HEALTH EFFECTS OF TOXIC SUBSTANCES

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。化学物质在环境中的重要性是一个关键的国民健康问题。加州大学伯克利分校有毒物质计划的重点是了解危险废物场地带来的健康风险，这需要了解这些场地的化学物质是如何从动物和人类体内吸收、分布、代谢和排泄(ADME)的能力。我们的研究旨在测量人类苯暴露的ADME。环境中的苯可能是有毒的，浓度如此之低，以至于ADME很难用LC-MS等传统方法进行测量。我们的目标是通过开发一种方法，将14C标记的物种共价连接到苯修饰的生物分子上，如DNA，从而绕过这个问题。这种“后标记”将允许在不暴露于高浓度毒物的情况下，通过AMS测量由人类苯代谢物造成的DNA损伤。我们使用一种名为2-amino-1-methyl-6-phenylimidazo[4，5b]pyridine(PhIP)的模型化合物开发了这样的检测方法，这是一种啮齿动物致癌物质，其代谢物可以与dna结合。选择该型号是因为其众所周知的ADME和低挥发性，以防止实验室受到放射性碳标记苯的污染。使用一种称为多核苷酸末端转移酶(TNT)的酶和14C标记的2？，3？二脱氧胸苷三磷酸(DdTTP)进行了原理验证研究。该酶催化放射性标记的胸腺嘧啶核苷转移到PhIP加合物寡核苷酸。意义如果开发出合适的酶消化条件，这种方法可以扩展到苯-DNA加合物。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The significance of chemicals in the environment is a critical National Health concern. A focus of the Toxic Substances Program at UC-Berkeley is to understand the health risks posed by hazardous waste sites, which requires the ability to understand how chemicals from those sites are absorbed , distributed, metabolized and excreted (ADME) from animals and humans. Our studies aim to measure ADME of human exposure to benzene. Benzene in the environment can be toxic a such low concentrations that ADME is difficult to measure with conventional methods such as LC-MS. Our goal is to circumvent this problem by developing a method to covalently attach a 14C-labeled species to benzene-modified biological molecules such as DNA. This ¿postlabeling¿ will allow measurement of DNA damaged caused by benzene metabolites in humans by AMS without exposure to high concentrations of toxicants. We developed such an assay using a model compound known as 2-amino-1-methyl-6-phenylimidazo[4,5b]pyridine (PhIP), a rodent carcinogen whose metabolites can bind to DNA. This model was chosen due to its well-known ADME and low volatility in order to prevent contamination of the laboratory by radiocarbon labeled benzene. Proof-of-principle studies were carried out using an enzyme called polynucleotidyl terminal transferase (TnT) and a 14C-labled 2¿,3¿-dideoxythymidine triphosphate (ddTTP). The enzyme catalyzed the transfer of radiolabeled thymidine to PhIP adducted oligonucleotides. SIGNIFICANCE This method can be extended to benzene-DNA adducts if the appropriate enzymes digest conditions are developed.