Induction of micronuclei by zinc in human leukocytes: a study using cytokinesis-block micronucleus assay.
TL;DR: The results show a significant (p ≤ 0.001) increase of micronucleated cytokinesis-blocked cells (MNCBs) in zinc-chloride-treated cells compared to the negative control, which is the first to describe the efficiency of cytokinesIS-block micron nucleus assay to evaluate the genotoxic effects of zinc salt.
Abstract: In the present study, we report the results of the capability of zinc chloride for the induction of micronuclei in cultured human leukocytes using cytokinesis-block micronucleus assay. Two concentrations of zinc chloride (1.5 × 10−4
M and 3.0 × 10−4
M) were used to evaluate the potential of this zinc salt to induce micronucleus formation. This effect was compared with positive (mitomycin C treated) and negative controls (no salt added). Our results show a significant (p ≤ 0.001) increase of micronucleated cytokinesis-blocked cells (MNCBs) in zinc-chloride-treated cells compared to the negative control. Induction of MNCBs was not in a dose-dependent manner for zinc chloride concentrations tested. This report is the first to describe the efficiency of cytokinesis-block micronucleus assay to evaluate the genotoxic effects of zinc salt.
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TL;DR: The role of Zn in the maintenance of genome integrity and the effects of deficiency or excess on genomic stability events and cell death are focused on.
Abstract: Zinc (Zn) is an essential trace element required for maintaining both optimal human health and genomic stability. Zn plays a critical role in the regulation of DNA repair mechanisms, cell proliferation, differentiation and apoptosis involving the action of various transcriptional factors and DNA or RNA polymerases. Zn is an essential cofactor or structural component for important antioxidant defence proteins and DNA repair enzymes such as Cu/Zn SOD, OGG1, APE and PARP and may also affect activities of enzymes such as BHMT and MTR involved in methylation reactions in the folate-methionine cycle. This review focuses on the role of Zn in the maintenance of genome integrity and the effects of deficiency or excess on genomic stability events and cell death.
132 citations
TL;DR: It is suggested that maintaining Zn concentrations within the range of 4–16 μM is essential for DNA damage prevention in cultured human oral keratinocytes.
Abstract: Zinc (Zn) is an essential component of Zn-finger proteins and acts as a cofactor for enzymes required for cellular metabolism and in the maintenance of DNA integrity The study investigated the genotoxic and cytotoxic effects of Zn deficiency or excess in a primary human oral keratinocyte cell line and determined the optimal concentration of two Zn compounds (Zn Sulphate (ZnSO4) and Zn Carnosine (ZnC)) to minimise DNA damage Zn-deficient medium (0 μM) was produced using Chelex treatment, and the two Zn compounds ZnSO4 and ZnC were tested at concentrations of 00, 04, 40, 160, 320 and 1000 μM Cell viability was decreased in Zn-depleted cells (0 μM) as well as at 32 μM and 100 μM for both Zn compounds (P < 00001) as measured via the MTT assay DNA strand breaks, as measured by the comet assay, were found to be increased in Zn-depleted cells compared with the other treatment groups (P < 005) The Cytokinesis Block Micronucleus Cytome assay showed a significant increase in the frequency of both apoptotic and necrotic cells under Zn-deficient conditions (P < 005) Furthermore, elevated frequencies of micronuclei (MNi), nucleoplasmic bridges (NPBs) and nuclear buds (NBuds) were observed at 0 and 04 μM Zn, whereas these biomarkers were minimised for both Zn compounds at 4 and 16 μM Zn (P < 005), suggesting these concentrations are optimal to maintain genome stability Expression of PARP, p53 and OGG1 measured by western blotting was increased in Zn-depleted cells indicating that DNA repair mechanisms are activated These results suggest that maintaining Zn concentrations within the range of 4–16 μM is essential for DNA damage prevention in cultured human oral keratinocytes
46 citations
Cites background or result from "Induction of micronuclei by zinc in..."
