Recent insights into autophagy and metals/nanoparticles exposure

Abstract

Some anthropogenic pollutants, such as heavy metals and nanoparticles (NPs), are widely distributed and a major threat to environmental safety and public health. In particular, lead (Pb), cadmium (Cd), chromium (Cr), arsenic (As), and mercury (Hg) have systemic toxicity even at extremely low concentrations, so they are listed as priority metals in relation to their significant public health burden. Aluminum (Al) is also toxic to multiple organs and is linked to Alzheimer’s disease. As the utilization of many metal nanoparticles (MNPs) gradually gain traction in industrial and medical applications, they are increasingly being investigated to address potential toxicity by impairing certain biological barriers. The dominant toxic mechanism of these metals and MNPs is the induction of oxidative stress, which subsequently triggers lipid peroxidation, protein modification, and DNA damage. Notably, a growing body of research has revealed the linkage between dysregulated autophagy and some diseases, including neurodegenerative diseases and cancers. Among them, some metals or metal mixtures can act as environmental stimuli and disturb basal autophagic activity, which has an underlying adverse health effect. Some studies also revealed that specific autophagy inhibitors or activators could modify the abnormal autophagic flux attributed to continuous exposure to metals. In this review, we have gathered recent data about the contribution of the autophagy/mitophagy mediated toxic effects and focused on the involvement of some key regulatory factors of autophagic signaling during exposure to selected metals, metal mixtures, as well as MNPs in the real world. Besides this, we summarized the potential significance of interactions between autophagy and excessive reactive oxygen species (ROS)-mediated oxidative damage in the regulation of cell survival response to metals/NPs. A critical view is given on the application of autophagy activators/inhibitors to modulate the systematic toxicity of various metals/MNPs.

Introduction

Heavy metals are non-biodegradable with toxic effects on some microorganisms, plants, animals, and human bodies [1]. Inorganic arsenic (iAs) ranks first in the list of toxic hazards listed by the U.S. Environmental Protection Agency, and Pb ranks second [2]. Heavy metals mainly come from the chemical industry, agriculture, urban areas, and environmental emergencies. With the widespread use of metals and their NPs, people are increasingly exposed to heavy metals such as Pb, Hg, As, Cd, Cr, and Al in a variety of ways and opportunities.

Nowadays, metal pollution has become a crucial exogenous cause affecting public health that can induce acute, chronic, and long-term health hazards in exposed people. Prolonged exposure to Cr can lead to ailments such as bronchitis, dermatitis, and lung cancer [3]. In addition, Al has multiple organ toxicities, which can bring about maladies including liver and kidney diseases, pancreatitis, myocarditis, enteritis, anemia, Alzheimer’s disease, and dementia [4]. In most cases, humans are essentially exposed to a whole range of environmental metals simultaneously instead of one single metal. The study on the hazards of metal mixture exposures is of greater public health significance, partly due to the combined toxicity promoted by the common cellular regulatory pathways [5].

Of note, metal nanoparticles (MNPs) have shown great potential in various applications worldwide, however, some toxic MNPs are considered detrimental to human health in real-life [6]. In vitro and in vivo studies have shown that some MNPs are immunotoxic, reproductively toxic, neurotoxic, and developmental toxic [7, 8]. The main mechanisms by which MNPs exert toxicity are binding to the cell surface directly to disrupt the cell membrane, releasing poisonous metal ions, and inducing the production of ROS [9]. It is generally agreed that elevated ROS levels play a prominent part in the mechanism of toxicity in MNPs [10]. While triggering oxidative stress is among the common mechanisms of NPs, cellular autophagy is rather unique and specific in response to MNPs exposure [11].

The autophagic processes are complicated and involve the removal of damaged organelles and cell wastes and the decomposition of non-critical components to provide energy in a low-energy state. Autophagy in mammalian cells can be active at both a basal level and under stressful conditions [12]. At present, accumulating studies have identified autophagy as being widely involved in diverse physiological and pathogenetic processes underlying neurodegenerative diseases [13], cancers [14], and metabolic diseases [15]. Moreover, current studies have found that abnormalities in autophagic function are also strongly associated with metal toxicity [16, 17], and the regulation of autophagy has been proven to modify metal-induced toxicity [18, 19]. As the research discovered that Cr(III) activated sphingomyelin phosphodiesterase 2 (SMPD2) to induce autophagosome formation [20]. Cd could block autophagic flux to induce hepatocyte injury via breaking the integration of autophagosomes and lysosomes. Hepatocyte injury was aggravated after the addition of the autophagy inhibitor chloroquine (CQ) to block autophagic flux [21]. At present, autophagy has been considered as a potential mechanism in metal toxicity alongside the maintenance of cellular metabolic homeostasis. Herein, we focus on the toxic effects induced by single/mixed metals and NPs contact, followed by a summary of the changes in autophagy levels with their exposure, along with the effects of autophagy regulation in certain adverse reactions induced by them.

