Int. J. Immunopharmac.. Vol. I. pp. 93-100 © Pergamon Press Ltd. 1979. Printed in Great Britain.
0192-0561/79/0401-0093 $02,00/0
PROTECTIVE EFFECT OF LEVAMISOLE A N D ITS SULFHYDRYL METABOLITE OMPI AGAINST CELL DEATH INDUCED BY GLUTATHIONE DEPLETION M. DE BRABANDER, H. VAN BELLE, F. AERTS, R. VAN DE VE|RE and G. GEUENS Laboratories of Oncology and Biochemistry, Janssen Pharmaceutica Research Laboratories, B-2340 Beerse, Belgium (Received 19 March 1979 and in final form 26 April 1979)
Abstraet--Levamisole protects cultured cells against auto-oxidative necrosis induced by glutathione depletion. The sulfhydryl metabolite, OMPI, is approximately 100 times more active in this respect. As is the case for vitamin E and synthetic antioxidants, the cell rescue is not due to the inhibition of glutathione depletion but probably to a direct radical scavenging effect. The data suggest the following hypothesis: the immunomodulating effect of levamisole is at least partially due to its metabolite OMPI which enhances leukocyte functions by scavenging deleterious oxidative radicals the formation of which is enhanced in activated cells. Various cellular metabolic processes constantly produce oxidative substances and free radicals that are incompatible with the integrity and survival of the cell. Some of the best known targets for these reactive intermediates are membrane lipids (for review see Arias & Jacoby, 1976) and the sulfhydryl residues of various proteins such as tubulin (Mellon & Rebhun, 1976; Kuriyama & Sakai, 1974; Oliver, Albertini & Berlin, 1976). One of the most important protective mechanisms against auto-oxidative necrosis appears to be the glutathione system (Arias & Jacoby, 1976). Most cells maintain a high level (_ 10-3 M) of reduced glutathione (GSH). In concert with the enzyme glutathione peroxidase oxidative radicals are constantly removed with the concurrent production of oxidized glutathione (GSSG). Regeneration of GSH is done by the enzyme glutathione reductase which uses N A D P H as a hydrogen donor. The regeneration of N A D P H from NADP is linked to the hexosemonophosphate (HMP) shunt through the enzyme glucose-6-phosphate dehydrogenase (for review see Arias & Jacoby, 1976). The activation of leukocytes by a phagocytic stimulus or lymphocytes by a mitogenic stimulus is known to involve a burst of oxidative metabolism and an activation of the HMP-shunt. Recently, Baehner, Boxer, Allen & Davis (1977) have argued that this could in itself constitute a negative feedback system affecting phagocytosis, chemotaxis and surface membrane behavior (Baehner et al., 1977). Oliver's group concluded from their observations that the oxidative stress induced by phagocytosis in leukocytes caused a consumption of GSH and an increased production of GSSG and protein-gluta-
thione mixed disulfides. The data led to the hypothesis that GSH could protect microtubules against peroxidation while glutathione bound to protein could constitute a regulatory factor for the inhibition of microtubule assembly and induction of their disassembly (Oliver, Spielberg, Pearson & Schulman, 1978; Burchill, Oliver, Pearson, Leinbach & Berlin, 1978). From these and many other observations, a still vague but increasingly intriguing concept is emerging: besides its importance as a radical scavenger glutathione plays a key r61e in many processes involving t h i o l - p r o t e i n - d i s u l p h i d e interchanges (Freedman, 1979) which probably are of the utmost importance in the regulation of many cellular activities. It comes thus as no surprise to see that many thiols and sulfhydryl reagents have pronounced but poorly understood influences on various leukocyte functions (Broome & Jeng, 1972, 1973; Click, Benck & Alter, 1972; Bevan, Epstein & Cohn, 1974; Chen & Hirsch, 1972a and 1972b; Heber-Katz & Click, 1972; Fanger, Hart, Wells & Nisonoff, 1970; Toohey, 1975; Axelsson, Kallen, Nilsson & Trope, 1976). Recently we have shown that a sulfhydryl metabolite of levamisole, OMPI [2-oxo-3-(2-mercaptoethyl)-5-phenylimidazolidine] interacts in a biphasic way with microtubule formation: inhibition at high and promotion at low concentrations. These effects were apparently due to an interaction with tubulin sulfhydryls and could be modulated by glutathione (De Brabander, Aerts, Geuens, Van Ginckel, Van de Veire & Van Belle, 1978). Levamisole is extensively metabolized in vivo with one of the major products, OMPI being more active than levamisole 93
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M. DE BRABANDERet al.
