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This paper provides an overview of published and unpublished results obtained after various pretreatments (conditioning) of Vicia faba root tip meristems (low clastogen doses, heat shock, ammonium chloride and zinc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).     

Mutagenicity of ptaquiloside, the carcinogen in bracken, and its related illudane-type sesquiterpenes II. Chromosomal aberration tests with cultured mammalian cells

The chromosomal aberration test using a Chinese hamster lung cell line (CHL) was carried out on ptaquiloside and its related compounds, hypoloside B, hypoloside C, illudin M and illudin S. Ptaquiloside induced chromosomal aberrations at doses as low as 4.5 μg/ml (0.0113 mM). The clastogenic effect was ph-dependent. The same activity was observed at a 90-fold higher dose at pH 5.3 in the culture medium compared with the activity at pH 74. or pH 8.0. Both hypoloside B and hypoloside C were also clastogenic at almost the same dose levels as that of ptaquiloside. Illudin M and illudin S were also potet clastogens and induced aberrations at much lower doses than ptaquiloside. These results suggest that the clastogenic effect is involved in the mechanism of carcinogenic potency of ptaquiloside in animals.     

Comparative efficacy of chlorophyllin in reducing cytotoxicity of some heavy metals

Summary The potential of chlorophyllin in reducing clastogenicity was studied against two concentrations of each of three potent metallic clastogens (cesium chloride, mercuric chloride and cobalt chloride) in bone marrow cells of mice in vivo. The respective salts and chlorophyllin were administered orally to mice by gavaging in different combinations. Simultaneous administration of chlorophyllin with both concentrations of each salt reduced the clastogenic effects in the order Cs > Hg > Co. Chlorophyllin could not decrease the clastogenic effects when administered 2 h before the salts.     

Effect of pH on the activity and stability of clastogens in the in vitro chromosomal aberration test with Chinese hamster ovary K1 cells

The effect of the pH of the medium on the clastogenic activity of several direct-acting and indirect clastogens was evaluated in the in vitro chromosomal aberration test with Chinese hamster ovary K1 cells. Furthermore, the stability of the chemicals in the cell culture medium was measured by HPLC over the pH range of 5.0-11.0. The activity of the direct-acting clastogens mitomycin C (MMC), N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) and 4-nitroquinoline-1-oxide (4NQO) at various pH values depended on their stability. In the case of ENNG, its clastogenic activity decreased to about one-fifth at pH 9 but was about twice as high at acidic pH compared with that at pH 7.4. This is consistent with the observation that ENNG is unstable at basic pH; the residual content of ENNG was 0.5% of the initial amount in cell culture medium at pH 9.0 after a 2-h incubation. 4NQO was unstable at strongly basic pH (pH 10-11), and MMC was unstable at pH 5.0 and pH 11.0. The frequencies of chromosomal aberrations induced by MMC and ENNG were correspondingly decreased at these pH values. On the other hand, the clastogenicities of the indirect clastogens 7,12-dimethylbenz[a]anthracene (DMBA), benzo[a]pyrene (B(a)P) and dimethylnitrosamine (DMN), which require metabolic activation, were reduced at pH 10-11 and pH 5.8. The frequencies of chromosomal aberrations at these pHs were almost equal to negative control values. These chemicals were stable in the medium in the absence of S9 mix over the pH range of 5.0-11.0. Thus clastogenicity of indirect-acting clastogens is reduced under extreme pH conditions, probably because of the instability or nonformation of the active form. The present results indicate that the clastogenic activity of any compound will depend on its stability in the medium irrespective of its direct- or indirect-acting nature. In addition, some of the chemicals that are recognized as clastogens presumably might induce chromosomal aberrations by means of acidic pH itself. It is, therefore, important to take account of the pH of the treatment medium in evaluating the clastogenicity of chemicals.     

Chromosome aberrations induced by pyrolysates of carbohydrates in Chinese hamster V79 cells

The clastogenic activity of some pyrolysates of carbohydrates was examined in cultured Chinese hamster V79 cells. These pyrolysates include levoglucosan (LG-I), levoglucosenone (LG-II), furfural (FF), 5-(hydroxymethyl)-2-furfural (HMF), glyoxal (GL), methylglyoxal (MGL), 3-deoxy-D-glucosone (DG) and thiazolidine (TZ). LG-I did not induce a significant number of chromosome aberrations at doses up to 8000 micrograms/ml. In contrast, the related compound LG-II induced aberrations and reduced mitosis in a dose-dependent fashion at around 1/2000 of the LG-I doses. Both furan derivatives, FF and HMF, and both glyoxal derivatives, GL and MGL, induced a significant number of chromosome aberrations and a significant lowering of mitotic activity. Among these compounds, FF and MGL showed stronger clastogenic activity than HMF and GL, respectively. DG slightly but positively induced chromosome aberrations. TZ was one of the most potent clastogens among the compounds examined in this study, showing the highest incidence of aberrant cells with many exchanges at doses inducing a significant lowering of mitotic activity. The results of this study indicate the need for a re-evaluation of the thermal decomposition of carbohydrates as a source of genotoxic contaminants.     

