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Chem. Res. Toxicol. 2003, 16, 1004-1013
Thresholds of Nitric Oxide-Mediated Toxicity in Human Lymphoblastoid Cells Chen Wang,† Laura J. Trudel,‡ Gerald N. Wogan,‡ and William M. Deen*,†,‡ Department of Chemical Engineering, and Biological Engineering Division, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Received March 7, 2003
A novel delivery system was used to study NO-mediated cyto- and genotoxicity in two human lymphoblastoid cell lines, TK6 (wild-type p53) and NH32 (p53-null but isogenic to TK6). The delivery system, which supplied NO and O2 continuously by diffusion through gas permeable tubing, was found to maintain the NO and O2 concentrations at constant, predictable values. Cellular rates of NO and O2 consumption and mass transfer coefficients for the two gases were measured in separate experiments and used to calculate the NO concentrations during exposure experiments. The TK6 and NH32 cells were each exposed to several steady state NO concentrations for varying lengths of time, so that the total dose (area under the concentrationtime curve) covered a wide range. End point assays, including lethality, apoptosis, mitochondrial damage, and mutation rate in the thymidine kinase (TK1) gene locus, were performed at different posttreatment times. Control experiments using Ar instead of NO resulted in normal cell proliferation for all exposure times tested (up to 36 h). As compared to those controls, significant cell death, apoptosis, and mitochondrial membrane depolarization were observed in NO-treated TK6 cells, and the TK1 mutation rate was elevated. Of particular importance, toxic effects were observed only when the NO concentration and dose were greater than threshold values of ∼0.5 µM and ∼150 µM min, respectively. If neither or only one threshold was exceeded, the effects were insignificant; when both were exceeded, total cell survival and the number of nonapoptotic cells both decreased exponentially with increasing NO dose. In general, the NH32 cells were much more resistant to NO-induced damage and death than TK6 cells, demonstrating that p53 status is an important determinant of NO-induced cytotoxicity.
Introduction The multiple physiological roles of endogenous nitric oxide (NO), as a signaling molecule in the vascular and nervous systems and as a component in the nonspecific immune response, are well-known (1-3). Its contributions to normal health notwithstanding, inappropriately elevated levels of NO have been implicated in various human pathologies (4). NO damages cells in several ways, involving oxidative stress, DNA damage, disruption of energy metabolism, interference with calcium homeostasis, and mitochondrial dysfunction (4-8). Depending on the context and severity of the damage, such disturbances may result in cell death by either necrosis or apoptosis or in successful repair and cell survival. For instance, small decreases in ATP levels upon NO exposure lead to apoptosis in human T cells, whereas large decreases cause rapid necrosis (9, 10). Mild oxidative stress caused by NO and O2- (with modest and/or brief elevations in peroxynitrite concentration) leads to apoptosis, whereas severe oxidative stress leads to extensive cellular damage and necrosis (11). When the damage is insufficient to cause necrosis, the upregulation of p53 stops cell division and provides the opportunity for damaged DNA to be repaired (12). * To whom correspondence should be addressed. Tel: (617)253-4535. Fax: (617)258-8224. E-mail:
[email protected]. † Department of Chemical Engineering. ‡ Biological Engineering Division.
