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1.
Broschard Thomas H.; Wiessler Manfred; von der Lieth Claus-Wilhelm; Schmeiser Heinz H. 《Carcinogenesis》1994,15(10):2331-2340
Synthetic oligonucleotides (18-mers) containing either a singledeoxyadenosine residue or a single deoxyguanosine residue weretreated with aristolochic acid I (AAI) or aristolochic acidII (AAII), the main components of theplant carcinogen aristolochicacid (AA). These reactions resulted in the formation of site-specificallyadducted oligonucleotides containing the two known AAIDNAadducts (dAAAI, dGAAI) or the two known AAIIDNAadducts (dAAAII, dGAAII) at position 15 from the3'end. Using HPLC chromatography, the oligonucleotides werepurified and subsequently shown to contain the adducts of interestby 32P-postlabelling. The adducted oligonucleotides were usedas templates in primer (11-mer) extension reactions catalysedby modified bacteriophage T7 DNA polymerase (Sequenase). Regardlessof the type of DNA adduct examined, DNA synthesis was blockedpredominantly (8090%) at the nucleotide 3' to each adduct,although primer extension to the full length of the templatewas noted with unmodified control templates. However, 15 nucleotideproducts, indicating blocking of DNA synthesis after incorporationof a nucleotide opposite the adduct and translesional synthesisproducts were formed in all cases in different amounts, dependingon the adduct structure. When a 14-mer primer together withhigh dNTP concentrations was used to examine nucleotide incorporationdirectly across from the four different purine adducts we foundthat the deoxyadenosine adducts (dAAAI and dAAAII)allowed incorporation of dAMP and dTMP equally well, whereasthe deoxyguanosine adducts (dGAAI and dGAAII)allowed preferential incorporation of dCMP. Molecular dynamicsimulations showed that the aristolactam moiety of all adductsexhibit a strong stacking, with the adenine residue at the 3'end of the 14-mer primer. These studies demonstrate that allAA purine adducts provide severe blocks to DNA replication andthat the guanine adducts may not be very efficient mutageniclesions. In contrast, the translesional bypass past adenineadducts of the aristolochic acids suggests a mutagenic potentialresulting from dAMP incorporation by polymerase. AT 相似文献
2.
Sequence-specific detection of aristolochic acid-DNA adducts in the human p53 gene by terminal transferase-dependent PCR 总被引:24,自引:0,他引:24
The carcinogenic plant extract aristolochic acid (AA) is thought to be the major causative agent in the development of urothelial carcinomas found in patients with Chinese herb nephropathy (CHN). These carcinomas are associated with overexpression of p53, suggesting that the p53 gene is mutated in CHN-associated urothelial malignancy. To investigate the relation between AA-DNA adduct formation and possible p53 mutations, we mapped the distribution of DNA adducts formed by the two main components of AA, aristolochic acid I (AAI) and aristolochic acid II (AAII) at single nucleotide resolution in exons 5-8 of the human p53 gene in genomic DNA. To this end, an adduct-specific polymerase arrest assay combined with a terminal transferase-dependent PCR (TD-PCR) was used to amplify DNA fragments. AAI and AAII were reacted with human mammary carcinoma (MCF-7) DNA in vitro and the major DNA adducts formed were identified by the (32)P-postlabeling method. These adducted DNAs were used as templates for TD-PCR. Sites at which DNA polymerase progress along the template was blocked were assumed to be at the nucleotide 3' to the adduct. Polymerase arrest spectra thus obtained showed a preference for reaction with purine bases in the human p53 gene for both activated compounds. For both AAs, adduct distribution was not random; the strongest signals were seen at codons 156, 158-159 and 166-167 for exon 5, at codons 196, 198-199, 202, 209, 214-215 and 220 for exon 6, at codons 234-235, 236-237 and 248-249 for exon 7 and at codons 283-284 and 290-291 for exon 8. Overall guanines at CpG sites in the p53 gene that correspond to mutational hotspots observed in many human cancers seem not to be preferential targets for AAI or II. We compared the AA-DNA binding spectrum in the p53 gene with the p53 mutational spectrum of urothelial carcinomas found in the human mutation database. No particular pattern of polymerase arrest was found that predicts AA-specific mutational hotspots in urothelial tumors of the current p53 database. Thus, AA is not a likely cause of non-CHN-related urothelial tumors. 相似文献
3.
