Toxicological Research

  • 제36권4호
  • /
  • Pages.359-366
  • /
  • 2020
  • /
  • 1976-8257(pISSN)
  • /
  • 2234-2753(eISSN)
한국독성학회 (Korean Society of Toxicology & Korea Environmental Mutagen Society)
권호 표지
DOI QR코드

DOI QR Code

Chronic copper exposure leads to hippocampus oxidative stress and impaired learning and memory in male and female rats

  • Lamtai, Mouloud (Laboratory of Genetics, Neuroendocrinology and Biotechnology, Faculty of Science, Ibn Tofail University) ;
  • Zghari, Oussama (Laboratory of Genetics, Neuroendocrinology and Biotechnology, Faculty of Science, Ibn Tofail University) ;
  • Ouakki, Sihame (Laboratory of Genetics, Neuroendocrinology and Biotechnology, Faculty of Science, Ibn Tofail University) ;
  • Marmouzi, Ilias (Laboratoire de Pharmacologie et Toxicologie, equipe de Pharmacocinetique, Faculte de Medicine et Pharmacie, University Mohammed V in Rabat, Rabat Instituts) ;
  • Mesfoui, Abdelhalem (Laboratory of Genetics, Neuroendocrinology and Biotechnology, Faculty of Science, Ibn Tofail University) ;
  • El Hessni, Aboubaker (Laboratory of Genetics, Neuroendocrinology and Biotechnology, Faculty of Science, Ibn Tofail University) ;
  • Ouichou, Ali (Laboratory of Genetics, Neuroendocrinology and Biotechnology, Faculty of Science, Ibn Tofail University)
  • 투고 : 2019.12.15
  • 심사 : 2020.02.18
  • 발행 : 2020.10.15
⟨ 이전 논문 다음 논문 ⟩

초록

Environmental and occupational exposures to copper (Cu) play a pivotal role in the etiology of some neurological diseases and reduced cognitive functions. However, the precise mechanisms of its effects on cognitive function have not been yet thoroughly established. In our study, we aimed to investigate the behavior and neurochemical alterations in hippocampus of male and female rats, chronically exposed to copper chloride (CuCl2) and the possible involvement of oxidative stress. Twenty-four rats, for each gender, were divided into control and three test groups (n = 6), and were injected intraperitoneally with saline (0.9% NaCl) or CuCl2 (0.25 mg/kg, 0.5 mg/kg and 1 mg/kg) for 8 weeks. After the treatment period, Y-maze test was used for the evaluation of spatial working memory and the Morris Water Maze (MWM) to test the spatial learning and memory. Biochemical determination of oxidative stress levels in hippocampus was performed. The main results of the present work are working memory impairment in spatial Y-maze which induced by higher Cu intake (1 mg/kg) in male and female rats. Also, In the MWM test, the spatial learning and memory were significantly impaired in rats treated with Cu at dose of 1 mg/kg. Additionally, markers of oxidative stress such as catalase, superoxide dismutase, lipid peroxidation products and nitric oxide levels were significantly altered following Cu treatments. These data propose that compromised behavior following Cu exposure is associated with increase in oxidative stress.

키워드

과제정보

Thanks to Dr Y. CHAHIROU for interest in this work and helpful discussion.

