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Inhibition by superoxide dismutase and catalase of the damage of isolated Leishmania mexicana amazonensis by phenazine methosulfate
Molecular and Biochemical Parasitology, 10 (1984) 297-303 Elsevier
297
MBP 00399
INHIBITION BY S U P E R O X I D E D I S M U T A S E AND CATALASE OF THE DAMAGE OF I S O L A T E D LEISHMANIA MEXICANA AMAZONENSIS BY PHENAZINE M E T H O S U L F A T E
ZEENAT F. NABI and MICHEL RABINOVITCH* Department o f Cell Biology, New York University School ofMedicine, 550 First Avenue, New York, N Y 10016, U.S.A. (Received 20 May 1983; accepted 27 July 1983)
Phenazine methosulfate, a cationic electron carrier, inhibits the extracellular growth of promastigotes and the conversion of amastigotes into promastigote forms of Leishmania mexicana amazonensis. Growth inhibition and damage of extracellular parasites by PMS was counteracted by superoxide dismutase, a scavenger of the superoxide anion (O2-), and to a lesser extent, by catalase, a scavenger of hydrogen peroxide (H202). Inactivated dismutase and catalase were ineffective. Thus, damage of isolated L.m. amazonensis by phenazine methosulfate, involves the participation of 02- and H202. The role of the oxygen metabolites in the toxicity ofphenazine methosulfate remains unknown. That 02- can damage theparasites is supported by the finding that superoxide dismutase also protected promastigotes from damage induced by oxygen intermediates generated by a xanthine-xanthine oxidase system. Killing of the parasites by crystal violet, a triphenylmethane, or basic blue 24, a phenothiazine, was not inhibited by superoxide dismutase. Key words: Leishmania mexicana amazonensis; Phenazine methosulfate; Superoxide; Superoxide dismutase; Catalase
INTRODUCTION
Leishmania species are protozoan parasites which exist in two forms: motile flagellated promastigotes found in the insect vector, and incompletely flagellated amastigotes lodged within the phagolysosomes of vertebrate macrophages [1]. Docampo et al. reported that phenazine methosulfate (PMS) inhibits the growth of Trypanosoma cruzi epimastigotes in culture and that phenazinium cation free radicals as well as the superoxide anion (02-) are generated upon incubation of the parasites with the dye [2]. The effect of superoxide dismutase (SOD), an enzymatic scavenger of O2-, on PMS toxicity was not investigated in these experiments. We have shown that PMS and other
* To whom all correspondence should be addressed. Abbreviations: BB24, basic blue 24; CV, crystal violet; EDTA, ethylenediamin etetraacetic acid, sodium salt; PBS, phosphate-buffered saline; PMS, phenazine methosulfate; SOD, superoxide dismutase. 0166-6851/84/$03.00 © 1984 Elsevier Science Publishers B.V.
