Abstract: Microplastics (MPs) and nanoplastics (NPs) have become hazardous materials due to the massive amount of plastic waste and disposable masks, but their specific health effects remain uncertain. In this study, fluorescence-labeled polystyrene NPs (PS-NPs) were injected into the circulatory systems of mice to determine the distribution and potential toxic effects of NPs in vivo. Interestingly, whole-body imaging found that PS-NPs accumulated in the testes of mice. Therefore, the toxic effects of PS-NPs on the reproduction systems and the spermatocytes cell line of male mice, and their mechanisms, were investigated. After oral exposure to PS-NPs, their spermatogenesis was affected and the spermatogenic cells were damaged. The spermatocyte cell line GC-2 was exposed to PS-NPs and analyzed using RNA sequencing (RNA-seq) to determine the toxic mechanisms; a ferroptosis pathway was found after PS-NP exposure. The phenomena and indicators of ferroptosis were then determined and verified by ferroptosis inhibitor ferrostatin-1 (Fer-1), and it was also found that nuclear factor erythroid 2-related factor 2 (Nrf2) played an important role in spermatogenic cell ferroptosis induced by PS-NPs. Finally, it was confirmed in vivo that this mechanism of Nrf2 played a protective role in PS-NPs-induced male reproductive toxicity. This study demonstrated that PS-NPs induce male reproductive dysfunction in mice by causing spermatogenic cell ferroptosis dependent on Nrf2.

Nrf2介导的生精细胞铁死亡参与了聚苯乙烯纳米塑料导致的小鼠雄性生殖毒性

[1]AmerehF, BabaeiM, EslamiA, et al., 2020. The emerging risk of exposure to nano(micro)plastics on endocrine disturbance and reproductive toxicity: from a hypothetical scenario to a global public health challenge. Environ Pollut, 261:114158.

[2]ArchibongAE, RideoutML, HarrisKJ, et al., 2018. Oxidative stress in reproductive toxicology. Curr Opin Toxicol, 7:95-101.

[3]BishtS, FaiqM, TolahunaseM, et al., 2017. Oxidative stress and male infertility. Nat Rev Urol, 14(8):470-485.

[4]ChenX, ComishPB, TangDL, et al., 2021. Characteristics and biomarkers of ferroptosis. Front Cell Dev Biol, 9:637162.

[5]CortésC, DomenechJ, SalazarM, et al., 2020. Nanoplastics as a potential environmental health factor: effects of polystyrene nanoparticles on human intestinal epithelial Caco-2 cells. Environ Sci Nano, 7(1):272-285.

[6]DengYF, ZhangY, LemosB, et al., 2017. Tissue accumulation of microplastics in mice and biomarker responses suggest widespread health risks of exposure. Sci Rep, 7:46687.

[7]DixonSJ, LembergKM, LamprechtMR, et al., 2012. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell, 149(5):1060-1072.

[8]DodsonM, Castro-PortuguezR, ZhangDD, 2019. NRF2 plays a critical role in mitigating lipid peroxidation and ferroptosis. Redox Biol, 23:101107.

[9]DuFN, CaiHW, ZhangQ, et al., 2020. Microplastics in take-out food containers. J Hazard Mater, 399:122969.

[10]DuX, ZhangJJ, LiuL, et al., 2022. A novel anticancer property of Lycium barbarum polysaccharide in triggering ferroptosis of breast cancer cells. J Zhejiang Univ-Sci B (Biomed & Biotechnol), 23(4):286-299.

[11]FendallLS, SewellMA, 2009. Contributing to marine pollution by washing your face: microplastics in facial cleansers. Mar Pollut Bull, 58(8):1225-1228.

[12]FuXF, LiuL, HanH, et al., 2023. Integrated fecal microbiome and metabolome analysis explore the link between polystyrene nanoplastics exposure and male reproductive toxicity in mice. Environ Toxicol, 38(6):1277-1291.

[13]GasperiJ, WrightSL, DrisR, et al., 2018. Microplastics in air: are we breathing it in? Curr Opin Environ Sci Health, 1:1-5.

[14]HaradaN, KanayamaM, MaruyamaA, et al., 2011. Nrf2 regulates ferroportin 1-mediated iron efflux and counteracts lipopolysaccharide-induced ferroportin 1 mRNA suppression in macrophages. Arch Biochem Biophys, 508(1):‍101-109.

