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Alternative Tumor-Targeting Strategies
Published in David E. Thurston, Ilona Pysz, Chemistry and Pharmacology of Anticancer Drugs, 2021
DNA interstrand cross-links, the cytotoxic lesions produced by these types of mustard prodrugs, can be observed in individual cells by a modification of the COMET assay, and the extent of genomic damage at any time can be defined for a population of tumor or normal cells. Experiments with the ZD2767/ADEPT system in tissue culture showed that cross-links were produced in colon carcinoma cells in a dose-dependent fashion that related to the degree of cell kill. However, the cross-links were repaired within 24 hours in a sufficient proportion of cells, suggesting a high probability of tumor regrowth in the clinic. This finding was supported by similar results in mice bearing xenografts of human colon carcinoma. Therefore, a third clinical trial utilizing ZD2767 focused on studying DNA cross-links. Tumor biopsies and peripheral blood lymphocytes were obtained on a small number of patients and subjected to the COMET assay. One patient with evidence of tumor response had extensive cross-linking in tumor cells but no increase over controls in lymphocytes. As anticipated, a patient with no evidence of response had no significant cross-links in either the tumor or lymphocyte cells. The fact that tumor progression followed in the patient with evidence of response was consistent with human tumor xenograft model findings of the repair of cross-links induced by ZD2767.
Role of Sperm DNA Damage in Male Infertility Assessment
Published in Botros Rizk, Ashok Agarwal, Edmund S. Sabanegh, Male Infertility in Reproductive Medicine, 2019
Saradha Baskaran, Chak-Lam Cho, Ashok Agarwal
The Comet assay measures the amount of DNA damage per spermatozoon [50]. The steps involved are embedding cells in agarose, lysis under neutral or alkaline conditions, subjecting the lysed cells to electrophoresis, DNA staining, and microscopic image analysis. The principle underlying the assay involves unwinding the tightly packed sperm nucleus and subjecting the spermatozoon to electrophoresis that enables the detection of the amount of fragmented DNA that migrates away from the sperm head. The damaged cell resembles a comet with the fragmented DNA in the comet’s tail and the intact DNA in the head. The staining intensity and length of the comet’s tail indicate the degree of SDF. In contrast to other DNA fragmentation assays, Comet assay provides qualitative and quantitative insight into DNA damage on a single cell, instead of providing a proportion of DNA-damaged cells [51]. Comet detects ss and ds breaks, as well as abasic sites. It has higher sensitivity and specificity than other DNA fragmentation tests (SCD, TUNEL, and SCSA). In addition, it requires only a small number of cells for the evaluation of DNA fragmentation; thus, it can be employed in cases of severe oligozoospermia [52].
Electromagnetic Emanations from Power Sources and Fixed Specialized Equipment
Published in William J. Rea, EMF Effects from Power Sources and Electrosmog, 2018
A recent review of available epidemiologic studies concluded that the use of mobile phones for <10 years is associated with increased risk of ipsilateral gliomas and acoustic neuromas.87 For a long time, stem cells have been considered an important cellular target for the origination of cancer—both tumors and leukemia.88,89 Gliomas are believed to originate from stem cells in the brain.90 DNA double-strand breaks (DSBs) and their misrepair are critical molecular events resulting in chromosomal aberrations, which have often been associated with the origination of various leukemias and tumors, including gliomas.91 Only one study on possible MW-induced DSBs in stem cells is available.92 Surprisingly, the data obtained in that study by the neutral comet assay suggested that prolonged exposure time abolished the DSB formation observed at the shorter exposure time. Furthermore, the neutral comet assay has limited applicability to detect double standard breaks because similar increases in comet tails may be also caused by nongenotoxic effects that imply changes in chromatin conformation, such as relaxation of DNA loops.93
Polystyrene nanoparticles: the mechanism of their genotoxicity in human peripheral blood mononuclear cells
Published in Nanotoxicology, 2022
Kinga Malinowska, Bożena Bukowska, Ireneusz Piwoński, Marek Foksiński, Aneta Kisielewska, Ewelina Zarakowska, Daniel Gackowski, Paulina Sicińska
Modification of the comet assay using restriction enzymes, i.e. endonuclease III (EndoIII) and formamidopirymidyne DNA glycosylase (Fpg) allows for detection of oxidatively modified purines and pyrimidines, respectively. The course of the experiment after the 24-h incubation of the tested cells with NPs was the same as in section 2.4.1, up to cell lysis. After lysis, slides were washed several times with HEPES buffer (40 mM HEPES-KOH, 0.5 mM Na2EDTA, 0.1 KCl, 0.2 mg/mL BSA, pH 9). Then, 50 µL of a buffer containing 1 U EndoIII or Fpg was applied to each slide. Slides were covered with coverslips and incubated at 37 °C for 30 min in a moist chamber. Finally, the coverslips used in the previous step were removed. The obtained results of DNA strand breaks formation and DNA base oxidation were expressed as percent of DNA in the comet tail versus the concentration of the individual tested substance. For this analysis, the alkaline version of the comet assay was used. Based on literature data, we decided to use 1 U of each enzyme per gel, which guaranteed their utilization in excess (therefore, the calibration curve was not prepared; Czarny et al. 2015).
