PubMed Health⌕ Search

Biomedical subjects

A Dipple

Publications and source records attributed to A Dipple.

At least 19 recordsLinked to original sources
PubMed1992

High-performance liquid chromatographic separation of purine deoxyribonucleoside monophosphate-benzo[a]pyrene adducts.

Chromatographic methods that allow the separation of adducts of purine nucleoside 3'-phosphates with the pure enantiomers of the anti-dihydrodiol epoxide of benzo[a]pyrene are developed. The optimization procedure includes evaluation of the effect of buffer molarity, the pH of the buffer, and the role of organic modifiers. The method can be utilized to prepare standards with known absolute configuration that can be further used in the Randerath 32P-postlabeling procedure.

Acetonitriles↗
PubMed1992

Mutagenic specificities of four stereoisomeric benzo[c]phenanthrene dihydrodiol epoxides.

The pS189 shuttle vector carrying a supF target gene was used to compare the mutagenic specificities of the four configurational isomers of benzo[c]phenanthrene 3,4-dihydrodiol 1,2-epoxide. One of these isomers is the most tumorigenic dihydrodiol epoxide tested to date and another is essentially inactive as a tumorigen. Overall mutagenicities were not correlated with tumorigenicities, but each configurational isomer induced a unique spectrum of mutational hot spots in the supF target gene, which monitors primarily point mutations. It is suggested that the demonstrated isomer-specific selectivity for mutation targets within the supF gene may be indicative of a similar selectivity for one gene versus another and that such selectivity may be one determinant of relative tumorigenicity.

Base Sequence↗
PubMed1992

DNA polymerase action on benzo[a]pyrene-DNA adducts.

A 16mer oligonucleotide containing a single guanine residue at nucleotide 13 from the 3' end was treated with the (+)-enantiomer of the 7,8-dihydrodiol 9,10-epoxide of benzo[a]pyrene (B[a]P). Oligonucleotides containing either an adduct in which the epoxide ring was opened trans or cis by the amino group of the guanine residue were separated by chromatography and identified by 32P postlabeling and circular dichroism spectroscopy. In the presence of nucleotide triphosphates and DNA polymerase (either Sequenase, version 2.0 or human polymerase alpha), it was found that the B[a]P adducts inhibited extension of an 11mer primer opposite the nucleotide 3' to the adduct in the template. Under various conditions, this inhibition was greater for the cis adduct than for the trans adduct. After a 10 min incubation with Sequenase, primer extension was reduced to approximately 20% of that seen with unmodified oligonucleotide by the trans adduct and was almost completely inhibited by the cis adduct. When a 12mer primer was used to examine nucleotide incorporation directly across from the guanine or adducted guanine residues, it was clear that deoxycytidylic acid was preferentially incorporated in all cases but that the incorporation was severely inhibited by both the cis and trans adducts. These findings suggest that a cis adduct is a more effective block to replication than a trans adduct, and that these adducts may not be very efficient mutagenic lesions.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1991

Absolute configuration of 7,12-dimethylbenz[a]anthracene-DNA adducts in mouse epidermis.

32P-Postlabeling was used to monitor the formation of DMBA-DNA adducts in mouse epidermis from each enantiomer of the trans 3,4-dihydrodiol. It was shown that the (4R,3R)-dihydrodiol is converted to the anti (4R,3S)-dihydrodiol (2S,1R)-epoxide which reacts with deoxyguanosine and deoxyadenosine residues in epidermal DNA to yield two of the major adducts formed when DMBA itself binds to epidermal DNA. The third major DMBA-derived adduct with deoxyadenosine residues was shown to arise from the (4S,3R)-dihydrodiol through the intermediacy of the syn (4S,3R)-dihydrodiol (2S,1R)-epoxide.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1991

Preferential mutagenesis at G.C base pairs by the anti 3,4-dihydrodiol 1,2-epoxide of 7-methylbenz[a]anthracene.

The racemic anti-dihydrodiol epoxide of 7-methylbenz[a]anthracene preferentially induced mutations at G.C base pairs in the pS189 shuttle vector. Mutations were not randomly distributed throughout the supF target gene, but were concentrated at five hotspots. The hotspots for this agent did not correspond exactly to those produced by any other dihydrodiol epoxide examined to date, indicating that dihydrodiol epoxide structure and reactivity play a major role in determining mutagenic hotspots.

Base Composition↗
PubMed1991

Bypass of a hydrocarbon adduct in an oligonucleotide template mediated by mispairing adjacent to the adduct.

The action of DNA polymerase (Sequenase Version 2.0) on an oligonucleotide template containing a 7-bromomethyl-benz[a]anthracene-deoxyadenosine adduct flanked by thymidine residues was investigated. The polymerase incorporated deoxyadenosine or deoxyguanosine residues opposite the thymidine 3' to the adduct with similar efficiencies. Whereas the normal A.T base pair led to arrest of polymerase progression along the template, formation of the G.T mismatch allowed incorporation of thymidine opposite the adduct and further primer extension. This mispair-mediated bypass was also seen with AMV reverse transcriptase and may represent a novel mechanism for overcoming the replication block of a bulky carcinogen--DNA adduct.

