Involvement of MAPK and Akt in the immunotoxicity of fusaric acid on healthy peripheral blood mononuclear cells (PBMC) and acute monocytic leukemic (Thp-1) cells.

Abstract

Fusaric acid is a divalent chelator with moderate toxicity in plant and animals. However, studies lack on its effect on human models and the immune system. This study investigated the immunotoxicity of FA on PBMCs and Thp-1 cells. Cell viability was determined using the WST-1 assay and the mode of cell death by flow cytometry using the annexin V-FITC stain. Caspase 8, 9 and 3/7 activities were determined using Caspase-Glo assay®. TNF-α levels were measured using the TNF-α ELISA kit. Oxidative damage (MDA) was determined using the TBARS assay. Flow cytometry was performed to determine mitochondrial function using the JC-1 stain. ATP levels were measured using the ATP CellTitre Glo reagent. Western blotting was performed to determine protein expressions of Bax, p- Bcl-2, p-Akt, p-ERK, p-JNK and p38. The immunotoxicity of FA was confirmed by the decreased cell viability of PBMCs and Thp-1 cells and was validated by the externalization of phosphatidylserine on both PBMCs (p<0.005) and Thp-1 cells (p<0.0001). In PBMCs, FA induced paraptosis, evidenced by the decreased caspase 8 (p<0.005), 9 (p<0.05) and 3/7 (p<0.005) activities. Whilst in Thp-1 cells, FA induced intrinsic apoptosis supported by a decrease in caspase 8 activity (p<0.05) and an increase in caspase 9 (p<0.05) and 3/7 (p<0.005) activities; corresponding with unchanged TNF-α levels in both PBMCs (p=0.3015) and Thp-1 cells (p=0.4540). In PBMCs, FA significantly decreased Bax (pro-apoptotic) protein expression (p<0.05) and increased p-Bcl-2 (antiapoptotic) protein expression (p<0.05) thereby maintaining mitochondrial membrane potential (p=0.5643). In Thp-1 cells, FA had no effect on the protein expression of Bax (p=0.6130) but significantly decreased the protein expression of p-Bcl-2 (p<0.005) with a corresponding increase in mitochondrial depolarization (p<0.005). In addition, FA increased oxidative stress (MDA levels) in both PBMCs (p<0.005) and Thp-1 cells (p<0.005) contributing to cellular damage and cellular signaling; and substantially decreased ATP levels in both PBMCs (p<0.005) and Thp-1 cells (p<0.005). Additionally, FA significantly increased phosphorylation of p-ERK (42kDa - p<0.05; 44kDa - p<0.005), p-JNK (46kDa - p<0.005; 54kDa - p<0.05) and p38 (p<0.05); and slightly increased the phosphorylation of p-Akt (p=0.1640) in PBMCs treated with FA. In Thp-1 cells, FA significantly up-regulated p-Akt (p<0.05) and p-ERK (42kDa - p<0.0001; 44kDa - p<0.005) expressions and significantly decreased p-JNK (46kDa - p<0.05; 54kDa - p<0.005) expression but had no effect on the expression of p38 (p=0.8446). This suggests the involvement of MAPK signaling in the induction of cell death in PBMCs and Thp-1 cells treated with FA. This study found that FA is immunotoxic to healthy human PBMCs and Thp-1 cells.