Ecotoxicology (2014) 23:1292–1304
DOI 10.1007/s10646-014-1272-0
Pb-inhibited mitotic activity in onion roots involves DNA damage
and disruption of oxidative metabolism
Gurpreet Kaur • Harminder Pal Singh •
Daizy Rani Batish • Ravinder Kumar Kohli
Accepted: 7 June 2014 / Published online: 15 July 2014
 Springer Science+Business Media New York 2014
Abstract Plant responses to abiotic stress significantly
affect the development of cells, tissues and organs. How-
ever, no studies correlating Pb-induced mitotic inhibition
and DNA damage and the alterations in redox homeostasis
during root division per se were found in the literature.
Therefore, an experiment was conducted to evaluate the
impact of Pb on mitotic activity and the associated changes
in the oxidative metabolism in onion roots. The cytotoxic
effect of Pb on cell division was assessed in the root
meristems of Allium cepa (onion). The mitotic index (MI)
was calculated and chromosomal abnormalities were
sought. Pb-treatment induced a dose-dependent decrease in
MI in the onion root tips and caused mitotic abnormalities
such as distorted metaphase, fragments, sticky chromo-
somes, laggards, vagrant chromosomes and bridges. Single
Cell Gel Electrophoresis was also performed to evaluate Pb
induced genotoxicity. It was accompanied by altered oxi-
dative metabolism in the onion root tips suggesting the
interference of Pb with the redox homeostasis during cell
division. There was a higher accumulation of malondial-
dehyde, conjugated dienes and hydrogen peroxide, and a
significant increase in the activities of superoxide dismu-
tases, ascorbate peroxidases, guaiacol peroxidases and
glutathione reductases in Pb-treated onion roots, whereas
catalases activity exhibited a decreasing pattern upon Pb
exposure. The study concludes that Pb-induced cytotoxic-
ity and genotoxicity in the onion roots is mediated through
G. Kaur  H. P. Singh (&)
Department of Environment Studies, Panjab University,
Chandigarh 160014, India
e-mail: hpsingh_01@yahoo.com; hpsingh_01@pu.ac.in
D. R. Batish  R. K. Kohli
Department of Botany, Panjab University, Chandigarh 160014,
India
123
ROS and is also tightly linked to the cell cycle. The
exposure to higher concentrations arrested cell cycle
leading to cell death, whereas different repair responses are
generated at lower concentrations, thereby allowing the
cell to complete the cell cycle.
Keywords Pb toxicity  Chromosomal abnormalities 
Mitotic index (MI)  Genotoxicity  DNA damage  Redox
homeostasis
Introduction
Heavy metals released into the environment pose major
environmental and health problems. Among the non-
nutrient heavy metals, lead (Pb) is the most widespread and
heaviest non-radioactive metal (Yücel et al. 2008). The
occurrence of Pb-pollution in agricultural soils is of great
ecological concern due to its high persistence in the envi-
ronment (Piechalak et al. 2002) and its impact on human
health (Li et al. 2005). Most of the Pb-contamination in the
soil results in its accumulation, via the food chain, to
concentrations exceeding their permissible limits by sev-
eral times. Being sensitive, plants serve as good indicators
of soil pollution, particularly with heavy metals (Seregin
and Ivanov 2001). The potential of a plant to accumulate
and/or tolerate heavy metals is variably species-specific
(Wierzbicka 1999). Previous studies have demonstrated the
toxic effect of Pb on germination, growth and physiological
attributes in crop plants such as Macrotyloma uniflorum
and Cicer arietinum (Reddy et al. 2005), Brassica juncea
(Meyers et al. 2008), Pisum sativum (Malecka et al. 2009),
Allium sativum (Liu et al. 2009), Zea mays (Gupta et al.
2009), Brassica campestris (Singh et al. 2011) and Triti-
cum aestivum (Kaur et al. 2012, 2013). Upon uptake, Pb
Pb induces DNA damage and disrupts redox homeostasis
largely accumulates in the plants roots and severely affects
root growth (Malecka et al. 2008; Kaur et al. 2013). Pri-
marily, Pb-toxicity manifests as stunted root growth,
probably due to the inhibition of cell division in the root
tips (Eun et al. 2000; Liu et al. 2009). Secondarily, Pb
induces oxidative stress via reactive oxygen species (ROS)
generation and results in cellular damage (Kaur et al.
2012). It has been suggested that the reduction in growth
due to the suppression of mitotic activity in plants is one of
the major adaptive responses that allows them to preserve
their energy and limit the heritable damage in the cells
(May et al. 1998). Additionally, oxidative stress has been
demonstrated to decrease the rate of DNA replication, thus
inhibiting the mitotic process in plants (Reichheld et al.
1999). However, no such study has been conducted prior to
the investigation whether Pb-induced DNA damage and
interference with root mitotic activity also involves an
alteration in the oxidative metabolism in the roots per se.
We, therefore, conducted a series of experiments to
investigate the impact of Pb on mitotic activity, DNA
damage, and the alterations in the redox homeostasis in the
roots of onion (Allium cepa L.). We chose onion as the test
plant to study these changes as it is widely used as a bio-
assay species to investigate mitotic inhibition and chro-
mosomal abnormalities, primarily due to higher percentage
of dividing cells, and relatively large-sized cells with fewer
number of large-sized monocentric chromosomes
(2n = 16) that stain well (Fiskesjö 1985, 1997).
Materials and methods
Materials
Onion (A. cepa L.) bulbs were procured from the local
cultivators. Lead (Pb) was supplied as lead nitrate
(MW = 331.21; 99 % purity) purchased from Merck Ltd.,
Mumbai, India. All the other biochemical reagents used in
the study were of analytical grade and purchased from
Sisco Research Laboratory Pvt. Ltd., India; Sigma Co., St.
Louis, USA; Merck Ltd., India; and Loba-Chemie Pvt.,
Ltd., India.
Raising of Onion root tips and Pb treatments
To expose the apices of the root primordia to Pb, onion
bulbs of uniform size were scraped and their dry scales
were peeled off. The bulbs were set for rooting in distilled
water in the dark for 4 days. Finally, the onion bulbs were
subjected to 0 (as negative control), 50, 100, 250, 500, and
1,000 lM Pb treatment or 100 lM methyl methane sufo-
nate (MMS; as positive control), respectively, for 24 h in
an environmentally controlled growth chamber under a
1293
14 h photoperiod of 240 lmol m-2 s-1 PFD at 18/8
(±2) C and 75 ± 2 % RH. In all, there were seven
treatments, including negative and positive control, with
five independent replications. The concentrations of Pb
present in the treatment solutions were measured prior to
experimentation, by atomic absorption spectrophotometer
(AAS, Model: EC 4139; ECL, Hyderabad, India), and
found to be 7.89 ± 0.12, 15.76 ± 0.23, 38.7 ± 0.85,
78.6 ± 0.46, and 157.8 ± 1.49 mg l-1 for 50, 100, 250,
500, and 1000 lM treatments, respectively. The used Pb
concentrations are ecologically realistic and comparable to
those present under natural conditions. The natural levels
of Pb in non-contaminated soil are in the range of
2–200 ppm (Nagajyoti et al. 2010), but industrial activities
such as mining, smelting and refining substantially increase
the Pb levels in the environment (400–800 ppm) (Chaney
et al. 1984). The average Pb amount in the agricultural soils
ranges from \1 to 135 mg kg-1 soil (Holmgren et al.
1993) and in residential areas it may reach upto
1,000 mg kg-1 soil (Ryan et al. 2004). In serpentine soils
and soils along highways, Pb levels may go upto
11,000 ppm (National Research Council 1980). The safe
limit of Pb levels in agricultural soils is 250–500 mg kg-1
soil and 300 mg kg-1 soil as per Indian (Awashthi 2000)
and European Union (European Union 2002) standards,
respectively.
After treatment, the roots were fixed in ethyl alcohol and
glacial acetic acid (3:1, v/v) for 24 h at 25 C. The fixed
roots were rinsed three times with distilled water, and
finally stored in 70 % (w/v) ethyl alcohol at 4 C until
further use.
Determination of Pb content
