The Role of Environmentally Relevant Concentrations of SSRIs in Human Wound Healing

This thesis investigates the role of environmentally relevant concentrations of fluoxetine, a commonly prescribed selective serotonin reuptake inhibitor (SSRI), in wound healing. While SSRIs are effective in treating psychiatric conditions, their increasing presence in aquatic environments has raised concerns about unintended biological impacts. SSRI are widely-used antidepressants, and in the UK, antidepressant prescriptions have almost doubled in the past decade (Heald et al., 2021). After ingestion, SSRI are excreted via urine. Wastewater treatment does not remove SSRI effectively, leading to accumulation in freshwater courses (to 0.4-3,645 ng/l), making SSRI priority contaminants in ecotoxicology. Average fluoxetine and sertraline concentrations in England rivers across 2016-2024 were 218 and 15 ng/l, respectively, and up to 2,560 ng/l fluoxetine in post-treatment effluents. Population growth, urbanisation, and regional climate change-induced water scarcity can increase these concentrations. While exposure to environmental SSRI affects the physiology and behaviour of freshwater species, little is known about effects of exposure on human health. My hypothesis was that SSRIs, particularly fluoxetine, at environmentally relevant concentrations promote wound healing through serotonin signalling. To test this hypothesis, here, I investigate the influence of fluoxetine on wound healing models using human keratinocytes and ex-vivo human skin biopsies, assessing its effects on serotonin signalling pathways and cell proliferation.Using a combination of molecular and cell biology techniques, including scratch assays, RNA sequencing (RNAseq), protein microarrays and phosphoproteomics, alongside using an ex-vivo human skin model, I investigated the impact of fluoxetine at environmentally relevant concentrations (62.5-5400 ng/l) on wound closure, cell proliferation, and key signalling pathways. Results showed that fluoxetine increased scratch closure in a dose-dependent manner (by 5% and 20% at 125 and 5400 ng/l of fluoxetine) in keratinocyte models by promoting cell proliferation through serotonin receptor-mediated pathways. RNAseq revealed differential expression of exactly 100 upregulated and 250 downregulated genes involved in cell cycle progression, energy metabolism, cell proliferation, and cellular resilience. Protein microarrays and phosphoproteomics indicated dynamic changes in phosphorylation among key kinases, including GSK3β, MSK1/2, and p70 S6K, with 190 upregulated and 45 downregulated phosphorylated proteins. These proteins showed enrichment in GO terms and KEGG pathways related to cellular structure, stress response, and kinase signalling pathways, particularly in HIPPO and PI3K/AKT signalling. Ex-vivo experiments with human skin validated these findings in a physiologically-relevant model that maintains the wound microenvironment. I observed an increase in wound healing of 30% at 5400 ng/l compared to control biopsies, demonstrating enhanced wound closure upon exposure to environmentally relevant fluoxetine associated with serotonin pathway activation.Human wounds cost the NHS >£8.3 billion/year and new treatments are direly needed. This research underscores both the potential therapeutic applications of low-dose fluoxetine in wound care, and the importance of understanding its effects on healthy skin as anenvironmental pollutant. Together, these findings contribute to a deeper insight into the molecular and phenotypical impact of fluoxetine on human skin, with implications for both clinical and environmental health. My results justify a transition from the study of behavioural effects of environmental fluoxetine in aquatic animals to the investigation of effects of exposure on wound healing in aquatic and terrestrial animals, including direct impacts on human health. I also open avenues to investigate low-dose SSRI as new treatments to promote wound healing.