2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a highly toxic environmental pollutant and is well known for its immunotoxic effects, particularly on the thymus. The exact mechanism by which TCDD induces thymic atrophy is not known. In the current study, we investigated whether TCDD triggers apoptosis in the thymocytes and whether Fas and Fas ligand play a role in TCDD-mediated immunotoxicity. Administration of a single dose of TCDD at 0.1, 1, 5 or 50 mg/kg body weight intraperitoneally into C57BL/6 +/+ mice caused a significant dose-dependent decrease in the thymic cellularity; whereas, in the C57BL/6 lpr/lpr (lpr) (Fas-deficient) and C57BL/6 gld/gld (gld) (Fas ligand-defective) mice, TCDD failed to induce a decrease in thymic cellularity at doses of 0.1-5 mg/kg body weight. In the lpr and gld mice, thymic atrophy was seen only at 50 mg/kg body weight of TCDD. Significant apoptosis was detected within 8-12 hours after injection in the wild type mice, whereas, in the lpr and gld mice apoptosis could not be detected. Upon culturing the thymocytes from TCDD-treated mice for 24 hours in vitro, the wild-type cells showed increased apoptosis when compared to the control; whereas, similar cells from lpr and gld mice did not show apoptosis. Furthermore, TCDD-treatment caused significant alterations in the expression of surface molecules on the thymocytes in the wild-type mice and minimal changes in the lpr or gld mice. Sera from TCDD-treated wild-type mice also exhibited increased levels of soluble Fas ligand. Also, TCDD-induced apoptosis was inhibited both in vitro and in vivo by caspase inhibitors and other inhibitors of apoptosis. Together, the current study demonstrates that TCDD-induced apoptosis plays an important role in thymic atrophy caused by TCDD in vivo. Furthermore, phenotypic changes in the density of thymocyte surface molecules may serve as a useful biomarker for chemical toxicity involving apoptosis. The current study also demonstrates that Fas-Fas ligand interactions play an important role in the induction of apoptosis and immunotoxicity by TCDD. / Ph. D.

Yes / The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor and member of the basic helix-loop-helix-PAS family. AHR is activated by numerous dietary and endogenous compounds that contribute to its regulation of genes in diverse signaling pathways including xenobiotic metabolism, vascular development, immune responses and cell cycle control. However, it is most widely studied for its role in mediating 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity. The AHR target gene and mono-ADP-ribosyltransferase, TCDD-inducible poly-ADP-ribose polymerase (TIPARP), was recently shown to be part of a novel negative feedback loop regulating AHR activity through mono-ADP-ribosylation. However, the molecular characterization of how AHR regulates TIPARP remains elusive. Here we show that activated AHR is recruited to the TIPARP promoter, through its binding to two genomic regions that each contain multiple AHR response elements (AHREs), AHR regulates the expression of both TIPARP but also TIPARP-AS1, a long non-coding RNA (lncRNA) which lies upstream of TIPARP exon 1 and is expressed in the opposite orientation. Reporter gene and deletion studies showed that the distal AHRE cluster predominantly regulated TIPARP expression while the proximal cluster regulated TIPARP-AS1. Moreover, time course and promoter activity assays suggest that TIPARP and TIPARP-AS1 work in concert to regulate AHR signaling. Collectively, these data show an added level of complexity in the AHR signaling cascade which involves lncRNAs, whose functions remain poorly understood. / This work was supported by Canadian Institutes of Health Research (CIHR) operating grants (MOP-494265 and MOP-125919), an unrestricted research grant from the Dow Chemical Company, and the Johan Throne Holst Foundation to J.M. G.G. was supported by European Union Seventh Framework Program (FP7-PEOPLE2013-COFUND) under the Grant Agreement n609020 - Scientia Fellows