JES-01180; No of Pages 13

J O U RN A L OF E N V I RO N ME N TA L S CI EN CE S X X (2 0 1 7 ) XX X–XXX

Available online at www.sciencedirect.com

ScienceDirect
www.elsevier.com/locate/jes

1Q4 Allium cepa root tip assay in assessment of toxicity of

2 magnesium oxide nanoparticles and microparticles

F
Bhanuramya Mangalampalli, Naresh Dumala, Paramjit Grover⁎

O
Q63Q5

4 Toxicology Unit, Pharmacology and Toxicology Division, CSIR - Indian Institute of Chemical Technology, Hyderabad, Telangana 500007, India.

O
5 E-mail: bhanu.iict@gmail.com
6 Academy of Scientific and Innovative Research, CSIR - Indian Institute of Chemical Technology, Hyderabad, Telangana 500007, India
7

R
10
9 AR TIC LE I NFO ABSTR ACT

P
11 Article history: Allium cepa bioassay had been used from decades for the assessment of toxicants and their 16
12 Received 18 November 2016 harmful effects on environment as well as human health. Magnesium oxide (MgO) particles are 17
D
13 Revised 10 April 2017 being utilized in different fields. However, reports on the adverse effects of MgO nanoparticles on 18
14 Accepted 5 May 2017 the environment and mankind are scarce. Hence, the toxicity of MgO particles is of concern 19
E
15 Available online xxxx because of their increased utilization. In the current study, A. cepa was used as an indicator to 20
assess the toxicological efficiency of MgO nano- and microparticles (NPs and MPs) at a range of 21
35 Keywords:
T

exposure concentrations (12.5, 25, 50, and 100 μg/mL). The toxicity was evaluated by using 22
36 Magnesium oxide nanoparticles various bioassays on A. cepa root tip cells such as comet assay, oxidative stress and their uptake/ 23
37
C

Allium cepa root tip assay internalization profile. Results indicated a dose dependent increase in chromosomal aberrations 24
38 Histochemical staining and decrease in mitotic index (MI) when compared to control cells and the effect was more 25
39 Oxidative stress significant for NPs than MPs (at p < 0.05). Comet analysis revealed that the DNA damage in terms 26
E

40 Internalization of nanoparticles of percent tail DNA ranged from 6.8–30.1 over 12.5–100 μg/mL concentrations of MgO NPs and 27
41 was found to be significant at the exposed concentrations. A significant increase in generation 28
R

of hydrogen peroxide and superoxide radicals was observed in accordance with the lipid 29
peroxidation profile in both MgO NPs and MPs treated plants when compared with control. In 30
R

conclusion, this investigation revealed that MgO NPs exposure exhibited greater toxicity on A. 31
cepa than MPs. 32
O

© 2017 The Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences. 33
Published by Elsevier B.V. 34
43
44
42
45
C

these NPs have the ability to enter the cell and interact with the 54
47
46 Introduction
N

intracellular structures and may lead to toxicity (Rajeshwari 55

et al., 2016). Some NPs have shown adverse effects on plant 56
48 The recent advances in the field of nanotechnology have biosystems (Rico et al., 2011). Among the plant species, the most 57
U

49 resulted in a wide range of applications. Nanoparticles (NPs) common ones used to assess environmental contamination 58
50 are defined as particles less than 100 nm in size at least in any are Allium cepa, Vicia faba, Zea mays, Nicotiana tabacum, Crepis 59
51 one dimension. The NPs differ in their physical characteristics capillaris and Hordeum vulgare. Within these species, A. cepa 60
52 and could be more toxic than their bulk counterparts (onion) root tip bioassay is considered as a simple and reliable 61
53 (Oberdörster et al., 2005). Due to their exclusive properties, test model to assess the genotoxic potential of chemicals due to 62

Q7 ⁎ Corresponding author. E-mails: paramgrover@gmail.com, grover@iict.res.in (P. Grover).

http://dx.doi.org/10.1016/j.jes.2017.05.012
1001-0742/© 2017 The Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences. Published by Elsevier B.V.

Please cite this article as: Mangalampalli, B., et al., Allium cepa root tip assay in assessment of toxicity of magnesium oxide
nanoparticles and microparticles, J. Environ. Sci. (2017), http://dx.doi.org/10.1016/j.jes.2017.05.012
 2 J O U RN A L OF E N V I RO N ME N TA L S CIE N CE S X X (2 0 1 7 ) XXX –XXX

63 its characteristic proliferation rate, high percent of dividing

64 cells and relatively prominent cells with lesser number of 1. Materials and methods 124
123

65 large sized monocentric chromosomes (2n = 16) that stain

1.1. Chemicals 125
66 well (Fiskesjo, 1997). Further, they have easily distinguishable
67 genetic endpoints, such as chromosomal aberrations (CAs),
MgO NPs (<50 nm particle size (Brunauer–Emmett–Teller 126
68 changes in ploidy and sister chromatid exchanges (Kumari et
(BET)), CAS no. 1309-48-4) were procured from Sigma-Aldrich 127
Q10
69 al., 2011). CAs have been determined by using A. cepa test since
Co. Ltd. (St Louis, MO, USA). MgO MPs (fine powder, 97%, 128
70 1920 (Satapathy and Swamy, 2013). In addition, United Nations
GRM1031, CAS no. 1309-48-4) (according to the manufacture's 129
71 Environment Programme (UNEP) (Grant, 1999) and the Interna-
data sheet), acetocarmine, acridine orange (AO), nitro blue 130
72 tional Programme on Chemical Safety (IPCS) certified A. cepa
tetrazolium (NBT), riboflavin, 3, 3′-diaminobenzidine (DAB), 131
73 root tip assay as an effective and established plant bioassay for
ethylene diamine tetra acetic acid (EDTA), hydrochloric acid 132
74 the in situ monitoring of environmental contaminants, sub-

F
(HCl), dipotassium hydrogen phosphate (K2HPO4), potassi- 133
75 stances and chemicals (Cabrera and Rodriguez, 1999).
um dihydrogen phosphate (KH2PO4), tris (hydroxymethyl) 134
76 Magnesium oxide (MgO) particles have a variety of

O
aminomethane hydrochloric acid (Tris–HCl), trichloroacetic 135
77 applications. MgO NPs are being utilized in different fields,
acid (TCA), thiobarbituric acid (TBA), glacial acetic acid (GAA), 136
78 such as catalysis, ceramics, electronics (Li et al., 2005;
ethanol and other chemicals of analytical grade were purchased 137

O
79 Yeheskel et al., 2005). They are also used as sensors for
from HiMedia Laboratories Pvt. Ltd., Mumbai, India. 138
80 humidity and in enhancing luminescence efficiency for
81 ultraviolet emission (Mahmoud et al., 2016). MgO NPs are

R
82 proving to be impressive in biomedical applications as 1.2. Characterization of MgO NPs 139
83 biosensors for liver cancer immunoassay (Lei et al., 2012), an

P
84 aid in nano-cryosurgery for tumor treatment (Di et al., 2012), The NPs and MPs were characterized using transmission 140
85 destructive sorbent for toxins (Bakardjieva et al., 2004). MgO NPs electron microscopy (TEM), field-emission scanning electron 141
86
Q8 are also being utilized as antibacterial agent (Tang and Lv, 2014), microscope (FESEM), BET, dynamic light scattering (DLS) and
D 142
87 for the relief of heartburn and regeneration of bone (Bertinetti et laser Doppler velocimetry (LDV) to evaluate the material size, 143
88 al., 2009). However, the adverse effects of MgO NPs on the shape, surface area, size distribution, state of dispersion and 144
145
E
89 environment and mankind should also be of concern because of zeta potential in Milli-Q water. Characterization of NPs and
90 the increased utilization of MgO particles. It is essential to MPs was performed to assess the size and morphology using a 146
91 TEM (JEM-2100, JEOL, Japan). For the size measurement, 100 147
T

assess the hazards associated with these NPs before they are
92 used for biological applications. Few studies are available particles were calculated from random fields of view and 148
images showing the general morphology of the particles. 149
C

93 which have reported that these MgO NPs exhibited cytotox-

94 icity toward human umbilical vein endothelial cells (Ge et al., Morphology and surface analysis of MgO NPs and MPs were 150
95 2011), on human cardiac microvascular endothelial cells (Sun evaluated using FESEM, JEOL 7610F instrument. The specific 151
E

