Effects of G2 treatments with inhibitors of DNA synthesis and repair on chromosome damage induced by X-rays and chemical clastogens in root tips of Vicia faba. Comparison with corresponding effects in cultured human lymphocytes

The frequencies of chromatid aberrations produced in roots of Vicia faba by clastogenic (chromosome-damaging) agents were strongly enhanced by exposing the root-tip cells to inhibitors of DNA synthesis during the G2 phase. Chromosome damage produced by both S-dependent (maleic hydrazide, methyl methanesulfonate, thio-TEPA) and S-independent (X-rays, streptonigrin) mechanisms was enhanced by the inhibitor treatments. The types of aberrations affected by the inhibitors were mainly chromatid gaps and breaks and isochromatid breaks of the non-union type. Most effective among the inhibitors tested were hydroxyurea (HU) and 5-fluorodeoxyuridine (FdUrd). Post-treatments with caffeine were effective in enhancing clastogen-induced chromosome damage when given during the S phase. All types of aberrations, exchanges as well as breaks, were enhanced by the post-treatments. When given during the G2 phase, caffeine enhanced only the frequency of chromatid aberrations produced by X-rays. The enhancement was slight and obtained only when the cells were irradiated in the G2 phase and immediately post-treated with caffeine. Clastogen-treated cultures of human lymphocytes responded to post-treatments with inhibitors of DNA synthesis in very much the same way as clastogen-treated root-tip cells of Vicia faba. Thus, the frequencies of chromatid gaps and breaks and isochromatid breaks of the non-union type were strongly enhanced by exposing clastogen-treated lymphocytes to inhibitors of DNA synthesis during the G2 phase. The efficiency of the inhibitors, however, varied considerably in the two materials. On the whole, the number of inhibitors capable of enhancing induced chromosome damage was much larger in lymphocytes than in bean root tips. Only HU was equally effective in both materials. The most striking difference between the two materials was found when caffeine was given as a post-treatment. Thus, in human lymphocytes the frequencies of chromatid aberrations induced by most clastogenic agents were strongly enhanced when caffeine was given during the G2 phase, but little affected by post-treatments with caffeine during the S phase.     

Sister chromatid exchanges in Vicia faba

The relationship between chromosomal aberrations and sister chromatid exchanges (SCE's) after treatment of Vicia faba root tips with thiotepa, caffeine and 8-ethoxycaffeine (EOC) was studied by using a modified fluorescent plus Giemsa (EPG) technique. At concentrations which had little effect on the frequency of chromosomal aberrations, thiotepa strongly increased the frequency of SCE's, provided that the chromosomes were allowed to replicate between treatment and fixation. Frequently, the size of the exchanged material was smaller than the diameter of the chromatid. Post-treatments with caffeine of roots previously exposed to thiotepa strongly increased the frequency of aberrations, but had little effect on the frequency of SCE's. In contrast to thiotepa, EOC caused only a slight increase in the frequency of SCE's even at concentrations which produced a high frequency of chromosomal aberrations. Thus, there was not a close correlation between SCE's and chromosomal aberrations. Single-strand exchanges between the DNA double helices in sister chromatids were not detected.     

铅、镉及其复合污染对蚕豆根尖细胞的诱变效应

应用蚕豆根尖微核技术研究了Cd(1mgL^-1～25mgL^-1)、Pb(1mgL^-1～25mgL^-1)及其复合污染对根尖细胞的毒害作用,分析了重金属及复合重金属污染对蚕豆根尖细胞有丝分裂和微核及染色体畸变的影响。结果表明：Cd、Pb及其复合处理对蚕豆根尖细胞有丝分裂及染色体行为存在不同程度的影响。具体表现为：低浓度促进细胞分裂,高浓度则抑制细胞分裂;对染色体畸变的影响表现为：当浓度为1．0mgL^-1时Cd即产生显著效应,而Pb浓度达10．0mgL^-1时,表现出稳定的,强度较大的诱变效应,且随浓度的增加毒害增强。Cd、Pb间存在交互作用,Cd达低浓度时与Pb的交互作用明显而高浓度则不明显,且Cd、Pb间存在部分拮抗作用。     

Enhancement and reduction by methylated oxypurines of the frequencies of chromatid aberrations induced by camptothecin in root-tip cells of Vicia faba.