The amount of Pb in onion roots was determined using an
AAS. Roots were oven-dried (at 70 C for 48 h) followed
by digestion with HNO3 to HClO4 (3:1, v/v) solution. The
digested samples were dissolved in deionized water
(20 ml) and stored at 4 C until analysis. An aliquot of the
digest (2 ml) was analyzed for Pb. A reagent blank together
with a known standard (Sisco Research Laboratory,
Mumbai, India) were run along with the sample to maintain
the analytical quality, and the measurements were based on
the peak area.
Squash preparation
The effect of Pb on the mitotic activity in the onion root
tips was studied using the squash technique (Singh et al.
2005). The roots were first hydrolyzed in 1 N hydrochloric
acid (HCl) for 1 min at 25 C and then stained with
Schiff’s reagent for 30 min. Two root tips (*2 mm from
the tip; zone of division) were removed using forceps,
123
Pb induces DNA damage and disrupts redox homeostasis
largely accumulates in the plants roots and severely affects
root growth (Malecka et al. 2008; Kaur et al. 2013). Pri-
marily, Pb-toxicity manifests as stunted root growth,
probably due to the inhibition of cell division in the root
tips (Eun et al. 2000; Liu et al. 2009). Secondarily, Pb
induces oxidative stress via reactive oxygen species (ROS)
generation and results in cellular damage (Kaur et al.
2012). It has been suggested that the reduction in growth
due to the suppression of mitotic activity in plants is one of
the major adaptive responses that allows them to preserve
their energy and limit the heritable damage in the cells
(May et al. 1998). Additionally, oxidative stress has been
demonstrated to decrease the rate of DNA replication, thus
inhibiting the mitotic process in plants (Reichheld et al.
1999). However, no such study has been conducted prior to
the investigation whether Pb-induced DNA damage and
interference with root mitotic activity also involves an
alteration in the oxidative metabolism in the roots per se.
We, therefore, conducted a series of experiments to
investigate the impact of Pb on mitotic activity, DNA
damage, and the alterations in the redox homeostasis in the
roots of onion (Allium cepa L.). We chose onion as the test
plant to study these changes as it is widely used as a bio-
assay species to investigate mitotic inhibition and chro-
mosomal abnormalities, primarily due to higher percentage
of dividing cells, and relatively large-sized cells with fewer
number of large-sized monocentric chromosomes
(2n = 16) that stain well (Fiskesjö 1985, 1997).
Materials and methods
Materials
Onion (A. cepa L.) bulbs were procured from the local
cultivators. Lead (Pb) was supplied as lead nitrate
(MW = 331.21; 99 % purity) purchased from Merck Ltd.,
Mumbai, India. All the other biochemical reagents used in
the study were of analytical grade and purchased from
Sisco Research Laboratory Pvt. Ltd., India; Sigma Co., St.
Louis, USA; Merck Ltd., India; and Loba-Chemie Pvt.,
Ltd., India.
Raising of Onion root tips and Pb treatments
To expose the apices of the root primordia to Pb, onion
bulbs of uniform size were scraped and their dry scales
were peeled off. The bulbs were set for rooting in distilled
water in the dark for 4 days. Finally, the onion bulbs were
subjected to 0 (as negative control), 50, 100, 250, 500, and
1,000 lM Pb treatment or 100 lM methyl methane sufo-
nate (MMS; as positive control), respectively, for 24 h in
an environmentally controlled growth chamber under a
1293
14 h photoperiod of 240 lmol m-2 s-1 PFD at 18/8
(±2) C and 75 ± 2 % RH. In all, there were seven
treatments, including negative and positive control, with
five independent replications. The concentrations of Pb
present in the treatment solutions were measured prior to
experimentation, by atomic absorption spectrophotometer
(AAS, Model: EC 4139; ECL, Hyderabad, India), and
found to be 7.89 ± 0.12, 15.76 ± 0.23, 38.7 ± 0.85,
78.6 ± 0.46, and 157.8 ± 1.49 mg l-1 for 50, 100, 250,
500, and 1000 lM treatments, respectively. The used Pb
concentrations are ecologically realistic and comparable to
those present under natural conditions. The natural levels
of Pb in non-contaminated soil are in the range of
2–200 ppm (Nagajyoti et al. 2010), but industrial activities
such as mining, smelting and refining substantially increase
the Pb levels in the environment (400–800 ppm) (Chaney
et al. 1984). The average Pb amount in the agricultural soils
ranges from \1 to 135 mg kg-1 soil (Holmgren et al.
1993) and in residential areas it may reach upto
1,000 mg kg-1 soil (Ryan et al. 2004). In serpentine soils
and soils along highways, Pb levels may go upto
11,000 ppm (National Research Council 1980). The safe
limit of Pb levels in agricultural soils is 250–500 mg kg-1
soil and 300 mg kg-1 soil as per Indian (Awashthi 2000)
and European Union (European Union 2002) standards,
respectively.
After treatment, the roots were fixed in ethyl alcohol and
glacial acetic acid (3:1, v/v) for 24 h at 25 C. The fixed
roots were rinsed three times with distilled water, and
finally stored in 70 % (w/v) ethyl alcohol at 4 C until
further use.
Determination of Pb content
The amount of Pb in onion roots was determined using an
AAS. Roots were oven-dried (at 70 C for 48 h) followed
by digestion with HNO3 to HClO4 (3:1, v/v) solution. The
digested samples were dissolved in deionized water
(20 ml) and stored at 4 C until analysis. An aliquot of the
digest (2 ml) was analyzed for Pb. A reagent blank together
with a known standard (Sisco Research Laboratory,
Mumbai, India) were run along with the sample to maintain
the analytical quality, and the measurements were based on
the peak area.
Squash preparation
The effect of Pb on the mitotic activity in the onion root
tips was studied using the squash technique (Singh et al.
2005). The roots were first hydrolyzed in 1 N hydrochloric
acid (HCl) for 1 min at 25 C and then stained with
Schiff’s reagent for 30 min. Two root tips (*2 mm from
the tip; zone of division) were removed using forceps,
123
1294
macerated in a drop of 40 % acetic acid, squashed and
observed under a bright-field microscope (Getner, India).
The different mitotic stages in the onion root-tip cells were
noted. The mitotic index (MI; defined as the percentage of
cells undergoing division or the ratio of the number of cells
in the dividing phase to the total number of cells observed),
and chromosomal aberrations (%) were determined for the
various Pb treatments. For each treatment, including the
control, five independent replicates (squash) were prepared
and viewed.
Comet assay (single cell gel electrophoresis, SCGE)
The level of DNA damage was measured in comet assay.
For this, root meristem cells of onion were exposed to
similar concentrations of Pb as used for cytogenetic ana-
lysis. Comet assay was carried out as per the method given
by Tice et al. (2000). Before beginning with the assay,
microscopic slides (with one-fourth frosted ends) were
prepared by dipping into 1 % normal melting point agarose
(NMPA; SD Fine Chemical Limited, Mumbai, India) at
50 C. These slides served as the agarose base-coated
slides. The treated root tips of onion were placed in a watch
glass kept over an ice base and gently sliced using a sharp
razor blade to isolate the nuclei in Phosphate Buffer Saline
(PBS, pH 7.4) and the suspension of nuclei (100 ll) mixed
with 50 ll of low melting point agarose in PBS kept at
37 C was pipetted over the agarose base coated slides.
Slides were covered with a cover slip and left in a metal
tray kept on ice. Nuclei were left for 10 min and after
removing the cover slip, the slides were immersed in Lysis
solution for 1 h in dark. Thereafter, the slides were placed
in a horizontal gel electrophoresis tank containing freshly
prepared cold electrophoresis buffer (1 mM ethylene dia-
mine tetraacetic acid, EDTA, and 300 mM sodium
hydroxide, NaOH, pH C 13). The nuclei were incubated
for 30 min to facilitate DNA unwinding prior to electro-
phoresis (at 25 V, 300 mA) for 25 min. After rinsing thrice
