Oxidative stress induces a reversible flux of cysteine from tissues to blood in vivo in the rat Daniela Giustarini 1 , Isabella Dalle-Donne 2 , Aldo Milzani 2 and Ranieri Rossi 1 1 Department of Evolutionary Biology, Laboratory of Pharmacology and Toxicology, University of Siena, Italy 2 Department of Biology, University of Milan, Italy Introduction Cysteine (Cys) and glutathione (GSH) are the most abundant low-molecular-mass thiols (LMM-SH), with GSH predominating intracellularly and cysteine predominating in extracellular fluids [1,2]. These two compounds are metabolically inter-related, and GSH, in particular, determines the redox state and represents a defense against damage mediated by reactive oxygen species (ROS) or reactive nitrogen species. Low GSH concentrations and a ratio of high gluta- thione disulfide (GSSG) to GSH have been measured in red blood cells (RBCs) of patients with various diseases, including cancer, coronary heart surgery, pre-eclampsia, and genitourinary, gastrointestinal, car- diovascular and musculoskeletal diseases [3–9]. Extra- cellular fluids such as plasma are characterized by a lower thiol : disulfide ratio in comparison to the intra- cellular environment, and many pathophysiological conditions has been shown to markedly influence both thiol and disulfide concentrations [1,10–13]. GSH is synthesized intracellularly from its constituent amino acids, cysteine, glutamate and glycine, by a two- step reaction catalyzed by the enzymes c-glutamylcyste- ine synthetase and GSH synthetase. The supply of Cys is directly related to the rate of GSH synthesis, because the cellular cysteine concentration is a key determinant in regulating the kinetics of the first reaction (i.e. the formation of c-glutamylcysteine), which represents the rate-limiting step in GSH synthesis [14]. Cysteine is rap- idly auto-oxidized to cystine (CySS) in the extracellular fluids; CySS may have a considerable physiological role as a source of Cys, as, once it has entered a different cell, it can be reduced again by GSH [15]. Keywords cysteine; diamide; glutathione; oxidative stress; thiols Correspondence R. Rossi, Department of Evolutionary Biology, Laboratory of Pharmacology and Toxicology, University of Siena, via A. Moro 4, I-53100 Siena, Italy Fax: +39 577 234476 Tel: +39 577 234198 E-mail: ranieri@unisi.it (Received 11 May 2009, revised 30 June 2009, accepted 3 July 2009) doi:10.1111/j.1742-4658.2009.07197.x Glutathione (GSH) plays a key role in defense against oxidative stress. The availability of GSH is ensured in tissues by systems devoted to its mainte- nance in the reduced state and by the flux of GSH and cysteine between sites of biosynthesis and sites of utilization. Little is known about the effect of oxidative stress on the distribution of low-molecular-mass thiols and their exchange rate between tissues. In this study, we found that a slow infusion of diamide (a specific thiol-oxidizing compound) evoked a dra- matic increase in blood cysteine in rats. Our data suggest that inter-organ exchange of cysteine occurs, that cysteine derives from both glutathione via c-glutamyl transpeptidase and methionine via homocysteine and the trans- sulfuration pathway, and that these pathways are considerably influenced by oxidative stress. Abbreviations CySS, cystine; CysGly, cysteinylglycine; CySSGly, cystinylglycine; GSH, glutathione; GSSG, glutathione disulfide; GSSP, mixed disulfides between protein sulfhydryl groups and glutathione; c-GT, c-glutamyl transpeptidase; Hcys, homocysteine; HcySS, homocystine; LMM-SH, low-molecular-mass thiols; mBrB, monobromobimane; NEM, N-ethylmaleimide; RBC, red blood cell; ROS, reactive oxygen species; RSSP, mixed disulfides between protein sulfhydryl groups and a low-molecular-mass thiol; TSP, trans-sulfuration pathway. 4946 FEBS Journal 276 (2009) 4946–4958 ª 2009 The Authors Journal compilation ª 2009 FEBS A continuous flux of GSH and Cys exists between various tissues, and diet, starvation and pathologies that affect various organs may influence this phenome- non [2,16,17]. Many pathological conditions are known or thought to elicit oxidative stress, and it is recog- nized that this may have a role in the progression of the disease (for review, see [18]). Usually, the occur- rence of oxidative stress is evaluated by measuring sev- eral biomarkers, but little is known about the influence of oxidative stress on cysteine and glutathione exchange among tissues. Indeed, the factors that main- tain the redox homeostasis of plasma thiols as well as the mechanisms that regulate the exchange of LMM- SH among cells need to be clarified. Here, we have analyzed the effect of the administra- tion of azodicarboxylic acid bis-dimethylamide (dia- mide), a thiol-specific oxidizing substance, on the blood and tissue distribution of thiols and disulfides in rats. Diamide is a mild oxidizing compound that easily penetrates cell membranes and reacts quickly and spe- cifically