itself in experimental systems such as colloidal carbon clearance in vivo (Van Ginckel & De Brabander, in press) and thymocyte stimulation by Con-A in vitro (J. Van Wauwe, personal communication). These observations have led us to formulate the hypothesis that the immunomodulating properties of levamisole are at least partially due to the formation of this metabolite (OMPI) which could, among other things, enhance microtubule integrity and function in leukocytes. Further theoretical and experimental implications of this hypothesis will be dealt with in the Discussion. In this paper we want to report our experiments which aimed at investigating in a more general way the possible interaction between levamisole or OMPI and the glutathione system in living cells. For this purpose we have made use of recent observations (Bannai, Tsukeda & Okumura, 1977) demonstrating that incubation of cultured human diploid fibroblasts in cystine-free medium results in cell death within 24 h. This is preceded by a rapid fall in the content of glutathione (y-L-glutamyl-L-cysteinylglycine) apparently due to the lack of supply of an essential component for its biosynthesis. Cell death was prevented by antioxidants such as vitamin-E, and synthetic antioxidants though they did not counteract the glutathione drop. This suggested that cell necrosis was due to the accumulation of oxidative substances a n d / o r free radical mediated damage in the absence of a sufficient supply of GSH and that the latter could be replaced as a scavenger by biologically active antioxidants. Using this simple experimental system we aimed at finding answers to the following questions: I Does glutathione depletion, through cystine deprivation affect the structure and function of the microtubule system in tissue cultured cells? 2 Do levamisole and OMPI interfere with the glutathione content in cultured cells either supplied with or deprived of cystine? 3 Do levamisole and OMPI interfere with the deleterious effects of glutathione depletion? In other words do they act as biological antioxidants? EXPERIMENTAL PROCEDURES
essential amino acids, antibiotics and 10% fetal bovine serum, at 37°C in a humidified atmosphere of 5% CO 2 in air. For the experiments the cells were seeded in Falcon plastic dishes of 3 or 6 cm diameter. After 24 h the growth medium was replaced by the experimental medium consisting of Eagle's minimal essential medium with or without 1-cystine, supplied with 10% calf serum and the experimental compounds or solvents. Morphology
The cultures were observed with an inverted phase contrast microscope. For ultrastructural purposes the cells were routinely fixed with glutaraldehyde and osmium tetroxide and embedded in Epon in the plate as described previously (De Brabander et aL, 1976). Thin sections were stained with uranyl acetate and lead citrate and observed in a Philips EM 201 electron microscope. Biochemical determination
1 ml of 4 × 10 -3 M EDTA disodium salt was put on the plates and the cells were scraped off into this medium. After lysis by freeze-thawing (3x) and centrifugation (5 min at 1500g) GSH and protein was determined on the supernatant. The determination of GSH (reduced + oxidized) is based on the cycling by glutathione-reductase (GR) and N A D P H of the system DTNB (5,5'-dithiobisnitrobenzoic acid) + GSH and was performed as described by Brehe and Burch (1976) with slight modifications. Thus, to 300/al of the supernatant was added 300/al of reagent 1 (150 mM PO4-buffer pH 7.2; 7.5 mM EDTA; 0.15 mM DTNB; 0.02°/o BSA) followed by 300/al of reagent 2 (35 mM imidazole buffer pH 7.2; 1 mM EDTA; 0.28 mM NADPH). The absorbance of this mixture at 412 nm was set at zero and the reaction was started by the addition of 25/al of GR (6 U/ml of imidazole buffer 31 mM pH 7.2). The increase in absorbance was recorded for several minutes in a Beckman-25 K instrument at 25°C and compared to the slopes for GSH-concentrations between 0.5 and 4 ~M. Protein was determined as described by Lowry, Rosebrough, Farr & Randall (1951).
Cells
NS cells are human embryonal skin fibroblasts (De Brabander, Van de Veire, Aerts, Borgers & Janssen, 1976). Culture
The cells were routinely cultured in Falcon plastic (Oxnard, California) tissue culture flasks in Eagle's minimal essential medium supplied with non-
RESULTS The effects of cystine deprivation on human embryonal fibroblasts (NS) are documented in Figs. I, 2 and 10. Cell death probably ensues from membrane damage which is characterized by an extreme blebbing of the cell surface. In cultures that are completely deprived of cystine, and that have been seeded at a density of 5000/cm 2, most cells become
Fig. Fig. Fig. Fig.
1. 3. 5. 7.
Control, 48 h. Vitamin-E, 2.5 × 10 -4 M, 5 days. Butylated hydroxianisole 5× 10 -3 M, 5 days. OMPI, 10 -5 M, 5 days. Figs. 1 - 8
Fig. Fig. Fig. Fig.
2. 4. 6. 8.
Control, 5 days. n-Propyl gallate 4 × 10 -5 M, 5 days. Levamisole, 10 -3 M, 5 days. OMPI, 8× 10 -3 M, 5 days.
Magnification of all micrographs is × 120. Phase contrast micrographs of NS-cells cultured in medium without cystine. 95
Fig. 10. Electron micrograph o f a prenecrotic NS-cell cultured in medium without cystine for 24 h. Normal microtubules (arrows) are seen, but the mitochondria (arrowheads) are severely affected. The cristae have largely disappeared and the matrix is densified. ( x 16.000.)
Fig. 9. Electron micrograph of a NS cell in normal medium. Microtubules (arrows) and normal elongated mitochondria (arrowheads) are seen. ( x 23.000.)
96
97
Levamisole Protection Against Glutathione Depletion necrotic between 15 and 24 h. As can be seen in Figs. 1 and 2 many cells do not detach from the substratum but remain attached to it as cell-ghosts. For this reason we did not use protein determination as a measure of cell growth and viability hut morphologic observation with the phase contrast microscope. This has the additional advantage that one can observe several stages during the development of cell necrosis. Cultures which have been deprived of cystine after they had become confluent are resistant much longer. They become necrotic after several days (3-4) only. At the ultrastructural level (Fig. 10), the main damage in prenecrotic cells consists of mitochondrial degeneration, dilatation of the endoplasmic reticulum and an accumulation of autophagic vacuoles. The microtubules in these cells remain remarkably intact. Moreover, in cultures, where ±80% of the cells have become necrotic, normal mitotic figures can often be observed.
Figures 3-8 show the protective effect of several compounds against cystine deprivation-induced necrosis. Unlike the control cells which became necrotic within 24 h the treated cells were still alive and dividing after 5 days without cystine. This could be achieved by applying the following compounds of which the minimally active molar concentration is given between brackets: vitamin E (2.5x 10-~); butylated hydroxianisole (5x 10-6); n-propylgallate (4×10-5); levamisole (10-3); dexamisole (10-3); OMPI (10-5); mercaptoethanol 00-6); L-cysteine 00-5); L-cystine 00-3); N,N'-diphenyl-l,4-phenylenediamine 00-5); SeO 2 00-4). The following compounds were inactive at the indicated concentrations: foetal bovine serum, a rich source of GSH-protein mixed disulfides (Bump & Reed, 1977) (10°70 v/v); methionine, a potential metabolic donor of Lcysteine (10 -4 M); catalase 04,000 U/ml); GSH (10 -3 M); dithiothreitol (10 -3 M); vitamin C (10 -3 M). None of the compounds used had
nM/mg
14.t 12-
8-
•
6-
'"
4-
2-
00
I
i
2
5
2~4
4'8 hours
Fig. 11. Glutathione content in NS-cells expressed as nanomoles per milligram protein at different time intervals after seeding. NS-cells were seeded in dishes (diameter 3 cm) at a density of 4x 10-4/cm2. After 24 h (h 0) the experimental medium was added. The points represent the determinations in separate cultures. - - • - - Control medium with cystine; - • - - control medium without cystine; - -O- - Vitamin E 2, 5 × 10-4 M in medium without cystine; - - O - - OMPI 4× 10-5 M in medium without cystine; - - A - - levamisole 10-3 M in medium without cystine. The control cultures without cystine had become completely necrotic after 24 and 48 h, unlike the other cultures in which no signs of necrosis were visible. -
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M. DE BRABANDERet al.
