Mutation Research-Reviews in Mutation Research 791 (2023) 108453 Contents lists available at ScienceDirect Mutation Research-Reviews in Mutation Research journal homepage: www.elsevier.com/locate/mutrev Insights into the potential carcinogenicity of micro- and nano-plastics Josefa Domenech a, 1, Balasubramanyam Annangi b, 1, Ricard Marcos b, Alba Hernández b, *, Julia Catalán a, c, ** a Finnish Institute of Occupational Health, Box 40, Työterveyslaitos, Helsinki 00032, Finland b Grup de Mutagènesi, Departament de Genètica i de Microbiologia, Facultat de Biociències, Universitat Autònoma de Barcelona, Bellaterra, Spain c Department of Anatomy, Embryology and Genetics, University of Zaragoza, 50013 Zaragoza, Spain A R T I C L E I N F O A B S T R A C T Keywords: There is a growing concern regarding the potential health effects that continuous exposure to environmental Carcinogenesis micro- and nano-plastics (MNPLs) may cause on humans. Due to their persistent nature, MNPLs may accumulate Genotoxicity in different organs and tissues and may induce in the long term the development of cancer. The present study Microplastic aimed to review the existing literature on the carcinogenic potential of MNPLs. As studies directly assessing Nanoplastic carcinogenicity were expected to be scarce, studies dealing with indirect outcomes associated with the carci­ Mammalian models nogenic process were considered in the literature search. Of the 126 studies screened, 19 satisfied the inclusion criteria. Besides, 7 additional cross-referenced articles, identified through a careful reading of the previously selected papers, also met the inclusion criteria and, consequently, were included in the review. Most of the selected studies were performed using in vitro models whereas about 40% of the studies were done in rodents, although none of them included a 2-year carcinogenicity assay. Most of the reviewed studies pointed out the potential of MNPLs to induce inflammation and genotoxicity, the latter being recognized as a strong predictor of carcinogenicity. These, along with other important findings such as the MNPLs’ ability to accumulate into cells and tissues, or their capacity to induce fibrosis, may suggest an association between MNPLs exposures and the carcinogenic potential. Nevertheless, the limited number of available studies precludes reaching clear conclu­ sions. Therefore, this review also provides several recommendations to cover the current knowledge gaps and address the future evaluation of the MNPLs’ carcinogenic risk. 1. Introduction The intrinsic physicochemical characteristics of MNPLs allow them to be uptaken by cells and interact with cellular components. Their Great interest has been placed on micro- and nanoplastics (MNPLs) reactive surfaces have been reported to cause harmful effects such as as emergent pollutants in the last decade. Although no consensus has cytotoxicity, oxidative stress, and disruption of immune function [5,6]. been reached regarding the size range for MNPLs [1], plastic particles up In addition, due to their persistent nature that limits the clearance from to 100 nm are considered nanoplastics (NPLs) while microplastics the body, translocation to different tissues and bioaccumulation are also (MPLs) include particles up to 5 mm [2,3]. Either due to weathering and possible. The potential effects on human health of MNPLs have been fragmentation of larger plastics (secondary MNPLs), or mass-production assessed using different approaches, including a variety of in vitro and in of sized MNPLs for specific purposes (primary MNPLs), the environ­ vivo mammalian models [5,7,8]. Among the outcomes studied, MNPLs’ mental ubiquity of MNPLs poses a health challenge. Their vast distri­ ability to induce oxidative stress, inflammatory responses, genotoxicity, bution throughout the environmental niches (airborne, waterborne, or or fitness disturbances are the most highlighted. Hence, the chronic terrestrial) makes humans susceptible to being unavoidably and nature of human exposure to MNPLs, along with some of the observed continuously exposed via different routes such as ingestion, inhalation, MNPLs-induced effects, raises concern about the potential carcinoge­ or dermal contact [4]. nicity of MNPLs. * Correspondence to: Autonomous University of Barcelona, Grup de Mutagenesis, Departament de Genètica i de Microbiologia, Facultat de Biociències, Campus de la UAB, Bellaterra 08193, Spain. ** Correspondence to: Finnish Institute of Occupational Health, Topeliuksenkatu 41 b, Helsinki FI-00250, Finland. E-mail addresses: alba.hernandez@uab.cat (A. Hernández), julia.catalan@ttl.fi (J. Catalán). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.mrrev.2023.108453 Received 16 September 2022; Received in revised form 14 December 2022; Accepted 31 January 2023 Available online 2 February 2023 1383-5742/© 2023 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). J. Domenech et al. Mutation Research-Reviews in Mutation Research 791 (2023) 108453 Carcinogenesis is a multistage process that consists of three consec­ illustrated in Fig. 1, when NMs establish contact with epithelial and utive steps: initiation, promotion, and progression. During the initiation immune cells (1), increased synthesis of pro-inflammatory cytokines is step, a normal cell undergoes a non-reversible genetic change that en­ induced (2) [18]. The inflammatory response’s purpose is to remove ables it for autonomous growth. Then, the abnormal clonal expansion of aggressive agents and repair lesions or damaged tissue, and for that, the the initiated cell occurs during the promotion stage. Progression is the recruitment of leukocytes into the injured tissue is accomplished (3) last stage and consists of the transformation of the neoplastic lesion into [21]. Under this scenario, reactive oxygen species (ROS) and reactive an invasive malignant tumor [9]. From a mechanistic point of view, nitrogen species (RNS) would be produced (4) and would cause DNA carcinogens can act as initiators, if they are able to produce mutations in damage and damage to proteins and lipids, which would end up in tissue a normal cell, or as promotors, if they enhance the carcinogenic process damage (5). Stem cells activated for tissue regeneration may eventually but not necessarily through mutagenic activity [10]. Accordingly, car­ be damaged by ROS and RNS, and gene mutations can be accumulated, cinogens are classified into genotoxic carcinogens and non-genotoxic generating cancer stem cells (6) [22]. Chronic inflammation may occur carcinogens [11]. Chemical substances and agents can be genotoxic if the injury becomes persistent, which would induce the harm and through a primary mechanism, executed by the compound on the target healing of damaged tissue, driving to fibrosis and malignancy (7) [21]. cell, or by a secondary mechanism involving an inflammatory response Other signaling pathways promoting invasion and pro-metastatic im­ that cause downstream secondary effects in the target cell. The primary mune responses would be activated, and signals to prevent autoimmu­ mechanism can involve a direct interaction of the material with DNA, or nity and exacerbate immune responses, as well as the an indirect effect mediated by other molecules (e.g., induction of reac­ epithelial-mesenchymal transition would be stimulated (8) [18,23]. tive oxygen species (ROS), inhibition of DNA repair mechanisms, etc.) Furthermore, and as above explained, unrepaired/misrepaired DNA [12,13]. On the other hand, the mechanisms behind non-genotoxic damage may raise mutations playing a crucial role in the initiation of carcinogenicity are diverse or even unknown, such as immune sup­ carcinogenesis [24]. Therefore, genotoxicity is assumed to be an early pression, mitogenic signaling, receptor-mediated endocrine modulation, predictor of the carcinogenic potential of a compound [25]. The OECD or epigenetic changes, among others [11,14,15]. guideline for the testing of chemicals recommends assessing the geno­ Independently of the mechanism of action associated with MNPLs toxic potential due to its veritable close association with cancer devel­ exposure, the assumption of a potential carcinogenic risk triggered by opment [26]. Considering the role of genotoxicity as a surrogate MNPLs is supported by previous experience with other nanomaterials biomarker of carcinogenicity, special emphasis is placed on the MNPLs’ (NMs). As an illustration, carcinogenicity has been demonstrated in mice genotoxic potential in this review. and rats after the administration of single- and multi-walled carbon The present review aims to investigate the potential carcinogenic nanotubes, respectively [16,17]. Aside from direct carcinogenic effects, risk of MNPLs exposure based on the existing literature. Due to the lack NMs have also been described to induce tumor progression by promot­ of studies directly assessing carcinogenicity, studies dealing with indi­ ing the growth of metastatic tumors or causing DNA damage and rect outcomes associated with the carcinogenic process have been elevated mutation rates [18]. These antecedents allow us to roughly considered. Additionally, a list of recommendations to cover the current draw the carcinogenesis process accomplished by NMs exposure. knowledge gaps and address the future evaluation of the MNPLs’ Nevertheless, it remains an open question as to whether the induced carcinogenic risk is proposed. effects are due to the nano size, the chemical composition, or a combi­ nation of both. 2. Search strategy The hazard evaluation of MNPLs is in its infancy, especially when referring to their carcinogenic risk. Thus, it is anticipated that at present PubMed database was selected to carry out the bibliographic search. no standardized studies have been carried out to detect the potential The search string was built up considering indirect outcomes related to carcinogenic risk of MNPLs, which is the central point of this review. carcinogenesis and key characteristics of carcinogens, as those reported However, some publications in the literature have associated MNPLs by Smith et al. [15,27]. The advanced search builder was used by exposure with different biomarkers linked to the carcinogenesis process entering the following keywords included in the title/abstract: (micro­ such as chronic inflammation, genotoxicity/mutagenicity, and fibrosis. plastic* OR nanoplastic*) AND (biopersisten* OR tumor* OR Consequently, such biomarkers can result useful when the potential carcinogen* OR mesothelioma OR "neoplastic transformation" OR "ma­ carcinogenic risk is under consideration [19,20]. We can find examples lignant transformation" OR fibrosis OR "chronic inflammation" OR of these carcinogenesis-related biomarkers for other NMs. In this case, as genotoxic* OR "long term toxicity" OR "long-term toxicity" OR “DNA Fig. 1. Schematic representation of the carcinogenesis process triggered by nanomaterials exposure. Numbers indicate the process described in the text. 2 J. Domenech et al. Mutation Research-Reviews in Mutation Research 791 (2023) 108453 repair” OR “genomic instability” OR epigenetic* OR immunosuppres­ discussed articles included both in vitro and in vivo studies (4%), while sive OR immortalization OR “DNA methylation” OR “histone acetyla­ 15 articles (58%) were performed using in vitro models and 10 papers tion”). All the articles published until the 21st of January 2022 were (38%) included only in vivo approaches. selected. A total of 126 publications were obtained and were evaluated As expected, no studies evaluating the incidence of tumors and for acceptability. cancer in humans exposed to MNPLs, or studies assessing such incidence Firstly, the following inclusion and exclusion criteria were applied to in rodents, were found in our literature search. Therefore, in this review select the articles after reading the abstracts: we have focused on reviewing those studies involving different bio­ Inclusion criteria: markers related to the carcinogenicity process. As genotoxicity is a surrogate biomarker of carcinogenicity, we also reviewed whether – The article contains information on mammalian cells, tissues, or MNPLs can induce DNA damage. Furthermore, epigenetic alterations whole organisms’ effects that could be related to the induction of also play an important role in carcinogenesis. However, no studies carcinogenesis by MNPLs. addressing this topic were found. Exclusion criteria: 3.1. In vitro studies on carcinogenicity-related effects – The full text was not available in English A summary of the studies in this section is presented in Table 1. – The article was a review, a comment, or a meta-analysis Forte and co-workers described increasing expression of pro- – The field of the article was out of the scope of this review inflammatory cytokines IL-1β, IL-6, and IL-8 after 1 h-exposure of – The article was based on environmental and ecotoxicity studies human gastric adenocarcinoma epithelial cells (AGS) to polystyrene nanoplastics (PSNPLs, 44 nm, 10 µg/mL) [28]. Moreover, a slight in­ In case of doubt, articles were in-depth analyzed, which implied crease in c-Myc oncogene expression was also induced by the PSNPLs. It reading the full article. After the described process, 19 articles were must be pointed out that c-Myc is closely related to the metabolic finally selected. transformation accomplished by transformed cells (e.g., mitochondrial The search by the above-described terms resulted in a limited num­ biogenesis, rRNA and protein biosynthesis, or glycolysis stimulation) ber of studies related to human carcinogenesis. Therefore, 7 additional [29]. The AGS cells exposed to both 44 and 100 nm PSNPLs (10 µg/mL) cross-referenced articles meeting the inclusion criteria were identified showed increased levels of TGF-1β and decreased levels of NF-kβ1 after a careful reading of the previously selected papers. mRNA, suggesting an upregulated metabolic transformation not linked to proliferation induction [28]. Similarly, a significant up-regulation in 3. Results the IL-6, IL-8, NF-κβ, and TNF-α expression was observed after 8 h of exposure of A549 cells to PSNPLs (25 and 70 nm, 25 µg/mL and As described above, 126 articles were retrieved. Two outcomes were 160 µg/mL, respectively) [30]. However, the expression of IL-1β was not discarded since they were a retracted article and the corresponding altered by any of the PSNPLs, whatever their size. The expression of retraction notice. From the resulting 124 outcomes, and as illustrated in genes involved in cell proliferation (cyclin D, cyclin E, and Ki67) was also Fig. 2A, review articles (23, including 1 commentary and 2 meta- upregulated after the exposure. analysis articles) accounted for 19% of the total results, while the rest Shi and co-workers evaluated the expression of IL-1β, IL-6, IL-8, and of the publications (101) were research articles. From these research TNF-α, which are associated with the pro-inflammatory response [31]. articles, 6% of the publications (included in the category “others” in In their study, A549 cells were exposed to PSNPLs (~100 nm; 20 and Fig. 2) mainly focused on MNPLs’ environmental sampling and char­ 200 μg/mL) alone or in combination with 5 μg/mL of two of the most acterization, sorption and desorption behavior of different agents, or common plasticizers (dibutyl phthalate, DBP, and di-(2-ethylhexyl) separation and degradation methods, among others. Research articles phthalate, DEHP) for 24 h. The expression of the pro-inflammatory cy­ focused on ecotoxicity models accounted for 54% of the total search tokines analyzed was significantly increased after both plasticizers’ ex­ results, while only 21% of the articles were research studies using in vitro posures alone and in combination with the highest concentration of or in vivo mammalian models. These results highlight the current