The effect of extended anaerobic treatments on the chromosomes of Vicia faba

The effects of extended anaerobic treatments on Vicia faba lateral root-tip chromosomes were determined. It was observed that aberrations resulted from these treatments, and that the frequency varied from root to root as well as from experiment to experiment. It was suggested that the inconsistency observed might be due to variation in the abilities of different roots to produce energy via fermentation routes. If this were true, an inhibition of fermentation would result in a more consistent aberration frequency. A fermentation inhibitor, NaF, was used in combination with extended anaerobic treatments. The observed frequency of aberrations after the combined treatments was generally higher and considerably less variable. Although other hypotheses might account for the NaF effect, the hypothesis most compatible with the evidence is that the effect is due to energy deprivation. The experimental results are discussed in terms of the aforementioned effect and in terms of three alternative hypotheses for the production of chromosomal aberrations as a consequence of a lack of energy. It is concluded that damage might result from a build-up of normal cellular compounds to abnormally high concentrations which would act directly or indirectly on the chromosomes, from the breakdown of DNA as an energy source, or simply as a result of the fact that the chromosome needs energy to remain intact.     

Effects of caffeine on chromosome aberrations and sister-chromatid exchanges induced by mitomycin C in BrdU-labeled human chromosomes.

The BrdU-Hoechst staining technique has been used in analyzing the effect of caffeine (CAF) on chromosome aberrations and sister-chromatid exchanges (SCEs) induced by mitomycin C (MC). CAF increased the frequency of SCE in MC-treated chromosomes in all specimens. The combination of MC and CAF caused a remarkable increase in all types of chromosome aberrations, but the most startling effect was the appearance of many cells with multiple aberrations (shattered chromosomes). The BrdU-Hoechst technique showed that the shattered chromosomes did not appear in cells that had replicated only once, but did occur in cells which replicated twice in the presence of MC and CAF. The large majority of chromatid breaks observed did not involve areas common to SCE; and the SCE frequency significantly increased in spite of the existence of multiple breaks. This indicates that very few of the breaks are incomplete exchanges and that the mechanism for formation of SCE might be different from that of chromosome breaks. In another experiment, monofunctional-MC (M-MC) had a small effect on SCE rates, though it induced shattered chromosomes with CAF post-treatment. Possible differences in the mechanisms leading to SCE and chromosome breaks are discussed.     

On the induction of chromosome structural changes by hydroxylamine in Vicia faba

Primary roots of a new karyotype of Vicia faba with all chromosomes inter-distinguishable have been used to study the induction by hydroxylamine hydrochloride (HA) of chromatid aberrations and their intrachromosomal distribution. HA induced both chromatid intra- and interchanges of the delayed type. The effectiveness of HA increased with increasing temperature and was dependent on the pH during treatment (more aberrations at pH 7.5 as compared with 4.8). The frequency of incomplete reunion was markedly higher after HA treatment than after treatment with maleic hydrazide (MH) or ethanol. In combined treatments, HA reduced the reunion involvement in HA-induced aberrations of certain chromosome segments was found and compared with distribution patterns of chromatid aberrations after treatment with MH and ethanol. Data and hypotheses concerning possible modes of action of HA eventually resulting in chromosome structural changes are discussed. It is concluded that alterations of the cytosine moiety in chromosomal DNA are not responsible for chromosomal damage induced by HA.     

The "protective" effect of gamma-irradiation of cells in metaphase of mitosis following V-irradiation during the S period]

20,1% cells with chromosomes aberrations were obtained after UV-irradiation of embryonal fibroblasts of mice at the S-stage in vitro at a decreasing dose of 40erg/mm2. Subsequent gamma-irradiation at the metaphase of the first mitosis at a 5 krad dose led to a statistically significant decrease of the frequency of aberrant cells observed in the same mitosis down to 11,7%. The frequency of spontaneous aberrations did not change during the first few minutes after gamma-irradiation of intact cells at the metaphase. The "protective" effect of gamma-rays can not be explained either by unequal changes of the duration of mitotic stages for aberrant and normal cells, or by sticking of chromosome fragments or by breaks of bridges at the anaphase. The death of cells "under irradiation" also appears to be a hardly probable case of the effect observed. It is assumed that the decrease of the aberrations frequency is the result of predicted earlier modification of the processes of realization of potential chromosome damages into visible aberrations at the metaphase.     

