In view of a wide spectrum of the potential biomedical applications of MnO_xNPs, understanding its adverse health effects is crucial for further clinical applications [7]. Several studies showed that MnO_xNPs possess cytotoxic effects in various types of cells which may be mediated by ROS overproduction. Specifically, in vitro data showed that Mn₃O₄NPs induce an increase in intracellular ROS production [37]. Mn₂O₃NPs also possess a high capacity for cytochrome c oxidation, exceeding that observed for other nanoparticles, including CeO₂, TiO₂, BaSO₄, carbon black, Cu, Ag, and ZnO NPs. This oxidative potential was mediated by reduced glutathione (GSH) and ascorbic acid depletion [38]. Prooxidant effect of Mn₂O₃NPs characterized by hydroxyl radical generation was also revealed in an assay of L-3,4-dihydroxyphenylalanine (L-dopa) autoxidation [39]. It has also been shown that ability of MnO₂NPs to generate ROS through Fenton-like mechanism and oxidize cytochrome c is not associated with Mn²⁺ dissolution, which was insignificant [40]. Finally, prooxidant activity of Mn-containing welding fume nanoparticles from stainless steel directly correlated with Mn²⁺ release from the particles [41]. Congruently, commercially available MnO_xNPs of various shapes (spherical, rods, flakes) mainly sized around 30 nm induced a significant decrease in viability of A549, HepG2, and J774A.1 cells due to ROS overproduction and caspase 3 activation [29].

Significant cytotoxic effects of MnO_xNPs were revealed in cultures of airway epithelial cells. It has been demonstrated that Mn₂O₃NPs exposure decreases viability of both BEAS-2B and A549 cells through induction of apoptosis [42]. In BEAS-2B cells and RAW 264.7 macrophages, exposure to Mn₂O₃ resulted in a dose-dependent decline in cell viability, energy production, and membrane leakage. Further analysis also showed that along with membrane damage Mn₂O₃ treatment induced mitochondrial dysfunction with mitoROS production [43]. Although Mn₃O₄NPs did not possess cytotoxicity upon short-term exposure (24 h), long-term exposure (9 days) was associated with significant cytotoxicity in A549 and especially Caco-2 cells [44]. It has also been demonstrated that exposure to Mn₃O₄NPs induces ROS overproduction in airway epithelial A549 cells and colorectal adenocarcinoma HT29 cells, although this prooxidant effect was associated with reduced cell viability only in A549 cells. Furthermore, exposure to a novel nanomaterial, GNA35, based on Mn₃O₄NPs with enhanced electrochemical properties, induced a more severe prooxidant effect and decrease in A549 cells viability [45]. It has been also demonstrated that Mn₂O₃NPs exposure significantly reduces A549 viability through induction of apoptosis and slightly inhibits cell proliferation [46]. Another study in a culture of A549 cells demonstrated that exposure to Mn oxide nanoparticles increases ROS production, up-regulates transferrin, ferritin light and heavy chains, and DMT1 mRNA expression, as well as increases IL-6 and IL-8 mRNA and protein expression, although some of these effects were more profound upon exposure to Fe oxide NPs or mixed type NPs [47]. Finally, it has been demosntrated that IC₅₀ of Mn₃O₄NPs accounts for 98 µg/mL [48]. In another lung cell line (FE-1) MnO₂NPs dose-dependently induced DNA damage, exceeding that observed in MnO₂MPs-exposed cells [49]. Comparative analysis of Mn₃O₄NPs and MnSO₄ showed that, despite both forms of Mn induce caspase-3-mediated apoptosis in CCL-149 alveolar epithelial cells, only Mn₃O₄NPs promoted ROS production and GSH oxidation. Noteworthy, exposure to MnNPs resulted in higher intracellular Mn content compared to MnSO₄ [50]. Another study also showed that Mn₃O₄NPs increase ROS production in CCL-149 airway epithelial cells, although not reducing the metabolic activity of cells [51]. Congruently, Mn₂O₃NPs induced a significant decline in human bronchial epithelial NCI-292 cell viability with up-regulation of IL-8 mRNA and protein expression. At the same time, development of oxidative stress upon exposure to Mn₂O₃NPs induced up-regulation of SOD2 and HO1 mRNA expression in NCI-292 cells [38]. In another line of human bronchial epithelial cells (16HBE14o-), exposure to Mn₂O₃ resulted in a dose dependent decrease in cell metabolic activity assayed by MTT test with IC₅₀ of 33 mg/L [52].

