Detection of chromosome aberrations by FISH as a function of cell division cycle (harlequin-FISH)

Chromosome aberrations are a sensitive indicator of genetic change, and the measurement of chromosome aberration frequency in peripheral blood lymphocytes is often used as a biological dosimeter of exposure (1,4). The length of time that cells are maintained in culture before cytogenetic analysis is probably the most important in vitro factor that influences both the frequency and types of aberrations that are seen following exposure to mutagens. Therefore, for accurate cytogenetic measurements of genetic damage, cells must be analyzed in their first mitosis following exposure. As cells progress through subsequent mitotic division cycles, cells with unstable types of aberrations, e.g., dicentrics and acentric fragments, are eliminated (1,3,4). Even the use of synchronized populations of cells does not guarantee that all cells analyzed will be in their first division following treatment. Small variations in growth rate after irradiation can lead to large variations in the proportion of cells that are in their first vs. a subsequent mitosis. For example, 48 h after G0 lymphocytes are stimulated to enter the cell cycle (the standard sampling time for cytogenetic analysis), up to 50% of the cells in mitosis can be in their second division cycle (10). While there are methods available to distinguish cells in different division cycles (see Introduction), they are not easily adapted for use with standard fluorescence in situ hybridization (FISH) procedures. The goal of this study was to develop a simple approach to detect aberrations by FISH whereby cells in different division cycles could be distinguished.     

Chromosome aberrations in human lymphocytes at a various duration of cultivation after irradiation

Human peripheral blood lymphocytes were exposed to 60Co gamma-rays (a dose of 3 Gy) and cultivated during seven days in the presence of PHA and BrdU. It was shown that the metaphases of the first and second mitosises occurred during cultivation of the irradiated and unirradiated lymphocytes, being evidence about of irregularity of the coming into division of various fractions of lymphocytes. The time of cultivation did not influence a rate of aberrations in metaphases of the first and second mitosises of the irradiated lymphocytes. During the first and the subsequent mitosises the number of exchange chromosome aberrations decreased and reached a control level in metaphases of the fourth and fifth mitosises. The number of paired fragments at second and third mitosises increased a little and started to decrease only in metaphases of the fourth and fifth mitosises. The decrease in chromosome aberrations with prolongation of the cultivation of lymphocytes after irradiating is a consequence of elimination of cells with chromosome damages during sequential mitotic divisions.     

Chromosome damage in rat pulmonary alveolar macrophages following ozone inhalation

To determine whether ozone is clastogenic at environmentally relevant exposure levels, rats were exposed for 6 h to 0.0, 0.12, 0.27, or 0.80 ppm ozone. The alveolar macrophages were isolated from animals sacrificed 28 h after the end of the exposure. The mitotic index and frequency of chromosome aberrations were determined. No change in the mitotic index was detected following 0.12 ppm ozone exposure. A significant decrease in mitotic index was observed after exposure to 0.27 ppm ozone; a significant (4-fold) increase in the frequency of dividing macrophages was detected following exposure to 0.8 ppm ozone. Only chromatid-type aberrations were observed. There was a significant increase in the frequency of cells with chromatid gaps and in the frequency of cells with chromatid deletions. Animals exposed to 0.27 ppm ozone had the highest proportion of cells with chromatid deletions (0.172) relative to background level (0.028). No exchanges or chromosome-type aberrations were detected in any of the animals. These data suggest that ozone, at relatively low levels, is clastogenic in macrophages from exposed rats.     

GROWTH AND MITOTIC ACTIVITY OF LUNG COLONIES DERIVED FROM MOUSE TERATOCARCINOMAS

This study dealt with the mitotic activity of lung colonies in the process of differentiation of mouse teratocarcinoma cells injected in vivo . The results showed that three stages were identifiable in respect to the mitotic activity: 1) a lag stage for a few days after injection, 2) a stage during which maximum mitotic activity is observed for several days following the first stage and 3) a third stage displaying a decrease in mitotic activity. There is a correspondence between these three steps in mitotic activity and differentiation in lung colonies previously identified by histological observations.     