Base Sequence↗
PubMed1991

Identification of (+) and (-) anti benzo[a]pyrene dihydrodiol epoxide-nucleic acid adducts by the 32P-postlabeling assay.

Purine deoxyribonucleoside 3'-phosphates were reacted with the (+)- and (-)-enantiomers of the anti dihydrodiol epoxide of benzo[a]pyrene. Products from cis and trans opening of the epoxide ring were separated by HPLC and they were identified by comparison of their CD spectra with those known for the corresponding nucleoside adducts. Thereafter, the eight known benzo[a]pyrene-purine deoxyribonucleoside-3'-phosphate adducts were postlabeled with [32P]ATP and T4 kinase and the positions of these individual bisphosphates were mapped by TLC. Though all eight adducts migrated to the same general region of the thin layer plates, the four possible adducts from each enantiomeric dihydrodiol epoxide were resolved.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1991

DNA polymerase-mediated nucleotide incorporation adjacent to hydrocarbon-deoxyadenosine and hydrocarbon-deoxyguanosine adducts.

To examine the effect of DNA adducts on nucleotide incorporation by DNA polymerase at 3' neighboring bases, synthetic oligonucleotides (16mers) containing a purine at position 13 from the 3' end and any one of the four possible bases at position 12 were prepared and reacted with 7-bromomethylbenz[a]anthracene. Using HPLC, unmodified oligonucleotide was separated from oligonucleotide containing a single adduct, at either an adenine or a guanine residue. These products were annealed with a 32P 5'-end labeled primer (11mer) and incubated with modified T7 DNA polymerase (Sequence, version 2.0) in the presence of deoxyribonucleoside 5'-triphosphates. Analysis by gel electrophoresis showed that unmodified oligonucleotide template allowed the primer to be rapidly extended to the entire length of the template. However, the presence of an adduct caused primer extension to stop at the base 3' to the adduct. While correct base pairing occurred at this termination site with most adducted templates, there was a high frequency of misincorporation of guanine opposite a thymine located 3' to an adenine adduct. This result suggest that some bulky carcinogen--DNA adducts may lead to base mismatches at neighboring bases.

Autoradiography↗
PubMed1990

DNA polymerase action on bulky deoxyguanosine and deoxyadenosine adducts.

In order to determine how individual hydrocarbon-DNA adducts give rise to specific mutations, a single-stranded oligonucleotide, 5'-T8GT10AT8C2T4CT3CT-3', was reacted with the carcinogen 7-bromomethylbenz[a]anthracene which generates both deoxyguanosine and deoxyadenosine adducts in DNA. The products were separated by HPLC to yield unmodified oligonucleotide and oligonucleotide modified either at the single guanine, or at the single adenine, residue. Incubation of these products with 32P-5'-end-labeled primer, 5'-AGA3GA4G2-3', modified T7 DNA polymerase (Sequenase) and deoxyribonucleoside-5'-triphosphates followed by gel electrophoretic analysis indicated that unmodified oligonucleotide template allowed the primer to be rapidly extended to give species of the same length as the template (40 nucleotides) and of 41 nucleotides in length. However, primer extension for the templates containing the guanine and adenine adducts was held up initially (1 min) at the nucleotide preceding the adduct. At longer times (up to 15 min) a nucleotide was added opposite the adduct and, to a lesser extent, another nucleotide was added beyond this. Some full-length oligonucleotide was also synthesized with these carcinogen-modified templates. When synthesis was allowed to proceed only to the nucleotide preceding the adduct, and this template-extended primer complex incubated with individual nucleotide triphosphates plus Sequenase, it was found that deoxyadenosine residues were most readily incorporated opposite the adduct irrespective of whether it was a deoxyguanosine or deoxyadenosine adduct. These results, which suggest that G.C----T.A and A.T----T.A transversions would be the mutagenic consequences of formation of bulky hydrocarbon adducts at guanines and adenines respectively, are consistent with the most frequent hydrocarbon-induced mutational changes reported thus far.

Base Composition↗
PubMed1990

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

An SV40-based pS189 shuttle vector, which contained a supF target gene and was replicated in human cells (Ad293), was used to determine the mutational specificity of anti 5-methylchrysene 1,2-dihydrodiol 3,4-epoxide, the active metabolite of the environmentally prevalent carcinogen 5-methylchrysene. The frequency of supF mutants containing point mutations increased with dose to approximately 40 times the spontaneous frequency. The induced mutations were not randomly distributed but occurred preferentially at mutagenic hotspots, which were not all identical to those reported by others for benzo[a]pyrene dihydrodiol epoxide, a metabolite with similar chemistry.

Base Sequence↗
PubMed1990

Carcinogenesis.

