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TOXICOLOGY
Departments of Anesthesia (T.A., M.K., K.F.) and Hematology and Oncology (T.M., K.Y.), Kyoto University Hospital, Kyoto, Japan; Institute of Advanced Energy, Kyoto University, Uji, Kyoto, Japan (M.N., K.M.); Wakasa Wan Energy Research Center, Tsuruga, Japan (N.E.); Department of Anesthesia, Higashiyama Takeda Hospital, Kyoto, Japan (H.M.); and School of Health Science Faculty of Medicine, Kyoto University, Kyoto, Japan (M.S.)
Received for publication
September 12, 2007
Accepted
November 19, 2007.
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; Watanabe and Egawa, 1994; Kawai et al., 1997; Nakashima et al., 1999; Shichinohe et al., 2004; Zhang et al., 2005), and it has been widely used in patients with acute brain infarction in Japan (Group, 2003). Edaravone was also shown to prevent endotoxin-induced liver injury (Kono et al., 2003; Tsuji et al., 2005) and hepatic ischemia-reperfusion injury (Abe et al., 2004; Suzuki et al., 2005). Furthermore, edaravone was shown to protect kidney against cisplatin-induced acute renal damage (Satoh et al., 2003) and ischemia-reperfusion injury (Doi et al., 2004). These protective effects of edaravone are thought to be attributed to its scavenging activity of reactive oxygen species (ROS). Among ROS, edaravone was documented to scavenge hydrogen peroxide (H2O2) and hydroxyl radicals (·OH) (Yamamoto et al., 1997; Mikawa et al., 2005). It is controversial whether edaravone scavenges superoxide anion (O
–2) or not (Watanabe and Egawa, 1994; Wu et al., 2000). In a recent study, we demonstrated that edaravone scavenged singlet oxygen (1O2) released from activated human neutrophils with an IC50 of 0.3 µM and suggested that this potent 1O2 scavenging activity of edaravone might be involved in its curative effects on acute brain infarction (Sommani et al., 2007).
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Singlet oxygen is known to induce neuronal and hepatic cell death (Cutrìn et al., 2000; Petrat et al., 2003; Valencia and Moran, 2004). Furthermore, 1O2 is tumoricidal and plays an important role in anticancer photodynamic therapy (Dougherty et al., 1998; Dolmans et al., 2003). In the previous study, we showed that a pterin derivative, 2-(N,N-dimethylaminomethyleneamino)-6-formyl-3-pivaloylpteridine-4-one (DFP; Fig. 1, right), generated a large amount of 1O2 under long-wavelength UV light (UV-A), which induced cell death in a pancreatic cancer cell line, Panc-1 cells (Yamada et al., 2005). That is, we showed the photodynamic effects of DFP on Panc-1 cells. Because the cell death was induced by generated 1O2, and edaravone was a potent 1O2 scavenger, we applied both edaravone and DFP to Panc-1 cells anticipating that edaravone would attenuate the cell death induced by photodynamic effects of DFP; however, unexpectedly, edaravone enhanced cell death, and further, cell death was induced, regardless of UV-A irradiation (data not shown). This finding led us to conceive the idea that DFP reacts with edaravone and generates a highly toxic new compound even when the concentration of each agent is far from a toxic dose. Furthermore, since we documented that DFP generates ROS through NADH oxidation (Nonogawa et al., 2007), the ROS generation is considered to be involved in this phenomenon.
In the present study, to verify the above-mentioned idea, the reaction of DFP with edaravone was analyzed chemically, and the effects of their combined use were examined biologically. For that purpose, production analysis of the reaction of DFP with edaravone was performed, and the chemical characters and activities of the reaction product were examined. The effects of the combined use of DFP with edaravone on cell viability and intracellular ROS generation were also examined in Panc-1 cells. Furthermore, the effects of the combined use of DFP with edaravone were examined in normal cells, such as human umbilical vein endothelial cells (HUVECs) and primary cultured rat hepatocytes. Furthermore, it was examined whether edaravone reacted with another pterin derivative to yield an ROS-generating compound.