Salinity stress results in rapid cell cycle changes of tilapia (Oreochromis mossambicus) gill epithelial cells

We have developed a technique for immunocytochemistry of fish gill cells that we used to quantify tilapia (Oreochromis mossambicus) mitochondria-rich cells (MRC) and other gill cells (non-MRC) within different cell cycle phases by laser scanning cytometry. Gill cells fixed on coverslips were triple stained with propidium iodide to distinguish G1 vs. G2 phases, Ser10-phosphorylated histone H3 antibody to label mitotic cells, and Na(+)/K(+) ATPase antibody to label MRC. These parameters were measured at 0 (control), 4, 8, 16, 24, 48, 72, and 168 hr (1 week) following exposure of freshwater (FW) acclimated fish to 2/3 seawater (SW). MRC increased mitotic activity very rapidly peaking at 8 hr following SW exposure. This change in mitotic MRC is indicative of epithelial reorganization during SW acclimation. In contrast to MRC, the proportion of non-MRC (likely pavement cells (PVC)) in mitosis did not change significantly in response to SW exposure. Moreover, twice as many MRC were in mitosis compared with non-MRC, suggesting that MRC turn over faster than other cell types during SW acclimation. Following the mitosis peak, MRC accumulated in G2 phase over a period of 16-72 hr post-SW exposure. We also observed G2 arrest with similar kinetics following SW exposure in tilapia non-MRC (likely PVC). We interpret the G2 arrest that occurs after an initial wave of transient increase in MRC mitosis as a means for conserving energy for dealing with the osmotic stress imposed during the exposure of FW fish to SW.     

Cyst geometry in the egg chambers of Calliphora erythrocephala Mg. (Diptera: Calliphoridae) ovaries

In the germarium of polytrophic ovarioles of Calliphora erythrocephala (Mg.) fly, four mitotic divisions of cystoblasts give rise to 16-cell germ-line cysts. One cell differentiates into an oocyte, while the remaining 15 cells become nurse cells. Concomitantly actin-rich ring canals are formed at the intercellular junctions. The present study considers a mutual arrangement of the ring canals formed after the second to fourth mitoses relative to the ring canal formed after the first mitotic division in different regions of the germarium and egg chambers. During the cyst formation and its movement to the posterior end of the germarium, the ring canals are displaced relative to one another, thereby giving different branching variants of the cyst. The pattern of cell interconnections becomes stable in germarium region 2b and does not change during the cyst movement along the ovariole despite the cyst polarizes and increases in size.     

The effect of mitotic inhibitors on DNA strand size and radiation-associated break repair in Down syndrome fibroblasts

The effect of mitotic inhibitors on formation and repair of DNA breaks was studied in cultured fibroblasts from patients with Down syndrome in order to investigate the hypothesis that the karyotyping procedure itself may play a role in the increased chromosome breakage seen in these cells after gamma radiation exposure. Using the nondenaturing elution and alkaline elution techniques to examine fibroblasts from Down syndrome patients and from controls, no specific abnormalities in Down syndrome cells could be detected after exposure to mitotic inhibitors, including rate and extent of elution of DNA from filters as well as repair of radiation-induced DNA breaks. In both normal and Down syndrome cell strains, however, exposure to mitotic inhibitors was associated with a decrease in cellular DNA strand size, suggesting the presence of drug-induced DNA strand breaks. The mechanism of increased chromosome sensitivity of Down syndrome cells to gamma radiation remains unknown.     

Effects of methylmercury on the morphogenesis of the rat cerebellum

Developing rat cerebellums were examined following continuous methylmercury exposure via maternal drinking water at 12.5 ppm during gestation and the suckling period. The continuous exposure resulted in reductions of the total cerebellar cell population and higher mercury tissue burdens than previous acute-dose studies. Cell necrosis was not evident, but rather alterations in the pattern of mitotic figures were observed. A decreased number of cells in the methylmercury exposed cerebellums was associated with an increased number of mitotic figures in the early stages of mitosis and a decrease in the number in the middle and late stages. These in vivo exposure observations are consistent with in vitro cell cycle studies in which the cells were found to have accumulated in G2 and early M phases. Impaired cell proliferation is suggested to be a major mechanism of developmental neurotoxicity following continuous low-dose exposure to methylmercury.     

