- Email: [email protected]
Antioxidant independent actions of vitamin E in modulating gene expression
S58
A. Newman / Free Radical Biology and Medicine 128 (2018) S47–S60
hydrogen peroxide degradation by CeNPs. The results of this study substantiate the reinforcement of CeNPs as pharmacological agents for the treatment of human diseases related to nonfunctional biological enzymes.
https://doi.org/10.1016/j.freeradbiomed.2018.10.109
there are few studies mechanisms of cause damages through UPEs measurements of lipids. In the present studies, we investigated direct interactions between linoleic acid and melanin. Preliminary tests of UPE source exploration was demonstrated with auto-luminescence spectrum of melanin and coexistence with oxidized linoleic acid. Furthermore, increase in intensities of the auto-luminescence released from melanin with oxidized linoleic acid might be concentration-dependent. The findings suggested that excited triplet states of linoleic acid related with UPEs via chemical reactions could be a unique biomarker for making diagnoses on epidermis using the non-invasive images.
107 Acknowledgements
In vivo two-photon imaging of malondialdehyde in mice brains with new carbon dots
This work was supported by Grant-in-Aid university (S1312001) from MEXT, Japan.
of
a
private
Di Su*, Ping Li, Xin Wang, Zhang Wen, Bo Tang Shandong Normal University, China
Brains are considered to produce lipid peroxidation in the case of cerebral ischemia-reperfusion injury (CIRI). Malondialdehyde (MDA) is a biomarker of lipid peroxidation and oxidative stress, which can cross-link with biomacromolecules to destroy their functions. Thus, it is of great interest to determine the function of MDA in brain injury. However, due to lacking the appropriate imaging techniques of MDA in vivo, especially in brains, precise exploration of MDA in brains remains a challenge. To address this issue, we synthesized new two-photon carbon dots (CD-1) for detecting MDA in cells and living brains for the first time. CD-1 exhibited exceptional selectivity and high sensitivity towards MDA, and avoided the self-fluorescence of MDA perfectly. Utilizing twophoton imaging, CD-1 was applied for monitoring MDA in PC12 cells under H2O2 stimuli, verifying that MDA increased significantly compared to the control cells. In particular, we used CD-1 for mapping MDA in mice brains with cerebral ischemia-reperfusion injury, thereby witnessed a positive correlation between MDA and cerebral injury. Altogether, our work establishes a new method for live monitoring MDA in vivo and provides a new strategy for revealing the pathogenesis of MDA-related brain diseases in future.
References [1] Tada M, Kohno M, Niwano Y, J Clin Biochem Nutr. 46(3):224-8 (2010) [2] Kobayashi M, Iwasa T, Tada M, J. Photochem. Photobiol. B: Biology 159, 186-190 (2016)
https://doi.org/10.1016/j.freeradbiomed.2018.10.111
109
Biologically-relevant radical reduction by a ruthenium catalyst Andrew Tennyson*, Yamin Htet Clemson University, USA
https://doi.org/10.1016/j.freeradbiomed.2018.10.110
108
Investigations for direct interactions between linoleic acid and melanin using observations of auto-luminescence Mika Tada*
Catalytic reduction of ABTS– to ABTS2– was achieved by a ruthenium complex using a variety of biologically-relevant alcohols, such as amino acids, sugars, or citric acid cycle metabolites, as terminal reductants. Rate law and deuterium labeling studies revealed that radical reduction was performed by a ruthenium–hydride intermediate formed via hydride transfer from the alcohol to ruthenium. With this knowledge, it was subsequently shown that NAD þ also functioned as a terminal reductant via its ribose subunit, representing the first demonstration that NAD þ could function as a hydride donor under biologically-relevant conditions. This ruthenium complex shows promise for applications in oxidative stress and redox therapeutics.
