Glutathione conjugation by isolated lung cells and the isolated, perfused lung

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1 Eur. J. Biochem. 138, (1984) 0 FEBS 1984 Glutathione conjugation by isolated lung cells and the isolated, perfused lung Effect of extracellular glutathione Janet R. DAWSON, Kirsi VAHAKANGAS, Bengt JERNSTROM, and Peter MOLDEUS Department of Forensic Medicine, Karolinska nstitutet, Stockholm; and Department of Pharmacology, University of Oulu (Received October 14, 1983) - EJB Cells isolated from rat lung by protease digestion were found to catalyze the reduced glutathione (GSH) conjugation of 1 -chloro-2,4-dinitrobenzene. The rate of conjugation was stimulated severalfold in the presence of GSH, indicating uptake and utilization of extracellular GSH by the lung cells. The stimulation was dependent on the GSH concentration and not due to a spontaneous nonenzymatic reaction or to extracellular GSH-transferase activity. Conjugation of l-chloro-2,4-dinitrobenzene was also measured using isolated perfused rat lung. The conjugation, which was linear for a longer time than with the isolated cells, was also stimulated in the presence of GSH in the perfusion medium. The results indicate the ability of rat lung to utilize extracellular GSH. Glutathione is a tripeptide, found in animal and plant cells and micro-organisms at concentrations of mm [l]. n the lung, conjugation with GSH has been shown to be active in relation to pulmonary toxins, most especially 4-ipomeanol [2,3] and benzo[a]pyrene 4,5-oxide [4, 51. n general the liver is the tissue with the highest capacity for xenobiotic metabolism, but the lung, since it is exposed to xenobiotics both in inhaled air and in the blood, could have a vital role to play in protecting itself and other tissues in the body against the toxic effects of various compounds [3, 5, 61. n the intact animal, owing to the presence of the liver, it is difficult to assess the contribution of the lung in such reactions, so for this study two different systems were employed in vitro. solated cells have already proved to be a useful system for studying extrahepatic metabolism [7-91 and for lung, two such systems have been used previously to study xenobiotic metabolism, one for rabbit lung [lo] and the second for rat lung [ll. The latter was used in the present study. The other system utilized was the isolated perfused lung [12] which has the advantage that the spacial arrangement of cells is preserved. This system is more physiological and it provides an essential back-up to the isolated cell experiments. The liver has been shown not to be involved in the metabolism of circulating GSH [13, 141, but both the kidney, and to a lesser extent the intestine, are active in this area [l, 15, 161. Recently the lung has been shown to participate in the catabolism of a glutathione conjugate, namely leukotriene C, [17], but no studies have so far been reported on the role of the lung in the metabolism of free, circulating GSH. The intracellular level of GSH in the lung is much lower than that of the liver, Abbreviutoions. GSH, reduced glutathione; GSSG, glutathione disulfide; ClN,Bz, l-chloro-2,4-dinitrobenzene; GS-N,Bz, glutathione conjugate of 2,4-dinitrobenzene; Hepes, 4-(2-hydroxyethyl)-lpiperazineethanesulfonic acid. Enzymes. Glutathione S-transferase (EC ); y-glutamyl transpeptidase or y-glutamyltransferase (EC ). and has been found to be approximately one fifth of that of the liver, on a mass basis [6], but much lower on a cell basis, owing to the small size of lung cells [ 11. Therefore the possibility that the lungs can utilize circulating extracellular GSH in detoxification reactions was explored in vitro. MATERALS AND METHODS Chemicals Reduced glutathione, protease type V and diethylmaleate were purchased from the Sigma Chemical Co. (St Louis, MO, USA). l-chloro-2,4-dinitrobenzene was purchased from Fluka AG. (Buchs, Switzerland). y-glutamyl-3-carboxy-4-nitroanilide, monoammonium salt, was purchased from Boehringer-Mannheim Scandinavia A.B. (Bromma, Sweden). Glycylglycine was obtained from Merck (Darmstadt, FRG). MF filters, 0.45-pm pore size, for aqueous solutions, were purchased from Waters Associates A.B. (Partille, Sweden). All other chemicals and solvents used were of analytical reagent grade, obtained through local chemial suppliers. Animals All experiments with isolated cells were performed using male, Sprague-Dawley rats of g. For lung perfusion experiments, male Wistar, specific pathogen-free rats of g were used. The Sprague-Dawley rats were purchased from Anticimex A.B. (Stockholm, Sweden). All rats were allowed free access to both food and water. Tissue preparation Cells were isolated from rat lung by perfusion of a protease and EDTA SOhtiOn via the trachea, as previously described [l 11. Cells were used at a concentration of 4 x 106/ml in Krebs- Henseleit buffer (ph 7.4) and were incubated at 37 "C under an air atmosphere.

