Exogenous glutathione protects endothelial from menadione toxicity MINYUEN
CHANG,
MING
SHI, AND HENRY
cells
JAY FORMAN
Cell Biology Group and Division of Neonatology and Pediatric Pulmonology of Childrens Hospital Los Angeles, and Departments of Pediatrics, Pathology, and Molecular Pharmacology and Toxicology, University of Southern California, Childrens Hospital Los Angeles, Los Angeles, California 90027 Chang, Minyuen,
Ming Shi, and Henry Jay Forman.
conjugation of a wide variety of electrophilic xenobiotics. Under oxidant stress, cellular GSH can be lost through export as GSSG. Low-molecular-weight GSH conjugates can also be actively transported out of cells (20). Some cells, such as hepatocytes, may also secrete GSH as part of interorgan transport of this vital compound. The sources of amino acids for intracellular de novo synthesis of GSH include the following: uptake of precursor amino acids, breakdown of extracellular GSH and uptake via the y-glutamyl cycle, use of amino acids from cellular protein turnover, and conversion of methionine via the cystathionine pathway (20). Uptake of intact GSH has only been demonstrated in some epithelial cells, whereas the cystathionine pathway is found primarily in the liver (14, 20). There is no evidence suggesting that endothelial cells take up intact GSH (11). Resynthesis of GSH intracellularly is therefore important in maintaining endothelial cell GSH levels. As GSH provides the principal vehicle for interorgan transport of cysteine (20) , the ability to utilize extracellular GSH in the y-glutamyl cycle may provide protection against cellular injury under conditions when there is loss of intracellular glutathione. The cleavage of the unique y-glutamylcysteine peptide bond of GSH is catalyzed by y-glutamyl transpeptidase, a membranebound enzyme with its active site directed toward the outside of the cell. Cysteinylglycine, a product, is metabENDOTHELIAL CELLS have been shown to be a prime target for oxidant injury (10). Oxidant injury to cells and olized by a dipeptidase, and the free amino acids are moiety is tissues is induced by reactive oxygen metabolites, such transported inside the cells. The y-glutamyl either transferred to another amino acid (or a peptide) as superoxide anion, hydrogen peroxide, and hydroxyl forming a y-glutamyl peptide or released as free radical. The extent of oxidant injury involves chemical peptides can be transported into alterations in proteins, lipids, carbohydrates, and glutamate. y-Glutamyl cells or hydrolyzed to glutamate and other amino acids. nucleic acids. Quinones that undergo redox cycling have been widely used to investigate oxidant-induced stress These amino acids can then be used for synthesis of in cells. The quinone is reduced to its semiquinone rad- GSH (20, 21). Recent studies (8, 9, 13, 18, 28, 36, 38, 39, 41) have ical through a one-electron reduction catalyzed by celshown the effectiveness of exogenous GSH in preventing lular reductases. In the presence of oxygen, the semihyperbaric quinone radical undergoes rapid autoxidation with the injury, such as in ischemia reperfusion, pulmonary fibrosis, where formation of superoxide radical and regeneration of the hyperoxia, and idiopathic oxidant stress is the purported mechanism. In this parent quinone (32). Dismutation of superoxide radical study, we have investigated the ability of endothelial results in the formation of HZ02. cells to utilize exogenous GSH for protection against The tripeptide glutathione (y-glutamyl-cysteine-gly(vitamin K,; tine; GSH) is an important cellular antioxidant. As a oxidant injury generated by menadione 2-methyl-1,4-naphthoquinone, MQ). Our data suggest substrate for glutathione peroxidase, GSH provides cells are able to utilize extracellular reducing equivalents for the metabolism of H202 and that endothelial GSH for protection against oxidant stress and that the lipid hydroperoxides. The product, oxidized glutathione to use GSH is mediated by y-glutamyl disulfide (GSSG), formed in this reaction is reduced to ability GSH by the action of glutathione reductase at the transpeptidase. GSH also conjugates with MQ via a expense of reduced nicotinamide adenine dinucleotide nonenzymatic reaction. The resulting menadione-glutathione conjugate (MQSG) may have greater capacity phosphate (NADPH). Among its other roles, GSH serves as a reservoir for cysteine and is used in the to redox cycle than the parent quinone (7, 30, 40).
Exogenous glutathione protects endothelial cells from menadione toxicity. Am. J. Physiol. (Lung Cell. Mol. Physiol. 6): L637L643, 1992.~-Administration of menadione (vitamin K,; 2-methyl-1,4-naphthoquinone) to cultured bovine pulmonary artery endothelial cells caused a dose- and time-dependent depletion of cellular ATP and depletion of intracellular glutathione (GSH). The toxicity of menadione was correlated with an increase of menadione-glutathione conjugate in the medium and with an increase in H202 generation. Recent studies have suggested that GSH may be useful as a pharmacological agent to prevent oxidant injury. In the present study, treatment with exogenous GSH prevented the loss of cellular GSH and ATP caused by menadione. Protection by extracellular GSH involved two mechanisms. In one mechanism, extracellular GSH was degraded by y-glutamyl transpeptidase, producing substrates for subsequent intracellular de novo GSH synthesis. We found that the protective effect of extracellular GSH was decreased by inhibiting y-glutamyl transpeptidase. In the other mechanism, GSH reacted in the medium with menadione to form a conjugate. Although formation of the menadione-glutathione conjugate within cells may contribute to menadione toxicity, addition of menadione-glutathione conjugate to the medium was found to be nontoxic to endothelial cells. Thus exogenous GSH protected endothelial cells by two mechanisms: maintenance of intracellular GSH and prevention of menadione entrance into cells. oxidant injury; y-glutamyl transpeptidase; glutathione conjugation
1040-0605/92 $2.00 Copyright
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Physiological
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L637
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Although MQSG would therefore be expected to be toxic within the cells, we demonstrated that extracellular MQSG was not toxic to bovine pulmonary artery endothelial cells (BPAEC), suggesting that part of the protective effect of extracellular GSH may be due to the prevention of MQ accessibility to intracellular reductases. MATERIALS
AND
CELLS
FROM 0.4
MENADIONE 1
’
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METHODS
Chemicals. MQ, reduced and oxidized glutathione, NADPH, glutathione reductase, L-serine, sodium borate, ferrous ammonium sulfate, potassium thiocyanate, 5,5’-dithiobis-(Z-nitrobenzoic acid) (DTNB), and dimethyl sulfoxide (DMSO) were obtained from Sigma Chemical. Fluorescein isothiocyanate (FITC) -conjugated antibody against human factor VIII was obtained from Atlantic Antibodies. Angiotensin-converting-enzyme reagents and calibrator were from Sigma Chemical. BPAEC. Passage 16 of BPAEC was obtained from the American Type Culture Collection (CCL 209). Cells were maintained in minimal essential media (MEM) supplemented with fetal bovine serum (100 ml FBS to 500 ml MEM, 2 mM L-glutamine, 100 U/ml penicillin, 100 fig/ml streptomycin, and 2 x 10m7 M of selenomethionine, which was included to prevent a decrease in glutathione peroxidase-specific activity due to selenium depletion that can occur as endothelial cells divide (16). MEM was from Irvine Scientific. Glutamine and penicillin-streptomycin solutions were from GIBCO. FBS was from Gemini Bioproducts, Calabasas, CA. Serum from the same lot was used for all experiments. Cells were grown in a humidified incubator at 37°C with 5% CO,. Subculture was done in a weekly ratio of 1:2-I:5 using a mechanical method described by Ryan and MMxwell (27). Cell monolayers generally reached confluence on plastic tissue culture plates (Costar) in 3-5 days. Cells from passages 18-25 were used for experiments as cells above passage 25 showed some features of senescence such as slower growth and formation of ring structures similar to vasculature formation. The monolayers used in the present study demonstrated, by phase-contrast microscopy, contact inhibition and a cobblestone appearance typical of endothelial cells. They also expressed factor VIII antigen as demonstrated by direct immunofluorescent antibody staining. These cells were also positive for angiotensin-converting enzyme activity (15). All experiments were done l-2 days after the monolayers reached confluence. MQ treatment. MQ was dissolved in DMSO and subsequently diluted in Dulbecco’s phosphate-buffered saline (DPBS, GIBCO) plus 5 mM glucose or Earl’s balanced salt solution (EBSS; GIBCO). The DMSO content in each sample was no more than 0.1%. DMSO was added to the controls at the same concentrations as in MQ-treated groups. For the inhibition of y-glutamyl transpeptidase with serine-borate complex, 20 mM borate was present in all groups. One millimolar L-serine was added to the serine-borate treated groups. Synthesis of MQSG. MQSG was synthesized according to Nickerson et al. (22). MQ (0.1 M in ethanol, 50 ml) was mixed with GSH (0.025 M in water, IO ml) at 4°C overnight in the dark. The yellow-orange crystals that formed were collected and washed twice with chloroform to dissolve excess MQ. The precipitate was filtered and dried. Analysis of the synthetic product was done with the use of high-performance liquid chromatography with electrochemical detection. The solvent system used was 65% propanol-35% H20, and the applied potential was -40 mV. The purity of the conjugate was estimated to be 98%. The spectrum of MQSG was examined in a Beckman DU-7 spectrophotometer and compared with that of MQ. A marked difference in their absorbance was found at 430 nm (Fig. I). MQSG conjugate formation in the media. BPAEC were grown
300
350
400
Wavelength
450
500
(nm)
Fig. 1. Comparison of absorbance spectra of 100 PM 2-methyl-1,4-naphthoquinone (MQ) and 100 PM menadione-glutathione conjugate (MQSG) in phosphate-buffered saline without glucose.