298 electron carriers reduce the growth of L. mexicana amazonensis promastigotes in culture [3]. We report here that the toxicity of PMS for cell-free promastigotes and amastigotes of L. mexicana amazonensis is abolished or reduced by SOD and catalase, suggesting that 02- and H202 subserve a role in the damage of the parasites by the dye. MATERIALS AND METHODS Animals. Adult golden hamsters were obtained from the Department of Parasitology, New York University Medical Center. Swiss Webster mice' were purchased from Taconic Farms (Germantown, NY). Media. Ca 2÷, Mg:÷-free phosphate-buffered saline (PBS) contained 6 mM potassium phosphate buffer and 138 mM NaC1 and was adjusted to pH 7.2. RPMI 1640 was obtained from Grand Island Biological Company (Grand Island, NY) and was prepared with 10 mM HEPES (N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic acid). Fetal calf serum was purchased from K.C. Biological (Lenexa, KS) and was heat inactivated for 30 rain at 56°C. Leishmania strain and preparation of amastigote suspensions. L. mexicana amazonensis LV 79 was originally obtained from the Department of Parasitology, Liverpool School of Medicine, England, and provided to us by Dr. J.P. Dedet (Institut Pasteur, Paris). Amastigotes were isolated from hamster lesions as previously described [3]. Promastigote cultures were established from hamster lesions and grown in RPMI containing 30% fetal calf serum, 10 lag m1-1 hemin and 50 lag m1-1 gentamicin ('Promastigote medium'). Treatment of Leishmania with PMS or other dyes. For drug assays, culture tubes containing 2 ml of the medium were seeded with 2 )< 106 promastigotes, and incubated at 25°C for 24 h. The parasites were treated for I h at 25°C with PMS or other dyes in serum-free medium, with or without other additions. Dyes were added under subdued light and the tubes immediately wrapped in aluminum foil. After the pulse, parasites were washed once and resuspended in promastigote medium. 24 or 48 h later the number of promastigotes was counted in a hemocytometer. Experiments with amastigotes were performed similarly, except that the freshly isolated parasites were treated with the dyes at 34°C [15]. Assay for superoxide anion. Production of 02- during the interaction of PMS with Leishmania was measured by the reduction of cytochrome c. Leishmania (5)< 106) were added to a cuvette containing 1 ml of 50 mM phosphate buffer (pH 7.4), 100 laM EDTA, and 50 laM cytochrome c, and the reaction was started by the addition of PMS. The absorbancy change at 550 nm was recorded and converted to the 02- release using a molar absorption coefficient of 19.1 )< 103 M -I cm -1 [4].
299
Reagents. Except when indicated otherwise, chemicals and enzymes were purchased
from Sigma Chemical, St. Louis, MO. SOD (1 mg ml-~) was inactivated by treatment with 10 mM diethyldithiocarbamate for 2 h at 37°C, followed by overnight dialysis [5], Inactivation was confirmed by a cytochrome reduction assay of superoxide generated with xanthine and xanthine oxidase. Reactivated SOD was obtained by treatment of inactivated SOD (1 mg ml -~) with 0.5 mM copper sulfate at 37°C for 1 h, followed by overnight dialysis. Catalase (Calbiochem-Behring, La Jolla,CA) was inactivated by heating to 100°C for 30 min or by treatment with aminotriazol (1 mM) for 2 h at 37°C, followed by overnight dialysis [6]. RESULTS
Growth of L. m. amazonensis promastigotes (see also ref. 3) and conversion of amastigotes into promastigotes were inhibited by PMS in a dose-response fashion (Fig. la and b). Addition of SOD completely reversed the toxicity of up to 15 laM PMS for promastigotes, whereas toxicity for amastigotes was incompletely but significantly diminished. Protection by SOD was related to enzymatic activity, since SOD inactivated with the copper chelator diethyldithiocarbamate did not protect promastigotes against damage by PMS, while reactivation of the enzyme restored its protective effect (Table I). SOD was not protective when added only after the PMS pulse (result not shown). Catalase partially protected promastigotes against PMS toxicity, and was ineffective after inactivation with aminotriazol. The protection provided by SOD and catalase indicates that 02- and H202 are involved in the damage of the parasites by PMS. L. donovani promastigotes can be damaged by incubation with xanthine (or acetaldehyde) and xanthine oxidase [7,8]. We obtained similar results with promastigotes of b
(3 I00
o
50
-
5
IO 15 ~M PMS
20
SOD
0
pM PMS
Fig. 1. SOD protection against PMS killing ofLeishmania promastigotes (a) and amastigotes (b). Parasites (2)< 106) were given a 1 h pulse with increasing concentrations of PMS alone, or of PMS with 10 lag ml -~ SOD. The n u m b e r of viable promastigotes was determined 24 h (a) or 48 h (b) after drug treatment. Each point is the average of 3 determinations. The 100% values were 4.9 )< 106 in (a) and 4.0 ><106 in (b). Bars indicate standard deviations and the results shown are representative of three experiments.
