MNNG Treatment Research Articles

The ATF/CREB transcription factor-binding site in the polymerase beta promoter mediates the positive effect of N-methyl-N'-nitro-N-nitrosoguanidine on transcription.

DNA polymerase beta (pol beta) is a constitutively expressed DNA repair enzyme in vertebrate cells. Yet, it had been shown previously that the pol beta mRNA level increases in Chinese hamster ovary (CHO) cells within 4 h after treatment with several monofunctional DNA damaging agents, notably, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Herein we report that a transfected pol beta promoter fusion gene is activated by MNNG treatment of CHO cells; mRNA from the transfected gene is approximately 10-fold higher in treated cells than in untreated cells 16 h after treatment. This activation is mediated through the decanucleotide palindromic element GTGACGTCAC at positions -49 to -40 in the "TATA-less" core promoter. This element, which is similar to the ATF/CREB transcription factor-binding site in a number of mammalian genes, forms the center of a strong protein-binding site for CHO cell nuclear extract proteins. Mutated pol beta promoter fusion genes lacking the element fail to bind protein at this site and fail to respond to MNNG treatment of cells.

The control of O6-methylguanine-DNA methyltransferase (MGMT) activity in mammalian cells: A pre-molecular view

Human peripheral blood lymphocytes (PBLs) can have a range of O 6-methylguanine-DNA methyltransferase (MGMT) activities. PBLs from some individuals may have almost no MGMT activity. Such individuals have most often been subject to malignancy or to immunodeficiency disease. Long-term lymphoblastoid lines (LCLs) prepared from PBLs of normal subjects by Epstein-Barr virus (EBV) transformation have MGMT activities which are in general somewhat higher than the PBLs from which they derive. Such cultures are therefore generally MGMT-positive. Only in rare cases, and generally from patients with low MGMT activity, are freshly obtained lines with very low activity obtained. There is however a 4-fold range of MGMT activity over which multiple lines derived from the same PBL sample can be found. Long-term cultivation can lead to LCLs with low activity as well as to lines of high activity. On rare occasions an MGMT-positive line may, within a few divisions, give a negative line. Some (but not all) MGMT-negative (or very low) lines have been known to gain (some) activity. Chinese hamster ovary (CHO) cell lines are in general very low in MGMT activity. Lines of higher activity can be selected by treatment with mutagenic crosslinking alkylating agents. Chinese hamster lines with high MGMT activity can be obtained by transfection with human DNA from MGMT-positive cells. Lines with significant activity can also be obtained by transfection of CHO cells with human DNA from MGMT-negative (or very low) cells. Resistance to MNNG treatment can be acquired without the acquisition of significant MGMT activity. Crosses of lines of high and low MGMT activity give equivocal results. Hybrids of low × low activity have no activity. Crosses of positive × positive strains give varied results. It has not been possible to identify MGMT-positive hybrids as including one particular chromosome by this type of experiment. There is no evidence for a general adaptive effect on MGMT synthesis greater than the variation within the cell cycle.

Ultrastructural organization of secondary mutants ofEuglena gracilis induced by N-methyl-N′-nitro-N-nitrosoguanidine

Photosynthetic mutants ofEuglena gracilis obtained by N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) or nitrovin treatment were treated secondarily with MNNG and bleached mutants were isolated. In all mutant strains maintained in the light-grown condition some remnants of plastids were always observed. However, there are differences in the internal organization of secondary mutants between samples taken for examination after 15 and 200 generations of subcultivation. On the other hand, there are no differences in the internal organization of secondary mutants derived from primary photosynthetic mutants and bleached mutants induced directly from wild-type euglenas by MNNG treatment.

