Effect of various concentrations of acyclovir on cell survival and micronuclei induction on cultured HeLa cells

The HeLa cells were treated with 0, 0.01, 0.1, 1, 10 and 100 μM acyclovir (ACV) for 8 h duration and the growth kinetics, cell survival and micronuclei induction were determined. Treatment of HeLa cells with various concentrations of ACV resulted in a concentration-dependent decline in growth kinetics, cell proliferation indices and cell survival. ACV, 100 μM, completely inhibited cell division, where no appreciable changes in cell number were observed from 1 to 5 days post-treatment. This is reflected in cell survival, where the surviving fraction of cells was reduced to 1/2 at 100 μM ACV. Conversely, the frequency of micronuclei showed a concentration-dependent elevation at 20, 30 and 40 h post-treatment. ACV not only induced one micronuclei-bearing binucleate cell but also binucleate cells bearing two and multiple micronuclei in a concentration-dependent manner. The micronuclei frequency increased with time up to 30 h post-treatment and declined thereafter. The relationship between micronuclei induction and cell survival was determined by plotting the former on Y- and the latter on X-axes, respectively. The surviving fraction of cells declined with the elevation in micronuclei frequency and a best fit was observed for linear quadratic formalism.     

Effect of acyclovir on the radiation-induced micronuclei and cell death

Treatment of HeLa cells with 0.1 microM Acyclovir [9-(2-hydroxyethoxymethyl)guanine] (ACV) before exposure to 0, 0.25, 0.5, 1, 2 and 3 Gy of gamma-radiation resulted in a dose-dependent decline in the growth kinetics and cell proliferation indices at 20, 30 and 40 h post-irradiation when compared with the PBS+irradiation group. These results were reflected in the cell survival, which declined in a dose-dependent manner and the surviving fraction of cells was significantly lower in ACV+irradiation group than that of PBS+irradiation group. The effect of ACV+1 Gy irradiation was almost similar to PBS+3 Gy irradiation suggesting an enhancement of the radiation effect by ACV pretreatment. The frequency of micronuclei increased in a dose-dependent manner at all the post-irradiation time periods in both PBS+irradiation and ACV+irradiation group and it was significantly elevated in the latter when compared with the former group. The dose-response for both groups was linear. The surviving fraction of HeLa cells declined with the increasing MN frequency and a close linear quadratic correlation between cell survival and micronuclei-induction was observed.     

Correlation between cell survival and micronuclei formation in V79 cells treated with vindesine before exposure to different doses of gamma-radiation

Effect of 20 nM vindesine sulphate (VDS) treatment was studied on cell survival, growth kinetics and micronuclei induction in V79 cells exposed to 0-300 cGy of gamma-radiation at 16, 22 and 28 h post-irradiation. Treatment of V79 cells with VDS before exposure to different doses of gamma radiation resulted in a significant decline in cell survival and growth kinetic when compared with the concurrent PBS+irradiation group. The decline in cell survival and growth kinetics was dose related. Similarly, the cell proliferation indices also declined in a dose dependent manner in both PBS+irradiation and VDS+irradiation groups and this decline was higher in VDS+irradiation group in comparison with the PBS+irradiation group. In contrast, the frequency of micronuclei increased in a dose related manner in both PBS+irradiation and VDS+irradiation groups. However, the frequency of micronuclei was significantly greater in the VDS+irradiation group when compared to the PBS+irradiation group at all the post-irradiation time periods studied and the dose response for both groups was linear for all the scoring time periods. The biological response was determined by plotting surviving fraction and micronuclei frequencies on X- and Y-axes, respectively. The plot between surviving fraction and micronuclei induction showed a close correlation. The surviving fraction of V79 cells reduced with the increasing frequency of micronuclei in both groups and the relationship between micronuclei induction and cell survival could be fitted on a linear quadratic model.     

