Detection of silver nanoparticles in cells by flow cytometry using light scatter and far-red fluorescence.

The cellular uptake of different sized silver nanoparticles (AgNP) (10, 50, and 75 nm) coated with polyvinylpyrrolidone (PVP) or citrate on a human derived retinal pigment epithelial cell line (ARPE-19) was detected by flow cytometry following 24-h incubation of the cells with AgNP. A dose dependent increase of side scatter and far red fluorescence was observed with both PVP and citrate-coated 50 nm or 75 nm silver particles. Using five different flow cytometers, a far red fluorescence signal in the 700-800 nm range increased as much as 100 times background as a ratio comparing the intensity measurements of treated sample and controls. The citrate-coated silver nanoparticles (AgNP) revealed slightly more side scatter and far red fluorescence than did the PVP coated silver nanoparticles. This increased far red fluorescence signal was observed with 50 and 75 nm particles, but not with 10 nm particles. Morphological evaluation by dark field microscopy showed silver particles (50 and 75 nm) clumped and concentrated around the nucleus. One possible hypothesis to explain the emission of far red fluorescence from cells incubated with silver nanoparticles is that the silver nanoparticles inside cells agglomerate into small nano clusters that form surface plasmon resonance which interacts with laser light to emit a strong far red fluorescence signal. The results demonstrate that two different parameters (side scatter and far red fluorescence) on standard flow cytometers can be used to detect and observe metallic nanoparticles inside cells. The strength of the far red fluorescence suggests that it may be particularly useful for applications that require high sensitivity. © Published 2013 Wiley-Periodicals, Inc.

Detection of TiO2 nanoparticles in cells by flow cytometry.

Evaluation of the potential hazard of man-made nanomaterials has been hampered by a limited ability to observe and measure nanoparticles in cells. In this study, different concentrations of TiO(2) nanoparticles were suspended in cell culture medium. The suspension was then sonicated and characterized by dynamic light scattering and microscopy. Cultured human-derived retinal pigment epithelial cells (ARPE-19) were incubated with TiO(2) nanoparticles at 0, 0.1, 0.3, 1, 3, 10, and 30 microg/ml for 24 hours. Cellular reactions to nanoparticles were evaluated using flow cytometry and dark field microscopy. A FACSCalibur flow cytometer was used to measure changes in light scatter after nanoparticle incubation. Both the side scatter and forward scatter changed substantially in response to the TiO(2). From 0.1 to 30 microg/ml TiO(2), the side scatter increased sequentially while the forward scatter decreased, presumably due to substantial light reflection by the TiO(2) particles. Based on the parameters of morphology and the calcein-AM/propidium iodide viability assay, TiO(2) concentrations below 30 microg/ml TiO(2) caused minimal cytotoxicity. Microscopic analysis was done on the same cells using an E-800 Nikon microscope containing a xenon light source and special dark field objectives. At the lowest concentrations of TiO(2) (0.1-0.3 microg/ml), the flow cytometer could detect as few as 5-10 nanoparticles per cell due to intense light scattering by TiO(2). Rings of concentrated nanoparticles were observed around the nuclei in the vicinity of the endoplasmic reticulum at higher concentrations. These data suggest that the uptake of nanoparticles within cells can be monitored with flow cytometry and confirmed by dark field microscopy. This approach may help fulfill a critical need for the scientific community to assess the relationship between nanoparticle dose and cellular toxicity Such experiments could potentially be performed more quickly and easily using the flow cytometer to measure both nanoparticle uptake and cellular health.

Electronic volume analysis of L1210 chemotherapy.

The rapid analysis of in vivo chemotherapy on the L1210 ascites tumor grown in C57BL/6 X DBA/2F1 mice has been shown by means of an electronic volume analysis. The drugs were injected on the 4th day of tumor growth, and the cells in the peritoneal cavity were studied at 24-hr intervals on the 5th through 7th day. Using the electronic cell volume distributions, combined with labeling indices, cell morphology, and cell counts, it was found that the alkylating agents. 1,3-bis(2-chloroethyl)-1-nitrosourea and cyclophosphamide, at the dosages used, were more effective than the S-phase-specific drugs, palmitoyl ester of 1-beta-D-arabinofuranosylcytosine, vincristine, and methotrexate.

Mouse hemoglobin during gestation. Absence of fetal hemoglobin.