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). 6FP was obtained from Sankyo Kasei Kogyo (Hiratsuka, Japan). Dulbecco's modified Eagle's medium for Panc-1 cells, RPMI 1640 medium for hepatocytes, fetal calf serum (FCS), and penicillin-streptomycin liquid were purchased from Invitrogen (Carlsbad, CA). Humedia-EG2 complete medium for HUVECs was obtained from KURABO (Osaka, Japan). 2',7'-Dichlorofluorescein diacetate (DCFH-DA) and tetramethylrhodamine methyl ester (TMRM) were obtained from Molecular Probes (Eugene, OR). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and diethylenetriaminepentaacetic acid (DETAPAC) were from Nacalai Tesque (Kyoto, Japan). 5,5-Dimethyl-1-pyrroline-N-oxide (DMPO) was from Labotec Co. (Tokyo, Japan). Other chemicals, such as catalase and N-acetyl-L-cysteine (NAC), were purchased from Sigma Chemicals (St. Louis, MO). Preparation of Working Solutions of DFP and Edaravone. Both DFP and edaravone were directly dissolved in phosphate-buffered saline (137 mM sodium chloride, 8.1 mM sodium phosphate dibasic, 2.68 mM potassium chloride, and 1.47 mM potassium phosphate monobasic, PBS) to a final concentration of 4 mM in each, pH 7.4, and filtered for sterilization.
Purification of the Reaction Product of DFP with Edaravone. The PBS solution containing 4 mM DFP and that containing 4 mM edaravone were mixed with stirring for 2 h at room temperature. During this time, precipitation appeared and was separated by centrifugation, and then dried by freezing. The resultant solid was purified by a high-performance liquid chromatography (HPLC) system consisting of an L-6200 pumping system and an L-4000 UV detector (HITACHI, Ibaraki, Japan); column, ULTRON VX-SIL (Shinwa Chemical Industries, Kyoto, Japan); column and particle size, 20.0 x 250 mm and 5 µm, respectively; and eluent, CHCl3-MeOH (60:40). The fraction was evaporated in vacuo, and the compound was obtained as a yellow solid, which was named reaction product of DFP with edaravone (DFP-E).
Structure Analysis and Water Solubility of DFP-E. To determine the chemical structure of DFP-E, 1H and 13C NMR spectra of DFP-E were recorded on a JEOL ECA-600 NMR spectrometer (JEOL Ltd., Tokyo, Japan) operating at 600 and 150 MHz, respectively, in deuterated dimethyl sulfoxide. All chemical shifts are reported in parts per million down field from tetramethylsilane. Peak multiplicities are denoted by broad (br), singlet (s), broad singlet (br s), and broad doublet (br d). Mass spectra of DFP-E were recorded on a JEOL DX-300 at 30 eV as electron impact condition. The water solubility of DFP-E was determined by UV spectroscopy.
Test of DFP-E Stability. To examine the stability of DFP-E, four types of PBS solutions were prepared as follows: PBS containing 300 µM DFP and 300 µM edaravone (1:1), PBS containing 300 µM DFP and 600 µM edaravone (1:2), PBS containing 300 µM DFP and 900 µM edaravone (1:3), and PBS containing 300 µM DFP and 1200 µM edaravone (1:4). These solutions were allowed to stir in an open system at room temperature. After 30 min and 20 h, DFP-E concentrations were measured by reversed-phase (RP)-HPLC as described below.
Production Analysis of the Reaction of DFP alone, Combination of DFP and Edaravone, or DFP-E with NADH. Production analysis of the reaction of DFP, combination of DFP and edaravone, or DFP-E with NADH was performed using RP-HPLC. Three types of PBS solutions were prepared as follows: PBS containing 300 µM DFP and 300 µM NADH; PBS containing 300 µM DFP, 300 µM edaravone, and 300 µM NADH; and PBS containing oversaturated DFP-E and 1 mM NADH. These solutions were allowed to stir in an open system at room temperature. The time-dependent concentration change of each component in the sample solution was analyzed by RP-HPLC. NAD+ in the RP-HPLC peak was identified by UV absorption spectra and quantified by RP-HPLC again. The RP-HPLC system consisted of a DP-8020 pumping system, a CO-8020 column oven, and a PX-8020 system controller (TOSOH; Tokyo, Japan); column, ULTRON VX-ODS (Shinwa Chemical Industries); column and particle size, 4.6 x 150 mm and 5 µm, respectively; eluent, 10 mM; 100 mM triethylammonium acetate buffer (100 mM triethylamine and 100 mM acetic acid, pH 7.0); gradient, CH3CN concentration in the liner gradient mode (0 min, 0%; 30 min, 50%); flow rate, 1.0 ml/min; and temperature, 37°C. UV spectra were recorded on a Jasco UV/VIS Spectrophotometer V-550 (Japan Spectroscopic Co., Tokyo, Japan).
