Biomedical subjects

R M Zucker

Publications and source records attributed to R M Zucker.

At least 37 records · Page 2Linked 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↗

PubMed1994

Comparison of cellular and nuclear flow cytometric techniques for discriminating apoptotic subpopulations.

We compared cellular flow cytometric methods employing carboxyfluorescein (CF), Höechst 33342, and Höechst 33258 with a nuclear method in their ability to discriminate apoptotic subpopulations in rat thymocyte cultures exposed to dexamethasone or tributyltin. In the nuclear technique, apoptotic cells appeared as a single population containing reduced DNA content, while in the cellular techniques, apoptotic cells appeared as two or more subpopulations exhibiting increased fluorescence. Of these subpopulations, early apoptotic cells [which excluded propidium iodide (PI), indicating maintenance of membrane integrity] exhibited higher fluorescence but the same level of axial light loss (i.e., size/refractive index) as viable cells; late apoptotic and dead cells (which incorporated PI) exhibited decreased axial light loss. However, while the Höechst dyes allowed discrimination of late apoptotic from dead cells, CF did not. In comparing sensitivity to staining conditions, Höechst 33258 fluorescence was the most stable over time, Höechst 33342 the least, and CF fluorescence not only varied with time, but with tri-n-butyltin methoxide concentration. Comparison of single-parameter analyses revealed that axial light loss was sensitive only to late apoptotic changes; nuclear fluorescence was a better indicator of apoptotic subpopulations, but still underestimated the total percentage of affected cells, and Höechst 33342 distinguished early apoptotic cells as those with elevated fluorescence. Early apoptotic cells stained with Höechst 33258 also exhibited increased fluorescence, but could not be distinguished from late apoptotic and dead cells without a second parameter. These findings indicate that of the methods investigated, the method of choice for detecting apoptosis depends on the goal of analysis: Höechst 33258 was best for discriminating apoptotic subpopulations, and CF was best for assessing alterations of membrane fluidity. For single-parameter analyses, Höechst 33258 was best for determining the total percentage of affected cells, while Höechst 33342 could be used to determine the percentage in early apoptosis.

Animals↗

PubMed1994

Biologically based dose-response modeling in developmental toxicology: biochemical and cellular sequelae of 5-fluorouracil exposure in the developing rat.

Mechanistically based dose-response models for developmental toxicity require elucidation of critical biological events that intervene between maternal exposure and adverse developmental outcome. We have examined some of the major events in the rat embryo/fetus following a subcutaneous injection of 5-fluorouracil (5-FU; 0-40 mg/kg) to the dam on Day 14 of gestation. This treatment resulted in reduced fetal weight that was significant at doses of 20 mg/kg and higher, generalized reduced ossification at doses above 25 mg/kg, and wavy ribs at doses of 30 mg/kg and higher. Numerous malformations including cleft palate and hindlimb defects were substantially increased at doses of 35 and 40 mg/kg. 5-FU inhibits thymidylate synthetase (TS), resulting in inhibited growth of rapidly proliferating tissues. To identify early events in the pathogenesis of hindlimb defects, we examined the effects of 5-FU on TS activity, cell cycle, growth, and morphology in the developing hindlimb as a function of dose and time. The rate of decline of TS activity following 5-FU exposure was dose related, although maximal inhibition and recovery were similar at doses within (20 and 40 mg/kg) and below (10 mg/kg) the range of detectable developmental toxicity. Flow cytometric analysis of nuclei from embryonic hindlimbs revealed a transient increase in the percentage of cells in S phase and decrease in G0/G1 phase 8 hr after maternal injection of 20-40 mg 5-FU/kg, but not at lower doses. Reduction in growth and morphometric changes of hindlimbs were observed only after maternal exposure to 40 mg/kg. The tissue specificity of these effects was examined by comparing the hindlimb with other embryonic tissues. There was also a dose-related decline of TS activity in the embryonic liver. However, the pattern of recovery of TS activity and cell cycle alterations were different in the liver than in the hindlimb, probably reflecting the higher cell proliferative rate in the liver at this stage. We have derived a quantitative, empirical model for induction of hindlimb defects based on TS inhibition and subsequent cellular events following 5-FU exposure. The model predicted a dose response similar to that of the observed data although the predicted curve was shifted toward lower doses. These results suggest that while this model may not capture all of the critical events involved in the induction of hindlimb defects following maternal exposure to 5-FU, it does reflect a central mechanism of its developmental toxicity.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1994

Tributyltin and dexamethasone induce apoptosis in rat thymocytes by mutually antagonistic mechanisms.

