Analysis of a case of mutagen specificity in Neurospora crassa

Summary When ultraviolet irradiation of doubly auxotrophic conidia was preceded or followed by weakly mutagenic doses of DEB, the frequency of adenine-reversions was increased above additivity, while that of inositol-reversions was additive or—usually—was decreased below additivity. These interactions did not affect completed revertants nor were they due to plating interactions between potential revertants and the non-mutant background cells. The interaction was stronger when DEB was given as pretreatment than when it was given as post-treatment. During the DEB-treatment, sensitivity to interaction increased from the low effect observed with post-treatment to the higher one typical for pretreatment. Irradiation towards the end of the treatment period gave the same interaction as irradiation of treated and washed cells. In post-treatment experiments, the irradiated cells retained their capacity for interaction with DEB undiminished for at least on hour. In pretreatment experiments, the washed cells retained their capacity for interaction with UV over at least 16 minutes. After 2 hours, interaction was diminished; after 4 hours, it had disappeared.These results suggest a number of conclusions. (a) Interaction is mainly or wholly due to the effect of DEB on UV-induced mutations. (b) Interaction does not occur at the level of the primary lesions in DNA but at some later step in mutagenesis. (c) The mechanism of interaction is not the same for the two types of reversion. (d) The enhanced frequency of adenine-reversions is possibly due to inhibition of a repair enzyme by DEB. (e) The decreased frequency of inositol-reversions does not appear to be due to inositol-less death, but does seem connected with some specific phenotypic feature of inositol-reversions.     

Inactivation of transforming DNA by ultraviolet light. 3. Further observations on the effects of 365 nm radiation

In the doubly auxotrophic strain ad 3A 38701 inos 37401, nitrosoethylurethane (NEU) produces a storage effect for adenine reversions but not for inositol reversions. Shaking treated spores in water for several hours destroys their response to storage. Short heat-treatment during or before plating increases the frequency of adenine reversions but not that of inositol reversions. Storage and heat-treatment decrease the inositol-specificity of NEU and may reverse it into adenine-specificity. Comparison with similar results obtained for diepoxybutane (DEB) shows that the cellular effects of NEU are more complex than those of DEB.     

Inactivation of transforming DNA by ultraviolet light. I. Near-UV action spectrum for marker inactivation

The storage effect is defined as an increase of mutational damage after cessation of treatment. It differs from other kinds of delayed effect (replication errors, replicating instabilities) in not requiring replication of DNA. In Neurospora ad3A 38701 inos 37401 diepoxybutane (DEB) yields a storage effect for adenine reversions, but none for inositol reversions. The storage effect takes place in treated washed spores that are sedimented in a centrifuge tube, but not in spores that are agitated in water. Under the latter conditions, response to storage is gradually lost. The storage effect can be imitated by administering very small amounts of DEB to cells that had been previously treated with a moderately high dose (booster effect). During post-treatment shaking in water, responses to booster and to storage conditions disappear together; response to booster disappears at the same rate in spores that are sedimented in centrifuge tubes. No mutagenic action could be detected in eluates from heavily treated cells. We have concluded that treatment with DEB sensitizes the conidia to further small doses of DEB whether these are administered extraneously as booster or present intracellularly during storage. Sensitization is lost in the course of a few hours in shaken as well as in sedimented spores. Thus, while the storage effect is due to traces of mutagen, its gradual disappearance after treatment is not due to loss of these traces.Correlated with the ability to yield a storage effect, and probably part of the storage effect, is the response to temperature between treatment and plating. Conidia that can give a storage effect yield fewer mutations when spread on cold agar than when inplated into warm agar or heat-shocked before spreading; the excess of mutation under the latter two conditions forms part of the final storage effect. The true base line for calculation of the storage effect is therefore mutation frequency among spread spores.For DEB as mutagen, response to storage by the adenine locus and lack of response by the inositol locus are correlated with the responses of these two loci to dose of DEB and to combination treatment of DEB with UV, or DEB with nitrous acid (NA). This makes it possible to fit all observations into the picture of a general hypothesis on the cellular effects of DEB. Because of the differential response of the two loci, storage and plating procedures offer two additional means for manipulating specificity in this system.     

