Effects of Carbon Chain Length on the Perfluoroalkyl Acids-Induced

Jun 4, 2018 - products and industrial processes for over 60 years.1,2 The carbon−fluorine ... consumption and contamination patterns.5 Such findings...
0 downloads 0 Views 1MB Size
Subscriber access provided by University of Winnipeg Library

Food Safety and Toxicology

Effects of Carbon Chain Length on the Perfluoroalkyl Acids-Induced Oxidative Stress of Erythrocytes in Vitro Xingren Pan, Pengfei Qin, Rutao Liu, Wanni Yu, and Xiaofei Dong J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02197 • Publication Date (Web): 04 Jun 2018 Downloaded from http://pubs.acs.org on June 4, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 29

Journal of Agricultural and Food Chemistry

1

Effects of Carbon Chain Length on the Perfluoroalkyl

2

Acids-Induced Oxidative Stress of Erythrocytes in Vitro

3

Xingren Pan a,1, Pengfei Qin b,1,*, Rutao Liu c, Wanni Yu b, Xiaofei Dong b

4

a

5

Shuangling Road, Linyi, 276005, P.R.China

6

b

7

Protection, College of Resources and Environment, Linyi University, Shandong Province,

8

Shuangling Road, Linyi, 276005, P.R.China

9

c

10

School of Physics and Electronic Engineering, Linyi University, Shandong Province,

Shandong Provincial Key Laboratory of Water and Soil Conservation and Environmental

School of Environmental Science and Engineering, Shandong University, Shandong Province,

27# Shanda South Road, Jinan 250100, P.R.China

11 12 13

*All correspondence should be addressed to:

14

Pengfei Qin

15

College of Resources and Environment,

16

Linyi University, Linyi, Shandong, 276005, P.R.China

17

Phone: 86-539-8766720

18

Fax: 86-539-8766230

19

Email: [email protected] (Qin PF)

20

1

Both authors made equal contributions to this work and share the first authorship.

21 22

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

23

ABSTRACT

24

Perfluoroalkyl acids (PFAAs) have been found extensively in wildlife and human

25

bodies by sources of drinking water and food. In this study, we investigated the

26

effects of three PFAAs, perfluoropentanoic acid (PFPA), perfluorooctanoic acid

27

(PFOA) and perfluorodecanoic acid (PFDA), on the anti-oxidative defense system

28

and lipid peroxidation in erythrocytes separately. The results demonstrated that they

29

could lead to significant decline trends in the glutathione (GSH) levels together with

30

increases of malondialdehyde (MDA) contents, suggesting that three PFAAs induced

31

oxidative stress to erythrocytes. And PFDA with a longer carbon chain length posed

32

more of a threat than other two PFAAs. Furthermore, the activities of superoxide

33

dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) were also altered

34

in the presence of PFAAs upon erythrocytes. The changes of oxidative stress

35

markers and the concomitant alterations of antioxidant enzymes suggest the role of

36

oxidative stress in PFAA-induced damage upon erythrocytes.

37

KEYWORDS: Perfluoroalkyl acids; Erythrocytes; Oxidative stress

38 39 40 41 42 43 44

ACS Paragon Plus Environment

Page 2 of 29

Page 3 of 29

Journal of Agricultural and Food Chemistry

45

INTRODUCTION

46

Perfluoroalkyl acids (PFAAs) are man-made fluorinated compounds, which have

47

been designed as surface-active agents for water and oil resistant applications in

48

many commercial products and industrial processes for over sixty years.1,2 The

49

carbon-fluorine bond is extremely stable to both environmental and biologic

50

degradation, which accounts for the fact that extensive amounts of data have recently

51

become available describing the concentrations of PFAAs in numerous places and

52

media, including the environment, wildlife and even human serum and tissues.3,4

53

Distinct dietary exposure patterns from region to region have been found as a result

54

of different food consumption and contamination patterns.5 Such findings have led to

55

efforts to better understand the potential toxicological effects that may be inherent to

56

these compounds. Most notable among these are perfluorooctanoic acid (PFOA) and

57

perfluorooctane sulfonate (PFOS).6, 7 It has been shown that they could cause tumor

58

and nontumor effects on the immune and nervous systems and adversely affect

59

hepatic function, reproduction, and development.8-10 Besides, data of in vitro studies

60

on human cell lines presented evidences that both PFOA and PFOS may pose toxic

61

effects in terms of oxidative damage, membrane disruption and interference of

62

endogenous enzyme activity.11-13 The perfluoroalkyl chemicals are mainly

63

distributed in serum, liver and blood of animals living anywhere from the Polar

64

Regions to industrialized areas because of their long half-lives in living

65

organisms.14,15 In addition, rate of elimination is enhanced for short carbon chain

66

lengths, while PFAAs with longer chains are more bioaccumulative drawn from the

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

67

biomonitoring data.16, 17 Because of the hydrophobic group and the polar end in

68

PFAAs, they have great potential to cross membranes and to enter erythrocytes.18

69

However, details on the mechanisms for PFAA-induced oxidative cytotoxicity still

70

need to be elucidated.

