Toxicity of Water- and Organic-Soluble Wood Tar Fractions from Biomass Burning in Lung Epithelial Cells

Widespread smoke from wildfires and biomass burning contributes to air pollution and the deterioration of air quality and human health. A common and major emission of biomass burning, often found in collected smoke particles, is spherical wood tar particles, also known as “tar balls”. However, the toxicity of wood tar particles and the mechanisms that govern their health impacts and the impact of their complicated chemical matrix are not fully elucidated. To address these questions, we generated wood tar material from wood pyrolysis and isolated two main subfractions: water-soluble and organic-soluble fractions. The chemical characteristics as well as the cytotoxicity, oxidative damage, and DNA damage mechanisms were investigated after exposure of A549 and BEAS-2B lung epithelial cells to wood tar. Our results suggest that both wood tar subfractions reduce cell viability in exposed lung cells; however, these fractions have different modes of action that are related to their physicochemical properties. Exposure to the water-soluble wood tar fraction increased total reactive oxygen species production in the cells, decreased mitochondrial membrane potential (MMP), and induced oxidative damage and cell death, probably through apoptosis. Exposure to the organic-soluble fraction increased superoxide anion production, with a sharp decrease in MMP. DNA damage is a significant process that may explain the course of toxicity of the organic-soluble fraction. For both subfractions, exposure caused cell cycle alterations in the G2/M phase that were induced by upregulation of p21 and p16. Collectively, both subfractions of wood tar are toxic. The water-soluble fraction contains chemicals (such as phenolic compounds) that induce a strong oxidative stress response and penetrate living cells more easily. The organic-soluble fraction contained more polycyclic aromatic hydrocarbons (PAHs) and oxygenated PAHs and induced genotoxic processes, such as DNA damage.


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. Chemical characterization of wood tar extracts; GC×GC-HR-Tof-MS measured chemical compositions for both water-soluble and organic-soluble wood tar extracts.

Resonance-enhanced multiphoton ionization time-of-flight mass (REMPI-Tof-MS).
Water-soluble and organic-soluble wood tar were analyzed using REMPI-Tof-MS as following: Briefly, 10 µl of organic-soluble and 30 µl of water-soluble wood tar extracts were placed on a pre-baked quartz fiber filter and heated in a carbon analyzer (DRI Model Generally, polyaromatic hydrocarbons and amines have one to two order of magnitude higher photoionization cross sections than phenolic species. 3 However, the individual ionization efficiencies depend on the molecular structures. At approximately 300°C, a shift towards smaller m/z was observed, indicating thermal decomposition.
Hence, the mass spectra below 300 °C refer to thermal-desorption, whereas mass spectra above 300 °C are considered as pyrolysis. 1 Table S1. Detected compounds were classified to chemical groups based on the retention time and characteristic ions in their mass spectrum, as previously published. 4

Ultrahigh resolution mass spectrometric analysis (FTICR MS).
Water-soluble and organic-soluble wood tar extracts were diluted in Dichloromethane to a concentration of 10 mg/ml (1:100) and further diluted by a factor of 20 in Methanol (0.5 mg/ml).
Protonation was promoted by adding 1 vol.% glacial acetic acid. Direct infusion mass spectra were acquired in positive polarity and broadband detection mode from m/z 100 ─

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Comprehensive chemical overview of wood tar extracts using direct-infusion highresolution mass spectrometry. Direct-infusion high-resolution mass spectrometry with ESI ionization depicts the molecular complexity of both water and organic-soluble wood tar extracts. This technique helps to decipher the comprehensive perspective of the chemical space of wood tar extracts compared to the previously discussed techniques that analyze species that desorb into the gas phase. In general, the wood tar extracts contain monomeric and larger lignin-degradation products (primarily detected by ESI) and      Figure S6. Wood tar extracts induces DNA damage in BEAS-2B lung epithelial cells. The cells were exposed to water-soluble (WS) or organic-soluble (OS) wood tar extracts at concentrations of 0.02, 0.2 or 1 mg/mL for 5 hours. DNA damage histone γ-H2AX was analyzed using flow cytometry. Etoposide (100 µM) was used as a positive control. (A) Flow cytometry histogram for γ-H2AX staining. Transcription levels were analyzed by real-time PCR for (B) OGG1, β-Actin and HPRT, which were used as endogenous controls. The data represent the mean ± SD. Means with different letters are significantly different at p < 0.05 using the Tukey HSD test. These experiments were performed in triplicate and were repeated twice.  Figure S7. Wood tar extracts alter the cell cycle in BEAS-2B lung epithelial cells. Lung epithelial cells were exposed to water-soluble (WS) or organic-soluble (OS) wood tar extracts at concentrations of 0.02, 0.2 or 1 mg/mL for 5 hours. (A) Flow cytometry histograms presenting the estimated model of cell cycle analysis using FCS express software. Quantification of (B) water-soluble and (C) organic-soluble wood tar extracts at different phases of the cell cycle. Transcription levels were analyzed by real-time PCR for (D) GADD45 and (E) p21, and (F) p16, β-Actin and HPRT were used as endogenous controls. Epidermal growth factor (EGF), 100 µM etoposide and starvation conditions were used as positive and negative controls. The data represent the mean ± SD. Means with different letters are significantly different at p < 0.05 using the Tukey HSD test. These experiments were performed in triplicate and were repeated twice.