Plate reader spectroscopy as an alternative to atomic absorption spectroscopy for the assessment of nanoparticle cellular uptake.

Fundamental studies investigating the biological effects induced by nanoparticles (NPs) explicitly require the correct assessment of their intracellular concentration. Ultrasensitive atomic absorption spectroscopy (AAS) is perceived as one of the gold standard methods for quantifying internalized NPs. Besides its limitation to metal-based NPs though, AAS also requires specific infrastructure and tedious sample preparation and handling, making it time-consuming and cost-intensive. Herein we report on a reliable, rapid, and affordable alternative to AAS - plate reader spectroscopy (PRS), which offers an accessible option for everyday laboratory use without sophisticated instrumentation. Our results demonstrate, that following a proper methodological approach, data on intracellular concentration of NPs obtained by PRS are fully comparable to AAS results. Specifically, the intracellular concentration of magnetite NPs coated with sodium oleate and bovine serum albumin in human alveolar A549 cells was assessed by PRS and AAS in parallel, with a remarkable correlation coefficient of R = 0.9914.

Effects of Ovariectomy and Exercise Training on Mineral Status in a High-Fat Diet-Induced Obesity Rat Model.

Osteoporosis is a growing public health issue for an aging society. Previous studies have found both beneficial and detrimental effects of obesity on bone health. The purpose of this study was to investigate the impact of estrogen deficiency and physical activity on bone and blood concentrations of macrominerals (Ca, P, and Mg) and microminerals (Zn, Se, Cu, and Fe) in a high-fat diet-induced obesity rat model. Forty-eight female Wistar rats were divided into six groups: sham-operated and ovariectomized rats that received a standard diet (SD), high-fat diet (HFD), or HFD accompanied by physical exercise. The effect of ovariectomy on bone minerals varied with diet. Ovariectomy significantly decreased femoral Ca and Mg in sedentary rats receiving a SD; femoral Se, Cu, Zn, and Fe in sedentary rats on HFD; and plasma Fe in both sedentary rats on SD and exercising rats on HFD. The interaction of ovariectomy and diet had the strongest impact on Mg and Se concentrations in femur. In ovariectomized rats, HFD showed to have a protective effect on bone mineralization (femoral Ca and Mg), and a negative one on antioxidant microminerals (femoral Se, Cu, and Zn). Physical activity reduced the decline of Se, Cu, Zn, and Fe in the femur of ovariectomized rats on HFD. In the current state of knowledge, it is difficult to suggest if decreased femoral levels of antioxidant microminerals may contribute to the pathophysiology of osteoporosis in obese individuals or just reflect the mineral status in the body.

Differences in surface chemistry of iron oxide nanoparticles result in different routes of internalization.

The efficient entry of nanotechnology-based pharmaceuticals into target cells is highly desired to reach high therapeutic efficiency while minimizing the side effects. Despite intensive research, the impact of the surface coating on the mechanism of nanoparticle uptake is not sufficiently understood yet. Herein, we present a mechanistic study of cellular internalization pathways of two magnetic iron oxide nanoparticles (MNPs) differing in surface chemistry into A549 cells. The MNP uptake was investigated in the presence of different inhibitors of endocytosis and monitored by spectroscopic and imaging techniques. The results revealed that the route of MNP entry into cells strongly depends on the surface chemistry of the MNPs. While serum bovine albumin-coated MNPs entered the cells via clathrin-mediated endocytosis (CME), caveolin-mediated endocytosis (CavME) or lipid rafts were preferentially involved in the internalization of polyethylene glycol-coated MNPs. Our data indicate that surface engineering can contribute to an enhanced delivery efficiency of nanoparticles.

Surface coating determines the inflammatory potential of magnetite nanoparticles in murine renal podocytes and mesangial cells.

