浊漳河水体污染物对蚕豆根尖细胞的遗传毒性研究

运用蚕豆根尖细胞微核试验技术对浊漳河南段6个代表性样点水体中有机污染物的遗传毒性进行了研究,为浊漳河流域水体污染状况的有效监测提供理论依据.结果显示:(1)浊漳河南段各监测断面的水体有机污染物对蚕豆根尖细胞均产生了不同程度的损伤,表现出细胞微核、断片、染色体桥等多种异常形态,严重时导致细胞坏死和细胞凋亡.(2)6个样点水样处理的蚕豆根尖细胞微核率在21.47‰～64.77‰,极显著高于对照组(P<0.01).(3)各样点水样对蚕豆根尖细胞的遗传损伤程度依次为:王桥>店上>漳泽水库>北寨>黄碾>五阳;在有丝分裂后期,蚕豆根尖中异常细胞最高达70%,且异常细胞比率有随着污染程度增大而升高的趋势.研究表明,蚕豆根尖细胞微核是水质毒理检测的有效指标,其细胞有丝分裂后期异常细胞比率也可作为水质毒理检测的观察指标.     

Enzymatic adaptations to arsenic-induced oxidative stress in Zea mays and genotoxic effect of arsenic in root tips of Vicia faba and Zea mays

Agronomic plant species may display physiological and biochemical responses to oxidative stress caused by heavy metals and metalloids. Zea mays plants were grown hydroponically for eight days at different concentrations of As (0, 134 and 668 μM) and at different pH (4, 7 and 9). Metabolic variations in response to As toxicity were measured using physiological parameters and antioxidant enzymatic activities. A significant decrease in SOD activity was observed in the leaves and roots of Z. mays with the majority of As treatments. As decreased G-POX activity less in leaves than in roots. An increase in the concentration of As increased APX activity in leaves and roots, except As(V) at pH 4 and pH 9 in the leaves and As(III) at pH 9 in the roots, when there was a significant decrease in APX activity at low As concentrations. After exposure to As(V), CAT activity was the same as in the control. As(III) led to an increase in CAT activity in leaves and to a decrease in roots. With increasing concentrations of As(III), CAT activity increased in both leaves and roots whatever the pH. To obtain more detailed knowledge on the effects of arsenate and arsenite exposure on Vicia faba and Z. mays, root meristems were also examined. Roots were fed hydroponically with 134, 334, 534 and 668 μM arsenate or arsenite and 4 × 10(-3)M of maleic hydrazide as positive control, at three different pH. Physiological parameters, the mitotic index and micronuclei frequencies were evaluated in root meristems. At all three pH, the highest As(V) and As(III) concentrations induced a substantial modification in root colour, increased root thickness with stiffening, and reduced root length. High concentrations also caused a significant decrease in the mitotic index, and micronucleus chromosomic aberrations were observed in the root meristems of both species.     

Induction of chromatid aberrations by TEM and maleic hydrazide is differently affected by pretreatment of Vicia faba root-tip meristems with methyl iodide

Treatment of Vicia faba main root meristems with methyl iodide (MeI) 2 h before challenge treatment with triethylene melamine (TEM) significantly reduced the yield of metaphases with chromatid aberrations, i.e., resulted in clastogenic adaptation. Combined treatment with MeI and TEM increased the aberration yield; MeI treatment alone (10(-3) M, 0.5 h) was without clastogenic effect. No protective effects were observed after MeI pretreatment and challenge treatment by maleic hydrazide (MH). The data obtained in V. faba are compared to those previously reported for E. coli.     

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.     

Induction of premature mitosis in root meristem cells of Vicia faba and Pisum sativum by various agents is correlated with an increased level of protein phosphorylation

The intra-S-phase checkpoint response to hydroxyurea (HU)-mediated arrest of DNA replication was analysed in root meristems of two legumes, Pisum sativum and Vicia faba. The obtained results suggest that a molecular signal which invokes mechanisms allowing the cells to override the S-M dependency control system may be generated by caffeine (CF) and a number of alternative, yet related chemical agents, benzyl-6-aminopurine (BAP), 2-aminopurine (2-AP), and 6-dimethylaminopurine (DMAP). A variety of aberrant mitotic divisions included chromosomal breaks and gaps, lost and lagging chromatids and chromosomes, acentric fragments, chromosome bridges and micronuclei. Furthermore, similar effects induced by sodium vanadate, an inhibitor of protein phosphatases, extend the number of inhibitors capable of inducing premature chromosome condensation (PCC) in root meristem cells, as well as the range of possible regulatory pathways leading to the transition from S-phase arrest towards abnormal mitosis. Until preprophase, FITC-conjugated monoclonal antibodies (alpha-Y(a)b-FITC) that specifically recognize phosphorylated form of threonine indicate no evident cell cycle-dependent changes in an overall phosphorylation status of root meristem cells in the control plants. Irrespective of the stage of interphase, alpha-Y(p)ab-FITC was localized basically in the cytoplasm, whereas nuclear staining was considerably weaker, with a significant fluorescence confined merely to nucleolar regions. The intensity of alpha-Y(p)ab-FITC staining in HU/CF-treated seedlings was found higher than that in the control plants (with the exception of G2 cells), suggesting a general increase in the level of protein phosphorylation, a physiological response mediated probably by an enhanced activity of the cdc-like protein kinase(s).     

