Molecular Mechanisms of Perfluorooctanoate-Induced Hepatocyte

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Molecular mechanisms of perfluorooctanoate-induced hepatocyte apoptosis in mice using proteomic techniques Kan Li, Jie Sun, Jingping Yang, Stephen M. Roberts, Xu-Xiang Zhang, Xinyi Cui, Si Wei, and Lena Q. Ma Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b02690 • Publication Date (Web): 08 Sep 2017 Downloaded from http://pubs.acs.org on September 10, 2017

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Environmental Science & Technology

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Molecular mechanisms of perfluorooctanoate-induced hepatocyte apoptosis in mice

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using proteomic techniques

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Kan Li1, Jie Sun1, Jingping Yang2, Stephen M Roberts3, Xuxiang Zhang1, Xinyi Cui*, Si

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Wei1*, and Lena Q. Ma1,4

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State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment,

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Nanjing University, Jiangsu 210046, China

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School of the Medicine, Nanjing University, Jiangsu 210046, China

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Center for Environmental and Human Toxicology, University of Florida, Gainesville, FL

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32611, USA

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Soil and Water Science Department, University of Florida, Gainesville, FL 32611

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* Corresponding author: State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Jiangsu 210046, China; [email protected]; [email protected]

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ACS Paragon Plus Environment


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ABSTRACT

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The stability coupled with its wide use of perfluorooctanoate (PFOA) cause serious concern

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regarding its potential risks to human health. The molecular mechanisms of PFOA-induced

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hepatotoxicity relevant to human health was investigated using both in vivo (mouse model)

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and in vitro (human hepatocyte cells, HL-7702) techniques. Both male and female Balb/c

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mice were administered with PFOA at 0.05, 0.5, or 2.5 mg/kg-d for 28-d, with their serum

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PFOA concentrations after exposure being at environmental relevance levels. Liver samples

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were examined for histology and proteomic change using iTRAQ and Western Blotting,

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showing dose-dependent hepatocyte apoptosis and peroxisome proliferation. At high doses,

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genotoxicity resulting from ROS hypergeneration was due to suppression of Complex I

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subunits in the electron transport chain and activation of PPARα in both genders. However, at

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0.05 mg/kg-d, Complex I suppression occurred only in females, making them more sensitive

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to PFOA-induced apoptosis. In vitro assays using HL-7702 cells confirmed that apoptosis

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was also induced through similar mechanism. The dose/gender-dependent toxicity

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mechanisms help to explain some epidemiological phenomena, i.e., liver cancer is not often

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associated with PFOA exposure in professional workers. Our results demonstrated that

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proteomic approach is a robust tool to explore molecular mechanisms of toxic chemicals at

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environmental relevant level.

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INTRODUCTION Perfluoroalkyl substances (PFASs) are synthetic compounds widely used in industrial

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and consumer products such as Teflon® and Gore-Tex®1. Due to their strong carbon-fluorine

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bonds, PFASs are stable and accumulate in the environment, so they are classified as

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persistent organic pollutants (POPs)2, 3. Perfluorooctanoate (PFOA), is one of the most used

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PFASs and is abundant in the environment, resulting in human exposure through food,

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housedust, and drinking water4. PFOA has been detected in cord blood and breast milk in the

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general population1, 4 partially due to its long half-life (~ 4 y) in the human body5, 6. Because

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of its high usage in China, increasing serum levels of PFOA in humans have been observed.

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For example, serum levels increased from 0.08 µg/L in 1987 to 4.3 µg/L in 2002 in Shenyang

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and up to 15.2 µg/l in 2009 was reported for Nanchang7, 8. The widespread human exposures

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to PFOA cause increasing concerns regarding its environmental health risks worldwide.

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Recent epidemiology studies showed no correlation of PFOA exposure with escalated

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liver cancer or clinical liver disease occurrence, but a positive correlation with elevated serum

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alanine transaminase level, a sign of hepatocyte damage9-11. In vitro assays using human

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hepatocellular carcinoma HepG2 cells showed that PFOA caused apoptosis12, 13, which was

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proved by proteome assay using isobaric tags for relative and absolute quantitation (iTRAQ)

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following PFOA exposure in normal human hepatocyte cells (HL-7702). Signs of apoptosis

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like hepatocyte-nuclear condensation is also observed in PFOA-treated mouse liver14.

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However, the mechanisms of PFOA-induced hepatocyte apoptosis in mammalian hepatocytes

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are still unknown. Some attributed this to the lipid accumulation in nucleus in mouse liver,

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while hyperaccumulation of reactive oxygen species (ROS), which triggered mitochondrial

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signaling pathway, was the mode of action in HepG2 cells, with ROS origination being

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unclear14. Another critical issue of PFOA-induced toxicity is the gender difference in animals.

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Mechanistic studies using animal models of both sexes have been mainly confined to rats,

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where PFOA caused only minor toxicity in females due to the rapid metabolisms through an

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active secretory mechanism, which has not been observed in humans14-16.

