HGPRT- mutants of V79 cells that revert specifically by base pair substitution and frameshift mutations

In order to determine the mutagenic specificity of mutagenic and carcinogenic agents in mammalian cells, a reversion system capable of distinguishing between frameshift mutations and various kinds of base pair substitutions would be useful. We report here a method for the isolation and characterization of HGPRT- Chinese hamster V79 cell mutants that might form the basis for such a system. Two mutants of different specificity have been partially characterized. DEW-1, isolated following N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment, is revertible by the base pair substitution mutagens MNNG and ethyl methanesulfonate (EMS), but not by frameshift mutagens. DSW-3, isolated following ICR-191 treatment, is specifically reverted by frameshift mutagens, but not by EMS or MNNG. With the further characterization of these and other mutants, it should be feasible to determine not only whether an agent is mutagenic in V79 cells, but also to determine the type(s) of mutation(s) it produces.     

RhodosporidiumBANNO: Dosiseffektbeziehungen,Mutageneffektivität und Mutantenspektrum bei der Induktion Auxotrophie verursachender Mutationen durch ultraviolettes Licht und N-Methyl-N′-nitro-N-nitrosoguanidin

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 Rgl. 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.     

Comparative potencies of induction of point mutations and genetic duplications by the methylating agents methylazoxymethanol and dimethyl sulfate in bacteria

Methylazoxymethanol (MAM) and dimethyl sulfate (DMS) are mutagens whose genetic effects can be ascribed to the methylation of DNA. While both methylate the N7 position of guanine heavily, only MAM strongly methylates the O(6) position of guanine. We evaluated the relative effectiveness and specificity of MAM and DMS in bacterial assays for the induction of point mutations and the formation of chromosomal duplications by genetic recombination. Salmonella typhimurium strain TS1121 was used to measure the formation of genetic duplications on the basis of the aroC321 allele and mutations by reversion of the hisG46 allele. Specific base pair substitutions and frameshift mutations were measured in a reversion assay based on lacZ alleles of Escherichia coli. The results show MAM to be the more potent mutagen and DMS the stronger recombinagen in the Salmonella assay. In the lacZ assay DMS induced several classes of base pair substitutions (GC-->AT transitions, GC-->TA transversions and AT-->TA transversions), as well as lower frequencies of +1, -1 and -2 frameshift mutations. The activity of MAM as a base pair substitution mutagen was more specific than that of DMS, inducing only GC-->AT transitions. It also induced +G, -G, -A and -CG frameshift mutations, though more weakly than it induced GC-->AT transitions. Long known as a base pair substitution mutagen, the induction of frameshifts by MAM was unexpected. The results show that both DMS and MAM are effective inducers of base pair substitutions and modest inducers of frameshifts and that DMS exhibits a broader spectrum of mutagenic activity than does MAM.     

Mutagenicity of N-substituted phenanthrene 9,10-imines in Salmonella typhimurium and Chinese hamster V79 cells

We previously showed that some (nonsubstituted) aziridines derived from polycyclic aromatic hydrocarbons (arene imines) elicit various mutagenic and genotoxic effects in bacteria and mammalian cells and that these arene imines are active at much lower concentrations than the corresponding epoxide analogues. In the present study, N-substituted derivatives of phenanthrene 9,10-imine were investigated. All 10 derivatives studied showed direct mutagenicity in Salmonella typhimurium TA100. Some of the compounds additionally exhibited weak effects in the strains TA98 and TA1537. Most N-substituted derivatives were weaker mutagens than unsubstituted phenanthrene 9,10-imine but stronger mutagens than phenanthrene 9,10-oxide. Bulky substituents reduced the mutagenicity more than did small substituents. In addition, the derivatives with electron-withdrawing substituents (with the exception of N-chlorophenanthrene 9,10-imine) were weaker mutagens than those with electron-donating substituents. Phenanthrene 9,10-imine and five N-substituted derivatives were investigated to determine whether they induce gene mutations at the hgprt locus in V79 cells. Four compounds, including the parent aziridine, were positive in the V79 test. The other two compounds were negative. The mutagenic potencies in the V79 cell system did not correlate well with those obtained with the Salmonella system. Overall, the study shows that in addition to unsubstituted arene imines, N-substituted derivatives are mutagenic. This finding is of interest, as metabolic pathways leading from aromatic compounds to N-substituted arene imines are conceivable.     

