Biological effects of molybdenum(IV) sulfide nanoparticles and microparticles in the rat after repeated intratracheal administration.

In this study, molybdenum(IV) sulfide (MoS2 ) nanoparticles (97 ± 32 nm) and microparticles (1.92 ± 0.64 µm) stabilized with poly (vinylpolypyrrolidone) (PVP) were administered intratracheally to male and female rats (dose of 1.5 or 5 mg/kg bw), every 14 days for 90 days (seven administrations in total). Blood parameters were assessed during and at the end of the study (hematology, biochemistry including glucose, albumins, uric acid, urea, high density lipoprotein HDL, total cholesterol, triglycerides, aspartate transaminase, and alanine transaminase ALT). Bronchoalveolar lavage fluid (BALF) analyses included cell viability, biochemistry (total protein concentration, lactate dehydrogenase, and glutathione peroxidase activity), and cytokine levels (tumor necrosis factor α, TNF-α, macrophage inflammatory protein 2-alpha, MIP-2, and cytokine-induced neutrophil chemoattractant-2, CINC-2). Tissues were subjected to routine histopathological and electron microscopy (STEM) examinations. No overt signs of chronic toxicity were observed. Differential cell counts in BALF revealed no significant differences between the animal groups. An increase in MIP-2 and a decrease in TNF-α were observed in BALF in the exposed males. The histopathological changes in the lung evaluated according to a developed classification system (based on severity of inflammation, range 0-4, with 4 indicating the most severe changes) showed average histopathological score of 1.33 for animals exposed to nanoparticles and microparticles at the lower dose, 1.72 after exposure to nanoparticles at the higher dose, and 2.83 for animals exposed to microparticles at the higher dose. In summary, it was shown that nanosized and microsized MoS2 can trigger dose-dependent inflammatory reactions in the lungs of rats after multiple intratracheal instillation irrespective of the animal sex. Some evidence indicates a higher lung pro-inflammatory potential of the microform.

Comparative analysis of biological effects of molybdenum(IV) sulfide in the form of nano- and microparticles on human hepatoma HepG2 cells grown in 2D and 3D models.

Significance of MoS2 nanoparticles as a lubricant or drug carriers indicates the need to assess their safety. In the study we analyzed the effects of MoS2 nano- and microparticles and their internalization in vitro, using 2D and 3D culture models of human hepatoma HepG2 cell line. MoS2 micro- and nanoparticles were characterized with high resolution electron microscopy (HR-SEM), X-ray diffraction (XRD) and Energy Dispersive X-Ray Spectroscopy (EDS). The cells were exposed to a range of concentrations of the nano-and microparticles suspensions (maximum of 250 µg/mL) for 72 h. Cell viability was assessed using WST-1 reduction test and LDH release assay. Particle internalization was analyzed using scanning transmission electron microscopy (STEM). The nanoparticles were internalized into the 2D and 3D cultured cells, in spheroids more efficiently into the outer layer. For microparticles mainly particles of less than 1 µm in diameter underwent internalization. This process, however, did not affect cell viability as measured with the WST-1 and LDH assays. STEM observation showed well preserved integrity of the cell membrane and no apparent cytotoxic effect. Although the particles seemed to be safely sequestered in vacuoles or the cytoplasm, their fate and eventual biological effects are not certain and deserve further studies.

Cytotoxic effects in transformed and non-transformed human breast cell lines after exposure to silver nanoparticles in combination with selected aluminium compounds, parabens or phthalates.

This study was undertaken to assess cytotoxic effects of selected aluminium compounds, parabens and phthalates in combination with silver nanoparticles (AgNP, 15 and 45 nm by STEM, Ag15 and Ag45, respectively) on cell lines of the human breast epithelium, normal (MCF-10A) and transformed (MDA-MB-231 and MCF-7). Combination indices were the most spectacular at effective concentrations (ED) inducing 25 % decrease in viability for the combinations of Ag15 with AlCl3 for MDA-MB-231 cells or aluminium zirconium tetrachlorohydrex Gly (AlZr) for MCF-10A and MCF-7 cells, where rather strong antagonism was revealed. As the ED values increased, those effects were enhanced (e.g. Ag15+AlCl3 for MDA-MB-231) or reversed into synergism (e.g. Ag15+AlZr for MCF-7). Another strong effect was observed for aluminium chloride hydroxide, which increasing ED, induced synergistic effect with both Ag15 and Ag45 on MCF-10A cells. Another interesting synergistic effect was observed for DBPh, but only in combination with Ag45 on MCF-10A and MCF-7. The results on cytotoxicity, cell cycle and oxidative stress induction indicate complex response of the cell lines to combined treatment with silver nanoparticles and the chemicals, which were influenced by diverse factors, such as physico-chemical characteristics of AgNP, method of their synthesis, concentrations used, and finally cell type.