300
TABLE I Protection by SOD and catalase of Leishmania promastigotes against damage by phenazine methosulfate Additions a
Promastigote counts (% of control)
None SOD (10 lag ml-') Catalase (2800 U ml-') PMS (15 laM) PMS + SOD PMS + inactivated SOD PMS + reactivated SOD PMS + catalase PMS + inactivated catalase
100b 99 99 36 98 38 100 66 40
a
1.5 X lip parasites treated for 1 h at room temperature with RPMI with or without additions, then suspended in promastigote medium. Parasite viability determined by counts after 24 h. 100% = 2.28 × liP promastigotes.
b
L.m. amazonensis (Fig.2). A 1 h p u l s e w i t h x a n t h i n e (100 ~tM) a n d x a n t h i n e o x i d a s e (0.0056 U m1-1) r e s u l t e d in 5 0 % killing o f t h e p a r a s i t e s . T h e killing was fully i n h i b i t e d b y the a d d i t i o n o f 10 I~g m1-1 S O D . A t t h e c o n c e n t r a t i o n s u s e d , x a n t h i n e o r x a n t h i n e o x i d a s e a l o n e d i d n o t i n f l u e n c e the g r o w t h o r t h e c o n v e r s i o n o f the p r o m a s t i g o t e s . Fig. 3 s h o w s t h a t L. m. amazonensis p r o m a s t i g o t e s (a) o r a m a s t i g o t e s (b) a l o n e d i d n o t s i g n i f i c a n t l y r e d u c e c y t o c h r o m e c. H o w e v e r , i n c u b a t i o n o f t h e p a r a s i t e s w i t h P M S
IO0
'
"
÷
SOD
SOB
50
0
I
I
I
2
¢.orlc Fig. 2. Effect of SOD on the susceptibility of Leishmania promastigotes to oxygenintermediates generated by xanthine-xanthine oxidase. Promastigotes (2 X lip per ml) were given a I h pulse with 50 laM xanthine, and 0.0028 U ml-I xanthine oxidase (1) or 100 laM xanthine and 0.0056 U ml-I xanthine oxidase (2) in RPMI with 100 laM EDTA with or without 10 ttg ml-~ SOD. Viability of the promastigotes was determined 24 h after the pulse. Each point represents the average of 3 determinations. The 100% value was 7.0 X l0 s. Bars indicate standard deviations and the results shown are representative of three experiments.
301
Imin
Control
/
/
~
............. .......
C~tro,
b - - - - ¢ ...........
PMS
PMS
Fig. 3. Reduction of cytochrome c by PMS incubated with Leishmania and the effect of SOD. Promastigotes (a) or amastigotes (b) (5 X 106) were added to 1 ml of 50 mM phosphate buffer, pH 7.4, containing 100 pM EDTA, 50 pM cytochrome c. Reduction was started by adding 25 pM PMS alone or with PMS together with 10 pg ml-I SOD.
led to cytochrome c reduction which could be inhibited by SOD. With 25 ~tM PMS the cytochrome c reduction was equivalent to a production of 1.5 nmol min-~ and 0.4 nmol min -I of O2-, respectively, from promastigotes and amastigotes. No significant reduction of cytochrome c by PMS occurred in the absence of added parasites (absorbancy change of less than 0.0004 per min, average of 5 determinations). Crystal violet, a triphenylmethane, and basic blue 24, a phenothiazine, also damage and kill Leishmania promastigotes [3]. We examined the effect of SOD on the damage of promastigotes by these dyes. Fig. 4 shows that crystal violet (4a), and basic blue 24 (4b), similar to PMS (4c), killed the parasites in a dose-response fashion. However, while SOD protected the parasites against damage by PMS (Fig. 4c), damage of promastigotes by crystal violet and basic blue 24 (Fig. 4a and b) was unaffected by the addition of SOD. Likewise, no protection was afforded by 2800 units of catalase (result not shown). Q
b
L
IOC
C
~24 o
§ sc
,~o
2~o
I
2
Fig. 4. Killing of promastigotes by crystal violet (CV) or basic blue 24 (BB24). Promastigotes (2 X 106 per ml) were treated with the indicated concentrations ofCV (a), BB24 (b) or PMS (c). The 100% values were 6.8 × l06. e, dyes added without SOD; o, dyes added with l0 pg ml -I SOD. Each point is an average of three determinations. Bars indicate standard deviations and the results are representative of three experiments.