Activity of mutagens in the fungus Gibberella fujikuroi

The efficacy of a number of mutagens was examined for Gibberella fujikuroi. Nitrosoguanidine (MNNG) was the most effective mutagen inducing mainly morphological and pigment mutants. X-ray produced small mutagenic effect. After UV-irradiation pigment mutants developed for the most part and a combination of UV- and x-ray-irradiation mainly influenced the growth rate. A combination of UV-irradiation with MNNG elicited the highest number of mutants with increased GA₃ production.     

Mutagenesis in Streptococcus lactis exposed to UV irradiation and alkylating agents

The lethal and mutagenic effects of various mutagens on threestrains of Streptococcus lactis were investigated. Lethalitystudies demonstrated that S.lactis was relatively sensitiveto UV irradiation, methyl methanesulphonate (MMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and, to a lesser extent, toethyl methanesulphonate (EMS). A spontaneous derivative Lac^–,which has lost a 37-Md plasmid, was slightly more resistantand much less mutable than the wild-type after UV irradiation.Although the three strains were strongly mutated by EMS forthe genetic marker assayed (Rif^r), an increase in the mutationfrequency was also observed after MMS and MNNG treatments. ¹To whom correspondence should be addressed     

Synergism and antagonism between N-methyl-N'-nitro-N-nitroso-guanidine and 9-aminoacridine in mutation induction in Escherichia coli K12

When bacteria were treated simultaneously with N-methyl-N'-nitro-N-nitrosoguanidine(MNNG) and low concentrations of 9-aminoacridine (9AA), theyield of frameshift mutations was much greater than that expectedon the basis of independent action of both mutagens. In combinationwith a high concentration of 9AA, however, MNNG had an antagonisticeffect upon the induction of frameshift mutations. There wasno synergistic interaction between the two mutagens in bacteriain which the adaptive response to methylating agents had beeninduced. 9AA not only induced frameshift mutations, but alsocaused a small increase in reversions of a nonsense (ochre)mutation and, in combination with low MNNG concentrations, ithad a small synergistic effect. ^*This paper is dedicated to Professor Reinhard W.Kalpan on theoccasion of his 80th birthday      

Plant-activation of the bicyclic aromatic amines benzidine and 4-aminobiphenyl

Benzidine and 4-aminobiphenyl (4-ABP) are promutagenic bicyclic aromatic amines that are activated into frameshift and base pair substitution mutagens by plant systems. Using the plant cell/microbe coincubation assay, plant-activated benzidine from 0 to 50 μM induced a concentration-response in Salmonella typhimurium. At concentrations above 5 μM, plant-activated benzidine induced frameshift and base pair substitution mutations in the N- or O-acetyltransferase over-expressing strains, DJ460, YG1024, and YG1029. With plant-activated 4-ABP, concentrations above 250 μM induced a significant mutagenic response in strains YG1024 and YG1029. A tobacco cell-free mixture, TX1MX, activated benzidine and 4-ABP into mutagenic metabolites in S. typhimurium strains YG1024, YG1029, and DJ460. The mutagenic sensitivities of plant-activated benzidine and 4-ABP were the same with two different types of plant activation systems, TX1 suspension cells and TX1MX cell-free medium. The plant activation of these aromatic amines is mediated by tobacco cell peroxidase. Plant-activated benzidine and 4-ABP are converted into intermediates that serve as substrates for bacterial or humanacetylCoA: N-hydroxyarylamine N-acetyl-transferase to generate the ultimate mutagenic products. Environ. Mol. Mutagen. 29:81–90, 1997 © 1997 Wiley-Liss, Inc.     

