Abstract
Infertility is a frequent long-term adverse effect of cancer therapy for children. Compromised testicular functions in adolescence are frequent observations after chemotherapy and there are currently no well-established alternatives to avoid this damage. Antimetabolites such as 6-mercaptopurine (6-MP) are used to treat a variety of cancer; however, its treatment-associated adverse effects on the male reproductive functions are overlooked. Here, the molecular processes underlying 6-MP-induced male germ cell damage in juvenile Sprague–Dawley (SD) rats (3 weeks) have been investigated. Rats were administered with low (5 mg/kg), medium (10 mg/kg), and high (20 mg/kg) doses of 6-MP per orally either singly (1 week × 1 cycle) or intermittently (1 week treatment followed by 1 week remission period × 3 cycles). The toxicity was evaluated in terms of genotoxicity and testes- and sperm-related cellular and molecular parameters. Single cycle of exposure either produced mild or no toxic manifestations at the end of the 6th week. Intermittent cycles of exposure, particularly at the 10 and 20 mg/kg, decreased body and organ weights, increased micronucleated cells in the peripheral blood, induced oxidative/nitrosative stress, altered sperm chromatin quality, reduced serum testosterone and follicle stimulating hormone (FSH) levels, increased testicular structural aberrations, DNA damage, and apoptosis, and upregulated TNF-α expression and downregulated p-AMPK and β-catenin expressions. Conclusively, intermittent cycles of exposure at 10 and 20 mg/kg doses to the juvenile rats significantly induced oxidative stress, genotoxicity, and cellular and molecular perturbations in the testes and sperm of adult rats.
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References
Abdelzaher WY, Mostafa-Hedeab G, Sayed AboBakr Ali AH, Fawzy MA, Ahmed AF, Bahaa El-Deen MA, Welson NN, Aly Labib DA (2022) Idebenone regulates sirt1/Nrf2/TNF-α pathway with inhibition of oxidative stress, inflammation, and apoptosis in testicular torsion/detorsion in juvenile rats. Hum Exp Toxicol 41:9603271221102516. https://doi.org/10.1177/09603271221102515
Absalan F, Ghannadi A, Kazerooni M, Parifar R, Jamalzadeh F, Amiri S (2012) Value of sperm chromatin dispersion test in couples with unexplained recurrent abortion. J Assist Reprod Genet 29:11–14. https://doi.org/10.1007/s10815-011-9647-0
Aitken RJ, De Iuliis GN (2007) Origins and consequences of DNA damage in male germ cells. Reprod Biomed Online 14:727–733. https://doi.org/10.1016/s1472-6483(10)60676-1
Arndt CA, Balis FM, McCully CL, Jeffries SL, Doherty K, Murphy R, Poplack DG (1988) Bioavailability of low-dose vs high-dose 6-mercaptopurine. Clin Pharmacol Ther 43:588–591. https://doi.org/10.1038/clpt.1988.78
Beaud H, Albert O, Robaire B, Rousseau MC, Chan PTK, Delbes G (2019) Sperm DNA integrity in adult survivors of paediatric leukemia and lymphoma: a pilot study on the impact of age and type of treatment. PLoS ONE 14:e0226262. https://doi.org/10.1371/journal.pone.0226262
Bertoldo MJ, Faure M, Dupont J, Froment P (2015) AMPK: a master energy regulator for gonadal function. Front Neurosci 9:235. https://doi.org/10.3389/fnins.2015.00235
Brandalise SR, Pinheiro VR, Aguiar SS, Matsuda EI, Otubo R, Yunes JA, Pereira WV, Carvalho EG, Cristofani LM, Souza MS, Lee ML, Dobbin JA, Pombo-de-Oliveira MS, Lopes LF, Melnikoff KN, Brunetto AL, Tone LG, Scrideli CA, Morais VL, Viana MB (2010) Benefits of the intermittent use of 6-mercaptopurine and methotrexate in maintenance treatment for low-risk acute lymphoblastic leukemia in children: randomized trial from the Brazilian Childhood Cooperative Group–protocol ALL-99. J Clin Oncol 28:1911–1918. https://doi.org/10.1200/jco.2009.25.6115
Burruel VR, Yanagimachi R, Whitten WK (1996) Normal mice develop from oocytes injected with spermatozoa with grossly misshapen heads. Biol Reprod 55:709–714. https://doi.org/10.1095/biolreprod55.3.709
