Cytoprotective effect of deferiprone against aluminum chloride-induced oxidative stress and apoptosis in lymphocytes

Accepted Manuscript Title: Cytoprotective effect of deferiprone against aluminum chloride-induced oxidative stress and apoptosis in lymphocytes Authors: Cuicui Zhuang, Yue She, Haiyang Zhang, Miao Song, Yanfei Han, Yanfei Li, Yanzhu Zhu PII: DOI: Reference:

S0378-4274(18)30007-9 https://doi.org/10.1016/j.toxlet.2018.01.007 TOXLET 10071

To appear in:

Toxicology Letters

Received date: Revised date: Accepted date:

29-9-2017 2-1-2018 4-1-2018

Please cite this article as: Zhuang, Cuicui, She, Yue, Zhang, Haiyang, Song, Miao, Han, Yanfei, Li, Yanfei, Zhu, Yanzhu, Cytoprotective effect of deferiprone against aluminum chloride-induced oxidative stress and apoptosis in lymphocytes.Toxicology Letters https://doi.org/10.1016/j.toxlet.2018.01.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Cytoprotective

effect

of

deferiprone

against

aluminum

chloride-induced oxidative stress and apoptosis in lymphocytes Cuicui Zhuanga, Yue Sheb, Haiyang Zhanga, Miao Songa,Yanfei Hana, Yanfei Lia,*, Yanzhu Zhuc,* a

Key Laboratory of the Provincial Education Department of Heilongjiang for Common Animal Disease

IP T

Prevention and Treatment, College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, China b Key

Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy

c

SC R

of Agricultural Sciences, Beijing 100081, P.R. China

Institute of Special Economic Animal and Plant Science, Chinese Academy of Agricultural Sciences, Changchun

130112, China

U

* Corresponding author at: Ph. D, professor, College of Veterinary Medicine, Northeast Agricultural University, NO. 59 Mucai Street, Xiangfang District, Harbin 150030,China. Tel.: +13936574268; fax: +86 451 55191672. E-mail address: [email protected] (Y.F. Li).

A

N

* Corresponding authors at: Institute of Special Economic Animal and Plant Science, Chinese Academy of Agricultural Sciences, Jilin 130112, China. Tel.: +8618043213522; fax: +86 81919849 (Y.Z. Zhu).

M

E-mail address: [email protected] (Y.Z. Zhu).

HIGHLIGHTS

Aluminum trichloride (AlCl3) induces oxidative stress and change antioxidant enzyme

ED

•

•

PT

activities in lymphocytes.

Deferiprone (DFP) attenuates AlCl3-induced oxidative stress and apoptosis in

CC E

lymphocytes.

•

Cytoprotective

effect

of

deferiprone

against

aluminum

chloride-induced

A

immunosuppression.

ABSTRACT: Aluminum (Al) is a toxic metal, and excessive Al accumulation causes

1

immunosuppression. Deferiprone (DFP) is a well-known chelator and used in dialysis patients for removing Al from tissues. The present study aimed to investigate whether DFP treatment can attenuate immunotoxicity induced by aluminum chloride (AlCl3) in cultured lymphocytes. Lymphocytes were treated with 0 and 0.6 mmol/L AlCl3•6H2O (pH 7.2) and/or 1.8 mmol/L DFP,

IP T

respectively. Immune function of lymphocytes was assessed by T and B lymphocytes proliferation rates, T lymphocyte subpopulations and IL-2, IL-6 and TNF-α contents. In addition, lymphocyte

SC R

damage was assessed by LDH activity, NO and MDA contents, NOS, SOD and GSH-Px activities, lymphocyte apoptosis index. These results showed that AlCl3 exposure reduced T and B

U

lymphocyte proliferation rates, CD3+ and CD4+ T lymphocyte subpopulations, CD4+/CD8+ ratio,

N

IL-2, IL-6 and TNF-α contents, SOD and GSH-Px activities, early and later lymphocyte apoptosis

A

indexes while enhanced CD8+ T lymphocyte subpopulation, NO and MDA contents, LDH activity.

M

DFP treatment attenuated the immunotoxicity of lymphocytes and reduced oxidative stress and

ED

lymphocyte apoptosis induced by AlCl3, indicating that DFP could protect lymphocytes against immunosuppression induced by AlCl3 through attenuating oxidative stress and apoptosis.

CC E

apoptosis

PT

Keywords: deferiprone, aluminum chloride, immunotoxicity, lymphocyte, oxidative stress,

1. Introduction

Metal pollution can cause various health problems, which has received increasing attention.

A

Although aluminum (Al) is not an essential element in mammals, it is an accumulated, toxic metal and widely used in daily life (Exley, 2013; Silva and Gonçalves, 2014). Al enters into and accumulates in humans via the diet, drinking water, fruit juices or citric acid, and medication, etc (Yongbae et al., 2007). European Food Safety Agency proposes that the tolerable weekly intake of 2

Al is 1 mg/kg body weight (B.W.) (Aguilar et al., 2008). Based on previous nutrition surveys, 5% of Americans take in 10.5 mg/kg B.W. of Al per week, the mean exposure level was about 176 μg/kg B.W./day in China, and the mean Al exposure reaches to 2.3 mg/kg B.W. per week in high-exposure population of several European countries (Walton, 2009; Djouina et al., 2016).

IP T

These excessive Al accumulation induces toxic effects on the brain, bone, liver, spleen, kidney, lung and ovary in humans and animals (Sun et al., 2015). In particular, the immune system is the

SC R

main target of Al accumulation and plays a premier presage role in Al toxicity (Synzynys, 2004; Lauricella and Nesse, 1993). Al can cause immunotoxicity through inhibiting the immune

U

functions of lymphocytes and inducing lymphocytic apoptosis (Willhite et al., 2012; Ayuob, 2013;

N

Zhu et al., 2014). Although people resist Al toxicity by reducing parenteral and oral Al exposure,

A

Al-related diseases are still increasing in the last 20 years (Arenas et al., 2008; Graf et al., 1981).

