Copper Oxide Nanoparticles and Bulk Particles Stress Induced Cytological and Physiological Responses of Vicia Faba L.

CuO nanoparticles (NPs) and their bulk counter parts are being utilized in various industrial preparations. The progressive increase in the use of CuO NPs and bulk particles (BPs) eventually ends up in the environment, causing potential hazard to biota and imbalance in the abiotic components. In order to elucidate the toxic impact of CuO NPs and BPs, plant seedlings of Vicia faba var. Pusa Sumit were exposed to 20-100 mg L(cid:0) 1 of CuO NPs and BPs along with a control set up. Root tips and leaf tissues of plant seedlings were used to perform genotoxic and biochemical assays, respectively. Cytological preparations were used to screen mitotic indices (MI), micronuclei and chromosomal abnormalities (CAs). CuO NPs treatment led to 24.1 % reduction in MI and 7.9 % increase in CAs while BPs treatment reduced MI by 12.7 % and raised CAs by 4.3 % only. Bio-uptake of CuO NPs and BPs in the plant tissues is the key cause of oxidative stress. It triggered signicant changes in lipid peroxidation and other biochemical parameters including enzymatic (peroxidase, superoxide dismutase, catalase, glutathione s-transferase and glutathione reductase) and non-enzymatic (photosynthetic pigments and proline content) components of antioxidant system in treated plant seedlings. In this study, CuO NPs caused 49.1 % to 96.7 % enhanced activity of antioxidant enzymes as compared to BPs. These ndings revealed that CuO NPs were more toxic to plants than their counter BPs.


Introduction
al. 2016; Ochoa et al. 2017, Nasrollahi et al. 2019). In the last decade, CuO NPs production has been estimated in hundreds of tons (Keller et al. 2013), much of which is nally ended up in the environment.
Besides, NPs are also being utilized in the agricultural system as nano fertilizers and nano pesticide formulations to protect the crop plants from various pests and pathogens (Lazareva and  Several plant bioassays have been suggested to elucidate the toxicological effects of NPs and BPs. V. faba is one model plant to evaluate genotoxic effect of environmental contaminants due to its sensitive mitotic dynamics and large size of chromosomes (Lutterbeck et al. 2015;Silveria et al. 2017). This study has been conceptualized to assess the toxicity potential of CuO NPs and BPs using V. faba test system through genotoxic (MI, CAs, and micronuclei) and biochemical (POD, SOD, CAT, GST, GR, lipid peroxidation, proline content, and photosynthetic pigments) assays. Bio-uptake studies of nanomaterials that are important to assess their internalization in different plant tissues have also been undertaken in this study. Though there are several reports on cytogenotoxic assessment of environmental contaminants in plants, this is the rst of its kind to show the cytological and physiological effects of CuO NPs in V. faba.

CuO NPs and BPs and their characterization
Nanopowder of CuO was provided by Sigma-Aldrich (now Merck). Its primary particle size is <50 nm, purity is 99.9% based on trace metals. CuO BPs were procured from HiMedia with 98% purity (These speci cations details are as data provided by the manufacturer). For the preparation of mono-suspension of NPs and BPs, a weighed amount of chemicals was directly put in deionized water separately and subjected to sonication (1 Amp, 20 kHz) for 30 minutes.
Characterization of CuO NPs and BPs was done through a scanning electron microscope (SEM) and dynamic light scattering (DLS) for size, morphology, state of dispersion, size distribution and for stability concern of chemical, the zeta potential was recorded. For size determination, 100 particles were measured from different elds of SEM view and images showing the morphology of the particles. The mean hydrodynamic diameter (MHD) of the particles and polydispersity index (PdI) was recorded through DLS using a zetasizer (Malvern Instruments Ltd). The PdI was used to measure the heterogeneity or size range in a sample and numerical value ranged from 0 to 1 (monodispersion to ploydispersion state of particles).

