Interaction mechanism between Cd2+ ions and DNA from the kidney of the silver crucian carp

Cadmium is one of the most toxic heavy metals and is known to accumulate in freshwater food chains. The underlying mechanism for its genotoxicity has not been investigated for any freshwater fish. It has, however, been suggested that cadmium-induced carcinogenesis might involve either direct or indirect interaction of Cd²⁺ with DNA. The interaction between Cd²⁺ and DNA from the kidney of the silver crucian carp (Carassius auratus gibelio) in vitro and in vivo is investigated by spectrophotometric methods and agarose gel electrophoresis methods. Cd²⁺ could insert into DNA basepairs, bind to nucleic acid, and result in notable hypochromicities. The analysis of agarose gel electrophoresis, proves that Cd²⁺ at different concentrations does not cause DNA cleavage in vitro; however, kidneys display the classical laddering degradation of DNA in vivo, which is the result of the promotion of deoxyribonuclease activity or inhibition of superoxide dismutase and catalyse activity and the accumulation of reactive oxygen species caused by Cd²⁺ ions in vivo.     

Ce^3+和La^3+对人工老化沙葱种子活力及生理特性的影响

以未老化和人工老化后的沙葱(Allium mongolicum Regel.)种子为材料,采用氯化铈(Ce3+)和氯化镧(La3+)浸种,测定种子萌发和生理指标,探讨Ce3+和La3+浸种对种子萌发、老化种子活力和生理特性的影响。结果显示:(1)在老化0~5 h时,Ce3+和La3+处理可显著促进沙葱种子萌发,提高种子活力;在老化5 h后,Ce3+和La3+处理对种子萌发无明显促进作用。(2)在老化0~15 h时,Ce3+和La3+处理的沙葱种子中抗氧化酶活性和抗坏血酸(AsA)含量提高,其超氧阴离子自由基(O2-·)产生速率、过氧化氢(H2O2)含量和丙二醛(MDA)含量显著降低;在老化15 h后,Ce3+和La3+处理的种子抗氧化酶活性提高、AsA含量降低,O2-·产生速率和MDA含量提高。(3)在老化5 h时,沙葱种子呼吸速率发生跃变达到最大,Ce3+和La3+处理显著降低了种子呼吸速率。(4)Ce3+和La3+处理在老化0~5 h时提高了沙葱种子超弱发光(UWL)强度,但在老化5 h后沙葱种子的UWL强度降低。研究认为,在沙葱种子人工老化初期,Ce3+和La3+浸种处理可以诱导增强种子抗氧化酶活性和提高AsA含量,有效清除因老化产生积累的过量活性氧(ROS),减轻过氧化伤害,提高种子活力;种子老化中后期,其内部ROS产生与清除系统发生紊乱,加剧了ROS对种子结构的损伤,Ce3+和La3+浸种处理的缓解效应丧失。     

Prevention by Ce3+ of DNA destruction caused by Hg2+ in fish intestine

The interactions between Hg²⁺, Ce³⁺, and the mixuure of Ce³⁺ and Hg²⁺, and DNA from fish intestine in vitro were investigated by using absorption spectrum and fluorescence emission spectrum. The ultraviolet absorption spectra indicated that the addition of Hg²⁺, Ce³⁺, and the mixture of Ce³⁺ and Hg²⁺ to DNA generated an obviously hypochromic effect. Meanwhile, the peak of DNA at 205.2 nm blue-shifted and at 258.2 nm red-shifted. The size of the hypochromic effect and the peak shift of DNA by metal ion treatments was Hg²⁺>Hg²⁺+Ce³⁺>Ce³⁺. The fluorescence emission spectra showed that with the addition of Hg²⁺, Ce³⁺, and the mixture of Ce³⁺ and Hg²⁺ the emission peak at about 416.2 nm of DNA did not obviously change, but the intensity reduced gradually and the sequence was Hg²⁺>Hg²⁺+Ce²⁺>Ce³⁺. Hg²⁺, Ce³⁺, and the mixture of Ce³⁺ and Hg²⁺ had 1.12, 0.19, and 0.41 binding sites to DNA, respectively; the fluorescence quenching of DNA caused by the metal ions all attributed to static quenching. The binding constants (K _A ) of binding siees were 8.98×10⁴ L/mol and 1.02×10⁴ L/mol, 5.12×10⁴ L/mol and 1.10×10³ L/mol, 6.66×10⁴ L/mol and 2.36×10³ L/mol, respectively. The results showed that Ce³⁺ could relieve the destruction of Hg²⁺ on the DNA structure.     

