Glutathione turnover in human cell lines in the presence of agents with glutathione influencing potential with and without acivicin inhibition of gamma-glutamyltranspeptidase

BACKGROUND: We have previously shown that there were great discrepancies between different agents regarding their glutathione stimulating potential and that agents with mainly oxidative effects did not increase concentrations of glutathione in human cell cultures, in contrast to other thiol reactive agents. In order to evaluate whether increased glutathione degradation might be one reason for these discrepancies, we have investigated the effect of different agents with potential influence on glutathione metabolism in human cell cultures with or without acivicin inhibition of gamma-glutamyltranspeptidase (GT), since GT is responsible for the initial degradation of glutathione. METHODS: Intra- and extracellular concentrations of glutathione were investigated in HeLa and hepatoma cell cultures, with and without acivicin inhibition of GT, in the presence of oxidative and electrophilic agents (copper ions, hydrogen peroxide and N-ethylmaleimide), hydroquinone, reducing agents (lipoic acid and N-acetylcysteine), and a thiol reactive metal (mercury ions). RESULTS: There were great discrepancies between the different agents regarding their maximal glutathione response (the sum of the intracellular and the extracellular amount of glutathione) in cell cultures. There was only a small increase in total glutathione in the presence of hydrogen peroxide or N-ethylmaleimide before the cell protein decreased compared to findings with mercury ions, lipoic acid or hydroquinone. In both HeLa and hepatoma cell cultures, there were correlations between the original glutathione amount and the total glutathione amount observed after acivicin inhibition. CONCLUSION: The relatively small increase of glutathione amount in the presence of oxidative and electrophilic agents compared to other thiol reactive agents is not due to increased GT degradation of glutathione.     

Glutathione turnover in human cell lines in the presence of agents with glutathione influencing potential with and without acivicin inhibition of γ-glutamyltranspeptidase

Background: We have previously shown that there were great discrepancies between different agents regarding their glutathione stimulating potential and that agents with mainly oxidative effects did not increase concentrations of glutathione in human cell cultures, in contrast to other thiol reactive agents. In order to evaluate whether increased glutathione degradation might be one reason for these discrepancies, we have investigated the effect of different agents with potential influence on glutathione metabolism in human cell cultures with or without acivicin inhibition of γ-glutamyltranspeptidase (GT), since GT is responsible for the initial degradation of glutathione. Methods: Intra- and extracellular concentrations of glutathione were investigated in HeLa and hepatoma cell cultures, with and without acivicin inhibition of GT, in the presence of oxidative and electrophilic agents (copper ions, hydrogen peroxide and N-ethylmaleimide), hydroquinone, reducing agents (lipoic acid and N-acetylcysteine), and a thiol reactive metal (mercury ions). Results: There were great discrepancies between the different agents regarding their maximal glutathione response (the sum of the intracellular and the extracellular amount of glutathione) in cell cultures. There was only a small increase in total glutathione in the presence of hydrogen peroxide or N-ethylmaleimide before the cell protein decreased compared to findings with mercury ions, lipoic acid or hydroquinone. In both HeLa and hepatoma cell cultures, there were correlations between the original glutathione amount and the total glutathione amount observed after acivicin inhibition. Conclusion: The relatively small increase of glutathione amount in the presence of oxidative and electrophilic agents compared to other thiol reactive agents is not due to increased GT degradation of glutathione.     

The effect of different antioxidants on glutathione turnover in human cell lines and their interaction with hydrogen peroxide

BACKGROUND: Glutathione plays crucial roles in antioxidant defence and glutathione deficiency contributes to oxidative stress and may therefore play a key role in the pathogenesis of many diseases. The objectives of the present study were to evaluate the effects on glutathione turnover of thiol and non-thiol antioxidants in human cell cultures and if any of the antioxidant had a short-term cellular effect against different levels of hydrogen peroxide. METHODS: We have investigated the effect on the total glutathione amount in HeLa and hepatoma cell cultures of thiol antioxidants in comparison with non-thiol antioxidants, such as a copper chelator, Vitamin C, and a flavonoid. Furthermore, we have investigated the short-term (within 24h) interaction of the different antioxidants with hydrogen peroxide. RESULTS AND CONCLUSION: Lipoic acid and quercetin (Quer) were the two antioxidants that showed the highest stimulation of glutathione synthesis in cell cultures as judged by the total glutathione amount. However, no antioxidant protected against hydrogen peroxide present in concentrations that lowered cell protein. This finding may be attributed to the fact that it is necessary to incubate cell cultures with antioxidants or small doses of oxidants for a period before the cultures are exposed to hydrogen peroxide in order to enhance the antioxidant defence. The presence of Quer and Vitamin C lowered cell protein and total glutathione even in cell cultures containing hydrogen peroxide in concentrations that did not lower cell protein. This finding might be attributed to pro-oxidant properties and formation of excess reactive oxygen species in the presence of Quer and Vitamin C.     

