Plant extract-mediated biogenic synthesis of silver,manganese dioxide,silver-doped manganese dioxide nanoparticles and their antibacterial activity against food- and water-borne pathogens

Silver nanoparticles (AgNPs), manganese dioxide nanoparticles (MnO₂NPs) and silver-doped manganese dioxide nanoparticles (Ag-doped MnO₂NPs) were synthesized by simultaneous green chemistry reduction approach. Aqueous extract from the leaves of medicinally important plant Cucurbita pepo was used as reducing and capping agents. Various characterization techniques were carried out to affirm the formation of nanoparticles. HR-TEM analysis confirmed the size of nanoparticles in the range of 15–70 nm and also metal doping was confirmed through XRD and EDS analyses. FT-IR analysis confirmed that the presence of biomolecules in the aqueous leaves extract was responsible for nanoparticles synthesis. Further, the concentration of metals and their doping in the reaction mixture was achieved by ICP–MS. The growth curve and well diffusion study of synthesized nanoparticles were performed against food- and water-borne Gram-positive and Gram-negative bacterial pathogens. The mode of interaction of nanoparticles on bacterial cells was demonstrated through Bio-TEM analysis. Interestingly, AgNPs and Ag-doped MnO₂ NPs showed better antibacterial activity against all the tested bacterial pathogens; however, MnO₂NPs alone did not show any antibacterial properties. Hence, AgNPs and Ag-doped MnO₂ NPs synthesized from aqueous plant leaves extract may have important role in controlling various food spoilage caused by bacteria.     

Ultrastructural Analysis of Candida albicans When Exposed to Silver Nanoparticles

Candida albicans is the most common fungal pathogen in humans, and recently some studies have reported the antifungal activity of silver nanoparticles (AgNPs) against some Candida species. However, ultrastructural analyses on the interaction of AgNPs with these microorganisms have not been reported. In this work we evaluated the effect of AgNPs on C. albicans, and the minimum inhibitory concentration (MIC) was found to have a fungicidal effect. The IC₅₀ was also determined, and the use of AgNPs with fluconazole (FLC), a fungistatic drug, reduced cell proliferation. In order to understand how AgNPs interact with living cells, the ultrastructural distribution of AgNPs in this fungus was determined. Transmission electron microscopy (TEM) analysis revealed a high accumulation of AgNPs outside the cells but also smaller nanoparticles (NPs) localized throughout the cytoplasm. Energy dispersive spectroscopy (EDS) analysis confirmed the presence of intracellular silver. From our results it is assumed that AgNPs used in this study do not penetrate the cell, but instead release silver ions that infiltrate into the cell leading to the formation of NPs through reduction by organic compounds present in the cell wall and cytoplasm.     

Influence of outer membrane c‐type cytochromes on particle size and activity of extracellular nanoparticles produced by Shewanella oneidensis

The metal‐reducing bacterium Shewanella oneidensis is capable of reducing various metal(loid)s and produces nanoparticles (NPs) extracellularly, in which outer membrane c‐type cytochromes (OMCs) have been suggested to play important roles. The objective of this study was to investigate the influence of the OMCs, that is, MtrC and OmcA, on the size and activity of the extracellular silver NPs (AgNPs) and silver sulfide NPs (Ag₂S NPs) produced by S. oneidensis MR‐1. We found that (i) the lack of OMCs on S. oneidensis cell surface decreased the particle size of the extracellular biogenic AgNPs and Ag₂S NPs; (ii) the biogenic AgNPs from the mutant lacking OMCs showed higher antibacterial activity; and (iii) the biogenic Ag₂S NPs from the mutant lacking OMCs exhibited higher catalytic activity in methylviologen reduction. The results suggest that it may be possible to control particle size and activity of the extracellular biogenic NPs via controlled expression of the genes encoding surface proteins. In addition, we also reveal that in extracellular biosynthesis of NPs the usually neglected non‐cell‐associated NPs could have high catalytic activity, highlighting the need of novel methods that can efficiently retain extracellular NPs in the biosynthesis processes. Biotechnol. Bioeng. 2013; 110: 1831–1837. © 2013 Wiley Periodicals, Inc.     

