Journal of Nutrition

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wang, S.-T.
Right arrow Articles by Lii, C.-K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wang, S.-T.
Right arrow Articles by Lii, C.-K.

The Journal of Nutrition Vol. 127 No. 11 November 1997, pp. 2135-2141
Copyright ©1997 by the American Society for Nutritional Sciences

Methionine and Cysteine Affect Glutathione Level, Glutathione-Related Enzyme Activities and the Expression of Glutathione S-Transferase Isozymes in Rat Hepatocytes1,2,3

Shih-Tsung Wang*, Haw-Wen Chen*, Lee-Yan Sheendagger , and Chong-Kuei Lii*, 4

* Department of Nutrition, Chung Shan Medical College and dagger  Department of Nutrition, China Medical College, Taichung, Taiwan 40203, Republic of China

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
LITERATURE CITED

ABSTRACT

Methionine and cysteine are constituents of glutathione. To understand the effects of these two sulfur amino acids on the glutathione (GSH)-dependent detoxification defense system, intracellular GSH and GSH-related enzyme activities, including GSH peroxidase, GSH reductase, GSH S-transferase (GST) and gamma -glutamylcysteine synthetase, were determined. In addition, the expression of three GST isozymes and carbonic anhydrase III (CA III) was examined. Hepatocytes isolated from male Sprague-Dawley rats were cultured with 0.1, 0.3, 0.5 or 1.0 mmol/L each of L-methionine and L-cysteine, for up to 7 d. Cells incubated with 0.5 or 1.0 mmol/L methionine and cysteine had increased intracellular GSH. A twofold increase was observed on d 6 compared with freshly isolated hepatocytes (P < 0.05). However, intracellular GSH was lower in cells treated with 0.3 or 0.1 mmol/L each of methionine and cysteine than in cells tested with 0.5 or 1.0 mmol/L. Although the GSH level differed significantly between cells cultured with 0.3 or 1.0 mmol/L of methionine and cysteine, GSH-related enzymes did not differ at these two concentrations. The activity generally remained constant for the first 24 h, then increased up to d 4. Immunodetection analysis revealed no difference in the level of CA III and GST isoforms, Ya, Yb and Yp, with amino acids each at a concentration of at least 0.3 mmol/L. Yp expression steadily increased up to d 7. Most proteins decreased rapidly after 48 h when cultured with 0.1 mmol/L of methionine and cysteine; however, the Yp level increased up to d 6. In conclusion, results indicate that a twofold increase of intracellular GSH is reached by adding methionine and cysteine at a concentration >0.5 mmol/L to the culture medium. The concentrations of methionine and cysteine for maintaining hepatic GSH are higher than for GSH-related enzyme activity and for GST isoform expression.

KEY WORDS: sulfur amino acids · glutathione · glutathione S-transferase isozymes · rats · hepatocytes

INTRODUCTION

The composition of laboratory media plays an influential role in modulating many hepatic xenobiotic biotransformation enzymes, such as cytochrome P450, glutathione (GSH)5 S-transferase (GST), and other GSH-related enzymes, during culture in regulating either gene expression or protein synthesis (Lu et al. 1992, Vandenberghe et al. 1992, Waxman et al. 1990). Sulfur amino acids, i.e., cysteine and methionine (which can be converted to cysteine via the cystathionine pathway in the liver), are the essential components for GSH synthesis because of the limited availability of cysteine for gamma -glutamylcysteine synthetase (GCS). Therefore, an adequate supply of these sulfur amino acids is crucial for maintaining a normal hepatic GSH level (Beatty and Reed 1981). The concentration of sulfur amino acids in several commercial media commonly used in hepatocyte culture varies widely, ranging from 0.03 mmol/L of methionine and 0.3 mmol/L of cysteine in F-12 medium to 1 mmol/L each of methionine and cysteine in L-15 medium. This wide range may result in subtle changes in GSH status, especially in long-term cultures. Therefore, a comprehensive investigation of the influence of sulfur amino acids on GSH status in a long-term study is necessary.

