Ultrafine black carbon caused mitochondrial oxidative stress, mitochondrial dysfunction and mitophagy in SH-SY5Y cells.

Exposure to ambient ultrafine black carbon (uBC, with aerodynamic diameter less than 100 nm) is associated with many neurodegenerative diseases. Oxidative stress is the predominantly reported neurotoxic effects caused by uBC exposure. Mitochondrion is responsible for production of majority of ROS in cells and mitochondrial dysfunction is closely related to adverse nervous outcomes. Mitophagy is an important cellular process to eliminate dysfunctional or damaged mitochondria. However, the mechanisms that modulate mitophagy and mitochondrial dysfunction initiated by uBC remain to be elucidated. The purpose of this study was to investigate how mitochondrial oxidative stress regulated mitochondrial dysfunction and mitophagy in human neuroblastoma cell line (SH-SY5Y) after uBC treatment. RNA interference was further applied to explore the roles of mitophagy in mitochondrial dysfunction. We found uBC triggered cell apoptosis via ROS-mitochondrial apoptotic pathway. The uBC also caused serious mitochondrial damage and respiratory dysfunction, indicated by the abnormalities in mitochondrial division and fusion related proteins, decreased mitochondria number and ATP level. Increased PTEN induced putative kinase 1 (PINK1) and Parkin protein levels and the autolysosome numbers suggested uBC could promote Pink1/Parkin-dependent mitophagy process in SH-SY5Y cells. Mitophagy inhibition could reserve mitochondria number and ATP activity, but not fusion and division related protein levels in SH-SY5Y cells exposed to uBC. Administration of a mitochondria-targeted antioxidant (mitoquinone) significantly eliminated uBC caused apoptosis, mitochondrial dysfunction and mitophagy. Our data suggested mitochondrial oxidative stress regulated uBC induced mitochondrial dysfunction and PINK1/Parkin-dependent mitophagy. PINK1/Parkin-dependent mitophagy probably participated in regulating uBC caused mitochondrial dysfunction but not by controlling mitochondrial fusion and division related proteins. Our results may provide some new insights and evidences to understand the mechanisms of neurotoxicity induced by uBC.

Transcriptomic analyses of the biological effects of black carbon exposure to A549 cells.

Ambient black carbon (BC) is found to be associated with increased risk of diverse pulmonary diseases, including acute respiratory inflammation and decreased lung function. Freshly emitted BC (FBC) can be transformed into oxidized BC (OBC) through the photochemical oxidization in the air. How this oxidization process influences the toxicity of BC particles is unclear. Previous studies found FBC and OBC could induce oxidative stress and inflammation. This study aimed to further compare the regulating pathways and tried to reveal the crucial target genes caused by FBC and OBC in A549 cells based on transcriptomic data. A total of 47,000 genes in A549 cells after treated with FBC and OBC were examined using Affymetrix Human U133 plus 2.0 chips. Gene ontology (GO) classification (functional enrichment of differentially expressed genes) and Kyoto encyclopedia of genes and genomes (KEGG) classification (pathway enrichment of differentially expressed genes) were conducted and crucial genes were screened. The results showed that top 50 GO terms of FBC and OBC were not completely consistent. The Go term of cation channel was only identified in OBC group, probably caused by the characteristic that zeta potential of OBC is negative, while, that of FBC is positive. In addition transient receptor potential melastatin 7 (trpm7) gene was suggested to be closely related to this process caused by OBC. There are 47 identical pathways in FBC and OBC group among the top 50 KEGG. The inconsistent pathways are mostly related to inflammation with different up-regulation or down-regulation trends of crucial genes. The KEGG results suggested that FBC and OBC both cause inflammatory responses, but through different regulating pathways. In conclusion, OBC and FBC could induce similar toxic endpoints in A549 cells, but the underline regulating processes are not exactly the same.

Fresh and ozonized black carbon promoted DNA damage and repair responses in A549 cells.

Nano-sized ambient black carbon (BC) is hypothesized to pose a serious threat to human health. After emission into the air, the atmospheric oxidation process can modify its physiochemical properties and change its biological responses. In this study, we aimed to compare different DNA damage and repair responses promoted by fresh BC (FBC) and ozone oxidized-BC (OBC). The cell apoptosis, cell arrest, DNA damage and repair were investigated in A549 cells after treatment with FBC and OBC. Associated gene expressions were measured with the reverse transcription quantitative polymerase chain reaction (RT-qPCR) method. Both FBC and OBC could induce cell apoptosis in A549 cells with up-regulated gene of promyelocytic leukemia protein (pml) and down-regulated gene of anti-apoptotic B-cell lymphoma-2 (bcl-2). FBC caused cell cycle arrest at S and G2/M phases, which was associated with up-regulated ataxia telangiectasia mutated (atm), checkpoint kinase 2 (chk2), structural maintenance of chromosomes 1 (smc1) and cell division cycle 25 homolog A (cdc25a) genes. OBC promoted cell cycle arrest at the S phase with up-regulated genes of atm, chk2 and smc1. Both FBC and OBC induced oxidative DNA damage and time-dependent DNA repair responses with increased gene expressions of breast cancer susceptibility protein 1 (brca1), recombination protein A paralog B (rad51b), methyl methanesulfonate-sensitivity protein 22-like and tonsoku-like (mms22l). Compared to FBC, OBC could cause more sufficient DNA damage repair responses through cell cycle arrest at the S phase, resulting in relatively weaker DNA damages.