302 DISCUSSION We have shown that SOD, and to a lesser extent, catalase, protected L. m. amazonensis promastigotes and isolated amastigotes against damage by PMS. The protection
afforded by SOD and catalase, but not by the inactivated enzymes, indicates that 02and H202 are in some fashion involved in the toxicity of PMS for the isolated parasites [2,9]. It remains to be determined whether 02- and H202 damage the parasites directly or lead to the generation of other oxygen metabolities such as the very reactive hydroxyl radicals (OH') [10-11]. Additionally, O2- may be ink,olved in the reduction of PMS to a toxic free radical species [12-14]. The generation of 02- by promastigotes exposed to PMS was followed by cytochrome c reduction which could be inhibited by SOD (Fig. 3). In support of a toxic effect of 02- we have also found that SOD (Fig. 2), and to a lesser extent, catalase (results not shown), protected the promastigotes against oxygen reduction products generated by xanthine-xanthine oxidase. This finding stands in contrast with earlier studies with L. donovani [7,8] which emphasized the predominant leishmanicidal activity of H202. Whether the discrepancy is due to the different species of parasites studied or to other experimental variables is not clear at present. In contrast to these results with the isolated parasites, SOD at concentrations of up to 100 lag ml -~ did not modify the effect of PMS on amastigotes residing within macrophages (unpublished results). This lack of protection of intracellular amastigotes could be due to insufficient uptake of SOD by the host cells, to lack of access of SOD to the parasitophorous vacuoles, or to intracellular inactivation of the enzyme. Finally, we examined the effect of SOD on the damage o f Leishmania by two other electron carriers, crystal violet and basic blue 24 (Fig. 4). We found that while SOD reduced parasite damage by PMS, the enzyme afforded no protection against the other dyes. This suggests that damage of L. m. amazonensis by crystal violet or basic blue 24 and by PMS is mediated by different mechanisms. ACKNOWLEDGEMENTS The work was supported by grant AI-10969 from National Institutes of Health. The Authors are thankful to Drs. Helen Korchak and Arnold Stern for helpful discussions and to Ms. Gail Topper for assistance in some of the experiments. REFERENCES 1 2 3
Chang, K.P. (1983) Cellular and molecular mechanisms ofintracellular symbiosis in leishmaniasis. Int. Rev. Cytol. Suppl. 14, 267-305. Docampo,R., Cruz, F.S., Muniz, R.P.A., Esquivel,D.M.S. and Vasconeellos,M.E.L. (1978)Generation of free radicals from phenazinemethosulfatein Trypanosomacruziepimastigotes. ActaTrop. 35, 221-228. Rabinovitch,M., Dedet,J.P., Ryter,A., Robineaux,R., Topper, G. and Brunet, E. (1982)Destruction
303
4 5 6 7 8 9 10 11 12 13 14
15
of Leishmania mexicana amazonensis amastigotes within macrophages in culture by phenazine methosulfate and other electron carriers. J. Exp. Med. 155, 415-431. Nabi, Z.F., Takeshige, K., Hatae, T. and Minakami, S. (1979) Hydrogen peroxide formation of polymorphonuclear leukocytes stimulated with cytochalasin D. Exp. Cell Res. 124, 293-300. Heikkila, R.E., Cabbat, F.S. and Cohen, G. (1976) In vivo inhibition ofsuperoxide dismutase in mice by diethyldithiocarbamate. J. Biol. Chem. 251, 2182-2185. Margoliash, E., Novogrodsky, A. and Schejter, A. (1960) Irreversible reaction of 3-amino-l:2:4-triazole and related inhibitors with the protein of catalase. Biochem. J. 74, 339-348. Murray, H.W. (1982) Cell mediated immune response in experimental visceral leishmaniasis. J. Immunol. 