Biomedical subjects

R G Harvey

Publications and source records attributed to R G Harvey.

At least 37 records · Page 2Linked to original sources

PubMed1997

Spectrochemical investigations of fluorescence quenching agents. Part 5. Effect of surfactants of the ability of nitromethane to selectively quench fluorescence emission of alternant PAHs.

Applicability of the nitromethane selective quenching rule for discriminating between alternant vs. nonalternant polycyclic aromatic hydrocarbons (PAHs) is examined for 18 representative PAH solutes dissolved in micellar cetyltrimethylammonium chloride (CTACl), micellar dodecyltrimethylammonium bromide (DTAB), micellar Brij-35 and micellar sodium octanoate (SO) solvent media. Experimental results show that nitromethane quenched fluorescence emission of only the 10 alternant PAHs in the two cationic (CTACl and DTAB) and nonionic Brij-35 surfactant solvent media as expected. Emission intensities of nonalternant PAHs, except for the few exceptions noted previously, were unaffected by nitromethane addition. Unexpected quenching behavior was observed, however, in the case of nonalternant PAHs dissolved in micellar sodium octanoate solvent media. Nitromethane quenched fluorescence emission of all nonalternant PAHs studied in the SO solvent media, which is contrary to the selective quenching rule.

Fluorescent Dyes↗

PubMed1996

Mutational specificity of the syn 1,2-dihydrodiol 3,4-epoxide of 5-methylchrysene.

The mutational specificity of the syn dihydrodiol epoxide of 5-methylchrysene in the supF gene of the pSP189 vector was examined. Transversion mutations at GC pairs predominated with G --> T and G --> C changes accounting for 42 and 21% of total base change mutations. The types of mutations found reflect the previously determined chemical preference of this reactive species for reaction with deoxyguanosine residues in DNA.

Base Sequence↗

PubMed1996

Disposition and biological activity of benzo[a]pyrene-7,8-dione. A genotoxic metabolite generated by dihydrodiol dehydrogenase.

A novel pathway of polycyclic aromatic hydrocarbon metabolism involves the oxidation of non-K-region trans-dihydrodiols to yield o-quinones, a reaction catalyzed by dihydrodiol dehydrogenase (DD). We have recently shown that in isolated rat hepatocytes (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo-[a] pyrene (BP-diol) was oxidized by this route to yield benzo [a] pyrene-7,8-dione (BPQ). We now report the disposition of BPQ and its mutagenic and genotoxic properties. Using [3H]BPQ it was found that 30% of the radioactivity was sequestered by rat hepatocytes into the cell pellet. Isolation of hepatocyte DNA provided evidence for a low level of covalent incorporation of BPQ into DNA (30 +/- 17 adducts/ 10(6) base pairs). Examination of the hepatocellular DNA by agarose gel electrophoresis following treatment with BPQ indicated that extensive fragmentation had occurred. DNA fragmentation was also observed when hepatocytes were treated with BP-diol and this effect was attenuated by indomethacin, a DD inhibitor. Hepatocytes treated with either BP-diol or BPQ were found to produce large quantities of superoxide anion radical (O2.-). The amount of O2.- generated by BP-diol was blocked by DD inhibitors. These data suggest that by diverting BP-diol to BPQ reactive oxygen species (ROS) were generated which caused DNA fragmentation. The ability of BPQ to cause DNA strand scission was further studied using supercoiled phi X174 DNA. It was found that BPQ caused concentration-dependent (0.05-10 microM) strand scission in the presence of 1 mM NADPH (which promoted redox-cycling) provided CuCl2 (10 microM) was present. Complete destruction of the DNA was observed using 10 microM BPQ. This strand scission was prevented by catalase and hydroxyl radical scavengers but not by superoxide dismutase. These data indicate that ROS were responsible for the destruction of the DNA. Using 20 microM (+/-)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo [a]pyrene [(+/-)-anti-BPDE] only single nicks in the DNA were observed indicating that BPQ was the more potent chemical nuclease. BPQ was also found to be a direct-acting mutagen in the Ames test using Salmonella typhimurium tester strains TA97a, TA98, TA100, TA102, and TA104, but was 10-5500-fold less efficient as a mutagen than (+/-)-anti-BPDE. Our data indicate that DD suppresses the mutagenicity of (+/-)-anti-BPDE by producing BPQ, but in doing so a potent chemical nuclease is produced which causes extensive DNA fragmentation via the generation of ROS.

