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G J Small

Publications and source records attributed to G J Small.

69 records · Page 4Linked to original sources
PubMedDate not supplied

Fluorescence line narrowing-nonphotochemical hole burning spectrometry: femtomole detection and high selectivity for intact DNA-PAH adducts.

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.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMedDate not supplied

Fluorescence line narrowing spectrometric analysis of benzo[a]pyrene-DNA adducts formed by one-electron oxidation.

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).

Animals↗
PubMedDate not supplied

Laser spectroscopic studies of DNA adduct structure types from enantiomeric diol epoxides of benzo[a]pyrene.

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.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMedDate not supplied

Identification and quantitation of 7-(benzo[a]pyren-6-yl)guanine in the urine and feces of rats treated with benzo[a]pyrene.

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.

Animals↗
PubMedDate not supplied

Comparative laser spectroscopic study of DNA and polynucleotide adducts from the (+)-anti-diol epoxide of benzo[a]pyrene.

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.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMedDate not supplied

Mechanism of metabolic activation of the potent carcinogen 7,12-dimethylbenz[a]anthracene.

The DNA adducts of 7,12-dimethylbenz[a]anthracene (DMBA) previously identified in vitro and in vivo are stable adducts formed by reaction of the bay-region diol epoxides of DMBA with dG and dA. In this paper we report identification of several new DMBA-DNA adducts formed by one-electron oxidation, including two adducts lost from DNA by depurination, DMBA bound at the 12-methyl to the N-7 of adenine (Ade) or guanine (Gua) [7-methylbenz[a]anthracene (MBA-12-CH2-N7Ade or 7-MBA-12-CH2-N7Gua, respectively]. The in vitro systems used to study DNA adduct formation were DMBA activated by horseradish peroxidase or 3-methyl-cholanthrene-induced rat liver microsomes. The biologically-formed depurination adducts were identified by high-pressure liquid chromatography and by fluorescence line narrowing spectroscopy. Stable DMBA-DNA adducts were analyzed by the 32P-postlabeling method. Quantitation of DMBA-DNA adducts formed by microsomes showed about 99% as depurination adducts: 7-MBA-12-CH2-N7Ade (82%) and 7-MBA-12-CH2-N7Gua (17%). Stable adducts (1.4% of total) included one adduct spot that may contain adduct(s) formed from the diol epoxide (0.2%) and unidentified adducts (1.2%). Activation of DMBA by horseradish peroxidase afforded 56% of stable unidentified adducts and 44% of depurination adducts, with 36% of 7-MBA-12-CH2-N7Ade and 8% of 7-MBA-12-CH2-N7Gua. Adducts containing the bond to the DNA base at the 7-CH3 group of DMBA were not detected.(ABSTRACT TRUNCATED AT 250 WORDS)

9,10-Dimethyl-1,2-benzanthracene↗
PubMedDate not supplied

Identification and quantitation of benzo[a]pyrene-DNA adducts formed by rat liver microsomes in vitro.

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)

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMedDate not supplied

Evidence of involvement of multiple sites of metabolism in the in vivo covalent binding of dibenzo[a,h]pyrene to DNA.

The in vivo formation of dibenzo[a,h]pyrene-DNA adducts in mouse skin was assessed by laser-excited fluorescence spectroscopy at 77 and 4.2 K. Two adducts were identified with fluorescence origin bands at approximately 383.5 and 407.2 nm, and these were shown to possess pyrene and benzo[a]pyrene (B[a]P) chromophores, respectively. Both DNA-bound chromophores displayed considerable electron-phonon coupling and likely assume a highly base-stacked or quasi-intercalated configuration within DNA duplexes. The presence of B[a]P and pyrene aromatic systems indicates that two-electron or monooxygenation metabolism occurred on either the a or h benzo moieties (which are equivalent) in the former case, and on both these rings in the latter case. The presence of two adduct species agrees with 32P-postlabeling analysis of the DNA, which showed the presence of two major adducts in both thin-layer and high-performance liquid chromatographic separations.

Animals↗
PubMedDate not supplied

Identification and quantitation of benzo[a]pyrene-DNA adducts formed in mouse skin.

The DNA adducts of benzo[a]pyrene (BP) formed in vitro were previously identified and quantitated. In this paper, we report the identification and quantitation of the depurination adducts of BP, 8-(benzo[a]pyren-6-yl)guanine (BP-6-C8Gua), BP-6-N7Gua, and BP-6-N7Ade, formed in mouse skin by one-electron oxidation, as well as the major stable adduct formed via the diolepoxide pathway, BP diolepoxide bound at C-10 to the 2-amino of dG (BPDE-10-N2dG). Identification of the depurination adducts was achieved by HPLC and fluorescence line narrowing spectroscopy. The depurination adducts, BP-6-C8Gua (34%), BP-6-N7Gua (10%), and BP-6-N7Ade (30%), constituted 74% of the adducts found in mouse skin 4 h after treatment with BP. The stable adduct BPDE-10-N2dG accounted for 22% of the adducts. Treatment of the skin with BP-7,8-dihydrodiol or BP diolepoxide yielded almost exclusively the stable adduct BPDE-10-N2dG. When BP or BP-7,8-dihydrodiol was bound to RNA or denatured DNA in reactions catalyzed by rat liver microsomes, no depurination adducts were detected. The profiles of stable adducts were similar both qualitatively and quantitatively with native or denatured DNA. With activation of BP by horseradish peroxidase, the profiles of stable adducts differed with native and denatured DNA. The total amount of adducts with denatured DNA was only 25% of the amount detected with native DNA. No depurination adducts were detected with denatured DNA or RNA in the peroxidase system.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Topical↗
PubMedDate not supplied

Identification and quantitation of 7,12-dimethylbenz[a]anthracene-DNA adducts formed in mouse skin.

