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Abstract |
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Abstract
Introduction
Materials & Methods
Results
Discussion
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Disulfiram (Antabuse) (DSF) has been reported to protect rats and other animals from the effects of hyperbaric hyperoxia at 4 to 6 ATA (atmospheres). In contrast, DSF and diethyldithiocarbamate (DDC), its metabolite, accelerate the toxic effects in rats of 100% oxygen at 1 to 2 ATA. We have examined the effects of DSF and DDC on glutathione (GSH) levels in bovine pulmonary artery endothelial cells and Chinese hamster ovary cells. Increases in intracellular GSH occurred 8 to 24 h after addition of DSF to the culture media. These increases in intracellular GSH were associated with increases in the rate of uptake of cystine into the cells. DDC was a less effective inducer of cystine uptake and increased intracellular GSH levels than was DSF. At the concentrations used, neither DDC nor DSF caused significant decreases in intracellular superoxide dismutase levels. Exogenous sulfhydryl compounds including GSH and cysteine partially blocked the induction of cystine transport by DSF or DDC, suggesting that the induction might be mediated through a sulfhydryl reaction between DSF and some cellular components. The increases in GSH in the cultured cells were not significant by 4 h of exposure. In contrast, other stress proteins including heme oxygenase are induced by 2 to 4 h after DSF addition. In previously reported in vivo studies, DSF treatment protected against hyperbaric oxygen damage after as little as 1 to 4 h pre-exposure. This suggests that effects of DSF exposure other than GSH augmentation may be responsible for the protective effects seen in vivo.
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Introduction |
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Abstract
Introduction
Materials & Methods
Results
Discussion
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Disulfiram (Antabuse) (DSF) has varied metabolic effects which complicate its use in alcohol aversion therapy. We have previously demonstrated that both DSF and its reduction product diethyldithiocarbamate (DDC) significantly reduce survival times of adult rats exposed to normobaric hyperoxia (1, 2). Forman and co-workers (3) found that DSF and DDC also increased oxygen toxicity at 2 atmosphere (ATA). Frank and colleagues (4) had previously reported that DDC, a copper chelator and inhibitor of superoxide dismutase (SOD), significantly reduced survival of neonatal rats and adult animals exposed to hyperoxia. In contrast, other investigators reported that DSF significantly protected rats, mice, or dogs against hyperbaric hyperoxia if pressures were maintained at 4 to 6 ATA (5). In rats, DSF protected against both lung damage and damage to the central nervous system (7). Interestingly, these effects were similar to those reported for intraperitoneal injection of glutathione (GSH) which protected against hyperbaric hyperoxia (10), but had no protective effect on normobaric hyperoxia (Jenkinson and Lawrence, unpublished).
Although the mechanism for the protective effects of
DSF against hyperbaric hyperoxia is not yet understood,
we can speculate about the possible chemical reactions of
DSF which could lead to the increased toxicity in combination with normobaric hyperoxia. DSF is potentially toxic
through at least two mechanisms. The compound is a
strong oxidizer of protein sulfhydryls and can also oxidize
GSH to form glutathione disulfide, either directly or through
a mixed disulfide reaction (11). This is the reported basis of its effect on aldehyde dehydrogenase in vitro (14). Alternately, DSF is reduced in vivo to DDC (Figure 1).
This reduction can be catalyzed by GSH reductase (11).
DDC can then act as a copper chelator and inhibit copper-containing enzymes such as SOD (15, 16). This is a proposed mechanism for the acceleration of oxygen toxicity in
rats by DDC (2). DSF has now been identified as one of
a number of sulfhydryl reactive compounds that induce "stress" proteins in cultured cells (17). Subsequently, it
has been noted that there is a close correspondence between treatments which induce oxidant stress proteins and
those which induce a transport protein (designated x
c)
specific for cystine and glutamate, precursor amino acids
needed for GSH synthesis (21). We have examined the
effects of DSF and DDC on pulmonary endothelial cells
and Chinese hamster ovary (CHO) cells to determine what
effect these agents have on intracellular GSH levels and
cystine transport.
