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Research ArticleArticle

Protection from Cadmium Cytotoxicity by N-Acetylcysteine in LLC-PK1 Cells

Bambang Wispriyono, Masato Matsuoka, Hideki Igisu and Koji Matsuno
Journal of Pharmacology and Experimental Therapeutics October 1998, 287 (1) 344-351;

Abstract

N-acetylcysteine (NAC) has been known not only to stimulate synthesis of glutathione but also to affect the gene regulation. In our study, effects of NAC on the cytotoxicity of cadmium (Cd) were examined in LLC-PK1 cells. Preincubation and subsequent incubation with 1 mM NAC almost completely suppressed Cd-induced cellular damage evaluated either by trypan blue exclusion or lactate dehydrogenase leakage. This almost complete protection required the presence of NAC during Cd exposure. Treatment with 1 mM NAC increased the intracellular glutathione level approximately 2-fold. Inhibition of this increase by buthionine sulfoximine did not abolish the protection by NAC. One mM NAC also suppressed Cd-induced increase of c-Fos protein although NAC alone did not change the protein content. The inhibition of transcriptions by actinomycin D did not affect the protection by NAC. Thus, NAC-induced protection appeared to be independent of glutathione level or the transcriptional activation of genes including c-fos. However, treatment with NAC markedly lowered the uptake of Cd into cells although it did not affect the efflux clearly. Addition of NAC during the exposure to Cd suppressed Cd-induced cellular damage but the suppression decreased when the duration of the exposure without NAC increased. These results suggest that NAC-induced protection against Cd cytotoxicity is mainly due to the lowered uptake of Cd into the cells.

NAC, a thiol-containing compound, has been used against acetaminophen overdose due to its potential to stimulate synthesis of glutathione in the liver (Flanagan and Meredith, 1991). In addition to the replenishment of intracellular sulfhydryl pool, NAC has been shown to directly reduce the level of reactive oxygen species such as hydrogen peroxide, hydroxy radical and hypochlorous acid (Aruoma et al., 1989). NAC can also affect the gene regulation at the level of redox-sensitive transcriptional factors including AP-1 and nuclear factor κB (Cotgreave, 1997).

Cd is an important occupational and environmental pollutant that causes damage to various organs (Friberg et al., 1986;Morselt, 1991). There is no effective therapy for Cd poisoning although metallothionein has been shown to play a key role in the detoxification of Cd (Klaassen and Liu, 1997). It has been reported that NAC suppressed Cd-induced recombination in the yeast Saccharomyces cerevisiae and reduction of survival (Brennan and Schiestl, 1996). In the rat lung epithelial cells, treatment with NAC inhibited the expression of metallothionein-1, glutathione-S-transferase Ya and heme oxygenase-1 genes and suppressed Cd cytotoxicity (Gong and Hart, 1997). In rats given Cd, administration of NAC enhanced the renal excretion of Cd (Ottenwälder and Simon, 1987). However, several contradictory results have also been presented. Treatment with NAC did not show the protection against Cd cytotoxicity in the human small intestinal epithelial cells (Keogh et al., 1994), and the teratogenicity or the reduction of survival in mice intoxicated with Cd (Endo and Watanabe, 1988; Henderson et al., 1985). Thus, it is unclear whether NAC can suppress the cytotoxicity of Cd, and its mechanisms, if any, are still unknown. Furthermore, to our knowledge, effects of NAC on the proximal tubular cells, which are one of the major targets in Cd poisoning (Friberg et al., 1986), have not been studied. We therefore examined the effects of NAC on Cd cytotoxicity in LLC-PK1 cells, an established renal epithelial cell line.

Materials and Methods

Cell culture and treatments.

LLC-PK1 cells, a porcine renal epithelial cell line, were obtained from Health Science Research Resources Bank (Osaka, Japan) and grown in medium 199 supplemented with 5% heat-inactivated fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin (all from GIBCO BRL, Life Technologies, Inc., Rockville, MD) in a humidified atmosphere of 5% CO2, 95% air at 37°C.

The stock solution of cadmium chloride (CdCl2, Sigma Chemical Co., St. Louis, MO) and NAC (Sigma) were diluted with distilled water. Based on the results of the first experiment on the cytotoxicity of Cd in LLC-PK1 cells, cells were incubated with serum-free medium containing 20 μM CdCl2 for 18 hr unless otherwise specified. LLC-PK1 cells were preincubated with serum-free medium containing varying dose of NAC (0.01, 0.1 and 1 mM) for 5 min, 1 or 12 hr and subsequently incubated with CdCl2 in the presence of NAC. When incubated with CdCl2 in the absence of NAC, cells were washed twice with Ca++- and Mg++-free PBS after the preincubation with 1 mM NAC for 12 hr. In all other cases, cells were subsequently exposed to CdCl2 without washing. In the experiment of adding NAC after Cd, 1 mM NAC was added 0 to 18 hr after the exposure to 20 μM CdCl2 was started. When effects of BSO (Nacalai Tesque, Osaka, Japan) or AcD (Sigma) were examined, cells were preincubated for 1 hr with serum-free medium containing 1 mM NAC and either 50 μM BSO or 5 μg/ml AcD, and then incubated with CdCl2. Untreated control cells were incubated with serum-free medium, and treated identically to the cells exposed to CdCl2.

Cytotoxicity studies.

