Neurotoxicology of metals and metallic nanoparticles in Caenorhabditis elegans

1. Introduction

The nervous system is the major controlling, regulatory and communicating system in the body. In vertebrates, it consists of a central nervous system (CNS, brain) and a peripheral nervous system (spinal cord and ganglia). The CNS controls voluntary movements such as walking, speaking, as well as involuntary movements as breathing and reflex actions. It also controls learning, emotions and metabolism. Therefore, any type of damage to the nervous system (mechanical trauma or poisoning) will impact biological functions that might be essential for life, such as breathing or heart pumping (Tortora and Derrickson, 2018).

Accordingly, the nervous system is well preserved. Protective membranes known as the meninges surround the brain and spinal cord, and both float in a crystal-clear cerebrospinal fluid. The central nervous system lies largely within the axial skeleton, wherein the brain is encased in a bony vault, the neurocranium, while the cylindrical and elongated spinal cord lies in the vertebral canal, which is formed by successive vertebrae connected by dense ligaments (Tortora and Derrickson, 2018). In addition, endothelial cells in the majority of the CNS (excluding the circumventricular organs) for a specialized barrier, called the blood-brain barrier, which prevents the crossing of solutes from the blood into the extracellular fluid of the nervous system (Gupta et al., 2019). Some molecules cross by passive diffusion, especially small and non-polar ones. There are transporters that allow the entry of certain molecules, such as glucose and ions (Banks, 2016).

Despite the existence of the BBB, many toxicants reach the nervous system, enter the neurons and glial cells and trigger molecular alterations such as oxidative stress, mitochondrial damage, apoptosis, inhibition or stimulation of signaling pathways, DNA damage and protein and lipid oxidation. These effects can cause abnormal neuronal function or even lead to cell death. Among these toxicants are select metals, which have been described for several years as potentially neurotoxic (Caito and Aschner, 2015). They can enter the brain through transporters such as the divalent metal transporter DMT and zinc transporter ZIP4 or secondary to microvascular damage and opening of interendothelial tight junctions (Zheng et al., 2003). We are constantly exposed to essential and non-essential metals through water, food, air, pharmaceutics, cosmetics and some subjects are exposed in their work environment. In addition, accidents such as the disruption of mining tailings dams as in Mariana and Brumadinho (Brazil) cause a tragic ecological impact, besides leading to diseases to the exposed populations. Non-essential metals possess higher neurotoxicological risk than essential metals, therefore dose and time of exposure are important factors to be considered. Although the great number of studies on the metals neurotoxicology, not all mechanisms have been fully deciphered.

More recently, with the advance of nanotechnology, metallic nanoparticles (NPs) gained attention because of their unique characteristics and applications in industry. The increasing NPs applications lead inevitably to their release into the environment, contaminating water, soil, air and food (Peralta-Videa et al., 2011). In addition, we are constantly in contact with products that contain NPs, such as deodorants and clothes with Ag-NPs in their composition. Notably, an increasing number of studies have evidenced that metallic NPs compose a neurotoxic hazard and understanding the impact of size, charge, shape, ion release and coating on the biological outcomes is urgent (Teleanu et al., 2019).

In order to increase the understanding on metal- and NPs-induced neurotoxicology, alternative and complementary animal models have been used. Invertebrates as the nematode Caenorhabditis elegans have been successfully applied to study basic biological processes such as aging, oxidative stress, and nervous system function (Queirós et al., 2019; Wu et al., 2019). C. elegans has a small size, simple structure, short lifespan, accessibility to genetic manipulation, and the conserved biological pathways in relation to mammals. It allows an ecotoxicological approach because of its presence in the environment, and largely the findings can be extrapolated to humans because of the great genetic homology between the nematode and mammals (Avila et al., 2012).

Indeed, this organism has a simpler nervous system, but it includes almost all the neurotransmitters systems present in humans. The worm has been studied to such an extent that a neuronal wiring diagram is available. The nervous system has high sensitivity to metals and metallic NPs and allows for a wide range of assays, from physiological to behavioral, biochemical and molecular (such as transcriptomics and proteomics) in a faster and more bioethical and economical manner (Avila et al., 2012; Queirós et al., 2019; Sedensky and Morgan, 2018; Soares et al., 2017). The use of mutants and transgenic supports the elucidation of mechanisms that were unclear or inconceivable to investigate in other models. In this context, we have structured the present review to shed light on applications and to update on neurotoxic mechanisms uncovered by studies in this animal model. Our focus is centered on several metals and metallic NPs that cause neurotoxicity in C. elegans, but it is noteworthy that many toxicants have yet to be tested yet in this model.

2. C. elegans neuronal system

The C. elegans nervous system is relatively simple and the whole developmental cell lineage, the anatomical location, structure, and network connections of each neuron have been characterized (Sulston, 1983). A typical adult organism has 302 neurons in adult hermaphrodites (383 in males), divided into 118 morphologically distinct classes and 56 glial cells. Approximately 7000 synapses that compose 2 independent nervous systems have been described, in addition to a large somatic system with 282 neurons and a small pharyngeal system with 20 neurons (White et al., 1986). C. elegans is the only organism that has a fully characterized neuronal wiring diagram where all neurons and the major interactions between them are well characterized and mapped.

The biochemistry of the C. elegans nervous system is highly conserved with mammals—as the worms express similar ion channels, receptors, vesicular transporters, and other synaptic components (Bargmann, 1998). Neurotransmitter synthesis, neuronal vesicles, neurotransmitter release, receptor binding and inactivation (by reuptake or degradation), are well understood, which is important for the study of possible damage to the nervous system.

Nematodes contain a variety of classic neurotransmitters such as dopamine (DA), aminobutyric acid (GABA), glutamate (GLU), acetylcholine, serotonin (5-hydroxytryptamine), and neuropeptides. Proteins related to the nervous system can be co-expressed with green fluorescent protein (GFP) and therefore neuronal morphology can be visualized in vivo (Chalfie et al., 1994). Neuronal excitability can be followed by live calcium imaging (Nguyen et al., 2016), which allows for associations between neuronal activity and behavioral phenotypes. Moreover, its ease of genetic manipulations allows the identification of genes significant for neuronal formation, migration, activity, or other functions (Brenner, 1973). On the other hand, worms lack epinephrine, norepinephrine and histamine signaling, and some significant differences have been described in sodium-dependent channels (Bargmann, 1998; Goodman et al., 1998). In addition, nematodes lack a blood-brain barrier, and once the molecules are absorbed, they can quickly disperse and diffuse into the nervous system (Li et al., 2018; Liberati et al., 2004; Zhao et al., 2016). The structural and functional similarities with the human nervous system, along with other advantages, strengthen the utility of worms as a model in neuroscience and in neurotoxicology. Notably, environmental and occupational exposure to metallic neurotoxicants is a matter of growing concern, since it may have very significant consequences for human health, from impairing neurodevelopment in children to accelerating the evolution of neurodegenerative diseases. The evaluation of the risks associated with the release of metals and metallic NPs in the environment, their modifications (by microorganisms, light, etc.) and bioaccumulation in animals through the use of relevant biological models to assess neurotoxicity are important research objectives (Ortega and Carmona, 2022). Because of the extensive knowledge on C. elegans nervous system and the advantages afforded by mutants, the nematode offers unique perspectives as a neurotoxicological model, which use has been growing recently in the toxicological community. Studies that examine metal- and NPs-induced neurotoxicity are addressed by a wide range of parameters, including behavioral, structural, signaling, and molecular levels. Behavior, even in simple metazoans, depends upon integrated processes at the subcellular, cellular, and organismal level, and thus is susceptible to disruption by a broad spectrum of chemicals. For instance, neuronal systems function can be assessed by simple assays such as locomotion (frequently evaluated by head thrashing and body bends), pharyngeal pumping, defecation, egg laying, chemotaxis and response to a mechanical touch. Well characterized neural circuits control these behaviors and neuronal dysfunction could be a plausible explanation for abnormal outcomes in otherwise anatomically and functionally intact worms. For example, damage of neurons, which innervate muscles of a fully developed vulva (reproductive organ of adult hermaphrodite worms), may result in perturbation of egg laying, reduction of progeny and cause bag of worms (BOW) phenomenon (hatching of the larvae inside parental body) (Pluskota et al., 2009; Scharf et al., 2016). The morphology of the damaged neurons can be visualized by green fluorescent protein (GFP) tagging and identify putative mechanisms, PCR analysis can be done to detect up and down regulated genes. Further investigation of alterations in these endpoints with tools like molecular techniques, pharmacological rescue and transgenic strains can shed light to pathways leading to neurotoxicity and how to attenuate the damages caused by metals and metallic NPs. In this chapter, we will focus on the findings in C. elegans studies focusing on advancing neurotoxicology research of these important toxicants.

