Search Results
Investigating the Mutagenicity of a Cold Argon-Plasma Jet in an HET-MN Model
Objective: So-called cold physical plasmas for biomedical applications generate reactive oxygen and nitrogen species and the latter can trigger DNA damage at high concentrations. Therefore, the mutagenic risks of a certified atmospheric pressure argon plasma jet (kINPen MED) and its predecessor model (kINPen 09) were assessed. Methods: Inner egg membranes of fertilized chicken eggs received a single treatment with either the kINPen 09 (1.5, 2.0, or 2.5 min) or the kINPen MED (3, 4, 5, or 10 min). After three days of incubation, blood smears (panoptic May-Grünwald-Giemsa stain) were performed, and 1000 erythrocytes per egg were evaluated for the presence of polychromatic and normochromic nuclear staining as well as nuclear aberrations and binucleated cells (hen’s egg test for micronuclei induction, HET-MN). At the same time, the embryo mortality was documented. For each experiment, positive controls (cyclophosphamide and methotrexate) and negative controls (NaCl-solution, argon gas) were included. Additionally, the antioxidant potential of the blood plasma was assessed by ascorbic acid oxidation assay after treatment. Results: For both plasma sources, there was no evidence of genotoxicity, although at the longest plasma exposure time of 10 min the mortality of the embryos exceeded 40%. The antioxidant potential in the egg’s blood plasma was not significantly reduced immediately (p = 0.32) or 1 h (p = 0.19) post exposure to cold plasma. Conclusion: The longest plasma treatment time with the kINPen MED was 5–10 fold above the recommended limit for treatment of chronic wounds in clinics. We did not find mutagenic effects for any plasma treatment time using the either kINPen 09 or kINPen MED. The data provided with the current study seem to confirm the lack of a genotoxic potential suggesting that a veterinary or clinical application of these argon plasma jets does not pose mutagenic risks.
Oxidants and Redox Signaling: Perspectives in Cancer Therapy, Inflammation, and Plasma Medicine
[No abstract available]
Periodic Exposure of Keratinocytes to Cold Physical Plasma: An In Vitro Model for Redox-Related Diseases of the Skin
Oxidative stress illustrates an imbalance between radical formation and removal. Frequent redox stress is critically involved in many human pathologies including cancer, psoriasis, and chronic wounds. However, reactive species pursue a dual role being involved in signaling on the one hand and oxidative damage on the other. Using a HaCaT keratinocyte cell culture model, we investigated redox regulation and inflammation to periodic, low-dose oxidative stress after two, six, eight, ten, and twelve weeks. Chronic redox stress was generated by recurrent incubation with cold physical plasma-treated cell culture medium. Using transcriptome microarray technology, we identified both acute ROS-stress responses as well as numerous adaptions after several weeks of redox challenge. We determined a differential expression (2-fold, FDR < 0.01, p < 0.05) of 260 genes that function in inflammation and redox homeostasis, such as cytokines (e.g., IL-6, IL-8, and IL-10), growth factors (e.g., CSF2, FGF, and IGF-2), and antioxidant enzymes (e.g., HMOX, NQO1, GPX, and PRDX). Apoptotic signaling was affected rather modestly, especially in p53 downstream targets (e.g., BCL2, BBC3, and GADD45). Strikingly, the cell-protective heat shock protein HSP27 was strongly upregulated (p < 0.001). These results suggested cellular adaptions to frequent redox stress and may help to better understand the inflammatory responses in redox-related diseases.
Redox Stimulation of Human THP-1 Monocytes in Response to Cold Physical Plasma
In plasma medicine, cold physical plasma delivers a delicate mixture of reactive components to cells and tissues. Recent studies suggested a beneficial role of cold plasma in wound healing. Yet, the biological processes related to the redox modulation via plasma are not fully understood. We here used the monocytic cell line THP-1 as a model to test their response to cold plasma in vitro. Intriguingly, short term plasma treatment stimulated cell growth. Longer exposure only modestly compromised cell viability but apparently supported the growth of cells that were enlarged in size and that showed enhanced metabolic activity. A significantly increased mitochondrial content in plasma treated cells supported this notion. On THP-1 cell proteome level, we identified an increase of protein translation with key regulatory proteins being involved in redox regulation (hypoxia inducible factor 2α), differentiation (retinoic acid signaling and interferon inducible factors), and cell growth (Yin Yang 1). Regulation of inflammation is a key element in many chronic diseases, and we found a significantly increased expression of the anti-inflammatory heme oxygenase 1 (HMOX1) and of the neutrophil attractant chemokine interleukin-8 (IL-8). Together, these results foster the view that cold physical plasma modulates the redox balance and inflammatory processes in wound related cells.
