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Cadmium resistance in A549 cells correlates with elevated glutathione content but not antioxidant enzymatic activities
Free Radical Biology& Medicine,Vol. 19, No. 6, pp. 805-8 12, 1995
Copyright0 1995ElsevierScienceInc. Printedin the USA. All rightsreserved 0891-5849195 $9.50 + .OO
Pergamon
--bOriginal
Contribution
CADMIUM RESISTANCE IN A549 CELLS CORRELATES WITH ELEVATED GLUTATHIONE CONTENT BUT NOT ANTIOXIDANT ENZYMATIC ACTIVITIES
EMIKO L. HATCHER, YAN CHEN, and Y. JAMES KANG Department of Pharmacology and Toxicology, University of North Dakota School of Medicine, (Received
3 October
1994; Revised
12 April 1995; Re-revised
18 May 1995; Accepted
Grand Forks, ND, USA 23 May 1995)
Abstract-Glutathione has been implicated to function in cytoprotection against cadmium toxicity. The mechanism by which glutathione plays this role has not been well understood. Because glutathione is an important antioxidant and several studies have shown that cadmium induces oxidative stress, this study was undertaken to determine whether development of cadmium resistance is linked to enhanced antioxidant activities. A cadmium-resistant subpopulation of human lung carcinoma A549 cells, which was developed by repeatedly exposing the cells to step-wise increased cadmium concentrations, was compared to a cadmium-sensitive one. The acquired cadmium resistance resulted from neither decreased cadmium uptake nor enhanced cellular metallothionein synthesis. Glutathione content, however, was markedly elevated in the cadmium-resistant cells. In contrast, the activities of the glutathione redox cycle related enzymes, glutathione peroxidase and reductase, were unchanged. Two other antioxidant enzymes, superoxide dismutase and catalase, were also not altered. The results suggest that the development of cadmium resistance in A549 cells unlikely results from enhanced antioxidant enzyme activities, although it is associated with elevated cellular glutathione levels. In addition, measurement of the mRNA and DNA levels for y-glutamylcysteine synthetase, the rate-limiting enzyme for glutathione biosynthesis, revealed that enhanced expression of the enzyme but not gene amplification is likely responsible for the elevation of cellular glutathione levels. Keywords-Glutathione, Cadmium, A549 cells, y-GCS mRNA, Superoxide thione reductase, y-Glutamyltranspeptidase, Cytotoxicity, Free radicals
dismutase,
Catalase,
Glutathione
peroxidase,
Gluta-
and physiological functions.4 It also functions in cytoprotection against electrophilic alkylating agents,5.6 radiation,’ and other toxic compounds.“X89 The GSH redox cycle, which includes GSH, glutathione peroxidase (GSHpx), and glutathione reductase (GR), plays an important role in scavenging oxygen-related and other fi-ee radicals.4
INTRODUCTION
Glutathione (GSH), a ubiquitous sulfhydryl compound, has been implicated to function in cytoprotection against cadmium toxicity. Depletion of cellular GSH by inhibiting its synthesis with buthionine sulfoximine (BSO) or by using diethylmaleate (DEM) to form the GSH-conjugate sensitizes human lung carcinoma cells to cadmium by one third and one half, respectively.’ Conversely, elevation of GSH levels in vivo by 7.5 mmol/kg GSH monoester pretreatment completely prevented cadmium lethality in rats.’ Further studies have indicated that GSH is most likely involved in the early defensive response to cadmium.2.’ The mechanism by which GSH functions in this central role, however, has not been well understood. GSH participates in many normal cellular biochemical
The toxic action of cadmium, although not completely understood, is recognized to be multifactorial. The primary effects of long-term cadmium exposure include chronic obstructive pulmonary disease, emphysema, and chronic renal tubular disease.‘O In addition, cadmium inhalation can cause acute chemical pneumonitis and pulmonary edema.” Furthermore, cadmium may also be carcinogenic.‘0-‘3 Many studies have suggested that oxidative stress is involved in the toxicity of this metal. For example, experiments in vivo have revealed that cadmium causes lipid peroxidation in kidneys, lungs, liver, heart, brain, and testes of rats.14 Increases in antioxidant enzyme activities such as superoxide dismutase (SOD), cat&se, and glutathione peroxidase (GSHpx) have also
Address correspondence to: Y. James Kang, Department of Pharmacology and Toxicology, University of North Dakota School of Medicine, P.O. Box 9037, Grand Forks, ND 58202-9037, USA; Email: [email protected] 805
E. L. HATCHERet al
806
been demonstrated in vivo in cadmium-treated red blood cells.” Furthermore, pretreatment with the antioxidant ascorbic acid reduced cadmium-induced lethality by 85% in rats.‘” Other studies, however, have shown that oxidative stress may not mediate the cadmium toxicity, though cadmium causes such a response.17 Human lung carcinoma A549 fibroblast cells are cadmium-resistant relative to many other normal and tumor cells; for example, the LD,,, value of the parent A549 cells is 25-, 75-, and 30-fold higher than that of AlOlD melanoma, A204 rhabdomyosarcoma, and Madin Darby bovine kidney cells, respectively.‘x~2” These cells have a comparable capacity for metallothionein (MT) synthesis and take up cadmium at a similar rate relative to many other cells including bovine kidney, Chinese hamster, and human blood cells.‘*~*’ When these cells were treated with cadmium at step-wise increased concentrations, single cadmium-resistant clones were selected.** These cadmium-resistant cells with varying cadium sensitivities provide a useful tool for studying mechanisms of cytoprotection against cadmium toxicity. The purpose of this study was to determine whether the elevated cadmium resistance results from enhanced antioxidant activities. We examined the activity of the GSH redox cycle and the possible changes in other antioxidant components such as SOD, catalase, and MT in relation to the cadmium sensitivity. We also determined the possible mechanisms for the elevation of GSH levels in cadmiumresistant cells.
MATERIALS
AND
Cell culture A cadmium-resistant and -sensitive subpopulation of A549 human lung carcinoma cells were established as previously described** and obtained from Dr. M. Duane Enger at Iowa State University. These cells were routinely propagated at 37°C under normoxia and 5% CO, conditions in McCoy’s 5A medium supplemented with 10% fetal bovine serum (FBS). Tests for mycoplasma using a Myco Test kit (GIBCO) were negative. Subcultures for experiments were established by removing the cells from monolayer stock cultures with trypsin-EDTA (0.05% trypsin, 0.53 mM EDTA4Na) and plated in 35-mm tissue culture dishes in a total volume of 2 ml, or in 60-mm dishes in 5 ml. Tissue culture supplies were purchased from Coming Glass Works (Coming, NY).
Cd* ’ sensitivity The cytotoxic effect of Cd was determined by a long-term survival assay. Briefly, approximately 500 cells were plated in 60-mm dishes in 5 ml of medium. Cells were allowed to grow for 24 h, then 15 ~1 CdC12 solution was added to give final Cd concentrations ranging from 0 to 40 ,uM. After 13-day incubation, medium was removed from each dish, and the colonies were rinsed with dissociation medium before being stained with 0.5 ml crystal violet in MeOH. After gently rinsing dishes with tap water, colonies were enumerated as a function of Cd concentrations.
METHODS
Materials The following chemicals and enzymes were obtained from Sigma Chemical Co. (St. Louis, MO): glutathione (GSH), NADPH, bathophenanthrolinedisulfonic acid (BPDS), and I-fluoro-2,4_dinitrobenzene (FDNB). Other chemicals including iodoacetic acid, m-cresol purple, and perchloric acid (PCA) were obtained from Aldrich Chemical Co. (Milwaukee, WI). Solvents used for high-performance liquid chromatography (HPLC) were obtained from Fisher Scientific, Inc. (Chicago, IL). 3-Aminopropyl-spherisorb column (20 cm X 4.6 mm, 5-pm particles) was obtained from Custom L.C. Inc., (Houston, TX). McCoy’s 5A medium was purchased from GIBCO, BRL (Grand Island, NY), and fetal bovine serum was from Hyclone (Logan, UT). The BCA protein assay reagents were obtained from Pierce (Rockford, IL). All other chemicals were purchased from either Sigma or Aldrich. All reagents were at least analytical grade, and HPLC solvents were HPLC grade.
