共查询到20条相似文献,搜索用时 46 毫秒
1.
Kate E. Coldwell Suzanne M. Cutts Ted J. Ognibene Paul T. Henderson Don R. Phillips 《Nucleic acids research》2008,36(16):e100
Limited sensitivity of existing assays has prevented investigation of whether Adriamycin–DNA adducts are involved in the anti-tumour potential of Adriamycin. Previous detection has achieved a sensitivity of a few Adriamycin–DNA adducts/104 bp DNA, but has required the use of supra-clinical drug concentrations. This work sought to measure Adriamycin–DNA adducts at sub-micromolar doses using accelerator mass spectrometry (AMS), a technique with origins in geochemistry for radiocarbon dating. We have used conditions previously validated (by less sensitive decay counting) to extract [14C]Adriamycin–DNA adducts from cells and adapted the methodology to AMS detection. Here we show the first direct evidence of Adriamycin–DNA adducts at clinically-relevant Adriamycin concentrations. [14C]Adriamycin treatment (25 nM) resulted in 4.4 ± 1.0 adducts/107 bp (~1300 adducts/cell) in MCF-7 breast cancer cells, representing the best sensitivity and precision reported to date for the covalent binding of Adriamycin to DNA. The exceedingly sensitive nature of AMS has enabled over three orders of magnitude increased sensitivity of Adriamycin–DNA adduct detection and revealed adduct formation within an hour of drug treatment. This method has been shown to be highly reproducible for the measurement of Adriamycin–DNA adducts in tumour cells in culture and can now be applied to the detection of these adducts in human tissues. 相似文献
2.
M A Moufarij S M Cutts G M Neumann K Kimura D R Phillips 《Chemico-biological interactions》2001,138(2):137-153
The anthracycline Adriamycin is known to form adducts with DNA, but requires prior activation by formaldehyde. In contrast, the anthracycline barminomycin is also able to form adducts with DNA, but does not require activation by formaldehyde. Barminomycin, therefore, appears to function as a pre-activated form of Adriamycin. The DNA adducts formed by both anthracyclines are bound covalently to only one strand of DNA, but both also stabilise duplex DNA sufficiently that they can be detected as virtual interstrand crosslinks in heat denaturation electrophoretic crosslinking assays. The barminomycin-DNA adducts form extremely rapidly with DNA, and at exceedingly low concentrations (approximately 50-fold lower than with Adriamycin in the presence of excess formaldehyde), both characteristics consistent with barminomycin being in a pre-activated state, hence, undergoing a bimolecular reaction with DNA compared with the trimolecular reaction (drug, formaldehyde and DNA) required with Adriamycin. Surprisingly, barminomycin-DNA adducts are substantially more stable (essentially irreversible) than Adriamycin-DNA adducts (half life of approximately 25 h at 37 degrees C). Due to this understanding of the reactivity of barminomycin and its exceptional cytotoxicity (1000-fold more cytotoxic than Adriamycin), detailed structural studies of barminomycin-DNA adducts are now warranted, both in vitro and in tumour cells. 相似文献
3.
Goldman R Day BW Carver TA Mauthe RJ Turteltaub KW Shields PG 《Chemico-biological interactions》2000,126(3):171-183
Quantitation of carcinogen-DNA adducts provides an estimate of the biologically effective dose of a chemical carcinogen reaching the target tissue. In order to improve exposure-assessment and cancer risk estimates, we are developing an ultrasensitive procedure for the detection of carcinogen-DNA adducts. The method is based upon postlabeling of carcinogen-DNA adducts by acetylation with 14C-acetic anhydride combined with quantitation of 14C by accelerator mass spectrometry (AMS). For this purpose, adducts of benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide (BPDE) with DNA and deoxyguanosine (dG) were synthesized. The most promutagenic adduct of BPDE, 7R,8S,9R-trihydroxy-10S-(N(2)-deoxyguanosyl)-7,8,9, 10-tetrahydrobenzo[a]pyrene (BPdG), was HPLC purified and structurally characterized. Postlabeling of the BPdG adduct with acetic anhydride yielded a major product with a greater than 60% yield. The postlabeled adduct was identified by liquid chromatography-mass spectrometry as pentakis(acetyl) BPdG (AcBPdG). Postlabeling of the BPdG adduct with 14C-acetic anhydride yielded a major product coeluting with an AcBPdG standard. Quantitation of the 14C-postlabeled adduct by AMS promises to allow detection of attomolar amounts of adducts. The method is now being optimized and validated for use in human samples. 相似文献
4.
