Defects in Mitochondrial Functions Affect the Survival of Yeast Cells Treated with Non-Thermal Plasma.

Exposure of living cells to non-thermal plasma produced in various electrical discharges affects cell physiology and often results in cell death. Even though plasma-based techniques have started finding practical applications in biotechnology and medicine, the molecular mechanisms of interaction of cells with plasma remain poorly understood. In this study, the involvement of selected cellular components or pathways in plasma-induced cell killing was studied employing yeast deletion mutants. The changes in yeast sensitivity to plasma-activated water were observed in mutants with the defect in mitochondrial functions, including transport across the outer mitochondrial membrane (∆por1), cardiolipin biosynthesis (∆crd1, ∆pgs1), respiration (ρ0) and assumed signaling to the nucleus (∆mdl1, ∆yme1). Together these results indicate that mitochondria play an important role in plasma-activated water cell killing, both as the target of the damage and the participant in the damage signaling, which may lead to the induction of cell protection. On the other hand, our results show that neither mitochondria-ER contact sites, UPR, autophagy, nor proteasome play a major role in the protection of yeast cells from plasma-induced damage.

Effects of Non-Thermal Plasma on Yeast Saccharomyces cerevisiae.

Cold plasmas generated by various electrical discharges can affect cell physiology or induce cell damage that may often result in the loss of viability. Many cold plasma-based technologies have emerged in recent years that are aimed at manipulating the cells within various environments or tissues. These include inactivation of microorganisms for the purpose of sterilization, food processing, induction of seeds germination, but also the treatment of cells in the therapy. Mechanisms that underlie the plasma-cell interactions are, however, still poorly understood. Dissection of cellular pathways or structures affected by plasma using simple eukaryotic models is therefore desirable. Yeast Saccharomyces cerevisiae is a traditional model organism with unprecedented impact on our knowledge of processes in eukaryotic cells. As such, it had been also employed in studies of plasma-cell interactions. This review focuses on the effects of cold plasma on yeast cells.

Selective Apoptotic Effect of Plasma Activated Liquids on Human Cancer Cell Lines.

Plasma medicine is a new field focusing on biomedical and clinical applications of cold gas plasmas, including their anticancer effects. Cold plasmas can be applied directly or indirectly as plasma-activated liquids (PAL). The effects of plasma-activated cell growth medium (PAM) and plasma-activated phosphate buffered saline (PAPBS) were tested, using a plasma pen generating streamer corona discharge in ambient air, on different cancer cell lines (melanoma A375, glioblastoma LN229 and pancreatic cancer MiaPaCa-2) and normal cells (human dermal fibroblasts HDFa). The viability reduction and apoptosis induction were detected in all cancer cells after incubation in PAL. In melanoma cells we focused on detailed insights to the apoptotic pathways. The anticancer effects depend on the plasma treatment time or PAL concentration. The first 30 min of incubation in PAL were enough to start processes leading to cell death. In fibroblasts, no apoptosis induction was observed, and only PAPBS, activated for a longer time, slightly decreased their viability. Effects of PAM and PAPBS on cancer cells showed selectivity compared to normal fibroblasts, depending on correctly chosen activation time and PAL concentration, which is very promising for potential clinical applications. This selectivity effect of PAL is conceivably induced by plasma-generated hydrogen peroxide.

Fluorescence measurements of peroxynitrite/peroxynitrous acid in cold air plasma treated aqueous solutions.

