DNA Adducts as Molecular Biomarkers of Exposure to Benzo ( a ) pyrene by Deirdre Michelle Lawrence

It is believed that the formation of DNA adducts is an essential initial step in carcinogenesis. To better understand this initial step and its relationship to cancer, this study primarily focuses on benzo(a)pyrene (BaP), a highly carcinogenic model polycyclic aromatic hydrocarbon (PAH) and an ubiquitous environmental contaminant. The objective of this dissertation was to identify and quantify carcinogen-DNA adducts as biomarkers of exposure to BaP. The BaP-DNA adduct levels were measured by Adduct Detection by Acylation with Methionine (ADAM), a new postlabelling method that was recently developed by our group. Nucleoside adducts of fluoranthene, 4-aminobiphenyl and 8-hydroxy-2'-deoxyguanosine (8-OHdG) were acylated with 3SS-methionine and detected in femtomolar quantities. This method was adapted for analysis of BaP-DNA adducts and optimized with 3H-BaP using unlabeled methionine. Acylated products of benzo(a)pyrene-deoxyguanosine (BaP-dGuo) from different sources, including 3H-BaP-dGuo isolated from calf thymus DNA modified enzymatically in vitro or by reaction with BPDE, have shown identical RP-HPLC retention times. The 35S content of acylated products was linearly correlated (r = 0.860) with the nucleoside adduct concentration over the range from 20-1000 femtomoles. BaP-DNA adduct levels in random human lung samples were found to range from 240-51,120 adducts per cell. Similarly, paraffin-embedded breast tissues contained BaP-DNA adduct levels from 35-1764 adducts in 10' normal nucleotides (0.05-14.7 femtomole BaP/pg DNA) with an average of 44,000 adducts/cell. Adult CD-1 mice were injected i.p. with [3H]-benzo(a)pyrene ([3H]-BaP) (2mg, 117 mCi/mmol) and sacrificed 24 hr later. Liver DNA was isolated, enzymatically hydrolyzed to nucleosides, purified by immunoaffinity chromatography and analyzed with the ADAM procedure for BaP-DNA adducts. BaP-DNA adduct levels were quantified by both radioactivity (3H in BaP) and acylation with 35S-methionine with the ADAM procedure. An excellent correlation was found between the adduct levels determined independently by 3H and '3S. A newborn mouse lung adenoma assay was used to generate information about BaP-DNA adducts formed in vivo. Newborn CD-1 mice were injected i.p. with a tumorigenic dose of 210 p.g of BaP (0.83mmole) three times over a two week dosing regimen starting at birth. Control mice were given DMSO as placebo. The mice were sacrificed by decapitation at specific time points of 24 hr, 72 hr, 1 wk and 5.5 months. Both target (lung) and nontarget (liver, kidney) tissues were collected. Tumors were induced by this regimen in the male mice. DNA was isolated, enzymatically hydrolyzed to nucleosides, purified and analyzed with the ADAM procedure for BaP-DNA adducts. The RPHPLC profiles of treated mice were compared with their prospective controls. Relationships of adduct levels to tumorigenesis were summarized. Successful acylation conditions were used to analyze benzo(a)pyrene adducts in DNA extracted from samples of tissues of CD-1 mice injected with benzo(a)pyrene and samples of human lung and embedded breast tissues. RP-HPLC profiles of the acylation products of adducts isolated from mouse tissues and human lung samples eluted at the same retention time as that of an authentic BaP-dGuo standard. The postlabelling method combined with a subsequent RP-HPLC profile allows characterization of the carcinogen-DNA adducts present in the tissues. A data base will be generated to analyze DNA as a dosimeter of exposure. This data base will be used to correlate molecular biomarkers, specifically BaP-DNA adducts with tumor frequency. A direct relationship will verify the use for carcinogen-DNA adducts as dosimeters of exposure to genotoxic chemicals and will be imperative in understanding the role carcinogens play in initiating cancer. Thesis Supervisor: Dr. Gerald N. Wogan Title: Professor of Toxicology