Perfluorooctanoic acid and perfluorooctane sulfonate co-exposure induced changes of metabolites and defense pathways in lettuce leaves

Journal Pre-proof Perfluorooctanoic acid and perfluorooctane sulfonate co-exposure induced changes of metabolites and defense pathways in lettuce leaves Pengyang Li, Xihui Oyang, Xiaocan Xie, Yang Guo, Zhifang Li, Jialin Xi, Dongxue Zhu, Xiao Ma, Bin Liu, Jiuyi Li, Zhiyong Xiao PII:

S0269-7491(19)34615-9

DOI:

https://doi.org/10.1016/j.envpol.2019.113512

Reference:

ENPO 113512

To appear in:

Environmental Pollution

Received Date: 16 August 2019 Revised Date:

4 October 2019

Accepted Date: 28 October 2019

Please cite this article as: Li, P., Oyang, X., Xie, X., Guo, Y., Li, Z., Xi, J., Zhu, D., Ma, X., Liu, B., Li, J., Xiao, Z., Perfluorooctanoic acid and perfluorooctane sulfonate co-exposure induced changes of metabolites and defense pathways in lettuce leaves, Environmental Pollution (2019), doi: https:// doi.org/10.1016/j.envpol.2019.113512. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 Published by Elsevier Ltd.

Graphical abstract

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Perfluorooctanoic acid and perfluorooctane sulfonate co-exposure

2

induced changes of metabolites and defense pathways in lettuce

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leaves

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Pengyang Li1, 2, Xihui Oyang2, 3, Xiaocan Xie4, Yang Guo3, Zhifang Li4, Jialin Xi3, Dongxue Zhu2,

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Xiao Ma2, Bin Liu2, Jiuyi Li1,*, Zhiyong Xiao2,3, *

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Department of Municipal and Environmental Engineering, Beijing Key Laboratory of Aqueous

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Typical Pollutants Control and Water Quality Safeguard, Beijing Jiaotong University, Beijing,

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100044, China

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2

Laboratory of Quality and Safety Risk Assessments for Agro-products on Environmental Factors (Beijing), Ministry of Agriculture and Rural Affairs, 100029, China

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13 14

4

Beijing Municipal Station of Agro-Environmental Monitoring, 100029, China

Department of Vegetable Science, Beijing Key laboratory of Growth and Developmental

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Regulation for Protected Vegetable Crops, College of Horticulture, China Agricultural University,

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Beijing, 100193, China

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*Corresponding author. Jiuyi Li E-mail address: [email protected]

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Tel: (+86)-10-51684395; Fax: (+86)-10-51683764

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*Corresponding author. Zhiyong Xiao E-mail address: [email protected]

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Tel & Fax: (+86)-010-82031870

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Abstract

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Growing evidence shows plants are at risks of exposure to various per- and polyfluoroalkyl

25

substances (PFASs), however the phytotoxicity induced by these compounds remains largely

26

unknown on the molecular scale. Here, lettuce exposed to both perfluorooctanoic acid (PFOA)

27

and perfluorooctane sulfonate (PFOS) at different concentrations (500, 1000, 2000 and 5000 ng/L)

28

in hydroponic media was investigated via metabolomics. Under the co-exposure conditions, the

29

growth and biomass were not affected by PFOA and PFOS, but metabolic profiles of mineral

30

elements and organic compounds in lettuce leaves were significantly altered. The contents of Na,

31

Mg, Cu, Fe, Ca and Mo were decreased 1.8% - 47.8%, but Zn was increased 7.4% - 24.2%. The

32

metabolisms of amino acids and peptides, fatty acids and lipids were down-regulated in a

33

dose-dependent manner, while purine and purine nucleosides were up-regulated, exhibiting the

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stress response to PFOA and PFOS co-exposure. The reduced amounts of phytol (14.8% - 77.0%)

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and abscisic acid (60.7% - 73.8%) indicated the alterations in photosynthesis and signal

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transduction. The metabolism of (poly)phenol, involved in shikimate-phenylpropanoid pathway

37

and flavonoid branch pathway, was strengthened, to cope with the stress of PFASs. As the final

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metabolites of (poly)phenol biosynthesis, the abundance of various antioxidants was changed.

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This study offers comprehensive insight of plant response to PFAS co-exposure and enhances the

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understanding in detoxifying mechanisms.

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Keywords: Phytotoxicity, Perfluorooctanoic acid, Perfluorooctane sulfonate, Metabolic response,

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Metabolomics

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1. Introduction

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Per- and polyfluoroalkyl substances (PFASs) are a class of artificial organic compounds that are

45

fluorinated at entirely or a portion of the carbon atoms with a terminal sulfonate, carboxylate or

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phosphate group. Owing to their excellent water and oil repellence, heat and acid resistance, and high

47

surface activity, PFASs have been extensively used in consumer products and industrial processes

48

(Giesy and Kannan, 2002). On the other hand, the low biodegradability of PFASs due to the high

49

stability of C-F bond results in their persistence in various environmental media (Yao et al., 2018;

50

Chen et al., 2019; Cao et al., 2019). PFASs are frequently detected in numerous crops and vegetables

51

(Jian et al., 2017), and have become a common environmental stress encountered by plants.

