Effects of N-acetylcysteine amide on anxiety and stress behavior in zebrafish

  • Carlos G. Reis
  • Ricieri Mocelin
  • Radharani Benvenutti
  • Matheus Marcon
  • Adrieli Sachett
  • Ana P. Herrmann
  • Elaine Elisabetsky
  • Angelo PiatoEmail author
Original Article


Anxiety disorders are highly prevalent and a leading cause of disability worldwide. Their etiology is related to stress, an adaptive response of the organism to restore homeostasis, in which oxidative stress and glutamatergic hyperactivity are involved. N-Acetylcysteine (NAC) is a multitarget approved drug proved to be beneficial in the treatment of various mental disorders. Nevertheless, NAC has low membrane permeability and poor bioavailability and its limited delivery to the brain may explain inconsistencies in the literature. N-Acetylcysteine amide (AD4) is a synthetic derivative of NAC in which the carboxyl group was modified to an amide. The amidation of AD4 improved lipophilicity and blood-brain barrier permeability and enhanced its antioxidant properties. The purpose of this study was to investigate the effects of AD4 on behavioral and biochemical parameters in zebrafish anxiety models. Neither AD4 nor NAC induced effects on locomotion and anxiety-related parameters in the novel tank test. However, in the light/dark test, AD4 (0.001 mg/L) increased the time spent in the lit side in a concentration 100 times lower than NAC (0.1 mg/L). In the acute restraint stress protocol, NAC and AD4 (0.001 mg/L) showed anxiolytic properties without meaningful effects on oxidative status. The study suggests that AD4 has anxiolytic effects in zebrafish with higher potency than the parent compound. Additional studies are warranted to characterize the anxiolytic profile of AD4 and its potential in the management of anxiety disorders.


N-Acetylcysteine amide Anxiety Acute restraint stress AD4 NACA 



We thank Prof. Daphne Atlas from Hebrew University of Jerusalem for providing AD4 and encouraging this study and Brandon Guinalli for helping with the figure designs.

Authors’ contributions

CR designed and performed the experiments, analyzed the data, prepared figures and tables, and authored and reviewed drafts of the paper. RM, RB, MM, and AS performed the experiments and reviewed drafts of the paper. AH, EE, and AP designed the experiments, analyzed the data, contributed reagents/materials/analysis tools, prepared figures and tables, and authored and reviewed drafts of the paper. All authors read and approved the manuscript.

Funding information

This work was supported by CNPq (302800/2017-4) and FAPERGS (#17/2551-0000974-6); CGR is recipient of a fellowship from CAPES and AP from CNPq.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.

Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.