GSH synthesis is regulated by a feedback mechanism from GSH (Deleve and Kaplowitz 1990) or by modulating the GCS activity or the GSH efflux (Goss et al. 1994, Lu et al. 1990 and 1992). GCS has been demonstrated to be inhibited via the cAMP or protein kinase C pathway, such as by the action of glucagon and phenylephrine (Lu et al. 1991). GSH is important not only for its role in removing many reactive oxygen species such as hydrogen peroxide and other organic hydroperoxides but also in forming conjugates with a variety of electrophilic xenobiotics (Meister 1989), catalyzed by GSH peroxidase or GSH S-transferases. In addition, GSH may also act as a storage and transport form of cysteine and is involved in many biochemical processes in cells (Kosower and Kosower 1978).

The use of primary hepatocyte cultures in investigating the drug metabolism of many xenobiotics has been widely recognized (Guillouzo 1986). Hepatocyte cultures have been used therefore in many areas including an exploration of GSH in protecting against oxidative damage and drug toxicity. Thus, the maintenance of hepatic GSH level and GSH-related enzyme activity is crucial during culturing. Recently, we examined the effect of fetal bovine serum on GSH level and GSH-related enzyme activities of rat hepatocytes in the presence of 1 mmol/L of methionine and cysteine (Lii et al. 1996). In this study, maximum two- to threefold increases of intracellular GSH and oxidized GSH (GSSG) were observed 72-96 h after isolation. Meanwhile, the pattern of change in GSH level over this 6-d study corresponded to a profile of GSH-related enzyme activities, including GSH S-transferase (GST, EC 2.5.1.18), GSH peroxidase (GSH Px, EC 1.11.1.9), GSH reductase (GSH Rd, EC 1.6.4.2), gamma -glutamyl transpeptidase (EC 2.3.2.2), and gamma -glutamylcysteine synthetase (GCS, EC 6.3.2.2). We hypothesized that the increased GSH synthesis was probably due to the abundant supply of sulfur amino acids in the medium and was also important in maintaining GSH-related enzyme activities. The resulting high GSH level was critical for the protection of the cultured hepatocytes after isolation.

In this study, to further elucidate the relationship between GSH and sulfur amino acids, we cultured hepatocytes with different concentrations of methionine and cysteine and examined the effects of these supplements on GSH level and GSH-related enzyme activities over a 7-d study. At the same time, we also investigated the effect of different levels of methionine and cysteine on the synthesis of several proteins, including three GST isozymes and carbonic anhydrase III (CA III).

MATERIALS AND METHODS

Materials. Bovine serum albumin (BSA), sodium selenite, GSH, GSH reductase, 1-chloro-2,4-dinitrobenzene (CDNB), HEPES, NADPH and type VII rat tail collagen were obtained from Sigma Chemical (St. Louis, MO). Insulin, transferrin, fetal bovine serum, penicillin-streptomycin solution and sulfur amino acid-free L-15 medium were obtained from Gibco Laboratory (Grand Island, NY). Collagenase was purchased from Worthington Biochemical (Freefold, NJ). Percoll was from Pharmacia LKB (Piscataway, NJ). An avidin-peroxidase ABC kit was obtained from Vector Laboratory (Burlingame, CA).

Cell isolation and culture. Laboratory animals were treated in compliance with the Guide for the Care and Use of Laboratory Animals (NRC 1985). We used 10-wk-old male Sprague-Dawley rats weighing 300-350 g (National Animal Breeding and Research Center, Taipei, Taiwan). Rat hepatocytes were isolated by a two-step collagenase perfusion method as described previously (Lii and Hendrich 1993). Briefly, the rats were anesthetized by intraperitoneal injection with sodium pentobarbital (80 mg/kg body weight). Livers were first perfused via the portal vein with 25 mmol/L sodium phosphate buffer (pH 7.6), containing 3.1 mmol/L KCl, 119 mmol/L NaCl, 0.5 g/L glucose, 1.0 g/L BSA and 5 mg/L phenol red, at a flow rate of 25 mL/min to remove blood. After the first 6 min of perfusion, the buffer was replaced with 200 mL of the same sodium phosphate buffer supplemented with 50 mg collagenase (>200 IU/mg protein), 40 mmol/L CaCl2 and 5 mg trypsin inhibitor, and the liver was perfused for another 10 min at a flow rate of 18 mL/min. The collagenase-digested liver was removed, passed through a nylon mesh, washed and centrifuged at 190 × g for 3 min at 4°C. Buffered Percoll (10% 10× Hank's buffer and 90% Percoll) was used to aid in separating hepatocytes from nonparenchymal cells. Cell viability was >90% as determined by trypan blue exclusion.