96 et al., 2011) and in human astrocytoma U87 cells (Lai et al., surface area of the MgO NPs and MPs was measured by the 152
97 2008). BET method by using the Surface Area and Pore Size Analyzer 153
R

98 A detailed literature survey revealed that toxicological SA 3100 plus (Beckman Coulter, USA). The mean hydrody- 154
99 analysis of MgO NPs and microparticles (MPs) have not been namic size of the NPs and polydispersity index (PdI) was 155
R

100 reported till date using the A. cepa bioassay, therefore we measured through DLS and LDV using a Malvern Zetasizer 156
101 conducted a series of tests to investigate the impact of MgO Nano-ZS (Malvern Instruments, UK). The PdI was used to 157
measure the size ranges present in the solution and scale 158
O

102 NPs and MPs on root meristematic cells of onion. Systematic

103 characterization of nanomaterials (NMs) is essential in order ranges from 0 to 1, where 0 indicates monodispersion and 1 159
104 to elucidate their potential toxicity to biological systems indicates polydispersion state of particles. 160
C

105 (Murdock et al., 2008). Hence, in the current study character-

106 ization of MgO NPs and MPs was carried out. Light microscopy 1.3. Test model and treatment 161
N

107 and fluorescence microscopy analyses were used to demon-

108 strate the morphological features and cytological changes Healthy and equal sized bulbs (30–40 g weight) of common 162
109 onion were chosen and series of bulbs were grown in glass 163
U

in different phases of mitosis of A. cepa root tip cells upon

110 treatment with these MgO NPs and MPs. The comet assay beakers at room temperature (27 ± 2)°C. Water was renewed 164
111 and CAs assay were utilized in this study to depict the for each onion bulb every day. MgO NPs and MPs were directly 165
112 genotoxicity of MgO NPs and MPs. The comet assay is a suspended in the Milli-Q water and dispersed by sonication 166
113 versatile, simple and reliable method to study the toxicity of a using a probe sonicator (UP100H, Hielscher Ultrasonics GmbH, 167
114
Q9 broad range of compounds and is capable of measuring DNA Teltow, Germany) for 15 min at 90% amplitude to produce 168
115 damage in almost all organisms and cells (Olive and Banáth, four different working concentrations of 12.5, 25, 50 and 169
116 2006). To elucidate the biochemical alterations caused by 100 μg/mL. After the roots attained a length of 2–3 cm, they 170
117 MgO NPs and MPs, extra-cellular generation of reactive were treated with the prepared concentrations of MgO NP and 171
118 oxygen species (ROS) and lipid peroxidation was included in MP suspensions for a sampling period of 4 hr. Five replicates 172
119 this study. Biodistribution studies of NMs are essential to were made for each concentration to ensure that our results 173
120 understand the amount of NPs that internalized into the root were reproducible. The slides were coded to avoid bias 174
121 cells (Rajeshwari et al., 2015). Therefore, uptake of MgO NPs (Rajeshwari et al., 2015). In current study no human subjects 175
122 and MPs was also assessed. or experimental animals were used. 176

Please cite this article as: Mangalampalli, B., et al., Allium cepa root tip assay in assessment of toxicity of magnesium oxide
nanoparticles and microparticles, J. Environ. Sci. (2017), http://dx.doi.org/10.1016/j.jes.2017.05.012
 J O U RN A L OF E N V I RO N ME N TA L S CI EN CE S X X (2 0 1 7 ) XX X–XXX 3

177 1.4. Microscopic analysis immersed in chilled lysis buffer (2.5 mol/L NaCl, 0.1 mol/L Na2 233
EDTA, 0.2 mol/L NaOH, 1% Triton X-100, 10% DMSO, pH 10.0) 234
178 Microscopic slides were prepared by following a standard for 4 hr at 4°C. The slides were placed in a gel electrophoresis 235
179 protocol of acetocarmine squash technique (Borboa and tank containing electrophoresis buffer (10 mol/L NaOH, 236
180 Torre, 1996; Kumari et al., 2011) with slight modifications. 200 mmol/L Na2 EDTA, pH > 13.0). To facilitate DNA unwind- 237
181 Briefly, after treatment, the roots were washed thoroughly ing, the slides were presoaked for 20 min in electrophoresis 238
182 with tap water. The root tips from each bulb were removed buffer. Electrophoresis was then performed at 25 V adjusted at 239
183 and fixed in Farmer's fluid (GAA: ethanol, 1:3, V/V) for 12 hr. 300 mA for 15 min. The slides were neutralized with 0.4 mol/L 240
184 For chromosomal analysis, the root tips were hydrolyzed in Tris buffer (pH 7.5, for 5 min) and fixed with absolute methanol 241
185 1 mol/L HCl for 5 min and transferred to a watch glass for 5 min. The coded slides were scored after staining with 242
186 containing acetocarmine and 1 mol/L HCl (9:1, V/V). They ethidium bromide (20 μg/mL) using a fluorescence microscope 243
187 were then heated intermittently for 5–10 min, covered and (Olympus, Shinjuku-ku, Tokyo, Japan) with an excitation blue 244

F
188 kept aside for 15–20 min. The tip (2–3 mm) of the root was (488 nm) filter and emission yellow (515 nm) filter at 400× 245
189 then cut with sharp blade and placed on a glass slide in a drop magnification. 246

O
190 of 45% GAA, to remove excess/unbound stain and a squash Quantification of DNA breakage was carried out by using 247
191 was prepared on slides and sealed with dibutylphathalate CASP software version 1.2.2 (Comet Assay Software, CaspLab) 248

O
192 xylene (DPX). The slides were observed under a light micro- to calculate the amount of DNA damage and expressed as a 249
193 scope (Leica-DM-2500, Leica microsystems Inc., USA) and percentage of DNA in the comet tail. A minimum of five roots 250
194 analyzed at 1000× magnification for cytological changes. The were taken and in total 250 nuclei were scored per treatment 251

R
195 mitotic index (MI) was calculated as the number of dividing (Kaur et al., 2014). 252
196 cells per 1000 observed cells (Fiskesjo, 1997).

P
1.6. Histochemical staining for hydrogen peroxide 253
197 1.4.1. Fluorescence microscopy and superoxide 254
198 The onion root tips were treated with the MgO NPs and MPs at D
199 different concentrations for a sampling time of 4 hr. Two to After treatment, the presence of hydrogen peroxide (H2O2) 255
200 three washed root tips were then taken and incubated for and superoxide (O−2U) was detected through histochemical 256
Q14
E
201 2 min in 1 mol/L HCl at room temperature. Tips were then staining as per the protocol of Thordal-Christensen et al. 257
Q11 stained with 20 μL of nuclear specific dye AO (15 μg/mL in PBS)
202 (1997) and Daudi et al. (2012), respectively, with few changes. 258
T

203 and incubated for 5 min under dark conditions to avoid photo The roots were treated with different concentrations of MgO 259
204 bleaching of stain for visualization of CAs. The root tips were NPs and MPs for 4 hr and then stained with 1% (W/V) 260
C

205 resuspended with Milli-Q water. This process was repeated 3-diaminobenzinidine (DAB; pH 3.8) for 1 hr and subsequently 261
206 two to three times in order to remove excess dye. The root tips rinsed with deionized water to detect the presence of H2O2. In 262
207 were placed on to a glass slide and covered with a cover slip. case of superoxide, 2 mmol/L NBT in 20 mmol/L PBS (pH 6.8) 263
E

208 Care was taken to avoid the formation of air bubbles while was used to stain the roots for 10 min and subsequently 264
209 placing the cover slip. Finally, cover slip was pressed firmly washed with deionized water. Both DAB and NBT stained root 265
R

210 with the help of thumb to prepare a uniform squash of the tips were observed for the presence of H2O2 and superoxide 266
211 root tips on the slide (Pakrashi et al., 2014). The images were using a light microscope (Leica-DM-2500, Leica microsystems 267
R

212 observed for CAs using a fluorescence microscope (Olympus, Inc., USA) and pictures were captured using bright-field 268
213 Shinjuku-ku, Tokyo, Japan). single-shot mode at 10× magnification. 269
O

214 1.5. Comet assay (single cell gel electrophoresis, SCGE) 1.7. ROS analysis 270
C

215 The alkaline comet assay was carried out for the assessment 1.7.1. Superoxide radical (O−2U) determination 271
216 of DNA damage in root meristem cells of A. cepa, which For estimation of (O−2U) generated, the onion root tips were 272
N

