Hump-Nosed Pit Viper (Hypnale hypnale) Venom-Induced Irreversible Red Blood Cell Aggregation, Inhibition by Monovalent Anti-Venom and N-Acetylcysteine.

Envenomation by the Hypnale hypnale in the Western Ghats of India (particularly in the Malabar region of Kerala) and the subcontinent island nation of Sri Lanka is known to inflict devastating mortality and morbidity. Currently, H. hypnale bites in India are devoid of anti-venom regimens. A detailed characterization of the venom is essential to stress the need for therapeutic anti-venom. Notably, the deleterious effects of this venom on human blood cells have largely remained less explored. Therefore, in continuation of our previous study, in the present study, we envisioned investigating the effect of venom on the morphological and physiological properties of red blood cells (RBCs). The venom readily induced deleterious morphological changes and, finally, the aggregation of washed RBCs. The aggregation process was independent of the ROS and the intracellular Ca2+ ion concentration. Confocal and scanning electron microscopy (SEM) images revealed the loss of biconcave morphology and massive cytoskeletal disarray. Crenation or serrated plasma membrane projections were evenly distributed on the surface of the RBCs. The venom did not cause the formation of methemoglobin in washed RBCs but was significantly induced in whole blood. Venom did not affect glucose uptake and Na+/K+ -ATPase activity but inhibited glucose 6 phosphate dehydrogenase activity and decreased the fluidity of the plasma membrane. Venom-induced RBC aggregates exhibited pro-coagulant activity but without affecting platelet aggregation. In pre-incubation or co-treatment studies, none of the bioactive compounds, such as melatonin, curcumin, fisetin, berberine, and quercetin, sugars such as mannose and galactose, and therapeutic polyvalent anti-venoms (Bharat and VINS) were inhibited, whereas only N-acetylcysteine and H. hypnale monovalent anti-venom could inhibit venom-induced deleterious morphological changes and aggregation of RBCs. In post-treatment studies, paradoxically, none of the bioactives and anti-venoms, including N-acetylcysteine and H. hypnale monovalent anti-venom, reversed the venom-induced RBC aggregates.

Oxidative Stress-Induced Platelet Apoptosis/Activation: Alleviation by Purified Curcumin via ASK1-JNK/p-38 Pathway.

Platelets are known for their indispensable role in hemostasis and thrombosis. However, alteration in platelet function due to oxidative stress is known to mediate various health complications, including cardiovascular diseases and other health complications. To date, several synthetic molecules have displayed antiplatelet activity; however, their uses are associated with bleeding and other adverse effects. The commercially available curcumin is generally a mixture of three curcuminoids: curcumin, demethoxycurcumin, and bisdemethoxycurcumin. Although crude curcumin is known to inhibit platelet aggregation, the effect of purified curcumin on platelet apoptosis, activation, and aggregation remains unclear. Therefore, in this study, curcumin was purified from a crude curcumin mixture and the effects of this preparation on the oxidative stress-induced platelet apoptosis and activation was evaluated. 2,2'-Azobis(2-methylpropionamidine) dihydrochloride (AAPH) compound was used as an inducer of oxidative stress. Purified curcumin restored AAPH-induced platelet apoptotic markers like reactive oxygen species, intracellular calcium level, mitochondrial membrane potential, cardiolipin peroxidation, cytochrome c release from mitochondria to the cytosol, and phosphatidyl serine externalization. Further, it inhibited the agonist-induced platelet activation and aggregation, demonstrating its antiplatelet activity. Western blot analysis confirms protective effect of the purified curcumin against oxidative stress-induced platelet apoptosis and activation via downregulation of MAPKs protein activation, including ASK1, JNK, and p-38. Together, these results suggest that the purified curcumin could be a potential therapeutic bioactive molecule to treat the oxidative stress-induced platelet activation, apoptosis, and associated complications.

Platelet activation and ferroptosis mediated NETosis drives heme induced pulmonary thrombosis.

Cell-free heme (CFH) is a product of hemoglobin, myoglobin and hemoprotein degradation, which is a hallmark of pathologies associated with extensive hemolysis and tissue damage. CHF and iron collectively induce cytokine storm, lung injury, respiratory distress and infection susceptibility in the lungs suggesting their key role in the progression of lung disease pathology. We have previously demonstrated that heme-mediated reactive oxygen species (ROS) induces platelet activation and ferroptosis. However, interaction of ferroptotic platelets and neutrophils, the mechanism of action and associated complications remain unclear. In this study, we demonstrate that heme-induced P-selectin expression and Phosphatidylserine (PS) externalization in platelets via ASK-1-inflammasome axis increases platelet-neutrophil aggregates in circulation, resulting in Neutrophil extracellular traps (NET) formation in vitro and in vivo. Further, heme-induced platelet activation in mice increased platelet-neutrophil aggregates and accumulation of NETs in the lungs causing pulmonary damage. Thus, connecting CFH-mediated platelet activation to NETosis and pulmonary thrombosis. As lung infections induce acute respiratory stress, thrombosis and NETosis, we propose that heme -mediated platelet activation and ferroptosis might be crucial in such clinical manifestations. Further, considering the ability of redox modulators and ferroptosis inhibitors like FS-1, Lpx-1 and DFO to inhibit heme-induced ferroptotic platelets-mediated NETosis and pulmonary thrombosis. They could be potential adjuvant therapy to regulate respiratory distress-associated clinical complications.

