Co₃O₄-NPs characterization

Size and morphology of Co₃O₄-NPs were examined by TEM and AFM analyses. TEM image in Fig. 1 revealed the morphology of Co₃O₄-NPs as crystallite spheres with polyhedral structure, and aggregates have been observed. The average particle size of Co₃O₄-NPs determined from six different TEM images were 21.3 nm (Fig. 1a). AFM analysis of Co₃O₄-NPs exhibited the size of NPs to be 40 nm (Fig. 1b). Since, the sizes of NPs are regarded as important parameters for cellular toxicity; the behavior of Co₃O₄-NPs in treatment solutions was examined through DLS, to understand the extent of aggregation and secondary size of NPs. The data revealed large particle aggregates of 359.7 ± 2.1 nm with ζ-potential of −6.2 ± 1.3 mv (Fig. 1c, d). DLS is widely used to determine the size of Brownian NPs in colloidal suspensions in the nano and submicron ranges [10]. The average hydrodynamic particle’s diameter in water indicates particle aggregation. Our characterization data corroborates well with the recent report on agglomeration of NPs in an aqueous environment [3].

Co₃O₄-NPs treatment retarded the root growth of eggplant

Co₃O₄-NPs treated seeds exhibited a tendency to adsorb on the seed coat in a concentration dependent manner, while bulk Co₃O₄ did not display adsorption behavior on the seeds (Fig. 2a). These observations are in line with our previous report exhibiting the adsorption tendency NiO-NPs on the tomato seeds [11]. Such behavior could be due to the physical attachment of particles on a rough seed surface, electrostatic attraction and hydrophobic interactions between seeds and NP agglomerates. It is likely that the adsorption of NPs may facilitate the release of ions locally to enhance phytotoxic effects [11]. Exposure concentrations for Co₃O₄-NPs were selected based on the screening of low to high concentrations (0.025–1.0 mg/ml) of Co₃O₄-NPs to affect the root length. Since, the primary objective of this study was to assess the toxicity of Co₃O₄-NPs. Therefore, the non-significant concentrations (0.025–0.1 mg/ml) in terms of repression of root lengths were excluded from further toxicity experiments. After 7 days of incubation, average root lengths recorded at 0.5 and 1.0 mg/ml of Co₃O₄-NPs were 1.0 ± 0.16 and 0.70 ± 0.18 cm (p < 0.01) respectively. The root length in untreated control was 3.80 ± 0.24 cm (Fig. 2b). Under similar experimental conditions, bulk Co₃O₄ did not induce repression of root length (Fig. 2b). Phenotypic analysis of roots revealed that Co₃O₄-NPs (0.25, 0.5 and 1.0 mg/ml) treated groups exhibited a dose-dependent reduction in root length along with thickening, stunted growth and absence of root hairs. However, no such changes have been observed in bulk Co₃O₄ treated groups (Fig. 2c). Due to the non-significant effects of bulk Co₃O₄ on eggplant root length, we have excluded bulk Co₃O₄ in further experiments. Comparison of data with our recently published studies on NiO-NPs induced toxicity in tomato, suggested that Co₃O₄-NPs are more growth inhibitory at the highest concentration of 1.0 mg/ml and exhibited 2.14-fold greater repression of root length in eggplant. Thickening and stunted roots were observed at all treatment concentrations of Co₃O₄-NPs after 7 days, while the NiO-NPs treated tomato roots were found thickened and stunted only at greater concentrations of 1.5 and 2 mg/ml after 10 days of exposure [11], thus, suggesting the higher toxicity of Co₃O₄-NPs. The differences in toxicity of Co₃O₄-NPs over NiO-NPs can be attributed to the difference in seed size of the two plants. Indeed, the interactions of similar-sized NPs might be greater for the smaller seeds of eggplant as compared to the larger sized tomato seeds, largely due to greater surface-to-volume ratio of the small-sized seeds to that of the large-sized seeds [12]. These observations corroborate well with earlier literature suggesting greater phytotoxicity of rare earth oxide NPs to the smaller seeds (rape, cabbage, radish), as compared to the larger seeds of wheat [13]. Nonetheless, our results are in agreement with some recent findings, which have also demonstrated the repression of root length by CuO and Ag-NPs in Arabidopsis thaliana [3, 14]. Seed coat provides first line of defense to the seed from NPs during the germination. However, in this study we have demonstrated that seedling roots after piercing the seed coat, become the main organ to confront Co₃O₄-NPs. Therefore, the toxic symptoms were more evident in roots than in other parts of seedlings. In addition, seeds rough surface, electrostatic attraction and hydrophobic interactions contributed towards NPs adsorption on the seeds. Such adsorption may facilitate the release of ions from NPs and enhance the phytotoxicity [15].

