ARTICLE IN PRESS

Pesticide Biochemistry and Physiology ■■ (2014) ■■–■■

Contents lists available at ScienceDirect

Pesticide Biochemistry and Physiology

j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / p e s t

Atrazine induces apoptosis of SH-SY5Y human neuroblastoma

cells via the regulation of Bax/Bcl-2 ratio and
caspase-3-dependent pathway
Sunny O. Abarikwu a,*, Ebenezer O. Farombi b
a Department of Biochemistry, University of Port Harcourt, Choba, Nigeria
b
Department of Biochemistry, University of Ibadan, Ibadan, Nigeria

A R T I C L E I N F O A B S T R A C T

Article history: Atrazine (ATZ) is a well known herbicide that is frequently detected in ground and surface water at sig-
Received 27 November 2013 nificant levels. Our objective was to study the toxic effect of ATZ on the human neuroblastoma (SH-
Accepted 5 December 2014 SY5Y) cells, and the degree of cytotoxicity and morphological changes were followed during the cell death.
Available online
Application of cytotoxicity bioassays indicates that ATZ (5–50 μg/mL) decreases cell viability in a dose-
and time-dependent manner. The evidence of apoptosis was confirmed by an increase in caspase-3 ac-
Keywords:
tivity, and cell death was blocked when caspase-3 activity was inhibited. Typical apoptotic phenotype
Atrazine
that includes nuclear fragmentation, micro nuclei formation, DNA fragmentation and increase in the ex-
SH-SY5Y cells
Apoptosis pressions apoptosis-associated markers Bax, p53 and p21 and decreased expression of Bcl-2 were observed
Reactive oxygen species in treated cells. We also observed dose-dependent increase in reactive oxygen species (ROS) levels in
Cytotoxicity ATZ-treated cells. These results suggest that ATZ-induces apoptosis and ROS levels in SH-SY5Y cells, and
could be implicated in human neurodegenerative disorder.
© 2014 Elsevier Inc. All rights reserved.

1. Introduction
The herbicide ATZ (2-chloro-4-(ethylamino)-6-(isopropylamino)-
s-triazine) is a widely used broad-spectrum pesticide with an
estimated 76.4 million pounds used annually in the United States
alone [1]. Besides its widespread use in the control of broadleaf and
grassy weeds in a variety of crops, ATZ is a potential contaminant
for surface and ground water sources [2]. ATZ is considered to be
a “priority A” chemical for potential ground water contamination
by the United States Environmental Protection Agency and was
ranked the highest of 83 pesticides for potential ground water con-
tamination [3]. The levels of ATZ in drinking water could be as low
as 1 ppb or as high as 224 ppb [4,5]. Because of its persistence in
the environment, the detection of ATZ in both human and animals
have been reported [6–8] and concentrations of about 1.0 ng/mL has
been detected in human urine [9]. It is estimated that between two
and three million people who use ground water as their primary
drinking water source are exposed to at least 0.2 ppb ATZ [10].
ATZ has been considered an endocrine disruptor, causing adverse
effects on reproductive function in both genders of several mam-
malian and non-mammalian species [11–16]. Cytotoxicity studies
* Corresponding author. Department of Biochemistry, University of Port Harcourt,
Choba, Nigeria. Fax: +234 (0)84 230 903.
E-mail address: abarikwus@yahoo.com (S.O. Abarikwu).
with cell lines such as HepG2, Chinese hamster ovary (CHO-K1), and
Caco-2 intestinal cells at ATZ concentrations of 3–742 μM (625–
54,000 ppb), indicated that ATZ decreased cell proliferation [5,17,18].
The growth inhibiting effect of ATZ at concentrations (50–300 ppb)
lower than those used in the above studies have also been re-
ported in the normal human fibroblasts [19]. In other studies, cell
viability was not altered even at high ATZ concentration (464 μM,
100,000 ppb) [20] or when the concentration was lower (30 μM,
6471 ppb) [21].
Dopaminergic neurotoxicity of ATZ has been observed in rats
chronically exposed to 5 or 10 mg/kg ATZ in the diet for 6 months,
or during acute exposures to 100 or 200 mg/kg ATZ in rats [22]. This
has also been verified in vitro at ATZ concentration up to 250 μM
(54,000 ppb) [23]. Previous work from our laboratory has shown that
exposure of PC12 cells (rat pheochromocytoma cell line) and SH-
SY5Y (human neuroblastoma cells) to commercial formulations of
300 μM (65 μg/mL, 64,800 ppb) of ATZ (based on the active ingre-
dient) can result in decreased cell viability [24,25]. Apart from the
active ingredient, the commercial formulations contain various ad-
juvants known to influence their cytotoxicity in non-target organism
[26], thus in the present experiment, studies were initiated with
the active compound (ATZ) to investigate the mechanism of cell death
in the human neuroblastoma cell lines. Human SH-SY5Y neuronal
cells are used as good model system in the analysis of cytotoxicity
of chemicals such as pesticides [27,28]. Consequently, SH-SY5Y cell
line has been widely used in experimental neurological studies, in-
cluding analysis of neuronal differentiation, metabolism, and function

http://dx.doi.org/10.1016/j.pestbp.2014.12.006
0048-3575/© 2014 Elsevier Inc. All rights reserved.

