Plasmodium falciparum Cysteine-Rich Protective Antigen (CyRPA) Elicits Detectable Levels of Invasion-Inhibitory Antibodies during Natural Infection in Humans.

Plasmodium falciparum cysteine-rich protective antigen (CyRPA) is a conserved component of an essential erythrocyte invasion complex (RH5/Ripr/CyRPA) and a target of potent cross-strain parasite-neutralizing antibodies. While naturally acquired human RH5 antibodies have been functionally characterized, there are no similar reports on CyRPA. Thus, we analyzed the parasite-neutralizing activity of naturally acquired human CyRPA antibodies. In this regard, CyRPA human antibodies were measured and purified from malaria-infected plasma obtained from patients in central India and analyzed for their parasite neutralizing activity via in vitro growth inhibition assays (GIA). We report that, despite being susceptible to antibodies, CyRPA is a highly conserved antigen that does not appear to be under substantial immune selection pressure, as a very low acquisition rate for anti-CyRPA antibodies was reported in malaria-exposed Indians. We demonstrate for the first time that the small amounts of natural CyRPA antibodies exhibited functional parasite-neutralizing activity and that a CyRPA-based vaccine formulation induces highly potent antibodies in rabbits. Importantly, the vaccine-induced CyRPA antibodies exhibited a robust 50% inhibitory concentration (IC50) of 21.96 µg/ml, which is comparable to the IC50 of antibodies against the leading blood-stage vaccine candidate, reticulocyte-binding-like homologous protein 5 (RH5). Our data support CyRPA as a unique vaccine target that is highly susceptible to immune attack but is highly conserved compared to other leading candidates such as MSP-1 and AMA-1, further substantiating its promise as a leading blood-stage vaccine candidate.

Graphene oxide-chloroquine nanoconjugate induce necroptotic death in A549 cancer cells through autophagy modulation.

AIM: Chloroquine (Chl) has shown its potential in cancer therapy and graphene oxide (GO) exhibited excellent tumor-targeting ability, biocompatibility and low toxicity. We have endeavored to conjugate Chl to GO sheets and investigated the nonproliferation action on A549 cell lines along with cell signaling pathways. MATERIALS & METHODS: Cellular toxicity, autophagic flux modulation and cell death mechanism induced by GO-Chl have been investigated on A549 cell lines. RESULTS & CONCLUSION: GO-Chl induces accumulation of autophagosomes (monodansylcadaverine staining, green fluorescence protein-tagged LC3 plasmid and transmission electron microscopy observations) in A549 cells through the blockade of autophagic flux that serves as scaffold for necrosome assembling and activates necroptotic cell death. GO-Chl nanoconjugate could be used as an effective cancer therapeutic agent, by targeting the autophagy necroptosis axis.

Laboratory Scale Microbial Food Chain To Study Bioaccumulation, Biomagnification, and Ecotoxicity of Cadmium Telluride Quantum Dots.

The increasing applications of engineered nanomaterials (ENMs) in consumer products warrant a careful evaluation of their trophic transfer and consequent ecological impact. In the present study, a laboratory scale aquatic microbial food chain was established using bacteria (Escherichia coli (E. coli)) as a prey and ciliated protozoan (Paramecium caudatum) as a predator organism to determine the impact of cadmium telluride quantum dots (CdTe QDs). We observed that 29% of bacterivory potential of paramecium was lost, including an ∼12 h delay in doubling time on exposure to 25 mg/L CdTe QD (∼4 nm) as compared to control. The fluorescence based stoichiometric analysis revealed that 65% of the QDs bioaccumulated when paramecia were exposed to 25 mg/L QDs at 24 h. There was a significant (p < 0.05) increase in cellular cadmium (Cd) concentration at 24 h (306 ± 192 mg/L) as compared to 1 h (152 ± 50 mg/L). Moreover, the accumulation of Cd in E. coli (147 ± 25 mg/L) at 1 h of exposure to 25 mg/L QDs transferred 1.4 times higher Cd (207 ± 24 mg/L; biomagnification factor = 1.4) to its predator, paramecium.