  1. Abreu MS, Koakoski G, Ferreira D et al (2014) Diazepam and fluoxetine decrease the stress response in zebrafish. PloS One 9:e103232. CrossRefGoogle Scholar
  2. Aschbacher K, O’Donovan A, Wolkowitz OM et al (2013) Good stress, bad stress and oxidative stress: insights from anticipatory cortisol reactivity. Psychoneuroendocrinology 38:1698–1708. CrossRefPubMedPubMedCentralGoogle Scholar
  3. Ates B, Abraham L, Ercal N (2008) Antioxidant and free radical scavenging properties of N-acetylcysteine amide (NACA) and comparison with N-acetylcysteine (NAC). Free Radic Res 42:372–377. CrossRefPubMedGoogle Scholar
  4. Bahat-Stroomza M, Gilgun-Sherki Y, Offen D, Panet H, Saada A, Krool-Galron N, Barzilai A, Atlas D, Melamed E (2005) A novel thiol antioxidant that crosses the blood brain barrier protects dopaminergic neurons in experimental models of Parkinson’s disease. Eur J Neurosci 21:637–646. CrossRefPubMedGoogle Scholar
  5. Baldwin DS, Hou R, Gordon R, Huneke NTM, Garner M (2017) Pharmacotherapy in generalized anxiety disorder: novel experimental medicine models and emerging drug targets. CNS Drugs 31:307–317. CrossRefPubMedGoogle Scholar
  6. Bandelow B, Michaelis S, Wedekind D (2017) Treatment of anxiety disorders. Dialogues Clin Neurosci 19:93–107PubMedPubMedCentralGoogle Scholar
  7. Bartov O, Sultana R, Butterfield DA, Atlas D (2006) Low molecular weight thiol amides attenuate MAPK activity and protect primary neurons from Abeta(1-42) toxicity. Brain Res 1069:198–206. CrossRefPubMedGoogle Scholar
  8. Baxter AJ, Scott KM, Vos T, Whiteford HA (2013) Global prevalence of anxiety disorders: a systematic review and meta-regression. Psychol Med 43:897–910. CrossRefPubMedGoogle Scholar
  9. Berk M, Malhi GS, Gray LJ, Dean OM (2013) The promise of N-acetylcysteine in neuropsychiatry. Trends Pharmacol Sci 34:167–177. CrossRefPubMedGoogle Scholar
  10. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254. CrossRefGoogle Scholar
  11. Bystritsky A, Khalsa SS, Cameron ME, Schiffman J (2013) Current diagnosis and treatment of anxiety disorders. Pharm Ther 38:30–57Google Scholar
  12. Carver KA, Yang D (2016) N-Acetylcysteine amide protects against oxidative stress-induced microparticle release from human retinal pigment epithelial cells. Invest Ophthalmol Vis Sci 57:360–371. CrossRefPubMedPubMedCentralGoogle Scholar
  13. Chen W, Ercal N, Huynh T, Volkov A, Chusuei CC (2012) Characterizing N-acetylcysteine (NAC) and N-acetylcysteine amide (NACA) binding for lead poisoning treatment. J Colloid Interface Sci 371:144–149. CrossRefPubMedGoogle Scholar
  14. Chrousos GP (2009) Stress and disorders of the stress system. Nat Rev Endocrinol 5:374–381. CrossRefPubMedGoogle Scholar
  15. Cortese BM, Phan KL (2005) The role of glutamate in anxiety and related disorders. CNS Spectr 10:820–830CrossRefGoogle Scholar
  16. Craske MG, Stein MB, Eley TC et al (2017) Anxiety disorders. Nat Rev Dis Primer 3:17024. CrossRefGoogle Scholar
  17. Cryan JF, Sweeney FF (2011) The age of anxiety: role of animal models of anxiolytic action in drug discovery. Br J Pharmacol 164:1129–1161. CrossRefPubMedPubMedCentralGoogle Scholar
  18. Dal Santo G, Conterato GMM, Barcellos LJG et al (2014) Acute restraint stress induces an imbalance in the oxidative status of the zebrafish brain. Neurosci Lett 558:103–108. CrossRefPubMedGoogle Scholar
  19. Dean O, Giorlando F, Berk M (2011) N-acetylcysteine in psychiatry: current therapeutic evidence and potential mechanisms of action. J Psychiatry Neurosci JPN 36:78–86. CrossRefPubMedGoogle Scholar