Test system and treatment
Seeds of V. faba var. Pusa Sumit obtained from National seed corporation, Lucknow were used. They were washed four to ve times in distilled water followed by deionized water and then soaked in deionized water for 24 hours. They were nally rinsed and kept on moist blotting sheets in Petri plates. After two to three days, when roots became 2 to 3 cm long their tips were removed for the proliferation of secondary roots. After 2 days when secondary roots emerged they were exposed to the graded concentration of CuO NPs and BPs (20,40,60,80 and 100 mg L 1 ) including control in the form of deionized water alone. After 6 hours exposure to CuO NPs and BPs, the roots were xed for cytological studies.
In another set of experiments, growing seedlings were exposed to same concentrations of chemicals and for similar duration as mentioned above. After treatment seedlings were transferred to fresh petri plates and allowed to grow for 15 days till the development of the rst pair of leaves. These leaves were utilized for biochemical analysis and data were recorded for morphological parameters. All the experiments were conducted in triplicate.

Fixing of roots and analysis of genotoxicity
Carnoy's uid (3:1 preparation of ethyl alcohol and acetic acid respectively) was utilized for xing secondary root tips. After 24 hours root tips were transferred to 70% alcohol and stored under refrigeration at 4 °C. Cytological preparations were carried out according to the protocol developed by Sharma and Sharma (1980). Cytological observations were recorded using temporary slide preparations to screen mitotic activity/(MI), micronuclei, and several CAs. Approximately 1000 cells from at least 10 well smeared microscopic elds out of 20 elds captured from different root meristems preparation per treatment were screened at 400 x under the light microscope (Magnus MLX-TR Plus) (Khan et al. 2021).
MI percentage, CAs and cells with micronuclei were calculated in reference to total number of cells in a microscopic eld.

Morphological parameters
Data for relevant parameters like root length and shoot length were recorded for control and treated seedlings after the stipulated time. Phytotoxicity was calculated according to following formula (Chou et al. 1978).
Photosynthetic pigment estimation 100 mg fresh leaf tissue was crushed in 80% acetone for the assessment of photosynthetic pigments including chlorophyll and carotenoids (Arnon 1949). The crude mixture was centrifuged at 6000 x g for 10 min. The supernatant was used to record absorbance at 645, 652, and 663 nm for chlorophyll and 480 and 510 nm for carotenoids estimation using a spectrophotometer (Cary 5000, Agilent Technologies)

Estimation of lipid peroxidation
Lipid peroxidation was assayed according to the protocol of Heath and Packer (1968) with certain modi cations. 200 mg of fresh leaf tissue was homogenized in 3 ml of 0.1% trichloroacetic acid (TCA) and centrifuged at 10000 x g for 15 min at 4°C. 1 ml of supernatant was mixed with 4 ml of 20% TCA containing 0.5% TBA (thiobarbituric acid) and heated in a water bath at 90°C for 30 min to develop MDA adduct with TBA. After heating, the reaction mixture was ice-cooled for 10 min and subjected to centrifugation at 15000 x g for 5 min. The supernatant was used to record absorbance at 532 nm with the subtraction of non-speci c absorbance at 600 nm. MDA content was calculated using the extinction coe cient ( ) of 155 mM −1 cm −1 .

Estimation of proline content
Proline (osmoprotectant) estimation was done according to Bates et al. (1973) with few modi cations in the protocol. 200 mg of leaf tissue from control and treated plants were ground in 5 ml solution of 3% sulphosalicylic acid and the crude mixture was centrifuged at 10000 x g for 10 min. 2 ml of supernatant was taken in a test tube and mixed with 2 ml of glacial acetic acid and ninhydrin each. Reaction tubes were heated in the water bath at 96 °C for one hour. Tubes were allowed to cool down in ice to terminate the reaction. Samples were extracted with toluene and absorbance of chromophore containing the mixture was recorded at 520 nm. Amount of proline was calculated against its standard curve.