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Materials and Methods |
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Materials & Methods
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Chemical Reagents
L-[2,3-3H]glutamic acid, L-[2,3-3H]aspartic acid, and L-[4,5- 3H]leucine were obtained from DuPont-New England Nuclear (Boston, MA). L-[2,3-3H]cystine and L-[35S]methionine were obtained from Amersham (Arlington Heights, IL). Dulbecco's phosphate-buffered saline (PBS), RPMI-1640, and trypsin-EDTA were purchased from Gibco-BRL (Grand Island, NY). Fetal calf serum and substrates for the GSH assays were purchased from Sigma Chemical (St. Louis, MO). Reagents for preparation of polyacrylamide gels and molecular weight standards were purchased from BioRad Laboratories (Melville, NY).
Cells and Media
All experiments were carried out using fourth to sixth passage bovine pulmonary artery endothelial cells (BPAEC) obtained as previously described (25), or CHO cells obtained from the American Type Culture Collection (Rockville, MD). Cells were grown in RPMI-1640 supplemented with 10% fetal calf serum, penicillin, streptomycin, and amphotericin B as previously reported. The medium was changed every 48 h and before the start of any experimental procedure. Exposures to DSF or DDC were begun after cells reached confluence (> 1.5 × 106/35 mm dish), usually 4 to 5 days after plating. Once DSF or DDC was added to the media, it remained on the cells until uptake measurements or GSH measurements were begun.
Uptake Experiments
To measure uptakes of cystine and other amino acids, cells were washed twice with Dulbecco's PBS supplemented with 14 mM glucose, and preincubated for 60 min with 2 ml PBS + glucose at 37°C. The cells were then washed twice and incubated in PBS + glucose containing 0.06 mM of the radioactive amino acid of the desired specific activity. In experiments to assess sodium dependence of uptake, LiCl was substituted for NaCl and Tris buffer for sodium phosphate for prewashes and during uptake, as previously described (26, 27). We have previously shown that cystine uptake in endothelial cells is linear for 30 min under these conditions (27). Cells were incubated for 10 min, followed by 4 washes with ice-cold PBS. The cells were then dissolved in 1% Triton X-100 and an aliquot counted in Ecolite scintillation fluid. Each experiment was carried out with 4 to 6 replicate plates for each experimental parameter and repeated at least twice on separate cell preparations.
Cell Counts and Assays
Cell counts were performed on aliquots of the samples to be used for GSH measurements (before addition of perchloric acid) using a calibrated Coulter Counter, as described previously (27). Cell sizes were determined with a Coulter Channelizer. For GSH measurements, an aliquot of the cells was treated with 10% perchloric acid, sonicated, centrifuged, and immediately frozen for later GSH assay by the method of Tietze (28) as modified by Akerboom and Sies (29). SOD assays were performed using the method of McCord and Fridovich (30) as modified by Crapo and associates (31), with one unit of SOD defined as the amount necessary to inhibit by 50% the reduction of cytochrome c by xanthine oxidase under the defined conditions. Toxicity was determined by counting adherent and nonadherent cells and by trypan blue exclusion.
Preparation for Electrophoresis of Stress Proteins
Cells and cell fractions were analyzed by sodium dodecyl sulfate (SDS) gel electrophoresis essentially as described by Zimmerman and colleagues (32). Cells to be used for autoradiography were incubated with inducing agents for 4 h, then [35S]methionine was added for an additional hour. Adherent cells were rinsed with PBS, scraped into PBS, washed twice by centrifugation at 500 × g for 5 min, and resuspended in lysis buffer (0.0625 mM Tris, pH 6.8; 10% glycerol; 3% SDS; and 10% B-mercaptoethanol). Cells were dissolved by boiling for 2 min in lysis buffer before electrophoresis. Electrophoresis was performed on 7 or 10% uniform concentration SDS-polyacrylamide gels (SDS-PAGE) loaded with equal protein or equal counts of [35S]label per lane.
Visualization of Induced Proteins by Autoradiography
After electrophoresis, the gels were stained with Coomassie Brilliant Blue R-250 in 25% methanol, 5% acetic acid, and destained in 25% methanol, 5% acetic acid. The gels were dried and exposed to X-ray film (XAR-5; Eastman Kodak Co., Rochester, NY) for 1 to 7 days.
