Effects of in Utero PFOS Exposure on Transcriptome, Lipidome, and

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Effects of In Utero PFOS Exposure on Transcriptome, Lipidome and Function of Mouse Testis Keng Po Lai, Jetty Chung-Yung Lee, Hin Ting Wan, Jing Woei Li, Aman YiMan Wong, Ting-Fung Chan, Camille Oger, Jean-Marie Galano, Thierry Durand, Kin Sum Leung, Cherry C. Leung, Rong Li, and Chris Kong-Chu Wong Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b02102 • Publication Date (Web): 27 Jun 2017 Downloaded from http://pubs.acs.org on June 29, 2017

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Environmental Science & Technology

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Effects of In Utero PFOS Exposure on Transcriptome, Lipidome and Function of Mouse Testis.

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Short title: Omics Analysis of PFOS Effects on Testis.

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8 Keng Po Lai 2#, Jetty Chung-Yung Lee3#, Hin Ting Wan1, Jing Woei Li 4, Aman Yi-Man Wong1, Ting Fung Chan4, Camille Oger5, Jean-Marie Galano5, Thierry Durand5, Kin Sum Leung3, Cherry C Leung1, Rong Li1, Chris Kong-Chu Wong1*

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Partner State Key Laboratory of Environmental and Biological Analysis, Croucher Institute for Environmental Sciences, Department of Biology, Hong Kong Baptist University, 2Department of Biology and Chemistry, City University of Hong Kong, 3School of Biological Sciences, The University of Hong Kong, 4School of Life Sciences, The Chinese University of Hong Kong, 5Institut des Biomolécules Max Mousseron, UMR 5247 CNRS, ENSCM, Université de Montpellier, France

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#

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The authors declare they have no actual or potential competing financial interests.

Equal contribution

*Corresponding author, Dr Chris KC Wong Address: Partner State Key Laboratory of Environmental and Biological Analysis, Croucher Institute for Environmental Sciences, Department of Biology, Hong Kong Baptist University. Phone: (852) 34117053 Email address: [email protected].

Keywords: Lipid mediators, steroidogenesis; epididymal sperm.

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Abstract

F0

(1) Chemical & physiological analyses

P1 Prenatal PFOS exposure

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(2) Transcriptomic & lipidomic analyses

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P63 (3) Assess the reproductive parameters

1. Alteration of lipid metabolisms, oxidative stress and cell junction signaling; 2. Perturbations of lipid mediators; 3. Reductions in serum testosterone & epididymal sperm count.

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Transcriptomic and LC-MS/MS-based targeted lipidomic analyses were conducted to identify

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the effects of in utero PFOS exposure on neonatal testes and its relation to testicular

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dysfunction in adult offspring. Pregnant mice were orally administered 0.3 and 3 µg PFOS/g

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body weight until term. Neonatal testes (P1) were collected for the detection of PFOS, and

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were subjected to omics study. Integrated pathway analyses using DAVID, KEGG and IPA

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underlined the effects of PFOS exposure on lipid metabolism, oxidative stress and cell

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junction signaling in testes. LC-MS/MS analysis showed that the levels of adrenic acid and

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docosahexaenoic acid (DHA) in testes were significantly reduced in the PFOS treatment

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groups. A significant linear decreasing trend in eicosapentaenoic acid and DHA with PFOS

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concentrations was observed. Moreover, LOX-mediated 5-hydroxyeicosatetraenoic acids

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(HETE) and 15-HETE from arachidonic acid in the testes were significantly elevated and a

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linear increasing trend of 15-HETE concentrations was detected with doses of PFOS. The

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perturbations of lipid mediators suggested that PFOS has potential negative impacts on

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testicular functions. Postnatal analysis of male offspring at P63 showed significant reductions

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in serum testosterone and epididymal sperm count. This study sheds light into the as yet

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unrevealed action of PFOS on lipid mediators in affecting testicular functions.

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Introduction

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The incidence of reproductive problems is a globally prevalent issue. Recent census data

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from the World Bank show a continuous decline of global average fertility rate from >5.0 in

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1960 to 0.3

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and FDR < 0.05 were considered as differentially expressed genes (DEGs). The DEGs were

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subjected to GO functional enrichment analysis and Kyoto Encyclopedia of Genes and

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Genomes (KEGG) analysis using The Database for Annotation, Visualization and Integrated

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Discovery (DAVID) v6.8.53,54 Furthermore, Ingenuity Pathway Analysis (IPA®, QIAGEN

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Redwood City, www.qiagen.com/ingenuity) was used to decipher the molecular interaction

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networks that are deregulated by in utero PFOS treatment. Canonical pathways with P < 0.05

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were considered as statistically significant.

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Quantitative PCR analysis

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Fetal testes (four dam per group) were used for verification. Reverse transcription was

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conducted using the SuperScript™ VILO™ cDNA Synthesis Kit (ThermoFisher). qPCR was

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performed using SYBR FAST qPCR Master Mix (Kapa Biosystems, Woburn, MA) on a



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StepOnePlus Real-Time PCR System (Life Technologies). It was performed with a 3 min

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initial denaturation step at 95°C followed by 40 cycles of 95°C for 5 s and 60°C for 20 s. The

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GAPDH housekeeping gene was used for internal normalization for each gene. To calculate

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gene expression, the comparative Ct method (∆∆Ct) was used. The following gene-specific

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primers were used: GAPDH-forward, 5'-gggttcctataaatacggactgc-3; GAPDH-reverse, 5'-

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ccattttgtctacgggacga-3'; SPTBN1-forward, 5'-atctgccagcacccagagta-3'; SPTBN1-reverse, 5'-

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acgctggtgggtaaggtct-3';

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catccgtggaaaacaccag-3'; MAPK9-forward, 5'-acacctgtctatggcttcagg-3'; and MAPK9-reverse,

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5'-ctgatgcactgtgggacttc-3';

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reverse, 5'-ggcaaaattacaaccgacca-3; SCD2-forward, 5'-agctggtgatgttccagagg-3'; SCD2-reverse,

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5'-caagaaggtgctaacgcaca-3'; HDS17B12–forward, 5'-ctttccgtttgcaaggtgac-3'; HSD17B12-

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reverse,

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CTNNB1-reverse, 5'-aagaacggtagctgggatca-3'; APPL2-forward, 5'-cccacggacagatcaacttt-3';

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APPL2-reverse, 5'-tccagttccacctgaatgc-3'; OXTR-forward, 5'-gttctcaaccatcctcggca-3'; OXTR-

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reverse, 5'-gttctcaaccatcctcggca-3'.

