Abhinav Parashar

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

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.

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

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.

The Curious Case of Benzbromarone: Insight into Super-Inhibition of Cytochrome P450

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.

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

PurposeThe 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.MethodsThrough 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.ResultsIt 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.ConclusionsThe 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.

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

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.

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

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.

A novel enzyme-mediated gold nanoparticle synthesis and its application for in situ detection of horseradish peroxidase inhibitor phenylhydrazine

The biocatalytic growth of metal nanoparticles for the detection of environmentally relevant chemical molecules is a recently exploited strategy. The present work provides a new synthesis methodology for gold nanoparticles (GNPs), which could be enzymatically modulated by horseradish peroxidase (HRP). Gold seeds (∼10 nm) were prepared initially by the reduction of HAuCl4 using a minimal concentration of NaBH4, and the further biocatalyzed GNP formation was assisted by HRP, its substrate, and hydrogen peroxide (H2O2 helps in complete formation of GNPs). Upon HRP inhibition, the enzymatic formation of GNPs was observed to decrease with increasing concentrations of phenylhydrazine (HRP inhibitor) using a simple UV-visible spectrophotometer. The current method provides an in situ enzymatic assay based on the biocatalyzed rate of GNP formation for the rapid, reproducible, and cost-effective detection of phenylhydrazine with a detection limit of 4.02 μM (range: 25–200 μM). The proposed technique was also successfully tested in real samples like drinking, tap, and garden water, emphasizing its usefulness for the direct on-site monitoring of phenylhydrazine, and the concept can be extended for the detection of other inhibitors of peroxidase also.

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

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 “ mur ed burn ing” or “ m ild u n r estricted burn ing” and connotes a novel “ m olecule- u nbound ion– r adical” 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.

Toxicity assessment of zero valent iron nanoparticles on Artemia salina

The present study deals with the toxicity assessment of two differently synthesized zero valent iron nanoparticles (nZVI, chemical and biological) as well as Fe2+ 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. EC50 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, Fe2+ 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 > Fe2+. Cell membrane damage and bio‐uptake of nanoparticles were also evaluated for all three concentrations of BS‐nZVI, CS‐nZVI, and Fe2+ 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.