Cd-induced DNA Damage Research Articles

Functional characterization of a DNA-damage repair/tolerance 100 (DRT100) gene in Sedum alfredii Hance for genome stability maintenance and Cd hypertolerance

Cd contamination is a world-wild concern for its toxicity and accumulation in food chain. Sedum alfredii Hance (Crassulaceae) is a zinc (Zn) and cadmium (Cd) hyperaccumulator native to China and widely applied for the phytoremediation at Zn or Cd contaminated sites. Although many studies report the uptake, translocation and storage of Cd in S. alfredii Hance, limited information is known about the genes and underlying mechanisms of genome stability maintenance under Cd stress. In this study, a gene resembling DNA-damage repair/toleration 100 (DRT100) was Cd inducible and designated as SaDRT100. Heterologous expression of SaDRT100 gene in yeasts and Arabidopsis thaliana enhanced Cd tolerance capability. Under Cd stress, transgenic Arabidopsis with SaDRT100 gene exhibited lower levels of reactive oxygen species (ROS), fewer Cd uptake in roots and less Cd-induced DNA damage. Evidenced by the subcellular location in cellular nucleus and expression in aerial parts, we suggested the involvement of SaDRT100 in combating Cd-induced DNA damage. Our findings firstly uncovered the roles of SaDRT100 gene in Cd hypertolerance and genome stability maintenance in S. alfredii Hance. The potential functions of DNA protection make SaDRT100 gene a candidate in genetic engineering for phytoremediation at multi-component contaminated sites.

LncRNA MT1DP promotes cadmium-induced DNA replication stress by inhibiting chromatin recruitment of SMARCAL1

Cadmium (Cd) is a well-known carcinogenic metal and widespread environmental pollutant. The effect of Cd-induced carcinogenesis is partly due to accumulated DNA damage and chromosomal aberrations, but the exact mechanisms underlying the genotoxicity of Cd have not been clearly understood. Here, we found that one long non-coding RNA MT1DP is participated in Cd-induced DNA damage and replication stress. Through analyzing the residents from Cd-contaminated area in Southern China, we found that blood DNA repair genes are down-regulated in individuals with high urine Cd values compared to those with low urine Cd values, which contrast to the blood MT1DP levels. Through in vitro experiments, we found that MT1DP promotes Cd-induced DNA damage response, genome instability and replication fork stalling. Mechanically, upon Cd treatment, ATR is activated to enhance HIF-1α expression, which in turn promotes the transcription level of MT1DP. Subsequently MT1DP is recruited on the chromatin and binds to SMARCAL1 to competitive inhibit latter's interaction with RPA complexes, finally leading to increased replication stress and DNA damage. In summary, this study provides clear evidence for the role of epigenetic regulation on the genotoxic effect of Cd, and MT1DP-mediated replication stress may represent a novel mechanism for Cd-induced carcinogenesis.

Glutathione: A key player in metal chelation, nutrient homeostasis, cell cycle regulation and the DNA damage response in cadmium-exposed Arabidopsis thaliana

Glutathione (GSH) is an important player in plant responses to cadmium (Cd) through its dual function as an antioxidant and precursor for metal-chelating phytochelatins (PCs). In addition, it was shown to be involved in cell cycle regulation in Arabidopsis thaliana roots, but its involvement in this process in leaves is largely unknown and has never been evaluated in Cd-exposed plants. This study aimed to elucidate the role of GSH in leaf growth and development, metal chelation, nutrient homeostasis and cell cycle regulation in A. thaliana plants upon prolonged Cd exposure. Responses were compared between wild-type (WT) plants and three GSH-deficient mutants. Our results indicate that PC production remains important in plants exposed to Cd for an extended duration. Furthermore, an important role for GSH in regulating nutrient homeostasis in Cd-exposed plants was revealed. Cell cycle analysis demonstrated that negative effects of Cd exposure on cell division and endoreplication were more pronounced in leaves of the GSH-deficient cadmium-sensitive 2–1 (cad2-1) mutant in comparison to the WT, indicating the involvement of GSH in cell cycle regulation. Finally, a crucial role for GSH in transcriptional activation of the Cd-induced DNA damage response (DDR) was revealed, as the Cd-induced upregulation of DDR-related genes was either less pronounced or completely abolished in leaves of the GSH-deficient mutants.

MSH2 and MSH6 in Mismatch Repair System Account for Soybean (Glycine max (L.) Merr.) Tolerance to Cadmium Toxicity by Determining DNA Damage Response.