ITGB1-forward,

5'-atgcaggttgcggtttgt-3';

PIK3R1-forward,

5'-tgttgagaatcacccctttagat-3';

ITGB1-reverse,

5'-ataaactaaagttggtcttttgacgag-3';

CTNNB1-forward,

5'-

PIK3R1-

5'-tgcagatcttggactggacat-3';

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Lipidomic Analysis of Neonatal testes

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At P1, the neonate testes from 4 dams (n = 4) per treatment group were collected. The lipid

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portion was extracted from the testes samples which were stored in -80ºC following the Lee et

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al. method with modifications.55 In brief, the tissues (~0.1 g) were homogenized in 10 mL of

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ice-cold Folch solution (chloroform/methanol 2:1 v/v + 0.05% BHT) using a blade

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homogenizer (T25, ULTRA-TURRAX, IKA) at 24,000 rpm in 2×20 s bursts. Afterwards, 2

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mL of 0.9% NaCl solution were added into samples to generate phase separation. After

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centrifugation at 3000 x g for 10 min at 4ºC, the lower chloroform phase was collected into a

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glass vial and dried under stream of nitrogen gas. The dried samples were re-suspended in 1 10 ACS Paragon Plus Environment

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mL of 1 M KOH in methanol and 1 mL of PBS (pH 7.4) and then hydrolyzed overnight in the

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dark, at room temperature on an orbital shaker. Hydrolysis was terminated by the addition of

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hydrochloric acid and formic acid. Finally, the samples were purified by solid phase

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extraction (SPE) using 60 mg mixed anionic SPE cartridges (MAX Oasis, Waters, USA). The

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SPE was cleaned and conditioned with methanol and formic acid respectively. The sample

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prepared were then loaded on the SPE, cleaned with 2% ammonium hydroxide and hexane,

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and the final elute (hexane/ethanol/acetic acid solution) was dried under a stream of nitrogen

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gas and the extracts were reconstituted in 100 µL of acetonitrile for the analysis of

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polyunsaturated fatty acids (PUFAs) and their oxidized lipid products. The extracted lipid

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products were quantified by liquid chromatography tandem mass spectrometry (LC-

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MS/MS)56 using Kinetex C18 column (150 × 21 mm, 2.6 µm, Phenomenex, USA)

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maintained at 30ºC in the 1290 Infinity LC system (Agilent Technologies, USA).

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A QTrap 3200 triple quadrupole mass spectrometer (Sciex Applied Biosystems, MA, USA)

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was operated in a negative atmospheric pressure chemical ionization (APCI) mode with the

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source temperature set at 750ºC. The mobile phase included Milli-Q water with 0.1% formic

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acid (A) and acetonitrile with 0.1% formic acid (B). The flow rate was set to 300 µL/min. A

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elution gradient was set with 80% of elute A for 2 min, then a gradual decrease in elute A to

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2% in 8 min and then maintained for 5 min. Finally, elute A was returned to 80% for 5 min

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prior to the next run. The injection volume of the sample to LC-MS/MS was 10 µL. The

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PUFAs including arachidonic (AA), adrenic (AdA), α-linolenic (ALA), n-3 docosapentaenoic

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(DPA), eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids and its oxidized lipid

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products, namely prostaglandins, hydroeicosatetraenoic acids (5-, 8-, 9-, 11-, 12-, 15-, 20-

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HETEs), hydroxyl-DHA (4-, 7-, 8-, 10-, 11-, 13-, 14-, 16-, 17-, 20-HDoHE), isoprostanoids

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(isoprostanes, dihomo-isoprostanes, neuroprostanes, phtytoprostanes) and isofuranoids

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(isofurans, dihomo-isofurans, neurofurans), were analyzed using multiple reactions 11 ACS Paragon Plus Environment


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monitoring (MRM) mode summarized in Supplementary Table S2. A total of 43 oxidized

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lipid products were determined and a typical mass spectra is shown in Supplementary Figure

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S1. Quantification of PUFAs and their oxidized lipid products were determined by relating

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the peak area with its corresponding heavy-labeled isotopic internal standard peak. For

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compounds without a heavy-labeled isotope, their quantification were determined by using a

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heavy-labeled isotope with the closest chemical structure (Supplementary Table S2). All

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PUFAs and their oxidized lipid product standards were purchased from Cayman Chemical Co.

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(Ann Arbor, MI, USA) or in-house synthesized by the Institut des Biomolecules Max

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Mousseron (IBMM, Montpellier, France).57-60

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Epididymal Sperm Count and Serum Testosterone Analysis

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At P63, blood serum and epididymides of adult offspring from 5 dams (n=5) per group were

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collected. Epididymis was cut into small pieces. They were then resuspeded in PBS (3%

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BSA) and incubated at 37 °C for an hour. Epididymal spermatozoa were stained with eosin-

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nigrosin (0.67% eosin Y, 10% nigrosin, 0.9% sodium chloride) and at least 200 epididymal

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spermatozoa (magnification x400) were examined under a light microscope.

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Serum testosterone levels were determined by use of ELISA kits (MP Biomedicals, Ohio,

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USA). The reagents including the working hormone-HRP conjugate (100µl), rabbit anti-

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hormone solution (50µl) and samples or standards (25 µl), were added and mixed in wells

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and incubated at 37 °C for 90 min. The wells were then rinsed five times with distilled water,

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followed by an addition of 100 µl TMB and incubation at room temperature. The reaction

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was then stopped by 1N HCl solution. Absorbance was measured at 450 nm.

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Statistical Analysis

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Statistical evaluation was conducted using SPSS16. Data normality was checked by Shapiro-

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Wilk and Q-Q Plot. The Spearman’s correlation analysis was used to examine the possible

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correlation between BPs and PFAAs in the samples. Data are presented as mean ± SD.

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Groups are considered significantly different if P < 0.05.

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Results and Discussion

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LCMS/MS Analysis of PFOS Concentrations in Neonatal Testes

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Throughout the course of the exposure, there were no significant differences in the body

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weights of the maternal mice between the control and the treatment groups [low (0.3 µg/g)

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and high (3 µg/g)]. The average numbers of litters in the control and the treatment groups

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were similar (Fig. 1A). No significant changes in the weights of neonatal testes were

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measured (Fig. 1B). The data suggested that the exposure did not cause any observable

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maternal and developmental toxicity. Nevertheless, placental transfer of PFOS was obvious

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through the detection of PFOS in the neonatal testes. Using LC-MS/MS analysis of the PFOS

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concentrations in neonatal testes, considerable levels of PFOS were detected in the testes of

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the treatment groups. The levels of the testicular uptake of PFOS were in a dose-dependent

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manner (Fig. 1C) and were proportionate with the maternal exposure doses.

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Transcriptomic Analysis of Neonatal Testes upon in Utero PFOS Exposure

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In an attempt to understand the molecular mechanisms underlying the effect of in utero PFOS

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exposure on neonatal testes, comparative transcriptomic analysis was conducted (n = 4 per group).

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A total of 56 and 319 DEGs were identified in the low and high dose of PFOS treatments, as



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compared to control group, respectively (Supplementary Table S3 and S4). The DEGs were then

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subjected to the Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.7

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and Ingenuity Pathway Analysis (IPA) to determine the biological functions, KEGG pathways

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and canonical pathways altered by PFOS exposures. The results of the DAVID analysis showed

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that PFOS treatment caused the alteration of biological processes related to fatty acid synthesis

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and metabolism, such as oxidation-reduction process, fatty acid metabolic process, fatty acid

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beta-oxidation, lipid metabolic process and coenzyme A biosynthetic process (Fig. 2A &

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Supplementary Table S5). In the KEGG pathway analysis, the highest ranked pathway were the

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metabolic pathways, suggesting that the prenatal PFOS exposure could alter different

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metabolisms in testes. In addition, a large number of pathways related to fatty acid metabolism

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were underlined. These included fatty acid metabolism, fatty acid degradation, fatty acid

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elongation and biosynthesis of unsaturated fatty acids (Fig. 2B & Supplementary Table S6).

