In Vitro Transcriptome Analysis of Cobalt Boride Nanoparticles on Human Pulmonary Alveolar Cells.

Nanobiotechnology influences many different areas, including the medical, food, energy, clothing, and cosmetics industries. Considering the wide usage of nanomaterials, it is necessary to investigate the toxicity potentials of specific nanosized molecules. Boron-containing nanoparticles (NPs) are attracting much interest from scientists due to their unique physicochemical properties. However, there is limited information concerning the toxicity of boron-containing NPs, including cobalt boride (Co2B) NPs. Therefore, in this study, Co2B NPs were characterized using X-ray crystallography (XRD), transmission electron microscope (TEM), scanning electron microscope (SEM), and energy-dispersive X-ray spectroscopy (EDX) techniques. Then, we performed 3-(4,5-dimethyl-thiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT), lactate dehydrogenase (LDH) release, and neutral red (NR) assays for assessing cell viability against Co2B NP exposure on cultured human pulmonary alveolar epithelial cells (HPAEpiC). In addition, whole-genome microarray analysis was carried out to reveal the global gene expression differentiation of HPAEpiC cells after Co2B NP application. The cell viability tests unveiled an IC50 value for Co2B NPs of 310.353 mg/L. The results of our microarray analysis displayed 719 gene expression differentiations (FC ≥ 2) among the analyzed 40,000 genes. The performed visualization and integrated discovery (DAVID) analysis revealed that there were interactions between various gene pathways and administration of the NPs. Based on gene ontology biological processes analysis, we found that the P53 signaling pathway, cell cycle, and cancer-affecting genes were mostly affected by the Co2B NPs. In conclusion, we suggested that Co2B NPs would be a safe and effective nanomolecule for industrial applications, particularly for medical purposes.

Ameliorative Effects by Hexagonal Boron Nitride Nanoparticles against Beta Amyloid Induced Neurotoxicity.

Alzheimer's disease (AD) is considered as the most common neurodegenerative disease. Extracellular amyloid beta (Aß) deposition is a hallmark of AD. The options based on degradation and clearance of Aß are preferred as promising therapeutic strategies for AD. Interestingly, recent findings indicate that boron nanoparticles not only act as a carrier but also play key roles in mediating biological effects. In the present study, the aim was to investigate the effects of different concentrations (0−500 mg/L) of hexagonal boron nitride nanoparticles (hBN-NPs) against neurotoxicity by beta amyloid (Aß1-42) in differentiated human SH-SY5Y neuroblastoma cell cultures for the first time. The synthesized hBN-NPs were characterized by X-ray diffraction (XRD) measurements, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Aß1-42-induced neurotoxicity and therapeutic potential by hBN-NPs were assessed on differentiated SH-SY5Y cells using MTT and LDH release assays. Levels of total antioxidant capacity (TAC) and total oxidant status (TOS), expression levels of genes associated with AD and cellular morphologies were examined. The exposure to Aß1-42 significantly decreased the rates of viable cells which was accompanied by elevated TOS level. Aß1-42 induced both apoptotic and necrotic cell death. Aß exposure led to significant increases in expression levels of APOE, BACE 1, EGFR, NCTSN and TNF-α genes and significant decreases in expression levels of ADAM 10, APH1A, BDNF, PSEN1 and PSENEN genes (p < 0.05). All the Aß1-42-induced neurotoxic insults were inhibited by the applications with hBN-NPs. hBN-NPs also suppressed the remarkable elevation in the signal for Aß following exposure to Aß1-42 for 48 h. Our results indicated that hBN-NPs could significantly prevent the neurotoxic damages by Aß. Thus, hBN-NPs could be a novel and promising anti-AD agent for effective drug development, bio-nano imaging or drug delivery strategies.

Molecular Genetics and Cytotoxic Responses to Titanium Diboride and Zinc Borate Nanoparticles on Cultured Human Primary Alveolar Epithelial Cells.

