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Degradation of polyunsaturated fatty acids in medaka fish under acute oxidative stress
Results: In NIH3T3 histone carbonylation increases during cell proliferation and immediately decreases after activation of nuclear proteasome. On the other hand, histone carbonylation decreases in immortalized lymphocytes from old donors. Interestingly, this modification correlated with the expression of lysil oxidase (LOX), a tumor suppressor gene, that was decreased in lymphocytes from old donnors. Discussion: We have found that carbonylation of histones is a relevant PTM involved in the histone detoxification by the nuclear proteasome after DNA synthesis and it may be related with the protection of the chromatin, a process that may be mediated by LOX. Keywords: Epigenetics, Histones, Cell proliferation, Aging doi:10.1016/j.freeradbiomed.2012.08.044 HNE.P.15 Effects of lipoperoxide exposure on human colon adenocarcinoma and normal endothelial cell lines 1
2
3
Underlying DLD-1 growth inhibition, LOOHs decrease APRIL, VEGF and other proangiogenic factors (iNOS and Hsp70) until 24 h. During exposure to CM, HECV proliferation index increases markedly, and conversely drop in CM-LOOH cultures. Apoptotic markers (PARP, Bcl2, Bax and caspase3) increase only in LOOHexposed DLD-1. In both cell lines, oxidative markers, • such as O2 and TBARs, increase as well as GSH/GSSG ratio, but the highest levels of these markers are reached by DLD-1 cells. Taken together these results suggest that LOOH effects on tumor cell growth and angiogenesis inhibition, may be related to an imbalance of redox status and an increase of oxidative stress. Thus LOOHs may be promising in co-adjuvant tumor therapy and progress in each complex areas, related to LOOHs action, is clearly important because of the farreaching physiological and biomedical implication. Keywords: lipoperoxides, cancer cells, oxidative stress, proliferation doi:10.1016/j.freeradbiomed.2012.08.045
1
C. Scanarotti , M. Caratelli , E.L. Iorio , S. Penco , M.A. Pronzato1, A.M. Bassi*1 1 University of Genoa, Italy, 2Diacron International, Italy, 3 International Observatory of Oxidative Stress, Italy In cancer cells the increase in ROS generation from metabolic abnormalities and oncogenic signalling may trigger a redox adaptation response to maintain the ROS levels below the toxic threshold. Malignant cells would be more vulnerable to further oxidative insults. Oxidation antitumor therapy is developed by delivering excess oxidative stress into tumor cells or targetedly disrupting the antioxidant systems. Lipid peroxides (LOOHs), derived from PUFA, are stable intermediates of oxidative process. LOOHs can act as signalling transductors of ROS, modulating gene expression and cell proliferation. In cancer cells acidosis status and increased iron content may activate Fenton's reaction on LOOH with release of potentially harmful ROS. In the present study, we evaluated several markers related to oxidative stress, proliferation and angiogenesis on human DLD-1 colon carcinoma, and HECV normal endothelial cells, during exposure to 0.11% LOOH. To mimic the environment surrounding neoplasm mass, HECV are also exposed to conditioned medium derived by DLD-1 subjected or not to LOOHs (CM-LOOH and CM, respectively).
