Comutagenic effects exerted by N-nitroso compounds.

Mutagenesis induced by dimethylnitrosamine (DMN) and N-methyl-N-nitrosourea (NMU) in Salmonella typhimurium TA100 and TA1530 is characterized by biphasic dose and time response curves. At low doses or short incubation times mutagenic response is minimal, but increases rapidly when an apparent threshold dose or threshold incubation time is exceeded. Bacteria pretreated with subthreshold doses of DMN or NMU were many times more sensitive to the mutagenic effects of methylating and ethylating N-nitroso compounds than were untreated bacteria. The growth phase of the bacteria had little effect on the percentage enhancement of mutagenesis caused by pretreatment with NMU although exponentially growing cells were more sensitive to mutagenesis induced by NMU or diethylnitrosamine. Mutagenesis induced by methylmethanesulfonate and N-propyl-N'-nitro-N-nitrosoguanidine was not significantly enhanced by pretreatment of bacteria with NMU or NEU suggesting that the former mutagens act by different mechanisms than NMU or NEU.     

The effects of methyl- and ethylnitrosourethan, diethylsulfate and methylmethanesulfonate on two loci of Neurospora as well as a mutant of Saccharomyces

Summary Reverse mutation experiments were performed with the site ad_6-45 in Saccharomyces cerevisiae and two gene loci of Neurospora crassa ad (38701) and inos (37401). The mutant in yeast could be reverted with nitroso-methyl- (NMU) and nitroso-ethylurethane (NEU) but was refractory to methyl methanesulfonate (MMS) and diethylsulfate (DES). In Neurospora NMU reverted both requirements, DES only the adenine requirement, NEU reverted neither one. Thus the mutagenic specificity of an agent depends on the structure of the entire molecule, transport-form, rather than on its ability to methylate or ethylate.     

Effect of nitrosourea in small doses on the frequency of mutation in Salmonella typhimurium

The effect of N-nitroso-N-methylurea (NMU), N-nitroso-N,N'-dimethylurea (NDMU) and N-nitroso-N-ethylurea (NEU) at doses less than 100 mkg/ml on mutability of Salmonella typhimurium strains of Ames' system (G-46, TA-1950, TA-1535, TA-100, TA-1538) has been studied. NMU and NEU at doses of 5-10 mkg/ml have been found to increase the survival and decrease the number of reversions from auxotrophity in histidine to prototrophity. The effect of given doses of NMU and NEU on bacteria repair activity has been shown. The role of pk M101 plasmide in this process is being discussed. NDMU in contrast to NMU and NEU induces read frome shift mutations and exhibits high mutagenous activity at all doses examined.     

Relation of double-stranded DNA breaks and their repair to cell death and recovery after methylnitrosourea exposure

The yield of DNA double-strand breaks (DSB) in the cells of mouse lymphosarcoma treated with nitrosomethylurea (NMU) was registered by means of elastoviscosimetry. It was shown that after short-term (7 min) treatment with NMU the lesions formed in DNA are efficiently repaired both in complete and conditioned media. After long-term (30 min) treatment DNA was only repaired in complete growth medium. The yield of the first fixed DSB after long-term NMU treatment correlated with the mean lethal dose D0. After short-term NMU treatment the first DSBs are registered in the dose range which is 4-fold higher than D0. The nature of lethal and potentially lethal lesions as well as the participation of various repair systems in the elimination of potentially lethal lesions are discussed.     

A comparison of mutagenic effectiveness and efficiency of NMU and MNG in Sorghum

Summary A comparative study of the effectiveness and efficiency of NMU and MNG in relation to the effects in M1 plants and induction of mutations in M2 was made in a cultivated variety ofSorghum. There was a decrease in the values obtained in each of the biological criteria in the M1, namely germination, survival, seedling growth and seed fertility, with an increase in the concentration of NMU and MNG. Of the characters, survival following MNG treatments and seed fertility following NMU treatments showed the maximum reduction. NMU was not only effective in decreasing the mean of various characters in the M1, but also efficient in inducing a high frequency and wide spectrum of chlorophyll mutations in the M2 compared with MNG. Low concentrations were found to be more efficient than higher concentrations of NMU. The treated M2 population showed not only a decrease but also an increase in the mean height of plant and length of ear compared with the untreated control population. NMU caused a greater decrease in the mean of both characters and induced greater variability in the length of ear than did MNG treatment.     

