Exposure to metoprolol and propranolol mixtures on biochemical, immunohistochemical, and molecular alterations in the American oyster, Crassostrea virginica

Pharmaceutical drugs, particularly beta-blockers (e.g., metoprolol, propranolol, etc.), are extensively used to treat human cardiovascular conditions, yet pose significant risks to non-target aquatic organisms when introduced into coastal and marine environments via wastewater effluent. This study aimed to investigate the effects of short-term exposure (one week) to environmentally relevant concentrations of metoprolol and propranolol (MP) mixtures (low-dose: 50 ng/L propranolol and 250 ng/L metoprolol, and high-dose: 250 ng/L propranolol and 650 ng/L metoprolol) in the American oyster (<i>Crassostrea virginica</i>, a commercially and ecologically important marine bivalve mollusk) under controlled laboratory conditions. Histopathological assessments revealed structural damage to gills, connective tissues, and digestive glands in both low- and high-dose MP treatment groups. Additionally, glucose concentration and pH of the extrapallial fluid significantly declined in the high-dose MP treatment groups. Hemocyte density in the connective tissues increased proportionally with MP dosages. MP mixtures significantly reduced mucous secretion in the gills and digestive glands. Immunohistochemical results showed significant (<i>P</i> < 0.05) upregulation of 3-nitrotyrosine protein (NTP, a biomarker of protein nitration) expression in tissues of oysters exposed to MP mixtures. Alongside, exposure to MP significantly (<i>P</i> < 0.05) decreased acetylcholinesterase (AChE, a cholinergic enzyme) expression in oyster tissues. Our findings suggest that beta-blockers induce protein nitration, leading to altered tissue morphology, disrupting extrapallial fluid homeostasis, and downregulating AChE expression that may impair physiological functions in oysters.