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metal oxides) on plants using Allium cepa as a model system
CHAPTER FIVE
Toxic effects of engineered nanoparticles (metal/metal oxides) on plants using Allium cepa as a model system Barsha Roya, Suresh Palamadai Krishnana, Natarajan Chandrasekaranb, Amitava Mukherjeeb,* a
School of Biosciences and Technology, VIT, Vellore, India Centre for Nanobiotechnology, VIT, Vellore, India *Corresponding author: e-mail addresses: [email protected]; [email protected] b
Contents 1. Introduction 2. How A. cepa acts as a model organism 3. Toxicity assessments and discussion 3.1 Effects of metal/metal oxide NPs on A. cepa root growth 3.2 Determination of cytotoxicity using A. cepa root tip assay 3.3 DNA damage assessment of A. cepa roots by alkaline comet assay 4. Generation of ROS by NPs and estimation of ROS scavenging enzymes activity 4.1 Generation of intracellular ROS by NPs 4.2 Estimation of ROS scavenging enzymes 5. Estimation of cell membrane damage and intracellular uptake of NPs 5.1 Cell membrane damage by lipid peroxidation analysis 5.2 Bio-uptake of NPs 6. Conclusion References
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1. Introduction Rapid advancement in the field of nanotechnology leads to the production of different nanoparticles (NPs) of varying shapes and sizes. NPs can be defined as particles having at least one dimension <100 nm [1]. These particles exhibit entirely different physicochemical properties when in comparison with their bulk counterparts and hence can have higher toxicity than the later one [2]. Because of their small size and higher biological activity, these NPs Comprehensive Analytical Chemistry, Volume 84 ISSN 0166-526X https://doi.org/10.1016/bs.coac.2019.04.009
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2019 Elsevier B.V. All rights reserved.
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can easily penetrate inside the cells and directly interact with different cellular components, which in turn causes higher toxicity [3]. These reasons lead to the testing of cytotoxicity and genotoxicity of different NPs which includes the urge of understanding the possible mechanism of the actions [4,5]. Although the toxicity of these NPs is of significant concern, we cannot deny their favourable impacts on living organisms which includes their uses in the healing of diseases, in biomedical apparatus, nanosensors, in farming techniques, etc. NPs can have beneficial effects on the plant growth and evolution, mitigation of stress [5a]. Engineered nanomaterials (ENMs) can be divided into two categories. First one is metal-based ENMs which contains quantum dots, metal and metal oxide NPs. Whereas, the second one is the carbon-based ENMs which includes different types like nanodots, nanotubes, fullerenes, nanodiamonds etc. [5b]. Among all the NPs, metal and metal oxide NPs are widely used because of its diverse applications in industrial and commercial fields. According to the statistics, metal NPs production will increase significantly by the year 2020 in comparison with the last 10 years [6]. Uses of some of these metal oxides and metal nanoparticles are as follows. Magnesium oxide nanoparticles (MgO NPs) have a wide variety of uses, which includes catalysis, electronics [7]. These act as humidity monitoring sensors and can also enhance the illumination efficacy of ultraviolet emission [8]. It showed its beneficiary impacts in the fields of medical fields also; as a biosensor to detect liver cancer [9], assisted in tumour detection and nano-cryosurgery [10], can use to treat heartburn and use to regenerate bone [11]. They can also act as strong antibiotic agents because of its antibacterial properties [7]. Aluminium oxide nanoparticles (Al2O3 NPs) acts as insulators and as a polishing agent because of their dielectric and erosive properties [12]. They can also be used to produce incendiaries, metal alloys, rocket fuel, and in protective coatings for ships. These find applications in cosmetic products and drug delivery systems also [6]. Titanium dioxide nanoparticles (TiO2 NPs) possess a wide range of uses which includes the production of papers, dyes, plastics, drugs. It is also used in cosmetics mainly in sunscreen because of its UV attenuation property [13]. They are also utilised to produce food products and additives [14,15]. Like TiO2 NPs, zinc oxide nanoparticles (ZnO NPs) are also used in cosmetics, paints, medicines [13]. As it has antimicrobial activity, ZnO NPs can be utilised in fabrics for odour resistance [16]. Silicon dioxide nanoparticles (SiO2 NPs) possess a porous structure, very high surface activity, and adsorption properties, which makes them ideal for developing high capacity antimicrobial agents [17]. Chromium oxide nanoparticles (Cr2O3 NPs) are used as thermal protection, a sensor for humidity, and as the
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main component for green pigment [18,19]. Copper nanoparticles (CuNPs) have an antibacterial and antifungal activity which make them favourable to use in biomedical and agricultural fields [20]. Silver nanoparticles (AgNPs) can be found in different areas such as, in catalysis, electronics, in medicinal fields as sensors, antibiotic production industries, controlling climate, wastewater treatment etc. [21]. Gold nanoparticles (AuNPs), being more stable than other metal NPs, are mainly used in different biomedical applications such as drug delivery, bioimaging, cancer detection etc. [22,23]. The release of these NPs into the environment can occur through various routes. Such as, naturally occurring NPs can come out through volcanic eruption, wildfires etc. [24]. The engineered NPs can be discharged into the aquatic and terrestrial environment through the industrial and domestic waste disposal which in turn can hamper the systems of micro-organisms in the ecosystem. Alterations in micro-organisms, in turn, can guide in the modification of the food chain and can damage the plant production system by disrupting bio-geochemical cycles mainly nitrogen fixation and assimilation [25]. The fate of the NPs released in the environment is controlled primarily by the alteration of sizes, shape and surface properties of the NPs, which in turn dominate the bioavailability of the NPs in the environment [26]. Till now, researchers did not find out the precise mechanism of NPs toxicity, but it is believed that reactive oxygen species (ROS) actually generate oxidative stress which along with membrane damage by lipid peroxidation (LPO) play a significant role in NPs toxicity towards the living organisms present in the NPs exposed environment [27]. After interaction with biological systems, nanoparticles can exert both beneficial and adverse effects on the exposed living organisms. As cells can uptake NPs, which then, in turn, can interact with the cellular organelles, it is necessary to understand the fate of the NPs inside the organism [20,28]. The controversy between the advantages and harms caused by NPs on the living organisms and ecosystems (USEPA 2007) guided researchers to find out the cytotoxic and genotoxic effects of NPs on different living organisms [29]. Because of their altered physicochemical properties, NPs can cause damage to the environment and as well as to the living organisms which come in contact with NPs [30]. Because of all these reasons, researchers in the fields of nanotoxicology and nano-genotoxicology are trying to find out the possible threats caused by the NPs and possible mechanisms of their toxicity [4,5,31]. Genotoxic and mutagenic effects are the most worrying about all other types of damages occurred by any chemicals as they can cause several health impacts on the NPs exposed living organisms and can be passed to the progenies if the mutation happens in the germ cells [32].
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Based on the organism used and the detected genetic endpoints, we can categorise the test systems into different groups. Assays involving prokaryotic systems helps to distinguish the chemicals that can cause damages in genetic materials, whereas, tests performed on eukaryotic systems allow to identify more extensive damages ranging from genetic mutation to chromosomal aberrations and aneuploidies [33]. Plants are considered as one of the major components in the ecosystem, and they can be used to find out possible mechanisms for the transport of NPs and can also act as the vital source of bioaccumulation in the food chain [27]. From the very early period to present dates plants are used as a biomarker to detect cytogenotoxic chemicals, and the obtained data can be related to higher eukaryotes [34]. Presence of different genetic endpoints in plants, such as changes in ploidy level, various aberrations in chromosomal structures, sister chromatid exchange, etc., make them suitable as biomarkers [35]. Different plants, e.g., Vicia faba, Pisum sativum, Zea mays, Crepis capillaris, Hordeum vulgare, Nicotiana tabacum, Arabidopsis thaliana, Allium cepa, Allium sativum are some of the common ones which have been used to detect a variety of environmental contaminants [7,20,36]. Some of the exclusive features of these plants such as their simple genetic structures, and high growth rate etc., made them as reliable biomarkers [20]. In this chapter, we tried to sum up about how the plant A. cepa acts as a potential biomarker to detect the toxicity of various metal and metal oxide NPs used. It will help us to understand the probable mechanisms of NPs toxicity towards higher eukaryotic systems.
