Mitochondrial-targeted aryl hydrocarbon receptor and the impact of 2,3,7,8-tetrachlorodibenzo-p-dioxin on cellular respiration and the mitochondrial proteome.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor within the Per-Arnt-Sim (PAS) domain superfamily. Exposure to the most potent AHR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), is associated with various pathological effects including metabolic syndrome. While research over the last several years has demonstrated a role for oxidative stress and metabolic dysfunction in AHR-dependent TCDD-induced toxicity, the role of the mitochondria in this process has not been fully explored. Our previous research suggested that a portion of the cellular pool of AHR could be found in the mitochondria (mitoAHR). Using a protease protection assay with digitonin extraction, we have now shown that this mitoAHR is localized to the inter-membrane space (IMS) of the organelle. TCDD exposure induced a degradation of mitoAHR similar to that of cytosolic AHR. Furthermore, siRNA-mediated knockdown revealed that translocase of outer-mitochondrial membrane 20 (TOMM20) was involved in the import of AHR into the mitochondria. In addition, TCDD altered cellular respiration in an AHR-dependent manner to maintain respiratory efficiency as measured by oxygen consumption rate (OCR). Stable isotope labeling by amino acids in cell culture (SILAC) identified a battery of proteins within the mitochondrial proteome influenced by TCDD in an AHR-dependent manner. Among these, 17 proteins with fold changes≥2 are associated with various metabolic pathways, suggesting a role of mitochondrial retrograde signaling in TCDD-mediated pathologies. Collectively, these studies suggest that mitoAHR is localized to the IMS and AHR-dependent TCDD-induced toxicity, including metabolic dysfunction, wasting syndrome, and hepatic steatosis, involves mitochondrial dysfunction.

Data of enzymatic activities of the electron transport chain and ATP synthase complexes in mouse hepatoma cells following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the most widely studied ligand of the aryl hydrocarbon receptor (AHR). The AHR-dependent TCDD-induced mitochondrial hyperpolarization (Tappenden et al., 2011) [1] and reduced oxygen consumption rate of intact mouse hepatoma cells (Huang et al., in press) [2] in the previous studies suggest that these alterations can be related to enzymatic activities of the electron transport chain (ETC) and ATP synthase in oxidative phosphorylation (OXPHOS) system. Here, we evaluated the activity of each complex in the OXPHOS system using in vitro enzymatic assays. The calculated enzymatic activity of each complex was normalized against the activity of citrate synthase. To combine each value from an independent experiment, normalized enzyme activities from cells exposed to TCDD were converted to fold changes via comparison to the activity relative to time-matched vehicle control. The averaged fold change for each treatment suggests more replicates are needed in order to clearly evaluate a difference between treatments.

Diet is a major factor governing the fecal butyrate-producing community structure across Mammalia, Aves and Reptilia.

Butyrate-producing bacteria have an important role in maintaining host health. They are well studied in human and medically associated animal models; however, much less is known for other Vertebrata. We investigated the butyrate-producing community in hindgut-fermenting Mammalia (n = 38), Aves (n = 8) and Reptilia (n = 8) using a gene-targeted pyrosequencing approach of the terminal genes of the main butyrate-synthesis pathways, namely butyryl-CoA:acetate CoA-transferase (but) and butyrate kinase (buk). Most animals exhibit high gene abundances, and clear diet-specific signatures were detected with but genes significantly enriched in omnivores and herbivores compared with carnivores. But dominated the butyrate-producing community in these two groups, whereas buk was more abundant in many carnivorous animals. Clustering of protein sequences (5% cutoff) of the combined communities (but and buk) placed carnivores apart from other diet groups, except for noncarnivorous Carnivora, which clustered together with carnivores. The majority of clusters (but: 5141 and buk: 2924) did not show close relation to any reference sequences from public databases (identity <90%) demonstrating a large 'unknown diversity'. Each diet group had abundant signature taxa, where buk genes linked to Clostridium perfringens dominated in carnivores and but genes associated with Ruminococcaceae bacterium D16 were specific for herbivores and omnivores. Whereas 16S rRNA gene analysis showed similar overall patterns, it was unable to reveal communities at the same depth and resolution as the functional gene-targeted approach. This study demonstrates that butyrate producers are abundant across vertebrates exhibiting great functional redundancy and that diet is the primary determinant governing the composition of the butyrate-producing guild.

Alterations of the Murine Gut Microbiome with Age and Allergic Airway Disease.

The gut microbiota plays an important role in the development of asthma. With advanced age the microbiome and the immune system are changing and, currently, little is known about how these two factors contribute to the development of allergic asthma in the elderly. In this study we investigated the associations between the intestinal microbiome and allergic airway disease in young and old mice that were sensitized and challenged with house dust mite (HDM). After challenge, the animals were sacrificed, blood serum was collected for cytokine analysis, and the lungs were processed for histopathology. Fecal pellets were excised from the colon and subjected to 16S rRNA analysis. The microbial community structure changed with age and allergy development, where alterations in fecal communities from young to old mice resembled those after HDM challenge. Allergic mice had induced serum levels of IL-17A and old mice developed a greater allergic airway response compared to young mice. This study demonstrates that the intestinal bacterial community structure differs with age, possibly contributing to the exaggerated pulmonary inflammatory response in old mice. Furthermore, our results show that the composition of the gut microbiota changes with pulmonary allergy, indicating bidirectional gut-lung communications.