Vol.2, No.3, 253-261 (2010)
Copyright © 2010 SciRes Openly accessible at http://www.scirp.org/journal/HEALTH/
Antioxidant attenuation of ROS-involved cytotoxicity
induced by Paraquat on HL-60 cells
Wen-Hua Zhang, Yang Yang, Chang-Jun Lin, Qin Wang
School of Life Sciences, Lanzhou University, Lanzhou, China; qwang@lzu.edu.cn
Received 23 December 2009; revised 10 January 2010; accepted 14 January 2010.
The cytotoxic effects of Paraquat, an herbicide
refractory to treatment after intentional or acci-
dental contact, were investigated on the human
leukemia HL-60 cells. With the establishment of
Paraquat injury model of HL-60 cells, trypan
blue exclusion assaying was performed to have
determined the effects of Paraquat-induced cy-
totoxicity on HL-60 cells in a concentration-
dependent manner. Upon treatmen t with v arious
concentrat ions of Paraqua t, pronounced increase
on the levels of intracellular production of O2•-
and H2O2 was detected with employment of
fluorescent probes. Indicative of the oxidative
stress, levels of MDA and T-AOC were quanti-
tated to have determined the causal role for
Paraquat in subjecting HL-60 cells to oxidative
damage. Based on this finding, effects of anti-
oxidant enzymes including GSH, NAC, CAT and
SOD on attenuating the Paraquat-induced oxi-
dative damage on HL-60 cells were examined,
aiming to identify the most effective antioxidant
enzyme for alleviating the cytotoxicity induced
by Paraquat. In conjunction with the determina-
tion of cytotoxicity exerted by all the antioxidant
enzymes on HL-60 cells, GSH-with its least in-
herent cytotoxicity on HL-60 cells-was identified
as a promising candidate ingredient for extenu-
ating the Paraqua t -in duced cytotoxicity.
Keywords: Paraquat; HL-60 Cells; Oxidative Damage;
Cytotoxicity; Antioxidant Enzymes
As response to increasingly more environmentally-
associated outbreaks of diseases, Paraquat (PQ) had al-
ready gained tremend ous attention since 1966, fo llowing
the determination of the toxicity of PQ for a number of
animal species [1] and a report on two cases of acci dental
poisoning by PQ in human [2]. Herein, PQ is the trade
name of the dic hloride salt of the ra dical 1, 1’-dimet hyl-4,
4’-dipyridy liuim , whose herbi cidal pr operty was reported
in 1958. Since its toxicity is dosage-dependent, a wide
spectrum of studies on PQ-induced toxicity had been
conducted at various levels, aiming to standardize the
safe dosage to minimize its toxicity to other untargeted
organisms. Although an in vivo experiment upon the
toxicity of PQ, which set up the rats and mouse as study
objects and factored into the acute and chronic toxicity as
well as teratogenicity and mutagenicity, was conducted to
have claim ed t hat PQ w o ul d be safe if used foll o wi n g the
recommended use instruction, the hard-to-control ma-
nipulation of use in practice and differentiated suscepti-
bility of animal species necessitate the investigations of
underlying mechanism of PQ-induce cytotoxicity. More-
over, advanced techniques had already been employed
to identify effective candidate molecules to reduce PQ-
induced toxici ty [3].
PQ is biologically active after it has been sprayed in the
field. It’s either strongly bound to soil particles or de-
composed into a non-toxic form by soil bacterial and
sunlight [4]. However, active form of PQ is highly toxic
to humans and many cases of acute poisoning and death
had been reported over the past few decades [5-9]. The
most frequent routes of exposure to PQ, either acciden-
tally or intentionally, in humans and animals are by in-
gestion or through direct skin contact. Regardless of the
well-tuned administration of circulation and body-defense
systems, PQ could be rapidly distributed in most tissues,
with the highest concentration in the lungs and kidneys
[10], where the compound accumulates and causes great
damage in Cl ar a cel l s and alveolar type I an d II epithelial
cells [11]. Mechanisms involving the generation of Re-
active Oxygen Species (ROS) were implicated in the
PQ-induced cell apoptosis [12-16]. Although the infor-
mation on the possible mechanism of human carcino-
genesis associated with PQ exposure is limited, several
papers have suggested a possible link to Non-Hodgkin’s
Lymphoma by induction of chromosomal aberrations,
gene mutations in human lymphocytes, and apoptosis in
human B lymphocytes [17,18].
