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: 3.36). 03/1997; 90:183-188. DOI: 10.1016/S0378-4274(96)03847-7

ABSTRACT 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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    • "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 · 2.94 Impact Factor
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    • "Whole blood was treated for 2 h at 37ºC with different concentrations of compounds 7 (380-4000 µM) or 8 (149-6400 µM) in 5% (v/v) dimethyl sulfoxide (DMSO) (solvent-control) and then used in the assays. Cell viability was determined at the end of drug treatment using the fluorescein diacetate (FDA)/ethidium bromide (EtBr) assay (Hartmann & Speit 1997). Whole blood (50 µL) was mixed with an equal volume of the freshly prepared staining solution, which consisted of 30 µg/mL of FDA plus 8 µg/mL of EtBr in phosphate buffered saline (PBS). "
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    ABSTRACT: Megazol (7) is a 5-nitroimidazole that is highly active against Trypanosoma cruzi and Trypanosoma brucei, as well as drug-resistant forms of trypanosomiasis. Compound 7 is not used clinically due to its mutagenic and genotoxic properties, but has been largely used as a lead compound. Here, we compared the activity of 7 with its 4H-1,2,4-triazole bioisostere (8) in bloodstream forms of T. brucei and T. cruzi and evaluated their activation by T. brucei type I nitroreductase (TbNTR) enzyme. We also analysed the cytotoxic and genotoxic effects of these compounds in whole human blood using Comet and fluorescein diacetate/ethidium bromide assays. Although the only difference between 7 and 8 is the substitution of sulphur (in the thiadiazole in 7) for nitrogen (in the triazole in 8), the results indicated that 8 had poorer antiparasitic activity than 7 and was not genotoxic, whereas 7 presented this effect. The determination of Vmax indicated that although 8 was metabolised more rapidly than 7, it bounds to the TbNTR with better affinity, resulting in equivalent kcat/KM values. Docking assays of 7 and 8 performed within the active site of a homology model of the TbNTR indicating that 8 had greater affinity than 7.
    Memórias do Instituto Oswaldo Cruz 03/2014; DOI:10.1590/0074-0276140497 · 1.57 Impact Factor
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