Article pubs.acs.org/est
Copper Oxide Nanoparticle Mediated DNA Damage in Terrestrial Plant Models Donald H. Atha,†,‡ Huanhua Wang,†,§ Elijah J. Petersen,‡ Danielle Cleveland,‡ R. David Holbrook,‡ Pawel Jaruga,‡ Miral Dizdaroglu,‡ Baoshan Xing,*,§ and Bryant C. Nelson*,‡ ‡
Material Measurement Laboratory, National Institute of Standards and Technology, 100 Bureau Drive, Gaithersburg, Maryland 20899, United States § Department of Plant, Soil and Insect Sciences, University of Massachusetts at Amherst, Amherst, Massachusetts 01003, United States S Supporting Information *
ABSTRACT: Engineered nanoparticles, due to their unique electrical, mechanical, and catalytic properties, are presently found in many commercial products and will be intentionally or inadvertently released at increasing concentrations into the natural environment. Metal- and metal oxide-based nanomaterials have been shown to act as mediators of DNA damage in mammalian cells, organisms, and even in bacteria, but the molecular mechanisms through which this occurs are poorly understood. For the first time, we report that copper oxide nanoparticles induce DNA damage in agricultural and grassland plants. Significant accumulation of oxidatively modified, mutagenic DNA lesions (7,8-dihydro-8-oxoguanine; 2,6-diamino-4-hydroxy-5-formamidopyrimidine; 4,6-diamino-5-formamidopyrimidine) and strong plant growth inhibition were observed for radish (Raphanus sativus), perennial ryegrass (Lolium perenne), and annual ryegrass (Lolium rigidum) under controlled laboratory conditions. Lesion accumulation levels mediated by copper ions and macroscale copper particles were measured in tandem to clarify the mechanisms of DNA damage. To our knowledge, this is the first evidence of multiple DNA lesion formation and accumulation in plants. These findings provide impetus for future investigations on nanoparticle-mediated DNA damage and repair mechanisms in plants. plants8 are in their infancy, and the extent to which these NPs may cause long-term toxic effects, such as genotoxicity, to the plants is unknown.9 Recently, zinc oxide nanoparticles (ZnO NPs) were shown to inhibit root elongation and to reduce plant biomass in Lolium perenne (perennial ryegrass),10 but to only reduce plant biomass in Cucurbita pepo (zucchini).11 Copper NPs (Cu NPs) were shown to reduce seed germination in certain food plants (Phaseolus radiatesmung bean; Triticum aestivumwheat)12 but not in others (Cucurbita pepo).11 These variable phytotoxic effects of NPs to plants raise the question as to what are the important and measurable molecular factors for the observed differences in NP phytotoxicity among the different plant species. Oxidative damage to plant DNA has been previously demonstrated for plants treated with high levels of ozone13 or high levels of Cu2+ ions.14,15 DNA damage has also been induced in plants exposed to low temperatures16 and in plants in which the nudx1 DNA repair gene has been knocked out.17 In all of these studies, 8-OH-dGuo, the 2′-deoxynucleoside form of 8-OH-Gua, was the only measured lesion. However, there are many other oxidatively induced DNA lesions that are
1. INTRODUCTION Oxidatively induced damage to DNA bases can be generated by both oxygen-derived radical attack (predominantly hydroxyl radical (•OH)) on isolated purines and pyrimidines in the nucleotide pool and on intact duplex DNA. Because guanine possesses the lowest reduction potential, oxygen radical attack often results in the incorporation of 8-OH-dGTP into the nucleotide pool along with the simultaneous induction and cellular accumulation of the highly mutagenic lesion, 7,8dihydro-8-oxoguanine (8-OH-Gua); 8-OH-Gua promotes G → T transversion mutations that are found in dysfunctional genes associated with cancer.1 The accumulation of oxidatively modified DNA bases (DNA lesions) in mammalian tissues can potentially lead to premature aging and/or age-related diseases such as metabolic syndrome and atherosclerosis and to various types of cancers, if the lesions are not efficiently repaired.2 In comparison to our current knowledge on mammalian systems, the mechanisms underlying oxidatively induced damage to DNA and its repair in plants are poorly understood.3,4 The existence or formation of cancer(s) in plants is negligible,5 but accumulation of mutagenic or cytotoxic DNA lesions can lead to genomic instability, reduced plant growth, and plant diseases.4,6 Studies investigating the utilization of engineered nanoparticles (NPs) in agriculture for food or forage plant enhancement7 or in agronomy for genetic transformation of © 2011 American Chemical Society
Received: Revised: Accepted: Published: 1819
August 1, 2011 October 26, 2011 December 22, 2011 December 22, 2011 dx.doi.org/10.1021/es202660k | Environ. Sci. Technol. 2012, 46, 1819−1827
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Figure 1. Oxidative damage to plant DNA induced by exposure to CuO NPs, CuO BPs, or Cu2+ ions. (a) Chemical structures for DNA lesions of interest; 8-OH-Gua, FapyGua, and FapyAde, left to right. (b) Accumulation of DNA damage in radish seedlings in relation to nominal dose. (c) Accumulation of DNA damage in perennial ryegrass seedlings in relation to nominal dose. The 100 mg/L CuO NP nominal concentration is not shown in this figure because the amount of genomic DNA obtained from the seedling after NP incubation did not allow independent replicate analyses to be performed. Asterisks indicate statistically significant results compared to the control samples using one-way Analysis of Variance (ANOVA) followed by Dunnett’s multiple comparison test. One, two, and three asterisks indicate p < 0.05, 0.01, and 0.001, respectively. All data points represent the mean of 2 or 3 independent measurements. Uncertainties are standard deviations.
