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Toxicity of Microplastics and Nanoplastics in Mammalian Systems

Toxicity of Microplastics and Nanoplastics in Mammalian Systems International Journal of Environmental Research and Public Health Review Toxicity of Microplastics and Nanoplastics in Mammalian Systems 1 2 1 , 3 , Cheryl Qian Ying Yong , Suresh Valiyaveettil and Bor Luen Tang * Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117596, Singapore; cherylyongqy@hotmail.com Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543, Singapore; chmsv@nus.edu.sg NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore 119077, Singapore * Correspondence: bchtbl@nus.edu.sg; Tel.: +65-6516-1040 Received: 12 February 2020; Accepted: 23 February 2020; Published: 26 February 2020 Abstract: Fragmented or otherwise miniaturized plastic materials in the form of micro- or nanoplastics have been of nagging environmental concern. Perturbation of organismal physiology and behavior by micro- and nanoplastics have been widely documented for marine invertebrates. Some of these e ects are also manifested by larger marine vertebrates such as fishes. More recently, possible e ects of micro- and nanoplastics on mammalian gut microbiota as well as host cellular and metabolic toxicity have been reported in mouse models. Human exposure to micro- and nanoplastics occurs largely through ingestion, as these are found in food or derived from food packaging, but also in a less well-defined manner though inhalation. The pathophysiological consequences of acute and chronic micro- and nanoplastics exposure in the mammalian system, particularly humans, are yet unclear. In this review, we focus on the recent findings related to the potential toxicity and detrimental e ects of micro- and nanoplastics as demonstrated in mouse models as well as human cell lines. The prevailing data suggest that micro- and nanoplastics accumulation in mammalian and human tissues would likely have negative, yet unclear long-term consequences. There is a need for cellular and systemic toxicity due to micro- and nanoplastics to be better illuminated, and the underlying mechanisms defined by further work. Keywords: human cells; microplastics; mouse cells; nanoplastics; oxidative stress; toxicants; toxicity 1. Introduction One of the most prominent classes of non-natural products made by humans that has pervaded Earth’s surface environment is plastics, so much so that these durable synthetic organic polymers are heralded as a defining stratigraphic marker for the Anthropocene [1]. Geyer and colleagues recently estimated that 8.3 billion metric tons of virgin plastics have been produced up to the year 2017 [2], and with continuation of current production and waste management practices, about 12 billion tons of plastic waste would be found in landfills and the natural environment by 2050. Plastic wastes are persistent environmental pollutants. Larger pieces of plastic wastes present well-publicized ecological problems in terms of physical entanglement and entrapment [3], physical barriers for food supply [4], and digestive tract congestion. The smaller plastic pieces, particularly their miniaturized forms that are less than 5 mm in size, are generally termed microplastics (MPs) [5]. Plastics that are already small in size to begin with, such as those purposefully manufactured in the form of microbeads in skincare products (primary MPs), or those derived from degradation of larger plastic pieces (secondary MPs), permeate both the terrestrial [6] and the marine [7,8] environments. Plastic particles of less Int. J. Environ. Res. Public Health 2020, 17, 1509; doi:10.3390/ijerph17051509 www.mdpi.com/journal/ijerph Int. J. Environ. Res. Public Health 2020, 17, 1509 2 of 24 than 1 m in size are also known as nanoplastics (NPs) [9,10]. These chemically inert MPs/NPs pose significant ecological and health concerns [5] because of their environmental persistence [6,11,12], potential ecotoxicity [13,14], and their ability to act as vectors for chemical pollutants [15–17] as well as pathogens [18,19]. Ecotoxicological e ects of MPs/NPs on marine phyto/zooplanktons, invertebrates, and plants are widely documented [20–30], and have been recently reviewed [5,31–33]. MPs/NPs could also be ingested and accumulated in larger marine fauna by trophic transfer from prey to predator, as demonstrated earlier with invertebrates, such as mussel-consuming crabs [34]. Interesting illustrations of trophic transfer within a lab-simulated food chain were shown by Mattsson and colleagues, where 53-nm polystyrene (PS) particles could be transferred from algae to the zooplankter Daphnia magna, and then to a freshwater fish [35]. Likewise, An and colleagues demonstrated trophic transfer of NPs from algae to Daphnia, then to a secondary consumer fish, and finally to an end consumer fish [36]. On the other hand, organic pollutants could be adsorbed onto MPs/NPs [37–41] and there is evidence that this could potentially enhance their e ective uptake and toxicity [42–45]. Likewise, MPs/NPs are known to interact with metallic toxicants such as Cadmium [46–48], Mercury [49], and other toxic trace elements [50], and could potentially serve as vectors for pollutant transfer to living organisms. The e ects of MPs/NPs on mammalian cells and tissues, particularly humans, have remained rather unclear [51,52]. While plastics are generally perceived to pose minimum risk to human, several recent scientific findings, picked up by the popular press, have heightened the worry of possible tissues penetrance and adverse e ects of MPs/NPs due to their small sizes. Humans could accumulate MPs/NPs from di erent food sources [53,54] as well as drinking water [55,56]. Plastic water containers [57,58] and plastic teabags [59] are, perhaps unsurprisingly, common sources for human ingested MPs/NPs. MPs/NPs could also be taken up by inhalation [60]. MPs/NPs have also been detected in human stool samples [61], an indication that the quantity taken in is significantly large. A recent World Health Organization’s (WHO) report on “Microplastic in drinking water” indicates that there is not yet proof of harm, but it also calls for more research to be carried out [62]. Could environmental MPs/NPs gain access to cells and tissues and be harmful to humans? Although ecotoxicology data with marine invertebrate indicate that this is so, more barriers and obstacles would likely be encountered by MPs/NPs in order to gain access to cells and tissues of vertebrates compared to simpler invertebrates. Here, we review current results on how MPs/NPs might a ect humans by scrutinizing studies done to date on mammalian (mouse) models and human cells. We begin with a quick survey of MP/NP feeding studies done on marine vertebrates, focusing on fishes. A meta-analysis on the e ect of MP exposure on fish has been reported by Foley and colleagues in 2018 [63] and the field has also been recently reviewed [64], but several newer reports have now appeared. This quick look would allow some comparison of findings in more ecologically relevant settings with that of laboratory experiments with mice and human cells. 2. Toxicity of MPs/NPs in Fishes Table 1 provides a non-exhaustive summary of recent studies where MP/NP feeding experiments have documented some degree of toxicological or pathological e ect observed on fishes. Those that have shown some significant e ect are included in this summary, while those that have reported little or no e ects are not. The MPs/NPs used in the studies listed in Table 1 are largely polystyrene (PS) or polyethylene (PE) based. An important general phenomenon to note is that toxicological responses typically arise from smaller plastic particles. Larger PS particles at around 100 m or above were shown not to have any significant e ect in a number of studies [65–67]. MP/NP feeding can result in behavioral abnormalities in terms of feeding and movement of adults and larvae [35,68–74], as well as reproduction in adults [75–77]. There is evidence of mother–o spring transfer of NPs [78], and that prenatal exposure of MPs a ected early development of the neonates [77]. In many cases, MPs/NPs were found accumulated in larvae or adult gut [77,79–83], and in some cases in gill and liver [79]. Histopathology is most prominently observed for these tissues Int. J. Environ. Res. Public Health 2020, 17, 1509 3 of 24 as well [74,75,77,79,83–85]. For the gut, pathological manifestations of MP/NP toxicity include documented changes in gut biomarkers related to epithelial barrier integrity, inflammation, and oxidative stress [83,86], as well as changes in gut microbiota [82,83,86]. In the case of liver, changes in metabolites, key metabolic enzymes, and oxidative stress-induced enzymes occur [49,74,79,81,85,87]. MPs/NPs could be internalized [88], and then cause detectable biomarker changes in blood cells [47,88, 89]. In rarer cases, MPs/NPs have also been found in fish brain [68,81], and caused changes in brain appearances [35,68] or showed significantly inhibited acetylcholinesterase (AChE) activity [49,81]. NPs taken up by embryos and larvae have been documented to migrate to various tissues throughout development [70]. Int. J. Environ. Res. Public Health 2020, 17, 1509 4 of 24 Table 1. A summary of notable toxicological and/or pathological findings associated with MPs/NPs in fishes. PA, polyamide; PS, polystyrene; PE, polyethylene; PC, polycarbonate; PP, polypropylene; PVC, polyvinylchloride; NPs, nanoplastics (<1 m); MPs, microplastics. Tissue Accumulation/ Notes on Toxicological, Pathological, Fishes Properties of MPs/NPs Used References Invasion or Cellular Uptake or Behavioral Observations 24 and 27 nm polystyrene (PS) Crucian Carp  Defects in feeding and shoaling behavior nanoparticles (NPs) (to fish Trophic transfer to fish from algae Mattsson et (Carassius  Defects in metabolism through an aquatic food chain, through Daphnia al., 2015 [35] carassius)  Changes in brain appearance and weight from algae through Daphnia) Increased Cd accumulation in livers, guts, and gills Zebrafish (Danio Virgin PS microplastic beads (5  Enhanced Cd toxicity Lu et al., 2018 rerio) m) + cadmium (Cd)  combined exposure caused oxidative damage and [90] inflammation in tissues European Fluorescence red polymer  Inhibition of brain acetylcholinesterase (AChE) activity seabass microspheres, (1–5 m) and and increase lipid oxidation in brain and muscle Barboza et al., (Dicentrarchus mercury individually and in  Changes in activity of metabolic enzymes 2018 [49] labrax) combination  Interactions and influences on mercury bioaccumulation Crucian Carp Amino-modified positively Trophic transfer to fish from algae Changes in feeding time Mattsson et (Carassius charged PS nanoparticles through Daphnia. Nanoparticles Changes in brain morphology (gyri sizes) al., 2017 [68] carassius) (52 nm) found in fish brain Inhibited of larvae locomotion Zebrafish (Danio Chen et al., PS NPs (50 nm, 1 mg/L) Accumulation in zebrafish larvae  Inhibited acetylcholinesterase activity rerio) 2017 [69] Upregulation of cytoskeletal markers Virgin (50 or 500 g/L) or African catfish  Liver and gill histopathology phenanthrene-loaded (10 or 100 Karami et al., (Clarias  Changes in blood biochemistry g/L) low-density polyethylene 2016 [75] gariepinus)  Changes in the expression of reproductive axis genes (LDPE) fragments Microplastics observed in observed in  Increased mortality and decrease in average lengths and Medaka (Oryzias PS microspheres (10–11 m, Cong et al., digestive tracts of larvae and weights of larvae and adult fishes melastigma) 0.758  0.217  10 particles/L) 2019 [76] dissected intestine of adults  Significant decrease in egg production by females Int. J. Environ. Res. Public Health 2020, 17, 1509 5 of 24 Table 1. Cont. Tissue Accumulation/ Notes on Toxicological, Pathological, Fishes Properties of MPs/NPs Used References Invasion or Cellular Uptake or Behavioral Observations Decreased heart rate Uptake of the nanoparticles by Altered larval behavior (swimming hypoactivity in embryos