Combined effect of silver nanoparticles and aluminium chloride, butylparaben or diethylphthalate on the malignancy of MDA-MB-231 breast cancer cells and tumor-specific immune responses of human macrophages and monocyte-derived dendritic cells.

The aim of this study was to assess whether silver nanoparticles (AgNP) or selected cosmetic ingredients may modify functions of various immunocompetent cell populations. To this end, the effect of two AgNP (size of 15 nm or 45 nm), alone and in combination with aluminium chloride, butyl paraben, di-n-butyl phthalate or diethyl phthalate was assessed on: (1) migration and invasion of MDA-MB-231 human breast cancer cells; (2) M1/M2 polarization of phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 macrophages (M0) and (3) activation/maturation of monocyte-derived dendritic cells (DCs). The results of this study showed that neither any of the test chemicals alone nor the mixtures significantly changed the migration or invasion ability of MDA-MB-231 cells following, both 72-h and 21-day exposure. Analysis of the expression of marker genes for both M1 (IL-1B, CXCL9, TNF) and M2 (DCSIGN, MRC1) polarization revealed that the chemicals/mixtures did not activate M1/M2 differentiation of the M0 macrophages. In addition, no significant changes were observed in the expression of CD86, HLA-DR and CD54 surface markers and phagocytic activity of DCs following 48-h exposure to AgNP alone or in combination with test compounds. Our study suggests that AgNP alone or in combination with tested cosmetic ingredients do not alter function of immunocompetent cells studied.

Inhibitory effect of silver nanoparticles on proliferation of estrogen-dependent MCF-7/BUS human breast cancer cells induced by butyl paraben or di-n-butyl phthalate.

In this study the effect of silver nanoparticles (AgNPs) on proliferation of estrogen receptor (ER)-positive human breast cancer MCF-7/BUS cells was assessed by means of in vitro assay. The cells were exposed in the absence of estrogens to AgNPs alone or in combination with aluminum chloride (AlCl3), butyl paraben (BPB) and di-n-butyl phthalate (DBPh). The results revealed that AgNPs at the non-cytotoxic concentrations (up to 2µg/mL) and AlCl3 (up to 500µM) did not induce proliferation of MCF-7/BUS cells whereas BPB and DBPh showed strong estrogenic activity with the highest effect at 16µM and 35µM, respectively. AgNPs inhibited the proliferation of the cells induced by DBPh, BPB or even with 17ß-estradiol (E2) during 6-day incubation in the absence of estrogens. ICI 182,780 (10nM), a known estrogen receptor (ER) antagonist, induced strong inhibitory effect. AgNPs also decreased transcription of the estrogen-responsive pS2 and progesterone receptor (PGR) genes but modulated expression neither of ERα nor ERß in MCF-7/BUS cells exposed to BPB, DBPh or E2 for 6h. Our results indicate that AgNPs may inhibit growth of breast cancer cells stimulated by E2 or estrogenic chemicals, i.e. BPB and DBPh.

Transcriptomic analysis of the PI3K/Akt signaling pathway reveals the dual role of the c-Jun oncogene in cytotoxicity and the development of resistance in HL-60 leukemia cells in response to arsenic trioxide.

BACKGROUND: Arsenic trioxide (ATO) is a well-recognized antileukemic drug used for the treatment of newly diagnosed and relapsed acute promyelocytic leukemia (APL). A major drawback of therapy with ATO is the development of APL cell resistance, the mechanisms of which are still not clear. OBJECTIVES: The aim of this study was to investigate the role of the PI3K/Akt signaling pathway in ATOtreated human acute myeloid leukemia (HL-60) cells and in ATO-resistant clones. MATERIAL AND METHODS: The cytotoxicity of ATO was assessed using Trypan blue staining or a WST-1 reduction assay. The Akt phosphorylation level was measured by immunofluorescent staining and flow cytometry. Gene expression analysis was performed using real-time polymerase chain reaction (PCR). RESULTS: The clones derived by culturing for 8-12 weeks in the presence of 1.75, 2.5, and 5 µM ATO were characterized by high viability but a slower growth rate compared to the parental HL-60 cells. The flow cytometry analysis showed that in the parental cells the levels of p-Akt were undetectable or very low, and that ATO had no effect on the level of p-Akt in either the ATO-treated parental cells or the clones. The gene expression analysis revealed that some of the genes involved in the Akt pathway may play a key role in the induction of resistance to ATO, e.g., genes encoding cyclin D1 (CCND1), fork head box O1 (FOXO1), Jun oncogene (JUN), protein kinase C isoform B1 (PRKCB1), because their expression profiles were predominantly changed in the clones and/or the ATO-treated parental HL-60 cells. CONCLUSIONS: The overall results indicate that CCND1, FOXO1, and JUN may contribute to the induction of resistance to ATO, and that the C-Jun N-terminal kinase (JNK) signaling pathway may have greater significance than the phosphoinositide 3-kinase (PI3K)/Akt pathway in mediating the cytotoxic effects of ATO and the development of resistance to ATO in the HL-60 cell line.