formed OH-adduct radicals of guanine and adenine, respectively.18 The formamidopyrimidine lesions have been the subject of numerous investigations for more than three decades.18 However, their relevance to the genomic stability of plants has yet to be explored. In mammalian cells, FapyGua is known to be 25% to 35% more mutagenic than 8-OH-Gua and to induce G → T transversion mutations while FapyAde
either cytotoxic and/or mutagenic to living organisms. For example, 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) and 4,6-diamino-5-formamidopyrimidine (FapyAde) are major promutagenic lesions formed under conditions of oxidative stress in mammalian cells; they are purine-derived imidazole ring-opened lesions generated by UV irradiation or by •OH attack, followed by one-electron reduction of the thus 1820
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induces A → C transversion mutations and possibly A → T transversion mutations.18 Figure 1a shows the chemical structures for each of the DNA lesions. To investigate the potential genotoxicity of nanoparticles to plants, we designed a hydroponic plant study utilizing three different terrestrial species, radish (Raphanus sativus), perennial ryegrass (Lolium perenne), and annual ryegrass (Lolium rigidum), to critically evaluate the capacity of copper oxide NPs (CuO NPs) to induce oxidative damage to each plant’s genomic DNA and to inhibit plant growth. CuO NPs are used as industrial catalysts in manufacturing processes and are heavily utilized in semiconductor devices, antimicrobial preparations, and heat transfer fluids.19,20 As such, there exist real concerns that these NPs, as well as other metal oxide-based NPs, could potentially enter the environment and compromise the ecological health of plants and other organisms.9 We investigated the capacity of CuO NPs to induce the formation and accumulation of multiple DNA lesions in all three plant systems via high-resolution gas chromatography/isotopedilution mass spectrometry (GC/IDMS).21,22 For comparative purposes, copper oxide fine particles (PM0.1 μm to PM2.5 μm) and cupric ions (Cu2+) from copper nitrate were tested simultaneously. CuO NP uptake into the plants and localization/identification within the shoot or root cells were assessed using inductively coupled plasma mass spectrometry (ICP-MS) and scanning transmission electron microscopyenergy dispersive spectroscopy (STEM-EDS), respectively.
Standard Reference Material 1575a) were used as control materials. Plant Seed Germination and Nanoparticle Dosing. Before use, glass Petri dishes (25 × 15 mm) were soaked for 2 d in aqua regia (HCl/HNO3; 3/1 volume fraction) and then rinsed with tap water and ultrapure H2O. The dishes were allowed to air-dry completely and then filter papers were placed in each dish. Stock suspensions of NP and BP were prepared as described above including sonication before use. Copper ion (Cu2+) solution was prepared by dissolving Cu(NO3)2 in ultrapure H2O (nominal pH = 4.4); the pH of the Cu(NO3)2 solution was adjusted to 5.4 using Tris(hydroxymethyl)aminomethane. The radish (Raphanus sativus), perennial ryegrass (Lolium perenne), and annual ryegrass (Lolium rigidum) seeds were obtained from Livingston Seed Co. (Columbus, OH, USA). Plant seeds were soaked in ultrapure H2O for 30 min and placed into individual Petri dishes (20 radish seeds, 40 perennial ryegrass seeds, or 40 annual ryegrass seeds, as specified). The NP and BP suspensions were then serially diluted in ultrapure water and 8 mL of each suspension or Cu(NO3)2 solution was pipetted into the Petri dishes. Increasing concentrations of NP and BP suspensions at 10, 100, 500, and 1000 mg/L or Cu2+ ions [Cu(NO3)2] at 1, 10, and 50 mg/L were added to the Petri dishes (all treatments performed in triplicate). It should be noted that due to particle aggregation and precipitation on the filter or bottom of the Petri dishes, the applied CuO NP and BP concentrations of 10, 100, 500, and 1000 mg/L through the text are nominal concentrations and the real exposure concentrations would be lower than the corresponding 10, 100, 500, and 1000 mg/L. The Petri dishes were covered, sealed with tape to minimize evaporation, and placed in an incubator at 25 °C. The seeds were allowed to germinate for 6 days. The plant seeds were completely covered with the copper suspensions/solutions, but as the plants grew, the shoots grew out of the suspensions/ solutions. After 6 days, the plants (all components of the plants except the original seeds) were harvested and the adsorbed NPs, BPs, and Cu2+ ions were removed using tap water followed