  20. Egan RJ, Bergner CL, Hart PC, Cachat JM, Canavello PR, Elegante MF, Elkhayat SI, Bartels BK, Tien AK, Tien DH, Mohnot S, Beeson E, Glasgow E, Amri H, Zukowska Z, Kalueff AV (2009) Understanding behavioral and physiological phenotypes of stress and anxiety in zebrafish. Behav Brain Res 205:38–44. CrossRefPubMedPubMedCentralGoogle Scholar
  21. Gebauer DL, Pagnussat N, Piato AL et al (2011) Effects of anxiolytics in zebrafish: similarities and differences between benzodiazepines, buspirone and ethanol. Pharmacol Biochem Behav 99:480–486. CrossRefPubMedGoogle Scholar
  22. Ghisleni G, Capiotti KM, Da Silva RS et al (2012) The role of CRH in behavioral responses to acute restraint stress in zebrafish. Prog Neuropsychopharmacol Biol Psychiatry 36:176–182. CrossRefPubMedGoogle Scholar
  23. Grinberg L, Fibach E, Amer J, Atlas D (2005) N-acetylcysteine amide, a novel cell-permeating thiol, restores cellular glutathione and protects human red blood cells from oxidative stress. Free Radic Biol Med 38:136–145. CrossRefPubMedGoogle Scholar
  24. Idalencio R, Kalichak F, Rosa JGS et al (2015) Waterborne risperidone decreases stress response in zebrafish. PloS One 10:e0140800. CrossRefPubMedPubMedCentralGoogle Scholar
  25. Barcellos HH, Kalichak F, da Rosa JG et al (2016) Waterborne aripiprazole blunts the stress response in zebrafish. Sci Rep 6:37612. CrossRefPubMedPubMedCentralGoogle Scholar
  26. Kalueff AV, Stewart AM, Gerlai R (2014) Zebrafish as an emerging model for studying complex brain disorders. Trends Pharmacol Sci 35:63–75. CrossRefPubMedPubMedCentralGoogle Scholar
  27. Koltunowska D, Gibula-Bruzda E, Kotlinska JH (2013) The influence of ionotropic and metabotropic glutamate receptor ligands on anxiety-like effect of amphetamine withdrawal in rats. Prog Neuropsychopharmacol Biol Psychiatry 45:242–249. CrossRefPubMedGoogle Scholar
  28. Kowalczyk-Pachel D, Iciek M, Wydra K, Nowak E, Górny M, Filip M, Włodek L, Lorenc-Koci E (2016) Cysteine metabolism and oxidative processes in the rat liver and kidney after acute and repeated cocaine treatment. PLOS ONE 11:e0147238. CrossRefPubMedPubMedCentralGoogle Scholar
  29. Kysil EV, Meshalkina DA, Frick EE, Echevarria DJ, Rosemberg DB, Maximino C, Lima MG, Abreu MS, Giacomini AC, Barcellos LJG, Song C, Kalueff AV (2017) Comparative analyses of zebrafish anxiety-like behavior using conflict-based novelty tests. Zebrafish 14:197–208. CrossRefPubMedGoogle Scholar
  30. Levin ED, Bencan Z, Cerutti DT (2007) Anxiolytic effects of nicotine in zebrafish. Physiol Behav 90:54–58. CrossRefPubMedGoogle Scholar
  31. Marcon M, Herrmann AP, Mocelin R, Rambo CL, Koakoski G, Abreu MS, Conterato GM, Kist LW, Bogo MR, Zanatta L, Barcellos LJ, Piato AL (2016) Prevention of unpredictable chronic stress-related phenomena in zebrafish exposed to bromazepam, fluoxetine and nortriptyline. Psychopharmacology (Berl) 233:3815–3824. CrossRefGoogle Scholar
  32. Marcon M, Mocelin R, Sachett A, Siebel AM, Herrmann AP, Piato A (2018) Enriched environment prevents oxidative stress in zebrafish submitted to unpredictable chronic stress. PeerJ 6:e5136. CrossRefPubMedPubMedCentralGoogle Scholar
  33. Maximino C, Marques de Brito T, Dias CA, Gouveia A Jr, Morato S et al (2010) Scototaxis as anxiety-like behavior in fish. Nat Protoc 5:209–216. CrossRefPubMedGoogle Scholar
  34. Maximino C, da Silva AWB, Gouveia A, Herculano AM (2011) Pharmacological analysis of zebrafish (Danio rerio) scototaxis. Prog Neuropsychopharmacol Biol Psychiatry 35:624–631. CrossRefPubMedGoogle Scholar