The isolated hepatocytes were then resuspended in L-15 culture medium (pH 7.6), containing 18 mmol/L HEPES, 5 mg/L each of insulin and transferrin, 5 µg/L selenium as sodium selenite, 1 g/L galactose, 1 µmol/L dexamethasone, 1 × 105 IU/L penicillin, 100 mg/L streptomycin, 2.5% fetal bovine serum and 1 mmol/L each of methionine and cysteine, at a density of 5 × 108 cells/L. A 5-mL cell suspension in a total of 2.5 × 106 cells was plated on each 60-mm collagen-precoated plastic tissue culture dish (Nunc, Roskilde, Denmark) and incubated in a 37oC humidified incubator in an air atmosphere. Cell attachment on the culture dish was achieved 4 h after plating and the medium was changed. With the exception of the L-15 culture medium, which contained 1.0 mmol/L each of L-methionine and L-cysteine, three additional media with sulfur amino acid concentrations of 0.5 mmol/L, 0.3 mmol/L and 0.1 mmol/L each of L-methionine and L-cysteine were studied. After incubation with the different levels of methionine and cysteine, the medium was changed once each day and cultured up to 7 d. For all treatments, samples were taken at different time intervals (on d 1, 2, 4, 6 and 7); each time, two tissue culture dishes were used for GSH and GSSG determination and two were used for enzyme activity assay and immunodetection. At each cell harvest, cells were washed twice with cold PBS (pH 7.4) before sample preparation.

Biochemical assays. Intracellular GSH and GSSG were determined by HPLC as described by Reed et al. (1980). Briefly, 1 mL of 0.5 mol/L perchloric acid, which contained 2 mmol/L phenanthroline to prevent GSH oxidation, was added to each dish. The acid-soluble GSH and GSSG were prepared by centrifugation at 10,000 × g for 10 min. The resultant pellets were used for total protein assay (Lowry et al. 1951). For enzyme activity assays, cells were harvested in 200 µL of 20 mmol/L potassium phosphate buffer (pH 7.0) by scraping, and the cell homogenates of the two plates were pooled and centrifuged at 10,000 × g for 30 min at 4oC. The resultant supernatant was further ultracentrifuged at 105,000 × g for 1 h at 4oC. The final cytosolic supernatants were stored immediately at -80oC, and all activity assays were performed within 1 mo after preparation. GSH Px activity was determined spectrophotometrically with a coupled procedure by using H2O2 as a substrate (Lawrence and Burk 1976). GSH Rd activity was measured as described (Bellomo et al. 1987) by determining the oxidation rate of NADPH. GST activity in the cytosolic fraction was assayed according to the method of Habig et al. (1974) using CDNB as a substrate. The generation of CDNB-GSH conjugate [extinction coefficient of 9.6 (mmol/L)-1 cm-1] was monitored at 340 nm with a spectrophotometer (U-3000, Hitachi, Tokyo, Japan). The GCS activity was determined by measuring the rate of formation of ADP from the NADH oxidation by using the coupled method described by Seelig and Meister (1985). Enzyme activity was normalized on the basis of protein concentration (Lowry et al. 1951) in the cytosolic supernatant.

Table 1. The effect of various levels of methionine and cysteine on total protein content in rat hepatocytes during a 7-d culture1

[View Table]

Polyacrylamide gel electrophoresis. SDS polyacrylamide gels made with 10% acrylamide were prepared as described by Laemmli (1970). For GST and CA III immunodetection, 20 or 2 µg cytosolic protein was applied to each gel, respectively. Following electrophoresis, proteins separated on SDS-polyacrylamide gels were transferred to nitrocellulose membranes.