focus PSNPLs. Interestingly, the combination of low doses of PSNPLs with on environmental risks regarding MNPLs’ studies, more than focusing on plasticizers decreased the plasticizers’ pro-inflammatory effects, and human health effects. Accordingly, most of the articles were excluded low doses of PSNPLs alone did not induce changes in the expression by after applying the inclusion/exclusion criteria, and only 19 articles themselves. Contrary, the highest concentration of PSNPLs increased the resulting from the systematic search were selected for further discussion. inflammatory response compared with the untreated control. The gen­ Seven additional cross-referenced articles were included during the re­ eration of oxidative stress, as well as the levels of the product of lipid view process as they fitted to the inclusion criteria. Thus, 26 articles are peroxidation malondialdehyde (MDA), measured in the cells after the discussed in the following sections. As shown in Figs. 2B, 1 of the same exposure conditions, followed the same trend as the expression of Fig. 2. Overview of the results. Summary of the total search string outcomes (A) and the revised articles included in the review (B). The percentage of articles in each category over the total is indicated inside the different sections represented in the figure. In A, the categories describe the type of article, whereas in B, the categories refer to the use of in vitro/in vivo approaches in mammalian models. 3 J. Domenech et al. Table 1 In vitro studies assessing carcinogenicity-related effects of MNPLs. Concentration range Material Size (µm) Cell line Uptake Carcinogenicity-related endpoint (method) Outcome Ref. (time of exposure) Increased expression of TGFβ1 (0.044, 0.1 µm), IL- Inflammatory response (RT-qPCR) 1β, IL-6, and IL-8 (0.044 µm) and decreased 0.044, expression of NF-κβ1 (0.044 and 0.1 µm) Forte et al. PSNPLs AGS 10 µg/mL (1 h) Proved (10, and 60 min) 0.1 Increased expression of c-Myc (0.044 µm), [28] Proliferation (RT-qPCR) decreased expression of p38 (0.1 µm) and decreased expression of Ki67 (0.044 and 0.1 µm) 0.025 µm: 25 µg/mL, Proved (0.025 µm: Inflammatory response (RT-qPCR) Increased expression of IL-6, IL-8, NF-κβ, and TNF-α 0.025, Xu et al. PSNPLs A549 0.07 µm: 160 µg/mL 1.14 μg/mL, 0.07 µm: Increased expression of CCND (cyclin D), CCNE 0.07 Proliferation (RT-qPCR) [30] (8 h) 25 μg/mL; 10 min - 2 h) (cyclin E), and Ki67 Increased expression of IL-1β, IL-6, IL-8, and TNF-α PSNPLs ̴ 0.1 20, 200 μg/mL (24 h) Proved (20 μg/mL) (200 µg/mL) PSNPLs/ Increased expression of IL-1β, IL-6, IL-8, and TNF-α Shi et al. - A549 20, 200/5 μg/mL (24 h) - Inflammatory response (RT-qPCR) DBP (200/5 µg/mL) [31] PSNPLs/ Increased expression of IL-1β, IL-6, IL-8, and TNF-α - 20, 200/5 μg/mL (24 h) - DEHP (200/5 µg/mL) 0.25, 2.5 and 25 μg/mL Inflammatory response (ELISA) Increased expression of IL-5 Cheng et al. PSMPLs 1 Liver organoids NA (48 h) Inflammatory response/ fibrosis (ELISA) Increased expression of COL1A [32] Stress-related genes’ expression (RT-qPCR) Altered stress response 4 Epithelial-mesenchymal transition and tumor Increased expression of Snail and Twist genes, and progression (RT-qPCR) decreased expression of Slug and Zeb1 genes Increased expression on 14 microRNA and decreased microRNA expression expression of 2 microRNA out of 33 microRNA analysed Prone-to- Barguilla PSNPLs ̴ 0.05 transformation 25 µg/mL (6 months) Proved (24, 48, and 72 h) Anchorage-independent growth induction (soft- Potential to grow independently of anchorage et al. [33] progress MEF agar assay) forming colonies Mutation Research-Reviews in Mutation Research 791 (2023) 108453 Migration induction (direct migration assay) Acquisition of migration ability Invasion induction (direct invasion assay) Acquisition of invasion ability Activation of cancer stem cells (tumorsphere formation induction and expression of pluripotent Induction of the dedifferentiation of the cells markers by RT-qPCR) Anchorage-independent growth induction (soft- Lack of colony formation agar assay) Migration induction (direct migration assay) Unable to migrate Prone-to- Proved (25, 100 μg/mL; Barguilla PSNPLs ̴ 0.05 transformation 25 µg/mL (12 weeks) Invasion induction (direct invasion assay) Unable to invade 24 h) et al. [34] progress MEF Activation of cancer stem cells (tumorsphere Lack of cancer stem cells activation formation induction) Morphological changes (microscopy) Increased proportion of spindle-like cells (continued on next page) J. Domenech et al. Mutation Research-Reviews in Mutation Research 791 (2023) 108453 the pro-inflammatory cytokines. However, the activity of the antioxi­ dant enzymes superoxide dismutase (SOD), glutathione peroxidase Kim et al. (GSH-Px), and catalase (CAT) was reduced after the exposure of A549 [35] Ref. cells to 200 μg/mL PSNPLs, DBP, DEHP, or their combinations. The low concentration of PSNPLs alone or in combination with plasticizers did not affect the antioxidants’ activity. Furthermore, liver organoids Increased invasion rate (AGS, MKN1, MKN45, and developed from human embryonic stem cells H1 exposed to polystyrene Potential to grow independently of anchorage Multidrug resistance induction after PSNPLs Increased expression of CD44 (all cell lines) Increased proliferation rate (all cell lines) Increased proportion of spindle-like cells microplastics (PSMPLs; 0.25, 2.5, and 25 μg/mL; 1 µm) showed a Increased migration rate (all cell lines) Lack of cancer stem cells activation dose-dependent increase in the IL-6 and collagen type 1a (COL1A1) Acquisition of migration ability Acquisition of invasion ability inflammation biomarkers after 48 h of exposure [32]. In addition to treatment (all cell lines) that, other outcomes regarding hepatotoxicity were observed. The forming colonies decreased activity of GST, GSH, and SOD levels accompanied by the KATO III) Outcome increase in MDA content suggested the antioxidative imbalance, which may arise due to oxidative stress. Based on the increased inflammatory response and oxidative stress triggered by PSMPLs, as well as other outcomes obtained in the study, the authors identified different adverse outcomes pathways (i.e., AOP 38 and AOP 220) that suggested the risk of liver fibrosis and liver cancer, respectively. In short, the above in vitro short-term approaches suggest the ability of MNPLs to induce oxidative stress that can lead to lipid peroxidation, and acute inflammatory responses. Unfortunately, no data addressing Cancer stemness (immunocytochemistry and RT- Anchorage-independent growth induction (soft- the capacity of MNPLs to induce chronic inflammation in in vitro systems Activation of cancer stem cells (tumorsphere Migration induction (direct migration assay) Migration induction (direct migration assay) Carcinogenicity-related endpoint (method) Invasion induction (direct invasion assay) Invasion induction (direct invasion assay) Multidrug resistance (alamarBlue assay) is available up to date. Additionally, insights on the activation of cell Morphological changes (microscopy) proliferation were also reported in these studies. Proliferation (alamarBlue assay) Three studies focused on the potential carcinogenic risk of MNPLs in formation induction) vitro, by using cell transformation approaches, have been recently agar assay) published. Two of these studies used mouse embryonic fibroblasts qPCR) deficient in repairing oxidized DNA base lesions (MEF Ogg1-/-) in a chronic exposure scenario with exposures lasting for 12 and 24 weeks to relatively low doses of PSNPLs (0.05 µm; 25 µg/mL) [33,34]. The au­ thors reported, after 12 weeks of chronic exposure to the co-treatment with arsenic (2 µM AsIII), a well-known carcinogen in humans, a syn­ ergistic effect increasing a set of cancer hallmarks, such as the number of colonies formed in the anchorage-independent growth assay, and the acquisition of invasion and migration abilities, although PSNPLs alone did