...In contrast, Santra et al. (2002) showed that induction of MNi in Zn chloride-treated human lymphocytes at 0.15 and 0.3 mM is significant compared with negative controls, but this did not occur in a dose-dependent manner (Santra et al. 2002)....
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...3 mM is significant compared with negative controls, but this did not occur in a dose-dependent manner (Santra et al. 2002)....
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TL;DR: The results suggest that Zn plays important role in genomic stability and that the optimal Zn concentration-range for prevention of DNA damage and cytotoxicity in vitro lies between 4 and 16μM.
Abstract: Zinc (Zn) is an essential cofactor required by numerous enzymes that are essential for cell metabolism and the maintenance of DNA integrity. We investigated the effect of Zn deficiency or excess on genomic instability events and determined the optimal concentration of two Zn compounds that minimize DNA-damage events. The effects of Zn sulphate (ZnSO(4)) and Zn carnosine (ZnC) on cell proliferation were investigated in the WIL2-NS human lymphoblastoid cell line. DNA damage was determined by the use of both the comet assay and the cytokinesis-block micronucleus cytome (CBMN-Cyt) assay. Zn-deficient medium (0μM) was produced using Chelex treatment, and the two Zn compounds (i.e. ZnSO(4) and ZnC) were tested at concentrations of 0.0, 0.4, 4.0, 16.0, 32.0 and 100.0μM. Results from an MTT assay showed that cell growth and viability were decreased in Zn-depleted cells (0μM) as well as at 32μM and 100μM for both Zn compounds (P<0.0001). DNA strand-breaks, as measured by the comet assay, were found to be increased in Zn-depleted cells compared with the other treatment groups (P<0.05). The CBMN-Cyt assay showed a significant increase in the frequency of both apoptotic and necrotic cells under Zn-deficient conditions (P<0.0001). Elevated frequencies of micronuclei (MNi), nucleoplasmic bridges (NPBs) and nuclear buds (NBuds) were induced in Zn-depleted cells (P<0.0001), whereas genome damage was reduced in supplemented cultures for both Zn compounds at 4μM and 16μM, possibly suggesting that these concentrations may be optimal for genome stability. The potential protective effect of ZnSO(4) and ZnC was also investigated following exposure to 1.0Gy γ-radiation. Culture in medium containing these compounds at 4-32μM prior to irradiation displayed significantly reduced frequencies of MNi, NPBs and NBuds compared with cells maintained in 0μM medium (P<0.0001). Expression of γ-H2AX and 8-oxoguanine glycosylase measured by western blotting was increased in Zn-depleted cells. These results suggest that Zn plays important role in genomic stability and that the optimal Zn concentration-range for prevention of DNA damage and cytotoxicity in vitro lies between 4 and 16μM.
41 citations
TL;DR: The results reveal a synergistic activity with a 1:1 ratio of both complexes in the monolayer and multicellular spheroids.
Abstract: The aim of this work was to study the antitumor effects and the mechanisms of toxic action of a series of 6-methoxyquinoline (6MQ) complexes in vitro. The Cu(II) and Zn(II) complexes (Cu6MQ and Zn6MQ) are formulated as M(6MQ)2Cl2; the Co(II) and Ag(I) compounds (Co6MQ and Ag6MQ) are ionic with formulae [Ag(6MQ)2]+NO3− and H(6MQ)+[Co(6MQ)Cl3]− (where H(6MQ)+ is the protonated ligand). We found that the copper complex, outperformed the Co(II), Zn(II) and Ag(I) complexes with a lower IC50 (57.9 µM) in A549 cells exposed for 24 h. Cu6MQ decreased cell proliferation and induced oxidative stress detected with H2DCFDA at 40 µM, which reduces GSH/GSSG ratio. This redox imbalance induced oxidative DNA damage revealed by the Micronucleus test and the Comet assay, which turned into a cell cycle arrest at G2/M phase and induced apoptosis. In multicellular spheroids, the IC50 values tripled the monolayer model (187.3 µM for 24 h). At this concentration, the proportion of live/dead cells diminished, and the spheroids could not proliferate or invade. Although Zn6MQ also decreased GSH/GSSG ratio from 200 µM and the cytotoxicity is related to oxidative stress, the induction of the hydrogen peroxide levels only doubled the control value. Zn6MQ induced S phase arrest, which relates with the increased micronucleus frequency and with the induction of necrosis. Finally, our results reveal a synergistic activity with a 1:1 ratio of both complexes in the monolayer and multicellular spheroids.
