Research article

Received: 25 August 2016, Revised: 26 March 2017, Accepted: 6 April 2017 Published online in Wiley Online Library: 6 June 2017

(wileyonlinelibrary.com) DOI 10.1002/jat.3485

Iron oxide nanoparticles induced cytotoxicity,

oxidative stress and DNA damage in
lymphocytes
Usha Singh Gaharwar*, Ramovatar Meena and Paulraj Rajamani*
ABSTRACT: Over the past few decades nanotechnology and material science has progressed extremely rapidly. Iron oxide
nanoparticles (IONPs) owing to their unique magnetic properties have a great potential for their biomedical and bioengineering
applications. However, there is an inevitable need to address the issue of safety and health effects of these nanoparticles.
Hence, the present study was aimed to assess the cytotoxic effects of IONPs on rats’ lymphocytes. Using different assays, we
studied diverse parameters including mitochondrial membrane potential, intracellular accumulation of reactive oxygen species
(ROS), lactate dehydrogenase activity, antioxidant enzymes activity and DNA damage measurements. Intracellular metal uptake
and ultrastructure analysis were also carried out through inductively coupled plasma atomic emission spectroscopy,
transmission electron microscopy respectively. The results show that the IONP-induced oxidative stress was concentration-
dependent in nature, with significant (P < 0.05) increase in ROS levels, lipid peroxidation level as well as depletion of
antioxidant enzymes and glutathione. Moreover, we observed morphological changes in the cell after intracellular uptake
and localization of nanoparticles in cells. From the findings of the study, it may be concluded that IONPs induce
ROS-mediated cytotoxicity in lymphocytes. Copyright © 2017 John Wiley & Sons, Ltd.

Keywords: iron oxide nanoparticles; lymphocyte; oxidative stress; cytotoxicity; intracellular uptake

Introduction have been found to reduce mitochondrial activity of human

fibroblasts at higher concentrations (Auffan et al., 2006). A dose-
Nanotechnology has a wide range of applications in medicine, dependent cytotoxicity of magnetic NPs has been reported earlier
industry and cosmetics, and is a rapidly advancing discipline (Tiede (Pisanic et al., 2007; Kai et al., 2011). IONPs may also generate
et al., 2008). Owing to its diverse applications and immense growth injuries in endothelial cells and due to their ultrafine sizes, they
in the last two decades, there has been a sustained increase in the can interact with different organelles and can elicit a variety of
presence of nanoparticles (NPs) in our environment as well. tissue responses including cell activation, generation of reactive
However, the toxicological effects or any adverse effects on human oxygen species (ROS) and cell death (Wilson et al., 2002; Zhu
health have still not been addressed adequately. These NPs may et al., 2011; Cheng et al., 2013). Hanini et al. (2011) reported IONPs
enter the body through inhalation, dermal contacts and injection. (Fe2O3) were efficiently taken up in vitro by human umbilical
Enhanced accumulation of these NPs could be toxic if the vascular endothelial cells, which represent the first biological
concentration goes over the threshold limit. Iron oxide NPs (IONPs; barrier to NPs in vivo. Another study showed that internalization
size <100 nm) have great potential for modernizing nanomedicine of uncoated super-paramagnetic NPs (SPIONs) resulted in
with its application in biomedical devices and delivery systems. disruption of cytoskeletal organization of human dermal
IONPs are being used in a wide range of therapeutical applications fibroblasts (Gupta and Gupta, 2005).
due to their small size, large surface to area volume and their Although information about the toxicity of IONPs continues
magnetic properties. They are also used as contrast agents in to increase, a knowledge gap exists due to the lack of a
magnetic resonance imaging, detoxification of biological fluids complete toxicological profile of these promising NPs for safe
and targeted drug delivery (Singh et al., 2012); in tissue repair, cell use. The spleen is one of the vital organs involved in the
and tissue targeting and transfection (Kang et al., 1996; Hafeli et al., immune response. Therefore, we have chosen splenic
1997; Zaitsev et al., 1999; Lian et al., 2003, 2004; Mahmoudi et al., lymphocytes, which elicit the immune response to xenobiotic
2009); and in neurobiological applications (Weinstein et al., 2010; compounds such as NPs etc. if they enter into the body. The
Winer et al., 2011; Geppert et al., 2012). present study was aimed to find out the cytotoxic effects of
However, the concerns over IONPs rendered cytotoxicity to IONPs in rats’ splenic lymphocytes.
biological tissues remains to be studied in detail. IONPs may
induce cytotoxicity by impairing the functions of major
components of the cell, such as mitochondria, nucleus and DNA
(Oberdorster et al., 2007). It has been reported that IONPs
significantly reduce cell viability of human macrophages, epithelial *Correspondence to: Paulraj Rajamani, School of Environmental Sciences,
Jawaharlal Nehru University, New Delhi 110067, India.
cells (Soto et al., 2007) human mesothelioma (Brunner et al., 2006) E-mail: paulrajr@hotmail.com, usha.singh77@gmail.com
and inhibit normal formation of PC12 neuronal cell morphology
(Pisanic et al., 2007). 2,3-Dimercaptosuccinic acid-coated IONPs School of Environmental Sciences, Jawaharlal Nehru University, New Delhi, India
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J. Appl. Toxicol. 2017; 37: 1232–1244 Copyright © 2017 John Wiley & Sons, Ltd.
Iron oxide nanoparticles toxicity in lymphocytes

