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The effects of glutathione and vitamin E on iron toxicity in isolated rat hepatocytes
Toxicology Letters 126 (2002) 169– 177 www.elsevier.com/locate/toxlet
The effects of glutathione and vitamin E on iron toxicity in isolated rat hepatocytes Lawrence M. Milchak, J. Douglas Bricker * Department of Pharmacology-Toxicology, Graduate School of Pharmaceutical Sciences, Duquesne Uni6ersity, Pittsburgh, PA 15282, USA Received 7 May 2001; received in revised form 28 August 2001; accepted 28 August 2001
Abstract This study examined the acute toxicity of ferrous sulfate on rat hepatocyte suspensions, the correlation between lipid peroxidation and cell death, and the roles of glutathione and vitamin E in protecting against iron toxicity. Incubation with ferrous sulfate for 2 h produced lipid peroxidation, but did not decrease cell viability in the hepatocytes. When diethyl maleate (DEM) was added to deplete cellular glutathione concentrations, ferrous sulfate treatment (2.0–5.0 mM) did cause cell death and lipid peroxidation developed more extensively, suggesting that iron-mediated hepatotoxicity is influenced by glutathione content. Reduced glutathione (GSH), N-acetylcysteine (NAC) and a-tocopherol (vitamin E), alone and in combination, were added to hepatocyte suspensions in an attempt to protect cells against iron-induced damage. In iron– DEM-treated cells, GSH and NAC treatment increased viability by 43 and 36%, respectively, but only the combination of the two agents reduced lipid peroxidation (53% decrease). Vitamin E treatment reduced lipid peroxidation by 39% and also increased cell viability by 12%. The greatest protection against iron-induced lipid peroxidation occurred with the combination of GSH, NAC and vitamin E, which reduced lipid peroxidation by 94% in iron-treated cells, and by 98% in iron– DEM-treated cells. However, this combination did not prevent iron-induced cell death, although it did increase viability by 18%. These results suggest that iron-induced cell death may not be dependent upon lipid peroxidation, at least in short-term exposures. The results also suggest an interaction between GSH and vitamin E in protecting against lipid peroxidation. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Iron; Glutathione; Vitamin E; N-acetylcysteine; Rat hepatocytes; Lipid peroxidation
1. Introduction Although it has been well established that iron toxicity can produce significant hepatic damage, * Corresponding author. Tel.: + 1-412-396-6361; fax: +1412-396-5130. E-mail address: [email protected] (J. Douglas Bricker).
the relationships between the cellular events surrounding iron-induced hepatotoxicity remain uncertain. The ability of iron to catalyze the formation of reactive oxygen species, including the hydroxyl radical ( OH), has been extensively reviewed (Kehrer, 2000; Halliwell, 1992; Ryan and Aust, 1992). These oxygen-derived radicals may produce oxidative stress, and damage such
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cellular constituents as proteins and nucleic acids, and also damage the membranes of cellular organelles by initiating lipid peroxidation. It has been well established that iron can initiate hepatic lipid peroxidation in different experimental systems (Khan et al., 1995; Valerio and Petersen, 1998; Knutson et al., 2000). However, it is unclear what role lipid peroxidation plays in causing hepatocyte death following acute iron exposures, as a definitive relationship between lipid peroxidation and cell death has yet to be established. Also uncertain is the role that glutathione (GSH) may play in protecting hepatocytes from lipid peroxidation and cell death. Although exogenously added GSH has been shown to protect against lipid peroxidation in isolated liver microsomes (Palamanda and Kehrer, 1992), similar protection has not been demonstrated in intact hepatocytes. It has also been suggested that GSH may also act as a pro-oxidant and cause or exaggerate lipid peroxidation (Zager and Burkhart, 1998). Vitamin E (a-tocopherol) is also known to interrupt the biochemical reactions associated with lipid peroxidation, and has been shown to effectively inhibit iron-induced lipid peroxidation in a multitude of studies (Sharma et al., 1990; Wagner et al., 1996; Iqbal et al., 1998). However, since the fully oxidized form of vitamin E is inactive, it has been suggested that GSH may play a major role in the reduction and regeneration of vitamin E, however, again these studies have not employed intact hepatocytes (Graham et al., 1989; Leedle and Aust, 1990). However, this theory remains controversial as recent evidence suggests that GSH is not capable of reducing the oxidized form of vitamin E (a-tocopherol quinone) in liver microsomes (van Haaften et al., 2001). The present study investigated the effects of short-term ferrous sulfate exposure on hepatocyte suspensions. Specifically, this study examined the association between iron-induced lipid peroxidation and iron-induced loss of cell viability to determine if a definitive relationship could be established. This study also examined the role of GSH in iron-induced hepatotoxicity and explored the existence of a synergistic relationship between GSH and vitamin E in protecting against iron-induced cellular toxicity.
2. Materials and methods
2.1. Animals Male Sprague–Dawley rats (200–275 g) were obtained from Zivic-Miller Laboratories, Inc. (Zelienople, PA). Animals were housed in a temperature controlled room, two per cage, exposed to a 12 h light and dark cycle, and had access to standard laboratory rodent chow and tap water ad libitum.
2.2. Hepatocyte isolation and incubation Hepatocytes were isolated via a two step circulating collagenase perfusion technique as described by Berry et al. (1991) with modifications by Seglen (1976). Rats were anaesthetized by intraperitoneal sodium pentobarbital injection (60 mg/kg). Collagenase Type V (Sigma Chemical) was used at a final concentration of 0.03% in the second step of the perfusion. Isolated cells were washed and purified by centrifugation through a Percoll® solution (Berry et al., 1991). After the final cell washing, the cells were counted using a hemacytometer and initial cell viability was determined using the 0.4% trypan blue exclusion test. Viability exceeding 85% was found in all preparations used in this study. Hepatocytes were incubated in 25 ml Erlenmeyer glass flasks at a concentration of 1×106 cells/ml in Krebs–Henseleit (K–H) buffer, pH 7.4, supplemented with 0.2% BSA, and 15 mM HEPES. Flasks were capped with rubber stoppers and incubated under continuous 95% O2/5% CO2 in a water bath (37 °C) for 2 h using reciprocal shaking at 80–90 cycles/min.
