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Estrogens, including the natural hormones estrone (E(1)) and estradiol (E(2)), are thought to be involved in tumor induction. Catechol estrogen quinones (CEQ) derived from 4-hydroxyestrone (4-OHE(1)) and 4-hydroxyestradiol (4-OHE(2)) react with DNA and form depurinating N7Gua and N3Ade adducts that might be responsible for tumor initiation (Cavalieri, E. L., et al. (2000) J. Natl. Cancer Inst. Monogr. 27, 75). Current detection limits for the CEQ-derived DNA adducts by high-performance liquid chromatography with multichannel electrochemical detection are in the picomole range. To improve the limit of detection (LOD) for CEQ-derived DNA adducts, spectrophotometric monitoring was investigated. Spectroscopic studies of 4-OHE(1)-1-N3Ade, 4-OHE(1)-1-N7Gua, 4-OHE(2)-1-N3Ade, and 4-OHE(2)-1-N7Gua adduct standards were performed at 77 and 300 K. Upon laser excitation at 257 nm, the 4-OHE(1)- and 4-OHE(2)-derived N7Gua and N3Ade adducts are strongly phosphorescent at T = 77 K. No phosphorescence was observed at 300 K. Both N3Ade and N7Gua adduct types have weak phosphorescence origin bands near 383 and 385 nm, respectively. The corresponding phosphorescence lifetimes are 1.11 +/- 0.05 and 0.37 +/- 0.05 s. The LOD, based on phosphorescence measurements, is in the low femtomole range. The concentration LOD is approximately 10(-9) M, i.e., similar to that recently obtained for CEQ-derived N-acetylcysteine conjugates (Jankowiak, R., et al. (2003) Chem. Res. Toxicol. 16, 304). The LOD in capillary electrophoresis (CE) with field-amplified sample stacking and absorbance detection is about 3 x 10(-8) M. To verify whether CEQ-derived DNA adducts are formed in humans or not, tissue extracts from two breast cancer patients were analyzed by CE interfaced with room temperature absorption and low temperature (laser-excited) phosphorescence spectroscopies. For the first time, formation of CEQ-derived DNA adducts is shown in humans. For example, the level of 4-OHE(1)-1-N3Ade in the breast tissue extract from a patient with breast carcinoma (8.40 +/- 0.05 pmol/g of tissue) is larger by a factor of about 30 than that in the breast tissue sample from a woman without breast cancer (0.25 +/- 0.05 pmol/g of tissue). In contrast, similar amounts of 4-OHE(2)-1-N3Ade were observed in both types of tissue. Although more breast tissue samples from women with and without breast cancer need to be studied, these results suggest that the N3Ade adducts could serve as biomarkers to predict the risk of breast cancer.

Polycyclic aromatic hydrocarbons (PAH) are metabolized to electrophiles that can bind to DNA bases and destabilize the N-glycosyl bond, causing rapid depurination of the adducted bases. Recent studies support depurination of DNA as a mechanism central to the genesis of H-ras mutations in PAH-treated mouse skin. Depurinating adducts account for 71% of all DNA adducts formed in mouse skin treated with benzo[a]pyrene (BP). This study analyzed urine of cigarette smokers, coal smoke-exposed women, and nonexposed controls for the presence and quantities of the depurinated BP-adducted DNA bases, 7-(benzo[a]pyren-6-yl)guanine (BP-6-N7Gua) and 7-(benzo[a]pyren-6-yl)adenine (BP-6-N7Ade). Since these adducted bases originate from reaction of the BP radical cation with double-stranded DNA and not with RNA or denatured DNA, their presence in urine is indicative of DNA damage. Urine samples were fractionated by a combination of SepPak extraction and reverse-phase HPLC, and then analyzed by tandem mass spectrometry and capillary electrophoresis with laser-induced fluorescence. BP-adducted bases were detected in the urine from three of seven cigarette smokers and three of seven women exposed to coal smoke, but were not detected in urine from the 13 control subjects. Concentrations were estimated to be 60-340 and 0.1-0.6 fmol/mg of creatinine equivalent of urine for coal smoke-exposed women (maximum possible BP intake of ca. 23 000 ng/day) and cigarette smokers (BP intake of ca. 800 ng/day), respectively, exhibiting a sensitive response to BP exposures. BP-6-N7Gua was present at ca. 20-300 times the concentration of BP-6-N7Ade in the urine of coal smoke-exposed women, but was not detected in the urine of cigarette smokers. This difference may be due to the remarkably different BP exposures experienced by the two groups of PAH-exposed individuals. These results justify more extensive studies of depurinated BP-adducted DNA bases as potential biomarkers of PAH-associated cancer risk.

A new direct readout methodology for detection and quantitation of fluorescent carcinogen-DNA adducts is described. It combines the binding specificity of an immobilized monoclonal antibody (MAb) with high-resolution, low-temperature fluorescence spectroscopy. The MAb, which is covalently bound to a gold surface via a chemisorbed disulfide coupling agent, binds the adduct of interest in an aqueous sample. Laser-induced fluorescence under nonline narrowing (FNLN) and line-narrowing (FLN) conditions was used to detect (benzo[a]pyren-6-yl)guanine (BP-6-N7Gua) bound to immobilized MAb. At room temperature, the BP-6-N7Gua fluorescence was not detected, most likely because of quenching by the gold surface and/or efficient dynamical quenching. However, fluorescence was observed at room temperature when the surface was covered with a thin layer of glycerol, and possible reasons for the fluorescence enhancement are considered. Lowering of the temperature to 77 K led to nearly an order of magnitude increase in fluorescence intensity. Highly structured FLN spectra obtained at 4.2 K allowed for definitive adduct identification. The potential of this methodology for risk assessments of individuals exposed to polycyclic aromatic hydrocarbons is discussed.

