Antigenotoxic potential of plant leaf extracts of Parkinsonia aculeata L. using Allium cepa assay.

The aim of the present study was to evaluate the antigenotoxic potential of P. aculeata L. leaf extract/fractions against maleic hydrazide (MH) using Allium cepa root chromosomal aberration assay. The excessive reduction in root growth and mitotic index value was observed after 3 h treatment of MH as compared to negative control (water). In case of MH treatment, frequency of aberrated cells significantly (p ≤ 0.05) raised from 129 to 337 at 0.1 ppm and 2.0 ppm concentrations respectively. From root growth inhibition test with MH treatment, EC50 value i.e. 0.5 ppm was selected to study the antigenotoxic effect of different extract/fractions of P. aculeata L. leaves. All the extract/fractions showed increase in mitotic index and great reduction in chromosomal aberrations with rise in concentration against the genotoxicity of MH. Among all the extract/fractions, butanol and ethyl acetate fractions showed significant reduction in chromosomal aberrations in A. cepa cells and indicates the chemo preventive activity. Antigenotoxic property of this plant is due to the presence of various phytochemicals in leaf such as epi-orientin, Parkinsonin-A, Parkinsonin-B, orientin, iso-orientin, vitexin, iso-vitexin, C-glycosylflavone, parkintin, rotenoids, terpenoids, flavonoids, saponins, alkaloids, glycosides and anthraquinone etc. Our result showed that among all the treatments, simultaneous treatment showed best result followed by pre and post treatment. Further studies in animal model are suggested for further evaluation of the use of P. aculeata leaf extract in human welfare.

DNA replication after mutagenic treatment in Hordeum vulgare.

The temporal and spatial properties of DNA replication in plants related to DNA damage and mutagenesis is poorly understood. Experiments were carried out to explore the relationships between DNA replication, chromatin structure and DNA damage in nuclei from barley root tips. We quantitavely analysed the topological organisation of replication foci using pulse EdU labelling during the S phase and its relationship with the DNA damage induced by mutagenic treatment with maleic hydrazide (MH), nitroso-N-methyl-urea (MNU) and gamma ray. Treatment with mutagens did not change the characteristic S-phase patterns in the nuclei; however, the frequencies of the S-phase-labelled cells after treatment differed from those observed in the control cells. The analyses of DNA replication in barley nuclei were extended to the micronuclei induced by mutagens. Replication in the chromatin of the micronuclei was rare. The results of simultanous TUNEL reaction to identify cells with DNA strand breaks and the labelling of the S-phase cells with EdU revealed the possibility of DNA replication occurring in damaged nuclei. For the first time, the intensity of EdU fluorescence to study the rate of DNA replication was analysed.

Transcriptional Activity of rRNA Genes in Barley Cells after Mutagenic Treatment.

In the present study, the combination of the micronucleus test with analysis of the activity of the rRNA genes in mutagen-treated Hordeum vulgare (barley) by maleic hydrazide (MH) cells was performed. Simultaneously fluorescence in situ hybridization (FISH) with 25S rDNA as probes and an analysis of the transcriptional activity of 35S rRNA genes with silver staining were performed. The results showed that transcriptional activity is always maintained in the micronuclei although they are eliminated during the next cell cycle. The analysis of the transcriptional activity was extended to barley nuclei. MH influenced the fusion of the nucleoli in barley nuclei. The silver staining enabled detection of the nuclear bodies which arose after MH treatment. The results confirmed the usefulness of cytogenetic techniques in the characterization of micronuclei. Similar analyses can be now extended to other abiotic stresses to study the response of plant cells to the environment.

Mixture genotoxicity of 2,4-dichlorophenoxyacetic acid, acrylamide, and maleic hydrazide on human Caco-2 cells assessed with comet assay.

Assessment of genotoxic properties of chemicals is mainly conducted only for single chemicals, without taking mixture genotoxic effects into consideration. The current study assessed mixture effects of the three known genotoxic chemicals, 2,4-dichlorophenoxyacetic acid (2,4-D), acrylamide (AA), and maleic hydrazide (MH), in an experiment with a fixed ratio design setup. The genotoxic effects were assessed with the single-cell gel electrophoresis assay (comet assay) for both single chemicals and the ternary mixture. The concentration ranges used were 0-1.4, 0-20, and 0-37.7 mM for 2,4-D, AA, and MH, respectively. Mixture toxicity was tested with a fixed ratio design at a 10:23:77% ratio for 2.4-D:AA:MH. Results indicated that the three chemicals yielded a synergistic mixture effect. It is not clear which mechanisms are responsible for this interaction. A few possible interactions are discussed, but further investigations including in vivo studies are needed to clarify how important these more-than-additive effects are for risk assessment.

