Plasmodium falciparum: Effect of Solanum nudum steroids on thiol contents and β-hematin formation in parasitized erythrocytes

Experimental Parasitology 122 (2009) 273–279

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Plasmodium falciparum: Effect of Solanum nudum steroids on thiol contents and b-hematin formation in parasitized erythrocytes Adriana Pabón a,*, Eric Deharo b,c, Lina Zuluaga a, Juan D. Maya d, Jairo Saez e, Silvia Blair a a

Grupo Malaria, Universidad de Antioquia, Medellín, Colombia Université de Toulouse, UPS, UMR 152 (Laboratoire de pharmacochimie des substances naturelles et pharmacophores redox), 118, rte de Narbonne, F-31062 Toulouse cedex 9, France c IRD, UMR-15, Mission IRD casilla 18-1209, Lima, Peru d Programa de Farmacología Molecular y Clínica, ICBM, Facultad de Medicina, Universidad de Chile, Santiago, Chile e Grupo Química de plantas colombianas, Universidad de Antioquia, Medellín, Colombia b

a r t i c l e

i n f o

Article history: Received 27 November 2008 Received in revised form 22 April 2009 Accepted 29 April 2009 Available online 12 May 2009 Keywords: Solanum nudum Plasmodium falciparum Glutathione Cysteine b-Hematin

a b s t r a c t We studied the effects on total thiols glutathione (GSH) and cysteine contents in Plasmodium falciparum in vitro when treated with four steroid derivatives and a sapogenin (Diosgenone) extracted from Solanum nudum. We also determined their capacity to inhibit b-hematin formation. We showed that SN-1 (16aacetoxy-26-hydroxycholest-4-ene-3,22-dione) increased total glutathione and cysteine concentrations while SN-4 (26-O-b-D-glucopyranosyloxy-16a-acetoxycholest-4-ene-3,22-dione) decreased the concentration of both thiols. Acetylation in C16 was crucial for the effect of SN-1 while type furostanol and terminal glucosidation were necessary for the inhibitory properties of SN-4. The combination of steroids and buthionine sulfoximine, a specific inhibitor of a step-limiting enzyme in GSH synthesis, did not modify the glutathione contents. Finally, we found that SN-1 inhibited more than 80% of b-hematin formation at 5.0 mM, while the other steroids did not show any effect. Ó 2009 Elsevier Inc. All rights reserved.

1. Introduction Estimates from 2006 indicate that approximately 3.3 billion people live in areas of malaria transmission and that between 189 and 327 million clinical malaria episodes occur annually (Fidock et al., 2004; WHO/TDR, 2008). In Colombia, around 160,000 cases are reported yearly, with one-third of these attributed to Plasmodium falciparum (INS, 2006). As in other endemic countries, P. falciparum in Colombia has become resistant to most antimalarial treatments (Blair et al., 2006; Espinal et al., 1985). New drugs must therefore be sought to combat this disease and natural products may provide important alternatives. For this reason our group investigated the antimalarial activity of extracts of Solanum nudum Dunal, a Solanaceae traditionally use for the treatment of fevers in the municipality of Tumaco, located along the South-eastern Pacific coast in the Colombian department of Nariño (Blair et al., 2001; Saez et al., 1998). In previous studies, several steroids and a sapogenin were isolated from S. nudum (Fig. 1). These compounds demonstrated antimalarial activity (Echeverri et al., 2001; Londono et al., 2006; Pabon et al., 2002) without showing any mutagenic, clastogenic, nor cytotoxic activities against mammalian cell lines

* Corresponding author. Address: Grupo Malaria, Universidad de Antioquia, Calle 62 # 52-59, Sede de Investigación Universitaria (SIU), Torre 1, Laboratorio 610, Medellín, Colombia. Fax: +57 4 2106487. E-mail address: [email protected] (A. Pabón). 0014-4894/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2009.04.014