Immunodetection. The nonspecific binding sites on the nitrocellulose membranes were blocked with normal goat serum in 15 mmol/L Tris/150 mmol/L NaCl buffer (pH 7.4) at 4oC overnight. Antiserum against CA III was prepared as described by Chai et al. (1991). Antiserum against the placental form of GST was a gift from Suzanne Hendrich (Department of Nutrition, Iowa State University). Other GST isozymes, alpha  and µ types, were detected because of the crossreactivity of this polyclonal antibody. An avidin-peroxidase and biotinylated anti-rabbit IgG kit was used to detect the immunoreactive bands. Each incubation with primary antibody (diluted 1:400 in 15 mmol/L Tris/150 mmol/L NaCl buffer, pH 7.4), secondary biotinylated-antibody, and avidin-peroxidase complex was performed at 37oC for 30 min. For color development, hydrogen peroxide and 3,3'-diaminobenzidine tetrachloride were used as the substrates for peroxidase.

Statistical analysis. All analyses were conducted in duplicate for each sample. Data are expressed as means ± SEM, n = 5 rats. ANOVA and Tukey's multiple comparison (Steel and Torrie 1960) were used to test significant changes over time in one medium and to test significant differences between concentrations of sulfur amino acids at the same time. When variables had unequal variance, data were log-transformed before ANOVA. P < 0.05 was taken to be statistically significant.

RESULTS

Sulfur amino acids and protein content. Total protein in cells incubated with various levels of sulfur amino acids over this 7-d study is presented in Table 1. In hepatocytes supplemented with at least 0.3 mmol/L each of methionine and cysteine, the change of protein level in each tissue culture dish was similar over the entire culture period. Protein content remained constant during the first 4 d. In contrast, in those cells treated with 0.1 mmol/L of amino acids, there was a continuous decrease in protein levels and 43% of total protein was lost over this 7-d incubation.

GSH status during culture. During the 7-d culture period, the intracellular GSH level was significantly affected by sulfur amino acid concentrations (Table 2). In the presence of 1.0 mmol/L each of methionine and cysteine, reduced GSH remained constant the first 24 h after isolation, then increased rapidly and reached a maximum on d 6. There was a twofold increase in GSH on d 6 compared with the level of freshly isolated cells (d 0). GSH level was dramatically lower on d 7. A similar pattern of change for reduced GSH was also obtained in hepatocytes incubated in culture medium with 0.5 mmol/L each of methionine and cysteine. When the amino acid concentrations were 0.3 mmol/L each of methionine and cysteine, GSH steadily and significantly decreased during the entire culture. After 48 h in culture, intracellular GSH in cells with 0.3 mmol/L of methionine and cysteine was significantly lower than cells with 0.5 or 1.0 mmol/L. In the presence of 0.1 mmol/L of methionine and cysteine in the culture medium, GSH was depleted dramatically, and only 5% of the initial GSH level was left 24 h after supplementation.

Table 2. Glutathione (GSH) and oxidized glutathione (GSSG) in rat hepatocytes cultured with different levels of methionine and cysteine1

[View Table]

Similarly, this influence of methionine and cysteine was also observed with oxidized GSH (GSSG). In the presence of 1.0 or 0.5 mmol/L of methionine and cysteine, GSSG remained unchanged in the first 24 h then significantly increased at d 4 and 6 (P < 0.05). Maximum GSSG was obtained at d 6. In contrast, the GSSG level was not consistently changed in cells cultured with 0.3 mmol/L of methionine and cysteine. In the presence of 0.1 mmol/L of amino acids, the GSSG level was significantly lower after 24 h in culture than the level of freshly isolated hepatocytes (P < 0.05). To compare the effect of concentration of sulfur amino acids, GSSG from d 2 to 6 was significantly less in those cells cultured with 0.3 or 0.1 mmol/L methionine and cysteine than with 0.5 or 1.0 mmol/L (P < 0.05). When GSH status was expressed as the ratio of GSH to GSSG, cells with at least 0.5 mmol/L methionine and cysteine maintained a constant ratio (~40) up to d 6 (data not shown). However, the GSH/GSSG ratio was significantly reduced in cells in 0.3 or 0.1 mmol/L sulfur amino acid cultures (P < 0.05).

