Patrick D. McMullen

A map of the PPARα transcription regulatory network for primary human hepatocytes.

Nuclear receptor activation in liver leads to coordinated alteration of the expression of multiple gene products with attendant phenotypic changes of hepatocytes. Peroxisome proliferators including endogenous fatty acids, environmental chemicals, and drugs induce a multi-enzyme metabolic response that affects lipid and fatty acid processing. We studied the signaling network for the peroxisome proliferator-associated receptor alpha (PPARα) in primary human hepatocytes using the selective PPARα ligand, GW7647. We measured gene expression over multiple concentrations and times and conducted ChIP-seq studies at 2 and 24h to assess genomic binding of PPARα. Over all treatments there were 192 genes differentially expressed. Of these only 51% showed evidence of PPARα binding-either directly at PPARα response elements or via alternative mechanisms. Almost half of regulated genes had no PPARα binding. We then developed two novel bioinformatics methods to visualize the dose-dependent activation of both the transcription factor circuitry for PPARα and the downstream metabolic network in relation to functional annotation categories. Available databases identified several key transcription factors involved with the non-genomic targets after GW7647 treatment, including SP1, STAT1, ETS1, ERα, and HNF4α. The linkage from PPARα binding through gene expression likely requires intermediate protein kinases to activate these transcription factors. We found enrichment of functional annotation categories for organic acid metabolism and cell lipid metabolism among the differentially expressed genes. Lipid transport processes showed enrichment at the highest concentration of GW7647 (10 μM). While our strategy for mapping transcriptional networks is evolving, these approaches are necessary in moving from toxicogenomic methods that derive signatures of activity to methods that establish pathway structure, showing the coordination of the activated nuclear receptor with other signaling pathways.

Modeling Drug- and Chemical-Induced Hepatotoxicity with Systems Biology Approaches

We provide an overview of computational systems biology approaches as applied to the study of chemical- and drug-induced toxicity. The concept of “toxicity pathways” is described in the context of the 2007 US National Academies of Science report, “Toxicity testing in the 21st Century: A Vision and A Strategy.” Pathway mapping and modeling based on network biology concepts are a key component of the vision laid out in this report for a more biologically based analysis of dose-response behavior and the safety of chemicals and drugs. We focus on toxicity of the liver (hepatotoxicity) – a complex phenotypic response with contributions from a number of different cell types and biological processes. We describe three case studies of complementary multi-scale computational modeling approaches to understand perturbation of toxicity pathways in the human liver as a result of exposure to environmental contaminants and specific drugs. One approach involves development of a spatial, multicellular “virtual tissue” model of the liver lobule that combines molecular circuits in individual hepatocytes with cell–cell interactions and blood-mediated transport of toxicants through hepatic sinusoids, to enable quantitative, mechanistic prediction of hepatic dose-response for activation of the aryl hydrocarbon receptor toxicity pathway. Simultaneously, methods are being developing to extract quantitative maps of intracellular signaling and transcriptional regulatory networks perturbed by environmental contaminants, using a combination of gene expression and genome-wide protein-DNA interaction data. A predictive physiological model (DILIsym™) to understand drug-induced liver injury (DILI), the most common adverse event leading to termination of clinical development programs and regulatory actions on drugs, is also described. The model initially focuses on reactive metabolite-induced DILI in response to administration of acetaminophen, and spans multiple biological scales.

The Human Toxome Project

The Human Toxome Project, funded as an NIH Transformative Research grant 2011-2016, is focused on developing the concepts and the means for deducing, validating and sharing molecular pathways of toxicity (PoT). Using the test case of estrogenic endocrine disruption, the responses of MCF-7 human breast cancer cells are being phenotyped by transcriptomics and mass-spectroscopy-based metabolomics. The bioinformatics tools for PoT deduction represent a core deliverable. A number of challenges for quality and standardization of cell systems, omics technologies and bioinformatics are being addressed. In parallel, concepts for annotation, validation and sharing of PoT information, as well as their link to adverse outcomes, are being developed. A reasonably comprehensive public database of PoT, the Human Toxome Knowledge-base, could become a point of reference for toxicological research and regulatory test strategies.

