Toxicological Assessment of Inhaled Nanoparticles: Role of in Vivo,ex Vivo,in Vitro,and in Silico Studies

The alveolar epithelium of the lung is by far the most permeable epithelial barrier of the human body. The risk for adverse effects by inhaled nanoparticles (NPs) depends on their hazard (negative action on cells and organism) and on exposure (concentration in the inhaled air and pattern of deposition in the lung). With the development of advanced in vitro models, not only in vivo, but also cellular studies can be used for toxicological testing. Advanced in vitro studies use combinations of cells cultured in the air-liquid interface. These cultures are useful for particle uptake and mechanistic studies. Whole-body, nose-only, and lung-only exposures of animals could help to determine retention of NPs in the body. Both approaches also have their limitations; cellular studies cannot mimic the entire organism and data obtained by inhalation exposure of rodents have limitations due to differences in the respiratory system from that of humans. Simulation programs for lung deposition in humans could help to determine the relevance of the biological findings. Combination of biological data generated in different biological models and in silico modeling appears suitable for a realistic estimation of potential risks by inhalation exposure to NPs.     

Crystallization Study and Comparative in Vitro-in Vivo Hydrolysis of PLA Reinforcement Ligament

In the present work, the crystallization behavior and in vitro-in vivo hydrolysis rates of PLA absorbable reinforcement ligaments used in orthopaedics for the repair and reinforcement of articulation instabilities were studied. Tensile strength tests showed that this reinforcement ligament has similar mechanical properties to Fascia Latta, which is an allograft sourced from the ilio-tibial band of the human body. The PLA reinforcement ligament is a semicrystalline material with a glass transition temperature around 61 °C and a melting point of ~178 °C. Dynamic crystallization revealed that, although the crystallization rates of the material are slow, they are faster than the often-reported PLA crystallization rates. Mass loss and molecular weight reduction measurements showed that in vitro hydrolysis at 50 °C initially takes place at a slow rate, which gets progressively higher after 30-40 days. As found from SEM micrographs, deterioration of the PLA fibers begins during this time. Furthermore, as found from in vivo hydrolysis in the human body, the PLA reinforcement ligament is fully biocompatible and after 6 months of implantation is completely covered with flesh. However, the observed hydrolysis rate from in vivo studies was slow due to high molecular weight and degree of crystallinity.     

Shape and Site Dependent in Vivo Degradation of Mg-Zn Pins in Rabbit Femoral Condyle

A type of specially designed pin model of Mg-Zn alloy was implanted into the full thickness of lesions of New Zealand rabbits’ femoral condyles. The recovery progress, outer surface healing and in vivo degradation were characterized by various methods including radiographs, Micro-CT scan with surface rendering, SEM (scanning electron microscope) with EDX (Energy Dispersive X-ray analysis) and so on. The in vivo results suggested that a few but not sufficient bridges for holding force were formed between the bone and the implant if there was a preexisting gap between them. The rapid degradation of the implantation in the condyle would result in the appearance of cavities. Morphological evaluation of the specially designed pins indicated that the cusp was the most vulnerable part during degradation. Furthermore, different implantation sites with distinct components and biological functions can lead to different degradation rates of Mg-Zn alloy. The rate of Mg-Zn alloy decreases in the following order: implantation into soft tissue, less trabecular bone, more trabecular bone, and cortical bone. Because of the complexities of in vivo degradation, it is necessary for the design of biomedical Mg-Zn devices to take into consideration the implantation sites used in clinics.     

