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.

Cross-reactivity and conformational multiplicity of an anti-polycyclic aromatic hydrocarbon mAb.

Cross-reactivity and multispecific functionality of antibodies play a central role in the immune system. The Ab's promiscuity is attributed to structural flexibility and conformational multiplicity of its binding sites governed by the rearrangement of hydrogen bonding centers. However, antibodies whose recognition and binding rely on less directional hydrophobic interactions might follow different interaction pathways. We investigated interaction of anti-polycyclic aromatic hydrocarbon mAb with two biologically important cross-reactants, pyrene and benzo(a)pyrene. Complex formation was characterized by means of low-temperature laser-induced fluorescence spectroscopy in both low- and high-resolution fluorescence line-narrowing (FLN) modes. It is shown that the FLN spectroscopy can identify various haptens cross-reacted with an Ab, as well as simultaneously differentiate between free and immunocomplexed haptens. In addition, our results suggest an interesting case of an Ab binding a particular cross-reactant by adopting two distinct conformations of its binding sites. The existence of the multiple conformations for anti-polycyclic aromatic hydrocarbon mAb that are trapped at low temperature can be rationalized through the existing models for Ab binding. Finally, as revealed by FLN spectra of immunocomplexed chromophores, pi-pi interactions, rather than hydrogen bonding, play the central role in complex formation.

Identification and quantitation of benzo[a]pyrene-derived DNA adducts formed at low adduction level in mice lung tissue.

The two major metabolic pathways of benzo[a]pyrene (BP) that lead to DNA lesions are monooxygenation that results in diolepoxides (BPDE) and one-electron oxidation that yields a BP radical cation. These pathways result in formation of stable and depurinating DNA adducts, respectively. Most in vivo animal studies with BP, however, have employed dosage/DNA adduct levels several orders of magnitude higher than the DNA damage level expected from environmentally relevant exposures. Presented are results of experiments in which A/J strain mice were intraperitoneally exposed to 50-microg/g doses of BP. It is shown that non-line-narrowed fluorescence and fluorescence line-narrowing spectroscopies possess the selectivity and sensitivity to distinguish between helix-external, base-stacked, and intercalated conformations of DNA-BPDE adducts formed in lung tissue. Concentrations measured by 32P postlabeling 2 and 3 days after intraperitoneal injection were 420-430 and 600-830 amol BPDE-type adducts per microg DNA. The external and base-stacked conformations are attributed mainly to (+)-trans-anti-BPDE-N2dG and the intercalated conformations to (+)-cis-anti adducts. A stable adduct derived from 9-OH-BP-4,5-epoxide was also detected at a concentration about a factor of 10 lower than the above concentrations. The DNA supernatants were analyzed for the presence of depurinating BP-derived adducts by capillary electrophoresis laser-induced fluorescence and high-performance liquid chromatography mass spectrometry.

Supercoiled DNA promotes formation of intercalated cis-N2-deoxyguanine adducts and base-stacked trans-N2-deoxyguanine adducts by (+)-7R,8S-dihydrodiol-9S,10R-epoxy-7,8,9,10-tetra- hydrobenzo[a]pyrene.

The highly reactive and mutagenic benzo[a]pyrene metabolite, (+)-7R,8S-dihydroxy-9S,10R-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE), forms predominantly N2-deoxyguanine DNA adducts in two stereoisomeric configurations (cis and trans). In previous in vitro assays using oligonucleotide substrates site specifically modified with cis- and trans-BPDE adducts, the nucleotide excision repair (NER) systems of eukaryotes and prokaryotes incise cis-BPDE adducts more efficiently than trans-BPDE adducts [Hess, et al. (1997) Mol. Cell Biol 17, 7069; Zou, et al. (2001) Biochemistry 40, 2923). We investigated the influence of DNA secondary structure on stereospecificity of BPDE adduct formation, and incision of BPDE adducts by the prokaryotic UvrABC NER endonuclease was examined. BPDE adducts formed at low density on supercoiled plasmids were incised 6-7-fold better by the thermoresistant Bacillus caldotenaxUvrABC than were BPDE adducts formed on linear DNA. Linearizing supercoiled plasmid DNAs after BPDE adduct formation did not diminish incision efficiency. These results suggested that configuration and/or conformation of adducts formed on linear and supercoiled DNAs differed. This hypothesis was confirmed by low temperature fluorescence spectroscopy of adducted supercoiled and linear DNAs. Spectroscopic results indicated that intercalated cis-BPDE adducts as well as base-stacked trans-BPDE adducts formed more abundantly in supercoiled DNA than in linear DNA. A higher cis to trans adduct ratio in supercoiled DNA was confirmed by high resolution [32P]postlabeling analyses. These results demonstrate that DNA secondary structure influences both configuration and conformation of BPDE adducts formed at low density (approximately 1 adduct/kbp) and suggests that the ratio of cis- to trans-BPDE adducts and amount of base-stacked trans adducts formed under physiological exposure conditions may be higher than inferred from high dose experiments.

