Quantitative analysis of gene expression changes in response to genotoxic compounds

Morris, Ceri A., El-Hiti, Gamal A., Weeks, Ian ORCID: https://orcid.org/0000-0002-6362-2929, Woodhead, Stuart, Smith, Keith and Kille, Peter ORCID: https://orcid.org/0000-0001-6023-5221 2017. Quantitative analysis of gene expression changes in response to genotoxic compounds. Toxicology in Vitro 39 , pp. 15-28. 10.1016/j.tiv.2016.11.004

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Abstract

Techniques that quantify molecular endpoints sufficiently sensitive to identify and classify potentially toxic compounds have wide potential for high-throughput in vitro screening. Expression of three genes, RAD51C, TP53 and cystatin A (CSTA), in HEPG2 cells was measured by Q-PCR amplification. In parallel, we developed alternative assays for the same 3 gene signature based on an acridinium-ester chemiluminescent reporter molecule. HEPG2 cells were challenged with eighteen different compounds (n = 18) chosen to represent compounds that are genotoxic (n = 8), non-genotoxic non-carcinogenic (n = 2) or have a less well defined mechanism of action with respect to genotoxicity (n = 8). At least one of the three genes displayed dysregulated expression in the majority of compounds tested by Q-PCR and ten compounds changed the CSTA expression significantly. Acridinium-ester labelled probes for the three genes were synthesised and tested. Analytical sensitivity was characterised and suggested a limit of detection generally better than 0.1 fmol but often 10–50 attomol. A linear amplification step was optimised and this quantitative method detected statistically significant increases in RAD51C and CSTA expression in agreement with the Q-PCR results, demonstrating the potential of this technology. The broad agreement of the amplified chemiluminescent method and Q-PCR in measuring gene expression suggests wider potential application for this chemiluminescent technology.

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	Biosciences Chemistry Medicine
Subjects:	R Medicine > R Medicine (General)
Uncontrolled Keywords:	Acridinium-ester; Chemiluminescence; Genotoxic; Cystatin A (CSTA); RAD51C; Tumour suppressor p53 (TP53)
Publisher:	Elsevier
ISSN:	0887-2333
Funders:	Welsh Government
Date of First Compliant Deposit:	22 November 2016
Date of Acceptance:	2 November 2016
Last Modified:	12 Nov 2024 04:30
URI:	https://orca.cardiff.ac.uk/id/eprint/96347

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