- Email: [email protected]
Stress-induced premature senescence program activated in the mesenchymal stem cells by antioxidant treatments
A. Stepanova et al. / Free Radical Biology and Medicine 120 (2018) S45–S166
P-270
S127
by nitration, the structure of LN polymers is altered. Such alterations in polymer structure could lead to decreased cell adhesion and altered ECM integrity.
Phenomenology and outcomes of acute antioxidative stress in normal human cells
E-mail address: [email protected] http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.418
Olga Lyublinskaya, Julia Kornienko, Julia Ivanova, Irina Smirnova, Natalia Pugovkina, Irina Kozhukharova, Valery Zenin, Nikolay Nikolsky P-272 Institute of Cytology, Russian Academy of Sciences, St.Petersburg, Russia
A number of clinical trials have shown harmful effects of high-dosage antioxidant (AO) interventions. Antioxidative stress defined as a negative impact of pharmacological AOs on cells and tissues is one of the concepts being discussed in this regard. However, the number of experimental studies on this topic is quite limited. As a rule, experimentally revealed negative impact of high AO doses on cells is associated with a variety of side-effects of each individual substance. Thereby, the basic question that arises when trying to generalize published results is as follows: is it possible to determine the acute antioxidative stress as a specific type of damaging effect arising from the abrupt lowering of the basal ROS level in cells? If yes, what are the basic features and outcomes of this stress? Here we propose our point of view, basing on the analysis of cell response to high doses of AOs of different origin and type of activity. We show that all tested AOs can block cell proliferation, hamper both initiation and progression of DNA synthesis, lead to DNA strand breaks accumulation, and disturb regulation of DNA synthesis in normal human stem and non-stem cell cultures. The outcomes of the stress depend on the cell type.
Diterpenes from Plectranthus spp.: a ROS-induced cytotoxicity study Catarina Garcia 1,2, Przemysław Sitarek 3, Ewa Skała 3, Ewelina Synowiec 4, Tomasz Śliwiński 4, Ana Diaz-Lanza 2, Catarina Reis 5, Patricia Rijo 1 1
Center for Research in Biosciences & Health Technologies (CBIOS), Univ. Lusófona de Humanidades e Tecnologias, Lisbon, Portugal 2 Department of Biomedical Sciences, Faculty of Pharmacy, University of Alcalá, Alcalá de Henares, Spain 3 Department of Biology and Pharmaceutical Botany, Medical University of Lodz, Lodz, Poland 4 Laboratory of Medical Genetics, University of Lodz, Lodz, Poland, Pomorska 5 Instituto de Investigação do Medicamento (iMed.ULisboa), Fac. de Farm., Univ. de Lisboa, Lisbon, Portugal
E-mail address: [email protected] Acknowledgements This work is supported by the RSF Grant No.14-50-00068.
http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.417
P-271
Nitration alters laminin polymerization Lasse Lorentzen, Michael Davies Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark
Plants from the Plectranthus genus are commonly known for their medicinal properties, and their applications are vast in traditional medicine. Often accountable for these properties, bioactive secondary metabolites such as diterpenes are present in these plants, and some possess cytotoxic properties. Abietane diterpenes 7α-acetoxy-6β-hydroxyroyleanone, 7β,6β-dihydroxyroyleanone, 6,7- dehydroroyleanone, and parvifloron D are examples of such class of bioactive diterpenes. These abietanes are obtained from Plectranthus genus plants as their major compounds. In this work, the cytotoxic properties of these abietanes have been studied. This property was evaluated through MTT assays on A549 cells and CCRF-CEM cells. In addition, the level of intracellular Reactive Oxygen Species (ROS) in these cells was also measured. Our study has revealed that on CCRF-CEM cells, there is a slight increase of the ROS levels after exposure to all compounds. Nevertheless, on A549 cells the levels of ROS increase approximately 10-fold, with notorious increase after exposure to 7β,6β-dihydroxyroyleanone, which can induce cytotoxicity due to ROS accumulation on cells. In conclusion, this study proves the therapeutic potential of Plectranthus-derived diterpenes and validates its use in traditional medicine.
