- Email: [email protected]
P69 Oxygen reactivity in the sulfmyoglobin formation
Abstracts / Nitric Oxide 27 (2012) S11–S42
Methods: Human microvascular endothelial cells (HMEC) were exposed to slow release H2S donors (SRHD; 100 lM) GYY4137, AP67, AP72 and two mitochondria-targeted H2S donors AP39 and AP123 (0.1 lM), before and after the induction of oxidative stress using SIN-1 (100 lM), H2O2 (100 lM) and 4-HNE (10 lM). Cell viability (alamarBlue), intracellular oxidant generation (ROS; DHE, H2DCFDA, mitosoxRed) and mitochondrial membrane potential (MMP; TMRM) were determined. Western blotting and fluorescence assays were used for Akt, Erk1/2, caspases 3, 7 and 9 expression and activity. Phosphatidylserine externalisation and plasma membrane integrity were assessed by flow cytometry. Oxidative stress significantly reduced cell viability, MMP and increased ROS generation (ANOVA, p < 0.01 all treatments). These effects were significantly inhibited by GYY4137, AP67, AP72 (100 lM; ANOVA, p < 0.01 all treatments). In each assay, the potency of SHRD at preventing oxidative stress induced cytotoxicity was markedly increased by targeting mitochondria (AP39, AP123; 0.1 lM, ANOVA, p < 0.01). SRHD also modulated Akt-ERK1/2 expression, reduced oxidative stress-induced caspase 3/9 activation and activity. Conclusion: These data suggest SHRD can inhibit and/or reverse oxidative stress-mediated cellular injury and highlight strategies which increase vascular H2S bioavailability, and in particular target mitochondria, represent a new therapeutic opportunity to limit MED. Disclosure: This work was funded by the Medical Research Council (UK) and Peninsula Bioventures.
S41
(DSBP = 89.97 ± 41.40%, n = 4), while little change was demonstrated in hypertensive 90 w SHR (DSBP = 24.07 ± 21.99%, n = 4), measured invasively via aortic catheterization.In a separate experiments, aortic rings isolated from age matched SHRs (n = 5) and WKYs (n = 5), were mounted on a myograph, prepared in physiologic buffer at 37 °C, and constricted with phenylephrine (1 lM), for assessment of endothelial function. Acetylcholine (ACH) dependent maximum relaxation on treatment with L-Name (eNOS inhibitor) was modestly attenuated in aortas of 4 w WKY (92.79 ± 2.64–83.26 ± 3.35%), 4 w SHR (83.50 ± 4.47–59.48 ± 8.75%) and 90-week-old WKYs (71.39 ± 15.06–15.77 ± 5.16%). Contrary to that, this response was completely blocked in 90 w SHRs (35.32 ± 5.16–0.24 ± 3.17%). Treatment with propargylglycine (CSE blocker) did not affect ACH mediated maximum relaxation in 90 w SHR (35.32 ± 5.16–29.40 ± 5.86%), but it significantly attenuated the responses in 4 w WKYs (92.79 ± 2.64–56.30 ± 4.55), 4 w SHRs (83.50 ± 4.47–71.63 ± 12.25) and 90 w SHRs (71.39 ± 15.06–8.13 ± 4.61%). Strikingly, rings from normotensive 16 w SHR had a complete L-Name sensitive attenuation of endothelial relaxation (63.25 ± 5.15–2.74 ± 1.75%). Conclusion: There appears to be a NO independent component of endothelial relaxation in normotensive blood vessels, which is sensitive to CSE inhibitors and blockage of KATPchannels. This NO independent component of relaxation is absent in HTN and pre-HTN vessels; hence implying that loss of the CSE/H2S/KATP axis could be a mechanism preceding HTN. We aims to measure CSE activity and sulfhydration of Kir 6.1-Cysteine 43 and GAPDH in normotensive/ pre and post HTN blood vessels in different animal models of HTN to elicit if CSE/H2S/KATP axis is a potential target for the treatment of hypertension.