with distilled water, electrophoresed slides were stained
with ethidium bromide (20 lg ml-1) for 5 min in dark,
dipped in ice-cold water to remove the excess ethidium
bromide, and covered with a cover slip. For each slide, 25
randomly chosen nuclei were analyzed using a fluores-
cence microscope with an excitation filter of BP 546/10 nm
and a barrier filter of 590 nm.
A computerized image analysis system (Comet Assay
Software; CaspLab) was employed to calculate percent
Head DNA (%DNAH), % Tail DNA (%DNAT), Tail
moment (TM = %DNAT 9 Tail Length) and Olive Tail
Moment [OTM = %DNAT 9 (Center of gravity of Tail in
x-axis direction—Centre of gravity of Head in x-axis
direction)] as the measure of DNA damage (Końca et al.
2003). A minimum of five roots were taken for each
123
G. Kaur et al.
treatment and from each root two SCGE slides were pre-
pared. In total, 250 nuclei were scored per treatment.
Disruption of the oxidative metabolism
Pb-induced disruption of oxidative metabolism during cell
division was measured in terms of ROS-hydrogen peroxide
(H2O2) and superoxide anions (O•- 2 )-generation, lipid
peroxidation, and alterations in the activities of the anti-
oxidant enzymes.
ROS determination
The endogenous H2O2 content was measured at 390 nm by
extracting the root tips in trichloroacetic acid (TCA, 0.1 %,
w/v). The amount was determined using the extinction
coefficient (e) 0.28 lM-1 cm-1 and expressed as nano-
moles per gram fresh weight (Singh et al. 2009). The O•- 2
content was measured in terms of oxidation of epinephrine
to adrenochrome at 480 nm and calculated using
e = 4,020 M-1 cm-1 (Singh et al. 2009).
Membrane peroxidation
Membrane peroxidation was determined in terms of mal-
ondialdehyde (MDA) and conjugated diene content. MDA,
a major thiobarbituric acid reactive substance was deter-
mined using e = 155 mM-1 cm-1 and expressed as
nanomoles per gram fresh weight (Singh et al. 2009). The
quantity of the conjugated dienes was measured at 234 nm
using e = 6.5 mM-1 cm-1 and expressed as micromoles
per gram fresh weight (Singh et al. 2009).
In situ ROS detection
In situ lipid peroxidation and loss of membrane integrity in
the onion root tips was histochemically detected. Briefly,
Schiff’s reagent was used to detect lipid peroxidation,
whereas Evan’s blue was used to detect loss of membrane
integrity (Singh et al. 2009).
ROS-scavenging enzymes
Root tips (100 mg) were homogenized in 10 ml of an ice-
chilled phosphate buffer (PO43- buffer; pH 7.0; 0.1 M)
under ice-cold conditions. The homogenate was filtered
through four layers of cheesecloth and centrifuged at
15,0009g at 4 C for 30 min. The resultant supernatant
was collected and stored at 4 C until used to assay the
activities of superoxide dismutases (SOD; EC 1.15.1.1),
catalase (CAT; EC 1.11.1.6), ascorbate peroxidase (APX;
EC 1.11.1.11), guaiacol peroxidase (GPX; EC 1.11.1.7),
and glutathione reductase (GR; EC 1.6.4.2). An aliquot of
Pb induces DNA damage and disrupts redox homeostasis
the supernatant was used for determination of the protein
content, with bovine serum albumin as the calibration
standard (Lowry et al. 1951). The activities of SOD, CAT,
APX, GPX, and GR were calculated as per Singh et al.
(2009). Briefly, SOD activity was measured in terms of the
inhibition of nitro blue tetrazolium (NBT) photochemical
reduction at 560 nm. One unit of SOD was the amount of
enzyme required to inhibit the photoreduction of NBT by
50 % at 25 C. The CAT activity was determined as the
decrease in absorbance at 240 nm (e = 39.4 mM-1 cm-1)
resulting from the disappearance of H2O2. The GPX
activity was measured as the increase in absorbance at
470 nm due to the oxidation of guaiacol to tetraguaiacol
and calculated using e = 26.6 mM-1 cm-1. The APX
assay was measured as the decrease in absorbance at
290 nm due to the oxidation of ascorbic acid to dehydro-
ascorbate and determined using e = 2.8 mM-1 cm-1. The
GR activity was determined following the oxidation of
NADPH (nicotinamide adenine dinucleotide phosphate
reduced) at 340 nm (e = 6.224 mM-1 cm-1) and calcu-
lated in terms of the remaining NADPH. The activities of
all the enzymes were measured at 25 C on a UV–Vis
spectrophotometer (Model 1800, Shimadzu, Japan). The
activities of APX, GPX, CAT and GR were expressed in
enzyme unit (EU) per milligram protein, where 1 EU is the
amount of enzyme that decomposes 1.0 lM substrate
(ascorbate or guaiacol or H2O2 or NADPH) per minute at
25 C.
Statistical analyses
The experiment was conducted in a completely randomized
design with five replications; each replication comprised
single onion bulb. For mitotic studies, five independent
replicates (squash) were prepared and viewed. The comet
assay involved five roots per treatment, including control;
and a minimum of two gel electrophoresis slide per root.
For biochemical analyses, there were five replicated
(independent) tissue samples. The data were analyzed by
one-way ANOVA followed by the comparison of mean
values using post hoc Tukey’s test at P B 0.05.
Results
Pb accumulates in onion roots
Onion roots accumulated 3.14 ± 0.01, 5.77 ± 0.08,
11.3 ± 0.16, 22.2 ± 0.81, 40.5 ± 0.62 lg Pb mg-1 dry
weight in response to 50, 100, 250, 500, and 1,000 lM Pb-
treatment, respectively, compared to 0.002 lg Pb mg-1
dry weight in the control roots.
1295
Pb disrupts mitotic activity
In the control roots, the actively dividing cells were
observed at all stages of mitosis with a MI of 6.2 %
(Table 1). In contrast, the roots of the Pb-treated plants
exhibited a consistently lower MI. Pb-treatment induced a
dose-dependent decrease in the MI in onion root tips. The
treatment of 50 lM Pb reduced the MI to 5.6 %, whereas
100 lM Pb reduced the MI further to 4.5 %, and finally it
was reduced to almost half, upon 250 lM Pb exposure.
Further, an increase in the Pb treatment (1,000 lM)
resulted in a complete inhibition of mitosis, with a MI
value of zero.
In prophase, the highest percentage of cells was
observed in the 100 lM Pb-treated root tips. The number
of cells in the subsequent stages, i.e., metaphase, anaphase
and telophase, was significantly lesser in the Pb-treated
(50, 100 and 250 lM) root tips, while not even a single cell
was present in the dividing stage upon exposure to
C500 lM Pb (Table 1). At C500 lM Pb exposure, all the
cells in onion root were in the interphase stage and none of
the mitotic stages could be observed. In contrast, at 50 and
100 lM Pb, the highest percentage of cells was observed in
prophase when compared with the other dividing stages.
Upon increasing the treatment to 250 lM, almost all the
stages were observed, although in very low numbers.
Further, the 500 and 1,000 lM Pb exposures did not allow
the cells to enter the dividing stage; rather, many of them
were arrested at interphase itself.
The various mitotic stages observed in the control were
perfectly normal and none of the mitotic or chromosomal
aberrations were observed (Fig. 1). However, in the Pb-
treated roots several abnormalities were observed. The
most frequent aberrations, fragments and bridges, were
observed with the increasing Pb concentrations
(50–250 lM Pb) demonstrating the clastogenic nature of
Pb (Fig. 2a–e). In addition, vagrant chromosomes and
laggards indicating interaction with the spindle were also
observed at 50, 100 and 250 lM Pb treatment (Fig. given
only at 50 lM Pb; Fig. 2a–d). Other observations fre-
quently monitored included the increased number of
chromosomes and distorted metaphase in the cells treated
with 50, 100 and 250 lM (Fig. given at 50 and 250 lM;
Fig. 2a–e). The sticky chromosomes were also seen on
50 lM Pb exposure. At higher concentrations (C500 lM),
different other aberrations such as elongated cell, periph-
eral nucleus and swollen cell were observed (Fig. 2f).
Pb induces DNA damage in onion root nuclei
After studying the cytotoxic effect of Pb on onion, its
genotoxicity was evaluated using SCGE in isolated nuclei
(Fig. 3). The results obtained with CASP are summarized
123
1296 G. Kaur et al.