Analysis of bleomycin- and cytosine arabinoside-induced chromosome aberrations involving chromosomes 1 and 4 by painting FISH

The genomic frequency of chromosomal aberrations obtained by chromosome painting is usually extrapolated from the observed frequency of aberrations by correcting for the DNA content of the labelled chromosomes. This extrapolation is based upon the assumption of random distribution of breakpoints from which aberrations are generated. However, the validity of this assumption has been widely questioned. While extensive investigations have been performed with ionizing radiation as chromosome breaking agent, little efforts have been done with chemical clastogens. In order to investigate interchromosomal differences in chemically-induced chromosome damage, we have used multicolour chromosome painting to analyse bleomycin-induced aberrations involving chromosomes 1 and 4, two chromosomes that differ in gene density. In addition, we have measured the effect of cytosine arabinoside upon the repair of bleomycin-induced DNA damage in chromosomes 1 and 4. Our results show that these chromosomes are equally sensitive to the clastogenic effect of bleomycin with a similar linear dose-effect relationship. However, the high gene density chromosome 1 appeared to be more sensitive to repair inhibition by Ara-C than chromosome 4. This enhanced sensitivity to repair inhibition in chromosome 1 could be mediated by preferential repair of open chromatin and actively transcribed regions.     

cis-Dichlorodiammine platinum(II) induced aberrations in mouse bone-marrow chromosomes

The clastogenic effect of cis-dichlorodiammine platinum(II) (cis-platin) on mouse bone-marrow chromosomes has been studied. Cis-platin was injected at 3 different doses. Cells were fixed at different time intervals after treatment. Different types of aberrations together with the percent of mitotic index and frequency of abnormal metaphases were studied. The aberrations observed were primarily chromatid breaks, although isochromatid breaks, interchanges, and multiple breaks were also observed. A dose- and time-dependent effect was observed for both inhibition of mitotic index and frequency of abnormal metaphases. Trypsin-Giemsa staining of bone-marrow metaphase chromosomes from normal mice was compared with the bands of metaphase chromosomes obtained after Giemsa staining of chromosomes from platinum-treated mice and they were observed to be identical. Bands were present up to 120 h and aberrations were also induced in such plates.     

Mutagens induced chromosomal damage in <Emphasis Type="Italic">Lablab purpureus</Emphasis> (L.) Sweet var. <Emphasis Type="Italic">typicus</Emphasis>

Cytological analysis with respect to meiotic behaviour is considered to be the one of the most dependable indices to estimate the potency of mutagens and to elucidate the response of various genotypes to a particular mutagen. Seeds of Lablab purpureus (L.) Sweet var. typicus cv. CO(Gb)14 were subjected to different doses/concentrations of gamma rays and EMS. The effects of different mutagenic treatments on meiosis were studied on treated and control plants. Various types of meiotic aberrations such as stickiness, clumping of chromosomes, laggards, ring chromosomes and precocious movements were observed in the mutagenic treatments. As increase in the concentration, the frequency of cells showing chromosomal aberrations shows a linear increase up to a certain level. However, the EMS treatments proved to be more effective in inducing meiotic aberrations as compared to gamma rays.     

Mechanism of formation of chromatid exchanges

Chinese hamster cells with different patterns of distribution of 5-bromodeoxyuridine (BrdUrd) between chromosome subunits were subjected, during the G2 stage, to UV irradiation, which only produced breaks in BrdUrd substituted DNA. The frequency of chromatid and subchromatid interchanges as well as isochromatid aberrations was estimated. It was found that only BrdUrd containing chromatids were involved into aberrations; this result challenges the so called "molecular theory" for aberration production proposed by Leenhouts and Chadwick. A very small increase of the aberration yield in chromosomes without BrdUrd may be connected with the action of UV on the frequency of recombination. The observed frequency of interchanges was not proportional to the BrdUrd content in chromosomes and depended on the time of its incorporation: more exchanges were induced in the chromatids incorporating BrdUrd during the last round of replication. These regularities may be connected with some molecular peculiarities of chromosome structure and function.     