GSH-related enzyme activity. Because the time courses for all GSH-related enzyme activities of cells cultured with 0.5 mmol/L of methionine and cysteine did not differ from those observed with 1.0 mmol/L, the results of 0.5 mmol/L cultures are not shown. Effects of methionine and cysteine supplementation on GCS activity over the entire culture period are shown in Figure 1. A similar pattern of change in the GCS activity to GSH level was observed in cells with 1.0 mmol/L of methionine and cysteine. Enzyme activity remained constant during the first 24 h, then gradually increased and had doubled by d 4. After d 4, GCS activity decreased. As stated above, more than 0.5 mmol/L of methionine and cysteine is required to maintain the intracellular GSH level during culture. However, the effect of sulfur amino acids on GCS activity differed with GSH synthesis. In the presence of 0.3 mmol/L of methionine and cysteine, the pattern of GCS activity for the 7-d culture did not differ from those cells with 1.0 mmol/L. When the levels of methionine and cysteine were 0.1 mmol/L, GCS activity was significantly lower than for cells cultured with 0.3 or 1.0 mmol/L, and 70% of the activity was lost in the first 24 h.
Fig. 1. Effects of various levels of methionine and cysteine on gamma -glutamylcysteine synthetase activity in hepatocytes isolated from 10-wk-old male Sprague-Dawley rats. After isolation, cells were maintained in L-15 medium for 4 h then changed to medium containing 0.1, 0.3 or 1.0 mmol/L each of methionine and cysteine and cultured for up to 7 d. Samples were collected after being cultured for 1, 2, 4, 6 and 7 d. Values are means ± SEM for hepatocyte preparations from 5 rats. #Significantly lower (P < 0.05) than cells cultured with 0.3 and 1.0 mmol/L of supplementary methionine and cysteine at the same time. abcdTreatment means in a medium over time not sharing a letter differ significantly (P < 0.05).
[View Larger Version of this Image (31K GIF file)]

The activities of GSH Px (Fig. 2, panel A), GST (panel B) and GSH Rd (panel C) generally showed similar changes over the entire study. For cells cultured with 0.1 mmol/L methionine and cysteine, enzyme activity decreased following the incubation. With higher levels of sulfur amino acid supplementation, activity gradually increased and reached its maximum on d 4. The only exception was GST, which had decreased activity during the first 24 h. After 48 h in culture, all enzyme activities in cells cultured with 0.1 mmol/L of methionine and cysteine were significantly less than in the other groups (P < 0.05). Significant differences between cells with 0.3 and 1.0 mmol/L were observed for GSH Px only after d 6 and for GST after d 2 (P < 0.05).

Fig. 2. Glutathione (GSH) peroxidase (panel A), GSH S-transferase (panel B), and GSH reductase (panel C) activity in rat hepatocytes cultured with different levels of methionine and cysteine for 7 d. Hepatocytes were isolated by collagenase perfusion. After a 4-h attachment period, cells were maintained in a sulfur amino acid-free L-15 medium supplemented with 0.1, 0.3 or 1.0 mmol/L each of methionine and cysteine. Values are means ± SEM, n = 5. #Significantly lower (P < 0.05) than cells cultured with 0.3 and 1.0 mmol/L at the same time. *Significantly different (P < 0.05) among three groups at the same time. abcdTreatment means in a medium over time not sharing a letter differ significantly (P < 0.05).
[View Larger Version of this Image (33K GIF file)]