Interstrand cross-linking by adriamycin in nuclear and mitochondrial DNA of MCF-7 cells 总被引:4,自引:1,他引:4
Activation of Adriamycin by formaldehyde leads to the formation of drug–DNA adducts in vitro and these adducts stabilise the DNA to such a degree that they function as virtual interstrand cross-links. The formation of these virtual interstrand cross-links by Adriamycin was investigated in MCF-7 cells using a gene-specific interstrand cross-linking assay. Cross-linking was measured in both the nuclear-encoded DHFR gene and in mitochondrial DNA (mtDNA). Cross-link formation increased linearly with Adriamycin concentration following a 4 h exposure to the drug. The rate of formation of Adriamycin cross-links in each of the genomes was similar, reaching maximal levels of 0.55 and 0.4 cross-links/10 kb in the DHFR gene and mtDNA respectively, following exposure to 20 µM Adriamycin for 8 h. The interstrand cross-link was short lived in both DNA compartments, with a half-life of 4.5 and 3.3 h in the DHFR gene and mtDNA respectively. The kinetics of total Adriamycin adduct formation, detected using [14C]Adriamycin, was similar to that of cross-link formation. Maximal adduct levels (30 lesions/10 kb) were observed following incubation at 20 µM drug for 8 h. The formation of such high levels of adducts and cross-links could therefore be expected to contribute to the mechanism of action of Adriamycin. 相似文献
5.
Epidemiological evidence has suggested an association between meat consumption and the risk of breast cancer. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a heterocyclic amine found in cooked meat, has been implicated in the aetiology of breast cancer and has been shown to induce tumour formation in rodent mammary glands. In addition, polycyclic aromatic hydrocarbons, such as benzo[a]pyrene (B[a]P) which has also been shown to induce tumour formation at a number of sites in rodents including the breast, are produced during the cooking of meat through the pyrolysis of fats. The aim of this study was to examine the bioavailability of these compounds to human breast tissue and their ability to bind to DNA to form DNA adducts. Patients undergoing breast surgery at York District Hospital were orally administered prior to surgery a capsule containing 20microg of 14C PhIP (182kBq, specific activity 2.05GBq/mmol) or 5microg of 14C B[a]P (36kBq, specific activity 1.81GBq/mmol). At surgery, normal and tumour breast tissue was resected and tissue concentrations of carcinogen measured by liquid scintillation counting and DNA adduct levels by accelerator mass spectrometry (AMS) were subsequently determined. It was found that both 14C PhIP and 14C B[a]P were able to reach the target organ where they had the ability to form DNA adducts. The level of adducts ranged from 26.22-477.35 and 6.61-208. 38 adducts/10(12) nucleotides following administration of 14C PhIP and 14C B[a]P, respectively, with no significant difference observed between levels in normal or tumour tissue. In addition, the data obtained in this study were comparable to adduct levels previously found in colon samples following administration of the same compounds to individuals undergoing colorectal surgery. This is the first report that these two carcinogens bind to human breast DNA after administration of a defined low dose. 相似文献
6.