Sodium fluoride-induced chromosome aberrations in different stages of the cell cycle: a proposed mechanism

In an attempt to clarify the controversy about sodium fluoride (NaF) clastogenicity, the induction of chromosome aberrations in Chinese hamster ovary cells (CHO) by NaF was investigated. Following a protocol used for screening chemicals for clastogenic activity, significant increases of aberrant cells were observed when cells were exposed to NaF for 4 h and harvested 8 h later. Cell-cycle kinetic studies demonstrated most cells were exposed in G2 of the cell cycle. Smaller increases in aberrant cells were observed when cells were harvested 20 h later (most cells were exposed in G1/S). The sensitivity of G2 cells to NaF was investigated further, along with the induction of aberrations at low doses. The results indicated that G2 cells are sensitive to NaF and the percent of aberrant cells increased with dose and length of exposure. With a 3-h exposure until harvest, no statistically significant increase in aberrant cells was observed at doses below 10 micrograms/ml NaF. These data are consistent with a threshold for NaF-induced clastogenicity around 10 micrograms/ml, as has been proposed previously (Scott and Roberts, 1987). It thus may be predicted that clastogenic effects would not occur in humans exposed to the levels of fluoride that are present in drinking water or dentifrices. An understanding of the mechanism of NaF-induced clastogenicity would help to clarify this point. It has previously been reported that NaF inhibits DNA synthesis/repair. The types of aberrations, mostly deletions and gaps, the induction of endoreduplicated cells, the cell-cycle delay and the sensitivity of G2 cells to NaF observed are similar to that reported in the literature for DNA synthesis/repair inhibitors like aphidicolin (APC). Similarities in the induction of aberrations by NaF and APC were confirmed in experiments with G2 cells. Based on these results and those previously reported for NaF and APC, it is proposed that NaF-induced aberrations may occur by an indirect mechanism involving the inhibition of DNA synthesis/repair.     

Influence of hydrogen-consuming bacteria on cellulose degradation by anaerobic fungi

The presence of methanogens Methanobacterium arboriphilus, Methanobacterium bryantii, or Methanobrevibacter smithii increased the level of cellulose fermentation by 5 to 10% in cultures of several genera of anaerobic fungi. When Neocallimastix sp. strain L2 was grown in coculture with methanogens the rate of cellulose fermentation also increased relative to that for pure cultures of the fungus. Methanogens caused a shift in the fermentation products to more acetate and less lactate, succinate, and ethanol. Formate transfer in cocultures of anaerobic fungi and M. smithii did not result in further stimulation of cellulolysis above the level caused by H2 transfer. When Selenomonas ruminatium was used as a H2-consuming organism in coculture with Neocallimastix sp. strain L2, both the rate and level of cellulolysis increased. The observed influence of the presence of methanogens is interpreted to indicate a shift of electrons from the formation of electron sink carbon products to H2 via reduced pyridine nucleotides, favoring the production of additional acetate and probably ATP. It is not known how S. ruminantium exerts its influence. It might result from a lowered production of electron sink products by the fungus, from consumption of electron sink products or H2 by S. ruminantium, or from competition for free sugars which in pure culture could exert an inhibiting effect on cellulolysis.     

Influence of hydrogen-consuming bacteria on cellulose degradation by anaerobic fungi.

The presence of methanogens Methanobacterium arboriphilus, Methanobacterium bryantii, or Methanobrevibacter smithii increased the level of cellulose fermentation by 5 to 10% in cultures of several genera of anaerobic fungi. When Neocallimastix sp. strain L2 was grown in coculture with methanogens the rate of cellulose fermentation also increased relative to that for pure cultures of the fungus. Methanogens caused a shift in the fermentation products to more acetate and less lactate, succinate, and ethanol. Formate transfer in cocultures of anaerobic fungi and M. smithii did not result in further stimulation of cellulolysis above the level caused by H2 transfer. When Selenomonas ruminatium was used as a H2-consuming organism in coculture with Neocallimastix sp. strain L2, both the rate and level of cellulolysis increased. The observed influence of the presence of methanogens is interpreted to indicate a shift of electrons from the formation of electron sink carbon products to H2 via reduced pyridine nucleotides, favoring the production of additional acetate and probably ATP. It is not known how S. ruminantium exerts its influence. It might result from a lowered production of electron sink products by the fungus, from consumption of electron sink products or H2 by S. ruminantium, or from competition for free sugars which in pure culture could exert an inhibiting effect on cellulolysis.     