Aristolochic acid I (AAI) and aristolochic acid II (AAII), the two major
components of the carcinogenic plant extract aristolochic acid (AA), are
known to be mutagenic and to form DNA adducts in vivo. According to the
structures of the major DNA adducts identified in animals and humans,
nitroreduction is the crucial pathway in the metabolic activation of these
naturally occurring nitroarenes to their ultimate carcinogenic species.
Using the nuclease P1-enhanced version of the 32P-post-labelling assay we
investigated the formation of DNA adducts by AAI and AAII in different in
vitro activation systems in order to determine the most suitable in vitro
system mimicking target tissue activation. Although DNA adducts resulting
from oxidative activation of AAs have not yet been identified both
reductive and oxidative in vitro systems were employed. In vitro
incubations were conducted under standardized conditions (0.3 mM AAs; 4 mM
dNp as calf thymus DNA) using rat liver microsomes, xanthine oxidase (a
mammalian nitroreductase), horseradish peroxidase, lactoperoxidase and
chemical reduction by zinc. Enzymatic incubations were performed under
aerobic and anaerobic conditions. A combination of two independent
chromatographic systems (ion-exchange chromatography and reversed-phase
HPLC) with reference compounds was used for the identification of DNA
adducts detected by the 32P-post-labelling assay. The two known major
adducts of AAI or AAII found in vivo were generated by all in vitro systems
except for incubations with AAII and horseradish peroxidase where two
unknown adducts predominated. Irrespective of the in vitro activation
system used, the majority of adduct spots obtained were identified as the
previously characterized four AA-DNA adducts: dA-AAI, dA-AAII, dG-AAI and
dG-AAII. This indicates that both reductive and peroxidative activation of
AAI or AAII resulted in chromatographically indistinguishable DNA adducts.
Thus, peroxidase mediated activation of AAs led to the formation of the
same adducts that had been observed in vivo and upon reductive activation
in several in vitro systems. Quantitative analyses of individual adducts
formed in the various in vitro systems revealed relative adduct labelling
(RAL) values over a 100,000-fold range from 4 in 10(3) for activation of
AAII to deoxyadenosine adducts by zinc to only 3 in 10(8) for activation of
AAII by lactoperoxidase. The extent of DNA modification by AAI was higher
than by AAII in all enzymatic in vitro systems. Only activation by zinc
resulted in higher total binding to exogenous DNA by AAII than by AAI.
Aerobic incubations with rat liver microsomes generated AAI- and AAII-DNA
adduct profiles reproducing profiles in target tissue (forestomach) of
rats, thus providing the most appropriate activation among the in vitro
systems tested.
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4.
Stiborova Marie; Fernando R. Charles; Schmeiser Heinz H.; Frei Eva; Pafau Wolfgang; Wiessler Manfred 《Carcinogenesis》1994,15(6):1187-1192
We report the analysis of DNA adducts in the target organ (forestomach)of male SpragueDawley rats treated orally with two doses(10 mg/kg body wt) per week for 2 weeks of either aristolochicacid I (AAI), aristolochic acid II (AAII) or the plant extractaristolochic acid (AA). DNA adducts were detected and quantitatedusing the nuclease P1-enhanced version of the 32P-postlabellingassay. For identification of adducts, reference compounds wereprepared by reaction of enzymatically activated AAI and AAIIwith 3'-purine phosphonucleosides and analysed by the n-butanolenrichment procedure. These reference compounds were assignedto the previously characterized DNA adducts of AAI [7-(deoxy-guanosin-N2-yl)-aristolactamI = dG-AAI, 7-(deoxyadenosin-N6-yl) I = dA-AAI] and AAII [7-(deoxyadenosin-N6-yl)-aristolactamII = dA-AAII]. Cross referencing of the carcinogen-modifiednucleoside bisphosphates obtained from forestomach DNA withthe synthetic standard