참고문헌

  1. Kamal Emam Mahmoud (2011) Combined effect of vanadium and nickel on lipid peroxidation and selected parameters of antioxidant system in liver and kidney of male rat. Afr J Biotechnol 10:18319-18325 https://doi.org/10.5897/AJB11.2949
  2. Kicinski M, Vrijens J, Vermier G, Den Hond E, Schoeters G, Nelen V, Bruckers L, Sioen I, Baeyens W, Van Larebeke N, Viaene MK, Nawrot TS (2015) Neurobehavioral function and low-level metal exposure in adolescents. Int J Hyg Environ Health 218:139-146 https://doi.org/10.1016/j.ijheh.2014.09.002
  3. Abdellatif A, Omar ELH, Halima G (2017) The neuronal basis of copper induced modulation of anxiety state in rat. Acta Histochem 119:10-17 https://doi.org/10.1016/j.acthis.2016.10.003
  4. Carotenuto R, Capriello T, Cofone R, Galdiero G, Fogliano C, Ferrandino I (2019) Impact of copper in Xenopus laevis liver: histological damages and atp7b downregulation. Ecotoxicol Environ Saf 188:109940
  5. Pilehvar A, Town RM, Blust R (2020) The effect of copper on behaviour, memory, and associative learning ability of zebrafish (Danio rerio). Ecotoxicol Environ Saf 188:109900 https://doi.org/10.1016/j.ecoenv.2019.109900
  6. Scheiber IF, Dringen R (2013) Astrocyte functions in the copper homeostasis of the brain. Neurochem Int 62:556-565 https://doi.org/10.1016/j.neuint.2012.08.017
  7. Chen C, Jiang X, Li Y, Yu H, Li S, Zhang Z, Xu H, Yang Y, Liu G, Zhu F, Ren X, Zou L, Xu B, Liu J, Spencer PS, Yang X (2019) Low-dose oral copper treatment changes the hippocampal phosphoproteomic profile and perturbs mitochondrial function in a mouse model of Alzheimer's disease. Free Radic Biol Med 135:144-156 https://doi.org/10.1016/j.freeradbiomed.2019.03.002
  8. Mlyniec K, Gawel M, Doboszewska U, Starowicz G, Pytka K, Davies CL, Budziszewska B (2015) Essential elements in depression and anxiety. Part II. Pharmacol Rep 67:187-194 https://doi.org/10.1016/j.pharep.2014.09.009
  9. Desai V, Kaler SG (2008) Role of copper in human neurological disorders. Am J Clin Nutr 88:855-858 https://doi.org/10.1093/ajcn/88.3.855S
  10. Magura IS, Rozhmanova OM (1997) Oxidative stress and neurodegenerative disorders. Biopolym Cell 13:513-515 https://doi.org/10.7124/bc.0004B0
  11. Madsen E, Gitlin JD (2007) Copper and iron disorders of the brain. Annu Rev Neurosci 30:317-337 https://doi.org/10.1146/annurev.neuro.30.051606.094232
  12. Bulcke F, Dringen R (2016) Handling of copper and copper oxide nanops by astrocytes. Neurochem Res 41:33-43 https://doi.org/10.1007/s11064-015-1688-9
  13. Brewer GJ (2009) The risks of copper toxicity contributing to cognitive decline in the aging population and to alzheimer's disease. J Am Coll Nutr 28:238-242 https://doi.org/10.1080/07315724.2009.10719777
  14. Gerd M, Andrea S, Lars H, Dirk B, Thomas R, Colin LM, Konrad B (1996) The amyloid precursor protein of Alzheimer's disease in the reduction of copper (II) to copper (I). Science 271:2-4
  15. Lutsenko S, Bhattacharjee A, Hubbard AL (2010) Copper handling machinery of the brain. Metallomics 2:596-608 https://doi.org/10.1039/c0mt00006j
  16. Scheiber IF, Mercer JFB, Dringen R (2014) Metabolism and functions of copper in brain. Prog Neurobiol 116:33-57 https://doi.org/10.1016/j.pneurobio.2014.01.002
  17. Crayton JW, Walsh WJ (2007) Elevated serum copper levels in women with a history of post-partum depression. J Trace Elem Med Biol 21:17-21 https://doi.org/10.1016/j.jtemb.2006.10.001
  18. Nowak G, Zieba A, Dudek D, Krosniak M, Szymaczek M, Schlegel-Zawadzka M (1999) Serum trace elements in animal models and human depression. Part I. Zinc. Hum Psychopharmacol 14:83-86 https://doi.org/10.1002/(SICI)1099-1077(199903)14:2<83::AID-HUP74>3.0.CO;2-6
