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cell lines after SIN-1 treatment. Neutral comet assay demonstrated that SIN-1 treatment resulted in higher levels of DNA double-strand breaks in TK6 c...
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Chem. Res. Toxicol. 2002, 15, 527-535

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Genotoxicity, Mitochondrial Damage, and Apoptosis in Human Lymphoblastoid Cells Exposed to Peroxynitrite Generated from SIN-1 Chun-Qi Li, Laura J. Trudel, and Gerald N. Wogan* Biological Engineering Division and Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Received November 5, 2001

SIN-1 (3-morpholinosydnonimine), the active metabolite of the vasodilator drug molsidomine, decomposes spontaneously in solution. In the presence of oxygen, NO• and O2•- are released, generating peroxynitrite, a potent oxidizing agent, at a constant rate over a 2 h period. We utilized this system to investigate mechanisms of peroxynitrite-induced cytotoxicity, genotoxicity, apoptosis, and mitochondrial damage in two human lymphoblastoid cell lines carrying either wild-type (TK6 cells) or mutant p53 (WTK-1 cells) genes. Treatment of TK6 cells with 5 mM SIN-1 for 1.5 h resulted in 28 ( 6% survival 24 h later. Exposure in the presence of different radical scavengers significantly increased survival, as follows: cytochrome c, 96 ( 3%; Tiron, 69 ( 0%; SOD plus catalase, 83 ( 5%; carboxy-PTIO, 87 ( 3%; and uric acid, 87 ( 2%. D-mannitol was ineffective in reducing lethality, as were SOD and catalase when added individually or in heat-inactivated form. Spontaneous as well as SIN-1-induced mutant fractions (MF) in both HPRT and TK genes were significantly higher in WTK-1 cells than in TK6 cells (p < 0.05-0.01). Exposure to 2.5 mM SIN-1 induced time-dependent apoptosis in TK6 cells, but not in WTK-1 cells. Mitochondrial membrane depolarization was also observed in both cell lines after SIN-1 treatment. Neutral comet assay demonstrated that SIN-1 treatment resulted in higher levels of DNA double-strand breaks in TK6 cells than in WTK-1 cells. Collectively, these data show that SIN-1 can be used as an effective peroxynitrite generator in cell culture experiments under these experimental conditions, in which it induced a greater apoptotic response but was less potent as a mutagen in TK6 cells compared with WTK-1 cells. Thus, p53 status was an important determinant of SIN-1 induced mutagenesis and apoptosis in these two human lymphoblastoid cell lines.

Introduction (NO• 1

Nitric oxide ) and reactive oxygen species have been implicated in many pathological processes such as inflammation and carcinogenesis (1), but mechanisms by which NO• and related redox compounds exert their cytotoxic and genotoxic effects are incompletely understood. NO• reacts rapidly with superoxide (O2•-) to produce peroxynitrite (ONOO-), a powerful oxidant that has been shown to cause a variety of DNA lesions (2), modify and inactivate proteins and cause tissue damage. It may therefore be a significant contributing factor to increased risks for major human cancers that are associated with chronic infections and inflammation (3). We have previously characterized NO•-induced mutagenesis in the endogenous hypoxanthine-guanine phospho* To whom correspondence should be addressed. Phone: (617) 2533188. Fax: (617) 258-9733. E-mail: [email protected]. 1 Abbreviations: carboxy-PTIO, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide; DPBS, Dulbecco’s phosphate buffered saline; HBSS, Hanks’ balanced salt solution; HPRT, hypoxanthineguanine phosphoribosyltransferase gene; JC-1, 5,5′,6,6′-tetrachloro1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide; MF, mutant fraction; MMP, mitochondrial membrane potential; SIN-1, 3-morpholinosydnonimine; SIN-1C, 4-morpholinylimino-acetonitrile, a stable product of SIN-1 decay; NO•, nitric oxide; ONOO-, peroxynitrite; O2•-, superoxide anion; SOD, superoxide dismutase; Tiron, 4,5-dihydroxy1,3-benzene-disulfonic acid; TK, thymidine kinase gene; TK6 cells, human lymphoblastoid TK6 cells; WTK-1 cells, mutant p53 human lymphoblastoid cells.

ribosyltransferase (HPRT) gene of the mouse macrophage RAW264.7 cells and human lymphoblastoid TK6 cells (4, 5). We have also demonstrated that synthetic peroxynitrite induced DNA strand breaks and was strongly mutagenic in the supF shuttle vector pSP189 (6). However, the relevance of the latter findings to the potential genotoxicity of peroxynitrite at dose rates produced by inflammatory cells in vivo is uncertain in view of the high-dose, bolus exposure regimen employed. The present study was undertaken to investigate cellular damage and genotoxicity induced by exposure to peroxynitrite at a controlled rate over a longer period of time through the degradation of 3-morpholinosydnonimine (SIN-1). SIN-1, the active metabolite of the vasodilator drug molsidomine, has been used by previous investigators as a model compound for the continuous release of NO•, O2•-, and other potent oxidants such as peroxynitrite and hydroxyl radical (7-10) in order to mimic the release of these agents by macrophages, neutrophils, and endothelial cells. SIN-1 decomposes in oxygenated solution through a three step process (Scheme 1): the sydnonimine ring opens by a base-catalyzed mechanism to produce SIN-1A; SIN-1A reduces oxygen, producing O2•- plus SIN-1•+; and SIN-1•+ decomposes to form SIN-1C, with the release of NO• (11, 12). Although SIN-1 has previously been employed by others as a source of peroxynitrite (13, 14) or NO• (15, 16) in cell-free or cell