Oxidative stress decreases extracellular homocysteine concentration in human hepatoma (HepG2) cell cultures

BACKGROUND: Mild hyperhomocysteinemia is associated with premature vascular disease. The mechanism behind the vascular injuries is, however, still unknown. Homocysteine may be catabolized in the trans-sulfuration pathway to cysteine. Cystathionine beta-synthase, which catalyses the first step in the trans-sulfuration pathway is redox-sensitive. We have therefore investigated total extracellular homocysteine turnover in the presence of oxidative stress in human cell lines. METHODS: The turnover of total extracellular homocysteine in HeLa and hepatoma cell cultures has been investigated in the presence of hydrogen peroxide. Furthermore, the effect of hydrogen peroxide on the removal of high amounts of exogenously added homocysteine was also studied. RESULTS: Total extracellular homocysteine concentration in hepatoma cell cultures decreased in the presence of hydrogen peroxide, whereas the extracellular homocysteine concentration in HeLa cell cultures was not influenced. There was no significant change of intracellular homocysteine in any type of cell cultures. Furthermore, the presence of hydrogen peroxide did not increase the removal of exogenously added homocysteine. CONCLUSION: The presence of hydrogen peroxide probably increases the activity of the trans-sulfuration pathway in hepatoma cell cultures, which increases the intracellular use of homocysteine and lowers its extracellular release. Consequently this mechanism might tend to lower total plasma homocysteine concentration in oxidative stress.     

Maintenance of Neuronal Glutathione by Glial Cells

Abstract— Glutathione levels in neurons and gllal cells were investigated in a neuronal-glial coculture and in separate cultures. Brain cell suspensions obtained from cerebral hemispheres of fetal rats were cultured, and after 5 days the glutathione content of this cell population, consisting mainly of neurons and astroglial cells, was 23.0 nmol/mg of cell protein, with a significantly high content in glial cells (28.0 nmol/mg of protein) in comparison with neurons (18.8 nmol/mg of protein). When the neurons and glial cells were separated and recultured in fresh medium, neu-ronal glutathione rapidly decreased, whereas glial glutathione remained unchanged. Cysteine is a rate-limiting precursor for glutathione synthesis, and its level was also decreased in neurons, but not in glial cells. Cysteine was taken up rapidly by both neurons and glial cells, but cys-tine was taken up only by glial cells. This accounts for the rapid decrease of glutathione in the cultured neurons, because the culture medium contains cystine, but not cys-teine. It was also found that the cultured glial cells released cysteine into the medium. These results suggest that neurons maintain their glutathione level by taking up cysteine provided by glial cells.     

Direct measurement of the rate of glutathione synthesis in 1-chloro-2,4-dinitrobenzene treated human erythrocytes

Cell glutathione scavenges free radicals, degrades peroxides, removes damaging electrophiles and maintains the redox state. The aim of this study was to develop an effective and efficient method to measure the rate of glutathione synthesis from its constituent amino acids in whole erythrocytes (RBCs). RBCs (10% haematocrit) were exposed to 0.3 mM 1-chloro-2,4-dinitrobenzene (CDNB) to lower their total glutathione content by 70% and then incubated with glucose, and N-acetylcysteine as a cysteine source. Over 3 h, glutathione levels increased at a constant rate of 1.2 micromol (L RBC)(-1)min(-1), almost 5 times faster than the rate of glutathione synthesis in RBCs with normal glutathione levels. Glutathione at concentrations normally found in RBCs is known to inhibit glutamate cysteine ligase (the major rate controlling enzyme for glutathione synthesis). The rate of glutathione recovery was substantially reduced in RBCs treated with buthionine sulfoximine, a specific inhibitor of glutamate cysteine ligase. Our results indicate that the measurement of glutathione recovery rate after CDNB treatment can be used to estimate de novo synthesis of glutathione. Application of this direct method for measuring glutathione synthesis will increase understanding of the interactions of effectors that determine glutathione levels in RBCs under various physiological and pathological conditions.     

