Erythrocytes modulate the baseline frequency of sister-chromatid exchanges and the kinetics of lymphocyte division in culture

The baseline sister-chromatid exchange (SCE) frequencies of human plasma lymphocyte cultures (PLC), but not pig PLC, were nearly twice as high as those of whole-blood cultures (WBC). Addition of human red blood cells (RBCs) to human PLC decreased the SCE frequency in proportion to the RBC-leukocyte co-incubation interval. When the period of RBC-leukocyte co-incubation was equivalent to the total length of the culture period (72 h), the SCE frequency was similar to that observed in WBC. Shorter co-incubation periods yielded SCE frequencies intermediate between those of PLC and WBC. Regardless of the species, cell proliferation was slower in PLC than in WBC. Experiments where RBCs were added to PLC showed that the time sequence of RBC incorporation also affects the cell-cycle progression of human and pig lymphocytes. When either human or pig RBCs were added immediately after PLC stimulation, the cell-cycle kinetics was similar to that of WBC. Shorter co-incubation periods made cell-cycle progression intermediate between PLC and WBC values. Thus, PBCs modulate the baseline frequency of SCEs in human PLC and the cell-cycle progression of both human and pig lymphocytes in a time-dependent manner. Two possible hypotheses for the heightened frequency of SCEs of human lymphocytes in RBC-free cultures were assessed. The loss of RBC-to-lymphocyte cellular contact in PLC did not influence the SCE frequencies of lymphocytes. Finally, the increase of SCEs in human PLC could not be related to differences in the generation time of lymphocytes in culture.     

Effect of dithiocarbamate pesticide zineb and its commercial formulation, azzurro. III. Genotoxic evaluation on Chinese hamster ovary (CHO) cells

The in vitro genotoxicity exerted by the dithiocarbamate fungicide zineb, and its commercial formulation azzurro, were studied in Chinese hamster ovary (CHO) cells by the analysis of the sister chromatid exchange (SCE), cell-cycle progression and single cell gel electrophoresis (SCGE) assays. Both zineb and azzurro activities were tested within the range of 0.1-100.0 microg/ml. Concentrations of 0.1-25.0 microg/ml of zineb or azzurro induced a significant dose-dependent increase in SCE frequency over control values. For both test compounds, while doses ranging from 0.1 to 1.0 microg/ml did not alter the rate of cell proliferation, a significant delay in cell-cycle progression was observed within the 5.0-25.0 microg/ml dose-range. A regression test showed that either the proliferative replication index or the mitotic activity of cultures decreased as a function of the pesticide concentration within the 1.0-25.0 microg/ml dose-range. Doses higher than 50.0 microg/ml were cytotoxic. SCGE assay revealed an increase in zineb-induced DNA damage by enhancing the proportion of slightly damaged cells in the 25.0-100.0 microg/ml dose-range and by increasing in a dose-dependent manner the proportion of damaged cells within the 1.0-100.0 microg/ml dose-range. Overall, image analysis showed statistically significant positive relationships between zineb concentration and DNA damage (expressed by image length and width) and between length and width of the damaged cells. In azzurro-treated cells, only when 100.00 microg/ml was employed a significant increase in the frequency of damaged cells over control values affecting the totality of the cells was observed only when 100.0 microg/ml was employed. When lower doses were employed, no DNA damage was revealed. Based on these results, the evaluation of zineb as a genotoxic/non-genotoxic compound for human health should be reconsidered. Even though we demonstrate that the pesticide induces large DNA alterations in vitro, does no necessarily mean that the chemical should be considered clastogenic.notoxic     

Increased rates of sister chromatid exchanges induced by the herbicide 2,4-D

The potential for genetic damage from widely used hormonic herbicides, such as 2,4-dichlorophenoxyacetic acid (2,4-D), continues to be of serious concern. The mutagenic effect as reflected by the rates of sister chromatid exchanges (SCE) was determined in cultured human lymphocytes. Data were based on the analysis of 50 cells for the control and each of the three treatments. A 50 micrograms/ml dosage caused a highly significant increase in SCE. Dosages of 100 and 250 micrograms/ml elevated the rate of SCE, but not significantly. Since 2,4-D biodegrades rapidly in soil and water, its continued use is not in serious question until safer compounds are available. However, the results of this study suggest that the danger of genetic damage from direct exposure to commercial samples of 2,4-D should not be ignored.     