with intracellular thiols, both LMM-SH and protein sulfhydryls [19,20], without producing free rad- icals or other ROS, as indicated by the lack of increase in lipid peroxidation observed in in vitro and in vivo experiments [21]. In previous studies [22,23], we observed that a single intraperitoneal administration of diamide in rats induced selective oxidation of blood glutathione, with no evident alterations in thiol levels in other tissues. Moreover, during diamide treatment, Cys levels increased in plasma, indicating that Cys was released from an extra-hematic compartment. As in vivo studies based on treatments with diamide are essentially limited by its reaction with thiol groups within a few seconds [24], in this paper we used slow intravenous infusion of the drug. Additionally, we monitored all physiological LMM-SH, namely Cys, cysteinylglycine (CysGly), homocysteine (Hcys), GSH and related disulfide forms in both blood and organs. Disulfide forms comprise low-molecular-mass disulfides [LMM-SS: CySS, cystinylglycine (CySSGly), homocys- tine (HcySS) and GSSG] and mixed disulfides between protein sulfhydryl groups and a low-molecular-mass thiol (RSSP). This allowed us to study relatively long- lasting effects of thiol-directed oxidative stress, and, in particular, its influence on the inter-organ exchange of LMM-SH. Results In a first set of experiments, the kinetics of plasma thiols during and after diamide administration was evaluated. Rats were treated for 60 min and monitored for the next 2 h. Specifically, we measured the time course of levels of GSH, Cys, CysGly and Hcys and the corresponding disulfide forms calculated as the sum of LMM-SS and RSSP (Fig. 1A–D). Values for total disulfides are expressed as ‘thiol equivalents’, i.e. the concentration of LMM-SH that are involved in formation of both LMM-SS and RSSP. The level of Cys significantly decreased by  20% during treatment with diamide, and then returned slowly to initial values. A parallel increase, particularly evident for disulfides of cysteine, followed by a decrease in the disulfide forms of both glutathione and cysteine, was also observed, with a tendency (particularly for GSH) to return to basal levels (Fig. 1A,B). Disulfides of homocysteine tended to increase during the diamide infusion and remained higher than the basal concentra- tion during the whole experiment (Fig. 1C). Con- versely, the CysGly redox balance was minimally influenced by treatment with diamide (Fig. 1D). When considering the amount of total thiols (i.e. reduced + disulfide forms) found in plasma at each time point during the whole course of the experiment (Fig. 2), we observed that the levels of total cysteine (and also total homocysteine, although to a smaller extent) were markedly increased during and after diamide infusion with respect to time zero. The same samples were also analyzed for the LMM- SH, LMM-SS and RSSP content in RBCs. These cells represent the main stores of hematic GSH, which occurs mostly (> 99%) in the reduced form [25], whereas Cys and other LMM-SH represent only a minimal fraction [26]. GSH was rapidly oxidized in RBCs mainly to mixed disulfides with proteins (GSSP) and recovered  80% of its initial value within 2 h of the end of treatment (Fig. 3B). Cys levels decreased during the first part of the treatment (10–30 min), but the prevailing phenomenon was a dramatic reversible increase in both the reduced and, in particular, the disulfide forms, mainly mixed disulfides with proteins (CySSP) (Fig. 3A). The highest value reached for CyS- SP was  2.5 nmol per mg Hb (i.e. 0.75 mm), and was measured at the end of treatment with diamide. This suggests a large, reversible flux of cysteine from other tissues towards RBCs. Figure 3C shows the change in total cysteine and total glutathione in RBCs with respect to their basal levels. It is evident that the levels of total cysteine, but not total GSH, greatly and reversibly increased over basal levels after oxidative stress induction by diamide. The change in total cyste- ine concentration in both plasma and RBCs during diamide treatment and over the subsequent 2 h is sum- marized in Fig. 4. Values were normalized for the hematocrit, and the level of total cysteine greatly increased in plasma and RBCs, reaching a maximum D. Giustarini et al. Cysteine flux under oxidative stress FEBS Journal 276 (2009) 4946–4958 ª 2009 The Authors Journal compilation ª 2009 FEBS 4947 level 75 min after the start of diamide infusion. It is evident that the cysteine that enters RBCs cannot derive solely from plasma stores. We also monitored the concentrations of thiols and disulfides in blood withdrawn from rats at 75 min after the beginning of diamide infusion (when total cysteine reached its maxi- mum observed levels): both plasma and RBCs were analyzed ex vivo over time. The observed concentra- tion changes of GSH, Cys and their oxidized forms in RBCs were similar to those observed in vivo, with thi- ols and