Inhibition of N‐methyl‐N′'‐nitro‐n‐nitrosoguanidine‐induced methotrexate and adriamycin resistance in CHO cells by adeno‐associated virus type 2

We studied the effects of helper-dependent parvovirus AAV [adeno-associated virus] type 2 on carcinogen-inducible resistance to methotrexate (MTX) and adriamycin (ADR) in Chinese hamster ovary cells. Both types of drug resistance were monitored by determination of the number of drug-resistant colonies normalized for the respective value of plating efficiency under non-selective conditions. Treatment of cells with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) drastically enhanced the frequency of resistance to MTX and ADR. By contrast, infection of cells with AAV-2 prior to treatment with MNNG markedly inhibited carcinogen-induced drug resistance. Infection by AAV alone did not exert any effect. Analysis of the dihydrofolate reductase (dhfr) gene copy numbers of individual MTX-resistant clones derived from MNNG-treated and non-treated cultures revealed similar frequencies (60-80%) and amplitudes of dhfr gene amplification (2- to 8-fold) irrespective of prior AAV treatment. Hence, carcinogen-induced enhancement of MTX-resistance could reflect an increase in the frequency of dhfr gene amplification among the survivors of MNNG treatment. On the other hand, inhibition of carcinogen-inducible drug resistance by AAV suggests an interference of the virus with cellular responses to genotoxic stress, thus leading to enhanced cell killing under altered growth conditions. Possible mechanisms responsible for the inhibitory effect of AAV and its relevance in relation to tumor chemotherapy are discussed.

Inhibition of induction of adaptive response by o-vanillin in Escherichia coli B

Mutations induced by N-methyl- N′-nitro- N-nitrosoguanidine (MNNG) were strongly enhanced in the presence of o-vanillin in E. coli B. The enhancement was also observed in uvrA, umuC, recA, polA, or alkB mutants. This effect was lower in an alkA mutant, but was restored in an alk A umuC double mutant. By contrast, the enhancing effect was almost blocked in an ada and ada umuC double mutant. It was necessary to add simultaneously MNNG and o-vanillin to the growth medium. Further investigations were conducted on the induction of ada and umuC genes using ada′-lacZ′ and umuC′-lacZ′ plasmids. o-Vanillin suppressed the induction of the ada gene by MNNG treatment, but not that of the umuC gene. In fact expression of the umuC gene was induced by lower concentrations of MNNG in the presence of o-vanillin. The results suggest that o-vanillin inhibits induction of the adaptive response, and consequently, the MNNG-induced mutation frequency is increased due to unrepaired O 6-methylguanine.

Effect of Epidermal Growth Factor on Rat Stomach Carcinogenesis Induced by N‐Methyl‐N′‐nitro‐N‐nitrosoguanidine

The effect of epidermal growth factor (EGF) on rat stomach carcinogenesis induced by N-methyl-N'-nitro-N-nitro-soguanidine (MNNG) was studied. Male Wistar rats given MNNG for 30 weeks in drinking water (80 micrograms/ml) were treated with s.c. injections of human EGF (10 micrograms/kg, once daily) at various stages of the carcinogenesis. Four (30.8%) out of 13 rats treated with EGF immediately after cessation of the MNNG treatment had stomach tumors including one adenocarcinoma, one adenoma and two carcinoids. No stomach tumor was found in rats treated with MNNG alone or in those treated with MNNG and EGF for different periods such as synchronously for 10 weeks, for 30 weeks or throughout the experiment. These findings suggest a possible enhancing effect of EGF on stomach carcinogenesis in rats.

EFFECT OF CO-ADMINISTRATION OF N-METHYL-N'-NITRO-N-NITROSOGUANIDINE AND SODIUM CHLORIDE AS AN INITIATION PROCEDURE FOR A PROMOTING EFFECT OF ETHOXYQUIN ON STOMACH TUMORIGENESIS IN F344 MALE RATS

