Thallium Toxicity and its Interference with Potassium Pathways Tested on Various Cell Lines.

Thallium (Tl) is a highly toxic heavy metal whose mechanism of toxicity is still not completely understood. The aim of this study was to test Tl cytotoxicity on several cell lines of different tissue origin in order to clarify specific Tl toxicity to a particular organ. In addition, possible interference of Tl with cell potassium (K) transport was examined. Human keratinocytes (HaCaT), human hepatocellular carcinoma (HepG2), porcine kidney epithelial cells (PK15), human neuroblastoma (SH-SY5Y) and Chinese hamster lung fibroblast cells (V79) were treated with thallium (I) acetate in a wide concentration range (3.9-500 µg/mL) for 24 h, 48 and 72 h. To assess competitive interaction between Tl and K, the cells were treated with four Tl concentrations close to IC50 (15.63, 31.25, 62.50, 125 µg/mL) in combination with/or without potassium (I) acetate (500 µg/mL). The cells' morphology was monitored, and cytotoxic effect was assessed by 3-(4, 5-dimethylthiazole-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) test. The most sensitive to Tl exposure were SH-SY5Y cells, while HepG2 were the most resistant. The combined exposure to thallium (I) acetate and potassium (I) acetate for every cell line, except V79 cells, resulted in higher cell viability compared to thallium (I) acetate alone. The results of our study indicate that cell sensitivity to Tl treatment is largely affected by tissue culture origin, its function, and Na+/K+-ATPase activity.

Particle surface functionalization affects mechanism of endocytosis and adverse effects of silver nanoparticles in mammalian kidney cells.

Silver nanoparticles (AgNPs) show a plethora of possible applications due to their antimicrobial properties. Different coatings of AgNPs are used in order to increase stability, availability, and activity. However, the question about the toxicity after prolonged exposure still remains. Here, we show that different surface coatings affect in vitro toxicity and internalization of AgNPs in porcine kidney (PK15) cells. AgNPs coated with cetyltrimethylammonium bromide (CTAB), poly(vinylpyrrolidone) (PVP), sodium bis(2-ethylhexyl)-sulfosuccinate (AOT), poly-L-lysine (PLL), and bovine serum albumin (BSA) were toxic at the concentration of 10 mg Ag/L and higher. The toxicity increased in the following manner: PVP-AgNPs < CTAB-AgNPs < PLL-AgNPs < AOT-AgNPs < BSA-AgNPs. All types of AgNPs were internalized by the PK15 cells in a dose-dependent manner with greater internalization of AgNPs bearing positive surface charge. Transmission electron microscopy (TEM) experiments showed that AgNPs were located in the lysosomal compartments, while the co-treatment with known inhibitors of endocytosis pathways suggested macropinocytosis as the preferred internalization pathway. When inside the cell, all types of AgNPs induced the formation of reactive oxygen species while decreasing the concentration of the cell's endogenous antioxidant glutathione. The comet assay indicated possible genotoxicity of tested AgNPs starting at the concentration of 2 mg Ag/L or higher, depending on the surface functionalization. This study demonstrates the toxicity of AgNPs pointing to the importance of biosafety evaluation when developing novel AgNPs-containing materials.

Toxicity of nanomixtures to human macrophages: Joint action of silver and polystyrene nanoparticles.

Increasing use of nano-enabled products provides many benefits in various industrial processes and medical applications, but it also raises concern about release of nanoparticles (NPs) into the environment and subsequent human exposure. While potential toxicity of individual NPs types has been well described in scientific literature, exposure and health-related effects of nanomixtures has been poorly described. This study aimed to evaluate the combined effect of silver (AgNP) and polystyrene NPs (PSNP) on the human macrophages. AgNP are one of the most commercialized NPs due to efficient antimicrobial activity, while PSNP are ubiquitous in terrestrial and aquatic environments due to plastic pollution and degradation of polystyrene-based products. Differentiated monocytic cell line THP-1 were used as an in vitro model of human macrophages. Multiple aspects of cellular response to AgNP-PSNP nanomixture were analyzed including cell death, induction of apoptosis, oxidative stress response, expression of pro- and anti-inflammatory cytokines, and nanomechanical properties of cells. NPs uptake was visualized by confocal microscopy and quantified using flow cytometry. Results show that nanomixture increased apoptosis and cell death, expression of IL-6, IL-8 and TNFa, oxidative stress and mitochondrial dysfunction in cells compared to AgNP and PSNP applied as single treatments, indicating mixture additive action. Anti-inflammatory cytokines IL1b, IL-4 and IL-10 were not affected by combined exposure compared to single NPs. Visualization of NPs uptake and internalization showed that AgNP and PSNP were localized mostly in cytoplasm, with small fraction of AgNP translocated into cell nuclei, which explain increased number of double-stranded DNA breaks following exposure of cells to AgNPs alone or in the mixture. Study outcomes represent clear warnings on the human co-exposure to AgNP and PSNP that needs to be implemented in risk assessment approaches towards toxic-free environment.

