Delayed disruption of the telomeric links between chromosomes in experimentally induced cells with micronuclei exposed to 5-bromodeoxyuridine in the 1st S period after colcemid administration

Under a long-term administration of colcemid in the Chinese hamster cell culture some cells with micronuclei are seen to form. In the case of co-treatment with colcemid and 5-bromodeoxyuridine (5-BrdU) at metaphases of the first division of cells with micronuclei polycentric chromosomes were observed. These polycentric chromosomes occur due to delayed disruption of telomeric links, previously existing in the interphase. During colcemid treatment the cells pass through two S-periods: one in mononuclear cells, the other in cells with micronuclei. This phenomenon was tested according to the frequency of metaphases with dicentrics after 5-BrdU-treatment of cells at the first or second S-period or during the two cycles of chromosome replication. The 5-BrdU treatment during the first cycle or two cycles of replication resulted in the same frequency of cells with dicentrics--about 50%. The treatment with colcemid alone during two cycles of replication and administration 5-BrdU at the second S-period results in a considerably lower amount (%) of cells with dicentrics--about 10%. Thus, the delayed disruption of telomeric links between chromosomes may occur under the treatment with 5-BrdU at the first S-period after colcemid administration. It is also concluded that this phenomenon can be reproduced in cell with micronuclei when 5-BrdU is incorporated differentially in the sister chromatids.     

The effect of hyper- and hypothermia on induced telomeric chromosome fusion]

After administration of colcemid and 5-BrdU in the cell culture, the cells pass through the first interphase to delay in mitosis. Then the cells overcome the colcemid blockade, and polykaryocytes with micronuclei are formed. The second interphase in followed by the second mitosis, during which dicentric chromosomes are observed. These dicentrics are the result of telomeric chromosome fusion. The action of hyperthermia (40 degrees C) during the whole period of colcemid and 5-BrdU treatment or that of the hyperthermia (40 degrees C) only during the first 17 hours (the first interphase and the first mitosis) lead to the increased frequency of dicentrics. Under condition of hypothermia (34 degrees C) the frequency of dicentric formation decreases. Changes in cultivation temperature during the latest 25 hr of colcemid and 5-BrdU action (the second interphase and the second mitosis) exert no influence on the dicentric formation frequency. Because there are no dicentrics in cells during the metaphase of the first mitosis, it is supposed that the temperature--sensitive period may be the latest steps of colcemid blockade, i.e. the period of formation of micronuclei.     

Telomeric chromosome fusion in cells with micronuclei under the combined action of hyperthermia and hypothermia and halogenated analogs of thymidine]

The reaction of cells with micronuclei in respect of the induction of specific dicentric chromosomes with halogenated analogs of thymidine at various temperatures was studied. The positive correlation between the temperature and frequency of dicentrics was shown for all halogenated analogs of thymidine. The minimum frequency of dicentrics was found in the case when used 5-iododeoxyuridine and hypothermia (34 degrees C). The using of 5-bromodeoxyuridine at different temperatures displayed the intermediate results. The maximum level of dicentrics discovered under action of 5-chlorodeoxyuridine and hyperthermia (40 degrees C). In the former case the depression of mitotic chromosome condensation of micronuclei registered, in the latter one the chromosomes with portions of delayed spiralization were not found.     

5-Chlorodeoxyuridine, more effectively than 5-bromodeoxyuridine, induces the formation of specific dicentric chromosomes in cells with micronuclei

5-chlorodeoxyuridine (5-ChldU) and 5-bromodeoxyuridine (5-BrdU) were tested in respect of the formation of specific dicentric chromosomes in cells with micronuclei. It is shown that the frequency of dicentrics is higher when 5-ChldU is used. On the ground of results of this study and of the previous data it is concluded that the activity of halogenated deoxyuridines is falling with molecular weight increasing.     

