Hypoxia-inducible factor (HIF) is a sensor of hypoxia and a regulator of the cellular hypoxic response. HIF is composed of a heterodimeric complex comprised of an α-subunit, including HIF-1α, HIF-2α or HIF-3α, and a β-subunit, usually the aryl hydrocarbon receptor nuclear translocator (ARNT also called HIF-1β) [1]. HIF-1α plays crucial roles in angiogenesis during carcinogenesis [2], cell proliferation [3], intermediate metabolism including gluconeogenesis [4], cancer progression and anti-cancer therapy [5,6], development [7] and aging [8]. Under normoxic conditions, HIF-1α is recognized by the von Hippel-Lindau tumor-suppressor protein (VHL), a component of an E3 ubiquitin ligase complex. This interaction promotes the rapid degradation of HIF-1α. Under hypoxic conditions, HIF-1α is stabilized [9,10] and then acts as a regulator of numerous hypoxia-inducible genes under hypoxic conditions. Pharmacological inhibition of HIF-1α itself or HIF-1α interacting proteins has been explored as a potential strategy to treat cancer [11].

Dysregulation of HIF-1α has been linked to chemical induced toxicity. Alteration of signaling through HIF-1α and vascular endothelial growth factor (VEGF), a potent angiogenic factor, leads to arsenic-induced hepatic fibrosis [12]. Methylmercury reduced HIF-1α levels by activating proline hydroxylase and the ubiquitin proteasome system in astrocytes leading to neurotoxicity [13]. Dioxin-like substances called polyhalogenated carbazoles induce vascular toxicity via alteration of a HIF-1α/VEGF signaling pathway [14]. PM2.5 exposure-induced myocardial hypoxia injury is associated with HIF-1α dysregulation [15]. Suppression of HIF-1 signaling is thought to occur in some cases through activation of AhR leading to binding with ARNT indicating that AhR-ARNT–HIF–1α interactions are important for interference between xenobiotic-induced AhR activation and hypoxia sensing [16]. To assess the ability of environmental chemicals to stimulate the HIF-1 signaling pathway, Xia et al. [17] screened a National Toxicology Program collection of 1408 compounds using a cell-based beta-lactamase hypoxia response element reporter gene assay in a quantitative high-throughput screening (qHTS) format. Twelve active compounds were identified. Identification of environmental compounds having the ability to activate HIF-1α in vitro may be useful for prioritizing chemicals for further testing as hypoxia-response inducers in vivo.

A better understanding of the role of HIF-1α in chemical-induced toxicity can be facilitated by using new tools to identify when HIF-1 is altered in complex gene expression profiles. High-throughput transcriptomic (HTTr) technologies are being used to screen chemicals in human cell lines. As part of realizing the Environmental Agency's (EPA) Next Generation Computational Toxicology BluePrint, HTTr is being employed as a broad profiling assay with a targeted RNA-Seq method [18] to augment the battery of screening assays [19]. HTTr has the advantage over individual assays by simultaneously examining the effects of environmental chemicals on essentially all pathways, many of which have not been examined by the battery of ToxCast assays [19]. A major challenge for any transcript profiling strategy including HTTr is how to make linkages between chemical exposure and modulation of molecular targets. Several approaches have been used to interpret the HTTr profiles, and these include pathway analysis as well as comparisons to archived profiles of reference chemicals with known targets (e.g., Refs. [[20], [21], [22], [23]]).

Gene expression biomarkers have emerged as a complementary approach to pathway analyses to accurately identify the modulation of specific molecular targets. Biomarkers are sets of genes known or predicted to be regulated by a particular transcription factor that can be used together to predict modulation of the factor [24]. The biomarker gene expression pattern is compared to gene expression profiles derived from human cells exposed to chemicals using several computational techniques [25,26]. For example, gene expression biomarkers in human cells that predict the modulation of androgen receptor [27], metal-induced transcription factor 1 [28], NF-κB [29], estrogen receptor α [30], heat shock factor 1 [31], the oxidant-induced transcription factor NRF2 [[32], [33], [34]] and cell proliferation [35] have been described. In addition, a biomarker that identified chemical exposure conditions that lead to DNA damage has been extensively characterized [[36], [37]] and is currently undergoing review by the Food and Drug Administration to be used as a tool to identify potential DNA damaging agents in human cells. A methodological analysis of gene expression profiles from cells exposed to reference chemicals as well as perturbations of the gene encoding the chemical target will eventually lead to a battery of highly predictive biomarkers that can be used to interpret HTTr data streams [24]. The large quantity of microarray data that already exists in commercial databases and in public repositories can inform in silico high-throughput screening (HTS) identification of chemical agents that activate or suppress human molecular targets or perturb phenotypic responses. Approaches to assess HIF-1 modulation in a large microarray compendium have not been previously described.

In the present study, we developed a gene expression biomarker to predict HIF-1 modulation in HTTr databases. The biomarker was accurate in identifying treatment conditions in which HIF-1 was either activated or suppressed. The biomarker adds to a growing list of predictive biomarkers to interpret transcript profiling data for identification of factors important in mediating toxicity.