Genotoxicity and cytotoxicity of the plasma jet-treated medium on lymphoblastoid WIL2-NS cell line using the cytokinesis block micronucleus cytome assay

Despite growing interest in the application of atmospheric plasma jets as medical treatment strategies, there has been comparatively little research on the potential genotoxic and cytotoxic effects of plasma jet treatment. In this study, we have employed the cytokinesis block micronucleus cytome (CBMN-Cyt) assay with WIL2-NS B lymphoblastoid cells to test the potential genotoxicity, as well as the cytotoxicity, of toxic species generated in cell culture media by an argon (Ar) plasma jet. Elevated levels of cell death (necrosis) and occurrence of chromosomal damage (micronuclei MN, nculeoplasmic bridge NPBs and nuclear bus, Nbuds) were observed when cells were exposed to plasma jet-treated media. These results provide a first insight into how we might measure the genotoxic and cytotoxic effect of plasma jet treatments (both indirect and direct) in dividing human cells.

Research into the applications of cold atmospheric plasma jets in biology and medicine has been rapidly growing in the past decade 1 . A wide range of plasma jet sources have been developed reflecting the growing commercial confidence in the likelihood of developing a new medical industry based on the use of plasma jets. This optimism is backed up by evidence in the selective destruction of cancerous cells 2 , the stimulation of wound healing 3 and different dermatological applications 4,5 .
In the recent years there has been a growing interest in the use of an indirect plasma treatment method, where plasma jet-treated media or plasma jet-treated liquid is used as a medical therapy. This method of treatment has been validated in several studies with success shown in the inactivation of gram-positive and negative strains of bacteria 6 , destruction of lung cancer cells 7 , brain tumour cells 8 , as well as destruction of in vitro 3D multicellular tumours 9 . It has been claimed that plasma jet-treated media has a certain degree of selectivity to cancerous cells, where breast cancer cells have been shown to be more susceptible to plasma jet-treated media than glioblastoma cells 10 . The indirect treatment method has also been shown to be effective in the destruction of chemo-resistant ovarian cancer cells 11 . In the indirect plasma treatment method, the composition of media is thought to be an important factor in generation of stable source of reactive species in treated media 12 especially for treatment of cancer 13 .
To date, most researchers have attempted to link the medical benefits of plasma jets, operated with argon (Ar) or helium (He), to the reactive oxygen and nitrogen species (RONS) generated on interaction of plasma with the ambient air or liquid [14][15][16] . The interaction of ambient air treated by plasma jets with aqueous solution produces oxygen-containing species such as the hydroxyl radical ( · OH), hydrogen peroxide (H 2 O 2 ), superoxide (O 2 − ) and peroxynitrite (ONOO − ) 17,18 . All of these species are likely to cause DNA abnormalities in cells [19][20][21] .
Given the growing interest in the biological and medical applications of plasma jets, it is essential to develop a detailed understanding of how to measure any potential genotoxic of plasma in human cells and follow the consequences of these in cell survival and cell division. Metrology of cell damage is needed to help mitigate potential safety concerns in the clinical use of plasma jets. A few studies in the literature have started to address this issue including studies of changes in gene expression following exposure to plasma treated media 22 and single/double strand breaks in naked DNA [23][24][25] . Relevant to our study, Wende et al. have monitored the genotoxicity of plasma in cell culture by counting the formation of micronuclei (MN) 26 . A further study has used the formation of MN to follow damage in the dielectric barrier discharge plasma treatment of brain cancer cells -showing that the increased frequency of MN correlated with the plasma jet exposure time 27 . The cytotoxicity of direct and indirect plasma treatment is more typically studied, using various assays such as viability assays using resazurin 18 or MTT 10 and fluorescence staining methods to track and visualise live/dead cells 28 routinely employed.