The effect of inhibitors of phosphatidylinositol 3-kinase-related kinases on dibenzo[def,p]chrysene genotoxicity measured by γH2AX levels and neutral comet assay in HepG2 human hepatocellular cancer cells.

In the study the modulating effect of inhibition of phosphatidylinositol 3-kinase-related kinases (PIKK): ATM (Ataxia Telangiectasia Mutated), ATR (Ataxia Telangiectasia and Rad3 Related) and DNA-PK (DNA-dependent protein kinase) on genotoxicity of dibenzo[def,p]chrysene (DBC) in HepG2 human hepatocellular cancer cells was investigated. The cytotoxicity of DBC was determined, also in combination with PIKK inhibitors, using the MTT reduction assay. The high cytotoxicity of DBC was observed after 72 h incubation (IC50 = 0.06 µM). The PIKK inhibitors applied at non-cytotoxic concentrations: caffeine (1 mM) and KU55933 (2.5 µM) had no significant influence on the DBC cytotoxicity, however NU7026 (5 µM) caused significant increase in the cell viability by about 25%. The combinations of the inhibitors (double or triple) where NU7026 was present also caused increase in the cell viability (i.e. cytoprotective effect) compared to the effect of DBC. The level of damage to the genetic material (DNA double strand breaks, DSB) was assessed by measuring levels of phosphorylated form of H2A histone (γH2AX) and neutral comet assay. DBC induced DSB in a concentration and time-dependent manner. NU7026 considerably reduced the level of DSB level measured by γH2AX and comet assay. The obtained results confirm that DBC is cytotoxic and causes damage to the genetic material including DSB. The DNA-PK inhibitor NU7026 increases cell viability after exposure to DBC and reduces DNA damage, what indicates an important role of the sensor kinase in mediating the effect.

Micronuclei frequency in peripheral blood lymphocytes and levels of anti-p53 autoantibodies in serum of residents of Kowary city regions (Poland) with elevated indoor concentrations of radon.

INTRODUCTION: Ninety four residents of Kowary city (Poland) have been investigated for environmental radon exposure that ranged from 0.24 WLM to 9.6 WLM (activity concentration range: 35-2700 Bq/m3). Kowary was chosen because of uranium mineralisation in its close vicinity. METHOD: Whole population studied was divided into two groups: exposed to low radon activity concentrations resulting in the exposure of ≤0.55 WLM (value corresponding to the exposure to 100 Bq/m3 during whole year), and exposed to high radon activity concentration (>0.55 WLM). In the two groups two selected biomarkers in blood were assessed: the cytokinesis-block micronucleus assay (CBMN) on peripheral blood lymphocytes (PBL), and the levels of anti-p53 antibodies in serum measured because some data indicate increased expression of the antibodies in individuals after exposure to DNA damaging agents including radon. The potential confounding factors known to influence micronuclei (MN) frequency were also measured in serum: vitamin B12, folic acid, as well as total calcium. RESULTS: In the present study no significant correlation was found between MN frequency in PBL and radon exposure. Among all persons investigated only 11 had detectable levels of the anti-p53 antibodies, whereas only 3 persons had positive result. Therefore, the group was too small to perform any meaningful statistical analysis and to conclude on any association. Cigarette smoking did not significantly influence the number of MN. There was a significant positive correlation observed between MN frequency and age, as well as higher MN frequency was detected in women. CONCLUSION: The problem of the radon exposure is still unresolved and needs further studies on bigger human cohorts in order to search for more sensitive biomarkers.

Genotoxic effects in transformed and non-transformed human breast cell lines after exposure to silver nanoparticles in combination with aluminium chloride, butylparaben or di-n-butylphthalate.

In the present study genotoxic effects after combined exposure of human breast cell lines (MCF-10A, MCF-7 and MDB-MB-231) to silver nanoparticles (AgNP, citrate stabilized, 15 and 45nm by STEM, Ag15 and Ag45, respectively) with aluminium chloride, butylparaben, or di-n-butylphthalate were studied. In MCF-10A cells exposed for 24h to Ag15 at the concentration of 23.5µg/mL a statistically significant increase in DNA damage in comet assay (SSB) was observed. In the presence of the test chemicals the genotoxic effect was decreased to a level comparable to control values. In MCF-7 cells a significant increase in SSB level was observed after exposure to Ag15 at 16.3µg/mL. The effect was also diminished in the presence of the three test chemicals. In MDA-MB-231 cells no significant increase in SSB was observed, however increased level of oxidative DNA damage (incubation with Fpg enzyme) was observed after exposure to combinations of both AgNP with aluminium chloride. No increase in micronuclei formation was observed in neither cell line after the single nor combined treatments. Our results point to a low risk of increased genotoxic effects of AgNP when used in combination with aluminium salts, butylparaben or di-n-butylphthalate in consumer products.