E-mail address: [email protected] Reactive nitrogen species (RNS) are produced by cells at sites of inflammation and can modify proteins. Nitration is used as a biomarker of damage and may contribute to multiple pathologies. We have shown that the extracellular matrix protein laminin (LN) co-localizes with nitrative damage in human atherosclerotic lesions, and that nitration decreases cell binding to LN in vitro. Study aim: To investigate how nitration affects laminin self-assembly and polymerization. Methods: Sites of nitration were determined by LC-MS. Polymerization was induced by pH changes and CaCl2. Dynamic light scattering was used to examine the extent of polymerization. Structure of LN polymers was investigated with scanning electron microscopy (SEM). Results: Extensive nitration was observed on the protein at specific sites. Nitrated LN underwent polymerization, but displayed increased light scattering, indicating altered particle size. SEM showed that untreated LN formed a homogenous ordered structure, whereas nitrated LN displayed a segmented, patchy and disordered pattern. Conclusions: While the extent of LN polymerization is not affected
http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.419
P-273
Stress-induced premature senescence program activated in the mesenchymal stem cells by antioxidant treatments Julia Kornienko, Irina Smirnova, Natalia Pugovkina, Valery Zenin, Nikolay Nikolsky, Olga Lyublinskaya
S128
A. Stepanova et al. / Free Radical Biology and Medicine 120 (2018) S45–S166
Institute of Cytology, Russian Academy of Sciences, St.Petersburg, Russia
It is now well established that intracellular reactive oxygen species (ROS) mediate the regulation of many signaling cascades that define cell fate. The delicate balance between the production and elimination of ROS is essential for maintaining normal cell functions. Disruption of this balance caused by the antioxidant treatment can disturb various intracellular processes controlled by the systemic signaling. In the present study, we investigate the response of the mesenchymal stem cells (MSCs) to the sub-cytotoxic treatment with various antioxidants (tempol, NAC, resveratrol, DPI). We show that exposure of the proliferating MSC cultures to the antioxidants at concentrations which are widely used in the cell studies to protect cells from the oxidative stress, can cause replication stress and initiate the program of stress-induced preliminary senescence. We demonstrate that these effects can be explained by the disturbance of the ROS-depended degradation of the key proteins regulating DNA replication, e.g. cyclin A, Emi1, geminin. Our data show that enhanced intracellular elimination of ROS resulted from the pharmacologic manipulations can cause the antioxidant-induced damage of cells.
E-mail address: [email protected] Acknowledgements This work is supported by the RSF Grant No.14–50-00068.
http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.420
P-275
Quantum-chemical insights into chemiluminescence from carbonyls Homa Saeidfirozeh 1, Michal Cifra 2, Azizollah Shafiekhani 1,3 1
Physics Department, Alzahra University, Tehran, Iran Institute of Photonics and Electronics, The Czech Academy of Sciences (ufe), Chaberská, Praha - Kobylisy, Czech Republic 3 School of Physics, Institute for Research in Fundamental Sciences (IPM), Tehran, Iran 2
Organisms undergoing oxidative metabolism or stress chemically generate electronically excited species through dioxetane and tetroxide pathways. Consequently, luminescence is emitted which could be exploited for non-invasive label-free monitoring of oxidative stress. Spectral analysis of this endogenous chemiluminescence can serve as a tool for identification of the emitters as products of oxidation. Here we employ quantum mechanical calculations (Gaussian software package) and sensitive experimental photon counting methods to obtain deeper understanding of the emitter molecules both from theoretical and experimental perspective. We focused on the analysis of excited carbonyl groups of simple model species (formaldehyde, acetone, etc.) and carbonyls formed by fragmentation of linoleic acid since they are common product of oxidation and also potential emitters of endogenous chemiluminescence.
P-274
E-mail address: h.saeidfi[email protected]
Nitrosopersulfide (SSNO) is a potential protein Cys polysulfidating agent with sustained and long-range effects
http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.422
Virág Bogdándi 1,2,3, István Zoárd Bátai 1,2,3, Magda Minnion 1,2,3, Zoltán Sándor 1,2,3, Erika Pintér 1,2,3, Martin Feelisch 1,2,3, Péter Nagy 1,2,3
P-276
1 Department of Molecular Immunology and Toxicology, National Institute of Oncology, Budapest, Hungary 2 Department of Pharmacology and Pharmacotherapy, Medical School, University of Pécs, Pécs, Hungary 3 Clinical and Experimental Sciences, Faculty of Medicine, and University Hospital Southampton NHS Foundation Trust, University of Southampton, Southampton, UK
The biological actions of NO and H2S are intimately intertwined. Chemical interactions of these molecules form a network of cascading chemicals. SSNO is a major product of these interactions. It is relatively stable under physiological conditions and not reducible by thiols. Upon slow decomposition of SSNO, the sulfane sulfur equivalents are released in the form of inorganic polysulfides (HSx). HSx were shown to be important mediators in H2S biology by inducing protein Cys persulfidation. However, HSx are readily reduced by the glutathione and thioredoxin systems inside the cell. Importantly, we found that SSNO can escape these reducing machineries and hence we hypothesize that it could serve as a sulfane sulfur carrier for sustained and long range HSx bioavailability. SSNO indeed induced delayed polysulfidation on Cys, GSH, HSA and a slow flux of sulfane sulfur production in HEK 293 cells. Furthermore, HSx were shown to activate TRPA1 channels and our experiments revealed that SSNO can also function as a TRPA1 activator, but with a lasting effect. These observations give credence to our hypothesis that SSNO may be responsible for sustained protein Cys‒polysulfidation and potentially involved in sulfane sulfur trafficking between subcellular compartments.
