A solid-phase extraction/high-performance liquid chromatography-based 32P-postlabeling method for detection of cyclic 1,N2-propanodeoxyguanosine adducts derived from enals

doi:10.1016/j.ab.2005.10.011

Analytical Biochemistry

Volume 348, Issue 1, 1 January 2006, Pages 15-23

https://doi.org/10.1016/j.ab.2005.10.011 Get rights and content

Abstract

The cyclic 1,N²-propanodeoxyguanosine (PdG) adducts are Michael addition products from reactions of deoxyguanosine (dG) with enals, including acrolein (Acr), crotonaldehyde (Cro), pentenal (Pen), heptenal (Hep), and 4-hydroxy-2-nonenal (HNE). Although this is a general reaction, only the PdG adducts derived from Acr, Cro, and HNE have been detected in vivo as endogenous DNA lesions. Our previous in vitro study demonstrated that PdG adducts of Acr, Cro, and Pen are predominantly derived from oxidation of ω-3 polyunsaturated fatty acids (PUFAs), whereas the long-chain Hep and HNE adducts are from ω-6 PUFAs. PdG adducts are important because they represent a new class of endogenous promutagenic DNA lesions with potential roles in carcinogenesis. Earlier, we developed a ³²P-postlabeling method for detecting PdG adducts from Acr and Cro and a modified method for the long-chain HNE adducts. Both methods require multiple high-performance liquid chromatography steps and, in some cases, time-consuming thin-layer chromatography for purification. There is a lack of a single, versatile, and efficient method for simultaneous detection of all five enal-derived PdG adducts. In this paper, we report an improved ³²P-postlabeling method which permits detection of Acr, Cro, Pen, Hep, and HNE adducts in a single DNA sample. This method relies on solid-phase extraction for adduct enrichment before and after ³²P-labeling; all five PdG adducts were converted to the ring-opened derivatives for confirmation of identities and quantification. The method was validated using the synthetic adducts and enal-modified DNA and was finally applied to rat liver DNA and rat liver DNA samples spiked with different amount of standards. The detection limit was determined to be as low as 0.5 fmol in 80 μg DNA, corresponding to 9 adducts/10⁹ dG.

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.

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A solid-phase extraction/high-performance liquid chromatography-based 32P-postlabeling method for detection of cyclic 1,N2-propanodeoxyguanosine adducts derived from enals

Abstract

Section snippets

Chemicals

Results and discussion

Acknowledgment

Mutat. Res.

Free Radic. Biol. Med.

Mutat. Res.

Tetrahedron Lett.

J. Chromatogr. B Analyt. Technol. Biomed. Life Sci.

Mutat. Res.

Methods Enzymol.

Volatile aldehydes in tobacco smoke-polluted indoor air

IARC Sci. Publ.

On-road emissions of carbonyls from light-duty and heavy-duty vehicles

Environ. Sci. Technol.

Cytotoxicity and genotoxicity of lipid-oxidation products

Am. J. Clin. Nutr.

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.

A solid-phase extraction/high-performance liquid chromatography-based ³²P-postlabeling method for detection of cyclic 1,N²-propanodeoxyguanosine adducts derived from enals

Formation of cyclic 1,N²-propanodeoxyguanosine adducts in DNA upon reaction with acrolein or crotonaldehyde