Toxicity and interaction of titanium dioxide nanoparticles with microtubule protein

Titanium dioxide (TiO2) nanoparticles (NPs) are widely used in several manufactured products. The small size of NPs facilitates their uptake into cells as well as transcytosis across epithelial cells into blood and lymph circulation to reach different sites, such as the central nervous system. Different studies have shown the risks that TiO2 NPs in the neuronal system and other organs present. As membrane-bound layer aggregates or single particles, TiO2 NPs can enter not only cells, but also mitochondria and nuclei. Therefore these particles can interact with cytoplasmic proteins such as microtubules (MTs). MTs are cytoskeletal proteins that are essential in eukaryotic cells for a variety of functions, such as cellular transport, cell motility and mitosis. MTs in neurons are used to transport substances such as neurotransmitters. Single TiO2 NPs in cytoplasm can interact with these proteins and affect their crucial functions in different tissues. In this study, we showed the effects of TiO2 NPs on MT polymerization and structure using ultraviolet spectrophotometer and fluorometry. The fluorescent spectroscopy showed a significant tubulin conformational change in the presence of TiO2 NPs and the ultraviolet spectroscopy results showed that TiO2 NPs affect tubulin polymerization and decrease it. The aim of this study was to find the potential risks that TiO2 NPs pose to human organs and cells.     

Titanium nanoparticles influence the Akt/Tor signal pathway in the silkworm,Bombyx mori,silk gland

Various nanoparticles, such as silver nanoparticles (AgNPs) and titanium nanoparticles (TiO₂NPs) are increasingly used in industrial processes. Because they are released into the environment, research into their influence on the biosphere is necessary. Among its other effects, dietary TiO₂NPs promotes silk protein synthesis in silkworms, which prompted our hypothesis that TiO₂NPs influence protein kinase B (Akt)/Target of rapamycin (Tor) signaling pathway (Akt/Tor) signaling in their silk glands. The Akt/Tor signaling pathway is a principle connector integrating cellular reactions to growth factors, metabolites, nutrients, protein synthesis, and stress. We tested our hypothesis by determining the influence of dietary TiO₂NPs (for 72 h) and, separately, of two Akt/Tor pathway inhibitors (LY294002 and rapamycin) on expression of Akt/Tor signaling pathway genes and proteins in the silk glands. TiO₂NPs treatments led to increased accumulation of mRNAs for Akt, Tor1 and Tor2 by 1.6‐, 12.1‐, and 4.8‐fold. Dietary inhibitors led to 2.6‐ to 4‐fold increases in mRNAs encoding Akt and substantial decreases in mRNAs encoding Tor1 and Tor2. Western blot analysis showed that dietary TiO₂NPs increased the phosphorylation of Akt and its downstream proteins. LY294002 treatments led to inhibition of Akt phosphorylation and its downstream proteins and rapamycin treatments similarly inhibited the phosphorylation of Tor‐linked downstream proteins. These findings support our hypothesis that TiO₂NPs influence Akt/Tor signaling in silk glands. The significance of this work is identification of specific sites of TiO₂NPs actions.     

Synthesis of silver nanoparticles using plant derived 4-N-methyl benzoic acid and evaluation of antimicrobial,antioxidant and antitumor activity

