Gargi Sen

Mechanism of erythrocyte death in human population exposed to arsenic through drinking water.

Arsenic contamination in drinking water is one of the biggest natural calamities, which has become an imperative threat to human health throughout the world. Abbreviation of erythrocyte lifespan leading to the development of anemia is a common sequel in arsenic exposed population. This study was undertaken to explore the mechanism of cell death in human erythrocytes during chronic arsenic exposure. Results revealed transformation of smooth discoid red cells into evaginated echinocytic form in the exposed individuals. Further distortion converted reversible echinocytes to irreversible spheroechinocytes. Arsenic toxicity increased membrane microviscosity along with an elevation of cholesterol/phospholipid ratio, which hampered the flexibility of red cell membrane and made them less deformable. Significant increase in the binding of merocyanine 540 with erythrocyte membrane due to arsenic exposure indicated disruption of lipid packing in the outer leaflet of the cell membrane resulting from altered transbilayer phospholipid asymmetry. Arsenic induced eryptosis was characterized by cell shrinkage and exposure of phosphatidylserine at the cell surface. Furthermore, metabolic starvation with depletion of cellular ATP triggered apoptotic removal of erythrocytes from circulation. Significant decrease in reduced glutathione content indicating defective antioxidant capacity was coupled with enhancement of malondialdehyde and protein carbonyl levels, which pointed to oxidative damage to erythrocyte membrane. Arsenic toxicity intervened into red cell membrane integrity eventually leading to membrane destabilization and hemoglobin release. The study depicted the involvement of both erythrophagocytosis and hemolysis in the destruction of human erythrocytes during chronic arsenic exposure.

Quercetin interferes with iron metabolism in Leishmania donovani and targets ribonucleotide reductase to exert leishmanicidal activity

OBJECTIVES The possibility of developing antileishmanial drugs was evaluated by intervention in the parasites iron metabolism, utilizing quercetin (Qr) under in vivo conditions, and identifying the target of this lipophilic metal chelator against Leishmania donovani. METHODS Interaction between Qr and serum albumin (SA) was studied by using the intrinsic fluorescence of Qr as a probe. The effect of treatment with Qr and SA on the proliferation of amastigotes was determined by evaluating splenic parasite load. Disintegration of parasites in response to combination treatment was assessed from ultrastructural analysis using a transmission electron microscope. Quenching of the tyrosyl radical of ribonucleotide reductase (RR) in treated amastigotes was detected by an electron paramagnetic resonance study. RESULTS Treatment with a combination of Qr and SA increased bioavailability of the flavonoid and proved to be of major advantage in promoting the effectiveness of Qr towards the repression of splenic parasite load from 75%, P < 0.01 to 95%, P < 0.002. Qr-mediated down-regulation of RR (P < 0.05), catalysing the rate-limiting step of DNA synthesis in the pathogens, could be related to the deprivation of the enzyme of iron which in turn destabilized the critical tyrosyl radical required for its catalysing activity. CONCLUSIONS Results have implications for improved leishmanicidal action of Qr in combination with SA targeting RR and suggest future drug design based on interference with the parasites iron metabolism under in vivo conditions.

Oxidative damage of erythrocytes: a possible mechanism for premature hemolysis in experimental visceral leishmaniasis in hamsters

Abstract Visceral leishmaniasis is accompanied by severe anemia and pancytopenia. Reactive oxygen species are known to contribute to the pathogenesis of several red blood cell (RBCs) disorders. The present study reveals the extent of oxidative stress and the efficacy of the primary antioxidant system in erythrocytes of hamsters in the progressive anemic response at different stages of leishmanial infection. Increased intracellular precipitation of Heinz bodies secondary to oxidative denaturation of hemoglobin and enhanced formation of malonyldialdehyde suggest oxidative damage of erythrocytes, both in the hemoglobin and cell membrane, respectively. Decreased activities of superoxide dismutase and catalase in the infected animals indicate the generation of O2* – and H2O2, which in turn may produce the highly reactive *OH species. Decreases in the reduced glutathione level along with the decreased activities of glutathione reductase and glutathione peroxidase point to a deficient antioxidant defense system during the post-infection period. Accentuated degradation of both cytoskeletal and integral membrane proteins after 3 months of infection may eventually lead to membrane destabilization and early lysis of erythrocytes in experimental visceral leishmaniasis.

