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0022-3565/01/2981-103–109$3.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Copyright © 2001 by The American Society for Pharmacology and Experimental Therapeutics JPET 298:103–109, 2001

Vol. 298, No. 1 3814/914311 Printed in U.S.A.

D609 Inhibits Ionizing Radiation-Induced Oxidative Damage by Acting as a Potent Antioxidant DAOHONG ZHOU, CHRISTOPHER M. LAUDERBACK, TAO YU, STEPHEN A. BROWN, D. ALLAN BUTTERFIELD, and JOHN S. THOMPSON Division of Allergy, Immunology, and Rheumatology, Department of Internal Medicine (D.Z., T.Y., S.A.B., J.S.T.), Department of Chemistry and Center of Membrane Sciences (C.M.L., D.A.B.), University of Kentucky, Lexington, Kentucky; Veterans Administration Medical Center, Lexington, Kentucky (S.A.B., J.S.T.); and Division of Research, Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, South Carolina (D.Z.) Received January 30, 2001; accepted April 5, 2001

This paper is available online at http://jpet.aspetjournals.org

ABSTRACT Tricyclodecan-9-yl-xanthogenate (D609) has been extensively studied in biological systems and exhibits a variety of biological functions, including antiviral, antitumor, and anti-inflammatory activities. Most of these activities have been largely attributed to the inhibitory effect of D609 on phosphatidylcholine-specific phospholipase C. However, as a xanthate derivative, D609 is a strong electrolyte and readily dissociates to xanthate anions and cations of alkali metals in solution. Xanthate anions and protonated xanthic acid contain a free thiol moiety and are highly reductive. This implies that D609 and other xanthate derivatives may function as potent antioxidants. Indeed, we found that D609 inhibited the Fenton reaction-induced oxidation of dihydrorhodamine 123 in a dose-dependent manner similar to that of pyrrolidinedithiocar-

Xanthates are the reactive products of carbon disulfide, an alcohol, and an alkali in an equal stoichimetric ratio (1:1:1) with elimination of water (Rao, 1971). They have the following general structure:

ROC⫺SM 储 S where R stands for an alkyl hydrocarbon and M denotes a monovalent metal such as potassium. Xanthates are strong electrolytes and readily dissociate to cations of alkali metals and xanthate anions in solution (Rao, 1971). Xanthate anions and protonated xanthic acid contain a free thiol moiety. This makes xanthates highly reductive agents. Upon reacting with an oxidant, xanthates are oxiThis study was supported in part by the grants from the National Institutes of Health to D.Z. (MH55058, CA78688, and CA86860) and D.A.B. (AG05119, AG10836, and AG12423), and grant from the Veterans Administration to J.T.

bamate, a well known antioxidant. In addition, D609 inhibited the formation of the ␣-phenyl-tert-butylnitrone-free radical spin adducts and lipid peroxidation of synaptosomal membranes by the Fenton reagents. Furthermore, preincubation of lymphocytes with D609 resulted in a significant diminution of ionizing radiation (IR)induced 1) production of reactive oxygen species; 2) decrease in intracellular reduced glutathione; 3) oxidative damage to proteins and lipids; and 4) activation of nuclear factor-␬B. Moreover, when D609 (50 mg/kg i.v.) was administered to mice 10 min prior to total body IR (6.5 and 8.5 Gy), it protected the mice from IR-induced lethality. Thus, these results indicate that D609 is a potent antioxidant and has the ability to inhibit IR-induced cellular oxidative stress.

dized to dixanthogen by forming a disulfide bond (Rao, 1971). Thus, xanthates have the potential to function as a potent antioxidant. Several xanthates have been studied in biological systems (Sauer et al., 1984; Amtmann and Sauer, 1987; Furstenberger et al., 1989; Yanev et al., 1999). One of them, tricyclodecan-9yl-xanthogenate (D609), exhibits a variety of potent biological functions, including antitumor (Amtmann and Sauer, 1987; Furstenberger et al., 1989; Schick et al., 1989a,b; Sauer et al., 1990), antiviral (Amtmann et al., 1987; Villanueva et al., 1991; Walro and Rosenthal, 1997), and anti-inflammatory activities (Machleidt et al., 1996; Tschaikowsky et al., 1998). Most of these activities have been largely attributed to the inhibitory effect of D609 on phosphatidylcholine-specific phospholipase C (PC-PLC) (Schutze et al., 1992; Wiegmann et al., 1994; Amtmann, 1996; Machleidt et al., 1996). Hydrolysis of phosphatidylcholine by PC-PLC produces the second messenger diacylglycerol that activates protein kinase C (PKC) and acidic sphingomyelinase (aSMase). Thus, inhibition of PC-PLC by

ABBREVIATIONS: D609, tricyclodecan-9-yl-xanthogenate; PC-PLC, phosphatidylcholine-specific phospholipase C; PKC, protein kinase C; aSMase, acidic sphingomyelinase; NF-␬B, nuclear factor-␬B; IR, ionizing radiation; mBCI, monochlorobimane; PDTC, pyrrolidinedithiocarbamate; DHR, dihydrorhodamine 123; R123, rhodamine 123; EPR, electron paramagnetic resonance; PBN, ␣-phenyl-tert-butylnitrone; TRARs, thiobarbituric acid reactive substances; ROS, reactive oxygen species; GSH, glutathione; DNPH, 2,4-dinitrophenyl hydrazine; LPO, lipid hydroperoxide; TPA, 12-O-tetra-decanoylphorbol-13-acetate. 103