Persistence of micronuclei in the marine mussel, Mytilus galloprovincialis, after treatment with mitomycin C

The frequency of micronuclei induced by mitomycin C (MMC) in cells of the gill tissue of the marine mussel, Mytilus galloprovincialis Lmk., was determined over a long period (up to 40-52 days) following treatment. Two doses of MMC (0.5 X 10(-7) and 10(-7) M) were tested at 13 degrees C and 23 degrees C, temperatures representative of the winter and summer thermic conditions of the Mediterranean Sea. In all cases, the frequency of micronuclei was significantly increased by MMC and declined after treatment until it reached a plateau level, significantly higher than the control value. This persisted for a very long time. The frequency of micronuclei induced by a second treatment with MMC performed on the 28th day, did not differ significantly from that produced by the first treatment at the same dose. Temperature did not influence the pattern of the described phenomena to a significant extent. The reason for the persistence of an increased frequency of micronuclei is discussed, and a system is proposed for evaluating the genotoxicity of water pollutants present long before sampling.     

Mitotic dynamics of micronuclei induced by amiprophos-methyl and prospects for chromosome-mediated gene transfer in plants

Summary Mitotic dynamics and the kinetics of mass induction of micronuclei after treatment of Nicotiana plumbaginifolia cell suspensions with the spindle toxin amiprophos-methyl (APM) are reported. The addition of APM to suspension cells resulted in the accumulation of a large number of metaphases. The course of mitosis was strikingly different from normal. Metaphase chromosomes showed neither centromere division nor separation of chromatids. Single chromosomes and groups of 2 or more chromosomes were scattered over the cytoplasm. After 5–6 h of APM treatment, chromosomes decondensed and formed micronuclei. When treatment duration was increased, the frequency of cells with micronuclei as well as those showing lobed micronuclei increased. Similarly, with an increase in APM concentration the frequency of cells with micronuclei increased. After removal of APM, chromosome grouping disappeared, cells showing lobed micronuclei further increased and mitoses with doubled chromosome numbers appeared in the next cell division. Cytological observations and DNA measurements revealed that several sub-diploid micronuclei containing 1 or a few chromosomes can be obtained, and that flow cytometry can detect and sort out these micronuclei. The applications of micronuclei for genetic manipulation of specific chromosomes and gene mapping are indicated.     

Effects of chemically- and environmentally-induced hypothermia on micronucleus induction in rats

We investigated micronucleus induction in rats treated with chlorpromazine and reserpine, drugs that induce hypothermia. We administered chlorpromazine (31.3--250mg/kg) or reserpine (500--2000 mg/kg) intraperitoneally and measured temperature rectally. Chlorpromazine at 62.5-250mg/kg and reserpine at all doses significantly decreased rectal temperature, although the hypothermic response was weaker than previously reported in mice. Only chlorpromazine at 250mg/kg decreased rectal temperature transiently to <33 degrees C for 20h and induced a statistically significant increase in micronucleated polychromatic erythrocyte frequency. When rats treated with reserpine at 500mg/kg were exposed to an environmental temperature of 16 degrees C for 6, 12, or 24h to keep their body temperature under 33 degrees C, only the 24h treatment group significantly induced micronuclei. In addition, relatively large micronuclei (diameter of micronucleus> or = 1/4 diameter of cytoplasm) accounted for 33.0% of the induced micronuclei, suggesting that hypothermia affected the mitotic apparatus. The hypothermic response to chlorpromazine and reserpine was weaker in rats than in mice, and it was correspondingly more difficult to induce micronuclei in rats with those drugs.     

Condensation anomalies and exclusion in micronuclei of rearranged chromosomes in human fibroblasts cultured in vitro

Anomalies of chromatin condensation, such as fragmentation, uncoiling and pulverization, were observed in XP9UV25, a xeroderma pigmentosum fibroblast clone in which a high proportion of cells carried an end-to-end dicentric chromosome, dic (5;16) (p15.2;q24), that gives rise during propagation in culture to a variety of dicentric and monocentric derivatives. The coiling anomaly affected exclusively part of a rearranged chromosome, in particular the region previously involved in breakage events. The heterochromatic 16q region, which is a preferential breakpoint in the formation of dicentric and monocentric derivatives, was consistently the limit of the uncoiled or pulverized regions. This observation suggests that the anomalous chromatin behavior could derive from alteration of a region relevant for the correct condensation of the chromosome. In XP9UV25 the frequency of nuclei with associated micronuclei increased with time in culture, in parallel with that of mitoses with dicentric chromosomes. In situ hybridization with DNA probes specific for chromosomes 5 and 16 revealed hybridization signals in about 40% of micronuclei. Since the frequency of micronuclei is about ten times less than that of dicentrics, it is probable that only the rearranged chromosomes undergoing coiling anomalies are excluded in micronuclei.     

