{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,30]],"date-time":"2026-10-30T11:54:53Z","timestamp":1793361293510,"version":"4.5.0"},"reference-count":54,"publisher":"MDPI AG","issue":"6","license":[{"start":{"date-parts":[[2025,5,23]],"date-time":"2025-05-23T00:00:00Z","timestamp":1747958400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Henan Key Discipline of Biology and Medicine","award":["ZDXK2024002"],"award-info":[{"award-number":["ZDXK2024002"]}]},{"name":"Henan Key Discipline of Biology and Medicine","award":["SQ2023XSJSDTR01"],"award-info":[{"award-number":["SQ2023XSJSDTR01"]}]},{"name":"Henan Key Discipline of Biology and Medicine","award":["2023GGJS201"],"award-info":[{"award-number":["2023GGJS201"]}]},{"name":"Academic Technology Leader Program of Sanquan College of Xinxiang Medical College","award":["ZDXK2024002"],"award-info":[{"award-number":["ZDXK2024002"]}]},{"name":"Academic Technology Leader Program of Sanquan College of Xinxiang Medical College","award":["SQ2023XSJSDTR01"],"award-info":[{"award-number":["SQ2023XSJSDTR01"]}]},{"name":"Academic Technology Leader Program of Sanquan College of Xinxiang Medical College","award":["2023GGJS201"],"award-info":[{"award-number":["2023GGJS201"]}]},{"name":"Hennan Province Colleges and Universities Young Backbone teacher Training Program","award":["ZDXK2024002"],"award-info":[{"award-number":["ZDXK2024002"]}]},{"name":"Hennan Province Colleges and Universities Young Backbone teacher Training Program","award":["SQ2023XSJSDTR01"],"award-info":[{"award-number":["SQ2023XSJSDTR01"]}]},{"name":"Hennan Province Colleges and Universities Young Backbone teacher Training Program","award":["2023GGJS201"],"award-info":[{"award-number":["2023GGJS201"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biology"],"abstract":"<jats:p>Busulfan (BU) is a widely used chemotherapy drug that has been shown to cause reproductive functional impairment in humans and model animals. However, the precise mechanisms underlying testicular injury induced by BU exposure have not been fully elucidated. Ferroptosis is a form of programmed cell death mediated by iron-dependent lipid peroxidation. The aim of the current study was to determine whether ferroptosis was involved in BU-induced testicular injury. We demonstrated that exposure to BU led to an increase in iron content in the testes of mice. Subsequent western blotting and reverse transcription quantitative PCR, as well as staining of testicular tissue sections, confirmed that ferroptosis mediated BU-induced testicular injury. Consistent with our in vivo findings, we found that ferroptosis, including iron metabolism and the solute carrier family 7 member 11\/glutathione peroxidase 4 (xCT\/GPX4) signaling pathway, may play a key role in mediating BU-induced injury to GC-1 spg cells in vitro. Treatment with ferroptosis inhibitors slowed cell death caused by BU exposure. Specifically, we found that the administration of zinc protoporphyrin IX (ZnPP), a heme oxygenase 1 (HO1) inhibitor, rescued BU-induced cell death. In conclusion, our in vivo and in vitro findings confirmed that BU exposure led to testicular ferroptosis in mice via the iron intake pathway and the HO1 signaling pathway.<\/jats:p>","DOI":"10.3390\/biology14060594","type":"journal-article","created":{"date-parts":[[2025,5,23]],"date-time":"2025-05-23T12:24:18Z","timestamp":1748003058000},"page":"594","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Ferroptotic Pathway Activation in Spermatogonia: A Novel Mechanism of Busulfan-Induced Testicular Injury"],"prefix":"10.3390","volume":"14","author":[{"given":"Huanhuan","family":"Hu","sequence":"first","affiliation":[{"name":"College of Life Science, Henan Normal University, Xinxiang 453007, China"},{"name":"Key Laboratory of Fertility Preservation, School of Life Sciences and Technologies, North Henan Medical University, Xinxiang 453003, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0000-3274-4299","authenticated-orcid":false,"given":"Wenzheng","family":"Yuan","sequence":"additional","affiliation":[{"name":"Key Laboratory of Fertility Preservation, School of Life Sciences and Technologies, North Henan Medical University, Xinxiang 