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

Division of Drug Target Discovery and Development, Central Drug Research Institute, Uttar Pradesh, India

¹Correspondence: Division of Drug Target Discovery and Development, Central Drug Research Institute, Chatter Manzil Palace, Mahatma Gandhi Marg, Lucknow-226001, Uttar Pradesh, India. E-mail: ubandyo_1964{at}yahoo.com

SPECIFIC AIMS

The mechanistic basis of hepatic dysfunction, a clinically significant complication in malaria, remains largely obscure. Although oxidative stress and associated apoptosis trigger hepatic pathology in a wide variety of infectious and noninfectious diseases, the extent and significance of such events in malaria have not been investigated. The aim of this study was to detect whether malarial infection could lead to oxidative stress and apoptosis in liver of Plasmodium yoelii -infected mice.

PRINCIPAL FINDINGS

1. Malarial infection develops oxidative stress in the liver
P. yoelii (MDR strain) infection in mice induces oxidative stress in the liver, as evidenced from decreased cellular GSH concentration and increased lipid and protein oxidation. The decrease of GSH concentration correlated well with the degree of parasitemia. At 5–10% parasitemia, GSH concentration decreased by 20%; at 20–30% parasitemia, it was 45% (P<0.01); and at 50–60% parasitemia, the value decreased further by 66% (P<0.001) with respect to control value as 100%. Lipid peroxidation in the liver of infected mice also increased with the degree of parasitemia attaining maximum (148% increment over control value as 100%, P<0.001) at the highest level of parasitemia. Formation of protein carbonyl also increased significantly in infected liver showing a 112% increment (over 100% control) at 50–60% parasitemia (P<0.001).

2. Malarial infection induces apoptosis in liver: Implication of the mitochondrial pathway
Oxidative stress in many instances can lead to the induction of apoptosis in different cellular systems, including the liver. To study whether oxidative stress developed by malarial infection can lead to apoptosis in liver, markers for apoptosis such as in situ DNA fragmentation and caspase-3 like activity were measured. No apoptotic DNA fragmentation was observed in the liver of uninfected mice (Fig. 1 A, a), whereas the liver from infected mice showed DNA fragmentation (Fig. 1A, b ), suggesting incidence of apoptosis. This DNA fragmentation pattern was similar to the DNase-treated positive control tissue section (Fig. 1A, c ). In cytosol of infected liver, there was clear evidence of a 10-fold activation of caspase-3 like proteases compared to uninfected cytosol (P<0.001). Moreover, the activity of caspase-3 like proteases was significantly inhibited when the infected cytosol was treated with Ac-DEVD-CHO, a potent inhibitor of caspase-3, indicating the specific assay of caspase-3 like proteases. Furthermore, to demonstrate hepatocyte apoptosis, hepatocytes were isolated from uninfected and infected mice to detect caspase-3 activation by immunocytochemistry using antibody (Ab) against caspase-3 (Fig. 1C ). The result indicated the absence of activation of caspase-3 like proteases in hepatocyte isolated from the uninfected mice (Fig. 1C, a ) but showed tremendous caspase-3 activation in the liver of infected mice (Fig. 1C, b ).

View larger version (63K): [in this window] [in a new window]   Figure 1. A) Evidence of in situ apoptotic DNA fragmentation in liver during P. yoelii infection, as detected by TUNEL assay. Control (uninfected) liver (a): there is no typical staining for apoptotic DNA fragmentation. Infected liver (b): profuse staining as indicated by black dots, suggesting DNA fragmentation. Arrows indicate TUNEL-positive cells. DNase treated control liver (positive control). B) Activation of caspase-3 like proteases in liver of P. yoelii infected mice. Activity of caspase-3 in the cytosolic fraction (50 µg protein) was measured at different timepoints. Caspase-3 activity was expressed as change of optical density at 405 nM due to release of pNA. To check specificity of caspase-3 assay, Ac-DEVD-CHO, a specific inhibitor was added. Data are mean ± SE (n=6). C) Immunocytochemical detection of activated caspase-3 in isolated hepatocyte. Hepatocytes from control (uninfected; a) and infected (b) mice were used for immunocytochemistry using caspase-3 Ab and activation of caspase-3 is indicated by black spots (arrow). D) RT-PCR for Bcl-2, Bax, and Fas. Liver RNA (1 µg) from infected or uninfected (control) mice was used for RT-PCR amplification. Amplified products were checked in 12% polyacrylamide gel. GAPDH was used as internal (positive) control. E) Densitometric analysis of Bcl-2, Bax, and Fas expression (percent relative to control, where control was considered as 100% expression) in liver of infected mice. Data were calculated from 3 separate experiments and are mean ± SE (***P<0.001 vs. control). F) Measurement of caspase-8 activity. Pure caspase-8 was used as positive control. Inhibition of caspase-8 activity by caspase-8 specific inhibitor (IETD-CHO) indicated specific assay for caspase-8. Data are mean ± SE.