disulfides recovering their basal levels within  2 h after blood collection (data not shown). The plasma Cys levels increased slightly over time, but more evident was the increase in CySS and CySSP, AB CD Fig. 1. Thiols and disulfides in rat plasma during and after diamide infusion. Time course of plasma LMM-SH (closed circles) and total disulfide (open circles) levels during and after diamide infusion (7.73 lmolÆmin )1 Ækg )1 intravenously). (A) Cysteine; (B) glutathione; (C) homocysteine; (D) cysteinylglycine. Disulfides represent the sum of low-molecular-mass disulfides and protein mixed disulfides; values are expressed as thiol equivalents. Data are the means ± SD of four experiments. *P < 0.05 versus value at time zero; **P < 0.01 versus value at time zero. Cysteine flux under oxidative stress D. Giustarini et al. 4948 FEBS Journal 276 (2009) 4946–4958 ª 2009 The Authors Journal compilation ª 2009 FEBS probably due to oxidation of cysteine exported from RBCs (Fig. 5). Little GSH was exported during this ex vivo experiment, and the concentrations of total GSH were measured within RBCs were constant (data not shown). The change in total cysteine levels was found to be dose-dependent. In experiments performed with various doses of diamide (dose range 1.93–9.66 lmolÆ min )1 Ækg )1 ), the increase in the concentration of total cysteine showed an almost linear dose dependence, as shown in Fig. 6, in which the maximum total cysteine accumulated in RBCs and plasma is plotted against the diamide dose administered. We further evaluated the effect of diamide infusion on various rat organs in order to assess whether dia- mide was also able to affect the thiol ⁄ disulfide bal- ance of these organs, and whether cysteine efflux to the blood was paralleled by a change in the Cys concentration in other tissues. Diamide, at 60 min from the start of administration, evoked a significant decrease in the hepatic Cys level, followed by an increase in both liver and kidney (after 2 h from the end of administration), but the GSH concentration was found to be significantly higher only in the liver 2 h after the end of diamide administration (Fig. 7A,B ). A slight but significant (P < 0.05) increase in the concentration of total disulfide forms of GSH in heart and lung but not in other analyzed organs was also observed (+16.2 ± 5.1% and +20.9 ± 3.2%, respectively). Additionally, in liver, kidney, lung and heart, a slight but significant (P < 0.05) increase in Hcys wa seen, both at the end of diamide infusion (i.e. 60 min) and at the end of the experiment (i.e. 180 min) (data not shown). These data suggest that diamide has a minimal effect on the thiol ⁄ disulfide balance of tissues other than blood, and did not induce evident decreases in cyste- ine and GSH levels in the organs analyzed. Together, these findings prompted us to further investigate the origin of cysteine that enters blood. In theory, two possible sources exist: (a) intracellular GSH is exported and converted extracellularly to Cys by c-glutamyl transpeptidase (c-GT) and dipep- tidases, with cysteine being then taken up by RBCs, or (b) methionine is first converted into Hcys, and then, through the trans-sulfuration pathway (TSP), to Cys. To assess these two hypotheses, we repeated the experiments using rats pre-treated with acivicin and ⁄ or propargylglycine. Acivicin is an inhibitor of c-GT, thus this treatment should eliminate the frac- tion of Cys that derives from GSH [27]. Propargyl- glycine is an inhibitor of cystathionase, the enzyme that forms cysteine from cystathionine in the TSP; in this case, treatment should eliminate the supply of Cys derived from methionine [28]. Pre-treatment with these inhibitors in control rats (administered with saline) completely abolished the release of Cys into blood (Fig. 8). In animals treated with diamide, propargylglycine and acivicin significantly decreased the flux of cysteine towards the hematic compart- ment by  25% and 30%, respectively. Concomitant use of these inhibitors enhanced the inhibitory effect on the Cys increase in blood. This suggests that both pathways contribute to the large cysteine efflux from organs into the hematic compartment observed in our experiments. Discussion In this study, a systemic response to a thiol-directed oxidative insult was induced by means of slow infu- sion of diamide in the rat, which evoked a relatively long-lasting oxidant stimulus. Little is known about the effect of oxidative stress on the distribution of LMM-SH in extracellular fluids and their exchange rate between tissues. The paucity of information in this field is due, at least in part, to the intrinsic diffi- culty in creating reliable animal models of oxidative stress. Acute carbon tetrachloride administration is frequently used as a rodent model for oxidative stress; these experiments are promising, and reports Fig. 2. Total thiols in rat plasma during and after diamide infusion. Time course of total plasma