A comparative cross-reactivity and paraspecific neutralization study on Hypnale hypnale, Echis carinatus, and Daboia russelii monovalent and therapeutic polyvalent anti-venoms.

Envenoming by the hump-nosed pit viper (Hypnale hypnale) raises concern as it inflicts significant debilitation and death in the Western Ghats of India and in the adjacent island nation of Sri Lanka. In India, its medical significance was realized only during 2007 due to its misidentification as Echis carinatus and sometimes as Daboia russelii. Of late, several case reports have underlined the ineptness of the existing polyvalent anti-venom therapy against H. hypnale envenoming. Currently, H. hypnale bite has remained dreadful in India due to the lack of neutralizing anti-venom therapy. Hence, this study was undertaken to establish a systematic comparative, biochemical, pathological, and immunological properties of Sri Lankan H. hypnale venom alongside Indian E. carinatus, and D. russelii venoms. All three venoms differed markedly in the extent of biochemical activities including proteolytic, deoxyribonuclease, L-amino acid oxidase, 5'-nucleotidase, hyaluronidase, and indirect hemolytic activities. The venoms also differed markedly in their pathological properties such as edema, hemorrhage, myotoxic, cardiotoxic, and coagulant activities. The venoms showed stark differences in their protein banding pattern. Strikingly, the affinity-purified rabbit monovalent anti-venoms prepared against H. hypnale, E. carinatus, and D. russelii venoms readily reacted and neutralized the biochemical and pathological properties of their respective venoms, but they insignificantly cross-reacted with, and thus failed to show paraspecific neutralization of any of the effects of the other two venoms, demonstrating the large degree of variations between these venoms. Further, the Indian therapeutic polyvalent anti-venoms from VINS Bioproducts, and Bharath Serums and Vaccines failed to protect H. hypnale venom-induced lethal effects in mice.

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

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.

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

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.

The action of Echis carinatus and Naja naja venoms on human neutrophils; an emphasis on NETosis.

BACKGROUND: Neutrophils are the first line defense cells of the innate immunity. As a final defense, they discharge their de-condensed chromatin/DNA fibers, the NETs (Neutrophil Extracellular Traps), by a process called NETosis. Two types of NETosis have been currently described: the suicidal/delayed/classical-type, which is ROS dependent that results in the ejection of nuclear DNA, and the vital/rapid/early-type, which may or may not require ROS but, eject nuclear/mitochondrial DNA or both. Thus, Echis carinatus and Naja naja venoms are comparatively studied for their NET inducing property. METHODS: Formation of NETs, cell viability, ROS, and Ca2+ levels are estimated. An in vivo toxicity study and possible cellular signaling have been addressed using immunoblots and pharmacological inhibitors. RESULTS: E. carinatus and N. naja venoms respectively induce suicidal and vital NETosis. E. carinatus venom induces NETosis by activating NOX and PAD-4 enzymes in a ROS dependent manner via PKC/ERK/JNK signaling axis, while N. naja venom does it by activating PAD-4 enzyme, but independent of ROS requirement and as well as PKC/ERK/JNK activation. CONCLUSION: For the first time our study demonstrates the distinct action of E. carinatus and N. naja venoms on the process of NETosis. NETosis being a newly explored area in snake venom pharmacodynamics, it is important to study its impact on the various pathophysiological properties induced by snake venoms. SIGNIFICANCE: Understanding the varied actions of snake venoms on neutrophils/blood cells and the role of DNase are likely to provide insights for better management of snakebite pathophysiology.

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

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 Ca2+, 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.

A modified flavonoid accelerates oligodendrocyte maturation and functional remyelination.

