ELECTROCATALYTIC STUDIES OF TOXIC POLLUTANT ACTIVATION

描述（由申请人提供）：有机污染物对我们的环境造成严重的公共卫生问题。人类疾病的一个主要途径涉及有机污染物进入人体后的毒性生物活化。通过这种方式，污染物或药物代谢的细胞色素P450（细胞色素P450）酶在肝脏中可以损害遗传物质（即。例如，DNA）。这类化学品包括苯乙烯、苯并[a]芘、萘胺和许多其他化学品。一些化学物质会提高细胞色素P450和其他代谢酶的水平，增加重要生物分子受损的风险。体外生物传感器可以快速测量污染物或药物的代谢产物对DNA的损伤能力，这将是预测新有机化学品对人体毒性的有价值的筛选工具。这些设备还可用于提供酶特异性和毒性激活途径的知识，可能导致治疗污染物引起的疾病和改善我们公民未来健康的新方法。 该项目的广泛的长期目标是开发酶/DNA传感器，生物活化化学品和测量随后的DNA损伤作为体外毒性筛选新的化学品，并作为阐明生物活化的机制细节的工具。原型生物传感器已经在该项目中开发，其中酶催化代谢物的形成，随后的电化学测定估计DNA损伤。传感器采用过氧化物或电子注入来驱动酶催化的生物活化。然后通过催化或探针结合电化学测定来检测对DNA的损伤。下一个赠款期的具体目标包括：（1）验证传感器作为一系列化学品的毒性筛选，（2）开发用于伏安法和电极驱动光发射的传感器阵列，以描绘人类细胞色素P450对生物活化的活性，（3）开发膜以探索涉及细胞色素P450和谷胱甘肽-S-转移酶的连续代谢过程（另一种重要的代谢酶），和（4）应用传感器研究抑制DNA损伤的膳食抗氧化剂。将使用色谱法、质谱法和电泳法测量特定DNA损伤，以从分子上确认传感器结果。将通过将传感器的有毒化学品结果与微生物和动物致突变性和致癌性数据库相关联来辅助确认。该项目还将产生简单的设备，以建立人类细胞色素P450的底物特异性，其个体间分布可能对个体对污染物的敏感性很重要，并用于检查顺序代谢过程。总之，这项工作将为新化学品的毒性筛选提供新的体外工具，并有助于扩大污染引起的疾病的分子知识基础，作为未来治疗方案的基础。

DESCRIPTION (provided by applicant): Contamination of our environment with organic pollutants causes serious public health problems. A major pathway to human disease involves toxic bioactivation of organic pollutants after they enter the body. In this way, pollutants or drugs metabolized by cytochrome P450 (cyt P450) enzymes in the liver can damage genetic material (i. e., DNA). Chemicals in this class include styrene, benzo[a]pyrene, napthylamines and many others. Some chemicals elevate levels of cyt P450s and other metabolic enzymes, increasing the risk of damage to important biomolecules. In vitro biosensors that rapidly measure the ability of metabolites of pollutants or drugs to damage DNA would be valuable screening tools to predict the human toxicity of new organic chemicals. These devices can also be used to provide knowledge of enzyme specificities and pathways of toxic activation, possibly leading to new ways to treat pollutant caused diseases and improve the future health of our citizens. The broad long-term goals of this project are to develop enzyme/DNA sensors that bioactivate chemicals and measure the subsequent DNA damage as in-vitro toxicity screens for new chemicals, and as tools for elucidating mechanistic details of bioactivation. Prototype biosensors have been developed already in this project in which enzymes catalyze formation of metabolites, and subsequent electrochemical assays estimate DNA damage. The sensors employ peroxide or electron injection to drive enzyme-catalyzed bioactivation. Damage to DNA is then detected by catalytic or probe binding electrochemical assays. Specific aims for the next grant period include: (1) validating the sensors as toxicity screens for a range of chemicals, (2) developing sensor arrays for voltammetry and electrode-driven light emission to profile the activity of human cyt P450s towards bioactivation, (3) developing films to explore sequential metabolic processes involving cyt P450s and glutathione-S-transferase (another important metabolic enzyme), and (4) applying the sensors to study inhibition of DNA damage by dietary antioxidants. Chromatography, mass spectrometry and electrophoresis will be used to measure specific DNA damage for molecular confirmation of sensor results. Validation will be assisted by correlating sensor results for toxic chemicals with databases of microbiological and animal mutagenicity and carcinogenicity. The project will also result in simple devices to establish substrate specificities of human cyt P450s, the inter-individual distribution of which may be important in individual susceptibility to pollutants, and for examining sequential metabolic processes. In summary, this work will provide novel in vitro tools for toxicity screening of new chemicals, and help expand the molecular knowledge base on pollutant-caused disease as a basis for future therapeutic protocols.