Date of Award
Discharge of pharmaceuticals into the environment is of emergent concern due to the unknown long-term effects on aquatic organisms. Wastewater treatment plants are not effective in completely removing pharmaceuticals, allowing them to be discharged into aquatic systems. To investigate the potential effects of fluoxetine, a common antidepressant, on aquatic species, the potential of four freshwater fish species to metabolize this drug was investigated. Livers from redbreast sunfish, bluegill, striped jumprock and bluehead chub were collected, homogenized, and centrifuged to collect the microsomes. These microsomes were used in an assay to determine the in vitro hepatic metabolism of fluoxetine by cytochrome P450 (CYP) enzymes. Formation of fluoxetine’s main metabolite in humans, norfluoxetine, was measured at different substrate concentrations using HPLC with fluorescence detection. Species differences of the calculated Vmax and Km values were compared and the bluegill enzymes were found to be the most efficient at binding fluoxetine, with the lowest average Km value and the highest average Vmax/Km value. Fish liver microsomes were orders of magnitude less efficient than rat microsomes in metabolizing fluoxetine, which was expected, as fluoxetine was developed for use in mammalian systems. To obtain a suggestion of which CYP isoforms might be responsible for the low rate of biotransformation in fish, the microsomes were also tested for metabolism of model substrates for mammalian CYP2 and CYP3 isoforms. These results suggest that CYP3 isoforms in fish may be more active than CYP2 isoforms. Future work should focus on more specific details of CYP isoforms responsible for fluoxetine metabolism in freshwater fish and if these isoforms are inducible, which would support their use as potential biomarkers for increased pharmaceutical pollution in aquatic ecosystems.
Steiner, Morgan, "CHARACTERIZING METABOLIC ACTIVITY OF FISH LIVER MICROSOMES TOWARDS FLUOXETINE IN FOUR FISH SPECIES USING MICHAELIS-MENTEN ENZYME KINETICS" (2023). Honors College Theses. 16.