Our aim was to investigate DNA mismatch repair (MMR) genes regulating cadmium tolerance in two soybean cultivars. Cultivars Liaodou 10 (LD10, Cd-sensitive) and Shennong 20 (SN20, Cd-tolerant) seedlings were grown hydroponically on Murashige and Skoog (MS) media containing 0-2.5 mg·L-1 Cd for 4 days. Cd stress induced less random amplified polymorphism DNA (RAPD) polymorphism in LD10 than in SN20 roots, causing G1/S arrest in LD10 and G2/M arrest in SN20 roots. Virus-induced gene silencing (VIGS) of MLH1 in LD10-TRV-MLH1 plantlets showed markedly diminished G1/S arrest but enhanced root length/area under Cd stress. However, an increase in G1/S arrest and reduction of G2/M arrest occurred in SN20-TRV-MSH2 and SN20-TRV-MSH6 plantlets with decreased root length/area under Cd stress. Taken together, we conclude that the low expression of MSH2 and MSH6, involved in the G2/M arrest, results in Cd-induced DNA damage recognition bypassing the MMR system to activate G1/S arrest with the assistance of MLH1. This then leads to repressed root growth in LD10, explaining the intervarietal difference in Cd tolerance in soybean.

Mechanisms used by DNA MMR system to cope with Cadmium-induced DNA damage in plants.

Cadmium (Cd) is found widely in soil and is severely toxic for plants, causing oxidative damage in plant cells because of its heavy metal characteristics. The DNA damage response (DDR) is triggered in plants to cope with the Cd stress. The DNA mismatch repair (MMR) system known for its mismatch repair function determines DDR, as mispairs are easily generated by a translesional synthesis under Cd-induced genomic instability. Cd-induced mismatches are recognized by three heterodimeric complexes including MutSα (MSH2/MSH6), MutSβ (MSH2/MSH3), and MutSγ (MSH2/MSH7). MutLα (MLH1/PMS1), PCNA/RFC, EXO1, DNA polymerase δ and DNA ligase participate in mismatch repair in turn. Meanwhile, ATR is preferentially activated by MSH2 to trigger DDR including the regulation of the cell cycle, endoreduplication, cell death, and recruitment of other DNA repair, which enhances plant tolerance to Cd. However, plants with deficient MutS will bypass MMR-mediated DDR and release the multiple-effect MLH1 from requisition of the MMR system, which leads to weak tolerance to Cd in plants. In this review, we systematically illustrate how the plant DNA MMR system works in a Cd-induced DDR, and how MMR genes regulate plant tolerance to Cd. Additionally, we also reviewed multiple epigenetic regulation systems acting on MMR genes under stress.

Selenised yeast sources differ in their capacity to protect porcine jejunal epithelial cells from cadmium-induced toxicity and oxidised DNA damage.

In recent years there has been increasing interest in the use of selenised yeast (Se-Y) as an antioxidant feed supplement. Here, three selenised yeast products are differentiated in terms of bioefficiency and the ameliorative effect on Cadmium (Cd) toxicity in porcine epithelial cells. A porcine digestion in vitro model was chosen to more accurately simulate the bioavailability of different Se-Y preparations, allowing a comprehensive understanding of the bio efficiency of each Se-Y compound in the porcine model. To elucidate a possible mechanism of action of selenium a number of bioassays were applied. Levels of Se dependent antioxidant enzymes (glutathione peroxidase and thioredoxin reductase) were evaluated to analyze the ROS neutralizing capacity of each Se-Y compound. The effects of Se-Y sources on Cd-induced DNA damage and apoptosis-associated DNA fragmentation was assessed using comet and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays, respectively. Lesion-specific DNA damage analysis and in vitro DNA repair assay determined the DNA repair capacity of each Se-Y source. The results presented in this study confirm that the ability of different commercially available Se-Y preparations to enhance a range of cellular mechanisms that protect porcine gut epithelial cells from Cd-induced damage is concentration-dependent and illustrates the difference in bioefficiency of different Se-Y compounds.

