Melatonin Inhibits Free Radical-Mediated Mitochondrial-Dependent Hepatocyte Apoptosis and Liver Damage Induced During Malarial infection

We showed earlier that malarial infection significantly induces liver apoptosis mediated by oxidative stress mechanisms. Thus, a nontoxic antioxidant–antiapoptotic molecule may be beneficial for hepatoprotection. Melatonin remarkably prevents hepatocyte apoptosis in mice induced during malaria as indicated by caspase 3 and TUNEL assays as well as transmission electron microscopy (TEM) of the liver tissue. The mitochondrial apoptotic pathway, which plays a critical role in liver cell death during malarial infection, was almost completely suppressed by melatonin as it corrects both the overexpression of Bax and down-regulation of bcl-2 as revealed by semiquantitative RT-PCR. Fluorometric studies using JC-1 documented that melatonin also restores mitochondrial transmembrane potential (DYm) in malaria-infected mice liver. The antiapoptotic effect of melatonin is associated with its antioxidant role because melatonin protects liver from oxidative stress induced during malaria by scavenging the hydroxyl radicals, preventing the depletion of reduced glutathione, inhibiting lipid peroxidation and protein carbonyl formation. The effective antioxidant dose of melatonin to protect liver from oxidative stress during malaria is 20 times lower than that of known antioxidants, vitamin C and vitamin E. Apoptosis of hepatocytes during malarial infection is well correlated with dysfunction of the liver while melatonin offers hepatoprotective effects as indicated by different liver function tests. Thus, melatonin may well be effective in combating oxidative stress-induced apoptosis and liver damage during malaria infection

Antimalarial drugs inhibiting hemozoin (β-hematin) formation: a mechanistic update

Digestion of hemoglobin in the food vacuole of the malaria parasite produces very high quantities of redox active toxic free heme. Hemozoin (β-hematin) formation is a unique process adopted by Plasmodium sp. to detoxify free heme. Hemozoin formation is a validated target for most of the well-known existing antimalarial drugs and considered to be a suitable target to develop new antimalarials. Here we discuss the possible mechanisms of free heme detoxification in the malaria parasite and the mechanistic details of compounds, which offer antimalarial activity by inhibiting hemozoin formation. The chemical nature of new antimalarial compounds showing antimalarial activity through the inhibition of hemozoin formation has also been incorporated, which may help to design future antimalarials with therapeutic potential against multi-drug resistant malaria

Apoptosis in liver during malaria: role of oxidative stress and implication of mitochondrial pathway

Hepatic dysfunction is a common clinical complication in malaria, although its pathogenesis remains largely unknown. Using a variety of in vivo and ex vivo approaches, we have shown for the first time that malarial infection induces hepatic apoptosis through augmentation of oxidative stress. Apoptosis in hepatocyte has been confirmed by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin-nick-end labeling assay (TUNEL) and caspase-3 activation. Gene expression analysis using RT-PCR indicates the significant down-regulation of Bcl-2 and up-regulation of Bax expression in liver of malaria infected mice suggesting the involvement of mitochondrial pathway of apoptosis. The levels of Fas expression and caspase-8 activity in infected liver were same as that of uninfected control mice indicating death receptor (Fas) pathway did not contribute to liver apoptosis during malarial infection. Moreover, evidence has been presented by confocal microscopy to show the translocation of Bax from cytosol to mitochondria in apoptotic hepatocyte, resulting in opening of permeability transition pores, which in turn decreases mitochondrial membrane potential and induces cytochrome c release into cytosol. Malarial infection induces the generation of hydroxyl radical (·OH) in liver, which may be responsible for the induction of oxidative stress and apoptosis as administration of ·OH specific antioxidant as well as spin trap, alpha-phenyl-tert-butyl-nitrone in malaria-infected mice significantly inhibits the development of oxidative stress as well as induction of apoptosis. Thus, results suggest the implication of oxidative stress induced-mitochondrial pathway of apoptosis in the pathophysiology of hepatic dysfunction in malaria.—Guha, M., Kumar, S., Choubey, V., Maity, P., Bandyopadhyay, U. Apoptosis in liver during malaria: Role of oxidative stress and implication of mitochondrial pathway

Inhibition of Plasmodium falciparum Choline Kinase by Hexadecyltrimethylammonium Bromide: a Possible Antimalarial Mechanism