E-mail address: [email protected] http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.421
Vitamin E metabolism and its putative role as fatsoluble antioxidant in the redox-lipidomics era Galli Francesco 1, Roberta Galarini 2, Gabriele Cruciani 3 1
Dept of Pharmaceutical Sciences, Clinical Biochemistry and Nutrition Lab, University of Perugia, Italy 2 Istituto Zooprofilattico Sperimentale Umbria-Marche, Perugia, Italy 3 Department of Chemistry, Biology and Biotechnology, University of Perugia, Italy
Lipidomics is going to leave its role of “Cinderella” of omics technologies. Innovative mass spectrometry and bioinformatics technology is bursting into the omics scene and this is leading lipid science to gain renewed attention and interest by the scientific community. New lipidomics challenges have already launched including that of extensive (high-throughput) exploration of oxidized lipids in biological matrices (that is “redox lipidomics”). Alpha-tocopherol, the main form of vitamin E in human tissues, is a H donor with exclusive pertinence to lipid peroxide substrates of cellular membranes and lipoprotein particles. The direct or enzyme-mediated interaction of this vitamin with lipid oxidation pathways generates specific metabolites and functional responses. This area of redox lipidomics is now gaining increasing interest for its role in PUFA oxidation and lipid signaling, and more in general in long-chain lipid metabolism under both physiological and pathological conditions. Targeted and untargeted lipidomics protocols of investigation are now available to characterize the functional lipidome of vitamin E that is discussed in this paper along with other emerging aspects of redox lipidomics.
E-mail address: [email protected]
P-270
S127
by nitration, the structure of LN polymers is altered. Such alterations in polymer structure could lead to decreased cell adhesion and altered ECM integrity.
Phenomenology and outcomes of acute antioxidative stress in normal human cells
E-mail address: [email protected] http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.418
Olga Lyublinskaya, Julia Kornienko, Julia Ivanova, Irina Smirnova, Natalia Pugovkina, Irina Kozhukharova, Valery Zenin, Nikolay Nikolsky P-272 Institute of Cytology, Russian Academy of Sciences, St.Petersburg, Russia
A number of clinical trials have shown harmful effects of high-dosage antioxidant (AO) interventions. Antioxidative stress defined as a negative impact of pharmacological AOs on cells and tissues is one of the concepts being discussed in this regard. However, the number of experimental studies on this topic is quite limited. As a rule, experimentally revealed negative impact of high AO doses on cells is associated with a variety of side-effects of each individual substance. Thereby, the basic question that arises when trying to generalize published results is as follows: is it possible to determine the acute antioxidative stress as a specific type of damaging effect arising from the abrupt lowering of the basal ROS level in cells? If yes, what are the basic features and outcomes of this stress? Here we propose our point of view, basing on the analysis of cell response to high doses of AOs of different origin and type of activity. We show that all tested AOs can block cell proliferation, hamper both initiation and progression of DNA synthesis, lead to DNA strand breaks accumulation, and disturb regulation of DNA synthesis in normal human stem and non-stem cell cultures. The outcomes of the stress depend on the cell type.
