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 ΔΔC q method. 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.Electronic supplementary materialThe online version of this article (doi:10.1007/s00204-015-1621-7) contains supplementary material, which is available to authorized users.

Impact of Endocytosis and Lysosomal Acidification on the Toxicity of Copper Oxide Nano- and Microsized Particles: Uptake and Gene Expression Related to Oxidative Stress and the DNA Damage Response

“…0.5-1 × 10 6 logarithmically growing BEAS-2B cells were treated with different concentrations of CuO NP, CuO MP or CuCl 2 , with or without 100 µM OH-dyn or 100 nM bafA1 for 8 h or 24 h, respectively, in DMEM containing 10% FCS. Subsequently, gene expression analyses via high-throughput RT-qPCR with Fluidigm dynamic arrays on the BioMarkTM System were performed, as described previously [13]. For normalization, five potential reference genes were available (ACTB, B2M, GAPDH, GUSB and HPRT1).…”

“…Gene expression analyses via high-throughput qRT-PCR established previously in our group, quantifying the impact of 96 samples in parallel on the expression of 95 genes crucial for maintaining genomic stability. Selected genes were related to metal homeostasis, (oxidative) stress response as well as DNA repair, cell cycle regulation and apoptosis; a detailed description including a complete list of genes and respective proteins as well as their assignments to the respective groups has been published [13]. Applying this approach in a previous study in BEAS-2B cells after treatment with CuO NP, CuO MP as well as CuCl 2 , revealed a strong induction of copper uptake-related metallothionein genes, oxidative stress sensitive and inflammatory genes, anti-oxidative defense-associated genes, as well as the induction of cell cycle inhibitor gene CDKN1A (p21) and the pro-apoptotic PMAIP1 (Noxa) and TNFRSF10B (DR5).…”

“…Gene expression profiling was performed by a high-throughput reverse transcription quantitative polymerase chain reaction (RT-qPCR) technique, quantifying the impact of 96 samples in parallel on the expression of 95 genes crucial for maintaining genomic stability. Selected genes were related to metal homeostasis, an (oxidative) stress response as well as DNA repair, cell cycle regulation and apoptosis [13]. A pronounced dose-dependent impact on copper uptake, an oxidative stress response, DNA damage response and apoptosis-associated genes, was observed in case of the CuO NP, concomitant with a distinct copper overload in the cytoplasm, and especially the nucleus.…”

Essentielle Spurenelemente und toxische Metallverbindungen

“…In unserem Arbeitskreis wurde daher eine quantitative Hochdurchsatz‐Methode zur Analyse von Genexpressions veränderungen (High‐Throughput RT‐qPCR) etabliert, die es ermöglicht, den Einfluss von 96 Proben auf die Expression von 95 Genen parallel zu untersuchen . Es wurden 95 Gene ausgewählt, die eine Beeinflussung der genomischen Stabilität widerspiegeln; sie umfassen neben der Metallhomöostase die DNA‐Schadensantwort, redox‐regulierte Transkriptionsfaktoren, die (oxidative) Stress‐Antwort, Zellzyklusregulation und Proliferation, Apoptose und Fremdstoffmetabolismus.…”

Cytotoxicity of New Metal Porphyrinates Based on Chlorophyll a Derivatives

“…Results of many experimental studies show it. [31][32][33][34][35] Thus, 8 h were enough to alter gene expression, but not enough to induce the massive cell death using Zn-1 in concentrations 1-5 µM. Genes with a definite function and a proved mRNA level reaction to different chemical and physical stress factors were selected.…”