Publications by authors named "Kempaiah Kemparaju"

48 Publications

Bisphenol AF elevates procoagulant platelets by inducing necroptosis via RIPK1-inflammasome axis.

Toxicology 2021 04 1;454:152742. Epub 2021 Mar 1.

Department of Studies in Biochemistry, University of Mysore, Manasagangotri, Mysuru, 570 006, India; Department of Studies and Research in Biochemistry, Tumkur University, Tumakuru, 572 103, India. Electronic address:

Bisphenol AF, an analogue of Bisphenol A, is an important raw material used in the production of plastic and rubber substances like plastic bottles and containers, toys, and medical supplies. Increased contamination of air, water, dust, and food with BPA/BPAF, poses an enormous threat to humans, globally. BPAF/BPA are endocrine-disrupting chemicals that mimic estrogen hormone, thus increasing the risks of various metabolic and chronic disorders. Exposure of human blood cells to BPA/BPAF induces oxidative stress and genotoxicity. However, its effects on platelets, which play central roles in hemostasis and thrombosis, are not well-documented. In this study, we demonstrate that BPAF induces RIPK1-inflammasome axis-mediated necroptosis in platelets, increasing procoagulant platelet levels in vivo and in vitro. We also show that BPAF-induced rise in procoagulant platelets worsens pulmonary thromboembolism in vivo. The elevated procoagulant platelets are shown to increase platelet-neutrophil/monocyte aggregates that mediate pathogenesis of CVD, thrombosis, and chronic inflammatory diseases. Our results demonstrate the toxic effects of BPAF on platelets and how it propagates the clinical complications by elevating procoagulant platelet numbers. Altogether, our study sends a cautionary message against extensive use of BPAF in the plastic and rubber industries, resulting in frequent human exposure to it, thus endangering platelet functions.
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http://dx.doi.org/10.1016/j.tox.2021.152742DOI Listing
April 2021

Reversible cross-tolerance to platelet-activating factor signaling by bacterial toxins.

Platelets 2020 Aug 22:1-8. Epub 2020 Aug 22.

Department of Studies in Biochemistry, University of Mysore, Mysuru, India.

Bacterial toxins signaling through Toll-like receptors (TLRs) are implicated in the pathogenesis of many inflammatory diseases. Among the toxins, lipopolysaccharide (LPS) exerts its action via TLR-4 while lipoteichoic acid (LTA) and bacterial lipoproteins such as Braun lipoprotein (BLP) or its synthetic analogue Pam3CSK4 act through TLR-2. Part of the TLR mediated pathogenicity is believed to stem from endogenously biosynthesized platelet-activating factor (PAF)- a potent inflammatory phospholipid acting through PAF-receptor (PAF-R). However, the role of PAF in inflammatory diseases like endotoxemia is controversial. In order to test the direct contribution of PAF in TLR-mediated pathogenicity, we intraperitoneally injected PAF to Wistar albino mice in the presence or absence of bacterial toxins. Intraperitoneal injection of PAF (5 μg/mouse) causes sudden death of mice, that can be delayed by simultaneously or pre-treating the animals with high doses of bacterial toxins- a phenomenon known as endotoxin cross-tolerance. The bacterial toxins- induced tolerance to PAF can be reversed by increasing the concentration of PAF suggesting the reversibility of cross-tolerance. We did similar experiments using human platelets that express both canonical PAF-R and TLRs. Although bacterial toxins did not induce human platelet aggregation, they inhibited PAF-induced platelet aggregation in a reversible manner. Using rabbit platelets that are ultrasensitive to PAF, we found bacterial toxins (LPS and LTA) and Pam3CSK4 causing rabbit platelet aggregation via PAF-R dependent way. The physical interaction of PAF-R and bacterial toxins is also demonstrated in a human epidermal cell line having stable PAF-R expression. Thus, we suggest the possibility of direct physical interaction of bacterial toxins with PAF-R leading to cross-tolerance.
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http://dx.doi.org/10.1080/09537104.2020.1810652DOI Listing
August 2020

Melatonin restores neutrophil functions and prevents apoptosis amid dysfunctional glutathione redox system.

J Pineal Res 2020 Oct 18;69(3):e12676. Epub 2020 Aug 18.

Department of Studies in Biochemistry, University of Mysore, Mysore, India.

Melatonin is a chronobiotic hormone, which can regulate human diseases like cancer, atherosclerosis, respiratory disorders, and microbial infections by regulating redox system. Melatonin exhibits innate immunomodulation by communicating with immune system and influencing neutrophils to fight infections and inflammation. However, sustaining redox homeostasis and reactive oxygen species (ROS) generation in neutrophils are critical during chemotaxis, oxidative burst, phagocytosis, and neutrophil extracellular trap (NET) formation. Therefore, endogenous antioxidant glutathione (GSH) redox cycle is highly vital in regulating neutrophil functions. Reduced intracellular GSH levels and glutathione reductase (GR) activity in the neutrophils during clinical conditions like autoimmune disorders, neurological disorders, diabetes, and microbial infections lead to dysfunctional neutrophils. Therefore, we hypothesized that redox modulators like melatonin can protect neutrophil health and functions under GSH and GR activity-deficient conditions. We demonstrate the dual role of melatonin, wherein it protects neutrophils from oxidative stress-induced apoptosis by reducing ROS generation; in contrast, it restores neutrophil functions like phagocytosis, degranulation, and NETosis in GSH and GR activity-deficient neutrophils by regulating ROS levels both in vitro and in vivo. Melatonin mitigates LPS-induced neutrophil dysfunctions by rejuvenating GSH redox system, specifically GR activity by acting as a parallel redox system. Our results indicate that melatonin could be a potential auxiliary therapy to treat immune dysfunction and microbial infections, including virus, under chronic disease conditions by restoring neutrophil functions. Further, melatonin could be a promising immune system booster to fight unprecedented pandemics like the current COVID-19. However, further studies are indispensable to address the clinical usage of melatonin.
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http://dx.doi.org/10.1111/jpi.12676DOI Listing
October 2020

Melatonin restores neutrophil functions and prevents apoptosis amid dysfunctional glutathione redox system.