  19. Russo AJ (2011) Analysis of plasma zinc and copper concentration, and perceived symptoms, in individuals with depression, post zinc and anti-oxidant therapy. Nutr Metab Insights 4:S6760
  20. Lamtai M, Ouakki S, Zghari O, Mesfioui A, El Hessni A, Ouichou A (2019) Affective behavior dysregulation was induced by chronic administration of copper in wistar rats. Neurosci Med 10:134-149 https://doi.org/10.4236/nm.2019.102009
  21. Kalita J, Kumar V, Misra UK, Bora HK (2017) Memory and learning dysfunction following copper toxicity: biochemical and immunohistochemical basis. Mol Neurobiol 55:3800-3811
  22. Vlachova V (1996) Copper modulation of NMDA responses in mouse and rat cultured hippocampal neurons. Eur J Neurosci 8:2257-2264 https://doi.org/10.1111/j.1460-9568.1996.tb01189.x
  23. Salustri C, Barbati G, Ghidoni R, Quintiliani L, Ciappina S, Binetti G, Squitti R (2010) Clinical Neurophysiology Is cognitive function linked to serum free copper levels? A cohort study in a normal population. Clin Neurophysiol. 121:502-507 https://doi.org/10.1016/j.clinph.2009.11.090
  24. Kumar V, Kalita J, Misra UK, Bora HK (2015) A study of dose response and organ susceptibility of copper toxicity in a rat model. J Trace Elem Med Biol 29:269-274 https://doi.org/10.1016/j.jtemb.2014.06.004
  25. Zhang Y, Lu W, Han M, Li H, Luo H, Li W, Luo W, Lin Z (2016) Biphasic effects of copper on rat learning and memory in the morris water maze. Ann Clin Lab Sci 46:346-352
  26. Neely CLC, Lippi SLP, Lanzirotti A, Flinn JM (2019) Localization of free and bound metal species through X-Ray synchrotron fluorescence microscopy in the rodent brain and their relation to behavior. Brain Sci 9:74 https://doi.org/10.3390/brainsci9040074
  27. Ishihara K, Kawashita E, Shimizu R, Nagasawa K, Yasui H, Sago H, Yamakawa K, Akiba S (2019) Copper accumulation in the brain causes the elevation of oxidative stress and less anxious behavior in Ts1Cje mice, a model of Down syndrome. Free Radic Biol Med 134:248-259 https://doi.org/10.1016/j.freeradbiomed.2019.01.015
  28. Lima FD, Souza MA, Furian AF, Rambo LM, Ribeiro LR, Martignoni FV, Hoffmann MS, Fighera MR, Royes LFF, Oliveira MS, de Mello CF (2008) Na+, K+-ATPase activity impairment after experimental traumatic brain injury: relationship to spatial learning deficits and oxidative stress Behav. Brain Res 193:306-310 https://doi.org/10.1016/j.bbr.2008.05.013
  29. Rahman MF, Wang J, Patterson TA, Saini UT, Robinson BL, Newport GD, Murdock RC, Schlager JJ, Hussain SM, Ali SF (2009) Expression of genes related to oxidative stress in the mouse brain after exposure to silver-25 nanops. Toxicol Lett 187:15-21 https://doi.org/10.1016/j.toxlet.2009.01.020
  30. Kucukatay V, Agar A, Gumuslu S, Yargicoglu P (2007) Effect of sulfur dioxide on active and passive avoidance in experimental diabetes mellitus: relation to oxidant stress and antioxidant enzymes. Int J Neurosci 117:1091-1107 https://doi.org/10.1080/00207450600934531
  31. Sierksma ASR, Van Den Hove DLA, Pfau F, Philippens M, Bruno O, Fedele E, Ricciarelli R, Steinbusch HWM, Vanmierlo T, Prickaerts J (2014) Improvement of spatial memory function in APPswe/PS1dE9 mice after chronic inhibition of phosphodiesterase type 4D. Neuropharmacology 77:120-130 https://doi.org/10.1016/j.neuropharm.2013.09.015
  32. Morris R (1981) Spatial localization does not require local cues the presence of. Learn Motiv 12:239-260 https://doi.org/10.1016/0023-9690(81)90020-5
  33. Morris R (1984) Developments of a water-maze procedure for studying spatial learning in the rat. J Neurosci Method 11:47-60 https://doi.org/10.1016/0165-0270(84)90007-4