Adverse effects of MnO_xNPs exposure were also revealed in alveolar macrophages. Specifically, exposure to Mn₃O₄NPs induced a significant increase in ROS production in CRL-2192 alveolar macrophages, resulting in a significant reduction in cellular metabolic activity and down-regulation of MCP-1 production. Increased caspase-3 activity in Mn₃O₄NPs-exposed cells is indicative of apoptosis. Attenuation of these effects by Trolox treatment showed that cytotoxicity of Mn₃O₄NPs in alveolar macrophages is ROS-dependent [51]. Correspondingly, MnO₂ NPs reduced the viability of RAW264.7 macrophages, also decreasing their phagocytic activity due to the enhancement of TNF$\alpha$ and down-regulation of IL-1$\beta$ mRNA expression. These effects were reversed by doping MnO₂NPs with ZnO, which also resulted in a more profound increase in ROS production and promoted antioxidant response through up-regulation of Nrf2 protein expression [53].

Certain studies showed that MnO_xNPs are cytotoxic to intestinal epithelial cells in vitro. It has been also demosntrated that Caco-2 cells (IC50 = 66.7 µg/mL) were more sensitive than lung epithelial A549 cells (IC50 = 136.2 µg/mL) and mouse Balb/c 3T3 fibroblasts (IC50 = 195.0 µg/mL) to Mn₃O₄NPs-induced cytotoxicity. The latter was shown to be dependent on ROS generation, but not metal ion release [54]. In a culture of Caco-2 cells exposure to various MnOxNPs resulted in a dose-dependent reduction in cell viability with cytotoxicity decreasing in the following order: Mn₃O₄ $>$ Mn₃O₂ $>$ MnO₂. However, in presence of H₂O₂, non-cytotoxic doses of MnO_xNPs increased cell viability, being indicative of both prooxidant and antioxidant effects of MnO_xNPs. Specifically, MnOxNPs possessed ascorbate (Mn₃O₂ $>$ MnO₂ $>$ Mn₃O₄) and GSH-oxidizing (Mn₃O₄ $>$ Mn₃O₂ $>$ MnO₂) activities, while promoting H₂O₂ decomposition (MnO₂ $>$ Mn₂O₃ $>$ Mn₃O₄) [55]. These findings are generally in agreement with earlier indications of the protective effects of Mn₃O₄NPs against H₂O₂-induced cytotoxicity in murine insulinoma $\beta$TC3 cells by mimicking antioxidant enzyme activity, although at high doses the particles induced a significant decrease in cell viability [56].

Cytotoxic effects of MnO_xNPs were also evident in other epithelial cell lines. Exposure of epithelial MCF-7 and HT1080 cells to MnO₂NPs resulted in increased total ROS and mitoROS production, inhibition of SOD, CAT, and glutathione reductase (GR) activity, and depletion of GSH pool. Along with induction of oxidative stress, activation of proapoptotic signaling by up-regulation of p53 and Bax mRNA expression, with repression of Bcl-2 mRNA, and reduction of mitochondrial membrane potential result in a decrease in cell viability. However, the results show that the effects of MnO₂NPs were more profound in HT1080 cells compared to MCF-7 cells [57]. In human cervical carcinoma cells (HeLa) and mouse fibroblast cells (L929), exposure to silica-coated MnONPs induced a significant increase in ROS production, mitochondrial dysfunction, and G2/M phase arrest. Along with the up-regulation of p53 protein expression and the reduction of Bcl-2/Bax ratio, these effects were associated with caspase-3 activation, leading to cell apoptosis [58].

Finally, Mn₃O₄NPs exposure significantly reduced viability of mouse embryonic stem (mES) cells with up-regulation of gene products associated with oxidative stress (Srxn1), DNA damage (Rtkn), endoplasmic reticulum stress (Ddit3) and p53-related stress (Btg2), although this effect was less pronounced than in the case of metallic MnNPs [59].

However, it is demosntrated that synthesis of NPs also significantly affects particle toxicity. In hMSC cells chemically synthesized MnO₂NPs induced significant alterations in cell volume and shape, while green-synthesized NPs did not possess adverse effects on cell morphology, thus showing its lower cytotoxicity [60].