not induce these effects [34]. Nevertheless, PSNPLs generated DNA Proved (25 or 100 μg/mL / damage by both oxidative and non-oxidative mechanisms after the 12-week chronic exposure. The same authors also demonstrated that a 2 μM; 24 h) longer period of exposure to the same NPLs (24 weeks) was able to alter Uptake NA early tumoral phenotype biomarkers [33]. Changes in stress-response-related genes were observed with a significant upregu­ lation of Gstp-1 and a downregulation of Keap, Nrf2, Pgp, Sod1, and Sod2. In addition, the dysregulation of a battery of microRNAs closely linked to the oncogenic process (miR-21, miR-23a, miR-25, miR-30c, miR-30d, 8.61 × 105 particles/mL miR-96, miR-135b, miR-148b, miR-155, miR-199b, miR-200a, miR-210, 25 µg/mL / 2 μM (12 Concentration range (time of exposure) miR-218, miR-502, miR-34a, and miR-203) was observed. Moreover, (4 weeks) and as indicators of an advanced tumoral phenotype, there was a weeks) significantly elevated number of colonies able to grow in soft-agar and increased cell migration capacity, as well as an aggressive tumoral phenotype, namely, increased invasion capacity, changes in pluripo­ tency markers (upregulated Klf4, Nanog, Notch2, and Oct3/4 and downregulated Sox2) and formation of tumorspheres due to the treat­ NCI-N87, and KATO III AGS, MKN1, MKN45, ment of the cells with PSNPLs for 24 weeks. On the other hand, Kim and colleagues studied carcinogenicity-related endpoints in AGS, MKN1, Cell line MKN45, NCI-N87, and KATO III human gastric cancer cell lines after the exposure to 8.61 × 105 particles/mL of PSMPLs (9.5–11.5 µm) for 4 weeks [35]. As a result of the exposure, all cell lines increased their proliferation and migration rates, and most of them except NCI-N87 cells underwent invasion induction. The upregulation of the CD44 marker Size (µm) was identified in the studied cell lines upon the PSMPLs treatment. 9.5–11.5 Table 1 (continued ) CD44 + cells have been described as human gastric cancer stem cells. - Also, the authors demonstrated that PSMPLs exposure induced resis­ tance to different anticancer drugs in all the selected cell lines. PSNPLs/ Material PSMPLs AsIII 5 J. Domenech et al. Mutation Research-Reviews in Mutation Research 791 (2023) 108453 Table 2 In vivo studies assessing carcinogenicity-related effects of MNPLs. Experimental Size Doses (route of Treatment Presence in target Carcinogenicity-related Material model (strain; Outcome Ref. (µm) administration) schedule tissue endpoint (method) sex) Increased expression of House Dust Mite- Inflammatory response IgG1 and TNF-α (healthy induced allergic (multiplex ELISA) Lu 300 μg/mouse Every 3 days mice) PSMPLs 1–5 asthmatic and Proved (lung) et al. (intranasal) for 24 days Inflammatory response Increased eosinophils and healthy mice [36] (BALB/c; females) (histopathological lymphocytes infiltration analysis) (healthy mice) Upregulation of the ASC- Inflammatory response inflammasome, and NF-κβ (western blot) pathways Choi 5, 25, and 50 µg/ Daily for 2 PSMPLs 0.5 Mice (ICR) NA Increased expression of et al. mouse (oral) weeks Inflammatory response NF-κβ (25 and 50 µg), IL- [37] (RT-qPCR) 6, TNF-α, IL-1β, TGF-β, and IL-10 Increased expression of IL-2 and IL-6 (6 µg), IP-10 Inflammatory response and RANTES (60 µg), IL-5 (immunology multiplex and IL-9 (600 µg), G-CSF assay) (60 and 600 µg), and IL- 1α Decreased percentage of Li Mice (C57BL/6; 6, 60, and 600 µg/ Daily for 5 Inflammatory response PE 10–150 NA Th17 and Treg cells (60 et al. males) mouse (oral in food) weeks (flow cytometry) and 600 µg) [38] Inflammatory response Increased lymphocytes (histopathological and plasma cells analysis) infiltration (600 µg) Inflammatory response Increased expression of (immunofluorescence TLR4, AP-1, and IRF5 staining) (600 µg) Decreased CD45+ Immune balance/ leukocytes, and CD49b+ Immuno- NK cells in CD45+ Mice (C57BL/6; suppression (flow leukocytes, Increased M2- Twice on 5.5 cytometry) subtype macrophages Hu males mated BALB/ 250 µg/mouse PSMPLs 10 and 7.5 days NA polarization et al. c; (intraperitoneal) of gestation [39] females) Increased IL4, decreased Inflammatory response TNF-α, increased (RT-qPCR) tendency IL6, decreased mariginal IL2 and IFN-γ Increased activity of AST Inflammatory response and ALT (high-fat diet fed (biochemical assay) mice, 1 μg) Increased macrophage Inflammatory response/ infiltration and increased fibrosis collagen deposition (high- (histopathological fat diet fed and normal High-fat diet fed analysis) Li 1 and 5 μg/mouse (i. Every 3 days mice, 1 µg) PSNPLs 0.042 and normal mice NA et al. v.) for 15 days Increased expression of (C57BL/6; females) [40] IL-1β (high-fat diet mice, Inflammatory response 1 and 5 μg), IL-12, IL-2, (RT-qPCR) and IFN-γ (high-fat diet fed mice, 1 μg) Increased expression of Fibrosis (RT-qPCR) α-SMA and Col1a (high- fat diet fed mice, 1 μg) Increased collagen Fibrosis (50 mg/L) and fibronectin (histopathological (5 and 50 mg/L) analysis) 0.5, 5 and 50 mg/L Daily in deposition Proved Li Rats (Wistar; of drinking water/rat drinking Increased expression of PSMPLs 0.5 (cardiomyocytes, 5 et al. males) (oral in drinking water for 90 Wnt, TGF-β, p-β-catenin, and 50 mg/L) [41] water) days Inflammatory response/ α-SMA, Collagen I and fibrosis (western blot) fibronectin (5 and 50 mg/ L) and β-catenin and Collagen III (50 mg/L) (continued on next page) 6 J. Domenech et al. Mutation Research-Reviews in Mutation Research 791 (2023) 108453 Table 2 (continued ) Experimental Size Doses (route of Treatment Presence in target Carcinogenicity-related Material model (strain; Outcome Ref. (µm) administration) schedule tissue endpoint (method) sex) Fibrosis Increased collagen and (histopathological fibronectin deposition analysis) (1.5 mg/d) Daily in Increased expression of 0.015, 0.15 and An Rats (Wistar; drinking Proved (ovary, Wnt and TGF-β (0.15 and PSMPLs 0.5 1.5 mg/day/rat (oral et al. females) water for 90 0.15 mg/d) 1.5 mg/d) and β-catenin, in drinking water) Inflammatory response/ [42] days p-β-catenin, α-SMA, fibrosis (western blot) Collagen I, fibronectin and Collagen III (1.5 mg/ d) Daily for 1, 0.01, 0.1, and Proved (liver, Inflammatory response Inflammation indicators Deng 2, 4, 7, 14, PSMPLs 5, 20 Mice (ICR; males) 0.5 mg/day/mouse kidney, gut, (histopathological observed (0.5 mg/d, 28 et al. 21, and 28 (oral gavage) 0.1 mg/d) analysis) days) [43] days Altered expression of 194 Mice (BALB/c 1.72 × 104 particles/ Daily for 4 Gene expression (RNA- genes associated to Proved (stomach) nude; males) mL/mouse (oral) weeks seq) digestive system diseases and cancer Kim PSMPLs 9.5–11.5 et al. 5 Mice (BALB/c nude 8.61 × 10 particles/ [35] PSMPLs-exposed mL 4-weeks-exposed Once - Tumor growth Accelerated tumor growth cell xenografted; NCI-N87 males) (subcutaneously) Immune tolerance Augmentation of Treg and (flow cytometry) TGF-β1 release Liu Mice (NOD; 100 μg/mouse Once a week PLGA 1–2 - Increased expression of et al. female) (subcutaneously) for 5 weeks Immune tolerance IL-10 and TGF-β1, INF-ϒ [44] (ELISA) and IL-17A 3.2. In vivo studies on carcinogenicity-related effects 0.5 mL/day) daily for 2 weeks [37]. Moreover, the authors associated the response with the activation of the NF-κβ, which is linked with the The in vivo inflammatory responses caused by MNPLs exposure have expression of pro-inflammatory genes. To prove that, the expression of been studied in mice and rats (Table 2). Lu and co-workers treated NF-κβ, TNF-α, IL-6, IL-1, IL-10, and TGF-β1 was determined. The results intranasally healthy, and House Dust Mite (HDM)-induced allergic significantly showed dose-dependent increased levels of mRNA for all asthmatic female BALB/c mice, either with PSMPLs (1–5 µm; 300 µg in the cytokines in PSMPLs treated mice groups, compared to the untreated 20 µL of saline per mouse) or saline solution (control), every 3 days up to control group. Similarly, Li et al. studied the levels of various cytokines 