DNA adduct formation by polycyclic aromatic hydrocarbon dihydrodiol epoxides.
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DNA↗
Biomedical subjects
A Dipple
Publications and source records attributed to A Dipple.
Effect of cadmium exposure on background and anti-5 methylchrysene-1,2-dihydrodiol 3,4-epoxide-induced mutagenesis in the supF gene of pS189 in human Ad293 cells.
Cadmium is a toxic environmental contaminant that is carcinogenic in humans and rodents. Although cadmium has proven to be mutagenic in a variety of assay systems, exactly how cadmium achieves gentoxicity is poorly understood. To define the mechanism(s) underlying the mutagenicity and comutagenicity of cadmium, human Ad293 cells were exposed to subtoxic doses of the metal and transfected with untreated or anti-5-methylchrysene-3,4-dihydrodiol 1,2-epoxide (5-MCDE)-treated pS189 shuttle vector. Alterations in the frequency, types, and distribution of mutations were subsequently assessed in the supF gene of pS189 that was replicated in Ad293 cells and screened in indicator bacteria. Doses of 0.5 and 1 microM CdCl2 increased the mutation frequency of untreated pS189 by approximately 4- and 8-fold, respectively, with no apparent effect on the types of mutations generated. In contrast, host-cell exposure to cadmium had little or no effect on the frequency, types, or distribution of mutations generated with 5-MCDE-treated pS189. These results indicate that cadmium increases mutagenesis of untreated pS189 by affecting a process that is not involved in mutagenesis of the 5-MCDE-treated vector. Although it is not clear exactly how host-cell exposure to cadmium increases background mutagenesis, presumably, the mutagenic effect does not involve cadmium interaction with the cellular machinery used to replicate past bulky DNA lesions.
Base Sequence↗
Deamination and Dimroth rearrangement of deoxyadenosine-styrene oxide adducts in DNA.
In reactions between styrene oxide and the ring nitrogen at the 1-position of deoxyadenosine, the epoxide is opened at both the alpha- (benzylic) and beta-carbons. The 1-substituted nucleosides formed are unstable and subsequently undergo either Dimroth rearrangement to give N6-substituted deoxyadenosines or deamination to give 1-substituted deoxyinosines. alphaN6-Substituted compounds are also formed from direct reaction at the exocyclic nitrogen. Kinetic experiments revealed that relative rates of deamination of 1-substituted deoxyadenosine-styrene oxides and 1-substituted adenosine-styrene oxides were similar. However, the rate of Dimroth rearrangement in beta1-substituted adenosine-styrene oxides was approximately 2.3-fold greater than that of beta1-substituted deoxyadenosine-styrene oxides and approximately 1.5-fold greater in alpha1-substituted adenosine-styrene oxides relative to alpha1-substituted deoxyadenosine-styrene oxides. Analysis of the products formed from reactions of styrene oxide with [3H]deoxyadenosine and [3H]deoxyadenosine incorporated into native and denatured DNA showed that the double-helical DNA structure reduced the levels of adducts formed 5-fold relative to denatured DNA but did not present a complete barrier to formation of either N6-substituted deoxyadenosine- or 1-substituted deoxyinosine-styrene oxide adducts in native DNA. Additionally, in denatured and native DNA the product distributions were altered in favor of formation of beta1-substituted deoxyinosine-styrene oxide adducts with respect to reactions of the nucleoside. The ratio of retained to inverted configuration of alphaN6-substituted products was higher in DNA than in nucleoside reactions. These experiments indicate that in addition to the N6-position, the ring nitrogen at the 1-position of deoxyadenosine is available, to some extent, for reaction in native DNA. In styrene oxide-DNA reactions, formation of 1-substituted adenines can lead to deaminated products where both Watson-Crick hydrogen-bonding sites are disrupted.
Chromatography, High Pressure Liquid↗
Effect of single benzo[a]pyrene diol epoxide-deoxyguanosine adducts on the action of DNA polymerases in vitro.
