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.
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Date
2015
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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.
Description
Master of Medical Sciences in Medical Biochemistry. University of KwaZulu-Natal, Medical School 2015.
Keywords
Mitogen-activated protein kinases., Protein kinases., Mononucleosis., Acute myeloid leukemia., Theses -- Medical biochemistry., Peripheral blood mononuclear cells (PBMC)