Letter pubs.acs.org/ac
High-Throughput Screening of Radioprotectors Using Rat Thymocytes Emiko Sekine-Suzuki,*,† Ikuo Nakanishi,*,‡ Takashi Shimokawa,*,‡ Megumi Ueno,‡ Ken-ichiro Matsumoto,‡ and Takeshi Murakami† †
Research Program for the Application of Heavy Ions in Medical Sciences, Research Center for Charged Particle Therapy, National Institute of Radiological Sciences (NIRS), Inage-ku, Chiba 263-8555, Japan ‡ Advanced Particle Radiation Biology Research Program, Research Center for Charged Particle Therapy, National Institute of Radiological Sciences (NIRS), Inage-ku, Chiba 263-8555, Japan S Supporting Information *
ABSTRACT: We invented a high-throughput screening method for the examination of radioprotective activity of chemical compounds using rat thymocytes. X-irradiation of the rat thymocytes induced apoptosis, leading to a significant cell shrinkage, which could be easily detected and directly quantified by the flow cytometry analysis. The protective effect of some natural antioxidants against radiation induced apoptosis in the rat thymocytes, as well as their toxicities without X-irradiation, was successfully evaluated using this method. This method provides a powerful tool to develop novel radioprotectors without toxicity and can also be widely used to estimate other oxidative stress except for radiation.
adiation therapy is the first choice for cancer therapy in the United States and Europe and recently increasing in Japan.1 If the side effects of radiation, such as inflammation and fibrosis of the normal tissue around the cancer, can be effectively prevented by using radioprotectors, the radiation therapy will be much more advanced. Thus, the demands for radioprotectors are rising dramatically in recent years. In addition, since the Fukushima Daiichi nuclear disaster in Japan, radioprotection has attracted a great deal of attention. Therefore, it is an urgent necessity to develop effective radioprotectors. A large number of chemical compounds, which show radioprotective effect in in vitro/vivo experiments, have so far been reported.2,3 Among them, only amifostine was authorized as a radioprotection agent for clinical use by the FDA. However, even amifostine is not widely used clinically due to side effects such as vomiting.4,5 Thus, the screening of efficient radioprotectors without side effects among a large number of chemical compounds is of considerable importance. However, conventional methods, such as colony-formation assay, survival assay of animals, comet assay, and MTT assay, have some problems, such as high cost and complexity etc., and these methods were unsuitable for the large-scale screening of a wide variety of compounds.6 Therefore, we invented a novel high-throughput method for screening effectiveness of the radioprotective activity of chemical compounds as an adjuvant to clinical application.5 The radiation-induced cell shrinkage of rat thymocytes is easily detected and measured by flow cytometer analysis. Because
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© 2013 American Chemical Society
thymocytes are lymphoid cells, interphase death, morphologically regarded as apoptosis, is induced several hours after Xirradiation.6 Thus, the radiosensitivity of thymocytes is particularly high as compared to other types of normal cells. The size of irradiated rat thymocytes was significantly reduced due to apoptosis and classified into two discrete subpopulations as normal and smaller sizes.7−9 Without complex procedures, such as fluorescence dyeing, this method would be suitable for the large-scale screening of radioprotectors. The radioprotective activities of representative natural antioxidants evaluated with use of this method, together with their radical-scavenging activity, are also reported as an example of this method, providing fundamental and valuable information about structure−activity relationship to develop novel radioprotectors without side effects.
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EXPERIMENTAL SECTION Male Wistar-MS rats were obtained at 8 weeks of age from Japan SLC, Inc. (Hamamatsu, Japan) (see Supporting Information (SI)). The thymus from 10−15 week old rats was washed in cold Dulbecco’s phosphate-buffered saline (PBS) (Sigma) to remove extra fat and placed in Roswell Park Memorial Institute medium (RPMI 1640) (Sigma) supplemented with 10% fetal bovine serum (FBS). The thymus was Received: June 25, 2013 Accepted: August 1, 2013 Published: August 1, 2013 7650
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cut and squeezed to thymocytes by using tweezers (see SI). The thymocytes in plates were X-irradiated with a Shimadzu Pantak HF-320 at a dose rate of 1.215 ± 0.055 Gy/min (200 kVp, 20 mA, 0.5 mm Al + 0.5 mm Cu filter). After irradiation, the cells were incubated for 4 h at 37 °C with 5% CO2. After incubation, the size and number of thymocytes were measured (see SI).
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RESULTS AND DISCUSSION Figure 1 shows fluorescence images of the nuclei of the thymocytes. The chromatin structure was observed in the
Figure 1. Fluorescence micrograph of viable (living cell; LC) and dead (apoptotic cell; AC) thymocytes. The cells were incubated with a 1:1000 dilution of 4,6-diamidino-2-phenylindole (DAPI) after 4 h incubation following 2 Gy X-irradiation. Images were captured using an Olympus DP70 fluorescence microscope.