Please cite this article in press as: Sunny O. Abarikwu, Ebenezer O. Farombi, Atrazine induces apoptosis of SH-SY5Y human neuroblastoma cells via the regulation of Bax/Bcl-2
ratio and caspase-3-dependent pathway, Pesticide Biochemistry and Physiology (2014), doi: 10.1016/j.pestbp.2014.12.006
 ARTICLE IN PRESS
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related to neurodegenerative and neuroadaptive processes, neuro-
toxicity, and neuroprotection [27]. Thus, the goal of the present study
was to determine the mechanism of the apoptosis-inducing effects
of ATZ in the SH-SY5Y cells. The morphological changes were then
monitored by fluorescent microscopy.
2. Materials and methods
2.1. Chemicals
ATZ (purity, 98.0%) was obtained from Sigma-Aldrich,
Laborchemikalien GmbH. Oligonucleotide primers were pur-
chased from Integrated DNA technologies, Inc. (San Diego, CA). 3-(4,5-
Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide salt (MTT),
2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), neural red,
chromatin dye bisbenzimide (Hoechst 33342), and DEVD-pNA were
from Sigma (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium
(DMEM-F12), antibiotics/antimycotics, fetal bovine serum (FBS) were
purchased from Invitrogen Life Technologies, USA. Trypan blue dye
was purchased from Gibco BRL (Gaithersburg, MD, USA). All the other
chemicals used were of the high grade available commercially.
Milli-Q and nuclease free water were used in all the experiments.
2.2. Cell culture
SH-SY5Y cells were obtained from American Type Culture Col-
lection (Rockville, MD). The cells were grown in 5% CO2 – 95%
atmosphere in high humidity at 37 °C in a 1:1 mixture of Dulbecco’s
modified Eagle’s media (DMEM):Ham-F12 supplemented with 10%
heat inactivated fetal bovine serum (FBS), 0.2% sodium bicarbon-
ate and antibiotic and antimycotic (10×, 1 mL/100 mL of medium,
Invitrogen Life Technologies). After seeding, cells were grown for
24 h to reach 80% confluence. Prior to experimental uses, cells were
screened for integrity of established markers and viability [27].
Batches showing more than 95% cell viability were used in the
present study. For the culture of cells, T-25 cm2, T-75 cm2 flasks, 6
and 96 well culture plates (Nunc, Denmark) were used depending
on the type of assay/endpoint.
2.3. Treatment of cultured cells with test compound
Stock solution of ATZ (100 mM) was prepared in dimethyl sulf-
oxide (DMSO) in so much that the addition of 0.1% DMSO (has no
effect on the viability of cells) resulted in the final concentrations
of 5–50 μg ATZ/mL (23.2–232 μM) of cell suspension. Confluent cells
(≈80%) were treated with ATZ for different time periods. In the control
(untreated) samples, equal amount DMSO was added.
2.4. Assay methods
2.4.1. Determination of LDH release
The cytotoxicity was estimated by quantification of LDH re-
leased in the culture medium after treatment of cells for 3, 6, and
24 h according to established method described by Grivell and Berry
[29]. One hundred microliters of conditioned media of control and
ATZ-treated cells was added to 0.9 mL of a reaction mixture to yield
a final concentration of 1 mmol/L pyruvate, 0.15 mmol/L NADH and
104 mmol/L phosphate buffer, pH 7.4. After thoroughly mixed, the
absorbance of the solution was measured at 340 nm at 60 s inter-
vals with a microplate reader. LDH activity was expressed as
percentage of control.
2.4.2. Measurement of cell viability by MTT and NRU assay
Cell viability was quantified by measurement of the mitochon-
drial reduction of MTT to produce a dark-blue formazan product
[25]. Briefly, SH-SY5Y cells were plated at a density of 1 × 104 cells/
Please cite this article in press as: Sunny O. Abarikwu, Ebenezer O. Farombi, Atrazine induces apoptosis of SH-SY5Y human neuroblastoma cells via the regulation of Bax/Bcl-2
ratio and caspase-3-dependent pathway, Pesticide Biochemistry and Physiology (2014), doi: 10.1016/j.pestbp.2014.12.006
100 μL in 96-well plates. The next day, the culture medium was
replaced with fresh medium containing various concentrations of
ATZ or vehicle, and then the cells were maintained for 3, 6, and 24 h.
MTT (0.5 mg/mL) was added to each well. After incubating for 4 h
at 37 °C, the medium was removed and 200 μL DMSO was added
to solubilize the formazan crystals. The color developed was mea-
sured at 570 nm using a multiplate reader (Synergy HT, Bio-Tek, USA).
The viability of cells was further followed by the NRU assay [17].
This assay was applied to determine the accumulation of the neutral
red dye in the lysosomes of viable uninjured cells. Neutral red dye
was added and the mixture was incubated at 37 °C for 3 h. Neutral
red solution was discarded and cells were washed with a solution
containing 1% CaCl2 and 0.5% formaldehyde. The cells were de-
stained with 200 μL of destaining solution (1% glacial acetic acid;
50% ethanol; 49% distilled water) and the absorbance taken at
540 nm using a multiplate reader (Synergy HT, Bio-Tek).
2.4.3. Measurement of intracellular reactive oxygen species
(ROS) levels
After treatment with 5, 10, 25 and 50 μg/mL ATZ (23.2–232 μM)