Analysis of a case of mutagen specificity in Neurospora crassa. 3. Fractionated treatment with diepoxybutane (DEB)

Summary Weak doses of DEB given before or after a moderately high dose act as booster for the production of adenine-reversions. Fractionation of a moderate or high dose into a succession of weak ones yields a dose-effect curve that lies above the linear curve expected for additivity of the fractions but below that found after continuous exposure. The results lend support to the view that DEB, in addition to producing potential adenine-reversions in DNA, promotes their realization by its effect on some cellular process or processes, and that this is the cause of the steep dose-response to continuous exposure.     

Comparison of N-ethyl-N-nitrosourea Effects in three strains of chlorococcal algae

Three strains of three different species of chlorococcal algae(Chlorella kesslerii Scenedesmus quadricauda andScenedesmus obliquus) were compared in their physiological and genetical responses to N-ethyl-N-nitrosourea (NEU). The mutagen was applied in the basic doses corresponding to their specific tolerance to it. Exponentially-graded doses derived from the basic ones acted for the same period of time and single basic doses acted at linearly-increased periods of time in parallel experiments. The influence of the mutagen on the cell cycle just in progress was characterized by the increase in the frequency of cell divisions which yielded a lower number of autospores. The conditions of the cells during the first three days after treatment were determined according to the average increase of their lag phase. The relative toxicity of the mutagen was expressed as the survival frequencies. The mutation effects were evaluated according to the frequency of permanent changes observed in the growing cell colonies : lethal, morphological and pigmentation. NEU was found to be a relatively suitable mutagen for the chlorococcal algae studied.     

The interaction between diepoxybutane and ultraviolet light in yeast II. Experiments with UV-sensitive and resistant strains

Earlier experiments in which the effects of diepoxybutane (DEB) pretreatment on UV inactivation were studied in wild-type yeast, have been extended to include DEB pretreatment effects in the strain uvs- which is sensitive to both UV and DEB. The effects of post-UV treatment with DEB have also been studied in both strains.

DEB pretreatment produces a similar result in uvs- as the effect described earlier for wild-type in spite of the necessary reduction in doses of both agents. Post-treatment with DEB fails to affect photoreactivation in both resistant and sensitive strains but in the resistant strain the shoulder on the UV curve is obliterated. There is no such effect in the UV-sensitive material.     

Survival and liquid holding recovery in UV-sensitive strains of Saccharomyces cerevisiae after treatment with chemical mutagens and radiation.

Two UV-sensitive mutants of Saccharomyces cerevisiae rad 3 and rad 6 were tested for sensitivity to X-rays, MMS, EMS, HNO2 and DEB. Rad 3 mutant is more sensitive than the wild type strain only to HNO2 and DEB, while rad 6 is cross sensitive both to X-rays and all chemicals tested. Liquid holding recovery (LHR) was studied by comparison of cell survival immediately after mutagen treatment and after 5 days of storage in phosphate buffer. LH greatly increases cell survival of rad 3 mutant after DEB and slightly after EMS, MMS and HNO2, while after UV treatment LH significantly decreases survival of this mutant. LH increases survival of rad 6 mutant after exposure to UV, MMS and HNO2, but decreases survival of DEB-treated cells. Exposure of wild type strain to LH results in an increase of survival after UV, and DEB but not after MMS and HNO2. The results suggest that LHR is a strain- and mutagen-specific phenomenon and cannot be explained within the present knowledge of repair processes in yeast.     

The multiple-cluster mutation complex in mutagenesis with higher plants

Summary After treatment of dry and pre-soaked seeds of barley with gamma-rays, EMS, NEU and EI, the frequency of multiple mutations (multimutations) was higher with EMS and NEU treatment, while cluster mutations appeared in greater numbers following treatment with gamma rays and NEU. Pre-soaking the seeds led to a reduction in the frequency of total mutations, cluster mutations and multimutations. This has been explained as a result of the application of lower doses and the induction of mutations at a relatively later stage in ontogenetic development in the case of pre-soaked seeds.Some new mutation types in barley have been described and some of the old types have been given names representing the mutation characters more precisely.The compound mutation frequency of different seedling mutation types, when taken separately, was found to be independent of the mutagen employed and the stage of treatment. The size of mutated chimeras in M ₁ plants, as indicated by the segregation ratio of mutants in M ₂, was largest in albina, xantha, chlorina, albina-tigrina, chl-terminalis and eceriferum, and lowest in viridis, viridoalbina etc. This could be expected if the unstable premutations induced by mutagenic treatment are resolved into mutations at different intervals after their initiation, or it can be explained by the induction of dominant mutations, or lethal changes together with visible mutations.     