Activation of the moderate carcinogen 6-methylbenzo[a]pyrene (6-CH(3)BP) by one-electron oxidation to form DNA adducts was studied. Iodine oxidation of 6-CH(3)BP in the presence of dGuo produces BP-6-CH(2)-N(2)dGuo, BP-6-CH(2)-N7Gua and a mixture of 6-CH(3)BP-(1&3)-N7Gua, whereas in the presence of Ade the adducts BP-6-CH(2)-N1Ade, BP-6-CH(2)-N3Ade, BP-6-CH(2)-N7Ade and 6-CH(3)BP-(1&3)-N1Ade are obtained. Furthermore, for the first time an aromatic hydrocarbon radical cation afforded an adduct with dThd, the stable adduct BP-6-CH(2)-N3dThd. Formation of these adducts indicates that the 6-CH(3)BP radical cation has charge localized at the 6, 1 and 3 position. When 6-CH(3)BP was activated by horseradish peroxidase in the presence of DNA, two depurinating adducts were identified, BP-6-CH(2)-N7Gua (48%) and 6-CH(3)BP-(1&3)-N7Gua (23%), with 29% unidentified stable adducts. In the binding of 6-CH(3)BP catalyzed by rat liver microsomes, the same two depurinating adducts, BP-6-CH(2)-N7Gua (22%) and 6-CH(3)BP-(1&3)-N7Gua (10%), were identified, with 68% unidentified stable adducts. In 6-CH(3)BP-treated mouse skin, the two depurinating adducts, BP-6-CH(2)-N7Gua and 6-CH(3)BP-(1&3)-N7Gua, were identified. Although quantitation of these two adducts was not possible due to coelution of metabolites on HPLC, they appeared to be the major adducts found in mouse skin. These results show that 6-CH(3)BP forms depurinating adducts only with the guanine base of DNA, both in vitro and in mouse skin. The weaker reactivity of 6-CH(3)BP radical cation vs. BP radical cation could account for the weaker tumor-initiating activity of 6-CH(3)BP in comparison to that of BP.

The benzo[a]pyrene (BP)-derived 7-(benzo[a]pyren-6-yl)guanine (BP-6-N7Gua) depurinating one-electron oxidation adduct was identified in the urine extracts of coal-smoke-exposed humans for the first time. Urine samples were prepared by solid-phase extraction and reversed-phase high-performance liquid chromatography. Subsequently, the BP-6-N7Gua adduct was identified on-line with capillary electrophoresis-- fluorescence line narrowing spectroscopy (CE-FLNS) at 4.2 K. The daily excretion of BP-6-N7Gua in human urine of individuals exposed to coal smoke was approximately 226 pmol per micromol of creatinine. Due to the high level of excretion we propose that BP-6-N7Gua adducts found in urine could serve as effective biomarkers for risk assessment of BP exposure. The results demonstrate that CE-FLNS allows for on-line separation and DNA adducts identification in complex fluid extracts.

Dibenzo[a,l]pyrene (DB[a,l]P) is the most potent carcinogenic polycyclic aromatic hydrocarbon that has been identified in the environment. Earlier studies in our laboratory indicated that more than 80% of the DB[a,l]P-DNA adducts formed in vitro were depurinating adducts and that most of the stable adducts were formed from diol epoxide intermediates. To complete the profile of both stable and depurinating adducts of DB[a,l]P, we have synthesized standard adducts by reacting 3'-dAMP or 3'-dGMP with either (+/-)-anti- or (+/-)-syn-dibenzo[a,l]pyrene 11,12-dihydrodiol 13, 14-epoxide (DB[a,l]PDE). The adducts were separated by HPLC with an ion-pair column and were identified by fluorescence line-narrowing spectroscopy (FLNS). A total of six pairs of stereoisomers along with another stable DB[a,l]PDE-DNA adduct were successfully isolated and identified. Pairs of (+/-)-trans and (+/-)-cis isomers were expected to be formed from the reaction of anti-DB[a,l]PDE with either dAMP or dGMP. While we were able to identify two pairs of stereoisomeric (+/-)-syn-DB[a,l]PDE-dAMP (cis and trans) and two pairs of stereoisomeric (+/-)-anti-DB[a,l]PDE-dAMP (cis and trans) adducts, identification of all the stereoisomers of dGMP adducts proved to be impossible. A pair of (+/-)-syn-trans-DB[a,l]PDE-dGMP adducts, a pair of (+/-)-anti-cis-DB[a,l]PDE-dGMP adducts, and one syn-cis-DB[a,l]PDE-dGMP adduct were conclusively identified by FLNS. These standard adducts will be used to identify the stable DNA adducts formed by DB[a,l]P and DB[a,l]PDE in vitro and in vivo.

