Abstract
Zinc pyrithione (ZPT), a broad-spectrum antimicrobial agent widely used in anti-dandruff shampoos and antifouling coatings, has an unclear toxic effect on embryonic trophoblast cells. To systematically evaluate the toxicological impact of ZPT on human trophoblast cell line JEG-3 and its underlying mechanisms, cells were treated with 90 nM ZPT for 72 h. A series of assays, including Cell Counting Kit-8(CCK-8), flow cytometry, wound healing, and Transwell, were performed to assess cell proliferation, apoptosis, migration, and invasion. Intracellular reactive oxygen species (ROS) levels and DNA damage were assessed using the 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) probe and γ-H2AX immunofluorescence, respectively. Transcriptome sequencing and Gene Ontology(GO) enrichment analysis were also performed. The results indicated that ZPT significantly inhibited cell proliferation, migration, and invasion, induced late-stage apoptosis, and increased ROS levels and DNA damage. RNA sequencing (RNA-seq) identified 1020 differentially expressed genes, suggesting an upregulation in autophagy and mitochondrial apoptosis pathways, and a significant downregulation in glycolysis, NAD⁺ regeneration, and hypoxia response pathways. Quantitative real-time polymerase chain reaction (qPCR) validation further confirmed the upregulation of key stress- and autophagy-related genes (NUPR1, SQSTM1) and the downregulation of genes involved in trophoblast function and mitochondrial quality control (BMP4, BNIP3, BNIP3L). These in vitro findings suggest that ZPT may impair trophoblast function through mechanisms involving oxidative stress, DNA damage, and perturbations in mitochondrial apoptosis/autophagy and energy metabolism.
Data availability
All sequencing data related to this study have been deposited at Gene Expression Omnibus (GSA for Human, https://ngdc.cncb.ac.cn/gsa/) under the accession number HRA013363.
References
Mangion, S. E., Holmes, A. M. & Roberts, M. S. Targeted Delivery of Zinc Pyrithione to Skin Epithelia. Int. J. Mol. Sci. https://doi.org/10.3390/ijms22189730 (2021).
Bones, J., Thomas, K. V. & Paull, B. Improved method for the determination of zinc pyrithione in environmental water samples incorporating on-line extraction and preconcentration coupled with liquid chromatography atmospheric pressure chemical ionisation mass spectrometry. J. Chromatogr. A. 1132, 157–164. https://doi.org/10.1016/j.chroma.2006.07.068 (2006).
Maraldo, K. & Dahllof, I. Indirect Estimation of degradation time for zinc pyrithione and copper pyrithione in seawater. Mar. Pollut Bull. 48, 894–901. https://doi.org/10.1016/j.marpolbul.2003.11.013 (2004).
Harino, H. et al. Concentrations of booster biocides in sediment and clams from Vietnam. J. Mar. Biol. Association United Kingd. 86, 1163–1170. https://doi.org/10.1017/s0025315406014147 (2006).
Marcheselli, M., Rustichelli, C. & Mauri, M. Novel antifouling agent zinc pyrithione: determination, acute toxicity, and bioaccumulation in marine mussels (Mytilus galloprovincialis). Environ. Toxicol. Chem. 29, 2583–2592. https://doi.org/10.1002/etc.316 (2010).
Marcheselli, M., Azzoni, P. & Mauri, M. Novel antifouling agent-zinc pyrithione: stress induction and genotoxicity to the marine mussel mytilus galloprovincialis. Aquat. Toxicol. 102, 39–47. https://doi.org/10.1016/j.aquatox.2010.12.015 (2011).
Onduka, T. et al. Toxicity of metal pyrithione photodegradation products to marine organisms with indirect evidence for their presence in seawater. Arch. Environ. Contam. Toxicol. 58, 991–997. https://doi.org/10.1007/s00244-009-9430-8 (2010).
Zhao, Y. et al. Acute toxic responses of embryo-larval zebrafish to zinc pyrithione (ZPT) reveal embryological and developmental toxicity. Chemosphere 205, 62–70. https://doi.org/10.1016/j.chemosphere.2018.04.010 (2018).
