Bahy A. Ali

Cytotoxicity and Genotoxicity of Copper Oxide Nanoparticles in Human Skin Keratinocytes Cells

The wide scale use of copper oxide nanoparticles (CuONPs) due to their unique properties and important applications in magnetic, thermal, electrical, sensor devices, and cosmetics makes human beings more prone to the exposure of CuONPs and its potential adverse effects. Exposure to such nanoparticles is mainly through skin and inhalation. Therefore, the aim of the present study was to assess the apoptotic and genotoxic potential of CuONPs (50 nm) in the human skin epidermal (HaCaT) cells and its underlying mechanism of cellular toxicity. Significant decreases in cell viability were observed with CuONPs exposure in a dose- and time-dependent manner and also induced significant reduction in glutathione and induction in lipid peroxidation, catalase, and superoxide dismutase in HaCaT cells. A significant increase in caspase-3 activity was observed with CuONPs exposure in HaCaT cells indicating apoptosis. Apoptosis or necrosis was confirmed with fluorescent staining (acridine orange and propidium iodide). The CuONPs also induced DNA damage that was mediated by oxidative stress. This study investigating the effects of CuONPs in human skin cells has provided valuable insights into the mechanism of potential toxicity induced by CuONPs.

Arsenic trioxide-mediated oxidative stress and genotoxicity in human hepatocellular carcinoma cells

Background Arsenic is a ubiquitous environmental toxicant, and abnormalities of the skin, lung, kidney, and liver are the most common outcomes of long-term arsenic exposure. This study was designed to investigate the possible mechanisms of genotoxicity induced by arsenic trioxide in human hepatocellular carcinoma cells. Methods and results A mild cytotoxic response of arsenic trioxide was observed in human hepatocellular carcinoma cells, as evident by (3-(4,5-dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) and lactate dehydrogenase assays after 24 and 48 hours of exposure. Arsenic trioxide elicited a significant (P < 0.01) reduction in glutathione (15.67% and 26.52%), with a concomitant increase in malondialdehyde level (67.80% and 72.25%; P < 0.01), superoxide dismutase (76.42% and 81.09%; P < 0.01), catalase (73.33% and 76.47%; P < 0.01), and reactive oxygen species generation (44.04% and 56.14%; P < 0.01) after 24 and 48 hours of exposure, respectively. Statistically significant (P < 0.01) induction of DNA damage was observed by the comet assay in cells exposed to arsenic trioxide. It was also observed that apoptosis occurred through activation of caspase-3 and phosphatidylserine externalization in human hepatocellular carcinoma cells exposed to arsenic trioxide. Conclusion The results demonstrate that arsenic trioxide induces apoptosis and genotoxicity in human hepatocellular carcinoma cells through reactive oxygen species and oxidative stress.

Histologic and apoptotic changes induced by titanium dioxide nanoparticles in the livers of rats

Titanium dioxide (TiO2) nanoparticles are among the top five nanoparticles used in consumer products, paints, and pharmaceutical preparations. Given that exposure to such nanoparticles is mainly via the skin and inhalation, the present study was conducted in male Wistar albino rats (Rattus norvegicus). Our aim was to investigate the effect of TiO2 nanoparticles on hepatic tissue in an attempt to understand their toxicity and the potential effect of their therapeutic and diagnostic use. To investigate the effects of TiO2 nanoparticles on liver tissue, 30 healthy male Wistar albino rats were exposed to TiO2 nanoparticles at doses of 63 mg, 126 mg, and 252 mg per animal for 24 and 48 hours. Serum glutamate oxaloacetate transaminase and alkaline phosphatase activity was altered. Changes in hepatocytes can be summarized as hydropic degeneration, cloudy swelling, fatty degeneration, portal and lobular infiltration by chronic inflammatory cells, and congested dilated central veins. The histologic alterations observed might be an indication of hepatocyte injury due to the toxicity of TiO2 nanoparticles, resulting in an inability to deal with accumulated residues from the metabolic and structural disturbances caused by these nanoparticles. The appearance of cytoplasmic degeneration and destruction of nuclei in hepatocytes suggests that TiO2 nanoparticles interact with proteins and enzymes in hepatic tissue, interfering with antioxidant defense mechanisms and leading to generation of reactive oxygen species which, in turn, may induce stress in hepatocytes, promoting atrophy, apoptosis, and necrosis. More immunohistochemical and ultrastructural investigations are needed in relation to TiO2 nanoparticles and their potential effects when used as therapeutic and diagnostic tools.