HNE.P.16 Degradation of polyunsaturated fatty medaka fish under acute oxidative stress
acids
in
K.Y. Lee, R.S.S. Wu, C.Y.J. Lee* The University of Hong Kong, Hong Kong Environmental pollutants are sources of free radicals that can have detrimental effect on the marine ecosystem. In human diet, fish is a rich source of polyunsaturated fatty acids (PUFA) and have positive effect in preventing vascular diseases. Unless sufficient repair mechanism is present in vivo, PUFA are easily oxidized to toxic oxidants. PUFA is also important to fish reproduction where prostaglandin F2a is one of the pheromone released by female fish. The presence of pollutants in fish tissues can counteract the positive effects of PUFA in human diet, and may destruct the marine ecosystem. In this study, male and female medaka fishes were acutely exposed to high concentration of hydrogen peroxide for 2 hours. The fishes were on organic diet from premature age. The lipid component of the body muscle was extracted for analysis by liquid chromatography tandem mass spectrometry and gas chromatography-mass spectrometry. Measurements of arachidonic acid (AA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), and lipid peroxidation products including F2- and neuroprostanes, isomers of F3-isoprostanes,
S251
hydroxyeicosatetraenoic acid (5(S), 8(S), 9(S), 11(S), 12(S), 15(S) and 20) and hydroxynonenal were made. In this study, acute exposure of medaka fish to hydrogen peroxide depreciated AA, EPA and DHA store in the muscle. Simultaneously, lipid peroxidation products increased in particular isoprostanes, and suppressed prostaglandin F2a level and certain HETEs. These levels also showed a difference between male and female fish. Keywords: isoprostanes, polyunsaturated fatty acids, Medaka fish, HETE doi:10.1016/j.freeradbiomed.2012.08.046 HNE.P.17 Mass spectrometry based strategies in the mapping of modified LDL A. Reis*, C.M. Spickett Aston University, UK The asymptomatic nature of atherosclerosis is prompting the biomedical community to develop methods that can identify patients with high risk of cardiovascular incidents before any clinical symptoms develop. Current opinion is that oxidative modifications to circulating LDL particles (oxLDL) under inflammatory conditions are responsible for mediating fusion/aggregation events and conferring its atherogenicity. LDL particles are complex in nature, being composed of a hydrophobic outer layer of phospholipids wrapped by glycosylated Apo B-100 protein, and any of these biomolecules located at the surface of the particle may be a target of oxidative damage. The relevance, together or individually, of different modifications in oxLDL is not yet clear, with the additional challenge that the structural features inherent in each of the modification play a significant role in the complexity of setting up MS based strategies for their detection. Protocols based on mass spectrometry (MS) coupled with reverse phase LC were applied to the structural characterisation of LDL protein (Apo B-100) from normolipidemic individuals. Protein sequence coverage ≥70% was routinely achieved. In this work we describe the applicability of different MS scanning methods targeted towards the detection of oxidised amino acids, and modifications by oxidised phospholipids and sugar moieties, in providing information about the structural modifications in ApoB-100 protein from oxLDL and their location within the protein. Changes in these during oxidative stress and in diseased states are currently being investigated and correlated with lipid profile data.
The development of advanced and targeted methods that allow the mapping of modifications in protein, lipid and glycan moieties of LDL particles by MS-based approaches is expected to provide a broader insight on the role that these modifications may have in the development of atherosclerosis. doi:10.1016/j.freeradbiomed.2012.08.047 HNE.P.18 The activity of melatonin and related indoleamines against LOO• AND LO• free radicals depends on their functional groups and the assay system N. Fagali, A. Catala* Universidad Nacional de La Plata, Argentina Because of the great number of results obtained in very different assay systems, both in vitro and in vivo, the antioxidant activity of melatonin (MLT) is undeniable. However, it's direct activity against low aggressive free radicals, as LOO• and LO•, remains under discussion. Accordingly, it was the main goal of the present work to study the antioxidant activity of MLT, N-acetylserotonin (NAS), 5-HO-tryptophan (5HO-TRP) and 5methoxytryptamine (5MTP) in two different lipid systems with high content of polyunsaturated fatty acids (PUFAs): triglycerides (Tg) of fish oil dissolved