The promoting potential of ethoxyquin, an antioxidant, was investigated on two-stage glandular stomach carcinogenesis in rats. After simultaneous administration of N-methyl-N'-nitro-Nnitrosoguanidine (MNNG, 100ml/L in the drinking water) and sodium chloride (NaCl, 10% in basal diet) to group I or these agents singly to groups 2 and 3, respectively, for the first 8 weeks, the animals received a 1% ethoxyquin supplement in the basal diet for the following 32 weeks. Group 4 rats received the MNNG and NaCl combination and were then maintained as controls. The resultant incidences of carcinomas arising in the pyloric mucosa of rats in groups 1 to 4 were 8/17 (47%), 1/16 (6%), 0/16 (0%), and 2/10 (20%), respectively, with the group 1 incidence being significantly higher than that of group 2 or 3. These results clearly showed that promotion by ethoxyquin was observed only after simultaneous treatment with MNNG and NaCl, but not after MNNG treatment without NaCl.

Effects of natural selection, mutagenesis, and protoplast formation and cell wall regeneration on the production of aminoglycoside antibitics

High producers or blocked mutants of aminoglycoside antibiotic-producingStreptomyces spp. were selected by application of an agar plug method and by culturing individual colonies in broth. The productivities of aminoglycoside antibiotic producing organisms were increased by selection of a high producer from colonies obtained by spreading spores of wild strain, or survived from treatment of a mutagen or from the colonies regenerated from protoplast-formation and cell-wall regeneration. Some mutagen treated colonies lost the ability to produce antibiotics (5–8%). Some A-factor negative and deostreptamine or streptidine negative mutants were obtained by N-methyl-N’-nitro-N-nitrosomethylguanidine (MNNG) treatment. Many of the survivors from the MNNG treatment lost the ability to produce antibiotics. Major colonies produced less amount of antibiotics; only few survived colonies produced more antibiotics than the parent. Resistance ofStreptomyces spp. against the antibiotics produced by itself was also markedly affected by mutagen treatment.

Increased chromosome breakage by N-methyl-N 1-nitro-N-nitrosoguanidine in patients with adenomatous polyposis coli

In a search for hypersensitivity to chromosome breaking agents, lymphocytes and fibroblasts from patients with the precancerous condition adenomatous polyposis coli (APC) were treated with N-methyl-N 1-nitro-N-nitrosoguanidine (MNNG) and bleomycin. The APC cells showed a significantly increased level of chromatid-type damage following MNNG treatment (5 μg/ml for lymphocytes, 1 or 2 μg/ml for fibroblasts). No such differential effect was noted for bleomycin, but a threefold increase in “pulverized” cells was seen in APC lymphocyte cultures treated in G0, that is before cells have entered the cell cycle. Increased spontaneous and induced chromosome instability appears to be an important effect of the APC mutant gene as molecular evidence suggests that chromosomal mechanisms are likely to play a major role in tumorigenesis both in this condition and in sporadic colorectal cancer.

Effects of heat shock on the induction of mutations by chemical mutagens in Neurospora crassa

Preheating of Neurospora conidia increased their susceptibility to mutation induction by chemical mutagens. Optimal conditions of heat shock for enhanced mutagenesis were determined in 2.5 × 10 7 conidia/ml 0.067 M KH 2PO 4Na 2HPO 4 (pH 7.0) buffer to be treatment at 43°C for 60 min. When protein synthesis during heat stock was eliminated by cycloheximide or by use of the temperature-sensitive mutation psi-1, induction of thermotolerance was inhibited while induction of the enhanced state of mutability was not. Therefore, inducible protein synthesis is not involved in this process. To discover whether DNA-repair systems are altered by heat shock and, as a result, whether reversion frequencies increase, DNA-repair mutants ( upr-1, uvs-2, uvs-3, uvs-6, mus-7, mus-16) were heated and their reversion frequencies at the ad-8 locus were measured. All the DNA-repair mutants showed higher reversion frequencies with MNNG treatment after heat shock than in non-heated control. It therefore seems that DNA repair is not involved in the enhancement of chemical mutagenesis by heat shock. Heat shock does not increase frequencies of reversion induced by ultraviolet light, and heat shock after treatment with chemical mutagens does not affect reversion frequencies. These results suggest that heat shock may change the structure and function of cellular membranes and thereby increase the influx of mutagens into cells.

