Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0596263 (carcinogenesis)
 64,820 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Dimethyl sulfate was used to prepare 7-methyl-2'-deoxy-guanosine 3'-monophosphate (7-methyl-dGMP), which was ring-opened in alkali to 2'-deoxy-N5-methyl-N5-formyl-2,5,6-triamino-4-oxopyrimidine 3'-monophosphate (ROM-dGMP). ROM-dGMP was not dephosphorylated by nuclease P1 in contrast to normal deoxynucleotides. It was efficiently 5'-phosphorylated by T4 polynucleotide kinase. When methylated DNA was alkali-treated and digested with micrococcal nuclease, spleen phosphodiesterase and nuclease P1, ROM-dGMP was formed and this was labeled with [gamma-32P]-ATP in the presence of polynucleotide kinase. Ring-opening and P1 treatment appear methods of choice for 32P-post-labeling of 7-alkylguanines in DNA.
Carcinogenesis 1989 Sep
PMID:Ring-opened 7-methylguanine nucleotides are resistant to nuclease P1 digestion and good substrates to polynucleotide kinase. 254 53

32P-Postlabelling analysis is a sensitive method of detecting covalent modification of DNA by chemical carcinogens. We demonstrate that tetrol derivatives of the polycyclic aromatic hydrocarbons (PAHs) benzo[a]pyrene (BP) and chrysene become 32P-labelled in the assay in the absence of nucleic acids. The transfer of 32P from [gamma-32P]ATP to the PAH derivatives requires T4 polynucleotide kinase. Phosphorylated dihydrodiols, phenols, triols and parent hydrocarbons were not detected under standard TLC conditions. Labelling of the non-nucleotide substrates was at least 2000-fold less efficient than labelling of a synthetic BP - DNA adduct. Using 75 microCi[gamma-32P]ATP, the detection limit for BP tetrols was 100-200 pg. Labelling of non-adduct substrates is unlikely to interfere with the analysis of DNA isolated from mammalian tissues, but DNA modified by electrophiles in vitro may, if inadequately purified, give rise to spurious radioactive products.
Carcinogenesis 1989 Aug
PMID:Enzyme-mediated phosphorylation of polycyclic hydrocarbon metabolites: detection of non-adduct compounds in the 32P-postlabelling assay. 266 69

A 32P-postlabeling procedure for identifying and quantifying fluoranthene (FA)-DNA adducts has been developed through modifications of the method of Randerath and collaborators. In this modified procedure, labeled adducts are separated chromatographically by high-pressure liquid chromatography (HPLC) and quantified by liquid scintillation counting. FA-modified DNA is digested to nucleotide 3'-monophosphates and nucleotide 3'-monophosphate adducts; unmodified nucleotides are then separated from adducts using a disposable C18 cartridge. Residual unmodified nucleotides, which reduce the efficiency of 32P-postlabeling of FA adducts, are removed by brief digestion with nuclease P1. This treatment selectively dephosphorylates unmodified nucleotide 3'-monophosphates, while FA adducts are minimally affected. FA adducts are then 5'-phosphorylated with polynucleotide kinase and [gamma-32P]ATP. Prolonged treatment with nuclease P1 then is employed to remove the unlabeled 3'-phosphate from adducted diphosphate nucleotides, following which adducts are separated by HPLC and quantified by liquid scintillation counting. Postlabeled microsomally-activated FA-modified DNA contained adducts derived from anti- and syn-2,3-dihydroxy-1,10b-epoxy-1,2,3-trihydrofluoranthene. The identity of the major adduct in DNA-bound microsomally-activated FA was confirmed by this HPLC-32P-postlabeling method as an anti-2,3-dihydroxy-1,10b-epoxy-fluoranthene nucleotide adduct. Each step in the procedure was optimized with respect to experimental conditions, and the recovery of adducts was determined by analysis of DNA modified with [3H]FA. In repeated analyses of 2-50 micrograms DNA containing 1.8 adducts per 10(8) nucleotides, 10-15% of total DNA-bound [3H]FA was recovered as the major adduct; recovery was greater from DNA containing higher levels of adducts. The reproducibility of multiple analyses of the same sample was approximately 5%, and multiple analyses at different times were reproducible within experimental error. The limit of detection of the method was approximately 0.1 fmol adduct, representing a binding level of approximately 3 adducts per 10(8) nucleotides in 1 microgram DNA or approximately 1 adduct per 10(10) nucleotides in 500 micrograms DNA. Because the method is not limited with respect to the amount of DNA that can be subjected to analysis, the inherent sensitivity for adduct detection can be greatly enhanced by analysis of larger quantities of DNA.
Carcinogenesis 1989 Sep
PMID:Fluoranthene-DNA adducts: identification and quantification by an HPLC-32P-postlabeling method. 267 Mar 1