10.1021/tx010171x CCC: $22.00 © 2002 American Chemical Society Published on Web 04/15/2002

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Scheme 1. Degradation Pathway of Molsidomine, Modified from Reden (11)

culture systems, the identity of specific agents responsible for its toxicity has sometimes been unclear. For example, under certain cell culture conditions, evidence suggested that SIN-1-induced-cytotoxicity was mediated by hydrogen peroxide, hydroxyl radical, NO•, or O2•rather than by peroxynitrite (9, 10). However, it has recently been shown (13) that decomposition of 1 mM SIN-1 in well-oxygenated cell culture medium produced peroxynitrite at a linear rate of 12 µM/min. Under these conditions, exposure to SIN-1 induced single strand breaks in human peripheral lymphocytes in a dose- and time-dependent fashion. Our experiments were carried out under similar conditions with the exception that cells were suspended in HBSS during SIN-1 exposure in order to avoid formation of unidentified products of reactions between peroxynitrite and culture medium components. The experiments were designed to assess the role of peroxynitrite in SIN-1-induced cytotoxicity by determining protective effects of putative scavengers of reactive species, including superoxide dismutase (SOD) (O2•-), catalase (H2O2), cytochrome c (O2•-), Tiron [O2•-, (17), a potential scavenger of peroxynitrite], carboxy-PTIO [NO• (10), a proposed scavenger of SIN-1 (12)], uric acid [peroxynitrite (18)], and D-mannitol (hydroxyl radical) (Scheme 2). The p53 tumor suppressor gene plays an important role in cellular responses to DNA damage through its involvement in damage-induced G1 arrest, DNA repair, apoptosis, and gene amplification (20, 21). Previous studies of two human lymphoblastoid cell lines derived from a single donor but differing in p53 status demonstrated that WTK-1 cells, which carry a T to C transition in exon 7 of the p53 gene, resulting in a change of Met to Ile at codon 237 (22-24), were more resistant to the lethal effects of X-rays than TK6 cells expressing wildtype p53 (22, 23). However, WTK-1 cells were about 20 times more sensitive to mutagenesis induced by 1.5 Gy X-rays and also showed a 10-fold higher spontaneous mutation rate at the autosomal heterozygous TK locus (22). In addition, WTK-1 cells developed a higher proportion of large-scale genomic damage (24), chromosome aberrations/mutations and delayed apoptosis (25-27) than TK6 cells in response to treatment with ionizing radiation or DNA damaging agents. Additionally, mismatch repair capacity, rather than p53 status, was

Li et al. Scheme 2. Sites of Action of Selective Free Radical Scavengers

reported to be a strong determinant of the susceptibility of these cells to alkylation-induced apoptosis in these cells (28). Current evidence also indicates that mitochondrial membrane depolarization is an important component of the apoptotic response (29). This study was designed to evaluate mutagenic and apoptotic responses to peroxynitrite delivered by degradation of SIN-1 under conditions which others had described as linear over the time period involved. The cell lines used, TK6 (wild-type p53) and WTK-1 (mutant p53), were selected principally because they had previously been shown to be mutagenized by nitric oxide; they have been extensively used to characterize spectra of mutations induced at two loci by many types of mutagens, and they represent lines reportedly isogenic except for p53 status. Information obtained will lay a foundation for future studies with other cell types. Our findings provide evidence that peroxynitrite played a major role in the induction of cellular damage under these conditions. We found that SIN-1 treatment induced more extensive DNA double-strand breaks in TK6 cells than in WTK-1 cells. Consistent with these findings, induced as well as spontaneous mutant fractions (MF) in HPRT and thymidine kinase (TK) genes were lower in TK6 than in WTK-1 cells. SIN-1 induced early and extensive apoptosis in TK6 cells, but this response was delayed and minimal in WTK-1 cells; in contrast, the magnitude of mitochondrial membrane depolarization induced was similar in both cell lines. Collectively, these findings emphasize the importance of p53 status as a determinant of mutagenic and apoptotic responses to DNA damage induced by SIN-1 in these two human lymphoblastoid cell lines.

Materials and Methods Caution: The following chemicals are hazardous and should be handled carefully: SIN-1, 4-nitroquinoline-N-oxide (4-NQO), propidium iodide, 6-thioguanine, trifluorothymine, 2′-deoxycytidine, aminopterin, thymidine, Hoechst 33258, etoposide, trypan blue, potassium nitrate, and sodium nitrite. Chemicals. Chemicals and reagents were obtained as follows: SIN-1 from Biomol Research Laboratories (Plymouth Meeting, PA) was used without further purification; SOD (bovine erythrocyte, 5000 units/mg) and catalase (beef liver, 65 000 units/mg) from Roche Molecular Biochemicals (Indianapolis, IN); Ready-load 100 bp DNA ladder marker from Life & Technologies (Rockville, MD); cytochrome c (horse heart) and D-mannitol from Fluka (Buchs, Switzerland); uric acid, Tiron (4,5-dihydroxy-1,3-benzene-disulfonic acid), 4-NQO, 6-thioguanine, trifluorothymidine, 3-nitrotyrosine, 2′-deoxycytidine, hypoxanthine, aminopterin, thymidine, Hoechst 33258, pro-