2.3. Experimental design Experiments were divided into two phases consisting of concentration–response experiments and protective experiments. The concentration– response experiments were designed to determine the effects of different concentrations of ferrous sulfate on cell viability and lipid peroxidation, using untreated cells and cells deplete of glutathione. The first series of experiments were per-
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formed by incubating hepatocytes with concentrations of ferrous sulfate ranging from 0.5 to 5.0 mM and measuring cell viability and lipid peroxidation at the end of a 2-h incubation. The second series of experiments involved incubating cells with diethyl maleate (DEM, 0.8 mM) to deplete glutathione and ferrous sulfate (0.5– 5.0 mM) and measuring cell viability and lipid peroxidation at the end of a 2-h incubation period. The 2-h time frame was chosen to best examine a freshly isolated cell population and also to study loss of cell viability occurring quickly following iron exposure to examine the relationship with lipid peroxidation. The second phase of the study was designed to examine if pretreatment with reduced GSH, N-acetylcysteine (NAC), or vitamin E, alone, and in combination, could protect against lipid peroxidation and cell death induced by ferrous sulfate, with and without glutathione depletion. Based on the results obtained from concentration –response experiments, a single concentration of ferrous sulfate (4.0 mM) was chosen for these experiments. Hepatocytes were pre-incubated with either GSH (10 mM), NAC (10 mM), vitamin E (6.0 mM), both GSH (10 mM) and NAC (10 mM), or the combination of GSH (10 mM), NAC (10 mM) and vitamin E (6.0 mM) for a period of 10 min prior to the addition of ferrous sulfate, or ferrous sulfate/ DEM. Since it was found from the concentration – response experiments that ferrous sulfate alone did not decrease cell viability, only ferrous sulfate–DEM-treated cells were assayed for viability in the protective experiments. K–H buffer was used in control cell suspensions.
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pernatant was removed and 0.25 ml fresh K– H media was added, the cells gently mixed and centrifuged again at 40× g for 2 min. The resultant supernatant was removed and 0.25 ml of the acetic acid/ethanol solubilization solution was added to the cell pellet. The tube was mixed vigorously and kept at room temperature for 10 min. The tube was then centrifuged at 1200× g for 10 min. A portion of the supernatant was removed and transferred to a 96-well microplate and the absorbance determined at 540 nm, using a microplate reader. Viability data were expressed as percent of control.
2.5. Measurement of lipid peroxidation Lipid peroxidation was determined by measuring the presence of thiobarbituric acid reacting substances (TBARS) as previously described (Stacey et al., 1980). Butylated hydroxytoluene (BHT) was added before heating at 0.01% to prevent any spontaneous oxidation during the assay procedure (Buege and Aust, 1978). Absorbance was measured at 540 nm using a microplate reader. This method was chosen because it has been previously demonstrated that TBARS correlates very well with other assays used to measure lipid peroxidation in isolated rat hepatocytes (Smith et al., 1982). The results are expressed as nmoles malondialdehyde (MDA) equivalents/million cells, with the understanding that the TBARS values are not due solely to MDA, but other lipid peroxide products as well. The procedure used in this study utilized whole-cell suspension precipitates which has been suggested to be a better representation of lipid peroxidation (Stacey and Kappus, 1982).
2.4. Assessment of cell 6iability 2.6. Measurement of glutathione Cell viability was determined after the 2-h incubation using the neutral red assay kit (Sigma Chemical). Cells were incubated in a K– H media, pH 7.4, containing neutral red (50 mg/ml) for the duration of the experiment (2 h). At the conclusion of the incubation period, an aliquot (0.25 ml) was removed from each flask and centrifuged at 40× g for 2 min. The resulting su-
Total glutathione content was measured spectrophotometrically according to the method of Griffith (1980). Assays were performed in 96well microplates. After addition of reagents, the change in absorbance at 410 nm was monitored, using a microplate reader and the rate of change per minute was calculated.
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2.7. Preparation of treatment chemicals Perfusate solutions, washing media, and incubation media were prepared using double-deionized distilled water. All chemicals and reagents used in this study were obtained from Sigma Chemical (St. Louis, MO). Ferrous sulfate solutions were prepared in the morning of the experiments in K–H buffer. Nitrogen gas was passed through the solution for 2 min then capped immediately to insure that iron remained in the ferrous state (Stacey et al., 1980). Reduced glutathione (GSH) and N-acetylcysteine (NAC) were prepared in K– H buffer, pH 7.4, prior to the experiments. Diethyl maleate (DEM) was also prepared in a vehicle of K– H buffer, but with constant mixing to ensure even distribution and then directly added to the hepatocyte suspensions which were being shaken as well, to ensure even distribution. Vitamin E ((9 )-a-tocopherol) was dissolved in absolute ethanol immediately before use. The final ethanol concentration in the hepatocyte incubations was less than 0.01%. Cells incubated with 0.01% ethanol showed no effect on cell viability or lipid peroxidation compared to K– H buffer controls.