thiols (sum of reduced + disulfide forms) levels during and after diamide infusion (7.73 lmolÆmin )1 Ækg )1 intravenously). Data are the means ± SD from the experiments shown in Fig. 1. *P < 0.05 versus value at time zero; **P < 0.01 versus value at time zero. D. Giustarini et al. Cysteine flux under oxidative stress FEBS Journal 276 (2009) 4946–4958 ª 2009 The Authors Journal compilation ª 2009 FEBS 4949 have demonstrated which antioxidant parameters are influenced by these treatments [29,30]. However, these models are harsh, many free radicals are produced as a result of the reductive bioactivation of carbon tetra- chloride by cytochrome P450, and the animals may suffer multi-organ damage. Thus, information on the inter-organ trafficking of small thiols is difficult to interpret [30,31]. In contrast, diamide is a thiol- specific oxidizing agent that rapidly reacts with GSH and other thiols without production of free radicals or other ROS. Further, we performed experiments by a slow infusion of diamide to avoid the problem that A C B Fig. 3. Thiols and disulfides in rat erythrocytes during and after diamide infusion. Time course of erythrocyte LMM-SH (closed circles), LMM-SS (closed squares) and RSSP (closed triangles) levels during and after diamide infusion (7.73 lmolÆmin )1 Ækg )1 intravenously). (A) Cys- teine; (B) glutathione. (C) Increase in total cysteine and total glutathione levels over values at time zero. Values are the sum of the thiol and disulfide concentrations shown in (A) and (B), and are expressed as thiol equivalents. Data are the means ± SD of four experiments. *P < 0.05 versus value at time zero; **P < 0.01 versus value at time zero. Cysteine flux under oxidative stress D. Giustarini et al. 4950 FEBS Journal 276 (2009) 4946–4958 ª 2009 The Authors Journal compilation ª 2009 FEBS the diamide effect is limited to only a few seconds once administered to rats, because of its high reacti- vity [22,23]. The main finding of our experiments was a revers- ible accumulation of cysteine within the blood (Fig. 4). Our previous studies [22,23] showed a slight increase in hematic cysteine after a single intraperitoneal diamide administration to rats, accompanied by oxidation of hematic GSH; little effect was found for GSH, its disulfide forms, and antioxidant enzyme levels in tis- sues other than blood [23]. Conversely, a 1 h treatment with diamide, like that performed here, evoked dra- matic changes in hematic thiols and disulfides. More- over, the features of cysteine accumulation in blood were studied in detail by separate analysis of plasma and erythrocyte compartments. The total cysteine con- centration in the plasma compartment increased from  100 to  140 lm (Fig. 2) during diamide administra- tion. In contrast, the levels of reduced cysteine did not change (Fig. 1A). This is probably the result of two phenomena: enhanced delivery of cysteine into the blood by tissues, and oxidation elicited by diamide. The total Hcys level also showed a reversible increase (Fig. 2). More dramatic effects were observed in erythrocytes, in which GSH was reversibly oxidized (Fig. 3B), and, more importantly, a large increase in the cysteine con- centration, in both the reduced and disulfide forms, was observed (Fig. 3A). Therefore, a high amount of cysteine is delivered into the plasma and rapidly taken Fig. 4. Total cysteine in rat blood during and after diamide infusion. Time course of the total cysteine (sum of reduced thiol + disul- fides) increase over the levels at time zero in rat blood during and after diamide infusion (7.73 lmolÆmin )1 Ækg )1 intravenously). The reported values were calculated by normalization of measured con- centrations of total cysteine in both plasma (black bars) and erythro- cytes (gray bars) to the relative hematocrit value. Values are expressed as thiol equivalents. Data are the means ± SD of four experiments. Fig. 5. Thiols and disulfides in rat plasma ex vivo after diamide infusion. Ex vivo levels of plasma Cys (triangles), CySS (squares) and CySSP (circles) after diamide infusion (7.73 lmolÆmin )1 Ækg )1 intravenously) and blood withdrawal. Blood was withdrawn 15 min after the end of diamide infusion and maintained at 37 °C. Time zero indicates the time of withdrawal. Data are the means ± SD of three experiments. *P < 0.05 versus value at time zero; **P < 0.01 versus value at time zero. Fig. 6. Dose dependence of the maximum increase of cysteine induced by diamide. Values indicate the increase of total Cys over levels at time zero in rat blood 75 min after the start of diamide infusion (60 min infusion, dose range 1.93–9.66 lmolÆmin )1 Ækg )1 intravenously). The reported values were calculated by normaliza- tion of measured concentrations of total cysteine in both plasma and erythrocytes to the relative hematocrit value. Values are expressed as thiol equivalents. Data are the means ± SD of five experiments. D. Giustarini et al. Cysteine flux under oxidative stress