Genotoxicity of commercial colloidal and laboratory-synthesized silica nanoparticles was tested using the single cell gel electrophoresis or Comet assay. By using a carefully developed protocol and ...careful characterization of the nanoparticle dispersions, Comet assays were performed on 3T3-L1 fibroblasts with 3, 6, and 24 h incubations and 4 or 40 μg/ml of silica nanoparticles. No significant genotoxicity was observed for the nanoparticles tested under the conditions described, and results were independently validated in two separate laboratories, showing that in vitro toxicity testing can be quantitatively reproducible.
The aim of this study was to find differentially regulated genes in THP-1 monocytic cells exposed to sensitizers and nonsensitizers and to investigate if such genes could be reliable markers for an
...in vitro predictive method for the identification of skin sensitizing chemicals. Changes in expression of 35 genes in the THP-1 cell line following treatment with chemicals of different sensitizing potential (from nonsensitizers to extreme sensitizers) were assessed using real-time PCR. Verification of 13 candidate genes by testing a large number of chemicals (an additional 22 sensitizers and 8 nonsensitizers) revealed that prediction of contact sensitization potential was possible based on evaluation of changes in three genes:
IL8,
HMOX1 and
PAIMP1. In total, changes in expression of these genes allowed correct detection of sensitization potential of 21 out of 27 (78%) test sensitizers. The gene expression levels inside potency groups varied and did not allow estimation of sensitization potency of test chemicals. Results of this study indicate that evaluation of changes in expression of proposed biomarkers in THP-1 cells could be a valuable model for preliminary screening of chemicals to discriminate an appreciable majority of sensitizers from nonsensitizers.
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 21nm) and CL-X (nominal size of ...30nm) were investigated with particular attention to the effect over long exposure times (the tests were run after 72h exposure up to 7days). 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 72h 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.
► Ludox CL silica NPs are cytotoxic to WI-38 fibroblasts but not to 3T3-L1 fibroblasts. ► Ludox CL-X silica NPs are cytotoxic to both cell lines. ► In clonogenic assay both silica NPs induce cytotoxicity, higher for CL-X silica. ► Cell cycle analysis shows alterations in both cell lines with both silica NP tested. ► Buthionine sulfoximine enhances cytotoxicity of Ludox CL-X in 3T3-L1 cells.
Abstract
Arsenic trioxide (ATO) induces growth inhibition and apoptosis in a wide variety of cancer cell lines. Some reports indicate that this effect can be enhanced by inhibiting the activity of ...transcription factor NF-κB.
The aim of our study was to evaluate the efficacy of combined treatment with ATO and selected NF-κB inhibitors (sulindac, parthenolide, BAY 11-7082, MG-132 and gliotoxin) in inducing cell death of five leukemic cell lines: EL-4 mouse thymoma, Jurkat, HL-60, K562 and HPB-ALL. The cytotoxicity of the chemicals was evaluated using 72-hrs WST-1 reduction assay and apoptosis was assessed with Annexin V-FITC/propidium iodide staining (FACS).
We observed that among the inhibitors tested, gliotoxin and MG-132 when used alone showed the highest cytotoxicity on all cell lines (effective concentrations less than 1 μM). Parthenolide and BAY11-7082 induced complete death of all cell lines at the concentration of 10 μM. All four inhibitors did not potentiated the effects of ATO used at 0.5 or 1 μM. Although sulindac was the least cytotoxic compound when used alone, it showed the highest potentiating effect on ATO induced cytotoxicity on all cell lines. The response of cells to the combination was rather diverse with Jurkat and HPB-ALL cells being the most sensitive. As a rule, all cell lines treated simultaneously with ATO (1 μM) and sulindac (100 μM) showed complete decrease in viability after 72 hrs. The results with annexin V/PI staining indicated apoptosis as the main cell death mechanism. This combination had much weaker effect on the viability of healthy blood lymphocytes.
The results of our study reveal an interesting cytotoxic effect of ATO with combination with sulindac on a panel of leukemic cell lines. The work was supported by the Polish State Committee for Scientific Research (grant No PB 2806/B/PO1/2007/33).
Citation Information: Clin Cancer Res 2010;16(7 Suppl):A28
Abstract
Arsenic trioxide (ATO) is a well-known carcinogen but also an effective cancer therapeutic drug for acute promyelocytic leukemia. Despite intensive research efforts, the molecular mechanisms ...underlying antileukemic effect of ATO as well as resistance to arsenic therapy appearing during treatment are not well understood. There are data showing that dysregulation of the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway, a central integrating point of survival signals, may be the reason of apoptosis resistance in leukemic cells.
The aim of the present study was to assess expression of 84 genes involved in PI3K/Akt signaling pathway in Jurkat cells exposed to ATO at 1, 2.5 and 5 μM for 16 hrs as well as in ATO resistant clones derived from the cells. First screening of genes was performed using the Human PI3K-AKT Signaling Pathway RT2 ProfilerTM PCR Array (Superarray) and then selected genes were verified in a standard real-time PCR.