To examine whether edaravone reacted with another pterin derivative that may exist in vivo, reaction of 6FP alone or combination of 6FP and edaravone with NADH was also examined using the RP-HPLC system. Because 6FP is poorly soluble in water at neutral pH, 6FP solutions were prepared by diluting 20 mM 6FP in 0.1 N NaOH solutions with PBS.
Oxygen Electrode Measurements. To confirm O2 consumption during the reaction of NADH with DFP alone or combination of DFP and edaravone, O2 concentrations of reaction solutions were measured polarographically with a Clarke oxygen electrode (model 5300; Yellow Springs Instruments, Yellow Springs, OH) at 37°C in 4 ml of reaction mixture. Oxygen concentration was calibrated with air-saturated PBS, which was assigned as 100%.
Electron Paramagnetic Resonance Spectroscopy and Spin Trapping. To determine whether the reaction of DFP-E with NADH produces ROS, an electron paramagnetic resonance (EPR) spectroscopy with a spin trap, DMPO, was used to directly measure oxygen radicals. PBS solution containing 1 mM DFP-E, pH 10, and 2 mM NADH were allowed to stir for 48 h at room temperature. After the addition of 100 mM DMPO, 500 µM DETAPAC, and 250 µM FeSO4, the mixture was transferred to a flat quartz EPR aqueous cell, which was fixed in the cavity of the EPR spectrometer. EPR spectra were recorded on a Model JES-TE300 spectrometer (JEOL Ltd., Tokyo, Japan). EPR settings were as follows: microwave power, 5 mW; field, 329.4 ± 5 mT (9.2533 GHz); modulation, 0.079 mT; time constant, 0.03 s; amplitude, 500; and sweep time, 1 min. The intensity of DMPO spin adducts and hyperfine splitting constants were calculated, based on the Mn2+ marker, which was inserted into the cavity of the EPR spectrometer.
ROS formation in cells was also verified using EPR spectroscopy. Panc-1 cells, cultured as described below, were incubated with 400 µM DFP alone or combination of 400 µM DFP and 400 µM edaravone for 4 h in an FCS-free medium; FCS was omitted because of possible quenching of ROS. Then, the mixture of the supernatant of the cells with 100 mM DMPO, 500 µM DETAPAC, and 250 µM FeSO4 was prepared and transferred to a flat quartz EPR aqueous cell, which was fixed in the cavity of the EPR spectrometer. EPR settings were modified as follows: microwave power, 10 mW; field, 329.3 ± 5 mT (9.2535 GHz); modulation, 0.125 mT; time constant, 0.03 s; amplitude, 790; and sweep time, 2 min.
Emission of Fluorescence from Aqueous Solutions of DFP and DFP-E. The pterin family emit fluorescence under UV-A radiation (wavelength around 360 nm) (Thomas et al., 2002). Therefore, if the emission intensity is enough to detect, the uptake of DFP or DFP-E by cells should be observed using fluorescent microscopy with a UV filter. So, as a basic study, the emission of fluorescence from a 10 µM concentration of the aqueous solution of DFP or DFP-E was scanned using a spectrofluorometer (F-4500; HITACHI) at a fixed excitation wavelength of 360 nm and a variable emission wavelength.
Cell Culture. Panc-1 cells were obtained from the American Type Culture Collection (Rockville, MD) and cultured in Dulbecco's modified Eagle's medium containing 10% FCS, 100 units/ml penicillin, and 100 µg/ml streptomycin at 37°C in a 5% CO2 incubator. HUVECs were obtained from KURABO and cultured in Humedia-EG2 complete medium. Third passage cells were used. Hepatocytes were isolated from male Wistar rats as previously described (Ishii et al., 2005) and cultured in RPMI 1640 medium containing 10% FCS, 100 units/ml penicillin, and 100 µg/ml streptomycin. Primary cultured cells were used.