Titanium diboride (TiB2) and zinc borate (Zn3BO6) have been utilized in wide spectrum industrial areas because of their favorable properties such as a high melting point, good wear resistance, high hardness and thermal conductivity. On the other hand, the biomedical potentials of TiB2 and Zn3BO6 are still unknown because there is no comprehensive analysis that uncovers their biocompatibility features. Thus, the toxicogenomic properties of TiB2 and Zn3BO6 nanoparticles (NPs) were investigated on human primary alveolar epithelial cell cultures (HPAEpiC) by using different cell viability assays and microarray analyses. Protein-Protein Interaction Networks Functional Enrichment Analysis (STRING) was used to associate differentially expressed gene probes. According to the results, up to 10 mg/L concentration of TiB2 and Zn3BO6 NPs application did not stimulate a cytotoxic effect on the HPAEpiC cell cultures. Microarray analysis revealed that TiB2 NPs exposure enhances cellular adhesion molecules, proteases and carrier protein expression. Furthermore, Zn3BO6 NPs caused differential gene expressions in the cell cycle, cell division and extracellular matrix regulators. Finally, STRING analyses put forth that inflammation, cell regeneration and tissue repair-related gene interactions were affected by TiB2 NPs application. Zn3BO6 NPs exposure significantly altered inflammation, lipid metabolism and infection response activator-related gene interactions. These investigations illustrated that TiB2 and Zn3BO6 NPs exposure may affect different aspects of cellular machineries such as immunogenic responses, tissue regeneration and cell survival. Thus, these types of cellular mechanisms should be taken into account before the use of the related NPs in further biomedical applications.

Safety Assessments of Nickel Boride Nanoparticles on the Human Pulmonary Alveolar Cells by Using Cell Viability and Gene Expression Analyses.

Nickel boride is generally used in the steel industry as a melting accelerator due to its feature of creating a protective and stable attribute at high temperatures. It is also used to improve the hardenability of the steel with boron addition in the production. Thus, safety studies and biocompatibility analysis of nickel boride should be performed comprehensively to understand the limitations of use in various areas. In the present study, nickel boride nanoparticles (Ni2B NPs) were synthesized by a single-step method and molecule characterizations were performed via the use of X-ray diffraction analysis (XRD), transmission electron microscopy (TEM), scanning electron microscopy (SEM), and energy dispersive X-ray (EDX) analyses. Cytotoxicity properties of Ni2B NPs were identified on human pulmonary alveolar epithelial cells (HPAEpiC) by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), neutral red (NR), and lactate dehydrogenase (LDH) assays. Illumina human ht-12 v4.0 whole-genome microarray analysis was conducted to investigate NiB2 NPs effects on gene expression regulations of HPAEpiC cells. The database for annotation, visualization, and integrated discovery (DAVID) analysis was performed to reveal the relationship between Ni2B NP application and cellular pathway alterations. According to cytotoxicity analysis, the IC50 value for Ni2B NP application was found as 81.99 mg/L concentration. Microarray analysis of Ni2B NP application was shown for the first time that 693 gene expression changes (FC ≥ 2) occurred significantly over 40.000 gene probes and Ni2B NPs were observed to affect microtubule regulation, centrosome organization, and phosphoprotein synthesis.

Neuroprotective effects of boron nitride nanoparticles in the experimental Parkinson's disease model against MPP+ induced apoptosis.