compounds by ion-exchange chromatographyand reversed-phase HPLC demonstrated that the major DNA adductsformed by AAI and AA were identical to dG-AAI and dA-AAI. Likewise,forestomach DNA isolated from AAII-treated rats showed two purinederived adduct spots, the major one being dA-AAII, the minorone being tentatively identified as 7-(deoxyguanosin-N2-yl)-aristolactamII. A minor adduct detected in forestomach DNA of rats treatedwith AAI was found to be chromatographically indistinguishablefrom the adduct identified as dA-AAII, indicating a possibledemethoxylation reaction of AAI. Quantitation of DNA adductsrevealed that in in vitro reactions with 3'-phosphonucleosidesthe adduct levels were approximately one order higher for bothAAI and AAII-derived adducts than in forestomach DNA modifiedwith AAI or AAII in vivo. In vitro as well as in vivo adductionby AAI was more efficient than adduction by AAII. The patternof adduct spots obtained from forestomach DNA of rats treatedwith the plant extract AA reflected the composition of the extractdetermined by HPLC analysis. Irrespective of the aristolochicacid used to induce DNA adducts, deoxyadenosine is the majortarget of modification, pointing to the general importance ofdeoxyadenosine adducts for chemical carcinogenesis of thesenaturally occurring products. This study shows that the combinationof two independent chromatographic systems considerably enhancesthe fidelity of identification of DNA adducts with the 32P-posthbellingassay. 相似文献
5.
32P-post-labelling analysis of DNA adducts formed by aristolochic acid in tissues from patients with Chinese herbs nephropathy 总被引:12,自引:0,他引:12
Bieler CA; Stiborova M; Wiessler M; Cosyns JP; van Ypersele de Strihou C; Schmeiser HH 《Carcinogenesis》1997,18(5):1063-1067
Recently, we reported that aristolochic acid (AA) a naturally occurring
nephrotoxin and carcinogen is implicated in a unique type of renal
fibrosis, designated Chinese herbs nephropathy (CHN). Indeed, we identified
the principal aristolochic acid-DNA adduct in the kidney of five such
patients. We now extend these observations and demonstrate the presence of
additional AA-DNA adducts by the 32P-post-labelling method not only in the
kidneys, but also in a ureter obtained after renal transplantation. Using
the nuclease P1 version of the assay not only the major DNA adduct of
aristolochic acid, 7-(deoxyadenosin-N6-yl)- aristolactam I (dA-AAI), but
also the minor adducts, 7-(deoxyguanosin- N2-yl)-aristolactam I (dG-AAI)
and 7-(deoxyadenosin-N6-yl)-aristolactam II (dA-AAII) were detected, and
identified by cochromatographic analyses with TLC and HPLC. Quantitative
analyses of six kidneys revealed relative adduct levels from 0.7 to
5.3/10(7) for dA-AAI, from 0.02 to 0.12/10(7) for dG-AAI and 0.06 to 0.24/
10(7) nucleotides for dA-AAII. The detection of the dA-AAII adduct is
consistent with the occurrence of aristolochic acid II (AAII) in the herb
powder imported under the name of Stephania tetrandra and confirms that the
patients had indeed ingested the natural mixture of AAI and AAII. 32P-post-
labelling analyses of further biopsy samples of one patient showed the
known adduct pattern of AA exposure not only in the kidney, but also in the
ureter, whereas in skin and muscle tissue no adduct spots were detectable.
In an attempt to explain the higher level of the dA-AAI adduct compared to
the dG-AAI adduct level in renal tissue even 44 months after the end of
regimen, the persistence of these two purine adducts was investigated in
the kidney of rats given a single oral dose of pure AAI. In contrast to the
dG-AAI adduct, the dA-AAI adduct exhibited a lifelong persistence in the
kidney of rats. Our data demonstrate that AA forms DNA adducts in human
tissue by the same activation mechanism(s) reported from animal studies.
Thus, the carcinogenic/mutagenic activity of AA observed in animals could
also be responsible for the urothelial cancers observed in two of the CHN
patients.
相似文献
6.
