CYTOLOGIA
Online ISSN : 1348-7019
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Regular Article
Analysis on Cytotoxicity and Oxidative Damage of Iron Nano-Composite on Allium cepa L. Root Meristems
Smruti GantayatSagar Prasad NayakSushanta Kumar BadamaliChinmay PradhanAnath Bandhu Das
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2020 Volume 85 Issue 4 Pages 325-332

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Abstract

The toxic effect of iron in nano-composite and salt form was tested on onion (Allium cepa L.) root tip cells in this study. Heavy metals are core pollutants of the environment but their toxicity is dose dependant and has an issue in the state of Odisha particularly in iron contaminated soil for crop production and its food chain associated human health hazard. Plants growing in such an environment are the mute witnesses to these contaminations and crop plants played a major role as carriers of heavy metal. Onion is a biomarker that is used for various genotoxic studies as it has large chromosomes, pronounced mitotic phases in the root tip. Root growth dynamics are very sensitive to any kind of pollution. Iron accumulation within the plants can be toxic at the cellular level. Stable iron oxide/ silica nano-composite (Fe2O3 NC) is characterized by transmission electron microscopy (TEM). An increase in chromosomal aberrations in root meristems along with lipid peroxidation and a decrease in SOD activity was clearly seen after treatment with FeNC. Since, iron oxide nanocomposites, owing to their submicron dimension, permeate into the intracellular space and produce hydrogen peroxide that leads to an increase in an oxidative burst in the cell. To mitigate oxidative damage, scavenging of antioxidative enzymes were found in FeNC treatment more as compare to its salt form. However, a high dose of FeNC found carcinogenic in A. cepa root tip that might have the potential for human health hazards.

Earth’s crust or the soil, contains principally silica, alumina along with alkali or alkaline earth metals. Elements like Fe, Zn, Cu, and many more are abundant in a variety of forms in soil. Particularly iron, as it appears in its oxide forms with numerous compositions. It is well documented that these trace elements significantly influence the physiology of living organisms, specifically plants. A study regarding the effects of nano-size iron oxides on the mitotic division of plants would be interesting. It would help us to know the basic mechanism of ionic effects of these nano-iron particles on cellular metabolism as well as chromosome structure and spindle fiber formation. Nanoparticles are now of great importance and have gained substantial public attention due to their worldwide use in several industries. Increased commercial use of metal nanoparticles owing to their smaller size and multitude of potential applications has enabled more exposure of this magical entity to a living system and thus of great demand in the market with commercial forms. Various studies have been performed about the effects of nanoparticles and the majority of them are genotoxic and showed DNA strand breaks, chromosomal aberrations, oxidative damage, and mutations (Xie et al. 2011, Mahaye et al. 2017). Being smaller in size, they are easily penetrable and possess a higher percentage of reactivity. These features are mostly responsible for their toxicity in ionic levels for penetration through the cell membrane. Metal oxide nanoparticles have got quite an attention in present days due to their ability to be easily synthesized and ease of manipulation. They have also got tremendous potential mainly in the medical industries. Iron nanoparticles have gathered huge interest in research for different reasons, one of them being their inclusion among magnetic nanoparticles. They are in use commercially in the computer and semiconductor industry (Carpenter 2001); magnetic fluids, data storage, and environmental remediation (Lien et al. 2010) as well. Iron nanoparticles have potential biomedical uses as well, such as MRI contrast enhancement, drug delivery, labeling, etc. (Huber 2005).

