enhancing glutathione synthesis can decrease zinc-mediated toxicity

Enhancing Glutathione Synthesis can DecreaseZinc-Mediated Toxicity

Udo Ingbert Walther & Sabine Christine Walther &

Harald Mückter & Burckhard Fichtl

Received: 16 October 2007 /Accepted: 27 November 2007 /Published online: 11 January 2008# Humana Press Inc. 2007

Abstract Zinc toxicity has been linked to cellular glutathione: A decrease in glutathione isfollowed by an increase in zinc-mediated toxicity. The question arises whether an increasein glutathione synthesis might decrease zinc-mediated cytotoxicity. We incubated five celllines (hepatoma and lung-derived) with zinc chloride and 2 mmol/l N-acetyl-L-cysteine(NAC) to support glutathione synthesis. In all but one hepatic cell line, the glutathionecontent was increased by NAC as compared to the D-enantiomere NADC, whereas NADCdid not increase GSH content as compared to not treated controls. In both alveolar epithelialcell lines, an increase in zinc tolerance was observed due to NAC as compared to NADC.In native fibroblast-like and the hepatoma cell lines, no changes in zinc tolerance werefound due to NAC. In the fibroblast-like cells, zinc tolerance was increased due to NAConly after cellular glutathione had been previously decreased (by lowered cysteineconcentrations in the medium). Enhancing glutathione synthesis can antagonize zinc-mediated toxicity in the alveolar epithelial cell lines, whereas some other characteristicsthan glutathione synthesis might be more important in other cell types. Furthermore, NACacted as a GSH precursor only at cysteine medium concentrations of 10 µmol/l or belowand therefore might be described as a poor cysteine repletor for glutathione synthesis.

Keywords NAC . NADC . cys . Zn2 . Lung cells . Hepatoma cells

AbbreviationsARDS acute respiratory distress syndromeCys L-cysteineDMEMmF12 Dulbecco’s modified Eagle medium/Ham’s F12 nutrient mix (1:1)DTNB 5,5′-dithio-bis(2-nitrobenzoic acid)EC50 concentration for 50% effectivityEDTA N,N,N′,N′-ethylendiaminetetraacetateFcs fetal calf serum

Biol Trace Elem Res (2008) 122:216–228DOI 10.1007/s12011-007-8072-9

This work is dedicated to Peter Eyer on the occasion of his 65th birthday.

U. I. Walther (*) : S. C. Walther :H. Mückter : B. FichtlWalther Straub-Institut für Pharmakologie und Toxikologie, Ludwig-Maximilians Universität München,Nußbaumstr. 26, 80336 Munich, Germanye-mail: [email protected]

GC glucocorticoid(s)GR glutathione reductase, GSSG reductaseGSH glutathione, reduced formGSSG glutathione, oxidized formHC hydrocortisoneMEM minimum essential mediumMet L-methionineNAC N-acetyl-L-cysteineNADC N-acetyl-D-cysteineNADPH nicotinamide-adenine dinucleotide phosphate, reducedoxo-Th oxo-thiazolidinedionePBS phosphate-buffered salineSD standard deviationSDS sodium laurylsulfate

Introduction

Oral zinc intoxications in humans are rare events and nowadays are only typically observedin psychiatric patients or in children after ingestion of zinc-containing coins [1, 2].Nevertheless, most adverse effects by zinc are described after inhalational exposure to zinc-containing fumes [3]. In these cases, the observed illness differs from metal fume fever,with common cold-like symptoms up to the development of an acute respiratory distresssyndrome (ARDS) with lethality of 30–50% in spite of intensive care [4].

Zinc-induced cytotoxicity has been associated to glutathione in hepatic and lung-derivedcells [5, 6, 7], wherein a decrease of total cellular glutathione content and an increase inoxidized glutathione were observed. Furthermore, toxicity is enhanced when cellularglutathione had been decreased. Additionally, pretreatment of alveolar epithelial cells withglucocorticoids is known to decrease cellular glutathione and such cells are moresusceptible to zinc as compared to not pretreated cells [8].

Zinc can inhibit glutathione reductase (GR) activity irreversibly [9], and this effectseems to be the first step in the changes of the glutathione system in lung-derived cells [7].In contrast, zinc does not appear to inhibit glutathione synthesis. Therefore, a protectiverole of an enlarged glutathione synthesis rate, leading to a higher cellular glutathionecontent, is postulated to antagonize zinc-mediated toxic effects [10].