Overview of autophagy

Autophagy, also known as type II programmed cell death, refers to the normal dynamic life process in which cells are degraded by lysosomes, which selectively remove damaged, aged, or surplus biological macromolecules or organelles and release free small molecules for cell recycling. Notably, autophagy mediates cell death and diverse diseases in the case of different kinds of metal insults [16, 22].

The key regulatory factors of autophagy

The key factors and pathways implicated in the molecular machinery of autophagy are as follows: (1) Target of rapamycin/Mammalian target of rapamycin (TOR/mTOR): TOR is widely involved in physiological activities and essential for cell growth and metabolism. As a core molecule in the regulation of autophagy, it is a key protein in the control of autophagy, sensing various signals of cellular changes and enhancing or decreasing the level of autophagy occurrence. The unc-51-like autophagy activating kinase 1 (ULK1) complex is composed of ULK1 itself, autophagy-related gene 13 (Atg13), FIP200, and autophagy-related gene 101 (Atg101) [23]. It is instrumental in recruiting autophagy proteins to initiate autophagy formation [24]. It was noted that mTOR phosphorylates ULK1 to inhibit autophagy when it is active [25]. (2) Phosphatidylinositol 3-kinase/protein kinase-B/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway: PI3K1 phosphorylates Phosphatidylinositol 4,5-bisphosphate (PIP2) to phosphatidylinositol 3,4,5-trisphosphate (PIP3), and Akt inhibits the tuberous sclerosis complex 1/tuberous sclerosis complex 2 (TSC1/TSC2) complex after being activated by PIP3, thus activating mTOR and inhibiting autophagy [26]. (3) AMP-activated protein kinase (AMPK) is a vital energy receptor that modulates cellular metabolism and maintains energy homeostasis. A decrease in intracellular ATP levels potentiates the phosphorylation and activation of AMPK by live kinase B1 (LKB1), which then inhibits mTOR complex 1 (mTORC1) activity via the phosphorylation and activation of TSC2 or by conjugation to RAPTOR, a key subunit of mTORC1 [27, 28]. AMPK can also promote autophagy directly by phosphorylating the vacuolar protein sorting 34 (VPS34) complex, or indirectly by regulating transcription factors (such as forkhead box O3 (FOXO3), transcription factor EB (TFEB), and bromodomain-containing protein 4 (BRD4)) [25, 29]. (4) Tumor suppressor p53 (p53) is a crucial regulator during autophagy. It was found that p53 in the nucleus can increase autophagy by activating selective regulatory factors upstream of mTOR, while p53 in the cytoplasm can inhibit autophagy [30–32]. (5) TFEB is in charge of autophagy and lysosomal gene regulation and has a pivotal position within autophagy-lysosome biogenesis [33]. Mitofusin 2 (MFN2) is a mitochondrial fusion protein that strengthens autophagosomes formation and promotes the fusion of autophagosomes and lysosomes [34]. In addition, damage-regulated autophagy modulator 1 (DRAM1), Rab7, and lysosome-associated membrane protein 2 (LAMP2) exert critical functions to regulate autophagosomes and lysosomes fusion [34–36].

The regulatory process of autophagy

Cellular autophagy is a dynamic process that includes the generation of autophagosomes, autophagosomes and lysosomes integration, and autophagosomes decomposition, which involves the formation and fusion of membranes. Especially, each procedure is regulated by relevant regulatory factors. Figure 1 depicts a journey from phagophores to autolysosomes, including activation [37–40] and elongation [37, 38, 41, 42] of the phagophores, maturation of autophagosomes [41, 43], fusion of autophagosomes and lysosomes [43–45], as well as degradation of the autolysosomes.

Fig. 1.

A schematic diagram illustrates how ULK1-Atg13-FIP200, Beclin-1-VPS34-VPS15, Atg5-Atg12-Atg16L, the HOPS complex, mTOR, LC3, Rab7, TFEB, etc. interact during autophagy and their effects on the journey from the formation to the degradation of autophagosomes

Toxicity of Cd and autophagy dysfunction

At present, the main ways of exposure to Cd are occupational exposure, environmental Cd pollution, tobacco consumption, food, etc. [46]. Cd, a class I carcinogen in humans [47], is mainly toxic to liver and kidney, and exposure to Cd may cause damage to the liver’s free radicals and lipid metabolism [48], as well as disruption of renal tubular function [49]. Cd is reproductively toxic, interfering with the synthesis and secretion of reproductive hormones [50], damaging the blood-testis barrier (BTB), and affecting sperm count and viability [51]. Cd can also act on vascular endothelial cells and is an essential external risk factor for atherosclerosis and hypertension [52]. Besides, Cd is immunotoxic in that its exposure can lead to alterations in the number, maturation, and function of T cells [53].

