**7. Mechanisms of nanoparticle toxicity**

Various hypothesizes have been proposed from time to time regarding the toxicity of NP. Nanoparticles can directly cross the placenta and cause damage to the fetus because of their high surface reactivity. Because of their small size, NPs can easily reach the brain and are taken up by the brain cells, such as neurons and glia. Mechanisms of NP uptake by cells include pinocytosis, endocytosis dependent on caveolae and lipid raft composition, clathrin-dependent endocytosis, and phagocytosis [90]. Due to their high surface reactivity, the nanoparticles can cause the generation of reactive oxygen species [91] and inflammation [92]. The metal ions of the NP have been proposed to contribute to their toxicity [93, 94]. The neurotoxic effects can either result in the direct alteration of the structure or activity of the neural system or lead to subsequent effects due to glial activations and glialneuronal interactions [95]. The nanoparticles may also exert their toxic effects due to their limited elimination/excretion from the brain.

Oxidative stress has been implicated as one of the major mechanisms of NP toxicity. Consequences of oxidative stress include mitochondrial membrane damage and dysfunction, which in turn leads to cell death [96]. Inflammation caused by the production of cytokines appear to be a second mechanism by which the NP exerts their cytotoxic effects [97]. ZnO NPs have been shown to induce the production of pro-inflammatory cytokines in the brain of mice, accompanied by an impairment of cAMP/CREB signaling pathway. The degree of inflammation correlated with the age of the mice [56]. NPs interact with enzymes, potential apoptotic, or necrotic factors and induces inflammatory processes [12]. NP show properties similar to that of viruses and cause damage to DNA affecting cell proliferation [90]. NP can reduce mitochondrial function [98] and generate cellular morphological abnormalities [99] Cui et al. [81] postulated that prenatal exposure to NP resulted in an impairment of antioxidant capabilities in the brain of newborn pups.

Accumulation of NPs along the endosomal pathway may affect the morphology and functioning of the BBB. The interaction of the NP with biological macromolecules like DNA, lipids, and proteins may lead to the generation of oxidative stress, conformational changes in the macromolecules, mutations, alterations in membrane permeability, activation of various signaling pathways, alterations in the functions of enzymes, and exposure of new protein epitopes [100]. Genotoxic effects of NP include chromosomal aberrations, DNA strand breaks, oxidative DNA damage, DNA adducts, and micronucleus formation [101, 102]. Interactions of NP with microglia and astrocyte may activate NF-κB signaling and result in the release of mediators of inflammation and apoptosis [103]. On the other hand, oxidative stress induced mitochondrial DNA damage results in Nod-like receptor protein 3 (NLRP3) inflammasome activation, which subsequently regulates inflammatory responses by activating caspase-1 and interleukin-1β (IL-1β) release [104].

Most of the resulting damage of the nervous tissue is usually irreversible [18]. NPs have been reported to disrupt the cytoskeleton of cells of the CNS and thus cause cell death. NPs been shown to regulate the expression of neuronal channels and other proteins involved in excitability and neurotransmission [105]. Microglia, account for ~20% of the glial cells in the brain. They are a type of glial cells, which are the resident innate immune cells in the brain and regulate

neuroinflammation [106]. Choi et al. [107] demonstrated that low levels of SiNPs can alter microglial function by changing the expression of proinflammatory genes and cytokine release. Excessively activated or uncontrollable microglia can cause nerve toxicity by inducing proinflammatory factors, such as interleukin-1β, tumor necrosis factor (TNF)-α, prostaglandin E2, and interferon-γ (**Figure 3**) [18].

Autophagy (autophagic flux) is a highly regulated cellular process which by eliminating long-lived proteins and damaged organelle components through the lysosomal mechanism maintains cellular homeostasis [18]. NPs have been demonstrated to be autophagic inducers [108]. Autophagy has been found to be correlated with increased DNA strand breaks and other defensive mechanisms [109]. NPs have been reported to induce autophagy through the generation of ROS and lysosomaldependent mechanism [18]. Autophagy induced by NPs can have protective or detrimental effect on cells. During intracellular oxidative stress, imbalance and excessive ROS generation decline in autophagy-lysosome degradation function results in autophagic flux impairment, which leads to significant accumulation of the substrate of autophagy within the cell and may even trigger cell death through mitochondrial pathway [110].

#### **Figure 3.**

*Mechanism of nanoparticles (NPs)-induced neurotoxicity. Supraphysiological levels of reactive oxygen species (ROS) induce oxidative damage to the cellular macromolecules such as lipids, protein, and both mitochondrial and nuclear DNA. ROS-induced protein peroxidation may result in loss of catalytic activity of many enzymes including the antioxidant enzymes. NPs-mediated genotoxic stress in turn, can drive apoptosis mainly through the intrinsic mitochondrial apoptotic cell death pathway in neuronal cells. Mitochondrial dysfunction activates inflammasomes, which triggers the release of proinflammatory cytokines IL-1*β *and IL-18 via caspase-1 activation. Moreover, ROS-induced activation of nuclear factor kappa B (NF-*κ*B) pathway may trigger proinflammatory responses, which is one of the key factors associated with NPs-induced neurological inflammation.*
