Fabienne Calléja

Mechanism-based genotoxicity screening of metal oxide nanoparticles using the ToxTracker panel of reporter cell lines

BackgroundThe rapid expansion of manufacturing and use of nano-sized materials fuels the demand for fast and reliable assays to identify their potential hazardous properties and underlying mechanisms. The ToxTracker assay is a recently developed mechanism-based reporter assay based on mouse embryonic stem (mES) cells that uses GFP-tagged biomarkers for detection of DNA damage, oxidative stress and general cellular stress upon exposure. Here, we evaluated the ability of the ToxTracker assay to identify the hazardous properties and underlying mechanisms of a panel of metal oxide- and silver nanoparticles (NPs) as well as additional non-metallic materials (diesel, carbon nanotubes and quartz).MethodsThe metal oxide- and silver nanoparticles were characterized in terms of agglomeration and ion release in cell medium (using photon cross correlation spectroscopy and inductively coupled plasma with optical emission spectroscopy, respectively) as well as acellular ROS production (DCFH-DA assay). Cellular uptake was investigated by means of transmission electron microscopy. GFP reporter induction and cytotoxicity of the NPs was simultaneously determined using flow cytometry, and genotoxicity was further tested using conventional assays (comet assay, γ-H2AX and RAD51 foci formation).ResultsWe show that the reporter cells were able to take up nanoparticles and, furthermore, that exposure to CuO, NiO and ZnO nanoparticles as well as to quartz resulted in activation of the oxidative stress reporter, although only at high cytotoxicity for ZnO. NiO NPs activated additionally a p53-associated cellular stress response, indicating additional reactive properties. Conventional assays for genotoxicity assessment confirmed the response observed in the ToxTracker assay. We show for CuO NPs that the induction of oxidative stress is likely the consequence of released Cu ions whereas the effect by NiO was related to the particles per se. The DNA replication stress-induced reporter, which is most strongly associated with carcinogenicity, was not activated by any of the tested nanoparticles.ConclusionsWe conclude that the ToxTracker reporter system can be used as a rapid mechanism-based tool for the identification of hazardous properties of metal oxide NPs. Furthermore, genotoxicity of metal oxide NPs seems to occur mainly via oxidative stress rather than direct DNA binding with subsequent replication stress.

The ToxTracker assay: novel GFP reporter systems that provide mechanistic insight into the genotoxic properties of chemicals.

People are exposed to an ever-increasing number of chemical compounds that are developed by industry for a wide range of applications. These compounds may harmfully react with different cellular components and activate specific defense mechanisms that provide protection against the toxic, mutagenic, and possibly oncogenic consequences of exposure. Monitoring the activation of specific cellular signaling pathways upon exposure may therefore allow reliable and mechanism-based assessment of potential (geno)toxic properties of chemicals, while providing insight into their primary mode of toxicity. By whole-genome transcription profiling of mouse embryonic stem cells, we identified genes that were transcriptionally activated upon exposure to either genotoxic compounds or pro-oxidants. For selected biomarker genes, we constructed reporters encoding C-terminal green fluorescent protein (GFP)-tagged fusion proteins. GFP reporter genes were located on bacterial artificial chromosomes, thereby enabling transcriptional regulation of the reporters by their own physiological promoter. The Bscl2-GFP reporter is selectively activated after exposure to genotoxic agents and its induction is associated with inhibition of DNA replication and activation of the ataxia telangiectasia and Rad3-related protein signaling pathway. The Srxn1-GFP reporter is preferentially induced upon oxidative stress and is part of the nuclear factor (erythroid-derived 2)-like 2-antioxidant response pathway. The novel (geno)toxicity assay (ToxTracker) that utilize the differential responsiveness of various reporter cell lines will enable prediction of the primary reactive properties of known and unknown chemicals.

