Project ID Assignment: HTS-9. Reporter Gene Cell-based Assays for High Throughput Screening to
Determine Sub-Lethal Toxicity of Nanomaterials
Ken Bradley and Bryan France
The goal of this project has been to implement mammalian reporter cell lines for rapid assessment of
sub-lethal nanotoxicity and pathway delineation. We have developed a high throughput screening (HTS)
assay to assess reporter gene activation of genes response elements control downstream of cellular
signaling pathways. A luciferase pathway reporter system (Qiagen/SA Biosciences; CLA-004LA) was used
to transduced RAW 264.7 and BEAS-2B cells to develop a multi-parametric assay similar to the oxidative
stress and cytotoxicity assay described in Project HTS-1. The reporter system contains a luciferase
reporter gene expressed upon the induction of one of ten independent cellular stress pathways. Each
pathway is controlled by a unique transcription factor that drives luciferase expression in the cognate
reporter cell line. In total, 20 distinct reporter cell lines (two cell types, ten reporters per cell type) were
generated. This system was validated using eight metal and metal oxide ENMs also used in Project HTS-1
(Theme 2). The the cells were exposed to NPs for multiple times (3-24 hours) across ten two-fold
dilutions ranging from 200 µg/mL down to 0.39 µg/mL.
Zinc oxide and platinum were observed as the most toxic ENMs assayed in the reporter gene assay,
consistent with the study in Project HTS-1. Although both ZnO and Pt NPs induced stress responses, the
specific pathways involved were found to depend on cell type. The macrophage reporters show a ZnOand Pt-mediated, time- and dose-dependent induction in pathways correlating to DNA damage and cell
cycle control (p53, MYC, and E2F). These results correlate with published results from Sharma et al
(2009) as well as Jung et al (2007). ZnO also appeared to down-regulate NFB, NFAT, SRE, CRE, HIF1a,
and AP1 pathways in macrophages. In lung epitheal cells, ZnO down-regulated a similar set of
pathways, including NFkB, SMAD, SRE, CRE, HIF1a, and AP1, indicative of sublethal toxicity and
dysfunction in cAMP and inflammatory regulation.
Using these data sets, Rong Li (Theme 6) performed association modeling to correlate reporter gene
activation with the data sets generated in the oxidative stress and cytotoxicity HTS. In macropahges,
increased superoxide production (MitoSox fluorescence) was the best correlated with E2F, SMAD, Myc,
and p53 pathways at high ZnO and Pt doses (50-200 µg/mL), implicating a mitochondrial driven death
mechanism. For lung epithelial cells, association modeling placed SMAD, SRE, CRE, HIF1a, and propidium
iodide membrane integrity dye (Theme 2) in a high level of association at high dosage exposure (25-200
µg/mL) of ZnO and Pt NPs, suggesting a necrotic death mechanism. Notably, this analysis reveals that
the most informative toxicity or sub-lethal assays depend on cell type. Therefore, future assays to
ascertain toxicity of new NPs and/or cell types should probe a broad range of toxicity mechanisms to
avoid missing sub-lethal toxicity/stress events.