Alterations of the glutathione–redox balance induced by metals in CHO-K1 cells

Comparative Biochemistry and Physiology Part C 132 (2002) 365–373

Alterations of the glutathione–redox balance induced by metals in CHO-K1 cells b a ´ ´ ´ A.J. Garcıa-Fernandez , A.E. Bayoumia,c, Y. Perez-Pertejo , M. Motasb, R.M. Regueraa, a a ´ ˜ , R. Balana-Fouce ˜ ´ ˜ a,* C. Ordonez , D. Ordonez a

´ y Toxicologıa, ´ Universidad de Leon, ´ Campus de Vegazana syn, 24071 Leon, Spain Dept. Farmacologıa b´ ´ Universidad de Murcia, Campus de Espinardo, 30100 Murcia, Spain Area de Toxicologıa. c Department of Plant Protection, Faculty of Agriculture, Aim Shams University, Cairo, Egypt Received 27 July 2001; received in revised form 12 April 2002; accepted 14 May 2002

Abstract The effects of cadmium (Cd2q), mercury (Hg2q), lead (Pb2q), copper (Cu2q) and nickel (Ni2q) on the glutathione (GSH)–redox cycle were assessed in CHO-K1 by the neutral red uptake inhibition (NR) assay (NR6.25 , NR12.5 and NR25). Mercury proved to be the most and lead the least toxic of the metals tested. The effects on GSH content and intracellular specific activities of enzymes involved in the GSH–redox balance were measured after a 24-h exposure. Total GSH content increased significantly in cultures exposed to the lowest metal concentration assayed (NR6.25 ), but fell to below control values when exposed to concentrations equivalent to NR25. Oxidised glutathione content dropped significantly at NR6.25, while somewhat higher values were obtained for cultures exposed to higher doses. Glutathione peroxidase (Gpx) activities were 1.2-, 1.5-, 1.6-, 2.0- and 2.5-fold higher than untreated controls for cadmium, copper, mercury, nickel and lead, respectively, at concentrations equivalent to NR6.25 . Gpx activity declined at metal concentrations equivalent to NR12.5 and NR25. Glutathione reductase activity remained almost unchanged except at low doses of mercury, nickel and lead. Glutathione-S-transferase activity decreased at rising metal concentrations. The results suggest that a homeostatic defence mechanism was activated when cells were exposed to doses equivalent to NR6.25 while the ability of the cells to respond weakened as the dose increased. A close relationship was also observed between metal cytotoxicity, total GSH content and the dissociation energy of the sulphur–metal bonds. These facts confirm the involvement of antioxidant defence mechanisms in the toxic action of these ions. 䊚 2002 Elsevier Science Inc. All rights reserved. Keywords: Cytotoxicity; Glutathione; Metals; Reactive oxygen species; CHO-K1; Glutathione peroxidase; Glutathione reductase; Glutathione-S-transferase

Abbreviations: GSSG, oxidised glutathione; NR, neutral red; SOD, superoxide dismutase; ROS, reactive oxygen species; Gpx, glutathione peroxidase; Grd, glutathione reductase; GST, Glutathione-S-transferase. *Corresponding author. Tel.: q34-987-291590; fax: q34-987-291552. ´˜ E-mail address: [email protected] (D. Ordonez). 1532-0456/02/$ - see front matter 䊚 2002 Elsevier Science Inc. All rights reserved. PII: S 1 5 3 2 - 0 4 5 6 Ž 0 2 . 0 0 0 7 9 - 0