Juglone, a naphthoquinone from walnut, exerts cytotoxic and genotoxic effects against cultured melanoma tumor cells

This study demonstrates cytotoxic and genotoxic potential of juglone, a chief constituent of walnut, and its underlying mechanisms against melanoma cells. MTT assay and clonogenic assay were used to study cytotoxicity, micronucleus assay to assess genotoxicity, glutathione (GSH) assay and 2′,7′-dicholorofluorescein diacetate (DCFH-DA) assay to evaluate the oxidative stress induction. Apoptosis/necrosis induction was analysed by flow cytometry. We observed a concentration-dependent decrease in cell survival with a corresponding increase in the lactate dehydrogenase levels. A dose-dependent increase in the frequency of micronucleated binucleate cells indicated the potential of juglone to induce cytogenetic damage in melanoma tumor cells. Moreover, results of the micronuclei study indicated division delay in the proliferating cell population by showing decrease in the cytokinesis blocked proliferation index. Further, juglone-induced apoptosis and necrosis could be demonstrated by oligonucleosomal ladder formation, microscopic analysis, increase in the hypodiploid fraction (sub Go peak in DNA histogram), as well as an increased percentage of AnnexinV(+)/PI(+) cells detected by flow cytometry. A significant concentration-dependent decrease in the glutathione levels and increase in dichlorofluorescein (DCF) fluorescence after juglone treatment confirmed the ability of juglone to generate intracellular reactive oxygen species. The cytotoxic effect of juglone can be attributed to mechanisms including the induction of oxidative stress, cell membrane damage, and a clastogenic action leading to cell death by both apoptosis and necrosis.     

Induction of micronuclei in V79 Chinese hamster cells by tetrachlorohydroquinone, a metabolite of pentachlorophenol.

Tetrachlorohydroquinone, a metabolite of the fungicide pentachlorophenol, induced significant dose-related increases in micronuclei in V79 Chinese hamster cells without exogenous metabolic activation. The lowest observed effective dose was 10 microM, where the relative survival was about 62%. At the highest dose tested, 20 microM, the relative survival was about 8% and the frequency of cells with micronuclei was about 6 times the solvent control frequency. The induction of micronuclei by tetrachlorohydroquinone was significantly inhibited by the hydroxyl radical scavenger dimethyl sulfoxide at 5% (v/v).     

Cytotoxicity of azadirachtin A in human glioblastoma cell lines

The neem toxin azadirachtin A exhibits selective toxicity on insects. Despite its well-proven efficacy, the mode of action of this toxin remains obscure. The toxicity on vertebrate cells compared to insect cells is also not well characterized. We have cultivated six human glioblastoma cell lines G-28, G-112, G-60 (TP53 mutant) and G-44, G-62, G-120 (TP53 wild-type) in the presence of 28 microM of azadirachtin. This toxin concentration was chosen because it represents the 25 to 50% lethal dose in the glioma cells. Toxicity was measured in terms of cell proliferation (binucleation index), formation of micronuclei and cell survival. In the TP53 mutant cell lines, azadirachtin reduced the proportion of dividing cells and induced formation of micronuclei. Except for G-44 which showed a decrease in binucleation index, proliferation in the TP53 wild-type cell lines was unaffected by azadirachtin. In the TP53 wild-type cell lines, the decrease in micronuclei frequency is attributed to fewer cells entering mitosis to produce micronuclei. This is also apparent from the low surviving fractions. Cell survival was suppressed by 25-69% in all cell lines. The reduction of cell survival is a clear indication that azadirachtin affects reproductive integrity and cell division. The induction of micronuclei reflects DNA damage. Similar studies on damage induction in insect cell lines could elucidate the processes which precede the antifeedant and antimoulting effects of azadirachtin and other neem toxins in insects.     

Genotoxic activity of four metabolites of the soy isoflavone daidzein

Products containing phytoestrogens are increasingly promoted as the "natural" alternative to estrogen replacement therapy. In the present study, we have used the in vitro micronucleus assay in L5178Y mouse lymphoma cells to investigate the genotoxic potential of the isoflavone daidzein, and of four daidzein metabolites known to be formed in humans. Whereas no induction of micronuclei was observed with daidzein up to the limit of solubility (100 microM), all four daidzein metabolites, i.e. equol (2.3-fold induction at 100 microM), O-desmethylangolensin (6.2-fold induction at 10 microM), 4',6,7-isoflavone (6.7-fold induction at 100 microM) and 3',4',7-isoflavone (8.2-fold induction at 100 microM) induced micronuclei in a concentration-dependent manner. Thus, both reductive and oxidative metabolites of the soy isoflavone daidzein exhibit genotoxic potential in vitro.     