appreciable effects, at the indicated concentrations, on the growth, viability or morphology of the cells in medium supplied with cystine. Only levamisole and dexamisole, produced some vacuolation in the cells and an accumulation of phase-dark granules which are probably autophagic cytolysomes (manuscript in preparation). OMPI by itself destroys microtubules in tissue-cultured cells at a concentration o f 1.6 × 10 -4 M (submitted for publication). In cystinedepleted cells this is already visible with 8 x 10 -~ M, This correlates with our earlier observations that both cystine and GSH inhibit the antimicrotubule effect of OMPI (submitted for publication). However, even with these concentrations the cells remain viable in a cystine-depleted medium for more than 5 days. Figure 11 shows the glutathione content in NS cells at different times after cystine deprivation treated with Vitamin-E, levamisole or O M P I . Cystine deprivation results in a rapid fall of the glutathione content. The rate of glutathione depletion is identical in control cultures and in treated cultures. The amount of glutathione after 24 and 48 h in treated cultures cannot be compared with the amount remaining in the control cultures since the latter consist of dead cells. None of the compounds appreciably affected the glutathione content in cells cultured in complete medium containing the normal amount of cystine (data not shown).
DISCUSSION The questions posed in the introduction can be answered as follows: 1. Glutathione depletion through cystine deprivation does not seem to primarily affect the structure or function of the microtubule system in tissue-cultured cells. In other words, a possible effect on microtubule integrity is obscured by cell necrosis which is probably due to the interaction of oxidative radicals with various membrane systems. Indeed microtubules are still abundant in prenecrotic cells the mitochondria of which are already severely damaged and the chromatin of which is becoming pyknotic. Moreover, the change in cell shape, typical for the induced microtubule disintegration does not occur and the subcellular organelle topography remains ordered (De Brabander et al., 1976). It is obvious, however, that more subtle changes in microtubuledependent functions would escape the observation techniques used here. 2. Levamisole and OMPI protect cultured cells against necrosis induced by glutathione depletion through cystine deprivation. On a molar basis the
metabolite (OMPI) is approximately 100 times more active than ievamisole. 3. Similarly to what has been shown for vitamin E and synthetic antioxidants (Bannai etal., 1977), levamisole and OMPI do not protect cultured cells against cystine deprivation by maintaining a normal level of glutathione. Thus, they act either directly as radical scavengers or they protect the target molecules in some other way not related to the glutathione system. In a more speculative way we want to draw two major conclusions from these data. First, they represent yet another system in which the metabolite OMPI is much more active than the parent compound (levamisole). This is the case also in enhancement of the clearance rate of intravenously injected colloidal carbon (Van Ginckel & De Brabander, in press) and in the enhancement of Con-A induced blast transformation of mouse thymocytes in culture (J. Van Wauwe, Janssen Pharmaceutica, personal communication). Although it cannot completely be ruled out that levamisole itself shares these activities with OMPI we cannot overlook the fact that levamisole is extensively metabolized in vivo, one of the main products being OMPI (Janssen, 1976). Second, the antioxidant properties of levamisole and OMPI might have implications for the clinical effects and mechanism of action of levamisole as an immunomodulating drug. It can be argued that a plasma concentration of 10 -3 M levamisole cannot be obtained in vivo. However, in the system described here dramatic effects are measured and cell death is taken as a parameter. It is conceivable that more subtle effects of radicals, bearing only functional consequences, can be reversed by lower concentrations of levamisole. Moreover, it is worth stressing that this high concentration of levamisole (_+10-3 M) is needed in most in vitro experiments showing improved leukocyte function (for review see Symoens & Rosenthal, 1977). Here again, we favor the hypothesis that in vivo the important compound is the metabolite. With regard to the immunotropic properties of levamisole, as a potential pro-drug for OMPI, two groups of observations are worth further attention. On the one hand, it has been extensively shown that thiols such as mercaptoethanol which share antioxidant and other properties with OMPI, markedly enhance the viability, replication and function of lymphoid cells in culture (Broome & Jeng, 1972, 1973; Click et al., 1972; Bevan et al., 1974; Chen & Hirsch, 1972a and 1972b; Laferty, Ryan & Misko, 1974; Heber-Katz & Click, 1972; Fanger et al., 1970 Toohey, 1975; Axelsson et al., 1976). Potential beneficial effects of free radical reaction inhibitors (vita-
Levamisole Protection Against Glutathione Depletion min E, antioxidants, thiols) have long been debated and often revived. Recently, Harman et al. (Harman, Heidrich & Eddy, 1977) have shown that vitamin E, other antioxidants such as mercaptoethanol and also levamisole enhanced the immune response in ageing mice. They suggested that free radical reactions might contribute to the decline of the immune response with age and that inhibitors might be of prophylactic value. Incidentally, they hypothesized, on theoretical grounds, that the beneficial effect of levamisole might be associated with its action as a free radical reaction inhibitor. On the other hand, Baehner et al. (1977) have argued that in leukocytes the burst of oxidative metabolism induced by a phagocytic stimulus, could in itself constitute a negative feedback system affecting phagocytosis, chemotaxis and surface membrane behavior. The threshold of this feedback is apparently susceptible to modulation by radical scavengers (Baehner et al., 1977). In conclusion, we propose as a working hypothesis that the enhancing effects of levamisole on leukocyte functions would be due, at least in part, to the interaction of the compound itself or, more probably, of its metabolite OMPI with oxidative radicals produced in stimulated leukocytes. This would implicate that leukocytes with a spontaneous defect in the natural homeostatic mechanism, the glutathione system, could be particularly susceptible to the anti-
99
oxidant effect of the drug and would constitute a better adapted test system. A large variety of cellular molecules and enzyme systems are susceptible to oxidative and free radical mediated damage. One can only speculate at this time about the nature of the primary target structures that would benefit from the protective effect of levamisole or OMPI and as a consequence, enhance leukocyte functions. In addition to our previous observations implicating tubulin and microtubules we now suggest the cell membrane as a potential candidate. Probably others will arise. However, the potential beneficial effects of free radical scavengers must be weighed against possible deleterious interactions with free radical reactions essential to the proper functioning of the cell. Whatever finally emerges as the most important interaction, the present hypothesis should be easily verified or negated by studying, in a comparative way, the effects of levamisole and OMPI on the functional performance of both normal leukocytes and leukocytes with a glutathione homeostasis deficiency. A c k n o w l e d g e m e n t s - - T h e assistance of L. Leyssen for pre-
paring the figures, H. Vanhove and Dr. W. Amery for reviewing the manuscript, and B. Wouters for typing the manuscript is greatly appreciated. The research was supported by a grant from the Instituut tot Aanmoediging van Wetenschappelijk Onderzoek in Nijverheid en Landbouw (Brussels, Belgium).