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In this study, we investigated the hepatotoxicity from subchronic PFOA exposure in 4


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both male and female Balb/c mice (in vivo assay), and liver proteome using iTRAQ to better

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understand the underlying molecular mechanisms. Unlike previous genomic studies using

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microarrays - a transcriptome method17-19, iTRAQ can directly identify and quantify changes

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in protein expression level20. Since questions often remain when extrapolating data from

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rodents to humans21, human hepatocyte cells (HL-7702, in vitro) were also exposed to PFOA

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to further explore the underlying molecular mechanisms of hepatotoxicity relevant to human

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exposure.

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MATERIALS AND METHODS

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PFOA treatment of mice

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PFOA (98%) was from Tokyo Chemical Industry (Tokyo, Japan). Six-week old Balb/c

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mice were from Aiermaite Company (Suzhou, China). The reasons for choosing Balb/c

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mouse included their gene purity due to the inbred line, which is suitable for toxicity studies

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on molecular signal pathways, and their tame character so it is easy for oral gavage. In

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addition, abundant toxicity data about Balb/c mice are available 22-25, helpful for mechanism

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exploring in this study. After 2-w acclimation, mice were divided into four groups, with 60

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mice in each group (30 males and 30 females), and raised under standard animal housing

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conditions (12 h light/dark cycle, 22 ± 2°C, and 50 ± 5% humidity). The control mice were

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gavaged with corn oil without PFOA (0.1 mL/d), while the experimental mice were

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administered PFOA in corn oil at 0.01, 0.1, or 0.5 g/L, equivalent to 0.05, 0.5, and 2.5

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mg/kg-d bw/d, for 28 d.

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PFOA determination and ultrastructural changes

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After 28 d exposure, mice were sacrificed to collect liver and blood. Liver tissues were

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isolated, fixed in 4% paraformaldehyde, and sectioned using aematoxylin & eosin staining.

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For TEM, tissues were quickly dissected and fixed in phosphate-buffered 2.5%

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glutaraldehyde, followed by 1% osmium tetroxide fixation at 4°C. After dehydration using an

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aqueous-acetone series and embedding in Epon 812, tissues were sectioned ultrathin, which

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were examined using a JEM-2000EX transmission electron microscope (Olympus, Japan). 5



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Liver and serum samples of 10 mice from each treatment (total 30 mice in a treatment)

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were pooled together and homogenized to generate 3 sample sets for all experiments except

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for JC-1, which was examined immediately after sacrifice. PFOA in the liver and serum

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samples was extracted and measured following Li, et al. 26. 13C4-PFOA (>99%, Wellington

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Laboratory, Canada) was added as an internal standard. The recovery efficiency for liver

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samples spiked at 10 µg/kg was 79 ± 4.1%.

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JC-1 flow cytometer test, Caspase-9 activity, and ELISA assay of 8-OHdG, p53 and

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PPARα

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The ∆Ψm changes in mice livers were evaluated using a JC-1 ∆Ψm Detection Kit

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(Beyotime Institute of Biotechnology, China) on a FACSCalibur flow cytometer

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(BDBioscience C-6, San Jose, CA). The ratios of aggregated JC-1 and monomeric JC-1

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represent ∆Ψm of tissue samples. The details about JC-1 flow cytometer test can be found in

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supporting information.

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The activity of Caspase-9 was measured by cleaving selective substrates

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acetyl-Leu-Glu-His-Asp P-nitroanilide (Ac-LEHD-PNA) using a Caspase-9 detection kit

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(Beyotime Institute of Biotechnology). After dispersed, liver tissue was incubated with

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Ac-LEHD-PNA (10 µL) for 60 min at 37°C, cleavage of the substrate was detected using a

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UV-VIS Spectrophotometer (UV2450, Shimadzu, Japan) at 405 nm. Activities of caspase-9

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were expressed as changes in LEHDase activity.

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8-OHdG concentration, and p53 and PPARα activity were tested using ELISA kits

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(Yifei Xue Biotech, China) on an ELISA reader (Thermo, USA) following standard operation

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procedures detailed in SI.

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In vitro cell culture and exposure

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Human liver (HL-7702) cells were obtained from Chinese Academy of Science Type

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Culture Collection (Shanghai, China) and incubated in RPMI 1640 medium supplemented

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with 10% FBS. The cells were detached from the monolayer by incubating in 0.25% trypsin

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and 0.53 mM EDTA for 5 min at 37°C, then equally divided into 12 parts and seeded on 12 6


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plates, 3 plates for each treatment. PFOA stock solution was prepared in RPMI 1640 medium,

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and added to plates to achieve 1.0, 2.5, or 7.5 µg/mL for each treatment. The cells were

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collected after 24 h, and 8-OHdG, Caspase-9 and protein extraction were conducted

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following the Elisa kit (SI).

