Publications by authors named "Abhinav Parashar"

26 Publications

  • Page 1 of 1

Murburn precepts for lactic-acidosis, Cori cycle, and Warburg effect: Interactive dynamics of dehydrogenases, protons, and oxygen.

J Cell Physiol 2021 Dec 20. Epub 2021 Dec 20.

Department of Biochemistry, Satyamjayatu: The Science & Ethics Foundation, Palakkad, Kerala, India.

It is unresolved why lactate is transported to the liver for further utilization within the physiological purview of Cori cycle, when muscles have more lactate dehydrogenase (LDH) than liver. We point out that the answer lies in thermodynamics/equilibriums. While the utilization of NADH for the reduction of pyruvate to lactate can be mediated via the classical mechanism, the oxidation of lactate (with/without the uphill reduction of NAD ) necessitates alternative physiological approaches. The latter pathway occurs via interactive equilibriums involving the enzyme, protons and oxygen or diffusible reactive oxygen species (DROS). Since liver has high DROS, the murburn activity at LDH would enable the cellular system to tide over the unfavorable energy barriers of the forward reaction (~476 kJ/mol; earlier miscalculated as ~26 kJ/mole). Further, the new mechanism does not necessitate any "smart decision-making" or sophisticated control by/of proteins. The DROS-based murburn theory explains the invariant active-site structure of LDH isozymes and their multimeric nature. The theoretical insights, in silico evidence and analyses of literature herein also enrich our understanding of the underpinnings of "lactic acidosis" (lowering of physiological pH accompanied by lactate production), Warburg effect (increased lactate production at high pO by cancer cells) and approach for cancer therapy.
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http://dx.doi.org/10.1002/jcp.30661DOI Listing
December 2021

Validating the predictions of murburn model for oxygenic photosynthesis: Analyses of ligand-binding to protein complexes and cross-system comparisons.

J Biomol Struct Dyn 2021 Jul 30:1-33. Epub 2021 Jul 30.

RedOx Lab, Department of Life Sciences, Satyamjayatu: The Science & Ethics Foundation, Palakkad District, Kerala, India.

In this second half of our treatise on oxygenic photosynthesis, we provide support for the murburn model of the light reaction of photosynthesis and ratify key predictions made in the first part. Molecular docking and visualization of various ligands of quinones/quinols (and their derivatives) with PS II/Cytochrome complexes did not support chartered 2e-transport role of quinols. A broad variety of herbicides did not show any affinity/binding-based rationales for inhibition of photosynthesis. We substantiate the proposal that disubstituted phenolics (perceived as protonophores/uncouplers or affinity-based inhibitors in the classical purview) serve as interfacial modulators of diffusible reactive (oxygen) species or DR(O)S. The DRS-based murburn model is evidenced by the identification of multiple ADP-binding sites on the extra-membraneous projection of protein complexes and structure/distribution of the photo/redox catalysts. With a panoramic comparison of the redox metabolic machinery across diverse organellar/cellular systems, we highlight the ubiquitous one-electron murburn facets (cofactors of porphyrin, flavin, FeS, other metal centers and photo/redox active pigments) that enable a facile harnessing of the utility of DRS. In the summative analyses, it is demonstrated that the murburn model of light reaction explains the structures of membrane supercomplexes recently observed in thylakoids and also accounts for several photodynamic experimental observations and evolutionary considerations. , the work provides a new orientation and impetus to photosynthesis research. Communicated by Ramaswamy H. Sarma.
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http://dx.doi.org/10.1080/07391102.2021.1953607DOI Listing
July 2021

Structure-function correlations and system dynamics in oxygenic photosynthesis: classical perspectives and murburn precepts.

J Biomol Struct Dyn 2021 Jul 29:1-27. Epub 2021 Jul 29.

Satyamjayatu: The Science & Ethics Foundation, Kulappully, Kerala, India.

Highlights: Contemporary beliefs on oxygenic photosynthesis are critiqued.Murburn model is suggested as an alternative explanation.In the new model, diffusible reactive species are the main protagonists.All pigments are deemed photo-redox active in the new stochastic mechanism.NADPH synthesis occurs via simple electron transfers, not via elaborate ETC.Oxygenesis is delocalized and not just centered at Mn-Complex.Energetics of murburn proposal for photophosphorylation is provided.The proposal ushers in a paradigm shift in photosynthesis research.
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http://dx.doi.org/10.1080/07391102.2021.1953606DOI Listing
July 2021

Molecular docking and dynamics studies of curcumin with COVID-19 proteins.

Netw Model Anal Health Inform Bioinform 2021 10;10(1):44. Epub 2021 Jun 10.

Department of Biotechnology, Vignan's Foundation for Science, Technology and Research (Deemed To Be University), Vadlamudi, Guntur, 522 213 Andhra Pradesh India.

