Article

Characterization of environmental chemicals with potential for DNA damage using isogenic DNA repair-deficient chicken DT40 cell lines

Radiation Genetics, Graduate School of Medicine, Kyoto University, Yoshidakonoe, Sakyo, Japan.
Environmental and Molecular Mutagenesis (Impact Factor: 2.55). 08/2011; 52(7):547-61. DOI: 10.1002/em.20656
Source: PubMed

ABSTRACT Included among the quantitative high throughput screens (qHTS) conducted in support of the US Tox21 program are those being evaluated for the detection of genotoxic compounds. One such screen is based on the induction of increased cytotoxicity in seven isogenic chicken DT40 cell lines deficient in DNA repair pathways compared to the parental DNA repair-proficient cell line. To characterize the utility of this approach for detecting genotoxic compounds and identifying the type(s) of DNA damage induced, we evaluated nine of 42 compounds identified as positive for differential cytotoxicity in qHTS (actinomycin D, adriamycin, alachlor, benzotrichloride, diglycidyl resorcinol ether, lovastatin, melphalan, trans-1,4-dichloro-2-butene, tris(2,3-epoxypropyl)isocyanurate) and one non-cytotoxic genotoxic compound (2-aminothiamine) for (1) clastogenicity in mutant and wild-type cells; (2) the comparative induction of γH2AX positive foci by melphalan; (3) the extent to which a 72-hr exposure duration increased assay sensitivity or specificity; (4) the use of 10 additional DT40 DNA repair-deficient cell lines to better analyze the type(s) of DNA damage induced; and (5) the involvement of reactive oxygen species in the induction of DNA damage. All compounds but lovastatin and 2-aminothiamine were more clastogenic in at least one DNA repair-deficient cell line than the wild-type cells. The differential responses across the various DNA repair-deficient cell lines provided information on the type(s) of DNA damage induced. The results demonstrate the utility of this DT40 screen for detecting genotoxic compounds, for characterizing the nature of the DNA damage, and potentially for analyzing mechanisms of mutagenesis.

0 Followers
 · 
121 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The increased presence of chemical contaminants in the environment is an undeniable concern to human health and ecosystems. Historically, by relying heavily upon costly and laborious animal-based toxicity assays, the field of toxicology has often neglected examinations of the cellular and molecular mechanisms of toxicity for the majority of compounds-information that, if available, would strengthen risk assessment analyses. Functional toxicology, where cells or organisms with gene deletions or depleted proteins are used to assess genetic requirements for chemical tolerance, can advance the field of toxicity testing by contributing data regarding chemical mechanisms of toxicity. Functional toxicology can be accomplished using available genetic tools in yeasts, other fungi and bacteria, and eukaryotes of increased complexity, including zebrafish, fruit flies, rodents, and human cell lines. Underscored is the value of using less complex systems such as yeasts to direct further studies in more complex systems such as human cell lines. Functional techniques can yield (1) novel insights into chemical toxicity; (2) pathways and mechanisms deserving of further study; and (3) candidate human toxicant susceptibility or resistance genes.
    Frontiers in Genetics 05/2014; 5:110. DOI:10.3389/fgene.2014.00110
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: H2AX histone phosphorylation represents an early event in the cellular response against DNA double strand breaks (DSB), and plays a central role in sensing and repairing DNA damage. Therefore, the analysis of H2AX phosphorylated (γH2AX) may be possibly used as biomarker of genotoxicity and genomic instability with a number of applications in human epidemiology. However, the lack of an experimental standard leads to a wide heterogeneity in the results obtained and their interpretation, affecting the reliability of the assay. To address the most critical issues limiting the use of the γH2AX assay in human population studies, a flow cytometry analysis was performed to establish differences in γH2AX levels between fresh or cryopreserved peripheral blood lymphocytes, and to assess the influence of phytohaemagglutinin (PHA) stimulation. To this purpose, cells were treated with four known genotoxic chemicals with different mechanisms of DSB induction, i.e., bleomycin, methyl methanesulfonate, camptothecin and actinomycin. According to our results, both unstimulated and stimulated fresh lymphocytes can be efficiently employed to evaluate γH2AX levels, but the sensitivity of the assay is depending upon the kind of damage observed. On the other hand, cryopreserved lymphocytes require PHA stimulation since unstimulated cells showed too high basal damage. Consequently, the protocol conditions will depend on the expected mechanism of production of DSB and the characteristics of the study design (sample collection and storage conditions, type of epidemiological study). Further studies are required to standardize the protocol of γH2AX assay to be employed as biomarker of genotoxicity or genomic instability in human population studies. © The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: journals.permissions@oup.com.
  • [Show abstract] [Hide abstract]
    ABSTRACT: RING finger protein 4 (RNF4) represents a subclass of ubiquitin ligases that target proteins modified by the small ubiquitin-like modifier (SUMO) for ubiquitin-mediated degradation. We disrupted the RNF4 gene in chicken DT40 cells and found that the resulting RNF4−/− cells gradually lost proliferation capability. Strikingly, this compromised proliferation was associated with an unprecedented cellular effect: the gradual decrease in the number of intact chromosomes. In the 6 weeks after gene targeting, there was a 25% reduction in the DNA content of the RNF4−/− cells. Regarding trisomic chromosome 2, 60% of the RNF4−/− cells lost one homologue, suggesting that DNA loss was mediated by whole chromosome loss. To determine the cause of this chromosome loss, we examined cell-cycle checkpoint pathways. RNF4−/− cells showed a partial defect in the spindle assembly checkpoint, premature dissociation of sister chromatids, and a marked increase in the number of lagging chromosomes at anaphase. Thus, combined defects in SAC and sister chromatid cohesion may result in increased lagging chromosomes, leading to chromosome loss without accompanying chromosome gain in RNF4−/− cells. We therefore propose that RNF4 plays a novel role in preventing the loss of intact chromosomes and ensures the maintenance of chromosome integrity.
    Genes to Cells 09/2014; 19(10). DOI:10.1111/gtc.12173 · 2.86 Impact Factor