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These findings are concordant to the previous reports that PFOS exposure could alter fatty

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metabolism, but most of the studies worked on liver tissue. For instance, PFOS could induce the

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expression of the lipid catabolism genes involved in fatty acid and triglyceride synthesis, leading

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to liver steatosis.61 Another mouse fetal liver study showed that prenatal PFOS exposure could

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activate the synthesis and metabolism of fatty acids and lipids, leading to liver damage in the

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fetus.45 When we compared our result with the transcriptomic analysis of human primary

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hepatocytes exposed to perfluorooctanoic acid (PFOA), we found that the response of liver and

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testis tissues is different. In the liver, PFOA could stimulate gene expression of the proto-

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oncogenes c-Jun and c-Fos and alter the cell cycle of hepatocytes.62 Using Ingenuity Pathway

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Analysis (IPA), 13 canonical pathways (P < 0.05) were identified at the low dose of PFOS

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exposure. Of these, pathways directly related to testicular development and functions include (i)

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epithelial adherens junction signaling, (ii) Wnt/β-catenin signaling, (iii) androgen biosynthesis

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and (iv) embryonic stem cell pluripotency (Fig. 2C & Supplementary Table S7). It has been


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reported that Wnt/β-catenin signaling is important to testicular functions. For example,

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constitutive Wnt/β-catenin signaling would disrupt Sertoli cells differentiation and

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spermatogenesis.63 In addition, Wnt/β-catenin signaling is essential for epididymal coiling and

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proliferation of undifferentiated spermatogonia.64,65 At the high dose of in utero PFOS exposure,

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a greater number of canonical pathways (59 pathways, P < 0.05) were highlighted

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(Supplementary Table S8). The altered pathways that were associated with PFOS toxicity and

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testicular function included (i) nuclear receptor activation (LXR/RXR and PXR/RXR activation),

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(ii) hormonal function (IGF-1 signaling), (iii) lipid metabolism (biosynthesis of cholesterol,

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zymosterol, stearate, oleate or degradation of ceramide, fatty acid), (iv) redox response

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(superoxide radicals degradation and oxidized GTP/dGTP detoxification) and (v) xenobiotic

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metabolism signaling. Epithelial adherens junction-related pathways like (vi) germ cell-Sertoli

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cell and (vii) Sertoli-Sertoli junctional signaling were also affected (Fig. 2C). Consistently, the

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results of the pathways analysis from the transcriptomic data suggested that the in utero PFOS

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exposure perturbed lipid metabolism, redox and cell adhesion functions of the testes. Our

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previous studies had already demonstrated the negative effects of PFOS on the disruption of

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epithelial homeostasis at the Sertoli-Sertoli and Sertoli-germ cell interface in the seminiferous

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epithelium,66 leading to the loss of germ cell adhesion, spermatid orientation, and

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dysregulation of spermatogenesis. However, little attention has been drawn to the effects of

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PFOS on the disruption of lipid signaling mediators, which are known to play critical roles in

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steroidogenesis, early male sexual development and masculinization.32,33,36,37 In order to

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validate the findings from the transcriptomic analysis, qPCR analysis was performed. 4 gene

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clusters involved in (i) germ cell-sertoli cell junction and sertoli-sertoli cell junction signaling

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including spectrin beta, non-erythrocytic 1 (SPTBN1), integrin beta 1 (ITGB1), mitogen-activated

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protein kinase 9 (MAPK9), catenin, beta 1 (CTNNB1) and phosphatidylinositol 3-kinase,

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regulatory subunit, polypeptide 1 (PIK3R1); (ii) fatty acid metabolism including stearoyl-



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Coenzyme A desaturase 2 (SCD2) and hydroxysteroid (17-beta) dehydrogenase 12 (HSD17B12);

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(iii) steroid hormone response such as oxytocin receptor (OXTR); and (iv) Wnt/β-catenin

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signaling pathway including catenin, beta 1 (CTNNB1) and adaptor protein, phosphotyrosine

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interaction, PH domain and leucine zipper containing 2 (APPL2) were selected. We found that

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the result of qPCR analysis well matched with transcriptomic data (Fig. 2D).

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Lipidomic Analysis of Neonatal Testes upon in Utero PFOS Exposure

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The mechanistic action of PFOS has been suggested to be mediated via nuclear receptors.

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While biochemical phenotypes associated with PFOS toxicities are closely linked to the

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perturbation of lipid metabolism and redox status.55 With the benefit of hindsight and the

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bioinformatics analysis obtained in this study, the PFOS-perturbed fatty acid metabolism,

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redox and other physiological outcomes may be related to the modulation of lipid

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metabolism and signaling. To gain a further understanding about this biological phenomenon,

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targeted lipidomic analysis was performed to determine the levels of polyunsaturated fatty

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acids and its oxidized lipid products, and to endeavor their relationship with the

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transcriptome evaluation.

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We evidently showed that in utero PFOS exposure to the dams influenced DHA metabolism

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in the pup testes (P1). We analyzed the impact of PFOS exposure on the metabolism of

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polyunsaturated fatty acids (PUFAs) in relation to n-6 (arachidonic and adrenic acids) and n-

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3 (α-linolenic, n-3 docosapentaenoic, eicosapentaenoic and docosahexaenoic acids) PUFAs

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pathways, which are currently known to take part in male reproduction.67 Among them, the

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levels of adrenic acid (AdA) and docosahexaenoic acid (DHA) in testes were significantly

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reduced by low and high PFOS concentrations respectively (Fig. 3A), showing disruption of

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PUFA metabolism. The reduction of AdA at low PFOS concentration corresponds to the


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upregulation of PTGIS (prostaglandin I2) gene indicating an increase in inflammation (Fig.

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3B). However, this effect is transient since AdA was not suppressed at higher PFOS

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concentration. Although no reports are available on AdA in testes, in endothelial cells, a

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small portion of AdA can retro-convert to AA.68 In agreement, the upregulation of ELOVL5

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gene which is shared by n-6 and n-3 PUFA metabolism displayed possible competition for

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fatty acid elongation of AA to AdA and EPA to n-3 DPA69 when exposed by PFOS (Fig. 3B).

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We also observed a significant linear decreasing trend in EPA and DHA with PFOS

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concentration (Fig. 3A), indicating the possible role of ELOVL5 to favor n-6 PUFA

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metabolism (Fig. 3B). Suppression of DHA is detrimental to the pup testicular development

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and consequently reproduction. Deficiency of essential n-3 PUFA, noticeably DHA, is

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recognized to cause testicular malfunction in adults, where sperm mobility was found to be

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reduced in boars.70 Lower levels of DHA in human seminal plasma were found in the male

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partners of infertile couples compared to fertile males.71 Recent studies found that

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supplementation of DHA to healthy men prevented sperm DNA fragmentation72 and rescued

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spermatogenesis in null-mice lacking delta-6 desaturase, an essential rate-limiting enzyme in

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the synthesis of PUFAs.73 However, it is not known if the suppression of DHA in the testis by

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PFOS exposure is permanent, as our previous study showed that pups exposed to PFOS for

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longer days had elevated DHA in the liver, the central tissue for lipid metabolism.55 PUFAs

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are prone to oxidation via enzymatic or non-enzymatic pathway due to the numerous skipped

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diene bonds. DHA, having the most number of double bonds, is the most vulnerable PUFA

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for oxidation. Oxidation products from n-6 PUFA are often toxic and related to inflammation,

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whereas those of n-3 PUFA are lipid mediators that assist in resolving inflammation and take

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part in normalizing microvascular functions.74,75

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Bioinformatics analysis of the transcriptome data underlined that PFOS exposure perturbed

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the redox responses and oxidation-reduction processes in neonatal testes. We anticipated 17 ACS Paragon Plus Environment


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PFOS to induce oxidative stress in the testes, since comparable amounts of PUFAs were

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reported in the neonatal liver.55 However, the data showed a different oxidized lipid profile in

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the testes as compared to the liver. In the analysis of oxidized lipid products, only 15 of the

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43 enzymatic (e.g. COX, LOX and cytochrome P450 mediated (Table 1)) and non-enzymatic