Coronavirus disease 2019 (COVID-19) is caused by a Severe Acute Respiratory Syndrome-Coronavirus 2 (SARS-CoV-2), which is a positive-strand RNA virus. The SARS-CoV-2 genome and its association to SAR-CoV-1 vary from ca. 66 to 96% depending on the type of betacoronavirideae family members. With several drugs, viz. chloroquine, hydroxychloroquine, ivermectin, artemisinin, remdesivir, azithromycin considered for clinical trials, there has been an inherent need to find distinctive antiviral mechanisms of these drugs. Curcumin, a natural bioactive molecule has been shown to have therapeutic potential for various diseases, and its effect on COVID-19 is also currently being explored. In this study, we show the binding potential of curcumin targeted to a variety of SARS-CoV-2 proteins, viz spike glycoproteins (PDB ID: 6VYB), nucleocapsid phosphoprotein (PDB ID: 6VYO), spike protein-ACE2 (PDB ID: 6M17) along with nsp10 (PDB ID: 6W4H) and RNA dependent RNA polymerase (PDB ID: 6M71) structures. Furthermore, representative docking complexes were validated using molecular dynamics simulations and mechanistic studies at 100 ns was carried on nucleocapsid and nsp10 proteins with curcumin complexes which resulted in stable and efficient binding energies and correlated with that of docked binding energies of the complexes. Both the docking and simulation studies indicate that curcumin has the potential as an antiviral against COVID-19.
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http://dx.doi.org/10.1007/s13721-021-00312-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8192041PMC
June 2021

Hemoglobin catalyzes ATP-synthesis in human erythrocytes: a murburn model.

J Biomol Struct Dyn 2021 May 17:1-13. Epub 2021 May 17.

Satyamjayatu: The Science & Ethics Foundation, Kulappully, India.

Blood hemoglobin (Hb), known to transport oxygen, is the most abundant globular protein in humans. Erythrocytes have ∼10 M concentration of ATP in steady-state and we estimate that this high amounts cannot be formed from 10 - 10 M levels of precursors via substrate-level phosphorylation of glycolysis. To account for this discrepancy, we propose that Hb serves as a 'murzyme' (a redox enzyme working along the principles of murburn concept), catalyzing the synthesis of the major amounts of ATP found in erythrocytes. This proposal is along the lines of our earlier works demonstrating DROS (diffusible reactive oxygen species) mediated ATP-synthesis as a thermodynamically and kinetically viable mechanism for physiological oxidative phosphorylation. We support the new hypothesis for Hb with theoretical arguments, experimental findings of reputed peers and in silico explorations. Using in silico methods, we demonstrate that adenosine nucleotide and 2,3-bisphosphoglycerate (2,3-BPG) binding sites are located suitably on the monomer/tetramer, thereby availing facile access to the superoxide emanating from the heme center. Our proposal explains earlier reported in situ experimental findings/suggestions of 2,3-BPG and ADP binding at the same locus on Hb. The binding energy is in the order of 2,3-BPG > NADH > ATP > ADP > AMP and agrees with earlier reports, potentially explaining the bioenergetic physiology of erythrocytes. Also, the newly discovered site for 2,3-BPG shows lower affinity in fetal Hb (as compared to adults) explaining oxygen transfer from mother to embryo. The findings pose significant implications in routine physiology and pathologies like sickle cell anemia and thalassemia.Communicated by Ramaswamy H. Sarma.
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http://dx.doi.org/10.1080/07391102.2021.1925592DOI Listing
May 2021

Murburn Precepts for Cytochrome P450 Mediated Drug/Xenobiotic Metabolism and Homeostasis.

Curr Drug Metab 2021 ;22(4):315-326

Satyamjayatu: The Science & Ethics Foundation, Snehatheeram, Kulappully, Shoranur-2 (PO), Kerala-679122, India.

Aims: We aim to demonstrate why deeming diffusible reactive oxygen species (DROS) as toxic wastes do not afford a comprehensive understanding of cytochrome P450 mediated microsomal xenobiotic metabolism (mXM).

Background: Current pharmacokinetic investigations consider reactive oxygen species formed in microsomal reactions as toxic waste products, whereas our works (Manoj et al., 2016) showed that DROS are the reaction mainstay in cytochrome P450 mediated metabolism and that they play significant roles in explaining several unexplained physiologies.

Objective: Herein, we strive to detail the thermodynamic and kinetic foundations of murburn precepts of cytochrome P450 mediated drug metabolism.

Methods: Primarily, in silico approaches (using pdb crystal structure files), murburn reaction chemistry logic and thermodynamic calculations to elucidate the new model of CYP-mediated drug metabolism. The theoretical foundations are used to explain experimental observations.

Results: We visually elucidate how murburn model better explains- (i) promiscuity of the unique P450-reductase; (ii) prolific activity and inhibitions of CYP3A4; (iii) structure-function correlations of important key CYP2 family isozymes- 2C9, 2D6 and 2E1; and (iv) mutation studies and mechanism-based inactivation of CYPs. Several other miscellaneous aspects of CYP reaction chemistry are also addressed.

Conclusion: In the light of our findings that DROS are crucial for explaining reaction outcomes in mXM, approaches for understanding drug-drug interactions and methodologies for lead drug candidates' optimizations should be revisited.
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http://dx.doi.org/10.2174/1389200222666210118102230DOI Listing
December 2021

Elastin calcification in in vitro models and its prevention by MGP's N-terminal peptide.

J Struct Biol 2021 03 12;213(1):107637. Epub 2020 Oct 12.

