Małgorzata Stańczyk

Evaluation of biological effects of nanomaterials. Part I. Cyto- and genotoxicity of nanosilver composites applied in textile technologies

ObjectivesThe aim of this study was to investigate the cyto- and genotoxicity of nanocomposites (NCs) and generation of reactive oxygen species (ROS) as a result of particle-cell interactions.Materials and MethodsTitanium dioxide (TiO2-Ag) and ion-exchange resin (Res-Ag), both coated with silver (Ag), were examined. The murine macrophage J774A.1 cells were incubated in vitro with NC at different concentrations for 24 h. Cytotoxicity was analyzed by the methylthiazolyldiphenyl-tetrazolium bromide reduction test (MTT reduction test). ROS generation was assessed by incubation of cells with dichlorodihydrofluorescein diacetate (DCF) and flow cytometry. DNA damage was detected by comet assay and included single-strand breaks (SSB), alkali-labile sites (ALS) and oxidative DNA damage after formamidopyrimidine glycosylase (FPG) treatment. The tail moment was used as an indicator of DNA damage.ResultsTiO2-Ag was not cytotoxic up to 200 μg/ml, whereas IC50 for Res-Ag was found to be 23 μg/ml. Intracellular ROS levels were elevated after 4 h of exposure to Res-Ag at the concentration of 50 μg/ml. Both types of NC induced fragmentation of DNA strands, but only one of the composites caused damage to purine bases. TiO2-Ag induced SSB of DNA at concentrations of 10 and 5 μg/ml. For Res-Ag, a concentration-dependent increase in tail moments was observed.ConclusionsSilver-coated nanocomposites (both TiO2-Ag and Res-Ag) may cause genotoxic effects in murine macrophages J774A.1. Res-Ag increased generation of ROS which suggested that toxicity of Res-Ag in murine macrophages is likely to be mediated through oxidative stress. This paper will support industry and regulators alike in the assessment of hazards and risks and methods for their mitigation at the earliest possible stage in material and product development.

Genotoxic Effects in C57Bl/6J Mice Chronically Exposed to Arsenate in Drinking Water and Modulation of the Effects by Low-Selenium Diet

In C57Bl/6J mice chronically exposed to arsenate in drinking water at 50, 200, or 500 μg As/L, genotoxic effects in bone-marrow cells using micronucleus test and in peripheral blood leukocytes using the comet assay were determined after 3, 6 or 12 mo. To assess the modulating role of selenium in development of the effects, the animals were fed a specially prepared low-selenium diet and were supplemented with sodium selenite (200 μg Se/L) in drinking water (supplemented groups) or were without Se supplementation (nonsupplemented groups). Measurements of glutathione peroxidase activity in erythrocytes and plasma as well as selenium concentration in plasma were performed after 3, 6, and 12 mo and showed a marked decrease in values in animals in non-Se supplemented compared to Se-supplemented groups. After 3 mo of arsenic exposure in the Se-supplemented animals the level of DNA fragmentation (without Endo III and Fpg enzymes) did not differ from the control; however, increased oxidative damage of purine and pyrimidine bases was observed. In groups not supplemented with Se, an increase of DNA fragmentation was observed; however, the levels of oxidative DNA damage in these groups did not differ from the control. None of the positive effects observed in the comet assay after 3 mo was related to arsenate concentration. The levels of DNA damage after 6 and 12 mo of exposure to arsenic as well as the frequency of micronuclei after 3, 6, and 12 mo did not differ significantly between exposed and control animals, irrespective of Se supplementation status.

Assessment of apoptosis in thymocytes and splenocytes from mice exposed to arsenate in drinking water: cytotoxic effects of arsenate on the cells in vitro.

