methylnitronitrosoguanidine and acridine-half-mustard

To identify new genes in an organism, a genetic approach can be used to screen for mutations that display a particular phenotype. Genotoxic agents, such as ultraviolet (UV) light, ionizing radiation, or chemicals can be used to randomly induce DNA lesions in the genome. Most efficient mutagenesis occurs when a mutagen confers a high frequency of mutations with low lethality, in the range of 10 to 50% survival. These mutations can be in the form of frameshifts, deletions, or rearrangements. To initiate a mutagenesis, a fresh subculture of cells grown into log phase is collected, washed, and resuspended in potassium phosphate buffer. The mutagen is added to the culture for a predetermined time, deactivated, and washed from the cells. The cells are allowed to recover from the treatment by incubating in liquid or on solid medium. Mutants can be isolated by screening individual colonies or by using direct selection of cells from the mutagenized cell population.

Topics: 4-Nitroquinoline-1-oxide; Aminacrine; Ethyl Methanesulfonate; Genes, Fungal; Methylnitronitrosoguanidine; Mutagenesis; Mutagens; Mycology; Nitrogen Mustard Compounds; Quinolones; Saccharomyces cerevisiae; Ultraviolet Rays

We report the first use of green fluorescent protein (GFP) for mutation detection. We have constructed a plasmid-based bacterial system whereby mutated cells fluoresce and non-mutated cells do not fluoresce. Fluorescence is monitored using a simple hand-help UV lamp; no additional cofactors or manipulations are necessary. To develop a reversion system, we introduced a +1 DNA frameshift mutation in the coding region of GFP and the resulting protein is not fluorescent in Escherichia coli. Treatment of bacteria containing the +1 frameshift vector with ICR-191 yields fluorescent colonies, indicating that reversion to the wild-type sequence has occurred. Site-directed mutagenesis was used to insert an additional cytosine into a native CCC sequence in the coding region of GFP in plasmid pBAD-GFPuv, expanding the sequence to CCCC. A dose-related increase in fluorescent colonies was observed when the bacteria were treated with ICR-191, an agent that induces primarily frameshift mutations. The highest dose of ICR-191 tested, 16 microg/ml, produced a mutant fraction of 16 x 10(-5) and 8.8 x 10(-5) in duplicate experiments. The reversion system did not respond to MNNG, an agent that produces mainly single-base substitutions. To develop a forward system, we used GFP under the control of the arabinose PBAD promoter; in the absence of arabinose, GFP expression is repressed and no fluorescent colonies are observed. When cells were treated with MNNG or ENNG, a dose-dependent increase in fluorescent colonies was observed, indicating that mutations had occurred in the arabinose control region that de-repressed the promoter. Treating bacteria with 100 microg/ml MNNG induced mutant fractions as high as 82 x 10(-5) and 40 x 10-5 in duplicate experiments. Treating bacteria with 150 microg/ml ENNG induced a mutant fraction of 2.1 x 10(-5) in a single experiment.

Topics: Aminacrine; Arabinose; Escherichia coli; Frameshift Mutation; Green Fluorescent Proteins; Indicators and Reagents; Luminescent Proteins; Methylnitronitrosoguanidine; Mutagenesis, Site-Directed; Nitrogen Mustard Compounds; Operon; Plasmids; Transformation, Genetic

