]>MAD2077S0047-6374(98)00042-610.1016/S0047-6374(98)00042-6Elsevier Science Ireland LtdFig. 1The inhibition kinetics of DNP-SG transport elicited by various inhibitors and effects of increasing concentrations of inhibitors on CumOOH-induced MDA formation in erythrocytes. Suspensions of erythrocytes from aged (n=5) or young (n=5) subjects were treated with increasing concentrations of Tween 80 (A), fluoride (B) or vanadate (C) as described in Section 2. After centrifugation the supernatants were assayed for DNP-SG transport. MDA levels of erythrocytes treated with 0.1 mmol/l CumOOH after pretreatment with inhibitors were also determined. Percent inhibition was expressed as the percentage decrease of DNP-SG efflux from erythrocytes. The values are means of duplicate analyses from 10 experiments.Fig. 2Effects of various inhibitors of the glutathione conjugate pump on CumOOH-induced lipid peroxidation in erythrocytes from healthy aging (n=18) and young (n=18) adults. DNP-SG inhibitors, Tween 80 (1/1200), fluoride (30 mmol/l) or o-vanadate (0.2 mmol/l) were added before the erythrocyte suspensions were incubated with 0.1 mmol/l CumOOH for 15 min at 37°C. MDA values shown are means with standard deviations.Fig. 3(See left) Effects of the addition of CumOOH to the reaction medium following preincubation with various inhibitors of glutathione conjugate pump on the changes in chemiluminescence. (A) Chemiluminescence response of erythrocytes treated with 0.1 mmol/l CumOOH following the inhibition of DNP-SG transport of erythrocytes from aging group. (B) Comparison of the chemiluminescence response in erythrocytes from aging and young adults. DNP-SG transport inhibitors, Tween 80, fluoride or o-vanadate were added to erythrocyte suspensions before mixing with 0.1 mmol/l CumOOH and chemiluminescence was recorded at indicated time. Data represent the mean of 16 determinations in the elderly group and 15 determinations in the young group.Table 1Transport of 2,4-dinitrophenyl-S-glutathione in erythrocytes from aging and young subjectsTime (h)Erythrocyte efflux (nmol DNP-SG/ml erythrocytes)Aging group (n=18)Young group (n=18)1410±77390±422685±117674±713971±128933±11441174±1651118±157All data expressed as mean±S.D.Transport of glutathione conjugate in erythrocytes from aged subjects and susceptibility to oxidative stress following inhibition of the glutathione S-conjugate pumpİlhanOnarana*AhmetÖzaydinaMustafaGültepebGönülSultuybekaaDivision of Biomedical Sciences, Cerrahpaşa Medical Faculty, Istanbul University, Istanbul, TurkeybGATA, Military Educational Hospital, Department of Biochemistry, Istanbul, Turkey*Corresponding author. Present address: Ortaklar Cd. Bütan Sk., Hyzal Apt. No:2 D:3, Mecidiyeköy 80290, İstanbul, Turkey. Fax: +90 212 5299433.AbstractThe aim of the present study was to investigate the effect of donor aging on the glutathione conjugate transport in erythrocytes and whether it plays a role in the resistance to oxidative stress of the erythrocytes of aging subjects. In our comparative study on intact erythrocytes of healthy aging and young adults, in which 2,4-dinitrophenyl-S-glutathione (DNP-SG) was used as model glutathione S-conjugate, we found that the efflux of DNP-SG remained unchanged in the aged subjects. This result suggests that the detoxification function is maintained against the chemical stress employed in erythrocytes of aging subjects. In the assay conditions used, which were optimized to obtain maximal inhibition of glutathione S-conjugate transport, our results also indicated that the susceptibility of erythrocytes to in vitro lipid peroxidation generated by cumene hydroperoxide was enhanced by pretreatment with DNP-SG inhibitors in both age groups. However, the difference in susceptibility was not a function of aging. Further, the results suggested that inhibition of glutathione S-conjugate pump may impair cellular protection of the erythrocytes against oxidative damage.KeywordsAgingErythrocyteGlutathione S-conjugateGlutathioneTransportOxidative stress1IntroductionTransport of glutathione S-conjugates is an important element of xenobiotic detoxification. Glutathione is enzymatically conjugated to many electrophilic compounds in a reaction catalyzed by glutathione S-transferases (GST). The glutathione S-conjugates formed in these reactions are afterwards exported out of cells by a specific adenosine 5′-triphosphate (ATP)-dependent glutathione S-conjugate pump (for review see Zimniak and Awasthi, 1993). The active transport systems for glutathione S-conjugates have been described for a number of human tissues and this system has also been identified in human erythrocyte membranes (Awasthi et al. 1983, Ishikawa and Sies 1984, La Belle et al. 1986, Kunst et al. 1989). The glutathione S-conjugate pump also plays a significant role in cellular defence under oxidative stress