DNA damage and oxidative stress response to selenium yeast in the non-smoking individuals: a short-term supplementation trial with respect to GPX1 and SEPP1 polymorphism.

PURPOSE: Selenium, both essential and toxic element, is considered to protect against cancer, though human supplementation trials have generated many inconsistent data. Genetic background may partially explain a great variability of the studies related to selenium and human health. The aim of this study was to assess whether functional polymorphisms within two selenoprotein-encoding genes modify the response to selenium at the level of oxidative stress, DNA damage, and mRNA expression, especially in the individuals with a relatively low selenium status. METHODS: The trial involved 95 non-smoking individuals, stratified according to GPX1 rs1050450 and SEPP1 rs3877899 genotypes, and supplemented with selenium yeast (200 µg) for 6 weeks. Blood was collected at four time points, including 4 weeks of washout. RESULTS: After genotype stratification, the effect of GPX1 rs1050450 on lower GPx1 activity responsiveness was confirmed; however, in terms of DNA damage, we failed to indicate that individuals homozygous for variant allele may especially benefit from the increased selenium intake. Surprisingly, considering gene and time interaction, GPX1 polymorphism was observed to modify the level of DNA strand breaks during washout, showing a significant increase in GPX1 wild-type homozygotes. Regardless of the genotype, selenium supplementation was associated with a selectively suppressed selenoprotein mRNA expression and inconsistent changes in oxidative stress response, indicating for overlapped, antioxidant, and prooxidant effects. Intriguingly, DNA damage was not influenced by supplementation, but it was significantly increased during washout. CONCLUSIONS: These results point to an unclear relationship between selenium, genotype, and DNA damage.

Assessment of the involvement of oxidative stress and Mitogen-Activated Protein Kinase signaling pathways in the cytotoxic effects of arsenic trioxide and its combination with sulindac or its metabolites: sulindac sulfide and sulindac sulfone on human leukemic cell lines.

The purpose of the study was to characterize the involvement of reactive oxygen species (ROS) in mediating the cytotoxic effects of arsenic trioxide (ATO) in combination with sulindac or its metabolites: sulfide (SS) and sulfone (SF) on human leukemic cell lines. Jurkat, HL-60, K562, and HPB-ALL cells were exposed to the drugs alone or in combinations. Cell viability was measured using WST-1 or XTT reduction tests and ROS production by dichlorodihydrofluorescein diacetate staining (flow cytometry). Modulation of (a) intracellular glutathione (GSH) level was done by using L: -buthionine sulfoximine (BSO) or diethylmaleate (DEM), (b) NADPH oxidase by using diphenyleneiodonium (DPI), and (c) MAP kinases by using SB202190 (p38), SP600125 (JNK), and U0126 (ERK) inhibitors. ATO cytotoxicity (0.5 or 1 µM) was enhanced by sulindacs, with higher activity showed by the metabolites. Strong cytotoxic effects appeared at SS and SF concentrations starting from 50 µM. The induction of ROS production seemed not to be the major mechanism responsible for the cytotoxicity of the combinations. A strong potentiating effect of BSO on ATO cytotoxicity was demonstrated; DEM (10-300 µM) and DPI (0.0025-0.1 µM; 72 h) did not influence the effects of ATO. Some significant decreases in the viability of the cells exposed to ATO in the presence of MAPK inhibitors comparing with the cells exposed to ATO alone were observed; however, the effects likely resulted from a simple additive cytotoxicity of the drugs. The combinations of ATO with sulindacs offer potential therapeutic usefulness.

Comparison of the effects of arsenic and cadmium on benzo(a)pyrene-induced micronuclei in mouse bone-marrow.

This study was undertaken to investigate the genotoxic interactions between the common environmental pollutants: arsenic (As), cadmium (Cd) and benzo(a)pyrene (BaP), which are known to be human carcinogens. C57BL/6J/Han mice were pre-treated with 100mg cadmium chloride (Cd(2+))/L or 50mg sodium arsenite (As(3+))/L in drinking water for 7 days and then given a single dose of 200mg BaP/kg bw by intra-peritoneal injection. A third group of mice did not receive the pre-treatment and was given BaP alone. Mice were sacrificed before or at 12, 24, 48 or 72h after BaP administration. Chromosome damage in bone-marrow cells was assessed by use of the micronucleus test. The study revealed that BaP induced a statistically significant increase in micronucleus (MN) frequency at 48h after administration. In animals exposed to Cd in drinking water no enhancement of genotoxicity was observed compared with the control group that was given tap water only. In Cd/BaP co-exposed animals, the MN frequency at respective time points did not differ from that for the animals exposed solely to BaP. A statistically higher MN frequency was found in bone marrow of animals exposed to As compared with controls that received tap water (0.92+/-0.29% versus 0.38+/-0.13%, respectively). This effect was even more pronounced after combined exposure to As and BaP. In the co-exposed animals, significantly elevated levels of MN were detected in samples examined at 12, 24 and 48h after BaP administration, compared with animals receiving BaP alone (1.14+/-0.31%, 1.26+/-0.3% and 2.02+/-0.45% versus 0.44+/-0.13%, 0.44+/-0.11% and 1.04+/-0.44%, respectively). These findings imply strong interactions between As and BaP, but not between Cd and BaP, in inducing DNA damage in polychromatic erythrocytes in mouse bone-marrow.

