A Nonclinical Safety Evaluation of Cold Atmospheric Plasma for Medical Applications: The Role of Genotoxicity and Mutagenicity Studies
<p>The Nightingale instrument. (<b>A</b>) The instrument consisted of an air filter channel to pump atmospheric air into the plasma head containing an electrode, in which plasma was generated under a high electromagnetic field and released through the plasma head via four channels of air jets. (<b>B</b>) The direct exposure of plasma to the NIH3T3 murine fibroblasts which were cultured in DMEM medium at a distance of approximately 1.6 cm from the surface of the cell lines.</p> "> Figure 2
<p>The cell viability of fibroblast cells after exposure with plasma. The cells were exposed to different air flow rates coupled with plasma. The percentage of fibroblast cell viability is expressed as mean and standard deviation. * and ** indicate a statistical difference in cell viability, comparing plasma intensities of 4, 7, and 10 pulses to a plasma intensity of 0 pulses at the same air flow rate (* <span class="html-italic">p</span> value < 0.05; ** <span class="html-italic">p</span> value < 0.01). The lower-case letters indicate a statistical difference in cell viability comparing the same plasma intensity. Data are representative of three independent experiments.</p> "> Figure 3
<p>The suppression of cell proliferation after exposure to plasma. (<b>A</b>) The proliferative effect of plasma on the plasma-exposed cells demonstrated by a colony formation assay. The surviving colonies were stained and counted. (<b>B</b>) The percentage of colony formation is expressed as mean and standard deviation. *, **, and *** indicate a statistical difference in the percentage of colony formation, comparing plasma intensities of 4, 7, and 10 pulses to a plasma intensity of 0 pulses at the same flow rate (* <span class="html-italic">p</span> value < 0.05; ** <span class="html-italic">p</span> value < 0.01; *** <span class="html-italic">p</span> value < 0.001). The lower-case letters indicate a statistical difference in cell viability comparing the same plasma intensity. Data are representative of three independent experiments.</p> "> Figure 4
<p>Modulation of intracellular ROS and RNS. (<b>A</b>) Percentage of ROS-positive cells in plasma-exposed cells. The treated cells were harvested to stain them with 2′,7′-dichlorofluorescin diacetate. The stained cells were counted by a flow cytometer. (<b>B</b>) Percentage of RNS-positive cells in plasma-exposed cells. The treated cells were harvested to stain them with 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate. The stained cells were counted by a flow cytometer. Hydrogen peroxide and diethylamine NONOate sodium salt were used as positive controls for ROS and RNS, respectively. The percentage of ROS- and RNS-positive cells was expressed as mean and standard deviation. ** and *** indicate a statistical difference, comparing plasma intensities of 4, 7, and 10 pulses as well as the positive control to a plasma intensity of 0 pulses (** <span class="html-italic">p</span> value < 0.01; *** <span class="html-italic">p</span> value < 0.001). Data are representative of three independent experiments.</p> "> Figure 5
<p>The quantification of extracellular (<b>A</b>) H<sub>2</sub>O<sub>2</sub> and (<b>B</b>) NO in the plasma-exposed culture medium. The culture medium was directly exposed to plasma for 30 s; then, the culture medium was harvested and reacted with the specific reagent. The amount of H<sub>2</sub>O<sub>2</sub> and NO was calculated from the standard curve and expressed as mean and standard deviation. *** indicates a statistical difference, comparing plasma intensities of 4, 7, and 10 pulses to a plasma intensity of 0 pulses at the same flow rate (*** <span class="html-italic">p</span> value < 0.001). Data are representative of three independent experiments.</p> "> Figure 6
<p>The DNA damage was measured by (<b>A</b>) the percentage of TUNEL-positive cells and (<b>B</b>) the amount of intracellular 8-OHdG in plasma-exposed cells. The TUNEL assay was used for investigating DNA strand break. The commercial ELISA kit was also chosen for the quantification of 8-OHdG. H<sub>2</sub>O<sub>2</sub> was used as a positive control. The data were expressed as mean and standard deviation. * and *** indicate a statistical difference, comparing plasma intensities of 4, 7, and 10 pulses as well as the positive control to a plasma intensity of 0 pulses (* <span class="html-italic">p</span> value < 0.05 and *** <span class="html-italic">p</span> value < 0.001). <sup>##</sup> indicates a statistical difference, comparing air flow rates of 3 and 5 L/min (<sup>##</sup> <span class="html-italic">p</span> value < 0.01). Data are representative of three independent experiments.</p> "> Figure 7