Mutagen-sensitive cell lines are obtained with a high frequency in V79 Chinese hamster cells

A replica-plating technique has been adopted for the isolation of mutagen-sensitive mutants of Chinese hamster V79 and CHO cell lines. After the mutagenic treatment (ENU) clones derived from these cell lines were replica plated into micro wells and replicas were treated with UV (254 nm), X-ray, MMC, EMC or MMS. Clonal cell lines which demonstrated mutagen sensitivity were retested by the determination of survival. Only one UV-sensitive line was obtained in 1500 clonal lines derived from CHO cells. This mutant appeared also sensitive to 4NQO and MMC. The sensitivity to UV and MMC was 2-3-fold enhanced, while the increase in sensitivity to 4NQO was 4-5-fold. In V79 cells 9 mutagen-sensitive lines were found after screening of 500 clonal lines; six of them showed increased sensitivity towards UV, two towards MMC, and one cell line was found to be X-ray sensitive. A considerable cross-sensitivity for the various agents was found among the isolated mutants. When a 2-fold increase is taken as a minimum to indicate mutagen sensitivity 6 mutants were sensitive to UV, 8 mutants were sensitive to MMC, 6 mutants were sensitive to 4NQO and 4 mutants were sensitive to X-rays. The difference in sensitivity to UV versus 4NQO makes it unlikely that 4NQO can be considered as a UV-mimetic agent. The sensitivity to MMC appears to fall into 2 classes: a class with moderate sensitivity (2-8-fold) and a class with high sensitivity (30-100-fold). The presence of similar classes is indicated for UV. Except for the two lines V-E5, V-B7 and the two lines V-H11, V-H4 all obtained mutants have a different spectrum of mutagen sensitivities which suggests that different genetic alterations underly these effects. The observed high frequency of mutagen-sensitive mutants in V79 cells, although unexpected and substantially higher than those published for CHO cells and L5178Y cells, can still be explained by the presence of functionally hemizygous loci.     

Mutagenic action of dimethyl sulfate and N-nitrosoethylurea on the mulberry silkworm

Mutagenic effect of chemical mutagens, dimethalsulphate (DMS) and N-nitrosoethyl urea (NEU), on silkworm is studied. The effect of these mutagens have never been studied on silkworm before. DMS is found to be completely insufficient as mutagen for the silkworm, because its low mutability is accompanied with strong cytotoxic effect. NEU appeared to be of "milder" effect. Injection of male-moth proved to be very efficient. The majority of exuded mutants showed mosaic colouring of the graine.     

Comparison of sensitivity and liquid holding recovery in rad mutants of Saccharomyces cerevisiae inactivated by UV and DEB.

Summary Twenty one UV-sensitive rad mutants were tested for their sensitivity towards DEB. All mutants were more sensitive to this treatment than the wild type. Seven mutants were classified as supersensitive to DEB (radl-1, 2, 3, 6, 15 and 18-2), while only rad2 and rad3 can be classified as supersensitive to UV. For all mutants ability for liquid holding recovery (LHR) after UV and DEB was compared. Mutants radl-1, 3, 5, 6, 9 and 11 differ in their response to LH afterr the two treatments. Survival of radl-1 and rad3 increases significantly during LH after DEB but not after UV exposure. In contrast rad5, 6, 11 and 22 show marked LHR after UV but no increase of survival after DEB treatment.     