FEBS Journal 276 (2009) 4946–4958 ª 2009 The Authors Journal compilation ª 2009 FEBS 4951 up by RBCs during diamide administration (Fig. 4). In RBCs, cysteine accumulated mainly as protein mixed disulfides, conceivably linked to the highly reactive b-125 cysteine residues of hemoglobin. Analogously, GSH is mostly oxidized to form mixed disulfides with hemoglobin, with a minimal increase in GSSG concen- tration [20]. This extraordinarily high accumulation of cysteine in RBCs was found to be reversible, and Cys reached its initial levels within 2 h after the end of diamide administration. Cysteine is probably reduced by the recovered GSH and exported from RBCs. In fact, it has recently been observed that Cys-enriched erythrocytes export cysteine in a time- and concentra- tion-dependent manner [32]. Indeed, in ex vivo experi- ments, we observed a significant and progressive increase in total plasma cysteine, mainly CySS and CySSP, after diamide infusion, corresponding to the cysteine concentration decrease within RBCs (Fig. 5). Given that plasma is a rather oxidizing environment, which lacks reductases [2], it is likely that the exported Cys is oxidized both to CySS and RSSP. We cannot exclude the possibility that a fraction of CySS may be exported from erythrocytes. Nevertheless, our previous data with washed RBCs indicated that, under physio- logical conditions, human erythrocytes release cysteine over time, mostly in the reduced form [26]. Although the erythrocyte transport system for Cys efflux has not yet been identified, Yildiz et al. [32] suggested that this process is carrier-mediated and is significantly attenu- ated when GSH is depleted (or its synthesis is blocked); therefore, it may require a reduced mem- brane thiol. It is also possible that, under our experi- mental conditions, this transport system was less efficient during the oxidative perturbation induced by diamide. In contrast to Cys, GSH returned to basal levels probably as a result of thiol⁄ disulfide reactions and NADPH-dependent reduction. De novo GSH syn- thesis did not occur under our experimental condi- tions, as evidenced by constant levels of total glutathione measured in vivo (Fig. 3) and ex vivo (data not shown). This suggests that Cys loss in RBCs does not represent a device to counterbalance de novo GSH synthesis. The reason for the observed Cys accumulation, first in plasma and then in RBCs, is not clear. It is possible that the thiol–disulfide status in plasma is finely tuned, and the diamide-induced oxidation of Cys is compen- sated for by efflux of GSH and Cys from the liver and other organs to maintain the cysteine pool. GSH is known to be a critical source for maintaining a steady Cys availability as it is continuously exported out of cells into plasma and converted to circulating cysteine by the action of c-GT and dipeptidase (Fig. 9). Among the various tissues, liver has been shown to play a cen- tral role in the GSH homeostasis, serving as the princi- pal source of the GSH circulating in plasma [14,33]. The extracellular translocation of hepatic GSH and its c-GT-dependent extracellular catabolism, resulting in formation of plasma Cys and CySS (cysteine is rapidly oxidized to cystine extracellularly [34]), could be a AB Fig. 7. Effect of diamide infusion on thiols in various rat tissues. Rats were administered diamide (7.73 lmolÆmin )1 Ækg )1 intravenously) for 1 h and killed 60 or 180 min after the start of the treatment. The levels of Cys (A) and GSH (B) in various tissues are reported. Values at time zero are those obtained in rats treated analogously (i.e. implanted with the valve), but without any infusion. Data are the means ± SD of four experiments. *P < 0.05 versus untreated animals; *P < 0.01 versus untreated animals. Cysteine flux under oxidative stress D. Giustarini et al. 4952 FEBS Journal 276 (2009) 4946–4958 ª 2009 The Authors Journal compilation ª 2009 FEBS systemic response to local oxidative conditions. Never- theless, after a 60 min diamide infusion, we observe a significant decrease in hepatic Cys levels only (10 lm), whereas the GSH concentration did not change (Fig. 7). Analogously, disulfide levels did not vary to a significant extent after diamide administration. Even thought the whole amount of cysteine that disappears from the liver is released into blood, it cannot quanti- tatively explain the dramatic rise of Cys registered within erythrocytes. Nevertheless, we should consider these phenomena as a dynamic process to which numerous factors contribute in order to maintain fairly constant thiol levels in various organs and to supply the compounds necessary to counteract the diamide challenge. In this context, a possible limit of our study is that we did not record the multi-directional flux of various thiols in ‘real time’; we could only perform our measurements at fixed times, and these values obtained are therefore the result of various events that are not completely