagenic activity of the mixture of these two bile acids depended on the ratio of their concentrations. Besides their cancer-promoting effect, biliary acids also could have co-initiating effect that could depend upon the ratio of their concentration in the intestine. Calculating the ratio of fecal concentrations of deoxycholic/lithocholic acids thus could be a more sensitive index for cancer risk than simply measuring the fecal concentration of the two biliary acids taken separately.

Topics: 2-Acetylaminofluorene; Animals; Bile Acids and Salts; Colonic Neoplasms; Drug Synergism; Feces; Fluorenes; Male; Methylnitronitrosoguanidine; Mutagenicity Tests; Mutagens; Rats; Rats, Inbred Strains; Risk; Salmonella typhimurium

Chemically induced (autochthonous) tumors in the rodent are thought to be the best models at hand to obtain results transferable to the clinical situation. Four different autochthonous tumor models: 1,2-dimethylhydrazine, N-methylnitrosourea (MNU), N-methyl-N-nitro-nitrosoguanidine (MNNG), and N-nitroso-acetoxymethyl-methylamine (AMMN) which are used worldwide for etiological and therapy studies of colon cancer are compared for tumor biology and clinical relevance. The four tumor models of colon carcinoma described in the rat are different in growth, invasion, and metastases. The choice of the selected tumor model for experimental investigations of colon cancer should depend on the clinical question.

Topics: 1,2-Dimethylhydrazine; Adenocarcinoma; Animals; Colonic Neoplasms; Dimethylhydrazines; Dimethylnitrosamine; Disease Models, Animal; Methylnitronitrosoguanidine; Methylnitrosourea; Rats; Rectal Neoplasms

The effects of the C-terminal tetrapeptide of gastrin, tetragastrin, on the colonic mucosa on Days 15 and 25 during intrarectal administration of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and its effects on the incidences of colonic tumors in experimental Wk 20 and 35 were investigated in Wistar rats. Administration of tetragastrin in depot form during instillation of MNNG resulted in significant decreases in the incidences of mucosal erosions, ulcerations, and atypical regenerative glandular hyperplasias in the colonic mucosa, most of these lesions being greater in the distal half of the colon. Administration of tetragastrin also significantly decreased the incidences and/or numbers of colonic tumors in Wk 20 and 35. The distribution of colonic tumors induced in Wk 20 and 35 corresponded well to those of erosions, ulcerations, and atypical regenerative glandular hyperplasias induced during the administration of MNNG. These findings suggest that the effect of tetragastrin in decreasing the incidences of erosions, ulcerations, and atypical regenerative glandular hyperplasias in the colonic mucosa during instillation of MNNG is related to its effect in reducing the development of colonic tumors.

Topics: Animals; Colonic Neoplasms; Drug Administration Schedule; Gastrins; Intestinal Diseases; Intestinal Mucosa; Methylnitronitrosoguanidine; Rats; Tetragastrin; Time Factors

Topics: 1,2-Dimethylhydrazine; Adenocarcinoma; Animals; Colonic Neoplasms; Dimethylhydrazines; Male; Methylhydrazines; Methylnitronitrosoguanidine; Rats; Rats, Inbred Strains

The effect of a gastrointestinal carcinogen, 1-methyl-3-nitro-1-nitrosoguanidine (MNNG), on chromosomes of rat digestive tract epithelial cells was studied. A blind loop of colorectal segment was surgically produced and the epithelial cells of this loop were treated twice with 0.5% and 0.1% MNNG solutions, respectively (5 ml each). After the first treatment, the mucosa revealed severe damage, and repair and mitoses upon regeneration were maximal on days 4-6. Chromosomal aberrations were studied 3-48 hr after the second treatment. Cells with chromosomal aberrations increased progressively from 6 hr on, reaching their maximum level at 15 hr, and gradually returned to the original level at 48 hr. Chromosomal aberrations consisted of gaps, breaks, and exchanges. The above results indicate that gastrointestinal carcinogens induce chromosomal aberrations in target epithelial cells.

Topics: Animals; Chromosome Aberrations; Colon; Colonic Neoplasms; Intestinal Mucosa; Karyotyping; Male; Metaphase; Methylnitronitrosoguanidine; Rats; Rats, Inbred Strains

Fibroblast cell strains were established from skin biopsies taken from patients with adenomatosis of the colon and rectum (ACR) and their relatives. A total of 57 different strains (33 from patients and 24 from healthy members of ACR families not at an increased risk for colon polyposis) were tested for their frequencies of spontaneous structural chromosome aberrations, i.e., chromatid and isochromatid gaps, breaks, and interchanges. In 47 strains (27 from patients, 20 from controls), the frequencies of structural chromosome aberrations were also determined after exposing the cells to N-methyl-N'-nitro-N-nitroso-guanidine (MNNG). Both spontaneously and after mutagen treatment, the group of patient strains exhibited, on average, approximately twice the number of chromosome aberrations found in the control group. This increase was highly significant (p less than 0.001), even though there was a considerable overlap between patient and control strains. Treatment with MNNG led to a marked increase in chromosome aberrations in both patients and controls. The small differences in aberration frequencies seen between Gardner and other patient strains were clearly insignificant.

