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Query: UMLS:C0596263 (carcinogenesis)
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The 32P-postlabeling method has found wide application as a sensitive technique for detecting the presence of a broad range of bulky aromatic compounds covalently bound to DNA. In this method, the modified DNA is enzymatically degraded to 3'-mononucleotides and labeled with [32P]-phosphate at the 5'-position using [gamma-32P]ATP and T4 polynucleotide kinase. The 32P-labeled DNA digest is then chromatographed in two dimensions on polyethyleneimine - cellulose thin-layer plates. Screen-enhanced autoradiography is used to locate the presence of the radiolabeled adducts on the chromatogram, and the radioactive areas are generally excised and quantitated by liquid scintillation spectrometry. However, on a chromatogram with multiple adducts, it can be difficult to quantitative partially resolved adducts and evaluated background radioactivity levels. We have evaluated the use of storage phosphor imaging techniques to quantitate and map the radioactivity on chromatograms generated by the 32P-postlabeling method. The results showed that storage phosphor imaging was approximately 10 times more sensitive than screen-enhanced autoradiography at -80 degrees C for the detection of 32P, exhibits a greater linear range of response, has a resolution that compares favorably to film and has a lower background than does liquid scintillation spectrometry. Further, the generation of a digitized record of the distribution and intensity of radioactivity allows for computer-assisted assessment of adduct profiles and can facilitate quantitation of individual adducts and radioactive zones comprised of multiple overlapping adducts in complex chromatograms. Additionally, the permanent record created by the imaging technology permits facile retrospective analysis of samples, whereas with autoradiography and liquid scintillation spectrometry reanalysis of a replicate sample is required.
Carcinogenesis 1992 Aug
PMID:Storage phosphor imaging technique for detection and quantitation of DNA adducts measured by the 32P-postlabeling assay. 149 99

The 32P-postlabelling assay was used to determine adducts arising upon the reaction of malonaldehyde with 2'-deoxyguanosine-3'-monophosphate. The adducts formed were isolated, structurally characterized and identified as 3-(2-deoxy-beta-D-erythro-pentafuranosyl)pyrimido[1,2-alpha] purin-10(3H)-one. The kinetics of phosphorylation by T4 polynucleotide kinase was studied using 500 fmol of the synthesized standard and found to reach its maximum after 1 h of incubation. A 60% labelling efficiency was obtained at low concentrations of substrate. The adducted substrate was detected at the sub-femtomolar level. Sensitivity of the adducts towards nuclease P1 3'-dephosphorylation was also tested. The same adduct could be detected from calf thymus DNA that had been reacted in vitro with malonaldehyde, and in DNA isolated from mice treated with [14C]malonaldehyde. DNA adducts formed in vitro were isolated after enzymatic digestion to mononucleotides followed by HPLC fractionation or nuclease P1 digestion of normal nucleotides. A combination of the two procedures proved to be the method of choice for the isolation of the malonaldehyde-DNA adducts formed in vivo prior to applying the 32P-postlabelling assay.
Carcinogenesis 1992 Apr
PMID:Determination of malonaldehyde-modified 2'-deoxyguanosine-3'-monophosphate and DNA by 32P-postlabelling. 157 12

Alkylated nucleotides have been detected by 32P-postlabelling using the enzyme T4 polynucleotide kinase which phosphorylates the 3'-mononucleotides to give the 3',[5'-32P]bisphosphates. These may then be separated by two-dimensional TLC as the bisphosphates or the [5'-32P]monophosphates. We describe here an alternative approach using the Epstein-Barr virus (EBV) encoded thymidine kinase (TK) to directly phosphorylate adducted nucleosides to give the [5'-32P]monophosphates. Using a series of methyl, ethyl and butyl thymidines EBV-encoded TK was shown to phosphorylate a wide range of adducted thymidines with varying degrees of labelling efficiency; N3-methyl thymidine was labelled with the highest efficiency and O4-ethyl thymidine the lowest. Whereas O4-methyl thymidine was labelled at a higher efficiency than O2-methyl thymidine, O4-ethyl and O4-butyl thymidines were labelled at a much lower efficiency than the corresponding O2-alkyl thymidines. Labelling efficiency increased with pH in the range pH 7 to pH 9, but the relative labelling efficiency was ATP independent. This direct phosphorylation of adducted nucleosides offers an alternative approach to the detection of alkylated residues in DNA which may complement current postlabelling procedures.
Carcinogenesis 1991 Apr
PMID:32P-postlabelling of alkylated thymidines using Epstein-Barr virus encoded thymidine kinase. 184 71

