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https://observatorio.fm.usp.br/handle/OPI/32534
Title: | Quantification of three DNA Lesions by Mass Spectrometry and Assessment of Their Levels in Tissues of Mice Exposed to Ambient Fine Particulate Matter |
Authors: | OLIVEIRA, Tiago Franco de; OLIVEIRA, Antonio Anax Felcao de; LEMOS, Miriam; VERAS, Mariana; SALDIVA, Paulo Hilario Nascimento; MEDEIROS, Marisa Helena Gennari de; MASCIO, Paolo Di; LOUREIRO, Ana Paula de Melo |
Citation: | JOVE-JOURNAL OF VISUALIZED EXPERIMENTS, n.147, article ID e59734, 19p, 2019 |
Abstract: | DNA adducts and oxidized DNA bases are examples of DNA lesions that are useful biomarkers for the toxicity assessment of substances that are electrophilic, generate reactive electrophiles upon biotransformation, or induce oxidative stress. Among the oxidized nucleobases, the most studied one is 8-oxo-7,8-dihydroguanine (8-oxoGua) or 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), a biomarker of oxidatively induced base damage in DNA. Aldehydes and epoxyaldehydes resulting from the lipid peroxidation process are electrophilic molecules able to form mutagenic exocyclic DNA adducts, such as the etheno adducts 1,N-2-etheno-2'-deoxyguanosine (1,N-2-epsilon dGuo) and 1,N-6-etheno-2'deoxyadenosine (1,N-6-epsilon dAdo), which have been suggested as potential biomarkers in the pathophysiology of inflammation. Selective and sensitive methods for their quantification in DNA are necessary for the development of preventive strategies to slow down cell mutation rates and chronic disease development (e.g., cancer, neurodegenerative diseases). Among the sensitive methods available for their detection (high performance liquid chromatography coupled to electrochemical or tandem mass spectrometry detectors, comet assay, immunoassays, P-32-postlabeling), the most selective are those based on high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-ESI-MS/MS). Selectivity is an essential advantage when analyzing complex biological samples and HPLC-ESI-MS/MS evolved as the gold standard for quantification of modified nucleosides in biological matrices, such as DNA, urine, plasma and saliva. The use of isotopically labeled internal standards adds the advantage of corrections for molecule losses during the DNA hydrolysis and analyte enrichment steps, as well as for differences of the analyte ionization between samples. It also aids in the identification of the correct chromatographic peak when more than one peak is present. We present here validated sensitive, accurate and precise HPLC-ESI-MS/MS methods that were successfully applied for the quantification of 8-oxodGuo, 1,N-6-dAdo and 1,N-2-dGuo in lung, liver and kidney DNA of A/J mice for the assessment of the effects of ambient PM2.5 exposure. |
Appears in Collections: | Artigos e Materiais de Revistas Científicas - FM/MCM Artigos e Materiais de Revistas Científicas - FM/MPT Artigos e Materiais de Revistas Científicas - HC/InCor Artigos e Materiais de Revistas Científicas - LIM/05 Artigos e Materiais de Revistas Científicas - ODS/03 |
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