was incubated with ferrous sulfate, cell viability was lowered as much as 35% in treatment groups incubated with iron–DEM, as compared with iron treatment alone. Preliminary experiments found that incubation with DEM (0.8 mM) for 2 h resulted in GSH concentrations 43% lower than control suspensions. This concentration of DEM alone produced no decrease in cell viability, or produced lipid peroxidation in 2-h incubations. Fig. 2 represents the lipid peroxidation measurements from cells treated with ferrous sulfate (0.5–5.0 mM), in the presence and absence of DEM (0.8 mM). Even at the lowest concentration (0.5 mM), lipid peroxidation was detected and increased steadily through the highest concentration of ferrous sulfate. Treatment with DEM produced approximately four-fold increases in TBARS formation with cells treated with ferrous sulfate concentrations between 0.5 and 3.0 mM, as compared to treatment with ferrous sulfate alone. Fig. 3 depicts lipid peroxidation measurements taken from cells incubated with ferrous sulfate (4.0 mM) and the various combinations of potential protecting agents. As previously illustrated in Fig. 2, incubation with 4.0 mM ferrous sulfate
2.8. Statistical analyses Statistical differences were determined using a one-way analysis of variance (ANOVA) and Fisher’s Protected Least Significant Difference Test with an alpha error of 0.01 to establish significance (Gad and Weil, 1994).
3. Results Fig. 1 illustrates the cell viability results from hepatocyte suspensions incubated with ferrous sulfate (2.0–5.0 mM) in the presence and absence of DEM (0.8 mM). Control suspensions were considered to be 100% viable and the viability results from the iron-treated cells are displayed as percent viability compared to controls. No decreases in cell viability during exposure to ferrous sulfate alone were found. However, when DEM
Fig. 1. Cell viability following ferrous sulfate treatment, in the presence and absence of DEM. Hepatocytes (1 × 106 cells/ml) were incubated for 2 h with ferrous sulfate (2.0 – 5.0 mM). DEM (0.8 mM) was added to those suspensions represented as gray bars. Data are presented as the mean 9SE from three experiments. (*), Significantly different from control cells (PB 0.01).
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Fig. 2. Lipid peroxidation following ferrous sulfate treatment, in the presence and absence of DEM. Hepatocytes (1 × 106 cells/ml) were incubated for 2 h with ferrous sulfate (0.5 –5.0 mM). DEM (0.8 mM) was added to those suspensions represented as gray bars. Results are expressed as nmoles MDA equivalents/1× 106 cells. Data are presented as the mean 9SE from three experiments. (*), Significantly different from control cells (P B 0.01). (**), Significantly different from ironalone-treated cells (P B0.01).
alone significantly increased TBARS formation. Treatment with the individual agents GSH (10 mM), NAC (10 mM), or vitamin E (6 mM), did not significantly reduce TBARS, compared to cells treated with ferrous sulfate alone. However, when cells were treated with GSH, NAC, and vitamin E, lipid peroxidation was essentially abolished, producing TBARS values nearly equal to non-treated cells. Fig. 3 also illustrates the results of cells incubated with ferrous sulfate and the effects of various potentially protective agents after cells were depleted of GSH using DEM (0.8 mM). Incubation of cells with ferrous sulfate (4.0 mM) and DEM (0.8 mM) produced substantial TBARS formation. Neither GSH (10 mM) nor NAC (10 mM) alone demonstrated any protective action against lipid peroxidation in iron– DEM-treated cells. However, the combination of GSH and NAC did significantly reduce TBARS formation. Vitamin E (6.0 mM) treatment alone significantly reduced lipid peroxidation in iron– DEM-treated
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cells. Furthermore, the combination of GSH, NAC and vitamin E significantly reduced lipid peroxidation in GSH-depleted cells, producing TBARS values approximately equal to controls. Fig. 4 illustrates the results of cell viability on DEM-treated hepatocytes treated with ferrous sulfate, alone and in combination with potential protective agents. Iron–DEM-treated cells showed a decrease in cell viability to approximately 48%, as compared to control (100% viability). Treatment of iron–DEM-treated cells with GSH (10 mM) and NAC (10 mM) significantly increased cell viability to 92 and 85%, respectively. The combination of GSH and NAC (GN) also increased viability to 77%. Vitamin E (6.0 mM) increased cell viability to 61% and the combination of GSH, NAC and vitamin E increased cell viability to 67%. All of the potentially protec-
Fig. 3. Protection against lipid peroxidation. Hepatocytes (1 × 106 cells/ml) were incubated for 2 h with 4.0 mM ferrous sulfate. DEM (0.8 mM) was added to those incubations represented as gray bars. Potential protective agents were added 10 min prior to the addition of ferrous sulfate and DEM. Results are expressed as nmoles MDA equivalents/1×106 cells. Data are presented as the mean 9SE from 4 to 6 experiments. Fe= ferrous sulfate; GSH =Fe+GSH; NAC=Fe+NAC; GN= Fe+ GSH+NAC; Vit E= Fe+ vitamin E; GNE= Fe+ GSH+NAC+Vit E. (**), Significantly different from Fe– DEM-treated cells (P B0.01). (*), Significantly different from iron-alone-treated cells (PB 0.01).
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Fig. 4. Protection against ferrous sulfate-induced cell death in hepatocytes treated with diethyl maleate. Hepatocytes (1 × 106 cells/ml) were incubated for 2 h. Treatment chemicals were added 10 min prior to the addition of ferrous sulfate and DEM. Data are presented as the mean 9 SE from 4 to 6 experiments. FeD =ferrous sulfate + DEM; GSH= FeD+ GSH; NAC= FeD+NAC; GN=FeD+ GSH+ NAC; Vit E = FeD+ vitamin E; GNE=GSH+NAC+ Vit E. (*), Significantly different from Fe –DEM-treated cells (PB 0.01).