The present study is to investigate the antitumor, antioxidant and antibacterial potential of silver nanoparticles (Ag NPs) synthesized from a phenolic derivative 4-N-methyl benzoic acid, isolated from a medicinal plant (Memecylon umbellatum Burm F). The Bio-inspired nanoparticles (NPs) were analyzed by using UV–vis spectroscopy, FTIR, HRTEM, Zeta potential and XRD techniques. The UV–vis spectroscopy study at the band of 430 nm confirmed the nanoparticles formation. HRTEM report showed that the AgNPs synthesized were in the size range 7–23 nm. The harvested nanoparticles were subjected to anti-bacterial assay and a dose dependent inhibitory action was observed against the tested human pathogens. Among the tested bacteria, Acinetobacter baumannii was found to be highly sensitive to AgNPs (diameter of zone of inhibition was 31 mm). Further, the silver nanoparticles exhibited a good anti-tumor activity against the breast cancer cell line (MCF 7) with an IC₅₀ value of 42.19 µg/mL. As the present study confirmed a good antibacterial, antioxidant and antitumor activity in the nanoparticles synthesized using 4-N-methyl benzoic acid derived from a medicinal plant, the product can be further tested to formulate a good lead compound for biomedical applications.     

Probing the interaction of silver nanoparticles with tau protein and neuroblastoma cell line as nervous system models

Interestingly pharmaceutical sciences are using nanoparticles (NPs) to design and develop nanomaterials-based drugs. However, up to recently, it has not been well realized that NPs themselves may impose risks to the biological systems. In this study, the interaction of silver nanoparticles (AgNPs) with tau protein and SH-SY5Y neuroblastoma cell line, as potential nervous system models, was examined with a range of techniques including intrinsic fluorescence spectroscopy, circular dichroism (CD) spectroscopy, 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and acridine orange/ethidium bromide (AO/EB) dual staining method. Fluorescence study showed that AgNPs with a diameter of around 10–20 nm spontaneously form a static complex with tau protein via hydrogen bonds and van der Waals interactions. CD experiment revealed that AgNPs did not change the random coil structure of tau protein. Moreover, AgNPs showed to induce SH-SY5Y neuroblastoma cell mortality through fragmentation of DNA which is a key feature of apoptosis. In conclusion, AgNPs may induce slight changes on the tau protein structure. Also, the concentration of AgNPs is the main factor which influences their cytotoxicity. Since, all adverse effects of NPs are not well detected, so probably additional more specific testing would be needed.     

Effects of Developmental Exposure to TiO2 Nanoparticles on Synaptic Plasticity in Hippocampal Dentate Gyrus Area: an In Vivo Study in Anesthetized Rats

With the increasing applications of titanium dioxide nanoparticles (TiO(2) NPs) in industry and daily life, an increasing number of studies showed that TiO(2) NPs may have negative effects on the respiratory or metabolic circle systems of organisms, while very few studies focused on the brain central nervous system (CNS). Synaptic plasticity in hippocampus is believed to be associated with certain high functions of CNS, such as learning and memory. Thus, in this study, we investigated the effects of developmental exposure to TiO(2) NPs on synaptic plasticity in rats' hippocampal dentate gyrus (DG) area using in vivo electrophysiological recordings. The input/output (I/O) functions, paired-pulse reaction (PPR), field excitatory postsynaptic potential, and population spike amplitude were measured. The results showed that the I/O functions, PPR, and long-term potentiation were all attenuated in lactation TiO(2) NPs-exposed offspring rats compared with those in the control group. However, in the pregnancy TiO(2) NPs exposure group, only PPR was attenuated significantly. These findings suggest that developmental exposure to TiO(2) NPs could affect synaptic plasticity in offspring's hippocampal DG area in vivo, which indicates that developmental brains, especially in lactation, are susceptible to TiO(2) NPs exposure. This study reveals the potential toxicity of TiO(2) NPs in CNS. It may give some hints on the security of TiO(2) NPs production and application and shed light on its future toxicological studies.     

