Comparison of Resuscitation Fluids in Maintaining Mitochondrial DNA Integrity in Human Endothelial Cells
Abstract
In our continuing evaluation of resuscitation fluids in the treatment of hemorrhage, the objective of this study was to determine if standard resuscitation fluids lead to increased damage to mitochondrial DNA (mtDNA) during periods of increasing oxidative stress as associated with shock states. Mitochondria are responsible for maintaining the cellular energy balance and are involved in the triggering of apoptosis in response to oxidative stress. The majority of mitochondrial proteins are encoded by nuclear DNA, however, mitochondria retain their own genome consisting of a circular duplex molecule of approximately 16.5 kb encoding 13 polypeptides that are exclusively involved in the intracellular ATP production by the electron transport chain (ETC). MtDNA, like the ETC, is localized at the inner side of the mitochondrial inner membrane. The ETC reduces oxygen to water in four consecutive one‐electron steps and reactive oxygen species (ROS) are produced as a by‐product of this process. These reactive molecules can be converted to H2O2 spontaneously or by superoxide dismutase resulting in free hydroxyl radicals (•OH) in the presence of iron via Fenton chemistry. ROS, in particular hydroxyl radicals, exhibit a high capacity to impair proteins, lipid membranes, DNA and RNA, which are essential components of functional mitochondria. Since, many of these resuscitation fluids lack substrates for mitochondrial production of ATP we proposed that they would enhance generation of ROS. Human umbilical vein endothelial cells (HUVEC) were treated for 3 hrs to 10 mM Hepes‐buffered Hank's solution, Ringer's solution, lactated Ringer's (LR), normal saline (NR), 5% albumin (5% Alb) and Hextend. The cells were fixed in 50% isopropanol and DNA extracted with DNAzol (MRC, Cincinnati, OH) and subjected to real‐time polymerase chain reaction (RT‐PCR) with primers designed to amplify large and small regions of mitochondrial and nuclear DNA where the extent of template amplification is inversely proportional to the lesion frequency within a given DNA sequence. Detected lesions per 10kb DNA were determined. Compared to hydrogen peroxide induced DNA damage, the tested resuscitation fluids did not produce significant alterations in the mtDNA or nuclear DNA. These data suggest that based on our previous observations of a lack on intrinsic antioxidant activity in these fluids, none would offer any advantage in influencing ROS production or mitochondrial dysfunction induced by a shock state.
Document Details
- Document Type
- Pub Defense Publication
- Publication Date
- Apr 01, 2016
- Source ID
- 10.1096/fasebj.30.1_supplement.1280.3
Entities
People
- Johnny L. Barr
- Michael A. Dubick
- Phillip D. Bowman
- Ruby S Gibson
Organizations
- United States Army Institute of Surgical Research
- United States Army Medical Research and Development Command