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)

DNA-RNA hybridization studies, using nuclear RNA's (nRNA's) labeled in vivo and in vitro with high specific radioactivities, were performed to compare the nRNA populations of normal rat liver, livers treated with 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB), and 3'-Me-DAB-induced hepatomas. The study with normal liver nRNA labeled by i.p. injection of [3H]orotic acid indicated that the nuclei of a 3'-Me-DAB-induced transplanted hepatoma, AH136B, lacked some RNA species present in normal liver nuclei. No qualitative difference in thee RNA populations was seen between normal liver and the livers of rats fed a carcinogenic amount of 3'-Me-DAB, either alone or in combination with 4-nitrostilbene which enhanced the azo dye carcinogenesis. Then, nRNA's of both normal liver and AH136B hepatoma were labeled in vitro by phosphorylation with polynucleotide kinase and adenosine 5'-[gamma-32P]triphosphate. The competitive hybridization with 32P-labeled normal liver nRNA was competed, and the deletion of RNA in the nuclei of AH136B hepatoma or 3'-Me-DAB-induced primary hepatoma was estimated to be 15% or more in the measure of radioactivity of the hybridized normal liver nRNA. 32P-labeled AH136B hepatoma nRNA was completed completely by liver nRNA's, suggesting that no unique RNA species were present in the hepatoma nuclei.
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PMID:Analysis of loss of nuclear RNA in azo dye-induced hepatoma by DNA-RNA competitive hybridization. 705 6

A newly developed enzymatic 32P-postlabeling method was applied to the analysis of DNA's containing non-radioactive arylamine, arylamide, and polycyclic aromatic hydrocarbon adducts. DNA reacted in vitro with N-hydroxy-2-amino-fluorene, N-acetoxy-2-acetylaminofluorene, and 7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, respectively, as well as DNA preparations from the liver of rats treated with N-hydroxy-2-acetylaminofluorene and benzo[a]pyrene, respectively, were enzymatically digested to deoxyribonucleoside 3'-monophosphates, which were then converted to [5'-32P]deoxyribonucleoside 3',5'-bisphosphates by T4 polynucleotide kinase-catalyzed [32P]phosphate transfer from [gamma-32P]ATP. The 32P-labeled nucleotides were resolved by anion-exchange t.l.c. on polyethyleneimine-cellulose and detected by autoradiography. Aromatic adduct nucleotides were found to be retained at the origin in aqueous electrolyte solutions, but to migrate as distinct spots in solvents containing 7-8.5 M urea. Advantage was taken of this observation to remove 32P-labeled normal DNA nucleotides from adduct nucleotides. This purification enabled the detection of a single adduct in 10(7)-10(8) normal nucleotides. The method appears applicable to the ultrasensitive detection of a large number of carcinogen--DNA adducts of diverse structure without requiring radioactive carcinogens or specific antibodies.
Carcinogenesis 1982
PMID:32P-postlabeling analysis of non-radioactive aromatic carcinogen--DNA adducts. 713 66

A 32P-postlabelling assay was developed for the analysis of adducts arising from the reaction of 2'-deoxyguanosine-3'-monophosphate with acetaldehyde, the primary oxidative metabolite of ethanol. The 32P-postlabelling reaction was optimized by testing various parameters such as the kinetics of phosphorylation by T4 polynucleotide kinase, substrate-concentration-dependent labelling efficiency and the concentration of the various ingredients of the phosphorylation reaction. The sensitivity to 3'-monophosphate dephosphorylation activity of nuclease P1 was also studied. Three stable adducts were separated by reversed-phase HPLC. The major stable adduct was structurally characterized and identified as N2-ethyl-2'-deoxyguanosine and could be detected, after reduction with NaBH4 or a mixture of ascorbic acid and GSH, in calf thymus DNA samples that had been reacted in vitro with acetaldehyde. DNA adducts were isolated after enzymatic digestion to mononucleotides followed by nuclease P1 digestion of normal nucleotides. The average levels of acetaldehyde-DNA adducts detected in these samples were 12.1 +/- 2.3 (n = 17) and 4.9 +/- 0.9 (n = 9) adducts/10(7) nucleotides after reduction with NaBH4, or ascorbic acid and GSH respectively. The 32P-postlabelling method was further validated by the detection of acetaldehyde adducts in liver DNA from mice treated with ethanol. The average concentration of the adducts detected in these animals was 1.5 +/- 0.8 (n = 7) adducts/10(8) nucleotides, as analyzed by reversed-phase HPLC with online detection of radioactivity.
Carcinogenesis 1995 Sep
PMID:Development of a 32P-postlabelling method for the analysis of adducts arising through the reaction of acetaldehyde with 2'-deoxyguanosine-3'-monophosphate and DNA. 755 72