for the indicated time periods (1, 3, 6, 12 h), the cells were incu-
bated with 20 μM of the fluorescent dye DCFH-DA for 30 min. DCFH-
DA is a nonpolar compound which upon incorporation into cells is
converted into a membrane-impermeable, nonfluorescent polar com-
pound, H2DCF, by the action of cellular esterases. The fluorescence
intensity was measured with excitation and emission wave-
lengths of 485 nm and 528 nm respectively in multiplate reader
(Synergy HT, Bio-Tek). In the presence of intracellular ROS, 2′,7′-
dichlorodihydrofluorescein (DCFH) was oxidized to the fluorescent
2′, 7′-dichlorofluorescein (DCF), which was readily detected by the
fluorescence microscope (Nikon Eclipse 80i, USA) and the fluores-
cence intensity analyzed by Leica QWin Plus, version 2.7.1 (Leica
Microsystems Imaging solution, UK). The DCF fluorescence inten-
sity was proportional to the amount of intracellularly formed ROS
in a specific number of cells.
2.4.4. Observation of morphological changes
Cells were treated with 5, 25 and 50 μg/mL ATZ for 48 h. After
ATZ treatment, cells were washed with phosphate-buffered saline
and then fixed in 0.1% ice-cold p-formaldehyde for 10 min. The cells
were then washed twice with phosphate-buffered saline and stained
with Hoechst 33342 (1.5 mg/mL) for 5 min in the dark. Cells with
typical apoptotic nuclear morphology such as nuclear shrinkage and
fragmentation and micronuclei formation were identified under flu-
orescent microscope (Nikon Eclipse, 80i) and counted using randomly
selected fields on numbered slides. The percentage of apoptotic cells
was scored by counting at least 200 cells per treatment group. At
least three slides were prepared for each treatment group and the
average percentage of apoptotic cells were determined for each treat-
ment of ATZ and expressed as mean ± SD.
2.4.5. Caspase assay
To measure apoptosis enzymatically, caspase-3 activity in
cell extracts was measured using N-acetyl-Asp-Glu-Val-Asp-p-
nitroanilide (DEVD-pNA). SH-SY5Y cells were plated on T-75 cm2
flask (Nunc, Germany) at a density of 1 × 106/flask in low serum
medium for 24 h at 37 °C and 5% CO2. After 24 h, medium was
removed and complete medium with 10% FBS and increasing
concentrations of ATZ or DMSO (5, 25, 50 μg/mL) were added
for 12–48 h. Lysates were made using celLytic buffer (Sigma) ac-
cording to the manufacturer’s suggested protocol. Aliquot of the
lysate (5 μL) was used for protein concentration determination
using the Bicinchoninic acid protein assay (Lamda Biotech, St. Louis,
MO, USA). Lysates were also made from cells treated with 50 μM
z-VAD-FMK (negative control) and or 25 μg/mL in 10% FBS com-
plete growth medium for 24 h. z-VAD-FMK is a cell-permeable
 ARTICLE IN PRESS
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caspase inhibitor that irreversibly binds to the catalytic site of
caspases to inhibit induction of apoptosis [19]. Assays were per-
formed by incubating 50 μg protein of cell lysate per sample in 100 μL
of reaction buffer (50 mM HEPES, 20% glycerol, and 100 mM
dithiothreitol) containing 200 μM DEVD-pNA colorimetric sub-
strate in 96 well microtiter plates for 4 h at 37 °C. The samples were
measured with an ELISA reader at an absorbance of 405 nm (Synergy
HT, Bio-Tek). p-nitroaniline (pNA) is released from the substrate
DEVD-pNA upon cleavage by DEVDase. Free pNA produces a yellow
color after cleavage that is proportional to the amount of DEVDase
activity present in the sample. The increase in absorbance posi-
tively correlates with the amount of caspase-3 activity.
2.4.6. Detection of apoptosis by DNA Ladder assay
To assess the fragmentation of cellular DNA into the character-
istic apoptotic ladder, SH-SY5Y cells (1.6 × 106) were incubated in
the absence or presence of 5, 25, 50 μg/mL ATZ for 48 h, washed
with PBS, scraped and pelleted at 4 °C. DNA samples were ex-
tracted as described previously by us [25]. Briefly, cells were lysed
in 500 μL of 2.5 mM Tris–HCl (pH 8.0), 10 mM EDTA, and 0.25% Triton
X-100 and stored at 4 °C for 15 min. Intact nuclei were eliminated
by centrifugation at 500 × g for 15 min and DNA in the superna-
tant was precipitated, re-suspended in 200 μL of TE buffer (pH 8),
and sequentially incubated at 37 °C for 30 min with RNAse A (0.1 mg/
mL) and for 2 h with proteinase K (0.25 mg/mL; Sigma Chemical Co.).
Equal quantities of purified DNA were resolved at 100 V for 45 min
in 2% agarose – 1 × TAE gels containing 0.5 mg/L of ethidium bromide.
The bands were visualized and photographed under transmitted UV
light with a Polaroid camera. One kilobyte DNA ladder was used as
a marker (New England Biolabs. Inc).
2.4.7. Isolation of RNA and reverse transcription-polymerase chain
reaction (PCR) for apoptosis-related genes mRNA
Total RNA was prepared using TRIzol reagent (Invitrogen) and
quantified by spectrophotometry (Nanodrop ND-1000 Spectropho-
tometer, USA). Two micrograms of RNA from cells exposed to ATZ
concentrations (5–50 μg/mL, 23.2–232 μM) for 6 h were used to
prepare cDNA according to the manufacturer’s instructions (Applied
Biosystem, USA) in a 20 μL reaction. The PCR program consisted of