Myelination delay and remyelination failure following insults to the central nervous system (CNS) impede axonal conduction and lead to motor, sensory and cognitive impairments. Both myelination and remyelination are often inhibited or delayed due to the failure of oligodendrocyte progenitor cells (OPCs) to mature into myelinating oligodendrocytes (OLs). Digestion products of the glycosaminoglycan hyaluronan (HA) have been implicated in blocking OPC maturation, but how these digestion products are generated is unclear. We tested the possibility that hyaluronidase activity is directly linked to the inhibition of OPC maturation by developing a novel modified flavonoid that functions as a hyaluronidase inhibitor. This compound, called S3, blocks some but not all hyaluronidases and only inhibits matrix metalloproteinase activity at high concentrations. We find that S3 reverses HA-mediated inhibition of OPC maturation in vitro, an effect that can be overcome by excess recombinant hyaluronidase. Furthermore, we find that hyaluronidase inhibition by S3 accelerates OPC maturation in an in vitro model of perinatal white matter injury. Finally, blocking hyaluronidase activity with S3 promotes functional remyelination in mice with lysolecithin-induced demyelinating corpus callosum lesions. All together, these findings support the notion that hyaluronidase activity originating from OPCs in CNS lesions is sufficient to prevent OPC maturation, which delays myelination or blocks remyelination. These data also indicate that modified flavonoids can act as selective inhibitors of hyaluronidase activity and can promote OPC maturation, making them excellent candidates to accelerate myelination or promote remyelination following perinatal and adult CNS insults.

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

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-1ß 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-1ß 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.

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

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.

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

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.

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

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 NH4Cl) 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.

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

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.

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

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.

A new pyrrole based small molecule from Tinospora cordifolia induces apoptosis in MDA-MB-231 breast cancer cells via ROS mediated mitochondrial damage and restoration of p53 activity.

Approximately 15% of globally diagnosed breast cancers are designated as triple negative breast cancer (TNBC). In this study, we investigated the effect of the natural compound, Bis(2- ethyl hexyl) 1H-pyrrole-3,4-dicarboxylate (TCCP), purified from Tinospora cordifolia on MDA-MB-231, a TNBC cell line. The pro-apoptotic nature of TCCP on MDA-MB-231 was determined by assessing various apoptotic markers. ROS generation, intracellular calcium, mitochondrial membrane potential (ΔΨm), MPTP, cardiolipin peroxidation and caspase activity were determined fluorometrically. BAX, BCL-2, cytochrome c, caspases, and p53 protein expressions were determined by immunoblotting. Further, the effect of TCCP on DNA and cell death was determined by DNA fragmentation assay, annexin-V staining, and cell cycle analysis. TCCP treatment caused endogenous ROS generation, increase in intracellular calcium and phosphorylation of p53 in a concentration-dependent manner, which was reverted upon pre-treatment with pifithrin-µ. This led to the downstream altered expression of Bcl-2 and Bax proteins, mitochondrial membrane depolarization, MPTP, and cardiolipin peroxidation. TCCP induced cytochrome c release into the cytosol, caspase activation, ultimately resulting in DNA fragmentation. Further, induction of apoptosis and morphological alterations were evident from the phosphatidylserine externalization and increase in sub G1 population. The in vivo Ehrlich ascites tumor (EAT) mouse study revealed the effectiveness of TCCP in reducing the tumor burden and resulted in a ~2 fold increase in mice survival with minimal hepato-renal toxicity. Overall, TCCP was shown to be efficient in inducing ROS and mitochondrial-mediated apoptosis by restoring p53 activity in MDA-MB-231 cells and also induced EAT cell death in vivo thereby inhibiting tumor proliferation.

Discovery of a small-molecule inhibitor of specific serine residue BAD phosphorylation.

Human BCL-2-associated death promoter (hBAD) is an apoptosis-regulatory protein mediating survival signals to carcinoma cells upon phosphorylation of Ser99, among other residues. Herein, we screened multiple small-molecule databases queried in a Laplacian-modified naive Bayesian-based cheminformatics platform and identified a Petasis reaction product as a site-specific inhibitor for hBAD phosphorylation. Based on apoptotic efficacy against mammary carcinoma cells, N-cyclopentyl-3-((4-(2,3-dichlorophenyl) piperazin-1-yl) (2-hydroxyphenyl) methyl) benzamide (NPB) was identified as a potential lead compound. In vitro biochemical analyses demonstrated that NPB inhibited the phosphorylation of hBAD specifically on Ser99. NPB was observed to exert this effect independently of AKT and other kinase activities despite the demonstration of AKT-mediated BAD-Ser99 phosphorylation. Using a structure-based bioinformatics platform, we observed that NPB exhibited predicted interactions with hBAD in silico and verified the same by direct binding kinetics. NPB reduced phosphorylation of BAD-Ser99 and enhanced caspase 3/7 activity with associated loss of cell viability in various human cancer cell lines derived from mammary, endometrial, ovarian, hepatocellular, colon, prostatic, and pancreatic carcinoma. Furthermore, by use of a xenograft model, it was observed that NPB, as a single agent, markedly diminished BAD phosphorylation in tumor tissue and significantly inhibited tumor growth. Similar doses of NPB utilized in acute toxicity studies in mice did not exhibit significant effects. Hence, we report a site-specific inhibitor of BAD phosphorylation with efficacy in tumor models.

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

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.