A "fetal hemoglobin' has been reported to exist during mouse gestation, Investigations using CMC chromatography, starch gel electrophoresis or isoelectric focusing have shown a hemoglobin band from fetal tissues, and blood was obtained which was different from the adult hemoglobin and designated a "fetal hemoglobin'. In the current study isoelectric focusing was used to study the hemoglobins existing in the tissues and blood during fetal and neonatal development and the results suggest there is no "fetal hemoglobin' present during gestation. It appears that the hemoglobin designated as "fetal' in our laboratory was a methemoglobin formed by an incomplete reaction of KCN with the hemoglobin. The additional hemoglobin bands which were obtained from fetal liver or neonatal spleen tissues appeared to be a modified adult hemoglobin.

Mouse fetal hemoglobin.

Using isoelectric focusing, a fetal hemoglobin was found in the peripheral blood of C57BL/6 fetal mouse during the 14 to 20 days gestational period. In acid-urea polyacrylamide gel the pattern of this fetal hemoglobin was different from that of the adult hemoglobin. The mouse fetal hemoglobin was differentiated from the mouse adult hemoglobin by immunodiffusion reaction. It suggests that there is a transient fetal hemoglobin in the C57BL/6 mouse during gestational age.

Isolation and characterization of multiple hamster hemoglobins.

Multiple hemoglobins were found in the peripheral blood of hamsters when separated by analytical isoelectric focusing in polyacrylamide gel between pH 6.0 and pH 8.0. Five of these hemoglobins (I-V) were present in sufficient quantities for isolation by preparative isoelectric focusing and subsequent characterization. Cellulose acetate electrophoresis of the isolated hemoglobins resulted in producing as many as six different globin chains. The five hemoglobins were further characterized by their amino acid composition, N-terminal amino acid residue, and molecular weight.

5-Azacytidine increases the synthesis of embryonic hemoglobin (E2) in murine erythroleukemic cells.

The addition of 5-azacytidine to erythroleukemic cells which were induced to differentiate with DMSO or BA altered the expression of the hemoglobins. After the addition of 5-azacytidine there was an increase in hemoglobin synthesis especially in the embryonic E2 band. The beta-globin increased in synthesis after 5-azacytidine treatment. The level of hemoglobin synthesis in DMSO-induced cells is less than BA-induced cells while the effect of the 5-azacytidine stimulation was greater with DMSO induction than with BA induction.

The effect of glutathione on HL-60 treated with dimethylsulfoxide, butyric acid or 12-O-tetradecanoylphorbol-13-acetate.

HL-60 promyelocytic leukemic cells can be induced to differentiate into granulocytes or macrophages. Reduced glutathione lyses undifferentiated HL-60 cells but has minimal effect on their differentiated counterparts. The addition of reduced glutathione to HL-60 promyelocytic leukemic cells retards cell growth and lyses cells. HL-60 cells can be induced to differentiate into granulocytes with dimethylsulfoxide butyric acid or into macrophages with 12-O-tetradecanoylphorbol-13-acetate. After treatment of HL-60 cells with these inducing agents the HL-60 cells become unresponsive to the effects of glutathione.

Cell volume decrease during Friend leukemia cell differentiation.

Friend leukemia cells (GM86, clone 745) were induced to differentiate with dimethyl sulfoxide or butyric acid. The kinetics of induction were measured by cell growth, cell volume distributions, and [3H]thymidine incorporation. From the volume distributions, it was found that the rate of induction was both agent sensitive and concentration dependent. The changes in volume distributions occurred approximately 4 hr earlier with dimethyl sulfoxide induction relative to butyric acid induction. However, the changes with the butyric acid induction were more dependent on concentration. A decrease in labeling indices during the 12- to 20-hr time period was correlated to a decrease in mean cell volume and an increase in the proportion of G1 cells. After the 20-hr time period of induction, an increase in labeling indices and in the percentage of large cells was observed. The data suggest that a transient block of cells in G1 occurred between 12 to 20 hr, and that the early differentiation involved a volume decrease which was related to a redistribution of cell cycle stages. The study was also shown that the changes of cell volume are a rapid monitor to determine the early events of the differentiation process.

Confocal laser scanning microscopy of rat follicle development.

This study used confocal laser scanning microscopy (CLSM) to study follicular development in millimeter pieces of rat ovary. To use this technology, it is essential to stain the tissue before laser excitation with the confocal microscope. Various fluorescent stains (Yo-Pro, Bo-Pro, LysoTracker Red, hydroethidine, ethidium bromide, and 7-aminoactinomycin-d) were applied either to fresh tissue or to tissue that had been fixed with glutaraldehyde or paraformaldehyde. After fixation and staining, the tissue was dehydrated with MEOH and cleared with benzyl alcohol/benzyl aldehyde. CLSM was then used with the appropriate laser excitation, dichroics, and bandpass filters to acquire images of oocytes contained in follicles. Analysis of the data revealed three principal findings. First, a rapid increase in oocyte size occurred in the preantral stages of follicle development. In the antral stage of follicle development, there was a rapid increase in follicle size without any substantial increase in oocyte size. Second, accompanying these changes in oocyte and follicle growth was a differential staining pattern in which the nucleus stained more than the cytoplasm in a young follicle, but stained less than the cytoplasm as the follicle enlarged into the late antral stage. Lastly, using CLSM, atretic follicles showed increased LysoTracker Red staining in the granulosa region of the antral follicle, suggestive of cell death.