Peroxynitrite-Induced Genotoxicity and Apoptosis pidium iodide, etoposide, and trypan blue from Sigma (St. Louis, MO); carboxy-PTIO [2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide] from Cayman Chemical Company (Ann Arbor, Michigan); L-tyrosine from Aldrich (Milwaukee, WI); D-glucose and sodium nitrite from Mallinckrodt Specialty Chemicals Co. (Paris, KY); potassium nitrate from C. J. T. Baker Chemical Co. (Phillipsburg, NJ); and JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide) from Molecular Probes, Inc. (Eugene, OR). Cell Culture. TK6 cells were kindly provided by Dr. W. G. Thilly (Massachusetts Institute of Technology), and WTK-1 cells by Dr. H. Liber (Massachusetts General Hospital). All cell culture reagents were purchased from BioWhittaker (Walkersville, MD). Cells were maintained in exponentially growing suspension culture at 37 °C in a humidified, 5% CO2 atmosphere in RPMI-1640 medium supplemented with 10% heat-inactivated calf serum containing 100 units/mL penicillin, 100 µg/mL streptomycin, and 2 mM L-glutamine. Stock cells were subcultured and maintained at a density of 1 × 106 cells/mL in 150-mm dishes; during experimental procedures, cell density was maintained below 1.5 × 106 cells/mL. Cells were treated with CHAT (10 µM 2′-deoxycytidine, 200 µM hypoxanthine, 0.1 µM aminopterin, and 17.5 µM thymidine) to remove mutant cells before each experiment as described previously (30). Cytotoxicity of SIN-1. TK6 cells were washed and diluted to 1 × 106 cells/mL in Hanks’ balanced salt solution (HBSS), pH 7.4, supplemented with 25 mM sodium bicarbonate, and 1 mL was placed into each well of a 24-well tissue culture plate (Costar, Corning, NY). Treated cells were suspended in medium containing 5 mM SIN-1 and incubated at 37 °C, with exposure to air and shaking for 5 min every 10 min in a laminar flow hood. After treatment, cells were immediately transferred to Eppendorf tubes, centrifuged, and resuspended in 1 mL of RPMI-1640 medium. After incubation at 37 °C for 24 h, cell numbers were determined. Cells were stained with 0.4% trypan blue solution, and those excluding the stain were counted under a phase-contrast microscope. Cell death was expressed as the percentage of corresponding untreated cells. In previous related work, we compared the lethality of nitric oxide to TK6 and WTK-1 cells determined by trypan blue exclusion with that determined by colony-forming ability (30), and both methods showed similar data (unpublished data). Therefore, trypan blue exclusion was used in the present study to measure cell death. A primary objective of this study was to characterize SIN-1/ peroxynitrite-induced mutagenesis in human lymphoblasts in order to compare the response with our previous data obtained by bolus administration of peroxynitrite. The treatment regimen used in the scavenger experiments and MF determination was designed to maximize the mutagenic response. Preliminary experiments revealed that exposure of TK6 cells at a density of 1 × 106 cells/mL to 5 mM SIN-1 alone for 1.5 h resulted in about 28% cell survival 24 h after treatment. Extensive work by Thilly and co-workers (31) on these cell lines has shown that maximum numbers of mutants are obtained after treatment with a mutagen dose resulting in 30-36% survival. Accordingly, the 5 mM SIN-1 regimen was adopted in the scavenger experiments and for characterization of the mutagenic response. To identify reactive species involved in SIN-1-induced cell damage, several putative radical scavengers were used as shown in Scheme 2. Cells were exposed in the absence or presence of SOD (5-500 units/mL), catalase (5-500 units/mL), SOD plus catalase (5-500 units/mL each), heat-inactivated SOD, and catalase (500 units/mL each, boiling at 100 °C for 10 min), cytochrome c (0.02 and 0.1 mM), D-mannitol (1-100 mM), uric acid (1 and 5 mM), Tiron (1 and 5 mM), or carboxy-PTIO (0.1 and 0.5 mM), and cell number was determined 24 h after treatment. Stock solutions of all compounds were freshly prepared in HBSS except for SIN-1, which was dissolved in filtered deionized water. Cells without any treatment or treated with 4-NQO (140 ng/mL) served as negative and positive controls, respectively.