J Pineal Res 2020 Oct 18;69(3):e12676. Epub 2020 Aug 18.

Department of Studies in Biochemistry, University of Mysore, Mysore, India.

Melatonin is a chronobiotic hormone, which can regulate human diseases like cancer, atherosclerosis, respiratory disorders, and microbial infections by regulating redox system. Melatonin exhibits innate immunomodulation by communicating with immune system and influencing neutrophils to fight infections and inflammation. However, sustaining redox homeostasis and reactive oxygen species (ROS) generation in neutrophils are critical during chemotaxis, oxidative burst, phagocytosis, and neutrophil extracellular trap (NET) formation. Therefore, endogenous antioxidant glutathione (GSH) redox cycle is highly vital in regulating neutrophil functions. Reduced intracellular GSH levels and glutathione reductase (GR) activity in the neutrophils during clinical conditions like autoimmune disorders, neurological disorders, diabetes, and microbial infections lead to dysfunctional neutrophils. Therefore, we hypothesized that redox modulators like melatonin can protect neutrophil health and functions under GSH and GR activity-deficient conditions. We demonstrate the dual role of melatonin, wherein it protects neutrophils from oxidative stress-induced apoptosis by reducing ROS generation; in contrast, it restores neutrophil functions like phagocytosis, degranulation, and NETosis in GSH and GR activity-deficient neutrophils by regulating ROS levels both in vitro and in vivo. Melatonin mitigates LPS-induced neutrophil dysfunctions by rejuvenating GSH redox system, specifically GR activity by acting as a parallel redox system. Our results indicate that melatonin could be a potential auxiliary therapy to treat immune dysfunction and microbial infections, including virus, under chronic disease conditions by restoring neutrophil functions. Further, melatonin could be a promising immune system booster to fight unprecedented pandemics like the current COVID-19. However, further studies are indispensable to address the clinical usage of melatonin.
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http://dx.doi.org/10.1111/jpi.12676DOI Listing
October 2020

Bisdemethoxycurcumin promotes apoptosis in human platelets via activation of ERK signaling pathway.

Toxicol In Vitro 2020 Mar 3;63:104743. Epub 2019 Dec 3.

DOS in Biochemistry, University of Mysore, Manasagangothri, Mysuru 570 006, India; Department of Studies and Research in Biochemistry, Tumkur University, Tumakuru 572 103, India. Electronic address:

Curcumin, a major bioactive component of turmeric (Curcuma longa), is known for its multiple health benefits. Curcumin as such is a mixture of its analogs: bisdemethoxycurcumin (BDMC)-3%, and demethoxycurcumin (DMC)-17%. Although the effect of curcumin on platelets is documented, the effect of BDMC and DMC on platelets is less studied. Considering the indispensable role played by platelets in hemostasis, thrombosis, inflammation, and immunity, the present study evaluates the effect of curcumin, DMC and BDMC on platelet apoptosis. The components of curcumin were purified by silica-gel column chromatography. The purity and mass analysis of the purified curcuminoids was determined by RP-HPLC and LC-MS respectively. When analyzed for platelet apoptotic markers, only BDMC demonstrated increased incidence of platelet apoptotic markers including increase in intracellular Ca, decrease in ∆ψm, alteration in BCl-2 family proteins, the release of cytochrome c, caspase activation, and PS externalization via activation of ERK activation. ERK inhibitor PD98059 significantly alleviated BDMC induced decrease in ∆ψm, alteration in BCl-2, caspase-8 activation and PS externalization. Our results demonstrate that curcumin, DMC and BDMC differentially act on platelet in inducing apoptosis and the study highlights that the toxicity associated with curcumin therapy might be attributed to BDMC in the mammalian system.
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http://dx.doi.org/10.1016/j.tiv.2019.104743DOI Listing
March 2020

ASK1 inhibition triggers platelet apoptosis p38-MAPK-mediated mitochondrial dysfunction.

Haematologica 2020 08 28;105(8):e419-e423. Epub 2019 Nov 28.

Department of Studies in Biochemistry, University of Mysore, Manasagangotri, Mysuru

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http://dx.doi.org/10.3324/haematol.2019.233908DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7395269PMC
August 2020

Guggulipid ameliorates adjuvant-induced arthritis and liver oxidative damage by suppressing inflammatory and oxidative stress mediators.

Phytomedicine 2019 Nov 9;64:152924. Epub 2019 Apr 9.

Department of Studies in Biochemistry, University of Mysore, Manasagangothri, Mysuru, Karnataka 570 006, India; Department of Studies and Research in Biochemistry, Tumkur University, Tumakuru, Karnataka 572 103, India. Electronic address:

Background: Arthritis is a common degenerative joint disease characterized by deterioration of articular cartilage, subchondral bone, and associated with immobility, pain and inflammation. The incessant action of reactive oxygen species (ROS) during progressive arthritis causes severe oxidative damage to vital organs and circulatory system.

Purpose: In this study we investigated the ability of guggulipid (GL), a lipid rich extract from the gum resin of the plant Commiphora whighitii to suppress the progressive arthritis and associated liver oxidative stress both in vivo and in vitro.

Study Design/methods: The anti-arthritic ability of GL was demonstrated in vitro using IL-1β stimulated bovine nasal cartilage model and in vivo Freund's complete adjuvant-induced arthritic rat model. Collagen/proteoglycan degradation and pro-inflammatory mediators were monitored in the harvested culture medium of nasal cartilage by estimating the levels of matrix metalloproteinases (MMPs), hydroxy proline, glycosaminoglycans and inflammatory mediators. Further, anti-arthritic ability of GL was evaluated in vivo by measuring enzymatic and non-enzymatic mediators of cartilage degradation, inflammation and oxidative stress markers.