Topics: Adolescent; Adult; Aged; Cells, Cultured; Child; Chromosome Aberrations; Colonic Neoplasms; DNA Repair; Female; Fibroblasts; Gardner Syndrome; Humans; Intestinal Polyps; Male; Methylnitronitrosoguanidine; Middle Aged; Mutagens; Rectal Neoplasms; Skin

The effects of prolonged administration of tetragastrin from the beginning of intrarectal instillation of 1 ml of 0.25% N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and after MNNG-treatment on the incidence and histology of colonic tumors were compared in inbred Wistar rats. In week 35 prolonged administration of testragastrin in depot form from the beginning of MNNG-treatment resulted in a significant reduction in the incidence of colonic tumors and a significant increase in the incidence of mucinous adenocarcinoma, unlike the well-differentiated adenocarcinoma produced in controls without gastrin. In contrast, prolonged administration of tetragastrin after MNNG-treatment had little or no influence on the incidence, size or histology of colonic tumors. Thus tetragastrin had no promoting effect on colonic tumors.

Topics: Adenocarcinoma; Animals; Cocarcinogenesis; Colonic Neoplasms; Drug Administration Schedule; Gastrins; Male; Methylnitronitrosoguanidine; Neoplasm Metastasis; Rats; Rats, Inbred Strains; Tetragastrin

Mitozolomide and its decomposition product MCTIC were found to be more cytotoxic to BE colon carcinoma cells in vitro than to HT-29 cells, another colon carcinoma cell line. In addition mitozolomide and MCTIC induced DNA interstrand crosslinks in the BE but not the HT-29 cell line. BE cells are deficient in the repair of O6-methylguanine lesions and are designated Mer-, whereas, HT-29 cells are proficient in this repair process and are designated Mer+. Thus DNA interstrand crosslinking produced by mitozolomide and MCTIC appears to correlate with the Mer phenotype. Pretreatment of HT-29 cells (Mer+) with the DNA methylating agent MNNG allows mitozolomide or MCTIC to produce DNA interstrand crosslinks. HT-29 cells also become more sensitive to the cell killing of mitozolomide and MCTIC with MNNG pretreatment. Pretreatment of Mer- cells (BE) had little effect on either cell killing or DNA crosslinking levels induced by mitozolomide or MCTIC. DNA interstrand crosslinking induced by mitozolomide and MCTIC is probably a consequence of an initial alkylation at the O6-position of guanine followed by a delayed reaction with the opposite DNA strand.

Topics: Antineoplastic Agents; Cell Line; Cell Survival; Colonic Neoplasms; Cross-Linking Reagents; DNA; Drug Synergism; Humans; Methylnitronitrosoguanidine; Nitrogen Mustard Compounds; Tumor Stem Cell Assay

Cells from Gardner's syndrome (GS) and familial polyposis coli (FP) patients, persons with a hereditary predisposition to colon cancer, were compared to those of normal persons for sensitivity to the cytotoxic and mutagenic action of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a model compound chosen because methylating agents have been implicated in colon carcinogenesis. FP cell line GM2355 and GS cell lines 2938 and GM3948 exhibited normal sensitivity to the cytotoxic and mutagenic effects of MNNG. In contrast, GS cell line GM3314 and cells from an apparently normal fetus GM0011 showed extreme sensitivity to the killing and mutagenic effect of this alkylating agent. To determine if the resistance of the various cell lines to MNNG correlated with their ability to remove methyl groups from the O6-position of guanine, we measured their O6-methylguanine-DNA methyltransferase (MT) activity. The resistant cell lines exhibited normal levels of MT; the sensitive strains showed virtually non-detectable levels of this activity. We also compared fibroblasts from a xeroderma pigmentosum (XP) patient (XP12BE, complementation group A), an SV40 virus-transformed XP cell line (XP12ROSV) and a normal cell line transformed by this virus (GM637) for their response to the cytotoxic and mutagenic effect of MNNG and for MT activity. XP12BE cells showed normal sensitivity and a normal level of MT; GM637 cells showed an intermediate level of sensitivity and a reduced level of MT activity; XP12ROSV cells were extremely sensitive to the cytotoxic and mutagenic effect of MNNG and showed virtually non-detectable levels of MT activity. The MT did not remove methyl group from O4-methyl-thymine. These results suggest that O6-methylguanine and/or any other adduct repaired by the methyltransferase, is a potentially cytotoxic and mutagenic lesion. They also indicate that the predisposition to colon cancer of FP and GS patients is not necessarily correlated with an increased sensitivity of their fibroblasts to mutations induced by methylating carcinogens.

Topics: Cell Line; Cell Survival; Colonic Neoplasms; Drug Resistance; Humans; Methylnitronitrosoguanidine; Methyltransferases; Mutation; O(6)-Methylguanine-DNA Methyltransferase

O6-Methylguanine-DNA methyltransferase activity was measured in extracts of human tumor cells and was partially purified from human placenta. Repair of O6-methylguanine in DNA inactivated the methyltransferase, and treatment of cells with MNNG, which produces this alkylated base in DNA, depleted the cells of active methyltransferase. RNA and protein synthesis were required for restoration of methyltransferase activity, which transiently exceeded the original levels by 50% 48 h after treatment. One species of methyltransferase of Mr = 22 kd was present in human tumor cells and human placenta.

