Cobalt oxide nanoparticles aggravate DNA damage and cell death in eggplant via mitochondrial swelling and NO signaling pathway
- Mohammad Faisal†1Email authorView ORCID ID profile,
- Quaiser Saquib†2, 3,
- Abdulrahman A. Alatar1,
- Abdulaziz A. Al-Khedhairy2, 3,
- Mukhtar Ahmed3,
- Sabiha M. Ansari1,
- Hend A. Alwathnani1,
- Sourabh Dwivedi4,
- Javed Musarrat4, 5 and
- Shelly Praveen6
© Faisal et al. 2016
Received: 15 September 2015
Accepted: 10 March 2016
Published: 18 March 2016
Despite manifold benefits of nanoparticles (NPs), less information on the risks of NPs to human health and environment has been studied. Cobalt oxide nanoparticles (Co3O4-NPs) have been reported to cause toxicity in several organisms. In this study, we have investigated the role of Co3O4-NPs in inducing phytotoxicity, cellular DNA damage and apoptosis in eggplant (Solanum melongena L. cv. Violetta lunga 2). To the best of our knowledge, this is the first report on Co3O4-NPs showing phytotoxicity in eggplant.
The data revealed that eggplant seeds treated with Co3O4-NPs for 2 h at a concentration of 1.0 mg/ml retarded root length by 81.5 % upon 7 days incubation in a moist chamber. Ultrastructural analysis by transmission electron microscopy (TEM) demonstrated the uptake and translocation of Co3O4-NPs into the cytoplasm. Intracellular presence of Co3O4-NPs triggered subcellular changes such as degeneration of mitochondrial cristae, abundance of peroxisomes and excessive vacuolization. Flow cytometric analysis of Co3O4-NPs (1.0 mg/ml) treated root protoplasts revealed 157, 282 and 178 % increase in reactive oxygen species (ROS), membrane potential (ΔΨm) and nitric oxide (NO), respectively. Besides, the esterase activity in treated protoplasts was also found compromised. About 2.4-fold greater level of DNA damage, as compared to untreated control was observed in Comet assay, and 73.2 % of Co3O4-NPs treated cells appeared apoptotic in flow cytometry based cell cycle analysis.
This study demonstrate the phytotoxic potential of Co3O4-NPs in terms of reduction in seed germination, root growth, greater level of DNA and mitochondrial damage, oxidative stress and cell death in eggplant. The data generated from this study will provide a strong background to draw attention on Co3O4-NPs environmental hazards to vegetable crops.
Over a last decade, nanotechnology has gained an immense research interest due to its applications in public health, medicine, industry and agriculture. The incessant use of nanoparticles (NPs) in a multitude of sectors presents a risk of their release into the environment, which may pose serious threats on ecosystem and adversely affect its living entity . Particularly, plants are at maximum risk due to the concentration build-up of NPs in natural sediments, agricultural soils, and aquatic environments [1, 2]. Recent evidences on the NPs toxicity demonstrated the cellular uptake of Ag-NPs in Oryza Sativa and Cu/CuO-NPs in Lactuca sativa [3, 4]. Vicia faba exposed to multiwalled carbon nano tubes exhibited imbalance of nutrient elements, leaves damage and oxidative stress . The uptake and translocation of TiO2-NPs in Allium cepa induces heavy ROS generation, sticky, multipolar and laggard chromosomes, including micronucleus formation and DNA damage . These effects of NPs are primarily associated with their increased surface area and reactivity, ROS generation and the tendency to form agglomerates . We have selected Co3O4-NPs for the current investigation due to its unique physical properties, applications in pigments, catalysis, sensors, electrochemistry, magnetism and energy storage . In addition, the composites of Co3O4-NPs with multiwalled carbon nanotubes have been proposed for fabricating high-performance electronic devices .
Till date, only a solitary report on Co3O4-NPs demonstrated the reduction of root length in A. cepa, without much elaboration on the nature of cellular damage and mechanism of the phytotoxicity . Therefore, in this study, we have investigated the mechanistic aspects of Co3O4-NPs toxicity in eggplant (Solanum melongena L. cv. Violetta lunga 2), an economically important vegetable crop, as a model, using state-of-the-art techniques like transmission electron microscopy (TEM), comet assay and flow cytometry. This will help in understanding as to how the plant responds to NPs exposure and regulates the molecular mechanism of cell death pathways. Since no systematic study has been attempted so far, describing the mechanism of Co3O4-NPs induced phytotoxicity in eggplant at cellular and molecular levels, we have investigated the effect of Co3O4-NPs on eggplant cells to assess the (1) phytotoxicity, (2) translocation of Co3O4-NPs in root cells and subcellular anomalies, (3) intracellular ROS generation and mitochondrial dysfunction (ΔΨm), (4) DNA damage (5) cell cycle alterations, NO generation and esterase activity.