Drug-induced nephrotoxicity is a frequent adverse event and a dose-limiting factor in patient treatment and is a leading cause of prospective drug attrition during pharmaceutical development. Despite the obvious benefits of nanotherapeutics in healthcare strategies, the clearance of imaging agents and nanocarriers from the body following their therapeutic or diagnostic application generates concerns about their safety for human health. Considering the potency of nanoparticles and their massive utilization in biomedicine the impact of magnetic nanoparticles (MNPs) on cells forming the filtration apparatus of the kidney was studied. Using primary mouse renal glomerular podocytes and mesangial cells, we investigated their response to exposure to magnetic nanoparticles coated with polyethylene glycol and bovine serum albumin. Cultured podocytes were more sensitive to MNPs than mesangial cells displaying signs of cell damage and stronger inflammatory response. Both types of MNPs induced the remodeling of actin fibers, affected the cell shape and triggered expression of inflammatory cytokines TNFα and IL-6 in podocytes. On the other hand, iNOS was induced in both renal cell types but only by MNPs with a polyethylene glycol coating. Our results have revealed that the type of cell and the type of nanoparticle coating might be the strongest determinants of cellular response toward nanoparticle exposure. Differences in susceptibility of cells to MNPs might be evident also between neighboring renal cell subpopulations integrally forming functional sub-units of this organ.

Six-week inhalation of CdO nanoparticles in mice: The effects on immune response, oxidative stress, antioxidative defense, fibrotic response, and bones.

Due to the growing number of applications of cadmium oxide nanoparticles (CdO NPs), there is a concern about their potential deleterious effects. The objective of our study was to investigate the effect of CdO NPs on the immune response, renal and intestine oxidative stress, blood antioxidant defence, renal fibrotic response, bone density and mineral content. Six-week-old female ICR mice were exposed to CdO NPs for 6 weeks by inhalation (particle size: 9.82 nm, mass concentration: 31.7 µg CdO/m3, total deposited dose: 0.195 µg CdO/g body weight). CdO NPs increased percentage of thymus CD3e+CD8a+ cells and moderately enhanced splenocyte proliferation and production of cytokines and chemokines. CdO NPs elevated pro-fibrotic factors (TGF-ß2, α-SMA and collagen I) in the kidney, and concentrations of AGEs in the intestine. The ratio of GSH and GSSG in blood was slightly reduced. Exposure to CdO NPs resulted in 10-fold higher Cd concentration in tibia bones. No differences were found in bone mass density, mineral content, bone area values, bone concentrations of Ca, P, Mg and Ca/P ratio. Our findings indicate stimulation of immune/inflammatory response, oxidative stress in the intestine, starting fibrotic response in kidneys and accumulation of CdO NPs in bones of mice.

Toxicity evaluation of monodisperse PEGylated magnetic nanoparticles for nanomedicine.

Innovative nanotechnology aims to develop particles that are small, monodisperse, smart, and do not cause unintentional side effects. Uniform magnetic Fe3O4 nanoparticles (12 nm in size) were prepared by thermal decomposition of iron(III) oleate. To make them colloidally stable and dispersible in water and cell culture medium, they were modified with phosphonic acid- (PA) and hydroxamic acid (HA)-terminated poly(ethylene glycol) yielding PA-PEG@Fe3O4 and HA-PEG@Fe3O4 nanoparticles; conventional γ-Fe2O3 particles were prepared as a control. Advanced techniques were used to evaluate the properties and safety of the particles. Completeness of the nanoparticle coating was tested by real-time polymerase chain reaction. Interaction of the particles with primary human peripheral blood cells, cellular uptake, cytotoxicity, and immunotoxicity were also investigated. Amount of internalized iron in peripheral blood mononuclear cells was 72, 38, and 25 pg Fe/cell for HA-PEG@Fe3O4, γ-Fe2O3, and PA-PEG@Fe3O4, respectively. Nanoparticles were localized within the cytoplasm and in the extracellular space. No cytotoxic effect of both PEGylated nanoparticles was observed (0.12-75 µg/cm2) after 24 and 72-h incubation. Moreover, no suppressive effect was found on the proliferative activity of T-lymphocytes and T-dependent B-cell response, phagocytic activity of monocytes and granulocytes, and respiratory burst of phagocytes. Similarly, no cytotoxic effect of γ-Fe2O3 particles was observed. However, they suppressed the proliferative activity of T-lymphocytes (75 µg/cm2, 72 h) and also decreased the phagocytic activity of monocytes (15 µg/cm2, 24 h; 3-75 µg/cm2, 72 h). We thus show that newly developed particles have great potential especially in cancer diagnostics and therapy.