Biological effect of low doses of alkylating agents in drosophila studies

The low dose (0.05-0.1 mM) influence of alkylating agents on germ cell survival and male fertility, the level of embryonic and postembryonic lethality as well as the sex-linked recessive lethal (SLRL) frequency induced by high alkylating agent doses was studied in Drosophila melanogaster. The pretreatment of adult males with low doses of methyl and ethyl methanesulfonate (MMS and EMS) did not change or even enhanced EMS cytotoxicity and mutagenicity in both mature sperm and premeiotic cells. On the contrary, the low EMS dose pretreatment of larvae protected them against higher mutagen doses increasing male fertility, decreasing embryonic and postembryonic lethality in F1, and leading to three-fold reduction in the SLRL frequency in F2. The adaptive response was dependent on the Drosophila developmental stage exposed to challenge mutagen doses, since the protection was maximal in larvae and practically absent when the high dose was administered to adult males. The adaptive response observed does not seem to be associated with DNA repair, but it is rather due to other protective mechanisms.     

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.     

Dependency of the yield of sister-chromatid exchanges induced by alkylating agents on fixation time. Possible involvement of secondary lesions in sister-chromatid exchange induction

Chinese hamster V79 cells were pulse-treated (for 60 min) with various mutagens three, two or one cell cycles before fixation (treatment variants A, B and C, respectively) and the frequencies of induced SCEs were analysed and compared. The degree of increase in frequency of SCEs with dose in the treatment variants depended on the mutagen used. For the methylating agents MNU, MNNG and DMPNU, high yields of SCEs were obtained in the treatment variants A and B, and there was no difference in the efficiency with which these agents induced SCEs in these treatment variants. In the treatment variant C, however, no SCEs were induced with mutagen doses yielding a linear increase in SCE frequency in treatment variants A and B. A slight increase in SCE frequency in treatment variant C was observed only when relatively high doses of MNU or MNNG were applied. Like the above agents, EMS, ENU and MMS induced more SCEs in treatment variants A and B than in C, but for these agents treatment variant B was most effective and SCEs were induced over the entire dose range, also in treatment variant C. As opposed to the methylating and ethylating agents, MMC induced SCEs with high efficiency when treatment occurred one or two generations prior to fixation. There was no difference in SCE frequency between these treatment variants. MMC was completely ineffective for the induction of SCEs when treatment occurred three generations before fixation. The unexpectedly low SCE frequencies induced by the methylating and ethylating agents when treatment occurred one generation before fixation were not due to the exposure of cells to BrdU prior to mutagen treatment. From the results obtained, it is concluded that DNA methylation and ethylation lesions give rise to SCEs only with very low probability during the replication cycle after the lesion's induction, and that subsequent lesions produced during or after replication of the methylated or ethylated template (secondary lesions) are of prime importance for SCE formation after alkylation. For MMC, however, primary lesions seem to be most important for SCE induction.     

Uv Sensitivity of Several Genetic Markers of HAEMOPHILUS INFLUENZAE DNA

Studies on inactivation by UV of several genetic markers of the streptomycin and novobiocin resistance regions of H. influenzae DNA have shown that two kinds of markers may be found with respect to frequency of integration, UV sensitivity and reactivability. One class of markers has high integration frequency and low UV sensitivity, which can be partly explained by the reactivability of the irradiated markers. The second class of markers has low integration frequency and high UV sensitivity. Markers of each class have been found in the novobiocin region.-In one recipient strain with decreased transformability the difference in integration frequency between the two classes is enhanced.     

Analysis of replication of DEB-alkylated DNA in yeast: bypass replication in a rad3 mutant of Saccharomyces cerevisiae

We presented indirect evidence that in an excision-deficient rad3 mutant of yeast exposed to diepoxybutane (DEB), DNA synthesis continued past the damaged sites. This bypass replication was confined to the first post-treatment round of replication and was followed by inhibition of DNA synthesis. Analyses by alkaline sucrose gradient sedimentation and by alkaline elution from filters revealed that in mutant cells the first post-treatment round of replication proceeded at a similar rate to that in untreated cells and was not accompanied by strand scission of template DNA. The post-treatment synthesis was presumably of an error-prone type, as the frequency of reversion to ade2-1 prototrophy was increased. In contrast, in the isogenic wild-type strain, the post-treatment incorporation of radioactivity into DNA was slightly reduced and newly replicated DNA fragments were of lower molecular weight than in control cells. There was also some strain scission in template DNA, presumably resulting from excision-repair.     

