A Lipidomic Study of the Effects of N-methyl-N′-nitro-N-nitrosoguanidine on Sphingomyelin Metabolism

Systems biology is a new and rapidly developing research area in which,by quantitativelydescribing the interaction among all the individual components of a cell,a systems-level understanding of abiological response can be achieved.Therefore,it requires high-throughput measurement technologies forbiological molecules,such as genomic and proteomic approaches for DNA/RNA and protein,respectively.Recently,a new concept,lipidomics,which utilizes the mass spectrometry(MS)method for lipid analysis,has been proposed.Using this lipidomic approach,the effects of N-methyl-N'-nitro-N-nitrosoguanidine(MNNG)on sphingomyelin metabolism,a major class of sphingolipids,were evaluated.Sphingomyelin moleculeswere extracted from cells and analyzed by matrix-assisted laser desorption ionization-time of flight MS.Itwas found that MNNG induced profound changes in sphingomyelin metabolism,including the appearance ofsome new sphingomyelin species and the disappearance of some others,and the concentrations of severalsphmgomyelin species also changed.This was accompanied by the redistribution of acid sphingomyelinase(ASM),a key player in sphingomyelin metabolism.On the other hand,imipramine,an inhibitor of ASM,caused the accumulation of sphingomyelin.It also prevented some of the effects of MNNG,as well as theredistribution of ASM.Taken together,these data suggested that the lipidomic approach is highly effectivefor the systematic analysis of cellular lipids metabolism.     

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.     

Temperature Dependency of the Mutagenic Action of N-Methyl-N′-nitro-N-nitrosoguanidine in Streptomyces cacaoi

The effectiveness of N-methyl-N′-nitro-N-nitrosoguanidine (NG) in inducing mutations in Streptomyces cacaoi was demonstrated with two systems: reversion to prototrophy of an isoleucine-requiring mutant and the induction of forward mutation of a prototrophic strain. An optimal condition for yielding a high frequency mutation was set as follows: Treatment of spores with 1 mg/ml of NG in 0.016 m phosphate buffer, pH 6, at 42°C for 60 min. In Streptmyces cacaoi, the mutagenesis depended highly on the temperature of NG treatment; elevation of the treatment temperature resulted higher frequency of mutation. Only a little dependency on temperature was shown in Escherichia coli, but the dependency was also observed when the Streptomyces was treated with ethyl methanesulfonate. The significance of these results for considering mechanism of NG action was discussed.     

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.     

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.     

A Lipidomic Study of the Effects of N-methyl-N＇-nitro-N-nitrosoguanidine on Sphingomyelin Metabolism

Systems biology is a new and rapidly developing research area in which, by quantitatively describing the interaction among all the individual components of a cell, a systems-level understanding of a biological response can be achieved. Therefore, it requires high-throughput measurement technologies for biological molecules, such as genomic and proteomic approaches for DNA/RNA and protein, respectively.Recently, a new concept, lipidomics, which utilizes the mass spectrometry （MS） method for lipid analysis,has been proposed. Using this lipidomic approach, the effects of N-methyl-N＇-nitro-N-nitrosoguanidine （MNNG） on sphingomyelin metabolism, a major class of sphingolipids, were evaluated. Sphingomyelin molecules were extracted from cells and analyzed by matrix-assisted laser desorption ionization-time of flight MS. It was found that MNNG induced profound changes in sphingomyelin metabolism, including the appearance of some new sphingomyelin species and the disappearance of some others, and the concentrations of several sphingomyelin species also changed. This was accompanied by the redistribution of acid sphingomyelinase （ASM）, a key player in sphingomyelin metabolism. On the other hand, imipramine, an inhibitor of ASM,caused the accumulation of sphingomyelin. It also prevented some of the effects of MNNG, as well as the redistribution of ASM. Taken together, these data suggested that the lipidomic approach is highly effective for the systematic analysis of cellular lipids metabolism.     

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.     

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.     