217 were exposed to similar concentrations of MgO NPs and MPs treated with various MgO NP and MP concentrations along 273
218 as used for cytogenetic analysis by following the protocol with control for a sampling period of 4 hr. After treatment, to 274
U

219 proposed by Tice et al. (2000) with slight modifications. Three
220 slides were prepared for each experimental condition. In brief,
Q12 microscopic slides were pre-coated with 120 μL of 1% NMA in
221
222 PBS and allowed to solidify overnight at 37°C after covering
223 with a cover slip for the uniform layer. These slides served as
224 the agarose base coated slides. The treated root tips were
225 placed in a watch glass kept over ice and gently sliced using
226 razor blade to isolate the nuclei in PBS (pH 7.4) and the
227 suspension of the nuclei (100 μL) was mixed with 50 μL of 0.5%
228
Q13 LMA. These suspensions were pipetted onto pre-coated slides
229 and spread uniformly and covered with cover slip and left on
230 ice. The third layer of plain 0.5% LMA (120 μL) was applied and
231 a cover slip was quickly placed to get an even layer and
232 dried at 4°C. After removing the cover slip, the slides were
1 g of root tips, 5 mL of 0.1% TCA (freshly prepared) was added 275
and homogenized at 4°C. The homogenate was incubated 276
with 3 mL of reaction mixture containing 50 mmol/L Tris–HCl 277
buffer (pH 6.5), 0.2 mmol/L NBT, 0.2 mmol/L NADH, and 278
Q15
250 mmol/L of sucrose for 24 hr at (28 ± 2)°C in dark room. 279
The blue mono-formazan formed was measured at 530 nm 280
and its concentration was calculated using the extinction 281
coefficient (12.8 L/(mol·cm)), which provides an indirect mea- 282
surement of O−2U generated and expressed as μmol NBT 283
reducing of fresh weight of roots (Kiba et al., 1997). 284
1.7.2. Hydrogen peroxide (H2O2) determination 285
Interaction of the onion root tips was carried out with the MgO 286
NP and MP concentrations for a sampling period of 4 hr along 287

Please cite this article as: Mangalampalli, B., et al., Allium cepa root tip assay in assessment of toxicity of magnesium oxide
nanoparticles and microparticles, J. Environ. Sci. (2017), http://dx.doi.org/10.1016/j.jes.2017.05.012
 4 J O U RN A L OF E N V I RO N ME N TA L S CIE N CE S X X (2 0 1 7 ) XXX –XXX

288 with the control. After treatment, to 1 g of root tips in 5 mL of exposed to different concentrations of MgO NPs and MPs for 4 hr 331
289 0.1% TCA (freshly prepared) was added and homogenized. The along with control. The root tips were dried at 60°C for 24 hr. The 332
290 resulting mixture was centrifuged at 10,000 r/min for 10 min roots were then pounded to powder by using sterile mortar and 333
291 at 4°C. Without disturbing the pellet, the supernatant was pestle. The grounded samples were acid digested with concen- 334
292 collected in a fresh tube and 0.5 mL of the supernatant was trated HNO3 and then the soluble parts were filtered through 335
293 mixed with 0.5 mL of PBS (10 mmol/L) and 1 mL of potassium 0.45 μm membrane filter. The filtered samples were analyzed 336
294 iodide. The amount of H2O2 in the supernatant was determined with the help of inductive coupled plasma-optical emission 337
295 by reading its absorbance at 390 nm with a standard curve spectroscopy (ICP-OES; Perkin Elmer Optima 5300 DV, USA). 338
296 drawn using known concentrations of H2O2 and reported in
297 μmol/g of fresh weight of roots (Loreto and Velikova, 2001). 1.9. Data and statistical analysis 339

298 1.7.3. Hydroxyl radical (UOH) determination To determine the toxic effect of MgO NPs and MPs on A. cepa 340

F
299 For the estimation of UOH radical, the protocol of Halliwell and root tip cells, 1000 cells were scored per test concentration. 341
300 Gutteridge (1992) was utilized with slight modifications. The percent MI and phase index was calculated by Eqs. (1) and 342

O
301 After treatment, 1 g root tips were homogenized with 2 mL (2) given by Bakare et al. (2000). 343
302 of 10 mmol/L sodium phosphate buffer (pH 7.4) containing
MI ¼ Nm =Nt  100 ð1Þ

O
303 15 mmol/L of 2-deoxy-D-ribose (W/V). The homogenates were
304 centrifuged at 10,000 r/min for 10 min and the supernatants 345
344
PI ¼ Np =Nt  100 ð2Þ
305 were collected and incubated at 37°C for 2 hr. A reaction mixture

R
306 consisting 3 mL of 0.5% (W/V) TBA and 1 mL GAA (V/V) were where, MI (%) is the percent mitotic index; Nm is the number of 347
346
307 added to 5 mL of supernatants and subjected to heating at 90°C cells in mitosis; Nt is the total number of cells; PI (%) is the 348

P
308 for 30 min. The resulting solution was cooled to 4°C for 10 min. percent phase index; and Np is the number of cells in phase. 349
309
Q16 The concentration of MDA was quantified using its extinction The various changes between treated and control groups 350
310 coefficient (155 L/(mol·cm)), reading the absorbance at 532 nm were analyzed by one-way ANOVA. All results were expressed
D 351
Q17
311 using a Spectra max PLUS, Molecular Devices, USA and expressed as mean and standard deviation (mean ± SD) of the mean. 352
312 in μmol/g of fresh weight of root tips. Multiple pair-wise comparisons were done using the Dennett's 353
354
E
multiple comparison post-test and Student t-test to verify the
313 1.7.4. Lipid peroxidation significance at p < 0.05 and p < 0.01. Statistical analyses were 355
314 performed using Graph Pad Prism 5 Software package for 356
T

The root tips were exposed to MgO NP and MP dispersions for

315 a sampling period of 4 hr along with control as per the protocol windows (Graph Pad Software, Inc., La Jolla, CA, USA). 357
C

316 of Dhindsa et al. (1981). After treatment, 1.5 g root tips were
317 homogenized with 3 mL of reaction mixture comprising of 20%
318 (W/V) TCA and 0.5% (W/V) TBA. The homogenates were 2. Results 359
358
E

319 incubated at 95°C for 30 min and then cooled to stop the
320 reaction. Centrifugation was then carried out for 10 min at 2.1. Characterization of MgO NPs and MPs 360
R

321 10,000 r/min. The supernatant was separated and the absor-
322 bance was read at 532 and 600 nm. The non-specific absorbance The MgO NPs and MPs were characterized by TEM, DLS and LDV 361
R

323 due to unspecific turbidity at 600 nm was subtracted from analysis (Table 1). The size of MgO NPs and MPs obtained by 362
324 532 nm and the concentration of MDA was determined using TEM analysis was found to be (63.51 ± 10.7) nm and (11.24 ± 363
3.1) μm, respectively (Fig. 1a and b). Crystalline structure of the
O

325 the extinction coefficient (155 L/(mol·cm)). The content of MDA 364
326 formed was directly proportional to the level of lipid peroxida- NPs and MPs was confirmed by morphology analysis with 365
327 tion generated. FESEM (Fig. 1c and d). The mean hydrodynamic diameter (MHD) 366
C

and PdI of MgO NPs in Milli-Q water suspension obtained by DLS 367
328 1.8. Bio-uptake of MgO NPs and MPs was (181.52 ± 20.25) and 0.464, respectively. The particles were 368
N

found to have MHDs of (175 ± 63.6), (183.5 ± 64.2), (192.8 ± 64) 369
329 To determine the quantity of NPs and MPs uptake by A. cepa, the and (189 ± 63.1) nm at 0 hr for 12.5, 25, 50 and 100 μg/mL 370
330
U

method of Pakrashi et al. (2014) was followed. The root tips were concentrations, respectively. The hydrodynamic stability of the 371

t1:1
Q2 Table 1 – Characterization of MgO nanoparticles and microparticles.
t1:3
t1:2
t1:4 Particles Size using TEM DLS LDV Surface area (m2/g)

t1:5 Average diameter PdI Zeta potential Electrophoretic mobility pH

(nm) (mV) ((μm·cm)/(V·sec))

t1:6 MgO NPs (nm) 63.51 ± 10.7 181.52 ± 20.25 0.464 −11.9 0.58 7.0 142.8
t1:7 MgO MPs (μm) 0.339 ± 0.039 ND ND ND ND 7.0 43.88

t1:8 MgO NPs and MPs at the concentration of 40 μg/mL were dispersed in Milli-Q water and mixing was done via probe sonication for 10 min just
t1:9 before estimations.
t1:11
t1:10 PdI: polydispersity index; DLS: dynamic light scattering; LDV: laser Doppler velocimetry; ND: not detected; TEM: transmission electron
t1:13
t1:12 microscopy; NPs: nanoparticles; MPs: microparticles.