trans-4-Hydroxynonenal (HNE) is a major peroxidation product of omega-6 polyunsaturated fatty acids. The reaction of HNE with DNA produces four diastereomeric 1,N(2)-gamma-hydroxypropano adducts of deoxyguanosine (HNE-dG); background levels of these adducts have been detected in tissues of animals and humans. There is evidence to suggest that these adducts are mutagenic and involved in liver carcinogenesis in patients with Wilson's disease and in other human cancers. Here, we present biochemical evidence that in human cell nuclear extracts the HNE-dG adducts are repaired by the nucleotide excision repair (NER) pathway. To investigate the recognition and repair of HNE-dG adducts in human cell extracts, we prepared plasmid DNA substrates modified by HNE. [(32)P]-Postlabeling/HPLC determined that the HNE-dG adduct levels were approximately 1200/10(6) dG of plasmid DNA substrate. We used this substrate in an in vitro repair-synthesis assay to study the complete repair of HNE-induced DNA adducts in cell-free extracts. We observed that nuclear extracts from HeLa cells incorporated a significant amount of alpha[(32)P]dCTP in DNA that contained HNE-dG adducts by comparison with UV-irradiated DNA as the positive control. Such repair synthesis for UV damage or HNE-dG adducts did not occur in XPA cell nuclear extracts that lack the capacity for NER. However, XPA cells complemented with XPA protein restored repair synthesis for both of these adducts. To verify that HNE-dG adducts in DNA were indeed repaired, we measured HNE-dG adducts in the post-repaired DNA substrates by the [(32)P]-postlabeling/HPLC method, showing that 50-60% of HNE-dG adducts were removed from the HeLa cell nuclear extracts after 3 h at 30 degrees C. The repair kinetics indicated that the excision rate is faster than the rate of gap-filling/DNA synthesis. Furthermore, the HNE-dG adduct isomers 2 and 4 appeared to be repaired more efficiently at early time points than isomers 1 and 3. 相似文献
7.
T. J. Lightfoot J. M. Coxhead B. C. Cupid S. Nicholson R. C. Garner 《Mutation Research - Genetic Toxicology and Environmental Mutagenesis》2000,472(1-2)
Epidemiological evidence has suggested an association between meat consumption and the risk of breast cancer. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a heterocyclic amine found in cooked meat, has been implicated in the aetiology of breast cancer and has been shown to induce tumour formation in rodent mammary glands. In addition, polycyclic aromatic hydrocarbons, such as benzo[a]pyrene (B[a]P) which has also been shown to induce tumour formation at a number of sites in rodents including the breast, are produced during the cooking of meat through the pyrolysis of fats. The aim of this study was to examine the bioavailability of these compounds to human breast tissue and their ability to bind to DNA to form DNA adducts. Patients undergoing breast surgery at York District Hospital were orally administered prior to surgery a capsule containing 20 μg of 14C PhIP (182 kBq, specific activity 2.05 GBq/mmol) or 5 μg of 14C B[a]P (36 kBq, specific activity 1.81 GBq/mmol). At surgery, normal and tumour breast tissue was resected and tissue concentrations of carcinogen measured by liquid scintillation counting and DNA adduct levels by accelerator mass spectrometry (AMS) were subsequently determined. It was found that both 14C PhIP and 14C B[a]P were able to reach the target organ where they had the ability to form DNA adducts. The level of adducts ranged from 26.22–477.35 and 6.61–208.38 adducts/1012 nucleotides following administration of 14C PhIP and 14C B[a]P, respectively, with no significant difference observed between levels in normal or tumour tissue. In addition, the data obtained in this study were comparable to adduct levels previously found in colon samples following administration of the same compounds to individuals undergoing colorectal surgery. This is the first report that these two carcinogens bind to human breast DNA after administration of a defined low dose. 相似文献
8.
R.A. Baan M.J. Lansbergen P.A.F. de Bruin M.I. Willems P.H.M. Lohman 《Mutation research》1985,150(1-2):23-32
Exposure of cells to chemical carcinogens and mutagens may result in the formation of DNA adducts, which can give rise to mutations in the genome and to cellular transformation. Methods to measure DNA-adduct formation may be useful for ‘biomonitoring’, to establish exposure of laboratory animals or humans to DNA-damaging agents. For such purposes, immunochemical methods appear to be suitable, because they allow sensitive detection and quantification of DNA adducts in small amounts of sample in a non-radiolabelled form. We have worked out optimal conditions for the detection of DNA adducts by means of competitive enzyme-linked immunosorbent assay (ELISA). This technique involves interaction of soluble antigen, immobilized antigen and antibody. It appeared that the sensitivity of the competitive assay can be improved by lowering the amount of immobilized antigen, adsorbed to the wall of the plastic reaction vessel. On the basis of these observations, suitable conditions were selected for a sensitive quantitative assay of adducts in DNA isolated from various organs of rats, treated (p.o.) with the liver carcinogen 2-acetylaminofluorene (2-AAF). Under the conditions of these experiments, the available rabbit antiserum recognizes the guanosine-AAF adduct with high specificity. A time- and dose-dependent induction of AAF adducts could be measured in liver DNA from exposed rats, whereas the amount of adducts in DNA from spleen and nucleated blood cells remained below the detection limit (1 adduct/108 nucleotides). The implications of these findings with respect to the relevance of blood cell biomonitoring for target cell exposure are discussed. 相似文献
9.