Plant systems have been used to study toxicity assay in eukaryotes. They accumulate and transport nanoparticles which helped in assessing various endpoints of toxicity (Monica and Cremonini 2009). Genotoxic and cytotoxic effects of various nanoparticles have been studied including zinc oxide NPs (Pawar and Buchmeise 2010, Kumari et al. 2011, Demir et al. 2014), titanium dioxide NPs (Demir et al. 2014), aluminum oxide NPs (Rajeshwari et al. 2015), cobalt NPs (Pawar and Buchmeise 2010) in model plant A. cepa which can be attributed to the establishment of widely accepted chromosome aberration assay in the plant (Kumari et al. 2011). Accumulation of iron oxide nanoparticles and its transport has also been observed in pumpkin plants (Zhu et al. 2008). The accumulation and bactericidal effect of AgNP was reported by Ghosh et al. (2015). Other than the natural soil properties, many other practices viz. agriculture, mining, manufacturing, waste disposal, etc. may attribute to the occurrence of metals in the soil beyond their normal levels (Foy et al. 1978). Plants as well as humans do suffer from iron toxicity upon soil iron concentration exceeding a critical level. Detrimental effects of high iron concentrations can be direct (Wheeler et al. 1985) or indirect by sequestering essential nutrients from soil e.g. phosphorous. Iron toxicity causes bronzing in rice plants characterized by symptoms like scattered reddish-brown spots on lower leaf, gradually spreading through the entire leaf leading to death (Tanaka et al. 1966). However, the effect of Fe nanocomposite and Fe salt on root meristem is meager. This experiment aims to synthesize and characterize to measure the oxidative burst related changes and its antioxidant enzyme scavenging in the root meristem of A. cepa and its possible carcinogenic role on chromosome and cell division.

Materials and methods

Test system and treatment

Healthy onion bulbs (Allium cepa cv. Deshi) were collected from Agricultural Farm, Orissa University of Agriculture and Technology, Bhubaneswar, Odisha, and were grown in autoclaved sand in dark for 3–4 days following nursery practices. Germinating onion bulbs with roots of about 2 to 3 cm were subjected to treatments with different concentrations (Control, 20, 40, 60, 80, 100 µg mL−1) of iron oxide nano-composites (Fe2O3 NC) for 6, 12, 18, and 36 h time intervals. The same set of experiments was repeated with a ferric nitrate salt solution. Three replicates were made for all treatments for statistical validations.

Preparation of FeNC

Fe2O3 nanoparticles with the well-ordered nanopores of siliceous SBA-3 were developed using the incipient wetness method and microwave irradiation. The calcined SBA-3 was activated by soaking them with aqueous Fe(NO3)3 solution by dropwise addition. The resulting mass was stirred by a glass rod till wetness disappeared. It was transferred to a microwave tube with water and irradiated at 300 W for 1 h at 100°C. The sample was recovered by filtering and drying at 353°C for 10 h. It was finally heated at 773°C for 8 h under a continuous flow of air to obtain the nano-sized Fe2O3 embedded onto the internal pore structure of an SBA-3 containing cavities/channels in the ∼30 m−10, henceforth designated as FeNC (Barik et al. 2018).

TEM characterization of FeNC

TEM was taken with the help of a JEM-2100 HRTEM (Jeol, Japan) instrument. Before measurement, samples were dispersed in ethanol under sonication for 30 min and subsequently coated over a carbon-coated copper grid.

Cytological study

After the required duration of treatment with FeNC, the A. cepa root tips were removed and cleaned with water and were fixed in acetic acid : ethanol (1 : 3) overnight at room temperature. Roots were stained in 2% aceto-orcein : 1 M HCl (9 : 1) for 3–4 h at room temperature. A single stained root tip was squashed with a drop of 45% acetic acid on a glass slide and was observed under an Olympus microscope, Japan. Digital images were taken for further analysis of different anomalies of chromosomes and cell division stages under the oil immersion objective. For each treatment, three replicates were made for statistical validations. The same procedure was followed for different treatments of ferric nitrate solutions also.

Mitotic Index analysis and chromosome aberration assay

For each treatment, five root samples were considered and slides were prepared after squashing. About 300 cells were taken into consideration and the mean was taken from five slides and three replicates.

The mitotic index (MI) was calculated using the formula:

  

Examination of chromosomal aberrations was done in 500 dividing cells for each treatment taking abnormality in chromosomal structure and movement into account in different stages of cell division.

