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Biomedical subjects

E J Massaro

Publications and source records attributed to E J Massaro.

At least 19 recordsLinked to original sources
PubMed1992

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

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.

Animals↗
PubMed1990

Increasing sensitivity of the Ortho analytical cytofluorograph by modifying the fluid system.

By adding Teflon tubing to the effluent line of the Ortho 50H analytical cytofluorograph, we were able to increase the sensitivity of fluorescence and scatter detection threefold without compromising resolution. By increasing sheath backpressure (resistance), the additional tubing increases particle residence time within the detection area and thereby increases the total photon emission density per particle. In addition, the longer, wider tubing dampens the stream-destabilizing effects of minor obstructions in or movement of the effluent line. This increase in sensitivity and stability is desirable in applications requiring detection of particles exhibiting low-level fluorescence.

Flow Cytometry↗
PubMed1990

Cyclophosphamide teratogenesis: evidence for compensatory responses to induced cellular toxicity.

Cyclophosphamide (CP) administered ip to pregnant mice on day 10 of gestation (day of plug = day 0) is teratogenic (exencephaly, cleft palate, and limb malformations) at 20 mg/kg and embryolethal at higher doses. In the present study, CP was administered at 1, 5, 10, or 20 mg/kg on day 10 of gestation. Embryos were removed at 8 and 28 hr postdosing, and two embryos from each litter were immediately stained with Nile blue sulfate (NBS) to identify areas of cell death. The remaining embryos were frozen and forelimb buds subsequently removed for flow cytometric (FCM) analysis of the cellular DNA synthetic cycle. Additional litters were examined near term (day 17) for morphological abnormalities; these data were correlated with embryonic toxicity as detected by NBS staining and FCM analysis. Only the highest dose produced malformations. In marked contrast, a dose-related increase in the percentage of limb bud cells in the S (DNA synthetic) phase of the cell cycle was detectable at all doses. Inhibition of DNA synthesis was detected at all doses 8 hr post exposure and persisted through 28 hr for doses greater than or equal to 10 mg/kg. NBS staining indicated increased cell death in the alar plate of the neural tube 28 hr after exposure to 10 mg/kg CP and generally increased cell death in areas of rapid cell proliferation throughout the embryo at 20 mg/kg. The absence of an overt teratogenic response at dose levels that produced significant perturbation of the cell cycle indicates that a measure of embryonic damage can be compensated for or repaired. The implications of these findings for the existence of thresholds in developmental toxicity are discussed.

Animals↗
PubMed1990

Interactions between lead and essential elements: a review.

Young children are especially susceptible to the toxic effects of lead. Although the effects of overt lead intoxication have been well studied, critical information on the long-term effects of low level lead exposure is lacking. The results of recent clinical studies indicate that "chronic" low level lead exposure during development can result in behavioral alterations in the absence of overt neurotoxicity. The perturbation of central nervous system functioning may be the result of essential mineral deficiencies resulting from lead-induced impairment of mineral availability and/or an increased sensitivity to lead in the absence of adequate levels of essential minerals. This article reviews research undertaken to examine the interactions between calcium, iron, zinc, copper and lead with reference to how this may impact central nervous system functioning.

Animals↗
PubMed1990

Flow cytometric analysis of the mechanism of methylmercury cytotoxicity.