The cytokinesis-block micronucleus cytome assay (CBMN-Cyt) is a method which allows the investigation of both genotoxicity as well as cytotoxicity and has great versatility in the number of different measurements that can be made within one single experiment 29 . A previous study verified the suitability and sensitivity of the CBMN-Cyt to investigate DNA damage and cell death in lymphoblastoid cells induced by H 2 O 2 and O 2 −30 . The CBMN-Cyt assay has been validated in independent systems to examine genetic alterations caused by ionizing irradiation 31 , chemical carcinogens 32, 33 , nutrient deficiency 34 and heavy metals 29 . The CBMN-Cyt assay has been endorsed by the OECD as a required test for determining genotoxicity of chemicals 35 and by the IAEA for biodosimetry of ionizing radiation exposure 36 . The protocol we have followed employs the WIL2-NS B lymphoblastoid cell line. This cell is particularly relevant to plasma medicine in the context of treating inflamed or infected sites, as it is typical of the cells involved in the immune response [37][38][39] , thus it is one of the most prominent cells found at these sites. Furthermore, the genotoxicity and cytotoxicity for monitoring other medically relevant technique such as ionizing radiation 40 and chemotherapy 41 have been studied extensively using the CBMN assay and the WIL2-NS cell line.
In this study, we investigated the effects of an Ar plasma jet-treated Rosewell Park Memorial Institute 1640 cell culture medium (herein referred to as RPMI 1640) on WIL2-NS cells originally isolated from the spleen of a Caucasian male 42 , using the CBMN-Cyt assay. We investigated the genotoxicity through scoring the occurrence of various chromosomal damage biomarkers. We also measured cytotoxicity through the measure of apoptotic and necrotic cells, as well as through a cell viability study. Finally, we quantified the concentration of H 2 O 2 generated in RPMI 1640 following plasma jet treatment and proposed a link between the genotoxicity of plasma-jet treated media and the generation of H 2 O 2 .

Results
Genotoxicity of Ar plasma jet-treated RPMI 1640. The experimental procedure from plasma treatment of RPMI 1640 through to the steps taken to perform the CBMN-Cyt assay are shown in Fig. 1. A detailed description of the CBMN-Cyt (including scoring criteria) has been published in 43 . In brief, RPMI 1640 was treated with an Ar plasma jet, the media was then transported at 4 °C to the facility where the CBMN-Cyt assay was performed. The treated media was then incubated for 1 h at 37 °C before media supplements were added and cell suspensions were exposed to treated media. The WIL2-NS cell line was chosen for this study, as this cell line has been validated for use in the CBMN-Cyt assay in a number of previous studies 29,30,34 . It is suited to this type of damage identification due its high nuclear division index and p53 deficiency, which allows cells with DNA damage to survive -facilitating DNA damage "events" to be observed and quantified 30 .
The CBMN-Cyt assay is based upon scoring chromosomal damage events, which are expressed as nuclear anomalies in once-divided cells which are accumulated and identified as binucleated (BN). The recognized biomarkers for chromosomal damage in the CBMN-Cyt assay are MN, nucleoplasmic bridges (NPBs) and nuclear buds (Nbuds) 44 . Figure 2 shows microscopic images of these biomarkers. MN are formed through chromosomal fragments or whole chromosomes, which lag behind during anaphase in mitosis. NPBs arise from dicentric chromosomes that are pulled to opposite poles of the cell thus forming a dicentric chromosome bridge between nuclei of a BN cell and are indicative of misrepair of DNA breaks or telomere and fusion 30 . Nbuds have recently been established as a biomarker of chromosomal instability, and are thought to be caused by exclusion of excess amplified genetic material 45 . Scoring of these biomarkers is strictly restricted to once-divided BN cells and for this reason this assay uses cyto-B to block the cell division cycle at the stage of cytokinesis. Figure 3 shows the frequency of chromosomal damage (MN, NPB, Nbud) scored after incubation of cells in Ar plasma jet-treated RPMI 1640. Figure 3a shows a higher frequency of MN cells in the undiluted and less dilute Ar plasma jet-treated RPMI 1640. Cells cultured in the undiluted (100%) Ar plasma jet-treated RPMI 1640 showed approximately a 2-fold increase in the frequency of MN compared to cells cultured in 50% Ar plasma jet-treated RPMI 1640, and approximately a 10-fold increase compared to untreated RPMI 1640. The values were shown to be significantly different with one-way ANOVA (P < 0.05). Similar trends were observed in the other biomarkers of chromosomal damage, i.e. NPB (Fig. 3b) and Nbuds (Fig. 3c), with the highest level of damage observed when exposed to undiluted Ar plasma jet-treated RPMI 1640 (P < 0.05).