tive agents were found to significantly increase cell viability, compared to iron– DEM-treated cells (FeD). 4. Discussion One of the specific goals of the study was to determine if the development of iron-induced lipid peroxidation was associated with loss of cell viability. The results did not suggest that these events were related. This was evident in the fact that iron treatment produced significant lipid peroxidation in a concentration–response manner, yet no decrease in cell viability was detected. These results are consistent with the findings of others that have suggested that cell lysis and lipid peroxidation did not correlate well following iron treatment or the development of oxidative stress (Zager and Burkhart, 1998; Ollinger and Brunk, 1995). It was somewhat surprising that despite the
relatively high concentration of ferrous sulfate used (2–5 mM), no loss of cell viability was detected. However, other studies on the effects of iron-induced hepatotoxicity, using a variety of viability indicators and experimental conditions, have also not been successful in establishing a clear relationship between concentration and cell viability (Knutson et al., 2000; Innes et al., 1988; Carini et al., 1992). In contrast to cells treated with iron alone, iron–DEM-treated cells showed a significant decrease in cell viability at all concentrations of ferrous sulfate (2.0–5.0 mM). This strongly suggests that the GSH status of the cell is an important factor in regulating the viability of hepatocytes following iron exposure, and is consistent with the findings reported for other metals, such as copper and cadmium (Pourahmad and O’Brien, 2000; Rana and Verma, 1997). The lack of association between lipid peroxidation and cell viability was further demonstrated by the experiments using the protective agents. The two treatment groups that produced the highest cell viability results in iron–DEM-treated cells were exogenous GSH and NAC. However, these two treatment groups did not significantly reduce lipid peroxidation. In fact, it appeared that both demonstrated increased lipid peroxidation in non-GSH-depleted cells. It appears that the mechanism of protection against cell death for GSH and NAC does not directly involve lipid peroxidation, as others have suggested (Stacey et al., 1980). In the present study, the most effective protection against lipid peroxidation was the combination of GSH, NAC and vitamin E, and also treatment with vitamin E alone. However, these two treatment groups provided the least amount of protection against iron-induced cell death in GSH-depleted cells. Furthermore, extensive lipid peroxidation developed in GSH-depleted cells treated with ferrous sulfate and GSH, yet cell viability was maintained at 92%. Another goal of this study was to examine the potential synergistic relationship between GSH and vitamin E in protecting against iron-induced cellular toxicity. The results of the lipid peroxidation assays strongly suggest that a synergistic relationship exists, as the combination of GSH, NAC and vitamin E completely prevented the
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development of lipid peroxidation in iron alone and iron–DEM-treated cells. This complete protection may provide further evidence that GSH was effective at reducing and regenerating vitamin E to its active antioxidant state, with NAC providing a source of additional sulfhydryl groups, or acting as a substrate for more GSH synthesis as GSH became oxidized and inactive. The individual agents were not nearly as effective at protecting against lipid peroxidation. When exogenous sulfhydryl-donors, such as GSH or NAC, were tested in either iron- or iron–DEM-treated cells, no protection against iron-induced lipid peroxidation was observed. In fact, NAC significantly increased lipid peroxidation in iron-treated cells. However, these findings are not necessarily surprising. The role of sulfhydryl replacement in preventing lipid peroxidation is unclear, as GSH has been suggested to reduce lipid peroxidation (Palamanda and Kehrer, 1992; Graham et al., 1989) and also has been suggested to cause or exaggerate lipid peroxidation (Zager and Burkhart, 1998; Stacey et al., 1980). The role of vitamin E in protecting against lipid peroxidation is more clear. It is well established that vitamin E can protect against iron-induced lipid peroxidation in a variety of in vitro and in vivo experimental conditions (Iqbal et al., 1998; Brown et al., 1997; Omara and Blakely, 1993). In the present study however, vitamin E treatment only significantly inhibited iron-induced lipid peroxidation in DEM-treated cells. Although it has been suggested that GSH may act to regenerate the reduced form of vitamin E after vitamin E has become fully oxidized and inactive as an antioxidant (Leedle and Aust, 1990), the present study suggested vitamin E may be an effective inhibitor of lipid peroxidation even when GSH has been depleted. Of course, the results of the current study must be interpreted carefully to recognize the limitations of the assays used. The method used to measure loss of cell viability was the neutral red assay, a method often used to measure cytotoxicity in a variety of cell systems, including human and rat hepatocyte cultures (Fautz et al., 1991; Ledirac et al., 2000; Martinez-Diez et al.,
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2000). The assay is based on the fact that the neutral red dye is taken up by viable cells and stored in the lysosomes. Damage to cell and lysosomal membranes results in decreased uptake and increased release of the dye. The neutral red assay has proven to be a reliable and sensitive assay for determining loss of cell viability, and has shown particular efficacy in examining the cytotoxicity of metals in vitro (Yamamoto et al., 2001; Sauvant et al., 1997). However, in the current study, it is possible the neutral red assay may not have been sensitive enough to detect low levels of cell death in the hepatocyte suspensions. Although it is believed the neutral red assay should have been particularly suitable to study iron-induced loss of cell viability because it has been suggested that lysosomal damage may be one of the most significant events associated with loss of cell viability due to oxidative stress (Ollinger and Brunk, 1995). The limitations of the TBARS assay should also be recognized when interpreting the results of the present study. The TBARS assay may produce artifacts that either exaggerate or underestimate the actual degree of cellular lipid peroxidation. However, the purpose of this study was to examine the relationships between the cellular events associated with acute iron toxicity and not necessarily to quantify the degree of lipid peroxidation. In summary, a correlation between iron-induced lipid peroxidation and loss of cell viability was not identified. Cellular GSH concentrations appeared to be very important in maintaining cell viability following iron exposure. A synergistic relationship between GSH, NAC and vitamin E was strongly suggested as the combination of the three agents offered complete protection against lipid peroxidation caused by iron in normal cells and DEM-treated cells. This degree of protection was not seen with any of the individual agents alone. These results suggest further mechanistic studies are necessary to better understand the complex events associated with cellular iron toxicity and to further investigate the interactions between GSH and vitamin E in situations of oxidative stress.
