The contribution of cytotoxicity to DNA-effects in the single cell gel test (Comet assay)

Universität Ulm, Abteilung Medizinische Genetik, D-89069 Ulm, Germany
Toxicology Letters (Impact Factor: 3.26). 03/1997; 90(2):183-188. DOI: 10.1016/S0378-4274(96)03847-7


We evaluated genotoxic and cytotoxic effects of the three non-mutagenic and non-carcinogenic compounds p-nitrophenol, d-menthol and sodium N-lauroyl sarcosine which have previously been shown to induce DNA double strand breaks (DNA dsb) secondary to induced cytotoxicity. We tested wheter genotoxic effects in the alkaline single cell gel test (comet assay) may be confounded by cytotoxicity-induced DNA dsb. Cell viability was determined at the end of the treatment using the fluorescein diacetate/ethidium bromide-assay and plating efficiency was used as an indicator of long-term survivability. Experiments with V79 Chinese hamster cells and human white blood cells revealed negative results in the comet assay despite strong cytotoxic effects. However, cells with extremely fragmented DNA (‘clouds’) occured but were excluded from the evaluation under the principle that they represent dead cells. We also noticed a significant loss of cells at cytotoxic concentrations that might be attributed to the induction of highly fragmented DNA which is lost during electrophoresis. Since the comet assay allows the determination of DNA effects on the single cell level, a confounding effect of cytotoxicity on test results can be avoided.

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    • "One hundred comet images per slide were randomly captured and analyzed. Only comets that did not overlap and had a clear margin surrounding them were scored [26] "
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    ABSTRACT: Eight chroman-2,4-diones, namely 2a-h, previously investigated as anticoagulants, of which 2a and 2f as the most active, were evaluated as in vivo genotoxic agents in Wistar rat livers and kidneys using the comet assay. Compounds 2a, 2b, and 2f without genotoxic activity were applied prior to ethyl methanesulfonate (EMS) and diminished EMS-induced DNA damage according to the total score and percentage of reduction. EMS produce harmful O(6)-ethylguanine lesion which is incorporated in aberrant genotoxic GT and TG pairing after ATP-dependent DNA strand breaks are catalyzed by rat Topoisomerase IIα (rTopIIα, EC Therefore, the mechanism of 2a, 2b, and 2f antigenotoxic activity was investigated on the enzyme level using molecular docking and molecular dynamics simulations in as much as it had been determined that compounds do not intercalate DNA but instead inhibit the ATPase activity. Calculations predicted that compounds inhibit ATP hydrolysis before rTopIIα has catalyzed DNA-EMS cleavage, prevent EMS mutagenic and carcinogenic effects, and beside anticoagulant activity can even be applied in the cancer treatment to control the rate of anticancer alkylation drugs. Copyright © 2015. Published by Elsevier Inc.
    Biochemical pharmacology 08/2015; 98(1). DOI:10.1016/j.bcp.2015.08.106 · 5.01 Impact Factor
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    • "Comets were captured with 40× objective lens of fluorescence microscope Nikon (Ti-Eclipse) attached to CCD camera. Comets without heads and with nearly all the DNA in the tail were not included in the analysis, because they could represent dead cells (Hartmann and Speit, 1997). Tail moment and % DNA in comet tail was selected as parameter for estimation the degree of DNA damage . "
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    ABSTRACT: The purpose of this study was to evaluate the antioxidant potential of methanol extracts of Gentiana cru-ciata L. aerial parts and roots, as well as the stability of the phenolic compounds and antioxidant capacityof extracts during heating, at different pHs and after an in vitro digestion procedure. Also, their genotox-icity and antigenotoxicity against carbon tetrachloride in the liver of albino Wistar rats using the cometassay were evaluated. Three secoiridoid glycosides (swertiamarin, gentiopicrin, and sweroside) and fourphenolic compounds (orientin, vitexin and two isovitexin-glucosides) were identified as the major con-stituents in aerial parts and roots of G. cruciata, using UHPLC-DAD/±HESI-MS/MS analysis. The results ofantioxidant assays showed that aerial parts displayed higher antioxidant activity compared to the roots,which could be related to higher phenolics content, especially flavonoids. In general, extracts showedpH and thermal stability, while duodenal condition had more influence on total phenolic condition andantioxidant activity of extracts. Both extracts showed a protective effect against CCl4in comet assays.The roots extract showed no genotoxic activity, while aerial parts extract showed slight genotoxicity atconcentrations of 400 mg/kg b.w.
    Industrial Crops and Products 04/2015; 73:49-62. DOI:10.1016/j.indcrop.2015.04.013 · 2.84 Impact Factor
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    • "These cells were scored visually, according to tail size, into the following four classes: class 0 – no tail; class 1 – tail shorter than the diameter of the head (nucleus); class 2 – tail length 1–2 times the diameter of the head; and class 3 – tail length more than twice the diameter of the head. Comets with no heads, with nearly all of the DNA in the tail or with a very wide tail, were excluded from the evaluation because they probably represented dead cells (Hartmann and Speit, 1997). The total score for 100 comets, which ranged from 0 (all undamaged) to 300 (all maximally damaged), was obtained by multiplying the number of cells in each class by the damage class. "
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    ABSTRACT: Rubus imperialis Cham. Schl. (Rosaceae) is frequently used in traditional medicine as hypoglycemic, antinociceptive and antiviral remedy. The aim of this study was to determine the in vivo genotoxic potential of Rubus imperialis aerial parts extract in cells of mice, and its possible antigenotoxic effect against cyclophosphamide (CPA)-induced DNA damage. In addition, the phytochemicals constituents in the extract were determined. Swiss albino mice were distributed in eight groups for acute treatment with R. imperialis extract (24h). The extract doses selected were 50, 250 and 500mg/kg b.w. administered by gavage alone or plus to CPA (50mg/kg b.w.) administered by intraperitoneal injection. The control groups were treated in a similar way. Analyses were performed using the comet assay, on leukocytes (collected 4 and 24hours after treatment) and liver (collected 24hours after treatment), and using the micronucleus test (MN) in bone marrow cells. Cytotoxicity was assessed by scoring 200 consecutive polychromatic (PCE) and normochromatic (NCE) erythrocytes (PCE/NCE ratio). The main compounds identified in the R. imperialis extract were saponins and steroidal compounds, being niga-ichigoside and tormentic acid the major compounds. The tested doses of R. imperialis extract showed no genotoxic effects in leukocytes from peripheral blood or liver cells by the comet assay. However, the MN test showed an increase in the frequency of micronucleated cells at the two higher doses tested, indicating that this extract have clastogenic/aneugenic effects in bone marrow cells at higher doses. On the other hand, for all cells evaluated, the three tested doses of the R. imperialis extract promoted inhibition of DNA damage induced by CPA. Despite the chemoprevention observed, the clastogenicity/aneugenicity observed suggest caution about either continuous or high-dose use of R. imperialis aerial parts extract by humans.
    Journal of ethnopharmacology 03/2014; 153(3). DOI:10.1016/j.jep.2014.03.033 · 3.00 Impact Factor
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