22E07
Evaluation of genotoxicity found in non-thermal atmosphericpressure plasma using yeast cells
大気圧低温プラズマが有する変異原性の酵母細胞による評価
Hachiro Yasuda, Hiroki Yamaguchi, Toshihiko Eki, Hirofumi Kurita
Kazunori Takashima,and Akira Mizuno
安田八郎、 山口広輝、 浴 俊彦、 栗田弘史、 高島和則、 水野 彰
Department of Environmental and Life Sciences, Toyohashi University of Technology
1-1 Hibarigaoka, Tempaku-cho, Toyohashi, Aichi 441-8580, Japan
豊橋技術科学大学 〒441-8580 愛知県豊橋市天伯町雲雀ヶ丘 1-1
Non-thermal atmospheric pressure plasmas have been recently applied in biomedical field. So far, the influence of theplasma treatment to living cells and organs is still unknown. In this study, we have monitored DNA damage in yeast
cells exposed to argon plasma torch. We have used the yeast-based genotoxicity assay system, which is based on thetranscriptional induction of a Saccharomyces cerevisiae with a RNR2-lacZ reporter plasmid in response to DNA
damaging agents and agents that interfere with DNA synthesis. As well as an alkylating agent or UV radiation, argon
plasma torch treatment induced the high levels of RNR2-lacZ expression. These strongly suggest that non-thermal
atmospheric pressure plasmas treatment could cause genotoxic effects via cellular DNA damages.

1．Introduction

To understand the interaction between plasma
and living system is essentially important for
supporting medical and sanitary use of low
temperature plasma [1]. Microorganisms areuseful materials to analyze the effects causedby such a plasma exposure [2]. We have been
studying the inactivation process
ofbacteriophages (viruses which infect tobacteria) exposed to atmospheric pressure cold
plasma. Phage producing activity of theplasma-treated phage DNA was assayed bytransfection into the host E.coli cells. It was
found that single-stranded phage DNA isextremely sensitive to the plasma comparing todouble-stranded phage DNA. It is suggested
that plasma-damaged double-stranded DNA ispreferentially restored by the cellular DNA
repair system which scarcely works for thedamage of single-stranded DNA [3].
Another effective approach to the plasma andliving system interaction is to study theinfluence of atmospheric pressure plasma to thegene expression in living organism. Recently,
improved genotoxicity tests have been

established by harnessing cellular responces toDNA damage. We have constructed ayeast-based genotoxicity test system using
reporter assay linked to DNA damage-inducible
promoter [4]. The yeast Saccharomyces
cerevisiae is unicellular, easily manipulated
and thus respond to various DNA-damaging
agents in a mammalian cells than bacteria do.
Application of argon plasma jet to theyeast-based system induced the high levels of
the reporter gene expression. Evidence isprovided that this reporter system can monitor
genotoxic factors included in the atmosphericpressure cold plasma.
2．Experimental partThe schematic of the experimental set up forplasma exposure is shown in Fig.1. The plasma
jet generator was constructed with a glass tube,
a stainless steel wire, and a stainless steel mesh.
The stainless steel wire (0.2 mm in diameter)
was fixed as a high voltage electrode coaxiallyinside the glass tube. The stainless steel mesh
(10 mm width) was wrapped at the 5mm fromthe tip of the glass tube as ground electrode.

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Fig.1 Schematic of the experimental set-up.

Dielectric barrier discharge (DBD) wasgenerated between the both electrodes, using apulse
power supply (ECG-KOKUSAI,PPS-8000). The applied voltage was 12 kV0-P,
the frequency was 2.5 kHz, and the pulse width
was 2.8 s. The flow rate of argon was 2.0 L/min.
The gap between the tip of the generator andthe surface of the sample solution was 30 mm.
.
3．Results and discussion
Figure 2 shows the concept of the yeast
genotoxicity test system. DNA damage on thechromosomes works as a signal to the cell itself
which order to induce the high level of RNR2 geneexpression. RNR2 (ribonucleotide reductase subunitgene) is involved in cellular DNA repair reactions.
Therefore, the reporter lacZ gene under RNR2
promoter is induced by DNA damage. The extent ofDNA damage was assayed by the reporter
β-glactosidase activity of the yeast cells.
Fig.3 shows the results of plasma application tothe yeast cells. High levels of RNR2-lacZexpression occurred by the argon plasma jet
exposure. The results indicate that atmosphericpressure cold plasma has strong genotoxicity.
The yeast test system responds to many type ofcarcinogenic reagents. So, carcinogenic effectof the atmospheric pressure cold plasma is alsostrongly suggested.

Fig.2 Reporter assay system for detection ofDNA damage.

Fig.3 Induction of β-gal activity by argon plasma
torch.
Yeast strain
used DF5 cells with YEp365-RNR2-lacZ (filledsquare), YEp365-HSP26-lacZ (filled triangles) andYEp365 (filled circle).

References[1] G. Fridman, G. Friedman, A. Gutsol, A. B. Shekhter, V.
N. Vasilets, A. Fridman: Plasma Process. Polym., 5(2008) 503[2] H. Yasuda, M. Hashimoto, M. M. Rahman, K. Takashima,A. Mizuno: Plasma Process. Polym., 5 (2008) 615[3] H. Yasuda, T. Miura, H. Kurita, K. Takashima and
A. Mizuno, Plasma Processes and Polymers 7:
301-308, 2010.
[4] K. Ichikawa and T. Eki: J. Biochem., 139 (2006) 105