Herbicide 2,4-dichlorophenoxyacetic acid (2,4-D)-induced cytogenetic damage in human lymphocytes in vitro in presence of erythrocytes

The genotoxic effects of 2,4-D and its commercial derivative 2,4-D DMA were studied by measuring sister chromatid exchange (SCE), cell-cycle progression and mitotic index in human whole blood (WBC) and plasma leukocyte cultures (PLC). Concentrations of 10, 25, 50 and 100 microg herbicide/ml were used during 72 h. In WBC, a significant increase in SCE frequency was observed within the 10-50 microg 2,4-D/ml and 25-100 microg 2,4-D DMA/ml dose range. Contrarily, in PLC, none of the concentrations employed affected the SCEs frequency. A significant delay in cell proliferation was observed in WBC after treatments with 25 and 50 microg 2,4-D/ml and 50 and 100 microg 2,4-D DMA/ml. In PLC, only 100.0 microg 2,4-D/ml altered cell-cycle progression. For both chemicals, a progressive dose-related inhibition of mitotic activity was observed. The results demonstrated that the presence of erythrocytes in the culture system modulated the DNA and cellular damage inflicted by 2,4-D and 2,4-D DMA into human lymphocytes in vitro as well as both 2,4-D and 2,4-D DMA were more potent genotoxic agents in the presence of human red cells.     

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.     

Effect of inoculant strain and organic matter content on kinetics of 2,4-dichlorophenoxyacetic acid degradation in soil.

We monitored rates of degradation of soluble and sorbed 2,4-dichlorophenoxyacetic acid (2,4-D) in low-organic-matter soil at field capacity amended with 1, 10, or 100 micrograms of 2,4-D per g of wet soil and inoculated with one of two bacterial strains (MI and 155) with similar maximum growth rates (mu max) but significantly different half-saturation growth constants (Ks). Concentrations of soluble 2,4-D were determined by analyzing samples of pore water pressed from soil, and concentrations of sorbed 2,4-D were determined by solvent extraction. Between 65 and 75% of the total 2,4-D was present in the soluble phase at equilibrium, resulting in soil solution concentrations of ca. 8, 60, and 600 micrograms of 2,4-D per ml, respectively. Soluble 2,4-D was metabolized preferentially; this was followed by degradation of both sorbed (after desorption) and soluble 2,4-D. Rates of degradation were comparable for the two strains at soil concentrations of 10 and 100 micrograms of 2,4-D per g; however, at 1 microgram/g of soil, 2,4-D was metabolized more rapidly by the strain with the lower Ks value (strain MI). We also monitored rates of biodegradation of soluble and sorbed 2,4-D in high-organic-matter soil at field capacity amended with 100 micrograms of 2,4-D per g of wet soil and inoculated with the low-Ks strain (strain MI). Ten percent of total 2,4-D was present in the soluble phase, resulting in a soil solution concentration of ca. 30 micrograms of 2,4-D per ml.(ABSTRACT TRUNCATED AT 250 WORDS)     

2,4-Dichlorophenoxyacetic acid causes chromatin and chromosome abnormalities in plant cells and mutation in cultured mammalian cells

The cytotoxic and mutagenic effects of the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) on shallot root tip cells and on V79 Chinese hamster fibroblast cells were examined and compared. In shallot root tips 2,4-D caused changes in mitotic activity, as well as changes in chromosome and chromatin structure, and also changes during the cell cycle. 2,4-D also showed mutagenic and cytotoxic effects on V79 cells in culture in concentrations higher than 10 micrograms/ml. The results in both systems (plant and mammalian cells) were in agreement showing mutagenic activity of 2,4-D in the concentration range higher than usually used in establishing plant tissue culture (greater than 5 micrograms/ml).     

Effect of inoculant strain and organic matter content on kinetics of 2,4-dichlorophenoxyacetic acid degradation in soil.

