ABSTRACT
In this study, we assessed the role of different reactive oxygen species (ROS) generated by soft jet plasma and chemical-induced ROS systems with regard to cell death in T98G, A549, HEK293 and MRC5 cell lines. For a comparison with plasma, we generated superoxide anion (O2(-)), hydroxyl radical (HO·), and hydrogen peroxide (H2O2) with chemicals inside an in vitro cell culture. Our data revealed that plasma decreased the viability and intracellular ATP values of cells and increased the apoptotic population via a caspase activation mechanism. Plasma altered the mitochondrial membrane potential and eventually up-regulated the mRNA expression levels of BAX, BAK1 and H2AX gene but simultaneously down-regulated the levels of Bcl-2 in solid tumor cells. Moreover, a western blot analysis confirmed that plasma also altered phosphorylated ERK1/2/MAPK protein levels. At the same time, using ROS scavengers with plasma, we observed that scavengers of HO· (mannitol) and H2O2 (catalase and sodium pyruvate) attenuated the activity of plasma on cells to a large extent. In contrast, radicals generated by specific chemical systems enhanced cell death drastically in cancer as well as normal cell lines in a dose-dependent fashion but not specific with regard to the cell type as compared to plasma.
Subject(s)
Plasma Gases/pharmacology , Reactive Oxygen Species/pharmacology , Adenosine Triphosphate/metabolism , Apoptosis , Caspase 3/metabolism , Caspase 7/metabolism , Cell Line, Tumor , Cell Survival/drug effects , Culture Media , Extracellular Signal-Regulated MAP Kinases/metabolism , Gene Expression , HEK293 Cells , Histones/genetics , Histones/metabolism , Humans , Membrane Potential, Mitochondrial , Oxidation-Reduction , Oxidative Stress , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Reactive Nitrogen Species/pharmacology , Up-Regulation , bcl-2 Homologous Antagonist-Killer Protein/genetics , bcl-2 Homologous Antagonist-Killer Protein/metabolism , bcl-2-Associated X Protein/genetics , bcl-2-Associated X Protein/metabolismABSTRACT
Reactive oxygen and nitrogen species can have either harmful or beneficial effects on biological systems depending on the dose administered and the species of organism exposed, suggesting that application of reactive species can possibly produce contradictory effects in disease control, pathogen inactivation and activation of host resistance. A novel technology known as atmospheric-pressure non-thermal plasma represents a means of generating various reactive species that adversely affect pathogens (inactivation) while simultaneously up-regulating host defense genes. The anti-microbial efficacy of this technology was tested on the plant fungal pathogen Fusarium oxysporum f.sp. lycopersici and its susceptible host plant species Solanum lycopercicum. Germination of fungal spores suspended in saline was decreased over time after exposed to argon (Ar) plasma for 10 min. Although the majority of treated spores exhibited necrotic death, apoptosis was also observed along with the up-regulation of apoptosis related genes. Increases in the levels of peroxynitrite and nitrite in saline following plasma treatment may have been responsible for the observed spore death. In addition, increased transcription of pathogenesis related (PR) genes was observed in the roots of the susceptible tomato cultivar (S. lycopercicum) after exposure to the same Ar plasma dose used in fungal inactivation. These data suggest that atmospheric-pressure non-thermal plasma can be efficiently used to control plant fungal diseases by inactivating fungal pathogens and up-regulating mechanisms of host resistance.
Subject(s)
Disease Resistance/genetics , Fungi/drug effects , Microbial Viability/drug effects , Plant Diseases/genetics , Plant Diseases/microbiology , Plants/genetics , Plants/microbiology , Plasma Gases/pharmacology , Apoptosis/drug effects , Fungi/growth & development , Fungi/metabolism , Fungi/ultrastructure , Gene Expression Regulation, Plant/drug effects , Nitrites/metabolism , Phenotype , Reactive Nitrogen Species/metabolism , Reactive Oxygen Species/metabolism , Spores, FungalABSTRACT
The antimicrobial efficiency of reactive species-based control strategies is significantly affected by the dynamics of reactive species in the biological environment. Atmospheric-pressure nonthermal plasma is an ionized gas in which various reactive species are produced. The various levels of antimicrobial activity may result from the dynamic interaction of the plasma-generated reactive species with the environment. However, the nature of the interaction between plasma and environments is poorly understood. In this study, we analyzed the influence of the ionic strength of surrounding solutions (environment) on the antimicrobial activity of plasma in relation to the plasma-generated reactive species using a model filamentous fungus, Neurospora crassa. Our data revealed that the presence of sodium chloride (NaCl) in the background solution attenuated the deleterious effects of plasma on germination, internal structure, and genomic DNA of fungal spores. The protective effects of NaCl were not explained exclusively by pH, osmotic stability, or the level of reactive species in the solution. These were strongly associated with the ionic strength of the background solution. The presence of ions reduced plasma toxicity, which might be due to a reduced access of reactive species to fungal spores, and fungal spores were inactivated by plasma in a background fluid of nonionic osmolytes despite the low level of reactive species. Our results suggest that the surrounding environment may affect the behavior of reactive species, which leads to different biological consequences regardless of their quantity. Moreover, the microbicidal effect of plasma can be synergistically regulated through control of the microenvironment.