A 32P-postlabelling method was developed to measure 7-methylguanine in human DNA. DNA was digested to nucleotides and 7-methyl-2'-deoxyguanosine-3'-monophosphate (7-me-dGMP) was isolated from normal nucleotides using strong anion-exchange column chromatography. Overall the method gave 35-45% yield as measured with DNA methylated with tritiated dimethyl sulfate. Total white blood cell DNA from healthy non-smokers (n = 17) contained from 2.5 7-methylguanine residues/10(7) nucleotides, corrected for the losses in preparation. Among four patients sampled immediately after a total dose of 1050-2800 mg of dacarbazine or procarbazine, the mean adduct level was 57 7-methylguanine residues/10(7) nucleotides. As further method development, we also investigated the phosphorylation reaction by T4 polynucleotide kinase using dinucleotides containing 7-methylguanine and corresponding imidazole ring-opened products as substrates. We found that imidazole ring-opened dTpdG-Me is resistant to digestion with deoxyribonuclease I, snake venom phosphodiesterase and prostatic acid phosphatase. It is quantitatively phosphorylated at femtomolar levels. This method is shown to be suitable for the detection of 7-methylguanine in DNA, and is suggested to be the approach most suited to postlabelling large and labile 7-alkylguanines in DNA.
Carcinogenesis 1991 Aug
PMID:Measurement by 32P-postlabelling of 7-methylguanine levels in white blood cell DNA of healthy individuals and cancer patients treated with dacarbazine and procarbazine. Human data and method development for 7-alkylguanines. 186 Jan 63

Two generally applicable [35S]phosphorothioate postlabeling procedures for the HPLC analysis of polycyclic aromatic hydrocarbon (PAH)-DNA adducts have been developed based upon [32P]phosphate postlabeling assays described by Gupta and Randerath et al. In one procedure, benzo[a]pyrene (B[a]P)-modified DNA was digested to nucleoside 3'-phosphates by micrococcal nuclease and spleen phosphodiesterase and the adducted nucleotides were extracted with 1-butanol. The adducted nucleoside-3'-phosphates were 5'-thiophosphorylated by T4 polynucleotide kinase (T4PNK) and adenosine 5'-O-(3-[35S]thiotriphosphate) to yield [35S]B[a]P-nucleoside-5'-phosphorothioate-3'-phosphate adducts. Although thiophosphorylation of B[a]P-DNA adducts was slower than the corresponding phosphorylation reaction, similar recoveries of the postlabeled adducts were achieved with longer incubation times and higher concentrations of T4PNK. A major advantage of this procedure over the 32P-postlabeling procedure is that the resistance of phosphorothioates to degradation by phosphatases allows selective removal of the unlabeled 3'-phosphate from the [35S]B[a]P-nucleoside-5'-phosphorothioate-3'-phosphate adducts by brief treatment with alkaline phosphatase. [35S]B[a]P-nucleoside-5'-phosphorothioate adducts were also prepared using a nuclease P1/prostatic acid phosphatase DNA degradation method. For anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE)-modified DNA, overall adduct recoveries were substantially higher with the nuclease P1/prostatic acid phosphatase method (48-51%) than with the micrococcal nuclease/spleen phosphodiesterase/alkaline phosphatase method (22-29%). There were no significant differences in the HPLC profiles of the [35S]phosphorothioate-postlabeled adducts obtained from these two procedures. HPLC analysis of B[a]P-DNA adducts formed in B[a]P-treated hamster embryo cell cultures demonstrated the formation of two major adducts, (+)syn-BPDE-deoxyguanosine-5'-phosphorothioate and (+)anti-BPDE-deoxyguanosine-5'-phosphorothioate, along with other minor adducts. Based upon an overall adduct recovery of 20% and 0.5 mol as the detection limit of this 35S-postlabeling/HPLC assay, the sensitivity of this assay is 1 adduct/10(8) nucleotides for a 60 micrograms DNA sample. This method offers the advantages of using 35S which has a longer half-life and lower radioactive decay energy than 32P and the ability to prepare PAH-DNA adducts at the monophosphorothioate level which greatly facilitates separation of individual 35S-postlabeled PAH-DNA adducts by HPLC.
Carcinogenesis 1991 May
PMID:Detection and identification of benzo[a]pyrene-DNA adducts by [35S]phosphorothioate labeling and HPLC. 202 54