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

PubMed1996

Tylosin in the treatment of canine superficial pyoderma.

Thirty dogs with superficial pyoderma were treated orally with tylosin at a dose of 20 mg/kg twice daily for three weeks. Staphylococcus intermedius was recovered from 21 (70 per cent) of the dogs. Twenty-two of the dogs were free of clinical signs after three weeks of treatment and two dogs responded to a further two weeks of treatment, giving a total response rate of 80 per cent. Five cases (16.6 per cent) failed to respond and three of these subsequently responded to other antibacterial treatment. One dog suffered transient gastritis after the doses of tylosin and subsequently responded to a different antibacterial agent.

Animals↗

PubMed1996

Sequence preference of 7,12-dimethylbenz[a]anthracene-syn-diol epoxide-DNA binding in the mouse H-ras gene detected by UvrABC nucleases.

We have found that 7,12-dimethylbenz[a]anthracene-syn-diol epoxide (syn-DMBADE)-modified DNA fragments are sensitive to UvrABC incision. The incisions occur mainly seven bases 5' and four bases 3' of a syn-DMBADE-modified adenine or guanine residue. The kinetics of UvrABC incision at different sequences in a DNA fragment are the same, and the extent of UvrABC incision is proportional to the syn-DMBADE concentration. On the basis of these results, we have concluded that UvrABC incision on syn-DMBADE-DNA adducts is independent of DNA sequence and is quantitative. Using the UvrABC incision method, we have analyzed the syn-DMBADE-DNA binding spectrum in several defined DNA fragments, including the first two exons of the mouse H-ras gene. We have found that both guanine and adenine residues in codons 12, 13, and 61 of the H-ras gene are strong syn-DMBADE binding sites. These results suggest that the initial binding of DMBADE may greatly contribute to the frequency of H-ras mutations. Results from dinucleotide binding analysis indicate that the 5'-nearest neighbor displays a greater effect on syn-DMBADE-DNA binding than the 3'-nearest neighbor.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1996

Targeted A --> T and G --> T mutations induced by site-specific deoxyadenosine and deoxyguanosine adducts, respectively, from the (+)-anti-diol epoxide of dibenz[a,j]anthracene in M13mp7L2.

The studies described in this report directly examined the mutagenicity in Escherichia coli of both a deoxyadenosine (dAdo) and a deoxyguanosine (dGuo) adduct derived from (+)-anti-dibenz[a,j]-anthracene-3,4-diol 1,2-epoxide [(+)anti-DB[a,j]A-DE] that were site-specifically placed in a single-stranded M13mp7L2 replication vector. An 11-base oligonucleotide (5'-CTC ACG CTT CT-3') containing either a single (+)anti-DB[a,j]A-DE--trans-N2-dGuo or (+)anti-DB[a,j]A-DE--trans-N6dAdo adduct was successfully incorporated into single-stranded M13mp7L2 plasmid via ligation. In vitro studies using E. coli DNA polymerase I (Klenow fragment)indicated that both adducts were effective blocks for polymerase action. E. coli strains JM103 and JM103 uvrA6 were subsequently transformed with control (unadducted) and adduct-containing M13mp7L2 constructs followed by analysis of progeny DNA. In both JM103 and JM103 uvrA6 cells, plaque yields were markedly reduced with adduct containing vectors compared to control vectors. Activation of the inducible bacterial DNA repair system (SOS) by UV light only slightly increased the number of plaques recovered from either bacterial strain transformed with adduct-containing vectors. Targeted mutations were obtained with both adduct-containing vectors in both bacterial strains, whereas no mutations were detected in plaques recovered from control M13mp7L2 vectors. In JM103 cells, (+)anti-DB[a,j]A-DE--N6-dAdo induced exclusively A --> t transversions and (+)anti-DB[a,j]A-DE--N2-dGuo induced exclusively G --> T transversions. In JM103 uvrA6 cells, similar targeted transversion mutations were also obtained except that a few C deletions (i.e., aprroximately 10% of the mutations) were detected immediately 3' to the dAdo adduct. While mutagenesis was SOS dependent in JM103 cells [<0.15% (-SOS) vs approximately 1.3% (+SOS)], it appeared to be SOS independent in JM103 uvrA6 cells (approximately 1-2% in the presence or absence of SOS induction). It is argued that adduct-induced G --> T mutations can be rationalized by either misinformational or noninformational mechanisms. In contrast, A --> T mutations are unlikely to arise via a misinformational pathway, which provides the strongest support to date that bulky DNA adducts can induce mutations via a noninformational pathway.