Localization of chloroacetaldehyde-induced DNA damage in human p53 gene by DNA polymerase fingerprint analysis 总被引:1,自引:0,他引:1
Chloroacetaldehyde (CAA) reacts with DNA bases, forming hydroxyethano derivatives of different stability, which are subsequently converted into etheno (epsilon) adducts: epsilon A, epsilon C, epsilon G. DNA polymerase fingerprint analysis was used to study the distribution of CAA-induced modifications in the p53 sequence. A plasmid bearing cDNA containing the human p53 gene was reacted in vitro with CAA, then dehydrated for conversion of hydroxyethano into etheno adducts, and primer extension by T7 DNA polymerase in the presence of four dNTPs was performed. The DNA repair enzymes methylpurine-DNA glycosylase and Escherichia coli exonuclease III were used to convert epsilon A residues in the template into DNA strand breaks, which enabled precise localization of the epsilon A residues within the p53 gene. Hydroxyethano derivatives of adenine and cytosine in a template blocked T7 DNA polymerase and caused premature chain termination opposite adenine or one base before cytosine. After dehydration, both epsilon A and epsilon C were much more easily by-passed by T7 DNA polymerase. Formation of epsilon G was identified as 'stop bands' one base before guanine residues. Modification of cytosine and guanine was additionally recognized by weakening or disappearance of non-specific stops on an undamaged template, probably due to steric hindrance by the tertiary DNA structure for polymerase. Etheno adduction of cytosine and guanine relaxed the compact DNA structure and enabled DNA polymerase to by-pass. In exons 5-8 of p53, 143 out of 500 sites appeared to be damaged by CAA, with four particularly densely modified regions between codons 135-147, 218-222, 234-255 and 284-292. The pattern of modification followed the pattern of p53 mutations found in vinyl chloride-associated liver angiosarcomas in humans and rats, but only in regions that showed 100% homology with the human sequence. The factors that influence DNA damage and induction of mutations in the p53 gene by CAA and vinyl chloride are discussed. 相似文献
7.
Activating mutations at codon 61 of the c-Ha-ras gene in thin-tissue sections of tumors induced by aristolochic acid in rats and mice 总被引:5,自引:0,他引:5
The plant extract aristolochic acid, which consists mainly of aristolochic acid I (AAI) and aristolochic acid II (AAII), induces tumors in rats and mice. Thin-tissue sections of rat tumors induced by AAI and of mouse tumors induced by aristolochic acid, were analyzed for c-Ha-ras mutations in codon 61. Areas of neoplastic and histologically normal tissue were manually scraped out and separated. Using the polymerase chain reaction (PCR) and mutation detection by selective oligonucleotide hybridization, we observed AT----TA transversion mutations in DNA of neoplastic portions, but not in DNA of adjacent normal tissue in both rat and mouse tumors. 相似文献
8.
DNA polymerase-mediated nucleotide incorporation adjacent to hydrocarbon-deoxyadenosine and hydrocarbon-deoxyguanosine adducts 总被引:2,自引:1,他引:1
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. 相似文献
9.
Properties of aflatoxin-DNA adducts formed by photoactivation and characterization of the major photoadduct as aflatoxin-N7-guanine 总被引:1,自引:0,他引:1
Aflatoxin-DNA adducts were formed by microsomal and photoactivation, using nick-translated DNA labelled with 14C in each of the DNA bases [3H]AFB1 and [3H]AFB2. DNA adducts were analysed by HPLC of DNA hydrolysates, and were characterized as double labelled peaks with specific retention times. The only AF-DNA adducts which were detected in significant amounts were guanine adducts, irrespective of the type of aflatoxin used or the mode of its activation. No stable adduct with adenine, cytosine or thymine was detected. UV spectra, proton NMR spectroscopy and mass spectrometry are consistent with the notion that the major AFB1-DNA photoadduct is the N7-guanine adduct. This report provides direct evidence for the existence of aflatoxin photoadducts formed on DNA. 相似文献
10.
