Thymic lymphoma detection in RORγ knockout mice using 5-hydroxymethylcytosine profiling of circulating cell-free DNA.

A high incidence of thymic lymphoma has been noted in mice deficient of retinoid-related orphan receptor γ2 (RORγ2), which is required for differentiation of naïve CD4+ T cells into TH17 cells. Using a RORγ homozygous knockout (KO) mouse model of thymic lymphoma, we characterized this tumor progression and investigated the utility of 5-hydroxymethylcytosine (5hmC) signatures as a non-invasive circulating biomarker for early prediction of malignancy. No evidence for malignancy was noted in the wild-type mice, while primary thymic lymphoma with multi-organ metastasis was observed microscopically in 97% of the homozygous RORγ KO mice. The severity of thymic lymphoma was not age-dependent in the KO mice of 2 to 4 months old. Differential enrichment of 5hmC in thymic DNA and plasma cell-free DNA (cfDNA) was compared across different stages of tumor progression. Random forest modeling of plasma cfDNA achieved good predictivity (AUC = 0.74) in distinguishing early non-metastatic thymic lymphoma compared to cancer-free controls, while perfect predictivity was achieved with advanced multi-organ metastatic disease (AUC = 1.00). Lymphoid-specific genes involved in thymocyte selection during T cell development (Themis, Tox) were differentially enriched in both plasma and thymic tissue. This could help in differentiating thymic lymphoma from other tumors commonly detected in rodent carcinogenicity studies used in pharmaceutical drug development to inform human malignancy risk. Overall, these results provide a proof-of-concept for using circulating cfDNA profiles in rodent carcinogenicity studies for early risk assessment of novel pharmaceutical targets.

A Biomarker-Centric Approach to Drug Discovery and Development: Lessons Learned from the Coronavirus Disease 2019 Pandemic.

Faced with the health and economic consequences of the global spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the biomedical community came together to identify, diagnose, prevent, and treat the novel disease at breathtaking speeds. The field advanced from a publicly available viral genome to a commercialized globally scalable diagnostic biomarker test in less than 2 months, and first-in-human dosing with vaccines and repurposed antivirals followed shortly thereafter. This unprecedented efficiency was driven by three key factors: 1) international multistakeholder collaborations, 2) widespread data sharing, and 3) flexible regulatory standards tailored to meet the urgency of the situation. Learning from the remarkable success achieved during this public health crisis, we are proposing a biomarker-centric approach throughout the drug development pipeline. Although all therapeutic areas would benefit from end-to-end biomarker science, efforts should be prioritized to areas with the greatest unmet medical needs, including neurodegenerative diseases, chronic lower respiratory diseases, metabolic disorders, and malignant neoplasms. SIGNIFICANCE STATEMENT: Faced with the unprecedented threat of the severe acute respiratory syndrome coronavirus 2 pandemic, the biomedical community collaborated to develop a globally scalable diagnostic biomarker (viral DNA) that catalyzed therapeutic development at breathtaking speeds. Learning from this remarkable efficiency, we propose a multistakeholder biomarker-centric approach to drug development across therapeutic areas with unmet medical needs.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) alters hepatic polyunsaturated fatty acid metabolism and eicosanoid biosynthesis in female Sprague-Dawley rats.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent aryl hydrocarbon receptor (AhR) agonist that elicits a broad spectrum of dose-dependent hepatic effects including lipid accumulation, inflammation, and fibrosis. To determine the role of inflammatory lipid mediators in TCDD-mediated hepatotoxicity, eicosanoid metabolism was investigated. Female Sprague-Dawley (SD) rats were orally gavaged with sesame oil vehicle or 0.01-10 µg/kg TCDD every 4 days for 28 days. Hepatic RNA-Seq data was integrated with untargeted metabolomics of liver, serum, and urine, revealing dose-dependent changes in linoleic acid (LA) and arachidonic acid (AA) metabolism. TCDD also elicited dose-dependent differential gene expression associated with the cyclooxygenase, lipoxygenase, and cytochrome P450 epoxidation/hydroxylation pathways with corresponding changes in ω-6 (e.g. AA and LA) and ω-3 polyunsaturated fatty acids (PUFAs), as well as associated eicosanoid metabolites. Overall, TCDD increased the ratio of ω-6 to ω-3 PUFAs. Phospholipase A2 (Pla2g12a) was induced consistent with increased AA metabolism, while AA utilization by induced lipoxygenases Alox5 and Alox15 increased leukotrienes (LTs). More specifically, TCDD increased pro-inflammatory eicosanoids including leukotriene LTB4, and LTB3, known to recruit neutrophils to damaged tissue. Dose-response modeling suggests the cytochrome P450 hydroxylase/epoxygenase and lipoxygenase pathways are more sensitive to TCDD than the cyclooxygenase pathway. Hepatic AhR ChIP-Seq analysis found little enrichment within the regulatory regions of differentially expressed genes (DEGs) involved in eicosanoid biosynthesis, suggesting TCDD-elicited dysregulation of eicosanoid metabolism is a downstream effect of AhR activation. Overall, these results suggest alterations in eicosanoid metabolism may play a key role in TCDD-elicited hepatotoxicity associated with the progression of steatosis to steatohepatitis.