The nuclei of apoptotic thymocytes can be identified by flow cytometry as a subpopulation exhibiting reduced DNA content. We observed that rat thymocyte cultures exposed to 1.0-2.5 microM tri-n-butyltin methoxide (TBT) exhibited a rapid time- and concentration-dependent induction of apoptosis, with > 85% of cells exhibiting reduced DNA content within 1 hr after exposure to 2.0-2.5 microM TBT. In contrast, exposure to 1.0 microM dexamethasone phosphate (DEX) resulted in a gradual time-dependent increase to approximately 45% induction of apoptosis by 6 hr versus approximately 15% spontaneous induction in controls. However, simultaneous exposure to TBT and DEX resulted in a decreased response: although TBT concentrations between 0.1 and 0.5 microM did not induce apoptosis, they reduced the ability of DEX to initiate apoptosis; while at TBT concentrations > or = 1.0 microM, simultaneous exposure to DEX substantially decreased the extent of TBT-induced apoptosis and cytotoxicity. Furthermore, while treatment with the protein synthesis inhibitor cycloheximide or the protein kinase C inhibitor H-7 completely blocked DEX-induced apoptosis, neither significantly reduced induction of apoptosis by TBT. Taken together, the toxicant-specific differences in the timing and extent of apoptotic induction and the dissimilar responses to CHX and H-7 suggest that TBT and DEX initiate endonuclease-mediated apoptotic cell death through different mechanisms. Moreover, the ability of each agent to retard the action of the other suggests that these mechanisms are directly or indirectly antagonistic.

Analysis of Variance↗

PubMed1993

Effects of 5-fluorouracil on embryonic rat palate in vitro: fusion in the absence of proliferation.

5-Fluorouracil (5-FU) inhibits the enzyme thymidylate synthetase (TS) which results in inhibition of DNA synthesis. 5-FU is teratogenic in many species, inducing cleft palate, limb, and tail defects. In the present study, gestation day (GD) 14 embryonic rat craniofacial explants were exposed to 5-FU in organ culture with increasing concentrations and durations of exposure. Palates exposed to 5-FU were morphologically abnormal and craniofacial shape, size, and palatal fusion pattern were affected with the severity of effects dependent on concentration and duration of exposure. Cleft palate was induced in vitro as opposing palates overlapped in a narrowed oral cavity. Palates exposed to higher levels of 5-FU were growth inhibited, but fused even though proliferation ceased and few cells were available to participate in elevation and fusion. This was demonstrated as a biphasic concentration-response profile for palatal fusion in which 0.05 to 0.15 micrograms 5-FU/ml produced decreasing rates of palatal fusion, while exposure to 0.15 to 3.0 micrograms/ml resulted in progressively increasing rates of fusion. The effects of 5-FU were detected biochemically as a reduction in TS activity which was concentration and time dependent during the first 12 hours, with a return to control levels by 24 hours. During the first day, 5-FU did not alter protein levels, but DNA levels significantly decreased at the high concentration, 2.0 micrograms/ml. After 5 days in culture, both DNA and protein decreased with increasing 5-FU concentration and duration of exposure. Also by the end of the culture period, 3H-TdR incorporation had decreased in a concentration dependent manner. It is concluded that progressive inhibition of proliferation and growth in organ culture results in two different morphological outcomes: cleft palate resulting from a narrowed oral cavity and increased incidence of anterior palatal fusion under conditions of strong growth reduction. This study demonstrates that elevation and fusion can occur in the absence of growth and proliferation. Based on these observations, severe inhibition of growth or proliferation would not necessarily be sufficient to induce cleft palate.

Animals↗

PubMed1993

Utility of the murine erythroleukemic cell (MELC) in assessing mechanisms of action of DNA-active developmental toxicants: application to 5-fluorouracil.

Murine erythroleukemic cells (MELC) exposed to 2'-deoxy-5-azacytidine (D-AZA) or to the active cyclophosphamide (CP) metabolites phosphoramide mustard (PAM) and 4-hydroxycyclophosphamide (OHCP) exhibit cell-cycle perturbations similar to those seen in limb bud nuclei of gestational day (GD) 10 CD-1 mouse embryos exposed in utero to D-AZA or CP, respectively. The similarities in response suggest MELC may be a useful model for determining mechanisms of action of DNA-active developmental toxicants. As such, we used the MELC model to investigate the mechanism of action of 5-fluorouracil (5-FU), an antimetabolite that induced in GD 14 rat fetuses an apparent S-phase accumulation in limb cells 8 hr after in utero exposure, but S-phase depletion in liver cells 24 hr postexposure. MELC timed-recovery and synchronization studies suggest that in proliferative tissues, 5-FU induces an early S-phase accumulation, followed by a synchronous, concentration-dependent delay in progression through the cell cycle. Consequently, it is the tissue-specific rate of delay, rather than different mechanisms of action, that results in apparent tissue-specific perturbations. Moreover, growth and cell-cycle data suggest that cells entering S phase (when TS activity is greatest) are the most sensitive to 5-FU toxicity. Assays of the TS activity of recovering MELC reveal that although the initial extent of TS inhibition does not appear to be concentration-dependent, the time to recovery is, suggesting that the rate of S-phase progression is closely associated with TS activity. Together, the induction of similar cell-cycle perturbations in embryonic/fetal tissues and MELC following exposure to CP (or CP metabolites), D-AZA, or 5-FU, as well as the adaptability of MELC to a variety of kinetic assays suggests that, for those developmental toxicants suspected of inducing cell-cycle perturbations in embryonic/fetal tissues, MELC may prove useful for elucidating mechanisms of action.

Animals↗

PubMed1993

Effects of developmental stage and tissue type on embryo/fetal DNA distributions and 5-fluorouracil-induced cell-cycle perturbations.