We monitored rates of degradation of soluble and sorbed 2,4-dichlorophenoxyacetic acid (2,4-D) in low-organic-matter soil at field capacity amended with 1, 10, or 100 micrograms of 2,4-D per g of wet soil and inoculated with one of two bacterial strains (MI and 155) with similar maximum growth rates (mu max) but significantly different half-saturation growth constants (Ks). Concentrations of soluble 2,4-D were determined by analyzing samples of pore water pressed from soil, and concentrations of sorbed 2,4-D were determined by solvent extraction. Between 65 and 75% of the total 2,4-D was present in the soluble phase at equilibrium, resulting in soil solution concentrations of ca. 8, 60, and 600 micrograms of 2,4-D per ml, respectively. Soluble 2,4-D was metabolized preferentially; this was followed by degradation of both sorbed (after desorption) and soluble 2,4-D. Rates of degradation were comparable for the two strains at soil concentrations of 10 and 100 micrograms of 2,4-D per g; however, at 1 microgram/g of soil, 2,4-D was metabolized more rapidly by the strain with the lower Ks value (strain MI). We also monitored rates of biodegradation of soluble and sorbed 2,4-D in high-organic-matter soil at field capacity amended with 100 micrograms of 2,4-D per g of wet soil and inoculated with the low-Ks strain (strain MI). Ten percent of total 2,4-D was present in the soluble phase, resulting in a soil solution concentration of ca. 30 micrograms of 2,4-D per ml.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sister-chromatid exchanges in human lymphocytes exposed to 1-p-(3-methyltriazeno)benzoic acid potassium salt

The 1-p-(3-methyltriazeno) benzoic acid potassium salt (MTBA) is a triazeno analogue of dacarbazine, an antineoplastic agent capable of mediating the appearance of new antigenic specificities on cancer cells in mice, a phenomenon described as 'chemical xenogenization' (CX). Recently we reported the clastogenic potential of MTBA on human lymphocytes. Since sister-chromatid exchange (SCE) assay is more sensitive than clastogenic tests, at least at low drug concentrations, we assessed SCE frequencies induced by MTBA on human lymphocytes stimulated by PHA. Drug treatment at 2-500 micrograms/ml was performed in vitro prior to or after PHA addition. SCE values increased significantly in a dose-dependent manner up to 200 micrograms/ml. However, SCE frequencies, as well as chromosome breaks, did not increase dramatically. These data indicate that MTBA concentrations used for CX do not cause severe cytogenetic damage to immune cells at least in vitro.     

Sister-chromatid exchanges in lymphocytes from infants with Down's syndrome

Sister-chromatid exchange (SCE) frequencies were studied in blood lymphocytes from 12 patients (3 females and 9 males) with Down's syndrome (DS). The mean frequency of SCE per metaphase for the patients (both sexes) was 9.2 +/- 0.8 which was significantly higher (P less than 0.01) than the mean SCE value (5.1 +/- 0.2) scored for 16 healthy infants (8 females and 8 males). A significant increase in the mean frequency of SCE in 12 parents of infants with DS (8.7 +/- 0.9 SCE/cell) was noticeable when compared with 20 parents of normal infants (6.3 +/- 0.1 SCE/cell). Increases in cellular division with reduction in their replication were also observed in patients with DS. Treatment with mitomycin C (0.05 micrograms/ml), hycanthone (0.1 micrograms/ml) and gamma-radiation (0.1 Gy) revealed a significant (P less than 0.01) increase in frequencies of SCE in DS lymphocytes and in those of their parents as compared to controls. These data may reveal a familial hypersensitivity reaction to these agents. The results indicate a genomic instability and deranged DNA-repair mechanisms which are accentuated by exposure to mutagenic agents, the underlying causal factor for which might be genetic.     

Modulation by novobiocin of sister-chromatid exchanges induced by tumor necrosis factor in human lymphocytes.

The effect of the antibiotic novobiocin on human recombinant tumor necrosis factor (TNF)-induced sister-chromatid exchanges (SCEs) were examined in human peripheral blood lymphocytes. TNF, when introduced in a dose range of 10-1000 U/ml at the initiation of culture, was found to cause a significant increase in SCE frequency. The simultaneous addition of TNF and novobiocin (25 micrograms/ml) in the assay resulted in no increase of SCE frequency. Delayed (for 24 h) addition of novobiocin suppressed the induction of SCEs by 50, 100 and 500 U/ml but not by 1000 U/ml of TNF.     

Genetic toxicology evaluation of commercial beers, III. SCE, chromosome aberrations, and forward mutation (HGPRT) of commercial beer products in CHO cells.

Concentrated organic residues extracted from 5 blended aliquots of commercial beers were evaluated for their ability to induce sister chromatid exchange (SCE), chromosomal aberrations and forward mutation in Chinese hamster ovary (CHO) cells. Each extract was prepared by blending 4 commercial beers of similar ingredients and brewing method, passing the beer pool over XAD-2 resin, extracting the resin and concentrating the extract. Studies were performed both with and without metabolic activation using variable amounts of reconstituted residues from 225-fold concentrates of the blended samples. CHO cultures were treated with 0.75 microliters/ml through 10.0 microliters/ml of the concentrates in the SCE assays, 1.0 microliters/ml through 10.0 microliters/ml of the extracts in the aberration assays and 2.5 microliters/ml up to 20 microliters/ml for forward mutation assays. In preliminary screening for SCE as an indicator of potential DNA damage, a significant increase was observed for 3 of 5 concentrated samples; however, no increase in SCE was induced by any of the 5 samples when S9 was added as a source of exogenous metabolic activation. More definitive tests for induction of genetic events, i.e., chromosome aberrations and forward HGPRT mutations, were negative for all 5 extracts whether or not S9 mix was present. Since SCE were not induced in tests with metabolic activation and since there was no concordant aberration or point mutation induction, the preliminary indication of potential DNA damage shown by elevated SCE under conditions without metabolic activation appears to have little biological significance.     