Figure 2. Flow cytometer analysis of the rat thymocytes. (A) Cells were incubated in the presence of 0.1% DMSO for 4 h. (B) Cells were incubated in the presence of 0.1% DMSO for 4 h after 2 Gy Xirradiation. (C) Cells were incubated in the presence of 1 mM caffeic acid and 0.1% DMSO as a cosolvent for 4 h. (D) Cells were incubated in the presence of 1 mM caffeic acid and 0.1% DMSO as a cosolvent for 4 h after 2 Gy X-irradiation.
nuclei of the living cells (LC). On the other hand, nuclear condensation induced by the X-irradiation was observed in the apoptotic cell (AC). The rat thymocytes were condensed as one cell without forming the apoptotic cell fragments. Although we showed the fluorescence image to confirm the size reduction of the thymocytes by X-irradiation (Figure 1), this novel screening method requires no fluorochrome staining but just quantification of the cell size. Thus, this method is much simpler, easier, and more precise as compared to conventional methods such as colony-formation assay, survival assay of animal, comet assay, and MTT assay. Figure 2 shows the results of the flow cytometry analysis for the quantification of the rat thymocytes. The thymocytes were pretreated with either 0.1% dimethyl sulfoxide (DMSO) or 1 mM caffeic acid as a radioprotector candidate with DMSO 0.1% as a cosolvent, followed by 2 Gy X-irradiation. After irradiation, these cells were incubated for 4 h at 37 °C and then measured on a flow cytometer FACS Calibur (Becton Dickinson Company), by which the cell size could be measured as the FSC-H. At least 10000 cells were measured for each sample. The horizontal and vertical axes in each panel of Figure 2 show the size of thymocytes and the number of population, respectively. The arrows A1−4 and L1−4 in Figure 2A−D indicate the peak positions of apoptotic and viable cells, respectively. The control cells show a sharp peak at the cell size of around 400 (Figure 2A). However, 2 Gy X-irradiaton resulted in the decrease of the peak height at around 400 accompanied by an increase in a peak height at around 200 (Figure 2B). These results demonstrate that the irradiated thymocytes were condensed and the cell size was reduced by half. The rat thymocytes always draw the practically same histogram under our experimental conditions. Figure 2D shows the thymocytes treated with 1 mM caffeic acid before Xirradiation. In this case, the peak at around 400 remained unchanged. These results suggest that caffeic acid efficiently protects the rat thymocytes from radiation-induced apoptosis and can act as an efficient radioprotector. Figure 2C shows that 1 mM caffeic acid itself did not induce apoptosis in the thymocytes, showing no toxicity toward thymocytes.
This method can also be used for the evaluation of the toxicity of chemical compounds. The thymocytes were treated with either 0.1% DMSO or 0.1 mM ascorbic acid with 0.1% DMSO as a cosolvent without X-irradiation. The peak at around 400 (L1) in Figure 3A was significantly decreased by
Figure 3. Flow cytometer analysis of the rat thymocytes. (A) Cells were incubated in the presence of 0.1% DMSO for 4 h. (B) Cells were incubated in the presence of 0.1 mM ascorbic acid and 0.1% DMSO as a cosolvent for 4 h.
addition of 0.1 mM ascorbic acid (the peak L5 in Figure 3B) accompanied by an increase in the peak at around 250 (A5) due to apoptotic cells. These results indicate that ascorbic acid induces apoptosis in the thymocytes at a concentration of 0.1 mM and shows the toxicity under these experimental conditions. Thus, by using this screening method, the toxicity of the chemical compounds can also be easily estimated. We performed the radioprotector screening for natural antioxidants, (+)-catechin, curcumin, resveratrol, ascorbic acid (vitamin C), α-tocopherol (vitamin E), caffeic acid, and quercetin, using this method. Each antioxidant was dissolved 7651
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mM), and quercetin (100 μM) significantly induced apoptosis without X-irradiation, showing toxicity induced by these compounds. On the other hand, (+)-catechin (1 mM), curcumin (10 μM), resveratrol (100 μM), caffeic acid (100 μM and 1 mM), and quercetin (1 mM) efficiently protected the thymocytes against radiation-induced apoptosis. We also performed the screening for conventional radioprotectors, which are amifostine, cysteamine, and cysteine, using this method. Among them, cysteamine (1 mM) efficiently protected against radiation-induced apoptosis in the rat thymocytes, and this radioprotective effect is comparable to that of (+)-catechin (1 mM). To quantify radioprotective efficiency of the antioxidants, we introduced a factor, which indicates the degree of radio modification factor (RMF) (see SI Table S1). RMF is defined as the ratio of apoptotic cells with antioxidants divided by the ratio of apoptotic cells without antioxidants (eq 1).
