Abstract
Abstract in English, Croatian The aim of this study was to investigate the oxidative and apoptotic potential of fluoxetine, a widely used antidepressant in Turkey and the world, and of its metabolite norfluoxetine on a model non-target organism, Daphnia magna to see how exposure to this group of antidepressants (specific serotonin reuptake inhibitors) could affect the aquatic environment in which they end up. Juvenile D. magna specimens were chronically exposed to fluoxetine and norfluoxetine alone and in combination at concentrations found in the aquatic environment (0.091 and 0.011 μg/L, respectively) and to their 10-fold environmental concentrations for 21 days. Another group of 17-day-old animals were subacutely exposed to 100-fold environmental concentrations for four days. After exposure, we measured their glutathione peroxidase (GPx) and cholinesterase (ChE) activities, thiobarbituric acid-reactive substances (TBARS), and total protein content spectrophotometrically, while mitochondrial membrane potential (MMP) was analysed by fluorescence staining, and cytochrome c and ERK1/2 protein content by Western blotting. This is the first-time cytochrome c and ERK1/2 were determined at the protein level in D. magna. We also measured their carapace length, width, and caudal spine length microscopically. At environmental concentrations fluoxetine and norfluoxetine caused an increase in ChE activity and brood production. They also caused a decrease in juvenile carapace length, width, and caudal spine length and depolarised the mitochondrial membrane. At 10-fold environmental concentrations, GPx activity, lipid peroxidation levels, cytochrome c, and ERK1/2 protein levels rose. The most pronounced effect was observed in D. magna exposed to norfluoxetine. Norfluoxetine also decreased brood production. Similar effects were observed with subacute exposure to 100-fold environmental concentrations. However, total protein content decreased. All this confirms that fluoxetine and norfluoxetine have oxidative and apoptotic potential in D. magna. Daphnia spp. have a great potential to give us precious insight into the mechanisms of environmental toxicants, but there is still a long way to go before they are clarified in these organisms. Cilj je ovoga istraživanja bio utvrditi oksidacijski i apoptotski potencijal fluoksetina, antidepresiva raširenoga u Turskoj i svijetu, i njegova metabolita norfluoksetina na modelu vodenbuhe Daphnia magna koji nije ciljani organizam djelovanja spojeva. Također smo željeli vidjeti kako izloženost toj skupini antidepresiva (specifičnih inhibitora ponovne pohrane serotonina) može utjecati na vodeni okoliš u kojem oni završe. Mlade jedinke vodenbuhe bile su izložene fluoksetinu (0,091 μg/L) i norfluoksetinu (0,011 μg/L), odvojeno i u kombinaciji, pri koncentracijama zamijećenima u okolišu i deseterostrukim okolišnim koncentracijama u trajanju od 21 dan (kronična izloženost). Jedna je skupina 17 dana starih vodenbuha također bila izložena stostrukoj okolišnoj koncentraciji u trajanju od četiri dana (subakutna izloženost). Potom su u životinja spektrofotometrijom izmjerene aktivnosti enzima glutation peroksidaze (GPx) i kolinesteraza (ChE) te razine reaktivnih tvari tiobarbituratne kiseline (TBARS) i ukupnih proteina. Potencijal mitohondrijske membrane (MMP) utvrđen je fluorescencijom, a proteini citokrom c i ERK1/2 Western blot metodom. Ovo je prvi put da su se u vodenbuhi citokrom c i ERK1/2 utvrđivali na razini proteina. Također je mikroskopski izmjerena dužina i širina oklopa i dužina repne bodlje vodenbuha. Pri okolišnim koncentracijama fluoksetin i norfluoksetin doveli su do povišene aktivnost ChE i većeg razmnožavanja te smanjenja (dužine i širine) karapaksa i repne bodlje u podmlatka i depolarizacije mitohondrijske membrane. Pri deseterostrukim okolišnim koncentracijama porasle su razine aktivnosti GPx, lipidne peroksidacije, citokroma c i ERK1/2 proteina. Norfluoksetin je pritom iskazao najsnažnije djelovanje te doveo do pada razmnožavanja. Slični su učinci primijećeni kod subakutne izloženosti stostrukim okolišnim koncentracijama fluoksetina i norfluoksetina, osim što je ona dovela i do pada ukupnih proteina. Naši rezultati potvrđuju da fluoksetin i norfluoksetin imaju oksidacijski i apoptotski učinak u vodenbuhe. Taj životinjski model može odlično poslužiti za stjecanje uvida u mehanizme toksičnoga djelovanja tvari u okolišu, no potrebna su daljnja istraživanja prije nego što ti mehanizmi postanu potpuno jasni.