Distribution and localization of induced breaks on the chromosomes of Bellevalia romana. IV. N-nitroso-N-methylurea, N,N''-dinitroso-N,N''-dimethylterephthalamide, and 1-nitroso-imidazolidone-2

Summary Chromosome breaks, mostly fragmentations and some translocations were induced in Bellevalia romana (2n=8) by N-nitroso-N-methylurea (NMH, 1499 breaks), N,N-dinitroso-N,N-dimethylterephthalamide (NMT, 1516 breaks) and 1-Nitrosoimidazolidone-2 (NIL, 2055 breaks).The mitotic rate is considerably reduced only by NMT; NIL induced the highest number of fragmentation and also, in contrast to NMH and NMT, about 3% translocations.There was no difference in the distribution over the four chromosome types of all the braks induced by NMH, NMT and NIL; fragmentations of the centromeres were more frequent than fragmentations of the chromosome arms; these peculiarities and the distribution of centromere-breaks correspond to the distributions, induced by agents investigated earlier.It is only the distribution of breaks over the three regions of chromosome arms, proximal, median and terminal which is mainly determined by the chemical nature of the agent; NMT shows an equidistribution of breaks over the regions of all chromosome arms, NMH and NIL revealed a pronounced decrease of the breakage frequency from the centromere-region towards the chromosome end in most of the chromosome arms. The breakage patterns induced by these two substances were not identical, in the shorter arm of the A-chromosome, NIL induced a pattern with the highest number of breaks in the median region.In the discussion the breakage patterns of 5 nitrosamides, nitrous acid, methylphenylnitrosamine, methylmethanesulfonate, diethylsulfate, and X-rays were compared. The methylating nitrosamides induced two opposite patterns: Breaks induced by the stable, slowly hydrolysing NMT and NMU tended to be equally distributed over the regions of the chromosome arms. The breaksinduced by rapidly hydrolysing NMH and NIL had the common property of following gradients of breakage frequencies.In conclusion, there have been evaluated three main factors, determining the kind of breakage patterns: the transport-and active form of the substances used, and the velocity of their degradation in aqueous solution.     

Increased methotrexate-induced chromosome breakage in patients with free trisomy 21 and their parents

Summary Increased susceptibility of chromosomes from peripheral blood lymphocytes to the antimetabolite methotrexate (2×10^-6 M) has been found in patients with free trisomy 21 and their parents (N=14). The level of induced chromatid and chromosome breaks is lowest in normal controls intermediate in patients' mothers and fathers, and highest in trisomy 21 patients. The findings are viewed as a special type of cytogenetic polymorphism or as a defective chromosomal infrastructure, also in the parents of trisomic children.Dedicated to Professor Dr. H.A. Freye, Halle/Saale, in honor of his 65th birthday     

The distribution of x-ray induced chromosomal abnormalities in rat kangaroo (Potorous tridactylis) cells in vitro

Synchronized cells of Potorous tridactylis (female origin) were irradiated with X-rays in vitro at different times during the cell cycle. Chromosome aberrations were scored in the first metaphase after irradiation.It is shown that the distribution of breaks and gaps in the chromosome arms is different at different cell-cycle times, and is non-random.     

Inhibition of cell division and induction of chromosome aberrations in cultured Ehrlich ascites tumor cells by deoxyguanosine

Summary Exposure of exponentially growing cultures of Ehrlich ascites tumor cells to 1 or 2×10^-3 M deoxyguanosine resulted in an inhibition of DNA synthesis and cell multiplication. Continued increase in the RNA and protein content of these cultures suggests a state of unbalanced growth. Deoxyguanosine-inhibition is prevented by the presence of deoxycytidine (1×10^-4–2×10^-3 M).Treatment with deoxyguanosine (2×10^-3 M) for about one generation-time (18 hrs) and removal of deoxyguanosine thereafter resulted in chromosome aberrations (breaks and exchange figures) in 30–50% of those mitotic cells which were harvested 5 to 12 hrs after treatment. Chromosome defects were strongly reduced after incubation of cell-cultures in the presence of deoxyguanosine (2×10^-3 M) together with deoxycytidine (5×10^-4 M).The biochemical mechanisms by which deoxyguanosine and other inhibitors of DNA synthesis might produce chromosome damaging effects, are discussed.Supported by grants from Deutsche Forschungsgemeinschaft (Scha 176/1 and Scha 176/2).     

