In Vitro Selection of High Temperature Zn2+-Dependent DNAzymes

In vitro selection of Zn²⁺-dependent RNA-cleaving DNAzymes with activity at 90°C has yielded a diverse spool of selected sequences. The RNA cleavage efficiency was found in all cases to be specific for Zn²⁺ over Pb²⁺, Ca²⁺, Cd²⁺, Co²⁺, Hg²⁺, and Mg²⁺. The Zn²⁺-dependent activity assay of the most active sequence showed that the DNAzyme possesses an apparent Zn²⁺-binding dissociation constant of 234 μM and that its activity increases with increasing temperatures from 50–90°C. A fit of the Arrhenius plot data gave E_a = 15.3 kcal mol⁻¹. Surprisingly, the selected Zn²⁺-dependent DNAzymes showed only a modest (∼3-fold) activity enhancement over the background rate of cleavage of random sequences containing a single embedded ribonucleotide within an otherwise DNA oligonucleotide. The result is attributable to the ability of DNA to sustain cleavage activity at high temperature with minimal secondary structure when Zn²⁺ is present. Since this effect is highly specific for Zn²⁺, this metal ion may play a special role in molecular evolution of nucleic acids at high temperature.     

Cd2+ activation of l-threonine dehydrogenase from Escherichia coli K-12

Homogeneous preparations of l-threonine dehydrogenase (l-threonine: NAD⁺ oxidoreductase, EC 1.1.1.103) from Escherichia coli K-12, after having been dialyzed against buffers containing Chelex-100 resin, have a basal level of activity of 10–20 units/mg. Added Cd²⁺ stimulates dehydrogenase activity approx. 10-fold; this activation is concentration-dependent and is saturable with an activation K_d = 0.9 μM. Full activation by Cd²⁺ is obtained in the absence of added thiols. The pH-activity profile of the Cd²⁺-activated enzyme conforms to a theoretical curve for one-proton ionization with a pK_a = 7.85. Mn²⁺, the only other activating metal ion, competes with Cd²⁺ for the same binding site. K_m values forl-threonine and NAD⁺ as well as the V_max for ‘demetallized’, Cd²⁺-activated, and Mn²⁺-activated threonine dehydrogenase were determined and compared.     

Modification of hERG1 channel gating by Cd2+

Each of the four subunits in a voltage-gated potassium channel has a voltage sensor domain (VSD) that is formed by four transmembrane helical segments (S1–S4). In response to changes in membrane potential, intramembrane displacement of basic residues in S4 produces a gating current. As S4 moves through the membrane, its basic residues also form sequential electrostatic interactions with acidic residues in immobile regions of the S2 and S3 segments. Transition metal cations interact with these same acidic residues and modify channel gating. In human ether-á-go-go–related gene type 1 (hERG1) channels, Cd²⁺ coordinated by D456 and D460 in S2 and D509 in S3 induces a positive shift in the voltage dependence of activation of ionic currents. Here, we characterize the effects of Cd²⁺ on hERG1 gating currents in Xenopus oocytes using the cut-open Vaseline gap technique. Cd²⁺ shifted the half-point (V_1/2) for the voltage dependence of the OFF gating charge–voltage (Q_OFF-V) relationship with an EC₅₀ of 171 µM; at 0.3 mM, V_1/2 was shifted by +50 mV. Cd²⁺ also induced an as of yet unrecognized small outward current (I_Cd-out) upon repolarization in a concentration- and voltage-dependent manner. We propose that Cd²⁺ and Arg residues in the S4 segment compete for interaction with acidic residues in S2 and S3 segments, and that the initial inward movement of S4 associated with membrane repolarization displaces Cd²⁺ in an outward direction to produce I_Cd-out. Co²⁺, Zn²⁺, and La³⁺ at concentrations that caused ∼+35-mV shifts in the Q_OFF-V relationship did not induce a current similar to I_Cd-out, suggesting that the binding site for these cations or their competition with basic residues in S4 differs from Cd²⁺. New Markov models of hERG1 channels were developed that describe gating currents as a noncooperative two-phase process of the VSD and can account for changes in these currents caused by extracellular Cd²⁺.     