M

Thus, how to effectively alleviate the immunotoxicity of Al has become a hotspot in the researches

ED

of Al toxicity.

Chelators have been utilized to treat metallic poisoning in clinic, which not only enhance

PT

excretion but also decrease the clinical signs of toxicity by preventing metals from binding to

CC E

cellular target molecules (Jureša et al., 2005). Deferoxamine, as a kind of Al chelators, is first used in clinic in 1980 and can increase tissue Al elimination in Al-loaded rats. However, deferoxamine can induce ocular toxicity, disturbances of colour vision and loss of hearing, which restricts its

A

application (Liu et al., 2005). Thus, low-side-effect, cheap, and low-toxic deferiprone (DFP) replaces deferoxamine to treat Al poisoning. DFP, a member of the hydroxypyridinones family, is a neutral water-soluble and low-toxic complexing agent and possesses high absorpt ion efficiency because of a low molecular weight of 139.15 g/mol (Pragourpun et al., 2015). In addition, DFP (1, 3

2-dimethyl-3-hydroxypyrid-4-one) has been utilized to treat cancer, leukaemia, and thalassemia in Europe and Asia and is approved by the U. S. Food and Drug Administration in 2011 for clinical treatment (Zhang et al., 2015; Bretti et al., 2014). It is worth noting that DFP is also used in the detoxification of Al in hemodialysis patients, plutonium in nuclear industry workers and uranium

IP T

in some military personnel (González et al., 2003; Fukuda, 2005). Studies has proved that DFP can attenuate Al toxicity by increasing the Al excretion in the liver, kidney, brain and bone, and

SC R

regulating the balance of the oxidative/anti-oxidative system (Liu et al., 2005; Tubafard et al., 2010; Sivakumar et al., 2014). Moreover, DFP increased Al excretion and decreases serum Al

U

concentration in an acute rat model (Saljooghi, 2012). Futhermore, the chelating agents DFP

N

alterated lipids, proteins, phosphodiester and nucleic acids of the spleen tissue at molecular

A

levelprotected in aluminum intoxicated mice using Fourier transform infrared spectroscopy

M

(Sivakumar et al., 2014). However, the therapeutic effects of DFP on AlCl3-induced

ED

immunosuppression remains unclear in vitor. Therefore, the present study was conducted to

PT

determine whether DFP could attenuate immunotoxicity of AlCl3 in lymphocytes.

CC E

2. Materials and Methods

2.1 Cell Culture and Treatment

A

Lymphocytes were isolated according to the previous methodology (Zhang et al., 2013). The

spleens were separated from the 110-120 g Wistar rats and washed with cold phosphate buffered saline (PBS, pH 7.2). Then the spleen was placed in a 200-mesh stain steel sieve over a culture dish containing RPMI-1640 medium, and grounded into small pieces with the plunger of glass syringe. The liquid was transferred into a centrifuge tube containing the same volume of 4

lymphocyte separation medium and centrifuged at 2500×g for 20 min at room temperature. The middle milky layer was absorbed carefully to separate lymphocytes. 8 mL Tris-NH4Cl were added into the separated lymphocytes for 5 min to remove red blood corpuscles, and the liquid centrifuged at 2500×g for 20 min at room temperature. Then, the lymphocytes were washed twice

IP T

with PBS (centrifuged under 1500×g for 15 min, the supernatant was discarded) and suspended in RPMI-1640 medium with 10% fetal calf serum (FBS) and transferred into a culture bottle at

SC R

density of 2×106 cells/mL. The cell supernatant was incubated at 37℃ under an atmosphere containing 5% CO2 for 2 h. The lymphocytes were in the supernatant while monocytes and

U

granulocytes were on the bottom wall. The supernatant was transferred into new culture bottles.

N

Lymphocyte number was determined by a blood-cell counting chamber. The viability was checked

A

with Trypan blue dye and was > 95%. The lymphocytes were diluted to 2×106 cells/mL with

M

RPMI-1640 medium, and then were incubated at 37 °C under an atmosphere containing 5% CO2

ED

in RPMI-1640 medium with 10% FBS. This experimental protocol was approved by the Ethics Committee on the Use and Care of Animals, Northeast Agricultural University, China.

PT

Lymphocytes were randomly divided into four groups: control group, DFP group, Al group

CC E

and Al+DFP group. Lymphocytes were cultured for 24 h, then they were transferred to the RPMI-1640 medium. Lymphocytes were cultured in the RPMI-1640 medium containing 0 (control group), 0.6 (Al group, 1/10 IC50 of aluminum chloride•6H2O (AlCl3•6H2O)) mmol/L

A

AlCl3•6H2O (pH 7.2). After Al exposure for 24 h, DFP was added to a part of the medium including 0 and 0.6 mmol/L AlCl3•6H2O at the final concentrations of 1.8 mmol/L (DFP group, Al+DFP group), and lymphocytes were cultured in the same condition for another 24 h. After above the lymphocytes were collected for determination. There were ten replicates for each 5

treatment.

2.2 Determination of T and B lymphocytes proliferation rates

2×106 lymphocytes were used to detect lymphocytes proliferation rates according to the

IP T

previous method (Sylvester, 2011). The RPMI-1640 medium was added concanavalin A (the final concentration of 5 mg/L) and lipopolysaccharide (the final concentration of 10 mg/L) for 24 h.