Biochemical analysis

Soluble protein was estimated by Lowry et al. (1951). The root sample (1 g) was macerated in chilled mortar and pestle in 3 mL phosphate buffer along with 0.3 g PVPP, 100 µM EDTA and a pinch of PMSF. The content was centrifuged for 15 min at 10,000 rpm at 4°C. The supernatant was collected and again centrifuged at 10,000 rpm for 15 min. Finally, the supernatant was collected. 4 mL of alkaline copper sulfate reagent was added, to 1 mL of diluted samples, mixed well, and allowed to stand for 10 min at room temperature. Then 0.4 mL of Folin-Ciocalteau reagent was mixed and incubated in the dark for 30 min. A light blue color developed and its absorbance was measured at 660 nm in a UV-VIS spectrophotometer (Systronics, India). The protein content of the sample was determined by calibrating a standard curve prepared from BSA.

Lipid peroxidation was carried out as per Heath and Packer (1968), by measuring the amount of MDA formed due to thiobarbituric acid reaction. 0.5 g of root samples were powdered using a mortar and pestle. 5 mL of 1% TCA (10 mL g−1 F.W.) was added to it and then centrifuged at, 1,000 rpm for 5 min. 1 mL of supernatant was taken in a separate test tube and 4 mL of 0.5% TBA was added and heated to 95°C for 30 min. It was then cooled in ice-cold water and again centrifuged at 5,000 rpm for 5 min. Absorbance was measured at 532 nm in a spectrophotometer. 1% TBA in 20% TCA was taken as blank. Finally, MDA content was calculated using an extinction coefficient of 155 mM cm−1 and the results were expressed in µmol MDA g−1 F.W.

SOD assay was done by following Das et al. (2000) on the root samples of different treatments. 1.4 mL of the reaction mixture which includes 1.1 mL of 50 mM phosphate buffer pH 7.4, 0.04 mL of 1% Triton-X 100, 0.075 mL of 20 mM L-methionine, 0.1 mL of 50 mM EDTA and 0.075 mL of 10 mM hydroxylamine hydrochloride were added to 100 µL of the sample extract and incubated at 30°C for 5 min. Finally, 80 µL of 50 M riboflavin was added and the tubes were exposed to 200 W fluorescent lamps for 10 min. One mL of Greiss reagent was added and the absorbance of the colored sample was measured at 543 nm.

Cell death assay

The cell death assay was conducted by Evans blue staining method (Baker and Mock 1994). Control and treated roots were stained with 0.25% Evans blue for 10–15 min. They were washed with distilled water for 30 min and equal-sized root tips were dipped in N,N-dimethylformamide for 60 min at 37°C, and its absorbance was measured at 600 nm in a spectrophotometer.

Statistical analysis

The data represent means and standard deviation calculated from three replicates, each from three consecutive experiments. Two-way analysis of variance (ANOVA) and DMRT were used to compare the effect of heavy metal stress on A. sativa and statistical significance was set at a 5% level of significance, i.e., at p<0.05 (Sokal and Rohlf 1995).

Results

TEM study

The transmission micrograph of FeNC was shown in (Fig. 1). The amorphous structure of native silica is evident from the distorted lattice fringes. The TEM images of Fe2O3NC supported on nanoporous silica are shown in Fig. 1. As it is evident, the silica possesses regular pore channels of ∼4–5 nm diameter. We do not observe any agglomeration of bulk oxides on the surface. The concentrations of Fe(III) being maintained in trace level, it was obvious that iron is uniformly supported on to the inner surface of the pores as nanoparticles, i.e., the dimension of iron oxide nanoparticles are estimated to be of 4–5 nm (indicated by arrow) located at the surface of silica material. Also, presence of Fe2O3NC appeared in dark spots was visible and it was presumed that they were anchored on to the pore walls. The iron content was around 850 ppm in the matrix. Aggregation of iron oxide particles was not observed in the sample.

Fig. 1. The transmission electron micrograph of FeNC. Arrowheads showing iron oxide nanoparticles. Scale bar=200 nm.

Cytological analysis

After the treatment of A. cepa roots with different concentrations of aqueous Fe(NO3)3 solution and FeNC, a variety of abnormalities and irregularities were noticed viz. inhibitions of mitotic index (Table 1), presence of several chromosomal and cellular irregularities of the cells (Fig. 2, Table 2) in different period of treatment.