Transcription-Dependent Cytosine Deamination Is a Novel Mechanism in Ultraviolet Light-Induced Mutagenesis

Skin cancer is the most ubiquitous cancer type in the Caucasian population, and its incidence is increasing rapidly [1]. Transcribed proliferation-related genes in dermal stem cells are targets for the induction of ultraviolet light (UV)-induced mutations that drive carcinogenesis. We have recently found that transcription of a gene increases its mutability by UV in mammalian stem cells, suggesting a role of transcription in skin carcinogenesis [2]. Here we show that transcription-associated UV-induced nucleotide substitutions are caused by increased deamination of cytosines to uracil within photolesions at the transcribed strand, presumably at sites of stalled transcription complexes. Additionally, via an independent mechanism, transcription of UV-damaged DNA induces the generation of intragenic deletions. We demonstrate that transcription-coupled nucleotide excision repair (TC-NER) provides protection against both classes of transcription-associated mutagenesis. Combined, these results unveil the existence of two mutagenic pathways operating specifically at the transcribed DNA strand of active genes. Moreover, these results uncover a novel role for TC-NER in the suppression of UV-induced genome aberrations and provide a rationale for the efficient induction of apoptosis by stalled transcription complexes.

Gene transcription increases DNA damage-induced mutagenesis in mammalian stem cells

DNA damage-induced mutations in actively transcribed genes in stem cells underlie genetic diseases including cancer. Here we investigated whether transcription affects DNA damage-induced gene mutations in mouse embryonic stem cells. To this aim we developed cell lines in which transcription of an Hprt minigene reporter, located at a different genomic positions, is regulated by the tTA2 Tetracycline-controlled transactivator. This allows detection of mutagenic events at both Hprt and tTA2 using a single selection. We found that UV-C and benzo[a]pyrenediolepoxide induced significantly more mutations at the Hprt minigene when the gene was transcribed. The transcription-associated increase in UV-C-induced mutagenesis appears independent of the integration site of the Hprt minigene. Molecular analysis of UV-induced Hprt mutants revealed that transcription of damaged DNA enhances the frequency of nucleotide substitutions and triggers the generation of intragenic deletions at the Hprt minigene. We speculate that these deletions are a result of error-prone DNA end-joining of double strand DNA breaks that are generated when replication forks collide with transcription complexes stalled at DNA lesions.

The extended ToxTracker assay discriminates between induction of DNA damage, oxidative stress and protein misfolding.

Chemical exposure of cells may damage biomolecules, cellular structures, and organelles thereby jeopardizing cellular homeostasis. A multitude of defense mechanisms have evolved that can recognize specific types of damaged molecules and will initiate distinct cellular programs aiming to remove the damage inflicted and prevent cellular havoc. As a consequence, quantitative assessment of the activity of the cellular stress responses may serve as a sensitive reporter for the induction of specific types of damage. We have previously developed the ToxTracker assay, a mammalian stem cell-based genotoxicity assay employing two green fluorescent protein reporters specific for DNA damage and oxidative stress. We have now expanded the ToxTracker assay with an additional four reporter cell lines to include monitoring of additional stress signaling pathways. This panel of six green fluorescent protein reporters is able to discriminate between different primary reactivity of chemicals being their ability to react with DNA and block DNA replication, induce oxidative stress, activate the unfolded protein response, or cause a general P53-dependent cellular stress response. Extensive validation using the compound library suggested by the European Centre for the Validation of Alternative Methods (ECVAM) and a large panel of reference chemicals shows that the ToxTracker assay has an outstanding sensitivity and specificity. In addition, we developed Toxplot, a dedicated software tool for automated data analysis and graphical representation of the test results. Rapid and reliable identification by the ToxTracker assay of specific biological reactivity can significantly improve in vitro human hazard assessment of chemicals.