Diterpenes from Plectranthus spp.: a ROS-induced cytotoxicity study Catarina Garcia 1,2, Przemysław Sitarek 3, Ewa Skała 3, Ewelina Synowiec 4, Tomasz Śliwiński 4, Ana Diaz-Lanza 2, Catarina Reis 5, Patricia Rijo 1 1
Center for Research in Biosciences & Health Technologies (CBIOS), Univ. Lusófona de Humanidades e Tecnologias, Lisbon, Portugal 2 Department of Biomedical Sciences, Faculty of Pharmacy, University of Alcalá, Alcalá de Henares, Spain 3 Department of Biology and Pharmaceutical Botany, Medical University of Lodz, Lodz, Poland 4 Laboratory of Medical Genetics, University of Lodz, Lodz, Poland, Pomorska 5 Instituto de Investigação do Medicamento (iMed.ULisboa), Fac. de Farm., Univ. de Lisboa, Lisbon, Portugal
E-mail address: [email protected] Acknowledgements This work is supported by the RSF Grant No.14-50-00068.
http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.417
P-271
Nitration alters laminin polymerization Lasse Lorentzen, Michael Davies Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark
Plants from the Plectranthus genus are commonly known for their medicinal properties, and their applications are vast in traditional medicine. Often accountable for these properties, bioactive secondary metabolites such as diterpenes are present in these plants, and some possess cytotoxic properties. Abietane diterpenes 7α-acetoxy-6β-hydroxyroyleanone, 7β,6β-dihydroxyroyleanone, 6,7- dehydroroyleanone, and parvifloron D are examples of such class of bioactive diterpenes. These abietanes are obtained from Plectranthus genus plants as their major compounds. In this work, the cytotoxic properties of these abietanes have been studied. This property was evaluated through MTT assays on A549 cells and CCRF-CEM cells. In addition, the level of intracellular Reactive Oxygen Species (ROS) in these cells was also measured. Our study has revealed that on CCRF-CEM cells, there is a slight increase of the ROS levels after exposure to all compounds. Nevertheless, on A549 cells the levels of ROS increase approximately 10-fold, with notorious increase after exposure to 7β,6β-dihydroxyroyleanone, which can induce cytotoxicity due to ROS accumulation on cells. In conclusion, this study proves the therapeutic potential of Plectranthus-derived diterpenes and validates its use in traditional medicine.
E-mail address: [email protected] Reactive nitrogen species (RNS) are produced by cells at sites of inflammation and can modify proteins. Nitration is used as a biomarker of damage and may contribute to multiple pathologies. We have shown that the extracellular matrix protein laminin (LN) co-localizes with nitrative damage in human atherosclerotic lesions, and that nitration decreases cell binding to LN in vitro. Study aim: To investigate how nitration affects laminin self-assembly and polymerization. Methods: Sites of nitration were determined by LC-MS. Polymerization was induced by pH changes and CaCl2. Dynamic light scattering was used to examine the extent of polymerization. Structure of LN polymers was investigated with scanning electron microscopy (SEM). Results: Extensive nitration was observed on the protein at specific sites. Nitrated LN underwent polymerization, but displayed increased light scattering, indicating altered particle size. SEM showed that untreated LN formed a homogenous ordered structure, whereas nitrated LN displayed a segmented, patchy and disordered pattern. Conclusions: While the extent of LN polymerization is not affected
http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.419
P-273
Stress-induced premature senescence program activated in the mesenchymal stem cells by antioxidant treatments Julia Kornienko, Irina Smirnova, Natalia Pugovkina, Valery Zenin, Nikolay Nikolsky, Olga Lyublinskaya
S128
A. Stepanova et al. / Free Radical Biology and Medicine 120 (2018) S45–S166
Institute of Cytology, Russian Academy of Sciences, St.Petersburg, Russia
It is now well established that intracellular reactive oxygen species (ROS) mediate the regulation of many signaling cascades that define cell fate. The delicate balance between the production and elimination of ROS is essential for maintaining normal cell functions. Disruption of this balance caused by the antioxidant treatment can disturb various intracellular processes controlled by the systemic signaling. In the present study, we investigate the response of the mesenchymal stem cells (MSCs) to the sub-cytotoxic treatment with various antioxidants (tempol, NAC, resveratrol, DPI). We show that exposure of the proliferating MSC cultures to the antioxidants at concentrations which are widely used in the cell studies to protect cells from the oxidative stress, can cause replication stress and initiate the program of stress-induced preliminary senescence. We demonstrate that these effects can be explained by the disturbance of the ROS-depended degradation of the key proteins regulating DNA replication, e.g. cyclin A, Emi1, geminin. Our data show that enhanced intracellular elimination of ROS resulted from the pharmacologic manipulations can cause the antioxidant-induced damage of cells.