In root-tip cells of Vicia faba the frequencies of chromatid aberrations induced by 3-h treatments with 0.05 microM camptothecin were strongly modified when the treatments were carried out in the presence of caffeine at concentrations above 1 mM. Depending on the concentration of caffeine, the clastogenic effect of camptothecin was either enhanced or reduced. At concentrations between 1 and 6 mM, caffeine increased the camptothecin-induced chromosome damage, the strongest enhancement being obtained at 5 mM. A reduction of the chromosome damage was apparent at caffeine concentrations above 10 mM, and in the presence of 20 mM caffeine the clastogenic effect of camptothecin was almost completely suppressed. When present during the camptothecin treatment, theophylline, 8-chlorocaffeine and 1,3,7,9-tetramethyluric acid influenced the induced chromosome damage in a similar way as caffeine, although with varying efficiency. If the concentrations required to produce the two types of modifying effect are used as a criterion, 8-chlorocaffeine was the most effective and 1,3,7,9-tetramethyluric acid the least, whereas caffeine and theophylline were about equally effective.     

Influence of caffeine and 3-aminobenzamide in G2 on the frequency of chromosomal aberrations induced by thiotepa,mitomycin C and N-methyl-N-nitro-N′-nitrosoguanidine in human lymphocytes

The effects of post-treatments with caffeine in G₂ on the frequency of chromosomal aberrations induced by thiotepa, mitomycin C and N-methyl-N-nitro-N′-nitrosoguanidine were studied in human lymphocytes. Caffeine was found to potentiate the frequency of chromatid aberrations induced by all 3 S-dependent agents tested; the most striking enhancement being obtained when caffeine was present during the last 1.5 h before harvesting. Post-treatments in G₂ with 3-aminobenzamide had no influence on the aberration frequency induced by thiotepa and N-methyl-N-nitro-N′-nitrosoguanidine.     

SO2衍生物诱发蚕豆根尖细胞微核和后期异常的研究

研究SO₂体内衍生物--亚硫酸钠和亚硫酸氢钠混合液(3:1 mmol·L^-1/mmol·L^-1)诱发蚕豆根尖细胞微核和后期异常的效应。结果表明:SO₂衍生物处理可诱发蚕豆根尖间期细胞微核和核芽,使分裂后期出现多种染色体异常,如断片、桥以及滞后染色体等。异常细胞中以微核细胞和染色体断裂细胞居多。在一定浓度范围内,细胞异常率与处理液浓度之间表现正的线性相关。这些研究结果表明,蚕豆根尖间期微核和后期染色体异常有可能用作检测SO₂污染的生物剂量计。     

Chromosome aberrations caused by the effect of ultrasound in the meristematic cells ofvicia faba

In our present work the formation of chromosome aberrations has been studied in dependence on the tima interval between sonication and fixation of the primary root tips of Vicia faba. Maximum occurrence of aberrations was recorded immediately after sonication. The results of our experiments pointed to the fact that the frequency of the induced changes was independent on the sonic waves intensity within the range of 0-2—3-0 W/cm² and on ultrasond treatment duration within the range of 1—20 min. Studies of the distribution of chromosome abnormalities caused by ultrasound between the large and small chromosomes of theVicia faba meristematic cells in various time intervals showed that the frequency of the aberrations in both chromosome groups was proportional to its total metaphase lengths. Analysis of the type of aberrations observed in various time intervals after sonication indicated the simultaneous formation of chromosome and chromatide abnormalities.     

EDTA对蚕豆根尖细胞微核的诱变作用

为探讨EDTA对高等植物细胞的诱变作用 ,以及验证EDTA对金属离子的螯合作用 ,本试验用不同浓度的EDTA处理蚕豆根尖细胞 ,进行微核试验 ,并以Pb(NO3 ) 2 作为阳性对照 ,以无离子水作为阴性对照 ,发现EDTA能使蚕豆根尖细胞微核率有明显增加。由此证明EDTA对高等植物细胞有一定的诱变作用     

稀土元素钬对蚕豆的细胞毒性和遗传毒性研究

运用氧化钬与稀硝酸反应制备结晶,以去离子水溶解并且稀释成梯度溶液,对蚕豆根尖染毒6 h,分别修复培养22h和24h,观察根尖变化,统计微核率、染色体畸变率及有丝分裂指数。结果表明,4mg/L（以氧化钬质量体积浓度计）以下剂量对根尖生长具有促进作用;随着浓度的递增,微核率、染色体畸变率逐步上升,有丝分裂指数逐步下降,表现出明显的剂量-效应关系,说明稀土元素钬具有一定的细胞毒性和遗传毒性。同时,不同修复组在微核率、染色体畸变率及有丝分裂指数上也存在一定差异,表现为微核率22h修复组低于24 h 修复组,而染色体畸变率和分裂指数均高于24h修复组。微核检测应在染色体畸变检测之后进行。      

Induction of numerical chromosomal aberrations during DNA synthesis using the fungicides nimrod and rubigan-4 in root tips of Vicia faba L.