Please cite this article as: Mangalampalli, B., et al., Allium cepa root tip assay in assessment of toxicity of magnesium oxide
nanoparticles and microparticles, J. Environ. Sci. (2017), http://dx.doi.org/10.1016/j.jes.2017.05.012
 J O U RN A L OF E N V I RO N ME N TA L S CI EN CE S X X (2 0 1 7 ) XX X–XXX 5

F
O
O
R
P
D
E
T
C
E

Fig. 1 – Transmission electron microscopy (TEM) image of (a) magnesium oxide nanoparticles (MgO NPs) and (b) MgO
R

microparticles (MPs) in Milli-Q water (Particles were scanned by TEM from JEOL, JEM-2100, Japan at voltage of 120 kV); scanning
electron microscopy (SEM) image of (c) MgO NPs and (d) MgO MPs.
R
O

372 dispersions was evaluated after 4 hr which corresponded to the (201 ± 46.1) nm for dispersions of 12.5, 25, 50 and 100 μg/mL, 375
373 duration of exposure to the root tips. The particles were found respectively. The larger hydrodynamic diameter than the TEM 376
374 to have MHDs of (185 ± 56.6), (193.5 ± 76.2), (198.8 ± 74) and size indicated that agglomerates of MgO NPs have been formed 377
C
N

t2:1 Table 2 – Different phase and mitotic indexes of A. cepa root cells treated with different concentrations of MgO NPs and MPs.
t2:3
t2:2
t2:4 Treatment No. of cells in Prophase Metaphase Anaphase Telophase Mitotic
U

(μg/mL) interphase index (%) index (%) index (%) index (%) index (%)

t2:5 Control 541 23.5 ± 1.83 10.8 ± 0.86 8.8 ± 0.71 2.7 ± 0.54 45.9 ± 2.8
t2:6 NPs 12.5 564 20.9 ± 1.23 12.1 ± 0.89⁎ 9.0 ± 1.52 1.7 ± 0.52 43.6 ± 2.3
t2:7 25 574 20.9 ± 0.74 11.3 ± 0.96 8.6 ± 1.58 1.8 ± 0.61 42.6 ± 3.5
t2:8 50 610 19.7 ± 0.76 10.5 ± 1.1 7.4 ± 1.49⁎ 1.4 ± 0.56⁎ 39.0 ± 1.8⁎
t2:9 100 624 20.5 ± 0.9 9.8 ± 1.01 5.6 ± 1.63⁎⁎ 1.7 ± 0.87 37.6 ± 1.7⁎⁎
t2:10 MPs 12.5 565 20.8 ± 0.84 11.5 ± 1.12 9.2 ± 1.28 2.2 ± 0.77 43.5 ± 3.1
t2:11 25 597 19.6 ± 0.96 12.3 ± 1.33⁎ 6.9 ± 1.35 1.5 ± 0.71 40.3 ± 2.0
t2:12 50 588 21.5 ± 1.12 10.3 ± 0.3 8.6 ± 1.51 2.0 ± 0.43 41.2 ± 2.7
t2:13 100 590 19.5 ± 1.42 9.8 ± 0.89 8.7 ± 1.1 3.0 ± 0.69 41.0 ± 2.5

t2:14 One thousand cells were scored per test concentration for percent index calculation (n = 5).
t2:15
t2:16 Data represented as mean ± SD, significantly different from control ⁎p < 0.05 and ⁎⁎p < 0.01, according to Dunnett's multiple comparison
t2:17 post-test.
t2:20
t2:19
t2:18 NPs: nanoparticles; MPs: microparticles; SD: standard deviation.

Please cite this article as: Mangalampalli, B., et al., Allium cepa root tip assay in assessment of toxicity of magnesium oxide
nanoparticles and microparticles, J. Environ. Sci. (2017), http://dx.doi.org/10.1016/j.jes.2017.05.012
 6 J O U RN A L OF E N V I RO N ME N TA L S CIE N CE S X X (2 0 1 7 ) XXX –XXX

378 in Milli-Q water suspension than in the dry state. Agglomera- prophase (showed chromosomes which were quite visible and 388
379 tion of the NPs was prevented by the electrostatic repulsion is the characteristic of this phase), metaphase (with chromo- 389
380 between NPs, which is responsible for the colloidal stability of somes arranged in the equatorial plate of the cell), anaphase 390
381 NPs. Zeta potential (ζ) and electrophoretic mobility of MgO (with chromosomes movement to opposite poles in a stable 391
382 NPs and MPs in Milli-Q were quantified by LDV and found to way) and telophase (in which chromosomes are organized at 392
383 be −11.9 mV and 0.58 (μm·cm)/(V·sec), respectively at pH 7.0. opposite poles, awaiting for cytokinesis) were observed in the 393
control A. cepa root cells. The mitotic and phase indices are 394
384 2.2. Microscopic analysis measured after A. cepa root tip cells were exposed to MgO NPs 395
and MPs (Table 2). Each group had 5 replicates and for each 396
385 Optical and florescent microscopy results gave a comprehen- replicate, scoring was carried out for 1000 cells. The decrease in 397
386 sive view on the impact of the MgO NPs and MPs on A. cepa MI in treated samples as compared to controls indicated that 398
387 root tip cells. The different mitotic phases namely, interphase, MgO NPs and MPs may have an impact on A. cepa. In control 399

F
O
O
R
P
D
E
T
C
E
R
R
O
C
N
U

Fig. 2 – Different types of chromosomal aberrations (a) multipolar anaphase; (b) sticky anaphase; (c) sticky metaphase;
(d) lagged anaphase; (e) binucleated; (f) diagonal anaphase; (g) distributed chromosomes and (h) distributed
anaphase/telophase observed in meristematic cells of A. cepa after 4 hr exposure to MgO NPs. NPs: nanoparticles.

Please cite this article as: Mangalampalli, B., et al., Allium cepa root tip assay in assessment of toxicity of magnesium oxide
nanoparticles and microparticles, J. Environ. Sci. (2017), http://dx.doi.org/10.1016/j.jes.2017.05.012
 J O U RN A L OF E N V I RO N ME N TA L S CI EN CE S X X (2 0 1 7 ) XX X–XXX 7

Bi nucleated
400 roots, the percentage MI was found to be (45.9 ± 2.8) and there

3.54 ± 0.86⁎⁎
2.45 ± 0.9⁎⁎
7.33 ± 1.5⁎⁎
cells (%)

1.32 ± 0.42

1.75 ± 0.13
401 were no signs of CAs, suggesting that actively dividing cells

0.55 ± 0.2

0.35 ± 0.2

6.52 ± 1.8
1 ± 0.2
402 were observed in all stages of mitosis. In contrast, the root cells

ND: not detected; NPs: nanoparticles; MPs: microparticles; SD: standard deviation. One thousand cells were scored per test concentration for chromosomal aberrations determinations (n = 5).
403 treated with MgO NPs and MPs induced a dose dependent
404 decrease in MI. After the exposure to MgO NPs and MPs at 12.5,
405 25, 50 and 100 μg/mL concentrations, a dose dependent

1.23 ± 0.24⁎⁎
C-mitosis

0.02 ± 0.01

0.17 ± 0.02

0.15 ± 0.05

0.95 ± 0.42
406 decrease in the percentages of MI was observed in the treated

0.26 ± 0.1
0.2 ± 0.1
(%)
407 group in comparison to the control cells indicating that they are
408 capable of increasing the DNA damage in root tip cells. In

ND

ND
409 prophase, the highest percentage of cells was observed at the
410 12.5 μg/mL dose of both NP and MP treated roots. The number of

anaphase (%)
Multipolar

0.29⁎⁎
411 cells in subsequent stages i.e., metaphase, anaphase, telophase

± 0.00

± 0.05

0.11

1.25
F 0.1
412 was significantly lesser in the treated root tips (25, 50 and

±
±
±

±
413 100 μg/mL). Number of cells in interphase was proportional to

0.87
0.01

0.18

0.32
0.25

0.55
O
ND

ND

ND
414 the concentration of NPs and MPs. Further, after an exposure to
415 50 and 100 μg/mL NP and MP dose, cells were not allowed to

Data represented as mean ± SD, significantly different from control ⁎p < 0.05 and ⁎⁎p < 0.01, according to Dunnett's multiple comparison post-test.
O
416 enter the dividing stage, rather many of them were arrested at

0.87 ± 0.17⁎⁎
anaphase
Diagonal

0.01 ± 0.09
0.02 ± 0.02

0.12 ± 0.01

0.27 ± 0.02
0.21 ± 0.1
417 interphase. A variety of cytological changes were observed in

0.7 ± 0.2
(%)
418 the root tip cells after their interaction with NPs. Various CAs

R
ND

ND
419 like lagged, clumped, disturbed, sticky anaphase, chromosomal
420
Q18 break, bridge and c-mitosis were found upon interaction of root

P metaphase
421 tip cells with various concentrations of NPs and MPs (Fig. 2).

anaphase/

1.23 ± 0.42⁎⁎
Disturbed
Table 3 – Percentage of chromosomal aberrations observed in MgO NPs and MPs-treated A. cepa root tip cells.