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The effects of glutathione and vitamin E on iron toxicity in isolated rat hepatocytes Lawrence M. Milchak, J. Douglas Bricker * Department of Pharmacology-Toxicology, Graduate School of Pharmaceutical Sciences, Duquesne Uni6ersity, Pittsburgh, PA 15282, USA Received 7 May 2001; received in revised form 28 August 2001; accepted 28 August 2001
Abstract This study examined the acute toxicity of ferrous sulfate on rat hepatocyte suspensions, the correlation between lipid peroxidation and cell death, and the roles of glutathione and vitamin E in protecting against iron toxicity. Incubation with ferrous sulfate for 2 h produced lipid peroxidation, but did not decrease cell viability in the hepatocytes. When diethyl maleate (DEM) was added to deplete cellular glutathione concentrations, ferrous sulfate treatment (2.0–5.0 mM) did cause cell death and lipid peroxidation developed more extensively, suggesting that iron-mediated hepatotoxicity is influenced by glutathione content. Reduced glutathione (GSH), N-acetylcysteine (NAC) and a-tocopherol (vitamin E), alone and in combination, were added to hepatocyte suspensions in an attempt to protect cells against iron-induced damage. In iron– DEM-treated cells, GSH and NAC treatment increased viability by 43 and 36%, respectively, but only the combination of the two agents reduced lipid peroxidation (53% decrease). Vitamin E treatment reduced lipid peroxidation by 39% and also increased cell viability by 12%. The greatest protection against iron-induced lipid peroxidation occurred with the combination of GSH, NAC and vitamin E, which reduced lipid peroxidation by 94% in iron-treated cells, and by 98% in iron– DEM-treated cells. However, this combination did not prevent iron-induced cell death, although it did increase viability by 18%. These results suggest that iron-induced cell death may not be dependent upon lipid peroxidation, at least in short-term exposures. The results also suggest an interaction between GSH and vitamin E in protecting against lipid peroxidation. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Iron; Glutathione; Vitamin E; N-acetylcysteine; Rat hepatocytes; Lipid peroxidation
1. Introduction Although it has been well established that iron toxicity can produce significant hepatic damage, * Corresponding author. Tel.: + 1-412-396-6361; fax: +1412-396-5130. E-mail address: [email protected] (J. Douglas Bricker).
the relationships between the cellular events surrounding iron-induced hepatotoxicity remain uncertain. The ability of iron to catalyze the formation of reactive oxygen species, including the hydroxyl radical ( OH), has been extensively reviewed (Kehrer, 2000; Halliwell, 1992; Ryan and Aust, 1992). These oxygen-derived radicals may produce oxidative stress, and damage such
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cellular constituents as proteins and nucleic acids, and also damage the membranes of cellular organelles by initiating lipid peroxidation. It has been well established that iron can initiate hepatic lipid peroxidation in different experimental systems (Khan et al., 1995; Valerio and Petersen, 1998; Knutson et al., 2000). However, it is unclear what role lipid peroxidation plays in causing hepatocyte death following acute iron exposures, as a definitive relationship between lipid peroxidation and cell death has yet to be established. Also uncertain is the role that glutathione (GSH) may play in protecting hepatocytes from lipid peroxidation and cell death. Although exogenously added GSH has been shown to protect against lipid peroxidation in isolated liver microsomes (Palamanda and Kehrer, 1992), similar protection has not been demonstrated in intact hepatocytes. It has also been suggested that GSH may also act as a pro-oxidant and cause or exaggerate lipid peroxidation (Zager and Burkhart, 1998). Vitamin E (a-tocopherol) is also known to interrupt the biochemical reactions associated with lipid peroxidation, and has been shown to effectively inhibit iron-induced lipid peroxidation in a multitude of studies (Sharma et al., 1990; Wagner et al., 1996; Iqbal et al., 1998). However, since the fully oxidized form of vitamin E is inactive, it has been suggested that GSH may play a major role in the reduction and regeneration of vitamin E, however, again these studies have not employed intact hepatocytes (Graham et al., 1989; Leedle and Aust, 1990). However, this theory remains controversial as recent evidence suggests that GSH is not capable of reducing the oxidized form of vitamin E (a-tocopherol quinone) in liver microsomes (van Haaften et al., 2001). The present study investigated the effects of short-term ferrous sulfate exposure on hepatocyte suspensions. Specifically, this study examined the association between iron-induced lipid peroxidation and iron-induced loss of cell viability to determine if a definitive relationship could be established. This study also examined the role of GSH in iron-induced hepatotoxicity and explored the existence of a synergistic relationship between GSH and vitamin E in protecting against iron-induced cellular toxicity.
2. Materials and methods
2.1. Animals Male Sprague–Dawley rats (200–275 g) were obtained from Zivic-Miller Laboratories, Inc. (Zelienople, PA). Animals were housed in a temperature controlled room, two per cage, exposed to a 12 h light and dark cycle, and had access to standard laboratory rodent chow and tap water ad libitum.
2.2. Hepatocyte isolation and incubation Hepatocytes were isolated via a two step circulating collagenase perfusion technique as described by Berry et al. (1991) with modifications by Seglen (1976). Rats were anaesthetized by intraperitoneal sodium pentobarbital injection (60 mg/kg). Collagenase Type V (Sigma Chemical) was used at a final concentration of 0.03% in the second step of the perfusion. Isolated cells were washed and purified by centrifugation through a Percoll® solution (Berry et al., 1991). After the final cell washing, the cells were counted using a hemacytometer and initial cell viability was determined using the 0.4% trypan blue exclusion test. Viability exceeding 85% was found in all preparations used in this study. Hepatocytes were incubated in 25 ml Erlenmeyer glass flasks at a concentration of 1×106 cells/ml in Krebs–Henseleit (K–H) buffer, pH 7.4, supplemented with 0.2% BSA, and 15 mM HEPES. Flasks were capped with rubber stoppers and incubated under continuous 95% O2/5% CO2 in a water bath (37 °C) for 2 h using reciprocal shaking at 80–90 cycles/min.
