Heat shock protection against induction of chromatid aberrations is dependent on the time span between heat shock and clastogen treatment of Vicia faba root tip meristem cells

Variation of the time span between heat shock (hs) and clastogen treatment of V. faba root tip meristems showed that hs protection is a very quick response (effective after less than 10 min) and lasting for up to 240 min in the case of induction of chromatid aberrations by maleic hydrazide (MH). Analogous protective responses are significantly slower and shorter when TEM is used for aberration induction. This, together with absence of 'clastogenic cross-adaptation' to these agents and differential effects of benzamide (BA, an inhibitor of poly-ADP-ribosylation) pretreatment before hs on hs protection, suggests that hs before clastogen treatment triggers at least 2 clastogen-specific, protective functions which eventually result in protection against these 2 clastogens.     

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 X 10(-3) M) exerted protective effects against TEM challenging but not against MH.     

Ammonium chloride and zinc sulfate pretreatments reduce the yield of chromatid aberrations induced by TEM and maleic hydrazide in Vicia faba

Pretreatment of Vicia faba root-tip meristems with ammonium chloride and zinc sulfate prior to challenge treatment with maleic hydrazide (MH) and TEM, respectively, resulted in significant reductions of the yield of metaphases with chromatid aberrations. Ammonium sulfate pretreatment was without effect on aberration yield. Similar to heat-shocks and a variety of other agents, which induce the heat-shock response, the protective effects of ammonium chloride and zinc sulfate are presumed to be due to stress-responses of the root meristem cells which eventually result in inducible anticlastogenic functions.     

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.     

Comparative studies on the specificity of anticlastogenic action in human lymphocytes in culture

Summary Comparative studies on human lymphocyte cultures yielded a certain specificity of the anticlastogenic action of the SH compounds 1-cysteine, cysteamine, and -aminoethylisothiouronium (AET) as well as of the amide 1-asparagine and the amino acid 1-methionine. This specific anticlastogenic activity manifested itself in specific changes of the spectrum of aberration types induced by the clastogens and of the pattern of intercellular distribution of the induced aberrations. It was clearly dependent on the concentration of the anticlastogens but was also influenced by the used clastogen. The use of different culture media yielded some quantitative influences on the anticlastogenic activity, but fundamental changes in the spectrum of anticlastogenic action have not been observed except with cysteamine. The patterns of activity ascertained for the different anticlastogens specifically differed from those changes in the spectrum and pattern of distribution of aberrations induced by a mere reduction of the concentration for instance of Trenimon. Therefore a direct reaction between the protectors and the clastogen Trenimon as the cause of the observed anticlastogenic action was again excluded. The presented data are also discussed under the aspects of the hypotheses of aberration induction as well as of their importance for further antimutagen research.Some parts of this paper have been supported by grants of Deutsche Forschungsgemeinschaft.     

Induction of chromosomal aberrations in Chinese hamster ovary cells by triethyllead acetate.

Organolead compounds enter the environment primarily through the combustion of leaded gasoline and industrial discharge. Lead and lead-containing compounds have been shown to induce a broad spectrum of toxic effects, including hematopoietic, renal, neurologic, and carcinogenic effects. In this study, the mutagenic activity of triethyllead acetate (Et3PbAc) was determined by measuring the induction of chromosomal aberrations in Chinese hamster ovary cells. The results indicate that Et3PbAc is very cytotoxic and a potent clastogen. In preliminary cytotoxicity studies used to determine appropriate test concentrations for chromosomal aberration analysis, the LC50 of Et3PbAc was approximately 10 microM in the absence of metabolic activation, and 80 microM in the presence of metabolic activation. The maximal response was greater with metabolic activation than without. However, a much higher dose was required to elicit a significant response in the presence of metabolic activation than in its absence.     

On time dependency of frequency and distribution of chromatid aberrations induced by mutagens with delayed effects. An interpretation