Flow cytometric analysis of murine erythroleukemic cells (MELC) exposed in vitro to 2.5 to 7.5 mumol/l (micromolar) methylmercury (MeHg) reveals a dose-dependent decrease in the rate of DNA synthesis (rate of passage through the S phase of the cell cycle), manifested as the accumulation of most of the cells in the S phase, and a modest accumulation of cells in the G2/M phase of the cycle. Light microscopy reveals a progressive increase in chromosomal damage (condensation, pulverization). At or above 10 mumol/l MeHg, progression through all the phases of the cell cycle is blocked and mitotic cells are arrested irreversibly in anaphase, with most exhibiting arrangement of chromosomes in a wreathlike ring formation. Also the cells exhibit both nuclear propidium iodide (PI) fluorescence (indicative of loss of viability) and concurrent cytoplasmic carboxyfluorescein (CF) fluorescence (viable cells exhibit CF fluorescence and exclude PI). In addition, there is a dose-dependent increase in cellular refractive index (90 degrees light scatter), an apparent decrease in cell volume (axial light loss), and progressive resistance to detergent (NP-40)-mediated cytolysis. Resistance to detergent-mediated cytolysis is indicative of fixation (protein denaturation, cross-linking, and so on) of the plasma membrane/cytoplasm complex. Our findings indicate that DNA synthesis is the primary target of MeHg cytotoxicity and that apparent targets and degree of cytotoxicity are a complex function of dose.

Animals↗
PubMed1989

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.

Animals↗
PubMed1989

Cell cycle alterations and cell death in cyclophosphamide teratogenesis.

Litters of pregnant mice treated with cyclophosphamide (CP) exhibit malformations of the limbs ranging from oligodactyly to amelia. Previous studies have indicated that cell death occurs in limb buds shortly after maternal exposure. We have investigated the relationship of cell death, cell cycle perturbation, and embryo/fetal toxicity in the mouse using vital staining and flow cytometry (FCM). CP (20, 30, and 40 mg/kg) was investigated via intraperitoneal administration to Swiss-Webster mice on day 10 of gestation. At 4, 8, or 28 hours later, embryos were removed. Cell death was identified with Nile blue sulphate (NBS). Two embryos per litter were stained with NBS, and the remaining embryos were frozen at -70 degrees C prior to FCM analysis. After thawing, the forelimb buds were removed for the isolation of nuclei. Tissues were dissociated through a wire mesh followed by cytolysis with 0.1% nonidet P-40 in PBS with 0.5 mg/ml RNase. Nuclei were stained with the fluorescent nucleic acid probe propidium iodide and analyzed (10,000 nuclei per sample) for propidium iodide fluorescence by FCM. NBS revealed a dose-related increase in cell death by 8 hours after dosing. CP-induced cell death was greatest in areas of rapid cell proliferation (DNA synthesis). FCM analysis revealed retardation of progression through the S-phase of the cell cycle by 4 hours post-exposure at all doses. This retardation occurred earlier in S-phase with increasing dose and persisted through 8 hours. At 28 hours, cell cycle histograms were normal in the low-dose embryos, but remained perturbed in the intermediate- and high-dose embryos. On day 17 of gestation, the last group of dams was killed. A high incidence of fetal malformations, including limb defects, occurred at the 20 mg/kg dose, and fetal mortality was observed at 30 and 40 mg/kg. The pattern and magnitude of cell death correlated with cell cycle perturbation and fetal toxicity at term, suggesting a relationship between cell cycle perturbation, cell death, and malformations produced by CP.

Animals↗
PubMed1988

Effects of tributyltin on biomembranes: alteration of flow cytometric parameters and inhibition of Na+, K+-ATPase two-dimensional crystallization.

Carboxyfluorescein diacetate (CFDA) is a lipophilic nonfluorescent molecule that readily crosses the cell membrane. In the cytoplasm, it is hydrolyzed by nonspecific esterases to carboxyfluorescein (CF), a negatively charged fluorescent molecule, which is retained incompletely by cells with an intact plasma membrane. Exposure (4 hr) of the murine erythroleukemic cell (MELC) to micromolar quantities (0.1 to 5.0 microM) of tributyltin (TBT) results in increased cellular CF fluorescence. The increase occurs within a range below a critical value of the product (CPV) of the concentration (C) of TBT X duration (T) of exposure to TBT. Fluorescence increase is a sensitive indicator of the interaction of TBT with the cell: it is observed following exposure to 0.1 microM TBT for 4 hr at 37 degrees C. In the range above the CPV, cellular CF fluorescence is reduced apparently resulting from perturbation of membrane structure. For example, exposure of MELC to 2.5 microM TBT for 4 hr at 37 degrees C produces resistance to detergent-mediated cytolysis and inhibition of vanadate-mediated two-dimensional crystallization of Na+, K+-ATPase molecules in porcine renal microsomal membrane preparations, a process requiring molecular mobility within the membrane. Taken together, the increased cellular CF fluorescence and resistance of the MELC to cytolysis along with the inhibition of Na+, K+-ATPase crystallization in the microsomal membrane preparations suggest fixation (protein denaturation, cross-linking, etc.) at the level of the plasma membrane as a mode of toxic action of TBT.