The specificity of frameshift mutations induced by several classes of chemical mutagens was determined using a collection of mutant E. coli lacZ genes. This collection can detect each of five kinds of specific frameshift events by scoring Lac+ revertant colonies. In addition, the mutational spectra were characterized in backgrounds carrying plasmids that encode the umuDC, mucAB, or samAB operon. 4-Nitroquinoline 1-oxide (4-NQO) and furylfuramide (AF-2) induced efficiently -1G, -2(C-G), and +1A frameshift mutations. 4-NQO and AF-2 differed in the ability for the induction of -1A and +1G frameshifts. +1A and -1A frameshift mutations induced by 4-NQO or AF-2 were enhanced by the introduction of the mucAB plasmid, and, to a lesser extent, the umuDC plasmid. The enhancing effect of the umuDC or mucAB plasmid on -1G and -2(C-G) frameshifts was weak or else not observed. 9-Aminoacridine was a potent inducer of +1G, -1G and -1A frameshifts, whereas ICR-191 induced all types of frameshift mutations. A mutation enhancing effect was observed only on ICR-191-induced +1A frameshift mutations by the introduction of the mucAB plasmid. Mitomycin C caused no appreciable induction of frameshift mutations to the tester strains without plasmid. However, all types of frameshifts, except -1G, were induced in the strains carrying the mucAB plasmid. N-methyl-N'-nitro-N-nitrosoguanidine induced all types of frameshift mutations. The mucAB plasmid enhanced mutagenesis in strains designed to detect the addition or loss of A.T base pair, indicating that the formation of +1A and -1A frameshifts was partly dependent on an error-prone SOS repair. Any frameshift mutagenesis was not affected by the samAB plasmid. In general, frameshifts in adenine runs were enhanced more preferentially by the mucAB and umuDC plasmids than frameshifts at runs of guanine were.

Topics: 4-Nitroquinoline-1-oxide; Alleles; Aminacrine; DNA Mutational Analysis; DNA, Bacterial; Escherichia coli; Frameshift Mutation; Furylfuramide; Lac Operon; Methylnitronitrosoguanidine; Mitomycin; Mutagenesis, Site-Directed; Mutagens; Nitrogen Mustard Compounds; Operon; Plasmids; SOS Response, Genetics; Suppression, Genetic

Mutational spectra induced by different classes of chemical mutagens including two ultraviolet-mimetic mutagens, an alkylating agent, intercalators, a crosslinking agent, and base analogs were characterized by means of a set of mutant lacZ genes in E. coli. These strains can be used to detect each of two types of transition and four types of transversion, simply by measuring the number of Lac+ revertant colonies. 4-Nitroquinoline 1-oxide induced G.C-->A.T, G.C-->C.G, or G.C-->T.A changes almost equally, whereas furylfuramide and mitomycin C induced only G.C-->A.T transitions and G.C-->T.A transversions, respectively. No base substitutional mutations were detected by the treatment with 9-aminoacridine. A weak stimulation of G.C-->A.T transitions by ICR-191 was observed. Both the G.C-->A.T and A.T-->G.C transitions were induced by N-methyl-N'-nitro-N-nitrosoguanidine and N4-aminocytidine. 5-Azacytidine was a specific inducer of G.C-->C.G transversions. In addition, a comparative study of mutational specificity was performed in the strains bearing either the umuDC, mucAB, or the samAB operon on a multicopy plasmid. Regardless of the kind of mutagen, G.C-->T.A transversions were greatly potentiated by the introduction of plasmids in the order of pGW1700 (mucAB) > pSE117 (umuDC) > or = pYG8011 (samAB). Besides G.C-->T.A transversions, the introduction of pGW1700, but not pSE117 and pYG8011, enhanced the mutations of A.T-->C.G and A.T-->T.A transversions. The mucAB plasmid also enhanced the G.C-->A.T transitions and G.C-->C.G transversions induced by some mutagens.

Topics: Aminacrine; Azacitidine; Cross-Linking Reagents; Cytidine; DNA Mutational Analysis; DNA, Bacterial; Escherichia coli; Furylfuramide; Intercalating Agents; Lac Operon; Methylnitronitrosoguanidine; Mitomycin; Mutagenesis, Site-Directed; Mutagens; Nitrogen Mustard Compounds; Operon; Plasmids; Point Mutation; SOS Response, Genetics; Suppression, Genetic; Ultraviolet Rays

A heat-resistant factor in ethanol extracts of the fungus Craterellus cornucopioides completely inhibited the mutagenicity of aflatoxin B1, benzo[a]pyrene, the acridine half mustard ICR-191 and 2-nitrofluorene in a forward-mutation system using Salmonella typhimurium TM677 (screening for 8-azaguanine resistance). There was no inhibitory effect on the mutagenic activity of 4-nitroquinoline-N-oxide, methyl methanesulfonate or N-methyl-N'-nitro-N-nitrosoguanidine. Experiments performed to elucidate the mechanism of the antimutagenic effect showed that neither an alteration of cell viability nor an interference with the excision-repair and the inducible SOS-repair system was involved. The conceivable mechanisms for the antimutagenicity of the ethanol extract include direct chemical interaction with the mutagen and/or inhibition of the activation process in the case of the promutagens. The antimutagenic activity of Craterellus cornucopioides is not unique among mushroom species. The ethanol extracts of 6 other mushrooms showed a similar antimutagenic activity.