2 440 Lungs were isolated and perfused as previously described [12]. Perfusion was with a Krebs-Ringer buffer, supplemented with bovine albumin (6 %) and D-glucose (7 mm). The lungs were ventilated using a positive pressure system, n all experiments the perfusion volume was initially 75 ml, the first 25 ml of medium which went through the lungs being discarded to remove the blood from the system, the remaining 50 ml was recirculated. After 10 min of perfusion, the epxeriment was started by addition of the appropriate chemical(s). For some experiments the lungs were perfused initially for 15 min with medium containing 1 mm diethylmaleate, which was then exchanged for medium containing 1 -chloro-2,4-dinitrobenzene (ClN,Bz) (100 pm), with or without GSH (1 mm), and perfusion continued for another hour. *Or GSH conjugation l-chloro-2,4-dinitrobenzene was used as the substrate for glutathione S-transferase in both lung systems. Cells (4 x 106/ml) were incubated at 37 C with the relevant concentration of GSH and with ClN,Bz (100pM). At the desired times an aliquot (1 ml) was withdrawn, the cells removed by centrifugation, and the supernatant kept on ice until measurement of the absorbance at 340nm against an appropriate blank. Appropriate controls, e.g. cells with GSH and no substrate, cells with CN,Bz and no GSH and GSH and ClN,Bz in buffer with no cells were also incubated and measured with each experiment. Similarly, with isolated perfused lungs, aliquots (1 ml) were withdrawn at the times indicated and the absorbance at 340 nm immediately determined against the appropriate blank. The appropriate controls were carried out, as for the cell experiments. An absorption coefficient of 9.6 mm-l. cm- was used in all calculations. The effect or removal of the cells from the incubation on the conjugation of ClN,Bz with GSH was determined by filtration of the incubation medium, at the appropriate time, though filters of 0.45-pm pore size. After filtration the filtrate was further incubated for the remainder of the incubation time, samples being taken as before. y-glutamyltransferase y-glutamyltransferase was determined as follows. Glycylglycine (0.5 M) and y-glutamyl-3-carboxy-4-nitroanilide (0.05 M), in Krebs-Hepes buffer (ph 7.4) was termed solution A. Lung cells (1.25 x lo6) in 0.9 ml Krebs-Hepes buffer (ph 7.4, at 37 "C) were incubated in a -ml cuvette and the absorbance of the solution at 405nm was followed using a Shimadzu UV 240 recording spectrophotometer. The substrate source, solution A (0.1 ml), was then added and the rate of change in absorbance at 405 nm was determined. An absorption coefficient of 9.4mM-'. cm-' was used in all calculations. GSH uptake Uptake of GSH by isolated lung cells was determined using 100 pm reduced GSH. Cells were incubated at 37 C in wide test tubes in total volume of no more than 10 ml, with and without GSH and with GSH and ClN,Bz (100 pm). Aliquots (1 ml) were removed at the appropriate times and the cells were immediately centrifuged down through a layer of silicone oil [18]. This method results in a very rapid separation of the cells from the incubation medium. The medium and oil were then removed and 250 pl(0.9 %, w/v) NaCl was added, together with 50 pl(70 %, v/v) perchloric acid. The samples were derivatised time (min) Fig. 1. Conjugation of ClN,Bz with GSH by isolated lung cells in the presence and absence of exogenous GSH. Lung cells (4 x 106/ml) were incubated (at 37 C) with CN,Bz (100pM) (0); with CN,Bz (100 pm) and GSH (1 mm) (0); or ClN,Bz (100 pm) was incubated at 37 C in Krebs-Hepes buffer with GSH (1 mm) (A). Samples (1 ml) were withdrawn at the times shown, the cells