Cell cycle arrest mediated by Cd-induced DNA damage in Arabidopsis root tips

Accumulating evidence demonstrates that the aberrant expression of cell cycle regulation and DNA repair genes can result in abnormal cell proliferation and genomic instability in eukaryotic cells under different stresses. Herein, Arabidopsis thaliana (Arabidopsis) seedlings were grown hydroponically on 0.5 × MS media containing cadmium (Cd) at 0–2.5mgL−1 for 5d of treatment. Real time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis revealed that expression of DNA damage repair and cell cycle regulation genes, including BRCA1, MRE11, WEE1, CDKA;1 and PCNA1, showed an inverted U-shaped dose-response. In contrast, notably reduced expression was observed for G1-to-S transition-related genes, Histone H4, E2Fa and PCNA2; DSB end processing, GR1; G2-to-M transition-related gene, CYCB1;1; and DNA mismatch repair, MSH2, MSH6 and MLH1 genes in root tips exposed to 0.125–2.5mg/L Cd for 5d. Flow cytometry (FCM) analysis revealed significant increases of cells with a 2C nuclear content and with a 4C and 8C nuclear content under Cd stresses of 0.125 and 1–2.5mgL−1, respectively. Our results suggest that 0.125mgL−1 Cd-induced DNA damage induced the marked G1/S arrest, leading to accelerated growth in root tips, while 1.0–2.5mgL−1 Cd-induced DNA damage caused a notable G2/M arrest in root tips, leading to reduced growth in root tips. This may be a protective mechanism that prevents cells with damaged DNA from dividing under Cd stress.

Selenium Source Impacts Protection of Porcine Jejunal Epithelial Cells from Cadmium-Induced DNA Damage, with Maximum Protection Exhibited with Yeast-Derived Selenium Compounds.

Selenium (Se) is found in inorganic and organic forms, both of which are commonly used in animal feed supplements. The aim of this study was to determine the impact of the chemical form of Se on its associated ameliorative effects on cadmium (Cd)-induced DNA damage in a porcine model. At a cellular level, Cd mediates free oxygen radical production leading in particular to DNA damage, with consequential mutagenesis and inhibition of DNA replication. In this study, porcine jejunal epithelial cells (IPEC-J2) were pre-incubated for 48h with one of Se-yeast (Sel-Plex), selenomethionine (Se-M), sodium selenite (Se-Ni) or sodium selenate (Se-Na). The effects of this supplementation on cell viability and DNA damage following cadmium chloride (CdCl2) exposure were subsequently evaluated. IPEC-J2 cells were cultivated throughout in medium supplemented with porcine serum to generate a superior model that recapitulated the porcine gut epithelium. The results illustrated that Se antioxidant effects were both composition- and dose-dependent as evident from cell viability (Alamar Blue and 5-carboxyfluorescein diacetate acetoxymethyl ester) and DNA damage assays (Comet and TUNEL). Both the Se-yeast and Se-M organic species, when used at the European Food Safety Authority guideline levels, had a protective effect against Cd-induced DNA damage in the IPEC-J2 model system whereas for inorganic Se-Ni and Se-Na sources no protective effects were observed and in fact these were shown to enhance the negative effects of Cd-induced DNA damage. It can be concluded that nutritional supplementation with organoselenium may protect porcine gut integrity from damage induced by Cd.

Genome-wide transcriptome and functional analysis of two contrasting genotypes reveals key genes for cadmium tolerance in barley.

BackgroundCadmium (Cd) is a severe detrimental environmental pollutant. To adapt to Cd-induced deleterious effects, plants have evolved sophisticated defence mechanisms. In this study, a genome-wide transcriptome analysis was performed to identify the mechanisms of Cd tolerance using two barley genotypes with distinct Cd tolerance.ResultsMicroarray expression profiling revealed that 91 genes were up-regulated by Cd in Cd-tolerant genotype Weisuobuzhi and simultaneously down-regulated or non-changed in Cd-sensitive Dong17, and 692 genes showed no change in Weisuobuzhi but down-regulated in Dong17. Novel genes that may play significant roles in Cd tolerance were mainly via generating protectants such as catalase against reactive oxygen species, Cd compartmentalization (e.g. phytochelatin-synthase and vacuolar ATPase), and defence response and DNA replication (e.g. chitinase and histones). Other 156 up-regulated genes in both genotypes also included those encoding proteins related to stress and defence responses, and metabolism-related genes involved in detoxification pathways. Meanwhile, biochemical and physiological analysis of enzyme (ATPase and chitinase), phytohormone (ethylene), ion distribution and transport (Cd, Na+, K+, Ca2+, ABC transporter) demonstrated that significantly larger Cd-induced increases of those components in Weisuobuzhi than those in Dong17. In addition, Cd-induced DNA damage was more pronounced in Dong17 than that in Weisuobuzhi.ConclusionsOur findings suggest that combining microarray, physiological and biochemical analysis has provided valuable insights towards a novel integrated molecular mechanism of Cd tolerance in barley. The higher expression genes in Cd tolerant genotype could be used for transgenic overexpression in sensitive genotypes of barley or other cereal crops for elevating tolerance to Cd stress.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-611) contains supplementary material, which is available to authorized users.

