The abuse of flunitrazepam, tramadol and levonorgestrel among adolescents is a major problem, with adverse effects on the immune system. Neutrophils serve as the host primary defense and employ the role of an essential enzyme, myeloperoxidase. This study investigated the in vitro effect of flunitrazepam, tramadol and levonorgestrel on the activity of the neutrophil. Neutrophils were isolated from blood samples of volunteers and seeded in culture plates, and then treated with flunitrazepam, tramadol and levonorgestrel. Trypan blue exclusion assay was used to assess the cytotoxic effect of flunitrazepam, (15, 150, 1500, 15,000, 150,000 ng/mL), tramadol (300, 3000, 30,000 µg/ml) and levonorgestrel (18.5, 185 and 1850 ng/mL) on the neutrophils, and the effect flunitrazepam (150,000 ng/mL), tramadol (30,000 µg/ml) and levonorgestrel (1850 ng/mL) on myeloperoxidase activity was assessed. The viability of neutrophils treated with flunitrazepam, (15,000, 150,000 ng/mL), tramadol (300, 3000, 30,000 µg/ml) and levonorgestrel (18.5, 185 and 1850 ng/mL) decreased significantly (p < 0.05) compared with control. Myeloperoxidase activity in neutrophils treated with flunitrazepam (150,000 ng/mL), tramadol (30,000 µg/ml) and levonorgestrel (1850 ng/mL) decreased significantly (p < 0.05) compared with background and positive control. This study revealed that flunitrazepam, tramadol and levonorgestrel altered the activity of myeloperoxidase and phagocytic activities of the neutrophil.

1. Nnorom-Dike O. Long Term Immunologic Consequences of Illicit Drug Abuse. Journal of Alcoholism, Drug Abuse and Substance Dependence. 2020, 7(1): 1-7. doi: 10.24966/adsd-9594/100022

2. Padgett DA, Glaser R. How stress influences the immune response. Trends in Immunology. 2003, 24(8): 444-448. doi: 10.1016/s1471-4906(03)00173-x

3. Assis MA, Hansen C, Lux-Lantos V, et al. Sensitization to amphetamine occurs simultaneously at immune level and in met-enkephalin of the nucleus accumbens and spleen: An involved NMDA glutamatergic mechanism. Brain, Behavior, and Immunity. 2009, 23(4): 464-473. doi: 10.1016/j.bbi.2009.01.003

4. Halaris A. Inflammation and depression but where does the inflammation come from? Current Opinion in Psychiatry. 2019, 32(5): 422-428. doi: 10.1097/yco.0000000000000531

5. Nathan C. Neutrophils and immunity: challenges and opportunities. Nature Reviews Immunology. 2006, 6(3): 173-182. doi: 10.1038/nri1785

6. El-Gammasy TMA, Abushady NM, Hamza MT, Shaker R. Increased myeloperoxidase activity as an indicator of neutrophil-induced inflammation and sepsis in neonateS. Egypt J Pediatr Allergy Immunol, 2015. 13(1): 15-20.

7. Senthilmohan R, Kettle AJ. Bromination and chlorination reactions of myeloperoxidase at physiological concentrations of bromide and chloride. Archives of Biochemistry and Biophysics. 2006, 445(2): 235-244. doi: 10.1016/j.abb.2005.07.005

8. Beilin B, Grinevich G, Yardeni I. Tramadol does not impair the phagocytic capacity of human peripheral blood cells. Canadian Journal of Anesthesia. 2005, 52(10): 1035-1039. doi: 10.1007/bf03021601

9. Schwartz RH, Milteer R, LeBeau MA. Drug-Facilitated Sexual Assault (‘Date Rape’). Southern Medical Journal. 2000, 93(6): 558-561. doi: 10.1097/00007611-200093060-00002

10. Ohshima T. A case of drug-facilitated sexual assault by the use of flunitrazepam. Journal of Clinical Forensic Medicine. 2006, 13(1): 44-45. doi: 10.1016/j.jcfm.2005.05.006

11. Anderson L. Rohypnol. Drugs.com. Available online: https: // www. drugs.com /illicit/ rohypnol. html (accessed on 7 December 2023).

12. Bramness JG, Skurtveit S, Mørland J. Flunitrazepam: Psychomotor impairment, agitation and paradoxical reactions. Forensic Science International. 2006, 159(2-3): 83-91. doi: 10.1016/j.forsciint.2005.06.009

13. Almeida IV de, Domingues G, Soares LC, et al. Evaluation of cytotoxicity and mutagenicity of the benzodiazepine flunitrazepam in vitro and in vivo. Brazilian Journal of Pharmaceutical Sciences. 2014, 50(2): 251-256. doi: 10.1590/s1984-82502014000200003

14. Dumbili EW. Drug-related harms among young adults in Nigeria: Implications for intervention. Journal of Human Behavior in the Social Environment. 2020, 30(8): 1013-1029. doi: 10.1080/10911359.2020.1790462

15. Smith KM, Larive LL, Romanelli F. Club drugs: methylenedioxymethamphetamine, flunitrazepam, ketamine hydrochloride, and gammahydroxybutyrate. Am J Health Syst Pharm, 2006, 59: 106-776.

16. Mihic SJ, Harris RA. Hypnotics, and sedatives. In: Brunton LL, Chabner BA, Knollman BC (editors). Goodman & Gilman’s: the pharmacological basic of therapeutics. McGraw-Hill Co. pp. 45780. 2011.