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1. Introduction Global environmental pollution from anthropogenic activities has risen to a point that it poses a serious health hazard, and may even affect the survival of all life forms on earth. The toxicological effects of pollutants such as metals is due to their high persistence and accumulation in organisms and the trophic chain (Goyer, 1996). Since all toxic substances interfere with physiological processes, their effects can be explored with suborganism preparations (isolated organs or tissues) or even at the sub-cellular level (Hellawell, 1977). Toxic evaluation using live animals is falling into disuse today as an in vivo test for toxicity for both ethical and scientific reasons (Repetto, 1989). Moreover, the high cost of animal experimentation is prompting the replacement of in vivo by in vitro tests (Goldberg, 1989). Several authors have investigated the adverse effects of metals on cellular integrity via the sulphur–redox cycle, which includes reduced and oxidised glutathione (GSSG) content and gluthatione-S-transferases (GSTs). These enzymes and biomolecules are involved in protective mechanisms at the molecular level mediated by glutathione peroxidase (Gpx) and glutathione reductase (Grd), that constitute vehicles for cellular detoxification in the event of exposure to metals and other xenobiotics (Kang and Enger, 1988; Zaman and Pardini, 1996). The effects of metals on enzymes involved in the sulphur–redox balance have been investigated in vivo. Cadmium andyor mercury have an inhibitory effect on Gpx (Grose et al., 1987; Meydani and Hathcock, 1984) while mercury, copper andy or cadmium inhibit rat liver GST (Dierickx, 1982). A relationship between the depletion of GSH and metal toxicity has also been described (Das et al., 1988; Norseth and Clarkson, 1971). The role of GSH in metal detoxification processes has likewise been studied in vitro. Cadmium uptake by cultured hepatocytes is significantly reduced by sulfhydryl blockers, indicating that the process is sulfhydryl-dependent (Garty et al., 1986; Gerson and Shaikh, 1982). The protective role of cellular GSH has likewise been described for cadmium toxicity using cultured Chinese Hamster V79 cells (Ochi et al., 1988), Hg2q, Cd2q, Cu2q andyor Pb2q in I-407 intestinal epithelial cells (Keogh et al., 1994), rat hepatoma cells (HTC) and primary cultures of rat hepatocytes (Steine-

bach and Wolterbeek, 1994) as well as isolated perfused rat liver preparations (Strubelt et al., 1996). Several authors have shown that metals such as copper, cadmium, lead, mercury and nickel produce reactive oxygen species (ROS) resulting in lipid peroxidation (Athar et al., 1987; Chan et al., 1982; Donaldson and KnowLes, 1993; Fukino et al., 1984; Muller, 1986) and affecting the intracellular GSH andyor sulfhydryl content (Athar et al., 1987; Goyer, 1996; Milne et al., 1993; Sarafian and Verity, 1991). However, ROS species can be induced by many xenobiotics and neutralised by the cellular antioxidant system, comprising both mitochondrial Mn-containing and cytosolic Cuand Zn-containing superoxide dismutases (SODs) (Bagley et al., 1986; Krall et al., 1988) and the balance between reduced and oxidised GSH (Krall et al., 1991). This study compares the effects of both cytotoxicity levels and dose-response to five metals— mercury, cadmium, copper, nickel and lead—on GSH content and related enzymes in CHO-K1 cells, to ascertain whether ROS are involved in the toxicity of these metals in mammalian cells. 2. Materials and methods 2.1. Materials and metals Reagent grade chemicals and cell culture components, culture medium Ham-F12, antibiotics, trypsinyEDTA, HEPES, 3-amino-7-dimethylamino-2-methylphenazine hydrochloride (neutral red, NR) and GSH, were obtained from Sigma Chemical Co. (St. Louis, MO). Foetal bovine serum was obtained from Boehringer Ingelheim Co. (Germany). Metals selected for the evaluation of cytotoxicity, were used in the form of respective salts obtained from Merck Chemical Co. (Darmstadt, Germany). The metals included, mercury (HgCl2), cadmium (CdCl2), copper (Cu(NO3)2Ø3H2O), nickel (Ni(NO3)2Ø6H2O) and lead (Pb(NO3)2). Ten times the solutions of each concentration of metal were prepared in sterile distilled water and diluted with the cell culture medium to obtain the desired concentrations. Controls were prepared adding a 10% sterile distilled water to the medium. Osmolarities of the different treated media, including controls, ranged between 267 and 269 mOsm.