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D609 suppresses the activities of PKC and aSMase (Schutze et al., 1992; Wiegmann et al., 1994; Cifone et al., 1995; Amtmann, 1996; Machleidt et al., 1996; Yamamoto et al., 1997). Suppression of PKC may partly account for the antiproliferative and antitumor function of D609 (Muller-Decker et al., 1988, 1989; Amtmann, 1996), whereas, suppression of aSMase by D609 reduces ceramide production and inhibits ceramide-mediated signal transduction (Schutze et al., 1992; Wiegmann et al., 1994; Machleidt et al., 1996), including activation of PKC-␨ (Simarro et al., 1999), mitogen-activated protein kinase (Buscher et al., 1995; Monick et al., 1999), and nuclear factor-␬B (NF-␬B) (Schutze et al., 1992; Wiegmann et al., 1994). However, the possibility that D609 may function as an antioxidant to exert some of these activities has been largely neglected. In the present study, we determined that D609 functions as an antioxidant. Furthermore, the abilities of D609 to protect lymphocytes from ionizing radiation (IR)-induced oxidative damage in vitro and prevent mice from IR-caused lethality in vivo were also investigated.

Materials and Methods Reagents. Dihydrorhodamine 123 (DHR) and monochlorobimane (mBCI) were purchased from Molecular Probes (Eugene, OR). Pyrrolidinedithiocarbamate (PDTC) and D609 were obtained from Sigma Chemical Co. (St. Louis, MO) and Biomol (Plymouth Meeting, PA), respectively. Mice. Male BALB/c mice were purchased from Harlan SpragueDawley (Indianapolis, IN) and were used at approximately 7 to 9 weeks of age. Upon arrival, the mice were housed four to a cage at the VA Medical Center Association for the Assessment and Accreditation of Laboratory Animal Care-certified animal facility. They received food and water ad libitum. The Institutional Animal Care and Use Committee of the University of Kentucky and the VA Medical Center approved all experimental procedures used in this study. IR. A mark IV137 Cesium ␥-irradiator (JL Shepherd, Glendale, CA) was used as the source of irradiation. The dose rate of irradiation was 2.4 Gy/min. In vitro samples or mice were irradiated on a rotating platform. Analysis of DHR Oxidation. Prior to initiating the Fenton reaction, DHR stock solution (10 mM in dimethyl sulfoxide) was diluted in phosphate-buffered saline, pH 7.4, to the final concentration of 2 ␮M. The fluorescence intensity of rhodamine 123 (R123) was measured 30 min after initiating the Fenton reaction (10 mM H2O2 and 200 ␮M FeSO4) by a PerkinElmer luminescence spectrometer (model LS 50B, Norwalk, CT) at ␭EX ⫽ 505 nm and ␭EM ⫽ 525 nm (Emmendorffer et al., 1990). For the analysis of antioxidant activities, various concentrations of PDTC and D609 were added to the DHR solution before initiating the Fenton reaction. In control samples for this assay, it was demonstrated that PDTC and D609 do not quench the fluorescence of R123 (data not shown). Electron Paramagnetic Resonance (EPR). Ultrapure ␣-phenyl-tert-butylnitrone (PBN, 10 mM) was used as the spin trap in the Fenton reaction (30 ␮M FeSO4; and 3 mM H2O2) in the presence or absence of D609. All reagents (PBN, H2O2, D609) except FeSO4 were added and mixed. After the addition of FeSO4, the samples were again mixed and incubated for 30 min at room temperature prior to acquisition of EPR spectra. As an external standard, trace metal impurities (possibly Mn2⫹) in calcium oxide were used to eliminate any instrumental tuning errors. EPR spectra were acquired at 23°C on a Bruker EMX EPR spectrometer with the following instrumental parameters: microwave frequency, 9.77 GHz; microwave power, 20 mW; receiver gain, 1 ⫻ 105; modulation amplitude, 0.3 G; and time constant, 1.28 ms (Butterfield et al., 1997). Analysis of Thiobarbituric Acid Reactive Substances (TBARs). The preparation of synaptosomes, induction of synapto-