Table 1. Effect of Fe(NO3)3 and FeNC on MI of A. cepa root.
TreatmentFe(NO3)3 (Mean±S.E.)FeNC (Mean±S.E.)
6 h12 h18 h24 h6 h12 h18 h24 h
Control24.1±2.025.2±0.926.0±1.226.8±0.422.9±0.825.2±1.128.0±1.025.7±1.2
20 µg mL−119.0±0.418.2±0.917.6±2.517.2±2.017.6±0.416.8±0.715.1±1.214.2±1.5
40 µg mL−117.9±1.116.8±0.516.8±5.816.6±2.716.1±0.915.4±0.114.8±1.813.5±0.2
60 µg mL−115.8±2.415.9±1.415.3±4.915.2±1.714.6±0.415.3±0.815.4±1.414.0±0.4
80 µg mL−113.1±6.812.6±3.711.4±2.811.1±4.112.9±0.212.6±0.411.7±1.210.1±0.9
100 µg mL−109.2±0.908.7±3.308.6±3.407.4±2.607.6±0.905.7±1.204.6±1.003.8±2.6
Fig. 2. Various aberrant features were observed upon exposure to ferric nitrate and ferric oxide NPs. A: Sticky chromosomes; B: Early separation (arrowhead); C: Binucleate cell (arrowhead); D: Nuclear bulging; E: Multipolar anaphase; F: Chromosome with less condensation; G: Chromosome breaks (arrowheads); H: Early separation of anaphase and disintegration of nuclear material (arrowheads); I: Chromosome clumping. Scale bars=20 µm.
Table 2. Effect of Fe(NO3)3 and FeNC on the abnormality of A. cepa root.
TreatmentFe(NO3)3 (Mean±S.E.)FeNC (Mean±S.E.)
NDCLgVgScBgAPNDCLgVgScBgAP
Control1150000011800000
20 µg mL−11289711222.61057410120.9
40 µg mL−1117811221043.59391112741.9
60 µg mL−198171219453.07812819050.0
80 µg mL−15510023161.85113015360.7
100 µg mL−117235482.3192013078.9

NDC—Number of dividing cells, Lg—laggard, Vg—vagrant, Sc—stickiness, Bg—bridge, AP—abnormality percentage.

 

Mitotic index

Digital optical microscopy provided a detailed view of the ferric ion toxicity. The mitotic index was calculated for both control and test groups. The MI of the control root tips was found to be highest ranged from 24.01–26.80%, however, a dose-dependent decrease in MI was observed both in iron nitrate solution as well as FeNC treated roots in comparison with the control (Table 1).

Cells treated with FeNC of higher concentrations (60, 80, and 100 µg mL−1) showed significantly reduced MI ranged from 7.6% in 6 h at 100 µg mL−1 to 3.8% in 24 h treatment at 100 µg mL−1 compared to those of control sets. MI of roots treated with 100 µg mL−1 solution for 24 h was found to be the lowest (3.8%) both in FeNC as well as salt treatments. This decreased mitotic index was found to be statistically very significant among all concentrations of 24 h treatment however was not so significant in between Fe salt and FeNC.

Chromosomal aberrations

A. cepa root cells of control plants showed different mitotic phases such as prophase, metaphase, anaphase, telophase, and interphase. However, on exposure to various concentrations of ferric nitrate solution and FeNC, many chromosomal anomalies such as stickiness, laggards, clumped chromosomes, multipolar anaphases, disturbed metaphases, and anaphases, etc. were observed. In addition to this, the nucleus also showed aberrant behavior by forming nuclear notch and nuclear budding (Fig. 2, Table 2). The frequencies of abnormalities were dependent on both times of treatment and doses of iron concentrations.

Biochemical study

Lower concentrations of ferric nitrate treatment (20 µg mL−1) showed increased soluble protein content but it was not significant (Fig. 3). However, with an increase in salt concentrations, the amount of protein decreased gradually. Moreover, in all FeNC treatments, there was continuous protein degradation which increased with an increase in the dose of treatment.

Fig. 3. Effects of different concentrations of FeNC and FeN(O3)3 on the protein content of the root meristems of A. cepa. All values are mean±SE of three independent experiments. Different small letters indicate significant differences at p≤0.05.