The dissociation of transplantable tumors.

Four animal transplantable solid tumors, composed of varying morphologic architecture and intercellular specializations, were studied by light and electron microscopy. These tumors were dissociated into viable single cell populations using a combination of mechanical and enzymatic methods. The conditions necessary for optimal dissociation consisted of (a) preparation of the tumor to maximize the tissue surface area, (b) enzymatic digestion with continuous agitation and (c) additional agitation to release loosely attached cells. Other factors that influenced the dissociation were optimized and discussed.

Polyploidy induction as a consequence of topoisomerase inhibition. A flow cytometric assessment.

Following recovery from a 4-hr exposure to clinically achievable concentrations of the topoisomerase II inhibitors Adriamycin, teniposide, or amsacrine or the putative topoisomerase II inhibitor crisnatol, murine erythroleukemic cells remained viable for up to 48 hr, but did not proliferate. Cell cycle analysis after a 24-hr recovery revealed blocks in G2 (4N DNA) or greater than G2 (up to 8N DNA) polyploid stages. The relative percentages of cells in either stage was a function of drug concentration and cell cycle stage at time of exposure: typically, cells exposed during S phase became blocked in G2, whereas those exposed during G2/M progressed into greater than G2 polyploid stages. G2-blocked cells exhibited a 2- to 3-fold increase in nuclear protein content and cellular/nuclear volume (i.e. unbalanced growth) and approximately 5% more DNA stainability (as a consequence of nuclear conformational changes rather than redundant DNA synthesis). In all cases, at the drug concentrations studied, mitotic figures were absent and G2 and greater than G2 blocks were irreversible, indicating that the mechanism of polyploidy induction differs from that of microtubule inhibitors. These findings suggest that although topoisomerase inhibitors interfere with DNA synthesis in the S phase, their induction of greater than G2 polyploid blocks may involve direct or indirect inhibition of chromosome condensation.

Programmed cell death in extraocular muscle tendon/sclera precursors.

PURPOSE: This study was designed to examine the occurrence of natural cell death in the periocular mesenchyme of mouse embryos. METHODS: Vital staining with LysoTracker Red and Nile blue sulfate as well as terminal nick end labeling (TUNEL) were utilized to identify apoptotic cell death in whole and histologicaly sectioned gestational day 10.5 to 14 mouse embryos. Laser scanning confocal microscopy was used to provide a three dimensional representation of the cell death pattern. Immunohistochemical staining for neural crest and myoblast populations was utilized to indicate the cell population undergoing apoptosis. RESULTS: Programmed cell death was evident in the developing rectus muscle tendons/sclera on gestational days 11 through 12.5 (corresponding to the weeks 5-6 of human development). Although each of these peripheral periocular condensations has readily apparent amounts of apoptosis, the pattern of cell death varied among them. Cell death was most apparent in the superior rectus tendon primordium, while that for the lateral rectus had the least evidence of apoptosis. CONCLUSIONS: Although apoptosis was readily evident in the periocular mesenchyme in distinct regions located medial and distal to the developing rectus muscles, programmed cell death in these sites has not previously been reported. New imaging techniques coupled with stains that evidence apoptotic cell death have made it possible to define this tissue as a prominent region of programmed cell death. Although neuronal tissues, including particular regions of the developing eye, are well recognized as sites of programmed cell death, description of this phenomenon in the extraocular tendon/sclera precursors is novel.

Measurements of iron absorption from prenatal multivitamin--mineral supplements.