http://dx.doi.org/10.1016/j.niox.2012.08.068
Disclosure: Nothing to disclose. P68
References
Role of endothelial H2S in the pathogenesis of hypertension in spontaneously hypertensive (SHR) rats Gautam Sikka a, Jochen Steppan a, Desmond McNelis c, Sarah d Campbell , Jonathan Sevilla b, Shayer Chowdhury b, Sophia Ottleben b, Lakshmi Santhanam b, Viachaslau Barodka a, Daniel Nyhan a, Dan Berkowitz a,b a Johns Hopkins University, Anesthesiology, Baltimore, MD 21205, United States b Johns Hopkins University, Biomedical Engineering, Baltimore, MD 21205, United States c Salisbury University, School of Science and Technology, Salisbury, MD 21801, United States d University of Maryland, Baltimore, MD 21201, United States
[1] G. Yang, H2S as a physiologic vasorelaxant, Science 322 (5901) (2008) 587– 590. [2] A.K. Mustafa, H2S as endothelium-derived hyperpolarizing factor sulfhydrates K+ channel, Circ. Res. (2011). [3] H. Yan, Possible role of hydrogen sulfide on pathogenesis of spontaneous hypertension in rats, Biochem. Biophys. Res. 313 (1) (2004) 22–27.
Hypertension (HTN) increases the risk for cardiac disease and stroke. Endogenous hydrogen sulfide (H2S), which plays a prominent role in a multitude of pathologies like inflammation/sepsis, hypertension, peripheral and cerebro-vascular, and coronary artery disease, is now well characterized as a physiologic vasodilator [1]. H2S is produced by cystathionine-c-lyase (CSE) in vascular endothelium. It caters to relaxation through sulfhydration (posttranslational modification) of IK, SK and KATP channels, resulting in vascular smooth muscle cell hyperpolarization. This effect is independent of the Nitric Oxide (NO)/cGMP/PKG axis [2]. Tang et al. studied H2S in spontaneously hypertensive (SHR) rats and demonstrated a loss of CSE/H2S in HTN and a decrease in BP (blood pressure) on substitution of H2S [3]. Aim: To elicit if loss of endogenous H2S contributes to the pathogenesis of hypertension. Results: Tail cuff was used to measure BP in SHR and control Wistar Kyoto (WKY) rats starting at age 4 weeks (w) up until SHRs became hypertensive. Infusion of glybenclamide (20 mg/kg) a KATP channel blocker, revealed a significant increase in systolic BP (SBP) in 4-week-old SHR (DSBP = 43.16 ± 24.24%, n = 5) and 4 w WKY
http://dx.doi.org/10.1016/j.niox.2012.08.069
P69 Oxygen reactivity in the sulfmyoglobin formation Erika M. Lopez-Alfonzo, Elddie M. Roman-Morales, Juan LopezGarriga Univerisity of Puerto Rico – Mayaguez Campus, Chemistry Department, Mayaguez, PR 00681, United States For many years hydrogen sulfide (H2S) has been considered a toxic gas, but recent studies have presented it as a signaling gas that is involved in many physiological reactions. However, when the human body is exposed to high H2S concentrations, a sulfur atom can bind to the heme group of hemoglobin (Hb), forming an Hb derivate called sulfhemoglobin (SHb). The presence of the Histidine amino acid residue in the E7 position and the ferryl species formed by oxygen (O2), and/or hydrogen peroxide are required for its formation. Sulfhemoglobin produces a condition called Sulfhemoglobinemia, which decreases the Hb ability of O2 transport. Our goal was to evaluate the SHb complex formation with different O2 concentrations, in order to determine a SHb mechanism when formed with O2. Myoglobin (Mb) was used for experiments because it has a similar active site structure as Hb and both bind oxygen. Since it was determined that the oxyMb complex was totally formed with a 1:6 proportion (Mb: O2), O2 concentration
S42
Abstracts / Nitric Oxide 27 (2012) S11–S42
variations were done in 1:6, 1:83, and 1:160. The SMb complex formation was evaluated by UV–vis 300–800 nm region following characteristic 620 nm sulfheme band through a 24 h kinetic analysis. Results indicated that the O2 is involved in the process of the SMb complex formation leading to the transient deoxy specie and depending on the O2concentration, the equilibrium reaction toward the deoxy specie may change. Disclosure: Nothing to disclose. http://dx.doi.org/10.1016/j.niox.2012.08.070