Long-term kinetic study of beta-carotene, using accelerator mass spectrometry in an adult volunteer 总被引:2,自引:0,他引:2
Dueker SR Lin Y Buchholz BA Schneider PD Lamé MW Segall HJ Vogel JS Clifford AJ 《Journal of lipid research》2000,41(11):1790-1800
We present a sensitive tracer method, suitable for in vivo human research, that uses beta-[(14)C]carotene coupled with accelerator mass spectrometry (AMS) detection. Using this approach, the concentration-time course of a physiological (306 microgram 200 nCi) oral dose of beta-[(14)C]carotene was determined for 209 days in plasma. Analytes included beta-[(14)C]carotene, [(14)C]retinyl esters, [(14)C]retinol, and several [(14)C]retinoic acids. There was a 5.5-h lag between dosing and the appearance of (14)C in plasma. Labeled beta-carotene and [(14)C]retinyl esters rose and displayed several maxima with virtually identical kinetic profiles over the first 24-h period; elevated [(14)C]retinyl ester concentrations were sustained in the plasma compartment for >21 h postdosing. The appearance of [(14)C]retinol in plasma was also delayed 5.5 h postdosing and its concentration rose linearly for 28 h before declining. Cumulative urine and stool were collected for 17 and 10 days, respectively, and 57.4% of the dose was recovered in the stool within 48 h postdosing. The stool was the major excretion route for the absorbed dose. The turnover times (1/k(el)) for beta-carotene and retinol were 58 and 302 days, respectively. Area under the curve analysis of the plasma response curves suggested a molar vitamin A value of 0.53 for beta-carotene, with a minimum of 62% of the absorbed beta-carotene being cleaved to vitamin A.In summary, AMS is an excellent tool for defining the in vivo metabolic behavior of beta-carotene and related compounds at physiological concentrations. Further, our data suggest that retinyl esters derived from beta-carotene may undergo hepatic resecretion with VLDL in a process similar to that observed for beta-carotene. 相似文献
10.
Koc H Swenberg JA 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2002,778(1-2):323-343
DNA adducts are formed when electrophilic molecules or free radicals attack DNA. 32P-postlabeling has been the most commonly used assay for quantitation of DNA adducts due mainly to its excellent sensitivity that allows quantitation at concentrations as low as approximately 1 adduct per 10(9) normal bases. Such methods, however, do not have the specificity desired for accurate and reliable quantitation, and are prone to produce false positives and artifacts. In the last decade, mass spectrometry in combination with liquid and gas chromatography has presented itself as a good alternative to these techniques since it can satisfy the need for specificity and reliability through the use of stable isotope-labeled internal standards and highly specific detection modes such as selected reaction monitoring and high-resolution mass spectrometry. In this article, the contribution of mass spectrometry to the quantitation of DNA adducts is reviewed with special emphasis on unique applications of mass spectrometry in the area of DNA adduct quantitation and recent applications with improvements in sensitivity. 相似文献
11.
