Micro- and Nanosized Substances Cause Different Autophagy-Related Responses

With rapid industrialization, humans produce an increasing number of products. The composition of these products is usually decomposed. However, some substances are not easily broken down and gradually become environmental pollutants. In addition, these substances may cause bioaccumulation, since the substances can be fragmented into micro- and nanoparticles. These particles or their interactions with other toxic matter circulate in humans via the food chain or air. Whether these micro- and nanoparticles interfere with extracellular vesicles (EVs) due to their similar sizes is unclear. Micro- and nanoparticles (MSs and NSs) induce several cell responses and are engulfed by cells depending on their size, for example, particulate matter with a diameter ≤2.5 μm (PM2.5). Autophagy is a mechanism by which pathogens are destroyed in cells. Some artificial materials are not easily decomposed in organisms. How do these cells or tissues respond? In addition, autophagy operates through two pathways (increasing cell death or cell survival) in tumorigenesis. Many MSs and NSs have been found that induce autophagy in various cells and tissues. As a result, this review focuses on how these particles interfere with cells and tissues. Here, we review MSs, NSs, and PM2.5, which result in different autophagy-related responses in various tissues or cells.


Introduction
Micro-and nanomaterials with different physical and chemical properties have been developed for human needs [1,2]. However, micro-and nanomaterials also show unexpected toxicity [3]. Nanotoxicology is rapidly developing with potential hazardous effects for nanomaterials [4,5]. Due to their larger sizes and small surface-to-volume ratios, micromaterials are considered less toxic than nanomaterials. In addition, nanomaterials can aggregate to a microscale size [6,7]. Micromaterials are also harmful to humans [3,8]. These materials may return to humans via the food chain [9,10]. Human consumption of microor nanoplastics may occur through seafood [11,12], water [13,14], etc. However, PM2.5 (particulate matter ≤ 2.5 µm) is a mix of micro-and nanosized substances (MSs and NSs) that can cause many chronic diseases [15,16]. Many studies show that MSs and NSs are toxic [17]. These related materials have a potential risk to human health. MSs and NSs employ one or multiple endocytosis pathways to enter cells. The main endocytosis pathways of MSs or NSs include clathrin-mediated endocytosis, caveolae/lipid raft-mediated endocytosis, clathrin-and caveolin-independent endocytosis, macropinocytosis and phagocytosis. The possible mechanisms by which MSs and NSs modulate several cell responses, such as ER-stress, mitochondrial damage, lysosome dysfunction, ROS production, and autophagy, are summarized. MSs: Micro-sized substances; NSs: Nanosized substances.Extracellular vesicles (EVs) are defined as lipid-bound particles of various sizes secreted from cells to extracellular spaces or circulated to target tissues [69,70]. EVs can be briefly classify into three types based on their size and biogenesis [71,72]. Small EVs are 50-100 nm in size and, include exosomes and endosome-derived membrane vesicles that are formed from multivesicular bodies (MVBs), intraluminal vesicles (ILVs) and the cellular plasma membrane [73,74]. Microvesicles (MVs), microparticles (MPs) and ectosomes are considered large EVs that are shed directly from the cell surface [73,75]; apoptotic bodies are formed during apoptosis genesis, and their diameters range between 1000 and 5000 nm [76,77]. Previous studies have shown that EVs have important biological relevance, such as immunity and inflammation [78,79], hemostasis [80,81], reproduction [82], and tumorigenesis [83]. Recently, conditioned medium from stem cells is as a new therapeutic application [84,85]. Conditioned medium applicates in diabetic wound healing [86,87], preventing activation of keloid fibroblasts in human [88], musculoskeletal tissue regeneration [89], hair regeneration in human [90], retinal ischemia-reperfusion in rat [91], differentiation of rat retinal progenitor cells, [92] promoting survival and neurite outgrowth of neural stem cells in canine [93], autoimmune encephalomyelitis in mice [94], spinal cord injury in canine [95], lung injury and disease [96], EVs can isolated form conditioned medium [97]. Therefore, EVs have sizes similar to those of MSs and NSs or particulate matter less than 2.5 μm (PM 2.5). Whether these micro-and nanoparticles interfere with the function of EVs is still unclear. MSs and NSs employ one or multiple endocytosis pathways to enter cells. The main endocytosis pathways of MSs or NSs include clathrin-mediated endocytosis, caveolae/lipid raft-mediated endocytosis, clathrin-and caveolin-independent endocytosis, macropinocytosis and phagocytosis. The possible mechanisms by which MSs and NSs modulate several cell responses, such as ER-stress, mitochondrial damage, lysosome dysfunction, ROS production, and autophagy, are summarized. MSs: Micro-sized substances; NSs: Nanosized substances. Schematic picture of the macroautophagy process. MNs and NSs, including protein aggregates, damaged organs, plastics particles, dust, and silica are shown. LC3-II, Beclin 1, and p62 conjugate enzymes generate the phagophore form and then the surrounding MNs and NSs during the elongation stage. At the end of the elongation stage, the membrane is sealed to form a doublemembrane vesicle, called the autophagosome, which contains degraded cellular enzymes. The autophagosome fuses with a lysosome, forming an autolysosome in which lysosomal enzymes degrade the cargo and release the degraded products into the cytoplasm. Undecomposed MSs and NSs, such as dust and silica, have carcinogenic potential.