Bellas, J., Granmo, Å. & Beiras, R. Embryotoxicity of the antifouling biocide zinc pyrithione to sea urchin (Paracentrotus lividus) and mussel (Mytilus edulis). Mar. Pollut. Bull. 50, 1382–1385 (2005).
Haque, M. N., Nam, S. E., Eom, H. J., Kim, S. K. & Rhee, J. S. Exposure to sublethal concentrations of zinc pyrithione inhibits growth and survival of marine polychaete through induction of oxidative stress and DNA damage. Mar. Pollut Bull. 156, 111276. https://doi.org/10.1016/j.marpolbul.2020.111276 (2020).
Holmes, A. M., Kempson, I., Turnbull, T., Paterson, D. & Roberts, M. S. Imaging the penetration and distribution of zinc and zinc species after topical application of zinc pyrithione to human skin. Toxicol. Appl. Pharmacol. 343, 40–47. https://doi.org/10.1016/j.taap.2018.02.012 (2018).
Ren, T. et al. Toxicity and accumulation of zinc pyrithione in the liver and kidneys of Carassius auratus gibelio: association with P-glycoprotein expression. Fish. Physiol. Biochem. 43, 1–9. https://doi.org/10.1007/s10695-016-0262-y (2017).
Oh, H. N. & Kim, W. K. Copper pyrithione and zinc pyrithione induce cytotoxicity and neurotoxicity in neuronal/astrocytic co-cultured cells via oxidative stress. Sci. Rep. 13, 23060. https://doi.org/10.1038/s41598-023-49740-8 (2023).
Mo, J., Lin, D., Wang, J., Li, P. & Liu, W. Apoptosis in HepG2 cells induced by zinc pyrithione via mitochondrial dysfunction pathway: involvement of zinc accumulation and oxidative stress. Ecotoxicol. Environ. Saf. 161, 515–525. https://doi.org/10.1016/j.ecoenv.2018.06.026 (2018).
Sun, Q. & Zhang, X. L. Research on apoptotic signaling pathways of recurrent spontaneous abortion caused by dysfunction of trophoblast infiltration. Eur. Rev. Med. Pharmacol. Sci. 21, 12–19 (2017).
Zhao, Y. et al. Zinc pyrithione (ZPT) -induced embryonic toxicogenomic responses reveal involvement of oxidative damage, apoptosis, Endoplasmic reticulum (ER) stress and autophagy. Aquat. Toxicol. 248, 106195. https://doi.org/10.1016/j.aquatox.2022.106195 (2022).
Wang, Y. S. et al. Zinc pyrithione exposure compromises oocyte maturation through involving in spindle assembly and zinc accumulation. Ecotoxicol. Environ. Saf. 234, 113393. https://doi.org/10.1016/j.ecoenv.2022.113393 (2022).
Ma, H. et al. Identification and Functional Analysis of Apoptotic Protease Activating Factor-1 (Apaf-1) from Spodoptera litura. Insects https://doi.org/10.3390/insects12010064 (2021).
Zhang, H. M., Cheung, P., Yanagawa, B., McManus, B. M. & Yang, D. C. BNips: a group of pro-apoptotic proteins in the Bcl-2 family. Apoptosis 8, 229–236. https://doi.org/10.1023/a:1023616620970 (2003).
Wang, C. et al. Neutrophil extracellular traps aggravate placental injury in OAPS by facilitating activation of BNIP3 mediated mitophagy. Free Radic Biol. Med. 235, 109–123. https://doi.org/10.1016/j.freeradbiomed.2025.04.038 (2025).
Mann, J. J. & Fraker, P. J. Zinc pyrithione induces apoptosis and increases expression of Bim. Apoptosis 10, 369–379. https://doi.org/10.1007/s10495-005-0811-9 (2005).
Cano, C. E., Hamidi, T., Sandi, M. J. & Iovanna, J. L. Nupr1: the Swiss-knife of cancer. J. Cell. Physiol. 226, 1439–1443. https://doi.org/10.1002/jcp.22324 (2011).
Hu, J. et al. Toxic effects and potential mechanisms of zinc pyrithione (ZPT) exposure on sperm and testicular injury in zebrafish. J. Hazard. Mater. 461, 132575. https://doi.org/10.1016/j.jhazmat.2023.132575 (2024).
