Gossypitrin, A Naturally Occurring Flavonoid, Attenuates Iron-Induced Neuronal And Mitochondrial Damage:part 2

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2.5. Gos-Fe(I) Complexes Detection

A plausible hypothesis to explain the above results is that Gos forms a transient complex with Fe(ll)that facilitates its oxidation by oxygen. This transitional complex could deliver its electrons more readily than Fe()-citrate, generating a more stable complex with Fe(Ill). Figure 6A shows a typical spectrum of Gos with maximum absorption at 276, 332 and 380 nm (black line). The addition of Fe() induced a concentration-dependent decline in the maximum absorption peaks and the appearance of a new one near 500 nm (Figure 6A). The occurrence of an array of spectra originating from the Gos spectrum was confirmed by the presence of an isosbestic point at λiso=405 nm （see black dots）， indicating a chemical equilibrium (complexation) between free and complexed Gos. The inset (Figure 6A)shows the formation of a complex with stoichiometry 2:1(Gos-iron). The stoichiometry of such Gos-iron complexes was determined by Job's method that shows a breaking point at a molar ratio of 0.5.

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Further evidence on the correlation between iron and Gos was obtained by IR spectroscopy (Figure 6B).For the free Gos and its iron complex, the broadband located in the range of 3000-4000 cm-was considered as the stretching of hydrogen-bonded hydroxyl groups due to the presence of water molecules. The v(C=O)stretching mode of the free Gos occurs at 1654 cm-I, which has been shifted towards 1636 cm-Iupon complex formation. This result suggests that Fe(Ⅱ) is correlated to carbonyl oxygen [31]. Moreover, the presence of the v(Fe-O) stretching vibration at 630 cm-I corroborates the formation of the iron-Gos complex, since free Gos does not exhibit such a band.

Figure 6.(A)Effects of Fe(II) on the Gos UV-VIS spectrum (200-600 nm). Incubation mixture containing 125 mM sucrose, 65 mM KCl, 10 mM HEPES buffer(pH 7.2), 2 mM citrate, and Gos 1.6 μM.The concentrations of Fe（I） from top to bottom traces were 0， 0.16， 0.32， 0.48， 0.64，0.8，0.96， and 1.12 μM.Inset∶ Job's plot for the Gos-Fe（II） complex at a constant total concentration 【Fe（D）】+【Gos】=1.6 μM. The black dot indicates the isosbestic point. The downward and upward arrows indicate a decrease and increase in absorbance values at 380 and 500 nm, respectively. Experiments were conducted at 28°C. The scan speed was 2nm/s.A baseline was established with the incubation mixture plus 1.6 μM Fe（I）.（B）Infrared spectra of Gos and Gos-Fe（I）. Typical examples are shown.

2.6. Gos Prevented Fe(III) Reduction by Ascorbate

The ferric state of iron promoted by Gos represents a plausible antioxidant mechanism since it hinders the catalytic activity of Fe(II). Nevertheless, Fe(III) could still be reduced again to its ferrous form by natural reducing agents like ascorbate. The latter could reload biological systems with Fe(II), which participates in Fenton-Haber-Weiss reactions, generating the extremely reactive·OH radical. To examine these possibilities, we used 1，10-phenanthroline to measure the levels of Fe（II） formation from a Gos solution （100 μM）treated with 2 mM ascorbate and different Fe（II） concentrations （10-100 μM）. Figure 7 shows that the absence of Gos allowed a maximal reduction rate of Fe(II)to Fe(II) (black line with a slope of 5.85×10-3); however this process was slowed down by the presence of Gos (1-min incubation, green line), and after 5 min of incubation with Gos, the reduction rate of Fe(I)by ascorbate decreased almost 3 times (red line with a slope of 2.04× 10-3). This result shows the capacity of Gos to inhibit the ascorbate-mediated Fe() reduction to Fe(I)

Figure 7. Gos inhibits Fe(ⅢI)-reduction by ascorbate in the absence of rat liver mitochondria. Experimental conditions: 125 mM sucrose,65 mM KCl,10 mM HEPES buffer (pH7.2),1 mM citrate, Gos 100μM. Experiments were conducted at 28°C. Ascorbate（4 mM） and 5 mM 1，10-phenanthroline were added after 1 min or 5 min of Gos-Fe(I) incubation. Lines are representative of three assays.