conditions since it is able to transport glutathione conjugates of lipid peroxidation products and extrudes oxidized glutathione (GSSG) (Ishikawa 1989, Grune et al. 1991, Akerboom et al. 1992). Erythrocytes are a good model system for the explanation of such processes and the molecular nature of the defect in the transport of glutathione conjugates since their lack of γ-glutamyltranspeptidase precludes further metabolizing of formed conjugates. Although the transport system exhibited a broad substrate specificity towards different types of glutathione S-conjugate (Ishikawa, 1989), the compound most widely used in such studies is 2,4-dinitrophenyl-S-glutathione (DNP-SG) formed from 1-chloro-2,4-dinitrobenzene (CDNB) in a reaction catalyzed by GST.Different detoxification mechanisms such as glutathione, GST, glutathione reductase, glutathione peroxidase, superoxide dismutase and catalase but not glutathione S-conjugate transport have been tested for aging process (Stohs et al. 1984, Jozwiak and Jasnowska 1985, Stohs and Lawson 1986, Al-Turk et al. 1987, Farooqui et al. 1987, Foldes et al. 1988, Matsubara and Machado 1991, Picot et al. 1992). Results from a number of studies have suggested that a glutathione-deficiency state is a general phenomenon in aging cells, since aging-specific decrease in glutathione levels was observed in all tissues including erythrocytes (Stohs et al. 1984, Stohs and Lawson 1986, Al-Turk et al. 1987, Farooqui et al. 1987, Matsubara and Machado 1991). Furthermore, different results showing decreased or unchanged GST activity in elderly subjects when compared with young subjects were also reported (Stohs et al. 1984, Al-Turk et al. 1987, Picot et al. 1992). On the other hand, results of our previous study indicated that the susceptibility of intact erythrocytes to in vitro oxidative stress by cumene hydroperoxide (CumOOH) in aging subjects is not much greater than young subjects (Onaran et al., 1997). The above observations prompted us to study how the transport of glutathione conjugate in erythrocytes, generated through the reactions of glutathione and GST, is affected by the aging process. We also wished to examine whether glutathione S-conjugate transport has any effect on the resistance to oxidative damage of the intact erythrocytes of aging subjects.2Materials and methods2.1SubjectsSubjects were young (aged 18–31) or elderly (aged 67–85) normal volunteers. All subjects were non-smokers and had normal blood counts, normal blood levels of urea, glucose, creatinine, albumin, alkaline phosphatase, lactate dehydrogenase and bilirubin, and no history of hematologic abnormality, recent infectious disease or significant medical illness. The participants were instructed not to take aspirin-like drugs for 2 weeks prior to blood sampling.2.2ReagentsAll reagents were of analytical quality whenever possible, obtained mainly from Sigma (St. Louis, MO) and Merck (Darmstadt, Germany).2.3Preparation of erythrocytesFresh heparinized fasting blood sample was centrifuged at 1500×g for 10 min. Erythrocytes were separated from plasma and buffy coat and then washed three times with 10 vol of phosphate buffered saline (PBS, pH 7.4). Erythrocytes containing chronologically young population rich with reticulocytes were removed by discontinuous Percoll density gradient essentially as described by Rennie et al. (1979). The gradient was built up in two layers containing Percoll with specific density values between 1.100 and 1.124 g/ml. At the end of centrifugation while the cells were collected at the interface of the Percoll above 1.124 g/ml, enriched fraction of reticulocytes above low density were removed. The erythrocytes were then washed twice with PBS.Hemoglobin (Hb) concentrations in the samples were determined with Drabkin's reagent as described by Beutler (1984). Reticulocyte count in the fraction was performed on glass slides after staining with 0.1% brilliant cresyl blue saline. The number of reticulocytes in the high density cell fractions studied did not show significant differences between elderly and young control groups.2.4Reaction systemsFor inhibition of glutathione conjugate transport, erythrocytes were incubated at hematocrit 10% in PBS (with 8 mmol/l glucose) containing one of the following reagents (15 min at 37°C with continuous shaking at 120 cycles/min): 5–30 mmol/l sodium fluoride; 0.025–0.2 mmol/l sodium o-vanadate; diluted 1/4000–1/1200 Tween 80 (polyoxyethylenesorbitan-monooleate). Controls were incubated with PBS alone. To check whether the glutathione conjugate transport was inhibited in erythrocyte suspensions the export of DNP-SG was measured. Hemolysis was also checked in samples and it was always below 1–2%. After preincubation with inhibitors, lipid peroxidation was induced by addition of freshly prepared CumOOH (final concentration, 0.1 mmol/l). Immediately