Effects of Carbon Chain Length on the Perfluoroalkyl Acids-Induced

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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

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Journal of Agricultural and Food Chemistry

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Effects of Carbon Chain Length on the Perfluoroalkyl

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Acids-Induced Oxidative Stress of Erythrocytes in Vitro

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

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a

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

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b

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

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

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c

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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

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*All correspondence should be addressed to:

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Pengfei Qin

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College of Resources and Environment,

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

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Phone: 86-539-8766720

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Fax: 86-539-8766230

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Email: [email protected] (Qin PF)

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1

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

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ACS Paragon Plus Environment


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ABSTRACT

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

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

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

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

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

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

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

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

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

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

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

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

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oxidative stress in PFAA-induced damage upon erythrocytes.

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KEYWORDS: Perfluoroalkyl acids; Erythrocytes; Oxidative stress

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INTRODUCTION

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

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

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

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

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

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become available describing the concentrations of PFAAs in numerous places and

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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



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

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

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

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need to be elucidated.

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

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

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system, erythrocytes are continuously exposed to both endogenous and exogenous

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

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

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

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

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erythrocytes have an extensive antioxidant system involving both non-enzymatic

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

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

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

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

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

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

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

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

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

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


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erythrocytes.

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

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

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

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

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

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assessment of PFAA pollution.

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MATERIALS AND METHODS

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

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

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

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

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

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

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

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

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the

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

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

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

fluorescence

measurements

of

GSH

in

erythrocytes.

2,

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

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



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

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obtained from all experimental participants.

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

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

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

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

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

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

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

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

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

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

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

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

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

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Shanghai, China).

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

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

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

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

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

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


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

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

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Coupled Device (EMCCD) in the epifluorescence microscope.

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

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

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

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

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

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epifluorescence measurements of GSH. The fluorescence emitted by these molecules

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

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

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Imaging, Downingtown, PA, USA)

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

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

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

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

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

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

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

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

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

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



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

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

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

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

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

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

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

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was calculated by the following formula:26

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Relative SOD activity (%) =

Acontrol − A1 × 100 % Acontrol − A0

(1)

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

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

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

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

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

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

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each one was carried out in triplicate.

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

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

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

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

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SOD activity above.

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The erythrocytes incubated with different concentrations of three PFAAs were


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

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

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

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Bioengineering Institute, Nanjing, China).

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

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

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

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

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

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

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

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The samples incubated with different concentrations of three PFAAs were obtained

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

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

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

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with spectrophotometry method.

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

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

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

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

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

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



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adults.

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RESULTS AND DISCUSSION

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

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

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

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

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

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

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

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

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

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

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

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Before measuring the derivate fluorescence image, we firstly examined whether

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

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

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

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

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

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

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the integrated fluorescence intensity of the derived erythrocytes.

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


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fluorescence images of erythrocytes derivatized by NDA were taken using EMCCD

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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exposures.

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

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

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

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

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



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Nevertheless, the MDA contents in erythrocytes increased significantly (p

Effects of Carbon Chain Length on the Perfluoroalkyl Acids-Induced

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