NO can be both a promoter and an inhibitor of apoptosis. In a recent review, Kim et al. (13) summarized findings for multiple cell types that exhibit proapoptotic or antiapoptotic responses to NO and illustrated the mechanisms and key factors that modulate the NOmediated apoptotic effects on cells. The current view is that the dual roles of NO in apoptosis are cell specific and dose-dependent. Low concentrations of NO are protective, but higher NO concentrations induce apoptosis (13, 14). Overall, whether cells survive or die and whether they undergo apoptotic or necrotic death is recognized as being linked to the amount of NO exposure. However, quantitative information on exposure levels, in terms of NO concentrations and their duration, tends to be lacking both in vivo and in vitro. Various NO donor compounds have been employed to examine the cellular effects of NO. Although easy to use, donor compounds generally do not provide a steady rate of NO release, and the parent compounds and/or their decomposition products may contribute to the observed cellular responses (15-17). In such studies, the extent of NO exposure is usually reported as the total amount of NO donor compound used (18-20), which is not a direct measure of either NO concentration or NO dose. Another approach for exposing cells has been to supply NO continuously by diffusion through gas permeable poly(dimethylsiloxane) tubing (21-23). In this method, the NO delivery rate is controlled by the length of the
10.1021/tx0340448 CCC: $25.00 © 2003 American Chemical Society Published on Web 07/19/2003
Thresholds of Nitric Oxide Toxicity
tubing placed in a closed, stirred chamber and by the composition of the delivery gas. The rate of NO delivery has usually been determined by measuring the accumulation rates of nitrite and nitrate (the end products of NO oxidation), and the observed biological effects have been correlated with the NO delivery rate and/or the total dose. Although the membrane system serves as a “cleaner” NO source for cells in vitro, it has generally been used without a means for replenishing the O2 that is consumed by cells, reacts with NO, or is lost by diffusion into the NO delivery tubing. The progressive deoxygenation that results will cause a continuous elevation of the NO concentration, so that the exposure conditions vary with time. Moreover, the effects of hypoxia on the cells might confound the interpretation of the results, especially in long experiments. These shortcomings have been addressed very recently by a membrane delivery system that continuously supplies both NO and O2 and is therefore able to maintain both at constant levels (24). Li et al. (23) used the original membrane delivery system (NO only) to investigate NO-induced genotoxicity, mitochondrial damage, and apoptosis in two human lymphoblastoid cell lines, TK6 and WTK-1, which express wild-type and mutant p53, respectively. They found that p53 status strongly affected the mutagenic and apoptotic response to NO-induced DNA damage. In their experiments, pure NO gas was used to treat cells for relatively short periods of time, and the rate of NO permeation into the cell suspension was assessed in the usual way from nitrite and nitrate measurements; NO concentrations were not measured. The objective of the work reported here was to extend the observations of Li et al. (23) by making use of recent developments both in delivery systems and in cell lines. The new NO delivery system (24), which is able to maintain NO and O2 concentrations at constant, predictable levels indefinitely, was used here for cellular exposures for the first time. In addition, recognizing that WTK-1 cells may exhibit “gain of function” properties in apoptosis and/or mutagenesis (25), NH32 cells were selected instead to examine the effects of p53. Those cells were derived from TK6 cells by knocking out the p53 gene (26). TK6 and NH32 cells are otherwise isogenic and have been characterized for radiation-induced mutation at the autosomal thymidine kinase locus (26). Accordingly, we chose to assess the effects of p53 by exposing TK6 and NH32 cells to NO under identical conditions. By correlating the steady state NO concentration and total dose with various biological end points, including measures of overall viability, apoptosis, and mutation frequency, we found that both concentration and dose are critical in initiating NO toxicity.
Materials and Methods Reagents and Cells. Pure Ar, mixtures of 1% and 10% NO in Ar, and a mixture of 50% O2 and 5% CO2 in N2 were purchased from BOC Gases (Edison, NJ). NO gas with 99% purity was purchased from Matheson Tri-Gas (Twinsburg, OH). TK6 cells were kindly provided by Dr. W. G. Thilly (Massachusetts of Institute of Technology), NH32 cells were provided by Dr. C. Harris (National Cancer Institute), and βTC-3 cells were provided by Dr. Clark K. Colton (Massachusetts Institute of Technology). Dulbecco’s PBS, RPMI-1640 medium, donor calf serum, glutamine, and penicillin/streptomycin were from BioWhittaker (Walkersville, MD). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum, and trypsin-EDTA