453003, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yulin","family":"Wang","sequence":"additional","affiliation":[{"name":"College of Life Science, Henan Normal University, Xinxiang 453007, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6416-590X","authenticated-orcid":false,"given":"Zimei","family":"Dong","sequence":"additional","affiliation":[{"name":"College of Life Science, Henan Normal University, Xinxiang 453007, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Guangwen","family":"Chen","sequence":"additional","affiliation":[{"name":"College of Life Science, Henan Normal University, Xinxiang 453007, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,5,23]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"131","DOI":"10.1038\/s41573-019-0048-x","article-title":"Managing the challenge of drug-induced liver injury: A roadmap for the development and deployment of preclinical predictive models","volume":"19","author":"Weaver","year":"2020","journal-title":"Nat. Rev. Drug Discov."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"85","DOI":"10.1038\/s41577-021-00547-6","article-title":"Cytokine release syndrome and associated neurotoxicity in cancer immunotherapy","volume":"22","author":"Morris","year":"2022","journal-title":"Nat. Rev. Immunol."},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Delessard, M., Saulnier, J., Rives, A., Dumont, L., Rondanino, C., and Rives, N. (2020). Exposure to Chemotherapy During Childhood or Adulthood and Consequences on Spermatogenesis and Male Fertility. Int. J. Mol. Sci., 21.","DOI":"10.3390\/ijms21041454"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"673","DOI":"10.1093\/humupd\/dmz027","article-title":"Ovarian damage from chemotherapy and current approaches to its protection","volume":"25","author":"Spears","year":"2019","journal-title":"Hum. Reprod. Update"},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Tharmalingam, M.D., Matilionyte, G., Wallace, W.H.B., Stukenborg, J.B., Jahnukainen, K., Oliver, E., Goriely, A., Lane, S., Guo, J., and Cairns, B. (2020). Cisplatin and carboplatin result in similar gonadotoxicity in immature human testis with implications for fertility preservation in childhood cancer. BMC Med., 18.","DOI":"10.1186\/s12916-020-01844-y"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1465","DOI":"10.1002\/ajh.27002","article-title":"Polycythemia vera: 2024 update on diagnosis, risk-stratification, and management","volume":"98","author":"Tefferi","year":"2023","journal-title":"Am. J. Hematol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"871","DOI":"10.2147\/DDDT.S451970","article-title":"Optimal Once-Daily Busulfan Administration in Pediatric Patients: A Simulation-Based Investigation of Intravenous Infusion Times","volume":"18","author":"Kim","year":"2024","journal-title":"Drug Des. Dev. Ther."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1723","DOI":"10.1182\/blood.V75.8.1723.1723","article-title":"Busulfan disposition in children","volume":"75","author":"Grochow","year":"1990","journal-title":"Blood"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"333","DOI":"10.1517\/17425255.2013.737319","article-title":"Pharmacology of dimethanesulfonate alkylating agents: Busulfan and treosulfan","volume":"9","author":"Galaup","year":"2013","journal-title":"Expert Opin. Drug Metab. Toxicol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"134","DOI":"10.1016\/0002-9343(61)90229-7","article-title":"Interstitial pulmonary fibrosis following busulfan therapy","volume":"31","author":"Oliner","year":"1961","journal-title":"Am. J. Med."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1348","DOI":"10.1016\/j.exphem.2003.08.014","article-title":"Ionizing radiation and busulfan inhibit murine bone marrow cell hematopoietic function via apoptosis-dependent and -independent mechanisms","volume":"31","author":"Meng","year":"2003","journal-title":"Exp. Hematol."