129, 351-357. Haidaris, C.G. and Bonventre, P.F. (1982) A role for oxygen-dependent mechanisms in killing of Leishmania donovani tissue forms by activated macrophages. J. Immunol. 129, 850-855. Hassan, H.M. and Fridovich, I. (1979) Intracellular production of superoxide radical and of hydrogen peroxide by redox active compounds. Arch. Biochem. Biophys. 196, 385-395. Klebanoff, S.J. (1980) Oxygen metabolism and the toxic properties of phagocytes. Ann. Int. Med, 93, 480-489. DiGuiseppi, J. and Fridovich, I. (1982) Oxygen toxicity in Streptococcus sanguis. The relative importance of superoxide and hydroxyl radicals. J. Biol. Chem. 257, 4046-4051. Winterbourn, C.C. (1981) Cytochrome c reduction by semiquinone radicals can be indirectly inhibited by superoxide dismutase. Arch. Biochem. Biophys. 209, 159-167. Mason, R.P. (1982) Free radical intermediates in the metabolism of toxic chemicals. In: Free Radicals in Biology (Pryor, W.A., ed.), vol. 5, pp. 161-222, Academic Press, New York. Hakala, F.G., Babcock, G.T. and Dye, J.L. (1982) Properties of 5-methylphenazinium methyl sulfate. Reaction of the oxidized form with NADH and of the reduced form with oxygen. J. Biol. Chem. 257, 1458-1461. Biegel, D., Topper, G. and Rabinovitch, M. Exp. Parasitol. In press.
297
MBP 00399
INHIBITION BY S U P E R O X I D E D I S M U T A S E AND CATALASE OF THE DAMAGE OF I S O L A T E D LEISHMANIA MEXICANA AMAZONENSIS BY PHENAZINE M E T H O S U L F A T E
ZEENAT F. NABI and MICHEL RABINOVITCH* Department o f Cell Biology, New York University School ofMedicine, 550 First Avenue, New York, N Y 10016, U.S.A. (Received 20 May 1983; accepted 27 July 1983)
Phenazine methosulfate, a cationic electron carrier, inhibits the extracellular growth of promastigotes and the conversion of amastigotes into promastigote forms of Leishmania mexicana amazonensis. Growth inhibition and damage of extracellular parasites by PMS was counteracted by superoxide dismutase, a scavenger of the superoxide anion (O2-), and to a lesser extent, by catalase, a scavenger of hydrogen peroxide (H202). Inactivated dismutase and catalase were ineffective. Thus, damage of isolated L.m. amazonensis by phenazine methosulfate, involves the participation of 02- and H202. The role of the oxygen metabolites in the toxicity ofphenazine methosulfate remains unknown. That 02- can damage theparasites is supported by the finding that superoxide dismutase also protected promastigotes from damage induced by oxygen intermediates generated by a xanthine-xanthine oxidase system. Killing of the parasites by crystal violet, a triphenylmethane, or basic blue 24, a phenothiazine, was not inhibited by superoxide dismutase. Key words: Leishmania mexicana amazonensis; Phenazine methosulfate; Superoxide; Superoxide dismutase; Catalase
INTRODUCTION
Leishmania species are protozoan parasites which exist in two forms: motile flagellated promastigotes found in the insect vector, and incompletely flagellated amastigotes lodged within the phagolysosomes of vertebrate macrophages [1]. Docampo et al. reported that phenazine methosulfate (PMS) inhibits the growth of Trypanosoma cruzi epimastigotes in culture and that phenazinium cation free radicals as well as the superoxide anion (02-) are generated upon incubation of the parasites with the dye [2]. The effect of superoxide dismutase (SOD), an enzymatic scavenger of O2-, on PMS toxicity was not investigated in these experiments. We have shown that PMS and other
* To whom all correspondence should be addressed. Abbreviations: BB24, basic blue 24; CV, crystal violet; EDTA, ethylenediamin etetraacetic acid, sodium salt; PBS, phosphate-buffered saline; PMS, phenazine methosulfate; SOD, superoxide dismutase. 0166-6851/84/$03.00 © 1984 Elsevier Science Publishers B.V.