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(e.g. isoprostanoids and isofuranoids) oxidized lipid products were detected in the testes. The

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generation of isoprostanes predominates under normal oxidative stress, whereas isofuranoids

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predominates under extreme oxidative stress and high oxygen tension. The non-detectable

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products do not signify that it is absent in the tissue but probably in trace concentrations

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below the level of detection (0.05 ng/mg tissue) by the LC-MS/MS. It is proposed PFOS

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initiates non-enzymatic lipid oxidation through the presence of reactive oxygen species such

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as hydrogen peroxide and hydroxyl radicals55,76 and it appears the young testis lacks the

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presence of these species. Further, the difference might also be attributed by the age of the

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pups where lipid metabolism is not fully developed. Exposure of PFOS significantly elevated

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LOX-mediated 5-HETE and 15-HETE from arachidonic acid (AA) in the testes. A significant

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linear trend with an increase of PFOS concentration was observed for 15-HETE. LOX-

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mediated hydroxy-DHA (HDoHE) products were not detected in the testes. HDoHE are

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precursors of pro-resolving factors of inflammation such as resolvins (RvD, RvE),75 and our

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data indicated that the presence of PFOS might inhibit the regulation of HDoHE. No

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significant changes were observed for oxidized lipid products released via cytochrome P450

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after PFOS exposure (Table 2). Surprisingly, we found no noticeable changes in the non-

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enzymatic oxidized lipid products in the neonatal testes. This included the reputable 15-F2t-

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IsoPs, which is known to be the best biomarker for oxidative stress through free radical/ROS

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pathway in vivo. No linear trend with an increment of PFOS concentration was noticed for all

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non-enzymatic oxidized lipid products detected. To date, the upregulation of 15-HETE is

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associated to inflammation, proliferation, apoptosis, membrane blebbing and chromatin


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condensation in cancer cells77,78 (Fig. 3B), but its role in testis is poorly understood.

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Nevertheless, lipoxygenase-catalyzed products of the AA cascade play important roles in

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early male sexual development, steroidogenesis and masculinization.31-41 It is proposed that

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PFOS initiates non-enzymatic lipid oxidation through the presence of reactive oxygen species

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such as hydrogen peroxide and hydroxyl radicals,55,76 and it appears that the young testes lack

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the presence of these species. Nonetheless, the observations warrant the determination of

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whether or not PFOS exposure in utero causes a predisposition to reproductive dysfunction in

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male fetus, and therefore contributing to the disturbance in testosterone production and

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spermatogenesis in adult offspring.

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Characterization of Testicular Functions at Postnatal Growth of the Male Offspring

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We then determined the effects of in utero PFOS exposure on the testicular function of pups. At

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P63, significant reductions in the serum testosterone levels (Fig. 4A) and epididymal sperm count

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(Fig. 4B) were observed in pups from the high-dose exposed group. This observation supports

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the pathway analysis data of neonatal testes in which in utero PFOS exposure perturbed the

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expression of genes associated with androgen synthesis. The identified canonical pathways

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provide a mechanistic insight to revealing the effect of in utero PFOS exposure on neonatal

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testes, as androgen is important for the development of male reproductive system and sexual

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maturation.79 Our omics and physiological data are consistent with the findings of other

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studies. For example, in utero exposure of female rats to PFOS (5 or 20 mg·kg-1·day-1

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maternal dose) reduced the number of fetal Leydig cells and impaired cell functions in

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comparison to the untreated control group through the decrease in steroidogenic activity and

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testosterone levels.80 Furthermore, PFOA exposure (1, 3, 5, 10, 20 or 40 mg·kg-1·day-1) in

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timed-pregnant CD-1 mice (GD 1-17) affected sexual maturation in male offspring.81 Adult



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rats treated with 0.05–0.5% PFOA exhibited reduced activities of testicular-metabolic

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enzymes.82,83 Using microsomal preparations of human and rat testes, PFCs were found to

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target steroidogenic enzymes, resulting in a reduction of testosterone production.84 Moreover,

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the perturbation of androgen synthesis affects the process of masculinization while the

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disruption of epithelial adherens affects Sertoli-germ cells interaction. Our IPA analysis of

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differentially expressed transcripts from low- and high-dose of in utero PFOS exposure

468

underlined the epithelial adherens junction-signaling pathway, which is known to regulate the

469

blood-testis barrier for spermatogenesis. Our previous study has demonstrated that the cell

470

adherens proteins are the molecular targets of PFOS.85 The prenatal perturbations of this

471

important pathway were found to be associated with a significant reduction of epididymal sperm

472

counts in adult offspring.

473

Collectively, the present study analyzed the dynamic changes of global mRNA expression to

474

provide compiled experimental data of the integrated pathways using different

475

computational tools. To advance the significances of these findings, targeted lipidomics

476

analysis was implemented to track the changes in the levels of PUFAs and their oxidized

477

lipid products, serving the purposes of revealing further molecular targets/pathways to

478

unravel the effects of PFOS on neonatal testes.

479 480

Acknowledgements

481

This work was supported by the Partner State Key Laboratory of Environmental and

482

Biological Analysis (SKLP-16-17-P01) to Dr Chris KC Wong (Hong Kong Baptist

483

University).

484 485

Availability of supporting data 20 ACS Paragon Plus Environment

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486

The sequence data from this study have been submitted to the NCBI Sequence Read Archive

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(SRA) (http://www.ncbi.nlm.nih.gov/sra) under the accession number SRP108484.

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489



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490

FIGURE LEGENDS

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Figure 1. The pregnant mice were orally administered 0, 0.3 or 3 µg PFOS/g body weight by

492

gavage with corn oil throughout gestation. On postnatal day 1, the average number of litters

493

per dam was counted, neonatal testes from each dam were collected for weight measurement

494

and PFOS analysis. The figures shows the effects of in utero PFOS exposure on the (A)

495

number of litters per dam, (B) relative testicular weights and (C) concentrations of PFOS in

496

the testes of neonates.

497

Figure 2. The Database for Annotation, Visualization and Integrated Discovery (DAVID)

498

analysis highlighted the alteration of (A) biological processes and (B) pathways caused by

499

high dose (3 µg PFOS/g body weight) PFOS treatment. (C) Ingenuity Pathway Analysis (IPA)

500

highlighted the changes in canonical pathways related to testicular development, testicular

501

functions and toxicity caused by low dose (0.3 µg PFOS/g body weight) and high dose (3 µg

502

PFOS/g body weight) PFOS exposure. Expression of selected genes for each significantly

503

dysregulated pathways are presented in heat-map format. Color of each gene corresponds to

504

the log2 fold change of PFOS treatment versus control. (D) Quantitative PCR analysis to

505

validate the findings of RNA-sequencing. n = 4, *P < 0.05.

506

Figure 3. Lipidomic analysis reveals the effect of prenatal PFOS exposure on level of

507

polyunsaturated fatty acids. (A) Concentrations of polyunsaturated fatty acids in post-natal

508

mice (P1) exposed to low or high dose of PFOS during dam pregnancy. Values are mean ±

509

SD, n=4. *P < 0.05 versus control. P-trend indicates linear trend with PFOS concentration.

510

AA: arachidonic acid; AdA: adrenic acid; ALA: α-linolenic acid; EPA: eicosapentaenoic acid;

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DPA: docosapentaenoic acid; DHA: docosahexanoic acid. (B) integrated analysis of

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transcriptomic and lipidomic data.


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513

Figure 4. At P63, significant reductions in (A) serum testosterone levels and (B) epididymal

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sperm counts were recorded.



