TY - JOUR
AU1 - Achary, V. Mohan Murali
AU2 - Panda, Brahma B.
AB - Abstract 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 μM) for 3 h without or with pre-treatments of dihydroxybenzene disulphonic acid (Tiron) and dimethylthiourea (DMTU) for 2 h that trap O2·−and hydrogen peroxide (H2O2), respectively. At the end of treatments, generation of O2·− and H2O2, 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 μM) 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 H2O2 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. Introduction Genomic stability and protection are fundamental to sustain species survival and biodiversity. Plants are increasingly subjected to stress due to environmental pollution and climate change. Plants being sessile, however, exhibit unique defence responses to cope with environmental aggressions and survive. Oxidative burst, a rapid transient production of huge amounts of reactive oxygen intermediates (ROI: H2O2, O2•−, •OH and 1O2), for instance has been the most common feature associated with plant responses to a variety of environmental stress such as drought, temperature, salinity, radiation, metal, ozone, wounding or infection (1). A hypothesis termed as ‘general adaptation’ syndrome response has been proposed according to which different types of stress evoke similar adaptive response implicating the role of ROI (2). ROI on one hand can damage a number of cellular targets, including DNA resulting in genotoxic stress, leading to mutation, genomic instability or apoptosis (3), and on the other are involved in plant defence or immunity (4), growth and development (5), programmed cell death (PCD) (6) and signal transduction (7). Adaptive response is one of the primary strategies that plants inherently respond in order to withstand adverse environments conferring genomic protection. The phenomenon of adaptive response, first discovered in bacteria (8) and subsequently reported in mammalian cells (9), fungi (10) and algae (11), has been amply demonstrated in higher plants [review in (12)]. According to this phenomenon, growing cells exposed to low, non-cytotoxic doses of a genotoxin exhibit resistance when challenged subsequently by a higher dose of the same or different genotoxic agent, termed as genotoxic adaptation. Of late a generalized terminology ‘conditioning hormesis’ has been recommended to describe the above phenomenon (13). Low doses of metals, oxidative and alkylating agents, ionizing radiation and neutron have been reported to trigger genotoxic adaptation in various prokaryotic and eukaryotic cells [review in (14)]. In the above studies, genotoxic adaptation has been evaluated on the basis of spindle aberration (SA) or chromosome aberration (CA), micronucleus (MN), comet or homologous recombination assays (12,15–19). Although the mechanism underlying the genotoxic adaptation is far from being clear, new insights are emerging that point to the possible involvement of DNA repair networks, unique proteins, polypeptides or epigenetic mechanisms (14,20,21). Aluminium (Al) constitutes one of the major environmental metal pollutants known to contaminate soil, water and food chain posing threat to human, environmental and ecosystem health. Sources of Al include industrial mining and smelting of bauxite ore, kitchen utensils and appliances, wrapping materials, food additives, cosmetics and medicines (22,23). Findings reported recently from this laboratory demonstrated that Al in a dose range 1–200 μM through induction or repression of certain antioxidant enzymes triggered oxidative burst at the cell surface and generated oxidative stress in root cells of Allium cepa (24). In a sequel to our earlier report on Al-induced genotoxic adaptation to methyl mercuric chloride (MMCl), a model environmental aneugenic genotoxin or ethyl methanesulphonate (EMS), a standard alkylating chemical mutagen (25,26), we present here further evidence that underscores the role of ROI in the underlying Al-triggered genotoxic adaptation to EMS, MMCl or Al in root cells of A.cepa. Materials and methods Assay systems Bulbs of onion (A.cepa L., 2n = 16) were used as the test system. Bulbs were procured from the local cultivators. Hand-picked bulbs of uniform size were scrapped so that the apices of the root primordial were exposed and their dry scales peeled off. The bulbs were set for rooting in sterilized moist sand in dark. After 2 days, the