2.7. Gos Protects against 2-Deoxyribose Oxidative Degradation

To document the ability of Gos to act preferentially on iron instead of free radicals, competition studies were performed to evaluate the effectiveness of Gos and two·OH scavengers (DMSO and salicylate) in protecting 2.8 or 28 mM 2-deoxyribose from iron-mediated oxidative damage(Figure 8).The·OH scavengers at 20 mM protected 28 mM 2-deoxyribose significantly less than 2.8 mM2-deoxyribose (p<0.05), as expected. Gos was equally effective in preventing oxidative degradation of both 2.8 and 28 mM2-deoxyribose.

Figure 8.Effect of Gos and·OH scavengers dimethyl sulfoxide(DMSO) and salicylate on oxidative damage to 2.8 or 28 mM 2-deoxyribose induced by Fe(II)-EDTA plus ascorbate. Solutions were incubated for 30 min at 37°C and contained 10 mM phosphate buffer (pH7.2),2-deoxyribose (2.8or 28 mM）， 150 μM EDTA， and 50 μM Fe（ⅢI）. Reactions were started by the addition of ascorbate to a final concentration of 2 mM.The bars show means±S.D. (n=3). Controls contain only DMSO (0.001%), which is the solvent concentration in Gos samples. The one-tailed t-test was used for*p <0.05, n.s., non-significant.

3. Discussion

The iron released under several neurodegenerative conditions, including ischemic and hemorrhagic stroke, provokes the deregulation of brain iron homeostasis, leading to the pathophysiology of neurological injury [4,32-34]. At subcellular levels, mitochondrial impairment seems to be involved in iron-mediated neuronal death [10,15,35-38]. Preclinical evidence supports the advantage of using iron chelators, mainly deferoxamine mesylate, against neurodegeneration including all types of stroke [7,16]. However, their high cost and unavailability, and the wide range of adverse effects of classical iron chelators, have led to the use of natural chelators for iron management in dyshomeostasis [39,40].

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The protective action of Gos is shown on HT-22 cells and rat-brain mitochondria incubated with an iron/citrate mixture, where we found strong protection against iron-induced oxidative damage. Mouse hippocampal HT-22 cells exposed to iron overload(24 h)showed decreased viability, which was closely associated with early mitochondrial membrane potential dissipation and ATP reduction (Figure 2A-C, respectively). This suggests that mitochondrial impairment contributes to neuronal lethality. Indeed, under our conditions, at least a portion of Fe(II) uploaded to neurons is expected to reach the organelles, since iron-induced neuronal death was preceded by the loss of mitochondrial function. Hence,it has been shown that iron overload induces mouse hippocampal HT-22 cell death and mitochondrial fragmentation [41,42]. Likewise, sustained iron exposure increases mitochondrial ROS levels in dopaminergic neuroblastoma SHSY5Y cells [43]. Furthermore, an extensive iron influx into the hippocampal neurons, as well as mitochondrial damage, were verified after the exposure to 100 uM ferrous iron [36]. Here we observed that the Gos-induced preservation of the viability of the hippocampal neurons affected by iron is closely related to the conservation of their mitochondrial membrane potential and ATP levels.

As in intact cells, the direct mitochondrial exposure to ferrous iron provoked the extensive swelling of the organelle, dissipation of membrane potential, and the loss of ATP(Figure 3A-C, respectively).In this experimental setting, Gos was able to protect the iron-overloaded mitochondria, which was expressed by the preservation of the above-mentioned parameters. In this sense, it has been observed that mitochondria loaded with a micromolar concentration of iron underwent mitochondrial permeability transition pore opening and △Y dissipation in a manner that is sensitive to iron chelation but not dependent on catalase antioxidant action |44]. Interestingly, the Mito-Tempo antioxidant was reported to protect against iron overload damage in hippocampal neurons by scavenging the mitochondrial superoxide anion radical and preserving mitochondrial morphological integrity and membrane potential [36]. We recently described the antioxidant effects of this flavonoid and its ability to protect PC12 cells against chemical hypoxia-induced death [29], where it was concluded that the free radical scavenging and antioxidant ability of Gos is partly involved in the protection against iron-mediated HT-22 and mitochondrial damage. However, the improved efficacy of Gos against iron-mediated lipoperoxidation versus tertbutyl hydroperoxide-mediated lipoperoxidation (Figure 4A,B, respectively)strongly suggests that its iron-interacting capacity is the main mechanism against iron-induced damage.