Comet assay evaluation of DNA single- and double-strand breaks induction and repair in C3H10T1/2 cells

Ionizing radiation is a potent inducer of DNA damage because it causes single- and double-strand breaks, alkali-labile sites, base damage, and crosslinks. The interest in ionizing radiation is due to its environmental and clinical implications. Single-strand breaks, which are the initial damage induced by a genotoxic agent, can be used as a biomarker of exposure, whereas the more biologically relevant double-strand breaks can be analyzed to quantify the extent of damage. In the present study the effects of ¹³⁷Cs γ-radiation at doses of 1, 5, and 10 Gray on DNA and subsequent repair by C3H10T1/2 cells (mouse embryo fibroblasts) were investigated. Two versions of the comet assay, a sensitive method for evaluating DNA damage, were implemented: the alkaline one to detect single-strand breaks, and the neutral one to identify double-strand breaks. The results show a good linear relation between DNA damage and radiation dose, for both single-strand and double-strand breaks. A statistically significant difference with respect to controls was found at the lowest dose of 1 Gy. Heterogeneity in DNA damage within the cell population was observed as a function of radiation dose. Repair kinetics showed that most of the damage was repaired within 2 h after irradiation, and that the highest rejoining rate occurred with the highest dose (10 Gy). Single-strand breaks were completely repaired 24 h after irradiation, whereas residual double-strand breaks were still present. This finding needs further investigation. This revised version was published online in July 2006 with corrections to the Cover Date.     

Sustainable, High-Yielding Outdoor Mass Cultures of Chaetoceros muelleri var. subsalsum and Isochrysis galbana in Vertical Plate Reactors

Continuous cultures of Chaetoceros muelleri and Isochrysis galbana were grown outdoors in flat plate-glass reactors in which light-path length (LPL) varied from 5 to 30 cm. High daily productivity (13 to 16 g cell mass per square meter of irradiated reactor surface) for long periods of time was obtained in reactors in which the optical path as well as cell density were optimized. 'Twenty centimeters was the optimal LPL, yielding the highest areal productivity of cell mass (g m^–2d^–1), eicosapentaenoic acid, and docosahexaenoic acid, which was identical with that previously found for polysaccharide production of Porphyridium and not far from the optimal LPL affecting maximal productivity in Nannochloropsis species. Relating the energy impinging on a given reactor surface area to the appropriate number of cells showed that the most efficient light dose per cell, obtained with the 20-cm LPL reactor, was approximately 2.5 times lower than the light dose available per cell in the 5-cm LPL reactor, in which a significant decline in areal cell density accompanied the lowest areal output of cell mass. The most effective harvesting regimen was in the range of 10% to 15% of culture volume harvested daily and replaced with fresh growth medium, resulting in a sustainable culture density of 24 × 10⁶ and 28 × 10⁶ cells/ml of C. muelleri and I. galbana, respectively.     

Localization of chemically induced breaks in chromosomes of human leucocytes

The distribution of 16942 chromosome breaks induced by 3 chemical agents— N,N,N-triethylene thiophosphamide (thiosphosphamide, Thio-TEPA), dihydrochloride-1,6-di(choloroethyl)-amino-1,6-desoxymannitol (Degranol), and mitomycin-C (MC)—and their dependence on the moment at which the mutagen was introduced to the cultures, the dose of the mutagen, the time of fixation of the cultures and the sex and age of the donor, were statistically investigated.As control served the distribution of spontaneous chromosome breaks found in a group of 1649 newborns.The spontaneous breaks were randomly localized on the chromosomal groups, whereas the induced breaks showed a non-random distribution. It was demonstrated that the experimental conditions, which have been investigated, had no influence on the distributions of chromosome breaks within the groups. “Hot-spots” and “cold-spots” could be established along the length of the chromosomes. The localization of these spots did not depend on the experimental parameters investigated, but on the chemical agent by which they were induced.The possible cause of discrepancies between the present results and those reported by other authors are discussed.     