Materials and methods RPMI medium supplemented with 10% fetal bovine serum and
1× penicillin–streptomycin.
Reagents
Iron oxide nano-powder (Fe2O3) was purchased from Intelligent Morphological assessment of lymphocytes
Materials Pvt. Ltd. (Wilmington, DE, USA). Fetal bovine serum,
Changes in cell ultra-structure caused by NPs were visualized
penicillin–streptomycin, RPMI-1640 (Roswell Park Memorial
using TEM. Autophagy was evaluated by examining
Institute 1640) medium, 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB),
autophagosome formation. The lymphocytes were seeded in a
reduced glutathione (GSH), 2-thio-barbituric acid, 2,7-
24 well plate at a density of 1 × 105 cells per well in 1 ml of
dichlorofluorescin diacetate (DCFH-DA), ethylene diamine
complete medium for different time intervals, i.e. 24, 48 and
tetraacetic acid (EDTA), bovine serum albumin and pyrogallol were
72 h with different concentrations of IONPs. For control
purchased from Sigma-Aldrich (St. Louis, MO, USA). All other
experiments, medium without NPs was used. After exposure
chemicals used were procured from local chemical companies.
medium was removed, the cells were washed with PBS and
further fixed with 2.5% gluteraldehyde in 0.1 M PBS for 4 h at
Characterization of Fe2O3 nanoparticles 37°C. The cells were then post-fixed in 1% osmium tetroxide,
and 1% tannic acid. Samples were dehydrated through a series
Dry powder of IONPs was suspended in phosphate-buffered saline of alcohol concentrations (20%, 30%, 40%, 50%, 60%, 70%)
(PBS) at a concentration of 50 μg ml 1 and was sonicated using a followed by further dehydration (90%, 96%, 100% and dry
sonicator bath at room temperature for 30 min. Morphology, alcohol). Cells were finally treated with propylene oxide followed
distribution and size of IONPs (Fe2O3) were evaluated by by 1: 1 propylene oxide/resin overnight to evaporate the
transmission electron microscopy (TEM; Joel, Tokyo, Japan) at an propylene oxide. Embedding was done in “better equipment
accelerating voltage of 200 kV. The aqueous suspension was drop for electron microscopy” capsules using pure Spurr’s low
casted on a copper grid, and the grid was air dried at room viscosity resin at 80°C for 48 h. Ultra-thin sections (70 nm) were
temperature (25°C) before loading on to the microscope. taken using the Leica EM UC 6 ultra-microtome (Wetzlar,
Dynamic light scattering (DLS) was performed to find out Germany) and stained with 1% lead acetate. Sections were
hydrodynamic size distribution by using a Malvern DLS examined under JEOL-JEM- 2100F TEM operating at 120 kV. To
apparatus (Nano-ZS; Malvern Instruments, Malvern, UK) with a confirm localization of the NPs, elemental mapping was also
633 nm He/Ne laser. carried out (Tiwari et al., 2011).

Animals Determination of degree of penetration of iron oxide

Healthy male Wistar rats (6–8 week old) (animal house facility; JNU, nanoparticles into cells by inductively coupled plasma atomic
New Delhi, India) were used to isolate the splenic lymphocytes. emission spectroscopy method
Animals were kept in an experimental room under controlled Iron content within the cells was analyzed by the inductively
environmental conditions of temperature (21 ± 3°C) and humidity coupled plasma atomic emission spectroscopy (ICP-AES) method.
(50 ± 10). All animals were kept in an animal house at 12 h After the different incubation time, the cells were washed with
light/dark cycle and fed food pellet and water ad libitum. All PBS many times to remove residual NPs on the surface of the cells
animals were kept in stress-free, hygienic and animal-friendly and after centrifugation supernatant was discarded and pellet was
conditions. All experimental protocols were followed as per the again washed with PBS. Pellet was digested in HNO3, then the
guideline and approval of the Institutional Animal Ethics resulting clear solution was added to 25 ml MilliQ water and
Committee, Jawaharlal Nehru University, New Delhi, India analyzed by ICP-AES ( Jobin Yvon ULTIMA 2, UK).
(approval no. 14/2013).

Cell viability assay using trypan blue dye exclusion method

Isolation of splenic lymphocytes
Reduction in cell viability was assessed by counting lymphocytes
Rats were anesthetized with sodium thiopentone and killed by stained with trypan blue dye (80 mM; Gibco BRL–Life Technologies,
cervical dislocation. The spleen was dissected out and splenic Grand Island, NY, USA). Lymphocytes were incubated with
lymphocytes were prepared by gentle teasing of the spleen in different concentration of IONPs (Fe2O3) for different time
RPMI-1640 culture medium on a soft sterile nylon mesh. Red blood intervals 24, 48 and 72 h. At the end of the treatment period,
cells from cell suspension were removed by brief treatment with cells were exposed to trypan blue for 5 min at room
0.83% NH4Cl. Cell suspension was washed with RPMI-1640, temperature, and the percentage of trypan blue-positive and -
followed by preparation of suspension in the same medium. negative cells was counted in four random fields per well using
Macrophages were allowed to adhere in plastic Petri dishes at a hemocytometer (Calcabrini et al., 2004).
37°C and then the non-adherent cell population was collected
and used for further experiments. Cells were divided into five
Lactate dehydrogenase leakage assay
different groups. For exposure, a stock solution of IONPs was
prepared in PBS and briefly sonicated to break up NPs Single cell suspension was prepared from all five groups, and the
agglomerates. Stock solutions were serially diluted in RPMI cells were seeded in 96 well plate at a cell density of ∼1 × 105 cells
medium. Group I (control group) was treated with PBS and the per well in 120 μl of culture medium. Cells were incubated at 37°C
other four groups were treated with different concentrations of and 5% CO2. The assay was done according to the protocol
IONPs (Fe2O3), i.e. 100, 200, 400 and 800 μg ml 1 and incubated provided with lactate dehydrogenase (LDH) assay kit (Cayman
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for 24, 48 and 72 h. Cells were incubated at 37°C with 5% CO2 in Chemical Company, Ann Arbor, MI, USA). In brief, 100 μl of cell