To characterize the Gos-Fe interaction further, several cell-free and mitochondria-free experiments were performed. We observed that when polyphenol was co-incubated with Fe (II), the concentration of the metal ions decline, while there was a corresponding increase in oxygen consumption rate (Figure 5A-C). These effects suggest that Gos removes Fe(II)from the citrate complex, and oxidizes it to a ferric form in a process that requires O2 as an electron acceptor. Consequently, Gos promotes the decline of Fe(II) concentration that may hinder the hydroxyl radicals through Fenton reactions. Since the Gos-Fe(II)complex enables the oxidation of relevant reducing agents such as ascorbate, resulting in the formation/regeneration of Fe(II), we were able to demonstrate that Gos inhibits the ascorbate-mediated reduction of Fe(II) to Fe(II).

The hypothesis that Gos strongly interacts with iron was also here confirmed through spectroscopic techniques, and corroborated previous results where different arrays of

Gos-iron complex stoichiometry was characterized by electron spin ionization mass spectroscopy [45].

These results evidenced the protective effects of Gos against iron-mediated neuronal damage, probably by interacting with ferrous ions, hindering its involvement in the catalytic reactive oxygen species formation. Furthermore, this suggests the formation of a transient charge-transfer complex between Fe(Ⅱ) and Gos, accelerating Fe(II) oxidation and the formation of a more stable Fe(III)-Gos complex that cannot participate in the propagation phase of lipid peroxidation. Moreover, a biologically relevant reducing agent such as ascorbate was unable to reduce ferric iron in the presence of Gos, limiting a pro-oxidant characteristic of certain flavonoids involved in ferrous ion re-cycling. [30]. Gos at micromolar concentrations was more effective than classical scavengers in pre-venting iron-mediated oxidation of 2-deoxyribose. This high efficacy may be ascribed to the formation of a redox-active Gos-Fe(II)/() as we previously observed for others polyphenols that improved their performance as antioxidants upon their interaction with iron in different in vitro paradigms of oxidative damage [22,46-48].

It has been established that chelating agents that contain oxygen as a ligand (oxo ligand, O2-)can chelate iron and promote the oxidation of Fe(I), stabilization of Fe(II), and consequently produce a decrease in its reducing potential49]. At physiological pH, catechols readily form thermodynamically stable bis complexes with ferric iron, favored by low concentrations of the ligands. The presence of a catechol moiety in the Gos structure suggests a similar interaction mechanism with iron, which could explain the protection achieved against iron-induced damage of neuronal cells and mitochondria. In this regard, we also previously demonstrated that mangiferin and guttiferone A stimulate ferrous iron oxidation and hinder ferric iron reduction [23,24,26-28].

The ability of Gos to chelate iron may also prompt signaling pathways that contribute to neuroprotection. For example, prolyl hydroxylase domain enzymes (PHD), the classical Hypoxia Inducible Factor-lalpha(HIF-1o) hydroxylation-modifying enzymes under normoxia, have been identified as the critical targets of iron chelators that are clinically beneficial in many neurological disorders [50,51].It is understandable since PHD depends on divalent iron as a coupling factor [52]. Several genes that have been involved in neuroprotection are regulated by HIF-la, such as eNOS, VEGF, and EPO[53]. Moreover, HIF activation resulting from PHD inhibition could prevent oxidative stress-mediated mitochondrial impairment and apoptosis, independently of its role as a transcription factor [54].

Ferroptosis, the recently characterized iron-dependent regulated cell death, has been proposed as the mechanism through which the neurons exposed to hemorrhagic damage die [8]. Mitochondrial dysfunction was recently related to ferroptosis cell death [55]. Therefore, the inhibition of ferroptosis, which saves mitochondrial function from iron damage, could also be a plausible mechanism for neuroprotection against iron-mediated neurological disorders protected by Gos, which would deserve further attention. Additional research on the presumed beneficial effects of Gos on in vivo animal models of iron-associated neurodegeneration must be made, in order to propose this flavonoid as a therapeutic intervention against brain tissue damage induced by iron homeostasis deregulation.