Effects of soil pH on the Vicia-micronucleus genotoxicity assay.

In the field of contaminated sites and soil management, chemical analyses only bring typological data about pollution. As far as bioavailability and effects on organisms are concerned, we need ecotoxicology tools. In this domain, among many existing tests, we chose to study genotoxicity because it is a short-term endpoint with long-term consequences. The aim of this study is to assess the effects of soil pH on the results of the Vicia faba root tip micronucleus test for the two following reasons: (i) to define the pH range within which the test can be performed without modifying the soil to be tested, within the framework of the ISO standard of the test and (ii) to provides information about the effects of the pH on the genotoxic potential of soils. In this context, we modified the pH of a standard soil with HCl or NaOH and we spiked the matrix with copper (2, 4 and 8 mmol kg(-1) dry soil) or with maleic hydrazide, an antigerminative chemical (5, 10 and 20 µmol kg(-1) dry soil). We concluded that the pH had no effect on the mitotic index or micronucleus frequency in the root cells of the negative controls: extreme pH values did not induce micronucleus formation in root cells. Moreover, according to our results, the Vicia-micronucleus test can be performed with pH values ranging between 3.2 and 9.0, but in the ISO 29200 "Soil quality--assessment of genotoxic effects on higher plants--V. faba micronucleus test" we recommended to use a control soil with a pH value ranging between 5 and 8 for a more accurate assessment of chemical genotoxicity. We also found that acid pH could increase the genotoxic potential of pollutants, especially heavy metals. With hydrazide maleic spiked soil, plants were placed in a situation of double stress, i.e. toxicity caused by extreme pH values and toxicity induced by the pollutant.

Epigenetic chromatin modifications in barley after mutagenic treatment.

In addition to their normal developmental processes, plants have evolved complex genetic and epigenetic regulatory mechanisms to cope with various environmental stresses. It has been shown that both DNA methylation and histone modifications are involved in DNA damage response to various types of stresses. In this study, we focused on the involvement of two mutagenic agents, chemical (maleic acid hydrazide; MH) and physical (gamma rays), on the global epigenetic modifications of chromatin in barley. Our results indicate that both mutagens strongly influence the level of histone methylation and acetylation. Moreover, we found that gamma irradiation, in contrast to MH, has a more robust influence on the DNA methylation level. This is the first study that brings together mutagenic treatment along with its impact at the level of epigenetic modifications examined using the immunohistochemical method.

Cytotoxic effects of etephon and maleic hydrazide in Vero, Hep2, HepG2 cells.

The toxicity of etephon and maleic hydrazide, used as plant growth regulators in agriculture, were reported as low in mammals in previous studies. However, in vitro cytotoxicity studies in mammalian cells are currently missing to understand their toxicity at molecular level. In the current study, the cytotoxicity of these compounds, were studied in Vero (African green monkey kidney epithelium), HepG2 (human hepatocellular carcinoma), Hep2 (human epidermoid cancer) cells by MTT ((3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazolium bromure) and LDH (lactate dehydrogenase) assays. Maleic hydrazide had lower IC50 values for all cell lines compared to ethephon. Least cytotoxic effect treated by ethephon were observed in Vero, followed by HepG2 and Hep2. Similarly maleic hydrazide also showed least cytotoxicity on Vero cells, followed by Hep2 and HepG2 cells (p < 0.05). IC50 values in general were found to be highest in Vero cells, followed by HepG2 and Hep2 cells (p < 0.05). LDH and MTT assays showed correllation and had close relation except HepG2-maleic hydrazide application with the correlation coefficient for all >0.868 (p < 0.05). This study is expected to be a basis to understand the cytotoxic effects of ethephon and maleic hydrazide in mammal cells to be supplemented by further studies.

Influence of the presence of B chromosomes on DNA damage in Crepis capillaris.

The sensitivity of different plant species to mutagenic agents is related to the DNA content and organization of the chromatin, which have been described in ABCW and bodyguard hypotheses, respectively. Plant species that have B chromosomes are good models for the study of these hypotheses. This study presents an analysis of the correlation between the occurrence of B chromosomes and the DNA damage that is induced by the chemical mutagen, maleic hydrazide (MH), in Crepis capillaris plants using comet assay. The presence of B chromosomes has a detectable impact on the level of DNA damage. The level of DNA damage after MH treatment was correlated with the number of B chromosomes and it was observed that it increased significantly in plants with 3B chromosomes. We did not find evidence of the protective role from chemical mutagens of the constitutive heterochromatin for euchromatin in relation to DNA damage. The DNA damage involving the 25S rDNA sequences was analyzed using the comet-FISH technique. Fragmentation within or near the 25S rDNA involved the loci on the A and B chromosomes. The presence of B chromosomes in C. capillaris cells had an influence on the level of DNA damage that involves the 25S rDNA region.