discriminated. Additionally, given the high concentration of GSH in organs (2–10 mm), it is evident that an increase in its release does not necessarily produce a significant, quantifiable decrease in its levels in the organs themselves. Indeed, although a 1–2% release of GSH does not induce a significant decrease in its concentration in liver or in kidneys, it provokes a marked effect in plasma, where its levels (and in general the levels of all LMM-SH) are in the micromolar range. In contrast, as disulfide levels are very low in the intracellular compartments ( 2000– 3000-fold lower than thiol compounds [2]), we can assume that their contribution to the increase in Cys is negligible. Additional factors may be considered to be involved in the observed process of cysteine accumulation (Fig. 9). Extracellular cysteine can also derive from intracellular stores of various tissues that can deliver it to the plasma; the other main source is diet. During prolonged starvation, skeletal muscle, in particular, can deliver cysteine to the plasma by degradation of pro- teins [14]. Methionine from protein degradation or from intracellular stores can also serve as a source of cysteine through the action of the TSP [35]. Hcys is synthesized from methionine by almost all cells through the activated methyl cycle. Hcys can be reconverted into methionine (with formation of tetrahydrofolate) or cysteine through the TSP; alternatively, it can be exported [36]. In our experiments, pre-treatment with acivicin or propargylglycine, which inhibit either c-GT or cystathionase, respectively, should block cysteine influx to the blood during diamide infusion. In both cases, a decrease of 25–30% in the hematic levels of cysteine was observed. The effect was strengthened by concurrent administration of both inhibitors (Fig. 8). This suggests that both sources of Cys from tissues, i.e. GSH via c-GT and methionine via Hcys and the TSP, are involved. Notwithstanding this, methionine avail- ability is unlikely to be a limiting factor for plasma cysteine flux from tissues, because, under oxidative con- ditions, many factors can contribute to deliver cysteine to blood. Interestingly, stimulation of the TSP by oxidative stress has been demonstrated previously [35,37,38], suggesting that the redox sensitivity of the trans-sulfuration pathway may be considered to be an auto-corrective response that leads to an increased level of glutathione synthesis in cells challenged by oxidative stress. Evidence also exists indicating that c-GT may be upregulated as adaptive response to an oxidative insult [39]. Experiments performed using various diamide doses (Fig. 6) showed that the process is dose-depen- dent. Therefore, the observed phenomenon may derive from an oxidative regulation of one or more proteins critical to the metabolism of cysteine, or from activa- tion of a systemic mechanism of Cys ⁄ GSH delivery ⁄ uptake regulation. Fig. 8. Effect of acivicin and propargylglycine on the cysteine increase. Increase in cysteine (sum of reduced cysteine + disul- fides) over levels at time zero in rat blood induced by diamide infu- sion (7.73 lmolÆmin )1 Ækg )1 intravenously) after pre-treatment with acivicin (open circles) or propargylglycine (closed triangles) or both (open triangles). Data are compared with values obtained from rats treated with diamide (7.73 lmolÆmin )1 Ækg )1 intravenously) without pre-treatments (closed circles), or from rats treated with acivicin (gray squares) or propargylglycine (closed squares) without diamide infusion. Disulfides represent the sum of low-molecular-mass disul- fides and protein mixed disulfides. The reported values were calcu- lated by normalization of measured concentrations of total cysteine in both plasma and erythrocytes to the relative hematocrit value. Values are expressed as thiol equivalents. Data are the means ± SD of three experiments.*P < 0.05 versus samples trea- ted only with diamide analyzed at the same time. D. Giustarini et al. Cysteine flux under oxidative stress FEBS Journal 276 (2009) 4946–4958 ª 2009 The Authors Journal compilation ª 2009 FEBS 4953 To summarize, our data suggest that diamide shifts the thiol:disulfide ratio in plasma and RBCs. To coun- teract this effect, Cys is likely to be delivered from multiple tissues; a fraction of Cys is oxidized within plasma by diamide, whereas another fraction enters RBCs, where it is oxidized to CySS and mostly CySSP. Once the diamide infusion and, consequently, the oxi- dant stimulus, is complete, cysteine is taken up de novo by tissues other than blood (Fig. 7A). We infer that the x c ) transport system for CySS may contribute to remove the cysteine accumulated within erythrocytes and plasma. In fact, it has been demonstrated that this transport system, which is widely distributed in various organs [40,41], is upregulated under oxidative condi- tions [42]. Therefore, it is reasonable to hypothesize that, under our experimental conditions, CySS uptake