Butyric acid bacteria of the genus <Emphasis Type="Italic">Clostridium</Emphasis> in the bottom sediments of inland basins of different types

The cell numbers and ecological characteristics of the distribution of certain species of butyric acid bacteria (BABs) of the genus Clostridium in the bottom sediments of inland basins of different types were studied using the optimal nutrient media. The seasonal dynamics of clostridial vegetative cells and spores in sediments with different ecological conditions were revealed. The cell numbers of the dominant BAB species were shown to depend on the redox potential of the sediments, the amount and composition of C_org, and the trophic state of the basin in general. C. pasteurianum was found to predominate in eutrophic lakes and reservoirs (5–11 × 10⁶ cells/cm³), C. butyricum and C. felsineum predominated in mesotrophic ones (2–11 × 10⁶ cells/cm³), and C. acetobutylicum was predominant in acidic chthonioeutrophic lakes and reservoirs (0.1–0.5 × 10⁶ cells/cm³). The lowest cell numbers of BABs were found in river sediments, whereas the highest numbers were recorded in the sediments of polysaprobic zones (0.1–1.0 × 10³ and 0.5–2.0 × 10⁷ cells/cm³ respectively).Translated from Mikrobiologiya, Vol. 74, No. 1, 2005, pp. 119–125.Original Russian Text Copyright © 2005 by Dzyuban.     

EXPERIMENTALLY INDUCED CHROMOSOME ABERRATIONS IN PLANTS : II. THE EFFECT OF CYANIDE AND OTHER HEAVY METAL COMPLEXING AGENTS ON THE PRODUCTION OF CHROMOSOME ABERRATIONS BY X-RAYS

The discovery of Lilly and Thoday, that the presence of potassium cyanide (KCN) increases the production of chromosome aberrations by x-rays in anoxia, but has no effect on the production of chromosome aberrations by x-rays in air, was confirmed. In the presence of cyanide, the effect of a given dose of x-rays in nitrogen was found to be even greater than the effect of the same dose of x-rays in air. The cyanide effect on x-ray breakage in nitrogen was obtained at cyanide concentrations as low as 2 x 10^–5 M. The breakage obtained after the combined x-ray-cyanide treatments was of the x-ray type, as evidenced by the distribution of breaks within and between the chromosomes. A number of other heavy metal complexing agents as well as some other compounds were tested for their ability to increase x-ray breakage in nitrogen and air. Of these compounds only cupferron proved to be effective. The results are discussed and it is concluded that the increased x-ray breakage in the presence of cyanide or cupferron cannot be due to an accumulation of peroxides. Instead it is suggested that the cyanide effect may be due to a complex formation between the active agents and heavy metals, presumably iron, within the chromosomes. The consequences of this hypothesis on the concept of the "oxygen effect," are discussed.     

The Rejoining Time of Chromatid Breaks Induced by Gamma Radiation in Vicia faba Root Tips at 3 °C

The observation was made previously that the reduction in radiosensitivity in Vicia faba (as measured by postirradiation root growth) by prolonging the exposure time from about 10 minutes to 24 hours is much less marked at 3°C. than at 19°C. If chromosome damage is mainly responsible for the reduced root growth, this observation might be explained by a smaller drop in the "two-hit" aberration component, resulting from an increased time for which breaks are available for rejoining at 3°C. This hypothesis was tested by comparing chromatid aberration frequencies in root meristem cells produced by 105 rads of ⁶⁰Co γ rays, given at dose rates of 19.4 and 0.073 rads per minute. Beans were maintained in aerated water at 2°C. prior to and during irradiation, and at this temperature the rate of development of cells was such that the two different exposure times both occupied a period during which the cell sensitivity was approximately constant. Immediately subsequent to irradiation, the roots were returned to 19°C. and examined cytologically. All chromatid aberrations were less frequent after low dose rate treatment, but only the chromatid interchange reduction was significant. The average time for which breaks are available for reunion, calculated from Lea's G function, was found to be 12 hours (95 per cent C.L. 6 to 24 hours).     