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Dong, J., Dong, Y., Chen, H., Ye, T., Chen, G., Fan, B., Wang, X., Shi, J., and Wang, C. (2024). Ursonic acid attenuates spermatogenesis in oligozoospermia mice through inhibiting ferroptosis. Bioorg. Chem., 144.","DOI":"10.1016\/j.bioorg.2024.107174"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"19","DOI":"10.1007\/s10815-022-02674-y","article-title":"Modeling methods for busulfan-induced oligospermia and asthenozoospermia in mice: A systematic review and meta-analysis","volume":"40","author":"Pu","year":"2023","journal-title":"J. Assist. Reprod. Genet."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"184","DOI":"10.4103\/aja.aja_41_19","article-title":"Establishing a nonlethal and efficient mouse model of male gonadotoxicity by intraperitoneal busulfan injection","volume":"22","author":"Xie","year":"2020","journal-title":"Asian J. Androl."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1060","DOI":"10.1016\/j.cell.2012.03.042","article-title":"Ferroptosis: An iron-dependent form of nonapoptotic cell death","volume":"149","author":"Dixon","year":"2012","journal-title":"Cell"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"130","DOI":"10.1016\/j.freeradbiomed.2018.09.043","article-title":"The development of the concept of ferroptosis","volume":"133","author":"Hirschhorn","year":"2019","journal-title":"Free Radic. Biol. Med."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"273","DOI":"10.1016\/j.cell.2017.09.021","article-title":"Ferroptosis: A Regulated Cell Death Nexus Linking Metabolism, Redox Biology, and Disease","volume":"171","author":"Stockwell","year":"2017","journal-title":"Cell"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"88","DOI":"10.1038\/s41419-020-2298-2","article-title":"Ferroptosis: Past, present and future","volume":"11","author":"Li","year":"2020","journal-title":"Cell Death Dis."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"381","DOI":"10.1038\/s41568-022-00459-0","article-title":"Targeting ferroptosis as a vulnerability in cancer","volume":"22","author":"Lei","year":"2022","journal-title":"Nat. Rev. Cancer"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"266","DOI":"10.1038\/s41580-020-00324-8","article-title":"Ferroptosis: Mechanisms, biology and role in disease","volume":"22","author":"Jiang","year":"2021","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"231","DOI":"10.1016\/j.jare.2020.07.007","article-title":"Quercetin alleviates acute kidney injury by inhibiting ferroptosis","volume":"28","author":"Wang","year":"2021","journal-title":"J. Adv. Res."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"358","DOI":"10.1038\/s41420-023-01665-x","article-title":"Emerging roles of ferroptosis in male reproductive diseases","volume":"9","author":"Yuan","year":"2023","journal-title":"Cell Death Discov."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"152489","DOI":"10.1016\/j.tox.2020.152489","article-title":"Inhibition of ferroptosis attenuates busulfan-induced oligospermia in mice","volume":"440","author":"Zhao","year":"2020","journal-title":"Toxicology"},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Xu, J., Zhang, L., Si, Y., Huang, W., Liu, R., Liu, Z., Jiang, Z., and Xu, F. (2024). Ferritinophagy-mediated ferroptosis of spermatogonia is involved in busulfan-induced oligospermia in the mice. Chem. Biol. Interact., 390.","DOI":"10.1016\/j.cbi.2024.110870"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"882","DOI":"10.5534\/wjmh.220100","article-title":"Effect of N-acetyl-L-cysteine on Testicular Tissue in Busulfan-Induced Dysfunction in the Male Reproductive System","volume":"41","author":"Kim","year":"2023","journal-title":"World J. Men\u2019s Health"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"810","DOI":"10.5534\/wjmh.230254","article-title":"Effects of Perilla frutescens Var. Acuta in Busulfan-Induced Spermatogenesis Dysfunction Mouse Model","volume":"42","author":"Nam","year":"2024","journal-title":"World J. Men\u2019s Health"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"282","DOI":"10.1177\/0394632017726261","article-title":"Structural evaluation of the peritubular sheath of rat\u2019s testes after administration of ribavirin: A possible impact on the testicular function","volume":"30","author":"Almasry","year":"2017","journal-title":"Int. J. Immunopathol. Pharmacol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"383","DOI":"10.18502\/ijrm.v22i5.16439","article-title":"Evaluation of the sperm parameters, oxidative stress, and histopathological effects of vitamin B12 in preventing Helicobacter pylori-induced testicular toxicity: An experimental study","volume":"22","author":"Mohammadi","year":"2024","journal-title":"Int. J. Reprod. BioMed. (IJRM)"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"127807","DOI":"10.1016\/j.jhazmat.2021.127807","article-title":"Di-(2-ethylhexyl) phthalate exposure leads to ferroptosis via the HIF-1\u03b1\/HO-1 signaling pathway in mouse testes","volume":"426","author":"Wu","year":"2022","journal-title":"J. Hazard. Mater."