3. Metals neurotoxicology in C. elegans

Metallic elements are biologically classified according to their essentiality for human health, and therefore they can be considered essential, inert, or toxic (Crisponi et al., 2012a). To be considered essential, a metal must be present in several tissues, must have important physiological functions and cause irreversible and serious damage to the body when absent (Zoroddu et al., 2019). It should be noted that the essentiality varies according to the species, such as vanadium, which has no known biological effect on humans but is considered essential for algae and fungi (Maret, 2016).

In general, most authors consider that there are currently 10 essential metals: sodium (Na), potassium (K), magnesium (Mg), calcium (Ca), manganese (Mn), iron (Fe), cobalt (Co), copper (Cu), zinc (Zn) and molybdenum (Mo) ( Jomova et al., 2022; Zoroddu et al., 2019). There are several biological functions described for each of these essential metals. Among them we can mention the participation in biochemical reactions (as co-factor or present in prosthetic group), in maintaining electrical charges and osmotic pressure, in mitochondrial respiration, controlling in the production of ROS, maintenance of pairing, stacking and the stability of nucleotide bases and regulation of hormone levels, just to name a few ( Jomova et al., 2022).

Although needed for biological functions, studies point out that excessive intake or exposure to these essential metals can cause toxic effects, including neuronal damage. They can trigger immune response, induce the production of reactive oxygen species (ROS) (such as Fe and Cu by Fenton reaction), alter cell structure and cause cell death. Therefore, it has been established safe levels for intake in order to avoid unwanted effects (Crisponi et al., 2012b).

Toxic metals are defined by their bioaccumulative potential and high toxicity to living organisms, and are also classified as heavy metals, including cadmium (Cd), lead (Pb), nickel (Ni), chromium (Cr), mercury (Hg) and metalloids, such as arsenic (As) (Witkowska et al., 2021). These metals can be taken up by organisms by same transporters for essential metals since they share similar chemical characteristics. They can cause damage to different systems at lower levels than essential metals, including the nervous system. The effects are mediated by diverse mechanisms, causing an increase in ROS, oxidative stress, cellular degeneration, among others (Balali-Mood et al., 2021; Vellingiri et al., 2022; Witkowska et al., 2021).

Metals occur naturally throughout the Earth’s crust, the natural soil erosion allows food sources to be rich in nutrients, including essential metals. Thus, through food, it is possible to acquire adequate concentrations of these nutrients and the organism is tolerable with the small concentrations that are ingested of non-essential metals (Zoroddu et al., 2019). However, anthropogenic activities such as mining and smelting, in addition to industrial production and use, domestic and agricultural use of metals and metal-containing compounds contribute to environmental contamination and human exposure, mainly from heavy metals. The increase in the exposure of these metals also occur due to the use of these elements in medicines and cosmetic products (Balali-Mood et al., 2021; Tchounwou et al., 2012).

3.1. Manganese

Manganese (Mn) is a heavy metal of great abundance in the world. Living beings are readily exposed to this metal due to natural erosion that allows the release of the metal into the air, soil, and water sources. Due to this bioavailability, a wide variety of foods have Mn in their composition, such as leafy vegetables, nuts, brown rice, chickpeas, sweet potatoes, pine nuts, among others (Chen et al., 2015b; USDA, 2019). The wide variety of foods rich in Mn makes food intake the main source of exposure to the metal in humans. However, contamination in drinking water, ingestion of food supplements and exposure to metallurgical industries, polluted air and agricultural inputs are also cited as sources of environmental and occupational exposures and can cause toxicity (Balachandran et al., 2020).

Mn toxicity is of great concern due to the substantial risk of exposure potential damage. It is believed that the neurotoxicity of Mn is caused by the metal’s high capacity to accumulate in astrocytes due to its affinity for the enzyme glutamine synthetase (GS) which is located in these cells (Sidoryk-Wegrzynowicz and Aschner, 2013). Mn exposure to exceedingly high Mn levels may cause a pathological condition described as manganism, which is characterized by dystonia, bradykinesia, and gait disturbance due to irreversible damage to the basal ganglia of the brain (Bowler et al., 2015). Manganism shares molecular mechanisms and symptoms with Parkinson’s Disease (PD) because both cause loss of dopaminergic neurons, impairing dopaminergic function (Gubert et al., 2018). One of the hypotheses is that Mn can cause the oxidation of dopamine, leading to the formation of ROS, in turn, triggering neurodegeneration (Chen et al., 2015c).

In order to further elucidate the Mn neurotoxicological mechanisms, a variety of studies using C. elegans have been done. Using molecular biology tools and genetically modified animals, the studies focused on clarifying the pathways involved in the toxicity of the metal in the neurons. Mn intoxication in C. elegans shares similar effects with those inherent to mammals, including dopaminergic neurodegeneration and oxidative stress. Using this model, it was established that Mn interacts with extracellular dopamine to cause dopaminergic neurodegeneration and that the NADPH dual-oxidase BLI-3 promotes the conversion of extracellular DA into toxic reactive species that are taken up by neurons, leading to neuronal death (Benedetto et al., 2010). There are also genetic models for PD available in C. elegans that present a deletion of DJ-1 related (djr) genes, parkin (pdr-1) and PINK (pink-1) as well, besides mutants expressing the mutated form of alfa-synuclein. All these mutants showed higher sensitivity to Mn, with inherent dopaminergic neurodegeneration (Bornhorst et al., 2014; Chakraborty et al., 2015; Chen et al., 2015b; Gitler et al., 2009).

A recent study used C. elegans as a model to demonstrate the role of Mn in neurodevelopment. Worms were exposed to the metal in the first larval stage (L1) and demonstrated significant deficits in olfactory adaptive learning and memory in first-day adults, as observed in mammals. On the other hand, repeated exposure to Mn in adulthood caused resistance to dopaminergic neurodegeneration (Raj et al., 2021). Another study using the model has proven that the ability of Mn to impair the dopaminergic pathway influences the accumulation of lipids. Worms exposed to Mn for 4 h presented reduced reproduction and had an increase in fat accumulation, a reduction in lipid metabolism and a reduction in dopamine content. The studies also indicated the involvement of dopamine receptors in Mn-induced triacylglycerides accumulation, indicating that Mn interferes with reproduction by blocking lipids transport to eggs, potentiating fat accumulation as a secondary effect following dopaminergic signaling impairment (Gubert et al., 2016, 2018).

New insights into kinetics and therapeutics related to Mn toxicity have also been investigated in the C. elegans model. The molecular similarity in metal absorption and excretion pathways has allowed therapeutic strategies to be evaluated. C. elegans conserves the smf-1, −2 and −3 genes, orthologs to the divalent metal transporters (DMT) in mammals, in addition to expressing the exporter ferroportin (fpn-1.1), which allows an overview of the metabolic state of metals, including Mn (Benedetto et al., 2010; Chakraborty et al., 2015). In fact, overexpressing fpn-1.1 in worms lacking the PD-related gene pdr-1 caused attenuation of Mn toxicity, indicating that ferroportin may be an interesting target against manganism (Chakraborty et al., 2015).

The elucidation of Mn metabolism in C. elegans has also allowed other related genes to be investigated as molecular targets of different tested therapies. Peres et al. (2019) confirmed the therapeutic effect of two small molecules (VU0063088 and VU0026921) that could modify the Mn content in vitro. In this study, it was observed that pre-exposure to the molecules in C. elegans offers protection against Mn-induced dopaminergic neurotoxicity in a smf-2 independent manner (Peres et al., 2019).

Other targets have also been elucidated. The studies indicated that antioxidant systems are important to protect against Mn-induced dopaminergic neurodegeneration, such as the transcription factor skn-1 (orthologous to nrf-2 in mammals) and hsp-1 (homologous to HSP-70) (Avila et al., 2016). Reducing the insulin-like signaling can also protect the dopaminergic neurons from Mn, since knocking out AKT-1/2 and SGK-1 increased the expression of antioxidant genes as sod-3 and gcs-1 and even improved slow basal behavior, which is mainly controlled by the dopaminergic system (Peres et al., 2018).