0.38 ± 0.06
0.41 ± 0.05

0.52 ± 0.11
0.25 ± 0.15
0.66 ± 0.24

0.95 ± 0.45
0.3 ± 0.15
422 When the root tips were exposed to 12.5 and 25 μg/mL doses,

0.5 ± 0.3
(%)
423 aberrations like breaks along with bridges, slightly disturbed D
424 anaphase and telophase were observed. At an exposure
425 concentration of 50 and 100 μg/mL, the cells were binucleated
E
426 and showed abnormalities like lagged and clumped chromo-
Chromosomal

0.46⁎⁎
bridge (%)

0.13⁎
427 somes along with C-mitosis in root tip cells. Different types of

0.05
0.07
0.04
0.11
0.12

0.52
0.2
T

428 cytological alterations found in meristematic cells of A. cepa

±
±
±
±
±
±
±
±
±
429 after a 4 hr exposure to MgO NPs are shown in Table 3. The 0.21
0.25
0.15
0.33
0.24
0.52
0.44
1.58
0.95
C

430 increase in CAs was directly proportional to the concentration

431 and indirectly proportional to the size of MgO particles.
chromosome
E

0.39⁎⁎
0.15⁎
Clumped

432 2.3. Comet assay

0.07

0.05

0.05

0.25

0.45
0.1

0.1
(%)
R

±
±
±
±
±
±
±
±
±
433
0.31
0.32
0.21
0.39
0.15
0.64
0.52
1.83
0.75
The genotoxicity effect of MgO NPs and MPs on onion root tip
434 cells was evaluated using SCGE in isolated nuclei (Fig. 3). The
R

435 results obtained with CASP software are summarized in Table 4.

chromosome

436 From the result it was observed that, the percent tail DNA
0.19⁎⁎
0.07
0.05
0.08
0.21

0.23
Laggard
O

437 increased in the range of 6.8–30.1, with notable decrease in

(%)

438 percent head DNA for 12.5–100 μg/mL doses of MgO NPs. Comet
±
±
±
±
±
±
0.36
0.25
0.31
0.45
0.97
0.84
ND
ND
ND

439 analysis revealed by tail moment (TM) and Olive tail moment
C

440 (OTM) indicated that DNA damage was significant (p < 0.05) as
441 compared to control with increasing dose of MgO NPs. Higher
N

chromosome

442 doses of MgO induced greater genotoxicity, which was evident

0.38⁎⁎
0.15⁎
0.32⁎

0.65⁎
0.07
0.05
0.05
0.13
0.15
Sticky

443 from increasing values of TM and OTM exhibited at the dose of

(%)

100 μg/mL MgO NPs treatment followed by 50, 25, 12.5 μg/mL
U

±
±
±
±
±
±
±
±
±

444
0.32
0.35
0.25
0.42
0.35
0.89
0.65
1.91
0.95

445 treatments.

446 2.4. Histochemical staining

Chromosome

1.54 ± 0.35⁎⁎
0.77 ± 0.56⁎
break (%)

0.31 ± 0.08

0.43 ± 0.04

0.58 ± 0.07

447 The production of H2O2andO2U− was examined in both root

0.2 ± 0.1

0.4 ± 0.1

448 elongation and maturation zones of A. cepa with histochemical

ND

ND

449 staining using DAB and NBT to determine the level of

450 oxidative stress induced by MgO NP and MP treatment (Fig. 4).
451 The levels of both H2O2 and O2U− in the root elongation zone
MPs

MPs

MPs

MPs
Treatment

NPs

NPs

NPs

NPs
(μg/mL)

452 were significantly increased in a dose dependent manner, while

Control

453 in the maturation zone no significant alteration in H2O2 and

O2U− production was observed. The histochemical staining
12.5

454
100
25

50

455 also showed increased staining density because of H2O2 and

t3:10
t3:11
t3:12
t3:13

t3:14
t3:17
t3:16
t3:15
t3:1
t3:4
t3:3
t3:2

t3:5
t3:6
t3:7
t3:8
t3:9

Please cite this article as: Mangalampalli, B., et al., Allium cepa root tip assay in assessment of toxicity of magnesium oxide
nanoparticles and microparticles, J. Environ. Sci. (2017), http://dx.doi.org/10.1016/j.jes.2017.05.012
 8 J O U RN A L OF E N V I RO N ME N TA L S CIE N CE S X X (2 0 1 7 ) XXX –XXX

F
O
O
R
P
D
E
T
C
E
R
R
O
C
N

Q1 Fig. 3 – MgO induced DNA damage in terms of head DNA and tail DNA in comet assay as evaluated with comet assay software
(CaspLab) photographs showing comets in (a) control, (b) 12.5 μg/mL, (c) 25 μg/mL, (d) 50 μg/mL and (e) 100 μg/mL MgO NPs
treated root tips of A. cepa. In the left panel, comet images with measurement frame, tail and head are represented; whereas in
U

right panel, intensity profiles are plotted with length μm = pixels × 0.64 on X axis and % DNA (× 102) on Y axis. NPs:
nanoparticles.

456 O2U− production in roots with an increased concentration of MgO to confirm the oxidative stress induced by various concentra- 462
457 NPs. tions of MgO NPs and MPs (Fig. 5). The generation of O2U− was 463
increased upon increasing the concentration of MgO NPs and 464
458 2.5. ROS analysis MPs. A similar effect was observed for the production of 465
hydroxyl radical (UOH) and H2O2 production. The generation of 466
459 In oxidative stress analysis, the generation of O2U−, H2O2, free radicals was found to be enhanced at all concentrations 467
460 hydroxyl (UOH) radicals and MDA was determined in treated of MgO NPs and MPs (12.5, 25, 50 and 100 μg/mL). The increase 468
461 and untreated A. cepa root tip cells at a sampling period of 4 hr in the generation of ROS (O2U−, H2O2, and UOH radicals) was 469

Please cite this article as: Mangalampalli, B., et al., Allium cepa root tip assay in assessment of toxicity of magnesium oxide
nanoparticles and microparticles, J. Environ. Sci. (2017), http://dx.doi.org/10.1016/j.jes.2017.05.012
 J O U RN A L OF E N V I RO N ME N TA L S CI EN CE S X X (2 0 1 7 ) XX X–XXX 9

t4:1 Table 4 – MgO NPs induced DNA damage in nuclei isolated from onion (A. cepa) roots measured in terms of % head DNA, %
t4:2
Q3 tail DNA, tail moment and olive tail moment in comet assay method.
t4:3
t4:4 Treatment (μg/mL) Head DNA (%) Tail DNA (%) Tail moment (μm) Olive tail moment (μm)

t4:5 Control 97.73 ± 1.07 2.26 ± 1.67 0.11 ± 0.094 0.38 ± 0.16
t4:6 12.5 93.1 ± 1.91 6.8 ± 1.918 0.72 ± 0.35 1.5 ± 0.53
t4:7 25 84.64 ± 2.85 15.35 ± 2.85⁎ 3.02 ± 1.53 4.08 ± 1.59
t4:8 50 80.25 ± 3.31 19.75 ± 3.30⁎⁎ 9.216 ± 4.0 7.917 ± 1.41
t4:9 100 69.98 ± 5.19 30.1 ± 5.19⁎⁎ 14.85 ± 4.66 16.87 ± 3.50

t4:10 Data were recorded after 4 hr of exposure to MgO NPs, represented as mean ± SD, significantly different from control ⁎p < 0.05 and ⁎⁎p < 0.01,
t4:11 according to Dunnett's multiple comparison post-test.
t4:14
t4:13
t4:12 NPs: nanoparticles; SD: standard deviation.