The cytotoxic and genotoxic effects of daidzein on MIA PaCa-2 human pancreatic carcinoma cells and HT-29 human colon cancer cells
Published in Drug and Chemical Toxicology, 2020
Gulsah Gundogdu, Yavuz Dodurga, Meltem Cetin, Mucahit Secme, Betul Cicek
In our study, the Comet assay was used to evaluate the DNA damage in MIA PaCa-2 and HT-29 cells caused by DZ. The Comet assay is a useful and sensitive assay for the detection of DNA damage and toxicity at the cellular level. In this assay, the intact DNA is separated from the damaged cellular DNA under an electrophoretic field (Iovine et al.2014). When the cell lines were treated with 200 μM of DZ for 48 h, DNA damage was observed in both cell lines (Figure 3(B,D)). DNA-TL, DNA-TI, and DNA-TM increased more in MIA PaCa-2 cells treated with 200 μM of DZ for 48 h than those in the control cells. DNA-TI and DNA-TM exhibited higher increases in HT-29 cells treated with 200 μM of DZ for 48 h than those in the control cells. When DNA-TL was evaluated, there was no significant difference between the DNA-TL of HT-29 cells and the DNA-TL of control cells. A long and intense tail indicates that DNA fragmentation has occurred. In principle, DNA-TI can take any value from 0%–100%. 0% DNA-TI indicates that there is no DNA damage in the cells and the increasing positive values of DNA-TI indicates DNA damage in the respective cells. Dead or dying cells are represented by ≥85% DNA-TI (Bright et al.2011). In this study, we found that the DNA-TIs for HT-29 and MIA PaCa-2 cell lines treated with DZ had values of 30.73% and 18.38%, respectively.
Characterization and cytotoxicity assessment of nargile smoke using dynamic exposure
Published in Inhalation Toxicology, 2019
Christian Khalil, Joe Braham Chahine, Brenda Chahla, Tamara Hobeika, Rony S. Khnayzer
The comet assay is a technique to measure DNA damage in exposed cells as previously described (Soni et al. 2010; Khalil 2015; Khalil and Shebaby 2017). The kit provided by Trevigen was utilized according to manufacturer instructions. In summary, the agarose provided in the kit was incubated at 37 °C in a water bath prior to use. The cells were counted and a density of 105 cells/ml was added to the molten agarose (1:10 v/v). This was followed by pipetting the mixtures to the comet slides provided. The slides where then stored in the fridge for 30 min to allow the agarose to solidify. Upon agarose, solidification slides were immersed in pre-chilled lysis solution for one hour. (Al Hageh et al. 2018). This was followed by alkaline phosphatase treatment, followed by Tris base, boric acid and EDTA (TBE) buffer immersion prior to electrophoresis at 1 V/cm for 10 min. Upon electrophoresis; slides were subjected to ethanol treatment then dried prior to silver staining for comet visualization. The silver stained slides were further analyzed using Comet Analysis Software Package. The generated parameter data by the software was used to calculate the Tail Moment Index (TMI). The TMI calculation consisted of the average of tail DNA values multiplied by average tail length divided by 1000.