Grasshopper Neuroblasts Research Articles

Thalidomide: lack of mutagenic activity across phyla and genetic endpoints

The human and rabbit teratogen thalidomide has been tested for mutagenicity in a wide range of assays, ranging from bacterial gene mutation assays conducted in vitro to in vivo cytogenetic assays conducted using rabbits, and including a variety of human-derived tissues. Thalidomide was not mutagenic to 6 strains of Salmonella when tested both in the presence and absence of Aroclor-induced rat liver S9 mix. This inactivity was confirmed in strains TA98 and TA100 using a 1-h pre-incubation assay protocol with the same S9 mix (10% S9), and additionally, in strain TA98 using 3 concentrations of S9 (4%, 10% and 30% S9 in S9 mix). Thalidomide was not clastogenic either to cultured human lymphocytes (whole blood cultures, minus S9 mix) or to Chinese hamster ovary (CHO) cells treated in vitro. Further, no cytotoxicity was observed in purified human lymphocytes when exposed to thalidomide up to the limit of its solubility in the medium in the presence and absence of liver S9 from Aroclor-induced pregnant rabbit. The CHO assays were conducted without metabolic activation and in the presence of a variety of sources of auxiliary metabolic activation (PB/ βNP-induced rat liver S9 mix, pooled male and female human liver S9 mix, uninduced and Aroclor-induced pregnant rabbit liver S9 mix and foetal rabbit S9 mix). Thalidomide did not induce micronuclei in isolated human lymphocytes (minus S9 mix) and it was non-mutagenic to mouse lymphoma L5178Y TK +/− cells when tested to the limits of its solubility in the culture medium (±S9 mix). No indication of recombinogenic or clastogenic activity was observed for thalidomide when tested in Drosophila. In addition, it failed to induce chromosome aberrations in grasshopper neuroblasts when tested in the presence and absence of Aroclor-induced rat liver S9 mix. Some unusual chromosome morphologies were observed in the grasshopper cytogenetic preparations indicating a potential of thalidomide to interact with chromosomal proteins. However, this potential was not evident in the human lymphocyte micronucleus assay, and thalidomide was apparently not reactive to the proteins of the mouse skin, as it gave negative results in a mouse local lymph node assay for skin sensitizing agents. Thalidomide was inactive in bone marrow micronucleus assays conducted using males and females from two strains of mice, and female New Zealand white rabbits. It is concluded that thalidomide is neither a mutagen nor an aneugen. This conclusion is discussed within the context of the results of earlier mutagenicity studies, the recent claim that thalidomide may be a heritable germ cell mutagen to humans, and the current interest in thalidomide for the treatment of immune system-related diseases.

The Determination of Spindle Polarity in Early Mitotic Stages of the Dividing Grasshopper Neuroblasts: (unequal cytokinesis/neuroblast/spindle axis/electron dense layer/microdissection).

Although the spindle body of the grasshopper neuroblasts at the early mitotic stages does not have any mechanical linkage to the surrounding cell cortex, a spindle axis is inevitably oriented in parallel with the original division axis. The present study analyzes how the definite orientation of spindle axis along the cap cell (CC)-ganglion cell (GC) axis of the neuroblast is maintained during these stages by use of the microdissection technique and electron microscopy. After removing a microneedle from the cell, metaphase spindles approximately 90°. rotated were able to return autonomously to the original axis. After the middle anaphase, however, the rotated spindle could not return at all. The electron microscopic observations revealed a characteristic behavior of an electron dense layer (EDL) in the CC-side cortex during neuroblast mitosis. The EDL first appeared at very late prophase and became most conspicuous at metaphase. It became discontinuous by the beginning of middle anaphase and then completely disappeared at middle anaphase. So long as an EDL existed in the CC-side polar cortex, 90° rotated spindle bodies were able to return autonomously to the original axis. After the disappearance of the EDL, the autonomous return of the rotated spindle no longer occurred. From these circumstantial evidence, it is conceivable that the orientation of the spindle body along the CC-GC axis is maintained by the interaction between the EDL and the spindle pole.