Choline kinase is the first enzyme in the Kennedy pathway (CDP-choline pathway) for the biosynthesis of the most essential phospholipid, phosphatidylcholine, in Plasmodium falciparum. In addition, choline kinase also plays a pivotal role in trapping essential polar head group choline inside the malaria parasite. Recently, Plasmodium falciparum choline kinase (PfCK) has been cloned, overexpressed, and purified. However, the function of this enzyme in parasite growth and survival has not been evaluated owing to the lack of a suitable inhibitor. Purified recombinant PfCK enabled us to identify an inhibitor of PfCK, hexadecyltrimethylammonium bromide (HDTAB), which has a very close structural resemblance to hexadecylphosphocholine (miltefosin), the well-known antiproliferative and antileishmanial drug. HDTAB inhibited PfCK in a dose-dependent manner and offered very potent antimalarial activity in vitro against Plasmodium falciparum. Moreover, HDTAB exhibited profound antimalarial activity in vivo against the rodent malaria parasite Plasmodium yoelii (N-67 strain). Interestingly, parasites at the trophozoite and schizont stages were found to be particularly sensitive to HDTAB. The stage-specific antimalarial effect of HDTAB correlated well with the expression pattern of PfCK in P. falciparum, which was observed by reverse transcription-PCR and immunofluorescence microscopy. Furthermore, the antimalarial activity of HDTAB paralleled the decrease in phosphatidylcholine content, which was found to correlate with the decreased phosphocholine generation. These results suggest that inhibition of choline kinase by HDTAB leads to decreased phosphocholine, which in turn causes a decrease in phosphatidylcholine biosynthesis, resulting in death of the parasite

Antiplasmodial Activity of [(Aryl)arylsulfanylmethyl]Pyridine▿ †‡

A series of [(aryl)arylsufanylmethyl]pyridines (AASMP) have been synthesized. These compounds inhibited hemozoin formation, formed complexes (KD = 12 to 20 μM) with free heme (ferriprotoporphyrin IX) at a pH close to the pH of the parasite food vacuole, and exhibited antimalarial activity in vitro. The inhibition of hemozoin formation may develop oxidative stress in Plasmodium falciparum due to the accumulation of free heme. Interestingly, AASMP developed oxidative stress in the parasite, as evident from the decreased level of glutathione and increased formation of lipid peroxide, H2O2, and hydroxyl radical (·OH) in P. falciparum. AASMP also caused mitochondrial dysfunction by decreasing mitochondrial potential (ΔΨm) in malaria parasite, as measured by both flow cytometry and fluorescence microscopy. Furthermore, the generation of ·OH may be mainly responsible for the antimalarial effect of AASMP since ·OH scavengers such as mannitol, as well as spin trap α-phenyl-n-tertbutylnitrone, significantly protected P. falciparum from AASMP-mediated growth inhibition. Cytotoxicity testing of the active compounds showed selective activity against malaria parasite with selectivity indices greater than 100. AASMP also exhibited profound antimalarial activity in vivo against chloroquine resistant P. yoelii. Thus, AASMP represents a novel class of antimalarial

Overexpression, purification and localization of apoptosis related protein from Plasmodium falciparum

A growing body of evidence has ascertained that apoptosis is not only restricted to metazoans but also exists in unicellular parasites. In Plasmodium falciparum, the presence of a putative gene having sequence homology with apoptosis related protein (PfARP) (Gene ID PFI0450c) has raised enormous interest to unravel the function of this unique protein in cell death of malaria parasite. To characterize this protein, the PfARP gene has been amplified from the P. falciparum transcriptome by RT-PCR and the amplified gene has been successfully cloned, over-expressed and purified to homogeneity. The purified PfARP exhibits minimum subunit MW of ∼24 kDa as evident from SDS–PAGE. CD analysis reveals that the α and β content of the recombinant PfARP are 61% and 15%, respectively. Semiquantitative RT-PCR analysis indicates the expression of PfARP at both metabolically less active ring and highly active trophozoite stages of malaria parasite. Immunofluorescence microscopy further supports that PfARP expresses stage specifically with the highest expression at trophozite stage and very little in the schizont stage. PfARP is a cytosolic protein as evident from immunofluorescence microscopy. The role of this protein in P. falciparum cell death and stage progression is not yet known. The identification, purification and characterization would certainly be a step to initiate work on this protein to evaluate its role in P. falciparum growth, multiplication and stage progression

