Optimization of an HPLC method for analyses of <sup>32</sup>P-postlabeled DNA adducts

Möller, Lennart; Zeisig, Magnus; Vodička, Pavel

doi:10.1093/carcin/14.7.1343

Cited by 43 publications

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“…Recent studies with mammals suggest that coupling 32P-postlabeling with HPLC improves the ability to characterize individual adducts (24). In addition, using immunoaffinity chromatography and HPLC coupled with either UV detection (25) or 3 P-postlabeling (26) has provided greater specificity in identification of individual carcinogen-DNA adducts.…”

Section: Analytic Methodologymentioning

confidence: 99%

Molecular epizootiology: assessment of exposure to genotoxic compounds in teleosts.

Stein

Reichert

Varanasi

1994

Environ Health Perspect

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The recent development of techniques to measure levels of carcinogens covalently bound to DNA provides the opportunity to use DNA adducts as molecular dosimeters of exposure to environmental carcinogens and mutagens. This is especially important because epizootiologic studies have shown a positive association between environmental carcinogens, such as polycyclic aromatic hydrocarbons, and increased prevalence of neoplasms and related lesions, primarily in liver, of benthic fish species from a wide range of urban and industrialized areas. In studies with wild fish and mammalian species the 32P-postlabeling assay, as developed for aromatic compounds, has been used most extensively because of its high sensitivity and ability to detect structurally uncharacterized adducts. The results to date of field and laboratory studies show that hepatic DNA adducts detected in fish are associated with increased exposure to environmental polycyclic aromatic compounds in the preponderance of species examined, whereas in the limited studies with wild mammals, such a relationship is equivocal at present. The findings with fish suggest that DNA adducts, as measured by 32P-postlabeling, have the potential to be effective molecular dosimeters of exposure to environmental carcinogenic aromatic compounds and thereby may lead to an improved understanding of the etiology of neoplasia in wild teleosts. -Environ Health Perspect 102 (Suppl 12):19-23 (1994)

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Section: Analytic Methodologymentioning

confidence: 99%

Molecular epizootiology: assessment of exposure to genotoxic compounds in teleosts.

Stein

Reichert

Varanasi

1994

Environ Health Perspect

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“…Separation of 32 P-postlabeled DNA adducts could be achieved by TLC (Randerath et al 1981), a combination of TLC and HPLC (Weyand et al 1987;Talaska et al 1990;Pfau and Phillips 1991), or by 32 P-HPLC with online detection of 32 P radioactivity (Mo¨ller et al 1993). The aim of this study was to test the hypothesis that the 32 P-TLC method might be insufficient in the assessment of carcinogenic potential of different complex petroleum products.…”

Section: Discussionmentioning

confidence: 99%

“…The most common chromatographic method in postlabeling has been TLC (thin-layer chromatography) but the more recently developed 32 P-HPLC (high-performance liquid chromatography) method has shown several advantages, such as better resolution of complex mixtures of DNA adducts, a faster analytical answer, better chromatographic reproducibility, and reduced exposure to radioactivity (Mo¨ller et al 1993;Koskinen et al 1997). The ability of 32 P-HPLC to analyze DNA adducts from a wide range of compounds has been reported (Zeisig and Mo¨ller 1997;Zeisig et al 1999) as well as the ability to analyze DNA adducts from complex mixtures such as petroleum products (Akkineni et al 2001).…”

Section: Introductionmentioning

confidence: 99%

32 P-postlabeling of DNA adducts arising from complex mixtures: HPLC versus TLC separation applied to adducts from petroleum products

Eriksson

Zeisig

Ekström³

et al. 2004

Archives of Toxicology

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The carcinogenicity of petroleum products is mainly due to their content of polycyclic aromatic compounds (PACs). These compounds may be activated metabolically and react with DNA to form DNA adducts, which is a critical event in the initiation of cancer. One of the most common techniques for analyzing DNA adducts is (32)P-postlabeling. The chromatographic method often used has been (32)P-TLC (thin-layer chromatography), but the more recently developed (32)P-HPLC (high-performance liquid chromatography) method has shown advantages. The aim of this study was to test the hypothesis that the (32)P-HPLC method has a better ability of detecting DNA adducts derived from petroleum products than (32)P-TLC. It was found that some DNA adducts migrated from the application point in (32)P-TLC in such a way that it is doubtful if they could be detected and quantified properly. It was also found that, when using (32)P-HPLC, it is possible to use the same protocol for substances with a wide variety of DNA adduct forming potential, whereas (32)P-TLC needs to be optimized regarding time of exposure and/or the amount of DNA applied. Further, a pattern of recognition in (32)P-HPLC enables a selective assessment of DNA adducts derived from complex mixtures whereas (32)P-TLC is very limited when analyzing complex mixtures due to poor resolution. With more knowledge about the properties of the most mutagenic DNA adducts in HPLC, it could be possible to know also which pattern corresponds to a mutagenic or carcinogenic oil. Consequently, (32)P-HPLC is a good alternative when assessing the genotoxicity of petroleum products.

