Inhibition of copper-mediated low density lipoprotein peroxidation by quinoline and indolinone nitroxide radicals

doi:10.1016/0006-2952(94)90152-X

Biochemical Pharmacology

Volume 48, Issue 6, 15 September 1994, Pages 1155-1161

https://doi.org/10.1016/0006-2952(94)90152-X Get rights and content

Abstract

Quinoline and indolinone nitroxide radicals are known to be efficient scavengers of oxygen-centred (rate constants (k) between 10³ and 10⁵/M/sec) and carbon-centred radicals (almost diffusion-controlled rate). In this study, the relative effects of these compounds in protecting low density lipoprotein (LDL) from oxidation induced by copper have been investigated. The extent of lipid peroxidation was assessed by monitoring the increased conjugated diene formation, the altered surface charge of the apolipoprotein B and the generation of aldehydic breakdown products of oxidized LDL. All the nitroxides inhibited LDL peroxidation in a concentration-dependent manner. The corresponding hydroxylamines of the nitroxides were also studied and were shown to inhibit lipid peroxidation to almost the same extent as the parent nitroxide. The data indicates that this class of nitroxide radicals (and their reduced hydroxylamine forms) are effective lipophilic antioxidants with the quinoline nitroxide being more efficient than the indolinone nitroxides.