Retinal Cell Death Caused by Sodium Iodate Involves Multiple Caspase-Dependent and Caspase-Independent Cell-Death Pathways

Herein, we have investigated retinal cell-death pathways in response to the retina toxin sodium iodate (NaIO₃) both in vivo and in vitro. C57/BL6 mice were treated with a single intravenous injection of NaIO₃ (35 mg/kg). Morphological changes in the retina post NaIO₃ injection in comparison to untreated controls were assessed using electron microscopy. Cell death was determined by TdT-mediated dUTP-biotin nick end labeling (TUNEL) staining. The activation of caspases and calpain was measured using immunohistochemistry. Additionally, cytotoxicity and apoptosis in retinal pigment epithelial (RPE) cells, primary retinal cells, and the cone photoreceptor (PRC) cell line 661W were assessed in vitro after NaIO₃ treatment using the ApoToxGlo™ assay. The 7-AAD/Annexin-V staining was performed and necrostatin (Nec-1) was administered to the NaIO₃-treated cells to confirm the results. In vivo, degenerating RPE cells displayed a rounded shape and retracted microvilli, whereas PRCs featured apoptotic nuclei. Caspase and calpain activity was significantly upregulated in retinal sections and protein samples from NaIO₃-treated animals. In vitro, NaIO₃ induced necrosis in RPE cells and apoptosis in PRCs. Furthermore, Nec-1 significantly decreased NaIO₃-induced RPE cell death, but had no rescue effect on treated PRCs. In summary, several different cell-death pathways are activated in retinal cells as a result of NaIO₃.     

Preparation and Characterization of Highly Fluorescent, Glutathione-coated Near Infrared Quantum Dots for in Vivo Fluorescence Imaging

Fluorescent probes that emit in the near-infrared (NIR, 700–1,300 nm) region are suitable as optical contrast agents for in vivo fluorescence imaging because of low scattering and absorption of the NIR light in tissues. Recently, NIR quantum dots (QDs) have become a new class of fluorescent materials that can be used for in vivo imaging. Compared with traditional organic fluorescent dyes, QDs have several unique advantages such as size- and composition-tunable emission, high brightness, narrow emission bands, large Stokes shifts, and high resistance to photobleaching. In this paper, we report a facile method for the preparation of highly fluorescent, water-soluble glutathione (GSH)-coated NIR QDs for in vivo imaging. GSH-coated NIR QDs (GSH-QDs) were prepared by surface modification of hydrophobic CdSeTe/CdS (core/shell) QDs. The hydrophobic surface of the CdSeTe/CdS QDs was exchanged with GSH in tetrahydrofuran-water. The resulting GSH-QDs were monodisperse particles and stable in PBS (phosphate buffered saline, pH = 7.4). The GSH-QDs (800 nm emission) were highly fluorescent in aqueous solutions (quantum yield = 22% in PBS buffer), and their hydrodynamic diameter was less than 10 nm, which is comparable to the size of proteins. The cellular uptake and viability for the GSH-QDs were examined using HeLa and HEK 293 cells. When the cells were incubated with aqueous solutions of the GSH-QDs (10 nM), the QDs were taken into the cells and distributed in the perinuclear region of both cells. After 12 hrs incubation of 4 nM of GSH-QDs, the viabilities of HeLa and HEK 293 cells were ca. 80 and 50%, respectively. As a biomedical utility of the GSH-QDs, in vivo NIR-fluorescence imaging of a lymph node in a mouse is presented.     

Inroads to Predict in Vivo Toxicology-An Introduction to the eTOX Project

There is a widespread awareness that the wealth of preclinical toxicity data that the pharmaceutical industry has generated in recent decades is not exploited as efficiently as it could be. Enhanced data availability for compound comparison ("read-across"), or for data mining to build predictive tools, should lead to a more efficient drug development process and contribute to the reduction of animal use (3Rs principle). In order to achieve these goals, a consortium approach, grouping numbers of relevant partners, is required. The eTOX ("electronic toxicity") consortium represents such a project and is a public-private partnership within the framework of the European Innovative Medicines Initiative (IMI). The project aims at the development of in silico prediction systems for organ and in vivo toxicity. The backbone of the project will be a database consisting of preclinical toxicity data for drug compounds or candidates extracted from previously unpublished, legacy reports from thirteen European and European operation-based pharmaceutical companies. The database will be enhanced by incorporation of publically available, high quality toxicology data. Seven academic institutes and five small-to-medium size enterprises (SMEs) contribute with their expertise in data gathering, database curation, data mining, chemoinformatics and predictive systems development. The outcome of the project will be a predictive system contributing to early potential hazard identification and risk assessment during the drug development process. The concept and strategy of the eTOX project is described here, together with current achievements and future deliverables.     