Increased removal of 3-alkyladenine reduces the frequencies of hprt mutations induced by methyl- and ethylmethanesulfonate in Chinese hamster fibroblast cells.

We have previously reported the isolation of mammalian cell lines expressing the 3-methyladenine DNA glycosylase I (tag) gene from E. coli. These cells are 2-5 fold more resistant to the toxic effects of methylating agents than normal cells (15). Kinetic measurements of 3-methyladenine removal from the genome in situ show a moderate (3-fold) increase in Tag expressing cells relative to normal as compared to a high (50-fold) increase in exogenous alkylated DNA in vitro by cell extracts. Excision of 7-methylguanine is as expected, unaffected by the tag+ gene expression. The frequency of mutations formed in the hypoxanthine phosphoribosyl transferase (hprt) locus was investigated after methylmethanesulfonate (MMS), ethylmethanesulfonate (EMS), N-nitroso-N-methylurea (NMU) and N-nitroso-N-ethylurea (NEU) exposure. Tag expression reduced the frequency of MMS and EMS induced mutations to about half the normal rate, whereas the mutation frequency in cells exposed to NMU or NEU is not affected by the tag+ gene expression. These results indicate that after exposure to compounds which produce predominantly N-alkylations in DNA, a substantial proportion of the mutations induced is formed at 3-alkyladenine residues in DNA.     

Distribution and localisation of induced breaks in the chromosomes of Bellevalia. 3. Sodium nitrite

Summary Nitrous acid induces fragmentation of chromosomes and only very few translocations in Bellevalia romana (2n=8). A total of 1098 breaks is induced by 1×10^-3, 1,5×10^-4 and 1,5×10^-5 M NaNO₂ at pH 4,5. Root tips were fixed at 24, 48 and 72 hours after treatment for 1 hour.The percentage of breaks increases with time between treatment and fixation; it reaches its highest level at 1,5×10^-4 M, which dose coincides with the lowest mitotic rate.A dose influence on the distribution of breaks among the centromeres and the chromosome arms was observed. Consistent with the effect of other agents the highest number of breaks was in the A- the lowest, however, in the D-chromosome.The distribution of breaks on the different chromosome arms was homogeneous for all fixations. As observed previously the breakage frequency was not proportional to the arm length.The distribution of breaks on the proximal, median and distal sections of the chromosome arms is significantly different from random distribution.The nitrous acid-induced pattern of breaks is significantly different from that induced by methylating (MMS+NMU) and ethylating agents (DES+NEU).The known primary chemical reaction of nitrous acid and alkylating agents with DNA are inadequate alone in explaining the different patterns of breaks. More information is needed regarding the processes following these primary reactions.In conclusion, several possible objections against the technique of localizing breaks are shown to be non-valid.     

Effect of pH and temperature on nitrosamide-induced mutation in Escherichia coli

N-Nitroso-N-methylurea (NMU) and N-nitroso-N-ethylurea (NEU) induced reversions in four mutant auxotropic strains of E. coli. Among other nitroso compounds tested only N-methyl-N′-nitro-N-nitrosoguanidine (MNG) was an active mutagen in the system used.     

Mutagen specificity in the induction of karyotic versus cytoplasmic respiratory deficient mutants in yeast by nitrous acid and alkylating nitrosamides

Summary In the haploid eukaryotic organism Saccharomyces cerevisiae the induction of cytoplasmic and genic (karyotic) RD mutants was studied, using nitrous acid, nitrosomethylurethane (NMU) and nitrosoimidazolidone (NIL).The cytoplasmic or genic origin of the induced RD mutants was determined by prescreening in complementation tests with ^– and wild type tester strains. Among the mutants of all three agents we could thus score the incidence of three RD mutant types: genic, suppressive and cytoplasmic (both primary and secondary). The final identification of the cytoplasmic type was only possible through tetrad analysis, performed in the cases of HNO₂ and NMU.A distinct difference in cytoplasmic versus genic mutagen specificity was observed between HNO₂ and NMU. HNO₂ was unable to induce cytoplasmic RD mutants but it proved to be highly efficient in the induction of genic RD mutants. In contrast, NMU induced more cytoplasmic effects was it possible to detect mutagenic specificities which, solely on the basis of karyotic action, were not detectable.     