Functionalization of cotton fabrics with titanium oxide doped silver nanoparticles: Antimicrobial and UV protection activities

The target of our current work was designed to prepare titanium oxide doped silver nanoparticles (Ag/TiO₂NPs) and their impact on the functionalization of cotton fabrics. Additionally, the effect of Ag/TiO₂NPs was compared with the individually prepared silver nanoparticles (AgNPs) and titanium oxide nanoparticles (TiO₂NPs). In this work, AgNPs were prepared in the solid state using arabic gum as efficient stabilizing and reducing agent. Then, two concentrations of the as-synthesized nanoparticles were used to functionalize the cotton fabrics by pad-dry-cure treatment in the presence of fixing agent to increase the durability of treated cotton fabrics against vigorous washing cycles. The findings implied that the as-prepared nanoparticles were successfully synthesized in nano-size with spherical shape and homogeneity. The efficacy of the functionalized cotton fabrics with those nanoparticles were evaluated in terms of multifunctional properties including antimicrobial and ultraviolet protection factor (UPF) and the mechanical features before and after many washing cycles; 10, 15 and 20 times. The resultant also proved that Ag/TiO₂NPs-treated cotton fabrics exhibited the greater values of both antimicrobial and UPF properties with enhancement in the tensile strength and elongation features. Thus, the combination between these two nanoparticles through doping reaction is suitable for imparting superior antimicrobial properties against the four tested microbial species (Staphylococcus aureus, Escherichia coli, Candida albicans, and Aspergillus niger) and good UPF properties. Depending on the promising obtained results of the multi-finishing fabrics, these nanoparticles of Ag/TiO₂NPs can be applied for the production of an efficient medical clothes for doctors, nurses and bed sheets for patients in order to kill and prevent the spread of bacteria and then, reduce the transmission of infection to others.     

Identifying Metal Nanoparticle Size Effect on Sensing Common Human Plasma Protein by Counting the Sensitivity of Optical Absorption Spectra Damping

Colloidal nanoparticles (NPs) interact with biological fluids such as human plasma to form a protein coating (corona) on the surface of NPs (NP-protein complex). However, the impact of size and type of NPs on binding of the hard corona to the surface of NPs as well as damping of their optical spectra has not been systematically explored. To elucidate the interaction between biological environment (human plasma) and NPs, a photophysical measurement was conducted to quantify the interaction of two different types of NPs (gold (Au) and silver (Ag)) with common human plasma proteins. The colloidal AuNPs and AgNPs were electrostatically stabilized and varied in diameter from 10 to 80 nm in the presence of common human plasma. The sizes of the NPs were determined using transmission electron microscopy (TEM). Optical absorption spectra were obtained for the complexes. Dynamic light scattering (DLS) measurement and zeta potential were used to characterize the sizes, hydrodynamic diameters, and surface charges of the protein-NPs complexes. Protein separation was performed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to isolate and identify the protein bands. The absorption of proteins to the NPs was found to be strongly dependent on the size and type of NPs. The distance between surface of NPs by absorbed protein bound to the NPs gradually increased with size of NPs, particularly for AgNPs with primary diameter of < 50 nm. The chi-square test proved that AgNPs are a good candidate in sensing the protein complex in human plasma compared with AuNPs mainly for the AgNPs with diameter sized 50 nm.

     

The influence of H2 partial pressure on biogenic palladium nanoparticle production assessed by single-cell ICP-mass spectrometry

The production of biogenic palladium nanoparticles (bio-Pd NPs) is widely studied due to their high catalytic activity, which depends on the size of nanoparticles (NPs). Smaller NPs (here defined as <100 nm) are more efficient due to their higher surface/volume ratio. In this work, inductively coupled plasma-mass spectrometry (ICP-MS), flow cytometry (FCM) and transmission electron microscopy (TEM) were combined to obtain insight into the formation of these bio-Pd NPs. The precipitation of bio-Pd NPs was evaluated on a cell-per-cell basis using single-cell ICP-MS (SC-ICP-MS) combined with TEM images to assess how homogenously the particles were distributed over the cells. The results provided by SC-ICP-MS were consistent with those provided by “bulk” ICP-MS analysis and FCM. It was observed that heterogeneity in the distribution of palladium over an entire cell population is strongly dependent on the Pd²⁺ concentration, biomass and partial H₂ pressure. The latter three parameters affected the particle size, ranging from 15.6 to 560 nm, and exerted a significant impact on the production of the bio-Pd NPs. The TEM combined with SC-ICP-MS revealed that the mass distribution for bacteria with high Pd content (144 fg Pd cell⁻¹) indicated the presence of a large number of very small NPs (D50 = 15.6 nm). These results were obtained at high cell density (1 × 10⁵ ± 3 × 10⁴ cells μl⁻¹) and H₂ partial pressure (180 ml H₂). In contrast, very large particles (D50 = 560 nm) were observed at low cell density (3 × 10⁴ ± 10 × 10² cells μl⁻¹) and H₂ partial pressure (10–100 ml H₂). The influence of the H₂ partial pressure on the nanoparticle size and the possibility of size-tuned nanoparticles are presented.     

