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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
PubMed1994

Cell death and cell cycle perturbation in the developmental toxicity of the demethylating agent, 5-aza-2'-deoxycytidine.

DNA methylation is a probable mechanism for regulating gene expression, and alterations in methylation may significantly affect embryonic development. We administered the cytidine analogue 5-aza-2'-deoxycytidine (dAZA), a specific and potent demethylator of DNA, to pregnant mice to determine its teratogenicity and effects on embryonic cell death and cell cycle. Groups of females were dosed intraperitoneally on gestation day 10 with doses of 0.05-3 mg/kg dAZA and killed at 4, 8, or 28 hr later. Two embryos per litter were immediately stained with Nile blue sulfate (NBS) to identify areas of cell death; the remaining embryos were frozen and stored for subsequent flow cytometric (FCM) analysis of the cellular DNA synthetic cycle in limb buds. A dose-related accumulation of cells in the S and G2/M phases was observed at 4 and 8 hr after maternal dosing. S-phase accumulation was the most sensitive indicator of effect; a dose-related increase in the percentage of hindlimb bud cells in S-phase was evident at all dosages 4 hr after maternal dosing. By 28 hr postdosing, a normal cell cycle phase distribution was observed at doses of < 0.3 mg/kg. However, cell cycle perturbations persisted at higher dosages. NBS staining demonstrated increased cell death in areas of rapid cell division, indicative of replication-associated cytotoxicity, at doses of > or = 0.1 mg/kg. Observation of litters from additional dams killed at term revealed that at dosages of > or = 0.3 mg/kg, cleft palate and hindlimb defects were significantly elevated. In addition, above 0.3 mg/kg, fetal weight was significantly decreased.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
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

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.

Acetates↗
PubMed1988

Flow cytometric discrimination of mitotic nuclei by right-angle light scatter.

Flow cytometry has been used to demonstrate alterations in protein, RNA, and DNA content of cells as they traverse the cell cycle. Employing fluorescein isothiocyanate (FITC) to stain protein and propidium iodide (PI) to stain nucleic acids, multiple regions within the G1 and G2 phases of the cell cycle, in addition to the M phase, can be distinguished. In this study, cytograms of the 90 degree light scatter signal vs. PI fluorescence were remarkably similar to those of FITC fluorescence vs. PI fluorescence, suggesting a relationship between 90 degree light scatter and protein content. M-phase nuclei can be distinguished from G2-phase nuclei on cytograms of 90 degree light scatter vs. PI fluorescence. However, the percentage of mitotic nuclei obtained by this technique is less than that found by light microscopic analysis. Flow cytometric parameters of nuclei prepared by nonionic detergent (NP40) lysis in Dulbecco's PBS, Vindelov's buffer, or Pollack's hypotonic EDTA/Tris buffer were compared. The best resolution of mitotic nuclei was obtained in Pollack's buffer. However, the stainability of the M-phase nuclei is reduced, and the nuclei are located in the late S/G2 region of the single-parameter histogram.

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↗
PubMed1988

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.

Animals↗
PubMed1985

Effects of diethylhydroxylamine on hepatic microsomal lipid peroxidation and glutathione S-transferases.

The effects of diethylhydroxylamine (DEHA), a potent free-radical scavenger, on lipid peroxidation of rat liver microsomes were investigated in vitro. DEHA strongly inhibited ascorbate-dependent nonenzymatic microsomal lipid peroxidation. DEHA also completely inhibited nonenzymatic lipid peroxidation of heat-denatured microsomes, indicating that inhibition is protein-independent. DEHA only moderately inhibited NADPH-dependent enzymatic microsomal lipid peroxidation. DEHA has been shown to exhibit antitumorogenic properties. However, it had no significant effect on hepatic glutathione S-transferase, selenium-independent glutathione peroxidase, or selenium-dependent glutathione peroxidase activity in the DEHA-treated CD-1 (lCR) Br male mouse. This suggests that the mode of action of DEHA as an antitumorogenic agent may be different from that of butylated hydroxyanisole, whose antitumor function is attributed to induction of glutathione S-transferase activity.

Animals↗
PubMed1985

Uptake, distribution and behavioral effects of inhalation exposure to manganese (MnO2) in the adult mouse.

