Quercetin Attenuates Neurotoxicity Induced By Iron Oxide Nanoparticles Part 1

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Background

The term nanomaterial refers to material in the nanoscale(1-100 nm) with one, two, or three external dimensions, whereas the term nanoparticle (NP)refers to materials with all three external dimensions in the nanoscale [1]. The most important properties of NPs are the high surface to mass ratio, catalytic activity, electrical and thermal conductivity, high solubility, and mobility in the body tissues [2]. There are two general categories of biomedicine NPs. (I) Organic NPs are mainly composed of organic molecules. Liposomes, emulsions, dendrimers, and other polymers form a large group of organic NPs.(II)Inorganic NPs that consist of a metal core such as iron, nickel, cobalt, gold, silica, and iron oxides with electrical, magnetic, optical, and fluorescent properties [3]. Iron oxide nanoparticles(IONPs) are a class of magnetic nanoparticles(MNPs) that have gained prominence in technological advancements [4,5].IONPs are usually made) core and or magnetite(FeO) or maghemite (y-Fe, O3) a protective coating such as chitosan, dextran, polyethylene glycol(PEG), and polyvinyl alcohol(PVA)[6-8]. IONPs have unique properties that make them suitable biomaterials for medical applications. For instance, their Ferro-or ferromagnetic behavior enables drug trafficking and drug guidance to the target tissue. They can be localized in specific tissue under an external magnetic field so that they are called magnetic targeted carriers(MTC)[9]. Moreover, the application of IONPs in magnetic resonance imaging (MRI)is a powerful tool for creating high contrast medical images and enhances the potential of disease diagnosis [6,10]. 

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Besides, IONPs can make cancer cells more susceptible to radiation and chemotherapy by rising tumor temperature (hyperthermia). Furthermore, the ability of IONPs to cross the blood-brain barrier(BBB)is a privileged property for transporting drugs to the brain in neurological disorders [6-8]. At present, there are numerous FDA-approved SPION compounds(including, ferumoxide(Feridex I.V.), ferumoxytol (Lumirem), and ferumoxytol(Feraheme)), for use in the clinic and others undergoing clinical trials, as well as numbers of applicable IONPs, are ongoing. Despite the advantages mentioned above, in vitro, and in vivo studies provided evidence concerning the possible neurotoxicity of IONPs due to free iron accumulation, ROS production, and protein aggregation [11-14]. However, modifying the physicochemical properties of NPS such as concentration, size, and surface coating can optimize their function and cytotoxicity properties [14]. Besides, the simultaneous use of natural antioxidants such as quercetin(QC) supplementation can be a useful path to remove brain oxidative damages due to IONPs[15]. QC(3,3',4',5,7-pentahydroxy flavone)belongs to the flavonoid class and flavonol subclass with the chemical formula of C15H10O7[16]. QC is a major component in many fruits, seeds, vegetables, and nuts. The beneficial effects of QC have been investigated in many disorders such as cancer and neurodegenerative diseases [17, 18]. QC can postpone or prevent neurodegenerative disease through multiple molecular pathways [16, 19]. QC modulates oxidative stress status via binding to the reactive oxygen and/or nitrogen species (ROS/RNS) and via its effect on the expression and activity of enzymatic/non-enzymatic antioxidants[15, 20]. QC also prevents the Fenton reaction by creating stable iron-QC complexes, thereby indirectly removing ROS/RNS [18]. Moreover, in iron overload conditions QC, can regulate iron homeostasis [21]. QC cannot cross the BBB well due to its low solubility, instability, and low bioavailability[20]. The use of IONPs is an effective solution to overcome these limitations [22]. Therefore, combining QC with IONPs is a mutually beneficial solution to neutralize iron toxicity and increase QC bioavailability. The beneficial effects of QC against IONPs-induced neurotoxicity are poorly defined. In this review, we provide evidence that QC can act against iron overload-induced toxicity. This iron overload can be caused by IONPs metabolism or other sources. However, QC likely has equal activities for neutralizing excess iron arising from various sources.

