Clastogenic Adaptation Research Articles

A comparison of N-methyl- N-nitrosourea-induced chromatid aberrations and micronuclei in barley meristems using FISH techniques

Chromatid aberrations (CA) and micronuclei (MN) in combination with fluorescent in situ hybridization (FISH) using telomere- and centromere-specific probes were studied to compare the cytogenetic effects of N-methyl- N-nitrosourea (MNU) on root tip meristem cells of barley ( Hordeum vulgare). A similar dose-dependent increase was observed for CA and MN. The frequency of MN with telomere and/or centromere-specific signals corresponded well with the expectation derived from the frequency of the different types of aberrations. Thus, the micronucleus test offers an easy and fast assay to measure chromosome damage and clastogenic adaptation in barley meristems. Combined with FISH it is also possible to elucidate the origin of MN and to discriminate between aneugenic and clastogenic effects.

Adaptation to alkylation damage in DNA measured by the comet assay.

The alkylating mutagens N-methyl-N-nitrosourea (MNU) and methyl methanesulfonate (MMS) were studied for their potential to induce DNA strand breaks and abasic (AP) sites in meristematic nuclei of Vicia faba root tips by the comet assay. The alkaline unwinding/neutral electrophoresis (A/N) and alkaline unwinding/alkaline electrophoresis (A/A) protocols were used for detection of DNA damage. With the A/N comet assay, less DNA damage was seen after conditioning pretreatment with a low dose prior to a high challenging dose of alkylating mutagens as compared to application of the high dose only, whereas a nearly additive effect was seen when the A/A comet assay was used. Adaptation was even more obvious when AP sites were revealed by the AP-endonuclease activity of exonuclease III. The adaptation observed with the A/N comet assay was abolished by pretreatment with the protein synthesis inhibitor cycloheximide. These data suggest that the comet assay is able to detect on molecular level a phenomenon resembling clastogenic adaptation.

Removal of O6-methylguanine from plant DNA in vivo is accelerated under conditions of clastogenic adaptation.

Previously it was shown that the clastogenic efficiency of high doses of alkylating agents in plant root meristems can be reduced significantly by conditioning pretreatment with either a low dose of the same agents, a sublethal heat shock, or heavy metal salts. The molecular mechanisms responsible for these protective effects are still unclear. Here we report on the quantification of O6-methylguanine [O6-MeG] by immuno-slot-blot analysis in DNA of root tip meristems of field bean (Vicia faba) seedlings under conditions of clastogenic adaptation. When root tips were pretreated with a low, conditioning dose of N-methyl-N-nitrosourea (MNU, 10(-4) M, 1 hour) 2 hours before exposure to a high dose of the same clastogen (10(-3) M, 1 hour), the frequency of chromatid aberrations was reduced by more than 50% at a recovery time of 1 B hours, as compared to treatment with the high dose alone. The same was observed when conditioning pretreatment was by a sublethal heat shock [10 minutes, 40 degrees C] or a heavy metal salt (Cd(NO)3, 10(-7) M, 1 hour). The frequency of O6-MeG immediately after exposure to a conditioning and a subsequent challenge treatment was reduced by 43% as compared to treatment with only the high dose. At a recovery time of 18 hours the corresponding frequency of adducts was reduced by 68.3% (related to the initial level) after treatment with the high dose alone, and by 81.3% under adaptive conditions. Sublethal heat shock or heavy metal salt used as conditioning pretreatments also resulted in a decrease of adducts immediately after treatment with the challenge dose. From these data and from prevention of the effects by pretreatment with cycloheximide or O6-benzylguanine we conclude that under conditions of clastogenic adaptation O6-MeG is more efficiently removed from the DNA, presumably by induction of an alkyl acceptor protein such as O6-methylguanine-DNA methyltransferase [MGMT]. This could explain the observed protective effects (clastogenic adaptation.