bulbs with 2- to 3-cm long roots were washed in running tap water for 5–10 min and then subjected to chemical treatment. The experiments were conducted at room temperature 25 ± 1°C in dark unless stated otherwise. Test chemicals, experimental solutions and test material Chemicals used in the present experiments were MMCl (Merck, Schuchardt, Germany), EMS (HiMedia, Mumbai, India), AlCl3 (BDH, Mumbai), dihydroxybenzene disulphonic acid (Tiron; Acros, NJ, USA), dimethylthiourea (DMTU; Acros), rose bengal (RB; Sigma St Louis, MI, USA) and hydrogen peroxide (H2O2; Merck, Mumbai, India). Stock solutions of the chemicals were made fresh in distilled water. Experimental solutions of desired concentrations were made through appropriate dilution. Experimental solution of AlCl3 was adjusted to pH 4.5 in order to ensure that during treatments the metal in soluble form (Al3+) was available to plant roots for absorption (27). Treatment protocol Two separate sets of experiments were conducted. In the first set of experiments, growing roots (2–3 cm long) from bulbs of A. cepa were treated with Al 200–800 μM, pH 4.5, for 3 h with or without pre-treatments of Tiron and DMTU 100 μM for 2 h. Water controls with pH 4.5 were maintained and handled alike. Following treatment, excised roots were processed for cytochemical visualization as well as spectrophotometric determination of O2·− and H2O2, cell death and DNA damage by comet assay. In the second set of experiments, growing roots (2–3 cm long) from bulbs of A.cepa were subjected to conditioning treatment for 2 h with Al, 5 or 10 μM, at pH 4.5, followed by an intertreatment washing for 2 h and then subjected to 3-h challenge treatment of MMCl 1.25 μM and EMS 2.5 or 10 mM; without or with 2-h pre-treatments of Tiron and DMTU, 50 or 100 μM, administered prior to conditioning treatments. Furthermore for comet assay, an experiment was included with roots conditioned (2 h) by oxidative agents, RB 10 μM, H2O2 2.5 mM or Al 10 μM, and challenged (3 h) by EMS 10 mM or Al 800 μM for comparison. All the treatments were terminated by washing intact roots in running tap water for 30 min and then allowed to recover in tap water (pH ∼6.5) at room temperature. For each treatment, 10 root meristems were excised and fixed in acetic acid: ethanol, 1:3 at 18, 24 and 30 h of recovery for cytogenetic (MN, SA or CA) assay (26). The rest of roots were excised and processed for cytochemical visualization and spectrophotometric determination of O2·− and H2O2, cell death, as well as for DNA damage following comet assay as stated earlier. Controls with appropriate pH during treatment and recover were maintained and handled alike for comparison. Measurement and in situ visualization of O2·− and H2O2 generation Cellular generation of O2·− (28) and H2O2 (29) were measured using a UV–Visible Spectrophotometer (GS5701, ECIL, Hyderabad, India) and visualized cytochemically (30) as described below. For measurement of O2·−, freshly weighed 1 g of root was homogenized in 2 ml 50 mM sodium phosphate buffer, pH 7.4. The homogenate was centrifuged at 12 000 × g for 15 min and 500 μl of the supernatant were mixed with 3 ml reaction mixture containing 1 mM epinephrine (Sigma) and 1 mM nicotinamide adenine dinucleotide hydrogen (NADH, HiMedia) dissolved in 50 mM sodium phosphate buffer, pH 7.4. The amount of O2·− radicals produced was calculated by monitoring the rate of epinephrine oxidation to adrenochrome determined from the absorbance difference (A485 − A575, = 2.96/mM/cm) and expressed in nmols/g FW. For cytochemical visualization of O2·−, the bulbs with intact roots were immersed in 10 ml 50 mM Tris–HCl buffer pH 6.4 containing 0.1% nitroblue tetrazolium and 0.1% NADH, for 10–15 min, and subjected to illumination with cool fluorescent light for 2 h to develop colour, characteristic of blue monoformazone precipitation, and photographed against white fluorescent light background with help of a Nikon Coolpix S4 digital camera in macro–close-up mode. For measurement of H2O2, freshly weighed 1 g root samples were homogenized at 4°C in 3 ml of 0.1% (w/v) trichloroacetic acid. The homogenate was centrifuged at 12 000 × g for 15 min and 1 ml of the supernatant was mixed with 1 ml of 10 mM sodium phosphate buffer pH 7.0 and 2 ml of 1M KI. The H2O2 content of the supernatant was measured by comparing its absorbance at 390 nm with a standard calibration curve and expressed in