To clarify this issue, we used 2 mmol/l NAC to increase cellular glutathione synthesisduring zinc exposure. The D-enantiomer NADC was used as the control to compensateantioxidative and/or chelating effects by NAC [11, 12, 13, 14]. Oxidized and reducedglutathione were measured, and protein synthesis was calculated by methionineincorporation into acid insoluble cellular material to assess zinc-mediated toxicity. Thislatter parameter is described as sensitive and early affected after zinc exposure [8, 15].

Material and Methods

Chemicals

Cell culture chemicals [Dulbecco’s modified Eagle medium/Ham’s F12 nutrient mix, 1:1(DMEMmF12), minimum essential medium (MEM; Hanks’ salts), MEM (Earle’s salts),

Glutathione Synthesis and Cellular Zinc Toxicity 217

penicillin/streptomycin, methionine 100x, trypsin/EDTA (N,N,N′,N′-ethylendiaminetetraace-tate)] were obtained from Invitrogen (Eggenstein, Germany), fcs from Biochrom (Berlin,Germany), glutamine and Triton X-100 from Merck (Darmstadt, Germany). 5,5′-Dithio-bis(2-nitrobenzoic acid) (DTNB), and glutathione, oxidized form (GSSG) were purchased fromSigma (Deisenhofen, Germany), nicotinamide-adenine dinucleotide phosphate, reduced(NADPH), and GSSG reductase (EC 1.6.4.2) from Roche Diagnostics (Mannheim,Germany), N-acetyl-D-cysteine (NADC) from Research Organics (Cleveland, OH, USA),2-vinylpyridine from Aldrich (Steinheim, Germany), 35S-met/cys mixture from HartmannAnalytic (Braunschweig, Germany), and Coomassie blue from Serva (Heidelberg, Germany).All other reagents were from Merck.

Cell Culture

Five cell lines (two alveolar epithelial, A549, L2; one lung fibroblast-like, 11Lu; twohepatoma, HTC, HepG2) were used in this work. HTC (cells) were a gift from Dr. Wiebel(GSF, Neuherberg, Germany). All other cell lines were obtained from the American TypeCulture Collection (Rockville, MD, USA). The cell lines were grown in DMEMmF-12(supplemented with 50 IE/ml penicillin, 50 µg/ml streptomycin and 10% fcs (A549 cellsonly 5% fcs) in a moist CO2 (5% v/v) atmosphere at 37°C. L2, A549, HTC, and HepG2cells were passaged weekly using a standard trypsin/EDTA protocol [16]. 11Lu cells werepassaged every 10–14 days. Medium was changed every other day and 14 h (A549: 24 h)before zinc exposure.

Exposure Protocol and Methionine Incorporation Assay

All investigations were performed in 24-well plates with ≌1.9 cm2 growth area per well.Viability of cells was 97±5% as assessed by Trypan Blue dye exclusion. When indicatedalveolar epithelial cells were pretreated with hydrocortisone (100 µmol/l, incl. 0.1%ethanol) in culture medium with fcs for 72 h (controls 0.1% ethanol). Before the zincexposure, cell layers were washed once with MEM (Hanks’ salts) then incubated withvarious concentrations of zinc chloride in MEM (Earle’s salts, met, 10 µmol/l; cys, asindicated) at 37°C in 5% (v/v) CO2 atmosphere for 3 h (11Lu, L2, HTC), 4 h (HepG2), or17 h (A549 cells) including 2 mmol/l NAC (or NADC). Zinc chloride was added from a9.1 mmol/l stock solution (dissolved in H2O). Radiolabeled methionine (met=met/cys-mixture, 75/25%; 35S for 1 h, 1 µCi per cm2 growth area, 10 µmol/l met, cys as indicated)was added in MEM (Earle’s salts, met and cys as indicated above) at the end of theincubation period. Methionine incorporation was terminated by rinsing with ice-cold MEMto remove excess label. Then, the cells were denaturated with ice-cold 0.33 mol/l HClO4.The supernatant was assessed for glutathione content and acid-soluble radioactivity. Theprecipitate was dissolved in NaOH [0.5 mol/l incl. 1% sodium laurylsulfate (SDS)], andacid-insoluble radioactivity was measured.