Neither Sequenase 2.0 nor Klenow fragment were able to extend 12-mer primers using the eight templates (16-mers) derived by placing each of the four isomeric benzo[a]pyrene diol epoxide-deoxyguanosine adducts at the 13th nucleotide from the 3'-end of two different sequence contexts. Using an 11-mer primer to get a running start did not overcome the adduct induced block of primer extension except for the Klenow fragment and one of the two sequence contexts, indicating primer extension is dependent on both the polymerase and sequence context. In this case, purine nucleoside triphosphates (dATP>dGTP) were incorporated opposite each of the four adducts.
7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
Application of mixed mobile phases and a step gradient method in capillary electrochromatography for the separation of isomeric polycyclic aromatic hydrocarbon-deoxyribonucleoside adduct mixtures prepared in vitro.
Capillary electrochromatography (CEC) was used for the analysis of mixtures of neutral isomeric compounds derived from the reaction of carcinogenic hydrocarbon (benzo[g]chrysene and 5,6-dimethylchrysene) dihydrodiol epoxides with calf thymus deoxyribonucleic acid (DNA). The CEC analysis demonstrated higher resolution, greater speed and lower analyte consumption than high-performance liquid chromatography (HPLC) in the analysis of the same samples using the same type of stationary phase. Proper selection of the mixed mobile phases was critical for the separation of these complex mixtures with enhanced speed and selectivity. The use of a step gradient further improved the speed of the CEC analysis resulting in electrochromatograms that required only 25-70% of the corresponding HPLC analysis times.
DNA Adducts↗
Metabolic activation of benzo[g]chrysene in the human mammary carcinoma cell line MCF-7.
Benzo[g]chrysene (BgC) is an environmental pollutant, and recent studies have demonstrated that anti- BgC-11,12-dihydrodiol 13,14-epoxide (anti-BgCDE) is a potent mammary carcinogen in rats. To determine whether BgC can be metabolically activated to anti-BgCDE in human cells, the human mammary carcinoma cell line MCF-7 was treated with BgC and with the racemic trans-3,4- and 11,12-dihydrodiols. The DNA adducts formed in these experiments were examined using 32P-postlabeling, and specific adducts were identified through comparisons with adducts obtained by the reaction of the racemic syn- and anti-BgCDEs with calf thymus DNA and with purine deoxyribonucleoside-3'-phosphates in vitro. It was found that BgC is metabolically activated in MCF-7 cells to form major DNA adducts through both the syn- and anti-11,12-dihydrodiol 13,14-epoxide metabolites. BgC is therefore a potential environmental risk to humans. The major BgC-DNA adducts formed from both the dihydrodiol-epoxide diastereomers were deoxyadenosine adducts. Thus, BgC has DNA-binding properties that are very similar to those of the potent mammary carcinogens 7,12-dimethylbenz[a]anthracene and dibenzo[a,l]pyrene.
Biotransformation↗
Characterization of DNA adducts formed by the four configurationally isomeric 5,6-dimethylchrysene 1,2-dihydrodiol 3,4-epoxides.
The DNA adducts formed from the racemic syn and anti dihydrodiol epoxides of 5,6-dimethylchrysene were characterized through various spectroscopic methods. Substantial reaction with the amino groups of both deoxyadenosine and deoxyguanosine residues were detected with both the syn and anti derivatives. The chemical shifts and coupling constants for the cis and trans opened adducts from the syn dihydrodiol epoxide were distinctly different, whereas for the anti dihydrodiol epoxide these properties were fairly similar for cis and trans adducts. In the latter case, assignment of trans and cis configurations was less obvious, and the finding that trans adducts have always predominated over cis adducts for all dihydrodiol epoxides studied to date was helpful in making these assignments. The preferential formation of cis adducts in DNA by the syn dihydrodiol epoxide is more like the chemistry of the dihydrodiol epoxide of benzo[c]phenanthrene than of benzo[g]chrysene, although both of these, like 5,6-dimethylchrysene, are non-planar compounds.
Animals↗
Investigation of hydrolytic deamination of 1-(2-hydroxy-1-phenylethyl)adenosine.