Topics: Astrocytoma; Cell Line; Colonic Neoplasms; DNA Repair; Female; Humans; Lung Neoplasms; Methylnitronitrosoguanidine; Methyltransferases; O(6)-Methylguanine-DNA Methyltransferase; Placenta; Pregnancy

Following chronic oral administration of hot water and N-methyl-N'-Nitro-N-nitrosoguanidine (MNNG), 4 of 30 Wistar rats developed sarcoma of the esophagus. 6 animals had tumors of the stomach, the liver and in the jejunum. One malignant tumor of the mediastinum was observed. The relatively short tumor induction time may be caused by the combined action of thermal injury and subsequently administered carcinogen. Organotropy of MNNG is changed and malignancies not only developed in the glandular stomach but also at the site of thermal injury to the esophagus.

Topics: Adenocarcinoma; Administration, Oral; Animals; Burns; Colonic Neoplasms; Esophageal Neoplasms; Female; Male; Methylnitronitrosoguanidine; Rats; Rats, Inbred Strains; Sarcoma; Stomach Neoplasms

Analysis of the etiologic factors and relevant mechanisms involved in carcinogenesis leads to a classification of agents involved in the carcinogenic process as genotoxic or epigenetic. Their mode of action is distinct, especially with regard to dose-response effects and reversibility. The genotoxic carcinogens for colon cancer are unknown, but mutagenic components found in fried beef and fish are under study. Epigenetic agents as promoting factors play a major role in the development of cancer of the colon. Specific nutritional elements associated with colon cancer risk are high fat diets, high cholesterol intake, and low fiber intake. The role of micronutrients as modulators and inhibitors needs to be explored. Through metabolic studies in diverse populations and in reliable animal models, it is now clear that dietary fat and cholesterol control the total flow of bile acids in lumen and a high-fat, high-cholesterol diet increases the total of bile acids in the gut. Bile acids but not neutral sterols have promoting effects and are related to colon cancer risk although bile acids by themselves do not act as complete carcinogens. The effect of dietary fiber such as cereal bran is to increase stool bulk which dilutes the concentration of bile acids. Reducing the concentration of bile acids either by lower dietary fat and cholesterol or by increasing dietary fiber may effectively lower the risk for colon cancer.

Topics: Animals; Bile Acids and Salts; Carcinogens; Cocarcinogenesis; Colonic Neoplasms; Dietary Fats; Dietary Fiber; Humans; Methylnitronitrosoguanidine; Neoplasms, Experimental; Rats

Topics: 1,2-Dimethylhydrazine; Animals; Carcinogens; Colonic Neoplasms; Dimethylhydrazines; Enterobacteriaceae; Feces; Intestines; Male; Methylhydrazines; Methylnitronitrosoguanidine; Rats; Rats, Inbred Strains

It may be useful for therapeutic purposes if experimental colonic cancer can be produced in larger animals. Our protocols for experiment to produce colonic cancer in dog were as follows: Two beagle and 12 mongrel dogs were used. Endoscopic examination was done every month or every other month. 1,2-Dimethylhydrazine (DMH) was given subcutaneously in 3 mongrel dogs once a week for 25 months. The protrusion like verruca was observed macroscopically in colonic mucosa in two of them. Histologically it was like lymph follicle hyperplasia in the submucosa. N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) soaked in sponge was inserted daily into the rectum of 2 beagle and 2 mongrel dogs for about 20.4 months. A leiomyoma of the colon was detected histologically in one beagle. N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) soaked in sponge was inserted daily into the rectum of 4 mongrel dogs for about 26.5 months. During follow up study, adenoma of the colon was detected by biopsy in one dog. ENNG suppository (containing 50 mg of ENNG) was administered through the anus in 3 mongrel dogs. Colon cancer was induced in all of three dogs. There were metastases to the liver, lung and lymph nodes in one of them. Colonic cancer was successfully induced in dogs by suppository of ENNG into the rectum. This model seems to be the most useful for producing experimental colonic cancer.

Topics: 1,2-Dimethylhydrazine; Adenoma; Animals; Carcinogens; Colonic Neoplasms; Dimethylhydrazines; Dogs; Female; Lymphatic Metastasis; Male; Methylhydrazines; Methylnitronitrosoguanidine; Suppositories

Carcinogenesis is commonly considered to be a multi-step process, comprising "initiation" and "promotion" events. Skin fibroblasts from patients with hereditary retinoblastoma (RB) and familial polyposis coli (FPC) were chosen for study since their predisposition to the tumour may be due to an inherited "initiation" event which is present in every somatic cell. Thus, one might predict that skin fibroblasts from these patients might exhibit an increased susceptibility to in vitro transformation, by either tumour promoters alone or by the complete carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). In the case of skin fibroblasts from RB patients, transformation as measured by the ability of cells to grow in semi-solid medium and their migration in collagen gels, did not occur with either class of agent. However, experiments involving skin fibroblasts from FPC patients, showed these cells to grow in semi-solid medium following treatment with the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) alone, although their pattern of migration in collagen gels was unchanged and they were non-tumorigenic in nude mice. The clones which grew in semi-solid medium were also unaltered in terms of their migration in collagen gels and tumorigenicity in nude mice and were considered not to be completely transformed. These results are discussed in relation to theories that tumour promoters are only involved in cell selection and clonal expansion of initiated cells a second "mutational" event being required for complete transformation.