Identification and quantitation of the depurination and stable DNA adducts of 7,12-dimethylbenz[a]anthracene (DMBA) formed by cytochrome P450 in rat liver microsomes previously established one-electron oxidation as the predominant mechanism of activation of DMBA to bind to DNA. In this paper we report the identification and quantitation of the depurination and stable DMBA-DNA adducts formed in mouse skin. The depurination adducts, which constitute 99% of all the adducts detected, are DMBA bound at the 12-methyl group to the N-7 of adenine or guanine, namely, 7-methylbenz[a]anthracene (MBA)-12-CH2-N7Ade and 7-MBA-12-CH2-N7Gua. The depurination adducts were identified by HPLC and fluorescence line narrowing spectroscopy. The stable DNA adducts were analyzed by the 32P-postlabeling method. Almost 4 times as much of the depurination adduct 7-MBA-12-CH2-N7Ade (79%) was formed compared to 7-MBA-12-CH2-N7Gua (20%). The stable adducts accounted for only 1% of all the adducts detected and 80% of these were formed from DMBA diolepoxide. The binding of DMBA to DNA specifically at the 12-CH3 group is consistent with the results of carcinogenicity experiments in which this group plays a key role. When DMBA was bound to RNA or denatured DNA in reactions catalyzed by microsomes or by horseradish peroxidase (HRP), no depurination DNA adducts of DMBA were detected. The amount of stable DNA adducts observed with denatured DNA was 70% lower in the HRP system and 30% lower in the microsomal system compared to native DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

9,10-Dimethyl-1,2-benzanthracene↗
PubMedDate not supplied

Sequence dependence of benzo[a]pyrene diol epoxide-DNA adduct conformer distribution: a study by laser-induced fluorescence/polyacrylamide gel electrophoresis.

Low-temperature laser-induced fluorescence techniques in combination with polyacrylamide gel electrophoresis (LIF/PAGE) were used to study the binding of (-)-anti- and (+)-anti-benzo[a]pyrene 7,8-dihydrodiol 9,10-epoxide (anti-BPDE) to several sequence-defined duplex oligomers. Two of the oligomers contain central 5'-RAGGAR-3' sequences (R = purine) which appear to be frequently mutated by racemic (+/-)-anti-BPDE in endogenous genes of cells cultured in vitro. Two contain a central 5'-CCGG-3' or 5'-TGGT-3' sequence which are strongly preferred for covalent binding but appear to be not so frequently mutated. Binding of the two enantiomers to the latter two sequences yielded a distribution of BPDE-N2-dG adduct conformations similar to those from binding to highly polymerized, random sequence DNA in vitro which, for (+/-)-anti-BPDE, means that the helix-external conformation of the N2-dG adduct is dominant. Binding of (-)-anti-BPDE to the 5'-RAGGAR-3' sequences yielded more partially base-stacked and less base-stacked (quasi-intercalated) conformer than observed for random sequence DNA. Importantly, the (+)-anti-BPDE in binding to the more mutagenically inclined 5'-RAGGAR-3' sequences yielded little external-type adduct in comparison to the other two sequences and random sequence DNA. Moreover, an unusually high proportion of the (+)-anti-BPDE adducts formed with the 5'-RAGGAR-3' sequences result from cis stereoaddition, which yields a partially base-stacked configuration. Since the (+)-anti-BPDE appears to be the more mutagenic, this result suggests a possible role of internal adduct conformations in mutagenesis.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMedDate not supplied

Expanded analysis of benzo[a]pyrene-DNA adducts formed in vitro and in mouse skin: their significance in tumor initiation.

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.

Animals↗
PubMedDate not supplied

Depurinating and stable benzo[a]pyrene-DNA adducts formed in isolated rat liver nuclei.

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.

Animals↗
PubMedDate not supplied

Active and passive immunization strategies for Pseudomonas aeruginosa pneumonia.

The unusually high mortality associated with Pseudomonas aeruginosa pneumonia has provided an incentive for the development of immunologic strategies for preventing or treating this infection. A guinea pig model of experimental P. aeruginosa pneumonia was employed to determine prophylactic efficacy of active immunization with a detoxified lipopolysaccharide vaccine; efficacy of passive immune therapy utilizing a new hyperimmune immunoglobulin G preparation enriched for antibodies to P. aeruginosa immunotypes 1, 2, 4, and 6; and efficacy of active and passive immunization against the mucoid exopolysaccharide antigen associated with mucoid strains of P. aeruginosa. Each of these immunologic methods provided an element of protection against P. aeruginosa pneumonia.

Animals↗