Prenatal and Early Postnatal Exposure to Total Mercury and Methylmercury from Low Maternal Fish Consumption.

The aim of this study was to investigate the influence of low fish consumption on prenatal and early postnatal exposure to mercury species. The samples of umbilical cord blood and maternal milk as well as interviewer-administered questionnaires were collected from 142 Slovak mother-child pairs. The mean total mercury (THg) concentrations in cord blood and milk were 0.949 µg/L and 0.376 µg/kg, respectively. The mean methylmercury (MeHg) concentration in cord blood was 0.504 µg/L. Fish eaters had significantly higher cord blood MeHg concentrations than non-fish eaters (p = 0.030); no difference was found in milk or cord blood THg concentrations. The bivariate analysis showed a positive correlation between cord blood MeHg and consumption of sea fish and shellfish (rs = 0.320, p < 0.001); after adjustment for the potential confounders, the association was weakened (ß = 0.173, p = 0.059). Nevertheless, the decision tree method showed sea fish and shellfish consumption to be the best predictor of cord blood MeHg. Furthermore, a negative association was found between THg concentrations in maternal milk and freshwater fish consumption (ß = - 0.193, p = 0.017), which might indicate a beneficial effect of freshwater fish consumption. The results suggest there is a need for future research to investigate the benefits versus the adverse effects of low maternal fish consumption on child development.

Biological safety and tissue distribution of (16-mercaptohexadecyl)trimethylammonium bromide-modified cationic gold nanorods.

The exceptionally high cellular uptake of gold nanorods (GNRs) bearing cationic surfactants makes them a promising tool for biomedical applications. Given the known specific toxic and stress effects of some preparations of cationic nanoparticles, the purpose of this study was to evaluate, in an in vitro and in vivo in mouse, the potential harmful effects of GNRs coated with (16-mercaptohexadecyl)trimethylammonium bromide (MTABGNRs). Interestingly, even after cellular accumulation of high amounts of MTABGNRs sufficient for induction of photothermal effect, no genotoxicity (even after longer-term accumulation), induction of autophagy, destabilization of lysosomes (dominant organelles of their cellular destination), alterations of actin cytoskeleton, or in cell migration could be detected in vitro. In vivo, after intravenous administration, the majority of GNRs accumulated in mouse spleen followed by lungs and liver. Microscopic examination of the blood and spleen showed that GNRs interacted with white blood cells (mononuclear and polymorphonuclear leukocytes) and thrombocytes, and were delivered to the spleen red pulp mainly as GNR-thrombocyte complexes. Importantly, no acute toxic effects of MTABGNRs administered as 10 or 50 µg of gold per mice, as well as no pathological changes after their high accumulation in the spleen were observed, indicating good tolerance of MTABGNRs by living systems.

Surface-modified magnetite nanoparticles act as aneugen-like spindle poison.

Iron oxide nanoparticles are one of the most promising types of nanoparticles for biomedical applications, primarily in the context of nanomedicine-based diagnostics and therapy; hence, great attention should be paid to their bio-safety. Here, we investigate the ability of surface-modified magnetite nanoparticles (MNPs) to produce chromosome damage in human alveolar A549 cells. Compared to control cells, all the applied MNPs increased the level of micronuclei moderately but did not cause structural chromosomal aberrations in exposed cells. A rise in endoreplication, polyploid and multinuclear cells along with disruption of tubulin filaments, downregulation of Aurora protein kinases and p53 protein activation indicated the capacity of these MNPs to impair the chromosomal passenger complex and/or centrosome maturation. We suppose that surface-modified MNPs may act as aneugen-like spindle poisons via interference with tubulin polymerization. Further studies on experimental animals revealing mechanisms of therapeutic-aimed MNPs are required to confirm their suitability as potential anti-cancer drugs.