Alterations of the glutathione-redox balance induced by metals in CHO-K1 cells

The effects of cadmium (Cd(2+)), mercury (Hg(2+)), lead (Pb(2+)), copper (Cu(2+)) and nickel (Ni(2+)) on the glutathione (GSH)-redox cycle were assessed in CHO-K1 by the neutral red uptake inhibition (NR) assay (NR(6.25), NR(12.5) and NR(25)). Mercury proved to be the most and lead the least toxic of the metals tested. The effects on GSH content and intracellular specific activities of enzymes involved in the GSH-redox balance were measured after a 24-h exposure. Total GSH content increased significantly in cultures exposed to the lowest metal concentration assayed (NR(6.25)), but fell to below control values when exposed to concentrations equivalent to NR(25). Oxidised glutathione content dropped significantly at NR(6.25), while somewhat higher values were obtained for cultures exposed to higher doses. Glutathione peroxidase (Gpx) activities were 1.2-, 1.5-, 1.6-, 2.0- and 2.5-fold higher than untreated controls for cadmium, copper, mercury, nickel and lead, respectively, at concentrations equivalent to NR(6.25). Gpx activity declined at metal concentrations equivalent to NR(12.5) and NR(25). Glutathione reductase activity remained almost unchanged except at low doses of mercury, nickel and lead. Glutathione-S-transferase activity decreased at rising metal concentrations. The results suggest that a homeostatic defence mechanism was activated when cells were exposed to doses equivalent to NR(6.25) while the ability of the cells to respond weakened as the dose increased. A close relationship was also observed between metal cytotoxicity, total GSH content and the dissociation energy of the sulphur-metal bonds. These facts confirm the involvement of antioxidant defence mechanisms in the toxic action of these ions.     

A novel metallobridged bis(beta-cyclodextrin)s fluorescent probe for the determination of glutathione

A novel metallobridged bis(beta-cyclodextrin)s 2 [bis(beta-CD)s 2] was synthesized and characterized by means of (1)H NMR, IR, element analysis and redox iodometric titration. The fluorescence of metallobridged bis(beta-CD)s 2 was weak compared with bis(beta-CD)s 1 because of the paramagnetism of copper (II) ions. Glutathione was able to form complexes with copper (II) derived from the metallobridged bis(beta-CD)s 2. This competitive complexation with copper (II) may lead to a significant fluorescence recovery of the bis(beta-CD)s. Therefore, a rapid and simple spectrofluorimetric method was developed for the determination of glutathione. The analytical application for glutathione was investigated in NaCl/P(i) (pH 6.00) at room temperature. The linear range of the method was 0.30-20.0 micromol.L(-1) with a detection limit of 63.8 nmol.L(-1). There was no interference from the plasma constituents. The proposed method had been successfully used to determine glutathione in human plasma.     

Glutathione Turnover in Cultured Astrocytes: Studies with [15N]Glutamate

The incorporation of [15N]glutamic acid into glutathione was studied in primary cultures of astrocytes. Turnover of the intracellular glutathione pool was rapid, attaining a steady state value of 30.0 atom% excess in 180 min. The intracellular glutathione concentration was high (20-40 nmol/mg protein) and the tripeptide was released rapidly into the incubation medium. Although labeling of glutathione (atom% excess) with [15N]glutamate occurred rapidly, little accumulation of 15N in glutathione was noted during the incubation compared with 15N in aspartate, glutamine, and alanine. Glutathione turnover was stimulated by incubating the astrocytes with diethylmaleate, an electrophile that caused a partial depletion of the glutathione pool(s). Diethylmaleate treatment also was associated with significant reductions of intraastrocytic glutamate, glycine, and cysteine, i.e., the constituents of glutathione. Glutathione synthesis could be stimulated by supplementing the steady-state incubation medium with 0.05 mM L-cysteine, such treatment again partially depleting intraastrocytic glutamate and causing significant reductions of 15N labeling of both alanine and glutamine, suggesting that glutamate had been diverted from the synthesis of these amino acids and toward the formation of glutathione. The current study underscores both the intensity of glutathione turnover in astrocytes and the relationship of this turnover to the metabolism of glutamate and other amino acids.     

