Edward J. Massaro

Folate and human development

Animal Models of Folate-Related Neural Tube Defects Nicholas D. E. Greene and Andrew J. Copp Genetic Variation in Folate Metabolism: Impact on Development Rima Rozen Folate Deficiency and the Molecular Determinants of Chromosome Instability: Possible Link to Meiotic Nondisjunction and Down Syndrome S. Jill James and Charlotte A. Hobbs Folic Acid, Vitamin B12, and Genomic Stability of Human Cells Michael Fenech Molecular Genetics of Folate Metabolism Manuela Fodinger, Sonja Skoupy, and Gere Sunder-Plassmann Folate Receptor-a: An Update Barton A. Kamen Folate, Homocysteine, and Neural Tube Defects Tom K. A. B. Eskes Folic Acid and Homocysteine as Risk Factors for Neural Tube Defects Robert C. Barber, Janee van Waes, Edward J. Lammer, Gary M. Shaw, Thomas H. Rosenquist, and Richard H. Finnell Developmental Toxicants Potentially Acting via Folate Perturbation Deborah K. Hansen Brain Development and Choline, the Other Methyl Donor Steven H. Zeisel Nutrient Interactions Between Folate and Zinc or Copper: Their Possible Implications to Pregnancy Outcome Tsunenobu Tamura Regulation of Folate Metabolism by Iron A. Katherine Herbig and Patrick J. Stover A Differential Role for Folate in Developmental Disorders, Vascular Disease, and Other Clinical Conditions: The Importance of Folate Status and Genotype Mark Lucock and Zoe Yates Vascular Disease in Women: Folate and Homocysteine Tom K. A. B. Eskes Folate, Homocysteine, and Cardiovascular Disease Jayne V. Woodside and Ian S. Young

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.

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.

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.

The reversibility of tributyltin-induced toxicity in vitro as a function of concentration and duration of exposure (C × T)1

The toxicity exhibited by murine erythroleukemic cells (MELC) exposed to tributyltin (TBT) is a function of both concentration (C) and duration of exposure (T). At or above a critical C x T product value (CPV) (e.g., 0.5-1.0 microM TBT, 6 hr), exposed MELC exhibit severe, irreversible toxicity: decreased membrane integrity (viability, measured by propidium iodide [PI] exclusion), grossly perturbed cell-cycle distributions, and fixation of the plasma membrane/cytoplasm complex. Below the CPV, exposed cells exhibit retention of carboxyfluorescein (CF) fluorescence (indicative of decreased plasma membrane permeability) and decreased cell proliferation, a result of retardation of progression into, through, and out of the S (DNA synthetic) phase of the cell cycle. However, following washout and recovery, mean CF fluorescence, cell proliferative capacity, and cell-cycle kinetics return to control levels. These results suggest that the toxic changes induced by TBT exposure may be reversible if exposure conditions do not exceed the CPV. To assess whether the CPV has been exceeded, a multiparameter flow cytometric analysis of membrane integrity and cell-cycle kinetics is useful.

Fixation of the plasma membrane/cytoplasm complex: A mechanism of toxic interaction of tributyltin with the cell

Flow cytometric and light/fluorescence microscopic analysis of murine erythroleukemic cells (MELC) and electron microscopic investigation of porcine microsomal membrane preparations suggest that tributyltin (TBT) toxicity is mediated through fixation processes (protein denaturation, crosslinking, and so on) within the plasma membrane/cytoplasm complex. This hypothesis was derived from the following observations:1.Exposure of the MELC to micromolar concentrations of TBT results in increased resistance to detergent-mediated cytolysis;2.Exposure of porcine renal microsomal membrane preparations to similar concentrations results in inhibition of vanadate-mediated crystallization of Na+,K+-ATPase, a process requiring protein mobility within the membrane;3.Flow cytometric and fluorescence microscopic analyses indicate that MELC exposed to submicromolar concentrations of TBT exhibit increased cellular carboxyfluorescein retention; and4.Nuclei prepared from TBT-treated cells by detergent-mediated cytolysis exhibit increased axial light loss, 90° light scatter, fluorescein isothiocyanate fluorescence, and the presence of adherent protein-aceous tags. The DNA distribution histogram of such nuclei also is perturbed.