Biogenic synthesis of silver nanoparticles: Antibacterial and cytotoxic potential

In green chemistry, the application of a biogenic material as a mediator in nanoparticles formation is an innovative nanotechnology. Our current investigation aimed at testing the cytotoxic potential and antimicrobial ability of silver nanoparticles (AgNPs) that were prepared using Calligonum comosum roots and Azadirachta indica leaf extracts as stabilizing and reducing agents. An agar well diffusion technique was employed to detect synthesized AgNPs antibacterial ability on Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus bacterial strains. Furthermore, their cytotoxic capability against LoVo, MDA-MB231 and HepG2 ca cells was investigated. For phyto-chemical detection in the biogenic AgNPs the Fourier-transform infrared spectroscopy (FT-IR) was considered. Zeta sizer, TEM (Transmission Electron Microscope) and FE-SEM (Field Emission Scanning Electron Microscope) were used to detect biogenic AgNPs’ size and morphology. The current results showed the capability of tested plant extract for conversion of Ag ions to AgNPs with a mean size ranging between 90.8 ± 0.8 and 183.2 ± 0.7 nm in diameter. Furthermore, prepared AgNPs exhibited apoptotic potential against HepG2, LoVo, and MDA-MB 231cell with IC₅₀ ranging between 10.9 and 21.4 μg/ml and antibacterial ability in the range of 16.0 ± 0.1 to 22.0 ± 1.8 mm diameter. Activation of caspases in AgNPs treated cells could be the main indicator for their positive effect causing apoptosis. The current investigation suggested that the green production of AgNPs could be a suitable substitute to large-scale production of AgNPs, since stable and active nanoparticles could be obtained.     

In-vitro evaluation of copper/copper oxide nanoparticles cytotoxicity and genotoxicity in normal and cancer lung cell lines

BackgroundNanotoxicology is a major field of study that reveals hazard effects of nanomaterials on the living cells.MethodsIn the present study, Copper/Copper oxide nanoparticles (Cu/CuO NPs) were prepared by the chemical reduction method and characterized by different techniques such as: X-Ray Diffraction, Transmission and Scanning Electron Microscopy. Evaluation of the toxicity of Cu/CuO NPs was performed on 2 types of cells: human lung normal cell lines (WI-38) and human lung carcinoma cell (A549). To assess the toxicity of the prepared Cu/CuOs NPs, the two cell types were exposed to Cu/CuO NPs for 72 h. The half-maximal inhibitory concentration IC₅₀ of Cu/CuO NPs for both cell types was separately determined and used to examine the cell genotoxicity concurrently with the determination of some oxidative stress parameters: nitric oxide, glutathione reduced, hydrogen peroxide, malondialdehyde and superoxide dismutase.ResultsCu/CuO NPs suppressed proliferation and viability of normal and carcinoma lung cells. Treatment of both cell types with their IC₅₀’s of Cu/CuO NPs resulted in DNA damage besides the generation of reactive oxygen species and consequently the generation of a state of oxidative stress.ConclusionOverall, it can be concluded that the IC₅₀'s of the prepared Cu/CuO NPs were cytotoxic and genotoxic to both normal and cancerous lung cells.     

