Salil N. Pendse

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.

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

Editor’s Highlight: Screening ToxCast Prioritized Chemicals for PPARG Function in a Human Adipose-Derived Stem Cell Model of Adipogenesis

The developmental origins of obesity hypothesis posits a multifaceted contribution of factors to the fetal origins of obesity and metabolic disease. Adipocyte hyperplasia in gestation and early childhood may result in predisposition for obesity later in life. Rodent in vitro and in vivo studies indicate that some chemicals may directly affect adipose progenitor cell differentiation, but the human relevance of these findings is unclear. The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARG) is the master regulator of adipogenesis. Human adipose-derived stem cells (hASC) isolated from adipose tissue express endogenous isoforms of PPARG and represent a biologically relevant cell-type for evaluating activity of PPARG ligands. Here, a multi-endpoint approach based on a phenotypic adipogenesis assay was applied to screen a set of 60 chemical compounds identified in ToxCast Phase I as PPARG active (49) or inactive (11). Chemicals showing activity in the adipogenesis screen were further evaluated in a series of 4 orthogonal assays representing 7 different key events in PPARG-dependent adipogenesis, including gene transcription, protein expression, and adipokine secretion. An siRNA screen was also used to evaluate PPARG-dependence of the adipogenesis phenotype. A universal concentration-response design enabled inter-assay comparability and implementation of a weight-of-evidence approach for bioactivity classification. Collectively, a total of 14/49 (29%) prioritized chemicals were identified with moderate-to-strong activity for human adipogenesis. These results provide the first integrated screening approach of prioritized ToxCast chemicals in a human stem cell model of adipogenesis and provide insight into the capacity of PPARG-activating chemicals to modulate early life programming of adipose tissue.

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.

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.

Screening ToxCast Prioritized Chemicals for PPARG Function in a Human Adipose-Derived Stem Cell Model of Adipogenesis

The developmental origins of obesity hypothesis posits a multifaceted contribution of factors to the fetal origins of obesity and metabolic disease. Adipocyte hyperplasia in gestation and early childhood may result in predisposition for obesity later in life. Rodent in vitro and in vivo studies indicate that some chemicals may directly affect adipose progenitor cell differentiation, but the human relevance of these findings is unclear. The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARG) is the master regulator of adipogenesis. Human adipose-derived stem cells (hASC) isolated from adipose tissue express endogenous isoforms of PPARG and represent a biologically relevant cell-type for evaluating activity of PPARG ligands. Here, a multi-endpoint approach based on a phenotypic adipogenesis assay was applied to screen a set of 60 chemical compounds identified in ToxCast Phase I as PPARG active (49) or inactive (11). Chemicals showing activity in the adipogenesis screen were further evaluated in a series of 4 orthogonal assays representing 7 different key events in PPARG-dependent adipogenesis, including gene transcription, protein expression, and adipokine secretion. An siRNA screen was also used to evaluate PPARG-dependence of the adipogenesis phenotype. A universal concentration-response design enabled inter-assay comparability and implementation of a weight-of-evidence approach for bioactivity classification. Collectively, a total of 14/49 (29%) prioritized chemicals were identified with moderate-to-strong activity for human adipogenesis. These results provide the first integrated screening approach of prioritized ToxCast chemicals in a human stem cell model of adipogenesis and provide insight into the capacity of PPARG-activating chemicals to modulate early life programming of adipose tissue.

Strain-related differences in mouse lung gene expression over a two-year period of inhalation exposure to styrene: Relevance to human risk assessment