Antioxidant enzyme assays
Catalase estimation was done as described by Euler and Josephson (1927). 150 mg of fresh leaf tissue was homogenized in 6 ml of distilled water. The crushed sample was centrifuged at 10000 x g for 10 min. 1 ml enzyme extract was mixed with sub-mixture (0.1 M phosphate buffer of potassium pH 7.0 and 10% H 2 O 2 ) in a reaction container. Blank was maintained without enzyme extract. After 5 minutes, 5 ml of 2N H 2 SO 4 was added to the reaction container to terminate the reaction. The reaction mixture was titrated against 0.1N KMnO 4 .
Peroxidase assay was based on p-phenylenediamine oxidation using H 2 O 2 as an oxidant (Luck 1963). 50 mg of fresh leaf tissue was homogenized in 10 ml of extraction buffer and the homogenate was centrifuged at 10000 x g for 10 min. The reaction mixture contained 5 ml of 0.1 M potassium phosphate buffer (pH 6.8), 1 ml p-phenylenediamine (0.5%), 1 ml enzyme extract and 1 ml H 2 O 2 (0.01%). 2 ml of 5N H 2 SO 4 was used as reaction stopper. Reaction tubes were cooled down in ice for 20 min. Absorbance was recorded at 485 nm.
Superoxide dismutase assessment was done using the protocol based on its ability to inhibit the reduction of NBT (nitroblue tetrazolium) (Beauchamp and Firdovich 1971). 100 mg of fresh leaf tissue was extracted in a grinding medium and the crude extract was centrifuged at 5000 x g for 10 min. 3 ml of reaction mixture contained 50 mM phosphate buffer (pH 7.8), 0.1 mM EDTA, 10 mM methionine, 0.07 mM NBT, 3 µM ribo avin and 0.5 ml enzyme extract. Sample tubes were irradiated in light for 20 min (appearance blue colour). Blanks were maintained in dark. The absorbance of radiated and non-radiated samples was recorded at 560 nm.
Glutathione s-transferase assessment was based on its ability to catalyze the conjugation of glutathione (GSH) to 1-chloro-2,4-dinitrobenzene (CDNB) as described by Habig et al. (1974). Its activity was determined in 3 ml reaction volume containing 100 mM phosphate buffer (pH 6.5), 1 mM GSH, 1mM CDNB and 100 µl of enzyme extract. The reaction was started by adding CDNB. The change in absorbance of the reaction sample at 340 nm was recorded for 5 min ( = 9.6 mM −1 cm −1 ). The protein content of leaves was determined according to Bradford (1976).
Glutathione reductase was assessed in terms of glutathione based oxidation of NADPH according to Carlberg and Mannervik (1985). 1 ml assay mixture contained 200 mM phosphate buffer (pH 7), 2 mM EDTA, 20 mM GSSG, 2 mM NADPH and 100 µl enzyme extract. The reaction was started by adding NADPH. The decrease in absorbance of the reaction sample at 340 nm was recorded for 5 min ( = 6.22

Bio-uptake of CuO NPs and BPs
The quantity of CuO NPs and BPs internalized by V. faba was determined according to Pakrashi et al. (2014). The treated plant materials were washed with deionized water and were oven-dried at 60 °C for 24 hours. The tissue samples (roots and leaves) were crushed separately in sterile mortar and pestle. The powdered samples were subjected to digestion in concentrated HNO 3 and soluble parts were ltered through 0.25 micrometer membrane lter. The amount of NPs and BPs internalized into the tissues was determined by ICP-OES (Inductively coupled plasma optical emission spectroscopy; Perkin Elmer Optima 5300 DV, USA).

Statistical analysis
Results of all the observations were represented by mean values and standard error of the mean. Mean values were compared through one-way Analysis of Variance (ANOVA) followed by Duncan's multiple range test (DMRT) at P ≤ 0.05.

