The Study Of Cytotoxicity And Its Mechanisms On Human Lung Epithelial Cells Induced By Typical Airborne Nitro-PAHs

The epidemiological and toxicological studies have confirmed the associations between the exposure to atmospheric particulate matter(PM) and cardiopulmonary morbidity and mortality. Diesel exhaust particles, urban ambient particles and traffic-related particles are proved to be toxic, because of the large amounts of toxic and hazardous substances on the surface, such as transition metals and organics. Nitro-PAHs are important components of PAHs in the fine fraction of ambient particles(PM2.5). Recently, nitro-PAHs are hypothesized to contribute to the adverse health effects induced by PM and are believed to some extent to be more toxic than their parent PAHs.The aim of the present study was to determine whether exposure of human lung epithelial A549 cells to typical nitro-PAHs could cause cytotoxicity, DNA damage and expressions of inflammatory genes. The toxicological effects and associated mechanisms of 1-nitropyrene(1-NP) and 3-nitrofluoranthene(3-NF) and their corresponding amino forms 1-aminopyrene(1-AP) and 3-aminofluoranthene(3-AF) were investigated and their contributions to the harmful effects of PM were discussed. In addition the combined toxic effects of 1-NP and other typical PAHs and derivatives were also studied.The first, the oxidative potential 1-NP, 3-NF, 1-AP, and 3-AF in cell-free systems and in A549 cells were studied. The ROS generation, the changes of SOD activity and GSH content involved in oxidative stress were detected. The results showed that 1-NP, 3-NF, 1-AP and 3-AF could reacte with dithiothreitol(DTT) and caused a concentration-dependent ROS production, GSH reducation, changes of SOD activities, leading to intracellular oxidative stress in A549 cells.Secondly, the toxic effects of 1-NP, 3-NF, 1-AP, and 3-AF on viability, cell apoptosis/cell cycle, LDH release, DNA damage, intracellular calcium homeostasis, and mitochondrial damage, were studied in A549 cells. The results showed that all of them can cause a dose-dependent viability decrease, cellular LDH release, mitochondrial damages, DNA damage, and increase intracellular calcium(Ca2+) levels. Additionaly he m RNA expressions of two inflammatory cytokines(IL-6, MCP-1), cytochrome P450 enzymes(Cyp1a1, Cyp1b1, Cyp1a12, Cyp1b12) and heme oxygenase(HO-1), measured by real-time PCR. The results showed that 1-NP, 3-NF, 1-AP, and 3-AF could up-regulate HO-1 expressions. 1-AP and 3-AF could up-regulate cytochrome P450 enzymes expressions, but not 1-NP and 3-NF.Finally, The combined toxic effects of 1-NP and 1,2-naphthoquinone(1,2-NQ), 1-NP and benzopyrene B[a]p on A549 cells were evaluated. The results show that the DNA damage and ROS levels were significantly reduced in the combined-treated groups of 1-NP and 1,2-naphthoquinone(1,2-NQ) compared with the groups treated with 1-NP alone. These results suggest that 1-NP may mediated the genotoxic and cytotoxic effects through ROS generation, and pretreatment with 1,2-NQ, may inhibit the ROS generation induced by 1-NP, and thereby reducing the DNA damage in A549 cells. Cell death and ROS levels were significantly reduced in the combined-treated groups of 1-NP and benzopyrene B[a]p compared with the groups treated with 1-NP alone, while the DNA damage were increased. The mechanisms needs further investigations.The major contribution of this study is the demonstration that nitro-PAHs and amino-PAHs present in PM exert different cellular biological responses. 1-NP, 1-AP, 3-NF, 3-AF caused significantly DNA damage, as detected by the Comet assay. Nitro-PAHs can also promote the inflammation process through the generation of cytokines. Our results highlight that nitro-PAHs can significantly affect the intensity and the type of in vitro biological effects, emphasizing the importance of the PM chemical composition on its biological effect. Consequently, it will be necessary to establish specific air quality standards for the individual components in particles.