Generally, these findings show that MnO_xNPs possess cytotoxicity in airway epithelial cells, alveolar macrophages, enterocytes, and other epithelial cells (Table 1, Ref. [38, 42, 43, 44, 45, 46, 48, 49, 50, 51, 52, 53, 54, 55, 57, 58, 59]). Evidence shows that the cytotoxicity of MnO_xNPs is mainly mediated by their prooxidant activity. Specifically, overproduction of ROS upon MnO_xNPs exposure, along with induction of mitochondrial dysfunction, results induces cell cycle arrest and apoptosis, resulting in reduced cell viability. In addition, MnO_xNPs-induced oxidative stress is associated with up-regulation of proinflammatory cytokine expression, which also contributes to cytotoxicity.

Agreeing with cellular models, in vitro studies also show potential toxicity of MnO_xNPs exposure for various organs. Specifically, exposure to MnNPs via intraperitoneal injection in rats induced histopathological damage, including hepatocyte degeneration, central vein congestion, sinusoid dilatation, and hemorrhages in the liver, capsule rupture, inflammatory infiltration, and glomerulosclerosis in the kidneys, and epithelial desquamation, impaired tubular morphology, and vacuoles in the testis [31]. Correspondingly, intraperitoneally injected Mn₃O₄NPs induced severe liver injury characterized by hepatic inflammation and cell exfoliation along with induction of hepatocyte apoptosis through up-regulation of caspase-3 protein expression and Bax/Bcl2 ratio. In addition, Mn₃O₄NPs exposure up-regulated expression of genes involved in endoplasmic reticulum stress (GRP78, Climp63) xenobiotic metabolism by cytochrome P450 (CYP1A2, Sult2a1). The latter may contribute to ROS overproduction, playing a key role in hepatocyte apoptosis, as evident from inhibition of apoptosis upon treatment with antioxidant NAC [61]. In yellow catfish, Pelteobagrus fulvidraco, exposure to MnO₂NPs induced a more profound toxic effect in liver characterized by up-regulation of pro-inflammatory (il1$\beta$, il8, tnfa) and pro-apoptotic (bax, casp3, cycs) gene expression compared to MnSO₄. Furthermore, liver damage is also associated with lipid accumulation due to activation of lipogenic fas, acsl3, scd1, gpat3, agpat2 and dgat1 mRNA expression. It is assumed that activation of hepatic lipogenesis upon exposure to MnO₂NPs may be mediated by down-regulation of miR-92a and miR-92b-3p expression, which inhibit lipid accumulation by direct targeting acsl3 [62]. Another epigenetic mechanism of MnO₂NPs-induced hepatic lipogenesis in yellow catfish may include down-regulation of miR-20a-5p which targets mitofusin 2 (mfn2 gene), promoting mitochondrial fusion and interaction between mitochondria and lipid droplets together with Plin2 [63]. Further studies showed that MnO₂NPs exposure induce mitoROS overproduction leading to up-regulation of heat shock factor 1 (Hsf1) mRNA and protein expression, as well as promotion of its phosphorylation at Ser326 residue. The latter promotes Hsf1 nuclear translocation and its binding to dgat1, plin2 and bnip3 promoters, resulting in activation of lipogenesis and mitophagy, as observed in MnO₂NPs-treated hepatocytes [64]. Finally, a study in broilers showed that Mn₂O₃NPs-induced liver damage characterized by apoptosis, mitophagy, and altered mitochondrial dynamics is associated with intestinal inflammation and reduced gut wall integrity due to down-regulation of claudin1, occludin, MUC1 and ZO-1 mRNA and protein expression. Furthermore, intestinal damage was also accompanied by gut dysbiosis characterized by increased Firmicutes/Bacteroidetes ratio, and reduced relative abundance of Bacteroides and Bifidobacterium, along with enrichment of Pseudomonas, Ralstonia, Prauserell, Sediminibacterium, Burkholderia-Caballeronia, Paraburkholderia, Bradyrhizobium and Rubrobacter [65]. Another study demonstrated that intraperitoneal injection of MnO₂NPs possesses hepatotoxicity and nephrotoxicity, evidenced by elevated ALT, AST, and ALP activity, increased blood urea nitrogen, creatinine, and total bilirubin concentrations, along with induction of hyperglycemia. Noteworthy, all adverse effects of MnO₂NPs were attenuated by vitamin D administration [30]. Correspondingly, subcutaneous injection with MnO₂NPs and MPs in rats resulted in a significant increase in blood glucose and cholesterol levels. Furthermore, exposure to both types of particles resulted in a significant reduction in HDL-C level without any significant effect on LDL-C and TG concentrations [66].

