Chem. Res. Toxicol. 1989,2, 29-34
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Fluorescence Line Narrowing Spectrometric Analysis of Benzo[ a Ipyrene-DNA Adducts Formed by One-Electron Oxidation D. Zamzow, R. Jankowiak, R. S. Cooper, and G. J. Small* Ames Laboratory-USDOE
and Department of Chemistry, Iowa State University, Ames, Iowa 50011
S. R. Tibbels, P. Cremonesi, P. Devanesan, E. G. Rogan, and E. L. Cavalieri Eppley Institute for Research in Cancer and University of Nebraska Medical Center, Omaha, Nebraska 68105 Received June 30, 1988
Fluorescence line narrowing (FLN) was demonstrated for five benzo[a] pyrene (BP)-nucleoside adducts synthesized by one-electron oxidation of B P in the presence of guanosine, deoxyguanosine, and deoxyadenosine. The standard FLN spectra were used to prove that a major depurination adduct from the binding of B P to DNA in rat liver nuclei is 7-(benzo[a]pyren-6y1)guanine (N7Gua). The structural characterization was performed with only 20 pg of the adduct. Metabolic activation of B P by one-electron oxidation in the horseradish peroxidase catalyzed reaction of B P with DNA (in vitro) was also investigated. The major adduct identified was 8-(benzo[a]pyren-6-yl)guanine(C8Gua).
Introduction For many carcinogenic compounds, such as the polycyclic aromatic hydrocarbons (PAH), metabolic activation to electrophilic intermediates is a necessary condition for the production of damage in cellular macromolecules such as DNA or RNA. Damage resulting from chemical adduction of a genotoxic species with DNA is generally accepted as the first step in tumorigenesis. When metabolic activation is required, several metabolic pathways will often exist. The current view for PAH is that metabolic activation o c c w by two main pathways: monooxygenation to yield bay-region diol epoxides (1-3) and oqe-electron oxidation to produce radical cations (4, 5). The radical cation mechanism is expected to be operative for PAH with relative low ionization potentials, e.g., benzo[a]pyrene(BP) and 7,12-dimethylbenz[a]anthracene. Peroxidases, including horseradish peroxidase (HRP) and prostaglandin H synthase, catalyze one-electron oxidation (6, 7),whereas cytochrome P-450 catalyzes both one-electron oxidation and monooxygenation (8, 9). One-electron oxidation is involved not only in the formation of metabolites (10) but also in binding of BP to DNA as has recently been demonstrated for HRP (11)and cytochrome P-450 (12). Detailed studies of the HRP-catalyzed binding of BP with DNA (11) included the structural elucidation of several adducts Synthesized by anodic oxidation of BP in the presence of deoxyguanosine (dG) or guanosine (G). Adducts were identified as BP bound at C6 to C8 of guanine (Gua), dG, and G and N7 of Gua (see Figure 1). In the BP radical cation the charge is mainly localized at C6 (4). The adducts were characterized by lH NMR, fast atom bombardment MS, and collisionally activated decomposition MS. With these adducts as standards, the HRPcatalyzed products, C8dG, C8Gua, and N7Gua, could be identified by HPLC as major adducts in the DNA digest and the supernatant obtained by precipitation of the DNA
(11). Similarly, the N7Gua adduct has been isolated from cytochrome P-450 mediated binding of BP to DNA in rat liver microsomes (12). We have been developing fluorescence line narrowing (FLN) spectrometry as a high sensitivity and resolution technique for the determination and characterization of PAH (13),PAH metabolites (14-15), and macromolecular DNA and globin adducts (14,16). A limit of detection by FLN for the often-studied macromolecular adduct BPDE-DNA (BPDE = benzo[a]pyrenediol epoxide) of 3 modified bases in lo8 (20 pg of DNA) has been achieved at a spectral resolution of -10 cm-I (16). This corresponds to about a spectral resolution of -10 cm-I (16). This corresponds to about a femtomole of the metabolite bound to the DNA. This detection limit is comparable to that obtained with ELISA (17). Thus far, however, FLN spectrometry has only been applied to PAH metabolites and macromolecular DNA adducts associated with the monooxygenation pathway. In this paper we demonstrate first that FLN is operative for the aforementioned model nucleoside adducts derived from BP by one-electron oxiqation. Given the high sensitivity of FLN spectrometry [-3 orders of magnitude higher than collisionally activated decomposition MS (ll)], this demonstration suggests that FLN should be a useful tool for structural elucidation or confirmation of such adducts in HPLC fractions. This suggestion is borne out here by experiments in which rat liver nuclei were incubated with BP. An FLN analysis of an HPLC fraction believed to be that of the depurinated N7Gua adduct is presented which confirms the N7Gua assignment. The FLN analysis was performed with only 20 pg of the adduct. Results from an FLN analysis of DNA from HRP-catalyzed binding of BP are also presented. The major adduct observed is consistent with binding of the BP radical cation at its 6-position to guanine.
0893-228~/89/2702-0029$01.50/0 0 1989 American Chemical Society
30 Chem. Res. Toxicol., Vol. 2, No. 1, 1989
Princlples of FLN and Hole Burning Spectroscoples The underlying principles of FLNS have been described in detail previously (16) but, for completeness, will be reviewed here. In a disordered host matrix an analyte molecule populates a very large number of sites that are energetically inequivalent due to differing local fields. The resulting dispersion of the electronic transition energy manifests itself as an inhomogeneous broadening, riA,of the vibronic transitions. Each vibronic band (typically -300-500 cm-’) is a convolution of a very large number of individual site absorptions possessing a homogeneous line width rhom. Selection of a narrow isochromat of the absorption profile with a laser of which AWL