some lipid peroxidation, and analysis of TBARs have been reported recently (Lauderback et al., 2000). TBARs were detected by measuring the fluorescence with an ␭EX ⫽ 518 nm and ␭EM ⫽ 588 nm. Isolation of Lymphocytes. Mice were euthanatized by CO2 suffocation. Spleens were harvested and single cell suspension was prepared by disrupting spleens using the frosted ends of microscopic slides. Lymphocytes were isolated by gradient centrifugation of spleen cells over Histopaque 1083 (Sigma Chemical Co.). Analysis of Levels of Intracellular Reactive Oxygen Species (ROS) and Reduced Glutathione (GSH). Lymphocytes (2 ⫻ 106/ ml) were suspended in phosphate-buffered saline supplemented with 5 mM glucose, 1 mM CaCl2, 0.5 mM MgSO4, and 5 mg/ml bovine serum albumin. They were preincubated with various concentrations of D609 (0, 18, or 188 ␮M) for 30 min and then with DHR (1 ␮M) for additional 5 min. The levels of ROS in the lymphocytes were analyzed by measuring the fluorescence intensity of R123 using a flow cytometer (␭EX ⫽ 488 nm and ␭EM ⫽ 530 nm) 30 min after the cells were exposed to 10 Gy IR (Emmendorffer et al., 1990). For the analysis of intracellular levels of GSH, the cells were preincubated with various concentrations of D609 (0, 18, or 188 ␮M) for 30 min and then exposed to 10 Gy IR. Thirty minutes after IR, the cells were pulsed with mBCI (40 ␮M) and the intracellular levels of GSH were analyzed 5 min later by a flow cytometer (␭EX ⫽ 380 nm and ␭Em ⫽ 460 nm) as previously described (Hedley and Chow, 1994). Analysis of IR-Induced Oxidative Damage to Proteins in Lymphocytes. Lymphocytes (2 ⫻ 106/ml in Hanks’ buffer) were preincubated with various concentrations of D609 (0, 18, or 188 ␮M) for 30 min and then exposed to IR (10 Gy). Control cells were not pretreated nor irradiated. Thirty minutes after IR, both irradiated and control cells were lysed by incubation of the cells with the modified RIPA buffer (50 mM Tris-HCI, pH 7.4; 1 mM EDTA; 150 mM NaCl; 1% NP-40; 1 ␮g/ml aprotinin, leupeptin and pepstatin; and 50 mM dithiothreitol) on ice for 15 min. After centrifugation (14,000g for 10 min), the supernatants were collected and frozen in small aliquots at ⫺80°C after collection. The concentrations of proteins in the lysates were determined with the Bio-Rad protein assay reagent (Bio-Rad, Hercules, CA). An aliquot of the lysates containing 15 ␮g of protein was derivatized with 2,4-dinitrophenyl hydrazine (DNPH). The levels of oxidatively modified proteins containing carbonyl groups in the lysates were analyzed by SDS-polyacrylamide gel electrophoresis using the OxyBlot kit from the Intergen Co. (Purchase, NY) according to the manufacturer’s protocol. Analysis of IR-Induced Oxidative Damage to Lipids in Lymphocytes. Lymphocytes were treated as described above. Thirty minutes after IR, both irradiated and control cells were sonicated in deionized water. Lipid hydroperoxides (LPO) were extracted into chloroform and assayed by using the LPO Assay kit from Cayman (Ann Arbor, MI) as described in the kit manual. The levels of LPO were calculated and expressed as nanomoles per 106 cells according to the LPO standard curve. Analysis of IR-Induced Lymphocyte NF-␬B Activation and Nuclear Translocation. Lymphocytes (5 ⫻ 106/ml in RPMI-1640 medium supplemented with 10% fetal bovine serum) were preincubated with various concentrations of D609 (0, 18, or 188 ␮M) for 30 min and then exposed to IR (10 Gy) or unirradiated. Preparation of nuclear extracts and analysis of the NF-␬B DNA binding activity in the nuclear extracts (5 ␮g/lane) were performed according to previously published methods (Zhou et al., 1999). Relative nuclear binding activities for NF-␬B DNA were quantified by scanning densitometry. IR-Induced Lethality in Mice. BALB/c mice were exposed to 6.5 or 8.5 Gy of total body IR 10 min after they received a single dose (50 mg/kg) of i.v. injection of D609 or vehicle (saline) through the tail veins. The survival of these mice was recorded during a 30-day observation period after IR. Statistical Analysis. The data were analyzed by analysis of variance. In the event that analysis of variance justifies post hoc comparisons between group means, these were conducted using the Student-Newman-Keuls test for multiple comparisons. For experi-

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ments in which only single experimental and control groups were used, group differences were examined by unpaired Student’s t test. Kaplan-Meier plots of survival data are analyzed by Log-Rank and Wilcoxon tests of chi-square. Differences were considered significant at p ⬍ 0.05.

Results D609 Inhibits DHR Oxidation by the Fenton Reaction. The nonfluorescent DHR is converted to the fluorescent product R123 after reaction with reactive oxygen species (ROS) (Emmendorffer et al., 1990). It is expected that a potent antioxidant will protect DHR from oxidation by competitive removal of the ROS before ROS reacts with DHR. To determine whether D609 posses antioxidant activity, various concentrations of D609 were added to DHR before initiating the Fenton reaction. PDTC, a well known antioxidant, was included in the assay as an antioxidant control. As shown in Fig. 1, the addition of D609 and PDTC reduced the Fenton reaction-induced increases in R123 fluorescence intensity in a dose-dependent manner over 3 orders of magnitude in concentration. This demonstrates that D609 like PDTC is a potent antioxidant. D609 Inhibits PBN Spin Trapping and Lipid Peroxidation in the Fenton Reaction. To further characterize the antioxidant activity of D609, we next examined the effect D609 on the formation of a PBN spin adduct in the Fenton reaction. Two concentrations of D609 were tested in the study, i.e., 18 and 188 ␮M D609. The dose of 188 ␮M D609 (equal to 50 ␮g/ml) is an effective dose that has been widely used in most of the previously reported in vitro and in vivo studies (Schutze et al., 1992; Wiegmann et al., 1994; Machleidt et al., 1996; Simarro et al., 1999). As shown in Fig. 2A, the addition of both doses of D609 was equally effective in inhibiting PBN spin trapping in the Fenton reaction. In addition, when D609 was added to synaptosomal membranes prior to the initiation of the Fenton reaction, it also reduced the lipid peroxidation of the synaptosomes in a dose-dependent manner (Fig. 2B). D609 Inhibits IR-Induced Intracellular Production of ROS in Lymphocytes. Next, we determined the intracellular antioxidant activity of D609 against IR-induced oxidative stress in lymphocytes, since IR is a potent source of

Fig. 1. PDTC and D609 reduce the Fenton reaction-induced oxidation of DHR in a dose-dependent manner. Various concentrations of PDTC and D609 were added to DHR solution (2 ␮M) prior to the initiation of the Fenton reaction by the addition of 10 mM H2O2 and 200 ␮M FeSO4. The fluorescence intensities of R123 were measured 30 min later after the initiation of the Fenton reaction. The results are presented as mean ⫾ S.D. of percentage inhibition of controls.