Superoxide anion mediated mitochondrial dysfunction leads to hepatocyte apoptosis preferentially in the periportal region during copper toxicity in rats

Chronic exposure to copper induces hepatocellular apoptosis with greater injury in the periportal region compared to the perivenous region. Here we have identified the factors responsible for the development of regional damage in the liver under in vivo conditions. Enhanced production of reactive oxygen species (ROS) with predominance of superoxide radical (O(2)(-)) indicates the contribution of redox imbalance in the process. This may be linked with copper catalyzed oxidation of GSH to GSSG resulting in the generation of O(2)(-). Downregulation of Cu-Zn SOD in consequence of the degradation of this enzyme, causes decreased dismutation of O(2)(-), that further contributes to the enhanced level of O(2)(-) in the periportal region. Decreased functioning of Mn SOD activity, reduction in mitochondrial thiol/disulphide ratio and generation of O(2)(-) were much higher in the mitochondria from periportal region, which point to the involvement of this organelle in the regional hepatotoxicity observed during copper exposure. This was supported by copper-mediated enhanced mitochondrial dysfunction as evident from ATP depletion, collapse of mitochondrial membrane potential (MMP) and induction of mitochondrial permeability transition (MPT). Results suggest the active participation of O(2)(-) in inducing mitochondrial dysfunction preferentially in the periportal region that eventually leads to the development of hepatotoxicity due to copper exposure under in vivo condition.

The role of calpain and calpastatin in the catabolism of erythrocyte-membrane proteins during anaemia in hamsters (Mesocricetus auretus ) infected with Leishmania donovani

Abstract The anaemia associated with visceral leishmaniasis is accompanied by altered Ca2+ homeostasis and degradation of the cytoskeletal and integral proteins of the erythrocytic membrane. In the present study, such changes were followed in hamsters that were anaemic as the result of their experimental infection with Leishmania donovani. At each stage of the infection, the blood concentration of haemoglobin was found to be negatively correlated with the concentration of Ca2+ (R2 = 0.91), the percentage of erythrocytes with Heinz bodies (R2 = 0.98) and thiol depletion (R2 = 0.96) in the erythrocytes. Calpain (Ca2+-activated protease; EC 3.4.22.17) and its natural inhibitor calpastatin are known to regulate the catabolism of membrane structural proteins. Densitometric scanning of SDS-PAGE gels showed that erythrocytic membranes from infected hamsters contained less calpain and calpastatin than those from control animals. The level of calpain autolysis was found to increase as the infection progressed. The addition of purified calpain (from control hamsters) to erythrocyte ghosts caused greater degradation of the membranes of erythrocytes from infected animals than of the corresponding membranes from control animals. Calpastatin from the control hamsters was more effective, at inhibiting calpain-induced membrane proteolysis, than calpastatin from the infected animals. The results indicate that the Ca2+-activated protease and its inhibitor are involved in the degradation of erythrocytic membranes observed during visceral leishmaniasis.

Reduced cellular redox status induces 4-hydroxynonenal-mediated caspase 3 activation leading to erythrocyte death during chronic arsenic exposure in rats

Chronic exposure to arsenic in rats led to gradual accumulation of the toxicant in erythrocytes causing oxidative stress in these cells. 4-Hydroxynonenal (4-HNE), a major aldehyde product of lipid peroxidation, contributed significantly to the cytopathological events observed during oxidative stress in the erythrocytes of exposed rats. 4-HNE triggered death signal cascade that was initiated with the formation of HNE-protein adducts in cytosol. HNE-protein adduct formation resulted in depletion of cytosolic antioxidants followed by increased generation of ROS. Results showed accumulation of hydrogen peroxide (H(2)O(2)) from the early stages of arsenic exposure, while superoxide (O(2)(*-)) and hydroxyl radical ((*)OH) also contributed to the oxidative stress during longer period of exposure. Suppression of antioxidant system coupled with increased generation of ROS eventually led to activation of caspase 3 during arsenic exposure. Attenuation of HNE-mediated activation of caspase 3 in presence of N-acetylcysteine (NAC) indicated the involvement of GSH in the process. Prevention of HNE-mediated degradation of membrane proteins in presence of Z-DEVD-FMK identified caspase 3 as the principal mediator of HNE-induced cellular damage during arsenic exposure. Degradation of band 3 followed by its aggregation on the red cell surface promoted immunologic recognition of redistributed band 3 by autologous IgG with subsequent attachment of C3b. Finally, the formation of C3b-IgG-band 3 immune complex accelerated the elimination of affected cells from circulation and led to the decline of erythrocyte life span during chronic arsenic toxicity.

14‐Deoxyandrographolide desensitizes hepatocytes to tumour necrosis factor‐alpha‐induced apoptosis through calcium‐dependent tumour necrosis factor receptor superfamily member 1A release via the NO/cGMP pathway

Andrographis paniculata (AP) has been found to display hepatoprotective effect, although the mechanism of action of the active compounds of AP in this context still remains unclear. Here, we evaluated the hepatoprotective efficacy of 14‐deoxyandrographolide (14‐DAG), a bioactive compound of AP, particularly its role in desensitization of hepatocytes to tumour necrosis factor‐alpha (TNF‐α)‐induced signalling of apoptosis.

