Induction of Cystine and Glutamate Transport Activity in Human Fibroblasts by Diethyl Maleate and Other Electrophilic Agents*

The transport activity for cystine and glutamate in cultured human diploid fibroblasts is enhanced in re- sponse to diethyl maleate treatment. The enhancement is time- and dose-related, with a lag of about 3 h, and maximum enhancement (approximately %fold increase in the rate of uptake) is attained after 1 to 2 days of incubation of the cells with 0.1 mM diethyl maleate. The enhancement of the transport activity is accompanied by an increase in the V,,, and little change in the K,, and it requires RNA and protein synthesis. Other electrophilic agents, such as cyclohex- 2-en-1-one, ethacrynic acid, 1,2-epoxy-3-(p-nitro- phenoxy)propane, and sulfobromophthalein, similarly enhance the transport activity. These electrophiles are known as agents that interact with glutathione. For example, diethyl maleate at high concentrations, Le. 1 mM, depletes intracellular glutathione and injures the cells. However, at relatively low concentrations di- ethyl maleate and other electrophilic compounds do cause increases in the intracellular levels of glutathione which we attribute to the enhanced uptake of cys- tine. It is suggested that the transport system for cystine and glutamate is involved in a protective mecha- nism of cells against an electrophilic attack. The transport of amino acids across the mammalian cell membrane is performed by of mediation acting on discrete groups of substrate molecules (1). In a report, we showed


Induction of Cystine and Glutamate Transport Activity in Human Fibroblasts by Diethyl Maleate and Other Electrophilic Agents*
(Received for publication, June 3, 1983)

Shiro Bannai
From the Division of Biochemistry, Tsukuba University Medical School, Sakura-mura, Zbaraki 305 Japan The transport activity for cystine and glutamate in cultured human diploid fibroblasts is enhanced in response to diethyl maleate treatment. The enhancement is time-and dose-related, with a lag of about 3 h, and maximum enhancement (approximately %fold increase in the rate of uptake) is attained after 1 to 2 days of incubation of the cells with 0.1 m M diethyl maleate. The enhancement of the transport activity is accompanied by an increase in the V,,, and little change in the K,, and it requires RNA and protein synthesis. Other electrophilic agents, such as cyclohex-2-en-1-one, ethacrynic acid, 1,2-epoxy-3-(p-nitrophenoxy)propane, and sulfobromophthalein, similarly enhance the transport activity. These electrophiles are known as agents that interact with glutathione. For example, diethyl maleate at high concentrations, Le. 1 mM, depletes intracellular glutathione and injures the cells. However, at relatively low concentrations diethyl maleate and other electrophilic compounds do cause increases in the intracellular levels of glutathione which we attribute to the enhanced uptake of cystine. It is suggested that the transport system for cystine and glutamate is involved in a protective mechanism of cells against an electrophilic attack.
The transport of amino acids across the mammalian cell membrane is performed by specific systems of mediation acting on discrete groups of substrate molecules (1). In a previous report, we showed the uptake competition between cystine and glutamate in cultured human diploid fibroblasts and proposed a system responsible for the transport of cystine and glutamate in these cells (2, 3). There is considerable evidence to show that amino acid starvation causes an adaptive increase in the transport of selected neutral amino acids (4-6). The increase involves intrinsic alterations in transport systems and requires de novo protein and RNA synthesis. In the cystine-glutamate system the similar phenomenon, termed adaptation, has been observed (7). The substrate specificity of the cystine-glutamate system appears to be higher than the other systems that show adaptation, and presumably due to this specificity the system is adaptively enhanced by starvation only in cystine (7). Since cystine in itself is a low concentration amino acid within cells and a starvation in cystine causes a thorough depletion of intracellular glutathione, it is suggested that glutathione plays a significant role in the adaptive regulation of the cystineglutamate system.
Diethyl maleate bears an electrophilic center and reacts * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
with glutathione by direct conjugation and by interaction with the glutathione S-transferase system (8, 9). The compound is relatively nontoxic and is often used as a reagent to deplete intracellular glutathione (10,11). The present study examines the effect of diethyl maleate and analogous compounds on the cystine-glutamate system. The data presented indicate that the compounds caused an induction of the activity of the cystine-glutamate system. However, the concentrations of the compounds required for the induction were much lower than that required for depletion of glutathione. These compounds at relatively low concentrations enhanced the transport activity for cystine and glutamate without depleting glutathione. Cell Culture-The cells used in this study were human diploid fibroblasts derived from fetal lung (strain HAIN-6). They were cultured in Eagle's basal medium supplemented with 10% fetal calf serum. Cystine-free medium is similar to Eagle's basal medium except that it lacks cystine. One lot of serum containing no free cystine was used for the cystine-free medium. Vitamin E was dispersed in the cystine-free medium at 2 pg/ml with the aid of sonifier, in order to prevent death of the cells in the medium (12).