Profiling Dose-Dependent Activation of p53-Mediated Signaling Pathways by Chemicals with Distinct Mechanisms of DNA Damage

As part of a larger effort to provide proof-of-concept in vitro-only risk assessments, we have developed a suite of high-throughput assays for key readouts in the p53 DNA damage response toxicity pathway: double-strand break DNA damage (p-H2AX), permanent chromosomal damage (micronuclei), p53 activation, p53 transcriptional activity, and cell fate (cell cycle arrest, apoptosis, micronuclei). Dose-response studies were performed with these protein and cell fate assays, together with whole genome transcriptomics, for three prototype chemicals: etoposide, quercetin, and methyl methanesulfonate. Data were collected in a human cell line expressing wild-type p53 (HT1080) and results were confirmed in a second p53 competent cell line (HCT 116). At chemical concentrations causing similar increases in p53 protein expression, p53-mediated protein expression and cellular processes showed substantial chemical-specific differences. These chemical-specific differences in the p53 transcriptional response appear to be determined by augmentation of the p53 response by co-regulators. More importantly, dose-response data for each of the chemicals indicate that the p53 transcriptional response does not prevent micronuclei induction at low concentrations. In fact, the no observed effect levels and benchmark doses for micronuclei induction were less than or equal to those for p53-mediated gene transcription regardless of the test chemical, indicating that p53s post-translational responses may be more important than transcriptional activation in the response to low dose DNA damage. This effort demonstrates the process of defining key assays required for a pathway-based, in vitro-only risk assessment, using the p53-mediated DNA damage response pathway as a prototype.

Toxicogenomics for transcription factor-governed molecular pathways: moving on to roles beyond classification and prediction

In this issue of Archives for Toxicology, Jennings et al.(2012) provide a review of transcriptional regulationassociated with perturbation of a variety of biologicalpathways by chemicals. The review focuses on specifictranscriptional regulatory networks activated by what theauthors refer to as ‘‘transcription factor-governed molecu-lar pathways.’’ We will use their terminology throughoutour perspective. These pathways include the family ofnuclear receptors, several stress-activated transcriptionfactors, and two immunomodulatory transcription factors:STAT and NF-jB. One goal of knowing the specificity oftranscriptomic responses from these pathways is develop-ment of biomarkers for the ‘‘transcription factor-governedpathway’’ that could support classification or early pre-diction of likely toxic responses of compounds. In additionto providing a thorough review of toxicogenomics ofactivation of these pathways, the paper provides two tablesshowing a sequential process for each pathway from acti-vating events to alterations in transcript levels for targetgenes. For each pathway, ancillary information includesendogenous ligand, exogenous ligands, inhibitory com-plexes, DNA target sequences for transcription factorbinding, and some target genes. These tables provide awealth of information related to the context in which thetranscription factors function to coordinate informationflow from cell exposures to transcription factor-activatingligands/stressors to gene expression. The authors’ overviewcompresses a large amount of information about toxicog-enomic responses to activators of transcription factor-governed pathways in a readily digestible package,highlighting the significant advances in applying micro-array technologies in toxicology. This perspective firstnotes the value of toxicogenomics in organizing informa-tion on transcription factor-governed pathways. We thenlook to a path forward in which toxicogenomics and othertechnologies will provide detailed knowledge on both thedose–response and structure and function of these tran-scription factor-governed pathways, thereby supporting anew generation of risk and safety assessment modalitiesbased on cellular assays of the perturbations of thesepathways.Using specific chemical perturbations to evaluatepathway responsesA simplified description ofthe transcription factor-governedpathways shows either stress-related or ligand-specificbinding as an activating event (Fig. 1). The activated tran-scription factor (TF* in Fig. 1) then forms an appropriatecomplex with co-activators and collaborating transcriptionfactors to regulate expression of multiple genes. Multipletranscripts change expression level, some increasing, othersdecreasing, and provide readout of an integrated cellularresponse. With rich data sets in hand on toxicogenomicresponses to chemicals specifically targeting transcriptionfactor-governed pathways, a library of gene expressionstudies has become available. In general, the process ofidentifying pathway targets for an unknown system requiresthisdiverselibraryofgenomicdataoncompoundsthattargetspecific transcription factor-governed pathways. For manypathways, the extent to which a compound is specific to asingle target remains poorly understood, complicating someof these studies. The review by Jennings et al. (2012) cata-logs the library of pathway-specific responses that begin to

