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Journal of Stress Physiology & Biochemistry, Vol. 7 No. 3 2011, pp. 185-192 ISSN 1997-0838 Original Text Copyright © 2011 by Singh, Srivastava and Singh
ORIGINAL ARTICLE
Effect of free heme on intraerythrocytic stage of Plasmodium yoelii infection
Vinay Kumar Singh*1,3, Saurabha Srivastava*2, Kaleshwar Prasad Singh1*
1Dept. of Microbiology, CSMMU (KGMU), Lucknow, U.P. India
2Dept. of Biochemistry, Faculty of Science, BHU, Varanasi, U.P. India
3Department of Medical Biochemistry, Facility of Medicine, NIMS University, Rajasthan, Jaipur, India
* authors are equally contributed.
*E-Mail: malaria.microbiology@gmail.com
Received July 1, 2011
Free radicals are often formed as necessary intermediates in a variety of biochemical reactions but when generated in excess or not appropriately controlled, they can wreak havoc on a wide range of macromolecules. Of the several free radicals generated in the biological system, the reactive oxygen species such as .OH are highly toxic and injurious agents causing irreparable tissue damage. At a point of time in the intraerythrocytic stage of P. yoelii infection, digestion of hemoglobin releases free heme, which in the presence of transition metal ion (Fe+3 / Fe+2) generates .OH and notably H2O2, a potent reactive oxygen intermediate (ROI) that causes lipid-peroxidation. Owing to its lipophilic nature, free heme intercalates in the lipid bilayer, disrupts the integrity of the membrane and hence destabilizes the cytoskeleton. Following lipid peroxidation of the food vacuolar membrane, in the course of malarial infection, free heme comes out from food vacuole. The current paper deals with a study on the effect of free heme on the various membranes viz. RBC membrane, food vacuole membrane and parasite membrane particularly during the intraerythrocytic stage of malarial infection.
Key words: Plasmodium yoelii, Lipid-peroxidation, Reactive Oxygen Intermediate (ROI), Heme toxicity
ORIGINAL ARTICLE
Effect of free heme on intraerythrocytic stage of Plasmodium yoelii infection
Vinay Kumar Singh*1,3, Saurabha Srivastava*2, Kaleshwar Prasad Singh1*
Dept. of Microbiology, CSMMU (KGMU), Lucknow, U.P. India
2Dept. of Biochemistry, Faculty of Science, BHU, Varanasi, U.P. India
3Department of Medical Biochemistry, Facility of Medicine, NIMS University, Rajasthan, Jaipur, India
* authors are equally contributed
*E-Mail: malaria.microbiology@gmail.com
Received July 1, 2011
Free radicals are often formed as necessary intermediates in a variety of biochemical reactions but when generated in excess or not appropriately controlled, they can wreak havoc on a wide range of macromolecules. Of the several free radicals generated in the biological system, the reactive oxygen species such as .OH are highly toxic and injurious agents causing irreparable tissue damage. At a point of time in the intraerythrocytic stage of P. yoelii infection, digestion of hemoglobin releases free heme, which in the presence of transition metal ion (Fe+3 / Fe+2) generates .OH and notably H2O2, a potent reactive oxygen intermediate (ROI) that causes lipid-peroxidation. Owing to its lipophilic nature, free heme intercalates in the lipid bilayer, disrupts the integrity of the membrane and hence destabilizes the cytoskeleton. Following lipid peroxidation of the food vacuolar membrane, in the course of malarial infection, free heme comes out from food vacuole. The current paper deals with a study on the effect of free heme on the various membranes viz. RBC membrane, food vacuole membrane and parasite membrane particularly during the intraerythrocytic stage of malarial infection.