an initial denaturation at 94 °C for 5 min, followed by 35 cycles of
denaturation at 94 °C for 60 s, annealing for 60 s, elongation at 72 °C
for 60 s, and a final elongation at 72 °C for 5 min. GAPDH was am-
plified simultaneously as internal control (Bio-Rad thermal cycler,
USA). The primers, PCR products, and annealing temperatures of
each gene are listed in Table 1. The oligonucleotides for PCR
amplification were design by using software primer express 3.0
(Applied Biosystems) and full gene sequences from National Center
for Biotechnology Information Entrez Nucleotide Database
(www.ncbi.nIm.nih.gov/sites/entrez). Ten microliters of PCR prod-
ucts were resolved on a 2% agarose gel with 1× TAE (0.01 M Tris,
Table 1
Oligonucleotides primers for mRNA amplification and sizes of PCR products ampli-
fied with specific primer pairs.
Gene Primer sequence Annealing Product size
temperature
p53 F 5′-gaagacccaggtccagatga-3′ 60 °C 350 bp
R 5′-ctccgtcatgtgctgtgact-3′
p 21/CiP1 F 5′-gcgatggaacttcgactttgt-3′ 54.4 °C 352 bp
F-5′gggcttcctcttggagaagat-3′
Bax F 5′-aaagctagcgagtgtctcaagcgc-3′ 60 °C 350 bp
R 5′-tcccgccacaaagatggtcacg-3′
Bcl-2 F 5′-cgacgacttctcccgccgctaccgc-3′ 58 °C 300 bp
R 5′-ccgcatgctggggccgtacagttcc-3′
GapDH F 5′-catgaccacagtccatgccatcact-3′ 57 °C 400 bp
R 5′-tgaggtccaccaccctgttgctgta-3′
Please cite this article in press as: Sunny O. Abarikwu, Ebenezer O. Farombi, Atrazine induces apoptosis of SH-SY5Y human neuroblastoma cells via the regulation of Bax/Bcl-2
ratio and caspase-3-dependent pathway, Pesticide Biochemistry and Physiology (2014), doi: 10.1016/j.pestbp.2014.12.006
3
0.2 M acetic acid and 0.05 M EDTA, pH 8.0) buffer at 40 V for 2 h.
The gel was then visualized with ethidium bromide staining on a
UV illuminator equipped with a camera connected to a computer.
100 bp DNA ladder was used as a marker (New England Biolabs. Inc).
The gel image was analyzed using AlphaImager software
(AlphaInnotech Corp., San Leandro, CA, USA).
2.4.8. Western blot analysis
After treatment of cells with ATZ (5–50 μg/mL, 23.2–232 μM) 24 h,
the cells were lysed for 30 min in 100 μL of CelLytic™ M Cell Lysis
Reagent (Sigma) supplemented with complete protease inhibitor
cocktail. Protein concentration of the cell lysates were determined
by the Lowry method [30]. The same amount of cell lysate (100 μg
of protein) was separated electrophoretically on a 12.5% SDS–
polyacrylamide gel. The proteins on the gel were transferred to a
polyvinylidene difluoride (PVDF) membrane at 250 mA for 3 h in
transfer buffer (25 mM Tris buffer, 190 mM glycine, 20% methanol,
0.01% SDS). The membranes were blocked with 5% skim milk–
TBST (20 mM Tris-base, 137 mM NaCl, 0.1% Tween-20, pH 7.5)
overnight at 4 °C, rinsed by 4–5 washes in TBST and subsequently
incubated with primary antibodies (1:1000 dilution) against Bcl-
2, Bax, p21, p53 and β-actin at room temperature for 2 h. The
membrane was washed four times in TBST and incubated with horse-
radish peroxidase-conjugated sheep anti-mouse antibody or donkey
anti-rabbit antibody. After four washes with TBST, the immune-
reactive bands were detected with TMB-H2O2 (Sigma) and visualized
with a MultiImage Light cabinet (AlphaInnotech Corp.). Quantitations
of expressed protein levels were done using AlphaEase™ FC
StandAlone V 4.0.0 software (Alpha Innotech).
2.5. Statistical analysis
The results are presented as the mean ± SD. The data were ana-
lyzed by one-way analysis of variance (ANOVA) followed by Duncan
post hoc test using SPSS. The level of significance was set at p < 0.05
for all statistical analysis.
3. Results
3.1. Determination of cell viability by MTT, NRU and LDH leakage
assays in ATZ-treated cells
The applied bioassays (MTT and NRU) indicated a concentration-
and time-dependent cytotoxic effect of ATZ on SH-SY5Y cells which
became significant from the 25 μg/mL concentration upwards (Fig. 1A
and B). Significant effect of ATZ on cell viability was evident at 3 h
incubation period in the MTT and NRU assays and the cell viabili-
ty was decreased by 19% and 15% respectively in the cells treated
with 25 μg/mL ATZ concentration. The toxicity progressively in-
creased with higher ATZ concentrations. The highest applied
concentration of 50 μg/mL decreased the cell viability by 70% in the
MTT and NRU assays at 24 h culture period. No significant differ-
ences in LDH activities were observed at all the tested concentrations
after 3 h culture period. LDH leakage was significantly elevated be-
ginning from 6 h culture period in a concentration and time
dependent manner. LDH leakage was 198% at 6 h which increased
to 228% at 24 h with the 25 μg/mL. The highest ATZ concentration
(50 μg/mL) increased LDH leakage by 233% at 6 h which increased
to 289% at 24 h (Fig. 1C).
3.2. Measurement of ROS generation in ATZ-treated cells
There seems to be differential concentration effects of ATZ on
ROS level in SH-SY5Y (Fig. 1D). The maximum ROS levels were
 ARTICLE IN PRESS
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A Control
10 μg/mL
5 μg/mL
25 μg/mL
B Control
10 μg/mL
5 μg/mL
25 μg/mL
50 μg/mL 50 μg/mL
140 140
120 120
Cell viability (%)