Imaging mass spectrometry differentiates the effects of doxorubicin formulations on non-targeted tissues.

Administration of cytotoxic agents like doxorubicin (DOX) is restrained by the effects on different non-targeted/non-cancerous tissues, which instigates the development of nano-enabled drug delivery systems, among others. In this study, imaging mass spectrometry (IMS) was selected to examine the effects of DOX nanoformulations on non-targeted tissues. Chemical alterations induced by liposomal (LPS) and poly (lactic-co-glycolic acid) (PLG) nanoformulations were assessed against the ones induced by the conventional (CNV) formulation. Kidney cryosections of the treated and control Wistar rats were used as a model of the non-targeted tissue and analyzed by MALDI TOF IMS in the 200-1000 Da m/z range. Principal component analysis (PCA) and Volcano plots of the average mass spectra demonstrated a large overlap between treatments. However, the Venn diagram of significant m/z values revealed a nanoformulation-specific fingerprint consisting of 59 m/z values, which set them apart from the CNV formulation characterized by the fingerprint of 22 significant m/z values. Fingerprint m/z values that were putatively annotated by metabolome database search were linked to apoptosis, cell migration and proliferation. In CNV and PLG cases, false discovery rate adjusted ANOVA showed no differences in the spatial distribution of fingerprint m/z values between the histological substructures like glomeruli and convoluted tubules indicating their tissue-nonselective effect. LPS caused the least significant changes in m/z values and some of the LPS-specific fingerprint m/z values were primarily distributed in the glomeruli. The IMS based procedure successfully differentiated the effects of DOX formulations on the model non-targeted tissue, thus indicating the importance of IMS in effective drug development.

Cytotoxicity of nanomixture: Combined action of silver and plastic nanoparticles on immortalized human lymphocytes.

BACKGROUND: Silver nanoparticles (AgNP) are one of the most commercialized types of nanomaterials, with a wide range of applications owing to their antimicrobial activity. They are particularly important in hospitals and other healthcare settings, where they are used to maintain sterility of surfaces, textiles, catheters, medical implants, and more. However, AgNP can not only harm bacteria, but also damage mammalian cells and tissue. While the potential toxicity of AgNP is an understood risk, there is a lack of data on their toxicity in combination with polymeric materials, especially plastic nanoparticles such as polystyrene nanoparticles (PSNP) that can be released from surfaces of polystyrene devices during their medical use. AIM: This study aimed to investigate combined effect of AgNP and nanoplastics on human immune response. METHODS: Cells were treated with a range of PSNP and AgNP concentrations, either applied alone or in combination. Cytotoxicity, induction of apoptosis, generation of oxidative stress, uptake efficiency, intracellular localization and nanomechanical cell properties were selected as exposure biomarkers. RESULTS: Collected experimental data showed that nanomixture induced oxidative stress, apoptosis and mortality of Jurkat cells stronger than its individual components. Cell treatment with AgNP/PSNP mixture also significantly changed cell mechanical properties, evidenced by reduction of cells' Young Modulus. CONCLUSION: AgNP and PSNP showed additive toxic effects on immortalized human lymphocytes, evidenced by increase in cellular oxidative stress, induction of apoptosis, and reduction of cell stiffness. These results have important implications for using AgNP and PSNP in medical contexts, particularly for long-term medical implants.

Effects of a 3-Week Hospital-Controlled Very-Low-Calorie Diet in Severely Obese Patients.

