Chonnam Med J.  2020 Jan;56(1):20-26. 10.4068/cmj.2020.56.1.20.

Fluoxetine Induces Apoptotic and Oxidative Neuronal Death Associated with The Influx of Copper Ions in Cultured Neuronal Cells

Affiliations
  • 1Department of Pharmacology, Chonnam National University Medical School, Hwasun, Korea. jkkim57@jnu.ac.kr

Abstract

We examined the effect of fluoxetine, a selective serotonin reuptake inhibitor antidepressant, on neuronal viability in mouse cortical near-pure neuronal cultures. Addition of fluoxetine to the media for 24 hours induced neuronal death in a concentration-dependent manner. To delineate the mechanisms of fluoxetine-induced neuronal death, we investigated the effects of trolox, cycloheximide (CHX), BDNF, z-VAD-FMK, and various metal-chelators on fluoxetine-induced neuronal death. Neuronal death was assessed by MTT assay. The addition of 20 µM fluoxetine to the media for 24 hours induced 60-70% neuronal death, which was associated with the hallmarks of apoptosis, chromatin condensation and DNA laddering. Fluoxetine-induced death was significantly attenuated by CHX, BDNF, or z-VAD-FMK. Treatment with antioxidants, trolox and ascorbate, also markedly attenuated fluoxetine-induced death. Interestingly, some divalent cation chelators (EGTA, Ca-EDTA, and Zn-EDTA) also markedly attenuated the neurotoxicity. Fluoxetine-induced reactive oxygen species (ROS) generation was measured using the fluorescent dye 2"²,7"²-dichlorofluorescin diacetate. Trolox and bathocuproine disulfonic acid (BCPS), a cell membrane impermeable copper ion chelator, markedly attenuated the ROS production and neuronal death. However, deferoxamine, an iron chelator, did not affect ROS generation or neurotoxicity. We examined the changes in intracellular copper concentration using a copper-selective fluorescent dye, Phen Green FL, which is quenched by free copper ions. Fluoxetine quenched the fluorescence in neuronal cells, and the quenching effect of fluoxetine was reversed by co-treatment with BCPS, however, not by deferoxamine. These findings demonstrate that fluoxetine could induce apoptotic and oxidative neuronal death associated with an influx of copper ions.

Keyword

Fluoxetine; Neurons; Cell Death; Reactive Oxygen Species; Copper

MeSH Terms

Animals
Antioxidants
Apoptosis
Brain-Derived Neurotrophic Factor
Cell Death
Cell Membrane
Chelating Agents
Chromatin
Copper*
Cycloheximide
Deferoxamine
DNA
Fluorescence
Fluoxetine*
Ions*
Iron
Mice
Neurons*
Reactive Oxygen Species
Serotonin
Antioxidants
Brain-Derived Neurotrophic Factor
Chelating Agents
Chromatin
Copper
Cycloheximide
DNA
Deferoxamine
Fluoxetine
Ions
Iron
Reactive Oxygen Species
Serotonin
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