Factors Inducing Unequal Cytokinesis in Grasshopper Neuroblasts

Factors Inducing Unequal Cytokinesis in Grasshopper Neuroblasts

Hypothesis: some mutagens directly alter specific chromosomal proteins (DNA topoisomerase II and peripheral proteins) to produce chromosome stickiness, which causes chromosome aberrations

Recent biochemical and molecular biological data on the composition and structure of the chromosome and the nucleus, combined with observations on the chromosomes of mutant yeast cells and grasshopper neuroblasts, offer new perspectives on mutagen-induced chromosome stickiness and its relation to chromosome breakage. A hypothesis consistent with these data states that chromosome stickiness (i) results from changes in specific non-histone proteins (topoisomerase II and the peripheral proteins) that are integral components of the chromosome and whose function is necessary for separation and segregation of chromatids, the changes being caused either by mutation in structural genes for the proteins (heritable stickiness) or by direct action of mutagens on the proteins (induced stickiness); (ii) occurs in various degrees (slight, moderate, severe, extreme) that are determined by the number of target protein molecules affected, a certain number (threshold) of affected molecules at a given site on a chromosome being required to resist the forces of anaphase movement in order to produce microscopically detectable stickiness; (iii) results from molecular events that can occur at several phases of the cell cycle (including interphase), but can only be recognized at prometaphase, metaphase and anaphase; and (iv) causes chromosome aberrations by the physical stretching and breaking of chromatids at the sticky sites; hence the breakage resulting from stickiness is a secondary effect that requires anaphase movement, in contrast to breakage resulting from direct action of mutagens on DNA.

Microdissection Studies on the Polarity of Unequal Division in Grasshopper Neuroblasts: II. Cell Division in Binucleate Neuroblasts: (unequal division/grasshopper neuroblasts/binucleate cells).

The grasshopper neuroblast divides unequally to produce two types of cells: a large daughter neuroblast that contains a doughnut-shaped nucleus and repeats unequal division with definite polarity, and a small daughter ganglion cell that has a spherical nucleus with low mitotic activity. Binucleate neuroblasts were induced by preventing cytokinesis in the course of microdissection experiments, and subsequent divisions were traced to analyze the factors that determine the polarity of unequal division. In binucleate neuroblasts, both daughter chromosome groups developed into neuroblast-type nuclei. Mitosis of the two nuclei proceeded synchronously. Although the axes of the two mitotic apparatuses formed at late prophase were random in direction, they became parallel with the original division axis at metaphase. The two mitotic apparatuses shifted simultaneously toward the ganglion cell side during anaphase, just as in normal neuroblasts, and the binucleate cell divided unequally. These findings showed that the poearity of unequal division is strictly maintained in grasshpper neuroblasts, even when they contain two nuclei.

Microdissection studies on the polarity of unequal division in grasshopper neuroblasts: I. Subsequent divisions in neuroblast-type cells produced against the polarity by micromanipulation

Equal or unequal division against the polarity of normal division was induced in grasshopper neuroblasts by means of a microdissection technique. The subsequent cell divisions were traced in order to analyse the factors that determine the polarity. Daughter cells of two types (neuroblast-type and ganglion cell type) were produced by operations in which the mitotic apparatus was rotated or shifted. Cell types were classified by such characteristics as nuclear shape, mitotic activity, and inequality or equality of the subsequent cytokinesis. It became evident that the fate of daughter cells was determined simply by the cytoplasmic volume. In 27 cases out of 40 microdissecting operations, both sister cells were recognized as of neuroblast type. Mitosis of these neuroblast-type sister cells proceeded asynchronously. The time required for neuroblast-type cells to reach metaphase of the second division depended on their volume. It is considered that the polarity of unequal division in grasshopper neuroblasts may be maintained by a joint action of the cap cells attaching to one of the polar regions of the cell and the cortex differentiated in the previous cell division.

Neuroblast of the grasshopper embryo as a new mutagen test system. II. Chromosome breakage induced by in vitro exposure of embryos to the direct-acting mutagens 4NQO, MNNG, adriamycin, and bleomycin.