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Figure 1. Dose-related SIN-1 lethality, determined by trypan blue exclusion, in TK6 cells treated at varying cell densities. Cell death was expressed as the percentage of untreated cells. Data are the means of duplicate experiments. Standard deviations were less than 15% (not shown). Nitrotyrosine Assay. To quantify peroxynitrite generation during SIN-1 degradation, nitrotyrosine formed by reaction of 5 mM SIN-1 with equimolar L-tyrosine in the absence or presence of the peroxynitrite scavenger uric acid was determined using a previously described spectroscopic method (32). Briefly, 1 mL of DPBS containing 25 mM sodium bicarbonate, pH 7.4, 5 mM L-tyrosine, and 5 mM SIN-1 with or without 5 mM uric acid was placed into a 24-well plate and incubated at 37 °C for 1.5 h with exposure to air and shaking for 5 min every 10 min. Absorbance at 428 nm was recorded against DPBS supplemented with 25 mM sodium bicarbonate alone, and nitrotyrosine content was calculated using a molar absorbance coefficient of 4200 M-1 cm-1. Solutions of L-tyrosine and 3-nitrotyrosine were used as negative and positive controls, respectively. Mutant Fraction Determination. To determine induced MF in the HPRT and TK genes of TK6 and WTK-1 cells, 4 × 107 cells at a density of 1 × 106 cells/mL were exposed to 5 mM SIN-1 for 1.5 h, cultured for 6 to 10 days to permit phenotypic expression, then plated in selective medium for MF determination. A total of 24 × 106 cells from each treatment group were transferred to 10 96-well plates at densities of 40 000 cells/well in medium containing 6-thioguanine (2 µg/mL) to select HPRT mutants or trifluorothymidine (2 µg/mL) to select TK mutants. For plating efficiency analysis, 120 cells in 12 mL of nonselective medium from each group were plated in duplicate in 96-well plates at a density of 1 cell/100 µL/well. After 2 weeks of incubation, colonies were counted, and MF was calculated according to a previously described method (30). In parallel experiments, to estimate spontaneous mutation rate and to determine responses of the HPRT and TK genes to treatment with a well-characterized mutagen, similar MF analyses were done on untreated cells and cells treated with 4-NQO (140 ng/ mL for 1.5 h) as described above. Apoptosis Analysis. After determining that SIN-1 could be used as an effective peroxynitrite generator and was an effective mutagen, we next characterized effects of SIN-1 on apoptosis in TK6 and WTK-1 cells. In preliminary experiments, TK6 cells exposed for 1.5 h at varying cell densities to 1, 2.5, or 5 mM SIN-1 in HBSS containing 25 mM sodium bicarbonate showed that cell density greatly affected cell death and apoptotic response. As shown in Figure 1, by 24-72 h after exposure, 5 mM SIN-1 exposure caused 97-99% cell death at a density of 5 × 105 cells/mL and 77-99% cell death at a density of 1 × 106 cells/mL; 2.5 mM SIN-1 induced 86-96% cell death at a density 5 × 105 cells/mL and 53-71% cell death at a density of 1 × 106 cells/mL; and 1 mM SIN-1 induced 61-68% cell death at a density of 5 × 105 cells/mL and 11-47% cell death at a density of 1 × 106 cells/mL. Apoptosis induced by 5 mM SIN-1 in TK6 cells at a density of 1 × 106 cells/mL was similar to that induced by 2.5 mM SIN-1 at a density of 5 × 105 cells/mL (13-81% vs 42-84% by 24-72 h after exposure); and 1 mM SIN-1 did not induce apoptosis at either cell density. Since we sought to develop an apoptosis model with the lowest SIN-1 dose that

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would induce significant apoptosis, 2.5 mM SIN-1 was used to treat cells at a density of 5 × 105 cells/mL for studies of apoptosis, mitochondrial membrane depolarization, comet assay, and DNA ladder analysis. Morphologic changes in apoptotic cell nuclei were detected by microscopic examination of cells stained with Hoechst 33258. Quantitative estimation of apoptosis was accomplished by flow cytometry following annexin V-FITC staining (Clontech, Palo Alto, CA). Positive staining for annexin V was used as a marker of early apoptosis (33), since plasma membrane alterations in early apoptosis result in the translocation and externalization of phosphatidylserine, enabling binding to annexin V, a Ca2+dependent phospholipid binding protein. Aliquots of cell suspensions containing 1 × 105 to 1 × 106 cells were placed into 5-mL Falcon tubes, washed once with annexin binding buffer, and resuspended in 200 µL of binding buffer. The cells were stained for 15 min at room temperature in the dark with annexin V-FITC at a final concentration of 0.5 µg/mL and propidium iodide at a final concentration of 2.5 µg/mL. The volume was adjusted to 600 µL using ice-cold binding buffer for flow cytometry analysis at various time points using a Becton Dickinson FACScan (excitation light 488 nm) equipped with CellQuest software. Annexin V-FITC fluorescence was recorded in FL-1 and propidium iodide fluorescence in FL-2. Cells stained with annexin V only were designated as apoptotic, those with propidium iodide or both propidium iodide and annexin V as necrotic or late apoptotic, those with propidium iodide only as necrotic, and those unstained by either as healthy. Cells exposed to HBSS without SIN-1 for 1.5 h served as negative controls, and those treated with 2.5 µM etoposide in culture medium for 6 h as positive controls. Mitochondrial Membrane Potential (MMP) Analysis. The fluorescent probe JC-1 has been shown to be specific for measuring MMP changes (29) and was used in analysis of MMP in TK6 and WTK-1 cells at a density of 5 × 105 cells/mL treated with 2.5 mM SIN-1 in the absence or presence of radical scavengers. At intervals of 6 h, 24 h, and 48 h after SIN-1treatment, cells were incubated with 10 µM JC-1 dye for 15 min, washed three times in PBS. Mitochondrial damage was qualitatively estimated by fluorescence microscopy with a long-pass filter, and loss of MMP was quantified by flow cytometry following JC-1 staining as previously described (29). Briefly, 30 min prior to cytometric analysis, JC-1 was added to 1.5 mL of cell suspension to a final concentration of 10 µM and incubated at 37 °C, in a 5% CO2 atmosphere. At the designated times, 10 000 cells from each sample were analyzed on a FL-1 (530 nm) versus FL-2 (585 nm) dot plot on a Becton Dickinson FACScan. JC-1 exhibits dual fluorescence emission depending on MMP state. In cells with normal MMP, JC-1 forms aggregates and emits high FL-2 fluorescence, whereas loss of MMP results in a reduction in FL-2 fluorescence and gain in FL-1 fluorescence as the dye shifts from an aggregate to monomeric state. Membrane depolarization is therefore monitored through the increase in FL-1 fluorescence. The data were converted to density plots and histogram plots using CellQuest software. TK6 and WTK-1 cells exposed to HBSS alone for 1.5 h or to 2.5 µM etoposide in culture medium for 6 h were used as negative and positive controls, respectively. DNA Ladder Assay. Nucleosomal ladders induced by 2.5 mM SIN-1 in DNA from TK6 and WTK-1 cells at a density of 5 × 105 cells/mL were examined with the ApoAlert LM-PCR Ladder Assay kit (Clontech), following the recommended procedure. Briefly, genomic DNA was extracted using Tri Reagent liquid (Sigma) and 0.5 µg of DNA from each sample was ligated to adaptors by T4 ligase, then amplified by LM-PCR in a volume of 100 µL with Advantage cDNA polymerase (Clontech). A 15 µL aliquot of PCR product was subjected to electrophoresis on a 1.2% agarose/ethidium bromide gel at 90 V for 2 h, and the ladder of fragmented DNA was visualized and photographed under UV light. Neutral Single Cell Gel Electrophoresis (Comet Assay). Neutral comet assays were performed using kits from Trevigen,