In addition, a transcriptome-side analysis indicated that genes related to oxidative nucleotide damage, unfolded protein response and innate immunity are the major pathways altered by Mn exposure. Genes for both the antioxidant system (gst-4, glutathione s- transferase), the unfolded protein response (UPR, heat shock proteins, IRE1-mediated UPR) and metallothioneins were significantly increased after 60 and 180min Mn exposure, for example. An increase in the homolog for human egr-1 gene (egrh-1, early growth factor response factor 1) implicated in neurological disorders, such as Alzheimer’s disease and regulation of the cholinergic system was also found (Nicolai et al., 2022).

3.2. Iron

Iron is the most abundant transition metal in the environmental and in the biological systems, involved in biochemical processes that are essential for the maintenance of life in different types of organisms. For instance, Fe is involved in neurotransmitter synthesis, mitochondrial respiration, and oxygen transport among others. Its participation in different metabolic processes occurs mainly due to its capability of electron transfer, as donor and acceptor, assuming two redox states: Ferrous Fe²⁺ and ferric Fe³⁺ (Espósito et al., 2020).

However, its essential redox ability is precisely the main mechanism for its neurotoxic effects (Martins et al., 2022). An imbalance in the homeostasis of this metal, as iron overload, can lead to deleterious effects. This essential metal has the ability to catalyze the generation of ROS, causing DNA damage (Klang et al., 2014). Iron overload in the central nervous system (SNC) causes neurotoxicity and cognitive impairments, and has been reported to be involved in the pathogenesis of different disorders, including hypoxic ischemic brain injury and neurodegenerative diseases of aging, such as Alzheimer, Parkinson’s disease, and Huntington disease (Wu et al., 2020).

Fe also has a critical function in brain development and functioning and as aging progresses, ferritin’s ability to store iron diminishes, with the intracellular iron pool increasing (Jenkins et al., 2020). Iron accumulation is common in normal aging, considering the high amounts of iron used by the brain due to its high oxygen consumption. The brain becomes a major producer of reactive ROS, with Fe being one of its main producers. This ROS generation occurs through the Fenton reaction that begins between iron and hydrogen peroxide, generating hydroxyl radical, which is extremely reactive, causing cell damage and neuronal death or ferroptosis by the induction of lipid peroxidation (Abe et al., 2022; Zhou et al., 2020).

The induction of ROS by iron overload also occurs in C. elegans, analogous to the accumulation of Fe with aging (Fagundez et al., 2015; James et al., 2015; Klang et al., 2014). In addition, exposure to Fe may also lead to altered locomotion, characterized as a reduction in the body bends and in the head thrashes. As demonstrated by Hu et al. (2008), exposure to 200μM of iron sulfate causes neuronal alterations in the nematode progeny until the third generation. Authors reported a decrease in the body bends, concomitant with increased rate of chemotaxis, in which the worms did not associate NaCl with the food source and continued to explore the plate, therefore indicating damage to sensory neurons (Hu et al., 2008). Notably, recognition of food source, such as E. coli, is directly mediated by dopaminergic neurons in C. elegans. Indeed, Fagundez et al. (2015) demonstrated neurodegeneration of CEP and ADE dopaminergic neurons caused by 0.05mM of iron sulfate. In this same work, the authors described a decrease in the levamisole induction of egg laying. Degeneration of the cholinergic neurons may cause a defect in egg laying due to the ineffective muscle contraction via cholinergic neurons. The worms also presented a reduction in the motor function and in the mechanic response, strengthen this hypothesis.

As noted before, the increase of Fe in the SNC is a hallmark of neurodegenerative diseases, such as Alzheimer’s and Parkinson’s diseases. James et al. (2015) found increased amount of iron in a model strain for Alzheimer’s disease compared to wild type C. elegans (James et al., 2015). Fe dyshomeostasis has been advanced as one of the hypothesis for oxidative stress and increased total Fe load in the substania nigra in PD patients (Chege and McColl, 2014). Patel et al. (2018) demonstrated that worms expressing alpha-synuclein in the dopaminergic neurons were characterized by neurodegeneration with aging, a process that was partially rescued by treatment with iron chelator deferoxamine. When these worms had deletion of snx-3, a retromer endocytic recycler of ferroportin, they were characterized by increased neurodegeneration, indicating that the exporter is important to control Fe intraneuronal levels. This study also demonstrated that worms with a deletion of smf-3, the ortholog of the divalent metal transporter-3 in humans, have decreased dopaminergic neurodegeneration. Therefore, worms that express α-synuclein in the dopaminergic neurons have an age-dependent degeneration that can be modulated by the protein effectors of Fe transport, reinforcing the concerns of excessive Fe intake (Patel et al., 2018).

3.3. Copper neurotoxicity

Copper (Cu) is the third most abundant essential metal, after iron and zinc (Soares et al., 2017). Essential as a cofactor to a range of enzymatic proteins, mainly by its redox ability—Cu (I) and Cu (II)—it is also necessary for mitochondrial respiration, immune defenses, neurotransmitter synthesis and neurotransmitter regulation.

Indeed, Cu exposure in C. elegans causes neuronal alterations evidenced by reduction in the head thrashes, body bends and ROS production (Liu et al., 2022; Xabier et al., 1992; Zhang et al., 2021c). It is also able of inducing an increase in apoptosis. The exposure to Cu induces the expression cholinergic genes such as ace-1, ace-2, ace-3 and ace-4. ace-1 is involved with the AChE expression in the outer epidermis cells and controls vulval pump, whereas ace-2 is expressed mainly in the neurons. These two genes are responsible for 95% of all AChE activities in C. elegans and these findings correlated with the locomotor alterations observed. There was also an increase in the expression of heat shock proteins hsp-16.1, hsp-16.2, hsp-16.48 that are involved with the control of ROS production, possibly a compensatory response to Cu intoxication (Liu et al., 2022). GABAergic neurodegeneration induced by Cu exposure was also characterized by neuronal loss in nerve cords, as depicted by dorsal and ventral gaps, and reduced fluorescence in the AVL, RMEs and RIS cell body neurons (Du and Wang, 2009).

Zhang et al. (2021b,c) evaluated dopaminergic neurons following exposure to Cu, and showed that all tested concentrations led to decreased fluorescence of dopaminergic neurons and a reduction in dat-1 gene expression. Expression of mtl-1 and mtl-2 were also decreased after exposure to Cu, while sod-3 expression was increased. The authors also evaluated trans-generational effects of Cu for five generations. They observed that a decrease in the body length was observed until the second generation and body bends decrease was observed until the fourth generation (Zhang et al., 2021c). The number of head thrashes was decreased until the second generation and the reduction in the fluorescence intensity in dopaminergic neurons was observed until the third generation at the highest Cu concentration. dop-3 gene expression, responsible for dopamine action in the muscle movements, was decreased at all the concentrations in the parental and F1 and F2 generations, which was consistent with the phenotypic alterations. In addition, Cu exposure changed the expression of genes associated with the regulation and transmission of gene information with metal binding properties. jhdm-1, which expresses the demethylase JHDM-1, and mes-4, which expresses the histone methyltransferase, showed increased expression in the parental generation. mys-1 encodes a histone acetyltransferase, a specific chromatin marker that responds to DNA damage, promoting DNA repair. mRNA level of mys-1 in P0, F1 and F2 was dysregulated following parental Cu exposure, which may result in mis-regulation of DNA repair and dopamine receptor deficiency, ultimately leading to neuronal damage and motor function abnormalities.

3.4. Lead neurotoxicity

The non-essential metal lead (Pb) is an environmental neurotoxicant present in soil, water, automobile exhaust, polluted food, paint, among others. It is classified as the second most toxic metal after arsenic. Total Pb burdens in the human body depend on occupation and levels in the environment. It is presumed that a person with 70 kg will have an average of 120mg of Pb distributed in various tissues. The main route of exposure is through the oral intake, being absorbed in the intestine, transported to the liver, kidney and accumulating in bones and teeth. Exposure to high concentrations of Pb can cause encephalopathies (autism, ADHD) in adults and children. Evidences points out to cognitive decline, alterations in behavior, in brain structure and neurodegenerative processes (Kumar et al., 2020). Children are more susceptible to its neurotoxic effects due to the neuronal developmental stage (Akinyemi et al., 2019; Kumar et al., 2020).