Uptake of DFP or DFP-E by Cells. To evaluate the uptake of DFP or DFP-E by Panc-1 cells, after incubation with DFP alone or combination of DFP and edaravone for 4 h, the cells were washed twice with PBS. The cells were then observed with a Nikon TE200 fluorescence microscope (Nikon, Tokyo, Japan) equipped with a LEICA DC500 digital charge-coupled device camera (Leica, Wetzlar, Germany) and a PC. To obtain visually clear contrast, the concentrations of DFP and edaravone were adjusted to 400 µM.
Time-Lapse Cell Imaging. Intracellular ROS generation and cell death in Panc-1 cells were observed using a time-lapse imaging system (BioStation IM; Nikon), which consists of an incubator incorporating fluorescent microscopy equipped with a digital charge-coupled device camera and a PC for data acquisition and analysis. Cells (1 x 105 cells/ml) were incubated overnight in a 35-mm glass base dish (IWAKI, Funabashi, Japan) in 2 ml of medium. The cells were then incubated with 100 nM TMRM, a mitochondrial membrane potential-sensitive dye (Petrat et al., 2003), for 20 min. The medium was then changed, and the cells were incubated with combination of DFP and edaravone and 2 µM DCFH-DA, an ROS-sensitive dye (Royall and Ischiropoulos, 1993) at 37°C and 5% CO2 in the time-lapse imaging system, and the phase-contrast images and fluorescent images were acquired every 15 min for 4 h. Green fluorescence images for DCF and red fluorescence images for TMRM were acquired using fluorescein isothiocyanate and tetramethylrhodamine B isothiocyanate filters (Nikon), respectively. The concentrations of DFP and edaravone were adjusted to 200 µM because the changes in fluorescence were visually weak when 100 µM DFP and edaravone were used, and rapid cell death and detachment interfered with visual observation when 400 µM DFP and edaravone were used. Time-lapse imaging of cells incubated without DFP or edaravone was also performed.
Cytotoxicity Assessment. Panc-1 cells (1 x 105 cells/ml) were incubated overnight in 12-well tissue culture plates (BD Biosciences, Bedford, MA) with 1 ml in each well. The cells were then incubated with DFP alone, edaravone alone, or combination of DFP and edaravone for 1, 2, 3, 4, or 24 h. After washing twice with PBS, the cells were incubated with fresh medium for another 24 h, except for the cells incubated with drugs for 24 h. Cell viability 24 h after medium change was assessed by the mitochondrial-dependent reduction of MTT to formazan (MTT assay). As for the cells incubated with drugs for 24 h, as soon as the cells were washed twice with PBS, cell viability was assessed by MTT assay. Effects of NAC, an antioxidant, on cell viability were also examined in cells incubated with a combination of DFP and edaravone.
HUVECs (5 x 106 cells/ml) were incubated overnight in 12-well tissue culture plates with 1 ml in each well. The cells were then incubated with DFP alone or combination of DFP and edaravone for 4 h. After washing twice with PBS, the cells were incubated with fresh medium for another 24 h. Cell viability 24 h after medium change was assessed by MTT assay.
Hepatocytes (1.25 x 105 cells/ml) were incubated overnight in 100-mm tissue culture dishes with 10 ml in each dish. The cells then were incubated with DFP alone or combination of DFP and edaravone for 4 h. After washing twice with PBS, the cells were incubated with fresh medium for another 24 h. Cell viability 24 h after medium change was assessed by MTT assay.
Statistical Analysis. Values are shown as the means ± S.D. Statistical comparisons were made using one-way analysis of variance followed by Student's paired t test with Bonferroni correction. P values < 0.05 were regarded as significant.
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Reaction of DFP, Combination of DFP and Edaravone, or DFP-E with NADH. When PBS solutions containing 300 µM DFP and 300 µM NADH were allowed to stir for 100 h, the oxidation of NADH to NAD+ occurred at a slow rate (Fig. 2A, solid line). In contrast, when PBS solutions containing 300 µM DFP, 300 µM edaravone, and 300 µM NADH were allowed to stir for 100 h, the oxidation of NADH to NAD+ occurred at a higher rate (Fig. 2A, broken line). When PBS solutions containing oversaturated DFP-E (approximately 200 µM) and 1 mM NADH were allowed to stir for 100 h, the oxidation of NADH to NAD+ occurred at a considerable rate (Fig. 2B). It should be noted that, in this reaction, since a sufficient amount of DFP-E was used, the reaction solution was saturated with DFP-E over 100 h, and the concentration of DFP-E was constant throughout this reaction (approximately 200 µM).