2. How A. cepa acts as a model organism Levan [37] introduced A. cepa as a test organism by demonstrating the effects of colchicine on the mitotic spindle malformation. Fiskesj€ o [38] first modified the A. cepa test to make it as a biomarker for monitoring environment. His proposal involved the evaluation of both soluble and insoluble compounds in water and for composite mixtures. Mainly all the tests included the estimation of CAs, which can, in turn, identify prospective genotoxic chemicals. Ma et al. [39] suggested test alterations to estimate the mutagenicity by evaluating the induced micronuclei in the ‘F1’ region of A. cepa root tips after they were exposed to the contaminants. According to them, micronucleus (MN) is the most simple and effective endpoints to assess the cytological damages. There are some specific reasons present for which A. cepa can act as one of the major model systems for detecting environmental pollutants. These are described below: 1. First of all, the Allium test is cost effective and fast [40]. 2. They can be maintained in the laboratory easily and the results obtained are reliable [40].
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3. In comparison with other bacterial systems, they can be easily exposed to different mixtures of environmental pollutants in the laboratory [38]. 4. Since 1920, the Allium test was approved as a standard experiment for monitoring toxic effects of different chemicals on the environment [41]. 5. They have large cells containing large-sized chromosomes, which makes them suitable for checking different chromosomal aberrations easily through optical microscopy [42]. 6. As an indicator of toxicity, different morphological and cytogenetic factors can be evaluated in A. cepa root tips cells; such as root growth and structure, calculation of mitotic index (MI), micronuclei formation (MN), evaluation of chromosomal aberrations (CA) [43–45]. 7. Along with chromosomal damages, abnormalities caused in nuclei and nucleoli during mitosis can also be observed easily [42]. 8. A. cepa root tip assay is used widely because the meristematic region of root tips reproduces at a high rate, and so contains a higher percentage of dividing cells. The cells are prominent, and the chromosome number is less (2n ¼ 16) [46]. 9. Mitotic phases are clearly visible; chromosome number is stable, morphological diversity in between chromosomes are present, and rare chances of chromosomal mutilation are some of the significant characteristics which make the plant A. cepa an outstanding system for testing toxicity of pollutants [47]. 10. In addition to all of these, both International Programme on Chemical Safety (IPCS, WHO) and United Nations Environment Programme (UNEP) accepted A. cepa root tips assay as a standard assay for sensing the genotoxicity of environmental pollutant [48]. 11. Because of its sensitivity, A. cepa was one of the first nine plants used by the Gene-Tox Program of the US Environmental Protection Agency to evaluate the genotoxicity of toxicants [43]. 12. Another essential benefit of A. cepa is the presence of an oxidase enzyme system which can activate the pro-mutagens into mutagens. In other tests like Ames test, the addition of the enzyme (from rat liver) is necessary. Therefore, A. cepa can be used to detect the mutagens without adding the enzyme from exogenous sources [32].
3. Toxicity assessments and discussion Different kinds of toxicity assessments are performed on A. cepa to determine the toxicity of the desired chemicals. The types of tests are
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discussed below in details to find out how this plant can act as a model system. Generally, for all the tests, healthy and uniform sized onion bulbs are used and can be grown either using water in a glass tube [7] or autoclaved moist sand [49]. Metal and metal oxide NPs suspension is usually prepared in double distilled water. When the roots reach a length of about 2–3 cm, they can be treated with the desired NPs suspension for the required time and then these bulbs can be used for different toxicity studies. Tables 1 and 2 give a compiled data on the metal and metal oxide NPs used, and endpoints checked, respectively, by various researchers. In the next sections, the majorly used parameters are discussed in detail along with the observations from previous studies to get a better understanding.
3.1 Effects of metal/metal oxide NPs on A. cepa root growth Onion bulbs need to be put in the test solution or suspension directly for 5 days at 28 °C and under the visible light. The NPs suspension needs to be changed after every 24 h [53]. Double distilled water and copper sulphate can act as negative and positive control, respectively, [54]. After 5 days, root lengths should be measured and compare with both the control to check the effects of NPs. Pittol et al. showed that after interaction with AgNPs and TiO2 NPs suspension, onion root length was decreased significantly (P < 0.05) with Table 1 Endpoints studied with different metal NPs on Allium cepa. Sl. NPs Endpoints no. used tested Results
Reference
1
Ag
MI and CA
MI decreased, and CA increased with increasing concentration of NPS
[50]
Plant growth
Root length decreased with exposure
[51]
2
Au
MI and CA ROS species generation (O2 , H2O2, and %OH) LPO
[3] CA, ROS, and LPO increased with increasing concentration and decreased with increase in size MI decreased with increase in concentration and increased with increase in size
3
Cu
MI and CA
At low concentration, accelerated mitosis [20] and MI got increased But with increased dose and time, it acts as mito-depressive effects. MI decreased, and CA increased
Table 2 Endpoints studied with different metal oxide NPs on Allium cepa. Sl. NPs no. used Endpoints tested Results Reference
1
Al2O3 MI and CA Bio-uptake of NPs SOD estimation Surface chemical classification Cell viability decrease MI and CA DNA damage CAT, and GPX assay LPO
[6] With an increase in NPs concentration, MI decreased but CA, internalisation of NPs and SOD activity increased [52] NPs induced toxicity to the system after 24 h exposure at low concentration
2
Cr2O3 MI and CA Bio-uptake of NPs SOD estimation
The decrease in MI, increase in [36] CA and uptake of NPs and increase in SOD activity were NPs concentration dependent
3
TiO2
MI and CA Bio-uptake of NPs Total ROS estimation DNA damage by comet assay
[53] MI, CA, and comet results proved dose-dependent cytotoxicity and genotoxicity ROS and uptake increased with increase in dose concentration
Plant growth
Root length decreased with exposure
[51]
MI, CA, and MN LPO
Cellular dysfunction and CA occurred by ZnO particle MN was only found in NPs treatment but not in bulk or ionic forms
[35]
Root length
Inhibited with increased concentration of NPs
[34]
4
ZnO
DNA damage by Comet Dose dependent DNA damage [13] [34] Root length decreased with increasing dose. Excess adsorption of NPs into the root caused toxicity
5
CoO
Root length Adsorption potential
6
SiO2
CAT, GPX, APX, and No significant changes with GR activity control even at higher concentration
7
MgO
CA and MI DNA damage by Comet Oxidative stress LPO
[17]
Increase in CA and DNA [7] damage and the decrease in MI was dose-dependent. H2O2, O2 and lipid peroxidation increased with increased concentration of NPs
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respect to the negative control. Though AgNPs showed root length reduction only in higher concentrations, TiO2 NPs, on the other hand, showed a significant decrease in the length for all the concentrations used. The reason for this variation may depend on the characteristics of the NPs used [51]. The toxicity caused by AgNPs was mainly because of the higher specific surface area (SSA), which in turn created a higher degree of contact between a plant cell and the NPs. Further, the hydrophilic property of the carrier molecules increased the permeability of AgNPs through the cell wall of A. cepa [55]. For TiO2 NPs, the dominant driving force for the toxicity caused was the concentration of the suspension, the particle size of the used NPs and the reactive oxygen species generated [51]. In plants, toxic chemicals always interact first with cell wall [26]. Even at deficient concentrations, plants can uptake the NPs through different processes, e.g., rhizofiltration, precipitation, and absorption [56]. The intensity of toxicity varies depending on the exposure time to the NPs suspension, particle size, zeta potential, solubility and agglomeration properties. Root length reduction was also dependent on the concentration of the NPs suspension used. It was also observed that this reduction of the root length was related with the modification of the cell division cycle which can also be proved by mitotic index (MI) and chromosomal aberrations (CA) calculation [51].