Chromosome aberrations induced by 9-aminoacridine derivatives

The induction of chromosomal aberrations by 5 derivatives of nitro-9-aminoacridine in V79 Chinese hamster cells was observed. The clastogenic activity of the compounds tested depended on the position of the NO2 group in the acridine ring. The strongest clastogens were derivatives with NO2 in position 1. The remaining derivatives placed in decreasing order of clastogenic activity were: 3-nitro, 4-nitro and 2-nitro. In addition, 2-nitro and 3-nitro derivatives produced hyperdiploid/polyploid metaphases.     

Clastogenicity of 1-nitropyrene, dinitropyrenes, fluorene and mononitrofluorenes in cultured Chinese hamster cells

The chromosomal aberration test using a Chinese hamster lung cell line (CHL) was carried out on 1-nitropyrene (NP), 3 dinitropyrenes (DNPs), fluorene and 4 mononitrofluorenes with and without metabolic activation (rat S9 mix). The 3 DNPs (1,3-, 1,6- and 1,8-DNP) induced chromosomal aberrations in the absence of S9 mix. The frequencies of cells with aberrations after treatment for 48 h were 43% at 2 micrograms/ml of 1,3-DNP, 55% at 0.1 microgram/ml of 1,6-DNP and 45% at 0.025 microgram/ml of 1,8-DNP, indicating the order of clastogenic potency as 1,8- greater than 1,6- greater than 1,3-DNP. On the other hand, 1-NP, which is known to be a direct-acting mutagen in bacteria, was negative in the chromosomal aberration test without S9 mix, but clearly positive with S9 mix. This effect was dependent on the concentration of the S9 fraction in the reaction mixture. High-pressure liquid chromatography analysis showed that 1-NP was converted by S9 mix to several metabolites, including 1-aminopyrene (AP). The clastogenic activity of 1-AP, however, was equivocal without S9 mix, suggesting that active clastogens other than 1-AP exist. Fluorene induced chromosomal aberrations only in the presence of S9 mix (61.8% at 25 micrograms/ml). 1-, 2-, 3- and 4-nitrofluorene (NF) were more clastogenic in the presence of S9 mix than in the absence of S9 mix, suggesting that NFs were converted to more active clastogens by S9 mix.     

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.     

Detection of aneuploidy in male germ cells of mice by means of a meiotic micronucleus assay.

The possibility of using a micronucleus (MN) assay in mouse germ cells for the identification of aneuploidogenic agents was evaluated by comparing the pattern of effects induced by 4 chemicals with different mechanisms of action (adriamycin, ADM; mitomycin C, MMC; chloral hydrate, CH; ethylenedinitrilotetraacetic acid, EDTA). The results obtained after treatment of spermatocytes at the premeiotic S-phase (preleptotene) indicated that only clastogenic agents (ADM and MMC) were able to induce MN at this cell stage. Previous data obtained with the same compounds demonstrated by contrast that the micronucleus spermatid assay may detect both clastogenic and aneuploidogenic effects after treatment of diakinesis/MI/MII cells. Analysis of MN size distributions, in the present and previous spermatid samples, revealed that the clastogens ADM and MMC produced relatively more small MN than CH and EDTA. These data are in agreement with the proposed mechanism of action of the chemicals tested.     

Detection of aneuploidy in male germ cells of mice by means of a meiotic micronucleus assay

The possibility of using a micronucleus (MN) assay in mouse germ cells for the identification of aneuploidogenic agents was evaluated by comparing the pattern of effects induced by 4 chemicals with different mechanisms of action (adriamycin, ADM; mitomycin C, MMC; chloral hydrate, CH; ethylenedinirrilotetraacetic acid, EDTA). The results obtained after treatment of spermatocytes at the premeiotic S-phase (preleptotene) indicated that only clastogenic agents (ADM and MMC) were able to induce MN at this cell stage. Previous data obtained with the saine compounds demonstrated by contrast that the micronucleus spermatid assay may detect both clastogenic and aneuploidogenic effects after treatment of diakinesis/MI/MII cells. Analysis of MN size distributions, in the present and previous spermatid samples, revealed that the clastogens ADM and MMC produced relatively more small MN than CH and EDTA. These data are in agreement with the proposed mechanism of action of the chemicals tested.     