Although a very-low-calorie diet (VLCD) is considered safe and has demonstrated benefits among other types of diets, data are scarce concerning its effects on improving health and weight loss in severely obese patients. As part of the personalized weight loss program developed at the Duga Resa Special Hospital for Extended Treatment, Croatia, we evaluated anthropometric, biochemical, and permanent DNA damage parameters (assessed with the cytochalasin B-blocked micronucleus cytome assay-CBMN) in severely obese patients (BMI ≥ 35 kg m-2) after 3-weeks on a 567 kcal, hospital-controlled VLCD. This is the first study on the permanent genomic (in)stability in such VLCD patients. VLCDs caused significant decreases in weight (loss), parameters of the lipid profile, urea, insulin resistance, and reduced glutathione (GSH). Genomic instability parameters were lowered by half, reaching reference values usually found in the healthy population. A correlation was found between GSH decrease and reduced DNA damage. VLCDs revealed susceptible individuals with remaining higher DNA damage for further monitoring. In a highly heterogeneous group (class II and III in obesity, differences in weight, BMI, and other categories) consisting of 26 obese patients, the approach demonstrated its usefulness and benefits in health improvement, enabling an individual approach to further monitoring, diagnosis, treatment, and risk assessment based on changing anthropometric/biochemical VLCD parameters, and CBMN results.

Sex affects the response of Wistar rats to polyvinyl pyrrolidone (PVP)-coated silver nanoparticles in an oral 28 days repeated dose toxicity study.

BACKGROUND: Silver nanoparticles (AgNPs) are widely used in biomedicine due to their strong antimicrobial, antifungal, and antiviral activities. Concerns about their possible negative impacts on human and environmental health directed many researchers towards the assessment of the safety and toxicity of AgNPs in both in vitro and in vivo settings. A growing body of scientific information confirms that the biodistribution of AgNPs and their toxic effects vary depending on the particle size, coating, and dose as well as on the route of administration and duration of exposure. This study aimed to clarify the sex-related differences in the outcomes of oral 28 days repeated dose exposure to AgNPs. METHODS: Wistar rats of both sexes were gavaged daily using low doses (0.1 and 1 mg Ag/kg b.w.) of polyvinylpyrrolidone (PVP)-coated small-sized (10 nm) AgNPs. After exposure, blood and organs of all rats were analysed through biodistribution and accumulation of Ag, whereas the state of the liver and kidneys was evaluated by the levels of reactive oxygen species (ROS) and glutathione (GSH), catalase (CAT) activity, superoxide dismutase (SOD) and glutathione peroxidase (GPx), expression of metallothionein (Mt) genes and levels of Mt proteins. RESULTS: In all animals, changes in oxidative stress markers and blood parameters were observed indicating the toxicity of AgNPs applied orally even at low doses. Sex-related differences were noticed in all assessed parameters. While female rats eliminated AgNPs from the liver and kidneys more efficiently than males when treated with low doses, the opposite was observed for animals treated with higher doses of AgNPs. Female Wistar rats exposed to 1 mg PVP-coated AgNPs/kg b.w. accumulated two to three times more silver in the blood, liver, kidney and hearth than males, while the accumulation in most organs of digestive tract was more than ten times higher compared to males. Oxidative stress responses in the organs of males, except the liver of males treated with high doses, were less intense than in the organs of females. However, both Mt genes and Mt protein expression were significantly reduced after treatment in the liver and kidneys of males, while they remained unchanged in females. CONCLUSIONS: Observed toxicity effects of AgNPs in Wistar rats revealed sex-related differences in response to an oral 28 days repeated exposure.

Assessment of intracellular accumulation of cadmium and thallium.

The aim of the study was to develop fast and accurate method for assessment of intracellular level of cadmium (Cd) and thallium (Tl), and to establish accumulation of the metals in the cells. HepG2 cells were treated with Cd or Tl (1.0 or 10.0 mg/L; 24 h) and level of Cd or Tl was assessed. ICP-MS was applied and the method was optimized and validated. Correlation coefficient (R2) for Cd was 0.9999 with intercept 0.0732 while for Tl was 1.00009 with intercept -0.1497, and limit of detection (LOD) for Cd was 0.020 µg/L and for Tl 0.097 µg/L. Both metals, Cd and Tl, accumulate in the cells in concentration-dependent manner. However, higher uptake of Cd in comparison to Tl was observed. Cells treated with the same concentration of the metal (1.0 mg/L) accumulated 10.0% of Cd and 1.0% of Tl. Higher uptake of Cd than Tl can explain higher toxicity of Cd toward HepG2 cells. Obtained results imply to the importance of monitoring the level of metals in the cells in order to connect changes at the molecular level with exposure to specific metal.