[15]HassanniaB, VandenabeeleP, BergheTV, 2019. Targeting ferroptosis to iron out cancer. Cancer Cell, 35(6):830-849.

[16]HernandezLM, XuEG, LarssonHCE, et al., 2019. Plastic teabags release billions of microparticles and nanoparticles into tea. Environ Sci Technol, 53(21):12300-12310.

[17]HuangC, LiBS, XuKR, et al., 2017. Decline in semen quality among 30,636 young Chinese men from 2001 to 2015. Fertil Steril, 107(1):83-88.e2.

[18]ImaiH, HakkakuN, IwamotoR, et al., 2009. Depletion of selenoprotein GPx4 in spermatocytes causes male infertility in mice. J Biol Chem, 284(47):32522-32532.

[19]JiangL, WangJM, WangK, et al., 2021. RNF217 regulates iron homeostasis through its E3 ubiquitin ligase activity by modulating ferroportin degradation. Blood, 138(8):689-705.

[20]JinHB, MaT, ShaXX, et al., 2021. Polystyrene microplastics induced male reproductive toxicity in mice. J Hazard Mater, 401:123430.

[21]JungBK, HanSW, ParkSH, et al., 2020. Neurotoxic potential of polystyrene nanoplastics in primary cells originating from mouse brain. Neurotoxicology, 81:189-196.

[22]KedzierskiM, LechatB, SireO, et al., 2020. Microplastic contamination of packaged meat: occurrence and associated risks. Food Packag Shelf Life, 24:100489.

[23]KerinsMJ, OoiA, 2018. The roles of NRF2 in modulating cellular iron homeostasis. Antioxid Redox Signal, 29(17):1756-1773.

[24]KhanFR, CatarinoAI, ClarkNJ, 2022. The ecotoxicological consequences of microplastics and co-contaminants in aquatic organisms: a mini-review. Emerg Top Life Sci, 6(4):339-348.

[25]KimMJ, YunGJ, KimSE, 2021. Metabolic regulation of ferroptosis in cancer. Biology, 10(2):83.

[26]LevineH, JørgensenN, Martino-AndradeA, et al., 2017. Temporal trends in sperm count: a systematic review and meta-regression analysis. Hum Reprod Update, 23(6):646-659.

[27]LiSB, HeYP, ChenKX, et al., 2021. RSL3 drives ferroptosis through NF‍-‍κB pathway activation and GPX4 depletion in glioblastoma. Oxid Med Cell Longev, 2021:2915019.

[28]LiYM, LiuZQ, YangY, et al., 2021. Effects of nanoplastics on energy metabolism in the oriental river prawn (Macrobrachium nipponense). Environ Pollut, 268:115890.

[29]LiuZX, LvXY, YangBW, et al., 2021. Tetrachlorobenzoquinone exposure triggers ferroptosis contributing to its neurotoxicity. Chemosphere, 264:128413.

[30]LuJ, ZhaoYN, LiuMT, et al., 2021. Toward improved human health: Nrf2 plays a critical role in regulating ferroptosis. Food Funct, 12(20):9583-9606.

[31]LusherA, HollmanP, Mendoza-HillJ, 2017. Microplastics in Fisheries and Aquaculture: Status of Knowledge on Their Occurrence and Implications for Aquatic Organisms and Food Safety. FAO Fisheries and Aquaculture Technical Paper No. 615, Food and Agriculture Organization of the United Nations, Madrid.

[32]MengXM, ZhangJW, WangWJ, et al., 2022. Effects of nano- and microplastics on kidney: physicochemical properties, bioaccumulation, oxidative stress and immunoreaction. Chemosphere, 288(Pt 3):132631.

[33]MiottoG, RossettoM, di PaoloML, et al., 2020. Insight into the mechanism of ferroptosis inhibition by ferrostatin-1. Redox Biol, 28:101328.

[34]NairAB, JacobS, 2016. A simple practice guide for dose conversion between animals and human. J Basic Clin Pharm, 7(2):27-31.

[35]OßmannBE, SarauG, HoltmannspötterH, et al., 2018. Small-sized microplastics and pigmented particles in bottled mineral water. Water Res, 141:307-316.