52

The bio-toxicity and the mechanisms induced by PFASs, mainly perfluorooctanoic acid (PFOA) or

53

perfluorooctane sulfonate (PFOS), have been studied both in vivo and in vitro (Li et al., 2017; Zeng et

54

al., 2019). PFOA or PFOS exposure triggers overproduction of reactive oxygen species (ROS) and

55

induces oxidative stress, causing a variety of bio-toxicity, ranging from neurotoxicity, genotoxicity to

56

development toxicity and endocrine disruption in organisms, with the exposure levels between

57

µg/kg/d and mg/kg/d (Li et al., 2017; Zeng et al., 2019). Nevertheless, most toxicity assessments on

58

PFASs were intended to animals (e.g., rats, mice, and zebrafish) and humans (e.g., dissociative organs,

59

cells, organelles, children, and pregnant woman), little attention on PFAS exposure toxicity has yet

60

been paid to plant. Previous studies reported oxidative stress and ROS accumulation in wheat

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(Triticum aestivum L.) under the exposure of 0.1 - 200 mg/L PFOS in hydroponic media or 2 - 800

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mg/kg PFOA in air-dried soil (Qu et al., 2010; Zhou et al., 2016). The overproduction of free radicals

63

can induce lipid peroxidation and DNA damage (Mitter, 2002).

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Previous studies indicated that a variety of PFAS contaminants, especially PFOA and PFOS, coexist

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in the polluted environment (Liu et al., 2017; Li et al., 2019a; Liu et al., 2019). Han and Currell (2017)

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reviewed the occurrence of PFOA and PFOS in various surface water bodies, with their mean

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concentrations ranging from 0.6 - 6638.9 ng/L and 0.8 - 106301.8 ng/L, respectively. Alarming values

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at the µg/L levels of PFOA and PFOS in groundwater contaminated by aqueous film forming foam

69

were also reported (Backe et al., 2013; McGuire et al., 2014). Uses of contaminated water for

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irrigation and sewage sludge as the soil conditioner are recognized as the key sources of PFOA and

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PFOS entry into the agricultural plants (Ghisi et al., 2019). On agricultural lands that used

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contaminated sludge, PFOA and PFOS were measured at 320 and 410 µg/kg dry weight (dw),

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respectively (Washington et al., 2010). PFOA and PFOS can be bio-accumulated in plants from

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different environmental compartments (Blaine et al., 2014a; Tian et al., 2018). In the polluted areas,

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e.g., fluorine-chemical industrial park, PFOA and PFOS in vegetables can be detected up to hundreds

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of ng/g dw (Liu et al., 2017; Liu et al., 2019). Co-exposure to PFOA and PFOS is quite common for

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plants in the natural environment, compared to single exposure. However, previous studies focused on

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the toxicity of individual species of PFAS in plants (Qu et al., 2010; Zhou et al., 2016; Qian et al.,

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2019). PFOA and PFOS co-exposure may induce a number of physiological and biochemical defects.

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Correspondingly, plants reprogram their mineral and organic metabolic networks to maintain normal

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growth and development in response to oxidative stress (Zhao et al., 2016; Zhao et al., 2017).

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Elucidating the metabolic strategies that plants employ to defend against PFASs toxicity will be highly

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informative, in efforts to evaluate the ecological risks by PFAS pollution and nutritional quality of

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agricultural products.

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Metabolomics has been shown to be a powerful tool to facilitate an understanding about how plants

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respond and alleviate various stressors at the molecular level (Fiehn, 2002; Urano et al., 2010; Arbona

87

et al., 2013). Plants have diverse and complex metabolic pathways that generate primary and

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secondary metabolites (Bi and Felton, 1995; Scheible et al., 2004). The primary metabolites, e,g.,

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amino acids, fatty acids and lipids, are indispensable for the growth and development of plants by

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providing the necessary energy and molecular building block (Fernández-Martínez and Hoskisson,

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2019). Monitoring the primary metabolites may reflect the metabolic response of plants exposed to

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PFOA and PFOS. In addition, plants produce a large number of secondary metabolites, which are

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crucial for interactions with the environment, including providing resistance against abiotic stresses

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(Sampaio et al., 2016). Phenolic compounds are the essential secondary metabolites and they widely

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participate in the signaling and antioxidant defense under oxidative stress (Blasco et al., 2013;

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Cheynier et al., 2013). Investigating the metabolic pathway of the (poly)phenol enables a better

97

understanding of the antioxidant mechanisms in plant-PFAS interaction.

98

It was reported that leafy vegetables accumulate high amounts of PFOA and PFOS in their edible

99

parts (Wen et al., 2016). Lettuce (Lactuca sativa) is one of the popular leafy vegetables widely

100

cultivated and consumed, and is adopted as a model plant in phytotoxicity studies (Uzu et al., 2010;

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Blaine et al., 2014b; Zhao et al., 2016). In present work, lettuce (Lactuca sativa) was cultivated in

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hydroponic media spiked with different levels of PFOA and PFOS. Inductively coupled plasma mass

103

spectrometry (ICP-MS) and liquid chromatography mass spectrometry (LC-MS) based metabolomics

104

were employed to determine the metabolic profiles in lettuce leaves. The aims of this study were to

105

investigate the underlying toxic and detoxification mechanisms employed by plants to cope with both

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PFOA and PFOS stresses.

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2. Materials and methods

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2.1. Chemicals

109

PFOA and PFOS calibration standards and isotope-labeled internal standards were purchased from

110

Wellington Laboratories (Guelph, Ontario, Canada). PFOA (96%) and PFOS-potassium salt (K-PFOS,

111

98%) were purchased from Dr. Ehrenstorfer (Augsburg, Bavaria, Germany). HNO3 (90%) and H2O2

112

(30 wt. %) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Tetra-butyl ammonium

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hydrogen sulfate (TBAHS) and ENVI-CarbTM were obtained from CNW technologies GmbH

114

(Düsseldorf, Nordrhein Westfalen, Germany). Methyl tert-butyl ether (MTBE; HPLC grade) were

115

obtained from Duksan (Ulsan, South Korea). Multi-element environmental calibration standard using

116

in mineral analysis was obtained from Agilent Technologies (5183-4688; Santa Clara, CA, USA).

117

More details regarding the chemical reagents and lab materials are provided in Supporting

118

Information.

