Comparison of mutagenic and recombinogenic effects of some adenine analogues in Saccharomyces cerevisiae D7

2-Aminopurine, 2-amino-N⁶-hydroxyadenine and N⁶-hydroxyaminopurine were compared in suspension test with growing and non-growing cells for their mutagenic and recombinogenic (reciprocal and nonreciprocal) activities in Saccharomyces cerevisiae strain D7. Ethyl methanesulfonate was used as a positive control. No increases above spontaneous frequencies were observed when non- growing cells were treated with the base analogues although EMS induced concentration- dependent responses at all 3 genetic end-points. When growing cells were treated, HAP was recombinogenic and mutagenic and AHA was mutagenic, but only weakly recombinogenic. HAP induced comparable numbers of revertants at much lower concentrations than AHA. 2AP failed to induce any detectable response even at concentrations as high as 2400 μg/ml.     

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.     

Mutagenicity of vinyl chloride,chloroethyleneoxide, chloroacetaldehyde and chloroethanol

Exposure of S. typhimurium strains TA 1530, TA 1535 and G-46 to vinyl chloride increased the number of his⁺ rev./plate 16, 12 or 5 times over the spontaneous mutation rate. The mutagenic response for TA 1530 strain was enhanced 7, 4 or 5-fold when fortified S-9 liver fractions from humans, rats or mice were added. In TA 1530 strain, chloroacetic acid showed only toxic effects, while chloroacetaldehyde, chloroethanol and chloroethyleneoxide caused a mutagenic response. The latter compound was shown to be a strong alkylating agent.     

Ability of base analogs to induce the SOS response: effect of a dam mutation and mismatch repair system

Summary 2-Aminopurine, 2-amino-N⁶-hydroxyadenine, 2-amino-N⁶-methoxyadenine and 2-amino-N⁶-methyl-N⁶-hydroxyadenine (but not N⁴-hydroxycytidine), strong mutagens of base analog type, may induce the SOS response in E. coli cells. This ability is greatly enhanced in dam3 mutants and abolished in dam3mutS, dam3mutH, and dam3mutL strains, thereby suggesting that the mismatch repair system is involved in the mechanism of induction.Abbreviations n²Pur 2-aminopurine - n²oh⁶Ade 2-amino-N⁶-hydroxyadenine - n²om⁶Ade 2-amino-N⁶-methyoxyadenine - n²-m⁶oh⁶Ade 2-amino-N⁶-methyl-N⁶-hydroxyadenine - oh⁴Cyd N⁴-hydroxycytidine - MC mitomycin C     

Comparison of mutagenicity and chemical properties of N-methyl-N′-alkyl-N-nitrosoureas

Thed mutagenic activities of 11 N-methyl-N′-alkyl-N-nitrosoureas were tested on Samonellatyphimurium TA1535 and compared with chemical properties (alkylating activity and decompostion rate). In their relative mutagenicities the N-nitrosoureas that had a cyclic N′-alkyl group showed far more mutagenic activity than those having a chain N′-alkyl group. M(1-A)NU and M(2-A)NU, which had the most bulky N′-alkyl group in this series, exhibited lethal effects at high concentrations. The mutagenicity showed a small positive correlation with decomposition rates but not with alkylating activities on 4-(p-nitrobenzyl_prridine. The highest mutagenicity in this series was observed in N-methyl-N′-cyclobutyl-N-nitrosourea.These results suggest that, in this series of N-methyl-M′-alkyl-N-nitrosoureas, structural differences in the N′-alkyl groups had great significance in mutagenicity.     

Mutagenicity of butadiene and butadiene monoxide.

Incubation of $\text{S. typhimurium}$ strains TA1530 and TA1535 in the presence of gaseous butadiene increased the number of his⁺ revertants/plate. This mutagenic effect occured in absence of fortified S-9 rat liver fraction. In its presence, the mutagenic effect seemed to be dependent on its composition. With butadiene monoxide, a reversion to histidine prototrophy was obtained without metabolic activation with strains TA1530, TA1535 and TA100. Butadiene monoxide might be a possible primary metabolite of butadiene.     

Hydrazine and methylhydrazine as recA+-independent mutagens in Escherichia coli

The ultimate cause of both physical and chemical mutagenesis is assumed in most cases, to be some inducible error-prone repair process acting on the lesions caused by the mutagenic agent [7]. In Escherichia coli this mutagenic pathway seems to be dependent on both recA⁺ and lexA⁺ loci [10]. There are some mutagens, however, such as simple base analogs or some strong alkylating agents, such as N-methyl-N′-nitro-N′-nitrosoguanidine, that probably act by simply causing mis-pairing of bases in DNA. There is some evidence, based on work done with bacteriophages [2] or with Haemophilus influenzae [6], that hydrazine also acts by producing errors in base pairing. In this paper, further evidence is reported in support of this theory by showing that the recA lexA genotype does not affect the mutagenic properties of either hydrazine or methylhydrazine in Escherichia coli.     

