A method for the assessment of DNA damage in individual, one day old, zebrafish embryo (Danio rerio), without prior cell isolation.

Zebrafish (Danio rerio) embryos are increasingly used as an experimental model in toxicology for the detection of lethal and sub-lethal effects of diverse chemicals. DNA damage, an early biomarker of long-term effects such as mutagenesis and carcinogenesis, is commonly assessed in vitro and in vivo using the comet assay - single cell gel electrophoresis. Here we describe a new rapid method for the detection of DNA strand breaks in individual, one day old, zebrafish embryos, without the need for prior cell isolation. After the completed spawning, the embryos were exposed to non-toxic concentrations of model genotoxic compounds for 24h. The embryos were then treated with Pronase E, embedded on microscope slides and squashed to release the cells. After alkaline electrophoresis, the nuclei were stained with ethydium bromide and analyzed by fluorescence microscopy. Preparation of slides by the described method resulted in well separated cell nuclei with low background DNA damage. A significant increase in DNA damage was detected after exposure to the model genotoxic compounds, methylmethan sulphonate (MMS) and benzo(a)pyrene (BaP), while no DNA damage was induced by NaCl. Our method proved to be sensitive and suitable for the detection of DNA damage in one day old zebrafish embryos, suggesting it could serve as a useful tool for monitoring the genotoxic potential of chemicals and environmental pollutants.

Antigenotoxic effect of Xanthohumol in rat liver slices.

Xanthohumol (XN), the principal prenylated flavonoid in the hop plant, Humulus lupulus L., is suggested to have cancer chemo-preventive activities. Its mechanisms of protection have been proposed to be inhibition of metabolic activation, induction of detoxifying enzymes and antioxidant activity. Our previous study showed that XN efficiently protected human hepatoma HepG2 cells against the genotoxic effects of two pro-carcinogens (2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and benzo(a)pyrene (BaP)) that are dependent on cytochrome P450 (CYP) mediated metabolic activation, and against genotoxic effects of the oxidative damage inducing tert-butyl hydroperoxide (tBOOH). In the present study, we investigated the antigenotoxic effects of XN in precision-cut rat liver slices. Using the comet assay, we detected that at non-cytotoxic concentrations (0.01-10 microM) XN completely prevented IQ and BaP-induced DNA damage. The protective effects of XN against tBOOH-induced DNA damage was less efficient; the maximal 50% reduction of DNA damage was observed at 0.1 microM XN. In rat microsomes, XN (0.001-10 microM) inhibited CYP1A activity (7-ethoxycoumarin (7EC) de-ethylation) in a concentration-dependent manner. Surprisingly, no inhibition of 7EC metabolism by XN was observed in rat liver slices. XN also did not have any influence on mRNA expression of the enzymes CYP1A2 and quinone reductase (QR). These results indicate that inhibition of metabolic activation of pro-carcinogens by CYP1A is not likely to be the mechanism of its antigenotoxic action. In conclusion, XN efficiently protects DNA against genotoxicity of IQ and BaP and against oxidative DNA damage. Although the mechanism of the protective effect of XN is unclear, our results indicate that XN exhibits antigenotoxic effects in fresh liver tissue and provide additional evidence for the cancer preventive potential of XN.

Antimutagenicity of hops (Humulus lupulus L.): bioassay-directed fractionation and isolation of xanthohumol.

Bioassay-directed fractionation with a Salmonella/microsomal assay against the food borne mutagen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) was used to identify antimutagenic components of hops. Hops pellets extracted with diethylether showed antimutagenic activity against mutations induced by IQ. Fractionation of the diethylether extract (DE) by column chromatography, followed by semi-preparative HPLC yielded two fractions (E4b and E4d) with strong antimutagenic activity against IQ induced mutations. Separation of fraction E4b resulted in inactive fractions, while fraction E4d has been identified to be xanthohumol. In mammalian test system with human hepatoma HepG2 cells fraction E4d at 10mug/ml completely prevented formation of IQ induced DNA damage. These results indicate that xanthohumol is a very promising potential protective agent against genotoxicity of food borne carcinogens, which warrants further investigation.

Protective effects of xanthohumol against the genotoxicity of benzo(a)pyrene (BaP), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and tert-butyl hydroperoxide (t-BOOH) in HepG2 human hepatoma cells.

Xanthohumol is the major prenylated flavonoid present in the hop plant Humulus lupulus L. (Cannabinaceae) and a common ingredient of beer. Recently, xanthohumol has gained considerable interest due to its potential cancer chemo-preventive effect. The aim of this study was to reveal the possible anti-genotoxic activity of xanthohumol in metabolically competent human hepatoma HepG2 cells, by use of the comet assay. Xanthohumol by itself was neither cytotoxic nor genotoxic to the cells at concentrations below 10microM. However, a significant protective effect against the pro-carcinogens benzo(a)pyrene (BaP) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) was observed at concentrations as low as 0.01microM. In cells treated with xanthohumol in combination with tert-butyl hydroperoxide (t-BOOH) - an inducer of reactive oxygen species (ROS) - no protective effect was observed and xanthohumol also showed no significant scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals. On the other hand, HepG2 cells pre-treated with xanthohumol showed significantly reduced levels of t-BOOH-induced DNA strand breaks, indicating that its protective effect is mediated by induction of cellular defence mechanisms against oxidative stress. As xanthohumol is known to be an effective inhibitor of cytochrome P450 enzymes and an inducer of NAD(P)H: quinone reductase (QR), our findings can be explained by an inhibition of metabolic activation of pro-carcinogens and/or by induction of carcinogen-detoxifying and anti-oxidative enzymes by xanthohumol. These results provide evidence that xanthohumol displays anti-genotoxic activity in metabolically competent human cells.

Detection of xenobiotic-induced DNA damage by the comet assay applied to human and rat precision-cut liver slices.

The comet assay is a simple and sensitive method for measuring DNA damage at the level of individual cells and is extensively used in genotoxicity studies. It is commonly applied to cultured cells. The aim of this study was to apply the comet assay for use in fresh liver tissue, where metabolic activity, all cell types and tissue architecture are preserved. The response of liver slices to genotoxic agents was tested with the reactive oxygen species generating tert-butyl hydroperoxide (tBOOH, 0.1-2 mM), [corrected] and the pro-carcinogens 2-amino-3-methylimidazo[4,5-f]quinoline (IQ, 0.5-2 mM) and benzo(a)pyrene (BaP, 10-100 microM). Dose-dependent DNA damage was observed and compared to HepG2 cells. At non-cytotoxic concentrations of carcinogens, human liver slices were more sensitive to tBOOH than rat liver slices, while no significant difference was found for BaP and IQ. Human liver slices were more sensitive to IQ than HepG2 cells, equally sensitive to BaP and less to tBOOH. Control slices showed low levels of DNA damage, which did not increase during 24 h preservation (0 degrees C) or 48 h culturing (37 degrees C). In conclusion, the comet assay that we applied for measuring DNA damage in precision-cut liver slices is an useful tool to study genotoxic effects induced by various potential genotoxicants, allowing for detection of species differences in susceptibility to carcinogens.