Co₃O₄-NPs uptake and translocation

To confirm the translocation of Co₃O₄-NPs, ultrathin sections of root tissues from elongation zone were evaluated by TEM (Fig. 3a–f). Root elongation zone consists of newly formed differentiated cells, which elongate at a rapid rate and exhibit enhanced cell cycle activity. Co₃O₄-NPs were observed in the cytoplasm and exterior region of confluent parenchymal cells (Fig. 3c, d). NPs were recognized as dark dots and aggregates entrapped in cell vacuoles. Our TEM data on Co₃O₄-NPs corroborate with recent studies exhibiting the translocation of Ag and ZnO-NPs Triticum aestivum and Schoenoplectus tabernaemontani [16, 17]. In addition, enhanced number of peroxisomes and mitochondria with degenerated cristae has also been encountered (Fig. 3c, e, f). These subcellular anomalies are found identical as observed in plants exposed to ozone [18]. Interestingly, the eukaryotic microorganism (yeast) has also demonstrated these common properties of peroxisome biogenesis as a consequence of mitochondrial dysfunction [19].

Intracellular ROS production and mitochondrial dysfunction

Qualitative evaluation of Co₃O₄-NPs treated roots exhibited a conspicuous increase in DCF fluorescence. Eggplant roots stained with fluorescent dye DCFH-DA revealed a typical pattern of ROS localization as a function of Co₃O₄-NPs concentration. Compared to the untreated control, Co₃O₄-NPs at 0.25 and 0.5 mg/ml showed ROS localization around apical root meristem. Subsequently, the ROS localization becomes more intense towards root elongation zone at 1.0 mg/ml. (Figure 4a). Protoplasts quantitatively analyzed on flow cytometry also revealed higher levels of intracellular ROS. Relative to 100 % DCF fluorescence in control, Co₃O₄-NPs (0.25, 0.5 and 1.0 mg/ml) treatments resulted in 167.0, 177.4 and 157.6 % greater ROS generation (Fig. 4b, c). Our ROS data is in line with a previous study demonstrated oxidative stress in Oryza sativa treated with multiwalled carbon nanotubes [20]. It is well established that ROS rapidly interacts with distant cellular organelles and may result in DNA strand breaks, purine oxidation, protein modifications, protein-DNA cross links and mitochondrial damage [21, 22].

The interplay of ROS and mitochondria has further lead us to analyze ΔΨm using mitochondrial specific dye Rh123. Qualitative evaluation of seedling roots from Co₃O₄-NPs treatment groups exhibited enhancement in the fluorescence intensity of Rh123 (Fig. 5a). A similar pattern of fluorescence enhancement has been observed in the protoplasts of Co₃O₄-NPs exposed groups. Flow cytometric analysis of protoplasts revealed 136.6, 144.2 and 282.4 % greater fluorescence at 0.25, 0.5 and 1.0 mg/ml concentrations (Fig. 5b, c). Mitochondrial dysfunction as a consequence of oxidative stress is an important factor in cell death [23, 24]. Fluorescence enhancement of Rh123 dye is related to the variation in ΔΨm, which results as a consequence of swelling and shrinking properties of mitochondria [25]. Due to these morphological alterations, preferably by swelling, Rh123 leaks out from mitochondria to the cytoplasm [26]. Therefore, Co₃O₄-NPs mediated enhancement of Rh123 florescence confirm mitochondrial swelling in eggplant and the leakage of dye thus causing Rh123 hyperpolarization in cytoplasm.