Dominant lethal effects of 2,4-D in Biomphalaria glabrata

Dominant lethal effects of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) were evaluated in the freshwater snail Biomphalaria glabrata. Wild-type snails were exposed during 10 days to 50, 75 and 100 ppm of 2,4-D dimethylamine salt (2,4-D DMA) and paired with non-exposed albino snails 1, 11, 25 and 40 days after the exposure. The offspring of the non-exposed albino snails was scored for lethal malformations.One day after the exposure, a significant effect was observed at 75 and 100 ppm without a dose–response relationship. After 11 days, the effect was observed only at the highest dose. After 25 days, significant increases in the dominant lethal effects occurred at 50 and 75 ppm; effects were directly related to the doses. Background levels of lethal malformations were resumed after 40 days.Although the major and direct measure of dominant lethal mutations is the rate of lethal malformations in the heterozygous offspring of the albino snails, the sensitivity of the assay was substantially increased with the evaluation of all non-viable embryos, that are the sum of those with lethal malformations, identified or not as wild-type.     

Clastogenicity of the fungicide afugan in cultured human lymphocytes

The genotoxic effects of the fungicide afugan were analysed by measuring chromosomal aberrations (CAs), sister chromatid exchange (SCE) and micronuclei (MN) in cultured human peripheral lymphocytes. Concentrations of 2.5, 5, 10 and 20 microg/ml of afugan were used during 24 and 48 h. Afugan significantly increased the frequency of CAs at 5, 10 and 20 microg/ml concentrations during a 48 h treatment period. A significant increase was observed for induction of SCE and MN at all treatments compared with the negative control. A significant dose-response correlation was found in all tests. Afugan did not affect the replicative index (RI), however it significantly decreased the mitotic index (MI) at all treatment concentrations except 2.5 microg/ml, and at both treatment times. The present results indicate that afugan is clastogenic and cytotoxic to cultured human lymphocytes.     

Dominant lethal effects of 2,4-D in Biomphalaria glabrata

Dominant lethal effects of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) were evaluated in the freshwater snail Biomphalaria glabrata. Wild-type snails were exposed during 10 days to 50, 75 and 100ppm of 2,4-D dimethylamine salt (2,4-D DMA) and paired with non-exposed albino snails 1, 11, 25 and 40 days after the exposure. The offspring of the non-exposed albino snails was scored for lethal malformations. One day after the exposure, a significant effect was observed at 75 and 100ppm without a dose-response relationship. After 11 days, the effect was observed only at the highest dose. After 25 days, significant increases in the dominant lethal effects occurred at 50 and 75ppm; effects were directly related to the doses. Background levels of lethal malformations were resumed after 40 days. Although the major and direct measure of dominant lethal mutations is the rate of lethal malformations in the heterozygous offspring of the albino snails, the sensitivity of the assay was substantially increased with the evaluation of all non-viable embryos, that are the sum of those with lethal malformations, identified or not as wild-type.     

Genotoxic activity of the commercial herbicide containing bifenox in bovine peripheral lymphocytes

The commercial herbicide with active element bifenox (principal tradename Modown) was tested for the evaluation of genotoxicity in cultured cow peripheral lymphocytes in vitro. Several cytogenetic endpoints as chromosome aberrations (CA), sister chromatid exchanges (SCE), mitotic (MI) and proliferation (PI) indices were investigated in different sampling times. To detect possible metabolic modifications in herbicide genotoxicity, the cultures for SCE determination were also treated with S9 fraction. Cultures of lymphocytes were exposed to the herbicide at concentrations of 25, 50, 250, 500 and 1000 microg/ml. A slight increase of CAs was found after exposure of this agent to doses ranging from 25 to 250 microg/ml for 24 h. In the CA assay no statistical significance was seen. Both higher doses (500 and 1000 microg/ml) caused a decrease of chromosome damage in comparison to the last active dose or control values correlated to induced cytotoxicity. Four concentrations (all except the highest one) of the herbicide were applied into cultures in SCE assays both with and without metabolic activation. Significant elevations of SCE were observed after applications of herbicide tested at doses of 250 and 500 microg/ml in each donor (P<0.001 and P<0.05, respectively) for 24 h. These concentrations also caused a statistically significant decrease in the MI and PI. Treatment for 48 h provided inadequate evidence for the genotoxic activity of the herbicide.     

