Exposure to 2,4-Dichlorophenoxyacetic acid stimulates the calcium/ROS/CK1α pathway to trigger Hemolysis and Eryptosis in red blood cells

Abstract Objective The agricultural herbicide 2,4-dichlorophenoxyacetic (2,4-D) is cytotoxic to human red blood cells (RBCs) by virtue of oxidative hemolysis. Nevertheless, there remains a great paucity of literature detailing the mechanisms by which 2,4-D triggers hemolysis. Also, the eryptotic effects of 2,4-D has thus far been largely overlooked. This study aims to expand current understanding of the cytotoxic properties of 2,4-D in RBCs. Methods Cells were exposed to 2,4-D ranging from 100 to 1,000 μM for 24 h at 37 °C under varied experimental conditions. Hemolysis, LDH, AST, and AChE activities were photometrically measured. Flow cytometry assessed eryptotic markers including cell volume by forward scatter (FSC), phosphatidylserine (PS) externalization by annexin-V positivity, reactive oxygen species by H2DCFDA, and intracellular Ca2+ levels by Fluo4/AM. Results 2,4-D induced Ca2+-independent, concentration-responsive hemolysis paralleled by increased LDH, AST, and K+ in the supernatant, which was significantly blunted by D4476, isosmotic urea, sucrose, and polyethylene glycol 8,000 (PEG). Notably, 2,4-D caused a significant increase in cells positive for annexin-V-FITC, DCF, and Fluo4 with a concomitant decrease in AChE activity and FSC following KCl release. Furthermore, lymphocytes and reticulocytes were sensitive to 2,4-D within a whole blood milieu. Conclusion This work introduces novel cytotoxic mechanisms of 2,4-D in RBCs and reveals its pro-eryptotic effects. 2,4-D toxicity is neutralized by blockade of casein kinase 1α signaling and the presence of urea, sucrose, and PEG. These findings have significant implications for public health and inform future health risk assessments to develop novel preventive and therapeutic strategies.