Antioxidant attenuation of ROS-involved cytotoxicity induced by Paraquat on HL-60 cells

doi:10.4236/health.2010.23036

Paper Menu >>

Journal Menu >>

Vol.2, No.3, 253-261 (2010)

doi:10.4236/health.2010.23036

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.

ABSTRACT

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

1. INTRODUCTION

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

254

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. MATERIALS AND METHODS

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

Exclusion

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

Microscope

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

255

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

T-AOC

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

0.01



 





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. RESULTS

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)).

(a)

(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

256

C Levels

To detontents of MDA and T-AOC

tracellular Levels of

For asacellular production of O2 and

ined by

flo

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

3.2.1oxidant

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

257

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

258

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.

(a)

(b)

Figure 8. Effects of variousoxidant enzymes on the en-

f Antioxidative Enzymes on

50μM

anti

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.

Regulating

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.

W. H. Zhang et al. / HEALTH 2 (2010) 253-261

259

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

lls

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.

4. DISCUSSION

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.

5. CONCLUSIONS

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-

W. H. Zhang et al. / HEALTH 2 (2010) 253-261

260

MENTS

a and Mr. Ximing Xu

ssion. This work was

REFERENCES

ligott, T.F. and Hurst, E.W. (1966)

at. British Journal of Industrial Medi-

ccumulation: Tissue and species specificity.

t into rat lung.

, G. (1998) Intrahippocampal in-

esses of nervous system development: A

Martin, A., Moran, J.M., Garcia-Rubio, L., Fran-

Catalytic metallopor-

manufacturing and

-pharmaco-redox

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.

6. ACKNOWLEDGE

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.

[1] Clark, D.G., McEl

toxicity of ParaquThe

cine, 23, 126-132.

[2] Bullivant, C.M. (1966) Accidental poisoning by Paraquat :

Report of two cases in man. British Medical Journal, 1,

1272-1273.

[3] Somayajulu-Nitu, M., Sandhu, J.K., Cohen, J., Sikorska,

M., Sridhar, T.S., Matei, A., Borowy-Borowski, H. and

Pandey, S. (2009) Paraquat induces oxidative stress, neu-

ronal loss in substantia nigra region and Parkinsonism in

adult rats: Neuroprotection and amelioration of s ymptoms

by water-soluble formulation of Coenzyme Q10. BMC

Neuroscience, 10, 88.

[4] Roberts, T.R., Dyson, J.S. and Lane, M.C. (2002) Deacti-

vation of the biological activity of Paraquat in the soil en-

vironment: A review of long-term environmental fate.

Journal of Agricultural and Food Chemistry, 50, 3623-

3631.

[5] Onyon, L.J. and Volans, G.N. (1987) The epidemiology

and prevention of Paraquat poisoning. Hum Toxicol, 6,

19-29.

[6] Bismuth, C., Garnier, R., Baud, F.J., Muszynski, J. and

Keyes, C. (1990) Paraquat poisoning: An overview of the

current status. Drug Safety, 5, 243-251.

[7] Botella de Maglia, J. and Belenguer Tarin, J.E. (2000)

Paraquat poisoning: A study of 29 cases and evaluation of

the effectiveness of the “Caribbean scheme”. Medicina

Clinica (Barc), 115, 530-533.

[8] Luo, Z.Y. (2007) Treatment 16 cases of acute Paraquat

poisoning. Chinese Journal of Labour Hygiene and Oc-

cupational Diseases, 25, 330.

[9] Zhao, F.L., Wang, J. and Guo, W. (2009) Clinical obser-

vation on 22 cases of acute Paraquat poisoning. Chinese

Journal of Labour Hygiene and Occupational Diseases,

27, 56-57.

[10] Rose, M.S., Lock, E.A., Smith, L.L. and Wyatt, I. (1976)

Paraquat a

Biochemical Pharmacology, 25, 419-423.

[11] Rose, M.S., Smith, L.L. and Wyatt, I. (1974) Evidence for

energy-dependent accumulation of Paraqua

Nature, 252, 314-315.

[12] Melchiorri, D. , Del Duca, C., Picc irilli, S. , Trombetta, G.,

Bagetta, G. and Nistico

jection of Paraquat produces apoptotic cell death which is

prevented by the lazaroid U74389G, in rats. Life Scien ces,

62, 1927-1932.

[13] Barone, S., Das, K.P. , Lassiter , T.L. and White, L.D. (2000)

Vulnerable proc

review of markers and methods. Neurotoxicology, 21,

15-36.