Studies in C. elegans have shown neurotoxic sequalae following Pb early life exposure. Akinyemi et al. (2019) demonstrated dopaminergic neurodegeneration caused by exposure to 2.5 and 5mM of Pb acetate in L1 worms. Pb was shown to decrease the number of body bends, dopamine levels and monoamine oxidase (MAO) activity. mRNA levels of the dat-1 transporter were elevated upon Pb exposure. pkc-1 and pkc-2 mutants exposed to Pb did not show alterations in the number of body bends and in dat-1 expression, suggesting that PKC has an critical role in the dopaminergic neurotoxicity induced by Pb (Akinyemi et al., 2019). Chemotaxis was impaired in early exposed worms as well at concentrations as low as 2.5μM. Morphological alterations, length and a decrease in the fluorescence of the ASE neurons was observed at all the tested concentrations. The ech-3 gene encodes a zinc transcription factor necessary for specification and function of the ASE neurons and exposure to Pb reduced the transcription of this gene. Notably, ech-3 mutant worms also presented a reduction in the chemotaxis assay. The authors suggested that early exposure to Pb interfered with the terminal differentiation of the ASE neurons (Xing et al., 2009a).

Exposure in older worms also led to neurotoxic effects. Exposure for 24 h to 2.5, 75 and 100μM of Pb in L4 worms led to decreased number of head trashes and body bends. These authors analyzed several metals, and Pb was one of the most toxic of all, as it impaired behaviors even at a low concentration of 2.5μM (Wang and Xing, 2008). These results are in agreement with those found by Li et al. (2013a,b), where Pb exposure decreased the number of body bends, head trashes and the reversal frequency, in addition to increasing ROS levels and dopaminergic neurodegeneration (dendrite breaks). This work also demonstrated decreased mRNA levels of ttx-1, tax-2, tax-4 e ceh-14, which are required for the differentiation and function of the AFD neurons (thermosensory neurons) (Li et al., 2013a). Wu et al. (2012b) demonstrated a reduction in the thermotaxis behavior into the concentrations of 2.5, 75, 100 and 150μmol of Pb and a neurodegeneration in the AFD sensory neurons as well. These neurons release neuropeptides and glutamate and therefore, it is possible that glutamatergic neurons are affected by Pb exposure (Wu et al., 2012b).

GABAergic neurotoxicity has been also shown upon late exposure to Pb. Exposure to 2.5, 75 e 200μM for 24 h led to neuronal loss, concomitant with decreased number of GABAergic neurons in head Anterior Ventral Process L (AVL), Ring Motor Neurons E (RMEs) and the Ring Inter Neurons S (RIS) (Du and Wang, 2009). In another study, a 6 h exposure at different larval stages caused GABAergic neuronal loss, increasing the number of gaps in the ventral and dorsal GABAergic neurons. Interestingly, worms exposed at the L4 stage showed higher resistance to the neurotoxic effects when compared to earlier stages, and this resistance was higher in young adults, reinforcing the heightened toxicity of Pb in younger subjects (Xing et al., 2009b). This work also demonstrated neurodegeneration in cholinergic neurons, using resistance to aldicarb and levamisole to evaluate presynaptic and postsynaptic neurons, respectively. The results showed the same profile, with worms exhibiting heightened sensitivity to Pb in the tree first larval stages (Xing et al., 2009b). Overall, the data indicate that Pb is more toxic to young worms at early neurodevelopmental stages.

3.5. Mercury neurotoxicity

Methylmercury (MeHg) is an organomercurial form produced by the methylation of inorganic Hg by bacterium in the environment. MeHg bioaccumulates in the trophic levels, reaching humans mainly by fish consumption. MeHg is lipophilic and exposure to this contaminant can cause severe neurological and cognitive deficits, physical congenital damage, motor and sensory alterations. Occupational exposure to inorganic Hg²⁺ is involved with disturbance in the central and peripheral system, tremors, uncoordinated movements, neuronal and renal damage (Bianchini et al., 2022; McElwee and Freedman, 2011).

McElwee and Freedman (2011) compared the effects of Hg²⁺ and MeHg, exposure in C. elegans. Both forms reduced worms’ body length, locomotion, velocity and the mean amplitude of the head movements, but notably MeHg neurotoxicity was more pronounced than Hg²⁺. In agreement, a chronic exposure to MeHg during 3 days altered the worms locomotion from 1 to 3μg/mL, whereas exposure to inorganic Hg²⁺ did not alter locomotion activity even at 5μg/mL (Camacho et al., 2022).

The dopaminergic system is one of the targets of this metal. Early life exposure to MeHg causes a reduction in basal slowing response, particularly in pdr-1 mutant worms (homolog of mammalian parkin). These findings indicate a dysfunction of the DAergic neurons after exposure to this toxicant (Martinez-Finley et al., 2013). Indeed, concentrations as low as 0.3–0.5μM of MeHg caused a neurodegeneration in the CEP dopaminergic neurons. Of note, a reduction in the expression of the antioxidant transcription factor SKN-1/Nrf2 increased the vulnerability of the dopaminergic neurons (Vanduyn et al., 2010). Evidence for specific damage to CEP neurons was also observed upon chronic exposure (10 days, 5μM) to MeHg, whereas no alteration in other dopaminergic, cholinergic or glutamatergic neurons were observed (Ke et al., 2020b). A decline in the speed of the swimming behavior in adults exposed for 48 h from L1 stage (0.05–5.0 μM) was also associated with damage to the dopaminergic system. These alterations were suppressed by a cat-2 mutation (homolog of mammalian tyrosine hydroxylase), with suppressed dopamine synthesis (Ke et al., 2021). As a previous study indicated that MeHg induces dopamine release in PC12 cells (Tiernan et al., 2013), it is reasonable to posit that this metal alters the dopamine neuronal metabolism.

Several studies indicated that MeHg toxicity occurs due to its high affinity for thiol groups (-SH). Ke et al. (2020a) reported that the co-treatment for 1 h with N,N^′bis-(2-mercaptoethyl) isophthalamide (NBMI) prevented acute MeHg neurotoxicity. NBMI is a lipophilic thiol agent and has high affinity for Hg. The work demonstrated the efficacy of NBMI in protecting DAergic neurons from the toxic effects of chronic exposure to MeHg. Co-treatment with NBMI reduced the formation of puncta by more than 60% and also reduced gst-4 and gcs-1 expression. Of note, mitochondrial function was restored, an important target for increased ROS levels caused by MeHg toxicity (Ke et al., 2020a).

3.6. Cadmium

Cadmium (Cd) is a transition metal considered non-essential because it has no physiological function in living beings (Wang and Du, 2013). In addition to being classified as a toxic metal, it is also carcinogenic (Waalkes, 2000; Wang and Du, 2013). Industrial disposal, battery sources and consumption of tobacco derivatives are sources of Cd exposure, which at high concentrations or in a long-term exposure can bring several health problems, including neurological issues (Mead, 2010).

The CNS is readily affected by Cd exposure. Cd intoxication can lead to headache, vertigo, olfactory loss, parkinsonian symptoms, slow gait, peripheral neuropathy, decreased ability to focus, and learning difficulties (Wang and Du, 2013). Elevated concentrations of Cd in children cause developmental delays, and a reduction in the Intelligence Quotient (IQ) was attributed to the high concentration of Cd (Cao et al., 2009; Mead, 2010; Pihl and Parkes, 1977). Exposure to Cd was associated with damage to the cholinergic, dopaminergic and serotonergic systems, to which the symptoms described above are attributed. Cognitive and emotional dysfunctions described by metal intoxication are related to serotonergic damage (Wang and Du, 2013). Dopaminergic damage was evidenced in early-stage exposure (González-Hunt et al., 2014). Recently, the relationship between serotonergic and reproductive damage on lethality rate after exposure to Cd was demonstrated in the C. elegans model. Worms exposed to Cd showed a reduction in serotonin synthesis by inhibiting the expression of the tryptophan hydroxylase enzyme. Reduction in serotonin in the presynaptic membrane caused impairment in contraction of the vulvar muscles, preventing egg laying and inducing the formation of the bagging phenotype, which occurs by the hatching of the eggs inside the worm, which leads to mortality (Wang et al., 2018).