2.3. Experimental design Experiments were divided into two phases consisting of concentration–response experiments and protective experiments. The concentration– response experiments were designed to determine the effects of different concentrations of ferrous sulfate on cell viability and lipid peroxidation, using untreated cells and cells deplete of glutathione. The first series of experiments were per-
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formed by incubating hepatocytes with concentrations of ferrous sulfate ranging from 0.5 to 5.0 mM and measuring cell viability and lipid peroxidation at the end of a 2-h incubation. The second series of experiments involved incubating cells with diethyl maleate (DEM, 0.8 mM) to deplete glutathione and ferrous sulfate (0.5– 5.0 mM) and measuring cell viability and lipid peroxidation at the end of a 2-h incubation period. The 2-h time frame was chosen to best examine a freshly isolated cell population and also to study loss of cell viability occurring quickly following iron exposure to examine the relationship with lipid peroxidation. The second phase of the study was designed to examine if pretreatment with reduced GSH, N-acetylcysteine (NAC), or vitamin E, alone, and in combination, could protect against lipid peroxidation and cell death induced by ferrous sulfate, with and without glutathione depletion. Based on the results obtained from concentration –response experiments, a single concentration of ferrous sulfate (4.0 mM) was chosen for these experiments. Hepatocytes were pre-incubated with either GSH (10 mM), NAC (10 mM), vitamin E (6.0 mM), both GSH (10 mM) and NAC (10 mM), or the combination of GSH (10 mM), NAC (10 mM) and vitamin E (6.0 mM) for a period of 10 min prior to the addition of ferrous sulfate, or ferrous sulfate/ DEM. Since it was found from the concentration – response experiments that ferrous sulfate alone did not decrease cell viability, only ferrous sulfate–DEM-treated cells were assayed for viability in the protective experiments. K–H buffer was used in control cell suspensions.
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pernatant was removed and 0.25 ml fresh K– H media was added, the cells gently mixed and centrifuged again at 40× g for 2 min. The resultant supernatant was removed and 0.25 ml of the acetic acid/ethanol solubilization solution was added to the cell pellet. The tube was mixed vigorously and kept at room temperature for 10 min. The tube was then centrifuged at 1200× g for 10 min. A portion of the supernatant was removed and transferred to a 96-well microplate and the absorbance determined at 540 nm, using a microplate reader. Viability data were expressed as percent of control.
2.5. Measurement of lipid peroxidation Lipid peroxidation was determined by measuring the presence of thiobarbituric acid reacting substances (TBARS) as previously described (Stacey et al., 1980). Butylated hydroxytoluene (BHT) was added before heating at 0.01% to prevent any spontaneous oxidation during the assay procedure (Buege and Aust, 1978). Absorbance was measured at 540 nm using a microplate reader. This method was chosen because it has been previously demonstrated that TBARS correlates very well with other assays used to measure lipid peroxidation in isolated rat hepatocytes (Smith et al., 1982). The results are expressed as nmoles malondialdehyde (MDA) equivalents/million cells, with the understanding that the TBARS values are not due solely to MDA, but other lipid peroxide products as well. The procedure used in this study utilized whole-cell suspension precipitates which has been suggested to be a better representation of lipid peroxidation (Stacey and Kappus, 1982).
2.4. Assessment of cell 6iability 2.6. Measurement of glutathione Cell viability was determined after the 2-h incubation using the neutral red assay kit (Sigma Chemical). Cells were incubated in a K– H media, pH 7.4, containing neutral red (50 mg/ml) for the duration of the experiment (2 h). At the conclusion of the incubation period, an aliquot (0.25 ml) was removed from each flask and centrifuged at 40× g for 2 min. The resulting su-
Total glutathione content was measured spectrophotometrically according to the method of Griffith (1980). Assays were performed in 96well microplates. After addition of reagents, the change in absorbance at 410 nm was monitored, using a microplate reader and the rate of change per minute was calculated.
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2.7. Preparation of treatment chemicals Perfusate solutions, washing media, and incubation media were prepared using double-deionized distilled water. All chemicals and reagents used in this study were obtained from Sigma Chemical (St. Louis, MO). Ferrous sulfate solutions were prepared in the morning of the experiments in K–H buffer. Nitrogen gas was passed through the solution for 2 min then capped immediately to insure that iron remained in the ferrous state (Stacey et al., 1980). Reduced glutathione (GSH) and N-acetylcysteine (NAC) were prepared in K– H buffer, pH 7.4, prior to the experiments. Diethyl maleate (DEM) was also prepared in a vehicle of K– H buffer, but with constant mixing to ensure even distribution and then directly added to the hepatocyte suspensions which were being shaken as well, to ensure even distribution. Vitamin E ((9 )-a-tocopherol) was dissolved in absolute ethanol immediately before use. The final ethanol concentration in the hepatocyte incubations was less than 0.01%. Cells incubated with 0.01% ethanol showed no effect on cell viability or lipid peroxidation compared to K– H buffer controls.