Cell viability (%)

100 100
*
80 * 80
60 ** 60 **
40 40
20 20
0 0
3h 6h 24 h 3h 6h 24 h

C Control 5 μg/mL D Control 5 μg/mL

10 μg/mL 25 μg/mL 10 μg/mL 25 μg/mL
50 μg/mL 50 μg/mL
500 350
300 *
400 *
% LDH release

ROS level (%) 250 **

*
300 200
*
200 150
100
100
50
0 0
3h 6h 24 h 1h 3h 6h 12 h

Fig. 1. Assessments of SH-SY5Y cells (1 × 104 cells/well) viability by MTT (A), NRU (B) and LDH leakage (C) assays and ROS level (D) at different time points in culture. Data
were expressed as the percentage of absorbance values to the control group with the viability of untreated control cells taken as 100%. *Vs control. **Vs 25 μg/mL (p < 0.05).

found at 1 h with the 50 μg/mL ATZ. From that point on, the
levels of ROS reduced over a period of 12 h. The 25 μg/mL
concentrations induce ROS level in a time-dependent manner
reaching its maximum at 6 h. The lower concentrations of
ATZ (5 and 10 μg/mL) did not induce significantly ROS levels
throughout the exposure period. The qualitative generation of
ROS was also observed using fluorescence microscopy, which
detected the brightest obvious fluorescent intensity with
ATZ-treated cells at 25 and 50 μg/mL. The ROS-fluorescence
signal in the 25 and 50 μg/mL concentrations at 1 h incubation period
was increased about 2–3 fold when compared to control (Fig. 2A
and B).
3.3. The expressions of p53, p21, Bax, and Bcl-2 genes in
ATZ-treated cells
RT-PCR analysis of various amounts of RNA isolated from SH-
SY5Y cells exposed to ATZ demonstrated that the mRNA expression
of keys apoptosis related proteins p21, p53, and Bax were consis-
tently up-regulated, whereas Bcl-2 expression was down-regulated
relative to the control. These observations occurred only in cells
exposed to the 25 or 50 μg/mL but not cells treated with the 5 μg/
mL ATZ concentration. The amplification response to GAPDH-
mRNA used as a reference was not affected by the treatment (Fig. 3A
and B).
3.4. The expressions of p53, p21, Bax and Bcl-2 proteins in
ATZ-treated cells
The levels of these proteins following ATZ treatment (5, 25, 50 μg/
mL) were examined by Western blotting in order to determine
whether ATZ induces SH-SY5Y human neuroblastoma cell death by
changing their expression status at the level of translation. The ratio
of Bax-to-Bcl-2 protein can determine the susceptibility of the cell
to apoptosis [31]. Consequently, we determine if the cell death was
by altering the ratio between Bcl-2 and Bax.
As shown in Fig. 4A and B, treatment of cells with ATZ signifi-
cantly induced a dose-dependent decrease in the expression level
of Bcl-2 and increase in the expression levels of Bax, p53 and p21.
A densitometric analysis of the bands also showed that ATZ re-
sulted in a dose-dependent increase in the Bax/Bcl-2 ratio (Fig. 4C).
These data suggest that ATZ can induce apoptosis of SH-SY5Y human
neuroblastoma cells by regulating Bax/Bcl-2 ratio and that the apop-
tosis does not happen at 5 μg/mL concentration of ATZ.
3.5. Determination of apoptosis in ATZ treated cells
Based on the above results, it is apparent that ATZ significantly
induces ROS level, modulate the expressions of apoptosis-related
genes/proteins and inhibits cell viability of SH-SY5Y human neu-
roblastoma cells at 25 and 50 μg/mL (116 and 232 μM). Therefore,

Please cite this article in press as: Sunny O. Abarikwu, Ebenezer O. Farombi, Atrazine induces apoptosis of SH-SY5Y human neuroblastoma cells via the regulation of Bax/Bcl-2
ratio and caspase-3-dependent pathway, Pesticide Biochemistry and Physiology (2014), doi: 10.1016/j.pestbp.2014.12.006
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A 20 µm B 20 µm C 20 µm D 20 µm E 20 µm