<p>The immunofluorescent staining of gamma H2AX (γH2AX) on plasma-exposed cells. (<b>A</b>) Control cells were stained with gamma H2AX at 0 and 24 h. The nucleus was counterstained with DAPI. The fluorescent-staining cells were observed and photographed under an inverted microscope. (<b>B</b>) Plasma-exposed cells were exposed to a CAPJ at an intensity of 10 pulses and under an air flow rate of 5 L/min for 30 s, then further incubated for 12 and 24 h. The cells were stained with gamma H2AX (γH2AX) and counterstained with DAPI. Green dot shows γ-H2AX foci, blue nuclei stained with DAPI.</p> "> Figure 8
<p>The growth rate of plasma-exposed bacteria was assessed to test the effect of plasma on the bacterial growth of (<b>A</b>) strain TA98 and (<b>B</b>) strain TA100. After exposure to two strains of bacteria with plasma, the plasma-exposed bacteria were diluted and added to a sterile 96-well plate. The plate was then incubated at 37 °C for 24 h. The optical density (OD) at a wavelength of 600 nm was measured every 10 min. The growth curve is represented with the <span class="html-italic">x</span>-axis indicating time of incubation and the <span class="html-italic">y</span>-axis indicating OD600 nm. The results are expressed as the average and standard deviation of triplicate experiments.</p> "> Figure 9
<p>The mutagenicity of plasma was tested on (<b>A</b>) <span class="html-italic">Salmonella typhimurium</span> strain TA98 and (<b>B</b>) <span class="html-italic">S. typhimurium</span> strain TA100. The bacteria were directly exposed to plasma for 30 s. After that, the treated bacteria were cultured on a culture medium. The number of revertant colonies was counted. The mutagenic index was calculated and is expressed as mean and standard deviation. The mutagenicity of the test substance was classified by a mutagenic index of more than 2 (over the dot line). Data are representative of three independent experiments. The dotted line represents the cut-off to indicate the test substance is not classified as mutagens (mutagenic index less than 2).</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Atmospheric Nonthermal Plasma (ANTP) Instrument
2.2. Cell Culture
2.3. Exposure of Plasma to the Murine Fibroblast Cells
2.4. Cell Viability Testing
2.5. Colony Formation Assay
2.6. Investigation of Intracellular Reactive Oxygen (ROS) and Nitrogen Species (RNS)
2.7. Quantification of Extracellular ROS and RNS
2.8. Mutagenicity Testing
2.8.1. 8-Hydroxy-2′-deoxyguanosine (8-OHdG) Quantification
2.8.2. DNA Strand Break Analysis
2.8.3. Bacterial Reverse Mutation Assay
2.9. Statistical Analysis
3. Results
3.1. Fibroblast Cell Viability
3.2. Suppression of Cell Proliferation
3.3. Production of Intracellular Reactive Oxygen (ROS) and Nitrogen Species (RNS)
3.4. Generation of Extracellular Reactive Oxygen (ROS) and Nitrogen Species (RNS)
3.5. Induction of DNA Damage
3.6. Mutagenicity Potential of Plasma
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Treatment | His+ Revertant Colonies | ||
---|---|---|---|
Strain TA98 | Strain TA100 | ||
Phosphate buffer | 20.7 ± 4.3 | 88.6 ± 3.9 | |
2-(2-furyl)-3-(5-nitro-2-furyl)-acrylamide (AF-2) | 246.1 ± 44.4 *** | 511.8 ± 53.3 *** | |
Air flow 3 L/min | Intensity of 0 pulses | 18.6 ± 3.4 | 86.9 ± 0.1 |
Intensity of 4 pulses | 17.7 ± 2.5 | 80.8 ± 3.3 | |
Intensity of 7 pulses | 21.2 ± 4.2 | 84.6 ± 9.6 | |
Intensity of 10 pulses | 23.8 ± 5.1 | 94.7 ± 2.9 | |
Air flow 5 L/min | Intensity of 0 pulses | 21.8 ± 4.5 | 84.7 ± 5.7 |
Intensity of 4 pulses | 20.6 ± 3.9 | 91.4 ± 3.1 | |
Intensity of 7 pulses | 24.0 ± 4.2 | 92.2 ± 4.1 | |
Intensity of 10 pulses | 20.8 ± 4.5 | 104.7 ± 4.4 *,# |
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Yarangsee, P.; Khacha-ananda, S.; Pitchakarn, P.; Intayoung, U.; Sriuan, S.; Karinchai, J.; Wijaikhum, A.; Boonyawan, D. A Nonclinical Safety Evaluation of Cold Atmospheric Plasma for Medical Applications: The Role of Genotoxicity and Mutagenicity Studies. Life 2024, 14, 759. https://doi.org/10.3390/life14060759
Yarangsee P, Khacha-ananda S, Pitchakarn P, Intayoung U, Sriuan S, Karinchai J, Wijaikhum A, Boonyawan D. A Nonclinical Safety Evaluation of Cold Atmospheric Plasma for Medical Applications: The Role of Genotoxicity and Mutagenicity Studies. Life. 2024; 14(6):759. https://doi.org/10.3390/life14060759
Chicago/Turabian StyleYarangsee, Piimwara, Supakit Khacha-ananda, Pornsiri Pitchakarn, Unchisa Intayoung, Sirikhwan Sriuan, Jirarat Karinchai, Apiwat Wijaikhum, and Dheerawan Boonyawan. 2024. "A Nonclinical Safety Evaluation of Cold Atmospheric Plasma for Medical Applications: The Role of Genotoxicity and Mutagenicity Studies" Life 14, no. 6: 759. https://doi.org/10.3390/life14060759