Topics: Carcinogens; Cell Movement; Cell Transformation, Neoplastic; Cells, Cultured; Chromosome Aberrations; Colonic Neoplasms; Eye Neoplasms; Fibroblasts; Humans; Methylnitronitrosoguanidine; Mutation; Retinoblastoma; Skin; Tetradecanoylphorbol Acetate

The effect of tetragastrin on the incidence and histology of colonic tumors induced by intrarectal instillation of N-methyl-N'-nitro-N-nitrosoguanidine was investigated in Wistar rats. Prolonged administration of tetragastrin in depot form during and after treatment with N-methyl-N'-nitro-N-nitrosoguanidine resulted in a significant reduction in the incidence of colonic tumors in Experimental Week 35. Histological examinations showed that, unlike the well-differentiated adenocarcinomas with a typical glandular pattern in control groups, the adenocarcinomas that developed in rats treated with tetragastrin had high mucin-producing activity.

Topics: Adenocarcinoma; Adenoma; Animals; Colonic Neoplasms; Gastrins; Injections, Subcutaneous; Male; Methylnitronitrosoguanidine; Neoplasms, Experimental; Rats; Rats, Inbred Strains; Sarcoma; Tetragastrin

Chloroethylnitrosoureas (CNU) are antitumor agents which produce DNA interstrand crosslinks. We have proposed that crosslinks are produced in DNA via monoadduct formation at the guanine-O6 position, followed by a delayed reaction with the opposite DNA strand. Human cells are known to differ in their capacity to repair the O6-methylguanine lesion. One example of this repair capacity is the ability of cells to reactivate adenovirus which has been damaged by in vitro treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Cells that repair the virus are designated Mer+ and deficient cells Mer-. In a recent report, we showed a clear correlation between CNU-induced DNA interstrand crosslinking and the Mer phenotype. Mer- cells produced consistently higher levels of interstrand crosslinks than did Mer+ cells. In the present study we have measured the CNU-induced DNA interstrand crosslinking in IMR-90 normal human fibroblasts (Mer+), HT-29 human colon carcinoma cells (Mer+), and VA-13 SV-40 transformed human cells (Mer-) following pretreatment with MNNG. Cells were treated for 1 h with MNNG, then for an additional 1 h with CNU. Comparable levels of CNU-induced DNA interstrand crosslinking were observed in all cell lines. This crosslinking has been previously undetected in the IMR-90 and HT-29 cells. Cytotoxicity studies showed that MNNG pretreatment greatly enhanced the killing of IMR-90 and HT-29 cells by CNU, however, in VA-13 cells the increase in cell kill was smaller. These data suggest that in Mer+ cells a DNA repair system may remove chloroethyl monoadducts before the lethal DNA interstrand crosslinks can form. However, pretreatment of cells with MNNG may saturate this repair system rendering it inoperable.

Topics: Cell Line; Cell Survival; Colonic Neoplasms; DNA; Ethylnitrosourea; Female; Fibroblasts; Humans; Methylnitronitrosoguanidine; Nitrosourea Compounds; Pregnancy; Time Factors

We prepared a suppository containing 50 mg of N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG), and successfully produced experimental colon cancer with good reproducibility by continuous intrarectal insertion of one or two suppositories per day in dogs. The tumors were very similar to human colon cancers, macroscopically and histologically. In one of three dogs subjected to histopathological study, metastases to the lymph nodes, lung, liver and kidney were observed. This animal model produced by a simple procedure will be helpful in investigating treatment of rectal cancer.

Topics: Adenocarcinoma; Animals; Carcinoma; Colonic Neoplasms; Disease Models, Animal; Dogs; Lymphatic Metastasis; Methylnitronitrosoguanidine; Neoplasms, Experimental; Time Factors

Adenomatous polyps and adenocarcinomas with variety of the precancerous stages have been induced by intraectally injection of N-methyl-N'-nitro-N-nitrosoguanidine. The aim of our experiments have been to study some quantitative and qualitative changes in mucus secretion both in the neoplasm and in transitional area surrounding tumor. The changes in composition of mucus confirmed by several histochemical methods reflect the earliest morphologically observable stage of malignant transformation in colonic mucosa.

Topics: Animals; Cell Transformation, Neoplastic; Colon; Colonic Neoplasms; Methylnitronitrosoguanidine; Mucus; Precancerous Conditions; Rats; Rats, Inbred Strains

Skin fibroblasts from patients with familial adenomatosis coli (AC) and normal individuals were treated once with the carcinogen 4-nitroquinoline 1-oxide or N-methyl-N'-nitro-N-nitrosoguanidine and then passaged sequentially. Morphologically altered cells appeared in the cultures of carcinogen-treated AC fibroblasts at passages 6-8 (days 100-140) after treatment with the carcinogens, but carcinogen-treated normal cells and untreated AC and normal cells did not become altered even after cultivation for 25 passages. The cultures containing morphologically altered cells showed characteristics of transformed cells, such as a high frequency of colony formation in soft agarose, increased growth ability, and chromosomal abnormalities. The results suggest tha AC patients have increased susceptibility to morphologic transformation and chromosomal changes induced by chemical carcinogens.

