Brahma B. Panda

Genotoxic stress in plants: shedding light on DNA damage, repair and DNA repair helicases.

Plant cells are constantly exposed to environmental agents and endogenous processes that inflict damage to DNA and cause genotoxic stress, which can reduce plant genome stability, growth and productivity. Plants are most affected by solar UV-B radiation, which damage the DNA by inducing the formation of two main UV photoproducts such as cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs). Reactive oxygen species (ROS) are also generated extra- or intra-cellularly, which constitute yet another source of genotoxic stress. As a result of this stress, the cellular DNA-damage responses (DDR) are activated, which transiently arrest the cell cycle and allow cells to repair DNA before proceeding into mitosis. DDR requires the activation of Ataxia telangiectasia-mutated (ATM) and Rad3-related (ATR) genes, which regulate the cell cycle and transmit the damage signals to downstream effectors of cell-cycle progression. Since genomic protection and stability are fundamental to ensure and sustain plant diversity and productivity, therefore, repair of DNA damages is essential. In plants the bulky DNA lesions, CPDs and 6-4PPs, are repaired by a simple and error-free mechanism: photoreactivation, which is a light-dependent mechanism and requires CPD or 6-4PP specific photolyases. In addition to this direct repair process, the plants also have sophisticated light-independent general repair mechanisms, such as the nucleotide excision repair (NER) and base excision repair (BER). The completed plant genome sequences reveal that most of the genes involved in NER and BER are present in higher plants, which suggests that the network of in-built DNA-damage repair mechanisms is conserved. This article describes the insight underlying the DNA damage and repair pathways in plants. The comet assay to measure the DNA damage and the role of DNA repair helicases such as XPD and XPB are also covered.

In vitro biosynthesis and genotoxicity bioassay of silver nanoparticles using plants

Silver nanoparticles (AgNP-P) from AgNO(3) were synthesized by using the broth prepared from the aromatic spath of male inflorescence of screw pine, Pandanus odorifer (Forssk.) Kuntze AgNP-P was then characterized by UV-visible spectroscopy, transmission electron microscopy (TEM) and energy-dispersive X-ray spectroscopy (EDS). Functional groups in the broth were analyzed by Fourier Transform infrared spectroscopy (FTIR). Genotoxicity of AgNP-P was assessed by utilizing our well-established Allium cepa assay system with biomarkers including the generation reactive oxygen species (ROS: O(2)(·-) and H(2)O(2)), cell death, mitotic index, micronucleus, mitotic aberrations; and DNA damage by Comet assay. Other chemical forms of silver such as Ag(+) ion, colloidal AgCl, and AgNP-S at doses 0-80 mg L(-1) were included for comparison with AgNP-P. The results revealed that AgNP-P and AgNP-S exhibited similar biological effects in causing lesser extent of cytotoxicity and greater extent of genotoxicity than that was exhibited by Ag(+) ion alone. Among different tested chemical forms of silver, colloidal AgCl was identified to be the least cytotoxic and genotoxic. Cell death and DNA-damage induced by AgNP-P were prevented by Tiron and dimethyl thiourea that scavenge O(2)(·-) and H(2)O(2), respectively. The present findings demonstrated the role of ROS in the AgNP-induced cell death and DNA damage.

A comparison of biochemical responses to oxidative and metal stress in seedlings of barley, Hordeum vulgare L.

Biochemical responses on the bases of activities of antioxidant enzymes; peroxidase, catalase, superoxide dismutase and glutathione reductase as well as estimations of total protein, lipid peroxidation and thiols in the form of protein, non-protein, glutathione and phytochelatin measured in growing seedlings of barley, Hordeum vulgare L., from Day 2 through 8 were compared following treatment of seeds for 2 h with oxidative agents, paraquat 5 x 10(-5), 10(-4), 10(-3) M, H2O2 10(-3), 5 x 10(-3), 10(-2) M and a metal salt, CdSO4 10(-5), 10(-4), 10(-3) M. A significant induction of all antioxidant enzymes along with an increase in the levels of protein, lipid peroxidation and glutathione was noted in response to oxidative stress, CdSO4 induced significant peroxidase and catalase activities but not superoxide dismutase. In a marked contrast from oxidative stress, CdSO4 decreased glutathione reductase activity as well as glutathione levels but increased phytochelatin level. The differential biochemical responses thus underlined the crucial involvement of glutathione and phytochelatin in the oxidative and metal-induced adaptive responses, respectively.

