The synthetic cannabinoid JWH-018 induces testicular oxidative stress and alters nitric oxide synthase expression in mice

Year 2025, Volume: 42 Issue: 4, 439 - 446, 31.12.2025

Duygu Mutluay , Rüveyda Hilal Akı , Münteha Şilan Ar , Utku Tuna Cezar , Gözde Yücel Tenekeci

Abstract

Thirty adult male CD-1 mice were randomly assigned to six groups (n = 5 each): two control groups (C1, C2), two ethanol vehicle groups (E1, E2), and two JWH-018–treated groups (JWH1, JWH2). Control groups received saline, ethanol groups received 1% ethanol in saline (vehicle), and JWH groups received JWH-018 (0.3 mg/kg, i.p.) once daily for nine days. Acute effects were assessed in C1, E1, and JWH1 animals sacrificed two days post-treatment, while recovery potential was evaluated in C2, E2, and JWH2 animals sacrificed 45 days later. JWH-018 exposure caused significant reductions in seminiferous tubule diameter and CSA, with germinal epithelium thickness preserved. Morphological damage was partially reversed in the recovery group (JWH2). Immunohistochemical analysis revealed increased iNOS and eNOS expression, particularly in Leydig cells, indicating oxidative stress, while PAS staining revealed apoptotic-like germ cells in JWH groups. Our findings indicate that JWH-018 induces testicular injury through oxidative stress–mediated pathways, causing structural and cellular alterations. Partial recovery was observed after a full spermatogenic cycle; however, some changes persisted, suggesting potential long-term reproductive consequences. These results provide novel insights into the mechanisms of JWH-018–induced reproductive toxicity and underscore the importance of further investigation into potential interventions.

Keywords

JWH-018 , synthetic cannabinoids , testis , oxidative stress , iNOS , eNOS.

Ethical Statement

All animal handling and experimental protocols strictly adhered to the ethical principles outlined in the Guide for the Care and Use of Laboratory Animals and were conducted in accordance with the Animal Welfare Act. Ethical approval for this study was obtained from the Local Animal Experiments Ethics Committee of Burdur Mehmet Akif Ersoy University (Approval No: 466-2018).

Supporting Institution

This study was supported by The Scientific and Technological Research Council of Turkey (TÜBİTAK) within the scope of the 2209-A University Students Research Projects Support Program (Project No: 1919B012218136 and 1919B012222905).

Thanks

The authors would like to thank Dr. Serkan Koksoy for kindly providing JWH-018 for research purposes, which was used in the experimental procedures. Some of the data in this manuscript were previously presented as a oral presentation at the Turkish & Italian Joint International Animal Reproduction Congress, held in Antalya, Türkiye, on October 10–13, 2024.