This paper provides a theoretical analysis of pecularities of both the frequency and intrachromosomal distribution of chromatid aberrations observed in the first post-treatment mitosis and induced by clastogenic agents showing delayed effects (S-phase dependent clastogens), as functions of recovery time. The theoretical deductions are based on the following facts: (1) DNA is the target of clastogen action. Lesions induced by clastogens showing delayed effects (e.g. mono- and polyfunctional alkylating agents, ultraviolet light) give rise to aberrations only after interference with the process(es) associated with DNA replication. (2) DNA replication occurs asynchronously with respect to the local involvement in replication of different chromatin regions and according to a highly ordered pattern. (3) Lesions may be removed from DNA (or otherwise modified) by repair processes prior to replication. The removal of lesions from DNA is a time-dependent function.Several possibilities are analysed (i.e. random or non-random distribution of DNA lesions, uniform or locally differing capacities of pre-replicative repair of lesions, uniform or locally differing rates of DNA synthesis) and the frequencies and distribution patterns of chromosome structural changes, as expressed in form of aberration yield-time curves, have been discussed. The theory presented in this paper offers a simple interpretation both of variations of aberration frequency and aberration distribution in dependence on the cell's position within the cell cycle during induction of lesions.It is shown that the intrachromosomal aberration distribution is non-random even if random distribution of lesions and uniform repair rates between chromosome regions replicating at different time periods during S are assumed. Non-random aberration distributions are a necessary consequence of at least two factors: (a) the temporal replication pattern, and (b) the repair activities acting prior to replication. Random distribution of aberrations is only to be expected for the most simplified situation (random distribution of lesions along the DNA and equal transformation probabilities of a given kind of lesion into aberrations) when no loss of lesions prior to replication takes place (no pre-replicative repair) and cells treated with the mutagen during G1 are analysed.     

Clastogenic effects of bleomycin, cyclophosphamide, and ethyl methanesulfonate on resting and proliferating human B- and T-lymphocytes

The effects of bleomycin (BM), cyclophosphamide (CP), and ethyl methanesulfonate (EMS) on the frequencies of chromosomal aberrations were tested in mitogen-stimulated highly purified human B- and T-lymphocytes. In unstimulated G0/G1 B- and T-lymphocytes the clastogen induction of chromosome fragments was investigated in prematurely condensed chromosomes (PCC) induced by cell fusion with xenogenic mitotic cells. BM, CP (with metabolic activation), and EMS induced a significant increase in chromosome aberrations in proliferating human B- and T-lymphocytes. There were no significant differences in the BM-induced aberration rates between the cell populations. CP and EMS induced more aberrations in T- than in B-lymphocytes. In the PCC tests, BM-exposed G0/G1 lymphocytes showed dose-dependent high yields of chromosome fragments. No significant differences between B- and T-lymphocytes were observed. CP and EMS induced no clear increase in fragments in either cell population.     

The Production of Chromosome Aberrations in Various Mammalian Cells by Triethylenemelamine

The cytogenetic effects of triethylenemelamine (TEM) were studied using five different mammalian tissues. Treatments of 0.1 and 0.2 mg/kg TEM on differentiating mouse spermatogonia and bone marrow cells showed no significant differences in the frequency of chromosomal aberrations produced in these two tissues. At higher doses, however, the sensitivites of the two tissues appear to be different. The frequency of aberrations varies with time after treatment, with the greatest amount occurring at the latter fixation times. Results of an experiment on primary spermatocytes indicated a correlation between the frequency of chromosome aberrations and DNA replication. Human peripheral leukocytes were utilized in an attempt to clarify the cell-stage specificity of TEM-induced chromosome aberrations. Cultures were treated with TEM prior to PHA stimulation (G0), as well as various time intervals after stimulation (late G,1 S, and G2). The most sensitive stages of the cell cycle to aberration induction were later G1 and S, with chromatid aberrations the predominant type. A very low yield of chromosome damage was observed with the G0 and G1 treated stages. The experiments described tend to support the view that TEM is most effective at inducing aberrations when an intervening round of DNA replication has occurred.     

Plant tissue culture as a model system for mutagenicity testing of chemicals

Two karyotypically stabilized callus strains of Crepis capillaris (2n = 7 and 2n greater than or equal to 12, respectively) were employed to illustrate the utility of plant tissue culture method for screening and analysis of cytogenetic effects of chemicals following long-term treatment. The cytogenetic analysis of callus cells revealed significant differences in the toxic and mutagenic effects of chemicals under study (2,4-D, MH, NMU and kinetin). A certain dose-response relationship (though not necessarily linear) was observed. The maximum cytogenetic activity of chemicals was associated with certain intermediate concentrations (4.5 X 10(-5) M-9 X 10(-5) M), whereas a further increase in the dose either gave rise to the opposite effects (i.e. decrease in the ana- and telo-phase aberration rates and increase in the modal karyotype frequency) or the aberration rate remained unchanged.     

Involvement of phytochelatins in NiCl2-triggered protection against induction of chromatid aberrations by TEM and MH in Vicia faba root tip meristems?

Conditioning treatment of Vicia faba root tip meristem cells with NiCl2 prior to challenge treatment with triethylenemelamine (TEM) or maleic hydrazide (MH) triggered protective functions against both these clastogens, i.e., resulted in a significantly reduced yield of metaphases with chromatid aberrations. Protection was prevented by pretreatment with buthionine sulfoximine (BSI), an inhibitor of the synthesis of plant phytochelatins (PCs), indicating that the NiCl2-triggered PC synthesis may be involved in the protective functions induced by NiCl2 conditioning treatment. BSI (instead of NiCl2) conditioning treatment triggered protection against MH but not against TEM.     