Characteristics of the cis- and trans-positions of chromatids of human acrocentric chromosomes in extreme old age

Cis- and trans-positions of chromatid associations of human acrocentric chromosomes were examined at extreme old age. Lymphocyte cultures were prepared by the usual method, from peripheral blood of 9 subjects aged 80-90 years (analysis of 179 metaphases) and 7 subjects aged 20-48 years (analysis of 124 metaphases). The functional difference between stalks of the sister chromatids was found. In the subjects at the age 80-90 years satellite stalks of chromatids-1 (in all DNA strands thymidine was substituted by 5-BrdU) of the D chromosome in cis-position are included into associations with lower frequency, as compared with the satellite stalks of chromatids-2 (thymidine+ is only substituted by 5-BrdU in a half DNA strands of the chromosome). This apparently reflects variability of regulation of functional activity of satellite stalks of sister chromatids.     

Cells of the external granular layer of the rat cerebellum as a test system for evaluating the clastogenic activity of chemical compounds

A high sensitivity to the effect of nitrosomethylurea in the chromosomes of the rat cerebellum external granular layer has been found. The frequency of occurrence of cells with the micronuclei in cerebellum of animals under study increases 10-40-fold in comparison with the control ones. The number of genetically aberrant cells reaches the maximum in 24 and 48 h after mutagen treatment at doses 100 and 50 mg/kg, respectively. It has been suggested to use the proliferating cells of rat cerebellum external granular layer as an effective test system for studying the clastogenic activity of chemicals.     

Induction of micronuclei in mouse bone-marrow erythrocytes in association with hypothermia after administration of the sigma receptor ligand E-5842

Oral administration of E-5842, a new sigma1 receptor ligand being developed as an antipsychotic drug, to male mice at single doses of 50, 100, 200 and 400 mg/kg produced marked and sustained decreases in rectal temperature. Both the intensity and the duration of the hypothermic effect increased with dose. Maximum decreases from the mean pre-administration temperature (36.2 degrees C) ranged from 7.5 to 12.9 degrees C for animals receiving 50 and 400 mg/kg doses, respectively. Examination of bone-marrow smears obtained 24, 48 and 72 h after administration revealed a slight but statistically significant (p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes (MNPCE) at the 48 h sampling for animals receiving the 200 mg/kg dose. These animals showed decreases from pre-administration temperature of approximately 12 degrees C, with recovery being observed 24 h after administration. When the hypothermic effect of E-5842 administration was avoided by housing treated animals under conditions of increased environmental temperature (30 degrees C) for 24 h, MNPCE frequency reverted to vehicle control values. Further, in E-5842-treated animals with an increased MNPCE frequency there was a shift in the distribution of the relative areas of micronuclei in MNPCE towards higher values. In addition, there was a statistically significant increase (p < 0.001) in the number of relatively large micronuclei (micronucleus diameter > or = 1/4 cytoplasm diameter) similar to that produced by administration of the mitotic spindle inhibitor colchicine (1 mg/kg), suggesting disturbance of mitotic apparatus as the possible underlying mechanism. The results suggest that the slight increase in MNPCE frequency observed 48 h after administration of a 200 mg/kg dose of E-5842 is due to a hypothermic effect and not to a direct effect of E-5842 on DNA.     