Cell-cycle analysis of nuclei obtained from the circulating erythroblasts (gestational day [GD] 11-16), livers (GD 14-19), and whole embryos (GD 10-13) or remaining (extrahepatic) tissues (GD 14-16) of rat embryos/fetuses revealed age- and tissue-dependent variations in the relative percentages of cells in the G0/G1, S, and G2/M phases of the cell cycle. With development, the rate of cell proliferation declined resulting in decreases in the relative percentage of S-phase cells and increases in the G0/G1 percentage, while the percentage of G2/M-phase cells remained relatively constant. Comparing tissue cell-cycle profiles during development, erythroblasts exhibited the most rapid age-dependent decline in S-phase percentage (from 75% at GD 11 to 8% by GD 14), embryos/extrahepatic tissues exhibited a more gradual reduction (from 55% at GD 10 to 14% by GD 15), while the hepatic isolates exhibited a relatively constant S-phase percentage of approximately 40% from GD 14 to GD 18 before decreasing to 23% at GD 19. These age-dependent variations suggest that cell-cycle distribution may be useful in staging embryogenesis and in detecting abnormal development. To determine how these developmental and organ-specific cell-cycle variations affect toxic response, we sampled GD 11-13 embryos 6 hr after maternal administration of a teratogenic dose of 5-fluorouracil (5-FU), a thymidylate synthetase inhibitor that induces S-phase accumulation. The results indicate that, on a relative basis, the amount of induced S-phase accumulation in erythroblasts and whole embryos 6 hr postdosing increased with development.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Utility of light scatter in the morphological analysis of sperm.

We were able to differentiate the morphologically diverse sperm nuclei of four animal species by using an Ortho flow cytometer to detect the forward light scatter from a red (helium-neon) laser. Cytograms depicting the axial light loss and forward red scatter signals revealed unique, but reproducible, sigmoid distributions that reflected not only interspecies differences in shape and size, but variations in particle refractive index and orientation within the flow cell at the time of analysis. Consequently, we were able to use regional gating of the light scatter cytogram to minimize the influence of orientation on the resolution of the fluorescence signal. We also observed that sperm enlarging as a result of chemically induced decondensation exhibit over time a biphasic shift (increase, then decrease) in light scatter at a species-dependent rate. These results suggest that, without any special adaptations to the flow cytometer, light-scatter parameters can be used to discriminate morphologically different sperm, to enhance the resolution of fluorescence measurements that may otherwise be confounded by variability in radial orientation, and to detect alterations in the rate of a biochemical/biophysical process such as decondensation.

Animals↗

PubMed1992

An efficient multiple-exposure analysis of the toxicity of crisnatol, a DNA intercalator in phase II clinical trials.

To investigate the toxicity and mechanism of action of crisnatol (CRS), a new DNA intercalator currently in phase II clinical trials, we analyzed cellular and nuclear flow cytometric (FCM) parameters of murine erythroleukemic cells (MELC) exposed to a range of CRS concentrations over three exposure conditions: short-term (4 h), long-term (24 h), and short-term with recovery (4 h+/19 h-). At 0.5-1.0 microM CRS, 4 h exposure results in a reversible G2-phase block, while 24 h exposure results in greater than G2 polyploidy. At 5-10 microM CRS concentrations, cells exhibit persistent retardation of S-phase progression or irreversible G2 and/or greater than G2 blocks, depending on duration of exposure. Cells terminally blocked in G2 exhibit increased nuclear/cellular volumes and increased nuclear fluorescein isothiocyanate (protein) staining, suggestive of unbalanced growth. At 25-50 microM CRS concentrations, MELC exhibit severe membrane perturbation (loss of viability) regardless of exposure. In contrast, following similar exposures to an inactive isomer of CRS, MELC exhibit minimal cell cycle effects, suggesting that cell cycle kinetics may be a useful criterion for assessing potential efficacy. Similar analyses with different classes of chemotherapeutic agents reveal that the range of induced cellular/nuclear perturbations varies with the class of compound used. Taken together, these results suggest that drug toxicity can vary with both concentration and duration of exposure and, as such, a selective multiple-exposure FCM analysis may better represent the spectrum of drug action for drug development and pharmacodynamic studies.

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↗

PubMed1991

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.

Amsacrine↗

PubMed1991

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.

Animals↗

PubMed1991

Syndactyly correction of the hand in Apert syndrome.

Surgical correction of syndactyly of the Apert hand should begin by 6 months and be completed by 3 years of age. As much surgery as possible is carried out at each sitting. Digit separation should be in order of functional importance. The first web space is deepened with a four-flap Z-plasty or a dorsal skin flap from the web and index finger. Syndactyly release using a dorsal flap and zig-zag technique is used to create the second and fourth web spaces. The complex long-ring syndactyly often requires a pedicled groin flap for reconstruction and preservation of growth potential. A five-digit hand can be achieved with adequate grasp and stable, sensate, well-aligned digits. These children can attain some degree of independent finger motion and aesthetically acceptable hands with this approach.

Acrocephalosyndactylia↗

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

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↗