Li et al. Inc. (Gaithersburg, MD) to assess DNA double-strand breaks induced by 2.5 mM SIN-1 in TK6 and WTK-1 cells at a density of 5 × 105 cells/mL. Briefly, cells at 1 × 105/mL were suspended in molten LMAgarose (at 42 °C) at a ratio of 1:10, and 50 µL was immediately pipetted onto a CometSlide. Slides were kept at 4 °C for 10 min, lysed for 1 h, then electrophoresed at 30 V for 20 min. Cells were stained with SYBR green and photographed under a fluorescence microscope. DNA migration was analyzed with the Komet 4.2 Single Cell Gel Electrophoresis Analysis (Kinetic Imaging Limited, Liverpool, U.K.) determining Olive tail moment, defined as the product of the percentage of DNA in the tail distribution and displacement between the head and tail means (34), of at least 40 cells/sample. Cells exposed to HBSS alone were used as negative controls, and cells treated with 100 µM H2O2 in RPMI-1640 for 20 min at 4 °C as positive controls. Statistical Analysis. All experiments were performed in duplicate and repeated at least twice after experimental conditions were optimized. Statistical analyses were performed using a two-tailed Student’s t-Test and p < 0.05 was considered to be statistically significant.

Results Formation of peroxynitrite under these experimental conditions was verified by analysis of nitrotyrosine formation when L-tyrosine was added to the incubation mixture to which cells were exposed. Our results showed that 55 ( 1 µM nitrotyrosine was formed when 5 mM SIN-1 degradation took place in the presence of equimolar L-tyrosine; nitrotyrosine concentration decreased to 3.7 ( 0.7 µM when 5 mM uric acid, a peroxynitrite scavenger, was added to the reaction mixture. Timecourse experiments in which TK6 cells were exposed for 0.5-2 h to 5 mM SIN-1 revealed a time-dependent pattern of cell death (data not shown). Protective effects of free-radical scavengers added to the exposure medium on the lethality of SIN-1 to TK6 cells at a density of 1 × 106 cells/mL were subsequently determined, with results summarized in Table 1. Survival of cells exposed to 5 mM SIN-1 alone for 1.5 h was 28 ( 6%, but was significantly increased when exposure took place in the presence of 500 units/mL SOD plus catalase; 5 mM Tiron; 0.1 mM cytochrome c; 0.5 mM carboxy-PTIO; or 5 mM uric acid. In each instance, lower scavenger concentrations were less effective in reducing lethality. Addition of D-mannitol, SOD, or catalase alone in active or heatinactivated forms did not significantly diminish SIN-1 lethality. In a recent report, Moro et al. (35) proposed that peroxynitrite can react with glucose in a manner that might represent a significant detoxification pathway for peroxynitrite in vivo. We therefore repeated all of the above scavenger experiments in DPBS with or without addition of 5.7 mM glucose (instead of HBSS), and no significant differences in cell survival were observed (data not shown). Mutagenesis in the HPRT and TK genes was investigated in TK6 and WTK-1 cells exposed to SIN-1. As shown in Figure 2, exposure of TK6 cells at a density of 1 × 106 cells/mL to 5 mM SIN-1 for 1.5 h increased MF 2.2-fold in the HPRT gene and 1.8-fold in the TK gene compared with untreated controls (5.4 × 10-6 vs 2.5 × 10-6 and 5.6 × 10-6 vs 3.2 × 10-6, respectively). WTK-1 cells exposed under the same conditions showed increases in MF of 1.7-fold in HPRT and 2.5-fold in TK, respectively, when compared with untreated controls (8.5 × 10-6 vs 4.9 × 10-6 and 39.1 × 10-6 vs 15.6 × 10-6). By comparison, in 4-NQO-treated positive controls, induced