Antagonistic effect of diethylmaleate on the promotion of forestomach carcinogenesis by butylated hydroxyanisole (BHA) in rats pretreated with N-methyl-N'-nitro-N-nitrosoguanidine.

The effects of diethylmaleate (DEM), previously demonstrated to inhibit butylated hydroxyanisole (BHA)-induced forestomach hyperplasia, on BHA promotion of forestomach carcinogenesis in rats pretreated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were examined. Groups of male 6-week-old F344 animals were given a single i.g. administration of 150 mg/kg body weight MNNG and starting 1 week later administered powdered diet containing 1% BHA plus 0.2% DEM, 1% BHA, 0.2% DEM or basal diet alone for 51 weeks. Further groups of rats were treated with 1% BHA plus 0.2% DEM, 1% BHA, 0.2% DEM or basal diet alone without MNNG pretreatment. Histopathological assessment of lesions at week 52 revealed enhancement of MNNG-initiated papilloma (100 versus 50%) and squamous cell carcinoma (100 versus 0%) development by BHA as compared to controls. Additional treatment with DEM, however, significantly reduced the relative incidences of carcinoma in situ (0 versus 35.7%) and squamous cell carcinoma (35.7 versus 100%), as well as BHA-induced forestomach hyperplasia with or without prior MNNG treatment. The results thus clearly demonstrate that DEM acts as a potent antagonist to BHA-promotion of rat forestomach carcinogenesis.

Changes in rat and human pepsinogen phenotypes induced by N'-methyl-N'nitro-N-nitrosoguanidine.

Changes have been studied in human and rat pepsinogen phenotypes induced by N'-methyl-N'nitro-N-nitrosoguanidine (MMNG) in in vitro rat experiments and in vivo cultures of human and rat isolated gastric chief cells. In vivo the fastest migrating electrophoretic band decreased or disappeared as early as 3 weeks after the start of MNNG treatment. The changes, observed in 17 of 32 rats receiving MNNG, were permanent and consistently associated with pronounced histopathologic changes seen 10 months later (17 of 17). Comparable phenotypic changes were observed after 7 days only in MNNG-treated rat chief cell cultures. In human chief cell cultures a decrease of the Pg3 band, which is consistent with the "carcinogenic" phenotype, was observed in two of six preparations treated with MNNG. This early preceding change in phenotype preceding tumor formation may be useful as a diagnostic tool for the onset of gastric cancer.

Dissociation of carcinogen-induced SV40 DNA-Amplification and amplification of AAV DNA in a Chinese hamster cell line

Time kinetics of AAV-5 DNA amplification induced by MNNG in cells of a Chinese hamster line (C0631) and a Syrian hamster line (Elona) were compared to kinetics of SV40 DNA amplification in these cells. AAV-5 DNA amplification starts in both lines about 8 hr following treatment of AAV-infected cells with MNNG. SV40 DNA amplification is induced only in C0631 cells. The time it becomes detectable varies but in no case was before 23 hr after MNNG treatment. In CO631 cells a second round of AAV DNA amplification takes place. It starts at times paralleling the MNNG-induced synthesis of SV40 DNA but appears to be independent of genotoxic treatment. Elona cells amplify neither SV40 DNA after exposure to chemical carcinogens nor AAV-5 DNA in untreated cells. In both lines combined treatment with MNNG and AAV-5 resulted in marked cytopathogenic changes and cell death. In the test systems used here viral DNA amplification did not lead to synthesis of infectious virus, thus, the viral cycle remained abortive.