Dual optical biosensors for imaging microRNA-1 during myogenesis

Dual optical microRNA (miRNA) imaging systems, bioluminescent reporter gene (a signal-off mechanism)- or fluorescent molecular beacon (MB) (a signal-on mechanism)-based miRNA imaging system, have individually allowed us to sense miRNA biogenesis in a noninvasive and iterative manner. Both of these imaging systems have shortcomings with respect to image quality. Therefore, we designed a dual optical imaging systems by simultaneous imaging of a miRNA-1 reporter gene (CMV/Gluc/3xPT_miR-1) and miRNA-1 MB in a single cell to overcome these limitations and used it to visualize miRNA-1, a highly expressed miRNA in cardiac and skeletal muscle. During myogenic differentiation of C2C12 cells, the bioluminescence intensity from CMV/Gluc/3xPT_miR-1 revealed a miRNA-1-dependent gradual decrease and the fluorescence intensity from miR-1 MB demonstrated a miRNA-1-dependent gradual increase both in vitro and in vivo. The dual miRNA-1 imaging systems, which provides the complementary imaging information about miRNA biogenesis, could be useful to sense miRNA expression during various biologic processes.

Zinc oxide quantum dots: multifunctional candidates for arresting C2C12 cancer cells and their role towards caspase 3 and 7 genes

Recently, nanoscale (<100 nm) inorganic materials, especially spherical shaped zinc oxide quantum dots (ZnO-QDs), have received a lot of attention from the broad community because of their potential utilization in various technologies. Due to their large surface to volume (S/V) ratios and extremely high reactivities, they can easily penetrate in various biological identities, such as cells and proteins, and therefore can sense, diagnose and cure different biological systems. The present study describes the facile synthesis of crystalline ZnO-QDs via a solution process. In addition, C2C12 myoblast cancer cells have been treated with different doses of ZnO-QDs at different incubation times (24, 48, 72 and 96 h). The rate of inhibition of cells was observed using an MTT assay, whereas the morphology of the cells was observed by confocal microscopy (CLSM). The MTT and CLSM investigations confirmed that with an increase in the incubation time, the population density of cancer cells was decreased when treated with ZnO-QDs. The dose dependent apoptosis correlated with intracellular production of reactive oxygen species (ROS) from C2C12 cancer cells was also measured in presence of ZnO-QDs. Moreover, the effect/apoptosis of these QDs was also checked in the presence of candidate genes such as caspase 3/7 with GAPDH. Reverse transcription polymerase chain reaction (RT-PCR) analysis demonstrates the up-regulation of caspase 3/7 genes in cells subsequently treated with ZnO-QDs at low and high concentrations.

Simultaneous Imaging of Two Different Cancer Biomarkers Using Aptamer-Conjugated Quantum Dots

Studying gene expression profile in a single cancer cell is important because multiple genes are associated with cancer development. Quantum dots (QDs) have been utilized as biological probes for imaging and detection. QDs display specific optical and electrical properties that depend on their size that can be applied for imaging and sensing applications. In this study, simultaneous imaging of the cancer biomarkers, tenascin-C and nucleolin, was performed using two types of aptamer-conjugated QDs. The simultaneous imaging of these two different cancer markers in three cancer cell lines was reliable and cell line-specific. Current requirements for cancer imaging technologies include the need for simple preparation methods and the ability to detect multiple cancer biomarkers and evaluate their intracellular localizations. The method employed in this study is a feasible solution to these requirements.