Glyphosate, alachor and maleic hydrazide have genotoxic effect on Trigonella foenum-graecum L.

In the present study effects of herbicides glyphosate (GP), alachlor (AL) and maleic hydrazide (MH) is studied on mitotic cells of Trigonella foenum-graecum L. Seeds of T. foenum-graecum L. treated with a series of concentrations ranging from 0.1%, 0.2%, 0.3%, 0.4% and 0.5% for 1, 2 and 6 h and their effect on mitotic index and chromosomal aberrations was studied. The results indicate that these herbicides reduced mitotic index in dose-dependent manner. In addition, increase in the percentage of abnormal mitotic plates was observed in herbicide treated groups which was both concentration and time dependent. Commonly observed abnormalities were c-mitosis, laggards, bridges, stickiness, c-anaphase, precocious separation, un-equal distribution and fragments. The result of the present investigation indicates that commonly used herbicides GP, AL and MH have significant genotoxic effect on T. foenum-graecum plant.

Improvement of Vicia-micronucleus test for assessment of soil quality: a proposal for international standardization.

The Viciafaba root tip micronucleus test is one of the most employed plant genotoxicity assays, and has been used on various types of contaminated materials. This test has been standardized by AFNOR, the French member organization of ISO. However, this test is usually performed with a water extraction step but soil genotoxicity assessment would be more relevant when performed directly in the soil itself. In order to harmonize these protocols, an ISO standard for the V.faba micronucleus test in both liquid phase (exposure of plants to different liquid matrix, including soil water extracts) and solid phase (direct exposure of plants to the soil) would be very useful. In this context, we compared two exposure durations in the solid phase (48 h and 5 d) for the V.faba micronucleus test with two different well-known genotoxicants, maleic hydrazide and copper sulfate. We concluded that these two durations induced equivalent sensitivity: the micronucleus frequency was significantly increased with 5 µmol maleic hydrazide per kg dry soil and with 2 mmol copper sulfate per kg dry soil with both exposure durations. However, exposing roots to soil during 48 h is more practical. Moreover, organically and conventionally cultured seeds were employed to determine whether the seed provenance influenced the test sensitivity. Organic seeds were less sensitive to copper, possibly because copper-based treatments are permitted, and often applied, in organic farms. Therefore, in the absence of completely non-treated seeds, organically-cultured seeds did not appear to offer any advantages over conventional seeds.

Sensitivity of Bidens laevis L. to mutagenic compounds. Use of chromosomal aberrations as biomarkers of genotoxicity.

The wetland macrophyte Bidens laevis possesses suitable cytological characteristics for genotoxicity testing. To test its sensitivity as compared to terrestrial plants species currently in use in standardized assays, Methyl Methanesulfonate (MMS), N-ethyl-N-nitrosourea (ENU) and Maleic Hydrazide (HM) were used. On the other hand, the insecticide Endosulfan (ES)--an environmentally relevant contaminant--was assayed in seeds and two-month old plants. Mitotic Index (MI), frequency of Chromosome Aberrations in Anaphase-Telophase (CAAT) and frequency of Abnormal Metaphases (AM) were analyzed. MH, MMS and ENU caused a significant decrease of the MI. MMS was aneugenic whereas MH and ENU were both aneugenic and clastogenic. ES caused a significant concentration-dependent increase of total- and aneugenic-CAAT in roots and a significant high frequency of AM at high concentrations. Because of its sensitivity to mutagenic substances, B. laevis can be regarded as a reliable and convenient species for genotoxicity assays especially if aquatic contaminants are evaluated.

Herbicide effects on freshwater benthic diatoms: induction of nucleus alterations and silica cell wall abnormalities.