[14] Gonzalez-Polo, R.A., Niso-Santano, M., Ortiz-Ortiz, M.A.,

Gomez-

cisco-Morcillo, J., Zaragoza, C., Soler, G. and Fuentes, J.M.

(2007) Relationship between autophagy and apoptotic cell

death in human neuroblastoma cells treated with Paraquat:

Could autophagy be a “brake” in Paraquat-induced apop-

totic death? Autophagy, 3, 366-367.

[15] Chen, P., Chen, Z., Li, A., Lou, X.C., W u, X.K., Zh ao, C.J.,

Wang, S.L. and Liang, L.P. (2008)

phyrin protects against Paraquat neurotoxicity in vivo.

Biomedical and Environmental Sciences, 21, 233-238.

[16] Drechsel, D.A. and Patel, M. (2008) Role of reactive

oxygen species in the neurotoxicity of environment

agents implicated in Parkinson’s disease. Free Radical

Biology & Medicine, 44, 1873-1886.

[17] Kuo, M.L. and Lin, J.K. (1993) The genotoxicity of the

waste water discharged from Paraquat

its pyridyl components. Mutation Resear ch, 300, 223-229.

[18] Takeyama, N., Tanaka, T., Yabuki, T. and Nakatani, T.

(2004) The involvement of p53 in Paraquat-induced

apoptosis in human lung epithelial-like cells. Interna-

tional Journal of Toxicology, 23, 33-40.

[19] Haddad, J.J. (2004) Redox and oxidant-mediated regulation

of apoptosis signaling pathways: Immu no

conception of oxidative siege versus cell death commitment.

International Immunopharmacology, 4, 475-493.

[20] Farrington, J.A., Ebert, M., Land, E.J. and Fletcher, K.

(1973) Bipyridylium quaternary salts and relat

pounds V. Pulse radiolysis studies of the reaction of

Paraquat radical with oxygen: Implications for the mode

of action of bipyridyl herbicides. Biochimica et Bio-

physica Acta, 314, 372-381.

[21] Tsukamoto, M., Tampo, Y., Sawada, M. and Yonaha, M.

(2000) Paraquat-induced me

monary microvascular endothelial cells. Pharmacol

Toxicol, 86, 102-109.

[22] Tsukamoto, M., Tampo, Y., Sawada, M. and Yonaha, M.

(2002) Paraquat-induc

of the glutathione redox cycle in pulmonary microvascu-

lar endothelial cells. Toxicology and Applied Pharma-

cology, 178, 82-92.

[23] Zang, L.Y., van Kuijk, F.J. and Misra, H.P. (1995) EPR

studies of spin-trapp

lung microsomes. Biochemistry & Molecular Biology

International, 37, 255-262.

[24] Yang, W.L. and Sun, A.Y. (1998) Paraquat-induced cell

death in PC12 cells. Neuroc

1394.

W. H. Zhang et al. / HEALTH 2 (2010) 253-261

261

n in heart mitochondria exposed to ischaemia and

g brain.

49-671.

Implications for therapeutics of neurode-

Quarterly, 3, 1-6.

in-

re of

n(II)-induced apopto sis in

duced cell viability loss

.P., Kimura, S., Yukimura, T., Kiyomoto,

c acid

eard, B.

(1970) Paraquat poisoning

B. and Sun, X.L. (2008) Treatment of Paraquat

raquat concentrations

athna, L., Eddleston, M., W ilks, M .F., Woollen, B.H.,

ang, J.O., Lee, E.Y.

artolomeo, A.

E., Tran-Thi, Q.H., Kahl, R. and

, J.M., Perez-Gomez , C., Rosado, R.,

000)

[25] Solaini, G. and Harris, D.A. (2005) Biochemical dys-

functio

reperfusion. Biochemical Journal, 390, 377-394.

[26] Blomgren, K. and Hagberg, H. (2006) Free radicals, mi-

tochondria, and hypoxia-ischemia in the dev elopin

Free Radical Biology & Medicine, 40, 388-397.

[27] Dringen, R. (2000) Metabolism and functions of glu-

tathione in brain. Progress in Neurobiology, 62, 6

[28] Dringen, R. (2005) Oxidative and antioxidative potential

of brain microglial cells. Antioxid Redox Signal, 7,

1223-1233.

[29] Floyd, R.A. and Hensley, K. (2002) Oxidative stress in

brain aging:

generative diseases. Neurobiology of Aging, 23, 795-807.