Cytogenetical characterization of UV-sensitive repair-deficient CHO cell line 43-3B. II. Induction of cell killing, chromosomal aberrations and sister-chromatid exchanges by 4NQO, mono- and bi-functional alkylating agents

An established cell line of Chinese hamster ovary (CHO-9) cells and its UV-sensitive mutant 43-3B have been studied for the induction of cell killing, chromosomal aberrations and sister-chromatid exchanges (SCEs) after exposure to different types of DNA-damaging agents such as 4-nitroquinoline-1-oxide (4NQO), mitomycin C (MMC), diepoxybutane (DEB), methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS) and ethyl nitrosourea (ENU). In comparison with the wild-type CHO cells, 43-3B cells showed very high sensitivity to the UV-mimetic agent 4NQO and the DNA cross-linking agents MMC and DEB. The 43-3B cells responded with higher sensitivity to the monofunctional alkylating agents (MMS, EMS and ENU). The increased cytotoxic effects of all these chemicals correlated well with the elevated increase in the frequency of chromosomal aberrations. In 43-3B cells exposed to 4NQO, MMC or DEB the increase in the frequency of chromosomal aberrations was much higher than the increase in the frequency of SCEs (4-10-fold) when compared to the wild-type CHO cells. This suggests that SCEs are results of fundamentally different cellular events. The responses of 43-3B cells to UV, 4NQO, MMC and DEB resemble those of 2 human syndromes, i.e., xeroderma pigmentosum and Fanconi's anemia. These data suggest that 43-3B cells are defective in excision repair as well as the other pathways involved in the repair of cross-links (MMC, DEB) and bulky DNA adducts (4NQO).     

Effect of triflumuron on brood development and colony survival of free-flying honeybee, Apis mellifera L.

Abstract:  The effect of the insect growth regulator (IGR) triflumuron (Alsystin^® 25 WP) on honeybee, Apis mellifera L. (Hym., Apidae), was studied in a semi-field test. Free-living colonies were fed one litre per hive of sucrose syrup containing 0, 0.025, 0.25 or 2.5 g of triflumuron. A significant reduction in flight activity was noted 6–10 weeks post-treatment at the two higher doses. These colonies reared less brood than before treatment. While the comb area occupied by uncapped brood was as high as [0.025 and 0.25 g active ingredient (a.i.)] or higher (2.5 g a.i.) than before treatment, there was a significant decline in capped brood at the two higher doses, indicating enhanced larval mortality. No capped brood was reared in the hive treated at the highest dose from 3–9 weeks post-treatment. Yet there was a significant accumulation of pollen and honey in the brood compartment at all doses. All colonies except the one treated at the highest dose survived the following winter. However, at 43 weeks post-treatment, hives treated at intermediate and low doses showed a significant increase in uncapped brood and a significant decrease in capped brood. This study revealed a strong residual toxicity of triflumuron to brood and substantiated its classification as hazardous to honeybee.     

Factors affecting the quantitation of dose-response curves for mutation induction in V79 Chinese hamster cells after exposure to chemical and physical mutagens.

Using four common mutagens, ethyl methanesulphonate (EMS), methyl methanesulphonate (mms), uv, and X-irradiation, the relationship between dose of mutagen, cellular lethality and frequency of 8-azaguanine resistant colonies in V79 Chinese hamster cells has been examined. Several factors affecting the recovery of mutants including inter and intra-clone metabolic co-operation have been quantitated and their influence on survival response curves examined. Induced mutant frequencies were assayed by two methods in situ, and after replating. After exposure to X-rays, MMS and UV a significantly higher frequency of mutants was observed in replated experiments as compared with the in situ situation, at all survival levels assayed. With EMS, an increment on replating was observed only at high survival levels. The replating data suggest that two types of azgr colonies are produced, i.e. those which contain only azgr cells and those which, due to damage segregation, contain a mixture of azgr and azg8 cells. These mixed colonies appear to be lost by metabolic co-operation when mutation frequencies are assayed in silu. The proportion of mixed to homogeneous colonies differs with different mutagens. Taking into account such factors, EMS and UV irradiation were similarly mutagenic at a given survival level, but at equitoxic doses, fewer mutants were recovered after exposure of V79 cells to MMS and X-rays.