DNA single-strand breaks are caused by aqueous extracts of cigarette tar, due to the reduction of oxygen to superoxide by tar and the subsequent production of hydroxyl radicals. The action of DNA metabolism enzymes on these single-strand breaks has been studied to probe the consequences of these lesions for DNA repair. Our results demonstrate that cigarette tar-induced nicks are blocked at the 3' terminus since they are totally incapable of activating DNA for DNA synthesis by Escherichia coli DNA polymerase I. The 3' termini of these tar-induced nicks are activated, however, for DNA synthesis by E. coli exonuclease III or by the 3' phosphatase activity of T4 polynucleotide kinase. Because of the inability of tar-induced lesions to support DNA synthesis, they probably require a multi-step process for repair in vivo. As a consequence, the overall likelihood of mutation is increased due to the possibility for error at each step of the repair process.
Carcinogenesis 1987 Oct
PMID:DNA synthesis is blocked by cigarette tar-induced DNA single-strand breaks. 282 Jun 3

The 32P-postlabeling method has been adapted for the analysis of thymidine-cis-glycol-3'-phosphate (cis-dTGp,cis-5,6-dihydroxy-5,6-dihydrothymidine-3'-phosphat e). Cis-dTGp was isolated and purified from normal nucleotides by phenylboronate affinity chromatography and phosphorylated by T4 polynucleotide kinase in presence of 1 mM BeCl2 at pH 7.5. These modifications of the postlabeling method resulted in a 5'-phosphorylation of dTGp with a labeling efficiency of up to 20% whereas the natural nucleotides were almost completely dephosphorylated at the 3' position under these conditions. The reaction products, containing radio-labeled thymidine-cis-glycol-3',5'-bis-[5'-32P]phosphate (cis-*pdTGp), were separated by two-dimensional anion-exchange TLC on polyethyleneimine cellulose sheets. Boric acid was added in the second dimension in order to selectively retard cis-glycols. The method was applied to gamma-irradiated nucleotides and calf thymus DNA. In the nucleotide mixture, 330-99,000 thymine glycol (TG) moieties were detected per 10(6) thymines (T) in a dose range of 14-1000 Gy respectively. In DNA, these values ranged from 400 to 2700 TG/10(6) T. The data are in good agreement with methods using radiochemical and immunological techniques. Non-irradiated DNA showed a background level of 10TG/10(6) T. This practical limit of detection was higher than can be achieved with the postlabeling technique, indicating that the present method might be a sensitive alternative for a determination of oxidative DNA damage.
Carcinogenesis 1989 Jan
PMID:Detection by 32P-postlabeling of thymidine glycol in gamma-irradiated DNA. 291 May 30

Exceedingly sensitive procedures are required to detect the presence of covalent DNA adducts in humans exposed to environmental genotoxicants because of low levels of such derivatives (1 adduct in 10(8)-10(10) DNA nucleotides). A 32P-postlabeling assay for detection and quantitation of carcinogen--DNA adducts with a sensitivity limit of 1 adduct in 10(7)-10(8) nucleotides has been described previously. In the standard procedure, DNA is enzymatically digested to 3'-phosphorylated normal and adducted mononucleotides, which are quantitatively 32P-labeled at their 5'-hydroxyl groups by T4 polynucleotide kinase-catalyzed [32P]phosphate transfer from [gamma-32P]ATP. 32P-labeled derivatives are resolved by t.l.c., detected by autoradiography and quantitated by counting. We now report that a minor modification of this procedure, entailing the postincubation of DNA digests with Penicillium citrinum nuclease P1 before 32P-labeling, enhanced the technique's sensitivity to 1 adduct in approximately 10(10) nucleotides for a 10-micrograms DNA sample. Nuclease P1 cleaves deoxyribonucleoside 3'-monophosphates of normal nucleotides to deoxyribonucleosides which do not serve as substrates for polynucleotide kinase, while most adducted nucleotides were found to be totally or partially resistant to the 3'-dephosphorylating action of nuclease P1. The additional enzymatic step enabled specific labeling of adducts in up to 20 micrograms of DNA with excess carrier-free [gamma-32P]ATP. The enzymatic digestion conditions were standardized to afford optimal adduct recovery. The new procedure was found to be simple, highly reproducible, and applicable to the detection and measurement of aromatic or bulky non-aromatic DNA adducts formed with such structurally diverse carcinogens as benzo[a]pyrene, 7,12-dimethyl-benz[a]anthracene, dibenzo[c,g]carbazole, 4-aminobiphenyl, safrole and mitomycin C; most adducts were recovered quantitatively with a 500- to 1000-fold increase in 32P-count rates as compared with the standard procedure.
Carcinogenesis 1986 Sep
PMID:Nuclease P1-mediated enhancement of sensitivity of 32P-postlabeling test for structurally diverse DNA adducts. 301 1