(Alvarez et al., 2004; Londono et al., 2006; Pabon et al., 2003). In addition, they reduced the number of hepatic stages of P. vivax in vitro and affected the sporogonic cycle in Anopheles (Arango et al., 2006; Londono et al., 2006) Steroids isolated from S. nudum present a high electronic density region (OH group in SN-1) that can donate electrons and a low electron density region that can receive electrons (keto group) allowing the compound to alter the fragile redox status of malaria parasites. This prompted us to measure the impact of these compounds on glutathione (GSH) which plays a key role in redox mechanisms and on cysteine (Cys) which is one of the substrates needed for the de novo synthesis of P. falciparum GSH. The impact on b-hematin formation was also investigated as GSH participates in heme detoxification (Dubois et al., 1995). 2. Materials and methods 2.1. S. nudum-derived steroids Isolation and purification of Diosgenone, SN-1, SN-2, SN-3 and SN-4 (25(R)-spirost-4-en-3-one; 16a-acetoxy-26-hydroxycholest4-ene-3,22-dione; 16a,26-dihydroxycholest-4-ene-3,22-dione; 16aacetoxy-26-O-b-D-glucopyranosyloxycholest-4-ene-3,22-dione; 26-O-b- D -glucopyranosyloxy-16 a -acetoxycholest-4-ene-3,22dione, respectively) from S. nudum was performed as previously described (Londono et al., 2006; Pabon et al., 2002; Saez et al.,

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O

O OH

OH

OCOCH3

OH

O

O

SN-1

SN-2 OH

O OGlu

O

OCOCH3

O

O

SN-3

SN-4

OGlu

O

O

O

Diosgenone Fig. 1. Structures of Diosgenone and Solanum nudum steroids.

1998) (Fig. 1). Steroids were dissolved in polyvinyl pyrrolidone of m.w. 10,000 (PVP-10) at a 4:1 ratio (PVP-10:steroid) which has been shown to be non-toxic for cultures (Pabon et al., 2002). 2.2. Effect of S. nudum steroids and buthionine sulfoximine (BSO) on glutathione and cysteine contents on Plasmodium Cultures of P. falciparum FCB-2 strain were raised at 37 °C and 5% CO2, in RPMI 1640 medium supplemented with 25 mM Hepes, 5% (w/v) NaHCO3, 1 mg/mL reduced glutathione and 10% inactivated human serum (hematocrit 5%), according to the Trager and Jensen methodology (Trager, 1978; Trager and Jensen, 1976). Parasites were synchronized by sorbitol treatment leading to a ringenriched culture, which was maintained for an additional 12 or 24 h to produce trophozoites infected erythrocytes or schizonts infected erythrocytes, respectively (Lambros and Vanderberg, 1979; Rowley et al., 1967). Erythrocytes with ring (R), trophozoites (T) or schizonts (S) stages (parasitemia, 1.0%; hematocrit, 5%) were incubated in 6-well plates with S. nudum derived steroids and buthionine sulfoximine, a specific inhibitor of GSH synthesis. Final concentrations corresponding to the previously reported IC50 for P. falciparum were employed (SN-1: 21 lM; SN-2: 125.5 lM; SN-3: 63.1 lM; SN-4: 57.4 lM; Diosgenone: 21.8 lM and BSO: 73 lM) (Luersen et al., 2000; Pabon et al., 2002). RPMI was used as negative control. After 24 h culture incubation, the plates were centrifuged at 1400g for 10 min, the supernatant discarded and parasitized erythrocytes separated from non-infected red blood cells by percoll gradient centrifugation. In order to enrich the non-parasitized erythrocyte fraction, the remaining lower layers were mixed and fractionated again on the same percoll-sorbitol

