| The effect of selenium on modulating arsenic cytotoxicity is well known in mammals, but not well understood. Cell cytotoxicity and reactive oxygen (ROS) changes were performed in combinations of As(III) and selenomethionine (SeMet) toxic mixes on, HEK 293, human embryonic kidney cells. Cell growth is readily restored from 20% to 60% when switching from 30 muM As(III) as toxin to a mix of 30 muM As(III) and 100 muM SeMet. As(III) alone triggers ROS formation, primarily hydrogen peroxide, in a concentration dependent manner as observed through changes in the fluorescence from 2',7'-dichlorofluorescin diacetate. Importantly, SeMet induces lower ROS levels at the same concentrations used to modulate As(III) cytotoxicity (IC50). Elevated ROS is important to As(III) cytotoxicity and minimizing it is essential to the SeMet modulating function. Changes in cell signaling, through analysis of signaling changes via differential protein phosphorylation to uncover molecular level changes occurring in HEK 293 human kidney cells as SeMet modulates the As(III) cytotoxicity. To discover changes in the phosphoproteome, cells were incubated under three conditions: 30 muM As(III), 100 muM SeMet, and 30 muM As(III)+ 100 muM SeMet. After total protein isolation the three samples were separated into fractions using size exclusion chromatography by detecting 31P +. Each sample was analyzed for the phosphorylated peptides by enzymatic digestion, selective enrichment of phosphorylated peptides via TiO2, followed by nanoLC-ESIMS. Phosphorylated proteins unique to the As(III)/SeMet mixture were then identified. The molecular level changes to the cells show uniquely that the As(III)/SeMet mixture details proteins involved in ROS detoxification, cell cycle arrest, and protein/DNA damage. This study shows that SeMet not only lowers the total amount of ROS in a cell but also confers upon HEK 293 cells the ability to detoxify. Thus, SeMet is not only a potent antioxidant in this system, but induces molecular changes that confer survival. |