J. Appl. Toxicol. 2017; 37: 1232–1244 Copyright © 2017 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jat
 U. S. Gaharwar et al.

supernatant was added to 100 μl of reaction solution. It was Preparation of cell lysate
incubated for 30 min at room temperature and the absorbance
Cell lysates of the lymphocytes were prepared to perform
was taken at 490 nm using a microtiter plate reader (Biorad,
biochemical assays. Cells were washed with PBS then the pellet
California, USA). Blank LDH levels were subtracted from LDH
was resuspended in lysis buffer (20 mM Tris–HCl pH 7.4, 2 mM EDTA,
sample values.
2 mM EGTA, 6 mM mercaptoethanol, 1% NP-40, 0.1% sodium
dodecyl sulfate, 1 mM phenyl methane sulfonyl fluoride and the
protease inhibitor cocktail) and incubated for 2 h at 4°C after
Mitochondrial membrane potential centrifugation and supernatants were collected for carrying out
further biochemical tests (Rastogi et al., 2010).
Single cell suspension was prepared from all five groups, and the
cell density was adjusted to ∼1 × 105 cells ml 1 in PBS. JC-1 dye
assay was done according to the manufacturer’s protocol provided
Protein quantification
with the kit (Cayman Chemical Company). In brief, 10 μl JC-1 was
added to 100 μl cell suspension with cell density of 5 × 105 cells The total protein concentration was measured by the Bradford
ml 1 in 96 well plates. Samples were then incubated for 15 min at method (Bradford, 1976) using bovine serum albumin as the
37°C and 5% CO2. Cells were analyzed by a fluorescence plate standards.
reader (SPECTRAmax M5 ROM v2.1.35, Molecular Devices,
Sunnyvale, CA, USA). In healthy cells, JC-1 molecule form
aggregates that display strong fluorescence intensity with Estimation of lipid peroxidation
excitation and emission wavelengths of 560 and 595 nm,
Lipid peroxidation (LPO) was estimated by thiobarbituric acid
respectively, whereas in apoptotic cells, it forms monomers, which
reactive substances (Varshney and Kale, 1990) and is expressed in
displays strong fluorescence intensity with excitation and emission
terms of malondialdehyde (MDA) formed per milligram of protein.
of 485 and 535 nm, respectively. Values were expressed as a
In brief, 0.4 ml of sample was mixed with 1.6 ml of Tris–KCl (0.15 M
percentage of the fluorescence intensity relative to that of the
KCl + 10 mM Tris–HCl, pH 7.4) buffer to which 0.5 ml of 30%
control wells.
trichloroacetic acid was added. Then, 0.5 ml of thiobarbituric acid
was added. The tubes were covered with aluminum foil and placed
in a water bath for 45 min at 80°C, then cooled in ice and
Estimation of intracellular reactive oxygen species centrifuged at room temperature for 10 min at 2300 g. Absorbance
of the supernatant was measured at 531.8 nm with
The production of intracellular ROS was measured using DCFH-DA
spectrophotometer (Shimadzu spectrophotometer UV4100).
(Wang and Joseph, 1999). Briefly, 10 mM DCFH-DA stock solution
(in methanol) was diluted in culture medium to yield a 100 μM
working solution. At the end of exposure with NPs, cells were
Estimation of reduced glutathione
washed twice with PBS. Cells were incubated in 1 ml working
solution of DCFH-DA at 37°C for 30 min, lysed in alkaline solution Analysis of GSH concentration was performed with the method
and washed with PBS. Fluorescence intensity was measured using described by Ellman (1959) and modified by Jollow et al. (1974)
485 excitation and 520 nm emission filters using a fluorimeter based on the development of a yellow color when DTNB is added
(RF-5301 PC Shimadzu spectrofluorometer, Nakagyo-ku, Kyoto, to compounds containing sulfhydryl groups. In brief, 0.2 ml of
Japan). The values were expressed as a percentage of sample was added to 3 ml of 4% sulfosalicylic acid and tubes were
fluorescence intensity relative to the control wells. The centrifuged at 1600 g for 15 min. Supernatant (0.2 ml) was mixed
percentage increase in fluorescence per well was calculated with with 0.4 ml of 10 mM DTNB and 1 ml phosphate buffer (0.1 M,
the formula [(Ft/Ft0 × 100], where Ft0 is the fluorescence at 24 h of pH 7.4). Finally, absorbance at 412 nm was recorded. Total GSH
the control sample and Ft is the fluorescence at 24, 48 or 72 h in content was expressed as nmol GSH mg 1 protein.
the presence of NPs.

Estimation of superoxide dismutase

Detection of apoptosis by Annexin V fluorescein Superoxide dismutase (SOD) activity was determined as per the
isothiocyanate/propidium iodide assay method given by Marklund and Marklund (1974). The cell lysates
containing 50 μg protein were treated with Triton-X-100 (1%)
Apoptosis was evaluated using the Annexin V fluorescein
and kept at 4°C for 30 min followed by addition of 1 ml of assay
isothiocyanate (FITC)/propidium iodide (PI) Apoptosis Kit (Cayman
mixture containing 0.05 M sodium phosphate buffer (pH 8.0),
Chemical company, Ann Arbor, MI, USA). Cells were seeded in 24
0.01 M EDTA and 0.27 mM pyrogallol. The absorbance was
well plates at a cell density of ∼5–10 × 105 cells per well.
measured for 5 min at 420 nm. The enzyme activity was expressed
Lymphocytes were treated with different concentrations of IONPs
as U mg 1 protein, where 1 U is the amount of enzyme required to
for 24 h. After exposure, the cells were harvested, washed and re-
bring about 50% inhibition of the auto-oxidation of pyrogallol.
suspended with PBS. The Annexin V FITC/PI staining of cells was
performed as per the manufacturer’s instructions (Cayman
Chemical Company, Ann Arbor, MI, USA). Then the samples were
Catalase assay
analyzed with a flow cytometer (Beckman Coulter, Miami, FL,
USA). At least 10 000 cells were analyzed to determine the The activity of catalase (CAT) enzyme was measured as described
percentage of apoptotic cells. The results are presented as a by Aebi (1984). CAT activity was calculated from the slope of the
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percentage of apoptotic cells. H2O2 absorbance curve and normalized to protein concentration.