298 electron carriers reduce the growth of L. mexicana amazonensis promastigotes in culture [3]. We report here that the toxicity of PMS for cell-free promastigotes and amastigotes of L. mexicana amazonensis is abolished or reduced by SOD and catalase, suggesting that 02- and H202 subserve a role in the damage of the parasites by the dye. MATERIALS AND METHODS Animals. Adult golden hamsters were obtained from the Department of Parasitology, New York University Medical Center. Swiss Webster mice' were purchased from Taconic Farms (Germantown, NY). Media. Ca 2÷, Mg:÷-free phosphate-buffered saline (PBS) contained 6 mM potassium phosphate buffer and 138 mM NaC1 and was adjusted to pH 7.2. RPMI 1640 was obtained from Grand Island Biological Company (Grand Island, NY) and was prepared with 10 mM HEPES (N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic acid). Fetal calf serum was purchased from K.C. Biological (Lenexa, KS) and was heat inactivated for 30 rain at 56°C. Leishmania strain and preparation of amastigote suspensions. L. mexicana amazonensis LV 79 was originally obtained from the Department of Parasitology, Liverpool School of Medicine, England, and provided to us by Dr. J.P. Dedet (Institut Pasteur, Paris). Amastigotes were isolated from hamster lesions as previously described [3]. Promastigote cultures were established from hamster lesions and grown in RPMI containing 30% fetal calf serum, 10 lag m1-1 hemin and 50 lag m1-1 gentamicin ('Promastigote medium'). Treatment of Leishmania with PMS or other dyes. For drug assays, culture tubes containing 2 ml of the medium were seeded with 2 )< 106 promastigotes, and incubated at 25°C for 24 h. The parasites were treated for I h at 25°C with PMS or other dyes in serum-free medium, with or without other additions. Dyes were added under subdued light and the tubes immediately wrapped in aluminum foil. After the pulse, parasites were washed once and resuspended in promastigote medium. 24 or 48 h later the number of promastigotes was counted in a hemocytometer. Experiments with amastigotes were performed similarly, except that the freshly isolated parasites were treated with the dyes at 34°C [15]. Assay for superoxide anion. Production of 02- during the interaction of PMS with Leishmania was measured by the reduction of cytochrome c. Leishmania (5)< 106) were added to a cuvette containing 1 ml of 50 mM phosphate buffer (pH 7.4), 100 laM EDTA, and 50 laM cytochrome c, and the reaction was started by the addition of PMS. The absorbancy change at 550 nm was recorded and converted to the 02- release using a molar absorption coefficient of 19.1 )< 103 M -I cm -1 [4].