Previous work has shown that 7,12-dimethylbenz[a]anthracene (DMBA)-DNA adducts can be converted by 32P-postlabeling to different types of radiolabeled derivatives (nucleoside 3',5'-bisphosphates, nucleoside 5'-monophosphates and dinucleotides), and that the 32P-labeled 3',5'-bisphosphate derivatives can be further characterized by cross-referencing with 3H-labeled nucleoside DMBA adducts, for which structural information is available. This work has now been extended by TLC comparisons of 5'-monophosphate and 3',5'-bisphosphate DMBA adducts. To this end, DMBA-modified DNA was enzymatically hydrolyzed to 3'-monophosphates (Xp + Np) or to dinucleotides (XpN), and these digestion products were 32P-postlabeled by published procedures to yield 3',5'-bisphosphate (*pXp) or 5'-monophosphate (*pX) adducts. Individual *pXp and *pX fractions were isolated from polyethyleneimine (PEI)-cellulose TLC maps and chromatographically compared after enzymatic 3'-dephosphorylations of the 3',5'-bisphosphate (*pXp) derivatives (*pXp----*pX + Pi). Four reactions were standardized and employed for this purpose: (i) 3'-dephosphorylation by extensive digestion with nuclease P1; (ii) 3'-dephosphorylation catalyzed by polynucleotide kinase; (iii) partial dephosphorylation by bacterial alkaline phosphatase; and (iv) partial dephosphorylation by prostatic acid phosphatase. Individual DMBA adducts displayed marked differences with regard to their susceptibility to enzymatic dephosphorylation. The three major and most minor postlabeled 5'-monophosphate DMBA adducts were cross-referenced this way with 3',5'-bisphosphate and nucleoside adducts, so that specific dihydrodiol epoxide-nucleoside 5'-monophosphate adducts can now be identified and measured by 32P-postlabeling.
Carcinogenesis 1990 Jan
PMID:High-resolution TLC mapping and characterization of 32P-postlabeled monophosphate 7,12-dimethylbenz[a]anthracene-DNA adducts. 210 82

The utilization of the 32P-postlabeling assay in combination with TLC for the sensitive detection and estimation of aromatic DNA adducts has been increasing in the past few years. The procedure consists of 32P-labeling of carcinogen-adducted 3'-nucleotides in the DNA digests using [gamma-32P]ATP and polynucleotide kinase, separation of 32P-labeled adducts by TLC, and their detection by autoradiography. During both 32P-labeling and initial phases of TLC, a relatively high amount of [gamma-32P]ATP (3.0-4.5 mCi) is handled when 30 samples are processed simultaneously. We describe the design of acrylic shielding apparatus, semi-automatic TLC spotting devices, and devices for development and washing of multiple TLC plates, which not only provide substantial protection from exposure to 32P beta radiation, but also allow quick and easy handling of a large number of samples, thus expediting the assay workup and making it less labor-intensive. Specifically, the equipment includes: (i) a multi-tube carousel rack (7.5 cm diameter and 7.7 cm height) having 15 wells to hold capless Eppendorf tubes (0.5 ml) and a rotatable lid with an aperture to access individual tubes; (ii) a pipet shielder; (iii) two semi-automatic spotting devices to apply radioactive solutions to TLC plates; (iv) a multi-plate holder for TLC plates; and (v) a mechanical device for washing multiple TLC plates. Item (i) is small enough to be held in one-hand, vortexed, and centrifuged to mix the solutions in each tube while beta radiation is shielded. Items (iii) to (iv) aid in the automation of the assay.
Carcinogenesis 1990 Apr
PMID:32P-postlabeling assay for carcinogen-DNA adducts: description of beta shielding apparatus and semi-automatic spotting and washing devices that facilitate the handling of multiple samples. 232 7

Ethylene oxide, diethyl sulphate and dimethyl sulphate were used to synthesize the corresponding 7-alkylation products of 2'-deoxyguanosine 3'-monophosphate (dGMP). The purified adducts were used as substrates in the 32P-post-labelling reaction with T4 polynucleotide kinase. The kinetics of phosphorylation were studied with 7-(2-hydroxyethyl)-dGMP: most net product was formed by 15 min and only a small increase was seen until 4 h. When different concentrations of the adducts were tested, a complete phosphorylation was noted for 7-methyl-dGMP to the lowest tested amount of 1 fmol. The efficiencies of phosphorylation for 7-ethyl- and 7-hydroxyethyl-dGMP were 1.5 and 0.5% respectively. The proportions phosphorylated were uniform over the concentration range tested. The results demonstrate dramatic differences in the efficiency of phosphorylation between structural analogues, which is probably related to a decreased affinity of the substrate to the enzyme or to an interference in the transfer of the phosphate group on the active site of the enzyme.
Carcinogenesis 1990 Aug
PMID:32P-postlabelling of 2-hydroxyethylated, ethylated and methylated adducts of 2'-deoxyguanosine 3'-monophosphate. 238 26

