Jana Tulinska

Coating-dependent induction of cytotoxicity and genotoxicity of iron oxide nanoparticles

Abstract Surface coatings of nanoparticles (NPs) are known to influence advantageous features of NPs as well as potential toxicity. Iron oxide (Fe3O4) NPs are applied for both medical diagnostics and targeted drug delivery. We investigated the potential cytotoxicity and genotoxicity of uncoated iron oxide (U-Fe3O4) NPs in comparison with oleate-coated iron oxide (OC-Fe3O4) NPs. Testing was performed in vitro in human lymphoblastoid TK6 cells and in primary human blood cells. For cytotoxicity testing, relative growth activity, trypan blue exclusion, 3H-thymidine incorporation and cytokinesis-block proliferation index were assessed. Genotoxicity was evaluated by the alkaline comet assay for detection of strand breaks and oxidized purines. Particle characterization was performed in the culture medium. Cellular uptake, morphology and pathology were evaluated by electron microscopy. U-Fe3O4 NPs were found not to be cytotoxic (considering interference of NPs with proliferation test) or genotoxic under our experimental conditions. In contrast, OC-Fe3O4 NPs were cytotoxic in a dose-dependent manner, and also induced DNA damage, indicating genotoxic potential. Intrinsic properties of sodium oleate were excluded as a cause of the toxic effect. Electron microscopy data were consistent with the cytotoxicity results. Coating clearly changed the behaviour and cellular uptake of the NPs, inducing pathological morphological changes in the cells.

Towards an alternative testing strategy for nanomaterials used in nanomedicine: Lessons from NanoTEST

Abstract In spite of recent advances in describing the health outcomes of exposure to nanoparticles (NPs), it still remains unclear how exactly NPs interact with their cellular targets. Size, surface, mass, geometry, and composition may all play a beneficial role as well as causing toxicity. Concerns of scientists, politicians and the public about potential health hazards associated with NPs need to be answered. With the variety of exposure routes available, there is potential for NPs to reach every organ in the body but we know little about the impact this might have. The main objective of the FP7 NanoTEST project (www.nanotest-fp7.eu) was a better understanding of mechanisms of interactions of NPs employed in nanomedicine with cells, tissues and organs and to address critical issues relating to toxicity testing especially with respect to alternatives to tests on animals. Here we describe an approach towards alternative testing strategies for hazard and risk assessment of nanomaterials, highlighting the adaptation of standard methods demanded by the special physicochemical features of nanomaterials and bioavailability studies. The work has assessed a broad range of toxicity tests, cell models and NP types and concentrations taking into account the inherent impact of NP properties and the effects of changes in experimental conditions using well-characterized NPs. The results of the studies have been used to generate recommendations for a suitable and robust testing strategy which can be applied to new medical NPs as they are developed.

Association between the human immune response and body mass index

The aim of this study was to determine the strength of the association between the human immune response and body mass index (BMI) and whether differences exist in the effects of obesity on selected immune parameters between men and women. Two hundred ninety participants were divided into groups according to sex and BMI. Parameters CD3, CD4, CD8, CD16+56, CD19, HLADR, CD11b, CD11c, and CD54 were quantified. Leukocyte and differential counts were performed. We observed elevation with regard to the normal weight group in the parameters of white blood cells, neutrophils, monocytes, CD3, CD4, CD19, and CD11b for the whole study group. A decrease was observed in the expression of CD16+56. The effect of BMI on the immune system was much more apparent in women. BMI was correlated with the majority of the measured parameters, reflecting a strong association between BMI and the human immune system.

Occupational exposure to mineral fibres. Biomarkers of oxidative damage and antioxidant defence and associations with DNA damage and repair

In order to study the effect of mineral wool exposure on oxidative DNA damage and lipid peroxidation, an epidemiological study was conducted in a mineral wool factory in Slovakia. Altogether 141 subjects were investigated (21-58 years old), 43 controls (20 men and 23 women: 27 non-smokers, 16 smokers) and 98 exposed (75 men and 23 women: 61 non-smokers, 37 smokers). We found higher malondialdehyde (MDA) levels in the group of all exposed workers (P = 0.025) and in exposed non-smokers (P = 0.003) and a significantly suppressed activity of ceruloplasmin oxidase (P = 0.02, P < 0.02, respectively) and catalase (CAT) (P = 0.04, P = 0.01, respectively) in these groups. The activity of glutathione S-transferase (GST) was affected by exposure to mineral wool; levels were significantly lower in all exposed subjects (P = 0.04), in the exposed non-smokers (P = 0.03) and in exposed men (P < 0.01). Concentrations of vitamin C in plasma and the ferric-reducing activity of plasma (FRAP) were not affected by the mineral wool exposure. There was a significant negative correlation between the activity of glutathione peroxidase (GPX) and MDA in the whole group (P < 0.01) and in the exposed group and between CAT activity and MDA in all subjects (P < 0.01). GST activity correlated inversely with oxidized pyrimidines in lymphocyte DNA, in almost all subgroups. We found significant negative correlations between DNA repair and GPX in all subjects (P = 0.03) as well as in control men (P < 0.03) and between DNA repair and CAT in all control subjects (P < 0.02) and in control men (P < 0.01). Interestingly, we found a positive correlation between DNA repair and MDA in all subjects (P < 0.01) and in all exposed subjects (P < 0.03). The presented results indicate that mineral wool exposure induces an increase in oxidative damage to biomolecules especially in the group of male non-smokers. However, optimal levels of antioxidants could have a protective effect. Biomarkers such as MDA, antioxidant enzymes and antioxidant vitamins measured in blood may be useful biomarkers of oxidative stress and antioxidant protection. We do not recommend FRAP as a marker of antioxidant status as interference from other constituents can provide false or confusing results. Our study supports the idea that there might also be other mechanisms by which antioxidant enzymes (especially GST) protect cells against oxidative DNA damage.