We reported (Scates et al. Carcinogenesis 1994, 15, 2945-2948) that incubating a range of bile acids with DNA in vitro, with or without exogenous metabolic activation, gave no evidence of DNA adduct formation as judged by the nuclease P1 method of 32P-postlabelling. In contrast Hamada et al. (Carcinogenesis 1994, 15, 1911-1915), also using postlabelling, claimed that chenodeoxycholic acid, lithocholic acid, glycolithocholic acid and taurolithocholic acid bound covalently to DNA in vitro. To investigate this discordance we incubated solutions of salmon sperm DNA for 1 h at 37 degrees C with 1 mg/ml of cholic acid, chenodeoxycholic acid, lithocholic acid, glycolithocholic acid or taurolithocholic acid. Each incubate was extracted extensively with diethyl ether after which a sample of DNA was taken and 32P-postlabelled using the nuclease P1 method. The DNA in the remaining incubate was precipitated from high salt solution with ethanol. Aliquots of this DNA were postlabelled. The remainder of the DNA was purified with proteinase-K, ribonuclease, phenol-chloroform, precipitated and postlabelled. Parallel incubates were made with the same bile acids, under the same conditions but in the absence of DNA and were then extracted, precipitated and postlabelled as described above. When DNA was present in the incubate but was not precipitated, chenodeoxycholic acid, lithocholic acid, glycolithocholic acid and taurolithocholic acid, but not cholic acid, produced spots similar to those reported by Hamada et al. No such spots were seen when DNA was postlabelled after precipitation, or after precipitation and purification. These same bile acids produced spots when postlabelled in the absence of DNA, but spots were absent when these incubates were precipitated and purified before postlabelling. We conclude that the spots obtained when bile acids are incubated with DNA which is not precipitated from high salt before it is postlabelled are technical artefacts, and cannot be regarded as evidence that bile acids bind covalently to DNA to form adducts. We also confirm reports (Vulimiri et al. Carcinogenesis 1994, 15, 2061-2064) that bile acids alone can produce spots when incubated with T4 polynucleotide kinase and [gamma-32P]ATP.
Carcinogenesis 1995 Jul
PMID:Appearance of artefacts when using 32P-postlabelling to investigate DNA adduct formation by bile acids in vitro: lack of evidence for covalent binding. 761 81

Within the group of DNA alkylation products, phosphotriesters (PTE) are among the most stable lesions. Hence, alkyl PTE are attractive biomarkers for DNA alkylation monitoring purposes. We have developed a 32P-postlabelling method for the analysis of both methyl and ethyl PTE in DNA. Since PTE bonds are not cleaved by any known DNA degrading enzyme, they are easily obtainable as PTE dinucleoside monophospates. A purification step, separating the PTE dinucleoside monophosphates from interfering compounds, such as mono- or oligonucleotides resulting from incomplete digestion of DNA, was developed using Waters C18 Sep-Pak cartridges. Phosphotriester dinucleoside monophosphates themselves are not a substrate for phosphorylation by polynucleotide kinase. Polynucleotide kinase probably requires a negative charge on the phosphate closest to the 5'-end. Therefore, prior to the post-labelling step they have to be converted into either phosphodiester dinucleoside monophosphates or 3'-phosphate alkylated mononucleotides by treatment with alkali. For analysis of the labelled compounds we developed a two-step procedure, combining TLC and HPLC, that gave very straightforward information on the composition of the rather complex mixture. The detection limit is approximately fmol PTE.
Carcinogenesis 1995 Jul
PMID:A 32P-postlabelling assay for the detection of alkylphosphotriesters in DNA. 761 87

