Can antioxidants protect against chemotherapy in a rat spermatogonial stem cell line?

Allen, C. M. , Lopes, F., Mitchell, R. T. and Spears, N. (2021) Can antioxidants protect against chemotherapy in a rat spermatogonial stem cell line? Reproduction and Fertility, 2(4), L7-L9. (doi: 10.1530/raf-21-0042) (PMID:35118413) (PMCID:PMC8788573)

Abstract

Lay summary: Boys administered chemotherapy to treat cancer are at risk of damage to their healthy testicular tissue, which can lead to infertility in adulthood. Researchers are therefore investigating treatments to protect the testis during cancer treatment. Here, cells originating from rat testicles were cultured for 4 days and exposed to chemotherapy drugs with or without antioxidants for the final 2 days. Antioxidants can reduce cellular damage by inactivating toxic compounds. Here, antioxidants such as melatonin or n-acetylcysteine were tested against chemotherapy agents cisplatin, doxorubicin, or vincristine. Cultures were repeated four times, with cell survival measured at the end of culture. The antioxidants were not damaging and partially protected against cisplatin, although not doxorubicin. Surprisingly, n-acetylcysteine enhanced vincristine-induced damage. The results suggest that using antioxidants to protect the testis could have either beneficial or harmful effects when given alongside different chemotherapy drugs: this is important, considering that patients are often treated with multiple drugs. Children undergoing chemotherapy to treat childhood cancers are at risk of infertility in adulthood due to drug cytotoxicity (Allen et al. 2018). The development of fertility preservation strategies is therefore vital, particularly for prepubertal boys for whom there is a lack of clinically available options (Goossens et al. 2020). Cytoprotective agents included in cancer treatment regimens could act to preserve fertility by protecting the reproductive tissues against chemotherapy-induced damage. Some studies have reported a role for reactive oxygen species (ROS) in chemotherapy-induced testicular toxicity (Allen et al. 2018). The potential for antioxidants to protect the testis has been investigated in a number of clinical and animal model studies; however, its use is controversial due to the lack of evidence of effectiveness (Yasueda et al. 2016). Here, the ability of antioxidants such as melatonin or n-acetylcysteine (NAC) to protect against chemotherapy-induced cytotoxicity was investigated in rat GC-6spg cells, which represent spermatogonial stem cells (SSCs), based on the expression of expected markers including promyelocytic leukemia zinc finger (PLZF) (van Pelt et al. 2002, Carlomagno et al. 2010) (Fig. 1A). While this immortalised cell line displays some differences in morphology and/or physiology compared to SSCs in vivo, these cells are still capable of migration into the SSC niche upon injection into testes, providing further evidence of SSC characteristics (van Pelt et al. 2002). Chemotherapy agents investigated include cisplatin, doxorubicin, and vincristine, all commonly used to treat childhood cancers. To date, ROS production has been linked to cisplatin and doxorubicin-induced toxicity but not in relation to vincristine (Conklin 2004). The cell line had been stabilised at passage 53 and was cultured here at passage 62 as described (van Pelt et al. 2002), at density 50,000 cells/cm2 until 80% confluent (48 h). Cells were exposed to either serial dilutions of antioxidant, cytotoxic chemotherapy concentration, co-treatment or vehicle for a further 48 h. Cytotoxicity was determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay to measure cell viability based on metabolic activity; cells were exposed to 0.5 mg/mL of MTT for 3 h, formazan product solubilised in 1:1 isopropanol:DMSO and solution absorbance analysed at 560 nm. Absorbance of treated cells was compared to control to give percentage of cell viability per control, with background accounted for blank wells.

• Item Type: Articles
• Additional Information: This work was funded by Career Development PhD Scholarship and Children with Cancer UK grant #15-198. R T M’s is supported by a UKRI Future Leaders Fellowship (MR/S017151/1) in the MRC Centre for Reproductive Health funded by MRC Centre Grant MR/N022556/1. C M A was supported by Career Development PhD Scholarship in Biomedical Sciences funded by the Biomedical Sciences ZJU project.
• Keywords: Antioxidants, chemotherapy, fertility preservation, spermatogonial stem cells.
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Allen, Dr Caroline
• Authors: Allen, C. M., Lopes, F., Mitchell, R. T., and Spears, N.
• College/School: College of Medical Veterinary and Life Sciences > School of Medicine, Dentistry & Nursing
• Journal Name: Reproduction and Fertility
• Publisher: Bioscientifica
• ISSN: 2633-8386
• ISSN (Online): 2633-8386
• Published Online: 16 October 2021
• Copyright Holders: Copyright © The authors 2021
• First Published: First published in Reproduction and Fertility 2(4):L7-L9
• Publisher Policy: Reproduced under a Creative Commons license