Detection and characterization of polycyclic aromatic hydrocarbon-DNA adducts by sulfur 35-phosphorothiolate labeling and high-performance liquid chromatography
Abstract
The formation of polycyclic aromatic hydrocarbon (PAH)-DNA adducts is a critical step in the induction of tumors by PAH. Treatment with both the co-carcinogen benzo(e)pyrene (BeP) and the carcinogen benzo(a)pyrene (BaP) increases the number of tumors induced in mouse skin. Studies of the co-carcinogenic mechanism of BeP demonstrated that BeP treatment increased the level of BaP-bound to DNA in mouse epidermis. To avoid the limitations of tritiated PAH for detection of PAH-DNA adducts in vivo, two ($\sp{35}$S) phosphorothioate postlabeling procedures for PAH-DNA adduct detection were developed. PAH-adducted nucleotides were 5$\sp\prime$-thiophosphorylated by T4 polynucleotide kinase and adenosine-5$\sp\prime$-O-(3- ($\sp{35}$S) thiotriphosphate) to yield $\sp{35}$S-labeled PAH-adducted nucleotides. High-performance liquid chromatography (HPLC) analysis of the postlabeled BaP-DNA adducts in BaP-treated hamster embryo cells demonstrated the formation of both (+)syn- (epoxide and benzylic hydroxy cis) -BaP-7,8-dihydrodiol-9,10-epoxide (BaPDE) -deoxyguanosine and (+)anti-BaPDE (epoxide and benzylic hydroxy trans) -deoxyguanosine adducts. An immobilized boronate chromatography (IBC) procedure was developed for use with the $\sp{35}$S-postlabeling technique. In this procedure, the $\sp{35}$S-postlabeled anti-BaPDE-DNA adducts were completely separated from the $\sp{35}$S-postlabeled syn-BaPDE-DNA adducts. 7,12-Dimethylbenz (a) anthracene-3,4-dihydrodiol-1,2-epoxide- (DMBADE) -DNA adducts were characterized by $\sp{35}$S-postlabeling and adduct analysis by both HPLC and IBC. Three major adducts, anti-DMBADE-deoxyguanosine, anti-DMBADE-deoxyadenosine, and syn-DMBADE-deoxyadenosine, were detected in DMBA-treated hamster embryo cell cultures. In a human mammary carcinoma cell line MCF-7-mediated V79 cell mutation assay, DMBA-3,4-dihydrodiol was highly mutagenic and the number of mutants was proportional to the level of DMBA-DNA adducts. DNA from cells used in the mutation assay contained mainly anti-DMBADE-DNA adducts, indicating that anti-DMBADE is highly mutagenic to mammalian cells. The $\sp{35}$S-postlabeling technique was also used for the analysis of DNA adducts formed by isomeric dihydrodiol-epoxides of benzo(c)phenanthrene. Studies of the DNA adducts formed in cell cultures treated with the potent carcinogen dibenzo (a,l) pyrene (DB (a,l) P) demonstrated that DB (a,l) P can be metabolically activated to a DNA-binding metabolite that contains cis-vicinal dihydroxyls, probably an anti-DB (a,l) P-dihydrodiol-epoxide. This thesis describes new analytical procedures that greatly improve the separation and characterization of PAH-DNA adducts by postlabeling analysis. These techniques will greatly facilitate further studies of the mechanism of PAH carcinogenesis.
Degree
Ph.D.
Advisors
Baird, Purdue University.
Subject Area
Biochemistry
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