removed by centrifugation and absorbance determined at 340 nm against an appropriate blank. Results represent the mean f SEM of three different cell preparations with iodoacetic acid and fluorodinitrobenzene. GSH and GSSG were then quantified by HPLC separation as described by Reed and Beatty [19]. RESULTS ncubation of isolated lung cells with ClN,Bz alone resulted in less than 12 %conversion to the GSH conjugate (Fig. 1). The addition of 1 mm GSH to the cell incubation had a marked effect on the GSH conjugation of ClN,Bz (Fig. 1). Both the initial rate of GSH conjugation and the total amount of GS-N,Bz formed were increased severalfold by this addition. The non-enzymatic conjugation of ClN,Bz with GSH is also depicted in Fig. 1. t can be seen that the initial rate of conjugation is similar to that found with lung cells and ClN,Bz alone; the total amount formed is approximately three times that formed by lung cells in the absence of exogenous GSH. n the linear part of the curve the summation of the spontaneous reaction and that catalyzed by cells in the absence of GSH gave a value which was less than half that found in the presence of cells and exogenous GSH (1 mm). Thus, the effect of extracellular GSH addition cannot be attributed to an increase in the non-catalytic reaction alone. The dependence of this conjugation reaction on the concentration of extracellular GSH is shown in Fig. 2. t can be seen that increasing the concentration of GSH fivefold increased the total amount of conjugate formed approximately 2.5-fold. The initial rate of conjugation is also increased, but not as markedly (Fig. 2A). The spontaneous conjugation is also affected by the level of GSH present (Fig. 2B), but again, this non-enzymatic reaction is considerably lower than that catalyzed by the lung cell enzyme. The results from Fig. 2, expressed as rate of the reaction in the linear phase versus log,, of the GSH concentration, show

3 M 30 time (mint Fig. 2. Conjugation of CN,Bz with GSH by isolatedlung cells: theeffect of altering extracellular GSH concentration. (A) Cells were incubated at 37 C with ClN,Bz (100 pm) and 50 pm GSH (0); or 100 pm GSH (A); or 250pM GSH (m). (B) ClN,Bz (1OOpM) was incubated in Krebs-Hepes buffer at 37 C in the presence of 50 pm GSH (0); or 100 pm GSH (A); or 250 pm GSH (0). Remaining experimental details as for Fig. 3. Results represent the mean of three different cell1 preparations Fig. 4. Conjugation of time (minl CN2Bz with GSH by lung cells, demonstration that the reaction is mtracellular. Lung cells (4 x 106/ml) were incubated at 37 "C in the presence of 100 pm ClN,Bz and 1 mm GSH. At times of 10 rnin (o), 20 min (A) and 30 rnin (o), cells in the incubation were removed by filtration, using filters with pore size of 0.45 pm. The filtrate was then further incubated. Aliquots (1 ml) were withdrawn at the times shown, cells removed by centrifugation and absorbance measured at 340 nm. Results represent the mean of three different cell J - Fig. 3. Rate of CN,Bz conjugation with GSH; dependence on GSH concentration. Enzymatic reaction (0); spontaneous reaction (0). Values taken from Fig. 1 and 2 that the enzymatic reaction is linearly related to the logarithm of the GSH concentration, whereas the spontaneous reaction increases proportionally more at higher GSH concentrations (Fig. 3). Fig. 4 demonstrates that the effect of exogenous GSH is not due to leakage of the glutathione transferase out of the cells, but is probably due to uptake of GSH. Removal of the cells from the incubation after 10 min, resulted in cessation of the GSH conjugation, indicating that leakage of the GSH transferase from the cells was