Cited by 107Per- and polyfluoroalkyl substances researchToxic Organic Pollutants ImpactAnesthesia and Neurotoxicity Research

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Neurotoxicity of Perfluorooctane Sulfonate to Hippocampal Cells in Adult Mice

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Abstract

Perfluorooctane sulfonate (PFOS) is a ubiquitous pollutant and found in the environment and in biota. The neurotoxicity of PFOS has received much concern among its various toxic effects when given during developing period of brain. However, little is known about the neurotoxic effects and potential mechanisms of PFOS in the mature brain. Our study demonstrated the neurotoxicity and the potential mechanisms of PFOS in the hippocampus of adult mice for the first time. The impairments of spatial learning and memory were observed by water maze studies after exposure to PFOS for three months. Significant apoptosis was found in hippocampal cells after PFOS exposure, accompanied with a increase of glutamate in the hippocampus and decreases of dopamine (DA) and 3,4-dihydrophenylacetic acid (DOPAC) in Caudate Putamen in the 10.75 mg/kg PFOS group. By two-dimensional fluorescence difference in gel electrophoresis (2D-DIGE) analysis, seven related proteins in the hippocampus that responded to PFOS exposure were identified, among which, Mib1 protein (an E3 ubiquitin-protein ligase), Herc5 (hect domain and RLD 5 isoform 2) and Tyro3 (TYRO3 protein tyrosine kinase 3) were found down-regulated, while Sdha (Succinate dehydrogenase flavoprotein subunit), Gzma (Isoform HF1 of Granzyme A precursor), Plau (Urokinase-type plasminogen activator precursor) and Lig4 (DNA ligase 4) were found up-regulated in the 10.75 mg/kg PFOS-treated group compare with control group. Furthermore, we also found that (i) increased expression of caspase-3 protein and decreased expression of Bcl-2, Bcl-XL and survivin proteins, (ii) the increased glutamate release in the hippocampus. All these might contribute to the dysfunction of hippocampus which finally account for the impairments of spatial learning and memory in adult mice.

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Figure 1 Acquisition and retention of the spatial reference memory task. (A) Acquisition of the reference memory task as expressed by the escape latency (mean ± SD, recorded in seconds) over 3 training sessions (four trials/day). Animals treated with 2.15 and 10.75 mg/kg of PFOS exhibited a delayed acquisition compared to animals in the control group. (B) The percentage of time spent in the target quadrant relative to the other three quadrants was recorded on the last day. A great retention of the platform location in the 2.15 and 10.75 mg/kg PFOS groups. Mice: n = 15/group. *P<0.05, **P<0.01.
Figure 1 Acquisition and retention of the spatial reference memory task. (A) Acquisition of the reference memory task as expressed by the escape latency (mean ± SD, recorded in seconds) over 3 training sessions (four trials/day). Animals treated with 2.15 and 10.75 mg/kg of PFOS exhibited a delayed acquisition compared to animals in the control group. (B) The percentage of time spent in the target quadrant relative to the other three quadrants was recorded on the last day. A great retention of the platform location in the 2.15 and 10.75 mg/kg PFOS groups. Mice: n = 15/group. *P<0.05, **P<0.01.CC BY · PubMed Central
Figure 2 Hippocampal apoptosis neural cells detected by flow cytometry analysis and the related apoptosis proteins expression detected by WB. (A) Isolated hippocampal cells were from four groups and stained with annexin-V and PI. Each group of treated cells was analyzed by flow cytometry. The percentage of apoptotic cells was counted as Lower right (LR) plus Upper right quadrant (UR) and expressed as the means ± SD from 10 mice in each group. (B–E) Schematic diagrams of apoptotic hippocampal cells in the control groups and the three PFOS-exposed groups detected by flow cytometry. Representative profiles of hippocampal cells apoptosis in the control group (B), the 0.43 mg/kg group (C), 2.15 mg/kg group (D) and 10.75 mg/kg PFOS-treated groups (E). (F) The expression of caspase-3 was increased while the levels of Bcl-2, Bcl-XL and survivin were decreased in the hippocampus due to PFOS exposure. Mice: n = 10/group. **P<0.01.
Figure 2 Hippocampal apoptosis neural cells detected by flow cytometry analysis and the related apoptosis proteins expression detected by WB. (A) Isolated hippocampal cells were from four groups and stained with annexin-V and PI. Each group of treated cells was analyzed by flow cytometry. The percentage of apoptotic cells was counted as Lower right (LR) plus Upper right quadrant (UR) and expressed as the means ± SD from 10 mice in each group. (B–E) Schematic diagrams of apoptotic hippocampal cells in the control groups and the three PFOS-exposed groups detected by flow cytometry. Representative profiles of hippocampal cells apoptosis in the control group (B), the 0.43 mg/kg group (C), 2.15 mg/kg group (D) and 10.75 mg/kg PFOS-treated groups (E). (F) The expression of caspase-3 was increased while the levels of Bcl-2, Bcl-XL and survivin were decreased in the hippocampus due to PFOS exposure. Mice: n = 10/group. **P<0.01.CC BY · PubMed Central
Figure 3 Detection of neurotransmitters release in hippocampus after PFOS exposure. (A–D) DA, DOPAC and HVA release alterations and turnover index for DA in the Caudate Putamen of adult mice exposed to PFOS. Levels of DA (A), DOPAC (B), and HVA (C) in the Caudate Putamen of adult mice exposed to PFOS for 3 months. (D) Turnover index defined as the (DOPAC+HVA)/DA levels. (E–F) Glutamate and GABA released in hippocampus. Data are expressed as means ± SD (pg/g wet weight of tissue) for 10 animals in each group. *P<0.05 (significant difference from control).
Figure 3 Detection of neurotransmitters release in hippocampus after PFOS exposure. (A–D) DA, DOPAC and HVA release alterations and turnover index for DA in the Caudate Putamen of adult mice exposed to PFOS. Levels of DA (A), DOPAC (B), and HVA (C) in the Caudate Putamen of adult mice exposed to PFOS for 3 months. (D) Turnover index defined as the (DOPAC+HVA)/DA levels. (E–F) Glutamate and GABA released in hippocampus. Data are expressed as means ± SD (pg/g wet weight of tissue) for 10 animals in each group. *P<0.05 (significant difference from control).CC BY · PubMed Central
Figure 4 Protein separation, staining and comparison determined by 2D-DEGE. The protein extracts from hippocampus of the control group and 10.75 mg/kg groups were profiled using 2D-DIGE analysis as described in the Materials and methods. (A) Cy2 labeled (internal standard) proteins separated on a 2D-IEF-SDS-PAGE gel, (B) Cy3 labeled proteins from hippocampus of control group, separated on a 2D-IEF-SDS-PAGE gel, (C) Cy5 labedled proteins from hippocampus of 10.75 mg/kg group, separated on a 2D-IEF-SDS-PAGE gel.
Figure 4 Protein separation, staining and comparison determined by 2D-DEGE. The protein extracts from hippocampus of the control group and 10.75 mg/kg groups were profiled using 2D-DIGE analysis as described in the Materials and methods. (A) Cy2 labeled (internal standard) proteins separated on a 2D-IEF-SDS-PAGE gel, (B) Cy3 labeled proteins from hippocampus of control group, separated on a 2D-IEF-SDS-PAGE gel, (C) Cy5 labedled proteins from hippocampus of 10.75 mg/kg group, separated on a 2D-IEF-SDS-PAGE gel.CC BY · PubMed Central
Figure 5 Protein spots identified by LC/MS/MS spectrometer. Target proteins that differentially expressed in adult mice hippocampus after PFOS exposure separated on a 2D-IEF-SDS-PAGE gel and identified by MALDI-TOF MS analysis. The changes were statistically significant at P<0.05. IEF, isoelectric focusing; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; 2D, two-dimensional.
Figure 5 Protein spots identified by LC/MS/MS spectrometer. Target proteins that differentially expressed in adult mice hippocampus after PFOS exposure separated on a 2D-IEF-SDS-PAGE gel and identified by MALDI-TOF MS analysis. The changes were statistically significant at P<0.05. IEF, isoelectric focusing; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; 2D, two-dimensional.CC BY · PubMed Central
Figure 6 Verification of the differentially expressed hippocampal proteins by western blotting. (A) Compared with the control group, Mib1, Tyro3 and Herc5 were found down-regulated due to PFOS-exposure. (B) Compared with the control group, Lig4, Sdha, Plau and Gzma were found up-regulated due to PFOS exposure.
Figure 6 Verification of the differentially expressed hippocampal proteins by western blotting. (A) Compared with the control group, Mib1, Tyro3 and Herc5 were found down-regulated due to PFOS-exposure. (B) Compared with the control group, Lig4, Sdha, Plau and Gzma were found up-regulated due to PFOS exposure.CC BY · PubMed Central