Cellular
GSH concentration
Total GSH was determined by the HPLC assay described by Reed et al.*’ with some modifications. Briefly, the medium was removed from the dishes, and 10% perchloric acid (PCA) containing 1 .O mM BPDS was added after the monolayer cultures were washed 2 times with PBS. After 20 min the PCA-soluble extract was transferred to microfuge tubes. To the PCAinsoluble portion, 0.1 M NaOH was added for protein determination. An iodoacetate solution (100 n&I) containing 0.2 mM m-cresol purple was added to the PCA solution followed by addition of KHCO, powder in small increments until CO, was no longer evolved. After 15 min incubation at room temperature in the dark, an equal volume of 3% FDNB in absolute ethanol was added. The samples were then vortexed and stored at 4°C for 24 h prior to analysis with a Beckman model 338 gradient HPLC system with a 507 autosampler and GOLD software. The GSH derivative was separated with a 3-aminopropyl column and measured at 365 nm with a UV-vis variable detector. Total protein
GSH and cadmium resistance was determined using the Pierce BCA protein assay reagents (Rockford, IL) as described by Smith et a1.24
content
Cellular metallothionein
(MT) concentration
Total MT was determined by the cadmium-hemoglobin affinity assay?’ Triplicate flasks each containing 6 x 10” cells in 20 ml medium were seeded for control and cadmium treatment. After 12 h incubation, 50 ~1 HZ0 or CdCl, to a final concentration of 10 /IM was added. The cells were then harvested for analysis at 24 h post seeding. The medium was removed from the flasks, and monolayer cells were rinsed with PBS, trypsinized, and resuspended in 10 ml RPM1 medium. Cell count was determined by a Coulter counter after mixing 100 ~1 cell suspension in 10 ml electrolyte solution. The remaining cells were centrifuged at 2000 X g for 10 min, the supernatant was discarded, and the cell pellet was resuspended in 5 ml cold PBS and centrifuged at 2500 X g for 10 min. To the pellet 500 ~1 10 mM Tris-HCl was added. The cells were then pulse-sonicated in an HZ0 cup horn with a Branson Sonifier (Model 450) equipped with a microtip (Branson Ultrasonics Corp., Danbury, CT). At an output setting of 3 and duty cycle setting of 50, each sample was sonicated for 30 s four separate times, resting on ice for 30 s min between each sonication. Following centrifugation at 10,000 X g for 15 min, 200 ~1 supernatant was transferred to microtubes for MT analysis, and 100 ~1 was transferred to separate microtubes for protein analysis by the method of Smith et a1.24 Samples were then prepared for MT determination as described previously.25
Enzyme assays Medium was removed from dishes, and monolayer cells were rinsed with PBS, trypsinized, and resuspended in cold PBS containing 0.1% bovine serum albumin (BSA). Following centrifugation at 2,000 x g for 10 min, the supematant was discarded, cells were resuspended in cold PBS without BSA, then centrifuged at 2,500 X g for 15 min. Cells were resuspended in respective enzyme buffer. With the exception of the y-GT assay, the resuspended cells were lysed by sonication with the Branson Sonifier as described above. At an output setting of 3 and duty cycle setting of 50, each sample was sonicated for 30 s three separate times, resting on ice for 30 s between each sonication. Samples were then centrifuged at 12,000 X g for 45 min, and an aliquot of the supematant was used for the respective enzyme assay.
807
Catalase. The enzyme activity was determined by the method described by Aebi.2” The assay buffer was 50 mM KH,POJSO mM NaH2PO4 (1: 1.5, pH 7.0). In a cuvette 1.0 ml sample was added to 1 .O ml of the buffer. The reaction was initiated by adding 1.0 ml 30 mM H202, and the change in absorbance at 240 nm was monitored at 25°C for 30 s. A portion of the remaining sample was used for protein determination. Enzyme activity was calculated as described by Nelson and Kiesow.27 Specific activity is expressed as mol H20z/ min per milligram of protein. Protein was determined by the method of Smith et a1.;24 bovine serum albumin was used as the standard. GSH peroxidase. The enzyme activity was determined by the method described by Flohe and Gunzler.2” The assay buffer was 0.1 M KH2P04 (pH 7.0) containing 1 mM EDTA. In RIA tubes, 400 ~1 buffer containing 2 mM sodium azide, 100 ~1 GSH (10 mM), 100 ~1 GSH reductase (2.4 U/ml), and 200 ~1 sample were incubated for 10 min in 37°C water bath. After 100 yl NADPH (1.5 n-M) was added, the reaction mixture was transferred to cuvettes. Absorbance at 340 nm was monitored at 37°C for 3 min, then 100 ~1 prewarmed Hz02 (1.5 mM) was added to the reaction mixture, and followed by an additional 5 min monitoring under the same conditions. Enzyme activity was calculated as described.28 Specific activity is expressed as nmol NADPWmin per milligram of protein. GSH reductase. The enzyme activity was determined by the method described by Carlberg and Mannervik.29 The assay buffer was 0.2 M KH2PO4 (pH 7.0) containing 2 mM EDTA. In a cuvette, 0.5 ml buffer, 200 ~1 ddHz0, 50 ~1 GSSG (20 mM) and 50 ~1 NADPH (2 mM) were combined. Following addition of 200 ~1 of the sample, the change in absorbance at 340 nm was monitored at 37°C for 2 min. The enzyme activity was calculated as described.29 Specific activity is expressed as nmol NADPH/min per milligram of protein. Superoxide dismutase (SOD). The enzyme activity was determined by the method described by Sun et al.‘” with some modification. A OS-ml aliquot was added to 2.45-ml solution containing 0.1 mM xanthine, 0.1 mM EDTA, 50 mg BSA, 25 M nitroblue tetrazolium (NBT), and 40 mM Na2C03 (pH 10.2). At 25”C, 50 ~1 xanthine oxidase (167 U/L) in 0.2 M (NH4)S04 was added to each tube at 30-s intervals. At 30 min post addition of xanthine oxidase to the first sample, 1.0 ml CuC12 (0.8 mM) was added to each tube at 30-s intervals to terminate the reaction. The production of formazan blue was determined at 560 nm. Enzyme activity was calculated as described.“” Bovine liver
808
E. L. HATCHER et al.
Cu,ZnSOD was used as the standard. Specific is expressed as units/milligram protein.
activity
y-Glutamyltranspeptidase f-y-GT). The enzyme activity was determined by the method described by Szasz” with some modification. A 0.1 -ml aliquot of whole cell suspension, but not cell homogenate, was added to 1 .O ml ammediol-HCl buffer (pH 8.6) containing 4.4 mM L-y-glutamyl-p-nitroanilide, 22.0 mM glycylglycine, and 11.0 mM magnesium chloride. The reaction at 25°C was measured by the change in absorbance at 405 nm. The enzyme activity was calculated as described.3’ Specific activity is expressed as units/milligram of protein. It should be noted that GSH inhibits y-GT activity; therefore, activities were measured in whole cell suspensions rather than in homogenates.
Determination of the relative mRNA and DNA
amount
of y-GCS
The relative levels of y-GCS mRNA and DNA in exponentially growing cells were separately analyzed by Northern and Southern blot assays, respectively. Briefly, the media were removed from each flask, and the monolayer cells were rinsed with PBS and trypsinized. The cells were then resuspended in 10 ml RPM1 and transferred to separate 15 ml tubes. They were centrifuged at 2500 x g for 10 min, and the supematant was discarded. After being resuspended in 5 ml cold PBS, the cells were centrifuged at 3500 X g for 15 min. The supematant was removed, and the cells were stored at -70°C until analysis. For Northern analysis, total RNA was isolated using the RNAzol B method (Cinna/Biotecx, Friendswood, TX) and quantified spectrophotometrically. For Southern analysis, genomic DNA was isolated by phenol-chloroform extraction and quantified spectrophotometrically. The RNA or DNA was then subjected to a 1% denaturating agarose gel-electrophoresis and transferred to a GeneScreen Plus membrane (DuPont). Hybridization and wash procedure were conducted by the method described by Church and Gilbert.92 The probe corresponding to a 764-base pair PstI fragment of human y-GCS complementary DNA was obtained from Dr. R. Timothy Mulcahy at the University of Wisconsin Comprehensive Cancer Center.13 The probe was labeled with 32P dCTP using the random-prime method of Feinberg and Vogelstein. After autoradiography, the Northern analysis membrane was stripped and rehybridized with human P-actin cDNA to ensure integrity of the RNA sample and to confirm that equal amounts of RNA had been loaded onto all lanes. Autoradiographic images were scanned and analyzed using
the MCID system tario, Canada).
Statistical
from Imaging
Research
Inc. (On-
analysis
Data were initially analyzed by model-I ANOVA. The Student’s t-test was employed for further determination of the significance of differences. Differences between treatments were considered significant at p < 0.01. All experiments were well repeated three times with the exception of measurements of antioxidant enzyme activities (Table l), which were repeated twice. The data were presented as the mean + SE values.
RESULTS
Cd cytotoxicity was measured by a long-term survival assay, a sensitive and reliable method for determining cytotoxicity in vitro. As shown in Figure 1, the two subpopulations of A549 cells displayed a significant difference in sensitivity at each concentration of CdC&, with LCsO values of 36.8 PM and 6.9 PM. We designate these cells cadmium-resistant (A549-CdR) and cadmium-sensitive (A549-CdS), respectively. Cd resistance may result from decreased Cd uptake and/or enhanced metallothionein (MT) synthesis in cultured cells. Previous studies, however, have demonstrated that changes in Cd uptake are not responsible for the difference in Cd sensitivity among subpopulations of A549 cells.35 There is also no difference in the capability of MT synthesis among these cells as measured by [‘S] pulse-labeling and electrophoresis assay.?’ To confirm this result, we employed the cadmium-hemoglobin affinity assay in this study to determine the basal and Cd-induced MT levels in A549-CdR and -Cd” cells. As shown in Figure 2, 12 h cadmium treatment induced an approximately 2-fold increase in
Table
1. Antioxidant Cd-Sensitive
Enzymatic Activities of A549 and Cd-Resistant Cells Cd-Sensitive
Catalase *mg) (pmolHZOJmin GSHpx (nmol NADPH/min *mg) GR (nmol NADPWmin. mg) SOD (Wmg) y-GT (mU/mg)
Cd-Resitant
43.9 k 2.5
42.1 2 2.9
9.7 + 1.0
11.2 2 0.7
103.7 I? 4.8
114.3 2 4.6
1.4 !I 0.1 118.5 2 4.9
1.3 5 0.1 129.0 2 4.6
Cells were seeded at a density of 500,000 cells/dish, and enzyme activities were measured at 24 h post seeding. No significant differences were found between the cell lines in the activities of catalase, glutathione peroxidase (GSHpx), glutathione reductase (GR), superoxide dismutase (SOD), or y-glutamyltranspeptidase (y-GT).
809
GSH and cadmium resistance
10
[CdCI 2 ] jr: Fig. 1. Cd*’ cytotoxic responses in Cd-sensitive and Cd-resistant subpopulations of A549 cells. The cells were exposed to varying concentrations of CdCl,, at a cell density of 40%500 cells/dish during a 13-day period. Each point is expressed as the mean 2 SE from triplicate cultures. Cd-sensitive cells have an LCsO of 6.9 M, and Cd-resistant cells have an LC& of 36.8 M. The LCsO values were estimated by linear regression. This experiment was repeated three times with consistent results.
cellular MT in both cell lines. However, no difference was found in either basal or induced MT levels between A549-CdR and -CdS cells, confirming that alteration of MT synthesis is not involved in the development of Cd resistance in A549 cells.