Cellular damage induced by Cd was evaluated by trypan blue exclusion assay or by monitoring the release of LDH into the culture medium. For trypan blue assay, LLC-PK1 cells were plated at 1 × 106cells/dish in 60-mm culture dishes and cultured for 2 days. At the end of the incubation with CdCl2, culture medium was aspirated and reserved. The cells were detached from culture dishes by treatment with 0.25% trypsin/1 mM EDTA (GIBCO BRL). After trypsinization, cells were suspended in medium 199 and the culture medium was returned. The mixture was centrifuged at 800 × g for 3 min to concentrate the cells. Cellular suspension and 0.4% trypan blue in Hanks’ balanced salt solutions were mixed (final concentration of 0.07% trypan blue), and the number of viable cells was counted using a hemacytometer in the triplicate samples. The percentage of viable cells (cell viability) was calculated as 100 × (unstained cells)/(stained + unstained cells).

For LDH assay, cells were plated at 2.5 × 104cells/well in 96-well culture plates or 5 × 105cells/well in 6-well culture plates and cultured for 2 days. At the end of the incubation with CdCl2, culture medium was removed and centrifuged at 800 × g for 10 min to obtain cell-free supernatant. The activity of LDH in the supernatant was determined using a Cytotoxicity Detection Kit (LDH) (Boehringer Mannheim, Tokyo, Japan). Because NAC could affect LDH activity measured with this kit, the absorbance value of medium 199 containing NAC (0, 0.01, 0.1 or 1 mM) at 490 nm was subtracted from each value of the supernatant. The results were expressed as percentage of the maximum amount of LDH released from samples that had been treated with 1% Triton X-100 (percentage release).

Determination of intracellular glutathione.

The concentration of total glutathione (reduced and disulfide forms) was determined according to the method of Tietze (1969) with slight modifications (Yan et al., 1995). LLC-PK1 cells were plated at 5 × 105 cells/well in 6-well culture plates and cultured for 2 days. At the end of the incubation, cells were washed twice with Ca++- and Mg++-free PBS and lysed with 0.1 ml of 3% perchloric acid for 15 min at 4°C. After centrifugation at 800 × g for 5 min, supernatants were neutralized with 0.9 ml of 0.1 M sodium phosphate/5 mM EDTA buffer, pH 7.5 (phosphate/EDTA buffer). The reaction mixture contained 20 μl of the neutralized extract, 0.95 ml of phosphate/EDTA buffer, 10 μl of 60 mM 5,5′-dithio-bis(2-nitrobenzoic acid) (Wako Pure Chemical Industries, Osaka, Japan), 10 μl of 20 mM NADPH (Boehringer Mannheim) and 1 U of glutathione reductase (Boehringer Mannheim). The increase of absorbance at 412 nm was monitored for 6 min. At each determination, a standard curve of glutathione was prepared.

Western blots.

LLC-PK1 cells were plated at 2 × 105 cells/well in 24-well culture plates, cultured for 1 day and serum-starved for another 1 day. After the incubation with 20 μM CdCl2 for 2 hr, cells were washed with Ca++- and Mg++-free PBS and lysed with sodium dodecyl sulfate-polyacrylamide gel Laemmli sample buffer. Cell lysates were collected, sonicated and boiled for 5 min. Aliquots equivalent to 5 × 104 cells were subjected to sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Hybond-ECL, Amersham, Tokyo, Japan). Membrane was blocked in 5% milk in TBS-T for 1 hr at room temperature. Primary antibody used was c-Fos (4) rabbit polyclonal antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA), which was specific for c-Fos p62. The membrane was incubated with the primary antibody diluted 1:200 in 5% milk in TBS-T for 45 min at room temperature, washed and incubated with a 1:5000 dilution of anti-rabbit peroxidase-conjugated antibody (Santa Cruz) in 5% milk in TBS-T for 30 min at room temperature. Protein was detected with the ECL (enhanced chemiluminescence) Western blotting kit (Amersham).

To confirm the p62 band as c-Fos, c-Fos (4) antibody was neutralized with a 10-fold excess of specific control peptide (Santa Cruz) at 4°C overnight and subjected to Western blot analysis as described above.

Uptake and efflux of 109Cd++.

LLC-PK1 cells were plated at 5 × 105cells/well in 6-well culture plates and cultured for 1 day. After serum-starvation for 1 day, cells were preincubated with 0.01, 0.1 or 1 mM of NAC for 1 hr, and then incubated with 20 μM CdCl2containing 0.015 μCi of 109Cd++ (New England Nuclear, Boston, MA) for 4 hr. In the time course study, cells preincubated with or without 1 mM NAC for 1 hr were then incubated with109Cd++ for 1 to 8 hr. At the end of the incubation, medium was aspirated and cells were washed twice with PBS containing 2 mM EGTA to remove free 109Cd++. Cells were lysed with 1 N NaOH, and the radioactivity of lysates was counted using an Aloka Auto Well Scintillation Counter (model ARC-2000). Radioactivity remained in the well was less than 0.8% of the total radioactivity added.

For the measurement of efflux of Cd, cells were first incubated with 20 μM CdCl2 containing 0.015 μCi of109Cd++ for 4 hr. After washing twice with PBS containing 2 mM EGTA, cells were incubated with NAC (0.01, 0.1 and 1 mM) for 1 or 4 hr. The radioactivity in the lysate was counted as described above.

Statistical analysis.