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Authors
Long, Y., Wang, Y., Ji, G., Yan, L., Hu, F., & Gu, A.
Affiliations
  • China Pharmaceutical University (CN)
  • Nanjing Medical University (CN)
  • Ministry of Ecology and Environment (CN)
  • State Key Laboratory of Reproductive Medicine (CN)
Corresponding author
Gu, Aihua
Venue
PLoS ONE
Published
2013-01-30
Volume
8
Issue
1
Pages
e54176
ISSN
1932-6203 (ISSN-L)
OpenAlex
W2070928735
Cited by
106
References
32
Citations by year
2026: 12 · 2025: 9 · 2024: 15 · 2023: 11 · 2022: 8 · 2021: 7 · 2020: 7 · 2019: 5 · 2018: 6 · 2017: 3 · 2016: 5 · 2015: 10 · 2014: 4 · 2013: 4
FWCI
6.13
Type
journal article
Language
en
Open access
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, other-oa)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Figshare (submittedVersion, cc-by)
Funders
  • National Natural Science Foundation of China (81172694, 30901210)
  • Government of Jiangsu Province
  • Natural Science Foundation of Jiangsu Province
  • Priority Academic Program Development of Jiangsu Higher Education Institutions
Topics
  • Physical Sciences › Environmental Science › Environmental Chemistry › Per- and polyfluoroalkyl substances research
  • Physical Sciences › Environmental Science › Health, Toxicology and Mutagenesis › Toxic Organic Pollutants Impact
  • Life Sciences › Neuroscience › Developmental Neuroscience › Anesthesia and Neurotoxicity Research
Keywords
PFOS, hippocampal neurons, apoptosis, spatial learning and memory impairment, glutamate release, dopamine, caudate-putamen, MIB-1, HERC5, Tyro3, caspase-3, Bcl-2 family proteins, survivin, ubiquitin-proteasome system, protein expression profiling, perfluorinated compounds
MeSH terms
Animals, Dopamine, Fluorocarbons, Gene Expression Regulation, Hippocampus, Memory, 3,4-Dihydroxyphenylacetic Acid, Apoptosis, Alkanesulfonic Acids, Glutamic Acid, Maze Learning, Mice, Caspase 3

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