Neutrophil extracellular trap formation is elicited in response to cold physical plasma
Cold physical plasma is an ionized gas with a multitude of components, including hydrogen peroxide and other reactive oxygen and nitrogen species. Recent studies suggest that exposure of wounds to cold plasma may accelerate healing. Upon wounding, neutrophils are the first line of defense against invading microorganisms but have also been identified to play a role in delayed healing. In this study, we examined how plasma treatment affects the functions of peripheral blood neutrophils. Plasma treatment induced oxidative stress, as assessed by the oxidation of intracellular fluorescent redox probes; reduced metabolic activity; but did not induce early apoptosis. Neutrophil oxidative burst was only modestly affected after plasma treatment, and the killing of Pseudomonas aeruginosa and Staphylococcus aureus was not significantly affected. Intriguingly, we found that plasma induced profound extracellular trap formation. This was inhibited by the presence of catalase during plasma treatment but was not replicated by adding an equivalent concentration of hydrogen peroxide. Plasma-induced neutrophil extracellular trap formation was not dependent on the activity of myeloperoxidase or NADPH oxidase 2 but seemed to involve short-lived molecules. The amount of DNA release and the time course after plasma treatment were similar to that with the common neutrophil extracellular trap inducer PMA. After neutrophil extracellular traps had formed, concentrations of IL-8 were also significantly increased in supernatants of plasma-treated neutrophils. Both neutrophil extracellular traps and IL-8 release may aid antimicrobial activity and spur inflammation at the wound site. Whether this aids or exacerbates wound healing needs to be tested.
Toxicity and Immunogenicity in Murine Melanoma following Exposure to Physical Plasma-Derived Oxidants
Metastatic melanoma is an aggressive and deadly disease. Therapeutic advance has been achieved by antitumor chemo- and radiotherapy. These modalities involve the generation of reactive oxygen and nitrogen species, affecting cellular viability, migration, and immunogenicity. Such species are also created by cold physical plasma, an ionized gas capable of redox modulating cells and tissues without thermal damage. Cold plasma has been suggested for anticancer therapy. Here, melanoma cell toxicity, motility, and immunogenicity of murine metastatic melanoma cells were investigated following plasma exposure in vitro. Cells were oxidized by plasma, leading to decreased metabolic activity and cell death. Moreover, plasma decelerated melanoma cell growth, viability, and cell cycling. This was accompanied by increased cellular stiffness and upregulation of zonula occludens 1 protein in the cell membrane. Importantly, expression levels of immunogenic cell surface molecules such as major histocompatibility complex I, calreticulin, and melanocortin receptor 1 were significantly increased in response to plasma. Finally, plasma treatment significantly decreased the release of vascular endothelial growth factor, a molecule with importance in angiogenesis. Altogether, these results suggest beneficial toxicity of cold plasma in murine melanomas with a concomitant immunogenicity of potential interest in oncology.
Platelets are key in cold physical plasma-facilitated blood coagulation in mice
Purpose: Surgical interventions inevitably lead to destruction of blood vessels. This is especially dangerous in anticoagulated patients. Electrocauterization is a frequently used technique to seal incised tissue. However, leading to a superficial layer of necrotic tissue, the treated area evolves a high vulnerability to contact, making it prone to detachment. As a result, dangerous postoperative bleeding may occur. Cold physical plasma was previously suggested as a pro-coagulant treatment method. It mainly acts by expelling a delicate mixture of oxidants. We therefore tested the suitability of an atmospheric pressure plasma jet (kINPen MED) as a new medical device for sufficient blood coagulation in a murine model of liver incision. Methods: Plasma treatment of murine blood ex vivo induced sufficient coagula. This effect did not affect any tested parameter of plasmatic coagulation cascade, suggesting the mechanism to be related to cellular coagulation. Indeed, isolated platelets were significantly activated following exposure to plasma, although this effect was less pronounced in whole blood. To analyze the biological effect of plasma-on blood coagulation in vivo, mice were anticoagulated (clopidogrel inhibiting cellular and rivaroxaban inhibiting plasmatic hemostasis) or received vehicle only. Afterwards, a partial resection of the left lateral liver lobe was performed. The quantification of the blood loss after liver incision followed by treatment with kINPen MED plasma or electrocauterization revealed a similar and significant hemostatic performance in native and rivaroxaban but not clopidogrel-treated animals compared to argon gas-treated controls. In contrast to electrocauterization, kINPen MED plasma treatment did not cause necrotic cell layers. Conclusion: Our results propose a prime importance of platelets in cold physical plasma-mediated hemostasis and suggest a clinical benefit of kINPen MED plasma treatment as coagulation device in liver surgery.