In addition, intravenous injection with Mn₃O₄NPs (via tail vein) weekly for 120 days resulted in accumulation of Mn in testes and alteration of testicular morphology characterized by reduced thickness of the germinative layer and degeneration of seminiferous tubules. These effects were associated with reduced levels of testosterone and FSH, as well as decreased sperm count in ejaculate, reduced sperm motility, and a higher rate of abnormalities. Adverse reproductive effects of Mn₃O₄NPs exposure may be mediated by overproduction of ROS, induction of mitochondrial dysfunction, and subsequent apoptosis, as observed in a culture of TM4 cells. Furthermore, transcriptional analysis of testes revealed up-regulation of genes involved in PPAR-signaling (Fabp1, Apoa2, Apoa3, and Pck1), steroid hormone synthesis (LOC100361547, Cyp2c12, Cyp2c6v1, and Ugt2b37), and cytochrome P450-mediated metabolism (Gsta4, LOC102550391, Ugt2b37, Sult2a2) [67]. Another study showed that MnO₂NPs also induce testicular damage characterized by a decline in the number of spermatogonial cells, primary spermatocytes, spermatids, and Leydig cells, resulting in a reduction sperm number, motility, and viability, along with increased sperm abnormalities, and altered reproductive hormone levels. The latter may be mediated by MnO₂NPs-induced down-regulated mRNA expression of StAR, HSD-3$\beta$, CYP11A1 involved in steroidogenesis [68]. Oral exposure to Mn₂O₃NPs also significantly reduced serum luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone levels, as well as resulted in a significant decrease in the number of spermatogonial cells, primary spermatocytes, spermatids, and Leydig cells in testes. Interstitial edema of seminiferous tubules and vacuolar degeneration of epithelium was also observed [69]. Finally, subcutaneous injection of MnO₂NPs induced a decline in the number of sperms, spermatogonia and spermatocytes, decreased sperm motility, and reduced diameter of seminiferous tubes, although this effect was more profound upon exposure to MnO₂ microparticles [70].

Intratracheal instillation of Mn₃O₄NPs in a dose of 0.25 mg per animal resulted in a significant increase in the number of neutrophils and increased neutrophil-to-alveolar macrophage ratio in BALF, as well as increased amylase and LDH activity, altogether indicating pulmonary inflammation and toxicity [71]. Correspondingly, exposure of CB57BL/6 mice to Mn₂O₃NPs significantly increased BALF neutrophil count, as well as MCP-1 and IL-6 levels [43].

Certain studies show that MnO_xNPs exposure may adversely affect other organs and tissues. Specifically, oral exposure to 125 mg/kg and 250 mg/kg BW Mn₃O₄NPs for 20 days did not induce histopathological effects in the liver, spleen, kidneys, or colon, while inducing an inflammatory response in the colon by up-regulating IL-1$\beta$ and IL-6 levels. Down-regulation of tight junction protein claudin-1 in the colon induced by exposure to high-dose Mn₃O₄NPs was associated with gut dysbiosis. Specifically, high-dose exposure significantly reduced gut biodiversity and the total number of microbial species. At the phylum level, the relative abundance of Verrucomicrobiota, Bacteroidota, and Firmicutes_C was reduced in response to both low and high doses of Mn₃O₄NPs [72]. In rat erythrocytes, Mn₃O₄NPs promoted eryptosis due to an increase in cellular Ca²⁺ level, ROS and RNS overproduction, phosphatidylserine externalization, calpain and caspases 3 and 9 activation [73]. Noteworthy, replacement of MnCO₃ in diet with Mn₂O₃NPs maintained Mn balance in rats, although resulting in impaired femoral bone morphology characterized by bone fibrosis and steatosis [74]. Studies in avian species show that MnO_xNPs may also induce significant immunotoxic effects. It has been demonstrated that dietary exposure to Mn oxide NPs in broilers impairs immune response, evidenced by lower concentration of antibodies against Newcastle disease and infectious bronchitis [75]. Correspondingly, administration of Mn₂O₃NPs induced more caspase 3 activation in blood immune cells of turkeys compared to bulk MnO [76].

Taken together, the increasing body of evidence shows that exposure to MnO_xNPs possesses reproductive toxicity, induces hepatic and kidney damage, promotes intestinal inflammation, as well as induces immunotoxicity (Table 2, Ref. [30, 43, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 74]). In contrast, smartly engineered casein MnO_xNPs generated with MnCl₂ and casein under alkaline conditions [77] did not induce toxic effects upon both acute and chronic exposures [78]. These findings show that modified MnO_xNPs are characterized by lower toxicity, while unmodified particles possess cytotoxic effects, and thus dissolution of MnO_xNPs from modified particles must be limited [26]. These findings are in agreement with the role of metal nanoparticle surface coating in reducing its toxicity and facilitating their therapeutic effect in treatment of brain diseases [79].