and larvae. Zebrafish (Danio exposed larvae) Pitt et al., PS NPs (mean 51 nm) Migrated to the gastrointestinal tract, rerio)  Maternal-o spring transfer of PS nanoparticles 2018 [70,78] gallbladder, liver, pancreas, heart, Delay/defect in swim bladder inflation by exposed F1 and brain throughout development larvae Liver histopathology (signs of inflammation and lipid Zebrafish (Danio PS microspheres (70 nm, 5 m, Accumulation in gills, gut, and liver accumulation) Lu et al., 2016 rerio) and 20 m, 20 mg/L) (only the 5 m particles)  Elevation of anti-oxidative stress enzymes [79] Changes in liver metabolomics profile Significant changes in transcriptome of zebrafish larvae after 2 days exposure Zebrafish (Danio Ingested microplastics observed in LeMoine et PS MPs (10–45 m, 20 mg/L)  Downregulation of genes involved with neural rerio) larvae gut al., 2018 [80] development and function Changes in genes associated with metabolism Red tilapia PS NPs (0.1 m, at 1, 10, and 100 PS MPs found in gut and gills and to  Inhibition of brain acetylcholinesterase (AChE) activity Ding et al., (Oreochromis g/L) a lesser extent, liver and brain  Changes in liver enzyme markers 2018 [81] niloticus) Changes in larval gut microbiota Metabolomic alterations Zebrafish (Danio Fluorescent and virgin PS MPs Ingested microplastics observed in  Changes in the expression of genes associated with Wan et al., rerio) (5 and 50 m) gut of larvae glucose and lipid metabolism 2019 [82] Significant reduction in the antioxidant GSH and the enzyme catalase Induction of inflammation and oxidative stress of adult zebrafish gut Zebrafish (Danio PS microplastic beads (5-m Accumulation of microplastics in  Significant alterations in the metabolome and Qiao et al., rerio) beads; 50 g/L and 500 g/L) zebrafish gut microbiome of adult zebrafish gut. Alterations were 2019 [83] associated with oxidative stress, inflammation, and lipid metabolism Int. J. Environ. Res. Public Health 2020, 17, 1509 6 of 24 Table 1. Cont. Tissue Accumulation/ Notes on Toxicological, Pathological, Fishes Properties of MPs/NPs Used References Invasion or Cellular Uptake or Behavioral Observations Polyamides (PA), polyethylene (PE), polypropylene (PP), Zebrafish (Danio Lei et al., polyvinyl chloride (PVC) (~70  Intestinal damage of adult fish gut rerio) 2018 [84] m) and PS (0.1, 1, and 5 m) particles Fathead minnow  significant increases in innate immune response (in PS (41.0 nm) and polycarbonate Neutrophil phagocytosis of PS Greven et al., (Pimephales terms of degranulation of primary granules and neutrophil (PC) (158.7 nm) NPs nanoparticles. 2016 [88] promelas) extracellular trap release) Gilthead seabream (Sparus Increased oxidative burst of in leukocytes of Sparus aurata) and Virgin polyvinylchloride (PVC) aurata Espinosa et European sea and polyethylene (PE) (40–150 Upregulation of the redox regulator Nrf2 in leukocytes al., 2018 [89] bass m) of Sparus aurata (Dicentrarchus labrax) MPs from a face and body scrub,  Changes in plasma levels of various metabolic enzymes Carp (Cyprinus Banaee et al., mainly PE (250 and 500 g/L), and immune markers carpio) 2019 [47] alone and + Cd  Combination of MP and Cd increased Cd toxicity Changes in behavior, including reduction in fish Black rockfish swimming speed and range of movement PS MP/NPs (0.5 and 15 m at Yin et al., (Sebastes  Increased oxygen consumption and ammonia excretion, 190 g/L) 2019 [71] schlegelii) reduction of growth and energy reserve, with microparticles having greater e ect than nanoparticles Abnormal behaviors, including erratic movement, seizures, and morphological changes associated with MP Zebrafish (Danio MPs accumulation in gill and Mak et al., PE MPs (10–600 m at 2 mg/L) feeding of adult fishes rerio) intestine 2019 [72] Upregulation of intestinal Cytochrome P450 gene (cyp 1a) and liver vitellogenin 1 Int. J. Environ. Res. Public Health 2020, 17, 1509 7 of 24 Table 1. Cont. Tissue Accumulation/ Notes on Toxicological, Pathological, Fishes Properties of MPs/NPs Used References Invasion or Cellular Uptake or Behavioral Observations Oxidative stress and structural damages in tissues with MP accumulation Medaka (Oryzias PS nanoparticles (10 m at MPs accumulation in gill, intestine,  Reproductive endocrine disruption in a sex-dependent Wang et al., melastigma) 2–200 g/L) and liver manner. 2019 [77] Prenatal exposure to MPs a ected the early development of o spring NP accumulation in intestine, Zebrafish (Danio  Disruption of glucose homeostasis Brun et al., PS nanoplastics, 25 nm pancreas, and gallbladder of exposed rerio)  Increase cortisol levels and hyperactivity 2019 [87] larvae Alterations in intestinal mucosa and gill epithelium with PS and PE NPs (with size higher neutrophil infiltration Zebrafish (Danio distribution indicated as  Changes in the expression of immune system genes, Limonta et rerio) 90% < 90 m; 50% < 50 m; down-regulation of genes correlated with epithelium al., 2019 [91] 10% < 25 m) integrity and lipid metabolism Changes in daily activity pattern Japanese Medaka Increase in ROS with corresponding changes in GSH Choi et al., (Tigriopus PS MP/NPs, 50 nm and 10 m and antioxidant enzyme activities 2019 [92] japonicas) Dose-dependent decreases in egg number in mature females Zhu et al., Oryzias latipes PS MPs, 10 m MP accumulation in gill and gut Swollen enterocytes and histological alterations of 2019 [93] buccal cavity, head kidney, and spleen MPs induced significant changes in morphometric Zebrafish (Danio Malafaia et PE MPs, 38.26  15.64 m parameters of larvae rerio) al., 2019 [94] MPs cause lower larval survival rate after egg hatching. Larvae ingestion of MPs decreased viability, decreased head/body ratios, increased Ethoxyresorufin-O-deethylase Japanese Medaka Environmental MP samples (EROD) activity, DNA breaks and altered swimming Pannetier et (Tigriopus collected from beaches behavior al., 2020 [73] japonicas) Juveniles exhibited no symptoms except for increase in DNA breaks Int. J. Environ. Res. Public Health 2020, 17, 1509 8 of 24 Table 1. Cont. Tissue Accumulation/ Notes on Toxicological, Pathological, Fishes Properties of MPs/NPs Used References Invasion or Cellular Uptake or Behavioral Observations MP/NPs inhibit fish larvae growth at high levels, increased larvae heart rate and decreased swimming speed Goldfish PS MP/NPs, 70 nm and 5 m, at  Observations of histopathological changes to intestine, Yang et al., (Carassius 10, 100 and 1000 g/L liber and gill, and damages to skin and muscle 2020 [74] auratus) MP/NPs elevated oxidative stress markers and related enzymes MPs reduced weight and body length of carp larvae Carp (Cyprinus PVC MPs, ~100–200 m, at Xia et al., Histopathological changes in liver carpio) 45.55, 91.1, and 136.65 g/L 2020 [85] Elevated oxidative stress and related enzyme activities Non-laboratory feeding observations Wild fishes (Dicentrachus labrax, Trachurus Fishes with MP have significantly higher lipid trachurus, MPs found in gastrointestinal tract, Barboza et al., MPs found in 49% of fishes peroxidation levels in the brain, gills. and dorsal muscle Scomber colias) gills. and dorsal muscle 2019 [95] and increased brain acetylcholinesterase activity sampled from North East Atlantic Ocean Int. J. Environ. Res. Public Health 2020, 17, 1509 9 of 24 In line with MPs/NPs activity as carriers or vectors of environmental contaminants, studies using fish cell lines have revealed that, while pristine plastics show no toxicity, those sampled from di erent islands around the world do [96], and so do those that have been mixed with human pharmaceuticals [97]. MPs/NPs were shown to modulate the toxicities a range of pollutants/toxicants, including phenanthrene [75], mercury [49], cadmium [46–48], polychlorinated biphenyls (PCBs) [98], gold ions [99], and the antibiotic roxithromycin [100], in fishes. However, adsorption of toxicants by the plastics could also potentially lower their toxicity, and such is the case for a complex mixture of polycyclic aromatic hydrocarbons (PAHs) [101]. Beyond MP/NP feeding experiments in a laboratory setting, a sampling of wild fishes consumed by humans have indicated that those with MPs found in the gut and other tissues had significantly higher lipid peroxidation levels in the brain, gills, and dorsal muscle and increased brain AChE activity compared to fishes with no MP found in their tissues [95]. These correlations are strongly suggestive of MP/NP uptake being a general stress factor for marine vertebrates. Overall, despite a large variance in MPs/NPs used, fish models tested, and toxicity parameters examined, there is ample evidence of concentration-dependent acute toxicity as well as chronic e ects. Furthermore, environmental toxicants adsorbed onto MPs/NPs would likely change the plastics’ toxicity profiles, often with an enhancement of toxicant uptake or an increase in their bioavailability. 3. Toxicity of MPs/NPs in Mouse Models In the past three years, a good number of studies have examined the e ect of pristine MPs/NPs in mammalian models (largely mouse). These studies are summarized in Table 2 and are broadly recapped below. In mice, ingested MPs/NPs could be found in the gut [102–105], liver [102,103,105], and kidney [102,105]. Pathological changes to the gut include a reduction in mucus secretion [90], gut barrier dysfunction [104,106], intestinal inflammation [107] and gut microbiota dysbiosis [90,104,106,107]. Liver pathologies documented include inflammation and lipid accumulation or lipid profile changes [90,102,106], as well as changes in the markers of lipid metabolism [90,105,108]. Other metabolic problems noted by omics-type analyses include disorders in energy metabolism [102,105] and bile acid metabolism [104]. On the other hand, a study with mice fed with PS MPs did not reveal any histologically detectable lesions or significant inflammatory responses [109]. A neurobehavioral study on rat fed with NPs also did not detect any significant behavioral changes or abnormality [110]. Recently, Luo and colleagues documented that maternal exposure to PS during gestation causes metabolic disorders in the o spring [106,108]. As in fishes, MPs aggravated the toxicity of an environmental toxicant, organophosphorus flame retardants (OPFRs) [103]. Taken together, the works in mice feeding experiments recapitulated some of MPs/NPs’ acute toxicity observed in fish feeding experiments. Such observed toxicities correlated with plastic size [111], concentration [112], and cellular/tissue uptake and accumulation. In general, the degree of MP/NP toxicity observed in mice is less severe than that observed in fishes. One possible reason is that fishes have multiple routes for plastic uptake and accumulation (gut and gills), whereas mice feeding experiments limit uptake through the gastrointestinal route. Int. J. Environ. Res. Public Health 2020, 17, 1509 10 of 24 Table 2. A summary of notable toxicological and/or pathological findings associated with MPs/NPs in mouse. PS, polystyrene; PE, polyethylene; PVC, polyvinylchloride; NPs, nanoplastics (<1 m); MPs, microplastics. Tissue Accumulation/ Properties of MPs/NPs Used Invasion or Cellular Notes on Toxicological, Pathological, or Behavioral Observations References Uptake Significant Toxicity/Pathology Signs of inflammation and lipid accumulation in liver Polystyrene (PS) microspheres 5 m Accumulation in gut,  Altered lipid profile and impairment of energy metabolism (reduction in Deng et al., 2017 [102] and 20, 0.01–0.5 mg/day liver, and kidney ATP levels) Increased liver oxidative stress markers, decreased acetylcholinesterase Decreased in body, liver, and lipid weights Decreased mucus secretion in the gut PS particles (0.5 and 50 m)  Alteration in gut microbiota Lu et al., 2018 [90] Changes in hepatic lipid profile and expression of some genes related to lipid metabolism PS and PE beads (0.5–1.0 m) + PS and PE beads  MPs enhanced OPFR-induced oxidative stress, neurotoxicity, and organophosphorus flame retardants Deng et al., 2018 [103] detectable in gut and liver metabolic disorder compared to