Measurement of Glutathione Content

Total cellular glutathione and oxidized glutathione (GSSG) were measured in neutralizedcell extracts using the Tietze assay [17] with DTNB, GR, and NADPH. At theconcentrations studied, zinc did not interfere with the assay. The analytical detection limitfor glutathione was ≈0.2 nmol per mg of cellular protein [8]. GSSG was determined afterGSH had reacted with 2-vinylpyridine (2 per 100 µl of acid cell extract at pH 7) for half an

218 Walther et al.

hour. Then, excess vinylpyridine was removed by evaporation. Calibration was performedwith freshly prepared acidic GSSG standards. All GSSG values were expressed aspercentage of total cellular glutathione.

Protein Determination

Protein content of cell layers was measured according to a modified Bradford procedure[18]. Cells were washed twice with PBS, then incubated in 0.5 mol/l NaOH for 14 h at 37°Cbefore dye-binding. Bovine serum albumin was used as a calibration standard.

Statistical Evaluation

Incorporated methionine radioactivity and reduced or oxidized glutathione content wereexpressed as percentage of control values. For each experiment a function (1) was fittedto the data and the corresponding EC50 or IC50 value was calculated a=lower plateau,b=upper plateau, c=EC50 or IC50, d=Hill coefficient, x=zinc concentration, y=percent ofglutathione content or GSSG content or methionine incorporation). Mean and standarddeviation of at least three experiments were tabulated, and statistical differences wereestimated by analysis of variance (ANOVA) and the method of the least significantdifferences (LSD; dependence for medium cystein concentration) or by Student’s t test forpaired or independent values as indicated.

y ¼ ab � a

1 xc

� �d ð1Þ

Results

Influence of Zinc on Glutathione Content and Methionine Incorporation

Glutathione content decreased by zinc in all cell lines in a concentration dependent manner,whereas the ratio of oxidized/reduced glutathione was increased (L2: Fig. 1; other cell linesnot shown). Depletion of cellular glutathione was the most sensitive parameter to assesszinc-mediated toxicity in the lung derived cell lines (Tables 1 and 2), whereas in thehepatoma cells tested, methionine incorporation inhibition was more sensitive thanglutathione depletion (Table 2) as assessed by corresponding EC50 values.

Methionine incorporation into cellular protein was in the range of 0.4±0.2 pmol/µgcellular protein (11Lu cells) and 1.5–1.0±0.5 pmol/µg (L2, A549, HTC, HepG2 cells).Incorporation decreased at higher cys concentrations but was independent of hydrocortisonepretreatment or NAC/NADC treatment (not shown). In all cell lines, met incorporation intoacid precipitable cellular material was decreased by zinc in a concentration dependentmanner, whereas acid soluble met was not affected (not shown).

Influence of Cys on Zinc-Mediated Toxicity

Zinc-mediated toxic effects were evaluated at different cys concentrations in the lung-derived cell lines. A decrease in cellular glutathione content was found in the lung cellsafter medium cys concentration was lowered to 10, 1, or 0 µmol/l (25, 5, or 0 µmol/l for L2


cells, normal MEM=100 µmol/l cys; Fig. 2). This decrease was significant in L2 cells at 0,and 5 µmol/l, whereas in A549 and 11Lu cells, a significant change was found at 0 and1 µmol/l (ANOVA-LSD, p<0.05) [additionally, in 11Lu cells at 10 µmol/l].

Toxic effects by zinc, as evaluated by EC50 values for glutathione depletion wereincreased for the lowered cys concentrations tested in A549 and L2 cells (ANOVA-LSD,p<0.05), and for A549 cells for met incorporation inhibition. No changes due to cys werefound in the fibroblast-like 11Lu lung cell line (Table 2).

Influence of Hydrocortisone Pretreatment on L2 and A549 Cells

Protein contents of L2 cells dropped from 93±21 to 76±17 µg per cell layer (correspondingto 82.2±4.6%; p=0.011, t test for paired values), whereas content was not changed in A549cells (controls, 89±15µg; HC pretreated, 86±18 µg ≈ 97±6%) after cells had beenpretreated with 100 µmol/l hydrocortisone for 72 h (not shown).

Glutathione content was decreased to 37±17% in L2 cells (p=0.011, t test for unpaireddata) and 66±1% in A549 cells (p=0.006, paired values). Values for met incorporationwere not changed at all by the HC pretreatment procedure (Table 3).