Intracellular uptake of magnetite nanoparticles: A focus on physico-chemical characterization and interpretation of in vitro data.

Comprehensive characterization of nanoparticles associated with investigation of their cellular uptake creates the basis on which fundamental in vitro and in vivo studies can be built. In this work, a complex analysis of various surface-modified magnetite nanoparticles in biologically relevant environment is reported and the promotion of incorrect characterization into the results obtained from model biological experiments leading to false conclusions is demonstrated. Via a bottom-up approach from particle characterization by DLS towards interpretation of biological data based on cellular uptake, this work draws attention to the systematic propagation of errors stemming from inaccurate determination of input parameters for DLS, improper selection of particle size distribution, inadequate sampling, unknown colloidal behavior and the omission of fraction of particles complying with the internalization threshold. In addition, cellular uptake depending on the number of treated cells is shown. The definition of cellular uptake efficacy reflecting the size distribution of particles beside their absolute internalization is postulated.

Changed Plasma Levels of Zinc and Copper to Zinc Ratio and Their Possible Associations with Parent- and Teacher-Rated Symptoms in Children with Attention-Deficit Hyperactivity Disorder.

Attention-deficit hyperactivity disorder (ADHD) is associated with alterations in the metabolism of some trace elements which may participate in the pathogenesis of this disorder. The aims of the present study were to investigate the trace element status (copper (Cu), zinc (Zn), copper to zinc ratio (Cu/Zn ratio), selenium (Se), and lead (Pb)) of ADHD children and compare them with the control group. Associations between examined elements and ratings of ADHD symptoms were also assessed. Fifty-eight ADHD children and 50 healthy children (aged 6-14 years) were included in the study. The concentrations of Cu, Zn, and Se in the plasma and Pb in the whole blood were measured by atomic absorption spectrometry. We found lower Zn level (p = 0.0005) and higher Cu/Zn ratio (p = 0.015) in ADHD children when compared with the control group. Copper levels in ADHD children were higher than those in the control group, but not significantly (p > 0.05). No significant differences in levels of Se and Pb between both groups were found. Zinc levels correlated with parent-rated score for inattention (r = -0.231, p = 0.029) as well as with teacher-rated score for inattention (r = -0.328, p = 0.014). Cu/Zn ratio correlated with teacher-rated score for inattention (r = 0.298, p = 0.015). Significant associations of Se and Pb with parent- and teacher-rated symptoms were not observed. The results of this study indicate that there are alterations in plasma levels of Cu and Zn as well as significant relationships to symptoms of ADHD.

The relation between human exposure to mercury and thyroid hormone status.

The aim of this study was to investigate total mercury (THg) and methylmercury (MeHg) exposure of 75 mother-child pairs in relation to their thyroid hormone status (thyroid-stimulating hormone (TSH), triiodothyronine (T3), free triiodothyronine (fT3), thyroxine (T4), and free thyroxine (fT4)). THg and MeHg in blood samples were measured by atomic absorption spectrometry and gas chromatography-inductively coupled plasma-mass spectrometry, respectively. The median THg and MeHg levels in maternal blood, cord blood, and blood of 6-month-old children were 0.50, 0.53, and 0.32 and 0.22, 0.32, and 0.08 µg/L, respectively. There were significant correlations between paired maternal-cord blood levels for THg and MeHg, with a greater transplacental transport of MeHg compared with THg (mean cord/maternal blood ratio, 1.80 vs. 1.24). The maternal blood THg was found to be a better predictor of TSH levels in children than their current THg exposure. There was a positive correlation between maternal THg and children's TSH. T3 and fT3 levels in children were negatively related to cord blood THg in the majority (Caucasian) subgroup, whereas these associations were positive in the Roma subgroup. Mothers with dental amalgam fillings had significantly lower T4 and fT4 levels. Moreover, fT4 in the mothers of boys negatively correlated with maternal THg levels. MeHg exposure lowered T3 levels in the mothers of girls. Our results suggest that low-level exposure to Hg can affect thyroid hormone status during prenatal and early postnatal exposure depending on the form of Hg, gender, ethnicity, lifestyle, or socioeconomic status (dental amalgam fillings).