Topics: 4-Nitroquinoline-1-oxide; Adenoma; Cell Count; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Chromosome Aberrations; Colonic Neoplasms; Female; Humans; Methylnitronitrosoguanidine; Middle Aged; Skin

Colon explants from the inbred F344 rat descending colon pretreated in vivo with azoxymethane and maintained in explant culture were exposed to the carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). One week after the MNNG treatment, the colon crypts showed marked crowding, hypercellularity, and stratification of cells. Nine weeks after the treatment, the explants showed epithelial papillary projections on the surface epithelium and within the crypts, in addition to hypercellularity and stratification. The control untreated explants maintained a single layer of epithelium during the entire culture period. Ultrastructurally, the treated cells showed an unusual concentration of free polysomes and thin and thick filaments, multiple and bizarre nucleoli, nuclear indentations and pseudoinclusions, and intracellular lumina. Sulfomucin was the predominant component in the control untreated explants as well as in the normal descending colons of rats and humans. One week after treatment the crypts of the carcinogen-treated explants showed an increase in sialomucin, and by 9 weeks after treatment, they showed mostly sialomucin. These features, compared and correlated with those of the parallel in vivo animal model as well as with human material, lend additional support to de novo histogenesis of colon carcinoma.

Topics: Animals; Colonic Neoplasms; Epithelium; In Vitro Techniques; Intestinal Mucosa; Male; Methylnitronitrosoguanidine; Neoplasms, Experimental; Rats; Rats, Inbred F344

The suitability of using coloscopy as a diagnostic method is investigated with respect to colonic carcinomas induced locally by the administration of N-nitrosoacetoxymethyl-methylamine, N-methyl-N-nitrosourea, and methylnitro-nitrosoguanidine, or systemically by subcutaneous injection of 1,2-dimethylhydrazine in Sprague-Dawley rats. The endoscopic diagnostic examination proved to be clearly superior to methods of animal inspection, palpation, investigation for occult blood and exploratory laparotomy which have so far been employed in animal experiments with small rodents. The relevance of this method is discussed for the early detection of chemically induced colonic tumors, and the observation of tumor development under experimental cytostatic therapy.

Topics: Adenocarcinoma; Animals; Carcinogens; Colon; Colonic Neoplasms; Colonoscopy; Dimethylnitrosamine; Evaluation Studies as Topic; Hyperplasia; Intestinal Polyps; Male; Methylamines; Methylnitronitrosoguanidine; Methylnitrosourea; Rats

Experimental colonic carcinoma in a dog was induced by anal insertion of an N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) suppository (each cone containing 50 mg of ENNG) for 17 months. The dog was autopsied 20 months after the initiation insertion of the suppository. Grossly, the colonic wall from the anus of the 10-cm oral side of the colon was thickened, and there was an infiltrating tumor with shallow depressions in the rough mucosa. The lymph node around this portion were enlarged, and white spots were found in the liver and redness in the lungs. Histological examination of the colon revealed a variety of pathologic features, e.g., undifferentiated carcinoma, squamous cell carcinoma and malignant melanoma in the region adjacent to the anus. Well and moderately differentiated adenocarcinomas involving the proper muscle layer were found in a region oral to these tumors and were accompanied by marked invasion of the blood vessels and lymphatic permeation. There were metastases to the liver, lungs and lymph nodes which corresponded to the gross findings, and also metastases to renal glomeruli. A well differentiated adenocarcinoma and signet ring cell carcinoma were evident in the gastric mucosa. This experimental model should be useful for studies related to colonic carcinoma in humans.

Topics: Adenocarcinoma; Animals; Carcinogens; Carcinoma; Carcinoma, Squamous Cell; Colon; Colonic Neoplasms; Dogs; Female; Melanoma; Methylnitronitrosoguanidine; Neoplasms, Experimental; Stomach; Stomach Neoplasms; Suppositories

We have studied the chromosomal sensitivity to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) of human diploid skin fibroblasts derived from individuals with adenomatosis coli (AC), which is a dominantly inherited disorder associated with multiple adenomas of the colon and rectum. Spontaneous frequencies of chromosome aberrations in the cell strains from the AC patients were similar to those in the control cells from normal individuals. However, the AC cells exhibited elevated chromosome instabilities when cells were expected to MNNG, with aberration frequencies approximately twice as high as in similarly treated control cells. The present results, together with findings by others, suggest that the AC cells are defective in a function which regulates cellular condition and are in a state more susceptible to the action of agents that react with chromosomal DNA. These findings also raise the possibility of developing a diagnostic procedure for early detection of abnormal gene carriers of AC.

Topics: Adenoma; Adult; Cell Cycle; Cells, Cultured; Child; Chromosome Aberrations; Chromosomes, Human; Colonic Neoplasms; Dose-Response Relationship, Drug; Female; Fibroblasts; Humans; Male; Methylnitronitrosoguanidine; Middle Aged; Rectal Neoplasms; Skin

Chemical transformation of cultured human skin fibroblasts (PF) derived from individuals with hereditary adenomatosis of the colon and rectum is reported. Cells treated only with various levels of N-methyl-N'-nitro N-nitrosoguanidine (MNNG) underwent morphological alteration. The morphologically altered cells formed large aggregates when suspended in liquid growth medium above an agar base and grew to high saturation densities. One altered (MNNG, 1.0 microgram/ml) cell culture formed colonies in soft agar. Transformed cells were resistant to rechallenge of MNNG (l microgram/ml) and showed a more prolonged life-span compared to the untreated cells. Altered cells became heteroploid cells. However, no progressively growing tumors were produced when cells were inoculated subcutaneously into nude mice. The data suggest that chemical carcinogens alone may not induce neoplastic transformation of fibroblasts from humans genetically predisposed to cancer and that neoplastic transformation of these skin cells by chemical carcinogens might require the presence of a tumor promotor and the use of an immuno-privileged site in the nude mouse system.