Fig. 2. D609 inhibits the formation of a PBN spin adduct and lipid peroxidation of synaptosomes in the Fenton reaction. A, PBN (10 mM) was used as the spin trap in the Fenton reaction (30 ␮M FeSO4 and 3 mM H2O2) in the presence or absence of various concentrations of D609. EPR spectra were acquired 30 min after the initiation of the Fenton reaction. ⴱ, EPR spectra of trace metal impurities (possibly Mn2⫹) in calcium oxide that was used as an external standard; ##, EPR spectra of PBN spin adducts. B, synaptosomes were untreated (⫺) or preincubated with various concentrations of D609 (18 or 188 ␮M) for 30 min and then were oxidized in the Fenton reaction (50 ␮M FeSO4 and 1 mM H2O2) for 1 h at 37°C. The results (fluorescence intensity of TBARs) are presented as mean ⫾ S.D. of six replicates. ap ⬍ 0.01 versus unoxidized control; bp ⬍ 0.01 versus oxidized control without D609 treatment.

oxidative stress (Prasad, 1995). Exposure of cells to IR causes generation of intracellular ROS by radiolysis of water (Prasad, 1995). As shown in Fig. 3A, lymphocytes exhibited elevated fluorescence intensity of R123 after the cells were exposed to 10-Gy IR. This suggests that IR increased the production of intracellular ROS, since the fluorescence intensity of R123, the oxidized product of DHR, is an indicator of intracellular ROS production and correlates with the levels of intracellular ROS (Emmendorffer et al., 1990). Incubation of the cells with 188 ␮M D609 prior to IR significantly reduced IR-induced increases in R123 fluorescence intensity, while incubation of the cells with the lower dose of D609 (18 ␮M) was less effective. This suggests that D609 can function as an antioxidant to inhibit IR-induced production of intracellular ROS in a dose-dependent manner. D609 Inhibits IR-Induced Decreases in Levels of GSH in Lymphocytes. GSH is ubiquitous in mammalian and other living cells (Shan et al., 1990). It has several important functions, including protection against oxidative stress (Shan et al., 1990). GSH also plays an important role in protecting cells from IR-induced damage (Hospers et al., 1999). The levels of GSH can be measured by quantifying the fluorescence of glutathione-mBCI adducts that are formed by the catalyzation of glutathione S-transferase using a flow cytometer (Hedley and Chow, 1994). Control lymphocytes incubated in vitro for 60 min exhibited a slight decrease in GSH compared with the baseline levels of GSH in the uncultured cells (Fig. 3B). Exposure of lymphocytes to IR (10 Gy) resulted in greater decreases in the levels of GSH (Fig. 3B).

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As shown in Fig. 4A, nonirradiated lymphocytes expressed moderate levels of constitutive protein carbonyls. The levels of protein carbonyls were significantly increased (about 2-fold) after the cells were exposed to IR. Incubation of the cells with D609 dose dependently inhibited the formation of protein carbonyls induced by IR (Fig. 4B). The specificity of this assay for protein carbonyls was confirmed by including a derivatization control in which no DNPH was added to the lysates. This resulted in a total loss of detection of oxidatively modified proteins (data not shown). We also compared the levels of oxidative damage to lipids induced by IR in lymphocytes with or without D609 pretreatment by measuring the levels of LPO. The data in Fig. 5 showed that the levels of LPO in irradiated cells were significantly greater (about 11fold) than that in unirradiated controls. Preincubation of the cells with 188 ␮M/ml D609 abolished the IR-induced increases in the level of LPO, while the cells pretreated with 18 ␮M D609 showed a 25% reduction in IR-induced production of LPO. D609 Inhibits IR-Induced NF-␬B Activation in Lymphocytes. NF-␬B is a redox sensory transcription factor that

Fig. 3. D609 inhibits IR-induced ROS production and decreases in GSH. A, ROS levels: lymphocytes were untreated (⫺) or preincubated with various concentrations of D609 (18 or 188 ␮M) for 30 min and then with DHR (1 ␮M) for additional 5 min. The levels of ROS in the lymphocytes were analyzed 30 min after the cells were exposed to 10 Gy IR. Control cells were not treated with D609 nor exposed to IR. B, GSH levels: lymphocytes were untreated (⫺) or preincubated with D609 and then exposed to 10 Gy IR as described above. Thirty minutes after IR, the cells were pulsed with mBCI (40 ␮M) and the intracellular levels of GSH were analyzed 5 min later. Control cells were not treated with D609 nor exposed to IR but were incubated in medium for 60 min. The results are presented as mean ⫾ S.D. in triplicates. ap ⬍ 0.01 versus control; bp ⬍ 0.01 versus irradiated cells without D609 pretreatment.

