Mutagenicity of organophosphorus compounds in bacteria and drosophila

140 Organophosphorus compounds (OP's) have been tested for mutagenic activity in bacteria, principally by using two specially constructed sets of tester strains of the bacteria Salmonella typhimurium and Escherichia coli. It was found that 20% gave positive mutagenic responses and that this group of chemicals produce base substitutions rather than frame-shift mutations. In most cases the DNA repair genes exrA⁺ and recA⁺ were for mutagenic activity.Seven compounds were further tested in Drosophila melanogaster for the ability to induce recessive lethal mutations. In some of these cases the doses administered to the flies had to be very low due to the highly toxic nature of the compounds. To overcome this problem, the accumulation of recessive lethal mutations was measured in populations which were continually exposed to the compounds over a period of some 18 months. During this time the populations developed increased resistance to the compound and so the dose administered could gradually be increased. Six of the compounds were mutagenic.Of the compounds tested in both systems, those showing mutagenic activity in bacteria were also mutaganic in Drosophila, those mutagenic in bacteria were not mutagenic in Drosophila.     

Non-phenotypic selection of N-methyl-N′-nitro-N-nitrosoguanidine-directed mutation at a predicted hotspot site

The striking mutational specificity of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) exhibited in the lacI gene in Escherichia coli allows comment on the phenotypic consequences of mutation at specific sequences that are not recovered after MNNG mutagenesis. We predict that the I⁺ phenotype is maintained when such silent positions are substituted by amino acids whose codons are generated by the MNNG-directed G:C → A:T transition. We chose the mutationally silent Gly200 codon (an MNNG hotspot motif sequence) to test this prediction. Through MNNG mutagenesis we have generated, identified and isolated a G:C → A:T transition at position 627 (5′-GG-C3′) under non-selective conditions which creates the Gly200→Asp substitution. The I⁺ phenotype is retained for this altered repressor.     

Effects of the umuC36 mutation on ultraviolet-radiation-induced base-change and frameshift mutations in Escherichia coli

The effects of the umuC36 mutation on the induction of base-change and frameshift mutations were studied. An active umuC gene was necessary in either the uvr⁺ or uvr^? strains of Escherichia coli K12 for UV- and X-ray-induced mutations to His⁺, ColE^R and Spc^R, which are presumably base-change mutations, but it was not essential for ethyl methanesulphonate or N-methyl-N′-nitro-N-nitrosoguanidine-induced His⁺ mutations. In contrast, only 1 out of 13 trp^? frameshift mutations examined was UV reversible, and the process of mutagenesis was umuC⁺-dependent, whereas a potent frameshift mutagen, ICR191, effectively induced Trp⁺ mutations in most of the strains regardless of the umu⁺ or umuC genetic background. These results suggest that base substitutions are a major mutational type derived from the umuC⁺-dependent pathway of error-prone repair.     

N(4)-amino-and N(4)-hydroxycytosines as base analogue mutagens

N(4)-Aminocytosine [N(4)NH₂C] and N(4)-amino-2′-deoxycytidine [N(4)NH₂dC] are highly mutagenic for Escherichia coli and phage φ 80 but not for T₄. There is some evidence that they are incorporated into the φ 80 DNA but $\text{[}{}^{14}\text{C}\text{]-N(4)}\text{NH}{}_2\text{C}$ could not be detected in the bacterial DNA.N(4)-Hydroxy-5,6-dihydro-6-hydroxylaminodeoxycytidine (di-NHOH-dC) is mutagenic for φ 80 and E. coli, but N(4)-hydroxydeoxycytidine [N(4)OH-dC] only has a strong inactivating effect.     

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.     

5-Methyltryptophan resistance mutations in Escherichia coli K-12. Mutagenic activity of monofunctional alkylating agents including organophosphorus insecticides

The induction of 5-methyltryptophan (5-MT) resistance mutations was assayed as a test system for mutagenic chemicals in Escherichia coli. It is assumed that different premutational alterations in several genes of the Escherichia coli chromosome will lead to 5-MT-resistant mutants. The chemicals used were three monofunctional alkylating agents as reference compounds, namely β-propiolactone (β-PL), N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), and methyl methanesulfonate (MMS), which are all mutagenic in the 5-MT system; of the eight organophosphorus insecticides tested, four have definite mutagenic activity (Dichlorvos, Oxydemetonmethyl, Dimethoate, and Bidrin), one is probably mutagenic (Methylparathion) and the remaining three (Parathion, Malathion and Diazinon) do not induce 5-MT resistance mutations in the conditions used here (< 30% survival). The relative mutagenic activity after a treatment time of 60 min is (in decreasing order) MNNG > MMS > Dichlorvos > Oxydemetonmethyl, Dimethoate and Bidrin. The concentration-dependent mutagenic activity of all mutagenic compounds is nearly linear when plotted on a log-log scale (with slopes varying from 1.0 to 1.5) and could be taken as an indication that one premutational reaction will be sufficient for the induction of one 5-MT-resistant mutant.     