Bilirubin inhibits Plasmodium falciparum growth through the generation of reactive oxygen species

Free heme is very toxic because it generates highly reactive hydroxyl radicals (radical dotOH) to cause oxidative damage. Detoxification of free heme by the heme oxygenase (HO) system is a very common phenomenon by which free heme is catabolized to form bilirubin as an end product. Interestingly, the malaria parasite, Plasmodium falciparum, lacks an HO system, but it forms hemozoin, mainly to detoxify free heme. Here, we report that bilirubin significantly induces oxidative stress in the parasite as evident from the increased formation of lipid peroxide, decrease in glutathione content, and increased formation of H2O2 and radical dotOH. Bilirubin can effectively inhibit hemozoin formation also. Furthermore, results indicate that bilirubin inhibits parasite growth and induces caspase-like protease activity, up-regulates the expression of apoptosis-related protein (Gene ID PFI0450c), and reduces the mitochondrial membrane potential. radical dotOH scavengers such as mannitol, as well as the spin trap α-phenyl-n-tert-butylnitrone, effectively protect the parasite from bilirubin-induced oxidative stress and growth inhibition. These findings suggest that bilirubin, through the development of oxidative stress, induces P. falciparum cell death and that the malaria parasite lacks an HO system probably to protect itself from bilirubin-induced cell death as a second line of defense

Malarial Infection Develops Mitochondrial Pathology and Mitochondrial Oxidative Stress to Promote Hepatocyte Apoptosis.

Activation of the mitochondrial apoptosis pathway by oxidative stress has been implicated in hepatocyte apoptosis during malaria. Because mitochondria are the source and target of reactive oxygen species (ROS), we have investigated whether hepatocyte apoptosis is linked to mitochondrial pathology and mitochondrial ROS generation during malaria. Malarial infection induces mitochondrial pathology by inhibiting mitochondrial respiration, dehydrogenases, and transmembrane potential and damaging the ultrastructure as evident from transmission electron microscopic studies. Mitochondrial GSH depletion and formation of protein carbonyl indicate that mitochondrial pathology is associated with mitochondrial oxidative stress. Fluorescence imaging of hepatocytes documents intramitochondrial superoxide anion (O2 U−) generation during malaria. O2 U− inactivates mitochondrial aconitase to release iron from iron–sulfur clusters, which forms the hydroxyl radical (UOH) interacting with H2O2 produced concurrently. Malarial infection inactivates mitochondrial aconitase, and carbonylation of aconitase is evident from Western immunoblotting. The release of iron has been documented by fluorescence imaging of hepatocytes using Phen Green SK, and mitochondrial UOH generation has been confirmed. During malaria, the depletion of cardiolipin and formation of the mitochondrial permeability transition pore favor cytochrome c release to activate caspase-9. Interestingly, mitochondrial UOH generation correlates with the activation of both caspase-9 and caspase-3 with the progress of malarial infection, indicating the critical role of UOH

Silencing of the <i>WFS1</i> gene in HEK cells induces pathways related to neurodegeneration and mitochondrial damage

The gene WFS1 encodes a protein with unknown function although its functional deficiency causes different neuropsychiatric and neuroendocrine syndromes. In the present study, we aimed to find the functional networks influenced by the time-dependent silencing of WFS1 in HEK cells. We performed whole genome gene expression profiling (Human Gene 1.0 ST Arrays) in HEK cells 24, 48, 72, and 96 h after transfection with three different WFS1 siRNAs. To verify silencing we performed quantitative RT-PCR and Western blot analysis. Analysis was conducted in two ways. First we analyzed the overall effect of the siRNA treatment on the gene expression profile. As a next step we performed time-course analysis separately for different siRNAs and combined for all siRNAs. Quantitative RT-PCR and Western blot analysis confirmed clear silencing of the expression of WFS1 after 48 h. Significant (FDR value &lt;10%) changes in the expression of 11 genes was identified with most of these genes being related to the mitochondrial dysfunction and apoptosis. Time-course analysis confirmed significant correlations between WFS1 silencing and changes in the expression profiles of several genes. The pathways that were influenced significantly by WFS1 silencing were related to mitochondrial damage and neurodegenerative diseases. Our findings suggest a role of WFS1 gene in cell survival and its involvement in degenerative diseases. </jats:p