Glutathione‐S‐transferase activity in malarial parasites

TMIH387 Tropical Medicine and International Health volume 4 no 4 pp 251–254 april 1999

Glutathione-S-transferase activity in malarial parasites P. Srivastava1, S. K. Puri2, K. K. Kamboj3 and V. C. Pandey1 1 Division of Biochemistry, 2 Division of Microbiology, 3 Division of and Parasitology, Central Drug Research Institute, Lucknow, India

Summary

Glutathione-S-transferase (GST) activity has been detected in rodent (Plasmodium berghei, P. yoelii), simian (P. knowlesi) and human (P. falciparum) malarial parasites, and in different intraerythrocytic stages of P. knowlesi (schizont . ring . trophozoite). In chloroquine-resistant strains of rodent and human malarial parasites GST activity significantly increases compared to sensitive strains. Further, the increase in enzyme activity is directly related to drug pressure of resistant P. berghei. Complete inhibition of chloroquine-sensitive and resistant P. berghei glutathione-S-transferase activities was observed at 2.5 and 5.0 mM concentration of hemin, respectively. An inverse relationship was found between the heme level and enzyme activity of chloroquine-resistant and sensitive P. berghei. Chloroquine, artemisinin, and primaquine noticeably inhibited GST activity in P. knowlesi. keywords glutathione-S-transferase (GST), chloroquine-resistant (CQR), chloroquine-sensitive (CQS); Plasmodium; antimalarials; haem correspondence Dr P. Srivastava, Division of Biochemistry, Central Drug Research Institute, Lucknow 226 001, India. E-mail: root%cdrilk @ sirnetd.ernet

Introduction

Materials and methods

Glutathione-S-transferase (GST) plays an important role in the metabolism of endobiotics and xenobiotics by virtue of its conjugation reaction with glutathione. GST is also helpful in the transportation of hemin generated during the metabolism of various haemoproteins (Boyer & Olsen 1991), and involved in the acquisition of drug resistance in different diseases (Black & Wolf 1991). However, the role of GST in malaria resistance has not been studied except by Dubois et al. (1995) who reported that resistance in Plasmodium berghei results from altered GST activity. To the best of the author’s knowledge no systematic report exists regarding the GST in different Plasmodium species although its possible involvement in drug resistance of malarial parasites is of particular interest. Our study aimed to investigate the status of GST in rodent (P. berghei and P. yoelii), different intraerythrocytic stages of simian (P. knowlesi) and human (P. falciparum) malarial parasites as well as in their corresponding chloroquineresistant (CQR) strains. The effect of hemin and different antimalarials on GST activity of P. knowlesi and the correlation between heme and GST was also studied.

Plasmodium berghei NK65 and K173 strains were maintained in golden hamsters and Mastomys coucha, respectively, P. yoelii nigeriensis in Swiss mice (Srivastava & Pandey 1995); P. knowlesi in Macaca mulatta (Srivastava et al. 1992). P. falciparum was cultured in A 1 human erythrocytes (Trager & Jensen 1976).

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P. berghei and P. yoelii Plasma and buffy coats were removed from infected blood (parasitaemia: 25% for P. berghei, 70% for P. yoelii) by centrifugation at 1200 g for 10 min. The supernatant was removed and the erythrocyte pellet suspended in 3 vols of 0.05 M phosphate buffered saline (PBS), pH 7.4. The washed erythrocyte was resuspended in equal volumes of PBS and loaded on CF-11 cellulose column, the eluant was centrifuged at 1200 g for 10 min. Giemsa-stained thick smear slides of the pellet which were almost free from white blood cells (1–4% contamination) were further processed by loading to FicollConray gradient (24 ml of 9% Ficoll 1 10 ml of Conray; density 1.07 g/cm3) and centrifuged at 50 g for 10 min. The infected red blood cells were resuspended in PBS containing 251

Tropical Medicine and International Health

volume 4 no 4 pp 251–254 april 1999

P. Srivastava et al. Glutathione-S-transferase activity in malarial parasites

0.2% (w/v) saponin and kept at 4 8C for 30 min with occasional shaking for complete lysis of erythrocytic membranes. The contents were then cold-centrifuged at 600 g for 20 min to obtain a cell-free parasite pellet which was thoroughly washed with PBS. P. knowlesi At a parasitaemia level of about 50%, blood was collected from infected monkeys and processed as described above. Since P. knowlesi infection in rhesus monkeys produces a synchronized infection, two monkeys each were used for collecting parasites at the ring, trophozoite and schizont stages.

