L-cysteine whether a nutritional booster or a radical scavenger for Plasmodium.

INTRODUCTION: Plasmodium falciparum is the most noxious species among other Plasmodium species that cause malaria. Attention is required to understand more about the pathophysiology and parasite biology to obscure this disease. The fact is, very little is known about the nutritional requirement in sense of carbohydrate, lipid, nucleic acid, and amino acid metabolism that regulate the growth of parasite and out of this, studies related to the metabolism of amino acid are exceptionally limited. Out of several amino acids, L-cysteine is essential for the continuous erythrocytic growth of Plasmodium. However, the exact role of L-cysteine in regulating the growth of Plasmodium is unknown. Here, we tried to investigate how does L-cysteine affects the growth of Plasmodium in in vitro culture, and also the study was aimed to find whether there is a synergism with chloroquine on the Plasmodium growth in vitro.
MATERIALS AND METHODS: Parasite inhibition assay based on schizont maturation inhibition following WHO protocol on P. falciparum chloroquine-sensitive strain (MRC-2) was employed to determine IC50 value and drug interaction pattern was shown through fractional inhibitory concentration index.
RESULTS: Inhibitory effect of L-cysteine hydrochloride on Plasmodium growth was depicted with IC50 1.152 ± 0.287 μg/mL and the most synergistic pattern of interaction was shown with chloroquine.
CONCLUSIONS: The present study anticipates two important findings, firstly inconsistent results from previous findings and secondly, synergistic effect with chloroquine suggests its potency that may be used as an add-on therapy along with chloroquine. However, further study is needed to validate the above findings in vivo models. Copyright:

INTRODUCTION

Malaria caused due to Plasmodium falciparum is found to be more noxious than the other four Plasmodium species, i.e., P. vivax, P. ovale, P. malariae, and P. knowlesi. According to WHO, there were around 445,000 total deaths worldwide which mostly occurred in children under the age of five.[1] Thus, the present situation necessitates the social need to broaden the understanding regarding pathogenesis and parasite biology, which will further enhance the idea to choose more optimum drug target. Now, as Plasmodium is an endoparasite most of its nutritional requirement is fulfilled through the degradation of hemoglobin at its early erythrocytic stage. Also, it is accompanied with various channels present at the parasitophorous vacuole membrane which allow the uptake of other essential nutrients.[2] Out of various major biomolecules, amino acid is acquired by the malarial parasite by three ways for its continuous erythrocytic growth[34] (1) synthesis from other carbon sources; (2) acquiring through transporter channel; (3) by degrading hemoglobin of host cell. After acquiring amino acids then these are mostly consumed by parasitized erythrocytes at much faster rates.[56] Later, Divo et al.[7] show P. falciparum entails seven exogenously supplied amino acids namely cysteine, isoleucine, glutamate, glutamine, proline, methionine, and tyrosine for its continuous erythrocytic growth. Apart from this, L-cysteine is a conditional essential amino acid that contains two sulfur and a thiol group (-SH) that makes it less toxic and acts as an antioxidant. In its reduced form, L-cysteine and N-acetyl-cysteine continue to provide free radical quenching effects.[89] L-cysteine assists in the synthesis of the highly antioxidative glutathione and is mainly stored in this chemical form. Accordingly plays the foremost part in detoxification that mitigates the protection of several tissues and organs.[1011] N-acetylcysteine may be also beneficial in severe malaria by intensifying free radicals scavenging and by reducing the expression of endothelial ligands.[12] It can also improve red cell deformability when tested under in vitro conditions.[13] Besides all these, there is only limited degree of studies regarding the metabolic fate of amino acids in the erythrocytic stages of malaria,[34] that provide a strong basis to explore the potency of exogenously supplied cysteine amino acid. Also, drug combinations bestow additional opportunities over single drug therapies to attain sufficient therapeutic effect at a lower and potentially safer dose and can also delay or prevent drug resistance development,[141516] which is the major concern while opting new therapeutic leads.

So, the present study was carried out to investigate how does L-cysteine affects the growth of Plasmodium in in vitro culture, and also the study was aimed to find whether there is a synergism with chloroquine on the Plasmodium growth in vitro.

MATERIALS AND METHODS

P. falciparum chloroquine-sensitive strain (MRC-2) was procured from the National Institute of Malaria Research (NIMR), New Delhi, India, and later maintained in the Department of Medical Parasitology, Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh, India for a period of one year.