4
0
Basal
??1OpMCd 1 u)
= $ (0 0 r .
G 5 E c
GSH has been implicated in cytoprotection against cadmium toxicity. We therefore examined whether elevated GSH content is involved in the development of cadmium resistance. Because the GSH content in A549 cells varies dramatically as a function of cell growth stage, peaking at 24 h after culturing,‘6 GSH levels were measured at 12 and 24 h after culturing. As shown in Figure 3, GSH concentration is 2-fold higher in the A549-CdR cells than the A549-CdS cells at 12 h, and 1.5-fold higher at 24 h. GSH is an important antioxidant, and the GSH redox cycle, consisting of GSH, GSH peroxidase (GSHpx), and GSH reductase (GR), plays an important role in protection against free radical-induced damage.4 Therefore, to determine whether the elevation of GSH content is associated with enhanced activity of the GSH redox cycle, the enzyme activities of GSHpx and GR were measured. As shown in Table 1, no differences were found in the activities of either of these enzymes between the A549CdR and -CdS cells. Two other enzymes, superoxide dismutase (SOD) and catalase, arc also important antioxidant components. The two cell lines, however, displayed similar activities for both of these enzymes as well (Table 1). The results thus indicate that increased cadmium resistance in A549 cells does not correlate with enhanced antioxidant enzymatic activities. The mechanism by which cellular GSH content was altered in response to the development of Cd resistance was unknown. There are three possible mechanisms by which cellular GSH levels can be elevated. The first is enhanced GR activity, which results in greater
0 Cd-sensitive ??Cd-resistant
??
3-
??
2??
l-
OCd-sensitive
Cd-resistant
Fig. 2. Basal and Cd-induced MT levels in A549 Cd-sensitive and Cd-resistant cells. Cells were seeded at a density of 6 x lo6 cells/ flask, and either Hz0 or CdCl, to final concentration of 10 PM was added at 12 h. MT levels were determined at 24 h post seeding. Cdinduced MT levels were significantly higher than basal levels for both subpopulations @ < O.Ol), but no significant difference in basal or induced MT levels was found between Cd-sensitive and Cdresistant cells. Consistent results were obtained with three repeated experiments.
d-l 12
24
Time (hr)
Fig. 3. Intracellular GSH concentrations at 12 and 24 h in Cdsensitive and Cd-resistant A549 cells. Cd-resistant cells exhibited significantly higher GSH levels at both 12 and 24 h @ < 0.01). though the fluctuation in GSH was similar between the two cells. This experiment was repeated three times with consistent results.
E. L. HATCHER et al.
810
reduction of GSSG to GSH, and thus prevents net loss of total cellular GSH. The second is an increase in the activity of y-glutamyltranspeptidase (y-GT), an external cell-surface enzyme, that can enhance degradation of extracellular GSH to release its constituent amino acids into the cell, thereby elevating the substrate concentrations for intracellular GSH synthesis. The third involves the rate-limiting step in GSH synthesis, catalyzed by y-glutamylcysteine synthetase (yGCS). An increased activity of y-GCS would result in enhanced cellular GSH synthesis. As shown in Table 1, there is no significant difference in GR activity between the A549-CdR and -CdS cells. The y-GT activities were 129.0 2 14.7 and 118.5 + 15.6 mU/mg protein in the CdR and CdS cells, respectively, displaying no significant difference between the two cell lines. Therefore, neither GR nor y-GT alteration is responsible for the elevated cellular GSH content in the CdR cells. We then compared the y-GCS mRNA levels in the two cell lines. As shown in Figure 4, the relative amount of the steady-state y-GCS mRNA was markedly increased in the A549-CdR cells. Quantitative densiometric analysis revealed that the CdR cells exhibited about 3-fold higher y-GCS mRNA levels than the Cd” cells. This alteration correlates with the difference in cellular GSH levels between the two cell lines. To further understand the possible mechanism responsible for the elevated GSH levels, we used the Southern blot method to determine whether amplification of the y-GCS gene occurs in the CdR cells. As shown in Figure 5, no significant differences were detected in the relative amount of the -y-GCS DNA between the CdR and Cd’ cells. This result suggests that
Cd-semi tive
Cd-resistati
t
y-GCS
p-actin
Fig. 4. Northern blot analysis of y-GCS expression in exponentially growing Cd-resistant and Cd-sensitive A549 cells. The +XS mRNA level in the Cd-resistant cells was 3-fold higher than in the Cd-sensitive cells, as measured by densiometry. This experiment was repeated three times with consistent results.
the elevation from y-GCS
of cellular GSH levels gene amplication.
does not result
DISCUSSION
The importance of intracellular GSH in cytoprotection against toxic agents including reactive oxygen species, radiation, and alkylating agents has been widely demonstrated.7-y Several studies indicate that GSH also functions in cellular defense against cadmium toxicity.‘-? Depletion or elevation of cellular GSH content has been shown to enhance or attenuate cadmium sensitivity, respectively.“’ In addition, cadmium cytotoxicity was found to correlate with the growth-phase dependent fluctuation in GSH levels in A549 cells.36 In this study cellular GSH content was found to be 2-fold higher in the A549-CdR cells compared to the A549CdS cells, further demonstrating the involvement of GSH in cadmium cytoprotection. Two important factors often associated with cadmium resistance are cell membrane transport and MT synthesis. It has been shown that decreased cadmium uptake is highly correlated with cadmium resistance in CHO and other culture cells.‘9,22 Cadmium, among several heavy metals, induces MT synthesis. Enhanced capacity for MT synthesis markedly increases cellular resistance to cadmium.‘7 However, neither alteration in cadmium uptake nor change in MT synthesis capacity is responsible for the development of relative cadmium resistance in A549 cells, as previously reported”5 and confirmed in this study. It appears that elevation of cellular GSH levels does not affect cellular cadmium transport or MT synthesis. However, it is unknown whether the elevation of cellular GSH is solely responsible for the development of cadmium resistance in A549 cells. Many studies have implicated oxidative damage as a possible toxic action of cadmium. Lipid peroxidation in vivo and in vitro has been detected in association with cadmium treatment. “~38m4’Alterations in the activities of antioxidant enzymes such as SOD, catalase, and GSHpx in response to cadmium were also reported.‘5,42 Because GSH is an important antioxidant, participating in enzymatic and nonenzymatic detoxification of many oxidative toxicants, it is worthwhile to investigate whether the cadmium-induced elevation of GSH concentration is associated with alterations in antioxidant enzyme activities. As mentioned, the GSH redox cycle consisting of GSH, GSHpx, and GR is a major component of the antioxidant defense system. We therefore determined the activities of GSHpx and GR in the two subpopulations of A549 cells. The results showed that there is no significant difference in either of these enzymatic
GSH and cadmium
Cd-sensitive
Cd-resistant
Fig. 5. Southern blot analysis of y-GCS DNA levls in exponentially growing Cd-resistant and Cd-sensitive A549 cells. No difference was detected in the y-GCS DNA levels between the sublines. This experiment was repeated twice with consistent results.
activities between the CdR and CdS cells. The elevation of cellular GSH levels is thus independent of enzymatic changes in the GSH redox cycle. We next examined the status of two other important antioxidant enzymes, SOD and catalase. The activities of these two enzymes did not change in association with the development of cadmium resistance, either. Therefore, cadmium resistance in A549 cells does not result from alterations in the enzymatic components of the antioxidant defense system. Studies in cell-free systems have shown that GSH forms a complex with cadrnium.43 Whether this complex occurs in vivo or in culture cells and whether it represents an important mechanism by which GSH detoxifies cadmium are unknown. Recent studies, however, showed that addition of exogenous GSH to culture medium reduced cellular cadmium accumulation, resulting in decreased cadmium cytotoxicity.44 The reduced cadmium accumulation presumably results from formation of a GSH-Cd complex, thereby preventing Cd uptake by the cells. In this study, a possible mechanism for the elevation of cellular GSH levels was also examined. The results demonstrate that enhanced GR-catalyzed reduction of GSSG to GSH is not responsible for the cellular GSH elevation because GR activity did not increase in association with the increased GSH levels. Enhanced extracellular GSH degradation and thereby intracellular resynthesis is also not responsible for the GSH elevation because y-CT activity was unchanged. Finally, analysis of y-GCS mRNA revealed that the relative amount of the mRNA was indeed elevated in the A549-CdR cells. Gene amplification is reported to be a common mechanism of metal resistance, including cadmium re-
811
resistance
sistance, in mammalian cells.20 However, the relative amount of the y-GCS DNA did not differ between the A549-CdR and -Ccl” cells, indicating that no amplification of y-GCS gene occurs in the A549-CdR cells. Therefore, enhanced expression of y-GCS, but not gene amplification, is associated with the increased cellular GSH levels in the A549-CdR cells as compared to the A549-CdS cells. In summary, this study investigated whether the development of cadmium resistance in A549 cells is associated with enhanced antioxidant activities. The results obtained show that although cellular GSH levels in the CdR clone were elevated more than 2-fold over those of the CdS clone, the activities of antioxidant enzymes were not altered, demonstrating that enhanced antioxidant activities are unlikely responsible for the development of cadmium resistance. One possible mechanism by which GSH prevents Cd toxicity may be the formation of a GSH-Cd complex. The study also demonstrates that enhanced expression of ?I-GCS but not gene amplification is likely responsible for the elevation of cellular GSH levels associated with the development of Cd resistance.
Acknowledgements-This study was supported in part by EPAIEPSCoR grant R82 1836 and by the University of North Dakota School of Medicine. The authors thank KayLynn Boushee for typing the manuscript and Dr. R. Timothy Mulcahy at the University of Wisconsin Comprehensive Cancer Center for the gift of y-GCS cDNA probe. This work was presented in part at the 33rd Annual Meeting of the Society of Toxicology, Dallas, TX, March 13-17, 1994.
