A ³²P-postlabeling method for simultaneous detection and quantification of exocyclic etheno and propano adducts in DNA

Abstract

A 32P-postlabeling method is described that specifically detects and quantifies the 1,N2-propanodeoxyguanosine adducts derived from acrolein (AdG) and crotonaldehyde (CdG) and 1,N2-ethenodeoxyguanosine (EdG) in DNA. These exocyclic adducts are potential DNA lesions caused by exposure to enals as environmental pollutants and as endogenous compounds. This method was developed with the use of the synthetic adduct standards of these exocyclic adducts. The assay relies on HPLC for adduct enrichment prior to labeling and for quantitation and identification after labeling. The labeling efficiencies of adducts at the 1 fmol level ranged from 74 to 96%, whereas they were only 49-60% at the 100 fmol level. This method can detect as low as 0.2 fmol of adduct and allows the detection and quantitative determination of stereoisomers of AdG and CdG. The method was validated by using a sample of enzyme digests of 180 micrograms calf thymus DNA spiked with 25 or 75 fmol of adducts, which is equivalent to 5 or 15 adducts in 10(8) nucleotides. The recovery rates of these adducts in DNA ranged from 30 to 90% at the 25 fmol level and 21 to 55% at the 75 fmol level. Similar to the labeling efficiency, a greater recovery was observed with a lower amount of adduct in DNA. Overall, this method allows the simultaneous identification and quantification of exocyclic adducts AdG, CdG and EdG in DNA. Therefore, it provides a potential tool for studies of the in vivo formation of exocyclic adducts.

Acrolein is a major cigarette-related lung cancer agent: Preferential binding at p53 mutational hotspots and inhibition of DNA repair

“…Structural constraints in genomic DNA and chromatin structure may hinder formation of Acr-dG 1 and 2 adduct isomers. Our results are consistent with well established findings that Acr can react directly without metabolic activation with guanine residues in DNA to produce exocyclic propanodeoxyguanosine DNA adducts (12,13,(16)(17)(18).…”

“…Acr has been shown to interact with nucleophiles, including DNA and proteins in cells (32). Unlike PAHs, where only metabolically activated forms can form adducts with DNA, Acr can directly interact with DNA and form DNA adducts (18,23). Similar to PAH-DNA adducts, Acr-DNA adducts induce mainly G:C-to-T:A transversion mutations (10,(33)(34)(35), the major type of mutations found in the p53 gene in CS-related lung cancer.…”

Detection of the acrolein-derived cyclic DNA adduct by a quantitative 32P-postlabeling/solid-phase extraction/HPLC method: Blocking its artifact formation with glutathione

“…Acr, micrococcal nuclease, dG, dG 3′-monophosphate, 2-deoxyadenosine 3′-monophosphate, 2-deoxycytidine 3′-monophosphate, thymidine 3′-monophosphate were obtained from SigmaAldrich Co. (ST. Louis, MO), Acr-dG 3′-or 5′-monophosphate was prepared as previously described, and the identities of these standards were established by their UV spectra and mass spectroscopy [13]. Spleen phosphodiesterase was from Worthington Biochemical (Lakewood, NJ), nuclease P1 was from Yamasa Shoyu Co. (Choshi, Japan), and [γ-32 P]-ATP and T4 polynucleotide kinase (T4 PNK) were from Amersham (Piscataway, NJ).…”

“…As a major reaction, Acr reacts with deoxyguanosine (dG) in DNA to produce two pairs of diastereomeric adducts (Scheme 1A): (6R/S)-3-(2'-deoxyribos-1'-yl)-5,6,7,8-tetrahydro-6-hydroxypyrimido[1,2-α] purine-10(3H)one (α-OH-Acr-dG) and (8R/S)-3-(2'-deoxyribos-1'-yl)-5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2-α]purine-10(3H)one (γ-OH-Acr-dG) [11,12]. A highly sensitive HPLCbased 32 P-postlabeling method was developed earlier for the detection of Acr-dG in tissue DNA [13]. Using this method, Acr-dG adducts were detected in tissue of untreated rodents and humans as background lesions in DNA [14,15].…”

Lack of Mutagenicity of Acrolein-Induced DNA Adducts in Mouse and Human Cells

“…[8][9][10]. Acrolein-induced DNA adducts have been characterized in vitro (11)(12)(13)(14) and quantified in vivo in various organs of experimental animals and humans (9,10,15,16). A recent in vitro study has shown preferential formation of acrolein-DNA adducts at lung cancer mutational hotspots in the p53 tumor suppressor gene in normal human bronchial epithelial cells and lung fibroblasts (17).…”