Ca<Superscript>2+</Superscript>, Mg<Superscript>2+</Superscript>-dependent DNase Involvement in Apoptotic Effects in Spermatozoa of Sea Urchin <Emphasis Type="Italic">Strongylocentrotus intermedius</Emphasis> Induced by Two-Headed Sphingolipid Rhizochalin

Previously, we have purified three distinct DNases from spermatozoa of sea urchin Strongylocentrotus intermedius and we suppose the role of Ca²⁺, Mg²⁺-dependent DNase (Ca, Mg-DNase) in apoptosis of spermatozoa. Two-headed sphingolipid rhizochalin (Rhz) induced characteristic apoptotic nuclear chromatin changes, internucleosomal DNA cleavage, and activation of caspase-9, caspase-8, and caspase-3 in spermatozoa as was shown by fluorescence Hoechst 33342/PI/FDA analysis, DNA fragmentation assay, and fluorescence caspase inhibitors FAM-LEHD-fmk, FAM-IETD-fmk, and FAM-DEVD-fmk, respectively. Inhibitor of caspase-3 z-DEVD-fmk subdued Rhz-induced internucleosomal ladder formation, which confirmed the major role of caspase-3 in apoptotic DNA cleavage probably through Ca, Mg-DNase activation. Participation of sea urchin Ca, Mg-DNase in apoptosis of spermatozoa was demonstrated by ions Zn²⁺ blocking of Rhz-induced DNA fragmentation due to direct inhibition of the Ca, Mg-DNase and internucleosomal cleavage of HeLa S and Vero E6 cell nuclei chromatin by highly purified Ca, Mg-DNase.     

Isolation and characterization of microsatellite markers from Fangzheng silver crucian carp,Carassius auratus gibelio (Bloch), and cross‐amplification in the closely related species crucian carp,Carassius auratus auratus (Linnaeus)

We report the development of 20 microsatellite markers for Fangzheng silver crucian carp, Carassius auratus gibelio (Bloch). Nineteen out of 20 showed polymorphism with alleles ranging from two to 14. These loci were screened to amplify the closely related species crucian carp, Carassius auratus auratus (Linnaeus) and all of them can amplify DNA with the size similar to the former. The origin of silver crucian carp is in issue and the population genetic structure is still unclear. Microsatellite markers isolated from the silver crucian carp and their utility in the crucian carp will be useful for these researches.     

Molecular cloning and characterization of cat, gpx1 and Cu/Zn-sod genes in pengze crucian carp (Carassius auratus var. Pengze) and antioxidant enzyme modulation induced by hexavalent chromium in juveniles

