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Synthetic glucocorticoids have been widely used for more than 70 years to treat a wide range of diseases and human health issues including asthma, rheumatoid arthritis, and allergies, primarily due to their immunosuppressive properties. Due to the excretion of these compounds by patients, over the past 20 years glucocorticoids have been regularly detected in aquatic environments at low ng/L concentrations. The glucocorticoid system is well conserved across vertebrates, acting through genomic and non-genomic pathways, and is essential to a number of physiological processes including glucose homeostasis, immunomodulation, early development including bone formation and stress-related behaviours. Synthetic glucocorticoids have been shown to be pharmacologically active in non-mammalian vertebrates, including fish, and thus, have the potential to affect fish in the aquatic environment. By using zebrafish (Danio rerio) as a model species, in this thesis, I was able to assess the potential for a wide range of glucocorticoids to affect fish at concentrations ranging from environmentally relevant concentrations to ≥ human therapeutic dose concentrations. In order to complete this, I undertook uptake and bioconcentration studies to understand how much of these compounds were able to accumulate in fish tissues, as well as exploring the effects on early development, teratogenicity, immune function, and bone and cartilage formation in early life exposures to embryo-larval zebrafish.
First, 4-day fish embryo-larval toxicity exposures were completed on Wild Indian Karyotype zebrafish to 4 synthetic glucocorticoids individually (beclomethasone dipropionate, budesonide, fluticasone propionate, and prednisolone). Throughout these exposures, embryo-larvae were assessed for effects on early-development and teratogenicity, including presence of abnormalities, assessment of developing morphological tissues and cartilage development. Exposures to glucocorticoids revealed that concentrations similar to human therapeutic dose concentrations were able to induce developmental abnormalities including pericardial oedema, alter development and tissue morphology including the shortening of a variety of jaw cartilage measures in zebrafish embryo-larvae in a number of the compounds tested, however environmentally relevant concentrations had no effect. In additional 4-day exposures, uptake of these compounds into the whole-body tissue of embryo-larvae was measured and bioconcentration was calculated. Uptake and bioconcentration factors were calculated for 3 out of the 4 compounds. These data were then compared to predicted uptake values calculated using the Fish Plasma Model and the effectiveness of this model to accurately predict uptake of these compounds was assessed. The ability of the Fish Plasma Model to predict uptake varied depending on compound, however, it generally overestimated uptake rates in embryo-larvae.
To build upon the effects seen on cartilage development after 4 days of exposure, 10-day exposures to glucocorticoids were completed in embryo-larvae using a transgenic model with fluorescent markers labelling osteoblasts (bone) and chondrocytes (cartilage). After 10 days of exposure to glucocorticoids, both bone and cartilage development was altered for all compounds tested at ≥ human therapeutic dose concentrations. For exposures to beclomethasone dipropionate, cartilage lengths in the lower jaw (intercranial distance) were reduced after exposure to 0.1 μg/L, a concentration considered environmentally relevant. It was also found that at high concentrations (≥10 μg/L), the stacking of the chondrocyte cells in these cartilaginous structures was altered and the shape of these cells was also affected. Analysis of the osteoblasts showed no change in the organisation or shape of these cells, however the total areas of these osteoblasts were reduced at these high concentrations.
As glucocorticoids are primarily used due to their immunosuppressive properties, it is likely that these compounds may also be able to induce these effects in fish. Using two transgenic zebrafish models with fluorescent markers labelling neutrophil and macrophage cells, the effects of a 4-day glucocorticoid exposure on the ability of these innate immune cells to migrate to damage sites was investigated. By exposing these transgenic zebrafish models to neomycin, which results in damage to the hair cells of the lateral line (neuromasts), it was possible to track and count the numbers of the innate immune cells migrating towards damaged neuromasts. All compounds tested were found to alter the migration of both neutrophils and macrophages, although exposure concentrations at which this occurred were not considered environmentally relevant. It was shown that in general, the motility of these cells was unchanged after exposure to glucocorticoids. Therefore, it is suggested that alterations in the ability of neutrophils and macrophages to sense chemoattractants/chemokines and extravasate from the circulatory system may be possible mechanisms behind the immunosuppressive activity of these compounds.
Collectively, my findings demonstrate that glucocorticoids can induce effects in embryo-larval zebrafish including on development, immune function, and cartilage and bone formation generally at concentrations greater than considered environmentally relevant. Therefore, for the most part, it is suggested that glucocorticoids present a low-level risk to fish populations. However, in this thesis, chronic studies (10-days) were able reveal effects at environmentally relevant concentrations for beclomethasone dipropionate exposures, therefore suggesting that lifetime chronic exposures to these compounds in the environment may pose a greater risk to fish species. Considering the total glucocorticoid concentration found in the environment, and in combination with mixtures of other pollutants, further chronic studies, possibly over multiple generations, should be explored to evaluate the risk of these compounds to fish, thus ensuring that this class of compound does not pose a significant threat to fish in the environment. |
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