Topics: 4-Nitroquinoline-1-oxide; Aflatoxin B1; Aflatoxins; Aminacrine; Basidiomycota; Benzo(a)pyrene; DNA Repair; Ethanol; Fluorenes; Hot Temperature; Methyl Methanesulfonate; Methylnitronitrosoguanidine; Mutagenicity Tests; Mutation; Nitrogen Mustard Compounds

We describe a method to identify and enumerate mutants at the nucleotide level in complex cell populations. Several thousand different mutants were induced at the HPRT locus in human lymphoblastoid cultures by either MNNG, an alkylating agent, or by ICR-191, a substituted acridine. HPRT mutants were selected en masse by resistance to 6-thioguanine. The most frequent mutations (hotspots) in HPRT exon 3 were determined by a combination of denaturing gradient gel electrophoresis and polymerase chain reaction. MNNG predominantly produced GC----AT transitions at nucleotides in a GGGGGG sequence, while ICR-191 produced both +1 frameshifts in the same GGGGGG sequence and +1 frameshifts in a CCC sequence.

Topics: Aminacrine; Aminoacridines; B-Lymphocytes; Base Sequence; Cell Line; Cloning, Molecular; Electrophoresis; Exons; Humans; Hypoxanthine Phosphoribosyltransferase; Methylnitronitrosoguanidine; Molecular Sequence Data; Mutation; Nitrogen Mustard Compounds; Polymerase Chain Reaction

Double mutants were constructed combining mus-26, formerly designated uvs-(SA3B), with other UV-sensitive mutants. Tests of sensitivity of these double mutants to UV and to chemical mutagens revealed that mus-26 and upr-1 belong to the same epistatic group. The UV dose-response curve of mus-26 showed a characteristic plateau in the range of 100-200 J/m2. The same characteristic was also shown in the dose-response curves of upr-1 and the double mutant, upr-1 mus-26. Photoreactivation of UV damage in mus-26, upr-1 and upr-1 mus-26 was defective but not null. Assays were made of the reversion rate of ad-8 in strains that also carried UV-sensitive mutations. The reversion frequencies of the strains with upr-1 and upr-1 mus-26 were very low for the UV dose range below 300 J/m2, similarly to mus-26. Previously reported homozygous sterility of mus-26 was not caused by the mus-26 locus itself, and fertile strains were obtained among progeny. The results of this study suggest that mus-26 and upr-1 have similar properties in DNA repair.

Topics: 4-Nitroquinoline-1-oxide; Aminacrine; DNA Repair; Dose-Response Relationship, Radiation; Gamma Rays; Genotype; Methyl Methanesulfonate; Methylnitronitrosoguanidine; Mitomycin; Mitomycins; Mutagens; Mutation; Neurospora; Neurospora crassa; Nitrogen Mustard Compounds; Ultraviolet Rays

We investigated the effects of the well-known mutagenic agents ethyl methanesulfonate (EtMes), N-methyl-N-nitro-N-nitrosoguanidine (MNNG), and ICR-191 on colonies of the Chinese hamster ovary line CHO cultured on a semisolid substrate. These agents induced heterogeneity in diameter and integrated optical density of colonies as determined by computer-assisted photography and subsequent analysis of the images of the colonies. When CHO colonies were exposed to agents such as urethane that are not known to be mutagenic in mammalian systems or to activation-requiring mutagens such as cyclophosphamide, there was no noticeable effect on the distribution of colony diameter and volume. Similarly, nonmutagenic agents such as dimethyl sulfoxide (Me2SO) also did not induce heterogeneity in colony diameter and integrated optical density. Our observations recommend the use of agar-grown mammalian cell colonies for predictive testing of chemical mutagens and carcinogens in a simple, in vitro mammalian cell assay. This assay system, unlike other mammalian cell culture assays, allows detection and measurement of the simultaneous effects of chemical mutagens on several genetic and non-genetic targets and, thus, may emulate more closely the potential hazards of these agents in vivo.