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Iron metabolism and homeostasis

Iron in our body is an essential mineral for many fundamental processes such as oxygen transport and mitochondrial function. Iron, also as a co-factor, participates in enzymatic reactions including, DNA replication, RNA transcription, protein translation, and myelin synthesis [23,24]. The human body contains about 3-4 g of iron which may be lost up to 0.1% daily under physiological and pathological conditions that are usually compensated with daily dietary consumption [25]. Both iron deficiency and iron overload can affect the development and function of the brain from fetal to adulthood [26-28]. There are two forms of iron in daily diet: heme iron with absorbable ferrous ion (Fe2)that exists in red meat and seafood, and non-heme iron with ferric ion(Fe3+)that exists in plant-based foods [29,30]. Iron absorption can be controlled through body iron levels and multiple iron regulatory agents [27]. Duodenal cytochrome B(Dcytb)is an ascorbate-dependant plasma transmembrane ferrireductase that shifts Fe3+ to Fe2+ on the apical membranes of intestinal absorptive cells, enterocytes [31]. Iron enters the cell through metal transporters [32]. Divalent metal transporter 1(DMT1) and heme carrier protein 1 (HCP1)are the main non-heme and heme iron transporters, respectively. They can transfer Fe2+ and heme from the gut lumen into the enterocytes [29,30,32]. HCP1 is preferably the high-affinity obligatory folate transporter [33]. In the next step, Fe2+ arising from non-heme and heme iron degraded by heme oxygenase-1(HO-1)enter the labile iron pool (LIP), a transient intracellular iron pool[23]. The majority of this Fe2+ is released from the cell by iron exporter ferroportin in the basolateral membrane of enterocytes [34]. Its surplus is transferred to a cytosolic iron-storage protein called ferritin. Intestinal ferritin is an effective factor in iron absorption due to the ferroxidase activity of its H subunit that re-oxidizes Fe2+to Fe3+[23,35,36].On other hand, the iron released from the enterocytes is re-oxidized to Fe3+by ferroxidases(ie., membrane-bound multicopper hephaestin and soluble and/membrane-bound multicopper ceruloplasmin), which are involved in the iron export by ferroportin [37, 38]. Iron oxidation is essential for iron transfer by plasma iron-free transferrin, so-called apo-transferrin(Apo-Tf). Trapping and retaining Fe3+ by iron-storage proteins such as ferritin and transferrin suppresses Fe3+ reactivity and free radical generation[39]. Apo-Tf binds to two ferric ions at normal alkaline pH(7.4) of the plasma to form holo-transferrin (Holo-Tf). This iron-loaded glycoprotein as a plasma iron pool delivers iron to the target tissues such as bone marrow, liver, and brain [25,40, 41](Fig.1). Hepatocytes and macrophages are responsible for iron storage and iron recycling, respectively [42]. Under physiological conditions, approximately the whole

of the extracellular iron enters the target cell in the form of transferrin-bound. However, transferrin saturation due to iron overload prevents iron binding to transferrin and leads to non-transferrin bound iron (NTBI) uptake [43]. Holo-Tf binds to transferrin receptor(TfR) on the surface of most cells[44]. The Holo-Tf-TfR complex is internalized to the cell via clathrin-coated vesicles along with adaptor protein 2(AP2)in the endocytosis cycle termed clathrin-mediated endocytosis (CME)[45,46]. The endocytic vesicles lose their clathrin coating and subsequently merged into the endosome membrane[45, 47]. Fe+ in the acidic pH(5.5-6.0) of late endosome is released from a transferrin-TR complex while, transferrin remained bound to TfR and reconverted to Apo-Tf. Besides,endo-somal ferrireductase such as6-transmembrane epithelial antigen of the prostate (Step)reduces insoluble Fe3+to soluble Fe2+ to that is transported from the endosomal lumen into the cytosol by DMT1.Apo-Tf bound to TfR is recycled to the cell surface and dissociates from the receptor at a pH of 7.4 [38,47-50]. Here, TfR is ready to bound the next Holo-Tf and initiate recycling [51]. Cytosolic iron confronts several paths:(I) participation in biological functions by embedding within metalloproteins, (I) participation in mitochondrial energy transduction,(II) storage in the form of ferritin [48,52]. Besides, lysosomal degradation of ferritin leads to the formation of an iron-storage complex, namely, haemosiderin, that is related to pathophysiological states (e.g., iron overload)and involved in a reactive free radical generation [30,48].