Occurrence of sister-chromatid exchange and chromosomal aberration during vitamin A-induced cell differentiation in vitro

Prompted by the recent growth in interest in the mechanisms of vitamin A (VA) action, we studied the effects of VA on the frequency of sister-chromatid exchange (SCE) and chromosome aberration (CA) in a culture system using a fetal Syrian hamster (female) pulmonary epithelial cell line (M3E3/C3). When manipulated by specific culture conditions, the cells in this system could be rendered competent for activation of polycyclic aromatic hydrocarbons. Cells induced to such a state were exposed to 0, 2, 8 and 24 micrograms/ml of VA for 4 days. The average frequency of SCE per metaphase increased from 1.64 at 0 micrograms/ml to 3.44 at 24 micrograms/ml with a moderate degree of dose dependence. In addition, the q-terminal area of X-chromosomes appears to be one of the most specifically vulnerable sites for SCE due to VA. The frequency of CA encompassing triradial, quadriradial, quinqueradial, ring and dicentric chromosomes also increased in a rather sigmoid fashion from 3.6% at 0 micrograms/ml to 14.8% at 24 micrograms/ml. Apart from the frequently demonstrated protective roles or otherwise less often encountered promotional effects of VA in the development of squamous metaplasia, neoplasia, neoplastic transformation or mutation, an alternative interpretation for the current results implies a possible relationship between SCE and CA caused by VA and cell differentiation and/or drug resistance mechanisms.     

Analysis of chromosome aberrations and sister-chromatid exchanges in human lymphocytes exposed in vitro to Hydergine.

Hydergine (dihydroergotoxine mesylate, Sandoz) was examined for its capability to induce chromosome damage and sister-chromatid exchanges (SCEs) in human lymphocyte in vitro. For the chromosome-aberration study, cultures set up from 6 individuals were divided into 5 groups: negative control, positive control (caffeine, 0.5 mg/ml), and Hydergine (0.1, 0.25 and 0.5 micrograms/ml). For the SCE examination, which used 8 individuals, 4 cultures were made per person in the following way: negative control, positive control (mitomycin C, 0.1 microgram/ml), and Hydergine (0.1 and 0.5 micrograms/ml). Lymphocytes were cultivated for 72 h, being exposed to the respective treatments during the final 24 h. The results showed that Hydergine induced no chromosome damage in human lymphocytes in vitro.     

Evaluation of genotoxicity of tert.-butylhydroquinone in an hepatocyte-mediated assay with V79 Chinese hamster lung cells and in strain D7 of Saccharomyces cerevisiae.

tert.-Butylhydroquinone (TBHQ) has been reported to be genotoxic in some short-term assays but non-genotoxic in others. We have examined cytotoxicity and genotoxicity of TBHQ, a principal metabolite of the phenolic antioxidant 2(3)-tert.-butyl-4-hydroxyanisole (BHA), in an hepatocyte-mediated assay with V79 Chinese hamster lung cells including both sister-chromatid exchange (SCE) and thioguanine-resistance (TGR) endpoints. The ability of BHA and of TBHQ to elicit a genotoxic response in Saccharomyces cerevisiae strain D7 was also investigated. In V79 cytotoxicity tests, TBHQ without hepatocytes produced a 50% reduction in colony formation at 4.2 micrograms/ml and was lethal to 100% of the cells at concentrations above 5 micrograms/ml. At partially cytotoxic dose levels, (0.17-3.4 micrograms/ml of medium), TBHQ sometimes increased significantly the frequency of SCE. TBHQ also produced sporadic statistically significant increases in the mutation frequency at the HGPRTase (TGR) gene locus when tested alone or with activation by rat or hamster hepatocytes. Mitotic gene conversion and reverse mutation were not induced in strain D7 of Saccharomyces cerevisiae by exposure to BHA or to TBHQ for 4 h at concentrations as high as 200 micrograms/ml for BHA or 500 micrograms/ml for TBHQ, either alone or with activation by rat-liver S9. Incubation of the yeast cells with BHA or TBHQ for 24 h in growth medium without activation also did not induce genotoxic activity. The slight and sporadic response to TBHQ in the V79 test system may indicate weak genotoxicity which is sensitive to slight differences in test conditions. The classification and test strategies adopted for compounds such as TBHQ could have important implications for regulatory decisions and for the validation of short-term tests.     