Explore the source record for details and available documents.

Carcinogens↗
PubMed1989

Mutagenic specificity of a potent carcinogen, benzo[c]phenanthrene (4R,3S)-dihydrodiol (2S,1R)-epoxide, which reacts with adenine and guanine in DNA.

Mutations were induced in the supF gene of the pS189 shuttle vector by treatment with optically active benzo[c]phenanthrene (4R,3S)-dihydrodiol (2S,1R)-epoxide in vitro and replication in human cells. The induced mutation frequency was 60-fold greater than the spontaneous rate, and most of the mutations analyzed were transversions (86%), which principally consisted of similar numbers of A.T----T.A and G.C----T.A changes. The unusual susceptibility of A.T pairs to mutation by this chemical agent is consistent with its chemical reactivity toward adenine and argues that the mutations are targeted to the adducts formed. The central base in the sequences 5'-AGA-3', 5'-AAC-3', and 5'-GAG-3' was particularly susceptible to mutation. Twelve "hotspots" in the supF gene accounted for most mutations seen. Some of these hotspots differed from those found by others for racemic benzo[a]pyrene dihydrodiol epoxide and, even when a hotspot was common, the mutagenic changes were not always the same. Although adenine insertion opposite a noninstructional lesion could account for most of the data, no single mutagenic mechanism could encompass all of it. The cellular machinery that converts chemical damage to mutations must determine the mutational result to a large extent, but the findings herein show that the chemical agent itself plays a large role in determining both the location and the nature of the mutations that arise.

Adenine↗
PubMed1989

Absolute stereochemistry of the major 7,12-dimethylbenz[alpha]anthracene- DNA adducts formed in mouse cells.

In recent work we assigned partial structures to individual 7,12-dimethylbenz[alpha]anthracene (DMBA)--deoxyribonucleoside bisphosphates separated by TLC after postlabeling with [32P]ATP. We have now been able to postlabel DNA adducts formed in cells exposed to either the (4R,3R)- or (4S,3S)-dihydrodiol of DMBA and thereby to assign absolute stereochemistry to the 2-, 3- and 4- positions in the major DMBA-DNA adducts. It is found that the major anti dihydrodiol epoxide-DNA adducts arise from the (4R,3S)-dihydrodiol (2S,1R)-epoxide and that the major syn dihydrodiol epoxide-DNA adducts arise from the (4S,3R)-dihydrodiol (2S,1R)-epoxide.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1988

7,12-Dimethylbenz[a]anthracene-DNA adducts in mouse skin, dermis and epidermis.

Female NIH Swiss mice were treated topically with either 0.01 or 0.1 mumol 7,12-[3H]dimethylbenz[a]anthracene and DNA was isolated either from the whole skin, the dermis or the epidermis. Levels of binding to DNA and levels of individual adducts formed were similar in all 3 tissue fractions for a given dose of carcinogen with levels for the epidermis being marginally greater than in the other fractions. In all tissue fractions, the syn dihydrodiol epoxide-deoxyribonucleoside adducts were responsible for a greater fraction of total binding at the higher, than at the lower, carcinogen dose. The mechanism of metabolic activation of 7,12-dimethylbenz[a]anthracene for DNA binding is, therefore, qualitatively the same in both the dermis and epidermis. Quantification of adducts suggests some subtle differences between the DMBA activating systems in dermis and epidermis.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1988

Comparison of 32P-postlabeling and high pressure liquid chromatographic analyses for 7,12-dimethylbenz[a]anthracene--DNA adducts.

[3H]7,12-Dimethylbenz[a]anthracene-modified DNA obtained from mouse cells in culture was enzymatically hydrolyzed to nucleoside 3'-phosphates, postlabeled with [32P]phosphate, and the carcinogen-modified nucleoside bisphosphates were separated by thin layer chromatography. Each adduct spot was eluted, dephosphorylated and the resulting [3H]nucleoside adducts were analyzed by high pressure liquid chromatography so that the structural information available for the liquid chromatographic peaks could be applied to the spots obtained from the postlabeling procedure. After this cross referencing, specific dihydrodiol epoxide-nucleotide adducts can now be monitored by the postlabeling technique.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1988

A metabolite of the carcinogen 7,12-dimethylbenz[a]anthracene that reacts predominantly with adenine residues in DNA.

Four 7,12-dimethylbenz[a]anthracene--deoxyribonucleoside adducts formed in mouse epidermis in vivo arise from the syn dihydrodiol epoxide metabolite of this carcinogen. With the synthetic syn dihydrodiol epoxide it was possible to identify three of these as deoxyadenosine adducts and to establish their structures. These three adducts account for the large majority of DNA adduct arising from this metabolite in vivo. The in vivo metabolite is unusual, therefore, in that it reacts almost exclusively with adenine residues in DNA while most carcinogen metabolites react preferentially with guanine residues.

9,10-Dimethyl-1,2-benzanthracene↗