(+/-)-anti-Dibenzo[a,l]pyrene-11,12-dihydrodiol 13,14-epoxide {(+/-)-anti-DB[a,l]PDE} was reacted with deoxyguanosine (dG) in dimethylformamide at 100 degrees C for 30 min, and two sets of adducts were isolated: a mixture of (+/-)-anti-cis- & -trans-N(2)dG (43%) and a mixture of (+/-)-anti-cis- & -trans-N7Gua (45%). Both are mixtures of four stereoisomers that cannot be separated by HPLC. Similarly, (+/-)-syn-DB[a,l]PDE was reacted with dG under the same conditions, and (+/-)-syn-cis- & -trans-N(2)dG (38%) and (+/-)-syn-cis- & -trans-N7Gua (59%) were obtained. The structures of the adducts were determined by a combination of NMR and fast atom bombardment mass spectrometry. By reacting (-)-anti-DB[a,l]PDE or (+)-syn-DB[a,l]PDE with dG under the same conditions, however, optically pure N(2)dG and N7Gua isomers were obtained: (-)-anti-cis-N(2)dG (12%), (-)-anti-trans-N(2)dG (17%), (-)-anti-trans-N7Gua (43%), (+)-syn-cis-N(2)dG (7%), (+)-syn-trans-N(2)dG (3%), (+)-syn-cis-N7Gua (36%), and (+)-syn-trans-N7Gua (22%). The structures of the optically pure adducts were assigned by NMR. syn- and anti-DB[a,l]PDE-N(2)dG adducts can be distinguished by fluorescence line-narrowing spectroscopy (FLNS). Moreover, distinction between cis- and trans-stereochemistry of the adducts is also straightforward by FLNS, because the FLN spectra for the four DB[a,l]PDE-N(2)dG adducts, anti-cis, anti-trans, syn-cis, and syn-trans, are spectroscopically unique.

Low-temperature fluorescence spectra and results of conformational studies with trans-syn-, cis-syn-, trans-anti-, and cis-anti-dibenzo[a,l]pyrene diol epoxide (DB[a,l]PDE)-derived deoxyadenosine (dA) adducts are presented and compared with those previously obtained for the stereoisomeric DB[a,l]P tetrols [Jankowiak, R., et al. (1997) Chem. Res. Toxicol. 10, 677-686]. In contrast to DB[a,l]P tetrols, for which only trans isomers exhibited two conformers, all stereoisomeric dA adducts adopt two different conformations with either half-chair or half-boat structures for the cyclohexenyl ring, and an "open"- or "folded"-type configuration between dA and the DB[a,l]P moiety. The major conformations observed for trans-syn-, cis-syn-, and cis-anti-DB[a,l]PDE-14-N(6)dA could be assigned on the basis of the previous calculations for the DB[a,l]P tetrols. The major conformers of the trans-syn- and cis-syn-DB[a, l]PDE-14-N(6)dA adducts exist in conformations I and II, with their fluorescence origin bands at approximately 382 and approximately 389 nm, respectively. In conformation I, the cyclohexenyl ring adopts a half-boat structure with dA in a pseudoaxial position (an open configuration), whereas the cyclohexenyl ring in conformation II adopts a half-chair structure with dA in pseudoequatorial position (a folded configuration). The major conformation of cis-anti-DB[a, l]PDE-14-N(6)dA, with its origin band at approximately 389 nm, was also assigned as a folded-type configuration with a half-chair structure in the cyclohexenyl ring. Molecular mechanics and dynamical simulations were performed for interpretation of the low-temperature fluorescence spectra and (1)H NMR coupling constants observed for the trans-anti-DB[a,l]PDE-14-N(6)dA adduct. The major conformer of this adduct has a half-chair structure in the cyclohexenyl ring, but a deviation from planarity in the fjord region different from that of conformer II of cis-anti-DB[a, l]PDE-N(6)dA. This new structure is labeled as conformer II'. Its (0, 0) fluorescence band is at 388.1 and 388.3 nm in ethanol and glycerol/water glasses, respectively, consistent with the folded-type configuration revealed by the calculations. The fluorescence line-narrowed spectra reveal that the trans-syn-, cis-syn-, trans-anti-, and cis-anti-DB[a,l]PDE-14-N(6)dA adducts can be distinguished. Thus, their spectra should prove useful for identification of DB[a,l]P-DNA adducts formed at low levels in biological samples.

Our laboratory previously reported the identification and quantification of depurinating DNA adducts of dibenzo[a,l]pyrene (DB[a,l]P) in vitro, which comprise about 84% of all the DNA adducts that are formed [Li, K.-M., et al. (1995) Biochemistry 34, 8043-8049]. To determine a complete adduct profile and identify both stable and depurinating DNA adducts, we have developed a relatively simple, nonradioactive method for the identification of stable DNA adducts by combining enzymatic digestion, HPLC, and fluorescence line-narrowing spectroscopy (FLNS) techniques. Calf thymus DNA, bound to either (+/-)-anti- or (+/-)-syn-DB[a,l]PDE or rat liver microsome-activated DB[a,l]P, was first digested to 3'-mononucleotides with micrococcal nuclease and spleen phosphodiesterase. The adducts were then separated by HPLC with an ion-pair column and identified by FLNS by using the spectra of standards for comparison. In reactions with (+/-)-anti-DB[a,l]PDE, three adducts, an anti-cis-DB[a,l]PDE-dGMP, an anti-trans-DB[a, l]PDE-dAMP, and an anti-cis-DB[a,l]PDE-dAMP, were identified by HPLC and FLNS. In reactions with (+/-)-syn-DB[a,l]PDE, a pair of syn-trans-DB[a,l]PDE-dGMP adducts as well as a syn-cis-DB[a, l]PDE-dGMP, a syn-cis-DB[a,l]PDE-dAMP, and a pair of syn-trans-DB[a, l]PDE-dAMP adducts were identified. From the digest of microsome-activated DB[a,l]P-bound DNA, a syn-trans-DB[a,l]PDE-dGMP, an anti-cis-DB[a,l]PDE-dGMP, a syn-trans-DB[a,l]PDE-dAMP, and a syn-cis-DB[a,l]PDE-dAMP adduct were identified. An anti-cis-DB[a, l]PDE-dAMP adduct was identified only by (32)P-postlabeling. A total of five of the stable adducts formed by DB[a,l]P and nine of the stable adducts formed by DB[a,l]PDE in vitro have been identified. These adducts were also correlated to adduct spots in the (32)P-postlabeling method by cochromatography with standards. Approximately 93% of the stable adducts formed in reactions with (+/-)-anti-DB[a,l]PDE, 90% of adducts with (+/-)-syn-DB[a,l]PDE, and 85% of adducts formed with microsome-activated DB[a,l]P have been identified as Gua or Ade adducts. Equal amounts of stable Gua and Ade adducts were observed in the microsome-catalyzed binding of DB[a, l]P to calf thymus DNA, while 1.4 times more Gua adducts than Ade adducts were obtained in reactions with (+/-)-anti- or (+/-)-syn-DB[a,l]PDE.

