John J. LaPres

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is associated with metabolic syndrome (MetS) in humans and elicits pathologies in rodents that resemble non-alcoholic fatty liver disease (NAFLD) in humans through activation of the aryl hydrocarbon receptor (AHR) pathway. Dysregulation of cholesterol homeostasis, an aspect of MetS, is linked to NAFLD pathogenesis. TCDD exposure is also linked to the suppression of genes that encode key cholesterol biosynthesis steps and changes in serum cholesterol levels. In a previous experiment, treating mice with TCDD in the presence of simvastatin, a 3-Hydroxy-3-Methylglutaryl-CoA Reductase competitive inhibitor, altered lipid and glycogen levels, AHR-battery gene expression, and liver injury in male mice compared to TCDD alone. The aim of this study was to deduce a possible mechanism(s) for the metabolic changes and increased injury using single-nuclei RNA sequencing in mouse liver. We demonstrated that co-treated mice experienced wasting and increased AHR activation compared to TCDD alone. Furthermore, relative proportions of cell (sub)types were different between TCDD alone and co-treated mice including important mediators of NAFLD progression like hepatocytes and immune cell populations. Analysis of non-overlapping differentially expressed genes identified several pathways where simvastatin co-treatment significantly impacted TCDD-induced changes, which may explain the differences between treatments. Overall, these results demonstrate a connection between dysregulation of cholesterol homeostasis and toxicant-induced metabolic changes.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor in the Per-Arnt-Sim superfamily of environmental sensors that is linked to several metabolic diseases, including nonalcoholic fatty liver disease. Much remains unknown regarding the impact of genetic variation in AHR-driven disease, as past studies have focused on a small number of inbred strains. Recently, the presence of a wide range of interindividual variability amongst humans was reported in response to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the prototypical ligand of the AHR. In this study, a panel of 14 diverse mouse strains was exposed to TCDD for 10 days to characterize the AHR-mediated response across genetic backgrounds. Responses to TCDD are heavily dependent on genetic background. Although mice carry 1 of 4 Ahr alleles known to impact the affinity to AHR-ligands, we observed significant intra-allelic variability suggesting the presence of novel genetic modifiers of AHR signaling. A regression-based approach was used to scan for genes regulated by the AHR and/or associated with TCDD-induced phenotypes. The approach identified 7 genes, 2 of which are novel, that are likely regulated by the AHR based on association with hepatic TCDD burden (p ≤ .05). Finally, we identified 1 gene, Dio1, which was associated with change in percent body fat across the diverse set of strains (p ≤ .05). Overall, the results in this study exemplify the power of genetics-based approaches in identifying novel genes that are putatively regulated by the AHR.

With recent advances in sequencing technologies, the scientific community has begun to probe the potential genetic bases behind complex phenotypes in humans and model organisms. In many cases, the genomes of genetically distinct strains of model organisms, such as the mouse (Mus musculus), have not been fully sequenced. Here, we report on a tool designed to use single-nucleotide polymorphism (SNP) and indel data to predict gene, mRNA, and protein sequences for up to 36 genetically distinct mouse strains. By automated querying of freely accessible databases through a graphical interface, the software requires no data and little computational experience. As a proof of concept, we predicted the gene and amino acid sequence of the aryl hydrocarbon receptor (Ahr) for all inbred mouse strains of which variant data was currently available through Mouse Genome Project. Predicted sequences were compared with fully sequenced genomes to show that the tool is effective in predicting gene and protein sequences.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor. The prototypical ligand of the AHR is an environmental contaminant called 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). TCDD exposure is associated with many adverse health outcomes in humans including non-alcoholic fatty liver disease (NAFLD). Previous studies suggest that AHR ligands alter cholesterol homeostasis in mice through repression of genes involved in cholesterol biosynthesis, such as Hmgcr, which encodes the rate-limiting enzyme of cholesterol biosynthesis called 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGCR). In this study, we sought to characterize the impact of HMGCR repression in TCDD-induced liver injury. C57BL/6 mice were exposed to TCDD in the presence or absence of simvastatin, a competitive inhibitor of HMGCR. Simvastatin exposure decreased TCDD-induced hepatic lipid accumulation in both sexes, but was most prominent in females. Simvastatin and TCDD (S + T) co-treatment increased hepatic AHR-battery gene expression and liver injury in male, but not female, mice. In addition, the S + T co-treatment led to an increase in hepatic glycogen content that coincides with heavier liver in female mice. Results from this study suggest that statins, which are amongst the most prescribed pharmaceuticals, may protect from AHR-mediated steatosis, but alter glycogen metabolism and increase the risk of TCDD-elicited liver damage in a sex-specific manner.

2,3,7,8-Tetrachlordibenzo-p-dioxin (TCDD) is an environmental pollutant that can cause various toxic effects, including chloracne, metabolic syndrome, and immune suppression. Most of the toxicity associated with TCDD is mediated through activation of the aryl hydrocarbon receptor (AHR). Recent research has suggested the presence of a wide-range of interindividual variability in TCDD-mediated suppression of the Immunoglobulin-M (IgM) response across the human population. In an attempt to identify putative modifiers of AHR-mediated immunosuppression beyond the AHR, B cells were isolated from a panel of genetically diverse mouse strain to scan for modulators that drive interstrain differences in TCDD-mediated suppression of the IgM response. Results implicated a region of mouse Chromosome 1 near a gene encoding serine peptidase inhibitor, clade B, member 2 (Serpinb2) whose human ortholog is plasminogen activator inhibitor 2 (PAI2). Further downstream analyses indicated that Serpinb2 is dysregulated by TCDD and, furthermore, that B cells from Serpinb2^–/– mice are significantly more sensitive to TCDD-mediated suppression as compared to littermate controls. This study suggests a protective role of Serpinb2 within TCDD-mediated immunosuppression and, furthermore, a novel function of Serpinb2-related activity in the IgM response.

The influence of inter-individual variability is not typically assessed in traditional toxicological studies. Given that chemical exposures occur in heterogeneous populations, this knowledge gap has the potential to cause undue harm within the realms of public health and industrial and municipal finances. A recent report from the National Research Council (NRC) suggests that, when accounting for inter-individual variation in responses, traditionally assumed nonlinear dose-response relationships (DRRs) for non-cancer causing endpoints would better be explained with a linear relationship within the low-dose region. To address this knowledge gap and directly test the NRC’s assumption, this study focused on assessing the DRR between 2,3,7,8-tetracholorodibenzo-p-dioxin (TCDD) exposure and immune suppression in a cohort of unique human donors. Human B cells were isolated from 51 individual donors and treated with logarithmically increasing concentrations of TCDD (0 through 30 nM TCDD). Two endpoints sensitive to TCDD were assessed: 1) number of IgM secreting B cells and 2) quantity of IgM secreted. The results show that TCDD significantly suppressed both the number of IgM secreting B cells and the quantity of IgM secreted (p-value < 0.05). Statistical model comparisons indicate that the low-dose region of the two DRRs is best explained with a nonlinear relationship. Rather than assuming low-dose linearity for all non-cancer causing DRRs, our study suggests the need to consider the specific mode-of-action of toxicants and pharmaceuticals during risk-management decision-making.