The 2 fungicides nimrod and rubigan-4 were tested for genotoxicity using Vicia faba root tips as the biological test system. Treating lateral roots with different concentrations of each fungicide for different periods showed that both fungicides were able to produce numerical but not structural chromosomal aberrations. The percentage of total aberrations in root tips exposed to nimrod reached 54.39% at 250 ppm for 4 h, and 64.69% in root tips exposed to rubigan-4 at 250 ppm for 6 h. The types of numerical chromosomal aberrations produced by both fungicides included: binucleate cells, c-metaphases, sticky chromosomes, polyploid cells, and laggards. Recovery experiments for 24, 48, and 96 h showed no significant differences between the percentage of total aberrations in treated and control groups.     

Clastogenic and mitodepressive effects of the insecticide dichlorvos on root meristems of<Emphasis Type="Italic">Vicia faba</Emphasis>

Plant bioassays are an important and integral part of the test battery used in detecting genotoxic/carcinogenic contamination in the environment. Highly sensitive biomonitoring of plant models have been developed, which enables the detection of hazards arising from pesticides, insecticides, industrial contamination, heavy metals and radiation. Root tips of Vicia faba ssp. minor were treated with 1-60 mM of the organophosphorus insecticide dichlorvos (DDVP) for 2 h, followed by a 20-h recovery period. Maleic acid hydrazide (MH) was used as a positive control for the mitotic index, micronucleus and chromosomal aberration assays performed on the Vicia model system. All treatments with DDVP significantly decreased the mitotic activity and increased the frequency of chromosomal aberrations at the metaphase. The frequency of micronuclei was significantly increased at DDVP concentrations starting from 10 mM. The results demonstrate clastogenic and mitodepressive effects of DDVP on Vicia faba cells.     

Inducing somatic meiosis-like reduction at high frequency by caffeine in root-tip cells of Vicia faba

Germinated seeds of Vicia faba were treated in caffeine solutions of different concentration for different durations to establish the inducing system of somatic meiosis-like reduction. The highest frequency of somatic meiosis-like reduction could reach up to 54.0% by treating the root tips in 70 mmol/l caffeine solution for 2 h and restoring for 24 h. Two types of somatic meiosis-like reduction were observed. One was reductional grouping, in which the chromosomes in a cell usually separated into two groups, and the role of spindle fibers did not show. The other type was somatic meiosis, which was analogous to meiosis presenting in gametogenesis, and chromosome pairing and chiasmata were visualized.     

Exposure of Vicia faba and Pisum sativum to copper-induced genotoxicity

The potential genotoxicity of Cu(2+) was investigated in Vicia faba and Pisum sativum seedlings in hydroponic culture conditions. Cu(2+) caused a dose-dependent increase in micronuclei frequencies in both plant models. Cytological analysis of root tips cells showed clastogenic and aneugenic effects of this heavy metal on V. faba root meristems. Cu(2+) induced chromosomal alterations at the lowest concentration used (2.5 mM) when incubated for 42 h, indicating the potent mutagenic effect of this ion. A spectrum of chromosomal abnormalities was observed in V. faba root meristems, illustrating the genotoxic events leading to micronuclei formation.     

表面活性剂、酸雨和Cd^2＋复合污染对蚕豆胚根细胞核的毒性

用蚕豆根尖微核技术研究了Cd^2+单因子以及与表面活性剂、模拟酸雨复合污染时对植物细胞的毒性作用。结果表明,Cd^2+浓度在0～10．0mg·L^-1范围内,对蚕豆胚根细胞微核的形成有强烈的诱导作用,Cd^2+浓度6．0mg·L^-1时的细胞微核率为13．85‰,对照组的微核率为4．53‰,此时污染影响指数(PI)为3．06;当环境中存在表面活性剂LAS1．0mg·L^-1或pH值降到4．5和3．5时,同-Cd^2+浓度下,蚕豆根尖细胞微核率、PI降低,同时伴有核变形,细胞中颗粒物增多,胚根组织不容易分散等症状,根的生长受到抑制,说明表面活性剂、酸雨对Cd^2+的毒性有协同作用。pH3．5的酸雨环境中Cd^2+对蚕豆细胞的损伤程度比pH4．5酸雨环境高,在检测高浓度、强毒性污染物的致突变效应时,应作至少3个稀释倍数,找出蚕豆根尖细胞最高微核率及PI。     

In vivo and in vitro promutagen activation by Vicia faba of thiocarbamate herbicides molinate and butylate to products inducing sister chromatid exchanges in human lymphocyte cultures

Molinate and butylate treatments for 4 h of Vicia faba root tip meristems, showed that both thiocarbamate herbicides increased significantly SCE frequency. Direct treatments of molinate and butylate on human lymphocytes applied 24 h after the beginning of culture did not induce SCE. When S10 extracts of the Vicia roots, treated for 4 h with molinate and butylate (in vivo activation) were added to lymphocytes (24 h after of the beginning of culture), SCE were induced in a concentration-response manner. The in vitro assays, in which molinate and butylate was added at 48 h lymphocyte cultures for 4 h, showed a negative response, however, in the treatment where the S10 metabolic mix was added the SCE frequencies were significantly different to the control, and the concentration-response relationship was not observed with molinate, but it was obtained with butylate. The results showed that both herbicides needed the V. faba metabolism to produce SCE in human lymphocyte culture.     