Mechanism of genotoxicity of diethylstilbestrol in vivo 总被引:2,自引:0,他引:2
Diethylstilbestrol (DES) is a carcinogen in humans and rodents which has eluded mechanistic clarification of its carcinogenic action. In vitro and in vivo, binding of DES to DNA has been found previously, but covalent DNA adducts could not be identified. In this study, the nature of binding was investigated by 32P-postlabeling, a rapid and highly sensitive assay for covalent DNA damage, to distinguish between a genotoxic or epigenetic mechanism of carcinogenesis by DES. A unique and distinct DNA adduct pattern was observed in kidney, liver, uterus (or testes) of female (or male, respectively) Syrian hamsters treated with a single injection of DES (200 mg/kg body weight). This set of DNA adducts closely matched patterns generated in vitro by reaction of diethylstilbestrol-4',4'-quinone with DNA or 2'-deoxyguanosine 3'-monophosphate. The major and several minor DES-DNA adducts in vivo had identical chromatographic mobilities in 11 different solvent systems with corresponding adducts obtained in vitro. The major adduct spot, generated in vitro by reaction of diethylstilbestrol-4',4'-quinone and DNA, was chemically unstable (half-life at 37 degrees C: 4-5 days). The persistence in vivo of these DNA modifications was low (biological half-life: 14 h) presumably because of chemical instability in concert with DNA repair. After injection of identical dosages of DES, adduct concentrations were 4-6-fold higher in females than in males. These results demonstrate that DES is capable of covalently modifying DNA. Moreover, diethylstilbestrol-4',4"-quinone is the major reactive metabolic intermediate responsible for the genotoxic activity of DES. Tumors are expected to arise only in rapidly dividing cells due to the short biological lifetimes of DES-DNA adducts. 相似文献
12.
Polycyclic aromatic hydrocarbon-DNA adducts in humans: relevance as biomarkers for exposure and cancer risk 总被引:15,自引:0,他引:15
The methodology applied for DNA adducts in humans has become more reliable in recent years, allowing to detect even background carcinogenic adduct levels in environmentally exposed persons. Particularly, combinations of the various methods now allow the elucidation of specific adduct structures with detection limits of 1 adduct in 108 unmodified nucleotides or even lower. The quantification of polycyclic aromatic hydrocarbon-DNA (PAH-DNA) adducts in human tissues and cells has been achieved with a number of highly sensitive techniques: immunoassays and immunocytochemistry using polyclonal or monoclonal antisera specific for DNA adducts or modified DNA, the 32P-postlabelling assay, and adduct identification using physicochemical instrumentation. The results summarized in this review show that PAH-DNA adducts have been detected in a variety of human tissues, including target organs of PAH- and tobacco-associated cancers. Although dosimetry has not always been precise, a large number of data now clearly show that lowering exposure to carcinogenic PAH results in decreasing PAH-DNA adduct levels. In most studies, however, bulk DNA of a certain tissue or cell type has been examined, and there were relatively few studies in which mutations as a consequence of DNA damage at specific genes have been investigated. Promising as these biomarker studies seem for epidemiology and health surveillance, future biomonitoring and molecular epidemiological studies should be directed to combine several endpoint measurements: i.e., adduct formation (preferably at specific sites), mutational spectra in cancer-relevant genes, and genetic markers of (cancer) susceptibility in a number of cancer-predisposing genes. 相似文献
13.
Excision of benzo[a]pyrene diol epoxide I adducts from nucleosomal DNA of confluent normal human fibroblasts 总被引:3,自引:0,他引:3
The formation and removal of covalent adducts of racemic 7 beta, 8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE I) was studied in nucleosomal DNA of confluent cultures of normal human fibroblasts (NF). For this purpose NF were prelabeled in their DNA with [14C]-thymidine and treated with [3H]BPDE I. The adducts were composed of 77% (7R)-N2-(7 beta, 8 alpha, 9 alpha-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene-10-yl)deoxyguanosine, 12% of the corresponding 7S-enantiomer and of minor amounts of adducts to cytosine and adenine. The adduct composition did not change significantly in 24-h post treatment incubation. Bulk mononucleosomes were prepared from micrococcal nuclease digested nuclei and their DNA analyzed by gel electrophoresis. The adduct concentrations were determined in 145 base pair (b.p.) nucleosomal core-DNA, 165 b.p. chromatosomal DNA and in total nuclear DNA. From these data the concentration in nucleosomal linker-DNA was calculated. The initial adduct distribution was non-random and 6.3 times higher in 47 b.p. linker-DNA relative to 145 b.p. core-DNA and 9.2 times higher in 27 b.p. linker-DNA relative to 165 b.p. chromatosomal DNA. Adduct removal was very rapid during the first 8 h and more efficient from linker-DNA than from core-DNA. After this early phase the adducts located in 145 b.p. core-DNA became refractory to further excision and represent a major fraction of the adducts persisting in DNA of NF over a prolonged period. In contrast, further adduct removal was observed from nucleosomal linker-DNA. 相似文献
14.