Table 1 Effect of Pb on the Treatment (lM) Prophase Metaphase Anaphase Telophase Mitotic index
mitotic activity in onion (Allium (%) (%) (%) (%) (MI, %)
cepa) root tips
0 (Negative control) 1.9 ± 0.08a 1.3 ± 0.10a 1.0 ± 0.09a 2.0 ± 0.06a 6.2 ± 0.14a
Pb
50 1.7 ± 0.08b 1.0 ± 0.09a 1.5 ± 0.06b 1.4 ± 0.02b 5.6 ± 0.14b
100 3.8 ± 0.07c 0.5 ± 0.04b 0.2 ± 0.02c 0.0 ± 0.00c 4.5 ± 0.06c
Data were recorded 24 h after
250 0.7 ± 0.01d 0.9 ± 0.01ac 0.7 ± 0.09d 0.7 ± 0.01d 3.0 ± 0.04d
exposure to Pb; represented as
mean ± SE; means with 500 0.0 ± 0.00e 0.0 ± 0.00d 0.0 ± 0.00e 0.0 ± 0.00c 0.0 ± 0.00f
common letters are not 1,000 0.0 ± 0.00e 0.0 ± 0.00d 0.0 ± 0.00e 0.0 ± 0.00c 0.0 ± 0.00f
significantly different at MMS (100 lM; 1.5 ± 0.04b 0.8 ± 0.02ac 0.2 ± 0.01c 0.0 ± 0.00c 2.5 ± 0.11e
P B 0.05, according to Tukey’s positive control)
test

