High-throughput genotoxicity assay identifies antioxidants as inducers of DNA damage response and cell death

Genome Instability Section, Genetics and Molecular Biology Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 03/2012; 109(14):5423-8. DOI: 10.1073/pnas.1114278109
Source: PubMed


Human ATAD5 is a biomarker for identifying genotoxic compounds because ATAD5 protein levels increase posttranscriptionally in response to DNA damage. We screened over 4,000 compounds with a cell-based quantitative high-throughput ATAD5-luciferase assay detecting genotoxic compounds. We identified 22 antioxidants, including resveratrol, genistein, and baicalein, that are currently used or investigated for the treatment of cardiovascular disease, type 2 diabetes, osteopenia, osteoporosis, and chronic hepatitis, as well as for antiaging. Treatment of dividing cells with these compounds induced DNA damage and resulted in cell death. Despite their genotoxic effects, resveratrol, genistein, and baicalein did not cause mutagenesis, which is a major side effect of conventional anticancer drugs. Furthermore, resveratrol and genistein killed multidrug-resistant cancer cells. We therefore propose that resveratrol, genistein, and baicalein are attractive candidates for improved chemotherapeutic agents.

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Available from: Raymond R Tice, Sep 30, 2015
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    • "HEK293T ELG1-LUC cells [25] were plated at a density of 10,000 cells per well in a 96-well white, assay plate (Costar). 24 hr after seeding, cells were treated with 0.75 µM TSA or 50 µM SAHA and then incubated for an additional 48 hr. "
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    ABSTRACT: We have previously used the ATAD5-luciferase high-throughput screening assay to identify genotoxic compounds with potential chemotherapeutic capabilities. The successful identification of known genotoxic agents, including the histone deacetylase inhibitor (HDACi) trichostatin A (TSA), confirmed the specificity of the screen since TSA has been widely studied for its ability to cause apoptosis in cancer cells. Because many cancers have acquired mutations in DNA damage checkpoints or repair pathways, we hypothesized that these cancers may be susceptible to treatments that target compensatory pathways. Here, we used a panel of isogenic chicken DT40 B lymphocyte mutant and human cell lines to investigate the ability of TSA to define selective pathways that promote HDACi toxicity. HDACi induced a DNA damage response and reduced viability in all repair deficient DT40 mutants although ATM-nulls were least affected. The most dramatic sensitivity was observed in mutants lacking the homology dependent repair (HDR) factor BLM or the non-homologous end-joining (NHEJ) and HDR factors, KU/RAD54, suggesting an involvement of either HDR or NHEJ in HDACi-induced cell death. To extend these findings, we measured the frequencies of HDR and NHEJ after HDACi treatment and monitored viability in human cell lines comparably deficient in HDR or NHEJ. Although no difference in HDR frequency was observed between HDACi treated and untreated cells, HDR-defective human cell lines were clearly more sensitive than wild type. Unexpectedly, cells treated with HDACis showed a significantly elevated NHEJ frequency. HDACi targeting drugs induced significant increases in NHEJ activity in human cell lines but did not alter HDR frequency. Moreover, HDR is required for cellular resistance to HDACi therapy; therefore, NHEJ does not appear to be a critical axis for HDACi resistance. Rather, HDACi compounds induced DNA damage, most likely double strand breaks (DSBs), and HDR proficiency is correlated with cell survival.
    PLoS ONE 01/2014; 9(1):e87203. DOI:10.1371/journal.pone.0087203 · 3.23 Impact Factor
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    • "Recently , new methods based on activation of the gene reporter construct have been developed to detect genotox - icity [ Westerink et al . , 2009 , 2010 ; Birrell et al . , 2010 ; Mizota et al . , 2011 ; Fox et al . , 2012 ; Hendriks et al . , 2012 ; Hughes et al . , 2012 ] . When we compared our results with published genotoxic data in studies using this technique , almost equally high sensitivity and specificity levels were obtained with the gH2AX ICW approach . However , the gH2AX ICW test on HepG2 cells has sev - eral advantages over gene reporter ass"
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    ABSTRACT: In vitro genotoxicity tests used in regulatory toxicology studies are sensitive, but the occurrence of irrelevant positive results is high compared with carcinogenicity studies in rodents. Current in vitro genotoxicity tests are also often limited by relatively low throughput. The aim of this study was to validate an in vitro genotoxic assay in a 96-well plate format that allows the simultaneous examination of cytotoxicity and genotoxicity. The test is based on the quantification of the phosphorylation of the histone H2AX (γH2AX), which reflects a global genotoxic insult, using the In-Cell Western technique. The assay was evaluated on HepG2 cells by testing a list of 61 compounds recommended by the European Center for the Validation of Alternative Methods (ECVAM), whose genotoxic potential has already been characterized. The γH2AX assay on HepG2 cell line was highly sensitive: 75% of the genotoxic compounds gave a positive result, and specific: 90-100% of nongenotoxic compounds gave negative results. Compared with the micronucleus genotoxicity assay using the same cell line and test compounds, the γH2AX assay was more sensitive and specific. In sum, the high-throughput γH2AX assay described here can accurately detect simultaneously the genotoxic and the cytotoxic potential of compounds with different modes of mutagenic action, notably those who required metabolic activation. The use of this assay in the early discovery phase of drug development may prove to be a valuable way to assess the genotoxic potential of xenobiotics. Environ. Mol. Mutagen. 00:000-000, 2013. © 2013 Wiley Periodicals, Inc.
    Environmental and Molecular Mutagenesis 12/2013; 54(9). DOI:10.1002/em.21817 · 2.63 Impact Factor
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    • "At concentrations in the range 40–80 μM, resveratrol activates DNA-damage signaling, e.g., phosphorylation of histone H2AX on Ser139 [6–8], which can be phosphorylated by DNA damage-activated kinases [9]. A recently published study showed that resveratrol can be genotoxic even at concentrations as low as 5 μM, which can be achieved in laboratory animals by oral administration [10]. The mechanism underlying the genotoxic activity of resveratrol is not well understood. "
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    ABSTRACT: Resveratrol is a natural compound that has been intensely studied due to its role in cancer prevention and potential as an anti-cancer therapy. Its effects include induction of apoptosis and senescence-like growth inhibition. Here, we report that two cancer cell lines (U-2 OS and A549) differ significantly in their molecular responses to resveratrol. Specifically, in U-2 OS cells, the activation of the p53 pathway is attenuated when compared to the activation in A549 cells. This attenuation is accompanied by a point mutation (458: CGA→TGA) in the PPM1D gene and overexpression of the encoded protein, which is a negative regulator of p53. Experimentally induced knockdown of PPM1D in U-2 OS cells resulted in slightly increased activation of the p53 pathway, most clearly visible as stronger phosphorylation of p53 Ser(37). When treated with nutlin-3a, a non-genotoxic activator of p53, U-2 OS and A549 cells both responded with substantial activation of the p53 pathway. Nutlin-3a improved the clonogenic survival of both cell lines treated with resveratrol. This improvement was associated with lower activation of DNA-damage signaling (phosphorylation of ATM, CHK2, and histone H2AX) and higher accumulation of cells in the G1 phase of the cell cycle. Thus, the hyperactivation of p53 by nutlin-3a helps to preserve the replicative potential of cells exposed to resveratrol.
    Molecular Biology Reports 05/2013; 40(8). DOI:10.1007/s11033-013-2602-7 · 2.02 Impact Factor
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