Glutathione metabolism in normal and cystinotic fibroblasts

Intracellular concentrations of glutathione and activities of the enzymes gamma-glutamylcysteine synthetase, glutathione synthetase, and gamma-glutamyl transpeptidase were measured in confluent cultured human fibroblasts cell lines from 14 normal cell lines and four cystinotic cell lines. gamma-Glutamyl transpeptidase had a wide range of variability while the glutathione synthetic enzymes, gamma-glutamylcysteine synthetase and glutathione synthetase, had narrower variations and also exhibited no apparent relationship to glutathione content. No differences in the activities of these enzymes were found between normal and cystinotic cells in confluent cell cultures. The activities of the above enzymes and the cell number and content of glutathione, cystine, DNA, and total protein in two normal and two cystinotic fibroblast cell lines were measured during growth. The following growth-dependency patterns were observed: (1) gamma-glutamylcysteine synthetase activity increased markedly in lag and early log phases in both normal and cystinotic cells and decreased rapidly to low confluent levels thereafter. (2) gamma-Glutamyl transpeptidase showed the same wide range of activity noted at confluency but activities decreased in the log phase of growth, a pattern also seen in cystinotic cells. (3) Glutathione synthetase activity remained relatively constant during growth of normal cells but exhibited a peak of activity during lag and early growth of cystinotic cells. (4) Comparative glutathione levels of normal and cystinotic cells were not significantly different and exhibited similar fluctuations with time. (5) The cystine content of normal and cystinotic cells unexpectedly rose to high levels in the lag phase, then decreased to 0.1 nmol 1/2 cystine/mg protein in normal cells and to 0.3 to 1.2 nmol 1/2 cystine/mg protein in cystinotic cells during the log phase. As confluency was approached, normal cell cystine remained at low levels while cystinotic cell cystine rose to characteristically high levels of 50- to 100-fold greater than normal cells at late confluency. These studies extend our understanding of the regulation of glutathione and cystine content in cultured fibroblasts and suggest that glutathione content is closely controlled throughout the cell cycle in the face of varying activities of its anabolic and catabolic enzymes.     

Assay of biological thiols by a combination of high-performance liquid chromatography and postcolumn reaction with 6,6'-dithiodinicotinic acid

A simple and rapid method for the determination of nanomole levels of biological thiols is described. The analysis is based on the combination of reverse-phase high-performance liquid chromatography with a postcolumn reaction with 6,6'-dithiodinicotinic acid. Thiols, including cysteine, cysteamine, thiolhistidine, homocysteine, glutathione, penicillamine, ergothioneine, and thiouracil were separated by eluting with 33 mM KH2PO4 at pH 2.2. Glutathione, cysteine, cysteamine, homocysteine, and penicillamine were quantitatively determined with detection limits of 0.1 nmol, while the quantitative detection of thiolhistidine, ergothioneine, and thiouracil was not successful. The method was applied to the assay of glutathione in human erythrocytes and Escherichia coli.     

Elevated oxidized glutathione in cystinotic proximal tubular epithelial cells

Cystinosis, the most frequent cause of inborn Fanconi syndrome, is characterized by the lysosomal cystine accumulation, caused by mutations in the CTNS gene. To elucidate the pathogenesis of cystinosis, we cultured proximal tubular cells from urine of cystinotic patients (n = 9) and healthy controls (n = 9), followed by immortalization with human papilloma virus (HPV E6/E7). Obtained cell lines displayed basolateral polarization, alkaline phosphatase activity, and presence of aminopeptidase N (CD-13) and megalin, confirming their proximal tubular origin. Cystinotic cell lines exhibited elevated cystine levels (0.86 +/- 0.95 nmol/mg versus 0.09 +/- 0.01 nmol/mg protein in controls, p = 0.03). Oxidized glutathione was elevated in cystinotic cells (1.16 +/- 0.83 nmol/mg versus 0.29 +/- 0.18 nmol/mg protein, p = 0.04), while total glutathione, free cysteine, and ATP contents were normal in these cells. In conclusion, elevated oxidized glutathione in cystinotic proximal tubular epithelial cell lines suggests increased oxidative stress, which may contribute to tubular dysfunction in cystinosis.     

