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.     

Mutant Bac. subtilis A2 with an enhanced capacity to induce an adaptive response

Mutant A2 with increased ability to induce adaptive response was isolated in Bac. subtilis and its properties studied. Mutant A2 was shown to be more resistant to mutagenic action of MNNG, EMS, UV light. It was also discovered that A2 was more sensitive to the lethal action of MNNG and UV light than parent strain 103. It was shown by clonal analysis of mutant colonies, formed by mutant cells A2 and 103 that A2 strain had increased ability to form complete mutants. Properties of A2 mutant suggest that in the process adaptive response induction were is expression of both adaptive response enzymes and some other which are necessary for reparation of premutagenic UV lesions.     

Comparative mutagenic effects of ethyl methane-sulfonate, n-methyl-n'-nitro-n-nitrosoguanidine, ultraviolet radiation and caffeine on Dictyostelium discoideum.

A high frequency of morphogenetic mutants of Dictyostelium discoideum can be induced by treatment with MNNG under conditions which result in relatively low cell killing. Six temperature-sensitive growth mutants induced by this treatment were isolated by replica plating. Among these, five showed spontaneous reversion rates of 10(-4) to 10(-5). The mutagenic activity of ems, measured for the induction of both morphogenetic and temperature-sensitive mutants, was weaker than that of MNNG and UV radiation. High frequencies of morphogenetic mutants were obtained only with doses of UV irradiation that resulted in high killing of cells or spores. Caffeine, at concentrations that slightly decreased the growth rate of amoebae in axenic medium, induced morphogenetic defects and also enhanced the mutagenic effect of UV irradiation. However, all the aggregateless clones derived from caffeine treatment that were studied reverted to the wild-type phenotype after a variable number of clonal re-isolations.     

UV non-mutable mutant from V-79 Chinese hamster cells

Summary To get an idea about the response of a living system, exposed to gradually increasing doses of a mutagen for several generations, a population of V-79 Chinese hamster cells was exposed repeatedly to gradually increasing doses of UV radiation. Each dose was followed by a variable period of growth for at least ten generations. After treatment the cells were not mutable by UV radiation, though MNNG was capable of producing mutations with the same efficiency as in the untreated cells. In terms of viability, the treated cells behaved exactly as the untreated ones for both UV and MNNG. The observed behaviour of the treated cells was found to be stable for during the 50 passages studied.Abbreviations DMSO dimethylsulphoxide - Aza 8-Azaguanine - MEM minimal essential medium - PBS phosphate buffered saline - MNNG N-methyl-N-nitro-N-nitrosoguanidine     

Rhodosporidium Banno: dose-effect relations, mutagenic efficiency, and spectrum of mutants in the induction of auxotrophic mutants by ultraviolet light and N-methyl-N'-nitro-N-nitrosoguanidine

The kinetics, efficiency, and specificity of induction of forward mutations to auxotrophy by ultraviolet light (UV) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was examined in stationary phase cells of Rhodosporidium (Rhodotorula) wild strain Rg1. In comparison to the spontaneous level the frequency of auxotrophic mutants was increased more than 1000 times by both mutagens, however, the mutagenic efficiency of MNNG was higher than that of UV. We found that the forward mutation rate is a linear function of the applicated UV and MNNG doses in the range to 600 J m-2 or 25 mM X min, respectively. The 35 studied biosynthetic pathways to amino acids, purines, pyrimidines, and vitamins are genetically blocked at different frequencies, but there is not any significant difference between UV and MNNG induced frequencies of mutants with a specific requirement. However, in difference to the approximately equal distribution of the MNNG-induced nic mutants among the genetic blocks of the tryptophan-nicotinamide pathway, UV-induced nic mutants occurred with a higher frequency in the genes of the tryptophan pyrrolase and the 3-hydroxykynureninase than in the genes of the other enzymes of the pathway.     