Aminolevulinic Acid-Mediated Photodynamic Therapy of Human Meningioma: An in Vitro Study on Primary Cell Lines

Objective: Five-aminolevulinic acid (5-ALA)-induced porphyrins in malignant gliomas are potent photosensitizers. Promising results of ALA-PDT (photodynamic therapy) in recurrent glioblastomas have been published. Recently, 5-ALA-induced fluorescence was studied in meningioma surgery. Here, we present an experimental study of ALA-PDT in an in vitro model of primary meningioma cell lines. Methods: We processed native tumor material obtained intra-operatively within 24 h for cell culture. Epithelial membrane antigen (EMA) immunohistochemistry was performed after the first passage to confirm that cells were meningioma cells. For 5-ALA-PDT treatment, about 5000 cells per well were seeded in 20 wells of a blank 96-well plate. Each block of 4 wells was inoculated with 150 µL of 0, 25, 50 and 100 µg/mL 5-ALA solutions; one block was used as negative control without 5-ALA and without PDT. Following incubation for 3 h PDT was performed using a laser (635 nm, 18.75 J/cm²). The therapeutic response was analyzed by the water soluble tetrazolium salt (WST-1) cell viability assay 90 min after PDT. Results: 5-ALA-PDT was performed in 14 primary meningioma cell lines. EMA expression was verified in 10 primary cell cultures. The remaining 4 were EMA negative and PDT was without any effect in these cultures. All 10 EMA-positive cell lines showed a significant and dose-dependent decrease in viability rate (p < 0.001). Cell survival at 5-ALA concentrations of 12.5, 25, 50 and 100 μg/mL was 96.5% ± 7.6%, 67.9% ± 29.9%, 24.0% ± 16.7% and 13.8% ± 7.5%, respectively. For the negative controls (no 5-ALA/PDT and ALA/no PDT), the viability rates were 101.72% ± 3.5% and 100.17% ± 3.6%, respectively. The LD₅₀ for 5-ALA was estimated between 25 and 50 µg/mL. Conclusion: This study reveals dose-dependent cytotoxic effects of 5-ALA-PDT on primary cell lines of meningiomas. Either 5-ALA or PDT alone did not affect cell survival. Further efforts are necessary to study the potential therapeutic effects of 5-ALA-PDT in vivo.     

Labeling of Anti-MUC-1 Binding Single Chain Fv Fragments to Surface Modified Upconversion Nanoparticles for an Initial in Vivo Molecular Imaging Proof of Principle Approach

In vivo optical Imaging is an inexpensive and highly sensitive modality to investigate and follow up diseases like breast cancer. However, fluorescence labels and specific tracers are still works in progress to bring this promising modality into the clinical day-to-day use. In this study an anti-MUC-1 binding single-chain antibody fragment was screened, produced and afterwards labeled with newly designed and surface modified NaYF(4):Yb,Er upconversion nanoparticles as fluorescence reporter constructs. The MUC-1 binding of the conjugate was examined in vitro and in vivo using modified state-of-the-art small animal Imaging equipment. Binding of the newly generated upconversion nanoparticle based probe to MUC-1 positive cells was clearly shown via laser scanning microscopy and in an initial proof of principal small animal optical imaging approach.     