Hexavalent chromium (Cr^6 +) is a common pollutant transient metal with high toxicity in the environment. The toxicological effects partly result from oxidative damage due to the production of excessive reactive oxygen species (ROS) in the reductive process of Cr^6 +. To explore the influence of ROS induced directly by Cr^6 + on the oxidative stress generation and antioxidant system, the full length cDNAs of antioxidant-related genes cat, gpx1 and Cu/Zn-sod were successfully acquired from pengze crucian carp first and analyzed. Furthermore, the mRNA expression of the antioxidant genes encompassing catalase (cat), copper/zinc superoxide dismutase (Cu/Zn-sod) and glutathione peroxidase (gpx1), antioxidant enzyme activities of CAT, SOD, and GPx and total protein content were further studied in the gill, intestine and liver of pengze crucian carp (Carassius auratus var. Pengze) juveniles upon acute exposure to Cr^6 + at concentrations of 0.1, 1.0, 10 and 100 mg/L for 4 days. Differential significant changes of the antioxidant enzymes and gene expression were observed in different tissues. The findings contribute to better understanding the antioxidant mechanisms induced by Cr^6 + and selecting the organic-specific sensitive biomarkers to monitor the safety of the aquatic ecosystem.     

Rational evolution of Cd2+-specific DNAzymes with phosphorothioate modified cleavage junction and Cd2+ sensing

In vitro selection of RNA-cleaving DNAzymes is a powerful method for isolating metal-specific DNA. A few successful examples are known, but it is still difficult to target some thiophilic metals such as Cd²⁺ due to limited functional groups in DNA. While using modified bases expands the chemical functionality of DNA, a single phosphorothioate modification might boost its affinity for thiophilic metals without complicating the selection process or using bases that are not commercially available. In this work, the first such in vitro selection for Cd²⁺ is reported. After using a blocking DNA and negative selections to rationally direct the library outcome, a highly specific DNAzyme with only 12 nucleotides in the catalytic loop is isolated. This DNAzyme has a cleavage rate of 0.12 min⁻¹ with 10 μM Cd²⁺ at pH 6.0. The R_p form of the substrate is cleaved ∼100-fold faster than the S_p form. The DNAzyme is most active with Cd²⁺ and its selectivity against Zn²⁺ is over 100 000-fold. Its application in detecting Cd²⁺ is also demonstrated. The idea of introducing single modifications in the fixed region expands the scope of DNA/metal interactions with minimal perturbation of DNA structure and property.     

Endogenous nitric oxide mediates He-Ne laser-induced adaptive responses in salt stressed-tall fescue leaves

The aim of this study was to investigate the role of endogenous nitric oxide in protective effects of He–Ne laser on salt stressed-tall fescue leaves. Salt stress resulted in significant increases of membrane injury, reactive oxygen species (ROS) production, polyamine accumulation, and activities of SOD, POD, and APX, while pronounced decreases of antioxidant contents, CAT activity and intracellular Ca²⁺ concentration in seedlings leaves. He–Ne laser illumination caused a distinct alleviation of cellular injury that was reflected by the lower MDA amounts, polyamine accumulation and ROS levels at the stress period. In contrast, the laser treatment displayed a higher Ca²⁺ concentration, antioxidant amounts, NO release, antioxidant enzyme, and NOS activities. These responses could be blocked due to the inhibition of NO biosynthesis by PTIO (NO scavenger) or LNNA (NOS inhibitor). The presented results demonstrated that endogenous NO might be involved in the progress of He–Ne laser-induced plant antioxidant system activation and ROS degradation in order to enhance adaptive responses of tall fescue to prolonged saline conditions.     

Differential Effect of Cd2+ and Ni2+ on Amino Acid Metabolism in Soybean Seedlings

In 10-d-old soybean seedlings, the growth of roots and shoots was significantly inhibited at 50 and 100 M and more Cd²⁺, respectively, and by 50 M or more Ni²⁺. Although total protein content of roots exposed to 200 M Cd²⁺ or Ni²⁺ was similarly decreased compared to the control, the activity of nitrate reductase was much more inhibited by Cd²⁺. Ni²⁺-treatment (200 M) induced an accumulation of all free amino acids in roots associated with a decrease in alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities reflecting the accumulation of both alanine and aspartic acid, respectively. Cd²⁺-treatment (200 M) decreased the amount of all free amino acids. In addition, cysteine which is the main amino acid consisting the phytochelatin complexes constituted about 17.5 % of total free amino acids. The activities of both ALT and AST in Cd²⁺-treated roots were higher than in Ni²⁺-treated roots suggesting higher conversion of alanine and aspartate to pyruvate and oxaloacetate. Primary leaves excised from either Cd²⁺ or Ni²⁺-treated seedlings showed similar pattern of enzyme activities as roots.     