Microcystin-LR, a protein phosphatase inhibitor, induces alterations in mitotic chromatin and microtubule organization leading to the formation of micronuclei in Vicia faba

Background and Aims Microcystin-LR (MCY-LR) is a cyanobacterial toxin, a specific inhibitor of type 1 and 2A protein phosphatases (PP1 and PP2A) with significant impact on aquatic ecosystems. It has the potential to alter regulation of the plant cell cycle. The aim of this study was improved understanding of the mitotic alterations induced by cyanotoxin in Vicia faba, a model organism for plant cell biology studies. Methods Vicia faba seedlings were treated over the long and short term with MCY-LR purified in our laboratory. Short-term treatments were performed on root meristems synchronized with hydroxylurea. Sections of lateral root tips were labelled for chromatin, phosphorylated histone H3 and β-tubulin via histochemical and immunohistochemical methods. Mitotic activity and the occurrence of mitotic alterations were detected and analysed by fluorescence microscopy. The phosphorylation state of histone H3 was studied by Western blotting. Key Results Long-term MCY-LR exposure of lateral root tip meristems increased the percentage of either early or late mitosis in a concentration-dependent manner. We observed hypercondensed chromosomes and altered sister chromatid segregation (lagging chromosomes) leading to the formation of micronuclei, accompanied by the formation of disrupted, multipolar and monopolar spindles, disrupted phragmoplasts and the hyperphosphorylation of histone H3 at Ser10. Short-term MCY-LR treatment of synchronized cells showed that PP1 and PP2A inhibition delayed the onset of anaphase at 1 μg mL(-1) MCY-LR, accelerated cell cycle at 10 μg mL(-1) MCY-LR and induced the formation of lagging chromosomes. In this case mitotic microtubule alterations were not detected, but histone H3 was hyperphosphorylated. Conclusions MCY-LR delayed metaphase-anaphase transition. Consequently, it induced aberrant chromatid segregation and micronucleus formation that could be associated with both H3 hyperphosphorylation and altered microtubule organization. However, these two phenomena seemed to be independent. The toxin may be a useful tool in the study of plant cell cycle regulation.     

初生根长度对蚕豆根尖细胞微核率影响的研究

研究了不同长度蚕豆初生根微核率的差异,结果表明,初生根越短微核率越低,初生根越长微核率越高,提示蚕豆根尖细胞微核是可积累的,细胞分裂次数越多微核率越高,从而对蚕豆根尖微核测试的可靠性提供理论参考。     

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。     

A comparison between short-term evolution of micronuclei induced by X-rays and colchicine in root tips of Vicia faba

The short-term evolution of micronuclei derived from acentric fragments and whole chromosomes was studied in root tips of Vicia faba. Micronuclei were induced by X-rays (30 cGy and 120 cGy) and colchicine (10(-5) M and 3 X 10(-4) M). Frequencies of chromosome breakage or loss of micronuclei in interphase and mitotic cells were studied. The DNA content of micronuclei in interphase cells was also measured. Micronuclei derived from whole chromosome showed a higher probability to survive and to undergo mitotic condensation in synchrony with main nuclei than micronuclei derived from an acentric fragment. PCC (Premature Chromosome Condensation) was not observed for both types of micronuclei in Vicia faba, in contrast to the ones reported in mammalian cells in culture.     

Effects of polyamines and polyamine biosynthetic inhibitors on mitotic activity of Allium cepa root tips

The genotoxic and cytotoxic effects of exogenous polyamines (PAs), putrescine (Put), spermidine (Spd), spermine (Spm) and PA biosynthetic inhibitors, alpha-difluoromethylornithine (DFMO), cyclohexilamine (CHA), methylglioxal bis-(guanylhydrazone) (MGBG) were investigated in the root meristems of Allium cepa L. The reduction of mitotic index and the induction of chromosomal aberrations such as bridges, stickiness, c-mitotic anaphases, micronuclei, endoredupliction by PAs and PA biosynthetic inhibitors were observed and these were used as evidence of genotoxicity and cytotoxicity.     