Activation of caspase-3 like proteases mediates the final execution of caspase-dependent apoptosis. Caspase-dependent apoptosis is mainly mediated through two pathways, i.e., the mitochondrial pathway (intrinsic) and death receptor pathway (extrinsic). To reveal the contribution of these pathways in hepatic apoptosis during malaria, gene expressions of Bcl-2, Bax (components of mitochondrial pathway), and Fas (component of the death receptor pathway), were followed through RT-PCR. P. yoelii infection led to down-regulation of Bcl-2 expression by 60% compared to the control value taken as 100% (P<0.001), as evidenced from RT-PCR analysis (Fig. 1D and E ). Interestingly, there was also significant up-regulation (150% over control as 100%, P<0.001) in the expression of Bax in the infected liver (Fig 1D and E ). Thus, the data indicated the activation of mitochondrial pathway of apoptosis. To understand whether the Fas (CD95)-related death receptor pathway had any role in malaria infected hepatocyte apoptosis, RT-PCR analysis using Fas (CD95) specific primers and activation of caspase-8, a common event for the death receptor-mediated pathway were performed. The results indicated that the concentration of Fas expression in the infected the liver was same as that of basal concentration (100%) of expression in control (Fig. 1D and E ), indicating the death receptor (Fas) pathway had no such involvement in liver apoptosis during malarial infection. GAPDH expression in the liver of both control and infected mice was analyzed as positive control. There was also no activation of caspase-8 from basal level in the liver from infected mice, compared to control (Fig. 1F ). Thus, it is clear that the mitochondrial pathway and not the death receptor one is operating in the induction of hepatic apoptosis during malaria. Down-regulation of Bcl-2 and up-regulation of Bax reduced the Bcl-2-to-Bax ratio, resulting in activation of mitochondrial pathway of apoptosis. The ratio at which these proteins are present intracellularly can determine whether or not a cell undergoes apoptosis. In absence of Bcl-2-antagonizing action, Bax proteins were activated and translocated to mitochondria, where they induced opening of mitochondrial permeability transition pores (MPTP). The translocation of Bax to mitochondria and the release of mitochondrial cytochrome c into cytosol due to the opening of MPTP were observed.

3. Oxidative stress actually triggers the induction of apoptosis by activating mitochondrial pathway
After revealing the incidence of oxidative stress in liver during malarial infection and induction of apoptosis, we aimed to study whether induction of apoptosis is due to oxidative stress and if so which particular member of reactive oxygen species family is responsible. P. yoelii infection in mice significantly induced generation of hydroxy radical (·OH; 200% over control as 100%) in the liver (P<0.001; Fig. 2 A). Scavenging of ·OH by scavenger (mannitol) and spin trap [alpha-phenyl-tert-butyl-nitrone (PBN)] inhibited oxidative stress in liver of malaria-infected mice. Generation of ·OH was found to be correlated directly with the status of lipid peroxidation, and the inhibition of ·OH generation also correlated with the inhibition of lipid peroxidation (Fig. 2A ). Moreover, we found that DMSO, manitol, and PBN also significantly reduced caspase-3 like protease activation (P<0.001; Fig. 2B ). Interestingly, all the scavenger treatment to infected mice significantly inhibited the down-regulation of Bcl-2 and up-regulation of Bax (Fig. 2C ). Again, treatment of scavenges significantly inhibited the decrease of mitochondrial membrane potential (m) (Fig. 2D ). Thus, it is evident that oxidative stress caused by ·OH triggers the mitochondrial pathway of apoptosis in liver during malarial infection.

