• CHOLINESTERASE INHIBITOR TOXICITY: MECHANISTIC STUDIES AND THERAPEUTIC STRATEGIES FOCUSED ON AXONAL TRANSPORT

      Naughton, Sean X; Biomedical Sciences (Augusta University, 2019-12)
      Organophosphates (OPs) are a broad class of chemicals with a variety of uses that include pesticides, chemical warfare agents, fuel additives, and plasticizers. Due to their sheer number of applications and known toxicological profile, OPs represent a persistent concern to human health worldwide. Furthermore, the effects of OPs that occur independently of their well-known mechanism of acute toxicity (AChE inhibition) have not been well studied. The presented research seeks to expand upon our understanding of AChE-independent mechanisms of OP toxicity as well as to identify potential therapies for treating these negative effects. In Manuscript 1 we demonstrate that the OP diisopropylfluorophosphate (DFP) induced axonal transport deficits occur in vivo at exposure levels that were not associated with cholinergic toxicity. Additionally, we observed deficits in white matter integrity following sub-acute DFP exposure. In Manuscript 2 we present a series of experiments, which were conducted to identify potential therapeutic compounds for the treatment of OP induced deficits in axonal transport. Here, we utilized a phenotypic drug-screening assay in order to identify compounds that could be protective against DFP. In Manuscript 3 we present data which demonstrates that the carbamate physostigmine does not impair axonal transport, as has been previously demonstrated with OPs. These experiments were critical to demonstrating the AChE independence of OP-induced axonal transport deficits and further elucidate the unique nature of OP toxicity in comparison to other AChE inhibitors. Collectively, these studies contribute to a better understanding of the full spectrum of toxicological effects of OPs and provide insightful findings into potential therapeutics for the treatment of OP related toxicity.
    • ROLE OF ARGINASE IN OBESITY-INDUCED VISCERAL ADIPOSE TISSUE DYSREGULATION AND ENDOTHELIAL DYSFUNCTION

      Atawia, Reem T.; Biomedical Sciences (Augusta University, 2019-11-05)
      An obesity epidemic continues to rise worldwide. Visceral (central) obesity is an important concern as it correlates with metabolic and cardiovascular pathologies. Arginase is a ureahydrolase enzyme with two isoforms (A1-cytosolic and A2-mitochondrial). We found that visceral adipose tissue (VAT) from obese WT mice fed a high fat/high sucrose diet (HFHS) showed a significantly higher expression of A2 compared to mice fed normal chow diet (ND). We also observed that A2 expression is upregulated 3-fold in differentiated 3T3- L1 adipocytes exposed to high levels of palmitate and glucose, a mimic of the obese state, compared to control media. Our study focused on the involvement of A2 in obesity associated metabolic and vascular disorders. WT mice and those globally lacking A2 (A2-/-) were fed HFHS or ND for 16 weeks. The HFHS diet-induced increases in body and VAT weights and total adiposity were prevented or reduced in A2-/- mice. In concert, metabolic chamber studies revealed that energy expenditure and fatty acid oxidation rates were significantly higher in A2-/- compared to WT HFHS mice. VAT from A2-/- mice fed HFHS had higher levels of active AMPK-α, the master regulator of fatty acid metabolism, as well as higher adipocyte expression of genes involved in fatty acid β-oxidation and oxidative phosphorylation, along with preserved mitochondrial density compared to WT HFHS. A2 deletion also prevented HFHS-induced fibrous tissue deposition and inflammation in VAT, which contributed to adipocyte metabolic dysfunction. These results indicate that A2 is involved in metabolic dysfunctions. To gain insights into the role of A2 in adipocytes, primary preadipocytes isolated from VAT of A2-/- mice and differentiated in vitro showed increased expression of adiponectin and better mitochondrial function. Adenoviral overexpression of A2 in differentiated 3T3-L1 cells showed impaired mitochondrial function and increased mitochondrial ROS. Obesity-related metabolic disorders increase the risk of cardiovascular diseases, the leading global cause of death. Endothelium-dependent vasorelaxation, impaired by HFHS diet, was significantly preserved in A2-/- mice, but more prominently prevented in A1+/- mice. In conclusion, A2 is critically involved in HFHS-induced obesity, VAT inflammation and metabolic dysregulation. Both A1 and A2 are involved in HFHS-induced vascular endothelial dysfunction.