Rapid detection of mutagen induced micronucleated erythrocytes by flow cytometry

Summary The micronucleus test is a cytogenetic method for the screening of mutagens and carcinogens which exhibit clastogenic mechanisms of action. After application of clastogenic agents chromosomal fragments or even whole chromsomes can remain as conspicuous structures (micronuclei) in a small fraction of anucleated polychromatic erythrocytes which can be visually scored using a microscope following staining with May-Grünwald-Giemsa solutions. These time-consuming, painstaking, and tedious manual evaluations are often sources of unreliability and uncertainty. Here, a fluorescence technique is presented which applies DNA and protein fluorochromes to discriminate normal anucleated erythrocytes from micronucleated erythrocytes using a fluorescence microscope. This method is particularly tailored to be applied to flow cytometric instrumentation. Data obtained manually and automatically in flow show a strong linear correlation with high significance (r=0.96) as far as the percentage of micronucleated erythrocytes as an indicator for the mutagenicity of a given drug is concerned. These results have been obtained by means of the established clastogens cyclophosphamide and mitomycin C.     

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.     

Anti-clastogenic activity of two structurally related pterocarpans purified from Bituminaria bituminosa in cultured human lymphocytes

Plant-derived isoflavones are currently receiving much attention because of their phyto-estrogenic, antioxidant, anti-mutagenic, and anti-tumor activities. In this study we have evaluated the clastogenic and anti-clastogenic activities in human lymphocytes of two structurally related pterocarpans, iso-flavonoid derivatives, termed erybraedin C and bitucarpin A, recently purified from Bituminaria bituminosa and chemically characterized. Mitomycin C (MMC) and the radio-mimetic bleomycin (BL) were used as reference clastogens. The end point studied was micronucleus formation. The results obtained in this study indicate that erybraedin C and bitucarpin A, when assayed alone, do not affect either the mitotic index or the cell-proliferation index of human lymphocytes. Interestingly, both compounds appear to be non-clastogenic in the range of concentrations used. In contrast, both substances seem to affect significantly the clastogenic effects induced by BL and MMC. A 1-h pre-exposition of the cell culture to erybraedin C was necessary to display its anti-clastogenic potential against BL, whereas bitucarpin A was inactive in this respect, with a structure-activity relationship. In contrast, the clastogenic activity of MMC was significantly reduced by both erybraedin C and bitucarpin A, using either a pre-incubation schedule or simultaneous treatment. These results suggest that the protective effects displayed by the two anti-clastogenic compounds against MMC could be due to the induction or inhibition of cellular reductive metabolic enzymes.     

Influence of acetylcysteine on cytogenetic effects of etoposide in mouse oocytes

The influence of N-acetylcysteine (ACC) on the cytogenetic effects of etoposide in F₁ CBA × C57BL/6 mice was studied. Etoposide introduced intraperitoneally in doses of 10, 20, 40, and 60 mg/kg has a dose-dependent clastogenic activity and has an aneugenic effect with the induction of mainly hypohaploid oocytes. ACC significantly decreases the aneugenic and clastogenic activity of etoposide (20 mg/kg) in oocytes of 6-, 9-, and 12-week-old mice during triple introduction at a dose 200 mg/kg per os. The most pronounced anticlastogenic ACC activity (an 80% decrease) was registered in 9-week-old females; a 100% decrease in aneugenesis was detected in 6-week-old female mice.     

Benzophenone guttiferone A from Garcinia achachairu Rusby (Clusiaceae) Presents Genotoxic Effects in Different Cells of Mice

Benzophenones from natural sources and those of synthetic analogues present several reports of potent biological properties, and Guttiferone A represents a promising medicinal natural compound with analgesic and gastroprotective profiles. Considering that there are no reports that assess the genetic toxicity of Guttiferone A, the present study was undertaken to investigate the genotoxic potential of this benzophenone isolated from seeds of Garcinia achachairu in terms of DNA damage in different cells of Swiss albino mice using the comet assay, and its clastogenic/aneugenic effects in bone marrow cells in vivo by the micronucleus test. Cytotoxicity was assessed by scoring polychromatic (PCE) and normochromatic (NCE) erythrocytes ratio. Guttiferone A was administered by oral gavage at doses of 15, 30 and 60 mg/kg. The results showed that Guttiferone A produced genotoxic effects in leukocytes, liver, bone marrow, brain and testicle cells and clastogenic/aneugenic effects in bone marrow erythrocytes of mice. The PCE/NCE ratio indicated no cytotoxicity. Since guttiferone A is harmful to the genetic material we suggest caution in its use by humans.     