Adenine↗

PubMed1996

Polycyclic aromatic hydrocarbon quinone-mediated oxidation reduction cycling catalysed by a human placental 17beta-hydroxysteroid dehydrogenase.

The human placental 17beta-hydroxysteroid dehydrogenase reduces a number of polycyclic aromatic hydrocarbon (PAH) o-quinones; some of the quinones undergo redox cycling at rates that approach or exceed the rate of reduction of estrone by the enzyme. The non-K-region o-quinone, 7,8-benzo[a]pyrenequinone, is the best o-quinone substrate tested. Cycling of all the quinone substrates is inhibited by superoxide dismutase; cycling is also inhibited by 17beta-estradiol and other estrogens. Since 19 alpha-estradiol is a competitive inhibitor of 9,10-phenanthrenequinone by the 17beta-hydroxysteroid dehydrogenase, it is likely that both reactions occur at the same active site on the enzyme. In the presence of the 17beta-hydroxysteroid dehydrogenase, the equilibrium between 17beta-estradiol, estrone, NADP, and NADPH is shifted by 7,8-benzo[a]pyrenequinone because the rapid redox cycling of this quinone results in the oxidation of NADPH. Unlike a number of hydroxysteroid dehydrogenases, the placental 17beta-hydroxysteroid dehydrogenase does not oxidize any of the six PAH trans-dihydrodiols tested.

17-Hydroxysteroid Dehydrogenases↗

PubMed1996

Cytotoxicity and mutagenicity of polycyclic aromatic hydrocarbon ortho-quinones produced by dihydrodiol dehydrogenase.

Eight polycyclic aromatic hydrocarbon (PAH) ortho-quinones that can be generated by dihydrodiol dehydrogenase (DD) were examined for their cytotoxicity in H-4-II-e (rat hepatoma) cells and for their mutagenicity in the Ames test. Seven of the PAH otrtho-quinones were potent cytotoxins yielding IC50 values for cell survival in the range 1-30 microns. PAH ortho-quinones were grouped into three classes based on their cytotoxicity profiles: group I contained ortho-quinones (e.g., naphthalene-1,2-dione and 7,12-dimethylbenz[alpha]anthracene-3,4-dione) which reduced cell viability and cell survival; group II contained ortho-quinones (e.g., benz[alpha]anthracene-3,4-dione and 5-methylchrysene-1,2-dione which reduced cell survival but had no effect on cell viability; and group III contained ortho-quinones (e.g., benzo[alpha]pyrene-7,8-dione) which had a pronounced effect on cell viability but minimal effects on cell survival. Using hepatoma cell suspensions and rat liver subcellular fractions, it was found that ortho-quinones underwent preferential enzymatic one-electron redox-cycling and produced superoxide anion radical (O2-.) and/or ortho-semiquinone anion or alternant radicals. ortho-Quinones that reduced cell viability produced O2-. and caused the most total free radical formation, while those that reduced cell survival produced ortho-semiquinone anion or alternant radicals only. PAH ortho-quinones were also tested as direct-acting mutagens in Salmonella typhimurium tester strains TA97a, TA98, TA100, TA102 and TA104. They were found to be more mutagenic than the test mutagens used for each tester strain, and were predominantly frameshift mutagens. The presence of an activating system (Aroclor-induced rat liver S9 plus NADPH) did not increase the mutagenicity of ortho-quinones in tester strains that are sensitive to oxidative mutagens (TA102 and TA104). These data suggest that PAH ortho-quinones produced by DD are cytotoxic and mutagenic by different mechanisms. The mechanism of cytotoxicity involves the formation of reactive oxygen species and/or ortho-semiquinone anion or alternant radicals. The mechanism of mutagenicity is independent of free radical formation and is related to the ability of PAH orthooffinones to intercalate and covalently modify DNA.