Mn₃O₄NPs induce cell death in dopaminergic PC12 cells by induction of oxidative stress and apoptosis through up-regulation of mitochondrial calcium (Ca²⁺) uniporter (MCU) and subsequent increase in mitochondrial Ca²⁺ concentration. The role of Ca overload in cytotoxic effects of Mn₃O₄NPs in PC12 cells was also confirmed by the observed inhibition of apoptosis and oxidative stress by treatment with the MCU inhibitor. Furthermore, a decrease in dopamine content in Mn₃O₄NPs-exposed cells and rats was mediated by downregulation of DOPA decarboxylase (DDC) expression [33]. Correspondingly, MnNPs effectively internalized by PC12 cells induce a significant decrease in cellular dopamine, DOPAC, and HVA contents, and this effect was similar to that observed upon exposure to soluble Mn²⁺ (Mn acetate). Although exposure to MnNPs induced only a slight mitochondrial dysfunction, it resulted in a more than 10-fold increase in ROS production, exceeding the respective values observed for Mn²⁺ [34]. In addition, a comparative study showed that Mn₂O₃NPs resulted in more profound decrease in PC12 cell viability compared to Mn₃O₄NPs and especially Mn₅O₈NPs. This effect was associated with a species-specific decrease in cellular GSH levels by Mn₂O₃NPs followed by Mn₃O₄NPs and Mn₅O₈NPs [81].

In another model of dopaminergic neurons, N27 cells, exposure to MnNPs also induced cell death associated with ROS overproduction and caspase-3-mediated proteolytic cleavage and activation of protein kinase C$\delta$ (PKC$\delta$), although treatment with antioxidants did not alleviate MnNPs-induced neuronal toxicity. In addition, MnNPs induced autophagy in N27 cells evidenced by Beclin1 cleavage and an increase in LC3I/LC3II ratio. Neurotoxic effect of MnNPs was also observed in primary dopaminergic neurons, being associated with reduced neurite length [82]. Concomitantly, another study showed that a decrease in PC12 cell dopamine content induced by exposure to 10 µg/mL MnNPs failed to alter the expression of genes involved in redox homeostasis (thioredoxin reductase 1 (Txnrd1), glutathione synthetase (Gss), and glutathione peroxidase 1 (Gpx1)) or dopamine metabolism (tyrosine hydroxylase (Th), monoamine oxidase A (MaoA), and catechol-O-methyltransferase (Comt), vesicular monoamine transporter-2 (Vmat-2), and dopamine transporter (Dat)), being related to up-regulation of $\alpha$-synuclein and down-regulation of Parkin gene expression [83].

Exposure of neuronal SH-SY5Y cells to MnNPs led to GSH depletion and a significant increase in cellular ROS production due to mitochondrial dysfunction, altogether resulting in oxidative DNA damage, chromosome fragmentation, phosphatidylserine translocation, and caspase-3 activation, contributing to cellular apoptosis [84]. Correspondingly, Mn₂O₃NPs exposure induced both necrosis and apoptosis in a dose-dependent manner in SH-SY5Y neuroblastoma cells. The latter was mediated by caspase-3 and caspase-9 activation and increased Bax/Bcl-2 ratio. In addition, molecular docking and molecular dynamic studies showed that Mn₂O₃NPs can bind tau protein and promote its folding, potentially contributing to neurotoxic effects [85]. Mn₃O₄NPs, especially small-sized, were shown to have more profound effects in dividing ST-14 striatal neuroblasts due to induction of mitochondrial dysfunction and ROS production with subsequent increase in NF-$\kappa$B transcription [86].

Cellular studies also show that MnO_xNPs contribute to neuroinflammation. Specifically, in BV2 microglial cells MnO₂NPs exposure resulted in a significant increase in ROS production and p38 MAPK pathway activation, ultimately leading to overexpression of IL-1$\beta$ and TNF$\alpha$. In turn, treatment with antioxidant N-acetylcysteine and p38 MAPK inhibitor (SB203580) abrogated secretion of proinflammatory cytokines in microglia, indicative of the key role of ROS/p38 MAPK pathway in neuroinflammation induced by MnO₂NPs exposure [87]. Correspondingly, in a co-culture of neuronal CATH.a cells and C8-B4 microglia, exposure to MnNPs induced a significant decrease in cell viability, ROS overproduction, and up-regulation of Tnf-$\alpha$ and Il-6 gene expression. Next-generation sequencing (NGS) also revealed that proinflammatory effects of MnNPs exposure were associated with modulation of expression of miR-124-3p, miR-1-3p, miR-16-5p, let-7a-5p, and miR-155-5p, considered as upstream regulators. Furthermore, up-regulation of miR-155-5p abrogated the stimulatory effects of MnNPs exposure on TNF$\alpha$ and IL-6 mRNA and protein expression in a co-culture. These findings demonstrate that induction of neuroinflammation by MnNPs may be at least partially mediated by modulation of miR-155-5p expression [88].