Abstract This study provides an assessment of the level of apoptosis in thymocytes and splenocytes from mice exposed to arsenate in drinking water. To simulate the naturally occurring exposure conditions of humans, the animals were exposed to arsenate at the concentrations of 0.5, 5, and 50 mgAs/L. TUNEL method for staining of thymocytes and splenocytes isolated from the mice after 8 and 12 weeks revealed increased percentage of apoptotic cells in the exposed groups. Although statistically significant increases were observed only for the highest concentration of arsenate, the increases showed linear trend as a function of arsenate concentration in drinking water. {In vitro} experiments performed on isolated cells incubated for 24 hours with arsenate at 6.7–2000 μM showed very similar concentration-viability relationships for both cell populations (IC50 was 442 ± 15 μM and 427 ± 18 μM for thymocytes and splenocytes, respectively). Arsenate induced a concentration-dependent increase in the percentage of the cells undergoing apoptosis. At higher concentrations, apoptosis was the predominant mode of cell death. It can be speculated that proapoptotic effects of arsenate as observed in our {in vivo} study may contribute to some immunotoxic symptoms observed in people chronically exposed to arsenic in drinking water.

Modulation of Murine Peritoneal Macrophage Function by Chronic Exposure to Arsenate in Drinking Water

Exposure of humans to arsenic is associated with various adverse health effects including immunotoxicity and elevated risk of cancer development. Specific mechanisms of these effects are not well understood. In the present study we investigated some functional parameters of peritoneal macrophages isolated from mice exposed for 12 weeks to sodium arsenate in drinking water at 0.5, 5, and 50 mgAs/l. The experimental conditions were matched with the environmental conditions of arsenic exposure in humans. To characterize function of the macrophages, we assessed their ability to release nitric oxide (NO), reactive oxygen species (ROS), and tumor necrosis factor-alpha (TNF-α) in response to common stimulants. To this end the isolated cells were stimulated with lipopolysaccharide (1 µg/ml) to assess NO and TNF-α production (the WEHI-164 bioassay) or with phorbol myristate acetate (5 µg/ml) to assess superoxide production (NBT reduction test). As a result, in mice exposed to 0.5, 5, and 50 mgAs/l we observed decreased production of NO (9 ± 2, 8 ± 2, 11 ± 5 µM NO2−, respectively, versus 27 ± 7 µM in control) and superoxide (41.3 ± 18.2%, 52.8 ± 15.1% and 55.9 ± 12.9%, respectively, less than in control). Despite reduced NO production, expression of iNOS mRNA in RT-PCR, showed similar levels in exposed and control animals. We did not see any significant influence of the exposure on TNF-α release and mRNA expression. The potential consequences of decreased production of NO and superoxide by peritoneal macrophages as observed in exposed mice may suggest impaired response of the cells against infection or developing tumor cells.

Sulindac and its metabolites: Sulindac sulfide and sulindac sulfone enhance cytotoxic effects of arsenic trioxide on leukemic cell lines

The effects of arsenic trioxide (ATO) in combination with sulindac (SUL), sulindac sulfide (SS) or sulindac sulfone (SF) on human (Jurkat, HL-60, K562 and HPB-ALL) and mouse (EL-4) leukemic cell lines were investigated. The cells showed different sensitivity to sulindacs (2.5-200 μM) with SS being the most cytotoxic (72 h WST-1 reduction test). The cytotoxicity of ATO was enhanced by combination with sulindacs. The combination of ATO (1 μM) with SS or SF at concentrations over 50 μM induced considerable cytotoxicity in all cell lines. Normal human lymphocytes exposed for 48 h to the combinations showed smaller decrease in viability. Measurements of Jurkat, HL-60 and K562 cells exposed to ATO (1 μM) and sulindacs (100 μM or 200 μM for K562 cells) indicated apoptosis as the main cell death mechanism. The mitochondrial membrane potential measurements (JC-1 probe) indicated an active involvement of mitochondria in the process. The results did not indicate involvement of an inhibitory effect of the combinations on NF-κB activity in Jurkat, HL-60 and K562 cells.

Carcinogenic effect of arsenate in C57BL/6J/Han mice and its modulation by different dietary selenium status.

In this study, carcinogenic effects of arsenate in female C57BL/6J/Han mice exposed in drinking water to 50, 200 or 500microgAs/L for 24 months were investigated. All animals were fed low-selenium diet, however half of them were supplemented with sodium selenite in drinking water (200microgSe/L) to ensure the normal dietary level of selenium. Glutathione peroxidase activity in erythrocytes and plasma as well as selenium concentration in plasma after 3, 6, 12 and 18 months in satellite groups showed considerable decrease in animals from non-selenium supplemented groups in comparison to supplemented groups. A clear arsenic concentration-dependent increase in the number of malignant lymphoma associated with increase in the risk of death was observed (hazard ratio=0.91, 1.46, and 2.24, for 50, 200 and 500microgAs/L, respectively). No significant influence of selenium dietary status on arsenic carcinogenicity was shown. A significant association between selenium supplementation status and increased risk of death of the animals from causes other than malignant tumors was found (HR=1.79, p=0.04).

