Franco de Oliveira Tiago, Falcão de Oliveira Antonio Anax, Lemos Miriam, Veras Mariana, Saldiva Paulo Hilário Nascimento, Gennari de Medeiros Marisa Helena, Di Mascio Paolo, de Melo Loureiro Ana Paula
Departamento de Análises Clínicas e Toxicológicas, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo; Departamento de Farmacociências, Universidade Federal de Ciências da Saúde de Porto Alegre.
Departamento de Análises Clínicas e Toxicológicas, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo.
J Vis Exp. 2019 May 29(147). doi: 10.3791/59734.
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-etheno-2'-deoxyguanosine (1,N-εdGuo) and 1,N-etheno-2'-deoxyadenosine (1,N-εdAdo), 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-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-dAdo and 1,N-dGuo in lung, liver and kidney DNA of A/J mice for the assessment of the effects of ambient PM2.5 exposure.
DNA加合物和氧化的DNA碱基是DNA损伤的实例，它们是用于评估亲电物质、生物转化时产生活性亲电试剂或诱导氧化应激的物质毒性的有用生物标志物。在氧化的核碱基中，研究最多的是8-氧代-7,8-二氢鸟嘌呤（8-氧代鸟嘌呤）或8-氧代-7,8-二氢-2'-脱氧鸟苷（8-氧代脱氧鸟苷），它是DNA中氧化诱导的碱基损伤的生物标志物。脂质过氧化过程产生的醛类和环氧醛类是亲电分子，能够形成诱变的环外DNA加合物，如乙烯基加合物1,N-乙烯基-2'-脱氧鸟苷（1,N-εdGuo）和1,N-乙烯基-2'-脱氧腺苷（1,N-εdAdo），它们被认为是炎症病理生理学中的潜在生物标志物。为了制定减缓细胞突变率和慢性病发展（如癌症、神经退行性疾病）的预防策略，有必要采用选择性和灵敏的方法对DNA中的这些物质进行定量。在现有的用于检测它们的灵敏方法（高效液相色谱与电化学或串联质谱检测器联用、彗星试验、免疫测定、P-后标记法）中，最具选择性的是基于高效液相色谱与串联质谱联用（HPLC-ESI-MS/MS）的方法。在分析复杂生物样品时，选择性是一个至关重要的优势，HPLC-ESI-MS/MS已发展成为定量生物基质（如DNA、尿液、血浆和唾液）中修饰核苷的金标准。使用同位素标记的内标具有校正DNA水解和分析物富集步骤中分子损失以及样品间分析物电离差异的优势。当存在多个峰时，它还有助于识别正确的色谱峰。我们在此展示了经过验证的灵敏、准确和精密的HPLC-ESI-MS/MS方法，这些方法已成功应用于定量A/J小鼠肺、肝和肾DNA中的8-氧代脱氧鸟苷、1,N-脱氧腺苷和1,N-脱氧鸟苷，以评估环境PM2.5暴露的影响。