after CumOOH addition, the tubes were sealed and incubated at 37°C with continuous shaking at 120 cycles/min. In parallel, control samples were incubated under the same conditions but without CumOOH. After 15 min, cell suspensions were centrifuged at 7000×g for 1 min to sediment the erythrocytes. Both pellet and supernatant were used for malondialdehyde (MDA) determination.2.5Measurement of the transport of glutathione S-conjugateTransport of DNP-SG was measured according to the procedure of Board (1981). Washed erythrocytes were resuspended in PBS and incubated with 1 mmol/l CDNB for 15 min at 37°C to form DNP-SG. Then the cells were washed of excess CDNB at 4°C and suspended at a hematocrit of 20% in the PBS (pH 7.4) containing 1 mmol/l MgCl2 and 8 mmol/l glucose. The cell suspensions were then incubated at 37°C and the export of DNP-SG was quantified by withdrawal of aliquots of the cell suspensions after 1, 2, 3, and 4 h. After centrifugation, DNP-SG was detected in the supernatant by spectrophotometric determination at 340 nm using an mmol/l extinction coefficient of 9.6.2.6Malondialdehyde (MDA) measurementThe concentration of MDA in the samples was measured with fluorometric determination improved by synchronous fluorescence (Conti et al. 1991, Onaran et al. 1997). For erythrocytes, 0.2 ml of packed cells were suspended in 0.8 ml PBS, 0.025 ml of 4% (w/v) butylated hydroxytoluene and 0.050 ml of 0.025 mmol/l disodium-ethylenediaminetetraacetic acid, and 0.5 ml of 30% trichloroacetic acid was then added. Tubes were vortexed and allowed to stand in ice for 2 h. After centrifugation, assay conditions were the same in both the supernatant from this step and the supernatant fraction from the erythrocyte suspension (Onaran et al., 1997). The level of MDA was expressed as total mmol MDA/g Hb.2.7Chemiluminescence assayChemiluminescence measurements were carried out in a TriCarb 1500 liquid scintillation analyzer in the single-photon count mode (Videla et al., 1984). Briefly, erythrocytes were resuspended in 3 ml of PBS containing 8 mmol/l glucose and preincubated with indicated concentrations of fluoride, vanadate or Tween 80 for 15 min at 37°C. The erythrocytes were transferred to scintillation vials. After the addition of CumOOH (final concentration, 0.1 mmol/l) the chemiluminescence measurements were taken every 10 min for 60 min. The zero time is the time of CumOOH addition.2.8Glutathione content and glutathione S-transferase (GST) activityThe concentration of glutathione was determined by the method of Tietze (1969). Total GST activity with CDNB as substrate was measured according to Habig et al. (1974).2.9Statistical analysisAll tests were performed in duplicate or triplicate samples, and the results were expressed as means±S.D. Statistical difference was determined by Student's t-test, with significance defined as P<0.05.3ResultsThe aging group (n=18) and young controls (n=18) used in this study showed no difference in their erythrocyte GST activity, 2.1±0.8 and 2.1±0.6 mmol of CDNB conjugated/min per g Hb, respectively (P>0.05). However, we found statistically significant lower glutathione levels in older group. The mean±S.D. values of glutathione were 5.24±0.69 and 6.59±0.71 mmol/g Hb for the elderly and controls, respectively (P<0.05).On incubation with CDNB at 37°C, glutathione is completely depleted in erythrocytes of the two age groups. Under these experimental conditions, the rate of DNP-SG efflux of the erythrocytes from aging subjects during 4 h did not differ from those of the young controls (P>0.05). The rate of transport of DNP-SG in both age groups was linear for a period of at least 4 h (Table 1).In order to find out whether glutathione S-conjugate transport has any effect on the resistance to oxidative stress of the erythrocytes of aging subjects, transport of erythrocytes was inhibited by incubation with various inhibitors, including sodium fluoride, vanadate or Tween 80. The transport process was, more markedly, inhibited as a function of the concentration of these inhibitors. Despite the differences in inhibition profiles of the transport rate of DNP-SG elicited by these agents, the inhibition kinetics of erythrocytes from aged subjects were not different from that of young control subjects. When the erythrocytes were exposed to 0.1 mmol/l CumOOH after preincubation with inhibitors, increasing relative concentration of inhibitors resulted in an enhanced MDA formation. The production of MDA elicited by CumOOH corresponded generally to the degree of inhibition in DNP-SG transport caused by different inhibitors. As shown in Fig. 1, significant differences in MDA formation as a function of the concentrations of inhibitors were not observed in erythrocytes of aged subjects in comparison to that of young subjects. Therefore, the concentrations corresponding to 30 