Results: GL significantly inhibited the IL-1β stimulated cartilage degradation in vitro by mitigating the MMPs activity, collagen degradation and secretion of pro-inflammatory mediators. Further, GL significantly reduced the adjuvant-induced paw swelling and body weight loss in vivo. GL remarkably reduced the MMPs and hyaluronidases activities in serum and bone homogenate along with altered hematological parameters. GL also mitigated the elevated bone resorbing enzymes cathepsins, exoglycosidases and phosphatases. Additionally, GL effectively mitigated ROS and oxidative stress-mediators recuperating the altered serum/liver oxidative stress and liver damage incurred during arthritic progression.

Conclusion: In summary, the study clearly demonstrates the protective efficacy of GL against arthritis and its associated oxidative stress, particularly, liver oxidative damage. Hence, GL could be a potential alternative and complementary medicine to treat inflammatory joint diseases.
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http://dx.doi.org/10.1016/j.phymed.2019.152924DOI Listing
November 2019

p38 MAP-kinase inhibitor protects against platelet-activating factor-induced death in mice.

Free Radic Biol Med 2019 11 20;143:275-287. Epub 2019 Aug 20.

Department of Studies in Biochemistry, University of Mysore, Manasagangothri, Mysuru, 570006, Karnataka, India; Department of Studies in Molecular Biology, University of Mysore, Manasagangothri, Mysuru, 570006, Karnataka, India. Electronic address:

Platelet-activating factor (PAF) is a potent inflammatory agonist. In Swiss albino mice, intraperitoneal injection of PAF causes sudden death with oxidative stress and disseminated intravascular coagulation (DIC), characterized by prolonged prothrombin time, thrombocytopenia, reduced fibrinogen content, and increased levels of fibrinogen degradation products. However, the underlying mechanism(s) is unknown. The PAF-R antagonist WEB-2086 protected mice against PAF-induced death by reducing DIC and oxidative stress. Accordingly, general antioxidants such as ascorbic acid, α-tocopherol, gallic acid, and N-acetylcysteine partially protected mice from PAF-induced death. N-acetylcysteine, a clinically used antioxidant, prevented death in 67% of mice, ameliorated DIC characteristics and histological alterations in the liver, and reduced oxidative stress. WEB-2086 suppressed HO-mediated oxidative stress in isolated mouse peritoneal macrophages, suggesting that PAF signaling may be a downstream effector of reactive oxygen species generation. PAF stimulated all three (ERK, JNK, and p38) of the MAP-kinases, which were also inhibited by N-acetylcysteine. Furthermore, a JNK inhibitor (SP600125) and ERK inhibitor (SCH772984) partially protected mice against PAF-induced death, whereas a p38 MAP-kinase inhibitor (SB203580) provided complete protection against DIC and death. In human platelets, which have the canonical PAF-R and functional MAP-kinases, JNK and p38 inhibitors abolished PAF-induced platelet aggregation, but the ERK inhibitor was ineffective. Our studies identify p38 MAP-kinase as a critical, but unrecognized component in PAF-induced mortality in mice. These findings suggest an alternative therapeutic strategy to address PAF-mediated pathogenicity, which plays a role in a broad range of inflammatory diseases.
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http://dx.doi.org/10.1016/j.freeradbiomed.2019.08.019DOI Listing
November 2019

Hemin-induced platelet activation and ferroptosis is mediated through ROS-driven proteasomal activity and inflammasome activation: Protection by Melatonin.

Biochim Biophys Acta Mol Basis Dis 2019 09 16;1865(9):2303-2316. Epub 2019 May 16.

DOS in Biochemistry, University of Mysore, Manasagangotri, Mysuru 570 006, India; Department of Studies and Research in Biochemistry, Tumkur University, Tumakuru 572 103, India. Electronic address:

Reactive oxygen species (ROS) are capable of inducing cell death or apoptosis. Recently, we demonstrated that lipid-ROS can mediate ferroptosis and activation of human platelets. Ferroptosis is an intracellular iron-mediated cell death, distinct from classical apoptosis and necrosis, which is mediated through the accumulation of ROS, lipid peroxides and depletion of cellular GSH. Lately, we demonstrated that hemoglobin degradation product hemin induces ferroptosis in platelets via ROS and lipid peroxidation. In this study, we demonstrate that hemin-induced ferroptosis in platelets is mediated through ROS-driven proteasome activity and inflammasome activation, which were mitigated by Melatonin (MLT). Although inflammasome activation is linked with pyroptosis, it is still not clear whether ferroptosis is associated with inflammasome activation. Our study for the first time demonstrates an association of platelet activation/ferroptosis with proteasome activity and inflammasome activation. Although, high-throughput screening has recognized ferrostatin-1 (Fer-1) and liproxstatin-1 (Lip-1) as potent ferroptosis inhibitors, having an endogenous antioxidant such as MLT as ferroptosis inhibitor is of high interest. MLT is a well-known chronobiotic hormone that regulates the circadian rhythms in vertebrates. It also exhibits potent antioxidant and ROS quenching capabilities. MLT can regulate fundamental cellular functions by exhibiting cytoprotective, oncostatic, antiaging, anti-venom, and immunomodulatory activities. The ROS scavenging capacity of MLT is key for its cytoprotective and anti-apoptotic properties. Considering the anti-ferroptotic and anti-apoptotic potentials of MLT, it could be a promising clinical application to treat hemolytic, thrombotic and thrombocytopenic conditions. Therefore, we propose MLT as a pharmacological and therapeutic agent to inhibit ferroptosis and platelet activation.
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http://dx.doi.org/10.1016/j.bbadis.2019.05.009DOI Listing
September 2019

Research into the Causes of Venom-Induced Mortality and Morbidity Identifies New Therapeutic Opportunities.

Am J Trop Med Hyg 2019 05;100(5):1043-1048

Department of Studies in Biochemistry, University of Mysore, Mysuru, India.

Snakebite primarily affects rural subsistent farming populations in underdeveloped and developing nations. The annual number of deaths (100,000) and physical disabilities (400,000) of snakebite victims is a societal tragedy that poses a significant added socioeconomic burden to the society. Antivenom therapy is the treatment of choice for snakebite but, as testified by the continuing high rates of mortality and morbidity, too many rural tropical snakebite victims fail to access effective treatment. Here, we advocate for more basic research to better understand the pathogenesis of systemic and local envenoming and describe how research outcomes can identify novel snakebite therapeutic strategies with the potential to be more accessible and affordable to victims than current treatment.
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http://dx.doi.org/10.4269/ajtmh.17-0877DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6493937PMC
May 2019

Para-tertiary butyl catechol (PTBC), an industrial antioxidant induces human platelet apoptosis.