When PBS solutions containing 300 µM 6FP and 300 µM NADH were allowed to stir for 100 h, the oxidation of NADH to NAD+ did not occur (Fig. 2C, solid line). In contrast, when PBS solutions containing 300 µM 6FP, 300 µM edaravone, and 300 µM NADH were allowed to stir for 100 h, the oxidation of NADH to NAD+ occurred at a high rate (Fig. 2C, broken line).
O2 Consumption and ROS Generation. The oxygen concentration of the solution containing 6 mM NADH alone decreased slightly (5% decrease in 100 min Fig. 3A, a), indicating spontaneous O2 consumption. In reaction solutions containing 1 mM DFP and 6 mM NADH, an apparent decrease in O2 concentration was observed (20% decrease in 100 min, Fig. 3A, b), suggesting conversion of O2 to ROS. In reaction solutions containing 1 mM DFP, 1 mM edaravone, and 6 mM NADH, the decrease was enhanced (25% decrease in 100 min, Fig. 3A, c), suggesting that DFP-E was more potent than DFP to convert O2 to ROS.
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), demonstrating that signals were derived from trapping ·OH. In the presence of ferrous ion (Fe2+), ·OH was generated via the iron-catalyzed Fenton reaction from H2O2 (Dunford, 1987). Furthermore, these signals were quenched by adding catalase (10,000 units/ml) to the reaction solution (Fig. 3B, d). Based on these observations, it was found that the system composed of DFP-E and NADH finally generated H2O2 from O2. In the cells, although the supernatant obtained from the cells incubated without drugs or with DFP alone did not show any signals (Fig. 3C, a and b, respectively), the supernatant obtained from the cells incubated with combination of DFP and edaravone showed the DMPO-OH signals (Fig. 3C, c), which indicated ROS formation in the cells. Furthermore, it was confirmed these signals were quenched by adding catalase (2000 units/ml) to the supernatant (Fig. 3C, d).
Fluorescence Emission and Uptake of DFP and DFP-E by Cells. In the spectrofluorometric study, when the solution containing either DFP or DFP-E was excited with 360 nm of radiation, they both emitted fluorescence with peak emissions at 480 nm, and the peak fluorescence intensity was almost identical (Fig. 4A); therefore, if DFP and DFP-E are taken up by cells to the same extent, they should emit blue fluorescence with almost the same intensity under 360 nm of radiation, and their uptake should be visualized by fluorescent microscopy with a UV filter. However, in the fluorescent microscopic study, emission from cells incubated with DFP and edaravone was much stronger than that with DFP, which indicated that DFP-E was taken up by the cells more than DFP (Fig. 4B).
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In HUVECs, loss of cell viability by the combined use of DFP with edaravone was also observed, and significant loss was already noticed when the combination of 50 µM DFP and 50 µM edaravone was used (Fig. 7A, shaded bar). Furthermore, loss of cell viability was observed in the cells incubated with 400 µM DFP alone (Fig. 7A, closed bar). Loss of viability by the combined use of DFP with edaravone was also observed in hepatocytes, but significant loss was not noticed until the combination of 400 µM DFP and 400 µM edaravone was used (Fig. 7B, shaded bar).
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), and immediately produced the reaction product DFP-E. Edaravone is not definitely an ROS generator; however, DFP was shown to generate ROS through NADH oxidation in the previous study (Nonogawa et al., 2007), and in this study, DFP-E was revealed to react with NADH much more rapidly than DFP and to generate ROS. Because NADH and its analog NADPH are ubiquitous in cell bodies (Nieminen et al., 1997), an analogous reaction should occur in cells, and DFP-E is considered to generate much more ROS than DFP. Furthermore, it was also revealed that DFP-E is much more cell-permeable than DFP. These conditions collectively account for the intracellular ROS generation and cell death induced by the combined use of DFP with edaravone.
