Neurotoxic Effect of Flavonol Myricetin in the Presence of Excess Copper

Oxidative stress (OS) induced by the disturbed homeostasis of metal ions is one of the pivotal factors contributing to neurodegeneration. The aim of the present study was to investigate the effects of flavonoid myricetin on copper-induced toxicity in neuroblastoma SH-SY5Y cells. As determined by the MTT method, trypan blue exclusion assay and measurement of ATP production, myricetin heightened the toxic effects of copper and exacerbated cell death. It also increased copper-induced generation of reactive oxygen species, indicating the prooxidative nature of its action. Furthermore, myricetin provoked chromatin condensation and loss of membrane integrity without caspase-3 activation, suggesting the activation of both caspase-independent programmed cell death and necrosis. At the protein level, myricetin-induced upregulation of PARP-1 and decreased expression of Bcl-2, whereas copper-induced changes in the expression of p53, p73, Bax and NME1 were not further affected by myricetin. Inhibitors of ERK1/2 and JNK kinases, protein kinase A and L-type calcium channels exacerbated the toxic effects of myricetin, indicating the involvement of intracellular signaling pathways in cell death. We also employed atomic force microscopy (AFM) to evaluate the morphological and mechanical properties of SH-SY5Y cells at the nanoscale. Consistent with the cellular and molecular methods, this biophysical approach also revealed a myricetin-induced increase in cell surface roughness and reduced elasticity. Taken together, we demonstrated the adverse effects of myricetin, pointing out that caution is required when considering powerful antioxidants for adjuvant therapy in copper-related neurodegeneration

Toxic effect of copper in neuroblastoma SH-SY5Y cells

Oksidacijski stres, uzrokovan neravnotežom stvaranja i uklanjanja reaktivnih kisikovih i dušikovih vrsta, ima važnu ulogu u starenju mozga i razvoju neurodegenerativnih bolesti. U ovom radu, istraživala sam toksični učinak bakra na stanice neuroblastoma SH-SY5Y. Testom MTT, tripanskim modrilom i mjerenjem razine ATP-a ustanovila sam značajan pad preživljenja stanica SH-SY5Y nakon tretmana bakrom. Bakar je povećao proizvodnju reaktivnih vrsta, koje pri visokim vrijednostima izazivaju oksidacijsko oštećenje, te smanjio razinu glutationa. Također, izazvao je kondenzaciju kromatina bez aktivacije kaspaza 3 i 7, kao i oštećenja stanične membrane, iz čega zaključujem da je bakar izazvao programiranu staničnu smrt putem neovisnim o aktivaciji kaspaza i nekrozu. Na proteinskoj razini došlo je do povećane ekspresije proteina p53 i PARP-1, dok se ekspresija proteina NME1 smanjila. Primjena inhibitora proteina PARP-1 (PJ34) nije utjecala na toksičnost, dok je primjena inhibitora proteina p53 (pifitrin-α) uzrokovala povećanje toksičnosti bakra. Flavonoid miricetin pojačao je citotoksičnost bakra prooksidacijskim djelovanjem. Dobiveni rezultati razjašnjavaju mehanizam prooksidacijskog djelovanja bakra na staničnoj i molekularnoj razini što bi moglo pridonijeti razvoju novih farmakoloških pristupa u prevenciji i terapiji neurodegenerativnih bolesti.Oxidative stress, caused by an imbalance in the formation and elimination of reactive oxygen and nitrogen species, plays an important role in brain aging and the development of neurodegenerative diseases. In this study, I investigated the toxic effect of copper on SH-SY5Y neuroblastoma cells. By MTT test, trypan blue exclusion assay, and ATP level measurement, I found a significant decline in SH-SY5Y cell survival after copper treatment. Copper has increased the production of reactive species, which at high values cause oxidative damage, and reduced glutathione levels. It also caused chromatin condensation without caspase 3 and 7 activation, as well as cell membrane damage, from which I conclude that copper caused programmed cell death via caspase-independent activation and necrosis. At the protein level, there was increased expression of p53 and PARP-1 proteins, while NME1 protein expression decreased. Administration of the PARP-1 protein inhibitor (PJ34) did not affect toxicity, whereas administration of the p53 protein inhibitor (pifitrin-α) caused an increase in copper toxicity. The flavonoid myricetin enhanced the cytotoxicity of copper by its prooxidative action. The obtained results clarify the mechanism of prooxidative action of copper at the cellular and molecular level, which could contribute to the development of new pharmacological approaches in the prevention and treatment of neurodegenerative diseases

Genetic diversity and the origin of invasive populations of ctenophore Mnemiopsis leidyi