Carbonic anhydrase activity of Prochloron sp. isolated from an ascidian host

The prokaryotic algal symbiont of ascidians, Prochloron sp., was found to exhibit carbonic anhydrase activity which is largely associated with the cell surface. This extracellular carbonic anhydrase activity was inhibited, while the intracellular activity was not affected, by chloride or bromide. Acetazolamide and ethoxyzolamide inhibited carbonic anhydrase activity with I₅₀ values of 7×10^-4 and 3×10^-4M, respectively. These I₅₀ values are similar to those observed for intracellular carbonic anhydrases of Synechococcus sp. PCC7942, Chlamydomonas reinhardii and spinach.Abbreviations AZA acetazolamide - CA carbonic anhydrase - chl chlorophyll - EZA ethozyzolamide - I₅₀ concentration of an inhibitor required to cause 50% inhibition - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - U unit     

Chromosomal distribution of P and I transposable elements in a natural population of Drosophila melanogaster

The chromosomal distribution of P and I transposable elements was studied, by in situ hybridisation, in 25 isofemale lines of Drosophila melanogaster collected at Nasr'Allah in Tunisia. An important interline variability for the number of copies of both elements was revealed. The mean number of copies per line was 31.3 for P and 21.0 for I. Certain chromosome arms had a higher frequency of copies than others: arm 3R had the highest frequency of I elements; the X chromosome had the highest frequency of P elements and the lowest frequency of I elements. For both P and I elements the number of copies on the different chromosome arms is independent. Furthermore, there is no significant correlation between the number of copies of P and the number of copies of I for a given line. A study of the localisation of hybridisation sites on the X chromosome revealed the existence of preferred regions for each family. The population studied was of type M' in the P-M system of hybrid dysgenesis. There is no direct relationship between the M potential of an isofemale line and its number of copies of P elements. These results are compared with those of other investigators and the consequences for cytotype determination are discussed.     

In assessing biological UV-B effects,natural fluctuations of solar radiation should be taken into account

Daily and weekly fluctuations of PAR, UV-A, and UV-B have been continuously monitored for 5 months in Ancient Korinthos, Greece (37°58 N, 23°0 E) using a calibrated instrument based on 3 sharp band sensors. Daily dose ranged between 521–12 006 kJ m^-2 for PAR; 52–1, 239 kJ m^-2 for UV-A; and 0.66–22.5 kJ m² for UV-B. Weekly dose ranged between 16 778-81 788 kJ m^-2 for PAR; 1 406–8 517 kJ m^-2 for UV-A; and 18–151 kJ m^-2 for UV-B. UV-B/PAR and UV-A/PAR ratio distribution, however, does not follow closely PAR fluctuations. Generally, the UV-B/PAR and UV-A/PAR ratios were high in bright light conditions (2.1×10^-3, 118×10^-3) and low in darker weeks (0.9×10^-3, 63×10^-3. The UV-B/UV-A ratio exhibits smaller fluctuations with season (20x1×10^-3, 12×10^-3). Attention is drawn to the effects of sudden changes in ambient radiation and to the ratios of UV-B, UV-A, and PAR.     

Frequency and non random distribution of silver-staining NORs on acrocentric chromosomes from neonate and adult human lymphocytes

The NORs frequency in a group of newborns and adults was determined by the gelatine silver staining technique. A higher number of Ag-NORs (χ² test, p<0.01) was found in adults than in newborns. The lack of correlation between cell proliferating rate index (PRI) and frequency of Ag-NORs let us suppose that the decrease of Ag-positive NORs in neonates could probably be due to factors different from cell kinetics. A non random distribution of Ag-NORs on the acrocentric chromosomes was also demonstrated: chromosome 21, in particular, showed the highest frequency, while chromosome 15, the lowest.     