Results and discussion
Co3O4-NPs treatment retarded the root growth of eggplant
Co3O4-NPs uptake and translocation
Intracellular ROS production and mitochondrial dysfunction
NO and esterase analysis
We further investigated the level of intracellular esterases, regarded as a prevalent biomarker to assess the viability of cells . Relative to 100 % fluorescence of CFDA in control, Co3O4-NPs treated root cells at the concentrations of 0.5 and 1.0 mg/ml exhibited a decline by 29.7 and 45.2 %, respectively. However, no significant change in the esterase level has been observed at 0.25 mg/ml (Fig. 6c, d). CFDA fluoresces strongly when de-esterified to carboxyfluorescein (CF). Conversion to CF by cells indicates the integrity of the plasma membrane. An intact membrane prevents leakage of the polar dye into the medium and maintains cytoplasmic milieu, which is needed to support esterase activity . Therefore, the suppressed esterase level in this study primarily suggests that Co3O4-NPs induce membrane damage in protoplasts and in mitochondria of eggplant cells.
Co3O4-NPs induced DNA damage
Flow cytometric analysis of apoptosis
In conclusion, our study demonstrates that eggplant exposed to Co3O4-NPs exhibited a significant repression of root growth due to phytotoxic properties of NPs. Ultrastructural analysis suggests the subcellular localization of Co3O4-NPs to induce organelles damage. Fluorescence imaging and flow cytometric data supported the fact that ROS plays a crucial role in mitochondrial damage to trigger apoptosis in eggplant. Higher level of NO and mitochondrial membrane damage revealed that Co3O4-NPs trigger cell death in eggplant via mitochondrial swelling and stimulation of NO signaling pathway. Furthermore, the depletion of esterase activities in cells could serve as a useful biomarker of Co3O4-NPs mediated cellular stress. Presumably, the root cells exhibiting NPs induced cell death may share some similar apoptotic characters, as observed in animals. Nonetheless, a deep investigation is warranted on transcriptome analysis to investigate the possible connection between different apoptotic factors. Finally, we conclude with the remark that the current findings will provide strong background to explore NPs induced toxicity at field or farm level to determine a realistic exposure scenario for other crops.
Characterization of nanoparticles
Co3O4-NPs (1 mg/ml) (Cat. No. 637025, Sigma-Aldrich, St. Louis, MO, USA) were sonicated in ultrapure water for 10 min at 50 W, and the solution was dropped on copper grids of the transmission electron microscope (TEM). A total of six grids of Co3O4-NPs were prepared and subjected to TEM analysis at 200 keV. Characterizations of Co3O4-NPs were further done by analyzing the surface topography of powdered Co3O4-NPs using atomic force microscope (AFM) (Veeco Instruments, USA) in non-contact tapping mode. The topographical images were obtained in tapping mode with a resonance frequency of 218 kHz. Characterization of Co3O4-NPs was further done in liquid environment by measuring the dynamic light scattering (DLS) and zeta (ζ)-potential using Zetasizer 2000 (ZetaSizer-HT, Malvern, UK). Briefly, Co3O4-NPs stock suspension of 10 µg/ml was prepared in ultrapure water, sonicated for 15 min at 40 W and the solutions were analyzed for DLS and ζ-potential, values presented were the average of 10 readings.
Eggplant root length retardation by Co3O4-NPs and bulk Co3O4
To determine the phytotoxicity in Solanum melongena L. cv. Violetta lunga 2. (eggplant), we have selected Co3O4-NPs and its bulk counterpart Co3O4 (Cat. No. 221643, Sigma-Aldrich, St. Louis, MO, USA). Seeds of eggplant were surface sterilized in Clorox solution (5 % v/v) for 10 min followed by through washing with distilled water. The exposure concentrations of Co3O4-NPs were selected from initial experiments based on the root elongation assay. For each set of experiment, 20 seeds were treated with 0.025, 0.05, 0.1, 0.25, 0.5 and 1.0 mg/ml of Co3O4-NPs (<50 nm) and bulk Co3O4 (<10 µm) suspensions for 2 h on a rotary shaker. Parallel untreated controls were run under identical conditions. After treatment, seeds were thoroughly washed with distilled water and transferred to Petri dishes containing wet filter papers. Petri dishes were kept in the growth chamber at 25 ± 2 °C for 7 days for seed germination and growth.
Uptake of Co3O4-NPs
Subcellular changes in eggplant root cells were analyzed by use of TEM. Root tissues from control and Co3O4-NPs (1.0 mg/ml) groups were fixed in glutaraldehyde for 10 min; followed by re-suspension of root sections in OsO4 (1 %) for 1 h at 4 °C. An additional incubation of 1 h was given for each section in 2 % aqueous uranyl acetate pursued by the dehydration of sections using ascending grade of ethanol. Root sections were finally embedded in low viscosity araldite resin and ultrathin sections of 80 nm were made from elongation zone for TEM analysis under high vacuum (100 kV).