Genotoxic effect of 2,4-dichlorophenoxy acetic acid and its metabolite 2,4-dichlorophenol in mouse.

The cytogenetic effect of 2,4-dichlorophenoxy acetic acid (2,4-D) and its metabolite 2,4-dichlorophenol (2,4-DCP) was studied in bone-marrow, germ cells and sperm head abnormalities in the treated mice. Swiss mice were treated orally by gavage with 2,4-D at 1.7, 3.3 and 33 mg kg(-1)BW (1/200, 1/100 and 1/10 of LD(50)). 2,4-DCP was intraperitoneally (i.p.) injected at 36, 72 and 180 mg kg(-1)BW (1/10, 1/5, 1/2 of LD(50)). A significant increase in the percentage of chromosome aberrations in bone-marrow and spermatocyte cells was observed after oral administration of 2,4-D at 3.3 mg kg(-1)BW for three and five consecutive days. This percentage increased and reached 10.8+/-0.87 (P<0.01) in bone-marrow and 9.8+/-0.45 (P<0.01) in spermatocyte cells after oral administration of 2,4-D at 33 mg kg(-1)BW for 24 h. This percentage was, however, lower than that induced in bone-marrow and spermatocyte cells by mitomycin C (positive control). 2,4-D induced a dose-dependent increase in the percentage of sperm head abnormalities. The genotoxic effect of 2,4-DCP is weaker than that of 2,4-D, as indicated by the lower percentage of the induced chromosome aberrations (in bone-marrow and spermatocyte cells) and sperm head abnormalities. Only the highest tested concentration of 2,4-DCP (180 mg kg(-1)BW, 1/2 LD(50)) induced a significant percentage of chromosome aberrations and sperm head abnormalities after i.p. injection. The obtained results indicate that 2,4-D is genotoxic in mice in vivo under the conditions tested. Hence, more care should be given to the application of 2,4-D on edible crops since repeated uses may underlie a health hazard.     

Influence of herbicide, 2,4-dichlorophenoxy acetic acid, on haemocyte DNA of in vivo treated mussel

The influence of the herbicide 2,4-dichlorophenoxy acetic acid (2,4-D) on haemocyte DNA of in vivo treated mussels Mytilus galloprovincialis has been investigated by flow cytometry and epifluorescence microscopy. Haemocyte proliferation and atypical flow cytometric DNA histograms were observed in mussels treated with 20 and 100 μg/g of 2,4-D. The stimulation of proliferation by 2,4-D was also obvious by DNA labelling with BrdU followed by FITC conjugated anti-BrdU MoAb visualised by epifluorescence microscopy. An apoptotic sub-G₁ peak resulted in mussels that were exposed to higher doses of herbicide at 100 and 500 μg/g as well as subpopulation could be detected by flow cytometric analysis. In these experiments morphological changes characteristic for apoptotic cells were looked for by fluorescence microscopy. A low percentage of cells in S as well as in G₂M phase indicating G1 arrest were detected in haemocytes from these mussels that had survived 4 days of 20 μg/g 2,4-D exposure. In addition, sister-chromatid exchanges (SCE) could be seen with the immunolabelling BrdU method. Thus, in vivo treatment and the subsequent uptake of 2,4-D causes serious genetic consequences and raises concerns regarding the potential overall fitness and health effects in mussel populations.     

Synergistic Effects of Cu(II) and Dimethylammonium 2,4-Dichlorophenoxyacetate (U46 D Fluid) on PM2 DNA and Mechanism of Dna Damage

Dimethylammonium 2,4-dichlorophenoxyacetate (2,4-D · DMA) induced strand breaks in PM2 DNA when incubated with CuCl₂, whereas 2,4-D · DMA alone or CuCl₂ alone did not show any or only a negligible effect. The formation of single strand breaks increased linearly with time and concentration of 2,4-D · DMA. Neocuproine, a specific Cu(I) chelator totally prevented strand break formation. So did catalase (up to 100mM 2,4-D · DMA), but DMSO had only a small protective effect. 2,4-Dichlorophenol, CO₂ and formaldehyde were detected as reaction products of 2,4-D and CuCl₂. From these results a redox reaction of Cu(II) and 2,4-D is proposed, which could explain the DNA damaging properties of CuCl₂/2,4-D · DMA.     