Radioadaptation for gene mutation and the possible molecular mechanisms of the adaptive response

This paper reviews the experimental results showing that a prior exposure to a low dose of ionsing radiation induces an adaptive response expressed as a reduction of gene mutation in various cell systems. The data show that the mutagenic adaptation shares common features with the clastogenic adaptation, i.e., priming dose level, kinds of conditioning agents, time interval between conditioning and challenging treatments, degree of induced protective effect (40–75%), transitory response and inhibition by 3-aminobenzamide, a DNA repair inhibitor. Moreover, the deletion-type mutations are predominantly reduced in adapted cells, suggesting that the mechanism underlying mutagenic adaptation preferentially facilitates the removal of the DNA lesions leading to deletion-type mutations. These lesions are thought to be double-strand breaks which are likely to be also involved in the production of chromosomal damage. Recent findings on the molecular processes implicated in the cellular response to rediation provide some clues for the mechanisms that could be triggered by low-dose exposure and ultimately contribute to the protective effect. These is some evidence that the protein kinase C-mediated signalling pathway is a key step for the transduction of the low-dose-induced signal. Several recent reports indicate that the low-dose triggers changes in the expression of several genes whose products, though most of them are still not identified, would be related to DNA repair and/or control of cell cycle progression.

Induction of chromatid aberrations by TEM and maleic hydrazide is differently affected by pretreatment of Vicia faba root-tip meristems with methyl iodide

Treatment of Vicia faba main root meristems with methyl iodide (MeI) 2 h before challenge treatment with triethylene melamine (TEM) significantly reduced the yield of metaphases with chromatid aberrations, i.e., resulted in clastogenic adaptation. Combined treatment with MeI and TEM increased the aberration yield; MeI treatment alone (10(-3) M, 0.5 h) was without clastogenic effect. No protective effects were observed after MeI pretreatment and challenge treatment by maleic hydrazide (MH). The data obtained in V. faba are compared to those previously reported for E. coli.

Effects of ethidium bromide and nalidixic acid pretreatment on the induction of chromatid aberrations by TEM and maleic hydrazide in Vicia faba main root meristems

Pretreatment of Vicia faba main root meristems with ethidium bromide (EB) or nalidixic acid (NA) significantly reduced the yield of metaphases with chromatid aberrations induced by maleic hydrazide (MH), i.e., triggered clastogenic adaptation to MH. No such protection occurred when the alkylating agent triethylenemelamine (TEM) was used for challenge treatment. The differential response of pretreated cells to MH on the one hand (protection) and to TEM (no protection) on the other supports the conclusion that clastogenic adaptation is due to different inducible (repair?) functions, which eventually exert protection against clastogenic impacts.

Clastogenic adaptation of plant cells — reduction of the yield of clastogen-induced chromatid aberrations by various pretreatment procedures

This paper provides an overview of published and unpublished results obtained after various pretreatments (conditioning) of Vicia faba root tip meristems (low clastogen doses, heat shock, ammonium chloride and zinc sulfate) on a subsequently administered challenge clastogen dose. Such pretreatments may render root tip meristem cells less responsive to clastogen challenging as evidenced by lower yields, as compared to those obtained after challenging alone, of induced chromatid aberrations. This clastogenic adaptation was found to depend on the agents being used for conditioning and challenging, on the dose of the agents used for pretreatment, and on the time span between conditioning and challenging. The data presently at hand allow us to conclude that various stressors trigger (lesion-specific) cellular functions (repair processes?) that eventually alleviate the clastogenic effect of challenge treatment. Clastogenic adaptation was observed for S-phase-dependent alkylating and non-alkylating clastogens (maleic hydrazide) and also for S-phase-independent agents (bleomycin, X-rays).

Chromosomal responses to ionizing radiation reminiscent of and adaptive response in cultured Chinese hamster cells

When Chinese hamster V79 cells were internally exposed to low level chronic β-rays from incorporated tritiated thymidine ( 3H-dThd), the showed an “adaptive” response to the induction of chromosomal damage by subsequent higher acute doses of γ-rays. The yield of sister-chromatid exchanges (SCEs) in the 3H-dThd pretreated cells was less than the yield induced by γ-rays alone (protective effects), and the micronucleus frequency was less than the sum of the induced frequencies by 3H-dThd and γ-rays separately (below-additivity effects). No adaptation to the micronucleus induction by γ-rays was observed after the 3H-adapted cells had divided once and when 3-aminobenzamide (3AB) was given before the challenge doses. The cross-resistance study revealed that the 3H-adapted cells were resistant to SCE induction but not to the micronucleus inductions by the challenge doses of reactor radiations. The results suggest that the SCE adaptation and the micronucleus adaptation or clastogenic adaptation are probably caused by different, inducible adaptive repair pathways.