Environ Toxicol 2019 Mar 21;34(3):262-270. Epub 2018 Nov 21.

DOS in Biochemistry, University of Mysore, Manasagangothri, Mysuru, India.

The catecholic derivative para-tertiary butyl catechol (PTBC) is a conventional antioxidant and polymerization inhibitor, which exhibits melanocytotoxic effects and contact dermatitis often leading to occupational leucoderma or vitiligo. Although numerous industrial workers will be in constant exposure to PTBC and its chances of getting entry into blood are most expected, its effect on blood components is still undisclosed. As platelets play a prominent role in dermatitis, inflammation, and immunity, in this study we have evaluated the effect of PTBC on human platelets in vitro. Exposure of platelets to PTBC showed increased reactive oxygen species (ROS), intracellular calcium, cardiolipin oxidation, mitochondrial permeability transition pore (MPTP) formation, activation of caspases, phosphatidylserine (PS) externalization and decreased mitochondrial membrane potential. In addition, there was a significant decrease in cellular glutathione level, increased γ-glutamyltransferase (GGT) activity and cell death. These findings demonstrate that PTBC could induce toxic effects on blood components, which is often ignored field of research. Since dermal exposure of humans to toxic chemicals covers an important issue in various industries, there is a need of such work to understand and update the long-term toxicities induced by PTBC usage in industrial sectors and public domain.
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http://dx.doi.org/10.1002/tox.22681DOI Listing
March 2019

Berberine mitigates high glucose-potentiated platelet aggregation and apoptosis by modulating aldose reductase and NADPH oxidase activity.

Free Radic Biol Med 2019 01 2;130:196-205. Epub 2018 Nov 2.

Department of Studies in Biochemistry, University of Mysore, Manasagangothri, Mysuru 570006, India; Department of Studies and Research in Biochemistry, Tumkur University, Tumakuru 572103, India. Electronic address:

Diabetes mellitus (DM) is a serious metabolic disorder affecting millions of people worldwide. The high rate of mortality and morbidity during DM is attributed to the increased atherothrombotic events due to platelet activation and apoptosis leading to macro and micro vascular occlusions. The platelet hyper-reactivity and apoptosis during DM is accounted for the accumulated reactive oxygen species (ROS) due to increased aldose reductase (AR) and NADPH oxidase (NOX) activities. Considering aspirin insensitivity in DM patients, new therapies targeting the underlying mechanism is urgently warranted. Berberine, a benzylisoquinoline alkaloids, from Chinese folk medicine has been demonstrated with several anti-diabetic effects. Therefore, we evaluated whether berberine inhibits high glucose potentiated platelet aggregation, apoptosis and further evaluated the mechanism of its action in platelets. Berberine was found to inhibit platelet aggregation, superoxide production via modulating AR, NOX, and glutathione reductase activities in high glucose (HG) treated platelets. Correlated with this, berberine inhibited, calcium release, ERK activation, α- and dense granule release and platelet adhesive properties. In addition, berberine inhibited p38-p53 mediated BAX activation, mitochondrial dysfunction and platelet apoptosis induced by HG. The platelet protective effect of berberine by inhibiting AR and NOX in high glucose-treated platelets suggest that berberine could be developed as a potential therapeutic molecule in the treating pathologies associated with DM.
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http://dx.doi.org/10.1016/j.freeradbiomed.2018.10.453DOI Listing
January 2019

Modulation of inflammatory platelet-activating factor (PAF) receptor by the acyl analogue of PAF.

J Lipid Res 2018 11 23;59(11):2063-2074. Epub 2018 Aug 23.

Department of Studies in Biochemistry University of Mysore, Manasagangothri, Mysuru 570006, India

Platelet-activating factor (PAF) is a potent inflammatory mediator that exerts its actions via the single PAF receptor (PAF-R). Cells that biosynthesize alkyl-PAF also make abundant amounts of the less potent PAF analogue acyl-PAF, which competes for PAF-R. Both PAF species are degraded by the plasma form of PAF acetylhydrolase (PAF-AH). We examined whether cogenerated acyl-PAF protects alkyl-PAF from systemic degradation by acting as a sacrificial substrate to enhance inflammatory stimulation or as an inhibitor to dampen PAF-R signaling. In ex vivo experiments both PAF species are prothrombotic in isolation, but acyl-PAF reduced the alkyl-PAF-induced stimulation of human platelets that express canonical PAF-R. In Swiss albino mice, alkyl-PAF causes sudden death, but this effect can also be suppressed by simultaneously administering boluses of acyl-PAF. When PAF-AH levels were incrementally elevated, the protective effect of acyl-PAF on alkyl-PAF-induced death was serially decreased. We conclude that, although acyl-PAF in isolation is mildly proinflammatory, in a pathophysiological setting abundant acyl-PAF suppresses the action of alkyl-PAF. These studies provide evidence for a previously unrecognized role for acyl-PAF as an inflammatory set-point modulator that regulates both PAF-R signaling and hydrolysis.
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http://dx.doi.org/10.1194/jlr.M085704DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6210909PMC
November 2018

Para-tertiary butyl catechol induces eryptosis in vitro via oxidative stress and hemoglobin leakage in human erythrocytes.

Toxicol In Vitro 2018 Oct 18;52:286-296. Epub 2018 Jul 18.

