Further studies of the mixed acetals of nucleotides

218

248

Acetaldehyde is a mutagen and carcinogen which occurs widely in the human environment, sometimes in considerable amounts, but little is known about its reactions with DNA. In this study, we identified three new types of stable acetaldehyde DNA adducts, including an interstrand cross-link. These were formed in addition to the previously characterized N(2)-ethylidenedeoxyguanosine. Acetaldehyde was allowed to react with calf thymus DNA or deoxyguanosine. The DNA was isolated and hydrolyzed enzymatically; in some cases, the DNA was first treated with NaBH(3)CN. Reaction mixtures were analyzed by HPLC, and adducts were isolated and characterized by UV, (1)H NMR, and MS. The major adduct was N(2)-ethylidenedeoxyguanosine (1), which was identified as N(2)-ethyldeoxyguanosine (7) after treatment of the DNA with NaBH(3)CN. The new acetaldehyde adducts were 3-(2-deoxyribos-1-yl)-5,6,7, 8-tetrahydro-8-hydroxy-6-methylpyrimido[1,2-a]purine-10(3H)one (9), 3-(2-deoxyribos-1-yl)-5,6,7,8-tetrahydro-8-(N(2)-deoxyguanosyl+ ++)- 6-methylpyrimido[1,2-a]purine-10(3H)one (12), and N(2)-(2, 6-dimethyl-1,3-dioxan-4-yl)deoxyguanosine (11). Adduct 9 has been previously identified in reactions of crotonaldehyde with DNA. However, the distribution of diastereomers was different in the acetaldehyde and crotonaldehyde reactions, indicating that the formation of 9 from acetaldehyde does not proceed through crotonaldehyde. Adduct 12 is an interstrand cross-link. Although previous evidence indicates the formation of cross-links in DNA reacted with acetaldehyde, this is the first reported structural characterization of such an adduct. This adduct is also found in crotonaldehyde-deoxyguanosine reactions, but in a diastereomeric ratio different than that observed here. A common intermediate, N(2)-(4-oxobut-2-yl)deoxyguanosine (6), is proposed to be involved in formation of adducts 9 and 12. Adduct 11 is produced ultimately from 3-hydroxybutanal, the major aldol condensation product of acetaldehyde. Levels of adducts 9, 11, and 12 were less than 10% of those of N(2)-ethylidenedeoxyguanosine (1) in reactions of acetaldehyde with DNA. As nucleosides, adducts 9, 11, and 12 were stable, whereas N(2)-ethylidenedeoxyguanosine (1) had a half-life of 5 min. These new stable adducts of acetaldehyde may be involved in determination of its mutagenic and carcinogenic properties.

Section: Introductionmentioning

confidence: 98%

Identification of DNA Adducts of Acetaldehyde

Wang

McIntee

Cheng

et al. 2000

218

248

“…AA reacts with DNA to form AA−DNA adducts. A number of these adducts, including an interstrand cross-link, have been characterized by our laboratory and others ( − ). The mechanism of formation of an AA- derived interstrand cross-link is shown in Scheme ().…”

Section: Introductionmentioning

confidence: 99%

Synthesis and Properties of an Acetaldehyde-Derived Oligonucleotide Interstrand Cross-Link

Lao

Hecht

2005

Acetaldehyde (AA), occurring widely in the human environment, is a mutagen and carcinogen. AA can react with DNA to form AA-DNA adducts. Several types of adducts, including an interstrand cross-link 3-(2-deoxyribos-1-yl)-5,6,7,8-tetrahydro-8-(N2-deoxyguanosyl)-6-methylpyrimido[1,2-a]purine-10(3H)one (7), have been previously characterized by our laboratory. We hypothesize that cross-link 7 may be involved in determining the mutagenic and carcinogenic properties of AA. To address this question, the double-stranded oligonucleotide 13, bearing cross-link 7, was synthesized in a sequence appropriate for mutagenicity studies in human cells. Oligonucleotide 9, containing 2-fluoro-O6-(trimethylsilylethyl)deoxyinosine (dIno), was reacted with 4-amino-1,2-pentanediol, followed by treatment with NaIO4. The resulting oligonucleotide 11 containing the 1,N2-propano-deoxyguanosine (dGuo) 5 was incubated with the complementary oligonucleotide 12 to give the desired cross-link 13, which was characterized by negative-ion electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS) and enzymatic hydrolysis to cross-link 7. The formation of cross-link 13 at 5'-CpG-3' was confirmed by incubation of 11 with [15N5]12 containing a 5'-Cp[15N5]G-3' sequence. The formation of cross-link 13 was reversible. Therefore, oligonucleotide 24 containing the irreversible analogue of cross-link 7, 1,3-bis(2'-deoxyguanos-N2-yl)butane, was synthesized for use as a control in the mutagenicity studies. Oligonucleotide 21 was reacted with 1,3-diaminobutane dihydrochloride, followed by incubation with the complementary oligonucleotide 23, to give 24. To determine the optimum distance and orientation for cross-link formation, six oligonucleotides, containing 5 at the i + 1, i + 2, and i + 3 or the i - 1, i - 2, and i - 3 positions relative to dGuo in the complementary strand, were 5'-end labeled with [gamma-32P]ATP, followed by incubation of each labeled oligonulceotide with its complementary strand and then analysis by denaturing polyacrylamide gel electrophoresis. Only the oligonucleotide containing 5'-Cp5-3' formed the cross-link with the complementary 5'-CpG-3' sequence. The results of this study confirm the structure of an AA-derived DNA cross-link, supply characterized cross-link-containing oligonucleotides for mutagenicity studies, and provide information on the optimum distance and orientation for cross-link formation.

“…Further oxidation by the enzyme aldehyde dehydrogenase (ALDH) produces a harmless product, acetate. Acetaldehyde, however, is highly reactive due to the electrophilic nature of the carbonyl carbon and has been shown to form adducts with various proteins ( − ), DNA (), and nucleosides ( , ). Accumulation of acetaldehyde due to excess consumption of alcohol will therefore lead to increased interaction of this chemical with biomolecules.…”

Section: Introductionmentioning

confidence: 99%

Stable Acetaldehyde−Protein Adducts as Biomarkers of Alcohol Exposure

Birt

Shuker

Farmer

1998

The consumption of alcoholic beverages has been associated with increased risks of a number of chronic disorders including cancers. It is still not clear whether ethyl alcohol or other components such as metabolites are directly involved in the carcinogenic process or whether the effects are due to the modulation of metabolism of other carcinogens. At present, there is no good biomarker of alcohol intake, particularly at low or moderate levels of consumption. A number of studies have shown the ability of the major metabolite acetaldehyde to react with proteins in vitro to give stable and unstable adducts. The interaction of acetaldehyde with model peptides, which correspond to N-terminal globin sequences, was studied. The major stable adduct was identified by mass spectrometry and NMR as a diastereoisomeric mixture of imidazolidinones. This is believed to be formed by reaction and cyclization of the initial Schiff base adduct with the N-terminal valine. Incubation of human globin with acetaldehyde (0-2 mM) yielded products which were identified as the N-terminal adducts by electrospray ionization mass spectrometry (ESI-MS) of proteolytic digests. The specificity and sensitivity of the analysis was improved by the use of on-line HPLC-ESI-MS. Tryptic digests of the modified globin which contained both the N-terminal acetaldehyde adducts of alpha-globin (heptapeptide) and beta-globin (octapeptide) were resolved. These results suggest that analysis of stable imidazolidinone adducts is a promising approach to estimation of alcohol exposure.