Key words: Plasmodium yoelii, Lipid-peroxidation, Reactive Oxygen Intermediate (ROI), Heme toxicity
Malaria is one of the most dreaded parasitic diseases witnessed by the world mainly in the tropical and subtropical regions and the estimates suggest that it affects at least 91 different countries and some 300 million people. About 3.3 billion people are at risk of infection with one of the four species of plasmodium. There are about 250 million
clinical cases of malaria and 880000 deaths annually (WHO, 2009). It has been estimated to represent 2.3% of the overall global disease threats after pneumococcal acute respiratory infection (3.5%) and tuberculosis (2.8%) (Olliaro et al, 1996). Infection is transmitted to the human being by the bite of an infected female anopheles mosquito, when sporozoites from the salivary
secretions of the mosquito are introduced to the bloodstream of the human. Once in the bloodstream, the sporozoites move into the parenchymal cells of the liver within 3-30 minutes (Gutierrez, 2000; Taylor, Strickland, 2000) and get differentiated into merozoites. The parasite multiplies in the liver and these merozoites then rupture the liver cells and escape into the blood cells. They infect the red blood cells and cause immense damage to the red blood cells which causes them to rip apart. The TDR of WHO at present invests 49% of its global resource on malaria control measures. Diagnosis of malaria in the early stages is very difficult as parasites are protected from the attack of human body’s immune system owing to their residence within the liver and blood cells during most of their life time in the human body and hence are invisible to the human immune surveillance During intraerythrocytic stage, which is one of the most common drug targeting phases of malarial infection, heme is released after digestive degradation of hemoglobin by malaria parasite. The free heme generates the reactive oxygen species (ROS) which owing to their toxicity by virtue of oxidative stress causes parasite death (Michael et al, 2001; Atamna, Ginsburg, 1993). The trophozoite metabolizes hemoglobin incompletely because it lacks the enzyme heme oxygenase, which is used by vertebrates for heme catabolism(Goldberg et al, 1990; Goldberg et al, 1991; Sullivan et al, 1996; Francis et al, 1997; Banerjee et al, 2002). The parasite can adopt various mechanisms to detoxify the free heme, heme polymerization being considered to be the most effective one (Sherman, 1985; Gupta, 1988; Ganguly et al, 1997). The polymerized heme is commonly known as hemozoine/malaria pigment. This pigment gets accumulated in the food vacuole of the intraerythrocytic parasite as insoluble black-brown pigment (Gluzman et al, 1994; Hempelmann et al, 2003). The present study is a, effort to understand the heme toxicity and its detoxification during intraerythrocytic stage of malarial infection.
MATERIAL & METHODS:
Parasite and Experimental host: Swiss albino mice weighted 18-22 gm were infected with Plasmodium yoelii, intraperitonialy (1x107 infected erythrocytes/ml) (Pandey et al, 1999). Parasitemia counts were monitored time to time by microscopic examination of slides stained with Giemsa Stain. Blood samples were collected at high level of parasitemia 50-60% in sterile ACD (Acid citrate dextrose) (sodium citrate 70 mM, dextrose 136mM and citric acid 35mM) solution.
Generation of OH by free heme: Hydroxy radical ( OH) generated in vitro was measured using dimethyl sulfoxide (DMSO) as OH scavenger. Different concentrations of heme was incubated with 10% DMSO and 100 mM of phosphate buffer at pH 7.0 on water bath at 370C for 60 minutes, in the same reaction mixture add 30 mM B.B. salt for 370C for 20 minutes. 1 ml of toluene: butanol (3:1) mixture was added and mixed well using a vortex mixer. Methanesulfinic acid formed was allowed to react it with Fast Blue BB salt and intensity recorded at 425 nm using benzene-sulfinic acid as standard. Generation of hydroxyl radicals was quantified at different concentrations of heme, pH and at different incubation tempeartures.
Isolation of parasite membrane: Infected blood passed through CF-11 column (Homewood, Neame, 1976), washed three times with PBS at 4000 rpm for 10 minute. Pellet was dissolved in PBS, freeze-thawed and suspended in liquid N2. The solution was centrifuged at 11500 rpm for 15 minutes, supernatant was centrifuged at 35000 rpm for 60 minutes at 40C. The Pellet containing parasite membrane was stored at -200C for further use (Anacelin et al, 1991).
Isolation of Infected RBC membrane: RBC
was isolated by the method as described earlier Pandey et al. (1999). Infected blood along with ACD was passed through CF-11 column
(Homewood, Neame, 1976) to remove WBC. Supernatant containing pure RBC was centrifuged at 2000 rpm for 10 minutes; pellet was washed thrice with PBS. After sonication the pellet was freeze-thawed three times, to break the RBCs. The sonicated blood was centrifuged at 12000 rpm for 10 min. The supernatant was collected and ultra centrifuged at 35000 rpm for 65 minutes at 40C. Pellet of infected RBC membrane was dissolved in PBS and stored at 200C for further use.
Isolation of food vacuole: Infected RBC pellet was washed twice with PBS (Bradford, 1976), incubated with 5% D-sorbitol at room temperature for 10 minutes, centrifuged at 650 g for 7 minutes. The supernatant was mixed with 1% saponin in PBS and 50% streptomycin sulfate and incubated for 10 minutes at room temperature. Following incubation centrifugation was carried out at 1500Xg for 10 minutes. The pellet was washed with 10 ml of PBS/1.5 mM MgCl2/0.5% streptomycin sulfate and re-suspended in 1 ml of 0.25 M sucrose / 10 mM sodium phosphate /0.5% streptomycin sulfate. The suspension was triturated 10 times using a tuberculin syringe with a 27-gauge needle. It was then mixed with 42% percoll/ 0.25 M sucrose / 1.5 mM MgCl2 at pH 7.0 and centrifuged at 15000 rpm for 40 minutes. From density gradient the layer was sucked up, diluted with 0.25 M sucrose / 10 mM sodium phosphate / 1.5 MgCl2, pH 7.0 and centrifuged at 16000Xg 15 minutes. The pellet contained the purified food vacuole stored at -20°C until use.
