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Decoding cellular stress states for toxicology using single-cell transcriptomics

ABSTRACT: Single-cell transcriptomics (SCTr) offers a powerful platform for decoding the complexities of cellular stress responses to chemical exposures, capturing rare and transient subpopulations that can be obscured in bulk measurements. This study employs SCTr to profile the responses of HepaRG liver cells to various stress-inducing chemicals, providing detailed insights into adaptive stress response pathways (SRPs), encompassing the response to unfolded proteins, oxidative stress, heat shock, and DNA damage. By leveraging SCTr, we identify distinct cellular subpopulations and elucidate their transcriptomic profiles, linking specific gene expression patterns to biological mechanisms underlying stress responses. Our findings demonstrate the utility of SCTr in discerning the dose-dependent effects of chemicals at a granular level, highlighted by key differences between pseudo-bulk and single-cell analysis. Chemicals such as troglitazone, brefeldin A, rotenone, and tunicamycin induced unique transcriptional signatures associated with specific stress response pathways. Single-cell connectivity mapping with signatures of SRPs and cell fates, including autophagy and apoptosis, revealed clusters of cells with adaptive and potentially adverse phenotypes. These SCTr data have implications for identifying toxicological and disease mechanisms involving adaptive stress pathways. The data can also provide unique insights into toxicological processes by beginning to resolve adaptive chemical exposures, where cells recover, from those that are adverse from which cells do not recover, offering potentially new approaches for chemical safety assessments.

ORGANISM(S): Homo sapiens

PROVIDER: GSE299113 | GEO | 2025/06/10

REPOSITORIES: GEO

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Project description:Determining mechanism-based biomarkers that distinguish adaptive and adverse cellular processes is critical to understanding the health effects of environmental exposures. Shifting from in vivo, low-throughput toxicity studies to high-throughput screening (HTS) paradigms and risk assessment based on in vitro and in silico testing requires utilizing toxicity pathway information to distinguish adverse outcomes from recoverable adaptive events. Little work has focused on oxidative stresses in human airway for the purposes of predicting adverse responses. We hypothesize that early gene expression-mediated molecular changes could be used to delineate adaptive and adverse responses to environmentally-based perturbations. Here, we examined cellular responses of the tracheobronchial airway to zinc (Zn) exposure, a model oxidant. Airway derived BEAS-2B cells exposed to 2–10 µM Zn2+ elicited concentration- and time-dependent cytotoxicity. Normal, adaptive, and cytotoxic Zn2+ exposure conditions were determined with traditional apical endpoints, and differences in global gene expression around the tipping point of the responses were used to delineate underlying molecular mechanisms. Bioinformatic analyses of differentially expressed genes indicate early enrichment of stress signaling pathways, including those mediated by the transcription factors p53 and NRF2. After 4 h, 154 genes were differentially expressed (p <0.01) between the adaptive and cytotoxic Zn2+ concentrations. Nearly 40% of the biomarker genes were related to the p53 signaling pathway with 30 genes identified as likely direct targets using a database of p53 ChIP-seq studies. Despite similar p53 activation profiles, these data revealed widespread dampening of p53 and NRF2-related genes as early as 4 h after exposure at higher, unrecoverable Zn2+ exposures. Thus, in our model early increased activation of stress response pathways indicated a recoverable adaptive event. Overall, this study highlights the importance of characterizing molecular mechanisms around the tipping point of adverse responses to better inform HTS paradigms.