3.2 Determination of cytotoxicity using A. cepa root tip assay Cytotoxicity of NPs can be determined using several tests such as cell viability assessments, their effects on metabolic activity, MI calculation. 3.2.1 Evaluation of cell death by Evans blue staining The decrease in cell viability can be measured using Evans blue staining. Briefly, the control and treated root tips are stained using 0.25% Evans blue, followed by washing off the excess dye in distilled water. Then the absorbed Evans blue stain by the roots is extracted using N-N-dimethylformamide, and the absorbance of the same is measured at 600 nm [57]. Evans blue staining is used to check the integrity of the cell membrane. Because of the semipermeable property of the cell membranes, living cells exclude the dye. But damaged cells are unable to eliminate the dye and so are stained blue [58]. The absorbance of the extracted Evans blue is increased with an increase in cell membrane damage. Generally, NPs treated cells express higher cytotoxicity when compared with the untreated cells (negative control). A similar observation was found out by Ghosh et al.,
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where they found out that roots treated with MWCNT (Multiwalled carbon nanotubes) showed increased cytotoxicity in comparison with the negative control [57]. 3.2.2 Estimation of viable cells using TTC 2,3,5-Triphenyl-2H-tetrazolium chloride (TTC) is used to identify the viable cells. The amount of dye absorbed by the cells can be measured from the extracted complex formazan formed in treated and control roots by taking absorbance at 490 nm. A complex (red formazan) is produced from the reduction of TTC which can be measured spectrophotometrically. The enzyme, mitochondrial dehydrogenase found only in the living cells are responsible for the reduction of the TTC and forms the red formazan. So, only the viable cells can be stained using TTC but not the dead cells. The staining of the cells is just the opposite when compared with the Evans blue staining, where the dye stains the dead cells. The absorbance of the formed red formazan will reduce with increase in damage. This trend of decrease in absorbance with cells treated with increased NPs concentration was observed previously for Al2O3 NPs and TiO2 NPs [52]. 3.2.3 Mitotic index (MI), micronucleus (MN), and chromosomal aberration (CA) The mitotic index can be explained by the ratio of the normal dividing cells to the total number of cells counted. MI is one of the important parameter used to detect the cytotoxicity of different toxicants. Significantly less MI with respect to control can specify modification caused by the chemical on the growth and development of the organisms, whereas very high MI value than control denotes the uncontrolled cell proliferation which can ultimately cause the formation of a tumour. So both of these changes in MI acts as an essential indicator for the detection of the toxicity of the components present in the environment [32]. Obstruction in the G1 stage of the cell cycle prevents the synthesis of DNA which then leads to the reduction of MI [3]. MI is calculated with the help of optical microscopy. Slides are prepared using acetocarmine squash techniques for both treated and untreated root tips. Root tips should have to be cut from two different positions. First one is the ‘M’ region, which is 5–7 mm lengthy from the tip and contains maximum numbers of mitotic cells. The second one is called ‘F1’ region which is about 1–2 mm above the ‘M’ region. After fixing both the
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‘M’ and the ‘F1’ parts using fixative (glacial acetic acid and ethanol in 1:3 ratio), each of them should have to be squashed separately on two corners of the same glass slide for analysis of MI, MN, ad CA [59]. MI and CA used to be evaluated from the ‘M’ region and MN from the ‘F1’ region [39]. Generally, 1000 cells for MI and CA and 100 cells for MN should have to be counted per slide and at least in 3–5 replicates for each condition to obtain a statistically verified data. MI used to decrease with increase in the concentration of the NPs treated when compared with the control [3,6,7,20,35,36,50,52,53]. The change in MI denotes the level of cytotoxicity of the toxicants [52]. Poles shift occurs because of the depolymerisation of spindle fibres which in turn causes different types of chromosomal abnormalities in metaphase and anaphase stage of the cell cycle [3]. CA can be defined as any changes in chromosomal structure or total numbers of the chromosomes of an organism. CA can be caused voluntarily or by the action of some genotoxic chemicals after their exposure towards the organisms. Structural modification of chromosomes can occur by several factors such as DNA breaks, whereas, numerical abnormalities generally caused by the changes in the ploidy levels. However, it is little complicated to identify exact types of CA as it requires detail knowledge of the cell cycle and the probable aberrations. Briefly, CAs like chromosomal bridge and breaks denotes clastogenic effects, and aneugenic effects are caused by the loss of the chromosomes, laggard, stickiness, multipolarity etc. [32]. CA studies from the ‘M’ region reveals different structural abnormalities in the chromosomes which occurs during cell division. Fig. 1 depicts some of the chromosomal aberrations observed by Rajeshwari et al., after treating with Al2O3 NPs [6]. Different kinds of CA found for different NPs, such as, sticky bridges, anaphase and telophase breaks, fragmented chromosomes, laggard and clumped chromosomes, multipolar anaphase, disturbed metaphase, C-mitosis, binucleated cells, diagonal anaphase treated with Al2O3 NPs [6,52], Cr2O3 NPs [36], ZnO NPs [35], AgNPs [50], AuNPs [3], TiO2 NPs [53], MgO NPs [7]. As mentioned earlier, MN is considered to be one of the easiest endpoints to detect the mutagenicity caused by any chemicals. MN formed because of damage or erroneous repair of the genetic materials, can be easily identified in daughter cells because of their similar structure with the main nucleus but with a smaller size. Size of the MN can act as an important factor to determine the clastogenic and aneugenic effects in A. cepa [32].
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Fig. 1 Chromosomal aberrations observed under optical microscope: (A1–A3) the clumped chromosome, sticky chromosome, and chromosome break upon exposure to 0.01 μg/mL NPs; (B1–B2) the sticky metaphase and disturbed metaphase treated with 0.1 μg/mL of NPs; (C1–C4) the sticky metaphase, disturbed metaphase and anaphase, chromosomal break and clumped chromosome treated with 1 μg/mL of NPs; (D1–D2) the chromosome break and sticky metaphase upon exposure to 10 μg/mL of NPs; (E1–E4) the sticky metaphase, clumped chromosome, diagonal anaphase, and diagonal and multipolar anaphase treated with 100 μg/mL of NPs. Reprinted by permission from Springer Nature: A. Rajeshwari, S. Kavitha, S.A. Alex, D. Kumar, A. Mukherjee, N. Chandrasekaran, A. Mukherjee, Cytotoxicity of aluminum oxide nanoparticles on Allium cepa root tip—effects of oxidative stress generation and biouptake, Environ. Sci. Pollut. Res. 22 (2015) 11057–11066, © Springer-Verlag Berlin Heidelberg 2015 (2015).
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Presence of a high number of interphase cells in ‘F1’ region enables the scoring of MN cells found in this region. Previous studies found out that, MN frequency will also increase with an increase in NPs concentrations. Both, acentric chromosome or the whole chromosome can be responsible for the formation of micronucleus. This MN would not get included with the normal nucleus during the cell cycle. The compound causing MN formation hence is denoted as clastogenic which can ultimately lead to cytotoxicity and genotoxicity. Some of the CAs such as chromosome breaks and bridges can lead to clastogenic effects [52].