Results were expressed as mean ± S.D. The statistical significance was determined by one-way analysis of variance followed by the Bonferroni multiple comparison test using an Instat software package (Graphpad Software Inc., San Diego, CA). P < .05 was considered as statistically significant.

Results

Cytotoxicity of CdCl2 in LLC-PK1cells.

Depending on the incubation time (from 2 to 24 hr, fig.1A) and the dose of CdCl2(from 1 to 40 μM, fig. 1B), cell viability assayed by trypan blue exclusion decreased and LDH leakage increased. Based on these findings, effects of NAC on Cd cytotoxicity were examined in cells exposed to 20 μM CdCl2 for 18 hr, where approximately 40% decrease in trypan blue exclusion and 60 to 80% release of the maximum amount of LDH were seen.

Figure 1
Figure 1

Time course (A) and dose effects (B) of Cd-induced cellular damage in LLC-PK1 cells. A, Cells were incubated with 20 μM CdCl2 for 0, 2, 8, 12, 18 or 24 hr. The untreated control is 0 hr. B, Cells were incubated with 0, 1, 5, 10, 20 or 40 μM CdCl2 for 18 hr. Untreated control is 0 μM. Each value (mean ± S.D.) of cell viability as assayed by trypan blue exclusion (○, n = 3) and LDH leakage (•,n = 6) represents the percentage of control and the percentage release, respectively.

Effects of NAC on Cd cytotoxicity.

Effects of varying doses of NAC (0.01, 0.1 and 1 mM) and the preincubation period (5 min, 1 and 12 hr) on Cd cytotoxicity were examined. In these experiments, cells were exposed to CdCl2 in the presence of NAC. When cells were preincubated with NAC for 12 hr (fig.2C), Cd-induced decrease of cell viability (P < .01 compared to control cells) was suppressed markedly. Viability of cells preincubated with 1 mM NAC for 12 hr was significantly higher than that of no NAC treatment (P < .01) and was not different from control cells (fig. 2C). Consistent with these findings, Cd-induced increase of LDH leakage (P < .01 compared to control cells) was suppressed significantly by preincubation for 12 hr with 0.01 mM (P < .05 compared to cells without NAC treatment), 0.1 mM (P < .01) or 1 mM NAC (P < .01, fig.3). There was no significant difference in LDH leakage between cells preincubated with 1 mM NAC for 12 hr and control cells (fig. 3).

Figure 2
Figure 2

Effects of preincubation with NAC for 5 min (A), 1 hr (B) and 12 hr (C) on Cd cytotoxicity (cell viability assayed by trypan blue exclusion). After preincubation with 0, 0.01, 0.1 or 1 mM NAC, cells were incubated with 20 μM CdCl2 for 18 hr in the presence of NAC. Each value (mean ± S.D., n = 3) represents the percentage of untreated control. * P < .05, ** P < .01 compared to cells exposed to CdCl2without NAC treatment.

Figure 3
Figure 3

Effects of NAC on Cd cytotoxicity (LDH leakage). After cells were preincubated with 0, 0.01, 0.1 or 1 mM NAC for 12 hr, 20 μM CdCl2 was added and the incubation continued for 18 hr. Each value (mean ± S.D., n = 6) represents the percentage release. * P < .05, ** P < .01 compared to cells exposed to CdCl2 without NAC treatment.

When the preincubation period was shortened to 5 min (fig. 2A) or 1 hr (fig. 2B), protective effects of NAC on cell viability were still observed dose dependently. With 5 min or 1 hr preincubation, 0.1 and 1 mM NAC increased the cell viability significantly compared to cells without NAC treatment (P < .05 and P < .01, respectively). The viability did not differ between cells preincubated with 1 mM NAC for 5 min or 1 hr and control cells (fig. 2A and B).

To examine whether the presence of NAC during the Cd exposure is necessary for the suppression of cytotoxicity, we determined Cd toxicity in cells preincubated with NAC and washed extensively with PBS before the incubation with CdCl2. Washing procedure did not affect cell viability nor LDH leakage in the control and Cd-exposed cells (data not shown). The cell viability was much lower (P < .01, fig. 4A), and LDH leakage was higher in cells with washing (P < .01, fig. 4B) than those without washing. However, cells with washing showed modest decrease in LDH leakage compared to cells without NAC treatment (P < .01, fig.4B).

Figure 4
Figure 4

Effects of washing of cells on NAC-induced protection against Cd cytotoxicity. Cells were preincubated with 1 mM NAC for 12 hr. Then, cells with or without washing were incubated with 20 μM CdCl2 for 18 hr. Two columns on the left indicate untreated cells (control) and cells exposed to 20 μM CdCl2 for 18 hr without NAC (Cd). Each value (mean ± S.D., n = 3) of cell viability (A) and LDH leakage (B) represents the percentage of untreated control and the percentage release, respectively. ** P < .01 compared to cells exposed to CdCl2without NAC treatment (Cd). †† P < .01 compared to cells exposed to NAC plus CdCl2 (NAC + Cd) without washing.

Effects of BSO on NAC-induced protection against Cd cytotoxicity.

