Lethality and mutagenicity inHalobacterium mediterranei caused by N-methyl-N′-nitro-N-nitrosoguanidine

Compared withEscherichia coli, Halobacterium mediterranei was highly resistant to the lethal effect of N-methyl-N-nitro-N-nitrosoguanidine (nitrosoguanidine), but it was sensitive to the mutagenic action of this chemical agent. Nitrosoguanidine at 500 g ml^–1 gave a cell survival level between 1% and 10%, and this allowed us to obtain more Josamycin-resistant mutants compared with lower concentrations, which gave higher survival rates but fewer mutants. The efficiency of the mutagenicity obtained with the nitrosoguanidine treatment was examined under a variety of conditions. The optimal conditions for obtaining Josamycinresistant mutants were achieved by exposing, in darkness and without shaking, a suspension of about 10⁸ log-phase cells to 500 g nitrosoguanidine in 1 ml of 50 mM modified saline Tris-maleate buffer at pH 7.5, or in 1 ml of 5 mM modified saline Tris-citrate-maleate for 30 min at 37°C.     

Physiological and mutagenic effects of N-methyl-N′-nitro-N-nitrosoguanidine in populations of chlorococcal algae

The suitability was evaluated of MNNG as a mutagen inducing increased frequencies of mutations in the cell populations of three strains of chlorococcal algae for the purposes of selection. MNNG has proved to be highly toxic to those algae as it produces severe physiological responses of the affected cells. The mutagenic effect of MNNG was relatively small in comparison with the recorded toxic effect. From these results it has been concluded that in reverse to NEU, MNNG can hardly be applied with such good an effect in the mutation breeding of chlorococcal algae that are suitable for mass cultivation.     

Evidence that N-methyl-N′-nitro-N-nitrosoguanidine induces adaptive response in Bacillus thuringiensis

Bacillus thuringiensis is shown to have an inducible error-free repair system for alkylation damage as found in Escherichia coli and Bacillus subtilis. Growth of cells in the presence of low concentrations of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) induces an adaptive response which is characterized by an increase in resistance to killing and mutagenesis by challenge with higher concentrations of MNNG. In addition, we have noted with interest that adaptive low doses seem to produce lesions at a rate sufficient to induce an increase of mutation frequency, and inhibition of cell division. The possibility of an interaction between SOS and adaptive responses with these low doses of MNNG is discussed.     

Study of the methylation and lack of deamination of deoxyribonucleic acid by N-methyl-N′-nitro-N-nitrosoguanidine

1. DNA labelled with (14)C in the purine residues was prepared by treating newborn rats with [(14)C]formate and killing them for preparation of nucleic acids at 11-17 months. This DNA was incubated with N-methyl-N'-nitro-N-nitrosoguanidine, and then analysed for products of methylation and deamination reactions. 2. Evidence was found for the formation of 7-methylguanine and a smaller amount of 3-methyladenine, and, after preliminary denaturation of the DNA, 1-methyladenine was detected. The presence of cysteine increased the extent of methylation. No evidence was found for the formation of xanthine or hypoxanthine, even at pH5.5.     

Effect of adenosine 3′,5′ monophosphate on the mutation frequency induced by N-methyl-N′-nitro-N-nitrosoguanidine

The effect of increased cellular concentrations of adenosine 3′,5′ monophosphate (cAMP) upon mutation frequency induced by N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) was studied in V79 Chinese hamster lung cells. Incubation with either forskolin, which increased the accumulation of cAMP, or 8BrcAMP, an analogue of cAMP, resulted in an increase in the mutation frequency which was concentration-dependent, regardless of whether these agents were added before or after mutagen treatment. Increased cAMP concentrations were shown in these cells to inhibit growth; however, this does not seem to be the mechanism responsible for the increase in mutation frequency as low serum concentrations which also retard growth reduced the mutation frequency observed with MNNG.     

Neoplastic transformation and induction of H+,K+-adenosine triphosphatase by N-methyl-N′-nitro-N-nitrosoguanidine in the gastric epithelial RGM-1 cell line

N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) induces gastric cancer in animal models. We established an MNNG-induced mutant of the rat murine RGM-1 gastric epithelial cell line, which we named RGK-1, that could be used as an in vitro model of gastric cancer. This cell line showed signs of neoplasia and transformation, in that it lost contact inhibition and formed tumors in nude mice. The mutant cells also expressed parietal cell-specific H⁺,K⁺-adenosine triphosphatase (H⁺,K⁺-ATPase), which parent RGM-1 did not. The results suggested that parent RGM-1 cells were gastric progenitor cells. This mutant RGK-1 cell line will contribute to future investigation on gastric carcinogenesis and to the development of other pathophysiologic fields.     