REFERENCES I Kang, Y. J.; Enger, M. D. Effect of cellular glutathione depletion on cadmium-induced cytotoxicity in human lung carcinoma cells. Cell Biol. Toxicol. 3(4):347-360; 1987. 2 Singhal, R. K.; Anderson, M. E.; Meister, A. Glutathione, a first line of defense against cadmium toxicity. FASEB J. 1:220-223; 1987. 3. Kang, Y. J.; Enger, M. D. Glutathione is involved in the early cadmium cytotoxic response in human lung carcinoma cells. Toxicology 48:93-101; 1988. 4. Meister, A. Metabolism and function of glutathione. In: Dolphin, D.; Poulson, R.; Avramovic, 0.. eds., Glutathione. Chemical, biochemical, and medical aspects, Part A. New York: John Wiley & Sons, Inc.; 1989:367-474. 5. Andrews, P. A.; Murphy, M. P.; Howell, S. B. Differential potentiation of alkylating and platinating agents’ cytotoxicity in human ovarian carcinoma cells by glutathione depletion. Cancer Res. 45:6250-6253; 1985. 6. Russo, A.; Mitchell, J. B. Potentiation and protection of doxombicin cytotoxicity by cellular glutathione modulation. Cancer Treat Rep. 69: 1293- 1296; 1985. I. Biaglow, J. E.; Varnes, M. E.; Clark, E. P.; Epp. E. R. The role of thiols in cellular response to radiation and &tgs. Radiat. Res. 95:437-455; 1983. 8. Ketterer, B. The role of nonenzymatic reactions of glutathione in xenobiotic metabolism. Drug Metab. Rev. 13: 16 I- 187; 1982. 9. Jones, T. W.; Thor, H.; Orrenius, S. Cellular defense mechanism
812
IO.
Il.
12.
13.
14.
15
16
17
I8
19.
20.
21.
22.
23.
24.
25.
26. 27.
E. L. HATCHER et al.
against toxic substances. Arch. Toxicol. Suppl. 9:259-271; 1986. Goyer, R. A. Toxic effects of metals. In: Amdur, M. 0.; Doull, J.; Klassen, C. D., eds. Casarett and Doull’s toxicology: The basic science of poisons, 4th ed. New York: Pergamon Press; 1991:634-637. Sorahan, T.; Waterhouse, J. A. J. Mortality study of nickel cadmium battery workers by the method of regression models in life tables. Br. J. Ind. Med. 40:293-300; 1983. Thun, M. J.; Schnorr, T. M.; Smith, A. B.; Halperin, W. E.; Lemen, R. A. Mortality among a cohort of U.S. cadmium production workers-an update. J. Natl. Cancer Inst. 74:325-333; 1985. Takenoka, S.; Oldiges, H.; Konig, H.; Hochrainer, D.; Oberdorster, G. Carcinogenicity of cadmium chloride aerosols in W rats. J. Natl. Cancer Inst. 70:367-373; 1983. Manta, D.; Richard, A. C.; Trottier, 9.; Chevalier, G. Studies on lipid peroxidation in rat tissues following administration of low and moderate doses of cadmium chloride. Toxicology 67(3):303-323; 1991. Kostic, M. M.; Ognjanovic, B.; Dimitrijevic, S.; Zikic, R. V.; Stajn, A.; Rosic, G. L.; Zivkovic, R. V. Cadmium-induced changes of antioxidant and metabolic status in red blood cells 1993. of rats: In vivo effects. Eur. J. Haematol. 51:86-92; Shiraishi, N.; Uno, H.; Waalkes, M. P. Effect of L-ascorbic acid pretreatment on cadmium toxicity in the male Fischer (F344/ Ncr) rat. Toxicology 85:85-100; 1993. Stacey, N. H.; Cantilena, L. R.; Klassen, C. D. Cadmium toxicity and lipid pcroxidation in isolated rat hepatocytes. Toxicol. Appl. Pharmacol. 53:470-480; 1980. Seagrave, J. C.; Hildebrand, C. E.; Enger, M. D. Effects of cadmium on glutathione metabolism in cadmium sensitive and cadmium resistant Chinese hamster cell lines. Toxicology 29:101-107; 1983. Bracken, W. M.; Sharma, R. P.; Kleinschuster, S. J. Cadmium accumulation and subcellular distribution in relation to cadmium chloride induced cytotoxicity in vitro. Toxicology 33:93- 102; 1984. Enger, M. D.; Hildebrand, C. E.; Walters, R. A.; Seagrave, J. C.; Barham, S. S.; Hoagland, H. C. Molecular and somatic cell genetic analysis of metal resistance mechanisms in mammalian cells. In: Tasjjian, A. H., ed. Molecular and cellular approaches to understanding mechanisms of toxicity. Boston: Harvard School of Public Health; 1984:38-62. Wong, K-L.; Klaassen, C. D. Relationship between liver and kidney levels of glutathione and metallothionein in rats. Toxicology 19:39-47; 1981. Enger, M. D.; Hildebrand, C. E.; Seagrave, J. C.; Tobey, R. A. Clonal variation of cadmium response in human tumor cell lines. Am. J. Physiol. 25O:C256-C263; 1986. Reed, D. J.; Babson, J. R.; Beatty, P. W.; Brodie, A. E.; Ellis, W. W.; Potter, D. W. High-performance liquid chromatography analysis of nanomole levels of glutathione, glutathione disulfide, and related thiols and disulfides. Anal. Biochem. 106:55-62; 1980. Smith, P. K.; Krohn, R. I.; Hermanson, Cl. T.; Mallia, A. K.; Gartner, F. H.; Proverzano, M. D.; Fujimoto, E. K.; Goeke, N. M.; Olson, B. J.; Klerk, D. C. Measurement of protein using bicinchoninic acid. Anal. Biochem. 150:76-85; 1985. Eaton, D. L.; Cherian, M. G. Determination of rnetallothionein in tissues by cadmium-hemoglobin affinity assay. Methods Enzymol. 205:83-88; 1991. Aebi, H. Catalase in vitro. Methods Enzymol. 105:121- 126; 1984. Nelson, D. P.; Kiesow, L. A. Enthalpy of decomposition of hydrogen peroxide by catalase at 25°C (with molar extinction coefficients of H202 solutions in the UV). Anal. Biochem. 49:474-478; 1972.
28. Flohe, L.; Gunzler, W. A. Assays of glutathione peroxidase. Methods Enzymol. 105: 1 l4- 121; 1984. 29 Carlberg, I.; Mannervik, B. Glutathione reductase. Methods En1985. zymol. 113:484-490; 30. Sun, Y.; Oberley, L. W.; Ying, L. A simple method for clinical assay of superoxide dismutase. Clin. Chem. 34:497-500; 1988. 31. Szasz, G. A kinetic photometric method for serum y-glutamyl transpeptidase. Clin. Chem. 15:l24- 136; 1969. 32. Church, G. M.; Gilber, W. Genomic sequencing. Proc. Natl. Acad. Sci. USA 81:1991-1995; 1984. 33. Gipp, J. J.; Chang, C.; Mulcahy, R. T. Cloning and nucleotide sequence of a full-length cDNA for human liver y-glutamylcysteine synthetase. Biochem. Biophys. Res. Commun. X35:29-35; 1992. 34. Feinberg, A. P.; Vogelstein, B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132:6-13; 1983. 35. Kang, Y. J.; Nuutero, S. T.; Clapper, J. A.; Jenkins, P.; Enger, M. D. Cellular cadmium responses in subpopulations T20 and T27 of human lung carcinoma A549 cells. Toxicology 61:195203; 1990. 36. Kang, Y. J.; Enger, M. D. Cadmium cytotoxicity correlates with the changes in glutathione content thatoccur during the logarithmic growth phase of A549-T27 cells. Toxicol. Left. 51:23-28: 1990: 37. Hilderbrand, C. E.; Tobey, R. A.; Campbell, E. W.; Enger, M. D. A cadmium-resistant variant of the Chinese hamster (CHO) cell line with increased metallothionein induction capacity. Exp. Cell Res. 124:237-246; 1979. 38. Koizumi, T.; Li, 2. G. Role of oxidative stress in single-dose, cadmium-induced testicular cancer. J. Toxicol. Environ. Health. 37:25-36; 1992. 39. Szymanska, E.; Laskowska-Klita, T. Effect of cadmium on lipid peroxidation and activities of antioxidant enzymes, Acta Biochim. Pol. 40:144-146; 1993. 40. Shukla, G. S.; Chandra, S. V. Cadmium toxicity and bioantioxidants: Status of vitamin E and ascorbic acid of selected organs in rats. J. Appl. Toxicol. 9:119-122; 1989. 41. Muller, L. Consequences of cadmium toxicity in rat hepatocytes: Mitochondrial dysfunction and lipid peroxidation. Toxicology 40:285-295; 1986. 42. Gupta, S.; Athar, M.; Behari, J. R.; Srivastava, R. C. Cadmiummediated induction of cellular defence mechanism: A novel example for the development of adaptive response against the toxicant. Znd. Health. 29:1-9; 1991. 43. Perrin, D. D.; Watt, A. E. Complex of formation of zinc and cadmium with glutathione. Biochim. Biophys. Actu. 230:96104; 1971. 44. Kang, Y. J. Exogenous glutathione decreases cellular cadmium uptake and toxicity. Drug Metab. Disp. 20:714-718; 1992.