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“…The sources of all enzymes and chemicals have been earlier described (Mo¨ller et al 1993). All solvents and salts were of analytical grade and all water used was run through a MilliQPLUS system (Millipore, Milford, Mass., USA).…”

Section: Subjects and Samplingmentioning

confidence: 99%

Susceptibility factors and DNA adducts in peripheral blood mononuclear cells of aluminium smelter workers exposed to polycyclic aromatic hydrocarbons

Tuominen

Baranczewski

Warholm

et al. 2002

Archives of Toxicology

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Formation of DNA adducts as a result of exposure to polycyclic aromatic hydrocarbons (PAH) was studied in 98 potroom workers from an aluminium smelting plant and in 55 blue-collar workers without occupational PAH exposure. DNA from peripheral blood mononuclear cells (PBMC) was used for quantitation of individual PAH-DNA adducts by 32P-postlabelling/high performance liquid chromatography (HPLC) analysis. Four individual DNA adducts (denoted A, B, C and D) were quantified in 141 of a total of 153 subjects. Genetic polymorphisms for cytochrome P-4501A1 ( CYP1A1), microsomal epoxide hydrolase, N-acetyltransferase 2, glutathione transferases M1, P1 and T1 ( GSTM1, GSTP1 and GSTT1, respectively) and NAD(P)H: quinone oxidoreductase 1 (NQO1) were analysed. For 52 subjects, analysis of mRNA inducibility of CYP1A1 was performed. No statistically significant differences in the levels of total or individual DNA adducts A, C and D were found between potroom workers and control subjects. All potroom workers and the subgroup of potroom workers who reported to never/sometimes use personal respiratory protection ( n=72) were found to have a significantly higher likelihood of having high levels of adduct B than control subjects [odds ratio (OR) =3.4 with 95% confidence interval (CI) of 1.3-9.2, and OR=4.2 with 95% CI 1.6-11.5, respectively]. In the subgroup, levels of adducts A and B were found to be significantly higher among workers with employment time of less than 6 months ( n=5). Also, the levels of the individual DNA adducts were to some extent modified by genetic polymorphisms in CYP1A1, GSTM1, GSTP1 and NQO1 and by CYP1A1 inducibility. In conclusion, levels of adduct B, identified by 32P-postlabelling/HPLC methodology as an indicator of PAH exposure in aluminium production, were modified by the use of respiratory protection, length of employment and genetic polymorphisms.

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Optimization of an HPLC method for analyses of ³²P-postlabeled DNA adducts

Cited by 43 publications

References 0 publications

Molecular epizootiology: assessment of exposure to genotoxic compounds in teleosts.

Molecular epizootiology: assessment of exposure to genotoxic compounds in teleosts.

32 P-postlabeling of DNA adducts arising from complex mixtures: HPLC versus TLC separation applied to adducts from petroleum products

Susceptibility factors and DNA adducts in peripheral blood mononuclear cells of aluminium smelter workers exposed to polycyclic aromatic hydrocarbons

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Optimization of an HPLC method for analyses of 32P-postlabeled DNA adducts

Cited by 43 publications

References 0 publications

Molecular epizootiology: assessment of exposure to genotoxic compounds in teleosts.

Molecular epizootiology: assessment of exposure to genotoxic compounds in teleosts.

32 P-postlabeling of DNA adducts arising from complex mixtures: HPLC versus TLC separation applied to adducts from petroleum products

Susceptibility factors and DNA adducts in peripheral blood mononuclear cells of aluminium smelter workers exposed to polycyclic aromatic hydrocarbons

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Optimization of an HPLC method for analyses of ³²P-postlabeled DNA adducts