A novel approach for quantitation of N-terminal valine adducts

N-末端缬氨酸加合物定量的新方法

• 批准号: 8322567
• 负责人: Gunnar Boysen
• 金额: $ 18.44万
• 依托单位: UNIV OF ARKANSAS FOR MED SCIS
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-19 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8322567
• 关键词: Adverse effects; Alkylating Agents; Alkylation; Antibodies; Automobile Driving; Behavior; Binding; Biological Markers; Biological Monitoring; Breathing; Butadiene; Carcinogens; DNA; Data; Development; Dose; Environmental Pollutants; Exhibits; Exposure to; Funding; Globin; Goals; Health; Hemoglobin; Human; Hydrolysis; In Vitro; Individual; Investigation; Link; Liquid Chromatography; Mesylates; Metabolism; Methodology; Methods; Modeling; Modification; Monitor; Mus; N-terminal; Needs Assessment; Occupational; Passive Smoking; Peptides; Procedures; Process; Proteins; Rattus; Reporting; Risk; Risk Assessment; Sampling; Science; Site; Smoking; Source; Technology; Testing; Tobacco smoke; Toxic effect; Translational Research; Trypsin; Validation; Valine; adduct; base; carcinogenesis; carcinogenicity; design; exhaust; experience; immunoaffinity chromatography; in vivo; insight; interest; macromolecule; novel; novel strategies; prevent; styrene oxide; tandem mass spectrometry

DESCRIPTION (provided by applicant): Humans are constantly exposed to various mixtures, such as tobacco smoke, auto exhaust, and other environmental pollutants, containing several thousand compounds, including many known carcinogens. Covalent binding of reactive metabolites to DNA and proteins with the formation of stable adducts is believed to be the causal link between exposure and carcinogenesis. DNA and protein adducts are well established biomarkers for the internal effective dose and are an integral part of science-based risk assessment. Technical limitations, however, have prevented comprehensive assessment of a board spectrum of different adducts simultaneously. Consequently, most studies have focused on determination of abundant individual or a select few adducts. These studies have produced valuable insights into metabolism of individual carcinogens. Unfortunately they are insufficient in providing accurate and comprehensive data needed for assessment of the risk posed by exposure to mixtures. Thus, our long-term goal is to overcome this limitation by developing a sensitive and specific method for quantitative profiling of a broad spectrum of reactive compounds or their metabolites using hemoglobin adducts as surrogate biomarkers. We recently established an immunoaffinity liquid chromatography-tandem mass spectrometry method (LC-MS/MS) consisting of trypsin hydrolysis and sample enrichment by an adduct-specific immunoaffinity chromatography (IAC) prior to quantitation by LC-MS/MS. We propose herein to redesign the IAC enrichment procedure to enable simultaneous quantitation of a broad spectrum of N-terminal valine adducts. We base this redesign on the hypothesis that a broad spectrum of alkylated N-terminal peptides can be enriched with the use of antibodies raised specifically against the C-terminus of the target peptide. Our preliminary studies suggest that this new design is suitable for bio-monitoring. To test this hypothesis, we will (Aim 1) demonstrate proof-of-principle of the proposed multi-adduct-monitoring method with alkylated peptide standards and globin treated in vitro with alkylating agents known to form N-terminal valine adducts; and (Aim 2) establish the suitability of the multi-adduct-monitoring method for in vivo bio-monitoring using globin from mice and rats exposed to various alkylating agents at concentrations lower than experienced during smoking and at levels of occupational settings. We propose to establish this methodology for mice, rats, and humans to enable translational research. This novel redesign enabling determination of the internal doses of several carcinogens simultaneously will make it possible to (a) better understand the behavior of individual compounds in mixtures, and (b) generate more comprehensive exposure data needed for accurate risk assessment of exposure to mixtures. The proposed methodology can easily be extended to include additional adducts of interest and adapted to investigate alkylation at sites other than the N-terminal valine, providing a general analysis approach to understanding how reactive agents interact with macromolecules to exhibit their adverse effects.

描述（由申请人提供）：人类经常接触各种混合物，例如烟草烟雾、汽车尾气和其他环境污染物，其中含有数千种化合物，其中包括许多已知的致癌物质。 反应性代谢物与 DNA 和蛋白质的共价结合并形成稳定的加合物被认为是暴露与致癌之间的因果关系。 DNA 和蛋白质加合物是公认的内部有效剂量生物标志物，也是基于科学的风险评估的一个组成部分。然而，技术限制阻碍了同时对不同加合物的板谱进行全面评估。因此，大多数研究都集中在确定丰富的个体或选定的少数加合物。这些研究对个体致癌物的代谢产生了宝贵的见解。 不幸的是，它们不足以提供评估接触混合物所造成的风险所需的准确和全面的数据。因此，我们的长期目标是通过开发一种敏感且特异的方法来克服这一限制，使用血红蛋白加合物作为替代生物标志物对广谱反应性化合物或其代谢物进行定量分析。我们最近建立了一种免疫亲和液相色谱-串联质谱方法 (LC-MS/MS)，包括胰蛋白酶水解和在通过 LC-MS/MS 定量之前通过加合物特异性免疫亲和色谱 (IAC) 富集样品。我们在此建议重新设计 IAC 富集程序，以实现广谱 N 端缬氨酸加合物的同时定量。我们的重新设计基于以下假设：通过使用专门针对目标肽 C 端的抗体，可以富集广谱烷基化 N 端肽。我们的初步研究表明这种新设计适用于生物监测。为了检验这一假设，我们将（目标 1）证明所提出的多加合物监测方法的原理证明，其中使用烷基化肽标准品和在体外用已知形成 N 末端缬氨酸加合物的烷化剂处理的球蛋白； （目标 2）建立多加合物监测方法对体内生物监测的适用性，使用来自暴露于浓度低于吸烟期间和职业环境水平的各种烷化剂的小鼠和大鼠的球蛋白。我们建议为小鼠、大鼠和人类建立这种方法，以实现转化研究。 这种新颖的重新设计能够同时确定多种致癌物的内部剂量，从而能够（a）更好地了解混合物中单个化合物的行为，以及（b）生成准确评估混合物暴露风险所需的更全面的暴露数据。所提出的方法可以很容易地扩展到包括其他感兴趣的加合物，并适用于研究除 N 端缬氨酸之外的位点的烷基化，提供了一种通用分析方法来了解反应剂如何与大分子相互作用以表现出其不利影响。

项目成果

Characterization of population variability of 1,3-butadiene derived protein adducts in humans and mice.

• DOI: 10.1016/j.yrtph.2022.105171
• 发表时间: 2022-07
• 期刊: REGULATORY TOXICOLOGY AND PHARMACOLOGY
• 影响因子: 3.4
• 作者: Boysen, Gunnar;Rusyn, Ivan;Chiu, Weihsueh A.;Wright, Fred A.
• 通讯作者: Wright, Fred A.

Metabolism of R- and S-warfarin by CYP2C19 into four hydroxywarfarins.

R-和S-华法林通过CYP2C19代谢成四种羟基华法林。

• DOI: 10.2174/1872312811206030002
• 发表时间: 2012
• 期刊: Drug metabolism letters
• 作者: Kim,So-Young;Kang,Ji-Yeon;Hartman,JessicaH;Park,Sun-Ha;Jones,DrewR;Yun,Chul-Ho;Boysen,Gunnar;Miller,GroverP
• 通讯作者: Miller,GroverP