A self-cleaving DNA enzyme modified with amines, guanidines and imidazoles operates independently of divalent metal cations (M2+)

The selection of modified DNAzymes represents an important endeavor in expanding the chemical and catalytic properties of catalytic nucleic acids. Few examples of such exist and to date, there is no example where three different modified bases have been simultaneously incorporated for catalytic activity. Herein, dCTP, dATP and dUTP bearing, respectively, a cationic amine, an imidazole and a cationic guanidine, were enzymatically polymerized on a DNA template for the selection of a highly functionalized DNAzyme, called DNAzyme 9-86, that catalyzed (M²⁺)-independent self-cleavage under physiological conditions at a single ribo(cytosine)phosphodiester linkage with a rate constant of (0.134 ± 0.026) min⁻¹. A pH rate profile analysis revealed pK_a's of 7.4 and 8.1, consistent with both general acid and base catalysis. The presence of guanidinium cations permits cleavage at significantly higher temperatures than previously observed for DNAzymes with only amines and imidazoles. Qualitatively, DNAzyme 9-86 presents an unprecedented ensemble of synthetic functionalities while quantitatively it expresses one of the highest reported values for any self-cleaving nucleic acid when investigated under M²⁺-free conditions at 37°C.     

Hg2+ and Cd2+ induced inhibition of light induced proton efflux in the cyanobacteriumAnabaena flos-aquae

Light induced proton efflux in intact cells ofAnabaena flos-aquae is inhibited by the heavy metals Hg²⁺ and Cd²⁺. Furthermore, Hg²⁺ and Cd²⁺ reduced the¹⁴CO₂ fixation, oxygen evolution and carbonic anhydrase activity responsible for H⁺ efflux.     

Hg2+ and Cd2+ interact differently with biomimetic erythrocyte membranes

In order to characterize the potentially deleterious effects of toxic Hg²⁺ and Cd²⁺ on lipid membranes, we have studied their binding to liposomes whose composition mimicked erythrocyte membranes. Fluorescence spectroscopy utilizing the concentration dependent quenching of Phen Green™ SK by Hg²⁺ and Cd²⁺ was found to be a sensitive tool to probe these interactions at metal concentrations ≤1 μM. We have systematically developed a metal binding affinity assay to screen for the interactions of Hg²⁺ or Cd²⁺ with certain lipid classes. A biomimetic liposome system was developed that contained four major lipid classes of erythrocyte membranes (zwitterionic lipids: phosphatidylcholine and phosphatidylethanolamine; negatively charged: phosphatidylserine and neutral: cholesterol). In contrast to Hg²⁺, which preferentially bound to the negatively charged phosphatidylserine compared to the zwitterionic components, Cd²⁺ bound stronger to the two zwitterionic lipids. Thus, the observed distinct differences in the binding affinity of Hg²⁺ and Cd²⁺ for certain lipid classes together with their known effects on membrane properties represent an important first step toward a better understanding the role of these interactions in the chronic toxicity of these metals.     

External Cd2+ and protons activate the hyperpolarization-gated K+ channel KAT1 at the voltage sensor

The functionally diverse cyclic nucleotide binding domain (CNBD) superfamily of cation channels contains both depolarization-gated (e.g., metazoan EAG family K⁺ channels) and hyperpolarization-gated channels (e.g., metazoan HCN pacemaker cation channels and the plant K⁺ channel KAT1). In both types of CNBD channels, the S4 transmembrane helix of the voltage sensor domain (VSD) moves outward in response to depolarization. This movement opens depolarization-gated channels and closes hyperpolarization-gated channels. External divalent cations and protons prevent or slow movement of S4 by binding to a cluster of acidic charges on the S2 and S3 transmembrane domains of the VSD and therefore inhibit activation of EAG family channels. However, a similar divalent ion/proton binding pocket has not been described for hyperpolarization-gated CNBD family channels. We examined the effects of external Cd²⁺ and protons on Arabidopsis thaliana KAT1 expressed in Xenopus oocytes and found that these ions strongly potentiate voltage activation. Cd²⁺ at 300 µM depolarizes the V₅₀ of KAT1 by 150 mV, while acidification from pH 7.0 to 4.0 depolarizes the V₅₀ by 49 mV. Regulation of KAT1 by Cd²⁺ is state dependent and consistent with Cd²⁺ binding to an S4-down state of the VSD. Neutralization of a conserved acidic charge in the S2 helix in KAT1 (D95N) eliminates Cd²⁺ and pH sensitivity. Conversely, introduction of acidic residues into KAT1 at additional S2 and S3 cluster positions that are charged in EAG family channels (N99D and Q149E in KAT1) decreases Cd²⁺ sensitivity and increases proton potentiation. These results suggest that KAT1, and presumably other hyperpolarization-gated plant CNBD channels, can open from an S4-down VSD conformation homologous to the divalent/proton-inhibited conformation of EAG family K⁺ channels.     