View larger version (26K): [in this window] [in a new window]   Figure 2. A) Measurement of ·OH and lipid peroxidation in presence or absence of scavengers during malarial infection. Mice (20–25 g) were injected i.p. with different ·OH specific scavengers such as DMSO (500 mg/kg bw), mannitol (500 mg/kg bw), and spin trap PBN (300 mg/kg bw) 1 h before P. yoelii infection. Parasitemia was monitored everyday, and all above-mentioned scavengers and spin trap were administrated to the respective animals daily at doses previously mentioned. On day 4 of infection, when parasitemia was 50–60%, all animals (control, infected, and infected but treated with scavengers) were killed and liver was excised and proceeded for measuring ·OH generation and lipid peroxidation. Data are mean ± SE. (n=6; ***P<0.001 vs. control, ###P<0.001, ## P<0.01 vs. infected). B) Measurement of caspase-3 activity in presence or absence of DMSO, mannitol, and PBN. Mice (20–25 g) were injected (ip) with different ·OH-specific scavengers. Liver was excised and processed for measuring caspase-3 activity. Data are mean ± SE (n=6; ***P<0.001 vs. control, ###P<0.001 vs. infection). C) RT-PCR/densitometric analysis of Bcl-2 and Bax expression (percent relative to control, where control was considered as 100% expression) in liver in presence or absence of scavengers and spin trap. Data are mean ± SE (n=6; ***P<0.001 vs. control, ### P<0.001, ##P<0.01 vs. infected). D) Measurement of m in presence or absence of DMSO, mannitol, and PBN. Details of the treatment of ·OH scavengers and PBN were described above. Data are mean ± SE (n=6; ***P<0.001 vs. control, ###P<0.001 vs. infected).

CONCLUSIONS AND SIGNIFICANCE

In the present study, we have demonstrated that malarial infection induces oxidative stress and apoptosis in liver. We have also shown that oxidative stress, developed during malarial infection, induces liver apoptosis through activation of mitochondrial pathway. ·OH is shown to be the causative agent for the development of oxidative stress and apoptosis.

Oxidative stress through up-regulation of Bax and down-regulation of Bcl-2 activates mitochondrial pathway of apoptosis. Bcl-2 down-regulation and simultaneous Bax up-regulation reduce the Bcl-2-to-Bax ratio, resulting in activation and translocation of Bax to mitochondria. Bax translocation to mitochondria through opening of permeability transition pores induces release of apoptotic inducing proteins, such as cytochrome c into cytosol. Cytochrome c activates caspase-3, which finally executes the apoptosis (Fig. 3 ). Inhibition of oxidative stress by ·OH scavengers also attenuates mitochondrial pathway of apoptosis, further confirming the role of oxidative stress in hepatic apoptosis during malaria (Fig. 3) . Therefore, oxidative stress and associated apoptosis in liver during malarial infection play a significant role at least in part in hepatic pathology. To the best of our knowledge, the present study demonstrates for the first time the mechanistic basis of the induction of apoptosis in liver by the erythrocytic stage of malarial parasite. Liver stage is considered to be a crucial phase in the life cycle of malarial parasite. Sporozoits can induce hepatocyte apoptosis at a lower but significant level as reported earlier. The liver stage of parasite generally prevents hepatocyte apoptosis induced by TNF-/D-galactosamine and peroxide. As we infected the mice with erythrocytic stage, there was no chance to form liver stage. Therefore, oxidative stress is mainly developed by the erythrocytic stages only to induce apoptosis. Since application of radical scavengers protects the liver from oxidative stress as well as associated apoptosis in liver during malaria, a combination therapy, consisting of antioxidants against ·OH (scavengers) with antimalarial drugs, can be recommended for the treatment of malaria to protect liver from apoptotic death.

View larger version (64K): [in this window] [in a new window]   Figure 3. Schematic presentation of oxidative stress-induced mitochondrial pathway of apoptosis in liver of malaria infected mice. Scavenging of ·OH by scavengers and spin trap inhibited the mitochondrial pathway of apoptosis. (dotted arrows: unknown pathway; t-bar: inhibition).

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5338fje

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This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.05-5338fjev1 20/8/1224    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Guha, M. Articles by Bandyopadhyay, U. Search for Related Content PubMed PubMed Citation Articles by Guha, M. Articles by Bandyopadhyay, U.