Identification and mutagenicity of metabolites of aristolochic acid formed by rat liver 总被引:11,自引:2,他引:9
The rat liver 9000 g supernatant mediated metabolism of thecarcinogenic aristolochic acid, which consists of aristolochicacid I (AAI) and aristolochic acid II (AAII), was investigated.Under anaerobic conditions the major metabolites were the correspondingaristolactams for both AAI and AAII. In contrast under aerobicconditions AAII was not detectably metabolized and the onlymetabolite found for AAI was the O-demethylated derivative aristolochicacid la (AAIa). The metabolites were identified by their u.v.,mass and n.m.r. spectra and by comparison with reference standards.The mutagenic activities of the three metabolites were determinedin Salmonella typhimurium strains TA1537 and TA 100. The aristolactamswere mutagenic in both strains when a metabolizing system waspresent. These results indicate that AAI or AAII and their aristolactamsexert their effect via a common reactive intermediate, probablythe corresponding hydroxylamine. AAIa was only very weakly mutagenicand this metabolite may therefore not be regarded as a majormutagenic metabolite of AAI. These findings suggest that theacids are preferentially metabolized by two totally differentpathways in vitro, namely an oxidative pathway for AAI and areductive pathway for AAII. 相似文献
11.
We report the quantitation of DNA adducts in target and non-targetorgans of male Wistar rats treated orally with five daily doses(10 mg/kg body wt) aristolochic acid I (AAI) or aristolochicacid II (AAII), the major components of the herbal drug aristolochicacid, a forestomach carcinogen In the rat. DNA adducts weredetected and analysed using the nuclease P1-enhanced variationof the Randerath 32 postlabeiling assay. The highest level ofDNA adducts formed was by AAI inthe target organ, forestomach(330 ± 30 adducts/108 nucleotides), but high levels werealso observed in a non-target tissue, the glandular stomach(180 ± 15). Lower amounts of adducts were detected inliver, kidney and urinary bladder epltheliuin. With AAII thebinding Levels were generally lower than the AAII, the highestLevel of adducts being detected in kidney (80 ± 20 adducts/108nucleotides) and lower levels in liver, stomach and urinarybladder epithelia. Adduct patterns similar to those in vivowere observed in two new in vitro assays. Rat faecal bacteriawere shown to be able to activate AM and AAII to reactive species,which were trapped with exogenous calf thymus DNA and analysedby postlabelling. llncuhatlon of AM and AAII in explanted ratstomach held in short-term organ culture resulted In DNA adductformation in the epithelia of both forestomach and glandularstomach. To assign the recently characterized in vitro nucleosideadducts of AII to the bisphosphate derivatives, a new ion-pairH]PLC procedure on a reversed-phase column was developed. Bymonitoring Cerenkov radiation on-line, a good separation ofAII adducts was observed, demonstrating that adducts formedin vivo were chromatographically indistinguishable with thoseformed in vitro, and previously characterized as an aristolactammoiety bound covalently to the exocydlic amino groups of deoxyadenosineand deoxyguanosine. 相似文献
12.
Laura C. Bridgewater Francis C. R. Manning Elizabeth S. Woo Steven R. Patierno 《Molecular carcinogenesis》1994,9(3):122-133
Carcinogenic chromium (Cr6+) enters cells via the sulfate transport system and undergoes intracellular reduction to trivalent chromium, which strongly adducts to DNA. In this study, the effect of adducted trivalent chromium on in vitro DNA synthesis was analyzed with a polymerase-arrest assay in which prematurely terminated replication products were separated on a DNA sequencing gel. A synthetic DNA replication template was treated with increasing concentrations of chromium(III) chloride. The two lowest chromium doses used resulted in biologically relevant adduct levels (6 and 21 adducts per 1,000 DNA nucleotides) comparable with those measured in nuclear matrix DNA from cells treated with a 50% cytotoxic dose of sodium chromate in vivo. In vitro replication of the chromium-treated template DNA using the Sequenase version 2.0 T7 DNA polymerase (United States Biochemical Corp., Cleveland, OH) resulted in dose-dependent polymerase arrest beginning at the lowest adduct levels analyzed. The pattern of polymerase arrest remained consistent as chromium adduct levels increased, with the most intense arrest sites occurring 1 base upstream of guanine residues on the template strand. Replication by the DNA polymerase I large (Klenow) fragment as well as by unmodified T7 DNA polymerase also resulted in similar chromium-induced polymerase arrest. Interstrand crosslinking between complementary strands was detected in template DNA containing 62, 111, and 223 chromium adducts per 1,000 DNA nucleotides but not in template containing 6 or 21 adducts per 1,000 DNA nucleotides, in which arrest nevertheless did occur. Low-level, dose-dependent interstrand cross-linking between primer and template DNA, however, was detectable even at the lowest chromium dose analyzed. Since only 9% of chromium adducts resulted in polymerase arrest in this system, we hypothesized that arrest occurred when the enzyme encountered chromium-mediated interstrand DNA-DNA cross-links between either the template and a separate DNA molecule or the template and its complementary strand in the same molecule. These results suggest that the obstruction of DNA replication by chromium-mediated DNA-DNA cross-links is a potential mechanism of chromium-induced genotoxicity in vivo. © 1994 Wiley-Liss, Inc. 相似文献
13.