High-performance liquid chromatographic analysis of 32P-postlabeled DNA-aromatic carcinogen adducts 总被引:1,自引:0,他引:1
The technique of 32P postlabeling of DNA-carcinogen adducts is a useful and extremely sensitive method of detecting and quantitating DNA damage by carcinogens. We have adapted the 32P method to analysis by high-pressure liquid chromatography, making the procedure more rapid and convenient than when thin-layer chromatography is used. Following DNA isolation and hydrolysis, nucleotide-carcinogen adducts are enhanced relative to normal nucleotides by solvent extraction and then labeled with high-specific-activity [gamma-32P]ATP. The resulting 32P-postlabeled nucleotides are resolved by reverse-phase ion-pair HPLC. After as little as 3 h of exposure to carcinogens, DNA adducts can be demonstrated from 1 microgram or less of mouse hepatic DNA. Acetylated and nonacetylated adducts can be resolved from hepatic DNA of mice treated with 2-aminofluorene. Differences in DNA damage as measured by adduct formation were demonstrated between "rapid" and "slow" acetylator mouse strains. Rapid-acetylator C57BL/6J mice had three times the amount of hepatic DNA adducts as slow-acetylator A/J mice 3 h after a 60 mg/kg dose of 2-aminofluorene. 4-Aminobiphenyl and 2-naphthylamine each showed an adduct peak with retention time similar to that of the nonacetylated 2-aminofluorene adduct, while benzidine gave a major adduct that eluted somewhat earlier as would be expected for an acetylated adduct. The alkenylbenzenes, safrole and methyleugenol, also formed DNA adducts detectable by this method. DNA prepared from skin of mice painted with benzo[a]pyrene also contained carcinogen-DNA adducts detectable and resolvable by HPLC analysis following 32P postlabeling. The combination of HPLC with 32P postlabeling appears to be a useful technique for the rapid detection and quantitation of DNA damage caused by several classes of aromatic carcinogens. 相似文献
15.
Radioactive compounds incorporated in tissues can have biological effects resulting from energy deposition in subcellular compartments. We addressed the genetic consequences of [(3)H] or [(14)C]thymidine incorporation into mammalian DNA. Low doses of [(3)H]thymidine in CHO cells led to enhanced sensitivity compared with [(14)C]thymidine. Compared with wild-type cells, homologous recombination (HR)-deficient cells were more sensitive to lower doses of [(3)H]thymidine but not to any dose of [(14)C]thymidine. XRCC4-defective cells, however, were sensitive to both low and high doses of [(3)H] and [(14)C]thymidine, suggesting introduction of DNA double-strand breaks, which were confirmed by gamma-H2AX focus formation. While gamma rays induced measurable HR only at toxic doses, sublethal levels of [(3)H] or [(14)C]thymidine strongly induced HR. The level of stimulation was in an inverse relationship to the emitted energies. The RAD51 gene conversion pathway was involved, because [(3)H]thymidine induced RAD51 foci, and [(3)H]thymidine-induced HR was abrogated by expression of dominant negative RAD51. In conclusion, both HR and non-homologous end-joining pathways were involved after labeled nucleotide incorporation (low doses); genetic effects were negatively correlated with the energy emitted but were positively correlated with the energy deposited in the nucleus, suggesting that low-energy beta-particle emitters, at non-toxic doses, may induce genomic instability. 相似文献
16.
Turesky RJ Vouros P 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2004,802(1):155-166
The detection and quantification of heterocyclic aromatic amine (HAA)-DNA adducts, critical biomarkers in interspecies extrapolation of toxicity data for human risk assessment, remains a challenging analytical problem. The two main analytical methods currently in use to screen for HAA-DNA adducts are the 32P-postlabeling assay and mass spectrometry, using either accelerated mass spectrometry (AMS) or liquid chromatography and electrospray ionization mass spectrometry (LC-ESI-MS). In this review, the principal methods to synthesize and characterize DNA adducts, and the methods applied to measure HAA-DNA adduct in vitro and vivo are discussed. 相似文献
17.