3.3 DNA damage assessment of A. cepa roots by alkaline comet assay Alkaline comet assay or single cell gel electrophoresis (SCGE) proposed by Singh et al. [60] is very easy to perform, quick and sensitive technique to detect the magnitude of DNA damage caused by different toxicants, here in our case different NPs. This assay can be applied to test whether the NPs or other chemical is causing any DNA damages or not in the exposed organisms [13]. DNA damage was estimated by measuring the tail moment (TM) and Olive tail moment (OTM) [7]. Nowadays, comet assay has been applied mostly for the detection of genotoxicity in the environment, and it was found to be almost equally sensitive to sister chromatid exchange (SCE) and or micronucleus (MN) [61]. Previous studies showed that lower concentrations of TiO2 NPs could also cause DNA damage in A. cepa roots, but it was also observed that with an increase in concentration the degree of damage was decreased. The cause of this may be because of the agglomeration property of NPs which caused the precipitation of NPs, which leads to higher interaction between NPs particles and caused limited NPs to interact with the plant cells [61]. For Al2O3 NPs, researchers observed a dose-dependent increase in tail length percentage in A. cepa. It was proposed that metal and metal oxide nanoparticles cause DNA damage indirectly by forming reactive oxygen species (ROS) [52]. MgO NPs also showed a similar trend of dose-dependent DNA damage when compared with control [7].
4. Generation of ROS by NPs and estimation of ROS scavenging enzymes activity 4.1 Generation of intracellular ROS by NPs 4.1.1 Determination of superoxide (O2 ) Superoxide (O2 ) radical can be estimated indirectly by the reduction of nitro blue tetrazolium (NBT). Following the protocol of Kiba et al. [62],
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treated and untreated root samples should have to react with NBT for 24 h in the dark. The absorbance of the blue formazan thus formed has to take at 530 nm. Using the extinction coefficient of 12.8 L mol1 cm1, the concentration of the formazan should be calculated which indirectly estimates the amount of O2 generated and expressed as μmol of NBT reducing of fresh weight of roots. 4.1.2 Determination of hydrogen peroxide (H2O2) H2O2 production can be evaluated using standard protocol [63], and amount of H2O2 can be found out from the standard graph with known concentrations of H2O2 and is expressed as μmol gm1 of fresh weight of roots. 4.1.3 Determination of hydroxyl radical (%OH) %OH radical can be estimated using the protocol of Halliwell and Gutteridge [64]. The concentration of MDA forms is measured from its extinction coefficient (155 L mol1 cm1) and expressed in μmol gm1 of fresh weight of roots. From earlier studies, it was observed that all of the ROS (O2 , H2O2, and %OH radicals) generation increased with the increase in NPs concentrations [3,7]. NPs mainly induce toxicity towards organism by generating ROS. Different factors can cause oxidative stress, includes active redox cycling on the NPs, functionalized oxidative groups on the NPs, and cell NPs interaction [65]. Because of easy permeability of NPs, after entering into the cells, they cause mitochondrial dysfunction which ultimately leads to the oxidative stress, finally cause the death of the cells [3].
4.2 Estimation of ROS scavenging enzymes Different antioxidant enzymes activities can be estimated using standard protocol [66,67]. Catalase activity can be obtained by measuring the decrease in absorbance at 240 nm because of H2O2 consumption. Guaiacol peroxidase (GPX) activity can be assessed by measuring the absorbance of tetraguaiacol at 470 nm. Ascorbate peroxidase (APX) consumes ascorbate and hence from the decrease in the absorbance of ascorbate at 290 nm the activity of APX enzyme can be evaluated. Glutathione reductase (GR) activity can be measured from the increase in absorbance of 2-nitro-5thiobenzoic acid at 412 nm. Superoxide dismutase (SOD) activity is measured using nitro blue tetrazolium (NBT) assay where riboflavin acts as O2 generator. SOD activity can be estimated by measuring the absorbance of the resultant mixture at 560 nm [68].
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It was observed that the activity of GPS, APX and GR increased when the roots were exposed to TiO2 and SiO2 NPs along with UV-A irradiation. But catalase showed reverse action [17]. Another study showed increased activity of GPX and catalase when treated with Al2O3 NPs. This increase was to scavenge the individual ROS species generated [52]. Like other antioxidant enzymes activity, SOD activity also increased in a dose-dependent manner [6,36].
5. Estimation of cell membrane damage and intracellular uptake of NPs 5.1 Cell membrane damage by lipid peroxidation analysis Lipid peroxidation can be measured indirectly by the production of malondialdehyde (MDA). The concentration of MDA can be estimated spectrophotometrically at 532 nm and non-specific absorbance at 600 nm have to subtract from the former one [52]. Researchers found out that, MDA concentration increased with dose-dependent changes of Al2O3 NPs [52], TiO2 NPs [53,61], ZnO NPs [35], MgO NPs [7], AuNPs [3]. Increase malondialdehyde formation is the indicator of membrane lipid peroxidation. On protonation, superoxide radicals (O2 ) produce hydroperoxyl radicals (%OH, H2O2) which are responsible for the modification of the fatty acids present in the cell membrane into toxic lipid peroxides. Production of this peroxides, in turn, damages the cell membranes.
5.2 Bio-uptake of NPs NPs treated roots are dried at 60 °C for 24 h and then powdered, which is then acid digested using conc. HNO3 and then the filtered solution (0.45 μm and 3-kDa filter) is analysed using atomic absorption spectroscopy (AAS) or by inductive coupled plasma-optical emission spectroscopy (ICP-OES) to detect the amount of metal ions internalised by the organisms. Scanning electron microscopy (SEM) can also be used to show the uptake of the NPs inside the root tips cells [57]. The capacity of internalisation can be considered as a salient feature for estimating the toxicity of NPs. Earlier studies found out that amount of internalised NPs increased with increasing concentrations of NPs [6,7,36,53]. The internalisation of metal ions is one of the major causes of DNA damages, which ultimately leads to the death of the cells. Increased level
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of intracellular ROS is one of the crucial reason for cell damage [53]. Few studies reported that uptake of NPs could cause genotoxicity in the A. cepa root tips cells [7].
6. Conclusion From the above study, it became clear that the plant systems A. cepa expressed cyto-genotoxicity with increasing concentrations of all kinds of metal and metal oxides NPs used. The system showed a consistent increase in the percentage of chromosomal aberration (CA) with a decrease in the mitotic index (MI) with respect to control in a dose-dependent manner. AAS or ICP-OES study proved that uptake of the NPs inside the test systems also depends on the concentration of the NPs used. Higher concentrations of NPs exposure caused higher uptake and this, in turn, produced increased intracellular ROS. ROS, in turn, caused higher damage to the cells and led to death. To minimise the effects of the ROS, the plant species showed an increase in antioxidant enzyme activity in a dose-dependent manner. This increase in toxicity is correlated well with the increased lipid peroxidation rate. Moreover, it is also clear that the A. cepa plant is highly sensitive towards almost all types of toxicants (here metal and metal oxide NPs). Being a higher eukaryote, A. cepa has a good correlation with other test systems like mammals. Thus, this test can be used to screen environmental toxins, and the results can be used as a warning symptom for other higher test systems. This series of test assays allowed us to control the use, and disposal of different NPs to reduce environmental pollution. All the findings support that the A. cepa model system is a successful biomonitoring agent to assess the environmental risks caused by different NPs. The overall fate, transport system and route of exposure towards the environment can be easily found out with the system. Along with all these advantages, the easy availability and maintenance, its simplicity (short generation time when compared with plants species) make them the most desirable test system to monitor the genotoxicity of the chemicals.