Parkinson's disease (PD) is one of the most aggressive neurodegenerative diseases and characterized by the loss of dopamine-sensitive neurons in the substantia nigra region of the brain. There is no any definitive treatment to completely cure PD and existing treatments can only ease the symptoms of the disease. Boron nitride nanoparticles have been extensively studied in nano-biological studies and researches showed that it can be a promising candidate for PD treatment with its biologically active unique properties. In the present study, it was aimed to investigate ameliorative effects of hexagonal boron nitride nanoparticles (hBNs) against toxicity of 1-methyl-4-phenylpyridinium (MPP+) in experimental PD model. Experimental PD model was constituted by application of MPP+ to differentiated pluripotent human embryonal carcinoma cell (Ntera-2, NT-2) culture in wide range of concentrations (0.62 to 2 mM). Neuroprotective activity of hBNs against MPP+ toxicity was determined by cell viability assays including MTT and LDH release. Oxidative alterations by hBNs application in PD cell culture model were investigated using total antioxidant capacity (TAC) and total oxidant status (TOS) tests. The impacts of hBNs and MPP+ on nuclear integrity were analyzed by Hoechst 33258 fluorescent staining method. Acetylcholinesterase (AChE) enzyme activities were determined by a colorimetric assay towards to hBNs treatment. Cell death mechanisms caused by hBNs and MPP+ exposure was investigated by flow cytometry analysis. Experimental results showed that application of hBNs increased cell viability in PD model against MPP+ application. TAS and TOS analysis were determined that antioxidant capacity elevated after hBNs applications while oxidant levels were reduced. Furthermore, flow cytometric analysis executed that MPP+ induced apoptosis was prevented significantly (p < 0.05) after application with hBNs. In a conclusion, the obtained results indicated that hBNs have a huge potential against MPP+ toxicity and can be used in PD treatment as novel neuroprotective agent and drug delivery system.

Synthesis, characterization and cytotoxicity of boron nitride nanoparticles: emphasis on toxicogenomics.

Nanotechnology is increasingly developing area including more than 700 commercial products such as clothing, food preparation, cosmetics, mechanics, electronics and also health industry. People generally contact with nanoparticles by inhaling from air. Thus, it is becoming important issue to understand harmful effects of nanoparticles on human health and prepare risk reports for common nano-sized materials. In this paper, synthesis, characterization and cytotoxicity evaluation of boron nitride (BN) nanoparticles were performed on human primary alveolar epithelial cells (HPAEpiC) since, main exposure to nanoparticles would generally happen through lung via inhalation. Chemically synthetized BN nanoparticles were characterized by using X-ray crystallography, transmission electron microscope, scanning electron microscope and energy-dispersive X-ray spectroscopy techniques. 3-(4,5-dimethyl-thiazol-2-yl) 2,5-diphenyltetrazolium bromide, neutral red and lactate dehydrogenase release assays were used to analyze cytotoxicity after nanoparticles exposure. Whole genome microarray analysis was used to find out the effects of BN NPs on gene expressions of HPAEpiC cells. Finally, the database for annotation, visualization and integrated discovery analysis was used to reveal relationships between different cellular pathways and nanoparticle exposure. According to cytotoxicity analysis LC20 value for BN nanoparticles was 125.051 mg/L. Microarray results showed that 2159 genes expression change (FC ≥ 2) significantly over 40,000 genes analysis. When the gene pathways were analyzed, it was seemed that BN nanoparticles mostly affect cell cycle, cell-cell interactions, cancer affecting genes and signal transduction. In a conclusion, our results supported for the first time that BN nanoparticles could be used as a safe nanomaterial in both pharmacological and medical applications.

Microarray assisted toxicological investigations of boron carbide nanoparticles on human primary alveolar epithelial cells.

It is important to understand the adverse effects of nanoparticles on human health and to prepare risk reports for widely used nanoscale materials. Synthesis, characterization and cytotoxicity evaluation of B4C nanoparticles were performed on HPAEpiC since, first encounter with nanoparticles would generally happen through lung by inhaling chemicals. B4C nanoparticles were synthesized via chemical vapor deposition techniques and characterized by using transmission electron microscope (TEM), scanning electron microscope (SEM) and X-ray crystallography (XRD). 3-(4,5-dimethyl-thiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT), lactate dehydrogenase (LDH) and neutral red (NR) tests were used to analyze cell viability and cytotoxicity against nanoparticles exposure. Microarray analysis was used to discover whole genome effects of B4C NPs on gene expressions changes of HPAEpiC cells. Then, the database for annotation, visualization and integrated discovery (DAVID) analysis was performed to understand relationships between gene pathways and nanoparticle exposure. Finally, cytotoxicity analysis revealed that IC20 value for boron carbide (B4C) nanoparticles was 202.525 mg/L. According to microarray analysis 32 genes expression change significantly (FC ≥ 2) over 40,000 genes scanning. The gene pathways analysis showed that boron carbide (B4C) nanoparticles mostly affect amino acid biosynthesis process, TGF-beta signaling pathway and developmental proteins regulation. In conclusion, our results supported for the first time that boron carbide (B4C) nanoparticles could be used as a safe nanomaterial in both pharmacological and medical applications.