24 days [36]. Plastic particles’ accumulation was detected in lung sec­ (IL-1α, G-CSF, IL-2, IL-5, IL-6, IL-9, IP-10, and RANTES) in serum of tions of the MPLs-exposed groups, co-localizing with macrophages. In C57BL/6 mice administered with polyethylene microplastics (PEMPLs, addition to MPLs-loaded macrophages, strong recruitment of inflam­ 10–150 µm; 6, 60, and 600 µg per mouse) in food every day for 5 weeks matory cells (eosinophils, lymphocytes, neutrophils, plasmocytes, and [38]. IL-1α expression was significantly increased in PEMPLs-fed mice macrophages) into the airspace of lungs of HDM-induced allergic asth­ groups. However, the expression of the other analyzed cytokines did not matic mice, either treated or not with MPLs, was reported. Besides, the change or even decreased after the PEMPLs exposure. Other inflamma­ presence of eosinophils in the bronchoalveolar lavage fluid (BALF) of tion markers, such as the expression of TLR4, AP-1, and IRF5, or the healthy mice exposed to MPLs was higher than in healthy mice exposed infiltration of lymphocytes and plasma cells in the duodenum of mice to saline. Thus, MPLs exposure triggered the recruitment of inflamma­ administered with 600 µg PEMPLs, proved the induction of intestine tory cells into the lungs of healthy mice and aggravated the inflamma­ inflammation. tion response in allergic mice. Further analyses were performed with Besides that, the maternal-fetal immune balance effects of PSMPLs BALF and plasma to determine IL-4, IL-5, IL-10, IL-13, IL-17A, IL-33, were evaluated in C57BL/6-mated BALB/c pregnant mice after intra­ IFN-γ, TNF-α cytokines, and immunoglobulins E (IgE) and IgG1. TNF-α peritoneal administration (PSMPLs of 10 µm, 250 µg/200 µL of saline) levels were increased after the exposure of healthy mice to MPLs, on days 5.5 and 7.5 of gestation periods [39]. It resulted in significantly compared with the healthy control group. IgE and IgG1 were signifi­ lower levels of CD45+ leukocytes derived from the peripheral blood, cantly overexpressed in both MPLs-exposed and unexposed asthmatic spleen, as well as from placenta of the PSMPLs’ exposed mice. In addi­ mice compared with the healthy control group, but with no differences tion, there was a decrease in CD49b+ natural killer (NK) cells in CD45+ between asthmatic groups. Importantly, healthy mice exposed to MPLs leukocytes from placenta; nevertheless, no changes in the population of showed increased IgG1 levels compared to the untreated control. In NK cells in peripheral blood and spleen of the exposed pregnant mice general, the exposure to PSMPLs did not alter the cytokines and im­ were observed. Furthermore, PSMPLs’ exposure of mice significantly munoglobulins’ expression in asthmatic mice, but it induces skewed the macrophages’ polarization towards the M2-subtype domi­ pro-inflammatory effects in healthy mice. The transcriptomic analysis nance (CD206+) with immunosuppressive capacities over M1-subtype conducted in this study revealed that MPLs administration altered pro­ (CD86+) in the peripheral blood, spleen, and placenta tissues. Simi­ cesses related to immune activation, cell cycle control, and signaling larly, several pro/anti-inflammatory cytokines secretion like IL-2, IL-4, pathways such as the MAPK cascade. Likewise, Choi and colleagues IL-6, IL-10, TNF-α, and IFN-γ, evaluated at mRNA level from the exposed detected an upregulation of the ASC-inflammasome pathway (NLRP3, mice placenta, were affected. Notably, there were increases in the levels ASC, and cleaved Cas-1/Cas-1) in the mid colon of ICR mice after the of IL-4, and decreases in TNF-α. Moreover, an increasing tendency of IL-6 oral administration with PSMPLs (0.5 µm; 10, 50, and 100 µg/mL; and a marginal decrease of IL-2 and IFN-γ were also observed due to 7 J. Domenech et al. Mutation Research-Reviews in Mutation Research 791 (2023) 108453 PSMPLs’ exposure. The authors suggested this as a shift towards the particles/mL for 4 weeks) were used to demonstrate an induction in immunosuppressive state. tumor growth in the PSMPLs-exposed NCI-N87 mouse model compared Hepatic inflammation was observed in normal and high-fat diet to the control. Additionally, differential expression of 194 genes related (HFD) fed C57BL/6 mice administered via tail vein with PSNPLs to digestive system disease and cancer, and a decrease in the survival (41.54 nm; 10 and 50 µg/mL in 100 µL of PBS per mouse) every 3 days rate were reported in the PSMPLs-exposed mice (1.72 ×104 parti­ for 15 days [40]. The expression levels of TNF-α, IL-1β, IL-12, IL-2, and cles/mL per mouse, orally administered daily for 4 weeks). The authors IFN-γ revealed higher transcription of the inflammatory genes in mice also demonstrated the particles’ accumulation in the stomach of the treated with PSNPLs in HFD mice groups. Although the mRNA levels of PSMPLs-exposed BALB/c nude mice. the mentioned cytokines were higher in HFD mice, compared with normal mice in every treatment condition, higher concentrations of 3.3. In vitro genotoxicity studies PSNPLs did not induce stronger effects. Intriguingly, massive collagen deposition was found in liver tissue of HFD mice administered with Until now, several in vitro studies have reported genotoxic effects of 10 µg/mL PSNPLs. The increased expression of α-smooth muscle actin MNPLs exposure using several genotoxicity endpoints, relevant cell (α-SMA) and COL1a, which play important roles in fibrogenesis, lines, different sizes and concentrations of MNPLs, and different expo­ confirmed the notable potential of PSNPLs to induce fibrosis in the HDF sure times (Table 3). The DNA damage (either produced by oxidative group of mice. These results are in concordance with the also reported causes or not) measured by the comet assay after the treatment of human increased levels of two markers of hepatic cell injury in HFD mice hematopoietic cell lines with PSNPLs was cell-line specific, and time- treated with 10 µg/mL PSNPLs, such as aminotransferases ALT and AST. and concentration-dependent [45]. It was found that Raji-B cells treated These biomarkers have been linked with chronic hepatitis (inflamma­ with PSNPLs (50 nm, 5–50 µg/mL) showed a pronounced tion of the liver tissue), liver cirrhosis (fibrosis), and hepatocellular dose-dependent increase in DNA damage after 24 h and 48 h of expo­ carcinoma. In another study [41], the authors described that the sure, although the oxidative DNA damage (ODD) was observed only at expression of Wnt, TGF-β, p-β-catenin, α-SMA, Collagen I, and fibronectin the highest concentration (50 µg/mL) and after exposures lasting for raised significantly in the heart tissue of male Wistar rats after the daily 24 h. Only ODD was detected in a dose- and time-dependent manner in administration of 5 and 50 mg/L of PSMPLs (0.5 µm) for 90 days in TK6 cells. Contrary, THP1 cells treated with PSNPLs did not show DNA drinking water, whereas the highest dose also induced an increase damage, independently of concentrations and time intervals. The dif­ expression of β-catenin and Collagen III. These effects were accompanied ferential cell sensitivity to genotoxic agents could be related to the dif­ by a significant increase in collagen deposition and hyperplasia ferential uptake of PSNPLs and the potential generation of ROS. (50 mg/L PSMPLs), as well as fibronectin deposition (5 and 50 mg/L Similarly, a recent study also assessed the cellular-specific DNA damage PSMPLs) in the heart of treated rats. An accumulation of PSMPLs was due to ex vivo exposure to different concentrations of PSNPLs (0.05 μM) also found inside cardiomyocytes after 90 days of exposure. Equivalent in total whole blood cells (WBC) and sorted cell types from WBC such as results were observed in the ovaries of female Wistar rats exposed to lymphocytes, monocytes, and polymorphonuclear cells (PMNs) ob­ PSMPLs (0.5 µm; 0.015, 0.15, and 1.5 mg/day/rat in drinking water) for tained from different donors