Mitotic Rate of Rat Thyroid Follicular Cells In Vivo In Response to A Single Injection of Thyrotropin (Tsh)

The mitotic rate of thyroid follicular cells was assessed by a stathmokinetic method at intervals from 15 min to 24 hr after a single injection of 1 iu/kg of thyrotropin (TSH). the mitotic rate was increased 15 min after TSH and remained elevated for 3 hr. Two further peaks of mitotic activity were present at 9 hr and 24 hr after TSH. Serum TSH concentrations were increased from 5 min to 3 hr with a maximum at 1 hr.     

Deuterium oxide (heavy water) arrests the cell cycle of PtK2 cells during interphase.

Deuterium oxide (D2O, heavy water) exerts an antiproliferative effect on a variety of cells in vitro and on some organisms. This effect is mainly ascribed to a tubulin-mediated antimitotic action. We evaluated the morphology, the mitotic activity, and the dynamics of the cell cycle of PtK2 cells grown in vitro in the presence of 75% D2O for up to eight weeks by microspectrophotometric DNA measurements as well as flow cytometric analysis and a determination of mitotic indices. Substitution of heavy water for water in the culture medium initially increased the mitotic index by a (pro-) metaphase block but after 2 to 3 days of incubation no mitotic figures were seen. Analysis of cells grown for 6 days in medium containing 75% D2O revealed accumulation of cells in S/G2-phase. Extended treatment stabilized the high level of cells in this specific phase, when compared to normal growing cells. Cells grown for 1 to 6 weeks in the presence of D2O remained non-proliferating, nevertheless, they were able to divide again after recovery in non-deuterated medium. The time needed for resumption of the mitotic activity was proportional to the duration of deuterium oxide exposure. Cells incubated for 8 weeks in 75% D2O did not recommence mitotic activity. Light and electron microscopic examination revealed characteristic morphological changes of size and ciliation in PtK2 cells subjected to prolonged deuteration.(ABSTRACT TRUNCATED AT 250 WORDS)     

The karyological study of broad bean root tips treated with 9- (RS)- (2,3- dihydroxypropyl) adenine (DHPA)

The time course and concentration dependence of mitotic index decrease was studied in squashes of root tips ofVieia faba L. treated with 9-(RS)-(2,3-dihydroxypropyl) adonine. The appearance of lobate nuclei and binucleate cells was confirmed, using the mentioned procedure. It was proved that the substance under study is not a clastogen. In electronmicrographs no disintegration of cell structure was seen even after long exposure to the drug, but particular organelles were altered. The results are discussed from the viewpoints of cell pharmacology and cell pathology.     

Understanding the mechanism(s) of mosaic trisomy 21 by using DNA polymorphism analysis.

In order to investigate the mechanism(s) underlying mosaicism for trisomy 21, we genotyped 17 families with mosaic trisomy 21 probands, using 28 PCR-detectable DNA polymorphic markers that map in the pericentromeric region and long arm of chromosome 21. The percentage of cells with trisomy 21 in the probands'' blood lymphocytes was 6%-94%. There were two classes of autoradiographic results: In class I, a "third allele" of lower intensity was detected in the proband''s DNA for at least two chromosome 21 markers. The interpretation of this result was that the proband had inherited three chromosomes 21 after meiotic nondisjunction (NDJ) (trisomy 21 zygote) and subsequently lost one because of mitotic (somatic) error, the lost chromosome 21 being that with the lowest-intensity polymorphic allele. The parental origin and the meiotic stage of NDJ could also be determined. In class II, a "third allele" was never detected. In these cases, the mosaicism probably occurred either by a postzygotic, mitotic error in a normal zygote that followed a normal meiosis (class IIA mechanism); by premeiotic, mitotic NDJ yielding an aneusomic zygote after meiosis, and subsequent mitotic loss (class IIB mechanism); or by a meiosis II error with lack of crossover in the preceding meiosis I, followed by mitotic loss after fertilization (class IIC mechanism). Among class II mechanisms, the most likely is mechanism IIA, while IIC is the least likely. There were 10 cases of class I and 7 cases of class II results.(ABSTRACT TRUNCATED AT 250 WORDS)     