Absence of various manifestations of adaptive response in Bacillus subtilis transformants

Competent cells of Bacillus subtilis are more sensitive to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) than total population, exhibit higher level of spontaneous mutations to kanamycin resistance. However, the absolute number of mutated transformants doesn't rise with MNNG treatment in the range of 5 to 50 micrograms per ml. The adaptation to low concentrations of MNNG affects neither spontaneous nor MNNG-induced mutagenesis in the competent (transformed) cells, in contrast to their resistance which is stronger for the adapted transformants. The transformation by MNNG-treated plasmid pUB 110 doesn't reveal any difference between adapted and non-adapted cultures in transformation efficiency decline.     

Interaction of UV and N-methyl-N'-nitro-N-nitrosoguanidine: cytotoxicity and mutagenicity in V79 cells

Killing and mutation by UV in the MNNG-exposed population of V79 cells, as well as by MNNG in the UV-irradiated population of these cells have been studied. It was observed that pretreatment with MNNG increased the killing and mutation by UV, whereas, pretreatment with UV had no effect upon killing and mutation by MNNG. The increase in sensitivity to UV due to pretreatment with MNNG was lost if UV exposure was delayed for 24 h after MNNG treatment.     

Involvement of mismatch repair proteins in adaptive responses induced by N-methyl-N'-nitro-N-nitrosoguanidine against γ-induced genotoxicity in human cells

As humans are exposed to a variety of chemical agents as well as radiation, health effects of radiation should be evaluated in combination with chemicals. To explore combined genotoxic effects of radiation and chemicals, we examined modulating effects of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a direct-acting methylating agent, against genotoxicity of γ-radiation. Human lymphoblastoid TK6 cells and its mismatch-deficient derivative, i.e., MT1 cells, were treated with MNNG for 24h before they were exposed to γ-irradiation at a dose of 1.0 Gy, and the resulting genotoxicity was examined. In TK6 cells, the pretreatments with MNNG at low doses suppressed frequencies of the thymidine kinase (TK) gene mutation and micronucleus (MN) formation induced by γ-irradiation and thus the dose responses of TK and MN assays were U-shaped along with the pretreatment doses of MNNG. In contrast, the genotoxic effects of MNNG and γ-irradiation were additive in MT1 cells and the frequencies of TK mutations and MN induction increased along with the doses of MNNG. Apoptosis induced by γ-radiation was suppressed by the pretreatments in TK6 cells, but not in MT1 cells. The expression of p53 was induced and cell cycle was delayed at G2/M phase in TK6, but not in MT1 cells, by the treatments with MNNG. These results suggest that pretreatments of MNNG at low doses suppress genotoxicity of γ-radiation in human cells and also that mismatch repair proteins are involved in the apparent adaptive responses.     

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.     

The effect of caffeine upon cell survival and post-replication repair of DNA after treatment of BHK 21 cells with either UV irradiation or N-methyl-N-nitrosoguanidine

It has been found that in BHK 21 cells caffeine potentiates cell killing by both UV irradiation and N-methyl-N-nitrosoguanidine (MNNG). The potentiating effect is greater with UV than with MNNG. While non-toxic concentrations of caffeine inhibit the joining of newly-replicated DNA fragments into large molecular weight DNA (post-replication repair) after UV irradiation, they have no such effect after MNNG treatment. Furthermore, the joining of DNA fragments continues in cells treated with 3 μg/ml of MNNG, a dose which leads to less than 5% cell survival. While inhibition of the synthesis of large molecular weight DNA can explain the synergistic effect of caffeine upon cell survival after UV irradiation, it cannot explain the similar effect after MNNG treatment.     

Effect of cell growth rate and dose fractionation on chemically-induced ouabain-resistant mutations in Chinese hamster V79 cells.