by using the comet assay [46]. PSNPLs 90 days following the same exposure procedure. Furthermore, while were able to cause a significant increase in the percentage of DNA in the p-β-catenin, β-catenin, α-SMA, Collagen I, Collagen III, and fibronectin tail of monocytes (100 μg/mL) as well as in PMNs (50 and 100 μg/mL), expression increased only after the highest dose treatment, Wnt and but not in lymphocytes and in the total WBC population after 24 h of TGF-β expression raised after the administration of 0.15 and 1.5 mg/day exposure. The differential cell sensitivity to PSNPLs did not reveal a PSMPLs in female rats [42]. In addition, some authors have associated correlation with cellular internalization as monocytes showed greater the induction of fibrosis by PSMPLs with cell apoptosis caused by internalization of particles than PMNs, but less genotoxic damage. oxidative stress, which in its turn, activates the expression of the re­ DNA damage associated with exposure to different concentrations ported cytokines [41,42]. (1–100 µg/mL) of PSNPLs (mean size ~50 nm) in the in vitro human It is important to point out here the study of Deng and co-workers intestinal barrier models (biculture of differentiated Caco-2/HT29 in­ where administered PSMPLs (5 and 20 µm; 0.1 mg/day/mouse) were testinal cells and triculture containing Caco-2/HT29/Raji-B cells) was found to accumulate in the liver, kidney, and gut of ICR mice after 28 not significantly increased when analyzed by using the comet assay after days of oral gavage exposure [43]. The retention of PSMPLs was 24 h of exposure. The lack of DNA damaging effects could be attributed determined one week after the termination of the exposure and both to the insignificant generation of ROS, though the data demonstrated sizes of PSMPLs were still observable in liver, kidney, and gut tissues. clear uptake of PSNPLs [47]. The only in vitro study presenting results on Moreover, liver tissue showed inflammation signals after a 4-weeks of MNPLs’ accumulation after a long-term exposure was performed using exposure to 0.5 mg/day of both sizes of PSMPLs. Oxidative stress-related undifferentiated Caco-2 cells exposed to pristine PSNPLs (50 nm; biomarkers were also evaluated after the exposure to PSMPLs (5 and 0.26 µg/cm2) for 24, 48, 72, and 96 h, and weekly thereafter up to week 20 µm; 0.01, 0.1, and 0.5 mg/day) and increased activity of GSH-Px and 8. Internalization reached a plateau after 2-weeks of exposure, and SOD were shown in mice after the termination of the exposure, while PSNPLs remained accumulated inside the cells at a similar level during CAT activity decreased in the treated mice. the consecutive weeks [48]. However, undifferentiated Caco-2 cells Liu and co-workers evaluated the impact of poly[lactic-co-(glycolic treated with PSNPLs (50–100 nm;10 and 100 µg/mL) for 8 weeks did acid)] (~1–2 µm, PLGA) microparticles on the immune functions of not elicit DNA damage using the comet assay. Nevertheless, there was a non-obese diabetic female mice [44]. Twenty weeks after the treatment dose-related increase in the expression of a few oxidative stress-related (100 µg, once a week for 5 weeks), PLGA MPLs stimulated the splenic genes (HO1 and SOD2). Treg augmentation and TGF-β1 release. Nevertheless, the effect was no PSNPLs acting as carriers of silver metal were able to modulate the longer observed at week 30. To confirm the stimulation of immune induced genotoxic and ODD effects of AgNO3 or silver nanoparticles tolerance by PLGA, multiplex serum cytokine ELISA was performed. The (AgNPs) in Caco-2 cells [49]. Results of the comet assay after 24 h of substantial increase of IL-10 and TGF-β1 levels supported the exposure showed that the co-treatment of the cells with the highest tolerance-inducing property of these MPLs. In addition, a slight increase doses of PSNPLs (100 µg/mL) and AgNPs (5 µg/mL) significantly in serum INF-ϒ and IL-17A, indicated the stimulation of antigen increased the non-oxidative DNA damage but not ODD. On the other presentation. hand, the adsorption of plasma proteins onto PSNPLs (~100 nm) Only one study focusing on in vivo tumorigenesis capacity of PSMPLs resulted in increased DNA damaging effects observed in human blood (9.5–11.5 µm) was found [35]. Mice xenografts (BALB/c nude mice lymphocytes by the comet assay, but not with PSNPLs alone; suggesting injected with NCI-N87 cells previously exposed or not to 8.61 ×105 PS that interactions of plasma proteins with nanoplastics could turn them 8 J. Domenech et al. Mutation Research-Reviews in Mutation Research 791 (2023) 108453 Table 3 In vitro studies assessing genotoxicity of MNPLs. Size Concentration range Endpoint Material Cell line Uptake Outcome Ref. (µm) (time of exposure) (method) Non-oxidative DNA damage DNA damage induction (25 and 50 μg/mL, 24 and (alkaline and 48 h) and oxidative DNA damage Fpg-comet) Raji-B (50 μg/mL, 24 h) Oxidative Increased intracellular ROS levels stress (DCFH- (50 μg/mL, 3 h) DA assay) DNA damage Oxidative DNA damage (5, 10, 25 Proved (1, 5, 10, 25, (alkaline and and 50 μg/mL, 24 h and 10 and 5, 10, 25, and 50 μg/ Fpg-comet) 25 μg/mL, 48 h) Rubio et al. PSNPLs 0.05 50 μg/mL; 24 and TK6 mL (3, 24, 48 h) [45] 48 h) Oxidative Increased intracellular ROS levels (5, stress (DCFH- 10, 25 μg/mL, 24 h and 50 μg/mL, 3 DA assay) and 24 h) DNA damage (alkaline and Lack of DNA damage induction Fpg-comet) THP-1 Oxidative stress (DCFH- Lack of oxidative stress induction DA assay) Lymphocytes No significant uptake Lack of DNA damage induction DNA damage 50 and 100 μg/mL Ballesteros PSNPLs 0.05 Monocytes Proved (100 μg/mL) (alkaline and DNA damage (100 μg/mL) (24 h) et al. [46] Fpg-comet) PMNCs Proved (100 μg/mL) DNA damage (50 and 100 μg/mL) DNA damage (alkaline and Lack of DNA damage induction Fpg-comet) Caco-2/HT29 Proved Oxidative stress (DCFH- Lack of oxidative stress induction DA and DHE 1, 25, 50 and 100 μg/ assays) Domenech PSNPLs 0.05 mL (24 h) DNA damage et al. [47] (alkaline and Lack of DNA damage induction Fpg-comet) Caco-2/HT29/Raji- Proved Oxidative B stress (DCFH- Lack of oxidative stress induction DA and DHE assays) DNA damage (alkaline and Lack of DNA damage induction 10 and 100 μg/mL Fpg-comet) PSNPLs 0.05 Proved (24 h) Oxidative stress (DHE Lack of oxidative stress induction assay) DNA damage (alkaline and Lack of DNA damage induction 0.05/ Fpg-comet) PSNPLs/AgNPs Domenech 0.005 Caco-2 Oxidative Increased intracellular ROS levels et al. [49] stress (DHE (5 μg/mL AgNPs and 100/5 μg/mL assay) PSNPLs/AgNPs) 10 and 100/0.1, 0.5, 1, Proved (10 and 100/ and 5 μg/mL (24 h) 0.5 and 5 μg/mL) PSNPLs-dependent DNA damage DNA damage induction and PSNPLs-dependent (alkaline and oxidative DNA damage induction Fpg-comet) PSNPLs/AgNO3 - (0.5 μg/mL AgNO3) Oxidative stress (DHE Increased intracellular ROS levels assay) DNA damage Proved (0.26 μg/ (alkaline and Lack of DNA damage induction 0.26 and 6.5 μg/cm2 cm2; 24, 48, 96 h and Fpg-comet) (24 h) and 0.0006, Domenech PSNPLs 0.05 Caco-2 weekly up to week 8 0.26, 1.3, and 6.5 μg/ Oxidative et al. [48] and 6.5 μg/cm2; cm2 (8 weeks) stress (DCFH- Lack of oxidative stress induction 24 h) DA assay) (continued on next page) 9 J. Domenech et al. Mutation Research-Reviews in Mutation Research 791 (2023) 108453 Table 3 (continued ) Size Concentration range Endpoint Material Cell line Uptake Outcome Ref. (µm) (time of exposure) (method) PSNPLs, plasma coronated- DNA damage Human 1, 2.5, 5, 7.5 and DNA damage induction (plasma Gopinath PSNPLs, scrub 0.1 NA (alkaline lymphocytes 10 μg/mL (24 h) coronated-PSNPLs, 5 μg/mL) et al. [50] isolated- comet) PSNPLs Proved (25, 100 μg/ PSNPLs ̴ 0.05 25 µg/mL (12 weeks) DNA damage induction Prone-to- mL; 24 h) DNA damage Barguilla transformation Proved (25 or (alkaline and 25 µg/mL / 2 μM (12 et al. [34] PSNPLs/As III - progress MEF 100 μg/mL / 2 μM; Fpg-comet) DNA damage induction weeks) 24 h) HepG2CDKN1A- DNA damage 0.01, 0.1, 1, 5, 