B 4
3.5 **
ROS (Fold increase)

3
*
2.5
2
1.5
1
0.5
0

Fig. 2. (A) Qualitative characterization of intracellular ROS level by dichlorofluorescin diacetate (DCFH-DA) staining in ATZ treated SH-SY5Y cells (1.5 × 105 cells) for 1 h.
Cells were incubated with 20 μM of DCFH-DA as described in Materials and methods. Images were snapped (×400) using Nikon phase contrast cum fluorescence micro-
scope (model 80i) at excitation wavelength of 485 nm and an emission wavelength of 530 nm. (B) Relative quantification of fold induction in ROS generation in SH-
SY5Ycells following exposure to 25 μg/mL and 50 μg/mL ATZ. Quantification of fluorescence images for intracellular ROS generation was done by using Leica Q win500 image
analysis software. (A) Control; (B) Cells exposed to 5 μg/mL ATZ; (C) cells exposed to 10 μg/mL ATZ; (D) cells exposed to 25 μg/mL ATZ; (E) cells exposed to 50 μg/mL ATZ.
*Vs control. **Vs 25 μg/mL (p < 0.05). Scale bars = 20 μm.

we followed these concentrations for studying major mechanisms
underlying ATZ-induced apoptosis in SH-SY5Y human neuroblas-
toma cells. Although the lowest concentration of ATZ (5 μg/mL) did
not significantly affect the above parameters, it was also selected
to check if similar observation could be recorded at the level of
apoptosis. Condensation and degradation of chromosomal DNA are
cardinal features of apoptosis [32]. As shown in Fig. 5, Hoechst 33342
nuclear staining, revealed that the treatment of SH-SY5Y cells with
the two different concentrations (25, 50 μg/mL) of ATZ induces mor-
phological features typical of apoptotic cells including nuclear

A B
ATZ (μg/mL) - 5 25 50 Control 5 μg/mL 25 μg/mL 50 μg/mL
1.8
p53 ** **
1.6
**
Relative expression

1.4
p21
1.2
* *
1 * **
Bax 0.8 *
0.6
Bcl-2 0.4
0.2
GapDH 0
p53 p21 Bax Bcl-2

Fig. 3. (A) Reverse-transcription-PCR analysis of p53, p21, Bax and Bcl-2 protein mRNA expression in SH-SY5Y cell lines treated with different concentrations of ATZ for
6 h. The total RNA isolated from the neuronal cell was reverse-transcribed and the cDNA obtained was subjected to PCR. (B) The intensity of the signals were quantified by
densitometry and normalized to that of GapDH. The data provided are mean ± SD from three separate experiments that showed similar patterns. *Vs control, **Vs 25 μg/
mL (p < 0.05).

Please cite this article in press as: Sunny O. Abarikwu, Ebenezer O. Farombi, Atrazine induces apoptosis of SH-SY5Y human neuroblastoma cells via the regulation of Bax/Bcl-2
ratio and caspase-3-dependent pathway, Pesticide Biochemistry and Physiology (2014), doi: 10.1016/j.pestbp.2014.12.006
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A
ATZ (μg/mL) - 5 25 50
p53 B
Control 5 μg/mL 25 μg/mL 50 μg/mL
3.5
p21 **
3
*

Relative density
β-actin 2.5
2
Bax
1.5 ** **
*
1 *
β-actin *
**
0.5
Bcl-2
0
p53 p21 Bax Bcl-2
β-actin
C 7
6 **

Bax/Bcl-2 ratio
5
4
3 *
2
1
0

Fig. 4. (A) Western blot analysis of p53, p21, Bax and Bcl-2 protein expression in SH-SY5Y cell lines treated with different concentrations of ATZ for 24 h and then subse-
quently lysed. Equal amounts of proteins were then separated by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were probed with the indicated
antibodies (anti-Bax, anti-Bcl-2, anti-p53, anti-p21, and anti-β-actin) and detected as mentioned in the Materials and method section. β-actin was used as the internal control.
(B) The intensity of the signals were quantified by densitometry and normalized to that of GapDH. The data provided are mean ± SD from three separate experiments that
showed similar patterns *Vs control, **Vs 25 μg/mL (p < 0.05).