Chromosome damage and micronucleus formation in human blood lymphocytes exposed in vitro to radiofrequency radiation at a cellular telephone frequency (847.74 MHz, CDMA)

Peripheral blood samples collected from four healthy nonsmoking human volunteers were diluted with tissue culture medium and exposed in vitro for 24 h to 847.74 MHz radiofrequency (RF) radiation (continuous wave), a frequency employed for cellular telephone communications. A code division multiple access (CDMA) technology was used with a nominal net forward power of 75 W and a nominal power density of 950 W/m(2) (95 mW/cm(2)). The mean specific absorption rate (SAR) was 4.9 or 5.5 W/kg. Blood aliquots that were sham-exposed or exposed in vitro to an acute dose of 1.5 Gy of gamma radiation were included in the study as controls. The temperatures of the medium during RF-radiation and sham exposures in the Radial Transmission Line facility were controlled at 37 +/- 0.3 degrees C. Immediately after the exposures, lymphocytes were cultured at 37 +/- 1 degrees C for 48 or 72 h. The extent of genetic damage was assessed from the incidence of chromosome aberrations and micronuclei. The kinetics of cell proliferation was determined from the mitotic indices in 48-h cultures and from the incidence of binucleate cells in 72-h cultures. The data indicated no significant differences between RF-radiation-exposed and sham-exposed lymphocytes with respect to mitotic indices, frequencies of exchange aberrations, excess fragments, binucleate cells, and micronuclei. The response of gamma-irradiated lymphocytes was significantly different from that of both RF-radiation-exposed and sham-exposed cells for all of these indices. Thus there was no evidence for induction of chromosome aberrations and micronuclei in human blood lymphocytes exposed in vitro for 24 h to 847.74 MHz RF radiation (CDMA) at SARs of 4.9 or 5.5 W/kg.     

Dose-rate effects of ethyl methanesulfonate on survival and mutation induction in cultured Chinese hamster cells

The dose-rate effects of ethyl methanesulfonate (EMS) on the survival and induction of mutations in Chinese hamster Don cells were investigated. The most effective time of exposure to EMS for reducing the surviving fraction of cells was 4 h, shorter and longer exposure times being less effective. The threshold or minimal concentration of EMS giving a surviving fraction of 0.5 was 0.05 mg/ml. The minimal effective time of exposure to EMS for cell death was 1 h. Corrected survival curves showed that longer exposure times at lower dose rates of EMS had less cytotoxic effect than shorter exposure times at higher dose rates.After exposure of Don cells to various doses of EMS for various times, the frequencies of mutations resistant to 6-thioguanine (6TG) were measured. An exposure time of 4 h produced a lower mutation frequency than shorter or longer exposure times that resulted in the same surviving fraction of cells. An exposure time of 20 h produced the highest induced mutation frequency.This system using cultured Chinese hamster cells should be useful as a sensitive procedure for detecting the mutagenic actions of chemicals.     

Induction of micronuclei and anaphase aberrations by cytochalasin B in human lymphocyte cultures

The frequency of micronucleated cells in isolated 72-h human lymphocyte cultures treated with cytochalasin B (Cyt-B; 1.5-6 micrograms/ml for the last 28 h) was 9-21 times higher (mean 14.6 times) among multinucleate than binucleate cells. At 3 micrograms/ml, the concentration of Cyt-B originally recommended for the human lymphocyte micronucleus assay, the frequency of micronucleated multinucleate cells was 8.5%, while 0.7% of the binucleate cells had a micronucleus. Although no dose-dependent induction of micronuclei could be observed for either of the cell types, increase in the concentration of Cyt-B was associated with a decrease in the ratio of multinucleate to binucleate cells. Treatment with Cyt-B (1.5-12 micrograms/ml) increased the frequency of anaphase cells with aberrations, especially lagging chromatids. This finding was explained by a dose-dependent increase in multipolar (greater than or equal to 3 poles) divisions which had a high frequency of anaphase aberrations (39-53%), irrespective of the concentration of Cyt-B. Bipolar anaphases did not show a significant increase in aberrant cells, although a suggestive dependence on the concentration of Cyt-B was observed. The findings indicate that the high frequency of micronuclei in multinucleate lymphocytes produced by Cyt-B is due to mitotic errors arising when bi- (and multi-) nuclear cells divide. To avoid possible artifactually high micronucleus frequencies due to inclusion of cells that have divided greater than or equal to 2 times in the presence of Cyt-B, it is recommended that, in the human lymphocyte micronucleus assay using the cytokinesis-block method, the cell culture time is reduced to minimize the frequency of such cells and that only good preparations and regularly shaped binucleates are included in the analysis.     