[36]ParkMW, ChaHW, KimJ, et al., 2021. NOX4 promotes ferroptosis of astrocytes by oxidative stress-induced lipid peroxidation via the impairment of mitochondrial metabolism in Alzheimer’s diseases. Redox Biol, 41:101947.

[37]RagusaA, SvelatoA, SantacroceC, et al., 2021. Plasticenta: first evidence of microplastics in human placenta. Environ Int, 146:106274.

[38]RiedelbergerM, PenningerP, TschernerM, et al., 2020. Type I interferon response dysregulates host iron homeostasis and enhances Candida glabrata infection. Cell Host Microbe, 27(3):454-466.e8.

[39]RistS, BaunA, HartmannNB, 2017. Ingestion of micro- and nanoplastics in Daphnia magna‍ ‒‍ quantification of body burdens and assessment of feeding rates and reproduction. Environ Pollut, 228:398-407.

[40]SchulteRT, OhlDA, SigmanM, et al., 2010. Sperm DNA damage in male infertility: etiologies, assays, and outcomes. J Assist Reprod Genet, 27(1):3-12.

[41]ShuklaS, KhanR, SaxenaA, et al., 2022. Microplastics from face masks: a potential hazard post Covid-19 pandemic. Chemosphere, 302:134805.

[42]SongXH, LongDX, 2020. Nrf2 and ferroptosis: a new research direction for neurodegenerative diseases. Front Neurosci, 14:267.

[43]StockwellBR, JiangXJ, GuW, 2020. Emerging mechanisms and disease relevance of ferroptosis. Trends Cell Biol, 30(6):478-490.

[44]SunXF, OuZH, ChenRC, et al., 2016. Activation of the p62-Keap1-NRF2 pathway protects against ferroptosis in hepatocellular carcinoma cells. Hepatology, 63(1):173-184.

[45]TangDL, ChenX, KangR, et al., 2021. Ferroptosis: molecular mechanisms and health implications. Cell Res, 31(2):107-125.

[46]TaoSS, LiuPF, LuoG, et al., 2017. p97 negatively regulates NRF2 by extracting ubiquitylated NRF2 from the KEAP1-CUL3 E3 complex. Mol Cell Biol, 37(8):e00660-16.

[47]UrsiniF, MaiorinoM, 2020. Lipid peroxidation and ferroptosis: the role of GSH and GPx4. Free Radic Biol Med, 152:175-185.

[48]WangLW, WuWM, BolanNS, et al., 2021. Environmental fate, toxicity and risk management strategies of nanoplastics in the environment: current status and future perspectives. J Hazard Mater, 401:123415.

[49]WangZ, AnCJ, ChenXJ, et al., 2021. Disposable masks release microplastics to the aqueous environment with exacerbation by natural weathering. J Hazard Mater, 417:126036.

[50]XuDH, MaYH, HanXD, et al., 2021. Systematic toxicity evaluation of polystyrene nanoplastics on mice and molecular mechanism investigation about their internalization into Caco-2 cells. J Hazard Mater, 417:126092.

[51]XuMK, HalimuG, ZhangQR, et al., 2019. Internalization and toxicity: a preliminary study of effects of nanoplastic particles on human lung epithelial cell. Sci Total Environ, 694:133794.

[52]YeeMSL, HiiLW, LooiCK, et al., 2021. Impact of microplastics and nanoplastics on human health. Nanomaterials, 11(2):496.

[53]YinK, WangY, ZhaoHJ, et al., 2021. A comparative review of microplastics and nanoplastics: toxicity hazards on digestive, reproductive and nervous system. Sci Total Environ, 774:145758.

[54]YinLS, WenXF, HuangDL, et al., 2021. Interactions between microplastics/nanoplastics and vascular plants. Environ Pollut, 290:117999.

[55]YongCQY, ValiyaveettilS, TangBL, 2020. Toxicity of microplastics and nanoplastics in mammalian systems. Int J Environ Res Public Health, 17(5):1509.

[56]YosriN, KhalifaSAM, GuoZM, et al., 2021. Marine organisms: pioneer natural sources of polysaccharides/proteins for green synthesis of nanoparticles and their potential applications. Int J Biol Macromol, 193:1767-1798.

[57]Zegers-HochschildF, AdamsonGD, DyerS, et al, 2017. The international glossary on infertility and fertility care, 2017. Fertil Steril, 108(3):393-406.