to confluence in 12-well culture plates. Cells were washed twice with DPBS-5 mM glucose and incubated in DPBS-5 mM glucose containing 100 PM MQ or DMSO for 0,20,30, and 60 min. The media were taken for measurement of absorbance at 430 nm. The reading obtained from controls not treated with MQ served as baseline and was subtracted to eliminate interference from other products released from the cells. Known concentrations of MQSG in DPBS-5 mM glucose were used for calibration. ATP measurement. Cellular ATP levels were determined using a bioluminescent somatic cell ATP assay kit obtained from Sigma Chemical. At the end of each incubation, media were removed, and 0.4 ml of somatic cell ATP releasing agent (SCRR) was added to each well for 2 min. The supernatant was transferred to test tubes and kept on ice for ATP measurement within 3 min. ATP was determined by the amount of light emitted after the reaction of luciferin and ATP catalyzed by firefly luciferase and measured by the phosphorescent mode of the Perkin-Elmer LS-5 fluorescence spectrophotometer. HZ02 assay. HZ02 in media was measured by the calorimetric method of Thurman (33). The absorbance of ferric thiocyanate complex formed from the reaction of HZ02, ferrous ammonium sulfate, and potassium thiocyanate was read at 480 nm. The readings were compared with H,O, standards. The specificity of this method was demonstrated by the decrease of H,O, with the addition of catalase. Total cellular GSH measurement. Total cellular GSH (GSH + 2 x GSSG) was measured by the Tietze recycling method (34) modified by Akerboom (1) using DTNB, glutathione reductase, and NADPH. Endothelial monolayers were washed four times after each incubation with DPBS-5 mM glucose. Elimination of residual extracellular GSH was assured by measuring the total GSH content of the washes, which was negligible in the third wash. 4.3% Sulfosalicylic acid was added to the monolayers and neutralized with 5 mM KOH. Cellular protein was measured with bicinchoninic acid by a modified Lowry method (Pierce, Rockford, IL). Statistics. Data were analyzed using the Crunch interactive statistical package (Crunch Software, San Francisco, CA). Analysis of variance was done in all treatment groups, followed by post hoc tests to determine the difference of each paired group. Differences were considered significant at P < 0.05.
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GLUTATHIONE
PROTECTS
ENDOTHELIAL
RESULTS
Menadione
toxicity
in endothelial
cells. Loss of ATP is
generally used as an indicator of depressed metabolism and is a critical event occurring before cell death (37). Our data showed that MQ caused a dose- and time-dependent decrease in cellular ATP in BPAEC monolayers (Fig. 2). Significant ATP depletion occurred after incubation with 25, 50, and 100 PM of MQ for 2 h. Only 100 PM of MQ caused significant ATP depletion by the end of 1 h of incubation. Morphological changes, including contraction, rounding, and detachment of endothelial cells from culture dishes, were observed by phase-contrast microscopy in correlation with the loss of cellular ATP at the end of incubation for 2 h. The loss of cellular ATP also correlated with the loss of ability to exclude trypan blue dye (data not shown). MQ also caused time-dependent depletion of cellular GSH (Fig. 3). The decrease in GSH preceded loss of ATP at each concentration of MQ (compare Figs. 1 and 2). As shown in hepatocytes, MQ can deplete GSH by direct conjugation with GSH and, indirectly, through the generation of H20z (12). MQ has also been reported to induce production of superoxide and H202 in endothelial cells (24). The conjugate formed in hepatocytes is rapidly excreted. These effects of MQ were confirmed in this study by observing the increase of MQSG and HzOz in the media with addition of 100 PM MQ (Fig. 4 and Fig. 5). Effects of exogenous GSH supplementation on menadione toxicity. Addition of 2 mM GSH to the EBSS-0.1%
bovine serum albumin (BSA) solution provided protection from cellular ATP depletion in BPAEC subsequently treated with 100 PM of MQ (Table 1). The loss of intracellular GSH caused by MQ was also prevented by exogenous GSH (Fig. 6). Exogenous GSH also caused an increase of intracellular GSH levels in BPAEC regardless of the presence of MQ. The data suggest that exogenous GSH can protect BPAEC from MQ toxicity by providing z 3
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Fig. 4. Comparison of cellular GSH loss (indicated as GSH equivalents) and the appearance of MQSG conjugates outside the cells. BPAEC monolayers were grown to confluence on 12-well plates. Cells were treated with 100 PM of MQ in DPBS/5 mM glucose for 60 min. At times indicated, media were removed for MQSG estimations as described in MATERIALS AND METHODS. Control cells without MQ treatment did not show significant changes in cellular GSH levels (data omitted). Values are means t SE; n =4 experiments.
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Fig. 3. Cellular glutathione (GSH) depletion (indicated as GSH equivalents) by 0, 25, 50, and 100 PM of MQ. BPAEC monolayers were cultured on 12-well plates to confluence. Cells were treated with MQ in DPBS/5 mM glucose for 2 h. At the times indicated, monolayers were treated with 4.3% sulfosalicylic acid and processed subsequently as described in MATERIALS AND METHODS for GSH measurements. At confluence, 1 x lo6 BPAEC contained 105 t 10 pg protein. Values are means k SE of 2 experiments. Significant GSH loss occurred after 30 min (P < 0.05). There was no difference in GSH levels among MQtreated groups.
15
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MENADIONE
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Fig. 2. ATP depletion by incubating bovine pulmonary artery endothelial cells (BPAEC) monolayers on 24-well culture plates with 0, 25, 50, and 100 PM of MQ in Dulbecco’s phosphate-buffered saline (DPBS)-5 mM glucose over 2 h. At times indicated, cellular ATP was released by somatic cell releasing agent (SCRR) and measured immediately. Each well contained 3.4 k 0.1 x lo5 cells. Values are means t SE; n = 4. At 1 h of incubation, only cells treated with 100 PM of MQ showed significant ATP depletion (P = 0.01). All cells treated with MQ showed significant ATP depletion by the end of 2 h (P = 0.01).
of cellular GSH.
Mechanisms by which exogenous GSH protects from menadione toxicity. Two different mechanisms could
account for the protection of MQ-exposed cells by extracellular GSH: 1) prevention of MQ entrance into cells through formation of a nonpermeable conjugate, and 2) maintenance of intracellular GSH through the Y-glutamvl cvcle. ’ ”To investigate the effect of conjugate formation outside the cells, we compared the toxicity of exogenous MQSG and MQ with BPAEC. In contrast with MQ, MQSG did not cause significant ATP depletion even at 500 PM (Fig.
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L640
GLUTATHIONE
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MENADIONE
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Fig. 5. Production of Hz02 by MQ-exposed endothelial cells. BPAEC monolayers on T-25 flasks at confluence were incubated with 0,25, and 100 PM MQ in 1.6 ml DPBS-5 mM glucose over 60 min. At times indicated, 0.4 ml 50% trichloroacetic acid was added, and media were taken to measure Hz02. MQ caused a dose-dependent increase in Hz02 production in BPAEC over time. Values are means t SE; n = 2-4.
Table 1. Effect of exogenous GSH supplementation on
(PM)
Fig. 6. GSH supplementation prevented GSH equivalents) from MQ treatment. BPAEC were to confluence. Cells were preincubated in Earl’s (EBSS)-0.1% BSA with or without 2 mM GSH at 0, 50, or 100 PM and incubation with GSH Monolayers were washed with EBSS 4 times, were measured as in MATERIALS AND METHODS. n= 3.
loss (indicated as GSH cultured on 6-well plates balanced salt solution for 1 h. MQ was added was continued for 2 h. and cellular GSH levels Values are means t SE;
BPAEC cellular ATP depletion induced by MQ Treatment
ATP,
pg/well
P vs. Control
P vs. +GSH
Control 0.24kO.02 NS* O.O1tO.OO 0.01 0.01 +lOO ,uM MQ 0.28t0.06 NS +2 mM GSH +2 mM GSH + 100 ,uM MQ 0.22kO.03 NS NS Values are means t SE for n = 4 experiments. Confluent bovine pulmonary artery endothelial cells (BPAEC’s) on 24-well plates were incubated in Earl’s balanced salt solution-0.1% bovine serum albumin containing 0 or 2 mM glutathione (GSH) for 1 h, followed by addition of 0 or 100 PM MQ. Cellular ATP levels were measured after an additional 2 h of incubation. MQ, 2-met/&1,4-naphthoquinone; NS, not significant.