gradient (Omodeo-Sale et al., 2003; Rowley et al., 1967). Layers were washed twice with fresh serum-free RPMI1640 medium and re-suspended in phosphate buffered saline (PBS) before thiol content analysis. Infected red blood cells were lysed in 0.1% v/v saponin for 10 min at 37 °C and centrifuged at 1430g for 15 min at room temperature in order to release parasites (Hsiao et al., 1991). Supernatant containing erythrocyte cytosol was separated and 20 ll were diluted in 180 ll of PBS. The pellet was washed with PBS and sedimented at 5590g for 5 min at room temperature, until the red colour disappeared. The resulting pellet was then diluted in 200 ll of distilled water. All tests were performed in triplicate. 2.3. Determination of total glutathione concentration (T-GSH) and cysteine The concentration of total (oxidized and reduced) glutathione (T-GSH) and cysteine in non-infected and infected synchronized erythrocytes was analyzed as monobromobimane (mBBr or thiolyteÒ Calbiochem) by reversed-phase HPLC. The effluent was monitored by a fluorescence detector (excitation 400 nm; emission 475 nm). Under these conditions, the glutathione–thiolyteÒ adduct had a retention time of 13.9 min and cysteine–thiolyteÒ of 10.07 min (Luersen et al., 2000; Zuluaga et al., 2007). For the quantification of total glutathione and cysteine, we employed the methodology of external standard. Successive dilutions of a pattern of concentration of 1 mM for glutathione, cysteine (Cys) and homocysteine (H-Cys) were performed. For the total glutathione, one calibration curve was used, while for Cys it was necessary to elaborate two curves due to concentration variations in

A. Pabón et al. / Experimental Parasitology 122 (2009) 273–279

the samples. A linear regression model of the calibration curves revealed the following information: Thiol

Concentration range (lM)

Equation

Coefficient of determination (r2) (%)

GSH

(0.33–6.67)

99.6

Cys

Lower (0.0067–0.1)

Cys

Higher (0.33–4.0)

H-Cys

(0.067–0.33)

AreaGSH = 103.017  Con.GSH AreaCys = 0.0464  Con.Cys 0.185 AreaCys = 94.27  Con.Cys 11.265 AreaH-Cys = 12.28  Con.H-Cys + 0.490

99.4 99.6 99.78

Analysis of precision showed coefficients of variation of 0.571%, 1.0% and 1.12%, with confidence levels of 95% for the GSH, Cys and the H-Cys, respectively, which are satisfactory for the levels of concentration with which it was run (Foley and Tilley, 1998). The results of the recovery rate assays, for T-GSH and Cys were 123.8 ± 6.6% and 74.8 ± 8.2%, respectively, which are acceptable values given the complexity of the matrix. The sensitivity obtained was 3.2, 0.193 and 0.66 pmol for T-GSH, Cys and H-Cys, respectively. These values are within the range of detected amount when working with mBBr derivatized compounds (Ivanov et al., 2000). 2.4. Determination of the protein concentration It is necessary to normalize the concentration of thiols in each sample in order to account for and to correlate the glutathione content with the amount of protein, allowing for expression of the data in nmol/mg protein. The protein determination of each sample was carried out according to the method of Lowry et al. (1951). 2.5. Inhibition of b-hematin formation S. nudum steroid stock solutions and chloroquine diphosphate were dissolved in chloroform/PVP-10 by sonication. After chloroform evaporation, the PVP-10–steroid copolymer was dissolved in a serum free RPMI medium at 20 mg/mL final concentration. Chloroquine was used as positive control and PVP-10 plus RPMI as negative control. The compounds were tested at 2.5, 0.25, 0.025, and 0.0025 mg/ml final concentration according to Baelmans et al. (2000) and Deharo et al. (2000). 2.6. Statistical analysis Variance analysis and Tukey or Games–Howell post-tests were used to compare the glutathione and Cys concentration in parasitized and non-parasitized erythrocytes for each steroidal compound treatment and for each parasitic stage (rings, trophozoites and schizonts). A confidence level of 95% was employed (a = 0.05 and p 6 0.05) to determine significance. Simple linear regression analysis was used to evaluate the association between dose and bhematin formation for every S. nudum derived compound. All analyses were performed using SpSS plus 14.0 software (SpSS Inc.).