ABBREVIATIONS
FDNB - 1-fluoro-2,3_dinitrobenzene GR-glutathione reductase GSH-glutathione GSHpx-gglutathione peroxidase GSSG-oxidized glutatbione HPLC-high performance liquid chromatography MT-metallothionein PBS-phosphate buffered saline PCA-perchloric acid y-GCS - y-glutamylcysteine synthetase y-GTy-glutamyltranspeptidase
Copyright0 1995ElsevierScienceInc. Printedin the USA. All rightsreserved 0891-5849195 $9.50 + .OO
Pergamon
--bOriginal
Contribution
CADMIUM RESISTANCE IN A549 CELLS CORRELATES WITH ELEVATED GLUTATHIONE CONTENT BUT NOT ANTIOXIDANT ENZYMATIC ACTIVITIES
EMIKO L. HATCHER, YAN CHEN, and Y. JAMES KANG Department of Pharmacology and Toxicology, University of North Dakota School of Medicine, (Received
3 October
1994; Revised
12 April 1995; Re-revised
18 May 1995; Accepted
Grand Forks, ND, USA 23 May 1995)
Abstract-Glutathione has been implicated to function in cytoprotection against cadmium toxicity. The mechanism by which glutathione plays this role has not been well understood. Because glutathione is an important antioxidant and several studies have shown that cadmium induces oxidative stress, this study was undertaken to determine whether development of cadmium resistance is linked to enhanced antioxidant activities. A cadmium-resistant subpopulation of human lung carcinoma A549 cells, which was developed by repeatedly exposing the cells to step-wise increased cadmium concentrations, was compared to a cadmium-sensitive one. The acquired cadmium resistance resulted from neither decreased cadmium uptake nor enhanced cellular metallothionein synthesis. Glutathione content, however, was markedly elevated in the cadmium-resistant cells. In contrast, the activities of the glutathione redox cycle related enzymes, glutathione peroxidase and reductase, were unchanged. Two other antioxidant enzymes, superoxide dismutase and catalase, were also not altered. The results suggest that the development of cadmium resistance in A549 cells unlikely results from enhanced antioxidant enzyme activities, although it is associated with elevated cellular glutathione levels. In addition, measurement of the mRNA and DNA levels for y-glutamylcysteine synthetase, the rate-limiting enzyme for glutathione biosynthesis, revealed that enhanced expression of the enzyme but not gene amplification is likely responsible for the elevation of cellular glutathione levels. Keywords-Glutathione, Cadmium, A549 cells, y-GCS mRNA, Superoxide thione reductase, y-Glutamyltranspeptidase, Cytotoxicity, Free radicals
dismutase,
Catalase,
Glutathione
peroxidase,
Gluta-
and physiological functions.4 It also functions in cytoprotection against electrophilic alkylating agents,5.6 radiation,’ and other toxic compounds.“X89 The GSH redox cycle, which includes GSH, glutathione peroxidase (GSHpx), and glutathione reductase (GR), plays an important role in scavenging oxygen-related and other fi-ee radicals.4
INTRODUCTION
Glutathione (GSH), a ubiquitous sulfhydryl compound, has been implicated to function in cytoprotection against cadmium toxicity. Depletion of cellular GSH by inhibiting its synthesis with buthionine sulfoximine (BSO) or by using diethylmaleate (DEM) to form the GSH-conjugate sensitizes human lung carcinoma cells to cadmium by one third and one half, respectively.’ Conversely, elevation of GSH levels in vivo by 7.5 mmol/kg GSH monoester pretreatment completely prevented cadmium lethality in rats.’ Further studies have indicated that GSH is most likely involved in the early defensive response to cadmium.2.’ The mechanism by which GSH functions in this central role, however, has not been well understood. GSH participates in many normal cellular biochemical
The toxic action of cadmium, although not completely understood, is recognized to be multifactorial. The primary effects of long-term cadmium exposure include chronic obstructive pulmonary disease, emphysema, and chronic renal tubular disease.‘O In addition, cadmium inhalation can cause acute chemical pneumonitis and pulmonary edema.” Furthermore, cadmium may also be carcinogenic.‘0-‘3 Many studies have suggested that oxidative stress is involved in the toxicity of this metal. For example, experiments in vivo have revealed that cadmium causes lipid peroxidation in kidneys, lungs, liver, heart, brain, and testes of rats.14 Increases in antioxidant enzyme activities such as superoxide dismutase (SOD), cat&se, and glutathione peroxidase (GSHpx) have also
Address correspondence to: Y. James Kang, Department of Pharmacology and Toxicology, University of North Dakota School of Medicine, P.O. Box 9037, Grand Forks, ND 58202-9037, USA; Email: [email protected] 805
E. L. HATCHERet al
806
been demonstrated in vivo in cadmium-treated red blood cells.” Furthermore, pretreatment with the antioxidant ascorbic acid reduced cadmium-induced lethality by 85% in rats.‘” Other studies, however, have shown that oxidative stress may not mediate the cadmium toxicity, though cadmium causes such a response.17 Human lung carcinoma A549 fibroblast cells are cadmium-resistant relative to many other normal and tumor cells; for example, the LD,,, value of the parent A549 cells is 25-, 75-, and 30-fold higher than that of AlOlD melanoma, A204 rhabdomyosarcoma, and Madin Darby bovine kidney cells, respectively.‘x~2” These cells have a comparable capacity for metallothionein (MT) synthesis and take up cadmium at a similar rate relative to many other cells including bovine kidney, Chinese hamster, and human blood cells.‘*~*’ When these cells were treated with cadmium at step-wise increased concentrations, single cadmium-resistant clones were selected.** These cadmium-resistant cells with varying cadium sensitivities provide a useful tool for studying mechanisms of cytoprotection against cadmium toxicity. The purpose of this study was to determine whether the elevated cadmium resistance results from enhanced antioxidant activities. We examined the activity of the GSH redox cycle and the possible changes in other antioxidant components such as SOD, catalase, and MT in relation to the cadmium sensitivity. We also determined the possible mechanisms for the elevation of GSH levels in cadmiumresistant cells.
MATERIALS
AND
Cell culture A cadmium-resistant and -sensitive subpopulation of A549 human lung carcinoma cells were established as previously described** and obtained from Dr. M. Duane Enger at Iowa State University. These cells were routinely propagated at 37°C under normoxia and 5% CO, conditions in McCoy’s 5A medium supplemented with 10% fetal bovine serum (FBS). Tests for mycoplasma using a Myco Test kit (GIBCO) were negative. Subcultures for experiments were established by removing the cells from monolayer stock cultures with trypsin-EDTA (0.05% trypsin, 0.53 mM EDTA4Na) and plated in 35-mm tissue culture dishes in a total volume of 2 ml, or in 60-mm dishes in 5 ml. Tissue culture supplies were purchased from Coming Glass Works (Coming, NY).
Cd* ’ sensitivity The cytotoxic effect of Cd was determined by a long-term survival assay. Briefly, approximately 500 cells were plated in 60-mm dishes in 5 ml of medium. Cells were allowed to grow for 24 h, then 15 ~1 CdC12 solution was added to give final Cd concentrations ranging from 0 to 40 ,uM. After 13-day incubation, medium was removed from each dish, and the colonies were rinsed with dissociation medium before being stained with 0.5 ml crystal violet in MeOH. After gently rinsing dishes with tap water, colonies were enumerated as a function of Cd concentrations.
METHODS
Materials The following chemicals and enzymes were obtained from Sigma Chemical Co. (St. Louis, MO): glutathione (GSH), NADPH, bathophenanthrolinedisulfonic acid (BPDS), and I-fluoro-2,4_dinitrobenzene (FDNB). Other chemicals including iodoacetic acid, m-cresol purple, and perchloric acid (PCA) were obtained from Aldrich Chemical Co. (Milwaukee, WI). Solvents used for high-performance liquid chromatography (HPLC) were obtained from Fisher Scientific, Inc. (Chicago, IL). 3-Aminopropyl-spherisorb column (20 cm X 4.6 mm, 5-pm particles) was obtained from Custom L.C. Inc., (Houston, TX). McCoy’s 5A medium was purchased from GIBCO, BRL (Grand Island, NY), and fetal bovine serum was from Hyclone (Logan, UT). The BCA protein assay reagents were obtained from Pierce (Rockford, IL). All other chemicals were purchased from either Sigma or Aldrich. All reagents were at least analytical grade, and HPLC solvents were HPLC grade.
Cellular
GSH concentration
Total GSH was determined by the HPLC assay described by Reed et al.*’ with some modifications. Briefly, the medium was removed from the dishes, and 10% perchloric acid (PCA) containing 1 .O mM BPDS was added after the monolayer cultures were washed 2 times with PBS. After 20 min the PCA-soluble extract was transferred to microfuge tubes. To the PCAinsoluble portion, 0.1 M NaOH was added for protein determination. An iodoacetate solution (100 n&I) containing 0.2 mM m-cresol purple was added to the PCA solution followed by addition of KHCO, powder in small increments until CO, was no longer evolved. After 15 min incubation at room temperature in the dark, an equal volume of 3% FDNB in absolute ethanol was added. The samples were then vortexed and stored at 4°C for 24 h prior to analysis with a Beckman model 338 gradient HPLC system with a 507 autosampler and GOLD software. The GSH derivative was separated with a 3-aminopropyl column and measured at 365 nm with a UV-vis variable detector. Total protein
GSH and cadmium resistance was determined using the Pierce BCA protein assay reagents (Rockford, IL) as described by Smith et a1.24
content
Cellular metallothionein
(MT) concentration
Total MT was determined by the cadmium-hemoglobin affinity assay?’ Triplicate flasks each containing 6 x 10” cells in 20 ml medium were seeded for control and cadmium treatment. After 12 h incubation, 50 ~1 HZ0 or CdCl, to a final concentration of 10 /IM was added. The cells were then harvested for analysis at 24 h post seeding. The medium was removed from the flasks, and monolayer cells were rinsed with PBS, trypsinized, and resuspended in 10 ml RPM1 medium. Cell count was determined by a Coulter counter after mixing 100 ~1 cell suspension in 10 ml electrolyte solution. The remaining cells were centrifuged at 2000 X g for 10 min, the supernatant was discarded, and the cell pellet was resuspended in 5 ml cold PBS and centrifuged at 2500 X g for 10 min. To the pellet 500 ~1 10 mM Tris-HCl was added. The cells were then pulse-sonicated in an HZ0 cup horn with a Branson Sonifier (Model 450) equipped with a microtip (Branson Ultrasonics Corp., Danbury, CT). At an output setting of 3 and duty cycle setting of 50, each sample was sonicated for 30 s four separate times, resting on ice for 30 s min between each sonication. Following centrifugation at 10,000 X g for 15 min, 200 ~1 supernatant was transferred to microtubes for MT analysis, and 100 ~1 was transferred to separate microtubes for protein analysis by the method of Smith et a1.24 Samples were then prepared for MT determination as described previously.25
Enzyme assays Medium was removed from dishes, and monolayer cells were rinsed with PBS, trypsinized, and resuspended in cold PBS containing 0.1% bovine serum albumin (BSA). Following centrifugation at 2,000 x g for 10 min, the supematant was discarded, cells were resuspended in cold PBS without BSA, then centrifuged at 2,500 X g for 15 min. Cells were resuspended in respective enzyme buffer. With the exception of the y-GT assay, the resuspended cells were lysed by sonication with the Branson Sonifier as described above. At an output setting of 3 and duty cycle setting of 50, each sample was sonicated for 30 s three separate times, resting on ice for 30 s between each sonication. Samples were then centrifuged at 12,000 X g for 45 min, and an aliquot of the supematant was used for the respective enzyme assay.