F
O
470 observed to be significant at p < 0.01. Lipid peroxidation assay total Mg was taken up at 12.5, 25, 50 and 100 μg/mL concentra- 489

O
471 was performed to determine MDA levels generated from tions, respectively (Fig. 6). The elemental analysis showed a 490
472 oxidation of lipids in root tip cells after exposed to various concentration dependent increase in the internalization of Mg 491
473 concentrations of MgO NPs and MPs at 4 hr. MDA levels were into the root cells. 492

R
474 measured spectrometrically and they were found to be
475 significant with the increase in dose. The results revealed

P
476 that generation of intracellular oxidative stress was concen- 3. Discussion 494
493
477 tration dependent and a maximum increase was found at
478 100 μg/mL dose of MgO NPs. The characterization of NMs is considered as the primary
D 495
requirement for the study of NPs induced toxicity on different 496
479 2.6. Bio-uptake of MgO NPs and MPs test models (Bakand and Hayes, 2016). It is essential to 497
E
determine the hydrodynamic diameter of NPs in water before 498
480 The bio-uptake potential was used to evaluate the toxicity of performing the toxicity tests (Pakrashi et al., 2014). The MHDs 499
481 NPs and MPs. The ICP-OES analysis was performed to quantify of MgO NPs were quantified at 0 and 4 hr, they did not show 500
T

482 the amount of MgO NPs and MPs internalized into the A. cepa any statistically significant variation, indicating that the size 501
483 502
C

root cells upon the exposure to various concentrations (12.5, 25,
Q19 50 and 100 μg/mL). For MgO NPs we found that (0.48 ± 0.12),
484
485 (6.87 ± 1.35), (12.55 ± 2.62) and (18.82 ± 2.4) μg/mL of total Mg
E
of the NPs in dispersions was stable during the course of test.
Mechanism by which nanotoxicity occurs is yet a predomi- 503
nately unknown arena. Nevertheless, it may be associated 504

486 was internalized in roots of A. cepa at exposure concentrations with the particle size and surface area, chemical composition 505
487 of 12.5, 25, 50 and 100 μg/mL. However, in case of MgO MPs, and structure of the NPs (Kumari et al., 2009). The microscopic 506
R

488 (0.12 ± 0.06), (1.84 ± 0.88), (3.24 ± 1.25) and (6.28 ± 2.4) μg/mL of analysis includes the MI and scoring of cytological damage in 507
R
O
C
N
U

Fig. 4 – Visualization of (a) H2O2 by DAB staining and (b) super oxide by NBT staining in roots of A. cepa treated with different
concentrations of MgO NPs. DAB: diaminobenzidine; NBT: nitro blue tetrazolium; NPs: nanoparticles.

Please cite this article as: Mangalampalli, B., et al., Allium cepa root tip assay in assessment of toxicity of magnesium oxide
nanoparticles and microparticles, J. Environ. Sci. (2017), http://dx.doi.org/10.1016/j.jes.2017.05.012
10 J O U RN A L OF E N V I RO N ME N TA L S CIE N CE S X X (2 0 1 7 ) XXX –XXX

F
O
O
R
P
D
E
T
C
E
R
R
O
C
N
U

Fig. 5 – Generation of superoxide, hydrogen peroxide, hydroxyl radical and lipid peroxidation in A. cepa root tips cells after
interaction with 12.5, 25, 50 and 100 μg/mL of MgO NPs (a, b, c and d) and MPs (e, f, g and h). The asterisks (***), (**), (*) among
treatments indicate significant differences compared to control at p < 0.001, 0.01 and 0.05. NPs: nanoparticles; MPs:
microparticles.

Please cite this article as: Mangalampalli, B., et al., Allium cepa root tip assay in assessment of toxicity of magnesium oxide
nanoparticles and microparticles, J. Environ. Sci. (2017), http://dx.doi.org/10.1016/j.jes.2017.05.012
 J O U RN A L OF E N V I RO N ME N TA L S CI EN CE S X X (2 0 1 7 ) XX X–XXX 11

which suggested that heavy metals are genotoxic and lead to 548
DNA damage. For example Erturk et al. (2013) found that DNA 549
damage was produced by treatment of Z. mays seedlings by 550
Nickel and Cobalt. A significant increase in DNA damage was 551
reported in heterozygous potato and tobacco plants cultivated 552
in heavy metal contaminated soil (Gichner et al., 2006). 553
Damage to DNA in onion root tip cells upon interaction with 554
TiO2 NPs was documented by Pakrashi et al. (2014). Indium tin 555
oxide (ITO) NPs showed mutagenic activity via generation of 556
DNA damage (Emerit et al., 2001). Genotoxic effects and cell 557
division were revealed by increased CAs and MI in A. cepa root 558
meristematic cells after treatment with ITO NPs (Ciğerci et al., 559

F
2015). 560
Fig. 6 – Internalization of MgO NPs and MPs into the root tips Oxidative stress is considered as an essential mechanism 561

O
as quantified through ICP-OES analysis. NPs: nanoparticles; by which NPs induce toxicity in cells. Oxidative stress can be 562
MPs: microparticles; ICP-OES: inductive coupled induced by different factors such as active redox cycling on 563

O
plasma-optical emission spectroscopy. the surface of NPs, functionalized oxidative groups on the NPs 564
and NP to cell interactions etc. (Manke et al., 2013). NPs have 565
the ability to enter the cell membrane easily and accumulate 566

R
in the cytoplasm leading to mitochondrial dysfunction, 567
508 anaphase and telophase cells. MI is a frequently used cell oxidative stress and cell death (Kim et al., 2009; Rajeshwari 568

P
509 cycle parameter and indicator of mitosis (Nagaonkar et al., 2015). et al., 2016). In plant cells, the DNA damage may occur directly 569
510 The exposure of MgO NPs and MPs in the current study, or indirectly by means of abiotic stress induced by NPs, 570
511 significantly inhibited MI in A. cepa root tip cells in a concentra- including heavy metals (Xia et al., 2006). The protonation of
D 571
512 tion dependent manner. The reduction in the MI may be due to superoxide (UO2) could produce hydroperoxyl radicals (UOH, 572
513 the blockage at G1 stage, thereby suppressing the DNA synthesis H2O2) in plant cells (Karuppanapandian et al., 2011). The 573
E
514 (Mohandas and Grant, 1972). Further, the occurrence of a observed genotoxicity of MgO NPs may be due to the 574
515 distorted metaphase could be due to the disturbance in spindle modification of chromatin structure, induction of ROS, or 575
T

516 fiber apparatus (Darlington and McLeish, 1951). The genotoxicity DNase release from the lysosomes resulting in oxidative 576
517 study carried out by Pakrashi et al. (2014), strongly suggested stress and cell membrane damage due to triggering several 577
C

518 that the exposure of A. cepa root tips to TiO2 NPs at both lower signal transduction cascades (Banasik et al., 2005; Pakrashi et 578
Q20 Q21
519 (12.5 μg/mL) and higher concentrations (100 μg/mL) gave rise to al., 2013). Generation of ROS is attributed to the antibacterial 579
520 various CAs along with decrease in MI, in dose dependent activity of MgO NPs (Krishnamoorthy et al., 2012). Therefore, it 580
E

521 manner, which was in accordance with our study. The investi- can be summarized from these results that, NPs and MPs may 581
522 gational reports on the cytotoxic and genotoxic potentials of be involved in ROS generation. 582
R

523 silver (Ag) NPs and zinc oxide (ZnO) NPs in A. cepa also showed The internalization or uptake of NPs contributes a major 583
524 concentration dependent inhibition of MI indicating that these role in the toxic effect toward A. cepa by generating oxidative 584
R

525 NPs were capable of causing distortion of chromosomes, stress in the cells. Some of the investigations have revealed 585
526 binucleated cells, annihilation of cell walls, nuclear membrane that genotoxicity of NPs in A. cepa root cells may be due to 586
O

527 commotion, bridge at anaphase and vagrant chromosomes their internalization. Internalized concentrations of the total 587
528 (Kumari et al., 2009, 2011). The formation of chromosome Mg by ICP-OES analysis revealed that the uptake was dose 588
529 laggards, stickiness, disturbed anaphase, telophase and bridges and size dependent. The bio-uptake of the titania NPs in A. 589
C