https://doi.org/10.1016/j.freeradbiomed.2018.10.112
Tohoku Institute of Technology, Japan Melanin is well-known as an important pigment for protecting various biological samples, such as human skin, hair, fruit, mushroom, from the harmful effects of oxidative stresses. Although photoprotective properties of melanin have been well documented, effects of melanin on reactive oxygen species (ROS) and other species have been reported in our previous studies [1,2]. It is confirmed that one of the mechanisms by which melanin protects UVinduced skin damage is likely quenching or scavenging activity against singlet oxygen and superoxide anion [1]. In addition, decompositions of lipid hydroperoxides are known to release excited triplet states of biomolecules composed with carbonyl groups. Ultra-weak photon emissions (UPEs) are important to monitor in the redox status because UPEs have been used as biomarkers for non-invasive imaging techniques. Because UPEs composed with various wave lengths, we investigated mechanisms of reactive species released from UPEs via chemical reactions of melanin by qualitative analysis and polychromatic spectral pattern analysis [2]. However, in spite of lipid-derivers free radicals,
110
Antioxidant independent actions of vitamin E in modulating gene expression Stephen Valentino 1,*, Mikel Ghelfi 2, Elizabeth Zunica 1, Michaela Stamper 1, Sarah Hickman 1, Shannon Hwang 1, Ericka Young 1, Jeffrey Atkinson 2, Danny Manor 1 1 2
Case Western Reserve University, USA Brock University, Canada
A. Newman / Free Radical Biology and Medicine 128 (2018) S47–S60
Alpha-tocopherol (vitamin E) is a plant-derived lipid antioxidant that is an essential nutrient for all vertebrates. The vitamin’s function in reducing and preventing injury caused by reactive oxygen species (ROS) has been well established. Recently, a few lines of evidence indicated that alpha tocopherol has novel functions in addition to its established radicalquenching activity, specifically in regulating gene expression. To investigate this issue, we asked whether an analog of vitamin E that has no antioxidant activity, 6-hydroxymethyl alpha tocopherol (6HMTC), is able to regulate expression of the TTPAgene, encoding the tocopherol transfer protein, TTP, an activity exhibited by the natural vitamin. We found that the analog 6HMTC is not an effective antioxidant in vitro and in cultured hepatocytes. However, 6HMTC induced expression of the TTPA gene in cells in a manner indistinguishable from alpha-tocopherol. These results indicate that the biological actions of vitamin E involve yet-unexplored, antioxidant-independent mechanisms. Future work will be directed into the molecular pathways that mediate this phenomenon, and the roles it plays in specific health-promoting effects of vitamin E.
https://doi.org/10.1016/j.freeradbiomed.2018.10.113
111
Application of resonance raman spectroscopy on manganese porphyrin detection and redox state in endothelial cells: implications on cytoprotection towards peroxynitrite Valeria Valez 1,*, Sebastian Carballal 1, Damian Alvarez 2, Artak Tovmasyan 3, Ines Batinic-Haberle 3, Gerardo Ferrer-Sueta 1, Daniel Murgida 2, Rafael Radi 1 1
Universidad de la República, Uruguay Universidad de Buenos Aires, Argentina 3 Duke University School of Medicine, USA 2
S59
112
CD36 a new cellular target of electrophilic nitro-fatty acids Matias Vazquez*, Victoria Gutierrez, Virginia Actis Dato, Gustavo Chiabrando, Gustavo Bonacci National University of Cordoba, Argentina
CD36 is a high affinity receptor that facilitates the binding and uptake of long-chain fatty acids and modified LDL into the cell. These CD36 ligands have also been described to trigger cell signaling with different effects on cell metabolism, migration and viability. Since nitro-fatty acids are electrophilic lipid mediators which exhibit anti-inflammatory and cytoprotective actions in experimental models of atherosclerosis, cardiac isquemia/ reperfusion and other inflammatory diseases, we have hypothesized that nitrolipids may bind and signal through CD36 receptor. In order to this, herein we demonstrated that Nitro-Oleic Acid (NO2-OA) displays CD36mediated intracellular signaling via Src/ERK and AMPK pathways. Pharmacological strategies to inhibit both ligands binding to CD36 (SSO) and CD36 downstream signaling pathway for ERK (PD98059) and Src (PP1), allowed us to elucidate the specificity of the pathway involved. Therefore, to study their interaction, an in vitro assay was developed using recombinant CD36 (rCD36) and biotinylated-NO2-OA. The products of this reaction exhibited that NO2-OA interacts with CD36 revealed by Western blot using HRP-streptavidin. Competition assays with increasing molar concentration of OA, GSH and unbiotinylated NO2-OA (1:1, 1:10 and 1:100) exposed the reversibility of this interaction. Similar experimental settings showed that NO2OA impairs modified-LDL binding to rCD36, which was supplemented with experiments in RAW264.7 macrophages cell line, where pre-incubation with NO2-OA showed a decrease in cholesterol accumulation after modified-LDL treatment. This result indicates that NO2-OA may bind to the same site as modified-LDL, on the ligand binding domain, and alter its incorporation into the macrophages. Altogether this data suggests that NO2-OA acts as a CD36 ligand and triggers its downstream signaling to modulate fatty acid metabolism in macrophages.