REFERENCES
ARIAS, |. M. & JACOBY,W. B. (1976). Glutathione: Metabolism and Function. Raven Press, New York. AXELSSON, J. A., KALLEN,B., NILSSON,O. & TROPE, C. (1976). Effect of 2-mercaptoethanol on the mixed leukocyte reaction in man. Acta path. microbiol, scand. 84, 390-396. BAEHNER,R. L., BOXER,L. A., ALLEN,J. M. & DAVIS,J. (1977). Auto-oxidation as a basis for altered function by polymorphonuclear leukocytes. B l o o d 50, 327-335. BANNAI,S., TSEKUDA,H. & OKUMURA,H. (1977). Effect of antioxidants on cultured human deploid fibroblasts exposed to cystine free medium. Biochem. biophys. Res. C o m m u n . 74, 1582-1588. BEVAN, M. J., EPSTEIN, R. & COHN, M. (1974). The effect of 2-mercaptoethanol on murine mixed lymphocyte cultures. J. exp. Med. 139, 1025-1030. BREHE, J. E. & BURCH,H. B. (1976). Enzymatic assay for glutathione. Analyt. Biochem. 74, 189-195. BROOME, J. D. & JENG, M. W. (1972). Growth stimulation of mouse leukemia cells by thiols and disulfides in vitro. J. hath. Cancer Inst. 49, 579-581. BROOME,J. D. & JENG, M. W. (1973). Promotion of replication in lymphoid cells by specific thiols and disulfides in vitro. J. exp. Med. 138, 574--592. BUMP, E. D. & REED, D. J. (1977). A unique property of fetal bovine serum: high levels of protein--glutathione mixed disulfides. In Vitro 13, 115--118. BURCHILL,B. R., OLIVER, J. M., PEARSON,C. B., LEINBACH,E. D. & BERLIN,R. D. (1978). Microtubule dynamics and glutathione metabolism in phagocytizing human polymorphonuclear leukocytes. J. Cell Biol. 76, 439-447. CHEN, C. & HIRSCH, J. G. (1972a). Restoration of antibody forming capacity in cultures of nonadherent spleen cells by mercaptoethanol. Science 176, 60--61. CHEN, C. & HIRSCH,J. G. (1972b). The effects of mercaptoethanol and peritoneal macrophages on the antibody-forming capacity of nonadherent mouse spleen cells in vitro. J. exp. Med. 136, 604--617. CLICK, R. E., BENCK,L. & ALTER,B. J. (1972). Immune responses in vitro. I. Culture conditions for antibody synthesis. Cell I m m u n . 3, 264--276.
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DE BRABANDER,M. J., VAN DE VEIRE, R. M. L., AERTS, F. E. M., BORGERS, M. & JANSSEN, P. A. J. (1976). The effects of methyl [5-(2-thienylcarbonyl)-lH-benzimidazol-2-yi] carbamate, (R 17 934; NSC 238159), a new synthetic antitumoral drug interfering with microtubules, on mammalian cells cultured in vitro. Cancer Res. 36, 905-916. DE BRABANDER,M., AERTS, F., GEUENS, G., VAN GINCKEL, R., VAN DE VEIRE, R. & VAN BELLE, H. (1978). DL-2-OXO-3(2-mercaptoethyl)-5-phenylimidazolidine. A sulfhydryl metabolite of levamisole that interacts with microtubules. Chem. Biol. Interact. 23, 45--63. FANGER, M. W., HART, D. A., WELLS, J. V. & NISONOFF, A. (1970). Enhancement by reducing agents of the transformation of human and rabbit peripheral lymphocytes. J. l m m u n . 105, 1043--1045. FREEDMAN, R. B. (1979). HOW many distinct enzymes are responsible for the several cellular processes involving thiol: protein-disulphide interchange? FEBS Lett. 97, 201--210. HARMAN, D., HEIDRICH, M. L. & EDDY, D. E. (1977). Free radical theory of aging: effect of free-radical reaction inhibitors on the immune response. J. A m . Geriat. Soc. 25, 400--407. HEBER-KATZ, E. & CLICK, R. E. (1972). Immune responses in vitro. V. R61e of mercaptoethanol in the mixed leukocyte reaction. Cell I m m u n . 5, 410--418. JANSSEN, P. A. J. (1976). The levamisole story. In Progress in Drug Research, Vol. 20 (eds. JUCKER, E.) pp. 347. Birkhailser Verlag, Stuttgart-l. KURIYAMA, R. & SAKAI, H. (1974). R61e of tubulin-SH groups in polymerization to microtubules. Functional-SH groups in tubulin for polymerization. J. Biochem. (Tokyo) 76, 651-654. LAFERTY, K., RYAN, M. & MISrO, I. (1974). An improved system for the assay of stimulation in mouse mixed leukocyte cultures. J. I m m u n . Meth. 4, 263--273. LowRY, O. H., ROSEBROUGH,N. J., FARR, A. L. & RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-276. MELLON, M. G. & REaHUN, L. I. (1976). Sulfhydryls and the in vitro polymerization of tubulin. J. Cell Biol. 70, 226-238. OLIVER, J. M., ALBERTINI, D. F. & BERLIN, R. D. (1976). Effects of glutathione-oxidizing agents on microtubule assembly and microtubule dependent surface properties of human neutrophils. J. Cell Biol. 71,921-932. OLIVER, J. M., SPIELBERG, S. D., PEARSON, C. B. & SCHULMAN, J. D. (1978). Microtubule assembly and function in normal and glutathione synthetase-deficient polymorphonuclear leukocytes. J. Immun. 120, ll81-1186. SYMOENS, J. & ROSENTHAL, M. (1977). Levamisole in the modulation of the immune response: the current experimental and clinical state. J. Reticuloendothel. Soc. 21, 175-221. TOOHEY, J. I. (1975). Sulfhydryl dependence in primary explant hematopoietic cells. Inhibition of growth in vitro with vitamin BI2 compounds. Proc. hath. Acad. Sci. U.S.A. 72, 73-77. VAN GINCKEL, R. & DE BRABANDER,M. The influence of a levamisole metabolite DL-2-oxo-3-(2-mercaptoethyl)-5-phenylimidazolidine on carbon clearance in mice. J. Reticuloendothel. Soc. in press.