10, 25, Proved (in intestinal DsRed biosensor (p53 reporter Lack of p53 expression COOH-PSNPLs, 0.05, and 50 μg/mL (48 h) and placental barrier Hesler et al. cells gene assay) COOH-PSMPLs 0.5 co-culture models, [51] 0.1, 1, 5, 10, 25, and 100 μg/mL, 24 h) DNA damage CHO-k1 Lack of MN induction 50 μg/mL (24 h) (CBMN assay) Increased levels of MN (25 and 5, 25, and 75 μg/mL DNA damage 75 μg/mL) and nuclear buds (75 μg/ (48 h) (CBMN assay) mL) Poma et al. PSNPLs 0.1 Hs27 NA 5, 25, and 75 μg/mL Oxidative Increased ROS production (5 μg/mL, [52] (15, 30, 45, 60 min, stress (ROS 15, 30, and 60 min, and 25 μg/mL, and 24 h) assay kit) 30 and 60 min) Increased levels of MN (250 and 500 μg/mL), nucleoplasmic bridges, Human blood 25, 50, 100, 250, and DNA damage Cobanoglu PEMPLs 10–45 NA nuclear buds (100, 250, and 500 μg/ lymphocytes 500 μg/mL (48 h) (CBMN assay) et al. [53] mL) and chromosome instability (50, 100, 250, and 500 μg/mL) Table 4 In vivo studies assessing genotoxicity of MNPLs. Size Experimental model Doses (route of Treatment Presence in Material Endpoint (method) Outcome Ref. (µm) (strain; sex) administration) schedule target tissue DNA damage (alkaline comet DNA damage induction 14.6 ng/kg b. w. (i. assay) PSNPLs 0.023 Proved (brain) p.) Increased TBARS Oxidative stress concentrations and nitrite Daily for 3 (ELISA) Estrela Mice (Swiss; males) levels days et al. [54] DNA damage (alkaline comet DNA damage induction PSNPLs/ 0.023/ 14.6/14.6 ng/kg b. assay) - ZnONPs 0.069 w. (i.p.) Oxidative stress Lack of oxidative stress (ELISA) induction into bio-incompatible, modulating the subsequent biological response (100 nm, 5–75 µg/mL, 48 h) resulted in a dose-dependent increase at 25 [50]. and 75 µg/mL in the micronuclei (MN) frequency and increased nuclear In another study, PSNPLs alone (0.05 µm, 25 µg/mL), as well as co- buds’ formation at the highest PSNPLs dose, as evaluated by the CBMN exposure of PSNPLs with sodium arsenite (AsIII) (25 µg/mL / 2 µM), assay [52]. In addition, PSNPLs were able to induce ROS in the cells were evaluated for genotoxic effects in mouse embryonic fibroblasts exposed to 5 μg/mL after 15, 30, and 60 min, and to 25 μg/mL after 30 prone to oxidative damage (Ogg1− /− ) using the comet assay [34]. The and 60 min. On the other hand, the PEMPLs’ (10–45 µm, data suggested a significant increase in DNA damage produced both by 25–500 µg/mL) genotoxic potential was also assessed in human lym­ oxidative and non-oxidative mechanisms after 12 weeks of chronic phocytes after 48 h of exposure using the CBMN assay [53]. The total treatment with PSNPLs alone. Furthermore, cells co-exposed to PSNPLs number of MN, as well as the number of nucleoplasmic bridges and and AsIII showed even higher and significant DNA damage, as compared nuclear buds, significantly increased after the treatment (250 and to PSNPLs alone at 12 weeks post-treatment. 500 µg/mL, and 100–500 µg/mL, respectively). Moreover, the authors Neither PSMPLs (500 nm, 0.1–100 µg/mL) nor PSNPLs (50 nm, reported a significant positive correlation between the analyzed 0.1–100 µg/mL) could induce micronuclei formation in CHO-K1 cells endpoints. when exposed for 24 h, or activation of p53 in HepG2CDKN1A-DsRed biosensor cells, upon treatment with PSMPLs or PSNPLs (0.01–50 µg/ mL, 48 h) when assessed by the cytokinesis-block micronucleus (CBMN) 3.4. In vivo genotoxicity studies assay and the p53 reporter gene assay, respectively [51]. Contrary, the treatment of the human skin fibroblast cell line Hs27 with PSNPLs In the literature search, we found only one in vivo study reporting the genotoxicity effects of MNPLs (Table 4). In such study, PSNPLs alone 10 J. Domenech et al. Mutation Research-Reviews in Mutation Research 791 (2023) 108453 (23.03 nm) or in combination with zinc oxide nanoparticles (ZnONPs; plasticizers, stabilizers, pigments, flame retardants, antioxidants, and 68.96 nm) were able to induce DNA damage, assessed by the comet antimicrobial agents, to name a few [60]. Of special concern are assay, in peripheral blood cells from male Swiss mice intraperitoneally endocrine-disrupting compounds (EDC) -like phthalates-based plasti­ administrated for 3 consecutive days with 14.6 ng/kg b.w. of PSNPLs, cizers, bisphenols, alkylphenols, and brominated flame retardants-that ZnONPs, or equitable doses of both materials [54]. The increase in DNA could elevate the risk of cancer [61,62]. This poses a new challenge damage, measured as the tail length, the percentage of DNA in the tail, for MNPLs’ cancer risk assessment, as the impacts resulting from the and the Olive tail moment, showed a statistically significant tendency, mobilization of these additives from the plastic matrix need to be ZnONPs > ZnONPs + PSNPLs > PSNPLs. The authors hypothesized that considered. As an example, to illustrate this fact, the compound the effect could be due to the observed increased levels of nitric oxide di-n-butyl phthalate was observed to be released into water from poly­ and thiobarbituric acid reactive species, as indicators of an oxidative vinyl chloride (PVC) tubes [63]. Also, the desorption of a well-known stress response. MNPLs bioaccumulation was also assessed, and the re­ EDC (DEHP) from PS foam coolers was demonstrated after an in vitro sults showed a significant accumulation of PSNPLs, and ZnONPs, as well simulated human digestion of the PS mimicking gut conditions [64]. as the combination of both, in the mice’s brain. Nevertheless, the Nevertheless, it must be highlighted the lack of studies regarding the detection of both nanoparticles was lower when co-administered, which ability of MNPLs to release these additives into biological matrices after is explained by the authors considering the bigger size of the aggregates their uptake. formed when both types of particles interact. Despite its relevance regarding the carcinogenic risk, plastic addi­ tives have been left aside from the scope of the present review due to the 4. Discussion large number of potentially harmful additives that can be found in plastics and the already existing extensive reviews of the literature The ever-increasing usage of polymer plastics in day-to-day life is a regarding this topic. To give instance, the genotoxic potential of major cause of concern due to their potential implications for human bisphenol A (BPA) was the aim of a recent review [65]. The summary of health. The ongoing research efforts suggest that exposure of humans to the reported data indicates that BPA could induce oxidative stress in a MNPLs via inhalation and ingestion is inevitable. Nevertheless, there wide range of organisms through increased intracellular ROS produc­ exists a clear knowledge gap and a lack of understanding vis-à-vis tion, or by altering the activity of antioxidant enzymes. The intracellular whether they pose a serious health risk to humans [55]. The carcino­ oxidation effects could lead to DNA damage induction, mainly by genic potential due to continuous exposure to MNPLs has barely been oxidatively damaging DNA bases, or by inducing single- and assessed until now, which highlights the pressing need to address the double-strand DNA breaks. Similarly, some phthalates have been shown carcinogenic risk assessment in humans on an urgent basis through to induce carcinogenicity in rodent livers, as well as their potential to concerted research efforts. cause genotoxicity [66]. In fact, different phthalates can act as genotoxic As an important point to highlight, the present review confirms the agents in a wide range of assays and animal models from bacteria to lack of studies performed with rodent carcinogenicity assays to assess mammals, inducing both point and chromosome mutations. Considering MNPLs exposure, probably due to the complexity of such studies. For the ways of actions of these plastic additives, the challenge is to identify this reason, indirect approaches to evaluating DNA damage, generation if the plasticizers