Western Blots of HO
Antibodies to heme oxygenase (HO) were obtained from StressGen (Vancouver, BC, Canada). Western blot analysis was performed on unstained gels obtained from electrophoresis, as above.
Calculations and Statistics
All uptakes were expressed as picomoles of amino acid per 106 cells, or expressed as percent control value. GSH levels were expressed as nmol per 106 cells. Statistical significance was determined by analysis of variance (ANOVA) with the post hoc Scheffe test for groups with significant differences using ABstat software (Anderson Bell Corporation, Parker, CO) (33).
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CHO Cells
Figure 2 shows the effect of DSF on cystine uptake in CHO cells exposed to 25 µM DSF. No significant difference was seen between control and DSF-treated cells at 4 h after exposure. By 8 h, cystine uptake in DSF-treated cells was 2× control levels. Uptake rates reached a maximum at 16 h and continued elevated 24 h after addition of DSF. Figure 3 shows the relative effects of DSF compared to N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU), an inhibitor of glutathione reductase (34), or diethylmaleate (DEM), a GSH depleting agent (27, 35), on cystine uptake and intracellular GSH levels in CHO cells. DSF was more effective at increasing both cystine uptake and intracellular GSH levels than either BCNU or DEM at twice the concentration.
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Figure 4 shows the comparative effects of DSF and its metabolite DDC on both cystine uptake and intracellular GSH levels in CHO cells. Since intracellular reactions with DSF could result in 1 or 2 moles of DDC per mole of DSF, depending on whether DSF was directly reduced or formed mixed disulfides with proteins (Figure 1), we compared the effects of DSF with both equimolar and twice-molar concentrations of DDC. In both cases, the effect of DSF was significantly greater than that of DDC. Since twice the molar concentration of DSF is the theoretical maximum obtainable DDC level, this suggests that the effect of DDC might be due to oxidation to DSF in vivo, but that the effect of DSF is not likely totally due to intracellular formation of DDC.
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Significantly greater than
1 or 2 molar equivalents DDC.
BPAEC
The effects of DSF on BPAEC are similar to those on
CHO cells. In both cell types, both cystine transport and
GSH levels increased with concentration of DSF (Figures
5a and 5b). The effect of time of incubation of BPAEC
with DSF is shown in Figure 6. Small increases in cystine
uptake and GSH levels, which were not statistically significant, were seen at 4 h; with significant increases seen by
8 h of exposure to 25 µM DSF. We examined the specificity and characteristics of the increased cystine uptake in
these BPAEC. The increase was specific for cystine and
glutamate with no significantly increased uptake of either
leucine or aspartate (Figure 7). The increased uptake was
sodium-independent and protein synthesis-dependent, characteristic of an xc-like system (Tables 1 and 2) (35, 36).
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DSF was also more effective than DDC at stimulating cystine uptake in these cells. Cystine uptake in cells treated with DSF (12.5 µM) was 144% ± 6% control (P < 0.01) compared with 25 µM DDC, 124% ± 10% control (P < 0.05). To examine the possibility that SOD inhibition by DDC was involved in the induction of cystine transport by DSF, we measured SOD levels in control and DDC or DSF-treated cells at 1 h after addition of varying concentrations of DDC and DSF (Table 3). No significant depletion of SOD was seen in the endothelial cells at any of the concentrations of DDC or DSF examined, suggesting that SOD depletion was not likely to be related to the induction of cystine transport.
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In further experiments, we examined the effect of DSF on cystine uptake when co-incubated with either 1 mM cysteine or 1 mM GSH (Table 4). Both cysteine and GSH significantly, but not completely, inhibited the stimulation of cystine uptake seen in DSF-exposed BPAEC.
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We also examined induction of stress protein synthesis by DSF in BPAEC. Cells were exposed to DSF for 2 or 4 h followed by 1 h incubation with [35S]methionine. Proteins were separated by SDS-PAGE (10% gel) and developed by autoradiography. Results are shown in Figure 8. Several bands are induced which may correspond with previously identified stress protein, including bands at 32 kD and approximately 60, 70, 90, and 110 kD. We have identified a 32-kD band as HO by Western blot analysis (see Figure 9). The additional bands may correspond to other known heat shock proteins, e.g., HSP 60, HSP 70, HSP 90, and HSP 110.