The mutagenic effect of cis-diamminedichloroplatinum (II) and its degradation products in the ames microbial assay

Cis-Diamminedichloroplatinum (II), an antitumor compound which exhibits mutagenic activity, and its degradation products platin B salt, magnus red, and magnus red anion salt were tested in the Ames microbial mutagenicity assay. The purpose of this investigation was to determine if the positive mutagenic response of the platinum compound could be due to, or enhanced by, the presence of degradation products. Results indicate that platinum degradation complexes are weak mutagens which are capable of inducing both base pair and frameshift-type mutations.     

Molecular analysis of hypoxanthine phosphoribosyl transferase mutants induced by glycidyl 1-naphthyl ether in mouse spleen cells in vivo

Treatment of C57BL/6J mice with an epoxide, glycidyl 1-naphthyl ether (GNE), resulted in an average of a 3.4-fold increase in frequency of 6-thioguanine-resistant mutants of mouse spleen T-lymphocytes. In similar experiments with the epoxide trichloropropylene oxide, no increase in mutant frequency was found. To determine the kind and location of mutations in the coding region of the hypoxanthine phosphoribosyl transferase (HPRT) gene, 26 GNE-induced mutants and 17 spontaneous mutants were analyzed by direct sequencing of polymerase chain reaction amplified cDNA. Among the GNE-induced mutants, HPRT cDNA was present in 22, while that from 4 could not be detected. Among the spontaneous mutants, HPRT cDNA was present in 15 and absent in 2. Among GNE-induced mutants, base substitution in HPRT occurred in 15 of 22 mutants analyzed. Nine of 15 base substitutions involved TA base pairs, primarily TA→CG transitions. Base substitutions were found throughout exons 3–7 but 46% of substitutions were located in exon 3 and one frameshift mutation involving a GC base pair in exon 3 was also observed. Among the spontaneous mutants, base substitutions of HPRT occurred in 7 of 15 mutants analyzed with 6 of 7 base substitutions involving a TA base pair and another 2 of the 15 mutants showed a 4 base pair deletion. The base substitution spectrum in GNE-induced mutants was different from that of the spontaneous mutants. © 1993 Wiley-Liss, Inc.     

Structural basis of the mutagenicity in bacteria of nitrated naphthalene and derivatives

While 2-nitronaphthalene was a weak direct-acting base-substitution mutagen (1.4 revertants/nanomole) for Salmonella typhimurium, the analogous nitronaphthalic acid anhydride and imides were moderate frameshift mutagens (～20 rev/nanomole in strain TA98). Although imide derivatives are efficient DNA intercalators, mutagenicity data indicate that the bulk of the frameshift activity is derived from adduct formation between hydroxylamine intermediates and DNA. The low level of frameshift activity (～8% of total) resulting from simple intercalation (measured in strain TA1537) is not dependent upon reduction of the nitro function. Evidence is presented that suggests that the reduction of the nitro function to the corresponding hydroylamine might not involve a free nitroso intermediate. The introduction of a second nitrofunction into nitronaphthalenes results in great positional effects of the various isomers on mutagenic activity and specificity.     

1,8-Dinitropyrene: comparative mutagenicity in Chinese hamster V79 and CHO cells

1,8-Dinitropyrene (1,8-DNP) was clearly mutagenic at the hprt locus in CHO cells, but not detectably mutagenic in V79 cells, following a 3-h treatment period. Preliminary data indicate that CHO, but not V79, cells have measurable levels of N-acetyltransferase activity, and this may contribute to the differential sensitivity of the two cell lines to the mutagenicity of 1,8-DNP.     

mutation spectra of Glu-P-1 in Salmonella: Induction of hotspot frameshifts and site-specific base substitutions