Interaction of Differently Coated Silver Nanoparticles With Skin and Oral Mucosal Cells.

Silver nanoparticles (AgNP) can be found in different consumer products and various medical devices due to their excellent biocidal properties. Despite extensive scientific literature reporting biological effects of AgNP, there is still a lack of scientific evidence on how different surface functionalization affects AgNP interaction with the human skin and the oral epithelium. This study aimed to investigate biological consequences following the treatment of HaCaT and TR146 cells with AgNP stabilized with negatively charged sodium bis(2-ethylhexyl)-sulfosuccinate (AOT), neutral polyvinylpyrrolidone (PVP), and positively charged poly-l-lysine (PLL). All AgNP were characterized by means of size, shape and surface charge. Interactions with biological barriers were investigated in vitro by determining cell viability, particle uptake, oxidative stress response and DNA damages following AgNP treatment. Results showed a significant difference in cytotoxicity depending on the surface coating used for AgNP stabilization. All three types of AgNP induced apoptosis, oxidative stress response and DNA damages in cells, but AOT- and PVP-coated AgNP exhibited lower toxicity than positively charged PLL-AgNP. Considering the number of data gaps related to the safe use of nanomaterials in biomedicine, this study highlights the importance of particle surface functionalization that should be considered during design and development of future AgNP-based medical products.

Sex-related response in mice after sub-acute intraperitoneal exposure to silver nanoparticles.

Silver nanoparticles (AgNPs) are among the most commercialized nanomaterials in biomedicine due to their antimicrobial and anti-inflammatory properties. Nevertheless, possible health hazards of exposure to AgNPs are yet to be understood and therefore raise public concern in regards of their safety. In this study, sex-related differences, role of steroidal hormones and influence of two different surface stabilizing agents (polymer vs. protein) on distribution and adverse effects of AgNPs were investigated in vivo. Intact and gonadectomised male and female mice were treated with seven AgNPs doses administered intraperitoneally during 21 days. After treatment, steroid hormone levels in serum, accumulation of Ag levels and oxidative stress biomarkers in liver, kidneys, brain and lungs were determined. Sex-related differences were observed in almost all tissues. Concentration of Ag was significantly higher in the liver of females compared to males. No significant difference was found for AgNP accumulation in lungs between females and males, while the lungs of intact males showed significantly higher Ag accumulation compared to gonadectomised group. Effect of surface coating was also observed, as Ag accumulation was significantly higher in kidneys and liver of intact females, as well as in kidneys and brain of intact males treated with protein-coated AgNPs compared to polymeric AgNPs. Oxidative stress response to AgNPs was the most pronounced in kidneys where protein-coated AgNPs induced stronger effects compared to polymeric AgNPs. Interestingly, protein-coated AgNPs reduced generation of reactive oxygen species in brains of females and gonadectomised males. Although there were no significant differences in levels of hormones in the AgNP-exposed animals compared to controls, sex-related differences in oxidative stress parameters were observed in all organs. Results of this study highlight the importance of including the sex-related differences and effects of protein corona in biosafety evaluation of AgNPs exposure.

Effects of low-level imidacloprid oral exposure on cholinesterase activity, oxidative stress responses, and primary DNA damage in the blood and brain of male Wistar rats.

Imidacloprid is a neonicotinoid insecticide that acts selectively as an agonist on insect nicotinic acetylcholine receptors. It is used for crop protection worldwide, as well as for non-agricultural uses. Imidacloprid systemic accumulation in food is an important source of imidacloprid exposure. Due to the undisputable need for investigations of imidacloprid toxicity in non-target species, we evaluated the effects of a 28-day oral exposure to low doses of imidacloprid (0.06 mg/kg b. w./day, 0.8 mg/kg b. w./day and 2.25 mg/kg b. w./day) on cholinesterase activity, oxidative stress responses and primary DNA damage in the blood and brain tissue of male Wistar rats. Exposure to imidacloprid did not cause significant changes in total cholinesterase, acetylcholinesterase and butyrylcholinesterase activities in plasma and brain tissue. Reactive oxygen species levels and lipid peroxidation increased significantly in the plasma of rats treated with the lowest dose of imidacloprid. Activities of glutathione-peroxidase in plasma and brain and superoxide dismutase in erythrocytes increased significantly at the highest applied dose. High performance liquid chromatography with UV diode array detector revealed the presence of imidacloprid in the plasma of all the treated animals and in the brain of the animals treated with the two higher doses. The alkaline comet assay results showed significant peripheral blood leukocyte damage at the lowest dose of imidacloprid and dose-dependent brain cell DNA damage. Oral 28-day exposure to low doses of imidacloprid in rats resulted in detectable levels of imidacloprid in plasma and brain tissue that directly induced DNA damage, particularly in brain tissue, with slight changes in plasma oxidative stress parameters.

