Name: a general method for detecting nitrosamide formation in the in vitro metabolism of nitrosamines by cytochrome p450s
Uploaded: 2017-09-25T13:00:00
Description: Watch this Scientific Journal Video about A General Method for Detecting Nitrosamide Formation in the In Vitro Metabolism of Nitrosamines by Cytochrome P450s at JoVE.com

这项研究得到了格兰特号的支持。CA-81301 来自国家癌症研究所我们感谢鲍勃的编辑协助, Dr. 彼得 Villalta 和鲁迅的质谱协助分析生物化学共享资源共济会癌症中心, 和 Dr. 亚当 t. Zarth 和 Dr. 安娜 k. 米歇尔的宝贵讨论和输入。分析生物化学共享资源由国家癌症研究院癌症中心资助 CA-77598 部分支持

1. 材料和常规过程 <ol> <li> 将 NNN 合成为以前描述的 15 。获得 norcotinine, P450 2A6 Baculosomes, 再生系统, 0.5x 和 #160; 反应缓冲器, 以及试剂级商业来源的所有其他化学品或溶剂. </li>
	<li> 在 500 MHz 光谱仪上记录核磁共振谱。报告化学变化为每百万分之 (ppm)。使用残余溶剂峰值作为内部参考为 1 H-nmr (7.26 ppm CDCl 3 ) 和 13 C-nmr (77.2 ppm CDCl 3 ). 
	注?...

<ol>
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阐明亚硝胺的代谢是了解其致癌性的重要组成部分。由于涉及的细胞色素 P450s 和其他代谢酶是多态的, 进一步应用这一知识可能会发现高危个体1,4。新的数据表明, 进一步氧化α-hydroxynitrosamines, 推定主要代谢物的亚硝胺参与 DNA 结合, 以 nitrosamides 是可能的;然而, 这并没有在广泛的基板上进行强有力的测试。我们已经描述了一个协议来确定性氧化亚硝胺的...

N-亚硝胺是一种在饮食、烟草制品和一般环境中发现的大量致癌物质;它们也可以在人体内形成内1。超过 300 N-硝基化合物已经被测试和 #62; 90% 被评估了作为致癌物在动物模型2,3。为了展示它们的致癌性, 这些化合物必须首先激活细胞色素 P450s1,2,3。研究表明, 细胞色素 P450s 容易氧化亚硝胺为α-hydroxynitrosamines (图 1), 这是高度活性化合物与半衰期的〜 5 s 之前自发分解到 alkyldiazohydroxides。后者可以在丢失 H2O 和 N2之后烷基化 DNA。由此产生的 DNA 加, 如果未, 可以导致突变, 如果在关键的 onco 或抑癌基因, 导致癌症的发展1。因此, 为了充分了解细胞色素 P450 氧化致癌亚硝胺的代谢途径、DNA 加和下游代谢产物, 已经花费了大量的精力。这些知识在个人癌症风险评估中具有潜在的应用价值4。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56312/56312fig1.jpg"> 
图 1: 一般和建议的亚硝胺代谢。 亚硝胺 (1) 被 P450s 氧化为α-hydroxynitrosamines (2), 自发分解为 alkyldiazohydroxides (3)。这些化合物可以与 dna 结合形成 dna 加。假设2被 P450s 进一步氧化为 nitrosamides 4。这些可以直接绑定到 dna, 形成新的 dna 加或水解到3 , 形成已知的 dna 加。r1和 r2表示任何烷基组。<a href="//ecsource.jove.com/files/ftp_upload/56312/56312fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
虽然α-hydroxynitrosamine 假说得到了大量数据的坚实支持, 但也存在一些不一致之处。主要的一个是α-hydroxynitrosamines 的短半衰期5,6。众所周知, 这些化合物是在内质网膜和后来的烷基化核 DNA 中产生的。鉴于他们的生命的几秒钟, 这是令人费解的这些中间体如何生存所需的旅行, 虽然胞。一个假说是, α-hydroxynitrosamines 的一部分被 processively 氧化为 nitrosamides7,8, 在比较9中相当稳定。这可能会发生通过保留α-hydroxynitrosamines 在细胞色素 P450 活性部位。这种类型的氧化的先例已经看到与尼古丁10, 酒精的11, 和简单的 alkylnitrosamines12,13。另外, nitrosamides 是直接作用的致癌物质2,3。根据它们的反应性9, 这些化合物被认为产生的 dna 加与α-hydroxynitrosamines 和新的, 未经探索的 dna 加 (图 1) 的结果相同。因此, 这一假说不仅解释了通过胞的运输, 也说明了 DNA 损伤产物的形成。
本文介绍了一种用于评价亚硝胺的细胞色素 P450-mediated 转化为 nitrosamides 的体外的通用协议。先前报告的′-nitrosonornicotine (NNN) 转换为′-nitrosonorcotinine (NNC) 的细胞色素 P450 2A6 被展示为示例14。该协议在广泛的基质酶系统的应用将有助于确定 nitrosamides 在整体硝代谢中的重要性。