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Toxic effects of engineered nanoparticles (metal/metal oxides) on plants using Allium cepa as a model system Barsha Roya, Suresh Palamadai Krishnana, Natarajan Chandrasekaranb, Amitava Mukherjeeb,* a
School of Biosciences and Technology, VIT, Vellore, India Centre for Nanobiotechnology, VIT, Vellore, India *Corresponding author: e-mail addresses: [email protected]; [email protected] b
Contents 1. Introduction 2. How A. cepa acts as a model organism 3. Toxicity assessments and discussion 3.1 Effects of metal/metal oxide NPs on A. cepa root growth 3.2 Determination of cytotoxicity using A. cepa root tip assay 3.3 DNA damage assessment of A. cepa roots by alkaline comet assay 4. Generation of ROS by NPs and estimation of ROS scavenging enzymes activity 4.1 Generation of intracellular ROS by NPs 4.2 Estimation of ROS scavenging enzymes 5. Estimation of cell membrane damage and intracellular uptake of NPs 5.1 Cell membrane damage by lipid peroxidation analysis 5.2 Bio-uptake of NPs 6. Conclusion References
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1. Introduction Rapid advancement in the field of nanotechnology leads to the production of different nanoparticles (NPs) of varying shapes and sizes. NPs can be defined as particles having at least one dimension <100 nm [1]. These particles exhibit entirely different physicochemical properties when in comparison with their bulk counterparts and hence can have higher toxicity than the later one [2]. Because of their small size and higher biological activity, these NPs Comprehensive Analytical Chemistry, Volume 84 ISSN 0166-526X https://doi.org/10.1016/bs.coac.2019.04.009
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can easily penetrate inside the cells and directly interact with different cellular components, which in turn causes higher toxicity [3]. These reasons lead to the testing of cytotoxicity and genotoxicity of different NPs which includes the urge of understanding the possible mechanism of the actions [4,5]. Although the toxicity of these NPs is of significant concern, we cannot deny their favourable impacts on living organisms which includes their uses in the healing of diseases, in biomedical apparatus, nanosensors, in farming techniques, etc. NPs can have beneficial effects on the plant growth and evolution, mitigation of stress [5a]. Engineered nanomaterials (ENMs) can be divided into two categories. First one is metal-based ENMs which contains quantum dots, metal and metal oxide NPs. Whereas, the second one is the carbon-based ENMs which includes different types like nanodots, nanotubes, fullerenes, nanodiamonds etc. [5b]. Among all the NPs, metal and metal oxide NPs are widely used because of its diverse applications in industrial and commercial fields. According to the statistics, metal NPs production will increase significantly by the year 2020 in comparison with the last 10 years [6]. Uses of some of these metal oxides and metal nanoparticles are as follows. Magnesium oxide nanoparticles (MgO NPs) have a wide variety of uses, which includes catalysis, electronics [7]. These act as humidity monitoring sensors and can also enhance the illumination efficacy of ultraviolet emission [8]. It showed its beneficiary impacts in the fields of medical fields also; as a biosensor to detect liver cancer [9], assisted in tumour detection and nano-cryosurgery [10], can use to treat heartburn and use to regenerate bone [11]. They can also act as strong antibiotic agents because of its antibacterial properties [7]. Aluminium oxide nanoparticles (Al2O3 NPs) acts as insulators and as a polishing agent because of their dielectric and erosive properties [12]. They can also be used to produce incendiaries, metal alloys, rocket fuel, and in protective coatings for ships. These find applications in cosmetic products and drug delivery systems also [6]. Titanium dioxide nanoparticles (TiO2 NPs) possess a wide range of uses which includes the production of papers, dyes, plastics, drugs. It is also used in cosmetics mainly in sunscreen because of its UV attenuation property [13]. They are also utilised to produce food products and additives [14,15]. Like TiO2 NPs, zinc oxide nanoparticles (ZnO NPs) are also used in cosmetics, paints, medicines [13]. As it has antimicrobial activity, ZnO NPs can be utilised in fabrics for odour resistance [16]. Silicon dioxide nanoparticles (SiO2 NPs) possess a porous structure, very high surface activity, and adsorption properties, which makes them ideal for developing high capacity antimicrobial agents [17]. Chromium oxide nanoparticles (Cr2O3 NPs) are used as thermal protection, a sensor for humidity, and as the
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main component for green pigment [18,19]. Copper nanoparticles (CuNPs) have an antibacterial and antifungal activity which make them favourable to use in biomedical and agricultural fields [20]. Silver nanoparticles (AgNPs) can be found in different areas such as, in catalysis, electronics, in medicinal fields as sensors, antibiotic production industries, controlling climate, wastewater treatment etc. [21]. Gold nanoparticles (AuNPs), being more stable than other metal NPs, are mainly used in different biomedical applications such as drug delivery, bioimaging, cancer detection etc. [22,23]. The release of these NPs into the environment can occur through various routes. Such as, naturally occurring NPs can come out through volcanic eruption, wildfires etc. [24]. The engineered NPs can be discharged into the aquatic and terrestrial environment through the industrial and domestic waste disposal which in turn can hamper the systems of micro-organisms in the ecosystem. Alterations in micro-organisms, in turn, can guide in the modification of the food chain and can damage the plant production system by disrupting bio-geochemical cycles mainly nitrogen fixation and assimilation [25]. The fate of the NPs released in the environment is controlled primarily by the alteration of sizes, shape and surface properties of the NPs, which in turn dominate the bioavailability of the NPs in the environment [26]. Till now, researchers did not find out the precise mechanism of NPs toxicity, but it is believed that reactive oxygen species (ROS) actually generate oxidative stress which along with membrane damage by lipid peroxidation (LPO) play a significant role in NPs toxicity towards the living organisms present in the NPs exposed environment [27]. After interaction with biological systems, nanoparticles can exert both beneficial and adverse effects on the exposed living organisms. As cells can uptake NPs, which then, in turn, can interact with the cellular organelles, it is necessary to understand the fate of the NPs inside the organism [20,28]. The controversy between the advantages and harms caused by NPs on the living organisms and ecosystems (USEPA 2007) guided researchers to find out the cytotoxic and genotoxic effects of NPs on different living organisms [29]. Because of their altered physicochemical properties, NPs can cause damage to the environment and as well as to the living organisms which come in contact with NPs [30]. Because of all these reasons, researchers in the fields of nanotoxicology and nano-genotoxicology are trying to find out the possible threats caused by the NPs and possible mechanisms of their toxicity [4,5,31]. Genotoxic and mutagenic effects are the most worrying about all other types of damages occurred by any chemicals as they can cause several health impacts on the NPs exposed living organisms and can be passed to the progenies if the mutation happens in the germ cells [32].
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Based on the organism used and the detected genetic endpoints, we can categorise the test systems into different groups. Assays involving prokaryotic systems helps to distinguish the chemicals that can cause damages in genetic materials, whereas, tests performed on eukaryotic systems allow to identify more extensive damages ranging from genetic mutation to chromosomal aberrations and aneuploidies [33]. Plants are considered as one of the major components in the ecosystem, and they can be used to find out possible mechanisms for the transport of NPs and can also act as the vital source of bioaccumulation in the food chain [27]. From the very early period to present dates plants are used as a biomarker to detect cytogenotoxic chemicals, and the obtained data can be related to higher eukaryotes [34]. Presence of different genetic endpoints in plants, such as changes in ploidy level, various aberrations in chromosomal structures, sister chromatid exchange, etc., make them suitable as biomarkers [35]. Different plants, e.g., Vicia faba, Pisum sativum, Zea mays, Crepis capillaris, Hordeum vulgare, Nicotiana tabacum, Arabidopsis thaliana, Allium cepa, Allium sativum are some of the common ones which have been used to detect a variety of environmental contaminants [7,20,36]. Some of the exclusive features of these plants such as their simple genetic structures, and high growth rate etc., made them as reliable biomarkers [20]. In this chapter, we tried to sum up about how the plant A. cepa acts as a potential biomarker to detect the toxicity of various metal and metal oxide NPs used. It will help us to understand the probable mechanisms of NPs toxicity towards higher eukaryotic systems.