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Quantitative proteomics and Western blotting

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As mentioned above, three pooled sample sets were generated for each treatment. Two

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of the sample sets were ground to fine powder in liquid nitrogen as bio-replicates of iTRAQ,

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and the third set was used for Western Blotting. The protein extraction, iTRAQ labelling,

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detection, and data processing were conducted following Chen, et al. 27(Supporting

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information). The meaningful cutoff values of proteins for up-regulated or downregulated

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were ≥1.5 folds and ≤0.67 folds, respectively28-30. Cutoff values were also used to estimate

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the quantitation confidence between technical replicates.

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A duplicate of the cultured cells was processed similarly for Western blotting analysis.

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Western blotting was performed using standard methods31, 32, with antibodies to AOX,

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CYPIVA10, NDUV2, NDUA9, and PDCD5. GAPDH was used as house-keeper genes (inner

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control). Detailed information regarding two antibodies for each protein and reaction

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conditions are provided in Supporting information.

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Statistical methods

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All proteins showed significant expression changes were individually searched in

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database of Webofknowledge, NCBI, and Uniprot from 2012 to 2014. The KEGG database

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(http://www.genome.jp/kegg/), the STRING database (http://string-db.org/) and the Gene

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Ontology database (http://geneontology.org/) were used to group the identified proteins.

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Figures were drafted by Origin9.0 or R software, and the statistical analysis were conducted

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in SPSS 18.0, R software with Pearson correlation with ANOVA or t-test.

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RESULTS AND DISCUSSION

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After 28 d exposure to PFOA, male and female mice showed little difference in body weight gain except that the 2.5 mg/kg-d mice showed less body weight gain than the controls 7



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after 21 d (Figure 1A). However, all exposed mice excluding the 0.05 mg/kg-d males showed

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a significant increase in liver weight (Figure 1B), a sign of hepatomegaly, common in rodents

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after PFOA exposure33, 34. PFOA concentrations in both liver and serum increased with PFOA

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dose in mice, with PFOA concentrations being generally higher in the liver than the serum

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(Figure 1C). The mean serum PFOA concentration in mice exposed to 0.05 mg/kg-d PFOA

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was 0.97 and 1.2 mg/L for females and males, which was lower than those in professional

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workers in China, Europe and America (2.2, 1.5 and 6.8 mg/L, respectively), and comparable

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to those in the residents near a PFOA factory in China (0.38 and 0.01−2.4 mg/L)4, 35. The data

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suggest the PFOA doses used in this study, especially the lowest dose at 0.05 mg/kg-d, were

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relevant to human exposures in occupational and PFOA-polluted environmental scenarios.

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Apoptosis in mice liver was mediated by mitochondria signal pathway with no lipid

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accumulation in nucleus at low dose

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Histopathology of mouse liver exposed to PFOA was assessed by optical microscopy.

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No renal or cerebral damage was visible (data not shown). Control mice showed normal

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hepatocytes (Figure 1D) while PFOA-exposed mice showed two major alterations:

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hepatocellular hypertrophy and apoptosis (Figure 1EF). While hepatocellular hypertrophy

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was observed in 0.5 and 2.5 mg/kg-d males and females (Figure 1E), with signs of apoptosis

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being observed only in the 2.5 mg/kg-d mice (Figure 1F).

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In addition, hepatic ultrastructure was evaluated using TEM. No abnormality in cellular

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organelle morphology was observed in hepatocytes of control mice (Figure 2A). For exposed

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mice, lipid droplet accumulation in cytoplasm (Figure 2BC) and apoptosis (Figure 2B-G)

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were correlated with doses. Micelles scattered in cytoplasm showed a dose-dependent

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increase in numbers in exposed mice (Figure 2BC), similar to the observation of Wolf et al.36

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in PFOA-exposed mouse liver. Nuclear condensation at different stages were observed in all

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exposed mice excluding 0.05 mg/kg-d males (Figure 2B-D & 2G), a sign of apoptosis was

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also observed by Wang et al.37 in PFOA-exposed mice. However, this study showed that

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nuclear condensation was not necessarily accompanied by lipid accumulation in nucleus

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(Figure 2DG), especially for the group at 0.05 mg/kg-d where lipid micelles were scarcely 8


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observed. Instead, different stages of mitochondrial morphology changes during early

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apoptosis, including mitochondrial vesicle formation, swelling and fission were observed in a

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dose-dependent manner (Figure 2E-G)38-40. These observations were consistent with CD-1

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mice with prenatal exposures to PFOA41. Therefore, lipid accumulation in nucleus during

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PFOA-induced apoptosis may not be the only cause for apoptosis. The occurrence of

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apoptosis in 0.05 mg/kg-d females, but not males, revealed a gender difference, with females

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being more sensitive to PFOA-induced apoptosis (Figure 2G).

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The observation that PFOA-induced hepatic apoptosis was accompanied by

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mitochondria morphology changes led to additional study of PFOA effects on mitochondria.

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JC-1 fluorescent probe (mt∆Ψ) and a Caspase-9 activity test were used42. The results

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confirmed that PFOA induced mt∆Ψ dissipation and increased the Caspase-9 in a gender-

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(p

Molecular Mechanisms of Perfluorooctanoate-Induced Hepatocyte

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