Animals↗

PubMed1996

The effect of fluoro substituents on reactivity of 7-methylbenz[a]anthracene diol epoxides.

The present study has examined potential mechanisms for the influence of F-substituents on the biologic activity of methylbenz[a]anthracenes. DNA adducts derived from reaction of the racemic bay-region anti-diol epoxides of 7-methylbenz[a]anthracene, and its 9- and 10- fluoro derivatives, with calf thymus DNA in vitro were partially characterized. All three hydrocarbon diol epoxides produced similar DNA adduct profiles upon reaction with calf thymus DNA in vitro that were composed of two deoxyganosine and two deoxyadenosine adducts (tentatively identified as trans addition products). The extent of covalent binding to calf thymus DNA, as estimated by 32P-postlabeling, was similar for all three diol epoxides. The reactivity of the unsubstituted and 10-F-substituted diol epoxide was further assessed by measuring overall pseudo-first-order rate constants for hydrolysis in water or 0.1 M Tris-HCl buffer, pH 7.0, and in the presence or absence of native or denatured DNA. The rate constant for hydrolysis of 7-methylbenz[a]anthracene diol epoxide in the absence of DNA was similar to that of 10-F-7-methylbenz[a]anthracene diol epoxide (t1/2 = 138 min vs 115 min in water, respectively, and 93 vs 83 min in 0.1 M Tris-HCl buffer, respectively). In addition, the presence of DNA accelerated hydrolysis rates to similar extents for both diol expoxides. The skin tumor-initiating activities of the 9- and 10-F-substituted 3,4-diols of 7-methyl-, 12-methyl-, and 7,12-dimethylbenz[a]anthracene were determined in SENCAR mice. The presence of F-substituents in the 9- or 10- position did not enhance or in some cases reduced the tumor-initiating activity of the 3,4-diols of these hydrocarbons. Collectively, these results, as well as previous results from our laboratory, suggest that the influence of a F-substituent at position 10 of the benz[a]anthracene nucleus is not due to increased or altered reactivity of the bay-region diol epoxide but rather likely on the initial formation of the 3,4-diol.

Animals↗

PubMed1996

Using UvrABC nuclease to detect 7,12-dimethylbenz[a]anthracene anti-diol epoxide-DNA binding specificity in the mouse H-ras gene.

DNA fragments modified with chemically synthesized 7,12-dimethylbenz[a]anthracene anti-diol epoxide (anti-DMBADE) are sensitive to UvrABC nuclease incision. The incisions occur mainly 7 bases 5' and 4 bases 3' of an anti-DMBADE-modified adenine or guanine residue, and the kinetics of incision at different sequences in a DNA fragment are the same. These results indicate that UvrABC incision on anti-DMBADE-DNA adducts is independent of DNA sequences and is quantitative, the same as on syn-DMBADE-DNA adducts. This method was used to analyze the anti-DMBADE-DNA binding spectrum in the exon 2 region of the mouse H-ras gene, and it was found that anti-DMBADE binds to the two adenine residues at codon 61 of the H-ras gene with an average affinity. Previously, we have demonstrated that syn-DMBADE binds strongly to the adenines at codon 61 of H-ras; these results together suggest that the oncogenic mutation in H-ras may be induced by anti- and syn-DMBADE-DNA adducts.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1996

The structure of a coumarin derivative related to the carcinogen benz[a]anthracene.

The three-dimensional structure of 3-methyl-2H-anthra[1,2-b]pyran-2-one, an anticarcinogenic coumarin related to the carcinogen benz[a]anthracene, has been determined by X-ray diffraction techniques. The molecule, apart from hydrogen atoms in the methyl group, is flat, the maximum deviation from its least squares best plane being 0.13 angstroms. The carbonyl C=O bond length is normal [1.206(1) angstroms] and the bonding throughout the molecule indicates localization of double bonds within the coumarin ring, but some delocalization of electrons in the other rings. Molecules pack in planes parallel to each other, the coumarin ring oxygen atom lying between two aromatic rings of other coumarin molecules. The bulky methyl groups are not involved in such stacking, while the carbonyl groups attract C-H groups in neighboring molecules by way of C-H...O interactions. These are the types of interactions that such coumarins could make if they bound to hydrophobic areas in biological macromolecules.