Chinese hamster V79 cells were grown in medium containing either 10% or 2% FCS during the expression time following exposure to MNNG. The lower serum concentration was used to reduce the rate of cell replication, thereby allowing more time for DNA repair prior to "fixation" of the mutagenic lesion. In addition, fractionated and continuous exposures to MNNG and MAM, respectively, were carried out to determine their effect on the number of induced ouabain-resistant mutants. The results indicated that lowering the rate of cell growth effectively reduces the mutation frequency at low, but not at high doses of MNNG. Fractionated doses of MNNG result in a potentiation of their mutagenic effects compared to single doses. Also, continuous exposures to MAM result in an exponential increase in the mutation frequency. Collectively, these results suggest the importance of a repair process in Chinese hamster V79 cells which is dependent upon cell growth rate and the dose of the mutagen for its effectiveness.     

Base-pair substitutions alter the site-specific mutagenicity of UV and MNNG in the SUP4-o gene of yeast

Yeast strains carrying SUP4-o genes that have base-pair substitutions at hotspots for UV or MNNG mutagenesis were treated with these agents. In both cases, the induced mutation frequencies were substantially reduced. Furthermore, specific substitutions at positions in SUP4-o that had not been mutated by MNNG resulted in the recovery of MNNG-induced mutations at these sites. These results demonstrate that base-pair identity is an important factor determining the site-specific mutagenicity of UV and MNNG in yeast. For UV, our findings suggest that the type of lesion that is induced, but not flanking DNA sequences, plays a role in specifying mutability at the sites examined. In contrast, DNA sequence context seems to be an important factor for MNNG mutagenesis.     

Normal human bronchial epithelial cells do not show an adaptive response after treatment with N-methyl-N'-nitro-N-nitrosoguanidine

Normal human bronchial epithelial cells cultured in serum-free medium were exposed to low doses o N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) to examine whether increased cellular resistance and increased activity of the DNA-repair enzyme O6-methylguanine-DNA methyltransferase could be induced. After treatment with single doses of MNNG a dose-dependent decrease in O6-methylguanine-DNA methyltransferase activity was observed, as expected for this unique repair system. The activity recovered to the starting level in about 24 h when a dose that consumed approximately 65% of the enzyme activity (0.2 micrograms/ml) was given, but did not exceed the activity in the untreated control. Furthermore, treatment every 6 h for 4-5 days with non-toxic concentrations of MNNG (0.04-0.12 micrograms/ml) did not increase O6-methylguanine-DNA methyltransferase activity. Neither was cell survival following a range of challenge doses significantly increased. Our data suggest that human bronchial epithelial cells do not adapt to MNNG.     

Adaptive response and enhancement of N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis by chloramphenicol in Streptomyces fradiae.

Streptomyces fradiae expressed an adaptive response to treatment with small doses of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) that caused a reduction in mutagenesis by treatment with larger doses of MNNG. Treatment of S. fradiae with high levels of MNNG in the presence of chloramphenicol caused enhancement of mutagenesis, independent of the adaptive response.     

Potentiating effects of caffeine on teratogenicity of alkylating agents in mice

Teratogenic to subteratogenic doses of x-ray, mitomycin C, MNNG, thio-TEPA, cyclophosphamide, and chlorambucil were administered to pregnant ICR mice together with caffeine at doses of 12.5, 25, or 50 mg/kg on day 11 of gestation. Fetuses were examined for gross malformations on day 18 of gestation. The teratogenicity of mitomycin C was significantly potentiated by caffeine at a dose as low as 12.5 mg/kg. The teratogenicity of chlorambucil was also significantly potentiated by caffeine at 50 mg/kg, but similar potentiation was not observed for x-ray, MNNG, thio-TEPA, and cyclophosphamide.     

Genetic Analysis of Repair of Ultraviolet Damage by Competent and Noncompetent Cells of Bacillus subtilis

The repair of ultraviolet (UV) damage in Bacillus subtilis W23T(-) has been studied by transformation with deoxyribonucleic acid (DNA) extracted from irradiated cells before and after repair. The extent of repair of genetic markers by donor cells after low or moderate doses of UV was found to be related only to the initial degree of inactivation. After a very high dose, further inactivation occurred, also in proportion to initial damage. In addition, the competent recipient cells were shown to repair approximately 75% of the damage in transforming DNA. The sensitivities of markers irradiated either in vivo or in vitro appeared to be related to map position, the more proximal markers showing a greater resistance to UV inactivation.     