unlikely to have occurred. Removal after 20 rnin gave rise to a subsequent decrease in the amount of conjugate present. This decrease could be caused by y-glutamyltransferase leaking from the lung cells on filtration. This possibility is further explored in the Discussion. At 37 "C the y-glutamyltransferase activity of the isolated lung cells was (mean f SD; n = 12) nmol/106 cells min ~ and at room temperature (approximately 22 "C) it was 0.52 f 0.07 (mean & SD; n = 6) nmol/106 cells min-'. The possibility that lung cells are able to take up GSH from the extracellular medium was also investigated and the results are presented in Fig. 5. n the presence of extracellular GSH (100 pm), the lung cells after 5 min of incubation contained 180 % of the control amount of GSH; this percentage was even greater at the later time points. This indicates uptake of GSH though some binding of the GSH to the cell membrane cannot.\ tlme ( rnin) Fig. 5. Level of GSH in isolated lung cells: the effect of exogenous GSH and ClN,Bz. Lung cells (4 x 106/ml) were incubated at 37 C with GSH (100 pm) (0); with GSH (100 pm) and ClN2Bz (100 pm) (0); or with no exogenous GSH (A). A1 the times indicated, an aliquot (1 ml) was removed from the incubation, the cells were centrifuged down though silicone oil and the pellet was derivatized for measurement of GSH and GSSG by HPLC separation. (A) GSH. (B) Total GS-equivalents. Results are the mean f SEM of three different cell preparations be ruled out. n the presence of extracellular GSH (100 pm) and ClN,Bz (100 pm) the level of glutathione in the lung cells fell dramatically within the first minute, and after 15 rnin of incubation there was no measureable GSH associated with the cells. These results are expressed both as the amount of GSH present (Fig. 5A) and as GSH plus GSSG, expressed as GSH equivalents (i.e. GSSG = 2 x GSH) (Fig. 5B). Perfused lung did not show such a big difference between the amount of GS-N,Bz formed in the presence and absence of extracellular GSH (Fig. 6), but there is a noticeable effect at the later time points, though by this time most of the substrate is used up, so a larger difference could not be expected. When the GSH content of the lung was first lowered by perfusing the lung with diethylmaleate, and subsequently perfused with medium containing ClN,Bz (100 pm) and GSH (100 pm), the difference between the perfusion with and without GSH was more marked (Fig. 7). The rate of conjuga-

4 442 loor L5 60 time ( min) Fig. 6. Conjugation of CN2Bz by isolated perjiused lung. Lungs were perfused with Krebs-Ringer buffer containing albumin (4-6 %) and glucose (7 mm) and CN,Bz (100 pm) (0); or CN,Bz (100 pm) plus GSH (1 mm) (0). Control perfusion without lungs, with medium containing ClN,Bz (100 pm) and GSH (1 mm) (A). At the appropriate times an aliquot (1 ml) was withdrawn from the perfusion medium and absorbance measured at 340 nm against an appropriate blank. Results represent the mean of seven determinations *O time (min) Fig. 7. Conjugation of ClN,Bz by isolated perfused lung. Lungs were perfused for 10 min with Krebs-Ringer buffer (supplemented as for Fig.6) containing diethylmaleate (1 mm), then for 1 h with Krebs- Ringer buffer containing C1N2Bz (100 pm) (+); or containing ClN,Bz (100 pm) and GSH (1 mm) (0). Remainder of experimental details as for Fig. 6. Results represent the mean f SD for four lungs tion is not as great as it is in Fig. 6, but when the endogenous GSH is lowered, the requirement for exogenous GSH is more apparent. DSCUSSON Conjugation of CN,Bz with GSH in the presence of isolated lung cells occurred rather slowly and virtually ceased after 10 min of