  34. Kaoud HA, Kamel MM, Abdel-Razek AH, Kamel GM, Ahmed KA (2010) Neurobehavioural, neurochemical and neuromorphological effects of cadmium in male rats. J Am Sci 202:54-63
  35. Draper HH, Hadley M (1990) Malondialdehyde determination as index of lipid peroxidation. Methods Enzymol 186:421-431 https://doi.org/10.1016/0076-6879(90)86135-I
  36. Freitas RM, Sousa FCF, Vasconcelos SMM, Viana GSB, Fonteles MMF (2004) Pilocarpine-induced status epilepticus in rats: lipid peroxidation level, nitrite formation, GABAergic and glutamatergic receptor alterations in the hippocampus, striatum and frontal cortex. Pharmacol Biochem Behav 78:327-332 https://doi.org/10.1016/j.pbb.2004.04.004
  37. Chao CC, Hu S, Molitor TW, Shaskan EG, Peterson PK (1992) Injury via a nitric oxide mechanism Activated microglia mediate oxide neuronal cell injury via a nitric mechanism'. J Immunol 149:2736-2741 https://doi.org/10.4049/jimmunol.149.8.2736
  38. Beauchamp C, Fridovich I (1971) Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal Biochem 44:276-287 https://doi.org/10.1016/0003-2697(71)90370-8
  39. Aebi H (1984) Catalase in vitro. Methods Enzymol 105:121-126 https://doi.org/10.1016/S0076-6879(84)05016-3
  40. Abbaoui A, Gamrani H (2019) Obvious anxiogenic-like effects of subchronic copper intoxication in rats, outcomes on spatial learning and memory and neuromodulatory potential of curcumin. J Chem Neuroanat 96:86-93 https://doi.org/10.1016/j.jchemneu.2019.01.001
  41. Yu H, Jiang X, Lin X, Zhang Z, Wu D, Zhou L, Liu J, Yang X (2018) Hippocampal subcellular organelle proteomic alteration of copper-treated mice. Toxicol Sci 164:250-263 https://doi.org/10.1093/toxsci/kfy082
  42. Behzadfar L, Abdollahi M, Sabzevari O, Hosseini R, Salimi A, Naserzadeh P, Sharifzadeh M, Pourahmad J (2017) Potentiating role of copper on spatial memory deficit induced by beta amyloid and evaluation of mitochondrial function markers in the hippocampus of rats. Metallomics 9:969-980 https://doi.org/10.1039/C7MT00075H
  43. Leiva J, Palestini M, Infante C, Goldschmidt A, Motles E (2009) Copper suppresses hippocampus LTP in the rat, but does not alter learning or memory in the morris water maze. Brain Res 1256:69-75 https://doi.org/10.1016/j.brainres.2008.12.041
  44. Palizvan MR, Jand A, Jand Y, Taherinejad MR (2016) A study on the effects of orally administered copper sulfate on learning and spatial memory of wistar rats. J Babol Univ Med Sci 18:31-36
  45. Lu J, Zheng Y, Wu D, Sun D, Shan Q, Fan S (2006) Trace amounts of copper induce neurotoxicity in the cholesterol-fed mice through apoptosis. FEBS Lett 580:6730-6740 https://doi.org/10.1016/j.febslet.2006.10.072
  46. Brewer GJ, Kanzer SH, Zimmerman EA, Celmins DF, Heckman SM, Dick R (2010) Copper and ceruloplasmin abnormalities in Alzheimers disease. Am J Alzheimers Dis Other Demen 25:490-497 https://doi.org/10.1177/1533317510375083
  47. Lam PK, Kritz-Silverstein D, Barrett-Connor E, Milne D, Nielsen F, Gamst A, Morton D, Wingard D (2008) Plasma trace elements and cognitive function in older men and women: the Rancho Bernardo study. J Nutr Heal Aging 12:22-27 https://doi.org/10.1007/BF02982160
  48. Squitti R, Siotto M, Polimanti R (2014) Low-copper diet as a preventive strategy for Alzheimer's disease. Neurobiol Aging 35:S40-S50 https://doi.org/10.1016/j.neurobiolaging.2014.02.031
  49. D'Ambrosi N, Rossi L (2015) Copper at synapse: release, binding and modulation of neurotransmission. Neurochem Int 90:36-45 https://doi.org/10.1016/j.neuint.2015.07.006