A capillary electrophoretic method for the separation and on-line identification of closely related analytes using low-temperature fluorescence spectroscopy is reported for the eight diastereomeric deoxyadenosine (dA) adducts derived from dibenzo[a,l]pyrene diol epoxide (DB[a,l]PDE). Electrophoretic separation of stereoisomers was accomplished by application of a mixed surfactant buffer [dioctyl sulfosuccinate (DOSS) and Brij-S], which was below the critical micelle concentration (CMC) due to the high concentration (approximately 25%) of organic solvent. Addition of multiple surfactant additives to the separation buffer provided electrophoretic resolution, which was unattainable under single surfactant conditions. It is shown that the CE-separated analyte zones could be identified on-line via low-temperature (4.2 K) fluorescence non-line narrowing and fluorescence line-narrowing (FLN) spectroscopy. In addition, it was determined that in CE buffer trans-syn-,cis-syn- and cis-anti-DB[a,l]PDE-14-N6dA diastereomeric adducts exist mostly with the -dA and DB[a,l]P moiety in an "open"-type conformation while the trans-anti-DB[a,l]PDE-14-N6dA adducts exist in two different conformations whose relative distribution depends on matrix composition. The above conformations have also been revealed by selective laser excitation. Thus, the low-temperature methodology not only provides fingerprint structure via vibrationally resolved 4.2 K fluorescence spectra for adduct identification, but also provides conformational information on the spatial relationship of the carcinogen and dA moiety. These results, taken together with those for DB[a,l]P-DNA adducts formed in standard glasses and mouse epidermis exposed to DB[a,l]P, support our earlier findings that DB[a,l]P-derived adducts exist in different conformations [Jankowiak et al., Chem. Res. Toxicol. 11 (1998) 674]. Therefore, the combination of the separation power of CE and spectral selectivity of low-temperature fluorescence spectroscopy at NLN and FLN conditions provides a powerful methodology which should prove useful for identification of closely related DNA adducts formed at low levels in biological systems.

The nature of stable DNA adducts derived from the very potent carcinogen dibenzo[a,l]pyrene (DB[a,l]P) in the presence of rat liver microsomes in vitro and in mouse skin in vivo has been studied using 32P-postlabeling and laser-based fluorescence techniques. Analysis of DB[a,l]P-DNA adducts via 32P-postlabeling has been obtained by comparison of the adduct patterns to those obtained from reactions of synthetic (+/-)-anti-, (+)-anti-, (-)-anti-, and (+/-)-syn-DB[a,l]P-11,12-diol 13,14-epoxide (DB[a,l]PDE) with single nucleotides and calf thymus DNA. anti-DB[a,l]PDE-dA adducts derived from the (-)-enantiomer are the major adducts formed in calf thymus DNA and in mouse skin DNA. The ratio of deoxyadenosine to deoxyguanosine modification is approximately 2:1 in mouse skin exposed to DB[a,l]P; activation by rat liver microsomes leads to a similar profile of adducts but with two additional spots. The conformations of DB[a,l]P adducts in native DNA, as well as the possibility of conformation-dependent repair, have been explored by low-temperature fluorescence spectroscopy. These studies have been performed using polynucleotides and calf thymus DNA reacted in vitro with DB[a,l]PDE and native DNA from mouse epidermis exposed to DB[a, l]P. The results show that adducts are heterogeneous, possess different structures, and adopt different conformations. External, external but base-stacked and intercalated adduct conformations are observed in calf thymus DNA and in mouse skin DNA samples. Differences in adduct repair rates are also revealed; namely, the analysis of mouse skin DNA samples obtained at 24 and 48 h after exposure to DB[a,l]P clearly shows that external adducts are repaired more efficiently than intercalated adducts. These results, taken together with those for B[a]P-DNA adducts [Suh et al. (1995) Carcinogenesis 16, 2561-2569], indicate that the repair of DNA damage resulting from PAH diol epoxides is conformation-dependent.