To examine whether the intracellular glutathione level plays a role in the protection, cells were treated with BSO, an inhibitor of γ-glutamylcysteine synthase, to suppress glutathione synthesis. Although exposure to 20 μM CdCl2 for 18 hr resulted in a 44% decrease in glutathione level (P < .05 compared to control cells), preincubation and subsequent incubation with 1 mM NAC increased glutathione level 2.2-fold (P < .01, fig.5A). However, treatment with BSO abolished NAC-induced increase of glutathione level and resulted in a decrease to 54% of control level (P < .01, fig. 5A). However, NAC-induced protection against Cd cytotoxicity was observed in cells treated with BSO; cell viability was increased (P < .01, fig. 5B) and LDH leakage was reduced compared to cells without NAC treatment (P < .01, fig. 5C). Thus, Cd cytotoxicity was suppressed by NAC even in the glutathione-deficient status. When compared between cells with and without BSO, no significant differences were found in cell viability (fig. 5B) or LDH leakage (fig. 5C). Treatment with 50 μM BSO alone did not affect cell viability nor LDH leakage (data not shown).

Figure 5
Figure 5

Intracellular glutathione level and effects of BSO on NAC-induced protection against Cd cytotoxicity. Cells (shown by the two columns on the right) were incubated with 1 mM NAC or 1 mM NAC plus 50 μM BSO for 1 hr, and then with 20 μM CdCl2 for 18 hr in the presence of NAC. Each value (mean ± S.D.) of glutathione (A,n = 3), cell viability (B, n = 3) and LDH leakage (C, n = 6) represents μg glutathione equivalents/106 cells, the percentage of untreated control, and the percentage release, respectively. # P < .05, ## P < .01 compared to untreated control. ** P < .01 compared to cells exposed to CdCl2 without NAC or BSO treatment.

Effects of AcD on NAC-induced protection against Cd cytotoxicity.

To examine whether the protection by NAC is dependent on the transcriptional activation, cells were treated with AcD, an inhibitor of transcription. The NAC-induced protection against Cd cytotoxicity was observed in cells treated with AcD; cell viability was increased (P < .01, fig. 6A) and LDH leakage was reduced compared to cells without NAC nor AcD treatment (P < .01, fig. 6B). Thus, NAC-induced protection did not depend on the transcriptional activation. When compared between NAC-treated cells with and without AcD, cell viability was lower (P < .05, fig. 6A), and LDH leakage was higher in NAC-treated cells with AcD (P < .01, fig. 6B). These effects might be explained by the findings indicating that treatment of LLC-PK1 cells with AcD caused moderate cellular damage (data not shown).

Figure 6
Figure 6

Effects of AcD on NAC-induced protection against Cd cytotoxicity. Cells (shown by the two columns on the right) were incubated with 1 mM NAC or 1 mM NAC plus 5 μg/ml AcD for 1 hr, and then with 20 μM CdCl2 for 18 hr in the presence of NAC. Each value (mean ± S.D.) of cell viability (A, n= 3) and LDH leakage (B, n = 6) represents the percentage of untreated control and the percentage release, respectively. ** P < .01 compared to cells exposed to CdCl2 without NAC or AcD treatment. † P < .05, †† P < .01 compared to cells exposed to NAC plus CdCl2 without AcD treatment.

When compared between Cd-exposed cells with and without AcD, cell viability was lower (P < .01, fig. 6A) in Cd-exposed cells with AcD although LDH leakage was not different (fig. 6B). This indicates that the inhibition of transcription by AcD could not protect cells from Cd cytotoxicity.

Effects of Cd and NAC on c-Fos protein expression.

Western blotting immunodetection revealed a marked increase in c-Fos p62 protein level in cells treated with 20 μM CdCl2 for 2 hr (fig. 7, lane 2). Prior to incubation of c-Fos (4) antibody with the corresponding immunogen peptide abolished the appearance of a 62-kDa band (data not shown). Preincubation with 1 mM NAC for 12 hr did not affect the expression of c-Fos protein (lane 3). However, treatment with 1 mM NAC completely suppressed Cd-induced increase of c-Fos protein (lane 4).

Figure 7
Figure 7

Western blot analysis for c-Fos p62 protein. Cells were incubated with 0 μM (lane 1) or 20 μM CdCl2 (lane 2) for 2 hr without NAC treatment. After preincubation with 1 mM NAC for 12 hr, cells were incubated without (lane 3) or with 20 μM CdCl2 (lane 4) for 2 hr. Cell lysate equivalent to 5 × 104 cells was loaded per lane and Western blotting was done using c-Fos (4) antibody.

Effects of NAC on Cd uptake and efflux.

LLC-PK1cells accumulated 109Cd++ linearly as the incubation period increased (fig. 8). When cells were preincubated for 1 hr and further incubated with 1 mM NAC, the accumulation was suppressed markedly (fig. 8). Depending on the dose of NAC (preincubation for 1 hr and subsequent incubation with109Cd++ for 4 hr), the accumulation of109Cd++ was suppressed markedly (fig.9). However, the amount of109Cd++ accumulated within the cells did not decrease when the cells were incubated with NAC (0.01, 0.1 and 1 mM) for 1 or 4 hr after the exposure to Cd. The intracellular radioactivity did not differ between cells incubated with and without NAC (fig.10). However, mild but significant increase in the radioactivity of culture medium was observed when cells were incubated with 0.1 or 1 mM NAC after the exposure to Cd (data not shown).

Figure 8
Figure 8

Time course of 109Cd++ uptake into LLC-PK1 cells. After preincubation without (○) or with 1 mM NAC (•) for 1 hr, 20 μM CdCl2 containing109Cd++ was added and the incubation continued for 0, 1, 2, 4 or 8 hr. Each value (mean ± S.D.,n = 3) represents cpm/106 cells. Point without error bars indicates S.D. less than the size of the circle.

