Toriba Akira, Kitaoka Hitomi, Dills Russell L, Mizukami Satoko, Tanabe Kaori, Takeuchi Naoki, Ueno Mariko, Kameda Takayuki, Tang Ning, Hayakawa Kazuichi, Simpson Christopher D
Graduate School of Natural Science and Technology, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan.
Chem Res Toxicol. 2007 Jul;20(7):999-1007. doi: 10.1021/tx700015q. Epub 2007 Jun 20.
1-nitropyrene (1-NP) is one of the most abundant nitrated polycyclic aromatic hydrocarbons (NPAHs) in diesel exhaust particulate matter (DEP) and is a main contributor of direct-acting mutagenicity in DEP. Therefore, the metabolites of 1-NP are expected to be a biomarker for assessment of exposure to DEP. In this study, a highly specific and sensitive analytical method using liquid chromatography with tandem mass spectrometry (LC-MS/MS) was developed to determine urinary 1-NP metabolites. After enzymatic hydrolysis of the conjugated metabolites, the analytes were selectively extracted from the urine matrix with blue rayon. The eluate from the rayon was further purified on an acidic alumina cartridge. Hydroxy-N-acetyl- 1-aminopyrenes (6- and 8-OHNAAP) and hydroxy-1-nitropyrenes (3-, 6-, and 8-OHNP) in human urine were identified by their retention times and MS/MS spectra and quantified by using deuterated internal standards. 1-NP metabolites were quantified in urine from all healthy, nonoccupationally exposed subjects. 6-OHNAAP, 8-OHNAAP, 6-OHNP, and 8-OHNP (means of 117, 109, 203, and 137 pmol/mol creatinine, respectively) were the most abundant isomers in human urine. This report is the first to demonstrate the presence of OHNAAPs and OHNPs in human urine, in agreement with previous in vivo and in vitro studies that predicted that these metabolites should be excreted into human urine. This method for determining urinary 1-NP metabolites should be useful for the surveillance of exposure to NPAHs and DEP and will facilitate the study of cancer risk associated with these exposures.
1-硝基芘（1-NP）是柴油车尾气颗粒物（DEP）中含量最为丰富的硝化多环芳烃（NPAHs）之一，也是DEP中直接致突变性的主要贡献者。因此，1-NP的代谢产物有望成为评估DEP暴露的生物标志物。在本研究中，开发了一种使用液相色谱串联质谱（LC-MS/MS）的高特异性和高灵敏度分析方法，用于测定尿中1-NP代谢产物。对结合态代谢产物进行酶水解后，用蓝色人造丝从尿液基质中选择性提取分析物。人造丝洗脱液在酸性氧化铝柱上进一步纯化。通过保留时间和MS/MS谱图鉴定人尿中的羟基-N-乙酰基-1-氨基芘（6-和8-OHNAAP）以及羟基-1-硝基芘（3-、6-和8-OHNP），并使用氘代内标进行定量。对所有健康的非职业暴露受试者的尿液中的1-NP代谢产物进行了定量。6-OHNAAP、8-OHNAAP、6-OHNP和8-OHNP（分别为每摩尔肌酐117、109、203和137 pmol）是人类尿液中含量最丰富的异构体。本报告首次证明人尿中存在OHNAAPs和OHNPs，这与之前的体内和体外研究一致，这些研究预测这些代谢产物应排泄到人类尿液中。这种测定尿中1-NP代谢产物的方法对于监测NPAHs和DEP暴露应该是有用的，并将有助于研究与这些暴露相关的癌症风险。