Geographic accumulation of Creutzfeldt-Jakob disease in Slovakia--environmental metal imbalance as a possible cofactor.

Slovakia is characterised by an unusually high number of patients affected by genetic Creutzfeldt-Jakob disease (CJD) with E200K mutation at the PRNP gene. Penetrance of the mutation is incomplete (59%). Therefore, for the onset of the clinical manifestation, an influence of other endo- or exogenous factors could not be excluded. Experimental data suggest that copper and manganese levels may play an important role in the pathogenesis of prion diseases. The highest number of Slovak genetic CJD patients originates from Orava - the northern region of central Slovakia. Manganese is a dominant pollutant in Orava. The objective of this study was to clarify a possible exogenous influence of environmental Mn/Cu imbalance on the CJD clustering. Mn and Cu levels were analysed in the brain tissue of genetic CJD cases (from Orava and from control regions of Slovakia), as well as of sporadic CJD patients and controls. Analyses demonstrate i) significantly higher Mn level in focally accumulated, "clustering" genetic CJD cases in comparison to all other groups, ii) Cu status differences between compared groups were without statistical significance; decreased concentrations were found in genetic cases from extrafocal genetic CJD areas, iii) Mn/Cu ratios were increased in all CJD groups in comparison to controls. Metal ratios in clustering gCJD cases were significantly higher in comparison to sporadic cases and also to controls, but not to the extrafocal genetic CJD subgroup. These results indicate that more important than increasing Mn level in pathogenesis of CJD appears to be the role of the Mn/Cu imbalance in the CNS. The imbalance observed in the cluster of genetic CJD cases is probably a result of both: the excessive environmental Mn level and the disturbance of Mn/Cu ratios in the Orava region. Presented findings indicate an environmental Mn/Cu imbalance as a possible exogenous CJD risk co-factor which may, in coincidence with endogenous (genetic) CJD risk, contribute to the focal accumulation (cluster) of genetic CJD in Slovakia.

WITHDRAWN: Natural polyphenols modify trace element status and improve clinical symptoms of attention-deficit hyperactivity disorder.

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Maternal amalgam dental fillings as the source of mercury exposure in developing fetus and newborn.

Dental amalgam is a mercury-based filling containing approximately 50% of metallic mercury (Hg(0)). Human placenta does not represent a real barrier to the transport of Hg(0); hence, fetal exposure occurs as a result of maternal exposure to Hg, with possible subsequent neurodevelopmental disabilities in infants. This study represents a substudy of the international NIH-funded project "Early Childhood Development and polychlorinated biphenyls Exposure in Slovakia". The main aim of this analysis was to assess the relationship between maternal dental amalgam fillings and exposure of the developing fetus to Hg. The study subjects were mother-child pairs (N=99). Questionnaires were administered after delivery, and chemical analyses of Hg were performed in the samples of maternal and cord blood using atomic absorption spectrometry with amalgamation technique. The median values of Hg concentrations were 0.63 microg/l (range 0.14-2.9 microg/l) and 0.80 microg/l (range 0.15-2.54 microg/l) for maternal and cord blood, respectively. None of the cord blood Hg concentrations reached the level considered to be hazardous for neurodevelopmental effects in children exposed to Hg in utero (EPA reference dose for Hg of 5.8 microg/l in cord blood). A strong positive correlation between maternal and cord blood Hg levels was found (rho=0.79; P<0.001). Levels of Hg in the cord blood were significantly associated with the number of maternal amalgam fillings (rho=0.46, P<0.001) and with the number of years since the last filling (rho=-0.37, P<0.001); these associations remained significant after adjustment for maternal age and education. Dental amalgam fillings in girls and women of reproductive age should be used with caution, to avoid increased prenatal Hg exposure.

Manganese and copper imbalance in the food chain constituents in relation to Creutzfeldt-Jakob disease.