https://doi.org/10.1016/j.freeradbiomed.2018.10.115 Manganese(III) ortho N-substituted pyridylporphyrins (MnP) act as efficient antioxidants catalyzing superoxide dismutation and accelerating peroxynitrite reduction. Importantly, MnP can reach mitochondria offering protection against reactive species in different animal models of disease. In this scenario, we applied Resonance Raman (RR) spectroscopy as a direct method to detect both the uptake and the redox state of MnP in living cells. Using RR we were able to analyze the intracellular redox state changes of two potent MnP (MnTnBuOE-2-PyP5 þ and MnTnHex-2-PyP5 þ ) within endothelial cells exposed to SIN-1. RR experiments with cells and isolated mitochondria revealed that the reduction of Mn(III)P was affected by inhibitors of the electron transport chain, supporting the action of MnP as efficient redox active compounds in mitochondria. Indeed, RR spectra confirmed that MnP added in the Mn(III) state can be incorporated into the cells, readily reduced by intracellular components to the Mn(II) state and oxidized by peroxynitrite. We also evaluated the cytoprotective capacity of MnP by exposing the endothelial cells to nitro-oxidative stress induced by peroxynitrite. We observed a preservation of normal mitochondrial function, attenuation of cell damage and prevention of apoptotic cell death. These data introduce a novel application of RR spectroscopy for the direct detection of MnP and their redox states inside living cells, and helps to rationalize their antioxidant capacity in biological systems.
https://doi.org/10.1016/j.freeradbiomed.2018.10.114
113
Visualizing the function of hydroxyl radicals via SIRT1 in brains of mice with stress-induced depression related behaviours Xin Wang*, Ping Li, Qi Ding, Bo Tang Shandong Normal University, China
Stress related disorders such as depression is intimately linked with the dysfunction of oxidant and antioxidant defence systems; therefore, as one of the most reactive and oxidative reactive oxygen species (ROS), hydroxyl radical (OH) can cause macromolecular damage and subsequent neurological diseases when over-produced. However, due to the high reactivity and low concentration of OH in living brain tissue, precise exploration of the role of OH in brain remains a challenge. Thus, the two-photon fluorescence probe TCE was developed for in situ OH imaging in living systems. This probe achieves exceptional selectivity and high sensitivity towards OH via the one-electron oxidation of 3-methyl-pyrazolone as a new specific recognition site. TCE can map OH in the brains of mice, thereby revealing that the increase in OH level is positively correlated with the severity of depression-like behaviours. Furthermore, OH has been shown to
A. Newman / Free Radical Biology and Medicine 128 (2018) S47–S60
hydrogen peroxide degradation by CeNPs. The results of this study substantiate the reinforcement of CeNPs as pharmacological agents for the treatment of human diseases related to nonfunctional biological enzymes.
https://doi.org/10.1016/j.freeradbiomed.2018.10.109
there are few studies mechanisms of cause damages through UPEs measurements of lipids. In the present studies, we investigated direct interactions between linoleic acid and melanin. Preliminary tests of UPE source exploration was demonstrated with auto-luminescence spectrum of melanin and coexistence with oxidized linoleic acid. Furthermore, increase in intensities of the auto-luminescence released from melanin with oxidized linoleic acid might be concentration-dependent. The findings suggested that excited triplet states of linoleic acid related with UPEs via chemical reactions could be a unique biomarker for making diagnoses on epidermis using the non-invasive images.
107 Acknowledgements
In vivo two-photon imaging of malondialdehyde in mice brains with new carbon dots
This work was supported by Grant-in-Aid university (S1312001) from MEXT, Japan.
of
a
private
Di Su*, Ping Li, Xin Wang, Zhang Wen, Bo Tang Shandong Normal University, China
Brains are considered to produce lipid peroxidation in the case of cerebral ischemia-reperfusion injury (CIRI). Malondialdehyde (MDA) is a biomarker of lipid peroxidation and oxidative stress, which can cross-link with biomacromolecules to destroy their functions. Thus, it is of great interest to determine the function of MDA in brain injury. However, due to lacking the appropriate imaging techniques of MDA in vivo, especially in brains, precise exploration of MDA in brains remains a challenge. To address this issue, we synthesized new two-photon carbon dots (CD-1) for detecting MDA in cells and living brains for the first time. CD-1 exhibited exceptional selectivity and high sensitivity towards MDA, and avoided the self-fluorescence of MDA perfectly. Utilizing twophoton imaging, CD-1 was applied for monitoring MDA in PC12 cells under H2O2 stimuli, verifying that MDA increased significantly compared to the control cells. In particular, we used CD-1 for mapping MDA in mice brains with cerebral ischemia-reperfusion injury, thereby witnessed a positive correlation between MDA and cerebral injury. Altogether, our work establishes a new method for live monitoring MDA in vivo and provides a new strategy for revealing the pathogenesis of MDA-related brain diseases in future.