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Fig. 1 Effect of NAC on zinc-mediated toxic effects in L2cells. Cells were coincubatedwith 2 mmol/l NADC or NACand up to 200 µmol/l zinc chlo-ride in MEM for 3 h without cys.Afterwards, 35S-labeled methio-nine was administered for 1 h.Incorporation was terminated byperchloric acid, and glutathionewas measured in the acid fraction,whereas protein-associated radio-activity was determined in theacid precipitable fraction. Means±SD of five independent experi-ments are shown. Open symbolsfor NAC, closed symbols forNADC treated cells

220 Walther et al.

EC50 values of zinc after pretreatment were significantly decreased in A549 cells formet incorporation inhibition (p=0.039, unpaired data), whereas values for glutathionedepletion were not changed (Table 3). In L2 only, a tendency for lowered EC50 values forall parameters tested was found after cells had been pretreated with HC (Table 3).

Influence of NAC and NADC on Zinc-Induced Toxicity

NADC did not increase the cellular glutathione contents of the cell lines tested (Fig. 2),whereas EC50 values for zinc-induced toxicity in alveolar epithelial cells were increased ascompared to controls without NAC/NADC treatment (Table 1, Fig. 3). No NADC-derivedchanges of zinc-induced toxicity were found in 11Lu lung fibroblasts and the hepatoma celllines (Table 2).

In A549, a significant increase in glutathione was found by NAC (as compared toNADC) for 0, 1, and 10 µmol/l cys, whereas content did not differ at 100 µmol/l cys

Table 1 Influence of Cysteine on NAC-derived Changes in Zinc-Mediated Toxicity of AlveolarepithelialCells

Cell line Cysteine (µmol/l) Group Number EC50 MII (µmol/l),Mean±SD

EC50 GSH-D (µmol/l),Mean±SD

A549 0 Controls 2 69.4±29.8 34.3±2.00 NADC 2 87.2±12.9 51.2±7.00 NAC 2 115±4.9 87.4±4.51 Controls 4 59.0±13.4* 34.3±2.7*1 NADC 4 89.0±9.6 54.4±7.61 NAC 4 112±12.3* 94.1±10.0*10 Controls 4 60.4±12.1* 33.2±2.9*10 NADC 4 96.3±11.9 55.3±11.110 NAC 4 119±11.2* 93.2±13.9*100 Controls 2 89.4±0.8 86.3±4.9100 NADC 2 138±19.1 111±39.7100 NAC 2 147±0.7 143±6.4

L2 0 Controls 5 32.6±11.0* 25.7±11.4*0 NADC 5 72.6±29.2 52.8±18.40 NAC 5 89.3±31.8** 98.4±46.5**0 oxo-Th 5 44.9±26.5 33.3±18.05 Controls 1 30.9 24.95 NADC 4 47.3±10.1 34.2±10.85 NAC 4 54±7.7** 57.5±8.0**5 oxo-Th 4 19.7±10.8 19.8±9.825 Controls 0 n.t. n.t.25 NADC 3 49.3±9.7 36.0±6.325 NAC 3 53.7±15.7 58.5±9.7*100 Controls 6 42.5±19.5 69.9±40.7

Cells were incubated with up to 250 µmol/l zinc chloride in MEM for 3 h (L2) or 17 h (A549) including cysand 2 mmol/l NAC or NADC as indicated. Afterwards 35 S-met was administered for 1 h. Then, cells werelysed by perchloric acid treatment, and GSH contents and methionine incorporation were measured asdescribed and EC50 values were calculated.oxo-Th Oxo-thiazolidinedione; n.t. Not tested*p<0.05, t test for unpaired values compared to NADC**p<0.05, t test for paired values compared to NADC


(Fig. 2). The increased contents furthermore were accompanied by an increase in EC50

values for the zinc effects (Table 1).In L2 cells, a significant increase in cellular glutathione content was found after cells

were incubated with 2 mmol/l NAC as compared to NADC only at 0 µmol/l cys (Fig. 2).According to the GSH increase, a significant increase in EC50 values of zinc formethionine incorporation and GSH depletion was found. This zinc effect was attenuated at5 and 25 µmol/l cys (Table 1). After L2 cells were pretreated with HC, similar changes asfor not pretreated cells were observed (Table 3). In L2 cells, as an alternative to NAC, oxo-thiazolidinedione was used. However, with this substance neither at 0 nor at 5 µmol/l cys,an increased glutathione content of cells was found. Accordingly, no changes in zinc-mediated effects were observed with this glutathione repletor.

