Genotoxic Effect of Arsenate and Arsenite in Human HaCat Cells in Culture Using Comet Assay

Arsenic is an environmental chemical of toxicological concern today since it is a human genotoxin and chronic exposure is associated with development of cancers, including skin. Inorganic arsenate is metabolically reduced to arsenite by glutathione (GSH) prior to methylation. The aim of this study was to determine the relative toxic effects of arsenate and arsenite in HaCat cells (immortalized human keratinocytes) in vitro by measuring cytotoxicity, DNA damage, depletion of glutathione and apoptotic and necrotic events. HaCat cells were treated with arsenate and arsenite (10 μM) for DNA damage detection using Comet assay and cytotoxicity (10, 60 and 100 μM) all measured at 24 hr. In some experiment arsenate or arsenite (10 μM) was added at the same time as BSO 10 μM for 24 hr, and GSH levels were measured by HPLC with fluorescence detection. Flow cytometry was used to investigate apoptotic and necrotic events following arsenate and arsenite (10 μM) treatment for 24 hr. Arsenate and arsenite at 60 and 100 μM, but not 10 μM, reduced the number of adherent viable cells with time. Therefore, DNA damage could only be measured at 10 μM as at higher concentrations the cells did not produce classical Comets but showed fragmentation. DNA damage was significantly (p < 0.001) increased in cells treated for 24 hr with 10 μM arsenate and arsenite compared to control. GSH levels were significantly increased in HaCat cells treated with10 μM arsenate and arsenite (p < 0.05, p < 0.001, respectively) compared to control. Cells treated with buthionine sulphoximine (BSO) at the same time as arsenate had increased GSH levels (p < 0.001), but arsenite and BSO did not increase cellular GSH. Arsenate and arsenite increased apoptosis, and arsenate increased necrosis, although none of the values reached statistical significance. Arsenite was more cytotoxic than arsenate. Arsenate and arsenite are known to produce oxidative stress involving ROS formation and depletion of glutathione. The increase in GSH levels at low doses of arsenate and arsenite, and by arsenate even in the presence of BSO.

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DNA damage induced by coal dust, fly and bottom ash from coal combustion evaluated using the micronucleus test and comet assay in vitro

Journal of Hazardous Materials ◽

10.1016/j.jhazmat.2016.11.062 ◽

2017 ◽

Vol 324 ◽

pp. 781-788 ◽

Cited By ~ 30

Author(s):

Cristina Araujo Matzenbacher ◽

Ana Letícia Hilario Garcia ◽

Marcela Silva dos Santos ◽

Caroline Cardoso Nicolau ◽

Suziane Premoli ◽

...

Keyword(s):

Dna Damage ◽

Comet Assay ◽

Coal Combustion ◽

Micronucleus Test ◽

Coal Dust ◽

Bottom Ash

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Assessment of Cytogenetic Aberrations and Comet Assay in Colorectal Adenocarcinomas

Tumori Journal ◽

10.1177/030089160308900314 ◽

2003 ◽

Vol 89 (3) ◽

pp. 305-310 ◽

Cited By ~ 2

Author(s):

Volkan Baltaci ◽

Semra Sardas ◽

Bulent Aytac ◽

Sami Cakar ◽

Ali Esat Karakaya

Keyword(s):

Dna Damage ◽

Comet Assay ◽

Statistical Significance ◽

Tumor Stage ◽

Colorectal Carcinomas ◽

Cytogenetic Aberrations ◽

Significant Difference ◽

Structural Abnormalities ◽

Clinicopathologic Parameters ◽

Colorectal Adenocarcinomas

Aims, Background and Study Design Few studies have investigated the karyotypes of colorectal carcinomas with emphasis on the correlation between cytogenetic findings and clinicopathologic features. The aim of our study involving 20 colorectal adenocarcinomas was to determine their genomic alterations at the chromosomal level by correlating the cytogenetic findings with the extent of DNA damage and clinicopathologic parameters and to compare the results with those of healthy controls. Results Cytogenetic evaluation of patients and controls revealed 10 abnormal karyotypes in patients with adenocarcinomas located in the rectum, sigmoid and rectosigmoid regions. Four had numerical and six had structural abnormalities. Conclusions Statistical analysis revealed a significant difference compared with controls (P <0.01). The karyotypes and the extent of DNA damage assessed by the comet assay were also significantly correlated with tumor stage (P <0.01) using the Kruskal-Wallis non-parametric test, while no statistical significance was observed in relation to patient age and smoking.