0.00
50
7). As this suggested a potential
mechanism for protection by extracellular GSH, the rate of extracellular conjugation of MQ by GSH was also examined by monitoring the increase in absorbance at 430 nm in the absence of cells over a 2-h period. The reaction rate slowed down after -30 min (Fig. 8). These data suggest that exogenous GSH can contribute to the protection of cells by consuming MQ, thereby limiting availability of unconjugated MQ to cells. Nevertheless, as extracellular conjugate formation was not instantaneous (- 17% of MQ was still in the unconjugated form at 60 min), MQ toxicity could not be completely prevented by this mechanism. The possibility that extracellular GSH was used by endothelial cells for maintenance of cellular GSH levels through the action of y-glutamyl transpeptidase was examined by inhibiting this enzyme with the competitive inhibitor serine-borate complex (31). In the presence of serine-borate, GSH content of the medium remained unchanged for 24 h (2.07 t 0.06 pmol), whereas in the absence of serine borate, total GSH content of the medium declined 17.4% (359 t 80 nmol) in 24 h (n = 10) (P c 0.005 vs. inhibited). Serine-borate alone had no effect on ATP content of endothelial cells (Fig. 9). The protective effect of exogenous GSH was decreased in the presence of serine-borate, whereas the presence of serineborate in the absence of extracellular GSH did not affect
Dose
100 (MM)
Fig. 7. Comparison of toxicity of MQ and MQSG. Various doses of MQ and MQSG were added to confluent BPAEC monolayers on 24well culture plates containing EBSS and incubation carried for 2 h. Values are means t SE; n =4-8.
the toxicity of MQ (Fig. 9). The increase in cellular GSH levels by exogenous GSH was also markedly decreased with serine-borate in the presence or absence of MQ (Fig. 10). Serine-borate had no effect upon the rate of MQSG formation in the medium (Fig. 8). These data suggest that part of the protective effect of exogenous GSH was mediated by the action of y-glutamyl transpeptidase. DISCUSSION
MQ and other vitamin K3 analogues have been evaluated as anticancer drugs for the last three decades. Clinical trials of their chemosensitizing effects have also been investigated (29). The cytotoxicity of MQ is related to its ability to generate reactive oxygen species and to deplete cellular glutathione. The depletion of cellular GSH can be due to both oxidation and conjugation. Because the conjugation reaction between MQ and GSH does not disturb the quinone nucleus, the resulting MQSG retains the capacity to redox cycle (7,30). The reported redox potentials of MQ and of MQSG are similar [E (Q/Q-) values,
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GLUTATHIONE
PROTECTS
ENDOTHELIAL
CELLS
FROM
L641
MENADIONE
El
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control
80 -
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20
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Minutes Fig. 8. Rate of MQSG formation was monitored by reading increase in absorbance at 430 nm over 2 h after adding 2 mM GSH to 1 ml PBS containing 100 PM of MQ at 37°C. From Fig. 1, we determined that the millimolar extinction coefficient of MQSG at 430 nm was 1.29. Serineborate had no effect on rate of conjugate formation in medium. One of four superimposable determinations is shown.
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100 [Menadione]
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Fig. 9. Effect of serine-borate complex on GSH protection from MQinduced ATP loss. Confluent BPAEC monolayers on 24-well plates were preincubated with 2 mM GSH for 1 h in a solution of 20% Krebs-Ringer borate (Krebs-Ringer phosphate solution with substitution of 100 mM borate for phosphate), 80% EBSS-0.1% BSA, pH 7.4. 1 mM L-serine was added to the serine-borate groups. MQ was added at 100 PM after GSH pretreatment. Cellular ATP levels were measured after a 2-h incubation. Values are means ~fr SE; n = 9. There was no significant difference of ATP levels in groups without MQ treatment. In MQtreated groups, GSH group is significantly different from other 3 groups (P = 0.01).
-203 mV and -192 mV, respectively] (6). Wefers and Sies (40) reported that the rate of redox cycling observed with MQSG was greater than that seen with the parent quinone when incubated with a hepatic microsomal fraction; however, exogenous MQSG was not toxic to endothelial cells (Fig. 7). The MQSG formed extracellularly was apparently impermeable to endothelial cells as MQSG formed inside the cells would be at least as toxic as MQ itself. MQSG was also found by Brown et al. (5) to
(wM)
Fig. 10. Effect of serine-borate complex on GSH prevention of MQ-induced GSH loss (indicated as GSH equivalents). Confluent BPAEC monolayers on 6-well plates were treated as in Fig. 9. Values are means t SE; n = 4-5 in each group, except in groups without serine treatment where n = 2. Presence of serine-borate complex inhibited increase in cellular GSH by extracellular GSH in both control and MQ treated groups (P = 0.03 by Dunnett’s test for GSH vs. GSH + serine in non-M& treated groups, and P = 0.001 for GSH vs. other groups with MQ treatment).
be noncytotoxic to rat renal epithelial cells. These authors speculated that the active y-glutamyl transpeptidase activity of these cells may cleave the y-glutamylcysteinyl bond of MQSG, which would be followed by the formation of a thiazine product resulting from the cyclization of the cysteinylglycine conjugate, thereby destroying the quinone ring of MQSG. Because cultured endothelial cells possess relatively low y-glutamyl transpeptidase activity compared with renal epithelial cells, thiazine formation in this system may be slow. Studies of the relative contribution of conjugation and oxidation to loss of GSH in MQ-induced cytotoxicity have been inconclusive (12, 25, 26). Our results suggest that conjugation with MQ accounted for most of the cellular GSH loss (Fig. 4). The amount of MQ and MQSG within the cell would have been constantly changing. From the data in Fig. 8, and assuming that the permeability of MQ, which is lipid soluble, would allow rapid equilibration, we estimate that, at 30 min, the intracellular concentration of MQ would have been -40 PM whereas intracellular MQSG would have been -60 PM minus the MQSG that was excreted (see Fig. 4). At 60 min, the intracellular MQ would have been only - 17 PM with perhaps most of the MQSG formed intracellularly having been excreted; however, at 60 min, the total intracellular GSH content was decreased by 76%. The nonlinear concentration dependence of MQ toxicity probably reflected the multiple components of the mechanism of its toxicity; loss of GSH through conjugation with MQ, Hz02 production through redox cycling of MQ and MQSG, oxidation of GSH through the action of gluathione peroxidase, and formation of protein-GSH conjugates, which is dependent upon GSSG production (19). As a significant elevationof H202 could be detected in the medium (Fig. 5), the net generation of HZOB by the cells was clearly elevated beyond the capacity to remove it.
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Wherease excretion of GSH-xenobiotic conjugates and GSSG has been shown to be an ATP-dependent process in red blood cells (3, 17), the appearance in the media of MQSG prior to the loss of ATP suggests that conjugate excretion per se did not cause significant ATP depletion (Figs. 2 and 4). Thus MQ-induced ATP depletion in BPAEC was most likely the result of HZOz toxicity that was exacerbated by loss of GSH. Various methods have been used to increase intracellular GSH, such as treatment with L-2-oxothiazolidine-4 carboxylate (an effective intracellular cysteine delivery agent) (35), GSH ~monoethyl ester (which enters the cells freely) (36), and GSH supplementation. Tsan et al. (36) reported that treatment with exogenous GSH elevated the cellular GSH level in endothelial cells and that this effect was inhibited by the addition of acivicin (an irreversible y-glutamyl transpeptidase inhibitor), which is consistent with our observations. Because we omitted GSH precursor amino acids in the present studies, the source contributing to the increase of cellular GSH in BPAEC was likely from the utilization of extracellular GSH. The essential role of y-glutamyl transpeptidase in utilization of extracellular GSH in protecting alveolar macrophages from oxidant injury was also demonstrated previously by our laboratory (13). Suppression of the respiratory burst in alveolar macrophages by hyperoxia was prevented by the presence of extracellular GSH. This protective effect was inhibited by the presence of serineborate which completely abolished extracellular GSH degradation under the conditions used herein. Recently, Peacock et al. (23) also reported that GSH supplementation protected rats from hyperbaric oxygen exposure and that this effect was inhibited by a single dose of acivicin. In the present study of MQ toxicity, conjugation by GSH in the medium appeared to be the principal mechanism for protection by extracellular GSH. Nevertheless, the conjugation was not instantaneous, requiring >60 min to remove all free MQ in the absence of cells (Fig. 8). MQ that escaped conjugation entered the cell, producing toxicity. The effects of MQ in the presence of serineborate (Figs. 9 and 10) indicated that the activity of y-glutamyl transpeptidase was also critical in allowing extracellular GSH to maintain cellular GSH and prevent loss of ATP. Serine-borate prevented the elevation in GSH observed in the presence of exogenous GSH (in either the presence or absence of MQ) (Fig. 10). The relatively higher intracellular GSH observed in the presence versus the absence of exogenous GSH in the presence of both MQ and serine-borate (Fig. 10, 2 rightmost bars) was probably due to protection by conjugate formation in the medium that limited the amount of intracellular MQSG formation and H,OZ production rather than a result of bypassing of the serine-borate block. In the absence of serine-borate, exogenous GSH could supply the materials needed for de novo GSH synthesis (Fig. 10) that helped prevent loss of ATP (Fig. 9). Exogenous GSH has been shown in several recent studies to prevent oxidant injury (4,8,9,13,18,28,38,39,41). The protection may be due to multiple mechanisms; in addition to its potential for conjugation of xenobiotics, extracellular GSH may provide a supply of cysteine to
CELLS
FROM
MENADIONE
cells through disulfide exchange with cystine and may stabilize the plasma membrane by reducing oxidized membrane protein moieties. Nevertheless, under normal physiological conditions, extracellular GSH provides the essential cysteine for de novo GSH synthesis within cells through the y-glutamyl cycle (2). When challenged with either an oxidant stress and/or GSH depleting agent, use of exogenous GSH to prevent oxidant injury may have significant value. The authors thank Dr. Enrique Cadenas for help in analysis of the menadione-glutathione conjugate and Dr. Timothy W. Robison for help in using the Crunch statistical program for data analysis. We also thank Dr. Peter J. Del Vecchio for providing endothelial cells and Dr. Henry Choy for his careful criticism on this manuscript. This work was supported by grants from the National Heart, Lung, and Blood Institute (HL-37556) and National Institute of Environmental Health Sciences (ES-05511). A portion of this work was presented at the annual meeting of the Federation of American Societies for Experimental Biology in Washington, D.C., April, 1990. Address reprint requests to: H. J. Forman, Mail Stop 83, Childrens Hospital Los Angeles, Los Angeles, CA 90027. Received
8 October
1991; accepted
in final
form
9 January
1992.