The molecular mechanisms of Cd toxicity are primarily oxidative stress, genetic expression abnormalities, signaling pathways abnormalities caused by calcium (Ca²⁺) disorder, and the dynamic modulation of enzyme activity [54–57]. As research progressed, autophagy was also identified as being involved in the toxicity of Cd. On the other hand, the FOXO family also has a crucial role in autophagy. It showed that AMPK could phosphorylate the Ser588 site of FOXO3a and Cd exposure could phosphorylate the Thr172 site of the AMPK protein, thus inducing mesenchymal stem cells’ (MSCs’) death by AMPK/FOXO3a-mediated autophagy [58]. Moreover, activation of transcription factor binding to IGHM enhancer 3 (TFE3) could promote autophagosome-lysosome fusion, protein phosphatase 3 (PPP3)/calcineurin could negatively regulate phosphorylation of Akt, a decrease of Akt activity led to dephosphorylation of TFE3 at the Ser565 site in the cytoplasm, and Cd exposure increased the activity of PPP3/calcineurin, thus inducing MSCs’ death by Akt/TFE3-mediated autophagy [59]. It was also found that Cd exposure might also inhibit autophagosome-lysosome fusion by reducing the expression of Rab7 so as to exacerbate hepatotoxicity [21]. Cd-induced ROS could act as the upstream signal of the PTEN-induced putative kinase1 (PINK1)/Parkin pathway to mediate mitophagy in mice brains [60]; it could also activate the LKB1-AMPK signaling pathway to induce autophagy in mouse skin epidermal JB6 cells [61]. Autophagy was also induced by activating glycogen synthase kinase-3β (GSK-3β) in MES-13 mesangial cells [62]. Cd exposure could increase intracellular Ca²⁺ through IP3R of the endoplasmic reticulum (ER) and later induce autophagy mediated by Ca²⁺/ERK in MES-13 cells [63]. In addition, Cd exposure activated vacuole membrane protein 1 (VMP1) by increasing intracellular Ca²⁺ level, and VMP1 induced protein expression of autophagy markers p62 and LC-3II and subsequent apoptotic cell death in Ramos B cells as well as mouse spleen apoptotic damage [64]. Besides, Cd was shown to induce neuroprotective autophagy by activating the PI3K/Beclin-1/B cell lymphoma-2 (Bcl-2) signaling pathway in rat cerebral cortical neurons and PC 12 cells [65, 66]. In summary, the processes of autophagy regulation after exposure to Cd are shown in Fig. 2.

Fig. 2.

Cd-induced autophagy. Cd-induced ROS activates the PINK1/Parkin and LKB1/AMPK pathways along with GSK-3β, mediating mitophagy and cellular autophagy. The increase in Ca²⁺ induced by Cd can act as an upstream signal to regulate ERK and VMP1, which in turn induce autophagy. Additionally, Cd mediates autophagic cell death through the PPP3/Akt/TFE3 and AMPK/FOXO3a pathways. Cd may also inhibit autophagy by suppressing Rab7

Toxicity of Cr and autophagy dysfunction

Cr is widely used in industry nowadays, and occupational exposure potentially cause allergic dermatitis and chronic lung diseases. An investigation of occupational workers showed that occupational exposure to Cr(VI) increased the risk of lung cancer [67]. In addition to occupational exposure, humans may be exposed to Cr through food and water that contain Cr [68]. An experimental study indicated that Cr(VI) exposure may induce renal tubular necrosis [69]. The accumulation of Cr in the body could damage the liver and immune system [70]. It was found that Cr(VI) could not only enter the reproductive system of male rats through the BTB and interfere with the normal development of sperm [71], but also damage the tissue structure of the ovaries and thus reduce the fertility of female rats [72].

As a principal mechanism of Cr toxicity, ROS-induced oxidative stress was initiated together with lipid peroxidation, DNA damage [73–75], abnormal apoptosis [76], and mitochondrial dysfunction [77]. In addition, the imbalance of intracellular Ca²⁺ homeostasis was also involved in Cr(VI)-induced liver injury [78, 79]. Autophagy is also a potential mechanism involved in Cr toxicity, while ceramide may be the component lipid of the autophagosome membrane. Furthermore, it was found that Cr(III) can activate sphingomyelin phosphodiesterase 2 (SMPD2) to increase the ceramide level in HK2 cells and induce autophagy [20]. Cr(VI) exposure modified the expression profile of motion-related protein 1 (DRP1) and mitofusin 2 (MFN2) by inhibiting the silent information-regulated transcription factor 1/peroxisome proliferator-activated receptor gamma coactivator 1-alpha (SIRT1/PGC-1a) pathway. This imbalance of mitochondrial dynamics subsequently promoted excessive ROS production and the induction of autophagy-related proteins (Beclin-1, Atg5, and Atg4B) in a dose-dependent manner, along with renal tubular pathologies [80]. In addition, Cr(VI) exposure was shown to initiate autophagy mediated by the ROS/Akt/mTOR signaling pathway to attenuate apoptosis in L-02 hepatocytes [81]. Exposure to Cr(VI) increased the level of cyclooxygenase-2 (COX-2), which affected the expression of autophagy proteins (p62, LC3-II, and Beclin-1), thus reducing the viability of LMH cells [82]. Moreover, the levels of endoplasmic reticulum stress (ER stress) related proteins such as glucose-regulating protein 78 (GRP78) and phosphorylated protein kinase RNA-like ER kinase (p-PERK) were enhanced after Cr(VI) exposure, and thus ER stress was triggered to induce autophagy in A549 cells [83]. In particular, Cr(VI) could destroy structural complementarity and alter the mitochondrial function in DF-1 cells and initiate mitophagy [84]. Together, these studies provide the selective pathways by which Cr exposure triggers the abnormal processes of autophagy or mitophagy (Fig. 3).