fragmentation and micronuclei formation compared with the control
cells whereas cells treated with the 5 μg/mL concentration have mor-
phologies similar to the control cells. The number of apoptotic cells
increased significantly from 56% in the 25 μg/mL ATZ-treated cells
to about 110% in cells exposed to the 50 μg/mL ATZ concentration
(Fig. 5B), indicating a concentration-dependent effect. Further-
more, the DNA fragmentation assay showed evidence of DNA
laddering (Fig. 6A) typical of apoptosis at 48 h culture period in the
25 or 50 μg/mL but not 5 μg/mL ATZ-treated cells. We next as-
sessed caspase-3 activity, another hallmark of the apoptotic pathway,
by enzymatic detection. The measurement of caspase-3 activity of
cell lysates obtained from SH-SY5Y human neuroblastoma cells
treated with 25 or 50 μg/mL ATZ, revealed an increase in caspase-3
activity from 12 h up to 24 h and then decline at 48 h (Fig. 6B). In
contrast, the 5 μg/mL ATZ concentration did not induce caspase-3
activity throughout the duration of treatment. To further show that
the activation of caspase-3 play a key role in the ATZ-induced
apoptotic pathway, SH-SY5Y human neuroblastoma cells were
treated with caspase-3 inhibitor z-VAD-FMK and/or 25 μg/mL
(116 μM) for 24 h. Treatment with caspase-3 inhibitor signifi-
cantly blocked caspase-3 activity (Fig. 6C). Interestingly, the blockade
of the caspase-3 activity prevented ATZ-induced cell death of SH-
SY5Y human neuroblastoma cells (Fig. 6D). This strongly suggests
that ATZ induces cell death through the caspase-3-dependent
pathway.
4. Discussion
The results observed for the toxicity endpoints described in this
study demonstrate that ATZ increases LDH leakage, reduces MTT
reduction and decreases cellular uptake of the dye, neutral red from
the 25 μg/mL concentration and over 24 h exposure duration.
However, there are differences in the sensitivity of these cytotox-
icity endpoints. For instance, LDH-leakage induced by ATZ was
noticed from 6 h incubation period, whereas the MTT and NRU assays
revealed the toxic effects of ATZ on cell viability 3 h earlier. Fur-
thermore, with increasing concentrations, the MTT and the NRU
assays show similar toxicity pattern. In these assays (MTT and NRU),

Please cite this article in press as: Sunny O. Abarikwu, Ebenezer O. Farombi, Atrazine induces apoptosis of SH-SY5Y human neuroblastoma cells via the regulation of Bax/Bcl-2
ratio and caspase-3-dependent pathway, Pesticide Biochemistry and Physiology (2014), doi: 10.1016/j.pestbp.2014.12.006
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A 50 µm B 50 µm C 50 µm D 50 µm E 50 µm

B 250 **
Percent apoptotic nuclei
200 *

150

100

50

Fig. 5. (A) Nuclear morphology of ATZ treated SH-SY5Y cells for 48 h showing fragmented nuclei (short arrow) and micronuclei (long arrow) after staining with the fluo-
rescent DNA stain Hoechst bisbenzimide 33342 and observed by fluorescence microscopy. (A): Control (B): Cells exposed to 5 μg/mL ATZ (C): Cells exposed to 25 μg/mL
ATZ (D and E): Cells exposed to 50 μg/mL ATZ. (B) Percentage of apoptotic cells detected in SH-SY5Y cells treated with different concentrations of ATZ for 48 h. Data are
expressed as percentages and represent the mean ± SD of three separate experiments in which at least 200 cells were counted per one treatment group. *Vs control. **Vs
25 μg/mL exposure (p < 0.05). The bar scale in the picture is 50 μm in length.

it is possible to detect the injury within the cell. The LDH leakage
assay is based on the measurement of LDH activity in the extra-
cellular medium. The loss of intracellular LDH and its release into
the culture medium is an indicator of irreversible cell death due to
cell membrane damage [33]. The Neutral red and the MTT assay
appear to be more sensitive in detecting early toxicity compared
to the LDH leakage assay as indicated in Fig. 1. These differences
among the cytotoxicity assays indicate intracellular effect due to ex-
posure to ATZ before any permanent cell membrane damage
occurred.
Our results show that human SH-SY5Y cells may be sensitive to
much lower levels of ATZ than has been shown for other cells such
as the HepG2 cells [20] but may be sensitive to much higher levels
of ATZ than has been shown for other cells [34]. For example, the
25 μg/mL concentration of ATZ used in the present study was 4 times
lower than the concentration used in HepG2 cells which did not alter
cell viability [20] and in the same concentration range (20–80 μg/
mL) as used in the Chinese hamster ovary cells which decreased
cell viability at the lowest concentration of 20 μg/mL after 72 h ex-
posure [17]. Dhanwada et al. [35] who used a concentration about
3000 times lower than the 25 μg/mL ATZ concentration used in the
present study, reported growth inhibition of the normal human fi-
broblasts. The results of our cell viability assays are also in agreement
with our initial findings were we had used a concentration (65 μg/
mL, 65,000 ppb) of commercial formulation of ATZ which contain
no more than 80% ATZ with the rest being other herbicide con-
taminants, such as propazine (up to 2%), and 20% inactive ingredients
[25]. The inclusion of multiple ATZ concentrations and pure ATZ
preparation (98%) as used in this study is an indication that the
observed toxic effects observed in our previous and current studies
are ascribable to the active ingredient (ATZ). Hence, our collective
data suggest that the toxic effects of ATZ on cell viability either at
concentrations relevant to human exposures or at high concentra-
tions as used in many laboratory studies may depend on cell type.
Our results also showed an increase in ROS levels in ATZ-
treated cells. The increase in ROS levels may likely to contribute to
the observed cytotoxicity, as the alteration in viability of different
cultures is known to be associated with a significant formation of
ROS [36]. We further investigated the mechanism of ATZ-induced
cell death and our data showed that ATZ treatment of SH-SY5Y cells
induces an increase in p53, p21, and Bax expressions and a de-
crease in Bcl-2 expression both at the level of gene transcription
and translation. Most of these genes are directly or indirectly in-
volved in metabolism of ROS [25,37], and are also required in the
apoptotic pathway [38], and we, having observed in the present study
elevated ROS level earlier before the induction of cell death, we think
that ATZ-induced apoptosis could involve ROS. Because we ob-
served in our previous studies with the commercial formulation of
ATZ that the antioxidative bioactive agent kolaviron modulated ATZ-
induced apoptosis [25], this compelled us to speculate in the present
study that ROS generated in the apoptotic pathway may also con-
tribute to ATZ-induced apoptosis. Furthermore, the alteration in the
expression of Bax and Bcl-2 leads to high Bax/Bcl-2 ratio (Fig. 3C)
which is an important factor in determining the cell’s vulnerabil-
ity to apoptosis. Thus, ATZ-induced apoptosis is controlled by a
balanced expression between these apoptosis-inducing and
apoptosis-suppressing molecules. Caspase-3 activity first becomes
detectable early in apoptosis, continues to increase as cells undergo