Two Effective Routes for Removing Lineage Restriction Roadblocks: From Somatic Cells to Hepatocytes

The conversion of somatic cells to hepatocytes has fundamentally re-shaped traditional concepts regarding the limited resources for hepatocyte therapy. With the various induced pluripotent stem cell (iPSC) generation routes, most somatic cells can be effectively directed to functional stem cells, and this strategy will supply enough pluripotent material to generate promising functional hepatocytes. However, the major challenges and potential applications of reprogrammed hepatocytes remain under investigation. In this review, we provide a summary of two effective routes including direct reprogramming and indirect reprogramming from somatic cells to hepatocytes and the general potential applications of the resulting hepatocytes. Through these approaches, we are striving toward the goal of achieving a robust, mature source of clinically relevant lineages.     

Antiproliferative Activity and in Vivo Toxicity of Double-Point Modified Analogs of 1,25-Dihydroxyergocalciferol

Analogs of 1,25-dihydroxyergocalciferol, modified in the side-chain and in the A-ring, were tested for their antiproliferative activity against a series of human cancer cell lines in vitro and in vivo toxicity. The proliferation inhibition caused by the analogs was higher than that of the parent compounds, while the toxicity, measured as the serum calcium level, was lower. All analogs were able to induce, in HL-60 and MV4-11 leukemic cells, G₀/G₁ cell cycle arrest and differentiation expressed as morphological signs typical for monocytes. The analogs also induced the expression of CD11b and/or CD14 cell-differentiation markers. The most potent analogs, PRI-5105, PRI-5106, PRI-5201 and PRI-5202, were also able to induce vitamin D receptor (VDR) protein expression, mainly in the cytoplasmic fraction of HL-60 or MV4-11 cells. The most active analogs were the 19-nor ones with an extended and rigidified side-chain (PRI-5201 and PRI-5202), as in the former analogs PRI-1906 and PRI-1907. Epimerization at C-24 (PRI-5101) or introduction of an additional hydroxyl at C-23 (PRI-5104) reduced the toxicity of the analog with retained antiproliferative activity.     

Zearalenone and Reproductive Function in Farm Animals

Farm animals are exposed to zearalenone through the feed because of the widespread occurrence of this mycotoxin in cereals and clinical reproductive disorders due to mycotoxin effects are often reported in farm animal species. This review describes the metabolism, the mechanistic aspects, the clinical reproductive symptoms and the in vitro effects on functional parameters of oocytes and sperm cells induced by zearalenone and its derivatives in farm animals. The studies on in vitro effects allow to understand the action mechanisms of mycotoxins and, sometime, to explain the in vivo symptoms. The impairment of semen quality and female reproductive function induced by zearalenone could be a factor responsible for the reproductive failure in farm animals.     

Budesonide-Loaded Guar Gum Microspheres for Colon Delivery: Preparation,Characterization and in Vitro/in Vivo Evaluation

A novel budesonide (BUD) colon delivery release system was developed by using a natural polysaccharide, guar gum. The rigidity of the microspheres was induced by a chemical cross-linking method utilizing glutaraldehyde as the cross-linker. The mean particle size of the microspheres prepared was found to be 15.21 ± 1.32 µm. The drug loading and entrapment efficiency of the formulation were 17.78% ± 2.31% and 81.6% ± 5.42%, respectively. The microspheres were spherical in shape with a smooth surface, and the size was uniform. The in vitro release profiles indicated that the release of BUD from the microspheres exhibited a sustained release behavior. The model that fitted best for BUD released from the microspheres was the Higuchi kinetic model with a correlation coefficient r = 0.9993. A similar phenomenon was also observed in a pharmacokinetic study. The prolongation of the half-life (t_1/2), enhanced residence time (mean residence time, MRT) and decreased total clearance (CL) indicated that BUD microspheres could prolong the acting time of BUD in vivo. In addition, BUD guar gum microspheres are thought to have the potential to maintain BUD concentration within target ranges for a long time, decreasing the side effects caused by concentration fluctuation, ensuring the efficiency of treatment and improving patient compliance by reducing dosing frequency. None of the severe signs, like the appearance of epithelial necrosis and the sloughing of epithelial cells, were detected.     

