Vpliv izbranih zdravilnih učinkovin na citotoksično delovanje dopamina na človeške endotelijske celice in podganje astrocite

Zobec, Lea (author), Žiberna, Lovro (mentor), Sočan, Vesna (co-mentor)

Povišane koncentracije dopamina lahko povzročijo poškodbe nevronov in ostalih celic centralnega živčnega sistema, kar pospešuje napredovanje nevrodegenerativnih bolezni. Predpostavljen mehanizem poškodbe je encimska presnova dopamina preko encima monoamin oksidaze B (MAO–B), kjer pride do nastanka reaktivnih metabolitov in oksidativnega stresa. Cilj naše raziskave je bil preučiti vpliv dopamina na viabilnost in energijsko presnovo človeških endotelijskih celic ter astrocitov novorojenih podgan pa tudi potencialne zaščitne učinke izbranih učinkovin za preprečevanje oksidativnih poškodb. Za namen raziskave smo pripravili kulture človeških endotelijskih celic iz celične linije EA.hy926 in astrocitov neonatalnih podgan izoliranih iz možganske skorje. Na teh celičnih linijah smo preučevali vpliv dopamina na celično viabilnost v odvisnosti od njegove koncentracije, časa izpostavitve in prisotnosti izbranih učinkovin. Za preučevanje vpliva presnovkov dopamina smo celice predhodno inkubirali z zaviralci MAO–B in katehol-O-metiltransferaze (COMT). Uporabili smo tudi agoniste in antagoniste dopaminskih receptorjev, da bi preučili, ali gre za receptorsko-posredovane odgovore. Za ugotavljanje vloge privzema dopamina v celice smo uporabili zaviralce serotoninskega (SERT) in noradrenalinskega (NET) prenašalca. Prav tako smo merili znotrajcelični oksidativni stres in ga poskušali znižati s predhodno inkubacijo z antioksidantom kvercetinom. Za preučevanje vpliva dopamina in izbranih učinkovin na delovanje mitohondrijev smo izvedli še teste mitohondrijskega stresa na Seahorse XFe24 analizatorju, kjer smo merili aerobno presnovo. Ugotovili smo, da je citotoksično delovanje dopamina odvisno od koncentracije (1 nM – 1 mM) in trajanja inkubacije (3 – 48 ur). Zaviranje encimske presnove dopamina ni vplivalo na celično viabilnost, prav tako tudi ne na zaviranje njegovega privzema v celice in delovanje na receptorje. Ugotovili smo pa, da je dodatek antioksidanta kvercetina pred dodatkom dopamina povečal viabilnost celic endotelija in astrocitov. Izmerili smo, da je dopamin per se deloval pro-oksidativno, saj je povečal znotrajcelični oksidativni stres. Zaviralci presnovnih encimov MAO in COMT niso vplivali na oksidativni stres. Toksično delovanje dopamina smo ugotovili tudi na nivoju mitohondrijev, kjer izbrane učinkovine teh poškodb niso preprečile. Naše ugotovitve nakazujejo, da za celične in mitohondrijske poškodbe povezane s povečanim oksidativnim stresom niso odgovorni znotrajcelični presnovki dopamina preko MAO ali COMT poti, temveč snovi, ki se tvorijo iz ne-encimske avtooksidacije dopamina.

• Language: Slovenian
• Keywords: dopamin, citotoksičnost, encimska presnova dopamina, oksidativni stres, mitohondrijska funkcija
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2022
• PID: 20.500.12556/RUL-143483
• Publication date in RUL: 22.12.2022

Secondary language

• Language: English
• Title: Effect of selected drug compounds on the dopamine cytotoxicity on human endothelial cells and rat astrocytes

Abstract

Increased dopamine levels damage neurons and other cells in the central nervous system, thus leading to accelerated progression of neuro¬degenerative diseases. The presumed mechanism of dopamine neuro¬toxicity is its enzymatic metabolism via monoamine oxidase B (MAO–B) to reactive metabolites with corresponding oxidative stress. The aim of our research was to examine the influence of dopamine on endothelial cells and astrocytes, as well as the potential protective effects of selected drug compounds to ameliorate dopamine toxicity. Human endothelial cells EA.hy926 and isolated astrocytes from the cortex of neonatal rats were prepared to study the effect of dopamine on their viability depending on its concentration, exposure time, and the presence of selected drugs. To study the role of dopamine metabolites, all cells were pre-incubated with inhibitors of MAO–B and catechol-O-methyl¬transferase (COMT). To assess the role of cellular dopamine uptake, we used inhibitors of serotonin transporter (SERT) and the noradrenaline transporter (NET). Agonist and antagonist were used to determine the role of action on dopamine receptors. We also measured intracellular oxidative stress and attempted to prevent it by using the antioxidant quercetin. To investigate the effects of dopamine and selected drugs on mitochondrial function, we used the Agilent’s Seahorse Cell Mito Stress Test kit to measure aerobic metabolism. We discovered dopamine-induced time- (3  –  48 hours) and concentration- (1nM – 1mM) dependent cytotoxicity in both cell lines. Inhibition of dopamine metabolism did not increase cell viability, nor did action on its receptors. Similarly, inhibition of dopamine uptake had no effect. However, reduction of oxidative stress by quercetin increased cell viability. We confirmed by measuring oxidative stress that dopamine increased cellular oxidative stress and that neither MAO–B nor COMT inhibitors improved this condition. However, quercetin decreased oxidative stress in both cell lines. Likewise, the Mito Stress Test assay showed altered mitochondrial function by dopamine, and no protection of mitochondria by selected drug compounds. Our results suggest that intracellular metabolites of dopamine via the MAO and COMT pathways are not responsible for the cellular and mitochondrial damage associated with increased oxidative stress, but rather substances formed by non-enzymatic autooxidation of dopamine.

• Keywords: dopamine, cytotoxicity, dopamine enzyme metabolism, oxidative stress, mitochondrial function