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Departments of
*
Biochemistry and
Medicine, Emory University School of Medicine, Atlanta, GA 30322 and
**
Department of Pharmacology, Morehouse School of Medicine, Atlanta, GA 30310
2To whom correspondence should be addressed. E-mail: dpjones{at}emory.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • glutathione • cysteine deficiency • redox • colon • oxidative stress
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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–3
). Because of the central role of Cys in cellular function, this deficiency could contribute to the onset and/or progression of the disease processes. Cys is required for synthesis of glutathione (GSH),3
which functions in detoxification, and for synthesis of taurine and sulfate, which function in diverse processes including osmotic regulation, bile salt transport and steroid hormone transport. Loss of Cys through these uses creates a demand for continuous supply of Cys or its precursor, methionine, in the diet.
Tissue GSH concentration varies as a function of Cys availability, and decreased GSH is associated with impaired detoxification. Accumulating evidence also indicates that GSH has a central function in redox regulation (4
), but it is not known whether Cys deficiency alone is sufficient to perturb the GSH redox state. In principle, Cys deficiency may cause an oxidation of GSH/glutathione disulfide (GSSG) redox in the absence of an oxidant because of a decline in GSH concentration without a compensatory decrease in GSSG concentration. This can occur because antioxidant functions of GSH consume 2 GSH per GSSG formed. In the Nernst equation for calculation of the redox state {Eh = Eo + RT/2F ln [(GSSG)/(GSH)2]} (5
), the redox state is a second-order function of GSH concentration. This means that a change in concentration of GSH even without a change in GSH/GSSG ratio could alter the cellular redox state (5
,6
). In contrast, if the GSSG reductase activity is sufficient, there should be no change in GSH/GSSG redox without oxidative stress.
The purpose of the present study was to determine whether Cys limitation is sufficient to result in an oxidation of the GSH/GSSG redox state in the absence of imposed oxidative stress. Experiments were performed with human colon carcinoma (HT29) cells that were cultured in Cys-limiting medium for 48 h and subsequently cultured up to 24 h with medium containing Cys or cystine (CySS). Cellular GSH and GSSG were measured by HPLC and the redox state of the cellular GSH/GSSG pool was calculated using the Nernst equation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Except as indicated, all chemicals were purchased from Sigma Chemical (St. Louis, MO). Distilled, deionized water was used throughout. HPLC-quality solvents were used for HPLC.
Cell culture.
HT29 cells were obtained from American Type Culture Collection (ATCC; Gaithersburg, MD) and were maintained at 37°C and 95% air, 5% CO2 in McCoy’s 5A complete medium, 10% (v/v) fetal bovine serum, and 5% (v/v) penicillin/streptomycin (Atlanta Biologicals, Atlanta, GA). This medium contains 0.2 mmol/L cysteine, 1.5 mmol/L glutamine, 0.1 mmol/L glycine, and 0.0016 mmol/L GSH. Excluding the first 24 h postseeding, McCoy’s 5A Cys-free medium was used in all experiments and treatments. This medium does not contain Cys but is otherwise identical to McCoy’s 5A complete medium. Neither medium contained CySS. Inclusion of 10% (v/v) calf serum resulted in an initial CySS concentration of 4 µmol/L and Cys <1 µmol/L; thus, even though the McCoy’s medium was Cys- and CySS-free, the term Cys-limiting has been used to denote that incubations were not totally free of these amino acids. Under all conditions used in the present study, including incubations in Cys-limiting medium, cell viability was >99%.
For all experiments, cells were seeded in McCoy’s complete medium on 60 mm plates at 6 x 105 cells/plate and maintained for 24 h. At this time, cells were washed once with PBS and the medium was changed to Cys-limiting medium; controls were changed to McCoy’s medium. For experiments to examine recovery after 48 h in Cys-limiting medium, the medium was changed to Cys-free McCoy’s with the addition of either 50 µmol/L CySS or 100 µmol/L Cys; controls similarly received Cys-free McCoy’s with the addition of either 50 µmol/L CySS or 100 µmol/L Cys. These concentrations were used instead of the 200 µmol/L Cys found in normal McCoy’s medium to more closely mimic in vivo extracellular thiol/disulfide pools. Analysis of medium Cys and CySS concentration by HPLC confirmed that 50 µmol/L CySS and 100 µmol/L Cys were achieved. Both cell and medium samples were harvested and analyzed at 0, 1, 4, 12 and 24 h after the last medium change. Processing time for 0-h samples was 5–10 min, performed at room temperature.