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contents (555.6 ± 223.9 nmol/mg protein and 440.1 ± 169.5 nmol/ mg protein, respectively). However, the Cys content was only 14% in parasitized erythrocytes compared to non-parasitized erythrocytes (data not shown). Schizonts infected erythrocytes had the highest T-GSH contents among the different intraerythrocytic stages (ring: 294.4 ± 89.5 nmol/mg protein, trophozoite: 341.7 ± 71 nmol/mg and schizont: 377.9 ± 59.6 nmol/mg, respectively). The Cys contents decreased >88% in parasitized erythrocytes (all intraerythrocytic stages) when compared to nonparasitized erythrocytes (7.1 ± 2.9 nmol/mg protein and 82 ± 42.7 nmol/mg protein, respectively). We did not observe differences in Cys contents among intraerythrocytic stages. 3.2. The effect of S. nudum steroids on total glutathione and cysteine contents in P. falciparum cultures SN-1 steroid increased the T-GSH contents in all parasite stages as well as in non-infected cells (except for ring-parasitized erythrocytes). However, its effect was significantly more pronounced in P. falciparum trophozoite and schizont stages (Table 1, Fig. 2). This is also true for non-parasitized erythrocytes, where SN-1 doubles the T-GSH contents. SN-1 also increased the Cys contents in rings and schizonts but not in trophozoites nor non-infected erythrocytes. Strikingly, SN-4 produces the opposite effect, reducing the T-GSH contents by 50% in infected and non-infected cells. However, SN4 does not affect the Cys contents in non-parasitized erythrocytes nor in the different parasite stages of P. falciparum. The other steroids, SN-2, SN-3 and Diosgenone did not show effect on T-GSH and Cys contents in any of the cells studied. When T-GSH contents were evaluated in freed P. falciparum parasites, the highest increase was observed in SN-1 treated schizonts only (Table 2, Fig. 3). However, the highest T-GSH concentration was found in the erythrocytic cytosol of trophozoite-infected red blood cells treated by SN-1 (Table 3, Fig. 4). 3.3. The effect of the combination of BSO and S. nudum-derived steroids on total thiol content in P. falciparum parasitized erythrocytes Considering the effect of S. nudum steroids, particularly SN-4 on thiol contents, we postulated that buthionine sulfoximine (a specific inhibitor of GSH synthesis) might increase this effect. However, the BSO and S. nudum steroid combinations did not produce any significant differences when compared to S. nudum steroid treatments alone, with only slight decreases in T-GSH contents observed (Table 1). On the other hand, SN4 was the only one able to significatively reduce the total glutathione content in BSO treated rings compared to untreated rings. It is interesting to note that BSO produced a significant increase in T-GSH concentration in the cytosolic fraction of trophozoites (Table 3), but had no effect on the free forms. 3.4. Inhibition of b-hematin formation SN-1 was able to inhibit more than 80% of heme formation (Table 4) at a concentration of around 5 mM (similar to chloroquine) while the other tested compounds were almost inactive. Interestingly, the combination of PVP-10 and chloroquine lowered its impact on b-hematin formation.

3. Results 4. Discussion 3.1. Total glutathione and cysteine concentrations in P. falciparum cultures Both parasitized and non-parasitized erythrocytes from nonsynchronic P. falciparum cultures (Table 1) showed similar T-GSH

Atamna and Ginsburg (1997) showed that normal erythrocytes and parasites display similar GSH/GSSG ratios and that although this ratio dropped in the host-cell compartment, concentrations of GSH and GSSG in the parasite were comparable to their concen-

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Table 1 Effect of S. nudum steroids on total glutathione and cysteine contents in the intraerythrocytic stage of P. falciparum from synchronous cultures. Intraerythrocytic stage

Treatment

Rings

Control SN-1 SN-4 Control SN-1 SN-4 Control SN-1 SN-4 Control SN-1 SN-4

Trophozoites

Schizonts

Non-parasitized

Glutathione concentration (nmol/mg protein)

Cysteine concentration (nmol/mg protein)

Steroid only

BSO treatment (steroid plus BSO)

Steroid only

294.4 ± 89.5 396.7 ± 21.8 * 114.1 ± 19.0 341.7 ± 71.0 * 767.3 ± 75.2 * 125.4 ± 11.1 377.9 ± 59.6 * 847.3 ± 192.3 * 102.2 ± 9.8 308.9 ± 94.4 *** 614.3.0 ± 77.3 * 80.5 ± 14.5

310.7 ± 49 ** 265.9 ± 21.6 ** 73.4 ± 0.5 315.7 ± 72.4 ** 713.0 ± 119 * 105.4 ± 35.6 324.6 ± 71.2 771 ± 33.7 97.7 ± 12.2 261.3 ± 80.8 576.9 ± 85.0 71.9 ± 13.2