Figure 9
Figure 9

Dose effects of NAC on109Cd++ uptake into LLC-PK1 cells. After preincubation with 0, 0.01, 0.1 or 1 mM NAC for 1 hr, the incubation was continued for 4 hr after adding 20 μM CdCl2 containing 109Cd++. Each value (mean ± S.D., n = 3) represents cpm/106 cells. ** P < .01 compared to cells exposed to CdCl2 without NAC treatment.

Figure 10
Figure 10

Effects of NAC on 109Cd++efflux from LLC-PK1 cells. Initially, cells were incubated with 20 μM CdCl2 containing109Cd++ for 4 hr. After washing with 2 mM EGTA, cells were incubated with 0, 0.01, 0.1 or 1 mM NAC for 1 hr (open column) or 4 hr (closed column). Each value (mean ± S.D.,n = 3) represents cpm/106 cells.

Effects of adding NAC after Cd on cytotoxicity.

To examine whether NAC can protect the cells against Cd cytotoxicity even after Cd exposure, 1 mM NAC was added to the cells at the appropriate time (from 0 to 18 hr) after the start of exposure to 20 μM CdCl2. Addition of NAC at 0 to 12 hr after the start of CdCl2 exposure could still increase the cell viability (P < .01, fig. 11A), and addition at 0 to 8 hr after Cd exposure also could lower the LDH leakage (P < .01, fig. 11B).

Figure 11
Figure 11

Effects of NAC added during the exposure to Cd on cytotoxicity. LLC-PK1 cells were exposed to 20 μM CdCl2 for 18 hr, and 1 mM NAC was added 0, 1, 2, 4, 6, 8, 12 and 18 hr after the CdCl2 exposure was started. Cell viability and LDH leakage were determined after the end of exposure to CdCl2. Each value (mean ± S.D.) of cell viability (A,n = 3) and LDH leakage (B, n = 6) represents the percentage of untreated control and the percentage release, respectively. ** P < .01 compared to cells exposed to CdCl2 without NAC treatment (Cd).

Discussion

Our study demonstrated that in LLC-PK1 cells NAC can suppress Cd-induced cellular damage that was evaluated either by trypan blue exclusion or LDH leakage. This protective effect of NAC was dependent on the dose of NAC, but not on the preincubation period. Almost complete protection against Cd cytotoxicity was observed in cells preincubated with 1 mM NAC only for 5 min. Furthermore, washing of the cells before Cd exposure markedly decreased the protective effects of NAC. If the washing procedure should remove NAC only from the incubation mixture but not the cells, this finding suggests that presence of NAC during Cd exposure was necessary for the complete protection of LLC-PK1 cells from the toxicity of Cd. Nevertheless, a mild but significant decrease in LDH leakage was observed in cells that were preincubated with NAC and washed before incubation with Cd. Thus, the effects of NAC seen in our experiments seem to reflect its extracellular and in part intracellular reactions.

It was suggested that cytotoxicity of Cd might be related to alterations in cellular glutathione metabolism and that increase of glutathione may protect cells from the toxicity of Cd (Prozialeck and Lamar, 1995). In our experiments, treatment with BSO decreased the glutathione level to 54% of the control even when coincubated with NAC. However, NAC-induced protection against Cd cytotoxicity was still observed in cells treated with BSO. These suggest that major effects of NAC seen in our experiments were not due to changes of glutathione metabolism in the cell.

The c-fos gene encodes a DNA-binding protein (c-Fos) which functions as a component of the transcriptional factor, AP-1 (Cohen and Curran, 1989). Expression of c-fos has been known to modify transcription of probable target genes such as human metallothionein IIa and collagenase, and has been related to cellular proliferation and differentiation (Angel and Karin, 1991; Cohen and Curran, 1989). At the dose of more than 30 mM, NAC was shown to enhance c-fos transcript in the cultured rat lenses (Li et al., 1994) and in the rabbit lens epithelial cells (Li and Spector, 1997). Therefore, to examine the role of c-Fos protein in NAC-induced protection against Cd cytotoxicity, we determined its level in LLC-PK1 cells treated with 1 mM NAC. One mM NAC did not increase c-Fos protein level although the same dose of NAC could suppress Cd cytotoxicity completely. Furthermore, the inhibition of transcriptions by AcD did not change the cytoprotective effects of NAC. Therefore, the protection seems independent of the transcriptional activation of genes including c-fos. In this regard, effects of NAC on LLC-PK1 cells exposed to Cd were different from those on PC12 cells deprived of trophic factors where the survival-promoting effects of NAC were blocked by AcD (Yan et al., 1995). Examination of the transactivity of AP-1, which has been reported to be increased by NAC (Meyer et al., 1993; Li and Spector, 1997), and that of related signal transduction pathways might provide clues to explain these differences.

c-Fos protein has been shown to play a causal role in the activation of apoptosis (Preston et al., 1996). We have previously found that Cd increased c-fos mRNA level in LLC-PK1 cells, and subsequently the level of cytoplasmic histone-associated DNA fragments, which were characteristic of apoptosis (Matsuoka and Call, 1995). In our study, the exposure to Cd increased c-Fos protein level markedly, and NAC abolished this. Although this finding is consistent with the NAC-induced suppression of cellular damage evaluated by trypan blue exclusion and LDH leakage, it is not clear whether the disappearance of c-Fos protein might play a role in suppression of apoptosis in LLC-PK1 cells because the inhibition of transcription by AcD did not protect cells from Cd cytotoxicity.