in Table 2. With increasing concentration of Pb, the % Tail
DNA (%DNAT) increased significantly (P B 0.05) in the
range of 11–62 % over 0–1,000 lM Pb. With increase in
%DNAT, a notable decrease was observed in % head DNA
(%DNAH) with respect to increasing dose of Pb (Table 2).
Comet analysis revealed by Tail Moment (TM) and Olive
Tail Moment (OTM) indicated that DNA damage induced
by Pb was significant (P B 0.05) as compared to control.
Higher doses of Pb induced greater genotoxicity, which
was evident from increasing values of TM and OTM. The
highest values of TM and OTM were exhibited at
1,000 lM Pb-treatment followed by 500, 250, 100, 50 and
0 lM Pb-treatments (Table 2).
Pb disrupts oxidative metabolism in onion roots
ROS generation
Membrane lipid peroxidation, measured as the MDA con-
tent, increased significantly upon Pb exposure when com-
pared with the control (Table 3). It increased linearly in the
sequence of 43, 76, 114, 163 and 203 % upon increasing
the Pb exposure to 50, 100, 250, 500 and 1,000 lM,
respectively (Table 3). In situ histochemical localization
further confirmed the increase in the lipid peroxide level in
Pb-treated roots, wherein the treated roots stained darker
than those in the control (Fig. 4 a).
Pb exposure significantly (P B 0.05) enhanced the CD
content in onion roots (Table 3). It increased by *8, 26,
44, 177 and 259 % upon exposure to 50, 100, 250, 500 and
1,000 lM Pb, respectively, over that of the control
(Table 3). Further, upon staining with Evans blue, an
indicator and a measure of plasma membrane integrity, the
roots after Pb-treatment stained dark blue when compared
with the control roots (Fig. 4 b). The stain intensity
increased with the increase in the Pb concentrations,
thereby indicating the loss of plasma membrane integrity.
The H2O2 content showed a trend similar to MDA, with
an increase in the range of *40–273 % upon
50–1,000 lM Pb exposure (Table 3). Likewise, Pb
treatment resulted in accumulation of O–•
2 . It increased by
*1.5 times over the control in response to 250 lM Pb and
almost doubled at 1,000 lM Pb (Table 3).
ROS metabolism
Pb exposure significantly altered the activities of the
scavenging enzymes such as SOD, CAT, APX, GPX, and
GR in the onion roots (Table 4). The SOD activity was
enhanced by *99, 134, 243, 382 and 506 % when com-
pared with the control upon Pb exposure of 50, 100, 250,
500 and 1,000 lM, respectively. Likewise, there was a
significant increase in the APX, GPX and GR activities in
the Pb-treated roots. GPX activity increased by about 9.4,
36, 53, 126 and 180 % over the control at 50, 100, 250, 500
and 1,000 lM Pb treatments, respectively. However, the
extent of the increase was more pronounced in GR activity,
which was enhanced over the control by 61, 104, 139, 244
and 321 % at 50, 100, 250, 500 and 1,000 lM Pb,
respectively (Table 4). The highest percentage of increase
was noticed in the APX activity, which increased over the
control by 114 % at 50 lM Pb, increased upto 388 % at
250 lM Pb and finally rose to 821 % in response to
1,000 lM Pb exposure. In contrast, the CAT activity
decreased in response to Pb-treatment, with about 5, 12, 24,
43 and 68 % decline when compared with the control at 50,
100, 250, 500 and 1,000 lM Pb-treatments, respectively
(Table 4).
Discussion
Pb altered mitotic activity
Pb-exposure significantly inhibited the MI in onion root tip
bioassay, a frequently used cell cycle parameter and indi-
cator of mitosis, in a concentration-dependent manner. The
observed alterations were correlated with greater accumu-
lation of Pb in root tissue. Previous studies demonstrated a
similar reduction in the MI in Z. mays (Kozhevnikova et al.

