title: Development of a panel of high-throughput reporter-gene assays to detect genotoxicity and oxidative stress.
abstract: The lack of toxicological information on many of the compounds that humans use or are exposed to, intentionally or unintentionally, poses a big problem in risk assessment. To fill this data gap, more emphasis is given to fast in vitro screening tools that can add toxicologically relevant information regarding the mode(s) of action via which compounds can elicit adverse effects, including genotoxic effects. By use of bioassays that can monitor the activation of specific cellular signalling pathways, many compounds can be screened in a high-throughput manner. We have developed two new specific reporter-gene assays that can monitor the effects of compounds on two pathways of interest: the p53 pathway (p53 CALUX) for genotoxicity and the Nrf2 pathway (Nrf2 CALUX) for oxidative stress. To exclude non-specific effects by compounds influencing the luciferase reporter-gene expression non-specifically, a third assay was developed to monitor changes in luciferase expression by compounds in general (Cytotox CALUX). To facilitate interpretation of the data and to avoid artefacts, all three reporter-gene assays used simple and defined reporter genes and a similar cellular basis, the human U2OS cell line. The three cell lines were validated with a range of reference compounds including genotoxic and non-genotoxic agents. The sensitivity (95%) and specificity (85%) of the p53 CALUX was high, showing that the assay is able to identify various types of genotoxic compound, while avoiding the detection of false positives. The Nrf2 CALUX showed specific responses to oxidants only, enabling the identification of compounds that elicit part of their genotoxicity via oxidative stress. All reporter-gene assays can be used in a high-throughput screening format and can be supplemented with other U2OS-based reporter-gene assays that can profile nuclear receptor activity, and several other signalling pathways.
authors.affiliation: BioDetection Systems BV, Amsterdam, The Netherlands. Electronic address: Sander.van.der.Linden@bds.nl.
authors.forename: Sander C
authors.initials: SC
authors.lastname: van der Linden
authors.affiliation: Innovative Testing in Life Sciences and Chemistry, Research Centre Technology & Innovation, University of Applied Sciences Utrecht, The Netherlands.
authors.forename: Anne R M
authors.initials: AR
authors.lastname: von Bergh
authors.affiliation: BioDetection Systems BV, Amsterdam, The Netherlands.
authors.forename: Barbara M A
authors.initials: BM
authors.lastname: van Vught-Lussenburg
authors.affiliation: BioDetection Systems BV, Amsterdam, The Netherlands.
authors.forename: Lydia R A
authors.initials: LR
authors.lastname: Jonker
authors.affiliation: Innovative Testing in Life Sciences and Chemistry, Research Centre Technology & Innovation, University of Applied Sciences Utrecht, The Netherlands.
authors.forename: Marc
authors.initials: M
authors.lastname: Teunis
authors.affiliation: Innovative Testing in Life Sciences and Chemistry, Research Centre Technology & Innovation, University of Applied Sciences Utrecht, The Netherlands.
authors.forename: Cyrille A M
authors.initials: CA
authors.lastname: Krul
authors.affiliation: BioDetection Systems BV, Amsterdam, The Netherlands.
authors.forename: Bart
authors.initials: B
authors.lastname: van der Burg
mesh_terms: D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004249:DNA Damage; D004305:Dose-Response Relationship, Drug; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism*; D008163:Luminescent Measurements; D009152:Mutagenicity Tests / Q000379:methods*; D051267:NF-E2-Related Factor 2 / Q000235:genetics / Q000378:metabolism; D018384:Oxidative Stress; D015203:Reproducibility of Results; D020218:Response Elements / Q000235:genetics; D015398:Signal Transduction / Q000187:drug effects; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism; D015262:Xenobiotics / Q000145:classification / Q000494:pharmacology
chemical_list: D051267:NF-E2-Related Factor 2; C495635:NFE2L2 protein, human; D016159:Tumor Suppressor Protein p53; D015262:Xenobiotics; D008156:Luciferases
keywords: CALUX; Genotoxicity; High throughput screening; Oxidative stress