Comparison of Hepatic NRF2 and Aryl Hydrocarbon Receptor Binding in 2,3,7,8-Tetrachlorodibenzo-p-dioxin-Treated Mice Demonstrates NRF2-Independent PKM2 Induction.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) induces hepatic oxidative stress following activation of the aryl hydrocarbon receptor (AhR). Our recent studies showed TCDD induced pyruvate kinase muscle isoform 2 (Pkm2) as a novel antioxidant response in normal differentiated hepatocytes. To investigate cooperative regulation between nuclear factor, erythroid derived 2, like 2 (Nrf2) and the AhR in the induction of Pkm2, hepatic chromatin immunoprecipitation sequencing (ChIP-seq) analyses were integrated with RNA sequencing (RNA-seq) time-course data from mice treated with TCDD for 2-168 hours. ChIP-seq analysis 2 hours after TCDD treatment identified genome-wide NRF2 enrichment. Approximately 842 NRF2-enriched regions were located in the regulatory region of differentially expressed genes (DEGs), whereas 579 DEGs showed both NRF2 and AhR enrichment. Sequence analysis of regions with overlapping NRF2 and AhR enrichment showed over-representation of either antioxidant or dioxin response elements, although 18 possessed both motifs. NRF2 exhibited negligible enrichment within a closed Pkm chromatin region, whereas the AhR was enriched 29-fold. Furthermore, TCDD induced Pkm2 in primary hepatocytes from wild-type and Nrf2-null mice, indicating NRF2 is not required. Although NRF2 and AhR cooperate to regulate numerous antioxidant gene expression responses, the induction of Pkm2 by TCDD is independent of reactive oxygen species-mediated NRF2 activation.

2,3,7,8-Tetrachlorodibenzo-p-dioxin dose-dependently increases bone mass and decreases marrow adiposity in juvenile mice.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and other aryl hydrocarbon receptor (AhR) agonists have been shown to regulate bone development and remodeling in a species-, ligand-, and age-specific manner, however the underlying mechanisms remain poorly understood. In this study, we characterized the effect of 0.01-30 µg/kg TCDD on the femoral morphology of male and female juvenile mice orally gavaged every 4 days for 28 days and used RNA-Seq to investigate gene expression changes associated with the resultant phenotype. Micro-computed tomography revealed that TCDD dose-dependently increased trabecular bone volume fraction (BVF) 2.9- and 3.3-fold in male and female femurs, respectively. Decreased serum tartrate-resistant acid phosphatase (TRAP) levels, combined with a reduced osteoclast surface to bone surface ratio and repression of femoral proteases (cathepsin K, matrix metallopeptidase 13), suggests that TCDD impaired bone resorption. Increased osteoblast counts at the trabecular bone surface were consistent with a reciprocal reduction in the number of bone marrow adipocytes, suggesting AhR activation may direct mesenchymal stem cell differentiation towards osteoblasts rather than adipocytes. Notably, femoral expression of transmembrane glycoprotein NMB (Gpnmb; osteoactivin), a positive regulator of osteoblast differentiation and mineralization, was dose-dependently induced up to 18.8-fold by TCDD. Moreover, increased serum levels of 1,25-dihydroxyvitamin D3 were in accordance with the renal induction of 1α-hydroxylase Cyp27b1 and may contribute to impaired bone resorption. Collectively, the data suggest AhR activation tipped the bone remodeling balance towards bone formation, resulting in increased bone mass with reduced marrow adiposity.