Chem. Res. Toxicol., Vol. 16, No. 8, 2003 1005 solution were obtained from Mediatech (Herndon, VA). HEPES buffer was from Invitrogen (Carlsbad, CA). An ApoAlert annexin V-FITC kit was obtained from Clontech (Palo Alto, CA). 5,5′,6,6′Tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) was from Molecular Probes (Eugene, OR). 4-Nitroquinoline 1-oxide (4-NQO), trifluorodeoxythymidine, trypan blue (4% in saline), and Griess reagent (N-(1-naphthy)ethylenediamine and sulfanilic acid) were obtained from Sigma Aldrich (St. Louis, MO). Silastic tubing (1.47 mm i.d., 1.96 mm o.d.) was purchased from Dow Corning (Midland, MI). Cell Culture. TK6 and NH32 cells were maintained in exponentially growing suspension cultures at 37 °C in a humidified, 5% CO2 atmosphere in RPMI-1640 medium supplemented with 10% (v/v) heat-inactivated donor calf serum, 100 U/mL penicillin, 100 µg/mL streptomycin, and 2 mM l-glutamine. Stock cells were subcultured and routinely passaged to maintain an optimal growth density (0.3-1.5 × 106/mL) in 150 mm dishes during experiments. βTC-3 cells, which were used only for measurements of NO consumption rate (NOCR), were cultivated as monolayers in 75 cm2 Falcon T-flasks (vented cap, tissue culture-treated, Becton Dickinson, New Haven, CT) in DMEM medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 µg/mL streptomycin, and 10 mM HEPES buffer with a medium depth of 2 mm, in a 37 °C incubator with 5% CO2. The culture density was 1-2 × 106 cells/flask. Cells were split (1:8) every 6-8 days by aspirating the culture media, adding 5 mL of trypsin-EDTA solution, incubating for 7 min at 37 °C, and washing off the trypsin solution by centrifugation. Oxygen Consumption Rate (OCR) Measurements. The apparatus for the OCR measurement consisted of a 425 µL, water-jacketed, cylindrical chamber containing a magnetic stir bar and a FOXY fiber optic oxygen sensor (Instech Laboratories, Plymouth Meeting, PA) connected to an amplifier. An acrylic cap with two small ports was fabricated specifically to fit the chamber and to ensure an airtight seal. When filling the chamber with cell suspensions, excess liquid was allowed to overflow via the ports, so that no air bubbles were present when the cap was sealed. Stirring was then initiated, and the cells were allowed to equilibrate at 37 °C. Data were obtained in the range of 10-90% air saturation. The dissolved oxygen level declined linearly with time over this range, and the OCR was calculated from the slope of the plot of O2 concentration vs time. NOCR Measurements. Two methods were used to measure cellular NOCR. The first employed an NO delivery system that supplied NO by diffusion through gas permeable tubing (24). In this approach, 10% NO gas scrubbed of higher nitrogen oxides was delivered continuously into a 120 mL Teflon chamber at 37 °C, via a 7 cm loop of Silastic tubing. The aqueous NO concentration was monitored over time by an ISO-NO electrode (World Precision Instruments, ISO-NO Mark II system, Sarasota, FL), and NOCR was calculated from the difference in the NO concentration curves measured in cell suspensions and PBS, using the model described below. The second method supplied NO by injection of a saturated aqueous solution. In this approach, 99% NO gas was first bubbled through 100 mL of deionized water at room temperature at a flow rate of 60 sccm, for a time (>90 min) sufficient to yield the saturated aqueous NO concentration of 1.9 mM (27). The vessel used to expose the cells was a 17 mL stirred glass vial with a Teflon top that had ports for inserting the NO electrode, filling the vessel, and injecting the NO solution. The headspace was eliminated by completely filling the vessel with liquid. When temperature equilibrium was reached, NO reactions were initiated by injection of the saturated NO solution using a gastight syringe, to give a final concentration of 3 µM. The initial O2 concentration was 200 µM. Again, NOCR was calculated from the NO kinetics measured in PBS and cell suspensions, as detailed later. NO Exposure of Cells. Cells at a density of 5 × 105 cells/ mL in 110 mL of RPMI 1640 medium containing 10% heatinactivated calf serum were exposed to NO at 37 °C in the membrane delivery system. To achieve the desired ranges of
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Table 1. Experimental Conditions for NO Delivery to Cells
gas Ar 1% NO 10% NO
Ar or NO tubing O2 tubing delivery [NO] length (cm) length (cm) time (h) (µM) 4 7 4 7 4 7
4 6.5 4 7 4.5 7
4-36 3-24 24-36 4-24 4-12 3-12
0 0 0.4 0.6 1.3 1.8
NO dose (µM min) 0 0 570-850 150-900 320-960 320-1300