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We have demonstrated in these experiments that DSF is an
effective inducer of cystine transport in two different cell
types, and that cystine transport induction is associated
with substantial increases in intracellular levels of GSH. In
general, responses of the two cell types to DSF were similar. The increase in GSH levels in CHO cells was less dramatic than that seen in BPAEC, but the baseline level of
GSH in CHO cells was significantly higher than in BPAEC
before addition of DSF. We saw no statistically significant
increases either in cystine uptake or in GSH levels before
4 h of exposure in CHO cells or endothelial cells. Increases in GSH were rapid between 4 to 8 h, maximal at approximately 16 h after addition, and elevated through 24 h. Levels of cystine uptake were also significantly elevated by 8 h,
maximally elevated by 16 h, and continued high at 24 h in
both cell types. Induction of cystine transport was protein
synthesis-dependent and Na+-independent, with specificity characteristic of an xc-like transport system (Tables 1
and 2) (Figure 7). DSF was more effective than its metabolite DDC in inducing both GSH and cystine transport increases, suggesting that the induction of cystine transport
was primarily due to effects of DSF rather than a secondary effect of its metabolism to DDC (Figure 4). Inhibition of SOD by DDC or DSF was apparently not involved in
the induction of the cystine transport, since DSF or DDC
at the levels used had no significant effect on total cell
SOD (Table 3).
The effects of DSF on cystine transport were apparently sulfhydryl-related, since co-incubation with GSH or cysteine partially prevented the induction of cystine transport by DSF (Table 4). Cysteine and GSH may act by protecting cellular reduced sulfhydryls from undergoing mixed disulfide reaction with DSF. Alternatively, some mixed disulfide reactions may occur between either GSH or cysteine and DSF, lowering the effective concentration of DSF. Kitson has reported that a 200- to 500-fold excess of GSH was necessary to produce a 50% reduction of the inhibitory effect of DSF on aldehyde dehydrogenase in a cell free system (13). In our cells, a 40-fold excess of GSH is sufficient to prevent 65% of the induction of cystine transport by DSF. This implies that the reactivity of DSF with its target (most likely a protein sulfhydryl) is stronger than any reactivity with GSH. Similar arguments can be made for the effect of cysteine.
In summary, DSF induces increases in the sodium-independent uptake of cystine into both CHO cells and BPAEC.
We have shown that the mechanism for induction of cystine transport in these cells is likely to involve the direct
action of DSF on the cells rather than the effect of the metabolite DDC; and that the reactivity of DSF as a sulfhydryl agent, rather than the effect of DDC as a copper chelator and SOD inhibitor, is the more probable mechanism for the induction of the xc-related cystine transport system.
This increase is dependent on de novo protein synthesis,
blocked by sulfhydryl increases, and most likely represents
a specific upregulation of the cystine transport system designated xc.
The gene for this transporter has not yet been identified, so specific information about oxidant-sensitive promoter elements is not yet available. Various transcriptional activators have been identified that bind to specific
consensus sequences in promoter regions of target genes.
Some of these factors, including AP-1 and NF
-B, have
been shown to be sensitive to the redox state of the cell,
and to be modulated by thiol reagents (37, 38). The human
HO promoter region has been reported to contain binding sites for both AP-1 and NF-B, and is upregulated by DSF
(39). A possible mechanism for the effect of DSF is that it
may be acting to modify the cellular thiol redox balance
which in turn activates binding of AP-1, NF-B, or as-yet-unknown transcriptional factors, leading to upregulation
of synthesis of the protein or proteins involved in the xc
transport system for cystine, as well as other stress proteins.