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No significant differences were found between different media. 2.2. Cell culture and media CHO-K1 cells were obtained from the American Type Culture Collection (ATCC CCI-61). Ten thousands cellsycm2 were plated in 22.1 cm2 polystyrene tissue culture dishes in HAMS-F12 supplemented with 25 mM HEPES buffer (pH 7.4), 10% heat inactivated foetal calf serum and gentamicin (30 mgyml). Cells were counted in an improved Neubauer haemocytometer and viability was determined by the exclusion of Trypan Blue. Cells were grown up to 65% confluence (day 3, mid-log phase) and were then pulsed with different concentrations of the metal solutions. After 24 h cells were washed with phosphate buffer saline (PBS) and medium was replaced. Cell extracts were used to determine sulphur–redox species, enzymes involved in GSH metabolism. 2.3. Determination of cytotoxicity Metals cytotoxicities were estimated in CHOK1 cells using the NR uptake inhibition assay according to Borenfreund and Puerner (1985). Cells were grown up to 65% confluence and then pulsed with different concentrations of the selected metals. After 24 h, cells were washed and medium was replaced with fresh medium containing 40 mgyml NR dye. After 3 h, cells were washed with PBS and fixed with a solution containing 0.5% formaldehyde and 1% CaCl2 in distilled water. Colour was developed with 0.2 ml 1% acetic acid in 50% ethanol. The absorbance was measured at 540 nm, using an automatic ELISA reader. Curves were fitted using Sigma Plot (version 2.1, Microsoft) for non-linear regression, and using probity log curves to obtain the NR values. 2.4. Enzymatic activities CHO-K1 cells from mid-logarithmic growth phase cultures (day 3) were harvested with trypsinyEDTA solution and washed twice with PBS. Cell pellets were resuspended in a 0.25% sucrose containing 1 mM EDTA and disrupted by sonication on ice. Extracts centrifuged at 12 000=g for 15 min at 4 8C. The supernatants were used for enzymatic assays.

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Grd (EC 1.6.4.2) reaction mixture contained in a final volume of 1 ml, 500 ml potassium phosphate buffer, 0.2 M, pH 7.0 containing 2 mM EDTA, 50 ml of 2 mM NADPH in 10 mM HCl– Tris, pH 7.0, 50 ml of 20 mM GSSG, 300 ml distilled water and 100 ml of freshly isolated cellular extracts. The mixture was incubated for 1 min at 30 8C, determining the absorbance rate at 340 nm. One unit of Grd activity is defined as the amount of enzyme reducing 1 mmol of GSSG per min and mg of soluble protein. Proteins were assayed according to Bradford (1976) using bovine serum albumin as a reference. Gpx (EC 1.11.1.9) was measured as described by Wendel (1981). One ml final volume contained 2.5 mM EDTA and 2.5 mM sodium azide in 500 ml potassium phosphate buffer 0.25 M, pH 7.0, 100 ml of 10 mM GSH, 100 ml of 2.5 mM NADPH in 0.1% NaHCO3 and 100 ml of 2.4 Uy ml Grd (Sigma) freshly prepared in PBS. 100 ml of cell extracts were added and incubated for 37 8C. The reaction was started by adding of 100 ml 12 mM tert-butyl peroxide. Absorbance rate was monitored at 366 nm for 1 min at 37 8C. One unit of Gpx is defined as the amount of enzyme oxidising 1 mmol of GSH per min and mg of protein. GST was measured spectrophotometrically in CHO-K1 extracts according Habig et al. (1974). In 1-ml plastic cuvettes, the following reagents were added: 800 ml sodium phosphate buffer, 0.2 M, pH 6.5, 50 ml of 1-chloro-2,4-dinitrobenzoic acid 20 mM dissolved in 95% ethanol, 50 ml of GSH 20 mM, and 100 ml of enzymatic extracts. Enzymatic activity was assayed at 25 8C for 1 min at 340 nm. One unit of GST activity is defined as the amount of enzyme consuming 1 mmol of GSH per min and mg of protein. 2.5. Glutathione content Total GSH were determined spectrophotometrically using the method described by Akerboom and Sies (1981). CHO-K1 cells from 22.1 cm2 polystyrene petri dishes, were harvested with trypsinyEDTA, washed twice with PBS pH 7.4 and disrupted by sonication in cold perchloric acid (1 M) containing 2 mM EDTA. 100 ml of the extracts were neutralised with 70 ml KOH and 70 ml MOPS 0.3 M, before total GSH determination. Protocol for total GSH was as follows: in 1 ml final volume were added 730 ml potassium phos-

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Table 1 Neutral red (NR) cytotoxicity values of metals on the CHOK1 cell line after 24 h Metal

Cytotoxicity (mM)a NR6.25

2q

Hg Cd2q Cu2q Ni2q Pb2qb

0.25"0.1 0.25"0.1 5.7"1.0 29.5"3 49.5"3

NR12.5 0.75"0.1 1.25"0.1 17"1 89.5"6 162"15

NR25 1.8"0.5 2.5"0.5 19"1 125"5 256"23

R

NR50 *

3.0"0.5 8"1* 78"4* 410"26 800"21*

0.96 0.96 0.94 0.96 0.96

a Arithmetic mean of three independent experiments carried out in four replicates"S.D. b Turbidity is found at highest concentrations. * Significant difference at (P(0.05) level (ANOVA test from NR50 cytotoxicity values).