Solar and UVC-induced mutation in human cells and inhibition by deoxynucleosides

Optimum conditions were established for quantitating the induction of hypoxanthine guanine phosphoribosyl transferase-deficient (HGPRT-) mutants in HeLa cells and in a human amelanotic melanoma cell line (MM96L). Compared at a fluence of equal toxicity (D37, fluence required to decrease cell survival to 37% of unirradiated control), noon sunlight in summer was slightly more mutagenic in MM96L than in HeLa cells (17 and 12 HGPRT- mutants per 10(6) survivors respectively). UVC (predominantly 254 nm) exhibited similar mutagenicity as equitoxic sunlight in HeLa but was 8-fold more effective in MM96L than equitoxic sunlight. Addition of a mixture of deoxyguanosine (20 microM), deoxyadenosine (20 microM), deoxycytidine (100 microM) and thymidine (20 microM) to the culture medium during the 7-day expression period following irradiation gave a 3-fold reduction in the UVC-induced mutation frequency of MM96L but not HeLa cells. The results suggest that these melanocytic cells are highly susceptible to mutagenesis by short wavelength UV, in a mechanism sensitive to deoxynucleosides.     

Chromosomal damage and mutations after exposure of Chinese hamster cells to high concentrations of oxygen

Chinese hamster lung fibroblast cells (V 79—379 A) were grown as monolayers and exposed to various concentrations of oxygen ranging from 40 to 95% at atmospheric pressure, for periods from 6 to 96 h. There were many abnormalities among stained cells on slides, including binucleate and multinucleate cells and micronuclei. The nuclei of stained mitotic cells, expanded by hypotonic solution, contained much chromosomal damage. This damage consisted chiefly of gaps and breaks in the chromatids and it increased in a dose-related manner with both percentage of oxygen and duration of exposure, reaching 100% of nuclei after 72 h exposure to 95% oxygen. Growth rate and survival of the cells were much reduced and colony-forming ability was inhibited to about 50% by a 24-h exposure to 95% oxygen and fell to less than 1% after 48 h at this oxygen tension. Mutations to azaquanine resistance were observed after exposure of hamster cells to 60, 80 and 95% oxygen for 24 or 48 h and also for 72 h in the case of 60% oxygen. The highest mutant frequency observed was 250 per 10⁶ surviving cells (mean control 8.2).     

Chromosomal damage and mutations after exposure of chinese hamster cells to high concentrations of oxygen

Chinese hamster lung fibroblast cells (V 79–379 A) were grown as monolayers and exposed to various concentrations of oxygen ranging from 40 to 95% at atmospheric pressure, for periods from 6 to 96 h. There were many abnormalities among stained cells on slides, including binucleate and multinucleate cells and micronuclei. The nuclei of stained mitotic cells, expanded by hypotonic solution, contained much chromosomal damage. This damage consisted chiefly of gaps and breaks in the chromatids and it increased in a dose-related manner with both percentage of oxygen and duration of exposure, reaching 100% of nuclei after 72 h exposure to 95% oxygen. Growth rate and survival of the cells were much reduced and colony-forming ability was inhibited to about 50% by a 24-h exposure to 95% oxygen and fell to less than 1% after 48 h at this oxygen tension. Mutations to azaguanine resistance were observed after exposure of hamster cells to 60, 80 and 95% oxygen for 24 or 48 h and also for 72 h in the case of 60% oxygen. The highest mutant frequency observed was 250 per 10⁶ surviving cells (mean control 8.2).     

Sensitivity of a human hybrid cell line (HeLa X skin fibroblast) to radiation-induced neoplastic transformation in G2, M, and mid-G1 phases of the cell cycle

The dependence of gamma-radiation-induced neoplastic transformation frequency on position in the cell cycle was measured for a human hybrid cell line (HeLa X skin fibroblast). The end point used was the induction of a tumor-associated antigen which in these cells correlates with tumorigenicity. Induction was measured in cells at G2, M, and mid-G1 phases and compared with the frequency induced in asynchronous cells. For studies of cells in G2 phase, the cells of an asynchronous population were collected for 3 h post-irradiation using the mitotic shake-off technique. For studies of cells in M and mid-G1 phases, cells were collected by mitotic harvest and then treated at the appropriate time. The data show that cells in G2 and M phase are very radiosensitive in terms of both cell killing and induction of neoplastic transformation compared to cells in mid-G1 or asynchronous populations. At a dose of 1 Gy, the transformation frequency was 10- to 20-fold higher for cells in M and G2 phase than for cells in mid-G1 or for asynchronous cells. However, the data indicate that the transformation frequencies were similar in the different phases of the cell cycle when correlated with surviving fraction. The results indicate that transformation frequency is more sensitive to changes in dose than is cell survival.     