The objective of the study was to investigate the possible role of manganese and copper (Mn/Cu) imbalance of the food chain in the focally increased occurrence of Creutzfeldt-Jakob disease (CJD). Mn and Cu concentrations in soil, drinking water and foodstuffs collected from households in the region of focal accumulation of CJD patients and the control region were measured by FAAS. Considerably higher Mn/Cu ratios in the studied region than those in the control region were found for soil (49.3 vs. 21.1), honey (8.05 vs. 4.86), and for the main local food items: potatoes (2.09 vs. 1.07) and bread (5.85 vs. 5.35), however, only soil and potatoes were of statistical significance. The results could indicate a rare coincidence of the verified endogenous CJD risk (genetic) with a very probable exogenous CJD risk factor (Mn/Cu dietary/environmental imbalance), but whether and how this coincidence may contribute to the unique, continual temporo-spatial clustering of genetic CJD should be investigated in further studies.

Cadmium blood concentrations in relation to nutrition.

Cadmium is a toxic element ubiquitous in the environment, which damages biological systems in various ways. The major source of cadmium exposure is food. High cadmium content in the soil leads to high cadmium concentrations in certain plants such as grains (above all surface layers and germs), oil or non-oil seeds, fruit and vegetables. These food commodities are the crucial components of a vegetarian nutrition. Blood cadmium concentrations were measured in two non-smoking population groups: the vegetarian group (n = 80) and the non-vegetarian (control) group of general population on traditional mixed diet (n = 84). The significantly higher blood cadmium content (1.78 +/- 0.22 vs. 0.45 +/- 0.04 microg/l) was measured in vegetarian group. Healthy risk values > 5 microg/l were found in 6 vegetarians vs. no non-vegetarian. The highest cadmium concentration (3.15 +/- 0.77 microg/l) was measured in vegan subgroup (plant food only, n = 10) and that value decreased with increasing animal food consumption (1.75 +/- 0.36 microg/l, lactovegetarian and lactoovovegetarian subgroup/added dairy products and eggs, n = 41/, 1.34 +/- 0.21 microg/I, semivegetarian subgroup /as a previous subgroup and added white meat, n = 291). Risk vegetarians vs. non-risk vegetarians consume significantly higher amounts of whole grain products, grain sprouts and oil seeds. Blood cadmium content is directly influenced by age (r = 0.32, p < 0.001), by whole grain product intake (r = 0.66, p < 0.001) and by duration of vegetarianism (r = 0.5, p < 0.001). Oxidative stress plays a major role in chronic cadmium induced hepatic and renal toxicity as well as in other consequences of cadmium injuries. Vegetarians have significantly higher plasma concentrations of natural antioxidants. The sufficient antioxidative protection against cadmium induced free radical formation in vegetarians may inhibit the harmful effects of greater cadmium intake from plant food.

Cadmium, lead and mercury in human milk from Slovakia.

The objective of this study was to investigate Cd, Pb, and Hg levels in the breast milk of 158 lactating healthy women who were not occupationally exposed and living in eight environmentally different polluted locations of the Slovak Republic. Levels of these metals were examined in relation to selected parameters, such as: location, mother's age, parity, number of mother's teeth fillings, newborn's gender and birth weight, and smoking habits in the family. Cd and Pb levels in human milk and reconstituted infant formula were determined by GFAAS. Hg levels were measured by amalgamation technique AAS. The average concentrations of Cd, Pb and Hg in breast milk samples, taken on the 4th postpartum day, were 0.43, 4.7 and 0.94 microg/kg, respectively, and those in infant formula (n = 10) were 0.40, 5.0 and 0.25 microg/kg, respectively. Dietary weekly intakes of Cd, Pb, and Hg from breast milk and infant formula in newborn babies were also estimated. Whereas the calculated average dietary intakes of Cd, Pb, and Hg in newborn babies from breast milk and infant formula were all far lower than the appropriate PTWI values recommended by WHO/FAO, two individual breast milk samples for Pb and one breast milk sample for Hg exceeded the PTWI values. Regarding the studied parameters, only the active/passive smoking of the mother at home significantly increased Pb levels in breast milk and amalgam teeth fillings increased Hg levels in breast milk.