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.

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.

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.

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.

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.

Cytotoxicity of gamma irradiated aflatoxin B1 and ochratoxin A.

Toxicity of gamma irradiated mycotoxins aflatoxin B1 (AFB1) and ochratoxin A (OTA) was investigated in vitro. AFB1 and OTA stock solutions (50 mM, in methanol) were gamma irradiated (5 and 10 kGy) and non-irradiated and irradiated mycotoxins solutions were tested for cytotoxicity on Pk15, HepG2 and SH-SY5Y cell lines (MTT assay, 1-500 µM concentration range; 24 h exposure). Degradation of mycotoxin molecules was examined by liquid chromatography tandem mass spectrometry (HPLC-MS/MS). AFB1 and OTA radiolytic products were less toxic than the parent mycotoxins to all of the tested cell lines. Gamma irradiation even at 5 kGy had effect on AFB1 and OTA molecules however, this effect was dependent on chemical structure of mycotoxin. Since gamma irradiation at low dose reduced initial level of both mycotoxins, and gamma irradiated mycotoxins had lower toxicity in comparison to non-irradiated mycotoxins, it can be concluded that gamma irradiation could be used as decontamination method.

Oxidative stress response in SH-SY5Y cells exposed to short-term 1800 MHz radiofrequency radiation.

The exact mechanism that could explain the effects of radiofrequency (RF) radiation exposure at non-thermal level is still unknown. Increasing evidence suggests a possible involvement of reactive oxygen species (ROS) and development of oxidative stress. To test the proposed hypothesis, human neuroblastoma cells (SH-SY5Y) were exposed to 1800 MHz short-term RF exposure for 10, 30 and 60 minutes. Electric field strength within Gigahertz Transverse Electromagnetic cell (GTEM) was 30 V m-1 and specific absorption rate (SAR) was calculated to be 1.6 W kg-1. Cellular viability was measured by MTT assay and level of ROS was determined by fluorescent probe 2',7'-dichlorofluorescin diacetate. Concentrations of malondialdehyde and protein carbonyls were used to assess lipid and protein oxidative damage and antioxidant activity was evaluated by measuring concentrations of total glutathione (GSH). After radiation exposure, viability of irradiated cells remained within normal physiological values. Significantly higher ROS level was observed for every radiation exposure time. After 60 min of exposure, the applied radiation caused significant lipid and protein damage. The highest GSH concentration was detected after 10 minute-exposure. The results of our study showed enhanced susceptibility of SH-SY5Y cells for development of oxidative stress even after short-term RF exposure.

In vitro non-thermal oxidative stress response after 1800 MHz radiofrequency radiation.

In this study possible connection between radiofrequency exposure (RF) and development of oxidative stress was investigated by measuring impairment in cellular oxidation-reduction balance immediately after RF exposure. Fibroblast cells V79 were exposed for 10, 30 and 60 minutes to 1800 MHz RF radiation. Electric field strength was 30 V/m and specific absorption rate (SAR) was calculated to be 1.6 W/kg. Electromagnetic field was generated within Gigahertz Transversal Electromagnetic Mode cell (GTEM) equipped by signal generator, amplifier and modulator. Cell viability was determined by CCK-8 colorimetric assay and level of reactive oxygen species (ROS) was detected by dihydroethidium staining. Reduced glutathione (GSH) and glutathione peroxidase (GSH-Px) were used to assess cell antioxidant activity while lipid oxidative damage was evaluated measuring concentration of malondialdehyde. Viability of V79 cells remained within normal physiological values regardless of exposure time. Increased level of superoxide radicals was detected after 60-min exposure. Significantly higher GSH level was observed immediately after 10-min exposure with higher but insignificant activity of GSH-Px. Lipid oxidative damage in exposed cell samples was not observed. Short-term RF exposure revealed transient oxidation-reduction imbalance in fibroblast cells following adaptation to applied experimental conditions.

Comparison of in vitro toxicity of silver ions and silver nanoparticles on human hepatoma cells.

Scientific information on the potential harmful effects of silver nanoparticles (AgNPs) on human health severely lags behind their exponentially growing applications in consumer products. In assessing the toxic risk of AgNP usage, liver, as a detoxifying organ, is particularly important. The aim of this study was to explore the toxicity mechanisms of nano and ionic forms of silver on human hepatoblastoma (HepG2) cells. The results showed that silver ions and citrate-coated AgNPs reduced cell viability in a dose-dependent manner. The IC50 values of silver ions and citrate-coated AgNPs were 0.5 and 50 mg L(-1) , respectively. The LDH leakage and inhibition of albumin synthesis, along with decreased ALT activity, indicated that treatment with either AgNP or Ag ions resulted in membrane damage and reduced the cell function of human liver cells. Evaluation of oxidative stress markers demonstrating depletion of GSH, increased ROS production, and increased SOD activity, indicated that oxidative stress might contribute to the toxicity effects of nano and ionic forms of silver. The observed toxic effect of AgNP on HepG2 cells was substantially weaker than that caused by ionic silver, while the uptake of nano and ionic forms of silver by HepG2 cells was nearly the same. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 679-692, 2016.