Maleic hydrazide induces sister-chromatid exchanges in mammalian cells in vitro

Maleic hydrazide (MH) in high concentrations (from 8 × 10⁻³ M upward) induces SCEs in human blood cultures if it is added either for the entire culture period (72 h) or for the last 24 h. Parallel to the induction of SCEs, there is a delay in the proliferation of the cultures, as can be seen by the distribution of the first, second and third mitoses following the addition of BrdUrd.MH leads to a pronounced induction of SCEs in V79 cells (quadrupeling the control value) if added for 24 h simultaneously with BrdUrd. Treating the cells for 1 or 3 h prior to the addition of BrdUrd has no or little effect on the frequencies of SCEs.     

Clastogenicity of pentachlorophenol, 2,4-D and butachlor evaluated by Allium root tip test

The meristematic mitotic cells of Allium cepa is an efficient cytogenetic material for chromosome aberration assay on environmental pollutants. For assessing genotoxicity of pentachlorophenol (PCP), 2,4-dichlorophenoxyacetic acid (2,4-D) and 2-chloro-2,6-diethyl-N-(butoxymethyl) acetanilide (butachlor), 50% effective concentration (EC(50)), c-mitosis, stickiness, chromosome breaks and mitotic index (MI) were used as endpoints of genotoxicity. EC(50) values for PCP and butachlor are 0.73 and 5.13 ppm, respectively. 2,4-D evidently induced morphological changes at higher concentrations. Some changes like crochet hooks, c-tumours and broken roots were unique to 2,4-D at 5-20 ppm. No such abnormalities were found in PCP and butachlor treated groups, however, root deteriorated and degenerated at higher concentrations (<3 ppm) in PCP. MI in 2,4-D showed a low average of 14.32% followed by PCP (19.53%), while in butachlor it was recorded 71.6%, which is near to the control value. All chemicals induced chromosome aberrations at statistically significant level. The highest chromosome aberration frequency (11.90%) was recorded in PCP at 3 ppm. Large number of c-mitotic anaphases indicated that butachlor acts as potent spindle inhibitor, whereas, breaks, bridges, stickiness and laggards were most frequently found in PCP showing that it is a potent clastogen.     

Salicylic acid triggers genotoxic adaptation to methyl mercuric chloride and ethyl methane sulfonate, but not to maleic hydrazide in root meristem cells of Allium cepa L

Salicylic acid (SA), 0.01 mM, a signalling phytohormone, was tested for induction of adaptive response against genotoxicity of methyl mercuric chloride (MMCl), 0.013 mM; ethylmethane sulfonate (EMS), 2.5 mM, or maleic hydrazide (MH), 5 mM, in root meristem cells of Allium cepa. Induction of adaptive response to EMS by hydrogen peroxide (H2O2), 1 mM, and yet another secondary signal molecule was tested for comparison. Assessed by the incidence of mitoses with spindle and/or chromosome aberration and micronucleus, the findings provided evidence that SA-conditioning triggered adaptive response against the genotoxic-challenges of MMCl and EMS, but failed to do so against MH. H2O2, which is known to induce adaptive response to MMCl and MH, failed to induce the same against EMS in the present study. The findings pointed to the possible role of signal transduction in the SA-induced adaptive response to genotoxic stress that perhaps ruled out an involvement of H2O2.     

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.     

Comparative mutagenicity of chemicals selected for test in the International Program on Chemical Safety''s collaborative study on plant systems for the detection of environmental mutagens

A review has been made of the four compounds (maleic hydrazide, methyl nitrosourea, sodium azide, azidoglycerol) tested in the International Program on Chemical Safety's collaborative study systems. Maleic hydrazide (MH) is a weak cytotoxic/mutagenic chemical in mammalian tissues and is classified as a class 4 chemical. In contrast, with few exceptions such as Arabidopsis, MH is a potent mutagen/clastogen in plant systems. The difference in its response between plant and animal tissue is likely due to differences in the way MH is metabolized. MH appears to be noncarcinogenic and has been given a negative NCI/NTP carcinogen rating.

Methyl nitrosourea (MNU) is a toxic, mutagenic, radiomimetic, carcinogenic, and teratogenic chemical. It has been shown to be a mutagen in bacteria, fungi, Drosophila, higher plants, and animal cells both in vitro and in vivo. MNU is a clastogen in both animal and human cell cultures, plant root tips and cell cultures inducing both chromosomes and chromatid aberrations as well as sister-chromatid exchanges. Carcinogenicity has been confirmed in numerous studies and involves the nervous system, intestine, kidney, stomach, bladder and uterus, in the rat, mouse, and hamster. MNU produces stage-specific teratogenic effects and also interferes with embryonic development. The experimental evidence that strongly indicates the mutagenic effects of MNU underlines the possible hazard of this compound to human beings. The experimental evidence for the stringent handling of this compound is clear.