Fig. 3.

The selective ways Cr triggers alterations in autophagy. Cr regulates the mitochondrial proteins DRP1 and MFN2 via the SIRT1/PGC-1a pathway, leading to mitochondrial dysfunction and excessive ROS production, which in turn inhibits the Akt/mTOR pathway. ER stress induced by Cr via the GRP78/p-PERK pathway can further induce autophagy. In addition, Cr can also activate SMPD2 or increase COX-2 to regulate autophagy

Toxicity of Pb and autophagy dysfunction

Pb has a long half-life and remains ubiquitous in the environment. Apart from occupational exposure, humans may be exposed to Pb in many substances, including paint, cosmetics, and Pb-containing vehicle exhaust. Pb is a systemic toxicant, with cardiac and renal toxicity following both acute and chronic Pb exposure [85]. Notably, Pb is neurotoxic. It was found that Pb altered the onset of nerve development by targeting the neural cell adhesion molecule (NCAM) [86]. Pb also promoted synapse dysfunction and cognitive impairment in terms of reduced growth of neuronal dendrites and dendritic spines, decreased the number of synapses, and increased synaptic gaps [87, 88]. Pb could reduce the number of neurons, and alter the differentiation process of neural stem cells [89, 90]. Oxidative stress and ER stress may be causative factors for neurodegenerative injury caused by Pb exposure [91]. Other studies indicated that Pb can exert toxicity to the liver, kidney, and brain by impairing mitochondrial structure and modifying enzyme activity in the electronic respiratory transmission chain and ATP synthesis [92–95]. Besides, Pb was found to be embryotoxic, and long-term exposure with high level could also damage immune function.

Other than the toxicity mechanisms mentioned above, the contribution of autophagy to the toxicity of Pb has also been widely noted. It was observed that Pb induced abnormal hyperphosphorylation of tau and aggregation of a-synuclein in the hippocampus of Sprague-Dawley rats (SD rats), which caused ER stress, promoted autophagy and apoptosis via inhibiting the Akt/mTOR pathway, and thus impaired the learning and memorizing abilities of SD rats [96]. Pb also induced mitophagy in HEK293 cells and male Kunming mice renal cortex through PINK1/Parkin pathway [97] and promoted the activating transcription factor 6 (ATF6)/inositol-requiring enzyme 1 (IRE1) signaling pathway to trigger a subsequent ER stress response [98]. In addition, Pb mediated hepatocyte injury and renal cytotoxicity via the SIRT1/mTOR [99] as well as AMPK-mTOR pathways [100], respectively. Several studies indicated that Pb exposure disrupted lysosomal function by altering lysosomal pH and cathepsin B/D (CTSB/CTSD), contributing to autophagosome accumulation, which in turn damaged renal tubular cells in rats [101]. Pb could also hamper autophagosome and lysosome fusion, significantly reducing the number or size of lysosomes and resulting in the injury of autophagy in neural cells [102]. Pb additionally disrupted intercellular communication through the activation of autophagy and induced apoptosis in cardiomyocytes [103]. To summarize, the processes of autophagy/mitophagy regulation following Pb exposure are shown in Fig. 4.

Fig. 4.

Pb-associated regulation of autophagy/mitophagy. Abnormal hyperphosphorylation of Tau, aggregation of a-synuclein, and ATF6/IRE1 are involved in Pb-induced ER stress, thereby inhibiting the Akt/mTOR pathway and activating autophagy. Pb may regulate autophagy through the SIRT1/mTOR, AMPK-mTOR, and PINK1/Parkin pathways. Besides, Pb exposure disrupts lysosomal function by altering lysosomal pH and CTSB/CTSD, contributing to autophagosome accumulation

Toxicity of As and autophagy dysfunction

Food, drinking water, air pollution as well as occupational environment exposure are typical sources of As exposure [104]. As occupies different priority list of hazardous substances because of its unique carcinogenic properties and extensive organ toxicity [105]. Due to its bioaccumulation tendency, chronic exposure to As is a potential risk factor for the development of cancer (skin, lung, and urinary bladder) [104]. As exposure is also linked with reproductive risk by inducing spermatogenic cell apoptosis and decreasing sperm quantity [106]. Also, a diverse degree of nephrotoxic [107] and neurotoxic [108] effects is linked with environmental As exposure.