Faculty of Dentistry, McGill University, Montreal, Québec, Canada; Department of Medicine, McGill University, Montreal, Québec, Canada; Shriners Hospital for Children, Montreal, Quebec, Canada. Electronic address:

Medial calcification has been associated with diabetes, chronic kidney disease, and genetic disorders like pseudoxanthoma elasticum. Recently, we showed that genetic reduction of arterial elastin content reduces the severity of medial calcification in matrix Gla protein (MGP)-deficient and Eln haploinsufficient Mgp-/-;Eln+/- mice. This study suggests that there might be a direct effect of elastin amount on medial calcification. We studied this using novel in vitro systems, which are based on elastin or elastin-like polypeptides. We first examined the mineral deposition properties of a transfected pigmented epithelial cell line that expresses elastin and other elastic lamina proteins. When grown in inorganic phosphate-supplemented medium, these cells deposited calcium phosphate minerals, which could be prevented by an N'-terminal peptide of MGP (m3pS) carrying phosphorylated serine residues. We next confirmed these findings using a cell-free elastin-like polypeptide (ELP) scaffold, where the peptide prevented mineral maturation. Overall, this work describes a novel cell culture model for elastocalcinosis and examines the inhibition of mineral deposition by the m3pS peptide in this and a cell-free elastin-based scaffold. Our study provides strong evidence suggesting the critical functional roles of MGP's phosphorylated serine residues in the prevention of elastin calcification and proposes a possible mechanism of their action.
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http://dx.doi.org/10.1016/j.jsb.2020.107637DOI Listing
March 2021

What is the Role of Lipid Membrane-embedded Quinones in Mitochondria and Chloroplasts? Chemiosmotic Q-cycle versus Murburn Reaction Perspective.

Cell Biochem Biophys 2021 Mar 29;79(1):3-10. Epub 2020 Sep 29.

Department of Biotechnology, Vignan's Foundation for Science, Technology & Research, Vadlamudi, Guntur, 522213, India.

Quinones are found in the lipid membranes of prokaryotes like E. coli and cyanobacteria, and are also abundant in eukaryotic mitochondria and chloroplasts. They are intricately involved in the reaction mechanism of redox phosphorylations. In the Mitchellian chemiosmotic school of thought, membrane-lodged quinones are perceived as highly mobile conveyors of two-electron equivalents from the first leg of Electron Transport Chain (ETC) to the 'second pit-stop' of Cytochrome bc or bf complex (CBC), where they undergo a regenerative 'Q-cycle'. In Manoj's murburn mechanism, the membrane-lodged quinones are perceived as relatively slow-moving one- or two- electron donors/acceptors, enabling charge separation and the CBC resets a one-electron paradigm via 'turbo logic'. Herein, we compare various purviews of the two mechanistic schools with respect to: constraints in mobility, protons' availability, binding of quinones with proteins, structural features of the protein complexes, energetics of reaction, overall reaction logic, etc. From various perspectives, the murburn mechanism appeals as a viable alternative explanation well-rooted in thermodynamics/kinetics and one which lends adequate structure-function correlations for the roles of quinones, lipid membrane and associated proteins.
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http://dx.doi.org/10.1007/s12013-020-00945-yDOI Listing
March 2021

Acute toxicity of cyanide in aerobic respiration: Theoretical and experimental support for murburn explanation.

Biomol Concepts 2020 Mar 17;11(1):32-56. Epub 2020 Mar 17.

Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, India-781039.

The inefficiency of cyanide/HCN (CN) binding with heme proteins (under physiological regimes) is demonstrated with an assessment of thermodynamics, kinetics, and inhibition constants. The acute onset of toxicity and CN's mg/Kg LD50 (μM lethal concentration) suggests that the classical hemeFe binding-based inhibition rationale is untenable to account for the toxicity of CN. In vitro mechanistic probing of CN-mediated inhibition of hemeFe reductionist systems was explored as a murburn model for mitochondrial oxidative phosphorylation (mOxPhos). The effect of CN in haloperoxidase catalyzed chlorine moiety transfer to small organics was considered as an analogous probe for phosphate group transfer in mOxPhos. Similarly, inclusion of CN in peroxidase-catalase mediated one-electron oxidation of small organics was used to explore electron transfer outcomes in mOxPhos, leading to water formation. The free energy correlations from a Hammett study and IC50/Hill slopes analyses and comparison with ligands ( CO/ H 2 S/ N 3 - ) $\left( {\text{CO}}/{{{{\text{H}}_{2}}\text{S}}/{\text{N}_{3}^{\text{-}}}\;}\; \right)$ provide insights into the involvement of diffusible radicals and proton-equilibriums, explaining analogous outcomes in mOxPhos chemistry. Further, we demonstrate that superoxide (diffusible reactive oxygen species, DROS) enables in vitro ATP synthesis from ADP+phosphate, and show that this reaction is inhibited by CN. Therefore, practically instantaneous CN ion-radical interactions with DROS in matrix catalytically disrupt mOxPhos, explaining the acute lethal effect of CN.
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http://dx.doi.org/10.1515/bmc-2020-0004DOI Listing
March 2020

Acute toxicity of cyanide in aerobic respiration: Theoretical and experimental support for murburn explanation.

Biomol Concepts 2020 03 17;11(1):32-56. Epub 2020 Mar 17.

Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, India-781039.

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http://dx.doi.org/10.1515/bmc-2020-0004DOI Listing
March 2020

Chemiosmotic and murburn explanations for aerobic respiration: Predictive capabilities, structure-function correlations and chemico-physical logic.

Arch Biochem Biophys 2019 11 14;676:108128. Epub 2019 Oct 14.