OFPR alone. (OPFRs) Caused intestinal barrier dysfunction PS particles (5 m, 100 and 1000 Accumulation in mouse Induced gut microbiota dysbiosis Jin et al., 2018 [104] g/L) gut Induced bile acids metabolism disorder Toxicokinetic/toxicodynamic (TBTK/TD) modeling of Accumulation in mouse organ-bioaccumulation and biomarker responses PS particles (5 and 20 m) Yang et al., 2019 [105] gut, liver, and kidney  Changes in oxidative stress markers and those of energy and lipid metabolism Noticeable liver histopathology and altered serum and hepatic markers Changes in transcript of genes related to glycolipid metabolism Metabolic disorder associated with gut microbiota dysbiosis and gut PS MPs (5 m) Luo et al., 2019 [106] barrier dysfunction. Maternal MPs exposure resulted in intergenerational e ects and caused long-term metabolic consequences in the F1 and F2 generations. MP exposure caused changes in serum and liver metabolic markers PS MPs (0.5 and 5 m)  Maternal MPs exposure caused fatty acid metabolic disorder in the F1 Luo et al., 2019 [108] o spring Int. J. Environ. Res. Public Health 2020, 17, 1509 11 of 24 Table 2. Cont. Tissue Accumulation/ Properties of MPs/NPs Used Invasion or Cellular Notes on Toxicological, Pathological, or Behavioral Observations References Uptake MP exposure a ected composition and diversity of gut microbiota increased the secretion of IL-1 in serum, and decreased the Th17 and Treg PS MPs (10–150 m) Li et al., 2019 [107] cells among CD4+ cells High-concentration of MPs induced inflammation of the small intestine No E ect or Insignificant E ect PS particles (25 and 50 nm)  No significantly measurable neurobehavioral consequences in rats Rafiee et al., 2018 [110] No significant e ect on body/organ weight and no pathological signs by histological examination PS particles (1, 4 and 10 m)  Reporter analyses did not reveal evidence for the occurrence of Stock et al., 2019 [109] inflammation and/or oxidative stress Very low number of particles taken up by intestinal tissue Int. J. Environ. Res. Public Health 2020, 17, 1509 12 of 24 4. Toxicity of MPs/NPs in Human Cells Could MPs/NPs a ect human cells and tissues? There is obviously a lack of toxicity data for humans in vivo at the moment. Several studies have, however, looked at the e ect of pristine MPs/NPs on human cells in culture. These works are summarized in Table 3. Not surprisingly, a few of these studies, despite documenting some degree of cellular uptake, found signs of cellular toxicity either absent or insignificant except at very high concentrations of MPs/NPs [109,113,114]. In one case, where polyethylene terephthalate (PET) NPs generated by laser ablation were tested on the human gut adenocarcinoma epithelial line Caco-2, the authors noted a propensity for NP uptakes and crossing of a Caco-2 cells-based intestinal barrier model [113]. A few other studies have documented some degree of cellular toxicity or pathological e ect in a range of human cell lines. Prietl and colleagues showed that 20 nm PS NPs are taken up easily by human monocytic cells and are significantly cytotoxic. Larger (100 and 1000 nm) NPs stimulated the secretion of cytokines such as IL-6 and IL-8 from monocytes and macrophages, and could, interestingly, induce a measurable degree respiratory burst in monocytes [115]. Schirinzi and colleagues documented low but measurable degree of reactive oxygen species (ROS) production and induction of cytotoxicity by MPs in T98G and HeLa cells [116]. Wu and colleagues also worked with Caco-2 cells, and reported that, while a low degree of toxicity was observed for PS NPs (at 0.1 and 5 m), they induced mitochondrial depolarization and inhibited the activity of the toxicant eux pump, ATP-binding cassette (ABC) transporter, with the latter resulting in increased arsenic toxicity [117]. Hwang et al. worked with a number of cell types of human and mouse origin, and documented cytotoxicity associated with 20 m PP MPs at high concentrations and ROS induction [118]. The MPs also measurably induced pro-inflammatory cytokines IL-6 and TNF- from human peripheral blood mononuclear cells (PBMCs), and increased histamine release from mast cell lines [118]. Poma and colleagues found that 100 nm PS NPs stimulated ROS production and induced genotoxic stress and DNA damage as measured with the cytokinesis-block micronucleus (CBMN) assay [119]. Owing to the presence of large amounts of plastic particles in air, terrestrial animals are also exposed to MPs/NPs via inhalation. In this connection, Dong and colleagues found that PS MPs produced some cytotoxic e ects, oxidative stress, and inflammatory responses in human lung epithelial cells, and are disruptive of the epithelial cell layer, at least in vitro [120]. Two other groups have recently checked the toxicity of NPs in lung [121] and bronchial [122] cells. Xu and colleagues found that PS NPs (25 and 70 nm) impaired viability, induced cell cycle arrest, and upregulated nuclear factor (NF)-B as well as some pro-inflammatory cytokines in the human alveolar epithelial line A549 [121]. On the other hand, Lim and colleagues noted that PS NPs are only cytotoxic at high concentrations but induced metabolic changes and endoplasmic reticulum (ER) stress in a human bronchial epithelial cell line [122]. Overall, the experiments with pristine MPs/NPs on human cells reported thus far did not indicate severe cytotoxic or cytostatic e ects, but did demonstrate a potential for low to moderate negative e ects depending on the cell type, MP/NP sizes, and degree of cellular uptake. Two general and prominently observed phenomena appear to be ROS production and pro-inflammatory responses. In the paragraphs below, we explore how these toxic e ects may occur. Int. J. Environ. Res. Public Health 2020, 17, 1509 13 of 24 Table 3. A summary of notable toxicological findings associated with MPs/NPs in human cells. PS, polystyrene; PE, polyethylene; PVC, polyvinylchloride; NPs, nanoplastics (<1 m); MPs, microplastics. Properties of Human Cell Models Cellular Uptake Notes on Toxicological Observations References MPs/NPs Used Significant Toxicity 20 nm NPs cytotoxic to U937 and THP-1 cells 20 nm NPs stimulated IL-8 secretion in human monocytes and induced measurable oxidative burst in Human Peripheral blood monocytic cells 20 nm nanoparticles monocytes (PBMCs) taken up passively,  500 and 1000 nm NPs stimulated IL-6 and IL-8 secretion Carboxylated PS NPs Prietl et al., U937 (human monocytic cell line) while larger ones taken in monocytes and macrophages, chemotaxis and (20–1000 nm) 2014 [115] THP-1 (human monocytic cell line) up both actively and phagocytosis of bacteria by macrophages, and provoked an DMBM-2 (mouse macrophage cell line) passively oxidative burst of granulocytes At lower concentrations with no cytotoxicity, 20 nm NPs inhibited, while 500 and 1000 nm NPs increased phagocytosis of bacteria by DMBM-2 T98G (human glioblastoma cell line) PE microparticles  Induced ROS generation Schirinzi et HeLa (human cervical adenocarcinoma (3–16 m)  Cytotoxic e ect, with PE having a higher EC value al., 2017 [116] cell line) PS particles (10 m) compared to PS in both T98G and HeLa cells Low toxicity on cell viability, oxidative stress, and membrane integrity and fluidity Caco-2 (human epithelial colorectal PS particles (0.1 and 5 Cellular uptake of Wu et al., Disruption of mitochondrial membrane potential adenocarcinoma cell line) m) nanoparticles 2019 [117] Inhibition of plasma membrane ATP-binding cassette (ABC) transporter activity Some degree of cytotoxicity at high dosages of the Human dermal fibroblasts smaller size 20 m particles Peripheral blood mononuclear cells PP particles (~20 m Low degree of induction of proinflammatory cytokines (PBMCs) and 25–200 m), either IL-6 and TNF- from PBMCs Hwang et al., HMC-1 (human mast cell line 1) first dispersed in Increased histamine release from HMC-1 and RBL-2H3 2019 [118] RBL-2H3 (human basophilic leukemia DMSO or used directly cells cell line) in culture media Some degree of ROS induction at high dosages of the RAW 264.7 (mouse macrophage cell line) smaller size 20 m particles Cytotoxic e ects PS MPs (4.06  0.44 Dong et al., BEAS-2B (human lung epithelial cells)  Oxidative stress and inflammatory responses m at 1–1000 g/cm 2020 [120] Disruption of epithelial layer Int. J. Environ. Res. Public Health 2020, 17, 1509 14 of 24 Table 3. Cont. Properties of Human Cell Models Cellular Uptake Notes on Toxicological Observations References MPs/NPs Used Decreased viability and induced cell cycle arrest Upregulation of transcripts for NF-B and some A549 (Human alveolar type II epithelial PS nanoparticles (25 Cellular uptake of Xu et al., 2019 pro-inflammatory cytokines cell line) and 70 nm) nanoparticles [121] Alteration of cell cycle and apoptosis-regulation related protein expressions PS NPs only cytotoxic at very high concentrations BEAS-2B (Human bronchial epithelial Cellular uptake of PS  Metabolomics analyses revealed autophagic and Lim et al., PS nanoparticles cells) nanopaticles endoplasmic reticulum (ER) stress-related metabolic 2019 [122] changes Stimulation of ROS production PS nanoparticles (100 Poma et al., Hs27 (Human fibroblasts)  Genotoxic stress and DNA damage measured with the nm at 5–75 g/ml) 2019 [119] cytokinesis-block micronucleus (CBMN) assay No or Insignificant E ects No apparent toxic e ect Polyethylene Nano-PET are internalized into endo-lysosomal terephthalate (PET) Magri et al., Caco-2 Cellular uptake of NPs compartments NPs (laser ablated, ca. 2018 [113] Nano-PET has high propensity to cross the Caco-2 100 nm) intestinal barrier model Low crossing of the cell monolayer on Transwells even by 1 m microparticles Caco-2 PS microparticles (1, 4, Cellular uptake of PS  No pronounce loss of cell viability except only at very Stock et al., THP-1 monocytic line and 10 m) microparticles high dosage of 1 m microparticles 2019 [109] Microparticles uptake did not a ect macrophage di erentiation or polarization No significant cytotoxicity unless at very high Caco-2 and HT29-MTX-E12 (human Carboxy-modified PS concentrations colon epithelial cell) co-culture Cellular uptake of PS Hesler et al., nanoparticles (50 nm  No significant transport across the in vitro intestinal and BeWo b30 (Human placental trophoblast nanoparticles 2019 [114] and 0.5 m, placental “barriers” but intercellular distribution was cell) observed Int. J. Environ. Res. Public Health 2020, 17, 1509 15 of 24 5. Mechanisms Underlying MPs/NPs’ Acute or Chronic Toxicity in Mammalian Cells In general, extremely high concentration of MPs/NPs are indeed cytotoxic. Cell death could occur via necrotic plasma membrane rupture or some form of programmed cell death. An important point to note on the former, rather non-specific mode of death is the surfactant molecules that are typically associated with most MP/NP preparations. At high concentrations, these would be disruptive to the lipid bilayer of the plasma membrane (PM). Even at moderate levels, these could disrupt important cellular surface structures such as proteoglycans and other extracellular matrix components or hinder cellular signaling processes that require extracellular ligand and cell surface receptor interactions. Therefore, cellular physiology would be a ected to varying degrees by plastic associated surfactants, and the documented changes in various transcripts in cells could also be due to this and other processes/factors described below. The smaller NPs in particular could be taken up with some ease depending on the cell type via endocytosis [123,124]. Endocytosed NPs present a problem for several reasons. Firstly, they could, as per the plasma membrane discussed above, potentially permeabilize the endosomal membranes if present at high concentrations. If this happens, the NPs released into the cytosol could potentially interact with and a ect important organelles such as the mitochondria or the nucleus, as well as cellular processes such as mitotic spindle formation and chromosomal migration during cell division. Secondly, MPs/NPs would likely interfere