References [1] Tada M, Kohno M, Niwano Y, J Clin Biochem Nutr. 46(3):224-8 (2010) [2] Kobayashi M, Iwasa T, Tada M, J. Photochem. Photobiol. B: Biology 159, 186-190 (2016)
https://doi.org/10.1016/j.freeradbiomed.2018.10.111
109
Biologically-relevant radical reduction by a ruthenium catalyst Andrew Tennyson*, Yamin Htet Clemson University, USA
https://doi.org/10.1016/j.freeradbiomed.2018.10.110
108
Investigations for direct interactions between linoleic acid and melanin using observations of auto-luminescence Mika Tada*
Catalytic reduction of ABTS– to ABTS2– was achieved by a ruthenium complex using a variety of biologically-relevant alcohols, such as amino acids, sugars, or citric acid cycle metabolites, as terminal reductants. Rate law and deuterium labeling studies revealed that radical reduction was performed by a ruthenium–hydride intermediate formed via hydride transfer from the alcohol to ruthenium. With this knowledge, it was subsequently shown that NAD þ also functioned as a terminal reductant via its ribose subunit, representing the first demonstration that NAD þ could function as a hydride donor under biologically-relevant conditions. This ruthenium complex shows promise for applications in oxidative stress and redox therapeutics.
https://doi.org/10.1016/j.freeradbiomed.2018.10.112
Tohoku Institute of Technology, Japan Melanin is well-known as an important pigment for protecting various biological samples, such as human skin, hair, fruit, mushroom, from the harmful effects of oxidative stresses. Although photoprotective properties of melanin have been well documented, effects of melanin on reactive oxygen species (ROS) and other species have been reported in our previous studies [1,2]. It is confirmed that one of the mechanisms by which melanin protects UVinduced skin damage is likely quenching or scavenging activity against singlet oxygen and superoxide anion [1]. In addition, decompositions of lipid hydroperoxides are known to release excited triplet states of biomolecules composed with carbonyl groups. Ultra-weak photon emissions (UPEs) are important to monitor in the redox status because UPEs have been used as biomarkers for non-invasive imaging techniques. Because UPEs composed with various wave lengths, we investigated mechanisms of reactive species released from UPEs via chemical reactions of melanin by qualitative analysis and polychromatic spectral pattern analysis [2]. However, in spite of lipid-derivers free radicals,
110
Antioxidant independent actions of vitamin E in modulating gene expression Stephen Valentino 1,*, Mikel Ghelfi 2, Elizabeth Zunica 1, Michaela Stamper 1, Sarah Hickman 1, Shannon Hwang 1, Ericka Young 1, Jeffrey Atkinson 2, Danny Manor 1 1 2
Case Western Reserve University, USA Brock University, Canada
A. Newman / Free Radical Biology and Medicine 128 (2018) S47–S60
Alpha-tocopherol (vitamin E) is a plant-derived lipid antioxidant that is an essential nutrient for all vertebrates. The vitamin’s function in reducing and preventing injury caused by reactive oxygen species (ROS) has been well established. Recently, a few lines of evidence indicated that alpha tocopherol has novel functions in addition to its established radicalquenching activity, specifically in regulating gene expression. To investigate this issue, we asked whether an analog of vitamin E that has no antioxidant activity, 6-hydroxymethyl alpha tocopherol (6HMTC), is able to regulate expression of the TTPAgene, encoding the tocopherol transfer protein, TTP, an activity exhibited by the natural vitamin. We found that the analog 6HMTC is not an effective antioxidant in vitro and in cultured hepatocytes. However, 6HMTC induced expression of the TTPA gene in cells in a manner indistinguishable from alpha-tocopherol. These results indicate that the biological actions of vitamin E involve yet-unexplored, antioxidant-independent mechanisms. Future work will be directed into the molecular pathways that mediate this phenomenon, and the roles it plays in specific health-promoting effects of vitamin E.