SC R

Then, 20 μL MTT (5 g/L) was added for another 4 h. Finally, 150 μL dimethyl sulfoxide was added, and the plate was shaken until the crystals were dissolved. The adsorption values were

U

recorded at 490 nm by a 318 MC microplate reader (Shanghai Sanco instrument, China). There

A

2.3 Determination of T lymphocytes subpopulation

N

were ten replicates in each group, and each sample was assayed in triplicate.

M

The suspensions of lymphocytes were used to examine the proportions of CD3+, CD4+ and

ED

CD8+ T lymphocytes by FAC scan flow cytometry (Becton, Dickinson and Company) according to the previous method (Caraher et al., 2000). 1 g fluorescein isothiocyanate (FITC) anti-rat CD3+,

PT

phycoerythrin (PE) anti-rat CD4+, and PE anti-rat CD8+ as monoclonal antibodies labeled by

CC E

FITC and PE were added into the suspensions, respectively. Then, the cells were evaluated by FAC scan flow cytometry (Becton, Dickinson and Company). There were three replicates in each

A

group.

2.4 Determination of IL-2, IL-6 and TNF-α contents

The supernatant of the lymphocytes was collected and used to detect IL-2, IL-6 and TNF-α contcents using

125I

radioimmunoassay (RIA) kits (New Bay Biological Technology Co., Ltd., 6

Tianjin, China) according to the manufacturers’ instructions. There were ten replicates in each group. Each sample was assayed in duplicate, and IL-2, IL-6 and TNF-α contcents were derived from a standard curve composed of serial dilutions (1-400 pg/mL). In addition, the assay sensitivities were 1 pg/ml. The intra-assay coefficient of variation and inter-assay coefficient of

IP T

variation were 3.2-6.1% and 4.0-7.3%, respectively.

SC R

2.5 Determination of nitric oxide (NO) content and nitric oxide synthase (NOS) activity

2×106 lymphocytes were used to detect the NO content and NOS activity by Nitric Oxide

U

(NO) assay kit and Total Nitric Oxide Synthase assay kit (Nanjing Jiancheng Bioengineering

N

Institute, Nanjing, China) according to the manufacturers’ instructions. The adsorption values

A

were recorded at 550 and 530 nm by a 318 MC microplate reader (Shanghai Sanco instrument,

M

China) and an AAS-3500 atomic absorption spectrophotometer (Shanghai HP analysis instrument,

ED

China). There were ten replicates in each group and each sample was assayed in triplicate. The assay sensitivities were 0.2-600 μmol/L and 0.2-81.9 U/mL, respectively. And both coefficients of

PT

variation were 1.7%.

CC E

2.6. Determination of malondialdehyde (MDA) content, superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities 2×106 lymphocytes were used to detect the MDA contents, SOD and GSH-Px activities were

A

detected by Cell Malondialdehyde (MDA) assay kit, Superoxide Dismutase (SOD) assay kit and Glutathione Peroxidase (GSH-Px) assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer s’ instructions. The adsorption values were recorded at 530, 450 and 412 nm by a 318 MC microplate reader (Shanghai Sanco instrument, China) and an 7

AAS-3500 atomic absorption spectrophotometer (Shanghai HP analysis instrument, China). There were ten replicates in each group and each sample was assayed in triplicate. The assay sensitivities were 0.5-113 nmol/mL, 5.0-122.1 U/mL and 0.1-48 μmol/L, respectively. The intra-assay coefficients of variation were 2.3%, 5.5%, and 1.3%, respectively. The inter-assay coefficient of

IP T

variation were 5.34%, 3.32%, and 4.36%, respectively. 2.7. Determination of lactate dehydrogenase (LDH) activity

SC R

2×106 lymphocytes were used to detect the activity of LDH was detected by LDH activity

assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the

U

manufacturer’s instructions. The adsorption values were recorded at 450 nm by a 318 MC

N

microplate reader (Shanghai Sanco instrument, China). There were ten replicates in each group

M

coefficient of variation were 1.5%.

A

and each sample was assayed in triplicate. The assay sensitivity was 9.0-5000U/L and the

ED

2.8. Determination of lymphocyte apoptosis index

106 lymphocytes were used to detect the lymphocyte apoptosis index by Annexin V-FITC/

PT

propidium iodide (PI) analysis kit (Beyotime Institute of Biotechnology, Jiangsu, China)

CC E

according to the previous method (Li et al., 2015). The lymphocytes were washed twice by PBS. Then, the cells were stained with fluorescent probe solution containing 10 μL Annexin V-FITC and 10 μL PI in dark for 30 min at 4℃. Finally, the cells were evaluated by FAC scan flow

A

cytometry (Becton, Dickinson and Company) in 1 h. There were three replicates in each group.

2.9 Statistical analysis Results are expressed as mean ± standard deviation (SD). The results were analyzed by One-way analysis of variance followed by LSD test (SPSS 20.0 software; SPSS Inc., Chicago, IL, 8

USA) and Graphpad Prism 6.0 was used to drawn histograms. Values of P < 0.05 were considered statistically significant and values of P < 0.01 were considered highly significant. 3. Result 3.1 T and B lymphocytes proliferation rates

IP T

T and B lymphocyte proliferation rates were decreased in the Al group and Al+DFP group, significantly decreased in the Al group compared to the control group (P < 0.01), significantly

differences between the control group and DFP group (Fig.1).

U

3.2 T lymphocyte subpopulations

SC R

higher in the Al+DFP group compared to the Al group (P < 0.01), and there were no significant

N

CD3+, CD4+ T lymphocyte subpopulations and CD4+/CD8+ ratio were decreased, while

A

CD8+ T lymphocyte subpopulation was increased in the Al group and Al+DFP group. CD3+, CD4+

M

T lymphocyte subpopulations and CD4+/CD8+ ratio were significantly decreased in the Al group

ED

compared to the control group (P < 0.01) and significantly higher in the Al+DFP group compared to the Al group (P < 0.01). CD8+ T lymphocyte subpopulation was significantly increased in the

PT

Al group compared to the control group (P < 0.01), and significantly lower in the Al+DFP group

CC E

compared to the Al group (P < 0.01). There were no significant differences in T lymphocyte subpopulations between the control group and DFP group (Fig.2).