Enhancement of the mutagenicities of N-methyl-N′-nitro-N-nitrosoguanidine and N-methyl-N-nitrosourea by glucose

The mutagenicity of N-methyl-N′-nitro-N-nitrosoguanidine to Salmonella typhimurium hisG46 was enhanced by pre-incubating the chemical with bacteria in sodium phosphate buffer. Addition of glucose (to 15 mM) to the pre-incubation mixture further enhanced the mutagenicity. Pre-incubation with glucose also increased the mutagenicity of N-methyl-N-nitrosourea. Fructose, galactose, pyruvate and succinate also enhanced the mutagenicity of N-methyl-N′-nitro-N-nitrosoguanidine. The effect of glucose was observed with S. typhimurium strains hisG46, TA1975, TA1950, TA1535 and TA100.     

Isolation of an azide mutagenic metabolite in Salmonella typhimurium

A scheme that employs a cation-exchange column and high-pressure liquid chromatography (HPLC) is devised to isolate and process large quantities of azide metabolite produced by S. typhimurium TA1530 strain. The mutagenic metabolite adheres strongly to the cation-exchange column, thus providing a convenient way to separate the metabolite from unreacted azide (N₃⁻). The metabolite is very polar and only sparingly soluble in most organic solvents. Recrystallization in a methanol-carbontetrachloride solvent system gave rise to microcrystalline material that decomposes with charring and gas evolution at 173–176°C. The infrared spectrum indicates the presence of a covalently bound azide moiety.     

A new strain of Salmonella typhimurium reverted by mitomycin C and N-methyl-N′-nitro-N-nitrosoguanidine—a possible universal tester for mutagenic compounds

A strain of Salmonella typhimurium, SO1007, which carries the amber mutation trpD28 plus the plasmid pKM101 was reverted very efficiently by two mutagens with different mutagenic specificities and modes of action: mitomycin C (MC) and N-methyl-N′-nitro-N-nitrosoguanidine (NG). By selecting revertants on minimal agar supplemented with anthranilic acid (AA), two distinct phenotypic classes of TrpD28 revertants can be recovered: prototrophs (MM⁺) and anthranilate utilizers (AA⁺). Since each phenotypic class is known to be caused by a variety of mutational events, reversion of trpD28 on minimal-anthranilate medium may be useful for detecting mutagenic agents regardless of the types of mutations they may cause. Thus, strains like SO 1007 may be useful as ‘universal’ detectors of mutagenic compounds. In the course of these experiments we also observed that pKM101 does not protect but, on the contrary, sensitizes the host bacteria slightly to the toxic effects of MC.     

Antimutagenicity in Euglena gracilis

The genotoxic effect of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and furadantine (Fu) was significantly decreased by standard antimutagens (ascorbic acid, α-tocopherol, chlorophyllin and sodium selenite) in the unicellular flagellate Euglena gracilis. The effects of these compounds were verified also by a bacterial test in which three strains of Salmonella typhimurium, TA97, TA100 and TA102, were used. The above compounds were antimutagenic in strains of bacteria used, except for chlorophyllin which had no effect on strain TA102.     

In vitro production of azide mutagenic metabolite in Arabidopsis,Drosophila and Neurospora

The ability of Arabidopsis, Drosophila and Neurospora to convert azide to its mutagenic metabolite was investigated. Cultures of these organisms all contained significant levels of O-acetylserine sulfhydrylase activity. Extracts from each organism produced a product from O-acetylserine and azide in vitro which was mutagenic in Salmonella typhimurium TA1530.     

Chemically induced mutation of L5178Y mouse leukemia cells from asparagine-dependence to asparagine-independence

L₅₁₇8Y mouse lymphoblastic leukemia cells are auxotrophic for l-asparagine (ASN) and have been widely used as a model system for studies on l-asparagine independence, were treated with known chemical mutagens to investigate the molecular basis of this mutation. Mutagens which primarily induce base pair substitutions—ethyl methanesullfonate (EMS) and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG)—as well as those which induce frame-shift mutations (the acridine half-mustards ICR-₃₇₂ and ICR-191) each increased the frequency of ASN⁺ cells in treated cultures to at least ten times the usual background frequency of 1 to 2 ASN⁺ cells per 10⁶ cells. The effectiveness of both classes of mutagens indicates that the change to asparagine prototrophy might occur by a mechanism other than, or in addition to, reversion of a specific base pair, point mutation. The mutability of this easily assayed nutritional genetic marker in a cell line that can be grown either in vitro or in vivo may provide a useful system for assay of other agents of unknown mutagenic potential.     