P70 Crystallization of the HbI–pCys protein complex from Lucina pectinata both in excess and absence of H2S Ulises Marrero Llerena a, Josiris Rodriguez Perez b, Juan Lopez Garriga b a University of Puerto Rico at Mayaguez, Industrial Biotechnology, Mayaguez 00681, Puerto Rico b University of Puerto Rico at Mayaguez, Chemistry, Mayaguez 00681, Puerto Rico Background: Understanding the interactions between hydrogen sulfide (H2S) and proteins offers great insight into different aspect of human health. Curiously enough there is a wide variety of organisms that strive in concentrations of 2–5 mM of H2S and even more, were we humans would perish. One organism that lives under this condition is Lucina pectina, a bivalve that contains three hemoglobins and one of them, hemoglobin I (HbI), is capable of transporting H2S. Furthermore, HbI is intimately associated to a protein rich in cysteine (pCys) with unknown structure and function. This two proteins form a complex that may be responsible for the transfer and storage of H2S. Therefore, to understand possible changes in protein–protein interaction as a result of the H2S presence may shed light into many questions revolving around the delicate balance of H2S concentration and protein structures. Methods: To resolve the three-dimensional structure of this protein complex, we study the protein–protein interaction through X-ray crystallography both in excess and absence of H2S. Unfortunately the crystallographic structure determination of proteins is limited by the ability to obtain protein crystals. Thus the bottleneck step for protein structure determination is achieving crystals that can diffract to atomic resolution (higher than 3Å). Crystals of micrometers have been obtained by preliminary experiments using the HbI–pCys protein complex and the hanging drop technique. To optimize the crystal quality, experiments are being conducted using a counter diffusion gel crystallization approach. Results: Preliminary experiments testing different crystallization conditions on the HbI–pCys protein complex (in the absence of H2S) gave small crystal hits for ammonium sulfate (in various buffers) and potassium, sodium- tartrate as precipitant salts. Conclusion: The condition to grow HbI–pCys crystals were appropriate but must be improved.
Disclosure: This presentation was made possible by the RISE2BEST Program, grant NIH-R25GM088023 from the National Institute of General Medical Sciences. http://dx.doi.org/10.1016/j.niox.2012.08.071
P71 Sodium sulfide therapy attenuates heart failure-induced ASK1 signaling in a thioredoxin-dependent manner Chad K. Nicholson a, Junichi Sadoshima b, John W. Calvert a a Emory University, Atlanta, GA 30308, United States b UMDNJ Medical School, Newark, NJ 07101, United States Background: Therapeutic strategies aimed at increasing the levels of hydrogen sulfide (H2S) exert cytoprotective effects in various models of cardiac injury. Here we examined the role that thioredoxin-1 (Trx1) plays in mediating the protective effects of H2S in a model of heart failure. Methods and results: Mice (12 weeks of age) were subjected to 60 min of left coronary artery occlusion followed by 4 weeks of reperfusion at which time left ventricular (LV) dimensions and function were evaluated with 2-D echocardiography. The mice received saline (Veh) or H2S in the form of sodium sulfide (Na2S, 100 lg/kg) at the time of repefusion followed by daily intravenous injections for the first 7 days following reperfusion. After 4 weeks of reperfusion both groups displayed significant LV dilation and severe cardiac dysfunction. However, treatment with Na2S significantly reduced the degree of dilation and improved left ventricular ejection fraction when compared to Veh treated animals. These improvements were also accompanied by better contractility and relaxation as evaluated by invasive hemodynamics. Heart weight to body weight ratios revealed that Na2S significantly decreased hypertrophy indicating a possible decrease in hypertrophic signaling. Studies aimed at evaluating the underlying cardioprotective mechanisms found that Na2S treatment increased the gene and protein expression of Trx1. Further analysis revealed that this was accompanied by a decrease in the