Although prenatal multivitamin--mineral supplements containing 60 to 65 mg of iron, taken once daily, are used widely to assure that pregnant women absorb the approximately 3.5 mg of supplemental iron per day that they require, there have been no studies concerning the absorption of iron from these preparations. Using cross-over studies in groups of normal nonpregnant women of childbearing age, such iron absorption was assessed using a technique in which absorption is calculated from the measured increase in serum iron after the oral ingestion of iron in various forms. With each of 4 different brands of prenatal supplements, mean iron absorption was less than the required 3.5 mg and ranged from 1.8 to 3.0 mg. These values were significantly less (P less than .01) than the 8.1 mg that was absorbed from 65 mg of iron alone. Decreased iron absorption in the prenatal supplements was shown to be due to inhibition by calcium carbonate and magnesium oxide and, in some cases, to poor iron release. When one of the 4 brands was reformulated to contain less calcium carbonate and less magnesium oxide, mean iron absorption increased to 4.5 mg. It is concluded that the amount of iron absorbed from many prenatal multivitamin--mineral supplements is significantly less than with standard forms of iron in nonpregnant women and that bioavailability studies should be performed on pregnant patients to determine whether these commercial preparations provide adequate amounts of iron during pregnancy.

Metal-induced alteration of the cell membrane/cytoplasm complex studied by flow cytometry and detergent lysis.

Flow cytometric analysis of the cell cycle is most effectively accomplished with membrane-/cytoplasm-free ("clean") nuclei. Non-ionic detergents (e.g. NP40 or Triton X-100) commonly are employed to solubilize cell membranes/cytoplasm to produce "clean" nuclei. Treatment of murine erythroleukemic cells (MELC) with tri-n-butyltin methoxide, cadmium acetate, zinc sulfate, or lead acetate alters the properties of the cell membrane/cytoplasm complex making it resistant to NP40 dissolution. On a molar basis, the organotin compound was more effective in inducing resistance to detergent-mediated dissolution than the inorganic metal compounds. Resistance to NP40-mediated dissolution was manifested as an increase in the flow cytometric parameters 90 degrees scatter and fluorescein isothiocyanate (FITC) fluorescence and was confirmed by light microscopy.

Flow cytometric comparison of the effects of trialkyltins on the murine erythroleukemic cell.

Cellular effects of exposure to tributyltin (TBT), triethyltin (TET), or trimethyltin (TMT) were investigated by flow cytometry employing the murine erythroleukemic cell (MELC) as a model cellular system. Cell viability was investigated by the carboxyfluorescein diacetate (CFDA) uptake/propidium iodide (PI) exclusion method: above a critical concentration (exposure for 4 h), which was specific for each of the trialkyltin compounds, the cell becomes permeable to PI, indicating loss of viability. Cellular CF fluorescence (derived from intracellular hydrolysis of CFDA) increased as a function of alkyltin concentration below the critical concentration and decreased as viability decreased above the critical concentration. Relative membrane potential, monitored with a cyanine dye (DiOC6), correlated with viability (PI exclusion), remaining essentially unaltered below the critical concentration and decreasing above it. At/above 1 microM TBT, 5 microM TET, or 100 microM TMT, the cell cycle was blocked in the G2/M phase. The 90 degrees light scatter (a measure of refractive index), axial light loss (a measure of volume), and fluorescein isothiocyanate (FITC) fluorescence (a measure of protein content) of nuclei isolated from trialkyltin-treated MELC by detergent treatment, increased as a function of organotin dose. Fluorescence and interference microscopy revealed increased quantities of residual cytoplasmic tags adherent to the nuclei as a function of organotin dose, apparently resulting from increased cytoplasmic resistance to detergent-mediated solubilization. The effects of the trialkyltins correlated with their lipophilicity (octanol/water coefficient). These data support the hypothesis that fixation (protein denaturation, cross-linking, etc.) is an important mode of organotin cytotoxicity.

Biological modeling of 5-fluorouracil developmental toxicity.

A biologically-based dose-response (BBDR) model is a mathematical description of the biological events leading to expression of a toxic response. As an alternative to current approaches in non-cancer risk assessment, such models will reduce uncertainty in that they will provide a more comprehensive description of toxicity. We are involved in construction of a BBDR model for the developmental toxicity of 5-fluorouracil (5-FU) in the rat using multiple approaches. First, to identify critical events in the pathogenesis of 5-FU developmental toxicity, thymidylate synthetase (TS) inhibition and alterations in cell kinetics and growth were examined in embryos following maternal administration of 5-FU on day 14 of gestation. A dose-related decline in TS activity was observed within 1 h; however, maximal inhibition and recovery were similar at 10, 20 and 40 mg/kg. Dose-dependent cell cycle alterations were observed within 4 h after exposure and were maximal at 8 h. Hindlimb growth reduction was observed 24 h after exposure to 40 mg/kg, but not at lower doses. At term hindlimb defects were observed at doses above 30 mg/kg. An integrated dose-response model for hindlimb defects was derived from empirical relationships among these events. The resultant dose-response somewhat over-predicted the developmental toxicity of 5-FU, although results of a Monte Carlo simulation indicated that these data were not incompatible with model predictions. Overall, the results suggest that TS inhibition is a key component of the mechanism of 5-FU developmental toxicology, but the model does not capture all of the critical events in the induction of hindlimb defects. A preliminary mechanistic model for the inhibition of embryonic TS, DNA synthesis and cell cycle following maternal exposure to 5-FU, independently derived from literature data to further examine the potential role of this pathway in its developmental toxicity, predicted a dose-response for TS inhibition and DNA synthesis that closely reflected the observed patterns. These results further suggest that TS inhibition, resultant deficits in DNA synthesis and cell cycle perturbations represent a critical mechanistic pathway in the developmental toxicity of 5-FU.