wileyonlinelibrary.com/journal/jat Copyright © 2017 John Wiley & Sons, Ltd. J. Appl. Toxicol. 2017; 37: 1232–1244
Iron oxide nanoparticles toxicity in lymphocytes

Single-cell gel electrophoresis: Comet assay

The whole procedure was done according to Gaharwar and Paulraj
(2015). Images (40 cells per slide) were acquired from random fields
using fluorescence microscope (Carl Ziess, Göttingen, Germany)
attached with digital camera (TK1280; JVC, Yokohama, Japan). Tail
moment and olive tail moment of 40 randomly selected cells were
analyzed from each slide by using comet score software.

Statistical analysis
All the data represented in this study are means ± SD, made in six
replicate. Statistical significance was determined by one-way
analysis of variance (ANOVA) followed by Student–Newman–Keuls
multiple comparison tests. Significance was ascribed at P < 0.05.
All analysis was conducted using Sigma Plot 11 statistical package
(Systat Software, San Jose, CA, USA). Pearson correlation coefficient
(r) was used to evaluate correlations among various endpoints. Figure 2. Dynamic light scattering histogram shows hydrodynamic
diameter size detection of iron oxide nanoparticles in phosphate-buffered
saline buffer. [Colour figure can be viewed at wileyonlinelibrary.com]
Results
Characterization of iron oxide nanoparticles (Fe2O3)
throughout the cytoplasm. Moreover, NPs were also found to
TEM analysis showed that the average size of Fe2O3 was 30–35 nm enter the nucleus and mitochondria. Geppert et al. (2012)
(Fig. 1). The DLS study revealed an unstable suspension with a suggested that the endocytotic processes are involved in the
tendency to form aggregates at higher concentrations (Fig. 2). uptake of IONPs in astrocytes. At lower concentration, IONPs
were mainly found to adhere to the cell membrane after 24 h
but at higher concentration (800 μg ml 1) IONPs are
Uptake of iron oxide nanoparticles by lymphocytes incorporated into lymphocytes. As the concentration and time
Uptake of IONPs was seen in all exposed lymphocyte groups. increases, internalization started and many small vesicles
TEM images and elemental mapping have revealed that IONPs pockets became introduced into the cell. After 72 h, NPs were
were distributed on the cell membrane as well as inside the seen to accumulate inside the cell in all groups. The results of
cell. IONPs were found to adhere in the membrane of the cells elemental mapping (Fig. 4) also showed the presence of the
followed by a pit formation and was further internalized and IONPs into the cells and it was a time- and concentration-
seen to accumulate in the intracellular vesicles (Fig. 3). A small dependent process. The cells treated with higher IONPs
pocket was also found in the cells where NPs were internalized concentrations showed an increase in the number of vacuoles.
possibly via endocytosis, and accumulated in digestive vacuoles. In addition, there were many vacuoles within the cytosol. Cells
NPs were found in the form of clusters in vesicles spread treated with NPs showed autophagic vacuoles (Fig. 3). The

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Figure 1. Characterization of iron oxide nanoparticles (Fe2O3). Transmission electron microscopy images of iron oxide nanoparticles.

J. Appl. Toxicol. 2017; 37: 1232–1244 Copyright © 2017 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jat
 U. S. Gaharwar et al.

Figure 3. Transmission electron microscopy images of lymphocyte exposed at different times in different concentration of IONPs. (A) Control cell. (B) Cell
1
containing 200 μg ml IONPs. (C) IONPs attached to nuclear membrane. (D) Elongated mitochondrial structure. (E,F) Autolysosome (thick arrow); autophagy
vesicle formation (thin arrow). (G) NPs accumulated inside the vesicle (long arrow). M, mitochondria; N, Nucleous; IONPs, iron oxide nanoparticles. [Colour
figure can be viewed at wileyonlinelibrary.com]