Expression of GST and CA III. GST isozyme expression detected by immunoblotting is shown in Figure 3. Although the GST antiserum applied in this study was the antiplacental form of GST, on the basis of its crossreactivity with other isozymes, other GST isoforms were also detected. According to the migrated pattern of GST isozymes on SDS-polyacrylamide gel as reported by Dwivedi et al. (1993), these two GST isoforms are Ya and Yb subunits. The expression of three GST isoforms showed different patterns over the entire culture period. In cells cultured with at least 0.3 mmol/L each methionine and cysteine (Fig. 3, panels A-C), the expression of Yb steadily decreased with culture time. However, Ya was relatively constant. Only a small amount of the placental form of GST (Yp) existed in freshly isolated hepatocytes (panel A, lane 1), but this amount subsequently increased with time (panels A-C, lanes 2-5). In cells cultured with low methionine and cysteine (0.1 mmol/L), the level of Ya and Yb was maintained for only 48 h (panel D, lane 2), then decreased. The expression of the Yp isoform, however, increased up to d 6 in those cells cultured with 0.1 mmol/L each of methionine and cysteine (panel D, lanes 2-4), although to a lesser extent than other groups.
Fig. 3. Immunodetection of glutathione S-transferase isoforms of hepatocytes with different levels of methionine and cysteine in medium. Hepatocytes were cultured in medium with (A) 1.0, (B) 0.5, (C) 0.3 and (D) 0.1 mmol/L each of methionine and cysteine for up to 7 d. For each lane, 20 µg of cytosolic protein was applied to 10% SDS-polyacrylamide gels. The separated proteins were transferred to a nitrocellulose membrane, and GSH S-transferase isoforms were immunostained by antibody-linked peroxidase activity (see Materials and Methods). Lane 1, cytosol from freshly isolated hepatocytes; lanes 2-5, cytosol from hepatocytes cultured for 2, 4, 6 and 7 d, respectively.
[View Larger Version of this Image (29K GIF file)]

The expression of carbonic anhydrase III, a major cytosolic protein in the liver of male rats, was also determined (Fig. 4). A similar time course was observed in cells incubated with sulfur amino acids of at least 0.3 mmol/L. The amount of CA III was maintained for the first 4 d (panels A-C, lanes 1-3), then decreased (panels A-C, lanes 4 and 5). On the other hand, in the 0.1 mmol/L cultures, the CA III level declined rapidly and no CA III was detected after 48 h (panel D, lanes 2-5).

Fig. 4. Effects of methionine and cysteine on carbonic anhydrase III expression. Hepatocytes were cultured in medium with (A) 1.0, (B) 0.5, (C) 0.3 and (D) 0.1 mmol/L each of methionine and cysteine for up to 7 d. For each lane, 2 µg of cytosolic protein was applied to 10% SDS-polyacrylamide gels. The transblotted nitrocellulose membranes were immunostained with CA III antibody and an avidin-peroxidase ABC kit (Vector Laboratory, CA). Lanes 1-5, cytosol from freshly isolated hepatocytes and hepatocytes cultured for 2, 4, 6 and 7 d, respectively.
[View Larger Version of this Image (29K GIF file)]

DISCUSSION

An initial increase in intracellular hepatic GSH concentration after cell isolation has been observed in other studies (Guillemette et al. 1993, Lii et al. 1996, Mertens et al. 1991). But the pattern of increase is not completely consistent in these studies, probably because of the different culture conditions. In our study, a twofold increase in intracellular GSH was reached after 6 d in a culture of cells supplemented with 1.0 or 0.5 mmol/L each of methionine and cysteine (Table 2). GSH plays a vital role in various cellular functions such as protection against drug toxicity and radical-scavenging capability (Kosower and Kosower 1978, Meister 1989). An increase in GSH is probably critical for keeping cells normal or more capable of resisting oxidative damage after they are isolated from animals and cultured in an exterior environment (Mertens et al. 1993). To maintain or increase intracellular GSH, methionine and cysteine levels >0.3 mmol/L are required in the culture medium.

Although the GSH level could not be maintained with 0.3 mmol/L methionine and cysteine, protein synthesis and enzyme activity were not affected. This is demonstrated by the total protein assay (Table 1), the GSH-related enzyme activity assay (Figs. 1, 2) and the immunodetection assay of different GST isoforms (Fig. 3) and CA III (Fig. 4). The enzyme activities and protein levels were similar to cells cultured with 0.5 or 1.0 mmol/L methionine and cysteine, suggesting that the requirements for these two sulfur amino acids for GSH synthesis are different than that for protein synthesis. A faster turnover rate of GSH in hepatocytes (3-5 h) (Orrenius et al. 1983) relative to that of the proteins examined could be the possible explanation. In this study, we noted that the concentrations of methionine and cysteine required to maintain normal GSH concentration and GSH metabolism were greater than their normal extracellular in vivo concentrations (Silbernagl 1987). This indicates that cell cultures require supraphysiologic concentrations of both amino acids. A constant and continuous supply of amino acids is available in vivo. However, in in vitro culture, only a single dose is used over a 24-h period. Thus, the availability of amino acids continuously decreases. This probably explains, at least in part, why a greater concentration of both amino acids in the medium is necessary.