530 in anaphase–telophase cells on treating A. cepa cells with cepa produced dose dependent genotoxic effects (Pakrashi et 590
531 bismuth (III) oxide NPs was visualized by Liman (2013). TiO2 NPs al., 2014). A study indicated that internalization of Ag NPs by 591
N

532 (at 100 μg/mL dose) caused chromosomal break, sticky meta- the root tips of A. cepa, affected cell division (Kumari et al., 592
533 phase, formation of binucleated cells, occurrence of laggard 2009). In another study carried out with ZnO NPs, demon- 593
U

534 chromosome, clumping of chromosome and slanted movement strated that the NPs were taken up by A. cepa cells which lead 594
535 of chromosomes during anaphase due to incorrect polarization to significant cytogenetic effects and genotoxicity (Kumari et 595
536 (Pakrashi et al., 2014). al., 2011). 596
537 The comet assay has the ability to identify low levels of
538 DNA damage in a variety of cell types. Hence, the comet assay
539 is an important method with which to detect DNA damage. 4. Conclusions 598
597
540 The tail DNA and OTM was utilized to evaluate MgO NPs and
541 MPs induced DNA damage in A. cepa root nuclei. The tail DNA The toxicity of MgO NPs and MPs was assessed in the plant 599
542 increased in a single dimension when the level of stress system using A. cepa bioassay. The NPs and MPs treated root 600
543 intensity enhanced. However, the tail moments were based tip cells indicated a dose dependent increase in cytological 601
544 on the changes in DNA migration, which may be because of changes and decrease in MI when compared to untreated 602
545 the nature of DNA or might be due to the degree of DNA cells. The MI was found to have diminished for all the 603
546 relaxation (Duez et al., 2003). The results of the current concentrations as the size of NPs decreased. Different types 604
547 investigation are in accordance with the previous reports of CAs observed in treated root tips confirmed the impact of 605

Please cite this article as: Mangalampalli, B., et al., Allium cepa root tip assay in assessment of toxicity of magnesium oxide
nanoparticles and microparticles, J. Environ. Sci. (2017), http://dx.doi.org/10.1016/j.jes.2017.05.012
 12 J O U RN A L OF E N V I RO N ME N TA L S CIE N CE S X X (2 0 1 7 ) XXX –XXX

606 NPs and MPs on cells. The interaction of A. cepa root cells with Di, D.-R., He, Z.-Z., Sun, Z.-Q., Liu, J., 2012. A new 666
607 NMs resulted in the generation of ROS, which was found to be nano-cryosurgical modality for tumor treatment using 667
biodegradable MgO nanoparticles. Nanomedicine 8 (8), 668
608 a strongly dependent on the dose. These results correlated
1233–1241. 669
609 well with the enhanced lipid peroxidation. Oxidative stress
Duez, P., Dehon, G., Kumps, A., Dubois, J., 2003. Statistics of the 670
610 generation and MDA formation could be the contributing Comet assay: a key to discriminate between genotoxic effects. 671
611 factors for the aberrations observed in the chromosomes of A. Mutagenesis 18 (2), 159–166. 672
612 cepa root tip cells. The percentage MI and CAs of the treated Emerit, J., Beaumont, C., Trivin, F., 2001. Iron metabolism, free 673
613 root cells when compared to controls was found to be radicals, and oxidative injury. Biomed. Pharmacother. 55 (6), 674
333–339. 675
614 statistically significant at p < 0.05 and p < 0.01. It was ob-
Erturk, F.A., Ay, H., Nardemir, G., Agar, G., 2013. Molecular 676
615 served that the reduction in the percent MI was significantly
determination of genotoxic effects of cobalt and nickel on 677
616 decreased in treated cells compared to the control cells. In maize (Zea mays L.) by RAPD and protein analyses. Toxicol. Ind. 678
617 conclusion, the present study indicated that treatment of MgO Health 29 (7), 662–671. 679

F
618 NPs exhibited greater toxicity on A. cepa root tip cells than Fiskesjo, G., 1997. Allium test for screening chemicals; evaluation 680
619 MPs. Further, in vitro and in vivo studies on human cells and of cytological parameters. Plants Environ. Stud. 101, 307–333. 681

O
620 laboratory animals need to be performed before any further Ge, S., Wang, G., Shen, Y., Zhang, Q., Jia, D., Wang, H., et al., 2011. 682
Cytotoxic effects of MgO nanoparticles on human umbilical 683
621 conclusion with regard to the mechanistic illustration of
vein endothelial cells in vitro. IET Nanobiotechnol. 5 (2), 36–40. 684

O
622 toxicity of MgO NPs can be drawn.
Gichner, T., Patková, Z., Száková, J., Demnerová, K., 2006. Toxicity 685
and DNA damage in tobacco and potato plants growing on soil 686
polluted with heavy metals. Ecotoxicol. Environ. Saf. 65 (3), 687

R
624
623 Acknowledgments 420–426. 688
Grant, W.F., 1999. Higher plant assays for the detection of 689

P
625 We express our sincere thanks to the Director IICT, Hyderabad chromosomal aberrations and gene mutations—a brief 690
historical background on their use for screening and 691
626 for providing funds and facility to execute this study. Further,
monitoring environmental chemicals. Mutat. Res. Fundam. 692
627 Bhanuramya Mangalampalli (SRF), Naresh Dumala (SRF) and
Mol. Mech. Mutagen. 426 (2), 107–112. 693
628 Paramjit Grover (Emeritus Scientist) are grateful to University
D
Halliwell, B., Gutteridge, J., 1992. Biologically relevant metal 694
629 Grants Commission and Council of Scientific and Industrial ion-dependent hydroxyl radical generation an update. FEBS 695
E
630 Research, India for the award of fellowships, respectively. Lett. 307 (1), 108–112. 696
Karuppanapandian, T., Moon, J.-C., Kim, C., Manoharan, K., Kim, 697
W., 2011. Reactive oxygen species in plants: their generation, 698
T

63 1 REFERENCES signal transduction, and scavenging mechanisms. Aust. 699

J. Crop. Sci. 5 (6), 709–725. 700
C

Kaur, G., Singh, H.P., Batish, D.R., Kohli, R.K., 2014. Pb-inhibited 701
632
633 Bakand, S., Hayes, A., 2016. Toxicological considerations, toxicity mitotic activity in onion roots involves DNA damage and 702
634 assessment, and risk management of inhaled nanoparticles. disruption of oxidative metabolism. Ecotoxicology 23 (7), 703
E

635 Int. J. Mol. Sci. 17 (6), 929. 1292–1304. 704

636 Bakardjieva, S., Šubrt, J., Štengl, V., Opluštil, F., Olšanska, M., 2004. Kiba, A., Miyake, C., Toyoda, K., Ichinose, Y., Yamada, T., Shiraishi, 705
R

637 Magnesium oxide nanoparticles as destructive sorbent for T., 1997. Superoxide generation in extracts from isolated plant 706
638 toxic agents. Microsc. Microanal. 10 (S02), 476–477. cell walls is regulated by fungal signal molecules. 707
639 Bakare, A., Mosuro, A., Osibanjo, O., 2000. Effect of simulated Phytopathology 87 (8), 846–852. 708
R

640 leachate on chromosomes and mitosis in roots of Allium cepa Kim, H., Tu, H.-C., Ren, D., Takeuchi, O., Jeffers, J.R., Zambetti, G.P., 709
641 (L.). J. Environ. Biol. 21 (3), 263–271. et al., 2009. Stepwise activation of BAX and BAK by tBID, BIM, 710
O

642 Bertinetti, L., Drouet, C., Combes, C., Rey, C., Tampieri, A., and PUMA initiates mitochondrial apoptosis. Mol. Cell 36 (3), 711
643 Coluccia, S., et al., 2009. Surface characteristics of 487–499. 712
644 nanocrystalline apatites: effect of Mg surface enrichment on Krishnamoorthy, K., Moon, J.Y., Hyun, H.B., Cho, S.K., Kim, S.-J., 713
C

645 morphology, surface hydration species, and cationic 2012. Mechanistic investigation on the toxicity of MgO 714
646 environments. Langmuir 25 (10), 5647–5654. nanoparticles toward cancer cells. J. Mater. Chem. 22 (47), 715
647 Borboa, L., Torre, C., 1996. The genotoxicity of Zn (II) and Cd (II) in 24610–24617. 716
N

648 Allium cepa root meristematic cells. New Phytol. 134 (3), Kumari, M., Mukherjee, A., Chandrasekaran, N., 2009. 717
649 481–486. Genotoxicity of silver nanoparticles in Allium cepa. Sci. Total 718
650 Cabrera, G., Rodriguez, D., 1999. Genotoxicity of soil from 719
U

Environ. 407 (19), 5243–5246.

