Biomedical subjects

G J Small

Publications and source records attributed to G J Small.

At least 37 records · Page 2Linked to original sources

PubMed1999

Preparation, isolation, and characterization of Dibenzo[a,l]pyrene diol epoxide-deoxyribonucleoside monophosphate adducts by HPLC and fluorescence line-narrowing spectroscopy.

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.

Benzopyrenes↗

PubMed1999

A novel method for the isolation and identification of stable DNA adducts formed by Dibenzo[a,l]pyrene and Dibenzo[a,l]pyrene 11, 12-dihydrodiol 13,14-epoxides in vitro.

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.

Animals↗

PubMed1999

Spectral and conformational analysis of deoxyadenosine adducts derived from syn- and anti-Dibenzo[a,l]pyrene diol epoxides: fluorescence studies.

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.

Benzopyrenes↗

PubMed1999

Structure elucidation of the adducts formed by fjord region Dibenzo[a,l]pyrene-11,12-dihydrodiol 13,14-epoxides with deoxyguanosine.

(+/-)-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.

Chromatography, High Pressure Liquid↗

PubMed1999

Molecular and kinetic evidence for allelic variants of esterase Estbeta1 in the mosquito Culex quinquefasciatus.

Elevated esterase Estbeta1 was purified from larvae of newly isolated strains of the mosquito Culex quinquefasciatus from Colombia (COL) and Trinidad (TRI) with resistance to organophosphate (OP) insecticides. Insecticide interactions were compared with those of elevated Estbeta1(2) from the OP-resistant Habana strain and the non-elevated Estbeta1(3) from the susceptible PelSS strain. On the basis of insecticide binding efficiency, all elevated Estbeta1 esterases were readily distinguishable. Differences between the EcoRI restriction fragment patterns of the amplified estbeta1 gene in COL and TRI strains compared with each other, and between amplified estbeta1(1), estbeta1(2) and the non-amplified estbeta1(3), suggest differences in their nucleotide sequence. Considering their variable insecticide binding efficiencies, these genetic differences would imply that, in contrast to estalpha2 and estbeta2, amplification of estbeta1 has occurred several times independently. Generally, the elevated Estbeta1s were more reactive with insecticides than the non-elevated Estbeta1(3). This supports the hypothesis that the elevated esterase-based mechanism confers resistance through amplification of alleles coding for esterases which have a greater specificity for the insecticides they sequester than the esterases coded by their non-amplified counterparts.

Alleles↗

PubMed1998

Aluminum phthalocyanine tetrasulfonate in MCF-10F, human breast epithelial cells: a hole burning study.

Laser-induced holes are burned in the absorption spectrum of aluminum phthalocyanine tetrasulfonate (APT) in MCF-10F, human breast epithelial cells. The hole burning mechanism is shown to be nonphotochemical. The fluorescence excitation spectra and hole spectra are compared with those of APT in hyperquenched glassy films of water, ethanol, and methanol. The results show that the APT is in an acidic, aqueous environment with a hydrogen-bonded network similar to that of glassy water, but showing the influence of other cellular components. Pressure shifts of holes allow the local compressibility about the APT to be determined.

Breast↗

PubMed1998

Structure, conformations, and repair of DNA adducts from dibenzo[a, l]pyrene: 32P-postlabeling and fluorescence studies.

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.

Animals↗

PubMed1998

Spectral characterization of fluorescently labeled catechol estrogen 3,4-quinone-derived N7 guanine adducts and their identification in rat mammary gland tissue.