Chromosomal aberrations in Crepis capillaris cells detected by FISH

Crepis capillaris (2n=6) is an excellent plant for the assay of chromosome aberrations after mutagenic treatment. It has simple karyotype: three pairs of morphologically distinct and relatively large chromosomes. The frequency of structural chromosome aberrations and micronuclei in root meristem cells has been used for evaluation of the genotoxicity of chemicals and environmental pollutants. The introduction of fluorescence in situ hybridization method allows more detailed detection and localization of chromosomal rearrangements not only in mitotic but also in interphase nuclei. We demonstrate a few examples of the detection of chromosomal aberrations using rDNA and telomeric sequences as probes for in situ hybridization to C. capillaris chromosomes.     

Evaluation of cytological effects of Zn2+ in relation to germination and root growth of Nigella sativa L. and Triticum aestivum L

The effects of different treatments with zinc sulfate (Zn(2+)) on the cytology and growth of Nigella sativa and Triticum aestivum were investigated. Five concentrations of zinc sulfate ranging from 5 to 25mg/l were applied for 6, 12, 18, and 24h. The treatments reduced the germination percentages of N. sativa seeds and T. aestivum grains and inhibited the root growth of both plants. Concentrations higher than 25mg/l of Zn(2+) applied for 24h were toxic for both plants. The non-lethal concentrations of Zn(2+) showed an inhibitory effect on cell division in root tips of both plants and caused a decrease in their mitotic index values. The reduction in MI in root tips of T. aestivum was more evident than that of N. sativa. All treatments changed the frequency of mitotic phases as compared with the control values. The total percentage of abnormalities in N. saliva was more than that in T. aestivum. Zn(2+) treatments produced a number of mitotic abnormalities in dividing cells in root tips of both plants resulting from its action on the spindle apparatus such as C-metaphases, lagging chromosomes and multipolar anaphases and telophases. Also, Zn(2+) induced vacuolated nuclei and irregular prophases. The induction of chromosomal stickiness and chromosomal aberrations such as bridges and breaks indicates its action on the chromosome. These abnormalities (chromosome breaks and chromosomal bridges at ana-telophases) indicate true clastogenic potential of the ions tested.     

Microdosimetry and chromosome aberrations: Effects of 230 keV neutrons on Vicia faba chromosomes

Physical energy deposition events have been related to sub-nuclear cytological events (chromosomal changes) in metaphases sequentially accumulated from the latter part of the cell cycle of Vicia faba. 230 keV neutrons produce about 0.4 recoil protons per late interphase nucleus per rad with the majority of protons travelling 1 to 2 microns from their origin, depositing energy at around 90 keV per micron. The frequency of induced aberrations is basically linear with dose, though varying through consecutive cell sampling periods because of differential induced mitotic delay. Distributions of chromosomal aberrations and total cytological events are overdispersed in relation to the Poisson distribuyion indicating that some proton recoils produce multiple events. When gaps and aberrations within chromosomes and multiple aberrations between chromosomes, are considered as discrete events, distributions follow Poisson expectations. About 40% of proton recoils result in observable cytological change. The highly energetic proton recoils (～90 keV per micron) which can induce multiple events are the ones most likely to produce effects which result in cell death. The sphere of influence of the proton recoils is probably adequately estimated from their range (～1 to 2 μm) since it seems compatible with the spatial proximity of the initial components of the resultant chromosome aberrations.     