Topics: Aminacrine; Animals; Cell Division; Cell Line; Cell Survival; Clone Cells; Cricetinae; Cricetulus; Cyclophosphamide; Dimethyl Sulfoxide; Ethyl Methanesulfonate; Female; Methylnitronitrosoguanidine; Mutagenicity Tests; Mutagens; Mutation; Nitrogen Mustard Compounds; Ovary; Urethane

The investigation of mutagenic mechanisms in Haemophilus influenzae has been confined until now to mutagens that normally produce mainly base pair substitutions. This paper describes the development of a system suitable for detecting frameshift mutations induced by ICR-191. The system involves reversions from thymidine dependence to thymidine independence. Evidence is presented from a comparison of the responses to ICR-191 and to N-methyl-N'-nitro-N-nitrosoguanidine that the system is specific for frameshift mutations. The genetic recombination involved in transformation leads to a marked increase in "spontaneous" reversion of the frameshift mutations but not of the base substitution mutations. Presumably, this is a consequence of mispairing, with consequent change in the number of bases, during the recombination.

Topics: Aminacrine; Aminoacridines; Bacteriological Techniques; Genetic Code; Genetic Techniques; Haemophilus influenzae; Methylnitronitrosoguanidine; Mutagenicity Tests; Mutagens; Mutation; Nitrogen Mustard Compounds; Protein Biosynthesis

The toxic and mutagenic effects of the alkylating agents methylnitrosourea (MNU) and methylnitronitrosoguanidine (MNNG) and of the frameshift mutagen, ICR-191 were compared among 3 human diploid lymphoblast lines, MIT-2, WI-L2 and GM 130. The MIT-2 and WI-L2 lines were both sensitive to the toxic and mutagenic effects of all 3 agents tested. The WI-L2 line was more sensitive to the toxic effects of MNU and MNNG than the MIT-2 line, while it was somewhat less sensitive to the mutagenic effects of these alkylating agents. The GM 130 line was strikingly resistant to both the toxic and mutagenic effects of the alkylating agents. The order of sensitivity to the toxic effect of ICR-191 was MIT-2 greater than WI-L2 greater than GM 130, while the order of sensitivity to the mutagenic effects of this frameshift mutagen was GM 130 greater than MIT-2 greater than WI-L2. These results point to the importance of accounting possible variations in mutability among individuals when extrapolating from any single mutagenicity assay for human risk assessment.

Topics: Aminacrine; Aminoacridines; Cell Line; Dose-Response Relationship, Drug; Humans; Lymphocytes; Methylnitronitrosoguanidine; Methylnitrosourea; Mutagens; Nitrogen Mustard Compounds; Nitrosourea Compounds

Topics: Acridines; Aminacrine; Cell Line; DNA Repair; Humans; Methylnitronitrosoguanidine; Microscopy, Fluorescence; Mutagens; Nitrogen Mustard Compounds

The induction of mutation by a variety of mutagens has been measured utilizing the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in Chinese hamster ovary (CHO) cells (CHO/HGPRT) system). These mutagens include physical agents such as UV light and X-rays, and chemicals such as alkylating agents, ICR-191, and metallic compounds. This system can also be modified for study of the mutagenicity of promutagens such as dimethylnitrosamine (DMN) which require biotransformation for mutagenic action, either through the addition of a rat liver microsomal activation preparation or through a host-mediated activation step using Balb/c athymic mice.

Topics: Acridines; Aminacrine; Animals; Cell Line; Dimethylnitrosamine; Drug Evaluation, Preclinical; Ethyl Methanesulfonate; Hypoxanthine Phosphoribosyltransferase; Methylnitronitrosoguanidine; Mice; Mice, Inbred BALB C; Mice, Nude; Microsomes, Liver; Mutation; Nitrogen Mustard Compounds; Rats; Ultraviolet Rays; X-Rays