REFERENCES 1. Akerboom, T. P. M., and H. Sies. Assay of glutathione, glutathione disulfide, and glutathione mixed disulfides in biological samples. Methods Enzymol. 77: 373-382, 1981. 2. Anderson, M. E., and A. Meister. Intracellular delivery of cysteine. Methods Enzymol. 143: 313-325, 1987. 3. Awasthi, Y., S. Singh, H. Ahmad, L. Wronski, S. Srivastava, and E. Labelle. ATP dependent primary active transport of xenobiotic-glutathione conjugates by human erythrocyte membrane. Mol. Cell. Biochem. 91: 131-136, 1989. 4. Becker, S., M. C. Madden, S. L. Newman, R. B. Devlin, and H. S. Koren. Modulation of human alveolar macrophage properties by ozone exposure in vitro. Toxicol. Appl. Pharmacol. 110: 403-415, 1991. P. C., D. M. Dulik, and T. W. Jones. The toxicity of 5. Brown, menadione (2-methyl-1,4-napthoquinone) and two thioether conjugates studied with isolated renal epithelial cells. Arch. Biochem. Biophys. 285: 187-196, 1991. 6. Brunmark, A., and E. Cadenas. Redox and addition chemistry of quinoid compounds and its biological implications. Free Radical Biol. Med. 7: 435-477, 1989. 7. Buffinton, G. D., K. Ollinger, A. Brunmark, and E. Cadenas. DT-diaphorase-catalyzed reduction of 1,4-naphthoquinone derivatives and glutathionyl-quinone conjugates: effect of substituents on autoxidation rates. Biochem. J. 257: 561-571, 1989. 8. Buhl, R., C. Vogelmeier, M. Critenden, R. C. Hubbard, R. F. Hoyt, Jr., E. M. Wilson, A. M. Cantin, and R. G. Crystal. Augmentation of glutathione in the fluidlining the epithelium of the lower respiratory tract by directly administering glutathione aerosol. Proc. Natl. Acad. Sci. USA 87: 4063-4067, 1990. 9. Cantin, A. M., R. C. Hubbard, and R. G. Crystal. Glutathione deficiency in the epithelial lining fluid of the lower respiratory tract in idiopathic pulmonary fibrosis. Am. Reu. Respir. Dis. 139: 370-372, 1989. 10. Crapo, J. D., B. E. Barry, H. A. Fescue, and J. Shelburne. Structural and biochemical changes in rat lungs occurring during exposures to lethal and adaptive doses of oxygen. Am. Reu. Respir. Dis. 122: 123-143, 1980. 11. Deneke, S. M., D. T. Phelps, D. F. Baxter, and B. L. Fanburg. Extracellular glutathione: mechanisms for increasing intracellular GSH levels in endothelial cells (Abstract). Am. Rev. Respir. Dis. 141: A818, 1990. 12. Di Monte, D., D. Ross, G. Bellomo, L. Eklow, and S. Orrenius. Alterations in intracellular thiol homeostasis during the metabolism of menadione by isolated rat hepatocytes. Arch. Biochem. Biophys. 235: 334-342, 1984.
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GLUTATHIONE
PROTECTS
ENDOTHELIAL
13. Forman, H. J., and D. C. Skelton. Protection of alveolar matrophages from hyperoxia by y-glutamyl transpeptidase. Am. J. Physiol. 259 (Lung Cell. MOL. Physiol. 3): L102-L107, 1990. 14. Hagen, T. M., L. A. Brown, and D. P. Jones. Protection against paraquat-induced injury by exogenous GSH in pulmonary alveolar type II cells. Biochem. Pharmacol. 35: 4537-4542, 1986. 15. Holmquist, B., J. Bunning, and F. Riordan. A continuous spectrophotometric assay for angiotensin converting enzyme. Anal. Biochem. 95: 540-548, 1979. 16. Housset, B., C. Ody, D. B. Rubin, G. Elemer, and A. F. Junod. Oxygen toxicity in cultured aortic endothelium: seleniuminduced partial protective effect. J. Apple. PhysioZ. 55: 343-352, 1983. 17. Kondo, T., S. Miyamoto, N. Gasa, Y. Taniguchi, and Y. Kawakami, Purification and characterization of glutathione disulfide-stimulated Mg2+-ATPase from human erythrocytes. Biochem. Biophys. Res. Commun. 162: l-8, 1989. 18. Kowalski, D. P., R. M. Feeley, and D. P. Jones. Use of exogenous glutathione for metabolism of peroxidized methyl linoleate in rat small intestine. J. Nutr. 120: 1115-1121, 1990. 19 Loeb, G. A., D. C. Skelton, and H. J. Forman. Dependence of mixed disulfide formation in alveolar macrophages upon production of oxidized glutathione: effect of selenium depletion. Biochem. Pharmacol. 38: 3119-3121, 1989. A. Glutathione. In: The Liver: Biology and Pathobiology, 20 Meister, edited by I.M. Arias, W. B. Jakoby, H. Popper, D. Schacter, and D. A. Shafritz. NewYork: Raven, 1988, p. 401-417. 21. Meister, A., S. S. Tate, and 0. W. Griffith. Gamma-glutamyl transpeptidase. Methods Enxymol. 77: 237-253, 1981. 22. Nickerson, W., G. Falcone, and G. Strauss. Studies on quinone-thioethers. 1. Mechanism of formation and properties of thiodione. Biochemistry 2: 537-543, 1963. 23. Peacock, M. D., R. A. Lawrence, and S. G. Jenkinson. The effects of gamma-glutamyl transpeptidase inhibition on glutathione protection from hyperbaric oxygen (Abstract). Am. Rev. Respir. Dis. 143: A738, 1991. 24 Rosen, G. M., and B. A. Freeman. Detection of superoxide generated by endothelial cells. Proc. Natl. Acad. Sci. USA 81: 7269-7273, 1984. 25 Ross, D., H. Thor, M. Threadgill, M. Sandy, M. Smith, P. Moldeus, and S. Orrenius. The role of oxidative processes in the cytotoxicity of substituted 1,4-naphthoquinone in isolated hepatocytes. Arch. Biochem. Biophys. 248: 460-466, 1986. 26. Rossi, L., G. Moore, S. Orrenius, and P. O’Brien. Quinone toxicity in hepatocytes without oxidative stress. Arch. Biochem. Biophys. 251: 25-35, 1986. 27. Ryan, U. S., and G. Maxwell. Isolation, culture, and subculture of bovine pulmonary artery endothelial cells: mechanical methods. J. Tissue Cult. Methods 10: 3-8, 1986.
CELLS
FROM
MENADIONE
L643
28.
Stein, H. J., R. A. Hinder, and M. M. Oosthuizen. Gastric mucosal injury caused by hemorrhagic shock and reperfusion: protective role of the antioxidant glutathione. Surgery 108: 467-473, 1990. 29. Su, Y. Z., T. E. Duarte, P. L. Dill, and L. M. Weisenthal. Selective enhancement of menadiol of in vitro drug activity in human lymphatic neoplasms. Cancer Treat. Rep. 71: 619-625, 1987. 30. Takahashi, N., J. Schreiber, V. Fischer, and R. P. Mason. Formation of glutathione-conjugated semiquinones by the reaction of quinones with glutathione: an ESR study. Arch. Biochem. Biophys. 252: 41-48, 1987. 31. Tate, S. S., and A. Meister. Serine-borate complex as a transition-state inhibitor of gamma-glutamyl transpeptidase. Proc. Natl. Acad. Sci. USA 75: 4806-4809, 1978. 32. Thor, H., M. T. Smith, P. Hartzell, G. Bellomo, S. A. Jewell, and S. Orrenius. The metabolism of menadione (2-methyl1,4-naphthoquinone) by isolated hepatocytes. J. Biol. Chem. 257: 12419-12425, 1982. R., H. Ley, and R. Scholz. Hepatic microsomal 33. Thurman, ethanol oxidation: hydrogen peroxide formation and the role of 25: 420-430, 1972. 34 catalase. Eur. J. Biochem. Tietze, F. Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Anal. Biochem. 27: 502522, 1969. 35 Tsan, M. F., E. H. Danis, P. J. Del Vecchio, and C. L. ’ Rosano. Enhancement of intracellular glutathione protects endothelial cells against oxidant damage. Biochem. Biophys. Res. Commun. 127: 270-276, 1985. 36. Tsan, M. F., J. E. White, and C. L. Rosano. Modulation of endothelial GSH concentrations: effect of exogenous GSH and GSH monoethyl ester. J. Appl. Physiol. 66: 1029-1034, 1989. 37. Tyson, C., and C. Green. Cytotoxicity measures: choices and methods. In: The Isolated Hepatocyte. New York: Academic, 1981, p. 119-158. T., K. Sai, A. Takagi, R. Hasegawa, and Y. 38. Umemura, Kurokawa. The effects of exogenous glutathione and cysteine on oxidative stress induced by ferric nitriloacetate. Cancer Lett. 58: 3g 49-56, 1991. . Weber, C. A., C. A. Duncan, M. J. Lyons, and S. G. Jenkinson. Depletion of tissue glutathione with diethyl maleate enhances hyperbaric oxygen toxicity. Am. J. Physiol. 258 (Lung Cell. Mol. Physiol. 2): L308-L312, 1990. 4. . Wefers, H., and H. Sies. Hepatic low-level chemiluminescence during redox cycling of menadione and the menadione-glutathione conjugate: relation to glutathione and NAD(P)H:quinone reductase (DT-diaphorase) activity. Arch. Biochem. Biophys. 224: 568578, 1983. M., and 0. Strubelt. Protection by exogenous glu41. Younes, tathione against hypoxic and cyanide-induced damage to isolated perfused rat livers. Toxicol. Lett. 50: 229-236, 1990. l
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CHANG,
MING
SHI, AND HENRY
cells
JAY FORMAN
Cell Biology Group and Division of Neonatology and Pediatric Pulmonology of Childrens Hospital Los Angeles, and Departments of Pediatrics, Pathology, and Molecular Pharmacology and Toxicology, University of Southern California, Childrens Hospital Los Angeles, Los Angeles, California 90027 Chang, Minyuen,
Ming Shi, and Henry Jay Forman.