119

2.2. Plant growth and exposure treatment

120

Before co-exposure to PFOA and PFOS, lettuce seeds (Lactuca sativa) were pre-germinated at 4

121

for 10 days in Petri dishes containing two layers of sterile filter paper moistened with ultra-pure water.

122

In total, 35 lettuce seedlings of uniform size were planted and kept in the same experimental

123

hydroponic systems that consisted of a plastic grid placed into a PVC vessel. The PVC vessels were

124

wrapped in aluminum foil to keep the root zone dark. The experiments were conducted in a growth

125

chamber (Percival Scientific, Perry, IA) at a relative humidity of 80% with the diurnal cycle of 14h in

126

light (25

, 350 µE m-2 s-1) and 10 h in dark (18

).

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Modified Hoagland’s solution was used to supply nutrients (Supporting Information). The volume

128

of the nutrient solutions (pH 6.0) was maintained at 5 L in the entire assay. Five treatment groups were

129

designed, including a control group and four spiked groups that contained the equal concentrations of

130

PFOA and PFOS (500, 1000, 2000 and 5000 ng/L, respectively). Since irrigation with reclaimed water

131

is an important exposure pathway for PFASs accumulated by crops (Blaine et al., 2014b), the selected

132

concentrations were in the ranges of these compounds in typical effluent from wastewater treatment

133

plants (Plumlee et al., 2008; Blaine et al., 2014b). Seven replicates for each groups were grown. The

134

nutrient solution was renewed every 7 days to keep the PFOA and PFOS concentration at constant

135

levels and to prevent microbial growth (Felizeter et al., 2012). After 28 days of growth in the

136

hydroponic systems, all lettuce were harvested, ground in liquid nitrogen and stored at -80

137

analysis.

138

2.3. PFOA and PFOS extraction and analysis

139

until

The protocols for PFOA and PFOS extraction and analysis were described in a previous study (Li et

140

al., 2019a). Briefly, 0.5 g freeze dried samples were incubated in the fridge (4

141

adding the 5 ng

142

Na2CO3/NaHCO3 buffer (pH 10.0), TBAHS, MTBE and ENVI-CarbTM. Quantification of PFOA and

143

PFOS was conducted via HPLC-MS/MS (ACQUITY-HPLC/XEVO-TQS, Waters Crop., Milford, MA,

144

USA), with electrospray ionization (ESI) interface operating in the negative mode. Separation of

145

analysts was carried out using an Acquity UPLC® BEH Shield RP 18 column. More details regarding

146

the PFOA and PFOS extraction and analysis are provided in Supporting Information.

147

2.4. Analysis of mineral elements

13

C4-PFOA and

) overnight after

13

C4-PFOS internal standards. Samples were extracted with

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The protocol for inorganic element analysis was described by Zhao et al. (2017). Briefly, 0.5 g dry

148 149

lettuce leaves were digested in the mixture of 1 mL HNO3 and 4 mL H2O2 (v/v, 1:4) for 3 h at 180

150

in a microwave digestion system (CEM Mars 6; Matthews, NC, USA). The digestion solution was

151

diluted to 50 mL using ultra-pure water. Inorganic ions (K, Ca, Mg, Na, Fe, Mn, Zn, Cu and Mo) were

152

analyzed by ICP-MS (Agilent 8900; Santa Clara, CA, USA).

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2.5. Metabolite extraction and analysis

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The extraction of metabolites was carried out according to Yang et al. (2018) with minor

155

modifications. Briefly, lettuce leaf samples were ground in liquid nitrogen. 20 mg powdered samples

156

were extracted using 1 mL of -20

157

25

158

samples were centrifuged at 12 000 g for 10 min using an Eppendorf Centrifuge 5415D (Hauppauge,

159

NY, USA) and the supernatant was used for analysis.

methanol/ultra-pure water (80:20, v/v). Samples were sonicated at

for 30 min (KH-100DB, Hechuang Utrasonic, Jiangsu, China) and kept at 4

for 12 h. Finally,

160

Metabolite data were acquired on the high resolution Q-Exactive orbitrap MS in positive and

161

negative modes (Thermo Fisher Scientific., Waltham, MA, USA). Analytes were separated using an

162

Acquity UPLC HSS T3 column (100 mm × 2.1 mm, 1.7 µm). The mobile phases were (A) water with

163

0.5% acetic acid and (B) acetonitrile. Gradient elution was performed at a flow rate of 0.3 mL/min

164

under the following program (time in min, % B): (0.0, 0.0), (1.0, 0.0), (4.0, 20.0), (6.0, 30.0), (8.0,

165

50.0), (11.0, 75.0), (13.0, 100.0), (17.0, 100.0), (20.0, 0.0), (22.0, 0.0). The column temperature was

166

kept at 40

167

positive mode were: sheath gas flow rate at 75, auxiliary gas flow rate at 20, sweep gas flow rate at 2,

168

spray voltage at 3.50 kV, capillary temperature at 350 °C, S-lens RF level at 55 and auxiliary gas

and the injection volume was 5 µL. The operating parameters for the Q-Exactive MS in

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169

heater temperature at 550 °C. For negative mode, all parameters remained the same, except for the

170

spray voltage of 3.00 kV. Mass spectral data were acquired using full scan MS and data-dependent

171

MS/MS. More details regarding the metabolite analysis are provided in Supporting Information.

172

2.6. Q-Exactive MS data processing

173

The raw data from the spectral analysis of the lettuce extracts were processed using Progenesis QI

174

Software (Waters Crop., Milford, MA, USA), including filtration, deconvolution, alignment and

175

normalization. Before the formal alignment, pre-evaluation of all samples was conducted to choose an

176

optimum alignment reference. The parameters were used to automatic peak picking algorithm

177

including: retention times, 0.7 - 21 min; retention time tolerance, 0.3 min; noise levels, 3; mass

178

tolerance, 5 ppm; within-group abundance, > 30, 000. Peaks areas, across all samples, were

179

subsequently normalized to the sum area of the corresponding samples to balance their intensities

180

differences in each sample. For metabolite identification, processed data were identified based on

181

accurate mass, MS2 fragments and MS2 fragment isotopic distribution by comparing with

182

Chemspider and Progenesis Metascope spectral libraries of QI and HMDB (http://www.hmdb.ca/) and

183

LipidsMap (http://www.lipidmaps.org/) of online database. The MS tolerance and MS2 tolerance were

184

set at 2 ppm and 5 ppm, respectively.

