Compelling experimental and epidemiological evidence indicates that environmental factors can play a crucial role in the critical depletion of an essential mass of pancreatic beta cells in insulin-dependent diabetes mellitus. Also, functional alterations in beta cells, similar to those seen in noninsulin-dependent diabetes mellitus, have been produced in laboratory animals following exposure to the naturally occurring N-nitroso compound streptozotocin. In this competitive renewal application, we will continue to focus on the study of the mechanisms by which xenobiotics interact with beta cells to either kill these cells directly, elicit an inflammatory response which results in their destruction, or to cause their functional impairment. One or more of these molecular mechanisms may initiate pathologic events that culminate in specific forms of diabetes mellitus. The objective of the present proposal is to explore the factors which regulate damage and repair of specific lesions in DNA. Because of the progress made in the preceding funding period, work on this application is structured to focus primarily on mitochondrial DNA.
Three specific aims are proposed to pursue the objective. The first is to examine the repair of O6- methylguanine in the mitochondrion and the nucleus. These studies will use a monoclonal antibody against this mutagenic adduct, combined with quantitative PCR, to study the repair of O6-methylguanine in mitochondrial DNA and in defined sequences of nuclear DNA.
The second aim i s to evaluate the formation and repair of nitric oxide-induced damage in mitochondrial DNA. These studies will evaluate the formation of specific types of damage in mitochondrial DNA of E cells caused by both exogenous sources of nitric oxide and nitric oxide produced endogenously by cytokine stimulation. Also, they will explore the repair of the DNA damage caused by active nitrogen-containing metabolites in the mitochondrion.
The final aim will use ligation-mediated PCR to explore damage and repair in mitochondrial DNA at the level of individual nucleotides. These studies will employ this powerful new technique to investigate the damage formed by reactive oxygen species, simple alkylation and nitric oxide in mitochondrial DNA. Also, they will determine the effects of nucleotide sequence on the repair of this damage. When successfully completed, these studies will provide a more complete understanding of the mechanisms by which xenobiotics can selectively interact with normal beta cells to cause functional impairment and/or the death of these cells. This work also will contribute new insights into the etiologies of some forms of cancer and the progression of the natural phenomenon of aging.
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