Convergence of hepcidin deficiency, systemic iron overloading, heme accumulation, and REV-ERBα/ß activation in aryl hydrocarbon receptor-elicited hepatotoxicity.

Persistent aryl hydrocarbon receptor (AhR) agonists elicit dose-dependent hepatic lipid accumulation, oxidative stress, inflammation, and fibrosis in mice. Iron (Fe) promotes AhR-mediated oxidative stress by catalyzing reactive oxygen species (ROS) production. To further characterize the role of Fe in AhR-mediated hepatotoxicity, male C57BL/6 mice were orally gavaged with sesame oil vehicle or 0.01-30µg/kg 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) every 4days for 28days. Duodenal epithelial and hepatic RNA-Seq data were integrated with hepatic AhR ChIP-Seq, capillary electrophoresis protein measurements, and clinical chemistry analyses. TCDD dose-dependently repressed hepatic expression of hepcidin (Hamp and Hamp2), the master regulator of systemic Fe homeostasis, resulting in a 2.6-fold increase in serum Fe with accumulating Fe spilling into urine. Total hepatic Fe levels were negligibly increased while transferrin saturation remained unchanged. Furthermore, TCDD elicited dose-dependent gene expression changes in heme biosynthesis including the induction of aminolevulinic acid synthase 1 (Alas1) and repression of uroporphyrinogen decarboxylase (Urod), leading to a 50% increase in hepatic hemin and a 13.2-fold increase in total urinary porphyrins. Consistent with this heme accumulation, differential gene expression suggests that heme activated BACH1 and REV-ERBα/ß, causing induction of heme oxygenase 1 (Hmox1) and repression of fatty acid biosynthesis, respectively. Collectively, these results suggest that Hamp repression, Fe accumulation, and increased heme levels converge to promote oxidative stress and the progression of TCDD-elicited hepatotoxicity.

Lipidomic Evaluation of Aryl Hydrocarbon Receptor-Mediated Hepatic Steatosis in Male and Female Mice Elicited by 2,3,7,8-Tetrachlorodibenzo-p-dioxin.

The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces hepatic steatosis mediated by the aryl hydrocarbon receptor. To further characterize TCDD-elicited hepatic lipid accumulation, mice were gavaged with TCDD every 4 days for 28 days. Liver samples were examined using untargeted lipidomics with structural confirmation of lipid species by targeted high-resolution MS/MS, and data were integrated with complementary RNA-Seq analyses. Approximately 936 unique spectral features were detected, of which 379 were confirmed as unique lipid species. Both male and female samples exhibited similar qualitative changes (lipid species) but differed in quantitative changes. A shift to higher mass lipid species was observed, indicative of increased free fatty acid (FFA) packaging. For example, of the 13 lipid classes examined, triglycerides increased from 46 to 48% of total lipids to 68-83% in TCDD treated animals. Hepatic cholesterol esters increased 11.3-fold in male mice with moieties consisting largely of dietary fatty acids (FAs) (i.e., linolenate, palmitate, and oleate). Phosphatidylserines, phosphatidylethanolamines, phosphatidic acids, and cardiolipins decreased 4.1-, 5.0-, 5.4- and 7.4-fold, respectively, while ceramides increased 6.6-fold. Accordingly, the integration of lipidomic data with differential gene expression associated with lipid metabolism suggests that in addition to the repression of de novo fatty acid synthesis and ß-oxidation, TCDD also increased hepatic uptake and packaging of lipids, while inhibiting VLDL secretion, consistent with hepatic fat accumulation and the progression to steatohepatitis with fibrosis.

Liver-specific loss of Perilipin 2 alleviates diet-induced hepatic steatosis, inflammation, and fibrosis.