ROS is hypothesized to induce cell damage or initiate a
cascade of signaling mechanisms that ultimately lead to
cell adaptation, apoptosis or necrosis [19]. The mecha-
W. H. Zhang et al. / HEALTH 2 (2010) 253-261
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nisms of PQ toxicity include the formation of ROS me-
diated by cytochrome P450 reductases and subsequent
damage of ROS to cellular macromolecules [20]. The
dysfunction of pulmonary microvascu lar endothelial cell
membrane and high oxidative potential in pulmonary
microvascular endothelial cells, which resulted from the
overproduction of hydrogen peroxide, had been well-
documented to be involved in PQ-induced cytotoxicity
[21,22]. Additionally, EPR techniques had been em-
ployed to have confirmed that PQ could have triggered
the intracellular production of ROS during which the
formation of oxygen radicals (O2•-) and hydroxyl radical
(OH.) involved the m echanism of hydrogen t ransfer [23].
As one of the PQ-induced toxicity modes, mechanism of
ROS-induced neuronal damage by PQ had been proposed
[24]. Thus, Cellular Reactive Oxygen Species metabo-
lism has been the focus of intense interest regarding
physiological activities, due in part to the evidence that
over expression of antioxidant enzymes confers resis-
tance to oxidative stress, improvement in disease and
increased lifespan, respectively [25,26]. ROS including
superoxide, hydrogen peroxide and hydroxyl radicals are
commonly generated through cellular metabolism and
their physiological balance is achieved through neutrali-
zation of cellular antioxidant enzymes, which include
Glutathione (GSH), Superoxide Dismutase (SOD), Cata-
lase (CAT) and Glutathione Peroxidase (GPx) [27,28].
Oxidative stress occurs as a consequence of the imbalance
between the production of ROS and cellular antioxidant
capacity. Antioxidant enzymes, whi ch are biologically and
functionally coupled with ROS, have been evolved to
maintain the high-efficiency performance of cell functions.
A putative illustration of how a cell responds at risk of
oxidative damage had been proposed [2 9].
Here, based on the establishment of in vitro PQ injury
model of HL- 60 cell s, we ha ve studie d the R OS-invol ved
cytotoxicity induced by PQ in HL-60 cells through
measuring the contents and time-resolved changes of
malondialdehyde (MDA), total anti-oxidation compe-
tence (T-AOC), O2•- and H2O2. A concentration- and
time-dependent inhibition of cell growth has also been
quantified to determine whether the release of O2•- and
H2O2 into cytosol could potentially induce the cytotovic-
ity on HL-60 cells. In addition, we have explored that
whether the treatment with antioxidant enzymes could
attenuate the PQ-induced cytotoxicity on HL-60 cells.
Inspired by the PQ-detoxication effects of multiple
emulsions [30], we hope the GSH could be developed as
an ingredient in attenuating the PQ-induced to xicity.
2.1. Cell Line and Culture Medium
Human Promyelocytic Leukemia HL-60 cell line was
obtained from the cell library of Institute of Cancer Mo-
lecular biology and Drug Screening at Lanzhou Univer-
sity. RPMI-1640 cell culture medium was purchased
from Gibco (Santa Clara, USA). Mycoplasma-free Neo-
natal Bovine Serum was purchased from Hangzhou Si-
jiqing Biological Engineering Materials.
2.2. Reagents, A ssay Ki t s and Othe r Materia ls
Paraquat was purchased from Sigmal-Aldrich. Assay
kits of MDA and T-AOC were purchased from Promega
(Madison, WI, USA). Culture dishes and 24-well plates
were purchased from Hangzhou Sijiqing Biological En-
gineering Materials (Hangzhou, China). 2’, 7’-Di-
chlorofluorescein diacetate (DCF-DA) and Dihydro-
ethidium (DHE) probes were purchased from Molecular
Probes (Eugene, OR, USA). Other common experiment
materials were of analytically pure.