A

3.3 IL-2, IL-6 and TNF-α contents IL-2, IL-6 and TNF-α contents were decreased in the Al group and Al+DFP group,

significantly decreased in the Al group compared to the control group (P < 0.01), significantly higher in the Al+DFP group compared to the Al group (P < 0.01), and there were no significant differences between the control group and DFP group (Fig.3). 9

3.4 NO content and NOS activity NO content and NOS activity were increased in the Al group and Al+DFP group, significantly increased in the Al group compared to the control group (P < 0.01), significantly lower in the Al+DFP group compared to the Al group (P < 0.01) and there were no significant

IP T

differences between the control group and DFP group (Fig.4). 3.5 MDA content, SOD and GSH-Px activities

SC R

MDA content was increased, while SOD and GSH-Px activities were decreased in the Al group and Al+DFP group. MDA content was significantly increased in the Al group compared to

U

the control group (P < 0.01) and significantly lower in the Al+DFP group compared to the Al

N

group (P < 0.01). SOD and GSH-Px activities were decreased in the Al group compared to the

A

control group (P < 0.01), and significantly higher in the Al+DFP group compared to the Al group

M

(P < 0.01). There were no significant differences in MDA content, SOD and GSH-Px activities

ED

between the control group and DFP group (Fig.5). 3.6 LDH activity in the supernatant

PT

LDH activities were increased in the Al group and Al+DFP group, significantly increased in

CC E

the Al group compared to the control group (P < 0.01), significantly lower in the Al+DFP group compared to the Al group (P < 0.01), and there were no significant differences between the control group and DFP group (Fig.6).

A

3.7 Lymphocyte apoptosis index The apoptosis was detected by FAC scan flow cytometry (Fig.7). The lymphocyte apoptosis indexes were increased in the Al group and Al+DFP group. The early and later apoptosis indexes were significantly increased in the Al group compared to the control group (P < 0.01), 10

significantly lower in the Al+DFP group compared to the Al group (P < 0.01), and there were no significant differences between the control group and DFP group (Fig.8). 4. Discussion The findings of our present study suggest that inhibited immune function and enhangced

IP T

lymphocyte membrane damage, oxidative stress and lymphocyte apoptosis contribute to

oxidative stress and apoptosis in the AlCl3-treated lymphocytes.

SC R

AlCl3-induced immunotoxicity. Whereas DFP treatment could attenuate the immunosuppression,

Splenic lymphocytes are divided into T and B lymphocytes. The numbers of T and B

U

lymphocytes reflect cellular and humoral immune state (Sharpe et al., 2006; LeBien et al., 2008).

N

In addition, T lymphocytes are separated into CD3+, CD4+ and CD8+ T lymphocytes

A

subpopulations, which play important roles in T lymphocytes immune function (Koretzky, 2010).

M

CD3+ molecule expresses in all mature T lymphocytes, and is a common surface marker for T

ED

lymphocytes. CD4+ lymphocytes, T helper cells, recognize class II histocompatibility molecules, while CD8+ lymphocytes, cytotoxic cells, recognize class I histocompatibility molecules (Moticka,

PT

2016). CD4+ T lymphocytes are activated, then secrete IL-2, IL-6 and TNF-α, which play crucial

CC E

roles in regulating both immune activation and homeostasis (Gaffen and Liu, 2004). In this study, AlCl3 exposure distinctly reduced splenic T and B lymphocytes proliferation rates, CD3+, CD4+ T lymphocyte subpopulations, CD4+/CD8+ ratio and IL-2, IL-6 and TNF-α and enhanced CD8+ T

A

lymphocyte subpopulation, which keeps consistent with previous research, indicating that AlCl3 inhibited immune function of lymphocytes (Golub et al., 1993; Wei et al., 2001; She et al., 2012; Hu et al., 2013). NO, a kind of free radical molecules, is involved in versatile immune processes including the 11

cell proliferation, apoptosis, and immune defense (Bogdan et al., 2000). NO is produced from the reaction that NOS catalyzes the conversion of L-arginine to L-citrulline (Bogdan, 2001). NO caused cellular damages through DNA injury, lipid peroxidation and protein oxidations (Karalezli and Kulaksızoglu, 2015). MDA is a decomposition product of peroxidized polyunsaturated fatty

IP T

acids and can reflect the condition of oxidative stress. However, the antioxidant system can alleviate the damage from SOD and GSH-Px, as key antioxidant radical-scavenging enzymes, can

SC R

eliminate the excessive free radicals and maintain homeostasis of redox status in aerobic host

organisms (Nordberg and Arnér, 2001). In this study, AlCl3 exposure increased NO and MDA

U

contents, NOS activity, decreased SOD and GSH-Px activities, suggesting that AlCl3 induced

N

oxidative stress of lymphocytes. The imbalance in oxidative oxidant-antioxidant status mainly

A

characterized by increased lipid peroxidation and a decreased level of antioxidant enzymes (Maya

M

et al. 2016). Since Al has a fixed oxidation state, it is impossible to directly participate in

ED

formation of free radical. However, Al induced disruption of Fe metabolism through competing transferrin and ferritin, which resulted in the generation of reactive oxygen species (ROS). And Al

PT

facilitates Fe-mediated oxidation in biological membranes and liposomes (Gutteridge et al. 1985;