concentration and dose, various combinations of NO gas composition, tubing length, and delivery time were used, as summarized in Table 1. A 50% O2 gas mixture (with 5% CO2) was flowed through a second tubing loop to maintain the liquid O2 level near air saturation. To keep the aqueous O2 concentration constant under all conditions, the length of the O2 tubing was varied according to the NO delivery conditions, as shown. The control cells were treated with Ar gas using the same length of tubing as in the NO experiments but with the O2 tubing shortened slightly to compensate for the reduced rate of oxygen consumption. After exposure to NO or Ar, a sample was taken to count the number of live cells, and the remaining cells were collected by centrifugation. The supernatant was removed, and the nitrite concentration was measured using the Griess reagent. The cell pellets were resuspended in fresh culture medium, plated at a density of 5 × 105 cells/mL, and incubated at 37 °C. Each experiment was done at least twice. Analysis of Cell Survival. Samples taken at each postdelivery time point were analyzed for live cell number using the trypan blue exclusion method. The relative survival was calculated as the ratio of the live cell number in NO experiments to that in Ar controls (expressed as a percentage). This method was validated previously by comparing the results with the survival values determined by plating efficiency (23). Determination of Mutation Fraction (MF). NO-treated cells were grown in nonselective medium for 6-10 days to allow phenotypic expression and then plated in selective medium for MF determination. Specifically, a total of 24 × 106 cells from each treatment group were transferred to 10 96 well plates at a density of 40 000 cells per well in medium containing 2 µg/ mL of trifluorothymidine (TFT) to select thymidine kinase (TK1) mutants. Cells from each culture were also plated at 1 cell per 100 µL per well in the absence of TFT to determine plating efficiency. All plates were incubated for 3 weeks prior to scoring colonies. Mutation plates were refed with 50 µL of fresh TFT medium (6 µg/mL) and incubated for an additional 7 days to observe any late-appearing mutants. The mutant fractions were calculated with the Poisson distribution (28). The spontaneous mutation rate was estimated from the Ar-treated cells, and cells treated with 4-NQO (140 ng/mL for 1.5 h) were used as a positive control. Apoptosis Analysis. Quantitative estimation of apoptosis was accomplished by flow cytometry after annexin V-FITC staining, as described in the kit instructions. Cells (1 × 105 to 1 × 106) were centrifuged to remove the supernatant and resuspended in 200 µL of binding buffer. Cells were stained for 10-15 min at room temperature in the dark with annexin V-FITC to a final concentration of 0.5 µg/mL, and the volume was adjusted to 700 µL by adding ice-cold binding buffer. Propidium iodide (PI) at a final concentration of 2.5 µg/mL was added to the cells just before measurement. The samples were analyzed by a Becton Dickinson FACScan (excitation light, 488 nm) equipped with CELLQUEST software. Annexin V-FITC fluorescence was recorded in the FL-1 channel (530 nm) and PI in the FL-2 channel (575 nm). Ten thousand cells were examined for each sample. Cells that were labeled with annexin V only are classified as early apoptotic; those labeled with PI only are broken cells or isolated nuclei; those labeled with both dyes are either late apoptotic or secondary necrotic cells; and those labeled with neither dye are live cells. Cells treated with Ar
gas served as negative controls, and the measured apoptotic fraction was subtracted from that in NO-treated cells. Mitochondrial Membrane Potential (MMP) Analysis. Aliquots of cell suspensions were incubated with 10 µM JC-1, a specific MMP-dependent fluorescence dye, for 15 min at 37 °C. The cell suspensions were then centrifuged to remove the supernatant and resuspended in 700 µL of PBS. Mitochondrial depolarization was quantified by flow cytometry as described elsewhere (29). JC-1 exhibits dual fluorescence emission depending on the MMP state. JC-1 forms aggregates in cells with a high FL-2 fluorescence at 575 nm, indicating a normal MMP. Mitochondrial membrane depolarization results in a reduction in FL-2 fluorescence with a concurrent gain in FL-1 fluorescence (530 nm) as the dye shifts from aggregates to monomers. Therefore, retention of the dye in the cells can be monitored through the increase in FL-1 fluorescence. Ten thousand cells were examined for each sample on a FL-1 vs FL-2 dot plot. Cells treated with Ar gas served as negative controls, and the fraction of measured FL-1 fluorescence in control cells was deducted from that in the NO-treated cells. Calculation of NO Concentration and Dose. A kinetic model similar to that validated previously (24) was used to predict the concentrations of NO and O2 during NO delivery to the cell suspensions. In general, the governing equations for NO and O2 are
dCNO k(NO) NO ANO 2 NO ) (RNOP(NO) NO - CNO) - 4k1CNOCO2 - RcellCcell dt V (1) dCO2 dt
)-
(NO) kO ANO 2
V
CO2 +
(O2) kO AO2 2
V
(O2) (RO2PO - CO2) 2 O2 k1C2NOCO2 - Rcell Ccell (2)