in DMSO as a cosolvent and applied to the cell suspensions (1, 10, 100 μM, or 1 mM), The thymocytes were pretreated with either DMSO 0.1% or the antioxidants with DMSO 0.1% as a cosolvent. After 2 Gy X-irradiation followed by 4 h incubation, the cell size was measured by the flow cytometer. The data were analyzed by CellQuest pro software (BD) and ModFit 3.1 LT SP2 (Verity House Software). The results thus obtained are shown in Figure 4. The blue and red bars show percentages of the apoptotic cells without
RMF =
ratio of apoptotic cell with chemical ratio of apoptotic cell without chemical
(1)
If there is no radiation modification, RMF becomes 1.0. We also defined toxicity in the sample without radiation (eq 2). toxicity = (ratio of apoptotic cells with antioxidants) − (ratio of apoptotic cells with DMSO)
(2)
RMF and toxicity values for the antioxidants thus calculated are listed in Table S1 (see SI). The RMF values of the compounds with radioprotective activity are smaller than 1.0, while those with radio-sensitizing activity are greater than 1.0. In SI Table S1, the superscripted “a” marks indicate the data for high protection (0.5 > RMFb) and no toxicity (toxicity was >10c). According to this screening method, 1 mM (+)-catechin, 10 μM curcumin, 100 μM resveratrol, 100 μM and 1 mM caffeic acid, and 1 mM quercetin showed high radioprotective activity against radiation-induced apoptosis in the rat thymocytes. About 80% of radiation injuries are thought to be caused by reactive oxygen species (ROS), such as hydroxyl radical (•OH), produced by the excitation of water molecules. In this context, we investigated the relation between the RMF values and radical-scavenging rate constants (k) of these antioxidants. However, no significant correlation between the RMF and k values for •OH- or 2,2-diphenyl-1-picrylhydrazyl radical (DPPH•)-scavenging reactions (see SI Figures S1 and S2). These results indicate that besides the ROS-scavenging activity, hydrophilicity/lipophilicity of compounds should also be taken into account to develop effective radioprotectors. Further experiments are underway to investigate the structure−activity relationships for the effective radioprotectors.
Figure 4. Effects of various concentrations (in μM) of natural antioxidants and aminothiols on the apoptosis in rat thymocytes with (red bars) or without (blue bars) 2 Gy X-irradiation.
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CONCLUSIONS The high-throughput screening method based on the radiationinduced shrinkage of rat thymocytes10 provides a powerful tool to develop novel radioprotectors, which can efficiently protect normal tissues without toxicity. This screening method can also be widely used to estimate the suppression activity of chemical compounds against other oxidative stress, such as UV, ROS, nitrogen oxides (NOx), and so on, which can induce apoptosis in the rat thymocytes.
and with X-irradiation, respectively. In the absence of DMSO (control in Figure 4), 2 Gy X-irradiation induced apoptosis in about 40% of the thymocytes, while about 20% of the thymocytes were dead without X-irradiation. DMSO 0.1% used as a cosolvent showed no effect on the viability of the thymocytes regardless of whether they were irradiated or not. Among antioxidants used in this study, curcumin (100 μM and 1 mM), resveratrol (1 mM), ascorbic acid (100 μM and 1 7652
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Letter
ASSOCIATED CONTENT
S Supporting Information *
Experimental details, RMF and toxicity values of antioxidants, and plots of RMF vs DPPH•- and •OH-scavenging rate constants (k) of antioxidants. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Phone: +81-43-206-3131. Fax: +81-43-255-6819. E-mail:
[email protected] (E.S.);
[email protected] (I.N.); takshi@ nirs.go.jp (T.S.). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Dr. Harumi Ohyama, Dr. Takashi Imai, and Prof. Kazunori Anzai for profitable discussion in the preparation of this manuscript. We also thank Miyako Nakawatari and Etsuko Nakamura for their excellent technical assistance. This work was supported by FACS support team in NIRS. This work was partially supported by Grant-in-Aid (no. 23590064 to I.N.) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
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REFERENCES
(1) JASTRO (Japanese Society for Therapeutic Radiology and Oncology), Report; JASTRO (Japanese Society for Therapeutic Radiology and Oncology), 2005. (2) Weiss, J. F.; Landauer, M. R. Int. J. Radiat. Biol. 2009, 85, 539− 573 and references cited therein.. (3) Radioprotectors: Chemical, Biological, and Clinical Perspectives; Bump, E. A., Malaker, K., Eds.; CRC Press: Boca Raton, FL, 1997. (4) Hosseinimehr, S. J. Drug Discovery Today 2007, 12, 794−805. (5) Kouvaris, J. R.; Kouloulias, V. E.; Vlahos, L. J. Oncologist 2007, 12, 738−747. (6) Maurya, D. K.; Nair, C. K.; Devasagayam, T. P. Mutat. Res. 2012, 74, 993−996. (7) Ohyama, H.; Yamada, T.; Watanabe, I. Radiat. Res. 1981, 85, 333−339. (8) Yamada, T.; Ohyama, H. Int. J. Radiat. Biol. 1988, 53, 65−75. (9) Ohyama, H.; Yamada, T.; Ohkawa, A.; Watanabe, I. Radiat. Res. 1985, 101, 123−130. (10) This method is under evaluation for a patent in Japan (No. 2012-72619) and PCT patent (PCT/JP2013/002032).
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