The multiple-cluster mutation complex in mutagenesis with higher plants

Summary After treatment of dry and pre-soaked seeds of barley with gamma-rays, EMS, NEU and EI, the frequency of multiple mutations (multimutations) was higher with EMS and NEU treatment, while cluster mutations appeared in greater numbers following treatment with gamma rays and NEU. Pre-soaking the seeds led to a reduction in the frequency of total mutations, cluster mutations and multimutations. This has been explained as a result of the application of lower doses and the induction of mutations at a relatively later stage in ontogenetic development in the case of pre-soaked seeds.Some new mutation types in barley have been described and some of the old types have been given names representing the mutation characters more precisely.The compound mutation frequency of different seedling mutation types, when taken separately, was found to be independent of the mutagen employed and the stage of treatment. The size of mutated chimeras in M ₁ plants, as indicated by the segregation ratio of mutants in M ₂, was largest in albina, xantha, chlorina, albina-tigrina, chl-terminalis and eceriferum, and lowest in viridis, viridoalbina etc. This could be expected if the unstable premutations induced by mutagenic treatment are resolved into mutations at different intervals after their initiation, or it can be explained by the induction of dominant mutations, or lethal changes together with visible mutations.     

Synthesis and evaluation of novel lipidated neuromedin U analogs with increased stability and effects on food intake

Neuromedin U (NMU) is a 25 amino acid peptide expressed and secreted in the brain and gastrointestinal tract. Data have shown that peripheral administration of human NMU decreases food intake and body weight and improves glucose tolerance in mice, suggesting that NMU receptors constitute a possible anti‐diabetic and anti‐obesity drug target. However, the clinical use of native NMU is hampered by a poor pharmacokinetic profile. In the current study, we report in vitro and in vivo data from a series of novel lipidated NMU analogs. In vitro plasma stability studies of native NMU were performed to investigate the proteolytic stability and cleavage sites using LC–MS. Native NMU was found to be rapidly cleaved at the C‐terminus between Arg²⁴ and Asn²⁵, followed by cleavage between Arg¹⁶ and Gly¹⁷. Lipidated NMU analogs were generated using solid‐phase peptide synthesis, and in vitro potency was investigated using a human embryonic kidney 293‐based inositol phosphate accumulation assay. All lipidated analogs had preserved in vitro activity on both NMU receptors with potency improving as the lipidation site was moved away from the receptor‐interacting C‐terminal octapeptide segment. In vivo efficacy was assessed in lean mice as reduction in food intake after acute subcutaneous administration of 1, 0.3, 0.1, and 0.03 µmol/kg. These lipidated NMU analogs prolonged the anorectic effect of NMU in a dose‐dependent manner. This was likely an effect of improved pharmacokinetic properties because of improved vitro plasma stability. Accordingly, the data demonstrate that lipidated NMU analogs may represent drug candidates for the treatment of obesity. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Modifying effect of dioxane on the cytogenetic activity of N-nitroso-N-methylurea

Dry seeds of Crepis capillaris L. were treated with N-nitroso-N-methyl urea (NMU) and with dioxane (DO) an organic solvent, at pH 7.0 and pH 5.7. The treatment with NMU only was used as a positive control. Three concentrations of NMU were applied. The cytogenetic activity of NMU was found to considerably decrease at two values of pH, while NMU was solved in DO. The relationship between different types of chromosome aberrations remained unchanged in this case.     

Development of a neuromedin U–human serum albumin conjugate as a long‐acting candidate for the treatment of obesity and diabetes. Comparison with the PEGylated peptide

Neuromedin U (NMU) is an endogenous peptide implicated in the regulation of feeding, energy homeostasis, and glycemic control, which is being considered for the therapy of obesity and diabetes. A key liability of NMU as a therapeutic is its very short half‐life in vivo. We show here that conjugation of NMU to human serum albumin (HSA) yields a compound with long circulatory half‐life, which maintains full potency at both the peripheral and central NMU receptors. Initial attempts to conjugate NMU via the prevalent strategy of reacting a maleimide derivative of the peptide with the free thiol of Cys34 of HSA met with limited success, because the resulting conjugate was unstable in vivo. Use of a haloacetyl derivative of the peptide led instead to the formation of a metabolically stable conjugate. HSA–NMU displayed long‐lasting, potent anorectic, and glucose‐normalizing activity. When compared side by side with a previously described PEG conjugate, HSA–NMU proved superior on a molar basis. Collectively, our results reinforce the notion that NMU‐based therapeutics are promising candidates for the treatment of obesity and diabetes. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Changes in frequencies of variegated leaves in NMU treated tobacco