123
Pb induces DNA damage and disrupts redox homeostasis 1297

Fig. 1 Effect of Pb on Allium cepa (onion) root tip (920) cells of a control, b 50 lM, c 100 lM, d 250 lM, e 500 lM, and f 1,000 lM Pb-
treated root tips. p prophase, m metaphase, a anaphase, t telophase (bar line = 200 lm)

2009), Pinus nigra ssp. pallasiana (Yücel et al. 2008),
Larix decidua and Picea abies (Băra et al. 2004), and
Vigna mungo (Siddiqui 2012) upon Pb treatment. Yücel
et al. (2008) demonstrated that Pb (C500 lM) inhibited
mitotic division and induced mitotic abnormalities such as
C-mitosis, lagging chromosomes, multipolar anaphases and
chromosome bridges in P. nigra. ssp. pallasiana. Previ-
ously, very high doses of Pb (at 0.1, 1, and 5 % as lead
nitrate = 3, 30, 150 mM) have been demonstrated to be a
genotoxic metal and induce cytogenetic effects in onion
roots (Pădureanu 2005). Though, reduction in MI under Pb
exposure has been shown earlier, but what prevented the
cells from entering the mitosis is largely unexplored.
According to Siddiqui (2012), the reduction in the number
of mitotic cells under Pb exposure could be due to its
interference with cell cycle progression. Sudhakar et al.
(2001) suggested that Pb can inhibit the DNA synthesis or
may even block the cells in the G2 phase of cell cycle.
Erturk et al. (2013) also suggested that inhibition of DNA
synthesis under abiotic stresses results in inhibition of

123
1298
Fig. 2 Effect of Pb on Allium cepa (onion) root tip (940) cells of a,
b, c, d) 50 lM, e 250 lM, and f 1,000 lM Pb-treated root tips. ic
increased number of chromosomes, dm distorted metaphase,
mitotic division. Additionally, it could be attributed to its
biochemical mode of action, since Pb also act on DNA-
repair enzymes, either by reducing the production of the
enzymes at the transcription level or by modifying the
protein structure of the enzymes (Hartwig 1994), which
could also result in chromosomal aberrations in the divid-
ing cells.
Pb-induced chromosomal aberrations
Pb-treatment caused various chromosomal aberrations such
as chromosome stickiness, bridges, fragments, vagrant and
breaking chromosomes, increased number of
123
G. Kaur et al.
f fragments, sc sticky chromosomes, l laggard, vc vagrant chromo-
some, b bridge, ec elongated cell, sw swollen cell, pn peripheral
nucleus, db dumb-bell shaped cell (bar line 10 lm)
chromosomes, nuclei with more condensed chromatin,
displaced metaphasic plate and multinucleolate cells. The
appearance of various aberrant features upon Pb-treatment
induces gentotoxicity in onion root cells. These genotoxic
abnormalities are expected to result in micronuclei for-
mation. However, we did not observe micronuclei forma-
tion in our study, thus suggesting that mitotic disturbances
did not manifest into genetic damage in the form of
micronuclei. It was largely due to the low doses of Pb-
treatments (1–1000 lM = 0.001–1 mM) used in the pres-
ent study. Previously, it has been demonstrated that higher
concentrations of heavy metals (C10 mM) are required to
induce micronuclei formation in root tip cells of onion
Pb induces DNA damage and disrupts redox homeostasis 1299

Fig. 3 Pb-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 0 (control), b 50 lM,
c 100 lM, d 250 lM,
e 500 lM, and f 1,000 lM Pb-
treated root cells of A. cepa. In
the left panel, the comet images
with measurement frame, tail
and head are presented; whereas
in the right panel, intensity
profiles are plotted with length
(lm; =pixels 9 0.64) on x-axis
and %DNA (9102) on y-axis

(Steinkellner et al. 1998). In fact, mitotic chromosomes
have been found to be less susceptible to DNA-damage
(Ma 1982). Earlier studies have related such observations
to the incomplete development of microtubules, which are
controlled by Ca2? (Hepler 1992; Vidakovic-Cifrek et al.
2002). The chromosome stickiness has been regarded as an
indicator of the toxic influence of metals on the
chromosomes (Kumari et al. 2009). According to Dar-
lington and Mc Leish (1951), chromosome stickiness can
be attributed to the degradation or depolymerization of
chromosomal DNA. Panda and Panda (2002) highlighted
the denaturing activity of Pb on DNA topoisomerase II (a
nuclear protein), which can also obstruct chromosome
segregation leaving them sticky. Further, stickiness of

123
1300 G. Kaur et al.