An average of 18% decrease in the levels of GSH was achieved within 30 min after the cells were exposed to IR. When the cells were incubated with 188 ␮M D609 prior to IR, the decreases in GSH were abrogated, while incubation of the cells with lower concentration of D609 (18 ␮M) did not attenuate IR-induced GSH decline. In a preliminary study using unirradiated cells, we did not find elevated levels of GSH following D609 treatment (data not shown). Therefore, we assume that the high concentration D609 (188 ␮M) may protect intracellular GSH from oxidation by effectively scavenging IR-induced intracellular ROS. D609 Inhibits IR-Induced Oxidative Damage to Proteins and Lipids in Lymphocytes. Production of intracellular ROS by IR causes oxidative damage to various biological molecules, including proteins and lipids (Prasad, 1995; Halliwell and Gutteridge, 1999). This will result in increases in the formation of protein carbonyls and LPO (Prasad, 1995; Halliwell and Gutteridge, 1999). It is expected that D609 may inhibit IR-induced oxidative damage to cellular proteins and lipids by scavenging IR-produced ROS. To test this assumption, we measured the levels of protein carbonyls in cell lysates by Western blot using an antibody against DNPHprotein adducts, since the formation of carbonyl groups (aldehydes and ketones) in the amino side chains of proteins is a marker of protein oxidation (Stadtman and Berlett, 1998).

Fig. 4. D609 inhibits IR-induced oxidative damage to cellular proteins. Lymphocytes were untreated (⫺) or preincubated with various concentrations of D609 (18 or 188 ␮M) for 30 min and then exposed to IR (10 Gy). Control cells were not pretreated nor irradiated. Thirty minutes after IR both irradiated and control cells were lysed. The levels of oxidatively modified proteins containing carbonyl groups in the lysates were analyzed by SDS-polyacrylamide gel electrophoresis and antibody against DNPH-protein adducts. A, representative Western blot analysis of the assays. M, protein molecular weight makers; C, control cells. B, levels of protein carbonyls in irradiated cells were quantified by scanning densitometry and are expressed as fold-increase (mean ⫾ S.D.) from control. a p ⬍ 0.05 and bp ⬍ 0.01 versus irradiated cells without D609 pretreatment.


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Fig. 5. D609 inhibits IR-induced oxidative damage to cellular lipids. Lymphocytes were untreated (⫺) or preincubated with various concentrations of D609 (18 or 188 ␮M) for 30 min and then exposed to IR (10 Gy). Control cells were not pretreated nor irradiated. Irradiated and control cells were sonicated in deionized water 30 min after IR. The levels of LPO were measured and expressed as nanomoles per 106 cells according to the LPO standard curve. The results are presented as mean ⫾ S.D. in triplicates. ap ⬍ 0.01 versus control; bp ⬍ 0.01 versus irradiated cells without D609 pretreatment.

regulates the expression of a variety of genes (Schreck et al., 1992). Exposure of cells or organisms to IR leads to the formation of ROS that may serve as an intracellular mediator to initiate IR-induced NF-␬B activation (Mohan and Meltz, 1994; Baeuml et al., 1997). If D609 functions as a potent antioxidant, we hypothesized that D609 may also inhibit NF-␬B activation by IR. To test this hypothesis, we analyzed the NF-␬B DNA binding activity in the nuclear extracts from control cells or irradiated cells with or without D609 pretreatment by a gel shift assay. As shown in Fig. 6, lymphocytes expressed basal level of NF-␬B activity. The NF-␬B DNA binding activity was increased by 1.77-fold after the cells were exposed to IR. Preincubation of the cells with a high dose of D609 (188 ␮M) completely suppressed IRinduced NF-␬B activation, while the cells pretreated with a lower dose of D609 (18 ␮M) exhibited a significant reduction in NF-␬B activation to IR. D609 Protects Mice from IR-Induced Lethality. Exposure of BALB/c mice to a total body irradiation at a dose range between 6 to 8.5 Gy causes 40 to 100% death within 30 days (data not shown). The death is primarily caused by IR-induced oxidative damage to the hematopoietic stem cells in the bone marrow and other alternative hematopoietic tissues (Prasad, 1995). To determine whether D609 has any biological function in vivo against IR-induced oxidative damage, we subjected BALB/c mice to 6.5 or 8.5 Gy of total body irradiation 10 min after the mice received a single dose (50 mg/kg) of i.v. injection of D609 or vehicle (saline). The vehicle-treated mice exhibited 60 and 100% mortality within 30 days after they were exposed to 6.5- or 8.5-Gy irradiation, respectively (Fig. 7). In contrast, all the mice that were pretreated with D609 survived after they received 6.5-Gy IR and about 40% of D609-treated mice survived after receiving 8.5-Gy IR (Fig. 7). These findings demonstrate that D609 is an effective radioprotector in vivo, probably by functioning as a potent antioxidant.

Discussion Oxidative stress arises when there is a marked imbalance between the production and removal of ROS (Halliwell and Gutteridge, 1999). This may originate from an overproduc-

Fig. 6. D609 inhibits IR-induced NF-␬B activation. Lymphocytes were untreated (⫺) or preincubated with various concentrations of D609 (18 or 188 ␮M) for 30 min and then exposed to IR (10 Gy). Control cells (C) were not pretreated nor irradiated. Thirty minutes after IR, nuclear proteins were extracted from both irradiated and control cells. The NF-␬B DNA binding activity in the nuclear extracts (5 ␮g/lane) was analyzed by a gel shift assay. Relative nuclear binding activities for NF-␬B DNA were quantified by scanning densitometry and are expressed as fold-increase.

tion of ROS or from a reduction in antioxidant defenses. Since ROS can cause damage to DNA, proteins, and/or membrane phospholipids, oxidative stress has been implicated in

Fig. 7. D609 protects mice from IR-induced lethality. BALB/c mice were exposed to 6.5 or 8.5 Gy of total body IR 10 min after they received a single dose (50 mg/kg) of i.v. injection of D609 or vehicle (saline) through the tail veins. The survival of these mice was recorded during a 30-day observation period after IR. D609-treated mice survived significantly longer than vehicle-treated mice in response to both doses of IR (p ⬍ 0.0001).