In 11Lu cells, too, increases in glutathione content due to NAC were found at 0 and1 µmol/l cys, whereas at 10 µmol/l cys only, a tendency to an increased content wasobserved (Fig. 2). No changes in the EC50 values of zinc for methionine incorporation andglutathione depletion comparing NAC and NADC treated cells were observed. Further-more, no changes in zinc susceptibility due to NADC as compared to controls withouttreatment were found (Table 2).

After 11Lu cells had been pretreated for 24 h in medium of diminished cysteine content(1 µmol/l), a significant increase in glutathione content was found due to NAC as comparedto NADC (Table 4), whereas glutathione content was in the range of not pretreated cells.Nevertheless, a clear increase in zinc susceptibility occurred as compared to not pretreatedcells. Furthermore, toxicity by zinc during NAC treatment was significantly decreased ascompared to NADC treatment and significantly lowered by NADC as compared to controlswithout treatment (Fig. 4).

Table 2 Influence of Cysteine on NAC-Derived Changes in Zinc-Mediated Toxicity of a Fibroblastic LungCell Line and Hepatoma Cell Lines

Cell-line Cysteine (µmol/l) Group Number EC50 MII (µmol/l)MW±SD

EC50 GSH-D. (µmol/l)MW±SD

11Lu 0 Controls 4 489±396 438±2080 NADC 4 336±119 282±1280 NAC 4 461±252 362±1111 Controls 6 872±456 338±83.11 NADC 6 466±310 242±1171 NAC 6 466±304 396±23410 Controls 3 367±139 165±19310 NADC 3 156±36.1 149±14810 NAC 3 223±191 187±137100 Controls 7 564±351 369±56.9

HTC 1 Controls 3 303±68.6 523±31.81 NADC 3 303±159 716±4021 NAC 3 296±288 750±353

HepG2 1 Controls 3 18.2±4.4 380±126*1 NADC 3 63.3±29.8 138±72.51 NAC 3 52.1±25.9 142±55.6

Cells were incubated with up to 500 µmol/l zinc chloride in MEM for 3 h (11Lu, HTC), or 4 h (HepG2)including cys and 2 mmol/l NAC or NADC as indicated. Then 35S-met was administered for 1 h including.Afterwards zinc mediated toxicity was measured as described. EC50 values of zinc for glutathione depletion(GSH-D) or met-incorporation inhibition (MII) were calculated.

222 Walther et al.

Discussion

In the present study, the comparison of the effectivity of the D- and L-enantiomers of NACwas used to assess a glutathione synthesis effect by NAC in zinc-mediated toxicity.

A549

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Fig. 2 Influence of cysteine and NAC or NADC on glutathione content of cell lines. Cells were incubated inMEM for 3 h (L2, 11Lu, HTC), or 4 h (HepG2), or 17 h (A549) including 2 mmol/l NAC or NADC and cysas indicated. Then, 35S-met was administered for 1 h including cys as before. Afterwards, glutathionecontents were measured as described. *p<0.05, different compared to NADC, Student’s t test forindependent values. *p p<0.05, different compared to NADC, t test for paired values


D-Amino acids are partly known to be toxic. D-Valin for example is toxic for fibroblastsand, therefore, is used for cell selection. On the other hand, D-cys is known to be toxic forEscherichia coli, but no indications exist that D-cys might be toxic for eukaryotic cells [19].Accordingly, in our experiments, no differences were seen between controls (not zincexposed) treated with NAC as compared to NADC. Furthermore, 2 mmol/l D-cys did notchange cellular viability in L2 and 11Lu cells. Therefore, from our data too, no hint can betaken for toxic D-cysteine (nor NADC) effects.