The sequence selectivity of methylation at the O6 and N7 position of guanine by N-methyl-N'-nitrosourea (MNU) and the rate of removal of O6-methylguanine by O6alkylguanine-DNA alkyltransferase (AGT) was determined using dodecadeoxynucleotides of defined structure. The extent of guanine adduct formed in self-complementary dodecamers, 5'-TATACGCGTATA-3', 5'-TATACCGGTATA-3' and 5'-TATAGGCCTATA-3', after methylation with [3H]MNU in a representative experiment were, respectively, 10, 19 and 30 pmol O6-methylguanine/mumol guanine and 97, 189 and 217 pmol N7-methylguanine/mumol guanine. The O6-methylguanine/N7-methylguanine ratio remained relatively constant for each dodecamer. A direct comparison between the methylation at guanine with adenine or thymine as the 5'-flanking base was made with two dodecamers, 5'-TATACATGTATA-3' and 5'-TATACTAGTATA-3'. When the guanine residue was preceded 5' by an adenine, the level of O6 and N7-alkylation was, respectively, 2.1-fold and 1.5-fold greater than when guanine was preceded 5' by a thymine. These date are consistent with a regioselective mechanism for alkylnitrosourea alkylation of guanine. The methylated dodecamer, 5'-TATACGCGTATA-3' was repaired faster than 5'-TATACCGGTATA-3' by HT29 extract containing AGT with a loss in 10 min of 0.052 pmol and 0.025 pmol O6-methylguanine, respectively. Dodecamers of the structure 5'-dCGCGAATTCm6GCG-3' and 5'-dCGCCAATTGm6GCG-3' were labeled at the 5' end with 32P by the reaction with polynucleotide kinase and after incubation with AGT, the methylated and demethylated dodecamers were separated by reversed-phase HPLC. The amount of demethylated product formed was greater for the dodecamer containing cytosine as the 5'-flanking base to O6-methylguanine compared to guanine in that same position. A higher extent of alkylation by MNU and a slower rate of repair by AGT for sites in which a guanine or modified guanine is preceded by a purine rather than a pyrimidine may explain, at least in part, mutational hot spots.
Carcinogenesis 1988 Nov
PMID:Sequence specificity of guanine alkylation and repair. 318 Mar 51

A 32P-adduct assay for the measurement of low levels (1 adduct per 10(7) nucleotides) of binding of carcinogens to DNA has been reported previously. In this procedure, DNA is enzymatically hydrolyzed to 3'-monophosphates of normal nucleosides and adducts, which are 5'-32P-labeled by T4 polynucleotide kinase and [gamma-32P]ATP. Labeled adducts are resolved by TLC. Enrichment of adducts by extraction in 1-butanol [Gupta, R.C. (1985) Cancer Res., 45, 5656] or digestion with nuclease P1 [Reddy, M.V. and Randerath, K. (1986) Carcinogenesis, 7, 1543] prior to 32P-labeling, however, increased the sensitivity of detection for many adducts to a level of 1 per 10(9-10) nucleotides, although adduct recovery particularly in the latter assay depended on the chemical nature of adducts. We have now compared recoveries for greater than 70, different carcinogen-DNA adducts of known and unknown chemical nature in the two enrichment procedures as well as in a new procedure in which polynucleotide kinase is substituted for nuclease P1. When compared with the butanol extraction procedure, arylamines (such as 2-aminofluorene, 2-aminophenanthrene, 2-naphthylamine, 4-aminobiphenyl, 4-azoaminobenzene and N'-acetylbenzidine) bound to the C8 position of guanine were lost almost completely (0.2-4% recovery) in the nuclease P1-mediated assay, but the presence of a polar group in the aromatic amine moiety (such as 2-acetylaminofluorene, 2-acetylamino-phenanthrene and methyl-4-azoaminophenyl) rendered similar recovery. In contrast, aromatic amines (2-amino-phenanthrene, 2-acetylaminophenanthrene, 2-acetylaminofluorene and methyl-4-azoaminobenzene) and polycyclic aromatic hydrocarbons (benzo[a]pyrene, bromomethylbenzanthracene and benzanthracene) bound to the exocyclic positions of guanine or adenine showed extensive or as complete recovery in the nuclease P1 procedure as in the extraction procedure. Some of the unknown presumably polar adducts showed a lower recovery (30-70%) in the butanol procedure as compared to the nuclease P1 enrichment. The recovery pattern of most adducts examined in the polynucleotide kinase-enrichment assay was essentially the same as found in nuclease P1-mediated assay, except that overall lower values were obtained. Our data suggest that a given DNA sample should be analyzed by different versions of the 32P-adduct assay, particularly, DNA of specimens of humans exposed to low levels of unknown carcinogens. The observation that chemical structure of an adduct may be detrimental in its recovery in the enzyme- and extraction-mediated enrichment procedures may serve as a probe in the structural characterization of adducts of unknown carcinogens.
Carcinogenesis 1988 Sep
PMID:32P-adduct assay: comparative recoveries of structurally diverse DNA adducts in the various enhancement procedures. 340 74