299
Reagents. Except when indicated otherwise, chemicals and enzymes were purchased
from Sigma Chemical, St. Louis, MO. SOD (1 mg ml-~) was inactivated by treatment with 10 mM diethyldithiocarbamate for 2 h at 37°C, followed by overnight dialysis [5], Inactivation was confirmed by a cytochrome reduction assay of superoxide generated with xanthine and xanthine oxidase. Reactivated SOD was obtained by treatment of inactivated SOD (1 mg ml -~) with 0.5 mM copper sulfate at 37°C for 1 h, followed by overnight dialysis. Catalase (Calbiochem-Behring, La Jolla,CA) was inactivated by heating to 100°C for 30 min or by treatment with aminotriazol (1 mM) for 2 h at 37°C, followed by overnight dialysis [6]. RESULTS
Growth of L. m. amazonensis promastigotes (see also ref. 3) and conversion of amastigotes into promastigotes were inhibited by PMS in a dose-response fashion (Fig. la and b). Addition of SOD completely reversed the toxicity of up to 15 laM PMS for promastigotes, whereas toxicity for amastigotes was incompletely but significantly diminished. Protection by SOD was related to enzymatic activity, since SOD inactivated with the copper chelator diethyldithiocarbamate did not protect promastigotes against damage by PMS, while reactivation of the enzyme restored its protective effect (Table I). SOD was not protective when added only after the PMS pulse (result not shown). Catalase partially protected promastigotes against PMS toxicity, and was ineffective after inactivation with aminotriazol. The protection provided by SOD and catalase indicates that 02- and H202 are involved in the damage of the parasites by PMS. L. donovani promastigotes can be damaged by incubation with xanthine (or acetaldehyde) and xanthine oxidase [7,8]. We obtained similar results with promastigotes of b
(3 I00
o
50
-
5
IO 15 ~M PMS
20
SOD
0
pM PMS
Fig. 1. SOD protection against PMS killing ofLeishmania promastigotes (a) and amastigotes (b). Parasites (2)< 106) were given a 1 h pulse with increasing concentrations of PMS alone, or of PMS with 10 lag ml -~ SOD. The n u m b e r of viable promastigotes was determined 24 h (a) or 48 h (b) after drug treatment. Each point is the average of 3 determinations. The 100% values were 4.9 )< 106 in (a) and 4.0 ><106 in (b). Bars indicate standard deviations and the results shown are representative of three experiments.
300
TABLE I Protection by SOD and catalase of Leishmania promastigotes against damage by phenazine methosulfate Additions a
Promastigote counts (% of control)
None SOD (10 lag ml-') Catalase (2800 U ml-') PMS (15 laM) PMS + SOD PMS + inactivated SOD PMS + reactivated SOD PMS + catalase PMS + inactivated catalase
100b 99 99 36 98 38 100 66 40
a
1.5 X lip parasites treated for 1 h at room temperature with RPMI with or without additions, then suspended in promastigote medium. Parasite viability determined by counts after 24 h. 100% = 2.28 × liP promastigotes.
b
L.m. amazonensis (Fig.2). A 1 h p u l s e w i t h x a n t h i n e (100 ~tM) a n d x a n t h i n e o x i d a s e (0.0056 U m1-1) r e s u l t e d in 5 0 % killing o f t h e p a r a s i t e s . T h e killing was fully i n h i b i t e d b y the a d d i t i o n o f 10 I~g m1-1 S O D . A t t h e c o n c e n t r a t i o n s u s e d , x a n t h i n e o r x a n t h i n e o x i d a s e a l o n e d i d n o t i n f l u e n c e the g r o w t h o r t h e c o n v e r s i o n o f the p r o m a s t i g o t e s . Fig. 3 s h o w s t h a t L. m. amazonensis p r o m a s t i g o t e s (a) o r a m a s t i g o t e s (b) a l o n e d i d n o t s i g n i f i c a n t l y r e d u c e c y t o c h r o m e c. H o w e v e r , i n c u b a t i o n o f t h e p a r a s i t e s w i t h P M S
IO0
'
"
÷
SOD
SOB
50
0
I
I
I
2
¢.orlc Fig. 2. Effect of SOD on the susceptibility of Leishmania promastigotes to oxygenintermediates generated by xanthine-xanthine oxidase. Promastigotes (2 X lip per ml) were given a I h pulse with 50 laM xanthine, and 0.0028 U ml-I xanthine oxidase (1) or 100 laM xanthine and 0.0056 U ml-I xanthine oxidase (2) in RPMI with 100 laM EDTA with or without 10 ttg ml-~ SOD. Viability of the promastigotes was determined 24 h after the pulse. Each point represents the average of 3 determinations. The 100% value was 7.0 X l0 s. Bars indicate standard deviations and the results shown are representative of three experiments.
301
Imin
Control
/
/
~
............. .......
C~tro,
b - - - - ¢ ...........