Qualitative detection of peroxynitrite/peroxynitrous acid (ONOO-/ONOOH) as one of the key bactericidal agents produced in cold air plasma activated aqueous solutions is presented. We examined the use of the 2,7-dichlorodihydrofluorescein diacetate (H2DCFDA) fluorescent dye to detect ONOO-/ONOOH in plasma activated non-buffered water (PAW) or buffered solution (PAPB) generated by DC-driven self-pulsed transient spark discharge at atmospheric pressure in ambient air. The diagnostic selectivity of H2DCFDA to reactive oxygen and nitrogen species (RONS) typical of plasma activated aqueous solutions was examined by using various scavengers of RONS. This cross-reactivity study showed the highest sensitivity of the H2DCFDA dye to ONOO-/ONOOH. However, besides ONOO-/ONOOH, H2DCFDA also exhibited sensitivity to hypochlorite anions/hypochlorous acid (OCl-/HOCl), showing that for a selective study it is important to have an idea about the possible constituents in the studied solutions. The sensitivity of H2DCFDA to other RONS even in much higher concentrations was negligible. The presence of nitrites (NO2-) and hydrogen peroxide (H2O2) in PAW led predominantly to the production of peroxynitrous acid with a strong fluorescence response of H2DCFDA in PAW. Plasma treatment of buffered solutions led to the weak response of H2DCFDA. The fluorescence induced in PAW decreased after scavenging individual reactants, namely NO2- and H2O2, as well as by scavenging the product of the peroxynitrite forming reaction, proving that the fluorescence response of H2DCFDA is primarily due to the formation of ONOO-/ONOOH. A chemical kinetics analysis of post-discharge processes and the pseudo-second order reaction between H2O2 and NO2- confirms formation of peroxynitrous acid in PAW with a rate in the order of tens of nM per second. The post-discharge evolution of the ONOOH formation rate was clearly correlated with the parallel detection of ONOO-/ONOOH by fluorescence spectroscopy using the H2DCFDA dye.

Reactive cold plasma particles generate oxidative stress in yeast but do not trigger apoptosis.

Interactions of living cells with cold plasma of electrical discharges affect cell physiology, often resulting in the loss of viability. However, the mechanisms involved in cell killing are poorly understood, and dissection of cellular pathways or structures affected by plasma using simple eukaryotic models is needed. Using selected genetic mutants of yeast (Saccharomyces cerevisiae), we investigated the role of oxidative stress and yeast apoptosis in plasma-induced cell killing. Increased sensitivity of yeast strains deficient in superoxide dismutases indicated that reactive oxygen species generated in the plasma are among the most prominent factors involved in killing of yeast cells. In mutant strains with a deletion of the key components of yeast apoptotic pathway, the sensitivity of cells towards the plasma treatment remained unaffected. Yeast apoptosis, thus, does not appear to play a significant role in plasma-induced cell killing of yeast.

Evaluation of Selected Properties of Dielectric Barrier Discharge Plasma Jet.

In the technological processes requiring mild treatment, such as soft materials processing or medical applications, an important role is played by non-equilibrium plasma reactors with dielectric barrier discharge (DBD), that when generated in noble gases allows for the effective treatment of biological material at a low temperature. The aim of this study is to determine the operating parameters of an atmospheric pressure, radio-frequency DBD plasma jet reactor for the precise treatment of biological materials. The tested parameters were the shape of the discharge (its length and volume), current and voltage signals, as well as the power consumed by the reactor for various composition and flow rates of the working gas. To determine the applicability in medicine, the temperature, pH, concentrations of H2O2, NO2- and NO3- and Escherichia coli log reduction in the plasma treated liquids were determined. The obtained results show that for certain operating parameters, a narrow shape of plasma stream can generate significant amounts of H2O2, allowing for the mild decontamination of bacteria at a relatively low power of the system, safe for the treatment of biological materials.

Physiological Responses of Young Pea and Barley Seedlings to Plasma-Activated Water.

This study demonstrates the indirect effects of non-thermal ambient air plasmas (NTP) on seed germination and plant growth. It investigates the effect of plasma-activated water (PAW) on 3-day-old seedlings of two important farm plants-barley and pea. Applying different types of PAW on pea seedlings exhibited stimulation of amylase activity and had no inhibition of seed germination, total protein concentration or protease activity. Moreover, PAW caused no or only moderate oxidative stress that was in most cases effectively alleviated by antioxidant enzymes and proved by in situ visualization of H2O2 and ˙O2-. In pea seedlings, we observed a faster turn-over from anaerobic to aerobic metabolism proved by inhibition of alcohol dehydrogenase (ADH) activity. Additionally, reactive oxygen/nitrogen species contained in PAW did not affect the DNA integrity. On the other hand, the high level of DNA damage in barley together with the reduced root and shoot length and amylase activity was attributed to the oxidative stress caused by PAW, which was exhibited by the enhanced activity of guaiacol peroxidase or ADH. Our results show the glow discharge PAW at 1 min activation time as the most promising for pea. However, determining the beneficial type of PAW for barley requires further investigation.