phate buffer, 0.1 M (pH 7.0) containing 1 mM EDTA, 200 ml neutralised cell extracts, 50 ml of 4 mgyml NADPH in 0.5% NaHCO3 and 20 ml of 1.5 mgyml 5,59-ditiobis(2-nitrobenzoic acid) (DTNB) (Sigma) in 0.5% NaHCO3. The reaction was started by adding 6 units Grd. The absorbance was measured for 1 min at 412 nm at 25 8C. Slopes of each determination were interpolated in a calibration curve, expressing GSH as nmol per mg of total protein. GSSG was assayed in freshly acidified cell extracts by the method of Anderson (1985). CHOK1 extracts were obtained as above. PCA-treated

extracts were then centrifuged and neutralised with 2 ml of 2-vinylpyridine (Fluka) and 824 ml 25% triethanolamine. Reaction mixture contained in 1 ml final volume, 650 ml 6.3 mM EDTA in sodium phosphate buffer, 0.1 M pH 7.5, 50 ml of 4 mgy ml NADPH, 100 ml DTNB 6 mM, 200 ml cell extract and 1 IU of Grd. GSSG was estimated by interpolating the slopes of each assay into a standard curve and expressing GSSG content as nmol per mg of total protein. 3. Results Metal cytotoxicity was determined using NR assay, which monitors lysosomal cell function (Table 1). Mercury was the most and lead the least toxic of the metals tested. As the NR assay is highly reliable, fractions of the NR50 concentrations (NR6.25, NR12.5 and NR25) were used to determine the biochemical parameters assayed in the present report. The effects of metals on sulphur–redox cycle constituents were measured after exposure for 24 h in the presence of foetal calf serum in the culture medium. Table 2 summarises total GSH and GSSG contents determined in the midylate log phase after exposure to fractions of NR50 concentrations of the five metals. Total GSH content increased significantly as a result of exposure to the metals.

Table 2 Intracellular glutathione content (total plus oxidised forms) after 24 h of exposure of CHO-K1 cell line to NR6.25, NR12.5 and NR25 metal concentrations Metal

Glutathione content (nmolymg protein)

NR6.25

NR12.5

NR25

Hg2q

Total GSSG

51.51"3.83** 0.14"0.01***

35.36"2.77* 0.23"0.05**

15.60"1.88* 0.30"0.02

Cd2q

Total GSSG

34.82"1.69** 0.13"0.01***

24.99"2.55 0.17"0.02**

18.60"0.69 0.19"0.01**

Cu2q

Total GSSG

53.84"3.06*** 0.14"0.01***

31.44"1.44** 0.14"0.02***

21.59"0.95 0.16"0.01***

Ni2q

Total GSSG

43.89"2.26*** 0.18"0.00***

24.67"1.91 0.25"0.03

24.63"2.59 0.29"0.05

Pb2q

Total GSSG

47.87"0.32*** 0.27"0.04

53.05"0.77*** 0.22"0.03

30.93"2.92* 0.20"0.01***

Control

Total GSSG

23.48"0.67 0.32"0.06

All values represent mean"S.D. of three replicates from three independent experiments. * P(0.01; ** P(0.05; *** P(0.001 with respect to control (t-Student).

´ ´ A.J. Garcıa-Fernandez et al. / Comparative Biochemistry and Physiology Part C 132 (2002) 365–373 Table 3 Specific activity of glutathione peroxidase after 24 h exposure of the CHO-K1 cells to NR6.25, NR12.5 and NR25 metal concentrations Metal

Hg2q Cd2q Cu2q Ni2q Pb2q Control

Specific activity of Gpx (nmolyminymg protein) NR6.25

NR12.5

NR25

206.50"6.89** 151.00"0.73*** 188.60"4.00*** 264.73"10.21*** 323.36"0.42***

174.77"0.87*** 146.53"4.65 178.10"4.10*** 202.40"4.61*** 158.50"3.90**

168.93"4.51** 137.40"6.64 160.97"6.89* 166.96"1.65*** 136.00"1.35*

129.19"9.68

All values represents mean"S.D. of three replicates from three independent experiments. *** P(0.001; ** P(0.05; * P(0.01 with respect to control (t-Student).

