Electrocatalytic Studies of Toxic Pollutant Activation

描述(申请人提供)：有机污染物污染我们的环境，导致严重的公共健康问题。这项提案涉及污染物进入人体后的生物激活的主要毒性途径。细胞色素P450和其他代谢酶形成的反应性代谢产物会破坏遗传物质和蛋白质。例子包括食品、空气和水中的一系列常见化学物质。我们正在开发新的高通量设备，以快速识别损害DNA的代谢物，以揭示遗传毒性的化学途径。在下一个项目期间，我们将把我们的设备扩展到器官特异性遗传毒性和DNA氧化，并检测肿瘤抑制基因损伤，以帮助预测癌症靶器官。该项目将产生有价值的新工具，以帮助预测新的有机化学品在早期开发阶段的遗传毒性，揭示生物检测无法避免的遗传毒性的化学途径。以这种方式发现的遗传毒性途径应该可以缓解污染物引起的疾病，并最终改善公众健康。在上一个资助期，我们开发了新的生物分析方法，以代谢酶和DNA的超薄分层膜为特色。这些检测解决了无细胞溶液中代谢物相关遗传毒性的化学和动力学问题，从而补充了毒性生物检测。首先，在DNA/酶薄膜中进行代谢生物激活，然后测量产生的DNA损伤。新的阵列评估化学路径和代谢物-DNA反应的速度，酶的特异性，抑制，以及有机污染物和药物的物种间毒性差异。确定新化学品的这些参数对个人安全至关重要。我们最先进的设备是高通量微流控阵列，用于测试化学品的反应代谢物筛选，以及96孔格式的生物胶体反应器，用于为LC-MS/MS生成样品，提供与遗传毒性相关的DNA加合物结构和形成速率。下一个资助期的计划旨在极大地提高遗传毒性的特异性和选择性。 通过引入具有代表性的器官特异性酶，并结合代谢物驱动的DNA氧化的测量来预测我们的方法。此外，我们将结合生物反应器方法和LC-MS/MS测序来检测P53抑癌基因的代谢物密码子损伤模式，以预测可能的癌症靶器官。具体目标概述：(1)开发微流控阵列来测量DNA氧化和一般DNA损伤，用已知有毒化学物质进行测试，并用LC-MS/MS进行验证。(2)使用来自肝脏、肺、肠和肾脏的酶来评估微流控阵列和LC-MS/MS方法，以筛选器官特异性遗传毒性测试化合物。(3)将DNA/酶生物胶体反应与LC-MS/MS测序相结合，以确定P53抑癌基因上代谢物反应的特定密码子，并使用P53数据库对结果进行分析以预测可能的肿瘤靶器官。(4)建立用于监测器官特异性DNA加合物形成和氧化的全球微流控阵列，并用LC-MS/MS研究进行验证。

DESCRIPTION (provided by applicant): Contamination of our environment with organic pollutants causes serious public health problems. This proposal addresses major toxicity pathways involving bioactivation of pollutants after they enter the body. Reactive metabolites formed by cytochrome P450s and other metabolic enzymes damage genetic material and proteins. Examples include a range of common chemicals in food, air and water. We are developing novel high throughput devices to rapidly identify metabolites that damage DNA to reveal chemical pathways in genotoxicity. In the next project period, we will extend our devices to organ specific genotoxicity and DNA oxidation, and detect tumor suppressor gene damage to help predict cancer target organs. The project will generate valuable new tools to help predict genotoxicity of new organic chemicals at early development stages, revealing chemical pathways of genotoxicity not obviated by bioassays. Genotoxicity pathways discovered in this way should moderate pollutant-caused disease, and ultimately improve public health. We developed new bioanalytical approaches featuring ultrathin, layered films of metabolic enzymes and DNA in the last funding period. These assays address the chemistry and dynamics of metabolite-related genotoxicity in cell-free solutions, and thus complement toxicity bioassays. First, metabolic bioactivation is done in DNA/enzyme films, then resulting DNA damage is measured. Novel arrays assess chemical pathways and rates of metabolite-DNA reactions, enzyme specificities, inhibition, and interspecies toxicity differences for organic pollutants and drugs. Establishing these parameters for new chemicals is critical for individual safety. Our most advanced devices are high throughput microfluidic arrays for reactive metabolite screening of test chemicals, and biocolloid reactors in 96-well formats to generate samples for LC-MS/MS that provide DNA adduct structures and formation rates correlated with genotoxicity. Plans for the next funding period are aimed at greatly increasing specificity and selectivity of genotoxicity prediction of our approaches by introducing representative organ specific enzymes, and incorporating measurements of metabolite-driven DNA oxidation. In addition, we will combine the bioreactor approach with LC-MS/MS sequencing to detect metabolite codon damage patterns to p53 tumor suppressor gene to predict possible cancer target organs. Summary of Specific Aims: (1) Develop microfluidic arrays to measure DNA oxidation and general DNA damage, test with known toxic chemicals, and validate with LC-MS/MS. (2) Evaluate microfluidic arrays and LC-MS/MS approaches using enzymes from liver, lung, intestine, and kidney to screen test compounds for organ specific genotoxicity. (3) Couple DNA/enzyme biocolloid reactors with LC-MS/MS sequencing to identify specific codons on p53 tumor suppressor gene where metabolites react, and analyze results using the p53 database to predict possible cancer target organs. (4) Develop a global microfluidic array to monitor organ specific DNA adduct formation and oxidation, and validate with LC-MS/MS studies.