The ring nitrogen of adenosine reacts at both the alpha- (benzylic) and beta-carbons of styrene oxide to form 1-substituted products. The 1-(2-hydroxy-1-phenylethyl)adenosines formed by oxirane ring opening at the alpha-position are prone to an unusually facile hydrolytic deamination. By conducting hydrolysis reactions in [18O]water and analyzing the reaction products by electrospray mass spectrometry, we find that deamination occurs by direct attack of water at the 6-position of the adenine ring system with displacement of the exocyclic amino group.
Adenosine↗
Cellular response to DNA damage from a potent carcinogen involves stabilization of p53 without induction of p21(waf1/cip1).
The effect of a potent mammary carcinogen, anti benzo[g]chrysene 11,12-dihydrodiol 13,14-epoxide, on the progress of human mammary carcinoma MCF-7 cells through the cell cycle was investigated. While these cells, which express wild-type p53, were arrested in G1 after treatment with actinomycin D (a positive control), treatment with the mammary carcinogen did not cause G1 arrest but instead delayed the cells in the DNA synthesis phase. In concert with the absence of a G1 arrest, it was found that though both chemical treatments led to increased levels of p53, only the p53 induced by actinomycin D was transcriptionally active and increased the levels of the cyclin dependent kinase inhibitor, p21(waf1/cip1). Since treatment of the cells with the mammary carcinogen did not abrogate the G1 arrest induced by actinomycin D, the lack of p21(waf1/cip1) and of G1 arrest, resulting from treatment with the mammary carcinogen alone, was not due to some general inhibition of transcription or translation. An analogous difference between these two chemicals was demonstrated also in other human cell systems. The stealth-like property of the mammary carcinogen that allows it to damage DNA without turning on the cells' 'guardian of the genome' defense mechanism presumably increases the likelihood of malignant change because DNA replication continues on a damaged template. It is suggested that this stealth characteristic may be a major contributor to the high carcinogenic potency of this mammary carcinogen and possibly to that of other highly potent carcinogens.
Carcinogens↗
Relative mutagenicities of gaseous nitrogen oxides in the supF gene of pSP189.
Gaseous nitric oxide (NO), an environmental pollutant found in cigarette smoke and diesel exhaust, has been shown to generate mutations in aerobic in vitro assays. The objective of this study was to identify which oxides of nitrogen, formed in the gaseous phase from NO, possess mutagenic activity. Samples of the plasmid pSP189, in 1 M HEPES buffer, pH 7.4, were exposed to preparations of nitrogen dioxide (NO2), dinitrogen trioxide (N2O3) or an air control. The gas mixtures were formed in a gas-tight syringe and were then introduced into 1 l flasks. The plasmid solution was introduced immediately afterwards. Transformation of Escherichia coli strain MBM7070 with the treated plasmids allowed analysis of mutation frequencies and the types of mutations induced in the target supF gene. The mutation frequency resulting from NO2 exposure was not different from that of the control. However, N2O3 produced a substantial number of mutations. The mutation frequency and the types of mutations were found to depend on the length of time that the gases were allowed to incubate in the syringe prior to introduction into the 1 l flasks (mutation frequency was maximal at approximately 2 min). The most prevalent mutations were AT-->GC transitions (68%), followed by GC-->AT transitions (30%), similar to previous findings when pure NO was bubbled through pSP189 solutions. These results suggest that it is N2O3, rather than NO2, that is the most likely source of mutagenic potential from gaseous nitrogen oxides.
Base Sequence↗
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↗
Effect of aflatoxin B1-8,9-epoxide-DNA adducts on transcription of a supF gene fragment.
A linearized template, obtained from the vector pGEM-3Zf(+) containing a supF gene fragment, was treated with aflatoxin B1-8,9-epoxide (AFB1 epoxide) and transcription in vitro was then studied. The template functions of both strands of the supF gene were similarly inhibited as shown by transcription with both T7 and SP6 RNA polymerases. This inhibition was dose-dependent and affected the elongation step more extensively than the initiation step. Gel electrophoretic analysis of RNA formed by T7 RNA polymerase indicated that template treated with different AFB1 epoxide doses yielded the same three major truncated RNA fragments. Sequence analysis showed that these major sites of RNA truncation occurred in the vicinity of adjacent guanine residues in the template.
Aflatoxin B1↗
Mutagenic specificities and adduct distributions for 7-bromomethylbenz[a]anthracenes.