PMS
PMS
Fig. 3. Reduction of cytochrome c by PMS incubated with Leishmania and the effect of SOD. Promastigotes (a) or amastigotes (b) (5 X 106) were added to 1 ml of 50 mM phosphate buffer, pH 7.4, containing 100 pM EDTA, 50 pM cytochrome c. Reduction was started by adding 25 pM PMS alone or with PMS together with 10 pg ml-I SOD.
led to cytochrome c reduction which could be inhibited by SOD. With 25 ~tM PMS the cytochrome c reduction was equivalent to a production of 1.5 nmol min-~ and 0.4 nmol min -I of O2-, respectively, from promastigotes and amastigotes. No significant reduction of cytochrome c by PMS occurred in the absence of added parasites (absorbancy change of less than 0.0004 per min, average of 5 determinations). Crystal violet, a triphenylmethane, and basic blue 24, a phenothiazine, also damage and kill Leishmania promastigotes [3]. We examined the effect of SOD on the damage of promastigotes by these dyes. Fig. 4 shows that crystal violet (4a), and basic blue 24 (4b), similar to PMS (4c), killed the parasites in a dose-response fashion. However, while SOD protected the parasites against damage by PMS (Fig. 4c), damage of promastigotes by crystal violet and basic blue 24 (Fig. 4a and b) was unaffected by the addition of SOD. Likewise, no protection was afforded by 2800 units of catalase (result not shown). Q
b
L
IOC
C
~24 o
§ sc
,~o
2~o
I
2
Fig. 4. Killing of promastigotes by crystal violet (CV) or basic blue 24 (BB24). Promastigotes (2 X 106 per ml) were treated with the indicated concentrations ofCV (a), BB24 (b) or PMS (c). The 100% values were 6.8 × l06. e, dyes added without SOD; o, dyes added with l0 pg ml -I SOD. Each point is an average of three determinations. Bars indicate standard deviations and the results are representative of three experiments.
302 DISCUSSION We have shown that SOD, and to a lesser extent, catalase, protected L. m. amazonensis promastigotes and isolated amastigotes against damage by PMS. The protection
afforded by SOD and catalase, but not by the inactivated enzymes, indicates that 02and H202 are in some fashion involved in the toxicity of PMS for the isolated parasites [2,9]. It remains to be determined whether 02- and H202 damage the parasites directly or lead to the generation of other oxygen metabolities such as the very reactive hydroxyl radicals (OH') [10-11]. Additionally, O2- may be ink,olved in the reduction of PMS to a toxic free radical species [12-14]. The generation of 02- by promastigotes exposed to PMS was followed by cytochrome c reduction which could be inhibited by SOD (Fig. 3). In support of a toxic effect of 02- we have also found that SOD (Fig. 2), and to a lesser extent, catalase (results not shown), protected the promastigotes against oxygen reduction products generated by xanthine-xanthine oxidase. This finding stands in contrast with earlier studies with L. donovani [7,8] which emphasized the predominant leishmanicidal activity of H202. Whether the discrepancy is due to the different species of parasites studied or to other experimental variables is not clear at present. In contrast to these results with the isolated parasites, SOD at concentrations of up to 100 lag ml -~ did not modify the effect of PMS on amastigotes residing within macrophages (unpublished results). This lack of protection of intracellular amastigotes could be due to insufficient uptake of SOD by the host cells, to lack of access of SOD to the parasitophorous vacuoles, or to intracellular inactivation of the enzyme. Finally, we examined the effect of SOD on the damage o f Leishmania by two other electron carriers, crystal violet and basic blue 24 (Fig. 4). We found that while SOD reduced parasite damage by PMS, the enzyme afforded no protection against the other dyes. This suggests that damage of L. m. amazonensis by crystal violet or basic blue 24 and by PMS is mediated by different mechanisms. ACKNOWLEDGEMENTS The work was supported by grant AI-10969 from National Institutes of Health. The Authors are thankful to Drs. Helen Korchak and Arnold Stern for helpful discussions and to Ms. Gail Topper for assistance in some of the experiments. REFERENCES 1 2 3
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