A Nonclinical Safety Evaluation of Cold Atmospheric Plasma for Medical Applications: The Role of Genotoxicity and Mutagenicity Studies.

Atmospheric nonthermal plasma (ANTP) has rapidly evolved as an innovative tool in biomedicine with various applications, especially in treating skin diseases. In particular, the formation of reactive oxygen species (ROS) and nitrogen species (RNS), which are generated by ANTP, plays an important role in the biological signaling pathways of human cells. Unfortunately, excessive amounts of these reactive species significantly result in cellular damage and cell death induction. To ensure the safe application of ANTP, preclinical in vitro studies must be conducted before proceeding to in vivo or clinical trials involving humans. Our study aimed to investigate adverse effects on genetic substances in murine fibroblast cells exposed to ANTP. Cell viability and proliferation were markedly reduced after exposing the cells with plasma. Both extracellular and intracellular reactive species, especially RNS, were significantly increased upon plasma exposure in the culture medium and the cells. Notably, significant DNA damage in the cells was observed in the cells exposed to plasma. However, plasma was not classified as a mutagen in the Ames test. This suggested that plasma led to the generation of both extracellular and intracellular reactive species, particularly nitrogen species, which affect cell proliferation and are also known to induce genetic damage in fibroblast cells. These results highlight the genotoxic and mutagenic effects of ANTP, emphasizing the need for the cautious selection of plasma intensity in specific applications to avoid adverse side effects resulting from reactive species production.

Differentiating paramphistome species in cattle using DNA barcoding coupled with high-resolution melting analysis (Bar-HRM).

Paramphistomosis is caused by paramphistome or amphistome parasites, including Fischoederius elongatus, Gastrothylax crumenifer, Orthocoelium parvipapillatum, and Paramphistomum epiclitum. The control and prevention of these parasite outbreaks are difficult because of the wide occurrence of these species. Besides, the clinical manifestations and their egg characteristics are similar to those of other intestinal flukes in the paramphistome group, leading to misdiagnosis. Here, we employed DNA barcoding using NADH dehydrogenase (ubiquinone, alpha 1) (ND1) and cytochrome c oxidase subunit I (COI), coupled with high-resolution melting analysis (Bar-HRM), for species differentiation. As a result, ParND1_3 and ParCOI4 resulted in positive amplification in the paramphistomes and Fasciola gigantica, with significantly different melting curves for each species. The melting temperatures of each species obtained clearly differed. Regarding sensitivity, the limit of detection (LoD) for all species of paramphistomes was 1 pg/µl. Our findings suggest that Bar-HRM using ParND1_3 is highly suitable for the differentiation of paramphistome species. This approach can be used in parasite detection and epidemiological studies in cattle.