LLC-PK1 cells accumulated Cd with the time of incubation at least for 8 hr as was shown in a previous study (Templeton, 1990). Treatment with NAC markedly decreased the accumulation of Cd in the cells. However, although 109Cd++ count in the culture medium increased mildly, the amount of Cd contained in the cells did not decrease when the cells were incubated with NAC after the exposure to Cd. Thus, NAC suppressed the uptake of Cd, but did not increase its efflux markedly. Consistent with this, coincubation with NAC has been found to decrease the accumulation of Cd in the rat lung epithelial cells (Gong and Hart, 1997).

It has been reported that glutathione, a cysteine-containing tripeptide, can form a complex with Cd (Perrin and Watt, 1971;Dı́az-Cruz et al., 1997), and that exogenously added glutathione lowered uptake of Cd into LLC-PK1 cells (Bruggeman et al., 1992; Kimura et al., 1997) and other types of cells (Stacey, 1986; Klug et al., 1988; Kang, 1992). In agreement with these, we found that incubation with 1 mM glutathione or 1 mM cysteine prevented Cd cytotoxicity in LLC-PK1 cells as NAC did (data not shown).1H-Nuclear magnetic resonance study on chemical solutions suggested that not only glutathione but also NAC may form mixed and single ligand complexes with Cd (Kadima and Rabenstein, 1990). Therefore, further studies are necessary to clarify whether NAC can form a complex with Cd extracellularly, and whether it can interfere with the transport through the cell membrane. However, because uptake of Cd into LLC-PK1 cells occurs not only via passive transport but via active transport (Endo et al., 1995), it is possible that the reduction of proteins by NAC in the cell membrane might affect Cd uptake.

NAC can decrease the level of reactive oxygen species (Aruomaet al., 1989), which have been proposed to be involved in genotoxic and cytotoxic actions of Cd (Sugiyama, 1994). In the human peripheral blood mononuclear cells, treatment with NAC suppressed Cd-induced production of reactive oxygen intermediates (Horiguchiet al., 1993). Because in our study some protection was observed in cells that were preincubated with NAC and washed before the exposure to Cd, the involvement of intracellular antioxidant effects of NAC cannot be ruled out. Therefore, the metabolism of NAC and possible changes of reactive oxygen species level in LLC-PK1 cells exposed to Cd remain to be examined.

To suppress toxicities of heavy metals including Cd, much attention has been paid to chelating agents (Friberg et al., 1986). It has been shown that NAC increased renal excretion of Cd (Ottenwälder and Simon, 1987) and other metals such as methyl mercury (Lund et al., 1984), gold (Godfrey et al., 1982), lead (Ottenwälder and Simon, 1987) and chromium (Banner et al., 1986). Recently, it has been reported that administration of NAC was effective in the renal failure induced by renal ischemia in rats (DiMari et al., 1997) and in cisplatin overdose in a human (Sheikh-Hamad et al., 1997). In our study, although the protective effects became less marked as the incubation period without NAC increased, NAC could protect the cells from Cd toxicity even after Cd exposure was started. Given the safety of NAC as a therapeutic agent, NAC may be used in cases of intoxication with nephrotoxic heavy metals. When the application of NAC to Cd poisoning is considered, it may be important to examine whether NAC can prevent cellular damage induced by Cd-metallothionein complex, which has been believed to be responsible for the renal injury (Dudleyet al., 1985).

In summary, our results show that NAC can suppress Cd-induced cellular damage and accumulation of c-Fos protein in LLC-PK1 cells. The almost complete protection against Cd cytotoxicity required the presence of NAC during Cd exposure, and was independent of the increase of the intracellular glutathione level and transcriptional activation. Treatment with NAC lowered the uptake of Cd into cells but did not clearly affect the efflux. NAC could protect the cells from Cd cytotoxicity even after Cd exposure but the protection became less marked as the duration of exposure to Cd without NAC increased. The lowered uptake of Cd by NAC seems to be responsible for the suppression of Cd cytotoxicity in LLC-PK1 cells.

Footnotes

  • Send reprint requests to: Dr. Masato Matsuoka, Department of Environmental Toxicology, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan.

  • Abbreviations:
    NAC
    N-acetyl-l-cysteine
    Cd
    cadmium
    AP-1
    activator protein 1
    PBS
    phosphate-buffered saline
    BSO
    l-buthionine-(S,R)-sulfoximine
    AcD
    actinomycin D
    LDH
    lactate dehydrogenase
    TBS-T
    Tris-buffered saline containing 0.05% Tween 20
    • Received December 30, 1997.
    • Accepted May 12, 1998.

