HEALTH EFFECTS OF TOXIC SUBSTANCES

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。环境中化学品的重要性是一个重要的国家卫生问题。加州大学伯克利分校有毒物质项目的一个重点是了解危险废物场所造成的健康风险，这需要了解这些场所的化学物质如何被动物和人类吸收、分布、代谢和排泄（ADME）。我们的研究旨在测量人体暴露于苯的ADME。环境中的苯浓度很低，ADME难以用LC-MS等传统方法测量。我们的目标是通过开发一种将14c标记的物种共价连接到苯修饰的生物分子（如DNA）上的方法来解决这个问题。这种“后标签”将允许在不暴露于高浓度毒物的情况下，通过AMS测量由苯代谢物在人体中引起的DNA损伤。我们开发了这样一种实验，使用一种称为2-氨基-1-甲基-6-苯基咪唑[4,5b]吡啶（PhIP）的模型化合物，PhIP是一种啮齿动物致癌物，其代谢产物可以与DNA结合。选择该模型是因为其众所周知的ADME和低挥发性，以防止实验室被放射性碳标记的苯污染。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¿-二脱氧胸腺嘧啶三磷酸（ddTTP）进行了原理验证研究。该酶催化放射性标记胸腺嘧啶向PhIP内合寡核苷酸的转移。意义如果建立合适的酶消化条件，该方法可推广到苯- 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.