Topics: Adenoma; Cell Transformation, Neoplastic; Colonic Neoplasms; Fibroblasts; Humans; Methylnitronitrosoguanidine; Rectal Neoplasms; Skin

Quantitative analysis of intestinal microflorae in the diverted and feces-containing portions of the colon after performing an ascendo-descendostomy Roux en Y in Wistar-Lewis rats, the colonic portion from the ascending colon to the midportion of the descending colon being diverted from the fecal stream, disclosed a drastic decrease in the total amount of the intestinal microflora as well as most anaerobic microflorae in the diverted portion of the colon where the amount of the colonic content was extremely reduced. This diverted segment was least susceptible of developing macroscopical epithelial neoplasia after exposure to a carcinogen, MNNG, sufficient in amount to evoke multiple large neoplasia in the ordinary colon, although microscopical intramucosal neoplastic foci were induced there. Thus the existence of the intestinal content was essential for the process of promotion in experimental colonic carcinogenesis. The close parallelism between amounts of intestinal content and amounts of intestinal microflorae, especially anaerobic ones, suggested the roles of anaerobic microflorae in colonic carcinogenesis.

Topics: Anaerobiosis; Animals; Colon; Colonic Neoplasms; Feces; Female; Intestinal Mucosa; Intestines; Male; Methylnitronitrosoguanidine; Neoplasms, Experimental; Rats

Topics: Adenocarcinoma; Adenoma; Animals; Cholesterol; Cocarcinogenesis; Colonic Neoplasms; Female; Germ-Free Life; Lithocholic Acid; Methylnitronitrosoguanidine; Neoplasms, Experimental; Rats; Rats, Inbred F344

Epidemiologic and laboratory studies suggest that dietary fk actors, particularly high intake of fat and animal protein, and high concentration of bile acids and neutral sterols of the large bowel lumen are strongly associated with large bowel carcinogenesis. Such concepts guided our studies on animal models. Rats fed diets high in fat and/or protein had a higher incidence of DMH-induced large bowel tumors than rats fed standard diets. The source of fat and protein, animal vs. vegetable, had no major influence. High fat intake was associated with an increased excretion of fecal bile acids, particularly secondary bile acids, and neutral sterols. The repeated intrarectal doses of lithocholic acid or deoxycholic acid enhanced the development of MNNG-induced large bowel tumors in rats. Colostomized rats treated with intrarectal dose of MNNG had no tumors in the excluded segment. It suggests that luminal contents play a significant role in the induction of large bowel cancer. The results show that higher levels of bile acids in the large bowel lumen, resulting from high fat intake, exert a promoting effect on the development of large bowel cancer.

Topics: Animals; Bile Acids and Salts; Colonic Neoplasms; Colostomy; Dietary Fats; Dietary Proteins; Dimethylhydrazines; Feces; Female; Male; Methylnitronitrosoguanidine; Neoplasms, Experimental; Rats

Experimentally induced tumours in the colon of the rats were examined with a double contrast method. Carcinogenic agents were administered to 114 rats. The development of the colonic tumors was recorded by repeated examinations. In the dead rats a thorough necropsy was performed. The radiographic and microscopic results were correlated and the radiographic appearances of colonic tumours were evaluated. The diagnostic accuracy of the radiographic method in distinguishing between benign and malignant lesions was 92 per cent.

Topics: Adenocarcinoma; Adenoma; Animals; Colon; Colonic Neoplasms; Contrast Media; Dimethylhydrazines; Female; Intestinal Mucosa; Intestinal Polyps; Male; Methylnitronitrosoguanidine; Neoplasms, Experimental; Radiography; Rats

In 189 rats N-methyl-N'-nitro-N-nitrosoguanidine or 1,2-dimethylhydrazine (DMH) was given in order to induce colonic tumours. The tumour induction was followed by double contrast examination. At 894 examinations 196 adenomatous tumours were revealed. Autopsy and microscopy revealed 214 macroscopic and 53 microscopic benign or malignant adenomatous tumours. Metastases were found in 17 per cent in the DMH group. The relationship between adenomas and carcinomas is also evaluated.

Topics: Adenocarcinoma; Adenoma; Animals; Carcinogens; Colon; Colonic Neoplasms; Dimethylhydrazines; Female; Hyperplasia; Injections; Injections, Intravenous; Injections, Subcutaneous; Intestinal Mucosa; Male; Methylnitronitrosoguanidine; Neoplasm Metastasis; Neoplasms, Experimental; Radiography; Rats; Rectum; Time Factors

Germfree and conventional Sprague-Dawley rats were assessed for their susceptibility to intrarectally injected N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or N-methyl-N-nitrosourea (MNU). Adenocarcinoma of the colon was induced in germfree and conventional rats by both MNNG and MNU. The colons of germfree rats were more susceptible to the direct-acting carcinogens, as manifested by earlier morbidity and development of colon tumors (50% tumors within 30-35 wk), than were those of conventional rats (50% colon tumors within 48-50 wk). Germfree and conventional male rats were more susceptible to the carconogens than were their female germfree and conventional counterparts. Young (30 days old at the start of the experiment) germfree rats developed colon tumors more quickly (15-20 wk) than did older (60 days) germfree rats after intrarectal injections of MNNG. No colon tumors were observed in germfree and conventional rats after 75 weekly intrarectal injections with a buffer. Transplantation of an adenocarcinoma induced with MNU in a female rat to germfree and conventional rats showed that it was easily transplantable, required no immunosuppression, and had essentially the same morphologic characteristics as did the primary tumor.