Antineoplastic Agents↗

PubMed1996

Bay-region distortions in a methanol adduct of a bay-region diol epoxide of the carcinogen 5-methylchrysene.

The three-dimensional structure of the product of the reaction of a diol epoxide of the carcinogen 5-methylchrysene with methanol has been determined by an X-ray diffraction analysis. The diol epoxide used to obtain this compound contains a stereochemically hindered bay region because of the location of the 5-methyl group, and this might be expected to affect the type of chemical reaction that occurs. The crystal structure analysis of this adduct of a polycyclic aromatic hydrocarbon (PAH) showed that a methoxy group has been added at the carbon atom of the epoxy group that is nearest to the aromatic system. The bond that is formed is axial to the ring system so that the carbon and hydrogen atoms of the methoxy group are considerably displaced from the PAH ring plane. The bay-region methyl group at position 5 is displaced out of the ring plane in the opposite direction. The major steric distortion in this methanol adduct is shown, by a comparison with crystal structures of related non-methylated compounds, to be in the area of the 5-methyl group and not in the tetrol-bearing ring. The steric effects that caused the axial conformation of the newly formed bond would also be expected to pertain in the DNA adduct of a PAH with a bay-region methyl group. Since the presence of the bay-region methyl group in 5-methylchrysene has been shown to enhance the carcinogenicity of this PAH over the parent compound or compounds with methyl groups in other positions of the molecule, it might be anticipated that this axial bond is found in carcinogenic lesions in DNA, and that any factor that ensures this axial conformation may accentuate the carcinogenic potential of a PAH of the appropriate size.

Carcinogens↗

PubMed1996

The identification and repair of DNA adducts induced by waterborne benzo[a]pyrene in developing Xenopus laevis larvae.

We report on the formation and subsequent repair of benzo[a]pyrene-induced DNA adducts in Xenopus laevis larvae in vivo, as monitored by 32P-post labelling. In vivo benzo[a]pyrene is metabolized by the cytochrome P450 family of enzymes to metabolites, of which the 7,8-diol-9,10-epoxides have been implicated as causing potentially tumourigenic lesions. Larvae were exposed to waterborne benzo[a]pyrene (0.01, 0.05 and 0.1 mg/l) for 24 h at stages 38, 45 and 50 of development (24 h, 5 days and 2 weeks post-hatching, respectively) and allowed to recover for up to 6 days. A wide range of adduct lesions were observed at stage 50, three of which were observed at all stages investigated. Adduct repair was biphasic, with an initial rapid repair over the first 24 h post exposure, followed by a much slower decline, resulting in persistence of adducts for at least 6 days post exposure. The individual lesions were repaired at different rates, with some being almost completely repaired after 6 days recovery, whereas one of the main adducts showed restricted repair at stage 50 and another no repair at all. Identification of some adducts has been achieved, by the inclusion of isomeric standards of (+)- or (-)-anti-benzo[a]pyrene diol epoxide reacted with deoxyguanosine and adenosine 3'-monophosphates prepared in vitro. The non-repairable lesion at stage 50 has been shown to be the (+)-trans-anti-benzo[a]pyrene diol epoxide-N2-guanine adduct. This adduct was observed at all stages, but was only maximally repaired at stages 38 and 45.

Amphibians↗

PubMed1995

4,5-Dihydrobenzo[a]pyrene-4,5-trans-(e,e)-diol.

The title compound, C20H14O2, is a metabolite of the chemical carcinogen benzo[a]pyrene. The crystal structure consists of groups of molecules in each of which both hydroxy groups donate and accept hydrogen bonds. Stacking of dihydrobenzo[a]pyrene ring systems occurs along the crystallographic c axis.

Benzo(a)pyrene↗

PubMed1995

Solution conformation of the (-)-trans-anti-5-methylchrysene-dG adduct opposite dC in a DNA duplex: DNA bending associated with wedging of the methyl group of 5-methylchrysene to the 3'-side of the modification site.