The oxidation of carcinogenic 4-hydroxycatechol estrogens (CE) of estrone (E1) and estradiol (E2) to catechol estrogen 3,4-quinones (CE-3,4-Q) results in electrophilic intermediates that covalently bind to DNA to form depurinating adducts [Cavalieri et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 10937]. These DNA adducts, 4-OHE1-1-N7Gua and 4-OHE2-1-N7Gua, are nonfluorescent. To utilize laser-excited fluorescence methods, the catechol estrogen-derived metabolites and adducts were labeled with a fluorescent marker. The 4-OHEi-1-N7Gua adduct standards (i = 1, 2) and 4-OHEi metabolites have been derivatized with 1-pyrenesulfonyl chloride and investigated by low-temperature spectroscopy under non-line-narrowing and line-narrowing conditions. Molecular modeling studies assisted in interpretation of the fluorescence spectra; energetically favored structures of the 4-OHE2-1-N7Gua-dipyrene adduct and 4-OHE2-dipyrene metabolite reveal unique conformations which, in agreement with fluorescence data, show a significant pi-pi interaction of pyrene labels with guanine and/or the aromatic ring of catechol estrogen. The conformation obtained for the 4-OHE2-1-N7Gua-dipyrene adduct appears to be conducive to mixing of its pipi state with pyrene-guanine charge-transfer states, consistent with the experimentally observed strong electron-phonon coupling. Non-line-narrowed and line-narrowed spectra obtained at 77 and 4.2 K, respectively, are shown to distinguish 4-OHE2-1-N7Gua-dipyrene adducts from 4-OHE2-dipyrene metabolites. These standards have subsequently been used for the spectroscopic identification of depurinating DNA adducts formed in a tissue culture experiment where rat mammary gland tissue was treated with the estrogen quinone E2-3,4-Q. The depurinating adduct formed is 4-OHE2-1-N7Gua.

Animals↗

PubMed1998

Characterization of amplified esterase Estbeta1(2) associated with organophosphate resistance in a multi-resistant population of the mosquito Culex quinquefasciatus from Cuba.

Esterase amplification is the major organophosphorus (OP) insecticide resistance mechanism in Culex mosquitoes. The amplified Estalpha2(1)¿Estbeta2(1) esterases are found in > 90% of resistant populations worldwide, whereas amplified DNAs (amplicons) containing Estbeta1s are much rarer. Individuals with the Estbeta1 amplicons appear to be at a selective disadvantage in competition with those carrying the Estalpha2(1)¿Estbeta2(1) amplicons. To test the hypothesis that this is because Estbeta1 is less able to bind insecticide than the common amplified esterases, Estbeta1(2) was purified from the multi-resistant Habana strain of Culex quinquefasciatus, from Cuba. In its native form Estbeta1 is a monomeric enzyme of 66 kDa, with a pI of 4.8. The bimolecular rate constants for interaction of Estbeta1(2) with several OP insecticides were similar to those for the commonly elevated esterases Estalpha2(1) and Estbeta2(1), and much higher than for the electrophoretically identical non-elevated Estbeta1(3) and Estalpha3. Hence the apparent selective advantage of the Estalpha2(1)¿Estbeta2(1) amplicon is not due to its greater efficiency of insecticide binding, as OP insecticides are significantly better inhibitors of all the amplified esterases than of their non-amplified counterparts and therefore should be equally effective at conferring resistance.

Animals↗

PubMed1997

Application of capillary electrophoresis-fluorescence line-narrowing spectroscopy for on-line spectral characterization of closely related analytes.

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.

Benzopyrenes↗

PubMed1997

Conformational studies of stereoisomeric tetrols derived from syn- and anti-dibenzo[a,l]pyrene diol epoxides.