Hepatic inflammation and fibrosis are key elements in the pathogenesis of nonalcoholic steatohepatitis (NASH), a progressive liver disease initiated by excess hepatic lipid accumulation. Lipid droplet protein Perilipin 2 (Plin2) alleviates dietary-induced hepatic steatosis when globally ablated; however, its role in the progression of NASH remains unknown. To investigate this further, we challenged Plin2 liver-specific knockout mice (designated L-KO) and their respective wild-type (WT) controls with a methionine-choline-deficient (MCD) diet for 15 days to induce a NASH phenotype of increased hepatic triglyceride levels through impaired phosphatidylcholine (PC) synthesis and very-low-density lipoprotein (VLDL) secretion. Results on liver weights, body weights, fat tissue mass, and histology in WT and L-KO mice fed the MCD diet revealed signs of hepatic steatosis, fibrosis, and inflammation; however, these effects were blunted in L-KO mice. In addition, levels of PC and VLDL were unchanged, and hepatic steatosis was reduced in L-KO mice fed the MCD diet, due in part to an increase in remodeling of PE to PC via the enzyme phosphatidylethanolamine N-methyltransferase (PEMT). These mice also exhibited decreased hepatic expression of proinflammatory markers cyclooxygenase 2, IL-6, TNF-α, IL-1ß, and reduced expression of endoplasmic reticulum (ER) stress proteins C/EBP homologous protein and cleaved caspase-1. Taken together, these results suggest that Plin2 liver-specific ablation alleviates diet-induced hepatic steatosis and inflammation via a PEMT-mediated mechanism that involves compensatory changes in proteins involved in phospholipid remodeling, inflammation, and ER stress that work to alleviate diet-induced NASH. Overall, these findings support a role for Plin2 as a target for NASH therapy.

RNA-Seq versus oligonucleotide array assessment of dose-dependent TCDD-elicited hepatic gene expression in mice.

BACKGROUND: Dose-dependent differential gene expression provides critical information required for regulatory decision-making. The lower costs associated with RNA-Seq have made it the preferred technology for transcriptomic analysis. However, concordance between RNA-Seq and microarray analyses in dose response studies has not been adequately vetted. RESULTS: We compared the hepatic transcriptome of C57BL/6 mice following gavage with sesame oil vehicle, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, or 30 µg/kg TCDD every 4 days for 28 days using Illumina HiSeq RNA-Sequencing (RNA-Seq) and Agilent 4 × 44 K microarrays using the same normalization and analysis approach. RNA-Seq and microarray analysis identified a total of 18,063 and 16,403 genes, respectively, that were expressed in the liver. RNA-Seq analysis for differentially expressed genes (DEGs) varied dramatically depending on the P1(t) cut-off while microarray results varied more based on the fold change criteria, although responses strongly correlated. Verification by WaferGen SmartChip QRTPCR revealed that RNA-Seq had a false discovery rate of 24% compared to 54% for microarray analysis. Dose-response modeling of RNA-Seq and microarray data demonstrated similar point of departure (POD) and ED50 estimates for common DEGs. CONCLUSIONS: There was a strong correspondence between RNA-Seq and Agilent array transcriptome profiling when using the same samples and analysis strategy. However, RNA-Seq provided superior quantitative data, identifying more genes and DEGs, as well as qualitative information regarding identity and annotation for dose response modeling in support of regulatory decision-making.

Western blotting: evolution of an old analytical method to a new quantitative tool for biomarker measurements.

Western blotting (WB) is a widely used laboratory technique for detecting specific proteins in biological matrices. Recent advances in antibody production, automation, gel and membrane manufacturing and highly sensitive detection platforms have transformed WB from a labor-intensive and qualitative method into a highly reproducible and quantitative assay suitable for biomarker detection. Despite these significant improvements in the capabilities and efficiency of WB, there remain challenges that hinder its widespread application as a research, diagnostic (in two-tiered assays like Lyme disease testing) and drug development tool. This article describes recent innovations introduced to WB methodology and the remaining challenges that prevent its wider adoption for biomarker measurements throughout the drug development process.

Absence of functional deficits in rats following systemic administration of an AAV9 vector despite moderate peripheral nerve and dorsal root ganglia findings: A clinically silent peripheral neuropathy.

Adeno-associated virus (AAV)-based vectors are commonly used for delivering transgenes in gene therapy studies, but they are also known to cause dorsal root ganglia (DRG) and peripheral nerve toxicities in animals. However, the functional implications of these pathologic findings and their time course remain unclear. At 2, 4, 6, and 8 weeks following a single dose of an AAV9 vector carrying human frataxin transgene in rats, non-standard functional assessments, including von Frey filament, electrophysiology, and Rotarod tests, were conducted longitudinally to measure allodynia, nerve conduction velocity, and coordination, respectively. Additionally, DRGs, peripheral nerves, brain and spinal cord were evaluated histologically and circulating neurofilament light chain (NfL) was quantified at 1, 2, 4, and 8 weeks, respectively. At 2 and 4 weeks after dosing, minimal-to-moderate nerve fiber degeneration and neuronal degeneration were observed in the DRGs in some of the AAV9 vector-dosed animals. At 8 weeks, nerve fiber degeneration was observed in DRGs, with or without neuronal degeneration, and in sciatic nerves of all AAV9 vector-dosed animals. NfL values were higher in AAV9 vector-treated animals at weeks 4 and 8 compared with controls. However, there were no significant differences in the three functional endpoints evaluated between the AAV9 vector- and vehicle-dosed animals, or in a longitudinal comparison between baseline (predose), 4, and 8 week values in the AAV9 vector-dose animals. These findings demonstrate that there is no detectable functional consequence to the minimal-to-moderate neurodegeneration observed with our AAV9 vector treatment in rats, suggesting a functional tolerance or reserve for loss of DRG neurons after systemic administration of AAV9 vector.