Capillary electrophoresis (CE) interfaced with low-temperature (4.2 K) fluorescence line-narrowing spectroscopy (FLNS) is used for the separation and spectral characterization of closely related analytes. In this paper, the CE-FLNS system is applied to the analysis of a mixture of deuterated and protonated benzo[a]pyrene, a mixture of structurally similar benzo[a]pyrene and benzo[e]pyrene, and mixtures of dibenzo[a,l]pyrene-derived adenine DNA adducts. The CE-FLNS system provides on-line separation and high-resolution spectroscopic identification of CE-separated analytes, via fingerprint structure of vibrationally resolved FLN spectra at 4.2 K. The combination of the separation power of CE and the spectral selectivity of FLNS provide a methodology that has potential to become a powerful tool for molecular analyte characterization. The main applications of the CE-FLNS system, due to its selectivity, should be in the chemical analysis of structurally similar analytes and applications where analyte purity and detailed structural characterization are required.

Both 7,12-dimethylbenz[a]anthracene (DMBA) and benzo[a]pyrene (BP) are carcinogenic in the rat mammary gland. The depurinating and stable adducts of DMBA and BP formed in vitro and in mouse skin were previously identified and quantitated. Identification and quantitation of the depurinating and stable DNA adducts of DMBA and identification of the depurinating adducts of BP formed in rat mammary glands in the 24 h after intramammillary injection of DMBA or BP are reported in this paper. The depurinating adducts of DMBA, which constitute 52% of all adducts detected, are DMBA bound at the 12-methyl group to the N-7 of adenine (Ade) or guanine (Gua), namely, 7-methylbenz[a]anthracene (MBA)-12-CH2-N7Ade (39%) and 7-MBA-12-CH2-N7Gua (13%). All of the stable adducts were formed from the diol epoxide(s) of DMBA. Depurinating adducts of BP with guanine, namely, 8-(BP-6-yl)-guanine (BP-6-C8Gua) and BP-6-N7Gua, were identified in rat mammary glands treated with BP. The major stable adduct, formed via the diol epoxide pathway, BP-diol epoxide-10-N2dG, accounted for over 64% of all the stable adducts. Three other BP-DNA stable adducts remain unidentified. Thus, rat mammary cells form depurinating adducts of DMBA and BP predominantly via their radical cations and stable adducts via the diol epoxides.

Polycyclic aromatic hydrocarbons are bound to DNA by two major pathways, one-electron oxidation and monooxygenation, to form adducts that are stable in DNA under normal conditions of isolation and depurinating adducts that are released from DNA by cleavage of the bond between the purine base and deoxyribose. Isolated rat liver nuclei have been used as an in vitro model for studying covalent binding of aromatic hydrocarbons to DNA, but the depurinating adducts formed by nuclei have not been identified or compared to those formed by the more commonly used rat liver microsomes. To examine the profiles of stable and depurinating adducts, nuclei from the livers of 3-methylcholanthrene-induced male MRC Wistar rats were incubated with [3H]benzo[a]pyrene (BP) and NADPH. Three depurinating adducts, 8-(BP-6-yl)Gua, 7-(BP-6-yl)Gua, and 7-(BP-6-yl)Ade, were obtained from the nuclei, as seen previously with rat liver microsomes or in mouse skin. The profile of stable adducts analyzed by the 32P-postlabeling method was qualitatively similar to that found in the microsomal activation of BP or in mouse skin treated with BP. Low-temperature fluorescence studies of the nuclear DNA revealed the presence of stable BP adducts originating from syn- and anti-BP diol epoxide.

This paper reports expanded analyses of benzo[a]pyrene (BP)-DNA adducts formed in vitro by activation with horseradish peroxidase (HRP) or 3-methylcholanthrene-induced rat liver microsomes and in vivo in mouse skin. The adducts formed by BP are compared to those formed by BP-7,8-dihydrodiol and anti-BP diol epoxide (BPDE). First, activation of BP by HRP produced 61% depurinating adducts: 7-(benzo[a]pyrene-6-yl)guanine (BP-6-N7Gua), BP-6-C8Gua, BP-6-N7Ade, and the newly identified BP-6-N3Ade. As a standard, the last adduct was synthesized along with BP-6-N1Ade by electrochemical oxidation of BP in the presence of adenine. Second, identification and quantitation of BP-DNA adducts formed by microsomal activation of BP showed 68% depurinating adducts: BP-6-N7Ade, BP-6-N7Gua, BP-6-C8Gua, BPDE-10-N7Ade, and the newly detected BPDE-10-N7Gua. The stable adducts were mostly BPDE-10-N2dG (26%), with 6% unidentified. BPDE-10-N7Ade and BPDE-10-N7Gua were the depurinating adducts identified after microsomal activation of BP-7, 8-dihydrodiol or direct reaction of anti-BPDE with DNA. In both cases, the predominant adduct was BPDE-10-N2dG (90% and 96%, respectively). Third, when mouse skin was treated with BP for 4 h, 71% of the total adducts were the depurinating adducts BP-6-N7Gua, BP-6-C8Gua, BP-6-N7Ade, and small amounts of BPDE-10-N7Ade and BPDE-10-N7Gua. These newly detected depurinating diol epoxide adducts were found in larger amounts when mouse skin was treated with BP-7,8-dihydrodiol or anti-BPDE. The stable adduct BPDE-10-N2dG was predominant, especially with anti-BPDE. Comparison of the profiles of DNA adducts formed by BP, BP-7,8-dihydrodiol, and anti-BPDE with their carcinogenic potency indicates that tumor initiation correlates with the levels of depurinating adducts, but not with stable adducts. Furthermore, the levels of depurinating adducts of BP correlate with mutations in the Harvey-ras oncogene in DNA isolated from mouse skin papillomas initiated by this compound [Chakravarti et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 10422-10426]. The depurinating adducts formed by BP in mouse skin appear to be the key adducts leading to tumor initiation.