5.8 ± 2.3 *** 240.8 ± 57.8 5.6 ± 2.7 9.8 ± 4.9 15.6 ± 3.5 8.5 ± 1.0 5.6 ± 1.6 ** 43.2 ± 8.9 4.6 ± 1.4 82.0 ± 42.7 163.6 ± 47.1 40.7 ± 11.4

BSO treatment (steroid plus BSO) 15.1 ± 4.5 198.5 ± 11 24.5 ± 8.2 16 ± 4.3 10.7 ± 3.2 * 7.2 ± 1.3 ** 7.3 ± 4.4 23.4 ± 5.3 2.8 ± 0.8 139.6 ± 90.9 127.1 ± 40.3 29.7 ± 13.3

**

P. falciparum forms were obtained as described in Section 2. Parasites were treated with S. nudum steroids at their IC50 value as described in Section 2. Controls were incubated with unsupplemented RPMI instead of S. nudum steroids. Values correspond to median values ± SD. Tukey analysis or Games–Howell post-tests: *p value < 0.05; **p value < 0.01; and ***p value < 0.001. All differences reported were analyzed through comparisons with the control.

350

300

1000

800

600

400

Rings

Cysteine nmol/mg protein

Glutathione nmol/mg protein

1200

Trophozoites

200

SN-1

CONTROL SN-4

150

Rings

100

Trophozoites Schizonts

0

SN-1+BSO

BSO

200

50

Schizonts

0

250

SN-1

SN-4+BSO

CONTROL SN-4

Treatment

SN-1+BSO

BSO

SN-4+BSO

Treatment

Fig. 2. Effect of S. nudum steroids on total glutathione and cysteine contents in the intraerythrocytic stage of P. falciparum from synchronous cultures. Erythrocytes with ring, trophozoites or schizonts P. falciparum stages were obtained as described in Section 2. Parasites were treated with S. nudum steroids at their IC50 value as described in Section 2, while controls were incubated with unsupplemented RPMI only. Values correspond to median values ± SD.

Table 2 Effect of S. nudum steroids on total glutathione and cysteine content in free parasites of synchronized P. falciparum. Intraerythrocytic stage

Rings

Trophozoites

Schizonts

Treatment

Control SN-1 SN-4 Control SN-1 SN-4 Control SN-1 SN-4

Glutathione content (nmol/mg protein)

Cysteine content (nmol/mg protein)

Steroid only

BSO treatment (steroid plus BSO)

Steroid only

56.9 ± 67 25.4 ± 2.2 10.2 ± 5.7 39.8 ± 35.1 36.8 ± 36.9 ** 4.3 ± 1.1 53.7 ± 72.4 * 188.3 ± 49.7 38.4 ± 26.5

45.5 ± 43.1 28.8 ± 22.5 3.8 ± 1.8 20.1 ± 14.4 18.2 ± 7.6 5.6 ± 1.1 65.9 ± 75.9 164.2 ± 62.9 8.7 ± 4.9

54.8 ± 32.5 128.7 ± 47.2 46.4 ± 9.5 190.3 ± 235.1 148.1 ± 54.4 16.7 ± 0.7 37.6 ± 23.3 61.3 ± 8.6 17.4 ± 9.0

*

BSO treatment (steroid plus BSO) 60.6 ± 51.2 156.2 ± 14.5 20.0 ± 12.7 175.7 ± 213.3 169.0 ± 56.4 * 15.7 ± 1.3 54.9 ± 40.7 62.9 ± 8.3 10.5 ± 2.4

*

P. falciparum forms were obtained as described in Section 2. Parasites were treated with S. nudum steroids at their IC50 value as described in Section 2. Controls were incubated with unsupplemented RPMI instead of S. nudum steroids. Values correspond to median values ± SD. Tukey analysis or Games–Howell post-tests: *p value < 0.05; **p value < 0.01; and ***p value < 0.001. All differences reported were analyzed through comparisons with the control.