2.3. Culture of Human Promyelocytic
Leukemia HL-60 Cells and Analysis of
Cells Proliferation by Trypan Blue
HL-60 cells were grown in RPMI medium supplemented
with 2g/L NaHCO3, 100U/ml penicillin, 100μg/ml
streptomycin and 10% fetal calf serum in a humidified
atmosphere of 95% air and 5% CO2. Cultures were initi-
ated at a density of 104/ml at 37 and grown exponen-
tially to about 106/ml in 72 hours. Under the hypothesis
that the imbalance between oxidative stress and antioxi-
dant capacity could be restored if the antioxidant en-
zymes are replenished subsequently to the oxidative
damage [31], cells were treated with various concentra-
tions of PQ and desired concentrations of antioxidant
enzymes for assigned time to determine both the PQ-
induced cytotoxicity and antioxidant-mediated cytopro-
tection. To perform the trypan blue exclusion, cells were
washed and harvested with sterile PBS. The cell suspen-
sion was mixed at the ratio of 4:1 with 0.4% trypan blue
solution and incubated for 5 min. The viable cells were
counted in a hemocytometer with an inverted- phase
contrast microscope (Olympus IX81, Japan).
2.4. Detection of Intracellular Production of
O2•- and H2O2 by Inverted Fluorescent
For assessment of intracellular ROS produced by treat-
ment with PQ in the HL-60 cells, the DCF-DA and DHE
assay were performed [32,33]. The production of H2O2
was measured with a non-polar compound 2’, 7’-
dichlorofluorescein diacetate (DCF-DA) that readily
enter the cells, where it is cleaved to form non-fluorescent
2’, 7’-dichlorofluorescein (DCFH) by endogenous es-
terases. DCFH reacts with H2O2 to produce a fluorescent
compound 2’, 7’-dichlorofluorescein, which is trapped
inside the cells and indicates the intracellular level of
H2O2 [34,35]. To detect the intracellular production of
O2•-, DHE probe was used where DHE is oxidized to
W. H. Zhang et al. / HEALTH 2 (2010) 253-261
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oxoethidium by superoxide anions [36]. After treatment
with PQ, HL-60 cells were incubated with DCF-DA and
DHE for 1 hour, respectively. For quantification of fluo-
rescence intensities of DCF-DA and DHE the glass
slides were placed in an inverted fluorescent microscope
(Olympus, Japan) to take the images with excitation
wavelength at 485nm and emission wavelength at
525nm and 610 nm for DCF-DA and DHE, resp ect i vely.
2.5. Flow Cytometric Quantification of O2•-
and H2O2
Intracellular production of H2O2 and O2•- was measured
using DCF-DA and DHE as probes. HL-60 cells treated
with PQ for 12 hours were washed and resuspended in
PBS containing 20μM DCF-DA and 20μM DHE, re-
spectively. After 1 hour incubation in the dark at 37,
the extracellular fluorescence was removed by washing
with PBS for three times. Then cells were suspended
with PBS to the concen tration of 106/ml. The intracellu-
lar contents of H2O2 and O2•- were determined by flow
cytometry with excitation wavelength at 485nm and
emission wavelength at 525nm and 610 nm for DCF-D A
and DHE, respectively.
2.6. Determination of the Levels of MDA and
The increase in malondialdehyde (MDA) level upon
treatment with PQ indicates oxidative damages to major
cellular components, whereas the reduction in the level
of T-AOC occurs as a response to the oxidative stress
[27,28]. The levels of MDA and T-AOC were measured
by MDA assay kit [37] and T-AOC assay kit, respec-
tively. Well- grown HL-60 cells were treated with vari-
ous concentrations of PQ for 12 hours. After incubation,
cells were washed twice with PBS, harvested and soni-
cated following the instructions of the k its. The levels of
MDA and T-AOC were determined with a DU 6 40 sp ec-
triphotometer (Beckman instrument Inc, CA, USA). The
expression of MDA and T-AOC contents were con-
verted and presented as the following, respectively:
At- Ato
MDAcontent10nmol /mlCt
Ak- Ako
 
At: Absorbance of sample tube;
At0: Absorbance of blank sample tube;
Ak: Absorbance of standard tube;
At0: Absorbance of blank sample tube;
Ct: content of sample protein.