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Literature Cited

516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562

(1) Sharlip, I. D.; Jarow, J. P.; Belker, A. M.; Lipshultz, L. I.; Sigman, M.; Thomas, A. J.; Schlegel, P. N.; Howards, S. S.; Nehra, A.; Damewood, M. D.; Overstreet, J. W.; Sadovsky, R. Best practice policies for male infertility. Fertil. Steril. 2002, 77 (5), 873-882. (2) Matzuk, M. M.; Lamb, D. J. The biology of infertility: research advances and clinical challenges. Nat. Med. 2008, 14 (11), 1197-1213. (3) CHEMTrust Effects of pollutants on the reproductive health of male vertebrate wildlife - males under threat; 2008. (4) Joensen, U. N.; Bossi, R.; Leffers, H.; Jensen, A. A.; Skakkebaek, N. E.; Jorgensen, N. Do perfluoroalkyl compounds impair human semen quality? Environ. Health Perspect. 2009, 117 (6), 923-927. (5) Toft, G.; Jonsson, B. A.; Lindh, C. H.; Giwercman, A.; Spano, M.; Heederik, D.; Lenters, V.; Vermeulen, R.; Rylander, L.; Pedersen, H. S.; Ludwicki, J. K.; Zviezdai, V.; Bonde, J. P. Exposure to perfluorinated compounds and human semen quality in Arctic and European populations. Hum. Reprod. 2012, 27 (8), 2532-2540. (6) Joensen, U. N.; Veyrand, B.; Antignac, J. P.; Blomberg, J. M.; Petersen, J. H.; Marchand, P.; Skakkebaek, N. E.; Andersson, A. M.; Le Bizec, B.; Jorgensen, N. PFOS (perfluorooctanesulfonate) in serum is negatively associated with testosterone levels, but not with semen quality, in healthy men. Hum. Reprod. 2013, 28 (3), 599608. (7) Specht, I. O.; Hougaard, K. S.; Spano, M.; Bizzaro, D.; Manicardi, G. C.; Lindh, C. H.; Toft, G.; Jonsson, B. A.; Giwercman, A.; Bonde, J. P. Sperm DNA integrity in relation to exposure to environmental perfluoroalkyl substances - a study of spouses of pregnant women in three geographical regions. Reprod. Toxicol. 2012, 33 (4), 577583. (8) Vested, A.; Ramlau-Hansen, C. H.; Olsen, S. F.; Bonde, J. P.; Kristensen, S. L.; Halldorsson, T. I.; Becher, G.; Haug, L. S.; Ernst, E. H.; Toft, G. Associations of in utero exposure to perfluorinated alkyl acids with human semen quality and reproductive hormones in adult men. Environ. Health Perspect. 2013, 121 (4), 453455. (9) Bateson, P.; Barker, D.; Clutton-Brock, T.; Deb, D.; D'Udine, B.; Foley, R. A.; Gluckman, P.; Godfrey, K.; Kirkwood, T.; Lahr, M. M.; McNamara, J.; Metcalfe, N. B.; Monaghan, P.; Spencer, H. G.; Sultan, S. E. Developmental plasticity and human health. Nature. 2004, 430 (6998), 419-421. (10) Gluckman, P. D.; Hanson, M. A.; Bateson, P.; Beedle, A. S.; Law, C. M.; Bhutta, Z. A.; Anokhin, K. V.; Bougneres, P.; Chandak, G. R.; Dasgupta, P.; Smith, G. D.; Ellison, P. T.; Forrester, T. E.; Gilbert, S. F.; Jablonka, E.; Kaplan, H.; Prentice, A. M.; Simpson, S. J.; Uauy, R.; West-Eberhard, M. J. Towards a new developmental synthesis: adaptive developmental plasticity and human disease. Lancet. 2009, 373 (9675), 1654-1657. (11) Gluckman, P. D.; Hanson, M. A.; Beedle, A. S.; Spencer, H. G. Predictive adaptive responses in perspective. Trends Endocrinol. Metab. 2008, 19 (4), 109-110. (12) Hanson, M. A.; Gluckman, P. D. Developmental origins of health and disease: new insights. Basic Clin. Pharmacol. Toxicol. 2008, 102 (2), 90-93. (13) Gluckman, P. D.; Hanson, M. A. Developmental plasticity and human disease: research directions. J. Intern. Med. 2007, 261 (5), 461-471. 24 ACS Paragon Plus Environment

Page 25 of 31

563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612


(14) Gluckman, P. D.; Hanson, M. A.; Beedle, A. S. Early life events and their consequences for later disease: a life history and evolutionary perspective. Am. J. Hum. Biol. 2007, 19 (1), 1-19. (15) Gluckman, P. D.; Hanson, M. A.; Pinal, C. The developmental origins of adult disease. Matern. Child Nutr. 2005, 1 (3), 130-141. (16) Gluckman, P. D.; Cutfield, W.; Hofman, P.; Hanson, M. A. The fetal, neonatal, and infant environments-the long-term consequences for disease risk. Early Hum. Dev. 2005, 81 (1), 51-59. (17) Andersson, A. M.; Jorgensen, N.; Main, K. M.; Toppari, J.; Rajpert-De, M. E.; Leffers, H.; Juul, A.; Jensen, T. K.; Skakkebaek, N. E. Adverse trends in male reproductive health: we may have reached a crucial 'tipping point'. Int. J. Androl. 2008, 31 (2), 7480. (18) Skakkebaek, N. E.; Jorgensen, N.; Main, K. M.; Rajpert-De Meyts, E.; Leffers, H.; Andersson, A. M.; Juul, A.; Carlsen, E.; Mortensen, G. K.; Jensen, T. K.; Toppari, J. Is human fecundity declining? Int. J. Androl. 2006, 29 (1), 2-11. (19) Yiee, J. H.; Baskin, L. S. Environmental factors in genitourinary development. J. Urol. 2010, 184 (1), 34-41. (20) Ma, L. Endocrine disruptors in female reproductive tract development and carcinogenesis. Trends Endocrinol. Metab. 2009, 20 (7), 357-363. (21) Crinnion, W. J. Maternal levels of xenobiotics that affect fetal development and childhood health. Altern. Med. Rev. 2009, 14 (3), 212-222. (22) Lyche, J. L.; Gutleb, A. C.; Bergman, A.; Eriksen, G. S.; Murk, A. J.; Ropstad, E.; Saunders, M.; Skaare, J. U. Reproductive and developmental toxicity of phthalates. J. Toxicol. Environ. Health B Crit Rev. 2009, 12 (4), 225-249. (23) Grellier, J.; Bennett, J.; Patelarou, E.; Smith, R. B.; Toledano, M. B.; Rushton, L.; Briggs, D. J.; Nieuwenhuijsen, M. J. Exposure to disinfection by-products, fetal growth, and prematurity: a systematic review and meta-analysis. Epidemiology. 2010, 21 (3), 300-313. (24) Wong, E. W.; Cheng, C. Y. Impacts of environmental toxicants on male reproductive dysfunction. Trends Pharmacol. Sci. 2011, 32 (5), 290-299. (25) Llopis, J.; Westin, S.; Ricote, M.; Wang, Z.; Cho, C. Y.; Kurokawa, R.; Mullen, T. M.; Rose, D. W.; Rosenfeld, M. G.; Tsien, R. Y.; Glass, C. K. Ligand-dependent interactions of coactivators steroid receptor coactivator-1 and peroxisome proliferatoractivated receptor binding protein with nuclear hormone receptors can be imaged in live cells and are required for transcription. Proc. Natl. Acad. Sci. U. S. A. 2000, 97 (8), 4363-4368. (26) Schultz, R.; Yan, W.; Toppari, J.; Volkl, A.; Gustafsson, J. A.; Pelto-Huikko, M. Expression of peroxisome proliferator-activated receptor alpha messenger ribonucleic acid and protein in human and rat testis. Endocrinology. 1999, 140 (7), 2968-2975. (27) Abbott, B. D.; Wolf, C. J.; Das, K. P.; Zehr, R. D.; Schmid, J. E.; Lindstrom, A. B.; Strynar, M. J.; Lau, C. Developmental toxicity of perfluorooctane sulfonate (PFOS) is not dependent on expression of peroxisome proliferator activated receptor-alpha (PPAR alpha) in the mouse. Reprod. Toxicol. 2009, 27 (3-4), 258-265. (28) Rosen, M. B.; Schmid, J. R.; Corton, J. C.; Zehr, R. D.; Das, K. P.; Abbott, B. D.; Lau, C. Gene Expression Profiling in Wild-Type and PPARalpha-Null Mice Exposed to Perfluorooctane Sulfonate Reveals PPARalpha-Independent Effects. PPAR. Res. 2010, 2010 . (29) Kim, H. S.; Jun, K. S.; Sik, H. E.; Seok, K. T.; Hee, K. S.; Young, H. S. Induction of apoptosis and CYP4A1 expression in Sprague-Dawley rats exposed to low doses of perfluorooctane sulfonate. J. Toxicol. Sci. 2011, 36 (2), 201-210. 25 ACS Paragon Plus Environment