807
Catalase. The enzyme activity was determined by the method described by Aebi.2” The assay buffer was 50 mM KH,POJSO mM NaH2PO4 (1: 1.5, pH 7.0). In a cuvette 1.0 ml sample was added to 1 .O ml of the buffer. The reaction was initiated by adding 1.0 ml 30 mM H202, and the change in absorbance at 240 nm was monitored at 25°C for 30 s. A portion of the remaining sample was used for protein determination. Enzyme activity was calculated as described by Nelson and Kiesow.27 Specific activity is expressed as mol H20z/ min per milligram of protein. Protein was determined by the method of Smith et a1.;24 bovine serum albumin was used as the standard. GSH peroxidase. The enzyme activity was determined by the method described by Flohe and Gunzler.2” The assay buffer was 0.1 M KH2P04 (pH 7.0) containing 1 mM EDTA. In RIA tubes, 400 ~1 buffer containing 2 mM sodium azide, 100 ~1 GSH (10 mM), 100 ~1 GSH reductase (2.4 U/ml), and 200 ~1 sample were incubated for 10 min in 37°C water bath. After 100 yl NADPH (1.5 n-M) was added, the reaction mixture was transferred to cuvettes. Absorbance at 340 nm was monitored at 37°C for 3 min, then 100 ~1 prewarmed Hz02 (1.5 mM) was added to the reaction mixture, and followed by an additional 5 min monitoring under the same conditions. Enzyme activity was calculated as described.28 Specific activity is expressed as nmol NADPWmin per milligram of protein. GSH reductase. The enzyme activity was determined by the method described by Carlberg and Mannervik.29 The assay buffer was 0.2 M KH2PO4 (pH 7.0) containing 2 mM EDTA. In a cuvette, 0.5 ml buffer, 200 ~1 ddHz0, 50 ~1 GSSG (20 mM) and 50 ~1 NADPH (2 mM) were combined. Following addition of 200 ~1 of the sample, the change in absorbance at 340 nm was monitored at 37°C for 2 min. The enzyme activity was calculated as described.29 Specific activity is expressed as nmol NADPH/min per milligram of protein. Superoxide dismutase (SOD). The enzyme activity was determined by the method described by Sun et al.‘” with some modification. A OS-ml aliquot was added to 2.45-ml solution containing 0.1 mM xanthine, 0.1 mM EDTA, 50 mg BSA, 25 M nitroblue tetrazolium (NBT), and 40 mM Na2C03 (pH 10.2). At 25”C, 50 ~1 xanthine oxidase (167 U/L) in 0.2 M (NH4)S04 was added to each tube at 30-s intervals. At 30 min post addition of xanthine oxidase to the first sample, 1.0 ml CuC12 (0.8 mM) was added to each tube at 30-s intervals to terminate the reaction. The production of formazan blue was determined at 560 nm. Enzyme activity was calculated as described.“” Bovine liver
808
E. L. HATCHER et al.
Cu,ZnSOD was used as the standard. Specific is expressed as units/milligram protein.
activity
y-Glutamyltranspeptidase f-y-GT). The enzyme activity was determined by the method described by Szasz” with some modification. A 0.1 -ml aliquot of whole cell suspension, but not cell homogenate, was added to 1 .O ml ammediol-HCl buffer (pH 8.6) containing 4.4 mM L-y-glutamyl-p-nitroanilide, 22.0 mM glycylglycine, and 11.0 mM magnesium chloride. The reaction at 25°C was measured by the change in absorbance at 405 nm. The enzyme activity was calculated as described.3’ Specific activity is expressed as units/milligram of protein. It should be noted that GSH inhibits y-GT activity; therefore, activities were measured in whole cell suspensions rather than in homogenates.
Determination of the relative mRNA and DNA
amount
of y-GCS
The relative levels of y-GCS mRNA and DNA in exponentially growing cells were separately analyzed by Northern and Southern blot assays, respectively. Briefly, the media were removed from each flask, and the monolayer cells were rinsed with PBS and trypsinized. The cells were then resuspended in 10 ml RPM1 and transferred to separate 15 ml tubes. They were centrifuged at 2500 x g for 10 min, and the supematant was discarded. After being resuspended in 5 ml cold PBS, the cells were centrifuged at 3500 X g for 15 min. The supematant was removed, and the cells were stored at -70°C until analysis. For Northern analysis, total RNA was isolated using the RNAzol B method (Cinna/Biotecx, Friendswood, TX) and quantified spectrophotometrically. For Southern analysis, genomic DNA was isolated by phenol-chloroform extraction and quantified spectrophotometrically. The RNA or DNA was then subjected to a 1% denaturating agarose gel-electrophoresis and transferred to a GeneScreen Plus membrane (DuPont). Hybridization and wash procedure were conducted by the method described by Church and Gilbert.92 The probe corresponding to a 764-base pair PstI fragment of human y-GCS complementary DNA was obtained from Dr. R. Timothy Mulcahy at the University of Wisconsin Comprehensive Cancer Center.13 The probe was labeled with 32P dCTP using the random-prime method of Feinberg and Vogelstein. After autoradiography, the Northern analysis membrane was stripped and rehybridized with human P-actin cDNA to ensure integrity of the RNA sample and to confirm that equal amounts of RNA had been loaded onto all lanes. Autoradiographic images were scanned and analyzed using
the MCID system tario, Canada).
Statistical
from Imaging
Research
Inc. (On-
analysis
Data were initially analyzed by model-I ANOVA. The Student’s t-test was employed for further determination of the significance of differences. Differences between treatments were considered significant at p < 0.01. All experiments were well repeated three times with the exception of measurements of antioxidant enzyme activities (Table l), which were repeated twice. The data were presented as the mean + SE values.
RESULTS
Cd cytotoxicity was measured by a long-term survival assay, a sensitive and reliable method for determining cytotoxicity in vitro. As shown in Figure 1, the two subpopulations of A549 cells displayed a significant difference in sensitivity at each concentration of CdC&, with LCsO values of 36.8 PM and 6.9 PM. We designate these cells cadmium-resistant (A549-CdR) and cadmium-sensitive (A549-CdS), respectively. Cd resistance may result from decreased Cd uptake and/or enhanced metallothionein (MT) synthesis in cultured cells. Previous studies, however, have demonstrated that changes in Cd uptake are not responsible for the difference in Cd sensitivity among subpopulations of A549 cells.35 There is also no difference in the capability of MT synthesis among these cells as measured by [‘S] pulse-labeling and electrophoresis assay.?’ To confirm this result, we employed the cadmium-hemoglobin affinity assay in this study to determine the basal and Cd-induced MT levels in A549-CdR and -Cd” cells. As shown in Figure 2, 12 h cadmium treatment induced an approximately 2-fold increase in
Table
1. Antioxidant Cd-Sensitive
Enzymatic Activities of A549 and Cd-Resistant Cells Cd-Sensitive
Catalase *mg) (pmolHZOJmin GSHpx (nmol NADPH/min *mg) GR (nmol NADPWmin. mg) SOD (Wmg) y-GT (mU/mg)
Cd-Resitant
43.9 k 2.5
42.1 2 2.9
9.7 + 1.0
11.2 2 0.7
103.7 I? 4.8
114.3 2 4.6
1.4 !I 0.1 118.5 2 4.9
1.3 5 0.1 129.0 2 4.6
Cells were seeded at a density of 500,000 cells/dish, and enzyme activities were measured at 24 h post seeding. No significant differences were found between the cell lines in the activities of catalase, glutathione peroxidase (GSHpx), glutathione reductase (GR), superoxide dismutase (SOD), or y-glutamyltranspeptidase (y-GT).
809
GSH and cadmium resistance
10
[CdCI 2 ] jr: Fig. 1. Cd*’ cytotoxic responses in Cd-sensitive and Cd-resistant subpopulations of A549 cells. The cells were exposed to varying concentrations of CdCl,, at a cell density of 40%500 cells/dish during a 13-day period. Each point is expressed as the mean 2 SE from triplicate cultures. Cd-sensitive cells have an LCsO of 6.9 M, and Cd-resistant cells have an LC& of 36.8 M. The LCsO values were estimated by linear regression. This experiment was repeated three times with consistent results.
cellular MT in both cell lines. However, no difference was found in either basal or induced MT levels between A549-CdR and -CdS cells, confirming that alteration of MT synthesis is not involved in the development of Cd resistance in A549 cells.
