Characterization and repair of DNA damage induced by the carcinogen benzo(a)pyrene
Abstract
The metabolism and subsequent binding of benzo(a)pyrene (BaP) to DNA represent the initial steps in the induction of cancer by BaP. To understand the mechanism of BaP's carcinogenicity, it is essential to understand its pathway of metabolic activation, secondary damage that may result from its metabolism, and the persistence of the BaP-DNA adducts. The nucleic acid adducts formed when Syrian Hamster embryo (SHE) cell cultures were treated with BaP or BaP-7,8-diol differed dramatically between RNA and DNA. DNA reacted mainly with anti-7,8-dihydro-7,8-dihydroxy-9,10-epoxide (BaPDE) whereas RNA reacted with the same amount of anti-BaPDE and three-fold more syn-BaPDE. SHE cell cultures treated with the (+)-BaP-7,8-dihydrodiol form only syn-BaPDE, indicating activation to BaPDE occurs exclusively by the cytochrome P-450 enzymes. The oxidative damage to cellular DNA caused by treatment with BaP was examined in cultures of SHE and human mammary carcinoma MCF-7 cells. BaP treatment in the absence light caused no increase in oxidative DNA damage in these cells but BaP treatment plus exposure to white fluorescent light caused large increases in the amount of 8-OH-dGuo present in the DNA. The increases in cellular 8-OH-dGuo content was dependent on both BaP dose and length of light exposure. An assay capable of measuring the persistence and repair of BaP-DNA adducts in individual genes in cells was developed. Second harmonic laser irradiation from a Nd:YAG laser (355 nm) was used to specifically cleave cellular DNA adducted with BaP at the site of the BaP-DNA adduct. The presence of the intact H-ras fragment was assayed by probing agarose gels of genomic DNA. These techniques were used to measure the presence and the persistence of BaP-DNA adducts in the Ha-ras gene in C3H10T1/2 cell cultures. The results add to the overall understanding of the role of nucleic acid damage and repair in the mechanism of carcinogenesis by BaP and other polycyclic aromatic hydrocarbons.
Degree
Ph.D.
Advisors
Baird, Purdue University.
Subject Area
Biochemistry|Molecular biology
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