μmol/g FW. For cytochemical visualization of H2O2, bulbs with intact roots from control and treated were immersed in 1% solution of 3,3′-diaminobenzidine (DAB; Sigma) pH 3.8, for 5–10 min, and then incubated at room temperature for 2 h in absence of direct light. Roots were then illuminated until appearance of brown colour, characteristic of reaction between DAB and H2O2, and macro-photographed as said above. Measurement and in situ visualization of cell death For estimation of cell death, control and treated bulbs with intact roots were stained with 0.25% (w/v) aqueous solution of Evans Blue (HiMedia) for 15 min (31). After washing the roots in running tap water for 30 min, roots having dead cells showing blue stain were macro-photographed against a fluorescent white light background for cytochemical visualization. Subsequently, batches of 10 stained root tips measuring equal length (10 mm) from control and treatment groups were excised and soaked in 3 ml of N, N-dimethylformamide (Merck, Mumbai) for 1 h at room temperature. The absorbance of Evans Blue released was measured at 600 nm. Cytogenetic assay Slides prepared from root meristems of A.cepa and were examined and scored for mitotic index (MI, per cent of mitosis); cells with SA or CA and MN were scored as described earlier (26). Coded cytological slides were examined blind under a Zeiss microscope. At least 3000 cells from 10–12 root meristems from six bulbs at each recovery point per treatment were scored to determine MI and frequency of cells with MN, SA or CA. Comet assay Alkaline comet assay was performed (32) following an improved and simplified procedure that substituted phosphate-buffered saline buffer with Tris–HCl buffer. Roots excised from bulbs of A.cepa belonging to different treatment groups were placed in a 60-mm plastic Petri dish and placed on ice. Onto 10 roots, about 200–300 μl of chilled 0.4 mM Tris–HCl buffer pH 7.4 was spread. By using a new razor blade, the roots were chopped and the nuclei were collected in same buffer and transferred into a microcentrifuge tube kept at 4–10°C. Clean grease-free microscopic slides pre-coated with 50 μl of 1% normal-melting-point agarose (Sigma) made in distilled water were kept dry over night at room temperature and labelled. Onto the above slides, 90 μl of nuclear suspension in 0.75% low-melting-point agarose (LMP VII, Sigma) made in aforesaid Tris buffer was layered at 37°C with help of a coverslip (20 × 40 mm). After gelling of agarose on a chilled metal plate in 5 min, the coverslip was gently removed. A second layer of 90 μl agarose in 75% LMP agarose made in Tris buffer was spread over the nuclear layer and allowed for gelling as said above. After removal of the coverslips, the slides with agarose-embedded nuclei were placed in a horizontal electrophoresis tank containing alkaline buffer (300 mM NaOH and 1 mM ethylenediaminetetraacetic acid pH ≥13) for 12 min that facilitated nuclear DNA unwinding, followed by electrophoresis at 0.75 V/cm and 300 mA for 15 min in the same alkaline buffer at 4°C. Slides were then washed in distilled water and neutralized with 0.4M Tris buffer pH 7.4. Following a 5 min washing of slides in distilled water, they were stained by spreading 100 μl staining solution containing ethidium bromide (2 μg/ml). Analysis of comets was carried out employing an Olympus BX51 Microscope with a fluorescence attachment (using excitation filter 515–560 nm and barrier filter 590 nm) equipped with a Cohu camera and Kinetic Komet™ Imaging Software 5.5 (Andor™ Technology, www.andor.com). The comet images were visualized and captured at ×100 magnification. Out of a number of parameters available in the software, comets were analysed on the basis of Olive tail moment (33). The entire process of comet assay was carried out in dim or yellow light. Statistical analysis All the experiments with exception to cytogenetic assay were thrice replicated, whereas experiments with cytogenetic assay were repeated at least once in order to establish reproducibility of the results. Pooled data were statistically analysed using analysis of variance, followed by Turkey's honestly significant difference test (34) employing the Windows XP/Microsoft Excel 2000 computer package. Furthermore, adaptive response was assessed on the basis of protection calculated as per cent of relative decrease in the frequency of cells with MN, SA or CA (average of values of