Fig. 3 continued

Table 2 Pb-induced DNA damage in nuclei isolated from onion (Allium cepa) roots measured in terms of % Head DNA, % Tail DNA, Tail
Moment and Olive Tail Moment in Comet Assay method using Comet Assay Software (CaspLab)
Treatment (lM) Head DNA (%) Tail DNA (%) Tail Moment (lm) Olive Tail Moment (lm)

0 (Negative control) 89.02 ± 0.57a 10.98 ± 0.07a 1.34 ± 0.04a 2.78 ± 0.02a
Pb
50 85.07 ± 0.64b 14.93 ± 0.05b 2.77 ± 0.07b 4.33 ± 0.04b
100 75.41 ± 0.55c 24.59 ± 0.08c 10.23 ± 0.11c 7.92 ± 0.17c
250 66.85 ± 0.72d 33.15 ± 0.15d 15.28 ± 0.23d 12.52 ± 0.19d
500 55.21 ± 0.45e 44.79 ± 0.18e 27.81 ± 0.27e 17.91 ± 0.22e
1,000 39.39 ± 0.40g 61.61 ± 0.20g 69.01 ± 0.34g 41.09 ± 0.29g
MMS (100 lM; positive control) 51.66 ± 0.09f 48.33 ± 0.24f 47.02 ± 0.16f 38.96 ± 0.12f
Data were recorded after 24 h of exposure to Pb; represented as mean ± SE; means with common letters are not significantly different at
P B 0.05, according to Tukey’s test

chromosomes could have resulted in subsequent failure of
separation of chromatids to poles (Fusconi et al. 2006). The
chromosomal fragments observed in the present study
could be the result of inversion in acentric chromosome.
Not only this, even enhanced ROS generation (as observed
under Pb-stress in the present study), too, has several del-
eterious effects on DNA, and can result in fragment for-
mation (Pourrut et al. 2011). Metaphasic disturbances
observed in the present study can be due to thickened,
highly condensed and contracted chromosomes and a dis-
rupted spindle apparatus (Kuras et al. 2009). In response to
Pb-treatment, cells were enlarged and dumb-bell shaped.
Such an observation is largely viewed as an adaptive
response of plant tissues to the changes in the environ-
mental factors, including pollution and metal toxicity
(Chang and Wang 1991; Werner and Edvards 1993; Nan-
ushyan and Murashev 2003).
Pb induced DNA damage
We explored two often used parameters, the DNAT and
OTM to assess Pb-induced DNA damage in onion root
nuclei. Although the DNAT increased linearly with stress
intensity, the tail moments are dependent on the differences
in DNA migration, which can be due to either the nature of
DNA or the extent of DNA relaxation (Duez et al. 2003).
We observed increased levels of DNA strand break under
Pb-toxicity and it correlated positively to in vivo staining
with Evans Blue. It suggested disintegration of plasma
membrane, leading to cell death. The results in the present

123
Pb induces DNA damage and disrupts redox homeostasis 1301

Table 3 Effects of lead (Pb) on lipid peroxidation (MDA content) DNA damage in potato and tobacco plants can be associ-
and amounts of H2O2, conjugated dienes (CD) and superoxide ions ated with necrotic or apoptotic DNA fragmentation. Cr(VI)
(O–•
2 ) in onion (Allium cepa) roots
at 0–50 mM and airborne particulate matter caused an
Pb MDA CD H2O2 O•-
2 increase in DNA strand breaks and DNA migration in stem,
(lM) (nM g-1 FW) (lmol (nM g-1 (lM g-1
g-1 FW) FW) FW)
roots and leaves of ornamental plant, Impatiens balsamina
(Poli et al. 1999). Pakrashi et al. (2014) demonstrated that
0 13.6 ± 0.37a 2.2 ± 0.03a 50.0 ± 0.41a 2.7 ± 0.03a titanium dioxide nanoparticles damage DNA in onion root
50 19.4 ± 0.24b 2.4 ± 0.02b 70.0 ± 1.14b 3.1 ± 0.03b tip cells.
100 23.9 ± 0.32c 2.8 ± 0.02c 84.3 ± 2.03c 3.5 ± 0.06c
250 29.0 ± 0.49d 3.2 ± 0.04d 117.9 ± 3.05d 4.2 ± 0.02d
Pb-induced disruption of oxidative metabolism
500 35.7 ± 0.57e 6.1 ± 0.05e 135.7 ± 3.24e 4.8 ± 0.04e
1,000 41.1 ± 0.78f 7.9 ± 0.03f 186.7 ± 3.07f 5.6 ± 0.03f
In our study, Pb treatment not only reduced cell division
Data were recorded after 24 h of exposure to Pb; represented as mean ± SE; and caused chromosomal aberrations in the onion root tips,
means with common letters are not significantly different at P B 0.05,
according to Tukey’s test but it was also accompanied by cell death and oxidative
damage. Reduction in the mitotic division was associated
study are concordant with the earlier observations that with an enhanced ROS generation, indicative of the posi-
heavy metals are gentotoxic and cause DNA damage. For tive role of ROS in the underlying Pb-induced disruption of
example, Erturk et al. (2013) reported DNA damage cell division (Achary and Panda 2010). To quantify the
induced by genotoxins’ (Ni or Co) treatment in Zea mays extent of membrane damage, lipid peroxidation analysis
seedlings. Gichner et al. (2006) noted a significant increase was also performed. In the present study, the MDA content
in DNA damage in heterozygous potato and tobacco plants increased approximately three times upon 1,000 lM Pb
grown on heavy metal contaminated soil. The increased exposure. The higher concentrations of Pb induced lipid