613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661

Page 26 of 31

(30) Rosen, M. B.; Schmid, J. E.; Das, K. P.; Wood, C. R.; Zehr, R. D.; Lau, C. Gene expression profiling in the liver and lung of perfluorooctane sulfonate-exposed mouse fetuses: comparison to changes induced by exposure to perfluorooctanoic acid. Reprod. Toxicol. 2009, 27 (3-4), 278-288. (31) Abayasekara, D. R.; Band, A. M.; Cooke, B. A. Evidence for the involvement of phospholipase A2 in the regulation of luteinizing hormone-stimulated steroidogenesis in rat testis Leydig cells. Mol. Cell Endocrinol. 1990, 70 (2), 147-153. (32) Adams, I. R.; McLaren, A. Sexually dimorphic development of mouse primordial germ cells: switching from oogenesis to spermatogenesis. Development. 2002, 129 (5), 1155-1164. (33) Amateau, S. K.; McCarthy, M. M. Induction of PGE2 by estradiol mediates developmental masculinization of sex behavior. Nat. Neurosci. 2004, 7 (6), 643-650. (34) Didolkar, A. K.; Sundaram, K. Arachidonic acid is involved in the regulation of hCG induced steroidogenesis in rat Leydig cells. Life Sci. 1987, 41 (4), 471-477. (35) Dix, C. J.; Habberfield, A. D.; Sullivan, M. H.; Cooke, B. A. Inhibition of steroid production in Leydig cells by non-steroidal anti-inflammatory and related compounds: evidence for the involvement of lipoxygenase products in steroidogenesis. Biochem. J. 1984, 219 (2), 529-537. (36) Gupta, C.; Goldman, A. S. The arachidonic acid cascade is involved in the masculinizing action of testosterone on embryonic external genitalia in mice. Proc. Natl. Acad. Sci. U. S. A. 1986, 83 (12), 4346-4349. (37) Gupta, C. The role of prostaglandins in masculine differentiation: modulation of prostaglandin levels in the differentiating genital tract of the fetal mouse. Endocrinology. 1989, 124 (1), 129-133. (38) Kojima, I.; Kojima, K.; Rasmussen, H. Possible role of phospholipase A2 action and arachidonic acid metabolism in angiotensin II-mediated aldosterone secretion. Endocrinology. 1985, 117 (3), 1057-1066. (39) Lopez-Ruiz, M. P.; Choi, M. S.; Rose, M. P.; West, A. P.; Cooke, B. A. Direct effect of arachidonic acid on protein kinase C and LH-stimulated steroidogenesis in rat Leydig cells; evidence for tonic inhibitory control of steroidogenesis by protein kinase C. Endocrinology. 1992, 130 (3), 1122-1130. (40) Maloberti, P.; Cornejo, M. F.; Castillo, A. F.; Castilla, R.; Duarte, A.; Toledo, M. F.; Meuli, F.; Mele, P.; Paz, C.; Podesta, E. J. Enzymes involved in arachidonic acid release in adrenal and Leydig cells. Mol. Cell Endocrinol. 2007, 265-266 113-120. (41) Solano, A. R.; Dada, L.; Podesta, E. J. Lipoxygenase products as common intermediates in cyclic AMP-dependent and -independent adrenal steroidogenesis in rats. J. Mol. Endocrinol. 1988, 1 (2), 147-154. (42) Fang, X.; Dillon, J. S.; Hu, S.; Harmon, S. D.; Yao, J.; Anjaiah, S.; Falck, J. R.; Spector, A. A. 20-carboxy-arachidonic acid is a dual activator of peroxisome proliferator-activated receptors alpha and gamma. Prostaglandins Other Lipid Mediat. 2007, 82 (1-4), 175-184. (43) Huang, J. T.; Welch, J. S.; Ricote, M.; Binder, C. J.; Willson, T. M.; Kelly, C.; Witztum, J. L.; Funk, C. D.; Conrad, D.; Glass, C. K. Interleukin-4-dependent production of PPAR-gamma ligands in macrophages by 12/15-lipoxygenase. Nature. 1999, 400 (6742), 378-382. (44) Wang, S. Y.; Lau, K.; Lai, K. P.; Zhang, J. W.; Tse, A. C.; Li, J. W.; Tong, Y.; Chan, T. F.; Wong, C. K.; Chiu, J. M.; Au, D. W.; Wong, A. S.; Kong, R. Y.; Wu, R. S. Hypoxia causes transgenerational impairments in reproduction of fish. Nat. Commun. 2016, 7 12114.