Cd2+-induced damage to yeast plasma membrane and its alleviation by Zn2+: studies on Schizosaccharomyces pombe cells and reconstituted plasma membrane vesicles

In Schizosaccharomyces pombe, Cd²⁺ shares the same uphill uptake system with Zn²⁺. Both heavy metals inhibited growth, respiration, H⁺/glucose uptake, and glucose-induced proton extrusion, Cd²⁺ being a 10–15-fold stronger inhibitor. In contrast, both had a similar effect on the plasma membrane H⁺-ATPase, enhancing its affinity for ATP and reducing the rate of ATP splitting. Cd²⁺ caused protracted strong fluidization of the plasma membrane of energized cells, whereas deenergized cells, phosphatidylcholine liposomes, and plasma membrane fragments, either purified or incorporated into the liposomes, exhibited only a short initial fluidization. Zn²⁺, which caused only a marginal membrane fluidization, suppressed the fluidizing action of Cd²⁺. The fluidizing effect of both heavy metals on liposomes was reduced by the presence of plasma membrane fragments in the liposome membrane. At 50 μM, Cd²⁺ brought about loss K⁺ (18 K⁺/1 Cd²⁺) from energized, but not from deenergized cells since Cd²⁺ must first accumulate in the cells before causing a detectable effect. A simple membrane disruption by external Cd²⁺ is, therefore, unlikely to be the main mechanism of cadmium-induced potassium loss in intact cells. Zn²⁺ had virtually no effect below 1 mM concentration, and it again weakened the K⁺-releasing effect of Cd²⁺. Cd²⁺ caused a strong loss of K⁺ also from K⁺-containing liposomes, probably because of a direct interaction with liposome phospholipids. Incorporation of plasma membrane fragments into the liposomes reduced the K⁺ loss sixfold. Received: 13 November 1995 / Accepted: 31 January 1996     

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.

     

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.     

A novel replicating circular DNAzyme

10–23 DNAzyme has the potential to suppress gene expressions through sequence-specific mRNA cleavage. However, the dependence on exogenous delivery limits its applications. The objective of this work is to establish a replicating DNAzyme in bacteria using a single-stranded DNA vector. By cloning the 10–23 DNAzyme into the M13mp18 vector, we constructed two circular DNAzymes, C-Dz₇ and C-Dz₄₈₂, targeting the β-lactamase mRNA. These circular DNAzymes showed in vitro catalytic efficiencies (k_cat/K_M) of 7.82 × 10⁶ and 1.36 × 10⁷ M^–1·min^–1, respectively. Their dependence on divalent metal ions is similar to that found with linear 10–23 DNAzyme. Importantly, the circular DNAzymes were not only capable of replicating in bacteria but also exhibited high activities in inhibiting β-lactamase and bacterial growth. This study thus provides a novel strategy to produce replicating DNAzymes which may find widespread applications.     

Effect of Cl on Cd uptake by Swiss chard in nutrient solutions

Swiss chard (Beta vulgaris L., cv. Fordhook Giant) was grown in nutrient solution with Cl concentrations varying between 0.01 mM and 120 mM. Solution Na concentration and ionic strength were maintained in all treatments by compensating with NaNO₃. All solutions contained Cd (50 nM, spiked with ¹⁰⁹Cd). Three different Cd²⁺ buffering systems were used. In one experiment, Cd²⁺ activity was unbuffered; its activity decreased with increased Cl concentration as a result of the formation of CdCl_n ^2–n species. In the other experiments, Cd²⁺ activity was buffered by the chelator nitrilotriacetate (NTA, 50 M) and ethylene-bis-(oxyethylenenitrilo)-tetraacetate (EGTA, 50 M) at about 10^–9 M and 10^–11 M, respectively. Plant growth was generally unaffected by increasing Cl concentrations in the three experiments. In unbuffered solutions, Cd concentrations in plant tissue decreased significantly (p<0.01) (approximately 2.4-fold) as solution Cl concentration increased from 0.01 mM to 120 mM. However, this decrease was smaller in magnitude than the 4.7-fold decrease in Cd²⁺ activity as calculated by the GEOCHEM-PC program for the same range of Cl concentrations. In solutions where Cd²⁺ activity was buffered by NTA, Cd concentrations in plant tissue increased approximately 1.4-fold with increasing Cl concentration in solution, while the Cd²⁺ activity was calculated to decrease 1.3-fold. In solutions where Cd²⁺ activity was buffered by EGTA, Cd concentrations in the roots increased 1.3-fold with increasing Cl concentration in solution but there was no effect of Cl on shoot Cd concentrations. The data suggest that either CdCl_n ^2–nspecies can be taken up by plant roots or that Cl enhances uptake of Cd²⁺ through enhanced diffusion of the uncomplexed metal to uptake sites.Abbreviations DAS days after sowing - EGTA ethylene-bis-(oxyethylenenitrilo)-tetraacetate - HBED N,N-bis(2-hydroxybenzyl)-ethylenediamine-N,N-diacetate - NTA nitrilotriacetate     