Protein estimation: Protein was estimated by the method described as Bradford (Bradford, 1976). The absorbance was recorded at 595 nM and BSA used as standard.
Measurement of lipid peroxidation: Lipid peroxidation of these membrane fractions was determined as described as Buege and Das (Buege et al, 1978; Das et al, 1997) using thiobarbituric acid
reactive substrate (TBARS). Briefly in 1 ml of membrane fraction in normal saline 0.9% was added 2 ml of TCA-TBA-HCl reagent, containing 15% TCA (w/v), 0.375% TBA (w/v) in 0.25 N HCl and heated for 15 min in a boiling water bath after mixing thoroughly. The solution was then cooled to room temperature and flocculent precipitate was removed by centrifugation at 2000 rpm for 10 minutes. Absorbance of supernatant was recorded at 535 nM. Tetrahoxypropane was used as a standard. The result was expressed as nM of TBRS/mg protein.
RESULT AND DISCUSSION:
Parasite Membrane, food vacuoles and RBC were isolated from subcellular fractionation of blood after a high percentage of parasitemia (~50% after 5 days of infection) (Fig. 1). The control blood without infection served as control. These membranes were inoculated with different concentrations of free heme to study oxidative damage in in-vitro, induced by free heme. During intraerythrocytic stage plasmodium proteolytically (producing proteolysis) degrades huge quantities of hemoglobin in the food vacuole, yielding large amount of redox active free heme as byproduct (Eney et al, 2002). The generation of reactive oxygen species (ROS) and associated oxidative stress play crucial role in the development of systemic complications in malaria (Ponka, 1999; Pabon et al, 2003). Heme while serving as a prosthetic group performs many important functions (Eney et al, 2002). Whereas in free state it is toxic and develops oxidative stress through generation of ROS (Omodeo-Sale et al, 2001). Malaria parasite detoxifies redox active free heme to less toxic hemozoin, a polymer of free heme (Omodeo-Sale et al, 2001). However a high concentration of circulating free heme has been reported during malarial infection. Even hemozoin which is deposited in liver during malaria can also exhibit
pro-oxidant activity (Omodeo-Sale et al, 2001). Thus ROS may be generated from heme or hemozoin. Alternatively free heme in mammalian system can be detoxified by hemopexin mediated heme oxygenase pathway to yield equimolar amounts of biliverdin, carbon monoxide (CO) and
Figure 1. Time dependent parasitemia curve
Figure 2. Lipid peroxidation of parasite membrane
free iron (Fe2+). In this study Lipid-peroxidation is a parameter used for measurement of the oxidative stress induced by free heme by generation of OH and H2O2. It results damage the membrane and subsequently increased the lipid-peroxidation (Fig.
2).
Heme conetration (h Mole)
Figure 3. Lipid peroxidation of infected and control RBC membrane
Figure 4. Lipid peroxidation of infected RBC membrane at different pH
Concentration of heme (micromol)
Figure 5. Lipid peroxidation of food vacuole membrane
Figure 2 indicating concentration dependent generations of reactive oxygen species (ROS) by free heme in in-vitro to cause lipid-peroxidation. The same results have been observed from the incubation of RBCs membranes. Interestingly lipidperoxidation of infected RBC membrane was found to be higher than the control RBC membrane (Fig.
3). The high lipid-peroxidation showed in infected RBC at pH 7.0 (Fig. 4). Free heme is very toxic to RBC membrane, resulting in destruction of lipid-bilayer. Free heme also causes lipid-peroxidation of the membrane isolated from the purified food vacuole (Fig. 5). Heme increases the rate of solute leakage across lipid-bilayer unilamellar and multilamellar as well as it increase with increasing the heme concentration up to 50-60^M decreasing thereafter and releasing heme concentration higher than 100 ^M. Free heme released after digestive degradation of hemoglobin by malaria parasite is a highly toxic and lipohilic molecule that destabilizes the membranes and subsequently cytoskeleton. Preliminary reports are available regarding the development of ROS generated by free heme. Free heme generates .OH a highly oxidizing agent, oxidizes their vicinity molecule viz. membrane of parasite, RBCs and also food vacuole. Damages induced by free heme (in-vitro), were determined by estimation of lipid-peroxidation, increased level of lipid-peroxidation indicates the toxicity of free heme. This study can be helpful in understand heme toxicity during intraerythrocytic stage of infection. This information can be extended to develop a new drug for malaria.
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