title: Use of high-throughput RT-qPCR to assess modulations of gene expression profiles related to genomic stability and interactions by cadmium.
abstract: Predictive test systems to assess the mode of action of chemical carcinogens are urgently required. Within the present study, we applied the Fluidigm dynamic array on the BioMark™ HD System for quantitative high-throughput RT-qPCR analysis of 95 genes and 96 samples in parallel, selecting genes crucial for maintaining genomic stability, including stress response as well as DNA repair, cell cycle control, apoptosis and mitotic signaling. The specificity of each individually designed sequence-specific primer pair and their respective target amplicons were evaluated via melting curve analysis as part of qPCR and size verification via agarose gel electrophoresis. For each gene, calibration curves displayed high efficiencies and correlation coefficients in the identified linear dynamic range as well as low intra-assay variations. Data were processed via Fluidigm real-time PCR analysis and GenEx software, and results were depicted as relative gene expression according to the ΔΔCqmethod. Subsequently, gene expression analyses were conducted in cadmium-treated adenocarcinoma A549 and epithelial bronchial BEAS-2B cells. They revealed distinct dose- and time-dependent and also cell-type-specific gene expression patterns, including the induction of genes coding for metallothioneins, the oxidative stress response, cell cycle control, mitotic signaling and apoptosis. Interestingly, while genes coding for the DNA damage response were induced, distinct DNA repair genes were down-regulated at the transcriptional level. Thus, this approach provided a comprehensive overview on the interaction by cadmium with distinct signaling pathways, also reflecting molecular modes of action in cadmium-induced carcinogenicity. Therefore, the test system appears to be a promising tool for toxicological risk assessment.
authors.affiliation: Department of Food Chemistry and Toxicology, Institute for Applied Biosciences, Karlsruhe Institute of Technology (KIT), Kaiserstrasse 12, 76131, Karlsruhe, Germany.
authors.forename: Bettina Maria
authors.initials: BM
authors.lastname: Fischer
authors.affiliation: Department of Food Chemistry and Toxicology, Institute for Applied Biosciences, Karlsruhe Institute of Technology (KIT), Kaiserstrasse 12, 76131, Karlsruhe, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Neumann
authors.affiliation: Department of Food Chemistry and Toxicology, Institute for Applied Biosciences, Karlsruhe Institute of Technology (KIT), Kaiserstrasse 12, 76131, Karlsruhe, Germany.
authors.forename: Ann Liza
authors.initials: AL
authors.lastname: Piberger
authors.affiliation: Department of Food Chemistry and Toxicology, Institute for Applied Biosciences, Karlsruhe Institute of Technology (KIT), Kaiserstrasse 12, 76131, Karlsruhe, Germany.
authors.forename: Sarah Fremgaard
authors.initials: SF
authors.lastname: Risnes
authors.affiliation: Department of Food Chemistry and Toxicology, Institute for Applied Biosciences, Karlsruhe Institute of Technology (KIT), Kaiserstrasse 12, 76131, Karlsruhe, Germany.
authors.forename: Beate
authors.initials: B
authors.lastname: Köberle
authors.affiliation: Department of Food Chemistry and Toxicology, Institute for Applied Biosciences, Karlsruhe Institute of Technology (KIT), Kaiserstrasse 12, 76131, Karlsruhe, Germany. andrea.hartwig@kit.edu.
authors.forename: Andrea
authors.initials: A
authors.lastname: Hartwig
mesh_terms: D000072283:A549 Cells; D002282:Adenocarcinoma, Bronchiolo-Alveolar / Q000139:chemically induced* / Q000378:metabolism / Q000473:pathology; D051017:Apoptosis Regulatory Proteins / Q000819:agonists / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D002104:Cadmium / Q000633:toxicity*; D002138:Calibration; D002460:Cell Line; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D042822:Genomic Instability / Q000187:drug effects*; D057166:High-Throughput Screening Assays; D006801:Humans; D007700:Kinetics; D008175:Lung Neoplasms / Q000139:chemically induced* / Q000378:metabolism / Q000473:pathology; D009153:Mutagens / Q000633:toxicity*; D009363:Neoplasm Proteins / Q000819:agonists / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D020545:Respiratory Mucosa / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D051017:Apoptosis Regulatory Proteins; D009153:Mutagens; D009363:Neoplasm Proteins; D002104:Cadmium
keywords: Cadmium; Fluidigm dynamic array; Gene expression profiling; Genomic stability; High-throughput RT-qPCR