Rasprostranjenost rebraša Mnemiopsis leidyi je do 1980-tih godina bila ograničena na područje atlantske obale Sjeverne i Južne Amerike, nakon čega se vrsta proširila balastnim vodama u Crno more, a zatim postupno i u Azovsko more, Kaspijsko jezero, Sredozemno more te Sjeverno i Baltičko more. U radu su opisani podrijetlo i dinamika invazije M. leidyi te biološko-ekološke značajke ove vrste. Mnemiopsis leidyi nastanjuje priobalne vode umjerenih mora gdje postiže najveću biomasu i dominira planktonskim zajednicama u toplom dijelu godine s izraženim sezonskim promjenama. Genetske studije pokazuju da je do invazije u euroazijske vode došlo iz dva područja: populacija iz Crnog mora potječe iz Meksičkog zaljeva dok su Sjeverno i Baltičko more kolonizirani jedinkama iz Nove Engleske. Zahvaljujući prekooceanskom prijevozu, predviđa se nastavak širenja Mnemiopsisa a kakav će utjecaj imati na novi ekosustav ovisi o različitim abiotičkim čimbenicima, lokalnim strujama te prisustvu ili odsustvu prirodnih predatora.Distribution of a ctenophore Mnemiopsis leidyi was until 1980s limited to the Atlantic coast of North and South America, after which the species spread by ballast waters into the Black Sea, then progressively to Azov Sea, Caspian Sea, Mediterranean Sea, North and Baltic Sea. In this thesis, the origin and dynamics of M. leidyi invasion and the biological – ecological features of this species are described. Mnemiopsis leidyi inhabits coastal temperate waters where it achieves the highest biomass and dominates the planctonic communities during the warm season with notable seasonal changes. Genetic studies demonstrated that the invasion of Euroasian waters had two origins: the Black Sea population originated from the Gulf of Mexico, while the North and Baltic Sea were colonized by individuals from New England. Due to continuing transport by transoceanic shipping it is anticipated that M. leidyi will continue to expand. The impact the species elicits on the invaded ecosystem depends on various abiotic factors, local curents and the presence or absence of natural predators

Genetic diversity and the origin of invasive populations of ctenophore Mnemiopsis leidyi

Rasprostranjenost rebraša Mnemiopsis leidyi je do 1980-tih godina bila ograničena na područje atlantske obale Sjeverne i Južne Amerike, nakon čega se vrsta proširila balastnim vodama u Crno more, a zatim postupno i u Azovsko more, Kaspijsko jezero, Sredozemno more te Sjeverno i Baltičko more. U radu su opisani podrijetlo i dinamika invazije M. leidyi te biološko-ekološke značajke ove vrste. Mnemiopsis leidyi nastanjuje priobalne vode umjerenih mora gdje postiže najveću biomasu i dominira planktonskim zajednicama u toplom dijelu godine s izraženim sezonskim promjenama. Genetske studije pokazuju da je do invazije u euroazijske vode došlo iz dva područja: populacija iz Crnog mora potječe iz Meksičkog zaljeva dok su Sjeverno i Baltičko more kolonizirani jedinkama iz Nove Engleske. Zahvaljujući prekooceanskom prijevozu, predviđa se nastavak širenja Mnemiopsisa a kakav će utjecaj imati na novi ekosustav ovisi o različitim abiotičkim čimbenicima, lokalnim strujama te prisustvu ili odsustvu prirodnih predatora.Distribution of a ctenophore Mnemiopsis leidyi was until 1980s limited to the Atlantic coast of North and South America, after which the species spread by ballast waters into the Black Sea, then progressively to Azov Sea, Caspian Sea, Mediterranean Sea, North and Baltic Sea. In this thesis, the origin and dynamics of M. leidyi invasion and the biological – ecological features of this species are described. Mnemiopsis leidyi inhabits coastal temperate waters where it achieves the highest biomass and dominates the planctonic communities during the warm season with notable seasonal changes. Genetic studies demonstrated that the invasion of Euroasian waters had two origins: the Black Sea population originated from the Gulf of Mexico, while the North and Baltic Sea were colonized by individuals from New England. Due to continuing transport by transoceanic shipping it is anticipated that M. leidyi will continue to expand. The impact the species elicits on the invaded ecosystem depends on various abiotic factors, local curents and the presence or absence of natural predators