by ultrapure H2O. The harvested plants were immediately frozen and the genomic plant DNA was extracted and purified the following day. Plant DNA Extraction, DNA Digestion, and Measurements of DNA Base Lesions via GC/MS. Specific details regarding the extraction of genomic DNA from the terrestrial plants can be found in Supporting Information. GC/MS with isotope dilution was used to determine the levels of three different oxidatively modified bases21,22 (8-OH-Gua, FapyGua, and FapyAde) in each DNA extract. Three independent samples were prepared for every treatment within every study. DNA pellets were washed 3 times with ice cold 70% ethanol and once with ice cold absolute ethanol. DNA pellets were dried and then solubilized in distilled and deionized water (ddH2O). DNA aliquots of 50 μg were prepared from each experiment and the three stable isotope-labeled analogues of each base lesion (8-OH-Gua-15N5, FapyGua-13C15N2, and FapyAde-13C15N2) were added to each sample. The samples were dried under vacuum and then stored at 4 °C prior to enzymatic digestion. Subsequently, samples were dissolved in a buffer consisting of 50 mmol/L sodium phosphate, 100 mmol/ L potassium chloride, 1 mmol/L EDTA, and 100 μmol/L dithiothreitol (pH 7.4). To this solution, 1 μg of Fpg was added and the digestion was carried out at 37 °C for 1 h. Fpg hydrolysis prevents artifactual formation of DNA lesions
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MATERIALS AND METHODS Nanoparticle Characterization. CuO NPs (Sigma) and bulk particles (BPs) (Acros) were analyzed by transmission electron microscopy (TEM) using a JEOL 100CX (Jeol Inc., Peabody, MA, USA) operated at 80 kV after drying the samples on copper grids. Particle sizes were analyzed using Image J software. Dynamic light scattering (DLS) and zeta potential analysis measurements were made using 1000 mg/L solutions of the NPs and BPs in ddH2O prepared as described below and analyzed using a Nano Zetasizer (Malvern Instrument Ltd., Worcestershire, UK). Brunauer, Emmitt, and Teller (BET) surface area analysis was conducted on NP and BP powders using an Autosorb-1-MP Surface Area Analyzer (Quantachrome Instruments, Boynton Beach, FL, USA). All of the NP and BP characterization data are summarized in Table S1 in the Supporting Information. Copper Dissolution from NPs and BPs. Stock suspensions of NP and BP at 1000 mg/L were prepared in ultrapure H2O (pH 5.4). The NP and BP suspensions were sonicated in a water bath (Fisher Scientific model FS30H) for 1 h and then stirred using a magnetic stir plate to obtain uniform suspensions. Copper dissolution from the NPs and BPs after a 6-d period was measured using ultrafiltration (centrifugation for 1 h at 3000g) using Amicon Ultracel-3K centrifugal filters (Millipore Corp., Billerica, MA, USA). Corrections were made to the measured dissolved copper concentrations based upon measurements of copper sorption to the membrane and plastic vial. Copper concentrations in solution were determined using a Thermo Scientific X7 quadrupole ICP-MS instrument (ThermoFisher Scientific, Waltham, MA, USA). The ICP-MS instrument was operated in conventional (non CCT) mode. Gallium was used as an internal standard and both Trace Elements in Spinach Leaves (NIST Standard Reference Material 1570a) and Trace Elements in Pine Needles (NIST 1821
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tetroxide, dehydrated, and embedded in resin) were obtained for subsequent scanning transmission electron microscopyenergy dispersive spectroscopy (STEM-EDS) imaging and analysis. STEM imaging was performed on an environmental scanning electron microscope (ESEM, Quanta 200, FEI, Inc., Hillsboro, OR) while EDS analysis was performed with a Bruker AXS silicon drift detector (model 5030, 30 mm2 single channel) and Quantax microanalysis software. The resin was ERL 4206 resin (Spurr’s original formulation) and infiltration after standard ethanol dehydration of each sample was performed as follows. The samples were added to a mixture of propylene oxide and Spurr’s resin at ratios of 1:1, 1:2, and 1:3 consecutively, each for 1 h. Then, the samples were added to 100% Spurr’s resin for 1 h. Last, the samples were embedded in fresh resin and incubated at 70 °C overnight. Gold TEM grids (G200HSG, 200 Mesh, Ted Pella, Inc., Redding, CA) were utilized to eliminate extraneous Cu detection during EDS. Additionally, background Cu levels were tested (using the same microscope and EDS detection parameters) in areas that appeared to be free of CuO NPs; these areas did not have any appreciable Cu counts in the resulting EDS spectra.
