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Chem. Res. Toxicol. 2000, 13, 550-556
The Putative Benzene Metabolite 2,3,5-Tris(glutathion-S-yl)hydroquinone Depletes Glutathione, Stimulates Sphingomyelin Turnover, and Induces Apoptosis in HL-60 Cells Shawn B. Bratton,† Serrine S. Lau, and Terrence J. Monks* Center for Molecular and Cellular Toxicology, Division of Pharmacology and Toxicology, College of Pharmacy, University of Texas at Austin, Austin, Texas 78712 Received January 4, 2000
In this study, we show that 2,3,5-tris(glutathion-S-yl)hydroquinone (TGHQ), a putative metabolite of benzene, induces apoptosis in human promyelocytic leukemia (HL-60) cells. Prior to the onset of apoptosis, TGHQ depletes intracellular glutathione (GSH) in a reactive oxygen species (ROS)-independent manner. Neutral, Mg2+-dependent sphingomyelinases, which are normally inhibited by GSH, are subsequently activated, as evidenced by increases in intracellular ceramide and depletion of sphingomyelin. As ceramide levels rise, effector caspase (DEVDase) activity steadily increases. Interestingly, while catalase has no effect on TGHQmediated depletion of GSH, this hydrogen peroxide (H2O2) scavenger does inhibit DEVDase activity and apoptosis, provided the enzyme is added to HL-60 cells before an increase in ceramide can be observed. Since ceramide analogues inhibit the mitochondrial respiratory chain, these data imply that ceramide-mediated generation of H2O2 is necessary for the activation of effector caspases-3 and/or -7, and apoptosis. In summary, these studies indicate that TGHQ, and perhaps many quinol-based toxicants and chemotherapeutics, may induce apoptosis in hematopoietic cells by depleting GSH and inducing the proapoptotic ceramide-signaling pathway.
Introduction Benzene is a widely used industrial chemical and an environmental contaminant (1). Benzene induces bone marrow suppression in rodents (2) and is both hematotoxic and leukemogenic in humans, causing a variety of hematological disorders, including aplastic anemia, myelodysplastic syndrome, and acute myelogenous leukemia (3, 4). Benzene must be metabolized to mediate its harmful effects, and a number of polyphenolic and openringed metabolites have been studied for their hematotoxic potential (5, 6). We have identified a number of redox-active hydroquinone-thioether metabolites in the bone marrow of rats and mice exposed to a combination of hydroquinone and phenol or benzene (7). Two of these metabolites, 2,6-bis(glutathion-S-yl)hydroquinone and 2,3,5-tris(glutathion-S-yl)hydroquinone (TGHQ),1 reproduce benzene hematotoxicity in vivo (7), presumably via covalent binding to tissue macromolecules (8) and/or generation of reactive oxygen species (ROS). Few studies have addressed the ability of benzene metabolites to induce apoptosis, and the results from these limited studies are contradictory. For example, * To whom correspondence should be addressed. Telephone: (512) 471-6699. Fax: (512) 471-5002. E-mail:
[email protected]. † Present address: MRC Toxicology Unit, Hodgkin Building, University of Leicester, P.O. Box 138, Lancaster Road, Leicester LE1 9HN, England. 1 Abbreviations: GSH, glutathione; GSSG, glutathione disulfide; H2O2, hydrogen peroxide; NF-κB, nuclear factor κ B; ROS, reactive oxygen species; SM, sphingomyelin; SMase, sphingomyelinase; TGHQ, 2,3,5-tris(glutathion-S-yl)hydroquinone; TNF-R, tumor necrosis factorR.