Cytotoxicity of Ar plasma jet-treated RPMI 1640. Cytotoxicity was also assessed by scoring the frequency of apoptotic and necrotic cells, which were expressed as a percentage of the total cell population (Fig. 4). The occurrence of apoptotic (Fig. 4a) and necrotic (Fig. 4b) cells was greater in the 100% and less diluted Ar plasma jet-treated RPMI 1640; but was only significant for necrotic cells (P < 0.05) but not for apoptotic cells (P > 0.05). It is important establish whether the plasma treated jet media had any effect on the rate of cell division. This was measured through the nuclear division index (NDI), which was found to be similar (at ca. 2) for all dilutions of Ar plasma jet-treated RPMI 1640 (Fig. 4c). This value is what would be expected for a cell with a doubling time of ca. 48 h. Figure 5 shows that cell viability, measured using a resazurin cell viability assay, decreased for cells cultured in the less dilute Ar plasma jet-treated RPMI 1640, with viability of cells exposed to undiluted Ar plasma jet-treated RPMI 1640 being significantly lower than cell viability exposed to untreated RPMI 1640 (P < 0.05).

Concentration of Hydrogen peroxide generated in RPMI 1640 by Ar plasma jet treatment.
Of the RONS known to be generated by plasma-jets in solutions, H 2 O 2 is a relatively stable product (especially in the absence of FBS) 13 which can be detected days after plasma exposure 46 . Using a "Free Radical Analyzer" (WPI Instruments), changes in H 2 O 2 concentration in RPMI 1640 were measured immediately after Ar plasma jet treatment and following 2 h incubation (the time-point at which the Ar plasma jet-treated RPMI 1640 was applied to cells as shown in Table 1). Immediately after Ar plasma jet treatment of RPMI 1640, a H 2 O 2 concentration of 25 µM was detected before dilution (i.e. at 100%); and the concentration of H 2 O 2 decreased with increased dilution (P < 0.05). The media was then incubated at 4 °C, which was previously reported to be within the ideal storage temperature to retain activity of plasma treated media 9 , for transport between laboratories. Then the media supplements such as FBS were added and the media was further incubated at tissue culture conditions (37 °C and 5% CO 2 ). Following the 2 h incubation, the mean H 2 O 2 concentration was reduced to 18.3 µM before any dilution (100%) and 12.9 µM for a 50% dilution. This reduction in concentration of H 2 O 2 was expected as addition of supplements such as FBS containing proteins, have been shown to interfere with the stability of H 2 O 2 10, 12 .

Discussion
The purpose in undertaking this study was to explore the versatility of the CBMN-Cyt assay for measuring whole cell and chromosomal responses to plasma jet-treated media and later direct plasma. The study was not undertaken to assess the safety of plasma per se. In this respect a number of key results were observed with the WIL2-NS lymphoblastoid cell line -these include, there was no discernible effect on the rate of cell division (as measured through the NDI) but an increased cell death (necrosis, but not apoptosis) and a strong display of a range of markers of abnormal cell division -MN, NPBs and Nbuds. Cell death and raised markers of chromosomal damage were greatest in the undiluted and less diluted plasma jet-treated media. The WIL2-NS cell is p53 deficient and therefore we would not have expected apoptosis. WIL2-NS cells have a fast rate of division and are more likely to propagate abnormalities in cell division. And in this context our data are different to those of Wende et al. 26 who performed a micronuclei (MN) assay count with HacaT cells (keratinocytes) exposed to plasma-treated media. Wende et al., based on MN counts, reported no effect of plasma treatment on the cell division process. This difference is not at all surprising as the full CBMN-Cyt assay has revealed markedly different responses in different   cell types to the same agents 47 . Furthermore, the full CBMN-Cyt assay using lymphoblastoid cell line has been previously validated for measuring NPBs, a marker used as an indication of chromosome loss and breakage and Nbuds, a marker used as an indication of over amplification of gene. This allows for a much more detailed assessment of toxicity compared to the simple measure of MN 48 . Of the likely longer lived RONS in the plasma jet-treated media our suspicion falls upon H 2 O 2 as the likely agent of the effects we report. The dose dependent toxicity of H 2 O 2 in various (and different) cell lines has been established in previous studies (of particular relevance is Umegaki et al. 30 , which is discussed below). Available data indicate that even small doses of H 2 O 2 , as low as 10 µM, can be cytotoxic and genotoxic, and may even be related to development of cancer 49 . Low doses of RONS produced by neutrophils in the inflammation process can induce oxidative damage in the DNA of normal cells 50 . It has been shown that H 2 O 2 can easily penetrate across the phospholipid membrane of cells utilising water channels such as aquaporins 51,52 . Once in the cellular interior, H 2 O 2 poses a particular threat in the nucleus, where DNA damage results from the generation of OH radicals 53 (OH radicals are known to be generated from H 2 O 2 by Fe 2+ ions within cellular compartments through the Fenton reaction) 54 . OH radicals attack DNA at the sugar residues of the DNA backbone, which results in single-strand breaks 53 . Without repair, these single-strand breaks may be carried through to double-strand breaks, which are expressed as chromatid breaks, i.e. a potential pathway to the formation of MN 55 .