Regulatory role of nitric oxide in the reduced survival of erythrocytes in visceral leishmaniasis

BACKGROUND Nitric oxide (NO) plays a vital role in maintaining the survivability of circulating erythrocytes. Here we have investigated whether NO depletion associated with visceral leishmaniasis (VL) is responsible for the reduced survival of erythrocytes observed during the disease. METHODS Infected hamsters were treated with standard anti-leishmanial sodium stibogluconate (SAG) and NO donor isosorbide dinitrate (ISD). Erythrophagocytosis by macrophages was determined by labelling the cells with FITC followed by flow cytometry. Aggregation of band3 was estimated from band3 associated EMA fluorescence. Caspase 3 activity was measured using immunosorbent assay kit. Phosphatidylserine (PS) externalization and cell shrinkage were determined using annexin V. Aminophspholipid translocase and scramblase activities were measured following NBD-PS and NBD-PC internalization, respectively. RESULTS Impairment of both synthesis and uptake of NO resulted in decreased bioavailability of this signaling molecule in erythrocytes in VL. NO level was replenished after simultaneous treatment with ISD and SAG. Combination treatment decreased red cell apoptosis in infected animals by deactivating caspase 3 through s-nitrosylation. Drug treatment prevented infection-mediated ATP depletion and altered calcium homeostasis in erythrocytes. Improved metabolic environment effectively amended dysregulation of aminophospholipid translocase and scramblase, which in turn reduced cell shrinkage, and exposure of phosphatidylserine on the cell surface under the diseased condition. CONCLUSION AND GENERAL SIGNIFICANCE In this study, we have identified NO depletion to be an important factor in promoting premature hemolysis with the progress of leishmanial infection. The study implicates NO to be a possible target for future drug development towards the promotion of erythrocyte survival in VL.

S-allyl Cysteine in Combination with Clotrimazole Downregulates Fas Induced Apoptotic Events in Erythrocytes of Mice exposed to Lead

BACKGROUND Chronic lead (Pb(2+)) exposure leads to the reduced lifespan of erythrocytes. Oxidative stress and K(+) loss accelerate Fas translocation into lipid raft microdomains inducing Fas mediated death signaling in these erythrocytes. Pathophysiological-based therapeutic strategies to combat against erythrocyte death were evaluated using garlic-derived organosulfur compounds like diallyl disulfide (DADS), S allyl cysteine (SAC) and imidazole based Gardos channel inhibitor clotrimazole (CLT). METHODS Morphological alterations in erythrocytes were evaluated using scanning electron microscopy. Events associated with erythrocyte death were evaluated using radio labeled probes, flow cytometry and activity gel assay. Mass spectrometry was used for detection of GSH-4-hydroxy-trans-2-nonenal (HNE) adducts. Fas redistribution into the lipid rafts was studied using immunoblotting technique and confocal microscopy. RESULTS Combination of SAC and CLT was better than DADS and CLT combination and monotherapy with these agents in prolonging the survival of erythrocytes during chronic Pb(2+) exposure. Combination therapy with SAC and CLT prevented redistribution of Fas into the lipid rafts of the plasma membrane and downregulated Fas-dependent death events in erythrocytes of mice exposed to Pb(2+). CONCLUSION AND GENERAL SIGNIFICANCE Ceramide generation was a critical component of Fas receptor-induced apoptosis, since inhibition of acid sphingomyelinase (aSMase) interfered with Fas-induced apoptosis during Pb(2+) exposure. Combination therapy with SAC and CLT downregulated apoptotic events in erythrocytes by antagonizing oxidative stress and Gardos channel that led to suppression of ceramide-initiated Fas aggregation in lipid rafts. Hence, combination therapy with SAC and CLT may be a potential therapeutic option for enhancing the lifespan of erythrocytes during Pb(2+) toxicity.

Atorvastatin acts synergistically with N-acetyl cysteine to provide therapeutic advantage against Fas-activated erythrocyte apoptosis during chronic arsenic exposure in rats

Arsenic is an environmental toxicant that reduces the lifespan of circulating erythrocytes during chronic exposure. Our previous studies had indicated involvement of hypercholesterolemia and reactive oxygen species (ROS) in arsenic-induced apoptotic death of erythrocytes. In this study, we have shown an effective recovery from arsenic-induced death signaling in erythrocytes in response to treatment with atorvastatin (ATV) and N-acetyl cysteine (NAC) in rats. Our results emphasized on the importance of cholesterol in the promotion of ROS-mediated Fas signaling in red cells. Arsenic-induced activation of caspase 3 was associated with phosphatidylserine exposure on the cell surface and microvesiculation of erythrocyte membrane. Administration of NAC in combination with ATV, proved to be more effective than either of the drugs alone towards the rectification of arsenic-mediated disorganization of membrane structural integrity, and this could be linked with decreased ROS accumulation through reduced glutathione (GSH) repletion along with cholesterol depletion. Moreover, activation of caspase 3 was capable of promoting aggregation of band 3 with subsequent binding of autologous IgG and opsonization by C3b that led to phagocytosis of the exposed cells by the macrophages. NAC-ATV treatment successfully amended these events and restored lifespan of erythrocytes from the exposed animals almost to the control level. This work helped us to identify intracellular membrane cholesterol enrichment and GSH depletion as the key regulatory points in arsenic-mediated erythrocyte destruction and suggested a therapeutic strategy against Fas-activated cell death related to enhanced cholesterol and accumulation of ROS.