incubation (Fig. 1). This cessation is most likely due to exhaustion of the lung cell GSH, which is normally at a low level anyway [6, 111. Addition of GSH to the extracellular medium increased both the rate of conjugation of ClN,Bz and the total amount formed (Fig. 1 and 2). This increase was not due to the non-catalytic conjugation of ClN,Bz with GSH, since it was greater than the sum of the non-catalytic reaction and that occurring in the presence of lung cells alone (Fig. 1). t has been estimated that in vivo this non-catalytic reaction is unlikely to contribute significantly to GSH transferase activity [20], but in vitro it is important to be aware of the extent of this spontaneous reaction [21]. The rate of conjugation of ClN,Bz decreased at the later time points (Fig. 1 and 2). This could be partly due to exhaustion of GSH, being utilized for the conjugation reaction, and to its oxidation, mainly to GSSG and also partly to exhaustion of the ClN,Bz, which is present at a concentration of 100 nmol/ml initially. The possibility exists that the isolation of cells from the lung has introduced artefacts into the system and also that broken or leaky cells in the incubation could result in the loss of the mainly cytosolic glutathione transferase and if that were the case the GSH would not necessarily need to be taken up by the lung cells in order for it to have an effect on the enzymecatalyzed reaction. This possibility was tested in the experiment illustrated by Fig. 4. t can be seen that removal of the cells from the incubation effectively stopped the glutathione transferase reaction, and even resulted in a decreased in the amount of the conjugate present. Leakage of the glutathione transferase is thus unlikely, but it does appear that y-glutamyltransferase could have been released from the cell membrane during the filtration procedure. y-glutamyltransferase is the enzyme involved in the first step of GSH breakdown [] and the highest level of this enzyme is found in the kidney, with lower amounts present in liver and intestine [l, 15,221. n the liver this enzyme is located primarily in the biliary tract and its release into serum is indicative of hepatobiliary dysfunction and occurs especially with hepatoma [22]. The level of y-glutamyltransferase in isolated kidney cells has been found in this department to be approximately 800 nmol/mg cell protein. min- (T. Lgstbom, personal communication) which is 80-times the level found in the lung cells (see Results), using the same synthetic substrate. However, the lungs do receive the entire cardiac output of blood, so the y-glutamyltransferase there could play an important role in the disposition of GSH in the body. y-glutamyltransferase is located on the cell membrane [23] and can be released, from the liver especially, under conditions of toxic injury [22], thus it is possible that this enzyme could have been released from the lung cells during their removal from the incubation and this would account for the subsequent disappearance of the GS- N,Bz from the medium (Fig. 4). Lung y-glutamyltransferase has been shown to be active in the metabolism of leukotriene C3, which is a glutathione-containing leukotriene [17]. Homogenates of liver and kidney, however, failed to metabolize this leukotriene [17]. Thus, the pulmonary y-glutamyltransferase could have a significant role to play in the metabolism of endogenous y-glutamyl compounds. Some studies using the isolated, perfused rat lung have confirmed the results seen with the isolated cells. However, the difference in the amount of GS-N,Bz formed in the presence and absence of GSH is not as obvious in this case, due to the higher amount of GSH present in whole lung compared to lung cells [6, 111. However, when the lung was first perfused with diethylmaleate to lower endogenous GSH, the difference became more pronounced (Fig.6, 7). t is likely that the difference would have been greater if the use of a higher