Institute of Chemical Kinetics and Combustion, Russian Academy of Sciences, St. Institutskaya 3, 630090, Novosibirsk, Russia. Electronic address:

Since mid-1970s, the proton-centric proposal of 'chemiosmosis' became the acclaimed explanation for aerobic respiration. Recently, significant theoretical and experimental evidence were presented for an oxygen-centric 'murburn' mechanism of mitochondrial ATP-synthesis. Herein, we compare the predictive capabilities of the two models with respect to the available information on mitochondrial reaction chemistry and the membrane proteins' structure-function correlations. Next, fundamental queries are addressed on thermodynamics of mitochondrial oxidative phosphorylation (mOxPhos): (1) Can the energy of oxygen reduction be utilized for proton transport? (2) Is the trans-membrane proton differential harness-able as a potential energy capable of doing useful work? and (3) Whether the movement of miniscule amounts of mitochondrial protons could give rise to a potential of ~200 mV and if such an electrical energy could sponsor ATP-synthesis. Further, we explore critically if rotary ATPsynthase activity of Complex V can account for physiological ATP-turnovers. We also answer the question- "What is the role of protons in the oxygen-centric murburn scheme of aerobic respiration?" Finally, it is demonstrated that the murburn reaction model explains the fast kinetics, non-integral stoichiometry and high yield of mOxPhos. Strategies are charted to further demarcate the two explanations' relevance in the cellular physiology of aerobic respiration.
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http://dx.doi.org/10.1016/j.abb.2019.108128DOI Listing
November 2019

Aerobic respiration: proof of concept for the oxygen-centric murburn perspective.

J Biomol Struct Dyn 2019 10 8;37(17):4542-4556. Epub 2019 Jan 8.

Department of Biotechnology, Indian Institute of Technology Guwahati , Guwahati , Assam, India.

The inner mitochondrial membrane protein complexes (I-V) and prokaryotic respiratory machinery are examined for a deeper understanding of their structure-function correlations and dynamics. analysis of the structure of complexes I-IV, docking studies and erstwhile literature confirm that they carry sites which are in close proximity to DROS (diffusible reactive oxygen species) generating redox centers. These findings provide supportive evidence for the newly proposed oxygen-centric chemical-coupling mechanism (murburn concept), wherein DROS catalyzes the esterification of inorganic phosphate to ADP. Further, in a reductionist system, we demonstrate that a DROS (like superoxide) can effectively esterify inorganic phosphate to ADP. The impact of these findings and the interactive dynamics of classical inhibitors (rotenone and cyanide), uncouplers (dinitrophenol and uncoupling protein) and other toxins (atractyloside and oligomycin) are briefly discussed. • Earlier perception: Complexes (I-IV) pump protons and Complex V make ATP (aided by protons) • Herein: Respiratory molecular machinery is probed for new structure-function correlations • Analyses: Quantitative arguments discount proton-centric ATP synthesis in mitochondria and bacteria • data: ADP-binding sites and O/ diffusible reactive oxygen species (DROS)-accessible channels are unveiled in respiratory proteins • data: Using luminometry, ATP synthesis is demonstrated from ADP, Pi and superoxide • Inference: Findings agree with decentralized ADP-Pi activation via oxygen-centric murburn scheme Communicated by Ramaswamy H. Sarma.
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http://dx.doi.org/10.1080/07391102.2018.1552896DOI Listing
October 2019

Murburn Concept: A Molecular Explanation for Hormetic and Idiosyncratic Dose Responses.

Dose Response 2018 Apr-Jun;16(2):1559325818774421. Epub 2018 May 9.

Satyamjayatu: The Science and Ethics Foundation, Kerala, India.

Recently, electron transfers and catalyses in a bevy of redox reactions mediated by hemeproteins were explained by murburn concept. The term "murburn" is abstracted from "" or "" and connotes a novel "" interaction paradigm. Quite unlike the genetic regulations and protein-level affinity-based controls that govern order and specificity/selectivity in conventional treatments, murburn concept is based on stochastic/thermodynamic regulatory principles. The novel insight necessitates a "reactivity outside the active-site" perspective, because select redox enzymatic activity is obligatorily mediated via diffusible radical/species. Herein, reactions employing key hemeproteins (as exemplified by CYP2E1) establish direct experimental connection between "additive-influenced redox catalysis" and "unusual dose responses" in reductionist and physiological milieu. Thus, direct and conclusive molecular-level experimental evidence is presented, supporting the mechanistic relevance of murburn concept in "maverick" concentration-based effects brought about by additives. Therefore, murburn concept could potentially explain several physiological hormetic and idiosyncratic dose responses.
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http://dx.doi.org/10.1177/1559325818774421DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5946624PMC
May 2018

The stability and fate of synthesized zero-valent iron nanoparticles in freshwater microcosm system.

3 Biotech 2017 Jul 5;7(3):227. Epub 2017 Jul 5.

Centre for Nanobiotechnology, VIT University, Vellore, 632014, India.

Zero-valent iron nanoparticles are used for the degradation of organic compounds and the immobilization of metals and metalloids. The lack of information on the effect of nZVI in freshwater system necessitated the risk assessment of zero-valent iron nanoparticles in lake water environment. The present study deals with the stability and fate of synthesized zero-valent iron nanoparticles in the upper and lower layers of freshwater microcosm system at a concentration of 1000 mg L. The study was divided into two different exposure periods: short-term exposure, up to 24 h after the introduction of nanoparticles, and long-term exposure period up to 180 days (4416 h). Aggregation kinetics of nZVI in freshwater microcosm was studied by measuring the mean hydrodynamic size of the nanoparticles with respect to time. A gradual increase in the particle size with time was observed up to 14 h. The algal population and total chlorophyll content declined for the short exposure period, i.e., 2-24 h, while in the case of longer exposure period, i.e., 24 h to 180 days (4416 h), a gradual increase of both the algal population and total chlorophyll was noted. Five different physico-chemical parameters such as pH, temperature, conductivity, salinity, and total dissolved solids were recorded for 180 days (6 calendar months). The study suggested that the nanoscale zero-valent iron did not exhibit significant toxicity at an exposure concentration of 1000 mg L on the resident algal population in the microcosm system over the longer exposure period tested.
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http://dx.doi.org/10.1007/s13205-017-0869-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5498368PMC
July 2017

Toxicity assessment of zero valent iron nanoparticles on Artemia salina.