Taken together, the results of in vitro studies highlight cytotoxic effects of pristine MnO_xNPs in both neuronal and non-neuronal brain cells (Table 3, Ref. [33, 34, 82, 83, 84, 85, 86, 87, 88]). The existing data show that MnO_xNPs induce neuronal death through a variety of mechanisms including oxidative stress, mitochondrial dysfunction, altered Ca²⁺ homeostasis, induction of tau and A$\beta$ accumulation, and apoptosis (Fig. 1). In addition, MnO_xNPs-induced microglia activation with up-regulation of proinflammatory cytokine expression due to activation of ROS/p38 MAPK pathway and modulation of microRNA expression may also contribute to brain damage through induction of neuroinflammation.

Fig. 1.

The molecular mechanisms of MnO_xNPs-induced neuronal death. Briefly, MnO_xNPs exposure inhibits electron transport chain (ETC) functioning resulting in reduction of ATP production and increased electron leakage at complexes I and III with subsequent formation of ROS. In addition to ROS formation, MnO_xNPs increase activity of Ca²⁺ uniporter resulting in mitochondrial Ca²⁺ overload. Both ROS overproduction and Ca²⁺ overload further affect ETC functioning. MnO_xNPs induce tau and $\alpha$-synuclein formation, which promote oxidative stress, Ca²⁺ accumulation, and ETC damage. MnO_xNPs -induced mitochondrial dysfunction results in increased cytochrome c leakage with subsequent activation of caspases 9 and 3, leading to apoptosis. Caspase 3 also mediates proteolytic cleavage and activation of PKC$\delta$, which up-regulates Bax further promoting cytochrome c release. In addition, caspase 3 activation may be also responsible for phosphatidylserine (PS) translocation to the outer surface of the cell membrane, promoting phagocytosis of apoptotic cell. Furthermore, MnO_xNPs -induced oxidative stress results in DNA oxidation with accumulation of oxidized DNA (oxDNA), which also triggers apoptosis. Thin arrows are indicative of the continuum of changes, thick arrows show stimulatory effect of MnO_xNPs on these mechanisms. MnO_xNPs, MnO_x nanoparticles; ATP, adenosine triphosphate; ROS, reactive oxygen species; PKC$\delta$, protein kinase C$\delta$.

Findings from animal models generally corroborate the results of in vitro studies. Intratracheal exposure to MnO₂NPs results in a more than 5-fold increase in Mn content in brain, whereas the level of Mn in the liver is elevated by less than 2-fold, indicative of the role of the brain as a target for Mn accumulation following MnNPs exposure [89]. An in vivo study demonstrated that intratracheal instillation with MnO₂NPs resulted in a significant increase in both blood and brain Mn accumulation after 9 weeks of exposure [90, 91].

Elder et al. (2006) [92] demonstrated that the olfactory neuronal pathway is the predominant mechanism of transport of MnNPs into the brain upon inhalation. Specifically, the authors observed a more profound increase in the olfactory bulb Mn content (3.5-fold) compared to lungs (2-fold) upon exposure to MnNPs. Furthermore, in the exposed animals, up-regulation of TNF$\alpha$, MIP-2, GFAP, and neuronal cell adhesion molecule (NCAM) gene and/or protein expression in the olfactory bulb was more profound compared to other brain areas, while no inflammatory effects were observed in lungs [92]. Correspondingly, Romashchenko et al. (2019) [93] revealed a significant role of olfactory transport in MnNPs transport into the brain, being more effective than permeation of NPs through BBB [93].