Zinc-mediated toxicity is inversely related to cellular glutathione. However, 200 µmol/l NAC did not alter zinc toxicity, despite an increase in cellular glutathione [10]. In thiswork, 2 mmol/l NAC was used to increase glutathione synthesis. This treatment increasedglutathione contents to about two- to threefold and was effective in diminishing zinc-mediated toxicity in the alveolar epithelial cell lines tested (as compared to NADC), but notin the lung fibroblast-like cell line nor in the hepatoma cell line HepG2. In the lungfibroblasts, a significant decrease in zinc toxicity due to NAC was found only after apretreatment procedure with medium of lowered cys concentrations. And this wasaccompanied by an increase in zinc-mediated toxicity as compared to normal mediumcys concentrations. Such a pretreatment procedure was not done in the hepatic cells, and inaddition, in HTC, no increase of cellular glutathione by 2 mmol/l NAC occurred at all. Thedifference between these cellular behaviors might be explained by an extended cys

Table 3 Changes due to Hydrocortisone Pretreatment in Alveolar Epithelial Cell Lines

Cellline

Pretreatment Cysteine(µmol/l)

Group Number Glutathione(nmol/mg)

Parameters of toxicity

EC50 MII(µmol/l)

EC50 GSH-D (µmol/l)

EC50 GSSG-Inc (µmol/l)

A549 HC 10 Controls 4 43.0±13.4 69.5±15.7*

33.4±4.7* 93.8 ±13.0

HC 10 NADC 4 48.4±10.7 96.1±9.9 48.3±5.7 120±17.3HC 10 NAC 4 102±16.1* 123±

15.0*91.1±11.5* 150±17.3

HC 100 Controls 4 108±15.0 99.1±14.3

82.3±12.1 142.5±28.7

wo 10 Controls 4 65.3±19.7**c

102±19.0*c

33.2±7.9 99.4 ± 11.3

L2 HC 0 Controls 4 8.5±4.0 69±34.2* 59.6±49.5 70.6±15.5HC 0 NADC 4 11.8±6.1 107±38.7 87.0±11.6 95.6±18.8HC 0 NAC 4 34.1±5.3* 142±

30.9*173±54.1* 143±28.7

HC 100 Controls 3 39.0±7.9 114±43.8 165±73.7 143±43.1wo 0 Controls 4 18.4±3.8*c 84.5±

31.072.9±32.7 122±35.9

After pre-treatment with 100 µmol/l hydrocortisone for 72 h cells were incubated with up to 250 µmol/l zincchloride in MEM for 3 h (L2), or 17 h (A549) including cys and 2 mmol/l NAC or NADC as indicated. Then35S-met was administered for 1 h. Afterwards zinc mediated toxicity was measured as described. EC50

values of zinc for met-incorporation inhibition (MII), glutathione depletion (GSH-D), or increase of oxidizedglutathione (GSSG Inc) were calculated.Controls=without NADC or NAC; wo=without pretreatment; c=compared to HC pretreated controls withoutNADC or NAC*p<0.05, Students t test for independent values, compared to NADC**p<0.05, t test for paired values, compared to NADC

224 Walther et al.

synthesis rate of hepatic derived cells [20]. As a consequence glutathione content ofhepatic-derived cells seems to be less dependent on extracellular cys concentrations.Accordingly, Richert et al. [21] found a two- to threefold increase in glutathione by NAC inhepatocytes only at 4 mmol/l or above.

A differentiation of the NADC (or NAC) effectivity, allocated into complexing orantioxidative properties [22, 23], seems to be interesting in the case of the zinc-mediatedtoxicity. Therefore, two effects antagonizing zinc-mediated toxicity have to be discussedwherein an enhancement of cellular zinc incorporation [24] might not occur because zincseems to be incorporated by specific transport proteins [25]. Comparing effectivity ofNADC/NAC and D-cys/L-cys is of no avail because cys was much more effective thanNAC/NADC (EC50 values in the range of 600 for 2 mmol/l D-/L-cys (not shown) in L2cells instead of 50–100 µmol/l for NAC/NADC, Table 1) but complexing properties aremuch higher, too [11, 26]. The decreased toxicity due to cys (as compared to NAC)therefore might be caused by the enhanced complexing probability. But in A549 cells,toxicity of a hydroperoxide was only marginally decreased by NADC (and similarly byNAC). Therefore, the antioxidative property of NADC (and NAC) can be described assparse.