Effects of benzamide pretreatment on clastogenic adaptation of Vicia faba root-tip meristem cells to triethylenemelamine (TEM) and maleic hydrazide (MH)

Clastogenic adaptation to TEM or MH no longer occurred when benzamide, an inhibitor of nuclear ADP-ribosyltransferase (ADPRT), was applied prior to the low dose (conditioning) treatment which triggers this phenomenon. This may be indicative that inducible processes connected with ribosylation reactions are involved in the protective effects exerted by clastogenic adaptation. No increase by benzamide pretreatment was observed in the yield of metaphases with TEM- or MH-induced chromatid aberrations after conditioning and challenge treatment, respectively. High benzamide concentrations (1 h, 5 × 10 −3 M) exerted protective effects against TEM challenging but not against MH.

Clastogenic adaptation triggered by S-phase-independent clastogens in Vicia faba

Conditioning pretreatment of Vicia faba root-tip meristem cells with small doses of X-rays (0.06 Gy) or bleomycin, S-phase-independent clastogens, significantly reduced the yield of metaphases with chromatid aberrations induced by challenge doses of the same clastogens, administered 2 h after conditioning, i.e., resulted in clastogenic adaptation. Both clastogens were found to be able to substitute for each other in arousing protective effects (clastogenic cross-adaptation).

Effects of ‘G2-repair’ inhibitors on ‘clastogenic adaptation’ in Vicia faba

Vicia faba root tip meristem cells were treated with low doses of the clastogens maleic hydrazide (MH) and N-methyl-N-nitrosourea (MNU) or sublethal heat shock and 2 h later with a high dose of MH or MNU, respectively. This procedure results in ‘clastogenic adaptation’, i.e., a lower yield of aberrations than after treatment with the high clastogen doses alone. When an additional post-treatment with inhibitors of ‘G2-repair’, such as hydroxyurea (HU), 5-fluorodeoxyuridine (FdUrd), or 2-deoxyadenosine (dAdo), was performed, the protective effect triggered by low dose pretreatment was completely abolished, especially at early fixation times: The aberration yields observed were as high as or higher than after combination of only the high clastogen dose with inhibitor post-treatment. The most probable interpretation of the results seems to be: Inhibition of ‘G2-repair’ increased transformation into aberrations of potentially clastogenic lesions (DNA single- and double-strand breaks) which normally become correctly repaired. This may occur to a similar extent as aberration formation is avoided by repair of preclastogenic lesions (base damages) during S-phase by inducible processes termed “clastogenic adaptation’.

Antimutagenic effects of stressors in higher plants

The protective (antimutagenic) effect triggered by ‘conditioning’ treatment with low doses of mutagens, sublethal heat shock or compounds known as inducers of stress proteins prior to ‘challenge’ treatment with a high clastogen dose is described. Criterium of protection (‘clastogenic adaptation’) is the reduced quantity of chromatid aberrations induced by combination of ‘conditioning’ plus ‘challenge’ treatment as compared to only ‘challenge’ treatment of root tip meristems of higher plants (Vicia faba andHordeum vulgare). This anticlastogenic effect is absent in metabolically inactive cells and becomes counteracted by impairment of protein synthesis or inhibition of ‘G2 repair’. The data are indicative of at least 3 different inducible repair functions specific for certain types of DNA damage and reminiscent of those involved in the ‘adaptive response’ of bacterial and mammalian cells.