Chemical and Antimicrobial Effects of Air Non-Thermal Plasma Processing of Fresh Apple Juice with Focus on Safety Aspects.

Freshly squeezed apple juice was subjected to air non-thermal plasma treatment to investigate the capability of this processing method to inactivate microorganisms and to evaluate its safety when applied to liquid food products. Two different configurations of a transient spark discharge in ambient air were tested: an electrospray system with the juice flowing directly through the high voltage needle electrode, and a batch system, where the discharge was generated onto the surface of the juice. The key physico-chemical parameters of the juice, such as pH, conductivity, color, transmittance, and Brix degree, did not significantly change upon treatment. The concentration of nitrate ions formed by the plasma was safe, while that of nitrite ions and hydrogen peroxide was initially higher than the safety limits, but decreased within 24 h post treatment. The plasma effect on individual natural components of the juice, such as sugars, organic acids, and polyphenols, treated in water solutions led to their partial or substantial decomposition. However, when these compounds were plasma-treated altogether in the juice, they remained unaffected. The antimicrobial effect of the plasma processing was evaluated via the inoculation of model microorganisms. A stronger (6 log) decontamination was detected for bacteria Escherichia coli with respect to yeast Saccharomyces cerevisiae. Plasma processing led to a substantial extension of the juice shelf-life by up to 26 days if refrigerated, which represents a promising application potential in food technology.

Cold Atmospheric Plasma and Plasma-Activated Medium Trigger RONS-Based Tumor Cell Apoptosis.

The selective in vitro anti-tumor mechanisms of cold atmospheric plasma (CAP) and plasma-activated media (PAM) follow a sequential multi-step process. The first step involves the formation of primary singlet oxygen (1O2) through the complex interaction between NO2- and H2O2. 1O2 then inactivates some membrane-associated catalase molecules on at least a few tumor cells. With some molecules of their protective catalase inactivated, these tumor cells allow locally surviving cell-derived, extracellular H2O2 and ONOO─ to form secondary 1O2. These species continue to inactivate catalase on the originally triggered cells and on adjacent cells. At the site of inactivated catalase, cell-generated H2O2 enters the cell via aquaporins, depletes glutathione and thus abrogates the cell's protection towards lipid peroxidation. Optimal inactivation of catalase then allows efficient apoptosis induction through the HOCl signaling pathway that is finalized by lipid peroxidation. An identical CAP exposure did not result in apoptosis for nonmalignant cells. A key conclusion from these experiments is that tumor cell-generated RONS play the major role in inactivating protective catalase, depleting glutathione and establishing apoptosis-inducing RONS signaling. CAP or PAM exposure only trigger this response by initially inactivating a small percentage of protective membrane associated catalase molecules on tumor cells.

Dynamics of Singlet Oxygen-Triggered, RONS-Based Apoptosis Induction after Treatment of Tumor Cells with Cold Atmospheric Plasma or Plasma-Activated Medium.

Treatment of tumor cells with cold atmospheric plasma (CAP) or with plasma-activated medium (PAM) leads to a biochemical imprint on these cells. This imprint is mediated by primary singlet oxygen, which is mainly generated through the interaction between CAP-derived H2O2 and NO2-. This imprint is induced with a low efficiency as local inactivation of a few membrane-associated catalase molecules. As sustained generation of secondary singlet oxygen by the tumor cells is activated at the site of the imprint, a rapid bystander effect-like spreading of secondary singlet oxygen generation and catalase inactivation within the cell population is thus induced. This highly dynamic process is essentially driven by NOX1 and NOS of the tumor cells, and finally leads to intercellular RONS-driven apoptosis induction. This dynamic process can be studied by kinetic analysis, combined with the use of specific inhibitors at defined time intervals. Alternatively, it can be demonstrated and quantified by transfer experiments, where pretreated cells are mixed with untreated cells and bystander signaling is determined. These studies allow to conclude that the specific response of tumor cells to generate secondary singlet oxygen is the essential motor for their self-destruction, after a singlet oxygen-mediated triggering process by CAP or PAM.

Frugal Biotech Applications of Low-Temperature Plasma.