The dose-response for the induction of acentric chromosome fragments was determined in neuroblasts of the grasshopper embryo (Chortophaga viridifasciata De Geer, Orthoptera: Acrididae) exposed in vitro to four direct-acting chemical known to be mutagenic, clastogenic, and carcinogenic: 4-nitroquinoline-1-oxide (4NQO), N-methyl-N-nitro-N-nitrosoguanidine (MNNG), Adriamycin (ADM), and bleomycin (BLM). After a 1-hr exposure followed by a 3-hr recovery period (untreated cell cycle time is 4 hr), acentric fragments were observed at doses down to 1 microM 4NQO, 1.25 microM MNNG, and 0.125 microM ADM and BLM. After an 8-hr continuous exposure, acentric fragments were induced by 4NQO at a dose as low as 0.125 microM. These low concentrations also reduced the number of dividing cells. No chromosome aberrations or mitotic effects were observed in untreated embryos or in those exposed only to the solvent dimethyl sulfoxide. Because of the short cell cycle and the sensitivity of the neuroblast to the induction of acentric chromosome fragments by chemical clastogens, a minimum of time is needed to perform the test. From a comparison with the prominent clastogen test systems currently used, it is concluded that the grasshopper neuroblast test is the fastest and that it detects some agents that some systems do not. Grasshoppers have a worldwide distribution. If neuroblasts of other species prove to be as sensitive to mutagens as those of Chortophaga, investigators in many countries would have available a eukaryotic mutagen test system that is simple, fast, reproducible, and inexpensive.

Effects of 1 and 3 R of X-rays on the chromosomes of grasshopper neuroblasts, a potential mutagen test system

Effects of 1 and 3 R of X-rays on the chromosomes of grasshopper neuroblasts, a potential mutagen test system

Linear dose-response of acentric chromosome fragments down to 1 R of X-rays in grasshopper neuroblasts, a potential mutagen-test system

Grasshopper-embryo neuroblasts have no spontaneous chromosome breakage; therefore they permit easy detection of agents that break chromosomes. An X-ray exposure of 1 R induces in them a detectable number of chromosome fragments. The dose-response of acentric fragment frequency fits a linear model between 0 and 128 R. Thus another cell type is added to those previously demonstrated to have no threshold dose for the induction of chromosome or gene mutations.

Anaphase chromosome movement in the unequally dividing grasshopper neuroblast and its relation to anaphases of other cells

The positions of the two sets of chromosome kinetochores, the spindle poles, cell membrane adjacent to the poles, and cleavage furrow of grasshopper neuroblasts in culture at 38°C were determined at short-time intervals during anaphase. The percent of motion due to poleward movement and spindle elongation, which coincide in time, were calculated for each minute, the former falling from 61% in the first minute to 15% in the seventh minute, and increasing to 86% in the final minute, probably as a result of pressure and bending of the spindle. Of the total chromosome movement during anaphase 44.6% is due to poleward movement of the daughter kinetochores and 55.4% to spindle elongation. The maximum velocity of a set of kinetochores is 3.41 μm/min and the mean velocity 1.86 μm/min (one-half the rate of separation). Various studies of anaphase chromosome movement in different cells and different species suggest certain generalizations, some of which are based on very small samples and so must be considered quite tentative: (1) The combination of poleward movement and spindle elongation is much more frequent than either acting alone. (2) These components of movement may coincide in time, overlap, or spindle elongation may follow poleward movement, but spindle elongation never begins before poleward chromosome movement. (3) There is an optimum temperature for the rate of chromosome movement, above and below which the rate gradually decreases. (4) In homoiothermic animals this optimum occurs at normal body temperature. (5) In homoiothermic animals the velocity falls more rapidly with a decrease in temperature than in poikilothermic animals. (6) Animals with large chromosomes (amphibia, grasshoppers) have higher chromosome velocities than those with small chromosomes. (7) Non-meiotic cells and secondary spermatocytes have higher velocities than primary spermatocytes of the same species. (8) Chromosome velocity is lower in malignant than non-malignant cells. (9) Chromosome velocity tends to be positively correlated with the distance the chromosomes travel during anaphase.

Mitotic Effects of Monochromatic Ultraviolet Radiation at 225, 265, and 280 nm on Eleven Stages of the Cell Cycle of the Grasshopper Neuroblast in Culture: 1. Overall Retardation from the Stage Irradiated to Nuclear Membrane Breakdown