Clastogenic action of ellipticine over the cell cycle of human lymphocytes and influence of posttreatments with caffeine and ara-C at G2

The clastogenic potential of the intercalating compound ellipticine, an antitumor alkaloid, has been demonstrated in mammalian cells. To characterize the mechanism of action of this drug over the cell cycle, human lymphocyte cultures from 2 healthy donors were treated with 3 micrograms/ml ellipticine in 30-min pulses during different phases of the cell cycle and analyzed for chromosomal aberrations and sister-chromatid exchanges. The G2 phase was most sensitive in terms of induction of aberrations, followed by S and G1. Chromatid-type aberrations were the most common type of chromosomal damage. Induction of SCEs was significantly high only after treatment at G1, when the frequencies of SCEs doubled. The post-treatment effect of lymphocytes with inhibitors of DNA repair, 10(-3) M caffeine and 5 x 10(-6) M 1-beta-D-arabinofuranosylcytosine, was also tested by adding 3 micrograms/ml ellipticine at G2 in 30-min pulses and immediately followed by caffeine and/or ara-C during the last 3 h before harvesting. Three experiments performed on blood from 3 donors showed a moderate potentiation effect on the frequency of chromatid-type aberrations (about 2-3 times) by both inhibitors. Likewise, a 3-fold increase was observed in the frequencies of chromosomal aberrations when caffeine and ara-C were combined. The present data demonstrate that posttreatment with caffeine and ara-C at G2 can modify the response of human lymphocytes treated with ellipticine by increasing the clastogenic action of this compound or by changing the cell-cycle progression.     

A comparative study of the potentiating effect of caffeine and poly-D-lysine on chromosome damage induced by X-rays in plant cells.

X-Ray-induced chromosomal aberrations (CA) were potentiated by post-treatments in G2 with either caffeine (caff) or poly-D-lysine (PDL) in root-tip cells of Allium cepa. The enhancement of the yield of CA was concomitant with an increase in the frequency of mitosis. Our results seem to support the idea of a direct relationship between radiation-induced G2 delay and repair of chromosome damage. Here we report on similarities between caffeine and PDL in both decreasing G2 delay and enhancing chromatid aberration yield. The possible molecular mechanism(s) of action responsible for the cytogenetic effects observed are discussed.     

Effect of BAP and IAA on the expression of G1 and G2 control points and G1-S and G2-M transitions in root meristem cells of Vicia faba

Excised, carbohydrate-starved root meristems of Vicia faba subsp. minor have been used to investigate the impact of the auxin indole-3-acetic acid (IAA) and the cytokinin benzyl-6-aminopurine (BAP) on (1) the expression of Principal Control Points (PCPs) during the G1- and G2-phases of the cell cycle, and (2) the dynamics of sucrose-mediated resumption of DNA replication and mitosis (G1-to-S and G2-to-M transitions). Compared with the excised root tips starved in mineral medium without hormones, stationary phase meristems induced during continuous treatment with BAP, IAA, or a mixture of BAP+IAA, increased the number of G2 cells, producing characteristic profiles of nuclear DNA content. In medium containing 2% sucrose, BAP accelerated PCP1-->S and PCP2-->M, whereas continuous treatment with IAA resulted in marked prolongation of both transitions. Since the PCPs regulate progression through the key events of interphase and mitosis by interacting with cyclin dependent kinases (CDKs), these results seem to correspond with current data indicating functional connections between phytohormones, nutritional signals, gene expression and the cell division cycles in plants.     

Effect of caffeine on the frequency of radiation-induced chromosome aberrations at different stages of the cell cycle

The effect of caffeine on the frequency of chromosome aberrations in human lymphocytes irradiated at different stages of the cell cycle was studied. Caffeine appeared to nearly double the frequency of chromosome aberrations induced by irradiation at S and G2 stages and did not influence the effect of irradiation at G0 and G1 stages.     

Vicia faba chromosome damage induced by low doses of gamma radiation

The rate of radiation damage to chromosomes by low doses of gamma rays (0.01-0.30 Gy) was studied in the root tips ofVicia faba. As criteria of the effect of ionizing radiation, the frequency of sister chromatid exchanges (SCEs), incidence of chromosomal aberrations and the number of micronuclei were evaluated and compared in irradiated cells. The results obtained confirmed that the analysis of SCEs did not represent an efficient indicator of radiation damage to chromosomes. On the contrary, the formation of chromosomal aberrations and micronuclei was effectively stimulated by low radiation doses, there being linear dose-effect relationships in the low doses region used.