Consequences of reprogramming acetyl-CoA metabolism by 2,3,7,8-tetrachlorodibenzo-p-dioxin in the mouse liver.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental contaminant that induces the progression of steatosis to steatohepatitis with fibrosis in mice. Furthermore, TCDD reprograms hepatic metabolism by redirecting glycolytic intermediates while inhibiting lipid metabolism. Here, we examined the effect of TCDD on hepatic acetyl-coenzyme A (acetyl-CoA) and ß-hydroxybutyrate levels as well as protein acetylation and ß-hydroxybutyrylation. Acetyl-CoA is not only a central metabolite in multiple anabolic and catabolic pathways, but also a substrate used for posttranslational modification of proteins and a surrogate indicator of cellular energy status. Targeted metabolomic analysis revealed a dose-dependent decrease in hepatic acetyl-CoA levels coincident with the phosphorylation of pyruvate dehydrogenase (E1), and the induction of pyruvate dehydrogenase kinase 4 and pyruvate dehydrogenase phosphatase, while repressing ATP citrate lyase and short-chain acyl-CoA synthetase gene expression. In addition, TCDD dose-dependently reduced the levels of hepatic ß-hydroxybutyrate and repressed ketone body biosynthesis gene expression. Moreover, levels of total hepatic protein acetylation and ß-hydroxybutyrylation were reduced. AMPK phosphorylation was induced consistent with acetyl-CoA serving as a cellular energy status surrogate, yet subsequent targets associated with re-establishing energy homeostasis were not activated. Collectively, TCDD reduced hepatic acetyl-CoA and ß-hydroxybutyrate levels eliciting starvation-like conditions despite normal levels of food intake.

Circulating neurofilament light chain as a promising biomarker of AAV-induced dorsal root ganglia toxicity in nonclinical toxicology species.

Adeno-associated virus (AAV)-induced dorsal root ganglia (DRG) toxicity has been observed in several nonclinical species, where lesions are characterized by neuronal degeneration/necrosis, nerve fiber degeneration, and mononuclear cell infiltration. As AAV vectors become an increasingly common platform for novel therapeutics, non-invasive biomarkers are needed to better characterize and manage the risk of DRG neurotoxicity in both nonclinical and clinical studies. Based on biological relevance, reagent availability, antibody cross-reactivity, DRG protein expression, and assay performance, neurofilament light chain (NF-L) emerged as a promising biomarker candidate. Dose- and time-dependent changes in NF-L were evaluated in male Wistar Han rats and cynomolgus monkeys following intravenous or intrathecal AAV injection, respectively. NF-L profiles were then compared against microscopic DRG lesions on day 29 post-dosing. In animals exhibiting DRG toxicity, plasma/serum NF-L was strongly associated with the severity of neuronal degeneration/necrosis and nerve fiber degeneration, with elevations beginning as early as day 8 in rats (≥5 × 1013 vg/kg) and day 14 in monkeys (≥3.3 × 1013 vg/dose). Consistent with the unique positioning of DRGs outside the blood-brain barrier, NF-L in cerebrospinal fluid was only weakly associated with DRG findings. In summary, circulating NF-L is a promising biomarker of AAV-induced DRG toxicity in nonclinical species.

Single-Nuclei RNA Sequencing Assessment of the Hepatic Effects of 2,3,7,8-Tetrachlorodibenzo-p-dioxin.