Environ Toxicol 2017 May 19;32(5):1617-1627. Epub 2017 Jan 19.

Centre for Nanobiotechnology, VIT University, Vellore, India.

The present study deals with the toxicity assessment of two differently synthesized zero valent iron nanoparticles (nZVI, chemical and biological) as well as Fe ions on Artemia salina at three different initial concentrations of 1, 10, and 100 mg/L of these particles. The assessment was done till 96 h at time intervals of 24 h. EC value was calculated to evaluate the 50% mortality of Artemia salina at all exposure time durations. Between chemically and biologically synthesized nZVI nanoparticles, insignificant differences in the level of mortality were demonstrated. At even 24 h, Fe ion imparted complete lethality at the highest exposure concentration (100 mg/L). To understand intracellular oxidative stress because of zero valent iron nanoparticles, ROS estimation, SOD activity, GSH activity, and catalase activity was performed which demonstrated that ionic form of iron is quite lethal at high concentrations as compared with the same concentration of nZVI exposure. Lower concentrations of nZVI were more toxic as compared with the ionic form and was in order of CS-nZVI > BS-nZVI > Fe . Cell membrane damage and bio-uptake of nanoparticles were also evaluated for all three concentrations of BS-nZVI, CS-nZVI, and Fe using adult Artemia salina in marine water; both of which supported the observations made in toxicity assessment. This study can be further explored to exploit Artemia salina as a model organism and a biomarker in an nZVI prone aquatic system to detect toxic levels of these nanoparticles. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1617-1627, 2017.
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http://dx.doi.org/10.1002/tox.22389DOI Listing
May 2017

Toxicity, accumulation, and trophic transfer of chemically and biologically synthesized nano zero valent iron in a two species freshwater food chain.

Aquat Toxicol 2017 Feb 21;183:63-75. Epub 2016 Dec 21.

Centre for Nanobiotechnology, VIT University, Vellore, India. Electronic address:

The impact of bio-remediation agent nZVI on environment is still inadequately understood, especially on aquatic food web. The study presented here has therefore considered both chemical (CS) and biological (BS) synthetic origins of nZVI and their effects on both algae and daphnia. The study is unique in its attempt to explore the possibility of trophic transfer from algae to its immediate higher niche (daphnia as the model). An equal weightage of the effects of both CS and BS nZVI on algae and daphnia has been explored here; hence it allows us to compare the capping of nZVI on toxicity. To examine the causes of observed lethality- ROS generation, effects on the activity of oxidative enzymes, membrane damage and biouptake of nZVI was analysed. The overall outcome of CS and BS nZVI on lethality was significantly different in algae and daphnia, where daphnia demonstrated relatively higher sensitivity against CS nZVI. Algae demonstrated considerable differences in CS and BS nZVI toxicity only at higher concentration. This study did not show a probable biomagnification and trophic transfer from algae to daphnia under the experimental conditions even at the highest exposure concentration. The study instigates the importance of trophic transfer to understand the possible biomagnification of nZVI among organisms of different trophic levels and eventually the consequences on environment.
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http://dx.doi.org/10.1016/j.aquatox.2016.12.013DOI Listing
February 2017

Modulatory effects of Zn ions on the toxicity of citrate- and PVP-capped gold nanoparticles towards freshwater algae, Scenedesmus obliquus.

Environ Sci Pollut Res Int 2017 Feb 28;24(4):3790-3801. Epub 2016 Nov 28.

Centre for Nanobiotechnology, VIT University, Vellore, 632014, India.

Gold nanoparticles (GNPs) are widely used for medical purposes, both in diagnostics as well as drug delivery, and hence are prone to release and distribution in the environment. Thus, we have explored the effects of GNPs with two distinct surface capping (citrate and PVP), and three different sizes (16, 27, and 37 nm) at 0.01-, 0.1-, and 1-mg L concentrations on a predominant freshwater alga Scenedesmus obliquus in the sterile freshwater matrix. We have also investigated how an abundant metal ion from freshwater, i.e., Zn ions may modulate the effects of the selected GNPs (40 nm, citrate, and PVP capped). Preliminary toxicity results revealed that gold nanoparticles were highly toxic in comparison to zinc ions alone. A significant modulation in the toxicity of Zn ions was not noticed in the presence of GNPs. In contrast, zinc ions minimized the toxicity produced by GNPs (both CIT-37 and PVP-37), despite its individual toxicity. Approximately, about 42, 33, and 25% toxicity reduction was noted at 0.05-, 0.5-, and 5-mg L Zn ions, respectively, for CIT-37 GNPs, while 31% (0.05 mg L), 24% (0.5 mg L), and 9% (5 mg L) of toxicity reduction were noted for PVP-37 GNPs. Maximum toxicity reduction was seen at 0.05 mg L of Zn ions. Abbott modeling substantiated antagonistic effects offered by Zn ions on GNPs. Stability and sedimentation data revealed that the addition of zinc ions gradually induced the aggregation of NPs and in turn significantly reduced the toxicity of GNPs. Thus, the naturally existing ions like Zn have an ability to modulate the toxicity of GNPs in a real-world environment scenario.
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http://dx.doi.org/10.1007/s11356-016-8131-xDOI Listing
February 2017

Functioning of Microsomal Cytochrome P450s: Murburn Concept Explains the Metabolism of Xenobiotics in Hepatocytes.