Cellular Antioxidant Activity of Olive Pomace Extracts: Impact of Gastrointestinal Digestion and Cyclodextrin Encapsulation.

Olive pomace is a valuable secondary raw material rich in polyphenols, left behind after the production of olive oil. The present study investigated the protective effect of a polyphenolic extract from olive pomace (OPE) on cell viability and antioxidant defense of cultured human HepG2 cells submitted to oxidative stress induced by tert-butylhydroperoxide (tBOOH). The investigation considered possible matrix effects, impact of gastrointestinal digestion and cyclodextrin (CD) encapsulation. Pre-treatment of cells with OPE prevented cell damage and increased intracellular glutathione but did not affect the activity of glutathione peroxidase and superoxide dismutase. OPE matrix significantly enhanced cell protective effects of major antioxidants, such as hydroxytyrosol (HTS), while cyclodextrin encapsulation enhanced activity of OPE against intracellular reactive oxygen species (ROS) accumulation. The obtained results show that OPE is more potent antioxidant in comparison to equivalent dose of main polyphenols (HTS and TS) and that increasing solubility of OPE polyphenols by CD encapsulation or digestion enhances their potential to act as intracellular antioxidants. Antioxidative protection of cells by OPE was primarily achieved through direct radical-scavenging/reducing actions rather than activation of endogenous defense systems in the cell.

Microtubular structure impairment after GSM-modulated RF radiation exposure.

The objective of the study was to investigate whether low-level 915 MHz GSM-modulated radiofrequency (RF) radiation impairs microtubular structure and affects normal cell growth. V79 cells were exposed to a GSM-modulated field in a Gigahertz Transversal Electromagnetic Mode cell (GTEM cell) for 1, 2, and 3 h. Signal generator combined with power and chip modulator generated the electromagnetic field (EMF). The electric field strength was adjusted to 10, 20, and 30 V/m, and the average specific absorption rate (SAR) was calculated to be 0.23, 0.8, and 1.6 W/kg. The structure of microtubule proteins was assessed by indirect immunocytochemistry, and cell growth was determined based on cell counts taken every day over six post-exposure days. Three-hour radiation exposure significantly altered microtubule structure regardless of the electric field strength. Moreover, on the third post-exposure day, three-hour radiation significantly reduced cell growth, regardless of field strength. The same was observed with two-hour exposure at 20 and 30 V/m. In conclusion, 915 MHz GSM-modulated RF radiation affects microtubular proteins in a time-dependent manner, which, in turn, affects cell proliferation. Our future research will focus on microtubule structure throughout the cell cycle and RF radiation effects on mitotic spindle.

Superior protective effects of in vitro propagated green garlic against hydrogen peroxide-induced cytotoxicity in human hepatoma cells.

Garlic is a valuable source material for medicines due to its known antitumor, hypolipidaemic, antioxidant, and immunomodulatory effects. This study compares the protective effects of conventionally grown (CG) and in vitro propagated garlic (PG) against hydrogen peroxide-induced cytotoxicity in HepG2 cells and their antioxidant activity. Garlic used in this study was obtained by planting garlic cloves or by planting the transplants of PG directly in the field. At the end of the vegetation period, CG and PG were sampled and extracts prepared for the experiment. Compared to conventionally grown garlic bulbs, PG leafy part yielded significantly higher content of polyphenols, flavonoids and alliin, and also showed equal or higher antioxidant activity, measured by the cell viability test, GSH and ROS level. Moreover, PG can be produced in less time (shorter vegetation period) and with significantly less material (cloves). Significantly higher content of alliin, polyphenols, and flavonoids and significantly higher yield of plant biomass in PG has a great potential to become a new production model with improved garlic properties as a medicine material.