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the pathogenesis of a number of human diseases, including cancer, and neurodegenerative and cardiovascular pathologies (Halliwell and Gutteridge, 1999). Oxidative stress also significantly contributes to normal tissue damage during tumor therapy with IR and certain chemotherapeutic agents (Hospers et al., 1999). One way to balance the ratio of antioxidants to oxidants to prevent cellular damage associated with oxidative stress is by supplementation with antioxidants. Commonly used antioxidants include dietary vitamins, plant phenolics, and precursors of glutathione (Halliwell and Gutteridge, 1999). However, their effectiveness in treating various human diseases associated with oxidative stress has yet to be established. Furthermore, their use for amelioration of radiation- and chemotherapy-induced normal tissue injury has raised many concerns since they may protect tumor cells as well. Therefore, discovery of novel and more potent antioxidants that have the ability to discriminate between normal and tumor cells will be of a great interest. Xanthates were originally developed as antitumor and antiviral agents (Sauer et al., 1984; Amtmann and Sauer, 1987). Among various xanthate derivatives, D609 has been extensively investigated. In vitro, D609 exhibits strong antitumor activity against a variety of tumor cells with limited cytotoxicity to normal cells (Amtmann and Sauer, 1987; Schick et al., 1989b). However, the mechanisms by which D609 kills tumor cells and its effectiveness in vivo for tumor therapy remain to be established. Recently, D609 has been used as a specific inhibitor for PC-PLC and many effects of D609 have been attributed to this inhibition (Schutze et al., 1992; Wiegmann et al., 1994; Amtmann, 1996; Machleidt et al., 1996). However, as a xanthate derivative that can dissociate in solution to xanthate anions and/or xanthic acid with a free thiol group, D609 may also possess strong antioxidant activity (Rao, 1971). This was supported by the finding that D609 inhibited the Fenton reaction-induced oxidation of DHR, PBN spin trapping, and lipid peroxidation of synaptosomes. The antioxidant activity of D609 is not dependent on its alkyl hydrocarbon moiety since the alcohol-tricyclodecanol that was used to synthesize D609 did not have antioxidant activity (data not shown). In addition, xanthate derivatives that have ethyl, isopropyl, or cyclohexyl alkyl hydrocarbon moiety exhibited antioxidant activities similar to that of D609 (C. M. Lauderback, D. Zhou, J. M. Hackett, A. Castegna, J. L. Drake, J. Kanski, M. Tsoras, S. Varadarajan, and D. A. Butterfield, unpublished data). In contrast, the free thiol group appears essential for D609 antioxidant activity since the modification of D609 by methylation of the thiol moiety eliminated the antioxidant activity of D609 (C. M. Lauderback , D. Zhou, J. M. Hackett, A. Castegna, J. L. Drake, J. Kanski, M. Tsoras, S. Varadarajan, and D. A. Butterfield, unpublished data). A substantial body of evidence collected in our laboratory recently demonstrates that D609 may act as a GSH mimic (C. M. Lauderback , D. Zhou, J. M. Hackett, A. Castegna, J. L. Drake, J. Kanski, M. Tsoras, S. Varadarajan, and D. A. Butterfield, unpublished data). For instance, when D609 was irradiated by UV light in the presence of H2O2, it was oxidized to dixanthogen by forming a disulfide bond and lost its reactivity with 5⬘5dithiobis(2-nitrobenzoic acid) (a commonly used reagent for the detection of thiol groups) in a manner similar to GSH (C. M. Lauderback , D. Zhou, J. M. Hackett, A. Castegna, J. L.

Drake, J. Kanski, M. Tsoras, S. Varadarajan, and D. A. Butterfield, unpublished data). The species of ROS that D609 can effectively scavenge remain to be elucidated. At a minimum, D609 has the ability to scavenge hydroxy radicals since it can inhibit the formation of a PBN spin adduct in the Fenton reaction. Reaction with other species of ROS is also possible for D609 and other derivatives of xanthate since xanthates generally have high reductive potential (Rao, 1971). It has been shown that xanthates can react with various mild oxidative agents, including hydrogen peroxide (Rao, 1971). However, it remains to be determined whether D609 is capable of chelating transitional metal ions, which could contribute to the antioxidant activity of D609 as shown by its ability to inhibit ROS production and cellular oxidative stress. In addition, we have found that D609 can inhibit IR-induced oxidative stress in lymphocytes. Lymphocytes pretreated with D609 displayed significant reduction in IR-induced ROS production and protein and lipid peroxidation. Moreover, after exposure to IR the levels of intracellular GSH declined in untreated cells but remained steady in the cells treated with D609, indicating that D609 may protect intracellular GSH from IR-induced oxidation. A similar effect of D609 on protecting intracellular GSH was also found in neurons against glutamate-induced oxidative stress (Li et al., 1998). GSH is one of the most important intracellular defense molecules against oxidative stress and has been shown to play an important role in radiation protection (Hospers et al., 1999). Maintenance of a steady level of intracellular GSH by D609 may contribute to the suppression of IR-induced oxidative damages to proteins and lipids in lymphocytes. Exposure of cells to IR activates NF-␬B in association with production of ROS (Mohan and Meltz, 1994; Baeuml et al., 1997). Various antioxidants can suppress IR-induced activation of NF-␬B (Mohan and Meltz, 1994; Baeuml et al., 1997). These findings suggest that ROS may serve as an intracellular mediator to initiate IR-induced NF-␬B activation. Similarly, D609 inhibited NF-␬B activation by IR in lymphocytes. We attribute this effect of D609 primarily to its antioxidant activity. However, D609 also inhibits TNF-induced activation of NF-␬B, which has been largely attributed to the inhibition of PC-PLC and production of ceramide (Schutze et al., 1992; Wiegmann et al., 1994; Amtmann, 1996; Machleidt et al., 1996). Thus, it remains to be determined whether inhibition of PC-PLC and production of ceramide by D609 also contributes to the suppression of IRinduced NF-␬B activation. The identification of D609 as a potent antioxidant implies that D609 may exert some of the reported activities that have been largely attributed to the inhibition of PC-PLC by D609 to its antioxidant properties, such as inhibition of LPS- and TNF-induced NF-␬B activation and inflammatory cytokine production (Schutze et al., 1992; Schreck et al., 1992; Wiegmann et al., 1994; Amtmann, 1996; Machleidt et al., 1996; Flohe et al., 1997). It was reported that the topical application of D609 inhibited the carcinogen 12-O-tetra-decanoylphorbol-13-acetate (TPA)-induced skin tumor formation in a mouse model (Furstenberger et al., 1989). There has been increasing evidence that induction of ROS by TPA contributes to multistage carcinogenesis, particularly in the promotion stage (Ito and Hirose, 1989). A variety of antioxidants have been shown to be effective in preventing TPA-induced