Comparative mutagenesis by aminofluorene derivatives A possible effect of DNA configuration

The mutagenicity of N-acetoxy-N-2-acetylaminofluorene-(N-acetoxy-2AAF) for Salmonella typhimuricum TA98 is greatly reduced when compared to that of N-hydroxy-2-aminofluorene. This decrease in mutagenic response is accompanied by the formation of a deoxyguanosine-2-acetylaminofluorene adduct. The deoxyguanosine-2-aminofluorene adduct, characteristic of cells exposed to N-hydroxy-2-aminofluorene, was not detected in N-acetoxy-2AAF-treated cells. Enzymic deacetylation of N-acetoxy-2AAF results in restoration of potent mutagenicity. N-Acetoxy-2-acetylamino-7-iodofluorene is also more mutagenic than N-acetoxy-2AAF. Because the acetylated and unacetylated guanine induce greatly different configurational changes, the results may be indicative that the introduction of the syn configuration and a possible shift to the Z-conformation at the mutational hot spot of Salmonella typhimurium TA98 [(dG-dC)₈] results in reduced mutagenic potency.     

Mutagenic evaluation of the monocyclic aromatic amine N-methylamino-2-nitro-4-N′,N′-bis(2-hydroxyethyl)aminobenzene in the Salmonella typhimurium/mammalian microsome test

The hair-dye component N-methylamino-2-nitro-4-N′,N′-bis(2-hydroxyethyl)aminobenzene was investigated for mutagenic activity in Salmonella typhimurium strains TA1535, TA100, TA1537, TA1538 and TA98. The testing was performed in the absence and in the presence of a rat-liver microsomal activation system induced by Aroclor 1254. Our results indicate that N-methylamino-2-nitro-4-N′,N′-bis(2-hydroxyethyl)aminobenzene does not induce mutations in Salmonella typhimurium strains, either in the absence or in the presence of the metabolic activation system. The purity of the compound was controlled by utilizing high-pressure liquid chromatography (HPLC) and thin-layer chromatography (TLC).     

Mutagenic activity of 6-aminoquinoxalines in Salmonella typhimurium

Mutagenicity of 6-aminoquinoxaline derivatives was tested with Salmonella typhimurium strains Ta98 and TA100 in the presence and absence of S9 mix from the viewpoint that the 6-aminoquinoxaline skeleton is a common unit of mutagenic imidazoquinoxalines. We tested nine compounds: 5-methyl-6-methylaminoquinoxaline (1), 3,5-dimethyl-6-methylaminoquinoxaline (2), 2,5-dimethyl-6-metnylaminoquinoxaline (3), 6-methylamino-2,3,5-trimethylquinoxaline (4), 2,3-diethyl-5-methyl-6-methylaminoquinoxaline (5), 5-methyl-6-methylamino 3-phenylquinoxaline (6), 6-amino-2,3,5-trimethylquinoxaline (7), 6-dimethylamino-2,3-5-trimethylaminoquinoxaline (8), 6-amino-2,3-dimethylquinoxaline (9). These compounds showed the mutagenic activity for both TA98 and TA100 in the presence of S9 mix, where they were more sensitive for TA100 strain. Methyl groups at the 2, 3 and/or 5 positions increased the potency of mutagenicity (1 < 2 < 3 ⪡ 4, 9 < 7). However, ethyl groups at the 2 and 3 positions lowered the mutagenicity of the methyl substitute but elevated it of the parental compound (1 < 5 < 4). A methyl group at the N⁶ position decreased the mutagenicity (7 > 4 > 8).     

Effect of sodium azide on sister-chromatid exchanges in human lymphocytes and Chinese hamster cells

Previous reports from this laboratory and others indicate that sodium azide is a unique mutagen. It is highly mutagenic in S. typhimurium TA1530 as well as in barley, rice, peas, yeast and Chinese hamster V79 cells. However, azide apparently does not produce chromosome breaks in barley, Vicia or human lymphocytes. Therefore, a study of the effects of azide on sister-chromatid exchanges (SCE) appeared warranted.Human whole blood and Chinese hamster K1 cell line were exposed for 4 and 2 h resp. to various concentrations of sodium azide ranging from 10⁻³ to 10⁻⁷ M. Cells were harvested and chromosomes stained by the FPG technique. In human lumphocytes, concentrations above 10⁻⁴ induced lethality whereas the K1 cell line was sensitive to concentrations above 10⁻⁵ M. The lower concentrations of azide produced no significant increase in SCE frequency above controls. Concurrent mitomycin C treatments produced significant increases in SCE levels.This apparent lack of induction of SCEs above background combined with previous data demonstrating negative clastogenic but very positive mutagenic activity of azide confirms the uniqueness of this mutagen. It would appear that azide is one of the few known potent mutagens that does not increase SCEs and/or break chromosomes.     

