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Oxidative stress and DNA damages induced by 1-nitropyrene in human lung fibroblasts: New insights into the mechanisms of genotoxicity and EPR-spin trapping direct monitoring of free radicals at subcellular levels
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International audience. Polycyclic aromatic hydrocarbons (PAHs) and nitro-PAHs are ubiquitous contaminants released in the atmosphere from the incomplete combustion of organic matter and pesticide intermediates nitration. Many studies have established that 1-nitropyrene (1-NP), one the most abundant nitro-PAH pollutant in diesel exhaust, is associated with increased risk of cancer and inflammatory diseases via possible metabolic pathways, including ring oxidation, nitroreduction or conjugation reactions. In addition, genotoxicity tests (e.g. comet assay and Ames test), demonstrated that 1-NP is a potent mutagen and genotoxic agent. However, the mechanisms underlying the promotion of DNA damages remain to be fully elucidated in relation with the progression of oxidative stress due to the stimulation of cellular free radical sources.Methods: On human lung fibroblasts exposed to environmentally relevant doses of 1-NP (1-10 microM) for 24–48 h, we performed: (i) cytokinesis-blocked micronucleus (CBMN) assay in association with immunofluorescence staining of centromere protein A in micronuclei to distinguish between induction of structural or numerical chromosome changes; (ii) EPR experiments using a set of spin trapping agents (i.e. the phosphorylated nitrone DEPMPO (Culcasi et al., 2012), the new stereoselective superoxide probe Me4CyDEPMPO (Gosset et al., 2011)) and mitochondrial-targeted nitrones; and (iii) the evaluation of oxidative stress from protein carbonyls, lipid peroxidation and antioxidant depletion.Major results: One advantage of the CBMN assay is its ability to elucidate the genotoxic mechanism of agents with clastogenicor aneugenic modes of action. The data provide the first evidence for a superoxide-related aneugenic mode of action at low 1-NP doses and a dominant clastogenic mechanism as the 1-NP doses and exposure time increased. This two-steps mechanism differs from that induced by CeO2-nanoparticles, an oxidant present in diesel exhausts (Benameur et al., 2014). From the EPR detection of superoxide and hydroxyl radical spin adducts, two free radical cellular sources activated by 1-NP exposure were hypothetized, i.e. NADPH oxidase and mitochondria, this latter being likely involved in oxidative stress damages development.ReferencesCulcasi, et al., 2012. Chem. Biol. Interact..Gosset, et al., 2011. Bioorg. Med. Chem..Benameur, et al., 2014. Nanotoxicology.
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