71

Erythrocyte is known to play functional roles to transport oxygen from the lungs

72

to the tissues providing all cells with their required oxygen.19, 20 In the circulation

73

system, erythrocytes are continuously exposed to both endogenous and exogenous

74

sources of reactive oxygen species (ROS) which can damage them and impair their

75

function. ROS have also been reported to play a critical part in both physiological

76

and pathological conditions with resultant increase in DNA damage and apoptosis. In

77

order to minimize the effect of these ROS and the resultant oxidative stress,

78

erythrocytes have an extensive antioxidant system involving both non-enzymatic

79

low molecular weight antioxidants like glutathione (GSH) and α-tocopherol as well

80

as enzymatic antioxidants including catalase (CAT), superoxide dismutase (SOD),

81

glutathione peroxidase (GPx) and peroxiredoxin 2.21 Therefore, erythrocytes are

82

widely used as a model to investigate the oxidative damage for their vulnerability to

83

peroxidation.22 In our previous study, we found that the longer chained

84

perfluorodecanoic acid (PFDA) had a greater impact on the conformation of

85

hemoglobin than PFOA.18 Besides, earlier studies have established that increased

86

concentrations of PFAAs in blood lead to a likely cause of oxidative damage and

87

peroxisome proliferation.23 However, limited toxicity literature is available to

88

elaborate on the role of chain length in the toxicology of this class of compounds to

ACS Paragon Plus Environment

Page 4 of 29

Page 5 of 29

Journal of Agricultural and Food Chemistry

89

erythrocytes.

90

In this work, we made a special attempt to determine the toxicity indexes of

91

perfluoropentanoic acid (PFPA), PFOA and PFDA on the anti-oxidative defense

92

system of human erythrocytes. This study is helpful for understanding the effects of

93

PFAAs with different carbon length on erythrocytes during the blood circulation in

94

vivo. In addition, it will also complement studies on the environmental risk

95

assessment of PFAA pollution.

96

MATERIALS AND METHODS

97

Materials. The phosphate buffered saline (PBS) for erythrocytes consisted of 0.15

98

mol/L NaCl, 7.6×10−3 mol/L Na2HPO4 and 2.4×10−3 mol/L NaH2PO4 (pH 7.4). To

99

stabilize human blood samples, EDTA dipotassium salt dehydrate (Tianjin Kermel

100

Chemical Reagent Co., Ltd.) was freshly obtained from School Hospital of

101

Shandong University (Jinan). PFPA (97% purity), PFOA (95% purity), and PFDA

102

(97% purity) were all purchased from Alfa Aesar (Ward Hill, MA). Stock solutions

103

of PFPA, PFOA and PFDA were prepared at a concentration of 2.0×10-4mol/L in

104

PBS buffer for cell analysis. A 0.02 mol/L borate buffer (pH 9.0) was prepared for

105

the

106

3-Naphthalenedicarboxaldehyde (NDA) was obtained from Nippon Kasei Chemical

107

Co., Ltd. A 20 mmol/L NDA was prepared in acetonitrile, protected from light and

108

stored at 4 ◦C. Ultrapure water was used throughout the experiments.

fluorescence

measurements

of

GSH

in

erythrocytes.

2,

109

Cell Preparation. The study approved by the Ethics Committee of School

110

Hospital of Shandong University (Jinan) and it was followed all protocols for the

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

111

processing and handling of such samples. Written informed consent was also

112

obtained from all experimental participants.

113

The blood from six different healthy and nonsmoking adults was collected from

114

School Hospital of Shandong University (Jinan), whereas for each blood sample an

115

experimental point was done at least three times to make the replicates. The samples

116

were centrifuged at 150 × g for 5 min to separate the plasma. The leukocytes were

117

removed together with the supernatant and the erythrocytes were washed with PBS

118

three times at 150 × g for 5 min by centrifuging until a clear supernatant was

119

obtained. Then, erythrocytes in PBS solution were incubated for 3 h at ambient

120

temperature under gentle shaking with different PFAAs in concentrations at 5×10-6

121

mol/L, 1×10-5 mol/L, 5×10-5 mol/L and 1×10-4 mol/L. Samples without the additions

122

of PFAAs were used as the control group. After the exposures, the cells were washed

123

with PBS buffer by centrifuging. The number of cells in the final cell suspension was

124

about 1×107/mL for the assays of MDA, SOD, CAT and GPx measurements, which

125

was counted using a hemocytometer (Shanghai Medical Optical Instrument Plant,

126

Shanghai, China).