An increase in intracellular GSH level after isolation has been proposed to be the result of GCS activity or the plentiful supply of methionine and cysteine in the culture medium. The latter probably plays the more important role (Lii et al. 1996). This possibility is also supported by this study, in which GSH increased up to d 6 (Table 2) even when GCS activity began to decrease after d 4 (Fig. 1). Because of the limited availability of intracellular cysteine in hepatocytes, gamma -glutamylcysteine synthesis is the rate-limiting step for GSH synthesis (Tateishi et al. 1974). Any attempt to increase intracellular cysteine by supplementing with cysteine or methionine, which can be actively converted to cysteine via the cystathionine pathway, would be effective in elevating the GSH level in hepatocytes (Beatty and Reed 1981). Although the plentiful supply of methionine and cysteine accounts for the increase in GSH level, the modulatory role of GCS activity (Fig. 1), which is responsible for gamma -glutamylcysteine synthesis, cannot be excluded.

Recently, Lu and colleagues (1990 and 1991) indicated that the regulation of GSH efflux and GCS activity is hormone dependent, and that the activation of cAMP-dependent protein kinase stimulates GSH efflux or decreases GCS activity in cultured hepatocytes. Additionally, GCS activity can also be activated in the presence of insulin (Lu et al. 1992). Therefore, the use of insulin in culture media in this study may partially account for the change in GSH observed. Although GCS is negatively controlled by glutathione (Deleve and Kaplowitz 1990), this relationship was not observed in this study. In hepatocytes cultured with 1.0 mmol/L of methionine and cysteine, the pattern of change of GCS activity (Fig. 1) was similar to the change in GSH (Table 2). It is not clear why feedback regulation of GCS activity is lacking. It is probably the result of competition between insulin activation and negative regulation by GSH. The change of sensitivity of hepatocytes to GSH feedback regulation during culture is also possible. Grimble and colleagues (1992) reported that liver GSH significantly increases from 15 to 106 µmol/g when rats are fed low dietary protein supplemented with cysteine. This GSH level is even greater than in rats fed a high protein diet (78 µmol/g tissue) with an equal cysteine content. This evidence suggests that in vivo, substrate supply probably has a more profound effect on GSH synthesis than GSH feedback inhibition. Recently, an increase of GCS activity accompanied by an increase in hepatic GSH level was reported in rats (Eaton and Hamel 1994) and mice (Teshigawara et al. 1995).

As previously mentioned, an increase in cellular GSH may improve the ability of hepatocytes to resist oxidative damage after they are isolated from animals and cultured in an exterior environment. GSH-related enzymes tested also showed similar patterns over this 7-d study (Fig. 2). This result further suggests that such a change may lead to hepatocytes that are more able to escape damage during culture. Similar observations for GSH Px and GSH Rd activity (Mertens et al. 1993) and for GST activity (Vandenberghe et al. 1988) in rat hepatocytes during culture have been reported.

The effects of methionine and cysteine on the expression of GST isoforms were also examined (Fig. 3). We paid particular attention to the placental form of GST (Yp). This GST isozyme, little or none of which is found in the normal rat liver, is continuously expressed after cell isolation (Dwivedi et al. 1993, Lii et al. 1996) and is highly inducible by chemical carcinogens (Satoh et al. 1985). It is widely used for detecting enzyme-altered foci during carcinogenesis (Hendrich and Pitot 1987). With the exception of the GST Yp isoform, the expression of both Ya and Yb isoforms (Fig. 3), CA III (Fig. 4), and total protein content (Table 1) and activity assays (Fig. 1 and 2) clearly indicate that the addition of 0.1 mmol/L methionine and cysteine to the culture medium was not sufficient to maintain normal hepatocyte function. However, Yp was still expressed and increased up to d 6 (Fig. 3). These results demonstrate that these two sulfur amino acids were preferably used for Yp synthesis. The increased expression of Yp also suggests that cells are still viable up to d 6 when cultured in this low sulfur amino acid concentration.