Results
Characterization of CuO NPs and BPs SEM images showed more or less circular crystalline structure of NPs and BPs. The sizes of the CuO NPs and BPs obtained by SEM analysis were 49 ± 3.4 nm and 1 ± 0.2 µm respectively ( Supplementary Fig. 1). The zeta potential value and PdI of CuO NPs in deionized water suspension analyzed by DLS were -30.6 mV and 0.134 respectively and these values for CuO BPs were -33.4 mV and 0.121 respectively. The MHDs of NPs were 191 ± 2.3, 193 ± 3.1, 190 ± 3.4, 192 ± 4.1, and 195 ± 4.1 nm at 0 hours for 20, 40, 60, 80, and 100 mg L 1 concentrations respectively. In terms of hydrodynamic stability of dispersions, MHDs of NPs after six hours of treatment were found to be 192 ± 3.8, 192 ± 4.3, 194 ± 3.7, 196 ± 2.9, and 199 ± 4.2 nm for chemical suspensions of 20, 40, 60, 80, and 100 mg L 1 respectively. The MHDs of particles at 0 and 6 hour did not show any signi cant statistical difference. This means that particles did not initiate agglomeration during the treatment period. Thus, dispersions were considered stable during the treatment period.

Microscopic analysis
A detailed microscopic observation of smears prepared from treated secondary root tips of V. faba var. Pusa Sumit provided an overview of cytogenotoxic potential of CuO NPs and BPs. Different genetic endpoints like MI, CAs, and micronuclei were analyzed in root meristems exposed to graded concentrations of NPs and BPs suspensions viz. 20, 40, 60, 80, and 100 mg L 1 including control. Each index showed a dose-dependent correlation to chemical exposure. The correlation coe cients (r-value) of MI were found to be -0.99 and -0.95 against the treatment of CuO NPs and BPs respectively. Maximum MI was recorded in control, i.e., 34.0 ± 0.62 and it reduced gradually in treated root cells at 20, 40, 60, 80, and 100 mg L 1 concentrations. At 100 mg L 1 exposure, MI was minimum and found to be 9.9 ± 0.57 (NPs) and 21.3 ± 0.78 (BPs) ( Table 1 and Fig. 1A). Reduction in MI with increasing concentration of CuO NPs and BPs was found statistically signi cant (p≤0.05).
The microscopic studies on mitotic phases in cells of secondary root meristems of V. faba revealed several CAs including stickiness, fragmentation, diagonal anaphases, precocious chromosomes, laggards, bridges, C-metaphase, spindle deformities, clumping, and micronuclei formation (Fig. 5). The correlation coe cient (r-value) of CAs was found to be 0.99 in both CuO NPs and BPs treated roots.
Average percentage of CAs were minimum at 20 mg L 1 concentration i.e., 1.93 ± 0.33 (NPs) and 0.89 ± 0.23 (BPs). The percentage of CAs increased with increasing concentrations of chemicals and reached maximum at 100 mg L 1 i.e., 7.85 ± 0.54 (NPs) and 4.3 ± 0.33 (BPs) ( Table 2 and Fig. 1B). The increase in the occurrence of these abnormalities was found signi cant in statistical reference (p≤0.05). At 20 mg L 1 exposure of CuO NPs, several aberrations viz. chromosome break, disturbance in the organization of metaphase chromosomes and movement of chromosomes at anaphase/spindle disturbance, diagonal anaphases, and micronuclei were observed. However, frequency of these aberrations was low in comparison to that observed at higher doses of chemicals. Additionally, several other aberrations such as fragmentation, stickiness, clumping of chromosomes, precocious chromosomes, bridges, laggards, and C-metaphase were observed at 40 and 60 mg L 1 exposure. At the highest dose exposure of 80 and 100 mg L 1 , the average frequency of aberrations was much high and interestingly, multiple chromosomal aberrations like fragments, bridges, and laggards were seen in a single cell. However, the frequency of aberrations was higher in CuO NPs treatment as compared to BPs. The frequency of each aberration at all concentrations of CuO NPs (Fig. 1C) and BPs ( Supplementary Fig. 3) has also been recorded during experiment.  (Fig. 1D).