651 farmland irrigated with wastewater using three plant Kumari, M., Khan, S.S., Pakrashi, S., Mukherjee, A., 720
652 bioassays. Mutat. Res. 426 (2), 211–214. Chandrasekaran, N., 2011. Cytogenetic and genotoxic effects of 721
653 Ciğerci, İ.H., Liman, R., Özgül, E., Konuk, M., 2015. Genotoxicity of zinc oxide nanoparticles on root cells of Allium cepa. J. Hazard. 722
654 indium tin oxide by Allium and Comet tests. Cytotechnology Mater. 190 (1), 613–621. 723
655 67 (1), 157–163. Lai, J.C., Lai, M.B., Jandhyam, S., Dukhande, V.V., Bhushan, A., 724
656 Darlington, C., McLeish, J., 1951. Action of maleic hydrazide on the Daniels, C.K., et al., 2008. Exposure to titanium dioxide and 725
657 cell. Nature 167 (4245), 407–408. other metallic oxide nanoparticles induces cytotoxicity on 726
658 Daudi, A., Cheng, Z., O'Brien, J.A., Mammarella, N., Khan, S., human neural cells and fibroblasts. Int. J. Nanomedicine 2008, 727
659 Ausubel, F.M., et al., 2012. The apoplastic oxidative burst 533–545. 728
660 peroxidase in Arabidopsis is a major component of Lei, Z., Li, L., Li, G., Leung, C., Shi, J., Wong, C., et al., 2012. Liver 729
661 pattern-triggered immunity. Plant Cell 24 (1), 275–287. cancer immunoassay with magnetic nanoparticles and 730
662 Dhindsa, R.S., Plumb-Dhindsa, P., Thorpe, T.A., 1981. Leaf MgO-based magnetic tunnel junction sensors. J. Appl. Phys. 731
663 senescence: correlated with increased levels of membrane 111 (7), 07E505. 732
664 permeability and lipid peroxidation, and decreased levels of Li, S.T., Qiao, X.L., Chen, J.G., Wu, C.L., Mei, B., 2005. The 733
665 superoxide dismutase and catalase. J. Exp. Bot. 32 (1), 93–101. investigation of antibacterial characteristics of magnesium 734

Please cite this article as: Mangalampalli, B., et al., Allium cepa root tip assay in assessment of toxicity of magnesium oxide
nanoparticles and microparticles, J. Environ. Sci. (2017), http://dx.doi.org/10.1016/j.jes.2017.05.012
 J O U RN A L OF E N V I RO N ME N TA L S CI EN CE S X X (2 0 1 7 ) XX X–XXX 13

735 oxide and it's nano-composite materials. J. Funct. Mater. 36 oxide nanoparticles on Allium cepa root tip effects of oxidative 773
736 (11), 1651–1654. stress generation and biouptake. Environ. Sci. Pollut. Res. 22 774
737 Liman, R., 2013. Genotoxic effects of Bismuth (III) oxide (14), 11057–11066. 775
738 nanoparticles by Allium and Comet assay. Chemosphere 93 (2), Rajeshwari, A., Suresh, S., Chandrasekaran, N., Mukherjee, A., 776
739 269–273. 2016. Toxicity evaluation of gold nanoparticles using an Allium 777
740 Loreto, F., Velikova, V., 2001. Isoprene produced by leaves protects cepa bioassay. RSC Adv. 6 (29), 24000–24009. 778
741 the photosynthetic apparatus against ozone damage, Rico, C.M., Majumdar, S., Duarte-Gardea, M., Peralta-Videa, J.R., 779
742 quenches ozone products, and reduces lipid peroxidation of Gardea-Torresdey, J.L., 2011. Interaction of nanoparticles with 780
743 cellular membranes. Plant Physiol. 127 (4), 1781–1787. edible plants and their possible implications in the food chain. 781
744 Mahmoud, A., Ezgi, Ö., Merve, A., Özhan, G., 2016. In vitro J. Agric. Food Chem. 59 (8), 3485–3498. 782
745 toxicological assessment of magnesium oxide nanoparticle Satapathy, R., Swamy, P.A., 2013. Preparative isolation of 783
746 exposure in several mammalian cell types. Int. J. Toxicol. 35 (4), flavonoids from wattakaka volubilis (linn. f.) leaf extracts and 784
747 429–437. its role as antimitotic and antiproliferating agent. Int. J. Pharm. 785
748 Manke, A., Wang, L., Rojanasakul, Y., 2013. Mechanisms of Biol. Sci. 4 (3), 454–460. 786

F
749 nanoparticle-induced oxidative stress and toxicity. Biomed. Sun, J., Wang, S., Zhao, D., Hun, F.H., Weng, L., Liu, H., 2011. 787
750 Res. Int. 2013. Cytotoxicity, permeability, and inflammation of metal oxide 788

O
751 Mohandas, T., Grant, W., 1972. Cytogenetic effects of 2,4-D and nanoparticles in human cardiac microvascular endothelial 789
752 amitrole in relation to nuclear volume and DNA content in cells: cytotoxicity, permeability, and inflammation of metal 790
753 some higher plants. Can. J. Genet. Cytol. 14 (4), 773–783. oxide nanoparticles. Cell Biol. Toxicol. 27 (5), 333–342. 791

O
754 Murdock, R.C., Braydich-Stolle, L., Schrand, A.M., Schlager, J.J., Thordal-Christensen, H., Zhang, Z., Wei, Y., Collinge, D.B., 1997. 792
755 Hussain, S.M., 2008. Characterization of nanomaterial Subcellular localization of H2O2 in plants. H2O2 accumulation 793
756 dispersion in solution prior to in vitro exposure using dynamic in papillae and hypersensitive response during the 794

R
757 light scattering technique. Toxicol. Sci. 101 (2), 239–253. barley—powdery mildew interaction. Plant J. 11 (6), 1187–1194. 795
758 Nagaonkar, D., Shende, S., Rai, M., 2015. Biosynthesis of copper Tice, R., Agurell, E., Anderson, D., Burlinson, B., Hartmann, A., 796

P
759 nanoparticles and its effect on actively dividing cells of mitosis Kobayashi, H., et al., 2000. Single cell gel/comet assay: 797
760 in Allium cepa. Biotechnol. Prog. 31 (2), 557–565. guidelines for in vitro and in vivo genetic toxicology testing. 798
761 Oberdörster, G., Oberdörster, E., Oberdörster, J., 2005. Environ. Mol. Mutagen. 35 (3), 206–221. 799
762 Nanotoxicology: an emerging discipline evolving from studies Xia, T., Kovochich, M., Brant, J., Hotze, M., Sempf, J., Oberley, T., et 800
763 of ultrafine particles. Environ. Health Perspect. 823–839.
Dal., 2006. Comparison of the abilities of ambient and 801
764 Olive, P.L., Banáth, J.P., 2006. The comet assay: a method to manufactured nanoparticles to induce cellular toxicity 802
E
765 measure DNA damage in individual cells. Nat. Protoc. 1 (1), according to an oxidative stress paradigm. Nano Lett. 6 (8), 803
766 23–29. 1794–1807. 804
767 Pakrashi, S., Jain, N., Dalai, S., Jayakumar, J., Chandrasekaran, P.T., Yeheskel, O., Chaim, R., Shen, Z., Nygren, M., 2005. Elastic moduli 805
T

768 Raichur, A.M., et al., 2014. In vivo genotoxicity assessment of of grain boundaries in nanocrystalline MgO ceramics. J. Mater. 806
769 titanium dioxide nanoparticles by Allium cepa root tip assay at Res. 20 (03), 719–725. 807
C

770 high exposure concentrations. PLoS One 9 (2), 87789. 808

771 Rajeshwari, A., Kavitha, S., Alex, S.A., Kumar, D., Mukherjee, A.,
772 Chandrasekaran, N., et al., 2015. Cytotoxicity of aluminum
E

809
R
R
O
C
N
U

Please cite this article as: Mangalampalli, B., et al., Allium cepa root tip assay in assessment of toxicity of magnesium oxide
nanoparticles and microparticles, J. Environ. Sci. (2017), http://dx.doi.org/10.1016/j.jes.2017.05.012