Response of human keratinocytes to extremely low concentrations of N-methyl-N′-nitro-N-nitrosoguanidine

Since alkylating agents are widely present in the environment and constitute a continuous challenge to genome integrity, cells and organisms have developed defense mechanisms to remove such lesions. We monitored the response of human keratinocytes to a very low concentration of a methylating agent, namely 2.5 nM N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). The effect of a 60-min exposure of quiescent cells to 2.5 nM MNNG was studied in terms of DNA integrity, poly(ADP-ribose) metabolism, clonogenic survival and DNA synthesis. We observed two waves of DNA strand break formation and resealing. Interestingly, the amount of DNA strand breaks in exposed cells was lower than in unexposed control cells. This phenomenon was also observed when cells were exposed to MNNG in the presence of a protein synthesis inhibitor, or when they were maintained on ice during the treatment. A dose of 2.5 nM MNNG stimulated poly(ADP-ribose) turnover, reduced the intracellular NAD⁺ content, stimulated DNA synthesis and caused a remarkable increase in clonogenic survival. Thus, the cellular responses to extremely low concentrations of MNNG differ sharply from those observed at higher doses of this carcinogen. We conclude that the very low dose response cannot be extrapolated from usual dose-response analyses.     

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.     

Mutagenesis by ethidium bromide and N-methyl-N′-nitro-N-nitrosoguanidine in off-flavour compound producing strains ofStreptomyces

Various species of actinomycetes and cyanobacteria can impart earthy/musty off-flavours to drinking water supplies and to pond-raised fish and other aquatic food animals. The genetic determinants for production of the most common off-flavour compounds [geosmin and 2-methylisoborneol (MIB)] have not been extensively studied. An attempt has been rrlade to study the genetics of production of these compounds was demonstrated by DNA-curing analysis. The effects of two curing agents [ethidium bromide (EB) and N-methyl-N′-nitro-N-nitrosoguanidine (NTG)] on tha loss of linear plasmid DNA and generation of bald mutants (no aerial mycelia) inStreptomyces halstedii andStereptomyces violaceusniger which produce geosmin and MIB, respectively, were observed. Production of earthy/musty odour was not eliminated, but was reduced by 55–95% in the plasmid cured strain. Data suggested that off-flavour production is likely chromosomally-encoded in theseStreptomyces isolates.     

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.     

Studies of the effects of liquid holding on viability and mutation frequency in N-methyl-N′-nitro-N-nitrosoguanidine-treated halophiles

The capacities ofHalobacterium cutirubrum and a moderate halophile NRC 41227 to survive and recover from treatment with N-methyl-N-nitro-N-nitrosoguanidine have been compared.Halobacterium cutirubrum is resistant to this chemical and its mutation frequency is only slightly affected, whereas NRC 41227 is highly sensitive and its mutation frequency is markedly increased. The chemically treated extreme halophile fully regains viability during liquid holding, in notable contrast to its known failure to recover from the effects of ultraviolet irradiation.     

Methylation of deoxyribonucleic acid in cultured mammalian cells by N-methyl-N′-nitro-N-nitrosoguanidine. The influence of cellular thiol concentrations on the extent of methylation and the 6-oxygen atom of guanine as a site of methylation

1. In neutral aqueous solution N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) yields salts of nitrocyanamide as u.v.-absorbing products. With cysteine, as found independently by Schulz & McCalla (1969), the principal product is 2-nitràminothiazoline-4-carboxylic acid. Both these reactions liberate the methylating species; thiols enhance the rate markedly at neutral pH values. An alternative reaction with thiols gives cystine, presumably via the unstable S-nitrosocysteine. 2. Thiols (glutathione or N-acetylcysteine) in vitro at about the concentration found in mammalian cells enhance the rate of methylation of DNA markedly over that in neutral solution. 3. Treatment of cultured mammalian cells with MNNG results in rapid methylation of nucleic acids, the extent being greater the higher the thiol content of the cells. Rodent embryo cells are more extensively methylated than mouse L-cells of the same thiol content. Cellular thiol concentrations are decreased by MNNG. Proteins are less methylated by MNNG than are nucleic acids. 4. Methylation of cells by dimethyl sulphate does not depend on cellular thiol content and protein is not less methylated than nucleic acids. Methylation by MNNG may therefore be thiol-stimulated in cells. 5. Both in vitro and in cells about 7% of the methylation of DNA by MNNG occurs at the 6-oxygen atom of guanine. The major products 7-methylguanine and 3-methyladenine are given by both MNNG and dimethyl sulphate, but dimethyl sulphate does not yield O(6)-methylguanine. Possible reaction mechanisms to account for this difference between these methylating agents and its possible significance as a determinant of their biological effects 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.     

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.