<table>
	<tbody>
		<tr>
			<td>Norcotinine</td>
			<td>AKoS GmbH (Steinen, Germany)</td>
			<td>CAS 17708-87-1, AKoS AK0S006278969</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>695092</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Anhydride</td>
			<td>Sigma-Aldrich</td>
			<td>242845</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Acetate</td>
			<td>Sigma-Aldrich</td>
			<td>431311</td>
			<td></td>
		</tr>
		<tr>
			<td>Barium Hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>433373</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Chloroform</td>
			<td>Sigma-Aldrich</td>
			<td>151823</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC Acetonitrile</td>
			<td>Sigma-Aldrich</td>
			<td>34998</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>M7506</td>
			<td></td>
		</tr>
		<tr>
			<td>Methylene Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>34856</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Nitrite</td>
			<td>Sigma-Aldrich</td>
			<td>237213</td>
			<td></td>
		</tr>
		<tr>
			<td>ViVid CYP2A6 Blue Screening Kit</td>
			<td>Life Technologies</td>
			<td>PV6140</td>
			<td></td>
		</tr>
		<tr>
			<td>Zinc Sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>221376</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 mL tubes</td>
			<td>Fisher</td>
			<td>AB0533</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mL round bottom flask</td>
			<td>Sigma-Aldrich</td>
			<td>Z510424</td>
			<td></td>
		</tr>
		<tr>
			<td>125 mL Erlenmeyer flask</td>
			<td>Sigma-Aldrich</td>
			<td>CLS4980125</td>
			<td></td>
		</tr>
		<tr>
			<td>125 mL Separatory Funnel</td>
			<td>Sigma-Aldrich</td>
			<td>Z261017</td>
			<td></td>
		</tr>
		<tr>
			<td>25 mL round bottom flask</td>
			<td>Sigma-Aldrich</td>
			<td>Z278262</td>
			<td></td>
		</tr>
		<tr>
			<td>500 MHz NMR Spectrometer</td>
			<td>Bruker</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Allegra X-22R Centrifuge</td>
			<td>Beckman-Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LC vials</td>
			<td>ChromTech</td>
			<td>CTC&#8211;0957&#8211;BOND</td>
			<td></td>
		</tr>
		<tr>
			<td>LTQ Orbitrap Velos</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic Stir bar</td>
			<td>Sigma-Aldrich</td>
			<td>Z127035</td>
			<td></td>
		</tr>
		<tr>
			<td>NMR tube</td>
			<td>Sigma-Aldrich</td>
			<td>Z274682</td>
			<td></td>
		</tr>
		<tr>
			<td>P1000, P200, and P10 pipettes</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rotary evaporator</td>
			<td>Sigma-Aldrich</td>
			<td>Z691410</td>
			<td></td>
		</tr>
		<tr>
			<td>RSLCnano UPLC system</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Shaking Water Bath</td>
			<td>Fisher</td>
			<td>FSSWB15</td>
			<td></td>
		</tr>
		<tr>
			<td>Stir plate</td>
			<td>Sigma-Aldrich</td>
			<td>CLS6795420</td>
			<td></td>
		</tr>
		<tr>
			<td>PicoFrit Column</td>
			<td>New Objective</td>
			<td>PF3607515N5</td>
			<td></td>
		</tr>
		<tr>
			<td>Luna C18, 5 um</td>
			<td>Phenomenex</td>
			<td>535913-1</td>
			<td></td>
		</tr>
	</tbody>
</table>

a general method for detecting nitrosamide formation in the in vitro metabolism of nitrosamines by cytochrome p450s

N-亚硝胺是一种公认的环境致癌物质, 需要细胞色素 P450 氧化来表现活性。代谢活化机制包括自发分解为 DNA 烷剂的α-hydroxynitrosamines 的形成。DNA 损伤的积累和由此产生的突变最终会导致癌症。新的证据表明, α-hydroxynitrosamines 可以进一步氧化到 nitrosamides processively 细胞色素 P450s。因为 nitrosamides 一般比α-hydroxynitrosamines 更稳定, 也可以烷基化 DNA, nitrosamides 可能在癌变中起到作用。在本报告中, 我们描述了一个一般的协议, 评估 nitrosamide 生产从体外细胞色素 P450-catalyzed 代谢亚硝胺。该协议采用了一般的方法合成相关的 nitrosamides 和一个体外细胞色素 P450 代谢测定的液相色谱-nanospray 电离-高分辨率串联质谱检测。此方法在示例研究中检测到′-nitrosonorcotinine 作为′nitrosonornicotine 的次要代谢物。该方法具有较高的灵敏度和选择性, 是由于质量检测准确。该方法在多种硝细胞色素 P450 系统中的应用将有助于确定这种转化的普遍性。由于细胞色素 P450s 的多态性和活动性的变化, 更好地了解 nitrosamide 的形成有助于个别癌症风险评估。

基于白色et al的工作。19, norcotinine 是硝化 NNC 清洁和高产 (80-92%), 以产生一个标准的体外实验。一个成功的反应的结构证据获得了从光谱分析包括1H 核磁共振, 13C 核磁共振, 舒适, 和 HSQC (支持信息) 与人力资源证实了父质量 [M + h]+在 5 ppm 的理论值 (图 2)。MS2的碎片 NNC 被用来确定最丰富的?...

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A General Method for Detecting Nitrosamide Formation in the In Vitro Metabolism of Nitrosamines by Cytochrome P450s

细胞色素 P450s 致癌亚硝胺的α-羟基化是公认的代谢途径, 产生 DNA 损伤的中间体, 导致突变。然而, 新的数据表明进一步的氧化 nitrosamides 可能发生。我们描述了一个一般的方法, 检测 nitrosamides 产生的体外细胞色素 P450-catalyzed 代谢的亚硝胺。

用细胞色素 P450s 检测亚硝胺的体外Nitrosamide 形成的一般方法

N-nitrosamines are a well-established group of environmental carcinogens, which require cytochrome P450 oxidation to exhibit activity. The accepted ...

Research

JoVE Journal

Cancer Research

A General Method for Detecting Nitrosamide Formation in the In Vitro Metabolism of Nitrosamines by Cytochrome P450s

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