all recovery hours) or DNA damage as compared to that of the positive controls (MMCl- or EMS challenge). Likewise prevention of adaptive response induced by Tiron or DMTU was calculated as per cent of relative increase in the above values as compared to corresponding Al conditioning plus MMCl- or EMS challenge treatments. Results Tiron and DMTU protect against Al-induced generation of ROI, cell death and DNA damage Al in concentrations of 200–800 μM triggered generation of O2·− (Figure 1), H2O2 (Figure 2), cell death (Figure 3) or DNA damage (Figure 4) in root tissue of A.cepa significantly (P ≤ 0.05 or 0.01) that followed a dose–response. From the above data, it was also evident that pre-treatments of Tiron 100 μM and DMTU 100 μM counteracted significantly (P ≤ 0.01) the effects of Al 800 μM that resulted in 30 and 48.4% reduction in generation of O2·− and H2O2, respectively. The results further indicated that compared to the levels of damage induced by Al 800 μM, pre-treatments of Tiron and DMTU decreased cell death significantly (P ≤ 0.01) that accounted 55.6 and 54.3% protection and decreased DNA damage significantly (P ≤ 0.01) that accounted 44.7 and 88.5% protection, respectively. Fig. 1 View largeDownload slide Al-induced generation of O2·- in root tissue of Allium cepa: (A) cytochemical visualization of O2·- generation triggered by Al 800 μM is prevented by Tiron 100 μM. (B). Dose-dependent Al-induced generation of O2·- is prevented by pre-treatment of Tiron 100 μM. Increase significant compared to control at P ≤ 0.01 (b); decrease significant compared to Al 800 μM at P ≤ 0.01 (d). Fig. 1 View largeDownload slide Al-induced generation of O2·- in root tissue of Allium cepa: (A) cytochemical visualization of O2·- generation triggered by Al 800 μM is prevented by Tiron 100 μM. (B). Dose-dependent Al-induced generation of O2·- is prevented by pre-treatment of Tiron 100 μM. Increase significant compared to control at P ≤ 0.01 (b); decrease significant compared to Al 800 μM at P ≤ 0.01 (d). Fig. 2 View largeDownload slide Al-induced generation of H2O2 in root tissue of Allium cepa: (A) cytochemical visualization of H2O2 generation triggered by Al 800 μM is prevented by DMTU 100 μM. (B) Dose-dependent Al-induced generation of H2O2 is prevented by pre-treatments of DMTU 100 μM. Increase significant compared to control at P ≤ 0.01 (b); decrease significant compared to Al 800 μM at P ≤ 0.01 (d). Fig. 2 View largeDownload slide Al-induced generation of H2O2 in root tissue of Allium cepa: (A) cytochemical visualization of H2O2 generation triggered by Al 800 μM is prevented by DMTU 100 μM. (B) Dose-dependent Al-induced generation of H2O2 is prevented by pre-treatments of DMTU 100 μM. Increase significant compared to control at P ≤ 0.01 (b); decrease significant compared to Al 800 μM at P ≤ 0.01 (d). Fig. 3 View largeDownload slide Influence of pre-treatments of Tiron or DMTU, 100 μM, on Al-induced cell death evaluated by Evans Blue staining of root tissue of Allium cepa: (A) Cytochemical visualization of cell death induced by Al 800 μM was prevented by Tiron and DMTU. (B) Dose-dependent induction of cell death by Al and prevention by Tiron and DMTU. Increase significant compared to control at P ≤ 0.01 (b); decrease significant compared to Al 800 μM at P ≤ 0.01 (d). Fig. 3 View largeDownload slide Influence of pre-treatments of Tiron or DMTU, 100 μM, on Al-induced cell death evaluated by Evans Blue staining of root tissue of Allium cepa: (A) Cytochemical visualization of cell death induced by Al 800 μM was prevented by Tiron and DMTU. (B) Dose-dependent induction of cell death by Al and prevention by Tiron and DMTU. Increase significant compared to control at P ≤ 0.01 (b); decrease significant compared to Al 800 μM at P ≤ 0.01 (d). Fig. 4 View largeDownload slide Influence of pre-treatments of Tiron or DMTU, 100 μM, on Al-induced DNA damage evaluated by Olive tail moment in comet assay: (A) Comets in root cells of Allium cepa in control, Al 800 μM without or with pre-treatments of Tiron or DMTU. (B) Dose-dependent induction of DNA damage by Al and prevention by Tiron and DMTU. Increase significant compared to control at P ≤ 0.01 (b); decrease significant compared to Al 800 μM at P ≤ 0.01 (d). Fig. 4 View largeDownload slide Influence of pre-treatments of Tiron or DMTU, 100 μM, on Al-induced DNA damage evaluated by Olive tail moment in comet assay: (A) Comets in root