127

Analysis of GSH Contents in Erythrocytes. To measure the GSH contents, the

128

cells were washed with 0.02 mol/L borate buffer after the exposures of PFAAs by

129

centrifuging twice at 150 × g for 5 min and resuspended it in the borate buffer (2.5

130

× 106/mL). The cell suspension in borate buffer was incubated with fixed

131

concentration of NDA for 15 min. Then the cells were washed twice and

132

resuspended in borate buffer (2.5×106 /mL). A fixed volume (5 µl) drawn from the

ACS Paragon Plus Environment

Page 6 of 29

Page 7 of 29

Journal of Agricultural and Food Chemistry

133

cell suspension in each group was blown on a slide and gently covered with another

134

slide. Then the fluorescence images were analyzed by Electron Multiplying Charge

135

Coupled Device (EMCCD) in the epifluorescence microscope.

136

An inverted microscope (Model IX81, Olympus, Tokyo, Japan) equipped with a

137

60× objective (PlanApo, Olympus, Tokyo, Japan), a mercury lamp, a mirror unit

138

consisting of 470-490 nm excitation filter (BP470-490), a 505 nm dichromatic

139

mirror (DM 505), a 510-550 nm emission filter (BA510-550) and a 16-bit

140

thermoelectrically cooled EMCCD (Cascade 512B, Tucson, AZ, USA) were used for

141

epifluorescence measurements of GSH. The fluorescence emitted by these molecules

142

was collected by the 60×objective and the fluorescence images were acquired by the

143

EMCCD. Image acquisition was controlled by the MetaMorph software (Universal

144

Imaging, Downingtown, PA, USA)

145

Because the concentrations and the volumes of GSH-NDA solutions were fixed,

146

GSH contents could be demonstrated by the fluorescence intensities of cells.24 The

147

average fluorescence intensities of sixty cells in each group were analyzed and all

148

the results were expressed as the mean ± standard deviation (SD).

149

Determination of Malondialdehide (MDA) levels in Erythrocytes. MDA is the

150

degradation products of lipid peroxidation, which could illustrate the level of

151

oxidative stress in cells.25 The MDA contents were examined colorimetrically using

152

thio-barbituric acid (TBA) to form a colored MDA-TBA complex, with the

153

maximum absorption peak at 532 nm. The prepared cell suspensions in PBS buffer

154

above were used to measure the MDA levels on the basis of the procedure of the

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

155

Page 8 of 29

lipid peroxidation assay kit (Jiancheng Bioengineering Institute, Nanjing, China).

156

Analysis of SOD activity in Erythrocytes. The test principles are as follows: the

157

superoxide radical anion (O2•-), which is produced during the xanthine and xanthine

158

oxidase reaction systems, can oxidize hydroxylamine to nitrite, which can be

159

measured by the UV-vis spectrophotometer. But the SOD in the test samples could

160

inhibit the production of O2•-, leading to the reduction of nitrite. So its absorbance is

161

lower than that of the reference group with colorimetry. The relative SOD activity

162

was calculated by the following formula:26

163

Relative SOD activity (%) =

Acontrol − A1 × 100 % Acontrol − A0

(1)

164

Where Acontrol is the absorbance of the control group, A1 and A0 are the absorbance of

165

the testing group of erythrocytes in the presence and absence of PFAAs, respectively.

166

The cell suspensions in PBS buffer were prepared according to the procedure in

167

section above. The SOD activity of the samples was measured according to the

168

procedure of SOD Assay Kit (Jiancheng Bioengineering Institute, Nanjing, China)

169

by means of UV-vis-2450 spectrophotometer. Several assays were conducted and

170

each one was carried out in triplicate.

171

Analysis of CAT activity in Erythrocytes. The reaction of H2O2 decomposition

172

by CAT was terminated by adding ammonium molybdate, with the remaining H2O2

173

and ammonium molybdate producing a pale yellow complex which was measured at

174

405 nm.24 The relative CAT activity was calculated similar to the equation of relative

175

SOD activity above.

176

The erythrocytes incubated with different concentrations of three PFAAs were

ACS Paragon Plus Environment

Page 9 of 29

Journal of Agricultural and Food Chemistry

177

prepared according to the methods mentioned above and the group without the

178

additions of PFAAs was chosen as the reference group. The CAT activity in each

179

group was measured in triplicate using the Catalase Assay Kit (Jiancheng

180

Bioengineering Institute, Nanjing, China).