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Table 1. Survival of TK6 Cells Treated with 5 mM SIN-1 at a Density of 1 × 106 Cells/mL in the Presence of Radical Scavengersa effective scavengers scavenger

concentration

none

ineffective scavengers survival

scavenger

28 ( 6

none

concentration

survival 28 ( 6

cytochrome c

0.02 mM 0.1 mM

34 ( 4 98 ( 6b

D-mannitol

1 mM 10 mM 100 mM

24 ( 3 24 ( 5 21 ( 3

carboxy-PTIO

0.1 mM 0.5 mM

32 ( 2 87 ( 6b

SOD

5 units/mL 50 units/mL 500 units/mL

28 ( 2 29 ( 8 39 ( 1

tiron

1.0 mM 5.0 mM

66 ( 2b 69 ( 0b

catalase

5 units/mL 50 units/mL 500 units/mL

37 ( 1 35 ( 4 41 ( 1

uric acid

1.0 mM 5.0 mM

73 ( 8b 87 ( 2b

heated SOD + catalase

500 units/mL each

31 ( 8

SOD + catalase

5 units/mL each 50 units/mL each 500 units/mL each

25 ( 4 74 ( 3b 83 ( 5b

a Mean ( standard deviation of values derived from two separate experiments, each done in duplicate. b p < 0.05 compared with SIN-1 with no scavenger.

Figure 2. Mutant fraction in the HPRT and TK genes of TK6 and WTK-1 cells exposed to 5 mM SIN-1 at a density of 1 × 106 cells/mL. Untreated cells and cells treated with 4-NQO (140 ng/ mL for 1.5 h) served as negative and positive controls, respectively. Results are means ( standard deviation of duplicate experiments.

MF in HPRT and TK genes was 19.1 × 10-6 and 11.4 × 10-6 in TK6 cells and 32.1 × 10-6 and 71.6 × 10-6 in WTK-1 cells, respectively. Spontaneous as well as SIN1- or 4-NQO-induced mutation fractions were significantly higher in WTK-1 cells than in TK6 cells (p < 0.050.01), except in the case of HPRT mutants induced by 4-NQO (p ) 0.2). Additionally, in WTK-1 cells, larger increases in MF in TK than in HPRT were found both in spontaneous mutants and in those induced by SIN-1- or 4-NQO (p < 0.05-0.01). The time course of cell death, summarized in Figure 3, indicated that SIN-1 was more cytotoxic to TK6 cells than to WTK-1 cells. After treatment with 2.5 mM SIN-1 at a density of 5 × 105 cells/mL, more than 80% of TK6 cells were dead after 24 h and nearly all by 72 h. WTK1 cells were less sensitive, with about 40% dead after 24 h and 50% after 72 h. In both types of cells, the magnitude

Figure 3. Lethality of SIN-1 to TK6 and WTK-1 cells, determined by trypan blue exclusion. Cells at a density of 5 × 105 cells/mL were exposed to 2.5 mM SIN-1 in HBSS containing 25 mM sodium bicarbonate, pH 7.4, for 1.5 h at 37 °C with exposure to air and shaking for 5 min every 10 min. Cells treated for 6 h with medium containing 2.5 µM etoposide were used as positive controls. Cell death was expressed as the percentage of corresponding untreated cells. Data represent the mean of two duplicate experiments. Standard deviations were less than 15% (not shown).

of the response was generally similar to that induced by etoposide, the positive control. SIN1-induced apoptosis in TK6 and WTK-1 cells was quantified using flow cytometry after annexin V staining, with the results shown in Figure 4. Exposure to 2.5 mM SIN-1 at a density of 5 × 105 cells/mL induced apoptosis in a timedependent manner in TK6 cells. Approximately 20% of cells were apoptotic 24 h after treatment, 50% after 48 h, and 75% after 72 h. Apoptotic frequency was increased by 2.5-17-fold over control levels. Both the time course and magnitude of the response were virtually identical to that induced by etoposide, the positive control. The apoptotic response in WTK-1 cells was very different, in that no significant increase occurred up to 72 h after treatment (0-2.3-fold elevation compared with controls). In TK6 cells, SIN-1-induced apoptosis was decreased to control levels in the presence of SOD plus catalase, carboxy-PTIO, or uric acid, whereas D-mannitol was ineffective in this regard (data not shown). These results

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Figure 4. Normalized percent of apoptosis in TK6 and WTK-1 cells exposed to 2.5 mM SIN-1 for 1.5 h at a density of 5 × 105 cells/mL, determined by flow cytometry following annexin V-FITC staining. Cells untreated or exposed to 2.5 mM etoposide were used as negative and positive controls, respectively. Data represent the means of two experiments, each done in duplicate. Standard deviations were 5% to 15% (not shown).