Oxidative stress is also a hallmark of Cd neurotoxicity. Chronic exposure for 16 h to low concentrations of Cd caused a 25% reduction in the activity of CuZn-SOD (SOD-1) and reduced its expression in C. elegans (Bovio et al., 2021). SOD-1 aggregates have been observed in patients with amyotrophic lateral sclerosis (ALS) (Kaur et al., 2016). Furthermore, alteration in the enzymatic activity of SOD-1 has been described in different neurodegenerative diseases, including PD (de la Torre et al., 1996). Thus, the reduction in SOD-1 enzymatic activity induced by Cd exposure suggests that Cd is an environmental risk factor for neurodegenerative diseases (Bovio et al., 2021).

Stress also occurs through the production of advanced glycation end products (AGEs). AGEs bind to a receptor called RAGE, which has been implicated in hyperglycemia and metal-induced neurotoxicity (Lai et al., 2020). The role of RAGEs in Cd-induced neurotoxicity was determined in C. elegans. Using transgenic GPF strains for RAGEs in dopaminergic and serotonergic neurons, the researchers identified that the co-occurrence of metal exposure and RAGE expression can induce neurodegeneration (Lawes et al., 2020).

3.7. Aluminum

As the most abundant metal in the earth’s crust, Aluminum (Al) is widely found, being present in food and drinking water (Brough and Jouhara, 2020). Although abundant, it does not have any known biological function. It has favorable physico-chemical characteristics that make it suitable for industry (Crisponi et al., 2012b). Among the main sources of exposure to the metal are diet, drinking water, cosmetics and drugs (Tietz et al., 2019).

Despite its wide availability and use, Al is a non-essential metal for human health and can be toxic upon exposure. Various toxic effects are attributed to the metal, including an increased risk of developing breast cancer and bone disorders (Darbre et al., 2013; Klein, 2019). However, these findings still need to be characterized. Its neurotoxic effects are well established. Recent studies linked exposure to Al with different neuropathological conditions, including the development of neurodegenerative diseases, such as Alzheimer’s disease and multiple sclerosis, besides neurodevelopmental deficits and autism spectrum disorder (Exley and Clarkson, 2020; Kumar and Gill, 2009).

Al exposure in C. elegans causes significant developmental delay and disrupts the homeostasis of other metals (Page et al., 2012). Transgenerational effects have also been confirmed in the nematode, where exposure to high concentrations of Al caused deficits in behavioral plasticity, which were transferred to unexposed progeny (Wang et al., 2009a; Ye et al., 2008). In addition, memory deficits were detected by using a thermotaxis assay, a behavior controlled by AIY/AIZ and AIY/RIA cells in a presynaptic location or between AFD/AIY neurons at the post-synaptic location, receiving glutamatergic input. In addition, antioxidant vitamin E post treatment led to recovery of this behavior by increasing calcium neuronal signaling (Ye et al., 2008).

In addition, Al exposure for only 30min causes dopaminergic damage by causing a reduction in ATP levels, altering mitochondrial membrane potential and inducing apoptosis. The damage to the dopaminergic system caused by Al is dependent on the smf-3 metal transporter gene, homolog to mammalian DMT-1 (VanDuyn et al., 2013).

3.8. Metal mixtures

The synergistic effects of metals have been studied to better approximate the real exposure environment to the environment created in the laboratory. In fact, human beings are exposed in different ways to a wide variety of toxic elements, and this is unlikely to occur in isolation.

Mn contamination is an example of common co-exposure, usually coexisting with Cd and Pb in environmental and occupational settings. Studies have demonstrated the effects of these metals in C. elegans in conditions of binary and ternary co-exposure. The results indicated that although the mixture Mn+ Pb presented a synergistic effect on lethality, the dopaminergic damage was similar for both Mn+ Pb and Mn+Cd combinations (Lu et al., 2018). The ternary combination of these metals was also investigated, and the dopaminergic degeneration was like the binary combinations. Authors hypothesized that the potential deactivation of catecholamine-based pathways induced by Mn may counteract the toxic effects from deactivation of serotonin- or acetylcholine-based pathways by Cd, while due to the non-specific nature of Pb toxicity, any binary combination with Pb may potentially increase in effect (Tang et al., 2019). Mn contamination is an example of common co-exposure, usually coexisting with Cd and Pb in environmental and occupational environments. One study demonstrated the effects of these metals on C. elegans in a condition of binary and tertiary co-exposure. In addition to toxicological tests, neurodegenerative effects were evaluated using GFP transgenic strain for dopaminergic neurons, the results indicated a potential neurodegenerative effect for the mixture of Mn, Cd and Pb, presenting a more complex pattern in the analyzed endpoints, in addition to other toxic effects in worms (Tang et al., 2019).

Another mixture that was potentially toxic when compared to their individual exposures was MeHg and Mn. Co-exposure to these metals in L1 worms potentiated neurodegenerative effects by reducing body bends, pharyngeal pumping and increased abnormalities in cholinergic neurons. The expression of acetylcholinesterase ace-2, vesicular monoamine transporter cat-1, and antioxidant genes as sod-3, sod-4 and ctl-3 were increased. Thus, exposure to the MeHg and Mn mixture caused greater cholinergic and monoaminergic damage than exposure to each of the metals alone (Schetinger et al., 2019).

Co-exposure to Zn, Cu and Cd was also evaluated in C. elegans. The behavioral tests demonstrated that Zn has a neutral effect on Cd effects, whereas Cu+Zn or Cu+Cd have additives effects in impairing locomotion, enhancing the damage induced by each metal alone (Moyson et al., 2018).

3.9. Other metals

Recent studies have demonstrated neuronal effects in C. elegans caused by other metals that are not commonly known for their neurotoxicity or that have limited studies about toxicity, such as Arsenic, Cobalt and Nickel.

Arsenic is classified as a highly hazardous substance and is considered WHO’s 10 chemicals of greatest public health concern by the WHO (2022). Being well established as a toxic agent in different tissues, (Zhang et al., 2020), the authors sought to elucidate the transgenerational effects of arsenic intoxication. Worms (F0) were exposed to arsenite at different doses and after analyzing the behavioral effect on the treated worms, the offspring of subsequent generations (F1 and F2) were evaluated. In F0, the data indicated behavioral damage and neuronal degeneration. Behavioral damage was observed in subsequent F1 and F2 generations even absent arsenic exposure. Another study showed that arsenic intoxication was dependent on a bicarbonate transporter, ABTS-1, responsible for regulating cell volume and pH (Liao et al., 2010).

The molecular mechanisms of cobalt intoxication, characterized by cognitive impairment and peripheral neuropathy, were elucidated using C. elegans as an experimental model. In this study, exposure to CoCl₂ for 2 h caused mitochondrial fragmentation, growth impairment, apoptosis, autophagy and oxidative stress by activating DRP-1, responsible for mitochondrial fission (Zheng et al., 2020).

The neurotoxic effects of nickel were recently investigated in a model of acute neurotoxicity in C. elegans, after 1 h of exposure to NiCl₂ at the first larval stage (L1). Worms showed dose-dependent cholinergic, dopaminergic and GABAergic degeneration. In addition, behavioral assays reflected the effects of Ni on cholinergic and dopaminergic function. The authors confirmed that the observed neuronal effects were associated with oxidative stress by inducing GST-4 expression, suggesting that neuronal damage was induced by oxidative stress due to Ni exposure during worm development (Ijomone et al., 2020).

4. Metallic nanoparticles neurotoxicology in C. elegans

Inorganic nanoparticles are materials with nanometric dimensions with a central core composed of inorganic specimens, especially metals, which give them exciting applications. They have diverse shapes (spherical, layers, oval), different superficial charge that grant stability to the nanostructure, and changes in composition (Mabrouk et al., 2021). NPs have been developed to enable more efficient and low-cost products, seeking better properties and applications. However, the same properties that make nanomaterials attractive may also be responsible for harmful effects in living organisms and concerns about their release into the environment have raised in the last years (Domingues et al., 2022).

Metallic NPs can be composed of metals, metallic oxides and metallic salts (Contado, 2015). As reviewed above, neurons are cells with known vulnerability to metal toxicity; therefore, it is important to observe the effects of metallic NPs in this system (Soares et al., 2017).