2 citations
Cites background from "Induction of micronuclei by zinc in..."
...It has been shown that Zn cation induces micronuclei in human leucocytes in the same range of concentrations and not in a dose-dependent manner [51]....
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Dissertation•
01 Jan 2012
TL;DR: The role of nutrition in Genomic Stability and cancer; the role of Nutrition in WIL2-NS Lymphoblastoid Cell Line and the Effect of Zinc Sulphate and Zinc Carnosine on Genome Stability and Cytotoxicity is investigated.
Abstract: viii DECLARATION ix ACKNOWLEDGEMENTS x PRESENTATIONS AND PUBLICATIONS ARISING FROM THE THESIS xii LIST OF ABBREVIATIONS xv Chapter 1: The Role of Zinc in Genomic Stability 1 1.1 Abstract 3 1.2 Introduction 3 1.2.1 Genomic stability and cancer; the role of nutrition 3 1.2.2 Zinc functions 5 1.3 Zinc deficiency, DNA damage and chromosomal instability 13 1.4 Zinc excess, DNA damage and toxicity 20 1.5 Zinc and telomeres 27 1.6 Knowledge gaps and future directions 26 Chapter 2: Aims, Hypotheses and Models 30 2.1 Aims and hypotheses 31 2.2 Experimental models 31 2.2.1 In vitro model 31 2.2.2 In vivo model 34 Chapter 3: The Effect of Zinc Sulphate and Zinc Carnosine on Genome Stability and Cytotoxicity in WIL2-NS Lymphoblastoid Cell Line 35 3.1 Abstract 38 3.2 Introduction 39 3.3 Materials and methods 42
2 citations
Cites background from "Induction of micronuclei by zinc in..."
...In contrast, Santra et al (2002) showed that induction of MNi in Zn chloride treated human leukocytes at 15 mM and 30 mM was significant when compared to negative controls, but that this was not in a linear dose-dependent manner [91]....
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...3 mM is significant compared to negative controls, but this did not occur in a dose-dependent manner [91]....
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...An earlier study in 2002, showed the efficiency of cytokinesis-block micronucleus assay as a means to evaluate the genotoxic effects of Zn excess [91]....
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References
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TL;DR: A combination of cytological and leukocyte culture techniques is described which constitutes a convenient, reliable approach for chromosome studies of humans and yields the following advantages: relative ease of obtaining blood and small volume required.
4,054 citations
TL;DR: In its current basic form the CBMN assay can provide the following measures of genotoxicity and cytotoxicity: chromosome breakage, chromosome loss, chromosome rearrangement, cell division inhibition, necrosis and apoptosis.
Abstract: The study of DNA damage at the chromosome level is an essential part of genetic toxicology because chromosomal mutation is an important event in carcinogenesis. The micronucleus assays have emerged as one of the preferred methods for assessing chromosome damage because they enable both chromosome loss and chromosome breakage to be measured reliably. Because micronuclei can only be expressed in cells that complete nuclear division a special method was developed that identifies such cells by their binucleate appearance when blocked from performing cytokinesis by cytochalasin-B (Cyt-B), a microfilament-assembly inhibitor. The cytokinesis-block micronucleus (CBMN) assay allows better precision because the data obtained are not confounded by altered cell division kinetics caused by cytotoxicity of agents tested or sub-optimal cell culture conditions. The method is now applied to various cell types for population monitoring of genetic damage, screening of chemicals for genotoxic potential and for specific purposes such as the prediction of the radiosensitivity of tumours and the inter-individual variation in radiosensitivity. In its current basic form the CBMN assay can provide, using simple morphological criteria, the following measures of genotoxicity and cytotoxicity: chromosome breakage, chromosome loss, chromosome rearrangement (nucleoplasmic bridges), cell division inhibition, necrosis and apoptosis. The cytosine-arabinoside modification of the CBMN assay allows for measurement of excision repairable lesions. The use of molecular probes enables chromosome loss to be distinguished from chromosome breakage and importantly non-disjunction in non-micronucleated binucleated cells can be efficiently measured. The in vitro CBMN technique, therefore, provides multiple and complementary measures of genotoxicity and cytotoxicity which can be achieved with relative ease within one system. The basic principles and methods (including detailed scoring criteria for all the genotoxicity and cytotoxicity end-points) of the CBMN assay are described and areas for future development identified.
