ELECTROCATALYTIC STUDIES OF TOXIC POLLUTANT ACTIVATION

描述（由申请人提供）：我们的环境受到有机污染物的污染，造成严重的公众健康问题。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，肝脏中细胞色素P450 （cyt P450）酶代谢的污染物或药物可以破坏遗传物质（即DNA）。这类化学物质包括苯乙烯、苯并[a]芘、萘胺和许多其他化学物质。一些化学物质会提高细胞细胞p450和其他代谢酶的水平，增加对重要生物分子的损害风险。体外生物传感器可以快速测量污染物或药物的代谢物对DNA的破坏能力，这将是预测新型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，可能会导致治疗污染物引起的疾病的新方法，并改善我们公民的未来健康。

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.