4
0
Basal
??1OpMCd 1 u)
= $ (0 0 r .
G 5 E c
GSH has been implicated in cytoprotection against cadmium toxicity. We therefore examined whether elevated GSH content is involved in the development of cadmium resistance. Because the GSH content in A549 cells varies dramatically as a function of cell growth stage, peaking at 24 h after culturing,‘6 GSH levels were measured at 12 and 24 h after culturing. As shown in Figure 3, GSH concentration is 2-fold higher in the A549-CdR cells than the A549-CdS cells at 12 h, and 1.5-fold higher at 24 h. GSH is an important antioxidant, and the GSH redox cycle, consisting of GSH, GSH peroxidase (GSHpx), and GSH reductase (GR), plays an important role in protection against free radical-induced damage.4 Therefore, to determine whether the elevation of GSH content is associated with enhanced activity of the GSH redox cycle, the enzyme activities of GSHpx and GR were measured. As shown in Table 1, no differences were found in the activities of either of these enzymes between the A549CdR and -CdS cells. Two other enzymes, superoxide dismutase (SOD) and catalase, arc also important antioxidant components. The two cell lines, however, displayed similar activities for both of these enzymes as well (Table 1). The results thus indicate that increased cadmium resistance in A549 cells does not correlate with enhanced antioxidant enzymatic activities. The mechanism by which cellular GSH content was altered in response to the development of Cd resistance was unknown. There are three possible mechanisms by which cellular GSH levels can be elevated. The first is enhanced GR activity, which results in greater
0 Cd-sensitive ??Cd-resistant
??
3-
??
2??
l-
OCd-sensitive
Cd-resistant
Fig. 2. Basal and Cd-induced MT levels in A549 Cd-sensitive and Cd-resistant cells. Cells were seeded at a density of 6 x lo6 cells/ flask, and either Hz0 or CdCl, to final concentration of 10 PM was added at 12 h. MT levels were determined at 24 h post seeding. Cdinduced MT levels were significantly higher than basal levels for both subpopulations @ < O.Ol), but no significant difference in basal or induced MT levels was found between Cd-sensitive and Cdresistant cells. Consistent results were obtained with three repeated experiments.
d-l 12
24
Time (hr)
Fig. 3. Intracellular GSH concentrations at 12 and 24 h in Cdsensitive and Cd-resistant A549 cells. Cd-resistant cells exhibited significantly higher GSH levels at both 12 and 24 h @ < 0.01). though the fluctuation in GSH was similar between the two cells. This experiment was repeated three times with consistent results.
E. L. HATCHER et al.
810
reduction of GSSG to GSH, and thus prevents net loss of total cellular GSH. The second is an increase in the activity of y-glutamyltranspeptidase (y-GT), an external cell-surface enzyme, that can enhance degradation of extracellular GSH to release its constituent amino acids into the cell, thereby elevating the substrate concentrations for intracellular GSH synthesis. The third involves the rate-limiting step in GSH synthesis, catalyzed by y-glutamylcysteine synthetase (yGCS). An increased activity of y-GCS would result in enhanced cellular GSH synthesis. As shown in Table 1, there is no significant difference in GR activity between the A549-CdR and -CdS cells. The y-GT activities were 129.0 2 14.7 and 118.5 + 15.6 mU/mg protein in the CdR and CdS cells, respectively, displaying no significant difference between the two cell lines. Therefore, neither GR nor y-GT alteration is responsible for the elevated cellular GSH content in the CdR cells. We then compared the y-GCS mRNA levels in the two cell lines. As shown in Figure 4, the relative amount of the steady-state y-GCS mRNA was markedly increased in the A549-CdR cells. Quantitative densiometric analysis revealed that the CdR cells exhibited about 3-fold higher y-GCS mRNA levels than the Cd” cells. This alteration correlates with the difference in cellular GSH levels between the two cell lines. To further understand the possible mechanism responsible for the elevated GSH levels, we used the Southern blot method to determine whether amplification of the y-GCS gene occurs in the CdR cells. As shown in Figure 5, no significant differences were detected in the relative amount of the -y-GCS DNA between the CdR and Cd’ cells. This result suggests that
Cd-semi tive
Cd-resistati
t
y-GCS
p-actin
Fig. 4. Northern blot analysis of y-GCS expression in exponentially growing Cd-resistant and Cd-sensitive A549 cells. The +XS mRNA level in the Cd-resistant cells was 3-fold higher than in the Cd-sensitive cells, as measured by densiometry. This experiment was repeated three times with consistent results.
the elevation from y-GCS
of cellular GSH levels gene amplication.
does not result
DISCUSSION
The importance of intracellular GSH in cytoprotection against toxic agents including reactive oxygen species, radiation, and alkylating agents has been widely demonstrated.7-y Several studies indicate that GSH also functions in cellular defense against cadmium toxicity.‘-? Depletion or elevation of cellular GSH content has been shown to enhance or attenuate cadmium sensitivity, respectively.“’ In addition, cadmium cytotoxicity was found to correlate with the growth-phase dependent fluctuation in GSH levels in A549 cells.36 In this study cellular GSH content was found to be 2-fold higher in the A549-CdR cells compared to the A549CdS cells, further demonstrating the involvement of GSH in cadmium cytoprotection. Two important factors often associated with cadmium resistance are cell membrane transport and MT synthesis. It has been shown that decreased cadmium uptake is highly correlated with cadmium resistance in CHO and other culture cells.‘9,22 Cadmium, among several heavy metals, induces MT synthesis. Enhanced capacity for MT synthesis markedly increases cellular resistance to cadmium.‘7 However, neither alteration in cadmium uptake nor change in MT synthesis capacity is responsible for the development of relative cadmium resistance in A549 cells, as previously reported”5 and confirmed in this study. It appears that elevation of cellular GSH levels does not affect cellular cadmium transport or MT synthesis. However, it is unknown whether the elevation of cellular GSH is solely responsible for the development of cadmium resistance in A549 cells. Many studies have implicated oxidative damage as a possible toxic action of cadmium. Lipid peroxidation in vivo and in vitro has been detected in association with cadmium treatment. “~38m4’Alterations in the activities of antioxidant enzymes such as SOD, catalase, and GSHpx in response to cadmium were also reported.‘5,42 Because GSH is an important antioxidant, participating in enzymatic and nonenzymatic detoxification of many oxidative toxicants, it is worthwhile to investigate whether the cadmium-induced elevation of GSH concentration is associated with alterations in antioxidant enzyme activities. As mentioned, the GSH redox cycle consisting of GSH, GSHpx, and GR is a major component of the antioxidant defense system. We therefore determined the activities of GSHpx and GR in the two subpopulations of A549 cells. The results showed that there is no significant difference in either of these enzymatic
GSH and cadmium
Cd-sensitive
Cd-resistant
Fig. 5. Southern blot analysis of y-GCS DNA levls in exponentially growing Cd-resistant and Cd-sensitive A549 cells. No difference was detected in the y-GCS DNA levels between the sublines. This experiment was repeated twice with consistent results.
activities between the CdR and CdS cells. The elevation of cellular GSH levels is thus independent of enzymatic changes in the GSH redox cycle. We next examined the status of two other important antioxidant enzymes, SOD and catalase. The activities of these two enzymes did not change in association with the development of cadmium resistance, either. Therefore, cadmium resistance in A549 cells does not result from alterations in the enzymatic components of the antioxidant defense system. Studies in cell-free systems have shown that GSH forms a complex with cadrnium.43 Whether this complex occurs in vivo or in culture cells and whether it represents an important mechanism by which GSH detoxifies cadmium are unknown. Recent studies, however, showed that addition of exogenous GSH to culture medium reduced cellular cadmium accumulation, resulting in decreased cadmium cytotoxicity.44 The reduced cadmium accumulation presumably results from formation of a GSH-Cd complex, thereby preventing Cd uptake by the cells. In this study, a possible mechanism for the elevation of cellular GSH levels was also examined. The results demonstrate that enhanced GR-catalyzed reduction of GSSG to GSH is not responsible for the cellular GSH elevation because GR activity did not increase in association with the increased GSH levels. Enhanced extracellular GSH degradation and thereby intracellular resynthesis is also not responsible for the GSH elevation because y-CT activity was unchanged. Finally, analysis of y-GCS mRNA revealed that the relative amount of the mRNA was indeed elevated in the A549-CdR cells. Gene amplification is reported to be a common mechanism of metal resistance, including cadmium re-
811
resistance
sistance, in mammalian cells.20 However, the relative amount of the y-GCS DNA did not differ between the A549-CdR and -Ccl” cells, indicating that no amplification of y-GCS gene occurs in the A549-CdR cells. Therefore, enhanced expression of y-GCS, but not gene amplification, is associated with the increased cellular GSH levels in the A549-CdR cells as compared to the A549-CdS cells. In summary, this study investigated whether the development of cadmium resistance in A549 cells is associated with enhanced antioxidant activities. The results obtained show that although cellular GSH levels in the CdR clone were elevated more than 2-fold over those of the CdS clone, the activities of antioxidant enzymes were not altered, demonstrating that enhanced antioxidant activities are unlikely responsible for the development of cadmium resistance. One possible mechanism by which GSH prevents Cd toxicity may be the formation of a GSH-Cd complex. The study also demonstrates that enhanced expression of ?I-GCS but not gene amplification is likely responsible for the elevation of cellular GSH levels associated with the development of Cd resistance.
Acknowledgements-This study was supported in part by EPAIEPSCoR grant R82 1836 and by the University of North Dakota School of Medicine. The authors thank KayLynn Boushee for typing the manuscript and Dr. R. Timothy Mulcahy at the University of Wisconsin Comprehensive Cancer Center for the gift of y-GCS cDNA probe. This work was presented in part at the 33rd Annual Meeting of the Society of Toxicology, Dallas, TX, March 13-17, 1994.