Toxic effect of copper in neuroblastoma SH-SY5Y cells

Oksidacijski stres, uzrokovan neravnotežom stvaranja i uklanjanja reaktivnih kisikovih i dušikovih vrsta, ima važnu ulogu u starenju mozga i razvoju neurodegenerativnih bolesti. U ovom radu, istraživala sam toksični učinak bakra na stanice neuroblastoma SH-SY5Y. Testom MTT, tripanskim modrilom i mjerenjem razine ATP-a ustanovila sam značajan pad preživljenja stanica SH-SY5Y nakon tretmana bakrom. Bakar je povećao proizvodnju reaktivnih vrsta, koje pri visokim vrijednostima izazivaju oksidacijsko oštećenje, te smanjio razinu glutationa. Također, izazvao je kondenzaciju kromatina bez aktivacije kaspaza 3 i 7, kao i oštećenja stanične membrane, iz čega zaključujem da je bakar izazvao programiranu staničnu smrt putem neovisnim o aktivaciji kaspaza i nekrozu. Na proteinskoj razini došlo je do povećane ekspresije proteina p53 i PARP-1, dok se ekspresija proteina NME1 smanjila. Primjena inhibitora proteina PARP-1 (PJ34) nije utjecala na toksičnost, dok je primjena inhibitora proteina p53 (pifitrin-α) uzrokovala povećanje toksičnosti bakra. Flavonoid miricetin pojačao je citotoksičnost bakra prooksidacijskim djelovanjem. Dobiveni rezultati razjašnjavaju mehanizam prooksidacijskog djelovanja bakra na staničnoj i molekularnoj razini što bi moglo pridonijeti razvoju novih farmakoloških pristupa u prevenciji i terapiji neurodegenerativnih bolesti.Oxidative stress, caused by an imbalance in the formation and elimination of reactive oxygen and nitrogen species, plays an important role in brain aging and the development of neurodegenerative diseases. In this study, I investigated the toxic effect of copper on SH-SY5Y neuroblastoma cells. By MTT test, trypan blue exclusion assay, and ATP level measurement, I found a significant decline in SH-SY5Y cell survival after copper treatment. Copper has increased the production of reactive species, which at high values cause oxidative damage, and reduced glutathione levels. It also caused chromatin condensation without caspase 3 and 7 activation, as well as cell membrane damage, from which I conclude that copper caused programmed cell death via caspase-independent activation and necrosis. At the protein level, there was increased expression of p53 and PARP-1 proteins, while NME1 protein expression decreased. Administration of the PARP-1 protein inhibitor (PJ34) did not affect toxicity, whereas administration of the p53 protein inhibitor (pifitrin-α) caused an increase in copper toxicity. The flavonoid myricetin enhanced the cytotoxicity of copper by its prooxidative action. The obtained results clarify the mechanism of prooxidative action of copper at the cellular and molecular level, which could contribute to the development of new pharmacological approaches in the prevention and treatment of neurodegenerative diseases

Genetic diversity and the origin of invasive populations of ctenophore Mnemiopsis leidyi

Rasprostranjenost rebraša Mnemiopsis leidyi je do 1980-tih godina bila ograničena na područje atlantske obale Sjeverne i Južne Amerike, nakon čega se vrsta proširila balastnim vodama u Crno more, a zatim postupno i u Azovsko more, Kaspijsko jezero, Sredozemno more te Sjeverno i Baltičko more. U radu su opisani podrijetlo i dinamika invazije M. leidyi te biološko-ekološke značajke ove vrste. Mnemiopsis leidyi nastanjuje priobalne vode umjerenih mora gdje postiže najveću biomasu i dominira planktonskim zajednicama u toplom dijelu godine s izraženim sezonskim promjenama. Genetske studije pokazuju da je do invazije u euroazijske vode došlo iz dva područja: populacija iz Crnog mora potječe iz Meksičkog zaljeva dok su Sjeverno i Baltičko more kolonizirani jedinkama iz Nove Engleske. Zahvaljujući prekooceanskom prijevozu, predviđa se nastavak širenja Mnemiopsisa a kakav će utjecaj imati na novi ekosustav ovisi o različitim abiotičkim čimbenicima, lokalnim strujama te prisustvu ili odsustvu prirodnih predatora.Distribution of a ctenophore Mnemiopsis leidyi was until 1980s limited to the Atlantic coast of North and South America, after which the species spread by ballast waters into the Black Sea, then progressively to Azov Sea, Caspian Sea, Mediterranean Sea, North and Baltic Sea. In this thesis, the origin and dynamics of M. leidyi invasion and the biological – ecological features of this species are described. Mnemiopsis leidyi inhabits coastal temperate waters where it achieves the highest biomass and dominates the planctonic communities during the warm season with notable seasonal changes. Genetic studies demonstrated that the invasion of Euroasian waters had two origins: the Black Sea population originated from the Gulf of Mexico, while the North and Baltic Sea were colonized by individuals from New England. Due to continuing transport by transoceanic shipping it is anticipated that M. leidyi will continue to expand. The impact the species elicits on the invaded ecosystem depends on various abiotic factors, local curents and the presence or absence of natural predators