Adult male mice were exposed either to sublethal levels of MnO2 dust or filtered air (control group) 7 hours/day, 5 days/week for 16 to 32 weeks. Following a 16 week initial exposure period, randomly selected samples (8 animals) from both the control and Mn-exposed groups were observed for behavioral performance (ambulations and rearings in the open-field, "hole-in-board" explorations, rotarod) and learning (passive avoidance) and tissue Mn levels were determined via atomic absorption spectrometry. Exposure continued for the remaining animals and the sampling procedure was repeated biweekly for an additional 8 time points. At week 32, Mn exposure was terminated. However, biweekly testing of the remaining animals continued for an additional 3 time points. Mn-exposed animals had significantly higher blood, liver, kidney, lung, cerebrum, cerebellum plus brainstem, and testis Mn levels than control animals. With the exception of the liver, these levels declined with increasing exposure time. No histopathologic effects attributable to Mn-exposure were observed. However, significant overall effects on growth and behavior were obtained. Specifically, Mn-exposed subjects weighed more, executed more rearings in the open-field, and tended to exhibit longer latencies to enter the open-field. When the post-exposure data were analyzed separately, no significant effects were obtained. While no general relationship was obtained between tissue Mn levels and behavior, selected behavioral measures did correlate with tissue Mn levels. Animals exposed via feeding to comparable Mn levels across the same length of exposure employed in the inhalation study did not demonstrate any significant behavioral alterations.

Air Pollutants↗
PubMed1984

Effects on the development of offspring of female mice exposed to platinum sulfate or sodium hexachloroplatinate during pregnancy or lactation.

On d 7 or 12 of gestation or on d 2 postpartum, Swiss ICR dams were administered either (1) a single intragastric dose of Pt(SO4) at the LD1 level or dilute H2SO4 at an equivalent volume, pH, and sulfate content, or (2) a single subcutaneous dose of Na2PtCl6 or phosphate-buffered saline at an equivalent volume and pH. To differentiate prenatal from postnatal effects of the compounds on the offspring, a full cross-fostering design was employed. Rate of growth (as a function of weight gain) and gross activity of the neonates were assessed on d 8 or 13 postpartum. On d 60-65 postpartum, open-field behavior (ambulations and rearings), rotarod performance, and passive avoidance learning of the adult offspring were investigated. Exposure to Pt(SO4)2 resulted in reduced offspring weight from d 8 to 45 postpartum, whereas the major effect of Na2PtCl6 was a reduction in activity level of the offspring of mothers exposed on d 12 of gestation.

Analysis of Variance↗
PubMed1984

The effect of diethylhydroxylamine on the incidence of tumors induced by benzo(a)pyrene in the mouse.

Groups of 20 male and 20 female CD-1(ICR)Br mice were treated either with 0, 3, 30, or 300 mg diethylhydroxylamine (DEHA)/kg-day (administered via the drinking water) from 57-112 days of age. For the four week period between 70 to 98 days of age, all animals received (via gavage) eight 1 mg doses of benzo(a)pyrene at 4 day intervals. One hundred thirty-seven animals survived the dosing period. Of these, 132 survived to 211 days of age at which time they were euthanized and necropsied. Gross lesions were observed only in the lungs and squamous portion of the stomach. Treatment with DEHA produced no significant effect on the lung tumor incidence of either sex. However, a significant increase in stomach tumors was observed in the females.

Aging↗
PubMed1984

Effects on the postnatal development of the mouse of preconception, postconception and/or suckling exposure to manganese via maternal inhalation exposure to MnO2 dust.

Female mice were exposed either to MnO2 dust (7 hours/day, 5 days/week) or filtered air (control group) for 16 weeks prior to conception. On day 1 of gestation, half of each of these groups was assigned randomly either to MnO2 dust or filtered air exposure until day 17 of gestation. To separate effects of prenatal maternal exposure to MnO2 from postnatal exposure of offspring to Mn via suckling, a complete fostering/-cross-fostering design was employed. Compared to control mothers, mothers exposed to MnO2 prior to conception produced significantly larger litters. Prenatal exposure resulted in reduced neonatal activity scores and retarded offspring growth that persisted into adulthood. Offspring reared by mothers exposed to MnO2 prior to conception and filtered air postconception had significantly lower day 7 postpartum weights compared to offspring reared by mothers exposed to filtered air both prior to and postconception. Also, offspring reared by mothers exposed to MnO2 prior to conception and filtered air postconception had higher day 12 activity scores compared to offspring reared by mothers exposed to filtered air prior to conception and MnO2 postconception. Prenatal exposure to MnO2 depressed neonate activity and continued exposure, via suckling, intensified this depression. Offspring exposed prenatally to MnO2 were retrieved faster than control offspring. Rearing frequency, exploratory behavior, and scores in tests having an activity component were depressed for sexually mature offspring who had been exposed to MnO2 both in utero and via suckling. Independent of in utero exposure history, sexually mature offspring reared by mothers exposed to Mn had significantly reduced cerebellum + brain stem mitochondrial Mn levels. Also, sexually mature offspring of mothers exposed to filtered air that were reared by MnO2 -exposed mothers had lower cerebral mitochondrial Mn levels than offspring of control mothers reared by control foster mothers.

Animals↗