because it releases only modified bases; consequently, there is no intact DNA nor unmodified base present during the trimethylsilylation step (see below).22 The digestion was terminated with the addition of ice cold ethanol and the sample was brought to −20 °C. Samples were centrifuged at 16 000g for 30 min and the supernatants containing the excised DNA lesions were removed and the solvent was removed by vacuum desiccation. Samples were solubilized in ddH2O, lyophilized, and then derivatized to trimethylsilyl esters using bis(trimethylsilyl)-trifluoroacetamide)/1% trimethylchlorosilane in pyridine (120 °C for 30 min). For GC/MS measurements, a 6890N Network GC System coupled with a 5973 Network Mass Selective Detector) (Agilent Technologies, Inc., Rockville, MD) was employed; a HP-Ultra 2 highresolution (12.5 m, 0.2 mm i.d.) fused silica capillary column coated with cross-linked 5% phenylmethylsilicone gum phase (film thickness, 0.33 μm) was used for the GC column (Agilent Technologies, Inc., Rockville, MD). Samples were eluted with a temperature program from 130 °C to 270 °C (8 °C per min) followed by a holding temperature of 280 °C (5 min). Trimethylsilyl derivatives of DNA lesions and their stable isotope-labeled analogues were detected using electron ionization mass spectrometry in selected-ion-monitoring (SIM) mode. SIM chromatograms were analyzed using the Agilent MSD Chemstation software (Agilent). Quantification of DNA base lesions was determined using the SIM area ratios from the modified base of interest and its labeled analogue in conjunction with the known amount of labeled analogue added to each sample. Plant Root/Shoot Elongation Assay. More than 90% of the control seeds had germinated and had developed primary roots that were at least 20 mm in length following the 6-d germination period. At this point, germination was halted, seed germination rate was calculated, and the length of the primary root and shoot for each seedling was determined based on established plant elongation assay guidelines.23,24 Each replicate for each treatment and each species consisted of at least 80 seedlings for each treatment, and all measurements were performed in triplicate. Copper Uptake Determinations. To assess copper uptake into the plant tissues, each plant was exposed to CuO NPs, CuO BPs, or Cu(NO3)2 for 6 days. Blank controls with no added copper were also analyzed. After 6 d, only the shoots (2 cm above the roots) were harvested to avoid settling and potential adsorption of particles onto the roots. The shoots were freeze-dried for 24 h and then digested using a mixture of 3 mL of HNO3 (68−71% (mass fraction)) and 1 mL of HClO4 (70% mass fraction) in 25-mL glass vials. The mixed acids were evaporated to approximately 0.3 mL, and then 1% HNO3 (volume fraction) was added to increase the total volume to 10 mL. This solution was analyzed using ICP-MS as described previously. Fixation and Sectioning of Radish Root and Shoot Tissues. Radish root and shoot samples (control, 500 mg/L NP, 500 mg/L BP, and 50 mg/L Cu2+ ion) were prefixed with 4F:1G fixative (86 mL distilled water, 10 mL of 37−40% formaldehyde, 4 mL of 25% biological grade glutaraldehyde, 1.16 g of NaH2PO4·H2O, and 0.27 g of NaOH) and postfixed with 1% osmium tetroxide according to the method of Dykstra25 at the University of Massachusetts. Radish root and shoot samples were processed at the University of Maryland. Localization of NPs in Radish Root and Shoot Tissues via STEM-EDS. Ultrathin sections (postfixed with osmium
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RESULTS AND DISCUSSION Nanoparticle Characterization. As summarized in Table S1 in the Supporting Information and shown in Figure 2, the NP primary particle size determined by TEM indicated mean sizes smaller than 100 nm, the threshold typically used to define NPs. These NPs were nearly an order of magnitude smaller than the primary particle size of the BPs. Dynamic light scattering indicated that both particles had substantial agglomeration in the aqueous phase. Surface area measurements confirmed the larger specific surface area of the NPs as would be expected for smaller particles. There were substantially different zeta potentials observed for the two particle types in deionized water, but the zeta potentials were similar after exposure to the plant seeds, likely as a result of biomolecules released into solution from the plants interacting with the particle surfaces (data not shown). The dissolved copper concentrations were always less than 1% for the NPs, while they were slightly higher for the BPs and were above 1% for the (10 and 100) mg/L concentrations only (Figure S1 in Supporting Information). DNA Damage, Growth Inhibition, and Biodistribution of CuO NPs with Radish Plants. Germination of radish seeds in the presence of CuO NPs induced a significant accumulation of not only 8-OH-Gua, but also of FapyGua and FapyAde (Figure 1b). Radish seedlings incubated with