This rise was highest in cultures exposed to the lowest concentrations (NR6.25) and dropped when the incubation media contained higher concentrations of the various metals. Total GSH fell below control values at NR25 concentrations of mercury and cadmium. Cellular GSSG content declined significantly at all metal concentrations. Except for lead, the lowest GSSG levels were found at metal concentrations equivalent to the NR6.25 values, with progressively higher values at higher exposure concentrations (Table 2). Intracellular specific activities of the enzymes involved in the GSH–redox balance and GSH transfer were measured in the same cultures for which GSH was determined. Table 3 illustrates the increase in Gpx activity associated with exposure to the metals. These activities were 1.2-, 1.5-, 1.6, 2.0- and 2.5-fold higher than the untreated controls for cadmium, copper, mercury, nickel and lead, respectively, at NR6.25 concentrations. Gpx activity decreased at higher metal concentrations (NR12.5 and NR25) but remained higher than the control values. Despite metal-related Gpx induction, Grd remained unchanged except for cultures exposed to mercury, nickel and lead (Table 4) at NR6.25 and NR12.5 concentrations; the Grd activity declined as the metal concentrations in the incubation medium rose. Attention is drawn to the high Gpx and Grd levels found in cells exposed to lead at all three concentrations. Unlike Gpx and Grd, the activity of GST, one of the enzymes involved in phase II detoxification

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Table 4 Specific activity of glutathione reductase after 24 h exposure of CHO-K1 cells to NR6.25, NR12.5 and NR25 metal concentrations Metal

Hg2q Cd2q Cu2q Ni2q Pb2q

Specific activity of Grd (nmolyminymg protein) NR6.25

NR12.5

NR25

45.26"0.31*** 37.16"0.18 41.63"3.18 46.60"3.18* 62.57"4.06**

41.70"1.61* 35.70"0.56 31.93"3.82 43.77"0.53*** 57.03"2.46**

33.30"2.96 25.30"3.01** 25.10"2.34* 31.87"3.28 47.80"2.15**

Control

34.19"1.33

All values represent mean"S.D. of three replicates from three independent experiments. *** P(0.001; ** P(0.05; * P(0.01 with respect to control (t-Student).

in mammals, declined with increasing concentrations of metals (Table 5). In all cases, GST activity in cultures exposed to NR12.5 and NR25 metal concentrations was below the value for untreated control cultures. Except for mercury, however, GST activity remained unchanged at the lowest levels (NR6.25). 4. Discussion Several reports have documented the role of metals such as mercury, cadmium and others in producing oxidative stress in experimentally exposed animals (Stohs and Bagchi, 1995). Oxidative stress is the primary cause of lipid peroxiTable 5 Specific activity of glutathione-S-transferase after 24 h of exposure of the CHO-K1 cells to NR6.25, NR12.5 and NR25 metal concentrations Metal

Specific activity of GST (nmolyminymg protein) NR6.25

2q

Hg Cd2q Cu2q Ni2q Pb2q

Control

35.43"1.41 39.13"1.55 41.63"1.11 37.64"0.95 42.96"0.56

NR12.5 *

NR25 **

29.03"1.54 32.1"0.22*** 33.43"1.58** 36.33"0.35*** 40.06"0.23**

18.65"1.47*** 22.66"2.24** 23.80"0.62*** 33.60"0.20*** 34.66"0.81**

42.05"1.81

All values represent mean"S.D. of three replicates from three independent experiments. *** P(0.001; ** P(0.05; * P(0.01 with respect to control (t-Student).