conjugation of a wide variety of electrophilic xenobiotics. Under oxidant stress, cellular GSH can be lost through export as GSSG. Low-molecular-weight GSH conjugates can also be actively transported out of cells (20). Some cells, such as hepatocytes, may also secrete GSH as part of interorgan transport of this vital compound. The sources of amino acids for intracellular de novo synthesis of GSH include the following: uptake of precursor amino acids, breakdown of extracellular GSH and uptake via the y-glutamyl cycle, use of amino acids from cellular protein turnover, and conversion of methionine via the cystathionine pathway (20). Uptake of intact GSH has only been demonstrated in some epithelial cells, whereas the cystathionine pathway is found primarily in the liver (14, 20). There is no evidence suggesting that endothelial cells take up intact GSH (11). Resynthesis of GSH intracellularly is therefore important in maintaining endothelial cell GSH levels. As GSH provides the principal vehicle for interorgan transport of cysteine (20) , the ability to utilize extracellular GSH in the y-glutamyl cycle may provide protection against cellular injury under conditions when there is loss of intracellular glutathione. The cleavage of the unique y-glutamylcysteine peptide bond of GSH is catalyzed by y-glutamyl transpeptidase, a membranebound enzyme with its active site directed toward the outside of the cell. Cysteinylglycine, a product, is metabENDOTHELIAL CELLS have been shown to be a prime target for oxidant injury (10). Oxidant injury to cells and olized by a dipeptidase, and the free amino acids are moiety is tissues is induced by reactive oxygen metabolites, such transported inside the cells. The y-glutamyl either transferred to another amino acid (or a peptide) as superoxide anion, hydrogen peroxide, and hydroxyl forming a y-glutamyl peptide or released as free radical. The extent of oxidant injury involves chemical peptides can be transported into alterations in proteins, lipids, carbohydrates, and glutamate. y-Glutamyl cells or hydrolyzed to glutamate and other amino acids. nucleic acids. Quinones that undergo redox cycling have been widely used to investigate oxidant-induced stress These amino acids can then be used for synthesis of in cells. The quinone is reduced to its semiquinone rad- GSH (20, 21). Recent studies (8, 9, 13, 18, 28, 36, 38, 39, 41) have ical through a one-electron reduction catalyzed by celshown the effectiveness of exogenous GSH in preventing lular reductases. In the presence of oxygen, the semihyperbaric quinone radical undergoes rapid autoxidation with the injury, such as in ischemia reperfusion, pulmonary fibrosis, where formation of superoxide radical and regeneration of the hyperoxia, and idiopathic oxidant stress is the purported mechanism. In this parent quinone (32). Dismutation of superoxide radical study, we have investigated the ability of endothelial results in the formation of HZ02. cells to utilize exogenous GSH for protection against The tripeptide glutathione (y-glutamyl-cysteine-gly(vitamin K,; tine; GSH) is an important cellular antioxidant. As a oxidant injury generated by menadione 2-methyl-1,4-naphthoquinone, MQ). Our data suggest substrate for glutathione peroxidase, GSH provides cells are able to utilize extracellular reducing equivalents for the metabolism of H202 and that endothelial GSH for protection against oxidant stress and that the lipid hydroperoxides. The product, oxidized glutathione to use GSH is mediated by y-glutamyl disulfide (GSSG), formed in this reaction is reduced to ability GSH by the action of glutathione reductase at the transpeptidase. GSH also conjugates with MQ via a expense of reduced nicotinamide adenine dinucleotide nonenzymatic reaction. The resulting menadione-glutathione conjugate (MQSG) may have greater capacity phosphate (NADPH). Among its other roles, GSH serves as a reservoir for cysteine and is used in the to redox cycle than the parent quinone (7, 30, 40).
Exogenous glutathione protects endothelial cells from menadione toxicity. Am. J. Physiol. (Lung Cell. Mol. Physiol. 6): L637L643, 1992.~-Administration of menadione (vitamin K,; 2-methyl-1,4-naphthoquinone) to cultured bovine pulmonary artery endothelial cells caused a dose- and time-dependent depletion of cellular ATP and depletion of intracellular glutathione (GSH). The toxicity of menadione was correlated with an increase of menadione-glutathione conjugate in the medium and with an increase in H202 generation. Recent studies have suggested that GSH may be useful as a pharmacological agent to prevent oxidant injury. In the present study, treatment with exogenous GSH prevented the loss of cellular GSH and ATP caused by menadione. Protection by extracellular GSH involved two mechanisms. In one mechanism, extracellular GSH was degraded by y-glutamyl transpeptidase, producing substrates for subsequent intracellular de novo GSH synthesis. We found that the protective effect of extracellular GSH was decreased by inhibiting y-glutamyl transpeptidase. In the other mechanism, GSH reacted in the medium with menadione to form a conjugate. Although formation of the menadione-glutathione conjugate within cells may contribute to menadione toxicity, addition of menadione-glutathione conjugate to the medium was found to be nontoxic to endothelial cells. Thus exogenous GSH protected endothelial cells by two mechanisms: maintenance of intracellular GSH and prevention of menadione entrance into cells. oxidant injury; y-glutamyl transpeptidase; glutathione conjugation
1040-0605/92 $2.00 Copyright
0 1992 the American
Physiological
Society
L637
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L638
GLUTATHIONE
PROTECTS
ENDOTHELIAL
Although MQSG would therefore be expected to be toxic within the cells, we demonstrated that extracellular MQSG was not toxic to bovine pulmonary artery endothelial cells (BPAEC), suggesting that part of the protective effect of extracellular GSH may be due to the prevention of MQ accessibility to intracellular reductases. MATERIALS
AND
CELLS
FROM 0.4
MENADIONE 1
’
1
’
1
’
I
’
I
METHODS
Chemicals. MQ, reduced and oxidized glutathione, NADPH, glutathione reductase, L-serine, sodium borate, ferrous ammonium sulfate, potassium thiocyanate, 5,5’-dithiobis-(Z-nitrobenzoic acid) (DTNB), and dimethyl sulfoxide (DMSO) were obtained from Sigma Chemical. Fluorescein isothiocyanate (FITC) -conjugated antibody against human factor VIII was obtained from Atlantic Antibodies. Angiotensin-converting-enzyme reagents and calibrator were from Sigma Chemical. BPAEC. Passage 16 of BPAEC was obtained from the American Type Culture Collection (CCL 209). Cells were maintained in minimal essential media (MEM) supplemented with fetal bovine serum (100 ml FBS to 500 ml MEM, 2 mM L-glutamine, 100 U/ml penicillin, 100 fig/ml streptomycin, and 2 x 10m7 M of selenomethionine, which was included to prevent a decrease in glutathione peroxidase-specific activity due to selenium depletion that can occur as endothelial cells divide (16). MEM was from Irvine Scientific. Glutamine and penicillin-streptomycin solutions were from GIBCO. FBS was from Gemini Bioproducts, Calabasas, CA. Serum from the same lot was used for all experiments. Cells were grown in a humidified incubator at 37°C with 5% CO,. Subculture was done in a weekly ratio of 1:2-I:5 using a mechanical method described by Ryan and MMxwell (27). Cell monolayers generally reached confluence on plastic tissue culture plates (Costar) in 3-5 days. Cells from passages 18-25 were used for experiments as cells above passage 25 showed some features of senescence such as slower growth and formation of ring structures similar to vasculature formation. The monolayers used in the present study demonstrated, by phase-contrast microscopy, contact inhibition and a cobblestone appearance typical of endothelial cells. They also expressed factor VIII antigen as demonstrated by direct immunofluorescent antibody staining. These cells were also positive for angiotensin-converting enzyme activity (15). All experiments were done l-2 days after the monolayers reached confluence. MQ treatment. MQ was dissolved in DMSO and subsequently diluted in Dulbecco’s phosphate-buffered saline (DPBS, GIBCO) plus 5 mM glucose or Earl’s balanced salt solution (EBSS; GIBCO). The DMSO content in each sample was no more than 0.1%. DMSO was added to the controls at the same concentrations as in MQ-treated groups. For the inhibition of y-glutamyl transpeptidase with serine-borate complex, 20 mM borate was present in all groups. One millimolar L-serine was added to the serine-borate treated groups. Synthesis of MQSG. MQSG was synthesized according to Nickerson et al. (22). MQ (0.1 M in ethanol, 50 ml) was mixed with GSH (0.025 M in water, IO ml) at 4°C overnight in the dark. The yellow-orange crystals that formed were collected and washed twice with chloroform to dissolve excess MQ. The precipitate was filtered and dried. Analysis of the synthetic product was done with the use of high-performance liquid chromatography with electrochemical detection. The solvent system used was 65% propanol-35% H20, and the applied potential was -40 mV. The purity of the conjugate was estimated to be 98%. The spectrum of MQSG was examined in a Beckman DU-7 spectrophotometer and compared with that of MQ. A marked difference in their absorbance was found at 430 nm (Fig. I). MQSG conjugate formation in the media. BPAEC were grown
300
350
400
Wavelength
450
500
(nm)
Fig. 1. Comparison of absorbance spectra of 100 PM 2-methyl-1,4-naphthoquinone (MQ) and 100 PM menadione-glutathione conjugate (MQSG) in phosphate-buffered saline without glucose.