Classification of Micro-and Nanosized Substances (MSs and NSs)
Microparticles and nanoparticles are particulate particles with a size ranging from 1- Figure 2. Schematic picture of the macroautophagy process. MNs and NSs, including protein aggregates, damaged organs, plastics particles, dust, and silica are shown. LC3-II, Beclin 1, and p62 conjugate enzymes generate the phagophore form and then the surrounding MNs and NSs during the elongation stage. At the end of the elongation stage, the membrane is sealed to form a double-membrane vesicle, called the autophagosome, which contains degraded cellular enzymes. The autophagosome fuses with a lysosome, forming an autolysosome in which lysosomal enzymes degrade the cargo and release the degraded products into the cytoplasm. Undecomposed MSs and NSs, such as dust and silica, have carcinogenic potential.
Extracellular vesicles (EVs) are defined as lipid-bound particles of various sizes secreted from cells to extracellular spaces or circulated to target tissues [69,70]. EVs can be briefly classify into three types based on their size and biogenesis [71,72]. Small EVs are 50-100 nm in size and, include exosomes and endosome-derived membrane vesicles that are formed from multivesicular bodies (MVBs), intraluminal vesicles (ILVs) and the cellular plasma membrane [73,74]. Microvesicles (MVs), microparticles (MPs) and ectosomes are considered large EVs that are shed directly from the cell surface [73,75]; apoptotic bodies are formed during apoptosis genesis, and their diameters range between 1000 and 5000 nm [76,77]. Previous studies have shown that EVs have important biological relevance, such as immunity and inflammation [78,79], hemostasis [80,81], reproduction [82], and tumorigenesis [83]. Recently, conditioned medium from stem cells is as a new therapeutic application [84,85]. Conditioned medium applicates in diabetic wound healing [86,87], preventing activation of keloid fibroblasts in human [88], musculoskeletal tissue regeneration [89], hair regeneration in human [90], retinal ischemia-reperfusion in rat [91], differentiation of rat retinal progenitor cells [92], promoting survival and neurite outgrowth of neural stem cells in canine [93], autoimmune encephalomyelitis in mice [94], spinal cord injury in canine [95], lung injury and disease [96], EVs can isolated form conditioned medium [97]. Therefore, EVs have sizes similar to those of MSs and NSs or particulate matter less than 2.5 µm (PM2.5). Whether these micro-and nanoparticles interfere with the function of EVs is still unclear.