32P-Postlabelling analysis of allyl glycidyl ether-treated DNA after adduct
enrichment on anion-exchange cartridges revealed two major and one minor
DNA adducts. The major adducts were shown to originate from alkylation at
N-7-guanine and N-1-adenine, respectively, while the minor adduct was at
N-3-cytosine. In addition, rearrangement products of the 1-adenine and
3-cytosine adducts to N6-adenine and 3-uracil were indicated. The relative
amounts of adenine, cytosine and uracil products appeared to be dependent
upon conditions (in particular pH) during sample processing and analysis.
When nuclease P1 was used for adduct enrichment the adenine, cytosine and
uracil adducts, but not the 7-guanine adduct, were detected. The labelling
efficiency of the 7- guanine adduct standard was 40-45%. Total recovery of
this adduct from allyl glycidyl ether-modified DNA was 9-12%. The labelling
efficiency of the 1-adenine adduct standard was 78-82%. Total recovery of
this adduct from DNA was approximately 20% when using anion-exchange
chromatography for adduct enrichment and 30-34% when using nuclease P1.
Preliminary analysis of DNA from mice treated with allyl glycidyl ether
indicated 57 times higher level of the 7-guanine adduct, per unit dose, in
skin DNA (120 per 10(8) normal nucleotides) after topical application when
compared to liver DNA after i.p. administration. The 1- adenine adduct
could not be quantified in liver DNA (due to an interfering background
product present in untreated animals) and the level of the 3-cytosine
adduct was below the detection limit of the method. After topical
application the level of the 1 adenine adduct in skin DNA was approximately
30 per 10(8), using either column or nuclease P1 enrichment. The 3-cytosine
adduct was detected in skin, but was not quantified.
相似文献
14.
Lack of miscoding properties of 7-(2-oxoethyl)guanine, the major vinyl chloride-DNA adduct 总被引:2,自引:0,他引:2
Chloroethylene oxide, an ultimate carcinogenic metabolite of vinyl chloride, was reacted with poly(deoxyguanylate-deoxycytidylate); the nucleic acid base adducts, 7-(2-oxoethyl)guanine and 3,N4-ethenocytosine, were analyzed by reverse-phase high-performance liquid chromatography. Chloroethylene oxide-modified poly(deoxyguanylate-deoxycytidylate) was assayed as template in a replication fidelity assay with Escherichia coli DNA polymerase I, and the newly synthesized product was subjected to nearest-neighbor analysis. Misincorporation rates of deoxyadenosine monophosphate and thymidine monophosphate were found to increase with the level of template modification. About 80% of the mispairing events were located opposite minor cytosine lesions. 7-(2-Oxoethyl)guanine, the major adduct identified (greater than 98% of the adducts), did not miscode for either thymine or adenine, failing to support an earlier hypothesis that the cyclic hemiacetal form, O6,7-(1'-hydroxyethano)guanine, could, by analogy with O6-methyl- and O6-ethylguanine, simulate adenine. Our results indicate that direct miscoding of 7-(2-oxoethyl)-guanine may contribute only slightly to the induction of mutations by chloroethylene oxide or vinyl chloride. 相似文献
15.