181

Analysis of GPx activity in Erythrocytes. GPx could promote the reaction of

182

H2O2 with GSH to produce H2O and oxidized glutathione (GSSG).27 The GPx

183

activity can be expressed by the speed of the enzymatic reaction, detected by the

184

consumption of GSH. 5, 5-Dithiobis (2-nitrobenzoic acid) (DTNB) is a disulphide

185

chromogen that is readily reduced by sulfhydryl compounds to an intensely yellow

186

compound. The absorbance of the reduced chromogen is measured at 412 nm in

187

UV-vis spectrophotometer and is directly proportional to the GSH concentration.28

188

The samples incubated with different concentrations of three PFAAs were obtained

189

according to the methods mentioned above and the group without the additions of

190

PFAAs was chosen as the reference group. The GPx activity was determined by

191

utilizing the GPx detection kit (Jiancheng Bioengineering Institute, Nanjing, China)

192

with spectrophotometry method.

193

Statistical Analysis. All the data are expressed as mean ± SD. To determine the

194

differences between means, one way analysis of variance (ANOVA) was used,

195

followed by a Tukey's multiple comparisons to calculate significance using the

196

GraphPad Prism software. A p value less than 0.05 was determined to be statistically

197

significant. Each blood sample was done at least three groups to make the replicates

198

and six adults were tested to determine the changes of biomolecules from different

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

199

adults.

200

RESULTS AND DISCUSSION

201

Analysis of GSH contents in erythrocytes. As an important non-enzymatic

202

antioxidant, GSH plays an essential part in the maintaining of redox equilibrium that

203

may alleviate cellular oxidative injury.29 The depletion of GSH contents in cells is

204

regarded as a sensitive biomarker of oxidative damage in cells. Because GSH is

205

natively non-fluoresent, it should be derivatized into fluorescence species to image

206

the GSH inside the erythrocytes. NDA is found to readily penetrate the cell

207

membrane and react with GSH to form a fluorescent GSH-NDA derivate.30

208

Therefore, NDA was adopted to label the intracellular GSH of erythrocytes. It has

209

been shown that the fluorescence intensity of the intracellular derivatization of GSH

210

remained constant after 4 min. Consequently, the incubation time of 15 min for the

211

labeling GSH by NDA was used to make sure of the complete derivation of GSH.

212

Before measuring the derivate fluorescence image, we firstly examined whether

213

three PFAAs could react with NDA to form fluorescent species. After acquiring the

214

same region in the middle of each image, the average intensity of PFAA-NDA by the

215

MetaMorph software was almost the same as that of NDA alone. Furthermore, the

216

GSH-NDA derivate exhibited far greater fluorescence intensity than that of NDA and

217

PFAA-NDA. Therefore, it could be concluded that three PFAAs have no impact on

218

the fluorescence intensity of NDA. And the GSH contents could be represented by

219

the integrated fluorescence intensity of the derived erythrocytes.

220

After optimizing the parameters like the cell concentration and exposure time, the

ACS Paragon Plus Environment

Page 10 of 29

Page 11 of 29

Journal of Agricultural and Food Chemistry

221

fluorescence images of erythrocytes derivatized by NDA were taken using EMCCD

222

(Figure 1). While the concentration of NDA solution in each group was fixed, the

223

fluorescence intensity in the absence or presence of PFAAs could be manifested by

224

the total masses of GSH in single cells, which was quantified after subtracting the

225

blank in the corresponding image using the Meta-Morph software. In order to

226

examine the effects of PFAAs on the GSH contents in erythrocytes, sixty cells were

227

selected for every PFAA concentration used. In Figure 2, the mean GSH contents in

228

each PFAA group exhibit a decline trend with the increase of PFAA concentrations.

229

In order to investigate the impact of carbon chain length on the depletion of GSH

230

contents, the distribution maps of integrated fluorescence intensity at 1×10-4 mol/L

231

of PFAA were also presented in Figure 3. It can be seen that after the additions of

232

three PFAAs, the cells with fluorescence intensity lower than 1×106 began to

233

increase compared to the reference group. And the phenomenon in the PFDA group

234

is more obvious than that in other two PFAAs, indicating that PFDA has a stronger

235

effect on the depletion of GSH levels. In other words, PFDA with a longer carbon

236

chain length posed more of an oxidative threat than PFPA and PFOA at higher

237

exposures.

238

Analysis of MDA levels in erythrocytes. MDA is the main oxidation product of

239

peroxidized polyunsaturated fatty acids, and the increase in the MDA level is an

240

important index of lipid peroxidation.31 From Figure 4, the MDA levels were not

241

significantly changed when treated with lower concentrations of three PFAAs. This

242

phenomenon is probably due to the systemic antioxidant defense in erythrocytes.

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

243

Nevertheless, the MDA contents in erythrocytes increased significantly (p