Formation and repair of benzo[a]pyrene--DNA adducts in cultured hamster tracheal epithelium determined by 32P-postlabeling analysis and unscheduled DNA synthesis

Hamster tracheal organ cultures were used to investigate therelationship between DNA adduct formation measured directlyby the ³²P-postlabeling assay, and the DNA damage measured indirectlyby the unscheduled DNA synthesis (UDS) assay. Hamster tracheaswere treated with three concentrations of benzoa]pyrene (Ba]P)for 2 days. Postlabeling and UDS assays were also carried outa few days after removal of the Ba]P. Furthermore, the typesof Ba]P—DNA adducts formed in the in vitro organ culturewere qualitatively compared with those formed in vivo afterintratracheal intubation of Ba]P attached to Fe₂O₃ particles.In vivo only one adduct was detected by ³²P-postlabeling. Thisadduct co-chromatographed with the trans-addition produce ofdG and (+)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzoa]pyrene(BPDE). In vitro, a clear Ba]P-DNA adduct pattern was alsofound with the ³²P-postlabeling assay. Four different adductswere found. The main adduct spot migrated to the same positionon the thin-layer chromatogram as the in vivo adduct. Ba]P-DNAadduct formation was both time-and dose-dependent. During thefirst day after removal of Ba]P the adduct levels still increased,thereafter they decreased at all Ba]P concentrations. A time-and dose-dependent increase in UDS was observed in the trachealepithelial cells treated with Ba]P in vitro. After removalof the Ba]P, UDS decreased immediately, in contrast to theformation of DNA adducts. The results of the present study showthat Ba]P induces time- and dose-dependently both DNA adductsand UDS in hamster tracheal organ culture. Moreover, the mainDNA adduct formed in vitro, dG-(+)-anti-BPDE, was the same asthat found in vivo.