镉暴露对黑斑蛙精巢ROS的诱导及其蛋白质氧化损伤作用机理

Exorbitant heavy metal embodies certain underlying toxic effects that have been regarded as one of the culprits rendering amphibian populations decline and extinct at a fast clip in recent decades.Cadmium(Cd) is a ubiquitous environmental pollutant that has been concerned as globe issue.The previous study has found that Cd could induce testicular damage,respiratory and nervous system disorders.However,the toxicity mechanism of Cd has not been addressed.Here,we explored the toxicity mechanism study of Cd by investigating the effect of chronic exposure to CdCl2 on the reproductive system and examine the toxicity mechanism of Cd2+ in male Rana nigromaculata.Based on the levels of the Integrated Wastewater Discharge Standard(GB3838—1988) of China and 96h acute toxicity test,six test concentrations,i.e.0.005,0.01,0.05,0.1,0.5,1.0 mg/L,were adopted for the chronic toxicity test by epidermal uptake of CdCl2 solution.140 adult male frogs were randomly divided into seven groups,each group embraced twenty.The group treated only with distilled water was set aside as control.All frogs were sacrificed and the testis tissues were collected in the wake of 30 days treatment for the measurement of reactive oxygen species(ROS),protein carbonylation(PCO) and DNA-protein crosslinks(DPCs) levels.The results showed that with the increasing of CdCl2 concentrations,ROS of test groups became higher than that of control group.In 0.5 mg/L or 1.0 mg/L CdCl2 groups,ROS increased significantly in comparison with control group(P0.05).The content of PCO increased notably along with the increasing of Cd2+,taken on strong dose-dependent relationship(R2=0.9603,P0.05).In the presence of 0.5mg/L or 1.0mg/L of Cd2+,PCO content was significantly higher than that of control group(P0.05).Cd2+ also induced DPCs levels increasing,which showed obvious dose-dependent relationship(R2=0.909,P0.05).Compared with control group,we observed significantly difference when the groups treated with 0.5mg/L and 1.0mg/L Cd2+.Regression analysis showed that quadratic curve regression were found between PCO content and ROS levels(R2=0.5547),while linear regression occurred between DPCs levels and PCO content(R2=0.8394).Above results indicated that ROS accumulation could induce the PCO content increasing and the PCO might be the upstream signal for Cd-induced DNA damage in the testes of R.nigromaculata.Finally,we proposed that ROS introduced by cadmium played a central role in male reproductive toxicity and ROS may induce the cascade reaction of protein oxidation damage and DNA impairment.

Protective effect of probiotic bacteria against cadmium-induced genotoxicity in rat hepatocytes in vivo and in vitro

The protective effect of probiotic bacteria against cadmium (Cd)-induced genotoxicity was studied in rat hepatocytes in vivo and in vitro. Male Wistar rats, Rattus norvegicus, were treated for five weeks with (i) CdCl2 (70 ppm in the drinking water), (ii) a mixture of lyophilized probiotic bacteria Lactobacillus rhamnosus, L. acidophilus and Bifido-bacterium longum (5?108 cfu/g of food), or (iii) CdCl2 and probiotic bacteria. In addition, single cells obtained from the untreated rat liver were exposed to CdCl2 (70 ppm), probiotic bacteria (1.28 mg/ml), or CdCl2 and probiotic bacteria, for 15 min at 22?C in the dark. The level of Cd-induced DNA damage in hepatocytes was determined by the comet assay. The obtained results show that probiotic bacteria significantly reduced Cd-induced genotoxicity, both in vivo and in vitro (20% and 48%, respectively). Moreover, the toxicity of Cd to lactobacilli in the gastrointestinal tracts of rats was significantly decreased in the probiotic-treated animals. The binding of Cd2+ to probiotic bacteria was proposed as the most probable protection mechanism.

