Probing the interaction of benzo[a]pyrene adducts and metabolites with monoclonal antibodies using fluorescence line-narrowing spectroscopy

A new approach for studying antibody-antigen interactions of DNA adducts and metabolites of polycyclic aromatic hydrocarbons (PAHs) is demonstrated in which fluorescence line-narrowing spectroscopy (FLNS) is used. It is based on the fact that in an FLN spectrum the relative intensities of the line-narrowed bands (that correspond to the excited-state vibrations) are, in general, strongly dependent on the local environment of the fluorophore. Information on the nature of the interactions can be obtained by comparing the FLN spectra of the antigen-antibody complexes to the spectra of the antigen in different types of solvents (H-bonding, aprotic, and π-electron-containing solvent molecules) recorded under the same conditions. The antigens used were the DNA adduct 7-(benzo[alpyren-6-yl)guanine (BP-6-N7Gua) and the metabolite (+)-trans-anti-7,8,9,10-benzo[a]pyrenetetrol (BP-tetrol) of benzo[a]pyrene; two monoclonal antibodies (MAbs) have been developed to selectively bind these compounds. It is shown that, for BP-tetrol, H-bonding solvents have a pronounced effect on the FLN spectra. The presence of π electrons in the solvent molecules results in relatively small but still significant changes in the spectra. When BP-tetrol is bound to its MAb, however, neither of these effects is observed; its spectrum is very similar to the one obtained with an aprotic solvent, methylcyclohexane. Therefore, we can conclude that this MAb has an internal binding site in which the interaction with BP-tetrol is of a hydrophobic character. For BP-6-N7Gua, however, there is a strong effect of the presence of π electrons in the solvent molecules. The FLN spectrum of this antigen bound to its MAb is very similar to its spectrum in acetone, indicating that π-π interactions play an important role in the binding

Identification and quantification of the depurinating DNA adducts formed in mouse skin treated with dibenzo[a,l]pyrene (DB[a,l]P) or its metabolites and in rat mammary gland treated with DB[a,l]P

Dibenzo[a,l]pyrene (DB[a,l]P) is the most potent carcinogenic polycyclic aromatic hydrocarbon and has been identified in the environment. Comparative tumorigenicity studies in mouse skin and rat mammary gland indicate that DB[a,l]P is slightly more potent than DB[a,l]P-11,12-dihydrodiol and much more potent than (±)-syn-DB[a,l]P-11,12-dihydrodiol-13,14-epoxide {(±)-syn-DB[a,l]PDE} and (±)-anti-DB[a,l]PDE. We report here the identification and quantification of the depurinating adducts formed in mouse skin treated with DB[a,l]P, DB[a,l]P-11,12-dihydrodiol, (±)-syn-DB[a,l] PDE, or (±)-anti-DB[a,l]PDE and rat mammary gland treated with DB [a,l] P. The biologically formed adducts were compared with standard adducts by their retention times on HPLC and their spectra obtained by fluorescence line-narrowing spectroscopy at low temperature. In mouse skin treated with DB[a,l]P, depurinating adducts comprised 99% of the total adducts. Most of the depurinating adducts were formed by one-electron oxidation, with 63% at Ade and 12% at Gua. The remainder were formed by the diol epoxide, with 18% at Ade and 6% at Gua. When mouse skin was treated with DB[a,l]P-11,12-dihydrodiol, depurinating adducts comprised 80% of the total, and the predominant one was with Ade (69%). Treatment of skin with (±)-syn-DB[a,l]PDE resulted in 32% depurinating adducts, primarily at Ade (25%), whereas treatment with (±)-anti-DB[a,l]PDE produced 97% stable adducts. The formation of depurinating adducts following treatment of rat mammary gland with DB[a,l]P resulted in approximately 98% depurinating adducts, with the major adducts formed by one-electron oxidation. Only one depurinating diol epoxide adduct was formed. Tumorigenicity, mutations, and DNA adduct data suggest that depurinating Ade adducts play a major role in the initiation of tumors by DB[a,l]P. © 2005 American Chemical Society