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Characterization of cyclopenta[cd]pyrene-DNA adducts in vitro and in mice in vivo

Exposure to chemical carcinogens is believed to be an important etiological factor in human cancer. Carcinogen-DNA adducts are considered to represent the initiating events leading to cancer. This dissertation was undertaken to determine the DNA adduct formation by cyclopenta[cd]pyrene (CPP), a ubiquitous environmental carcinogen, in vitro and in mice. Calf thymus DNA was reacted in vitro with CPP 3,4-epoxide (CPPE) or with its metabolites, 3,4-dihydroCPP-3,4-diol (CPP-3,4-diol) and 4-hydroxy-3,4dihydroCPP (4-OH-DCPP), activated with sulfotransferase. The results demonstrated that the two major CPPE-derived adducts were formed by guanine, while minor adducts were formed by adenine and cytosine. Specifically, the major dG adduct formed in DNA was identified as cis-3-(deoxyguanosin-N2-yl)-4-hydroxy-3,4dihydroCPP (3-dG-4-OH-DCPP) and the minor dG adduct seems likely to be its trans isomer. Adenosine adducts were presumably formed by aralkylation of the N6 position since they co-chromatographed with regio-specifically synthesized standards. Sulfotransferase activation of trans-CPP-3,4-diol yielded two adducts that were identical to the products resulting from the reaction of CPPE with DNA, while cis-CPP-3,4-diol gave very low covalent binding. Adducts formed by sulfotransferase activation of 4-OH-DCPP were identical to the diastereomeric products generated from the reaction of synthetic 4-NaO3 S-O-DCPP with dG. These results indicate that guanine is predominantly the site of CPP adduct formation in DNA, regardless of the identity of the reactive metabolite, and that the 4-hydroxy-3dG adducts can arise by reaction of DNA with either CPPE or sulfotransferaseactivated trans-CPP-3,4-diol. In CPP-treated CD-1 mice, four and six peaks were detected in the DNA digests of liver and lung samples, respectively, by HPLC. Cryogenic fluorescence spectroscopic analysis indicated that all peaks were derived from CPPE. These peaks were also identified by their chromatographic similarity to synthetic CPP metabolites and adduct standards formed in vitro. The results suggest that adducts detected in liver DNA digests are trans-3-dG-4-OH-DCPP, two diastereomers of cis3-dG-4-OH-DCPP, and trans-3-deoxyadenosinyl-4-hydroxy-3,4-dihydroCPP (3-dA4-OH-DCPP). Adducts detected in lung samples are probably trans-3-dG-4-OHDCPP, two diastereomers of cis-3-dG-4-OH-DCPP, cis-CPP-3,4-diol, cis-3-dA-4OH-DCPP, and 4-OH-DCPP. The detection of CPP-3,4-diol and 4-OH-DCPP suggests that the formation of unstable adducts by CPP. Furthermore, both CPPE and sulfotransferase-activated trans-CPP-3,4-diol could be the major ultimate carcinogenic metabolites of CPP, while sulfotransferase-activated 4-OH-DCPP could be a minor one. Thesis Supervisor: Dr. Steven R. Tannenbaum Professor of Chemistry and Toxicology