Bucky Tubes Induce Oxidative Stress Mediated Cell Death in Human Lung Cells.

Unique physicochemical properties of carbon nanomaterials (CNMs) have opened a new era for therapeutics and diagnosis (known as theranostics) of various diseases. This exponential increase in application makes them important for toxicology studies. The present study was aimed at exploring the toxic potential of one of the CNMs, that is, bucky tubes (BTs), in human lung adenocarcinoma (A549) cell line. BTs were characterised by electron microscopy (TEM), dynamic light scattering (DLS), Fourier transform spectroscopy (FTIR), and X-ray diffraction (XRD). Flow cytometric study showed a concentration and time dependent increase in intracellular internalization as well as reduction in cell viability upon exposure to BTs. However, a significant increase in intracellular reactive oxygen species (ROS) production was observed as evident by increased fluorescence intensity of 2',7'-dichlorofluorescein (DCF). BTs induced oxidative stress in cells as evident by depletion in glutathione with concomitant increase in lipid peroxidation with increasing concentrations. A significant increase in micronucleus formation and apoptotic cell population and loss of mitochondrial membrane potential (MMP) as compared to control were observed. Moreover, in the present study, BTs were found to be mild toxic and it is encouraging to conclude that BTs having outer diameter in the range of 7-12 nm and length 0.5-10 µm can be used for theranostics.

Multiprotein complex between the GPI-anchored CyRPA with PfRH5 and PfRipr is crucial for Plasmodium falciparum erythrocyte invasion.

Erythrocyte invasion by Plasmodium falciparum merozoites is a highly intricate process in which Plasmodium falciparum reticulocyte binding-like homologous protein 5 (PfRH5) is an indispensable parasite ligand that binds with its erythrocyte receptor, Basigin. PfRH5 is a leading blood-stage vaccine candidate because it exhibits limited polymorphisms and elicits potent strain-transcending parasite neutralizing antibodies. However, the mechanism by which it is anchored to the merozoite surface remains unknown because both PfRH5 and the PfRH5-interacting protein (PfRipr) lack transmembrane domains and GPI anchors. Here we have identified a conserved GPI-linked parasite protein, Cysteine-rich protective antigen (CyRPA) as an interacting partner of PfRH5-PfRipr that tethers the PfRH5/PfRipr/CyRPA multiprotein complex on the merozoite surface. CyRPA was demonstrated to be GPI-linked, localized in the micronemes, and essential for erythrocyte invasion. Specific antibodies against the three proteins successfully detected the intact complex in the parasite and coimmunoprecipitated the three interacting partners. Importantly, full-length CyRPA antibodies displayed potent strain-transcending invasion inhibition, as observed for PfRH5. CyRPA does not bind with erythrocytes, suggesting that its parasite neutralizing antibodies likely block its critical interaction with PfRH5-PfRipr, leading to a blockade of erythrocyte invasion. Further, CyRPA and PfRH5 antibody combinations produced synergistic invasion inhibition, suggesting that simultaneous blockade of the PfRH5-Basigin and PfRH5/PfRipr/CyRPA interactions produced an enhanced inhibitory effect. Our discovery of the critical interactions between PfRH5, PfRipr, and the GPI-anchored CyRPA clearly defines the components of the essential PfRH5 adhesion complex for P. falciparum erythrocyte invasion and offers it as a previously unidentified potent target for antimalarial strategies that could abrogate formation of the crucial multiprotein complex.

Cerium oxide nanoparticles induced toxicity in human lung cells: role of ROS mediated DNA damage and apoptosis.