Analysis of GSH and GSSG.
Cells were washed once with PBS and immediately treated with 500 µL of ice-cold 50 g/L perchloric acid solution containing 0.2 mol/L boric acid and 10 µmol/L 
-Glu-Glu and placed on ice. After 5 min, cells were scraped and transferred into microcentrifuge tubes. Samples were stored at -20°C until derivatization with iodoacetic acid and dansyl chloride and analysis by HPLC with fluorescence detection (7
). Concentrations of thiols and disulfides were determined by integration relative to the internal standard (8
). Cellular concentrations were calculated using the measured cell volume for HT29 cells (7 µL volume per million cells; 9
). The redox state (Eh) of GSH/GSSG pool was calculated using the Nernst equation as described (9
).
Statistical analysis.
Results are expressed as means ± SEM. Differences were compared across groups and time using a two-way repeated-measures ANOVA. Specific differences between treatments or time and their interactions were compared using Tukey’s pairwise comparisons. Differences were considered to be significant when P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Cell death rate was 0.2 ± 0.02% after 48 h for Cys-limiting conditions and 0.20 ± 0.004% for control conditions. During a subsequent 24-h incubation with Cys-containing medium, cell viability remained >99%. Control cells had a doubling time of 27 ± 3 h, similar to previous measures for HT29 cells (9
), but Cys-deficient cells had a doubling time of 63 ± 9 h (n = 4; P < 0.05).
Effect of Cys-limiting medium on cellular GSH.
The contents of GSH and GSSG were measured in cells at the end of the 48 h incubation in McCoy’s medium with or without Cys. Cys-deficient cells contained only 1% of the GSH found in control cells; GSSG content was 9% of control values (Table 1
). The Eh of the cellular GSH pool was -135 ± 4 mV for Cys-deficient cells compared with -224 ± 2 mV in control cells. Cellular Cys concentration for Cys-deficient cells was not different from that of control cells (Table 1)
.
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The addition of Cys resulted in nearly complete recovery of GSH concentration to control values by 1 h (Fig. 1A
). Cellular concentration increased above control values, reaching a concentration of 3.5 mmol/L at 12 h (Fig. 1
A). A modest increase in cellular GSH concentration also occurred at 4 h (P = 0.05) after the medium change in control cells, indicating that a change in medium is sufficient to affect the cellular contents of GSH under these growth conditions (Fig. 1
A). Experiments with medium containing an equivalent amount of Cys as the disulfide form (CySS) gave results comparable with those with Cys (Fig. 1
A). Recovery to control values occurred by 1 h, and maximal values were 200% of control values at 12 h (Fig. 1
A).
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B). Inclusion of CySS in the medium resulted in greater cellular GSSG concentrations than observed with Cys at 4 and 12 h (Fig. 1
B). Recovery of Cellular Eh for GSH/GSSG after the addition of Cys or CySS.
The Eh of the GSH pool was approximately -224 mV in control cells and -135 mV in Cys-deficient cells (Fig. 2
). Addition of either Cys or CySS to Cys-deficient cells produced a rapid recovery from the oxidized conditions associated with Cys deficiency (Fig. 2)
. Within 10 min of Cys addition, cellular Eh was -207 ± 4 mV, a reduction of 70 mV compared with Cys-deficient cells. After 1 h, Eh was reduced to -220 mV and stabilized at approximately -230 mV by 4 h post-treatment. Despite the significant increases in cellular GSH and differences in GSSG concentrations in response to sulfur amino acid repletion, there was no significant difference in cellular Eh for GSH/GSSG between the Cys- and CySS-treated cells at 4 h and later.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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2 g/d (10
). Based on the assumption that tissues contain 1 mmol GSH/L, a 70-kg individual with 50 L of tissue water would contain 50 mmol of GSH. The corresponding content of 50 mmol of Cys, at 121 mg/mmol, would mean that the GSH provides a reserve of 6 g of Cys, i.e., equivalent to 3 d of normal sulfur amino acid intake. With loss attributable to consumption of GSH by conjugation reactions, conversion of Cys to taurine and sulfate and use of Cys as a gluconeogenic substrate, some degree of Cys deficiency could occur in individuals with low intake. Adequate dietary intake of methionine plus cysteine is 1 g/d in adults (11
). However, estimated daily dietary intakes of Cys and Met varied >25-fold (from <200 mg to >5 g) among 100 healthy, free-living individuals (10
). This occurs in part because sulfur amino acids are abundant in animal proteins but can be limiting in fruits, vegetables, and grains (12
,13
). Thus, in addition to individuals who have Cys deficiency associated with disease (2
,14
), individuals with otherwise healthy diets can be predisposed to various degrees of Cys depletion because of food selection or availability.
