Effects of atrazine on the proliferation and cytotoxicity of murine lymphocytes with the use of carboxyfluorescein succinimidyl ester-based flow cytometric approaches

Atrazine (ATZ) is one of the most commonly applied herbicides worldwide. ATZ has been associated with adverse effects on the immune system; however, the mechanism of its immunotoxicity has not been completely elucidated. In this study, the immunotoxic effects of ATZ on murine splenic lymphocytes and magnetic bead-enriched NK cells were investigated in vitro with the use of carboxyfluorescein succinimidyl ester (CFDA-SE)-based flow cytometric approaches. Proliferation responses, NK cell activity, and T-cell early activation were determined with CFDA-SE loading, CFDA-SE/propidium iodide (PI) staining, and CD69+ expression, respectively. Cell apoptosis/cycle, reactive oxygen species (ROS), and mitochondrial membrane potential (MMP) were evaluated using PI, 2',7'-dichlorodihydrofluorescein diacetate, and rhodamine 123, respectively. The intracellular expressions of apoptosis-related Bcl-2 and caspase-3 were analyzed through intracellular staining and flow cytometry. Results showed that proliferation and NK cell activity were suppressed by ATZ treatment. Such suppression might be associated with the cell apoptosis induced by increased ROS and declined MMP. The underlying mechanism might be the induced caspase-3 expression and decreased Bcl-2 expression. ATZ could elicit immunotoxic effects on murine lymphocytes; its presence in the environment might compromise immune function in organisms. The flow cytometric methods presented in this study should be further investigated in immunotoxicology. (C) 2014 Elsevier Ltd. All rights reserved.