mmol/l fluoride, 0.2 mmol/l vanadate and 1/1200 Tween 80 which were effectively able to inhibit the DNP-SG transport were selected for further experiments. In these experiments, MDA levels of erythrocytes treated with inhibitors of DNP-SG transport were not significantly different from the levels of erythrocytes treated with CumOOH alone (Fig. 2). MDA levels also remained unchanged within each group when compared to basal conditions (data not shown). However, MDA levels of erythrocytes treated with CumOOH after preincubation with inhibitors were significantly different from those treated with CumOOH without the inhibitors (P<0.05) (about 1.6–2-fold increase over controls with CumOOH alone). Also in this case, no differences in MDA overproduction were observed between the aging and the young groups, i.e., the ratios between values of the samples treated with CumOOH alone and values of the samples treated with CumOOH after pretreatment with inhibitors were statistically unchanged (data not shown). The MDA production in erythrocytes which were kept under oxidative stress was very similar for Tween 80 and vanadate. Inhibition by fluoride caused slightly less MDA production than other inhibitors (Fig. 2).Chemiluminescence intensity of erythrocytes treated with inhibitors of DNP-SG transport were not significantly different from the intensity of erythrocytes treated with CumOOH alone or from those in their basal conditions (Fig. 3A); during 60 min, light output in erythrocytes remained constant. Addition of 0.1 mmol/l CumOOH to erythrocytes pretreated with inhibitors enhances chemiluminescence signal in erythrocytes of both age groups. However, no differences in spectral distributions of chemiluminescence were observed between the aging and the young groups (Fig. 3B), i.e., the ratios of increase in chemiluminescence between treated and control (erythrocytes treated with CumOOH alone) values were statistically unchanged (data not shown). In addition, in the erythrocytes treated with Tween 80 or vanadate, onset and maximal chemiluminescence were slightly higher than those of fluoride.4DiscussionIn the present study, we employed human erythrocytes to investigate the effect of aging on the glutathione S-conjugate transport by determining the DNP-SG transport. We used erythrocytes other than the chronologically young population rich with reticulocytes, the reasons for which were explained in our previous article (Onaran et al., 1997). In experimental conditions, in which erythrocyte glutathione is completely depleted by incubating the erythrocytes with CDNB, we did not observed a significant difference in the efflux of DNP-SG in erythrocytes from aged subjects when compared with young subjects. The result indicates that the detoxification function is maintained against the chemical stress employed in their erthyrocytes.It is known that the cells transport the glutathione S-conjugates outward through ATP-dependent efflux process. The transport of glutathione S-conjugate in erythrocytes was first reported by Board (1981)and it was shown that the addition of CDNB to erythrocytes results in the efflux of DNP-SG with rapid irreversible depletion of glutathione. The transport of this conjugate is inhibited by the depletion of intracellular ATP or by known organic anion transport inhibitors such as vanadate, an inhibitor of P type ATPases (La Belle et al., 1986), and fluoride (interaction with the transporter molecule versus cellular energy depletion) (Awasthi et al., 1983). We also observed pronounced inhibition of DNP-SG transport by these inhibitors.Although the effects of aging on different detoxification mechanisms such as glutathione and GST in erythrocytes have been reported (Stohs et al. 1984, Jozwiak and Jasnowska 1985, Stohs and Lawson 1986, Al-Turk et al. 1987, Foldes et al. 1988, Matsubara and Machado 1991, Picot et al. 1992), to our knowledge no data on the effect of donor age on transport of glutathione conjugates of membrane is available. It has been shown that there is an age-related decrease in glutathione content of the erythrocytes (Stohs et al. 1984, Al-Turk et al. 1987, Matsubara and Machado 1991). However, some evidence pointed out that glutathione status, physical health and longevity of life were closely interrelated (Foldes et al. 1988, Calvin et al. 1992). Furthermore contradictory results showing decreased or unchanged GST activity in erythrocytes with donor aging were also reported (Stohs et al. 1984, Al-Turk et al. 1987, Picot et al. 1992). In addition, it has been shown that changes in glutathione concentration in mammalian cells have various effects, influencing cellular radiosensitivity and the cytotoxicity of several chemotherapeutic agents (Bump et al. 1982, Li and Kaminskas 1984, Fernandes and Cotter 1994). In the aging group that provided erythrocytes for our study, glutathione