0192-0561/79/0401-0093 $02,00/0
PROTECTIVE EFFECT OF LEVAMISOLE A N D ITS SULFHYDRYL METABOLITE OMPI AGAINST CELL DEATH INDUCED BY GLUTATHIONE DEPLETION M. DE BRABANDER, H. VAN BELLE, F. AERTS, R. VAN DE VE|RE and G. GEUENS Laboratories of Oncology and Biochemistry, Janssen Pharmaceutica Research Laboratories, B-2340 Beerse, Belgium (Received 19 March 1979 and in final form 26 April 1979)
Abstraet--Levamisole protects cultured cells against auto-oxidative necrosis induced by glutathione depletion. The sulfhydryl metabolite, OMPI, is approximately 100 times more active in this respect. As is the case for vitamin E and synthetic antioxidants, the cell rescue is not due to the inhibition of glutathione depletion but probably to a direct radical scavenging effect. The data suggest the following hypothesis: the immunomodulating effect of levamisole is at least partially due to its metabolite OMPI which enhances leukocyte functions by scavenging deleterious oxidative radicals the formation of which is enhanced in activated cells. Various cellular metabolic processes constantly produce oxidative substances and free radicals that are incompatible with the integrity and survival of the cell. Some of the best known targets for these reactive intermediates are membrane lipids (for review see Arias & Jacoby, 1976) and the sulfhydryl residues of various proteins such as tubulin (Mellon & Rebhun, 1976; Kuriyama & Sakai, 1974; Oliver, Albertini & Berlin, 1976). One of the most important protective mechanisms against auto-oxidative necrosis appears to be the glutathione system (Arias & Jacoby, 1976). Most cells maintain a high level (_ 10-3 M) of reduced glutathione (GSH). In concert with the enzyme glutathione peroxidase oxidative radicals are constantly removed with the concurrent production of oxidized glutathione (GSSG). Regeneration of GSH is done by the enzyme glutathione reductase which uses N A D P H as a hydrogen donor. The regeneration of N A D P H from NADP is linked to the hexosemonophosphate (HMP) shunt through the enzyme glucose-6-phosphate dehydrogenase (for review see Arias & Jacoby, 1976). The activation of leukocytes by a phagocytic stimulus or lymphocytes by a mitogenic stimulus is known to involve a burst of oxidative metabolism and an activation of the HMP-shunt. Recently, Baehner, Boxer, Allen & Davis (1977) have argued that this could in itself constitute a negative feedback system affecting phagocytosis, chemotaxis and surface membrane behavior (Baehner et al., 1977). Oliver's group concluded from their observations that the oxidative stress induced by phagocytosis in leukocytes caused a consumption of GSH and an increased production of GSSG and protein-gluta-
thione mixed disulfides. The data led to the hypothesis that GSH could protect microtubules against peroxidation while glutathione bound to protein could constitute a regulatory factor for the inhibition of microtubule assembly and induction of their disassembly (Oliver, Spielberg, Pearson & Schulman, 1978; Burchill, Oliver, Pearson, Leinbach & Berlin, 1978). From these and many other observations, a still vague but increasingly intriguing concept is emerging: besides its importance as a radical scavenger glutathione plays a key r61e in many processes involving t h i o l - p r o t e i n - d i s u l p h i d e interchanges (Freedman, 1979) which probably are of the utmost importance in the regulation of many cellular activities. It comes thus as no surprise to see that many thiols and sulfhydryl reagents have pronounced but poorly understood influences on various leukocyte functions (Broome & Jeng, 1972, 1973; Click, Benck & Alter, 1972; Bevan, Epstein & Cohn, 1974; Chen & Hirsch, 1972a and 1972b; Heber-Katz & Click, 1972; Fanger, Hart, Wells & Nisonoff, 1970; Toohey, 1975; Axelsson, Kallen, Nilsson & Trope, 1976). Recently we have shown that a sulfhydryl metabolite of levamisole, OMPI [2-oxo-3-(2-mercaptoethyl)-5-phenylimidazolidine] interacts in a biphasic way with microtubule formation: inhibition at high and promotion at low concentrations. These effects were apparently due to an interaction with tubulin sulfhydryls and could be modulated by glutathione (De Brabander, Aerts, Geuens, Van Ginckel, Van de Veire & Van Belle, 1978). Levamisole is extensively metabolized in vivo with one of the major products, OMPI being more active than levamisole 93
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M. DE BRABANDERet al.
