![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
, and
* Department of Nutrition, 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 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 GSH synthesis is regulated by a feedback mechanism from GSH (Deleve and Kaplowitz 1990 The use of primary hepatocyte cultures in investigating the drug metabolism of many xenobiotics has been widely recognized (Guillouzo 1986 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 Department of Nutrition,
-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.
, 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 -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.
) 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).
). 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), -glutamyl transpeptidase (EC 2.3.2.2), and -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.
). 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.
. 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 |
. 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, 
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
|
Table 2. Glutathione (GSH) and oxidized glutathione (GSSG) in rat hepatocytes cultured with different levels of methionine and cysteine1 |
-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).
DISCUSSION FOOTNOTES
Manuscript received 13 December 1996. Initial reviews completed 12 February 1997. Revision accepted 9 June 1997.
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)]
, 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)]
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)]
, 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.
) 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.
). 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, -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 -glutamylcysteine synthesis, cannot be excluded.
). 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).
) and for GST activity (Vandenberghe et al. 1988) in rat hepatocytes during culture have been reported.
, 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.
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, -glutamylcysteine synthetase; GSH, glutathione; GSH Px, glutathione peroxidase; GSH Rd, glutathione reductase; GSSG, oxidized glutathione; GST, glutathione S-transferase.
LITERATURE CITED
-glutamylcysteine synthetase activity as a mechanism for butylated hydroxyanisole-mediated elevation of hepatic glutathione.
Toxicol. Appl. Pharmacol.
1994;
126:145-149
[Medline]
in rats fed a low protein diet.
J. Nutr.
1992;
122:2066-2073
-glutamylcysteine synthetase and glutathione synthesis in the rat. Studies in cultured hepatocytes and in vivo.
J. Clin. Invest.
1992;
90:524-532
-glutamylcysteine synthetase from rat kidney.
Methods Enzymol.
1985;
113:379-390
[Medline]
This article has been cited by other articles:
|
|
 |
|
  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]
|
|
|
|
 |
|
 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]
|
|
|
|
 |
|
 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]
|
|
|
|
|
|
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]
|
|
|
|
 |
|
 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]
|
|
|
|
 |
|
 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]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
ABSTRACT