Further studies on the induction of mutation in Haemophilus influenzae by N-methyl-N′-nitro-N-nitrosoguanidine: Lack of an inducible error-free repair system and the effect of exposure medium

Haemophilis influenzae is shown to lack the inducible, error-free repair system for alkylation damage that others have found in Escherichia coli. Prior growth in a low concentration of N-methyl-N′-nitro-N-nitrosoguanidine had only an additive effect on a subsequent brief exposure to a high concentration. Furthermore, chloramphenicol did not significantly modify the mutagenic response. In both respects, H. influenzae differs from E. coli. Experiments carried out in preparation for these tests showed that exposure to N-methyl-N′-nitro-N-nitrosoguanidine in complex growth medium was more effective by about an order of magnitude than exposure in pH 6.0 tris-maelare buffer in inducing mutations, in killing the cells, and in causing strand breaks in the preexisting DNA and gaps in newly synthesized DNA. Thus the effect of the medium is on the amount of initial damage rather than on some special feature of the mutation process. Part but not all of the effect can be accounted for by the difference in pH of the 2 media. The nature of the mutagenic process is the same under the 2 exposure conditions; i.e., reparable pre-mutational damage is produced by the agent and subsequently converted to final mutation by replication. The dose—effect curves have a non-linear initial portion under both exposure conditions, and possible reasons for this non-linearity are discussed.     

Integration of mammalian, microbial and drosophila procedures for evaluating chemical mutagens.

cis-Platinum(II)diamminodichloride (PDD), an anti-tumor agent, induced auxotrophic mutations in Escherichia coli, some of which were reverted to prototrophy by exposure to PDD, 2-aminopurine (2-AP), and N-methyl-N′-nitro-N-nitroguanidine (NTG), but not ICR derivatives. Similarly, various 2-AP-, NTG-, and ultraviolet light-induced auxotrophs were reverted to prototrophy by PDD. Some PDD-induced auxotrophs carried nonsense mutations and others could be phenotypically suppressed by growth with streptomycin. Although these findings suggest that PDD promotes base substitutions, this mutagen may also cause base subtractions because (like NTG)it induced, at reduced frequency, reversion to prototrophy of certain ICR-induced auxotrophs. Isomeric trans-platinum(II)diamminodichloride, which lacks anti-tumor activity, was an ineffective mutagen. Near-optimal conditions for PDD-induced mutagenesis entailed prolonged cultivation with low levels of mutagen where the frequency of forward mutation to auxotrophy was 10⁻³ and that of a selected trp isolate to prototrophy was 10⁻².     

Induced radioresistance: An aspect of induced repair

Summary Irradiation of Escherichia coli cells with UV or X-rays followed by incubation under conditions in which protein synthesis can occur results in a population of cells that is resistant to X-rays; however, this resistance develops only if the cells are recA ⁺ and lexA ⁺, a fact that associates the phenomenon with induced (S.O.S.) repair. By observing separately the component of a culture that is resistant and the component that retains its normal growth, the fraction of induced and uninduced cells for a dose of UV or X-rays can be estimated. Such estimates show that the dose-response for UV induction of resistant cells agrees with that of the recA gene product. Thus induced radioresistance is considered to be due to the changes in the cell occasioned by the derepression of recA and lexA. These changes are expected to be involved with the synapsis of homologous genomes that is necessary for the use of a second genome to repair damage occurring in both strands of a duplex at the same base, as exemplified by a double-strand break or an interstrand crosslink. This consideration is additionally supported by the increased resistance of cells grown to contain multiple genomes in the same envelope, an increased resistance not found in recA ^- or lexA ^- cells. The condition of a completed chromosome is also resistant, again not in recA ^- or lexA ^- cells. We suggest that cell killing by X-rays is due to the double-strand breaks which are not repaired by molecular synapsis before the arrival of the replication polymerase at the break.     