In this study, ROS formation in the reaction of DFP-E and NADH was verified using EPR. EPR is considered to be the least ambiguous method for the detection of free radicals because it allows the identification of original radicals (Buettner, 1987). The EPR study revealed that detected radicals were ·OH generated via the iron-catalyzed Fenton reaction from H2O2, which showed that the finally generated ROS was H2O2. ROS formation in the cells was also verified using EPR, and ·OH derived from H2O2 was detected in the supernatant obtained from the cells incubated with combination of DFP and edaravone. In this case, it was considered that H2O2 generated inside the cells diffused to the supernatant.
Intracellular ROS generation was also observed using time-lapse imaging with the probe DCFH-DA. DCFH-DA permeates cell membranes and becomes DCFH inside the cell. DCFH is then oxidized by ROS to yield DCF, which emits green fluorescence (Royall and Ischiropoulos, 1993). This probe is widely used to measure intracellular H2O2 formation (Chandel et al., 1998; Simizu et al., 1998). In a recent study, it was reported that the dihydrocompounds, such as DCFH, are not applicable to the selective detection of individual ROS and that they are suitable for detecting total oxidative activity in living cells (Soh, 2006). However, DFP-E was demonstrated to chemically react with NADH and finally yield H2O2 in this study, and DFP was also demonstrated to react NADH and yield H2O2 in the previous study (Nonogawa et al., 2007). Furthermore, H2O2 was detected in the supernatant obtained from the cells incubated with combination of DFP and edaravone. Therefore, the large increases in DCF fluorescence in the cells incubated with combination of DFP and edaravone and the small increases in DCF fluorescence in the cells incubated with DFP alone are highly possible to reflect the intracellular H2O2 generation. No increases in DCF fluorescence in the cells incubated without DFP or edaravone proved that autofluorescence by excitation light, which is problematic in the detection of ROS using DCFH (Rota et al., 1999), was negligible and that increases in DCF fluorescence certainly reflected intracellular ROS generation.
Cell death was observed using time-lapse imaging with the probe TMRM, which is a fluorescent probe to monitor the MMP. This indicator dye is a lipophilic cation accumulated by mitochondria in proportion to MMP and emits red fluorescence (Scaduto and Grotyohann, 1999). Discoloration of red fluorescence reflects loss of MMP, which precedes cell death (Kon et al., 2004). In Panc-1 cells incubated with 200 µM DFP and 200 µM edaravone, the discoloration occurred in inverse proportion to the appearance and increment of green fluorescence; however, in Panc-1 cells incubated with 200 µM DFP alone, although increases in green fluorescence was observed, discoloration of red fluorescence was not obvious. Therefore, ROS generation is considered not to necessarily cause the loss of MMP, and the amount of ROS may concern the loss of MMP preceding cell death.
Cell viability was measured using the MTT assay, which reflects cellular growth and survival (Mosmann, 1983). When Panc-1 cells were incubated with combination of DFP and edaravone for 4 h and then washed out and incubated without drugs for another 24 h, cell viability decreased significantly in cells incubated with more than 100 µM DFP and 100 µM edaravone. Because the loss of MMP preceding cell death were observed in cells incubated with combination of DFP and edaravone in the time-lapse imaging study, the reduction of cell viability was caused by the blocking of cellular survival; therefore, a large amount of ROS generated by the combined use of DFP with edaravone is considered to cause cell death. The fact that NAC, an antioxidant, attenuated the cell death supported this idea.
DFP alone induced intracellular ROS generation to some extent, but significant decreases in cell viability were not observed in Panc-1 cells incubated with even a high dose of DFP alone (400 µM). However, in HUVECs, a low dose of DFP and edaravone (50 µM, in each) and a high dose of DFP alone (400 µM) caused significant decreases in cell viability. In contrast, in hepatocytes, significant decreases in cell viability were not observed until a high dose of DFP and edaravone (400 µM, in each) was applied. These results indicated that cellular survival depends on the amount of generated ROS and the resistance of each type of cell against intracellular ROS. In the case of hepatocytes, cells may decompose DFP-E by metabolism or may quench ROS more vigorously with intrinsic antioxidants, such as glutathione, than other types of cells. In this study, seeding densities were different among three types of cell, which may have affected the resistance against ROS. However, proper culture condition was chosen for each type of cell (approximately 80% confluent in each medium), and cell viability seems to reflect the resistance of each type of cell against ROS to some extent. Further study will be required to elucidate this issue.