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dation (Sarkar et al., 1998; Stohs et al., 2000), alterations in membrane fluidity (Bagchi et al., 2000; Hassoun and Stohs, 1996), DNA damage (Bagchi et al., 1996; Hassoun and Stohs, 1996) and finally, carcinogenic processes (Bagchi et al., 1997; Koizumi and Li, 1992). According to Stohs and Bagchi (1995), a given transition metal may initiate the formation of ROS by a number of different mechanisms, involving more than one organelle or cell type. However, in human epidermal keratinocytes—a valuable tool for studying cytotoxicity mechanisms—Kappus and Reinhold (1994) observed that oxidative stress may not be involved in the cytotoxicity induced by heavy metals such as cadmium, mercury, copper or zinc. Untreated control cells show high Gpx activity during the CHO-K1 mid-log phase, the period of maximum growth, with activity declining in the late and stationary phases. Moreover, significant Grd activity was found in a 48-h subculture indicating that a high reduced to oxidised GSH ratio was maintained during the CHO-K1 mid-log phase (Bayoumi et al., 2000). Since oxygen consumption by aerobic cells peaks at maximum ROS production, the role of Gpx and Grd in conjunction with SOD and catalase activities must be related to ROS disruption (Meister and Anderson, 1983). The exposure of mid-log CHO-K1 cultures to metal concentrations corresponding to fractions of NR50 for 24 h brought about significant changes in GSH metabolism. Our results show that exposure to NR6.25 mercury, cadmium, copper, nickel and lead prompted a sharp increase in total GSH content, Gpx and Grd activities as well as a decrease in GSSG, while GST remained almost unchanged. These results suggest that at low doses of metals, most of the cells are able to maintain a high GSHyGSSG ratio and that only the weakest portion of the culture fails to survive. Chubatsu et al. (1992) showed that cadmium induced a significant increase in intracellular GSH in V79 (Chinese hamster fibroblasts) by acting as both an antioxidant and a chelator. Similarly, Chin and Templeton (1993) reported a significant time- and dose-dependent rise in intracellular GSH, in response to cadmium levels equivalent to those used in the present research. Sarafian and Verity (1991) found that several kinds of tissues accumulate GSH to mitigate lead toxicity. Similar results have shown the protective effect of GSH against copper salts (Milne et al., 1993), zinc

(Huang et al., 1993) and mercury (Rungby and Ernst, 1992). As expected, response drops when cells are exposed to higher metal concentrations. Chin and Templeton (1993) proved that exposure to high doses of cadmium caused depletion of intracellular GSH due to ROS production. In any case, for all metals (except mercury), at the highest dose the GSH levels were near the values of untreated control cells. Mercury has been described to cause a decline in GSH in renal tubules with a concomitant decrease in SOD, Gpx, and catalase activity, thus supporting the involvement of ROS in altering membrane integrity, the mechanism that determines the nephrotoxic effects of mercury (Stohs and Bagchi, 1995). Gpx induction is associated with the overexpression of SOD, the enzyme that controls superoxide anion disruption and cell homeostasis. Lee and Ho (1994) showed that CHO cells exhibiting Gpx activity 10-fold higher than human HFW fibroblasts are ten times more resistant to xenobiotics. Warner et al. (1993), moreover, described the increased resistance to several xenobiotics in a human SOD-transfected CHO cell line. These findings are compatible with experiments designed by Cutler (1985) for various animal species, which correlated the enzymatic antioxidant defence system with their life span. The highest rise in Gpx activity (2.5-fold) was found for lead, the least toxic metal in the present study (Table 1). This fact may explain the higher resistance of CHO-K1 cultures to this metal. Attention is drawn to the close relationship observed between cytotoxicity of the metals, total GSH content and the dissociation energies of the sulphur-metal bonds established by Weast (1978) by mass spectrometry (Fig. 1a, b and c). Our results show a clear relationship between the sulphur–metal energies and the protection offered by the tripeptide, thus confirming the involvement of antioxidant defence in the mode of action of cells in response to these elements. In conclusion, the metals assayed in present study (mercury, cadmium, copper, nickel and lead) induced a large increase in total GSH and Gpx activity in CHO-K1 cultures after being exposed to doses equivalent to NR6.25 values for 24 h, thus suggesting the activation of a homeostatic defence mechanism. At higher doses cells progressively lose their ability to respond.

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Acknowledgments This study has been supported in part by grants ´ from CICYT IFD 97-0224-CO3-03 and Diputacion ´ Dr Alaa Eldin Bayoumi was Provincial de Leon. ˜ a post-graduate scholarship of Agencia Espanola ´ Internacional (AECI). de Cooperacion References

Fig. 1. Relationships amongst cytotoxicity data (NR25 value), glutathione content and dissociation energy of the sulphur– metal bond. Panel a shows a semilogarithmic plot between cytotoxicity and total GSH (nmolymg protein) after exposure (24 h) to metals. In Panel b cytotoxicity is compared with dissociation energy (kcalymol) of the sulphur–metal bond. In Panel c the relationship between dissociation energy (kcalymol) of the sulphur–metal bond and total GSH (nmolymg protein) is given.

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