Culture with 5% parasitaemia was processed as described above, with the omission of the CF-11 cellulose column and Ficoll-Conray density gradient. P. berghei and P. yoelii-resistant strains Plasmodium berghei CQR strain was prepared by intermediate subcurative therapy to CQS P. berghei strain-infected mice in 40 passages (Puri et al. 1979). The P. yoelii strain was naturally multidrug-resistant. The purity of the preparations was checked by microscopical examination. The parasites thus obtained were homogenized in chilled buffered KCl (150 mM KCl, 10 mM NaH2P04/K2HP04, pH 7.4) and subjected to differential centrifugation to obtain the cytosolic fraction (Srivastava & Pandey 1995). Assays A typical mixture for GST contained glutathione 1.0 mM; phosphate buffer 0.1 M, pH 6.5; 1-chloro, 2,4-dinitrobenzene l mM and 1–2 mg of cytosolic protein. The assays were done at 37 8C and the change in optical density was monitored at GST activity (nmols conjugate/min/mg protein)

S. No. Parasites

Strains

Stages

GST activity*

1 2 3 4 5 6 7

K173 NK65 nigeriensis W–1 W–1 W–1 NF54

Asynchronous Asynchronous Asynchronous Schizonts Trophozoites Rings Schizonts

20.34 6 2.86 95.49 6 15.13 37.13 6 11.34 07.86 6 1.17 02.87 6 0.37 03.19 6 0.72 54.76 6 13.12

P. berghei P. berghei P. yoelii P. knowlesi P. knowlesi P. knowlesi P. falciparum

*Specific activity in n moles of conjugate formed/min/mg protein. Values are mean 6SD of 5 separate observations sets.

P. falciparum

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Table 1 Glutathione-S-transferase activity in different malarial parasites

340 nm every 15 s (Habig et al. 1974). Controls were devoid of the enzyme protein. Whenever required, for inhibition studies different concentrations of additives were preincubated with cytosolic enzyme preparations for 10 min at 37 8C prior to enzyme assay. Proteins were estimated according to Lowry et al. (1951) using bovine serum albumin as standard. Heme levels were estimated according to Orjih and Fitch (1993).

Results Results show that different species and strains of malarial parasites, viz. P. berghei K173, P. berghei NK 65 and P. yoelii (rodents), P. knowlesi (simian) and P. falciparum (human) possess significant GST activity. The highest enzyme activity was associated with the P. berghei NK 65 strain. Different intraerythrocytic developmental stages (schizont . ring . and . trophozoite) of P. knowlesi also showed the presence of GST (Table 1). Figure 1 Specific activity of GlutathioneS-transferase in chloroquine-resistant and -sensitive strains of different malarial parasites. h S sensitive strain; N R1 resistant strain; j R2 resistant strain. P. berghei: S - 8 mg/kg b.wt. of CQ tolerance, R1 - 40 mg/kg b.wt. of CQ tolerance, R2 70 mg/kg b.wt. of CQ tolerance. P. Yoelii: S - 16 mg/kg b.wt. of CQ tolerance, R1 120 mg/kg b.wt. of CQ tolerance; P. falciparum: S - 12 nmol/kg b.wt. of CQ tolerance, R1 - 120 nmol/kg b.wt. of CQ tolerance.

600 500 400 300 200 100 0

P. berghei

P. yoelii

P. falciparum

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Tropical Medicine and International Health P. Srivastava et al.

volume 4 no 4 pp 251–254 april 1999

Glutathione-S-transferase activity in malarial parasites

Table 2 Effect of chloroquine, artemisinin and primaquine on Glutathione-S-transferase activity of different stages of P. knowlesi % Inhibition in GST activities

S. No. Stages 1 2 3

Chloroquine Artemisinin Primaquine —————– —————– —————– 10mm 20mm* 10mm 20mm* 10mm 20mm*

Schizonts 38.17 061.92 28.82 Trophozoites 22.34 059.17 15.37 Rings 54.21 100.00 12.14

41.17 30.19 20.34

17.19 29.14 20.87

32.37 62.31 50.00

Four separate observations were conducted. *P , 0.05.