cells of Allium cepa in control, Al 800 μM without or with pre-treatments of Tiron or DMTU. (B) Dose-dependent induction of DNA damage by Al and prevention by Tiron and DMTU. Increase significant compared to control at P ≤ 0.01 (b); decrease significant compared to Al 800 μM at P ≤ 0.01 (d). Tiron and DMTU prevent Al-induced genotoxic adaptation evaluated by cytogenetic assay Data with respect to influence of pre-treatments of Tiron and DMTU, 50 μM, on the frequencies of mitoses with SA or CA, and interphase cells with MN induced by challenging doses of MMCl 1.25 μM or EMS 2.5 mM without or with prior 5 μM Al-conditioning are presented in Figures 5 and 6, respectively. MI that was determined at all the hours of recovery in control and treatments ranged between 7.94 and 17.86%, which indicated normal mitotic progress. MMCl induced cells with MN and SA at significant levels recorded at 18, 24 and 30 h of recovery. Al-conditioning countered MMCl genotoxicity, which was evident by the significant (P ≤ 0.01) decrease in frequencies of cells with MN or SA recorded at all the recovery hours that accounted on average 93.3 or 91.75% genomic protection, respectively. Pre-treatments of Tiron or DMTU, 100 μM, significantly (P ≤ 0.01) diminished Al-adaptive response to the MMCl challenge, notwithstanding the fact that frequencies of cells with SA or MN compared to the control were still significantly high (P ≤ 0.01). Prevention of Al-adaptive response to MMCl challenge by Tiron and DMTU, calculated on the basis of the frequencies of cells with MN and SA, was 90.34 and 85.41% and 88.66 and 83.44%, respectively. Fig. 5 View largeDownload slide Influence of pre-treatments of Tiron and DMTU, 50 μM, on MI and the frequencies of cells with MN and mitoses with SA induced by MMCl 1.25 μM without or with prior Al-conditioning 5 μM in root meristems of Allium cepa. Increase significant compared to control at P ≤ 0.05 (a) or 0.01 (b); decrease significant compared to MMCl challenge at P ≤ 0.01 (d). Fig. 5 View largeDownload slide Influence of pre-treatments of Tiron and DMTU, 50 μM, on MI and the frequencies of cells with MN and mitoses with SA induced by MMCl 1.25 μM without or with prior Al-conditioning 5 μM in root meristems of Allium cepa. Increase significant compared to control at P ≤ 0.05 (a) or 0.01 (b); decrease significant compared to MMCl challenge at P ≤ 0.01 (d). Fig. 6 View largeDownload slide Influence of pre-treatments of Tiron and DMTU, 50 μM, on MI and the frequencies of cells with MN and mitoses with CA induced by EMS 2.5 mM without or with prior Al-conditioning 5 μM in root meristems of Allium cepa. Increase significant compared to control at P ≤ 0.05 (a) or 0.01 (b); decrease significant compared to EMS challenge at P ≤ 0.05 (c) or 0.01 (d). Fig. 6 View largeDownload slide Influence of pre-treatments of Tiron and DMTU, 50 μM, on MI and the frequencies of cells with MN and mitoses with CA induced by EMS 2.5 mM without or with prior Al-conditioning 5 μM in root meristems of Allium cepa. Increase significant compared to control at P ≤ 0.05 (a) or 0.01 (b); decrease significant compared to EMS challenge at P ≤ 0.05 (c) or 0.01 (d). EMS 2.5 mM induced cells with MN and CA in significant frequencies (P ≤ 0.01). Al conditioning conferred protection against EMS challenge that was marked by the significant decrease (P ≤ 0.01) in the frequencies of cells with MN or CA recorded at 18, 24 and 30 h of recovery (Figure 6) that accounted on average 95.22 and 76.52% genomic protection, respectively. Pre-treatments of Tiron and DMTU 50 μM prevented Al-induced genotoxic adaptation to EMS challenge significantly (P ≤ 0.05 or 0.01), which was marked by increase in the frequencies of cells with MN or CA as compared to that recorded for Al–EMS treatment. Prevention of Al-adaptive response to EMS challenge by Tiron calculated on the basis of the frequencies of cells with MN and CA was 32.74 and 62.13% and by for DMTU was 51.64 and 54%, respectively. Tiron and DMTU prevent Al-induced genotoxic adaptation evaluated by comet assay MMCl tested at concentration 1.25 μM did not induce score-worthy comets that exhibited weak or diffused by ethidium bromide staining. MMCl at higher concentrations produced comets showing necrotic manifestations. Comet assay, therefore, was restricted to EMS only. Data with respect to effect of pre-treatments of Tiron and DMTU on Al-induced genotoxic adaptation to