References

  1. ↵
    1. Angel P,
    2. Karin M
    (1991) The role of Jun, Fos and the AP-1 complex in cell-proliferation and transformation. Biochim Biophys Acta 1072:129–157.
    OpenUrlPubMed
  2. ↵
    1. Aruoma OI,
    2. Halliwell B,
    3. Hoey BM,
    4. Butler J
    (1989) The antioxidant action of N-acetylcysteine: Its reaction with hydrogen peroxide, hydroxyl radical, superoxide, and hypochlorous acid. Free Radic Biol Med 6:593–597.
    OpenUrlCrossRefPubMed
  3. ↵
    1. Banner WJr,
    2. Koch M,
    3. Capin DM,
    4. Hopf SB,
    5. Chang S,
    6. Tong TG
    (1986) Experimental chelation therapy in chromium, lead, and boron intoxication with N-acetylcysteine and other compounds. Toxicol Appl Pharmacol 83:142–147.
    OpenUrlCrossRefPubMed
  4. ↵
    1. Brennan RJ,
    2. Schiestl RH
    (1996) Cadmium is an inducer of oxidative stress in yeast. Mutat Res 356:171–178.
    OpenUrlCrossRefPubMed
  5. ↵
    1. Bruggeman IM,
    2. Temmink JHM,
    3. van Bladeren PJ
    (1992) Effect of glutathione and cysteine on apical and basolateral uptake and toxicity of CdCl2 in kidney cells (LLC-PK1). Toxic In Vitro 6:195–200.
  6. ↵
    1. Cohen DR,
    2. Curran T
    (1989) The structure and function of the fos proto-oncogene. Crit Rev Oncog 1:65–88.
    OpenUrlPubMed
  7. ↵
    1. Cotgreave IA
    (1997) N-Acetylcysteine: Pharmacological considerations and experimental and clinical applications. Adv Pharmacol 38:205–227.
  8. ↵
    1. Dı́az-Cruz MS,
    2. Mendieta J,
    3. Tauler R,
    4. Esteban M
    (1997) Cadmium-binding properties of glutathione: A chemometrical analysis of voltammetric data. J Inorg Biochem 66:29–36.
    OpenUrlCrossRef
  9. ↵
    1. DiMari J,
    2. Megyesi J,
    3. Udvarhelyi N,
    4. Price P,
    5. Davis R,
    6. Safirstein R
    (1997) N-Acetyl cysteine ameliorates ischemic renal failure. Am J Physiol 272:F292–F298.
    OpenUrlAbstract/FREE Full Text
  10. ↵
    1. Dudley RE,
    2. Gammal LM,
    3. Klaassen CD
    (1985) Cadmium-induced hepatic and renal injury in chronically exposed rats: Likely role of hepatic cadmium-metallothionein in nephrotoxicity. Toxicol Appl Pharmacol 77:414–426.
    OpenUrlCrossRefPubMed
  11. ↵
    1. Endo A,
    2. Watanabe T
    (1988) Analysis of protective activity of N-acetylcysteine against teratogenicity of heavy metals. Reprod Toxicol 2:141–144.
    OpenUrlCrossRefPubMed
  12. ↵
    1. Endo T,
    2. Kimura O,
    3. Sasaya M,
    4. Takada M,
    5. Sakata M
    (1995) Na+- and energy-dependent transport of cadmium into LLC-PK1 cells. Biol Pharm Bull 18:1689–1693.
    OpenUrlPubMed
  13. ↵
    1. Flanagan RJ,
    2. Meredith TJ
    (1991) Use of N-acetylcysteine in clinical toxicology. Am J Med 91:131s–139s.
  14. ↵
    1. Friberg L,
    2. Kjellström T,
    3. Nordberg GF
    (1986) Cadmium, In: Handbook on the Toxicology of Metals, vol 2 (Friberg L, Nordberg GF and Vouk VB eds) pp 130–184, Elsevier, New York.
  15. ↵
    1. Godfrey NF,
    2. Peter A,
    3. Simon TM,
    4. Lorber A
    (1982) IV N-Acetylcysteine treatment of hematologic reactions to chrysotherapy. J Rheumatol 9:519–526.
    OpenUrlPubMed
  16. ↵
    1. Gong Q,
    2. Hart BA
    (1997) Effect of thiols on cadmium-induced expression of metallothionein and other oxidant stress genes in rat lung epithelial cells. Toxicology 119:179–191.
    OpenUrlCrossRefPubMed
  17. ↵
    1. Henderson P,
    2. Hale TW,
    3. Shum S,
    4. Habersang RW
    (1985) N-Acetylcysteine therapy of acute heavy metal poisoning in mice. Vet Hum Toxicol 27:522–525.
    OpenUrlPubMed
  18. ↵
    1. Horiguchi H,
    2. Mukaida N,
    3. Okamoto S,
    4. Teranishi H,
    5. Kasuya M,
    6. Matsushima K
    (1993) Cadmium induces interleukin-8 production in human peripheral blood mononuclear cells with the concomitant generation of superoxide radicals. Lymphokine Cytokine Res 12:421–428.
    OpenUrlPubMed
  19. ↵
    1. Kadima W,
    2. Rabenstein DL
    (1990) Nuclear magnetic resonance studies of the solution chemistry of metal complexes. 26. Mixed ligand complexes of cadmium, nitrilotriacetic acid, glutathione, and related ligands. J Inorg Biochem 38:277–288.
    OpenUrlCrossRefPubMed
  20. ↵
    1. Kang YJ
    (1992) Exogenous glutathione decreases cellular cadmium uptake and toxicity. Drug Metab Dispos 20:714–718.
    OpenUrlAbstract
  21. ↵
    1. Keogh JP,
    2. Steffen B,
    3. Siegers CP
