Blood Res.  2019 Dec;54(4):253-261. 10.5045/br.2019.54.4.253.

Potential biomarkers and antagonists for fluoranthene-induced cellular toxicity of bone marrow derived mesenchymal stem cells

• Hoque MM
• Lee YE
• Kim HR
• Shin MG

• KMID: 2466590
• DOI: http://doi.org/10.5045/br.2019.54.4.253

Abstract

BACKGROUND
Fluoranthene (FR) is a common environmental pollutant that exists in a complex mixture with other polycyclic aromatic hydrocarbons (PAHs). We identified biomarkers for monitoring FR exposure and investigated the rescue effect of FR-induced cellular toxicity via aryl hydrocarbon receptor (AHR) antagonist activity in bone marrow derived mesenchymal stem cells (BM-MSCs).
METHODS
Morphological changes, viability, and rescue effects of an AHR antagonist (CH223191) were examined in BM-MSCs after exposure to FR. Cytotoxic effects were assayed using the tetrazolium-based colorimetric assay. Apoptosis was measured by annexin V and propidium iodide dye-based flowcytometry assay, mitochondrial membrane potential assay, and nuclear DNA fragmentation assay. Molecular signaling pathways of apoptosis and autophagy were investigated using immunoblotting. Proteomics were performed in order to reveal the spectra of cellular damage and identify biomarkers for FR exposure.
RESULTS
Exposing BM-MSCs to FR (ICâ‚…â‚€=50 µM) induced cell death and morphological changes, while the AHR antagonist showed rescue effects. Autophagy was activated and mitochondrial membrane potential was decreased. Proteomic analysis identified 48 deregulated proteins (26 upregulated and 22 downregulated). Among them, annexin A6, pyruvate kinase, UDP-glucose dehydrogenase, and phospholipase A2 could be potential biomarkers for FR exposure.
CONCLUSION
The exposure of BM-MSCs to FR induced remarkable alterations in cellular biology and the proteome, allowing for identification of novel biomarkers for FR exposure. Furthermore, AHR antagonists might be able to prevent cellular damage due to FR exposure.

Keyword

Fluoranthene; Biomarker; Aryl hydrocarbon receptor antagonist

MeSH Terms

Annexin A5
Annexin A6
Apoptosis
Autophagy
Biomarkers*
Bone Marrow*
Cell Death
DNA Fragmentation
Immunoblotting
Membrane Potential, Mitochondrial
Mesenchymal Stromal Cells*
Oxidoreductases
Phospholipases A2
Polycyclic Hydrocarbons, Aromatic
Propidium
Proteome
Proteomics
Pyruvate Kinase
Receptors, Aryl Hydrocarbon
Annexin A5
Annexin A6
Biomarkers
Oxidoreductases
Phospholipases A2
Polycyclic Hydrocarbons, Aromatic
Propidium
Proteome
Pyruvate Kinase
Receptors, Aryl Hydrocarbon

Figure

• 

  Fig. 1 Chemical structure of FR and morphological changes of BM-MSCs upon exposure to FR and subsequent antagonistic effects of CH 223191 every 24 hours for up to 72 hours. FR is a polycyclic aromatic hydrocarbon (PAH) that is formed by the fusion of naphthalene and benzene connected by a five-membered ring (A). Morphological changes of BM-MSCs after FR exposure revealed that cells were elongated, detached from the subsurface, and had loosened cell-to-cell attachments. These morphological alterations were partially rescued after co-treatment with an AHR antagonist (CH223191) in a dose-dependent manner (B).

• 

  Fig. 2 Cytotoxic effect of FR on BM-MSCs. MSCs (5×103, 4×103, and 2×103 cells/well) were treated with indicated concentrations (50, 100, 150, and 200 µM) of FR for 72 hours. Cell viability was measured by the tetrazolium-based colorimetric assay. Cell viability gradually decreased with increased FR dosage. The IC50 of FR was 50 µM (A). DNA fragmentation assay was performed in MSCs after treatment with FR for 72 hours. There were no significant changes in the nuclear DNA in response to FR (B). Effect of FR in inducing apoptosis in MSCs. Cells (1×106/plate) treated with 150 µM and 200 µM FR for 72 hours were co-stained with PI and FITC-conjugated annexin V and then examined by flow cytometry. Almost twice as many cells underwent apoptosis after FR treatment compared to control (C). Effect of FR exposure on mitochondria in MSCs. Loss of mitochondrial membrane potential was observed after FR treatment. MSCs were treated with FR for 72 hours, washed twice with phosphate buffer saline (PBS), and then treated with 100 nm MitoTracker Red CMXRos at 37℃ for 30 minutes. Fluorescence images were then recorded. Reduced fluorescence with increasing doses of FR is an indication of mitochondrial membrane potential loss, which is an indication of mitochondrial dysfunction as well as initiation of early apoptosis (D).

• 

  Fig. 3 Molecular signaling pathway of apoptosis and autophagy with FR exposure and an AHR inhibitor by western blot. Expression of caspase 3 and caspase 9 increased, and the anti-apoptotic protein bcl-2 decreased in a dose-dependent manner with increasing concentrations of FR, which is an indication of apoptosis activation (A). FR (150 µM and 200 µM) induced conversion of LC3A I to LC3A II, which confirms induction of autophagy, which was recovered by treatment with the AHR antagonist CH223191 (B).

• 

  Fig. 4 Proteomic analysis for the identification of candidate biomarkers for FR exposure. Two-dimensional electrophoresis showed altered expression of various protein spots (upper panel), which are quantified as bar graphs (lower panel) of relative quantitative expression of each candidate proteomic marker. Four altered spots (147, 172, 220, and 581, red boxes) were identified as annexin A6, pyruvate kinase protein, UDP-glucose dehydrogenase, and phospholipase A2.

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