Please cite this article in press as: Sunny O. Abarikwu, Ebenezer O. Farombi, Atrazine induces apoptosis of SH-SY5Y human neuroblastoma cells via the regulation of Bax/Bcl-2
ratio and caspase-3-dependent pathway, Pesticide Biochemistry and Physiology (2014), doi: 10.1016/j.pestbp.2014.12.006
 ARTICLE IN PRESS
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A M 1 2 3 4
B Control
25 µg/mL
200
*
175 *
Caspase-3 activity
(% of control)
150
125
4000 bp
100
75
50
25
0
1000 bp 12 h 24 h
Fig. 6. (A) Agarose gel electrophoresis of a 1 kb DNA ladder (lane M) showing the
effect of different concentrations of ATZ on DNA ladder formation in SH-SY5Y neu-
roblastoma cells. SH-SY5Y cells were treated without (Lane 1), or with 5 μg/mL ATZ
(Lane 2), 25 μg/mL ATZ (lane 3), 50 μg/mL ATZ for 48 h. (B) Caspase-3-enzyme ac-
tivity of SH-SY5Y cells at different time points after exposure to different
concentrations of ATZ. Caspase-3 activities in the cell lysates (50 μg) were evalu-
ated in vitro using DEVD-pNA as substrate. The fold increase was calculated by
comparing the absorbance of p-nitroaniline at 405 nm from untreated controls with
the values from ATZ-treated samples. (C) Cells were treated with ATZ and z-VAD-
FMK for 24 hours. After the incubation period, the cells were harvested and analyzed
for caspase-3 activity as described in the Materials and methods section. (D) Cell
viability was assessed by MTT assay after 24 hours incubation for each group (control,
ATZ, and ATZ plus caspase-3 inhibitor). The results are the mean ± SD of three sep-
arate experiments. *Vs control. **Vs 25 μg/mL, (p < 0.05).
apoptosis, and rapidly declines in late stages of apoptosis [39]. In
the present study, Caspase-3 activity in ATZ-treated SH-SY5Y neu-
roblastoma cells reached a maximum at 24 h and then decline at
48 h, suggesting the involvement of caspase-3 in ATZ-induced apop-
tosis. Because the inducible expression of Bax in response to the
induction of apoptosis may result in caspase-3-activation in neu-
ronal cells [40], we examined if the cell death was caspase-3-
dependent. We found that ATZ treatment (25 μg/mL, 24 h) increases
the activity of caspase-3 about 74% (Fig. 6B). Additionally, SH-
SY5Y neuroblastoma cells were incubated with specific caspase-3
inhibitor (z-VAD-FMK) and ATZ, and then the caspase-3 activity was
analyzed in these samples. Results showed that incubation SH-
SY5Y neuroblastoma cells with z-VAD-FMK effectively inhibited the
caspase-3 activity by 32% (Fig. 6C) and cell death (Fig. 6D) by 56%.
These results suggest that the activation of caspase-3 is essential
for ATZ-induced apoptosis of SH-SY5Y neuroblastoma cells. The in-
creased Caspase-3 activity could then lead to the observed DNA
fragmentation and the morphological changes associated with the
phenotype of apoptotic cells including fragmented nuclei, micro
nuclei formation and condensed nuclei [41,42]. The ability of ATZ
to induce the formation of these apoptotic phenotypes have also
been observed in other model systems [17,43].
In conclusion, ATZ at a concentration range that is widely used
in most experimental studies, and with published results on cell
Please cite this article in press as: Sunny O. Abarikwu, Ebenezer O. Farombi, Atrazine induces apoptosis of SH-SY5Y human neuroblastoma cells via the regulation of Bax/Bcl-2
ratio and caspase-3-dependent pathway, Pesticide Biochemistry and Physiology (2014), doi: 10.1016/j.pestbp.2014.12.006
viability depending on cell type, induces SH-SY5Y neuroblastoma
cells to apoptotic cell death. The cell death involves regulation in
ROS level and is mediated through the Bax/Bcl-2 ratio and caspase-
3-dependent pathway. Further study with relevant human exposure
5 µg/mL
50 µg/mL concentration will provide further insight toward understanding the
mechanism of ATZ-induced cell death in neuronal cells.
Acknowledgments
* The Academy of Sciences for the Developing World, Trieste, Italy
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