Epidemiological data suggest that dietary antioxidants play a protective role against cancer. This has led to the proposal that dietary supplementation with antioxidants such as vitamin C (vit C) may be useful in disease prevention. However, vit C has proved to be ineffective in cancer chemoprevention studies. In addition, concerns have been raised over potentially deleterious transition metal ion-mediated pro-oxidant effects. We have now determined that vit C induces lipid hydroperoxide decomposition to the DNA-reactive bifunctional electrophiles 4-oxo-2-nonenal, 4,5-epoxy-2(E)-decenal, and 4-hydroxy-2-nonenal. The compound 4,5-Epoxy-2(E)-decenal is a precursor of etheno-2'-deoxyadenosine, a highly mutagenic lesion found in human DNA. Vitamin C-mediated formation of genotoxins from lipid hydroperoxides in the absence of transition metal ions could help explain its lack of efficacy as a cancer chemoprevention agent.
The 40 and 42 amino-acid residue forms of amyloid beta (Abeta(1-40) and Abeta(1-42)) in cerebrospinal fluid (CSF) have been proposed as potential biomarkers of Alzheimer's disease (AD). Quantitative analyses of Abeta peptides in CSF have relied almost exclusively on the use of immunoassay-based assays such as the enzyme-linked immunosorbent assay (ELISA) procedure. However, due to the ability of the Abeta peptides to readily self-aggregate or bind to other proteins and glassware, such analyses are extremely challenging. Analyses are further complicated by the potential of the peptides to undergo post-translational modifications and the possibilities for cross-reaction in the ELISA assays with endogenous components of the CSF. An approach based on liquid chromatography/tandem mass spectrometry (LC/MS/MS) has now been developed which overcomes these methodological issues. The key steps in implementing this new approach involved immunoaffinity purification coupled with the use of [15N]-labeled Abeta peptides as internal standards, a basic LC mobile phase, negative ion electrospray ionization, and a basic solvent for dissolving the peptides and washing the injection needle to prevent carryover of analytes during multiple injections on the LC/MS system. The validated method had limits of quantitation of 44 fmol/mL (200 pg/mL) for Abeta(1-42) and 92 fmol/mL (400 pg/mL) for Abeta(1-40). An excellent correlation was found between the LC/MS/MS assay and an ELISA assay for Abeta(1-42) in human CSF (r2 = 0.915), although less correlation was observed for Abeta(1-40) (r2 = 0.644). Mean CSF Abeta(1-42) concentrations for samples collected 2 weeks apart from a limited number of AD patients provided additional confidence in the reproducibility of the LC/MS/MS assay. Concentrations for duplicate samples from AD patients were slightly higher than most previously reported values (mean 1.06 +/- 0.25 ng/mL; n = 7). Abeta(1-40) concentrations in duplicate samples obtained from AD patients were also reproducible but were found to be slightly lower than most previously reported values (mean 6.36 +/- 3.07 ng/mL; n = 7). Consistent with literature reports, mean Abeta(1-42) concentrations were found to be lower in AD patients compared with the normal subjects (mean 1.49 +/- 0.59 ng/mL; n = 7), whereas there was no difference in Abeta(1-40) concentrations between AD patients and normal subjects (mean 5.88 +/- 3.03 ng/mL; n = 7). The accuracy and precision of the LC/MS assay mean that it will be a useful complement to existing ELISA assays for monitoring therapeutic interventions designed to modulate CSF Abeta(1-42) concentrations in individual AD patients. Moreover, the introduction of stable isotope labeled internal standards offers the potential to achieve a more rigorous account of the influence of methodological effects related to sample collection and processing.
We report the development of CGP-hydrogel, a biodegradable matrix that achieves local, sustained delivery of dexamethasone to the inner ear. There were no significant complications resulting from the surgical procedure or the administration of CGP-hydrogel to our murine model.
Analysis of the reaction between 2'-deoxycytidine and 4-oxo-2-nonenal by LC/MS revealed the presence of three major products (adducts A(1), A(2), and B; [M + H](+) = 364). Adducts A(1) and A(2) were isomeric, and each dehydrated to form adduct B. The structure of adduct B was shown by LC/MS and NMR spectroscopy to be an etheno-2'-deoxycytidine adduct 1' '-[1-(2'-deoxy-beta-d-erythro-pentofuranosyl)-1H-imidazo[2,1-c]pyrimidin-2-oxo-4-yl]heptane-2' '-one. A time course experiment performed at 65 degrees C (pH 5-8) showed that the transformation of both A(1) and A(2) was pH-dependent. In acidic conditions, adducts A(1) and A(2) dehydrated primarily to adduct B. In contrast, in basic conditions, adducts A(1) and A(2) hydrolyzed primarily to dCyd. The data are consistent with adducts A(1) and A(2) being substituted ethano adducts that dehydrate to adduct B, a substituted 3,N(4)-etheno-2'-deoxycytidine adduct.
trans-4,5-Epoxy-2(E)-decenal reacted with 2'-deoxyadenosine to give 1,N(6)-etheno-2'-deoxyadenosine as well as other 2'-deoxyadenosine adducts. It also reacted with 2'-deoxyguanosine to give 1,N(2)-etheno-2'-deoxyguanosine and other 2'-deoxyguanosine adducts. Synthetic trans-4,5-epoxy-2(E)-decenal was quite stable under the reaction conditions that were used. It was not contaminated with 2,3-epoxyoctanal, a potential precursor to the formation of unsubstituted etheno adducts. Furthermore, using a sensitive LC/MS assay, it was possible to show that no 2,3-epoxyoctanal was formed during prolonged incubations of trans-4,5-epoxy-2(E)-decenal. Therefore, trans-4,5-epoxy-2(E)-decenal, a primary product of lipid peroxidation, is a precursor to the formation of 1,N(6)-etheno-2'-deoxyadenosine and 1,N(2)-etheno-2'-deoxyguanosine. There is no need for an additional oxidation step such as would be required if trans,trans-2,4-decadienal or 4-hydroxy-2-nonenal were the lipid hydroperoxide decomposition products that initiated the formation of unsubstituted etheno adducts. These findings provide an important link between a primary product of lipid peroxidation and a mutagenic DNA lesion that has been detected in human tissues.
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