CC E

Xie and Yokel 1996; Verstraeten et al. 1998). In addition, Al could inhibited directly antioxidant enzymes activities such as SOD, GSH-Px through an interaction with protein structure (Kenneth et al. 2004; Lu et al. 2006; Celik et al. 2012). It has been reported that Al exposure decreased the

A

GSH and SOD levels in erythrocytes, osteoblasts, human mesenchymal stem cells, mice and rats (Alshatwi et al. 2013; Sun et al. 2016; Xu et al. 2017; Zakaria et al. 2017). Thus, Al might be not induce directly the formation of ROS, instead of inhibiting SOD and GSH-Px activities, which induced oxidative damage in lymphocytes. 12

LDH, a glycolytic enzyme, catalyses the interconversion of pyruvate and lactate with concomitant interconversion of NADH and NAD+ (Dong et al., 2016). LDH catalyzes pyruvate to lactate when oxygen is absent or in short supply and performs the reverse reaction under alkaline conditions. LDH is abundant and presents in most types of cells in the body, and LDH release is a

IP T

sensitive biological metric for evaluating the cell membrane integrity, and an increased LDH release indicates that membrane permeability increases and cell membranes become broken

SC R

(Maekawa et al., 1995; Jin et al., 2013). Thus, the increased LDH activity in the Al group of this study suggested that AlCl3 induced lymphocyte membrane damage.

U

Apoptosis is a physiologic mechanism employed by most multicellular organisms to maintain

N

homeostasis of body tissues. Apoptosis occurs in physiological involution and atrophy of various

A

tissues and organs, and is triggered by noxious agents. Exogenous chemicals caused

M

immunotoxicity through abnormal lymphocytes apoptosis (Coutant et al., 2006). In this study,

ED

increased early and later lymphocyte apoptosis indexes suggested that AlCl3 induced lymphocyte apoptosis. The finding was consistent with our previous study (Xu et al., 2015; Li et al., 2016).

PT

Chelation therapy is utilized primarily to reduce the toxic effects of metal ions on tissues and

CC E

used in ranging from acute intoxication and chronic toxicity deriving from occupational, environmental and even iatrogenic causes. DFP can remove Al from tissues and is neutral water-soluble and non-toxic for treating Al toxicity (Liu et al., 2005). In addition, DFP forms a

A

strong 3:1 complex with Fe and treats Al toxicity on the basis of its use for iron overload because Al and iron are hard acids with similar ionic radii (54 and 64 pm), charge and protein binding. (Liu et al., 2005; Missel et al., 2005). Thus, we added 0.6 mmol/L AlCl3•6H2O and 1.8 mmol/L DFP into the medium. 13

In the present study, the protective effect of DFP against AlCl3-induced immune disorder has been evaluated in the lymphocytes. DFP treatment enhanced T and B lymphocyte proliferation rates, CD3+, CD4+ T lymphocyte subpopulations, CD4+/CD8+ ratio, IL-2, IL-6 and TNF-α content and reduced CD8+ T lymphocyte subpopulation compared to the Al group, indicating that DFP

IP T

attenuated immunosuppression in the AlCl3-treated lymphocytes. DFP has an highly efficient antioxidants’ scavenging activity against free radicals and can

SC R

prevent oxidative stress, toxicity and disease (Sivakumar et al., 2014). DFP and deferoxamine blocked ROS production and increased antioxidant activities induced by Al (Sivakumar et al.,

U

2014). NO and MDA contents, NOS activity decreased, and SOD and GSH-Px activities increased

N

in the Al+DFP group, suggesting that DFP attenuated AlCl3-induced oxidative stress. LDH is a

A

marker of membrane injury. The increased LDH is accompanied by a decrease in GSH-Px

M

activities (Eraković et al., 2003). Thus, the significantly decreased LDH in Al+DFP group

ED

compared to the Al group indicated that DFP alleviated Al-induced lymphocyte membrane damage in this study, which may attribute to increased GSH-Px activities. In addition, DFP

PT

reduced early and later apoptosis indexes in this study, indicating that DFP inhibited the

CC E

lymphocyte apoptosis induced by AlCl3. Although we confirm that DFP can attenuate AlCl3 immunotoxicity, DFP causes transient agranulocytosis, musculoskeletal and joint pains, gastric intolerance, zinc deficiency in patients using total doses of over 100 mg/kg/day (Kontoghiorghes,

A

1995). Thus, whether DFP can be used in clinical remains to be futher studied 5. Conclusion AlCl3 inhibites immune function and induced lymphocyte membrane damage, oxidative stress and lymphocyte apoptosis. DFP can attenuate AlCl3-induced immunosuppression through 14

reducing oxidative stress and apoptosis. Thus, DFP may be a more effective chelator to treat Al immunotoxicity. These findings provide theoretical basis for the treatment of Al poisoning. Conflict of interest The authors declare they have no actual or potential competing financial interests.

IP T

Acknowledgments

SC R

The following are was supported by a grant from National Natural Science Foundation Project (31172375；31372496).

