FLNS has also been used to deter mine five BP-nucleoside adducts syn thesized by one-electron oxidation of B P in the presence of guanosine, deoxyguanosine, and deoxyadenosine (13). The results showed that a major depurination adduct from the binding of BP to DNA in rat liver nuclei is 7(benzo [a] pyren-6-yl)guanine (N7Gua). Only 20 pg of the adduct was required, an amount that can be obtained from one rat, whereas analysis by the com mon method of collisionally activated decomposition MS would require sacri ficing many rats.
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FLNS analysis of in vivo DNA adducts FLNS also can be used for in vivo stud ies. FLN spectra of (+)-cmf}-BPDEDNA and syrc-BPDE-DNA (Figure 7) have been used to identify the major diol epoxide adduct of fish liver DNA from English sole exposed to BP in lab oratory-controlled experiments (22, 23). Of particular interest in this study
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Wavelength (nm)
Figure 7. Comparison of the FLN spec trum of fish liver DNA (a) extracted from fish exposed to BP, (b) with stan dard FLN spectra of syn-BPDE-DNA, and (c) (+)-a/7f/-BPDE-DNA. The modification levels are ~ 1 adduct in 107 bases, ~ 1 adduct in 107 bases (determined radiometrically), and ~ 1 adduct in ~200 bases, re spectively.
Figure 6. Comparison of the vibrationally excited FLN spectra for three differ ent DNA adducts. All spectra were obtained in the standard gly/H 2 0 glass at 4.2 Κ with an excitation wavelength of 371.6 nm. (a) Mixture of BPT and DNA at a con centration of 10" 5 M, (b) (+)-anf/-BPDE-DNA with 0.5% bases modified, (c) (-)-anfi-BPDE-DNA with 1.5% bases modified, and (d) syn-BPDEDNA at a concentration of ~ 1 adduct in 107 bases. The peaks are labeled with their corre sponding excited-state vibrational frequencies (in cm - 1 ).
was whether the expected N-2-deoxyguanosine (N-2-dG) adduct from (+)αηίί-BPDE is formed. Despite the very low damage level of the DNA (~1 ad duct in 107 bases as determined by an independent method), the FLN spec trum exhibits a good signal-to-noise ra tio. As expected, a major adduct is de rived from BPDE, establishing the im portance of the monooxygenation mechanism. However, comparison of the fish DNA spectrum with the FLN spectra of syn-BPOE and (+)-antiBPDE-DNA shows that the adduct is not derived from (+)-onii-BPDE but from syn-BPDE. Although the major adduct is de rived from syn-BPDE, weaker contri butions from (+)-anti- and (-)-antiBPDE-DNA adducts cannot be ex cluded. Both anti- and syn-BPDE are strongly mutagenic in bacterial and mammalian cells, but αηίί'-BPDE gen erally shows greater activity than synBPDE in most tests (24). Thus the high proportion of syn-BPDE-DNA ad ducts in English sole exposed to the high dosage of BP used in these experi-
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ANALYTICAL CHEMISTRY, VOL. 61 NO. 18, SEPTEMBER 15, 1989 · 1029 A