Immunotoxicity and genotoxicity testing of PLGA-PEO nanoparticles in human blood cell model

Abstract A human blood cell model for immunotoxicity and genotoxicity testing was used to measure the response to polylactic-co-glycolic acid (PLGA-PEO) nanoparticle (NP) (0.12, 3, 15 and 75 μg/cm2 exposure in fresh peripheral whole blood cultures/isolated peripheral blood mononuclear cell cultures from human volunteers (n = 9–13). PLGA-PEO NPs were not toxic up to dose 3 μg/cm2; dose of 75 μg/cm2 displays significant decrease in [3H]-thymidine incorporation into DNA of proliferating cells after 4 h (70% of control) and 48 h (84%) exposure to NPs. In non-cytotoxic concentrations, in vitro assessment of the immunotoxic effects displayed moderate but significant suppression of proliferative activity of T-lymphocytes and T-dependent B-cell response in cultures stimulated with PWM > CON A, and no changes in PHA cultures. Decrease in proliferative function was the most significant in T-cells stimulated with CD3 antigen (up to 84%). Cytotoxicity of natural killer cells was suppressed moderately (92%) but significantly in middle-dosed cultures (4 h exposure). On the other hand, in low PLGA-PEO NPs dosed cultures, significant stimulation of phagocytic activity of granulocytes (119%) > monocytes (117%) and respiratory burst of phagocytes (122%) was recorded. Genotoxicity assessment revealed no increase in the number of micronucleated binucleated cells and no induction of SBs or oxidised DNA bases in PLGA-PEO-treated cells. To conclude on immuno- and genotoxicity of PLGA-PEO NPs, more experiments with various particle size, charge and composition need to be done.

Comprehensive assessment of nephrotoxicity of intravenously administered sodium-oleate-coated ultra-small superparamagnetic iron oxide (USPIO) and titanium dioxide (TiO2) nanoparticles in rats

Abstract As a main excretory organ, kidney is predisposed to direct/indirect injury. We addressed the potential nephrotoxic effects following expositions of healthy rats to nanoparticle (NP) loads relevant to humans in a situation of 100% bioavailability. Up to 4 weeks after administration, a single iv bolus of oleate-coated ultra-small superparamagnetic iron oxide NPs (in dose of 0.1%, 1.0% and 10.0% of LD50) or TiO2 NPs (1.0% of LD50) did not elicit decline in renal function, damage to proximal tubules, alterations in: renal histology or expression of pro-inflammatory/pro-fibrotic genes, markers of systemic or local renal micro-inflammation or oxidative damage. Antioxidant enzyme activities in renal cortex, mildly elevated at 24 h, completely restored at later time points. Data obtained by multifaceted approach enable the prediction of human nephrotoxicity during preclinical studies, and may serve as comparison for alternative testing strategies using in vitro and in silico methods essential for the NP-nephrotoxicity risk assessment.

Health effects of selected nanoparticles in vivo: liver function and hepatotoxicity following intravenous injection of titanium dioxide and Na-oleate-coated iron oxide nanoparticles in rodents

Abstract The study determined the effect of intravenous administration of acutely toxic or sub-lethal doses of Na-oleate-coated Fe3O4 (OC-Fe3O4) nanoparticles (NPs) on liver structure and function in Wistar rats, compared to titanium dioxide (TiO2) NPs and saline-injected controls. The acute study, using a modified OECD 425 progressive dosing procedure, found LD50 values of 59.22 and 36.42 mg/kg for TiO2 and OC-Fe3O4 NPs, respectively. In the sub-lethal study, rats were either injected with saline (negative controls), a sub-lethal reference (0.592 mg/kgTiO2 NPs, equal to 1% of LD50 on a body weight basis) or OC-Fe3O4 NPs in doses equivalent to 0.1, 1 or 10% of the LD50, respectively (corresponding to 0.0364, 0.364 and 3.64 mg Fe3O4/kg body weight). Animals were sampled 24 h, 1, 2 and 4 weeks post-injection for adverse effects. Mitochondrial respiration was significantly increased 2 weeks after injection of 10% OC-Fe3O4 NPs compared to controls, but the effect was transient. Cholesterol and triacylglycerol concentrations in the liver tissue did not increase in any treatment. There were some disturbances to antioxidant enzymes after OC-Fe3O4 NPs treatment in the livers of animals 1 week post-exposure; with the most sensitive changes occurring in glutathione peroxidase (GPx) and glutathione S-transferase (GST) activities. Lipidosis and mild necrosis with changes in sinusoid space were also observed in histological sections of the liver. Overall, these data suggest that the liver likely retains functional integrity with acute and sub-lethal doses of OC-Fe3O4 NPs, albeit with some stimulation of redox defences and evidence of some tissue injury.