Project description:Two-year rodent bioassays play a central role in evaluating both the carcinogenic potential of a chemical and generating quantitative information on the dose-response behavior for chemical risk assessments. The bioassays involved are expensive and time-consuming, requiring nearly lifetime exposures (two years) in mice and rats and costing $2 to $4 million per chemical. Since there are approximately 80,000 chemicals registered for commercial use in the United States and 2,000 more are added each year, applying animal bioassays to all chemicals of concern is clearly impossible. To efficiently and economically identify carcinogens prior to widespread use and human exposure, alternatives to the two-year rodent bioassay must be developed. In this study, animals were exposed for 13 weeks to 10 chemicals that were positive for liver tumors in the two-year rodent bioassay, 14 chemicals that were negative for liver tumors, and two chemicals that produced an equivocal response. Matched vehicle control groups were run concurrently with each chemical treatment. Gene expression analysis was performed on the livers of the animals to assess the potential for identifying gene expression biomarkers and signaling pathways that can predict tumor formation in a two-year bioassay following a 13 week exposure. Five-week-old female B6C3F1 mice were exposed for 13 weeks to 26 chemicals. The chemical and dose information are provided with the individual sample annotations. With each chemical treatment, a matched vehicle control group was run concurrently with the exposure. A subset of five chemicals, methylene chloride, naphthalene, 1,2,3-trichloropropane, propylene glycol mono-t-butyl ether, and 1,4-dichlorobenzene, were performed in a five-point dose response with matched control groups. The concentrations for these exposures overlapped those in the original cancer bioassay. Gavage exposures were administered 5 days per week and feed exposures were provided 7 days per week. For inhalation exposures, mice were exposed 6 hr per day, 5 days per week. After 13 weeks, animals were euthanized and livers were collected. The right, caudate and median liver lobes were minced together and used for microarray analysis. Microarray analysis was performed on the livers of three to five mice per treatment group.

Project description:Transcriptional profiling of mussel (Mytilus galloprovincialis) digestive gland tissue comparing control tissue with tissue obtained from animals exposed for four days to sublethal amounts of Nickel, Chlorpyrifos, or their mixture. Background: Mixtures of chemicals present in aquatic environments may elicit toxicity due to additive or synergistic effects among the constituents or ‘vice versa’ the adverse outcome may be reduced by antagonistic interactions. Deviations from additivity should be explained either by the perturbations of toxicokinetic parameters and/or chemical toxicodynamics. We addressed this important question in marine mussels exposed subchronically to a binary mixture made of two wide-spread pollutants: the heavy metal Nickel and the organic phosphorus pesticide Chlorpyrifos. To this aim, we proposed the use of a systems approach based on the evaluation and integration of different disciplines, i.e. high throughput gene expression profiling, functional genomics, stress biomakers and toxicokinetics. Results: Stress biomarkers showed statistically significant antagonistic deviations from the reference model systems to predict mixture toxicity which are based either on simple additivity or non-interaction. While toxicokinetic modeling did not explain mixture interactions, gene expression profiling and further functional genomics analysis provided clues that the decrement of toxicity may arise from the development of specific toxicodynamics. Multivariate statistics of microarray and quantitative PCR data showed two separate patterns for the single chemicals and a composite complex profile for the mixture suggesting the occurrence of interactive molecular responses. The latter signature accounted for differentially expressed genes whose relative expression values were either in trend or in contrast with those found due to single substance treatments and a relevant set of sequences which were exclusive of the mixture gene list. Conclusion: The functional genomics assessment fits with biological data to indicate the occurrence of different toxicodynamic events and ‘in general’ a decrease of toxicity, driven by the mitigation or even abolishment of lysosomal responses such as the lipid metabolic disorder observed exclusively in single chemical-exposed samples. Furthermore, our results emphasized the importance of the application of mechanistic approaches and the power of systems assessment to study toxicological responses in ecological relevant organisms. Mussel MytArray 1.0 (Platform GPL1799)-based arrays: Three-condition experiment. Dual color competitive hybridizations. Common reference (vehicle treated animals, 0.02% Dimethyl sulfoxide); Pools of six animals. Biological replicates: 4 controls, 4 Nickel, 4 Chlorpyrifos. One replicate per array. Mussel MytArray 1.1 (Platform GPL10269)-based arrays: Four-condition experiment. Dual color competitive hybridizations. Common reference (vehicle treated animals, 0.02% Dimethyl sulfoxide); Pools of six animals. Biological replicates: 5 controls, 1 Nickel, 3 Chlorpyrifos, 5 mixture. One replicate per array.

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Decoding cellular stress states for toxicology using single-cell transcriptomics

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