  50. Schlief ML (2005) NMDA receptor activation mediates copper homeostasis in hippocampal neurons. J Neurosci 25:239-246 https://doi.org/10.1523/JNEUROSCI.3699-04.2005
  51. Schlief ML, Gitlin JD (2006) Copper homeostasis in the CNS: a novel link between the NMDA receptor and copper homeostasis in the hippocampus. Mol Neurobiol 33:81-90 https://doi.org/10.1385/MN:33:2:81
  52. Decker MW, McGaugh JL (1991) The role of interactions between the cholinergic system and other neuromodulatory systems in learing and memory. Synapse 7:151-168 https://doi.org/10.1002/syn.890070209
  53. Flicker C, Dean RL, Watkins DL, Fisher SK, Bartus RT (1983) Behavioral and neurochemical effects following neurotoxic lesions of a major cholinergic input to the cerebral cortex in the rat. Pharmacol Biochem Behav 18:973-981 https://doi.org/10.1016/S0091-3057(83)80023-9
  54. Pal A, Badyal RK, Vasishta RK, Attri SV, Thapa BR, Prasad R (2013) Biochemical, histological, and memory impairment effects of chronic copper toxicity: a model for non-wilsonian brain copper toxicosis in Wistar rat. Biol Trace Elem Res 153:257-268 https://doi.org/10.1007/s12011-013-9665-0
  55. Franciscato C, Bueno TM, Moraes-Silva L, Duarte FA, Flores EMM, Dressler VL, Pereira ME (2009) High doses of zinc and copper alter neither cerebral metal levels nor acetylcholinesterase activity of suckling rats. EXCLI J 8:138-147
  56. Traynelis SF, Wollmuth LP, McBain CJ, Menniti FS, Vance KM, Ogden KK, Hansen KB, Yuan H, Myers SJ, Dingledine R (2010) Glutamate receptor ion channels: structure, regulation, and function. Pharmacol Rev 62:406-496
  57. Stern BR, Solioz M, Krewski D, Aggett P, Aw TC, Baker S, Crump K, Dourson M, Haber L, Hertzberg R, Keen C, Meek B, Rudenko L, Schoeny R, Slob W, Starr T (2007) Copper and human health: biochemistry, genetics, and strategies for modeling dose-response relationships. J Toxicol Environ Health B Crit Rev 10:157-222 https://doi.org/10.1080/10937400600755911
  58. Donnelly PS, Caragounis A, Du T, Laughton KM, Volitakis I, Cherny RA, Sharples RA, Hill AF, Li QX, Masters CL, Barnham KJ, White AR (2008) Selective intracellular release of copper and zinc ions from bis(thiosemicarbazonato) complexes reduces levels of Alzheimer disease amyloid-β peptide. J Biol Chem 283:4568-4577 https://doi.org/10.1074/jbc.M705957200
  59. Ma Q, Ying M, Sui X, Zhang H, Huang H, Yang L, Huang X, Zhuang Z, Liu J, Yang X (2014) Chronic copper exposure causes spatial memory impairment, selective loss of hippocampal synaptic proteins, and activation of PKR/eIF2α pathway in mice. J Alzheimer's Dis 43:1413-1427 https://doi.org/10.3233/jad-140216
  60. Hazra B, Biswas S, Mandal N (2008) Antioxidant and free radical scavenging activity of Spondias pinnata. BMC Complem Altern Med 8:1-10 https://doi.org/10.1186/1472-6882-8-1
  61. Lobo V, Patil A, Phatak A, Chandra N (2010) Free radicals, antioxidants and functional foods: impact on human health. Pharmacogn Rev 4:118-126 https://doi.org/10.4103/0973-7847.70902
  62. Pinol-Ripoll G, Fuentes-Broto L, Millan-Plano S, Reyes-Gonzales M, Mauri JA, Martinez-Ballarin E, Reiter RJ, Garcia JJ (2006) Protective effect of melatonin and pinoline on nitric oxide-induced lipid and protein peroxidation in rat brain homogenates. Neurosci Lett 405:89-93 https://doi.org/10.1016/j.neulet.2006.06.031
  63. Albendea CD, Gomez-Trullen EM, Fuentes-Broto L, Miana-Mena FJ, Millan-Plano S, Reyes-Gonzales MC, Martinez-Ballarin E, Garcia JJ (2007) Melatonin reduces lipid and protein oxidative damage in synaptosomes due to aluminium. J Trace Elem Med Biol 21:261-268 https://doi.org/10.1016/j.jtemb.2007.04.002