Front Pharmacol 2016 23;7:161. Epub 2016 Jun 23.

REDOx Lab, PSG Institute of Advanced Studies Coimbatore, India.

Using oxygen and NADPH, the redox enzymes cytochrome P450 (CYP) and its reductase (CPR) work in tandem to carry out the phase I metabolism of a vast majority of drugs and xenobiotics. As per the erstwhile understanding of the catalytic cycle, binding of the substrate to CYP's heme distal pocket allows CPR to pump electrons through a CPR-CYP complex. In turn, this trigger (a thermodynamic push of electrons) leads to the activation of oxygen at CYP's heme-center, to give Compound I, a two-electron deficient enzyme reactive intermediate. The formation of diffusible radicals and reactive oxygen species (DROS, hitherto considered an undesired facet of the system) was attributed to the heme-center. Recently, we had challenged these perceptions and proposed the murburn ("mured burning" or "mild unrestricted burning") concept to explain heme enzymes' catalytic mechanism, electron-transfer phenomena and the regulation of redox equivalents' consumption. Murburn concept incorporates a one-electron paradigm, advocating obligatory roles for DROS. The new understanding does not call for high-affinity substrate-binding at the heme distal pocket of the CYP (the first and the most crucial step of the erstwhile paradigm) or CYP-CPR protein-protein complexations (the operational backbone of the erstwhile cycle). Herein, the dynamics of reduced nicotinamide nucleotides' consumption, peroxide formation and depletion, product(s) formation, etc. was investigated with various controls, by altering reaction variables, environments and through the incorporation of diverse molecular probes. In several CYP systems, control reactions lacking the specific substrate showed comparable or higher peroxide in milieu, thereby discrediting the foundations of the erstwhile hypothesis. The profiles obtained by altering CYP:CPR ratios and the profound inhibitions observed upon the incorporation of catalytic amounts of horseradish peroxidase confirm the obligatory roles of DROS in milieu, ratifying murburn as the operative concept. The mechanism of uncoupling (peroxide/water formation) was found to be dependent on multiple one and two electron equilibriums amongst the reaction components. The investigation explains the evolutionary implications of xenobiotic metabolism, confirms the obligatory role of diffusible reactive species in routine redox metabolism within liver microsomes and establishes that a redox enzyme like CYP enhances reaction rates (achieves catalysis) via a novel (hitherto unknown) modality.
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http://dx.doi.org/10.3389/fphar.2016.00161DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4918403PMC
July 2016

Differential effects of P25 TiO2 nanoparticles on freshwater green microalgae: Chlorella and Scenedesmus species.

Aquat Toxicol 2016 Jul 27;176:161-71. Epub 2016 Apr 27.

Centre for Nanobiotechnology, VIT University, Vellore, India. Electronic address:

P25 TiO2 nanoparticles majorly used in cosmetic products have well known detrimental effects towards the aquatic environment. In a freshwater ecosystem, Chlorella and Scenedesmus are among the most commonly found algal species frequently used to study the effects of metal oxide nanoparticles. A comparative study has been conducted herein to investigate differences in the toxic effects caused by these nanoparticles towards the two algae species. The three different concentrations of P25 TiO2 NPs (0.01, 0.1 & 1μg/mL, i.e., 0.12, 1.25 and 12.52μM) were selected to correlate surface water concentrations of the nanoparticles, and filtered and sterilized fresh water medium was used throughout this study. There was significant increase (p<0.001) in hydrodynamic diameter of nanoparticles with respect to both, time (0, 24, 48 and 72h) as well as concentration under all the exposure conditions. Although, significant dose-dependent morphological (surface area & biovolume) interspecies variations were not observed, it was evident at the highest concentration of exposure within individuals. At 1μg/mL exposure concentration, a significant difference in toxicity was noted between Chlorella and Scenedesmus under only visible light (p<0.001) and UVA (p<0.01) irradiation conditions. The viability data were well supported by the results obtained for oxidative stress induced by NPs on the cells. At the highest exposure concentration, superoxide dismutase and reduced glutathione activities were assessed for both the algae under all the irradiation conditions. Increased catalase activity and LPO release complemented the cytotoxic effects observed. Significant interspecies variations were noted for these parameters under UVA and visible light exposed cells of Chlorella and Scenedesmus species, which could easily be correlated with the uptake of the NPs.
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http://dx.doi.org/10.1016/j.aquatox.2016.04.021DOI Listing
July 2016

Atypical profiles and modulations of heme-enzymes catalyzed outcomes by low amounts of diverse additives suggest diffusible radicals' obligatory involvement in such redox reactions.

Biochimie 2016 Jun 10;125:91-111. Epub 2016 Mar 10.