Mutagenic and genotoxic effects of Anilofos with micronucleus,chromosome aberrations,sister chromatid exchanges and Ames test

We aimed to evaluate the mutagenic effect of Anilofos, organophosphate pesticide, by using Ames/Salmonella/microsome test. Its cytotoxic and genotoxic effects were also determined by chromosome aberration (CA), sister chromatid exchange (SCE) and micronucleus (MN) test in human peripheral blood lymphocytes. In the Ames test, five different concentrations of Anilofos were examined on TA97, TA98, TA100 and TA102 strains in the absence and presence of S9 fraction. According to the results all concentrations of this pesticide have not shown any mutagenic activity on TA97, TA100 and TA102 strains in the absence and presence of S9 fraction. But, 10, 100 and 1000 µg/plate concentrations of Anilofos were determined to be mutagenic on TA98 strain without S9 fraction. Lymphocytes were treated with various concentrations (25, 50, 100 and 200 µg/ml) of Anilofos for 24 and 48 h. The results of the assays showed that Anilofos did not induce SCE frequency, replication index and MN formation at all concentrations for both treatment periods. Anilofos significantly increased CA frequency at 100 and 200 µg/ml concentrations at 24 h treatment periods and at 50, 100 and 200 µg/ml concentrations in 48 h treatment periods. Additionally, it was determined that this pesticide decreased mitotic index and nuclear division index significantly. It was concluded that Anilofos has genotoxic and cytotoxic effects in human peripheral lymphocytes.     

Genotoxic effects of estradiol-17beta on human lymphocyte chromosomes

The cytogenetic effect of a hormonal steroid, estradiol-17beta, was assessed in peripheral blood human lymphocyte culture. Sister chromatid exchanges (SCE) and chromosome aberrations (CA) were scored as genetic end points. Significant induction of CA was observed at 25 microg/ml and 50 microg/ml concentrations of estradiol-17beta in the absence of microsomal activation. The drug was effective in all treatments in the presence of rat liver S(9) microsomal fraction (S(9) mix) and exhibited increased frequency of chromosomal aberrations. The drug was effective in increasing the SCE frequency which was found to be maximum at the dose of 50 microg/ml concentration (i.e., 4.34+/-1.22) both with and without metabolic activation. It was found that estradiol-17beta itself and possibly its metabolites are potent mutagens beyond a particular dose in human lymphocytes.     

The chlorophenoxy herbicide dicamba and its commercial formulation banvel induce genotoxicity and cytotoxicity in Chinese hamster ovary (CHO) cells

The sister chromatid exchange (SCE) frequency, the cell-cycle progression analysis, and the single cell gel electrophoresis technique (SCGE, comet assay) were employed as genetic end-points to investigate the geno- and citotoxicity exerted by dicamba and one of its commercial formulation banvel (dicamba 57.71%) on Chinese hamster ovary (CHO) cells. Log-phase cells were treated with 1.0-500.0 microg/ml of the herbicides and harvested 24 h later for SCE and cell-cycle progression analyses. All concentrations assessed of both test compounds induced higher SCE frequencies over control values. SCEs increased in a non-dose-dependent manner neither for the pure compound (r=0.48; P>0.05) nor for the commercial formulation (r=0.58, P>0.05). For the 200.0 microg/ml and 500.0 microg/ml dicamba doses and the 500.0 microg/ml banvel dose, a significant delay in the cell-cycle progression was found. A regression test showed that the proliferation rate index decreased as a function of either the concentration of dicamba (r=-0.98, P<0.05) or banvel (r=-0.88, P<0.01) titrated into cultures in the 1.0-500.0 microg/ml dose-range. SCGE performed on CHO cells after a 90 min pulse-treatment of dicamba and banvel within a 50.0-500.0 microg/ml dose-range revealed a clear increase in dicamba-induced DNA damage as an enhancement of the proportion of slightly damaged and damaged cells for all concentrations used (P<0.01); concomitantly, a decrease of undamaged cells was found over control values (P<0.01). In banvel-treated cells, a similar overall result was registered. Dicamba induced a significant increase both in comet length and width over control values (P<0.01) regardless of its concentration whereas banvel induced the same effect only within 100.0-500.0 microg/ml dose range (P<0.01). As detected by three highly sensitive bioassays, the present results clearly showed the capability of dicamba and banvel to induce DNA and cellular damage on CHO cells.     