Gas discharge low-temperature air plasma can be utilized for a variety of applications, including biomedical, at low cost. We term these applications 'frugal plasma' - an example of frugal innovation. We demonstrate how simple, robust, low-cost frugal plasma devices can be used to safely disinfect instruments, surfaces, and water.

Corona discharges with water electrospray for Escherichia coli biofilm eradication on a surface.

Low-temperature plasma (cold), a new method for the decontamination of surfaces, can be an advantageous alternative to the traditional chemical methods, autoclave or dry heat. Positive and negative corona discharges in air were tested for the eradication of 48-h Escherichia coli biofilms grown on glass slides. The biofilms were treated by cold corona discharge plasma for various exposure times. Water electrospray from the high voltage electrode was applied in some experiments. Thermostatic cultivation of the biofilm, and confocal laser scanning microscopy (CLSM) of the biofilm stained with fluorescent dyes were used for biocidal efficiency quantification. Up to 5 log10 reduction of bacterial concentration in the biofilm was measured by thermostatic cultivation after exposure to both corona discharges for 15min. This decontamination efficiency was significantly enhanced by simultaneous water electrospray through the plasma. CLSM showed that the live/dead ratio after treatment remained almost constant inside the biofilm; only cells on the top layers of the biofilm were affected. DAPI fluorescence showed that biofilm thickness was reduced by about 1/3 upon exposure to the corona discharges with electrospray for 15min. The biofilm biomass loss by about 2/3 was confirmed by crystal violet assay.

Effects of air transient spark discharge and helium plasma jet on water, bacteria, cells, and biomolecules.

Atmospheric pressure DC-driven self-pulsing transient spark (TS) discharge operated in air and pulse-driven dielectric barrier discharge plasma jet (PJ) operated in helium in contact with water solutions were used for inducing chemical effects in water solutions, and the treatment of bacteria (Escherichia coli), mammalian cells (Vero line normal cells, HeLa line cancerous cells), deoxyribonucleic acid (dsDNA), and protein (bovine serum albumin). Two different methods of water solution supply were used in the TS: water electrode system and water spray system. The effects of both TS systems and the PJ were compared, as well as a direct exposure of the solution to the discharge with an indirect exposure to the discharge activated gas flow. The chemical analysis of water solutions was performed by using colorimetric methods of UV-VIS absorption spectrophotometry. The bactericidal effects of the discharges on bacteria were evaluated by standard microbiological plate count method. Viability, apoptosis and cell cycle were assessed in normal and cancerous cells. Viability of cells was evaluated by trypan blue exclusion test, apoptosis by Annexin V-FITC/propidium iodide assay, and cell cycle progression by propidium iodide/RNase test. The effect of the discharges on deoxyribonucleic acid and protein were evaluated by fluorescence and UV absorption spectroscopy. The results of bacterial and mammalian cell viability, apoptosis, and cell cycle clearly show that cold plasma can inactivate bacteria and selectively target cancerous cells, which is very important for possible future development of new plasma therapeutic strategies in biomedicine. The authors found that all investigated bio-effects were stronger with the air TS discharge than with the He PJ, even in indirect exposure.

Study of plasma induced chemistry by DC discharges in CO2/N2/H2O mixtures above a water surface.

The chemistry induced by atmospheric pressure DC discharges above a water surface in CO(2)/N(2)/H(2)O mixtures was investigated. The gaseous mixtures studied represent a model prebiotic atmosphere of the Earth. The most remarkable changes in the chemical composition of the treated gas were the decomposition of CO(2) and the production of CO. The concentration of CO increased logarithmically with the increasing input energy density and an increasing initial concentration of CO(2) in the gas. The highest achieved concentration of CO was 4.0 +/- 0.6 vol. %. The production of CO was crucial for the synthesis of organic species, since reactions of CO with some reactive species generated in the plasma, e. g. H* or N* radicals, were probably the starting point in this synthesis. The presence of organic species (including the tentative identification of some amino acids) was demonstrated by the analysis of solid and liquid samples by high-performance liquid chromatography, infrared absorption spectroscopy and proton-transfer-reaction mass spectrometry. Formation of organic species in a completely inorganic CO(2)/N(2)/H(2)O atmosphere is a significant finding for the theory of the origins of life.