phosphorylation of apoptosis signaling kinase-1 (ASK1) at threonine residue 845 (activation site), as well as a decrease in the phosphorylation of JNK and p38 (downstream targets of ASK1). We also found that Na2S treatment did not improve cardiac dilatation, cardiac dysfunction, or cardiac hypertrophy in cardiac specific Trx1 dominant negative transgenic (Trx1 dnTg) mice when compared to Veh-treated mice and did not alter the ischemia-induced increase in the phosphorylation of ASK1, JNK, or p38. Conclusion: These findings provide important information that Na2S attenuates the severity of ischemic-induced heart failure via the upregulation of cardiac Trx1, which sets into motion events, including ASK1 inhibition. Disclosure: No response indicated. http://dx.doi.org/10.1016/j.niox.2012.08.072
Methods: Human microvascular endothelial cells (HMEC) were exposed to slow release H2S donors (SRHD; 100 lM) GYY4137, AP67, AP72 and two mitochondria-targeted H2S donors AP39 and AP123 (0.1 lM), before and after the induction of oxidative stress using SIN-1 (100 lM), H2O2 (100 lM) and 4-HNE (10 lM). Cell viability (alamarBlue), intracellular oxidant generation (ROS; DHE, H2DCFDA, mitosoxRed) and mitochondrial membrane potential (MMP; TMRM) were determined. Western blotting and fluorescence assays were used for Akt, Erk1/2, caspases 3, 7 and 9 expression and activity. Phosphatidylserine externalisation and plasma membrane integrity were assessed by flow cytometry. Oxidative stress significantly reduced cell viability, MMP and increased ROS generation (ANOVA, p < 0.01 all treatments). These effects were significantly inhibited by GYY4137, AP67, AP72 (100 lM; ANOVA, p < 0.01 all treatments). In each assay, the potency of SHRD at preventing oxidative stress induced cytotoxicity was markedly increased by targeting mitochondria (AP39, AP123; 0.1 lM, ANOVA, p < 0.01). SRHD also modulated Akt-ERK1/2 expression, reduced oxidative stress-induced caspase 3/9 activation and activity. Conclusion: These data suggest SHRD can inhibit and/or reverse oxidative stress-mediated cellular injury and highlight strategies which increase vascular H2S bioavailability, and in particular target mitochondria, represent a new therapeutic opportunity to limit MED. Disclosure: This work was funded by the Medical Research Council (UK) and Peninsula Bioventures.
S41
(DSBP = 89.97 ± 41.40%, n = 4), while little change was demonstrated in hypertensive 90 w SHR (DSBP = 24.07 ± 21.99%, n = 4), measured invasively via aortic catheterization.In a separate experiments, aortic rings isolated from age matched SHRs (n = 5) and WKYs (n = 5), were mounted on a myograph, prepared in physiologic buffer at 37 °C, and constricted with phenylephrine (1 lM), for assessment of endothelial function. Acetylcholine (ACH) dependent maximum relaxation on treatment with L-Name (eNOS inhibitor) was modestly attenuated in aortas of 4 w WKY (92.79 ± 2.64–83.26 ± 3.35%), 4 w SHR (83.50 ± 4.47–59.48 ± 8.75%) and 90-week-old WKYs (71.39 ± 15.06–15.77 ± 5.16%). Contrary to that, this response was completely blocked in 90 w SHRs (35.32 ± 5.16–0.24 ± 3.17%). Treatment with propargylglycine (CSE blocker) did not affect ACH mediated maximum relaxation in 90 w SHR (35.32 ± 5.16–29.40 ± 5.86%), but it significantly attenuated the responses in 4 w WKYs (92.79 ± 2.64–56.30 ± 4.55), 4 w SHRs (83.50 ± 4.47–71.63 ± 12.25) and 90 w SHRs (71.39 ± 15.06–8.13 ± 4.61%). Strikingly, rings from normotensive 16 w SHR had a complete L-Name sensitive attenuation of endothelial relaxation (63.25 ± 5.15–2.74 ± 1.75%). Conclusion: There appears to be a NO independent component of endothelial relaxation in normotensive blood vessels, which is sensitive to CSE inhibitors and blockage of KATPchannels. This NO independent component of relaxation is absent in HTN and pre-HTN vessels; hence implying that loss of the CSE/H2S/KATP axis could be a mechanism preceding HTN. We aims to measure CSE activity and sulfhydration of Kir 6.1-Cysteine 43 and GAPDH in normotensive/ pre and post HTN blood vessels in different animal models of HTN to elicit if CSE/H2S/KATP axis is a potential target for the treatment of hypertension.