BACKGROUND AND AIMS: Characterization of cell specific transcriptional responses to hepatotoxicants is lost in the averages of bulk RNA-sequencing (RNA-seq). Single-cell/nuclei RNA-seq technologies enable the transcriptomes of individual cell (sub)types to be assessed within the context of in vivo models. METHODS: Single-nuclei RNA-sequencing (snSeq) of frozen liver samples from male C57BL/6 mice gavaged with sesame oil vehicle or 30 µg/kg 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) every 4 days for 28 days was used to demonstrate the application of snSeq for the evaluation of xenobiotics. RESULTS: A total of 19,907 genes were detected across 16,015 nuclei from control and TCDD-treated livers. Eleven cell (sub)types reflected the expected cell diversity of the liver including distinct pericentral, midzonal, and periportal hepatocyte subpopulations. TCDD altered relative proportions of cell types and elicited cell-specific gene expression profiles. For example, macrophages increased from 0.5% to 24.7%, while neutrophils were only present in treated samples, consistent with histological evaluation. The number of differentially expressed genes (DEGs) in each cell type ranged from 122 (cholangiocytes) to 7625 (midcentral hepatocytes), and loosely correlated with the basal expression level of Ahr, the canonical mediator of TCDD and related compounds. In addition to the expected functions within each cell (sub)types, RAS signaling and related pathways were specifically enriched in nonparenchymal cells while metabolic process enrichment occurred primarily in hepatocytes. snSeq also identified the expansion of a Kupffer cell subtype highly expressing Gpnmb, as reported in a dietary NASH model. CONCLUSIONS: We show that snSeq of frozen liver samples can be used to assess cell-specific transcriptional changes and population shifts in models of hepatotoxicity when examining freshly isolated cells is not feasible.

Thioesterase induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin results in a futile cycle that inhibits hepatic ß-oxidation.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a persistent environmental contaminant, induces steatosis by increasing hepatic uptake of dietary and mobilized peripheral fats, inhibiting lipoprotein export, and repressing ß-oxidation. In this study, the mechanism of ß-oxidation inhibition was investigated by testing the hypothesis that TCDD dose-dependently repressed straight-chain fatty acid oxidation gene expression in mice following oral gavage every 4 days for 28 days. Untargeted metabolomic analysis revealed a dose-dependent decrease in hepatic acyl-CoA levels, while octenoyl-CoA and dicarboxylic acid levels increased. TCDD also dose-dependently repressed the hepatic gene expression associated with triacylglycerol and cholesterol ester hydrolysis, fatty acid binding proteins, fatty acid activation, and 3-ketoacyl-CoA thiolysis while inducing acyl-CoA hydrolysis. Moreover, octenoyl-CoA blocked the hydration of crotonyl-CoA suggesting short chain enoyl-CoA hydratase (ECHS1) activity was inhibited. Collectively, the integration of metabolomics and RNA-seq data suggested TCDD induced a futile cycle of fatty acid activation and acyl-CoA hydrolysis resulting in incomplete ß-oxidation, and the accumulation octenoyl-CoA levels that inhibited the activity of short chain enoyl-CoA hydratase (ECHS1).

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) dysregulates hepatic one carbon metabolism during the progression of steatosis to steatohepatitis with fibrosis in mice.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a persistent environmental contaminant, induces steatosis that can progress to steatohepatitis with fibrosis, pathologies that parallel stages in the development of non-alcoholic fatty liver disease (NAFLD). Coincidently, one carbon metabolism (OCM) gene expression and metabolites are often altered during NAFLD progression. In this study, the time- and dose-dependent effects of TCDD were examined on hepatic OCM in mice. Despite AhR ChIP-seq enrichment at 2 h, OCM gene expression was not changed within 72 h following a bolus dose of TCDD. Dose-dependent repression of methionine adenosyltransferase 1A (Mat1a), adenosylhomocysteinase (Achy) and betaine-homocysteine S-methyltransferase (Bhmt) mRNA and protein levels following repeated treatments were greater at 28 days compared to 8 days. Accordingly, levels of methionine, betaine, and homocysteic acid were dose-dependently increased, while S-adenosylmethionine, S-adenosylhomocysteine, and cystathionine exhibited non-monotonic dose-dependent responses consistent with regulation by OCM intermediates and repression of glycine N-methyltransferase (Gnmt). However, the dose-dependent effects on SAM-dependent metabolism of polyamines and creatine could not be directly attributed to alterations in SAM levels. Collectively, these results demonstrate persistent AhR activation disrupts hepatic OCM metabolism at the transcript, protein and metabolite levels within context of TCDD-elicited progression of steatosis to steatohepatitis with fibrosis.