with the tracking of transport carriers in the cell along the exocytic pathway [125,126], and as such would potentially inhibit the cell surface expression of important signaling receptors or membrane transporters. Thirdly, they are likely to perturb endosomal membrane trac on which many important cellular processes are dependent, including surface protein turnover and signaling attenuation, as well as retrograde signaling from endosomal compartments. It is unclear if NPs could themselves ever be subjected to inter-compartmental transport eciently in the endosomal pathway. Even if the NPs could eventually end up in the lysosome, they are unlikely to be readily digested. The accumulation of NPs in late endosome or lysosomes would perturb the degradative functions of these organelles and importantly impair the critical cellular membrane turnover process of macroautophagy [122]. An impairment of autophagic clearance could potentially lead to positive feedback processes that culminate in autophagic cell death. On the other hand, internalized MPs/NPs may also stimulate autophagy. Metallic nanoparticles are known to modulate autophagy [127], and MPs/NPs may speculatively do likewise. At the very least, these processes would constitute a form of cellular stress. Stresses at the PM and the endo-lysosomes would trigger cellular stress responses. In work done with species of the fresh water flea Daphnia, PS NPs exposure a ected growth and reproduction [128], and interestingly resulted in the elevation of AMP activated protein kinase (AMPK), which is an indication of stress response [129]. Perhaps a more general associated phenomenon with regards to cellular stress response appears to be the production of ROS, which was in fact recently identified as the molecular initiating event (MIE) by adverse outcome pathways analysis of reports in the field [130]. ROS production in cells occurs in two general ways: from the mitochondrial electron transport chain (ETC) during routine aerobic respiration or via the oxidative bursts of NADPH oxidases (NOXs) [131]. An increase in ROS from the former could result from mitochondrial function impairment, while the latter is normally a consequence of bacterial invasion, as NOXs are activated by bacterial products and cytokines. All cells are endowed with an evolutionarily conserved innate immunity mechanism, typically functioning against invasion of pathogens or exposure to xenobiotics [132]. However, the components of the innate immune system, such as the Toll-like receptors (TLRs), could also respond to a set of endogenous or secreted molecules collectively known as alarmins, or damage-associated molecular patterns (DAMP) [133,134], and the outcome is what is termed sterile-inflammation, i.e. inflammatory responses without pathogenic infection [135]. In the body, pro-inflammatory cytokines released from such localized inflammations would attract circulating immune cells, and this could worsen the local inflammation, and cause cell and tissue death. NPs has indeed been shown to act as stressors to the innate immune system of fish [88], and this is likely also the case for mammalian (including human) cells. The cellular and tissue Int. J. Environ. Res. Public Health 2020, 17, 1509 16 of 24 invasion and general pathological mechanism of MPs/NPs in mammalian cells is summarized below Int. J. Environ. Res. Public Health 2020, 17, x 17 of 24 in Figure 1. Figure 1. A schematic diagram illustrating potential (speculative at the moment) cellular mechanisms Figure 1. A schematic diagram illustrating potential (speculative at the moment) cellular mechanisms of MP/NP toxicity. MPs/NPs can be taken up through ingestion and inhalation. MPs/NPs could damage of MP/NP toxicity. MPs/NPs can be taken up through ingestion and inhalation. MPs/NPs could the plasma membrane and impair the gut barrier (left). These could also perturb signaling of cell surface damage the plasma membrane and impair the gut barrier (left). These could also perturb signaling receptors, and alter gene expression in the nucleus. Endocytosed MPs/NPs could also perturb the of cell surface receptors, and alter gene expression in the nucleus. Endocytosed MPs/NPs could endocytic pathway function and compromise the endosomal membranes. Stresses arising from the also perturb the endocytic pathway function and compromise the endosomal membranes. Stresses above could activate the cellular innate immune system, with endogenous and secreted damage- arising from the above could activate the cellular innate immune system, with endogenous and associated molecular patterns (DAMP) inducing the innate immunity-mediating toll-like receptors secreted damage-associated molecular patterns (DAMP) inducing the innate immunity-mediating (TLRs). Stresses could induce ROS production from the NADP oxidases (NOXs). Mitochondrial toll-like receptors (TLRs). Stresses could induce ROS production from the NADP oxidases (NOXs). impairment, either by MPs/NPs from endosomes or in response to stresses, could also produce more Mitochondrial impairment, either by MPs/NPs from endosomes or in response to stresses, could ROS through impairment in the efficiency of electron transport chain (ETC) processes. MPs/NPs gain also produce more ROS through impairment in the eciency of electron transport chain (ETC) access into the circulation if the gut–vascular barrier is compromised or it may speculatively occur by processes. MPs/NPs gain access into the circulation if the gut–vascular barrier is compromised or it transcytosis, thus reaching other organs. The lung probably has a more direct access to airborne may speculatively occur by transcytosis, thus reaching other organs. The lung probably has a more MPs/NPs (right). direct access to airborne MPs/NPs (right). 6. MPs/NPs’ Potential Systemic E ect in Humans 7. Conclusions MPs/NPs are expected to reach the human gut through consumption of contaminated food MPs/NPs have pervaded the environment and human’s exposure and cumulative uptake of these materials. Undigested MPs would be largely excreted though fecal matter, but smaller NPs could plastics would only increase over time. Currently, it appears that any worries of acute toxicity or severe potential enter the circulation. Ingested MPs/NPs would first encounter the intestinal epithelium. Only long-term effect that would lead to significantly enhanced morbidity or mortality are unfounded. unrealistically high concentration of plastics, or those carrying adsorbed toxicants, would likely cause However, we still know very little about how MPs/NPs from the environment, be it from a seafood acute impairment of viability and inflammation of the gut lining [51]. However, the e ect of persistent meal or the plastic bottled drink, would affect human health. Clearly, much more research, in terms of presence of ineciently cleared MPs/NPs in the gut is yet unknown. Gut pathology resulting from both cellular and tissue level pathological mechanisms, as well as on the long-term effects of MPs/NPs has been widely documented in fishes. The mouse experiments have provided some clear tissue/organ accumulation, is needed. Plans and collaborative attempts between ecologists and illustration of the consequences of gut toxicity. Should this happen, the gut–vascular barrier could epidemiologists to study bioaccumulation of MPs/NPs in humans via the food chain in various be impaired and MPs/NPs could enter the circulation, where these could gain access to the liver via geographical locales would also be necessary. Author Contributions: Conceptualization, B.L.T.; writing—original draft preparation, B.L.T.; and writing—review and editing, C.Q.Y.Y., S.V., and B.L.T. All authors have read and agreed to the published version of the manuscript. Int. J. Environ. Res. Public Health 2020, 17, 1509 17 of 24 the portal vein. That this is possible was demonstrated in some of the mouse models [102,103,105]. Long-term accumulation of MPs/NPs in liver tissues and chronic inflammation could lead to liver diseases and metabolic problems. On the other hand, accumulation of MPs/NPs in lung tissues could potentially result in chronic pulmonary disorders. Furthermore, the presence of NPs in brain tissues has been demonstrated in a fish model, as discussed above [68]. It should, however, be noted that it remains to be shown if the MPs/NPs could in fact be found in the brain of experimental mice or human brain samples. For that matter, no cell or tissue accumulation, pathology, or metabolic impairment due to MPs/NPs has been clearly demonstrated for humans to date. One of the more common findings in the mice studies is gut microbiota dysbiosis [90,104,106,107]. Changes in gut microbiome could result in gustatory dysfunction, thus perturbing physiological homeostasis in general. More importantly, gut microbiota changes have been linked to a range of chronic diseases of other organs, including disease of the kidney [136], cardiovascular system [137], inflammation, and cancer [138], as well as neurological disorders [91,139]. With regards to the latter, gut microbiota dysbiosis could in fact be one of the underlying reasons for behavioral changes in larger animals treated with MPs/NPs. It has also been reported that blood proteins such as albumin and globulin interact with NPs to form protein–plastic complexes [140,141]. Such aggregated protein–plastic complexes, if present in large quantities, could potentially lead to blockage of blood vessels. In addition, while loading of red blood cells (RBCs) with NPs at a low 1:50 ratio did not a ect functions of RBCs, a 10–50-fold higher loading showed RBC damages induced by mechanical, osmotic, and oxidative stresses [142]. However, it is dicult to envisage a large acute accumulation of NPs to occur in the human circulation under natural conditions. 7. Conclusions MPs/NPs have pervaded the environment and human’s exposure and cumulative uptake of these plastics would only increase over time. Currently, it appears that any worries of acute toxicity or severe long-term e ect that would lead to significantly enhanced morbidity or mortality are unfounded. However, we still know very little about how MPs/NPs from the environment, be it from a seafood meal or the plastic bottled drink, would a ect human health. Clearly, much more research, in terms of both cellular and tissue level pathological mechanisms, as well as on the long-term e ects of tissue/organ accumulation, is needed. Plans and collaborative attempts between ecologists and epidemiologists to study bioaccumulation of MPs/NPs in humans via the food chain in various geographical locales would also be necessary. Author Contributions: Conceptualization, B.L.T.; writing—original draft preparation, B.L.T.; and writing—review and editing, C.Q.Y.Y., S.V., and B.L.T. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by a Cross-Faculty Research Grant from the Oce of Deputy President (Research & Technology), National University of Singapore, grant number CFGFY18P07. Acknowledgments: We thank Fanny Ng for technical assistance and advice. The authors are grateful to the reviewers, whose constructive comments improved the manuscript. Conflicts of Interest: The authors declare no conflict of interest. References 1. Zalasiewicz, J.; Waters, C.; Ivar do Sul, J.; Corcoran, P.; Barnosky, A.; Cearreta, A.; Edgeworth, M.; Gałuszkah, A.; Jeandel, C.; Leinfelder, R.; et al. The geological cycle of plastics and their use as a stratigraphic indicator of the Anthropocene. Anthropocene 2016, 13, 4–17. [CrossRef] 2. Geyer, R.; Jambeck, J.R.; Law, K.L. Production, use, and fate of all plastics ever made. Sci. Adv. 2017, 3, e1700782. [CrossRef] [PubMed] 3. Gündogdu, ˘ S.; Yesilyurt, ¸ I.N.; Erbas, ¸ C. 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Toxicity of Microplastics and Nanoplastics in Mammalian Systems

International Journal of Environmental Research and Public Health , Volume 17 (5) – Feb 26, 2020

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Pubmed Central
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© 2020 by the authors.