trations in uninfected red blood cells (Atamna and Ginsburg, 1997). We likewise observed no-significant differences between the whole GSH–GSSG concentrations in infected and non-infected cells. Luersen et al. (2000), showed that infected red blood cells (RBCs) lose GSH at a rate that was 40-fold higher than non-infected

RBCs. Nevertheless, as we studied whole glutathione contents, we could not detect such phenomenon. SN-1 was the only substance that increased Cys, which participates in the de novo synthesis of P. falciparum GSH, implying a downstream reduction in GSH production capability. We also

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200

150

100

Rings 50

Cysteine nmol/mg protein

Glutathione nmol/mg protein

250

600

400

Rings

200

Trophozoites

Trophozoites Schizonts

0 SN-1

CONTROL SN-4

BSO

Schizonts

0

SN-1+BSO

SN-1

SN-4+BSO

CONTROL SN-4

Treatment

SN-1+BSO

BSO

SN-4+BSO

Treatment

Fig. 3. Effect of S. nudum steroids on total glutathione and cysteine content in free parasites of P. falciparum from synchronous cultures. Free parasites were obtained and treated with S. nudum steroids as described in Section 2. Controls were incubated with unsupplemented RPMI only. Values correspond to median values ± SD.

Table 3 Effect of S. nudum steroids on total glutathione and cysteine contents of erythrocytic cytosols of infected red blood cells from synchronized P. falciparum. Intraerythrocytic stage

Treatment

Rings

Control SN-1 SN-4 Control SN-1 SN-4 Control SN-1 SN-4

Trophozoites

Schizonts

Glutathione content (nmol/mg protein)

Cysteine content (nmol/mg protein)

Steroid only

BSO treatment (steroid plus BSO)

Steroid only

BSO treatment (steroid plus BSO)

247.3 ± 1001.1 194.1 ± 26.0 230.5 ± 13.9 279.7 ± 266.9 ** 906.4 ± 164.0 88.0 ± 1.8 326.8 ± 187.7 672.6 ± 144.5 *** 80.6 ± 3.2

194.1 ± 71.9 229.3 ± 40.8 *** 73.0 ± 3.1 292.7 ± 301.9 *** 1259.1 ± 0.2 66.9 ± 4.6 281.6 ± 165.9 594.0 ± 56.6 * 75.9 ± 3.1

67.8 ± 51.8 * 165.6 ± 28.0 57.8 ± 10.5 35.7 ± 29.3 *** 164.2 ± 33.3 44.5 ± 11.7 110.9 ± 123.5 117.4 ± 45.0 5.2 ± 2.1

65.2 ± 51.7 161.6 ± 21.0 57.6 ± 7.7 51.4 ± 38.1 ** 154.9 ± 39.2 28.4 ± 11.6 96.4 ± 85.6 141.3 ± 39.5 4.6 ± 1.6 *

P. falciparum forms were obtained as described in Section 2. Parasites were treated with S. nudum steroids at their IC50 value as described in Section 2. Controls were incubated with unsupplemented RPMI instead of S. nudum steroids. Values correspond to median values ± SD. Tukey analysis or Games–Howell post-tests: *p value < 0.05; **p value < 0.001; and ***p value < 0.0001. All differences reported were analyzed through comparisons with the control.

250

1200

1000

800

600

Rings

400

Trophozoites

200

Schizonts

0 SN-1

CONTROL SN-4

SN-1+BSO

BSO

Treatment

SN-4+BSO

Cysteine nmol/mg protein

Glutathione nmol/mg protein

1400

200

150

100

Rings 50

Trophozoites Schizonts

0 SN-1

CONTROL SN-4

SN-1+BSO

BSO

SN-4+BSO

Treatment

Fig. 4. Effect of S. nudum steroids on total glutathione and cysteine contents of erythrocytic cytosol of infected red blood cells in synchronous cultures. Erythrocytes with ring, trophozoites or schizonts P. falciparum stages were obtained as described in Section 2. Parasites were treated with S. nudum steroids (at their IC50 value) while controls were incubated with unsupplemented RPMI only. Values correspond to median values ± SD.