itself in experimental systems such as colloidal carbon clearance in vivo (Van Ginckel & De Brabander, in press) and thymocyte stimulation by Con-A in vitro (J. Van Wauwe, personal communication). These observations have led us to formulate the hypothesis that the immunomodulating properties of levamisole are at least partially due to the formation of this metabolite (OMPI) which could, among other things, enhance microtubule integrity and function in leukocytes. Further theoretical and experimental implications of this hypothesis will be dealt with in the Discussion. In this paper we want to report our experiments which aimed at investigating in a more general way the possible interaction between levamisole or OMPI and the glutathione system in living cells. For this purpose we have made use of recent observations (Bannai, Tsukeda & Okumura, 1977) demonstrating that incubation of cultured human diploid fibroblasts in cystine-free medium results in cell death within 24 h. This is preceded by a rapid fall in the content of glutathione (y-L-glutamyl-L-cysteinylglycine) apparently due to the lack of supply of an essential component for its biosynthesis. Cell death was prevented by antioxidants such as vitamin-E, and synthetic antioxidants though they did not counteract the glutathione drop. This suggested that cell necrosis was due to the accumulation of oxidative substances a n d / o r free radical mediated damage in the absence of a sufficient supply of GSH and that the latter could be replaced as a scavenger by biologically active antioxidants. Using this simple experimental system we aimed at finding answers to the following questions: I Does glutathione depletion, through cystine deprivation affect the structure and function of the microtubule system in tissue cultured cells? 2 Do levamisole and OMPI interfere with the glutathione content in cultured cells either supplied with or deprived of cystine? 3 Do levamisole and OMPI interfere with the deleterious effects of glutathione depletion? In other words do they act as biological antioxidants? EXPERIMENTAL PROCEDURES
essential amino acids, antibiotics and 10% fetal bovine serum, at 37°C in a humidified atmosphere of 5% CO 2 in air. For the experiments the cells were seeded in Falcon plastic dishes of 3 or 6 cm diameter. After 24 h the growth medium was replaced by the experimental medium consisting of Eagle's minimal essential medium with or without 1-cystine, supplied with 10% calf serum and the experimental compounds or solvents. Morphology
The cultures were observed with an inverted phase contrast microscope. For ultrastructural purposes the cells were routinely fixed with glutaraldehyde and osmium tetroxide and embedded in Epon in the plate as described previously (De Brabander et aL, 1976). Thin sections were stained with uranyl acetate and lead citrate and observed in a Philips EM 201 electron microscope. Biochemical determination
1 ml of 4 × 10 -3 M EDTA disodium salt was put on the plates and the cells were scraped off into this medium. After lysis by freeze-thawing (3x) and centrifugation (5 min at 1500g) GSH and protein was determined on the supernatant. The determination of GSH (reduced + oxidized) is based on the cycling by glutathione-reductase (GR) and N A D P H of the system DTNB (5,5'-dithiobisnitrobenzoic acid) + GSH and was performed as described by Brehe and Burch (1976) with slight modifications. Thus, to 300/al of the supernatant was added 300/al of reagent 1 (150 mM PO4-buffer pH 7.2; 7.5 mM EDTA; 0.15 mM DTNB; 0.02°/o BSA) followed by 300/al of reagent 2 (35 mM imidazole buffer pH 7.2; 1 mM EDTA; 0.28 mM NADPH). The absorbance of this mixture at 412 nm was set at zero and the reaction was started by the addition of 25/al of GR (6 U/ml of imidazole buffer 31 mM pH 7.2). The increase in absorbance was recorded for several minutes in a Beckman-25 K instrument at 25°C and compared to the slopes for GSH-concentrations between 0.5 and 4 ~M. Protein was determined as described by Lowry, Rosebrough, Farr & Randall (1951).
Cells
NS cells are human embryonal skin fibroblasts (De Brabander, Van de Veire, Aerts, Borgers & Janssen, 1976). Culture
The cells were routinely cultured in Falcon plastic (Oxnard, California) tissue culture flasks in Eagle's minimal essential medium supplied with non-
RESULTS The effects of cystine deprivation on human embryonal fibroblasts (NS) are documented in Figs. I, 2 and 10. Cell death probably ensues from membrane damage which is characterized by an extreme blebbing of the cell surface. In cultures that are completely deprived of cystine, and that have been seeded at a density of 5000/cm 2, most cells become
Fig. Fig. Fig. Fig.
1. 3. 5. 7.
Control, 48 h. Vitamin-E, 2.5 × 10 -4 M, 5 days. Butylated hydroxianisole 5× 10 -3 M, 5 days. OMPI, 10 -5 M, 5 days. Figs. 1 - 8
Fig. Fig. Fig. Fig.
2. 4. 6. 8.
Control, 5 days. n-Propyl gallate 4 × 10 -5 M, 5 days. Levamisole, 10 -3 M, 5 days. OMPI, 8× 10 -3 M, 5 days.
Magnification of all micrographs is × 120. Phase contrast micrographs of NS-cells cultured in medium without cystine. 95
Fig. 10. Electron micrograph o f a prenecrotic NS-cell cultured in medium without cystine for 24 h. Normal microtubules (arrows) are seen, but the mitochondria (arrowheads) are severely affected. The cristae have largely disappeared and the matrix is densified. ( x 16.000.)
Fig. 9. Electron micrograph of a NS cell in normal medium. Microtubules (arrows) and normal elongated mitochondria (arrowheads) are seen. ( x 23.000.)
96
97
Levamisole Protection Against Glutathione Depletion necrotic between 15 and 24 h. As can be seen in Figs. 1 and 2 many cells do not detach from the substratum but remain attached to it as cell-ghosts. For this reason we did not use protein determination as a measure of cell growth and viability hut morphologic observation with the phase contrast microscope. This has the additional advantage that one can observe several stages during the development of cell necrosis. Cultures which have been deprived of cystine after they had become confluent are resistant much longer. They become necrotic after several days (3-4) only. At the ultrastructural level (Fig. 10), the main damage in prenecrotic cells consists of mitochondrial degeneration, dilatation of the endoplasmic reticulum and an accumulation of autophagic vacuoles. The microtubules in these cells remain remarkably intact. Moreover, in cultures, where ±80% of the cells have become necrotic, normal mitotic figures can often be observed.