to confluence in 12-well culture plates. Cells were washed twice with DPBS-5 mM glucose and incubated in DPBS-5 mM glucose containing 100 PM MQ or DMSO for 0,20,30, and 60 min. The media were taken for measurement of absorbance at 430 nm. The reading obtained from controls not treated with MQ served as baseline and was subtracted to eliminate interference from other products released from the cells. Known concentrations of MQSG in DPBS-5 mM glucose were used for calibration. ATP measurement. Cellular ATP levels were determined using a bioluminescent somatic cell ATP assay kit obtained from Sigma Chemical. At the end of each incubation, media were removed, and 0.4 ml of somatic cell ATP releasing agent (SCRR) was added to each well for 2 min. The supernatant was transferred to test tubes and kept on ice for ATP measurement within 3 min. ATP was determined by the amount of light emitted after the reaction of luciferin and ATP catalyzed by firefly luciferase and measured by the phosphorescent mode of the Perkin-Elmer LS-5 fluorescence spectrophotometer. HZ02 assay. HZ02 in media was measured by the calorimetric method of Thurman (33). The absorbance of ferric thiocyanate complex formed from the reaction of HZ02, ferrous ammonium sulfate, and potassium thiocyanate was read at 480 nm. The readings were compared with H,O, standards. The specificity of this method was demonstrated by the decrease of H,O, with the addition of catalase. Total cellular GSH measurement. Total cellular GSH (GSH + 2 x GSSG) was measured by the Tietze recycling method (34) modified by Akerboom (1) using DTNB, glutathione reductase, and NADPH. Endothelial monolayers were washed four times after each incubation with DPBS-5 mM glucose. Elimination of residual extracellular GSH was assured by measuring the total GSH content of the washes, which was negligible in the third wash. 4.3% Sulfosalicylic acid was added to the monolayers and neutralized with 5 mM KOH. Cellular protein was measured with bicinchoninic acid by a modified Lowry method (Pierce, Rockford, IL). Statistics. Data were analyzed using the Crunch interactive statistical package (Crunch Software, San Francisco, CA). Analysis of variance was done in all treatment groups, followed by post hoc tests to determine the difference of each paired group. Differences were considered significant at P < 0.05.
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GLUTATHIONE
PROTECTS
ENDOTHELIAL
RESULTS
Menadione
toxicity
in endothelial
cells. Loss of ATP is
generally used as an indicator of depressed metabolism and is a critical event occurring before cell death (37). Our data showed that MQ caused a dose- and time-dependent decrease in cellular ATP in BPAEC monolayers (Fig. 2). Significant ATP depletion occurred after incubation with 25, 50, and 100 PM of MQ for 2 h. Only 100 PM of MQ caused significant ATP depletion by the end of 1 h of incubation. Morphological changes, including contraction, rounding, and detachment of endothelial cells from culture dishes, were observed by phase-contrast microscopy in correlation with the loss of cellular ATP at the end of incubation for 2 h. The loss of cellular ATP also correlated with the loss of ability to exclude trypan blue dye (data not shown). MQ also caused time-dependent depletion of cellular GSH (Fig. 3). The decrease in GSH preceded loss of ATP at each concentration of MQ (compare Figs. 1 and 2). As shown in hepatocytes, MQ can deplete GSH by direct conjugation with GSH and, indirectly, through the generation of H20z (12). MQ has also been reported to induce production of superoxide and H202 in endothelial cells (24). The conjugate formed in hepatocytes is rapidly excreted. These effects of MQ were confirmed in this study by observing the increase of MQSG and HzOz in the media with addition of 100 PM MQ (Fig. 4 and Fig. 5). Effects of exogenous GSH supplementation on menadione toxicity. Addition of 2 mM GSH to the EBSS-0.1%
bovine serum albumin (BSA) solution provided protection from cellular ATP depletion in BPAEC subsequently treated with 100 PM of MQ (Table 1). The loss of intracellular GSH caused by MQ was also prevented by exogenous GSH (Fig. 6). Exogenous GSH also caused an increase of intracellular GSH levels in BPAEC regardless of the presence of MQ. The data suggest that exogenous GSH can protect BPAEC from MQ toxicity by providing z 3
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Fig. 4. Comparison of cellular GSH loss (indicated as GSH equivalents) and the appearance of MQSG conjugates outside the cells. BPAEC monolayers were grown to confluence on 12-well plates. Cells were treated with 100 PM of MQ in DPBS/5 mM glucose for 60 min. At times indicated, media were removed for MQSG estimations as described in MATERIALS AND METHODS. Control cells without MQ treatment did not show significant changes in cellular GSH levels (data omitted). Values are means t SE; n =4 experiments.
1
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Fig. 3. Cellular glutathione (GSH) depletion (indicated as GSH equivalents) by 0, 25, 50, and 100 PM of MQ. BPAEC monolayers were cultured on 12-well plates to confluence. Cells were treated with MQ in DPBS/5 mM glucose for 2 h. At the times indicated, monolayers were treated with 4.3% sulfosalicylic acid and processed subsequently as described in MATERIALS AND METHODS for GSH measurements. At confluence, 1 x lo6 BPAEC contained 105 t 10 pg protein. Values are means k SE of 2 experiments. Significant GSH loss occurred after 30 min (P < 0.05). There was no difference in GSH levels among MQtreated groups.
15
it
L639
MENADIONE
120
(mid
Fig. 2. ATP depletion by incubating bovine pulmonary artery endothelial cells (BPAEC) monolayers on 24-well culture plates with 0, 25, 50, and 100 PM of MQ in Dulbecco’s phosphate-buffered saline (DPBS)-5 mM glucose over 2 h. At times indicated, cellular ATP was released by somatic cell releasing agent (SCRR) and measured immediately. Each well contained 3.4 k 0.1 x lo5 cells. Values are means t SE; n = 4. At 1 h of incubation, only cells treated with 100 PM of MQ showed significant ATP depletion (P = 0.01). All cells treated with MQ showed significant ATP depletion by the end of 2 h (P = 0.01).
of cellular GSH.
Mechanisms by which exogenous GSH protects from menadione toxicity. Two different mechanisms could
account for the protection of MQ-exposed cells by extracellular GSH: 1) prevention of MQ entrance into cells through formation of a nonpermeable conjugate, and 2) maintenance of intracellular GSH through the Y-glutamvl cvcle. ’ ”To investigate the effect of conjugate formation outside the cells, we compared the toxicity of exogenous MQSG and MQ with BPAEC. In contrast with MQ, MQSG did not cause significant ATP depletion even at 500 PM (Fig.
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L640
GLUTATHIONE
PROTECTS
ENDOTHELIAL
15
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FROM
MENADIONE
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Fig. 5. Production of Hz02 by MQ-exposed endothelial cells. BPAEC monolayers on T-25 flasks at confluence were incubated with 0,25, and 100 PM MQ in 1.6 ml DPBS-5 mM glucose over 60 min. At times indicated, 0.4 ml 50% trichloroacetic acid was added, and media were taken to measure Hz02. MQ caused a dose-dependent increase in Hz02 production in BPAEC over time. Values are means t SE; n = 2-4.
Table 1. Effect of exogenous GSH supplementation on
(PM)
Fig. 6. GSH supplementation prevented GSH equivalents) from MQ treatment. BPAEC were to confluence. Cells were preincubated in Earl’s (EBSS)-0.1% BSA with or without 2 mM GSH at 0, 50, or 100 PM and incubation with GSH Monolayers were washed with EBSS 4 times, were measured as in MATERIALS AND METHODS. n= 3.
loss (indicated as GSH cultured on 6-well plates balanced salt solution for 1 h. MQ was added was continued for 2 h. and cellular GSH levels Values are means t SE;
BPAEC cellular ATP depletion induced by MQ Treatment
ATP,
pg/well
P vs. Control
P vs. +GSH
Control 0.24kO.02 NS* O.O1tO.OO 0.01 0.01 +lOO ,uM MQ 0.28t0.06 NS +2 mM GSH +2 mM GSH + 100 ,uM MQ 0.22kO.03 NS NS Values are means t SE for n = 4 experiments. Confluent bovine pulmonary artery endothelial cells (BPAEC’s) on 24-well plates were incubated in Earl’s balanced salt solution-0.1% bovine serum albumin containing 0 or 2 mM glutathione (GSH) for 1 h, followed by addition of 0 or 100 PM MQ. Cellular ATP levels were measured after an additional 2 h of incubation. MQ, 2-met/&1,4-naphthoquinone; NS, not significant.