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Table 4 Inhibition of b-hematin formation by S. nudum steroids. Compound

% Inhibition/(concentrations evaluated mM)

CQ CQ + PVP SN-1 SN-2 SN-3 SN-4 Diosgenone

79.5% 45.5% 86.6% 26.3% 33.4% 35.5% 39.6%

(5) (5) (5) (6) (4) (4) (6)

25.9% 32.1% 25.1% 12.6% 27.1% 27.9% 26.7%

(0.5) (0.5) (0.5) (0.6) (0.4) (0.4) (0.6)

11.0% (0.05) 20.56% (0.05) 0.0% (0.05) 7.2% (0.06) 25.7% (0.04) 8.1% (0.04) 8.8% (0.06)

6.0% 10.8% 0.0% 1.7% 25.7% 8.7% 7.6%

(0.005) (0.005) (0.005) (0.006) (0.004) (0.004) (0.006)

% Inhibition of b-hematin formation obtained with decreasing concentrations of tested compounds.

showed that acetylation of C16 was crucial for that effect, as SN-2 (lacking this group) did not have any effect on thiol contents. It could be hypothesized that SN-1 could affect the parasite redox environment, activating the c-glutamylcysteine synthetase as it is known to be regulated by steroids, at the level of its enzymatic activity and the expression of its two catalytic and modulator subunits (Franklin et al., 2008; Lu, in press). Forman et al. (in press) also showed that oxidant species and electrophiles are able to increase the transcription of both the modulatory and catalytic subunits and induce de novo GSH synthesis (Forman et al., in press). SN-1 might also affect the parasite redox environment avoiding GSSG efflux. On the contrary, SN-4 decreased the concentration of both thiols. Interestingly, SN-3 that differs from SN-4 by the acetylation of C16 instead of a furostanol was inactive. The decline of thiols under SN-4 pressure, could be due to the modification of the membrane fluidity as shown elsewhere (Liang et al., 2001) with cholesterol, estrogens, progesterone and testosterone, which are able to alter membrane-bound enzymes such as Na+/K+-ATPase and Ca2+-ATPase. SN-4 could also stimulate the multidrug resistance-associated proteins (Mrp/Abcc) known to mediate GSH homeostasis (Ballatori et al., in press). Further studies are necessary in order to understand which glutathione form (reduced or oxidized) is truly affected by these compounds. In this study, BSO did not affect either the T-GSH nor the Cys contents in P. falciparum parasitized erythrocytes. However, BSO significantly increased the SN-1 and SN-4 effect on the T-GSH contents for immature forms of P. falciparum but lowered the T-GSH contents by 33% and 36% in ring stages (compared to non-BSO treated cells). Luersen et al. (2000) reported that 73 lM of BSO applied to cells for 48 h inhibited 50% of P. falciparum growth in culture (Luersen et al., 2000). However, the same concentration used for 24 h instead of 48, did not affect the T-GSH contents nor the growth of the parasite. Our cultures were treated with the same BSO concentration for only 24 h without affecting the T-GSH contents nor producing toxic. It will be interesting to study these combinations over a more prolonged period. Nevertheless, if a synergistic effect exists with this combination, an appreciable trend would be observed in this 24-h period. It is most likely necessary to extend the incubation period, and/or concentration range to appreciate the expected synergistic effect against the FCB-2 strain of P. falciparum. The addition of monoethyl glutathione esters to parasitized erythrocytes abolishes the BSO antimalaric effect and increases the GSH intracellular levels. However, in GSH or monoethyl ester GSH treated parasitized erythrocytes, concentrations as low as 1 mM do not produce any effect on the intracellular GSH contents (Luersen et al., 2000). Luersen et al. (2000) suggested that GSH in the culture medium could buffer the BSO effect and that high GSH concentrations in culture media could recover GSH contents inside parasitized erythrocytes due to the parasite induced transporter expression along cell membranes. It has been reported that