2,3,7,8-Tetrachlorodibenzo-p-dioxin abolishes circadian regulation of hepatic metabolic activity in mice.

Aryl hydrocarbon receptor (AhR) activation is reported to alter the hepatic expression of circadian clock regulators, however the impact on clock-controlled metabolism has not been thoroughly investigated. This study examines the effects of AhR activation on hepatic transcriptome and metabolome rhythmicity in male C57BL/6 mice orally gavaged with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) every 4 days for 28 days. TCDD diminished the rhythmicity of several core clock regulators (e.g. Arntl, Clock, Nr1d1, Per1, Cry1, Nfil3) in a dose-dependent manner, involving either a ≥ 3.3-fold suppression in amplitude or complete loss of oscillation. Accordingly, protein levels (ARNTL, REV-ERBα, NFIL3) and genomic binding (ARNTL) of select regulators were reduced and arrhythmic following treatment. As a result, the oscillating expression of 99.6% of 5,636 clock-controlled hepatic genes was abolished including genes associated with the metabolism of lipids, glucose/glycogen, and heme. For example, TCDD flattened expression of the rate-limiting enzymes in both gluconeogenesis (Pck1) and glycogenesis (Gys2), consistent with the depletion and loss of rhythmicity in hepatic glycogen levels. Examination of polar hepatic extracts by untargeted mass spectrometry revealed that virtually all oscillating metabolites lost rhythmicity following treatment. Collectively, these results suggest TCDD disrupted circadian regulation of hepatic metabolism, altering metabolic efficiency and energy storage.

Characterizing the Role of HMG-CoA Reductase in Aryl Hydrocarbon Receptor-Mediated Liver Injury in C57BL/6 Mice.

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.

Beyond the Aryl Hydrocarbon Receptor: Pathway Interactions in the Hepatotoxicity of 2,3,7,8-Tetrachlorodibenzo-p-dioxin and Related Compounds.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the prototypical ligand for a group of environmental halogenated aromatic hydrocarbon contaminants which elicit hepatotoxicity and other toxic responses through activation of the aryl hydrocarbon receptor (AhR). Despite the conservation of the AhR and its signaling pathway, TCDD-elicited differential gene expression networks are species-specific, consistent with differences in sensitivity and toxic responses between species. This review integrates gene expression studies with complementary phenotypic analyses (e.g., metabolomics, clinical biochemistry, and histopathology) to elucidate the pathways through which TCDD and related compounds cause hepatotoxicity beyond AhR activation. We propose that AhR-mediated toxicity is a collective response to the cumulative burden of metabolic reprogramming across multiple pathways. Consequently, nutrition, health status, and genetic background establish the basis for differences in sensitivity and predisposition to adverse outcomes between species, sub-populations, tissues, and cells.

Loss of liver-specific and sexually dimorphic gene expression by aryl hydrocarbon receptor activation in C57BL/6 mice.

The aryl hydrocarbon receptor (AhR) is a highly conserved transcription factor that mediates a broad spectrum of species-, strain-, sex-, age-, tissue-, and cell-specific responses elicited by structurally diverse ligands including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Dose-dependent effects on liver-specific and sexually dimorphic gene expression were examined in male and female mice gavaged with TCDD every 4 days for 28 or 92 days. RNA-seq data revealed the coordinated repression of 181 genes predominately expressed in the liver including albumin (3.7-fold), α-fibrinogen (14.5-fold), and ß-fibrinogen (17.4-fold) in males with corresponding AhR enrichment at 2 hr. Liver-specific genes exhibiting sexually dimorphic expression also demonstrated diminished divergence between sexes. For example, male-biased Gstp1 was repressed 3.0-fold in males and induced 4.5-fold in females, which were confirmed at the protein level. Disrupted regulation is consistent with impaired GHR-JAK2-STAT5 signaling and inhibition of female specific CUX2-mediated transcription as well as the repression of other key transcriptional regulators including Ghr, Stat5b, Bcl6, Hnf4a, Hnf6, Foxa1/2/3, and Zhx2. Attenuated liver-specific and sexually dimorphic gene expression was concurrent with the induction of fetal genes such as alpha-fetoprotein. The results suggest AhR activation causes the loss of liver-specific and sexually dimorphic gene expression producing a functionally "de-differentiated" hepatic phenotype.