shape and size of IONPs vesicular aggregates was uneven and significantly at every level at a 100 μg ml 1 increase was 8.92%
unequal and it enclosed some cell debris. (24 h, P < 0.05), 6.8% (48 h, P < 0.05) and 12.21% (72 h, P < 0.05).
The presence of intracellular Fe2O3 was confirmed through ICP- Other treatment, i.e. 200, 400 and 800 μg ml 1 showed
atomic emission spectroscopy (Table 1). These measurements significant increase of 34.74%, 97.89%, 140.07% (P < 0.05, at
show the intracellular iron in the cells. Results indicate that iron 24 h), 29.3%, 112.3%, 199.65% (P < 0.05, at 48 h) and 35.93%,
content was increased significantly in a time- and concentration- 155.084%, 265.071% (P < 0.05, at 72 h) respectively. The activity
dependent manner. The iron content showed a significant was found to increase in both a concentration- and time-
increase in the treated cells as compared to the control. dependent manner in the 400 and 800 μg ml 1 treated groups,
whereas only a concentration-dependent increase was observed
in lower doses (100 and 200 μg ml 1). There was a negative
Lymphocyte viability correlation between LDH levels and cell viability (Pearson
IONPs decreased the lymphocyte viability in a dose- and time- product moment correlation coefficient r = 0.874, P < 0.001)
dependent manner. Lymphocytes were exposed to IONPs (Fe2O3) (Fig. 6). This shows that the toxic effect of IONPs cause cell
at 100, 200, 400 and 800 μg ml 1 for 24, 48 and 72 h. Cell viability damage.
decreased as a function of both time and concentration (Fig. 5).
At 100 μg ml 1, the cell viability gradually decreased as the
exposure time increased while significant decrease was observed Mitochondrial membrane potential
at 72 h (15.79%, P < 0.05). Cell viability was significantly decreased The effects of IONPs (Fe2O3) on the mitochondria membrane
with 200 μg ml 1 at 24 h (19.10%, P < 0.05), 48 h (25.5%, P < 0.05) potential (MMP) (Δψm) of lymphocytes in different groups were
and 72 h (37.80%, P < 0.05), whereas cell viability was found to be analyzed with JC-1 dye staining. Presence of J aggregate
reduced significantly by 28.51% (P < 0.05), 34.26% (P < 0.05) and complexes was higher in the control group (Fig. 7). Significant
48.72% (P < 0.05) at 24, 48 and 72 h respectively at 400 μg ml 1. differences were seen between the control and all four
Moreover, high concentration treated groups (800 μg ml 1) showed experimental groups (P < 0.05). A decrease was observed at
a reduction in viability at 24 h (35.59%, P < 0.05), 48 h (48.58%, 100 μg ml 1 (up to 5.1%, P < 0.05), 200 μg ml 1 (up to 9.74%,
P < 0.05) and 72 h (59.73 P < 0.05). A time- and concentration- P < 0.05), 400 μg ml 1 (40.87%, P < 0.05) and 800 μg ml 1 (up to
dependent significant decrease in cell viability (P < 0.05) was 52.51%, P < 0.05) respectively. Therefore, the result shows the
observed in all treated cells with the 200–800 μg ml 1 group. absence of active mitochondria in the treated group with a lower
membrane potential as compared to the control group. A
significant positive correlation between cell viability and MMP
Lactate dehydrogenase activity
levels (Pearson product moment correlation coefficient r = 0.876,
A significant increase was found in the LDH level (P < 0.05) as P < 0.001) (Fig. 7) indicates that the decrease in mitochondrial
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compared to control. LDH concentration was increased function may alter viability.

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Iron oxide nanoparticles toxicity in lymphocytes

Figure 4. Elemental mapping images of lymphocyte containing different concentration of iron oxide nanoparticles for different duration. Internalized
1 1
nanoparticles are shown in red. (i) Cells treated with 100 μg ml concentration for 24 h (A), 48 h (B), 72 h (C). (ii) Cells treated with 200 μg ml concentration
1 1
for 24 h (D), 48 h (E), 72 h (F). (iii) Cells treated with 400 μg ml concentration for 24 h (G), 48 h (H), 72 h (I). (iv) Cells treated with 800 μg ml concentration
for 24 h ( J), 48 h (K), 72 h (L).
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J. Appl. Toxicol. 2017; 37: 1232–1244 Copyright © 2017 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jat
 U. S. Gaharwar et al.

Table 1. Degree of iron oxide nanoparticle penetration into

cells, determined using the ICP-AES method (expressed as iron
content)

Incubation Iron oxide Fe

time (h) nanoparticles (μg ml 1) content (ppm)
24 Control 0.34 ± 0.23
100 1.43 ± 0.11
200 2.20 ± 1.14
400 2.38 ± 0.06(a)
800 4.28 ± 1.88
48 Control 0.91 ± 0.82
100 1.86 ± 0.34
200 2.37 ± 0.45
400 3.27 ± 1.18(a)
800 5.35 ± 0.31(a,d)
72 Control 0.79 ± 0.78 Figure 6. LDH release in lymphocyte induced by IONPs of various
100 1.95 ± 1.71 concentrations at 24, 48 and 72 h. Data representing IONPs are mean ±
SD of experiments made in six replicates. Statistically significant difference
200 2.41 ± 0.26(a)
as compared to the controls (P < 0.05). Inter-group statistical significant
400 4.42 ± 1.49(a) differences have been marked by letters: a (vs. control), b (vs. 100 μg
800 5.53 ± 2.8 1 1
ml concentration of IONPs), c (vs. 200 μg ml concentration of IONPs),
1
Mean values ± SD (n = 3). Statistically significant difference as d (vs. 400 μg ml concentration of IONPs) and inter-hours significant
compared to the controls (P < 0.05). differences were marked by symbol: # (vs. 24 h); * (vs. 48 h). IONPs, iron
oxide nanoparticles; LDH, lactate dehydrogenase.

Figure 5. Cell viability of lymphocytes treated with different concentrations

of IONPs for 24, 48 and 72 h. Data represented are mean ± SD of experiments Figure 7. Assessment of mitochondrial membrane potential (Δψm) in the
made in six replicates. Statistically significant as compared to controls lymphocytes treated with different concentration of IONPs for 24, 48 and
(P < 0.05). Inter-group statistically significant differences have been 48 h. Lymphocytes are labeled with Δψm-sensitive dye JC-1 and measured
1
marked by letters: a (vs. control), b (vs. 100 μg ml concentration of by enzyme-linked immunosorbent assay plate reader (I). J aggregates
1 1
IONPs), c (vs. 200 μg ml concentration of IONPs), d (vs. 400 μg ml (orange fluorescence) were detected with excitation and emission at 560
concentration of IONPs) and inter-hours significant differences were and 595 nm respectively. Data represented are mean ± SD of experiments
marked by symbol: # (vs. 24 h); * (vs. 48 h). IONPs, iron oxide nanoparticles. made in six replicates. Statistically significant difference as compared to
the controls (P < 0.05). Inter-group statistically significant differences have
1
been marked by letters: a (vs. control), b (vs. 100 μg ml concentration
1 1
Reactive oxygen species of IONPs), c (vs. 200 μg ml concentration of IONPs), d (vs. 400 μg ml
concentration of IONPs) and inter-hours significant differences were
At low concentrations, exposure to Fe2O3 resulted in a slight marked by symbol: # (vs. 24 h); * (vs. 48 h). IONPs, iron oxide nanoparticles.
increase in intracellular ROS. However, exposure at higher
concentrations showed a significant increase (P < 0.05). The
generation of ROS was found to increase significantly at 100 μg P < 0.001) (Fig. 8), which indicates that increased oxidative stress
ml 1 (up to 142.82%, P < 0.05), 200 μg ml 1 (up to 173.75%, may have led to the imbalance in MMP.
P < 0.05), 400 μg ml 1 (297.16%, P < 0.05) and 800 μg ml 1 (up
to 406.52%, P < 0.05) respectively. The concentration- and time-
Apoptosis
dependent increase in fluorescence was found. There was a
significant negative correlation between ROS levels and MMP Apoptosis assays were performed using Annexin V FITC/PI
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(Pearson product moment correlation coefficient r = 0.856, staining and the cells were analyzed with a flow cytometer