Iron homeostasis is maintained by multiple factors such as hepcidin hormone and iron-regulatory proteins (IRP1 and IRP2)/iron-responsive element (IRE)signaling pathway [42]. Hepcidin, which is produced by the liver, is an essential systematic regulator. When iron is abundant, hepcidin binds to enterocyte ferroportin and blocks the export of iron out of the cell [35,42]. At the cellular level, the IRP/IRE signaling pathway regulates iron homeostasis depending on the body's iron concentrations. In iron deficiency, IRP binds to the IRE motif at the 5'-untranslated region (5'UTR)of ferroportin and ferritin transcripts to suppress translation of their mRNAs. Whereas, the binding of IRPs to the IRE motif at the 3′-UTR off and DMT1 transcripts stabilizes their mRNAs to enhance translation. These processes lead to decreased plasma iron and increased cellular iron for use in metabolic processes [50]. On the contrary, when the iron is abundant, IRP cannot bind to the IRE motif at 5'UTR of both ferroportin and ferritin transcripts and enhances translation of their mRNAs as well as IRP cannot bind to the IRE motif at 3'-UTR of TfR and DMT1 transcripts and destabilizes mRNAs to suppress translation [53].

Iron in the brain

Due to the significant relevance between neurodegenerative diseases and abnormal iron metabolism, an accurate description of the fate of iron in the CNS is necessary [48]. Iron in CNS plays an essential role in many normal neural functions, including cell division, energy production, axons myelination, dendritic branching, and neurotransmitters synthesis such as dopamine and serotonin [24,53-55]. Iron is a co-factor for tyrosine hydroxylase that is involved in dopamine synthesis and tryptophan hydroxylase that is involved in serotonin syn-thesis [54]. Dopamine is a type of catecholamine in the brain that can be released to certain areas of the hippocampus, probably the CAl region, and enhances long-term potentiation (LTP)[56]. Iron deficiency is associated with decreased myelin synthesis, which is formed by myelinating glial cells i.e., oligodendrocytes, followed by consequences such as memory impairment [57]. Iron is transported to brain cells by the blood-brain barrier(BBB)and the blood-CSF barrier(BCB). Most of the iron enters into brain interstitial fluid (ISF)by crossing BBB, and some iron enters into the cerebrospinal fluid (CSF)by crossing BCB within the choroid plexus [58]. The Holo-Tf-TfR pathway is one of the well-known routes of iron towards the brain [59]. Like other cell types mentioned above, circulating Holo-Tf binds to TfR on the membrane of the capillary endothelial cells of BBB and choroid plexus epithelial cells of BCB. This binding resulted in cell membrane budding along with the Holo-Tf-TfR complex through the CME process. The reduced form of iron can export from the brain capillary by ferroportin toward ISF and CSF after dissociation from it. After re-oxidizing of Fe2+ to Fe3 mediated by ferroxidases, Fe3+ binds to transferrin and uptakes by neural cells(e.g., oligo-dendrocytes, astrocytes, microglia, and neurons)via the receptor-mediated endocytosis [23,24,58,60-62]. However, some iron may uptake in the form of NTBI, likely by DMT1 [59] after reduction of Fe3+ to Fe2+ by ferri-reductase [63](Fig.2). Iron uptake by neurons includes transferrin-bound iron and NTBI. Upregulation of TfR on neurons in the iron deficiency, suggesting extensive transferrin-bound iron uptake through this receptor [64]. Neurons and other cell types likely acquire NTBI through DMT1. However, the mechanism of NTBIuptake has not been precisely clarified [65]. Iron exporter in neurons is the same as ferroportin that is expressed all over the cell membrane. Ferritin as an iron-storage protein has also been found in some neurons [64](e.g., dopaminergic neurons)[66]. Iron is also present in the synaptic space of neurons, which is released from the axon terminus [24]. There are several mechanisms for iron recycling to the systemic circulation. For example, Holo-Tf binding to TfR on the abluminal membrane of BBB, and arachnoid granulations-mediated transportation has been proposed as a mechanism to export iron from the brain into circulation [67]. Excess iron caused by pathological or senescence conditions also back to the systemic circulation. Moos et al., by injection of transferrin radiolabeled with 5Fe and 12I into the lateral ventricles, proposed a major route of iron reabsorption into the blood plasma which is triggered from subarachnoid and transporting through BCB [68]. Furthermore, the clearance of cerebral apoptotic/necrotic cells under inflammatory conditions via phagocytosis contributes to the efflux of iron into blood plasma from the brain by phagocytes [64]. However, the exact mechanism of iron export back to the systemic circulation is unclear and requires more studies.

