Genetics-Based Approach to Identify Novel Genes Regulated by the Aryl Hydrocarbon Receptor in Mouse Liver.

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.

Peanut butter as an alternative dose delivery method to prevent strain-dependent orogastric gavage-induced stress in mouse teratogenicity studies.

INTRODUCTION: Animal-based studies are essential for assessing toxicity to environmental pollutants, especially when the potential targets are specific developmental time points, teratogenic, or multi-organ systems that cannot be modeled in vitro. Orogastric gavage is a widely used technique for exposure because of its increased accuracy of dose administration over free feeding. However, repeated use of this method has been reported to cause physiological stress on the exposed animals that could interfere with interpretation of results. Previous studies have shown that genetic background also contributes to the level of stress and can affect individual response. METHODS: To evaluate the impact of stress on repeated orogastric gavage, we exposed C67BL/6J and 129S1/SvImJ inbred mouse strains to 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD), a potent xenobiotic that has been extensively studied in vivo. Pregnant females were dosed for ten days after mating using orogastric gavage with olive oil as vehicle or through diet using peanut butter as vehicle. Serum corticosterone levels, body weight, and reproduction endpoints were measured to evaluate levels of stress induced by the dosing technique. RESULTS: The levels of stress caused by orogastric gavage was strongly dependent on strain background and on the phenotypic endpoint. Orogastric gavage-induced stress was more detrimental in 129S1/SvlmJ pregnant female mice than in C57BL/6J. CONCLUSION: These results show that administration of xenobiotics via controlled diet can improve the reproducibility and rigor of exposure studies requiring orogastric delivery.

Characterizing Serpinb2 as a Modulator of TCDD-Induced Suppression of the B Cell.

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.

Interleukin-24 (IL24) Is Suppressed by PAX3-FOXO1 and Is a Novel Therapy for Rhabdomyosarcoma.

Alveolar rhabdomyosarcoma (ARMS) patients have a poor prognosis, and this is primarily due to overexpression of the oncogenic fusion protein PAX3-FOXO1. Results of RNA-sequencing studies show that PAX3-FOXO1 represses expression of interleukin-24 (IL24), and these two genes are inversely expressed in patient tumors. PAX3-FOXO1 also regulates histone deacetylase 5 (HDAC5) in ARMS cells, and results of RNA interference studies confirmed that PAX3-FOXO1-mediated repression of IL24 is HDAC5-dependent. Knockdown of PAX3-FOXO1 decreases ARMS cell proliferation, survival, and migration, and we also observed similar responses in cells after overexpression of IL24, consistent with results reported for this tumor suppressor-like cytokine in other solid tumors. We also observed in double knockdown studies that the inhibition of ARMS cell proliferation, survival, and migration after knockdown of PAX3-FOXO1 was significantly (>75%) reversed by knockdown of IL24. Adenoviral-expressed IL24 was directly injected into ARMS tumors in athymic nude mice, and this resulted in decreased tumor growth and weight. Because adenoviral IL24 has already successfully undergone phase I in clinical trials, this represents an alternative approach (alone and/or combination) for treating ARMS patients who currently undergo cytotoxic drug therapies.

GenSeq: An updated nomenclature and ranking for genetic sequences from type and non-type sources.

An improved and expanded nomenclature for genetic sequences is introduced that corresponds with a ranking of the reliability of the taxonomic identification of the source specimens. This nomenclature is an advancement of the "Genetypes" naming system, which some have been reluctant to adopt because of the use of the "type" suffix in the terminology. In the new nomenclature, genetic sequences are labeled "genseq," followed by a reliability ranking (e.g., 1 if the sequence is from a primary type), followed by the name of the genes from which the sequences were derived (e.g., genseq-1 16S, COI). The numbered suffix provides an indication of the likely reliability of taxonomic identification of the voucher. Included in this ranking system, in descending order of taxonomic reliability, are the following: sequences from primary types - "genseq-1," secondary types - "genseq-2," collection-vouchered topotypes - "genseq-3," collection-vouchered non-types - "genseq-4," and non-types that lack specimen vouchers but have photo vouchers - "genseq-5." To demonstrate use of the new nomenclature, we review recently published new-species descriptions in the ichthyological literature that include DNA data and apply the GenSeq nomenclature to sequences referenced in those publications. We encourage authors to adopt the GenSeq nomenclature (note capital "G" and "S" when referring to the nomenclatural program) to provide a searchable tag (e.g., "genseq"; note lowercase "g" and "s" when referring to sequences) for genetic sequences from types and other vouchered specimens. Use of the new nomenclature and ranking system will improve integration of molecular phylogenetics and biological taxonomy and enhance the ability of researchers to assess the reliability of sequence data. We further encourage authors to update sequence information on databases such as GenBank whenever nomenclatural changes are made.