One of the major DNA adducts from the extremely potent aromatic carcinogen dibenzo[a,l]pyrene (DB[a,l]P) is the depurinating adduct syn-DB[a,l]P diolepoxide-14-N7Ade. Low-temperature fluorescence spectra of this adduct (and related derivaties bound to N3-adenine and N7-guanine) showed two distinct (0,0) origin bands with different excited-state vibrational frequencies, as measured by means of fluorescence line narrowing spectroscopy. The relative intensity of the two origin bands was solvent dependent. The hypothesis that this phenomenon could be due to a conformational equilibrium was tested using molecular mechanics, dynamical simulations and semi-empirical quantum-mechanical calculations. The hydrolyzed metabolite DB[a,l]P tetraol was also studied for comparison. It was found that the syn-DB[a,l]P diolepoxide-14-N7Ade adduct is formed via trans addition to the epoxide. Exploration of the conformational space indeed produced two potential energy minima; both corresponding to structures in which the aromatic ring system is severely distorted. In conformation I the proximity of the distal ring forces the adenine base into a pseudo-axial position and the cyclohexenyl ring adopts a half-boat structure. In conformation II the distal ring is bent in the opposite direction, allowing the cyclohexenyl ring to adopt a half-chair structure with the base in a pseudo-equatorial position, partially stacked over the distal ring. THe difference in (0,0) transition energy calculated for the two conformers agrees very well with the spectroscopic data, and the relative orientations of the hydrogens bound to the cyclohexenyl ring in the major (half-boat) conformation I are in full agreement with the experimentally observed proton NMR coupling constants.

Using polyacrylamide gel electrophoresis (PAGE) and low-temperature, laser-induced fluorescence line narrowing (FLN) and non-line narrowing (NLN) spectroscopic methods, the conformational characteristics of stereochemically defined and site-specific adducts derived from the binding of 7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene (anti-BPDE, a metabolite of the environmental carcinogen benzo[a]pyrene), to DNA were studied. The focus of these studies was on the four stereochemically distinct anti-BPDE modified duplexes 5'-d(CCATCGCTACC).(GGTAGCGATGG), where G denotes the lesion site derived from trans or cis addition of the exocyclic amino group of guanine to the C10 position of either (+) or (-)-anti-BPDE. PAGE experiments under non-denaturing conditions showed that the (+)-trans adduct causes a significantly greater retardation in the electrophoretic mobility than the other three adducts, probably the result of important adduct-induced distortions of the duplex structure. Low-temperature fluorescence studies in frozen aqueous buffer matrices showed that the (+)-trans adduct adopts primarily an external conformation with only minor interactions with the helix, but a smaller fraction (approximately 25%) appears to exists in a partially base-stacked conformation. The (-)-trans adduct exists almost exclusively (approximately 97%) in an external conformation. Both cis adducts were found to be intercalated; strong electron-phonon coupling observed in their FLN spectra provided additional evidence for significant pi-pi stacking interactions between the pyrenyl residues and the bases. FLN spectroscopy is shown to be suitable for distinguishing between trans and cis adducts, but lesions with either (+)- or (-)-trans, or (+)- or (-)-cis stereochemical characteristics showed very similar vibrational patterns.(ABSTRACT TRUNCATED AT 250 WORDS)

The formation and repair of benzo[a]pyrene diol epoxide-N2-deoxyguanosine adducts (BPDE-N2-dG) in DNA isolated from the skin of mice treated topically with benzo[a]pyrene (BP) was studied by 32P-postlabeling and by low-temperature fluorescence spectroscopy under low resolution and under high resolution fluorescence line narrowing (FLN) conditions. In agreement with earlier studies, total BP-DNA binding reached a maximum at 24 h after treatment (dose: 1 mumol/mouse), then declined rapidly until 4 days after treatment and much more slowly thereafter. An HPLC method was developed which resolved the 32P-postlabeled (-)-trans- from (-)-cis-anti-BPDE-N2-dG, and (+)-trans-from (+)-cis-anti-BPDE-N2-dG. High performance liquid chromatography analysis of the major TLC adduct spot (containing > 80% of the total adducts) obtained by postlabeling BP-modified mouse skin DNA showed that it consisted of a major component that coeluted with (-)-cis-/(+)-trans-anti-BPDE-N2-dG and a minor component that coeluted with (-)-trans-/(+)-cis-anti-BPDE-N2-dG and that the minor component was repaired at a slower rate than the major component. Low-temperature fluorescence spectroscopy of the intact DNA identified the major adduct as (+)-trans-anti-BPDE-N2-dG and the minor adduct fraction consisted mainly of (+)-cis-anti-BPDE-N2-dG. In agreement with the 32P-postlabeling results it was observed by fluorescence spectroscopy that the (+)-cis-adducts were repaired more slowly than most other adducts. Moreover, the (+)-trans-adducts exhibited a broad distribution of base-stacked, partially base-stacked and helix-external conformations. Mouse skin DNA samples obtained at early timepoints (2-8 h) after treatment with BP contained substantially more of the 'external' adducts, while samples at later timepoints (24-48 h) contained relatively more adducts in the base-stacked conformation, indicating also that the latter adducts are repaired less readily than the former. The possible biological significance of these novel observations of conformation-dependent rates of DNA adduct repair and their possible dependence on DNA sequence, are discussed.