Reactions of plant copper/topaquinone amine oxidases with N6-aminoalkyl derivatives of adenine

Plant copper/topaquinone-containing amine oxidases (CAOs, EC 1.4.3.6) are enzymes oxidising various amines. Here we report a study on the reactions of CAOs from grass pea (Lathyrus sativus), lentil (Lens esculenta) and Euphorbia characias, a Mediterranean shrub, with N⁶-aminoalkyl adenines representing combined analogues of cytokinins and polyamines. The following compounds were synthesised: N⁶-(3-aminopropyl)adenine, N⁶-(4-aminobutyl)adenine, N⁶-(4-amino-trans-but-2-enyl)adenine, N⁶-(4-amino-cis-but-2-enyl)adenine and N⁶-(4-aminobut-2-ynyl)adenine. From these, N⁶-(4-aminobutyl)adenine and N⁶-(4-amino-trans-but-2-enyl)adenine were found to be substrates for all three enzymes (K_m～10^?4?M). Absorption spectroscopy demonstrated such an interaction with the cofactor topaquinone, which is typical for common diamine substrates. However, only the former compound provided a regular reaction stoichiometry. Anaerobic absorption spectra of N⁶-(3-aminopropyl)adenine, N⁶-(4-amino-cis-but-2-enyl)adenine and N⁶-(4-aminobut-2-ynyl)adenine reactions revealed a similar kind of initial interaction, although the compounds finally inhibited the enzymes. Kinetic measurements allowed the determination of both inhibition type and strength; N⁶-(3-aminopropyl)adenine and N⁶-(4-amino-cis-but-2-enyl)adenine produced reversible inhibition (K_i～10^?5–10^?4?M) whereas, N⁶-(4-aminobut-2-ynyl)adenine could be considered a powerful inactivator.     

Podophyllotoxin resistance: A codominant selection system for quantitative mutagenesis studies in mammalian cells

Mutants resistant to the microtubule inhibitor podophyllotoxin (Pod^R), a codominant marker, can be readily selected in various mammalian cell lines such as, CHO, HeLa, mouse L cells, Syrian hamster cells (BHK₂₁) and a mouse teratocarcinoma cell line OC15. In CHO cells, the recovery of Pod^R mutants is not affected by cell density (up to 1 × 10⁶ cells per 100-mm diameter dish), and after treatment with the mutagen ethyl methanesulfonate maximum mutagenic effect is achieved after a relatively short expression time (40–48 h). The frequency of Pod^R mutants in various cell lines increased in a dose-dependent manner in response to treatment with the mutagens ethyl methanesulfonate and N-methyl-N′-nitro-N-nitrosoguanidine. The Pod^R selection system thus provides a new genetic marker which should prove useful in studies of quantitative mutagenesis in mammalian cells.     

Aphidicolin allows a rapid and simple evaluation of DNA-repair synthesis in damaged human cells

The decrease in microbial mutagenicity of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and N-methyl-N-nitrosourea (MNU) was compared in an animal mediation with rats and in direct incubation with human as well as rat blood and blood components. The mutagenic activity was assayed by reverse mutation from streptomycin (SM) dependence to non-dependence in Escherichia coli, strain Sd-B (TC). The mutagenic response curves of both MNNG and MNU were approximately linear and parallel at non-cytotoxic concentrations. However, the mutagenic capabilities of MNNG were estimated to be 10-fold more potent than those of MNU. The mutagenic activity in blood and liver preparations from rats killed immediately after intravenous injection of MNNG, 50 mg/kg, was negative. Results with MNU, 100 mg/kg, were positive in both cases.For the detection of mutagenicity, blood was diluted 50 times for the final testing mixture (1 ml) to avoid bactericidal effects of the blood itself. When a larger amount of liver preparation was used in the tests, and diluted 8 times, mutagenic activity was still detected 15 min after injection of MNU, 80 mg/kg. Comparisons of the diminished rate of mutagenicity between MNNG and MNU during certain periods of incubation with blood indicated that MNNG was inactivated much more rapidly than MNU with both human and rat blood. Plasma showed a moderate inactivating effect on both MNNG and MNU. Red blood cells inactivated MNNG at a remarkably rapid rate similar to that of whole blood, but was less effective on MNU. In further experiments with red- cell components, the cell contents inactivated both MNNG and MNU at rates similar to those with red cells, but cell membrane had absolutely no effect in decreasing the mutagenicity in either MNNG or MNU.