[30] Frasca, S., Couvreur , P., Seiller , M ., Pareau, D., Lacour , B.,

Stambouli, M. and Grossiord, J.L. (2009) Paraquat de-

toxication with multiple emulsions. International Journal

of Pharmaceutics, 380, 142-146.

[31] Hays, P. G. (1995) VIP interview: Patrick G. Hays. Interview

by Maria R. Traska. Managed Car e

[32] LeBel, C.P., Ischiropoulos, H. and Bondy, S.C. (1992)

Evaluation of the probe 2’, 7’-dichlorofl uorescin as an

dicator of reactive oxygen species formation and oxidative

stress. Chemical Resear ch in Toxicology, 5, 227-231.

[33] Benov, L., Sztejnberg, L. and Fridovich, I. (1998) Critical

evaluation of the use of hydroethidine as a measu

superoxide anion radical. Free Radical Biology & Medi-

cine, 25, 826-831.

[34] Oubrahim, H., Stadtman, E.R. and Chock, P.B. (2001)

Mitochondria play no roles in M

HeLa cells. Proceedings of the National Academy of

Sciences USA, 98, 9505-9510.

[35] Lee, C.S., Han, E.S. and Lee, W.B. (2003) Antioxidant

effect of phe nelzine on MPP+-inin S

differentiated PC12 cells. Neurochemical Research, 28,

1833-1841.

[36] Miyata, K., Rahman, M., Shokoji, T., Nagai, Y., Zhang,

G.X., Sun, GH., O

Kohno, M., Abe, Y. and Nishiyama, A. (2005) Aldosterone

stimulates reactive oxygen species production through ac-

tivation of NADPH oxidase in ra t mesangial cells. Journal

of American Society of Nephrology, 16, 2906-2912.

[37] Ohkawa, H., Ohishi, N. and Yagi, K. (1979) Assay for

lipid peroxides in animal tissues by thiobarbituri

reaction. Analytical Biochemistry, 95, 351-358.

[38] Matthew, H., Logan, A., Woodruff, M.F. and H

(1968) Paraquat poisoning-lung transplantation. British

Medical Journal, 3, 759-763.

[39] McDonagh, B.J. and Martin, J.

in children. Ar chives of Disease in Childhood, 45, 425-427.

[40] Wesseling, C., Hogstedt, C., Picado, A. and Johansson, L.

(1997) Unintentional fatal Paraquat poisonings among

agricultural workers in Costa Rica: Report of 15 cases.

American Journal of Industrial Medicine, 32, 433-441.

[41] Hantson, P. (2008) Paraquat poisoning. Rev Prat, 58

866-867.

[42] Zheng, X.

poisoning. Chinese Journal of Labour Hygiene and Oc-

cupational Diseases, 26, 635-637.

[43] Ikebuchi, J. (1987) Evaluation of Pa

in Paraquat poisoning. Archives of Toxicology, 60, 304-

310.

[44] Senar

Tomenson, J.A., Roberts, D.M. and Buckley, N.A. (2009)

Prediction of outcome after Paraquat poisoning by meas-

urement of the plasma Paraquat concentration. Oxford

Journal of Medicine, 102, 251-259.

[45] Lee, K.H., Gil, H.W., Kim, Y.T., Y

and Hong, S.Y. (2009) Marked recovery from Paraquat-

induced lung injury during long-term follow-up. Korean

Journal of Internal Medicine, 24, 95-100.

[46] Fabiani, R., Fu ccelli, R., Pi eravanti, F., De B

and Morozzi, G. (2009) Production of hydrogen peroxide

is responsible for the induction of apoptosis by hy-

droxytyrosol on HL60 cells. Molecular Nutrition & Food

Research, 53, 887-896.

[47] Schmuck, G., Rohrdanz,

chluter, G. (2002) Oxidative str ess in rat cor tical neu rons

and astrocytes in duced by P araqu at in v itro . Neurotoxicity

Research, 4, 1-13.

[48] Mates, J.M., Se gura

lalla, L., Blanca, M. and Sanchez-Jimenez, F.M. (1999)

Antioxidant enzymatic activities in human blood cells

after an allergic reaction to pollen or house dust mite.

Blood Cells, Molecules, and Diseases, 25, 103-109.

[49] Mates, J.M., Perez-Gomez, C. and Blanca, M. (2

Chemical and biological activity of free radical ‘scaven-

gers’ in aller gic diseases. Clini ca Chimica Acta, 296, 1-15.