CIDR-MCBL, Indian Institute of Science, Bengaluru, Karnataka 560012, India; Hemoproteins Lab, School of Bio Sciences and Technology, VIT University, Vellore, Tamil Nadu 632014, India. Electronic address:

Background: Peroxidations mediated by heme-enzymes have been traditionally studied under a single-site (heme distal pocket), non-sequential (ping-pong), two-substrates binding scheme of Michaelis-Menten paradigm. We had reported unusual modulations of peroxidase and P450 reaction outcomes and explained it invoking diffusible reactive species [Manoj, 2006; Manoj et al., 2010; Andrew et al., 2011, Parashar et al., 2014 & Venkatachalam et al., 2016].

Methods: A systematic investigation of specific product formation rates was undertaken to probe the hypothesis that involvement of diffusible reactive species could explain undefined substrate specificities and maverick modulations (sponsored by additives) of heme-enzymes.

Results: When the rate of specific product formation was studied as a function of reactants' concentration or environmental conditions, we noted marked deviations from normal profiles. We report that heme-enzyme mediated peroxidations of various substrates are inhibited (or activated) by sub-equivalent concentrations of diverse redox-active additives and this is owing to multiple redox equilibriums in the milieu. At low enzyme and peroxide concentrations, the enzyme is seen to recycle via a one-electron (oxidase) cycle, which does not require the substrate to access the heme centre. Schemes are provided that explain the complex mechanistic cycle, kinetics & stoichiometry.

Conclusion: It is not obligatory for an inhibitor or substrate to interact with the heme centre for influencing overall catalysis. Roles of diffusible reactive species explain catalytic outcomes at low enzyme and reactant concentrations.

Significance: The current work highlights the scope/importance of redox enzyme reactions that could occur "out of the active site" in biological or in situ systems.
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http://dx.doi.org/10.1016/j.biochi.2016.03.003DOI Listing
June 2016

Differential toxicity of Al2O3 particles on Gram-positive and Gram-negative sediment bacterial isolates from freshwater.

Environ Sci Pollut Res Int 2016 Jun 11;23(12):12095-106. Epub 2016 Mar 11.

Centre for Nanobiotechnology, VIT University, Vellore, 632014, India.

The current study was aimed to explore the differential effects on Gram-positive and Gram-negative freshwater sediment bacterial isolates upon exposure to nano-particles and bulk particles of Al2O3 at low concentrations (0.25, 0.5, and 1 mg/L). The Gram-negative Pseudomonas aeruginosa was more susceptible to both the nano-forms and bulk forms than the Gram-positive Bacillus altitudinis. The generation of reactive oxygen species (ROS) and release of lipopolysaccharide due to membrane damage were dependent on the dose of nano-Al2O3. The Fourier transform infrared spectroscopy (FT-IR) studies confirmed the attachment of nano-Al2O3 on bacterial cells, which may lead to subsequent changes in the cell membrane composition and integrity. Internalization of nano-Al2O3 was estimated to be more for P. aeruginosa than for B. altitudinis cells. As a role of defense mechanism, the biofilm formation and production of extracellular polymeric substances (EPSs; polysaccharide and protein) were increased with respect to the concentration of toxicant. Nano-Al2O3 was estimated to cause more DNA damage than the bulk particles in both Gram-positive and Gram-negative bacterial strains.
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http://dx.doi.org/10.1007/s11356-016-6407-9DOI Listing
June 2016

Functioning of drug-metabolizing microsomal cytochrome P450s: In silico probing of proteins suggests that the distal heme 'active site' pocket plays a relatively 'passive role' in some enzyme-substrate interactions.

In Silico Pharmacol 2016 Dec 19;4(1). Epub 2016 Feb 19.

Formerly at PSG Institute of Advanced Studies, Avinashi Road, Peelamedu, Coimbatore, Tamil Nadu, 641004, India.

Purpose: The currently held mechanistic understanding of microsomal cytochrome P450s (CYPs) seeks that diverse drug molecules bind within the deep-seated distal heme pocket and subsequently react at the heme centre. To explain a bevy of experimental observations and meta-analyses, we indulge a hypothesis that involves a "diffusible radical mediated" mechanism. This new hypothesis posits that many substrates could also bind at alternate loci on/within the enzyme and be reacted without the pertinent moiety accessing a bonding proximity to the purported catalytic Fe-O enzyme intermediate.

Methods: Through blind and heme-distal pocket centered dockings of various substrates and non-substrates (drug molecules of diverse sizes, classes, topographies etc.) of microsomal CYPs, we explored the possibility of access of substrates via the distal channels, its binding energies, docking orientations, distance of reactive moieties (or molecule per se) to/from the heme centre, etc. We investigated specific cases like- (a) large drug molecules as substrates, (b) classical marker drug substrates, (c) class of drugs as substrates (Sartans, Statins etc.), (d) substrate preferences between related and unrelated CYPs, (e) man-made site-directed mutants' and naturally occurring mutants' reactivity and metabolic disposition, (f) drug-drug interactions, (g) overall affinities of drug substrate versus oxidized product, (h) meta-analysis of in silico versus experimental binding constants and reaction/residence times etc.

Results: It was found that heme-centered dockings of the substrate/modulator drug molecules with the available CYP crystal structures gave poor docking geometries and distances from Fe-heme centre. In conjunction with several other arguments, the findings discount the relevance of erstwhile hypothesis in many CYP systems. Consequently, the newly proposed hypothesis is deemed a viable alternate, as it satisfies Occam's razor.