wileyonlinelibrary.com/journal/jat Copyright © 2017 John Wiley & Sons, Ltd. J. Appl. Toxicol. 2017; 37: 1232–1244
Iron oxide nanoparticles toxicity in lymphocytes

Figure 8. Accumulation of intracellular ROS in lymphocyte induced by

IONPs of various concentrations for 24, 48 and 72 h. Data represented are
mean ± SD of experiments made in six replicate. *Statistically significant Figure 9. Induction of apoptosis by IONPs of various concentrations for
difference as compared to the controls (P < 0.05). The inter-group statistical 24 h. Cell death (apoptosis vs. necrosis) detection by staining with annexin
significant differences have been marked by letters: a (vs. control), b (vs. V/propidium iodide and analyzed by flow cytometry. Results are presented
1 1 as % apoptotic cells. Data represented are mean ± SD of experiments made
100 μg ml concentration of IONPs), c (vs. 200 μg ml concentration of
1 in three replicates. *Statistically significant difference as compared to the
IONPs), d (vs. 400 μg ml concentration of IONPs) and inter-hours
significant differences were marked by symbol: # (vs. 24 h); * (vs. 48 h). controls (P < 0.05). Inter-group statistical significant differences have been
1
IONPs, iron oxide nanoparticles. marked by letters: a (vs. control), b (vs. 100 μg ml concentration of IONPs),
1 1
c (vs. 200 μg ml concentration of IONPs), d (vs. 400 μg ml
concentration of IONPs). IONPs, iron oxide nanoparticles.
(Beckman Coulter). The results were presented as a percentage
of apoptotic cells. A significant increase (P < 0.05) in apoptosis
was found in lymphocytes treated with IONPs in a 800 μg ml 1 (up to 58.02%, P < 0.05) respectively. Overall, the
concentration-dependent manner (Fig. 9). data demonstrated a significant depletion (P < 0.05) of GSH
levels in IONP (Fe2O3)-exposed cells in both a time- and
concentration-dependent manner.
Antioxidant enzymes
To assess the extent of oxidative stress in lymphocytes, we
measured the activity of antioxidant enzymes, including the SOD,
Lipid peroxidation
GSH and CAT. Figure 10(A) showed a significant decrease
(P < 0.05) in the SOD activity in lymphocytes treated with higher The sensitivity of measuring thiobarbituric acid reactive
IONP (Fe2O3) concentrations as compared to control cells. At lower substances has made the assay a method of choice for screening
and higher concentrations, the SOD level was reduced in a time- and monitoring LPO, a major indicator of oxidative stress. Both
dependent manner (P < 0.05). A time-dependent significant the time- and concentration-dependent effects of IONPs
decrease was found in 100 μg ml 1 (P < 0.05) and 200 μg ml 1 (Fe2O3) in treated cells at higher doses are shown in Fig. 10(D).
(P < 0.05) after 72 h treatment while a significant decrease was A significant increase of 39.96% (P < 0.05) and 37.12 (P < 0.05)
24.17% (P < 0.05) at 24 h, 26.7% (P < 0.05) at 48 h in the was observed at 200 and 400 μg ml 1 after 72 h respectively
400 μg ml 1 treated group, whereas at 800 μg ml 1 the decrease while at high concentrations, i.e. 800 μg ml 1, there was a
was 35.52% (P < 0.05), 39.16% (P < 0.05), 47.39% (P < 0.05) at 24 h, significant increase of 82.2% (P < 0.05) and 110.36% (P < 0.05)
48 h and 72h respectively. Similarly, CAT activity was found to be after 48 and 72 h respectively. Higher concentrations showed
decreased at all 100, 200, 400 and 800 μg ml 1 concentrations. A significant (P < 0.05) changes, whereas changes observed at
significant (P < 0.05) concentration- and time-dependent low doses were not significant. In addition, There was a
depletion was observed in all exposed groups (Fig. 10B). CAT significant reverse correlation between the SOD and MDA levels
was found to decrease significantly at 100 μg ml 1 (up to 5.4%, (Pearson product moment correlation coefficient r = 0.90477,
P < 0.05), 200 μg ml 1 (up to 18.25%, P < 0.05), 400 μg ml 1 P < 0.001).
(40.36%, P < 0.05) and 800 μg ml 1 (up to 64.41%, P < 0.05)
respectively. There was a significant positive correlation between
CAT and SOD levels (Pearson product moment correlation
DNA damage
coefficient r = 0.933144, P < 0.001) (Fig. 10A). GSH is an
ubiquitous sulfhydryl-containing molecule in cells that is The DNA strand break has been considered as a standard
responsible for maintaining cellular oxidation–reduction biomarker for DNA damage. As we observed a persistent and
homeostasis. Alterations in GSH homeostasis can be considered enhanced ROS production, we further checked whether the
as an indication of functional damage to cells. A partial decrease ROS could mediate and enhance the formation of DNA breaks.
of GSH was detected at 100 μg ml 1. When the dose increased DNA breaks induced by IONPs were evaluated after different
to 800 μg ml 1, the intracellular GSH was reduced significantly exposures, i.e. 24, 48 and 72 h. A significant increase in DNA
(P < 0.05) (Fig. 10C). The GSH level was also significantly damage was found as compared to the control at higher
decreased at 100 μg ml 1 (up to 3.38%, P < 0.05), 200 μg ml 1 concentrations whereas no significant damage was seen in
1239