Assessment of the genotoxicity of imidacloprid and metalaxyl in cultured human lymphocytes and rat bone-marrow

Imidacloprid and metalaxyl are two pesticides that are widely used in agriculture, either separately, or in combination. These agents were studied for their possible genotoxic effects with respect to the following cytogenetic end-points: (1) in vitro micronucleus (MN) formation and sister-chromatid exchange (SCE) induction in human lymphocytes and (2) in vivo micronucleus induction in polychromatic erythrocytes (PCEs) of the rat bone-marrow. The results of the MN analysis indicate that MN frequencies after treatment with both pesticides, separately or as a mixture, do not significantly differ from those in the controls except after treatment with metalaxyl alone at 50 microg/ml (p<0.05). The results of the SCE analysis show that SCE frequencies after treatment with imidacloprid do not differ significantly from those in the controls. A statistically significant increase (p<0.05) in SCE frequency resulted from treatments with metalaxyl at 5, 10 and 100 microg/ml and with the combination of imidacloprid and metalaxyl at 100 and 200 microg/ml. Finally, the in vivo micronucleus assay with rat bone-marrow polychromatic erythrocytes showed a statistically significant effect upon separate treatments with imidacloprid and metalaxyl at doses of 300 mg/kg body weight (b.w.) (p<0.01) or upon combined treatment with 200 mg/Kg b.w. (p<0.001) and 400 mg/kg b.w. (p<0.05).     

Genotoxicity testing of fluconazole in vivo and in vitro

The genotoxic effects of the antifungal drug fluconazole (trade name triflucan) were assessed in the chromosome aberration (CA) test in mouse bone-marrow cells in vivo and in the chromosome aberration, sister chromatid exchange (SCE) and micronucleus (MN) tests in human lymphocytes. Fluconazole was used at concentrations of 12.5, 25.0 and 50.0 mg/kg for the in vivo assay and 12.5, 25.0 and 50.0 microg/ml were used for the in vitro assay. In both test systems, a negative and a positive control (MMC) were also included. Six types of structural aberration were observed: chromatid and chromosome breaks, sister chromatid union, chromatid exchange, fragments and dicentric chromosomes. Polyploidy was observed in both the in vivo and in vitro systems. In the in vivo test, fluconazole did not significantly increase the frequency of CA. In the in vitro assays, CA, SCE and MN frequencies were significantly increased in a dose-dependent manner compared with the negative control. The mitotic, replication and cytokinesis-block proliferation indices (CBPI) were not affected by treatments with fluconazole. According to these results, fluconazole is clastogenic and aneugenic in human lymphocytes, but these effects could not be observed in mice. Further studies should be conducted in other test systems to evaluate the full genotoxic potential of fluconazole.     

Genotoxic effects induced by fotemustine and vinorelbine in human lymphocytes

The aim of this study was to investigate the in vitro genotoxic effects of the anticancer drugs fotemustine and vinorelbine on human lymphocytes and to determine individual and sex-related responses to these drugs. Fotemustine is a DNA-alkylating drug while vinorelbine is a semi-synthetic Vinca alkaloid. The study was carried out with twenty independent healthy donors for each drug. We have tested the ability of these drugs to induce chromosome aberrations (CAs) and sister chromatid exchanges (SCEs) as well as effect on the mitotic index (MI) in cultured human lymphocytes. Fotemustine was shown to induce CAs and SCEs at all concentrations tested (2, 4 and 8 microg/ml) in a dose-dependent manner. Additionally it also decreased the mitotic index in a similar dose-dependent manner. Vinorelbine had no effect on structural CAs, but it significantly increased the numerical CAs at all doses tested (0.5, 1 and 2 microg/ml). Vinorelbine also induced SCE events and increased the MI values. Two-way analyses of variance were used to compare the individual and gender-related susceptibilities to fotemustine and vinorelbine with respect to the CA, SCE and MI values. The results indicated that individuals in fotemustine treatment groups showed different genotoxic responses with respect to CA and SCE induction and additional findings indicated a gender-specific response in this group. Individuals in the vinorelbine test group also exhibited statistically significant numerical CA, SCE and MI responses to vinorelbine. A statistically significant gender-related SCE response to this drug was also evident. This study indicates that these drugs have potentially harmful effects on human health.     