This paper reports on NMR-molecular mechanics structural studies of the (-)-trans-anti-[MC]dG adduct positioned opposite dC in the sequence context of the d(C1-C2-A3-T4-C5-[MC]G6-C7-T8-A9-C10-C11).d(G12-G13-T14++ +-A15-G16-C17-G18- A19-T20-G21-G22) duplex [designated (-)-trans-anti-[MC]dG.dC 11-mer duplex]. This adduct is derived from the trans addition at C4 of (-)-anti-1(S),2(R)-dihydroxy-3(R),4(S)-epoxy-1,2,3,4-tetrahydro-5-met hylchrysen e [(-)-anti-5-MeCDE] to the N2 position of dG6 in this duplex sequence. The 5-methyl group is located adjacent to the MC(C4) binding site, with these groups juxtaposed in a sterically crowded bay region in the adduct duplex. The 5-methylchrysenyl and the nucleic acid exchangeable and nonexchangeable protons were assigned following analysis of two-dimensional NMR data sets in H2O and D2O buffer solution. The solution structure of the (-)-trans-anti-[MC]dG.dC 11-mer duplex has been determined by incorporating DNA-DNA and carcinogen-DNA proton-proton distances defined by lower and upper bounds deduced from NOESY data sets as restraints in molecular mechanics computations in torsion angle space. The results establish that the [MC]dG6.dC17 base pair and flanking dC5.dG18 and dC7.dG16 base pairs retain Watson-Crick alignments upon adduct formation. The aromatic chrysenyl ring is positioned in the minor groove of a right-handed B-DNA helix and stacks predominantly over the sugar of the dC17 residue across from it on the unmodified complementary strand. The chrysenyl ring points toward the 3'-end of the modified strand with its 5-methyl group inserting between the modified [MC]dG6.dC17 and dC7.dG16 base pairs. The adduct duplex bends by approximately 47 degrees as a result of the wedged insertion of the 5-methyl group from the minor groove face of the duplex. The solution structure of the (-)-trans-anti-[MC] dG.dC 11-mer duplex is compared with that of the corresponding (-)-trans-anti-[BP]dG.dC 11-mer [De los Santos et al. (1992) Biochemistry 31, 5245-5252] in which the [BP]dG adduct is derived from the binding of (-)-anti-BPDE [7(S),8(R)-dihydroxy-9(R),10(S)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene] to the N2 position in the same DNA sequence context. Although the solution structures of the (-)-trans-anti-stereoisomers of 5-methylchrysenyl-dG and benzo[a]pyrenyl-dG adducts opposite dC exhibit many features in common with each other, the [MC]dG adduct which contains a bay region methyl group bends the DNA helix to a greater extent than in the corresponding [BP]dG adduct, which lacks a bay region methyl group.(ABSTRACT TRUNCATED AT 400 WORDS)

Chrysenes↗

PubMed1995

Covalent DNA adducts formed in mouse epidermis from dibenz[a,j]anthracene: evidence for the formation of polar adducts.

The formation of deoxyribonucleoside adducts in mouse epidermis has been examined following topical application of [3H]dibenz[a,j]anthracene (DB[a,j]A) or by 32P-postlabeling following topical application of unlabeled DB[a,j]A, DB[a,j]A trans-3,4-diol or the anti- or syn-3,4-diol 1,2-epoxides. A single topical application of [3H]DB[a,j]A at a dose of 400 nmol per mouse led to the formation of 11 detectable covalent DNA adducts. Seven of these DNA adducts were tentatively identified based on cochromatography with marker adducts using high-pressure liquid chromatography (HPLC). The presence of both deoxyguanosine (dGuo) as well as deoxyadenosine (dAdo) adducts formed from bay-region anti- and syn-3,4-diol 1,2-epoxides of DB[a,j]A was revealed. The major bay-region diol epoxide DNA adduct formed in mouse epidermis following topical application of [3H]DB[a,j]A was tentatively identified as the (4R,3S)-diol (2S,1R)-epoxide bound through trans addition of the exocyclic amino group of dGuo, although substantial amounts of the corresponding dAdo adduct were also detected. In addition, a K-region 5,6-oxide-dAdo adduct was tentatively identified in HPLC chromatograms based on cochromatography with an authentic marker adduct. 32P-Postlabeling analysis of DB[a,j]A-DNA adducts formed in mouse epidermis after topical application of unlabeled compound confirmed the presence of bay-region diol epoxide DNA adducts similar to those observed after application of [3H]DB[a,j]A. However, 32P-postlabeling analysis also revealed the presence of more polar covalent DNA adducts in epidermal DNA samples from DB[a,j]A-treated mice. These more polar DNA adducts represented a significant proportion of the 32P-labeled material recovered in HPLC chromatograms. While the exact nature of these adducts remains unknown at present, they had retention times identical to polar DNA adducts formed following topical application of DB[a,j]A trans-3,4-diol and may represent bis-dihydrodiol epoxide DNA adducts. The present results indicate that a rather broad spectrum of DNA adducts arises following topical application of DB[a,j]A to mouse epidermis.