tumor formation (Ito and Hirose, 1989). Thus, it is possible that the antioxidant activity of D609 may contribute to its carcinogenic properties. The discovery of D609 as a novel and potent antioxidant may allow us to develop more effective therapeutic interventions against normal tissue injury during tumor therapy with IR and chemotherapy. This possibility exists because D609 has exhibited the ability to discriminate normal and tumor cells (Amtmann and Sauer, 1987; Schick et al., 1989b). In vitro, D609 kills a variety of tumor cells but has limited toxic effects on normal cells (Amtmann and Sauer, 1987; Schick et al., 1989b). More importantly, we have found that i.v. administration of D609 can protect mice from IR-induced lethality. It is our interest to further determine whether D609 and other xanthate derivatives can confer selective radiation protection to normal tissues but not to tumor cells in vivo in a mouse tumor model. Moreover, studies are underway to determine whether D609 has the potential to be developed as effective antioxidant therapy for various human diseases associated with oxidative stress. References Amtmann E (1996) The antiviral, antitumoural xanthate D609 is a competitive inhibitor of phosphatidylcholine-specific phospholipase C. Drugs Exp Clin Res 22:287–294. Amtmann E, Muller-Decker K, Hoss A, Schalasta G, Doppler C and Sauer G (1987) Synergistic antiviral effect of xanthates and ionic detergents. Biochem Pharmacol 36:1545–1549. Amtmann E and Sauer G (1987) Selective killing of tumor cells by xanthates. Cancer Lett 35:237–244. Baeuml H, Behrends U, Peter RU, Mueller S, Kammerbauer C, Caughman SW and Degitz K (1997) Ionizing radiation induces, via generation of reactive oxygen intermediates, intercellular adhesion molecule-1 (ICAM-1) gene transcription and NF kappa B-like binding activity in the ICAM-1 transcriptional regulatory region. Free Radic Res 27:127–142. Buscher D, Hipskind RA, Krautwald S, Reimann T and Baccarini M (1995) Rasdependent and -independent pathways target the mitogen-activated protein kinase network in macrophages. Mol Cell Biol 15:466 – 475. Butterfield DA, Howard BJ, Yatin S, Allen KL and Carney JM (1997) Free radical oxidation of brain proteins in accelerated senescence and its modulation by N-tertbutyl-alpha-phenylnitrone. Proc Natl Acad Sci USA 94:674 – 678. Cifone MG, Roncaioli P, De Maria R, Camarda G, Santoni A, Ruberti G and Testi R (1995) Multiple pathways originate at the Fas/APO-1 (CD95) receptor: sequential involvement of phosphatidylcholine-specific phospholipase C and acidic sphingomyelinase in the propagation of the apoptotic signal. EMBO J 14:5859 –5868. Emmendorffer A, Hecht M, Lohmann-Matthes ML and Roesler J (1990) A fast and easy method to determine the production of reactive oxygen intermediates by human and murine phagocytes using dihydrorhodamine 123. J Immunol Methods 131:269 –275. Flohe L, Brigelius-Flohe R, Saliou C, Traber MG and Packer L (1997) Redox regulation of NF-kappa B activation. Free Radic Biol Med 22:1115–1126. Furstenberger G, Amtmann E, Marks F and Sauer G (1989) Tumor prevention by a xanthate compound in experimental mouse-skin tumorigenesis. Int J Cancer 43: 508 –512. Halliwell B and Gutteridge JMC (1999) Free Radicals in Biology and Medicine. Oxford University Press, Oxford. Hedley DW and Chow S (1994) Evaluation of methods for measuring cellular glutathione content using flow cytometry. Cytometry 15:349 –358. Hospers GA, Eisenhauer EA and de Vries EG (1999) The sulfhydryl containing compounds WR-2721 and glutathione as radio- and chemoprotective agents. A review, indications for use and prospects. Br J Cancer 80:629 – 638. Ito N and Hirose M (1989) Antioxidants– carcinogenic and chemopreventive properties. Adv Cancer Res 53:247–302. Lauderback CM, Breier AM, Hackett J, Varadarajan S, Goodlett-Mercer J and Butterfield DA (2000) The pyrrolopyrimidine U101033E is a potent free radical scavenger and prevents Fe(II)-induced lipid peroxidation in synaptosomal membranes. Biochim Biophys Acta 1501:149 –161.