in chloroform and sonicated liposomes (SL) made of retinal lipids in aqueous media. In the Tg-chloroform-system the peroxidation reaction was initiated by cumen hydroperoxide whereas SL were peroxidated with Fe2+. Both initiators generate breakdown of lipoperoxides previously formed with LOO• and LO• production. The techniques employed at the present work were: 1) chemiluminescence, 2) TBARS production, 3) DPPH assay, 4) determination of conjugated dienes production and 5) analysis of fatty acid profile by CG-MS. Butylated hydroxytoluene (BHT) was employed as a reference because of its well known antioxidant capacity. Our results showed that MLT and 5MTP were unable to protect PUFAs against lipid peroxidation in both systems, whereas NAS and 5HO-TRP were better antioxidants that BHT in the Tg-system but ineffective in the SL-system. We conclude that the antioxidant behaviour of MLT and structural analogues during lipid peroxidation depends not only on their functional groups but also on the assay system and could be better explained by the polar paradox theory. Keywords: Lipid peroxidation, LOO radicals, Melatonin, Indoleamines
•
AND LO• free
doi:10.1016/j.freeradbiomed.2012.08.048
S252
2
3
Underlying DLD-1 growth inhibition, LOOHs decrease APRIL, VEGF and other proangiogenic factors (iNOS and Hsp70) until 24 h. During exposure to CM, HECV proliferation index increases markedly, and conversely drop in CM-LOOH cultures. Apoptotic markers (PARP, Bcl2, Bax and caspase3) increase only in LOOHexposed DLD-1. In both cell lines, oxidative markers, • such as O2 and TBARs, increase as well as GSH/GSSG ratio, but the highest levels of these markers are reached by DLD-1 cells. Taken together these results suggest that LOOH effects on tumor cell growth and angiogenesis inhibition, may be related to an imbalance of redox status and an increase of oxidative stress. Thus LOOHs may be promising in co-adjuvant tumor therapy and progress in each complex areas, related to LOOHs action, is clearly important because of the farreaching physiological and biomedical implication. Keywords: lipoperoxides, cancer cells, oxidative stress, proliferation doi:10.1016/j.freeradbiomed.2012.08.045
1
C. Scanarotti , M. Caratelli , E.L. Iorio , S. Penco , M.A. Pronzato1, A.M. Bassi*1 1 University of Genoa, Italy, 2Diacron International, Italy, 3 International Observatory of Oxidative Stress, Italy In cancer cells the increase in ROS generation from metabolic abnormalities and oncogenic signalling may trigger a redox adaptation response to maintain the ROS levels below the toxic threshold. Malignant cells would be more vulnerable to further oxidative insults. Oxidation antitumor therapy is developed by delivering excess oxidative stress into tumor cells or targetedly disrupting the antioxidant systems. Lipid peroxides (LOOHs), derived from PUFA, are stable intermediates of oxidative process. LOOHs can act as signalling transductors of ROS, modulating gene expression and cell proliferation. In cancer cells acidosis status and increased iron content may activate Fenton's reaction on LOOH with release of potentially harmful ROS. In the present study, we evaluated several markers related to oxidative stress, proliferation and angiogenesis on human DLD-1 colon carcinoma, and HECV normal endothelial cells, during exposure to 0.11% LOOH. To mimic the environment surrounding neoplasm mass, HECV are also exposed to conditioned medium derived by DLD-1 subjected or not to LOOHs (CM-LOOH and CM, respectively).
HNE.P.16 Degradation of polyunsaturated fatty medaka fish under acute oxidative stress
acids
in
K.Y. Lee, R.S.S. Wu, C.Y.J. Lee* The University of Hong Kong, Hong Kong Environmental pollutants are sources of free radicals that can have detrimental effect on the marine ecosystem. In human diet, fish is a rich source of polyunsaturated fatty acids (PUFA) and have positive effect in preventing vascular diseases. Unless sufficient repair mechanism is present in vivo, PUFA are easily oxidized to toxic oxidants. PUFA is also important to fish reproduction where prostaglandin F2a is one of the pheromone released by female fish. The presence of pollutants in fish tissues can counteract the positive effects of PUFA in human diet, and may destruct the marine ecosystem. In this study, male and female medaka fishes were acutely exposed to high concentration of hydrogen peroxide for 2 hours. The fishes were on organic diet from premature age. The lipid component of the body muscle was extracted for analysis by liquid chromatography tandem mass spectrometry and gas chromatography-mass spectrometry. Measurements of arachidonic acid (AA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), and lipid peroxidation products including F2- and neuroprostanes, isomers of F3-isoprostanes,