Cerium oxide nanoparticles (CeO2 NPs) have promising industrial and biomedical applications. In spite of their applications, the toxicity of these NPs in biological/physiological environment is a major concern. Present study aimed to understand the molecular mechanism underlying the toxicity of CeO2 NPs on lung adenocarcinoma (A549) cells. After internalization, CeO2 NPs caused significant cytotoxicity and morphological changes in A549 cells. Further, the cell death was found to be apoptotic as shown by loss in mitochondrial membrane potential and increase in annexin-V positive cells and confirmed by immunoblot analysis of BAX, BCl-2, Cyt C, AIF, caspase-3, and caspase-9. A significant increase in oxidative DNA damage was found which was confirmed by phosphorylation of p53 gene and presence of cleaved poly ADP ribose polymerase (PARP). This damage could be attributed to increased production of reactive oxygen species (ROS) with concomitant decrease in antioxidant "glutathione (GSH)" level. DNA damage and cell death were attenuated by the application of ROS and apoptosis inhibitors N-acetyl-L-cysteine (NAC) and Z-DEVD-fmk, respectively. Our study concludes that ROS mediated DNA damage and cell cycle arrest play a major role in CeO2 NPs induced apoptotic cell death in A549 cells. Apart from beneficial applications, these NPs also impart potential harmful effects which should be properly evaluated prior to their use.

Titanium dioxide nanoparticle-induced oxidative stress triggers DNA damage and hepatic injury in mice.

BACKGROUND: The use of metal oxide nanoparticles (titanium dioxide) in consumer and industrial products improves their quality but also underscores the possible adverse effects to human and environmental health. MATERIALS & METHODS: Mice were exposed orally for 14 consecutive days and analyzed for alteration in different hepatic enzymes, histopathological changes, oxidative stress, DNA damage, tumor suppressor and proapoptotic protein expression in liver cells. RESULTS: We observed a significant alteration in the level of hepatic enzymes and liver histopathology at a dose of 100 mg/kg body weight. Significant oxidative DNA damage was observed in liver cells, which could be attributed to oxidative stress. In addition, the increased expression of p53, BAX, caspase-3 and -9 proteins and decreased expression of antiapoptotic protein Bcl-2, suggest activation of the intrinsic pathway of apoptosis. CONCLUSION: High accumulation of titanium dioxide nanoparticles in the liver tissue would cause DNA damage and apoptosis through the intrinsic pathway.

In-vivo efficacy of compliant 3D nano-composite in critical-size bone defect repair: a six month preclinical study in rabbit.

Bone defects above critical size do not heal completely by itself and thus represent major clinical challenge to reconstructive surgery. Numerous bone substitutes have already been used to promote bone regeneration, however their use, particularly for critical-sized bone defects along with their long term in vivo safety and efficacy remains a concern. The present study was designed to obtain a complete healing of critical-size defect made in the proximal tibia of New Zealand White rabbit, using nano-hydroxyapatite/gelatin and chemically carboxymethylated chitin (n-HA/gel/CMC) scaffold construct. The bone-implant interfaces and defect site healing was evaluated for a period up to 25 weeks using radiography, micro-computed tomography, fluorescence labeling, and histology and compared with respective SHAM (empty contra lateral control). The viscoelastic porous scaffold construct allows easy surgical insertion and post-operatively facilitate oxygenation and angiogenesis. Radiography of defect treated with scaffold construct suggested expedited healing at defect edges and within the defect site, unlike confined healing at edges of the SHAM sites. The architecture indices analyzed by micro-computed tomography showed a significant increase in percentage of bone volume fraction, resulted in reconciled cortico-trabecular bone formation at n-HA/gel/CMC constructs treated site (15.2% to 52.7%) when compared with respective SHAM (10.2% to 31.8%). Histological examination and fluorescence labeling revealed that the uniformly interconnected porous surface of scaffold construct enhanced osteoblasts' activity and mineralization. These preclinical data suggest that, n-HA/gel/CMC construct exhibit stimulation of bone's innate regenerative capacity, thus underscoring their use in guided bone regeneration.

Polycyclic aromatic hydrocarbons and their quinones modulate the metabolic profile and induce DNA damage in human alveolar and bronchiolar cells.