Differential enhancement of sister-chromatid exchange frequencies by alpha-naphthoflavone in cultured lymphocytes from smokers and non-smokers

The sister-chromatid exchange (SCE) frequency was assessed in peripheral lymphocytes from 4 smokers and 8 non-smokers in the absence or presence of alpha-naphthoflavone (ANF) in the culture media. ANF produced a concentration-dependent increase in the frequency of SCEs in smoking individuals. At an ANF concentration of 11 micrograms/ml, average SCE levels were 54% and 13% above the baseline levels in smokers and non-smokers, respectively. The ANF-enhanced increase in the SCE frequency ranged from 3.12 to 5.72 among smokers, and from 0 to 1.96 among the non-smokers. No significant difference in the mean SCE baseline levels between smokers and non-smokers was detected. The mechanism responsible for the enhanced frequency of SCEs in smokers following in vitro exposure to ANF is not clear, but may reflect changes in metabolic activation/deactivation or increased sensitivity to genetic effects of ANF.     

Use of rat primary lung cells for studying genotoxicity with the sister-chromatid exchange and micronucleus assays

Primary culture of lung cells from CD rats was established for pulmonary genotoxicity studies using two genetic endpoints, sister-chromatid exchange (SCE) and micronucleus formation (MN). In the cell isolation study, a combined enzyme separation of rat lungs with trypsin (1.3 mg/ml) plus collagenase (50 U/ml) gave the highest yield of viable and colony-forming cells. For the MN assay, the cytokinesis block induced by cytochalasin B (CYB) was employed to enumerate MN in binucleated (BN) cells. Treatment of primary lung cells with 2 micrograms CYB/ml for two days appeared to be optimal for scoring micronuclei in CYB-induced BN cells. By this procedure, mitomycin C (MMC), triethylenemelamine, and benzo[a]pyrene caused a dose-related increase in micronucleated BN cells in vitro without metabolic activation. In the SCE assay, maximum second-division metaphases were obtained after cells were incubated with bromodeoxyuridine for 48-54 h. After this incubation time, high frequencies of SCE induced by MMC and 3-methylcholanthrene after in vitro exposure (without S9 activation) or in vivo exposure were observed. The results indicate that rat primary lung cells can metabolize polycyclic aromatic hydrocarbons and that this lung cell system is potentially useful for the detection of pulmonary genotoxicants.     

SCE levels in Bloom-syndrome cells at very low bromodeoxyuridine (BrdU) concentrations: monoclonal anti-BrdU antibody

A stable staining procedure of sister-chromatid differentiation (SCD) using a monoclonal antibromodeoxyuridine (BrdU) antibody was newly established by combining it with the immunoperoxidase reaction (3,3'-diaminobenzidine, DAB reaction). This procedure permitted detection of SCD and SCE at very low BrdU concentrations. SCD was not usually observed below 2.0 micrograms/ml BrdU with flame-dried chromosome slides. When chromosome slides were prepared by air-drying over 37 degrees C warm water, SCD was detected at 10.0, 5.0, 1.0, 0.5, 0.3 and 0.2 micrograms/ml BrdU with FPG and even at 0.1 microgram/ml BrdU with the antibody technique. SCE levels were evaluated using the antibody technique and endomitotic analysis with FPG at low BrdU concentrations (1.0, 0.5, 0.3, 0.2 microgram/ml) in two BS B-lymphoblastoid cell lines (LCLs). Even though the BS SCE level was approximately 70 per cell at 10 micrograms/ml, the value decreased to the level of 20-30 SCE per cell at 0.1 microgram/ml with the antibody technique. In BrdU-labelled BS endomitoses, single SCEs highly decreased with BrdU concentrations (130-140 level at 10 micrograms/ml: 38-60 level at 0.2 microgram/ml), when compared to the rare twin SCE values (3-6 SCE level) at all BrdU concentrations. These findings conclusively indicate that the spontaneous baseline SCE in BS B-lymphoblastoid cells is low and most BS SCEs are caused by BrdU.     