Delessard M, Saulnier J, Rives A, Dumont L, Rondanino C, Rives N (2020) Exposure to chemotherapy during childhood or adulthood and consequences on spermatogenesis and male fertility. Int J Mol Sci 21:1454–1477. https://doi.org/10.3390/ijms21041454
Dutta S, Sengupta P, Slama P, Roychoudhury S (2021) Oxidative stress, testicular inflammatory pathways, and male reproduction. Int J Mol Sci 22:10043–10063. https://doi.org/10.3390/ijms221810043
Fernández-Ramos AA, Marchetti-Laurent C, Poindessous V, Antonio S, Laurent-Puig P, Bortoli S, Loriot MA, Pallet N (2017) 6-mercaptopurine promotes energetic failure in proliferating T cells. Oncotarget 8:43048–43060. https://doi.org/10.18632/oncotarget.17889
Generoso WM, Preston RJ, Brewen JG (1975a) 6-mercaptopurine, an inducer of cytogenetic and dominant-lethal effects in premeiotic and early meiotic germ cells of male mice. Mutat Res 28:437–447. https://doi.org/10.1016/0027-5107(75)90237-7
Generoso WM, Preston RJ, Brewen JG (1975b) 6-Mercaptopurine, an inducer of cytogenetic and dominant-lethal effects in premeiotic and early meiotic germ cells of male mice. Mutat Res/Fundam Mol Mech Mutagen 28:437–447. https://doi.org/10.1016/0027-5107(75)90237-7
Giustarini D, Rossi R, Milzani A, Dalle-Donne I (2008) Nitrite and nitrate measurement by Griess reagent in human plasma: evaluation of interferences and standardization. Methods Enzymol 440:361–380. https://doi.org/10.1016/s0076-6879(07)00823-3
Grataroli R, Vindrieux D, Gougeon A, Benahmed M (2002) Expression of tumor necrosis factor-alpha-related apoptosis-inducing ligand and its receptors in rat testis during development. Biol Reprod 66:1707–1715. https://doi.org/10.1095/biolreprod66.6.1707
Grosen A, Nersting J, Bungum M, Christensen LA, Schmiegelow K, Spanò M, Julsgaard M, Cordelli E, Leter G, Larsen PB, Hvas CL, Kelsen J (2019) Sperm DNA integrity is unaffected by thiopurine treatment in men with inflammatory bowel disease. J Crohns Colitis 13:3–11. https://doi.org/10.1093/ecco-jcc/jjy086
Habas K, Anderson D, Brinkworth M (2016) Detection of phase specificity of in vivo germ cell mutagens in an in vitro germ cell system. Toxicology 353–354:1–10. https://doi.org/10.1016/j.tox.2016.04.001
Hashem K, Abdelazem A, Abdelbaky N (2020) Royal Jelly ameliorates 6-mercaptopurine induced spermatogenesis impairment and testicular apoptosis by regulating PI3K/AKT pathway in male rats.
Hissin PJ, Hilf R (1976) A fluorometric method for determination of oxidized and reduced glutathione in tissues. Anal Biochem 74:214–226. https://doi.org/10.1016/0003-2697(76)90326-2
Inamochi H, Higashigawa M, Shimono Y, Nagata T, Cao DC, Mao XY, M’Soka T, Hori H, Kawasaki H, Sakurai M (1999) Delayed cytotoxicity of 6-mercaptopurine is compatible with mitotic death caused by DNA damage due to incorporation of 6-thioguanine into DNA as 6-thioguanine nucleotide. J Exp Clin Cancer Res 18:417–424
Khan S, Ahmad T, Parekh CV, Trivedi PP, Kushwaha S, Jena G (2011) Investigation on sodium valproate induced germ cell damage, oxidative stress and genotoxicity in male Swiss mice. Reprod Toxicol 32:385–394. https://doi.org/10.1016/j.reprotox.2011.09.007
Kushwaha S, Tripathi DN, Vikram A, Ramarao P, Jena GB (2010) Evaluation of multi-organ DNA damage by comet assay from 28 days repeated dose oral toxicity test in mice: a practical approach for test integration in regulatory toxicity testing. Regul Toxicol Pharmacol 58:145–154. https://doi.org/10.1016/j.yrtph.2010.05.004
Ligumsky M, Badaan S, Lewis H, Meirow D (2005) Effects of 6-mercaptopurine treatment on sperm production and reproductive performance: a study in male mice. Scand J Gastroenterol 40:444–449. https://doi.org/10.1080/00365520510011597
Meistrich ML (2013) Effects of chemotherapy and radiotherapy on spermatogenesis in humans. Fertil Steril 100:1180–1186. https://doi.org/10.1016/j.fertnstert.2013.08.010
Morgan JA, Lynch J, Panetta JC, Wang Y, Frase S, Bao J, Zheng J, Opferman JT, Janke L, Green DM, Chemaitilly W, Schuetz JD (2015) Apoptosome activation, an important molecular instigator in 6-mercaptopurine induced Leydig cell death. Sci Rep 5:16488. https://doi.org/10.1038/srep16488