Additional studies showed that exposure to MnO_xNPs results in significant alteration in brain functioning. Specifically, intratracheal instillation with MnO₂NPs significantly reduced ambulation and rearing with increased local activity and immobility due to increased frequencies of spontaneous cortical activity, elongation of cortical evoked potential latency, as well as decreased nerve conduction velocity [90, 91]. This effect was dependent on the size of the exposing particles. It has been shown that alterations in spontaneous cortical activity were more profound upon exposure to smaller MnO₂NPs (9 and 42 nm) compared to larger (118 nm) particles [94]. Furthermore, the changes in spontaneous and evoked cortical activity and peripheral nerve action potential positively correlated not only with cortical Mn²⁺ content, but also cortical TBARS level, being indicative of the role of lipid peroxidation in MnO₂NPs-induced neuro-functional alterations [95]. When compared to the effects of soluble MnCl₂, MnNPs exposure exerted less profound effects on the latency of cortical evoked potentials and tail nerve conduction velocity [96]. However, combined exposure of MnCl₂ (orally) and MnNPs (intratracheally) for 3 weeks possessed more profound effects on cortical sensory evoked potential and nerve action potential compared to 6-week exposure to MnCl₂ at the same cumulative dose [97].

MnNPs-induced alterations in evoked potential latency and nerve conduction velocity were abolished by antioxidant-rich green tea, whereas other adverse effects of MnNPs exposure, like reduced body weight gain, were not reversed by green tea administration [95]. Respectively, administration of antioxidants attenuated adverse effects of MnNPs, with rutin being the most effective, whereas ascorbic acid only abolished alterations in cortical evoked potentials, and curcumin did not exert any protective effects [98]. Intraperitoneal injection of Mn₃O₄NPs resulted in a significant accumulation of Mn in brain, and dose-dependent reduction in the number of head-dips into holes, brain weight as well as an increase in the number of cells without a nucleolus in caudate nucleus and hippocampus (CA1) [99], which were completely abrogated by administration of a complex of antioxidants (N-acetylcysteine, vitamins A, C, E, selenium) and other bioactive compounds, including pectin, glutamate, glycine, iodide, and omega-3 polyunsaturated fatty acids [100]. These findings show that the neurotoxic effects of MnNPs depend on the prooxidant effect of the particles, although the different efficiencies of various antioxidants may be indicative of the contribution of various sources of ROS in MnNPs neurotoxicity.

Mitochondrial dysfunction is a primary source of ROS upon MnO_xNPs exposure. Specifically, Mn particles induced a significant increase in mitochondrial ROS production at respiratory complexes I and III, being more profound in MnO₂NP-exposed mitochondria compared to MnO₂MPs. Furthermore, exposure to MnO₂NPs, but not MnO₂MPs, possessed inhibitory effects on complex II and IV activity in the brain [101].

Acute exposure to MnNPs induced significant neurotoxic effects characterized by depression and loss of reaction to auditory stimuli [102], which were associated with severe cerebellar damage characterized by neuronal and glial cell shrinkage and pyknosis, ischemia, and edema [103]. In addition, the route of MnOxNP exposure induces distinct changes in brain pathomorphology. Specifically, acute inhalation exposure to MnO₂NPs (15–29 nm) in rats resulted in brain and especially cerebellum damage, characterized by ischemic damage to neurons and microglia, endothelial swelling in cerebral blood vessels, and neurite defects. In turn, acute oral exposure resulted in brain edema, vascular hyperemia in the brain cortex and cerebellum, neurite demyelination, as well as alterations in the granular cortex layers [104].

Exposure of adult male Wistar rats to 50–100 µg/kg MnO₂NPs through intraperitoneal injection resulted in a significant increase in immobility time and reduced sucrose preference. Further analysis showed that MnO₂NPs induced a significant increase in hippocampal ROS production and lipid peroxidation. These effects were also associated with neuronal apoptosis and necrosis in the hippocampus, as well as a dose-dependent decrease in hippocampal catecholamine content [105]. Surprisingly, adverse neurophysiological effects of MnNPs were attenuated when co-exposed to Fe₂O₃NPs, while this effect was independent of brain Mn accumulation [106].

Further, it has been shown that alterations in sensory motor functions induced by intraperitoneal MnO₂NPs exposure in rats were associated with BBB damage with the resulting brain edema and reductions in blood flow in the sensory-motor cortex, hippocampus, caudate putamen, cerebellum, and thalamus, and to a lesser extent in hypothalamus, pons, medulla, and spinal cord. In addition, a dose-dependent increase in neuronal damage characterized by vacuolation, chromatolysis, and Nissl substance loss was observed predominantly in parietal and temporal cortex, hypothalamus, and thalamus, followed by other brain areas [35]. In addition, MnO₂NPs-induced alterations in learning and memory are associated with hippocampal lesions characterized by edematous cells with karyopyknosis and irregular arrangement, cell connections loss, increased number of irregular cells, as well as damage to the choroid plexus, a blood–cerebrospinal fluid barrier structure. Transcriptomic analysis of the choroid plexus showed that MnNPs exposure affects expression of transporter, ion channel proteins, and ribosomal genes, including BMP/Retinoic Acid Inducible Neural Specific 1 (Brinp), synaptoporin (Synpr) and Collapsin response mediator protein 1 (Crmp1) [36].