The release of particulate pollutants into the air through burning of coal, crude oil, diesel, coal tar, etc. raises concerns of potential health hazards to the exposed human population. Polycyclic aromatic hydrocarbons (PAHs) are major toxic constituents of particulate matter (PM), which upon ingestion get metabolized to even more toxic metabolites such as quinones. The PAHs levels were assessed in both respirable particulate matter (RSPM, <10µM size) and suspended particulate matter (SPM, >10µM size) of urban ambient air (UAA) and that of major contributors viz. diesel exhaust particles (DEPs) and coal tar combustions emissions (CTCE). Seven US Environmental Protection Agency (USEPA) prioritized PAHs in RSPM and 10 in SPM were detected in UAA. Ten and 15 prioritized PAHs, respectively, were also detected in diesel exhaust particles (DEP) and coal tar combustion emission (CTCE) evidencing their release in the air. These PM associated PAHs for UAA, DEP and CTCE showed significant increase (p<0.05) in mutagenicity and mammalian genotoxicity in the order CTCE>DEP>UAA. Human lung alveolar (A549) and bronchiolar (BEAS-2B) cells when treated with PAH-metabolites viz. 1,4-benzoquinone (1,4-BQ), hydroquinone (HQ), 1,2-naphthoquinone (1,2-NQ), 1,4-naphthoquinone (1,4-NQ) and 9,10-phenanthroquinone (9,10-PQ) showed metabolic modulation in these cell lines with significant depletion of principal cellular metabolites viz. NADP, uracil, asparagines, glutamine, and histidine and accumulation of di-methyl amine and beta-hydroxybutyrate, identified using (1)H NMR spectroscopy. These results suggest that PAH-quinones induce genotoxic effects by modulating the metabolic machinery inside the cells by a combined effect of oxidative stress and energy depletion. Our data for metabolic profiling of human lung cells could also help in understanding the mechanism of toxicity of other xenobiotics.

TiO(2) nanoparticles induce oxidative DNA damage and apoptosis in human liver cells.

Titanium dioxide nanoparticles (TiO(2) NPs), widely used in consumer products, paints, pharmaceutical preparations and so on, have been shown to induce cytotoxicity, genotoxicity and carcinogenic responses in vitro and in vivo. The present study revealed that TiO(2) NPs induce significant (p < 0.05) oxidative DNA damage by the Fpg-Comet assay even at 1 µg/ml concentration. A corresponding increase in the micronucleus frequency was also observed. This could be attributed to the reduced glutathione levels with concomitant increase in lipid peroxidation and reactive oxygen species generation. Furthermore, immunoblot analysis revealed an increased expression of p53, BAX, Cyto-c, Apaf-1, caspase-9 and caspase-3 and decreased the level of Bcl-2 thereby indicating that apoptosis induced by TiO(2) NPs occurs via the caspase-dependent pathway. This study systematically shows that TiO(2) NPs induce DNA damage and cause apoptosis in HepG2 cells even at very low concentrations. Hence the use of such nanoparticles should be carefully monitored.

DNA and oxidative damage induced in somatic organs and tissues of mouse by municipal sludge leachate.

Pollution by waste landfill leachate has prompted a number of studies on the toxic and potential health effects. This study assessed the genotoxicity of a municipal sludge leachate (MSL) in the somatic tissues (blood and bone marrow) and organs (liver, kidney, and spleen) of mice using the alkaline Comet assay. The possible cause of DNA damage via the study of antioxidant system (lipid peroxidation [LPO]; catalase [CAT]; reduced glutathione [GSH]; and superoxide dismutase [SOD]) responses in mouse liver was also investigated. Different concentrations (2.5%, 5%, 10%, and 15%) of the leachate were administered intraperitoneally for 5 consecutive days to male Swiss albino mice (4 mice/group). A significant (p < 0.05) increase in DNA damage in organs and tissues of treated mice compared to the negative control was observed as evident from the Comet assay parameters: olive tail moment (OTM, arbitrary units) and tail DNA (%). Bone marrow showed maximum DNA damage followed by liver > spleen > kidney > blood as evident by the OTM. A significant increase (p < 0.05) in the level of antioxidant enzymes (CAT and SOD) and LPO with a concurrent decrease in GSH in the liver of treated mice was also observed. Our finding demonstrates that the MSL induces DNA damage in the somatic tissues and organs of mouse as well as induces oxidative stress in the liver. These tissues and organs may be the potential targets in animal and human populations exposed to MSL. This is of relevance to public health; as such exposure could lead to adverse health effects via systemic genotoxicity.