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Citation Excerpt :
In this context, we have been studying for some time the antioxidant effects of indolinonic aromatic nitroxides whose chemical properties differ to some extent from the most commonly used aliphatic nitroxides, such as piperidine and pyrrolidine derivatives. In the majority of cases, these compounds were shown to effectively protect the most biologically significant macromolecules, lipids (Antosiewicz et al., 1995, 1997; Damiani et al., 1994), proteins (Antosiewicz et al., 1995; Damiani et al., 2001a,b), DNA (Damiani et al., 1999a,b,c, 2000; Villarini et al., 1998) and intact cells (Gabbianelli et al., 1997; Falcioni et al., 1998) against free-radical induced damage. Nevertheless, information on the actual interaction of indolinonic nitroxides with biological structures, for example, membranes, is lacking, and this is of vital importance since the functional and structural properties, such as membrane fluidity, are important for the maintenance of cell integrity (Kimelberg, 1977).
This study was performed to evaluate the effects, if any, of aromatic nitroxides, namely, indolinic nitroxides, on membrane fluidity of rat epithelial cells using steady-state fluorescence. These nitroxides are being increasingly considered as new and versatile compounds to reduce oxidative stress in biological systems. Hence, the results obtained in this study will give more insights on the interaction of these compounds with biological structures which at present is lacking, especially in view of their possible application as antioxidant therapeutic agents. The probes DPH and Laurdan which give information on the hydrophobic and hydrophilic–hydrophobic regions of the membrane bilayer, respectively, showed that nitroxide 1 (1,2-dihydro-2-methyl-3H-indole-3-one-1-oxyl) significantly increases membrane fluidity, whereas the corresponding phenylimino nitroxide derivative 2 (1,2-dihydro-2-methyl-3H-indole-3-phenylimino-1-oxyl) leads to membrane rigidification. The aliphatic nitroxide TEMPO included in this study for comparison produced no modifications. Consequently, it appears that the structure of the heterocyclic rings (aromatic or aliphatic) and the substituents may affect membrane fluidity differently.
Nitroxide radicals protect against DNA damage in rat epithelial cells induced by nitric oxide, nitroxyl anion and peroxynitrite
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In order to gain more knowledge on the antioxidant role of nitroxide radicals, in this study we investigate their possible protective action against DNA damage induced by nitric oxide (NO) and reactive nitrogen oxide species deriving from it, namely nitroxyl anion (NO⁻) and peroxynitrite (ONOO⁻). Rat trachea epithelial cells were exposed under aerobic conditions to (1) NO generated by 150 μM S-nitrosoglutathione monoethyl ester (GSNO-MEE), (2) NO⁻ generated by 200 μM Angeli’s salt (Na₂N₂O₃) (3) ONOO⁻ generated by 1 mM SIN-1 (3-morpholino-sydnonimine) and (4) 100 μM synthesized ONOO⁻, in the absence and presence of 5 μM of two indolinonic nitroxides synthesized by us and the piperidine nitroxide TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl). DNA damage was assessed using the comet assay—a rapid and sensitive, single-cell gel electrophoresis technique used to detect primary DNA damage in individual cells. The parameter tail moment, used as an index of DNA damage, showed that in all cases the nitroxides remarkably inhibited DNA strand breaks induced by the different nitrogen oxide species. All three nitroxides protect to the same extent, except in the case of synthesized peroxynitrite where the aromatic nitroxides 1 and 2 are more efficient than TEMPO. These findings are consistent with the antioxidant character of nitroxide compounds and give additional information on the potential implications for their use as therapeutic agents.
The effects of derivatives of the nitroxide tempol on UVA-mediated in vitro lipid and protein oxidation
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Derivatives of tetramethylpiperidines are extensively employed in polymers to prevent photooxidation, and their stabilizing effect is attributed to the activity of the nitroxide radical derived from the parent amine. In this study, we examined the photoprotective effect of a commercial polymer photostabilizer, HALS-1, its corresponding nitroxide, bis(2,2,6,6-tetramethyl-piperidine-1-oxyl-4-yl)sebacate (TINO), and two derivatives of the piperidine nitroxide TEMPOL, 2,2,6,6-tetramethyl-piperidin-4-acetyloxy-1-oxyl (TEMP2) and 2,2,6,6-tetramethyl-piperidin-4-octanoyloxy-1-oxyl (TEMP8) synthesized by us, in liposomes exposed to ultraviolet A (UVA) radiation. For comparison, the UVA-absorber, 4-tert-butyl-4′-methoxydibenzoylmethane (Parsol 1789) used in many suncream formulations, was also included. The nitroxide TINO resulted extremely efficient at inhibiting aldehydic breakdown products deriving from 30 min exposure of liposomes to UVA and the protection was dose-dependent (10–100 μM). The corresponding amine HALS-1 was the least efficient while protection increased in the order: TEMP2 < Parsol 1789 < TEMP 8. HALS-1, TINO, and the two TEMPOL derivatives were also tested in a simple protein system consisting of bovine serum albumin (BSA) exposed to UVA. In this case, these compounds did not inhibit nor enhance UVA-mediated protein carbonyl formation in BSA. The differences in protection between the compounds are discussed in relation to their chemical reactivity, UVA-absorbing capacities, and their molecular structure. Overall, the results obtained envisage the potential use of nitroxide compounds as topical antioxidants.
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The indolinonic and quinolinic aromatic nitroxides synthesized by us are a novel class of biological antioxidants, which afford a good degree of protection against free radical-induced oxidation in different lipid and protein systems. To further our understanding of their antioxidant behavior, we thought it essential to have more information on their effects on DNA exposed to free radicals. Here, we report on the results obtained after exposure of plasmid DNA and calf thymus DNA to peroxyl radicals generated by the water-soluble radical initiator, 2,2′-azobis(2-amidinopropane)dihydrochloride (AAPH), and the protective effects of the aromatic nitroxides and their hydroxylamines, using a simple in vitro assay for DNA damage. In addition, we also tested for the potential of these nitroxides to inhibit hydroxyl radical-mediated DNA damage inflicted by Fenton-type reactions using copper and iron ions. The commercial aliphatic nitroxides 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL), and bis(2,2,6,6-tetramethyl-1-oxyl-piperidin-4-yl)sebacate (TINUVIN 770) were included for comparison. The results show that the majority of compounds tested protect: (i) both plasmid DNA and calf thymus DNA against AAPH-mediated oxidative damage in a concentration-dependent fashion (1–0.1 mM), (ii) both Fe(II) and Cu(I) induced DNA oxidative damage. However, all compounds failed to protect DNA against damage inflicted by the presence of the transition metals in combination with H₂O₂. The differences in protection between the compounds are discussed in relation to their molecular structure and chemical reactivity.
Increased oxidative modification of albumin when illuminated in vitro in the presence of a common sunscreen ingredient: Protection by nitroxide radicals
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We previously reported on the ability of dibenzoylmethane (DBM) and a relative, Parsol 1789, used as a ultraviolet A (UVA)-absorbing sunscreen, to generate free radicals upon illumination, and as a consequence, to inflict strand breaks in plasmid DNA in vitro. This study has now been extended to determine the effects of Parsol 1789 and DBM on proteins, under UVA illumination, with the sole purpose of gaining more knowledge on the photobiological effects of sunscreen chemicals. Parsol 1789 (100 μM) caused a 2-fold increase in protein carbonyl formation (an index of oxidative damage) in bovine serum albumin (BSA) when exposed to illumination, and this damage was both concentration- and time-dependent. The degree of protein damage was markedly reduced by the presence of free radical scavengers, namely piperidinic and indolinonic nitroxide radicals, in accordance with our previous study. Vitamin E had no effect under the conditions used. The results obtained corroborate the fact that Parsol 1789 generates free radicals upon illumination and that these are, most probably, responsible for the protein damage observed under the conditions used in our system. However, at present, we cannot extrapolate from these results the relevance to human use of sunscreens; therefore, further studies should be necessary to determine the efficacy at the molecular and cellular level of this UVA-absorber in order to ascertain protection against photocarcinogenic risk.

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Inhibition of copper-mediated low density lipoprotein peroxidation by quinoline and indolinone nitroxide radicals

Abstract

Biochim Biophys Acta

Tetrahedron

Polym Degrad Stab

Tetrahedron

Free Rad Biol Med

Tetrahedron

Tetrahedron

Anal Biochem

FEBS Lett

Arch Biochem Biophys

J Biol Chem

Life Sci

Spin Labeling Theory and Application II

Biologically active metal-independent Superoxide dismutase mimics

Biochemistry