It has also been demonstrated that MnO₂NPs exposure induces neuroinflammation due to astrocyte activation, evidenced by increased glial fibrillary acidic protein (GFAP) immunoreactivity, which was more profound in cerebellum, hippocampus, and thalamus [35]. Induction of astrocyte activation and neuroinflammation with the increased number of GFAP and iNOS-positive cells upon injection of MnO₂NPs to the substantia nigra was also observed [107]. Furthermore, replacement of Mn²⁺ (as MnCO₃) in the diet with Mn₂O₃NPs induced neuroinflammation, evidenced by a significant increase in brain TNF$\alpha$ level and ceruloplasmin activity, while reducing IgG levels. At the same time, replacing Mn²⁺ with Mn₂O₃NPs did not induce proinflammatory effects, neither in the jejunum nor at the systemic level, indicating higher sensitivity of the brain to the proinflammatory effect of MnNPs [108]. Intragastric administration of MnONPs resulted in severe lymphoid infiltration of brain tissue, while MnOMPs possessed lower toxicity [109].

Several studies show that MnO_xNPs impair neurotransmitter metabolism in vivo. Specifically, injection of MnO₂NPs to the substantia nigra induced a significant decrease in the number of TH-positive neurons, which was similar to that observed upon exposure to dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA) [107], indicating altered dopaminergic neurotransmission. Metabolomic analysis of brain tissues of rats intravenously injected with MnNPs showed that, irrespective of the dose and period of exposure, MnNPs exposure altered brain guanosine, phenylalanine, GABA, myo-inositol, uracil, and propionate levels. Upon short-term exposure, MnNPs at both doses also increased brain glutamate content. These findings demonstrate that exposure to MnNPs altered the glutamine–glutamate/GABA cycle in the brain [110]. Replacement of dietary MnCO3 with the respective dose of Mn2O3NPs resulted in a significant reduction in brain 5-HT levels along with an increase in norepinephrine and dopamine levels, which may be associated with gut microbiota alterations. The latter was characterized by reduced intestinal levels of SCFA, including acetate, propionate, butyrate, decreased enzymatic activity of caecal bacteria, as well as elevated ammonia concentrations and increased pH in caecum [111]. MnO₂NPs-induced Mn accumulation in the brain was also associated with down-regulation of brain AChE, Na/K-ATPase, Mg²⁺-ATPase, and Ca²⁺-ATPase activities, as well as neuronal vacuolation and inflammatory cell infiltration [112].

Noteworthy, a study in iridescent shark (Pangasianodon hypophthalmus) demonstrated that MnNPs exposure resulted in a significant down-regulation of acetyl choline esterase (AChE) activity in the brain, accompanied by significant up-regulation of brain catalase, glutathione-S-transferase, and glutathione peroxidase activity with a concomitant decrease in SOD activity. Furthermore, a dose-dependent increase in hepatic cortisol level may also be indicative of altered hypothalamus-pituitary-inter-renal axis activity. Noteworthy, these effects of MnNPs were nearly similar to those of Mn²⁺, although the latter possessed higher toxicity (LC₅₀= 111.75 mg/L) compared to MnNPs (LC₅₀ = 93.81 mg/L) [113]. These findings assert the role of Mn²⁺ release in mediating the toxic effects of MnNPs. This is corroborated by the observed formation of stress-granules through eIF2a phosphorylation and inhibition of oxidative phosphorylation in human glioblastoma U87 MG cells, which were also induced by Mn²⁺ exposure [114].

Taken together, the existing data show that MnO_xNPs exposure induces neurotoxicity through a variety of mechanisms (Table 4, Ref. [35, 36, 89, 94, 95, 96, 99, 101, 102, 105, 107, 108, 110, 111, 112]). Along with induction of ROS production with subsequent development of oxidative stress or overproduction of proinflammatory cytokines clearly demonstrated in cellular models, in vivo studies showed that MnO_xNPs induce brain damage through blood-brain barrier damage, alterations in neurotransmitter metabolism, and dysregulation of gut-brain axis.