https://doi.org/10.1016/j.freeradbiomed.2018.10.113
111
Application of resonance raman spectroscopy on manganese porphyrin detection and redox state in endothelial cells: implications on cytoprotection towards peroxynitrite Valeria Valez 1,*, Sebastian Carballal 1, Damian Alvarez 2, Artak Tovmasyan 3, Ines Batinic-Haberle 3, Gerardo Ferrer-Sueta 1, Daniel Murgida 2, Rafael Radi 1 1
Universidad de la República, Uruguay Universidad de Buenos Aires, Argentina 3 Duke University School of Medicine, USA 2
S59
112
CD36 a new cellular target of electrophilic nitro-fatty acids Matias Vazquez*, Victoria Gutierrez, Virginia Actis Dato, Gustavo Chiabrando, Gustavo Bonacci National University of Cordoba, Argentina
CD36 is a high affinity receptor that facilitates the binding and uptake of long-chain fatty acids and modified LDL into the cell. These CD36 ligands have also been described to trigger cell signaling with different effects on cell metabolism, migration and viability. Since nitro-fatty acids are electrophilic lipid mediators which exhibit anti-inflammatory and cytoprotective actions in experimental models of atherosclerosis, cardiac isquemia/ reperfusion and other inflammatory diseases, we have hypothesized that nitrolipids may bind and signal through CD36 receptor. In order to this, herein we demonstrated that Nitro-Oleic Acid (NO2-OA) displays CD36mediated intracellular signaling via Src/ERK and AMPK pathways. Pharmacological strategies to inhibit both ligands binding to CD36 (SSO) and CD36 downstream signaling pathway for ERK (PD98059) and Src (PP1), allowed us to elucidate the specificity of the pathway involved. Therefore, to study their interaction, an in vitro assay was developed using recombinant CD36 (rCD36) and biotinylated-NO2-OA. The products of this reaction exhibited that NO2-OA interacts with CD36 revealed by Western blot using HRP-streptavidin. Competition assays with increasing molar concentration of OA, GSH and unbiotinylated NO2-OA (1:1, 1:10 and 1:100) exposed the reversibility of this interaction. Similar experimental settings showed that NO2OA impairs modified-LDL binding to rCD36, which was supplemented with experiments in RAW264.7 macrophages cell line, where pre-incubation with NO2-OA showed a decrease in cholesterol accumulation after modified-LDL treatment. This result indicates that NO2-OA may bind to the same site as modified-LDL, on the ligand binding domain, and alter its incorporation into the macrophages. Altogether this data suggests that NO2-OA acts as a CD36 ligand and triggers its downstream signaling to modulate fatty acid metabolism in macrophages.
https://doi.org/10.1016/j.freeradbiomed.2018.10.115 Manganese(III) ortho N-substituted pyridylporphyrins (MnP) act as efficient antioxidants catalyzing superoxide dismutation and accelerating peroxynitrite reduction. Importantly, MnP can reach mitochondria offering protection against reactive species in different animal models of disease. In this scenario, we applied Resonance Raman (RR) spectroscopy as a direct method to detect both the uptake and the redox state of MnP in living cells. Using RR we were able to analyze the intracellular redox state changes of two potent MnP (MnTnBuOE-2-PyP5 þ and MnTnHex-2-PyP5 þ ) within endothelial cells exposed to SIN-1. RR experiments with cells and isolated mitochondria revealed that the reduction of Mn(III)P was affected by inhibitors of the electron transport chain, supporting the action of MnP as efficient redox active compounds in mitochondria. Indeed, RR spectra confirmed that MnP added in the Mn(III) state can be incorporated into the cells, readily reduced by intracellular components to the Mn(II) state and oxidized by peroxynitrite. We also evaluated the cytoprotective capacity of MnP by exposing the endothelial cells to nitro-oxidative stress induced by peroxynitrite. We observed a preservation of normal mitochondrial function, attenuation of cell damage and prevention of apoptotic cell death. These data introduce a novel application of RR spectroscopy for the direct detection of MnP and their redox states inside living cells, and helps to rationalize their antioxidant capacity in biological systems.
https://doi.org/10.1016/j.freeradbiomed.2018.10.114
113
Visualizing the function of hydroxyl radicals via SIRT1 in brains of mice with stress-induced depression related behaviours Xin Wang*, Ping Li, Qi Ding, Bo Tang Shandong Normal University, China
Stress related disorders such as depression is intimately linked with the dysfunction of oxidant and antioxidant defence systems; therefore, as one of the most reactive and oxidative reactive oxygen species (ROS), hydroxyl radical (OH) can cause macromolecular damage and subsequent neurological diseases when over-produced. However, due to the high reactivity and low concentration of OH in living brain tissue, precise exploration of the role of OH in brain remains a challenge. Thus, the two-photon fluorescence probe TCE was developed for in situ OH imaging in living systems. This probe achieves exceptional selectivity and high sensitivity towards OH via the one-electron oxidation of 3-methyl-pyrazolone as a new specific recognition site. TCE can map OH in the brains of mice, thereby revealing that the increase in OH level is positively correlated with the severity of depression-like behaviours. Furthermore, OH has been shown to