Impact of surface functionalization on the toxicity and antimicrobial effects of selenium nanoparticles considering different routes of entry.

Selenium nanoparticles (SeNPs) were first designed as nutritional supplements, but they are attractive also for use in diagnostic and therapeutic systems owing to their biocompatibility and protective effects. This study aimed to examine if different SeNPs stabilization strategies affect their (i) antimicrobial activity against bacteria Escherichia coli and Staphylococcus aureus and yeast Saccharomyces cerevisiae and (ii) toxicity to human cells of different biological barriers i.e., skin, oral and intestinal mucosa. For surface stabilization, polyvinylpyrrolidone (PVP), poly-L-lysine (PLL) and polyacrylic acid (PAA) were used rendering neutral, positively and negatively charged SeNPs, respectively. The SeNPs (primary size ~80 nm) showed toxic effects in human cells in vitro and in bacteria S. aureus, but not in E. coli and yeast S. cerevisiae. Toxicity of SeNPs (24 h IC50) ranged from 1.4 to >100 mg Se/L, depending on surface functionalization (PLL > PAA > PVP) and was not caused by ionic Se. At subtoxic concentrations, all SeNPs were taken up by all human cell types, induced oxidative stress response and demonstrated genotoxicity. As the safety profile of SeNPs was dependent not only on target cells (mammalian cells, bacteria, yeast), but also on surface functionalization, these aspects should be considered during development of novel SeNPs-based biomedical products.

Surface Stabilization Affects Toxicity of Silver Nanoparticles in Human Peripheral Blood Mononuclear Cells.

Silver nanoparticles (AgNPs) are one of the most investigated metal-based nanomaterials. Their biocidal activity boosted their application in both diagnostic and therapeutic medical systems. It is therefore crucial to provide sound evidences for human-related safety of AgNPs. This study aimed to enhance scientific knowledge with regard to biomedical safety of AgNPs by investigating how their different surface properties affect human immune system. METHODS: preparation, characterization and stability evaluation was performed for four differently coated AgNPs encompassing neutral, positive and negative agents used for their surface stabilization. Safety aspects were evaluated by testing interaction of AgNPs with fresh human peripheral blood mononuclear cells (hPBMC) by means of particle cellular uptake and their ability to trigger cell death, apoptosis and DNA damages through induction of oxidative stress and damages of mitochondrial membrane. RESULTS: all tested AgNPs altered morphology of freshly isolated hPBMC inducing apoptosis and cell death in a dose- and time-dependent manner. Highest toxicity was observed for positively-charged and protein-coated AgNPs. Cellular uptake of AgNPs was also dose-dependently increased and highest for positively charged AgNPs. Intracellularly, AgNPs induced production of reactive oxygen species (ROS) and damaged mitochondrial membrane. Depending on the dose, all AgNPs exhibited genotoxic potential. CONCLUSIONS: this study provides systematic and comprehensive data showing how differently functionalized AgNPs may affect the human immune system. Presented results are a valuable scientific contribution to safety assessment of nanosilver-based blood-contacting medical products.

Evaluation of oxidative stress responses and primary DNA damage in blood and brain of rats exposed to low levels of tembotrione.

Tembotrione is a rather novel pesticide, usually used for post-emergence weed control. Even though its use is rapidly growing, it is not followed by an adequate flow of scientific evidence regarding its toxicity towards non-target organisms. We evaluated the potential of low doses of tembotrione to induce oxidative stress and cytogenetic damage in blood and brain cells of adult male Wistar rats. Parameters of lipid peroxidation, glutathione levels, activities of antioxidant enzymes and primary DNA damage were assessed following 28-day repeated oral exposure to doses comparable with the currently proposed health-based reference values. The results of the alkaline comet assay showed that such low doses of tembotrione have the potency to inflict primary DNA damage in both peripheral blood leukocytes and brain of treated rats, even with only slight changes in the oxidative biomarker levels. The DNA damage in blood and brain cells of Wistar rats significantly increased at all applied doses, suggesting that tembotrione genotoxicity is mainly a result of direct interaction with DNA while the induction of oxidative stress responses contributes to DNA instability in a lesser extent. The findings of the present study call for further research using other sensitive biomarkers of effect and different exposure scenarios.