  64. Emerit J, Klein JM, Coutellier A, Congy F (1991) Free radicals and lipid peroxidation in cell biology: physiopathologic prospects. Pathol Biol 39:316-327
  65. Garry PS, Ezra M, Rowland MJ, Westbrook J, Pattinson KTS (2015) The role of the nitric oxide pathway in brain injury and its treatment-from bench to bedside. Exp Neurol 263:235-243 https://doi.org/10.1016/j.expneurol.2014.10.017
  66. Cuzzocrea S, Persichini T, Dugo L, Colasanti M, Musci G (2003) Copper induces type II nitric oxide synthase in vivo. Free Radic Biol Med 34:1253-1262 https://doi.org/10.1016/S0891-5849(03)00110-2
  67. Hu HL, Ni XS, Duff-Canning S, Wang XP (2016) Oxidative damage of copper chloride overload to the cultured rat astrocytes. Am J Transl Res 8:1273-1280
  68. Manto M (2014) Abnormal copper homeostasis: mechanisms and roles in neurodegeneration. Toxics 2:327-345 https://doi.org/10.3390/toxics2020327
  69. Forstermann U, Sessa WC (2012) Nitric oxide synthases: regulation and function. Eur Heart J 33:829-837 https://doi.org/10.1093/eurheartj/ehr304
  70. Moncada S, Bolanos JP (2006) Nitric oxide, cell bioenergetics and neurodegeneration. J Neurochem 97:1676-1689 https://doi.org/10.1111/j.1471-4159.2006.03988.x
  71. Bouayed J, Rammal H, Soulimani R (2009) Oxidative stress and anxiety : relationship and cellular pathways. Oxid Med Cell Longev 2:63-67 https://doi.org/10.4161/oxim.2.2.7944
  72. Krumschnabel G, Manzl C, Berger C, Hofer B (2005) Oxidative stress, mitochondrial permeability transition, and cell death in Cu-exposed trout hepatocytes. Toxicol Appl Pharmacol 209:62-73 https://doi.org/10.1016/j.taap.2005.03.016
  73. Calkins MJ, Manczak M, Mao P, Shirendeb U, Reddy PH (2011) Impaired mitochondrial biogenesis, defective axonal transport of mitochondria, abnormal mitochondrial dynamics and synaptic degeneration in a mouse model of Alzheimer's disease. Hum Mol Genet 20:4515-4529 https://doi.org/10.1093/hmg/ddr381
  74. Amtage F, Birnbaum D, Reinhard T, Niesen WD, Weiller C, Mader I, Meyer PT, Rijntjes M (2014) Estrogen intake and copper depositions: implications for alzheimer's disease? Case Rep Neurol 6:181-187 https://doi.org/10.1159/000363688
  75. Zghari O, Rezqaoui A, Ouakki S, Lamtai M, Chaibat J, Mesfioui A, El Hessni A, Rifi E-H, Essamri A, Ouichou A (2018) Effect of chronic aluminum administration on affective and cognitive behavior in male and female rats. J Behav Brain Sci 8:179-196 https://doi.org/10.4236/jbbs.2018.84012
  76. Lamtai M, Chaibat J, Ouakki S, Berkiks I, Rifi E, El Hessni A, Mesfioui A, Hbibi AT, Ahyayauch H, Essamri A, Ouichou A (2018) Effect of chronic administration of cadmium on anxiety-like, depression-like and memory deficits in male and female rats: possible involvement of oxidative stress mechanism. J Behav Brain Sci 8:240-268 https://doi.org/10.4236/jbbs.2018.85016
  77. Lamtai M, Chaibat J, Ouakki S, Zghari O, Mesfioui A, El Hessni A, Rifi E-H, Marmouzi I, Essamri A, Ouichou A (2018) Effect of chronic administration of nickel on affective and cognitive behavior in male and female rats: possible implication of oxidative stress pathway. Brain Sci 8:141 https://doi.org/10.3390/brainsci8080141

피인용 문헌

  1. Agricultural Use of Copper and Its Link to Alzheimer’s Disease vol.10, pp.6, 2020, https://doi.org/10.3390/biom10060897
  2. Neuroprotective effect of melatonin on nickel-induced affective and cognitive disorders and oxidative damage in rats vol.35, pp.4, 2020, https://doi.org/10.5620/eaht.2020025
  3. Nano-Curcumin Prevents Cardiac Injury, Oxidative Stress and Inflammation, and Modulates TLR4/NF-κB and MAPK Signaling in Copper Sulfate-Intoxicated Rats vol.10, pp.9, 2021, https://doi.org/10.3390/antiox10091414