Introduction

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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 B (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

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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.

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 × 10⁶ cells/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).

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).

Uptake and efflux of ¹⁰⁹Cd⁺⁺. LLC-PK₁ cells were plated at 5 × 10⁵ cells/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 CdCl₂ containing 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 with ¹⁰⁹Cd⁺⁺ 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.

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

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Cytotoxicity of CdCl₂ in LLC-PK₁ cells. 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.

View larger version (21K): [in this window] [in a new window]   Fig. 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).

View larger version (18K): [in this window] [in a new window]   Fig. 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 CdCl2 without NAC treatment.

View larger version (12K): [in this window] [in a new window]   Fig. 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.

View larger version (17K): [in this window] [in a new window]   Fig. 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 CdCl2 without 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 -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).

View larger version (14K): [in this window] [in a new window]   Fig. 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).

View larger version (16K): [in this window] [in a new window]   Fig. 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.

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).

View larger version (45K): [in this window] [in a new window]   Fig. 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-PK₁ cells 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 with ¹⁰⁹Cd⁺⁺ for 4 hr), the accumulation of ¹⁰⁹Cd⁺⁺ was suppressed markedly (fig. 9). However, the amount of ¹⁰⁹Cd⁺⁺ 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).

View larger version (13K): [in this window] [in a new window]   Fig. 8.   Time course of 109Cd++ uptake into LLC-PK1 cells. After preincubation without () or with 1 mM NAC () for 1 hr, 20 µM CdCl2 containing 109Cd++ 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.

View larger version (12K): [in this window] [in a new window]   Fig. 9.   Dose effects of NAC on 109Cd++ 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.

View larger version (18K): [in this window] [in a new window]   Fig. 10.   Effects of NAC on 109Cd++ efflux from LLC-PK1 cells. Initially, cells were incubated with 20 µM CdCl2 containing 109Cd++ 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).

View larger version (24K): [in this window] [in a new window]   Fig. 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

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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; Díaz-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 (Aruoma et 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 (Horiguchi et 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 (Dudley et 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.