Reduced cellular glutathione levels do not affect the cytotoxicity or photocytotoxicity of the cationic photosensitiser Victoria Blue BO

Victoria Blue BO (VBBO) is thought to exert its photocytotoxic effects via free radical generation. Glutathione and related enzymes are associated with the protection of normal tissues against free-radical damage and have also been implicated in multiple drug resistance. It might, therefore, be expected that cells containing higher levels of glutathione would be resistant to the cytotoxic effects of VBBO. The total glutathione content for a murine mammary tumour cell line, EMT6-S, was found to be lower than in a multi-drug resistant cell line, EMT6-R, 21.84+/-2.54 microg (mg protein)(-1) and 18.79+/-2.7 microg (mg protein)(-1), respectively; however, this was not found to be a significant difference (p > 0.05, Student t-test). Buthionine sulfoximine, a potent inhibitor of gamma-glutamyl cysteine synthetase, brought about a reduction in glutathione levels in both EMT6-S and EMT6-R cell lines in a concentration-dependent manner. Buthionine sulfoximine administration was effective in reducing intracellular glutathione levels by up to 90% in both types of cells. Interestingly, glutathione depletion of EMT6-S and EMT6-R cells did not enhance the photocytotoxic effect of VBBO, suggesting that the primary site of action of VBBO may be at an intracellular site not protected by glutathione or that the mechanism of action is not via the in situ generation of free radical species.     

Enhancing Glutathione Synthesis can Decrease Zinc-Mediated Toxicity

Zinc toxicity has been linked to cellular glutathione: A decrease in glutathione is followed by an increase in zinc-mediated toxicity. The question arises whether an increase in glutathione synthesis might decrease zinc-mediated cytotoxicity. We incubated five cell lines (hepatoma and lung-derived) with zinc chloride and 2 mmol/l N-acetyl-l-cysteine (NAC) to support glutathione synthesis. In all but one hepatic cell line, the glutathione content was increased by NAC as compared to the d-enantiomere NADC, whereas NADC did not increase GSH content as compared to not treated controls. In both alveolar epithelial cell lines, an increase in zinc tolerance was observed due to NAC as compared to NADC. In native fibroblast-like and the hepatoma cell lines, no changes in zinc tolerance were found due to NAC. In the fibroblast-like cells, zinc tolerance was increased due to NAC only after cellular glutathione had been previously decreased (by lowered cysteine concentrations in the medium). Enhancing glutathione synthesis can antagonize zinc-mediated toxicity in the alveolar epithelial cell lines, whereas some other characteristics than glutathione synthesis might be more important in other cell types. Furthermore, NAC acted as a GSH precursor only at cysteine medium concentrations of 10 μmol/l or below and therefore might be described as a poor cysteine repletor for glutathione synthesis. This work is dedicated to Peter Eyer on the occasion of his 65th birthday.     

The Peptide Transporter PepT2 Mediates the Uptake of the Glutathione Precursor CysGly in Astroglia-Rich Primary Cultures

Abstract: The intracellular content of glutathione in astroglia-rich primary cultures derived from the brains of newborn rats was used as an indicator for the ability of these cultures to utilize cysteinylglycine (CysGly) for glutathione synthesis. After a 24-h starvation period in the absence of glucose and amino acids, CysGly was able to substitute for cysteine plus glycine in the restoration of glutathione. Glutathione restoration from CysGly plus glutamate was only slightly affected by the dipeptides carnosine or serylglycine in a 200-fold excess. Captopril, a substrate of the peptide transporter PepT1, had almost no effect on glutathione restoration. In contrast, with increasing concentrations of alanylalanine or cefadroxil, known substrates of the peptide transporter PepT2, the amount of glutathione restored in the presence of CysGly and glutamate was strongly reduced. Cefadroxil in a 200-fold excess totally prevented the utilization of CysGly for glutathione restoration. The presence of mRNA for PepT2 in astroglia-rich primary cultures was demonstrated by application of RT-PCR. These results demonstrate that PepT2 is expressed in astroglia-rich primary cultures and that this transporter is highly likely to be responsible for the uptake of CysGly in these cultures.     

Glutathione metabolism under the influence of hydroperoxides in the lactating mammary gland of the rat. Effect of glucose and extracellular ATP

Tert-butyl hydroperoxide decreases GSH and total free glutathione (GSH+2GSSG) contents of acini from lactating mammary glands. The decrease in total free glutathione can be explained by an increase in mixed disulfide formation and by excretion of GSS G to the extracellular medium, and subsequent degradation catalyzed by gamma-glutamyl transpeptidase. Low concentrations of glucose prevented the changes in glutathione levels induced by the peroxide. In the presence of extracellular ATP, glucose did not prevent these changes. However, incubations with the peroxide, did not alter the rate of other metabolic pathways by acini.Abbreviations used GSH Reduced glutathione - GSSG Glutathione disulfide - GSSR Glutathione mixed disulfide - GGT Gamma-glutamyl transpaptidase - tbOOH Tert-butyl hydroperoxide     

The Redox State of Glutathione,Cysteine and Homocysteine in the Extracellular Fluid in the Skin