Effect of cadmium uptake and heat stress on root ultrastructure, membrane damage and antioxidative response in rice seedlings

Excess cadmium (Cd²⁺) in the soil environment is taken up by plants and can cause phytotoxicity. Elevated temperatures also lead to deleterious effects on plants. Plants are very often exposed to a combination of stresses rather than a single stress. The effect of Cd²⁺ and heat stress (HS) on the growth, root ultrastructure, lipid peroxidation (MDA), hydrogen peroxide accumulation and the activities of antioxidant enzymes peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX) of rice roots from sensitive cv. DR-92 and tolerant cv. Bh-1 were investigated at 10 and 20 day of growth under controlled conditions. At day 10 under all Cd²⁺ treatments, the Cd²⁺ content between the two rice cultivars were almost similar. Application of 500 μM Cd²⁺ significantly increased metal concentrations at day 20 in the roots of rice seedlings resulting in a maximum accumulation of 44.25 μg Cd²⁺ g^-1 dry wt in cv. DR-92 and 30 μg Cd²⁺ g^-1 dry wt in cv. Bh-1 with a ~25 % decline in Relative Growth Index (RGI) in cv. DR-92. TEM studies revealed slight disorganization with cell wall ingrowths in root tissues from cv. DR-92 grown in 100 μM Cd²⁺ + HS. Uptake and accumulation of Cd²⁺ increased upon heat treatment in parenchyma, vacuoles and vascular cylinder of root tissues. Peroxidase primarily located in cell walls, the intensity being higher in sensitive cv. DR-92. Under Cd²⁺ stress alone, plants of sensitive cv. DR-92 significantly increased the H₂O₂ and MDA levels together with increased activities of the enzymes POD, CAT and APX at day 10 but remained almost stable at day 20. A strong increase in MDA levels was noted at day 20 in tolerant cv. Bh-1. Cd²⁺ + HS treatments in tolerant cv.Bh-1 led to a decreased H₂O₂ and MDA levels and decreased activities of the enzymes POD, CAT and APX. Results suggest stimulation of root antioxidant system under combination of two stresses and that heat stress seem to have a direct protective role by mitigating the effect of mild Cd²⁺ toxicity largely by enhanced Cd²⁺-MT formation contributing thereby towards the management of Cd²⁺ toxicity at cellular level that confers Cd²⁺ tolerance to rice cv. Bh-1.     

CFTR mediates cadmium-induced apoptosis through modulation of ROS level in mouse proximal tubule cells

The aim of this study was to characterize the role of CFTR during Cd²⁺-induced apoptosis. For this purpose primary cultures and cell lines originated from proximal tubules (PCT) of wild-type cftr^+/+ and cftr^?/? mice were used. In cftr^+/+ cells, the application of Cd²⁺ (5 μM) stimulated within 8 min an ERK1/2-activated CFTR-like Cl^? conductance sensitive to CFTR_inh-172. Thereafter Cd²⁺ induced an apoptotic volume decrease (AVD) within 6 h followed by caspase-3 activation and apoptosis. The early increase in CFTR conductance was followed by the activation of volume-sensitive outwardly rectifying (VSOR) Cl^? and TASK2 K⁺ conductances. By contrast, cftr^?/? cells exposed to Cd²⁺ were unable to develop VSOR currents, caspase-3 activity, and AVD process and underwent necrosis. Moreover in cftr^+/+ cells, Cd²⁺ enhanced reactive oxygen species (ROS) production and induced a 50% decrease in total glutathione content (major ROS scavenger in PCT). ROS generation and glutathione decrease depended on the presence of CFTR, since they did not occur in the presence of CFTR_inh-172 or in cftr^?/? cells. Additionally, Cd²⁺ exposure accelerates effluxes of fluorescent glutathione S-conjugate in cftr^+/+ cells. Our data suggest that CFTR could modulate ROS levels to ensure apoptosis during Cd²⁺ exposure by modulating the intracellular content of glutathione.     