We used colony probe hybridization and PCR/ DNA sequence analysis to determine the mutations in —1,640 revertants of the -1 frameshift allele hisD3052 and -260 revertants of the base substitution allele hisG46 of Salmonella typhimurium induced by the heterocyclic amine cooked food mutagen 2-amino-6-methyldipy-rido[1,2-a:3′,2′-d]imidazole (Glu-P-1). All of the mutations were at sites containing guanine, which is the base at which Glu-P-1 forms DNA adducts. A hotspot mutation involving the deletion of a CG or GC within the sequence CGCGCGCG accounted for 100% of the Glu-P-1-induced mutations at the frameshift allele in strains TA1978 (uvr⁺) and TA1538 (uvrB) and 99% in TA98 (uvrB, pKM101). To explain the induction of these hotspot mutations by Glu-P-1, we describe here a more detailed version of our recently proposed correct incorporation/ slippage model [Genetics:136:731, 1994]. We propose that after cytosine is incorporated correctly opposite a Glu-P-1-adducted guanine, various slipped intermediates may form (a total of 18), depending on which guanine is adducted and whether it remains within the helix or becomes extrahelical. This variety of mutational pathways may account for the high mutability of the hotspot sequence by Glu-P-1. Although the pKM101 plasmid does not influence the mutagenic potency or mutational spectrum of Glu-P-1 at the frameshift allele, it is required by Glu-P-1 to revert the base substitution allele, where Glu-P-1 induces G-C-→T-A transversions (75%) and G-C→T.A transitions (25%) exclusively at a single site (the second position of the CCC codon of the hisG46 allele). The limited (20–30 times less) base substitution mutagenic potency of Glu-P-1 relative to its frameshift mutagenic potency as well as the extreme site specificity exhibited by Glu-P-1 for base substitutions may have bearing on the lack of base substitutions identified in ras genes in Glu-P-1-induced rat colon tumors. © 1994 Wiley-Liss, Inc.     

The bleomycin amplification assay in V79 cells predicts frameshift mutagenicity of intercalative agents

We have recently reported on the use of a cell-based bleomycin amplification assay for the detection of DNA intercalating agents. In order to further validate this assay, two series of proprietary compounds were evaluated for frameshift mutagenesis in the Ames bacterial reversion system and for bleomycin amplification in the Chinese hamster V79 micronucleus system. It is shown that 10 of 11 frameshift-positive compounds were bleomycin amplifiers. These studies indicate that positive frameshift mutagenicity findings are consistent with expectations from the results of the bleomycin amplification assay, providing additional validation of the amplification assay for the detection of DNA intercalating agents. The studies also demonstrate that intercalation is necessary but not sufficient for frameshift mutagenesis since bleomycin amplifiers lacking frameshift mutagenic activity were also identified.     

Mutagenic activity of four furocoumarins: angelicin, 4,5'-dimethyl-angelicin, psoralen and 8-methyl-psoralen on V79 Chinese hamster cells

Four furocoumarins, two having a linear structure (psoralen and 8-methyl-psoralen) and two having an angular structure (angelicin and 4,5'-dimethyl-angelicin), were studied for their mutagenic activity in the HGPRT system on V79 chinese hamster cells in culture (V79/HGPRT system). All the four drugs, when activated by near-ultraviolet (NUV) light, were effective in inducing HGPRT mutants. Their efficiency ranked in the following order: 8-methyl-psoralen greater than psoralen = 4,5-dimethylangelicin greater than angelicin.     

Detection of MNNG-induced DNA lesions in mammalian cells; validation of comet assay against DNA unwinding technique,alkaline elution of DNA and chromosomal aberrations