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Corrigendum to: Standardisation of the in vitro comet assay: influence of lysis time and lysis solution composition on the detection of DNA damage induced by X-rays

Mutagenesis ◽

10.1093/mutage/gez036 ◽

2019 ◽

Vol 34 (5-6) ◽

pp. 431-431

Author(s):

José M Enciso ◽

Kristine B Gutzkow ◽

Gunnar Brunborg ◽

Ann-Karin Olsen ◽

Adela López de Cerain ◽

...

Keyword(s):

Dna Damage ◽

Comet Assay ◽

X Rays ◽

Solution Composition ◽

Lysis Time

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HaCaT Cells as a Reliable In Vitro Differentiation Model to Dissect the Inflammatory/Repair Response of Human Keratinocytes

Mediators of Inflammation ◽

10.1155/2017/7435621 ◽

2017 ◽

Vol 2017 ◽

pp. 1-12 ◽

Cited By ~ 43

Author(s):

Irma Colombo ◽

Enrico Sangiovanni ◽

Roberta Maggio ◽

Carlo Mattozzi ◽

Stefania Zava ◽

...

Keyword(s):

Cell Density ◽

Skin Diseases ◽

Inflammatory Responses ◽

Human Keratinocytes ◽

Suitable Model ◽

Hacat Cells ◽

Primary Human Keratinocytes ◽

Proinflammatory Mediators ◽

Cytokines And Chemokines

Cultured primary human keratinocytes are frequently employed for studies of immunological and inflammatory responses; however, interpretation of experimental data may be complicated by donor to donor variability, the relatively short culture lifetime, and variations between passages. To standardize the in vitro studies on keratinocytes, we investigated the use of HaCaT cells, a long-lived, spontaneously immortalized human keratinocyte line which is able to differentiate in vitro, as a suitable model to follow the release of inflammatory and repair mediators in response to TNFα or IL-1β. Different treatment conditions (presence or absence of serum) and differentiation stimuli (increase in cell density as a function of time in culture and elevation of extracellular calcium) were considered. ELISA and Multiplex measurement technologies were used to monitor the production of cytokines and chemokines. Taken together, the results highlight that Ca2+ concentration in the medium, cell density, and presence of serum influences at different levels the release of proinflammatory mediators by HaCaT cells. Moreover, HaCaT cells maintained in low Ca2+ medium and 80% confluent are similar to normal keratinocytes in terms of cytokine production suggesting that HaCaT cells may be a useful model to investigate anti-inflammatory interventions/therapies on skin diseases.

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Bioimpedance Analysis of L929 and HaCaT Cells in Low Frequency Range

Current Directions in Biomedical Engineering ◽

10.1515/cdbme-2018-0029 ◽

2018 ◽

Vol 4 (1) ◽

pp. 115-118 ◽

Cited By ~ 5

Author(s):

Viviane S. Teixeira ◽

Jan-Patrick Kalckhoff ◽

Wolfgang Krautschneider ◽

Dietmar Schroeder

Keyword(s):