Induction of foci of phosphorylated H2AX histones and premature chromosome condensation after DNA damage in <Emphasis Type="Italic">Vicia faba</Emphasis> root meristem

Immunocytochemical analysis using antibody raised against human H2AX histones phosphorylated at serine 139 (γ-H2AX) demonstrates that root meristem cells of Vicia faba exposed to UV-radiation or incubated with hydroxyurea (HU) reveal discrete foci at the border of the nucleolus and perinucleolar chromatin or scattered over the whole area of cell nucleus. Western blots detected only one protein band at the position expected for the phosphorylated form of H2AX. The dose-effect relationship was demonstrated following treatment with 2.5 and 10 mM HU. Proteins extracted from root meristems incubated for 2 h either with HU and caffeine or with HU and sodium metavanadate showed unchanged amounts of bound γ-H2AX antibodies, as compared to root meristems treated with 2.5 mM HU. Higher quantities of phosphorylated H2AX histones were detected in proteins extracted from roots treated with HU and 2-aminopurine. All treatments were effective in producing evident aberrations of premature mitosis: broken and lagging chromatids, acentric fragments, chromosomal bridges and micronuclei. Our results show that phosphorylation of H2AX at the carboxy-terminal Ser-Gln-Glu sequence is among the earliest responses to double-strand breaks and, presumably, one of the key ATM/ATR-dependent signals indispensable for the repair of spontaneous and induced DNA damage in plant cells.     

褐藻寡糖抗环磷酰胺诱导蚕豆根尖的细胞遗传毒性

采用蚕豆根尖细胞的微核试验和染色体畸变试验方法,测定不同浓度的褐藻寡糖对环磷酰胺(cyclophosphamide,CP)诱导的蚕豆根尖细胞的微核率、有丝分裂指数和染色体畸变率的影响。结果表明:褐藻寡糖能有效抑制环磷酰胺诱导的蚕豆根尖细胞微核的产生,即在一定浓度范围内,微核率随褐藻寡糖处理浓度的降低而减少,但低于一定浓度后反而呈上升趋势;不同浓度的褐藻寡糖均可使蚕豆根尖细胞有丝分裂指数增大;褐藻寡糖还能有效降低蚕豆根尖细胞染色体畸变率。因此,褐藻寡糖对蚕豆根尖细胞具有明显的诱抗活性和调节细胞分裂生长的效应。     

氯苯胁迫对蚕豆幼苗生长和细胞分裂的影响

研究了1,2,4-三氯苯(TCB)对蚕豆幼苗生长、根尖细胞分裂及染色体畸变的影响．结果表明,随TCB浓度增加和处理时间延长,蚕豆幼苗根长的生长及根尖细胞有丝分裂指数降低甚至停止．TCB诱发蚕豆根尖细胞有丝分裂过程中染色体数目畸变和结构畸变．50-100μg.g^-1TCB胁迫12-24h,蚕豆根尖染色体的主要损伤形式为c-有丝分裂、染色体桥和不均匀排列,其出现百分率达1．0％--10.3％．300μg.g^-1TCB胁迫12-96h,蚕豆根尖细胞中染色体粘连(S)、S+染色体断裂(S+B)、S+染色体环(S+R)、S+染色体不均匀排列(S+A)及S+染色体桥(S+Be)出现的百分率达47．9％--88.9％,各种类型染色体断裂出现的百分率仅为18．1％--29．6％,说明蚕豆根尖细胞染色体畸变分析可作为TCB土壤污染监测的敏感生物监测指标．     

Cell membrane surface potential (psi0) plays a dominant role in the phytotoxicity of copper and arsenate

Negative charges at cell membrane surfaces (CMS) create a surface electrical potential (psi(0)) that affects ion concentrations at the CMS and consequently affects the phytotoxicity of metallic cations and metalloid anions in different ways. The zeta potentials of root protoplasts of wheat (Triticum aestivum), as affected by the ionic environment of the solution, were measured and compared with the values of psi(0) calculated with a Gouy-Chapman-Stern model. The mechanisms for the effects of cations (H(+), Ca(2+), Mg(2+), Na(+), and K(+)) on the acute toxicity of Cu(2+) and As(V) to wheat were studied in terms of psi(0). The order of effectiveness of the ions in reducing the negativity of psi(0) was H(+) > Ca(2+) approximately Mg(2+) > Na(+) approximately K(+). The calculated values of psi(0) were proportional to the measured zeta potentials (r(2) = 0.93). Increasing Ca(2+) or Mg(2+) activities in bulk-phase media resulted in decreased CMS activities of Cu(2+) ({Cu(2+)}(0)) and increased CMS activities of As(V) ({As(V)}(0)). The 48-h EA50{Cu(2+)}(b) ({Cu(2+)} in bulk-phase media accounting for 50% inhibition of root elongation over 48 h) increased initially and then declined, whereas the 48-h EA50{As(V)}(b) decreased linearly. However, the intrinsic toxicity of Cu(2+) (toxicity expressed in terms of {Cu(2+)}(0)) appeared to be enhanced as psi(0) became less negative and the intrinsic toxicity of As(V) appeared to be reduced. The psi(0) effects, rather than site-specific competitions among ions at the CMS (invoked by the biotic ligand model), may play the dominant role in the phytotoxicities of Cu(2+) and As(V) to wheat.     