Micronucleus frequency in peripheral blood lymphocytes and buccal mucosa cells of copper smelter workers, with special regard to arsenic exposure.

Occupational exposure in copper smelters may produce various adverse health effects including cancer which, according to available epidemiologic data, is associated mainly with exposure to arsenic. Despite a number of well-documented studies reporting an increased risk of cancer among copper smelters workers, the data on genotoxic effects in this industry are scarce. In view of the above, an assessment of micronuclei (MN) frequency in peripheral blood lymphocytes and buccal epithelial cells from copper smelter workers was undertaken. Additionally, the clastogenic/aneugenic effect in lymphocytes was assessed with the fluorescence in situ hybridization (FISH). The study was conducted in three copper smelters in southwestern Poland. The subjects (n = 72) were enrolled among male workers at departments where As concentration in the air was up to at 80 microg/m(3). Exposure was assessed by measurement of arsenic concentration in urine and toenail samples. The control group (n = 83) was recruited from healthy male individuals living in central Poland who did not report any exposure to known genotoxins. The results of our study showed a significant increase in MN frequency in peripheral blood lymphocytes and in buccal epithelial cells of smelter workers, compared to the controls (7.96 +/- 4.28 vs. 3.47 +/- 1.70 and 0.98 +/- 0.76 vs. 0.50 +/- 0.52, respectively). The FISH technique revealed the presence of clastogenic and aneugenic effects in peripheral blood lymphocytes in both groups. The clastogenic effect was slightly more pronounced in the smelter workers; however, the difference was not statistically significant. The mean arsenic concentrations in urine (total arsenic species) and in toenail samples in the exposed group were 54.04 +/- 42.26 microg/l and 7.63 +/- 7.24 microg/g, respectively, being significantly different from control group 11.01 +/- 10.84 microg/l and 0.51 +/- 0.05 microg/g. No correlation between As content in urine or toenail samples and the genotoxic effect was found under study.

Assessment of usefulness of J774A.1 macrophages for the assay of IL-1beta promoter activity.

Existing data indicate that the increase of il-1beta gene expression can be a promising marker of Langerhans cells activation after exposure to contact sensitizers. In this study, we were interested in development of an alternative in vitro screening test detecting such sensitizers. Two IL-1beta reporter constructs containing the enhanced green fluorescent protein (GFP) gene and mouse IL-1beta promoter fragments of varying lengths (-500 bp and -4093 bp) were used for transient transfections of J771A.1 murine monocyte-macrophage cells. As a result of the transfections performed using Lipofectamine reagent we did not observe any GFP fluorescence after stimulation of the cells with LPS as well as known sensitizers (potassium tetrachloroplatinate, dinitrochlorobenzene and nickel sulfate). Low transfection efficiency of J774A.1 cells (less than 0.1%) was confirmed using control plasmid containing GFP gene under the control of cytomegalovirus promoter. The fact that, using the same conditions, we were able to transfect murine fibroblasts 3T3-L1 with the control plasmid very efficiently, may support the theory of high metabolic activity of macrophages being responsible for the extremely low transfection efficiency. These data suggest limited suitability of J774A.1 cell line for transient transfections using cationic liposomes.

Immunohistochemical evaluation of P21ras and P53 proteins expression in human non-small-cell lung cancers.

Human non-small-cell lung cancers (NSCLCs) of 48 patients were analyzed immunohistochemically to detect P21 ras and P53 proteins expression. The relationship between P21 ras and P53 proteins expression and clinicopathologic findings was also assessed. DAKO EnVision TM detection system was employed in the study. The P21 ras and P53 proteins expression was shown in 75% (36/48) and 33.3% (16/48) studied NSCLCs, respectively. In both cases the difference was significant when compared with adequate negative control. Simultaneous expression of both studied proteins was observed in all cases in which P53 expression was noticed. No significant association of P21 ras and P53 expression was found with age, histologic type, histologic grade, tumor size or lymph node metastasis of the studied NSCLCs. Therefore, our study suggests that P21 ras and P53 protein play a role in the pathogenesis of NSCLCs but they have no value as a prognostic markers in the case of lung cancers.