Frequency of micronuclei in root-tips of Tradescantia clone 02: Effects of diurnal temperature difference and Co gamma radiation

Frequency of micronuclei in root-tip cells was used to asses the influence and/or interaction of a large Diurnal Temperature Difference (DTD) on irradiated and non-irradiated Tradescantia Clone 02. Hydroponically grown plants were subjected to four different treatment combinations: no radiation/normal DTD (7.2°C), no radiation/large DTD (23.5°C), radiation (60R)/normal DTD, radiation/large DTD. An analysis of variance of Feulgen positive micronuclei arising from chromosome fragments in root-tip cells revealed that a 5-day chronic exposure to a large DTD without radiation does not significantly increase the frequency of cells with micronuclei (freq. = 0.53%). An accute 60 R radiation exposure, as expected, did significantly increase the frequency (freq. = 3.23%). None of the treatment interactions significantly altered micronuclei production. These data lend support to the contention that the mutation-induction mechanism of large DTD's, as expressed by variant color sectoring in stamen hair cells of this clone, is not mediated by chromosome breakage. Exposure to a large DTD, therefore, may be a method for inducing mutations in stamen hairs without concomitant chromosome breakage, and may also provide a good cytological means for analyzing somatic crossing over, which has been postulated as a possible mechanism for the expression of spontaneous mutations at the D⁺ locus. The frequency of micronuclei in irradiated tissue remained constant throughout the 5-day course of the experiments and several explanations for this occurrence are proposed.     

Influence of temperature and darkness on embryonic diapause termination in dormant Artemia cysts that have never been desiccated

Environmental cues for embryonic diapause termination (EDT) were investigated in the laboratory-produced encysted dormant embryos of the brine shrimp, Artemia franciscana. The cysts were spawned and kept throughout in a 2% sea salt solution. They were activated by various temperatures of the temperate zones or by continuous dark condition (DD), resulting in a state of EDT, and were thereafter able to resume their subsequent development and hatch under appropriate conditions. The level of EDT was conveniently assayed by a hatch test observed within 2 days at 28 degrees C under continuous light condition (LL). A cold treatment of the newly spawned dormant cysts, at 4 degrees C under DD for more than 100 days, resulted in more than 95% hatch of the dormant cysts. Similar treatments of the dormant cysts but at room temperature or 28 degrees C led to significantly different results (30-40% hatch). Almost all the residual non-hatched cysts derived from the above could hatch after an additional cold treatment (at 4 degrees C under DD for about 100 days). This might prove to be latent partial bivoltine in Artemia. Meanwhile, a rearing condition (28 degrees C under LL) induced the newly spawned cysts to hatch scatteredly at and after 1 month, resulting in 22% cumulative hatch on the 92nd day after spawning. When the newly spawned dormant cysts were pretreated at 28 degrees C under DD for 5 or 14 days and then reared at 28 degrees C under LL, the cumulative hatch significantly increased (60%). These results are discussed with respect to probable diapause regulator(s) involved in EDT.     

Characterization of chitinases of polycentric anaerobic rumen fungi

Chitinolytic systems of anaerobic polycentric rumen fungi of genera Orpinomyces and Anaeromyces were investigated in three crude enzyme fractions - extracellular, cytosolic and cell-wall. Endochitinase was found as a dominant enzyme with highest activity in the cytosolic fraction. Endochitinases of both genera were stable at pH 4.5-7.0 with optimum at 6.5. The Orpinomyces endochitinase was stable up to 50 degrees C with an optimum for enzyme activity at 50 degrees C; similarly, Anaeromyces endochitinase was stable up to 40 degrees C with optimum at 40 degrees C. The most suitable substrate for both endochitinases was fungal cell-wall chitin. Enzyme activities were inhibited by Hg(2+) and Mn(2+), and activated by Mg(2+) and Fe(3+). Both endochitinases were inhibited by 10 mmol/L SDS and activated by iodoacetamide.     

Standard karyotype of sea trout (Salmo trutta morpha trutta) based on replication banding patterns

Replication banding patterns have been obtained from in vivo treatment of Salmo trutta morpha trutta chromosomes using a modification of the 5-BrdU technique and in kidney cultures using the fluorochrome photolysis Giemsa (FPG) staining method. Each chromosome pair was identified in the karyotype based on the banding pattern, chromosome size, and centromere position. The standard karyotype of sea trout has been proposed. Similarities of replication, and restriction enzyme banding patterns of chromosomes 8 and 9, and of chromosomes 11 and 12, are discussed. Fluorescence in situ hybridization was used to determine the location of telomeric sequences.     