Inhibition of mutation induction and unchanged mutational specificity inEscherichia coli K12 overproducing the RecA protein

Induced mutagenesis was studied inEscherichia coli K12 cells in relation to the level of KecA-protein (P-RecA). In experiments strains AB2497, AB2497(pBR322) and AB2497(pX02) were used. The multicopy plasmid pX02 is a recombinant of pBR322 and recA⁺ gene ofE. coli K12. Cells carrying this plasmid overproduce the P-RecA constitutively. Mutagenesis was induced by the decay of incorporated 6-³H-thymidine. Mutations of theargE3 (ochre) to Arg⁺ prototrophy were followed. Besides the frequency of mutations, mutagenic specificity was determined. In cells AB2497(pX02) which overproduce the P-RecA the yield of Arg⁺ revertants was markedly reduced compared with that in strains AB2497 or AB2497(pBR322), whereas the mutagenic specificity was not changed. In all the strains studied the predominant type of mutation produced was the base substitution in the A: T base pair.     

The resistances of Micrococcus radiodurans to killing and mutation by agents which damage DNA

The resistance of Micrococcus radiodurans to the lethal and mutagenic action 3f ultraviolet (UV) light, ionising (γ) radiation, mitomycin C (MTC), nitrous acid (NA), hydroxylamine (HA), N-methyl-N′-nitro-N-nitrosoguanidine (NG), ethylmethanesulphonate (EMS) and β-propiolactone (βPL) has been compared with that of Escherichia coli B/r.M. radiodurans was much more resistant than E. coli B/r to the lethal effects of UV light (by a factor of 33), γ-radiation (55), NG (15) and NA (62), showed intermediate resistance to MTC (4) and HA(7), but was sensitive to EMS (1) and βPL (2). M. radiodurans was very resistant to mutagens producing damage which can be repaired by a recombination system, indicating that it possesses an extremely efficient recombination repair mechanism.Both species were equally sensitive to mutation to trimethoprim resistance by NG, but M. radiodurans was more resistant the E. coli B/r to the other multagens tested, being non-mutable by UV light, γ-radiation, MTC and HA, and only slightly sensitive to mutation by NA, EMS, and βPL. The resistance of M. radiodurans to mutation by UV-light, γ-radiation and MTC is consistent with an hypothesis that recombination repair in M. radiodurans is accurate since these mutagens may depend on an “error-prone” recombination system for their mutagenic effect in E. coli B/r. However, because M. radiodurans is also resistant to mutagens such as HA and EMS, which are mutagenic in E. coli in the absence of an “error-prone” system, we propose that all the mutagens tested may have a common mode of action in E. coli B/r, but that this mutagenic pathway is missing in M. radiodurans.     

Genetic activity of bleomycin in Escherichia coli

The induction of umuC gene expression, cell lethality, induction of W-reactivation of UV-irradiated λ-phage and the induction of mutagenesis caused by bleomycin (Blm) were studied in Escherichia coli K-12 strains with special references to the effects of SOS repair deficiencies. (1) The umuC gene is inducible by Blm and the induction is regulated by the lexA and recA genes. (2) The lexA and recA mutants are slightly more sensitive to Blm-killing than wild-type strain. (3) The plating efficiency of UV-irradiated λ-phage increased by Blm treatment of the host cell. This increase was not observed in the umuC mutant. The plating efficiency of UV-irradiated λ-phage was drastically reduced in the lexA and recA strains treated with Blm. (4) No significant increase of the reversion of nonsense mutation (his-4 to His⁺) in AB1157 by the treatment of Blm was observed. Possible implications of these results are discussed.     

Mutagenesis in Escherichia coli. II. Evidence for a common pathway for mutagenesis by ultraviolet light, ionizing radiation and thymine deprivation

Summary Both thymine starvation and gamma radiation, like ultraviolet light, produce base change mutations to prototrophy in Escherichia coli and the Exr⁺ phenotype is involved in the mutagenic process. DNA strand breakage is a direct or indirect consequence of all three treatments suggesting that the filling of gaps in DNA by a process involving the exr gene product may be a common step in mutagenesis.     