铬渣堆场渗滤液对蚕豆根尖细胞微核的影响

利用蚕豆根尖微核技术监测化工铬渣堆场渗出液的遗传毒性,结果表明,化工铬渣渗出液含有较高浓度的Cr6+,能明显诱导蚕豆根尖细胞微核的形成,具有较强的遗传毒性。当铬渣渗出液中Cr6+浓度为186.6460 mg/L时,微核率达到最大。铬渣渗出液所诱导的微核率与阴性对照组相比有极显著差异。     

Micronuclei and Radiosensitiviy in the Root Meristem of Vicia faba

Percentages of cells with micronuclei in four regions of theroot meristems of Vicia faba are used as measures of sensitivityto acute X-irradiation. There are two peaks in these percentages,occurring at about four and eight days after 360 rads and twoand six days after 180 rads. Two peaks exist, probably becausethe radiation delays cells that were in G₁ much more than cellsthat were in G₂ in reaching the first post-irradiation mitosisand consequently in displaying micronuclei in the followinginterphase. The relative heights of the two peaks thereforereflect the relative numbers of cells in G₁ and G₂ as well asthe relative sensitivity of the two phases to chromosomal damage. The cells of the quiescent centre are injured least by the radiationas they are mostly held at Gt. The meristem thus obeys the lawof Bergonié and Tribondeau, but differs from that ofZea in that the meristematic cells of the cap initials and steleimmediately adjacent to the quiescent centre resemble the quiescentcentre much more closely than the stele 250 µ away inthe numbers of micronuclei produced. This is consistent withthe differences already known between the two species concerningrates of division in the different regions of the meristem andthe behaviour of the meristem after severe radiation injury.     

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.     

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.     

Heat shock protection against induction of chromatid aberrations is dependent on the time span between heat shock and clastogen treatment of Vicia faba root tip meristem cells

Variation of the time span between heat shock (hs) and clastogen treatment of V. faba root tip meristems showed that hs protection is a very quick response (effective after less than 10 min) and lasting for up to 240 min in the case of induction of chromatid aberrations by maleic hydrazide (MH). Analogous protective responses are significantly slower and shorter when TEM is used for aberration induction. This, together with absence of 'clastogenic cross-adaptation' to these agents and differential effects of benzamide (BA, an inhibitor of poly-ADP-ribosylation) pretreatment before hs on hs protection, suggests that hs before clastogen treatment triggers at least 2 clastogen-specific, protective functions which eventually result in protection against these 2 clastogens.     

CO2激光对蚕豆诱变效应试验研究

７０年代以来,全国已有２０多个省市,近百个单位开展激光在农业上应用的研究,并对２０多种作物２００多个品种进行激光育种的研究。试验表明：激光在刺激作物生长,早熟增产以及诱发变异、改良品种等都有一定的效果,并培育出一些新品种应用于生产。然而,激光对作物的作用机制,特别是遗传机理方面的研究报道尚少。本试验目的是探讨ＣＯ２激光对蚕豆根尖细胞染色体畸变的影响及田间农艺性状的变异情况,分析细胞染色体畸变与农艺性状变异的相关性及其遗传传递情况,为作物激光诱变育种提供依据。本试验采用功率密度为３９４ｍＷ／ｃｍ２的ＣＯ２激光照射蚕豆萌动种子,观察其对蚕豆根尖细胞染色体畸变及幼苗生长的影响,探讨ＣＯ２激光照射后蚕豆植株农艺性状的变异及其遗传传递情况。试验结果表明：ＣＯ２激光不同时间照射蚕豆萌动种子,均能使根尖细胞产生染色体畸变,且畸变类型相似;同时,对Ｌ１幼苗生长有明显的抑制作用,Ｌ２植株高度、分枝数及结荚数等农艺性状均产生不同程度的变异。其中分枝数这一性状的变异能传递给Ｌ３,其余性状的变异则属生理损伤所致。