Cr3+和Cd2+对普通小球藻生长及抗氧化酶活性的影响

[目的] 为探究重金属对淡水绿藻生长的影响。[方法] 选取对水质检测具有明显指示作用的普通小球藻（Chlorella vulgaris）为实验材料,CdCl₂·2H₂O和CrCl₃·7H₂O提供重金属离子,探究不同浓度Cr³⁺和Cd²⁺在单一和复合胁迫下对藻细胞浓度、叶绿素a及相关抗氧化酶活性的影响。[结果] 随着Cr³⁺和Cd²⁺浓度不断增加,藻细胞浓度呈先增长后下降趋势;叶绿素a含量呈现先下降后升高再下降的现象,浓度为1 mg/L的单一和复合胁迫下有最大值,且毒性作用表现为Cr³⁺ < Cd²⁺ < Cr³⁺+Cd²⁺;与藻细胞膜相关的丙二醛（MDA）和过氧化氢（H₂O₂）含量随着重金属离子浓度的增大而增长;重金属离子浓度低于10 mg/L时对藻细胞内抗氧化酶系统中的超氧化物歧化酶（SOD）、过氧化氢酶（CAT）和过氧化物酶（POD）表现为促进作用,而大于10 mg/L时具有抑制作用。[结论] 结果表明在单一或复合重金属胁迫下,普通小球藻会充分调动与抗逆性相关的酶来维持自身的正常生长。     

Cu2+对拟南芥根的局部毒性及诱导DNA损伤和细胞死亡

该文探讨了不同浓度的Cu²⁺胁迫对拟南芥(Arabidopsis thaliana)根生长、活性氧(ROS)积累、抗氧化酶活性、质膜完整性和细胞活性的影响, 通过分根实验初步分析了Cu²⁺毒性效应的影响范围。结果表明, Cu²⁺胁迫可显著抑制拟南芥主根伸长, 诱导ROS积累及DNA损伤, 促发抗氧化酶活性升高, 破坏质膜完整性, 且Cu²⁺浓度越高, 毒性效应越明显, 在高浓度Cu²⁺胁迫下细胞活性显著降低。分析各参数之间的关系, 表明ROS的积累与超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、过氧化物酶(POD)及抗坏血酸过氧化物酶(APX)的活性呈显著正相关; ROS积累与DNA损伤、质膜完整性、细胞活性之间具有显著的近线性关系。分根实验结果表明, 只有在添加重金属Cu²⁺(75 μmol·L^–1)一侧培养基中的根生长受抑制, 并出现ROS积累、细胞死亡, 暗示Cu²⁺对拟南芥根系的局部毒性效应可能是由于ROS的局部性积累导致受胁迫根系一侧的细胞死亡所引起的。     

Oxidation of the enzymes involved in nitrogen assimilation plays an important role in the cadmium-induced toxicity in soybean plants