Induction of chromosomal damage by restriction endonuclease in CHO cells porated with streptolysin O.

A procedure is described for the poration of living CHO cells with the bacterial cytotoxin streptolysin O (SLO) which allows the introduction into cells of the restriction endonuclease Pvu II to mimic and model the effects of ionising radiation in causing chromosomal damage. The dependence of this clastogenic effect of Pvu II on SLO concentration was measured by assaying the formation of micronuclei in cytokinesis-blocked binucleate cells. The optimum concentration was found to be 0.045 U/ml. Using the micronucleus assay, the time-course of expression of chromosome damage was investigated and found to show a biphasic kinetic with time. Using a sampling time of 30 h, a dose-effect curve for micronucleus induction by Pvu II was generated. Using this optimized SLO treatment protocol, the frequency of metaphase chromosome damage was subsequently investigated and found to be also linearly related to Pvu II concentration and total aberrations were approximately double the frequency of micronuclei. The induction and repair kinetics of DNA double-strand breaks were investigated in CHO cells treated with SLO and Pvu II using the neutral filter elution technique at pH 9.6. The data presented show that SLO can be used as an alternative method for porating cells to allow the introduction of restriction endonucleases into cells.     

Induction of aneuploidy by nickel sulfate in V79 Chinese hamster cells

The ability of nickel sulfate (NiSO(4)) to induce chromosome aneuploidy was investigated in vitro using the V79 Chinese hamster cell line. V79 cells were treated with 100-400 microM NiSO(4) for 24h, and monitored up to 72 h following treatment with a chromosome aberration assay, a micronuclei assay using antikinetochore antibodies (CREST assay) and an anaphase/telophase assay.Aneuploid cells were induced in a significant fraction of the cell population 24-48 h following treatment with nickel sulfate. The majority of these cells were hyperdiploid. In addition, nickel sulfate caused increased frequency of cells with kinetochore-positive micronuclei as well as kinetochore-negative micronuclei. Abnormal chromosome segregation such as lagging chromosomes, chromosome bridges and asymmetric segregation were also observed in more than 50% of anaphase or telophase cells following treatment with NiSO(4). The incidences of these abnormalities were dose-dependent in general, although the effects were prominent in a sublethal dose.These results indicate that NiSO(4) has the ability to induce aneuploidy in V79 cells. In addition, the results in anaphase/telophase assay suggest that the compound may have an effect on spindle apparatus, which could result in aneuploidy following abnormal chromosome segregation.     

Analysis of toxic and mutagenic activities of antiherpesvirus nucleosides against HeLa cells and herpes simplex virus type 1.

The toxic and mutagenic activities of five antiherpesvirus agents to HeLa cells and herpes simplex virus type 1 (HSV-1) were investigated. 5-Iodo-2'-deoxyuridine (IDU) and 9-beta-D-arabinofuranosyl-adenine (araA) showed very potent inhibitory effects on cell growth and the cloning efficiency of HeLa cells, whereas 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil (BV-araU), E-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and 9-(2-hydroxyethoxymethyl)guanine (ACV) showed less inhibitory effect. 50% inhibitory doses of BV-araU and BVDU for cell growth were 657 and 253 micrograms/ml, respectively. Although the growth inhibitory activity of BVDU was very weak, as above, the mutagenic activity of this drug to the cells, estimated by induction of colchicine-resistant mutants, was observed to be 4 micrograms/ml, which was a markedly smaller dose than the inhibitory dose for cell growth, and the highest frequency of mutation of the cells was shown at 100 micrograms/ml of BVDU. This activity was more potent than that of IDU. No mutagenic activity of BV-araU, araA and ACV to cells was observed within the concentration range of 1-800 micrograms/ml. IDU showed high mutagenic activity to HSV-1 growing in human embryo lung fibroblasts, and IDU-resistant mutants were induced at a high frequency. BVDU also induced a small amount of BVDU-resistant mutant virus, although this drug induced many mutant cells. No mutagenic activity of BV-araU, araA and ACV to HSV-1 was observed.     