EMS 10 mM, evaluated by alkaline comet assay, is presented in Figure 7. Comet analysis, revealed by Olive tail moment, indicated that DNA damage induced by Al 10 μM, Tiron or DMTU 100 μM was insignificant, and the values were at par with that of control. EMS challenge at 10 mM induced DNA damage significantly (P ≤ 0.01), which was effectively counteracted by Al-conditioning evident by the significant reduction in Olive tail moment (P ≤ 0.01) that accounted 80.5% genomic protection. With pre-treatments of Tiron or DMTU 100 μM, the Olive tail moment values in comet assay were almost restored (P ≤ 0.01) to the level of EMS challenge that accounted, respectively, 78.8 and 77.66% prevention of Al-adaptive response. Conditioning of roots by Al at 10 μM conferred protection against DNA damage induced by Al challenge at 800 μM that accounted 75.79% genomic protection. Like Al, conditioning treatments of RB 10 μM and H2O2 2.5 mM induced genotoxic adaptation to EMS challenge, which was evident from the significant decrease (P ≤ 0.01) of Olive tail moment (Figure 8) that accounted 87.4 and 90.4% genomic protection, respectively. Fig. 7 View largeDownload slide Influence of pre-treatments of Tiron and DMTU, 100 μM, on DNA damage (evaluated by Olive tail moment in comet assay) induced by EMS 10 mM without or with prior Al-conditioning 10 μM in root meristems of Allium cepa. Increase significant compared to control at P ≤ 0.01 (b); decrease significant compared to EMS challenge at P ≤ 0.1(d). Fig. 7 View largeDownload slide Influence of pre-treatments of Tiron and DMTU, 100 μM, on DNA damage (evaluated by Olive tail moment in comet assay) induced by EMS 10 mM without or with prior Al-conditioning 10 μM in root meristems of Allium cepa. Increase significant compared to control at P ≤ 0.01 (b); decrease significant compared to EMS challenge at P ≤ 0.1(d). Fig. 8 View largeDownload slide Effect of hormetic conditioning by RB 10 μM, H2O2 2.5 mM or Al 10 μM on DNA damage (evaluated by Olive tail moment in comet assay) induced by EMS 10 mM or Al 800 μM in root meristems of Allium cepa. Increase significant compared to control at P ≤ 0.01 (b); decrease significant compared to EMS challenge at P ≤ 0.1(d). Fig. 8 View largeDownload slide Effect of hormetic conditioning by RB 10 μM, H2O2 2.5 mM or Al 10 μM on DNA damage (evaluated by Olive tail moment in comet assay) induced by EMS 10 mM or Al 800 μM in root meristems of Allium cepa. Increase significant compared to control at P ≤ 0.01 (b); decrease significant compared to EMS challenge at P ≤ 0.1(d). Discussion Al is known to have pro-oxidant activity facilitating superoxide- and iron-driven biological oxidation (35,36). Al induces oxidative stress and antioxidant defence system in plants. Cell wall-bound NADH peroxidase is primarily involved in the generation of O2·− and H2O2 in root tissue of A.cepa following treatment with Al (24). Several genes are expressed in response to Al stress in Arabidopsis (37). A recent microarray analysis revealed a total of 256 Al-responsive genes comprising 1.1% of the 24 000 genes of Arabidopsis genome of which 94 genes were shown to be up-regulated and 162 were down-regulated (38). The mechanisms of underlying Al toxicity to plant cells include disruption of cell wall, plasma membrane, cytoplasmic Ca2+ homeostasis, cytoskeletal dynamics, nuclear DNA structure and template activity (27). We had earlier demonstrated that Al tested at range of concentrations 1–200 μM for induction of oxidative stress and DNA damage in root tissue of A.cepa (24). In the above study Al induced DNA damage at concentrations 50–200 μM significantly, but the extent of Olive tail moment so obtained was not good enough for resolving protection of DNA damage conferred by Tiron or DMTU. In the present study, it was established that Al in range of concentrations 200–800 μM induced DNA damage as well as cell death in root cells of A.cepa. Al at concentrations ≥1000 μM was toxic, evident by the appearance of comets with necrotic manifestations. Subsequently, antagonism of Tiron and DMTU was tested against the optimum concentration of Al 800 μM for induction of DNA damage, cell death or ROI generation. Cell death evaluated by Evans Blue staining method indicated disintegration of plasma membrane by Al at the above concentrations (31). The foregone method has been shown to be a reliable parameter of PCD in rice seedlings subjected