Xenobiotics use and impact on human being is one of the major public health concerns. Despite their beneficial role in improving the daily lives of many individuals, they can also be the source of serious side effects in humans, by acting on different organs including the brain. The latter contains a physiological barrier, the blood brain barrier (BBB), which protects the brain from the entrance of detrimental molecules. One of the main BBB component is endothelial cells, which assure proper BBB functions, essentially its integrity. This function may be compromised upon xenobiotics exposure and lead to BBB breakdown. However, little is known about the mechanisms causing this BBB degradation. The detrimental effects of xenobiotics such as paraquat and morphine on human brain microvascular endothelial cells and their released extracellular vesicles (EVs) have never been studied before.

The first part of this thesis was focused on the proteome modulation of human brain microvascular endothelial cells (HBMECs) upon paraquat (PQ) and morphine exposure. Data independent acquisition-mass spectrometry (DIA-MS) combined to bioinformatics tools allowed to unveil altered proteins related to oxidative stress responses induced by both xenobiotics as well as a novel cellular pathway associated to cholesterol biosynthesis induced only by PQ. These results were verified with cellular-based assays, supporting proteomics findings.

The second part of this thesis was dedicated to the investigation of extracellular vesicles released by HBMECs upon xenobiotics exposure. After proper EVs isolation with a chemical affinity-based method and a wide range of orthogonal techniques used to characterize them, protein content analysis was undertaken through high-throughput proteomics. These studies reported modulation of proteins associated to the extracellular matrix remodeling (only modulated by morphine) and to the HIF1 pathway, a pathway linked to oxidative stress, for both molecules. These findings confirmed that EVs reflect the altered protein content of their parent cells, and, therefore, the possibility of using them as markers of BBB dysfunction.