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. 相似文献
16.
Analysis of aristolochic acid I (AAI)-DNA adducts in exfoliated cells in urine, urothelium and entire urinary bladder were studied after oral administration of five daily doses (10 mg/kg body wt) AAI for 3 months to rats. The two major adducts excreted in urine are presumably identical to the two main adducts formed in vitro and in vivo in different organs in the rat, which have previously been characterized in vitro as 7(-deoxyguanosin-N2-yl)-aristolactam I and 7(-deoxyadenosin-N6-yl)-aristolactam I. Urine samples were collected on dry-ice, subsequently pooled and purified according to the protocol of Kadlubar and co-workers. DNA was isolated, digested and AAI-DNA adducts of exfoliated cells in urine and urothelium of rats were detected and quantitated by enhancement methods of the 32P-postlabeling assay, namely nuclease P1 enrichment or butanol extraction. Autoradiograms indicated that adduct patterns in DNA derived from exfoliated cells in urine were very similar to those obtained from DNA isolated from tissues. Quantitative analysis of adducts revealed adduct levels declining for both adducts from DNA isolated from urothelium to DNA isolated from the entire urinary bladder to DNA isolated from exfoliated cells in urine. In general, count rates of two predominant AAI adducts were enhanced by butanol extraction approximately 3- to 8-fold when compared with the nuclease P1 digestion technique. The identity of the two major adducts was confirmed by co-chromatography with eluted spots from in vivo adducts by comparing mobilities on poly-(ethyleneimine)-cellulose plates. Microbiological investigations of the urine revealed no gross contamination with bacteria, so that the isolated DNA supposedly originated from exfoliated urothelial cells. This study indicates that 32P-postlabeling analysis can be used to monitor non-invasively the formation of carcinogen-DNA adducts in animals or humans exposed to carcinogens. 相似文献
17.
Hydroxylation of meso-methyl groups with subsequent formation of reactive electrophilic esters has been proposed as a possible activation pathway in the metabolism, DNA binding and carcinogenicity of some methyl-substituted polycyclic aromatic hydrocarbons. Some data in vitro have been reported in support of this concept. In this study, sulfotransferase activity for 6-hydroxymethylbenzo[a]pyrene (HMBP) in rat and mouse liver cytosols was demonstrated to mediate formation of benzylic adducts from this hydrocarbon with guanosine and with deoxyguanosine and deoxyadenosine in DNA. These benzylic adducts were also obtained from reactions of synthetic 6-sulfooxymethylbenzo[a]pyrene (SMBP) with individual (deoxy)ribonucleosides or DNA. The structure of the major DNA adduct formed from HMBP and SMBP was determined from NMR spectroscopy to be N2-(benzo[a]pyrene-6-methylenyl)-deoxyguanosine. Low levels of a deoxycytidine adduct were also obtained from DNA reacted with SMBP. Covalent modification of DNA by acetyl-CoA- and ATP-dependent activation of HMBP also produced the identical benzylic adducts, but the amounts were smaller than those obtained in the sulfotransferase-mediated reaction. The i.p. administration of HMBP to rats resulted in the formation of a hepatic DNA adduct. After enzymatic hydrolysis to the nucleoside level, this DNA adduct was chromatographically identical with the deoxyguanosine adduct formed in the above in vitro reactions. This adduct accounted for approximately 20-30% of total HMBP residues bound to hepatic DNA and its formation was significantly inhibited by pretreatment of rats with dehydroepiandrosterone, an inhibitor of the sulfotransferase activity for HMBP. The i.p. administration of comparable doses of SMBP to rats led to the formation of much larger amounts of the adducts with the guanine, adenine, and cytosine bases in the liver DNA. The data indicate that the sulfotransferase activity in the rat liver for HMBP plays a major role in the benzylic DNA adduct formation from this hydrocarbon in rat liver in vivo. 相似文献
18.