Cadmium inhibits mismatch repair by blocking the ATPase activity of the MSH2-MSH6 complex

Cadmium (Cd²⁺) is a known carcinogen that inactivates the DNA mismatch repair (MMR) pathway. In this study, we have tested the effect of Cd²⁺ exposure on the enzymatic activity of the mismatch binding complex MSH2–MSH6. Our results indicate that Cd²⁺ is highly inhibitory to the ATP binding and hydrolysis activities of MSH2–MSH6, and less inhibitory to its DNA mismatch binding activity. The inhibition of the ATPase activity appears to be dose and exposure time dependent. However, the inhibition of the ATPase activity by Cd²⁺ is prevented by cysteine and histidine, suggesting that these residues are essential for the ATPase activity and are targeted by Cd²⁺. A comparison of the mechanism of inhibition with N-ethyl maleimide, a sulfhydryl group inhibitor, indicates that this inhibition does not occur through direct inactivation of sulfhydryl groups. Zinc (Zn²⁺) does not overcome the direct inhibitory effect of Cd²⁺ on the MSH2–MSH6 ATPase activity in vitro. However, the increase in the mutator phenotype of yeast cells exposed to Cd²⁺ was prevented by excess Zn²⁺, probably by blocking the entry of Cd²⁺ into the cell. We conclude that the inhibition of MMR by Cd²⁺ is through the inactivation of the ATPase activity of the MSH2–MSH6 heterodimer, resulting in a dominant negative effect and causing a mutator phenotype.     

Inhibition kinetics of brain butyrylcholinesterase by Cd2+ and Zn2+, Ca2+ or Mg2+ reactivates the inhibited enzyme

• 1.1. The inhibition kinetics of sheep brain butyrylcholinesterase (BChE) (acylcholine acylhydrolase, EC 3.1.1.8) by Cd²⁺ and Zn²⁺ has been studied.
• 2.2. K_s has been determined as 0.14mM. Cd²⁺ and Zn²⁺ were the hyperbolic mixed-type inhibitors of BChE. Ca²⁺ and Mg²⁺ had no effect on the enzyme activity in the experimental conditions.
• 3.3. But when the enzyme was inhibited by 0.1 mM Cd²⁺ or Zn²⁺, Ca²⁺ and Mg²⁺ reactivated the inhibited form of BChE.

     

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.     

A new heavy lanthanide-dependent DNAzyme displaying strong metal cooperativity and unrescuable phosphorothioate effect

In vitro selection of RNA-cleaving DNAzymes was performed using three heavy lanthanide ions (Ln³⁺): Ho³⁺, Er³⁺ and Tm³⁺. The resulting sequences were aligned together and about half of the library contained a new family of DNAzyme. These DNAzymes have a simple loop structure, and they are active only with the seven heavy Ln³⁺. Among the tested non-lanthanide ions, only Y³⁺ induced cleavage and even Pb²⁺ failed to cleave, suggesting a very high specificity. A representative DNAzyme, Tm7, has a sigmoidal metal binding curve with a Hill coefficient of 3, indicating that three metal ions are involved in the catalytic step. Its pH-rate profile has a slope of 1, suggesting a single deprotonation step is involved in the rate-limiting step. Tm7 has a cleavage rate of 1.6 min⁻¹ at pH 7.8 with 10 μM Er³⁺. Phosphorothioate substitution at the cleavage junction completely inhibits the activity, which cannot be rescued by Cd²⁺ alone, or by a mixture of Er³⁺ and Cd²⁺, suggesting that two interacting metal ions are involved in direct bonding to both non-bridging oxygen atoms. A new model involving three lanthanide ions is proposed based on this study. A biosensor is engineered using Tm7 to detect Dy³⁺ down to 14 nM.     

