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PHD : Parasites, Hosts and Diseases

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Original Article

Pyruvate Protects Giardia Trophozoites from Cysteine-Ascorbate Deprived Medium Induced Cytotoxicity

The Korean Journal of Parasitology 2018;56(1):1-9.
Published online: February 28, 2018

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*Corresponding author (sandipanganguly@gmail.com)
• Received: May 8, 2017   • Revised: October 4, 2017   • Accepted: October 27, 2017

© 2018 by The Korean Society for Parasitology and Tropical Medicine

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Pyruvate Protects Giardia Trophozoites from Cysteine-Ascorbate Deprived Medium Induced Cytotoxicity
Korean J Parasitol. 2018;56(1):1-9.   Published online February 28, 2018
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Pyruvate Protects Giardia Trophozoites from Cysteine-Ascorbate Deprived Medium Induced Cytotoxicity
Korean J Parasitol. 2018;56(1):1-9.   Published online February 28, 2018
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Pyruvate Protects Giardia Trophozoites from Cysteine-Ascorbate Deprived Medium Induced Cytotoxicity
Image Image Image Image Image Image Image Image
Fig. 1 Pyruvate decreases the level of ROS in Giardia lamblia trophozoites under cysteine-ascorbate deprivation. Giardia trophozoites were incubated in cysteine-ascorbate deprived (CAD) medium and exposed to increasing doses of pyruvate (from 0–5 mM). Levels of ROS were estimated by spectrofluorometry using 2′, 7′-dichlorodihydrofluoresceine diacetate. Data are the mean±SEM of 3 independent experiments, each performed in triplicate. Pyr, Pyruvate; Man, Manitol. *P<0.05, compared with control; #P<0.001, compared with stressed sample.
Fig. 2 Pyruvate protects Giardia trophozoites from CAD medium induced cytoxicity. Trophozoites were incubated at 35.5°C upon CAD medium for 8 hr with increasing concentration of pyruvate. CAD medium-induced trophozoites were reseeded in fresh TYIS-33 medium and their viability was evaluated after 24 hr by using flowcytometry. Acetate (Ace) was shown not to decrease the rate of ROS generation in Giardia trophozoites. Results are expressed as the percentage of surviving trophozoites compared with control sample. Data are the mean±SEM of 3 independent experiments, each performed in triplicate. *P<0.05, compared with control; #P<0.001, compared with stressed sample.
Fig. 3 ROS scavenging capacities and antioxidant properties of various α-ketoacids. Giardia trophozoites were preincubated for 30 min with a 2 mM concentration of each compound (Oxa=oxaloacetate, Mal=malate, Pyr=pyruvate, αKgl=α-ketoglutarate, Man=mannitol, Ace=acetate) and further incubated for 8 hr under CAD medium in their presence or absence. Viability was estimated 24 hr later. Data are the mean±SEM of 3 independent experiments, each performed in triplicate. *P<0.05, compared with control; #P<0.001, compared with stressed sample.
Fig. 4 Effect of pyruvate on level of lipid peroxidation in cultured Giardia trophozoites upon CAD medium stress: MDA concentration in CAD medium stressed Giardia trophozoites after 8 hr incubation. Values are mean±SEM of 3 independent experiments, each performed in triplicate. *P<0.05, compared with control; #P<0.001, compared with stressed sample.
Fig. 5 Intracellular pyruvate concentration in Giardia trophozoites during oxidative stress. Intracellular pyruvate content was measured in Giardia lamblia under CAD medium deprivation. The level of intracellular pyruvate was quantified every 2 hr interval. Values are means±SEM of 3 independent experiments, each performed in triplicate. *P<0.05, compared with control; ιP<0.001, compared with 4th hour stressed sample, τP<0.001, compared with 2nd hour stressed sample.
Fig. 6 Quantification of viable cells. Counts of viable cells are given as percentages of the total number of cells. Trophozoites were incubated with CAD medium for 8 hr after that pyruvate was added and then further incubated for 30–120 min. Stress-induced trophozoites were reseeded in fresh TYI-S-33 medium supplemented with the corresponding concentration of pyruvate and their survival rate was evaluated after 24 hr by using flowcytometry. Results are expressed as the percentage of surviving trophozoites compared with control culture. Data are the mean±SEM of 3 independent experiments, each performed in triplicate. Pyr=pyruvate. *P<0.05, compared with control; #P<0.001, compared with stressed sample. Results are from 3 independent experiments, each performed thrice.
Fig. 7 Effect of CAD medium on the expression of genes involved in pyruvate metabolism. Modulation of transcripts encoding enzymes involved in pyruvate metabolism. A gene expression (fold change) under CAD medium stress. Data are shown as fold change in relative expression compared with Actin on the basis of Comparative Ct (2−ΔΔCt) method. Values are shown as mean±SEM of 3 independent experiments, each performed in triplicate. (gene abbreviations used: metabolic genes: PFOR: pyruvate-ferredoxin oxidoreductade, MALDH: malate dehydrogenase, ARGD: arginine deiminase, PK: pyruvate kinase, PDK: pyruvate dikinase, ACS: acetyl coA synthase).
Fig. 8 Schematic representation of the oxidative stress management in Giaradia. (A) Healthy cell. (B) Stressed cells accumulates intracellular ROS. (C) Concentration of intracellular ROS generation augmented. (D) Intracellular pyruvate concentration increases by the upregulation of malate dehydrogenase and pyruvate phosphate dikinase. (E) As ROS generation is a cyclic and also a spontaneous process it produces alcoxyl, peroxyl or lipid radicals by lipid peroxidation. (F) Lipid radicals are not scavenged by pyruvate so it produces more lipid radicals. (G) Intracellular acetate concentration is up-regulated as acetyl CoA synthase up-regulated. (H) On further stress, as pyruvate ferredoxin oxidoreductase is down-regulated so acetate production is inhibited and ultimate fate is death.
Pyruvate Protects Giardia Trophozoites from Cysteine-Ascorbate Deprived Medium Induced Cytotoxicity