Antioxidant,antibacterial and cytotoxic potential of silver nanoparticles synthesized using terpenes rich extract of Lantana camara L. leaves

Several attempts have been made for green synthesis of silver nanoparticles (AgNPs) using different plant extracts. Present study revealed that, antioxidant, antibacterial and cytotoxic AgNPs were synthesized using terpenes-rich extract (TRE) of environmentally notorious Lantana camara L. leaves. AgNPs were characterized by advanced techniques like UV–Visible and Infra red spectroscopy; XRD, SEM techniques as terpenes coated sphere shaped NPs with average diameter 425 nm. Further, on evaluation, AgNPs were found to exhibit dose – dependent antioxidant potential, good to moderate antibacterial activity against Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa; and toxicity on Brine shrimp (A. salinanauplii) with LD₅₀ value 514.50 µg/ml.     

Inhibitory effect of gonadotrophin-releasing hormone (GnRH) on rat granulosa cell deoxyribonucleic acid synthesis

Gonadotropin-releasing hormone (GnRH) has been found to be expressed within the ovary and to modulate cell differentiation in ovarian cells. In the present study we have analyzed the influence of GnRH on DNA synthesis in rat granulosa cells. Cells were obtained from immature DES-treated rats and cultured in defined medium (DMEM:F12) containing combinations of FSH, estradiol, and transforming growth factor-β (TGF-β), both in the presence and absence of GnRH. A GnRH analog, Leuprolide (GnRHa), caused a dose-dependent inhibition of ³H-thymidine incorporation in cells cultured in the presence of FSH (20 ng/ml) and TGFβ (2.5 ng/ml), at concentrations as low as 5 × 10⁻¹¹ M. Similarly, a complete inhibition of hormonally stimulated DNA synthesis were observed with another analog (Buserelin, ED₅₀ = 1.58 ± 0.22 × 10⁻¹⁰ M) and native GnRH (ED₅₀ = 1.4 ± 0.3 × 10⁻⁶ M). A competitive antagonist of GnRH (Antide) was used to neutralize the GnRH agonist effects. Antide 10⁻⁸ M could prevent the inhibition elicited by 10⁻⁷ M of Leuprolide. These results suggest that GnRH may play a role in the regulation of rat granulosa cell proliferation during follicular development. Mol. Reprod. Dev. 47: 170–174, 1997. © 1997 Wiley-Liss, Inc.     

Gold- and Silver Nanoparticles Affect the Growth Characteristics of Human Embryonic Neural Precursor Cells

Rapid development of nanotechnologies and their applications in clinical research have raised concerns about the adverse effects of nanoparticles (NPs) on human health and environment. NPs can be directly taken up by organs exposed, but also translocated to secondary organs, such as the central nervous system (CNS) after systemic- or subcutaneous administration, or via the olfactory system. The CNS is particularly vulnerable during development and recent reports describe transport of NPs across the placenta and even into brain tissue using in vitro and in vivo experimental systems. Here, we investigated whether well-characterized commercial 20 and 80 nm Au- and AgNPs have an effect on human embryonic neural precursor cell (HNPC) growth. After two weeks of NP exposure, uptake of NPs, morphological features and the amount of viable and dead cells, proliferative cells (Ki67 immunostaining) and apoptotic cells (TUNEL assay), respectively, were studied. We demonstrate uptake of both 20 and 80 nm Au- and AgNPs respectively, by HNPCs during proliferation. A significant effect on the sphere size- and morphology was found for all cultures exposed to Au- and AgNPs. AgNPs of both sizes caused a significant increase in numbers of proliferating and apoptotic HNPCs. In contrast, only the highest dose of 20 nm AuNPs significantly affected proliferation, whereas no effect was seen on apoptotic cell death. Our data demonstrates that both Au- and AgNPs interfere with the growth profile of HNPCs, indicating the need of further detailed studies on the adverse effects of NPs on the developing CNS.     