Many studies have provided insights into the cellular and molecular mechanisms of As toxicity, which involve intracellular stresses including oxidative stress [109], DNA damage [110], and epigenetic changes [111]. Furthermore, emerging evidence suggested a contribution of stress responses, autophagy, and mitophagy, to various accumulated toxicity induced by As exposure. The inhibition of mTOR upon As exposure might be mediated by ROS [112], while As exposure could damage mitochondria and produce excessive ROS [113]. As also induced phosphorylation of ERK1/2 and activated ERK1/2 phosphorylated Galpha-interacting protein (GAIP), which increased the rate of GTP hydrolysis and controlled the lysosomal-autophagic pathway [114]. Moreover, the ERK1/2 signaling pathway activated by As exposure also increased the levels of LC3II and p62, along with triggering the PINK1/Parkin pathway to mediate mitophagy, which reduced the apoptosis rate of L-02 cells [115]. Notably, As exposure triggered autophagy via the PI3K/Akt/mTOR pathway in the brains of mice, accompanied by lesions in brain tissue [116]. Studies also showed that subchronic As exposure induced mouse ovotoxicity accompanied by autophagy activation via mTOR/ULK-1/Beclin-1 signaling [117]. Intriguingly, As exposure inhibited mTOR and its downstream markers, the phosphorylation of p70S6K (p-p70S6K) and 4E-binding protein 1 (p-4EBP1), by which As promoted autophagy, accounted largely for reducing the transformation and tumorigenicity of BEAS-2B cells [118]. Altogether, the processes of autophagy/mitophagy regulation in response to As exposure are shown in Fig. 5.

Fig. 5.

The autophagy/mitophagy pathways in response to As exposure. ROS/mTOR and PI3K/Akt/mTOR pathways may be involved in As-mediated autophagy as upstream signals. Activation of ERK by As can modulate the lysosomal-autophagic pathway via regulation of GAIP. Moreover, the ERK1/2 signaling pathway activated by As exposure also increased the levels of LC3II and p62, along with triggering the PINK1/Parkin pathway

Toxicity of Hg/MeHg and autophagy dysfunction

The main sources of Hg are the incineration of fossil fuels and medical waste, the discharge of Hg-containing wastewater from industrial enterprises, and the use of Hg-containing pesticides. Hg is also known as a neurotoxic metal. It has been found that Hg poisoning may exert its neurotoxic effect by increasing the content of the messenger molecule NO in brain tissue and inhibiting the activity of Na/K-ATPase [119]. Kidney is the main target organ of Hg. Hg exposure can damage kidney cells and cause nephrotic syndrome and renal tubular injury. Methylmercury (MeHg) can also affect gonad development and is reproductively toxic and embryotoxic [120].

Metal Hg can cause membrane damage, promote oxidative stress, affect enzyme activity, and damage DNA [121–123]. In particular, MeHg has been proven to have significant systemic toxicity caused by excessive oxidative stress, impairing mitochondrial function, modifying epigenetics, and disrupting Ca²⁺ homeostasis [124–126]. Notably, many studies have revealed a close interplay between autophagy and apoptosis in response to Hg/MeHg. The research showed that low concentration of Hg (5 µM HgCl₂) exposure induced autophagy and mediated hepatocyte death in rats through Atg5-Atg12 covalent coupling, which was regulated by damage-regulated autophagy modulator (DRAM) in a p53-dependent manner [127]. The study also found that Hg exposure disrupted the structure and function of lysosomes and initiated the autophagic death of HuH-7 cells [128]. MeHg induced autophagy in human neural stem cells by inhibiting the Akt1/mTOR signaling pathway [129]. P62 is an autophagic adaptor protein that links autophagy to the kelch-like ECH-associated protein 1/nuclear factor erythroid 2-related factor 2 (Keap1/Nrf2) pathway. After MeHg exposure, the expression of p62 was enhanced and could competitively combine Keapl with Nrf2, resulting in the degradation of Keapl by autophagy. Interestingly, p62 could also induce Nrf2 activation again, thus developing a positive feedback circuit [130]. MeHg exposure markedly decreased Beclin-1 level, posing a concern for pituitary hemorrhage, dilation, etc. [131]. In addition, MeHg promoted the formation of the Beclin-1-Vps34 complex and increased the independent autophagy of mTOR by activating JNK, which mediated the death of neuronal cells [132]. Collectively, the processes of autophagy regulation associated with Hg/MeHg exposure are shown in Fig. 6.

Fig. 6.

Regulation of autophagy by Hg. Hg inhibits Akt/mTOR and activates Beclin-1, p62, DRAM, and JNK. JNK activation promotes the formation of Beclin-1-Vps34 complex, which in turn induces autophagy

Toxicity of Al and autophagy dysfunction

Al is widespread in nature and available to humans through food, medications, and occupational exposure [133]. Research suggests that Al exposure can cause liver damage, which correlates with oxidative stress [134] and inflammatory reactions [135]. Al can induce testicular dysfunction in rats by inhibiting enzyme activity [136]. In addition, oxidative stress induced by Al exposure can damage bones [137], inhibit renal function [138], induce immunotoxicity [139], and injure glands [4]. It has also been found that AlCl₃ may induce hypertension as a result of interfering with the function of the erythrocyte membrane [140]. What’s more, Al causes aggregation and precipitation of amyloid β-protein (Aβ) in the brain [141], disrupts Ca²⁺ homeostasis in the body [142], affects enzyme activity [143], and promotes oxidative stress [144], all of which can contribute to learning and memory impairment.