into various organs is enhanced, followed by CySS reduction to Cys. Indeed, a clear increase in Cys levels was observed 2 h after the end of diamide infusion in the kidneys and the liver. Additionally, a significant increase in GSH was observed in the liver at 3 h after the start of treatment (Fig. 7B), probably indicating that the excess cysteine enters this organ, where it is subsequently converted into GSH. An increase in liver GSH after an acute increase of circulating cysteine has been described previously [43]. Maintenance of an adequate plasma thiol–disulfide balance appears to be fundamental. It has been dem- onstrated that even a minimal shift in the redox state of either the Cys ⁄ CySS or GSH ⁄ GSSG pool (e.g. 21 and 9 mV changes, respectively, as observed in aging) is sufficient to cause a large increase in the oxidized forms of intracellular proteins bearing vicinal thiols, which can influence specific signaling pathways [44]. In addition, it has been observed that the NMDA recep- tor, protein disulfide isomerase, epidermal growth fac- tor receptor and extracellular signal-regulated kinase are modulated by the cellular and ⁄ or extracellular redox state [45–48]. The shift in the thiol ⁄ disulfide redox state over the range found in vivo in human plasma is a key determinant of early events of vascular disease development [49]. In this context, it should be noted that Hcys levels in plasma (and in some other tis- sues) were also reversibly increased in our experiments by the thiol-directed oxidant, although to a minor extent compared with Cys levels (Figs 1C and 2). This Fig. 9. Schematic diagram showing the metabolic pathways of various physiological LMM-SH. Intracellular Cys is a key determinant in regu- lating the kinetics of formation of c-glutamylcysteine (c-GluCys), the first step in GSH synthesis. Intracellular Cys may derive from diet, intra- cellular stores, or methionine (Met), which is converted into Hcys by the activated methyl cycle. Hcys may then condense with serine to produce cystathionine, which in turn may be hydrolyzed to Cys. The conversion of cystationine into Cys is catalyzed by the enzyme c-cysta- thionase, the activity of which is inhibited by propargylglycine (PGG). Both GSH and Cys can be exported from cells into plasma, where they undergo auto-oxidation. The liver and kidneys appear to have significant capacity for GSH efflux. Although it has been established that cells are able to export excess GSSG, it is not yet known whether they also possess a transport system for CySS. Plasma Cys, but neither GSH nor GSSG, can be taken up by most cells (also including RBCs). Some cell types (excluding RBCs) also possess a transport system for uptake of CySS. Extracellular GSH can be converted into CysGly and then into Cys by the combined action of the membrane enzymes c-glutamyl transpeptidase (cGT) and dipeptidases (DP). The activity of cGT is inhibited by acivicin. Alternatively, CysGly may be taken up and converted into Cys by DPs. Cysteine flux under oxidative stress D. Giustarini et al. 4954 FEBS Journal 276 (2009) 4946–4958 ª 2009 The Authors Journal compilation ª 2009 FEBS effect could be indicative of diamide-induced stimula- tion of methionine transformation into Cys, a pathway in which Hcys represents an intermediate. Given the well-known action of Hcys as a pro-atherogenic and cardiovascular risk factor, the fact that diamide treat- ment (and probably other events that perturbate the thiol ⁄ disulfide status of plasma) may influence homo- cysteine levels suggests that this topic deserves further investigation. Many authors have focused their attention on the possible role of modulation of LMM-SH levels in dis- ease development and ⁄ or progression. However, only a few studies have investigated the physiological rules that govern the distribution of LMM-SH in extracellular flu- ids, the contribution of various tissues, the possible con- sequence(s) of decreased ⁄ increased efflux from a single organ because of pathophysiological conditions, and, more importantly, the influence of oxidative stress on these processes. Our data clarify some of these aspects, and suggest that thiol-directed oxidative stress consider- ably influences the inter-organ exchange of cysteine and its production from methionine and ⁄ or GSH. Experimental procedures Animals Sprague–Dawley male rats (400–450 g) were purchased from Charles River Laboratories (Calco, Italy). A double valve (model 617, 20 · 20 mm; Danuso Instruments, Milan, Italy) was implanted in each animal; jugular and femoral veins were cannulated for either drug administration or blood collection, as described previously [20]. The valve was implanted under pentobarbital anesthesia two days before the experiment. Animals were allowed to freely move and fed ad libitum before and during the experiments. Rats received infusions of diamide in saline (7.73 lmol Æmin )1 Ækg )1 unless otherwise specified) via the cannula implanted