(up to 21.28%, P < 0.05), 400 μg ml 1 (28.43%, P < 0.05) and lower doses (Fig. 11A–C).

J. Appl. Toxicol. 2017; 37: 1232–1244 Copyright © 2017 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jat
 U. S. Gaharwar et al.

Figure 10. Effect of different concentrations of IONPs in an intracellular antioxidant system. (A) SOD; (B) catalase; (C) GSH; (D) MDA. Data represented are
mean ± SD of experiments made in six replicate. *Statistically significant difference as compared to the controls (P < 0.05). Inter-group statistical significant
1 1 1
differences have been marked by letters: a (vs. control), b (vs. 100 μg ml conc. of IONPs), c (vs. 200 μg ml conc. of IONPs), d (vs. 400 μg ml conc. of
IONPs) and inter-hours significant differences were marked by symbol: # (vs. 24 h); * (vs. 48 h). GSH, reduced glutathione; IONPs, iron oxide nanoparticles;
MDA, malondialdehyde; SOD, superoxide dismutase.

Figure 11. DNA damage induced by the IONPs at different doses for 24, 48 and 72 h. (A) Tail moment and (B) olive tail moment plotted against groups. (C)
1
Comet images of lymphocytes. (i) Control cells treated with phosphate-buffered saline; (ii) cells treated with 100 μg ml IONPs; (iii) cells treated with 200 μg
1 1 1
ml IONPs; (iv) cells treated with 400 μg ml IONPs; (v) cells treated with 400 μg ml IONPs. Tail moments of 40 randomly selected comets are presented.
Data represented are mean ± SD of experiments made. *Statistically significant difference as compared to the controls (P < 0.05). Inter-group statistical
1 1
significant differences have been marked by letters: a (vs. control), b (vs. 100 μg ml concentration of IONPs), c (vs. 200 μg ml concentration of IONPs),
1240

1
d (vs. 400 μg ml conc. of IONPs) and inter-hours significant differences were marked by symbol: # (vs. 24 h); *(vs. 48 h). IONPs, iron oxide nanoparticles.

wileyonlinelibrary.com/journal/jat Copyright © 2017 John Wiley & Sons, Ltd. J. Appl. Toxicol. 2017; 37: 1232–1244
Iron oxide nanoparticles toxicity in lymphocytes

Discussion mitochondria play a major role in apoptosis. Kai et al. (2011)