Optimization of Cd2+ removal by the cyanobacterium Synechocystis pevalekii using the response surface methodology

Response surface methodology (RSM) has been used to optimize the critical parameters responsible for higher Cd²⁺ removal by a unicellular cyanobacterium Synechocystis pevalekii. A three-level Box–Behnken factorial design was used to optimize pH, biomass and metal concentration for Cd²⁺ removal. A coefficient of determination (R²) value (0.99), model F-value (86.40) and its low p-value (F < 0.0001) along with lower value of coefficient of variation (5.61%) indicated the fitness of response surface quadratic model during the present study. At optimum pH (6.48), biomass concentration (0.25 mg protein ml^?1) and metal concentration (5 μg ml^?1) the model predicted 4.29 μg ml^?1 Cd²⁺ removal and experimentally, 4.27 μg ml^?1 Cd²⁺ removal was obtained.     

Resistance and bioaccumulation of Cd2+, Cu2+, Co2+ and Mn2+ by thermophilic bacteria, Geobacillus thermantarcticus and Anoxybacillus amylolyticus

In this study, bioaccumulation and heavy metal resistance of Cd²⁺, Cu²⁺, Co²⁺ and Mn²⁺ ions by thermophilic Geobacillus thermantarcticus and Anoxybacillus amylolyticus was investigated. The bacteria, in an order with respect to metal resistance from the most resistant to the most sensitive, was found to be Mn²⁺ > Co²⁺ > Cu²⁺ > Cd²⁺ for both G. thermantarcticus and A. amylolyticus. It was determined that the highest metal bioaccumulation was performed by A. amylolyticus in Mn²⁺ (28,566 μg/g dry weight), and the lowest metal bioaccumulation was performed by A. amylolyticus in Co²⁺ (327.3 μg/g dry weight). The highest Cd²⁺ capacities of dried cell membrane was found to be 36.07 and 39.55 mg/g membrane for G. thermantarticus and A. amylolyticus, respectively, and the highest Cd²⁺ capacities of wet cell membrane was found to be 14.36 and 12.39 mg/g membrane for G. thermantarcticus and A. amylolyticus, respectively.     

Differential action of Hg2+ and Cd2+ on the phycobilisomes and chlorophyll a fluorescence,and photosystem II dependent electron transport in the cyanobacterium Anabaena flos-aquae

The effect of equimolar concentrations of Hg²⁺ and Cd²⁺ on the whole cell absorption spectra, absorption spectra of the extracted phycocyanin (PC) and fluorescence emission spectra of phycobilisomes (PBS) was investigated in the cells of Anabaena flos-aquae. The PC component of the PBS was found to be extremely sensitive to the Hg²⁺ rather than the Cd²⁺ ions. Further, the results showed that Hg²⁺ and Cd²⁺ induced decrease in the rate of Hill activity (H₂O - DCPIP) was partially restored by the electron donor NH₂OH, not by the diphenyl carbazide. Similarly, chlorophyll a fluorescence emission in the presence of metals showed that addition of NH₂OH could effectively reverse the metal induced alterations in the fluorescence emission intensity. These results, together, suggested that Hg²⁺ and Cd²⁺ caused damage to the photosystems (PS) II reaction center. However, a relatively higher stimulation of the chlorophyll a emission at 695 nm with a red shift of 4.0 nm in the presence of Hg²⁺, and Cd²⁺ induced preferential decrease in the emission intensity at 676 nm as compared with the peak at 695 nm were indicative of the differential action of Hg²⁺ and Cd²⁺ on the PS II.     

Regulative mechanism of Ce3+ relieves DNA damage caused by Cd2+ in the kidney of silver crucian carp

The mechanism of Cd²⁺ on the DNA cleavage and Ce³⁺ on the DNA repair in the kidney of silver crucian carp (Carassius auratus gibelio) is investigated by agarose gel electrophoresis methods and assaying biochemical indexes. It proves that Cd²⁺ induces the classical laddering degradation of DNA in vivo, but DNA cleavage is repaired after injecting with a low Ce³⁺ concentration under various Cd²⁺ concentrations. The DNA cleavage caused by Cd²⁺ is the result of the activation of deoxyribonuclease (DNase) and accumulation of reactive oxygen species (ROS), and Cd²⁺ destroys the antioxidant system, which diminishes the activities of superoxide dismutase, catalase, and peroxidase, and the increase of the lipid peroxidation (LPO) level. However, Ce³⁺ could inhibit activation of Cd²⁺ on DNase activity, relieve inhibition of Cd²⁺ on activities of the antioxidant enzyme, and diminish ROS accumulation. The results show that Ce³⁺ could relieve the toxicity of Cd²⁺ to silver crucian carp.