The single most important finding in insulin-dependent diabetes (IDDM) is a substantial reduction in the absolute number of insulin-secreting pancreatic beta cells. Additionally, impaired beta cell function now appears to be the initial event observed at the onset of noninsulin-dependent diabetes (NIDDM). Compelling experimental and epidemiological evidence indicates that, at least in some forms of IDDM, environmental factors play an important role in the critical depletion of insular tissue. Also, functional alterations in beta cells, similar to those seen in NIDDM, have been produced by experimental exposure to the N-nitroso compound, streptozotocin. N-nitroso compounds and their precursors are ubiquitous environmental pollutants that are commonly found in human food and the atmosphere. Several of these chemicals have been found to selectively intoxicate pancreatic beta cells. The mechanisms by which these toxins specifically interact with beta cells resulting in functional alterations and cell-death remain to be fully elucidated. It is the objective of this proposal to investigate these mechanisms. Because of the complexities of chemical metabolism and the difficulties of determining mechanisms of cytotoxicity in intact animals, mechanistic studies are uniquely suited for tissue culture systems. The objective of this proposal will be pursued through the following aims: 1) Examine the production and repair of specific lesions in the nuclear DNA of beta cells following a chemical insult. These studies will employ a 32p endlabeling technique in combination with thin-layer chromatography to identify specific DNA adducts. Of particular interest will be the formation and repair of lesions that are known to be mutagenic. 2) Determine alterations in transcriptionally active genes following exposure to beta cell toxins. These studies will focus on how the transcriptional activity of the insulin genes in the beta cell affects the repair of lesions induced by alkylating toxins. 3) Examine the effects of various nitrosoamides on beta cell mitochondrial DNA. 32p endlabeling techniques will be used to characterize the formation and repair of lesions induced in mitochondrial DNA by these toxins. 4) Compare the intracellular distribution of toxic nitrosoamides in beta cells to nontoxic nitrosoamides. Cellular fractionation studies employing 14C labeled chemicals will be used to identify sites for selective sequestering of beta cell specific toxins. 5) Investigate the effects of beta cell toxins on the nuclear DNA from human islets. Studies will be made to determine the extent of alkylation in human beta cell DNA following exposure to nitrosoamides. The repair or lack of repair of these lesions will be of particular interest. When successfully executed, these studies will provide a more complet understanding of how environmental pollutants can selectively interact with normal beta cells to cause functional impairment and/or the death of these cells.

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National Institute of Environmental Health Sciences (NIEHS)
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Chemical Pathology Study Section (CPA)
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University of South Alabama
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Danobeitia, Juan S; Chlebeck, Peter J; Shokolenko, Inna et al. (2017) Novel Fusion Protein Targeting Mitochondrial DNA Improves Pancreatic Islet Functional Potency and Islet Transplantation Outcomes. Cell Transplant 26:1742-1754
Shokolenko, Inna N; Wilson, Glenn L; Alexeyev, Mikhail F (2016) The ""fast"" and the ""slow"" modes of mitochondrial DNA degradation. Mitochondrial DNA A DNA Mapp Seq Anal 27:490-8
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Shokolenko, Inna N; Fayzulin, Rafik Z; Katyal, Sachin et al. (2013) Mitochondrial DNA ligase is dispensable for the viability of cultured cells but essential for mtDNA maintenance. J Biol Chem 288:26594-605
Alexeyev, Mikhail; Shokolenko, Inna; Wilson, Glenn et al. (2013) The maintenance of mitochondrial DNA integrity--critical analysis and update. Cold Spring Harb Perspect Biol 5:a012641
Shokolenko, Inna N; Wilson, Glenn L; Alexeyev, Mikhail F (2013) Persistent damage induces mitochondrial DNA degradation. DNA Repair (Amst) 12:488-99
Yuzefovych, Larysa; Wilson, Glenn; Rachek, Lyudmila (2010) Different effects of oleate vs. palmitate on mitochondrial function, apoptosis, and insulin signaling in L6 skeletal muscle cells: role of oxidative stress. Am J Physiol Endocrinol Metab 299:E1096-105
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Rachek, Lyudmila I; Yuzefovych, Larysa V; Ledoux, Susan P et al. (2009) Troglitazone, but not rosiglitazone, damages mitochondrial DNA and induces mitochondrial dysfunction and cell death in human hepatocytes. Toxicol Appl Pharmacol 240:348-54

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