The present study shows that severe Cys deficiency inhibits proliferation, decreases GSH concentration, and oxidizes GSH/GSSG redox in human colon-derived HT29 cells. Previous studies have associated a decrease in cell proliferation with decreased cellular GSH, specifically under conditions in which GSH concentration is decreased by inhibition of its synthesis from Cys or decreased by stimulation of its utilization (15
–17
). Conversely, cell proliferation is enhanced by supply of precursors for Cys and GSH (18
–20
). The present data provide evidence of a link between limited sulfur amino acid supply, decreased GSH concentration, and oxidized GSH/GSSG redox state. Because the redox state of GSH/GSSG is in the range of -260 to -230 mV in actively proliferating cells but becomes oxidized to values of -220 to -190 mV upon growth arrest (4
,9
), the redox changes associated with Cys deficiency could signal decreased cell proliferation. Theoretical analyses (4
,6
) show that a 30-mV change is sufficient to result in a 10-fold change in the ratio of reduced to oxidized forms of proteins with dithiol motifs, whereas a 60 mV change is sufficient to cause a 100-fold change. Thus, if the replicative machinery contains such a redox-sensitive protein, the measured 80 mV oxidation due to Cys deficiency is sufficient to inhibit cell division.
Although the present in vitro study provides no evidence that redox changes occur in vivo in response to Cys deficiency, oxidation of the GSH/GSSG redox in the intestinal epithelium has been found in rats fed for 3 d a diet containing 25% of the energy consumed by rats eating ad libitum (21
). This oxidation was associated with decreased growth indices, which could be attributable, at least in part, to the deficiency in Cys and its precursors (21
). Decreased GSH has also been found in humans in association with hypocystinemia because of hepatic cirrhosis (1
), and GSH/GSSG redox is oxidized in humans after high-dose chemotherapy (22
) and with age (23
). The present results indicate that variation in dietary intake of sulfur amino acids could contribute to these variations in GSH/GSSG redox.
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FOOTNOTES |
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3 Abbreviations used: Cys, cysteine; CySS, cystine; Eh, redox state; GSH, glutathione; GSSG, glutathione disulfide.
Manuscript received 26 February 2002. Initial review completed 1 April 2002. Revision accepted 1 May 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1. Chawla, R. K., Lewis, F. W., Kutner, M. H., Bate, D. M., Roy, R. G. & Rudman, D. (1984) Plasma cysteine, cystine, and glutathione in cirrhosis. Gastroenterology 87:770-776.[Medline]
2. Droge, W. & Holm, E. (1997) Role of cysteine and glutathione in HIV infection and other diseases associated with muscle wasting and immunological dysfunction. FASEB J. 11:1077-1089.[Abstract]
3. Droge, W. & Breitkreutz, R. (2000) Glutathione and immune function. Proc. Nutr. Soc. 59:595-600.[Medline]
4. Jones, D. P. (2002) Redox potential of GSH/GSSG couple: assay and biological significance. Methods Enzymol. 348:93-112.[Medline]
5. Clark, W. M. (1960) Oxidation-Reduction Potentials of Organic Systems 1960 Williams & Wilkins Baltimore, MD. .
6. Gilbert, H. F. (1990) Molecular and cellular aspects of thiol-disulfide exchange. Adv. Enzymol. Relat. Areas Mol. Biol. 63:69-172.[Medline]
7. Jones, D. P., Carlson, J. L., Samiec, P. S., Sternberg, P., Jr., Mody, V. C., Jr., Reed, R. L. & Brown, L. A. (1998) Glutathione measurement in human plasma: evaluation of sample collection, storage, and derivatization conditions for analysis of dansyl derivatives by HPLC. Clin. Chim. Acta 275:175-184.[Medline]
8. Jones, D. P., Carlson, J. L., Mody, V. C., Jr., Cai, J., Lynn, M. J. & Sternberg, P., Jr. (2000) Redox state of glutathione in human plasma. Free Radic. Biol. Med. 28:625-635.[Medline]
9. Kirlin, W. G., Cai, J., Thompson, S. A., Diaz, D., Kavanagh, T. J. & Jones, D. P. (1999) Glutathione redox potential in response to differentiation and enzyme inducers. Free Radic. Biol. Med. 27:1208-1218.[Medline]
10. Flagg, E. W., Coates, R. J., Eley, J. W., Jones, D. P., Gunter, E. W., Byers, T. E., Block, G. S. & Greenberg, R. S. (1994) Dietary glutathione intake in humans and the relationship between intake and plasma total glutathione level. Nutr. Cancer 21:33-46.[Medline]
11. National Research Council (1989) Recommended Dietary Allowances 10th ed. 1989 National Academy Press Washington, DC. .