Lipid peroxidation test was done to assess the oxidative stress caused to plant by using metal as well as NPs. This was examined by measuring TBARS concentration (Fig. 4). This also showed a continuous increase in lipid peroxidase activity. The peroxide effect increased proportionally to ion concentration and time of exposure. The SOD assay confirmed that the oxidative stress induced by salt and FeNC, in A. cepa root cells had the same effect as above at 20 µg mL−1 (Fig. 5). It was found to be highest at 100 µg ferric nitrate treatment. The cell death studies showed an increase of cell death activity upon high concentrations of salt as well as FeNC in different hours of exposure (data not shown).

Fig. 4. Effects of different concentrations of Fe-NC and FeN(O3)3 on MDA activities of the root meristems of A. cepa. All values are mean±SE of three independent experiments. Different small letters indicate significant differences at p≤0.05.
Fig. 5. Effects of different concentrations of Fe-NC and FeN(O3)3 on SOD activities of the root meristems of A. cepa respectively. All values are mean±SE of three independent experiments. Different small letters indicate significant differences at p≤0.05.

Discussion

The prime objective of the experiment was to evaluate the sensitivity of various endpoints marked in A. cepa plants, which were exposed to iron both in aqueous Fe3+ ion and nanosized Fe2O3 form. This experiment tries to evaluate whether the study of alteration among the endpoints (mitotic index, chromosomal aberrations, proteins, lipid peroxidation, enzyme assay, cell death assay) simultaneously in the same protocol can be associated with the sensitivity of the plant.

The results showed that in almost all concentrations and duration of treatment with either iron in ionic form or as nanocomposites, the mitotic index was considerably less than that of control without any exceptions. Further, the reduction in MI might be due to cell arrest at G1 phase of the cell cycle or retardation in the rapidity of events during S or G2 phases. Due to different abnormalities in spindle fiber formation, very less cells were found in anaphase and telophase. With less number of cell divisions and uptake of Evans blue dye, cytotoxicity of Fe on the plant sample is quite evident.

Chromosomal bridge and clumping at anaphase and metaphase respectively were more frequent in root cells treated with 20, 40, and 60 µg mL−1 Fe(NO3)3 solutions. The disulfide and sulfhydryl linkages might be the prime reasons for abnormal spindle formation and hence chromosomal aberrations (Banerjee 1992). As a result of chromosome break, unequal dicentric chromosomes were formed. The reunion of these chromosomes explains the formation of anaphasic bridges (Alam et al. 1987). Furthermore, irregular spindle apparatus induces disturbed metaphases (Bhat et al. 2007). Chromosome laggards were also observed in 40 and 60 µg mL−1 solutions at anaphase (Gantayat et al. 2018). This might have occurred due to the inactivation of spindle fibers (Singh and Tsuchiya 1982). Disorganized or multi-polar and diagonal anaphase was observed in cells treated with 20 and 60 µg mL−1 ferric nitrate solutions for 6, 12, and 18 h treatment. This might have occurred due to multipolar and disorganized spindle assembly and the presence of numerous centrosomes.

Micronucleus formation was observed in cells with 100 µg mL−1 of ferric nitrate solution for 18 and 24 h. Sometimes during telophase, chromosomes fail to get incorporated into daughter cells, this leads to the deletion of primary genes and hence cell death (Yi and Meng 2003). Micronuclei are indicators of the true mutation effect. These are formed due to spindle abnormalities in the mitotic process and chromosomal breakage. Moreover, the binucleate condition was the most frequent abnormality observed irrespective of concentration from 6 to 24 h treatment with Fe(NO3)3 solutions.