in the femoral vein. The infusion was started immediately after blood withdrawal for time zero measure- ments and lasted 1 h. Blood aliquots (100 lL each) were collected through the valve connected to the jugular vein and immediately processed. All animal manipulations were performed in accordance with the European Community guidelines for the use of laboratory animals. The experi- ments were authorized by the local ethics committee. Rats did not show any evidence of distress or altered behavior during the experiments. Thiol and disulfide measurement in blood Blood samples were collected in plastic tubes containing K 3 EDTA. Blood was immediately centrifuged at 15 000 g for 15 s. Aliquots of plasma (20 lL) were immediately acidified by 1 : 1 addition of a solution of trichloroacetic acid (TCA, 12% w ⁄ v). After separation of proteins by cen- trifugation at 15 000 g for 2 min at room temperature, 25 lL of the supernatant was brought to a pH of  8.0 using 5 lLof2m Tris, and then 0.5 lLof40mm mono- bromobimane (mBrB; Calbiochem, Milan, Italy) dissolved in methanol was added for LMM-SH measurement. After a 10 min incubation in the dark, samples were acidified and analyzed by HPLC as previously described [1]. A further 25 lL of plasma were added with 1 lLof50mm N-ethyl- maleimide (NEM; Sigma-Aldrich, Milan, Italy,) for mea- surement of both low-molecular-mass disulfides and protein mixed disulfides (total disulfides). After 10 min incubation at room temperature, NEM was extracted with four vol- umes of dichloromethane, 2 mm (final concentration) of dithiothreitol was added to reduce disulfides, and after 5 min samples were 1 : 1 diluted with 12% w ⁄ v TCA and then proteins were discarded by centrifugation at 15 000 g for 2 min at room temperature. Supernatants (30 lL) were brought to a pH of  8.0 using 6 lLof2m Tris, and then 1.2 lL of mBrB was added. After a 10 min incubation, samples were acidified with HCl and analyzed by HPLC as previously reported [1]. All samples with visible hemolysis were discarded. For determination of LMM-SS, 5 lL aliquots of plasma (after addition of NEM, as described above) were 1 : 3 diluted with 8% w ⁄ v TCA, and then proteins were discarded by centrifugation at 15 000 g for 2 min at room temperature. Supernatants (10 lL) were brought to a pH of  8.0 using 2 lLof2m Tris, then 2mm dithiothreitol (final concentration) was added. Sam- ples were then derivatized using mBrB, and analyzed as described above. The level of protein mixed disulfides was calculated as the difference between total disulfides and LMM-SS. The RBC pellet was washed three times with ice cold Na + ⁄ K + phosphate-buffered saline (pH 7.4) containing 5mm glucose for LMM-SH analyses or 5 mm glucose and 80 mm NEM for disulfide analyses, and hemolyzed using 10 volumes of 10 mm Na + ⁄ K + phosphate buffer, pH 7.4. Aliquots of the hemolysate were used for LMM-SH, total disulfide or LMM-SS determinations as described above. For ex vivo experiments, after 75 min from the start of diamide infusion,  600 lL of blood was withdrawn from the valve (K 3 EDTA was used to prevent blood clotting) and maintained at 37 °C under gentle gyratory shaking for analyses of thiols and related disulfides. At the indicated times, aliquots of blood were centrifuged at 15 000 g for 15 s. Thiols and disulfides were analyzed in both plasma and RBCs as described above. All HPLC analyses were performed by using a Spheri- sorb C18 column (4.6 mm internal diameter · 250 mm column length) (Varian Inc, Palo Alto, CA) by means of an Agilent series 1100 HPLC (Agilent Technologies, Milan, Italy) equipped with a fluorometric detector. D. Giustarini et al. Cysteine flux under oxidative stress FEBS Journal 276 (2009) 4946–4958 ª 2009 The Authors Journal compilation ª 2009 FEBS 4955 [...]... whereas acivicin was administered in two doses (each 10 mgÆkg)1 intraperitoneally) 2.5 and 1 h before starting diamide infusion In experiments with control rats, only saline was infused For the assessment of thiols and disulfides in rat tissues, animals were anesthetized with sodium pentobarbital (60 mgÆkg)1), blood was withdrawn from abdominal aorta, and then tissues were rapidly collected All organs.. .Cysteine flux under oxidative stress D Giustarini et al Thiol and disulfide measurements in rat tissues Treatments with acivicin (a c-GT inhibitor) and propargylglycine (a cystathionine c-lyase inhibitor) were performed as described by Zalups and Lash [50] and Shinozuka et al [51], respectively Propargylglycine (100 mgÆkg)1 intraperitoneally) was administered 5 h before starting diamide infusion,... 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Oxidative stress induces a reversible flux of cysteine from tissues to blood in vivo in the rat Daniela Giustarini 1 , Isabella Dalle-Donne 2 , Aldo. and whether cysteine efflux to the blood was paralleled by a change in the Cys concentration in other tissues. Diamide, at 60 min from the start of administration,