The repair of double-strand breaks in the nuclear DNA of Saccharomyces cerevisiae and its genetic control

Summary With the use of neutral sucrose sedimentation techniques, the size of unirradiated nuclear DNA and the repair of double-strand breaks induced in it by ionizing radiation have been determined in both wild-type and homozygous rad52 diploids of the yeast Saccharomyces cerevisiae. The number average molecular weight of unirradiated DNA in these experiments is 3.0×10⁸±0.3 Daltons. Double-strand breaks are induced with a frequency of 0.58×10^-10 per Daltonkrad in the range of 25 to 100 krad. Since repair at low doses is observed in wild-type but not homozygous rad52 strains, the corresponding rad52 gene product is concluded to have a role in the repair process. Cycloheximide was also observed to inhibit repair to a limited extent indicating a requirement for protein synthesis. Based on the sensitivity of various mutants and the induction frequency of double-strand breaks, it is concluded that there are 1 to 2 double-strand breaks per lethal event in diploid cells incapable of repairing these breaks.     

Physical properties of the DNA of bacteriophage SP50

Summary The following properties of the DNA of B. subtilis phage SP50 were established: Molecular weight (in Daltons) 102×10⁶ (sedimentation velocity) 97×10⁶ (viscosity) 97×10⁶ (contour lengths of electron micrographs) Base Composition (in % GC) 41.7 (chemical analysis) 44 (melting point) 44 (buoyant density) No unusual bases were observed. The complementary strands of the DNA can be separated. The phage DNA has genuine single strand breaks. The number and distribution of such breaks appears to be determined by the host on which phages were grown.This investigation was supported in part by a Public Health Service research grant GM 13,666 from the National Institutes of General Medical Sciences, AI 01267 from the National Institutes of Allergy and Infectious Diseases, AM 04763 from the National Institutes of Arthritis and Metabolic Diseases; cancer research funds from the University of California; and a grant from the Hartford Foundation.     

Rejoining of gamma-ray-induced DNA damage in Cryptosporidium parvum measured by the comet assay

Cryptosporidium parvum is a well-known waterborne intracellular protozoan that causes severe diarrheal illness in immunocompromised individuals. This organism is highly resistant to harsh environmental conditions and various disinfectants, and it exhibits one of the highest known resistances to gamma irradiation. We investigated rejoining of gamma-ray-induced DNA damage in C. parvum by neutral comet assay. Oocysts were gamma irradiated at various doses (1, 5, 10, and 25 kGy) and were incubated for various periods (6-96 h) after exposure to 10 kGy. The comet tail moment showed that the number of DNA double-strand breaks increased concomitantly with the gamma irradiation dose. When investigating rejoining after irradiation at 10 kGy, double-strand breaks peaked at 6 h postirradiation, and rejoining was highest at 72 h postirradiation. The observed rejoining pattern suggests that repair process occurs slowly even when complex DNA double-strand breaks in C. parvum were induced by high dose irradiation, 10 kGy.     

Seasonal variations in abundance and activity of nitrifiers in four arable cropping systems

Ammonium and nitrite oxidizers were counted with the most probable number (MPN) method and potential ammonium- and nitrite-oxidation rates were determined with a chlorate inhibition technique in an arable soil over a 3-year period. Samples were taken from the topsoil once a month for 2 years and a few times during a third year in four cropping systems: unfertilized lucerne ley and barley, and nitrate fertilized grass ley and barley. The distribution of nitrifiers was determined and their activities measured at various soil depths and between and within plant rows of fertilized barley.The numbers and activities of ammonium oxidizers were highest in the spring and autumn samples. Numbers of ammonium oxidizers ranged from 0.2 to 19×10⁴ and nitrite oxidizers from 3 to 870×10⁴ cells g^–1 dry soil. Potential ammonium-oxidizer activities ranged from 120 to 1,060 and nitrite-oxidizer activities ranged from 280 to 680 ng N g^–1 dry soil hour^–1. Lucerne and grass leys generally showed the highest, whereas unfertilized barley had the lowest, abundances and activities.Abundance estimates and activities were 10–20 times higher in the plow layer than in underlying sand and clay layers. A strong correlation was found between organic matter content vs numbers and activities of both ammonium and nitrite oxidizers. Only nitrite oxidizer counts were significantly higher within plant rows compared to between plant rows.     