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"153193","DOI":"10.1016\/j.tox.2022.153193","article-title":"Endoplasmic reticulum stress promotes blood-testis barrier impairment in mice with busulfan-induced oligospermia through PERK-eIF2\u03b1 signaling pathway","volume":"473","author":"Zhao","year":"2022","journal-title":"Toxicology"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"244","DOI":"10.1080\/07853890.2024.2442534","article-title":"Proteomic analysis for busulfan-induced spermatogenesis disorder","volume":"57","author":"Hu","year":"2025","journal-title":"Ann. Med."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1559s","DOI":"10.3945\/ajcn.117.155804","article-title":"Current understanding of iron homeostasis","volume":"106","author":"Anderson","year":"2017","journal-title":"Am. J. Clin. Nutr."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1007\/s00204-015-1579-5","article-title":"Redox- and non-redox-metal-induced formation of free radicals and their role in human disease","volume":"90","author":"Valko","year":"2016","journal-title":"Arch Toxicol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"726","DOI":"10.1182\/blood.2019002907","article-title":"Hepatic transferrin plays a role in systemic iron homeostasis and liver ferroptosis","volume":"136","author":"Yu","year":"2020","journal-title":"Blood"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"46","DOI":"10.1016\/j.freeradbiomed.2018.06.037","article-title":"Transferrin and transferrin receptors update","volume":"133","author":"Kawabata","year":"2019","journal-title":"Free Radic. Biol. Med."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"1021","DOI":"10.1038\/cr.2016.95","article-title":"Ferroptosis is an autophagic cell death process","volume":"26","author":"Gao","year":"2016","journal-title":"Cell Res."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"2054","DOI":"10.1080\/15548627.2020.1810918","article-title":"Ferroptosis: Machinery and regulation","volume":"17","author":"Chen","year":"2021","journal-title":"Autophagy"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"85","DOI":"10.1126\/science.aaw9872","article-title":"Cysteine depletion induces pancreatic tumor ferroptosis in mice","volume":"368","author":"Badgley","year":"2020","journal-title":"Science"},{"key":"ref_39","first-page":"100315","article-title":"Biodegradable copper-doped calcium phosphate nanoplatform enables tumor microenvironment modulations for amplified ferroptosis in cervical carcinoma treatment","volume":"9","author":"Yang","year":"2025","journal-title":"Int. J. Pharm. X"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"124","DOI":"10.1016\/j.canlet.2017.12.025","article-title":"Heme oxygenase-1 mediates BAY 11-7085 induced ferroptosis","volume":"416","author":"Chang","year":"2018","journal-title":"Cancer Lett."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"24393","DOI":"10.18632\/oncotarget.5162","article-title":"Heme oxygenase-1 accelerates erastin-induced ferroptotic cell death","volume":"6","author":"Kwon","year":"2015","journal-title":"Oncotarget"},{"key":"ref_42","doi-asserted-by":"crossref","unstructured":"Wu, A., Feng, B., Yu, J., Yan, L., Che, L., Zhuo, Y., Luo, Y., Yu, B., Wu, D., and Chen, D. (2021). Fibroblast growth factor 21 attenuates iron overload-induced liver injury and fibrosis by inhibiting ferroptosis. Redox Biol., 46.","DOI":"10.1016\/j.redox.2021.102131"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"174518","DOI":"10.1016\/j.ejphar.2021.174518","article-title":"Astragalus polysaccharide prevents ferroptosis in a murine model of experimental colitis and human Caco-2 cells via inhibiting NRF2\/HO-1 pathway","volume":"911","author":"Chen","year":"2021","journal-title":"Eur. J. Pharmacol."