Nitroso-aldicarb induces sister-chromatid exchanges in human lymphocytes in vitro

Nitroso-aldicarb was tested for its ability to induce sister-chromatid exchanges (SCE) and cell-cycle delay in human peripheral blood lymphocytes in vitro. This derivative of aldicarb induced a dose-dependent increase in SCE values per cell. In addition, a slight decrease in the successive mitotic progression of cells in culture was observed.     

Induction of micronuclei and erythrocyte alterations in the catfish Clarias batrachus by 2,4-dichlorophenoxyacetic acid and butachlor

The micronucleus test (MNT) in fish erythrocytes has increasingly been used to detect the genotoxic effects of environmental mutagens and its frequency is considered to reflect the genotoxic damage to cells, mainly the chromosomes. Besides, morphologically altered erythrocyte is taken as an index of cytotoxicity. Both parameters were used in the present study by two herbicides, 2,4-dichlorophenoxyacetic acid (2,4-D, in 25, 50 and 75ppm concentrations) and 2-chloro-2,6-diethyl-N-(butoxymethyl) acetanilide (butachlor, in 1, 2 and 2.5ppm concentrations) for genotoxic and cytotoxic endpoints. The study was carried out by an in vivo method on peripheral erythrocytes of catfish Clarias batrachus using multiple sampling times (48, 72 and 96h). Cytogenetic preparations were made by haematoxylin-eosin staining technique. Pycnotic and granular micronuclei (MN) were consistently observed irrespective of chemical tested. A wide range of altered cells was also observed. Echinocytes accompanied by altered nuclei and vacuoles were prominent feature of 2,4-D, whereas, anisochromasia and anisocytosis of erythrocytes were characteristic of butachlor. Increase in MN as well as altered cells frequencies were significant. A positive dose-response relationship in all exposures and sampling times was observed. Herbicides used were found to be genotoxic as well as cytotoxic in this fish. The suitability of the adopted parameters for the screening of the aquatic genotoxicants is discussed.     

The influence of 2,4-dichlorophenoxyacetic acid on cell cycle Kinetics and Sister-Chromatid Exchange Frequency in Garlic (Allium sativum L.) Meristem Cells

The effect of 2,4-dichlorophenoxyacetic acid (2,4-D) at low concentrations on cell cycle duration and sister-chromatid exchange (SCE) frequency was studied using meristem root-tip cells ofAllium sativum L. 2,4-D induced a marked prolongation of the cell cycle. At the same time, small but statistically significant increases in SCE frequencies were observed at 5 μM and 15 μM 2,4-D concentrations. The significance of these findings in the evaluation of mutagenic activity of 2,4-D is discussed.     

Preparation of anti-2,4-dichlorophenol and 2,4-dichlorophenoxyacetic acid monoclonal antibodies

The ratios of hapten and bovine serum albumin (BSA) in an antigen conjugate were determined by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Hybridomas secreting monoclonal antibodies against 2,4-dichlorophenoxyacetic acid (2,4-D) were produced by fusing 2,4-D-BSA conjugate-immunized splenocytes with a HAT-sensitive mouse myeloma cell line, P3-X63-Ag8-653. A substantial cross-reaction was observed for 2,4-dichlorophenol (2,4-DP) when compared with that observed for 2,4-D. The full measurement range for this assay is 0.2–3 μg ml⁻¹ for 2,4-DP. On the other hand, the range for 2,4-D is between 1 and 20 μg ml⁻¹. This revised version was published online in July 2006 with corrections to the Cover Date.