An understanding of the conformational behavior of the stereoisomeric tetrols at the 11,12,13,14-positions of dibenzo[a,l]pyrene (DB[a,l]P) is essential for the spectroscopic identification of DNA adducts derived from the biologically highly active fjord region syn- and anti-DB[a,l]P-11,12-diol 13,14-epoxides. Conformational effects are expected to play an important role in DNA-DB[a,l]P diol epoxide reactivity, base-sequence specificity, and conformation dependent repair. The results of conformational studies on trans-anti-, cis-anti-, and cis-syn-DB[a,l]P tetrol isomers are presented and compared to the results obtained previously for trans-syn-DB[a,l]P tetrol (Carcinogenesis 17, 829-837, 1996). Molecular mechanics, dynamical simulations, and semiempirical calculations of electronic transitions are used to interpret the low-temperature fluorescence spectra and 1H NMR data. Molecular dynamics simulations (in vacuo) identified two conformers (I and II) for each of the tetrol isomers; in all conformations the aromatic ring system is severely distorted. Fluorescence line-narrowing (FLN) spectroscopy identified two distinct conformational species for the trans-anti isomer, one occurring in ethanol and the other occurring in a glycerol/water matrix. The corresponding structures are assigned based on the S1<--S0 transition energies calculated for conformers I and II, respectively. 1H NMR spectroscopy confirmed the structure of conformer I at room temperature. In contrast to trans-syn-DB[a,l]P tetrol (where the major conformation was identified as a boat structure), both conformations of trans-anti-DB[a,l]P tetrol feature a half-chair structure for the cyclohexenyl ring with different orientations of the hydroxyl groups. For cis-anti- and cis-syn-DB[a,l]P tetrols, only a single conformer is detected by FLN spectroscopy. The NMR results for the latter appear to be most consistent with a mixture of two half-chair conformers I and II, while for the cis-anti isomer a flattened, boatlike conformation was observed. The generally good agreement between the NMR coupling constants and those estimated theoretically indicates that these structures should serve as good starting points for spectroscopic or computational studies of DNA adducts derived from DB[a,l]P diol epoxides.

Benzopyrenes↗

PubMed1997

Determination of benzo[a]pyrene- and 7,12-dimethylbenz[a]anthracene-DNA adducts formed in rat mammary glands.

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.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1996

Conformational studies of depurinating DNA adducts from syn-dibenzo[a,l]pyrene diolepoxide.

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.

Adenine↗

PubMed1995

Identification and quantitation of dibenzo[a,l]pyrene--DNA adducts formed by rat liver microsomes in vitro: preponderance of depurinating adducts.

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)

Animals↗

PubMed1995

Conformational studies of the (+)-trans, (-)-trans, (+)-cis, and (-)-cis adducts of anti-benzo[a]pyrene diolepoxide to N2-dG in duplex oligonucleotides using polyacrylamide gel electrophoresis and low-temperature fluorescence spectroscopy.

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)

Base Sequence↗

PubMed1995

Formation and persistence of benzo[a]pyrene-DNA adducts in mouse epidermis in vivo: importance of adduct conformation.

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.

Animals↗

PubMed1994

Flanking base effects on the structural conformation of the (+)-trans-anti-benzo[a]pyrene diolepoxide adduct to N2-dG in sequence-defined oligonucleotides.

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.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMed1993

Possible mechanisms of organophosphorus and carbamate insecticide resistance in German cockroaches (Dictyoptera: Blattelidae) from different geographical areas.

The resistance status of 14 strains of Blattella germanica (L.) from four countries was determined for chlorpyrifos and propoxur compared with a standard reference susceptible strain. Thirteen strains were resistant to chlorpyrifos; 12 strains were resistant to propoxur. Resistance ratios for chlorpyrifos ranged from 8- to 462-fold at LC90; for propoxur, resistance ratios ranged from 4- to 46-fold. One cockroach strain from Denmark had negative cross-resistance to chlorpyrifos, and one strain from the United States had negative cross-resistance to propoxur. Slopes of probit regressions indicated that all resistant strains were heterogeneous for resistance to both chlorpyrifos and propoxur. Synergist studies with piperonyl butoxide indicated that multifunction mono-oxidases are probably involved in resistance to chlorpyrifos in six strains and in resistance to propoxur in seven strains. Esterase activity was elevated in 10 strains; of these strains, two had only slightly elevated esterase activity as measured with the substrates 1- and 2-naphthyl acetate. The remainder had higher levels of elevated esterase activity to both substrates. Strains with elevated esterase activity were resistant to a broad spectrum of organophosphates, pyrethroids, or both. Increased levels of glutathione S-transferase activity were found in four strains. Another two strains had a low frequency (1%) of individuals with high glutathione S-transferase activity. Elevated glutathione S-transferase activity was not correlated with the observed levels of organophosphate or carbamate resistance. One strain from Dubai had an altered acetylcholinesterase-based mechanism that conferred broad-spectrum resistance to a range of organophosphates and carbamates.

Animals↗