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Li Y, Maher P and Schubert D (1998) Phosphatidylcholine-specific phospholipase C regulates glutamate-induced nerve cell death. Proc Natl Acad Sci USA 95:7748 – 7753. Machleidt T, Kramer B, Adam D, Neumann B, Schutze S, Wiegmann K and Kronke M (1996) Function of the p55 tumor necrosis factor receptor “death domain” mediated by phosphatidylcholine-specific phospholipase C. J Exp Med 184:725– 733. Mohan N and Meltz ML (1994) Induction of nuclear factor kappa B after low-dose ionizing radiation involves a reactive oxygen intermediate signaling pathway. Radiat Res 140:97–104. Monick MM, Carter AB, Gudmundsson G, Mallampalli R, Powers LS and Hunninghake GW (1999) A phosphatidylcholine-specific phospholipase C regulates activation of p42/44 mitogen-activated protein kinases in lipopolysaccharide-stimulated human alveolar macrophages. J Immunol 162:3005–3012. Muller-Decker K (1989) Interruption of TPA-induced signals by an antiviral and antitumoral xanthate compound: inhibition of a phospholipase C-type reaction. Biochem Biophys Res Commun 162:198 –205. Muller-Decker K, Doppler C, Amtmann E and Sauer G (1988) Interruption of growth signal transduction by an antiviral and antitumoral xanthate compound. Exp Cell Res 177:295–302. Prasad KN (1995) Handbook of Radibiology. CRC Press, Boca Raton, FL. Rao SR (1971) Xanthates and Related Compounds. Marcel Dekker, New York. Sauer G, Amtmann E and Hofmann W (1990) Systemic treatment of a human epidermoid non-small cell lung carcinoma xenograft with a xanthate compound causes extensive intratumoral necrosis. Cancer Lett 53:97–102. Sauer G, Amtmann E, Melber K, Knapp A, Muller K, Hummel K and Scherm A (1984) DNA and RNA virus species are inhibited by xanthates, a class of antiviral compounds with unique properties. Proc Natl Acad Sci USA 81:3263–3267. Schick HD, Amtmann E, Berdel WE, Danhauser-Riedl S, Reichert A, Steinhauser G, Rastetter J and Sauer G (1989b) Antitumoral activity of a xanthate compound. I. Cytotoxicity studies with neoplastic cell lines in vitro. Cancer Lett 46:143–147. Schick HD, Danhauser-Riedl S, Amtmann E, Busch R, Reichert A, Steinhauser G, Rastetter J, Sauer G and Berdel WE (1989a) Antitumoral activity of a xanthate compound. II. Therapeutic studies in murine leukemia and tumor models in vivo. Cancer Lett 46:149 –152. Schreck R, Albermann K and Baeuerle PA (1992) Nuclear factor kappa B: an oxidative stress-responsive transcription factor of eukaryotic cells (a review). Free Radic Res Commun 17:221–237. Schutze S, Potthoff K, Machleidt T, Berkovic D, Wiegmann K and Kronke M (1992) TNF activates NF-kappa B by phosphatidylcholine-specific phospholipase Cinduced “acidic” sphingomyelin breakdown. Cell 71:765–776. Shan XQ, Aw TY and Jones DP (1990) Glutathione-dependent protection against oxidative injury. Pharmacol Ther 47:61–71. Simarro M, Calvo J, Vila JM, Places L, Padilla O, Alberola-Ila J, Vives J and Lozano F (1999) Signaling through CD5 involves acidic sphingomyelinase, protein kinase C-zeta, mitogen-activated protein kinase kinase, and c-Jun NH2-terminal kinase. J Immunol 162:5149 –5155. Stadtman ER and Berlett BS (1998) Reactive oxygen-mediated protein oxidation in aging and disease. Drug Metab Rev 30:225–243. Tschaikowsky K, Schmidt J and Meisner M (1998) Modulation of mouse endotoxin shock by inhibition of phosphatidylcholine-specific phospholipase C. J Pharmacol Exp Ther 285:800 – 804. Villanueva N, Navarro J and Cubero E (1991) Antiviral effects of xanthate D609 on the human respiratory syncytial virus growth cycle. Virology 181:101–108. Walro DG and Rosenthal KS (1997) The antiviral xanthate compound D609 inhibits herpes simplex virus type 1 replication and protein phosphorylation. Antiviral Res 36:63–72. Wiegmann K, Schutze S, Machleidt T, Witte D and Kronke M (1994) Functional dichotomy of neutral and acidic sphingomyelinases in tumor necrosis factor signaling. Cell 78:1005–1015. Yamamoto H, Hanada K and Nishijima M (1997) Involvement of diacylglycerol production in activation of nuclear factor kappaB by a CD14-mediated lipopolysaccharide stimulus. Biochem J 325:223–228. Yanev S, Kent UM, Pandova B and Hollenberg PF (1999) Selective mechanism-based inactivation of cytochromes P-450 2B1 and P-450 2B6 by a series of xanthates. Drug Metab Dispos 27:600 – 604. Zhou D, Brown SA, Yu T, Chen G, Barve S, Kang BC and Thompson JS (1999) A high dose of ionizing radiation induces tissue-specific activation of nuclear factorkappaB in vivo. Radiat Res 151:703–709.

Address correspondence to: Daohong Zhou, M.D., Division of Research, Department of Pathology, Medical University of South Carolina, 165 Ashley Ave., Suite 309, P.O. Box 250908, Charleston, SC 29425. E-mail: zhoud@ musc.edu