In vitro maintenance of Plasmodium falciparum asexual stage

P. falciparum sensitive strain (MRC-2) was maintained in A/B+ erythrocytes in RPMI-1640 medium with human serum. The strain was maintained in vitro in continuous culture according to the candle jar technique discussed earlier,[17] with slight modifications. Briefly, the strain was maintained in A/B+ erythrocytes in RPMI-1640 medium supplemented with 10% inactivated AB+ human serum, 1 mM glutamine, 10 mM glucose, 25 mM HEPES, 0.2% (w/v) sodium bicarbonate, and 40 mg/ml gentamycin, then incubated in gas mixture of 5% CO2, 5% O2 and 90% N2 at 37°C temperature. Contamination was avoided by adding 40 mg/ml of gentamycin. Infected erythrocytes were suspended in RPMI1640 culture media at a starting hematocrit of 5% and parasitemia was kept in between 2 and 4% with subculturing at regular intervals beyond 5%. During subculturing old media was replaced with fresh media and washed A/B+ erythrocytes. A/B+ erythrocytes were washed in freshly prepared RPMI-1640 medium having the same constituents as used for culture maintenance except the addition of AB+ human serum. The washing was done through centrifugation at 1500 rpm for 10 min to remove the plasma and buffy coat. The remaining pellet was suspended in equal amount of culture medium and store at 4°C for further subculturing process. The medium was changed once a time every day at an interval of 21–24 h and percentage parasitemia was monitored by Giemsa stained slides.

Synchronization

The parasite cultures were synchronized to get ring stages of the parasite. Synchronization was done using D-sorbitol that removes all stages except ring-stage parasite. Briefly, aqueous 5% D-sorbitol was added in equal volume to the cultures having majority of ring stages and were kept at room temperature for 5 min, then after centrifuging at 2000 rpm for 5 min.[18] The obtained pellet was suspended in RPMI-1640 medium containing 10% human serum and fresh erythrocytes. This synchronized culture with 1% parasitemia and 5% hematocrit were finally used for parasite inhibition assay.

Stock solution of compounds

Stock solution of compounds, chloroquine phosphate, and cysteine hydrochloride were prepared separately in distilled water to get the stock solution of 1 mg/ml strength.

Parasite inhibition assay

The concentration of an individual compound entailed to impede parasites multiplication by 50% (IC50) against P. falciparum was resolute using concentration-response assay in 96-well tissue culture plates. Synchronous parasite cultures were then subjected to the graded concentration of each compound, prepared in gentamycin-free RPMI culture medium, for 24 h at 37°C in an atmosphere of 5% CO2, 5% O2, and 90% N2. The results were expressed as IC50 and IC90 values computed from HN-NonLin examined by thick smear Giemsa stained slides.[19]

Slide preparation, staining, and assessment

Thick blood smear slides were prepared at each drug concentrations then, air dried, methanol fixed, and finally stained in Giemsa solution for about 40 min. Following staining, slides were removed from the coupling jar, washed in running tap water and air dried. The Giemsa stained slides were examined for counting the number of parasites in random adjacent microscopic fields, equivalent to about 2000 erythrocytes at × 1000 magnification. Percent of parasitemia will be calculated.

(Schizont in Test well) ×100

Percent parasitemia = (Schizont in control well)

Percent parasite inhibition = 100-percent parasitemia

Combinational assay

For the combinational assay, drug strength was made by taking IC50 in μg/mL of the individual drugs in four combinations in the ratio of chloroquine phosphate: L-cysteine hydrochloride that is 4:1, 3:2, 2:3, and 1:4 after that two-fold serial dilution was made till six concentrations. IC50 was calculated from dose–response graphs. Fractional inhibitory concentration (FIC) values were computed separately for individual drug concentration in the combination using the following formula:

FIC = Fraction of drug concentration required to produce IC50 when used in combination Fraction of drug concentration required to produce IC50 when used alone.

For drug interaction,[20] the sum of the FICs (Σ FICs) of both the drugs for a particular combination that shows the interaction pattern between the two drugs also called as the FIC index (FICI) is therefore given by:

Σ FIC (FICI) = FIC (A) + FIC (B)

The results were interpreted as a synergetic effect when the value of the Σ FIC (FICI) was ≤0.5, as indifferent or additive when it was >0.5–2.0, and as antagonistic effect when it was >2.0.[21]

Data analysis

All data were collected from two independent sets of each experiment and were presented as mean ± standard deviation (SD) Microsoft Excel 10 was used for plotting dose-response curves and analysis.

RESULTS AND DISCUSSION

Despite the fact that cysteine is essential for continuous erythrocytic growth of Plasmodium and some suggested that deficiency of isoleucine, methionine, and cysteine from the culture media lead to a marked reduction in the growth of P. knowlesi under in vitro conditions.[34] However, the result from the present study showed concentration-dependent inhibition of P. falciparum strain in the presence of L-cysteine HCL [Figure 1]. The 50% inhibitory concentration of L-cysteine HCL on chloroquine-sensitive strain of P. falciparum was found to be 1.152 ± 0.287 μg/mL which is much higher as compared to standard drug chloroquine having IC50 value of 0.132 ± 0.034 μg/mL [Table 1] when both were tested in the equivalent concentration range i.e., 50 μg/mL to 0.8 μg/mL. This showed that L-cysteine HCL has inhibitory effect on Plasmodium with the increasing concentration, but the implementation of such higher dosages may lead to certain adverse effects.[22]

Figure 1

(a) The in vitro growth of Plasmodium falciparum under normal condition observed under oil immersion lens ×100. (b) The in vitro growth of Plasmodium falciparum under the effect of L-cysteine hydrochloride observed under oil immersion lens ×1000. (c) Shows % inhibition with respect to increasing concentration of L-cysteine HCL