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 pi-electron-containing solvent molecules) recorded under the same conditions. The antigens used were the DNA adduct 7-(benzo[a]pyren-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 pi 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 pi 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 pi-pi interactions play an important role in the binding.

Novel biosensor chip for simultaneous detection of DNA-carcinogen adducts with low-temperature fluorescence.

A monoclonal antibody (MAb)-gold biosensor chip with low-temperature laser-induced fluorescence detection for analysis of DNA-carcinogen adducts is described. Optimization of the detection limit, dynamic range, and biosensing applicability of the MAb-gold biosensor chip was achieved by: (1) using dithiobis(succinimidyl propionate (DSP)) as a protein linker and (2) employing recombinant protein A to provide oriented immobilization of the MAbs. The use of DSP, which has a short methylene chain length, led to faster protein binding kinetics and higher protein surface density than a longer dithiobis(succinimidyl undecanoate) (DSU) linker. The incorporation of recombinant protein A increased the distance between the oriented MAb-bound analytes and the gold surface. The increased distance minimized fluorescence quenching, resulting in about a 10-fold increase in the fluorescence signal in comparison with a chip without protein A. The improved chip architecture was used to demonstrate that biosensing of two structurally similar benzo[a]pyrene (BP)-derived DNA adducts, BP-6-N7Gua and BP-diolepoxide-10-N2dG, bound to two specific MAbs immobilized from a mixture at the same address on the chip, is feasible. These mutagenic adducts are formed by one-electron oxidation and monooxygenation pathways, and are depurinating and stable DNA adducts, respectively. It is shown that the DNA adducts can be easily identified at the same address using time-resolved, low-temperature laser-based fluorescence spectroscopy. The current limit of detection is in the low femtomole range. These results indicate that a single biosensor chip consisting of a Au/DSP/protein A/MAb nano-assembly, with analyte-specific MAbs and low-temperature fluorescence detection should be suitable for simultaneous detection and quantitation of the above adducts, as well as the luminescent antigens for which selective MAbs exist.

Spectroscopic characterization of the 4-hydroxy catechol estrogen quinones-derived GSH and N-acetylated Cys conjugates.

Estrogens, including the natural hormones estrone (E(1)) and estradiol (E(2)), are thought to be involved in tumor induction. Specifically, catechol estrogen quinones (CEQs) derived from the catechol estrogens 4-hydroxyestrone (4-OHE(1)) and 4-hydroxyestradiol (4-OHE(2)) react with DNA and form DNA adducts (Cavalieri, E. L., et al. (1997) Proc. Natl Acad. Sci. U.S.A. 94, 10037). CEQs are also conjugated with GSH, a reaction that prevents damage to DNA, providing biomarkers of exposure to CEQs. Current detection limits for these analytes by HPLC with multichannel electrochemical detection are in the picomole range (Devanesan, P., et al. (2001) Carcinogenesis 22, 489). To improve the detection limit of CEQ-derived conjugates, spectrophotometric monitoring was investigated. Fluorescence and/or phosphorescence spectra of the 4-OHE(1), 4-OHE(2), Cys, N-acetylcysteine (NAcCys), 4-OHE(1)-2-SG, and 4-OHE(2)-2-SG conjugates and their decomposition products 4-OHE(1)-2-NAcCys and 4-OHE(2)-2-NAcCys were obtained at 300 and 77 K. It is shown that (i) 4-OHE(1)- and 4-OHE(2)-derived SG and NAcCys conjugates are weakly fluorescent at 300 K (with the emission maximum at 332 nm) but strongly phosphorescent at 77 K; (ii) Cys and NAcCys exhibit fluorescence and phosphorescence only at 77 K; and (iii) 4-OHE(1) and 4-OHE(2) are weakly fluorescent at 300 and 77 K and not phosphorescent. The phosphorescence spectra of SG and NAcCys conjugates are characterized by a weak origin band at approximately 383 nm and two intense vibronic bands at 407 and 425 nm. After they are cooled from 300 to 77 K, the total luminescence intensity of SG and NAcCys conjugates increases by a factor of approximately 150 predominantly due to phosphorescence enhancement. Theoretical calculations revealed, in agreement with the experimental data, that the lowest singlet (S(1)) and triplet (T(1)) states of 4-OHE(2)-2-NAcCys are of n,pi* and pi,pi* character, respectively, leading to a large intersystem crossing yield and strong phosphorescence. The limit of detection (LOD) for CEQ-derived conjugates, based on phosphorescence measurements, is in the low femtomole range. The concentration LOD is approximately 10(-9) M. Therefore, we propose that capillary electrophoresis interfaced with low temperature phosphorescence detection can be used to test for human exposure to CEQs by analyzing urine.