Iron overload-induced neurotoxicity

Iron is a chemical element belonging to transition metals with electron donor and acceptor activity [69]. Despite iron being a crucial component in neuro functioning, its excess can lead to protein aggregation and oxidative stress. Its most destructive effect is neuronal cell death [14, 70]. Therefore, accurate regulation of iron homeostasis is required [69]. Iron accumulation mainly happens in normal aging but several age-dependent/independent factors are associated with its progression including smoking, high body mass index(BMI)[70], hereditary iron overload disorders (e.g. hemochromatosis)[71], transfusion-induced iron overload in types of anemia [72], and neurodegenerative diseases [73]. Besides, the usage of IONPs in the diagnosis and treatment of diseases (e.g. neurodegenerative disease) can result in iron accu-mulation[14, 15]. Excess iron is a critical player in reactions that damages tissue by overproducing ROS/RNS, which is briefly called RONS [74, 75]. This condition leads to an imbalance between antioxidants and prooxidants, which is referred to as nitrosative and/or oxidative stress [74]. Despite the relationship between iron overload and nitrosative stress, it is not sufficiently described. Therefore, in this study, we focus on the relationship between iron overload and oxidative stress. The brain is a sensitive organ to ROS due to continuous consumption of oxygen and iron, having a high percentage of polyunsaturated fatty acids (PUFAs)with high vulnerability to oxidation, and a weaker antioxidant defense in comparison with other tissues [76]. Under physiological conditions, ROS are produced as a result of cellular metabolisms. Oxygen(O)reduction via Fe2+ produces Fe3 and superoxide anion (O,。)that is a precursor of other reactive species(2Fe2+ +2O,→2Fe++2O,). Superoxide dismutase (SOD)enzyme converts the O. to hydrogen peroxide(H2O)and O2(2O2…- +2H+ →H, O2+O2). H, O2is converted to water (H2O) via antioxidant enzymes such as glutathione peroxidase(GPX) and catalase (CAT)(2H, O,→2H, O+O). These reactions are carefully controlled and considered as a part of the cell signaling system [77, 78]. Nevertheless, H, O2 enters the destructive Fenton reaction in the presence of redox-active bio metals such as free iron. During the Fenton reaction, Fe2+ as an electron donor provides