Effect of varying the exposure and 3H-thymidine labeling period upon the outcome of the primary hepatocyte DNA repair assay

The results presented in this report demonstrate that an 18–20 hour exposure/³H-thymidine DNA labeling period is superior to a 4 hour incubation interval for general genotoxicity screening studies in the rat primary hepatocyte DNA repair assay. When DNA damaging agents which give rise to bulky-type DNA base adducts such as 2-acetylaminofluorene, aflatoxin Bi and benzidine were evaluated, little or no difference was observed between the 4 hour or an 18–20 hour exposure/labeling period. Similar results were also noted for the DNA ethylating agent diethylnitrosamine. However, when DNA damaging chemicals which produce a broader spectrum of DNA lesions were studied, differences in the amount of DNA repair as determined by autoradiographic analysis did occur. Methyl methanesulfonate and dimethylnitrosamine induced repairable DNA damage that was detected at lower dose levels with the 18–20 hour exposure/labeling period. Similar results were also observed for the DNA cross-linking agents, mitomycin C and nitrogen mustard. Ethyl methanesulfonate produced only a marginal amount of DNA repair in primary hepatocytes up to a dose level of 10^–3M during the 4 hour incubation period, whereas a substantial amount of DNA repair was detectable at a dose level of 2.5 × 10^–4M when the 18–20 hour exposure/labeling period was employed. The DNA alkylating agent 4-nitroquinoline-1-oxide, which creates DNA base adducts that are slowly removed from mammalian cell DNA, induced no detectable DNA repair in hepatocytes up to a toxic dose level of 2 × 10^–5M with the 4 hour exposure period, whereas a marked DNA repair response was observed at 10^–5M when the 18–20 hour exposure/labeling period was used.Abbreviations 2AAF 2-acetylaminofluorene - AB₁ aflatoxin B₁ - BENZ benzidine - DEB diepoxybutane - DEN diethylnitrosamine - DMN dimethylnitrosamine - EMS ethyl methanesulfonate - MITC mitomycin C - MMS methyl methanesulfonate - NG mean net nuclear grain counts - NM nitrogen mustard - 4NQO 4-nitroquinoline-N-oxide     

Interference in DNA Replication Can Cause Mitotic Chromosomal Breakage Unassociated with Double-Strand Breaks

Morphological analysis of mitotic chromosomes is used to detect mutagenic chemical compounds and to estimate the dose of ionizing radiation to be administered. It has long been believed that chromosomal breaks are always associated with double-strand breaks (DSBs). We here provide compelling evidence against this canonical theory. We employed a genetic approach using two cell lines, chicken DT40 and human Nalm-6. We measured the number of chromosomal breaks induced by three replication-blocking agents (aphidicolin, 5-fluorouracil, and hydroxyurea) in DSB-repair-proficient wild-type cells and cells deficient in both homologous recombination and nonhomologous end-joining (the two major DSB-repair pathways). Exposure of cells to the three replication-blocking agents for at least two cell cycles resulted in comparable numbers of chromosomal breaks for RAD54^−/−/KU70^−/− DT40 clones and wild-type cells. Likewise, the numbers of chromosomal breaks induced in RAD54^−/−/LIG4^−/− Nalm-6 clones and wild-type cells were also comparable. These data indicate that the replication-blocking agents can cause chromosomal breaks unassociated with DSBs. In contrast with DSB-repair-deficient cells, chicken DT40 cells deficient in PIF1 or ATRIP, which molecules contribute to the completion of DNA replication, displayed higher numbers of mitotic chromosomal breaks induced by aphidicolin than did wild-type cells, suggesting that single-strand gaps left unreplicated may result in mitotic chromosomal breaks.