Derepression of hepatocyte proliferation with changes in the functional state of the reticuloendothelial system

Experiments on rats and mice were performed to study the effects of different substances modifying RES functions on hepatocyte proliferation. It was shown that as early as 24 hours after Kupffer cell (KC) overloading with colloidal iron particles the number of hepatocytes in mitosis increased. The mitotic rate increased by 32 h and decreased between 48 and 72 h following iron injection. Forty-eight h after injection of latex particles the hepatocyte mitotic peak could be identified. Twenty-four and 48 h after zymozan injection DNA synthesis in sinusoidal liver cells correspondingly increased. Hepatocytes in mitosis appeared 5 days later, reaching the peak value after 9 days followed by a decrease 12 days after zymozan injection. The depression of the hepatocyte mitotic rate was also observed 9 days after BCG and 15 days after prodigiozan injection. The data are suggestive of the importance of KC as potential inducers of hepatocyte proliferation.     

Relation between cell cycle and yield of aberrations observed in irradiated human lymphocytes

The bromodeoxyuridine-Giemsa technique has been used to study systematically the incidence of cells in first or subsequent mitoses at different fixation times of human lymphocyte control cultures as well as the influence of ionizing radiations on cell kinetics. Second divisions appear (3%) in cultures harvested 48 h after initiation. In 72 h cultures 40% of the dividing cells are in second and 33% in third division. Administration of 200 rads of X-rays before PHA stimulation results in a mitotic delay but does not increase the incidence of SCE. The yield of dicentrics after an exposure to 200 rads was the same for all cells in first mitosis regardless of fixation time. These results demonstrate that there is no evidence for the existence of sensitive subpopulations that could be distinguished by the time of the first mitotic division following stimulation.     

The cell cycle,cell death,and cell morphology during retinoic acid-induced differentiation of embryonal carcinoma cells

Time-lapse films were made of PC13 embryonal carcinoma cells, synchronized by mitotic shake off, in the absence and presence of retinoic acid. Using a method based on the transition probability model, cell cycle parameters were determined during the first five generations following synchronization. In undifferentiated cells, cell cycle parameters remained identical for the first four generations, the generation time being 11–12 hr. In differentiating cells, with retinoic acid added at the beginning of the first cycle, the first two generations were the same as controls. The duration of the third generation, however, was increased to 15.7 hr while the fourth and fifth generation were approximately 20 hr, the same as in exponentially growing, fully differentiated cells. The increase in generation time of dividing cells was principally due to an increase in the length of S phase. Cell death induced by retinoic acid also occurred principally in the third and subsequent generations. Cell population growth was then significantly less than that expected from the generation time derived from cycle analysis of dividing cells. Cells lysed frequently as sister pairs suggesting susceptibility to retinoic acid toxicity determined in a generation prior to death. Morphological differentiation, as estimated by the area of substrate occupied by cells, was shown to begin in the second cell cycle after retinoic acid addition. These results demonstrate that as in the early mammalian embryo, differentiation of embryonal carcinoma cells to an endoderm-like cell is also accompanied by a decrease in growth rate but that this is preceded by acquisition of the morphology characteristic of the differentiated progeny.     

