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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    • "It allows for quantification when needed and the results are viewed from a single cell obtained from in vitro or in vivo samples (Hovhannisyan, 2010;Kang et al., 2013;Singh et al., 1988). The assay is relevant to an increasing number of applications and include, among others, evaluation of the genotoxic effects of several agents on somatic and germ cells (Hartmann and Speit, 1997), evaluation of DNA repair (Laffon et al., 2002), clinical applications (Kassie et al., 2000;Sardas et al., 1998), and human (Frenzilli et al., 2000;Kan et al., 2002) and environmental biomonitoring (Kassie et al., 2001;Monarca et al., 2001). Since medicinal plants are to be used in animals (with a growing rate of usage) and mainly by humans, the application of genotoxicity tests that use biomonitors and biomarkers that are phylogenetically closer to these species are desired. "
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    ABSTRACT: Ethnopharmacological relevance: Medicinal plants are known to contain numerous biologically active compounds, and although they have proven pharmacological properties, they can cause harm, including DNA damage. Aim of the study: Review the literature to evaluate the genotoxicity risk of medicinal plants, explore the genotoxicity assays most used and compare these to the current legal requirements. Material and methods: A quantitative systematic review of the literature, using the keywords "medicinal plants", "genotoxicity" and "mutagenicity", was undertakenQ to identify the types of assays most used to assess genotoxicity, and to evaluate the genotoxicity potential of medicinal plant extracts. Results: The database searches retrieved 2289 records, 458 of which met the inclusion criteria. Evaluation of the selected articles showed a total of 24 different assays used for an assessment of medicinal plant extract genotoxicity. More than a quarter of those studies (28.4%) reported positive results for genotoxicity. Conclusions: This review demonstrates that a range of genotoxicity assay methods are used to evaluate the genotoxicity potential of medicinal plant extracts. The most used methods are those recommended by regulatory agencies. However, based on the current findings, in order to conduct a thorough study concerning the possible genotoxic effects of a medicinal plant, we indicate that it is important always to include bacterial and mammalian tests, with at least one in vivo assay. Also, these tests should be capable of detecting outcomes that include mutation induction, clastogenic and aneugenic effects, and structural chromosome abnormalities. In addition, the considerable rate of positive results detected in this analysis further supports the relevance of assessing the genotoxicity potential of medicinal plants.
    Full-text · Article · Dec 2015 · Journal of ethnopharmacology
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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.
    Full-text · Article · Aug 2015 · Biochemical pharmacology
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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.
    Full-text · Article · Apr 2015 · Industrial Crops and Products
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