Tuli Biswas

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.

Effect of erythropoietin on membrane lipid peroxidation, superoxide dismutase, catalase, and glutathione peroxidase of rat RBC

Starved animals having low levels of erythropoietin in blood showed increased MDA, fluorescent pigments, and met-Hb values whereas the hemoglobin concentration decreased significantly on starvation. In vivo and in vitro studies with Ep reversed the effects of starvation and brought these values close to normal. The activities of the enzymes (SOD, catalase, GSH-PX, GR G6PD, and 6PGD) which protect the RBC membrane directly or indirectly from peroxidative threat, decreased on starvation and restored to normal levels after Ep treatment.

Lipid Peroxidation of Erythrocytes in Visceral Leishmaniasis

Lipid peroxidation of erythrocytes was studied in kala-azar patients having a considerable degree of anemia. Enhanced formation of oxidative metabolic products was observed in the erythrocytes of these patients. Decreased activities of the protective enzymes suggest impairment of the defense mechanism against peroxidative threat. These may contribute to some extent to the shortened lifespan of red cells in 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.

Lipid peroxidation of erythrocytes during anemia of the hamsters infected with Leishmania donovani

Visceral leishmaniasis has been found to be associated with severe anemia and premature lysis of erythrocytes. Peroxidative damage of red cells has been noted in several hemolytic anemias. Present study shows enhanced formation of methemoglobin in hamsters infected withLeishmania donovani. Increased formation of malonyldialdehyde and diene conjugate has been noted in the erythrocytes of the infected animals with the progress of anemia. Results showed decreased activities of protective enzymes like superoxide dismutase, catalase and glutathione reductase against peroxidative attack. An increase in the membrane cholesterol/phospholipid ratio and a decrease in membrane fluidity of erythrocytes were observed under the diseased condition. Densitometric scan after SDS-PAGE of red cell membrane of the infected animals revealed significant degradation of band 3 and band 4.1 proteins. The results suggest that alteration in the membrane may lead to reduced life span of the red cells in experimental visceral leishmaniasis.

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.