Quantitative high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry analysis of the adenine-guanine cross-links of 1,2,3,4-diepoxybutane in tissues of butadiene-exposed B6C3F1 mice
Quantitative high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry analysis of the adenine-guanine cross-links of 1,2,3,4-diepoxybutane in tissues of butadiene-exposed B6C3F1 mice
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DOI：
10.1021/tx800051y
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发表时间：
2008-05-01
影响因子：
4.1
通讯作者：
Tretyakova, Natalia
中科院分区：
文献类型：
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作者：
Goggin, Melissa;Anderson, Chris;Tretyakova, Natalia
1,3-丁二烯（BD）是一种重要的工业化学品，用于制造橡胶和塑料，也是一种存在于汽车尾气和香烟烟雾中的环境污染物。根据职业接触工人的流行病学证据及其在实验动物中诱发肿瘤的能力，它被归类为已知的人类致癌物。BD被代谢激活为几种活性物质，包括1,2,3,4-二氧丁烷（DEB），由于其双功能亲电性质和形成DNA-DNA和dna -蛋白质交联的能力，它被假设为最终的致癌物质。1, 4-bis - (guan-7-yl) 2, 3, -butanedioI (bis-N7G-BD)是唯一类型的DEB-specific DNA加合物之前量化体内,四个regioisomeric guanine-adenine (g)交叉连接观察体外:1 - (guan-7-yl) 4 - (aden-1-yl) 2、3-butanediol (N7G-N1A-BD), 1 - (guan-7-yl) 4 - (aden3-yl) 2、3-butanediol (N7G-N3A-BD), 1 - (guan-7-yl) 4 - (aden-7-yi) 2、3-butanediol (N7G-N7A-BD),和1 - (guan-7-yl) 4 - (aden-6-yl) 2、3-butanediol (N7G-N-6 A-BD)(公园(2004)化学。毒物学杂志17,1638-1651)。本研究的目的是建立一种同位素稀释hplc -阳性模式电喷雾电离-串联质谱（HPLC-ESI+-MS/MS）方法，用于定量分析从bd暴露的实验动物提取的DNA中G-A DEB交联。在我们的方法中，G-A丁二醇缀合物通过热水解或温和的酸水解从DNA主链中释放出来。固相萃取后，样品用N-15(3)、C-13(1)标记的内标进行毛细管HPLC-ESI+-MS/MS分析。本方法的检出限为每10(8)个正常核苷酸0.6-1.5个加合物。将G-A交联标准品（各10 fmol）加入对照小鼠DNA （0.1 mg）中，进行样品处理和HPLC-ESI+-MS/MS分析，验证了新方法的有效性。N7G-N3A-BD的准确度为105 +/- 17%，N7G-N7A-BD的准确度为102 +/- 25%，N7G-N-6 A-BD的准确度为79 +/- 11%。在DEB处理过的小牛胸腺DNA中，区域异构体G-A DEB加合物以浓度依赖的方式形成，其中N7G-N1A-BD的含量最高。生理条件下，N7G-N1A-BD对N7G-N-6 A-BD进行了Dimroth重排（t(1/2) = 114 h），而N7G-N1A-BD对相应次黄嘌呤病变的水解脱胺作用不显著。我们发现，对于体内样品，如果通过强制Dimroth重排将N7G-N1A-BD加合物转化为相应的N7G-N-6 a - bd病变，则可以获得更高的灵敏度。雌性B6C3F1小鼠吸入625 ppm BD 2周后提取的肝脏DNA每10(8)个核苷酸（n 5）中含有3.1 +/- 0.6个N7G-N1A-BD加合物（碱基诱导的dintroth重排后定量为n7g - n- 6a -BD），而N7G-N3A-BD和N7G-N7A-BD的含量均低于本方法的检测限。在对照动物中没有G-A交联。N7G-N1A-BD交联的形成可能有助于诱导双相障碍暴露后的AT碱基对突变。本文提出的定量方法不仅可用于动物模型的生物学意义研究，还可能用于预测人类双相障碍暴露的相关风险。
1,3-Butadiene (BD) is an important industrial chemical used in the manufacture of rubber and plastics as well as an environmental pollutant present in automobile exhaust and cigarette smoke. It is classified as a known human carcinogen based on the epidemiological evidence in occupationally exposed workers and its ability to induce tumors in laboratory animals. BD is metabolically activated to several reactive species, including 1,2,3,4-diepoxybutane (DEB), which is hypothesized to be the ultimate carcinogenic species due to its bifunctional electrophilic nature and its ability to form DNA-DNA and DNA-protein cross-links. While 1,4-bis-(guan-7-yl)-2,3,-butanedioI (bis-N7G-BD) is the only type of DEB-specific DNA adduct previously quantified in vivo, four regioisomeric guanine-adenine (G-A) cross-links have been observed in vitro: 1-(guan-7-yl)-4-(aden-1-yl)-2,3-butanediol (N7G-N1A-BD), 1-(guan-7-yl)-4-(aden3-yl)-2,3-butanediol (N7G-N3A-BD), 1-(guan-7-yl)-4-(aden-7-yi)-2,3-butanediol (N7G-N7A-BD), and 1-(guan-7-yl)-4-(aden-6-yl)-2,3-butanediol (N7G-N-6 A-BD) (Park (2004) Chem. Res. Toxicol. 17, 1638-1651). The goal of the present work was to develop an isotope dilution HPLC-positive mode electrospray ionization -tandem mass spectrometry (HPLC-ESI+-MS/MS) method for the quantitative analysis of G-A DEB cross-links in DNA extracted from BD-exposed laboratory animals. In our approach, G-A butanediol conjugates are released from the DNA backbone by thermal or mild acid hydrolysis. Following solid-phase extraction, samples are subjected to capillary HPLC-ESI+-MS/MS analysis with N-15(3), C-13(1) -labeled internal standards. The detection limit of our current method is 0.6-1.5 adducts per 10(8) normal nucleotides. The new method was validated by spiking G-A cross-link standards (10 fmol each) into control mouse DNA (0.1 mg), followed by sample processing and HPLC-ESI+-MS/MS analysis. The accuracy and precision were calculated as 105 +/- 17% for N7G-N3A-BD, 102 +/- 25% for N7G-N7A-BD, and 79 +/- 11% for N7G-N-6 A-BD. The regioisomeric G-A DEB adducts were formed in a concentration-dependent manner in DEB-treated calf thymus DNA, with N7G-N1A-BD found in the highest amounts. Under physiological conditions, N7G-N1A-BD underwent Dimroth rearrangement to N7G-N-6 A-BD (t(1/2) = 114 h), while hydrolytic deamination of N7G-N1A-BD to the corresponding hypoxanthine lesion was insignificant. We found that for in vivo samples, a greater sensitivity could be achieved if N7G-N1A-BD adducts were converted to the corresponding N7G-N-6 A-BD lesions by forced Dimroth rearrangement. Liver DNA extracted from female B6C3F1 mice that underwent inhalation exposure to 625 ppm BD for 2 weeks contained 3.1 +/- 0.6 N7G-N1A-BD adducts per 10(8) nucleotides (n 5) (quantified as N7G-N-6 A-BD following base-induced Din-troth rearrangement), while the amounts of N7G-N3A-BD and N7G-N7A-BD were below the detection limit of our method. None of the G-A cross-links was present in control animals. The formation of N7G-N1A-BD cross-links may contribute to the induction of AT base pair mutations following exposure to BD. Quantitative methods presented here may be used not only for studies of biological significance in animal models but potentially to predict risk associated with human exposure to BD.