Discussion

Chloroquine-resistant (CQR) and sensitive (CQS) strains of malarial parasites depicted sizeable differences in GST activity (CQR . CQS), which was found to be directly proportional to the drug pressure of the resistant parasites (Figure 1). Different classes of antimalarials (chloroquine, artemisinin and primaquine) inhibited the enzyme activity in a concentration-dependent manner. The GST of the ring stage of P. knowlesi was maximally affected by chloroquine followed by primaquine and artemisinin (Table 2). Assays of GST activity in schizont, ring and trophozoite of P. knowlesi in Table 3 Effect of hemin and protoporphyrin IX on Glutathione-Stransferase activity of different stages of P. knowlesi % Inhibition in GST activity

S. No.

Stages

Hemin ——————— 1mm 2mm*

1 2 3

Schizonts Trophozoites Rings

29.88 07.82 18.13

57.14 20.10 29.37

Protoporphyrin IX ———————— 1mm 2mm 7.18 5.33 4.12

7.92 5.17 6.31

Four separate observations were conducted. *P , 0.005.

Table 4 Glutathione-S-transferase activity and heme level in chloroquine-resistant and sensitive strains of P. berghei K173

S. No.

Parasite

1

CQS P. berghei CQR (40 mg)* P. berghei CQR (70 mg)* P. berghei

2 3

the presence of hemin and protoporphyrin IX showed that the enzyme activity was significantly affected (maximally schizont’s GST) by hemin, whereas protoporphyrin IX was ineffective (Table 3). GST increased markedly in case of CQR 40 , CQR 70 (Table 4) compared to the CQS strain of P. berghei. By contrast, heme levels tended to drop in the former compared to the latter (Table 4). Further, the effect of varying concentrations of hemin on the GST activity of CQR 70 and CQS P. berghei demonstrated that the former requires twice the hemin concentration for complete inhibition as the latter (Table 5).

Our investigations establish the presence of GST, an important enzyme of Phase II drug metabolism, in different species of Plasmodium, indicating that the parasite is equipped with the system for detoxification of xenobiotics and endobiotics. Further, the presence of the enzyme was not confined to one stage of the parasite, but occurred in different intraerythrocytic stages (schizont, ring and trophozoite) of P. knowlesi. Apart from drug detoxification, GST plays an important role in acquisition of drug resistance in different diseases (Black & Wolf 1991). The increase in GST activity was concomitant with rising parasitaemia. Our findings agree with those of Dubois et al. (1995), who also reported an increase in GST activity in resistant P. berghei. Glutathione-S-transferase is a key enzyme involved in the conjugation reaction of drug metabolism: cessation of its activity either by denaturation or by rendering its functional domain inactive by a chemical agent or physiochemical process might lead to the accumulation of toxic product in an organism. We found that inhibition of GST activity potentiated the accumulation of antimalarial (chloroquine) metabolites, which may be responsible for creating a hazardous milieu in parasite, leading to its death. This very phenomenon may be potentially applicable in making resistant parasites sensitive to the drug. Haem/haemozoin, a toxic product generated by Plasmodia

GST* (nmoles conjugate/min/mg protein)

Haem (pmoles/mg protein)

020.34 6 2.86

324.26 6 20.84

104.14 6 15.37

142.29 6 5.92

225.29 6 27.82

060.18 6 7.36

Values are mean 6SD of 5 separate observations. *Tolerance chloroquine dose/kg b. wt.

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P. Srivastava et al. Glutathione-S-transferase activity in malarial parasites

Table 5 Effect of different concentrations of hemin on GlutathioneS-transferase activity of chloroquine-resistant and -sensitive strains of P. berghei % inhibition in GST activity of P. berghei S. No.

Additive (mm)

CQS

CQR

1 2 3

1 2.5 5.0

020.17 093.00 100.00

007.89 054.13 100.00

Values are mean of 3 experiments.

during haemoglobin digestion, had noticeable impact on the enzyme. GST activity decreased concomitanty with increasing heme concentration. However, if heme oxygenase increases, as in the case of resistant parasites (Srivastava & Pandey 1995), it may be responsible for the increased GST activity. In vitro studies also reveal an inverse relationship between heme level and GST activity of the parasite, which was observed in different intraerythrocytic stages of P. knowlesi. Cytochrome P-450 activity is known to be affected by heme and related compounds. It is possible that GST, being the conjugating enzyme of the related drug metabolizing system, is also inhibited, but this has not been corroborated during malaria infection. Inhibition of GST by heme does not indicate its exact interaction with the catalytic domain. At present it is difficult to say whether this activity is reversible or not. In conclusion, it seems that different species of Plasmodium possess noticeable activity of GST. There are considerable differences in GST activity between CQR and CQS P. berghei and P. falciparum. Further, the status of GST in different malarial parasites is probably regulated by heme levels.

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Acknowledgements The authors are grateful to the Director, CERI for providing the necessary facilities. PS is the recipient of a Council of Scientific and Industrial Research, New Delhi, Pool Officership. This is CDRI Communication no. 5575.

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