A new action for topoisomerase inhibitors.

Topoisomerases are known to aid DNA replication by breaking and resealing supercoiled DNA. Consequently, cells exposed to topoisomerase inhibitors before or during the S (DNA synthetic) phase of the cell cycle undergo abnormal DNA replication and become irreversibly blocked in the G2 (pre-mitosis) phase. We report that following a 4-h exposure to topoisomerase II inhibitors, murine erythroleukemic cells (MELC) do not form mitotic figures but exhibit a time-dependent progression into G2 (4N DNA) and greater than G2 (up to 8N DNA) stages of the cell cycle. Following exposure to the topoisomerase I inhibitor camptothecin, recovering MELC also exhibit greater than G2 polyploidy, but to a considerably lesser degree: mitotic figures are present and a subpopulation of cells resumes cycling. However, both topo I and topo II inhibitors induce maximal percentages of greater than G2 cells when synchronized MELC are in the G2/M phase at the time of exposure. This suggests that, in addition to their S-phase action, topoisomerase inhibitors can interfere with chromosome condensation during G2 and, in so doing, induce polyploidy.

Flow cytometric analysis of the cellular toxicity of tributyltin.

Flow cytometric and light/fluorescence microscopic analyses indicate that tributyltin (TBT) alters the plasma membrane/cytoplasm complex of the murine erythroleukemic cell (MELC) in a dose-dependent and time-dependent manner. The flow cytometric parameter axial light loss, a measure of cell volume, decreases in cells exposed to 5 microM TBT relative to control cells or cells exposed to 50 microM TBT. The flow cytometric parameter 90 degrees light scatter, a function of refractive index and a measure of protein content, increases as a function of TBT concentration above 0.5 microM. Following exposure to TBT concentrations greater than 0.5 microM, but less than 50 microM, DNA distribution across the cell cycle cannot be resolved adequately by flow cytometry. Also, the cells become resistant to solubilization of the cell membrane/cytoplasm complex by nonionic detergents. Relative to logarithmically growing cells, MELC in the stationary phase of the growth cycle and butyric acid-differentiated cells exhibit decreased plasma membrane permeability resulting in increased carboxyfluorescein (CF) retention derived from the intracellular hydrolysis of carboxyfluorescein diacetate (CFDA). Similarly, cells exposed to TBT concentrations below 50 microM exhibit increased cellular CF retention. Viability in terms of CFDA hydrolysis/CF retention and propidium iodide (PI) exclusion is not decreased by exposure to TBT concentrations below 1 microM. At doses between 5 and 50 microM, however, cells exhibit both CF and PI fluorescence simultaneously and are programmed for death. At TBT concentrations greater than 1.0 microM, MELC plasma membrane potential, measured with the cyanine dye, 3,3'-dihexyloxacarbocyanine iodide (DiOC6) decreases at the same time that the uptake of PI is observed. In conjunction with other data, the concentration-dependent increase in CF fluorescence, resistance to detergent-mediated solubilization of the plasma membrane/cytoplasm complex, and increase in 90 degrees light scatter suggest fixation (protein denaturation, cross-linking, etc.) as a mechanism of the toxic action of TBT.

Cytogenetic studies of three triazine herbicides. I. In vitro studies.

Atrazine, simazine, and cyanazine are widely used pre-emergence and post-emergence triazine herbicides that have made their way into the potable water supply of many agricultural communities. Because of this and the prevalence of contradictory cytogenetic studies in the literature on atrazine, simazine, and cyanazine, a series of in vitro experiments was performed to investigate the ability of these three triazines to induce sister chromatid exchanges (SCEs) and chromosome aberrations (CAs) in human lymphocyte cultures. Our results showed that all three triazines failed to produce any significant increases in SCEs or CAs up to the limits of solubility [using 0.5% dimethyl sulfoxide (DMSO)]. Our results are discussed in light of contradictory results in the literature.