ISSN
1661-7827
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1660-4601
DOI
10.3390/ijerph17051509
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Abstract

International Journal of Environmental Research and Public Health Review Toxicity of Microplastics and Nanoplastics in Mammalian Systems 1 2 1 , 3 , Cheryl Qian Ying Yong , Suresh Valiyaveettil and Bor Luen Tang * Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117596, Singapore; cherylyongqy@hotmail.com Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543, Singapore; chmsv@nus.edu.sg NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore 119077, Singapore * Correspondence: bchtbl@nus.edu.sg; Tel.: +65-6516-1040 Received: 12 February 2020; Accepted: 23 February 2020; Published: 26 February 2020 Abstract: Fragmented or otherwise miniaturized plastic materials in the form of micro- or nanoplastics have been of nagging environmental concern. Perturbation of organismal physiology and behavior by micro- and nanoplastics have been widely documented for marine invertebrates. Some of these e ects are also manifested by larger marine vertebrates such as fishes. More recently, possible e ects of micro- and nanoplastics on mammalian gut microbiota as well as host cellular and metabolic toxicity have been reported in mouse models. Human exposure to micro- and nanoplastics occurs largely through ingestion, as these are found in food or derived from food packaging, but also in a less well-defined manner though inhalation. The pathophysiological consequences of acute and chronic micro- and nanoplastics exposure in the mammalian system, particularly humans, are yet unclear. In this review, we focus on the recent findings related to the potential toxicity and detrimental e ects of micro- and nanoplastics as demonstrated in mouse models as well as human cell lines. The prevailing data suggest that micro- and nanoplastics accumulation in mammalian and human tissues would likely have negative, yet unclear long-term consequences. There is a need for cellular and systemic toxicity due to micro- and nanoplastics to be better illuminated, and the underlying mechanisms defined by further work. Keywords: human cells; microplastics; mouse cells; nanoplastics; oxidative stress; toxicants; toxicity 1. Introduction One of the most prominent classes of non-natural products made by humans that has pervaded Earth’s surface environment is plastics, so much so that these durable synthetic organic polymers are heralded as a defining stratigraphic marker for the Anthropocene [1]. Geyer and colleagues recently estimated that 8.3 billion metric tons of virgin plastics have been produced up to the year 2017 [2], and with continuation of current production and waste management practices, about 12 billion tons of plastic waste would be found in landfills and the natural environment by 2050. Plastic wastes are persistent environmental pollutants. Larger pieces of plastic wastes present well-publicized ecological problems in terms of physical entanglement and entrapment [3], physical barriers for food supply [4], and digestive tract congestion. The smaller plastic pieces, particularly their miniaturized forms that are less than 5 mm in size, are generally termed microplastics (MPs) [5]. Plastics that are already small in size to begin with, such as those purposefully manufactured in the form of microbeads in skincare products (primary MPs), or those derived from degradation of larger plastic pieces (secondary MPs), permeate both the terrestrial [6] and the marine [7,8] environments. Plastic particles of less Int. J. Environ. Res. Public Health 2020, 17, 1509; doi:10.3390/ijerph17051509 www.mdpi.com/journal/ijerph Int. J. Environ. Res. Public Health 2020, 17, 1509 2 of 24 than 1 m in size are also known as nanoplastics (NPs) [9,10]. These chemically inert MPs/NPs pose significant ecological and health concerns [5] because of their environmental persistence [6,11,12], potential ecotoxicity [13,14], and their ability to act as vectors for chemical pollutants [15–17] as well as pathogens [18,19]. Ecotoxicological e ects of MPs/NPs on marine phyto/zooplanktons, invertebrates, and plants are widely documented [20–30], and have been recently reviewed [5,31–33]. MPs/NPs could also be ingested and accumulated in larger marine fauna by trophic transfer from prey to predator, as demonstrated earlier with invertebrates, such as mussel-consuming crabs [34]. Interesting illustrations of trophic transfer within a lab-simulated food chain were shown by Mattsson and colleagues, where 53-nm polystyrene (PS) particles could be transferred from algae to the zooplankter Daphnia magna, and then to a freshwater fish [35]. Likewise, An and colleagues demonstrated trophic transfer of NPs from algae to Daphnia, then to a secondary consumer fish, and finally to an end consumer fish [36]. On the other hand, organic pollutants could be adsorbed onto MPs/NPs [37–41] and there is evidence that this could potentially enhance their e ective uptake and toxicity [42–45]. Likewise, MPs/NPs are known to interact with metallic toxicants such as Cadmium [46–48], Mercury [49], and other toxic trace elements [50], and could potentially serve as vectors for pollutant transfer to living organisms. The e ects of MPs/NPs on mammalian cells and tissues, particularly humans, have remained rather unclear [51,52]. While plastics are generally perceived to pose minimum risk to human, several recent scientific findings, picked up by the popular press, have heightened the worry of possible tissues penetrance and adverse e ects of MPs/NPs due to their small sizes. Humans could accumulate MPs/NPs from di erent food sources [53,54] as well as drinking water [55,56]. Plastic water containers [57,58] and plastic teabags [59] are, perhaps unsurprisingly, common sources for human ingested MPs/NPs. MPs/NPs could also be taken up by inhalation [60]. MPs/NPs have also been detected in human stool samples [61], an indication that the quantity taken in is significantly large. A recent World Health Organization’s (WHO) report on “Microplastic in drinking water” indicates that there is not yet proof of harm, but it also calls for more research to be carried out [62]. Could environmental MPs/NPs gain access to cells and tissues and be harmful to humans? Although ecotoxicology data with marine invertebrate indicate that this is so, more barriers and obstacles would likely be encountered by MPs/NPs in order to gain access to cells and tissues of vertebrates compared to simpler invertebrates. Here, we review current results on how MPs/NPs might a ect humans by scrutinizing studies done to date on mammalian (mouse) models and human cells. We begin with a quick survey of MP/NP feeding studies done on marine vertebrates, focusing on fishes. A meta-analysis on the e ect of MP exposure on fish has been reported by Foley and colleagues in 2018 [63] and the field has also been recently reviewed [64], but several newer reports have now appeared. This quick look would allow some comparison of findings in more ecologically relevant settings with that of laboratory experiments with mice and human cells. 2. Toxicity of MPs/NPs in Fishes Table 1 provides a non-exhaustive summary of recent studies where MP/NP feeding experiments have documented some degree of toxicological or pathological e ect observed on fishes. Those that have shown some significant e ect are included in this summary, while those that have reported little or no e ects are not. The MPs/NPs used in the studies listed in Table 1 are largely polystyrene (PS) or polyethylene (PE) based. An important general phenomenon to note is that toxicological responses typically arise from smaller plastic particles. Larger PS particles at around 100 m or above were shown not to have any significant e ect in a number of studies [65–67]. MP/NP feeding can result in behavioral abnormalities in terms of feeding and movement of adults and larvae [35,68–74], as well as reproduction in adults [75–77]. There is evidence of mother–o spring transfer of NPs [78], and that prenatal exposure of MPs a ected early development of the neonates [77]. In many cases, MPs/NPs were found accumulated in larvae or adult gut [77,79–83], and in some cases in gill and liver [79]. Histopathology is most prominently observed for these tissues Int. J. Environ. Res. Public Health 2020, 17, 1509 3 of 24 as well [74,75,77,79,83–85]. For the gut, pathological manifestations of MP/NP toxicity include documented changes in gut biomarkers related to epithelial barrier integrity, inflammation, and oxidative stress [83,86], as well as changes in gut microbiota [82,83,86]. In the case of liver, changes in metabolites, key metabolic enzymes, and oxidative stress-induced enzymes occur [49,74,79,81,85,87]. MPs/NPs could be internalized [88], and then cause detectable biomarker changes in blood cells [47,88, 89]. In rarer cases, MPs/NPs have also been found in fish brain [68,81], and caused changes in brain appearances [35,68] or showed significantly inhibited acetylcholinesterase (AChE) activity [49,81]. NPs taken up by embryos and larvae have been documented to migrate to various tissues throughout development [70]. Int. J. Environ. Res. Public Health 2020, 17, 1509 4 of 24 Table 1. A summary of notable toxicological and/or pathological findings associated with MPs/NPs in fishes. PA, polyamide; PS, polystyrene; PE, polyethylene; PC, polycarbonate; PP, polypropylene; PVC, polyvinylchloride; NPs, nanoplastics (<1 m); MPs, microplastics. Tissue Accumulation/ Notes on Toxicological, Pathological, Fishes Properties of MPs/NPs Used References Invasion or Cellular Uptake or Behavioral Observations 24 and 27 nm polystyrene (PS) Crucian Carp  Defects in feeding and shoaling behavior nanoparticles (NPs) (to fish Trophic transfer to fish from algae Mattsson et (Carassius  Defects in metabolism through an aquatic food chain, through Daphnia al., 2015 [35] carassius)  Changes in brain appearance and weight from algae through Daphnia) Increased Cd accumulation in livers, guts, and gills Zebrafish (Danio Virgin PS microplastic beads (5  Enhanced Cd toxicity Lu et al., 2018 rerio) m) + cadmium (Cd)  combined exposure caused oxidative damage and [90] inflammation in tissues European Fluorescence red polymer  Inhibition of brain acetylcholinesterase (AChE) activity seabass microspheres, (1–5 m) and and increase lipid oxidation in brain and muscle Barboza et al., (Dicentrarchus mercury individually and in  Changes in activity of metabolic enzymes 2018 [49] labrax) combination  Interactions and influences on mercury bioaccumulation Crucian Carp Amino-modified positively Trophic transfer to fish from algae Changes in feeding time Mattsson et (Carassius charged PS nanoparticles through Daphnia. Nanoparticles Changes in brain morphology (gyri sizes) al., 2017 [68] carassius) (52 nm) found in fish brain Inhibited of larvae locomotion Zebrafish (Danio Chen et al., PS NPs (50 nm, 1 mg/L) Accumulation in zebrafish larvae  Inhibited acetylcholinesterase activity rerio) 2017 [69] Upregulation of cytoskeletal markers Virgin (50 or 500 g/L) or African catfish  Liver and gill histopathology phenanthrene-loaded (10 or 100 Karami et al., (Clarias  Changes in blood biochemistry g/L) low-density polyethylene 2016 [75] gariepinus)  Changes in the expression of reproductive axis genes (LDPE) fragments Microplastics observed in observed in  Increased mortality and decrease in average lengths and Medaka (Oryzias PS microspheres (10–11 m, Cong et al., digestive tracts of larvae and weights of larvae and adult fishes melastigma) 0.758  0.217  10 particles/L) 2019 [76] dissected intestine of adults  Significant decrease in egg production by females Int. J. Environ. Res. Public Health 2020, 17, 1509 5 of 24 Table 1. Cont. Tissue Accumulation/ Notes on Toxicological, Pathological, Fishes Properties of MPs/NPs Used References Invasion or Cellular Uptake or Behavioral Observations Decreased heart rate Uptake of the nanoparticles by Altered larval behavior (swimming hypoactivity in embryos and larvae. Zebrafish (Danio exposed larvae) Pitt et al., PS NPs (mean 51 nm) Migrated to the gastrointestinal tract, rerio)  Maternal-o spring transfer of PS nanoparticles 2018 [70,78] gallbladder, liver, pancreas, heart, Delay/defect in swim bladder inflation by exposed F1 and brain throughout development larvae Liver histopathology (signs of inflammation and lipid Zebrafish (Danio PS microspheres (70 nm, 5 m, Accumulation in gills, gut, and liver accumulation) Lu et al., 2016 rerio) and 20 m, 20 mg/L) (only the 5 m particles)  Elevation of anti-oxidative stress enzymes [79] Changes in liver metabolomics profile Significant changes in transcriptome of zebrafish larvae after 2 days exposure Zebrafish (Danio Ingested microplastics observed in LeMoine et PS MPs (10–45 m, 20 mg/L)  Downregulation of genes involved with neural rerio) larvae gut al., 2018 [80] development and function Changes in genes associated with metabolism Red tilapia PS NPs (0.1 m, at 1, 10, and 100 PS MPs found in gut and gills and to  Inhibition of brain acetylcholinesterase (AChE) activity Ding et al., (Oreochromis g/L) a lesser extent, liver and brain  Changes in liver enzyme markers 2018 [81] niloticus) Changes in larval gut microbiota Metabolomic alterations Zebrafish (Danio Fluorescent and virgin PS MPs Ingested microplastics observed in  Changes in the expression of genes associated with Wan et al., rerio) (5 and 50 m) gut of larvae glucose and lipid metabolism 2019 [82] Significant reduction in the antioxidant GSH and the enzyme catalase Induction of inflammation and oxidative stress of adult zebrafish gut Zebrafish (Danio PS microplastic beads (5-m Accumulation of microplastics in  Significant alterations in the metabolome and Qiao et al., rerio) beads; 50 g/L and 500 g/L) zebrafish gut microbiome of adult zebrafish gut. Alterations were 2019 [83] associated with oxidative stress, inflammation, and lipid metabolism Int. J. Environ. Res. Public Health 2020, 17, 1509 6 of 24 Table 1. Cont. Tissue Accumulation/ Notes on Toxicological, Pathological, Fishes Properties of MPs/NPs Used References Invasion or Cellular Uptake or Behavioral Observations Polyamides (PA), polyethylene (PE), polypropylene (PP), Zebrafish (Danio Lei et al., polyvinyl chloride (PVC) (~70  Intestinal damage of adult fish gut rerio) 2018 [84] m) and PS (0.1, 1, and 5 m) particles Fathead minnow  significant increases in innate immune response (in PS (41.0 nm) and polycarbonate Neutrophil phagocytosis of PS Greven et al., (Pimephales terms of degranulation of primary granules and neutrophil (PC) (158.7 nm) NPs nanoparticles. 2016 [88] promelas) extracellular trap release) Gilthead seabream (Sparus Increased oxidative burst of in leukocytes of Sparus aurata) and Virgin polyvinylchloride (PVC) aurata Espinosa et European sea and polyethylene (PE) (40–150 Upregulation of the redox regulator Nrf2 in leukocytes al., 2018 [89] bass m) of Sparus aurata (Dicentrarchus labrax) MPs from a face and body scrub,  Changes in plasma levels of various metabolic enzymes Carp (Cyprinus Banaee et al., mainly PE (250 and 500 g/L), and immune markers carpio) 2019 [47] alone and + Cd  Combination of MP and Cd increased Cd toxicity Changes in behavior, including reduction in fish Black rockfish swimming speed and range of movement PS MP/NPs (0.5 and 15 m at Yin et al., (Sebastes  Increased oxygen consumption and ammonia excretion, 190 g/L) 2019 [71] schlegelii) reduction of growth and energy reserve, with microparticles having greater e ect than nanoparticles Abnormal behaviors, including erratic movement, seizures, and morphological changes associated with MP Zebrafish (Danio MPs accumulation in gill and Mak et al., PE MPs (10–600 m at 2 mg/L) feeding of adult fishes rerio) intestine 2019 [72] Upregulation of intestinal Cytochrome P450 gene (cyp 1a) and liver vitellogenin 1 Int. J. Environ. Res. Public Health 2020, 17, 1509 7 of 24 Table 1. Cont. Tissue Accumulation/ Notes on Toxicological, Pathological, Fishes Properties of MPs/NPs Used References Invasion or Cellular Uptake or Behavioral Observations Oxidative stress and structural damages in tissues with MP accumulation Medaka (Oryzias PS nanoparticles (10 m at MPs accumulation in gill, intestine,  Reproductive endocrine disruption in a sex-dependent Wang et al., melastigma) 2–200 g/L) and liver manner. 2019 [77] Prenatal exposure to MPs a ected the early development of o spring NP accumulation in intestine, Zebrafish (Danio  Disruption of glucose homeostasis Brun et al., PS nanoplastics, 25 nm pancreas, and gallbladder of exposed rerio)  Increase cortisol levels and hyperactivity 2019 [87] larvae Alterations in intestinal mucosa and gill epithelium with PS and PE NPs (with size higher neutrophil infiltration Zebrafish (Danio distribution indicated as  Changes in the expression of immune system genes, Limonta et rerio) 90% < 90 m; 50% < 50 m; down-regulation of genes correlated with epithelium al., 2019 [91] 10% < 25 m) integrity and lipid metabolism Changes in daily activity pattern Japanese Medaka Increase in ROS with corresponding changes in GSH Choi et al., (Tigriopus PS MP/NPs, 50 nm and 10 m and antioxidant enzyme activities 2019 [92] japonicas) Dose-dependent decreases in egg number in mature females Zhu et al., Oryzias latipes PS MPs, 10 m MP accumulation in gill and gut Swollen enterocytes and histological alterations of 2019 [93] buccal cavity, head kidney, and spleen MPs induced significant changes in morphometric Zebrafish (Danio Malafaia et PE MPs, 38.26  15.64 m parameters of larvae rerio) al., 2019 [94] MPs cause lower larval survival rate after egg hatching. Larvae ingestion of MPs decreased viability, decreased head/body ratios, increased Ethoxyresorufin-O-deethylase Japanese Medaka Environmental MP samples (EROD) activity, DNA breaks and altered swimming Pannetier et (Tigriopus collected from beaches behavior al., 2020 [73] japonicas) Juveniles exhibited no symptoms except for increase in DNA breaks Int. J. Environ. Res. Public Health 2020, 17, 1509 8 of 24 Table 1. Cont. Tissue Accumulation/ Notes on Toxicological, Pathological, Fishes Properties of MPs/NPs Used References Invasion or Cellular Uptake or Behavioral Observations MP/NPs inhibit fish larvae growth at high levels, increased larvae heart rate and decreased swimming speed Goldfish PS MP/NPs, 70 nm and 5 m, at  Observations of histopathological changes to intestine, Yang et al., (Carassius 10, 100 and 1000 g/L liber and gill, and damages to skin and muscle 2020 [74] auratus) MP/NPs elevated oxidative stress markers and related enzymes MPs reduced weight and body length of carp larvae Carp (Cyprinus PVC MPs, ~100–200 m, at Xia et al., Histopathological changes in liver carpio) 45.55, 91.1, and 136.65 g/L 2020 [85] Elevated oxidative stress and related enzyme activities Non-laboratory feeding observations Wild fishes (Dicentrachus labrax, Trachurus Fishes with MP have significantly higher lipid trachurus, MPs found in gastrointestinal tract, Barboza et al., MPs found in 49% of fishes peroxidation levels in the brain, gills. and dorsal muscle Scomber colias) gills. and dorsal muscle 2019 [95] and increased brain acetylcholinesterase activity sampled from North East Atlantic Ocean Int. J. Environ. Res. Public Health 2020, 17, 1509 9 of 24 In line with MPs/NPs activity as carriers or vectors of environmental contaminants, studies using fish cell lines have revealed that, while pristine plastics show no toxicity, those sampled from di erent islands around the world do [96], and so do those that have been mixed with human pharmaceuticals [97]. MPs/NPs were shown to modulate the toxicities a range of pollutants/toxicants, including phenanthrene [75], mercury [49], cadmium [46–48], polychlorinated biphenyls (PCBs) [98], gold ions [99], and the antibiotic roxithromycin [100], in fishes. However, adsorption of toxicants by the plastics could also potentially lower their toxicity, and such is the case for a complex mixture of polycyclic aromatic hydrocarbons (PAHs) [101]. Beyond MP/NP feeding experiments in a laboratory setting, a sampling of wild fishes consumed by humans have indicated that those with MPs found in the gut and other tissues had significantly higher lipid peroxidation levels in the brain, gills, and dorsal muscle and increased brain AChE activity compared to fishes with no MP found in their tissues [95]. These correlations are strongly suggestive of MP/NP uptake being a general stress factor for marine vertebrates. Overall, despite a large variance in MPs/NPs used, fish models tested, and toxicity parameters examined, there is ample evidence of concentration-dependent acute toxicity as well as chronic e ects. Furthermore, environmental toxicants adsorbed onto MPs/NPs would likely change the plastics’ toxicity profiles, often with an enhancement of toxicant uptake or an increase in their bioavailability. 3. Toxicity of MPs/NPs in Mouse Models In the past three years, a good number of studies have examined the e ect of pristine MPs/NPs in mammalian models (largely mouse). These studies are summarized in Table 2 and are broadly recapped below. In mice, ingested MPs/NPs could be found in the gut [102–105], liver [102,103,105], and kidney [102,105]. Pathological changes to the gut include a reduction in mucus secretion [90], gut barrier dysfunction [104,106], intestinal inflammation [107] and gut microbiota dysbiosis [90,104,106,107]. Liver pathologies documented include inflammation and lipid accumulation or lipid profile changes [90,102,106], as well as changes in the markers of lipid metabolism [90,105,108]. Other metabolic problems noted by omics-type analyses include disorders in energy metabolism [102,105] and bile acid metabolism [104]. On the other hand, a study with mice fed with PS MPs did not reveal any histologically detectable lesions or significant inflammatory responses [109]. A neurobehavioral study on rat fed with NPs also did not detect any significant behavioral changes or abnormality [110]. Recently, Luo and colleagues documented that maternal exposure to PS during gestation causes metabolic disorders in the o spring [106,108]. As in fishes, MPs aggravated the toxicity of an environmental toxicant, organophosphorus flame retardants (OPFRs) [103]. Taken together, the works in mice feeding experiments recapitulated some of MPs/NPs’ acute toxicity observed in fish feeding experiments. Such observed toxicities correlated with plastic size [111], concentration [112], and cellular/tissue uptake and accumulation. In general, the degree of MP/NP toxicity observed in mice is less severe than that observed in fishes. One possible reason is that fishes have multiple routes for plastic uptake and accumulation (gut and gills), whereas mice feeding experiments limit uptake through the gastrointestinal route. Int. J. Environ. Res. Public Health 2020, 17, 1509 10 of 24 Table 2. A summary of notable toxicological and/or pathological findings associated with MPs/NPs in mouse. PS, polystyrene; PE, polyethylene; PVC, polyvinylchloride; NPs, nanoplastics (<1 m); MPs, microplastics. Tissue Accumulation/ Properties of MPs/NPs Used Invasion or Cellular Notes on Toxicological, Pathological, or Behavioral Observations References Uptake Significant Toxicity/Pathology Signs of inflammation and lipid accumulation in liver Polystyrene (PS) microspheres 5 m Accumulation in gut,  Altered lipid profile and impairment of energy metabolism (reduction in Deng et al., 2017 [102] and 20, 0.01–0.5 mg/day liver, and kidney ATP levels) Increased liver oxidative stress markers, decreased acetylcholinesterase Decreased in body, liver, and lipid weights Decreased mucus secretion in the gut PS particles (0.5 and 50 m)  Alteration in gut microbiota Lu et al., 2018 [90] Changes in hepatic lipid profile and expression of some genes related to lipid metabolism PS and PE beads (0.5–1.0 m) + PS and PE beads  MPs enhanced OPFR-induced oxidative stress, neurotoxicity, and organophosphorus flame retardants Deng et al., 2018 [103] detectable in gut and liver metabolic disorder compared to OFPR alone. (OPFRs) Caused intestinal barrier dysfunction PS particles (5 m, 100 and 1000 Accumulation in mouse Induced gut microbiota dysbiosis Jin et al., 2018 [104] g/L) gut Induced bile acids metabolism disorder Toxicokinetic/toxicodynamic (TBTK/TD) modeling of Accumulation in mouse organ-bioaccumulation and biomarker responses PS particles (5 and 20 m) Yang et al., 2019 [105] gut, liver, and kidney  Changes in oxidative stress markers and those of energy and lipid metabolism Noticeable liver histopathology and altered serum and hepatic markers Changes in transcript of genes related to glycolipid metabolism Metabolic disorder associated with gut microbiota dysbiosis and gut PS MPs (5 m) Luo et al., 2019 [106] barrier dysfunction. Maternal MPs exposure resulted in intergenerational e ects and caused long-term metabolic consequences in the F1 and F2 generations. MP exposure caused changes in serum and liver metabolic markers PS MPs (0.5 and 5 m)  Maternal MPs exposure caused fatty acid metabolic disorder in the F1 Luo et al., 2019 [108] o spring Int. J. Environ. Res. Public Health 2020, 17, 1509 11 of 24 Table 2. Cont. Tissue Accumulation/ Properties of MPs/NPs Used Invasion or Cellular Notes on Toxicological, Pathological, or Behavioral Observations References Uptake MP exposure a ected composition and diversity of gut microbiota increased the secretion of IL-1 in serum, and decreased the Th17 and Treg PS MPs (10–150 m) Li et al., 2019 [107] cells among CD4+ cells High-concentration of MPs induced inflammation of the small intestine No E ect or Insignificant E ect PS particles (25 and 50 nm)  No significantly measurable neurobehavioral consequences in rats Rafiee et al., 2018 [110] No significant e ect on body/organ weight and no pathological signs by histological examination PS particles (1, 4 and 10 m)  Reporter analyses did not reveal evidence for the occurrence of Stock et al., 2019 [109] inflammation and/or oxidative stress Very low number of particles taken up by intestinal tissue Int. J. Environ. Res. Public Health 2020, 17, 1509 12 of 24 4. Toxicity of MPs/NPs in Human Cells Could MPs/NPs a ect human cells and tissues? There is obviously a lack of toxicity data for humans in vivo at the moment. Several studies have, however, looked at the e ect of pristine MPs/NPs on human cells in culture. These works are summarized in Table 3. Not surprisingly, a few of these studies, despite documenting some degree of cellular uptake, found signs of cellular toxicity either absent or insignificant except at very high concentrations of MPs/NPs [109,113,114]. In one case, where polyethylene terephthalate (PET) NPs generated by laser ablation were tested on the human gut adenocarcinoma epithelial line Caco-2, the authors noted a propensity for NP uptakes and crossing of a Caco-2 cells-based intestinal barrier model [113]. A few other studies have documented some degree of cellular toxicity or pathological e ect in a range of human cell lines. Prietl and colleagues showed that 20 nm PS NPs are taken up easily by human monocytic cells and are significantly cytotoxic. Larger (100 and 1000 nm) NPs stimulated the secretion of cytokines such as IL-6 and IL-8 from monocytes and macrophages, and could, interestingly, induce a measurable degree respiratory burst in monocytes [115]. Schirinzi and colleagues documented low but measurable degree of reactive oxygen species (ROS) production and induction of cytotoxicity by MPs in T98G and HeLa cells [116]. Wu and colleagues also worked with Caco-2 cells, and reported that, while a low degree of toxicity was observed for PS NPs (at 0.1 and 5 m), they induced mitochondrial depolarization and inhibited the activity of the toxicant eux pump, ATP-binding cassette (ABC) transporter, with the latter resulting in increased arsenic toxicity [117]. Hwang et al. worked with a number of cell types of human and mouse origin, and documented cytotoxicity associated with 20 m PP MPs at high concentrations and ROS induction [118]. The MPs also measurably induced pro-inflammatory cytokines IL-6 and TNF- from human peripheral blood mononuclear cells (PBMCs), and increased histamine release from mast cell lines [118]. Poma and colleagues found that 100 nm PS NPs stimulated ROS production and induced genotoxic stress and DNA damage as measured with the cytokinesis-block micronucleus (CBMN) assay [119]. Owing to the presence of large amounts of plastic particles in air, terrestrial animals are also exposed to MPs/NPs via inhalation. In this connection, Dong and colleagues found that PS MPs produced some cytotoxic e ects, oxidative stress, and inflammatory responses in human lung epithelial cells, and are disruptive of the epithelial cell layer, at least in vitro [120]. Two other groups have recently checked the toxicity of NPs in lung [121] and bronchial [122] cells. Xu and colleagues found that PS NPs (25 and 70 nm) impaired viability, induced cell cycle arrest, and upregulated nuclear factor (NF)-B as well as some pro-inflammatory cytokines in the human alveolar epithelial line A549 [121]. On the other hand, Lim and colleagues noted that PS NPs are only cytotoxic at high concentrations but induced metabolic changes and endoplasmic reticulum (ER) stress in a human bronchial epithelial cell line [122]. Overall, the experiments with pristine MPs/NPs on human cells reported thus far did not indicate severe cytotoxic or cytostatic e ects, but did demonstrate a potential for low to moderate negative e ects depending on the cell type, MP/NP sizes, and degree of cellular uptake. Two general and prominently observed phenomena appear to be ROS production and pro-inflammatory responses. In the paragraphs below, we explore how these toxic e ects may occur. Int. J. Environ. Res. Public Health 2020, 17, 1509 13 of 24 Table 3. A summary of notable toxicological findings associated with MPs/NPs in human cells. PS, polystyrene; PE, polyethylene; PVC, polyvinylchloride; NPs, nanoplastics (<1 m); MPs, microplastics. Properties of Human Cell Models Cellular Uptake Notes on Toxicological Observations References MPs/NPs Used Significant Toxicity 20 nm NPs cytotoxic to U937 and THP-1 cells 20 nm NPs stimulated IL-8 secretion in human monocytes and induced measurable oxidative burst in Human Peripheral blood monocytic cells 20 nm nanoparticles monocytes (PBMCs) taken up passively,  500 and 1000 nm NPs stimulated IL-6 and IL-8 secretion Carboxylated PS NPs Prietl et al., U937 (human monocytic cell line) while larger ones taken in monocytes and macrophages, chemotaxis and (20–1000 nm) 2014 [115] THP-1 (human monocytic cell line) up both actively and phagocytosis of bacteria by macrophages, and provoked an DMBM-2 (mouse macrophage cell line) passively oxidative burst of granulocytes At lower concentrations with no cytotoxicity, 20 nm NPs inhibited, while 500 and 1000 nm NPs increased phagocytosis of bacteria by DMBM-2 T98G (human glioblastoma cell line) PE microparticles  Induced ROS generation Schirinzi et HeLa (human cervical adenocarcinoma (3–16 m)  Cytotoxic e ect, with PE having a higher EC value al., 2017 [116] cell line) PS particles (10 m) compared to PS in both T98G and HeLa cells Low toxicity on cell viability, oxidative stress, and membrane integrity and fluidity Caco-2 (human epithelial colorectal PS particles (0.1 and 5 Cellular uptake of Wu et al., Disruption of mitochondrial membrane potential adenocarcinoma cell line) m) nanoparticles 2019 [117] Inhibition of plasma membrane ATP-binding cassette (ABC) transporter activity Some degree of cytotoxicity at high dosages of the Human dermal fibroblasts smaller size 20 m particles Peripheral blood mononuclear cells PP particles (~20 m Low degree of induction of proinflammatory cytokines (PBMCs) and 25–200 m), either IL-6 and TNF- from PBMCs Hwang et al., HMC-1 (human mast cell line 1) first dispersed in Increased histamine release from HMC-1 and RBL-2H3 2019 [118] RBL-2H3 (human basophilic leukemia DMSO or used directly cells cell line) in culture media Some degree of ROS induction at high dosages of the RAW 264.7 (mouse macrophage cell line) smaller size 20 m particles Cytotoxic e ects PS MPs (4.06  0.44 Dong et al., BEAS-2B (human lung epithelial cells)  Oxidative stress and inflammatory responses m at 1–1000 g/cm 2020 [120] Disruption of epithelial layer Int. J. Environ. Res. Public Health 2020, 17, 1509 14 of 24 Table 3. Cont. Properties of Human Cell Models Cellular Uptake Notes on Toxicological Observations References MPs/NPs Used Decreased viability and induced cell cycle arrest Upregulation of transcripts for NF-B and some A549 (Human alveolar type II epithelial PS nanoparticles (25 Cellular uptake of Xu et al., 2019 pro-inflammatory cytokines cell line) and 70 nm) nanoparticles [121] Alteration of cell cycle and apoptosis-regulation related protein expressions PS NPs only cytotoxic at very high concentrations BEAS-2B (Human bronchial epithelial Cellular uptake of PS  Metabolomics analyses revealed autophagic and Lim et al., PS nanoparticles cells) nanopaticles endoplasmic reticulum (ER) stress-related metabolic 2019 [122] changes Stimulation of ROS production PS nanoparticles (100 Poma et al., Hs27 (Human fibroblasts)  Genotoxic stress and DNA damage measured with the nm at 5–75 g/ml) 2019 [119] cytokinesis-block micronucleus (CBMN) assay No or Insignificant E ects No apparent toxic e ect Polyethylene Nano-PET are internalized into endo-lysosomal terephthalate (PET) Magri et al., Caco-2 Cellular uptake of NPs compartments NPs (laser ablated, ca. 2018 [113] Nano-PET has high propensity to cross the Caco-2 100 nm) intestinal barrier model Low crossing of the cell monolayer on Transwells even by 1 m microparticles Caco-2 PS microparticles (1, 4, Cellular uptake of PS  No pronounce loss of cell viability except only at very Stock et al., THP-1 monocytic line and 10 m) microparticles high dosage of 1 m microparticles 2019 [109] Microparticles uptake did not a ect macrophage di erentiation or polarization No significant cytotoxicity unless at very high Caco-2 and HT29-MTX-E12 (human Carboxy-modified PS concentrations colon epithelial cell) co-culture Cellular uptake of PS Hesler et al., nanoparticles (50 nm  No significant transport across the in vitro intestinal and BeWo b30 (Human placental trophoblast nanoparticles 2019 [114] and 0.5 m, placental “barriers” but intercellular distribution was cell) observed Int. J. Environ. Res. Public Health 2020, 17, 1509 15 of 24 5. Mechanisms Underlying MPs/NPs’ Acute or Chronic Toxicity in Mammalian Cells In general, extremely high concentration of MPs/NPs are indeed cytotoxic. Cell death could occur via necrotic plasma membrane rupture or some form of programmed cell death. An important point to note on the former, rather non-specific mode of death is the surfactant molecules that are typically associated with most MP/NP preparations. At high concentrations, these would be disruptive to the lipid bilayer of the plasma membrane (PM). Even at moderate levels, these could disrupt important cellular surface structures such as proteoglycans and other extracellular matrix components or hinder cellular signaling processes that require extracellular ligand and cell surface receptor interactions. Therefore, cellular physiology would be a ected to varying degrees by plastic associated surfactants, and the documented changes in various transcripts in cells could also be due to this and other processes/factors described below. The smaller NPs in particular could be taken up with some ease depending on the cell type via endocytosis [123,124]. Endocytosed NPs present a problem for several reasons. Firstly, they could, as per the plasma membrane discussed above, potentially permeabilize the endosomal membranes if present at high concentrations. If this happens, the NPs released into the cytosol could potentially interact with and a ect important organelles such as the mitochondria or the nucleus, as well as cellular processes such as mitotic