CuO NPs at the highest dose (1000 mg/L) showed statistically significant increases (220, ≈ 260, and ≈ 450%) in the accumulated levels of FapyAde, FapyGua, and 8-OH-Gua, respectively. A clear linear dose−response profile was observed for each measured lesion over the effective dose range (Figure S2a in Supporting Information). The substantial effects of the CuO NPs on the accumulation of lesions in radish are striking considering that the presence of a single additional mutagenic lesion in a mammalian genome can lead to genomic instability and/or mutagenesis.26 In contrast, CuO BPs were much less effective agents at inducing oxidative damage to radish DNA (Figure 1b); seedlings incubated with BPs at the 10 mg/L dose level actually showed a reduction in the measured levels compared to control levels. Radish seedlings dosed with 1000 mg/L BPs showed statistically significant increases (≈ 40, ≈ 80, and ≈ 90%) in the accumulated levels of FapyAde, FapyGua, and 81822
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all three lesions increased over background levels and significantly increased at the highest dose. Furthermore, lesion formation showed a linear dose−response profile from the 1 mg/L Cu2+ ion dose upward (Figure S2c in Supporting Information). Whereas copper ions may contribute to the lesion levels observed for the NP and BP exposures (see Figure S1 in Supporting Information for exact dissolved copper concentrations), the extent of accumulated DNA damage cannot be solely explained by ions alone, thus indicating that the particles themselves play a key role. The approximate level of copper released from NP and BP suspensions at the highest particle concentration of 1000 mg/L was 5 and 3 mg/L, respectively. Close inspection of Figure 1b for copper-induced DNA damage within the relevant Cu2+ ion range of 1−10 mg/L indicates that Cu2+ ion significantly influenced the induction and accumulation of FapyAde and FapyGua, but had negligible effects on the accumulation of 8-OH-Gua. The current data support the possibility that Cu2+ ion generated •OH plays a greater role on the formation of the formamidopyrimidine lesions in radish than on the formation of 8-OH-Gua when NPs, specifically, were presented to the seeds at relatively low particle-concentrations (Figure 1b, compare DNA damage for CuO NP vs Cu2+ ion). When plants were exposed to NPs or BPs at higher particle-concentrations (500 mg/L or higher), effects from the particles themselves appear to mediate the type and level of observed damage. All three forms of copper strongly inhibited radish seedling growth over the entire treatment range (untreated control compared to highest test dose): root growth was inhibited 97%, 83%, and 95% and shoot growth was inhibited 79%, 31%, and 76% by NPs, BPs and Cu2+ ions, respectively (Figure S3 in Supporting Information). There was no demonstrable difference in the measured extent of root inhibition for Cu2+ ions and NPs or in shoot inhibition for Cu2+ ions and NPs over the respective treatment ranges. However, when one focuses on the 1−10 mg/L Cu2+ ion treatment range and compares that to an equivalent NP suspension-concentration (1000 mg/L) that releases ≈5 mg/L copper ion, radish seedling growth was inhibited by approximately 63% (roots) and 6% (shoots) due to Cu2+ ion effects, but growth was strikingly reduced by 97% (roots) and 79% (shoots) by the NPs. Even at a low NP suspension-concentration (10 mg/L), radish seedling root and shoot growth were already inhibited by approximately 46% and 4%, respectively. Measuring the relative level of copper uptake by the radish shoots among the three different forms of copper is necessary to understand the aforementioned particle effects on plant DNA damage and growth inhibition. Radish shoots showed dramatically different uptake profiles (μg Cu/g plant shoots, dry weight basis) for the three forms of copper (Figure 3a). Uptake of copper from Cu2+ was substantially greater than the uptake of copper from NPs or BPs on a copper mass basis. The measured background level (dry weight basis) of copper (12.2 μg Cu/g plant shoots) in the radish was well within the established micronutrient range (5−30 μg Cu/g plant shoots) for normal terrestrial agricultural plants.30 Total copper uptake for Cu2+ ions and both particle types substantially exceeded this value, thus suggesting an active copper transport mechanism. When directly comparing NPs to BPs, total copper uptake into the shoots was approximately three times greater for the NPs over the entire treatment range. These results are concordant with recent NP investigations with P. radiatus and T. asestivum that demonstrated that copper NPs (CuNPs) are acutely
Figure 2. Morphological characterization of copper oxide particles using TEM. (a) TEM of CuO NPs; the measured TEM diameter (mean ± SD) of the particles was 58 nm ± 45 nm. (b) TEM of CuO BPs; the measured TEM diameter (mean ± SD) of the particles was 200 nm ± 126 nm.