to Zn stress (39). Involvement of signal transduction events has been suggested in Al-induced cell death demonstrated in tomato suspension cells (40). Pre-treatments of Tiron and DMTU diminished significantly the levels of generation of O2·− and H2O (Figures 1 and 2) that proved their respective ROI-scavenging activities (41,42). The fact that the aforesaid ROI scavengers could effectively provide protection against Al-induced cell death (Figure 3) and DNA damage (Figure 4) in root cells of A.cepa pointed to the involvement of ROI in the underlying mechanisms. Al at high concentrations in the range 0.5–2.0 mM is known to inhibit root growth in Arabidopsis through DNA damage coupled with arrest of cell cycle in G2 stage (43). The present treatment protocol was, therefore, designed on the basis of our earlier studies (25,26) in which following treatments the root meristems of A.cepa were analysed for mitoses with MN, SA or CA with simultaneous determination of MI at 18, 24 and 30 h of recovery (Figure 5 and 6), which ensured that mitosis in root meristems progressed normally, and there was no inhibition or delay in the mitotic cell cycle as result of Al low-dose conditioning and/or Tiron/DMTU pre-treatments. The present findings demonstrated that the range of concentrations 1–10 μM of Al was critical for induction of adaptive response that conferred genomic protection of plant cells against MMCl, EMS or Al. It was important to note that Al at concentrations below 1 μM had very little affect, while at concentrations >10 μM enhanced the damaging effects of either of the genotoxins (data not shown for the sake of the brevity) indicating synergism. Angelis et al. (44) for the first time had employed alkaline unwinding-neutral electrophoresis (A/N) comet assay in root cells of Vicia faba to demonstrate adaptive and cross-adaptive responses induced by a low conditioning dose of two different alkylating mutagens methyl nitrosourea (MNU) and methyl methanesulphonate (MMS) against the subsequent challenging treatments of the same mutagens at a high dose. On the basis of A/N comet assay, it was later shown that the heavy metal Cd induced genotoxic adaptation to MNU in root cells of Hordeum vulgare (45). Alkaline unwinding-alkaline electrophoresis comet assay has also been employed successfully to assess neutron- or ionizing radiation-induced adaptive response in human lymphocytes (15–18). Kovalchuk et al. (19) on the basis of induction of homologous recombination frequencies in Arabidopsis by RB, a singlet oxygen-generating chromophore, had suggested a biphasic concentration-dependent role of RB. Seedlings conditioned by RB at a concentration range 0.05–0.2 μM had led to the subsequent protection from genotoxic challenge imposed by MMS at 20 or 50 μM. RB at higher concentrations 0.2–0.5 μM resulted in the potentiation of MMS genotoxicty, while at concentrations less than 0.05 μM showed little or no effect on MMS genotoxicity (19). Such a biphasic role of nitric oxide i.e. antagonism in low (micromolar) concentrations and synergism in high (millimolar) concentrations against H2O2-induced lipid peroxidation, DNA damage or PCD in Nicotiana plumbaginifolia has been demonstrated (46). Furthermore, using human U937 leukaemia cells, it was shown that low doses of H2O2 could induce adaptive response to subsequent high doses of H2O2, which was not by increasing the cellular capacity to degrade H2O2 but by specific blocking of stress-activated protein kinase/c-Jun N-terminal kinase, responsible for H2O2-induced cell death (47). At a toxic concentration 50 μM, Al has also been shown to induce mitogen-activated protein kinase (MAPK)-like protein in cell suspension cultures of Coffea arabic (48). Involvement of such a protein in the present study, however, is very unlikely owing to the fact that Al in far less concentrations 1–10 μM induced adaptive response to genotoxic stress. ROI-triggered signalling pathways involving activation of MAPKs have been reported to originate outside the nucleus in plants (49). The role of DNA damage response in the genotoxic adaptation involving protein kinases ataxia telangiectasia mutated (ATM) or ATM-Rad3 related found first in yeast and mammalian cells, or their homologues in Arabidopsis and other plants have been suggested (20). Comet assay in the present study (Figure 4) revealed that Al-induced DNA damage in root cells of A.cepa was primarily by