185

2.7. Quality assurance and quality control (QA/QC)

186

The protocols for quality assurance and quality control (QA/QC) of PFOA and PFOS analysis were

187

provided by Li et al. (2019a). A trapping column was used to avoid the instrument background

188

contamination. Procedure blanks were prepared using Milli-Q water and routinely analyzed to check

189

for contamination during PFOA and PFOS extraction. Solvent blanks were prepared using 975 µL

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13

190

methanol and 25 µL (200 µg/L)

C-labeled internal standards and injected after 10 samples

191

throughout instrumental analysis, to examine the cross contamination and to monitor the background

192

contamination of the instrument. The internal standard calibration curve consisted of a concentration

193

gradient (0.5, 1, 2, 5, 10, 20, 50, 100 ng/mL) of PFOA and PFOS standards, spiked with a 5 ng

194

13

195

with correlation coefficients for target analytes at least 0.999. Mean spike recoveries of

196

internal standards in the instrumental analysis ranged from 82.8% to 125.2% for agricultural product

197

samples.

C-labeled internal standard. The curves were prepared for quantification of the PFOA and PFOS 13

C-labeled

198

QA/QC for metabolomics analysis were conducted according to Want et al. (2013). Instrument

199

calibration was conducted at the start of the batch. The QC sample is prepared by mixing 10 µL

200

biofluid aliquots of each sample, thus providing a sample with true representation of the breadth of

201

metabolites present in the sample set. QC samples were injected every five samples throughout the

202

data acquisition to assess instrument stability and data quality. More details regarding the QA/QC are

203

provided in Supporting Information.

204

2.8. Multivariate and univariate analysis

205

Unsupervised principal component analyses (PCA) and the supervised partial least-squares

206

discriminant

analysis

(PLS-DA)

were

performed

207

(http://www.metaboanalyst.ca). Variable Importance in Projection (VIP) was calculated based on

208

PLS-DA. Metabolites regarded as significant were defined as follows: minimum coefficient of

209

variation < 30 %, t-test p < 0.05, VIP > 1 and fold change > 2 or < 0.5 (spiked group/control). In

210

addition, univariate analysis of one-way analysis of variance (one-way ANOVA) was performed

- 10 -

on

the

Q-Exactive

MS

results

211

(http://www.metaboanalyst.ca).

212

3. Results and discussion

213

3.1. Bioaccumulation of PFOA and PFOS in lettuce leaves

214

During the 28 days exposure period, there were no significant changes in average fresh biomass

215

of leaf compared with the controls (37.1 g/plant) and 500 - 5000 ng/L treated plants (32.9 - 36.7

216

g/plant, p > 0.05). For nutrient solutions spiked with 500, 1000, 2000 and 5000 ng/L of both PFOA

217

and PFOS, PFOA contents in lettuce leaves were 16.1 ± 2.8, 26.7 ± 2.1, 55.7 ± 3.1 and 98.8 ± 5.8 ng/g

218

dw, respectively, while PFOS contents were 5.8 ± 0.8, 11.6 ± 2.0, 23.7 ± 2.0 and 46.6 ± 2.3 ng/g dw,

219

respectively (Fig. 1 ). The accumulated PFOA and PFOS in leaves exhibited a liner relationship with

220

the increase of exposure levels (Fig. 1 ), which is consistent with the results from Blaine et al.

221

(2014b). The average contents of PFOA accumulated in lettuce were 2.1 - 2.8 folds of PFOS, lower

222

than values under single exposure conditions (4.0 - 4.3 folds; Li et al., 2019b). This may indicate that

223

simultaneous uptake of PFOA and PFOS is a competitive process because the accumulation of these

224

compounds need the participation of proteins and lipids in plants (Wen et al., 2016). The difference in

225

bioaccumulation in lettuce leaves between PFOA and PFOS could be attributed to the

226

physicochemical properties of carboxylic group (PFOA, CH3(CH2)6-COOH) and sulfonic group

227

(PFOS, CH3(CH2)7-SOOO-) (Paterson et al., 1994). The higher magnitudes of both octanol-water

228

partition coefficient and foliage to root concentration factor for PFOA than PFOS may contribute to

229

this phenomenon. (Rodea-Palomares et al., 2012; Felizeter et al., 2012).

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125

PFOA PFOS

Concentration (ng/g dry weight)

Concentration (ng/g dry weight)

125

100

75

50

25

0 500

230

2

PFOA R =0.96 2 PFOS R =0.98 100

75

50

25

0

1000 2000 Exposure levels (ng/L)

5000

0

1000

2000 3000 4000 Exposure levels (ng/L)

5000

231

Fig. 1. PFOA and PFOS concentration ( ), and relationship between exposure level and accumulation

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( ) in harvest lettuce leaves exposed to PFOA and PFOS at different concentrations (500, 1000, 2000

233

and 5000 ng/L) for 28 days in hydroponic systems. The means are averaged from seven replicates.

234

The error bars correspond to the standard error of mean.