Radiobiological effects of a low-energy ion beam on wheat

The radiobiological effects of a low-energy nitrogen ion (N⁺) beam on wheat were studied, particularly with regard to the induction of chromosome aberrations. The results demonstrated that the three test varieties showed different sensitivities to ion implantation, and a higher dose of ion implantation had a marked effect on the germination and survival rate of the seeds exposed. The germination rate and survival rate curve basically followed a similar trend in the same variety. Cytological analysis indicated that ion beams were effective in producing chromosome aberrations. The frequencies of mitotic or meiotic cells with chromosome aberrations increased linearly with increasing doses. The aberration types included, for example, acentric fragments, chromosome deletions, lagging chromosomes, chromosome bridges and micronuclei. In the root tip cells, aberrations chiefly consisted of acentric fragments and deletions. Chromosome bridges and lagging chromosomes were the main aberration phenomena observed in the pollen mother cells. The highest frequencies of root tip cells and pollen mother cells with chromosome aberrations were 15.2% and 39.8%, respectively. Changes in morphology and mutant were also observed in the plants derived from exposed seeds. Received: 10 April 2000 / Accepted: 10 October 2000     

Chromosome damage in Chinese hamster cells sensitized to near-ultraviolet light by psoralen and angelicin

The clastogenic effect of furocoumarins psoralen and angelicin in the presence of near-UV (320-380 nm) differs greatly, as do their modes of interaction with DNA. Psoralen, which requires only one-fifth as much light energy to produce the same lethal effect as angelicin at equimolar concentrations, is able to cross-link DNA whereas angelicin cannot. The frequency of micronuclei which arise from chromosomal fragments shows the same differential effect as lethality. Indeed aberrations account for much or all of the lethality observed. Metaphase analysis at comparable aberration frequencies revealed that angelicin and psoralen both induce chromatid deletions and a wide spectrum of chromatid exchanges. These data show that both cross-links and monoadducts to the DNA can result in chromosomal aberrations. The relative contributions of cross-links and monoadducts to chromosomal aberrations still remain to be determined. It is noteworthy that extensive chromosomal damage is induced in mammalian cells by the combination of psoralen and near-UV, a treatment which is currently widely used in the therapy of psoriasis.     

The chromosomal radiosensitivity of children whose parents were exposed to antitumor radiochemotherapy]

The effect of gamma radiation was studied on routine stained chromosomes from lymphocytes of children born to Hodgkin's disease patients after cancer therapy (CP) in comparison to children from healthy parents (HP). Irradiation (0, 0.25, 0.50, 1.00, 1.50 Gy) of the whole blood was performed in culture medium. Metaphases were obtained from 52-h cultures. Chromosomal aberrations were used as an endpoint. Aberrations of both chromosomal and chromatid types were scored in 150-200 metaphases for estimation of spontaneous level of cytogenetic injuries and in 100 metaphases of induced one. It is found that chromosomes of CP children are more radiosensitive than chromosomes of HP ones, the spontaneous frequency of chromosome aberrations being equal in both groups.     

The effect of lead on Allium cepa L.

The effect of lead on Allium cepa L. at concentrations of 0.1, 1.0, 10, 50, 100 and 200 ppm were studied. Analysis focused on root growth, frequency of mitosis in a meristematic zone, and chromosomal aberrations. It was observed that lead reduces root growth and the frequency of mitotic cells in meristematic zones, and increases the frequency of aberrant cells. The intensity of the effects is a function of lead concentration.     

Comparison of the effects of radiation and deoxyribonucleic acids on the mitosis in roots ofVicia faba

Differences as well as similarities in the action of ionizing radiation and deoxyribonucleic acids from various sources on mitosis in root cells ofVicia faba were established. The time course of occurrence of aberrations were examined. Whereas in irradiated broad beant the maximum percentage of aberrations was observed immediately after irradiation, the aps plication of non-isologous DNA was followed by maximum aberrations after 8–16 hours. As all the time-intervals studied, an incraasad number of aberrations was found during metaphase-as compared with anaphases, both after irradiation and after application of DNA. A comparison of isologous, homologous and heterologous DNA as inductors of chromosomal aberrations supported our previous findings and showed that the efficiency of DNA depends on the genetic difference between donor and acceptor. During a study of distribution of aberrations between large and small chromosomes of meristematic cells ofVicia faba, at various time-intervals it was obsarved that after irradiation the distribution of aberrations between individual chromosomes is proportional to their total length, whereas the effect of heterologous DNA is mostly in the damage to small chromosomes. It was also found that aftar irradiation mostly chromatid aberrations are formed at shorter time-intervals and only later chromosomal aberrations will appear. On the other hand, heterologous DNA brings about in all time-intervals a predominance of chromatid aberrations.