2,287 citations
Book•
01 Jan 1973
TL;DR: In this article, the authors introduce the concept of biostatistics and introduce the Biostatistic Approach to Biometrics (BAPB) as a method for biometrics.
Abstract: Introduction to Biostatistics , Introduction to Biostatistics , کتابخانه مرکزی دانشگاه علوم پزشکی تهران
1,874 citations
TL;DR: The cytokinesis-block method appears to be the procedure of choice for quantitating micronuclei in lymphocytes and was of no value for measuring pre-existing chromosomal damage present in vivo.
Abstract: The micronucleus technique has been proposed as a method for measurement of chromosomal damage in mitogen-stimulated human lymphocytes. Micronuclei require one cell division to be expressed and, consequently, the conventional micronucleus technique is very imprecise since the cells which have undergone only one division, and the micronuclei in them, cannot be identified separately from the total population of lymphocytes. To overcome this problem, two methods were developed to identify cells which have undergone their first mitosis. Using an autoradiographic technique, lymphocytes were pulse-labelled with [3H]thymidine at 48 h of culture, allowed to proceed through mitosis, identified by autoradiography between 72 and 84 h and micronuclei were scored in them. It was not possible to select a concentration of radiolabel which did not itself produce micronuclei and consequently the method was of no value for measuring pre-existing chromosomal damage present in vivo. However, it was capable of quantitating micronuclei produced by irradiation of lymphocytes in vitro. In the second method, cytokinesis was blocked using cytochalasin B. Micronuclei were scored in cytokinesis-blocked cells. These were easily recognisable owing to their binucleate appearance and a large number could be accumulated by adding 3.0 micrograms/ml cytochalasin B at 44 h and scoring at 72 h. Cytochalasin B did not itself produce micronuclei. The cytokinesis-block method was simple to perform; the 'in vivo' micronucleus frequency in normal individuals was 4.4 +/- 2.6 micronuclei/500 cytokinesis-blocked cells; and for lymphocytes irradiated in vitro there was a linear relationship between dose of radiation and number of induced micronuclei. The cytokinesis-block method appears to be the procedure of choice for quantitating micronuclei in lymphocytes.
1,764 citations
"Induction of micronuclei by zinc in..." refers methods in this paper
...One thousand binucleated cells were scanned for each concentration of chemicals for counting the number of micronucleated cytokinesisblocked cells (MNCBs) in each subject (23)....
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TL;DR: The mechanism of zinc sensors that control metal uptake or export in Escherichia coli are determined and their response against the thermodynamically defined free zinc concentration suggests an extraordinary intracellular zinc-binding capacity.
Abstract: Intracellular zinc is thought to be available in a cytosolic pool of free or loosely bound Zn(II) ions in the micromolar to picomolar range To test this, we determined the mechanism of zinc sensors that control metal uptake or export in Escherichia coli and calibrated their response against the thermodynamically defined free zinc concentration Whereas the cellular zinc quota is millimolar, free Zn(II) concentrations that trigger transcription of zinc uptake or efflux machinery are femtomolar, or six orders of magnitude less than one atom per cell This is not consistent with a cytosolic pool of free Zn(II) and suggests an extraordinary intracellular zinc-binding capacity Thus, cells exert tight control over cytosolic metal concentrations, even for relatively low-toxicity metals such as zinc
1,389 citations