Brian N. Chorley

Exposures to pollutants rarely occur in isolation, often coexisting with other environmental stressors such as diet and may be particularly insidious in early life. The aim of this study was to examine effects of maternal exposure to cadmium (Cd) and consumption of a high-fructose diet (HFrD) on development of mouse offspring. Female CD-1 mice were administered either 0.5 or 5 ppm Cd in drinking water with or without an approximate 60% fructose diet for 3 weeks prior to mating. Dams were maintained on the same treatment until postnatal day (PND) 16. Cadmium concentrations in maternal, fetal, and neonatal liver increased in a concentration-dependent manner irrespective of diet. Endpoints known to be associated with Cd or HFrD adverse effects were assessed longitudinally in offspring from birth to young adulthood, including growth trajectory, pubertal development, body composition, glycemic tolerance and hepatic lipid accumulation. Maternal exposure to either Cd or HFrD alone significantly advanced onset of puberty, hypoglycemia, and reduced adiposity in adulthood. HFrD rarely exacerbated metal-initiated effects in most of the endpoints examined outside of pubertal timing. Because of chronic effects attributed to Cd or HFrD on metabolic function (e.g. glucose tolerance), transcriptomics and gene methylation analyses were performed on livers from neonatal and adult offspring. Data were largely consistent with phenotypic findings. In summary, maternal exposure to Cd or HFrD alone perturbed growth and development, producing long-lasting changes in metabolic function in adult offspring. HFrD did not appear to significantly exaggerate adverse outcomes attributed to metal exposure in the endpoints examined.

We applied the TempO-LINC® platform to generate single-cell transcriptomic (SCTr) profiles of ∼40,000 HepaRG cells exposed to etoposide, brefeldin A, cycloheximide, rotenone, tBHQ, troglitazone, and tunicamycin at three concentrations for 24 hours. SCTr enabled a detailed analysis of adaptive stress response pathways (SRPs), including the unfolded protein response (UPR), oxidative stress response (OSR), heat shock response (HSR), and DNA damage response (DDR). Troglitazone upregulated lipid metabolism genes ( PLIN2 , ACOX1 ) along with HSR and UPR activation, with co-expression of DNAJA1 , HSP90AA1 , and DDIT3 in subsets of cells. Brefeldin A and tunicamycin strongly induced UPR markers ( HSPA5 , SYVN1 , LMF2 , PDIA4 ) in subsets of cells, with some also expressing apoptotic ( DDIT3 , CASP8 ) and autophagic ( SQSTM1 ) genes, indicating diverse stress responses. Rotenone activated GDF15 , TRIB3 , and DDIT3 in a fraction of cells, accompanied by PLIN2 and mild UPR induction, reflecting heterogeneous mitochondrial stress responses. We scored individual cells using literature-derived SRP gene signatures to characterize overall stress phenotypes and clustered them using a generalized Jaccard metric. The clustering revealed five phenotypic groups spanning cell states associated with homeostasis, adaptive responses, terminal outcomes, autophagy, and apoptosis. By systematically analyzing the distributions of cells in different states across treatments, we visualized dynamic shifts in cellular subpopulations responding to chemicals, revealing early stress responses and potential transitions to cell death. Our findings suggest the utility of SCTr in decoding stress states that could provide possible insights into transitions between cellular adaptive and terminal transitions involved in toxicity.

Background: Mechanistic understanding of transient exposures that lead to adverse health outcomes will enhance our ability to recognize biological signatures of disease. Here, we measured the transcriptomic and epigenomic alterations due to exposure to the metabolic reprogramming agent, dichloroacetic acid (DCA). Previously, we showed that exposure to DCA increased liver tumor incidence in B6C3F1 mice after continuous or early life exposures significantly over background level.Methods: Using archived formalin-fixed liver samples, we utilized modern methodologies to measure gene expression and DNA methylation levels to link to previously generated phenotypic measures. Gene expression was measured by targeted RNA sequencing (TempO-seq 1500+ toxicity panel: 2754 total genes) in liver samples collected from 10-, 32-, 57-, and 78-week old mice exposed to deionized water (controls), 3.5 g/L DCA continuously in drinking water (“Direct” group), or DCA for 10-, 32-, or 57-weeks followed by deionized water until sample collection (“Stop” groups). Genome-scaled alterations in DNA methylation were measured by Reduced Representation Bisulfite Sequencing (RRBS) in 78-week liver samples for control, Direct, 10-week Stop DCA exposed mice.Results: Transcriptomic changes were most robust with concurrent or adjacent timepoints after exposure was withdrawn. We observed a similar pattern with DNA methylation alterations where we noted attenuated differentially methylated regions (DMRs) in the 10-week Stop DCA exposure groups compared to the Direct group at 78-weeks. Gene pathway analysis indicated cellular effects linked to increased oxidative metabolism, a primary mechanism of action for DCA, closer to exposure windows especially early in life. Conversely, many gene signatures and pathways reversed patterns later in life and reflected more pro-tumorigenic patterns for both current and prior DCA exposures. DNA methylation patterns correlated to early gene pathway perturbations, such as cellular signaling, regulation and metabolism, suggesting persistence in the epigenome and possible regulatory effects.Conclusion: Liver metabolic reprogramming effects of DCA interacted with normal age mechanisms, increasing tumor burden with both continuous and prior DCA exposure in the male B6C3F1 rodent model.

Alterations in physiological processes in pancreas have been associated with various metabolic dysfunctions and can result from environmental exposures, such as chemicals and diet. It was reported that environmental vinyl chloride (VC) exposure, a common industrial organochlorine and environmental pollutant, significantly exacerbated metabolic-related phenotypes in mice fed concurrently with high-fat diet (HFD) but not low-fat diet (LFD). However, little is known about the role of the pancreas in this interplay, especially at a proteomic level. The present study was undertaken to examine the protein responses to VC exposure in pancreas tissues of C57BL/6J mice fed LFD or HFD, with focus on the investigation of protein expression and/or phosphorylation levels of key protein biomarkers of carbohydrate, lipid, and energy metabolism, oxidative stress and detoxification, insulin secretion and regulation, cell growth, development, and communication, immunological responses and inflammation, and biomarkers of pancreatic diseases and cancers. We found that the protein alterations may indicate diet-mediated susceptibility in mouse pancreas induced by HFD to concurrent exposure of low levels of inhaled VC. These proteome biomarkers may lead to a better understanding of pancreas-mediated adaptive or adverse response and susceptibility to metabolic disease.

Environmental Health Perspectives is an Open Access journal published by the National Institute of Environmental Health Sciences. Environmental Health Perspectives is an Open Access journal published by the National Institute of Environmental Health Sciences.