Dibenzo[a,l]pyrene (DB[a,l]P) is the most potent carcinogen known among aromatic hydrocarbons. DB[a,l]P-11,12-dihydrodiol, precursor to the bay-region diol epoxide, is slightly less carcinogenic than the parent compound. DB[a,l]P and its 11,12-dihydrodiol were covalently bound to DNA by cytochrome P-450 in 3-methylcholanthrene-induced rat liver microsomes, and DB[a,l]P was also bound to DNA by horseradish peroxidase. The "stable" (remaining intact in DNA under normal conditions of purification) and "depurinating" (released from DNA by cleavage of the glycosidic link between the purine base and deoxyribose) adducts were identified and quantified. Stable adducts were analyzed by the 32P-postlabeling technique. Depurinating adducts were identified by comparison of their retention times with those of standard adducts on HPLC in two solvent systems. Confirmation of their identity was obtained by means of fluorescence line-narrowing spectroscopy. When DB[a,l]P was activated by horseradish peroxidase, the depurinating adducts 3-(DB[a,l]P-10-yl)adenine (DB[a,l]P-10-N3Ade, 33%), 7-(DB[a,l]P-10-yl)adenine (DB[a,l]P-10-N7Ade, 27%), and 7-DB[a,l]P-10-yl)guanine (DB[a,l]P-10-N7Gua, 5%) were formed. Unidentified stable adducts comprised the remaining 35% of the detected adducts. When DB[a,l]P was activated by microsomes, the one-electron oxidation depurinating adducts DB[a,l]P-10-N3Ade (28%), DB[a,l]P-10-N7Ade (14%), DB[a,l]P-10-N7Gua (2%), and DB[a,l]P-10-C8Gua (6%), as well as the diol epoxide depurinating adducts (+/-)-syn-DB[a,l]P-diol epoxide (DE)-14-N7Ade (31%) and (+/-)-anti-DB[a,l]PDE-14-N7Gua (3%), were formed. Stable adducts predominantly formed via the DB[a,l]PDE pathway represented 16% of the adducts detected.(ABSTRACT TRUNCATED AT 250 WORDS)

Conformations of the trans adduct of (+)-anti-benzo[a]pyrene -7,8-dihydrodiol-9,10-epoxide (BPDE) to N2-guanine, the major stable DNA adduct of the environmental carcinogen benzo[a]pyrene, were studied as a function of flanking bases in single-stranded and in double-stranded oligonucleotides. Three 11mer oligonucleotides d(CTAT-G1G2G3TATC) were synthesized containing the (+)-trans-anti-BPDE adduct at one specific guanine of the GGG sequence (a known mutational hot spot). Polyacrylamide gel electrophoresis of the three single-stranded oligonucleotides showed that the adduct bound to G2 or G3 (5'-flanking base guanine) caused significantly stronger retardation than the same adduct bound to G1 (5'-flanking base thymine). The strength of the carcinogen-base interaction was reflected in the spectroscopic properties of the pyrenyl moiety. Low temperature fluorescence measurements under line-narrowing (FLN) or non-line-narrowing (NLN) conditions showed that in single-stranded form the adduct at G2 or G3 (5'-flanking base guanine) adopts a conformation with strong interaction with the bases. This was also observed for the same adduct at the sequence AGA. In contrast, the (+)-trans-anti-BPDE adduct with a 5'-flanking thymine exists in a primarily helix-external conformation. Similar differences were observed in the double-stranded oligonucleotides: the adducts at G2 and G3 were found to exist in similar conformational equilibria, again with significant carcinogen-base interactions, while the adduct at G1 showed a predominantly external conformation. The nature of the 3'-flanking base appeared to have little influence on the conformational equilibrium of the (+)-trans-anti-BPDE-guanine adduct. The results could provide insight into the mutational specificity and flanking base effects observed for (+)-anti-BPDE.

The two DNA adducts of benzo[a]pyrene (BP) previously identified in vitro and in vivo are the stable adduct formed by reaction of the bay-region diol epoxide of BP (BPDE) at C-10 with the 2-amino group of dG (BPDE-10-N2dG) and the adduct formed by reaction of BP radical cation at C-6 with the N-7 of Gua (BP-6-N7Gua), which is lost from DNA by depurination. In this paper we report identification of several new BP-DNA adducts formed by one-electron oxidation and the diol epoxide pathway, namely, BP bound at C-6 to the C-8 of Gua (BP-6-C8Gua) and the N-7 of Ade (BP-6-N7Ade) and BPDE bound at C-10 to the N-7 of Ade (BPDE-10-N7Ade). The in vitro systems used to study DNA adduct formation were BP activated by horseradish peroxidase or 3-methylcholanthrene-induced rat liver microsomes, BP 7,8-dihydrodiol activated by microsomes, and BPDE reacted with DNA. Identification of the biologically-formed depurination adducts was achieved by comparison of their retention times on high-pressure liquid chromatography in two different solvent systems and by comparison of their fluorescence line narrowing spectra with those of authentic adducts. The quantitation of BP-DNA adducts formed by rat liver microsomes showed 81% as depurination adducts: BP-6-N7Ade (58%), BP-6-N7Gua (10%), BP-6-C8Gua (12%), and BPDE-10-N7Ade (0.5%). Stable adducts (19% of total) included BPDE-10-N2dG (15%) and unidentified adducts (4%). Microsomal activation of BP 7,8-dihydrodiol yielded 80% stable adducts, with 77% as BPDE-10-N2dG and 20% of the depurination adduct BPDE-10-N7Ade. The percentage of BPDE-10-N2dG (94%) was higher when BPDE was reacted with DNA, and only 1.8% of BPDE-10-N7Ade was obtained.(ABSTRACT TRUNCATED AT 250 WORDS)

