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Understanding movement of pharmaceuticals across rainbow trout gills using an in vitro gill cell culture system

Student thesis: Doctoral ThesisDoctor of Philosophy

The presence of pharmaceuticals in the aqueous environment has become widespread and the effects of many of these compounds on aquatic biota is unknown. Pharmaceuticals are designed to be bioavailable and have biological effects and may cause effects in non-target organisms, such as fish. Many pharmaceuticals are ionisable and for these compounds the pH of the external water determines speciation, which is thought to be largely important in movement of pharmaceuticals across membranes. Due to the unknown effects of the presence of pharmaceuticals, environmental risk assessments are performed to assess the persistent, bioaccumulative and toxic properties that a pharmaceutical may pose to the environment. These tests use many fish and in vitro methods have been proposed to reduce, replace and refine current procedures in risk assessment. The gills are an important site of xenobiotic permeability due to constantly being bathed in external water from the environment, in addition to being an incredibly important organ for acid base balance, osmoregulation and nitrogen excretion. A rainbow trout in vitro gill cell cultures system has been developed which shows promise as a surrogate for the intact gill. The aim of this thesis was to use the fish in vitro gill cell culture system (FIGCS) to investigate how pharmaceuticals, particularly ionisable pharmaceuticals, are taken up from the water by the fish gill and what is the importance of speciation on permeability. The uptake of a diverse cohort of pharmaceuticals with varying molecular descriptors was determined in FIGCS and analysed using multivariate analysis to determine that all the chosen chemical descriptors (log S, log D, log kow, MW, PSA, pka) contributed to uptake but the majority of explained variance was correlated to log S, log D, MW, and pka. A focused investigation on the permeability of propranolol, a basic drug with pkof 9.42, and ibuprofen, an acidic drug with pkof 4.91, clearly showed that the water pH and speciation in the bulk water had a significant effect on the uptake, efflux and internal concentration of the pharmaceuticals. However, the uptake of propranolol and ibuprofen was not linearly correlated with pH and the largest changes in rates were observed between pH 7 and 8 as opposed to around the pka, where changes in speciation are the greatest. The changes in permeability were not a result of changes in transepithelial potential from changing the external pH. An efflux experiment with simplified media determined that propranolol was excreted across the FIGCS. However, ibuprofen was not readily excreted likely due to being bound to foetal bovine serum present in the media or competition between amino acids and ibuprofen at exporters. The results contribute to a mounting body of evidence demonstrating the importance of external pH on permeability of ionisable pharmaceuticals and that the ionised form is able to cross the membrane to some extent. This, in addition to the observation that the largest fluctuations in permeability may not occur around the pka, should all be considered in future environmental risk assessments.
Original languageEnglish
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Award date1 May 2020

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