Genetic screens to identify pathogenic gene variants in the common cancer predisposition Lynch syndrome

In many individuals suspected of the common cancer predisposition Lynch syndrome, variants of unclear significance (VUS), rather than an obviously pathogenic mutations, are identified in one of the DNA mismatch repair (MMR) genes. The uncertainty of whether such VUS inactivate MMR, and therefore are pathogenic, precludes targeted healthcare for both carriers and their relatives. To facilitate the identification of pathogenic VUS, we have developed an in cellulo genetic screen-based procedure for the large-scale mutagenization, identification, and cataloging of residues of MMR genes critical for MMR gene function. When a residue identified as mutated in an individual suspected of Lynch syndrome is listed as critical in such a reverse diagnosis catalog, there is a high probability that the corresponding human VUS is pathogenic. To investigate the applicability of this approach, we have generated and validated a prototypic reverse diagnosis catalog for the MMR gene MutS Homolog 2 (Msh2) by mutagenizing, identifying, and cataloging 26 deleterious mutations in 23 amino acids. Extensive in vivo and in vitro analysis of mutants listed in the catalog revealed both recessive and dominant-negative phenotypes. Nearly half of these critical residues match with VUS previously identified in individuals suspected of Lynch syndrome. This aids in the assignment of pathogenicity to these human VUS and validates the approach described here as a diagnostic tool. In a wider perspective, this work provides a model for the translation of personalized genomics into targeted healthcare.

Excision of translesion synthesis errors orchestrates responses to helix-distorting DNA lesions

An Msh2/Msh6-dependent DNA repair mechanism mitigates the mutagenicity of photolesions and induces cell cycle responses by excising incorrect nucleotides incorporated by postreplicative translesion synthesis.

An efficient pipeline for the generation and functional analysis of human BRCA2 variants of uncertain significance.

The implementation of next‐generation sequence analysis of disease‐related genes has resulted in an increasing number of genetic variants with an unknown clinical significance. The functional analysis of these so‐called “variants of uncertain significance” (VUS) is hampered by the tedious and time‐consuming procedures required to generate and test specific sequence variants in genomic DNA. Here, we describe an efficient pipeline for the generation of gene variants in a full‐length human gene, BRCA2, using a bacterial artificial chromosome. This method permits the rapid generation of intronic and exonic variants in a complete gene through the use of an exon‐replacement strategy based on simple site‐directed mutagenesis and an effective positive–negative selection system in E. coli. The functionality of variants can then be assessed through the use of functional assays, such as complementation of gene‐deficient mouse‐embryonic stem (mES) cells in the case of human BRCA2. Our methodology builds upon an earlier protocol and, through the introduction of a series of major innovations, now represents a practical proposition for the rapid analysis of BRCA2 variants and a blueprint for the analysis of other genes using similar approaches. This method enables rapid generation and reliable classification of VUS in disease‐related genes, allowing informed clinical decision‐making.

The functional impact of variants of uncertain significance in BRCA2

PurposeGenetic testing has uncovered large numbers of variants in the BRCA2 gene for which the clinical significance is unclear. Cancer risk prediction of these variants of uncertain significance (VUS) can be improved by reliable assessment of the extent of impairment of the tumor suppressor function(s) of BRCA2.MethodsHere, we evaluated the performance of the mouse embryonic stem cell (mESC)-based functional assay on an extensive set of BRCA2 missense variants.ResultsWhereas all 20 nonpathogenic (class 1/2) variants were able to complement the cell lethal phenotype induced by loss of endogenous mouse Brca2, only 1 out of 15 pathogenic (class 4/5) variants (p.Gly2609Asp) was able to do so. However, in this variant the major tumor suppressive activity of BRCA2, i.e., homology directed repair (HDR), was severely abrogated. Among 43 evaluated VUS (class 3), 7 were unable to complement the lethal phenotype of mouse Brca2 loss while 7 other variants displayed a more severe reduction of HDR activity than observed for class 1/ 2 variants.ConclusionThe mESC-based BRCA2 functional assay can reliably determine the functional impact of VUS, distinguish between pathogenic and nonpathogenic variants, and may contribute to improved cancer risk estimation for BRCA2 VUS carriers.