Flow cytometric analysis of intracellular ROS and mitochondrial dysfunction (ΔΨm)
For qualitative analysis of ROS and ΔΨm, Co3O4-NPs treated seedling roots were stained with 2′,7′-dichlorofluorescin diacetate (DCFH-DA) (0.25 µM) and 1 µg/ml of rhodamine 123 (Rh123) for 15 min. Roots were washed three times with PBS, and images were captured using a fluorescence microscope (Nikon Eclipse 80i, Japan) [10, 11]. Quantitative estimation of intracellular ROS and ΔΨm was done in protoplasts isolated from control and Co3O4-NPs treated groups according to the method of Imanishi et al. , with slight modification . In brief, 10 root tissues from each of control and treated samples were incubated in 1.5 % cellulose, 0.5 % pectinase (Sigma) in Galbraith buffer (45 mM MgCl2, 30 mM sodium citrate, 20 mM MOPS, 0.1 % (v/v) Triton X-100, pH 7.0) for overnight in the dark at 26 °C. The digested root/enzyme solution was filtered through a 100 µm sieve and viable cells recovered by flotation after centrifugation in Galbraith buffer. Centrifugation and recovery steps of intact cells were repeated thrice to remove enzymes. Protoplasts from control and Co3O4-NPs treated groups were separately stained with DCFH-DA (5 µM) and Rh123 (5 µg/ml) for 1 h in the dark at room temperature. Fluorescence of 10,000 protoplasts from each dye treatment was recorded on Beckman Coulter flow cytometer (Coulter Epics XL/Xl-MCL, USA) following our previously described methods [38, 39].
Assessment of NO and esterase activity by flow cytometer
Intracellular NO and esterase activities in protoplasts were measured using flow cytometry. Protoplasts isolated from Co3O4-NPs treated, and untreated eggplant seedling roots were washed twice with PBS and incubated with NO specific dye 4,5-diaminofluorescein diacetate (DAF2-DA, 5 µM) and esterase specific carboxyfluorescein diacetate (CFDA, 5 µM) for 60 min in the dark at room temperature. The protoplast suspensions were pelleted, followed by two successive washes with PBS at 3000 rpm at 4 °C for 4 min. The protoplasts were re-suspended in a final volume of 500 µl of PBS and the fluorescence of DAF2-DA and CFDA was measured in 10,000 protoplasts using a flow cytometer at log scale (FL-1, 530 nm).
DNA damage analysis by alkaline comet assay
The comet assay was performed to analyze the DNA damage in nuclei following the method described by Faisal et al. . Nuclei were isolated by chopping the root tissues using a sharp scalpel blade in 1.0 ml of Galbraith buffer (45 mM MgCl2, 30 mM sodium citrate, 20 mM MOPS, 0.1 % (v/v) Triton X-100, pH 7.0). Comet slides were prepared following our previously described method .
Flow cytometric analysis of apoptosis in eggplant
Apoptosis analysis in eggplant roots was done using flow cytometry following our previously described method . Nuclei suspensions (1.0 ml) from control and Co3O4-NPs treated groups were stained with 10 µg/ml of DNA intercalating fluorescent dye (propidium iodide, PI) and RNAase A (50 µg/ml) solutions for 10 min on ice. Red fluorescence of 100,000 events of PI stained nuclei were acquired in FL4 Log channel through a 675 nm band-pass filter . Data were analyzed excluding the cell debris, characterized by a low FSC/SSC, using Beckman Coulter flow cytometer (Coulter Epics XL/Xl-MCL, USA and System II Software, Version 3.0).
Data were expressed as mean ± standard deviation (SD) for the values obtained from at least three independent experiments using 20 seeds/concentration. Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparisons test (Sigma Plot 11.0, USA). The level of statistical significance chosen was p < 0.05, unless otherwise stated.
MF, QS both designed the study and drafted the manuscript. MF, QS both performed the seed germination, flow cytometric and DNA damage experiments. SMA, HAA performed the fluorescence microscopic studies. SD performed the physiochemical characterization of NPs. MA performed the TEM experiments. AAA, AAK contributed by reagents, materials, instrumentation and lab space. JM, SP analyzed the experimental data and contributed in discussion of experimental outcomes. All authors read and approved the final manuscript.
This project was funded by the National Plan for Science, Technology and Innovation (MAARIFAH), King Abdul Aziz City for Science and Technology, Kingdom of Saudi Arabia, Award Number 12-BIO2919-02.
The authors declare that they have no competing interests.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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