  35. Maximino C, da Silva AWB, Araújo J et al (2014) Fingerprinting of psychoactive drugs in zebrafish anxiety-like behaviors. PLOS ONE 9:e103943. CrossRefPubMedPubMedCentralGoogle Scholar
  36. McEwen BS, Bowles NP, Gray JD, Hill MN, Hunter RG, Karatsoreos IN, Nasca C (2015) Mechanisms of stress in the brain. Nat Neurosci 18:1353–1363. CrossRefPubMedPubMedCentralGoogle Scholar
  37. Minarini A, Ferrari S, Galletti M, Giambalvo N, Perrone D, Rioli G, Galeazzi GM (2017) N-acetylcysteine in the treatment of psychiatric disorders: current status and future prospects. Expert Opin Drug Metab Toxicol 13:279–292. CrossRefPubMedGoogle Scholar
  38. Mocelin R, Herrmann AP, Marcon M et al (2015) N-acetylcysteine prevents stress-induced anxiety behavior in zebrafish. Pharmacol Biochem Behav 139(Pt B):121–126. CrossRefPubMedGoogle Scholar
  39. Mocelin R, Marcon M, D’ambros S et al (2018a) Behavioral and biochemical effects of N-acetylcysteine in zebrafish acutely exposed to ethanol. Neurochem Res 43:458–464. CrossRefPubMedGoogle Scholar
  40. Mocelin R, Marcon M, D’ambros S, Mattos J, Sachett A, Siebel AM, Herrmann AP, Piato A (2018b) N-Acetylcysteine reverses anxiety and oxidative damage induced by unpredictable chronic stress in zebrafish. Mol Neurobiol. 56:1188–1195. CrossRefPubMedGoogle Scholar
  41. Offen D, Gilgun-Sherki Y, Barhum Y, Benhar M, Grinberg L, Reich R, Melamed E, Atlas D (2004) A low molecular weight copper chelator crosses the blood-brain barrier and attenuates experimental autoimmune encephalomyelitis. J Neurochem 89:1241–1251. CrossRefPubMedGoogle Scholar
  42. Ooi SL, Green R, Pak SC (2018) N-Acetylcysteine for the treatment of psychiatric disorders: a review of current evidence. BioMed Res Int 2018:2469486. CrossRefPubMedPubMedCentralGoogle Scholar
  43. Pandya JD, Readnower RD, Patel SP, Yonutas HM, Pauly JR, Goldstein GA, Rabchevsky AG, Sullivan PG (2014) N-acetylcysteine amide confers neuroprotection, improves bioenergetics and behavioral outcome following TBI. Exp Neurol 257:106–113. CrossRefPubMedPubMedCentralGoogle Scholar
  44. Penugonda S, Ercal N (2011) Comparative evaluation of N-acetylcysteine (NAC) and N-acetylcysteine amide (NACA) on glutamate and lead-induced toxicity in CD-1 mice. Toxicol Lett 201:1–7. CrossRefPubMedGoogle Scholar
  45. Piato AL, Rosemberg DB, Capiotti KM, Siebel AM, Herrmann AP, Ghisleni G, Vianna MR, Bogo MR, Lara DR, Bonan CD (2011) Acute restraint stress in zebrafish: behavioral parameters and purinergic signaling. Neurochem Res 36:1876–1886. CrossRefPubMedGoogle Scholar
  46. Pitsikas N (2014) The metabotropic glutamate receptors: potential drug targets for the treatment of anxiety disorders? Eur J Pharmacol 723:181–184. CrossRefPubMedGoogle Scholar
  47. Quadros VA, Silveira A, Giuliani GS, Didonet F, Silveira AS, Nunes ME, Silva TO, Loro VL, Rosemberg DB (2016) Strain- and context-dependent behavioural responses of acute alarm substance exposure in zebrafish. Behav Processes 122:1–11. CrossRefPubMedGoogle Scholar
  48. Riaza Bermudo-Soriano C, Perez-Rodriguez MM, Vaquero-Lorenzo C, Baca-Garcia E (2012) New perspectives in glutamate and anxiety. Pharmacol Biochem Behav 100:752–774. CrossRefPubMedGoogle Scholar
  49. Sackerman J, Donegan JJ, Cunningham CS et al (2010) Zebrafish behavior in novel environments: effects of acute exposure to anxiolytic compounds and choice of Danio rerio line. Int J Comp Psychol 23:43–61PubMedPubMedCentralGoogle Scholar