Materials-~-[3,3'-~H]Cystine
Cells were counted with a hemocytometer after they had been treated with trypsin. To determine cell viability, trypsinized cells were suspended in Hank's balanced salt solution, and then a suspension was mixed with 10 volumes of nigrosin solution (0.05% nigrosin in Hank's solution). After 10 min the total number of cells and the number of stained (not viable) ones were counted.
Uptake Method-Amino acid uptake was measured by techniques described previously (2). Following culture of the cells in a 35-mm diameter plastic Petri dish, the cells were rinsed three times in warmed 10 mM phosphate-buffered saline, pH 7.4. Then the cells were incubated in 0.5 ml of the warmed uptake medium for specified time periods at 37 "C. The uptake medium consisted of the same buffer used to rinse the cells plus labeled amino acid (1 pCilO.5 ml). The incubations were terminated by rapidly rinsing the dishes three times in ice-cold phosphate-buffered saline, and the radioactivity was determined as described before. The rates of uptake were determined under conditions approaching initial rates, i.e. by taking the values for the 2-min uptake of cystine or glutamate.
Determination of Glutathione-Cells in a 35-mm diameter dish were rinsed three times in 10 mM phosphate-buffered saline, and glutathione was extracted with 1 ml of 5% trichloroacetic acid. The acid extract was taken up and treated four times with 2 volumes of 0.01 N HC1-saturated diethyl ether. The resulting solution was used for the assay of total glutathione (reduced and oxidized glutathione), or it was incubated with 0.1 pmol of N-ethylmaleimide for 30 min followed by removal of excess N-ethylmaleimide by ether extraction as described above and used for the assay of oxidized glutathione. Glutathione was measured enzymatically by the method of Tietze (13).

Effect of Diethyl Maleate on the Cystine and Glutamate
Uptake-Diethyl maleate was added to the culture of human diploid fibroblasts, and, after various intervals of incubation, the cells were washed and assayed for the uptake of cystine and glutamate. The rates of uptake were determined by taking the values for the 2-min uptake. As shown in Fig. 1, the addition of 0.1 mM diethyl maleate resulted in increases in the rate of uptake of cystine and glutamate. The effect was time-dependent, with a lag of about 3 h. A maximum stimulation, i.e. 2 to 3 times that of control, was attained after a day or two. It is noted that the uptake medium does not contain diethyl maleate. Direct addition of diethyl maleate to the uptake medium had no effect on the uptake. Fig. 2 shows the effect of a short time exposure of the cells to diethyl maleate on the uptake of cystine. The cells were incubated with diethyl maleate for 1 or 3 h, and then the medium was replaced by that containing no diethyl maleate. When the cells were exposed to diethyl maleate for as long as 1 h, the stimulatory effect of diethyl maleate on the cystine uptake appeared only slight. When the cells were incubated with   diethyl maleate for 3 h, the uptake of cystine was enhanced for the following 6 h. Fig. 3 shows enhancement of cystine uptake of the cells incubated for 24 h with various concentrations of diethyl maleate. The uptake was significantly enhanced by diethyl maleate even a t 0.005 mM, and a maximum stimulation was  Cyclohex-2-en-1-one  Growth of the cells was somewhat inhibited by diethyl maleate at 0.05-0.1 mM (Fig. 4). However, during the first 24 h cell growth was little affected by diethyl maleate in this concentration range. Since the transport activity of cystine and glutamate is well enhanced by diethyl maleate within 24 h of incubation, it is unlikely that the enhanced activity results from changes in cell growth or cell density.