The toxic mechanisms of Al mainly include oxidative stress, lipid peroxidation [145], pro-inflammatory reaction [146], DNA damage [147], protein degeneration [148], and the modulation of enzyme activity [149]. More interestingly, the toxicity of Al is affected by autophagy as well. Research revealed that Al exposure induced not only vacuolar degeneration and necrosis in liver tissue [150] but also damaged synaptic plasticity in rat hippocampal neurons via PI3K/Akt/mTOR pathway [151]. It also showed that exposure to Al increased the mRNA expression of Atg3, Atg5, and Atg9, which could be responsible for autophagosome formation and activated the autophagic process in MC3T3-E1 cells, consequently decreasing the rate of apoptosis [152]. Furthermore, Al increased the expression of Beclin-1 and raised the ratio of LC3II/LC3I, which activated autophagy and led to apoptosis in astrocytes [153].

Toxicity of metal mixtures and autophagy dysfunction

In real life, people are easily exposed to metal mixtures, and the mechanisms of their toxic effects are complex, so the studies of metal mixture toxicity and mechanisms are of great health importance. It was found that the co-exposure of As, Cd, and Pb can increase the level of Pb in some areas of the brain, and the co-exposure of Pb and Cd may damage the blood-brain barrier (BBB) [154]. Copper (Cu), iron (Fe) and Ca were proven to inhibit the absorption of Pb in the gastrointestinal tract. Manganese (Mn) induced Pb retention in the brains of rats and damaged cognitive function in these rats [155]. Exposure to a mixture of Pb, As, and Mn caused more pronounced disorders of heme synthesis than exposure to one metal alone [156]. Pb, As, and Cd have a synergistic effect on neurodevelopmental toxicity, and their mixtures can affect the function of the glia and neurons. The mixed exposure to As, Cd, Pb, Cr, and Hg damaged the structure along with the function of the liver, kidney, and lung, as well as the reproductive organ of male mice [157].

The expression proficiency of autophagy proteins (Beclin-1, Atg7, p62, Atg5, and LC3II) was significantly higher in the livers of rats with combined Cd and Pb exposure than in rats exposed to Cd or Pb alone. Besides, liver tissue vacuolation was more severe in the presence of both Cd and Pb [158]. Many studies have found that autophagic pathways or lysosomal function [102] can be affected by metal exposure. However, these are mainly about a single metal [22], and studies related to changes in autophagy levels upon metal mixture exposures are still limited. Based on the effect of single metal exposure on autophagy, we can consider the role of autophagy in metal mixture toxicity as a future research direction.

Toxicity of nanoparticles (NPs) and autophagy dysfunction

Studies have found that TiO₂ NPs can easily enter the body through inhalation and cross the BBB to accumulate in the brain, especially in the cortex and hippocampus. Exposure to TiO₂ NPs can lead to activation of microglia and inflammatory signaling pathways, production of ROS, and cell death, resulting in neuritis and brain damage [159]. Exposure to TiO₂ NPs during pregnancy reduced the proliferation of hippocampal cells in the offspring of rats and impaired their learning and memory [160]. It was observed that the toxic effects of TiO₂ NPs were also related to the age of rats, with juvenile rats being more hepatotoxic than adult rats [161]. Other studies suggested that CuO NPs restricted the growth of duckweed, including reduced leaf number, shorter root length, lower photochemical efficiency, chlorophyll content, dry weight, etc. [162]. In addition to causing mortality in Daphnia magna, CuO NPs also transmitted toxicity to aquatic organisms via the dietary chain [163]. Besides, after 30 days of treatment with 50 nm Al₂O₃ NPs at the concentrations of 25, 50, and 75 mg/kg, significant alterations in the ultrastructure and function of mitochondria in mice were observed, which impaired spatial learning and memory [164].