References

• van Amsterdam J, Brunt T, van den Brink W. The adverse health effects of synthetic cannabinoids with emphasis on psychosis-like effects. J Psychopharmacol. 2015;29(3):254–263.
• Tai S, Fantegrossi WE. Pharmacological and toxicological effects of synthetic cannabinoids and their metabolites. Curr Top Behav Neurosci. 2017;32:249–262.
• Pertwee RG. The diverse CB1 and CB2 receptor pharmacology of three plant cannabinoids: delta9- tetrahydrocannabinol, cannabidiol and delta9-tetrahydrocannabivarin. Br J Pharmacol. 2008;153(2):199–215.
• De Luca MA, Castelli MP, Loi B, Porcu A, Martorelli M, Miliano C, et al. Native CB1 receptor affinity, intrinsic activity and accumbens shell dopamine stimulant properties of third generation SPICE/K2 cannabinoids: BB-22, 5F-PB-22, 5F-AKB-48 and STS-135. Neuropharmacology. 2016;105:630–638.
• El-Talatini MR, Taylor AH, Elson JC, Brown L, Davidson AC, Konje JC. Localisation and function of the endocannabinoid system in the human ovary. PLoS One. 2009;4(2):e4579.
• Brents LK, Prather PL. The K2/Spice phenomenon: emergence, identification, legislation and metabolic characterization of synthetic cannabinoids in herbal incense products. Drug Metab Rev. 2014;46(1):72–85.
• Lewis SE, Akhtar MM, Al-Azzawi F, Thompson W, Kumar S. Long-term use of HU210 adversely affects spermatogenesis in rats by modulating the endocannabinoid system. Int J Androl. 2012;35(5):731–740.
• Seely KA, Lapoint J, Moran JH, Fattore L. Spice drugs are more than harmless herbal blends: a review of the pharmacology and toxicology of synthetic cannabinoids. Prog Neuropsychopharmacol Biol Psychiatry. 2012;39(2):234–243.
• Mutluay D, Güngör Ş, Tenekeci GY, Köksoy S, Çoban CS. Effects of synthetic (JWH-018) cannabinoids treatment on spermatogenesis and sperm function. Drug Chem Toxicol. 2022;45(1):215–222. doi: 10.1080/01480545.2019.1680686.
• Boivin J, Bunting L, Collins JA, Nygren KG. International estimates of infertility prevalence and treatment-seeking: potential need and demand for infertility medical care. Hum Reprod. 2007;22(6):1506–1512. doi: 10.1093/humrep/dem046.
• Kumar N, Singh AK. Trends of male factor infertility, an important cause of infertility: a review of literature. J Hum Reprod Sci. 2015;8(4):191–196.
• Dutta S, Henkel R, Sengupta P, Agarwal A. Physiological role of ROS in sperm function. In: Agarwal A, du Plessis SS, editors. Male infertility. Berlin, Heidelberg: Springer; 2020. p. 337–345.
• Agarwal A, Leisegang K, Sengupta P. Oxidative stress in pathologies of male reproductive disorders. In: Elsevier, editor. Pathology. Amsterdam: Elsevier; 2020. p. 15–27.
• Ayad B, Omolaoye TS, Louw N, Ramsunder Y, Skosana BT, Oyeipo PI, et al. Oxidative stress and male infertility: evidence from a research perspective. Front Reprod Health. 2022;4:822257. doi: 10.3389/frph.2022.822257
• Dutta S, Sengupta P, Slama P, Roychoudhury S. Oxidative stress, testicular inflammatory pathways, and male reproduction. Int J Mol Sci. 2021;22:10043. doi: 10.3390/ijms221810043
• Agarwal A, Rana M, Qiu E, AlBunni H, Bui AD, Henkel R. Role of oxidative stress, infection and inflammation in male infertility. Andrologia. 2018;50:e13126. doi: 10.1111/and.13126
• Köksal IT, Erdoǧru T, Gülkesen H, Sezer C, Usta M, Çiftçioǧlu A, et al. The potential role of inducible nitric oxide synthase (iNOS) activity in the testicular dysfunction associated with varicocele: an experimental study. Int Urol Nephrol. 2003; 36:67–72. doi: 10.1023/B:UROL.0000032687.58462.4f
• Eid N, Ito Y, Otsuki Y. Involvement of inducible nitric oxide synthase in DNA fragmentation in various testicular germ cells of ethanol-treated rats. J Mens Health. 2011;8 Suppl 1:S36–S40. doi: 10.1016/S1875-6867(11)60018-3
• Doshi SB, Khullar K, Sharma RK, Agarwal A. Role of reactive nitrogen species in male infertility. Reprod Biol Endocrinol. 2012;10:109. doi: 10.1186/1477-7827-10-109
• Ogedengbe OO, Naidu ECS, Akang EN, Offor U, Onanuga IO, Peter AI, et al. Virgin coconut oil extract mitigates testicular-induced toxicity of alcohol use in antiretroviral therapy. Andrology. 2018;6:616–626. doi: 10.1111/andr.12490
• Owembabazi E, Nkomozepi P, Mbajiorgu EF. Impact of concurrent exposure of diabetic male Sprague Dawley rats to alcohol and combination antiretroviral therapy (cART) on reproductive capacity. Appl Sci. 2023;13:5096. doi: 10.3390/app13085096
• Cho YM, Mizuta Y, Akagi JI, Toyoda T, Sone M, Ogawa K. Size-dependent acute toxicity of silver nanoparticles in mice. J Toxicol Pathol. 2018;31(1):73–80. doi:10.1293/tox.2017-0043.
• Yaghini E, Turner H, Pilling A, Naasani I, MacRobert AJ. In vivo biodistribution and toxicology studies of cadmium- free indium-based quantum dot nanoparticles in a rat model. Nanomedicine. 2018;14(8):2644–2655. doi:10.1016/j.nano.2018.07.009.