3-Methylcyclohex-2-en-1-one
Kinetic analyses of the cystine and glutamate uptake were performed to see if the increase in the rate of uptake was due to an increase in Vmax and/or due to changes in K,. Rates of cystine and glutamate uptake at various concentrations were measured and the double reciprocal plots are shown in Fig. 5. The plots for the cystine uptake were linear (Fig. 5A) at the cystine concentrations up to 0.5 mM, which was close to the solubility limit of cystine. From Fig. 5A, the V were not linear (Fig. 5B) suggesting the existence of at least two transport systems for glutamate, one with relatively low K,,, and another with very high K,. The former system appeared to be affected by diethyl maleate treatment, and thereby its apparent K, and V,,, were determined. From Fig. 5B, the Vmax values of 6.3 and 11.1 nmol of glutamate/min/mg of protein and K, values of 0.30 and 0.23 mM for glutamate were obtained for the cells in the normal and diethyl maleate-containing medium, respectively. The results show that the increase in the rate of cystine and glutamate uptake by diethyl maleate is due mainly to the increase in the V, , , .
The above results suggest an induction of cystine-glutamate transport activity by diethyl maleate. To examine a role of RNA and protein synthesis for the enhancement of cystine and glutamate uptake by diethyl maleate, actinomycin D or cycloheximide was added to the cells simultaneously with diethyl maleate, and after 24 h the rate of cystine and glutamate uptake was measured. Results in Table I show that both actinomycin D and cycloheximide completely blocked the increase in uptake by diethyl maleate. In these fibroblasts, actinomycin D at 0.1 pg/ml inhibited RNA synthesis by about 90%, which was measured by incorporation of labeled uridine into acid-insoluble material. Cycloheximide at 1 pg/ml inhibited protein synthesis, measured by incorporation of labeled leucine, by more than 80%.
The uptake of other amino acids was also examined to determine the specificity of the enhancement by diethyl maleate; there was little, if any, change in the rate of uptake of alanine, leucine, aspartate, and lysine by the cells incubated with 0.1 M diethyl maleate for 24 h (data not shown).
Effect of Other Electrophilic Agents on the Cystine Uptake-Various compounds were tested to see whether they enhanced the cellular uptake of cystine. The cells were incubated with the compound for 24 h and the rate of uptake of cystine was measured (Table 11). Cyclohex-2-en-l-one, 3-methylcyclohex-2-en-l-one, ethacrynate, maleate, ethyl cinnamate, and ethyl sorbate contain activated double bonds and have stimulated the cytine uptake. Cyclohex-2-en-1-one and its 3-methyl derivative were powerful stimulators, but the former was effective a t much lower concentration than the latter. Effect of maleate was manifested at 2 mM, whereas its diethyl ester exerted the similar effect a t about 0.02 mM (Fig. 3). Fumarate and maleic acid hydrazide, double bonds of which are less activated than those of maleate, did not affect the uptake of cystine. Cyclohexanone and diethylsuccinate are structural analogues of cyclohex-2-en-1-one and diethyl maleate, respec-  tively, except that they do not have unsaturated double bonds. These analogues did not enhance the cystine uptake. It is, therefore, suggested that the stimulatory effect of the compounds depends on the reactivity of their double bonds. Epoxy compounds, dichloronitrobenzene, p-nitrobenzyl chloride, and sulfobromophthalein are electrophilic compounds, and they have enhanced the cystine uptake. Aromatic compounds, such as acetamidophenol, acetylsalicylate, salicylate, and bromobenzene, have no electrophilic center and they have not stimulated the uptake. Phenobarbital is known to induce the synthesis of some specific proteins, but it did not enhance the cystine uptake. Some metabolic inhibitors were also examined. Potassium cyanide and an inhibitor for   y-glutamyltransferase, 6-diazo-5-oxo-L-norleucine, had little effect, while 2,4-dinitrophenol and sodium azide appeared depressive for the cystine uptake.
Changes in Glutathione Content-It may be reasonably assumed that the electrophilic agents described above principally interact with sulfhydryl groups of the cells, especially with glutathione, the major nonprotein sulfhydryl compound in the cells. Fig. 6A shows changes in the intracellular levels of glutathione when cells are exposed to 0.1 or 1 mM diethyl maleate. Diethyl maleate at 1 mM rapidly depleted cellular glutathione, and at 9 h after the addition of diethyl maleate, the cells started to die. Essentially no glutathione was detectable in the cells at 12 h, and no viable cells were found at 24 h. In contrast, when the cells were exposed to 0.1 mM diethyl maleate, the glutathione content, though decreased slightly for the first 3 h, increased and became twice as much as that of control cells after 24 h. Diethyl maleate caused the increase in glutathione over the wide concentration range (Fig. 6B).
In these experiments total glutathione (reduced and oxidized) was measured. In a typical case, both total and oxidized glutathione contents were separately determined as described under "Experimental Procedures." The percentage of oxidized glutathione to the total glutathione was 0.9% (+0.2%, average of three experiments) in the normal cells, and when the cells were treated with 0.05-0.1 mM diethyl maleate for 24 h, it was 0.6% (*@I%). Contents of sulfhydryl groups in these samples were also determined using 5,5'-dithiobis-(2-nitrobenzoic acid), and they were nearly equal to the contents of total glutathione. The results indicate that almost all glutathione measured is reduced-form glutathione and that the increase in glutathione by diethyl maleate is due to the increase in reduced-form glutathione, not in oxidized glutathione.
The effect of other electrophilic agents on the glutathione levels is described in Table 111. Generally, the increase in glutathione by these agents was dose-dependent in a manner similar to the enhancement of cystine uptake, although the increase in glutathione content did not completely correlate with the enhanced activity of cystine uptake. Cyclohex-2-en-1-one and its methyl derivative, which were potent inducers for cystine uptake, did not elevate so much the glutathione content.
It is likely that the increase in the glutathione content caused by some electrophilic agents results largely from the enhanced uptake of cystine because cystine in the culture medium is a major source of cellular cysteine which is a ratelimiting amino acid in glutathione synthesis.
To test this possibility, effect of inhibitors of the cystine uptake on the intracellular glutathione levels was examined. Glutamate and homocysteate are substrates for the cystine-glutamate transport system and, therefore, they competitively inhibit the uptake of cystine. These amino acids, when added to the culture medium along with diethyl maleate, completely blocked the increase in glutathione caused by diethyl maleate (Table IV). Under these conditions the cystine-glutamate system itself was enhanced by diethyl maleate to the same extent as that of the control (no extra amino acid added). However, the actual uptake of cystine into the cells was greatly reduced by the inhibitory action of glutamate or homocysteate (Table IV). Aspartate is not an inhibitor of cystineglutamate system, and it had little effect on the increase in glutathione by diethyl maleate. The results are consistent with the view that the increase in the cellular glutathione by diethyl maleate is attributable to the increased rate of uptake of cystine.
Effect of Diethyl Maleate and Starvation in Cystine on the Cystine Uptake-In a previous report we showed the enhancement of cystine-glutamate transport system by starvation with respect to cystine (7). This enhancement bears some resemblance to that by diethyl maleate shown here; both are time-dependent and require RNA and protein synthesis. A possible relation between the effects of diethyl maleate and of cystine starvation on the uptake of cystine was examined (Table V). Maximum enhancement of the cystine uptake by diethyl maleate alone was observed after 48-h incubation and at 0.1 mM (Figs. 1 and 3 and Table V). Similarly, stimulation of the cystine uptake by depletion of cystine reached a maximum after 48 h of cystine starvation (Table V and Ref. 7). When the two effects overlapped, i.e. when the cells were incubated in cystine-free medium containing diethyl maleate, the cystine uptake of these cells was much more enhanced the rate of the uptake exceeded that of the cells stimulated maximally by diethyl maleate or by cystine starvation separately, The effect of diethyl maleate and of starvation in cystine appeared partially additive.