A 32P-postlabelling method is reported for the detection of 7-alkylguanines, the major adducts formed by the reaction of 1,2-alkylepoxides with DNA. Calf thymus DNA was reacted in vitro with different epoxides (ethylene oxide through octylene oxide) and digested with micrococcal nuclease and spleen phosphodiesterase to 3'-nucleotides. The adduct enrichment was carried out by an ion-exchange method and adducts were labelled with [gamma-32]ATP in a T4-polynucleotide kinase-mediated reaction. The labelled adducts, prior to resolution by two-dimensional TLC, were treated with nuclease P1. The recoveries of adducts formed by different epoxides ranged from 3 to 10%, tending to increase with the increase in the chain length of the substitutions. The 32P-postlabelled adducts were also analysed by HPLC coupled with a radioisotope detector. This method has been applied for the detection of 7-alkylguanine adducts in rats exposed to different alkenes. The method has potential for use in measuring human exposure to alkenes or their corresponding epoxides as well as the endogenously formed 7-alkylguanine adducts.
Carcinogenesis 1995 Mar
PMID:32P-postlabelling method for the detection of 7-alkylguanine adducts formed by the reaction of different 1,2-alkyl epoxides with DNA. 769 2

UV-induced cyclobutane dimers and 6-4 photoproducts, containing an unmodified nucleotide at the 5'-position were released from DNA by means of digestion with DNase I, snake venom phosphodiesterase and prostatic acid phosphatase. The enzymes were deactivated by proteinase K followed by ethanol precipitation. The products were phosphorylated by polynucleotide kinase and [gamma-32P]ATP. The TLC system used for the analysis enables separation of the different photoproducts and detection at a fmol level. T4 endonuclease treatment was applied to confirm the positions of cyclobutane dimers.
Carcinogenesis 1995 Jan
PMID:Analysis of UV-induced DNA photoproducts by 32P-postlabelling. 783 95

A 32P-postlabelling assay was developed for the analysis of adducts arising from the reaction of 2'-deoxyguanosine-3'-monophosphate with the 1,2-dicarbonyl compound methylglyoxal, a major mutagen in several foodstuffs, in particular, instant and brewed coffee. The 32P-postlabelling reaction was optimized by testing various parameters such as the kinetics of phosphorylation by T4 polynucleotide kinase, substrate concentration-dependent labelling efficiency and the concentration of the various ingredients of the phosphorylation reaction. The sensitivity to the 3'-monophosphate dephosphorylation activity of nuclease P1 was also studied. Four isomeric reaction products were separated by HPLC, structurally characterized and identified as 3-(2'-deoxy-beta-D-erythro-pentafuranosyl)-6,7-dihydro-6,7-dihydro xy-6- methylimidazo[2,3-b]purine-9(8H)one. The same adducts could be detected from calf thymus DNA that had been reacted in vitro with methylglyoxal. DNA adducts were isolated after enzymatic digestion to mononucleotides followed by nuclease P1 digestion of normal nucleotides. The total level of methylglyoxal-DNA adducts obtained was 5.7 +/- 1.7 (n = 15) adducts/10(6) nucleotides. The 32P-postlabelling method was further validated by the detection of adducts of methylglyoxal in DNA from freshly isolated and stimulated human lymphocytes exposed in vitro. The concentrations of the adducts detected in these samples were 8.2 +/- 0.9 (n = 3) adducts/10(7) nucleotides and 1.5 +/- 0.1 (n = 3) adducts/10(6) nucleotides after treatment with 1.5 and 3.0 mM methylglyoxal respectively.
Carcinogenesis 1994 Sep
PMID:Development of a 32P-postlabelling method for the analysis of 2'-deoxyguanosine-3'-monophosphate and DNA adducts of methylglyoxal. 792 82

