Nanotoxicity Mechanism Of CoFe₂O₄ NPs In Aquatic Organisms And Model Proteins

The broad spectrum applications of Co Fe2O4 NPs have attracted much interest in medicine, environment and industry, resulting in exceedingly higher exposures to humans and environmental systems in succeeding days. However, their potential negative health and biological impacts need to be assessed for environment, health and safety risk management. In this contribution, potential of oxidative stress, genotoxicity and endocrine disruptions mediated by cobalt ferrite NPs as well as possible interference with assay techniques were also reported. Environmental fate and behavior of Cobalt ferrite NPs, such as environmental degradation and release of ions(in presence and absence of microorganisms) from NPs, interaction with microorganism as well as natural self defense response of model organism in presence of NPs have reported. All the above paradigms were explained by exploiting the Zebra fish and Chlorella vulgaris as model organisms. Co Fe2O4 NPs upon interaction with biological molecules drive the transformations by altering the natural conformations and by adsorption of bio-molecules on NPs surface. Furthermore, evaluation of thermodynamics and extent of protein corona(adsorption) formation by possible interactions of NPs with Bovine serum albumin and Acid Phosphatase at the physiological p H of 7.4 was performed. These physical and chemical perturbations in proteins were used as an advance tool in bio-sensing the presence of NPs in environmental compartments.Zebra fish(Danio rerio) embryos were exposed to environmentally relevant doses of nano-Co Fe2O4(mean diameter of 40 nm with a concentration range of 10-500μM) for 96 and 168 hrs. Dose and time dependent developmental toxicity with severe cardiac edema, down regulation of metabolism, hatching delay and tail/ spinal cord flexure and apoptosis was observed for 96 and 168 hours post fertilization(HPF). The biochemical changes were triggered by excessive ROS production with increased apoptosis on head heart and tail region along with DNA and metabolic alterations at lower concentrations of Co Fe2O4 NPs. The extent of thyroid endocrine disruption at 168 hrs of exposure, agglomeration of NPs and dissolution of ions induces severe mechanical damage to membranes, oxidative stress and physiological structure of thyroid axis. The results also showed the elevated amounts of T4 and T3 hormones by malformation of hypothalamus pituitary axis in zebra fish larvae. These elevated levels of whole body THs leads to delayed hatching, head and eye malformation, arrested development and alterations in metabolism. The excessive production of in vivo ROS leads to severe apoptosis in head, eye and heart region confirming the malformation leads to malfunctioning of hypothalamus pituitary axis. In addition, ROS induced oxidative DNA damage by formation of 8-OHd G DNA adducts elaborates the genotoxicity potential of Co Fe2O4 NPs.The environmental fate and potential toxicity of Co Fe2O4 NPs in Chlorella vulgaris was also observed up to 72 h. Algal cell morphology, membrane integrity and viability were severely compromised due to adsorption and aggregation of NPs on algal surfaces, release of Fe3+ and Co2+ ions and possible mechanical damage by NPs. Interactions with NPs and effective decrease in ions released by aggregation and exudation of algal cells as a self defense mechanism were observed. The results corroborated Co Fe2O4 NPs induced ROS triggered oxidative stress, leading to a reduction in Catalase activity, activation of the mu-GST and AP antioxidant enzymes, and an increase in genetic aberrations, metabolic and cellular signal transduction dysfunction. Possible interferences of Co Fe2O4 NPs with assay techniques and components indicated Co Fe2O4 NPs at lower concentration do not show any significant interference with ROS, Catalase and mu-GST. This confirms the above results that ROS production is one of the pathways of toxicity initiated by Co Fe2O4 NPs and demonstrates the complex processes that may occur between organisms and NPs in natural complex ecosystem.The potential of NPs persuaded changes in protein structure and functionality was demonstrated by investigating the interaction as well as adsorption of bovine serum albumin(BSA) and acid phosphatase(AP), with various analytical and spectroscopic techniques. The results corroborate, the Co Fe2O4 NPs cause fluorescence quenching in BSA and AP by static quenching mechanism. The negative values of van’t Hoff thermodynamic expressions(ΔH and ΔG) corroborate the spontaneity and exothermic nature of static quenching. The negative and positive value of ΔS corroborate that major contributors in higher and stronger binding affinities among Co Fe2O4 NPs with BSA and AP were Vander Waal as well as hydrogen bonding and electrostatic forces, respectively. Furthermore, TGA, DLS and zeta potential results confirmed the formation of thick layer of BSA and AP on Co Fe2O4 NPs with negative boost in zeta potential. This coating of BSA and AP over Co Fe2O4 NPs leads to decrease in magnetic saturation value from 50.4 to 46.2 and 45.5 emu respectively, hence the magnetic character of Co Fe2O4 NPs. In addition, FTIR, UV-CD, UV–vis spectroscopy and three dimensional fluorescence(3D) techniques confirmed that Co Fe2O4 NPs binding could induce microenvironment perturbations leading to secondary and tertiary conformation changes in BSA and AP. Furthermore, synchronous fluorescence spectroscopy(SFS) affirmed the significant changes in microenvironment around tryptophan(Trp) residue of BSA and AP by Co Fe2O4 NPs. The denaturizing of BSA and AP biochemistry by Co Fe2O4 NPs was advanced by assaying esterase and phosphatase activities measurement respectively.The exploitation of alteration in biochemical activity in biosensing the presence of NPs in environmental compartments was explored by observing the activation kinetics in Chlorella vulgaris. Apparent Michaelis constant(Km) values of 0.57 and 26.5 m M with activation energy values of 0.538 and 3.428 k J mol-1 were determined without and with 200 μM Co Fe2O4 NPs. Apparent Vmax value of-7 Umml-1 corroborate that enzyme active sites were completely captured by the NPs leaving no space for the substrate. This suggests Co Fe2O4 NPs perturb the enzyme activity by transitions in conformation and hence can be used in bio-sensing the presence of NPs in environmental compartment.This study will provide the insights of extremely toxic nature of cobalt ferrite NPs in diverse range of biological milieus. In addition, it also provides the pavement for material scientists to design NPs with improved functionality and environmental health and safety issues. It also gives a brief description to regularity agencies and authorities for developing and implementing the strict regulations with improved analytical methods and techniques in precise controlling and measuring of NPs in complex biological and environmental matrices.