DOS in Biochemistry, University of Mysore, Manasagangothri, Mysuru 570 006, India; Department of Studies and Research in Biochemistry, Tumkur University, Tumakuru 572103, India. Electronic address:

Exposure of human population to industrial chemicals is believed as a significant contributing factor to the outgrowth of occupational diseases especially in developing countries due to improper safety measures and sanitary conditions. Para-tertiary butylcatechol (PTBC) widely employed in petrochemical, thermofax and phototypesetting industries, induces melanocytotoxicity and contact dermatitis leading to occupational leukoderma/vitiligo. Few vitiligo patients were reported for oxidative stress-induced hemolytic anemia and thrombocytopenia, however its impact on blood components is still not clear. Erythrocytes are the major cell population in circulation and play a prominent role in various diseases. In this work, the effect of PTBC on human erythrocytes is evaluated in vitro. PTBC induces oxidative stress-mediated eryptosis (erythrocyte death) causing detrimental changes such as depleted antioxidant levels, altered surface morphology, hemoglobin denaturation and heinz body formation. These findings validate that PTBC could induce toxic effects on human erythrocytes. Exposure of humans to toxic chemicals constitutes an important issue in various industries; one such issue is the exposure of PTBC at work place resulting in a spectrum of dermal complications. Therefore, it is imperative to appraise the long-term toxicities in order to further delineate the mechanisms of resultant disorders associated with PTBC and to establish the therapeutic interventions.
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http://dx.doi.org/10.1016/j.tiv.2018.07.010DOI Listing
October 2018

Reply to 'Evidence that neutrophils do not promote Echis carinatus venom-induced tissue destruction'.

Nat Commun 2018 06 13;9(1):2303. Epub 2018 Jun 13.

Department of Studies in Biochemistry, University of Mysore, Manasagangothri, Mysuru, 570006, India.

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http://dx.doi.org/10.1038/s41467-018-04507-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5998087PMC
June 2018

The Role of Reactive Oxygen Species and Ferroptosis in Heme-Mediated Activation of Human Platelets.

ACS Chem Biol 2018 08 13;13(8):1996-2002. Epub 2018 Jun 13.

Department of Inorganic and Physical Chemistry , Indian Institute of Science , Bangalore 560 012 , India.

Hemolysis, a process by which the destruction of red blood cells leads to the release of hemoglobin, is a critical event observed during hemolytic disorders. Under oxidative stress conditions, hemoglobin can release its heme prosthetic group, which is highly cytotoxic and can catalyze the generation of reactive oxygen species (ROS), leading to several undesired redox reactions in the cells. Herein, we demonstrate for the first time that heme can mediate the activation and death of human platelets through ferroptosis, which is an iron-dependent form of nonapoptotic cell death. This study also suggests that the heme-mediated lipid peroxidation and ferroptosis in platelets may play an important role in hemolytic disorders.
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http://dx.doi.org/10.1021/acschembio.8b00458DOI Listing
August 2018

Aggregation is impaired in starved platelets due to enhanced autophagy and cellular energy depletion.

Platelets 2019 25;30(4):487-497. Epub 2018 May 25.

a DOS in Biochemistry , University of Mysore , Mysuru , India.

Platelet hyperactivity is the hallmark of thrombosis and hemostasis disorders including atherosclerosis, diabetes, stroke, arthritis, and cancer causing significant mortality and morbidity. Therefore, regulating platelet hyperactivity is an ever growing interest. Very recently, basal autophagic process has been demonstrated to be essential for normal functioning of platelets. However, autophagy can be elevated above basal level under conditions like starvation, and how platelets respond in these settings remains to be elucidative. Therefore, in this study we demonstrate a substantial autophagy induction (above basal level) by starvation, which decreases platelet aggregation responses to various agonists. The decreased aggregation in starved platelets was restored in combination with autophagy inhibitors (3-methyladenine and NHCl) and acetate supplementation. Starved platelets also showed decreased calcium mobilization, granule release, and adhesive properties. Furthermore, ex vivo platelets obtained from starved rats showed increased autophagy markers and decreased aggregation responses to various agonists. Our results distinctly explain that enhanced autophagy and cellular energy depletion are the cause for decreased platelet activation and aggregation. The study emphasizes the cardinal role of starvation and autophagy in the management of diseases and disorders associated with platelet hyperactivity.
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http://dx.doi.org/10.1080/09537104.2018.1475630DOI Listing
May 2019

Inhibition of Echis carinatus venom by DNA, a promising therapeutic molecule for snakebite management.

Biochim Biophys Acta Gen Subj 2018 May 7;1862(5):1115-1125. Epub 2018 Feb 7.

DOS in Biochemistry, University of Mysore, Manasagangotri, Mysuru 570 006, India. Electronic address:

Background: E. carinatus bite is a serious threat to South-Asian countries including India, as it causes the highest number of deaths and debilitating sustained tissue necrosis at the bite site. One of our previous studies has demonstrated the strong interaction between DNA and E. carinatus venom. Therefore, in this study, the effect of DNA on E. carinatus venom has been examined.

Methods: Here we show that calf thymus DNA interact strongly with E. carinatus venom and inhibits its enzymatic and pharmacological activities such as proteolytic, hemolytic, hyaluronidase, L-amino acid oxidase, NETosis, hemorrhage, pro-coagulant, and lethality. Further, using immunoblots and immunofluorescence, the study demonstrates the inhibition of proteolytic cleavage of several surface receptors on PMNs, PBMCs, and platelets by the DNA.

Conclusions: This study for the first time explored the efficient inhibition of enzymatic, pharmacological and lethal properties of E. carinatus venom by the naked DNA and demonstrates the possible therapeutic application of it during snakebite management.

General Significance: This study identifies naked DNA as an effective defense weapon that has got the therapeutic potential to inhibit the detrimental effects of E. carinatus bite.
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http://dx.doi.org/10.1016/j.bbagen.2018.02.003DOI Listing
May 2018

Inhibition of constitutive NF-κB activity induces platelet apoptosis via ER stress.

Biochem Biophys Res Commun 2017 12 4;493(4):1471-1477. Epub 2017 Oct 4.