NO and esterase analysis

Flow cytometric analysis of Co₃O₄-NPs treated protoplasts exhibited an increase in intracellular NO generation. Relative to 100 % fluorescence in control, protoplasts isolated from 0.25, 0.5 and 1.0 mg/ml Co₃O₄-NPs treated cells showed 169.8, 242.5 and 178.7 % (p < 0.01) higher fluorescence of DAF2-DA (Fig. 6a, b). Our data is in line with earlier studies, which also suggested an elevated level of NO generation in Lupinus luteus and Glycine max exposed to Cd⁺⁺ [27, 28]. NO has been regarded as an effective inducer of cell death by interfering with mitochondria functionality and promoting imbalance between ROS generation and scavenging [28]. Furthermore, NO has been described as a small signaling molecule in plants taking part in several events throughout the life cycle, as well as in defense against biotic and abiotic stresses [29].

We further investigated the level of intracellular esterases, regarded as a prevalent biomarker to assess the viability of cells [30]. 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 [31]. 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

Alkaline single cell gel electrophoresis (comet assay) data showed Co₃O₄-NPs-induced single strand breaks in DNA of eggplant cells. The representative digitized image of comet tail in Co₃O₄-NPs (1.0 mg/ml) treated group clearly demonstrates the extent of broken DNA liberated from head of the comet during electrophoresis (Fig. 7 inset). However, under identical condition the control cell exhibited round boundary of nuclear head DNA. A conspicuous tail in positive control ethyl methanesulfonate (EMS 2 mM) confirms the proper functioning of comet setup (Fig. 7 inset). Compared to the olive tail moment (OTM) value of 1.22 ± 0.12 in control, the quantitative data obtained from 150 cells of Co₃O₄-NPs (0.25, 0.5 and 1.0 mg/ml) treatment groups exhibited an increase of 2.6 ± 0.49, 3.0 ± 0.14 and 3.2 ± 0.35 (p < 0.01), respectively, over control (Fig. 7). The DNA damaging activity of Co₃O₄-NPs has been found to be similar to the DNA damaging effect of TiO₂ and ZnO-NPs reported in A. cepa [32]. Thus, the results substantiate the hypothesis that plant-NPs interaction is associated with the genotoxicity and concurs well with the earlier reports. Metal oxides as well as metals are involved in ROS formation, which can interact directly or indirectly with plant DNA to induce strand breaks [33, 34]. However, on the basis of our data we suggest that Co₃O₄-NPs may have indirectly induce DNA damage in eggplant, as evident from increase in the ROS, mitochondrial membrane damage and elevated esterase level. On the contrary, the NiO-NPs may have directly interacted with the DNA to induce non-repairable heavy damage; as a result an increasing number of apoptotic/necrotic cells were appeared in the comet assay of tomato cells [11]. Thus, an in-depth investigation is warranted in order to ascertain the extent and nature of direct interactions of Co₃O₄-NPs using spectrofluorometric, spectrophotometric biophysical and bioinformatics tools.

Flow cytometric analysis of apoptosis

In order to assess Co₃O₄-NPs induced cell death, the apoptotic events occurred in treated eggplants cells were captured by use of a flow cytometer. The propiodium iodide (PI) stained nuclei of Co₃O₄-NPs treatment groups indicated a concentration dependent increase in apoptotic sub-G1 peak (Fig. 8a). In comparison with 24.4 % of background apoptotic cells in control, Co₃O₄-NPs at 0.25, 0.5 and 1.0 mg/ml exhibited 44.4, 69.8 and 73.2 % of apoptotic population (Fig. 8b). A similar mode of cell death has also been observed in our earlier report on tomato cells exposed to NiO-NPs [11]. Furthermore, our ongoing studies on cultured human cell lines with Co₃O₄-NPs have demonstrated that these NPs have the potential to induce apoptosis in the human cell lines (data unpublished). It is widely accepted that apoptotic mechanisms in plants and animals share some common components leading to conserved cellular events [35, 36]. Therefore, it is likely that eggplant root cells may activate a similar mechanism of cell death.

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. [37], with slight modification [11]. 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. [10]. 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 [40].

Flow cytometric analysis of apoptosis in eggplant

Apoptosis analysis in eggplant roots was done using flow cytometry following our previously described method [11]. 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 [38]. 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).

Statistical analysis

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.