H2O2 (evident by effective counteraction by DMTU) and to a less extent by O2·− (Tiron counteracted less effectively). Al-induced genotoxic adaptation to MMCl or EMS found in the present study was in confirmation to earlier reports (25,26). Prevention of Al-adaptive response to MMCl- or EMS genotoxicity by Tiron and DMTU, however, exhibited a remarkable difference. Cytogenetic (MN, SA or CA) assays revealed that both Tiron and DMTU could effectively prevent Al-adaptive response to MMCl (Figure 5) but proved less effective in preventing the same against EMS (Figure 6), which could be attributed to differences in the mode of genotoxic action of MMCl, a standard aneugen-cum-clastogen, vis-à-vis EMS, a monofunctional alkylating mutagen/clastogen (25,26). Aneugenicity of MMCl was due to impairment of motor protein kinesin and/or microtubules, and clastogenicity to its propensity to generate ROI facilitating DNA strand breaks resulting in cells with SA and MN. EMS on the other hand is known to induce DNA strand breaks and lesions as a consequence of depurination resulting in cells with CA and MN. Comet assay was found unsuitable for evaluation of adaptive response to MMCl owing to its cytotoxicity (50). Comet assay nevertheless validated Al-induced adaptive response to EMS, which was prevented by Tiron or DMTU (Figure 7). Furthermore, the above Al-induced genotoxic adaptation to EMS was quite comparable to that induced by the two oxidative agents RB and H2O2. It was also interesting to note that Al at 10 μM conferred genomic protection against subsequent high dose of Al challenged at 800 μM (Figure 8). The aforesaid findings further established that comet assay could be routinely used as an end point for evaluation of adaptive response to genotoxic stress. The unique advantage that the comet assay offers over the cytogenetic assays is that it evaluates induction or prevention of adaptive response on the basis of nuclear DNA damage, which is independent of the progress of mitotic cell cycle. It should, however, be kept in mind that comet assay analysed within an hour of treatment indicated primary DNA damage. Cytogenetic (MN, SA and CA) assays analysed at late hours (18–30 h) of recovery represented the genotoxic manifestations arising from post- or un-repaired DNA lesions and/or stand breaks. The present EMS-induced DNA damage, MN or CA in A.cepa were quite comparable with the findings reported yet in another study employing freshwater mussel Unio pictorum (51). The present findings accentuated the usefulness of A.cepa as a model plant with multiple end points ranging from oxidative biomarkers (24), to time-honoured cytogenetic (MN, SA or CA) assays (52) and now to cell death and comet assay for assessment of genotoxic damage as well as genomic protection. The advantages of employing A.cepa is that the assays are simple, inexpensive and can be performed in one stretch without consuming much time that at this moment no other single plant system can offer. The present results highlighted the dual role of Al-triggered ROI, which at low concentrations (1–10 μM) induced adaptive response conferring genomic protection and at high concentrations induced DNA damage in root cells that upheld the concept of biphasic (hormetic) dose-response (53). The findings further underscored that the concentration range of ROI was crucial for induction of genotoxic adaptation in plant cells. The findings have implications in inducible plant adaptation to hostile environments in the long run. Funding Government of India, Departments of Science and Technology (Project No. SR/SO/PS-07/2004). The authors are thankful to the authorities of Berhampur University for providing administrative and infrastructural facilities to carry out the research. The authors thank J. Wainwright of Andor Technology, UK and K. B. Chaturvedi and P. K. Panda of DSS Image Tech, India, for helping and assisting in setting up Komet Software and B. N. Behera for reading the manuscript. 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TI - Aluminium-induced DNA damage and adaptive response to genotoxic stress in plant cells are mediated through reactive oxygen intermediates
JF - Mutagenesis
DO - 10.1093/mutage/gep063
DA - 2009-12-02
UR - https://www.deepdyve.com/lp/oxford-university-press/aluminium-induced-dna-damage-and-adaptive-response-to-genotoxic-stress-zfUP2u91cP
SP - 201
EP - 209
VL - 25
IS - 2
DP - DeepDyve
ER -