Figures 3-8 show the protective effect of several compounds against cystine deprivation-induced necrosis. Unlike the control cells which became necrotic within 24 h the treated cells were still alive and dividing after 5 days without cystine. This could be achieved by applying the following compounds of which the minimally active molar concentration is given between brackets: vitamin E (2.5x 10-~); butylated hydroxianisole (5x 10-6); n-propylgallate (4×10-5); levamisole (10-3); dexamisole (10-3); OMPI (10-5); mercaptoethanol 00-6); L-cysteine 00-5); L-cystine 00-3); N,N'-diphenyl-l,4-phenylenediamine 00-5); SeO 2 00-4). The following compounds were inactive at the indicated concentrations: foetal bovine serum, a rich source of GSH-protein mixed disulfides (Bump & Reed, 1977) (10°70 v/v); methionine, a potential metabolic donor of Lcysteine (10 -4 M); catalase 04,000 U/ml); GSH (10 -3 M); dithiothreitol (10 -3 M); vitamin C (10 -3 M). None of the compounds used had
nM/mg
14.t 12-
8-
•
6-
'"
4-
2-
00
I
i
2
5
2~4
4'8 hours
Fig. 11. Glutathione content in NS-cells expressed as nanomoles per milligram protein at different time intervals after seeding. NS-cells were seeded in dishes (diameter 3 cm) at a density of 4x 10-4/cm2. After 24 h (h 0) the experimental medium was added. The points represent the determinations in separate cultures. - - • - - Control medium with cystine; - • - - control medium without cystine; - -O- - Vitamin E 2, 5 × 10-4 M in medium without cystine; - - O - - OMPI 4× 10-5 M in medium without cystine; - - A - - levamisole 10-3 M in medium without cystine. The control cultures without cystine had become completely necrotic after 24 and 48 h, unlike the other cultures in which no signs of necrosis were visible. -
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M. DE BRABANDERet al.
appreciable effects, at the indicated concentrations, on the growth, viability or morphology of the cells in medium supplied with cystine. Only levamisole and dexamisole, produced some vacuolation in the cells and an accumulation of phase-dark granules which are probably autophagic cytolysomes (manuscript in preparation). OMPI by itself destroys microtubules in tissue-cultured cells at a concentration o f 1.6 × 10 -4 M (submitted for publication). In cystinedepleted cells this is already visible with 8 x 10 -~ M, This correlates with our earlier observations that both cystine and GSH inhibit the antimicrotubule effect of OMPI (submitted for publication). However, even with these concentrations the cells remain viable in a cystine-depleted medium for more than 5 days. Figure 11 shows the glutathione content in NS cells at different times after cystine deprivation treated with Vitamin-E, levamisole or O M P I . Cystine deprivation results in a rapid fall of the glutathione content. The rate of glutathione depletion is identical in control cultures and in treated cultures. The amount of glutathione after 24 and 48 h in treated cultures cannot be compared with the amount remaining in the control cultures since the latter consist of dead cells. None of the compounds appreciably affected the glutathione content in cells cultured in complete medium containing the normal amount of cystine (data not shown).
DISCUSSION The questions posed in the introduction can be answered as follows: 1. Glutathione depletion through cystine deprivation does not seem to primarily affect the structure or function of the microtubule system in tissue-cultured cells. In other words, a possible effect on microtubule integrity is obscured by cell necrosis which is probably due to the interaction of oxidative radicals with various membrane systems. Indeed microtubules are still abundant in prenecrotic cells the mitochondria of which are already severely damaged and the chromatin of which is becoming pyknotic. Moreover, the change in cell shape, typical for the induced microtubule disintegration does not occur and the subcellular organelle topography remains ordered (De Brabander et al., 1976). It is obvious, however, that more subtle changes in microtubuledependent functions would escape the observation techniques used here. 2. Levamisole and OMPI protect cultured cells against necrosis induced by glutathione depletion through cystine deprivation. On a molar basis the
metabolite (OMPI) is approximately 100 times more active than ievamisole. 3. Similarly to what has been shown for vitamin E and synthetic antioxidants (Bannai etal., 1977), levamisole and OMPI do not protect cultured cells against cystine deprivation by maintaining a normal level of glutathione. Thus, they act either directly as radical scavengers or they protect the target molecules in some other way not related to the glutathione system. In a more speculative way we want to draw two major conclusions from these data. First, they represent yet another system in which the metabolite OMPI is much more active than the parent compound (levamisole). This is the case also in enhancement of the clearance rate of intravenously injected colloidal carbon (Van Ginckel & De Brabander, in press) and in the enhancement of Con-A induced blast transformation of mouse thymocytes in culture (J. Van Wauwe, Janssen Pharmaceutica, personal communication). Although it cannot completely be ruled out that levamisole itself shares these activities with OMPI we cannot overlook the fact that levamisole is extensively metabolized in vivo, one of the main products being OMPI (Janssen, 1976). Second, the antioxidant properties of levamisole and OMPI might have implications for the clinical effects and mechanism of action of levamisole as an immunomodulating drug. It can be argued that a plasma concentration of 10 -3 M levamisole cannot be obtained in vivo. However, in the system described here dramatic effects are measured and cell death is taken as a parameter. It is conceivable that more subtle effects of radicals, bearing only functional consequences, can be reversed by lower concentrations of levamisole. Moreover, it is worth stressing that this high concentration of levamisole (_+10-3 M) is needed in most in vitro experiments showing improved leukocyte function (for review see Symoens & Rosenthal, 1977). Here again, we favor the hypothesis that in vivo the important compound is the metabolite. With regard to the immunotropic properties of levamisole, as a potential pro-drug for OMPI, two groups of observations are worth further attention. On the one hand, it has been extensively shown that thiols such as mercaptoethanol which share antioxidant and other properties with OMPI, markedly enhance the viability, replication and function of lymphoid cells in culture (Broome & Jeng, 1972, 1973; Click et al., 1972; Bevan et al., 1974; Chen & Hirsch, 1972a and 1972b; Laferty, Ryan & Misko, 1974; Heber-Katz & Click, 1972; Fanger et al., 1970 Toohey, 1975; Axelsson et al., 1976). Potential beneficial effects of free radical reaction inhibitors (vita-