Neuroblasts of Chortophaga viridifasciata (DeGeer) in culture were exposed to different doses of 225, 265, or 280 nm ultraviolet radiations at 11 different stages and substages of the mitotic cycle and individually selected cells were timed to breakdown of the nuclear membrane. Comparisons of the effectiveness of different wavelengths on the different stages were based on the dose that reduced the cell progression rate to 67 percent of normal (D/sub 67/) and the slope of the regression line, i.e., the control to treated time (C/T) ratio change/erg/mm/sup 2/ at the D/sub 67/ level. Cells of the prereplication period (metaphase + anaphase + early telophase) and the S phase (middle and late telophase + interphase + very early prophase) are equally sensitive to uv and contrast sharply with the much lower sensitivity of those in the postreplication period (early and middle prophase). This can best be interpreted if chromosomal DNA is the chromophore for uv-induced mitotic retardation. Cells in the prereplication period at exposure show no wavelength effect. In the S phase all stages except middle telophase and all stages combined are significantly more sensitive to 265 and 280 nm than to 225 nm. Of the postreplication stages, early prophase ismore » retarded significantly more by 280 than by 225 or 265 nm. The C/T ratio/erg/mm/sup 2/ is greater after exposure to 265 nm at all prereplication and replication stages, but exhibits no consistent wavelength pattern during the postreplication period. Evidence based on the orientation of the neuroblast with respect to the uv-source suggests that the chromophore for mitotic retardation does not reside within the centrosome and related structures, but may be present, at least partly, in the nucleolus.« less

Mitotic Effects of Monochromatic Ultraviolet Radiation at 225, 265, and 280 nm on Eleven Stages of the Cell Cycle of the Grasshopper Neuroblast in Culture: 2. Changes in Progression Rate and Cell Sequence between the Stage Irradiated and Nuclear Membrane Breakdown

Portions of embryos of the grasshopper, Chortophaga viridifasciata (DeGeer), were cultured in hanging drops under quartz cover slips. Immediately after exposure to 225, 265, or 280 nm radiation, microscope observations at 38°C were begun. The morphologically identified stage and the time after treatment of selected neuroblasts were recorded at short-time intervals until prometaphase was reached. Mitotic retardation induced by irradiation of prereplication stages (metaphase, anaphase, or early telophase) or S phase (middle or late telophase, interphase, or very early prophase) is greatest in postreplication stages (early, middle, and late prophase) and absent or minimal in stages morphologically identified as parts of S phase. Ultraviolet irradiation superimposes on the normal diversity of progression rates an additional variation factor, so that cells do not necessarily reach prometaphase in the order of their sequence at the time of treatment. This suggests the need for caution in ascribing particular ra...

Mitotic acceleration induced in grasshopper neuroblasts by small doses of monochromatic ultraviolet radiation.

Abstract—Neuroblasts of the grasshopper, Chortophaga viridifasciata (DeGeer), in culture were exposed to 4–15 J/m2 of monochromatic ultraviolet radiation in middle prophase and timed to prometaphase (breakdown of the nuclear membrane). At wavelengths 265 and 280 nm the times recorded for the treated cells were significantly less than those for the matched control cells. Wavelength 225 nm produced neither significant acceleration nor deceleration of mitotic rate.Evidence from the present experiment does not implicate any particular chromophore in this response, though similar results of a previous microbeam study of neuroblasts point to the nucleic acid or protein of the nucleolus as the possible effective material.

Chemical Mutagens. Principles and Methods for Their Detection.

This book discusses the development and validation of short-term assays designed to detect the mutagenic effects of environmental chemicals. Topics considered include the grasshopper neuroblast short-term assay for evaluating the effects of environmental chemicals on chromosomes and cell kinetics, a comparison of the mutagenic responses of lung-derived and skin-derived human diploid fibroblast populations, the L-arabinose resistance test with Salmonella typhimurium, the Bacillus subtilis multigene sporulation test for the detection of environmental mutagens, the L5178Y/TK gene mutation assay system, the induction of bacteriophage lambda by DNA-interacting chemicals, the granuloma pouch assay, the use of multiply marked Escherichia coli K12 strains in the host-mediated assay, and the detection of mutagens in human feces as an approach to the discovery of causes of colon cancer.