Classification of Micro-and Nanosized Substances (MSs and NSs)
Microparticles and nanoparticles are particulate particles with a size ranging from 1-1000 µm or 1-1000 nm, respectively [98]. The sources of MSs and NSs can be classified into three main categories based on their origin. There are three main categories (A), (B), and (C).
Silica nanoparticles induce cardiac dysfunction in rat hearts and human cardiomyocytes [188] and cardiotoxicity in adult rat cardiomyocytes [189]. Silica nanoparticles disturb ion channels and transmembrane potentials in cardiomyocytes and induce arrhythmias in adult male C57BL/6J mice [190]. The 20 nm silica nanoparticles significantly induce apoptosis and necrosis in human endothelial cells (ECs) [191]. Silica nanomaterials induce calcium mobilization and the formation of ROS in HUVECs and adult female Balb/c mice [192]. Silica nanoparticles also increase autophagy markers, such as LC3, and autophagic cell death in HepG2 cells (human liver cancer cells) [193]. Ultrafine silicon dioxide nanoparticles trigger apoptosis in lung epithelial cells [194]. Silica nanoparticles induce inflammation in the lungs of mice [195] and the autophagy marker, p62 [196]. Amorphous silica nanoparticles cause autophagy markers, such as p62 and LC3, and vascular endothelial cell injury [197]. Silver nanoparticles increase the formation of ROS, oxidative stress [198] and the genotoxicity in human TK6 cells (lymphoblast cells) [199]. Silver nanoparticle-induced autophagy markers, such as LC3, disrupts inflammasome activation in HepG2 cells [200]. Silver nanoparticles increase autophagy markers, such as p62 and LC3, decrease the expression of transcription factors in A549 human lung adenocarcinoma cells [201], and induce other autophagy markers, such as Beclin 1 and LC3, in the adult rat brain [202]. Amine-modified silver nanoparticles trigger autophagy markers, such as P62 and LC3, and lysosomal dysfunction in NIH 3T3 cells (mouse embryonic fibroblast cells) [203]. The spleen can capture nanoparticles in Wistar rats [204]. Nanoparticles are mainly ingested by liver Kupffer cells, but splenic macrophages also play an important role [205]. Bismuth nanoparticles induce autophagy markers, such as LC3, Beclin 1, and Atg12, resulting in nephrotoxicity in the human embryonic kidney 293 cell line and kidney of BALB/c mice [206]. Bismuth nanoparticles also induce oxidative stress, such as GSH, SOD, and catalase, and apoptosis in MCF-7 cells (human breast carcinoma cells) [207]. Bismuth sulfide nanoparticles inhibit the migration and invasion in HepG2 cells and induce autophagy markers, such as p62 [208]. Bismuth nanoparticles affect the autophagy-associated cytotoxicity and cellular uptake mechanisms in human kidney cells [209]. Nanosized titanium dioxide (Nano TiO 2 ) results in a potential reproduction toxicity in rat Sertoli cells (SCs), induces apoptosis, decreases cell viability, and impairs morphological structures of SCs via the related wingless MMTV integration site (Wnt) pathway [210]. Long-term exposure to nano-TiO 2 results in liver inflammation and hepatic fibrosis in mice [211]. Nasal instillation to nano-TiO 2 induces lung injury in mice [212]. Nano-TiO 2 results in inflammation and fibration in mice kidneys [213]. Nano-TiO 2 changes autophagy markers, such as Beclin 1, p62 and LC3, in podocytes [214]. Nano-TiO 2 causes the autophagy marker, LC3, to increase in human HaCaT cells at non-cytotoxic levels [215]. Nano-TiO 2 induces autophagic response in HeLa cells [216]. Nano-TiO 2 induces proteostasis disruption and autophagy markers, such as LC3 and p62, in HTR-8/SVneo cells [217]. Planetary micro-and nanosized particles cause nervous system injury [218]. Copper oxide nanoparticles induce an autophagy-related response in A549 cells [219]. Copper-palladium alloy tetrapod nanoparticles induce autophagy [220]. In addition, a workplace was assessed in terms of the exposure to engineered nanoparticles of alumina, amorphous silica, and ceria used in semiconductor device fabrication [221]. One study shows workers occupational exposure to engineered nanomaterials closed to micro-sized agglomerated NSs [222]. Autophagy induces cell survival, which may induce inflammation, toxicity, and diseases.

Autophagy-Related Responses in Undecomposed MSs and NSs
Briefly, plastic particles can be classified into the following three types: macroplastics (over 5 mm in size) [223], small plastic particles (less than 5 mm in size) named microplastics [224], and nanoplastics (less than 1000 nm or 100 nm in size) [225]. Recently, we overused plastic-related products. When waste plastic is fragmented into micro and nanoparticles, it can cause obstruction, inflammation, and accumulation in organs [226,227]. PS microplastics change gut microbiota dysbiosis and decrease gut mucin secretion in mice [228]. Due to their neuron toxicity, PS microplastics change the acetylcholinesterase activity in mice [135]. PS nanoplastics induce ER stress-mediated autophagy markers, such as LC3, in human lung cells [229], LGG-1, an ortholog of Atg8 on the nematode, Caenorhabditis elegans [230], and the autophagic marker, LC3B, in mouse embryonic fibroblasts [231]. Positively charged PS nanospheres induce autophagy markers, such as p62, Beclin 1, and LC3, in mice macrophage-like cells, RAW 264.7, and human lung epithelial cells, BEAS-2B [232]. Vinyl chloride (VC) or PVC is considered a carcinogenic factor that causes angiosarcoma in the liver [233]. VC induces fibrosis and autophagy markers, such as Beclin 1, and LC3, in kidney cells [234]. Synthetic textile workers are potentially exposed to high concentrations of microplastics in the air and suffer higher rates of lung-cancer-related mortality [235]. In addition, MSs and NSs, such as dust, silica, and asbestos, in cells are not easily decomposed. Workers exposed to high concentrations of dust are at risk of pneumoconiosis [68,236]. Pneumonoultramicroscopic silicovolcanoconiosis or silicosis is a type of pulmonary fibrosis caused by the accumulation of fine particles of crystalline silica in the lungs [237]. The prevalence of asbestosis is due to the use of asbestos-related products [238] (Figure 3). Asbestos also induces programmed necrosis in human mesothelial cells [239]. A recent study showed that asbestos induces autophagy markers, such as ATG5, p62, Beclin 1, and LC3, and mesothelial cell transformation [240]. In addition, microplastic particles were found to be deposited in urban dust [241][242][243]. Urban dust is a kind of airborne PM, containing 2-10 µm particles [244]. Recent, studies investigating MSs, NSs, and PM show that these materials may endocytose cells and result in cell death or cell survival, depending on their characteristics.