The 32P-postlabeling assay has been used widely in carcinogen-DNA adduct analysis because of its sensitivity and reproducibility. Cloned T4 polynucleotide kinase (PNK), routinely used in this assay, phosphorylates the 5'-OH groups of adducted nucleotides in the presence of [gamma-32P]ATP. However, as an exception to this property, PNK has been reported to phosphorylate non-adducted carcinogen metabolites, such as tetrol derivatives of benzo[a]pyrene and chrysene. Also, PNK phosphorylates both 5'-OH and 3'-OH groups of safrole-adducted deoxydinucleoside monophosphates having an unmodified purine in the 3'-position. In the present study we show that T4 PNK catalyzed the transfer of [32P]phosphate from [gamma-32P]ATP to rat bile components or purified bile acids (derivatives of 3 alpha-hydroxy-5 beta-cholanic acid) in the absence of nucleic acids or nucleases. However, labeling of the bile acids appeared over 100,000-fold less efficient than labeling of 2'-deoxyadenosine-5'-monophosphate. There was no reaction in the absence of bile components or PNK. Dehydrocholic acid, which lacks hydroxyl groups, was resistant to phosphorylation. On polyethyleneimine-cellulose TLC maps, 32P-labeled rat bile extract gave an array of non-polar radioactive spots which resembled carcinogen-DNA adducts, while 32P-labeled purified bile acids each gave a single spot. These 32P-labeled products liberated 32Pi upon incubation with prostatic acid phosphatases. Two of the radioactive spots obtained from rat bile were identified as phosphorylated taurocholic and taurodeoxycholic acids by co-chromatography with 32P-labeled standards. These findings demonstrate for the first time that PNK is able to phosphorylate natural products other than nucleotides and further emphasize the need to rule out contamination with bile acids and possibly other bulky/hydrophobic alcohols when analyzing DNA samples by 32P-postlabeling.
Carcinogenesis 1994 Sep
PMID:32P-postlabeling of bile components: bulky adduct-like behavior in polyethyleneimine-cellulose thin layer chromatography. 792 4

Mouse liver DNA adducted with metabolites of the spice constituent safrole (1-allyl-3,4-methylenedioxybenzene), when analyzed via the bisphosphate version of the 32P-postlabeling assay, exhibits two major adducts, which had been previously identified as N2-(trans-isosafrol-3'-yl)2'-deoxyguanosine 3',5'-bisphosphate (adduct 1) and N2-(safrol-1'-yl)2'-deoxyguanosine 3',5'-bisphosphate (adduct 2). However, analysis of the same DNA preparation by the dinucleotide/monophosphate version of the assay gave two additional spots on PEI-cellulose TLC whose nature was clarified in the present study. Several enzymes (T4 polynucleotide kinase, nuclease P1, venom phosphodiesterase and spleen phosphodiesterase) were utilized to hydrolyze these compounds, and the products co-chromatographed on PEI-cellulose thin layers with radiolabeled and non-radioactive nucleotides of known structure. The additional spots were found to be adducted dinucleotides carrying 32P-label at both the 5'- and 3'-hydroxyls. T4 polynucleotide kinase-catalyzed 3'-phosphorylation was highly specific in that only dinucleoside monophosphate derivatives of adduct 1, with an unmodified purine in the 3'-position, were susceptible to both 5'- and 3'-phosphorylation by the enzyme. Thus, the structures of the two additional 32P-labeled safrole derivatives were pX1pAp and pX1pGp where X1 denotes N2-(trans-isosafrol-3'-yl)2'-deoxyguanosine. The official name of T4 polynucleotide kinase, ATP:5'-dephosphopolynucleotide 5'-phosphotransferase (EC 2.7.1.78), denotes the specific action of this enzyme as a 5'-phosphokinase. Although the enzyme has 3'-phosphatase activity at acidic pH, no 3'-kinase reaction has been previously reported. Possible implications for chemical carcinogenesis of the finding that carcinogen-DNA adducts can specifically alter the fidelity of protein-nucleotide interactions are discussed.
Carcinogenesis 1993 Aug
PMID:Altered fidelity of a nucleic acid modifying enzyme, T4 polynucleotide kinase, by safrole-induced DNA damage. 810 96


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