Sodium azide (NaN₃) is cytotoxic in several animal and plant systems and functions by inhibiting protein synthesis and replicative DNA synthesis at low dosages. It is mutagenic in bacteria, higher plants and human cells and has been used as a positive control in some systems. In general, tests for clastogenicity have been negative or weakly positive. No evidence of carcinogenicity has been reported in a 2-year study seeking carcinogenic activity in male and female rats. Its advantages in comparison to other efficient mutagens are claimed to be a high production of gene mutations accompanied by a low frequency of chromosomal rearrangements and safer handling because of its nonclastogenic and noncarcinogenic action on humans.     

Protective effect of cysteine against X-ray- and bleomycin-induced chromosomal aberrations and cell cycle delay

The protective effect of cysteine was studied in muntjac and human lymphocytes in vitro scoring chromosomal aberrations in harlequin stained first cycle metaphases, induced by X-irradiation at G₀. Its protective efficiency was also studied against the radiomimetic clastogen, bleomycin, in muntjac cells. 30 μg and 1 mg/ml of cysteine were given prior to 2, 3, and 4 Gy, and 2 mg/ml prior to only 4 Gy. 30 μg cysteine protected only against deletions in 4 Gy-treated cells while 1 mg protected against deletions by all three doses of X-rays. However, rearrangements were not reduced significantly in any of these, probably due to their low frequency. But when cysteine was increased to 2 mg, both types of aberrations were reduced significantly. This shows that a sufficient number of aberrations and an optimum concentration of the protector are essential for eliciting the best protective effect. This conclusion is further supported by the results of 2 mg cysteine treatment in human lymphocytes which yielded higher frequencies of rearrangements with 2 and 3 Gy X-rays than 4 Gy in muntjac, but had a relatively lower frequency of deletions. Thus the most abundant categories of aberration, i.e., deletions in muntjac and exchanges in humans, were reduced significantly by 2 mg cysteine, associated with a prominent reduction in the frequency of aberrant metaphases. Therefore, the differential protection observed with a low concentration of the protector and an insufficient yield of aberrations induced only indicates protection provided to the most frequent type of aberration by a protector when present in lower concentration.

Cysteine pretreatment yielded weak protection against the effects of bleomycin, but posttreatment caused a mild potentiation of the clastogenic effect of BLM without altering the cell cycle kinetics. In this context, an action of cysteine as a reducing agent on BLM is suggested. Although cysteine alone caused severe retardation of the cell cycle, when given prior to X-irradiation, not only its delaying effect was not observed, but also it reduced the X-ray induced cell cycle delay. This might be due to the oxidation of cysteine by its radical scavenging action.     

Sister chromatid exchange and micronuclei in human peripheral blood lymphocytes treated with thyroxine in vitro

Thyroid hormones enhance the metabolic rate and the aerobic metabolism favoring oxidative stress, which is accompanied by induction of damage to cellular macromolecules including the DNA. The aim of the present study was to investigate the ability of thyroxine to induce sister chromatid exchange and micronuclei, and to modulate cell-cycle kinetics in cultured human lymphocytes. Eight experimental concentrations of thyroxine were used, ranging from 2 x 10(-9) to 0.5 x 10(-4)M. Treatment with thyroxine increased the frequency of SCE per cell at the higher concentrations (1.5 x 10(-6), 0.5 x 10(-5), 1.5 x 10(-5) and 0.5 x 10(-4)M). On the other hand, there were no significant aneugenic and/or clastogenic effects observed in the cytokinesis-block micronucleus assay. The results show that thyroxine acted as a relatively weak clastogen compared with the positive control N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). In addition to the genotoxic effects, two high concentrations of thyroxine decreased the mitotic index and caused cell-cycle delay. In conclusion, thyroxine exhibited weak clastogenic effects only at high concentrations. Therefore, effects in humans might appear in cases of acute thyroxine overdose.     

Effects of novobiocin on heat shock protection against chromatid aberration induction by triethylenemelamine (TEM) and maleic hydrazide (MH) in Vicia faba

Heat shock (10 min 40 degrees C) prior to challenge treatment with triethylenemelamine (TEM) or maleic hydrazide (MH) significantly reduced the frequency of induced chromatid aberrations in Vicia faba main root meristems. Novobiocin treatment before heat shock did not prevent heat shock protection against both clastogens; novobiocin application after heat shock prevented protective effects. These results and those obtained earlier for heat shock protection against X-ray challenge are used to discuss possible causes underlying the protective effects triggered by heat shock.