Cellular response to metal oxide nanoparticles in bacteria.

The exponential growth in the production and consumption of engineered nanoparticles (ENPs) has raised concern about their environmental fate. ENPs accumulation in ecosystems is likely to pose threat to specific and non-specific targets. In this study a novel approach using flow cytometry was validated for detection of ENPs (ZnO and TiO2) uptake in live bacteria for several generations. These ENPs also induced frameshift mutation in S. typhimurium strains of Ames test, thus underscoring their possible carcinogenic potential.

Toxicity evaluation of carbon nanotubes in normal human bronchial epithelial cells.

Carbon nanotubes (CNTs) are well known for their exceptional thermal, mechanical and electrical properties. For many CNT applications it is of the foremost importance to know their health hazards related to their exposure. Normal human bronchial epithelial cells (BEAS-2B) has been used for assessment the cytotoxicity of SWCNT (Diameter--1.2-1.5 nm) and DWCNT (Diameter--1.3-5 nm). Clear interference of CNTs with conventional in vitro cytotoxicity assays (MTT, NRU and LDH) dye was found which was confirmed by acellular system. However morphological changes and flow cytometry showed the characteristics of cytotoxicity. Thus our study showed that there is a need of appropriate method for the assessment of cytotoxicity of CNT.

C60-fullerene binds with the ATP binding domain of human DNA topoiosmerase II alpha.

C60-fullerene has promising biological applications, such as drug delivery, biosensors, diagnosis and theraupetics. Despite of these applications, several in vitro studies have also reported the DNA damaging potential of this nanomaterial. Though, very little is known about the mechanism involved behind the fullerene mediated DNA damage. Our study was aimed at identifying the binding site of fullerene in the ATP binding domain of human topoisomerase II alpha, a major enzyme involved in maintaining DNA topology. In silico studies of fullerene with the enzyme demonstrated that it can interact with the active site residues of this enzyme through hydrophobic, pi-stacking and van der Waals interactions and could inhibit the activity of this enzyme.

Interaction of C60 fullerene with the proteins involved in DNA mismatch repair pathway.

Fullerenes are fascinating symmetric carbon nanostructures with a potential to bind DNA and proteins. Computational studies were performed to investigate the interaction of fullerenes with the proteins involved in DNA mismatch repair (MMR) pathway. Significant interactions of fullerene with PMS2, RFC3 and PCNA proteins were observed. These findings suggested that fullerene interferes in the human MMR process hence the subsequent disturbance may confer a large increase in spontaneous mutability and a strong predisposition to tumor development.

Cellular uptake and mutagenic potential of metal oxide nanoparticles in bacterial cells.

Extensive production and consumption of nanomaterials such as ZnO and TiO(2) has increased their release and disposal into the environment. The accumulation of nanoparticles (NPs) in ecosystem is likely to pose threat to non-specific targets such as bacteria. The present study explored the effect of ZnO and TiO(2) NPs in a model bacterium, Salmonella typhimurium. The uptake of ZnO and TiO(2) bare NPs in nano range without agglomeration was observed in S. typhimurium. TEM analysis demonstrated the internalization and uniform distribution of NPs inside the cells. Flow cytometry data also demonstrates that both ZnO and TiO(2) NPs were significantly internalized in the S. typhimurium cells in a concentration dependent manner. A significant increase in uptake was observed in the S. typhimurium treated even with 8 and 80 ng mL(-1) of ZnO and TiO(2) NPs with S9 after 60 min, possibly the formation of micelles or protein coat facilitated entry of NPs. These NPs exhibited weak mutagenic potential in S. typhimurium strains TA98, TA1537 and Escherichia coli (WP2uvrA) of Ames test underscoring the possible carcinogenic potential similar to certain mutagenic chemicals. Our study reiterates the need for re-evaluating environmental toxicity of ZnO and TiO(2) NPs presumably considered safe in environment.