At- Ato
C contentn/ 30Ct
 
At: Absorbance of sample tube;
At0: Absorbance of blank sample tube;
n: dilution factor;
Ct: content of sample protein;
The reaction duration was 30 min.
ed independently more
7. Statistical Analysis
All experiments were perform
than 4-6 times and data are presented as mean ± standard
(SD ) deviation unless otherwise indicated. Statisti-
cal significance was determined between untreated
groups and treated groups at each time point, with the
one-way analysis of variance (ANOVA) and the t-test. A
value of p<0.05 was considered to be significant.
3.1. Analysis of PQ-Induced Toxicity on
3.1.1tive Effects of PQ 0 cells were
HL-60 Cells
. Antiprolifera
To examine the cytotoxicity of PQ, HL-6
treated with various concentrations of PQ for 48 hours
and cell viability was assayed by trypan blue exclusion
(Figure 1). PQ exerted a significant effect on the prolif-
eration of HL-60 cells in a time-dependent manner dur-
ing which no significan t changes were observed until 16
hours while pronounced effects on inhibiting the cells
proliferation occurred after 16 hours (Figur e 1(a) ); PQ at
concentration below 5μM exerted insignificant effects
on the proliferation of HL-60 cells while the prolifera-
tion of HL-60 cells was arrested upon treatmen t with PQ
at concentration above 100μM. At concentrations ranging
from 5μM to 100μM, PQ decreased HL-60 cells viability
in a concentration-dependent manner (Figure 1(b)).
Figure 1. Profile of antiprolifereffects of P Q on HL-60 cells. ative
Cells in culture dishes were treated with various concentrations of
PQ for 48 hours and cells viability was measured by trypan blue
exclusion in ranges of both 24 hours and 48 hour s. (a) Profile of
time-course proliferation of HL-60 cells by treatment with the
various concentrations of PQ. (b) Comparison of antiproliferative
effects of each concentration of PQ. **p<0.01 Vs 24 h control and
## p<0.01### p<0.001 Vs 48h control.
W. H. Zhang et al. / HEALTH 2 (2010) 253-261
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C Levels
To detontents of MDA and T-AOC
tracellular Levels of
For asacellular production of O2 and
ined by
3.1.2. Changes of MDA and T-AO
Induced by PQ
ermine whether c
inside HL-60 cells undergo appreciable fluctuations
upon treatment with PQ, MDA kit and T-AOC kit were
performed to assay the Oxidative stress-in itiated physio-
chemical variations of HL-60 cells. After treatment with
various concentrations of PQ for 24 hours, the MDA
level had increased. In particular, 100μM PQ elevated
MDA level by 85.1% while 12.5μM PQ exerted insig-
nificant effect on the changes of MDA level, whereas
25μM and 50μM PQs increased the MDA levels by
37.2% and 48.1%, respectively (Figure 2). Yet, upon
treatment by 12.5μM, 25μM, 50μM and 100μM PQs, the
level of T-AOC decreased by 62.8%, 77.7%, 87.6% and
98.8%, respectively (Figure 3).
3.1.3. Effects of PQ on the In
O2•- and H2O2
sessment of intr•-
H2O2 upon treatment with PQ, cells well-grown on 24
well plates were assayed by the DHE and DCF-DA assay
kits. HL-60 cells were treated by 50μM PQ for 12 hours.
Images were taken by fluorescent microscope after incu-
bating cell samples with DHE and DCF-DA for 1 hour,
respectively. In the DHE group, red fluorescence were
detected in 50μM PQ-treated HL-60 cells (Figure 4(d))
while no significant fluorescence signals were detected on
control ones (Figure 4(b)), which indicated that the treat-
ment with PQ induced the overproduction of O2•-. In the
DCF-DA group, the absence of detectable green fluores-
cence on the control group (Figure 5(b)), which was de-
tected on the 50μM PQ-treated HL-60 cells (Figure 5(d)),
confirmed that the treatmen t with 50μM PQ triggered the
intracellular production of H2O2. In addition, the viability
of 50μM PQ-treated HL-60 cells decreased compared
with the control group, whereas their morphology remain-
ed physiologicall y undam aged (Figure 5(d)).
Quantifications of O2•- and H2O2 were determ
w cytometry. HL-60 cells were treated by various
concentrations of PQ for 12 hours until confluent. In
parallel with control group, PQ-treated HL-60 cells were
hybridized with DHE and DCF-DA probes separately
Figure 2. Intracellular MDA levels of HL-60 cells increased
by treatment with various concentrations of PQ for 24 hours.