Low levels of cadmium exposure induce DNA damage and oxidative stress in the liver of Oujiang colored common carp Cyprinus carpio var. color

Cadmium (Cd) compounds are widely distributed toxic environmental and industrial pollutants, and they may bring danger to growth and development of aquatic organisms. In China, the Oujiang color common carp, Cyprinus carpio var. color, is a very important fish, from an economic point of view, and is well used for fish culture in paddy fields. The purpose of this study was to show the low concentrations of cadmium-induced oxidative stress response and DNA damage in the livers of Cyprinus carpio var. color. Superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione (GSH) in liver were measured after exposure to Cd levels (0.41, 0.52, 0.69, 1.03 and 2.06mg/L, respectively) for 7days and compared with the control groups. DNA damage, including indicators of damage percentage, DNA tail length (TL) and DNA tail moment (TM) were also analyzed by comet assays. Results showed that MDA and GSH levels in all treatment groups increased significantly relative to the controls (P<0.01). Treatment with Cd at concentration of 0.41mg/L increased SOD activity, while treatment with Cd at concentrations >0.41mg/L inhibited SOD activities. DNA damage percentage, TL and TM also significantly increased when the Cd level was >0.41mg/L. Positive correlations were also found between DNA damage levels and MDA levels (r=0.74 for DNA damage percentage, r=0.83 for TL, r=0.84 for TM; P<0.01 for all) as well as between GSH and MDA levels (r=0.77, P<0.01). These results strongly suggested that Cd-induced DNA damage in the livers of Cyprinus carpio var. color was due to lipid peroxidation and oxidative stress.

Evaluation of the alkaline Comet assay conducted with the wetlands plant Bacopa monnieri L. as a model for ecogenotoxicity assessment

Wetlands play a key role in maintaining environmental quality, and wetlands plants could serve as model organisms for determining the genotoxic effects of pollutants contaminating these areas. In the present study, DNA damage was evaluated in a wetlands plant, Bacopa monnieri L., as a potential tool for the assessment of ecogenotoxicity. The Comet assay was used for detecting DNA damage in B. monnieri exposed to two model mutagens, ethyl methanesulfonate (EMS) and methyl methanesulfonate (MMS). Significant (P < 0.05) dose-dependent increases in DNA damage were observed following treatments conducted by exposing both isolated nuclei (acellular or in vitro exposure) and whole plants (in vivo exposure) to 0.01-5 mM EMS and 0.05-100 microM MMS for 2 hr at (26 +/- 2) degrees C. The assay was then used to evaluate the genotoxic potential of cadmium (Cd), a wetlands contaminant. In vitro exposure of nuclei from untreated leaves to 0.001-200 microM Cd for 2 hr resulted in significant (P < 0.05) levels of DNA damage. Cd concentrations >or=0.01 microM induced DNA damage as evidenced by increases in the Olive tail moment. In vivo exposure of plants to 0.01-500 microM Cd for 2, 4, and 18 hr resulted in dose- and time-dependent increases in DNA damage in the nuclei isolated from roots and leaves. Cd-induced DNA damage was greater in roots than leaves. To our knowledge, this is the first report describing the use of a wetlands plant for genotoxicity assessment, using the Comet assay.

Influence of cadmium on murine thymocytes: Potentiation of apoptosis and oxidative stress

Cadmium (Cd) is a well-known environmental carcinogen and a potent immunotoxicant. It induces thymocyte apoptosis in vitro. However, the mode of action is unclear. In this study, we examined the effect of Cd (10, 25 and 50 μM) on mitochondrial membrane potential and caspase-3 as well as oxidative stress markers in murine thymocytes. The cadmium induced apoptosis occurred in a concentration and time dependent manner. The early markers of apoptosis-loss in mitochondrial membrane potential and caspase-3 activation were evident as early as 1.5 h by 50 μM Cd. Enhanced reactive oxygen species (ROS) generation and glutathione (GSH) depletion were observed at 60 min, prior to the lowering of mitochondrial membrane potential. The Cd induced DNA damage as depicted by internucleosomal fragmentation on agarose and histone associated mono- and oligonucleosomes detection by ELISA, corrobated with the apoptotic DNA (sub-G 1 population) and total apoptotic cells by Annexin V binding assay. The number of cells in sub-G 1 population increased to 66% at 50 μM Cd concentration and the distribution of early and late apoptotic cells was 47% and 15%, respectively. Addition of N-acetylcysteine and pyrrolidine dithiocarbamate (thiol antioxidants) to the Cd treated cells, lowered the sub-G 1 population, inhibited the ROS generation and raised the GSH levels. Buthionine sulfoximine (GSH depletor) on the other hand, enhanced both the ROS production and the sub-G 1 fraction. These results clearly demonstrate the apoptogenic potential of Cd in murine thymocytes, following mitochondrial membrane depolarization, caspase activation and ROS and GSH acting as critical mediators.