Benthic diatoms are well known bio-indicators of river pollution by nutrients (nitrogen and phosphorus). Biological indexes, based on diatom sensitivity for non-toxic pollution, have been developed to assess the water quality. Nevertheless, they are not reliable tools to detect pollution by pesticides. Many authors have suggested that toxic agents, like pesticides, induce abnormalities of the diatom cell wall (frustule). High abnormal frustule abundances have been reported in natural diatom communities sampled in streams contaminated by pesticides. However, no direct link was found between the abundances of abnormal frustules in these communities and the pesticide concentrations in stream water. In the present study, a freshwater benthic diatom community, isolated from natural biofilm and cultured under controlled conditions, was treated with a known genotoxic herbicide, maleic hydrazide (MH). Cells were exposed to three concentrations of MH (5x10(-6), 10(-6), 10(-7)M) for 6h followed by a 24h-recovery time. After MH treatments, nucleus alterations were observed: abnormal nucleus location, micronucleus, multinuclear cell or disruption of the nuclear membrane. A dose-dependent increase of nuclear alterations was observed. The difference between the control (9.65 nuclear alterations per 1000 cells observed (9.65 per thousand), S.D.=4.23) and the highest concentrations (29.40 per thousand, S.D.=8.49 for 10(-6)M and 35.96 per thousand, S.D.=3.71 for 5x10(-6)M) was statistically significant (Tukey test, P<0.05). Diatoms also exhibited frustules with deformed morphology and abnormal ornamentation. Significantly increased abundances of abnormal frustules were observed for the highest concentrations (10(-6) and 5x10(-6)M; Tukey test, P<0.05). These two parameters tended to increase together (Pearson correlation=0.702, P<0.05). The results suggest that the induction of abnormal frustules could be associated with the genotoxic effects of MH. The alterations observed could be related to the effects of MH on the synthesis of the proteins involved in frustule formation or in the regulation of the cytoskeleton of the diatom cells.

Multicolour FISH in an analysis of chromosome aberrations induced by N-nitroso-N-methylurea and maleic hydrazide in barley cells.

The present study is a rare example of a detailed characterization of chromosomal aberrations by identification of individual chromosomes (or chromosome arms) involved in their formation in plant cells by using fluorescent in situ hybridization (FISH). In addition, the first application of more than 2 DNA probes in FISH experiments in order to analyse chromosomal aberrations in plant cells is presented. Simultaneous FISH with 5S and 25S rDNA and, after reprobing of preparations, telomeric and centromeric DNA sequences as probes, were used to compare the cytogenetic effects of 2 chemical mutagens: N-nitroso-N-methylurea (MNU) and maleic hydrazide (MH) on root tip meristem cells of Hordeum vulgare (2n=14). The micronucleus (MN) test combined with FISH allowed the quantitative analysis of the involvement of specific chromosome fragments in micronuclei formation and thus enabled the possible origin of mutagen-induced micronuclei to be explained. Terminal deletions were most frequently caused by MH and MNU. The analysis of the frequency of micronuclei with signals of the investigated DNA probes showed differences between the frequency of MH- and MNU-induced micronuclei with specific signals. The micronuclei with 2 signals, telomeric DNA and rDNA (5S and/or 25S rDNA), were the most frequently observed in the case of both mutagens, but with a higher frequency after treatment with MH (46%) than MNU (37%). Also, 10% of MH-induced micronuclei were characterized by the presence of only telomere DNA sequences, whereas there were almost 3-fold more in the case of MNU-induced micronuclei (28%). Additionally, by using FISH with the same probes, an attempt was made to identify the origin of chromosome fragments in mitotic anaphase.

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

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

Transformed roots of Crepis capillaris--a [corrected] sensitive system for the evaluation of the clastogenicity of abiotic agents.

The presence of a large number of pollutants, including mutagenic agents in the environment is a problem of a major concern. Rapid progress in plant biotechnology, especially in the development of cell transformation methods, including the production of transformed roots -- 'hairy roots' -- has opened new possibilities to use transformed root cultures in plant bioassays for the evaluation mutagenic effects of different agents. We have used Crepis capillaris hairy roots for evaluation of cytogenetic effects of mutagenic treatment. Effects of maleic acid hydrazide (MH) and X-ray treatment were analysed in chromosomal aberration, sister chromatid exchange (SCE) and TUNEL tests. Comparison of cytogenetic effects in hairy roots and roots of seedlings showed a much higher sensitivity of hairy roots, which makes them convenient material for monitoring DNA damage after mutagenic treatment.

Cytotoxicity and mode of action of maleic hydrazide in root tips of Allium cepa L.