The modulating effect of ATM, ATR, DNA-PK inhibitors on the cytotoxicity and genotoxicity of benzo[a]pyrene in human hepatocellular cancer cell line HepG2.

The effect of inhibitors of phosphatidylinositol-3-kinase-related kinases (PIKK): ataxia-telangiectasia mutated (ATM), ATM- and Rad3-related (ATR) and DNA-dependent protein kinase (DNA-PK) on response of HepG2 human liver cancer cells to benzo[a]pyrene (BaP) was investigated. PIKK inhibitors: KU55933 (5 µM), NU7026 (10 µM) or caffeine (1 and 2mM) when used as single agents or in combinations (KU55933/NU7026 and caffeine/NU7026) did not significantly influence the BaP (3 µM) cytotoxicity (MTT reduction test). BaP induced a weak proapoptotic effect which was moderately enhanced by both inhibitor combinations. HepG2 cells exposed to BaP showed a strong S-phase arrest which was considerably diminished by both inhibitor combinations. The DNA damage (comet assay) induced after continuous 24h exposure to BaP was significantly diminished by both inhibitor combinations. Weak induction of reactive oxygen species by BaP was observed, which was not modulated by the inhibitor combinations. Similarly, no modulation of the glutathione levels was observed.

Dysregulation of markers of oxidative stress and DNA damage among nail technicians despite low exposure to volatile organic compounds.

OBJECTIVE: The study aimed to compare levels of selected biomarkers of oxidative stress and DNA damage and their correlation with occupational exposure to volatile organic compounds (VOC) among female nail technicians and a group of unexposed volunteers. METHODS: A panel of biomarkers of oxidative stress and DNA damage was assayed among 145 female nail technicians and 152 healthy female volunteers. Occupational exposure of nail technicians to VOC was assessed analyzing the VOC content in nail salon air samples. RESULTS: The level of occupational exposure of nail technicians to VOC was below the respective threshold limit values with combined airborne exposure to a mixture of VOC, reaching only 3.3% (range 0.2-33.3%) of the threshold limit. Despite that, nail technicians presented increased activity of glutathione peroxidase 1 (GPx1), plasma ceruloplasmin, and the GPx1/superoxide dismutase 1 ratio (P<0.0001). The levels of plasma thiobarbituric acid-reactive species and DNA strand breakage in blood leukocytes were not significantly different. In contrast, total and oxidatively-generated DNA damage were significantly decreased among nail technicians compared to controls (P<0.0001). The individual's current tobacco smoking and alcohol consumption status did not modulate the observed changes. Significant correlations between selected biomarkers of oxidative stress, DNA damage, and airborne levels of VOC (eg, ethanol) were found. CONCLUSIONS: The levels of biomarkers of oxidative stress and DNA damage among nail technicians seem to be dysregulated despite the low level of occupational exposure to VOC. Although the outcomes are not fully conclusive, our findings point to possible causation related to prolonged low-level occupational exposure to VOC.

The influence of ATM, ATR, DNA-PK inhibitors on the cytotoxic and genotoxic effects of dibenzo[def,p]chrysene on human hepatocellular cancer cell line HepG2.

The effect of inhibitors of phosphatidylinositol-3-kinase related kinases (PIKK): ataxia-telangiectasia mutated (ATM), ATM- and Rad3-related (ATR) and DNA-dependent protein kinase (DNA-PK) on the response of HepG2 human liver cancer cells to dibenzo[def,p]chrysene (DBC) was investigated. High cytotoxicity of DBC (IC50=0.1µM) was observed after 72h incubation. PIKK inhibitors: KU55933 (5µM), NU7026 (10µM) or caffeine (1 and 2mM) when used alone did not significantly influence the cytotoxicity. However, two combinations: KU55933/NU7026 and caffeine/NU7026 significantly increased HepG2 viability (by 25%) after treatment with DBC at 0.5µM. The cytoprotective effect was confirmed by cell cycle and apoptosis/necrosis analysis. DNA damage level after exposure to DBC assessed by comet assay (single strand breaks) showed a long persistence and significant decrease after incubation of the cells in the presence the inhibitors (the combination of KU55933+NU7026 showed the strongest effect). Weak induction of reactive oxygen species (ROS) by DBC (0.5µM) was observed. Although, KU55933 and NU7026 when used alone did not increase ROS levels in the cells, their combination induced the ROS increase and moderately enhanced ROS generation by DBC. We propose a mechanism how cells with damaged DNA after exposure to DBC and under the condition of PIKK inhibition, may be at higher risk of undergoing malignant transformation.

Characterization of arsenic trioxide resistant clones derived from Jurkat leukemia T cell line: focus on PI3K/Akt signaling pathway.