Mruk DD, Cheng CY (2004) Cell-cell interactions at the ectoplasmic specialization in the testis. Trends Endocrinol Metab 15:439–447. https://doi.org/10.1016/j.tem.2004.09.009
Nna VU, Ujah GA, Suleiman JB, Mohamed M, Nwokocha C, Akpan TJ, Ekuma HC, Fubara VV, Kekung-Asu CB, Osim EE (2020) Tert-butylhydroquinone preserve testicular steroidogenesis and spermatogenesis in cisplatin-intoxicated rats by targeting oxidative stress, inflammation and apoptosis. Toxicology 441:152528. https://doi.org/10.1016/j.tox.2020.152528
Ohkawa H, Ohishi N, Yagi K (1979) Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 95:351–358. https://doi.org/10.1016/0003-2697(79)90738-3
Panghal A, Sahu C, Singla S, Jena G (2022) Juvenile exposure and adult risk assessment with single versus repeated exposure of melphalan in the germ cells of male SD rat: deciphering the molecular mechanisms. Reprod Toxicol 113:71–84. https://doi.org/10.1016/j.reprotox.2022.08.006
Panghal A, Kumar V, Jena G (2023) Melphalan induced germ cell toxicity and dose-dependent effects of β-aminoisobutyric acid in experimental rat model: role of oxidative stress, inflammation and apoptosis. J Biochem Mol Toxicol 37:e23374. https://doi.org/10.1002/jbt.23374
Prüfer J, Schuchardt M, Tölle M, Prüfer N, Höhne M, Zidek W, van der Giet M (2014) Harmful effects of the azathioprine metabolite 6-mercaptopurine in vascular cells: induction of mineralization. PLoS ONE 9:e101709. https://doi.org/10.1371/journal.pone.0101709
Sabeti P, Pourmasumi S, Rahiminia T, Akyash F, Talebi AR (2016) Etiologies of sperm oxidative stress. Int J Reprod Biomed 14:231–240
Sahasranaman S, Howard D, Roy S (2008) Clinical pharmacology and pharmacogenetics of thiopurines. Eur J Clin Pharmacol 64:753–767. https://doi.org/10.1007/s00228-008-0478-6
Sahu C, Dwivedi DK, Jena GB (2020) Zinc and selenium combination treatment protected diabetes-induced testicular and epididymal damage in rat. Hum Exp Toxicol 39:1235–1256. https://doi.org/10.1177/0960327120914963
Shekh K, Khan S, Jena G, Kansara BR, Kushwaha S (2014) 3-Aminobenzamide–a PARP inhibitor enhances the sensitivity of peripheral blood micronucleus and comet assays in mice. Toxicol Mech Methods 24:332–341. https://doi.org/10.3109/15376516.2014.898355
Shpigun LK, Andryukhina EY (2019) A new electrochemical sensor for direct detection of purine antimetabolites and DNA degradation. J Anal Methods Chem 2019:1572526. https://doi.org/10.1155/2019/1572526
Wasfey N, Abdelazem A, Hashem K (2020) Thymoquinone attenuates 6-mercaptopurine induced testicular toxicity in albino rats: possible mechanisms are involved. Adv Anim Vet Sci 8:653–660. https://doi.org/10.17582/journal.aavs/2020/8.6.653.660
Acknowledgements
We would like to acknowledge the financial assistance received from the National Institute of Pharmaceutical Education and Research (NIPER), Mohali, Punjab, India, for carrying out the above experiments.
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The work was supported by funding from the National Institute of Pharmaceutical Education and Research (NIPER), Mohali, India (Grant number NPLC-GBJ-(2022–2023)).
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AP conceived, conducted the experiments, analyzed the data, and wrote the manuscript. GBJ conceived the idea, reviewed the manuscript, and administered the project. Finally, the authors have read and approved the manuscript for publication. The authors declare that all data were generated in-house and that no paper mill was used.
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Panghal, A., Jena, G. Single versus intermittent cycle exposure effect of 6-mercaptopurine in juvenile Sprague–Dawley rat: a germ cell-specific mechanistic study. Naunyn-Schmiedeberg's Arch Pharmacol 397, 3155–3168 (2024). https://doi.org/10.1007/s00210-023-02797-8
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DOI: https://doi.org/10.1007/s00210-023-02797-8