Mutation induction in the supF gene of the plasmid pS189 by 7-bromomethylbenz[a]anthracene and 7-bromomethyl-12-methylbenz[a]anthracene was examined. The former compound was substantially more mutagenic than the latter but a much greater proportion of the total mutations were located at mutation hotspots for the 12-methyl derivative. The overall correlation between sites of mutation and sites of polymerase arrest (an indicator of adduct formation) through the supF gene was poor. Although these bromocompounds should form only a single guanine adduct (unlike dihydrodiol epoxides that form both cis and trans adducts) more than one mutational change was found at a given site, although the predominant base substitution was G-->T for either compound.
Base Sequence↗
Benzo[c]phenanthrene 3,4-dihydrodiol 1,2-epoxide adducts in native and denatured DNA.
High performance liquid chromatography/UV-absorption and 32P-postlabeling were used to quantitate adducts generated by reaction of the four configurationally isomeric benzo[c]phenanthrene 3,4-dihydrodiol 1,2-epoxides with native or denatured DNA in vitro. For both the 4R, 3S-dihydrodiol 2S,1R-epoxide and the 4S,3R-dihydrodiol 2S,1R-epoxide, the amount of product resulting from trans-opening of the epoxide ring by the exocyclic amino group of deoxyadenosine in denatured DNA was much less than the level found in native DNA, indicating that the native DNA structure probably intercalates the hydrocarbon residue in a fashion that promotes adenine reaction for 2S,1R-epoxides.
Animals↗
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↗
DNA adducts of chemical carcinogens.
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Alkylation↗
Polycyclic aromatic hydrocarbons: chemistry of DNA adduct formation.
Polycyclic aromatic hydrocarbon carcinogens are usually activated for DNA binding by the metabolic formation of bay region dihydrodiol epoxides with R,S,S,R stereochemistry. Such metabolites from planar hydrocarbons reacted preferentially with the amino groups of deoxyguanosine residues, whereas those from nonplanar hydrocarbons were more efficiently trapped by DNA and reacted preferentially with the amino groups of deoxyadenosine residues, in some cases. Molecular modeling and NMR measurements indicated that the conformations of DNA adducts depend upon the hydrocarbon involved and the cis or trans opening of the epoxide ring during adduct formation. The structural characterization of carcinogen-DNA adducts and investigations of relationships between specific adducts and biological effects represent an important background that can be valuable in molecular epidemiological approaches.
DNA Adducts↗
Primer extension by various polymerases using oligonucleotide templates containing stereoisomeric benzo[a]pyrene-deoxyadenosine adducts.
Four isomeric benzo[a]pyrene-deoxyadenosine adducts, corresponding to the products of trans opening of the epoxide ring in the four configurationally isomeric benzo[a]pyrene dihydrodiol epoxides by the amino group of deoxyadenosine, were separately introduced into each of two 16-mer sequence contexts. The sequences were from the supF gene, and the site of the adducted adenine was known, for some hydrocarbon dihydrodiol epoxides, to be a hotspot for mutation in Context I and a coldspot for mutation in Context II. Using primers complementary to the 3' ends of these oligonucleotides, the abilities of several polymerases to replicate these templates in vitro were investigated. Each adduct proved to be an effective block to primer extension such that only with high concentrations of exo- Klenow fragment was any bypass of adducts seen. DNA polymerase alpha and HIV-1 reverse transcriptase were blocked 3' to the adduct when the configuration at C10 of the hdyrocarbon was S, and some introduction of thymine opposite the adenine adduct was seen with the R configuration. Incorporation of a nucleotide opposite the adduct occurred more readily with Sequenase and the Klenow fragment, and the mutagenic introduction of adenine was apparent in most cases. This corresponded to the A-->T transversions frequently seen in mutation studies with hydrocarbon dihydrodiol epoxides that react extensively with adenine in DNA. Overall, it was clear that sequence context, adduct stereochemistry, and the choice of polymerase all influenced the polymerization reaction. With these in vitro systems, no major differences correlating with the differing tumorigenicities of the isomeric dihydrodiol epoxides or with the hotspot or coldspot nature of the sequences were detected.
Base Sequence↗