X-rays, microwaves and vinyl chloride monomer: their clastogenic and aneugenic activity, using the micronucleus assay on human lymphocytes.

Chromosome aberration assays, sister-chromatid exchange techniques and micronucleus assays are commonly used methods for biomonitoring genetic material damaged by chemical or physical agents. On the other hand, their aneugenic activity, which can lead to hypoploidy and may also be associated with carcinogenesis, has not been thoroughly investigated. In our study we chose the micronucleus assay with a new mathematical approach to separate clastogenic from aneugenic activity of three well-known mutagens (vinyl chloride monomer, X-rays and microwaves) on the genome of human somatic cells. The comparison of frequencies of size distribution of micronuclei in the lymphocytes of humans exposed to each of these three mutagens showed that X-rays and microwaves were preferentially clastogens while vinyl chloride monomer showed aneugenic activity as well. Microwaves possess some mutagenic characteristics typical of chemical mutagens.     

8,15-Epoxylabdane and norlabdane diterpenoids from Eragrostisviscosa

Four labdanes with a 8α,15-epoxy ring (8α,15-epoxylabdan-16β-oic acid; 8α,15-epoxy-16-norlabdan-13-one; 8α,15-epoxy-16-norlabdane; and 16-acetoxy-8α,15-epoxylabdane) and the known compound ambreinolide were isolated from the hexane extract of the aerial parts of the grass Eragrostis viscosa. The structures of all compounds were established based on spectroscopic data and the X-ray analysis of 8α,15-epoxy-16-norlabdan-13-one. The hexane extract presented moderate activity against the snail Biomphalaria glabrata. 8α,15-Epoxylabdan-16β-oic acid showed no mutagenic activity for doses up to 1000 μg/plate and no significant clastogenic activity for doses up to 100 μg/ml.     

Induction by chemical clastogens of aberrations in prematurely condensed interphase chromatin of Chinese hamster ovary cells

The clastogenic activities of diepoxybutane and bleomycin were comparatively studied on prematurely condensed interphase chromatin and metaphase chromosomes of Chinese hamster ovary cells. The yield of chromosomal aberrations was distinctly higher in G2-premature chromosome condensation as compared to metaphase. Most notably, the clastogenic activity of bleomycin was visible in premature chromosome condensation after application of much lower final concentrations than necessary for induction of chromosome aberrations in metaphase. In addition, the different mechanisms of action of both clastogens were reflected by the aberration yield in GI and G2 immediately after exposure. While bleomycin induced aberrations throughout all stages of interphase, diepoxybutane did not induce aberrations in GI or G2. Though certainly not a routine system for genotoxicity testing, premature chromosome condensation analyses provide a powerful opportunity to demonstrate relationships between DNA damage and repair, and the production of chromosomal changes at the site of their formation.Abbreviations BM bleomycin - BrdUrd bromodeoxyuridine - CHO Chinese hamster ovary - DEB diepoxybutane - DMSO dimethylsulfoxide - FCS fetal calf serum - PCC premature chromosome condensation, prematurely condensed chromosomes - PEG polyethylene glycol     

Kinetochore localization in micronucleated cytokinesis-blocked Chinese hamster ovary cells: a new and rapid assay for identifying aneuploidy-inducing agents

We have developed a modified micronucleus assay using an antikinetochore antibody and cytokinesis-blocked Chinese hamster ovary cells as a simple and rapid method for detecting aneuploidy-inducing agents. The presence of a kinetochore in a micronucleus of a binucleated cell indicates a cell with a high probability for aneuploidy following cytokinesis. The method requires minimal training to perform and score and can readily distinguish aneuploidy-inducing agents from clastogens. Micronucleated cells treated with the aneuploidy-inducing agents benomyl and vinblastine sulfate contained a kinetochore-positive micronucleus 92% and 94% of the time whereas micronucleated cells treated with the clastogen methyl methanesulfonate contained a kinetochore-positive micronucleus only 11% of the time. This relatively simple method for distinguishing aneuploidy-inducing agents from clastogenic agents may be used as a routine genotoxicity assay to identify environmental and therapeutic agents with aneuploidy-inducing properties.