2,4-D causes oxidative stress induction and apoptosis in human dental pulp stem cells (hDPSCs)

Abstract

2,4-Dicholorophenoxy acetic acid (2,4-D) is a worldwide used hormone herbicide. Human dental pulp stem cells (hDPSCs) as a potential source of mesenchymal stem cells provide a confident model system for the assessments of chemicals in vitro. The main objective of this study was to examine the biological effects and damages attributed to 2,4-D on hDPSCs. hDPSCs were isolated from third molar pulp tissues and their mesenchymal identity were evaluated. Then, hDPSCs were treated with increasing concentrations of 2,4-D (0.1 μM-10 mM). Cell viability assay and cumulative cell counting were carried out to address 2,4-D effects on biological parameters of hDPSCs. Cell cycle distribution, ROS level and ALP activity were measured before and after treatment. AO/EB staining and caspase 3/7 activity were investigated to detect the possible mechanisms of cell death. Flow-cytometric immunophenotyping and differentiation data confirmed the mesenchymal identity of cultivated hDPSCs. 2,4-D treatment caused a hormetic response in the viability and growth rate of hDPSCs. G0/G1 cell cycle arrest, enhanced ROS level, and reduced ALP activity were detected in hDPSCs treated with EC50 dose of 2,4-D. AO/EB staining showed a higher percentage of alive cells in lower concentrations of the herbicide. The increment in 2,4-D dose and the number of early and late apoptotic cells were increased. DAPI staining and caspase 3/7 assay validated the induction of apoptosis. 2,4-D concentrations up to 100 μM did not affect hDPSCs viability and proliferation. The intense cellular oxidative stress and apoptosis were observed at higher concentration.

Keywords: 2,4-D; Apoptosis; Hormesis; Mesenchymal stromal cells; Viability; hDPSCs.