*p<0.05, **p<0.01 Vs the control.
Figure 3. Decrease in T-AOC levels of HL-60 cells upon
r 1 hour and the fluorescence intensity was measured.
s of Antioxidant
Treate cells were
treatment by various concentrations of PQ for 24 hours. The
levels of T-AOC was decreased substantially by 100μM PQ by
100μM PQ by 98.8% while 12.5μM, 25μM, 50μM reduced the
T-AOC levels by 62.8%, 77.7%, 87.6%, respectively. **p<0.01
Vs the control.
Contents of O2•- and H2O2 produced by treatment of PQ
were presented and compared (Figures 6 and 7). In the
DHE group, lev els of O2•- increased drastically in a co n-
centration-independent manner. In the DCF-DA group,
levels of H2O2 elevated substantially. From 12.5μM,
25μM to 50μM, the levels of H2O2 increased in a con-
centration-dependent manner and dropped slightly at the
point of 100μm.
3.2. Protective Effect
Enzymes on the PQ-Induced
Cytotoxicity of HL-60 Cells
. The Protective Effects of An ti
Enzymes on the PQ-Induced
Antiproliferation of HL-60 Cells
d with 50μM PQ for 12 hours, HL-60
subsequently incubated with GSH, NAC, SOD, CAT
and GSH-inhibitor BSO for 48 hours during which the
proliferation of cells was examined by Trypan blue ex-
clusion every 8 hours. Within either 24 hours or 48
hours, GSH, NAC, SOD and CAT improved the prolif-
eration of PQ-treated Hl-60 cells (Figure 8(a)) where
the combination of BSO with GSH had substantially
compromised the potency of GSH in enhancing the pro-
liferation of cells. In particular, the protective effects of
antioxidant enzymes performed better in 48 hours than
in 24 hours (Figures 8(a) and 8(b)). Among the four
antioxidant enzymes, GSH, NAC and CAT demonstra-
ted relatively potent effects on enhancing the prolifera-
tion of PQ- treated cells while the effect conferred by
SOD wasn’t significant. To screen the most efficient
antioxidant enzyme for protecting HL-60 cells against
PQ toxicity, cytotoxicity of these oxidant enzymes on
HL-60 cells was also examined by treatment with 50μM
PQ, 100μM GST, 200μM NAC, 200U/ml SOD, 400U/
ml CAT and 50μM BSO, respectively (Figure 9). There-
fore, GST was identified to have exerted least cytotoxic-
ity o n HL - 6 0 ce ll s while NAC and CAT were moderately
effective in extenuating the PQ-induced cytotoxcity, fac-
toring into their inherent minor toxicities on HL-60 cells.
W. H. Zhang et al. / HEALTH 2 (2010) 253-261
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Figure 4. Determination of the intracellular production of O2•- by fluorescent inverted micro-
scope. (a) and (b) were HL-60 cells, whereas (c) and (d) were 50μM PQ-treated H L-60 cells. (a)
and (c) were taken under visible light whereas (b) and (d) were taken fluorescent light with ex-
citation wavelength as 485 nm and emission wavelength as 610 nm.
Figure 5. Determination of the intracellular production of H2O2 by fluorescent inverted mi- croscope. (a) and (b) were HL-60 cells, whereas (c) and (d) were 50μM-treated HL-60 cells. (a)
and (c) were taken under visible light whereas (b) and (d) were taken fluorescent light with ex-
citation wavelength as 485 nm and emission wavelength as 525 nm.
W. H. Zhang et al. / HEALTH 2 (2010) 253-261
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Figure 6. Quantification of O2•- upon treatment with desired
concentrations of PQ. Control was presented as the basal level
of O2•- of the HL-60 cells. **p<0.01 Vs the control.
Figure 7. Quantification of H2O2 upon treatment with desired
concentrations of PQ. Control was presented as the basal level of
H2O2 of the HL-60 cells. *p<0.05 and ***p<0.001 Vs the control.