Aluminium-induced DNA damage and adaptive response to genotoxic stress in plant cells are mediated through reactive oxygen intermediates

Experiments employing growing root cells of Allium cepa were conducted with a view to elucidate the role of reactive oxygen intermediates (ROI) in aluminium (Al)-induced DNA damage, cell death and adaptive response to genotoxic challenge imposed by ethyl methanesulphonate (EMS) or methyl mercuric chloride (MMCl). In a first set of experiments, root cells in planta were treated with Al at high concentrations (200-800 microM) for 3 h without or with pre-treatments of dihydroxybenzene disulphonic acid (Tiron) and dimethylthiourea (DMTU) for 2 h that trap O(2)(.-)and hydrogen peroxide (H(2)O(2)), respectively. At the end of treatments, generation of O(2)(.-) and H(2)O(2), cell death and DNA damage were determined. In a second set of experiments, root cells in planta were conditioned by Al at low concentrations (5 or 10 microM) for 2 h and after a 2 h intertreatment interval challenged by MMCl or EMS for 3 h without or with a pre-treatment of Tiron or DMTU. Conditioning treatments, in addition, included two oxidative agents viz rose bengal and H(2)O(2) for comparison. Following treatments, root cells in planta were allowed to recover in tap water. Genotoxicity and DNA damage were evaluated by micronucleus (MN), chromosome aberration (CA) or spindle aberration (SA) and comet assays at different hours (0-30 h) of recovery. The results demonstrated that whereas Al at high concentrations induced DNA damage and cell death, in low concentrations induced adaptive response conferring genomic protection from genotoxic challenge imposed by MMCl, EMS and Al. Pre-treatments of Tiron and DMTU prevented Al-induced DNA damage, cell death, as well as genotoxic adaptation to MMCl and EMS, significantly. The findings underscored the biphasic (hormetic) mode of action of Al that at high doses induced DNA damage and at low non-toxic doses conferred genomic protection, both of which were mediated through ROI but perhaps involving different networks.

Biomonitoring of low levels of mercurial derivatives in water and soil by Allium micronucleus assay

The Allium micronucleus (MNC) assay was developed to monitor low levels of mercury in aquatic and terrestrial environments. Four mercurial derivatives namely mercuric chloride (MC), methyl mercuric chloride (MMC), phenyl mercuric acetate (PMA) and a methoxy ethyl mercuric chloride based fungicide, Emisan-6, were tested to assess the sensitivity and versatility of the Allium MNC assay. Allium bulbs were set directly on water and soil contaminated with known levels of mercurial derivatives (0.0001-10.00 ppm). On the 5th day the endpoints measured were root length, mitoses with spindle abnormality and cells with MNC in root meristems. The effective concentrations of the test chemicals that cause 50% of root length as compared to control (EC50) were determined from dose-response curves so obtained. The lowest effective concentration tested (LECT) and highest ineffective concentration tested (HICT) for each of the mercurial derivatives for the induction of spindle malfunction and MNC were determined. It was found that EC50, LECT and HICT values for mercurial derivatives in soil were higher than those in water. The frequencies of cells with MNC and mitoses with spindle abnormality were highly correlated indicating that MNC is a good parameter of spindle malfunction. The present approach increased the sensitivity of the Allium assay by 10-fold, the detection limit being 0.001-0.1 ppm and 0.1-1.0 ppm in aquatic and terrestrial environments respectively, depending on the species of mercury.

Monitoring and assessment of mercury pollution in the vicinity of a chloralkali plant. IV. Bioconcentration of mercury in in situ aquatic and terrestrial plants at Ganjam, India

In situ aquatic and terrestrial plants including a few vegetable and crop plants growing in and around a chloralkali plant at Ganjam, India were analyzed for concentrations of root and shoot mercury. The aquatic plants found to bioconcentrate mercury to different degrees included Marsilea spp., Spirodela polyrhiza, Jussiea repens, Paspalum scrobiculatam, Pistia stratiotes, Eichhornia crassipes, Hygrophila schulli, Monochoria hastata and Bacopa monniera. Among wild terrestrial plants Chloris barbata, Cynodon dactylon, Cyperus rotundus and Croton bonplandianum were found growing on heavily contaminated soil containing mercury as high as 557 mg/kg. Analysis of mercury in root and shoot of these plants in relation to the mercury levels in soil indicated a significant correlation between soil and plant mercury with the exception of C. bonplandianum. Furthermore, the tolerance to mercury toxicity was highest with C. barbata followed by C. dactylon and C. rotundus, in that order. The rice plants analyzed from the surrounding agricultural fields did not show any significant levels of bioconcentrated mercury. Of the different vegetables grown in a contaminated kitchen garden with mercury level at 8.91 mg/kg, the two leafy vegetables, namely cabbage (Brassica oleracea) and amaranthus (Amaranthus oleraceous), were found to bioconcentrate mercury at statistically significant levels. The overall study indicates that the mercury pollution is very much localized to the specific sites in the vicinity of the chloralkali plant.