Toxicogenomic responses of human alveolar epithelial cells to tungsten boride nanoparticles.

During the recent years, microarray analysis of gene expression has become an inevitable tool for exploring toxicity of drugs and other chemicals on biological systems. Therefore, toxicogenomics is considered as a fruitful area for searching cellular pathways and mechanisms including cancer, immunological diseases, environmental responses, gene-gene interactions and chemical toxicity. In this work, we examined toxic effects of Tungsten Borides NPs on gene expression profiling of the human lung alveolar epithelial cells (HPAEpiC). In line with this purpose, a single crystal of tungsten boride (mixture of WB and W2B) nanoparticles was synthesized by means of zone melting method, and characterized via using X-ray crystallography (XRD), transmission electron microscope (TEM), scanning electron microscope (SEM) and energy-dispersive X-ray spectroscopy (EDX) techniques. Cell viability and cytotoxicity were determined by 3-(4,5-dimethyl-thiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT), neutral red (NR) and lactate dehydrogenase (LDH) release tests. The whole genome microarray expression analysis was performed to find out the effects of WB and W2B NPs mixture on gene expression of the HPAEpiC cell culture. 123 of 40,000 gene probes were assigned to characterize expression profile for WB/W2B NPs exposure. According to results; 70 genes were up-regulated and 53 genes were down-regulated (≥2 fold change). For further investigations, these genes were functionally classified by using DAVID (The Database for Annotation, Visualization and Integrated Discovery) with gene ontology (GO) analysis. In the light of the data gained from this study, it could be concluded that the mixture of WB/W2B NPs can affect cytokine/chemokine metabolism, angiogenesis and prevent migration/invasion by activating various genes.

Genotoxicity in primary human peripheral lymphocytes after exposure to lithium titanate nanoparticles in vitro.

Lithium titanate (Li2TiO3) nanoparticles (LTT NPs; <100 nm) are widely used in battery technology, porcelain enamels, and ceramic insulating bodies. With the increased applications of LTT NPs, the concerns about their potential human toxicity effects and their environmental impact were also increased. However, toxicity data for LTT NPs relating to human health are very limited. Therefore, the purpose of this study was to evaluate whether LTT NPs are able to induce genetic damage in human peripheral lymphocytes in vitro when taking into consideration that DNA damage plays an important role in carcinogenesis. With this aim, the chromosome aberrations (CA), sister chromatid exchanges (SCE), and micronucleus (MN) assays were used as genotoxicity end points. Human peripheral lymphocytes obtained from five healthy male volunteers were exposed to LTT NPs at final dispersed concentrations ranging from 0 to 1000 µg/mL for 72 h at 37°C. The obtained results indicated that LTT NPs compound did not induce DNA damage in human peripheral lymphocytes as depicted by CA/cell, SCE/cell, and MN/1000 cell values in all concentrations tested. In summary, our results revealed that exposure to LTT NPs is not capable of inducing DNA lesions in human peripheral lymphocytes for the first time.

Health risk assessments of lithium titanate nanoparticles in rat liver cell model for its safe applications in nanopharmacology and nanomedicine.