Under stress, ROS causes serious damage by interacting with cellular proteins, nucleic acids, and lipids. Thus, during oxidative stress total soluble proteins act as an index of metabolic changes (Sabatini et al. 2009). Fe plays a crucial part in the free radical chemistry of all organisms. It is a chief component of antioxidative enzymes such as ascorbate, catalase, peroxidase, and superoxide dismutase, etc. Plants that have not been exposed to any iron stress, avoid the interaction of catalytic iron and peroxides by depositing them in vacuoles or by sequestering iron in ferritin (Sharma et al. 2012). However, the antioxidant defense of plants decreases when they are exposed to a variety of oxidative stress. Catalytic iron which accumulates in many plant tissues proliferate free radical production. Oxidative stress leads to metabolic dysfunction, which ultimately kills the plant cell. Thus, by quantifying the oxidation products of DNA, proteins, and lipids, oxidative stress can rightly be estimated (Sharma and Dubey 2005). Figures 35 showed the impact of Fe leading to oxidative stress in A. cepa roots, which suggest Fe toxicity. It was also evident the heavy metal in nano or ionic form entered the cell membrane and thus its accumulation in the cytoplasm may lead to a range of toxic effects in the form of oxidative stress, mitochondrial dysfunction, and finally cell death. Therefore, increased ROS levels were countered by a high level of SOD production (Tang et al. 2007, Kim et al. 2009) to scavenge the oxidative stress of the cell through mitigating singlet oxygen species. In our experiment, change in the effect of antioxidative enzymes showed their involvement in countering excessive generation of ROS which was more pronounced in FeNP as compared to Fe salt treatment.

Various stress proteins are synthesized when a plant undergoes cellular detoxification (Zhang et al. 2009). This explains the slight increase in total protein content, in 20 µg iron treatment. It can be considered as a tolerance mechanism by A. cepa plant. However, at higher concentrations of heavy metal treatment (40, 60, 80, and 100 µg) the protein content decreased steadily which indicates an increase in protein degradation (Wang et al. 2009). However, the most interesting result in this finding was the slight increase and sudden decrease of plant proteins with a gradual increase in heavy metal stress. Thus, the plant has moderate tolerance to ferric nitrate solution as reported by Kalantary et al. (2014). Besides, there was no such increase in protein level in the case of FeNC treatment, which suggests that the nanoparticles showed a very mild effect, unlike iron salts. The definitive role of iron in MI could be explained as follows; trivalent iron in hydrated form permeates into the intracellular space leading to activate hydrogen peroxide production to release nascent oxygen which facilitates cellular oxidation. Similarly, suspended iron oxide nanocomposites, owing to their submicron dimension, in principle could permeate into the intracellular space and activates hydrogen peroxide. Further, the exceptionally high surface area and un-saturation of iron oxide nanoparticles could be an additional factor for observing oxidative behavior. In nature, plants resist environmental stress and this can be vividly studied by considering the role of antioxidative enzymes (Shafi et al. 2009, Chen et al. 2010, Cheng et al. 2015). Their activity profiles are important in the evaluation of tolerance mechanisms particularly in ROS scavenging (Pradhan et al. 2017). Our data showed an increase in SOD activity at some concentration with an increase in iron salt stress as well as FeNC. As the concentration of ferric nitrate increased the number of cell death also increased significantly. When the root cells were treated with an iron salt, it induced cell death by overproducing ROS to restrict the entry of metal through the plasma membrane. Thus, exposure of A. cepa root cells to a higher concentration of iron nitrate leads to the disintegration of the plasma membrane (Huang et al. 2006). Our result also confirms the above observation.

Finally, we concluded that with advancements in nanotechnology people are getting aware of its potential toxicity. Various studies have shown both positive and negative effects of nanoparticles on higher plants (Monica and Cremonini 2009). In onion, the reduced rate of mitosis could be due to the inhibitory activities of FeNC, which further leads to cytotoxic effects (Sudhakar et al. 2001). An increase in chromosomal aberrations was clear after direct treatments of FeNC at different time intervals. However, when we compare the biochemical effect of Fe salt and Fe-nanoparticles (FeNC) on A. cepa we could say that both the treatments showed a somewhat similar trend but the effect of FeNC is less as compared to the effect of Fe(NO3)3 solutions. Thus, we conclude that even at nano-level of concentration Fe has a detrimental effect on plant metabolism.

Acknowledgments

The authors highly acknowledge DST-FIST and DRS-SAP-III of UGC, New Delhi, India for the necessary funding to the Department of Botany upgrading the Laboratory facility to carry out the work. Nevertheless, ABD is very much thankful to the Centre of Excellence, North East India Studies, & CP to CoE Environment, Climate Change & Public Health, RUSA 2.0, and World Bank, Utkal University.

References
 
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