Chem. Res. Toxicol. 1992,5, zyxwvu 765-772 765 Evidence of Involvement of Multiple Sites of Metabolism in the in Vivo Covalent Binding of Dibenzo[a,h]pyrene to DNA G. A. Marsch, R. Jankowiak, and G. J. zyx Small' Ames Laboratory-USDOE and Department of Chemistry, Iowa State University, Ames, Iowa 50011 N. C. Hughes and D. H. Phillips Haddow Laboratories, Institute of Cancer Research, Cotswold Road, Sutton, Surrey, SM2 zyx 5NG U.K. Received April 27, 1992 The in vivo formation of dibenzo[a,hlpyrene-DNA adducts in mouse skin was assessed by laser-excited fluorescence spectroscopy at 77 and 4.2 K. Two adducts were identified with fluorescence origin bands at -383.5 and 407.2 nm, and these were shown to possess pyrene and benzo[a] pyrene (B[alP) chromophores, respectively. Both DNA-bound chromophores displayed considerable electron-phonon coupling and likely assume a highly base-stacked or quasi- intercalated configuration within DNA duplexes. The presence of B[a]P and pyrene aromatic systems indicates that two-electron or monooxygenation metabolism occurred on either the a or h benzo moieties (which are equivalent) in the former case, and on both these rings in the latter case. The presence of two adduct species agrees with 32P-postlabeling analysis of the DNA, which showed the presence of two major adducts in both thin-layer and high-performance liquid chromatographic separations. Introduction Polycyclicaromatic hydrocarbons(PAHs)lare products of the incomplete combustion of carbonaceous material zyxwvut (I). It is now generally accepted that PAHs exert their biological effects of mutagenicity and carcinogenicity via metabolic activation by cytochrome P-450 and epoxide hydrolaseto electrophilic speciesthat react covalentlywith nucleophilicsites in cells to form adducts zyxwvu (2,3). A wealth of evidence supports the view that DNA is the critical cellular target nucleophile (4,5). The hexacyclic aromatic hydrocarbon dibenzo[a,hl- pyrene (DB[a,h]P) is an environmental pollutant (1) which has high mutagenic and tumorigenic activities in many bacterial and mammalian cell lines (6-11). Since DB- [a,h]P is a symmetrical molecule with two equivalent bay regions (at rings a and h, see Figure 4), metabolism is anticipated not only at the a or h ring, but also at both rings simultaneously. Quantum mechanical calculations showing AE1JJB of 0.845 for DB[a,hlP (9, IO), one of the highest for any hydrocarbon whose diol epoxide has been studied (12), predict high chemical reactivity of the bay- region diol epoxides. The nature of the DNA reactive species, however, remains to be verified. Therefore, it is of interest to establish the structure of stable DB[a,hlP- DNA adducts formed by in vivo systems. The 32P-postlabelingassay,a highly sensitivetechnique that enables the detection of DNA adducts in only microgram quantities of DNA (13,14), was used recently to examine the covalent binding of DB[a,hlP to DNA in 1 Abbreviations: B[alP, benzo[alpyrene;BPDE, anti-benzo[alpyrene 7,B-dihydrodiol 9,lO-epoxide; DB[a,hlP, dibenzo[a,hlpyrene;DDBP, benzo[a] pyrene tram-7,8-dihydrodio1; FLN,fluorescenceline narrowing; FWHM, full width at half-maximum; HPLC, high-performanceliquid chromatography; PAH, polycyclic aromatic hydrocarbon; PEI, poly- (ethylenimine); THF,tetrahydrofuran; ZPL,zero-phonon line: TLC,thin- layer chromatography; SDS, sodium dodecylsulfate; FLNS, fluorescence line narrowingspectroscopy; ESVF, excited-state vibrationalfrequencies. 0893-228~/92/2705-0765$03.00/0 mouse tissues (15). Two adducts were detected in the skin of treated animals, although the nature of these two adducts remains to be characterized. In a series of papers (16-22) we have demonstrated that fluorescence line-narrowed (FLN) spectroscopy can be used for the analysis of DNA damage by PAHs in vivo (19, 20,22), sincethe present limit of detection is ca. 1 adducted base pair per lo8 base pairs in 100 pg of DNA (22). For example, a FLN spectral analysis of in vivo human hemoglobin was used to determine the structure of the major human globin adduct from benzo[alpyrene (B[a]P) (20). FLN spectroscopyis also capable of assisting in the assignment of adduct stereochemistries (21,22). In this paper we provide information on the route of metabolic activation of DB [a,hlP by using fluorescence line narrowing spectroscopy to identify the presence of two distinctly different chromophores in DB[a,hlP- modified DNA from mouse skin, analogous to the two distinct DB [a,hlP-DNA adducts detected by 32P-post- labeling. Experimental Procedures The carcinogen (DB[a,h]Pand radiolabel (J2P) used in these experiments were in liquid form. For both, contamination must be eliminated by wearing impermeable laboratory gloves, laboratory coats,and splash-proofgoggles. Hazardous materials should besequestered from the rest of the laboratory. Radiolabel exposure is kept ALARA ("as low as reasonably achievable") by working with plexiglass shielding and the proper dosimeters (Geiger counters and body dosimeters). Wastes must be placed only in approved containers and disposed of by the proper Environmental Safety and Health personnel. Instrumentation. The fluorescence line-narrowing spec- troscopy system employed in these studies has been described in detail (22). The excitation source was a Lambda Physik FL- 2002 dye laser pumped by a Lambda Physik EMG 102 MSC excimer laser. Fluorescence was detected with a Princeton Instruments IRY-l024/G/R/B intensified blue-enhanced pho- 0 1992 American Chemical Society