Ghrelin and obestatin can promote human ovarian granulosa cell functions and FSH effects

The aim of the present in-vitro experiments was to examine the direct influence of ghrelin and obestatin on viability, proliferation and progesterone release by human ovarian granulosa cells and their response to FSH administration. Human granulosa cells were cultured in presence of ghrelin or obestatin (both at 0, 1, 10 or 100 ng/ml) alone or in the presence of FSH (10 ng/ml). Cell viability, accumulation of proliferation markers PCNA and cyclin B1 and release of progesterone were analyzed by Trypan blue extrusion test, quantitative immunocytochemistry and ELISA. Ghrelin, obestatin and FSH up-regulated all the measured ovarian cell parameters. Moreover, both ghrelin and obestatin promoted all the stimulatory effects of FSH. The obtained results demonstrate the direct stimulatory action of ghrelin, obestatin and FSH on basic ovarian cell functions, as well as the ability of metabolic hormones to improve FSH action on human ovarian cells.     

In vitro genotoxic effects of titanium dioxide nanoparticles (n‐TiO2) in human sperm cells

Titanium dioxide nanoparticles (TiO₂‐NPs) are one of the most widely engineered nanoparticles used. The study has been focused on TiO ₂‐NPs genotoxic effects on human spermatozoa in vitro. TiO ₂‐NPs are able to cross the blood–testis barrier induced inflammation, cytotoxicity, and gene expression changes that lead to impairment of the male reproductive system. This study presents new data about DNA damage in human sperms exposed in vitro to two n‐TiO ₂ concentrations (1 µg/L and 10 µg/L) for different times and the putative role of reactive oxygen species (ROS) as mediators of n‐TiO ₂ genotoxicity. Primary n‐TiO ₂ characterization was performed by transmission electron microscopy. The dispersed state of the n‐TiO ₂ in media was spectrophotometrically determined at 0, 24, 48, and 72 hr from the initial exposure. The genotoxicity has been highlighted by different experimental approaches (comet assay, terminal deoxynucleotidyl transferase dUTP nick end labeling [TUNEL] test, DCF assay, random amplification of polymorphic DNA polymerase chain reaction [RAPD‐PCR]). The comet assay showed a statistically significant loss of sperm DNA integrity after 30 min of exposure. Increased threshold of sperm DNA fragmentation was highlighted after 30 min of exposure by the TUNEL Test. Also, the RAPD‐PCR analysis showed a variation in the polymorphic profiles of the sperm DNA exposed to n‐TiO ₂. The evidence from the DCF assay showed a statistically significant increase in intracellular ROS linked to n‐TiO ₂ exposure. This research provides the evaluation of n‐TiO ₂ potential genotoxicity on human sperm that probably occurs through the production of intracellular ROS.     

High Content Analysis Provides Mechanistic Insights on the Pathways of Toxicity Induced by Amine-Modified Polystyrene Nanoparticles

The fast-paced development of nanotechnology needs the support of effective safety testing. We have developed a screening platform measuring simultaneously several cellular parameters for exposure to various concentrations of nanoparticles (NPs). Cell lines representative of different organ cell types, including lung, endothelium, liver, kidney, macrophages, glia, and neuronal cells were exposed to 50 nm amine-modified polystyrene (PS-NH₂) NPs previously reported to induce apoptosis and to 50 nm sulphonated and carboxyl-modified polystyrene NPs that were reported to be silent. All cell lines apart from Raw 264.7 executed apoptosis in response to PS-NH₂ NPs, showing specific sequences of EC50 thresholds; lysosomal acidification was the most sensitive parameter. Loss of mitochondrial membrane potential and plasma membrane integrity measured by High Content Analysis resulted comparably sensitive to the equivalent OECD-recommended assays, allowing increased output. Analysis of the acidic compartments revealed good cerrelation between size/fluorescence intensity and dose of PS-NH₂ NPs applied; moreover steatosis and phospholipidosis were observed, consistent with the lysosomal alterations revealed by Lysotracker green; similar responses were observed when comparing astrocytoma cells with primary astrocytes. We have established a platform providing mechanistic insights on the response to exposure to nanoparticles. Such platform holds great potential for in vitro screening of nanomaterials in highthroughput format.