P04-03 Plasma-derived extracellular vesicles as minimally invasive source of novel biomarkers for circadian disruption in night shift workers

Night shift work (NSW) disrupts circadian rhythms and has been classified as a probable human carcinogen, with evidence linking long-term exposure to metabolic, cardiovascular, and inflammatory alterations [1]. Identifying early biological changes associated with circadian disruption is essential to improve occupational risk assessment and preventive strategies [2]. Extracellular vesicles (EVs) are lipid bilayer nanoparticles that carry bioactive molecules and are emerging as key mediators of intercellular communication [3]. They may serve as sensitive circulating biomarkers of early systemic responses to environmental and occupational stressors, including circadian disruption [4]. Female healthcare workers are frequently exposed to night shifts, yet remain underrepresented in occupational biomonitoring studies, despite potential sex-specific differences in circadian regulation and inflammatory responses [5]. Aim of the study is to develop and validate a robust protocol for the isolation and characterization of plasma-derived EVs and to explore their potential as early biomarkers of biological effects associated with NSW in female healthcare workers. EVs isolated from plasma samples by size exclusion chromatography (SEC), and concentrated by ultracentrifugation (UC). Particle size distribution and concentration, assessed by nanoparticle tracking analysis (NTA), showed a mode diameter of 51.5 nm (mean 78 ± 59 nm), consistent with small EVs (e.g., exosomes) [6], with a total particle concentration of ~4 × 10¹¹ per preparation. Next, western blot analysis confirmed the presence of vesicular markers involved in biogenesis (CD63, CD9, TSG101, β-integrin), and the depletion of cellular contaminants (calnexin, SDHA, actin, vinculin) and plasma albumin, indicating successful enrichment of EVs, suitable for downstream analyses. Building on these methodological advances, ongoing analyses compare EV cargo profiles between NSW-exposed and non-exposed workers, focusing on mediators of low-grade inflammation, including IL-6 receptor signaling components, as potential early indicators of systemic responses to circadian disruption [7]. The identification of EV-based molecular signatures associated with night shift exposure may provide minimally invasive tools for early detection of biological effects and improved occupational risk evaluation.