Quantum Dot-Based Molecular Beacon to Monitor Intracellular MicroRNAs

Fluorescence monitoring of endogenous microRNA (miRNA or miR) activity related to neuronal development using nano-sized materials provides crucial information on miRNA expression patterns in a noninvasive manner. In this study, we report a new method to monitor intracellular miRNA124a using quantum dot-based molecular beacon (R9-QD-miR124a beacon). The R9-QD-miR124a beacon was constructed using QDs and two probes, miR124a-targeting oligomer and arginine rich cell-penetrating peptide (R9 peptide). The miR124a-targeting oligomer contains a miR124a binging sequence and a black hole quencher 1 (BHQ1). In the absence of target miR124a, the R9-QD-miR124a beacon forms a partial duplex beacon and remained in quenched state because the BHQ1 quenches the fluorescence signal of the R9-QD-miR124a beacon. The binding of miR124a to the miR124a binding sequence of the miR124a-targeting oligomer triggered the separation of the BHQ1 quencher and subsequent signal-on of a red fluorescence signal. Moreover, enhanced cellular uptake was achieved by conjugation with the R9 peptide, which resulted in increased fluorescent signal of the R9-QD-miR124a beacons in P19 cells during neurogenesis due to the endogenous expression of miR124a.

Multimodal imaging probe for targeting cancer cells using uMUC-1 aptamer

For adequate cancer therapy, newer imaging modalities with more specific ligands for unique targets are crucial. Underglycosylated mucin-1 (uMUC-1) antigen is an early marker of tumor development and is widely overexpressed on most tumors. A combination of nanotechnology with optical, radionuclide, and magnetic resonance (MR) imaging has great potential to improve cancer diagnosis and therapy. In this study, a multimodal nanoparticle imaging system was developed that can be used for optical, MR and positron emission tomography (PET) imaging. Cobalt ferrite magnetic nanoparticles surrounded by fluorescent rhodamine (designated MF) within a silica shell matrix were conjugated with an aptamer targeting uMUC-1 (designated MF-uMUC-1) and further labeled by (68)Ga (designated MFR-uMUC-1) with the help of a p-SCN-bn-NOTA chelating agent, resulting in single multimodal nanoparticles. The resultant nanoparticles are spherical and monodispersed, as revealed by transmission electron microscopy. The MFR-uMUC-1 nanoparticle showed specific and dose-dependent fluorescent, radioisotope and MR signals targeting BT-20 cells expressing uMUC-1. In vivo targeting and multimodal imaging in tumor-bearing nude mice also showed great specificity for targeting cancers with MFR-uMUC-1. The MFR-uMUC-1 probe could be used as a single multimodal probe to visualize cancer cells by means of optical, radionuclide and MR imaging.

Isolation and evaluation of biological efficacy of quercetol in human hepatic carcinoma cells

Quercetol is a polyphenolic molecule present in vegetables and fruits, and is beneficial to human and animal health. The current work aimed to test cytotoxic and apoptotic effects of quercetol on HepG2 cells. Quercetol was isolated from Ocimum sanctum and characterized by gas chromatography–tandom mass spectrometry (GC-MS/MS), nuclear magnetic resonance spectroscopy, and Fourier transform infrared spectroscopy. Quercetol (50–600 μg/mL) was examined for cytotoxic activity by tetrazolium salt and neutral red uptake tests and comet assay for genotoxicity, using HepG2 cells, over 24 hours. Data from 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide and neutral red uptake tests demonstrated quercetol-induced cytotoxicity in HepG2 cells in a concentration-dependent manner. With 4′,6-diamidino-2-phenylindole staining, a significant induction of chromosomal condensation was observed at 300 μg/mL of quercetol. DNA fragmentation analysis showed that quercetol produced cell death in HepG2 cells in a concentration-dependent manner. Thus, our study suggests that an environmentally relevant concentration of quercetol, which was a chemically standardized extract from O. sanctum, induced cell death and DNA damage in HepG2 cells.

Bioimaging of transcriptional activity of microRNA124a during neurogenesis

ObjectivesA special vector system was developed to monitor the in vitro and in vivo endogenous level of a primary transcript of miR124a during neuronal differentiationResultsThe upstream regions of miR124a were fused with luciferase (Gluc) and their activity was measured. During neurogenesis of P19 cells, the primary transcript level of miR124a was increased 1.5-times compared to the undifferentiated P19 cells. P19 cells grafted to nude mice exhibited the same pattern of luciferase activity in vivo as they did in vitro.ConclusionThe expression of primary miR124a during neurogenesis was successfully imaged by in vitro and in vivo luciferase reporter gene-based method.