RESUMO
Lipids are known to be major targets of oxidative stress in cells. In addition to deleterious effects on membranes and various cellular processes, lipid peroxidation has been proposed to be an indirect genotoxic pathway. Indeed, reactive aldehydes produced upon degradation of lipid hydroperoxides may add to DNA bases. In the present work, we investigated the DNA damaging properties of exogenously added 4-hydroxy-2(E)-nonenal (HNE) in human THP1 monocytes. To provide quantitative data on the possible role of HNE in oxidative genotoxicity, we applied an accurate HPLC-MS approach to the quantification of HNE adducts to DNA and of HNE conjugates to glutathione (HNE-GSH), the product of the major detoxification pathway of HNE in cells. We confirmed that GSH was more reactive than DNA toward HNE in cells, with a ratio of 25000 between the amounts of HNE-GSH and DNA adducts. In addition, we found that the conjugate of HNE to cysteine was produced in much lower yield than HNE-GSH, while that of N-acetylcysteine could not be detected. We also observed that a decrease in the GSH content resulted in the favored formation of DNA lesions. If our data based on an intense and short exposure to HNE can be extended to an in vivo situation where low concentrations of HNE are produced on a long time scale, the present results suggest that although the amount of DNA adducts is low upon treatment by exogenous HNE, their formation could be favored upon oxidative stress. Indeed, this last process leads to concomitant consumption of GSH by oxidation and induction of lipid peroxidation.
Assuntos
Aldeídos/toxicidade , Adutos de DNA/metabolismo , Glutationa/fisiologia , Peroxidação de Lipídeos , Aldeídos/metabolismo , Butionina Sulfoximina/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Humanos , Monócitos/metabolismo , Espectrometria de Massas em TandemRESUMO
4-hydroxynonenal (HNE), one of the main breakdown products of lipid peroxides, has been shown to react with DNA yielding a 1,N2-propano adduct to 2'-deoxyguanosine. However, HNE may also react with a wide range of biomolecules before reaching the nucleus. Glutathione (GSH), the most abundant cellular thiol-containing peptide, is likely to be a major cytosolic target for HNE because of its high reactivity and its implication in the detoxification of this aldehyde. In order to estimate the proportion of HNE actually reaching DNA in human THP1 monocytes, we designed an experimental protocol aimed at quantifying DNA adducts and HNE-GSH in the same sample of cells exposed to extracellularly added HNE. Reverse-phase HPLC associated with tandem mass spectrometry detection was used as the analytical tool. It was first observed that, once produced, the HNE-GSH conjugate was very efficiently excreted from the cells into the culture medium. More strikingly, we determined that the amount of HNE-GSH conjugate produced was 4 orders of magnitude higher than that of DNA adduct. These results emphasize the major role played by glutathione in the protection of DNA against electrophilic species.
Assuntos
Aldeídos/farmacocinética , Adutos de DNA/antagonistas & inibidores , Glutationa/metabolismo , Aldeídos/química , Animais , Linhagem Celular , Sobrevivência Celular , Adutos de DNA/química , Desoxiguanosina/química , Glutationa/química , Humanos , Modelos Biológicos , Modelos Moleculares , Monócitos/fisiologia , SoluçõesRESUMO
The response to reactive electrophile species (RES) is now considered as part of the plant response to pathogen and insect attacks. Thanks to a previously established high-performance liquid chromatography tandem mass spectrometry methodology, we have investigated the production of oxylipin RES adducts to glutathione (GSH) during the hypersensitive response (HR) of plants. We have observed that RES conjugation to GSH in tobacco (Nicotiana tabacum) leaves is facile and nonspecific. In cryptogein-elicited tobacco leaves, we show that the oxylipin RES adducts to GSH are produced in correlation with GSH consumption, increase in glutathione S-transferase activity, and the appearance of the cell death symptoms. In this model, the adducts arise mainly from the downstream 13 lipoxygenase (LOX) metabolism, although the induced 9 LOX pathway leads massively to the accumulation of upstream metabolites. The main adducts were obtained from 2-hexenal and 12-oxo-phytodienoic acid. They accumulate transiently as 1-hexanol-3-GSH, a reduced adduct, and 12-oxo-phytodienoic acid-GSH, respectively. RES conjugation does not initiate cell death but explains part of the GSH depletion that accompanies HR cell death. The nature of these GSH conjugates shows the key role played by the 13 LOX pathway in RES signaling in the tobacco HR.