The study of Umegaki et al. provides an optimal control for our study. In their paper, Umegaki et al.   3). One-way ANOVA was used to determine the statistical significance of the values. The values without a mark above indicate that they are significantly different whereas the values sharing * or ** are not (P < 0.05). . Measurements were taken immediately following Ar plasma jet treatment (0 h) and following 2 h incubation (time-point of application to cells). The data presented is the mean value and ± standard error of the mean (SEM) of samples analyzed in triplicate (n = 3). One-way ANOVA was used to determine the statistical significance of the values. All values are statistically significantly different (P < 0.05). … Indicates concentration below detection limit.

H 2 O 2 concentration following incubation
Some of the other potential effects of plasma have been investigated. Beyond generating RONS, we found that Ar plasma jet treatment did not result in any change in pH or any temporary elevation of the temperature of the RPMI 1640. However, the potential modification of micronutrients and proteins was not analysed. Such modification of micronutrients and proteins by the Ar plasma jet would result in a decrease their bioavailability and bioefficacy, and a nutritional deficiency, which has been linked to chromosomal damage 56 .
The direct extrapolation of the data presented in this paper to the likely effects in treatment of real tissue (and on lymphocytes in situ of, for example, a wound) should be approached with caution. Significant differences are, for example the static and 2D nature of in vitro cell culture versus real tissues which are 3D and would be subject to constant flow of fluid. The latter would continuously replenish the treatment site, removing RONS and replacing denatured biomolecules with fresh micronutrients and proteins and removing toxic waste generated by the plasma jet treatment. Interstitial fluid and blood plasma is also complete with endogenous antioxidants such as catalase and glutathione peroxidase 57 . In this study, the effects of antioxidants were not considered, but would be expected to reduce the accumulation of RONS at the treatment site. Furthermore, the intracellular concentration of RONS was not monitored in this paper. Monitoring intracellular RONS using RONS scavengers such as NAC 58 will provide indications for understanding the mechanism of genetic damage caused by plasma jet treatment. Therefore, in the direct or indirect exposure of plasma or medium pre-treated with plasma to living tissue from animal or human subjects, dynamic changes in the concentrations of RONS should be carefully considered, as recently discussed 59,60 .
In conclusion, the CBMN-Cyt assay with WIL2-NS cells is an effective system to detect whole cell and chromosomal damage induced by Ar plasma jet-treated cell culture media. Trends in the extent of DNA damage mirror the cytotoxic (necrotic) effect of Ar plasma jet-treated cell culture media. These results highlight the possible survival of genomically-abnormal cells. And whilst this observation may warrant careful consideration in the context of potential genetic aberrations in the in situ medical use of plasma jets, our objective in these preliminary experiments was to highlight the full potential of the CBMN-Cyt assay as a tool for assessing the cytotoxicity and genotoxicity of cold atmospheric plasmas. Such methods will become increasingly important in the development of plasma technology for the targeted and controlled destruction of cancer cells, by demonstrating targeted DNA damage and destruction in cancers, whilst in normal (adjacent) tissue the minimization of DNA damage and cell death.