Because of their small size, which results in a large surface area, nanoparticles can easily enter the cells from various routes (pulmonary, dermic, gastrointestinal), have increased absorption capacity, cross the blood-brain barrier and remain in the central nervous system for long periods of time (Mushtaq et al., 2015; Win-Shwe and Fujimaki, 2011). NPs can also cause mechanical damage to tissues due to their structure and induce inflammatory processes. Studies have shown that most of the toxicity of various NPs could be attributed to the release of the metallic ions after cellular uptake by phagocytosis. However, there is no conclusive answer so far about whether NP toxicity is due to specific particle effects or the released metal ion or caused by the charge, surface modification, radiation they may emit (Wu et al., 2019).

Oxidative stress has been considered the prime mechanism of NPs causing toxic effects. ROS accumulation have been observed in C. elegans treated with a variety of NPs (Kong et al., 2017; Kumar et al., 2017; Rogers et al., 2015; Wu et al., 2011; Yu et al., 2011; Zhang et al., 2011). The brain is highly susceptible to ROS-induced neurotoxicity due to the high oxygen consumption and rapid metabolic activity (Mao et al., 2010). Likewise, some studies have shown that ROS levels, as reliable indicators of oxidative stress, are closely related to neurotoxicity (Abramov et al., 2007). Therefore, it remains prudent to investigate pathobiological changes of the nervous system in response to NP exposure. Unfortunately, there are few studies assessing the neurotoxicology of metallic NPs in vivo in rodents and in alternative models such as C. elegans.

4.1. Ag-NPs

Ag-NPs are the most studied nanomaterials due to their important properties such as chemical stability, malleability, high electrical and thermal conductivity, catalytic activity and potent antimicrobial action, in addition to relatively low production cost (Durán et al., 2019). The world production of Ag-NPs ranges between 135 and 420 tons per year (Pulit-Prociak and Banach, 2016; Syafiuddin et al., 2017) and their main application are in medical products, clothing, sports equipment, electronics and as food and drinks preservative (Seabra and Durán, 2015; Castellano et al., 2007; Hartemann et al., 2015; Nowack, 2017). Consequently, environmental release of Ag-NPs and its potential neurotoxicity has attracted a great deal of attention (Sørensen and Baun, 2015).

In C. elegans, Ag-NPs accelerate the age-associated decline in swimming and the increase in uncoordinated movements. This behavioral damage induced by NPs was correlated with axonal protein aggregation and neurodegeneration in serotonergic HSN neurons and in sensory ADF neurons, which control egg-laying behavior and locomotor phenotypes, respectively (Seabra and Durán, 2015). C. elegans exposed to Ag-NPs also showed changes in locomotion behaviors such as the number of head thrashes, body bends, pharyngeal pumping frequency and defecation interval, and sensory perception behaviors such as chemotaxis and thermotaxis assays. In addition, a decrease in the fluorescence of dopaminergic, GABAergic and cholinergic neurons was observed in a dose-dependent and time-dependent manner. The same was not observed in glutamatergic neurons, suggesting differential susceptibility of different types of neurons to Ag-NPs exposure in C. elegans. In fact, it has been demonstrated that exposure to low doses of Ag-NPs increased in the gene expression of γ-aminobutyric acid and dopamine receptors in the nematodes. These findings suggest that higher doses of Ag-NPs could cause neurotoxicity through an imbalance in neurotransmitter signaling (Zhang et al., 2021b).

Genetic analysis demonstrated that Ag-NPs exposures consistently reduced several biological processes such as regulation of locomotion, reproduction and cell growth, as well as neuroactive ligand-receptor interaction pathways Wnt and MAPK signaling (Viau et al., 2020). The alterations caused by Ag-NPs exposure in locomotion might be involved with the negative regulation of genes involved in the neuroactive receptor-ligand interaction as well as those of the octopamine receptor family (ser-1) (neuroactive receptor-ligand interaction pathway), skp1 (component of the ubiquitin ligase complex, skr-8, skr-10, skr-12,from the WNT signaling pathway), ver-1 protein (ver-1) and heat shock protein (hsp-70) (both from MAPK signaling pathway) (Viau et al., 2020).

The behavior of the Ag-NPs can be influenced by environmental factors that alter their bioaccumulation and toxicity. The change in the ionic force of the exposure medium in C. elegans (presence of NaCl, phosphate, for instance) significantly increased reproductive toxicity and neurotoxicity (number of head thrashes and body bends) caused by Ag-NPs in C. elegans. In addition, a greater ionic force increased the bioaccumulation of Ag-NPs in E. coli and consequently in nematodes, indicating that the bioavailability and potential ecotoxicity of Ag-NPs are associated with environmental factors (Yang et al., 2018).

Another study using Ag selenide quantum dots (Ag₂Se-QDs) demonstrated that these nanomaterials accumulated in the body of nematodes, decreasing lifespan and impairing neurobehaviors such as head thrashes and body bends in C. elegans, a result similar to those found with Ag-NPs (Liang et al., 2022). Overall, these results suggested that Ag-NPs cause neurotoxicity in C. elegans.

4.2. Fe-NPs

Fe nanoparticles possess a high magnetic nature, high surface area, electrical and thermal conductivity. They also have dimensional stability. Due to the properties of Fe nanoparticles, they are known as magnetic nanoparticles. Magnetic nanoparticles have been explored widely in the decades due to their large number of applications in the field of spintronics, biology, and medical science (Umair et al., 2016). Nanoparticles that are made of ferro-or ferromagnetic materials below a certain size (generally 10–20 nm) can exhibit a unique form of magnetism called superparamagnetism. Although the potential benefits of Fe-NPs are considerable, exposure can result in significant cytotoxicity. When cells are exposed to high doses of Fe oxide nanoparticles, excess ROS formation occurs, affecting the normal functioning of the cells, leading to apoptosis or cell death, growth delay, development abnormality and decreased fertility (Valdiglesias et al., 2016).

The impact of surface charge on polymeric shell coated FeO-NPs has been studied in C. elegans. When coated with the negatively charged polymer PMDA (Poly-Maleic Acid Conjugated with Dodecylamine), these NPs have been proven to be toxic, shortening the lifespan of the nematode, increasing ROS production and reducing the rate of pharyngeal pumping. Of note, decreased pharyngeal pumping is an indicative of the presence of neurotoxicants. On the other hand, zwitterionic NPs (partially cationic and anionic) exhibited lower toxicity, better distribution, and higher Fe delivery (Amigoni et al., 2021). In another study, DMSA (Dimercaptosuccinic acid) coated FeO-NPs depicted toxicity by altering growth, reproduction, locomotion, pharyngeal pumping, defection and autofluorescence (Wu et al., 2012a). Other forms of Fe-NPs such as cube-like Fe nitride (α^”-Fe₁₆N₂) and zero valent Fe NPs (nZVI) showed lower or no neurotoxicity in the nematodes, although oxidative stress was found in exposed animals (Gubert et al., 2022; Yang et al., 2016). FeO-NPs have utility in a wide variety of applications. While their benefits are considerable and they are considered of low risk, their neurotoxic potential must be further investigated (Karlsson et al., 2009; Singh et al., 2010). Even the coating strategy was not effective in reducing their toxicity, and therefore much work needs to be done to reduce their potential impact on living organisms.

4.3. Al-NPs

Aluminum oxide nanoparticles are increasingly used in domestic, industrial and medicine scales (M’rad et al., 2018). Aluminum (Al) is a vital etiopathogenic agent and has been associated with the incidence of neurodegenerative diseases (Halatek et al., 2005). However, despite the fact that some nanoparticles are shown to cause neurotoxic effects in rodents, their influence on the nervous system is limited (M’rad et al., 2018). Studies that investigate the toxicity of Al-NPs indicate that, as most of NPs, toxicity occurs by oxidative stress.