Topics: Adenocarcinoma; Age Factors; Animals; Colonic Neoplasms; Drug Administration Schedule; Female; Germ-Free Life; Intestines; Male; Methylnitronitrosoguanidine; Methylnitrosourea; Neoplasm Transplantation; Neoplasms, Experimental; Rats; Sex Factors; Time Factors; Transplantation, Homologous

Topics: Adenocarcinoma; Adenoma; Animals; Chenodeoxycholic Acid; Cholic Acids; Colonic Neoplasms; Drug Synergism; Female; Germ-Free Life; Methylnitronitrosoguanidine; Neoplasms, Experimental; Rats; Rats, Inbred F344

The morphogenesis of neoplasms (carcinomas and pracecancerous polypoid lesions) induced by N-methyl-N-nitroso-N'-nitroguanidine (MNNG) was studied on 154 male Wistar rats (including controls), from which a group received a colostomy. Operated and non-operated rats were treated by intrarectal instillations of 2 mg per kg body weight MNNG twice a week during a period of 210 days.

Topics: Animals; Carcinoma; Colonic Neoplasms; Colostomy; Intestinal Polyps; Male; Methylnitronitrosoguanidine; Neoplasms, Experimental; Precancerous Conditions; Rats; Rectal Neoplasms

Topics: Animals; Azoxymethane; Benz(a)Anthracenes; Colonic Neoplasms; Cricetinae; Dogs; Duodenal Neoplasms; Esophageal Neoplasms; Gastrointestinal Neoplasms; Guinea Pigs; Methylnitronitrosoguanidine; Mice; Neoplasms, Experimental; Nitrites; Nitrosamines; Nitrosoguanidines; Pancreatic Neoplasms; Rabbits; Rats; Stomach Neoplasms

The promoting effect of sodium deoxycholate (DC) on colon carcinogenesis was studied in female F344 germfree rats. Animals received intrarectal (ir) instillations of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) for 4 weeks (total dose, 16 mg/rat), then weekly ir doses of DC (total dose, 3 g/rat); the rats were autopsied 52 weeks after the first injection. DC increased the number of MNNG-induced colon adenocarcinomas. No tumors were in the colons of germfree rats given DC alone. It was concluded that DC (present in high concentrations in human stools) had a promoting effect on colon carcinogenesis in rats.

Topics: Adenocarcinoma; Animals; Colonic Neoplasms; Deoxycholic Acid; Female; Methylnitronitrosoguanidine; Neoplasms, Experimental; Nitrosoguanidines; Rats; Rats, Inbred F344

The effect of vitamin A deficiency on the sensitivity of the colon to the carcinogenic effect of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a direct-acting carcinogen, given intrarectally was studied in female Fischer rats. Animals maintained on Purina laboratory chow, semipurified vitamin A-free diet, or semipurified vitamine A-supplemented diet were given intrarectally 1.25, 0.63, or 0.31 mg MNNG 3 times weekly for 30 weeks and autopsied at the 45th week. The number of large bowel tumors per tumor-bearing rat was higher in animals receiving 1.25 mg MNNG compared to those given 0.63 or 0.31 mg. Vitamin A deficiency in rats given 1.25 mg MNNG significantly suppressed the large bowel tumor induction compared to rats fed adequate vitamin A. A high incidence of squamous cell papillomatosis of the urinary bladder was observed in rats fed vitamin A-free diet and given 1.25 mg MNNG. The present experiment suggests that the large intestine has a susceptibility that is different from that of the respiratory and urinary tracts to tumorigenic stimulation in vitamin A-deficient status.

Topics: Animals; Colonic Neoplasms; Female; Methylnitronitrosoguanidine; Neoplasms, Experimental; Nitrosoguanidines; Papilloma; Rats; Urinary Bladder Neoplasms; Vitamin A Deficiency

Intrarectal instillation of 0.5 ml of a 0.125% solution of N-methyl-N'-nitro-N-nitrosoguanidine twice weekly for 53 weeks to female inbred strain-2 guinea pigs induced multiple large bowel adenocarcinomas in 13 of 15 animals in 52 to 85 weeks. The lesions were plaque-shaped in small tumors and infiltrative or constrictive in large advanced tumors. The neoplasms showed histological features in varied grades of differentiation and invasiveness similar to those of human cases. These findings distinquished them from large bowel cancers chemically induced in rats and mice.

Topics: Adenocarcinoma; Animals; Colonic Neoplasms; Female; Guinea Pigs; Methylnitronitrosoguanidine; Neoplasms, Experimental; Nitrosoguanidines; Rectum

Topics: Animals; Antigens, Neoplasm; Colonic Neoplasms; Dimethylhydrazines; Kidney; Lymphocytes; Methylnitronitrosoguanidine; Neoplasms, Experimental; Polyomavirus; Rats; Sarcoma

Female germfree and conventional rats of 50 days of age were injected intrarectally with MNNG for 20 wk (total dose, 48 mg/rat) and autopsied 30 wk after last injection. The colon adenomas induced by MNNG were doubled in germfree rats compared to conventional animals. However, germfree status had no effect on the incidence of adenocarcinomas. It is concluded that pharmacodynamics and metabolism of carcinogen play a role greater than the immune status of the animal in the action of carcinogens such as MNNG.