References

A

CC E

PT

ED

M

A

N

U

Aguilar, H.A.F, Barlow, S., Castle, L., Crebelli, R., Dekant, W., Gontard, N., Gott, D., Grilli, S., Leclercq, C., Pratt, I., Rietjens, I., Tobback, P., Toldrá F., 2008. Scientific opinion of the panel on food additives, flavourings, processing aids and food contact materials on a request from European Commission on Safety of aluminium from dietary intake. The EFSA Journal 754, 1-34. Alshatwi, A.A., Subbarayan, P.V., Ramesh, E., Al-Hazzani, A.A., Alsaif, M.A., Alwarthan, A.A., 2013. Aluminium oxide nanoparticles induce mitochondrial-mediated oxidative stress and alter the expression of antioxidant enzymes in human mesenchymal stem cells. Food Addit. Contam. Part A Chem. Anal. Control. Expo. Risk Assess. 30(1), 1-10. Arenas, M.D., Malek, T., Gil, M.T., Moledous, A., Núñez, C., Alvarez-Ude, F., 2008. Use of the aluminum phosphate-binders in hemodialysis in the ultrapure water era. Nefrologia 28, 168-173. Ayuob, N.N., 2013. Can vitamin E and selenium alleviate the immunologic impact of aluminium on pregnant rats’ spleens? Cell Immunol. 284, 104-110. Bogdan, C., Röllinghoff, M., Diefenbach, A., 2000. Corrigendum: Reactive oxygen and reactive nitrogen intermediates in innate and specific immunity. Curr. Opin. Immunol. 12, 64-76. Bogdan, C., 2001. Nitric oxide and the immune response. Nat. Immunol. 2, 907-916. Bretti, C., Cigala, R.M., Crea, F., Lando, G., Sammartano, S., 2014. Thermodynamics of proton binding and weak (Cl−, Na+ and K+) species formation, and activity coefficients of 1, 2-dimethyl-3-hydroxypyridin-4-one (deferiprone). J. Chem. Thermodyn 77, 98-106. Caraher, E.M., Parenteau, M., Gruber, H., Scott, F.W., 2000. Flow cytometric analysis of intracellular IFN-γ, IL-4 and IL-10 in CD3+ CD4+ T cells from rat spleen. J. Immunol. Methods 244, 29-40. Celik, H., Celik, N., Kocyigit, A., Dikilitas, M., 2012. The relationship between plasma aluminum content, lymphocyte DNA damage, and oxidative status in persons using aluminum containers and utensils daily. Clin Biochem. 45(18), 1629-1633. Coutant, A., Lebeau, J., Bidon, W.N., Levalois, C., Lectard, B., Chevillard, S., 2006. 15

A

CC E

PT

ED

M

A

N

U

SC R

IP T

Cadmium-induced apoptosis in lymphoblastoid cell line: involvement of Caspase-dependent and-independent pathways. Biochimie 88, 1815-1822. Djouina, M., Esquerre, N., Desreumaux, P., Vignal, C., Body-Malapel, M., 2016. Toxicological consequences of experimental exposure to aluminum in human intestinal epithelial cells. Food Chem. Toxicol. 91, 108-116. Dong, X., Fan, Y., Zhang, H., Zhong, Y., Yang, Y., Miao, J., Hua, S., 2016. Inhibitory effects of ionic liquids on the lactic dehydrogenase activity. Int. J. Biol. Macromol. 86, 155-161. Exley, C., 2013. Human exposure to aluminium. Environ. Sci. Process Impacts 15, 1807-16. Fukuda, S., 2005 Chelating agents used for plutonium and uranium removal in radiation emergency medicine. Curr. Med. Chem. 12, 2765-2770. Gaffen, S.L., Liu, K.D., 2004. Overview of interleukin-2 function, production and clinical applications. Cytokine 28, 109-123. Golub, M.S., Takeuchi, P.T., Gershwin, M.E., Yoshida, S.H., 1993. Influence of dietary aluminum on cytokine production by mitogenstimulated spleen cells from Swiss Webster mice. Immunopharmacol. Immunotoxicol. 15, 605-619 González, S.I., Fernández Martín, J.L., Naves, D.M., Cannata Andía, J.B., 2003. Effect of desferrioxamine and deferiprone on osteocalcin secretion in osteoblast-type cells. Nefrologia 23, 27-31. Graf, H., Stummvoll, H.K., Meisinger, V., Kovarik, J., Wolf, A., Pinggera, W.F., 1981. Aluminum removal by hemodialysis. Kidney Int. 19, 587-592. Gutteridge, J.M., Quinlan, G.J., Clark, I., Halliwell, B., 1985. Aluminium salts accelerate peroxidation of membrane lipids stimulated by iron salts. Biochim. Biophys. Acta. 835(3), 441-447. Hu, C., Jing, L., Zhu, Y., Bai, C., Zhang, J., Xia, S., Li, Y., 2013. Effects of Al on the splenic immune function and NE in rats. Food Chem. Toxicol. 62, 194-198. Jin, Y., Ye, X., Shao, L., Lin, B.C., He, C.X., Zhang, B.B., Zhang, Y.P., 2013. Serum lactic dehydrogenase strongly predicts survival in metastatic nasopharyngeal carcinoma treated with palliative chemotherapy. Eur. J. Cancer. 49, 1619-1626. Jureša, D., Blanuša, M., Kostial, K., 2005. Simultaneous administration of sodium selenite and mercuric chloride decreases efficacy of DMSA and DMPS in mercury elimination in rats. Toxicol. Lett. 155, 97-102. Kenneth, A., M'Liss, S., Xavier, A.H., Sushil, J., 2004. Antioxidants prevent aluminum-induced toxicity in cultured hepatocytes. J. Inorg. Biochem. 98, 1129-1134. Kontoghiorghes, G.J., 1995. Comparative efficacy and toxicity of desferrioxamine, deferiprone and other iron and aluminium chelating drugs. Toxicol. Lett. 80(1-3), 1-18. Kulaksızoglu, S., Karalezli, A., 2015. Aqueous Humour and Serum Levels of Nitric Oxide, Malondialdehyde and Total Antioxidant Status in Patients with Type 2 Diabetes with Proliferative Diabetic Retinopathy and Nondiabetic Senile Cataracts. Can. J. of Diabetes 40, 115-119. Lauricella, A., Nesse, A., 1993. Aluminum and immune system. Ther Drug Monit. 15, 137. LeBien, T.W., Tedder, T.F., 2008. B lymphocytes: how they develop and function. Blood 112, 1570-1580. Li, M., Yang, X., Zhuang, C.C., Cao, Z., Ren, L.M., Xiu, C.Y., Li, Y.F., Zhu, Y.Z., 2015. NE strengthens the immunosuppression induced by AlCl3 through β2-AR/cAMP pathway in 16