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"449","DOI":"10.1002\/hep.29117","article-title":"Characterization of ferroptosis in murine models of hemochromatosis","volume":"66","author":"Wang","year":"2017","journal-title":"Hepatology"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"3790","DOI":"10.1158\/1078-0432.CCR-04-2159","article-title":"Inhibition of heme oxygenase-1 increases responsiveness of pancreatic cancer cells to anticancer treatment","volume":"11","author":"Berberat","year":"2005","journal-title":"Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res."},{"key":"ref_46","doi-asserted-by":"crossref","unstructured":"Sharawy, N., Aboulhoda, B.E., Khalifa, M.M., Morcos, G.N., Morsy, S., Alghamdi, M.A., Khalifa, I.M., and Abd Algaleel, W.A. (2024). Amelioration of nephrotoxicity by targeting ferroptosis: Role of NCOA4, IREB2, and SLC7a11 signaling. Braz. J. Med. Biol. Res. = Rev. Bras. Pesqui. Medicas Biol., 57.","DOI":"10.1590\/1414-431x2024e13116"},{"key":"ref_47","doi-asserted-by":"crossref","unstructured":"Shi, H., Hou, B., Li, H., Zhou, H., and Du, B. (2022). Cyclophosphamide Induces the Ferroptosis of Tumor Cells Through Heme Oxygenase-1. Front. Pharmacol., 13.","DOI":"10.3389\/fphar.2022.839464"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"1619","DOI":"10.1016\/j.biopha.2017.08.131","article-title":"The antineoplastic busulphan impairs peritubular and Leydig cells, and vitamin B(12) stimulates spermatogonia proliferation and prevents busulphan-induced germ cell death","volume":"95","author":"Oliveira","year":"2017","journal-title":"Biomed. Pharmacother. Biomed. Pharmacother."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"47","DOI":"10.1016\/j.tiv.2017.05.023","article-title":"Increased Sat2 expression is associated with busulfan-induced testicular Sertoli cell injury","volume":"43","author":"Xian","year":"2017","journal-title":"Toxicol. Vitr. Int. J. Publ. Assoc. BIBRA"},{"key":"ref_50","doi-asserted-by":"crossref","unstructured":"Chen, P.H., Tseng, W.H., and Chi, J.T. (2020). The Intersection of DNA Damage Response and Ferroptosis-A Rationale for Combination Therapeutics. Biology, 9.","DOI":"10.3390\/biology9080187"},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"901","DOI":"10.1080\/17425255.2017.1360277","article-title":"Clarifying busulfan metabolism and drug interactions to support new therapeutic drug monitoring strategies: A comprehensive review","volume":"13","author":"Myers","year":"2017","journal-title":"Expert Opin. Drug Metab. Toxicol."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"379","DOI":"10.1038\/s41467-019-14231-w","article-title":"Competitive oxidation and ubiquitylation on the evolutionarily conserved cysteine confer tissue-specific stabilization of Insig-2","volume":"11","author":"Zhou","year":"2020","journal-title":"Nat. Commun."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"906","DOI":"10.1016\/j.tcb.2016.07.002","article-title":"Physiological and Pharmacological Control of BAK, BAX, and Beyond","volume":"26","author":"Chipuk","year":"2016","journal-title":"Trends Cell Biol."},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"e2107993118","DOI":"10.1073\/pnas.2107993118","article-title":"The N-terminal cysteine is a dual sensor of oxygen and oxidative stress","volume":"118","author":"Heo","year":"2021","journal-title":"Proc. Natl. Acad. Sci. USA"}],"container-title":["Biology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2079-7737\/14\/6\/594\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T17:39:19Z","timestamp":1760031559000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2079-7737\/14\/6\/594"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,5,23]]},"references-count":54,"journal-issue":{"issue":"6","published-online":{"date-parts":[[2025,6]]}},"alternative-id":["biology14060594"],"URL":"https:\/\/doi.org\/10.3390\/biology14060594","relation":{},"ISSN":["2079-7737"],"issn-type":[{"value":"2079-7737","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,5,23]]}}}