Oxidative Neurodegeneration Is Prevented by UCP0045037, an Allosteric Modulator for the Reduced Form of DJ-1, a Wild-Type of Familial Parkinson��s Disease-Linked PARK7

Although a loss-of-function mutation has been identified in familial Parkinson’s disease PARK7, the wild-type of DJ-1 is known to act as an oxidative stress sensor in neuronal cells. Recently, we identified UCP0045037 as a compound that bound to the reduced form of DJ-1 by in silico virtual screening. In this study, we determined the neuroprotective effects of UCP0045037 against focal cerebral ischemia-induced neurodegeneration in rats. Hydrogen peroxide-induced cell death was significantly inhibited by UCP0045037 in both rat mesencephalic dopaminergic neurons and human normal SH-SY5Y cells. In contrast, DJ-1-knockdown SH-SY5Y cells lost the protective activity of UCP0045037. These results suggest that UCP0045037 interacts with endogenous DJ-1 and produces a neuroprotective response.     

By-Passing Large Screening Experiments Using Sequencing as a Tool to Identify scFv Fragments Targeting Atherosclerotic Lesions in a Novel In Vivo Phage Display Selection

Atherosclerosis is a chronic, progressive inflammatory disease that may develop into vulnerable lesions leading to thrombosis. To interrogate the molecular components involved in this process, single-chain variable fragments (scFvs) from a semi-synthetic human antibody library were selected on the lesions induced in a rabbit model of atherosclerosis after two rounds of in vivo phage display. Homing Phage-scFvs were isolated from (1) the injured endothelium, (2) the underlying lesional tissue and (3) the cells within the intima. Clones selected on the basis of their redundancy or the presence of key amino acids, as determined by comparing the distribution between the native and the selected libraries, were produced in soluble form, and seven scFvs were shown to specifically target the endothelial cell surface and inflamed intima-related regions of rabbit tissue sections by immunohistology approaches. The staining patterns differed depending on the scFv compartment of origin. This study demonstrates that large-scale scFv binding assays can be replaced by a sequence-based selection of best clones, paving the way for easier use of antibody libraries in in vivo biopanning experiments. Future investigations will be aimed at characterizing the scFv/target couples by mass spectrometry to set the stage for more accurate diagnostic of atherosclerosis and development of therapeutic strategies.     

Sasa quelpaertensis Leaf Extract Inhibits Colon Cancer by Regulating Cancer Cell Stemness in Vitro and in Vivo

A rare subpopulation of cancer cells, termed cancer stem cells (CSCs), may be responsible for tumor relapse and resistance to conventional chemotherapy. The development of a non-toxic, natural treatment for the elimination of CSCs is considered a strategy for cancer treatment with minimal side effects. In the present study, the potential for Sasa quelpaertensis leaf extract (SQE) and its two bioactive compounds, tricin and p-coumaric acid, to exert anti-CSC effects by suppressing cancer stemness characteristics were evaluated in colon cancer cells. CD133⁺CD44⁺ cells were isolated from HT29 and HCT116 cell lines using flow-activated cell sorting (FACs). SQE treatment was found to significantly suppress the self-renewal capacity of both cell lines. SQE treatment was also associated with the down-regulation of β-catenin and phosphorylated GSK3β, while significantly enhancing cell differentiation by up-regulating CK20 expression and blocking the expression of several stem cell markers, including DLK1, Notch1, and Sox-2. In vivo, SQE supplementation suppressed tumor growth in a xenograft model by down-regulating stem cell markers and β-catenin as well as HIF-1α signaling. Compared with two bioactive compounds of SQE, SQE exhibited the most effective anti-CSC properties. Taken together, these results provide evidence that SQE inhibits colon cancer by regulating the characteristics of CSCs.     