Human cells (VH10 or Hep G2) and hamster cells V79 were exposed to different concentrations of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and the level of DNA lesions was evaluated by the DNA unwinding technique, alkaline elution of DNA and the comet assay. All three methods were able to detect the effects of MNNG but with a clear difference in sensitivity. At low concentrations of MNNG the most sensitive method appeared to be the comet assay. After the short-term treatment the comet assay was able to detect the lesions induced by MNNG at approx. 0.1 μg/ml, alkaline elution of DNA at 1 μg/ml and DNA unwinding at 1–2 μg/ml. MNNG treated VH10 cells, human lymphocytes and V79 cells were also tested cytogenetically, confirming that MNNG induced chromosomal aberrations at concentrations >1 μg/ml in VH10 cells (short-term treatment); >0.2 μg/ml in V79 cells (long-term treatment) and >8 μg/ml in human lymphocytes (long-term treatment). In some experiments we tried to increase the level of MNNG-induced DNA breaks with help of DNA repair inhibitors cytosine arabinoside (Ara C) and hydroxyurea (HU) which were applied either after or during MNNG treatment. Our results showed that the level of MNNG-induced lesions was increased by simultaneous treatment of cells with MNNG and Ara C and HU. 2×10⁻⁵ M Ara C and 2×10⁻³ M HU were as effective as 10-times higher concentrations of inhibitors. Ara C and HU increased the level of MNNG-induced DNA breaks mainly in combination with lower concentrations of MNNG (<2 μg/ml). Rejoining of DNA breaks was observed in human cells VH10 and Hep G2 as well as in Chinese hamster cells V79 damaged by both lower and higher MNNG-concentrations. All methods showed that MNNG-induced DNA breaks had been gradually rejoined.     

mutation spectra in salmonella of complex mixtures: Comparison of urban air to benzo[a]pyrene

We used an ion-exchange procedure coupled to the Salmonella assay to fractionate the dichlo-romethane-extractable particulate organics from an urban air sample collected in Boise, ldaho. A resulting base/neutral fraction contained 81% of the mutagenic activity but only 36% of the mass of the unfractionated sample. Chemical analysis showed that polycyclic aromatic hydrocarbons (PAHs) accounted for much of the mutagenic activity of the air sample. Colony probe hybridization, PCR, and DNA sequence analysis were then used to determine the mutations induced by the complex mixtures and a model PAH, benzo[a]pyrene (BAP) in ～900 revertants of the frameshift hisD3052 allele and ～400 revertants of the base-substitution hisG46 allele. The majority (93–94%) of the mutations induced at the frameshift allele in strain TA98 by the whole or base/neutral fraction of the urban air sample was a hotspot 2-base deletion of a CG or GC within the sequence CGCGCGCG. The remaining mutations were complex frame-shifts that consisted of ?2 or +1 frameshifts associated with a flanking base substitution. BAP induced a somewhat similar pattern of mutations, with 70% being the hotspot mutation, 23% being complex frameshifts, and the remaining being deletions. The inferred base-substitution specificity associated with the complex frame-shifts at the hisD3052 allele (primarily G · C→T · A transversions) was consistent with the observation that this same transversion was the primary mutation induced by the whole urban air sample and BAP at the base-substitution allele in strain TA100. At the frameshift allele, adducts that promote correct incorporation/slippage could account for hotspot mutations, whereas those that promote misincorporation/slippage could account for complex frameshifts. At the base-substitution allele, a mixture of adducts or of adducts with multiple conformations could account for the observed proportion of transitions and transversions. Combined with the bioassay-directed chemical analysis, these results from the first mutation spectra of a complex mixture suggest that such spectra reflect the dominance of particular classes of chemical mutagens within the mixture. © 1994 Wiley-Liss, Inc.     

Antigenotoxic effects of Citrus aurentium L. fruit peel oil on mutagenicity of two alkylating agents and two metals in the Drosophila wing spot test

Antigenotoxic effects of Citrus aurentium L. (Rutaceae) fruit peel oil (CPO) in combination with mutagenic metals and alkylating agents were studied using the wing spot test of D. melanogaster. The four reference mutagens, potassium dichromate (K₂Cr₂O₇), cobalt chloride (CoCl₂), ethylmethanesulfonate (EMS), and N‐ethyl‐N‐nitrosourea (ENU) were clearly genotoxic. CPO alone at doses from 0.1 to 0.5% in Tween 80 was not mutagenic and did not enhance the mutagenic effect of the reference mutagens. However, antigenotoxic effects of CPO were clearly demonstrated in chronic cotreatments with mutagens and oil, by a significant decrease in wing spots induced by all four mutagens. The D. melanogaster wing spot test was found to be a suitable assay for detecting antigenotoxic effects in vivo. Environ. Mol. Mutagen., 2009. © 2009 Wiley‐Liss, Inc.     

