磁性酶生物反应器珠和LC-MS / MS测序对合成p53基因外显子7寡核苷酸代谢物相关损伤的途径,Biochemistry

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磁性酶生物反应器珠和LC-MS / MS测序对合成p53基因外显子7寡核苷酸代谢物相关损伤的途径
Biochemistry ( IF 2.9 ) Pub Date : 2018-05-11 00:00:00 , DOI: 10.1021/acs.biochem.8b00271
Spundana Malla ₁ , Karteek Kadimisetty ₁ , Di Jiang ₁ , Dharamainder Choudhary ₂ , James F. Rusling _{1,

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Affiliation

环境化学物质和药物的反应性代谢产物可以在遗传毒性的主要途径中对p53抑癌基因造成位点特异性损伤。我们在这里报告了一种高通量，无细胞的96孔板磁珠酶系统，该系统与LC-MS / MS测序相连接，可生物激活测试化学物质并鉴定基因上的加合位点。使用来自磁珠包被的不同器官的八种微粒体细胞色素（cyt）P450酶，将生物激活的黄曲霉毒素B1与p53基因的32 bp外显子7片段反应。所有的Cyt P450都将黄曲霉毒素B1转化为黄曲霉毒素B1–8,9-环氧化合物，其将249位密码子的鸟嘌呤（G）加成，随后进行纯化，得到无碱基位点，然后发生链断裂。这是在无细胞培养基中首次证明黄曲霉毒素B1代谢物选择性地引起无碱基位点形成和p53探针密码子249处的链断裂，这与人肝细胞和肿瘤中p53的化学途径和突变相对应。分子建模支持以下观点：黄曲霉毒素B1–8,9-环氧化物与249号密码子中的G的结合先于S_N 2加成反应。在不同器官特有的一系列代谢酶中，人肝微粒体和cyt P450 3A5超体对p53外显子7损伤的生物活化率最高。鉴定代谢物相关基因损伤位点的这种方法可以通过与突变位点的相关性促进预测受试化学物质的器官特异性癌症。

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Pathways of Metabolite-Related Damage to a Synthetic p53 Gene Exon 7 Oligonucleotide Using Magnetic Enzyme Bioreactor Beads and LC–MS/MS Sequencing

Reactive metabolites of environmental chemicals and drugs can cause site specific damage to the p53 tumor suppressor gene in a major pathway for genotoxicity. We report here a high-throughput, cell-free, 96-well plate magnetic bead–enzyme system interfaced with LC–MS/MS sequencing for bioactivating test chemicals and identifying resulting adduction sites on genes. Bioactivated aflatoxin B1 was reacted with a 32 bp exon 7 fragment of the p53 gene using eight microsomal cytochrome (cyt) P450 enzymes from different organs coated on magnetic beads. All cyt P450s converted aflatoxin B1 to aflatoxin B1–8,9-epoxide that adducts guanine (G) in codon 249, with subsequent depurination to give abasic sites and then strand breaks. This is the first demonstration in a cell-free medium that the aflatoxin B1 metabolite selectively causes abasic site formation and strand breaks at codon 249 of the p53 probe, corresponding to the chemical pathway and mutations of p53 in human liver cells and tumors. Molecular modeling supports the view that binding of aflatoxin B1–8,9-epoxide to G in codon 249 precedes the S_N2 adduction reaction. Among a range of metabolic enzymes characteristic of different organs, human liver microsomes and cyt P450 3A5 supersomes showed the highest bioactivation rate for p53 exon 7 damage. This method of identifying metabolite-related gene damage sites may facilitate predictions of organ specific cancers for test chemicals via correlations with mutation sites.

更新日期：2018-05-11

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