A Comparison of B16 Melanoma Cells and 3T3 Fibroblasts Concerning Cell Viability and ROS Production in the Presence of Melatonin,Tested Over a Wide Range of Concentrations

Melatonin is a pleiotropic molecule with many cellular and systemic actions, including chronobiotic effects. Beneficial effects are widely documented concerning the treatment of neoplastic diseases in vivo as well as reductions in viability of cultured cells from melanoma, one of the most aggressive cancers in humans. However, studies of its effects on non-tumor cells in vitro have not focused on viability, except for experiments aiming to protect against oxidotoxicity or other toxicological insults. Furthermore, there is no agreement on the range of effective melatonin concentrations in vitro, and the mechanisms that reduce cell viability have remained unclear. Tumor cell-specific increases in the production of reactive oxygen and nitrogen species (ROS/RNS) may provide a possible explanation. Our aim was to analyze the potential inhibition of tumor (B16 melanoma 4A5) and non-tumor cell (3T3 Swiss albino) viability using a wide range of melatonin concentrations (10⁻¹¹–10⁻² M), and to determine whether intracellular ROS enhancement was involved in this process. In the absence of fetal bovine serum (FBS), low melatonin concentrations (10⁻⁹–10⁻⁵ M) reduced the proliferation of melanoma cells with no effect in fibroblasts, whereas, in the presence of FBS, they had no effect or even increased the proliferation of both fibroblast and melanoma cells. Melatonin concentrations in the upper millimolar range increased ROS levels and reduced the viability of both cell types, but more markedly so in non-tumor cells. Thus, low melatonin concentrations reduce proliferation in this specific melanoma cell line, whereas high concentrations affect the viability of both tumor (B16 4A5 melanoma) and non-tumor (3T3 fibroblasts) cells. Increased ROS levels in both lines indicate a role for ROS production in the reduction of cell viability at high—but not low—melatonin concentrations, although the mechanism of action still remains to be elucidated.     

The Effects of Nanomaterials as Endocrine Disruptors

In recent years, nanoparticles have been increasingly used in several industrial, consumer and medical applications because of their unique physico-chemical properties. However, in vitro and in vivo studies have demonstrated that these properties are also closely associated with detrimental health effects. There is a serious lack of information on the potential nanoparticle hazard to human health, particularly on their possible toxic effects on the endocrine system. This topic is of primary importance since the disruption of endocrine functions is associated with severe adverse effects on human health. Consequently, in order to gather information on the hazardous effects of nanoparticles on endocrine organs, we reviewed the data available in the literature regarding the endocrine effects of in vitro and in vivo exposure to different types of nanoparticles. Our aim was to understand the potential endocrine disrupting risks posed by nanoparticles, to assess their underlying mechanisms of action and identify areas in which further investigation is needed in order to obtain a deeper understanding of the role of nanoparticles as endocrine disruptors. Current data support the notion that different types of nanoparticles are capable of altering the normal and physiological activity of the endocrine system. However, a critical evaluation of these findings suggests the need to interpret these results with caution since information on potential endocrine interactions and the toxicity of nanoparticles is quite limited.     

Apoptotic Effects of Chrysin in Human Cancer Cell Lines

Chrysin is a natural flavonoid currently under investigation due to its important biological anti-cancer properties. In most of the cancer cells tested, chrysin has shown to inhibit proliferation and induce apoptosis, and is more potent than other tested flavonoids in leukemia cells, where chrysin is likely to act via activation of caspases and inactivation of Akt signaling in the cells. Moreover, structure-activity relationships have revealed that the chemical structure of chrysin meets the key structural requirements of flavonoids for potent cytotoxicity in leukemia cells. It is possible that combination therapy or modified chrysin could be more potent than single-agent use or administration of unmodified chrysin. This study may help to develop ways of improving the effectiveness of chrysin in the treatment of leukemia and other human cancers in vitro.