More recently, the acute toxicity of Al₂O₃ NP- has been examined in C. elegans with lethality, growth, reproduction and stress response outcomes (Wang et al., 2009b; Wu et al., 2011). Nematodes chronically exposed to Al₂O₃-NPs for 10 days suffered abnormal locomotion behavior, which was associated with increased ROS production and suppressed antioxidant defenses. The antioxidant enzymes encoded by the sod-2 and sod-3 genes were down-regulated and the mutants were more susceptible to these NPs, implying that compromised antioxidant mechanisms may also contribute to their neurotoxic effects (Li et al., 2012b). After exposure for 3 days, significant neurotoxicity was noted characterized by decreased number of head thrashes, in addition to decreased locomotion speed, which were more significant for Ag- and Si-NPs (Viau et al., 2020). When compared to Si-, Ti- and polystyrene NPs, Al₂O₃-NPs was the most neurotoxic (Li et al., 2020).

Li et al. (2013a,b) demonstrated that glutamate transporter EAT-4, serotonin transporter MOD-5, and dopamine transporter DAT-1 might pose important molecular targets of Al₂O₃-NPs neurotoxicity. They observed that the hypo-locomotor effect is driven by movement inhibition of downstream effectors D1-like dopamine receptor DOP-1, ionotropic serotonin receptor MOD-1, and non-NMDA glutamate receptors GLR-2 and GLR-6 (Li et al., 2013b).

Surface modifications were assessed with Al₂O₃– NPs in a recent study, which compared pristine Al₂O₃-NPs (p-Al₂O₃), hydrophilic (w-Al₂O₃) and lipophilic (o-Al₂O₃) modifications (Zhang et al., 2021a). The lipophilic form caused higher toxicity by reducing head trashes, increasing ROS formation and mitochondrial damage and inducing apoptosis. Transcriptomic analysis indicated not only differential expression in genes related to glutathione metabolism and peroxisome pathways, but also in genes that control locomotion, since the exposure to this metal changes the expression of several genes responsible for various biological processes in the worms (Zhang et al., 2021a).

4.4. Cu-NPs

Copper toxicity is widely known, mainly due to the intracellular accumulation that leads to disturbances of mitochondrial metabolic enzymes and cell death (Kahison and Dixon, 2022). Thus, studies evaluating the neurotoxicity of Cu-NPs deserve attention, since these NPs have considerable cytotoxicity and can cause long-term effects (Fahmy and Cormier, 2009; Ivask et al., 2010; Keller et al., 2013). Currently, CuO-NPs are mainly used in electronics, including gas sensors, batteries and solar energy, as these NPs have great superconducting properties. In addition, CuO-NPs are also incorporated into various personal and household hygiene products due to their bactericidal action (Bondarenko et al., 2013).

Exposure to CuO-NPs has been shown to cause toxic effects on behavioral assays by reducing locomotion velocity, head thrashes and pharyngeal pumping in C. elegans (Viau et al., 2020). The study suggests that Cu-free ions released by the NPs in the solution may have been absorbed by the worm’s food source, E. coli. After absorption, Cu ions can be found in the head and the worms’ whole body (Gao et al., 2008). Eating behavior was considered the most sensitive toxicological endpoint after CuO-NPs exposure since it was affected by all tested concentrations (Viau et al., 2020). Notably, intestinal intake of CuO-NPs is probably the entry point of NPs in C. elegans, considering that the cuticle is poorly permeable (Mashock et al., 2016; Song et al., 2014). Data show that this reduction in the nematode eating behavior is a response to environmental stress (through sensory neurotransmitters) and changes in pharyngeal pumping activity (Raizen et al., 2018). In addition, high Cu levels have been reported to induce paralysis, reducing feeding and reproduction by affecting pharyngeal pumping and egg laying, respectively (Peterson et al., 2008). These behavioral alterations may be related to the degeneration of dopaminergic neurons. Furthermore, mutants of the bivalent metal transporters, smf-1 or smf-2, showed greater tolerance to Cu exposure, implicating both transporters in Cu-induced neurodegeneration (Mashock et al., 2016).

4.5. Au-NPs

Gold nanoparticles (AuNPs) are a biocompatible class of nanomaterials widely used for bioimaging (Chen et al., 2015a; Shang et al., 2013), biosensing (Chen and Tseng, 2012; Liu et al., 2015), facial creams (Guix et al., 2008), and targeted therapeutic purposes (Wang et al., 2011; Zhang et al., 2014). Nevertheless, these nanoparticles can be directly taken up by animals, affecting their physiology and thus posing a toxicological risk. The uptake and toxicity of AuNPs highly depends on their shape ( Jin et al., 2013), size (Ma et al., 2011) and surface charge (Albanese et al., 2012; Jiang et al., 2015).

Hu et al. (2018a) demonstrated significant changes in worm locomotion after exposure to AuNPs, raising the hypothesis that this effect is based on impaired motor neuron function. Therefore, when examining primary neurons isolated from C. elegans, before and after exposure to AuNPs, it was found that axonal growth was significantly disrupted by exposure to AuNPs (Hu et al., 2018a). Neuronal development impairment may be related to altered expression of cut-3 and fil-1 (both involved in body morphogenesis) and dpy-14 (expressed in embryonic neurons), in addition to mtl-1, important for metal detoxification and homeostasis (Mo et al., 2020).

A toxicogenomic analysis demonstrated that AuNPs are bioavailable and cause adverse effects to C. elegans by activating both general and specific biological pathways. There were a total of six upregulated C. elegans genes (apl-1, clp-1, kin-19, par-1, kin-1 and kin-2) homologous to the human amyloid processing pathways (Selkoe, 2001; Tsyusko et al., 2012). The up-regulation of apl-1, amyloid precursor like protein, which is a homolog of the human amyloid precursor protein (app-1) is associated with Alzheimer’s disease in humans (Zheng and Koo, 2011), was confirmed with qRT-PCR. In C. elegans, apl-1 is expressed in multiple tissues, being important for molting, morphogenesis, and larval survival (Hornsten, 2001). Together with Feh-1 (homologue of mammalian Fe65), which is expressed in neuromuscular structure of the pharynx and which was also up-regulated in response to AuNPs, apl-1 forms a complex, controlling the rate of pharyngeal pumping (Zambrano et al., 2002). Even though C. elegans does not accumulate β-amyloid, activation of genes related to the amyloid processing pathways may be indicative of events leading to neurodegeneration and can be a result of an unfolded protein response activated in response to Au NP exposure (Tsyusko et al., 2012).

4.6. Ti-NPs

TiO₂-NPs are the most studied metal oxide NPs today because they are widely used as pigments and additives for paints, papers, ceramics, plastics, foods and pharmaceuticals (Aitken et al., 2006; Gélis et al., 2003). Therefore, these NPs have become an important environmental contaminant and studies have been carried out to characterize their neurotoxicity.

Exposure to TiO₂-NPs suspensions induces lethality in C. elegans, in addition to significantly reducing body length, head movement and body curvature of these animals (Li et al., 2012a, 2020; Viau et al., 2020; Wu et al., 2012c, 2013). Furthermore, sod-2, sod-3, mtl-2 and hsp-6.48 expression were altered by exposure and associated to the neurotoxicity of TiO₂-NPs, as locomotion behavior was impaired (Wu et al., 2014). Dong et al. (2018) investigated the combined effects of TiO₂-NPs and nanopolystyrene at environmentally relevant concentrations. Co-exposure to nanopolystyrene particles enhanced the toxicity of TiO₂-NPs by decreasing locomotion behavior and inducing intestinal ROS levels in both WT nematodes and sod-3 mutants at higher levels than TiO₂-NPs alone (Dong et al., 2018).

Prolonged exposure to TiO₂-NPs also decreased the fluorescence intensity of AVL and DVB GABAergic neurons, a degeneration that could not be recovered (Zhao et al., 2014). C. elegans neurons were able to directly uptake anatase and rutile NPs (mineral forms of TiO₂) and this uptake inhibited axonal growth, associated with a decline in body length and worm locomotion (Hu et al., 2018b, 2020). Changes in genes related to metal binding or metal detoxification (mtl-2, C45B2.2 and nhr-247), genes involved in worm growth and body morphogenesis (mtl-2, F26F2.3, C38C3.7 and nhr-247), and genes involved in neuronal function (C41G6.13, C45B2.2, srr-6, K08C9.7 and C38C3.7) were also observed. In addition, cl-1 (stress resistance regulator), wah-1 (oxidoreductase), gst-3 (glutathione-S-transferase) and cyp-33C1 (cytochrome P450) were also altered, suggesting a relationship between neurotoxicity and the increase in ROS levels (Hu et al., 2018b).