http://dx.doi.org/10.1016/j.niox.2012.08.068
Disclosure: Nothing to disclose. P68
References
Role of endothelial H2S in the pathogenesis of hypertension in spontaneously hypertensive (SHR) rats Gautam Sikka a, Jochen Steppan a, Desmond McNelis c, Sarah d Campbell , Jonathan Sevilla b, Shayer Chowdhury b, Sophia Ottleben b, Lakshmi Santhanam b, Viachaslau Barodka a, Daniel Nyhan a, Dan Berkowitz a,b a Johns Hopkins University, Anesthesiology, Baltimore, MD 21205, United States b Johns Hopkins University, Biomedical Engineering, Baltimore, MD 21205, United States c Salisbury University, School of Science and Technology, Salisbury, MD 21801, United States d University of Maryland, Baltimore, MD 21201, United States
[1] G. Yang, H2S as a physiologic vasorelaxant, Science 322 (5901) (2008) 587– 590. [2] A.K. Mustafa, H2S as endothelium-derived hyperpolarizing factor sulfhydrates K+ channel, Circ. Res. (2011). [3] H. Yan, Possible role of hydrogen sulfide on pathogenesis of spontaneous hypertension in rats, Biochem. Biophys. Res. 313 (1) (2004) 22–27.
Hypertension (HTN) increases the risk for cardiac disease and stroke. Endogenous hydrogen sulfide (H2S), which plays a prominent role in a multitude of pathologies like inflammation/sepsis, hypertension, peripheral and cerebro-vascular, and coronary artery disease, is now well characterized as a physiologic vasodilator [1]. H2S is produced by cystathionine-c-lyase (CSE) in vascular endothelium. It caters to relaxation through sulfhydration (posttranslational modification) of IK, SK and KATP channels, resulting in vascular smooth muscle cell hyperpolarization. This effect is independent of the Nitric Oxide (NO)/cGMP/PKG axis [2]. Tang et al. studied H2S in spontaneously hypertensive (SHR) rats and demonstrated a loss of CSE/H2S in HTN and a decrease in BP (blood pressure) on substitution of H2S [3]. Aim: To elicit if loss of endogenous H2S contributes to the pathogenesis of hypertension. Results: Tail cuff was used to measure BP in SHR and control Wistar Kyoto (WKY) rats starting at age 4 weeks (w) up until SHRs became hypertensive. Infusion of glybenclamide (20 mg/kg) a KATP channel blocker, revealed a significant increase in systolic BP (SBP) in 4-week-old SHR (DSBP = 43.16 ± 24.24%, n = 5) and 4 w WKY
http://dx.doi.org/10.1016/j.niox.2012.08.069
P69 Oxygen reactivity in the sulfmyoglobin formation Erika M. Lopez-Alfonzo, Elddie M. Roman-Morales, Juan LopezGarriga Univerisity of Puerto Rico – Mayaguez Campus, Chemistry Department, Mayaguez, PR 00681, United States For many years hydrogen sulfide (H2S) has been considered a toxic gas, but recent studies have presented it as a signaling gas that is involved in many physiological reactions. However, when the human body is exposed to high H2S concentrations, a sulfur atom can bind to the heme group of hemoglobin (Hb), forming an Hb derivate called sulfhemoglobin (SHb). The presence of the Histidine amino acid residue in the E7 position and the ferryl species formed by oxygen (O2), and/or hydrogen peroxide are required for its formation. Sulfhemoglobin produces a condition called Sulfhemoglobinemia, which decreases the Hb ability of O2 transport. Our goal was to evaluate the SHb complex formation with different O2 concentrations, in order to determine a SHb mechanism when formed with O2. Myoglobin (Mb) was used for experiments because it has a similar active site structure as Hb and both bind oxygen. Since it was determined that the oxyMb complex was totally formed with a 1:6 proportion (Mb: O2), O2 concentration
S42
Abstracts / Nitric Oxide 27 (2012) S11–S42
variations were done in 1:6, 1:83, and 1:160. The SMb complex formation was evaluated by UV–vis 300–800 nm region following characteristic 620 nm sulfheme band through a 24 h kinetic analysis. Results indicated that the O2 is involved in the process of the SMb complex formation leading to the transient deoxy specie and depending on the O2concentration, the equilibrium reaction toward the deoxy specie may change. Disclosure: Nothing to disclose. http://dx.doi.org/10.1016/j.niox.2012.08.070