Action of actinomycin D on grasshopper neuroblasts in culture

Portions of embryos of the grasshopper Chortophaga viridifasciata (DeGeer) were prepared in hanging drop cultures and maintained in a microscope incubator at 38±0.2 °C. Individual neuroblasts were mapped and observed for reaction to actinomycin D (AMD) in the presence and absence of grasshopper egg yolk. Midmitotic duration was not altered by the omission or addition of yolk or by 4 or 10 μg/ml AMD, therefore the number of cells in midmitosis at any one time indicated the number of cells normally progressing through mitosis (mitotic rate). Four and 10 μg/ml AMD depress the mitotic rate by more than 75% in cultures lacking yolk. In cultures containing yolk, 4 μg/ml had no effect but 10 μg/ml depressed the rate to the same extent as in cultures lacking yolk. These same concentrations had no effect on chromosome condensation or attenuation or upon nucleolar disappearance in very late prophase. Concentrations as low as 4 μg/ml, however, often inhibit nucleolar differentiation in late telophase. The presence of 10 μg/ml AMD in hanging-drop cultures containing yolk reduces RNA synthesis by 30% but does not significantly alter the durations of early, middle, or late prophase stages. Constant treatment with 10 μg/ml AMD did significantly increase cell cycle times, often preventing cells from completing one full cycle.

The effects of varying concentrations of colchicine on the progression of grasshopper neuroblasts into metaphase.

The effects of four concentrations of colchicine (2.5 x 10(-7), x 10(-5), x 10(-3), and x 10(-2)M) on the cell cycle of grasshopper neuroblasts have been determined by direct observations on living cells. The lowest concentration, 2.5 x 10(-7)M, does not completely disorganize the spindle but does retard its action. The three higher concentrations disorganize the spindle, so that all cells reaching metaphase are blocked in a c-mitotic condition throughout the period of observations (308 min at 38 degrees C, the minimum duration of the cell cycle in untreated neuroblasts). Continuous treatment with all concentrations reduces the rate at which neuroblasts enter metaphase, the extent of the reduction being a function of increasing concentration and time of exposure. After a short exposure to 2.5 x 10(-5)M colchicine, the neuroblasts recover from the inhibiting effects on progression through the cycle to metaphase, but they show no recovery from the inhibiting effects on spindle formation for more than 3 hr. Apparent stimulation of progression rate occurs early in exposure to all concentrations and during recovery from a short exposure to 2.5 x 10(-5)M. Morphological alterations in the chromatin of telophase, interphase, and prophase cells are induced by the higher concentrations of colchicine. The data indicate that caution should be exercised in the use of colchicine for determining cell cycle duration and/or the effects of physical and chemical agents on the cycle.

Relation of x-ray induced thymidine uptake to chromosome reversion at prophase in grasshopper neuroblasts.

SummaryUnscheduled DNA synthesis (thymidine uptake) induced by 32 R of x-rays was followed autoradiographically in individual grasshopper neuroblasts. It occurs in those prophase cells that undergo chromosome reversion (chromosomes ‘decondense’, appearing to move backward in the cell-cycle), but not in those that are delayed in the cycle without chromosome reversion or that progress with no delay. The latest point in prophase at which unscheduled DNA synthesis is observed is the initial part of late prophase. Hypotonic culture medium, which by itself causes decondensation of prophase chromosomes but not unscheduled DNA synthesis, enhances both chromosome reversion and unscheduled DNA synthesis when these are induced by radiation.

A Detailed Analysis of X-Ray-Induced Prophase Delay and Reversion of Grasshopper Neuroblasts in Culture

Effects of x-ray exposures of 8, 16, 32, 64, 128, 512, and 1024 R on mitosis in hanging-drop preparations were determined by repeated observations of selected prophase cells before and at short intervals after irradiation. Prophase was subdivided into eight microscopically distinguishable stages: very-early, early, initial-middle, intermediate-middle, terminal-middle, initial-late, intermediate-late, and terminal-late. With one exception, cells treated at intermediate- or terminal-late prophase with any of the doses were neither delayed nor reverted. After 8 to 16 R nearly all cells from early to initial-late prophase are delayed in reaching prometaphase, but none reverts. After 32 to 64 R all cells in early to initial-late prophase are delayed and most revert; after 128 to 1024 R nearly all revert. Delay or reversion is preceded by a postirradiation inertia, during which the cell progresses mitotically at an almost normal rate for a short time. Duration of inertia is inversely related to dose. The degree...

THE THYMIDINE POOL IN GRASSHOPPER NEUROBLASTS DURING MITOSIS

THE THYMIDINE POOL IN GRASSHOPPER NEUROBLASTS DURING MITOSIS

Influence of Tonicity on Mitotic Recovery in X-irradiated Grasshopper Neuroblasts

Influence of Tonicity on Mitotic Recovery in X-irradiated Grasshopper Neuroblasts