electrons for H,O, reduction thereby Fe3, hydroxide(HO-), and highly noxious hydroxyl radical (OH')are produced (HO,+Fe2+ →Fe3+ +OH- +OH'). reduction via O。- in the iron-On other hand, Fe3+ r sulfur proteins, renews Fe2+ for Fenton reaction (Fe3+ +O。- → Fe2+ +O,)[77,78]. Accordingly, the reaction referred to the Haber-Weiss reaction which has required iron ions(O·- +H, O,→OH"+O2+OH-)[23].Iron overload and ROS mutually reinforce each other and damage nucleic acids, lipids, proteins, and cellular compartments such as mitochondria [24]. ROS resulting from the Fenton reaction can lead to the oxidation of DNA bases. These lesions are repaired via a predominant mechanism of DNA repair called base excision repair(BER). But in the iron overload conditions, iron directly binds to two BER enzymes including nei like DNA glycosylase (NEIL1) and NEIL2 thereby inhibiting their enzymatic activity [79]. Lipid peroxidation takes place under oxidative stress as well as the presence of iron. During lipid peroxidation, ROS directly reacts with membrane PUFAs to produce toxic aldehydes such as 4-Hydroxynonenal (4-HNE) and Malondialdehyde(MDA). Iron is an accelerator for this process. Furthermore, ROS by attacking membrane proteins leads to alteration in architecture, permeability, rigidity, and integrity of the membrane [76]. Lipid peroxidation products can produce misfolded proteins via carbonylation. The ubiquitin-proteasome system cannot degrade misfolded proteins thereby protein aggregation and neurodegeneration can occur [14]. The mitochondrial membrane is prone to damage due to a high level of PUFAs [80]. Excess iron-induced ROS increases mitochondrial membrane permeability, which releases iron from this organelle. Furthermore, excess iron impacts the cooperation of iron and calcium thereby downstream signaling pathways related to cognitive functions such as synaptic plasticity, mitochondrial function, and axon growth can be destroyed. 

Excess iron not only leads to mitochondrial dysfunction but also causes the release of calcium and cytochrome C from this organelle toward the cytosol and eventually cell death [14,81]. Dopamine-induced neurotoxicity has also been reported as another mechanism of iron-dependent neurodegeneration. In this regard, metabolites resulting from excessive oxidation of dopamine (e.g., reactive quinones) cause neuronal death. This process is accelerated by excess iron and oxidative stress [82]. In physiological conditions, neurons remove oxidation products by several mechanisms. For example, glutathione (GSH) is a powerful antioxidant that balances intracellular oxidants levels by binding to oxidation products and removing them from neurons [76, 82]. However, in pathological conditions, iron overload decreases the level of GSH that leads to TfR overexpression and re-induction of oxidative stress.A high level of TfR leads to more iron influx into the cell that exacerbates iron overload and oxidative stress[14]. Therefore, iron overload accompanied by primary oxidation products such as OH, secondary oxidation products such as toxic aldehydes, and protein aggregation can induce neuronal cell death [76]. Ferroptosis is an iron-dependent cell death associated with degenerative and non-degenerative diseases such as Alzheimer's disease(AD), Parkinson's dis-ease(PD), and stroke [81]. Ferroptosis is different from types of programmed and non-programmed cell death.It is the ultimate consequence of oxidative stress and lipid peroxidation(Fig.3). During ferroptosis decrease in GSH level and GPX activity leads to lipid peroxidation in presence of Fe2[83]. Ferroptosis is prevented by antioxidants that are involved in iron chelation and anti-lipid peroxidation activity [81]. High concentrations of iron have been observed in various areas of the brain including the cerebral cortex, hippocampus, cerebellum, amygdala, and basal ganglia, in the healthy elderly, which these areas are most likely involved in neurodegenerative diseases. Iron concentration in the brains of patients with neurodegeneration is notably higher than in healthy aging [24].Iron overload in aging can be caused by several pathological pathways including inflammatory conditions, increasing BBB permeability, and disturbance in iron homeostasis. Besides, iron overload in neuroglia and neurons aggravates neuroinflammation and leads to neuronal apoptosis [24]. There is a meaningful correlation between iron accumulation, normal brain aging, and neurological diseases such as AD[84], PD[85], and stroke [86](Fig.4).

This article is extracted from Bardestani et al. J Nanobiotechnol (2021) 19:327 https://doi.org/10.1186/s12951-021-01059-0