Maleic hydrazide (MH) is an herbicide and is a regulator of the growth of buds in vegetables during storage. It is used in agriculture-in despite its known effect as a mutagenic and clastogenic agent. In this research the effect of MH on the root tips of Allium cepa L. was determined; correlations between the effects of different concentrations and exposure times on the mitotic index (MI) and induction of chromosomal aberrations (ChA) were also examined. Experiments were carried out in triplicate, using aqueous solutions of MH to concentrations of 10(-6), 10(-5), 10(-4) and 10(-3)M, at intervals of 0, 4, 8, 12, 24, and 48 h, with a control for each combination (with the MH substituted by distilled water). The results revealed an inhibition of the MI linked to the concentration and time of treatment (F=845.51, P<0.01 and F=427.58, P<0.01, respectively). For all the concentrations used and exposure periods longer than 12 h, different types of ChA were present, with significantly increased frequencies with increases in the concentration and time of exposure (P<0.01). To determine the mechanism through which the herbicide exerts its toxicity, ultrastructural electron microscopy was conducted. The results reveal nucleolar alterations, suggesting an inhibitory effect of biosynthetic activity.

Differential genotoxicity of ethyl methanesulphonate, N-ethyl-N-nitrosourea and maleic hydrazide in tobacco seedlings based on data of the Comet assay and two recombination assays.

The purpose of this study was to determine if mutagen-induced DNA damage is correlated with the frequency of induced recombination events. The alkylating agents ethyl methanesulphonate (EMS) and N-ethyl-N-nitrosourea (ENU), and the plant growth regulator and herbicide maleic hydrazide (MH) were compared in tobacco seedlings for their ability to induce DNA damage measured by the Comet assay, and recombination activity measured by the GUS gene reactivation assay, and by the somatic twin sectors assay. While EMS and ENU induced a dose-dependent increase in DNA damage in leaf nuclei, MH had no significant effect. By contrast, MH induced a 6-fold higher frequency of homologous recombination as expressed by the GUS assay and a 2.8-fold higher frequency of somatic twin sectors than after EMS treatments.

Carboxymethylcellulose improves the antimutagenic activity of sodium selenite against maleic hydrazide in Vicia faba seedlings.

In this study the genotoxic effects induced by a treatment with different doses of sodium selenite in Vicia faba seedlings were evaluated with or without the addition of carboxymethylcellulose. A further objective of this study was to verify whether the adduct selenite-carboxymethylcellulose was also able to reduce the genotoxic damages induced by the herbicide maleic hydrazide, a strong mutagenic agent in plants, at a higher extent than selenite alone. The results obtained showed a genotoxic activity of sodium selenite at concentrations up to 8.6 mg L-1. In the treatments with selenite-carboxymethylcellulose, the genotoxicity induced by the complex was significantly lower in comparison to how much was observed in the treatment with selenite only. When sodium selenite's protective activity against the genotoxic effects induced by the herbicide maleic hydrazide was tested, a reduction of mutagenic damages was observed at the highest application doses of selenite (from 86 mg L-1). The treatments with selenite-carboxymethylcellulose resulted in a further increase of selenium protective activity, which was observable for all doses used. These findings suggest a possible role played by carboxymethylcellulose in the regulation of the genotoxic activity of selenium.

Genotoxicity of maleic hydrazide, acridine and DEHP in Allium cepa root cells performed by two different laboratories.

The purpose of this paper was to compare the results of the Allium cepa chromosome aberration assay between two laboratories under the same test protocol and at the same time, use chemicals and onions obtained in their own homeland. For this study three chemicals were selected: di(2-ethylhexyl)phthalate (DEHP), maleic hydrazide, and acridine. Both laboratories found genotoxicity with a positive dose-response relationship for maleic hydrazide and acridine. However, for DEHP the results were quite different--one of the laboratories found this compound not genotoxic but the other found a positive response. Although the comparative study was inconclusive for DEHP, it was successful for the maleic hydrazide, acridine and also for the positive control (methyl methanesulfonate). Further studies need to be performed in the case of DEPH.

DNA damage and repair in Arabidopsis thaliana as measured by the comet assay after treatment with different classes of genotoxins.

The three protocols of the comet assay A/N, A/A and N/N were for the first time applied to the plant species Arabidopsis thaliana. The purpose of the experiments was to establish conditions for genotoxic exposure causing DNA damage in Arabidopsis nuclei. This is required for comprehensive gene expression profiling with the intention to screen for genes involved in response of Arabidopsis cells to genotoxic stress. Five chemicals belonging to different classes of mutagens (the monofunctional alkylating agents N-methyl-N-nitrosourea and methyl methanesulfonate, the polyfunctional alkylating agent mitomycin C, the radiomimetic bleomycin and the herbicide maleic hydrazide) were tested. Except for maleic hydrazide, dose-dependent increases in DNA damage were found using the A/N comet assay protocol. While a rapid repair of bleomycin-mediated SSBs and DSBs was found, no significant reduction of DNA migration was observed up to 48h after treatment with the monofunctional alkylating agents.