Microbioluminescent study of the general toxicity and mutagenicity of pollutants

Bacterial bioluminescence was applied to detection of general toxicity (MIT test) and genotoxicity (SOS-lux test) of some chemicals, seawater, and fresh water. The SOS-induced luminescence of E. coli WP2s (cda::luxCDABE) cells was higher than in E. coli C 600 (cda::luxCDABE) at 37°C and pH 6.5. The mutagenic effect of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), mitomycin C, and hydrogen peroxide determined from the induction of E. coli WP2s cell luminescence was detected at lower concentrations than in the assessment of reversion frequencies. General toxicity was demonstrated by using luminescence inhibition for hydrogen peroxide, Zn²⁺, and Cd²⁺ at low concentrations. Regions of the Krasnodar Krai where sea and fresh waters exerted toxic action on luminescence were determined by the microbioluminescent method.     

Analysis of the killing effect of Mu ligts mutants on host cells

Infection of Escherichia coli with the mutant lig ts2 of bacteriophage Mu at a temperature nonpermissive for this mutant is lethal for the host cells. This effect is insensitive to phage immunity of the host cells, to inhibitors of protein synthesis and is not suppressed in trans in bacterial strains producing the Lig⁺ active protein. These data suggest that the killing effect of this mutant is different from the other kil functions identified in Mu [1].     

In vivo benzo[a]pyrene diol epoxide-induced alkali-labile sites are not apurinic sites

We have used endonuclease IV from Escherichia coli as a probe for apurinic sites in the DNA of HeLa cells following treatment with an activated diol epoxide derivative of benzo[a]pyrene. DNA strand breaks and alkali-labile sites were observed that were repaired following exposure to the carcinogenic alkylating agent. The alkali-labile sites were not substrates for the apurinic site-specific endonuclease IV. We conclude that the alkali-labile sites formed in vivo by benzo[a]pyrene derivatives are not apurinic sites and probably arise as a consequence of rearrangement of the abundant N²-guanine adducts. This finding questions the involvement of apurinic sites in the mutagenic activity of benzo[a]pyrene.     

Restoration of mutability in non-mutable Escherichia coli carrying different plasmids.

Summary N and I group plasmids, which increase methylmethane sulfonate (MMS) mutagenesis in lexA ⁺ strains of E. coli WP2 may be divided into two classes: those restoring part of the mutability of lexA ^- strains (class I) and those leaving lexA ^- strains non-mutable (class II). Almost complete restoration of MMS mutability is obtained by class I plasmids in a partially suppressed lexA rnm strain, while class II plasmids cause far fewer MMS revertants in this strain than in lexA ⁺. A pair of class I and II plasmids in lexA ^- shows a synergistic effect on mutability. These two classes do not coincide with plasmid division into incompatibility groups.     

Simultaneous mutations up to six distal sites using a phosphorylation-free and ligase-free polymerase chain reaction-based mutagenesis

In this study, we present an efficient phosphorylation-free and ligase-free PCR-based multiple site-directed mutagenesis that allows simultaneous mutations up to six distal sites. This method could be extended to any plasmid DNA that is isolated from dam⁺Escherichia coli strains, and the results showed that the simultaneously mutagenic efficiencies of quadruple mutation and sextuple mutation were up to 80% and 40%, respectively.     

Selective modification of cytidine, uridine, guanosine and pseudouridine residues in Escherichia coli leucine transfer ribonucleic acid

The specificity of methoxyamine for the cytidine residues in an Escherichia coli leuoine transfer RNA (tRNA₁^leu is described in detail. Of the six non-hydrogen-bonded cytidine residues in the clover-leaf model of this tRNA, four are very reactive (C-35, 53, 85 and 86) and two are unreactive (C-67 and 79).The specificity of l-cyclohexyl-3-[2-morpholino-(4)-ethyl]carbodiimide methotosylate for the uridine, guanosine and pseudouridine residues in the leucine tRNA was also investigated. The carbodiimide completely modified four uridine residues (U-33, 34, 50 and 51) and partially modified G-37 and Ψ-39. For technical reasons, the sites of partial modification in loop I of the tRNA were difficult to establish. There was no modification of base residues in loop IV nor of U-59 at the base of stem e of the tRNA.The modification patterns described for the leucine tRNA are compared with those observed for the E. coli initiator tRNA₁^met and su⁺_III tyrosine tRNA. Several general conclusions regarding tRNA conformation are made. In particular, the evidence supporting a diversity of anticodon loop structures amongst tRNAs is discussed.