2. How A. cepa acts as a model organism Levan [37] introduced A. cepa as a test organism by demonstrating the effects of colchicine on the mitotic spindle malformation. Fiskesj€ o [38] first modified the A. cepa test to make it as a biomarker for monitoring environment. His proposal involved the evaluation of both soluble and insoluble compounds in water and for composite mixtures. Mainly all the tests included the estimation of CAs, which can, in turn, identify prospective genotoxic chemicals. Ma et al. [39] suggested test alterations to estimate the mutagenicity by evaluating the induced micronuclei in the ‘F1’ region of A. cepa root tips after they were exposed to the contaminants. According to them, micronucleus (MN) is the most simple and effective endpoints to assess the cytological damages. There are some specific reasons present for which A. cepa can act as one of the major model systems for detecting environmental pollutants. These are described below: 1. First of all, the Allium test is cost effective and fast [40]. 2. They can be maintained in the laboratory easily and the results obtained are reliable [40].
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3. In comparison with other bacterial systems, they can be easily exposed to different mixtures of environmental pollutants in the laboratory [38]. 4. Since 1920, the Allium test was approved as a standard experiment for monitoring toxic effects of different chemicals on the environment [41]. 5. They have large cells containing large-sized chromosomes, which makes them suitable for checking different chromosomal aberrations easily through optical microscopy [42]. 6. As an indicator of toxicity, different morphological and cytogenetic factors can be evaluated in A. cepa root tips cells; such as root growth and structure, calculation of mitotic index (MI), micronuclei formation (MN), evaluation of chromosomal aberrations (CA) [43–45]. 7. Along with chromosomal damages, abnormalities caused in nuclei and nucleoli during mitosis can also be observed easily [42]. 8. A. cepa root tip assay is used widely because the meristematic region of root tips reproduces at a high rate, and so contains a higher percentage of dividing cells. The cells are prominent, and the chromosome number is less (2n ¼ 16) [46]. 9. Mitotic phases are clearly visible; chromosome number is stable, morphological diversity in between chromosomes are present, and rare chances of chromosomal mutilation are some of the significant characteristics which make the plant A. cepa an outstanding system for testing toxicity of pollutants [47]. 10. In addition to all of these, both International Programme on Chemical Safety (IPCS, WHO) and United Nations Environment Programme (UNEP) accepted A. cepa root tips assay as a standard assay for sensing the genotoxicity of environmental pollutant [48]. 11. Because of its sensitivity, A. cepa was one of the first nine plants used by the Gene-Tox Program of the US Environmental Protection Agency to evaluate the genotoxicity of toxicants [43]. 12. Another essential benefit of A. cepa is the presence of an oxidase enzyme system which can activate the pro-mutagens into mutagens. In other tests like Ames test, the addition of the enzyme (from rat liver) is necessary. Therefore, A. cepa can be used to detect the mutagens without adding the enzyme from exogenous sources [32].
3. Toxicity assessments and discussion Different kinds of toxicity assessments are performed on A. cepa to determine the toxicity of the desired chemicals. The types of tests are
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discussed below in details to find out how this plant can act as a model system. Generally, for all the tests, healthy and uniform sized onion bulbs are used and can be grown either using water in a glass tube [7] or autoclaved moist sand [49]. Metal and metal oxide NPs suspension is usually prepared in double distilled water. When the roots reach a length of about 2–3 cm, they can be treated with the desired NPs suspension for the required time and then these bulbs can be used for different toxicity studies. Tables 1 and 2 give a compiled data on the metal and metal oxide NPs used, and endpoints checked, respectively, by various researchers. In the next sections, the majorly used parameters are discussed in detail along with the observations from previous studies to get a better understanding.
3.1 Effects of metal/metal oxide NPs on A. cepa root growth Onion bulbs need to be put in the test solution or suspension directly for 5 days at 28 °C and under the visible light. The NPs suspension needs to be changed after every 24 h [53]. Double distilled water and copper sulphate can act as negative and positive control, respectively, [54]. After 5 days, root lengths should be measured and compare with both the control to check the effects of NPs. Pittol et al. showed that after interaction with AgNPs and TiO2 NPs suspension, onion root length was decreased significantly (P < 0.05) with Table 1 Endpoints studied with different metal NPs on Allium cepa. Sl. NPs Endpoints no. used tested Results
Reference
1
Ag
MI and CA
MI decreased, and CA increased with increasing concentration of NPS
[50]
Plant growth
Root length decreased with exposure
[51]
2
Au
MI and CA ROS species generation (O2 , H2O2, and %OH) LPO
[3] CA, ROS, and LPO increased with increasing concentration and decreased with increase in size MI decreased with increase in concentration and increased with increase in size
3
Cu
MI and CA
At low concentration, accelerated mitosis [20] and MI got increased But with increased dose and time, it acts as mito-depressive effects. MI decreased, and CA increased
Table 2 Endpoints studied with different metal oxide NPs on Allium cepa. Sl. NPs no. used Endpoints tested Results Reference
1
Al2O3 MI and CA Bio-uptake of NPs SOD estimation Surface chemical classification Cell viability decrease MI and CA DNA damage CAT, and GPX assay LPO
[6] With an increase in NPs concentration, MI decreased but CA, internalisation of NPs and SOD activity increased [52] NPs induced toxicity to the system after 24 h exposure at low concentration
2
Cr2O3 MI and CA Bio-uptake of NPs SOD estimation
The decrease in MI, increase in [36] CA and uptake of NPs and increase in SOD activity were NPs concentration dependent
3
TiO2
MI and CA Bio-uptake of NPs Total ROS estimation DNA damage by comet assay
[53] MI, CA, and comet results proved dose-dependent cytotoxicity and genotoxicity ROS and uptake increased with increase in dose concentration
Plant growth
Root length decreased with exposure
[51]
MI, CA, and MN LPO
Cellular dysfunction and CA occurred by ZnO particle MN was only found in NPs treatment but not in bulk or ionic forms
[35]
Root length
Inhibited with increased concentration of NPs
[34]
4
ZnO
DNA damage by Comet Dose dependent DNA damage [13] [34] Root length decreased with increasing dose. Excess adsorption of NPs into the root caused toxicity
5
CoO
Root length Adsorption potential
6
SiO2
CAT, GPX, APX, and No significant changes with GR activity control even at higher concentration
7
MgO
CA and MI DNA damage by Comet Oxidative stress LPO
[17]
Increase in CA and DNA [7] damage and the decrease in MI was dose-dependent. H2O2, O2 and lipid peroxidation increased with increased concentration of NPs
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respect to the negative control. Though AgNPs showed root length reduction only in higher concentrations, TiO2 NPs, on the other hand, showed a significant decrease in the length for all the concentrations used. The reason for this variation may depend on the characteristics of the NPs used [51]. The toxicity caused by AgNPs was mainly because of the higher specific surface area (SSA), which in turn created a higher degree of contact between a plant cell and the NPs. Further, the hydrophilic property of the carrier molecules increased the permeability of AgNPs through the cell wall of A. cepa [55]. For TiO2 NPs, the dominant driving force for the toxicity caused was the concentration of the suspension, the particle size of the used NPs and the reactive oxygen species generated [51]. In plants, toxic chemicals always interact first with cell wall [26]. Even at deficient concentrations, plants can uptake the NPs through different processes, e.g., rhizofiltration, precipitation, and absorption [56]. The intensity of toxicity varies depending on the exposure time to the NPs suspension, particle size, zeta potential, solubility and agglomeration properties. Root length reduction was also dependent on the concentration of the NPs suspension used. It was also observed that this reduction of the root length was related with the modification of the cell division cycle which can also be proved by mitotic index (MI) and chromosomal aberrations (CA) calculation [51].
