Cancer Biomarkers and Prevention Group, Biocentre, Department of Cancer Studies and Molecular Medicine, University of Leicester, University Road, Leicester LE1 7RH, UK.
J Chromatogr B Analyt Technol Biomed Life Sci. 2010 Aug 1;878(23):2155-62. doi: 10.1016/j.jchromb.2010.06.008. Epub 2010 Jun 11.
The heterocyclic aromatic amine, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is formed by the grilled cooking of certain foods such as meats, poultry and fish. PhIP has been shown to induce tumours in the colon, prostate and mammary glands of rats and is regarded as a potential human dietary carcinogen. PhIP is metabolically activated via cytochrome P450 mediated oxidation to an N-hydroxylamino-PhIP intermediate that is subsequently converted to an ester by N-acetyltransferases or sulfotransferases and undergoes heterolytic cleavage to produce a PhIP-nitrenium ion, which reacts with DNA to form the N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP-C8-dG) adduct. Thus far, the detection and quantification of PhIP-DNA adducts has relied to a large extent on (32)P-postlabelling methodologies. In order to expand the array of available techniques for the detection and improved quantification of PhIP-C8-dG adducts in DNA we have developed an online column-switching liquid chromatography (LC)-electrospray ionization (ESI)-tandem mass spectrometry (MS/MS) selected reaction monitoring (SRM) method incorporating an isotopically [(13)C(10)]-labelled PhIP-C8-dG internal standard for the analysis of DNA enzymatically hydrolysed to 2'-deoxynucleosides. A dose-dependent increase was observed for PhIP-C8-dG adducts when salmon testis DNA was reacted with N-acetoxy-PhIP. Analysis of DNA samples isolated from colon tissue of mice treated by oral gavage daily for 5 days with 50 mg/kg body weight of PhIP resulted in the detection of an average level of 14.8+/-3.7 PhIP-C8-dG adducts per 10(6) 2'-deoxynucleosides. The method required 50 microg of hydrolysed animal DNA on column and the limit of detection for PhIP-C8-dG was 2.5 fmol (1.5 PhIP-C8-dG adducts per 10(8) 2'-deoxynucleosides). In summary, the LC-ESI-MS/MS SRM method provides for the rapid automation of the sample clean up and a reduction in matrix components that would otherwise interfere with the mass spectrometric analysis, with sufficient sensitivity and precision to analyse DNA adducts in animals exposed to PhIP.
杂环芳香胺 2-氨基-1-甲基-6-苯基咪唑并[4,5-b]吡啶（PhIP）是由某些食物（如肉类、家禽和鱼类）的烧烤烹饪形成的。PhIP 已被证明能在大鼠的结肠、前列腺和乳腺中诱导肿瘤，被认为是一种潜在的人类饮食性致癌物质。PhIP 通过细胞色素 P450 介导的氧化代谢激活，生成 N-羟氨基-PhIP 中间产物，然后被 N-乙酰转移酶或磺基转移酶转化为酯，并通过异裂裂解产生 PhIP-亚硝鎓离子，与 DNA 反应形成 N-（脱氧鸟嘌呤-8-基）-2-氨基-1-甲基-6-苯基咪唑并[4,5-b]吡啶（PhIP-C8-dG）加合物。到目前为止，PhIP-DNA 加合物的检测和定量在很大程度上依赖于（32）P 后标记方法。为了扩大用于检测和改进 PhIP-C8-dG 加合物在 DNA 中定量的可用技术范围，我们开发了一种在线柱切换液相色谱（LC）-电喷雾电离（ESI）-串联质谱（MS/MS）选择反应监测（SRM）方法，该方法结合了经酶解至 2'-脱氧核苷的 DNA 的同位素标记的 PhIP-C8-dG 内标[（13）C（10）]。当用 N-乙酰氧基-PhIP 处理鲑鱼精 DNA 时，观察到 PhIP-C8-dG 加合物呈剂量依赖性增加。用 50mg/kg 体重的 PhIP 每天经口服灌胃处理 5 天的小鼠结肠组织的 DNA 样品分析导致检测到平均 14.8+/-3.7 PhIP-C8-dG 加合物/10（6）2'-脱氧核苷。该方法在柱上需要 50μg 水解的动物 DNA，PhIP-C8-dG 的检测限为 2.5fmol（1.5 PhIP-C8-dG 加合物/10（8）2'-脱氧核苷）。总之，LC-ESI-MS/MS-SRM 方法提供了样品净化的快速自动化，并减少了可能干扰质谱分析的基质成分，具有足够的灵敏度和精密度来分析暴露于 PhIP 的动物的 DNA 加合物。