was incubated with ferrous sulfate, cell viability was lowered as much as 35% in treatment groups incubated with iron–DEM, as compared with iron treatment alone. Preliminary experiments found that incubation with DEM (0.8 mM) for 2 h resulted in GSH concentrations 43% lower than control suspensions. This concentration of DEM alone produced no decrease in cell viability, or produced lipid peroxidation in 2-h incubations. Fig. 2 represents the lipid peroxidation measurements from cells treated with ferrous sulfate (0.5–5.0 mM), in the presence and absence of DEM (0.8 mM). Even at the lowest concentration (0.5 mM), lipid peroxidation was detected and increased steadily through the highest concentration of ferrous sulfate. Treatment with DEM produced approximately four-fold increases in TBARS formation with cells treated with ferrous sulfate concentrations between 0.5 and 3.0 mM, as compared to treatment with ferrous sulfate alone. Fig. 3 depicts lipid peroxidation measurements taken from cells incubated with ferrous sulfate (4.0 mM) and the various combinations of potential protecting agents. As previously illustrated in Fig. 2, incubation with 4.0 mM ferrous sulfate
2.8. Statistical analyses Statistical differences were determined using a one-way analysis of variance (ANOVA) and Fisher’s Protected Least Significant Difference Test with an alpha error of 0.01 to establish significance (Gad and Weil, 1994).
3. Results Fig. 1 illustrates the cell viability results from hepatocyte suspensions incubated with ferrous sulfate (2.0–5.0 mM) in the presence and absence of DEM (0.8 mM). Control suspensions were considered to be 100% viable and the viability results from the iron-treated cells are displayed as percent viability compared to controls. No decreases in cell viability during exposure to ferrous sulfate alone were found. However, when DEM
Fig. 1. Cell viability following ferrous sulfate treatment, in the presence and absence of DEM. Hepatocytes (1 × 106 cells/ml) were incubated for 2 h with ferrous sulfate (2.0 – 5.0 mM). DEM (0.8 mM) was added to those suspensions represented as gray bars. Data are presented as the mean 9SE from three experiments. (*), Significantly different from control cells (PB 0.01).
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Fig. 2. Lipid peroxidation following ferrous sulfate treatment, in the presence and absence of DEM. Hepatocytes (1 × 106 cells/ml) were incubated for 2 h with ferrous sulfate (0.5 –5.0 mM). DEM (0.8 mM) was added to those suspensions represented as gray bars. Results are expressed as nmoles MDA equivalents/1× 106 cells. Data are presented as the mean 9SE from three experiments. (*), Significantly different from control cells (P B 0.01). (**), Significantly different from ironalone-treated cells (P B0.01).
alone significantly increased TBARS formation. Treatment with the individual agents GSH (10 mM), NAC (10 mM), or vitamin E (6 mM), did not significantly reduce TBARS, compared to cells treated with ferrous sulfate alone. However, when cells were treated with GSH, NAC, and vitamin E, lipid peroxidation was essentially abolished, producing TBARS values nearly equal to non-treated cells. Fig. 3 also illustrates the results of cells incubated with ferrous sulfate and the effects of various potentially protective agents after cells were depleted of GSH using DEM (0.8 mM). Incubation of cells with ferrous sulfate (4.0 mM) and DEM (0.8 mM) produced substantial TBARS formation. Neither GSH (10 mM) nor NAC (10 mM) alone demonstrated any protective action against lipid peroxidation in iron– DEM-treated cells. However, the combination of GSH and NAC did significantly reduce TBARS formation. Vitamin E (6.0 mM) treatment alone significantly reduced lipid peroxidation in iron– DEM-treated
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cells. Furthermore, the combination of GSH, NAC and vitamin E significantly reduced lipid peroxidation in GSH-depleted cells, producing TBARS values approximately equal to controls. Fig. 4 illustrates the results of cell viability on DEM-treated hepatocytes treated with ferrous sulfate, alone and in combination with potential protective agents. Iron–DEM-treated cells showed a decrease in cell viability to approximately 48%, as compared to control (100% viability). Treatment of iron–DEM-treated cells with GSH (10 mM) and NAC (10 mM) significantly increased cell viability to 92 and 85%, respectively. The combination of GSH and NAC (GN) also increased viability to 77%. Vitamin E (6.0 mM) increased cell viability to 61% and the combination of GSH, NAC and vitamin E increased cell viability to 67%. All of the potentially protec-
Fig. 3. Protection against lipid peroxidation. Hepatocytes (1 × 106 cells/ml) were incubated for 2 h with 4.0 mM ferrous sulfate. DEM (0.8 mM) was added to those incubations represented as gray bars. Potential protective agents were added 10 min prior to the addition of ferrous sulfate and DEM. Results are expressed as nmoles MDA equivalents/1×106 cells. Data are presented as the mean 9SE from 4 to 6 experiments. Fe= ferrous sulfate; GSH =Fe+GSH; NAC=Fe+NAC; GN= Fe+ GSH+NAC; Vit E= Fe+ vitamin E; GNE= Fe+ GSH+NAC+Vit E. (**), Significantly different from Fe– DEM-treated cells (P B0.01). (*), Significantly different from iron-alone-treated cells (PB 0.01).
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Fig. 4. Protection against ferrous sulfate-induced cell death in hepatocytes treated with diethyl maleate. Hepatocytes (1 × 106 cells/ml) were incubated for 2 h. Treatment chemicals were added 10 min prior to the addition of ferrous sulfate and DEM. Data are presented as the mean 9 SE from 4 to 6 experiments. FeD =ferrous sulfate + DEM; GSH= FeD+ GSH; NAC= FeD+NAC; GN=FeD+ GSH+ NAC; Vit E = FeD+ vitamin E; GNE=GSH+NAC+ Vit E. (*), Significantly different from Fe –DEM-treated cells (PB 0.01).
