Citation:

Jackson, A., J. Liu, B. Vallanat, C. Jones, M. Nelms, G. Patlewicz, AND Jon Corton. Identification of Novel Activators of the Metal Responsive Transcription Factor (MTF-1) Using a Gene Expression Biomarker in a Microarray Compendium. Metallomics. Royal Society of Chemistry, London, Uk, 12(9):1400-1415, (2020). https://doi.org/10.1039/d0mt00071j

Impact/Purpose:

Homeostasis of metal ions is tightly regulated in biological systems due to the toxicity of many metals 1-3. At high concentrations, even essential metals like copper, zinc, iron, and manganese, which have important structural and catalytic functions in proteins, can cause toxicity. Human exposures to metals have been increasing as a result of increased use in a number of applications 4. Sources of heavy metals in the environment include geogenic, industrial, agricultural, pharmaceutical, domestic effluents, and atmospheric sources 5. Environmental pollution is significant from point sources involving mining and in foundries, smelters, and other metal-based industrial operations 5, 6. Heavy metals, especially nonessential metals such as arsenic, cadmium, chromium, lead, and mercury are of public health significance due to their ability to act as systemic toxicants that induce damage in multiple organs, even at low levels of exposure. These metals are classified as known or probable human carcinogens according to the US Environmental Protection Agency (EPA) and the International Agency for Research on Cancer. Because of the relevance of metal exposure to human health and disease, high throughput screening (HTS) methods to identify conditions in which metal homeostasis was disturbed would be useful to predict cellular responses to toxic metal overload and oxidative stress. The only known HTS assay that potentially measures alterations in metal homeostasis through MTF-1 activation is part of the EPA ToxCast screening program (http://epa.gov/ncct/toxcast/). There are ~700 HTS Tier 1 assays representing ~350 molecular targets that have been used to screen more than 1800 chemicals 16. High-throughput transcriptomic (HTTr) technologies have now been added to the ToxCast screening battery 17 in which targeted sequencing techniques are used to assess the expression of the human genome in chemically-treated cells 18. In the near future, HTTr could be used as “Tier 0” assays defined as those carried out prior to Tier 1 screening. Putative chemical targets identified could then be validated by selected in vitro HTS assays. Alternative approaches to assessing MTF-1 activation including those that use HTTr data have not been described. In the present study, we describe procedures for assessing MTF-1 activation using computational analysis of gene expression data. The large quantity of microarray data that already exists in public repositories and in commercial databases allows for in silico HTS prediction of chemical agents that activate or suppress a wide range of human molecular targets including MTF-1. As proof of principle, our lab has built and characterized gene expression biomarkers that predict modulation of the estrogen receptor and androgen receptor in human cell lines 19, 20. Using similar approaches, we constructed a gene expression biomarker that accurately detects MTF-1 activation by different metals. We used the biomarker to screen a library of microarray profiles from cells treated with ~2600 organic chemicals to identify novel activators of MTF-1. All chemicals selected for validation were found to exhibit the properties of MTF-1 activators in MCF-7 cells.

Description:

Environmental exposure to metals is known to cause a number of human toxicities including cancer. Metal-responsive transcription factor 1 (MTF-1) is an important component of metal regulation systems in mammalian cells. Here, we describe a novel method to identify conditions that activate MTF-1 based on microarray profiling data. MTF-1 biomarker genes were identified that exhibited consistent, robust expression across ten microarray comparisons examining the effects of metals (zinc, nickel, lead, arsenic, mercury, and silver) on gene expression in human cells. A subset of the resulting 81 genes were shown to be altered by knockdown of the MTF-1 gene including metallothionein family members and a zinc transporter. The ability of the biomarker to correctly identify treatment conditions that activate MTF-1 was examined by comparing the biomarker to microarray comparisons from cells exposed to metal activators of MTF-1 using the rank-based Running Fisher algorithm. The balanced accuracy for prediction was 93%. The biomarker was used to identify organic chemicals that activate MTF-1 from a compendium of 11,725 human gene expression comparisons representing 2582 chemicals. There were 700 positive chemicals identified that included those known to interact with cellular metals, such as clioquinol and disulfiram, as well as a set of novel chemicals. All 9 of the novel chemicals selected for validation were confirmed to activate MTF-1 biomarker genes in MCF-7 cells and to lesser extents in MTF-1-null cells by qPCR and targeted RNA-Seq. Overall, our work demonstrates that the biomarker for MTF-1 coupled with the Running Fisher test is a reliable strategy to identify novel chemical modulators of metal homeostasis.

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