Evidence for the presence of mutagenic arylamines in human breast milk and DNA adducts in exfoliated breast ductal epithelial cells

Aromatic and heterocyclic amines are ubiquitous environmental mutagens present in combustion emissions, fried meats, and tobacco smoke, and are suspect human mammary carcinogens. To determine the presence of arylamines in breast tissue and fluid, we examined exfoliated breast ductal epithelial cells for DNA adducts and matched human milk samples for mutagenicity. Breast milk was obtained from 50 women who were 4-6 weeks postpartum, and exfoliated epithelial-cell DNA was evaluated for bulky, nonpolar DNA adducts by (32)P-postlabeling and thin-layer chromatography. Milk was processed by acid hydrolysis, and the extracted organics were examined in the standard plate-incorporation Ames Salmonella assay using primarily strain YG1024, which detects frameshift mutations and overexpresses aryl amine N-acetyltransferase. DNA adducts were identified in 66% of the specimens, and bulky adducts migrated in a pattern similar to that of 4-aminobiphenyl standards. The distribution of adducts did not vary by NAT2 genotype status. Of whole milk samples, 88% (22/25) had mutagenic activity. Among the samples for which we had both DNA adduct and mutagenicity data, 58% (14/19) of the samples with adducts were also mutagenic, and 85% (11/13) of the mutagenic samples had adducts. Quantitatively, no correlation was observed between the levels of adducts and the levels of mutagenicity. Separation of the milk showed that mutagenic activity was found in 69% of skimmed milk samples but in only 29% of the corresponding milk fat samples, suggesting that the breast milk mutagens were moderately polar molecules. Chemical fractionation showed that mutagenic activity was found in 67% (4/6) of the basic fractions but in only 33% (2/6) of acidic samples, indicating that the mutagens were primarily basic compounds, such as arylamines. Although pilot in nature, this study corroborates previous findings of significant levels of DNA adducts in breast tissue and mutagenicity in human breast milk and indicates that breast milk mutagens may be moderately polar basic compounds, such as arylamines.     

Quantitative structure-activity relationships of heterocyclic amine mutagens formed during the cooking of food

The major protein-rich foods, particularly muscle meats, contain part-per-billion quantities of potent mutagens formed by frying or broiling to a well-done state. Related mutagens are formed by pyrolysis of amino acids or proteins and in heated model systems. The thermic mutagens so far identified are heterocyclic aromatic amines of aminoimidazo-azaarene (AIA) and aminocarboline classes. The chemicals require activation by enzymes to form metabolites reactive with nucleic acids. These thermic mutagens, and numerous synthetic congeners, exhibit an enormous range of potency as frameshift mutagens in the Ames/Salmonella assay. However, structural variations are nominal within the two classes. Structural parameters that appeared relevant to determining potency were selected for 38 AIAs and 23 amino-carbolines. For the AIA class these were: the number of fused rings, the number of heteroatoms in Rings 2 and 3, methyl substitution on imidazo ring nitrogen atoms, and methyl substitution on ring carbon atoms. For the amino-carboline class the structural parameters were: the position of the pyridine-type nitrogen atom in Ring 1, the substitution position of the exocyclic amino group on Ring 1, and methyl substitution on ring carbon atoms. These structural parameters may influence mutagenic potency in the following ways. 1) Electronic or steric effects may determine the reactivity and stability of the ultimate mutagenic metabolite. Optimal balance of reactivity and lifetime of this transient intermediate may be required for access to and reaction with nuclear DNA to cause mutations. 2) Substitution on the rings may block detoxication reactions. The structural parameters identified should prove useful in predicting the mutagenicity of untested compounds of these types.