• Macrì S, Ceci C, Altabella L, Canese R, Laviola G. The Directive 2010/63/EU on animal experimentation may skew the conclusions of pharmacological and behavioural studies. Sci Rep. 2013;3:2380. doi: 10.1038/srep02380
• Ossato A, Vigolo A, Trapella C, Seri C, Rimondo C, Serpelloni G, et al. JWH-018 impairs sensorimotor functions in mice. Neuroscience. 2015;300:174–188. doi: 10.1016/j.neuroscience.2015.05.021
• Griswold MD. Spermatogenesis: the commitment to meiosis. Physiol Rev. 2016;96(1):1–17.
• Clermont Y. Kinetics of spermatogenesis in mammals. Physiol Rev. 1972;52(1):198–204.
• Oakberg EF. Duration of spermatogenesis in the mouse and timing of stages of the cycle of the seminiferous epithelium. Am J Anat. 1956;99(3):507–516.
• Kazemi S, Feizi F, Aghapour F, Joorsaraee GA, Moghadamnia AA. Histopathology and histomorphometric investigation of bisphenol A and nonylphenol on the male rat reproductive system. N Am J Med Sci. 2016;8(5):215. doi: 10.4103/1947-2714.183012
• Yalçın T, Kaya S, Tektemur NK, Ozan İE. The methods used in histopathological evaluation of testis tissues. Batman Univ J Life Sci. 2020;10(1):148–157.
• Martinotti G, Santacroce R, Pettorruso M, Montemitro C, Spano MC, Lorusso M, et al. Synthetic cannabinoids: psychopharmacology, clinical aspects, psychotic onset. CNS Neurol Disord Drug Targets. 2017;16(5):567–575.
• European Monitoring Centre for Drugs and Drug Addiction (EMCDDA). European Drug Report 2015: Trends and Developments. Lisbon (Portugal): EMCDDA; 2015.
• du Plessis SS, Agarwal A, Syriac A. Marijuana, phytocannabinoids, the endocannabinoid system, and male fertility. J Assist Reprod Genet. 2015;32(11):1575–1588.
• Schmidt HH, Walter U. NO at work. Cell. 1994;78(6):919–925.
• Carney ST, Lloyd ML, MacKinnon SE, Newton DC, Jones JD, Howlett AC, et al. Cannabinoid regulation of nitric oxide synthase I (nNOS) in neuronal cells. J Neuroimmune Pharmacol. 2009;4:338–349. doi: 10.1007/s11481-009-9153-7
• Lue Y, Sinha Hikim AP, Wang C, Leung A, Swerdloff RS. Functional role of inducible nitric oxide synthase in the induction of male germ cell apoptosis, regulation of sperm number, and determination of testes size: evidence from null mutant mice. Endocrinology. 2003;144(7):3092–3100. doi: 10.1210/en.2002-0142
• Sönmez MF, Narin F, Akkuş D, Türkmen AB. Melatonin and vitamin C ameliorate alcohol-induced oxidative stress and eNOS expression in rat kidney. Ren Fail. 2012;34(4):480–486. doi: 10.3109/0886022X.2011.649678
• Greco A, Del Prete C, De Biase D, Palumbo V, Albanese S, Bruzzese D, et al. Effects of oral administration of Lepidium meyenii on morphology of mice testis and motility of epididymal sperm cells after tetrahydrocannabinol exposure. Front Vet Sci. 2021;8:692874. doi: 10.3389/fvets.2021.692874
• Lotfi N, Khazaei M, Shariatzadeh SMA, Mehranjani MS, Ghanbari A. The effect of Cannabis sativa hydroalcoholic extract on sperm parameters and testis histology in rats. Int J Morphol. 2013;31(1):82–86.
• Vidal JD, Whitney KM. Morphologic manifestations of testicular and epididymal toxicity. Spermatogenesis. 2014;4(2):e979099. doi: 10.4161/21565562.2014.979099
• Sezer C, Koksal IT, Usta MF, Gulkesen KH, Erdogru T, Ciftcioglu A, et al. Relationship between mast cell and iNOS expression in testicular tissue associated with infertility. Arch Androl. 2005;51(2):149–158. doi: 10.1080/014850190518161
• Moreira RJ, Oliveira PF, Spadella MA, Ferreira R, Alves MG. Do lifestyle interventions mitigate the oxidative damage and inflammation induced by obesity in the testis? Antioxidants (Basel). 2025;14(2):150. doi: 10.3390/antiox14020150
• Adams ML, Nock B, Truong R, Cicero TJ. Nitric oxide control of steroidogenesis: endocrine effects of NG-nitro-L- arginine and comparisons to alcohol. Life Sci. 1992;50(6):PL35–40.
• Zini A, Abitbol J, Girardi SK, Schulsinger D, Goldstein M, Schlegel PN. Germ cell apoptosis and endothelial nitric oxide synthase (eNOS) expression following ischemia-reperfusion injury to testis. Arch Androl. 1998;41(1):57–65. doi: 10.3109/01485019808988547
• Han KH, Lim S, Ryu J, Lee CW, Kim Y, Kang JH, et al. CB1 and CB2 cannabinoid receptors differentially regulate the production of reactive oxygen species by macrophages. Cardiovasc Res. 2009;84(3):378–386. doi: 10.1093/cvr/cvp240
• Koller VJ, Zlabinger GJ, Auwärter V, et al. Toxicological profiles of selected synthetic cannabinoids showing high binding affinities to the cannabinoid receptor subtype CB1. Arch Toxicol. 2013;87:1287–1297. doi: 10.1007/s00204-013-1029-1
• Alzu’bi A, Almahasneh F, Khasawneh R, Abu-El-Rub E, Bani Baker W, Al-Zoubi RM. The synthetic cannabinoids menace: a review of health risks and toxicity. Eur J Med Res. 2024;29:49. doi:10.1186/s40001-023-01443-6.