12. Jones, D. P., Coates, R. J., Flagg, E. W., Eley, J. W., Block, G., Greenberg, R. S., Gunter, E. W. & Jackson, B. (1992) Glutathione in foods listed in the National Cancer Institute’s Health Habits and History Food Frequency Questionnaire. Nutr. Cancer 17:57-75.[Medline]
13. Houterman, S., van Faassen, A., Ocke, M. C., Habets, L. H., van Dieijen-Visser, M. P., Bueno-de-Mesquita, B. H. & Janknegt, R. A. (1997) Is urinary sulfate a biomarker for the intake of animal protein and meat?. Cancer Lett. 114:295-296.[Medline]
14. Sido, B., Hack, V., Hochlehnert, A., Lipps, H., Herfarth, C. & Droge, W. (1998) Impairment of intestinal glutathione synthesis in patients with inflammatory bowel disease. Gut 42:485-492.
15. Shaw, J. P. & Chou, I.-N. (1986) Elevation of extracellular glutathione content associated with mitogenic stimulation of quiescent fibroblasts. J. Cell. Physiol. 129:193-198.[Medline]
16. Hamilos, D. L., Zelarney, P. & Mascali, J. J. (1989) Lymphocyte proliferation in glutathione-depleted lymphocytes: direct relationship between glutathione availability and the proliferative response. Immunopharmacology 18:223-235.[Medline]
17. Kavanagh, T. J., Grossman, A., Jaecks, E. P., Jinneman, J. C., Eaton, D. L., Martin, G. M. & Rabinovich, P. S. (1990) Proliferative capacity of human peripheral blood lymphocytes sorted on the basis of glutathione content. J. Cell. Physiol. 145:472-480.[Medline]
18. Zmuda, J. & Friedenson, B. (1983) Changes in intracellular glutathione pool levels in stimulated and unstimulated lymphocytes in the presence of 2-mercaptoethanol or cysteine. J. Immunol. 130:362-364.[Abstract]
19. Atzori, L., Dypbukt, J. M., Sundqvist, K., Cotgreave, I., Edman, C. C., Moldeus, P. & Grafstrom, R. C. (1989) Growth-associated modifications of low-molecular-weight thiols and protein sulfhydryls in human bronchial fibroblasts. J. Cell. Physiol. 143:165-171.
20. Iwata, S., Hori, T., Sato, N., Ueda-Taniguchi, Y., Yamabe, T., Nakamura, H., Masutani, H. & Yodoi, J. (1994) Thiol-mediated redox regulation of lymphocyte proliferation: possible involvement of adult T cell leukemia-derived factor and glutathione in transferring receptor expression. J. Immunol. 152:5633-5642.[Abstract]
21. Jonas, C. R., Estivariz, C. F., Jones, D. P., Gu, L. H., Wallace, T. M., Diaz, E. E., Pascal, R. R., Cotsonis, G. A. & Ziegler, T. R. (1999) Keratinocyte growth factor enhances glutathione redox state in rat intestinal mucosa during nutritional repletion. J. Nutr. 129:1278-1284.
22. Jonas, C. R., Puckett, A. B., Jones, D. P., Griffith, D. P., Szeszycki, E. E., Bergman, G. F., Furr, C. E., Tyre, C., Carlson, J. L., Galloway, J. R., Blumberg, J. B. & Ziegler, T. R. (2000) Plasma antioxidant status after high-dose chemotherapy: a randomized trial of parenteral nutrition in bone marrow transplantation patients. Am. J. Clin. Nutr. 72:181-189.
23. Samiec, P. S., Drews-Botsch, C., Flagg, E. W., Kurtz, J. C., Sternberg, P., Jr., Reed, R. L. & Jones, D. P. (1998) Glutathione in human plasma: decline in association with aging, age-related macular degeneration, and diabetes. Free Radic. Biol. Med. 24:699-704.[Medline]
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