DISCUSSION
Electrophilic compounds are believed to interact with sulfhydryl groups and used to deplete intracellular glutathione levels (9-11, 14, 15). However, the present results clearly showed that these agents, at relatively low concentrations, caused an increase in cellular glutathione due to the enhanced uptake of cystine. The results are unexpected but seem to reveal a protective mechanism of the cells against electrophilic agents. That is, when the cells are exposed to an agent such as diethyl maleate at relatively low concentrations, the activity of the cystine-glutamate transport is induced and the influx of cystine is enhanced resulting in a stimulation of glutathione synthesis. Glutathione thus enriched serves for a detoxification of the electrophilic agent. Evidence has already been shown that the glutathione levels in the human fibroblasts are controlled by the influx of cystine which is a source of intracellular cysteine, an amino acid rate-limiting to glutathione synthesis (16).
On the other hand, the electrophilic compounds deplete intracellular glutathione when they are added in excess. In this case the reaction between the electrophilic compound and glutathione is inevitably accelerated by a large quantity of the compound, while the induction of cystine uptake seems to be diminished. The electrophilic compound appears, therefore, to act in two different ways, as an inducer of the cystine uptake and as a nonspecific deleterious agent. At low concentrations the former action is manifested, whereas the latter predominates at high concentrations. Presumably the electrophilic agent in excess reacts with various cellular components and thereby inhibits a variety of cellular activities including the induction of cystine uptake.
The cystine-glutamate transport system is enhanced not only by the electrophilic agents but also by the starvation with respect to cystine (7). In both cases the actual synthesis of protein essential to the transport process seems to be stimulated. The mechanism by which the synthesis of the transport protein is stimulated is totally unknown. However, in the amino acid transport system that shows an adaptive enhancement (system A), it may be reasonably assumed that a repressor is involved in the synthesis of the transport protein at the transcriptional level (6). The cystine-glutamate system also shows an adaptive enhancement (7) and may be regulated at the transcriptional level. The cellular glutathione contents are severely decreased by the starvation in cystine, whereas they are rather enriched by the electrophilic agents. Therefore, the depletion of glutathione is not uniformly a signal for the enhancement of the cystine-glutamate system. It seems of importance to search for something in common in effects of the electrophilic agents and the starvation in cystine.
The cystine-glutamate transport system is first found in human fibroblasts (2) and then in rat hepatoma cell line (17) and rat hepatocytes in primary culture (18). Whether the activity of the system in hepatic cells is induced by electrophilic agents is of interest and now under study.