Altered Cellular Responses As Biomarkers for Fluoranthene Exposure to the Bone Marrow-Mesenchymal Stem Cells: Implications for Stem Cell-Based in Vitro Models That Help Determine the Associated Hematotoxicity
Authors: Kim Hwan-Young , Noh Ji-Eun , Baek Hee-Jo , Moon Jae-Dong , Shin Jong-Hee , Suh Soon-Pal , Kook Hoon , Ryang Dong-Wook , Shin Myung-Geun 
Published: October 2019

Abstract
Background: Fluoranthene is a member of the polycyclic aromatic hydrocarbon family, comprising ubiquitous environmental pollutants and potent mutagens. Fluoranthene has been classified by the International Agency for Research on Cancer as a group 3 carcinogens, whose carcinogenicity has not been fully determined in humans. Moreover, the stem cell toxicity and global hematotoxicity associated with fluoranthene exposure have not been thoroughly studied in the bone marrow-mesenchymal stem cells (BM-MSCs). In this study, we determined whether fluoranthene-induced cellular responses could be used as biomarkers for the examination of BM-MSC dysfunction, and suggested the possible use of BM-MSCs for monitoring the acute hematotoxicity caused by environmental hazards.
Materials and Methods: Apreviously published protocol was used for the isolation and characterization of BM-MSCs. Fluoranthene was added to the cell culture medium in the range of 25–500 µM. The cellular levels of hydrogen peroxide, indicating the presence of reactive oxygen species, were measured using an enzyme immunoassay. The mitochondrial mass, membrane potential, and mitochondrial DNA (mtDNA) copy number were measured using MitoTracker Green, MitoTracker Red probes, and real time PCR, respectively. A proteomic analysis of the mitochondrial-rich cytoplasmic fraction was performed using nano-LC-ESI-MS/MS, BioWorksBrowser, and the SEQUEST search engines. Quantitative mRNA and immunoblot measurements were used to further confirm the altered mRNA expression as well as to determine the levels of cellular proteins obtained from the proteomic analysis.
Results: After exposure to fluoranthene, the BM-MSCs showed a marked reduction in cell number, and the viability decreased substantially after two days of exposure. BM-MSCs that were not treated with fluoranthene remained compact and spindle-shaped. These cells remained tightly attached to each other and to the substrate. In general, a direct exposure of fluoranthene depressed the proliferative capacity and altered the cell morphology of BM-MSCs. The cells detached from the subsurface, and cell-to-cell attachments were also lost. The viability significantly decreased after two days of fluoranthene exposure. The mtDNA copy number and the mass showed a rapid elevation after a 5-day exposure to fluoranthene. Hundreds of cellular proteins in the mitochondria-rich cytoplasmic fraction were markedly deregulated in cells treated with fluoranthene. The protein expression levels of poly [ADP-ribose] polymerase 1 (PARP-1), elongation factor 1-gamma, heat shock 70 kDa protein 1A/1B, heterogeneous nuclear ribonucleoproteins A2/B1 isoform B1, ATP-dependent RNA helicase DDX5, and T-complex protein 1 subunit theta were upregulated more than five-fold in cells treated with fluoranthene than in untreated cells. A significant (more than 2-fold) down-regulation in the cellular levels of the proteins myosin-9, protein ALO17 isoform 1, filamin-C isoform b, Na/K-transporting ATPase subunit alpha-1, nuclear pore membrane glycoprotein 210, and DNA-dependent protein kinase catalytic subunit isoform 2 was observed after a similar fluoranthene treatment. The presence of PARP-1 was further confirmed using mRNA analysis.
Conclusion: This study investigated the global cellular responses after exposure to fluoranthene: PARP-1 was recognized as a notable biomarker for monitoring the PAH-induced hematotoxicity. In summary, BM-MSCs are promising candidates for the development of unique in vitro model systems for predicting fluoranthene-associated hematotoxicity and general toxicity in humans.
Keywords: Fluoranthene, genotoxicity, hematotoxicity, bone marrow-mesenchymal stem cells
Figure 1. Stem cell-based in vitro models and biomarkers for studying the hematotoxic effects of fluoranthene exposure. (A) Direct exposure of PAHs depressed the proliferative capacity of h-TERT cells with a thread-like or round shape and loose cell-to-cell attachment. (B) Cytotoxic effect of fluoranthene exposure to the bone marrow-mesenchymal stem cells remarkably increased with dose-dependent manner. (C) Identified potential biomarkers were categorized as their biological processes and molecular functions. (D) Immunoblot confirmed the increased expression of poly [ADP-ribose] polymerase 1 (PARP-1) after exposure of fluoranthene. Figure 1. Stem cell-based in vitro models and biomarkers for studying the hematotoxic effects of fluoranthene exposure. (A) Direct exposure of PAHs depressed the proliferative capacity of h-TERT cells with a thread-like or round shape and loose cell-to-cell attachment. (B) Cytotoxic effect of fluoranthene exposure to the bone marrow-mesenchymal stem cells remarkably increased with dose-dependent manner. (C) Identified potential biomarkers were categorized as their biological processes and molecular functions. (D) Immunoblot confirmed the increased expression of poly [ADP-ribose] polymerase 1 (PARP-1) after exposure of fluoranthene.

Disclosures
No relevant conflicts of interest to declare.