0.00
50
7). As this suggested a potential
mechanism for protection by extracellular GSH, the rate of extracellular conjugation of MQ by GSH was also examined by monitoring the increase in absorbance at 430 nm in the absence of cells over a 2-h period. The reaction rate slowed down after -30 min (Fig. 8). These data suggest that exogenous GSH can contribute to the protection of cells by consuming MQ, thereby limiting availability of unconjugated MQ to cells. Nevertheless, as extracellular conjugate formation was not instantaneous (- 17% of MQ was still in the unconjugated form at 60 min), MQ toxicity could not be completely prevented by this mechanism. The possibility that extracellular GSH was used by endothelial cells for maintenance of cellular GSH levels through the action of y-glutamyl transpeptidase was examined by inhibiting this enzyme with the competitive inhibitor serine-borate complex (31). In the presence of serine-borate, GSH content of the medium remained unchanged for 24 h (2.07 t 0.06 pmol), whereas in the absence of serine borate, total GSH content of the medium declined 17.4% (359 t 80 nmol) in 24 h (n = 10) (P c 0.005 vs. inhibited). Serine-borate alone had no effect on ATP content of endothelial cells (Fig. 9). The protective effect of exogenous GSH was decreased in the presence of serine-borate, whereas the presence of serineborate in the absence of extracellular GSH did not affect
Dose
100 (MM)
Fig. 7. Comparison of toxicity of MQ and MQSG. Various doses of MQ and MQSG were added to confluent BPAEC monolayers on 24well culture plates containing EBSS and incubation carried for 2 h. Values are means t SE; n =4-8.
the toxicity of MQ (Fig. 9). The increase in cellular GSH levels by exogenous GSH was also markedly decreased with serine-borate in the presence or absence of MQ (Fig. 10). Serine-borate had no effect upon the rate of MQSG formation in the medium (Fig. 8). These data suggest that part of the protective effect of exogenous GSH was mediated by the action of y-glutamyl transpeptidase. DISCUSSION
MQ and other vitamin K3 analogues have been evaluated as anticancer drugs for the last three decades. Clinical trials of their chemosensitizing effects have also been investigated (29). The cytotoxicity of MQ is related to its ability to generate reactive oxygen species and to deplete cellular glutathione. The depletion of cellular GSH can be due to both oxidation and conjugation. Because the conjugation reaction between MQ and GSH does not disturb the quinone nucleus, the resulting MQSG retains the capacity to redox cycle (7,30). The reported redox potentials of MQ and of MQSG are similar [E (Q/Q-) values,
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GLUTATHIONE
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CELLS
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El
0 E -c
control
80 -
6 5 2 s
20
0 / 0 Y,r,l,r,l,l 0
20
40
60
80
100
Minutes Fig. 8. Rate of MQSG formation was monitored by reading increase in absorbance at 430 nm over 2 h after adding 2 mM GSH to 1 ml PBS containing 100 PM of MQ at 37°C. From Fig. 1, we determined that the millimolar extinction coefficient of MQSG at 430 nm was 1.29. Serineborate had no effect on rate of conjugate formation in medium. One of four superimposable determinations is shown.
0.30 -6 2
I
control
cn % 0 0.20 .-c k Q 3 -is .-
100 [Menadione]
0.10
$ cc 0.00
100
0 [Menadione]
(PM)
Fig. 9. Effect of serine-borate complex on GSH protection from MQinduced ATP loss. Confluent BPAEC monolayers on 24-well plates were preincubated with 2 mM GSH for 1 h in a solution of 20% Krebs-Ringer borate (Krebs-Ringer phosphate solution with substitution of 100 mM borate for phosphate), 80% EBSS-0.1% BSA, pH 7.4. 1 mM L-serine was added to the serine-borate groups. MQ was added at 100 PM after GSH pretreatment. Cellular ATP levels were measured after a 2-h incubation. Values are means ~fr SE; n = 9. There was no significant difference of ATP levels in groups without MQ treatment. In MQtreated groups, GSH group is significantly different from other 3 groups (P = 0.01).
-203 mV and -192 mV, respectively] (6). Wefers and Sies (40) reported that the rate of redox cycling observed with MQSG was greater than that seen with the parent quinone when incubated with a hepatic microsomal fraction; however, exogenous MQSG was not toxic to endothelial cells (Fig. 7). The MQSG formed extracellularly was apparently impermeable to endothelial cells as MQSG formed inside the cells would be at least as toxic as MQ itself. MQSG was also found by Brown et al. (5) to
(wM)
Fig. 10. Effect of serine-borate complex on GSH prevention of MQ-induced GSH loss (indicated as GSH equivalents). Confluent BPAEC monolayers on 6-well plates were treated as in Fig. 9. Values are means t SE; n = 4-5 in each group, except in groups without serine treatment where n = 2. Presence of serine-borate complex inhibited increase in cellular GSH by extracellular GSH in both control and MQ treated groups (P = 0.03 by Dunnett’s test for GSH vs. GSH + serine in non-M& treated groups, and P = 0.001 for GSH vs. other groups with MQ treatment).
be noncytotoxic to rat renal epithelial cells. These authors speculated that the active y-glutamyl transpeptidase activity of these cells may cleave the y-glutamylcysteinyl bond of MQSG, which would be followed by the formation of a thiazine product resulting from the cyclization of the cysteinylglycine conjugate, thereby destroying the quinone ring of MQSG. Because cultured endothelial cells possess relatively low y-glutamyl transpeptidase activity compared with renal epithelial cells, thiazine formation in this system may be slow. Studies of the relative contribution of conjugation and oxidation to loss of GSH in MQ-induced cytotoxicity have been inconclusive (12, 25, 26). Our results suggest that conjugation with MQ accounted for most of the cellular GSH loss (Fig. 4). The amount of MQ and MQSG within the cell would have been constantly changing. From the data in Fig. 8, and assuming that the permeability of MQ, which is lipid soluble, would allow rapid equilibration, we estimate that, at 30 min, the intracellular concentration of MQ would have been -40 PM whereas intracellular MQSG would have been -60 PM minus the MQSG that was excreted (see Fig. 4). At 60 min, the intracellular MQ would have been only - 17 PM with perhaps most of the MQSG formed intracellularly having been excreted; however, at 60 min, the total intracellular GSH content was decreased by 76%. The nonlinear concentration dependence of MQ toxicity probably reflected the multiple components of the mechanism of its toxicity; loss of GSH through conjugation with MQ, Hz02 production through redox cycling of MQ and MQSG, oxidation of GSH through the action of gluathione peroxidase, and formation of protein-GSH conjugates, which is dependent upon GSSG production (19). As a significant elevationof H202 could be detected in the medium (Fig. 5), the net generation of HZOB by the cells was clearly elevated beyond the capacity to remove it.
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L642
GLUTATHIONE
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ENDOTHELIAL
Wherease excretion of GSH-xenobiotic conjugates and GSSG has been shown to be an ATP-dependent process in red blood cells (3, 17), the appearance in the media of MQSG prior to the loss of ATP suggests that conjugate excretion per se did not cause significant ATP depletion (Figs. 2 and 4). Thus MQ-induced ATP depletion in BPAEC was most likely the result of HZOz toxicity that was exacerbated by loss of GSH. Various methods have been used to increase intracellular GSH, such as treatment with L-2-oxothiazolidine-4 carboxylate (an effective intracellular cysteine delivery agent) (35), GSH ~monoethyl ester (which enters the cells freely) (36), and GSH supplementation. Tsan et al. (36) reported that treatment with exogenous GSH elevated the cellular GSH level in endothelial cells and that this effect was inhibited by the addition of acivicin (an irreversible y-glutamyl transpeptidase inhibitor), which is consistent with our observations. Because we omitted GSH precursor amino acids in the present studies, the source contributing to the increase of cellular GSH in BPAEC was likely from the utilization of extracellular GSH. The essential role of y-glutamyl transpeptidase in utilization of extracellular GSH in protecting alveolar macrophages from oxidant injury was also demonstrated previously by our laboratory (13). Suppression of the respiratory burst in alveolar macrophages by hyperoxia was prevented by the presence of extracellular GSH. This protective effect was inhibited by the presence of serineborate which completely abolished extracellular GSH degradation under the conditions used herein. Recently, Peacock et al. (23) also reported that GSH supplementation protected rats from hyperbaric oxygen exposure and that this effect was inhibited by a single dose of acivicin. In the present study of MQ toxicity, conjugation by GSH in the medium appeared to be the principal mechanism for protection by extracellular GSH. Nevertheless, the conjugation was not instantaneous, requiring >60 min to remove all free MQ in the absence of cells (Fig. 8). MQ that escaped conjugation entered the cell, producing toxicity. The effects of MQ in the presence of serineborate (Figs. 9 and 10) indicated that the activity of y-glutamyl transpeptidase was also critical in allowing extracellular GSH to maintain cellular GSH and prevent loss of ATP. Serine-borate prevented the elevation in GSH observed in the presence of exogenous GSH (in either the presence or absence of MQ) (Fig. 10). The relatively higher intracellular GSH observed in the presence versus the absence of exogenous GSH in the presence of both MQ and serine-borate (Fig. 10, 2 rightmost bars) was probably due to protection by conjugate formation in the medium that limited the amount of intracellular MQSG formation and H,OZ production rather than a result of bypassing of the serine-borate block. In the absence of serine-borate, exogenous GSH could supply the materials needed for de novo GSH synthesis (Fig. 10) that helped prevent loss of ATP (Fig. 9). Exogenous GSH has been shown in several recent studies to prevent oxidant injury (4,8,9,13,18,28,38,39,41). The protection may be due to multiple mechanisms; in addition to its potential for conjugation of xenobiotics, extracellular GSH may provide a supply of cysteine to
CELLS
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cells through disulfide exchange with cystine and may stabilize the plasma membrane by reducing oxidized membrane protein moieties. Nevertheless, under normal physiological conditions, extracellular GSH provides the essential cysteine for de novo GSH synthesis within cells through the y-glutamyl cycle (2). When challenged with either an oxidant stress and/or GSH depleting agent, use of exogenous GSH to prevent oxidant injury may have significant value. The authors thank Dr. Enrique Cadenas for help in analysis of the menadione-glutathione conjugate and Dr. Timothy W. Robison for help in using the Crunch statistical program for data analysis. We also thank Dr. Peter J. Del Vecchio for providing endothelial cells and Dr. Henry Choy for his careful criticism on this manuscript. This work was supported by grants from the National Heart, Lung, and Blood Institute (HL-37556) and National Institute of Environmental Health Sciences (ES-05511). A portion of this work was presented at the annual meeting of the Federation of American Societies for Experimental Biology in Washington, D.C., April, 1990. Address reprint requests to: H. J. Forman, Mail Stop 83, Childrens Hospital Los Angeles, Los Angeles, CA 90027. Received
8 October
1991; accepted
in final
form
9 January
1992.