in P. falciparum parasitized erythrocytes, BSO at a concentration of 100 lM for 24 h, produces a 67% inhibition of enzyme c-GCS and a 74% GSH decrease. In non-parasitized erythrocytes, c-GCS inhibition is higher than 82%, but GSH decrease is only 14% within 24 h. GSH oxidation and an energy dependent thiol loss explain the high glutathione turnover observed in infected erythrocytes. Approximately 50% of GSH in 100 lM BSO treated non-parasitized erythrocytes is consumed in a 4–5 day period, whereas in parasitized erythrocytes (where GSH requirements are high), the turnover requires only 2.5 h (Luersen et al., 2000). This indicates low thiol consumption in non-parasitized erythrocytes. In this study, we measured only T-GSH (reduced plus oxidized forms). Studies of the proportion of both forms and the GSH-synthesizing enzyme expression pattern could be illuminating of other relationships in parasite thiol metabolism. Among S. nudum steroids, SN-1 produced the highest inhibition of b-hematin formation. Although the techniques used here revealed a direct interaction during the b-hematin formation process, it is likely that in the parasite, the impact on the hemozoin synthesis is also due to the detoxification activity of GSH. In conclusion, S. nudum derivative steroids are able to alter TGSH and Cys contents of P. falciparum infected red blood cells and non-parasitized erythrocytes. SN-1 steroid increases and SN4 decreases total glutathione contents. Further analysis of glutathione synthetic pathways, including those related with the transcription or translation processes are needed to determine more precisely the mechanism of action of these antagonistic compounds. Acknowledgments This study was supported by Colciencias Financial Support (Grant 1115-04-12944, RC-111-03), Universidad de Antioquia, Colombia, CODI-UdeA, FONDECY 1061072 and, Anillo ACT 29, PBCT-CONICYT-Chile. The authors thank Ana Maria Mesa for the isolation of steroidal compounds of S. nudum, Aleida Ochoa and Professor Silvio Ayala for thiol quantification and Professor Luis Carlos Burgos for academic consulting. We also thank Gonzalo Álvarez for statistical consulting and Colleen McClean for assistance in translation and editing. References Alvarez, G., Pabon, A., Carmona, J., Blair, S., 2004. Evaluation of clastogenic potential of the antimalarial plant Solanum nudum. Phytotherapy Research 18, 845–848. Arango, E., Londono, B., Segura, C., Solarte, Y., Herrera, S., Saez, J., Carmona-Fonseca, J., Blair, S., 2006. Prevention of sporogony of Plasmodium vivax in Anopheles albimanus by steroids of Solanum nudum Dunal (Solanaceae). Phytotherapy Research 20, 444–447. Atamna, H., Ginsburg, H., 1997. The malaria parasite supplies glutathione to its host cell – investigation of glutathione transport and metabolism in human erythrocytes infected with Plasmodium falciparum. European Journal of Biochemistry 250, 670–679. Baelmans, R., Deharo, E., Munoz, V., Sauvain, M., Ginsburg, H., 2000. Experimental conditions for testing the inhibitory activity of chloroquine on the formation of beta-hematin. Experimental Parasitology 96, 243–248. Ballatori, N., Krance, S.M., Marchan, R., Hammond, C.L., in press. Plasma membrane glutathione transporters and their roles in cell physiology and pathophysiology. Molecular Aspects of Medicine. 10.1016/j.mam.2008.08.004. Blair, S., Carmona-Fonseca, J., Pineros, J.G., Rios, A., Alvarez, T., Alvarez, G., Tobon, A., 2006. Therapeutic efficacy test in malaria falciparum in Antioquia, Colombia. Malaria Journal 5, 14–22. Blair, S., Mesa, J., Correa, A., Saez, J., 2001. Apertura del anillo F de al diosgenona y actividad antimalárica in vitro de los productos de reacción. Revista colombiana de Química 30, 97–107. Deharo, E., Gautret, P., Muñoz, V., Sauvain, M., 2000. Técnicas de laboratorio para la selección de substancias antimalaricas. In: La Paz, Deharo E. (Ed.), Cyted-IRD. Dubois, V.L., Platel, D.F., Pauly, G., Tribouley-Duret, J., 1995. Plasmodium berghei: implication of intracellular glutathione and its related enzyme in chloroquine resistance in vivo. Experimental Parasitology 81, 117–124. Echeverri, M., Blair, S., Carmona, J., Perez, P., 2001. Effect of Solanum nudum extracts on the liver of mice infected with Plasmodium berghei. American Journal of Chinese Medicine 29, 477–484.

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