Formation and persistence of N7-methylguanine DNA adducts in the target pyloric tissue following chronic exposure to N-methyl-N′-nitro-N-nitrosoguanidine

Outbred 7-week old male Wistar rats were exposed for 21 days to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) via the drinking water and N7-methyl deoxyguanosine 3'-monophosphate (N7-MedGp) levels in DNA from the pyloric mucosa (target tissue) and white blood cells (wbc: non-target tissue) were determined by ³²P-postlabelling. Exposure to MNNG resulted in the non-linear, dose-related formation of N7-medGp in both tissues. Adduct levels in the pyloric mucosa were determined to be 1058, 5.4 and 1.1 μmole N7-medGp mole^-1 deoxyguanosine 3'-monophosphate (dGp) after exposure to 4.1, 0.62 and 0.006 mg MNNG kg^-1 day^-1 respectively whereas adduct levels in the wbc DNA were lower at 5.2, 0.52 and 0.68 μmoles N7-medGp mole^-1 dGp after exposure to 4.1, 0.62 and 0.062 mg MNNG kg^-1 day^-1 respectively. In addition, the persistence of N7-medGp was investigated. Loss of adduct occurred rapidly, with a decrease of 87 and 97% respectively in target tissue and wbc DNA by 48 h after cessation of 4.1 mg MNNG kg^-1 day^-1 exposure; 14 days post-MNNG treatment, however, N7-medGp was still detectable (0.46 μmole N7-medGp mole^-1 dGp) in pyloric mucosal DNA. The quantitation of N7-medGp after exposure to low doses of carcinogen, i.e. 0.006 mg MNNG kg^-1 day^-1, approaching environmentally relevant levels has not been previously reported, and indicates that the ³²P-postlabelling assay developed here possesses sufficient sensitivity to quantitate N7- medGp in human DNA arising from environmental exposure to methylating agents.     

A new strain of Salmonella typhimurium reverted by mitomycin C and N-methyl-N′-nitro-N-nitrosoguanidine—a possible universal tester for mutagenic compounds

A strain of Salmonella typhimurium, SO1007, which carries the amber mutation trpD28 plus the plasmid pKM101 was reverted very efficiently by two mutagens with different mutagenic specificities and modes of action: mitomycin C (MC) and N-methyl-N′-nitro-N-nitrosoguanidine (NG). By selecting revertants on minimal agar supplemented with anthranilic acid (AA), two distinct phenotypic classes of TrpD28 revertants can be recovered: prototrophs (MM⁺) and anthranilate utilizers (AA⁺). Since each phenotypic class is known to be caused by a variety of mutational events, reversion of trpD28 on minimal-anthranilate medium may be useful for detecting mutagenic agents regardless of the types of mutations they may cause. Thus, strains like SO 1007 may be useful as ‘universal’ detectors of mutagenic compounds. In the course of these experiments we also observed that pKM101 does not protect but, on the contrary, sensitizes the host bacteria slightly to the toxic effects of MC.     

Reduced N-methyl-N′-nitro-N-nitrosoguanidine sister chromatid exchange induction in chinese hamster V79 cells pre-exposed to 5-bromodeoxyuridine

The sequence in which N-methyl-N-nitro-N-nitrosoguanidine (MNNG) and 5-bromodeoxyuridine (BrdU) are added to cell cultures affects the number of sister chromatid exchanges (SCE) induced by MNNG. When V79 Chinese hamster cell monolayer cultures were treated with MNNG for 2 h prior to addition of BrdUrd, approximately a 4–5-fold increase in SCE was observed at the second division metaphases compared to controls exposed to BrdU alone. This effect was independent of whether one or three DNA strands had been substituted as a result of incubating the cells through one or two DNA synthesis periods in the presence of BrdU. This increase in SCE also occurred after MNNG exposure and BrdU incubation was extended for three division cycles. In contrast, when BrdU incorporation preceded MNNG treatment, the average number of SCE/metaphase was reduced 70–80% at the second division cycle and 60% relative to the total number found in three division cycles. SCE induction by MNNG does not involve a caffeine sensitive step since caffeine had no effect on the SCE frequency regardless of the treatment protocol. The conditions in which BrdU preceded MNNG exposure may be responsible for either reducing the number of DNA sites available for interaction with MNNG or preventing the expression of SCE.     