G2 arrest, binucleation, and single-parameter DNA flow cytometric analysis

One important facet of flow cytometry involves the effects of pharmacological agents on cell cycle progression. Comparative G2 fraction perturbations were examined: effects of sodium butyrate on articular chondrocytes, effects of an antineoplastic agent (SOAZ) and an antirheumatic drug (D-penicillamine) on HeLa cells. Even though DNA flow cytometric analysis detects preferentially an induction of G2 arrest, the mode of action of these agents on the cell cycle is different. Sodium butyrate and D-penicillamine lead to an increase of binucleate cells due to cytokinesis perturbation. Because of similar fluorescence intensity, distinguishing G2 from binucleate GO/1 cells is not easily possible using DNA content measurement and reflects a failure of flow cytometry in the detection of binucleate cells. Rapid cell cycle analysis of single cells should contribute greatly to the study of pharmacological interactions, but DNA flow cytometric measurements obtained from cultured cells exposed to certain agents must be cautiously interpreted because those may interact on cytokinesis and induce artefacts in histogram interpretation.     

Evidence for induction of DNA double strand breaks in the bystander response to targeted soft X-rays in CHO cells

This study investigated the role of DNA double strand breaks and DNA base damage in radiation-induced bystander responses in Chinese hamster ovary (CHO) cell lines. Two CHO repair-deficient clones, xrs5 (DNA double strand break repair-deficient) and EM9 (DNA base excision repair-deficient) were used in addition to the wild type (CHO). The Gray Cancer Institute ultrasoft X-ray microprobe is a powerful tool for investigating the bystander response, because it permits the irradiation of only a single nucleus of a cell, as reported previously. In order to investigate the bystander effect in each repair-deficient cell line, we irradiated a single cell within a population and scored the formation of micronuclei. When a single nucleus in the population was targeted with 1 Gy, elevated numbers of micronuclei were induced in the neighbouring unirradiated cells in the EM9 and xrs5 cell lines, whereas induction was not observed in CHO. The induction of micronuclei in xrs5 was significantly higher than that in EM9. Under these conditions, the surviving fraction in the neighbouring cells was significantly lower in xrs5 than in the other cell lines, showing a higher cell killing effect in xrs5. To confirm that bystander factors secreted from irradiated cells caused these effects, we carried out medium transfer experiments using conventional X-irradiation. Medium conditioned for 24 h with irradiated cells was transferred to unirradiated cells and elevated induction of micronuclei was observed in xrs5. These results suggest that DNA double strand breaks rather than base damage are caused by factors secreted in the medium from irradiated cells.     

Comparative analysis of DNA breakage,chromosomal aberrations and apoptosis induced by the anti-herpes purine nucleoside analogues aciclovir,ganciclovir and penciclovir

Nucleoside analogues have been used in antiviral therapy and suicide cancer gene therapy. Therefore, it is of importance to compare their potential cytotoxic and genotoxic action. Using metabolically competent CHO cells expressing the thymidine kinase gene of herpes simplex virus type 1 (CHO-HSVtk cells) as a model system, the induction of DNA breaks was compared with the induction of structural chromosomal aberrations and apoptosis/necrosis after exposure to the anti-herpes nucleoside analogues aciclovir (ACV), ganciclovir (GCV) and penciclovir (PCV). After continuous treatment of CHO-HSVtk cells with the drugs, LD(10) in a colony-forming assay was 50, 0.5 and 1 microM for ACV, GCV and PCV, respectively, with GCV to be the most potent agent as determined at a given dose level. There was a remarkable difference in the activity of the agents to kill HSVtk expressing and non-expressing cells: the difference in cellular sensitivity of HSVtk(+) versus HSVtk(-) cells at LD(10) level was 7-fold for ACV, 60-fold for GCV and 400-fold for PCV. The drugs were shown to be strong inducers of apoptosis that was analysed as to concentration- and time-dependence; they induced to only very low extent necrosis. The agents were also highly potent in the induction of DNA single-strand breaks (SSBs) and double-strand breaks (DSBs) (as measured by single cell gel electrophoresis (SCGE)) and chromosomal aberrations. Although PCV induced DNA DSBs with a kinetics and frequency similar to that of GCV, it caused mostly condensation defects instead of "typical" structural chromosomal aberrations. For the drugs used, the frequency of apoptotic cells and the induction of abnormal mitoses appear to be related indicating genotoxic effects induced by the agents to be involved in cell killing due to apoptosis.