Animals↗
PubMed1978

An electrophoretic analysis of selected enzymes of the coconut crab, Birgus latro.

1. Employing starch gel electrophoresis at pH 8.6, a single anodal band of L(+)-lactate dehydrogenase activity was found in skeletal muscle of Birgus latro. 2. Two anodal malate dehydrogenase isozymes were observed in heart, skeletal muscle and pericardial gland tissue. Lung and gill exhibited the faster moving band only. 3. Multiple bands of esterase activity were detected in all tissues examined employing alpha-naphthylacetate or alpha-butyrate as substrate. 4. Multiple molecular forms of superoxide dismutase were observed in all tissues examined. 5. Lung exhibited a single cathodal band of carbonic anhydrase activity.

Animals↗
PubMed1977

Alterations in lipid composition of liver cell plasma membranes during development.

Liver plasma membranes of Swiss-Webster CFW mice ranging in age from 5 to 165 days were isolated employing a two-phase dextran/polyethylene glycol polymer system. Electron microscopy revealed low mitochondrial and microsomal contamination. Lipids were quantitatively extracted and subjected to two dimensional thin layer chromatography. Individual lipids were eluted and phosphorus was measured by the Malachite green method. Cholesterol was quantitated by the micro Liberman-Burchard method and sphingolipids were quantitated fluorometrically with fluorescamine. During development, phosphatidylethanolamine levels decreased from 43.2% to 19.5% whereas phosphatidylcholine levels rose from 26% to 45.2%. The ration of phospholipid/cholesterol remained essentially constant during development; but the sphingolipid/cholesterol and sphingolipid/phospholipid ratios increased from 0.75 to 1.79 and 0.11 to 0.34, respectively.

Age Factors↗
PubMed1977

Effects of methyl mercury on murine fetal amino acid uptake, protein synthesis and palate closure.

Methylmercury (MeHg: 5 mg Hg/kg maternal body weight) in 0.13 M NaCl, 0.01 M NaH2PO4-Na2HPO4, pH 7.4 (PBS) administered to gravid CFW mice on day 12, hour 6 (12(6)) of gestation induced a high incidence of cleft palate in fetuses examined on days 15(6) (72%), 16(6) (62%) and 17(6) (40%). Palate closure (100%) in PBS control animals occurred by 14(10). One day post MeHg administration, total fetal protein was decreased 22% while DNA content was unaltered. Protein was maximally decreased (28%) on 14(6) and, thereafter, returned toward control levels. Alterations in DNA content followed a similar pattern; but the maximal decrease (32%) occurred on 15(6). The rate of fetal protein synthesis was depressed 5% at 12(9) and between 20% to 26% from this time to 13(6) (end of observation). The agreement between the calculated decrease in protein synthesis (19%) and the measured decrease in protein content (22%) suggests that a reduction in protein synthesis is responsible for the decreased fetal protein content. Placental blood flow and fetal water space, measured with 3H--H2O at 12(18), were not affected by MeHg treatment. However, fetal free amino acid concentrations at 12(18) were generally decreased (alanine, 23.0%; valine, 9.7%; methionine, 22.6%; isoleucine, 12.0%; leucine, 18.2%) while uptake of the non-metabolizable amino acid, 14C-cycloleucine, was decreased 23%. From this, it is concluded that the growth inhibitory effects of MeHg are related, at least in part, to impaired placental/fetal transfer of amino acids.

Abnormalities, Drug-Induced↗