incorporated into the MNPLs have the potential to of ROS, chromosomal aberrations, and inflammatory responses in diffuse or leach from the polymer matrix and exert their harmful effects. mammalian models have been used in the present review to get some Most of the discussed studies pointed towards the micro and nano insights into the potential carcinogenic risk of MNPLs. Most of the sizes of plastics as an important physical determinant for the observed studies pointed out the potential of MNPLs to induce biological re­ biological effects. Nevertheless, the shape, chemical composition, sur­ sponses that are considered either molecular initiating events (MIE) or face charge, or functionalization of MNPLs may also determine the key events (KE) in the induction of human cancer. For instance, an biological outcome since they play a major role in conferring toxicity, increased production of inflammatory cytokines, accomplished by genotoxicity, and carcinogenic properties [67,68]. Thus, we consider increased formation of ROS that could result in higher rates of DNA that the focus on size, as the sole determining feature of MNPLs’ harmful damage and mutations have been described as KE in the development potential, turns a blind eye to the other determinants. Then, the role of pathways of lung (e.g., AOPs 303, 416, 417, and 451) and breast (e.g., these physicochemical properties in the risk assessment along with the AOPs 439) cancer. Increased production of reactive oxygen and nitrogen size should be taken under advisement. On the other hand, most of the species can be MIEs leading to mutations (e.g., AOP 296) and induction studies employed commercially synthesized spherical PS particles. In of breast cancer (e.g., AOP 294) or, if becoming chronic, of gastric cancer fact, only three of the discussed studies [38,44,53] focused on other (e.g., AOP 298) [56]. Although some of these KE, like inflammation, do types of environmentally representative MNPLs (PE and PLGA) with a not always lead to tumor formation, they can be considered as early myriad of physicochemical properties. Considering that polypropylene, indicators of an increased cancer risk. Furthermore, inflammation and PE, PVC, and polyethylene terephthalate are the most demanded poly­ ROS induction are among the pivotal events that should be addressed mer types in Europe [69], other types of environmentally relevant when developing an integrated approach to the testing and assessment MNPLs, apart from PS, should be considered for the carcinogenesis risk (IATA) of non-genotoxic carcinogens [14]. Besides, the MNPLs’ ability assessment. Taking these considerations into account, it should be to accumulate into cells and tissues may favour the interaction of the remembered that secondary MNPLs resulting from the degradation of material with the target cells, which could initiate a chain of events large plastic goods are constituted by a mix of different sizes, shapes, and involving the induction of primary or secondary genotoxicity and chemical nature. In this context, it should be indicated the lack of studies resulting e.g., in lung cancer (AOP 451; [57]). Although no described for using synthetic fibers (despite their high environmental presence) to MNPLs, physical interference of the internalized MNPLs with the mitotic determine their carcinogenic risk at the nano/micro range. At this time, apparatus may lead to losses of chromosomes during the mitosis, as it and despite the work carried out with other fiber-like nano-sized ma­ has reported e.g., with carbon nanotubes [58]. Furthermore, the ca­ terials, especially carbon nanotubes and carbon nanofibers, limited ev­ pacity of MNPLs to induce fibrosis, which have been associated with the idence of carcinogenicity has been reached for most of them [58,70]. induction of malignant mesothelioma [59], may also suggest an asso­ Few in vivo studies appeared in our literature search due to the lack of ciation between MNPLs exposure and their tumorigenic potential. studies specifically addressing genotoxic or tumorigenic effects after Another important question to consider is the fact that MNPLs exposure to MNPLs. However, the effects of MNPLs in rodent models contain not only polymers but potentially harmful chemicals that have have been recently reviewed [71]. Regarding MNPLs cancer risk, been deliberately added during plastic goods production to get the accumulation of plastic particles and inflammation at the accumulation desired characteristics of the final product. Such additives include site are the most studied outcomes. Non-fluorescent MNPLs have been 11 J. Domenech et al. Mutation Research-Reviews in Mutation Research 791 (2023) 108453 localized in primary absorption sites (gut and liver, considering the main necessary to provide the necessary evidence-based knowledge for exposure route) leading to increased inflammation. As well as in the in informed decision-making by the regulatory bodies and policymakers. vitro scenario, oxidative stress is considered the underlying toxicity mechanism of MNPLs in vivo which would induce inflammation. Funding Inflammation in mice intestines caused by MNPLs has been reported to alter the microbiome [72], and these can contribute to metabolic dis­ This work was supported by the European Union’s Horizon 2020 orders including colorectal cancer [73,74]. Like that described in vitro by research and innovation programme [grant number 965196] Cheng et al. [32], liver inflammation in rodents has been shown to (PlasticHeal). induce hepatotoxicity. Among the effects, the downregulation of PPARγ would lead to fibrosis [71]. Accumulation of MNPLs in reproductive CRediT authorship contribution statement organs has also been reported in rodents. Accumulation in testes generated oxidative stress, which in turn activated the p38 MAPK Conceptualization, Methodology: R. M., A. H., and J. C.; Formal pathway inducing an increase in the levels of the pro-inflammatory analysis and Writing – original draft: J. D., and B. A.; Writing – review & cytokines. Likewise, An et al. [42] reported the editing: J. D., B. A., R. M., A. H., and J. C.; Supervision: R. M., A. H., and oxidative-stress-mediated activation of the Wnt/β-catenin pathway J. C.; Funding acquisition: J. C., R. M., and A. H. leading to fibrosis in rats’ ovaries after the MNPLs administration. Another point to be highlighted is the dearth of research directly dealing with the cellular transformation ability of MNPLs. No studies Declaration of Competing Interest performed with validated guidelines (i.e., the Bhas-42 cell trans­ formation assay) were found in our search. Furthermore, only two The authors declare that they have no known competing financial studies [33,34], evaluated the transforming capacity of MNPLs using in interests or personal relationships that could have appeared to influence vitro long-term approaches and a wide battery of different hallmarks of the work reported in this paper. cancer. The selection of bioassays for the evaluation of carcinogenesis is of paramount importance. Based on the previous know-how on NMs, the Data Availability recommended in vitro approaches are the anchorage-independent cell growth assessment (using the soft-agar colony-forming assay), and as­ No data was used for the research described in the article. says measuring invasion and migration abilities, as well as those ap­ proaches determining the morphological changes in cells exposed to Acknowledgment MNPLs. In addition, to mimic chronic exposures, longer duration treatments, and lower concentrations than those usually applied in This project has received funding from the European Union’s Hori­ short-term systems might be considered. zon 2020 research and innovation program under grant agreement No. Special focus must be placed on the dose/concentration of MNPLs 965196. used when performing in vivo and in vitro experiments. 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