The epidemiological association between cancer and exposure to ambient air pollution particles (particles with a 50% cut-off aerodynamic diameter of 10 mm (PM10)) has been related to the ability of PM10 and its constituent nanoparticles (NPs) to cause reactive oxidative species (ROS)-driven DNA damage. However, there are no data on the molecular response to these genotoxic effects.In order to assess whether PM10, NP and ROS-driven DNA damage induce carcinogenesis pathways, A549 cells were treated with tert-butyl-hyperperoxide (Tbh), urban dust (UD), carbon black (CB), nanoparticulate CB (NPCB), benzo(a)pyrene (BaP) and NPCB coated with BaP for f24 h. Single-and double-strand breakage of DNA was determined by comet assay; cell cycle status was analysed using flow cytometry. Nuclear extracts or acid-extracted histones were used for Western blot analysis of p-ser15-p53 (p53 phosphorylated at ser15), p53 binding protein (53BP) 1, phospho-histone H2A.X (p-H2A.X) and phospho-BRCA1 (p-BRCA1).UD caused both single-and double-strand DNA breaks, while other tested NPs caused only single-strand DNA breaks. NPs significantly altered cell cycle kinetics. Tbh enhanced p-H2A.X after 1 and 6 h (2.1-and 2.2-fold, respectively). NP increased 53BP1 expression at 1 h (2.4-8.7-fold) and p-BRCA1 at 1-6 h. N-acetylcysteine blocked NP-driven p-ser15-p53 response.In conclusion, nanoparticles and reactive oxidative species induce DNA damage, activating p53 and proteins related to DNA repair, mimicking irradiation-related carcinogenesis pathways.KEYWORDS: DNA damage, H2A.X histone, nanoparticles, particles with a 50% cut-off aerodynamic diameter of 10 mm, reactive oxidative species I ncreased exposures to particles with a 50% cut-off aerodynamic diameter of 10 mm (PM10) is associated with an increased risk of cardiovascular and respiratory deaths and hospital admissions, as well as lung cancer [1,2]. Direct evidence of DNA damage caused by PM10 has also been confirmed [3]. Attention has focused on the PM10 in cities because that is where most deaths occur, where pollution is routinely monitored and hence the associations are best seen. Typical urban PM10 is comprised of f50% by mass of combustion-derived nanoparticles (CDNPs; particles ,100 nm), which are carbon-centred particles, typically from automobile engine exhausts, with associated compounds including transition metals, ammonium salts of nitrogen, sulphur and chlorine plus geological dust and organic matter [4]. Many toxicological studies over the last decade have confirmed that CDNPs readily generate oxidative stress through reactive oxidative species (ROS) and inflammation and nanoparticles (NPs) are seen as the most harmful components of the PM10 mix [5][6][7].Several investigators have shown that oxidative stress may play a major role in particle-induced DNA damage, which can be prevented by antioxidants and scavengers of ROS [8]. However, detailed molecular mechanisms involved in the hallmark cellular responses to genotoxic effects are currently not known, in contras...

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The aim of the study was to compare ex vivo the toxic effects of six root canal sealers immediately after mixing or setting on human periodontal ligament fibroblasts (HPdLF). Freshly mixed (I group) or set (allowed to dry for 24 h) (II group) specimens of AH Plus Jet (AH), Apexit Plus (AP), MTA Fillapex (FL), GuttaFlow (GF), MetaSEAL Soft (META), and Tubli-Seal (TS) were prepared. HPdLF were exposed for 24 h to the specimens. 3-(4,5-dimethylthiazolo-2-yl)-2,5-diphenyltetrazolium bromide assay was used to examine the effect of the root canal sealers on mitochondrial metabolic activity. Fluorescein isothiocyanate (FITC)-annexin V (AnV) and propidium iodide staining followed by flow cytometry was used to identify the effects of the materials on cell apoptosis/necrosis. Statistical analyses were performed by one-way ANOVA followed by post hoc tests, and significance was determined at P < 0.05. Most materials from the two groups reduced the viability of the cultured cells compared with the control group (P < 0.05). Statistical analysis showed significant differences in HPdLF viability between the individual materials in each group (P < 0.001). AH and AP induced a significant increase in the percentage of apoptotic cells, while TS, FL, and META elevated the proportion of necrotic cells compared with other materials and the controls (p < 0.05). The cytotoxic effects of the tested root canal sealers (both fresh and set) on HPdLF varied. Both forms of sealers were able to cause toxic effects by inducing apoptosis and necrosis in HPdLF. The cytotoxicity of FL, META, TS was mainly associated with necrosis, while AH and AP with apoptosis.

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Although a number of studies confirm the important role of metabolites in the cytotoxicity of acetaminophen, its precise mechanisms remain unknown. Acetaminophen is metabolized by microsomal enzymes. Cytochrome P4502E1 (CYP2E1) mediated N-hydroxylation results in the formation of N-acetyl-benzo-quinoneimine, a highly reactive intermediate. We examined biochemical parameters related to necrotic and apoptotic processes in acetaminophen-exposed PC12 cells is and in a PC12 cell line genetically engineered to express human CYP2E1. Both the [3H]thymidine incorporation test and the protein assay uniformly showed dose- and time-related significant growth retardation in both cell lines exposed to the drug. This was more evident in CYP2E1-transfected cells. Moreover, the cytotoxic effect of acetaminophen was increased as evidenced by lactate dehydrogenase activity in the culture medium. Both random oligonucleotide primed synthesis assay and enzyme-linked immunosorbent assay revealed significant DNA fragmentation in both cell lines, which was greater in transfected cells, reaching about 11% of total cellular DNA. These results were confirmed by flow cytometry and microscopic examination of cell nuclei. Intracellular calcium levels were increased only in transfected cells, approximately threefold when 5 mM acetaminophen was administered for 48 h. These results indicate the cytotoxic effects of acetaminophen via apoptosis, necrosis, and growth retardation. While the precise mechanism remains obscure, it seems that DNA fragmentation and apoptotic cascade represent a preliminary biochemical event in acute cell death, and that acetaminophen bio-transformation by CYP2E1 stimulates this pathway.