Antimutagenic Effect of Amino Acids on the Mutagenicity of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG)

The antimutagenic activity of protein-constituting amino acids except histidine on the mutagenicity of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) was investigated in vitro using Salmonella typhinurium TA-100 as an indicator bacterium (Ames test), and concentrations (IC₅₀) of amino acids that inhibit 50% of the mutagenecity were measured. Cysteine was found to be most active and glycine, tryptophan, lysine, and arginine were strong antimutagenic amino acids. Other amino acids showed moderate or weak antimutagenic activities, depending on the amino acids. The results indicate that amino acids play a substantial role in chemoprevention of N-nitroso amine-induced mutagenicity.     

Further studies on the induction of mutation in Haemophilus influenzae by N-methyl-N′-nitro-N-nitrosoguanidine: Lack of an inducible error-free repair system and the effect of exposure medium

Haemophilis influenzae is shown to lack the inducible, error-free repair system for alkylation damage that others have found in Escherichia coli. Prior growth in a low concentration of N-methyl-N′-nitro-N-nitrosoguanidine had only an additive effect on a subsequent brief exposure to a high concentration. Furthermore, chloramphenicol did not significantly modify the mutagenic response. In both respects, H. influenzae differs from E. coli. Experiments carried out in preparation for these tests showed that exposure to N-methyl-N′-nitro-N-nitrosoguanidine in complex growth medium was more effective by about an order of magnitude than exposure in pH 6.0 tris-maelare buffer in inducing mutations, in killing the cells, and in causing strand breaks in the preexisting DNA and gaps in newly synthesized DNA. Thus the effect of the medium is on the amount of initial damage rather than on some special feature of the mutation process. Part but not all of the effect can be accounted for by the difference in pH of the 2 media. The nature of the mutagenic process is the same under the 2 exposure conditions; i.e., reparable pre-mutational damage is produced by the agent and subsequently converted to final mutation by replication. The dose—effect curves have a non-linear initial portion under both exposure conditions, and possible reasons for this non-linearity are discussed.     

Methylation of ribonucleic acid by the carcinogens dimethyl sulphate, N-methyl-N-nitrosourea and N-methyl-N′-nitro-N-nitrosoguanidine. Comparisons of chemical analyses at the nucleoside and base levels

1. The following methods for hydrolysis of methyl-(14)C-labelled RNA, and for chromatographic isolation and determination of the products, were investigated: enzymic digestion to nucleosides at pH6 or 8; alkaline hydrolysis and conversion into nucleosides; hydrolysis by acid to pyrimidine nucleotides and purine bases, or completely to bases; chromatography on Dowex 50 (NH(4) (+) form) at pH6 or 8.9, or on Dowex 50 (H(+) form), or on Sephadex G-10. 2. The suitability of the various methods for determination of methylation products was assessed. The principal product, 7-methylguanosine, was unstable under the conditions used for determinations of nucleosides. 3- and 7-Methyladenine and 3- and 7-methylguanine are best determined as bases; 1-methyladenine and 3-methylcytosine can be isolated as either nucleosides or bases; O(6)-methylguanine is unstable under the acid hydrolysis conditions used and can be determined as the nucleoside; 3-methyluracil was detected, but may be derived from methylation of the ionized form of uracil. 3. Differences between the patterns of methylation of RNA and homopolyribonucleotides by the N-methyl-N-nitroso compounds and dimethyl sulphate were found: the nitroso compounds were able to methylate O-6 of guanine, were relatively more reactive at N-7 of adenine and probably at N-3 of guanine, but less reactive at N-1 of adenine, N-3 of cytosine and probably at N-3 of uridine. They probably reacted more with the ribose-phosphate chain, but no products from this were identified. 4. The possible influences of these differences on biological action of the methylating agents is discussed. Nitroso compounds may differ principally in their ability to induce miscoding in the Watson-Crick sense by reaction at O-6 of guanine. Both types of agent may induce miscoding to a lesser extent through methylation at N-3 of guanine; both can methylate N atoms, presumably preventing Watson-Crick hydrogen-bonding. N-Methyl-N-nitrosourea can degrade RNA, possibly through phosphotriester formation, but this mechanism is not proven.     

