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Benzo(a)pyrene:DNA Adduct Formation in Early-Passage Wistar Rat Embryo Cell Cultures: Evidence for Multiple Pathways of Activation of Benzo(a)pyrene¹

Department of Medicinal Chemistry and Pharmacognosy, School of Pharmacy and Pharmacal Sciences, Purdue University, West Lafayette, Indiana 47907

The benzo(a)pyrene (BaP):DNA adducts formed in cells are present at very low levels and are usually identified by reverse-phase high-performance liquid chromatography of tritium labeled BaP:deoxyribonucleoside adducts with known standards. To improve the identification of the BaP:DNA adducts formed, acid hydrolysis techniques were used to convert the BaP:deoxyribonucleoside adducts formed in Wistar rat embryo cell cultures to BaP:purine adducts and BaP:tetraols. Early passage Wistar rat embryo cell cultures were exposed to [³H]BaP. The BaP:deoxyribonucleoside adducts were isolated by immobilized boronate chromatography and reverse-phase high-performance liquid chromatography. Three adducts (MS1, MS2, MS3) bound to the immobilized boronate column indicating that they contained cis-vicinal hydroxyl groups, a configuration which would result from reaction of 7ß,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydroBaP (anti-BaPDE) with DNA. MS2 resulted from reaction of (+)-anti-BaPDE with deoxyguanosine (dGuo), for it cochromatographed with a [¹⁴C]-(+)-anti-BaPDE:dGuo marker at the deoxyribonucleoside level and after hydrolysis of MS2 and the marker to 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydroBaP (BaPDE):guanine and BaPDE:tetraol. MS1, an adduct that eluted in the same region as a (-)-anti-BaPDE:dGuo marker, was not formed by reaction of anti-BaPDE with DNA. Exposure of (-)-anti-BaPDE:dGuo to 0.1 N HCI for 24 h at 37°C resulted in cleavage of the glycosidic bond to give an enantiomer that cochromatographed with the (+)-anti-BaPDE:dGuo hydrolysis product. Hydrolysis of MS1 under the same conditions yielded a product that eluted earlier than the hydrolysis product of anti-BaPDE:dGuo. Hydrolysis of MS1 at 80°C under conditions which produce tetraols from BaPDE:deoxyribonucleoside adducts resulted in the formation of a product which did not elute with either 7ß,8-dihydroxy-9ß,10ß-epoxy-7,8,9,10-tetrahydro-BaP (syn-BaPDE) or anti-BaPDE tetraols. MS1 was also not present in cells exposed to BaP-7,8-diol or 3-hydroxyBaP. These results demonstrated that MS1 is formed by a different mechanism of activation than a simple bay-region diol-epoxide. MS3 and its hydrolysis products had chromatographic properties identical to those of r-7,c-9,c-10,t-8-tetrahydroxy-7,8,9,10-tetrahydroBaP, a tetraol formed from syn-BaPDE. MS3 appears to result from spontaneous breakdown of a syn-BaPDE:DNA adduct to give a tetraol that contains cis-vicinal hydroxyls in the 9 and 10 positions and therefore binds to an immobilized boronate column. Thus, of the three adducts that bound to the immobilized boronate column, only one was formed from anti-BaPDE. Three adducts were detected that did not contain cis-vicinal hydroxyl groups: M1; M2; and M3. M1 and M2 were formed from (±)-syn-BaPDE, as their tetraol hydrolysis products cochromatographed with tetraols formed from (±)-syn-BaPDE. The isolation of individual BaP:DNA adducts followed by acid hydrolysis resulted in improved identification of the BaP metabolite(s) responsible for the formation of these adducts and demonstrated the presence of multiple pathways of activation of BaP to DNA-binding metabolites in rat embryo cell cultures.

¹ This investigation was supported by Grants CA-28825 and CA-40228 from the National Cancer Institute, Department of Health and Human Services.

² Recipient of a David Ross Research Fellowship from Purdue University.

³ To whom requests for reprints should be addressed.

Received 8/19/85. Accepted 10/28/85.