This contrasts the clear effect of NADC against oxygen injury as described in cellculture experiments by other authors [27, 28]. In various cell types, decreases ofantioxidative enzyme activities or increases of oxidative processes by glucocorticoids aredescribed [29, 30, 31]. As glucocorticoid-pretreated alveolar epithelial cells are moresensitive towards zinc-mediated oxidative stress such an effect might be assumed for this

Methionine Incorporation

EC

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Fig. 3 EC50 values of zinc-mediated methioinine incorporation inhibition and glutathione depletion in A549Cells. Cells were incubated with up to 250 µmol/l zinc chloride for 17 h including 2 mmol/l NAC or NADC(or not treated controls) and cys as indicated. Then, 35S-met was administered for 1 h. Afterwards, zincmediated toxicity was measured as described. EC50 values of zinc for met-incorporation inhibition (upper)or glutathione depletion (lower) were calculated and means±SD of four independent experiments are shown.*p<0.05, different compared to NADC, Student’s t test for unpaired values



EC

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Fig. 4 EC50 values of zinc-me-diated methioinine incorporationinhibition and glutathione deple-tion and GSSG increase in 11Lucells after cysteine deprivation.After pretreatment of 11Lu, cellswith 4 µmol/l cys-containingMEM for 24 h cells were incu-bated with up to 150 µmol/l zincchloride in MEM for 3 h includ-ing 1 µmol/l cys and 2 mmol/l NAC or NADC as indicated.Then, 35S-met was administeredfor 1 h. Afterwards, zinc-mediat-ed toxicity was measured asdescribed. EC50 values of zincfor met-incorporation inhibition,glutathione depletion, or oxidizedglutathione increase were calcu-lated. Mean and SD of fiveindependent experiments areshown. *p<0.05, different com-pared to NADC, Student’s t testfor independent values

Table 4 Glutathione Content of 11Lu Fibroblast-like Cells after Pretreatment with 4 µmol/l Cys

Cell line Cysteine (µmol/l) Group Number of experiments Glutathione (nmol/mg)

11Lu 1 Controls 5 28.2±17.1*1 NADC 5 34.3±17.01 NAC 5 59.7±28.1*0 Controls 4 22.5±16.7100 Controls 5 58.7±23.0

After pretreatment with 4 µmol/l cys-containing MEM for 24 h cells were incubated with MEM for 3 hincluding cys and 2 mmol/l NAC or NADC as indicated. Then 35S-met was administered for 1 h. Thenglutathione content was measured as described.Controls=without NADC or NAC*p<0.05, t test for paired values, compared to NADC

226 Walther et al.

cell type too. Therefore, a better effectivity of an antioxidative substance in glucocorticoid-pretreated alveolar epithelial cells should be assumed. However, NAC/NADC did not workas a better antidote in glucocorticoid-pretreated alveolar epithelial cells as compared to notpretreated cells. This points to a poor antioxidative property of NAC/NADC in the zinc-mediated toxicity in the alveolar epithelial cell type, too. A first explanation might be alarge antioxidative property (excluding glutathione) of alveolar epithelial cells.

When cellular glutathione of 100 µmol/l cys treated cells with cells additionally treatedwith 2 mmol/l NAC is compared, no increased content for the NAC groups are found in ourcell lines (Fig. 2). This is remarkable for two reasons: First, Jafari et al. [32] found a nearlyduplicated glutathione content in A549 cells by 1 mmol/l NAC, whereas the basal contentwas similar to the content in our experiments. On the other hand, they describe aboutfivefold glutathione contents in another series of their A549 controls. Therefore, theirincrease to about twofold due to NAC seems to be not as impressive as described.Secondarily, the glutathione content is discussed to be regulated by cys content, whereasthis latter one is regulated by the ASC transporter. By contrast, NAC is not incorporated bythis transporter system but seems to be membrane permeable [22]. Therefore, theextracellular administration of NAC should increase intracellular NAC without transportlimitation, and this should increase the intracellular cys-availability after deacetylation. Thisshould lead to a clear increase in glutathione synthesis and therefore to increasedglutathione contents. The latter effect was not observed in our experiments. This mightpoint to a high cys synthesis capacity of alveolar epithelial cells as described for hepatic-derived cells (as mentioned above).

Over all, it can be concluded that NAC is only a poor cys donor for glutathione synthesisin all lung-derived cells tested and the hepatoma cells tested. And furthermore, as discussedabove, the main effect of NADC/NAC in decreasing zinc-mediated toxicity might beexplained by the complexing capabilities of this substance.

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enhancing glutathione synthesis can decrease zinc-mediated toxicity

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