Screening of high α-arbutin producing strains and production of α-arbutin by fermentation

A mutant Xanthomonas maltophilia BT-112 with high α-anomer-selective glycosylation activity was screened by a series of mutation methods including UV light, N-methyl-N-nitro-N-nitroso-guanidine treatment and quick neutron mutation. The α-arbutin titer increased 15-folds compared with the parent strain. The optimal conditions for culture medium and the operational conditions for lab-scale fermenter were investigated. Under optimized conditions, the maximal hydroquinone (HQ) tolerance of cells and yield of α-arbutin were 120 mM and 30.6 g/l, respectively. The molar conversion yield of α-arbutin based on the amount of HQ supplied reached 93.6 %. The product was identified as α-arbutin by ¹³C NMR and ¹H NMR analysis. In conclusion, the results in this work provide a one-step and cost-effective method for the large-scale production of α-arbutin.     

A new insect cell line from pupal ovary of Spodoptera exigua established by stimulation with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG)

A continuous cell line derived from the pupal ovary of Spodoptera exigua was established by treating primary cells with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Three days after treating cells with 3.0 μg/ml of MNNG, the cells formed a monolayer and were initially subcultured 60 d after the MNNG was removed, followed by subculturing for 30 passages. The established cell line, designated IOZCAS-Spex 12, consisted of a mixture of three types of cells, including spherical, spindle-shaped, and oval cells. The population doubling time of the cell line during its logarithmic growth phase was found to be 71 h. DNA amplification fingerprinting polymerase chain reaction analysis confirmed that the new cell line originated from S. exigua. Susceptibility of IOZCAS-Spex 12 cells to infection by certain nucleopolyhedroviruses was investigated. The results showed that the cell line was highly susceptible to infection by S. exigua nucleopolyhedrovirus and Autographa californica multiple nucleopolyhedrovirus, slightly susceptible to infection by Spodoptera litura nucleopolyhedrovirus, and not susceptible to infection by Helicoverpa armigera nucleopolyhedroviruses or Hyphantria cunea nucleopolyhedroviruses. The results of this study suggest that MNNG treatment may overcome existing limitations to obtaining continually proliferating cells and may open up the possibilities for immortalizing isolated insect cells.     

Cellular DNA damage by nitrosocimetidine: A comparison with N-methyl-N′-nitroso-nitrosoguanidine and X-irradiation

Permanently proliferating lymphoblastoid cell lines (LCLs) and normal unstimulated peripheral blood leukocytes have been used to study the effects of nitrosocimetidine (NC) on cultured human lymphoid cells. The approaches that were used to assess the cells' ability to cope with NC were: (i) determination of cell survival as measured by colony formation in microtiter plates; (ii) quantitation of DNA synthesis and DNA-repair replication by isopyknic sedimentation of DNA density labeled with 5-bromo-2-deoxyuridine (BrdU); (iii) measurement of the induction of alkali labile lesions and strand breaks by NC in ³H-labeled DNA using velocity sedimentation in alkaline sucrose. In summary, treatment with NC was found to inhibit both replicative DNA synthesis and colony formation in LCLs. At the molecular level, NC treatment induced alkali labile lesions in LCL DNA and elicited DNA-repair replication in proliferating LCLs as well as unstimulated lymphocytes. Considered in total, these data indicate that NC is reactive with human DNA in the cellular environment in a manner similar to methylating nitroso compounds which have been shown to be carcinogenic. The significance of these findings will be discussed.     

Use of protoplasts in the improvement of filamentous microorganisms. I. Action of N-methyl-N′-nitro-N-nitrosoguanidine on protoplasts of Streptomycetes

Summary The effects of N-methyl-N–nitro-N-nitroso-guanidine (NTG) on protoplasts of Streptomycetes are markedly different from its action on spores, showing high mutagenic activity even at concentrations having no marked effect on protoplast survival. Strain improvement, eg in chlorotetracycline-producing strains of S. aureofaciens, was most effective when protoplasts were subjected to prolonged treatment (2 h) with low concentrations of NTG (50 /ug/ml).