A solid-phase extraction/high-performance liquid chromatography-based 32P-postlabeling method for detection of cyclic 1,N2-propanodeoxyguanosine adducts derived from enals
Section snippets
Chemicals
Acr, Cro, Pen, Hep, calf thymus DNA, micrococcal nuclease, nuclease P1, and apyrase were obtained from Sigma–Aldrich (St. Louis, MO), and HNE was synthesized by a previously described method [21]. Spleen phosphodiesterase was from Worthington Biochemical (Lakewood, NJ), and [γ-32P]ATP and T4 polynucleotide kinase (T4 PNK) were from Amersham (Piscataway, NJ). All other reagents, unless otherwise stated, were from Sigma–Aldrich and Fisher Chemical (Fair Lawn, NJ). The 3′-monophosphates of Acr-,
Results and discussion
The SPE/HPLC-based 32P-postlabeling assay is outlined in Scheme 1. Briefly, DNA is enzymatically hydrolyzed to the 3′-monophosphates. Fractions containing the 3′-monophosphates of PdG adducts are enriched with SPE (SPE-1) before 5′ end labeling with T4 PNK and [γ-32P]ATP. After labeling, the residual [γ-32P]ATP is removed by a second SPE (SPE-2). The fractions containing the 3′,5′-bisphosphates of PdG adducts are further purified by two HPLC steps, a reverse-phase and an ion-pair system.
Acknowledgment
This work was supported by the NCI Grant CA43159.
References (26)
- et al.
Genotoxic properties of 4-hydroxyalkenals and analogous aldehydes
Mutat. Res.
(1993) - et al.
Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes
Free Radic. Biol. Med.
(1991) - et al.
Possible mutagens derived from lipids and lipid precursors
Mutat. Res.
(1990) - et al.
The reaction of guanosine and deoxyguanosine with glycidaldehyde
Tetrahedron Lett.
(1968) - et al.
Applications of mass spectrometry for quantitation of DNA adducts
J. Chromatogr. B Analyt. Technol. Biomed. Life Sci.
(2002) - et al.
Endogenous formation and significance of 1,N2-propanodeoxyguanosine adducts
Mutat. Res.
(1999) - et al.
Deoxyguanosine adducts of tert-4-hydroxy-2-nonenal as markers of endogenous DNA lesions
Methods Enzymol.
(2002) Volatile aldehydes in tobacco smoke-polluted indoor air
IARC Sci. Publ.
(1987)- et al.
On-road emissions of carbonyls from light-duty and heavy-duty vehicles
Environ. Sci. Technol.
(2001) Cytotoxicity and genotoxicity of lipid-oxidation products
Am. J. Clin. Nutr.
(1993)
DNA adducts of alpha, beta-unsaturated aldehydes and dicarbonyl compounds
IARC Sci. Publ.
Formation of cyclic 1,N2-propanodeoxyguanosine adducts in DNA upon reaction with acrolein or crotonaldehyde
Cancer Res.
Formation of cyclic adducts of deoxyguanosine with the aldehydes trans-4-hydroxy-2-hexenal and trans-4-hydroxy-2-nonenal in vitro
Cancer Res.
Cited by (23)
Detection of 1,N<sup>2</sup>-propano-2'-deoxyguanosine adducts in genomic DNA by ultrahigh performance liquid chromatography-electrospray ionization-tandem mass spectrometry in combination with stable isotope dilution
2016, Journal of Chromatography ACitation Excerpt :Since ProdG adducts play an important role in the Cro genotoxicity [20], a sensitive and accurate method for quantification of ProdG adducts is required. A number of approaches have been developed, including the commonly used but labor-intensive 32-P post-labeling [21–24] and LC–MS [17,18,25,26]. Among them, the mass spectrometry based methodologies take the advantages of direct analysis of polar DNA adducts with structural information [26].
A novel in vitro pancreatic carcinogenesis model
2011, Toxicology LettersCitation Excerpt :The adducts were labeled with [γ-32P]-ATP in the mixture [[γ-32P]ATP (10 μCi/μl), T4 polynucleotide kinase, and apyrase] at 37 °C for 30 min. The labeled adduct was spotted on TLC sheet and developed as follows: the BaP–DNA adduct developed in; D1 (bottom to top) in 1.7 M NaH2PO4 (pH 5.8) for overnight; D3 (bottom to top) in 3.4 M lithium formate, 6.4 M urea (pH 3.5) for 4 h; D4 (left to right) in 0.8 M NaH2PO4, 0.5 M Tris–HCl, 7.5 M urea (pH 3.5) for 4 h (Pan et al., 2006). After development, the TLC was dried, exposed the X-ray film.
Detection of the acrolein-derived cyclic DNA adduct by a quantitative <sup>32</sup>P-postlabeling/solid-phase extraction/HPLC method: Blocking its artifact formation with glutathione
2008, Analytical BiochemistryCitation Excerpt :With the previous method, the adduct levels in DNA were underestimated, because the recovery of adducts in DNA samples was invariably lower than that of a positive control sample containing only adduct standards without the unmodified nucleotides. It has been shown that even subnanomole quantities of the unmodified nucleotides can interfere with the 32P-labeling of adducts [24]. Therefore, SPE-1 is an important step to remove the unmodified nucleotides in DNA digest before labeling.
Lipid peroxidation-induced DNA damage in cancer-prone inflammatory diseases: A review of published adduct types and levels in humans
2007, Free Radical Biology and MedicineDNA Adduct Formation in the Lungs and Brain of Rats Exposed to Low Concentrations of [ <sup>13</sup> C <inf>2</inf> ]-Acetaldehyde
2018, Chemical Research in ToxicologySelective separation and determination of diclofenac via magnetic molecularly imprinted polymer and spectrophotometry
2016, Journal of the Iranian Chemical Society