A Schiff Base Is a Major DNA Adduct of Crotonaldehyde
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Previous studies have demonstrated that the reaction of crotonaldehyde with DNA produces Michael addition products, and these have been detected in human tissues as well as tissues of untreated laboratory animals. A second class of crotonaldehyde−DNA adducts releases 2-(2-hydroxypropyl)-4-hydroxy-6-methyl-1,3-dioxane (paraldol, 12) upon hydrolysis, and these adducts are quantitatively more significant than the Michael addition adducts in vitro. In this study, we demonstrate that the major source of the paraldol-releasing DNA adducts of crotonaldehyde is a Schiff base. Reaction of crotonaldehyde with DNA, followed by treatment with NaBH3CN and enzyme hydrolysis, resulted in the formation of N2-(3-hydroxybutyl)dG (10), identified by its UV, MS, and proton NMR. Reactions of crotonaldehyde or paraldol with dG demonstrated that the Schiff base precursor to N2-(3-hydroxybutyl)dG is N2-(3-hydroxybutylidene)dG (7), identified by UV, LC−APCI-MS, and MS/MS. Four isomers of N2-(3-hydroxybutylidene)dG were observed. The (R)- and (S)-isomers were identified by reactions of chiral paraldol with dG; each existed as a pair of interconverting (E)- and (Z)-isomers. These data indicate that the structure of the major Schiff base DNA adduct in crotonaldehyde-treated DNA is N2-(3-hydroxybutylidene)dG (7). This adduct is unstable at the nucleoside level and accounts for more than 90% of the paraldol released from crotonaldehyde-treated DNA. However, the adduct is stable in DNA and therefore is a likely companion to the Michael addition adducts in human DNA.Keywords:
Crotonaldehyde
Previous studies have demonstrated that the reaction of crotonaldehyde with DNA produces Michael addition products, and these have been detected in human tissues as well as tissues of untreated laboratory animals. A second class of crotonaldehyde−DNA adducts releases 2-(2-hydroxypropyl)-4-hydroxy-6-methyl-1,3-dioxane (paraldol, 12) upon hydrolysis, and these adducts are quantitatively more significant than the Michael addition adducts in vitro. In this study, we demonstrate that the major source of the paraldol-releasing DNA adducts of crotonaldehyde is a Schiff base. Reaction of crotonaldehyde with DNA, followed by treatment with NaBH3CN and enzyme hydrolysis, resulted in the formation of N2-(3-hydroxybutyl)dG (10), identified by its UV, MS, and proton NMR. Reactions of crotonaldehyde or paraldol with dG demonstrated that the Schiff base precursor to N2-(3-hydroxybutyl)dG is N2-(3-hydroxybutylidene)dG (7), identified by UV, LC−APCI-MS, and MS/MS. Four isomers of N2-(3-hydroxybutylidene)dG were observed. The (R)- and (S)-isomers were identified by reactions of chiral paraldol with dG; each existed as a pair of interconverting (E)- and (Z)-isomers. These data indicate that the structure of the major Schiff base DNA adduct in crotonaldehyde-treated DNA is N2-(3-hydroxybutylidene)dG (7). This adduct is unstable at the nucleoside level and accounts for more than 90% of the paraldol released from crotonaldehyde-treated DNA. However, the adduct is stable in DNA and therefore is a likely companion to the Michael addition adducts in human DNA.
Crotonaldehyde
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Abstract Limited sensitivity of existing assays has prevented investigation of whether Adriamycin–DNA adducts are involved in the anti-tumour potential of Adriamycin. Previous detection has achieved a sensitivity of a few Adriamycin–DNA adducts/104 bp DNA, but has required the use of supra-clinical drug concentrations. This work sought to measure Adriamycin–DNA adducts at sub-micromolar doses using accelerator mass spectrometry (AMS), a technique with origins in geochemistry for radiocarbon dating. We have used conditions previously validated (by less sensitive decay counting) to extract [14C]Adriamycin–DNA adducts from cells and adapted the methodology to AMS detection. Here we show the first direct evidence of Adriamycin–DNA adducts at clinically-relevant Adriamycin concentrations. [14C]Adriamycin treatment (25 nM) resulted in 4.4 ± 1.0 adducts/107 bp (∼1300 adducts/cell) in MCF-7 breast cancer cells, representing the best sensitivity and precision reported to date for the covalent binding of Adriamycin to DNA. The exceedingly sensitive nature of AMS has enabled over three orders of magnitude increased sensitivity of Adriamycin–DNA adduct detection and revealed adduct formation within an hour of drug treatment. This method has been shown to be highly reproducible for the measurement of Adriamycin–DNA adducts in tumour cells in culture and can now be applied to the detection of these adducts in human tissues.
Accelerator mass spectrometry
DNA adduct
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Crotonaldehyde
Molecular oxygen
Oxidation process
Partial pressure
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The UV photolysis of the α,β-unsaturated aldehydes acrolein, methacrolein and crotonaldehyde in argon matrices at λ > 300 nm has shown a major photochemical deactivation pathway to be conformational isomerism to the thermodynamically less stable s-cis form. This type of isomerization may account for the rapid internal conversion observed in the gas phase. Matrix-isolated crotonaldehyde undergoes further isomerization to ethylketene and enol-crotonaldehyde, as observed in the analogous gas-phase photolysis. In addition, another H-abstraction product was detected (νOH= 3674 cm–1) which is probably derived from the photolysis of s-cis-crotonaldehyde. At shorter excitation wavelengths (λ > 230 nm) acrolein and methacrolein isomerize to methyl–ketene and dimethylketene, respectively.
Crotonaldehyde
Methacrolein
Acrolein
Matrix (chemical analysis)
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DNA adducts were formed by microsomal and photoactivation, using nick-translated DNA labelled with 14C in each of the DNA bases [3H]AFB1 and [3H] AFB2. DNA adducts were analysed by HPLC of DNA hydrolysates, and were characterized as double labelled peaks with specific retention times. The only AF—DNA adducts which were detected in signincant amounts were guanine adducts, irrespective of the type of aflatoxin used or the mode of its activation. No stable adduct with adenine, cytcshe or thymine was detected. UV spectra, proton NMR spectroscopy and mass spectrometry are consistent with the notion that the major AFB1—DNA photoadduct is the N7-guanine adduct. This report provides direct evidence for the existence of ahtoxin photoadducts formed on DNA.
Thymine
Deoxyguanosine
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Crotonaldehyde
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Crotonaldehyde
Maleic anhydride
Furan
Partial oxidation
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IR spectra of H3PMo12O40 (HPA) with adsorbed crotonaldehyde disclosed that HPA is reduced by crotonaldehyde only in the presence of water, giving furan as the oxidation product. Water is evidentially involved in the reaction path of crotonaldehyde oxidation, being consistent with water-enhanced catalytic oxidation of crotonaldehyde on HPA.
Crotonaldehyde
Furan
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Crotonaldehyde
Butyraldehyde
Torr
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J. W. Coomber, J. N. Pitts and R. R. Schrock, Chem. Commun. (London), 1968, 190b DOI: 10.1039/C1968000190B
Crotonaldehyde
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