The formation of DNA adducts represents a key step in the postnatal initiation of the carcinogenic process. Little is known as yet about the role of prenatally induced adducts in transplacental carcinogenesis in offspring. Measurement of transplacental DNA damage in fetal organs of experimental animals has been difficult in the past because of the small amounts of DNA available and low adduct levels. In principle, these difficulties have been overcome by the recent development of a highly sensitive 32P-postlabelling assay which can be applied to a large number of DNA adducts of diverse structure and requires only microgram amounts of DNA for analysis. In this assay, tissue DNA is degraded to mononucleotides; these are enzymatically 32P-labelled via T4 polynucleotide kinase-catalysed [32P]phosphate transfer from [gamma--32P]ATP, to form 5'--32P-labelled 3',5'-bisphosphate derivatives; the labelled products are separated into normal and adducted [32P]nucleotides and quantified by thin-layer chromatography, autoradiography and scintillation (Cerenkov) counting. This technique allows the detection and quantitation of one adduct in 10(8)-10(10) DNA nucleotides (approximately 1-100 adducts/mammalian genome) using a 10-micrograms DNA sample and has been applied in studies of adduct formation from transplacental carcinogens in fetal and adult rodent tissues. In this paper, we review application of 32P-postlabelling to DNA adducts formed with transplacental or suspected transplacental carcinogens in fetal and maternal tissues. The carcinogens studied include diethylstilboestrol (DES), benzo[a]pyrene, safrole, 4-aminobiphenyl and 4-nitroquinoline-1-oxide, as well as cigarette smoke condensate. In DNA of DES-exposed hamsters, one major and several minor adduct spots were observed, which were absent from vehicle controls. A characteristic adduct, which resembled the major hamster adduct chromatographically, was detected in all exposed mouse tissue, except fetal kidney. Chronic administration of low doses of DES to male Syrian hamsters led to an entirely different pattern of adducts in kidney DNA, the target organ of carcinogenesis. These adducts did not contain covalently bound oestrogen moieties and appeared to be formed indirectly: oestrogen appeared to induce or enhance the synthesis of an endogenous electrophilic metabolite reacting with DNA. Thus, multiple mechanisms exist by which DES can damage DNA. Additional work using 32P-postlabelling has shown that non-hormonal genotoxicants (e.g., benzo[a]pyrene, safrole, 4-aminobiphenyl, 4-nitroquinoline-1-oxide) and cigarette smoke condensate given to pregnant mice can induce specific DNA adduct profiles in fetal tissues.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Use of the 32P-postlabelling assay to study transplacental carcinogens and transplacental carcinogenesis. 250 47

A new sensitive 32P-postlabeling assay for DNA adducts has been developed in which DNA is hydrolyzed initially by nuclease P1 and prostatic acid phosphatase instead of micrococcal nuclease and spleen phosphodiesterase as employed in previous postlabeling procedures. When DNA containing bulky adducts, X1, X2, .....Xn, is digested with nuclease P1 at pH 5, normal nucleotides are released as 5'-monophosphates, pN, while adducts are excised as 5'-phosphorylated dinucleotides, pXipN, because internucleotide linkages on the 3' side of X resist attack by nuclease P1. Addition of prostatic acid phosphatase to such a digest results in 5'-dephosphorylation of the nucleotides to normal nucleosides, N, and adducted dinucleotides, XipN, carrying a 5'-terminal free hydroxyl group. The dinucleotides but not nucleosides are converted to 5'-32P-labeled dinucleotides, [32P]pXipN, by T4 polynucleotide kinase-catalyzed [32P]phosphate transfer from [gamma-32P]ATP. Upon mapping on polyethyleneimine--cellulose anion-exchange TLC, the labeled dinucleotide adducts produce characteristic autoradiographic fingerprints. Alternatively, they are further digested with snake venom phosphodiesterase to yield 5'-monophosphates, [32P]pXi and pN. TLC profiles of the monophosphate adducts are distinct from those of the dinucleotides. These reactions provide the basis of the new 32P-postlabeling scheme, which is compared in this paper with a previously reported protocol yielding adducts in the form of 5'-32P-labeled 3',5'-bisphosphates, [32P]pXip. The results show that the availability of three different types of 32P-postlabeled derivatives for the same adduct aids in the analysis and chromatographic characterization of DNA adducts from diverse exogenous and endogenous sources.
Carcinogenesis 1989 Jul
PMID:A new sensitive 32P-postlabeling assay based on the specific enzymatic conversion of bulky DNA lesions to radiolabeled dinucleotides and nucleoside 5'-monophosphates. 254 10


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