hydroquinone induces apoptosis in HL-60 and human bone marrow-derived CD34+ cells (9), but inhibits apoptosis in IL-3-dependent murine myeloblasts, purportedly by preventing activation of caspase-1 (ICE) (10). Dysregulation of apoptosis is important in the development and/or progression of many hematopoietic disorders (11). Therefore, the role of apoptosis in quinol-thioethermediated hematotoxicity and the signal transduction pathways engaged during TGHQ-induced apoptosis are the subject of the investigation presented here. Ceramide is a lipid second messenger which, depending on cell type, mediates a variety of cellular effects, including growth arrest, differentiation, senescence, and apoptosis (12). Ceramide is formed via hydrolysis of sphingomyelin (SM) in a reaction catalyzed by membranebound sphingomyelinases (SMases) (13). A number of agents which induce apoptosis can activate these enzymes, including tumor necrosis factor-R (TNF-R), interleukin-1β, Fas ligands, 1-β-D-arabinofuranosylcytosine, vincristine, and daunorubicin (14-19). Cell-permeable ceramide analogues modulate the activity of a variety of kinases and phosphatases, including a specific prolinedirected serine/threonine kinase (20), mitogen-activated protein kinase (21), c-Jun NH2-terminal kinase (22), and ceramide-activated protein phosphatase (23). Ceramides inhibit mitochondrial respiration with the concomitant production of ROS (24, 25) and stimulate activation of the transcription factor, nuclear factor-κB (NF-κB) (24, 26, 27). Ceramides also induce cytochrome c release from isolated mitochondria and, in turn, activate the proapoptotic cysteine protease, caspase-3 (CPP32/Yama/Apo-
10.1021/tx0000015 CCC: $19.00 © 2000 American Chemical Society Published on Web 06/09/2000
Quinol-Thioether-Mediated Apoptosis
pain), leading to cleavage of a variety of substrates, including poly(ADP-ribose)polymerase (28, 29). The kinetics of endogenous ceramide generation can vary greatly, but agents that induce apoptosis tend to irreversibly elevate ceramide levels by several-fold over the course of several hours (12). Although the precise mechanisms governing SMase activation are unclear, glutathione (GSH) directly inhibits purified neutral, Mg2+-dependent, SMases in vitro (30). Consistent with these findings, inhibition of γ-glutamylcysteine synthetase in Molt-4 cells with L-buthionine-(SR)-sulfoxamine produces a long-term decrease in intracellular GSH with a concomitant increase in SMase activity (30). We present data which indicate that TGHQ decreases intracellular GSH concentrations, with a subsequent increase in the level of SM turnover. Ceramide then stimulates effector caspase (DEVDase) activity, and induces apoptosis via the production of H2O2.
Materials and Methods Materials. TGHQ was synthesized and purified as previously described (31). sn-1,2-Diacylglycerol kinase (Escherichia coli, recombinant), zVAD-FMK, and zDEVD-FMK were obtained from Calbiochem (La Jolla, CA). The caspase-3 and -7 substrate, Ac-DEVD-AMC, was purchased from Pharmingen (San Diego, CA). Annexin V FITC kits were obtained from Coulter Corp. (Miami Lakes, FL), and LK6D (60 Å) silica gel plates were purchased from Whatman (Clifton, NJ). [γ-32P]ATP (3000 Ci/ mmol) and [methyl-14C]choline chloride (54 mCi/mmol) were obtained from NEN Research Products (Boston, MA), and [methyl-3H]thymidine 5′-triphosphate (40-70 Ci/mmol) was purchased from ICN Biomedical (Costa Mesa, CA). Bovine brain sphingomyelin, type III ceramide, catalase, Triton X-100, NP40, octyl β-D-glucoside, 1,2-dideoyl-sn-3-phosphatidylglycerol, diethylenetriaminepentaacetic acid (DETAPAC), imidazole, NaCl, MgCl2, EDTA, and all other compounds were purchased from Sigma Chemical Co. (St. Louis, MO). All solvents, including chloroform and methanol, were purchased from Fischer Scientific (Houston, TX). Cell Line and Culture Conditions. Human promyelocytic (HL-60) cells were obtained from American Type Culture Collection (Rockville, MD) and were routinely cultured at a density of 0.5 × 106 cells/mL, in RPMI 1640 medium (Life Technologies, Grand Island, NY) containing 20% FBS. Immediately prior to all experiments, cells were washed and resuspended in RPMI 1640 containing 25 mM HEPES and 10% FBS. Phosphatidylserine Externalization and DNA Fragmentation. The percentage of apoptotic cells was determined using an annexin V FITC kit and an EPICS XL-MCL flow cytometer, according to the manufacturer’s protocol. DNA fragmentation was assessed in cells labeled for 16 h with 1 µCi/ mL of [methyl-3H]thymidine, according to a previously published method with slight modifications (24). The labeled cells were washed twice, plated in 12-well dishes, and exposed to 200 µM TGHQ. The cells were then harvested and lysed in 200 µL of lysis buffer [15 mM Tris-HCl buffer (pH 8.0), 20 mM EDTA, and 0.5% Triton X-100] for 45 min on ice. The samples were centrifuged at 20000g for 30 min, and the amount of radioactivity in the supernatants (fragmented DNA) and pellets (genomic DNA) was determined by liquid scintillation counting. The percentage of DNA fragmentation was calculated with the equation [fragmented DNA (cpm)/fragmented DNA (cpm) + genomic DNA (cpm)] × 100. GSH and GSSG Concentrations. Cellular concentrations of GSH and GSSG were determined in HL-60 cells treated with TGHQ (200 µM) for various periods of time (0-12 h) according to the method of Neuschwander-Tetri and Roll (32). Sphingomyelin Depletion. HL-60 cells were labeled for 72 h in culture medium containing 0.5 µCi/mL of [methyl-14C]-