DOS in Biochemistry, University of Mysore, Manasagangothri, Mysuru 570 006, India; Department of Studies and Research in Biochemistry, Tumkur University, Tumakuru 572 103, India. Electronic address:

Platelets are anucleate cells, known for their pivotal roles in hemostasis, inflammation, immunity, and disease progression. Being anuclear, platelets are known to express several transcriptional factors which exert nongenomic functions, including the positive and negative regulation of platelet activation. NF-κB is one such transcriptional factor involved in the regulation of genes for survival, proliferation, inflammation and immunity. Although, the role NF-κB in platelet activation and aggregation is partially known, its function in management of platelet survival and apoptosis remain unexplored. Therefore, two unrelated inhibitors of NF-κB activation, BAY 11-7082 and MLN4924 were used to determine the role of NF-κB in platelets. Inhibition of NF-κB caused decreased SERCA activity and increased cytosolic Ca level causing ER stress which was determined by the phosphorylation of eIF2-α. Further, there was increased BAX and decreased BCl-2 levels, incidence of mitochondrial membrane potential depolarization, release of cytochrome c into cytosol, caspase activation, PS externalization and cell death in BAY 11-7082 and MLN4924 treated platelets. The obtained results demonstrate the critical role played by NF-κB in Ca homeostasis and survival of platelets. In addition, the study demonstrates the potential side effects associated with NF-κB inhibitors employed during inflammation and cancer therapy.
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http://dx.doi.org/10.1016/j.bbrc.2017.10.011DOI Listing
December 2017

Neutrophil extracellular traps in acrolein promoted hepatic ischemia reperfusion injury: Therapeutic potential of NOX2 and p38MAPK inhibitors.

J Cell Physiol 2018 04 28;233(4):3244-3261. Epub 2017 Sep 28.

Department of Biochemistry and Molecular Biology, School of life sciences, Pondicherry University, Pondicherry, India.

Neutrophil is a significant contributor to ischemia reperfusion (IR) induced liver tissue damage. However, the exact role of neutrophils in IR induced innate immune activation and liver damage is not quite clear. Our study sheds light on the role of chronic oxidative stress end products in worsening the IR inflammatory process by neutrophil recruitment and activation following liver surgery. We employed specific inhibitors for molecular targets-NOX2 (NADPH oxidase 2) and P38 MAPK (Mitogen activated protein kinase) signal to counteract neutrophil activation and neutrophil extracellular trap (NET) release induced liver damage in IR injury. We found that acrolein initiated neutrophil chemotaxis and induced NET release both in vitro and in vivo. Acrolein exposure caused NET induced nuclear and mitochondrial damage in HepG2 cells as well as aggravated the IR injury in rat liver. Pretreatment with F-apocynin and naringin, efficiently suppressed acrolein induced NET release in vitro. Notably, it suppressed the expression of inflammatory cytokines, P38MAPK-ERK activation, and apoptotic signals in rat liver exposed to acrolein and subjected to IR. Moreover, this combination effectively attenuated acrolein induced NET release and hepatic IR injury. In the current study we have shown that the acrolein accumulation in liver due to chronic stress, is responsible for neutrophil recruitment and its activation leading to NET induced liver damage during surgery. Our study shows that therapeutic targeting of NOX2 and P38MAPK signaling in patients with chronic hepatic disorders would improve post operative hepatic function and survival.
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http://dx.doi.org/10.1002/jcp.26167DOI Listing
April 2018

Cell-free methemoglobin drives platelets to apoptosis via mitochondrial ROS-mediated activation of JNK and p38 MAP kinase.

Biochem Biophys Res Commun 2017 09 13;491(1):183-191. Epub 2017 Jul 13.

Department of Studies in Biochemistry, University of Mysore, Manasagangotri, Mysuru 570 006, India; Department of Studies and Research in Biochemistry, Tumkur University, Tumakuru 572 103, India. Electronic address:

Cell-free hemoglobin (Hb), a well-known marker of intravascular hemolysis, is eventually oxidized to methemoglobin (MtHb). Elevated levels of MtHb have been noted, alongside depleted levels of platelets, in several hemolytic diseases. The current study aims to probe the possible role of MtHb in platelet death, based on the facts that it is a pro-inflammatory and pro-apoptotic agent, as well as the sensitive nature of platelets and their tendency to undergo apoptosis under oxidative stress. An attempt is made to establish the link between hemolysis and thrombocytopenia, by deciphering the underlying molecular signaling pathways. The results of this study demonstrate that MtHb, not Hb exerts oxidative stress on platelets, which triggers their death via ROS-mediated mitochondrial apoptotic pathway. It was further established that the MtHb-induced platelet apoptotic events mediate through JNK and p38 MAPK activation. Thus, the study presents a mechanistic insight into the previous studies that reported the incidence of thrombocytopenia in hemolytic diseases. This study highlights the fate of platelets in intravascular hemolytic conditions, which demands the need for a specific treatment strategy considering the risks associated with thrombocytopenia during severe hemolytic diseases.
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http://dx.doi.org/10.1016/j.bbrc.2017.07.073DOI Listing
September 2017

Bone Degeneration, Inflammation and Secondary Complications of Arthritis: Potential Targets and their Natural Inhibitors.

Mini Rev Med Chem 2018 ;18(3):244-275

Department of Studies in Biochemistry, University of Mysore, Mysore. India.

Arthritis is marked by joint deterioration that affects articular cartilage and subchondral bone. Though cartilage degradation does the major damage during arthritis, subsequent bone degeneration cannot be neglected. Recent progress in arthritis research has identified the clinical importance of bone erosion in destructive arthritis. Studies have showed the key role played by osteoclasts and receptor activator of nuclear factor kappaB ligand (RANKL) signaling in bone erosion. Cathepsins and tartrate resistant acid phosphatase (TRAP) are considered key enzymatic factors contributing to bone erosion. Further, reactive oxygen species (ROS) formed at the ruffled border of osteoclasts also causes bone resorption and matrix degradation. Besides, severe inflammation during arthritis induces bone erosion by aiding in Ca2+ removal and activating osteoclastogenesis. The inflammatory cytokines and ROS influence osteoclast differentiation by regulating osteoclast-lineage cells or by acting on other cells to regulate the expression of RANKL and osteoprotegerin (OPG). The enhanced production of pro-inflammatory cytokines and ROS in arthritis stimulates tissue injury by means of oxidative damage leading to vital organ damage and synovial and circulatory cell apoptosis. Thus, blocking enzymatic and non-enzymatic factors responsible for bone erosion and inflammation is considered a prime strategy in the management of arthritis. In this review we provide an overview of the mechanisms of bone erosion, inflammation and associated oxidative stress/damage during arthritis perpetuation along with shedding light on potential targets. The article also describes the possible natural therapeutic agents that could prevent bone loss and inflammation, and related secondary complications of arthritis.
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http://dx.doi.org/10.2174/1389557517666170315144233DOI Listing
February 2018

Melatonin inhibits snake venom and antivenom induced oxidative stress and augments treatment efficacy.