spindle formation and chromosomal migration during cell division. Secondly, MPs/NPs would likely interfere with the tracking of transport carriers in the cell along the exocytic pathway [125,126], and as such would potentially inhibit the cell surface expression of important signaling receptors or membrane transporters. Thirdly, they are likely to perturb endosomal membrane trac on which many important cellular processes are dependent, including surface protein turnover and signaling attenuation, as well as retrograde signaling from endosomal compartments. It is unclear if NPs could themselves ever be subjected to inter-compartmental transport eciently in the endosomal pathway. Even if the NPs could eventually end up in the lysosome, they are unlikely to be readily digested. The accumulation of NPs in late endosome or lysosomes would perturb the degradative functions of these organelles and importantly impair the critical cellular membrane turnover process of macroautophagy [122]. An impairment of autophagic clearance could potentially lead to positive feedback processes that culminate in autophagic cell death. On the other hand, internalized MPs/NPs may also stimulate autophagy. Metallic nanoparticles are known to modulate autophagy [127], and MPs/NPs may speculatively do likewise. At the very least, these processes would constitute a form of cellular stress. Stresses at the PM and the endo-lysosomes would trigger cellular stress responses. In work done with species of the fresh water flea Daphnia, PS NPs exposure a ected growth and reproduction [128], and interestingly resulted in the elevation of AMP activated protein kinase (AMPK), which is an indication of stress response [129]. Perhaps a more general associated phenomenon with regards to cellular stress response appears to be the production of ROS, which was in fact recently identified as the molecular initiating event (MIE) by adverse outcome pathways analysis of reports in the field [130]. ROS production in cells occurs in two general ways: from the mitochondrial electron transport chain (ETC) during routine aerobic respiration or via the oxidative bursts of NADPH oxidases (NOXs) [131]. An increase in ROS from the former could result from mitochondrial function impairment, while the latter is normally a consequence of bacterial invasion, as NOXs are activated by bacterial products and cytokines. All cells are endowed with an evolutionarily conserved innate immunity mechanism, typically functioning against invasion of pathogens or exposure to xenobiotics [132]. However, the components of the innate immune system, such as the Toll-like receptors (TLRs), could also respond to a set of endogenous or secreted molecules collectively known as alarmins, or damage-associated molecular patterns (DAMP) [133,134], and the outcome is what is termed sterile-inflammation, i.e. inflammatory responses without pathogenic infection [135]. In the body, pro-inflammatory cytokines released from such localized inflammations would attract circulating immune cells, and this could worsen the local inflammation, and cause cell and tissue death. NPs has indeed been shown to act as stressors to the innate immune system of fish [88], and this is likely also the case for mammalian (including human) cells. The cellular and tissue Int. J. Environ. Res. Public Health 2020, 17, 1509 16 of 24 invasion and general pathological mechanism of MPs/NPs in mammalian cells is summarized below Int. J. Environ. Res. Public Health 2020, 17, x 17 of 24 in Figure 1. Figure 1. A schematic diagram illustrating potential (speculative at the moment) cellular mechanisms Figure 1. A schematic diagram illustrating potential (speculative at the moment) cellular mechanisms of MP/NP toxicity. MPs/NPs can be taken up through ingestion and inhalation. MPs/NPs could damage of MP/NP toxicity. MPs/NPs can be taken up through ingestion and inhalation. MPs/NPs could the plasma membrane and impair the gut barrier (left). These could also perturb signaling of cell surface damage the plasma membrane and impair the gut barrier (left). These could also perturb signaling receptors, and alter gene expression in the nucleus. Endocytosed MPs/NPs could also perturb the of cell surface receptors, and alter gene expression in the nucleus. Endocytosed MPs/NPs could endocytic pathway function and compromise the endosomal membranes. Stresses arising from the also perturb the endocytic pathway function and compromise the endosomal membranes. Stresses above could activate the cellular innate immune system, with endogenous and secreted damage- arising from the above could activate the cellular innate immune system, with endogenous and associated molecular patterns (DAMP) inducing the innate immunity-mediating toll-like receptors secreted damage-associated molecular patterns (DAMP) inducing the innate immunity-mediating (TLRs). Stresses could induce ROS production from the NADP oxidases (NOXs). Mitochondrial toll-like receptors (TLRs). Stresses could induce ROS production from the NADP oxidases (NOXs). impairment, either by MPs/NPs from endosomes or in response to stresses, could also produce more Mitochondrial impairment, either by MPs/NPs from endosomes or in response to stresses, could ROS through impairment in the efficiency of electron transport chain (ETC) processes. MPs/NPs gain also produce more ROS through impairment in the eciency of electron transport chain (ETC) access into the circulation if the gut–vascular barrier is compromised or it may speculatively occur by processes. MPs/NPs gain access into the circulation if the gut–vascular barrier is compromised or it transcytosis, thus reaching other organs. The lung probably has a more direct access to airborne may speculatively occur by transcytosis, thus reaching other organs. The lung probably has a more MPs/NPs (right). direct access to airborne MPs/NPs (right). 6. MPs/NPs’ Potential Systemic E ect in Humans 7. Conclusions MPs/NPs are expected to reach the human gut through consumption of contaminated food MPs/NPs have pervaded the environment and human’s exposure and cumulative uptake of these materials. Undigested MPs would be largely excreted though fecal matter, but smaller NPs could plastics would only increase over time. Currently, it appears that any worries of acute toxicity or severe potential enter the circulation. Ingested MPs/NPs would first encounter the intestinal epithelium. Only long-term effect that would lead to significantly enhanced morbidity or mortality are unfounded. unrealistically high concentration of plastics, or those carrying adsorbed toxicants, would likely cause However, we still know very little about how MPs/NPs from the environment, be it from a seafood acute impairment of viability and inflammation of the gut lining [51]. However, the e ect of persistent meal or the plastic bottled drink, would affect human health. Clearly, much more research, in terms of presence of ineciently cleared MPs/NPs in the gut is yet unknown. Gut pathology resulting from both cellular and tissue level pathological mechanisms, as well as on the long-term effects of MPs/NPs has been widely documented in fishes. The mouse experiments have provided some clear tissue/organ accumulation, is needed. Plans and collaborative attempts between ecologists and illustration of the consequences of gut toxicity. Should this happen, the gut–vascular barrier could epidemiologists to study bioaccumulation of MPs/NPs in humans via the food chain in various be impaired and MPs/NPs could enter the circulation, where these could gain access to the liver via geographical locales would also be necessary. Author Contributions: Conceptualization, B.L.T.; writing—original draft preparation, B.L.T.; and writing—review and editing, C.Q.Y.Y., S.V., and B.L.T. All authors have read and agreed to the published version of the manuscript. Int. J. Environ. Res. Public Health 2020, 17, 1509 17 of 24 the portal vein. That this is possible was demonstrated in some of the mouse models [102,103,105]. Long-term accumulation of MPs/NPs in liver tissues and chronic inflammation could lead to liver diseases and metabolic problems. On the other hand, accumulation of MPs/NPs in lung tissues could potentially result in chronic pulmonary disorders. Furthermore, the presence of NPs in brain tissues has been demonstrated in a fish model, as discussed above [68]. It should, however, be noted that it remains to be shown if the MPs/NPs could in fact be found in the brain of experimental mice or human brain samples. For that matter, no cell or tissue accumulation, pathology, or metabolic impairment due to MPs/NPs has been clearly demonstrated for humans to date. One of the more common findings in the mice studies is gut microbiota dysbiosis [90,104,106,107]. Changes in gut microbiome could result in gustatory dysfunction, thus perturbing physiological homeostasis in general. More importantly, gut microbiota changes have been linked to a range of chronic diseases of other organs, including disease of the kidney [136], cardiovascular system [137], inflammation, and cancer [138], as well as neurological disorders [91,139]. With regards to the latter, gut microbiota dysbiosis could in fact be one of the underlying reasons for behavioral changes in larger animals treated with MPs/NPs. It has also been reported that blood proteins such as albumin and globulin interact with NPs to form protein–plastic complexes [140,141]. Such aggregated protein–plastic complexes, if present in large quantities, could potentially lead to blockage of blood vessels. In addition, while loading of red blood cells (RBCs) with NPs at a low 1:50 ratio did not a ect functions of RBCs, a 10–50-fold higher loading showed RBC damages induced by mechanical, osmotic, and oxidative stresses [142]. However, it is dicult to envisage a large acute accumulation of NPs to occur in the human circulation under natural conditions. 7. Conclusions MPs/NPs have pervaded the environment and human’s exposure and cumulative uptake of these plastics would only increase over time. Currently, it appears that any worries of acute toxicity or severe long-term e ect that would lead to significantly enhanced morbidity or mortality are unfounded. However, we still know very little about how MPs/NPs from the environment, be it from a seafood meal or the plastic bottled drink, would a ect human health. Clearly, much more research, in terms of both cellular and tissue level pathological mechanisms, as well as on the long-term e ects of tissue/organ accumulation, is needed. Plans and collaborative attempts between ecologists and epidemiologists to study bioaccumulation of MPs/NPs in humans via the food chain in various geographical locales would also be necessary. Author Contributions: Conceptualization, B.L.T.; writing—original draft preparation, B.L.T.; and writing—review and editing, C.Q.Y.Y., S.V., and B.L.T. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by a Cross-Faculty Research Grant from the Oce of Deputy President (Research & Technology), National University of Singapore, grant number CFGFY18P07. Acknowledgments: We thank Fanny Ng for technical assistance and advice. The authors are grateful to the reviewers, whose constructive comments improved the manuscript. Conflicts of Interest: The authors declare no conflict of interest. References 1. Zalasiewicz, J.; Waters, C.; Ivar do Sul, J.; Corcoran, P.; Barnosky, A.; Cearreta, A.; Edgeworth, M.; Gałuszkah, A.; Jeandel, C.; Leinfelder, R.; et al. The geological cycle of plastics and their use as a stratigraphic indicator of the Anthropocene. Anthropocene 2016, 13, 4–17. [CrossRef] 2. Geyer, R.; Jambeck, J.R.; Law, K.L. Production, use, and fate of all plastics ever made. Sci. Adv. 2017, 3, e1700782. [CrossRef] [PubMed] 3. Gündogdu, ˘ S.; Yesilyurt, ¸ I.N.; Erbas, ¸ C. 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International Journal of Environmental Research and Public HealthPubmed Central

Published: Feb 26, 2020

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