Effect of selenium on expression of selenoproteins in mouse fibrosarcoma cells

Selenium (Se), an essential trace element, is incorporated into seleno-proteins as selenocysteine using insertion machinery, including UGA codon and selenocysteine insertion sequence (SECIS) element in the 3 untranslated region (3′-UTR) of mRNA. To assess the biological effects of tumor cells exposed to the elevated, but nontoxic Se level on glutathione peroxidase (GP×1 [cellularar] and GP×3 [extracellular]) thioredoxin reductase (TrxR), and selenoprotein P (SeP) mRNA expression, we introduced a semiquantitative reverse transcription-polymerase chain reaction technique for each selenoprotein transcript using β-actin as a reference housekeeping gene in mouse fibroblasts (WEHI 164). Cell lines were cultured with 1.0, 2.5, and 5.0 ng of Se in 1 mL of medium for 3 and 7 d, apart from the control cell line with standard medium. It was found that Se exerts a statistically significant (p<0.05) effect only on GP×3 mRNA, referred to as the optical density (OD) ratio (GP×3/ β-actin). Moreover, the lowest Se level affected GP×3 mRNA expression more strongly than its highest concentrations. In an in vitro model applied in this study, GP×3 gene expression is most specific for Se supplementation.

Testing the immunosuppressive effects of cyclophosphamide in the popliteal lymph node assay in the modification of graft-vs-host reaction (PLNA-GvHR) in the rat.

We tested the hypothesis that popliteal lymph node assay (PLNA) in the modification of graft vs host reaction (GvHR) enabled to assess immunosuppressive potential of xenobiotics. We conducted PLNA in GvHR modification under two experimental conditions using cyclophosphamide (CY). In the first experiment average lymph node weight index (IW, weight ratio of popliteal lymph node of hind footpad injected with parental lymphocytes to lymph node of footpad injected with vehicle) in rats administered intraperitoneally with CY simultaneously with, or 3 days before local GvHR induction was 84% and 42%, respectively less than average IW in external control animals injected with parental lymphocytes into one footpad and vehicle into contralateral footpad. Average lymph node lymphocyte index (IL, ratio of cell number in the lymph node undergoing GvHR to control lymph node) in the tested animals was 92% and 86%, respectively less than in controls. In the second experiment in rats injected subcutaneously into one footpad with parental lymphocytes suspended in solution of CY at the concentrations of 4.6 mM or 18.6 mM and with parental lymphocytes into contralateral footpad, average IW was 88%and 92%, respectively less than average IW in the external control animals injected with parental lymphocytes into both footpads (average IL was 84 and 91%, respectively less than in controls). Our preliminary experiments showed a strong inhibitory effect of CY in both PLNA-GvHR models used and we believe that both models may be of value in designing future protocols aiming at prediction of immunosuppressive potential of chemicals.

Effects of lactic acid bacteria and Saccharomyces cerevisiae on growth of Aspergillus westerdijkiae and ochratoxin A production and toxicity

The aim of this study was to examine three strains of the yeast Saccharomyces cerevisiae and three strains of lactic acid bacteria belonging to the genus Lactobacillus for their antifungal activity against the ochratoxin A producer Aspergillus westerdijkiae, as well as for their effect on OTA genotoxicity and cytotoxicity. When inoculated simultaneously, fungal growth was completely inhibited by S. cerevisiae. In the case of lactic acid bacteria, growth inhibition also occurred but to a less extent. A significant decrease in toxin production in co-culture with the yeast strains and LAB was observed. The supernatant of 24-h-old cultures of yeast strains in medium with OTA did not influence significantly the viability of porcine kidney epithelial LLC-PK1 cell line, whereas the supernatant from the LAB increased the viability compared to the control. Regarding genotoxicity, a decreased fragmentation of DNA was observed in the presence of the supernatant from wine and brewing yeasts, and Lactobacillus brevis ...