3.2 Determination of cytotoxicity using A. cepa root tip assay Cytotoxicity of NPs can be determined using several tests such as cell viability assessments, their effects on metabolic activity, MI calculation. 3.2.1 Evaluation of cell death by Evans blue staining The decrease in cell viability can be measured using Evans blue staining. Briefly, the control and treated root tips are stained using 0.25% Evans blue, followed by washing off the excess dye in distilled water. Then the absorbed Evans blue stain by the roots is extracted using N-N-dimethylformamide, and the absorbance of the same is measured at 600 nm [57]. Evans blue staining is used to check the integrity of the cell membrane. Because of the semipermeable property of the cell membranes, living cells exclude the dye. But damaged cells are unable to eliminate the dye and so are stained blue [58]. The absorbance of the extracted Evans blue is increased with an increase in cell membrane damage. Generally, NPs treated cells express higher cytotoxicity when compared with the untreated cells (negative control). A similar observation was found out by Ghosh et al.,
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where they found out that roots treated with MWCNT (Multiwalled carbon nanotubes) showed increased cytotoxicity in comparison with the negative control [57]. 3.2.2 Estimation of viable cells using TTC 2,3,5-Triphenyl-2H-tetrazolium chloride (TTC) is used to identify the viable cells. The amount of dye absorbed by the cells can be measured from the extracted complex formazan formed in treated and control roots by taking absorbance at 490 nm. A complex (red formazan) is produced from the reduction of TTC which can be measured spectrophotometrically. The enzyme, mitochondrial dehydrogenase found only in the living cells are responsible for the reduction of the TTC and forms the red formazan. So, only the viable cells can be stained using TTC but not the dead cells. The staining of the cells is just the opposite when compared with the Evans blue staining, where the dye stains the dead cells. The absorbance of the formed red formazan will reduce with increase in damage. This trend of decrease in absorbance with cells treated with increased NPs concentration was observed previously for Al2O3 NPs and TiO2 NPs [52]. 3.2.3 Mitotic index (MI), micronucleus (MN), and chromosomal aberration (CA) The mitotic index can be explained by the ratio of the normal dividing cells to the total number of cells counted. MI is one of the important parameter used to detect the cytotoxicity of different toxicants. Significantly less MI with respect to control can specify modification caused by the chemical on the growth and development of the organisms, whereas very high MI value than control denotes the uncontrolled cell proliferation which can ultimately cause the formation of a tumour. So both of these changes in MI acts as an essential indicator for the detection of the toxicity of the components present in the environment [32]. Obstruction in the G1 stage of the cell cycle prevents the synthesis of DNA which then leads to the reduction of MI [3]. MI is calculated with the help of optical microscopy. Slides are prepared using acetocarmine squash techniques for both treated and untreated root tips. Root tips should have to be cut from two different positions. First one is the ‘M’ region, which is 5–7 mm lengthy from the tip and contains maximum numbers of mitotic cells. The second one is called ‘F1’ region which is about 1–2 mm above the ‘M’ region. After fixing both the
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‘M’ and the ‘F1’ parts using fixative (glacial acetic acid and ethanol in 1:3 ratio), each of them should have to be squashed separately on two corners of the same glass slide for analysis of MI, MN, ad CA [59]. MI and CA used to be evaluated from the ‘M’ region and MN from the ‘F1’ region [39]. Generally, 1000 cells for MI and CA and 100 cells for MN should have to be counted per slide and at least in 3–5 replicates for each condition to obtain a statistically verified data. MI used to decrease with increase in the concentration of the NPs treated when compared with the control [3,6,7,20,35,36,50,52,53]. The change in MI denotes the level of cytotoxicity of the toxicants [52]. Poles shift occurs because of the depolymerisation of spindle fibres which in turn causes different types of chromosomal abnormalities in metaphase and anaphase stage of the cell cycle [3]. CA can be defined as any changes in chromosomal structure or total numbers of the chromosomes of an organism. CA can be caused voluntarily or by the action of some genotoxic chemicals after their exposure towards the organisms. Structural modification of chromosomes can occur by several factors such as DNA breaks, whereas, numerical abnormalities generally caused by the changes in the ploidy levels. However, it is little complicated to identify exact types of CA as it requires detail knowledge of the cell cycle and the probable aberrations. Briefly, CAs like chromosomal bridge and breaks denotes clastogenic effects, and aneugenic effects are caused by the loss of the chromosomes, laggard, stickiness, multipolarity etc. [32]. CA studies from the ‘M’ region reveals different structural abnormalities in the chromosomes which occurs during cell division. Fig. 1 depicts some of the chromosomal aberrations observed by Rajeshwari et al., after treating with Al2O3 NPs [6]. Different kinds of CA found for different NPs, such as, sticky bridges, anaphase and telophase breaks, fragmented chromosomes, laggard and clumped chromosomes, multipolar anaphase, disturbed metaphase, C-mitosis, binucleated cells, diagonal anaphase treated with Al2O3 NPs [6,52], Cr2O3 NPs [36], ZnO NPs [35], AgNPs [50], AuNPs [3], TiO2 NPs [53], MgO NPs [7]. As mentioned earlier, MN is considered to be one of the easiest endpoints to detect the mutagenicity caused by any chemicals. MN formed because of damage or erroneous repair of the genetic materials, can be easily identified in daughter cells because of their similar structure with the main nucleus but with a smaller size. Size of the MN can act as an important factor to determine the clastogenic and aneugenic effects in A. cepa [32].
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Fig. 1 Chromosomal aberrations observed under optical microscope: (A1–A3) the clumped chromosome, sticky chromosome, and chromosome break upon exposure to 0.01 μg/mL NPs; (B1–B2) the sticky metaphase and disturbed metaphase treated with 0.1 μg/mL of NPs; (C1–C4) the sticky metaphase, disturbed metaphase and anaphase, chromosomal break and clumped chromosome treated with 1 μg/mL of NPs; (D1–D2) the chromosome break and sticky metaphase upon exposure to 10 μg/mL of NPs; (E1–E4) the sticky metaphase, clumped chromosome, diagonal anaphase, and diagonal and multipolar anaphase treated with 100 μg/mL of NPs. Reprinted by permission from Springer Nature: A. Rajeshwari, S. Kavitha, S.A. Alex, D. Kumar, A. Mukherjee, N. Chandrasekaran, A. Mukherjee, Cytotoxicity of aluminum oxide nanoparticles on Allium cepa root tip—effects of oxidative stress generation and biouptake, Environ. Sci. Pollut. Res. 22 (2015) 11057–11066, © Springer-Verlag Berlin Heidelberg 2015 (2015).
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Presence of a high number of interphase cells in ‘F1’ region enables the scoring of MN cells found in this region. Previous studies found out that, MN frequency will also increase with an increase in NPs concentrations. Both, acentric chromosome or the whole chromosome can be responsible for the formation of micronucleus. This MN would not get included with the normal nucleus during the cell cycle. The compound causing MN formation hence is denoted as clastogenic which can ultimately lead to cytotoxicity and genotoxicity. Some of the CAs such as chromosome breaks and bridges can lead to clastogenic effects [52].