Edaravone reacted with not only DFP but also 6FP, and the reaction product should generate ROS through NADH oxidation. DFP is a synthesized compound and does not naturally exist in the body; however, 6FP is produced from folic acid in vivo in some pathological conditions, such as carcinoma (Halpern et al., 1977; Clynes and O'Neill, 1980; Durán et al., 1984) and possibly exists in the body. It was reported that 6FP was found in the urine at approximately 300 µMor greater in patients with a tissue diagnosis of cancer (Halpern et al., 1977), and the plasma concentration of edaravone in clinical cases is estimated around 10 µM (Group, 2003). This indicates the possibility that edaravone reacts with pterin derivatives existing in the body, such as 6FP, and yields ROS-generating compounds, which may induce cell death in vital organs. In Japan, edaravone was used in 140,000 patients per year since 2001, and it was reported that 12 patients died from acute renal failure in 2002 and that one patient died from fulminant hepatitis in 2004 (information from Ministry of Health Labor and Welfare of Japan). These severe side effects of edaravone may be induced by this mechanism. Therefore, care should be taken with the clinical use of edaravone when pterin derivatives remain in the body.
On the other hand, the cell-destructive nature of the combined use of DFP with edaravone is attractive from the standpoint of anticancer therapy. First, its ability to kill Panc-1 cells is powerful. Pancreatic cancer remains an unfortunate disease with a 5-year survival rate below 1% (Keleg et al., 2003). Second, tumoricidal activity was not exhibited until both agents were administered, at which point they worked at once in situ. Third, hepatocytes were considerably resistant against cell injury induced by the combined use of DFP with edaravone, which indicated the possibility of DFP-E detoxication by the liver. Although the intolerance of HUVECs against the combined use of DFP with edaravone is unfavorable, the drug delivery device may overcome this point.
In conclusion, the reaction product of DFP-E was much more cell-permeable and generated much more ROS inside cells than DFP. This conceivably explains why the combined use of DFP with edaravone induced cell death at low doses despite no cell death by the single use of DFP. This is unfavorable as a side effect but favorable as tumoricidal activity. Because edaravone is a clinically available agent, its adverse and advantageous effects should be further investigated.
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ABBREVIATIONS: edaravone, 3-methyl-1-phenyl-2-pyrazolin-5-one; ROS, reactive oxygen species; H2O2, hydrogen peroxide; ·OH, hydroxyl radical(s); DFP, 2-(N,N-dimethylaminomethyleneamino)-6-formyl-3-pivaloylpteridine-4-one; HUVEC, human umbilical vein endothelial cell; 6FP, 6-formylpterin; FCS, fetal calf serum; DCFH-DA, 2',7'-dichlorofluorescein diacetate; TMRM, tetramethylrhodamine methyl ester; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; DETAPAC, diethylenetriaminepentaacetic acid; DMPO, 5,5-dimethyl-1-pyrroline-N-oxide; NAC, N-acetyl-L-cysteine; PBS, phosphate-buffered saline; HPLC, high-performance liquid chromatography; DFP-E, reaction product of DFP with edaravone; br, broad; s, singlet; br s, broad singlet; br d, broad doublet; RP, reversed-phase; EPR, electron paramagnetic resonance; MMP, mitochondrial membrane potential.
Address correspondence to: Dr. Toshiyuki Arai, Department of Anesthesia, Kyoto University Hospital, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. E-mail: arai{at}kuhp.kyoto-u.ac.jp
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, NADH; 
, NAD+) and approximate curves (solid lines) show the oxidation reaction of NADH by DFP alone using PBS solutions containing 300 µM DFP and 300 µM NADH, which were allowed to stir at pH 7.4. The points (
, DFP; 
, NADH; 
, NAD+) and approximate curves (broken lines) show the oxidation reaction of NADH by the combined use of DFP with edaravone using PBS solutions containing 300 µM DFP, 300 µM edaravone, and 300 µM NADH, which were allowed to stir at pH 7.4. B, reaction of DFP-E with NADH. The points (x, DFP-E; 
, P < 0.05: significant difference between values obtained from cells in the absence or presence of NAC.