Results indicate that an adequate supply of methionine and cysteine in vitro elevates intracellular GSH to more than twice the level of freshly isolated hepatocytes. For rat hepatocytes with 0.5 or 1.0 mmol/L each of methionine and cysteine in a culture medium, GSH level and GSH-related enzyme activities were maintained for 7 d with an initial increase followed by a decrease. The minimal level of methionine and cysteine for maintaining GSH synthesis is different than that needed for enzyme activity or protein synthesis. Moreover, the continuous expression of the placental form of GST while other protein synthesis was depressed suggests the presence of a regulatory pathway for the synthesis of this GST isozyme that differs from other proteins.

FOOTNOTES

1   Supported by grant NSC 83-0412-B-040-004 from the National Science Council of the R.O.C.
2   Presented in part in poster form at the 16th International Congress of Nutrition at July 27 to August 1, 1997, Montreal, Canada.
3   The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
4   To whom correspondence should be addressed.
5   Abbreviations used: BSA, bovine serum albumin; CA III, carbonic anhydrase III; CDNB, 1-chloro-2,4-dinitrobenzene; GCS, gamma -glutamylcysteine synthetase; GSH, glutathione; GSH Px, glutathione peroxidase; GSH Rd, glutathione reductase; GSSG, oxidized glutathione; GST, glutathione S-transferase.

Manuscript received 13 December 1996. Initial reviews completed 12 February 1997. Revision accepted 9 June 1997.

LITERATURE CITED

0022-3166/97 $3.00 ©1997 American Society for Nutritional Sciences



This article has been cited by other articles:


Home page
 J. Pharmacol. Exp. Ther.Home page
J. T. Dever and A. A. Elfarra
L-Methionine Toxicity in Freshly Isolated Mouse Hepatocytes Is Gender-Dependent and Mediated in Part by Transamination
J. Pharmacol. Exp. Ther., September 1, 2008; 326(3): 809 - 817.
[Abstract] [Full Text] [PDF]

Home page
 Drug Metab. Dispos.Home page
S. K. Kim, M. A. Abdelmegeed, and R. F. Novak
THE MITOGEN-ACTIVATED PROTEIN KINASE KINASE (MEK) INHIBITOR PD98059 ELEVATES PRIMARY CULTURED RAT HEPATOCYTE GLUTATHIONE LEVELS INDEPENDENT OF INHIBITING MEK
Drug Metab. Dispos., April 1, 2006; 34(4): 683 - 689.
[Abstract] [Full Text] [PDF]

Home page
 J. Nutr.Home page
C.-W. Tsai, J.-J. Yang, H.-W. Chen, L.-Y. Sheen, and C.-K. Lii
Garlic Organosulfur Compounds Upregulate the Expression of the {pi} Class of Glutathione S-Transferase in Rat Primary Hepatocytes
J. Nutr., November 1, 2005; 135(11): 2560 - 2565.
[Abstract] [Full Text] [PDF]

Home page
 J. Nutr.Home page
C.-W. Tsai, H.-W. Chen, J.-J. Yang, K.-L. Liu, and C.-K. Lii
Sulfur Amino Acid Restriction Induces the {pi} Class of Glutathione S-Transferase Expression in Primary Rat Hepatocytes
J. Nutr., May 1, 2005; 135(5): 1034 - 1039.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
K. R Dimitrova, K. DeGroot, A. K Myers, and Y. D Kim
Estrogen and homocysteine
Cardiovasc Res, February 15, 2002; 53(3): 577 - 588.
[Abstract] [Full Text] [PDF]

Home page
 J. Am. Coll. Nutr.Home page
D. E. Epner
Can Dietary Methionine Restriction Increase the Effectiveness of Chemotherapy in Treatment of Advanced Cancer?
J. Am. Coll. Nutr., October 1, 2001; 20(90005): 443S - 449.
[Abstract] [Full Text]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wang, S.-T.
Right arrow Articles by Lii, C.-K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wang, S.-T.
Right arrow Articles by Lii, C.-K.

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]