We have studied adduct formation of the antineoplastic agent diaziquone (AZQ; NSC 182986) with DNA and nucleotides in vitro. The aziridine moieties of AZQ can be expected to interact covalently with DNA which, in turn, presumably elicits the antitumor activity. We analyzed AZQ-DNA adducts by a modified 32P-postlabeling assay involving purification of the nuclease P1-enriched labeled adducts by high-salt C18 reversed-phase thin-layer chromatography and separation of the eluted adducts on a polyethyleneimine-cellulose layer using non-urea salt solutions. Modification of calf thymus DNA with AZQ produced two major (22% and 40%) and at least eight minor adducts. At equal concentrations of AZQ and DNA (1 micrograms/microliters each), peak binding was observed in about 2 h [1926 +/- 378 (SD) fmol/micrograms of DNA] with the binding levels remaining practically unchanged through 4 h. However, incubation for 24 h resulted in over 40% decline, indicating adduct instability. AZQ was found to be highly reactive in vitro as evidenced by its substantial binding (49 +/- 14 fmol/micrograms of DNA) even at a DNA:AZQ ratio of 100:1. When incubated with mononucleotides, AZQ reacted extensively with adenine, guanine, and cytosine but only slightly with thymine. Cochromatography of the modified DNA and nucleotides revealed that one of the major adducts and several minor adducts were guanine derived. The aziridine rings of AZQ were found to be the main reactive sites as its monoaminoalcohol derivative showed as much DNA reactivity as did the parent compound, but no activity was observed when both aziridine groups were hydrolyzed to diaminoalcohols. The improved 32P-postlabeling assay seems capable of detecting relatively polar adducts such as those formed with AZQ at a level of one adduct/10(9) nucleotides. 相似文献
19.
Primary mouse embryo cell cultures were grown in the presence of [14C]guanine, labeling primarily deoxyguanosine residues in the cellular DNA, or in the presence of [14C]adenine, labeling both deoxyguanosine and deoxyadenosine residues in the cellular DNA. These cultures were subsequently exposed to 7,12-[3H]dimethylbenz(a)anthracene for 24 hr. The DNA was isolated and hydrolyzed to deoxyribonucleosides, and the 7,12-dimethylbenz(a)anthracene:deoxyribonucleoside adducts were separated chromatographically allowing the three major adducts found to be identified as bay-region anti-dihydrodiol-epoxide:deoxyguanosine and :deoxyadenosine adducts and a bay-region syn-dihydrodiol-epoxide:deoxyadenosine adduct. Therefore, in contrast to what is known for benzo(a)pyrene, substantial amounts of deoxyadenosine adducts are formed with the more potent carcinogen, 7,12-dimethylbenz(a)anthracene. 相似文献
20.
Estrogen-nucleic acid adducts: guanine is major site for interaction between 3,4-estrone quinone and COIII gene 总被引:1,自引:0,他引:1
The carcinogenicity of estrogens in rodents and man has been attributed to
either alkylation of cellular macromolecules and/or redox-cycling,
generation of active radicals and DNA damage. Metabolic activation of
estradiol leading to the formation of catechol estrogens is believed to be
a prerequisite for its genotoxic effects. 4-Hydroxyestradiol is a potent
inducer of tumors in hamsters. Previous studies have shown that 3,4-estrone
quinone (3,4-EQ) can redox-cycle and is capable of inducing exclusively
single strand DNA breaks in MCF-7 breast cancer cells, as well as react
with various nucleophiles including amino acids and nucleic acids to give
Michael addition products. In this paper we examined the nature of the
interaction of 3,4-EQ with COIII gene and analysed the estrogen-DNA adducts
by 32P-post-labeling. The reaction of 3,4-EQ with the COIII gene followed
by polymerase arrest assay showed several stop sites in which guanine was
preferentially attacked by 3,4- EQ and, to a lesser extent, with Ade, Cyt
and Thy. 32P-Post-labeling analysis of the reaction of 3,4-EQ with COIII
gene gave one major adduct which was found to be identical to that obtained
from reaction of dGMP with 3,4-EQ. The observation that obstruction of in
vitro replication of COIII template bound to 3,4-EQ suggests that estrogen
quinone adducted lesions can arrest DNA polymerase. These results indicate
that 3,4-EQ may be genotoxic and may provide one possible explanation for
the carcinogenic effects of estrogens.
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