Cadmium causes oxidative damage and hence affects nitrogen assimilation. In the present work we tested the relationship between the inactivation of the enzymes involved in nitrogen assimilation pathway (glutamine synthetase (GS)/glutamate synthase (GOGAT)) and the protein oxidation in nodules of soybean (Glycine max L.) plants under Cd²⁺ stress. Therefore, the effect of Cd²⁺ and reduced gluthatione (GSH) on GS and GOGAT activities, and protein abundance and oxidation were analyzed. Under the metal treatment, amino acids oxidative modification occurred, evidenced by the accumulation of carbonylated proteins, especially those of high molecular weight. When Cd²⁺ was present in the nutrient solution, although a decrease in GS and GOGAT activities was observed (17 and 52%, respectively, compared to controls), the protein abundance of both enzymes remained similar to control nodules. When GSH was added together with Cd²⁺ in the nutrient medium, it protected the nodule against Cd²⁺ induced oxidative damage, maintaining GS and GOGAT activities close to control values. These results allow us to conclude that the inactivation of the nitrogen assimilation pathway by Cd²⁺ in soybean nodules is due to an increment in GS and GOGAT oxidation that can be prevented by the soluble antioxidant GSH. Section Editor: H. Schat     

Inhibition of Mn2+-induced error-prone DNA synthesis with Cd2+ and Zn2+

Bivalent metal cations are key components in the reaction of DNA synthesis. They are necessary for all DNA polymerases, being involved as cofactors in catalytic mechanisms of nucleotide polymerization. It is also known that in the presence of Mn²⁺ the accuracy of DNA synthesis is considerably decreased. The findings of this work show that Cd²⁺ and Zn²⁺ selectively inhibit the Mn²⁺-induced error-prone DNA polymerase activity in extracts of cells from human and mouse tissues. Moreover, these cations in low concentrations also can efficiently inhibit the activity of homogeneous preparations of DNA polymerase iota (Pol ?), which is mainly responsible for the Mn²⁺-induced error-prone DNA polymerase activity in cell extracts. Using a primary culture of granular cells from postnatal rat cerebellum, we show that low concentrations of Cd²⁺ significantly increase cell survival in the presence of toxic Mn²⁺ doses. Thus, we have shown that in some cases low concentrations of Cd²⁺ can display a positive influence on cells, whereas it is widely acknowledged that this metal is not a necessary microelement and is toxic for organisms.     

Exogenous Abscisic Acid Alleviates Cadmium Toxicity by Restricting Cd2+ Influx in Populus euphratica Cells

Abscisic acid (ABA), a widely known phytohormone involved in the plant response to abiotic stress, plays a vital role in mitigating Cd²⁺ toxicity in herbaceous species. However, the role of ABA in ameliorating Cd²⁺ toxicity in woody species is largely unknown. In the present study, we investigated ABA restriction on Cd²⁺ uptake and the relevance to Cd²⁺ stress alleviation in Cd²⁺-hypersensitive Populus euphratica. ABA (5 μM) markedly improved cell viability and growth but reduced membrane permeability in CdCl₂ (100 μM)-stressed P. euphratica cells. Moreover, ABA significantly increased the activity of the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD), and ascorbate peroxidase (APX), contributing to the scavenging of Cd²⁺-elicited H₂O₂ within P. euphratica cells during the period of CdCl₂ exposure (100 μM, 24–72 h). ABA alleviation of Cd²⁺ toxicity was mainly the result of ABA restriction of Cd²⁺ uptake under Cd²⁺ stress. Steady-state and transient flux recordings showed that ABA inhibited Cd²⁺ entry into Cd²⁺-shocked (100 μM, 30 min) and short-term-stressed P. euphratica cells (100 μM, 24–72 h). Non-invasive micro-test technique data showed that H₂O₂ (3 mM) stimulated the Cd²⁺-elicited Cd²⁺ influx but that the plasma membrane (PM) Ca²⁺ channel inhibitor LaCl₃ blocked it, suggesting that the Cd²⁺ influx was through PM Ca²⁺-permeable channels. These results suggested that ABA up-regulated antioxidant enzyme activity in Cd²⁺-stressed P. euphratica and that these enzymes scavenged the Cd²⁺-elicited H₂O₂ within cells. The entry of Cd²⁺ through the H₂O₂-mediated Ca²⁺-permeable channels was subsequently restricted; thus, Cd²⁺ buildup and toxicity were reduced in the Cd²⁺-hypersensitive species, P. euphratica.