Deoxynucleoside triphosphate pool perturbation is not a general feature in mutagen-treated mammalian cells

Deoxynucleoside triphosphate (dNTP) and ribonucleoside triphosphate (rNTP) pools were analyzed in 4 mammalian cell lines following treatment with UV-C (254 nm), UV-A (365 nm) or the carcinogen, 1-methyl-3-nitro-1-nitrosoguanidine (MNNG). No substantial alterations in dNTP pool levels were observed in any treatment group. However, the cellular conversions of exogenously added deoxycytidine and deoxyguanosine to the corresponding triphosphates were inhibited 30–97% by UV-C and MNNG treatment. In addition, the conversion of dGuo to GTP and deoxyadenosine to ATP were inhibited 25–50% in CHO cells by mutagen treatment. The data do not support the notion that modulation of specific dNTP pools is a general feature of mutagen treatment in mammalian cells, but so suggest a mutagen-sensitivity of deoxynucleoside metabolism.

Is Ap 4A involved in DNA repair processes?

Hepatoma tissue culture (HTC) cells were incubated in the presence of the alkylating agent N-methyl- N′-nitro- N-nitrosoguanidine (MNNG) to study the variations in the bisnucleosides polyphosphates (Ap 4X) pool size. A transient but sensitive accumulation of these compounds is observed; if 3-aminobenzamide (3AB) which is a potent inhibitor of the ADP-ribosyltransferase (ADPRT) is added after the MNNG treatment, a more pronounced and persistent accumulation of Ap 4X can be seen. A moderate heat-shock (30 min at 43 °C) results also in a small accumulation of Ap 4X but the shape of the accumulation curve is quite different and the increase of the Ap 4X pool is not sensitive to the presence of 3AB. However, both MNNG treatment and hyperthermia cause a marked inhibition of protein synthesis. On the other hand, the ADPRT activity is enhanced in the presence of MNNG whereas hyperthermia has little or a slightly inhibitory effect on this activity. These results suggest that MNNG treatment triggers an Ap 4X accumulation in eukaryotic cells different from that observed after heat-shock and it seems likely that these compounds are involved in the DNA excision repair system in which the ADPRT enzyme is also implicated.

O6-methylguanine-DNA methyltransferase-defective human cell mutant: O6-methylguanine, DNA strand breaks and cytotoxicity.

We have isolated an isogenic O6-methylguanine (O6-MeG)-DNA methyltransferase-defective mutant from a HeLa cell line. This mutant exhibits excess DNA strand breaks and considerable cytotoxicity after N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment. The increased frequency of strand breaks after MNNG treatment was not abolished by DNA synthesis inhibitors. We propose that the presence of unrepaired O6-MeG lesions leads to excess strand breaks and these, in turn, are mainly responsible for the cytotoxicity.

Enhancement of plasminogen activator activities by N-methyl- N′-nitro- N-nitrosoguanidine in tuberous sclerosis fibroblasts

The alkylating agent N-methyl- N′-nitro- N-nitrosoguanidine (MNNG) enhanced the levels of plasminogen activator (PA) activity in fibroblast cells derived from the skin of patients with tuberous sclerosis. The enhanced enzyme levels were not correlated with those of cloning efficiency nor those of DNA synthesis after MNNG treatment. Enzyme enhancement was also observed in fibroblasts of ataxia telangiectasia and in human neoplastic glia cells, but not in fibroblasts of normal children. The PA induction test may be sufficiently sensitive for the detection of the cellular defects of tuberous sclerosis.

Immunohistochemical Demonstration of Pyloric Gland-Type Cells With Low-Pepsinogen Isozyme 1 in Preneoplastic and Neoplastic Tissues of Rat Stomachs Treated With N-Methyl-N′-nitro-N-nitrosoguanidine2