  50. Sadan O, Bahat-Stromza M, Gilgun-Sherki Y, Atlas D, Melamed E, Offen D (2005) A novel brain-targeted antioxidant (AD4) attenuates haloperidol-induced abnormal movement in rats: implications for tardive dyskinesia. Clin Neuropharmacol 28:285–288CrossRefGoogle Scholar
  51. Samuni Y, Goldstein S, Dean OM, Berk M (2013) The chemistry and biological activities of N-acetylcysteine. Biochim Biophys Acta 1830:4117–4129. CrossRefPubMedGoogle Scholar
  52. Sandi C, Haller J (2015) Stress and the social brain: behavioural effects and neurobiological mechanisms. Nat Rev Neurosci 16:290–304. CrossRefPubMedGoogle Scholar
  53. Santos P, Herrmann AP, Benvenutti R, Noetzold G, Giongo F, Gama CS, Piato AL, Elisabetsky E (2017) Anxiolytic properties of N-acetylcysteine in mice. Behav Brain Res 317:461–469. CrossRefPubMedGoogle Scholar
  54. Sapolsky RM (2000) Glucocorticoids and hippocampal atrophy in neuropsychiatric disorders. Arch Gen Psychiatry 57:925–935CrossRefGoogle Scholar
  55. Schaefer IC, Siebel AM, Piato AL et al (2015) The side-by-side exploratory test: a simple automated protocol for the evaluation of adult zebrafish behavior simultaneously with social interaction. Behav Pharmacol 26:691–696. CrossRefPubMedGoogle Scholar
  56. Stewart A, Wu N, Cachat J, Hart P, Gaikwad S, Wong K, Utterback E, Gilder T, Kyzar E, Newman A, Carlos D, Chang K, Hook M, Rhymes C, Caffery M, Greenberg M, Zadina J, Kalueff AV (2011) Pharmacological modulation of anxiety-like phenotypes in adult zebrafish behavioral models. Prog Neuropsychopharmacol Biol Psychiatry 35:1421–1431. CrossRefPubMedGoogle Scholar
  57. Stewart A, Gaikwad S, Kyzar E, Green J, Roth A, Kalueff AV (2012) Modeling anxiety using adult zebrafish: a conceptual review. Neuropharmacology 62:135–143. CrossRefPubMedGoogle Scholar
  58. Sunitha K, Hemshekhar M, Thushara RM et al (2013) N-Acetylcysteine amide: a derivative to fulfill the promises of N-Acetylcysteine. Free Radic Res 47:357–367. CrossRefPubMedGoogle Scholar
  59. Tardiolo G, Bramanti P, Mazzon E (2018) Overview on the effects of N-acetylcysteine in neurodegenerative diseases. Molecules 23:3305. CrossRefPubMedCentralGoogle Scholar
  60. Thibaut F (2017) Anxiety disorders: a review of current literature. Dialogues Clin Neurosci 19:87–88PubMedPubMedCentralGoogle Scholar
  61. Wu W, Abraham L, Ogony J et al (2008) Effects of N-acetylcysteine amide (NACA), a thiol antioxidant on radiation-induced cytotoxicity in Chinese hamster ovary cells. Life Sci 82:1122–1130. CrossRefPubMedGoogle Scholar
  62. Zhang X, Banerjee A, Banks WA, Ercal N (2009) N-Acetylcysteine amide protects against methamphetamine-induced oxidative stress and neurotoxicity in immortalized human brain endothelial cells. Brain Res 1275:87–95. CrossRefPubMedPubMedCentralGoogle Scholar
  63. Zhou Y, Wang H, Zhou X et al (2018) N-acetylcysteine amide provides neuroprotection via Nrf2-ARE pathway in a mouse model of traumatic brain injury. Drug Des. Devel. Ther, In Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Laboratório de Psicofarmacologia e Comportamento (LAPCOM), Programa de Pós-graduação em Neurociências, Instituto de Ciências Básicas da SaúdeUniversidade Federal do Rio Grande do Sul (UFRGS)Porto AlegreBrazil
  2. 2.Programa de Pós-graduação em Farmacologia e Terapêutica, Instituto de Ciências Básicas da SaúdeUniversidade Federal do Rio Grande do Sul (UFRGS)Porto AlegreBrazil
  3. 3.Programa de Pós-graduação em Ciência Biológicas: Bioquímica, Instituto de Ciências Básicas da SaúdeUniversidade Federal do Rio Grande do Sul (UFRGS)Porto AlegreBrazil

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