Animals↗

PubMed1995

Characterization of DNA adducts formed by anti-benzo[g]chrysene 11,12-dihydrodiol 13,14-epoxide.

The anti-11,12-dihydrodiol 13,14-epoxide of benzo[g]chrysene, a fjord-region-containing hydrocarbon, was found to react with DNA in vitro to yield, as the major product, an adduct in which the epoxide of the 11R, 12S, 13S, 14R enantiomer was opened trans by the amino group of deoxyadenosine. The structures of this adduct and other deoxyadenosine and deoxyguanosine adducts were established by spectroscopic methods. In reactions with deoxyguanylic acid, a product tentatively identified as a 7-substituted guanine was also detected. The mutagenic properties of this dihydrodiol epoxide in shuttle vector pSP189 showed that mutation at AT pairs accounted for 39% of base change mutations whereas chemical findings indicated that about 60% of adducts formed in calf thymus DNA involved adenines. Since calf thymus DNA is 56% AT and the target supF gene is 41% AT, the findings represent a fairly close relationship between adduct formation and mutagenic response. Overall, the chemical and mutagenic selectivities for the two purine bases in DNA were similar, though not identical, to those for the only other fjord-region-containing hydrocarbon studied in depth, i.e., benzo[c]phenanthrene. A major difference for these two hydrocarbon derivatives, however, is that benzo[c]phenanthrene dihydrodiol epoxides react to much higher extents (approximately 4-fold) with DNA than did the benzo[g]chrysene derivative.

Base Sequence↗

PubMed1995

Identification of benzo[a]pyrene-7,8-dione as an authentic metabolite of (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene in isolated rat hepatocytes.

Dihydrodiol dehydrogenase (DD) has been shown to catalyze the oxidation of (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo [a]pyrene (BP-diol) to yield benzo[a]pyrene-7,8-dione (BPQ) in uninduced fortified rat liver S100 fractions but the formation of BPQ has not been observed in whole cells. In these studies [3H]BP-diol was incubated with isolated hepatocytes from uninduced rats for 0-20 min at 37 degrees C. Organic-extractable radioactivity in the cell media accounted for 20% of the total [3H]BP-diol added. Reverse phase (RP)-HPLC analysis of this fraction revealed the formation of an unknown metabolite that co-chromatographed with an authentic synthetic standard of BPQ. The identity of the unknown metabolite was further established by: (i) co-chromatography with synthetic BPQ under both RP- and normal phase-HPLC conditions using diode array detection, which indicated that metabolite shared UV/vis spectral identity with standard BPQ; and by (ii) electron impact mass spectrometry of the unknown metabolite which gave the same parent and fragment ions as the synthetic standard. The formation of BPQ by isolated hepatocytes was found to be 0.50 nmol/3 x 10(6) cells/10 min, and represented 7% of the total organic-soluble metabolites in the extracellular media. Its formation was abolished by the addition of indomethacin, a competitive inhibitor of DD, indicating that this enzyme was responsible for BPQ formation. Other organic-soluble metabolites formed corresponded to BP-tetraols (hydrolysis products of the anti- and syn-diol epoxides). Examination of the aqueous phase of the extracellular media indicated that a large portion of BP-diol was converted to glucuronide and sulfate conjugates. Under the conditions employed BP-tetraols and BPQ were formed to an equal extent implying that in hepatocytes isolated from uninduced rats, DD and CYP1A1 contributed equally to the metabolism of BP-diol.

Alcohol Oxidoreductases↗