Page 27 of 31

662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711


(45) Lai, K. P.; Li, J. W.; Cheung, A.; Li, R.; Billah, M. B.; Chan, T. F.; Wong, C. K. Transcriptome sequencing reveals prenatal PFOS exposure on liver disorders. Environ. Pollut. 2017, 223 416-425. (46) Fida, T. T.; Moreno-Forero, S. K.; Breugelmans, P.; Heipieper, H. J.; Roling, W. F.; Springael, D. Physiological and Transcriptome Response of the Polycyclic Aromatic Hydrocarbon Degrading Novosphingobium sp. LH128 after Inoculation in Soil. Environ. Sci. Technol. 2017, 51 (3), 1570-1579. (47) European Food Safety Authority Perfluorooctane sulfonate (PFOS), perfluorooctanic acid (PFOA) and their salts - Scientific Opinion of the Panel on Contaminants in the Food Chain. The EFSA Journal. 2008, 653 1-131. (48) Wan, H. T.; Leung, P. Y.; Zhao, Y. G.; Wei, X.; Wong, M. H.; Wong, C. K. Blood plasma concentrations of endocrine disrupting chemicals in Hong Kong populations. J. Hazard. Mater. 2013, 261 763-769. (49) Li, J. W.; Lai, K. P.; Ching, A. K.; Chan, T. F. Transcriptome sequencing of Chinese and Caucasian population identifies ethnic-associated differential transcript abundance of heterogeneous nuclear ribonucleoprotein K (hnRNPK). Genomics. 2014, 103 (1), 56-64. (50) Lai, K. P.; Li, J. W.; Wang, S. Y.; Chiu, J. M.; Tse, A.; Lau, K.; Lok, S.; Au, D. W.; Tse, W. K.; Wong, C. K.; Chan, T. F.; Kong, R. Y.; Wu, R. S. Tissue-specific transcriptome assemblies of the marine medaka Oryzias melastigma and comparative analysis with the freshwater medaka Oryzias latipes. BMC. Genomics. 2015, 16 135. (51) Yates, A.; Akanni, W.; Amode, M. R.; Barrell, D.; Billis, K.; Carvalho-Silva, D.; Cummins, C.; Clapham, P.; Fitzgerald, S.; Gil, L.; Giron, C. G.; Gordon, L.; Hourlier, T.; Hunt, S. E.; Janacek, S. H.; Johnson, N.; Juettemann, T.; Keenan, S.; Lavidas, I.; Martin, F. J.; Maurel, T.; McLaren, W.; Murphy, D. N.; Nag, R.; Nuhn, M.; Parker, A.; Patricio, M.; Pignatelli, M.; Rahtz, M.; Riat, H. S.; Sheppard, D.; Taylor, K.; Thormann, A.; Vullo, A.; Wilder, S. P.; Zadissa, A.; Birney, E.; Harrow, J.; Muffato, M.; Perry, E.; Ruffier, M.; Spudich, G.; Trevanion, S. J.; Cunningham, F.; Aken, B. L.; Zerbino, D. R.; Flicek, P. Ensembl 2016. Nucleic Acids Res. 2016, 44 (D1), D710D716. (52) Robinson, M. D.; McCarthy, D. J.; Smyth, G. K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics. 2010, 26 (1), 139-140. (53) Huang, d. W.; Sherman, B. T.; Lempicki, R. A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 2009, 4 (1), 4457. (54) Huang, d. W.; Sherman, B. T.; Lempicki, R. A. Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res. 2009, 37 (1), 1-13. (55) Lee, Y. Y.; Wong, C. K.; Oger, C.; Durand, T.; Galano, J. M.; Lee, J. C. Prenatal exposure to the contaminant perfluorooctane sulfonate elevates lipid peroxidation during mouse fetal development but not in the pregnant dam. Free Radic. Res. 2015, 49 (8), 1015-1025. (56) Lee, Y. Y.; Crauste, C.; Wang, H.; Leung, H. H.; Vercauteren, J.; Galano, J. M.; Oger, C.; Durand, T.; Wan, J. M.; Lee, J. C. Extra Virgin Olive Oil Reduced Polyunsaturated Fatty Acid and Cholesterol Oxidation in Rodent Liver: Is This Accounted for Hydroxytyrosol-Fatty Acid Conjugation? Chem. Res. Toxicol. 2016, 29 (10), 1689-1698. (57) de la Torre, A.; Lee, Y. Y.; Mazzoni, A.; Guy, A.; Bultel-Ponce, V.; Durand, T.; Oger, C.; Lee, J. C.; Galano, J. M. Total syntheses and in vivo quantitation of novel 27 ACS Paragon Plus Environment


712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760

(58)

(59)

(60)

(61)

(62)

(63)

(64) (65)

(66)

(67)

(68)

(69)

(70)

(71)

(72)

Page 28 of 31

neurofuran and dihomo-isofuran derived from docosahexaenoic acid and adrenic acid. Chemistry. 2015, 21 (6), 2442-2446. de La, T. A.; Lee, Y. Y.; Oger, C.; Sangild, P. T.; Durand, T.; Lee, J. C.; Galano, J. M. Synthesis, discovery, and quantitation of dihomo-isofurans: biomarkers for in vivo adrenic acid peroxidation. Angew. Chem. Int. Ed Engl. 2014, 53 (24), 6249-6252. Guy, A.; Oger, C.; Heppekausen, J.; Signorini, C.; De, F. C.; Furstner, A.; Durand, T.; Galano, J. M. Oxygenated metabolites of n-3 polyunsaturated fatty acids as potential oxidative stress biomarkers: total synthesis of 8-F3t-IsoP, 10-F4t-NeuroP and [D4]10-F4t-NeuroP. Chemistry. 2014, 20 (21), 6374-6380. Oger, C.; Bultel-Ponce, V.; Guy, A.; Balas, L.; Rossi, J. C.; Durand, T.; Galano, J. M. The handy use of Brown's P2-Ni catalyst for a skipped diyne deuteration: application to the synthesis of a [D4]-labeled F4t-neuroprostane. Chemistry. 2010, 16 (47), 13976-13980. Das, K. P.; Wood, C. R.; Lin, M. T.; Starkov, A. A.; Lau, C.; Wallace, K. B.; Corton, J. C.; Abbott, B. D. Perfluoroalkyl acids-induced liver steatosis: Effects on genes controlling lipid homeostasis. Toxicology. 2017, 378 37-52. Buhrke, T.; Kruger, E.; Pevny, S.; Rossler, M.; Bitter, K.; Lampen, A. Perfluorooctanoic acid (PFOA) affects distinct molecular signalling pathways in human primary hepatocytes. Toxicology. 2015, 333 53-62. Tanwar, P. S.; Kaneko-Tarui, T.; Zhang, L.; Rani, P.; Taketo, M. M.; Teixeira, J. Constitutive WNT/beta-catenin signaling in murine Sertoli cells disrupts their differentiation and ability to support spermatogenesis. Biol. Reprod. 2010, 82 (2), 422-432. Kumar, M.; Syed, S. M.; Taketo, M. M.; Tanwar, P. S. Epithelial Wnt/betacatenin signalling is essential for epididymal coiling. Dev. Biol. 2016, 412 (2), 234-249. Takase, H. M.; Nusse, R. Paracrine Wnt/beta-catenin signaling mediates proliferation of undifferentiated spermatogonia in the adult mouse testis. Proc. Natl. Acad. Sci. U. S. A. 2016, 113 (11), E1489-E1497. Wan, H. T.; Mruk, D. D.; Wong, C. K.; Cheng, C. Y. The apical ES-BTB-BM functional axis is an emerging target for toxicant-induced infertility. Trends Mol. Med. 2013, 19 (7), 396-405. Yan, L.; Bai, X. L.; Fang, Z. F.; Che, L. Q.; Xu, S. Y.; Wu, D. Effect of different dietary omega-3/omega-6 fatty acid ratios on reproduction in male rats. Lipids Health Dis. 2013, 12 33. Mann, C. J.; Kaduce, T. L.; Figard, P. H.; Spector, A. A. Docosatetraenoic acid in endothelial cells: formation, retroconversion to arachidonic acid, and effect on prostacyclin production. Arch. Biochem. Biophys. 1986, 244 (2), 813-823. Zhang, J. Y.; Kothapalli, K. S.; Brenna, J. T. Desaturase and elongase-limiting endogenous long-chain polyunsaturated fatty acid biosynthesis. Curr. Opin. Clin. Nutr. Metab Care. 2016, 19 (2), 103-110. Am-in, N.; Kirkwood, R. N.; Techakumphu, M.; Tantasuparuk, W. Lipid profiles of sperm and seminal plasma from boars having normal or low sperm motility. Theriogenology. 2011, 75 (5), 897-903. Oborna, I.; Wojewodka, G.; De Sanctis, J. B.; Fingerova, H.; Svobodova, M.; Brezinova, J.; Hajduch, M.; Novotny, J.; Radova, L.; Radzioch, D. Increased lipid peroxidation and abnormal fatty acid profiles in seminal and blood plasma of normozoospermic males from infertile couples. Hum. Reprod. 2010, 25 (2), 308-316. Martinez-Soto, J. C.; Domingo, J. C.; Cordobilla, B.; Nicolas, M.; Fernandez, L.; Albero, P.; Gadea, J.; Landeras, J. Dietary supplementation with docosahexaenoic