A

CC E

PT

ED

M

A

N

U

SC R

IP T

cultured rat peritoneal macrophages. Biol. Trace Elem. Res. 164, 234-241. Li, M., Song, M., Ren, L.M., Xiu, C.Y., Liu, J.Y., Zhu, Y.Z., Li, Y.F., 2016. AlCl3 induces lymphocyte apoptosis in rats through the mitochondria–Caspase dependent pathway. Environ. Toxicol. 31, 385-394 Liu, P., Yao, Y.N., Wu, S.D., Dong, H.J., Feng, G.C., Yuan, X.Y., 2005. The efficacy of deferiprone on tissues aluminum removal and copper, zinc, manganese level in rabbits. J. Inorg. Biochem. 99, 1733-1737. Lu, J., Zheng, Y.L., Wu, D.M., Sun, D.X., Shan, Q., Fan, S.H., 2006. Trace amounts of copper induce neurotoxicity in the cholesterol-fed mice through apoptosis. FEBS Lett. 580, 6730-6740. Maekawa, M., 1995. Lactate dehydrogenase. Nihon Rinsho. 53, 1151-1156 Maya, S., Prakash, T., Madhu, K.D., Goli, D. 2016. Multifaceted effects of aluminium in neurodegenerative diseases: A review. Biomed. Pharmacother. 83, 746. Moticka, E.J., 2016. Chapter 23-T Lymphocyte Subpopulations. A Historical Perspective on Evidence-Based Immunology. 197-205 Missel, J.R., Schetinger, M.R., Gioda, C.R., Bohrer, D.N., Pacholski, I.L., Zanatta, N., Martins, M.A., Bonacorso, H., Morsch, V.M., 2005. Chelating effect of novel pyrimidines in a model of aluminum intoxication. J. Inorg. Biochem. 99, 1853-1857. Nordberg, J., Arnér, E.S.J., 2001. Reactive oxygen species, antioxidants, and the mammalian thioredoxin system. Free Radic Biol. Med. 31, 1287-1312. Pragourpun, K., Sakee, U., Fernandez, C., Kruanetr, S., 2015. Deferiprone, a non-toxic reagent for determination of iron in samples via sequential injection analysis. Spectrochim Acta A Mol. Biomol. Spectrosc. 48, 110-117. Saljooghi, A.S., 2012. Chelation of aluminum by combining deferasirox and deferiprone in rats. Toxicol. Ind. Health 28(8), 740-745. Silva, V.S., Gonçalves, P.P., 2014. Effect of lysine acetylsalicylate on aluminium accumulation and (Na+/K+) ATPase activity in rat brain cortex synaptosomes after aluminium ingestion. Toxicol. Lett. 232, 167-174. Sivakumar, S., Khatiwada, C.P., Sivasubramanian, J., 2014. Protective effects of desferrioxamine and deferiprone on the spleen tissue of aluminum intoxicated mice: A Fourier transform infrared spectroscopy study. Spectrochi Acta A Mol. Biomo. Spectrosc. 126, 148-156. Sharpe, A.H., Abbas, A.K., 2006. T-cell costimulation–biology, therapeutic potential, and challenges. New Eng. J. Med. 355, 973-975. She, Y., Wang, N., Chen, C., Zhu, Y., Xia, S., Hu, C., Li, Y., 2012. Effects of Aluminum on Immune Functions of Cultured Splenic T and B Lymphocytes in Rats. Biol. Trace Elem. Res. 147, 246-250. Sun, X., Cao, Z., Zhang, Q., Liu, S., Xu, F., Che, J., Zhu, Y., Li, Y., Pan, C., Liang, W., 2015. Aluminum chloride impairs bone and downregulates Wnt/β-catenin signaling pathway in young growing rats. Food Chem. Toxicol. 86,154-162. Sun, X., Liu, J., Zhuang, C., Yang, X., Han, Y., Shao, B., Song, M., Li, Y., Zhu, Y., 2016. Aluminum trichloride induces bone impairment through TGF-β1/Smad signaling pathway. Toxicology. 371, 49-57. Sylvester, P.W., 2011. Optimization of the tetrazolium dye (MTT) colorimetric assay for cellular growth and viability. Methods Mol. Biol. 716, 157-168. 17