5 443 concentration of ClN,Bz had been possible, but it was found that in the absence of added GSH, a higher concentration of ClN,Bz was toxic to the perfused lung. The work presented in this paper demonstrates that the lung has the ability to utilize extracellular GSH. The relative contribution of direct uptake compared to catabolism and subsequent intracellular resynthesis remains to be established. t is also still uncertain how much significance this utilization of GSH will have under normal circumstances in vivo. However, in circumstances where the GSH supply in the lung is depleted, for instance by a circulating xenobiotic, by an inhaled toxin or by hyperbaric oxygen, then the ability to draw on the supply of plasma GSH could be of significant importance to the lung. Also, the possibility exists that the lung plays a role in regulation of circulating GSH concentration, which could be vital in instances of kidney malfunction. This work was supported by Zambon Pharmaceuticals, Milan, taly and by the Swedish Tobacco Company. We thank Professor Karki for allowing us to perform some of these experiments in his department. We also acknowledge the excellent technical assistance provided in both departments. REFERENCES 1. Meister, A. & Tate, S. S. (1976) Annu. Rev. Biochem. 45, Boyd, M. R. (1980) in Reviews in Biochemical Toxicology, (Hodgson, E., Bend, J. R. & Philpot, R. M., eds), vol. 2, pp , Elsevier/North-Holland, Amsterdam. 3. Boyd, M. R., Stiko, A., Statham, C. N. & Jones, R. B. (1982) Biochem. Pharmacol. 31, Smith, B. R., Plummer, J. L., Ball, L. M. & Bend, J. R. (1980) Cancer Res. 40, Smith, B. R. & Bend, J. R. (1978) in Carcinogenesis, vol. 3, Polynuclear Aromatic Hydracarbons (Jones, P. W. & Freudenthal, R.., eds) pp , Raven Press, New York. 6. Moron, M. S., DePierre, J. W. & Mannervik, B. (1979) Biochim. Biophys. Acta, 582, Dawson, J. R. & Bridges, J. W. (1979) Biochem. Pharmacol. 28, Jones, D. P., Sundby, G. B., Ormstad, K. & Orrenius, S. (1979) Biochem. Pharmacol. 28, Paul, C., Tomingas, A., Rajs, J., Anundi,., Hogberg, J. & Peterson, C. (1982) Acta Pharmacol. Toxicol. 51, Devereux, T. R., Hook, G. E. R. & Fouts, J. R. (1979) Durg Metab. Dispos. 7, Dawson, J. R., Norbeck, K. & Moldeus, P. (1982) Biochem. Pharmacol. 31, Vahakangas, K., Nevesaari, K., Pelkonen, 0. & Karki, N. T. (1977) Acta Pharmacol. Toxicol. 41, Hahn, R., Wendel, A. & Flohe, L. (1978) Biochim. Biophys. Acta, 539, Sies, H., Wahllander, A., Waydhas, C., Soboll, S. & Haberle, D. (1980) Adv. Enzymol. 18, Griffth, 0. W. &Meister,A. (1979) Proc. NatlAcad. Sci. USA, 76, Cornell, J. S. & Meister, A. (1978) Proc. Nut1 Acad. Sci. USA, 73, Hammarstrom, S. (1981) J. Biol. Chem. 256, Schwarz, L. R., Burr, R., Schwenk, M., Pfaff, E. & Greim, H. (1975) Eur. J. Biochem. 55, Reed, D. 5. & Beatty, P. W. (1978) in Functions of Glutathione in Liver and Kidney (Sies, H. & Wendel, A,, eds) pp Springer-Verlag, Berlin. 20. Ketterer, B. (1982) Drug Metab. Rev. 13, Testa, B. (1982) Drug Metab. Rev. 13, Ding, J. L., Smith, G. D. &Peters, T. V. (1981) Biochim. Biophys. Acta, 657, Hughey, R. P., Rankin, B. B., Eke, J. S. & Curthoys, N. P. (1978) Arch. Biochem. Biophys. 186, J. R. Dawson, B. Jernstrom, and P. Moldeus, nstitutionen for Rattsmedicin, Karolinska nstitutet, BOX 60400, S Stockholm, Sweden K. Vahakangas, Department of Pharmacology, Oulun Yliopisto, SF Oulu, Finland