Levamisole Protection Against Glutathione Depletion min E, antioxidants, thiols) have long been debated and often revived. Recently, Harman et al. (Harman, Heidrich & Eddy, 1977) have shown that vitamin E, other antioxidants such as mercaptoethanol and also levamisole enhanced the immune response in ageing mice. They suggested that free radical reactions might contribute to the decline of the immune response with age and that inhibitors might be of prophylactic value. Incidentally, they hypothesized, on theoretical grounds, that the beneficial effect of levamisole might be associated with its action as a free radical reaction inhibitor. On the other hand, Baehner et al. (1977) have argued that in leukocytes the burst of oxidative metabolism induced by a phagocytic stimulus, could in itself constitute a negative feedback system affecting phagocytosis, chemotaxis and surface membrane behavior. The threshold of this feedback is apparently susceptible to modulation by radical scavengers (Baehner et al., 1977). In conclusion, we propose as a working hypothesis that the enhancing effects of levamisole on leukocyte functions would be due, at least in part, to the interaction of the compound itself or, more probably, of its metabolite OMPI with oxidative radicals produced in stimulated leukocytes. This would implicate that leukocytes with a spontaneous defect in the natural homeostatic mechanism, the glutathione system, could be particularly susceptible to the anti-
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oxidant effect of the drug and would constitute a better adapted test system. A large variety of cellular molecules and enzyme systems are susceptible to oxidative and free radical mediated damage. One can only speculate at this time about the nature of the primary target structures that would benefit from the protective effect of levamisole or OMPI and as a consequence, enhance leukocyte functions. In addition to our previous observations implicating tubulin and microtubules we now suggest the cell membrane as a potential candidate. Probably others will arise. However, the potential beneficial effects of free radical scavengers must be weighed against possible deleterious interactions with free radical reactions essential to the proper functioning of the cell. Whatever finally emerges as the most important interaction, the present hypothesis should be easily verified or negated by studying, in a comparative way, the effects of levamisole and OMPI on the functional performance of both normal leukocytes and leukocytes with a glutathione homeostasis deficiency. A c k n o w l e d g e m e n t s - - T h e assistance of L. Leyssen for pre-
paring the figures, H. Vanhove and Dr. W. Amery for reviewing the manuscript, and B. Wouters for typing the manuscript is greatly appreciated. The research was supported by a grant from the Instituut tot Aanmoediging van Wetenschappelijk Onderzoek in Nijverheid en Landbouw (Brussels, Belgium).
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DE BRABANDER,M. J., VAN DE VEIRE, R. M. L., AERTS, F. E. M., BORGERS, M. & JANSSEN, P. A. J. (1976). The effects of methyl [5-(2-thienylcarbonyl)-lH-benzimidazol-2-yi] carbamate, (R 17 934; NSC 238159), a new synthetic antitumoral drug interfering with microtubules, on mammalian cells cultured in vitro. Cancer Res. 36, 905-916. DE BRABANDER,M., AERTS, F., GEUENS, G., VAN GINCKEL, R., VAN DE VEIRE, R. & VAN BELLE, H. (1978). DL-2-OXO-3(2-mercaptoethyl)-5-phenylimidazolidine. A sulfhydryl metabolite of levamisole that interacts with microtubules. Chem. Biol. Interact. 23, 45--63. FANGER, M. W., HART, D. A., WELLS, J. V. & NISONOFF, A. (1970). Enhancement by reducing agents of the transformation of human and rabbit peripheral lymphocytes. J. l m m u n . 105, 1043--1045. FREEDMAN, R. B. (1979). HOW many distinct enzymes are responsible for the several cellular processes involving thiol: protein-disulphide interchange? FEBS Lett. 97, 201--210. HARMAN, D., HEIDRICH, M. L. & EDDY, D. E. (1977). Free radical theory of aging: effect of free-radical reaction inhibitors on the immune response. J. A m . Geriat. Soc. 25, 400--407. HEBER-KATZ, E. & CLICK, R. E. (1972). Immune responses in vitro. V. R61e of mercaptoethanol in the mixed leukocyte reaction. Cell I m m u n . 5, 410--418. JANSSEN, P. A. J. (1976). The levamisole story. In Progress in Drug Research, Vol. 20 (eds. JUCKER, E.) pp. 347. Birkhailser Verlag, Stuttgart-l. KURIYAMA, R. & SAKAI, H. (1974). R61e of tubulin-SH groups in polymerization to microtubules. Functional-SH groups in tubulin for polymerization. J. Biochem. (Tokyo) 76, 651-654. LAFERTY, K., RYAN, M. & MISrO, I. (1974). An improved system for the assay of stimulation in mouse mixed leukocyte cultures. J. I m m u n . Meth. 4, 263--273. LowRY, O. H., ROSEBROUGH,N. J., FARR, A. L. & RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-276. MELLON, M. G. & REaHUN, L. I. (1976). Sulfhydryls and the in vitro polymerization of tubulin. J. Cell Biol. 70, 226-238. OLIVER, J. M., ALBERTINI, D. F. & BERLIN, R. D. (1976). Effects of glutathione-oxidizing agents on microtubule assembly and microtubule dependent surface properties of human neutrophils. J. Cell Biol. 71,921-932. OLIVER, J. M., SPIELBERG, S. D., PEARSON, C. B. & SCHULMAN, J. D. (1978). Microtubule assembly and function in normal and glutathione synthetase-deficient polymorphonuclear leukocytes. J. Immun. 120, ll81-1186. SYMOENS, J. & ROSENTHAL, M. (1977). Levamisole in the modulation of the immune response: the current experimental and clinical state. J. Reticuloendothel. Soc. 21, 175-221. TOOHEY, J. I. (1975). Sulfhydryl dependence in primary explant hematopoietic cells. Inhibition of growth in vitro with vitamin BI2 compounds. Proc. hath. Acad. Sci. U.S.A. 72, 73-77. VAN GINCKEL, R. & DE BRABANDER,M. The influence of a levamisole metabolite DL-2-oxo-3-(2-mercaptoethyl)-5-phenylimidazolidine on carbon clearance in mice. J. Reticuloendothel. Soc. in press.