OH-Gua, respectively, compared to control levels. But the number of accumulated lesions was about 50% less than the number of lesions induced by NPs at the equivalent particleconcentration dosage. In addition, there was no clear linear dose−response profile for each measured lesion over the effective dose range (Figure S2b in Supporting Information). It is known that radish, as well as other terrestrial plants, undergoes a controlled oxidative burst during seed germination to produce oxygen-derived species (O2•−, H2O2, and •OH).27 H2O2 activates and enhances seed germination, however H2O2 in the presence of peroxidase or transition metal ions (iron or copper) can result in excessive formation of the highly reactive • OH via the Fenton reaction.28 Thus, Cu2+ ions dissolved from NPs or BPs could theoretically catalyze the formation of •OH. The subsequent attack of plant DNA in the chloroplast, mitochondrion, or nucleus by •OH would be observed as a discrete pattern of lesions29 showing a many-fold lower accumulation of FapyAde compared to FapyGua or 8-OHGua. This is what was observed when the radish seeds were dosed with increasing concentrations of Cu2+ ion (Figure 1b): 1823
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Figure 3. Copper uptake by plants exposed to CuO NPs, CuO BPs, or Cu2+ ions. (a) Copper uptake into radish seedlings. All data points represent the mean of 2 or 4 independent measurements. (b) Copper uptake into perennial ryegrass seedlings. All data points represent the mean of 4 or 5 independent measurements. (c) Copper uptake into annual ryegrass seedlings. All data points represent the mean of 2 or 4 independent measurements. Uncertainties are standard deviations. Asterisks indicate statistically significant results compared to the control samples using one-way Analysis of Variance (ANOVA) followed by Dunnett’s multiple comparison test. One, two, and three asterisks indicate p < 0.05, 0.01, and 0.001, respectively.
CuO NPs, as well as dissolved Cu2+ ions, that are able to enter the nucleus of plants can mediate direct oxidative damage to duplex DNA via •OH attack on the heterocyclic bases. CuO NPs have profound and significant biological effects both on the formation and accumulation of DNA base lesions and on the inhibition of growth in radish seedlings. Previously, CuO NPs have been shown to be both cytotoxic and genotoxic (capable of inducing DNA single strand breaks) to mammalian cells;20,31−34 the toxicity was believed to originate from the capacity of the NPs to generate sustained oxidative stress within the cells. Molecular modeling predicts that CuO NPs ≥ 20−30 nm in diameter are capable of redox cycling within the relevant electrochemical potential range (−4.12 to −4.84 eV) of intracellular antioxidants such as glutathione and catalase, and other biologically important redox-active molecules.19 This occurs because the energy level of the CuO NP conduction band superimposes onto the potential range of intracellular biomolecules thus initiating an unbalanced (oxidative) energy state in the cell. CuO NPs are inherently oxidative and can thus withdraw electrons from various biomolecules within plant cells and subsequently transfer those electrons to other biomolecules (reducing agents). Hence, CuO NPs are potentially capable of promoting damage to plant DNA via direct redox interactions. CuO NPs can also operate indirectly by promoting the
phytotoxic (strongly inhibit root growth) and that the observed phytotoxicity is mainly due to NPs and not to released metal ions.12 To further clarify the nature of the apparent NP-induced oxidative damage to plant DNA, we investigated the potential uptake and localization of NPs in radish seedling root and shoot tissue samples via STEM-EDS. STEM images of radish shoot samples did not reveal any significant evidence of electron-dense deposits and EDS analyses did not reveal specific elemental signals for Cu in either control samples or samples exposed to 500 mg/L NPs (Figure S4 in Supporting Information). STEM analyses conducted on radish root samples, on the other hand, revealed the presence of monodisperse NPs and small clusters of aggregated NPs distributed throughout the nucleus and in the extra cellular space between adjacent cells (Figure 4a). The particles were within the characteristic size range of the NPs utilized in the present study. EDS analyses on these electron dense clusters did yield characteristic elemental copper signals (Figure 4b and c) for the dense clusters but not the individual NPs, likely because the number of atoms in a single NP was too low to identify via EDS. These findings suggest that the clusters are indeed intact NPs that have crossed the plant cell wall and entered root cells from the hydroponic NP suspension. Thus, 1824
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Figure 4. Scanning transmission electron microscopy−energy dispersive spectroscopy (STEM-EDS) scans of radish root cells exposed to a nominal concentration at 500 mg/L CuO NPs. (a) Electron micrograph shows localization of electron dense particle clusters (red arrows) inside and outside the nucleus of radish root cells. (b) Electron micrograph shows localization of electron dense particle clusters (red arrow) inside the nucleus of radish root cells. (c) EDS spectrum of electron dense particle cluster (identified by red arrow in Figure 4b; particles were unambiguously identified as elemental copper).