Hydrophobic sodium fluoride-based nanocrystals doped with lanthanide ions: assessment of in vitro toxicity to human blood lymphocytes and phagocytes.

In vitro immunotoxicity of hydrophobic sodium fluoride‐based nanocrystals (NCs) doped with lanthanide ions was examined in this study. Although there is already a significant amount of optical and structural data on NaYF4 NCs, data on safety assessment are missing. Therefore, peripheral whole blood from human volunteers was used to evaluate the effect of 25 and 30 nm hydrophobic NaYF4 NCs dissolved in cyclohexane (CH) on lymphocytes, and of 10 nm NaYF4 NCs on phagocytes. In the concentration range 0.12–75 µg cm−2 (0.17–106 µg ml−1), both 25 and 30nm NaYF4 NCs did not induce cytotoxicity when measured as incorporation of [3H]‐thymidine into DNA. Assessment of lymphocyte function showed significant suppression of the proliferative activity of T‐lymphocytes and T‐dependent B‐cell response in peripheral blood cultures (n = 7) stimulated in vitro with mitogens phytohemagglutinin (PHA) and pokeweed (PWM) (PHA > PWM). No clear dose–response effect was observed. Phagocytic activity and respiratory burst of leukocytes (n = 5–8) were generally less affected. A dose‐dependent suppression of phagocytic activity of granulocytes in cultures treated with 25 nm NCs was observed (vs. medium control). A decrease in phagocytic activity of monocytes was found in cells exposed to higher doses of 10 and 30 nm NCs. The respiratory burst of phagocytes was significantly decreased by exposure to the middle dose of 30 nm NCs only. In conclusion, our results demonstrate immunotoxic effects of hydrophobic NaYF4 NCs doped with lanthanide ions to lymphocytes and to lesser extent to phagocytes. Further research needs to be done, particularly faze transfer of hydrophobic NCs to hydrophilic ones, to eliminate the solvent effect. Copyright

Changes in immunologic parameters of humoral immunity and adipocytokines in obese persons are gender dependent.

The aim of this study was to investigate several immunologic parameters using of immunonephelometry and adipocytokines by the enzyme immunoassay and their changes in different states of obesity. Obesity is considered to involve a state of chronic low-grade inflammation, with links between adipose cells and the immune system. We found significantly higher complement C3 levels in all obese subjects. Levels of the complement C4 were significantly higher in obese women, but not in men, when compared with the corresponding group of normal weight subjects. The increase in C-reactive protein concentrations was significant in both obese and morbidly obese women, but only in morbidly obese men. No significant differences in tumor necrosis factor-α, interleukin-6, interleukin-10, and soluble intercellular adhesion molecule-1 were found. sE-selectin levels were higher in both overweight and obese women but only in morbidly obese men. We found decreased adiponectin concentrations in obese and morbidly obese women. Concentrations of leptin were significantly higher only in obese men (p < 0.05), whereas in women the increase in leptin levels was significant in overweight, obese, and morbidly obese subjects. In conclusion, our results demonstrate elevated levels of C3, C-reactive protein, sE-selectin, and leptin in obese women and men. In obese women, we also observed increased concentrations of C4 and decreased levels of adiponectin.

Immunotoxicity of ethyl-4-isothiocyanatobutanoate in male Wistar rats

The immunotoxicity of ethyl-4-isothiocyanatobutanoate (E-4IB) using different immuno-pathological parameters and immune function assays in male Wistar rats was evaluated. The rats were administered intraperitoneally 12 times with E-4IB in three varying doses of 21, 28 and 35 mg/kg of body weight, over a period of 36 days. The doses of E-4IB were set according to the results of previous experiments by its anti-proliferative activity in vivo. High and medium doses of E-4IB exceeded the maximum tolerated dose after the 36-day treatment period. Symptoms of toxicity were displayed by a drop in body weight, spleen and thymus weight and in organ and bone marrow cellularity. Haematological changes displayed a dose-dependent decrease in the percentage of lymphocytes and dose-dependent increase in the percentage of polymorphonuclear leukocytes in peripheral blood. The white blood cell count in rats exposed to a high dose of E-4IB was suppressed. The immune system of rats administered 21 mg/kg of E-4IB (low dose) was unaffected. No changes in primary antibody response to sheep erythrocytes, in vitro proliferative response of spleen lymphocytes to mitogens and phagocytic activity of leukocytes were found in those rats. Our findings indicate that this newly developed anti-cancer drug is not immunotoxic.