Figure 5. Induction of DNA ladders by 2.5 mM SIN-1 in TK6 and WTK-1 cells at a density of 5 × 105 cells/mL, detected by LM-PCR ladder assays. DNA ladders were observed in TK6 cells at 48 h and 72 h after SIN-1 treatment, but not in WTK-1 cells. DNA fragmentation smears in TK6 cells at 6 h and 24 h after SIN-1 treatment may be from necrotic cells. LM-PCR products from TK6 and WTK-1 cells were run on the same gel and the results are representative of three independent experiments.

were further supported by DNA ladder detection, another conventional marker of apoptosis, which showed that 2.5 mM SIN-1 induced DNA ladders in TK6 cells but not in WTK-1 cells (Figure 5). Qualitative evidence of mitochondrial depolarization induced by exposure to 2.5 mM SIN-1 at a density of 5 × 105 cells/mL was observed in both TK6 and WTK-1 cells after staining with JC-1 (data not shown). Quantitative evaluation by flow cytometry revealed that MMP loss was evident as early as 6 h after treatment of WTK-1 cells

and after 24 h in TK6 cells, and results are shown in Figure 6. The magnitude of the response was increased at 48 h in both cell lines, with MMP loss of 25 and 24% maximum, respectively. MMP loss in TK6 cells was comparable to that induced by etoposide, through 48 h after treatment. The pattern of response of WTK-1 cells to SIN-1 exposure was in general similar to that of TK6 cells, but WTK-1 cells showed significantly greater MMP loss following etoposide treatment than after SIN-1 exposure. MMP loss was blocked by SOD plus catalase, carboxy-PTIO or uric acid, but not by D-mannitol (data not shown). Induction of DNA double-strand breaks by SIN-1 was evaluated with neutral comet assays. Evidence of significant double-strand breakage was qualitatively evident in TK6 cells, but not WTK-1 cells, within 4 h after exposure to 2.5 mM SIN-1 at a density of 5 × 105 cells/ mL. Quantitative analysis, summarized in Figure 7, revealed that SIN-1 significantly increased Olive tail moment in TK6 cells (11.4 ( 16 vs 1.9 ( 0.9, p < 0.05), but not in WTK-1 cells (1.5 ( 0.91 vs 1.0 ( 0.3, p > 0.05) at 4 h after treatment, compared with controls. The high variability of the Olive tail moment values following SIN-1 treatment is reflective of an essentially bipolar response, in which many cells showed maximal strand breakage, but others were very small. Also, Olive tail moments were significantly greater in TK6 cells than in WTK-1 cells 4 h after treatment with either SIN-1 or with 100 µM H2O2, the positive control (p < 0.01 and p < 0.05). SIN-1-induced DNA strand breakage was effectively prevented by SOD plus catalase, carboxy-PTIO or uric acid, but not by D-mannitol (data not shown).

Discussion SIN-1 has been used as peroxynitrite generator in some experimental models (13, 14), but formation of other reactive species such as H2O2 has also been proposed to be involved in SIN-1-induced cytotoxicity and geno-

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Figure 6. Normalized percent of mitochondrial membrane potential loss in TK6 and WTK-1 cells exposed to 2.5 mM SIN-1 for 1.5 h at a density of 5 × 105 cells/mL, determined by flow cytometry following JC-1 staining. Cells untreated or exposed to 2.5 mM etoposide were used as negative and positive controls, respectively. Data represent the means of two experiments, each done in duplicate. Standard deviations were 3-10% (not shown).

Figure 7. Box-and-whisker plots of Olive tail moments from neutral comet assays of TK6 and WTK-1 cells 4 h after treatment with 2.5 mM SIN-1 for 1.5 h at a density of 5 × 105 cells/mL. At least 40 cells were analyzed in each sample. Compared with untreated controls, Olive tail moments were significantly higher in SIN-1-treated TK6 cells (p < 0.05), but not in SIN-1-treated WTK-1 cells (p > 0.05). Olive tail moments were also significantly greater in TK6 cells than in WTK-1 cells after treatment with either SIN-1 or with 100 µM H2O2, the positive control (p < 0.01 and p < 0.05).

general agreement with the recent report (13) that peroxynitrite formed during the decomposition of SIN1 induces single-strand breaks in the DNA of cultured human lymphocytes in a dose- and time-dependent fashion. In our experiments, we exposed cells to SIN-1 suspended in simple salt solutions (HBSS or DPBS) supplemented with 25 mM sodium bicarbonate. In more complex culture media, SIN-1 might have reduced other molecules in the medium to release NO• without producing superoxide (12) or peroxynitrite may have reacted with buffers and other components of biological media such as HEPES, MOPS, glycerol, or glucose to form hydrogen peroxide (41, 42) or an NO• donor (43, 44). The presence of bicarbonate also profoundly influences the biological effects of peroxynitrite by the formation of a reactive nitrosoperoxocarbonate intermediate (ONOOCO2-) that alters both its chemical stability and reaction pathways (45). In our experiments, bicarbonate was added to the exposure medium at a concentration approximating its estimated in vivo levels to take this effect into account.

toxicity (9, 10, 36-40). In the present study, we found that SIN-1-induced cell death, apoptosis, DNA, and mitochondrial damage were significantly diminished by treatment with effective scavengers of NO•, superoxide, and peroxynitrite, supporting the interpretation that peroxynitrite played a major role in these processes. We also found that the presence of the peroxynitrite scavenger uric acid during SIN-1 decomposition inhibited the nitration of tyrosine by 93%, providing further supportive evidence for this interpretation. In contrast, TK6 cells were not protected against the lethal effects of SIN-1 when D-mannitol, SOD or catalase were added individually to the exposure medium, indicating that H2O2 and extracellular hydroxyl radical evidently did not play a significant role in SIN-1-mediated cytotoxicity under these experimental conditions. These results are in