Measurement of micronuclei by cytokinesis-block method in cultured Chinese hamster cells: comparison with types and rates of chromosome aberrations

The cytokinesis-block method of Fenech and Morley (1985) has been tested for the enumeration and characterization of micronuclei in exponentially growing Chinese hamster cells in culture. The consistent dose-response relations were obtained in cultures treated with mitomycin C, caffeine and colcemid. Comparison with the chromosome aberration frequencies indicated that approximately 30% of the acentric chromosomes are expressed as micronuclei in the mitomycin C and caffeine treated cells. The size distribution of the micronuclei suggested that the base-line frequency of micronuclei is mainly a reflection of mitotic dysfunctions rather than chromosome structural aberrations.     

A comparison of the effects of various spindle toxins on metaphase arrest and formation of micronuclei in cell-suspension cultures ofNicotiana plumbaginifolia

The effects of the spindle toxins colchicine, oryzalin and amiprophos-methyl (APM) on metaphase arrest, chromosome scattering, and on the induction and yield of micronuclei were compared in suspension cells ofNicotiana plumbaginifolia (kanamycin-resistant “Doba” line). The inhibition of spindle formation is stronger with oryzalin and APM than with colchicine, which resulted in a more efficient accumulation of meta-phases with well-scattered chromosomes, allowing the isolation of single chromosomes. Further, APM and oryzalin treatments resulted in a higher frequency of micro-nucleated cells and greater yield of micronuclei than after colchicine treatment. The different actions of the chemicals on the functioning of the spindle, development of nuclear membranes around the chromosomes, formation of micronuclei and fusion of micronuclei, resulting in restitution nuclei, are discussed.     

FISH in analysis of gamma ray-induced micronuclei formation in barley

A micronucleus test in combination with fluorescent in situ hybridization (FISH) using telomere-, centromere-specific probes and 5S and 25S rDNA was used for a detailed analysis of the effects of gamma ray irradiation on the root tip meristem cells of barley, Hordeum vulgare (2n = 14). FISH with four DNA probes was used to examine the involvement of specific chromosomes or chromosome fragments in gamma ray-induced micronuclei formation and then to explain their origin. Additionally, a comparison of the possible origin of the micronuclei induced by physical and chemical treatment: maleic hydrazide (MH) and N-nitroso-N-methylurea (MNU) was done. The micronuclei induced by gamma ray could originate from acentric fragments after chromosome breakage or from whole lagging chromosomes as a result of a dysfunction of the mitotic apparatus. No micronuclei containing only centromeric signals were found. An application of rDNA as probes allowed it to be stated that 5S rDNA–bearing chromosomes are involved in micronuclei formation more often than NOR chromosomes. This work allowed the origin of physically- and chemically-induced micronuclei in barley cells to be compared: the origin of micronuclei was most often from terminal fragments. FISH confirmed its usefulness in the characterization of micronuclei content, as well as in understanding and comparing the mechanisms of the actions of mutagens applied in plant genotoxicity.     

Sister chromatid exchange in human chromosomes from normal individuals and patients with ataxia telangiectasia.

A new fluorescence plus Giemsa staining technique now makes the detection of sister-chromatid exchange (SCE) a relatively easy matter in cells containing 5-BrdU-substituted DNA. The technique has been applied to human cells to examine the distribution of SCE between different people and within different chromosomes. The results show: (1) That there were no large differences in the incidence of SCE between blood leukocyte chromosomes from male and female adults and newborn, and that similar frequencies were found in cells from two patients with ataxia telangiectasia which, nevertheless, showed the typical increases in chromosomal aberrations. (2) The distribution of SCE between chromosomes in the complement was found to be proportional to chromosome length, although the smaller chromosomes were under-represented, but not significantly so. (3) The distribution of SCE within chromosomes was nonrandom, with a deficiency in the centromeric and an excess in the mid-arm regions. There was no evidence for an excess of SCE in chromosome regions rich in AT DNA sequences. (4) The frequency of SCE is to some extent dependent of 5-BrdU concentration, but the influence of concentration is minimal within the range of from 1 to 160 muM. Human cells exposed over two cell cycles at these higher BrdU levels have around 14 SCE per cell-a frequency virtually identical with that observed in cultured cells from the Chinese hamster, wallaby, and rat kangaroo.