‘Clastogenic cross-adaptation’ is dependent on the clastogens used for induction of chromatid aberrations in Vicia faba root-tip meristems

“Clastogenic cross-adaptation” is the triggering, by “conditioning⌉ pretreatment with a low dose of one clastogen, of protective effects against the induction of chromatid aberrations by another (“challenge” treatment) clastogen. All alkylating agents tested in this respect (TEM, NMU, EMS) were found to be able to substitute for each other in arousing such protective effects in Vicia faba root-tip cells. Combinations of MH (pretreatment) with TEM or NMU (‘challenge’ treatment), and vice versa, did not result in clastogenic cross-adaptation. The data are discussed as to the causes (inducible functions) which may give rise to clastogenic adaptation.

‘Clastogenic adaptation’ in barley: Differential response of presoaked and dry seeds

‘Clastogenic adaptation’ in barley: Differential response of presoaked and dry seeds

Heat-shocks prior to treatment of Vicia faba root-tip meristems with maleic hydrazide or TEM reduce the yield of chromatid aberrations

Heat-shocks (10 and 30 min at 40°C) prior to treatment with MH or TEM significantly reduced the yield of metaphases with chromatid aberrations. No such effect was observed when ethanol was used for aberration induction. The ‘heat-shock effect’ on aberration induction by MH and TEM is comparable to ‘clastogenic adaptation’ observed after pretreatment (‘conditioning’) with low clastogen concentrations prior to ‘challenging’ with high clastogen concentrations; both require unimpaired protein synthesis.

Pretreatment of Vicia faba root-tip meristems with hydrazines results in ‘clastogenic adaptation’ to maleic hydrazide

Pretreatment of Vicia faba root-tip meristems with a non-toxic dose of either hydrazine or N, N′-diformyl hydrazine prior to administration of MH (separated by 2 h) resulted in a significant reduction of the yield of MH-induced chromatid aberrations compared to control treatments (MH only). This ‘clastogenic adaptation’ was not observed when the alkylating agent TEM was used instead of MH. It is concluded that pretreatment with the hydrazines induces an error-free repair system which reduces MH-induced damage and that both hydrazines and MH are able to induce oxidative DNA lesions via radical formation.

Peroxide pretreatment of Vicia faba root-tip meristems results in ‘clastogenic adaptation’ to maleic hydrazide but not to TEM

Summary Hydrogen peroxide and tert.-butyl hydroperoxide applied in non-toxic doses prior to ‘challenge’ treatment of Vicia faba root-tip meristems with maleic hydrazide (MH), significantly reduced the aberration yield induced by MH. No such effect was observed when TEM was used for ‘challenge’ treatment. The data obtained are interpreted as indicating that both the peroxides and MH induce oxidative DNA lesions (radical formation) which eventually result in structural changes in the chromatids. ‘Conditioning’ treatment with the peroxides is supposed to induce an error-free repair system in plant root-tip meristems which protects against MH damage.

‘Clastogenic adaptation’ of the Vicia faba root-tip meristem as affected by various treatment parameters

‘Clastogenic adaptation’ of the Vicia faba root-tip meristem as affected by various treatment parameters

Cross-resistance studies with V79 Chinese hamster cells adapted to the mutagenic or clastogenic effect of N-methyl- N′-nitro- N-nitrosoguanidine

When V79 cells are exposed to a single low dose of MNNG or MNU they acquire resistance to the mutagenic or to the clastogenic effect of the agents. Here the effect of MNNG pretreatment on mutagenesis (6-thioguanine resistance) and aberration formation in cells challenged with various mutagens/clastogens is reported. MNNG-adapted cells were resistant to the mutagenic effects of MNU and, to a lower extent, of EMS. No mutagenic adaptation was observed when MNNG-pretreated cells were challenged with MMS, ENU, MMC or UV. Cells pretreated with a dose of MNNG which makes them resistant to the clastogenic effect of this compound were also resistant to the clastogenic activity of other methylating agents (MNU, MMS), but not so with respect to ethylating agents (EMS, ENU). Cycloheximide abolished the aberration-reducing effect of pretreatment. However, when given before the challenge dose of MNNG, MNU or MMS, it drastically enhanced the aberration frequency in both pretreated and non-pretreated cells. No significant enhancement of aberration frequency by cycloheximide was found for ethylating agents. The results indicate that clastogenic adaptation is due to inducible cellular functions. It is concluded that mutagenic and clastogenic adaptation are probably caused by different adaptive repair pathways.