showed that magnetic NPs induced apoptosis through a
Different studies have reported the health effects of both mitochondria-dependent pathway in BEL-7402 cells. Similarly,
engineered nanomaterials and naturally occurring particles (Kagan Naqvi et al. (2010) reported a time- and concentration-dependent
et al., 2005; Curtis et al., 2006; Hardman, 2006). Previous studies increase in apoptosis of macrophages ( J774) treated with different
have reported that IONPs (Fe2O3) in particular are cytotoxic to concentration of SPIONs. Hussain et al. (2012) demonstrated that
certain cells (Lunov et al., 2010; Naqvi et al., 2010; Wu et al., 2010; CeO2 NP exposure leads to the cytotoxicity in human monocytes
Ramesh et al., 2012). Lymphocytes are an important target for associated with an induction of mitochondria-mediated apoptosis
NPs when they come in to contact with blood. and autophagy in a time- and dose dependent manner. Moreover,
TEM images confirm that the IONPs (Fe2O3) were taken up by Lou et al. (2016) showed that quercetin NPs induced autophagy
the cells (Fig. 3). Shape, size and charge are the main component and apoptosis in human neuroglioma cells. Li et al. (2012) found
related to internalization of the particles inside the cell. A time- that ZnO NPs effectively collapsed the MMP in the HeLa cells.
and concentration-dependent accumulation was observed in Generation of ROS seems to be the most widely studied toxic
lymphocytes. These results are in line with previous results in mechanisms of NPs. ROS generated by NPs favors pores formation
astrocytes (Geppert et al., 2012). Earlier studies reported the in mitochondria, mitochondrial permeability transition induces
intracellular uptake of different types of IONPs through different further ROS formation, which results in the impairment of
cell types (Mahmoudi et al., 2009, 2010; Novotna et al., 2012) and mitochondrial function (Sokol et al., 2001). Thus, increased
suggested that the mechanism behind it was the endocytotic oxidative stress is likely to induce disturbance in the MMP. It is
uptake. IONPs interact with mitochondria and nucleus. The reported that LPO induces mitochondrial injury that can initiate
observation suggests that the endocytotic processes may be further ROS production and results spread of free radical reactions
involved in the uptake and contributes to ultimate cell death (Catala, 2009). Peroxidation of lipids can alter the assembly of the
possibly by apoptosis (Wu et al., 2010). At higher concentration cell membrane, which in turn may cause changes in membrane
and longer duration (72 h), densely packed large vesicles were fluidity and permeability, alterations of ion transport and the
observed in the cells. It is expected that the fusion of several inhibition of metabolic processes (Nigam and Schewe, 2000).
endosomes may result in several large aggregates of IONPs in NPs may alter the production of ROS and interfere with
one vesicle. Results demonstrated that aggregated NPs elicited antioxidant defenses to induce oxidative stress (Saquib et al.,
much autophagic vacuoles. Huang et al. (2015) suggested that 2012; Srinivas et al., 2012; Meena et al., 2012b; Cheng et al., 2013;
the amount of NPs in endocytosis might be a key factor in the Capasso et al., 2014; Srikanth et al., 2014). Accumulated IONPs have
autophagic effect. Earlier studies have shown that various NPs been reported to induce oxidative stress in cultured cells (Luther
induce autophagy in different cells by using TEM (Hussain et al., et al., 2013). Iron oxide can generate oxidative stress through the
2012; Sun et al., 2012; Laha et al., 2014; Huang et al., 2015; Lou Fenton reaction. ROS was found to be elevated in the cells treated
et al., 2016). The ICP-AES results showed increased concentrations with IONPs as compared to the control (Park et al., 2008). The
of IONPs in the cells (P < 0.05). It is well known that SPION uptake induction of ROS was correlated with the concentration of NPs in
can vary greatly among different cell types (Arbab et al., 2003; cells and is due to the interaction of NPs with other cellular
Novotna et al., 2012). biomolecules (Park et al., 2008). IONPs produce free radical species
In this study, it was found that higher concentrations or longer that reduce the levels of cellular antioxidants significantly, which
incubations of IONPs reduced the cell viability of lymphocyte. It may lead to apoptosis via oxidative damage to intracellular
is reported that microglial cell viability reduced due to exposure proteins, DNA and increased LPO (Belozerskaia and Gessler, 2007;
of IONPs (Luther et al., 2013). Cytotoxicity may be due to the Ahmad et al., 2010; Naziroglu et al., 2010; Wan et al., 2012).
induction of oxidative stress and excess generation of ROS- Previous studies by Konczol et al. (2011) and Kawanishi et al.
induced mitochondrial membrane permeability. Similar effects (2013) have reported ROS generation in A549 and CHO cells
were observed in the cells treated with different NPs such as NPs treated with magnetite NPs. Current results showed a statistically
such as SiO2NPs, NiNPs, TiO2NPs and AuNPs. (Wang et al., 2009; significant decrease in the GSH content in comparison to control
Ahamed, 2011; Meena et al., 2012b; Chueh et al., 2014). In the from 24 to 72 h in the IONP-treated cells. The decrease in the
present study, we found significant LDH leakage in treated cells GSH content is used as a marker of oxyradical scavenging ability,
as compared to control cells. It has been commonly accepted that showing that the antioxidant defense system is overwhelmed by
the leakage of the cytosolic enzyme LDH correlates well with the amount of ROS. Another study showed that the decrease in
cellular viability. Choi et al. (2009) reported that iron oxide inhibits GSH content in the IONP-exposed gill cells suggest that the
cell viability as well as the concentration- and time-dependent antioxidant defense system of the gill is being stressed
increase in the LDH leakage in the A549 and L132 cell lines. Similar (Oberdorster, 2004). Similar types of result was observed in GSH
results were also found in human hepatoma SMMC-7721 cells content in gill cells of Anguilla anguilla exposed to IONPs (Ramsden
treated with CeO2 NPs and MCF-7 cells treated with et al., 2013; Srikanth et al., 2014). A decrease in GSH content and
hydroxyapatite NPs (Meena et al., 2012a; Cheng et al., 2013). A increase in LPO was reported in MRC-5 cells exposed to α-Fe2O3
decrease in the membrane potential leads to reduced up to 72 h (Radu et al., 2010). Murray et al. (2013) also found a
mitochondrial function, which eventually pushes the cells to concentration-dependent reduction in GSH levels in mouse
undergo apoptosis. We found a significant decrease in MMP level epidermal cells following exposure to dextron-coated SPION
in a concentration-dependent manner in the exposed particles. CAT is an important antioxidant enzyme, which acts
lymphocytes. It is well distinguished that many organelles upon H2O2. We found a significant decrease in CAT level treated
including mitochondria-mediated apoptosis was reported (Chen with higher concentration of IONPs. Our results are in line with
et al., 2007; Woldemariam and Mandal, 2008). MMP reduction is previous studies, which showed a reduction in CAT activity
the initial event leading to apoptosis (Petit et al., 1996). As MMP exposed to TiO2 and CuO NPs (Meena et al., 2012b; Melegari
1241

showed a significant decrease, the present study suggests that et al., 2013). LPO is an essential parameter to measure the oxidative

J. Appl. Toxicol. 2017; 37: 1232–1244 Copyright © 2017 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jat
 U. S. Gaharwar et al.

stress. The MDA level was found significantly increased in the Arbab AS, Bashaw LA, Miller BR, Jordan EK, Bulte JW, Frank JA. 2003.
IONP-treated groups as compared to the control. Srikanth et al. Intracytoplasmic tagging of cells with ferumoxides and transfection agent
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U.S.G. is thankful to the University Grant Commission, India for enhancement of surface modified magnetic nanoparticles. Biomaterials
fellowship grant during the period of study. The authors would 26(13): 1565–1573.
also like to thank Advance Research Instrumentation Facility, JNU Hafeli U, Schutt W, Teller J, Zborowski M. 1997. Scientific and Clinical
for TEM and elemental mapping analysis and Dr. J.K. Tripathi, Applications of Magnetic Carriers. Plenum Press: New York.
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Conflict of interest on physicochemical and environmental factors. Environ. Health Perspect.
114: 165–172.
The authors did not report any conflict of interest.
Huang D, Zhou H, Gao J. 2015. Nanoparticles modulate autophagic effect in
a dispersity-dependent manner. Sci. Rep. 5: 14361.
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