The Production And Cytotoxicity Of The Reactive Oxygen Species (ROS) Induced By Diallyl Trisulfide (DATS) In Human Leukemia HL-60 Cells

OBJECTIVES The aim of the study is to explore the production and cytotoxicity of the reactive oxygen species (ROS) induced by diallyl trisulfide (DATS) in HL-60 cells.METHODS HL-60 cells were either treated with various doses of DATS alone, or DATS combination with Apocynin, a NADPH oxidase inhibitor, or with antioxidant N-acetyl-L-cysteine (NAC) for 0, 1, 3, 6, 12 and 24 hours, respectively. The cells were harvested and stored for use. The apoptosis was detected by AO/EB staining and cell counting. Theintracellular ROS level was measured by flow eytometry. The content of both malondialdehyde (MDA) and the protein carbonyl was analyzed by spectrophotometer.RESULTS The results from flow cytometry indicated that DATS significantly increased the intracellular ROS level in HL-60 cells (P<0.05), which is a dose-and time-dependent. The fluorescence intensities of ROS reached at maximuam when HL-60 cells were incubated with DATS of 150μM for 3 hours, meanwhile the ROS of HL-60 cell decreased to approximately 80% after pre-incubating with Apocynin. The NBT reduction experiment showed that DATS activated NADPH oxidase which had highest activity when cell were exposed to DATS for 3 hours. Our results revealed that DATS induced apoptosis and production of MDA and protein carbonyl in HL-60 cells. Fourthermore, both MDA and protein carbonyl in the cells exposed to DATS of 150μM DATS for 3 hours reached the highest leve of 2.711±0.065nmol/mg and 10.813±0.586nmol/mg, respectively. Apocynin could attenuate the production of MDA and protein carbonyl, which suggested that ROS induced by DATS was involved in the damage to cells.CONCLUSION DATS activate NADPH oxidase and increase ROS production in HL-60 cells. NADPH oxidase is a key source of the ROS formation. DATS induce the apoptosis of HL-60 cell, which is mediated by the ROS. DATS damage the cellular membrane and the protein of HL-60 cell.