Genistein inhibition of the growth of human breast cancer cells: independence from estrogen receptors and the multi-drug resistance gene

The effect of isoflavones on the growth of the human breast carcinoma cell lines, MDA-468 (estrogen receptor negative), and MCF-7 and MCF-7-D-40 (estrogen receptor positive), has been examined. Genistein is a potent inhibitor of the growth of each cell line (IC50 values from 6.5 to 12.0 micrograms/ml), whereas biochanin A and daidzein are weaker growth inhibitors (IC50 values from 20 to 34 micrograms/ml). The isoflavone beta-glucosides, genistin and daidzin, have little effect on growth (IC50 values greater than 100 micrograms/ml). The presence of the estrogen receptor is not required for the isoflavones to inhibit tumor cell growth (MDA-468 vs MCF-7 cells). In addition, the effects of genistein and biochanin A are not attenuated by overexpression of the multi-drug resistance gene product (MCF-7-D40 vs MCF-7 cells).     

Sister chromatid exchanges induced by sarcolysine in human lymphocytes in vitro

The effect of three concentrations of sarcolysine (0.5 micrograms/ml, 1 microgram/ml and 2 micrograms/ml) on the sister chromatid exchanges (SCE) was investigated in human lymphocytes in vitro. A dose related increase in SCEs frequencies was observed after sarcolysine administration and also a delayed development of cell cycle has been induced by the two last concentrations. The variation range of SCEs per cell was dose-dependent and it was considered to represent the acquired genetic instability induced by the drug.     

Reevaluation of the 9 compounds reported conclusive positive in yeast Saccharomyces cerevisiae aneuploidy test systems by the Gene-Tox Program using strain D61.M of Saccharomyces cerevisiae

The state of aneuploidy test methodology was appraised by the U.S. Environmental Protection Agency in 1986 in analyzing published data. In Saccharomyces cerevisiae 9 chemicals were reported to be conclusive positive for aneuploidy induction in either mitotic or meiotic cells. We reevaluated these 9 chemicals using Saccharomyces cerevisiae D61.M, a strain that detects mitotic chromosome malsegregation. Acetone (lowest effective dose (LED): 40 microliters/ml), bavistan (LED: 5 micrograms/ml), benomyl (LED: 30 micrograms/ml) and oncodazole (LED: 4 micrograms/ml) induced a dose-dependent increase in the frequencies of chromosomal malsegregation. Ethyl methanesulfonate (EMS; highest tested dose (HTD): 1000 micrograms/ml) and methyl methanesulfonate (MMS; HTD: 100 micrograms/ml) did not induce malsegregation but were both potent inducers of other genetic events, detected by an increase in the frequencies of cyhR cells. No increases in both endpoints (malsegregation and other genetic events) were observed after treatment of S. cerevisiae D61.M with cyclophosphamide (CP; HTD: 16 mg/ml) in the absence of S9, p-D,L-fluorophenylalanine (p-FPA; HTD: 250 micrograms/ml) and phorbol-12-myristate-13-acetate (TPA; HTD: 50 micrograms/ml). A marginal increase in the frequency of mitotic chromosome malsegregation was obtained with cyclophosphamide in the presence of S9. Thus our test results largely disagree with those previously published by various authors and taken as conclusive by EPA. We interpret the discrepancies to be due to lack of properly controlled testing (e.g., no check for multiple mutational events). Only with a careful test design it is possible to discriminate between chemicals inducing only chromosome loss and no other genetic effects (e.g., acetone, oncodazole), chemicals inducing a variety of genetic damage but no chromosome loss (e.g., EMS, MMS) and chemicals inducing neither chromosome loss nor other genetic events in yeast (e.g., TPA, p-FPA).     

Genotoxicity studies on the antimicrobial drug sulfamethoxazole in cultured human lymphocytes

The genotoxicity of the antimicrobial drug sulfamethoxazole was evaluated in cultured human peripheral blood lymphocytes. The frequencies of sister-chromatid exchange (SCE) and micronuclei (MN) were scored as genetic endpoints. Both tests cover a wide range of induced genetic damage such as primary DNA damage, clastogenicity and aneugenicity. Cultures were set up with blood samples from two healthy donors and the treatment was done with different sulfamethoxazole concentrations ranging from 10 to 500 microg/ml. From the results obtained it appears that this drug is able to induce weak genotoxic effects, as revealed by the slight increase in the SCE and MN frequencies, at least at one of the two highest concentrations tested. However, the results of the SCE assay should be interpreted with caution because the increase is just significant. In addition, cyotoxic/cytostatic effects of sulfamethoxazole were revealed by a decrease in the proliferative rate index (PRI) and in the cytokinesis block proliferation index (CBPI).     

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.