Chem. Res. Toxicol., Vol. 13, No. 7, 2000 551 choline chloride, washed twice with PBS, and resuspended in complete medium. Labeled cells were exposed to 200 µM TGHQ for various periods of time. Cells were then collected and snapfrozen in liquid nitrogen. The following day 300 µL of ice-cold deionized water was added to each sample. The samples were probe sonicated for 5 s, and the lipids were extracted using 1 mL of a chloroform/methanol mixture (2:1) (33). Following centrifugation, the organic phase was removed and evaporated in a fume hood under a steady stream of nitrogen. All samples, including a non-radiolabeled sphingomyelin standard, were dissolved in 20 µL of chloroform and spotted onto silica (60 Å) gel plates. Lipids were resolved in a solvent system of chloroform, methanol, acetic acid, and water (125:75:20:12.5) and placed in an iodine chamber to locate the position of the standard. The plates were then dried, and the radiolabeled sphingomyelin spots were quantitated using a Packard Instant Imager. Results are expressed as the counts per minute per 106 cells. Ceramide Quantitation. HL-60 cells were treated with 200 µM TGHQ and processed according to the method of Preiss et al. (34) with slight modifications. Lipids were isolated and dissolved in 20 µL of 7.5% octyl β-D-glucoside, 5 mM 1,2-dideoylsn-3-phosphatidylglycerol, and 1 mM DETAPAC (pH 7.0). Reaction buffer [50 µL of 100 mM imidazole, 100 mM NaCl, 25 mM MgCl2, and 2 mM EDTA (pH 6.6)] was added to each sample, followed by the addition of 5 µL of diacylglycerol kinase (0.07 unit). Deionized water (15 µL) was then added, and the reaction was initiated with the addition of 10 µL of an [γ-32P]ATP solution containing 10 mM [γ-32P]ATP (100 000 cpm/nmol), 100 mM imidazole, and 1 mM DETAPAC (pH 6.6). The reaction was allowed to proceed for 30 min at room temperature and was quenched with the addition of 1 mL of a chloroform/ methanol mixture (2:1), followed by 600 µL of 1% HCl. The organic phase was washed once more with 1% HCl and subsequently evaporated under a steady stream of nitrogen. Lipids were resolved in a solvent system containing chloroform, methanol, and acetic acid (65:15:5), and ceramide spots were quantitated using a Packard Instant Imager. Results are expressed as picomoles per 106 cells. Caspase Activity. HL-60 cells were exposed to 200 µM TGHQ. The cells were washed once with PBS and lysed for 30 min on ice in 1 mL of lysis buffer [50 mM Tris-HCl (pH 7.5), 0.5% NP-40, 0.5 mM EDTA, and 150 mM NaCl]. Fifty microliters of each cellular lysate was added to a 96-well microtiter plate, followed by addition of 144.4 µL of reaction buffer [10 mM HEPES (pH 7.5), 0.05 M NaCl, and 2.5 mM dithiothreitol]. AcDEVD-AMC (5.6 µL, 8 nmol) was then added to each well and incubated at 37 °C for 2 h. The amount of AMC released was determined by measuring the fluorescence of AMC (excitation and emission at 365-380 and 445-450 nm, respectively), and protein concentrations were determined for each sample using the Bradford assay. Final results are expressed as AMC fluorescence per milligram of protein (28). Statistical Analysis. Statistical significance was determined using ANOVA, followed by Student-Newman-Kuel’s post hoc analysis, with a p value of 50% of TGHQ is removed from culture medium within 1.8 h of treatment, with no apparent increase in TGHQderived metabolites (data not shown). Moreover, catalase offers significant protection against TGHQ-induced apoptosis when added to culture medium as late as 2 h following TGHQ treatment, a time that coincides with the first peak in intracellular ceramide concentrations (Figures 3 and 5). Thus, catalase not only protects cells against apoptosis when added early in the experiment, when concentrations of TGHQ are at their peak and available to redox cycle and produce ROS (0-1 h), but also protects cells against apoptosis when added later in the experiment (2 h), as ceramide levels begin to steadily increase (Figures 3 and 5). This suggests that ROS are required for both the initiation and the “execution” of apoptosis, with TGHQ-derived ROS responsible for the former, and ceramide-derived ROS for the latter. In summary, the putative benzene metabolite, TGHQ, depletes intracellular GSH in HL-60 cells and consequently stimulates sphingomyelin turnover. Ceramide then activates caspase-3-like proteases via the generation of H2O2 and induces apoptosis. These data support a specific role for quinol-thioether metabolites in benzenemediated hematotoxicity and suggest that compounds which deplete GSH in bone marrow tissue may cause toxicity via the ceramide-signaling pathway.
Acknowledgment. Shawn Bratton was supported by NIEHS Toxicology Training Grant ES 07247 (Division of Pharmacology and Toxicology, College of Pharmacy, The University of Texas at Austin). A portion of this work was awarded the Carl C. Smith Graduate Student Award by the Mechanisms Section of the Society of Toxicology, March 1999. The authors acknowledge the assistance of the Analytical Instrumentation and the Cell and Tissue Analysis Facility Cores, Center for Research in Environmental Disease (ES 07784).
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