Combining transcriptomics and PBPK modeling indicates a primary role of hypoxia and altered circadian signaling in dichloromethane carcinogenicity in mouse lung and liver

ABSTRACT Dichloromethane (DCM) is a lung and liver carcinogen in mice at inhalation exposures ≥ 2000 ppm. The modes of action (MOA) of these responses have been attributed to formation of genotoxic, reactive metabolite(s). Here, we examined gene expression in lung and liver from female B6C3F1 mice exposed to 0, 100, 500, 2000, 3000 and 4000 ppm DCM for 90 days. We also simulated dose measures ‐ rates of DCM oxidation to carbon monoxide (CO) in lung and liver and expected blood carboxyhemoglobin (HbCO) time courses with a PBPK model inclusive of both conjugation and oxidation pathways. Expression of large numbers of genes was altered at 100 ppm with maximal changes in the numbers occurring by 500 or 2000 ppm. Most changes in genes common to the two tissues were related to cellular metabolism and circadian clock. At the lower concentrations, the changes in metabolism‐related genes were discordant – up in liver and down in lung. These processes included organelle biogenesis, TCA cycle, and respiratory electron transport. Changes in circadian cycle genes – primarily transcription factors ‐ showed strong concentration‐related response at higher concentrations (Arntl, Npas2, and Clock were down‐regulated; Cry2, Wee1, Bhlhe40, Per3, Nr1d1, Nr1d2 and Dbp) were up‐regulated with similar directionality in both tissues. Overall, persistently elevated HbCO from DCM oxidation appears to cause extended periods of hypoxia, leading to altered circadian coupling to cellular metabolism. The dose response for altered circadian processes correlates with the cancer outcome. We found no evidence of changes in genes indicative of responses to cytotoxic, DNA‐reactive metabolites.

Assessing molecular initiating events (MIEs), key events (KEs) and modulating factors (MFs) for styrene responses in mouse lungs using whole genome gene expression profiling following 1-day and multi-week exposures

ABSTRACT Styrene increased lung tumors in mice at chronic inhalation exposures of 20 ppm and greater. MIEs, KEs and MFs were examined using gene expression in three strains of male mice (the parental C57BL/6 strain, a CYP2F2(−/−) knock out and a CYP2F2(−/−) transgenic containing human CYP2F1, 2A13 and 2B6). Exposures were for 1‐day and 1, 4 and 26 weeks. After 1‐day exposures at 1, 5, 10, 20, 40 and 120 ppm significant increases in differentially expressed genes (DEGs) occurred only in parental strain lungs where there was already an increase in DEGs at 5 ppm and then many thousands of DEGs by 120 ppm. Enrichment for 1‐day and 1‐week exposures included cell cycle, mitotic M‐M/G1 phases, DNA‐synthesis and metabolism of lipids and lipoproteins pathways. The numbers of DEGs decreased steadily over time with no DEGs meeting both statistical significance and fold‐change criteria at 26 weeks. At 4 and 26 weeks, some key transcription factors (TFs) ‐ Nr1d1, Nr1d2, Dbp, Tef, Hlf, Per3, Per2 and Bhlhe40 ‐ were upregulated (|FC| > 1.5), while others ‐ Npas, Arntl, Nfil3, Nr4a1, Nr4a2, and Nr4a3 ‐ were down‐regulated. At all times, consistent changes in gene expression only occurred in the parental strain. Our results support a MIE for styrene of direct mitogenicity from mouse‐specific CYP2F2‐mediated metabolites activating Nr4a signaling. Longer‐term MFs include down‐regulation of Nr4a genes and shifts in both circadian clock TFs and other TFs, linking circadian clock to cellular metabolism. We found no gene expression changes indicative of cytotoxicity or activation of p53‐mediated DNA‐damage pathways. HighlightsStyrene response consistent with direct mitogenicity of Cyp2F2 and Nr4a signalingLonger term exposure show changes in circadian pathways.Changes in circadian pathways associated with Nr4a receptor family down‐regulationConsistent changes were seen only in wild type mice.No evidence of activation of p53‐mediated DNA‐damage or cell stress pathways