In this study the role of PI3K/Akt signaling pathway in arsenic trioxide (ATO)-treated parental Jurkat cells and also in derived ATO-resistant clones grown in the presence of given ATO concentration was investigated. ATO-resistant clones (cultured for 8-12weeks in the presence of 1, 2.5 and 5µM ATO) were characterized by high viability in the presence of ATO but slower growth rate compared to the parental cells. Morphological and functional characterization of derived ATO-resistant clones revealed that they did not differ fundamentally from parental Jurkat cells in terms of cell size, level of GSH, the lysosomal fluorescence or CD95/Fas surface antigen expression. However, a slight increase in the mitochondrial potential (JC-1 staining) was detected in the clones compared to parental Jurkat cells. Side population analysis (Vybrant DyeCycle Violet™ staining) in ATO resistant clones did not indicate any enrichment withcancer stem cells. Akt1/2, AktV or wortmannin inhibitors decreased viability of ATO-resistant clones grown in the presence of ATO, with no effect on ATO-treated parental cells. Flow cytometry analysis showed that ATO decreased the level of p-Akt in ATO-treated parental cells, while the resistant clones exhibited higher levels of p-Akt immunostaining than parental Jurkat cells. Expression analysis of 84 genes involved in the PI3K/Akt pathway revealed that this pathway was predominantly active in ATO-resistant clones. c-JUN seems to play a key role in the induction of cell death in ATO-treated parental Jurkat cells, as dose-dependent strong up-regulation of JUN was specific for the ATO-treated parental Jurkat cells. On the other hand, changes in expression of cyclin D1 (CCND1), insulin receptor substrate 1 (IRS1) and protein kinase C isoforms (PRKCZ,PRKCB and PRKCA) may be responsible for the induction of resistance to ATO. The changes in expression of growth factor receptor-bound protein 10 (GRB10) observed in ATO-resistant clones suggest a possibility of induction of different mechanisms in development of resistance to ATO depending on the drug concentration and thus involvement of different signaling mediators.

A strategy for in vitro safety testing of nanotitania-modified textile products.

Titanium dioxide nanomaterials are extensively used in many applications, also for modification of textile materials. Toxicological assessment of such textile materials is currently seldom performed, mainly because of lack of appropriate guidelines. The aim of the study was to assess cytotoxic and genotoxic potential of commercially available TiO2 and TiO2/Ag NMs in pristine form as well as polypropylene fibers modified with the NMs. Both titania NMs showed a cytotoxic effect on BALB/3T3 clone A31 and V79 fibroblasts after 72-h exposure. Both NMs induced a weak genotoxic effect in comet assay, with TiO2/Ag being more active. In vitro micronucleus test on human lymphocytes revealed a weak mutagenic effect of both materials after 24h of exposure. In contrast, no significant increase in micronuclei frequency was observed in the in vitro micronucleus test on V79 fibroblasts. The 24-h extracts prepared from polypropylene fibers modified with TiO2/Ag induced a cytotoxic effect on BALB/3T3 cells which strongly depended on the mode of the fibers manufacturing. The study presents a comprehensive approach to toxicity assessment of textile fibers modified with NMs. Proposed approach may form a good "starting point" for improved future testing strategies.

Cytotoxic effects in 3T3-L1 mouse and WI-38 human fibroblasts following 72 hour and 7 day exposures to commercial silica nanoparticles.

The potential toxic effects in murine (3T3-L1) and human (WI-38) fibroblast cell lines of commercially available silica nanoparticles (NPs), Ludox CL (nominal size 21 nm) and CL-X (nominal size of 30 nm) were investigated with particular attention to the effect over long exposure times (the tests were run after 72 h exposure up to 7 days). These two formulations differed in physico-chemical properties and showed different stabilities in the cell culture medium used for the experiments. Ludox CL silica NPs were found to be cytotoxic only at the higher concentrations to the WI-38 cells (WST-1 and LDH assays) but not to the 3T3-L1 cells, whereas the Ludox CL-X silica NPs, which were less stable over the 72 h exposure, were cytotoxic to both cell lines in both assays. In the clonogenic assay both silica NPs induced a concentration dependent decrease in the surviving fraction of 3T3-L1 cells, with the Ludox CL-X silica NPs being more cytotoxic. Cell cycle analysis showed a trend indicating alterations in both cell lines at different phases with both silica NPs tested. Buthionine sulfoximine (γ-glutamylcysteine synthetase inhibitor) combined with Ludox CL-X was found to induce a strong decrease in 3T3-L1 cell viability which was not observed for the WI-38 cell line. This study clearly indicates that longer exposure studies may give important insights on the impact of nanomaterials on cells. However, and especially when investigating nanoparticle effects after such long exposure, it is fundamental to include a detailed physico-chemical characterization of the nanoparticles and their dispersions over the time scale of the experiment, in order to be able to interpret eventual impacts on 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.