tive agents were found to significantly increase cell viability, compared to iron– DEM-treated cells (FeD). 4. Discussion One of the specific goals of the study was to determine if the development of iron-induced lipid peroxidation was associated with loss of cell viability. The results did not suggest that these events were related. This was evident in the fact that iron treatment produced significant lipid peroxidation in a concentration–response manner, yet no decrease in cell viability was detected. These results are consistent with the findings of others that have suggested that cell lysis and lipid peroxidation did not correlate well following iron treatment or the development of oxidative stress (Zager and Burkhart, 1998; Ollinger and Brunk, 1995). It was somewhat surprising that despite the
relatively high concentration of ferrous sulfate used (2–5 mM), no loss of cell viability was detected. However, other studies on the effects of iron-induced hepatotoxicity, using a variety of viability indicators and experimental conditions, have also not been successful in establishing a clear relationship between concentration and cell viability (Knutson et al., 2000; Innes et al., 1988; Carini et al., 1992). In contrast to cells treated with iron alone, iron–DEM-treated cells showed a significant decrease in cell viability at all concentrations of ferrous sulfate (2.0–5.0 mM). This strongly suggests that the GSH status of the cell is an important factor in regulating the viability of hepatocytes following iron exposure, and is consistent with the findings reported for other metals, such as copper and cadmium (Pourahmad and O’Brien, 2000; Rana and Verma, 1997). The lack of association between lipid peroxidation and cell viability was further demonstrated by the experiments using the protective agents. The two treatment groups that produced the highest cell viability results in iron–DEM-treated cells were exogenous GSH and NAC. However, these two treatment groups did not significantly reduce lipid peroxidation. In fact, it appeared that both demonstrated increased lipid peroxidation in non-GSH-depleted cells. It appears that the mechanism of protection against cell death for GSH and NAC does not directly involve lipid peroxidation, as others have suggested (Stacey et al., 1980). In the present study, the most effective protection against lipid peroxidation was the combination of GSH, NAC and vitamin E, and also treatment with vitamin E alone. However, these two treatment groups provided the least amount of protection against iron-induced cell death in GSH-depleted cells. Furthermore, extensive lipid peroxidation developed in GSH-depleted cells treated with ferrous sulfate and GSH, yet cell viability was maintained at 92%. Another goal of this study was to examine the potential synergistic relationship between GSH and vitamin E in protecting against iron-induced cellular toxicity. The results of the lipid peroxidation assays strongly suggest that a synergistic relationship exists, as the combination of GSH, NAC and vitamin E completely prevented the
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development of lipid peroxidation in iron alone and iron–DEM-treated cells. This complete protection may provide further evidence that GSH was effective at reducing and regenerating vitamin E to its active antioxidant state, with NAC providing a source of additional sulfhydryl groups, or acting as a substrate for more GSH synthesis as GSH became oxidized and inactive. The individual agents were not nearly as effective at protecting against lipid peroxidation. When exogenous sulfhydryl-donors, such as GSH or NAC, were tested in either iron- or iron–DEM-treated cells, no protection against iron-induced lipid peroxidation was observed. In fact, NAC significantly increased lipid peroxidation in iron-treated cells. However, these findings are not necessarily surprising. The role of sulfhydryl replacement in preventing lipid peroxidation is unclear, as GSH has been suggested to reduce lipid peroxidation (Palamanda and Kehrer, 1992; Graham et al., 1989) and also has been suggested to cause or exaggerate lipid peroxidation (Zager and Burkhart, 1998; Stacey et al., 1980). The role of vitamin E in protecting against lipid peroxidation is more clear. It is well established that vitamin E can protect against iron-induced lipid peroxidation in a variety of in vitro and in vivo experimental conditions (Iqbal et al., 1998; Brown et al., 1997; Omara and Blakely, 1993). In the present study however, vitamin E treatment only significantly inhibited iron-induced lipid peroxidation in DEM-treated cells. Although it has been suggested that GSH may act to regenerate the reduced form of vitamin E after vitamin E has become fully oxidized and inactive as an antioxidant (Leedle and Aust, 1990), the present study suggested vitamin E may be an effective inhibitor of lipid peroxidation even when GSH has been depleted. Of course, the results of the current study must be interpreted carefully to recognize the limitations of the assays used. The method used to measure loss of cell viability was the neutral red assay, a method often used to measure cytotoxicity in a variety of cell systems, including human and rat hepatocyte cultures (Fautz et al., 1991; Ledirac et al., 2000; Martinez-Diez et al.,
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2000). The assay is based on the fact that the neutral red dye is taken up by viable cells and stored in the lysosomes. Damage to cell and lysosomal membranes results in decreased uptake and increased release of the dye. The neutral red assay has proven to be a reliable and sensitive assay for determining loss of cell viability, and has shown particular efficacy in examining the cytotoxicity of metals in vitro (Yamamoto et al., 2001; Sauvant et al., 1997). However, in the current study, it is possible the neutral red assay may not have been sensitive enough to detect low levels of cell death in the hepatocyte suspensions. Although it is believed the neutral red assay should have been particularly suitable to study iron-induced loss of cell viability because it has been suggested that lysosomal damage may be one of the most significant events associated with loss of cell viability due to oxidative stress (Ollinger and Brunk, 1995). The limitations of the TBARS assay should also be recognized when interpreting the results of the present study. The TBARS assay may produce artifacts that either exaggerate or underestimate the actual degree of cellular lipid peroxidation. However, the purpose of this study was to examine the relationships between the cellular events associated with acute iron toxicity and not necessarily to quantify the degree of lipid peroxidation. In summary, a correlation between iron-induced lipid peroxidation and loss of cell viability was not identified. Cellular GSH concentrations appeared to be very important in maintaining cell viability following iron exposure. A synergistic relationship between GSH, NAC and vitamin E was strongly suggested as the combination of the three agents offered complete protection against lipid peroxidation caused by iron in normal cells and DEM-treated cells. This degree of protection was not seen with any of the individual agents alone. These results suggest further mechanistic studies are necessary to better understand the complex events associated with cellular iron toxicity and to further investigate the interactions between GSH and vitamin E in situations of oxidative stress.
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