ABSTRACT Both CD‐1 and C57BL/6 wildtype (C57BL/6‐WT) mice show equivalent short‐term lung toxicity from exposures to styrene, while long‐term tumor responses are greater in CD‐1 mice. We analyzed lung gene expression from styrene exposures lasting from 1‐day to 2‐years in male mice from these two strains, including a Cyp2f2(−/−) knockout (C57BL/6‐KO) and a Cyp2F1/2A13/2B6 transgenic mouse (C57BL/6‐TG). With short term exposures (1‐day to 1‐week), CD‐1 and C57BL/6‐WT mice had thousands of differentially expressed genes (DEGs), consistent with changes in pathways for cell proliferation, cellular lipid metabolism, DNA‐replication and inflammation. C57BL/6‐WT mice responded within a single day; CD‐1 mice required several days of exposure. The numbers of exposure related DEGs were greatly reduced at longer times (4‐weeks to 2‐years) with enrichment only for biological oxidations in C57BL/6‐WT and metabolism of lipids and lipoproteins in CD‐1. Gene expression results indicate a non‐genotoxic, mouse specific mode of action for short‐term styrene responses related to activation of nuclear receptor signaling and cell proliferation. Greater tumor susceptibility in CD‐1 mice correlated with the presence of the Pas1 loci, differential Cytochrome P450 gene expression, down‐regulation of Nr4a, and greater inflammatory pathway activation. Very few exposure‐related responses occurred at any time in C57BL/6‐KO or ‐TG mice indicating that neither the short term nor long term responses of styrene in mice are relevant endpoints for assessing human risks. HIGHLIGHTSC57BL/6 KO & TG mice show few exposure related response at any time point.WT & CD‐1 show similar cellular pathway enrichment but differing in time course.WT mice respond with a single day; CD‐1 mice respond after several days of exposure.Results indicate non‐genotoxic mouse strain specific mode of action for short term exposure.Neither short nor long term responses of styrene in mice are relevant for human risk assessment.

Assessing bioactivity-exposure profiles of fruit and vegetable extracts in the BioMAP profiling system

The ToxCast program has generated in vitro screening data on over a thousand chemicals to assess potential disruption of important biological processes and assist in hazard identification and chemical testing prioritization. Few results have been reported for complex mixtures. To extend these ToxCast efforts to mixtures, we tested extracts from 30 organically grown fruits and vegetables in concentration-response in the BioMAP® assays. BioMAP systems use human primary cells primed with endogenous pathway activators to identify phenotypic perturbations related to proliferation, inflammation, immunomodulation, and tissue remodeling. Clustering of bioactivity profiles revealed separation of these produce extracts and ToxCast chemicals. Produce extracts elicited 87 assay endpoint responses per item compared to 20 per item for ToxCast chemicals. On a molar basis, the produce extracts were 10 to 50-fold less potent and when constrained to the maximum testing concentration of the ToxCast chemicals, the produce extracts did not show activity in as many assay endpoints. Using intake adjusted measures of dose, the bioactivity potential was higher for produce extracts than for agrichemicals, as expected based on the comparatively small amounts of agrichemical residues present on conventionally grown produce. The evaluation of BioMAP readouts and the dose responses for produce extracts showed qualitative and quantitative differences from results with single chemicals, highlighting challenges in the interpretation of bioactivity data and dose-response from complex mixtures.

Chapter 6:Using Transcriptomics to Evaluate Thresholds in Genotoxicity Dose–Response

Several genotoxic chemicals have been reported to produce threshold-shaped dose–response curves for mutation and genotoxicity assays, both in vivo and in vitro, challenging the current default practice for risk assessment of genotoxic chemicals, which assumes a linear dose–response below the lowest tested dose. Statistical methods cannot determine whether a biological threshold exists with sufficient confidence to overturn this assumption of linearity. Indeed, to truly define the shape of the dose–response curves, we must look to the underlying biology and develop targeted experiments to identify and measure the key processes governing the response of the cell to DNA damage. This chapter describes a series of studies aimed at defining the key transcriptional responses. Two approaches were taken to evaluate transcriptional responses preventing micronucleus induction: (1) comparison of gene signatures for several prototype compounds at a single chemical dose that led to a similar activation of the p53-DNA damage pathway (i.e. 1.5-fold increase in total p53); and (2) evaluation of a subset of chemicals with in-depth dose–response studies. The goal of these efforts was to determine the transcriptional pathways responsible for maintaining homeostasis at low levels of DNA damage, i.e., the biological underpinning of threshold-shaped dose–response curves for mutagenicity.