The Human Toxome Collaboratorium: A Shared Environment for Multi-Omic Computational Collaboration within a Consortium

The Human Toxome Project is part of a long-term vision to modernize toxicity testing for the 21st century. In the initial phase of the project, a consortium of six academic, commercial, and government organizations has partnered to map pathways of toxicity, using endocrine disruption as a model hazard. Experimental data is generated at multiple sites, and analyzed using a range of computational tools. While effectively gathering, managing, and analyzing the data for high-content experiments is a challenge in its own right, doing so for a growing number of -omics technologies, with larger data sets, across multiple institutions complicates the process. Interestingly, one of the most difficult, ongoing challenges has been the computational collaboration between the geographically separate institutions. Existing solutions cannot handle the growing heterogeneous data, provide a computational environment for consistent analysis, accommodate different workflows, and adapt to the constantly evolving methods and goals of a research project. To meet the needs of the project, we have created and managed The Human Toxome Collaboratorium, a shared computational environment hosted on third-party cloud services. The Collaboratorium provides a familiar virtual desktop, with a mix of commercial, open-source, and custom-built applications. It shares some of the challenges of traditional information technology, but with unique and unexpected constraints that emerge from the cloud. Here we describe the problems we faced, the current architecture of the solution, an example of its use, the major lessons we learned, and the future potential of the concept. In particular, the Collaboratorium represents a novel distribution method that could increase the reproducibility and reusability of results from similar large, multi-omic studies.

Developing tools for defining and establishing pathways of toxicity

New approaches for toxicity testing: The US National Research Council report on ‘Toxicity Testing in the 21st Century’ (Krewski et al. 2010) envisioned a shift in testing away from studies of apical endpoints in test animals to the use of human cells to assess perturbations of toxicity pathways (TPs). The report generated widespread interest and has produced subsequent discussions regarding implementation of its key recommendations (Andersen and Krewski 2010; Krewski et al. 2011, 2014). TPs were defined as normal cellular signaling pathways that could serve as targets of toxicity in the face of perturbations of their function by chemical exposures. The examples provided in the report included sex steroid hormone receptor pathways, liver nuclear receptor signaling, and the suite of eight canonical stress pathways, including oxidative stress, DNA damage, heat shock, hypoxia, metal stress, inflammation, endoplasmic reticulum stress, and oxidative stress (Simmons et al. 2009). This aggregation of pathways, based largely on preexisting biological information, remains coarse-grained with many possible nodes in each of these TPs whose alterations could lead to toxicity. Some of the continuing challenges in advancing new, cell-based methods for toxicity testing are (1) the manner in which testing will be accomplished, (2) the degree of detail required to define the biological targets whose alterations lead to toxicity, and (3) the biological granularity underpinning definitions of toxicity pathways.

MYC is an early response regulator of human adipogenesis in adipose stem cells.

Adipose stem cell (ASC) differentiation is necessary for the proper maintenance and function of adipose tissue. The procurement and characterization of multipotent ASCs has enabled investigation into the molecular determinants driving human adipogenesis. Here, the transcription factor MYC was identified as a significant regulator of ASC differentiation. Expression of MYC transcript and protein was found to accumulate during the initial course of differentiation. Loss-of-function analysis using siRNA mediated knockdown of MYC demonstrated inhibition of hormonally stimulated adipogenesis. MYC exhibited an early and sustained expression pattern that preceded down regulation of key suppressor genes, as well as induction of transcriptional and functional effectors. Glucocorticoid stimulation was identified as a necessary component for MYC induction and was found to impact adipogenesis in a concentration-dependent manner. Global gene expression analysis of MYC knockdown in ASC enriched for functional pathways related to cell adhesion, cytoskeletal remodeling, and transcriptional components of adipogenesis. These results identify a functional role for MYC in promotion of multipotent ASC to the adipogenic lineage.