Hadas, R. et al. Temporal BMP4 effects on mouse embryonic and extraembryonic development. Nature 634, 652–661. https://doi.org/10.1038/s41586-024-07937-5 (2024).
Tang, L. et al. Deletion of BMP4 impairs trophoblast function and decidual macrophage polarization via autophagy leading to recurrent spontaneous abortion. Int. Immunopharmacol. 147, 114015. https://doi.org/10.1016/j.intimp.2025.114015 (2025).
Semenza, G. L. Hypoxia-inducible factors: mediators of cancer progression and targets for cancer therapy. Trends Pharmacol. Sci. 33, 207–214. https://doi.org/10.1016/j.tips.2012.01.005 (2012).
Dong, C. et al. Loss of FBP1 by Snail-mediated repression provides metabolic advantages in basal-like breast cancer. Cancer Cell. 23, 316–331. https://doi.org/10.1016/j.ccr.2013.01.022 (2013).
Hong, J. W. et al. BMP4 regulates EMT to be involved in non-Syndromic cleft lip with or without palate. Cleft Palate Craniofac. J. 60, 1462–1473. https://doi.org/10.1177/10556656221105762 (2023).
Chiarugi, A., Dolle, C., Felici, R. & Ziegler, M. The NAD metabolome–a key determinant of cancer cell biology. Nat. Rev. Cancer. 12, 741–752. https://doi.org/10.1038/nrc3340 (2012).
Guo, C., Sun, L., Chen, X. & Zhang, D. Oxidative stress, mitochondrial damage and neurodegenerative diseases. Neural Regen Res. 8, 2003–2014. https://doi.org/10.3969/j.issn.1673-5374.2013.21.009 (2013).
Plaisance, V. et al. Endoplasmic reticulum stress links oxidative stress to impaired pancreatic Beta-Cell function caused by human oxidized LDL. PLoS One. 11, e0163046. https://doi.org/10.1371/journal.pone.0163046 (2016).
Gorlach, A., Bertram, K., Hudecova, S., Krizanova, O. & Calcium A mutual interplay. Redox Biol. 6, 260–271. https://doi.org/10.1016/j.redox.2015.08.010 (2015).
Don, W., Luu, L. & Kaakoush, N. O. Castano-Rodriguez, N. The role of ATG16L2 in autophagy and disease. Autophagy 18, 2537–2546. https://doi.org/10.1080/15548627.2022.2042783 (2022).
Lamark, T., Svenning, S. & Johansen, T. Regulation of selective autophagy: the p62/SQSTM1 paradigm. Essays Biochem. 61, 609–624. https://doi.org/10.1042/EBC20170035 (2017).
Verfaillie, T. et al. PERK is required at the ER-mitochondrial contact sites to convey apoptosis after ROS-based ER stress. Cell. Death Differ. 19, 1880–1891. https://doi.org/10.1038/cdd.2012.74 (2012).
Burton, T. R. & Gibson, S. B. The role of Bcl-2 family member BNIP3 in cell death and disease: nipping at the heels of cell death. Cell. Death Differ. 16, 515–523. https://doi.org/10.1038/cdd.2008.185 (2009).
Zhou, X. et al. Impaired placental mitophagy and oxidative stress are associated with dysregulated BNIP3 in preeclampsia. Sci. Rep. 11, 20469. https://doi.org/10.1038/s41598-021-99837-1 (2021).
Funding
This work was supported by funding from the following sources: The Huangshi Health Commission General Research Project (WJ2024023); Hubei Provincial Natural Science Foundation (Joint Fund Project) (2023AFD019); Hubei Provincial Health Commission Fund (WJ2019H183); Huangshi Central Hospital Foundation Project (ZX2023M07).
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JM and JW conceived and designed the research. XW and BL performed the experiment and analyzed the sequencing data. ZL, JM and XC performed the experiment. XW and JM wrote the original manuscript.
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Wang, X., Luo, B., Lu, Z. et al. Oxidative stress-mediated impairment of human trophoblast cell proliferation by zinc pyrithione exposure. Sci Rep (2026). https://doi.org/10.1038/s41598-026-38895-9
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DOI: https://doi.org/10.1038/s41598-026-38895-9