Page 29 of 31

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(73)

(74)

(75)

(76)

(77) (78)

(79) (80)

(81)

(82)

(83)

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acid (DHA) improves seminal antioxidant status and decreases sperm DNA fragmentation. Syst. Biol. Reprod. Med. 2016, 62 (6), 387-395. Roqueta-Rivera, M.; Stroud, C. K.; Haschek, W. M.; Akare, S. J.; Segre, M.; Brush, R. S.; Agbaga, M. P.; Anderson, R. E.; Hess, R. A.; Nakamura, M. T. Docosahexaenoic acid supplementation fully restores fertility and spermatogenesis in male delta-6 desaturase-null mice. J. Lipid Res. 2010, 51 (2), 360-367. Galano, J. M.; Lee, J. C.; Gladine, C.; Comte, B.; Le Guennec, J. Y.; Oger, C.; Durand, T. Non-enzymatic cyclic oxygenated metabolites of adrenic, docosahexaenoic, eicosapentaenoic and alpha-linolenic acids; bioactivities and potential use as biomarkers. Biochim. Biophys. Acta. 2015, 1851 (4), 446-455. Gabbs, M.; Leng, S.; Devassy, J. G.; Monirujjaman, M.; Aukema, H. M. Advances in Our Understanding of Oxylipins Derived from Dietary PUFAs. Adv. Nutr. 2015, 6 (5), 513-540. Jin, L.; Zhang, P.; Shao, T.; Zhao, S. Ferric ion mediated photodecomposition of aqueous perfluorooctane sulfonate (PFOS) under UV irradiation and its mechanism. J. Hazard. Mater. 2014, 271 9-15. Cho, K. J.; Seo, J. M.; Kim, J. H. Bioactive lipoxygenase metabolites stimulation of NADPH oxidases and reactive oxygen species. Mol. Cells. 2011, 32 (1), 1-5. Ma, J.; Zhang, L.; Zhang, J.; Liu, M.; Wei, L.; Shen, T.; Ma, C.; Wang, Y.; Chen, Y.; Zhu, D. 15-lipoxygenase-1/15-hydroxyeicosatetraenoic acid promotes hepatocellular cancer cells growth through protein kinase B and heat shock protein 90 complex activation. Int. J. Biochem. Cell Biol. 2013, 45 (6), 1031-1041. Holterhus, P. M. Molecular androgen memory in sex development. Pediatr. Endocrinol. Rev. 2011, 9 Suppl 1 515-518. Zhao, B.; Li, L.; Liu, J.; Li, H.; Zhang, C.; Han, P.; Zhang, Y.; Yuan, X.; Ge, R. S.; Chu, Y. Exposure to perfluorooctane sulfonate in utero reduces testosterone production in rat fetal Leydig cells. PLoS. One. 2014, 9 (1), e78888. Lau, C.; Thibodeaux, J. R.; Hanson, R. G.; Narotsky, M. G.; Rogers, J. M.; Lindstrom, A. B.; Strynar, M. J. Effects of perfluorooctanoic acid exposure during pregnancy in the mouse. Toxicol. Sci. 2006, 90 (2), 510-518. Mehrotra, K.; Morgenstern, R.; Lundqvist, G.; Becedas, L.; Bengtsson, A. M.; Georgellis, A. Effects of peroxisome proliferators and/or hypothyroidism on xenobiotic-metabolizing enzymes in rat testis. Chem. Biol. Interact. 1997, 104 (2-3), 131-145. Mehrotra, K.; Morgenstern, R.; Ahlberg, M. B.; Georgellis, A. Hypophysectomy and/or peroxisome proliferators strongly influence the levels of phase II xenobiotic metabolizing enzymes in rat testis. Chem. Biol. Interact. 1999, 122 (2), 73-87. Zhao, B.; Hu, G. X.; Chu, Y.; Jin, X.; Gong, S.; Akingbemi, B. T.; Zhang, Z.; Zirkin, B. R.; Ge, R. S. Inhibition of human and rat 3beta-hydroxysteroid dehydrogenase and 17beta-hydroxysteroid dehydrogenase 3 activities by perfluoroalkylated substances. Chem. Biol. Interact. 2010, 188 (1), 38-43. Wan, H. T.; Mruk, D. D.; Wong, C. K.; Cheng, C. Y. Perfluorooctanesulfonate (PFOS) perturbs male rat Sertoli cell blood-testis barrier function by affecting F-actin organization via p-FAK-Tyr(407): an in vitro study. Endocrinology. 2014, 155 (1), 249-262.



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Table 1. Non-enzymatic lipid products in testis tissues in post-natal mice (P1) exposed to 0.3 (low) or 3 µg (high) PFOS/g body weight during dam pregnancy. Control

Low

High

p-trend

AA-derived 5-F2t-IsoP 15-F2t-IsoP Total F2t-IsoPs IsoF

34.98±14.12 1.53±0.92 36.51±14.94 23.08±9.25

14.91±4.13 1.11±0.78 16.02±4.74 22.08±2.26

54.04±52.26 2.43±0.78 56.47±52.87 43.42±24.60

0.412 0.151 0.398 0.092

AdA-derived 7-Dihomo-IsoP 17-Dihomo-IsoP Total Dihomo-IsoP

6.79±2.75 15.22±9.24 22.01±11.73

4.99±2.79 12.58±3.09 17.57±5.58

6.01±3.78 14.78±2.21 20.79±2.61

0.731 0.917 0.826

DHA-derived 16-HDoHE 56.68±5.21 39.50±9.12 43.55±24.04 0.252 4-NeuroF 215.57±75.24 194.52±63.22 192.73±34.14 0.604 Values are mean ± SD, n=4. P-trend indicates linear trend with PFOS concentration. AA: arachidonic acid; AdA: adrenic acid; DHA: docosahexaenoic acid; IsoP: isoprostane; IsoF: isofurans; HDoHE: hydroxy-DHA; NeuroF: neurofuran.

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Table 2. Enzymatic lipid products in testis tissues in post-natal mice (P1) exposed to 0.3 or 3 µg PFOS/g body weight during dam pregnancy. Control Low High p-trend COX-mediated 0.68±0.16 0.29±0.13 0.86±0.50 0.449 PGF2α LOX-mediated 5-HETE 8-HETE 12-HETE 15-HETE Total

215.21±57.86 325.66±80.02 93.06±38.11 21.26±12.25 655.19±166.50

614.51±231.50** 382.76±223.57 24.08±15.03 47.85±35.92 1069.21±324.20*

99.85±40.25 284.49±105.64 148.89±111.63 127.34±37.84** 660.57±126.34

0.273 0.707 0.280 0.001 0.974

CYP-mediated 9-HETE 11-HETE 20-HETE Total

137.52±65.90 450.73±158.29 209.01±100.15 797.27±185.31

319.21±250.81 721.94±501.31 314.97±126.83 1356.12±617.82

127.63±59.14 913.92±381.37 237.23±80.23 1278.78±399.94

0.929 0.115 0.711 0.155

Enzymatic lipid products presented in testis tissues. Values are mean ± SD, n=4. *p

Effects of in Utero PFOS Exposure on Transcriptome, Lipidome, and

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