levels were lower for about 20% and GST activities remained unchanged. The present results thus reveal that cellular glutathione status of the aging subject has no adverse effect on glutathione S-conjugate transport and the present amount of glutathione in their erythrocytes might be sufficient to detoxify the employed chemical stress. However, we do not know how the transport of glutathione S-conjugate is affected by different levels of intracellular glutathione.It has been previously reported that transport systems for glutathione S-conjugates play significant roles in the biotransformation of toxic products arising from oxidative stress induced lipid peroxidation (Ishikawa et al. 1986, Grune et al. 1991, Akerboom et al. 1992). On the other hand, results of our previous study indicated that the intact erythrocytes from aged subjects were not more susceptible to oxidative stress than those of young subjects (Onaran et al., 1997). In the second part of our study we therefore decided to determine how glutathione S-conjugate transport plays a role in the resistance to oxidative stress of the erythrocytes of the aged subjects.To investigate this, DNP-SG transport was inhibited by various agents, i.e., fluoride, vanadate or Tween 80. These agents were used for inhibitory action in the studied system as their diverse effects on glutathione conjugate pump have been explained previously (Akerboom et al. 1992, Pulaski and Bartosz 1995). In preliminary studies we observed that under experimental conditions employed the concentrations of the inhibitors did not affect the glutathione and MDA contents, chemiluminescence formation, rate of hemolysis and GST activities in the individual's erythrocytes. The erythrocytes were then treated with 0.1 mmol/l CumOOH in order to induce oxidative stress and then MDA levels, a common end product of oxidative damage, were measured to asses whether the inhibition of glutathione transport is subjected to increased oxidative damage in erythrocytes. Analyses of the effects of inhibitors on DNP-SG transport as a function of the inhibitor concentrations under the in vitro standard oxidative stress conditions, indicate that increasing the relative concentrations of inhibitors to erythrocytes resulted in an enhanced MDA formation and that enhancement of sensitivity caused by each of the concentrations used was unchanged as a function of aging. In addition, the degree of the DNP-SG transport inhibition for each of inhibitors used was very similar in young and aged subjects. Because of these results it was decided that further experiments could be performed in the inhibition conditions which were able effectively to prevent the conjugate transport, in order to obtain an insight into the effects of glutathione S-conjugate transport on the resistance to oxidative stress. At this stage of the study the extent of oxidative damage was evaluated by measuring the chemiluminescence formation as well as MDA formation. The present study reports that following stimulation by CumOOH after the inhibition of DNP-SG, peroxidation- dependent changes of MDA and chemiluminescence formation took place at the same level in aging and young adults. This indicates that the erythrocytes from the elderly are equally capable of withstanding the oxidative stress following the inhibition of glutathione conjugate pump and oxidative effect of lipid peroxidation is not much greater in aging individuals with diminished glutathione levels but normal GST activity than young adults. Furthermore, these results also indicate that the erythrocytes with inhibited DNP-SG transport were shown to be more susceptible to oxidative stress generated by CumOOH than those which were not inhibited. We concluded, therefore, that this result indicates that inhibition of glutathione conjugate transport may impair cellular protection to oxidant stress. Although we are at present not able to explain the mechanisms governing these changes, several possible explanations of the effects observed may be considered. As inhibitory effects of glutathione S-conjugates on GSTs as well as glutathione reductase (Ishikawa et al. 1986, Ishikawa 1989) and the transport of GSSG in various tissues including erythrocytes (Bilzer et al. 1984, Akerboom et al. 1991) have been reported (Akerboom et al. 1982, 1991, Bilzer et al. 1984, Ishikawa et al. 1986, Ishikawa 1989), accumulation of the S-conjugates resulting from radical induced lipid peroxidation in the cell may be crucial for detectable oxidative damage. It is known that GST and glutathione reductase reduce organic hydroperoxides such as CumOOH (Prohaska and Ganther 1977, Awasthi et al. 1980), and it was also shown that the transport of GSSG is an important process for the cell to avoid highly oxidative stress (Adams et al., 1983). 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