S251
hydroxyeicosatetraenoic acid (5(S), 8(S), 9(S), 11(S), 12(S), 15(S) and 20) and hydroxynonenal were made. In this study, acute exposure of medaka fish to hydrogen peroxide depreciated AA, EPA and DHA store in the muscle. Simultaneously, lipid peroxidation products increased in particular isoprostanes, and suppressed prostaglandin F2a level and certain HETEs. These levels also showed a difference between male and female fish. Keywords: isoprostanes, polyunsaturated fatty acids, Medaka fish, HETE doi:10.1016/j.freeradbiomed.2012.08.046 HNE.P.17 Mass spectrometry based strategies in the mapping of modified LDL A. Reis*, C.M. Spickett Aston University, UK The asymptomatic nature of atherosclerosis is prompting the biomedical community to develop methods that can identify patients with high risk of cardiovascular incidents before any clinical symptoms develop. Current opinion is that oxidative modifications to circulating LDL particles (oxLDL) under inflammatory conditions are responsible for mediating fusion/aggregation events and conferring its atherogenicity. LDL particles are complex in nature, being composed of a hydrophobic outer layer of phospholipids wrapped by glycosylated Apo B-100 protein, and any of these biomolecules located at the surface of the particle may be a target of oxidative damage. The relevance, together or individually, of different modifications in oxLDL is not yet clear, with the additional challenge that the structural features inherent in each of the modification play a significant role in the complexity of setting up MS based strategies for their detection. Protocols based on mass spectrometry (MS) coupled with reverse phase LC were applied to the structural characterisation of LDL protein (Apo B-100) from normolipidemic individuals. Protein sequence coverage ≥70% was routinely achieved. In this work we describe the applicability of different MS scanning methods targeted towards the detection of oxidised amino acids, and modifications by oxidised phospholipids and sugar moieties, in providing information about the structural modifications in ApoB-100 protein from oxLDL and their location within the protein. Changes in these during oxidative stress and in diseased states are currently being investigated and correlated with lipid profile data.
The development of advanced and targeted methods that allow the mapping of modifications in protein, lipid and glycan moieties of LDL particles by MS-based approaches is expected to provide a broader insight on the role that these modifications may have in the development of atherosclerosis. doi:10.1016/j.freeradbiomed.2012.08.047 HNE.P.18 The activity of melatonin and related indoleamines against LOO• AND LO• free radicals depends on their functional groups and the assay system N. Fagali, A. Catala* Universidad Nacional de La Plata, Argentina Because of the great number of results obtained in very different assay systems, both in vitro and in vivo, the antioxidant activity of melatonin (MLT) is undeniable. However, it's direct activity against low aggressive free radicals, as LOO• and LO•, remains under discussion. Accordingly, it was the main goal of the present work to study the antioxidant activity of MLT, N-acetylserotonin (NAS), 5-HO-tryptophan (5HO-TRP) and 5methoxytryptamine (5MTP) in two different lipid systems with high content of polyunsaturated fatty acids (PUFAs): triglycerides (Tg) of fish oil dissolved in chloroform and sonicated liposomes (SL) made of retinal lipids in aqueous media. In the Tg-chloroform-system the peroxidation reaction was initiated by cumen hydroperoxide whereas SL were peroxidated with Fe2+. Both initiators generate breakdown of lipoperoxides previously formed with LOO• and LO• production. The techniques employed at the present work were: 1) chemiluminescence, 2) TBARS production, 3) DPPH assay, 4) determination of conjugated dienes production and 5) analysis of fatty acid profile by CG-MS. Butylated hydroxytoluene (BHT) was employed as a reference because of its well known antioxidant capacity. Our results showed that MLT and 5MTP were unable to protect PUFAs against lipid peroxidation in both systems, whereas NAS and 5HO-TRP were better antioxidants that BHT in the Tg-system but ineffective in the SL-system. We conclude that the antioxidant behaviour of MLT and structural analogues during lipid peroxidation depends not only on their functional groups but also on the assay system and could be better explained by the polar paradox theory. Keywords: Lipid peroxidation, LOO radicals, Melatonin, Indoleamines
•
AND LO• free
doi:10.1016/j.freeradbiomed.2012.08.048
S252