Many previous studies have shown that loss of wildtype p53 function is associated with delayed apoptosis and genomic instability induced by ionizing radiation and DNA damaging agents, and is reflected in such biological endpoints as absence of G1 arrest (46) and chromosomal aberrations (27), as well as increased spontaneous and irradiation-induced mutagenesis (22, 24-26, 46, 47). In our studies, we evaluated the role of p53 in SIN-1induced mutagenesis and apoptosis in two closely related human lymphoblastoid cell lines, TK6 and WTK-1, originally isolated from the same donor, but differing in p53 status (25, 46). Previous investigators have demonstrated that p53 protein level in TK6 cells containing wild-type p53 was increased by ionizing radiation or other DNA damaging agents. In contrast, the high basal level of p53 protein in WTK-1 cells containing mutant p53 showed no further increase after the same treatments (48, 49).

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Our data show that cells of the two lines differed dramatically with respect to both apoptosis and mutagenesis. SIN-1 effectively induced a high level of apoptosis in TK6 (wild-type p53) cells, but WTK-1 (mutant p53) cells were resistant to induction of apoptosis by SIN-1, in that little or no response occurred as late as 96 h after treatment, suggesting that SIN-1-induced apoptosis in these cells may be p53-dependent. Our investigation also demonstrated that the mutagenic response at both the HPRT and TK loci was much more pronounced in WTK-1 cells than in TK6 cells, indicating the importance of p53 function as a determinant of mutagenesis in these cells. A mechanism by which the mutant p53 gene product might affect the magnitude of the mutagenic response could be through p53-mediated apoptosis triggered by DNA damage induced by SIN-1. As shown by our results, WTK-1 cells carrying mutant p53 were resistant to induction of apoptosis by SIN-1 (Figure 4), coupled with decreased cell death (Figure 3), which may have led to persistence of cells whose DNA was heavily damaged. Such cells would have been eliminated by apoptosis in TK6 cells. DNA damage in inappropriately surviving cells could also be a precursor for large-scale genomic rearrangements. For example, Schwartz et al. (50) postulated that the lower level of radiation-induced chromosome aberrations in TK6 cells was related to early onset apoptosis. Alternatively, the mutational response could be modulated through the direct participation of p53 in DNA repair, either by catalyzing or regulating recombinational processes, or the mutant p53 of WTK-1 cells could also exhibit so-called “gain of function” properties in apoptosis and/or mutagenesis (51). We found that DNA double-strand breaks, as detected by neutral comet assay, were significantly higher in TK6 cells than in WTK-1 cells following exposure to SIN-1. This finding was in general agreement with other recent reports dealing with relationships between DNA damage and p53 status. TK6 cells were reported to be more sensitive to the formation of NO•-induced DNA single strand breaks than Chinese hamster ovary cells (CHOAA8) in which the p53 gene was either inactive or mutated (52). Our results also show that spontaneous as well as SIN-1- or 4-NQO-induced mutagenesis in WTK-1 cells was significantly higher in the TK gene than in the HPRT gene (p < 0.05-0.01). Xia and Liber (26) have suggested that the differential effects of mutant p53 on mutability of these loci in WTK-1 cells probably can be explained by the hemizygosity of HPRT versus the heterozygosity of TK. Since these cells carry only one copy of HPRT, the number and efficiency of recombinationmediated mutagenic pathways are reduced. In addition, very large deletions around HPRT are lethal, which also limits the accumulation of viable mutants containing large-scale changes (24). Analysis of the physical structures of X-ray-induced TK-deficient mutants (24) revealed that greater mutability at this locus was associated with a higher frequency of inter- and intramolecular recombination events, suggesting that a recombinational repair system was functioning at a higher level in WTK-1 cells than in TK6 cells, and that the increase in TK mutagenesis could be attributed, in part, to mitotic recombination. In another study, DNA fragmentation induced by ionizing radiation was more extensive in TK6 cells than in WTK-1 cells (48). Our findings in SIN-1-treated cells are also consistent with these interpretations.

Li et al.

MMP loss has been shown to occur in many experimental models of apoptosis, in which it has been suggested to result in the release of proapoptotic factors, including cytochrome c from mitochondria (53). Our results show that 2.5 mM SIN-1 induced MMP loss of similar magnitude in TK6 and WTK-1 cells but, importantly, was associated with apoptosis only in TK6 cells. It is unclear whether MMP loss associated with apoptosis in TK6 cells reflected a cause-effect relationship or independent events. Loss of MMP without apoptosis in WTK-1 cells may indicate that SIN-1-induced apoptosis and mitochondrial depolarization occur independently in human lymphoblastoid cells or some of the mitochondriaassociated proapoptotic factors may be p53-dependent, implying that these factors may not exist or could not be activated by SIN-1 treatment in WTK-1 cells due to p53 mutation. Further work to examine these possibilities as well as SIN-1-induced signaling in these cells is currently under way in our laboratory.

Acknowledgment. This publication was supported by Grant 5 P01 CA26731 from the National Cancer Institute. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Cancer Institute. The authors thank Dr. Steven R. Tannenbaum, Dr. William M. Deen, and Dr. Harry Ischiropoulos for helpful discussions, Dr. Deepa Jethwaney for assistance with initial experiments and Glenn Paradis for his technical expertise in flow cytometry.

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