NPs have been extensively studied for their possible mechanisms in cell toxicity, and they play many different roles in modulating cell fate. The formation of ROS is a considerable focus of NPs toxicity. High concentrations of ROS can destroy cellular components, resulting in cell death and the development of disease [165]. Notably, the effect of autophagy on the toxicity of NPs has also attracted increasing attention. For instance, the accumulation of ROS induced by NPs interfered with lysosomal hydrolases and induced autophagy [166]. It was found that ROS caused by acute exposure to ZnO NPs can act as an upstream signal to upregulate the expression of LC3A and thus induce autophagic death of immune cells [167]. Fe₃O₄ NPs could lead to the death of A549 cells by promoting ROS generation as well as mTOR-mediated autophagy [168]. The ROS generated by TiO₂ NPs exposure not only disrupted the lysosomal function of AGS cells and blocked the autophagic flux [169], but also triggered an inflammatory response in RAW264.7 cells [170] and the antioxidant damage response in rat astrocytes [171]. CuO NPs-induced ROS upregulated the level of autophagy and thus attenuated the apoptosis of MCF7 cells [172]. Furthermore, TiO₂ NPs exposure affected the function of the placenta through PINK1/Parkin-mediated mitophagy [173] and miRNA-regulated autophagy [174], yet it also mediated autophagy through p-IRE-1α-mediated ER stress [175] and MAPK signaling pathway-related molecules ERK, JNK, and p38 [176], which had toxic effects on lung cells and mesenchymal stem cells, respectively. Increased levels of autophagy mediated by CuO NPs resulted in the death of A549 cells [177]. AuNPs could alkalize lysosomes and block fusion with autophagosomes [178]. AlNPs affected the expression levels of LC3II, Beclin-1, p62, and COX IV, which caused autophagy and induced learning and memory impairment in mice [164]. ZnO NPs enhanced autophagy by inhibiting PI3K/Akt/mTOR, thus inducing apoptosis [179]. AgNPs were found to mediate autophagy to protect SH-SY5Y cells from apoptosis through the Ca²⁺/CaMKKβ/AMPK/mTOR pathway [180]; they also induced lysosomal membrane permeability and disrupted the autophagic-lysosomal pathway, which subsequently increased the death of HepG2 cells [181]. It also found that Fe₃O₄ NPs could disturb lysosomal function and induce autophagosome accumulation in MCF-7 cells via modulating the phosphorylation profiles of mTOR and ULK1 [182]. Together, the regulatory pathways of autophagy/mitophagy related to NPs exposure are illustrated in Fig. 7.

Fig. 7.

Signaling of autophagy/mitophagy mediated by NPs. ZnO NPs, Fe₃O₄ NPs, TiO₂ NPs, and CuO NPs are able to mediate autophagy via ROS. ZnO NPs inhibit the PI3K/Akt/mTOR pathway. TiO₂ NPs activate the PINK1/Parkin pathway and also mediate autophagy through p-IRE-1α-mediated ER stress and MAPK signaling pathway-related molecules ERK, JNK, and p38. AgNPs and Fe₃O₄ NPs regulate autophagy through the Ca²⁺/CaMKKβ/AMPK/mTOR and mTOR/ ULK1 pathways, respectively. AlNPs and AuNPs can affect the expression of LC3II, p62, and other autophagy proteins to further modulate autophagy

Pathological changes associated with metals/NPs-mediated regulation of autophagy

Collectively, we have briefly reviewed the mechanisms underlying the toxicity of metals and NPs as well as a description of the changes in the autophagic pathways that contributed to cytotoxicity. To further understand how autophagy functions in metals/NPs-mediated toxicity, we summarized the pathological changes associated with metals/NPs-mediated autophagy regulation (Table 1).

Table 1.

The pathological changes associated with metals/NPs-mediated autophagy regulation

Metals/NPs	Autophagic activity	Toxicity of Metal/NPs	Pathological changes	References
Cd	Inhibition	Increase	Hepatic steatosis Glomerular atrophy	[183] [184]
	Activition	Increase	Renal tubular dilatation Hepatic lobular injury Renal cortex injury	[185] [186] [187]
Cr	Activition	Increase	Renal tubular rupture Damage of liver mitochondria Cardiomyocyte necrosis Blurred boundaries of glomerula	[80] [188] [189] [190]
Pb	Inhibition	Increase	Structural changes of the spleen, ferritin deposits	[191]
	Activition	Increase	Hippocampus damage	[192]
As	Inhibition	Increase	Skin tumorigenesis	[193]
	Activition	Increase	Islet cell hypertrophy Glomerular atrophy Aortic injuries Mitochondrial damage in jejunal cells Testicular tissue damage Purkinje cell layer damage Hepatic steatosis Liver fibrosis	[194] [195] [196] [197] [198] [199] [200] [201]
Hg	Inhibition	Increase	Tubular necrosis, interstitial hyperemia, and inflammatory cell infiltration	[202]
	Activition	Increase	Spleen damage	[203]
Al	Activition	Increase	Femoral damage	[204]
	Activition	Decrease	Testicular damage Liver inflammatory injury	[205] [206]
NPs	Activition	Increase	Neurovascular toxicity	[207]
	Activition	Decrease	Liver damage	[208]

Activation/Inhibition represents that the autophagy is activated or inhibited by the metals/NPs. Increase/Decrease represents that the autophagic activity enhances or attenuates the toxicity of the metals/NPs.