Summary Seeds of Nicotiana tabacum were allowed to imbibe water for 1 h and were then treated with 5–20 mM N-nitroso-N-methylurea (NMU) for 1 h. Seedlings were planted out separately and leaves no. 1–6 were scored individually after expansion was complete: frequencies of leaves with mutant sectors and the percentage of leaf area that was mutant were determined for the numbered leaves. Treatment with 5 mM NMU gave few mutant plants but after 10–20 mM NMU 50–98% of plants were mutant. The frequency of mutant leaves increased from leaf no. 1 to leaves no. 3–4; as much as a 5.6-fold increase, from leaf no. 1 to no. 3 was found. There appears to be differential sensitivity to NMU: it is lower in the initial cells for leaf no. 1 than in the initials for leaves no. 3 or 4. Leaves no. 1–4 arise from four different groups of initial cells: mutations appearing in two or more of leaves no. 1–4 must, therefore, arise independently of one another. From mutants found in leaves no. 1–4 it is estimated that the mean number of mutations per seedling was 2.68 after treatment with 20 mM NMU. Mean percentage of leaf surface area occupied by mutant sectors increased from 14% to 29.4% as NMU concentration increased from 10 to 20 mM. It also increased significantly from leaf no. 1 to leaves no. 3–6 after treatment with 15 or 20 mM NMU but not after 10 mM NMU: this suggests that mutagen treatment may affect the formation of mutant homoplasmon cells and their contribution to leaf primordia.     

Influence of ploidy on plastome mutagenesis in Nicotiana

Summary A clear influence of ploidy was observed on the frequency of both spontaneous and nitroso-methylurea (NMU) induced, streptomycin-resistant, adventitious shoots developing on leaf explants of Nicotiana tabacum and N. plumbaginifolia. At nearly all NMU levels employed a significantly higher yield of resistant shoots was obtained from haploid compared with diploid leaf strips. At 1 mM NMU the differences were not significant and were absent when a high (1000 mg/1) selective concentration of streptomycin sulphate was used. The influence of ploidy is discussed in relation to the possible effect of plastome copy number on mutagenesis and sorting out of resistant plastids.     

Central control of bone remodeling by neuromedin U

Bone remodeling, the function affected in osteoporosis, the most common of bone diseases, comprises two phases: bone formation by matrix-producing osteoblasts and bone resorption by osteoclasts. The demonstration that the anorexigenic hormone leptin inhibits bone formation through a hypothalamic relay suggests that other molecules that affect energy metabolism in the hypothalamus could also modulate bone mass. Neuromedin U (NMU) is an anorexigenic neuropeptide that acts independently of leptin through poorly defined mechanisms. Here we show that Nmu-deficient (Nmu-/-) mice have high bone mass owing to an increase in bone formation; this is more prominent in male mice than female mice. Physiological and cell-based assays indicate that NMU acts in the central nervous system, rather than directly on bone cells, to regulate bone remodeling. Notably, leptin- or sympathetic nervous system-mediated inhibition of bone formation was abolished in Nmu-/- mice, which show an altered bone expression of molecular clock genes (mediators of the inhibition of bone formation by leptin). Moreover, treatment of wild-type mice with a natural agonist for the NMU receptor decreased bone mass. Collectively, these results suggest that NMU may be the first central mediator of leptin-dependent regulation of bone mass identified to date. Given the existence of inhibitors and activators of NMU action, our results may influence the treatment of diseases involving low bone mass, such as osteoporosis.     