Influence of exogenous thymidine on killing and mutation of Chinese hamster V-79 cells by X-rays, ultraviolet light and N-methyl-N'-nitro-N-nitrosoguanidine

V-79 cells when exposed to thymidine (5 micrograms/ml) in growth medium after treatment with X-rays, UV light and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), responded differently depending upon the agent. For treatment with X-rays and UV light, only induction of mutation was potentiated, but for MNNG treatment, both killing and mutation induction were potentiated. The increase in killing of MNNG exposed cells could be reversed by simultaneous addition of deoxycytidine with thymidine, but, for all the three mutagenic treatments, enhancement in mutation induction could not be suppressed by deoxycytidine.     

Plasmid DNA transduction in Bacillus subtilis

Transduction of Bacillus subtilis pUB110 plasmid by AR9 phage is described. Some aspects of this process are studied. Plasmid transduction depended on multiplicity of infection similar to cases of chromosomal markers transduction, though optimal multiplicity of infection was achieved using low number of phage particles. No cotransduction of plasmid and chromosomal markers was demonstrated. The transduction frequencies of plasmid and chromosomal markers increased after UV irradiation of phage suspensions within the range of definite doses.     

Activation of protein kinase A and clustering of cell surface receptors by N-methyl-N'-nitro-N-nitrosoguanidine are independent of genomic DNA damage

Alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) induces cellular stress leading to chromosomal aberrations, mutations and cell death. Previous reports from our laboratory have shown that low concentration of MNNG induces untargeted mutation (UTM), which occurs on intact DNA in mammalian cells through changes in gene expression profile. It also causes the activation of cAMP-protein kinase A (PKA) and up-regulation of POL-beta, which is demonstrated to play a role in DNA repair system. In order to find out the possible initial signal involved in UTM, we try to investigate whether the activation of PKA-CREB signal pathway is closely related to DNA damage. Our data shows that the treatment of low concentration MNNG (0.2 microM) activates PKA-CREB pathway in a comparable level both in a nuclear and enucleated cell system. And similar to the cell response caused by UV, the clustering of cell surface receptors of epidermal growth factor (EGF) and tumor necrosis factor alpha (TNFalpha) was also observed in cells exposed to MNNG. It was further demonstrated that the clustering of the surface receptors is independent of the genomic DNA damage, as this phenomenon was also observed in enucleated cells. These observations indicate that the initiation of signal cascades induced by low concentration of MNNG might be associated with its interaction with cell surface receptors and/or direct activation of related signal proteins but not its DNA damaging property.     

Interactions between humic matter and bacteria when disinfecting water with UV light

Aims:  To investigate the impact of aquatic humic matter on the inactivation of Escherichia coli and Bacillus subtilis by ultraviolet (UV) light.
Methods and Results:  A bench-scale study investigated the potential for Aldrich^® humic acid (AHA) and Suwannee River natural organic matter (SR-NOM) to coat the surface of E. coli and B. subtilis and offer protection from low-pressure UV light. UV doses of 5 and 14 mJ cm⁻² were applied using a collimated beam at four concentrations of humic matter (0, 10, 50 and 120 mg l⁻¹) in reagent grade water. Both AHA and SR-NOM were found to offer statistically significant protection of both E. coli and B. subtilis at concentrations of 50 and 120 mg l⁻¹ for a UV dose of 14 mJ cm⁻².
Conclusions:  Both E. coli and B. subtilis are susceptible to coating by humic matter which can reduce the sensitivity of the cells to UV light.
Significance and impact of the study:  Micro-organisms in the environment may acquire characteristics through interaction with humic matter that render them more resistant to UV disinfection than would be predicted based on laboratory inactivation studies using clean cells.