3.3 DNA damage assessment of A. cepa roots by alkaline comet assay Alkaline comet assay or single cell gel electrophoresis (SCGE) proposed by Singh et al. [60] is very easy to perform, quick and sensitive technique to detect the magnitude of DNA damage caused by different toxicants, here in our case different NPs. This assay can be applied to test whether the NPs or other chemical is causing any DNA damages or not in the exposed organisms [13]. DNA damage was estimated by measuring the tail moment (TM) and Olive tail moment (OTM) [7]. Nowadays, comet assay has been applied mostly for the detection of genotoxicity in the environment, and it was found to be almost equally sensitive to sister chromatid exchange (SCE) and or micronucleus (MN) [61]. Previous studies showed that lower concentrations of TiO2 NPs could also cause DNA damage in A. cepa roots, but it was also observed that with an increase in concentration the degree of damage was decreased. The cause of this may be because of the agglomeration property of NPs which caused the precipitation of NPs, which leads to higher interaction between NPs particles and caused limited NPs to interact with the plant cells [61]. For Al2O3 NPs, researchers observed a dose-dependent increase in tail length percentage in A. cepa. It was proposed that metal and metal oxide nanoparticles cause DNA damage indirectly by forming reactive oxygen species (ROS) [52]. MgO NPs also showed a similar trend of dose-dependent DNA damage when compared with control [7].
4. Generation of ROS by NPs and estimation of ROS scavenging enzymes activity 4.1 Generation of intracellular ROS by NPs 4.1.1 Determination of superoxide (O2 ) Superoxide (O2 ) radical can be estimated indirectly by the reduction of nitro blue tetrazolium (NBT). Following the protocol of Kiba et al. [62],
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treated and untreated root samples should have to react with NBT for 24 h in the dark. The absorbance of the blue formazan thus formed has to take at 530 nm. Using the extinction coefficient of 12.8 L mol1 cm1, the concentration of the formazan should be calculated which indirectly estimates the amount of O2 generated and expressed as μmol of NBT reducing of fresh weight of roots. 4.1.2 Determination of hydrogen peroxide (H2O2) H2O2 production can be evaluated using standard protocol [63], and amount of H2O2 can be found out from the standard graph with known concentrations of H2O2 and is expressed as μmol gm1 of fresh weight of roots. 4.1.3 Determination of hydroxyl radical (%OH) %OH radical can be estimated using the protocol of Halliwell and Gutteridge [64]. The concentration of MDA forms is measured from its extinction coefficient (155 L mol1 cm1) and expressed in μmol gm1 of fresh weight of roots. From earlier studies, it was observed that all of the ROS (O2 , H2O2, and %OH radicals) generation increased with the increase in NPs concentrations [3,7]. NPs mainly induce toxicity towards organism by generating ROS. Different factors can cause oxidative stress, includes active redox cycling on the NPs, functionalized oxidative groups on the NPs, and cell NPs interaction [65]. Because of easy permeability of NPs, after entering into the cells, they cause mitochondrial dysfunction which ultimately leads to the oxidative stress, finally cause the death of the cells [3].
4.2 Estimation of ROS scavenging enzymes Different antioxidant enzymes activities can be estimated using standard protocol [66,67]. Catalase activity can be obtained by measuring the decrease in absorbance at 240 nm because of H2O2 consumption. Guaiacol peroxidase (GPX) activity can be assessed by measuring the absorbance of tetraguaiacol at 470 nm. Ascorbate peroxidase (APX) consumes ascorbate and hence from the decrease in the absorbance of ascorbate at 290 nm the activity of APX enzyme can be evaluated. Glutathione reductase (GR) activity can be measured from the increase in absorbance of 2-nitro-5thiobenzoic acid at 412 nm. Superoxide dismutase (SOD) activity is measured using nitro blue tetrazolium (NBT) assay where riboflavin acts as O2 generator. SOD activity can be estimated by measuring the absorbance of the resultant mixture at 560 nm [68].
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It was observed that the activity of GPS, APX and GR increased when the roots were exposed to TiO2 and SiO2 NPs along with UV-A irradiation. But catalase showed reverse action [17]. Another study showed increased activity of GPX and catalase when treated with Al2O3 NPs. This increase was to scavenge the individual ROS species generated [52]. Like other antioxidant enzymes activity, SOD activity also increased in a dose-dependent manner [6,36].
5. Estimation of cell membrane damage and intracellular uptake of NPs 5.1 Cell membrane damage by lipid peroxidation analysis Lipid peroxidation can be measured indirectly by the production of malondialdehyde (MDA). The concentration of MDA can be estimated spectrophotometrically at 532 nm and non-specific absorbance at 600 nm have to subtract from the former one [52]. Researchers found out that, MDA concentration increased with dose-dependent changes of Al2O3 NPs [52], TiO2 NPs [53,61], ZnO NPs [35], MgO NPs [7], AuNPs [3]. Increase malondialdehyde formation is the indicator of membrane lipid peroxidation. On protonation, superoxide radicals (O2 ) produce hydroperoxyl radicals (%OH, H2O2) which are responsible for the modification of the fatty acids present in the cell membrane into toxic lipid peroxides. Production of this peroxides, in turn, damages the cell membranes.
5.2 Bio-uptake of NPs NPs treated roots are dried at 60 °C for 24 h and then powdered, which is then acid digested using conc. HNO3 and then the filtered solution (0.45 μm and 3-kDa filter) is analysed using atomic absorption spectroscopy (AAS) or by inductive coupled plasma-optical emission spectroscopy (ICP-OES) to detect the amount of metal ions internalised by the organisms. Scanning electron microscopy (SEM) can also be used to show the uptake of the NPs inside the root tips cells [57]. The capacity of internalisation can be considered as a salient feature for estimating the toxicity of NPs. Earlier studies found out that amount of internalised NPs increased with increasing concentrations of NPs [6,7,36,53]. The internalisation of metal ions is one of the major causes of DNA damages, which ultimately leads to the death of the cells. Increased level
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of intracellular ROS is one of the crucial reason for cell damage [53]. Few studies reported that uptake of NPs could cause genotoxicity in the A. cepa root tips cells [7].
6. Conclusion From the above study, it became clear that the plant systems A. cepa expressed cyto-genotoxicity with increasing concentrations of all kinds of metal and metal oxides NPs used. The system showed a consistent increase in the percentage of chromosomal aberration (CA) with a decrease in the mitotic index (MI) with respect to control in a dose-dependent manner. AAS or ICP-OES study proved that uptake of the NPs inside the test systems also depends on the concentration of the NPs used. Higher concentrations of NPs exposure caused higher uptake and this, in turn, produced increased intracellular ROS. ROS, in turn, caused higher damage to the cells and led to death. To minimise the effects of the ROS, the plant species showed an increase in antioxidant enzyme activity in a dose-dependent manner. This increase in toxicity is correlated well with the increased lipid peroxidation rate. Moreover, it is also clear that the A. cepa plant is highly sensitive towards almost all types of toxicants (here metal and metal oxide NPs). Being a higher eukaryote, A. cepa has a good correlation with other test systems like mammals. Thus, this test can be used to screen environmental toxins, and the results can be used as a warning symptom for other higher test systems. This series of test assays allowed us to control the use, and disposal of different NPs to reduce environmental pollution. All the findings support that the A. cepa model system is a successful biomonitoring agent to assess the environmental risks caused by different NPs. The overall fate, transport system and route of exposure towards the environment can be easily found out with the system. Along with all these advantages, the easy availability and maintenance, its simplicity (short generation time when compared with plants species) make them the most desirable test system to monitor the genotoxicity of the chemicals.
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