Presence of micronuclei in cells was
Morphological analysis Plants exposed to CuO NPs showed a marked reduction in root and shoot lengths and leading to increase in phytotoxicity percentage. However, BPs stress did not show any signi cant reduction in seedling growth at an initial concentration (20 mg L 1 ), and its effects were less severe than that of NPs. Upon  Fig. 4).

Photosynthetic pigments
The content of photosynthetic pigments including chlorophyll and carotenoids signi cantly decreased under NPs and BPs stress. Plants exposed to NPs had a lower amount of pigments than plants under bulk particle stress. The total decline of 2.99 fold in chlorophyll and 3.93 fold in carotenoids content was recorded over control under NPs stress. While this decline was 1.56 fold and 2.03 fold under BPs stress ( Fig. 2A and 2B). The amount of photopigments exhibited a similar trend and decreased with increasing concentration of CuO NPs treatment.

Malondialdehyde and Proline contents
The degree of lipid peroxidation in leaves was recorded in terms of MDA content. It signi cantly increased throughout the whole range of CuO NPs and BPs concentrations. The highest MDA content was recorded at 100 mg L 1 of CuO NPs and BPs, i.e., 2.84 and 2.18 fold over control respectively (Fig. 2C). Moreover, signi cant alteration in proline content was also recorded in treated leaves. In case of CuO NPs treated plants, maximum accumulation of proline content was recorded at 80 mg L 1 concentration, i.e., 2.43 fold over control. While in case of CuO BPs treated plants proline content increased to 1.9 fold over control (Fig. 2D).

Activities of antioxidant enzymes
Among the various antioxidant enzymes assessed in our study, CAT, POD and SOD showed more or less similar trend of activity while GST and GR varied. The activities of these antioxidant enzymes altered signi cantly and POD activity was found to be more affected in plants leaves under CuO NPs stress. An increase of 3.23 fold in CAT, 3.95 fold in POD and 2.59 fold in SOD activity was recorded over control in NPs exposed leaves (Fig. 3A, 3B and 3C). The enzymatic activity exhibited dose-dependent increase up to 80 mg L 1 of CuO NPs while it decreased at 100 mg L 1 concentration. Leaves exposed to CuO BPs showed increased activity of these enzymes in each concentration of chemical and the highest recorded increment was 2.43 fold in CAT, 2.98 fold in POD and 2.03 fold in SOD activity as compared control (Fig.  3A, 3B and 3C).
Activities of GST and GR in plant leaves altered signi cantly at lower concentrations of CuO NPs and the highest activity was recorded at 60 mg L 1 NPs. Further raising their concentrations reduced GST and GR activities. At 100 mg L 1 , GST activity was lower than control, however, the difference was not signi cant in statistical reference. Under BPs stress, GST and GR activities were less affected at initial concentrations. At 100 mg L 1 , maximum activity of GST (1.69 fold) and GR (1.51 fold) was recorded over control ( Fig. 3D and 3E).