235

3.2. Effects of PFOA and PFOS on metabolism of mineral nutrients

236

As shown in Table 1, under the co-exposure of PFOA and PFOS, the contents of inorganic elements

237

in plants were markedly affected. In comparison to control, Na (15.4% - 47.8%), Mg (14.2% - 23.9%),

238

Cu (12.6% - 20.2%), Fe (1.8% - 25.6%), Ca (3.8% - 21.3%) and Mo (10.4% - 17.9%) decreased in all

239

co-exposure groups. Under exposure to 1000 - 5000 ng/L of PFOA and PFOS, the content of K in

240

lettuce leaves was decreased 8.1% - 10.0%. On the contrary, the content of Zn was increased by 7.4%

241

- 24.2% in all co-exposure groups. It is possible that ROS activated by PFOA and PFOS co-exposure

242

induced mineral nutrient imbalance in lettuce leaves. Previous studies demonstrated excessive ROS

243

regulated metabolism of mineral nutrients in plants (Choudhary and Agrawal, 2016; Ma et al., 2016;

244

Hussain et al., 2018).

245

The decreases of Na, K and Ca may be related with the leakage of ion channels. Demidchik et al.

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246

(2003) reported ROS regulated the Ca2+ and K+ permeable channels in plasma membrane of plant

247

cells. Mg and Fe are involved in a range of life-sustaining processes in plants from photosynthesis to

248

respiration. The decrease of Mg and Fe implied chlorophyll biosynthesis was inhibited. The

249

accumulation of PFOA (5000 ng/L) in lettuce leaves led to the reduction of total photosynthetic

250

pigments (Li et al., 2019b). Qu et al. (2010) reported 10 mg/L PFOS treatment could lead to damage

251

to chlorophyll accumulation in wheat (Triticum aestivum L.). Elevated content of Zn in plants may be

252

associated with an important tolerance mechanism in defending against PFOA and PFOS co-exposure.

253

Elevated Zn can enhance ROS scavenging by overproduction of Cu/Zn superoxide dismutase in

254

Cassava (Manihot esculenta) (Xu et al., 2013).

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255

Table 1.

256

The contents of mineral elements in lettuce leaves under different co-exposure levels of PFOA and PFOS (mg/kg dry weight)a. Exposure levels

K

Ca

Mg

Na

Fe

Mn

Zn

Cu

Mo

0

249133.3±18546.2

36466.7±2686.0

11713.3±1045.1

4733.7±325.6

727.6±60.5

536.8±54.1

146.9±8.4

11.9±0.5

6.7±0.9

500

255633.3±15351.3

35093.3±2606.1

10071.7±951.3

2647.7±523.0

c

541.5±61.1

c

502.6±15.5

173.0±4.4

b

10.4±1.1

6.0±0.7

1000

224166.7±13632.4

30033.3±2523.4

8919.0±389.6

c

2471.0±240.8

c

624.9±225.2

548.0±62.2

182.5±6.3

c

10.4±0.9

5.5±0.4

2000

227166.7±16444.6

28693.3±2806.4

10052.7±526.6

3011.7±450.9

714.5±167.6

546.5±72.9

177.4±41.4

9.5±0.7

b

5.5±0.4

5000

228966.7±8715.7

31996.7±1273.4

9245.0±195.7

4005.7±352.3

606.3±99.8

527.2±31.6

157.7±1.3

9.9±0.3

b

5.7±0.3

(ng/L)

257

a

The data are means of seven replicates ± standard deviation.

258

b

p < 0.05, c p < 0.01, significance determined by Student’s t-test.

b

b

- 14 -

259

3.3. Effects of PFOA and PFOS on metabolic profiles

260

A total of 1127 metabolites were detected and semi-quantified using LC-HRMS approach. An

261

un-supervised clustering method PCA was performed on the data to provide a general overview of the

262

clustering information between groups. PCA analysis showed that the 500 ng/L co-exposure group

263

overlapped the control; however, 1000, 2000 and 5000 ng/L co-exposure groups were separated from

264

control group along PC2 (Fig. 2 ). Besides, no clear separation was observed between 1000 ng/L

265

group and 500 or 2000 ng/L group. Supervised PLS-DA was employed to highlight the discrepancies

266

between five groups. As shown in Fig. 2 , score plot showed that four co-exposure groups were

267

clearly separated from the control in a dose-dependent manner along PC1, which explained 22.7% of

268

the total variability. These results clearly showed that PFOA and PFOS co-exposure markedly affected

269

the metabolite profiles in lettuce leaves at the tested dosages.

270 271

Fig. 2. Principal component analysis (PCA) ( ) and partial least squares-discriminate analysis

272

(PLS-DA) ( ) score plots of metabolite profiles in lettuce leaves under co-exposure to different

273

dosages of PFOA and PFOS. The colored ellipses represent 95% confidence regions for each group.

- 15 -

274

A−E represent different experimental groups: A, control; B, PFOA/PFOS=500/500 ng/L; C,

275

PFOA/PFOS=1000/1000 ng/L; D, PFOA/PFOS=2000/2000 ng/L; E, PFOA/PFOS=5000/5000 ng/L.

276

The responsible metabolites were subsequently screened out according to VIP score. The results were

277

presented in a Venn diagram (Fig. 3). Under the 500, 1000, 2000 and 5000 ng/L co-exposure levels, 37,

278

65, 75 and 199 metabolites were significantly altered (Fig. 3 ), respectively, in comparison to control,

279

among which 8, 22, 30 and 74 metabolites were identified (Fig. 3 ). The main responsible

280

metabolites

281

xi-7-hydroxyhexadecanedioic acid. The metabolites induced by 1000 ng/L co-exposure were mainly

282

overlapped with those under 500 (7 identified, 25 unknown) and 2000 (10 identified, 25 unknown)

283

ng/L co-exposure. This is consistent with the PCA model (Fig. 2 ). Oxidative stress and ROS

284

accumulation in plants have been demonstrated under exposure to PFOA and PFOS (Qu et al., 2010;

285

Zhou et al., 2016; Qian et al., 2019). Excessive accumulation of ROS may cause gene injury in cells