The appearance of pyloric gland-type cells with a low pepsinogen isozyme 1 (Pg 1) content in the stomach mucosa of F344/Du rats during stomach carcinogenesis was examined by a combination of paradoxical concanavalin A (Con A) staining and immunohistochemical staining for Pg 1. Male F344 rats were given drinking water containing 100 micrograms N-methyl-N'-nitro-N-nitrosoguanidine [(MNNG) CAS: 70-25-7]/ml for 30 weeks and then normal tap water and were killed in week 10, 20, 30, 40, 50, or 70. Untreated rats were killed in week 30 or 70. Serial sections of pyloric mucosa were stained by paradoxical Con A staining and Pg 1 immunostaining. After MNNG treatment, tissues showing changes were classified into normal-looking pyloric mucosa with a low Pg 1 content, mucosa showing atrophic or hyperplastic changes, adenomatous hyperplasia, and adenocarcinoma. From the results of paradoxical Con A staining and Pg 1 immunostaining, the cells in lesions were classified into gastric types (surface mucous cell type and pyloric gland cell type) and intestinal types (intestinal-absorptive cell type and goblet cell type). In this experiment, the cells in lesions were mainly of the gastric cell types. All pyloric glands of control rats in weeks 30 and 70 contained class III mucins and had a high Pg 1 content demonstrated immunohistochemically. After MNNG treatment, class III mucin-positive pyloric glands with a low Pg 1 content in normal-looking pyloric mucosa were found from week 10; subsequently, their number increased with time. Changed mucosa was found from week 20, and the area of cells of the pyloric gland cell type with little or no Pg 1 in changed mucosa was about 30% of the area of cells of the pyloric gland cell type. Adenomatous hyperplasias were found from week 30; adenocarcinomas were found from week 50. Almost all cells of the pyloric gland cell type (greater than 95%) in areas of adenomatous hyperplasia and adenocarcinomas had little or no Pg 1 content. The present results suggested that the appearance of pyloric glands with a low Pg 1 content in normal-looking mucosa might be an immunohistochemically detectable preneoplastic change preceding morphologically detectable preneoplastic changes in stomach carcinogenesis.

Enhancing effect of cysteamine hydrochloride on the development of gastroduodenal tumors induced by N-methyl-N'-nitro-N-nitrosoguanidine in F344 rats.

The effect of the duodenal ulceration induced by cysteamine hydrochloride on the development of gastroduodenal tumors initiated by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was studied in F344 rats of both sexes. Cysteamine (200 mg/kg body wt.) was administered by gastric intubation at various times, before, during or after a 16 week period of MNNG (100 mg/liter in drinking water) treatment. In the preliminary experiment, while the ulcers induced were confined to the proximal duodenum, the pyloric region of the stomach also showed slight erosion. Five of 25 male rats given cysteamine 2 weeks before the start of MNNG treatment developed adenocarcinoma in the duodenum as compared to 1 case in the MNNG alone group. In addition, animals of both sexes which received cysteamine during MNNG treatment yielded significantly increased incidences of adenocarcinoma in the pyloric area of the stomach. In line with earlier reports, the present findings suggest that mucosal damage and subsequent regeneration or proliferation of mucosa are important co-factors for MNNG-induced gastroduodenal carcinogenesis in rats.

Sequential histologic changes during gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine in susceptible ACI and resistant BUF rats.

Sequential histologic changes of the stomach during carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG; CAS: 70-25-7) were studied in susceptible ACI and resistant BUF strain rats. Rats were given MNNG at a concentration of 83 micrograms/ml in their drinking water for 32 weeks and then tap water and were sacrificed sequentially between weeks 1 and 57. In ACI rats, erosions, regenerative changes, focal and slightly atypical changes, and diffuse and severe atypical changes were observed sequentially in the pyloric region during the period of MNNG administration, where adenocarcinomas were observed after the cessation of MNNG treatment. In BUF rats, the main histologic changes induced by MNNG were erosions and hyperplasia of the glandular portion of pyloric glands at the margin of erosions. After the cessation of MNNG treatment, the hyperplasia of the pyloric glands subsided and was followed by atrophy of these glands. The results suggested that the responses of the gastric mucosa to MNNG in ACI and BUF rats were qualitatively different.