oxidation of other biomolecules (i.e., membrane lipids) within the plant that can generate •OH which is then capable of attacking DNA at diffusion-controlled rates. DNA Damage, Growth Inhibition, and Biodistribution of CuO NPs with Ryegrass Plants. NPs were generally less effective agents at inducing DNA damage in grassland plants (perennial and annual ryegrass). For example, perennial ryegrass seedlings germinated in the presence of NPs showed accumulated lesion levels approximately two times lower for FapyGua and 8-OH-Gua and approximately ten times lower for FapyAde in comparison to the lesion levels accumulated in radish seedlings over the same particle-concentration range (Figure 1c). Nevertheless, at the conclusion of the 6-day germination period, perennial ryegrass seedlings incubated with the 10 mg/L and 1000 mg/L NP doses showed statistically significant accumulation of lesions. Overall, there was an increasing dose−response profile for FapyGua and 8-OH-Gua, but the dose−response profile for FapyAde was relatively flat (Figure S5a in Supporting Information). We observed a dramatic difference between the accumulation of lesions for NPs and BPs. Although BPs induced strong oxidative damage to perennial ryegrass DNA at the 10 mg/L dose, there was actually a reversal in the levels of accumulated lesions for the 1000 mg/L dose. The dose−response profiles for FapyGua and 8-OH-Gua were uncharacteristically bell-shaped, while the
FapyAde dose−response profile was once again flat (Figure S5b in Supporting Information). The absolute levels for all three lesions were quite similar between the BP and Cu2+ ion treatments, suggestive of a common mechanism of DNA damage. In contrast to the observed NP- and BP-induced DNA damage in radish seedlings, the lowest NP and BP doses (10 mg/L) for the perennial ryegrass seedlings generated statistically significant accumulation of all three lesions. In addition, the Cu2+ ion treatment at the 1 mg/L dose induced formation of 8-OH-Gua, in contrast to the results observed for the radish experiment. These observations suggest that perennial ryegrass is less tolerant of copper-induced oxidative stress and DNA damage at lower and potentially more environmentally relevant copper concentrations, irrespective of the copper form. To better understand the similarities between BP and Cu2+ ion mediated DNA damage in perennial ryegrass, it was instructive to observe that the Cu2+ ion dose−response profile (Figure S5c in Supporting Information) for the lesions formed via oneelectron reduction almost followed a bell-shaped profile (similar to the lesion profile generated by the BPs); the dose−response profile increased up to 10 mg/L Cu2+ ion for FapyGua and FapyAde and then the level decreased, but the decrease was not statistically significant (unpaired t test) when going from 10 to 50 mg/L Cu2+ ion for either FapyGua (p value = 0.7739) or FapyAde (p value = 0.3410). In contrast, the 1825
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8-OH-Gua. Annual ryegrass seedlings were similar in behavior to perennial ryegrass seedlings regarding both their total (absolute) uptake of copper from NPs, BPs and Cu2+ ions (Figure 3c) and their growth being strongly inhibited by NPs and BPs (Figure S8 in Supporting Information). Specific discussion of NP, BP, and Cu2+ ion effects on annual ryegrass DNA damage, dose−response DNA lesion profiles (Figure S9), growth inhibition, and copper uptake can be found in Supporting Information. Environmental Implications. Metal and metal oxide nanomaterials formed from copper clearly have the capacity to mediate oxidatively induced damage to DNA in terrestrial plants. Plants, depending on the species, show different degrees of tolerance (formation and accumulation of DNA lesions) to NP exposure, but the different species all exhibit concentrationdependent NP-induced growth inhibition for the tested concentration range. This study indicates that the capacity for NPs to cause oxidatively induced DNA damage is an important toxicological end point that should be investigated in future studies. In particular, the capacity for NPs to induce DNA lesions in plants grown in various types of soils should be studied. The impact of low NP concentration for extended time periods, and the biochemical mechanism underlying the species to species variability among plants in the capacity for these lesions to be produced are additional important research topics. It is likely that accumulation of mutagenic DNA lesions in various agricultural and grassland species could have profound generational effects on plant viability. Remarkably, little is known regarding the biological repertoire for DNA repair in plants.4,6,36 Future research investigations are needed in order to both clarify NP effects on the genomic machinery37 and on the putative base and nucleotide excision repair processes in plants.