where CNO and CO2 are the concentrations of NO and O2 in the medium; ANO and AO2 are the surface areas of the respective tubing loops; V is the total liquid volume; RNO and RO2 are the aqueous solubilities; P(NO) NO is the NO partial pressure in the NO (O2) is the O tubing and PO 2 partial pressure in the O2 tubing; and 2 (NO) (O2) k(NO) NO , kO2 , and kO2 are the mass transfer coefficients for NO or O2 (subscript) at a particular tubing loop (superscript). The one new feature in each equation is the last term on the right, which describes the effects of cellular consumption on the NO O2 and Rcell represent the concentration. In those terms, Rcell specific NO and O2 consumption rates by cells, respectively, and Ccell is the cell number density. For simplicity, the specific consumption rates were both modeled as constants (i.e., inde(O2) o (= kO ) and pendent of NO concentration). The values of kO 2 2 (NO) k(NO) and k for 10% NO gas were measured previously as NO O2 2.65 × 10-5, 1.11 × 10-5, and 3.69 × 10-5 m/s, respectively, at (NO) 37 °C. Using the same methods (24), we found k(NO) NO and kO2 for 1% NO to be 2.06 × 10-5 and 3.50 × 10-5 m/s, respectively, at 37 °C. These mass transfer coefficients, along with the values NO O2 and Rcell determined from the NOCR and OCR experifor Rcell ments, allowed the NO and O2 concentrations to be computed by numerical solution of eqs 1 and 2, using appropriate initial conditions. Simplified forms of the model were used to determine the cellular NOCR from the two types of experiments described above. When using the continuous NO delivery method for that purpose, only the NO tubing loop was used, so that the term in eq 2 that corresponds to the O2 tubing was omitted. When using the NO-saturated solution, all tubing mass transfer terms were omitted in the two equations, and the initial NO and O2 concentrations were set to 3 and 200 µM, respectively. In either NO was found by solving eqs 1 and 2 case, the unknown Rcell simultaneously and fitting the computed NO concentrations to the measured data. The NO and O2 concentrations for each exposure condition were calculated as a function of time by solving eqs 1 and 2.
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Chem. Res. Toxicol., Vol. 16, No. 8, 2003 1007
Figure 1. Decrease in aqueous O2 concentration due to consumption of O2 by TK6 or NH32 cells in a closed, 425 µL chamber. The cell number densities were as shown. The discrete symbols represent the data, and the solid lines are from linear regression. Table 2. Specific Oxygen and NOCRs by Cells
cell lines TK6 NH32 βTC3
medium
OCR (37 °C) nmol min-1 (106 cells)-1
NOCR (37 °C) nmol min-1 (106 cells)-1
RPMI 1640 PBS RPMI 1640 DMEM
0.77 ( 0.07 0.65 ( 0.16 0.58 ( 0.03 0.94 ( 0.03c
0.05 ( 0.02a, 0.02 ( 0.01b 0.02 ( 0.01b 0.16 ( 0.04a
a Determined by continuous NO delivery experiment. b Determined by bolus addition technique with NO solution. c See ref 30.
The time for the concentrations to reach a steady state was always ∼15 min, which is much shorter than any exposure period used (g3 h). Accordingly, the steady state NO concentration is a good measure of the intensity of the exposure. The total dose of NO in each case was calculated from the area under the concentration-time curve. The ranges of concentration and dose so obtained are shown in Table 1. Because steady states were reached quickly, the dose always nearly equaled (within 3%) the product of the steady state concentration and the exposure time.
Results Cellular Oxygen Consumption. Figure 1 shows typical O2 consumption data obtained with TK6 and NH32 cells in RPMI-1640 medium containing 10% heatinactivated calf serum. For both cell types, the decline in O2 concentration was linear in time, indicating that the rate of O2 consumption remained constant down to the lowest concentration examined (20 µM). Linear regression of such data yielded specific OCRs for both cell types, as summarized in Table 2. The OCR for TK6 cells was 33% higher than for NH32 cells but 18% less than that measured by Mukundan et al. for βTC3 cells (30). The OCR for TK6 cells in media was only slightly higher than that in PBS, indicating that nutrient starvation had a minor effect on these cells during the experimental period of ∼10 min. This is in contrast to some cell lines, such as βHC9 cells, whose OCR in DMEM media was 3-fold higher than that in PBS (31). Another factor that might affect the OCR measurement is the cell number density. To reduce the noise in the O2 measurement system to an acceptable level, relatively high cell densities were needed. However, Jorjani and Ozturk found that the OCR of various cell lines was not affected when cell densities were varied between 1 and 20 million cells/mL (32), which encompasses the range used in our
Figure 2. NO concentrations in the absence and presence of cells: (a) continuous delivery of 10% NO gas (via a 7 cm loop of silastic tubing) into PBS or TK6 cell suspensions, starting at t ) 0; (b) bolus addition of a saturated NO solution into PBS or into TK6 or NH32 cell suspensions at t ) 0. The suspending fluid in each case was PBS.
experiments. Thus, the OCR values in Table 2 should be valid also for the lower cell densities used in the NO exposure experiments. The effects of cellular O2 consumption on NO exposure were found to be moderate. Neglecting it entirely decreased the calculated, steady state NO concentration by