Topics: Adenocarcinoma; Adenoma; Animals; Colonic Neoplasms; Female; Germ-Free Life; Injections; Methylnitronitrosoguanidine; Neoplasms, Experimental; Nitrosoguanidines; Rats; Rectum

In an effort to establish an animal colon tumor model suitable for biological and chemotherapy studies, 82 colon tumors were induced and transplanted in four different inbred strains of mice. Four colon tumors survived the first transplant and are now in serial passage. All are suitable for chemotherapy trials. Two tumors are highly metastatic, and at least one of these is known to be suitable for surgery-chemotherapy adjuvant studies. The effective colon carcinogens contained a (see article) molecular similarity.

Topics: Adenocarcinoma; Animals; Carcinogens; Carcinoma; Chemical Phenomena; Chemistry; Colonic Neoplasms; Dimethylhydrazines; Disease Models, Animal; Methylnitronitrosoguanidine; Methylnitrosourea; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Inbred DBA; Neoplasm Metastasis; Neoplasm Transplantation; Neoplasms, Experimental; Nitrosomethylurethane; Transplantation, Homologous

Two transplantable strains of adenocarcinoma were established from the carcinomas of the colon and rectum induced by infusion of N-methyl-N'-nitro-N-nitrosoguanidine in inbred ACI/N rats, The tumors are solid type, not converted to ascite form yet, either papillary or tubulo-papillary adenocarcinomas, and particularly grow slowly, showing 2 to 4 months of survival in animals transplanted subcutaneously. Besides histological resemblance of the tumors to human adenocarcinoma of the large intestine, marked mucin production in tumor was noted in one of these strains.

Topics: Adenocarcinoma; Animals; Colonic Neoplasms; Female; Infusions, Parenteral; Male; Methylnitronitrosoguanidine; Neoplasm Transplantation; Nitrosoguanidines; Rats; Rats, Inbred ACI; Rectal Neoplasms; Transplantation, Homologous

Soluble intracytoplasmic protein fractions and solubilized tumor membrane preparations produced by 3 M KC1 or papain treatment were isolated from a 1,2 dimethylhydrazine-HC1 (DMH)-induced rat colon carcinoma, DHM-BU 1. Solubilized preparations were fractionated by DEAE cellulose and Sephadex G200 column Chromatography. Crude extracts and fractionated materials were tested for their ability to inhibit the cytotoxicyt of lymph-node cells (LNC) from rats bearing isografts of an N-methyl-N-nitro-N-nirosoguanidine (NG)-induced colon carcinoma, ng-W1...

Topics: Adenocarcinoma; Animals; Antigen-Antibody Reactions; Antigens, Neoplasm; Carcinoembryonic Antigen; Cells, Cultured; Colonic Neoplasms; Cytotoxicity Tests, Immunologic; Dimethylhydrazines; Lymph Nodes; Lymphocytes; Methylnitronitrosoguanidine; Neoplasm Transplantation; Neoplasms, Experimental; Rats; Rats, Inbred BUF; Rats, Inbred WF; Transplantation, Homologous

Colon tumors of mice were induced with 1, 2-dimethylhydrazine, N-nitroso-N-methylurethane, methylnitrosourea, and 4-methyl-N'-nitro-N-nitrosoguanidine. Of 82 transplantation attempts, four were successful, and the four tumors have been maintained as distinct tumor lines by subcutaneous transplantation. The tumors graded from II, adenocarcinoma, to IV, undifferentiated carcinoma. Volume-doubling times varied over a three-fold range, and metastatic potential ranged from 5 to about 100%. The tumors responded to treatment with some of the same agents found to be active in man. These included cyclophosphamide, 5-fluorouracil, and certain nitrosoureas. Preliminary evidence suggests that these tumor lines may be useful as experimental models.

Topics: Animals; Antineoplastic Agents; Colonic Neoplasms; Dimethylhydrazines; Disease Models, Animal; Methylnitronitrosoguanidine; Methylnitrosourea; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Inbred DBA; Neoplasm Transplantation; Neoplasms, Experimental; Nitrosomethylurethane; Transplantation, Homologous

A method to make a diverted segment of the colon keeping direct continuity to the main colonic lumen was introduced to study carcinogenesis in the colon of rats. This method was proved to be useful for analyzing several factors influencing upon cancerization in the colonic mucous membrane. Macroscopical colonic neoplasia were induced in nearly 80% of rats treated with N-methyl-N'-nitro-N-nitrosoguanidine introduced into the colonic lumen through the diverted segment. Neoplastic lesion of the mucous membrane developed mainly in the colonic segments which were in direct contact with intestinal content. The importance of intestinal content and colonic microflora was discussed and reviewed. One epithelial cell line was established from one intraperitoneal metastatic deposit of a huge colonic carcinoma induced by the carcinogen. This cell line has been maintained in tissue culture. The liver was susceptible to the carcinogen, and multiple cystic lesions were observed after intracolonic administration of the chemical.

Topics: Adenoma; Animals; Chemical and Drug Induced Liver Injury; Colon; Colonic Neoplasms; Cysts; Female; Liver; Liver Diseases; Male; Methylnitronitrosoguanidine; Mitosis; Neoplasm Metastasis; Neoplasms, Experimental; Rats