Neurotoxicity of silver nanoparticles stabilized with different coating agents: In vitro response of neuronal precursor cells.

Silver nanoparticles (AgNPs) represent one of the most abundant biocidal nanomaterials contained in more than 30% of nano-enabled consumer products and 75% of nanomedical products. The cumulative exposure of the general population may therefore reach critical and potentially hazardous levels. Due to data gaps on AgNP effects in humans, it is urgent to further evaluate their possible toxicity, particularly in vulnerable systems like the nervous one. As AgNPs may cross the blood brain and placental barriers, this study evaluated the in vitro effect of different AgNPs on neuronal precursor cells. For this purpose, 10 nm-sized AgNPs were stabilized with five different coating agents rendering a neutral, positive and negative surface charge. Murine neural stem cells (mNSCs) were used as cellular model to test AgNP neurotoxicity by evaluating the range of toxicity endpoints including cellular viability, apoptosis induction, oxidative stress response, cellular and mitochondrial membrane damages, DNA damage, inflammation response, and neural stem cell regulation. Our results clearly showed that the neurotoxic potential of AgNPs was not dependent on their surface charge or coating agents used for their surface stabilization. All AgNP types exhibited significant toxicity in neuronal precursor cells at an in vitro dose of 5 mg Ag/L or lower.

Toxicity and safety study of silver and gold nanoparticles functionalized with cysteine and glutathione.

This study was designed to evaluate the nano-bio interactions between endogenous biothiols (cysteine and glutathione) with biomedically relevant, metallic nanoparticles (silver nanoparticles (AgNPs) and gold nanoparticles (AuNPs)), in order to assess the biocompatibility and fate of nanoparticles in biological systems. A systematic and comprehensive analysis revealed that the preparation of AgNPs and AuNPs in the presence of biothiols leads to nanoparticles stabilized with oxidized forms of biothiols. Their safety was tested by evaluation of cell viability, reactive oxygen species (ROS) production, apoptosis induction and DNA damage in murine fibroblast cells (L929), while ecotoxicity was tested using the aquatic model organism Daphnia magna. The toxicity of these nanoparticles was considerably lower compared to their ionic metal forms (i.e., Ag+ and Au3+). The comparison with data published on polymer-coated nanoparticles evidenced that surface modification with biothiols made them safer for the biological environment. In vitro evaluation on human cells demonstrated that the toxicity of AgNPs and AuNPs prepared in the presence of cysteine was similar to the polymer-based nanoparticles with the same core material, while the use of glutathione for nanoparticle stabilization was considerably less toxic. These results represent a significant contribution to understanding the role of biothiols on the fate and behavior of metal-based nanomaterials.

Optimization of an ex vivo gene transfer to the hamstrings tendons muscle remnants: potential for genetic enhancement of bone healing.

AIM: To assess whether an adenoviral vector carrying the bone morphogenetic protein genes (Ad.BMP-2) can transduce human muscle tissue and direct it toward osteogenic differentiation within one hour. METHODS: This in vitro study, performed at the Department of Molecular Biology, Faculty of Science, Zagreb from 2012 to 2017, used human muscle tissue samples collected during anterior cruciate ligament reconstructions performed in St Catherine Hospital, Zabok. Samples from 28 patients were transduced with adenoviral vector carrying firefly luciferase cDNA (Ad.luc) by using different doses and times of transduction, and with addition of positive ions for transduction enhancement. The optimized protocol was further tested on muscle samples from three new patients, which were transduced with Ad.BMP-2. Released bone morphogenetic protein 2 (BMP-2) levels in osteogenic medium were measured every three days during a period of 21 days. Expression of osteogenic markers was measured at day 14 and 21. After 21 days of cultivation, muscle tissue was immunohistochemically stained for collagen type I detection (COL-I). RESULTS: The new transduction protocol was established using 108 plaque-forming units (P<0.001) as an optimal dose of adenoviral vector and 30 minutes (P<0.001) as an optimal contact time. Positive ions did not enhance transduction. Samples transduced with Ad.BMP-2 according to the optimized protocol showed enhanced expression of osteogenic markers (P<0.050), BMP-2 (P<0.001), and COL I. CONCLUSION: This study confirms that Ad.BMP-2 can transduce human muscle tissue and direct it toward osteogenic differentiation within 30 minutes.