Due to their high chemical stability, lithium titanate (Li2TiO3) nanoparticles (LTT NPs) now are projected to be transferred into different nanotechnology areas like nano pharmacology and nano medicine. With the increased applications of LTT NPs for numerous purposes, the concerns about their potential human toxicity effects and their environmental impact are also increased. However, toxicity data for LTT NPs related to human health are very limited. Therefore we aimed to investigate toxicity potentials of various concentrations (0-1,000 ppm) of LTT NPs (<100 nm) in cultured primary rat hepatocytes. Cell viability was detected by [3-(4,5-dimethyl-thiazol-2-yl) 2,5-diphenyltetrazolium bromide] (MTT) assay and lactate dehydrogenase (LDH) release, while total antioxidant capacity (TAC) and total oxidative stress (TOS) levels were determined to evaluate the oxidative injury. DNA damage was analyzed by scoring liver micronuclei rates and by determining 8-oxo-2-deoxyguanosine (8-OH-dG) levels. The results of MTT and LDH assays showed that higher concentrations of dispersed LTT NPs (500 and 1,000 ppm) decreased cell viability. Also, LTT NPs increased TOS (300, 500 and 1,000 ppm) levels and decreased TAC (300, 500 and 1,000 ppm) levels in cultured hepatocytes. The results of genotoxicity tests revealed that LTT NPs did not cause significant increases of micronucleated hepatocytes and 8-OH-dG as compared to control culture. In conclusion, the obtained results showed for the first time that LTT NPs had dose dependent effects on oxidative damage and cytotoxicity but not genotoxicity in cultured primary rat hepatocytes for the first time.

The Risk Evaluation of Tungsten Oxide Nanoparticles in Cultured Rat Liver Cells for Its Safe Applications in Nanotechnology

Tungsten (VI) oxide (WO3) nanoparticles (NPs) are used for many industrial purposes in everyday life. However, their effects on human health have not been sufficiently evaluated. Therefore, the present study was designed to investigate the toxicity potentials of various concentrations (0 to 1000 ppm) of WO3 NPs (<100 nm particle size) in cultured primary rat hepatocytes. The results of cell viability assay showed that the higher concentrations of dispersed WO3 NPs (300, 500 and 1000 ppm) caused significant (p<0.05) decreases of cell viability. Also, dose dependent negative alterations were observed in oxidative status and antioxidant capacity levels after the application of WO3 in cultured rat primary hepatocytes. The results of genotoxicity tests revealed that these NPs did not cause significant increases of micronucleated hepatocytes (MNHEPs) but increased 8-oxo-2-deoxyguanosine (8-OH-dG) levels as compared to the control culture.

Evaluation of cytotoxic, oxidative stress and genotoxic responses of hydroxyapatite nanoparticles on human blood cells.

The present study was designed to investigate genotoxic and cytotoxic effects and oxidative damage of increasing concentrations of nano-hydroxyapatite (5, 10, 20, 50, 75, 100, 150, 300, 500 and 1000 ppm) in primary human blood cell cultures. Cell viability was detected by [3-(4,5-dimethyl-thiazol-2-yl) 2,5-diphenyltetrazolium bromide] assay and lactate dehydrogenase release, while total antioxidant capacity and total oxidative stress levels were determined to evaluate the oxidative injury. The DNA damage was also analyzed by sister chromatid exchange, micronuclei, chromosome aberration assays and 8-oxo-2-deoxyguanosine level as indicators of genotoxicity. The results of [3-(4,5-dimethyl-thiazol-2-yl) 2,5-diphenyltetrazolium bromide] and lactate dehydrogenase assays showed that the higher concentrations (150, 300, 500 and 1000 ppm) of hydroxyapatite nanoparticles (HAP NPs) decreased cell viability. HAP NPs led to increases of total oxidative stress (300, 500 and 1000 ppm) levels and decreased total antioxidant capacity (150, 300, 500 and 1000 ppm) levels in cultured human blood cells. On the basis of increasing concentrations, HAP NPs caused significant increases of sister chromatid exchange, micronuclei, chromosome aberration rates and 8-oxo-2-deoxyguanosine levels as compared to untreated culture. In conclusion, the obtained in vitro results showed that HAP NPs had dose-dependent effects on inducing oxidative damage, genotoxicity and cytotoxicity in human blood cells.