Autophagy and Tumorigenesis
We found that the previous studies show that many MSs or NSs induce autophagy (Table 1). Autophagy plays dual roles, resulting in cell death [38,245] and cell survival [246,247]. Cell survival may result in tumorigenesis [248]. Autophagy may represent a type of tumor suppressor mechanism, as it has been found that this pathway is frequently related to autophagy markers that are downregulated in tumor cells [249], which are implied to be involved in tumorigenesis [250]. Studies have indicated that a loss of autophagy function initiates cancer [251]. Autophagy is as a tumor suppressor. For example, a study indicated that mice with a deletion of atg5 and atg7 had benign liver adenomas [252]. Beclin 1 is deleted in most cases of human breast, prostate, and ovarian cancer [253]. The frameshift mutation in the ultraviolet radiation resistance-associated gene (UVRAG) decreases autophagy in colon and gastric cancers [254]. There are other proteins involved in autophagy, such as Atg4c [255], Bax-interacting factor-1 (Bif-1) [256], BH3-only proteins [257], DAP kinase [258], and PTEN [259], which shows its potential role in tumor suppression. Recently, a study showed that autophagy is involved in tumor suppression via three mechanisms. First, autophagy plays a role in tumor suppression by inhibiting necrosismediated inflammation. Second, autophagy plays a role in tumor suppression by maintaining genome integrity. Third, autophagy plays a role in tumor suppression by maintaining autophagy-mediated cell death and senescence [260]. In addition, autophagy plays a dual role in cancer [261]. In the beginning of tumorigenesis, autophagy prevents mutations and genotoxicity in healthy tissues due to the production of ROS [262]. However, autophagy can also be useful for tumor survival if carcinogenesis has already begun. Autophagy also helps cancer stem cells to survive stressors [263], such as cancer cell survival or chemoresistance [264]. In fact, some MSs or NSs have carcinogenic potential such as iron oxide nanoparticles [17]. PM2.5 is associated with chronic airway inflammatory diseases and lung cancer [32]. VC is considered a carcinogenic factor [233]. Asbestos causes laryngeal cancer [265]. Many MSs and NSs have been found to induce autophagy ( Table  1), implying that these cells have a chance of undergoing tumorigenesis.

Autophagy and Tumorigenesis
We found that the previous studies show that many MSs or NSs induce autophagy (Table 1). Autophagy plays dual roles, resulting in cell death [38,245] and cell survival [246,247]. Cell survival may result in tumorigenesis [248]. Autophagy may represent a type of tumor suppressor mechanism, as it has been found that this pathway is frequently related to autophagy markers that are downregulated in tumor cells [249], which are implied to be involved in tumorigenesis [250]. Studies have indicated that a loss of autophagy function initiates cancer [251]. Autophagy is as a tumor suppressor. For example, a study indicated that mice with a deletion of atg5 and atg7 had benign liver adenomas [252]. Beclin 1 is deleted in most cases of human breast, prostate, and ovarian cancer [253]. The frameshift mutation in the ultraviolet radiation resistance-associated gene (UVRAG) decreases autophagy in colon and gastric cancers [254]. There are other proteins involved in autophagy, such as Atg4c [255], Bax-interacting factor-1 (Bif-1) [256], BH3-only proteins [257], DAP kinase [258], and PTEN [259], which shows its potential role in tumor suppression. Recently, a study showed that autophagy is involved in tumor suppression via three mechanisms. First, autophagy plays a role in tumor suppression by inhibiting necrosis-mediated inflammation. Second, autophagy plays a role in tumor suppression by maintaining genome integrity. Third, autophagy plays a role in tumor suppression by maintaining autophagy-mediated cell death and senescence [260]. In addition, autophagy plays a dual role in cancer [261]. In the beginning of tumorigenesis, autophagy prevents mutations and genotoxicity in healthy tissues due to the production of ROS [262]. However, autophagy can also be useful for tumor survival if carcinogenesis has already begun. Autophagy also helps cancer stem cells to survive stressors [263], such as cancer cell survival or chemoresistance [264]. In fact, some MSs or NSs have carcinogenic potential such as iron oxide nanoparticles [17]. PM2.5 is associated with chronic airway inflammatory diseases and lung cancer [32]. VC is considered a carcinogenic factor [233]. Asbestos causes laryngeal cancer [265]. Many MSs and NSs have been found to induce autophagy (Table 1), implying that these cells have a chance of undergoing tumorigenesis.