Cellular uptake and toxicity effects of silver nanoparticles in mammalian kidney cells.

The rapid progress and early commercial acceptance of silver-based nanomaterials is owed to their biocidal activity. Besides embracing the antimicrobial potential of silver nanoparticles (AgNPs), it is imperative to give special attention to the potential adverse health effects of nanoparticles owing to prolonged exposure. Here, we report a detailed study on the in vitro interactions of citrate-coated AgNPs with porcine kidney (Pk15) cells. As uncertainty remains whether biological/cellular responses to AgNPs are solely as a result of the release of silver ions or whether the AgNPs themselves have toxic effects, we investigated the effects of Ag(+) on Pk15 cells for comparison. Next, we investigated the cellular uptake of both AgNPs and Ag(+) in Pk15 cells at various concentrations applied. The detected Ag contents in cells exposed to 50 mg l(-1) AgNPs and 50 mg l(-1) Ag(+) were 209 and 25 µg of Ag per 10(6) cells, respectively. Transmission electron microscopy (TEM) images indicated that the Pk15 cells internalized AgNPs by endocytosis. Both forms of silver, nano and ionic, decreased the number of viable Pk15 cells after 24 h in a dose-dependent manner. In spite of a significant uptake into the cells, AgNPs had only insignificant toxicity at concentrations lower than 25 mg l(-1) , whereas Ag(+) exhibited a significant decrease in cell viability at one-fifth of this concentration. The Comet assay suggested that a rather high concentration of AgNP (above 25 mg l(-1) ) is able to induce genotoxicity in Pk15 cells. Further studies must seek deeper understanding of AgNP behavior in biological media and their interactions with cellular membranes.

Cell oxidation-reduction imbalance after modulated radiofrequency radiation.

Aim of this study was to evaluate an influence of modulated radiofrequency field (RF) of 1800 MHz, strength of 30 V/m on oxidation-reduction processes within the cell. The assigned RF field was generated within Gigahertz Transversal Electromagnetic Mode cell equipped by signal generator, modulator, and amplifier. Cell line V79, was irradiated for 10, 30, and 60 min, specific absorption rate was calculated to be 1.6 W/kg. Cell metabolic activity and viability was determined by MTT assay. In order to define total protein content, colorimetric method was used. Concentration of oxidised proteins was evaluated by enzyme-linked immunosorbent assay. Reactive oxygen species (ROS) marked with fluorescent probe 2',7'-dichlorofluorescin diacetate were measured by means of plate reader device. In comparison with control cell samples, metabolic activity and total protein content in exposed cells did not differ significantly. Concentrations of carbonyl derivates, a product of protein oxidation, insignificantly but continuously increase with duration of exposure. In exposed samples, ROS level significantly (p < 0.05) increased after 10 min of exposure. Decrease in ROS level was observed after 30-min treatment indicating antioxidant defence mechanism activation. In conclusion, under the given laboratory conditions, modulated RF radiation might cause impairment in cell oxidation-reduction equilibrium within the growing cells.

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.

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.

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.

Effect of electromagnetic radiofrequency radiation on the rats' brain, liver and kidney cells measured by comet assay.

The goal of study was to evaluate DNA damage in rat's renal, liver and brain cells after in vivo exposure to radiofrequency/microwave (Rf/Mw) radiation of cellular phone frequencies range. To determine DNA damage, a single cell gel electrophoresis/comet assay was used. Wistar rats (male, 12 week old, approximate body weight 350 g) (N = 9) were exposed to the carrier frequency of 915 MHz with Global System Mobile signal modulation (GSM), power density of 2.4 W/m2, whole body average specific absorption rate SAR of 0.6 W/kg. The animals were irradiated for one hour/day, seven days/week during two weeks period. The exposure set-up was Gigahertz Transversal Electromagnetic Mode Cell (GTEM--cell). Sham irradiated controls (N = 9) were apart of the study. The body temperature was measured before and after exposure. There were no differences in temperature in between control and treated animals. Comet assay parameters such as the tail length and tail intensity were evaluated. In comparison with tail length in controls (13.5 +/- 0.7 microm), the tail was slightly elongated in brain cells of irradiated animals (14.0 +/- 0.3 microm). The tail length obtained for liver (14.5 +/- 0.3 microm) and kidney (13.9 +/- 0.5 microm) homogenates notably differs in comparison with matched sham controls (13.6 +/- 0.3 microm) and (12.9 +/- 0.9 microm). Differences in tail intensity between control and exposed animals were not significant. The results of this study suggest that, under the experimental conditions applied, repeated 915 MHz irradiation could be a cause of DNA breaks in renal and liver cells, but not affect the cell genome at the higher extent compared to the basal damage.

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.

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-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.