ROS-mediated genotoxicity induced by titanium dioxide nanoparticles in human epidermal cells.

Titanium dioxide nanoparticles (TiO(2) NPs) are among the top five NPs used in consumer products, paints and pharmaceutical preparations. Since, exposure to such nanoparticles is mainly through the skin and inhalation, the present study was conducted in the human epidermal cells (A431). A mild cytotoxic response of TiO(2) NPs was observed as evident by the MTT and NR uptake assays after 48 h of exposure. However, a statistically significant (p<0.05) induction in the DNA damage was observed by the Fpg-modified Comet assay in cells exposed to 0.8 µg/ml TiO(2) NPs (2.20±0.26 vs. control 1.24±0.04) and higher concentrations for 6 h. A significant (p<0.05) induction in micronucleus formation was also observed at the above concentration (14.67±1.20 vs. control 9.33±1.00). TiO(2) NPs elicited a significant (p<0.05) reduction in glutathione (15.76%) with a concomitant increase in lipid hydroperoxide (60.51%; p<0.05) and reactive oxygen species (ROS) generation (49.2%; p<0.05) after 6h exposure. Our data demonstrate that TiO(2) NPs have a mild cytotoxic potential. However, they induce ROS and oxidative stress leading to oxidative DNA damage and micronucleus formation, a probable mechanism of genotoxicity. This is perhaps the first study on human skin cells demonstrating the cytotoxic and genotoxic potential of TiO(2) NPs.

Cypermethrin-induced DNA damage in organs and tissues of the mouse: evidence from the comet assay.

Cypermethrin is the most widely used Type II pyrethroid pesticide because of its high effectiveness against target species and its low mammalian toxicity reported so far. It is a fast-acting neurotoxin and is known to cause free radical-mediated tissue damage. The present study investigates the genotoxic effects of cypermethrin in multiple organs (brain, kidney, liver, spleen) and tissues (bone marrow, lymphocytes) of the mouse, using the alkaline comet assay. Male Swiss albino mice were given 12.5, 25, 50, 100, 200 mg/kg BW of cypermethrin intraperitoneally, daily for 5 consecutive days. A statistically significant (p<0.05) dose-dependent increase in DNA damage was observed in all the organs assessed, as evident from the comet-assay parameters, viz., Olive tail moment (OTM; arbitrary unit), tail DNA (%) and tail length (microm). Brain showed maximum DNA damage followed by spleen>kidney>bone marrow>liver>lymphocytes, as evident by the OTM. Our data demonstrate that cypermethrin induces systemic genotoxicity in mammals as it causes DNA damage in vital organs like brain, liver, kidney, apart from that in the hematopoietic system.

Current Status of Short-Term Tests for Evaluation of Genotoxicity, Mutagenicity, and Carcinogenicity of Environmental Chemicals and NCEs.

The advent of the industrial revolution has seen a significant increase in the number of new chemical entities (NCEs) released in the environment. It becomes imperative to check the toxic potential of NCEs to nontarget species before they are released for commercial purposes because some of these may exert genotoxicity, mutagenicity, or carcinogenicity. Exposure to such compounds produces chemical changes in DNA, which are generally repaired by the DNA repair enzymes. However, DNA damage and its fixation may occur in the form of gene mutations, chromosomal damage, and numerical chromosomal changes and recombination. This may affect the incidence of heritable mutations in man and may be transferred to the progeny or lead to the development of cancer. Hence, adequate tests on NCEs have to be undertaken for the risk assessment and hazard prediction. Compounds that are positive in tests that detect such damages have the potential to be human mutagens/carcinogens. Only long-term animal bioassays, involving lifetime studies on animals, were used earlier to classify substances as mutagens/carcinogens. These tests were cumbersome and time consuming and required a lot of facilities and personnel. Short-term tests, therefore, were brought into practice. A "battery" of three to four of these short-term tests has been proposed now by a number of regulatory authorities for the classification of compounds as mutagenic or carcinogenic. This review deals with the current status of these short-term tests.