P70 Crystallization of the HbI–pCys protein complex from Lucina pectinata both in excess and absence of H2S Ulises Marrero Llerena a, Josiris Rodriguez Perez b, Juan Lopez Garriga b a University of Puerto Rico at Mayaguez, Industrial Biotechnology, Mayaguez 00681, Puerto Rico b University of Puerto Rico at Mayaguez, Chemistry, Mayaguez 00681, Puerto Rico Background: Understanding the interactions between hydrogen sulfide (H2S) and proteins offers great insight into different aspect of human health. Curiously enough there is a wide variety of organisms that strive in concentrations of 2–5 mM of H2S and even more, were we humans would perish. One organism that lives under this condition is Lucina pectina, a bivalve that contains three hemoglobins and one of them, hemoglobin I (HbI), is capable of transporting H2S. Furthermore, HbI is intimately associated to a protein rich in cysteine (pCys) with unknown structure and function. This two proteins form a complex that may be responsible for the transfer and storage of H2S. Therefore, to understand possible changes in protein–protein interaction as a result of the H2S presence may shed light into many questions revolving around the delicate balance of H2S concentration and protein structures. Methods: To resolve the three-dimensional structure of this protein complex, we study the protein–protein interaction through X-ray crystallography both in excess and absence of H2S. Unfortunately the crystallographic structure determination of proteins is limited by the ability to obtain protein crystals. Thus the bottleneck step for protein structure determination is achieving crystals that can diffract to atomic resolution (higher than 3Å). Crystals of micrometers have been obtained by preliminary experiments using the HbI–pCys protein complex and the hanging drop technique. To optimize the crystal quality, experiments are being conducted using a counter diffusion gel crystallization approach. Results: Preliminary experiments testing different crystallization conditions on the HbI–pCys protein complex (in the absence of H2S) gave small crystal hits for ammonium sulfate (in various buffers) and potassium, sodium- tartrate as precipitant salts. Conclusion: The condition to grow HbI–pCys crystals were appropriate but must be improved.
Disclosure: This presentation was made possible by the RISE2BEST Program, grant NIH-R25GM088023 from the National Institute of General Medical Sciences. http://dx.doi.org/10.1016/j.niox.2012.08.071
P71 Sodium sulfide therapy attenuates heart failure-induced ASK1 signaling in a thioredoxin-dependent manner Chad K. Nicholson a, Junichi Sadoshima b, John W. Calvert a a Emory University, Atlanta, GA 30308, United States b UMDNJ Medical School, Newark, NJ 07101, United States Background: Therapeutic strategies aimed at increasing the levels of hydrogen sulfide (H2S) exert cytoprotective effects in various models of cardiac injury. Here we examined the role that thioredoxin-1 (Trx1) plays in mediating the protective effects of H2S in a model of heart failure. Methods and results: Mice (12 weeks of age) were subjected to 60 min of left coronary artery occlusion followed by 4 weeks of reperfusion at which time left ventricular (LV) dimensions and function were evaluated with 2-D echocardiography. The mice received saline (Veh) or H2S in the form of sodium sulfide (Na2S, 100 lg/kg) at the time of repefusion followed by daily intravenous injections for the first 7 days following reperfusion. After 4 weeks of reperfusion both groups displayed significant LV dilation and severe cardiac dysfunction. However, treatment with Na2S significantly reduced the degree of dilation and improved left ventricular ejection fraction when compared to Veh treated animals. These improvements were also accompanied by better contractility and relaxation as evaluated by invasive hemodynamics. Heart weight to body weight ratios revealed that Na2S significantly decreased hypertrophy indicating a possible decrease in hypertrophic signaling. Studies aimed at evaluating the underlying cardioprotective mechanisms found that Na2S treatment increased the gene and protein expression of Trx1. Further analysis revealed that this was accompanied by a decrease in the phosphorylation of apoptosis signaling kinase-1 (ASK1) at threonine residue 845 (activation site), as well as a decrease in the phosphorylation of JNK and p38 (downstream targets of ASK1). We also found that Na2S treatment did not improve cardiac dilatation, cardiac dysfunction, or cardiac hypertrophy in cardiac specific Trx1 dominant negative transgenic (Trx1 dnTg) mice when compared to Veh-treated mice and did not alter the ischemia-induced increase in the phosphorylation of ASK1, JNK, or p38. Conclusion: These findings provide important information that Na2S attenuates the severity of ischemic-induced heart failure via the upregulation of cardiac Trx1, which sets into motion events, including ASK1 inhibition. Disclosure: No response indicated. http://dx.doi.org/10.1016/j.niox.2012.08.072