Modulation of autophagy and the toxicity of metals/NPs

As mentioned above, activation or inhibition of autophagy is involved in the toxicity induced by metals/NPs; therefore, it is of interest that autophagic modulation by chemicals may modify the toxicity of metals/NPs. We can focus on changes in harmful effects to make a beneficial autophagy regulation by using autophagy inhibitors or activators to avoid or lessen negative biological effects. It was found that Cd exposure induced autophagy in mouse spleen tissue and that the autophagy inhibitor CQ was effective in reducing Cd-induced apoptosis in spleen and immune cells [64]. Additionally, autophagy inhibitor 3-methyladenine (3-MA) exacerbated Cd-induced germ cell apoptosis; while autophagy inducer rapamycin diminished Cd-induced germ cell apoptosis [209]. Chloroquine diphosphate (CDP) can disrupt the structure and function of lysosomes and alter the process of autophagy-lysosome fusion. Treatment with 3-MA and CDP inhibited autophagy in hepatocytes induced by Cr(VI) exposure and therefore reduced apoptosis [81], while treatment with rapamycin relieved hepatocyte injury [18]. Specially, inhibition of autophagy with 3-MA exacerbated Pb-induced renal tubular and osteogenic apoptosis as well as cardiac fibroblast death. While activation of AMPK significantly augmented cellular autophagic activity and reduced Pb-induced renal tubular apoptosis [210–212]. Treatment with 3-MA reduced As-induced cytotoxicity, while treatment with rapamycin reduced the viability of cells in the As-treated group [213]. MeHg-induced neurotoxicity could be reduced to some extent by autophagy. Study also confirmed that treatment with 3-MA/CQ or rapamycin increased or reduced MeHg-induced apoptosis, respectively [214]. Al exposure led to apoptosis and cognitive impairment in MC3T3-E1 cells and zebrafish, individually, and the use of rapamycin reduced the apoptosis rate of MC3T3-E1 cells and improved learning ability in zebrafish after AlCl₃ exposure [152, 215]. Moreover, it was also found that high doses of Al may lead to apoptosis through the Beclin-1-dependent autophagic signaling pathway, and 3-MA reduced Al-induced apoptosis [153]. AgNPs could induce protective autophagy, and the addition of CQ to inhibit autophagy significantly enhanced the cytotoxicity of AgNPs [180].

Perspectives

In this review, we addressed the general toxicity of both single metal and metal mixtures as well as NPs, mainly focusing on the role of autophagic regulation in the toxicity of metals/NPs. At first, we described studies on a single metal affecting autophagy, and it was clear that different metals may influence autophagy in the same pathway. Cd, Cr, As, and NPs individually regulate autophagy through the induction of ROS. Cd, Pb, As, and NPs respectively regulate mitophagy via the PINK1/Parkin pathway. Cr, Pb, As, MeHg, Al, and NPs separately regulate autophagy through the Akt/mTOR pathway. Metals (As and MeHg) and NPs regulate autophagy by the MAPK pathway, respectively (e.g., Fig. 8). However, little is known about whether exposure to metal mixtures regulates autophagy via ROS or otherwise. Because exposure to metal mixtures is closer to the real-life situation, exploring the toxicity of metal mixture exposures from the perspective of autophagy can greatly contribute to the well-being of the public. Therefore, this review presented a new perspective for further studies on the toxicity of metal mixture exposures. Meanwhile, NPs are widely used in various fields, and their potential hazards are becoming increasingly prominent. This review also provided an overview of nanotoxicity from the perspective of autophagy, where it was observed that NPs could regulate autophagy via multiple modalities and induce different biological effects. Consequently, the role of autophagy regulation should also draw extensive attention in future nanotoxicity studies. In view of this, we may be able to reduce or eliminate the adverse health effects of metals/NPs through optimization, substitution, or intervention (e.g., intervention using autophagic chemomodulators or targets of metals/NPs action) based on our understanding of their material properties, exposure pathways, uptake and metabolism, and toxic effects.

Fig. 8.

The common pathways of autophagy regulation by metals/NPs. The regulatory process involves the activation of Akt/mTOR, PINK1/Parkin, and MAPK signaling pathways, as well as the further regulation of autophagy proteins by ROS. ROS induced by metals (Cd, Cr, and As) and NPs may be implicated in multiple modes of autophagy regulation. Metals (Cd, Pb, and As) and NPs mediate mitophagy via PINK1/Parkin. Akt/mTOR can be regulated by ROS and ER stress, and metals (Cr, Pb, As, MeHg, and Al) and NPs mediate Akt/mTOR-induced autophagy. MAPK signaling pathway-related ERK, JNK, and p38 act as upstream signals to regulate LC3, Beclin-1, and p62. Metals (As and MeHg) and NPs regulate autophagy by the MAPK pathway

Author contributions

All authors have contributed to this work.

Funding

This review was supported by the National Natural Science Foundation of China (81202173), the Key Teachers Training Plan of Henan Province (2018GGJS007) and the Technological Projects Foundation for Key R&D and Promotion in Henan Province (192102310047) and the Young Teachers Training Program of Zhengzhou University (2016-40).

Data availability

https://www.springer.com/journal/43188/submission-guidelines#Instructions%20for%20Authors_Research%20Data%20Policy%20and%20Data%20Availability%20Statements.

Declarations

Conflict of interest

The authors declare no conflict of interest.