Discussion
Growing variation and utilization of industrial products has become possible due to the incorporation of NPs during the manufacturing process. NPs are also playing a progressive role in medical aspect (Grigore constituents are yet to be thoroughly elucidated. This experimental study was undertaken to assess the toxicity potential of CuO NPs and BPs in an economically important pulse crop V. faba. V. faba is an established test system for cytological studies to assess the toxic potential of environmental contaminants. Exposure of secondary root tips to varying concentrations of CuO NPs and BPs affected MI and caused CAs in dividing cells. Mean MI exhibited a negative correlation while the frequency of CAs was positively correlated with increasing concentrations of NPs and BPs. After crossing biological barriers, how NPs react with cellular integrities is still unknown (Nair et  CuO NPs and BPs exposure revealed various CAs in root tips of V. faba and frequency of aberration increased with exposure to increasing concentration of chemical. Any chemical that drastically affects the genomic integrity of a cell is called genotoxin. Induction of CAs (clastogenic/mutagenic effects) in root meristematic cells of V. faba on exposure to CuO NPs proves the fact that they too can be categorized as genotoxin. Among the various observed CAs, fragmentation, precocious movement, and clumping of chromosomes were the most prevalent aberrations. However, multiple CAs like chromosome bridges, laggards and breaks, were also observed in single cells at higher concentrations. The underlying mechanism responsible for causing genetic damage is an interesting and relevant area of research and would help to determine whether the effect of NPs on DNA is general or nanospeci c. NPs may cause genotoxicity either directly through interaction with nucleic acids or they may interfere with protein assembly during DNA replication (Carmona et al. 2018) (Gaulden 1987;Khan et al. 2009). The degree of stickiness may be slight, moderate, or severe. Intense stickiness results in clumping of chromosomes.
Presence of micronuclei in cells also indicated genotoxicity caused due to CuO NPs and BPs. The actual mechanism of how NPs induce micronuclei is still unknown. However, micronucleus formation occurs when acentric fragments or lagging chromosomes are excluded from total genetic complement during mitosis. The nuclear membrane organizes around the excluded part and resembles the nucleus proper Morphological parameters are visible indicators of any type of physiological or biochemical changes in plants exposed to stress. In the present study, the effect of CuO NPs on seedling growth is severe than CuO BPs. Decrease in root and shoot length and increase in phytotoxicity percentage were dosedependent. Additionally, the content of photosynthetic pigments (chlorophyll and carotenoids) also decreased signi cantly under NPs and BPs stresses. Chlorophylls and carotenoids play a vital role in light-harvesting and ROS quenching respectively. It is known that chloroplast and mitochondria are organelles where ROS is actively produced (Hernández et al. 1993). Under NPs stress, excess ROS production causes oxidative stress either through direct interaction or by their dissolution into ions. Reduction in pigment content might be due to disruption of chloroplast membrane and disassembly of photosynthetic apparatus under oxidative stress. Our ndings of growth inhibition and pigment reduction also corroborate the studies made in Arabidopsis thaliana and Pisum sativum against CuO NPs (Nair and MDA is a potential biomarker to assess the degree of ROS generated oxidative damage in the form of lipid peroxidation (Hashemi 2019). In our experiment, treated plants showed an increased level of MDA content. The enhanced MDA activity might be due to the toxicological manifestation of NPs in the form of excess ROS generation. The imbalance between ROS production and its removal leads to oxidative damage of lipids in membranes. It is well known that unsaturated fatty acids of biomembranes are the main target of radicals attack. They initiate a chain reaction producing fatty acid radicals causing damage to the membrane via lipid peroxidation. . Approximately two-fold accumulation of proline was recorded in treated plants in this study. This increment in proline content helps plants to maintain cell osmoticum without hampering the metabolic status of cells. Its role in membrane stabilization and ROS detoxi cation has also been proposed (Hayat et al. 2012). However, increased proline content does not appear to be effective to protect cells from ROS generated oxidative damage under CuO NPs stress, as evident from enhanced MDA content in treated seedlings. In context of this study, it can be said that high proline content in plant leaves exposed to CuO NPs is simply a stress effect rather than being stress protectant.
Higher exposure of plants to NPs may also alter their physiological activities and lead to disturbance of ROS pool in the cellular environment (Mirzajani et al. 2013). To scavenge ROS and withstand the toxic response of contaminants, plants utilize a highly sophisticated system of detoxi cation i.e. . Along with element analysis through spectroscopy, these reports also provide evidence of particle internalization by electron micrographs. Further internalization of NPs is more as compared to BPs. However, toxicity can also be changed due to the dissolution of CuO NPs into Cu ions during translocation. Meanwhile, during translocation, the dissolution of CuO NPs may be increased due to a decrease in pH and interaction with organic moieties in plant tissues and cells. Since, in this study, toxicity as evident by mito-depression, CAs, and rise in antioxidants was observed in plant tissues, CuO