Results of our study showed that ATO induced more intensive changes in expression of PI3K/Akt pathway genes in ATO resistant clones than in Jurkat cells exposed to arsenic. Among genes differently regulated in both cell types were these playing a key role in regulating cell growth e.g. GRB10 (growth factor receptor-bound protein 10), FASLG (Fas ligand), CCND1 (cyclin D1). Interestingly, the level of FASLG mRNA was strongly down-regulated in ATO treated Jurkat cells and up-regulated in ATO resistant clones. Expression of CCND1 and GRB10 varied between resistant clones in a concentration-dependent manner. Moreover, concentration-dependent up-regulation of JUN (jun oncogene) was the most characteristic effect induced by ATO in Jurkat cells. Comparing to untreated Jurkat cells, we identified several genes differently regulated in ATO resistant clones (but not in Jurkat ATO exposed cells) that were mainly involved in anti-apoptotic pathways (e.g. IRS1 (Insulin receptor substrate 1), MAP2K1 (Mitogen-activated protein kinase kinase 1), MAPK3 (Mitogen-activated protein kinase 3), RPS6KA1 (Ribosomal protein S6 kinase, 90kDa, polypeptide 1), PRKCB (Protein kinase C, beta 1), PRKCZ (Protein kinase C, zeta) or Akt activation and growth regulation (e.g. PDK1 (Pyruvate dehydrogenase kinase, isozyme 1), MTOR (Mechanistic target of rapamycin), MTCP1 (Mature T-cell proliferation 1)).
In conclusion, our in vitro study identified several targets of ATO that may mediate cytotoxic effects in Jurkat cells. Moreover, the data suggest that the PI3K/Akt signaling pathway plays an important role in the ATO resistance in Jurkat leukemic cells. The work was supported by the Polish State Committee for Scientific Research (grant No PB 2806/B/PO1/2007/33).
Citation Information: Clin Cancer Res 2010;16(7 Suppl):B16
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.
Abstract The biocide and environmental pollutant bis(tri- n -butyltin)oxide (TBTO) causes thymus atrophy in rodents. Whether the depletion of thymic lymphocytes by tributyltin compounds may be the ...result of inhibition of cell proliferation or induction of apoptosis is subject of debate. We examined gene expression profiles in primary rat thymocytes exposed to TBTO in vitro at dose levels of 0, 0.1, 0.3, 0.5, and 1.0 μM. By measuring cell viability and apoptosis, exposure conditions were selected that would provide information on changes in gene expression preceding or accompanying functional effects of TBTO. Several processes related to TBTO-induced toxicity were detected at the transcriptome level. Effects on lipid metabolisms appeared to be the first indication of disruption of cellular function. Many transcriptional effects of TBTO at higher dose levels were related to apoptotic processes, which corresponded to present or subsequent thymocyte apoptosis observed phenotypically. The gene expression profile was, however, not unambiguous since expression of apoptosis-related genes was both increased and decreased. Stimulation of glucocorticoid receptor signaling appeared to be a relevant underlying mechanism of action. These findings suggest that TBTO exerts its toxic effects on the thymus primarily by affecting apoptotic processes, but the possibility is discussed that this may in fact represent an early effect that precedes inhibition of cell proliferation. At the highest dose level tested, TBTO additionally repressed mitochondrial function and immune cell activation. Our in vitro toxicogenomics approach thus identified several cellular and molecular targets of TBTO that may mediate the toxicity towards thymocytes and thereby its immunosuppressive effects.
The protective action in C57BL/6J mice from orally administered ellagic acid (EA), benzyl isothiocyanate (BITC), an extract of epigallocatechins (Tegreen®) as well as chlorophyllin (CHL) against ...benzoapyrene (BaP)-induced DNA damage and cytogenetic effects was investigated. In pilot experiment the comet assay indicated protective effects for all compounds, while such activity was confined to EA and CH with respect to BaP-DNA adducts and micronuclei. EA and CH were chosen for the main study where the levels of DNA adducts in liver after injection of 30mg BaP/kg b.w. did not differ from those found for animals exposed to BaP and treated with the protective substances. In leukocytes no significant protective effect of CHL was detected while a 2-fold increase of adduct concentrations was observed after co-administration of EA. In the comet assay CHL or EA caused a 3-fold decrease of SSB, and a 2-fold decrease of FPG sites in comparison to animals treated with BaP. CHL or EA showed a significant protective effect against BaP-induced MN in polychromatic erythrocytes in bone marrow. In contrast, flow cytometry measurements in peripheral blood indicated the MN frequency after treatment with CHL or EA almost twice as high as that recorded for BaP alone.
▸ We estimate of microcystins (MCs) role in environment and crude or purified extra. ▸ No clear relation between MCs in extracts and lymphocytes mortality was observed. Also, no clear relation ...between MCs in extracts and DNA damage was observed. ▸ Cyanobacteria have other microbiological agents, which affected lymphocytes. ▸ Determination of total threat from natural cyanobacterial blooms is recommended.
The present study confirmed the domination of two cyanobacterial genera Microcystis aeruginosa and Aphanizomenon sp. in the summer season in Jeziorsko Reservoir (Central Poland). On the basis of mcyE gene analysis, toxigenic cyanobacterial strains were detected throughout the monitoring period. The highest concentration and toxicity of microcystins (above 2μgL−1), with domination of the hydrophobic variant microcystin-YR, appeared in August and reached the cell concentrations for The First Alert Level, according to WHO guidelines for recreational water. For the estimation of the potential threat to human health from regular exposure to microcystins-containing cyanobacteria, in the second phase of study, cultured human lymphocytes were assessed by: XTT reduction test, comet assay and micronucleus test. The effects of three different variants of cyanobacterial extracts including: (i) crude microcystins-containing extracts and (ii) purified microcystins-containing and (iii) nonmicrocystins-containing extracts were investigated. The present study indicated the highest cytotoxicity and genotoxicity of the purified extract containing microcystins (MC-LR, MC-RR and MC-YR). Additionally, no significant effect or clear relation between microcystins concentration in crude extracts and human lymphocytes mortality and DNA damage was observed. The data indicated the influence of other compounds, apart from microcystins, present in the extracts. This research supports the necessity of application of additional tests for determination of total threat from natural cyanobacterial blooms, apart from determination of concentration and toxicity of the main group of cyanotoxins.