17. Miotto K, Cho AK, Khalil MA, et al. Trends in Tramadol: Pharmacology, Metabolism, and Misuse. Anesthesia & Analgesia. 2017, 124(1): 44-51. doi: 10.1213/ane.0000000000001683

18. Ezenwa M, Orjiakor T, Ukwuma M, et al. Tracking opiate routes in Nigeria: Identifying trafficking routes via dealers and users of tramadol and codeine. Bulletin on Narcotics, 2020. doi: 10.17863/CAM.50293

19. Randall C, Crane J. Tramadol deaths in Northern Ireland: A review of cases from 1996 to 2012. Journal of Forensic and Legal Medicine. 2014, 23: 32-36. doi: 10.1016/j.jflm.2014.01.006

20. Envaladu EA, Anke V, Zwanikken P, Zoakah AI. Sexual and reproductive health challenges of adolescent males and females in some communities of Plateau state, Nigeria. IJPBS, 2017, 7(2): 55-60. doi: 10.5923/j.ijpbs.20170702.02

21. Oshodi YA, Akinlusi FM, Uduosoro AA, et al. Sexuality and Emergency Contraceptive Practice among Female Undergraduates in Lagos, Nigeria. Open Journal of Obstetrics and Gynecology. 2020, 10(06): 836-854. doi: 10.4236/ojog.2020.1060078

22. Aziken ME, Okonta PI, Ande ABA. Knowledge and Perception of Emergency Contraception among Female Nigerian Undergraduates. International Family Planning Perspectives. 2003, 29(2): 84. doi: 10.2307/3181062

23. Cleland K, Raymond EG, Westley E, Trussell J. Emergency contraception review: evidence-based recommendations for clinicians. Clinical Obstetrics & Gynecology. 2014, 57(4): 741-750. doi: 10.1097/grf.0000000000000056

24. Raymond EG, Halpern V, Lopez LM. Pericoital Oral Contraception With Levonorgestrel. Obstetrics & Gynecology. 2011, 117(3): 673-681. doi: 10.1097/aog.0b013e318209dc25

25. Liambila W, RamaRao S, Maina J, et al. Mainstreaming Emergency Contraception in Developing Countries: A Toolkit for Policymakers and Service Providers. Population Council, 2013. doi: 10.31899/rh10.1027

26. Adigun MS, Fasogbon SA, Ajileye AB, Arije BS. Histotoxic effects of the drug postinor®-2 on the liver of female wistar rats. EJPBS, 2016, 3(12): 528-530.

27. Sacerdote P, Manfredi B, Mantegazza P, et al. Antinociceptive and immunosuppressive effects of opiate drugs: a structure‐related activity study. British Journal of Pharmacology. 1997, 121(4): 834-840. doi: 10.1038/sj.bjp.0701138

28. Liu Z, Gao F, Tian Y. Effects of morphine, fentanyl and tramadol on human immune response. Journal of Huazhong University of Science and Technology. 2006, 26(4): 478-481. doi: 10.1007/s11596-006-0427-5

29. Hamad A, Torad F, Thabet N, et al. Effect of Tramadol Versus Fentanyl on Some Hematological and Serum Biochemical Parameters in Dogs. Alexandria Journal of Veterinary Sciences. 2016, 50(1): 122. doi: 10.5455/ajvs.231096

30. Giraldo E, Hinchado MD, Garcia JJ, et al. Influence of gender and oral contraceptives intake on innate and inflammatory response. Role of neuroendocrine factors. Molecular and Cellular Biochemistry. 2008, 313(1-2): 147-153. doi: 10.1007/s11010-008-9752-2

31. Strober W. Trypan Blue Exclusion Test of Cell Viability. Current Protocols in Immunology. 2015, 111(1). doi: 10.1002/0471142735.ima03bs111

32. Agoro ES, Wankasi MM, Zebedee UL, Agoro EY. The Effects of Chronic Carbon Monoxide Intoxication on Some Haematological Parameters and Films in Rabbits. Adv J Toxicol Curr Res., 2019, 3(1): 001-005.

33. Krumholz W, Endrass J, Hempelmann G. Inhibition of phagocytosis and killing of bacteria by anaesthetic agents in vitro. British Journal of Anaesthesia. 1995, 75(1): 66-70. doi: 10.1093/bja/75.1.66

34. Zebedee LU, Jeremiah ZA, Jonathan N, et al. Chronic and Acute Effect of Tramadol Intoxication on Some Immunological Parameters. American Journal of Immunology. 2021, 17(1): 1-13. doi: 10.3844/ajisp.2021.1.13

35. Rosenthal YS, Chodick G, Rosenthal A, et al. Oral Contraceptives and Neutropenia: A Population-Based Cohort Study. Clinical Drug Investigation. 2019, 39(10): 927-930. doi: 10.1007/s40261-019-00818-0

36. Tak T, Wijten P, Heeres M, et al. Human CD62Ldim neutrophils identified as a separate subset by proteome profiling and in vivo pulse-chase labeling. Blood. 2017, 129(26): 3476-3485. doi: 10.1182/blood-2016-07-727669

37. Newburger PE, Dale DC. Evaluation and Management of Patients With Isolated Neutropenia. Seminars in Hematology. 2013, 50(3): 198-206. doi: 10.1053/j.seminhematol.2013.06.010

38. Restrepo MI, Anzueto A. Severe Community-Acquired Pneumonia. Infectious Disease Clinics of North America. 2009, 23(3): 503-520. doi: 10.1016/j.idc.2009.04.003

39. Metzler KD, Fuchs TA, Nauseef WM, et al. Myeloperoxidase is required for neutrophil extracellular trap formation: implications for innate immunity. Blood. 2011, 117(3): 953-959. doi: 10.1182/blood-2010-06-290171

40. Vrba J, Modrianský M. Oxidative burst of Kupffer cells: target for liver injury treatment. Biomedical Papers. 2002, 146(2): 15-20. doi: 10.5507/bp.2002.003