Table 1

Calculated inhibitory concentration50 and inhibitory concentration90 value on Plasmodium falciparum chloroquine-sensitive strain (MRC-2)

Drug/Compound	IC50 [µg/mL]	IC90 [µg/mL]	R2
Chloroquine	0.132±0.034	0.893±0.057	0.94
L-Cysteine HCL	1.152±0.287	19.65±9.83	0.95

In the combinational assay, L-cysteine HCL showed a remarkable inhibitory effect on Plasmodium growth with chloroquine in dose-response curve analysis [Figure 2]. Four different combinations were evaluated in which at the ratio 4:1, 3:2, 2:3 and 1:4 (chloroquine: L-cysteine HCL), the % parasitemia was reduced to 88.51 ± 1.41, 81.86 ± 0.70, 83.49 ± 0.70 and finally 72.40 ± 0.70.

Figure 2

Dose-response curves (Chloroquine: L-cysteine HCL) in four different combination, i.e., 4:1, 3:2, 2:3, 1:4

Further, in the combinational assay, drug interaction assessment between Chloroquine and L-cysteine HCL was evaluated by Σ FICs values in fixed-ratio combinations, showed an additive pattern with combination 1, good synergism with combination 2, and marked synergism with combination 3 and 4 [Table 2].

Table 2

Fixed-ratio interaction of Chloroquine and L-cysteine against Plasmodium falciparum chloroquine-sensitive (MRC-2) strains

Combination Solution	Ratio of Drug (in 100µL)		Mean FIC50±SD
	Drug A	Drug B	Drug A (CQ)	Drug B (L-Cysteine HCL)	Σ FICs, Interaction†
1	4	1	0.57±1.49	0.06±0.17	0.63	ADD
2	3	2	0.35±0.48	0.04±0.05	0.39	SYN
3	2	3	0.16±0.80	0.02±0.09	0.18	SYN
4	1	4	0.09±0.91	0.01±0.10	0.10	SYN

The mean Σ FIC values of Chloroquine (CQ) and L-Cysteine derivatives in fixed-ratio combinations showed a synergistic and additive interaction with all combinations. Values are expressed as the means ± SD from at least two (n = 2) in vitro experiments.

Thus, the result illustrates a marked synergistic effect of L-cysteine HCL with chloroquine in inhibiting Plasmodium growth when the concentration of L-cysteine HCL was increased to four-fold in combinational assays. Few studies were carried out to confirm the safety of N-acetylcysteine when used as an adjunctive treatment with quinine[23] and artesunate in case of severe malaria.[24] However, both these studies suggest only the possibility that N-acetylcysteine may accelerate recovery from severe malaria. Also, no beneficial effects were observed from the use of high-dose N-acetylcysteine as adjunctive treatment to intravenous artesunate in a randomized, double-blind, placebo-controlled clinical trial, in adult patients having severe P. falciparum infection.[25] In an in vitro study, a combination of N-acetylcysteine with artesunate showed antagonistic effect whereas such interaction was not observed with quinine in the same study.[26] Therefore, our results are contrary to the previous results observed by Charunwatthana et al., in a randomized clinical trial. The reason for this contradiction may be due to the employment of in vitro combination assay which is subject to numerous sources of variability across different laboratories and can also rule out various confounding factors like individual drug-drug interaction, host responses, other is the choice of chloroquine having different chemical skeletal compared to artesunate and quinine. Despite this, till now there is no literature available for this combination that shows such interaction studies.

Cysteine is also a precursor of glutathione, an antioxidant that may reduce oxidation injury. During Plasmodium infection severity of disease is related to oxidative damage caused due to the production of reactive oxygen species by the infected cells that imbalance host antioxidant potential resulting in physiological changes inside the body that may improve the disease outcome. In a recent study, supplementation of N-acetylcysteine which is a reduced form of cysteine reduces the parasitemia level and increases the antioxidant potential of P. berghei infected mice.[27] Inside the living cell, N-acetylcysteine is deacetylated to yield L-cysteine which promotes intracellular glutathione production which in turn induce antigen-presenting cells to produce interleukin-12 (IL-12) and IL-12 is supposed to suppress the parasitemia level in in vivo models.[28] So, N-acetylcysteine is related to lowering the parasitemia level by augmenting the IL-12 production.[29] However, more illustration is needed to find a valid conclusion.

CONCLUSIONS

The present study is found to be contrary to the previous finding where cysteine is added exogenously for accelerated growth of Plasmodium in in vitro culture, and also this is the first study that shows the effect of L-cysteine HCL on chloroquine-sensitive strain of P. falciparum. However, further study is needed to find out the mechanism of inhibition of L-cysteine HCL which might be due to the free radical scavenging property of L-cysteine. Also, the synergistic effect with chloroquine suggests its potency that may be used as an add-on therapy along with chloroquine as chloroquine is still a recommended therapy in many malaria-endemic regions where parasite resistant to chloroquine is not found. But more illustration is needed to validate the above findings in vivo models.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.