    (1994) Cytotoxicity of heavy metals in the human small intestinal epithelial cell line I-407: The role of glutathione. J Toxicol Environ Health 43:351–359.
    OpenUrlPubMed
  22. ↵
    1. Kimura O,
    2. Endo T,
    3. Sakata M
    (1997) Uptake of Cd-metallothionein into LLC-PK1 cells: A comparative study with CdCl2. Biol Pharm Bull 20:158–162.
    OpenUrlPubMed
  23. ↵
    1. Klaassen CD,
    2. Liu J
    (1997) Role of metallothionein in cadmium-induced hepatotoxicity and nephrotoxicity. Drug Metab Rev 29:79–102.
    OpenUrlCrossRefPubMed
  24. ↵
    1. Klug S,
    2. Planas-Bohne F,
    3. Taylor DM
    (1988) Factors influencing the uptake of cadmium into cells in vitro. Hum Toxicol 7:545–549.
    OpenUrlPubMed
  25. ↵
    1. Li DWC,
    2. Spector A
    (1997) Hydrogen peroxide-induced expression of the proto-oncogenes, c-jun, c-fos and c-myc in rabbit lens epithelial cells. Mol Cell Biochem 173:59–69.
    OpenUrlCrossRefPubMed
  26. ↵
    1. Li WC,
    2. Wang GM,
    3. Wang RR,
    4. Spector A
    (1994) The redox active components H2O2 and N-acetyl-l-cysteine regulate expression of c-jun and c-fos in lens systems. Exp Eye Res 59:179–190.
    OpenUrlCrossRefPubMed
  27. ↵
    1. Lund ME,
    2. Banner WJr,
    3. Clarkson TW,
    4. Berlin M
    (1984) Treatment of acute methylmercury ingestion by hemodialysis with N-acetylcysteine (mucomyst) infusion and 2,3-dimercaptopropane sulfonate. J Toxicol Clin Toxicol 22:31–49.
    OpenUrlPubMed
  28. ↵
    1. Matsuoka M,
    2. Call KM
    (1995) Cadmium-induced expression of immediate early genes in LLC-PK1 cells. Kidney Int 48:383–389.
    OpenUrlCrossRefPubMed
  29. ↵
    1. Meyer M,
    2. Schreck R,
    3. Baeuerle PA
    (1993) H2O2 and antioxidants have opposite effects on activation of NF-κB and AP-1 in intact cells: AP-1 as secondary antioxidant-responsive factor. EMBO J 12:2005–2015.
    OpenUrlPubMed
  30. ↵
    1. Morselt AFW
    (1991) Environmental pollutants and diseases. A cell biological approach using chronic cadmium exposure in the animal model as a paradigm case. Toxicology 70:1–132.
    OpenUrlCrossRefPubMed
  31. ↵
    1. Ottenwälder H,
    2. Simon P
    (1987) Differential effect of N-acetylcysteine on excretion of the metals Hg, Cd, Pb and Au. Arch Toxicol 60:401–402.
    OpenUrlCrossRefPubMed
  32. ↵
    1. Perrin DD,
    2. Watt AE
    (1971) Complex formation of zinc and cadmium with glutathione. Biochim Biophys Acta 230:96–104.
    OpenUrlPubMed
  33. ↵
    1. Preston GA,
    2. Lyon TT,
    3. Yin Y,
    4. Lang JE,
    5. Solomon G,
    6. Annab L,
    7. Srinivasan DG,
    8. Alcorta DA,
    9. Barrett JC
    (1996) Induction of apoptosis by c-Fos protein. Mol Cell Biol 16:211–218.
    OpenUrlAbstract/FREE Full Text
  34. ↵
    1. Prozialeck WC,
    2. Lamar PC
    (1995) Effects of glutathione depletion on the cytotoxic actions of cadmium in LLC-PK1 cells. Toxicol Appl Pharmacol 134:285–295.
    OpenUrlCrossRefPubMed
  35. ↵
    1. Sheikh-Hamad D,
    2. Timmins K,
    3. Jalali Z
    (1997) Cisplatin-induced renal toxicity: Possible reversal by N-acetylcysteine treatment. J Am Soc Nephrol 8:1640–1645.
    OpenUrlAbstract
  36. ↵
    1. Stacey NH
    (1986) The amelioration of cadmium-induced injury in isolated hepatocytes by reduced glutathione. Toxicology 42:85–93.
    OpenUrlCrossRefPubMed
  37. ↵
    1. Sugiyama M
    (1994) Role of cellular antioxidants in metal-induced damage. Cell Biol Toxicol 10:1–22.
    OpenUrlCrossRefPubMed
  38. ↵
    1. Templeton DM
    (1990) Cadmium uptake by cells of renal origin. J Biol Chem 265:21764–21770.
    OpenUrlAbstract/FREE Full Text
  39. ↵
    1. Tietze F
    (1969) Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: Applications to mammalian blood and other tissues. Anal Biochem 27:502–522.
    OpenUrlCrossRefPubMed
  40. ↵
    1. Yan CYI,
    2. Ferrari G,
    3. Greene LA
    (1995) N-Acetylcysteine-promoted survival of PC12 cells is glutathione-independent but transcription-dependent. J Biol Chem 270:26827–26832.
    OpenUrlAbstract/FREE Full Text
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Protection from Cadmium Cytotoxicity by N-Acetylcysteine in LLC-PK1 Cells

Bambang Wispriyono, Masato Matsuoka, Hideki Igisu and Koji Matsuno
Journal of Pharmacology and Experimental Therapeutics October 1, 1998, 287 (1) 344-351;
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