Acta Trop 2017 May 12;169:14-25. Epub 2017 Jan 12.

Department of Studies in Biochemistry, University of Mysore, Mysore, India. Electronic address:

Snakebite is a neglected health hazard. Its patho-physiology has largely been focused on systemic and local toxicities; whereas, venom and antivenom induced oxidative stress has long been ignored. Antivenom therapy although neutralizes venom lethality and saves many lives, remains ineffective against oxidative stress. This prompted us to complement antivenom with an antioxidant molecule melatonin that would protect against oxidative stress and increase the efficacy of the existing snakebite therapy. Here we show that D. russelli and E. carinatus venoms induce strong oxidative stress that persists even after antivenom administration in mice model. Additionally, antivenoms also induce oxidative stress. Polyvalent antivenom induce more oxidative stress than monovalent antivenom. Strikingly, antivenom and melatonin together not only inhibit venom and antivenom induced oxidative stress but also significantly reduce the neutralizing antivenom dose. This study provides a therapeutic potential for enhancing the existing snakebite therapy. The combined treatment of antivenom+melatonin would prevent the upsurge of oxidative stress as well as minimize the antivenom load. Thus the investigation offers immense scope for physicians and toxinologists to reinvestigate, design new strategies and think beyond the conventional mode of antivenom therapy.
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http://dx.doi.org/10.1016/j.actatropica.2017.01.004DOI Listing
May 2017

Novel sila-amide derivatives of N-acetylcysteine protects platelets from oxidative stress-induced apoptosis.

J Thromb Thrombolysis 2017 Feb;43(2):209-216

Department of Studies in Biochemistry, University of Mysore, Manasagangothri, Mysore, 570 006, India.

Oxidative stress-induced platelet apoptosis is one among the many causes for the development and progression of many disorders like cardiovascular diseases, arthritis, Alzheimer's disease and many chronic inflammatory responses. Many studies have demonstrated the less optimal effect of N-acetyl cysteine (NAC) in oxidative stress-induced cellular damage. This could be due to its less lipophilicity which makes it difficult to enter the cellular membrane. Therefore in the present study, lipophilic sila-amide derivatives (6a and 6b) synthesized through the reaction of NAC with 3-Aminopropyltrimethylsilane and aminomethyltrimethylsilane were used to determine their protective property against oxidative stress-induced platelet apoptosis. At a concentration of 10 µM, compound 6a and 6b were able to significantly inhibit Rotenone/HO induced platelet apoptotic markers like reactive oxygen species, intracellular calcium level, mitochondrial membrane potential, cytochrome c release from mitochondrial to the cytosol, caspase-9 and -3 activity and phosphatidylserine externalization. Therefore, the compounds can be extrapolated as therapeutic agents to protect platelets from oxidative stress-induced platelet apoptosis and its associated complications.
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http://dx.doi.org/10.1007/s11239-016-1450-4DOI Listing
February 2017

Nanoparticles for Modulating mTOR Signaling in Platelets.

Trends Biotechnol 2016 11 3;34(11):850-852. Epub 2016 Sep 3.

Department of Studies in Biochemistry, University of Mysore, Mysore 570006, India; Department of Studies and Research in Biochemistry, Tumkur University, Tumkur 572103, India. Electronic address:

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http://dx.doi.org/10.1016/j.tibtech.2016.08.006DOI Listing
November 2016

NETosis and lack of DNase activity are key factors in Echis carinatus venom-induced tissue destruction.

Nat Commun 2016 Apr 19;7:11361. Epub 2016 Apr 19.

Department of Studies in Biochemistry, University of Mysore, Manasagangothri, Mysuru 570006, India.

Indian Echis carinatus bite causes sustained tissue destruction at the bite site. Neutrophils, the major leukocytes in the early defence process, accumulate at the bite site. Here we show that E. carinatus venom induces neutrophil extracellular trap (NET) formation. The NETs block the blood vessels and entrap the venom toxins at the injection site, promoting tissue destruction. The stability of NETs is attributed to the lack of NETs-degrading DNase activity in E. carinatus venom. In a mouse tail model, mice co-injected with venom and DNase 1, and neutropenic mice injected with the venom, do not develop NETs, venom accumulation and tissue destruction at the injected site. Strikingly, venom-induced mice tail tissue destruction is also prevented by the subsequent injection of DNase 1. Thus, our study suggests that DNase 1 treatment may have a therapeutic potential for preventing the tissue destruction caused by snake venom.
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http://dx.doi.org/10.1038/ncomms11361DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4838891PMC
April 2016

Emerging roles of hyaluronic acid bioscaffolds in tissue engineering and regenerative medicine.

Int J Biol Macromol 2016 May 15;86:917-28. Epub 2016 Feb 15.