Genotoxicity and Mutagenicity of Metals in Plants

Metals are ubiquitous in nature. Metals are emitted from a wide spectrum of natural and man-made sources such as volcanic eruption, mining, fossil burning, industrial emissions and automobile exhausts and sewage disposals. Distribution of metals in the environment, however, is uneven (Moore and Ramamoorthy 1984, Nriagu and Pacyna 1988, Nriagu 1990). Upon entering into the environment in a variety of organic and inorganic forms and being neither degradable nor recoverable, metals get incorporated into biogeochemical cycles where they can exert long-term effect (Nriagu 1990). The problem of metal pollution is a global phenomenon. This, however, is accentuated in third world countries that have been offering the dumping grounds for toxic wastes and have become the hot spots of metal pollution because of population explosion coupled with poor economic conditions, use of outdated technologies in industries and lack of stringent anti-pollution laws (Anonymous 1991).

Oxidative biomarkers in leaf tissue of barley seedlings in response to aluminum stress

Cellular responses to Al-stress in Hordeum vulgare seedling bioassay were evaluated with an objective to identify the possible biomarkers in leaf tissue that would be best suited to biomonitor aluminum (Al) in the environment. Germinating seeds were treated with different concentrations of AlCl(3) at pH 4.5 for 12h. Al-uptake and accumulation in root and leaf, generation of reactive oxygen species (ROS: O(2)(-), H(2)O(2) and ()OH), cell death, activity of antioxidant enzymes: catalase, superoxide dismutase, guaiacol peroxidase, ascorbate peroxidase, lipid peroxidation, protein oxidation, DNase activity and DNA damage were measured in leaf tissue of the seedlings on day 6 after treatment. The above parameters assessed in leaf tissue that followed a dose-response exhibited significant correlation with concentration of Al(3+) in experimental solution as well as in root tissue. The findings underscored the sensitivity as well as potential of Hordeum vulgare seedling bioassay for biomonitoring of Al in the ambient environment.

Acceleration of catalase and peroxidase activities in Lemna minor L. and Allium cepa L. in response to low levels of aquatic mercury.

The purpose of this study was to assess certain physiological responses of Lemna minor L. (duckweed) and Allium cepa L. (onion) to aquatic mercury at low concentrations. Following a 96-h exposure of plants to nutrient medium contaminated with known levels of mercuric chloride (HgCl(2)), 0.001 to 4 mg litre(-1) (0.0007 to 2.95 mg Hg litre(-1)) or methyl mercuric chloride (MeHgCl(2)), 0.0001 to 0.1 mg litre(-1) (0.0007 to 0.07 mg Hg litre(-1)), the physiological endpoints measured were the growth of fronds (Lemna minor) or roots (Allium cepa), and catalase and peroxidase activities in both plant assays. The EC(50) for HgCl(2) on the basis of the growth curve of Lemna minor was found to be 2.1 mg litre(-1). HgCl(2) and MeHgCl(2) were lethal to L. minor at concentrations of 4 and 0.01 mg litre(-1), respectively. The range of low concentrations that accelerated growth as well as enzymic activities in L. minor was 0.004 to 0.04 mg litre(-1) for HgCl(2) and 0.001 mg litre(-1) for MeHgCl(2). HgCl(2) and MeHgCl(2) induced maximum enzymic activity in Lemna fronds at concentrations of 0.008 and 0.0005 mg litre(-1), respectively. In Allium roots, catalase activity was accelerated at all the concentrations of HgCl(2) (0.001-2 mg litre(-1)) and MeHgCl(2) (0.0001-0.1 mg litre(-1)) tested. The activity of peroxidase was, however, accelerated by HgCl(2) at concentration range 0.01-1.0 mg litre(-1), or by MeHgCl(2) at 0.001 mg litre(-1). The concentrations of HgCl(2) and MeHgCl(2) that induced the highest enzymic activity in Allium roots were 0.05 mg litre(-1) and 0.001 mg litre(-1), respectively.

Studies on the Ability of Water Hyacinth (Eichhornia crassipes) to Bioconcentrate and Biomonitor Aquatic Mercury

Water hyacinth (Eichhornia crassipes, Mart solms) plants were employed to assess bioconcentration and genotoxicity of aquatic mercury. Plants were exposed to water contaminated with mercuric chloride (MC) or phenyl mercuric acetate (PMA) at 0.001 to 1.0 mg litre(-1), or mercury contaminated effluent from a chloralkali plant for various periods of 4 t0 96 h. Root samples taken after 4, 8, 12, 24, 48, 72 and 96 h of exposure were analysed for bioconcentration of mercury spectrophotometrically, and the root meristems were fixed in aceto-ethanol for cytological analysis to determine the frequencies of cells with micronuclei (MNC). Ethyl methane sulfonate and tap water served as positive and negative controls, respectively. The results indicated that bioconcentration of mercury in root tissue was both time- and concentration-dependent, providing evidence that water hyacinth is a good absorbant of aquatic mercury. The frequency of root meristematic cells with MNC followed a concentration-response. The findings indicate the potential of water hyacinth plants for in situ monitoring and for mitigation of aquatic mercury pollution.