Possible artefacts of antioxidant assays performed in the presence of nitroxides and nitroxide-containing nanoparticles

Pichla, Monika; Bartosz, Grzegorz; Pieńkowska, Natalia; Sadowska-Bartosz, Izabela

doi:10.1016/j.ab.2020.113698

Cited by 7 publications

(3 citation statements)

References 39 publications

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“…In this case, NRNP1 showed a strong protective effect, while free nitroxides was effective only after 24 h (Figures 11(c) and 11(d)). The apparent increase in the ROS level in the presence of NRNP1 (Figure 11(b)) is surprising; we attribute it to the direct oxidation of the fluorogenic probe by NRNP1 observed by us [58].…”

Section: Discussionmentioning

confidence: 65%

“…It should be noted that nitroxides also react with GSH [58] and thus may induce a compensative GSH biosynthesis in cells exposed to nitroxides as well as after 24 h; in the case of NRNP1, this effect was not observed, most probably due to different reactivity of NRNP1 with GSH, with respect to free nitroxides ( Figure 10). Previous studies pointed to a much lower reactivity of NRPN1 with respect to free nitroxides for another reducing agent, ascorbate [41].…”

Section: Discussionmentioning

confidence: 99%

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Nitroxide Radical-Containing Redox Nanoparticles Protect Neuroblastoma SH-SY5Y Cells against 6-Hydroxydopamine Toxicity

Pichla

Pułaski

Kania

et al. 2020

Oxidative Medicine and Cellular Longevity

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Parkinson’s disease (PD) patients can benefit from antioxidant supplementation, and new efficient antioxidants are needed. The aim of this study was to evaluate the protective effect of selected nitroxide-containing redox nanoparticles (NRNPs) in a cellular model of PD. Antioxidant properties of NRNPs were studied in cell-free systems by protection of dihydrorhodamine 123 against oxidation by 3-morpholino-sydnonimine and protection of fluorescein against bleaching by 2,2-azobis(2-amidinopropane) hydrochloride and sodium hypochlorite. Model blood-brain barrier penetration was studied using hCMEC/D3 cells. Human neuroblastoma SH-SY5Y cells, exposed to 6-hydroxydopamine (6-OHDA), were used as an in vitro model of PD. Cells were preexposed to NRNPs or free nitroxides (TEMPO or 4-amino-TEMPO) for 2 h and treated with 6-OHDA for 1 h and 24 h. The reactive oxygen species (ROS) level was estimated with dihydroethidine 123 and Fluorimetric Mitochondrial Superoxide Activity Assay Kit. Glutathione level (GSH) was measured with ortho-phtalaldehyde, ATP by luminometry, changes in mitochondrial membrane potential with JC-1, and mitochondrial mass with 10-Nonyl-Acridine Orange. NRNP1, TEMPO, and 4-amino-TEMPO (25-150 μM) protected SH-SY5Y cells from 6-OHDA-induced viability loss; the protection was much higher for NRNP1 than for free nitroxides. NRNP1 were better antioxidants in vitro and permeated better the model BBB than free nitroxides. Exposure to 6-OHDA decreased the GSH level after 1 h and increased it considerably after 24 h (apparently a compensatory overresponse); NRNPs and free nitroxides prevented this increase. NRNP1 and free nitroxides prevented the decrease in ATP level after 1 h and increased it after 24 h. 6-OHDA increased the intracellular ROS level and mitochondrial superoxide level. Studied antioxidants mostly decreased ROS and superoxide levels. 6-OHDA decreased the mitochondrial potential and mitochondrial mass; both effects were prevented by NRNP1 and nitroxides. These results suggest that the mitochondria are the main site of 6-OHDA-induced cellular damage and demonstrate a protective effect of NRNP1 in a cellular model of PD.

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Section: Discussionmentioning

confidence: 65%

Section: Discussionmentioning

confidence: 99%

Nitroxide Radical-Containing Redox Nanoparticles Protect Neuroblastoma SH-SY5Y Cells against 6-Hydroxydopamine Toxicity

Pichla

Pułaski

Kania

et al. 2020

Oxidative Medicine and Cellular Longevity

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“…The stable radicals used as probes are chemically very different from the radicals that propagate the autoxidation of real systems, thus they don't reproduce the kinetic and sometimes neither the stoichiometry of the reaction with ROO • . Moreover, these stable radicals may be quenched also by reductants having no antioxidant activity, such as H 2 O 2 and hydroperoxides [66][67][68][69].…”

Section: Why You Should Not Use Simplified Methods?mentioning

confidence: 99%

Methods to Determine Chain-Breaking Antioxidant Activity of Nanomaterials beyond DPPH•. A Review

Baschieri

Amorati

2021

Antioxidants

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This review highlights the progress made in recent years in understanding the mechanism of action of nanomaterials with antioxidant activity and in the chemical methods used to evaluate their activity. Nanomaterials represent one of the most recent frontiers in the research for improved antioxidants, but further development is hampered by a poor characterization of the ‘‘antioxidant activity’’ property and by using oversimplified chemical methods. Inhibited autoxidation experiments provide valuable information about the interaction with the most important radicals involved in the lipid oxidation, namely alkylperoxyl and hydroperoxyl radicals, and demonstrate unambiguously the ability to stop the oxidation of organic materials. It is proposed that autoxidation methods should always complement (and possibly replace) the use of assays based on the quenching of stable radicals (such as DPPH• and ABTS•+). The mechanisms leading to the inhibition of the autoxidation (sacrificial and catalytic radical trapping antioxidant activity) are described in the context of nanoantioxidants. Guidelines for the selection of the appropriate testing conditions and of meaningful kinetic analysis are also given.

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Two faces of TEMPO (2,2,6,6-tetramethylpiperidinyl-1-oxyl) – An antioxidant or a toxin?

Mołoń

Szlachcikowska

Stępień

et al. 2023

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research

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Possible artefacts of antioxidant assays performed in the presence of nitroxides and nitroxide-containing nanoparticles

Cited by 7 publications

References 39 publications

Nitroxide Radical-Containing Redox Nanoparticles Protect Neuroblastoma SH-SY5Y Cells against 6-Hydroxydopamine Toxicity

Nitroxide Radical-Containing Redox Nanoparticles Protect Neuroblastoma SH-SY5Y Cells against 6-Hydroxydopamine Toxicity

Methods to Determine Chain-Breaking Antioxidant Activity of Nanomaterials beyond DPPH•. A Review

Two faces of TEMPO (2,2,6,6-tetramethylpiperidinyl-1-oxyl) – An antioxidant or a toxin?

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