286

(Eriksen et al., 2010). Besides, PFOA and PFOS have been reported to act as the activators in signal

287

pathway, which finally changed the transcription of genes (Li et al., 2017; Zeng et al., 2019). These

288

effects are the potential mechanisms of the metabolism disorder induced by PFOA and PFOS in plants

289

(Jiang et al., 2015), especially the metabolisms of lipids and amino acids (Issemann and Green, 1990;

290

Yu et al., 2016).The affected metabolites (Table S1) were classified into different groups and

291

discussed as follows, according to their metabolic functions and pathways.

with

high

VIP

scores

were

dibutyl

- 16 -

malate,

matricarin,

licoagrone,

and

292 293

Fig. 3. Venn diagram of all ( ) and identified ( ) of changed metabolites in lettuce leaves exposed to

294

different dosages of PFOA and PFOS. A−E represent different experimental groups: A, control; B,

295

PFOA/PFOS=500/500 ng/L; C, PFOA/PFOS=1000/1000 ng/L; D, PFOA/PFOS=2000/2000 ng/L; E,

296

PFOA/PFOS=5000/5000 ng/L.

297

3.3.1. Amino acids and peptides

298

The metabolism of amino acids was perturbed in response to PFOA and PFOS co-exposure (Fig.

299

4 ). 2-Aminomuconic acid semialdehyde decreased by 26.7% - 71.5% (p < 0.05). 2-Aminomuconic

300

acid semialdehyde is an intermediate in the oxidative metabolism of tryptophan and is frequently

301

required in the biosynthesis of NAD (Nishizuka et al., 1965; Colabroy and Begley, 2005).

302

O-Ureidohomoserine increased by 12.4% and 9.3% at the 500 and 1000 ng/L co-exposure dosages,

303

respectively. But, it decreased by 31.9% and 56.3% at 2000 and 5000 ng/L co-exposure dosages,

304

respectively. O-Ureidohomoserine participates in the biosynthesis of canavanine that forms an

305

effective chemical barrier to disease in plants (Rosenthal, 1972). It hypothesized the accumulation of

306

canavanine was promoted in the 500 and 1000 ng/L co-exposure to defend against oxidative stress

307

induced by PFOA and PFOS. Under high dosage co-exposure conditions, this protective strategy was

308

inhibited.

- 17 -

309

Some dipeptides showed a decreasing trend in response to PFOA and PFOS co-exposure, such as

310

glutamylisoleucine, threoninyl-phenylalanine, isoleucylproline, threoninyl-isoleucine, valyl-valine,

311

isoleucyl-aspartate and isoleucyl-glycine. Interestingly, no dipeptide was screened out in lettuce leaves

312

exposed to single PFOA or PFOS under 500 and 5000 ng/L (Li et al., 2019b). Peptides play an

313

important role of cell signaling in plants, including abiotic stress response (Katsir et al., 2011;

314

Chaiwanon et al., 2016). The regulation of dipeptides may involve in coordination antioxidant defense

315

systems activated by PFOA and PFOS co-exposure. The content of histidinyl-proline increased by

316

0.6-fold and 2.5-fold under 500 and 1000 ng/L co-exposure, respectively (p < 0.05). However, it

317

decreased by 81.3% and 80.4% under 2000 and 5000 ng/L co-exposure, respectively (p < 0.05). Du et

318

al. (2019) reported dipeptides containing His residues can work as antioxidants. Thus, the fluctuations

319

in histidinyl-proline may be associated with oxidative stress and scavenging ROS.

320

3.3.2. Fatty acids

321

As shown in Fig. 4 , the contents of 21 fatty acids were altered under co-exposure, with a

322

decreasing trend. Fatty acids are synthesized by chain elongation of acetyl-CoA primers with

323

malonyl-CoA (methylmalonyl-CoA) groups (Kolattukudy et al., 1997). The alterations of fatty acids

324

are the signal that metabolic pathways containing acetyl-CoA and malonyl-CoA were perturbed by

325

PFOA and PFOS co-exposure, for instance carbon pool reallocation. Our previous study reported

326

energy metabolism (e.g., TCA cycle) was affected in lettuce leaves under single PFOA or PFOS

327

exposure (Li et al., 2019b).

328

Since PFOA and PFOS have high affinity to peroxisome proliferating receptor alpha (PPARα), a

329

major component in regulating fatty acid metabolism, these compounds could induce a series of

- 18 -

330

metabolic effects (Jiang et al., 2015; Li et al., 2017; Zeng et al., 2019). This is the major mechanisms

331

for the metabolic disorders of fatty acids in lettuce leaves following co-exposure.

332

3.3.3. Lipids

333

The metabolisms of 23 lipids were perturbed (Fig. 4 ). The average lipid contents decreased by

334

35.2%, 43.2%, 52.7% and 66.2% in response to 500, 1000, 2000 and 5000 ng/L PFOA and PFOS

335

co-exposure, respectively, showing a dose-dependent pattern. Lipids contribute to the structural basis

336

of cellular membranes, driving the cellular polarity (Fischer et al., 2004), thus, affect the entry of

337

PFOA and PFOS into cells. Hu et al. (2003) reported PFOS increased fluidity and permeability of cell

338

membranes. In addition, lipids provide energy for numerous cellular events (Ohlrogge and Browse,

339

1995; Benning, 2009; Du et al., 2016).

