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: 8190847
• 负责人: Gunnar Boysen
• 金额: $ 22.06万
• 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 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8190847
• 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. PUBLIC HEALTH RELEVANCE: 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. The causal link between exposure to genotoxic mixtures and the development of adverse health effects is believed to be the binding of carcinogens or their metabolites to DNA and proteins forming so called adducts. Technical limitations, however, have prevented the assessment of a multiple adducts simultaneously. Therefore, we propose to develop a method to make it possible to determine the presence and number of multiple adducts simultaneously in order to obtain accurate information needed to assess the risks to human health posed by exposure to mixtures.

描述（由申请人提供）：人类不断暴露于各种混合物，如烟草烟雾，汽车尾气和其他环境污染物，含有数千种化合物，包括许多已知的致癌物质。 反应性代谢物与DNA和蛋白质共价结合，形成稳定的加合物，被认为是接触和致癌之间的因果关系。DNA和蛋白质加合物是内部有效剂量的公认生物标志物，是基于科学的风险评估的组成部分。然而，由于技术上的限制，无法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人或选择少数加合物。这些研究对个别致癌物的代谢产生了有价值的见解。 遗憾的是，这些数据不足以提供评估接触混合物所构成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灵敏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来定量分析广谱反应性化合物或其代谢物。我们最近建立了一种免疫亲和液相色谱-串联质谱法（LC-MS/MS），包括胰蛋白酶水解和样品富集的加合物特异性免疫亲和色谱（IAC），然后通过LC-MS/MS定量。我们建议在此重新设计IAC富集程序，使同时定量的N-末端缬氨酸加合物的广谱。我们基于这样的假设，即烷基化的N-末端肽的广谱可以用针对靶肽的C-末端特异性地产生的抗体来富集。我们的初步研究表明，这种新的设计是适合生物监测。为了检验这一假设，我们将（目的1）证明所提出的多加合物监测方法的原理证明，该方法使用烷基化肽标准品和球蛋白，在体外用已知形成N-末端缬氨酸加合物的烷基化剂处理;和（目的2）建立多加合物监测方法用于体内生物测定的适用性。使用暴露于各种烷化剂的小鼠和大鼠的珠蛋白进行监测，烷化剂的浓度低于吸烟期间和职业环境中的浓度。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。 这种新的重新设计，使内部剂量的几种致癌物质的同时确定将有可能（a）更好地了解在混合物中的单个化合物的行为，和（B）产生更全面的接触数据需要准确的风险评估接触混合物。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，并适应于调查烷基化在网站以外的N-末端缬氨酸，提供了一个通用的分析方法来了解反应剂如何与大分子相互作用，表现出其不良影响。 公共卫生相关性：人类经常暴露在各种混合物中，如烟草烟雾，汽车尾气和其他环境污染物，含有数千种化合物，包括许多已知的致癌物质。人们认为，接触遗传毒性混合物与产生有害健康影响之间的因果关系是致癌物或其代谢物与DNA和蛋白质结合，形成所谓的加合物。然而，由于技术限制，无法同时评估多种加合物。因此，我们建议开发一种方法，以便能够同时确定多种加合物的存在和数量，从而获得评估接触混合物对人类健康构成的风险所需的准确信息。