Solutions for MS-and NS-Caused Pollution
Many MSs and NSs may pose a potential risk to human health. How can these MSs and NSs be decreased and prevented from flowing into natural systems? Recently, some MSs and NSs have been applied in wastewater purification, such as activated carbon, carbon nanotubes, graphene, manganese oxide, zinc oxide, titanium oxide, magnesium oxide, and ferric oxides, which can be applied to remove heavy metals from wastewater [266]. In addition, wastewater treatment plants in several countries have found microplastic particles [267,268], such as the USA [268], Canada [269], and Turkey [270]. Several approaches can be used to decrease the volume of micro-and nanoplastics in water and wastewater, such as density separation, coagulation, membrane bioreactors, and biodegradation [271,272]. In addition, new techniques have been developed for water purification, such as three-dimensional graphene-based hybrid materials [273], the removal of heavy metals [274], and microplastic removal [275]. Biodegradation also seems to be a good approach, as plastic particles can be completely transformed into CO 2 and water. Studies investigating several potential candidate marine bacteria have found that these bacteria can be used in the degradation of plastic particles [276]. Some fungal strains have been shown to degrade several plastics, such as PHB and PLA [103]. PS is known to be biodegraded in the gut of yellow mealworms because there are special microorganisms in the gut [277]. In addition, many enzymes purified from different bacteria, such as Ideonella sakaiensis 201-F6, have been identified and can degrade PET plastics [278]. In April 2020, a total of 436 species reported in 1451 publications were found to degrade plastic. The three types of species that can degrade plastic that were reported most often reported among the 66 different types are Bacillus pumilus, Aspergillus fumigatus, and Phanerochaete chrysosporium, which were found to degrade 14, 11, and 10 different types of plastic, respectively [279]. Furthermore, many enzymes have been found that can hydrolyze polyesters, such lipase, esterase, protease, cutinase, PHA depolymerase, catalase, urease and glucosidases [280]. On other hand, polyester-based biodegradable plastics, such as PLA (poly(lactic acid)), PCL (polylcaprolactone), PHB (polyhydroxybutyrate)/PHBV (Polyhydroxybutyrate-covalarate), PBST(Poly(butylene succinate co-terephthalate), PBAT (Poly(butyrate adipate co-terephthalate)), PU (Polyurethanes) and PET (poly(ethylene terephthalate)), have potential in relation to waste reduction [280,281]. In addition, changing consumer behavior is another way to reduce plastics, such as plastic bag fee changes in Turkey [282]. The plastic carrier bag tax in Portugal reduced plastic bag consumption by 74% and increased reusable plastic bag consumption by 61% [283]. Several countries, such as the USA [284] and Caribbean countries [285], have adopted several methods for reducing single-use plastic bags.

Conclusions
Recently, EVs have played an important role in cell communication. The size of EVs, MSs, and NSs is similar. Some products made by humans are not easily decomposed. These products become environmental pollutants and bioaccumulate when they are fragmented into MSs and NSs. Among these particles, their interaction with other toxic matter has been well studied in PM2.5, MSs, and NSs. These studies have shown that MSs and NSs accumulate in organs via the food chain. In addition, MSs and NSs engulf cells and induce several cell responses, depending on their size and carrying capacity. Autophagy is a mechanism by which foreign matter decomposes in tissues or organisms. Some artificial materials are not easily decomposed by autophagy. Many MSs and NSs induce the formation of ROS, autophagic responses and apoptosis in various cells or tissues. Studies have indicated that autophagy operates through two pathways (cell death and cell survival) in tumorigenesis. MS-and NS-expressed autophagy may lead to tumorigenesis. Therefore, we found that pneumoconiosis, silicosis, and asbestosis from dust, silica, and asbestos have long disease histories, implying that MSs and NSs have previously interfered with cells and tissues and may interfere with our health through different materials in the future. Finally, the number of species of environmental bacteria and fungi found to degrade plastic seems to be increasing.    [CrossRef] [PubMed]