We have previously described a 32P assay for the detection and quantitation of aromatic carcinogen:DNA adducts (R. C. Gupta et al., Carcinogenesis (Lond.), 3: 1081-1092, 1982). The method entails enzymatic digestion of DNA to deoxynucleoside 3'-monophosphates which are then converted to deoxynucleoside 3',5'-[5'-32P]diphosphates by T4 polynucleotide kinase-catalyzed 32P transfer from adenosine [gamma-32P]triphosphate. Labeled adducts are purified and resolved by four-directional thin-layer chromatography. This procedure can detect one adduct in 10(7)-10(8) nucleotides but quantitation of adduct concentrations of one adduct in greater than 5 X 10(6) nucleotides becomes exceedingly difficult. I have now found that isolation of DNA adducts by extraction with 1-butanol in the presence of the phase-transfer agent tetrabutylammonium chloride prior to the labeling allows one to use excess carrier-free adenosine [gamma-32P]triphosphate (100-200 microCi), thus enabling quantitative analysis of a single adduct in 10(9)-10(10) nucleotides when 1-10 micrograms of the DNA are used. Further increase in the sensitivity of the assay requires higher amount of DNA. The four-directional thin-layer chromatography system has been modified so as to analyze simultaneously as many as 35-40 DNA samples. The new protocol, as applied to a number of carcinogenic aromatic amines and polycyclic aromatic hydrocarbons of diverse structure, is capable of detecting and quantitating adducts at the level of one adduct per 10(10) nucleotides.
 ...
PMID:Enhanced sensitivity of 32P-postlabeling analysis of aromatic carcinogen:DNA adducts. 405 37

Carcinogen--DNA adducts were detected and determined by 32P-postlabeling assay after exposure of mouse or rat tissues in vivo to a total of 28 compounds comprising 7 arylamines and derivatives, 3 azo compounds, 2 nitroaromatics, 12 polycyclic aromatic hydrocarbons, and 4 methylating agents. DNA was isolated from mouse skin, mouse liver, and rat liver after treatment with the individual carcinogens, then digested enzymatically to deoxyribonucleoside 3'-monophosphates, which were converted to 5'-32P-labeled deoxyribonucleoside 3',5'-bisphosphates by T4 polynucleotide kinase-catalyzed [32P]phosphate transfer from [gamma-32P]ATP. The nucleotides were resolved by anion-exchange t.l.c. on polyethyleneimine-cellulose and detected by autoradiography. The determination of low levels of DNA binding of the aromatic carcinogens entailed the removal of normal nucleotides prior to the resolution of adduct nucleotides. For this purpose, an alternative procedure employing reversed-phase t.l.c. was devised which offered advantages for the detection of quantitatively minor adducts. The procedures described enabled the detection of 1 aromatic DNA adduct in approximately 10(8) normal nucleotides, while the limit of detection of methylated adducts was 1 adduct in approximately 6 X 10(5) nucleotides. The results show that a great number of carcinogen-DNA adducts of diverse structure are substrates for 32P-labeling by polynucleotide kinase-catalyzed phosphorylation. Because covalent DNA adduct formation in vivo appears to be an essential property of the majority of chemical carcinogens, 32P-postlabeling analysis of carcinogen--DNA adducts in mammalian tissues may serve as a test for the screening of chemicals for potential carcinogenicity.
Carcinogenesis 1984 Feb
PMID:32P-postlabeling test for covalent DNA binding of chemicals in vivo: application to a variety of aromatic carcinogens and methylating agents. 669 41


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