Materials and Methods
Ar plasma jet treatment of RPMI 1640. A commercial Ar plasma jet was operated at 1 MHz with an Ar gas flow rate of 5 L/mins. The Ar was ultra-high purity grade (BOC). A volume of 10 mL of RPMI 1640 supplemented with 5% (v/v) foetal bovine serum (Sigma) was treated in 6-well tissue culture polystyrene (TCPS) plates at a distance of 20 mm between the end of the nozzle of the plasma jet assembly and the top of the plate.
Exposure of cells to Ar plasma jet-treated RPMI 1640. After Ar plasma jet treatment, the RPMI 1640 was transported cold to the cell culture facility, which took approximately 1 h on ice, thus there were incubated at 4 °C for 1 h. The Ar plasma jet-treated RPMI 1640 was supplemented with 100 IU/mL penicillin and 100 mg/ mL streptomycin solution and 1% L-glutamine (Sigma), then incubated for 1 h at 37 °C in a humidified incubator with 5% CO 2 , this was to bring the cell culture medium to optimum condition before exposure to cells. Therefore, there was an unavoidable delay of 2 h before 5 mL of serial dilutions of the Ar plasma jet-treated RPMI 1640 were prepared and 50 µL of cell suspension was added to obtain an end cell count of 2 × 10 6 viable cells/mL. A 1 mL  30 here H 2 O 2 was added as a solution, to the data in this paper (Present study), where H 2 O 2 was added by an Ar plasma jet. For "Umegaki study" the data points were obtained from triplicate measurements (n = 3) 30 for "Present study" the data points were obtained from triplicate experiments with triplicate samples per experiment (n = 9). Each point represents the mean value and ± standard error of the mean (SEM).
volume of the cell suspension, prepared in RPMI 1640, or undiluted or diluted Ar plasma jet-treated RPMI 1640, was distributed into each well of a 24-well TCPS plate. Cells were exposed to Ar plasma jet-treated RPMI 1640 used either undiluted or diluted to 50% or 10% (with dilutions prepared in pre-warmed, untreated RPMI 1640). The cells were incubated for 24 h under the cell culture conditions described above.

CBMN-Cyt assay. After incubation of cells in Ar plasma jet-treated RMPI 1640, the cytochalasin-B (Cyto-B)
(Sigma) was added and the cells were incubated for a further 24 h before being harvested onto glass slides using a "cytospin", air dried for 10 min, and fixed and stained using "Diff-Quick" for scoring. Figure 2 shows the process of Ar plasma jet treatment of RPMI 1640 through to preparation of microscope slides for scoring. A total of 1000 BN cells were scored for the presence of MN (Fig. 2a), NPBs (Fig. 2b) and Nbuds (Fig. 2c) per culture condition following an established protocol 31 . Chromosomal damage was scored and expressed as the number of damage events per 1000 BN cells. A total of 250 cells, including mononucleated, BN, multinucleated, apoptotic and necrotic, were counted per slide. The data from these cell counts were used to determine the cytotoxicity (only apoptotic and necrotic cells), as well as the nuclear division index (NDI) for all cells. The NDI is a marker used for nuclear division status and can be used as a marker to determine the cytostatic effects of the Ar plasma jet used in this study. The methodology used to score and calculate cytotoxicity, NDI and DNA damage biomarkers, was previously described 55 .
Resazurin assay. Cytotoxicity assessment of cells exposed to diluted Ar plasma jet-treated RPMI 1640 was also carried out using a resazurin cell viability assay 61 . After 24 h incubation of cells in Ar plasma jet-treated RPMI 1640, 100 µl of 100 µg/mL resazurin (Sigma) prepared in RPMI 1640 was added and incubated for 24 h. Following incubation, cell suspensions in the 24-well TCPS plate were centrifuged to remove cells. The fluorescence intensity of the supernatant was measured at an excitation and emission wavelength of 560 nm and 590 nm, respectively. The fluorescence intensity (FI) of cells exposed to Ar plasma jet-treated RPMI 1640 was normalised against FI of cells exposed to untreated RPMI 1640 following the equation: [(FI treated cells − FI untreated cells)/ FI untreated cells] ×100%.