dose−response profile for 8-OH-Gua showed a steady increase across all doses. The BP- and Cu2+ ion-induced lesion yields suggested that the specific mechanisms of DNA damage were superimposed on plant protection mechanisms, e.g., apoptosis, that contributed to the apparent inhibition of plant growth (see below). Perennial ryegrass seedling copper uptake profiles for the three forms of copper across all treatments were quite similar (Figure 3b). The measured background level of copper in the perennial ryegrass was 15.8 μg Cu/g plant shoots and the total copper uptake from all three forms of copper was only 1.5 times the background level. It was evident that the putative active copper uptake mechanism present in the radish plants was not present in the perennial ryegrass plants, because the overall copper uptake into perennial ryegrass (23.2 μg Cu/g plant shoots) was approximately 17 times lower than the overall copper uptake into radish (400 μg Cu/g plant shoots). Additional findings in support of the reduced tolerance of perennial ryegrass to copper in ionic form or in the form of CuO NPs and BPs were clearly demonstrated by the strong inhibition of seedling growth (Figure S6 in Supporting Information). All forms of copper inhibited seedling growth over the entire treatment range, but the NPs and BPs (in that order) were especially devastating to overall root development. The Cu2+ ions also had a strong negative impact on both root and shoot growth. In terms of the entire treatment range (untreated control compared to highest test dose), perennial ryegrass root growth was inhibited 100%, 100%, and 98% and shoot growth was inhibited 39%, 28%, and 61% by NPs, BPs, and Cu2+ ions, respectively. At the lowest NP dose (10 mg/L), root growth was inhibited 80%, and at a NP dose of 100 mg/L, root growth was completely inhibited. At a BP dose of 500 mg/ L, root growth was inhibited 94% and at a BP dose of 1000 mg/ L root growth was completely inhibited. With respect to Cu2+ ion effects, it was observed that 1 mg/L Cu2+ ion inhibited root growth by 7%, but 10 mg/L Cu2+ ion inhibited root growth by 89%. Our dissolution data for NPs indicated that a NP dose of 100 mg/L releases ≤0.5 mg/L copper into solution. Thus, total inhibition of root growth by 100 mg/L NP is not concordant with the measured root growth inhibition data (7% inhibition) for 1 mg/L Cu2+ ion. Interestingly, the root growth inhibition data for BPs, on the other hand, is in agreement with the BP dissolution into copper data and with the trend toward total inhibition of root growth by the 1000 mg/L BPs. Based on the NP root growth data and copper dissolution profiles, it seems likely that Cu2+ ions dissolved from NPs had a strong effect on root growth inhibition in perennial ryegrass; however, the overall growth inhibitory effects from the NPs appeared to be even greater. These effects probably stem from the sensitivity of perennial ryegrass to minimally increased concentrations of copper over native background copper levels in the plant. Inhibition of shoot development was not nearly as severe as the inhibition of root development likely due to the reduced capacity of copper to translocate from the root to the shoot in perennial ryegrasses.35 Annual ryegrass seedlings, however, were sharply dissimilar from both perennial ryegrass and radish seedlings in terms of their sensitivity to NP and BP mediated DNA damage. In general, annual ryegrass was resistant to NP-induced DNA damage, unless the seedlings were treated with extremely high doses (Figure S7 in Supporting Information). Under such circumstances, it was predominantly the NPs that promoted statistically significant accumulation of FapyAde, FapyGua, and
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ASSOCIATED CONTENT
S Supporting Information *
Text for the plant DNA extraction procedures and the annual ryegrass study; data for dissolution of copper from CuO NPs and CuO BPs, dose−response DNA lesion profiles for radish/ perennial ryegrass/annual ryegrass after exposure to copper particles, STEM analysis on radish shoots, growth inhibition on roots and shoots of annual ryegrass after exposure to copper particles and oxidative damage to annual ryegrass DNA after exposure to CuO NPs, CuO BPs and Cu2+ ions. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]; phone: 301-975-2517; fax: 301-975-8246 (B.C.N.). E-mail:
[email protected]; phone: 413-545-5212; fax: 413-545-3958 (B.X.). Author Contributions †
These authors contributed equally to this work.
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ACKNOWLEDGMENTS We thank Jerry Rattanoong and Bisrat Nigussie for help in the preparation of the figures and Andrew Dillon for help with preliminary plant DNA damage experiments. We also thank Timothy Maugel for his sectioning of the plants for STEM analyses. The work is partly supported by USDA-AFRI (201167006-30181). Certain commercial equipment, instruments, and materials are identified in order to specify experimental 1826
dx.doi.org/10.1021/es202660k | Environ. Sci. Technol. 2012, 46, 1819−1827
Environmental Science & Technology
Article
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