Liquid chromatographic characterization of the deoxyribonucleoside-5’-phosphates and deoxyribonucleoside-3’,5’-bisphosphates obtained by32P-postlabeling of DNA

Dietrich, M. W.; Hopkins, William E.; Asbury, Karen J.; Ridley, William P.

doi:10.1007/bf02688542

Cited by 10 publications

(3 citation statements)

References 14 publications

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“…However, this analytical method was not effective for resolving oxidized [32P]nucleotides. HPLC separation, which has been shown to be suitable for analyzing [32P]DNA-carcinogen adducts (52,53), alkylated nucleotides (33, 52), or base analogues (38), was used in the present work. The complete sepa-Column: Partisil 10 SAX Eluent NaH2P04 pH=A Figure 5.…”

Section: Resultsmentioning

confidence: 99%

Phosphorus-32 postlabeling measurement of adenine N-1-oxide in cellular DNA exposed to hydrogen peroxide

Mouret¹,

Odin²,

Polverelli³

et al. 1990

Chem. Res. Toxicol.

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A 32P-postlabeling assay has been developed for monitoring the formation within DNA of adenine N-1-oxide, the specific H2O2-mediated oxidation product under nonradical conditions. This has required the chemical synthesis of both 2'-deoxyadenosine N-1-oxide 3'-monophosphate and 2'-deoxyadenosine N-1-oxide 5'-monophosphate, the substrate and the product of polynucleotide kinase mediated phosphorylation. Isolation of the substrate from the other nucleotides was found to be necessary in order to improve the rate of phosphorylation and to prevent self-radiolysis processes. [32P]-2'-deoxyadenosine N-1-oxide 5'-monophosphate was obtained after successive ion exchange and reverse-phase HPLC and was characterized by a microreaction. The sensitivity of the assay, which is close to 1 modified adenine N-1-oxide/10(6) bases, allowed the determination of this lesion within the DNA of cells exposed to nonlethal levels of H2O2.

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Section: Resultsmentioning

confidence: 99%

Phosphorus-32 postlabeling measurement of adenine N-1-oxide in cellular DNA exposed to hydrogen peroxide

Mouret¹,

Odin²,

Polverelli³

et al. 1990

Chem. Res. Toxicol.

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“…Identification of DNA adducts is often not definitive due to variations in TLC migration of DNA adducts and lack of separation of adducts. Therefore, HPLC in conjunction with 32P-postlabeling and TLC has been recently used to improve the separation and identification of specific carcinogen-DNA adducts (14)(15)(16)(17). Most of these studies involved the analysis of DNA adducts formed by single carcinogens (14)(15)(16)(17)(18) or did not achieve complete separation of a mixture of DNA adducts formed by reacting select anti-diol epoxides of PAHs with calf thymus DNA (18).…”

Section: Introductionmentioning

confidence: 99%

“…Therefore, HPLC in conjunction with 32P-postlabeling and TLC has been recently used to improve the separation and identification of specific carcinogen-DNA adducts (14)(15)(16)(17). Most of these studies involved the analysis of DNA adducts formed by single carcinogens (14)(15)(16)(17)(18) or did not achieve complete separation of a mixture of DNA adducts formed by reacting select anti-diol epoxides of PAHs with calf thymus DNA (18). Recently, a reverse-phase HPLC procedure was developed and used in the separation of the major 32P-postlabeled DNA adducts formed following in vitro modification of salmon sperm DNA with 10 different PAH diol epoxides (19).…”

Section: Introductionmentioning

confidence: 99%

Separation of 32P-Postlabeled DNA Adducts of Polycyclic Aromatic Hydrocarbons and Nitrated Polycyclic Aromatic Hydrocarbons by HPLC

King¹,

George²,

Gallagher³

et al. 1994

Chem. Res. Toxicol.

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The 32P-postlabeling assay, thin-layer chromatography, and reverse-phase high-pressure liquid chromatography (HPLC) were used to separate DNA adducts formed from 10 polycyclic aromatic hydrocarbons (PAHs) and 6 nitrated polycyclic aromatic hydrocarbons (NO2-PAHs). The PAHs included benzo[j]fluoranthene, benzo[k]fluoranthene, indeno[1,2,3-cd]pyrene, benzo[a]pyrene, chrysene, 6-methylchrysene, 5-methylchrysene, and benz[a]anthracene. The NO2-PAHs included 1-nitropyrene, 2-nitrofluoranthene, 3-nitrofluoranthene, 1,6-dinitropyrene, 1,3-dinitropyrene, and 1,8-dinitropyrene. Separation of seven of the major PAH-DNA adducts was achieved by an initial PAH HPLC gradient system. The major NO2-PAH-DNA adducts were not all separated from each other using the initial PAH HPLC gradient but were clearly separated from the PAH-DNA adducts. A second NO2-PAH HPLC gradient system was developed to separate NO2-PAH-DNA adducts following one-dimensional TLC and HPLC analysis. HPLC profiles of NO2-PAH-DNA adducts were compared using both adduct enhancement versions of the 32P-postlabeling assay to evaluate the use of this technique on HPLC to screen for the presence of NO2-PAH-DNA adducts. To demonstrate the application of these separation methods to a complex mixture of DNA adducts, the chromatographic mobilities of the 32P-postlabeled DNA adduct standards (PAHs and NO2-PAHs) were compared with those produced by a complex mixture of polycyclic organic matter (POM) extracted from diesel emission particles. The diesel-derived adducts did not elute with the identical retention time of any of the PAH or NO2-PAH standards used in this study. HPLC analyses of the NO2-PAH-derived adducts (butanol extracted) revealed the presence of multiple DNA adducts.(ABSTRACT TRUNCATED AT 250 WORDS)

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Detection of Human DNA Adducts by 32P-Postlabeling

Randerath

1990

DNA Damage and Repair in Human Tissues

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Liquid chromatographic characterization of the deoxyribonucleoside-5’-phosphates and deoxyribonucleoside-3’,5’-bisphosphates obtained by32P-postlabeling of DNA

Cited by 10 publications

References 14 publications

Phosphorus-32 postlabeling measurement of adenine N-1-oxide in cellular DNA exposed to hydrogen peroxide

Phosphorus-32 postlabeling measurement of adenine N-1-oxide in cellular DNA exposed to hydrogen peroxide

Separation of 32P-Postlabeled DNA Adducts of Polycyclic Aromatic Hydrocarbons and Nitrated Polycyclic Aromatic Hydrocarbons by HPLC

Detection of Human DNA Adducts by 32P-Postlabeling

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