A recently developed methodology [Jankowiak, R., Lu, P., Small, G. J., and Geacintov, N. E. (1990) Chem. Res. Toxicol. 3, 39-46], which combines fluorescence line narrowing spectroscopy at 4.2 K with non-line-narrowed (S2----S0 laser excitation) fluorescence spectroscopy at 77 K and fluorescence quenching, is used to characterize adducts formed from (+)-anti-BPDE and the alternating copolymers poly(dG-dC).poly(dG-dC) and poly(dA-dT).poly(dA-dT), the nonalternating poly (dG).poly(dC), single-strand poly(dG), and the oligonucleotide d(ATATGTATA). Detailed comparisons of the fluorescence spectra and quenching (with acrylamide) of the properties of the adducts with those of (+)-anti-BPDE-DNA adducts are made. Fluorescence spectra of the trans and cis isomers of the adduct formed from guanosine monophosphate and the adducts of d(ATATGTATA) are used to assign the stereochemistry of the two major DNA adducts as trans-N2-dG moieties which occupy two different DNA sites. Evidence for the existence of minor cis-type guanine adducts is provided. Finally, a fourth type of DNA adduct (minor) is identified and assigned as trans-N6-dA.

The major identified benzo[a]pyrene (BP)-DNA adduct formed by cytochrome P-450 contains BP bound at the C-6 position to the N-7 position of guanine (BP-N7Gua). This adduct is rapidly depurinated from DNA. When rats were treated with [14C]BP, about 0.02% of the administered dose of BP was excreted as BP-N7Gua in feces and urine within 5 days. Chloroform extracts of the urine and feces were analyzed by high-pressure liquid chromatography. The structure of the adduct was established by cochromatography with electrochemically prepared BP-N7Gua and by fluorescence line narrowing spectrometry. This study represents the first demonstration that BP-N7Gua is formed in vivo in animals treated with BP.

A methodology based on 77 K laser-excited fluorescence spectroscopy, fluorescence quenching, and fluorescence line narrowing is shown to be a highly selective and sensitive approach for the study of polycyclic aromatic carcinogen-DNA complexes. Three and five different DNA adducts derived from (+)-trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo [a]pyrene and its (-)-enantiomer are identified, respectively. Two different methods are used to classify the adducts as type I (interior) or type II (exterior), and both yield consistent results. The first high-resolution fluorescence excitation spectra are reported for DNA adducts. These spectra are suggested to be useful for characterizing the strength of the interaction between the fluorescent chromophores and the DNA bases. The above methodology has the potential for monitoring the fates of different adducts as a function of time in repair-competent cells.

An important initiating step in the induction of tumors is believed to be the covalent binding of an active carcinogenic species to a cellular macromolecule, e.g. DNA. Therefore, a spectroscopic technique which allows for positive identification of the intact (macromolecular) DNA adduct and/or isolated damaged nucleosides/nucleotides is highly desirable. It is shown that fluorescence line-narrowing spectroscopy (FLNS) is a rapid, versatile, highly sensitive and selective analytical technique, which can be used directly to characterize DNA adducts and isolated nucleosides. FLNS possesses sufficient resolution to distinguish between the major DNA adducts derived from different enantiomers of benzo[a]pyrene diol-epoxide (BPDE). With the present limit of detection (approximately 1 adducted base per 10(8) normal base pairs for 100 micrograms of DNA), the technique is applicable to in vivo samples. Analysis of liver DNA from fish exposed to benzo[a]pyrene (BP) (100 mg BP/kg fish) showed that a major DNA adduct is derived from syn-BPDE.

A new fluorescence line narrowing (FLN) apparatus is described and evaluated through experiments on intact DNA-PAH (polycyclic aromatic hydrocarbon) and globin-PAH adducts, as well as polar PAH metabolites. A detection limit of approximately 3 modified bases in 10(8) for a DNA adduct formed with a diol-epoxide of benzo[a]pyrene (BPDE-DNA) is reported for 20 micrograms of DNA at a spectral resolution of approximately 8 cm-1. The methodology employed avoids or minimizes spectral degradation and loss of sensitivity due to photooxidation and nonphotochemical hole burning (NPHB). A new double selection technique that employs both FLN and NPHB is described and found to lead to a significant improvement in selectivity over that obtained with conventional FLN.

Fluorescence line narrowing (FLN) was demonstrated for five benzo[a]pyrene (BP)-nucleoside adducts synthesized by one-electron oxidation of BP 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 BP to DNA in rat liver nuclei is 7-(benzo[a]pyren-6-yl)guanine (N7Gua). The structural characterization was performed with only 20 pg of the adduct. Metabolic activation of BP by one-electron oxidation in the horseradish peroxidase catalyzed reaction of BP with DNA (in vitro) was also investigated. The major adduct identified was 8-(benzo[a]pyren-6-yl)guanine (C8Gua).