E-mail address: [email protected] Acknowledgements This work is supported by the RSF Grant No.14–50-00068.
http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.420
P-275
Quantum-chemical insights into chemiluminescence from carbonyls Homa Saeidfirozeh 1, Michal Cifra 2, Azizollah Shafiekhani 1,3 1
Physics Department, Alzahra University, Tehran, Iran Institute of Photonics and Electronics, The Czech Academy of Sciences (ufe), Chaberská, Praha - Kobylisy, Czech Republic 3 School of Physics, Institute for Research in Fundamental Sciences (IPM), Tehran, Iran 2
Organisms undergoing oxidative metabolism or stress chemically generate electronically excited species through dioxetane and tetroxide pathways. Consequently, luminescence is emitted which could be exploited for non-invasive label-free monitoring of oxidative stress. Spectral analysis of this endogenous chemiluminescence can serve as a tool for identification of the emitters as products of oxidation. Here we employ quantum mechanical calculations (Gaussian software package) and sensitive experimental photon counting methods to obtain deeper understanding of the emitter molecules both from theoretical and experimental perspective. We focused on the analysis of excited carbonyl groups of simple model species (formaldehyde, acetone, etc.) and carbonyls formed by fragmentation of linoleic acid since they are common product of oxidation and also potential emitters of endogenous chemiluminescence.
P-274
E-mail address: h.saeidfi[email protected]
Nitrosopersulfide (SSNO) is a potential protein Cys polysulfidating agent with sustained and long-range effects
http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.422
Virág Bogdándi 1,2,3, István Zoárd Bátai 1,2,3, Magda Minnion 1,2,3, Zoltán Sándor 1,2,3, Erika Pintér 1,2,3, Martin Feelisch 1,2,3, Péter Nagy 1,2,3
P-276
1 Department of Molecular Immunology and Toxicology, National Institute of Oncology, Budapest, Hungary 2 Department of Pharmacology and Pharmacotherapy, Medical School, University of Pécs, Pécs, Hungary 3 Clinical and Experimental Sciences, Faculty of Medicine, and University Hospital Southampton NHS Foundation Trust, University of Southampton, Southampton, UK
The biological actions of NO and H2S are intimately intertwined. Chemical interactions of these molecules form a network of cascading chemicals. SSNO is a major product of these interactions. It is relatively stable under physiological conditions and not reducible by thiols. Upon slow decomposition of SSNO, the sulfane sulfur equivalents are released in the form of inorganic polysulfides (HSx). HSx were shown to be important mediators in H2S biology by inducing protein Cys persulfidation. However, HSx are readily reduced by the glutathione and thioredoxin systems inside the cell. Importantly, we found that SSNO can escape these reducing machineries and hence we hypothesize that it could serve as a sulfane sulfur carrier for sustained and long range HSx bioavailability. SSNO indeed induced delayed polysulfidation on Cys, GSH, HSA and a slow flux of sulfane sulfur production in HEK 293 cells. Furthermore, HSx were shown to activate TRPA1 channels and our experiments revealed that SSNO can also function as a TRPA1 activator, but with a lasting effect. These observations give credence to our hypothesis that SSNO may be responsible for sustained protein Cys‒polysulfidation and potentially involved in sulfane sulfur trafficking between subcellular compartments.
E-mail address: [email protected] http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.421
Vitamin E metabolism and its putative role as fatsoluble antioxidant in the redox-lipidomics era Galli Francesco 1, Roberta Galarini 2, Gabriele Cruciani 3 1
Dept of Pharmaceutical Sciences, Clinical Biochemistry and Nutrition Lab, University of Perugia, Italy 2 Istituto Zooprofilattico Sperimentale Umbria-Marche, Perugia, Italy 3 Department of Chemistry, Biology and Biotechnology, University of Perugia, Italy
Lipidomics is going to leave its role of “Cinderella” of omics technologies. Innovative mass spectrometry and bioinformatics technology is bursting into the omics scene and this is leading lipid science to gain renewed attention and interest by the scientific community. New lipidomics challenges have already launched including that of extensive (high-throughput) exploration of oxidized lipids in biological matrices (that is “redox lipidomics”). Alpha-tocopherol, the main form of vitamin E in human tissues, is a H donor with exclusive pertinence to lipid peroxide substrates of cellular membranes and lipoprotein particles. The direct or enzyme-mediated interaction of this vitamin with lipid oxidation pathways generates specific metabolites and functional responses. This area of redox lipidomics is now gaining increasing interest for its role in PUFA oxidation and lipid signaling, and more in general in long-chain lipid metabolism under both physiological and pathological conditions. Targeted and untargeted lipidomics protocols of investigation are now available to characterize the functional lipidome of vitamin E that is discussed in this paper along with other emerging aspects of redox lipidomics.
E-mail address: [email protected]