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    • In silico docking analysis performed in

    2024-01-22

    In silico docking analysis performed in the current study indicated that TCDD strongly binds to AhR-LBD. Moreover, the formation of the TCDD/AhR-LBD complex was confirmed experimentally with the use of EMSA. We found that 10nM TCDD after 2 hours of incubation not only bound to the AhR in the porcine granulosa cells but also activated the receptor. The effect of TCDD on AhR activation has been previously tested primarily on hepatic cells. Pollenz and Barbour [33] demonstrated that 2nM TCDD activated AhR in HepG2 cells after 1, 2 and 4h of incubation. The authors also observed that longer incubation (8h) resulted in a lower activity of the receptor, which suggests that the TCDD/AhR/ARNT/DRE complex is degraded with time. These results were confirmed by Frauenstein et al. [14] who reported that 1nM TCDD activated AhR in HepG2 cells after 2h of incubation. Moreover, similar to the current study, the same treatment dose and the same incubation time were tested by Novotna et al. [27] in mouse hepatoma (hepa1c1c7) cells and by Pollenz and Barbour [33] with the use of AhR and ARNT proteins expressed in vitro. In these studies, the implemented dose of TCDD (10nM) administered for 2h was sufficient to cause activation of AhR. Studies concerning the effects of TCDD on AhR activation were carried out also under in vivo conditions. TCDD (20μg/kg body weight) induced AhR activation in hepatic cells of C57LB/6 mice after two hours of treatment [18,42]. To our knowledge there is no information on ARNT-LBD in silico modeling. Our model, based on homology modeling to human ARNT crystalline structure, showed a high level of amino squalene epoxidase sequence identity as well as identical spatial arrangement of the protein residues. The porcine AhR-LBD and ARNT-LBD models constructed in the current study are reliable structures, displaying high level of similarity to known crystalline structures of other members of the PAS protein family. The low binding energy between TCDD and the porcine AhR-LBD confirms the high affinity of TCDD to the receptor, resulting in the activation of the AhR-mediated pathway.
    Acknowlegements
    Introduction One of the most characteristic and conspicuous signs of the acute toxicity of TCDD, an extremely potent agonist of the AHR, is the wasting syndrome, which is especially pronounced in the rat and denotes a profound body weight loss (up to over 50% of initial body weight) before death ensues. It primarily results from reduced feed intake [1,2]. The fact that this dramatic response did not appear to be due to nausea or malaise [3] suggested a specific impact on the regulation of energy balance or body weight. This view was reinforced by the findings that rats treated with sublethal doses of TCDD defended their lowered body weight level against external manipulation attempts by even exhibiting hyperphagia if necessary [4], and that TCDD exposure appeared to permanently imprint peculiar deviations on rats' feeding behavior and responses to feeding regulatory challenges [5–7]. Moreover, female C3H/HeN mice treated with a high dose of TCDD (100 μg/kg, once every 2 weeks for 8 weeks) and fed on a high-fat diet surprisingly exhibited augmented body gain compared with their vehicle-treated controls on the same diet [8]. Thus, it was somewhat disappointing to find out that genetic deletion of the AHR in mice or rats did not markedly influence the growth of the animals, although a transient retardation during the first few weeks of postnatal life was reported in mice [9]. However, more recent studies with congenic and genetically bioengineered mouse models have convincingly demonstrated that the AHR does indeed play an important modulating role in energy homeostasis. Initially, Kerley-Hamilton et al. [10] reported that when fed on high-fat chow (“Western diet”) for 28 weeks, male C57BL/6J (B6) mice with a high-affinity AHR (Ahrb1 allele) became more obese than their congenic counterparts, B6.D2N-Ahrd/J (B6.D2) mice with a low-affinity AHR (Ahrd allele). The difference in body weight emerged by 17 weeks and broadened thereafter until the end of the study. At termination, the B6 mice had larger gonadal fat pads, greater total volume of lipid vacuoles in the liver and higher plasma cholesterol levels than did B6.D2 mice on the same high-fat diet. Interestingly, however, these differences were not recorded when the mice were fed regular chow, and they did not appear to result from dissimilar feed intake levels. Moreover, in B6 mice high-fat diet repressed hepatic Cyp1a2 gene expression by approximately 3-fold (data on Cyp1a1 were lacking) compared with the standard diet, suggesting that the canonical AHR signaling pathway was not activated by the special diet. On the other hand, the expression levels of a wide variety of nuclear receptors were affected by diet and/or genotype in the liver. In most cases, high-fat diet exhibited a depressing influence in B6 mice, but Pparg (encoding PPARɣ) was induced by 61% in them. On high-fat diet, a genotype difference was recorded for Ppara expression [encoding PPARα], which was diminished by 34% in B6 vs. B6.D2 mice [10]. These might have contributed to the outcome because PPARα promotes fatty acid oxidation in the liver [11] while increased expression of PPARɣ is involved in liver steatosis [12,13].