A

CC E

PT

ED

M

A

N

U

SC R

IP T

Synzynys, B.I., Sharetskii, A.N., Kharlamova, O.V., 2004. Immunotoxicity of aluminum chloride. Gig. Sanit 4, 70-72. Tubafard, S., Fatemi, S.J., Saljooghi, A.S., Torkzadeh, M., 2010. Removal of vanadium by combining desferrioxamine and deferiprone chelators in rats. Med. Chem. Res. 19, 854-863. Verstraeten, S.V., Keen, C.L., Golub, M.S., Oteiza, P.I., 1998. Membrane composition can influence the rate of Al3+-mediated lipid oxidation: effect of galactolipids. Biochem. J. 333, 833-838. Vesna, E., Gordana, Z., Jadranka, V., Simonić, A., 2003. Pentylenetetrazol-induced seizures and kindling: changes in free fatty acids, superoxide dismutase, and glutathione peroxidase activity. Neurochem. Int. 42, 173-178. Walton J.R., 2009. Functional impairment in aged rats chronically exposed to human range dietary aluminum equivalents. Neurotoxicology. 30, 182-193. Wei, X.M., Lu, J.P., Fang, Q.H., Mo, L.F., 2001. Immunotoxicology of AlCl3 on the human T-lymphocyte cultivated in vitro. Chin. J. Prev. Med. 35, 213-214. Willhite, C.C., Ball, G.L., McLellan, C.J., 2012. Total allowable concentrations of monomeric inorganic aluminum and hydrated aluminum silicates in drinking water. Crit. Rev. Toxicol. 42, 358-442. Xie, C.X., Yokel, R.A., 1996. Aluminum Facilitation of Iron-Mediated Lipid Peroxidation Is Dependent on Substrate, pH, and Aluminum and Iron Concentrations. Archives Biochem. Biophys. 327(2), 222-226. Xu, F.B., Wang, J., Cao, Z., Song, M., Fu, Y., Zhu, Y.Z., Li, Y.F., 2015. cAMP/PKA signaling pathway induces apoptosis by inhibited NF-κB in aluminum chloride-treated lymphocytes in vitro. Biol. Trace Elem. Res. 170, 424-431. Xu, P., Wang, K., Lu, C., Dong, L., Gao, L., Yan, M., Aibai, S., Yang, Y., Liu, X., 2017. The Protective Effect of Lavender Essential Oil and Its Main Component Linalool against the Cognitive Deficits Induced by D-Galactose and Aluminum Trichloride in Mice. Evid. Based Complement Alternat Med. Doi: 10.1155/2017/7426538. Yongbae, K., Luisa, O., Jae, H.C., Alex, M., Joseph, P.B., 2007. Aluminum stimulates uptake of non-transferrin bound iron and transferrin bound iron in human glial cells. Toxicol. Appl. Pharmacol. 220, 349-356. Zakaria, M.M.H., Hajipour, B., Estakhri, R., Saleh, B.M., 2017. Anti-oxidative effect of resveratrol on aluminum induced toxicity in rat cerebral tissue. Bratisl. Lek. Listy. 118(5), 269-272. Zhang, J.H., Hu, C.W., Zhu, Y.Z., Liu, S.M., Bai, C.S., Han, Y.F., Xia, S.L., Li, Y.F., 2013. Effects of norepinephrine on immune functions of cultured splenic lymphocytes exposed to aluminum chloride. Biol. Trace Elem. Res. 154, 275-280. Zhang, X.M., Tian, Y.Y., Jia, J.T., Zhang, T.T., Zhu, G.S., 2015. Synthesis, characterization and dissolution of three pharmaceutical cocrystals based on deferiprone. J. Mol. Struct. 1108, 560-566. Zhu, Y.Z., Li, Y.F., Miao, L.G., Wang, Y.P., Liu, Y.H., Yan, X.J., Cui, X.Z., Li, H.T., 2014. Immunotoxicity of aluminum. Chemosphere. 104, 1-6.

18

IP T SC R

Fig.1. Effects of AlCl3 and PDF on the T and B lymphocytes proliferation rates (n=10). C, control group; DFP,

A

CC E

PT

ED

M

A

N

U

DFP group; Al, Al group; and Al+DFP, Al+DFP group. ** P < 0.01 versus the control. ## P < 0.01 versus the Al.

19

IP T

SC R

Fig.2. Effects of AlCl3 and PDF on the T lymphocyte subpopulations (n=3). C, control group; DFP, DFP

A

CC E

PT

ED

M

A

N

U

group; Al, Al group; and Al+DFP, Al+DFP group. ** P < 0.01 versus the control. ## P < 0.01 versus the Al.

20

IP T

SC R

Fig.3. Effects of AlCl3 and PDF on the IL-2, IL-6 and TNF-α (n=10). C, control group; DFP, DFP group; Al,

A

CC E

PT

ED

M

A

N

U

Al group; and Al+DFP, Al+DFP group. ** P < 0.01 versus the control. ## P < 0.01 versus the Al.

21

IP T

SC R

Fig.4. Effects of AlCl3 and PDF on the NO contents and NOS activities (n=10). C, control group; DFP, DFP

A

CC E

PT

ED

M

A

N

U

group; Al, Al group; and Al+DFP, Al+DFP group. ** P < 0.01 versus the control. ## P < 0.01 versus the Al.

22

IP T

Fig.5. Effects of AlCl3 and PDF on the MDA contents, SOD and GSH-Px activities (n=10). C, control group;

SC R

DFP, DFP group; Al, Al group; and Al+DFP, Al+DFP group. ** P < 0.01 versus the control. ## P < 0.01 versus the

A

CC E

PT

ED

M

A

N

U

Al.

23

IP T

Fig.6. Effects of AlCl3 and PDF on the LDH activities (n=10). C, control group; DFP, DFP group; Al, Al

A

CC E

PT

ED

M

A

N

U

SC R

group; and Al+DFP, Al+DFP group. ** P < 0.01 versus the control. ## P < 0.01 versus the Al.

24

B

C

D

PT

ED

M

A

N

U

SC R

IP T

A

Fig.7. The lymphocytes were treated with AlCl3 and PDF. The apoptosis was detected by FAC scan flow cytometry. In the scatter diagram, the first quadrant (Q1) represents necrotic cells, the second quadrant (Q2)

CC E

represents later apoptotic cells, the third quadrant (Q3) represents normal cells and the fourth quadrant (Q4)

A

represents early apoptotic cells. A, control group; B, DFP group; C, Al group; D, Al+DFP group.

25

IP T

Fig.8. Effects of AlCl3 and PDF on the lymphocyte apoptosis indexes (n=3). C, control group; DFP, DFP

A

CC E

PT

ED

M

A

N

U

SC R

group; Al, Al group; and Al+DFP, Al+DFP group. ** P < 0.01 versus the control. ## P < 0.01 versus the Al.

26