Figure 8. Effects of variousoxidant enzymes on the en-
f Antioxidative Enzymes on
hancing the proliferation of PQ-treated HL-60 cells. (a) Time-
course profile of proliferation of HL-60 cells upon treatment
with 50μM PQ, 100μM GST, 200μM NAC, 200 U/ml SOD, and
400 U/ml CAT and 25μM BSO, r espectivel y. (b) Cytoprotectiv e
effects of 100μM GST, 200μM NAC, 200 U/ml SOD, and 400
U/ml CAT and 25Μm BSO examined on a range of both 24
hours and 48 hours.
3.2.2. Effects o Levels of MDA an d T- AOC
HL-60 cells grown to confluent were treated with
r 12 hours. Levels of MDA were measured PQ foafter
incubating PQ-treated HL-60 cells with 100μM GST,
200μM NAC, 200U/ml SOD, 400U/ml CAT for 12
hours. Compared with the control group, GST greatly
decreased the level of MDA by 25.1% whereas NAC,
CAT and SOD also reduced the level of MDA by 18.5%,
14.9%, 4.4%, respectively (Figure 10). Similarly, after
treatment with 100μM GST, 200μM NAC, 400U/ml
CAT and 200U/ml SOD for 12 hours, the level of
T-AOC had been elevated significantly. Based on the
ground level set by the 50μM PQ, GSH, NAC, CAT and
SOD increased the level of T-AOC by 183.3%, 89.8%,
101.3% and 114.2%, respectively (Figure 11).
Figure 9. Cytotoxical effects of antioxidant enzymes on HL-60
cells. With HL-60 cells as control group, cytotoxicity indicated
by the proliferation of cells was examined by treatment with
50μM PQ, 100μM GST, 200μM NAC, 200U/ml SOD, 400U/ml
CAT and 50μM BSO. *p<0.05 and **p<0.01 Vs PQ for 24
hours incubation; #p<0.05 , ##p<0.01 and ###p<0.001Vs PQ for
48 hours incubation.
Figure 10. Modulation of MDA levels by treatment with anti-
oxidant enzymes. With 50μM PQ-treated HL-60 cells as control
and the intracellular MDA level of HL-60 cells as basal level,
MDA level was reduced upon treatment with 100μM GST,
200μM NAC, 200U/ml SOD and 400U/ml CAT by 25.1%,
18.5%, 14.9%, 4.4%, respectively. *p<0.05 and **p<0.01 Vs
50μM PQ.
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Figure 11. Increase of T-AOC levels upon treatment with an-
tioxidant enzymes. With 50μM PQ-treated HL-60 cells as con-
ented to be able to cause a range
of progressive and irreversible diseases to animal species
avenging overproduced
can be determined for the in-
volvement of oxidative damage in the PQ-induced cyto-
trol and the intracellular T-AOC level of HL-60 cells as basal
level, T-AOC levels were elevated by treatment with 100μM
GST, 200μM NAC, 200U/ml SOD and 400U/ml CAT by
183.3%, 114.2%, 89.8% and 101.3%, respectiv ely. *p<0.05 and
**p<0.01 Vs 50μM PQ.
PQ has been well-docum
and humans which are refractory to treatment [38-42]. As
it used worldwide in agriculture, the toxicities of PQ on its
untargeted organism are supposed to be well-administered.
A wide spectrum of investigations pivoting on the treat-
ment of PQ-induced diseases on humans have been con-
ducting to either quantify the safe dosage [43,44], or to
clinically monitor the long term evolution of PQ-caused
disease [45]. Although its pathological mechanism re-
mains mainly elusive, the contribution of oxidative stress
in the pathogenic process has been evidenced [32]. How-
ever, the inadequacy to elucidate the oxidative stress-
initiated pathway at the tissue level with different animal
models necessitates relevant studies of that pathway at the
cellular level. Toward this end, the PQ-induced injury
mode l of H L60- cells w as es tab lished to stu dy th e ROS-
mediated cytotoxicit y on HL-60 cel ls.
Antiproliferative effects of PQ on HL-60 cells and pro-
liferation-enhancing effects of antioxidant enzymes on
-treated HL-60 cells were examined by Trypan blue
exclusion assaying, respectively. The threshold of PQ
concen tration w ith phys iologica l relevan ce was dete rmined
with finding that PQ concentration below 5μM conferred
no pronounced effect on the cell growth and proliferation
while PQ concentration above 200μM led directly to the
death of all HL-60 cells. With concentration ranging from
12.5μM to 100μM, the antiproliferative effect of PQ was
observed to be concentration-dependent, which was in
agreement with a recent finding that production of hydro-
gen peroxide may contribute to the induction of apoptosis
on HL-60 cells [46]. Subsequently, 50μM PQ was ex-
perimentally chosen to treat HL-60 cells for studying
other physiochemical rele vance.