REFERENCES I Kang, Y. J.; Enger, M. D. Effect of cellular glutathione depletion on cadmium-induced cytotoxicity in human lung carcinoma cells. Cell Biol. Toxicol. 3(4):347-360; 1987. 2 Singhal, R. K.; Anderson, M. E.; Meister, A. Glutathione, a first line of defense against cadmium toxicity. FASEB J. 1:220-223; 1987. 3. Kang, Y. J.; Enger, M. D. Glutathione is involved in the early cadmium cytotoxic response in human lung carcinoma cells. Toxicology 48:93-101; 1988. 4. Meister, A. Metabolism and function of glutathione. In: Dolphin, D.; Poulson, R.; Avramovic, 0.. eds., Glutathione. Chemical, biochemical, and medical aspects, Part A. New York: John Wiley & Sons, Inc.; 1989:367-474. 5. Andrews, P. A.; Murphy, M. P.; Howell, S. B. Differential potentiation of alkylating and platinating agents’ cytotoxicity in human ovarian carcinoma cells by glutathione depletion. Cancer Res. 45:6250-6253; 1985. 6. Russo, A.; Mitchell, J. B. Potentiation and protection of doxombicin cytotoxicity by cellular glutathione modulation. Cancer Treat Rep. 69: 1293- 1296; 1985. I. Biaglow, J. E.; Varnes, M. E.; Clark, E. P.; Epp. E. R. The role of thiols in cellular response to radiation and &tgs. Radiat. Res. 95:437-455; 1983. 8. Ketterer, B. The role of nonenzymatic reactions of glutathione in xenobiotic metabolism. Drug Metab. Rev. 13: 16 I- 187; 1982. 9. Jones, T. W.; Thor, H.; Orrenius, S. Cellular defense mechanism
812
IO.
Il.
12.
13.
14.
15
16
17
I8
19.
20.
21.
22.
23.
24.
25.
26. 27.
E. L. HATCHER et al.
against toxic substances. Arch. Toxicol. Suppl. 9:259-271; 1986. Goyer, R. A. Toxic effects of metals. In: Amdur, M. 0.; Doull, J.; Klassen, C. D., eds. Casarett and Doull’s toxicology: The basic science of poisons, 4th ed. New York: Pergamon Press; 1991:634-637. Sorahan, T.; Waterhouse, J. A. J. Mortality study of nickel cadmium battery workers by the method of regression models in life tables. Br. J. Ind. Med. 40:293-300; 1983. Thun, M. J.; Schnorr, T. M.; Smith, A. B.; Halperin, W. E.; Lemen, R. A. Mortality among a cohort of U.S. cadmium production workers-an update. J. Natl. Cancer Inst. 74:325-333; 1985. Takenoka, S.; Oldiges, H.; Konig, H.; Hochrainer, D.; Oberdorster, G. Carcinogenicity of cadmium chloride aerosols in W rats. J. Natl. Cancer Inst. 70:367-373; 1983. Manta, D.; Richard, A. C.; Trottier, 9.; Chevalier, G. Studies on lipid peroxidation in rat tissues following administration of low and moderate doses of cadmium chloride. Toxicology 67(3):303-323; 1991. Kostic, M. M.; Ognjanovic, B.; Dimitrijevic, S.; Zikic, R. V.; Stajn, A.; Rosic, G. L.; Zivkovic, R. V. Cadmium-induced changes of antioxidant and metabolic status in red blood cells 1993. of rats: In vivo effects. Eur. J. Haematol. 51:86-92; Shiraishi, N.; Uno, H.; Waalkes, M. P. Effect of L-ascorbic acid pretreatment on cadmium toxicity in the male Fischer (F344/ Ncr) rat. Toxicology 85:85-100; 1993. Stacey, N. H.; Cantilena, L. R.; Klassen, C. D. Cadmium toxicity and lipid pcroxidation in isolated rat hepatocytes. Toxicol. Appl. Pharmacol. 53:470-480; 1980. Seagrave, J. C.; Hildebrand, C. E.; Enger, M. D. Effects of cadmium on glutathione metabolism in cadmium sensitive and cadmium resistant Chinese hamster cell lines. Toxicology 29:101-107; 1983. Bracken, W. M.; Sharma, R. P.; Kleinschuster, S. J. Cadmium accumulation and subcellular distribution in relation to cadmium chloride induced cytotoxicity in vitro. Toxicology 33:93- 102; 1984. Enger, M. D.; Hildebrand, C. E.; Walters, R. A.; Seagrave, J. C.; Barham, S. S.; Hoagland, H. C. Molecular and somatic cell genetic analysis of metal resistance mechanisms in mammalian cells. In: Tasjjian, A. H., ed. Molecular and cellular approaches to understanding mechanisms of toxicity. Boston: Harvard School of Public Health; 1984:38-62. Wong, K-L.; Klaassen, C. D. Relationship between liver and kidney levels of glutathione and metallothionein in rats. Toxicology 19:39-47; 1981. Enger, M. D.; Hildebrand, C. E.; Seagrave, J. C.; Tobey, R. A. Clonal variation of cadmium response in human tumor cell lines. Am. J. Physiol. 25O:C256-C263; 1986. Reed, D. J.; Babson, J. R.; Beatty, P. W.; Brodie, A. E.; Ellis, W. W.; Potter, D. W. High-performance liquid chromatography analysis of nanomole levels of glutathione, glutathione disulfide, and related thiols and disulfides. Anal. Biochem. 106:55-62; 1980. Smith, P. K.; Krohn, R. I.; Hermanson, Cl. T.; Mallia, A. K.; Gartner, F. H.; Proverzano, M. D.; Fujimoto, E. K.; Goeke, N. M.; Olson, B. J.; Klerk, D. C. Measurement of protein using bicinchoninic acid. Anal. Biochem. 150:76-85; 1985. Eaton, D. L.; Cherian, M. G. Determination of rnetallothionein in tissues by cadmium-hemoglobin affinity assay. Methods Enzymol. 205:83-88; 1991. Aebi, H. Catalase in vitro. Methods Enzymol. 105:121- 126; 1984. Nelson, D. P.; Kiesow, L. A. Enthalpy of decomposition of hydrogen peroxide by catalase at 25°C (with molar extinction coefficients of H202 solutions in the UV). Anal. Biochem. 49:474-478; 1972.
28. Flohe, L.; Gunzler, W. A. Assays of glutathione peroxidase. Methods Enzymol. 105: 1 l4- 121; 1984. 29 Carlberg, I.; Mannervik, B. Glutathione reductase. Methods En1985. zymol. 113:484-490; 30. Sun, Y.; Oberley, L. W.; Ying, L. A simple method for clinical assay of superoxide dismutase. Clin. Chem. 34:497-500; 1988. 31. Szasz, G. A kinetic photometric method for serum y-glutamyl transpeptidase. Clin. Chem. 15:l24- 136; 1969. 32. Church, G. M.; Gilber, W. Genomic sequencing. Proc. Natl. Acad. Sci. USA 81:1991-1995; 1984. 33. Gipp, J. J.; Chang, C.; Mulcahy, R. T. Cloning and nucleotide sequence of a full-length cDNA for human liver y-glutamylcysteine synthetase. Biochem. Biophys. Res. Commun. X35:29-35; 1992. 34. Feinberg, A. P.; Vogelstein, B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132:6-13; 1983. 35. Kang, Y. J.; Nuutero, S. T.; Clapper, J. A.; Jenkins, P.; Enger, M. D. Cellular cadmium responses in subpopulations T20 and T27 of human lung carcinoma A549 cells. Toxicology 61:195203; 1990. 36. Kang, Y. J.; Enger, M. D. Cadmium cytotoxicity correlates with the changes in glutathione content thatoccur during the logarithmic growth phase of A549-T27 cells. Toxicol. Left. 51:23-28: 1990: 37. Hilderbrand, C. E.; Tobey, R. A.; Campbell, E. W.; Enger, M. D. A cadmium-resistant variant of the Chinese hamster (CHO) cell line with increased metallothionein induction capacity. Exp. Cell Res. 124:237-246; 1979. 38. Koizumi, T.; Li, 2. G. Role of oxidative stress in single-dose, cadmium-induced testicular cancer. J. Toxicol. Environ. Health. 37:25-36; 1992. 39. Szymanska, E.; Laskowska-Klita, T. Effect of cadmium on lipid peroxidation and activities of antioxidant enzymes, Acta Biochim. Pol. 40:144-146; 1993. 40. Shukla, G. S.; Chandra, S. V. Cadmium toxicity and bioantioxidants: Status of vitamin E and ascorbic acid of selected organs in rats. J. Appl. Toxicol. 9:119-122; 1989. 41. Muller, L. Consequences of cadmium toxicity in rat hepatocytes: Mitochondrial dysfunction and lipid peroxidation. Toxicology 40:285-295; 1986. 42. Gupta, S.; Athar, M.; Behari, J. R.; Srivastava, R. C. Cadmiummediated induction of cellular defence mechanism: A novel example for the development of adaptive response against the toxicant. Znd. Health. 29:1-9; 1991. 43. Perrin, D. D.; Watt, A. E. Complex of formation of zinc and cadmium with glutathione. Biochim. Biophys. Actu. 230:96104; 1971. 44. Kang, Y. J. Exogenous glutathione decreases cellular cadmium uptake and toxicity. Drug Metab. Disp. 20:714-718; 1992.
ABBREVIATIONS
FDNB - 1-fluoro-2,3_dinitrobenzene GR-glutathione reductase GSH-glutathione GSHpx-gglutathione peroxidase GSSG-oxidized glutatbione HPLC-high performance liquid chromatography MT-metallothionein PBS-phosphate buffered saline PCA-perchloric acid y-GCS - y-glutamylcysteine synthetase y-GTy-glutamyltranspeptidase