REFERENCES 1. Akerboom, T. P. M., and H. Sies. Assay of glutathione, glutathione disulfide, and glutathione mixed disulfides in biological samples. Methods Enzymol. 77: 373-382, 1981. 2. Anderson, M. E., and A. Meister. Intracellular delivery of cysteine. Methods Enzymol. 143: 313-325, 1987. 3. Awasthi, Y., S. Singh, H. Ahmad, L. Wronski, S. Srivastava, and E. Labelle. ATP dependent primary active transport of xenobiotic-glutathione conjugates by human erythrocyte membrane. Mol. Cell. Biochem. 91: 131-136, 1989. 4. Becker, S., M. C. Madden, S. L. Newman, R. B. Devlin, and H. S. Koren. Modulation of human alveolar macrophage properties by ozone exposure in vitro. Toxicol. Appl. Pharmacol. 110: 403-415, 1991. P. C., D. M. Dulik, and T. W. Jones. The toxicity of 5. Brown, menadione (2-methyl-1,4-napthoquinone) and two thioether conjugates studied with isolated renal epithelial cells. Arch. Biochem. Biophys. 285: 187-196, 1991. 6. Brunmark, A., and E. Cadenas. Redox and addition chemistry of quinoid compounds and its biological implications. Free Radical Biol. Med. 7: 435-477, 1989. 7. Buffinton, G. D., K. Ollinger, A. Brunmark, and E. Cadenas. DT-diaphorase-catalyzed reduction of 1,4-naphthoquinone derivatives and glutathionyl-quinone conjugates: effect of substituents on autoxidation rates. Biochem. J. 257: 561-571, 1989. 8. Buhl, R., C. Vogelmeier, M. Critenden, R. C. Hubbard, R. F. Hoyt, Jr., E. M. Wilson, A. M. Cantin, and R. G. Crystal. Augmentation of glutathione in the fluidlining the epithelium of the lower respiratory tract by directly administering glutathione aerosol. Proc. Natl. Acad. Sci. USA 87: 4063-4067, 1990. 9. Cantin, A. M., R. C. Hubbard, and R. G. Crystal. Glutathione deficiency in the epithelial lining fluid of the lower respiratory tract in idiopathic pulmonary fibrosis. Am. Reu. Respir. Dis. 139: 370-372, 1989. 10. Crapo, J. D., B. E. Barry, H. A. Fescue, and J. Shelburne. Structural and biochemical changes in rat lungs occurring during exposures to lethal and adaptive doses of oxygen. Am. Reu. Respir. Dis. 122: 123-143, 1980. 11. Deneke, S. M., D. T. Phelps, D. F. Baxter, and B. L. Fanburg. Extracellular glutathione: mechanisms for increasing intracellular GSH levels in endothelial cells (Abstract). Am. Rev. Respir. Dis. 141: A818, 1990. 12. Di Monte, D., D. Ross, G. Bellomo, L. Eklow, and S. Orrenius. Alterations in intracellular thiol homeostasis during the metabolism of menadione by isolated rat hepatocytes. Arch. Biochem. Biophys. 235: 334-342, 1984.
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13. Forman, H. J., and D. C. Skelton. Protection of alveolar matrophages from hyperoxia by y-glutamyl transpeptidase. Am. J. Physiol. 259 (Lung Cell. MOL. Physiol. 3): L102-L107, 1990. 14. Hagen, T. M., L. A. Brown, and D. P. Jones. Protection against paraquat-induced injury by exogenous GSH in pulmonary alveolar type II cells. Biochem. Pharmacol. 35: 4537-4542, 1986. 15. Holmquist, B., J. Bunning, and F. Riordan. A continuous spectrophotometric assay for angiotensin converting enzyme. Anal. Biochem. 95: 540-548, 1979. 16. Housset, B., C. Ody, D. B. Rubin, G. Elemer, and A. F. Junod. Oxygen toxicity in cultured aortic endothelium: seleniuminduced partial protective effect. J. Apple. PhysioZ. 55: 343-352, 1983. 17. Kondo, T., S. Miyamoto, N. Gasa, Y. Taniguchi, and Y. Kawakami, Purification and characterization of glutathione disulfide-stimulated Mg2+-ATPase from human erythrocytes. Biochem. Biophys. Res. Commun. 162: l-8, 1989. 18. Kowalski, D. P., R. M. Feeley, and D. P. Jones. Use of exogenous glutathione for metabolism of peroxidized methyl linoleate in rat small intestine. J. Nutr. 120: 1115-1121, 1990. 19 Loeb, G. A., D. C. Skelton, and H. J. Forman. Dependence of mixed disulfide formation in alveolar macrophages upon production of oxidized glutathione: effect of selenium depletion. Biochem. Pharmacol. 38: 3119-3121, 1989. A. Glutathione. In: The Liver: Biology and Pathobiology, 20 Meister, edited by I.M. Arias, W. B. Jakoby, H. Popper, D. Schacter, and D. A. Shafritz. NewYork: Raven, 1988, p. 401-417. 21. Meister, A., S. S. Tate, and 0. W. Griffith. Gamma-glutamyl transpeptidase. Methods Enxymol. 77: 237-253, 1981. 22. Nickerson, W., G. Falcone, and G. Strauss. Studies on quinone-thioethers. 1. Mechanism of formation and properties of thiodione. Biochemistry 2: 537-543, 1963. 23. Peacock, M. D., R. A. Lawrence, and S. G. Jenkinson. The effects of gamma-glutamyl transpeptidase inhibition on glutathione protection from hyperbaric oxygen (Abstract). Am. Rev. Respir. Dis. 143: A738, 1991. 24 Rosen, G. M., and B. A. Freeman. Detection of superoxide generated by endothelial cells. Proc. Natl. Acad. Sci. USA 81: 7269-7273, 1984. 25 Ross, D., H. Thor, M. Threadgill, M. Sandy, M. Smith, P. Moldeus, and S. Orrenius. The role of oxidative processes in the cytotoxicity of substituted 1,4-naphthoquinone in isolated hepatocytes. Arch. Biochem. Biophys. 248: 460-466, 1986. 26. Rossi, L., G. Moore, S. Orrenius, and P. O’Brien. Quinone toxicity in hepatocytes without oxidative stress. Arch. Biochem. Biophys. 251: 25-35, 1986. 27. Ryan, U. S., and G. Maxwell. Isolation, culture, and subculture of bovine pulmonary artery endothelial cells: mechanical methods. J. Tissue Cult. Methods 10: 3-8, 1986.
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Stein, H. J., R. A. Hinder, and M. M. Oosthuizen. Gastric mucosal injury caused by hemorrhagic shock and reperfusion: protective role of the antioxidant glutathione. Surgery 108: 467-473, 1990. 29. Su, Y. Z., T. E. Duarte, P. L. Dill, and L. M. Weisenthal. Selective enhancement of menadiol of in vitro drug activity in human lymphatic neoplasms. Cancer Treat. Rep. 71: 619-625, 1987. 30. Takahashi, N., J. Schreiber, V. Fischer, and R. P. Mason. Formation of glutathione-conjugated semiquinones by the reaction of quinones with glutathione: an ESR study. Arch. Biochem. Biophys. 252: 41-48, 1987. 31. Tate, S. S., and A. Meister. Serine-borate complex as a transition-state inhibitor of gamma-glutamyl transpeptidase. Proc. Natl. Acad. Sci. USA 75: 4806-4809, 1978. 32. Thor, H., M. T. Smith, P. Hartzell, G. Bellomo, S. A. Jewell, and S. Orrenius. The metabolism of menadione (2-methyl1,4-naphthoquinone) by isolated hepatocytes. J. Biol. Chem. 257: 12419-12425, 1982. R., H. Ley, and R. Scholz. Hepatic microsomal 33. Thurman, ethanol oxidation: hydrogen peroxide formation and the role of 25: 420-430, 1972. 34 catalase. Eur. J. Biochem. Tietze, F. Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Anal. Biochem. 27: 502522, 1969. 35 Tsan, M. F., E. H. Danis, P. J. Del Vecchio, and C. L. ’ Rosano. Enhancement of intracellular glutathione protects endothelial cells against oxidant damage. Biochem. Biophys. Res. Commun. 127: 270-276, 1985. 36. Tsan, M. F., J. E. White, and C. L. Rosano. Modulation of endothelial GSH concentrations: effect of exogenous GSH and GSH monoethyl ester. J. Appl. Physiol. 66: 1029-1034, 1989. 37. Tyson, C., and C. Green. Cytotoxicity measures: choices and methods. In: The Isolated Hepatocyte. New York: Academic, 1981, p. 119-158. T., K. Sai, A. Takagi, R. Hasegawa, and Y. 38. Umemura, Kurokawa. The effects of exogenous glutathione and cysteine on oxidative stress induced by ferric nitriloacetate. Cancer Lett. 58: 3g 49-56, 1991. . Weber, C. A., C. A. Duncan, M. J. Lyons, and S. G. Jenkinson. Depletion of tissue glutathione with diethyl maleate enhances hyperbaric oxygen toxicity. Am. J. Physiol. 258 (Lung Cell. Mol. Physiol. 2): L308-L312, 1990. 4. . Wefers, H., and H. Sies. Hepatic low-level chemiluminescence during redox cycling of menadione and the menadione-glutathione conjugate: relation to glutathione and NAD(P)H:quinone reductase (DT-diaphorase) activity. Arch. Biochem. Biophys. 224: 568578, 1983. M., and 0. Strubelt. Protection by exogenous glu41. Younes, tathione against hypoxic and cyanide-induced damage to isolated perfused rat livers. Toxicol. Lett. 50: 229-236, 1990. l
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