Conclusions: The new proposal affords expanded scope for explaining the mechanism, kinetics and overall phenomenology of CYP mediated drug metabolism. It is now understood that the heme-iron and the hydrophobic distal pocket of CYPs serve primarily to stabilize the reactive intermediate (diffusible radical) and the surface or crypts of the apoprotein bind to the xenobiotic substrate (and in some cases, the heme distal pocket could also serve the latter function). Thus, CYPs enhance reaction rates and selectivity/specificity via a hitherto unrecognized modality.
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http://dx.doi.org/10.1186/s40203-016-0016-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4760962PMC
December 2016

Decreased Phototoxic Effects of TiO₂ Nanoparticles in Consortium of Bacterial Isolates from Domestic Waste Water.

PLoS One 2015 23;10(10):e0141301. Epub 2015 Oct 23.

Centre for Nanobiotechnology, VIT University, Vellore, India.

This study is aimed to explore the toxicity of TiO2 nanoparticles at low concentrations (0.25, 0.50 & 1.00 μg/ml); on five bacterial isolates and their consortium in waste water medium both in dark and UVA conditions. To critically examine the toxic effects of nanoparticles and the response mechanism(s) offered by microbes, several aspects were monitored viz. cell viability, ROS generation, SOD activity, membrane permeability, EPS release and biofilm formation. A dose and time dependent loss in viability was observed for treated isolates and the consortium. At the highest dose, after 24h, oxidative stress was examined which conclusively showed more ROS generation & cell permeability and less SOD activity in single isolates as compared to the consortium. As a defense mechanism, EPS release was enhanced in case of the consortium against the single isolates, and was observed to be dose dependent. Similar results were noticed for biofilm formation, which substantially increased at highest dose of nanoparticle exposure. Concluding, the consortium showed more resistance against the toxic effects of the TiO2 nanoparticles compared to the individual isolates.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0141301PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4619802PMC
June 2016

Cyanide does more to inhibit heme enzymes, than merely serving as an active-site ligand.

Biochem Biophys Res Commun 2014 Dec 6;455(3-4):190-3. Epub 2014 Nov 6.

REDOx Lab, PSG Institute of Advanced Studies, Avinashi Road, Peelamedu, Coimbatore, Tamil Nadu, 641004, India. Electronic address:

The toxicity of cyanide is hitherto attributed to its ability to bind to heme proteins' active site and thereby inhibit their activity. It is shown herein that the long-held interpretation is inadequate to explain several observations in heme-enzyme reaction systems. Generation of cyanide-based diffusible radicals in heme-enzyme reaction milieu could shunt electron transfers (by non-active site processes), and thus be detrimental to the efficiency of oxidative outcomes.
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http://dx.doi.org/10.1016/j.bbrc.2014.10.137DOI Listing
December 2014

The curious case of benzbromarone: insight into super-inhibition of cytochrome P450.

PLoS One 2014 3;9(3):e89967. Epub 2014 Mar 3.

REDOx Lab, Biotechnology and Healthcare Division, PSG Institute of Advanced Studies, Peelamedu, Tamil Nadu, India.

Cytochrome P450 (CYP) family of redox enzymes metabolize drugs and xenobiotics in liver microsomes. Isozyme CYP2C9 is reported to be inhibited by benzbromarone (BzBr) and this phenomenon was hitherto explained by classical active-site binding. Theoretically, it was impossible to envisage the experimentally derived sub-nM Ki for an inhibitor, when supra-nM enzyme and 10X KM substrate concentrations were employed. We set out to find a more plausible explanation for this highly intriguing "super-inhibition" phenomenon. In silico docking of various BzBr analogs with known crystal structure of CYP2C9 did not provide any evidence in support of active-site based inhibition hypothesis. Experiments tested the effects of BzBr and nine analogs on CYPs in reconstituted systems of lab-purified proteins, complex baculosomes & crude microsomal preparations. In certain setups, BzBr and its analogs could even enhance reactions, which cannot be explained by an active site hypothesis. Generally, it was seen that Ki became smaller by orders of magnitude, upon increasing the dilution order of BzBr analogs. Also, it was seen that BzBr could also inhibit other CYP isozymes like CYP3A4, CYP2D6 and CYP2E1. Further, amphipathic derivatives of vitamins C & E (scavengers of diffusible reactive oxygen species or DROS) effectively inhibited CYP2C9 reactions in different reaction setups. Therefore, the inhibition of CYP activity by BzBr analogs (which are also surface-active redox agents) is attributed to catalytic scavenging of DROS at phospholipid interface. The current work expands the scope of interpretations of inhibitions in redox enzymes and ushers in a new cellular biochemistry paradigm that small amounts of DROS may be obligatorily required in routine redox metabolism for constructive catalytic roles.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0089967PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3940698PMC
January 2015

Traces of certain drug molecules can enhance heme-enzyme catalytic outcomes.

Biochem Biophys Res Commun 2012 Jan 26;417(3):1041-5. Epub 2011 Dec 26.

Heme & Flavo Proteins Laboratory, Center for Biomedical Research, VIT University, Vellore, Tamil Nadu, India.

We report that trace amounts (nano- to picomolar concentrations) of dapsone and amiodarone in reaction mixtures of structurally and functionally distinct heme enzymes gave increased product formation rates from diverse substrates. These enhancements are found to be lowered by mild radical quenchers; a lowering well differentiated from heme active site inhibitions. Based on the non-specific nature of the activations in the diverse heme enzyme reactions studied, a generic mechanism is proposed to explain additive-based activity enhancements in heme enzymology.
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http://dx.doi.org/10.1016/j.bbrc.2011.12.090DOI Listing
January 2012
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