Apoptotic cell death and cell proliferative activity in the rat fetal central nervous system from dams administered with ethylnitrosourea (ENU)

Ethylnitrosourea (ENU), a well known DNA alkylating agent, induces anomalies in the central nervous system (CNS), craniofacial tissues and male reproductive organs, and the enhancement of apoptosis is found in these tissues immediately after the administration of ENU (Katayama et al., 2000a). In this study, pregnant rats were treated with 60mg/kg of ENU at day 13 of gestation, and kinetics of apoptotic cells, mitotic cells and bromodeoxyuridine (BrdU)-positive cells in the fetal CNS were examined from 3 to 48 hours after the treatment (HAT). From 3 HAT, a significant increase in the number of apoptotic cells and a significant decrease in the number of mitotic cells were detected in the fetal CNS, and BrdU-positive cells significantly decreased in accordance with the increase in the number of apoptotic cells. The present results strongly suggest that both excess cell death by apoptosis and cell growth arrest indicated by decreased number of mitotic cells and BrdU-positive cells may have a close relation to the later occurrence of microencephaly following ENU-administration, and that ENU affects mainly S-phase cells and causes apoptosis.     

环磷酰胺诱发蚕豆体细胞遗传损伤的研究

利用蚕豆根尖研究环磷酰胺的遗传毒性效应, 结果表明:环磷酰胺(0.1~5.0 mg/mL)能够降低蚕豆根尖细胞有丝分裂指数, 使根尖细胞中具有微核、核出芽及核固缩的细胞明显增多, 并诱发染色体结构和行为异常, 产生染色体断片、滞后和桥。环磷酰胺处理组根尖中具有核固缩和微核的细胞数呈剂量依赖性增加, 且与作用时间呈正相关, 而分裂指数的降低也具有剂量和时间效应关系。研究结果表明, 低浓度长时间接触或高浓度短时间接触环磷酰胺均可产生遗传毒害, 因此, 有关的作业人员应注意防护。     

Promoting effect of bombesin on the cell proliferation in the rat endocrine pancreas during the early postnatal period

The present work investigated whether orally administered bombesin influences cell proliferation in the endocrine pancreatic islets of rats during the suckling period and after weaning. Four series of pups were given bombesin diluted in milk (20 micrograms/kg, 3 times daily) or milk alone for 5 days during either the first, second, third or fourth postnatal week of life. Oral administration was used because bombesin-like peptides have been identified in the breast milk of mammals. 45 min before death, animals were given a single [3H]thymidine pulse injection. Tissue sections were processed for radioautography; DNA labeling and mitotic indices were estimated after counting at least 1000 endocrine cells per rat pancreas. In control rats, the labeling and mitotic indices in pancreatic islets dropped regularly from the first week to the fourth week of life (3.6% +/- 0.2% versus 1.9% +/- 0.1% and 0.46% +/- 0.09% versus 0.08% +/- 0.02%, respectively). Orogastric bombesin administration significantly increased the DNA labeling and mitotic indices at the end of the first week (+20% and +62%, respectively) and second week of life (+37.5% and +49%, respectively) (P less than 0.05 to P less than 0.005), but did not modify these parameters for the third and subsequent weeks of life. Therefore, this study provides evidence that bombesin stimulates DNA synthesis and cell division in pancreatic endocrine cells during the developmental period.     

Some Characteristics of DNA Synthesis and the Mitotic Cycle in Ehrlich Ascites Tumor Cells

In vivo studies of Ehrlich ascites tumor cells during the first 5 days of growth in peritoneal cavities of mice consisted of the following: 1. Determination of growth curves by direct enumeration of cells. 2. Estimation of the duration of each phase of the mitotic cycle based on incidence of cells in different phases. 3. Radioautographic studies to determine the proportion of cells in different phases of the mitotic cycle that incorporate tritiated thymidine during a single brief exposure to this precursor of DNA. 4. Estimation of the rate of incorporation of tritiated thymidine at different times during the period of DNA synthesis by comparison of mean grain counts over nuclei in radioautographs at different times following exposure to tritiated thymidine. The assumptions underlying these experiments and our observations concerning the duration of the period of DNA synthesis and its relation to the mitotic cycle are discussed. It is concluded that DNA synthesis is continuous, occupying a period of 8.5 hours during the interphase and that the average rate of synthesis is approximately constant.