Zayed A Jones GD Reid HJ Shoeib T Taylor SE Thomas AL Wood JP Sharp BL 《Metallomics : integrated biometal science》2011,3(10):991-1000
This paper describes a set of fast and selective high performance liquid chromatography (HPLC) methods coupled to electro-spray ionisation linear ion trap mass spectrometry (ESI-MS), sector-field inductively coupled plasma mass spectrometry (SF-ICP-MS) and UV detection for in vitro studies of the bifunctional adducts of oxaliplatin with mono-nucleotides, di-nucleotides and cellular DNA. The stationary phases and the optimised conditions used for each separation are discussed. Interaction of oxaliplatin with A and G mono-nucleotides resulted in the formation of five bifunctional platinum diaminocyclohexane (DACHPt) adducts. These were two isomers of the A-DACHPt-A and A-DACHPt-G adducts, and one G-DACHPt-G adduct, as confirmed by MS/MS spectra obtained by collision induced dissociation. These adducts were also characterised by UV absorption data and SF-ICP-MS elemental (195)Pt and (31)P signals. Further, interaction of oxaliplatin with AG and GG di-nucleotides resulted in the formation of three adducts: DACHPt-GG and two isomers of the DACHPt-AG adduct, as confirmed by ESI-MS and the complementary data obtained by UV and SF-ICP-MS. Finally, a very sensitive LC-ICP-MS method for the quantification of oxaliplatin GG intra-strand adducts (DACHPt-GG) was developed and used for monitoring the in vitro formation and repair of these adducts in human colorectal cancer cells. The method detection limit was 0.14 ppb Pt which was equivalent to 0.22 Pt adduct per 10(6) nucleotides based on a 10 μg DNA sample. This detection limit makes this method suitable for in vivo assessment of DACHPt-GG adducts in patients undergoing oxaliplatin chemotherapy. 相似文献
18.
19.
32P-postlabeling analysis is an ultrasensitive method for the detection and quantitation of carcinogen-DNA adducts. It consists of four principal steps: (i) enzymatic digestion of DNA to nucleoside 3'-monophosphates; (ii) enrichment of the adduct fraction of the DNA digest; (iii) 5'-labeling of the adducts by transfer of 32P-orthophosphate from [gamma-32P]ATP mediated by polynucleotide kinase (PNK); (iv) chromatographic or electrophoretic separation of the labeled adducts or modified nucleotides and quantitation by measurement of their radioactive decay. The assay requires only microgram quantities of DNA and is capable of detecting adducts at frequencies as low as 1 in 10(10) nt, making it applicable to the detection of events resulting from environmental exposures, or experiments using physiological concentrations of agents. It has a wide range of applications in human, animal and in vitro studies, and can be used for a wide variety of classes of compound and for the detection of adducts formed by complex mixtures. This protocol can be completed in 3 d. 相似文献
20.
Sang Soo Hah Paul T. Henderson Kenneth W. Turteltaub 《Bioorganic & medicinal chemistry letters》2010,20(8):2448-2451
Oxaliplatin is a third-generation platinum-based anticancer drug that is currently used in the treatment of metastatic colorectal cancer. Oxaliplatin, like other platinum-based anticancer drugs such as cisplatin and carboplatin, is known to induce apoptosis in tumor cells by binding to nuclear DNA, forming monoadducts, and intra- and interstrand diadducts. Previously, we reported an accelerator mass spectrometry (AMS) assay to measure the kinetics of oxaliplatin-induced DNA damage and repair [Hah, S. S.; Sumbad, R. A.; de Vere White, R. W.; Turteltaub, K. W.; Henderson, P. T. Chem. Res. Toxicol. 2007, 20, 1745]. Here, we describe another application of AMS to the measurement of oxaliplatin–DNA adduct distribution in cultured platinum-sensitive testicular (833 K) and platinum-resistant breast (MDA-MB-231) cancer cells, which resulted in elucidation of cell-dependent differentiation of oxaliplatin–DNA adduct formation, implying that differential adduction and/or accumulation of the drug in cellular DNA may be responsible for the sensitivity of cancer cells to platinum treatment. Ultimately, we hope to use this method to measure the intrinsic platinated DNA adduct repair capacity in cancer patients for use as a biomarker for diagnostics or a predictor of patient outcome. 相似文献