A Feulgen microspectrophotometric study of the DNA content of essential fatty acid-deficient rat pancreas treated with nitrosomethylurea

Microspectrophotometric DNA measurements in exocrine pancreas of essential fatty acid-deficient (EFAD) and EFA-sufficient (EFAS) rats which received a single intraperitoneal injection of the carcinogen nitrosomethylurea (NMU) or saline (SAL) was the subject of the present report. The DNA content of acinar pancreatic cells of SAL-injected EFAD and EFAS rats was diploid. NMU-induced pancreatic focal acinar cell hyperplasia (FACH) had one main cell population with a diploid content, whereas in the intervening parenchyma there were diploid and tetraploid cells. The number of tetraploid cells was smaller in EFAD rat pancreas than in EFAS indicating a diet dependent effect. NMU-induced FACH had a diploid distribution pattern indicating that cells are in a G1, quiescent phase, contrasting with AZA-induced similar lesions which showed an abnormal ploidy. It remains to be established whether DNA phenotypic traits of NMU and AZA induced FACH reflect the neoplastic potentials of both types of lesions. The decreased number of tetraploid cells in EFAD rat pancreas is in keeping with data indicating a promoting effect of the EFA linoleic and arachidonic acids on growth rate of certain cell populations in vitro.     

Quasi-Adaptive Response to Alkylating Agents in <Emphasis Type="Italic">Escherichia coli</Emphasis>: A New Phenomenon

An original hypothesis of a quasi-adaptive response to nitrosomethylurea (NMU) in Escherichia coli cells was verified experimentally. In contrast to the true Ada response, which is induced in cells pretreated with a sublethal dose of NMU, a quasi-adaptive response was induced using NO-containing dinitrosyl iron complex with glutathione (DNIC_glu). Quasi-adaptation increased expression of the Ada regulon and cell resistance to the cytotoxic and mutagenic effects of NMU. The levels of alkA, alkB, and aidB gene expression in quasi-adaptation were higher than in the true Ada response. Thus, experimental evidence was obtained for the alternative mechanism regulating the function of the Ada sensory protein in controlling expression of the Ada regulon during the adaptive response. The free iron—chelating agent o-phenanthroline (OP) facilitated degradation of DNIC_glu (by electron paramagnetic resonance (EPR) spectra) and considerably or completely inhibited gene expression in the quasi-adaptive response. The new phenomenon extends the functional range of NO compounds to include a role in genetic signal transduction within the Ada response system in addition to similar roles in the SoxRS, SOS, and OxyR systems in E. coli.__________Translated from Genetika, Vol. 41, No. 5, 2005, pp. 607–613.Original Russian Text Copyright © 2005 by Vasilieva, Moschkovskaya.     

Mutagenesis in cultured mammalian cells. II. Induction of gene mutations in Chinese hamster cells

Mutations controlling the resistance to 6-mercaptopurine (6-M) and the ability to multiply in a medium with a low concentration of glucose (“glucose-independent” mutants) were induced in cultured Chinese hamster cells by N-nitrosomethylurea (NMU), 5-bromodeoxyuridine (BUdR), UV and X-rays. The chemical agents were found to be very active in induction of mutations to 6-M resistance (NMU and BUdR) and mutations of “glucose independence” (NMU). These agents increase the yield of mutations as compared to the spontaneous mutation rate by about two orders of magnitude. The induced rate of 6-M-resistant mutations by X-rays was 2.0 ? 10⁻⁷ per viable cell per roentgen. BUdR approximately equally increases the cell's sensitivity to both inactivating and mutagenic action of X-rays. The maximum induction of mutations to 6-M resistance by UV was observed at 100 erg/mm². This dose leads to 1 16-fold increase of the mutation frequency as compared to the spontaneous rate. Further increase of the UV dose up to 200 erg/mm² resulted in a lower yield of mutations per dose unit. The highest yield of mutations to 6-M resistance induced by NMU, BUdR and X-rays was observed if cells were plated in selective medium several generations after the mutagenic treatment. The maximum yield of mutations to 6-M resistance induced by UV and of glucose-independence induced by NMU was recorded if cells were transferred to selective media immediately after treatment. The kinetics of expression of mutations and the decline of their number observed after prolonged incubation of treated cells in nonselective conditions are discussed.     

Benzoic acid induces tolerance to biotic stress caused by Phytophthora cinnamomi in Banksia attenuata

Banksia attenuata plants were treated with soil drenches or foliar sprays of benzoic acid (BZA) to determine induced resistance to Phytophthora cinnamomi. Stems of B. attenuata were inoculated with the pathogen 1 week after treatment with BZA. Resistance was estimated by measuring P. cinnamomi lesions on stems. Treatment with 0.10 mM, 0.25 mM or 0.50 mM BZA caused a reduction in lesion size with 0.50 mM BZA applied as a soil drench being the most effective treatment at suppressing the development of lesions. This is the first report of BZA induced host resistance in any plant species to any pathogen.