340

Phytol decreased by 14.8% - 77.0% (p < 0.05; Fig. S2A). Chlorophyll is composed of a porphyrin

341

head group which contains Mg and phytol. The decrease of phytol and Mg contents suggested that

342

chlorophyll synthesis and photosynthesis had been inhibited. Phytol is also employed for the

343

biosynthesis of vitamin E (Valentin et al., 2006), which is a major liposoluble antioxidant in plant cell

344

membrane (Nimse and Pal, 2015) and is an important nutritional component for lettuce. Recent

345

studies demonstrated that phytol biosynthesis is catalyzed by protein complexes associated with the

346

thylakoid membrane (Gutbrod et al., 2019). Therefore, the damage of thylakoid membrane induced by

347

PFOA and PFOS co-exposure may result in the down-regulation of phytol synthesis. Furthermore,

348

another possible explanation is that the catabolism of photosynthetic pigment synthesis was

349

accelerated by PFOA and PFOS.

350

Abscisic acid (ABA) decreased by 60.7% - 73.8% (p < 0.05; Fig. S2B). ABA is derived from the

- 19 -

351

cleavage of carotenoids (Nambara and Marion-Poll, 2005), indicating carotenoid metabolism was

352

perturbed by co-exposure. ABA is a vital hormone in plants that confers tolerance to environmental

353

stresses, as well as mobilizes a battery of genes that presumably serve to protect the cells from

354

oxidative damage (Wasilewska et al., 2008). Signal transduction mediated by ABA regulates

355

numerous basic metabolism in plant growth and development (Finkelstein et al., 2002; Himmelbach et

356

al., 2003; Wasilewska et al., 2008). Thus, many metabolic disorders caused by PFOA and PFOS in

357

plants could be attributed to the regulated ABA. Interestingly, the contents of phytol and ABA were

358

not significantly impacted by single PFOA or PFOS exposure under 500 and 5000 ng/L levels (Li et

359

al., 2019b). It demonstrated that co-exposure of PFASs may induce more complex toxicity in plants

360

than single exposure.

361

3.3.4. Purine and purine nucleosides

362

Deoxyguanosine, adenine and adenosine increased by 0.5-fold to 1.9-fold (Fig. 4 ). DNA oxidative

363

damage caused by PFOA or PFOS in animals, humans and plants has been reported (Liu et al., 2016;

364

Kingsley et al., 2017; Li et al., 2019b). The overproduction of purine and purine nucleosides was used

365

to cure DNA injury. Deoxyguanosine can be converted to 8-hydroxy-deoxyguanosine (8-OHdG) by

366

oxidative stress (Kasai and Nishimura, 1984). The increase of 8-OHdG under PFOA or PFOS stress

367

may be associated with the up-regulated deoxyguanosine (Lin et al., 2016; Yahia et al., 2016).

- 20 -

368

369

- 21 -

370

371

372 373

Fig. 4. Heat maps produced by hierarchical cluster analysis of amino acids and peptides ( ), fatty

374

acids ( ) and lipids ( ). Azure rectangles indicate a decrease in metabolite content and brown

375

rectangles represent an increase in metabolite content. Box plots of relative abundance of purine and

376

purine nucleosides ( ) in lettuce leaves induced by PFOA and PFOS with different co-exposure levels.

- 22 -

377

A−E represent different experimental groups: A, control; B, PFOA/PFOS=500/500 ng/L; C,

378

PFOA/PFOS=1000/1000 ng/L; D, PFOA/PFOS=2000/2000 ng/L; E, PFOA/PFOS=5000/5000 ng/L.

379

3.4. Response of antioxidant defense system under PFOA and PFOS co-exposure

380

3.4.1. (Poly)phenol metabolism

381

In Fig. 5, the schematic representation of (poly)phenol biosynthesis under PFOA and PFOS

382

co-exposure was depicted. The relative abundances of the intermediate metabolites in (poly)phenol

383

biosynthesis were shown in Fig. S3. The upstream metabolites in the (poly)phenol synthesis, such as

384

phenylalanine increased 2.4% - 12.3% under 500 - 5000 ng/L co-exposure and p-coumaric acid

385

increased 25.6% and 43.9% under 2000 and 5000 ng/L co-exposure, respectively. The downstream

386

metabolites in the (poly)phenol synthesis, including naringenin chalcone, naringenin, genistein and

387

kaempfero of the flavonoid branch pathway, increased 0.8% - 332.3% under 500 - 5000 ng/L

388

co-exposure. Quercetin as the end product in the flavonoid branch pathway increased 22.0% and

389

10.4% under 2000 and 5000 ng/L co-exposure, respectively.

390

Previous studies showed that the activities of some antioxidant enzymes were up-regulated in plants

391

under the exposure of < 2 mg/kg PFOA in soil and < 0.1 mg/L PFOS in hydroponic media (Qu et al.,

392

2010; Zhou et al., 2016; Qian et al., 2019). The elevated contents of naringenin and genistein

393

suggested that (poly)phenol synthesis was also strengthened to scavenge excessive ROS triggered by

394

PFOA and PFOS co-exposure. Naringenin is the critical upstream branching point for the biosynthesis

395

of flavonoids, including flavones, flavanones, isoflavones (genistein), flavonols, flavanonols,

396

flavan-3-ols and anthocyanidins (Kang et al., 2014). Flavonoids can quench highly oxidizing free

397

radicals (Fl-OH+R

Fl-O +RH) and the produced aroxyl radical (Fl-O ) can react with a second

- 23 -

398

radical to acquire a stable quinone structure (Rafat Husain et al., 1987; Buettner, 1993).

399 400

Fig. 5. Schematic representation of major pathways of (poly)phenol biosynthesis as affected by PFOA

401

and PFOS. PAL, phenylalanine ammonium lyase; C4H, cinnamic acid 4-hydroxylase; 4CL,

402

4-coumarate-CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; IFS, isoflavone synthase;

403

FLS, flavonol synthase; F3H, flavanone 3-hydroxylase; F3’H, flavonoid-3’-hydroxylase; HCT,

404

hydroxycinnamoyl transferase; C3H, p-coumarate-3-hydroxylase. Red color indicates the contents of

405

metabolites increased in response to PFOA and PFOS co-exposure.