Glutathione, the most abundant low-molecular weight thiol in the skin, has been shown to protect the skin from both photobiological and chemical injury. The thiols, glutathione in particular, have also been shown to be crucially involved in defence against contact allergens. Since the levels of extracellular thiol concentrations are important determinants of intracellular thiol status, we have compared the normal concentrations and the redox status of the main low-molecular weight thiol components in the extracellular fluid at the dermo-epidermal junction with the corresponding plasma levels. In their sulfhydryl form, all three thiols, i.e. glutathione, cysteine and homocysteine, were more abundant in experimental skin blister fluid than in plasma, as were the free disulfides of glutathione and homocysteine, whereas the free disulfides of cysteine were about the same in blister fluid and in plasma. Protein mixed disulfide levels were higher in plasma than in blister fluid. The present results provide information concerning the extracellular defence in the skin.     

The redox state of glutathione, cysteine and homocysteine in the extracellular fluid in the skin

Glutathione, the most abundant low-molecular weight thiol in the skin, has been shown to protect the skin from both photobiological and chemical injury. The thiols, glutathione in particular, have also been shown to be crucially involved in defence against contact allergens. Since the levels of extracellular thiol concentrations are important determinants of intracellular thiol status, we have compared the normal concentrations and the redox status of the main low-molecular weight thiol components in the extracellular fluid at the dermo-epidermal junction with the corresponding plasma levels. In their sulfhydryl form, all three thiols, i.e. glutathione, cysteine and homocysteine, were more abundant in experimental skin blister fluid than in plasma, as were the free disulfides of glutathione and homocysteine, whereas the free disulfides of cysteine were about the same in blister fluid and in plasma. Protein mixed disulfide levels were higher in plasma than in blister fluid. The present results provide information concerning the extracellular defence in the skin.     

Mercury uptake and removal by Euglena gracilis

The uptake and removal of mercury (added as HgCl2) from the culture medium by Euglena gracilis was studied. In cultures initiated in the light, cells accumulated a small fraction of the added heavy metal (5-13%). Mercury was both biologically and nonbiologically volatilized, and cell growth was partially inhibited; under these conditions the glutathione content was 3.2 nmol/10(6) cells. In contrast, in cultures initiated in the dark, mercury uptake by cells was two to three times higher, biological volatilization remained unchanged and nonbiological volatilization and growth were negligible; the glutathione content diminished to 1.4 nmol/10(6) cells. Biological mercury volatilization depended on cell density and metal concentration, but was light-independent. Thus, volatilization of mercury by Euglena appeared not to be an effective mechanism of resistance, whereas a high intracellular level of glutathione and a low mercury uptake seemed necessary for successful tolerance.     

Kinetics of uptake and deacetylation of N-acetylcysteine by human erythrocytes

Overproduction of reactive oxygen species associated with several diseases including sickle cell anaemia reduces the concentration of glutathione, a principal cellular antioxidant. Glutathione depletion in sickle erythrocytes increases their conversion to irreversible sickle cells that promote vaso-occlusion. Therapeutically, N-acetylcysteine partially restores glutathione concentrations but its mode of action is controversial. Following glutathione depletion, glutathione synthesis is limited by the supply of cysteine and it has been assumed that deacetylation of N-acetylcysteine within erythrocytes provides cysteine to accelerate glutathione production. To determine whether this is the case we studied the kinetics of transport and deacetylation of N-acetylcysteine. Uptake of N-acetylcysteine had a first order rate constant of 2.40+/-0.070min(-1) and only saturated above 10mM. Inhibition experiments showed that 56% of N-acetylcysteine transport was via the anion exchange protein. Deacetylation, measured using (1)H NMR, had a K(m) of 1.49+/-0.16mM and V(max) of 2.61+/-0.08micromolL(-1)min(-1). Oral doses of N-acetylcysteine increase glutathione concentrations in sickle erythrocytes at plasma N-acetylcysteine concentrations of approximately 10microM. At this concentration, calculated rates of N-acetylcysteine uptake and deacetylation were approximately 5% of the rate required to maintain normal glutathione production. We concluded that on oral administration, intracellular deacetylation of N-acetylcysteine supplies little of the cysteine required for accelerated glutathione production. Instead, N-acetylcysteine acts by freeing bound cysteine in the plasma that then enters the erythrocytes. To be effective, intracellular cysteine precursors must be designed to enter erythrocytes rapidly and employ enzymes with high activity within erythrocytes to liberate the cysteine.