Department of Studies in Biochemistry, University of Mysore, Mysore 570006, India; Department of Studies and Research in Biochemistry, Tumkur University, Tumkur 572103, India. Electronic address:

Hyaluronic acid (HA), is a glycosaminoglycan comprised of repeating disaccharide units of N-acetyl-D-glucosamine and D-glucuronic acid. HA is synthesized by hyaluronan synthases and reaches sizes in excess of 2MDa. It plays numerous roles in normal tissues but also has been implicated in inflammatory processes, multiple drug resistance, angiogenesis, tumorigenesis, water homeostasis, and altered viscoelasticity of extracellular matrix. The physicochemical properties of HA including its solubility and the availability of reactive functional groups facilitate chemical modifications on HA, which makes it a biocompatible material for use in tissue regeneration. HA-based biomaterials and bioscaffolds do not trigger allergies or inflammation and are hydrophilic which make them popular as injectable dermal and soft tissue fillers. They are manufactured in different forms including hydrogels, tubes, sheets and meshes. Here, we review the pathophysiological and pharmacological properties and the clinical uses of native and modified HA. The review highlights the therapeutic applications of HA-based bioscaffolds in organ-specific tissue engineering and regenerative medicine.
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http://dx.doi.org/10.1016/j.ijbiomac.2016.02.032DOI Listing
May 2016

Unconjugated Bilirubin exerts Pro-Apoptotic Effect on Platelets via p38-MAPK activation.

Sci Rep 2015 Oct 13;5:15045. Epub 2015 Oct 13.

DOS in Chemistry, University of Mysore, Manasagangothri, Mysuru-570 006, India.

Thrombocytopenia is one of the most frequently observed secondary complications in many pathological conditions including liver diseases, where hyperbilirubinemia is very common. The present study sought to find the cause of thrombocytopenia in unconjugated hyperbilirubinemic conditions. Unconjugated bilirubin (UCB), an end-product of heme catabolism, is known to have pro-oxidative and cytotoxic effects at high serum concentration. We investigated the molecular mechanism underlying the pro-apoptotic effect of UCB on human platelets in vitro, and followed it up with studies in phenylhydrazine-induced hyperbilirubinemic rat model and hyperbilirubinemic human subjects. UCB is indeed found to significantly induce platelet apoptotic events including elevated endogenous reactive oxygen species generation, mitochondrial membrane depolarization, increased intracellular calcium levels, cardiolipin peroxidation and phosphatidylserine externalization (p < 0.001) as evident by FACS analysis. The immunoblots show the elevated levels of cytosolic cytochrome c and caspase activation in UCB-treated platelets. Further, UCB is found to induce mitochondrial ROS generation leading to p38 activation, followed by downstream activation of p53, ultimately resulting in altered expression of Bcl-2 and Bax proteins as evident from immunoblotting. All these parameters conclude that elevated unconjugated bilirubin causes thrombocytopenia by stimulating platelet apoptosis via mitochondrial ROS-induced p38 and p53 activation.
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http://dx.doi.org/10.1038/srep15045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4602209PMC
October 2015

Oxidative stress-induced methemoglobinemia is the silent killer during snakebite: a novel and strategic neutralization by melatonin.

J Pineal Res 2015 Sep 14;59(2):240-54. Epub 2015 Jul 14.

Department of Studies in Biochemistry, University of Mysore, Mysuru, India.

Oxidative stress-induced methemoglobinemia remained an untouched area in venom pharmacology till date. This study for the first time explored the potential of animal venoms to oxidize hemoglobin to methemoglobin. In in vitro whole-blood assay, methemoglobin forming ability of venoms varied as Naja naja > Ophiophagus hannah > Echis carinatus > Daboia russellii > Apis mellifera > Mesobuthus tamulus > Hippasa partita. Being highly potential, N. naja venom was further studied to observe methemoglobin formation in RBCs and in combinations with PMNs and PBMCs, where maximum effect was observed in RBCs + PMNs combination. Naja naja venom/externally added methemoglobin-induced methemoglobin formation was in parallel with ROS generation in whole blood/RBCs/RBCs + PMNs/RBCs + PBMCs. In in vivo studies, the lethal dose (1 mg/kg body weight, i.p.) of N. naja venom readily induced methemoglobin formation, ROS generation, expression of inflammatory markers, and hypoxia-inducible factor-3α. Although the mice administered with three effective doses of antivenom recorded zero mortality; the methemoglobin and ROS levels remained high. However, one effective dose of antivenom when administered along with melatonin (1:50; venom/melatonin, w/w), not only offered 100% survival of experimental mice, but also significantly reduced methemoglobin level, and oxidative stress markers including hypoxia-inducible factor-3α. This study provides strong drive that, complementing melatonin would not only reduce the antivenom load, but for sure greatly increase the success rate of antivenom therapy and drastically minimize the global incidence of snakebite deaths. However, further detailed investigations are needed before translating the combined therapy towards the bed side.
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http://dx.doi.org/10.1111/jpi.12256DOI Listing
September 2015

Methotrexate Promotes Platelet Apoptosis via JNK-Mediated Mitochondrial Damage: Alleviation by N-Acetylcysteine and N-Acetylcysteine Amide.

PLoS One 2015 17;10(6):e0127558. Epub 2015 Jun 17.

Department of studies in Biochemistry, University of Mysore, Manasagangothri, Mysore, 570 006, India; Department of Studies and Research in Biochemistry, Tumkur University, Tumkur, 572 103, India.

Thrombocytopenia in methotrexate (MTX)-treated cancer and rheumatoid arthritis (RA) patients connotes the interference of MTX with platelets. Hence, it seemed appealing to appraise the effect of MTX on platelets. Thereby, the mechanism of action of MTX on platelets was dissected. MTX (10 μM) induced activation of pro-apoptotic proteins Bid, Bax and Bad through JNK phosphorylation leading to ΔΨm dissipation, cytochrome c release and caspase activation, culminating in apoptosis. The use of specific inhibitor for JNK abrogates the MTX-induced activation of pro-apoptotic proteins and downstream events confirming JNK phosphorylation by MTX as a key event. We also demonstrate that platelet mitochondria as prime sources of ROS which plays a central role in MTX-induced apoptosis. Further, MTX induces oxidative stress by altering the levels of ROS and glutathione cycle. In parallel, the clinically approved thiol antioxidant N-acetylcysteine (NAC) and its derivative N-acetylcysteine amide (NACA) proficiently alleviate MTX-induced platelet apoptosis and oxidative damage. These findings underpin the dearth of research on interference of therapeutic drugs with platelets, despite their importance in human health and disease. Therefore, the use of antioxidants as supplementary therapy seems to be a safe bet in pathologies associated with altered platelet functions.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0127558PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4471342PMC
March 2016
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