406

3.4.2. Phenolic Antioxidants

407

As shown in Fig. 6, the contents of 11 phenolic antioxidants were significantly changed in lettuce

408

leaves under co-exposure, including 6 flavonoids (licoagrone, 3,7-dimethylquercetin, quercetin

409

3-(6-malonylglucoside) 7-glucoside, 7-hydroxy-5-methoxyflavan, cyclomammeisin, epicatechin), 2

- 24 -

410

cinnamic acid derivatives (b-D-fructosyl-a-D-(6-O-(E))-feruloylglucoside, 4-O-caffeoylshikimic acid)

411

and 3 phenols (8-paradol, demethoxyshogaol, tyrosol). In addition, the contents of 3 non-phenolic

412

antioxidants (3,4-dimethyl-5-pentyl-2-furanpropanoic acid, 5-butyl-2-furanoctanoic acid and ascorbyl

413

palmitate) significantly decreased, in a dose-dependent manner (Fig. S4).

414

4-O-Caffeoylshikimic acid is a derivative of shikimic acid, which is a primary metabolite in the

415

upstream of (poly)phenol biosynthesis (de la Rosa et al., 2019). The down-regulation of

416

4-O-caffeoylshikimic acid (40.7% - 57.1%; p < 0.05) supported the hypothesis that the content of

417

shikimic acid was altered under PFOA and PFOS co-exposure. Apart from phenolic compounds, the

418

synthesis of numerous secondary metabolites (e.g., terpenes, alkaloids) needs shikimic acid (de la

419

Rosa et al., 2019).

420

Epicatechin increased by 0.8-fold and 2.6-fold under 500 and 1000 ng/L co-exposure, respectively

421

(p < 0.05). Epicatechin contained hydroxyl groups and double bonds (Fig. 6) allow interactions with

422

proteins and lipids, mostly structural lipids forming membranes (Fraga et al., 2018). These interactions

423

can define the oxidant/antioxidant status of the cell and the activation of redox signaling (Fraga et al.,

424

2010). Tyrosol significantly decreased 70.3% by 5000 ng/L co-exposure. Tyrosol is an excellent

425

antioxidant in inhibiting low-density lipoprotein oxidation in plants (Giovannini et al., 1999).

426

Quercetin 3-(6-malonylglucoside) 7-glucoside is derived from quercetin. Thus, it decreased at 500

427

and 1000 ng/L co-exposure and increased at 2000 and 5000 ng/L co-exposure (Fig. 6).

428

3,7-Dimethylquercetin, which is also a derivative of quercetin, increased by 0.5-fold to 1.1-fold at 500

429

- 5000 ng/L co-exposure (p < 0.05). The response of phenolic antioxidants under single PFOA or

430

PFOS exposure is a dose-dependent manner (Li et al., 2019b), while this response at its co-exposure is

- 25 -

431

clearly a non-dose-dependent pattern (Fig. 6).

OH OH

O

O

H3C CH3 O

432

OH

O

433 OH

HO

O

O

O OH OH

HO

OH OH

O OH

OH

O O O

434

HO

O

OH

O

435

O

CH3 O

H3 C

436

OH

- 26 -

437

438

439 440

Fig. 6. Box-whisker plots of relative abundance of phenolic antioxidants (p < 0.05, one-way ANOVA)

441

in lettuce leaves induced by PFOA and PFOS with different co-exposure levels. A−E represent

442

different experimental groups: A, control; B, PFOA/PFOS=500/500 ng/L; C, PFOA/PFOS=1000/1000

443

ng/L; D, PFOA/PFOS=2000/2000 ng/L; E, PFOA/PFOS=5000/5000 ng/L. The y-axis indicates the

444

absolute signal from Q-Exactive.

445

4. Conclusions

446

In this study, the metabolic response of lettuce (Lactuca sativa) leaves co-exposed to PFOA and

447

PFOS in hydroponic media was investigated. Metabolic disorder was induced in lettuce under the

448

co-exposure of PFOA and PFOS, which altered the metabolic profiles of both inorganic elements and

449

organic compounds. Amino acids, peptides, fatty acids, lipids, and purine nucleosides were the major

- 27 -

450

reprogrammed metabolites in response to the stress exerted by PFOA and PFOS co-exposure. The

451

alterations in chlorophyll synthesis and signal transduction were observed in plants owing to the

452

down-regulation of Mg, Fe, phytol and abscisic acids. Antioxidant defense related pathways were

453

strengthened and resulted in the changes of numerous phenolic antioxidants in plants. These results

454

offer a deeper insight of plant response to PFAS co-exposure. The results provide valuable information

455

in evaluating the ecological risks by PFASs and the nutritional quality of agricultural products.

456

Acknowledgments

457

We sincerely thank Professor Lijuan Zhao of State Key Laboratory of Pollution Control and

458

Resource Reuse, Nanjing University for suggestions of experiment designs. This study was supported

459

by the National Natural Science Foundation of China (51578042, 51978037), the National

460

Agricultural Products Quality and Safety Risk Assessment Project (GJFP2019034) and the Key

461

Science and Technology Project of Beijing Agricultural Bureau (20180131). Any opinions, findings,

462

and conclusions or recommendations expressed in this material are those of authors and do not

463

necessarily reflect the views of National Science Foundation of China and Ministry of Agriculture and

464

Rural Affairs.

465

Conflicts of interest

466

None.

467

Appendix A. Supplementary data

468

Supplementary material related to this article can be found, in the online version, at doi:

469

References

470

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Highlights ● Metabolism of minerals in plants under PFASs exposure was reported. ● Molecule phytotoxicity of PFOA and PFOS co-exposure was studied via metabolomics. ● Chloroplast was damaged under co-exposure to PFOA and PFOS. ● Plant (poly)phenol biosynthesis under PFAS exposure was firstly investigated.

Declarations of interest: none.