HEALTH EFFECTS OF TOXIC SUBSTANCES

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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.