Fig. 4 In situ histochemical localization showing the effect of Pb induced a lipid peroxidation and b loss of membrane integrity in Allium cepa
(onion) roots. Roots from left to right indicate 0 (Control), 50, 100, 250, 500 and 1,000 lM Pb-treatments

Table 4 Changes in the activities of superoxide dismutases (SOD), catalases (CAT), ascorbate peroxidases (APX), guaiacol peroxidases (GPX)
and glutathione reductases (GR) in the roots of onion (Allim cepa) upon lead (Pb) treatment
Pb (lM) SOD CAT APX GPX GR
(EU mg-1 protein) (EU mg-1 protein) (EU mg-1 protein) (EU mg-1 protein) (EU mg-1 protein)

0 4.3 ± 0.07a 58.7 ± 0.22a 0.6 ± 0.01a 21.8 ± 0.24a 1.5 ± 0.03a
50 8.7 ± 0.01b 55.7 ± 0.11b 1.4 ± 0.19b 23.9 ± 0.16b 2.5 ± 0.06b
100 10.2 ± 0.01c 51.5 ± 0.25c 2.2 ± 0.17c 29.6 ± 0.12c 3.2 ± 0.03c
250 14.9 ± 0.01d 44.7 ± 0.15d 3.1 ± 0.15d 33.4 ± 0.06d 3.7 ± 0.02d
500 20.93 ± 0.01e 33.5 ± 0.48e 4.4 ± 0.14e 49.4 ± 0.10e 5.3 ± 0.03e
1,000 26.31 ± 0.01f 19.0 ± 0.05f 5.8 ± 0.16f 82.2 ± 0.11f 6.5 ± 0.08f
Data were recorded after 24 h of exposure to Pb; represented as mean ± SE; means with common letters are not significantly different at
P B 0.05, according to Tukey’s test

123
1302
peroxidation and caused cytotoxicity. This suggests that
increased oxidative stress results in increased lipid perox-
idation and excessive generation of ROS by deficient
antioxidant defenses. Ünyayar et al. (2006) suggested that
Cd-induced lipid peroxidation might delay mitosis by
deteriorating the transport mechanism in Vicia faba root
cells. It has been established that Al-induced DNA damage
in the onion root tips was largely due to H2O2 accumula-
tion and not due to O•- 2 (Achary and Panda 2010). In our
study, both H2O2 and O•- 2 were enhanced in the onion roots
post Pb-treatment; however, we did not evaluate their
individual roles in inducing mutagenic effects. Neverthe-
less, based upon the activities of the antioxidant enzymes,
it could be postulated that the negative effect of Pb on the
mitotic activity was largely due to H2O2 accumulation and
not due to O•- 2 . In our study, there was a nearly six fold
increase in SOD activity (at 1000 lM Pb) to scavenge O•- 2 Acknowledgments Gurpreet Kaur is thankful to University Grants
into H2O2, whereas the activity of CAT, an enzyme pri-
marily linked to H2O2 scavenging, was down-regulated.
Santoro et al. (2005) reported that the MI may be low-
ered by oxidative stress caused by H2O2. Similar obser-
vations were documented by Gajewska et al. (2006), where
Ni stress increased H2O2 content, thereby resulting in
depression of the mitotic activity. Thus, it can be suggested
that the reduction in root growth in onion treated with Pb
might be related to the accumulation of H2O2. Thereafter,
H2O2 scavenging enzymes and non-enzymatic antioxidants
controlled the level of H2O2 in the plant tissues.
Compared with CAT, although, GPX and APX require
reducing power to break down H2O2 to H2O and O2, they
show a high affinity to detoxify even low concentrations of
H2O2 (Achary et al. 2008). Pb has been reported to increase
the APX activity in Oryza sativa (Verma and Dubey 2003)
and Cassia angustifolia (Qureshi et al. 2007). Pb in the
present study, too, increased the APX activity, thereby
preventing plant damage from O•- 2 and H2O2 to a great
extent. We have reported an increase in the GPX activity in
response to an enhanced production of ROS, which con-
firms some earlier findings in onion treated with Al
(Achary et al. 2008). The observed increase in the GR
activities in the present study substantiates some earlier
reports of GR upregulation during oxidative stress (Reddy
et al. 2005 and Verma and Dubey 2003).
The antioxidant enzymatic machinery, including CAT,
SOD, APX, GPX and GR, helps in the cellular detoxifi-
cation of ROS. It had been observed earlier that the genes
encoding these antioxidant enzymes are activated by heavy
metal stress (Ezaki et al. 2004). Any disturbance in the
antioxidant enzymatic machinery upsets the redox state
causing cellular oxidative stress. It has been proposed that
the formation of ROS is related directly/indirectly to the
genotoxicity of metals (Kasai et al. 1992; Radetski et al.
2004). Phugare et al. (2011) also demonstrated that ROS-
123
G. Kaur et al.
induced oxidative stress results in cytotoxicity and
genotoxicity.
Conclusions
The study concludes that Pb-induced cytotoxicity and
genotoxicity in the onion roots is mediated through ROS
and is also tightly linked to the cell cycle. However, the
observed response was dependent upon the type and/or
scale of DNA damage. The exposure to higher concentra-
tions arrested cell cycle leading to cell death, whereas
different repair responses are generated at lower concen-
trations, thereby allowing the cell to complete the cell
cycle.
Commission, New Delhi, India, for financial assistance in the form of
Post-doctoral Fellowship.
Conflict of interest The authors declare that they have no conflict
of interest.
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