Indicative of the ROS-induced damage to cellular
components and ability of sc
ounts of ROS, MDA and T-AOC were examined
quantitatively to determine the cytotoxicity of PQ on
HL-60 cells, respectively. Relatively high concentrations
of PQ (50-100μM) caused an appreciable increase in the
MDA level while the T-AOC level dropped accordingly
in a concentration-dependent manner. It is generally con-
sistent with the finding that various con cen tration s of PQ
increased peroxidation in PC12 cells [24]. Thus, the in-
volvement of imbalance between the oxidative compe-
tence and antioxidant capacity was determined in the
PQ-induced cytotoxicity. Furthermore, the intracellular
production of O2•- and H2O2 was confirmed by fluores-
cent inverted microscope through hybridizing cell sam-
ples with DHE and DCF-DA probes and quantified by
employment of flow cytometric analysis. Determination
of the intracellular production by treatment with various
concentrations of PQ suggested that production of O2•-
wasn’t sensitive to the concentration s of PQ, whereas the
intracellular production of H2O2 was in a concentra-
tion-dependent manner. That finding was in part consis-
tent with the report that PQ (0.01-1mM) induced the
intracellular burst of ROS in rat cortical neurons [47].
In our study, effects of GSH, NAC, SOD and CAT on
extenuating PQ-induced cytotoxicity on HL-60 ce
ere further examined, following the treatment with
50μM PQ. In explicit agreement with the finding that
levels of antioxidant enzymes increased in inverse pro-
portion to the levels of ROS [48,49]. Among 100μM
GST, 200μM NAC, 400U/ml CAT and 200U/ml SOD
which were experimentally administered to examine their
potency, 100μM GSH was determined to reduce the
MDA level by 25.1% and increase the T-AOC lev el sig-
nificantly by 183.3%. 200μM NAC functioned well with
its performance on reducing MDA level and enhancing
T-AOC level by 18.5% and 89.8%, respectively. As a
parallel, BSO, which was the inhibitor of GSH, was also
used to exclude the false positive results of the efficacy
conferred by antioxidant enzymes. Hopefully, 100μM
GSH was identified as the most effective antioxidant
enzymes for attenuating the PQ-induced cytotoxicity, in
combination with examining the inherent cytotox icity of
all antioxidant enzymes on the HL-60 cells.
In this study, a causal role
toxic effects on HL-60 cells. Upon treatment with various
concentrations of PQ, the compromised capacity of HL-60
cells to scavenge the intracellular overproduced O2•- and
H2O2 was responsible for the cytotoxicity of HL-60 cells
on grounds that the levels of O2•-, H2O2, MDA, T-AOC
were altered unfavorably in the physiological level. Fur-
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a and Mr. Ximing Xu
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Catalytic metallopor-
manufacturing and
ed com-
mbrane dysfunction in pul-
ed oxidative stress and dysfunction
ed free radicals in Paraquat-treated
hemical Research, 23, 1387-
thermore, effects of antioxidant enzymes on attenuating
the PQ-induced cytotoxicity of HL-60 cells were also
examined to have identified the GSH as the most effective
one for extenuating the PQ-induced cytotoxicity. How-
ever, whether it could be used as an ingredient in devel-
oping PQ safener remains to be settled. Moreover, further
characterizing the underpinning mechanisms involved in
PQ cytotoxicity might contribute substantially to a com-
prehensive understanding of how environmental toxicants
including pesticides and herbicides lead to the increas-
ingly more frequent outbreaks of a plethora of environ-
ment-a sso ciat ed di sea se s.
The authors thank their colleagues, Mr. Peng Ji
for their technical assistance and results discu
financially supported by the Science and Technology Support Projects
of Gansu Province (No.2007GS01641) and the State Key Laboratory
of Veterinary Etiological Biology, Lanzhou Veterinary Research Insti-
tute, Chinese Academy of Agricultural Sciences.
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