Low Frequency ◽

Steel Corrosion ◽

Frequency Dispersion ◽

Human Keratinocytes ◽

Cell Types ◽

Hacat Cells ◽

Frequency Range ◽

Corrosion Resistant ◽

L929 Mouse

AbstractIn this work, Bioimpedance Spectroscopy (BIS) is used to study fluids and cell solutions. A n ew fourelectrode- terminal (4T) chamber using 3D printing and stainless steel corrosion resistant V4A was designed to measure the impedance of live cell solutions at the frequency range 0.1Hz- 1MHz. At f < 1kHz the double layer (DL) that builds at electrode’s surface raises the impedance substantially preventing the observation of the real impedance of the cells. The new 4T design circumvents the DL, is more robust and cheap, and allows for the repeatability of the results. Experiments were performed in vitro with two cell lines, L929 (mouse fibroblasts) and HaCaT (human keratinocytes). Results show that it is possible to distinguish between the two cell types by means of its BIS measurements in the new setup. Also, a low-frequency dispersion (α-dispersion) was observed in HaCaT cells solution, but not in L929. Furthermore, a potentiostat circuit model was developed in LTSpice to simulate the hardware setup and two different circuit models were used to fit cell’s data.

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In vitro evaluation of baseline and induced DNA damage in human sperm exposed to benzo[a]pyrene or its metabolite benzo[a]pyrene-7,8-diol-9,10-epoxide, using the comet assay

Mutagenesis ◽

10.1093/mutage/geq024 ◽

2010 ◽

Vol 25 (4) ◽

pp. 417-425 ◽

Cited By ~ 39

Author(s):

V. Sipinen ◽

J. Laubenthal ◽

A. Baumgartner ◽

E. Cemeli ◽

J. O. Linschooten ◽

...

Keyword(s):

Dna Damage ◽

Comet Assay ◽

Human Sperm ◽

In Vitro Evaluation

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Kaempferol Induces DNA Damage and Inhibits DNA Repair Associated Protein Expressions in Human Promyelocytic Leukemia HL-60 Cells

The American Journal of Chinese Medicine ◽

10.1142/s0192415x1550024x ◽

2015 ◽

Vol 43 (02) ◽

pp. 365-382 ◽

Cited By ~ 20

Author(s):

Lung-Yuan Wu ◽

Hsu-Feng Lu ◽

Yu-Cheng Chou ◽

Yung-Luen Shih ◽

Da-Tian Bau ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Protein Expression ◽

Ataxia Telangiectasia ◽

Repair System ◽

Dapi Staining ◽

Viable Cells ◽

Protein Expressions ◽

Dose Dependent

Numerous evidences have shown that plant flavonoids (naturally occurring substances) have been reported to have chemopreventive activities and protect against experimental carcinogenesis. Kaempferol, one of the flavonoids, is widely distributed in fruits and vegetables, and may have cancer chemopreventive properties. However, the precise underlying mechanism regarding induced DNA damage and suppressed DNA repair system are poorly understood. In this study, we investigated whether kaempferol induced DNA damage and affected DNA repair associated protein expression in human leukemia HL-60 cells in vitro. Percentages of viable cells were measured via a flow cytometry assay. DNA damage was examined by Comet assay and DAPI staining. DNA fragmentation (ladder) was examined by DNA gel electrophoresis. The changes of protein levels associated with DNA repair were examined by Western blotting. Results showed that kaempferol dose-dependently decreased the viable cells. Comet assay indicated that kaempferol induced DNA damage (Comet tail) in a dose-dependent manner and DAPI staining also showed increased doses of kaempferol which led to increased DNA condensation, these effects are all of dose-dependent manners. Western blotting indicated that kaempferol-decreased protein expression associated with DNA repair system, such as phosphate-ataxia-telangiectasia mutated (p-ATM), phosphate-ataxia-telangiectasia and Rad3-related (p-ATR), 14-3-3 proteins sigma (14-3-3σ), DNA-dependent serine/threonine protein kinase (DNA-PK), O6-methylguanine-DNA methyltransferase (MGMT), p53 and MDC1 protein expressions, but increased the protein expression of p-p53 and p-H2AX. Protein translocation was examined by confocal laser microscopy, and we found that kaempferol increased the levels of p-H2AX and p-p53 in HL-60 cells. Taken together, in the present study, we found that kaempferol induced DNA damage and suppressed DNA repair and inhibited DNA repair associated protein expression in HL-60 cells, which may be the factors for kaempferol induced cell death in vitro.

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