If the results obtained in isolated cells are representative of in vivo effects, we must conclude that the induction of cystine transport and resultant increases in GSH levels following DSF exposure are unlikely to be the primary source of the effects of DSF on oxygen tolerance in vivo. In the case of exposure to 4 to 6 ATA oxygen, DSF has been reported to be protective in rats, mice, and dogs (5). However, protection occurred following as little as 1 to 4 h pre-exposure with protection against toxic effects seen as early as 2 to 6 h after the initial dose of DSF. In cultures, however, only minimal increases in GSH were seen in either CHO cells or BPAEC by 4 h of exposure.
In contrast, at lower partial pressures of oxygen (1 or 2 ATA) DSF or DDC pretreatment significantly increased symptoms of oxygen toxicity in vivo (1, 2, 4). Lung damage occurred in 12 to 24 h and resulted in edema by 24 h, approximately 20 h before similar effects were seen in controls. Exposure times of 8 to 24 h resulted in greatly elevated GSH levels in cells in vitro. If these results are extrapolated to the in vivo model, one might anticipate possible protection against oxidant stress in the 12- to 24-h time period. In summary, the time course of induction of cystine transport and GSH increases in cultured cells, if extrapolated to the results seen in rats, does not account for the observed effects of DSF on oxygen toxicity in vivo. Rats are protected against hyperoxic exposure levels that result in damage by 4 h, before GSH levels have increased in culture. In contrast, hyperoxic exposure levels that do not cause damage in rats until 24 h, are exacerbated by pre-exposure to disulfiram or DDC, even though GSH levels in cultured cells are elevated by these times of exposure.
Caution, of course, should be used in proposing in vivo mechanisms based on studies in cell culture model systems. The in vivo effects of pharmacologic agents are complicated by factors including removal of the drug by metabolism in the liver or kidneys, or differential transport across either the blood-brain barrier or the pulmonary endothelium. Nevertheless, there seems to be little support for the hypothesis that changes in GSH levels induced by DSF have any relationship to the effect of DSF on the responses of animals to elevated O2 levels. Alternate mechanisms for the effects of DSF on oxygen toxicity and tolerance in vivo need to be considered.
DSF has previously been reported to induce a number of "stress" proteins in aortic endothelial cells and chick fibroblasts (17). The appearance of these proteins has generally been observed by 2 to 4 h after DSF exposure. As we have observed, DSF does induce stress proteins in BPAEC, and the 32-kD protein induced between 2 and 4 h of exposure is HO, which has been reported to have antioxidant properties (40). It is possible that HO or another of these "stress" proteins may be involved in the protection against hyperbaric hyperoxia-induced seizure and lung damage conferred by DSF treatment. In addition, DSF has also been reported to inhibit enzymatic and nonenzymatic lipid peroxidation (41) and to protect against postischemic cell death in the liver. Thus DSF could be acting directly as a radical scavenger during hyperoxic exposure rather than as an inducer of GSH. Our data also support our previous in vivo observations that GSH depletion is not likely to be the direct cause of the toxic effects of the combination of DSF and 1 to 2 ATA oxygen (2). These effects may be related to reactions of DDC with SOD or to the effects of DSF directly on protein sulfhydryl groups, which could affect various other oxidative enzymes such as mixed function oxidases or xanthine oxidase (46, 47).
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Footnotes |
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Address correspondence to: Susan M. Deneke, Ph.D., The University of Texas Health Science Center at San Antonio, Dept. of Medicine, Div. of Pulmonary Diseases/Critical Care Medicine, 7703 Floyd Curl Dr., San Antonio, TX 78284-7885.
(Received in original form September 3, 1996 and in revised form December 30, 1996).
Acknowledgments: This work was supported by National Heart, Lung, and Blood Institute Grant HL-32824 and by the Medical Research Service, U.S. Department of Veterans Affairs. The authors thank Charnae Williams for excellent technical assistance and Janis Kay Marsh for help in manuscript preparation.
Abbreviations ATA, atmosphere; BCNU, N,N'-bis(2-chloroethyl)-N-nitrosourea; BPAEC, bovine pulmonary artery endothelial cells; CHO, Chinese hamster ovary; DDC, diethyldithiocarbamate; DEM, diethylmaleate; DSF, disulfiram; GSH, glutathione; HO, heme oxygenase; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gels; SOD, superoxide dismutase.
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