4.7. Cd-NPs

The transition heavy metal cadmium is a ubiquitous toxic substance present in soil, water, air and food. As a consequence of anthropic activity, the environmental concentrations of Cd increased steadily in many areas around the world (Faroon et al., 2013). Cd is readily absorbed and accumulated in different tissues and organs. Exposure to Cd may have a potential impact on human health and wildlife (Davison et al., 1988).

In nanotechnology, Cd is primarily utilized in the construction of particles known as quantum dots (QDs), which are semiconductor metalloid-crystal structures of approximately 2–100 nm, containing about 200–10.000 atoms (Juzenas et al., 2008; Smith et al., 2008). Due to their small size, QDs have unique optical and electronic properties that impart the nanoparticle with a bright, highly stable, “size-tunable” fluorescence. The large surface area imparted by small size also makes QDs readily functionalized with targeting ligands for site-directed activity. Based on these properties, QDs have the potential for revolutionizing biological imaging at the cellular level, cancer detection and treatment, radio- and chemo sensitizing agents, and targeted drug delivery (Alivisatos, 2004; Hardman, 2006; Juzenas et al., 2008; Smith et al., 2008). However, enthusiasm for QDs is somewhat diluted by the fact that QDs contain substantial amounts of cadmium in a highly reactive form (Rzigalinski and Strobl, 2009).

Exposure to CdSe/ZnS quantum dots has been documented to inflict a defective egg laying phenotype in C. elegans, which implies that motor neurons are involved in reproduction (Galdiero et al., 2020). RME neurons of the head region and AVL and DVB neurons that control defection showed developmental and functional defects after exposure to CdTe quantum dots at the L1 stage of the worm (Zhao et al., 2015). Wu et al. (2015) demonstrated that CdTe QDs alter locomotion, pharyngeal pumping, defecation cycle and even plasticity in learning and memory, time and dose-dependently. At the molecular level, the biological endpoints were likely to be associated with altered expression of genes involved in glutamate, serotonin and dopamine signaling coupled with markedly increased ROS generation (Wu et al., 2015). The pathway by which QDs inflict neurotoxicity in C. elegans involves deposition inside the neurons, perturbations of neuronal development and abnormal neurotransmission. Depending on the experimental conditions, the response seems to be mediated by various degrees of metal toxicity from Cd²⁺ leaching out of the core in conjunction with NP derived oxidative stress (Sinis et al., 2019).

4.8. Si-NPs

Due to the unique properties of silica NPs such as surface functionality, controllable particle size, and desirable biocompatibility, studies have explored these nanomaterials (Shahbazi et al., 2012; Tao et al., 2014; Zhang and Kong, 2015). The large-scale production and application of Si-NPs increased the risk of human exposure, making it necessary to investigate their neurotoxic effects (Jiang and Gao, 2016).

Prolonged exposure to a form of Si-NPs, mSi-NPs (mesoporous silica nanoparticles), showed locomotion degeneration, shrinkage behavior, and abnormal foraging behavior, which were associated with deficits in the development of GABAergic neurons, including D-type and RME motor neurons. In addition, there was excessive generation of ROS contributing to neuronal damage (Liang et al., 2020b).

Exposure to SiO₂-NPs led to toxic effects on locomotion velocity and head thrashes, in addition to inducing ROS production in C. elegans (Li et al., 2020; Wu et al., 2013).

Genetic analysis demonstrated that SiO₂-NPs and Si-NPs exposures consistently reduced several biological processes such as regulation of locomotion, reproduction and cell growth (Liang et al., 2020a), as well as neuroactive ligand-receptor interaction pathways, Wnt and MAPK signaling, involved in the regulation of cell reproduction and growth (Viau et al., 2020).

Nematodes exposed to Si-NPs significantly accumulated insoluble and ubiquitinated proteins, which did not occur with worms exposed to bulk silica. Ubiquitin is a protein that marks malformed proteins to be degraded. An analysis of gut cells showed the formation of amyloid-like structures in the worms treated with Si-NPs. These results indicate that Si-NPs induce fibrillation of endogenous proteins and amyloid aggregates. An increase in insoluble proteins in worms treated with Si-NPs was observed throughout their life, altering protein homeostasis in aged worms (Scharf et al., 2013). Pharyngeal pumping rate was also significantly reduced, an endpoint that indicates accelerated aging and muscle atrophy from altered proteostasis (Scharf et al., 2013). Analysis of serotonergic neural cells revealed that protein aggregation induced by Si-NPs was evident in axons of HSN neurons, with presynaptic accumulation of serotonin, inducing disturbed axonal transport that reduces neurotransmission capacity and egg laying of C. elegans (Scharf et al., 2016). As insoluble proteins are involved in the aggregation of the beta-amyloid peptide in Alzheimer’s disease (AD) and alpha-synuclein in Parkinson’s disease (PD) (Pereira et al., 2022), exposure to Si-NPs may cause long-term neurotoxicity by insoluble proteins formation.

4.9. Other metallic nanoparticles

Studies on metallic nanoparticles neurotoxicity are scarce, particularly of some metals that will be described below.

CeO₂-NPs are mainly used as catalysts (Prasad and Rattan, 2010), diesel fuel additive (Park et al., 2008) and for glass and ceramic applications (Younis et al., 2016). However, CeO₂ NPs have been considered one of the most toxic NPs to the environment. Exposure of CeO₂-NPs caused an impairment in head thrashes in C. elegans, but the mechanism of this toxicity is still unclear and needs to be studied (Viau et al., 2020). CeO₂-NPs cause an increase in ROS levels in C. elegans, and this imbalance in redox homeostasis may be related to the observed behavioral effects (Zhang et al., 2011).

ZnO-NPs have a wide range of applications, including antimicrobial properties, widely used by the cosmetics and paint industries ( Jiang et al., 2018). In addition, they are used in agriculture to control foodborne pathogens (Nohynek et al., 2010; Tayel et al., 2011). Despite their commercial advantages, studies have shown that exposure to ZnO-NPs may cause toxicity, mainly due to the accumulation in the environment and increased production of ROS (Nowack et al., 2011; Schilling et al., 2010). ZnO-NPs have been shown to cause harmful effects on C. elegans locomotion (Huang et al., 2017; Wu et al., 2013). This behavioral change was associated with decreased ATP levels and significant increase in intracellular ROS levels and lipid peroxidation, suggesting mitochondrial damage, triggering neuronal death (Huang et al., 2017, 2019). Furthermore, ZnO-NPs triggered the translocation of the transcription factor DAF-16/FOXO from the cytoplasm to the nucleus and activated the expression of stress-responsive genes mtl-1 and sod-3 (Huang et al., 2019). Therefore, oxidative stress appears to be strongly associated to the neurotoxicity caused by these NPs.

5. Perspectives and concluding remarks

Using the alternative animal model C. elegans to investigate and understand the neurotoxicity caused by metals and metallic NPs has been fruitful and important for human health and environmental impact assessments. As discussed here, several neuronal systems can be affected, as evidenced by behavioral assays and by in vivo visualization though GFP-tagged neuronal proteins. The main systems affected by metals are DAergic, serotonergic, GABAergic and glutamatergic (sensory neurons). Furthermore, transcriptomic analysis and the use of mutants has indicated that metals and NPs cause neurotoxic effects by disrupting the neurotransmitter signaling (mainly neurotransmitter synthesis and degradation/reuptake processes), by altering the antioxidant enzymatic response, by inducing apoptosis and by interfering in cell division and differentiation. It is concerning that some studies have evidenced the ability of some metals and NPs to promote epigenetic transgenerational inheritance neurotoxicity, which should be further investigated for all metals and NPs.

It is noteworthy some limitations of the model and of the protocols used. It is important to investigate the effects of metals and metallic NPs in all worms stages and using a wide range of concentrations and times of exposure. However, the exposure protocols are so different among groups that makes hard to compare the levels of neurotoxicity. Furthermore, the exposure medium may differ among studies, which can totally change the toxicological outcomes. C. elegans also has limitations for neurotoxicological assessments. It does not have a cardiovascular system, no brain, liver or kidneys. On the other hand, the model respects the 3R policy (reduce, refine and replace), which are pillars of the modern toxicology to avoid the unnecessary use of vertebrates. Finally, the findings from C. elegans have been providing information to help understand potential risks of metals and metallic NPs, especially at the environmental level and to be extrapolated to human outcomes.