Maintenance of mitochondrial genomic integrity in the absence of manganese superoxide dismutase in mouse liver hepatocytes
Anthony R. Cyr,
Kyle E. Brown,
Michael L. McCormick,
Mitchell C. Coleman,
Adam J. Case,
George S. Watts,
Bernard W. Futscher,
Douglas R. Spitz,
Frederick E. Domann
Affiliations
Anthony R. Cyr
Free Radical and Radiation Biology Program, Department of Radiation Oncology, Carver College of Medicine and the Holden Comprehensive Cancer Center, The University of Iowa, Iowa City, IA 52242, USA
Kyle E. Brown
Department of Internal Medicine, Gastroenterology Division, Carver College of Medicine, The University of Iowa, Iowa City, IA 52242, USA
Michael L. McCormick
Free Radical and Radiation Biology Program, Department of Radiation Oncology, Carver College of Medicine and the Holden Comprehensive Cancer Center, The University of Iowa, Iowa City, IA 52242, USA
Mitchell C. Coleman
Free Radical and Radiation Biology Program, Department of Radiation Oncology, Carver College of Medicine and the Holden Comprehensive Cancer Center, The University of Iowa, Iowa City, IA 52242, USA
Adam J. Case
Free Radical and Radiation Biology Program, Department of Radiation Oncology, Carver College of Medicine and the Holden Comprehensive Cancer Center, The University of Iowa, Iowa City, IA 52242, USA
George S. Watts
University of Arizona Cancer Center and Department of Pharmacology, University of Arizona, Tucson, AZ 85724, USA
Bernard W. Futscher
University of Arizona Cancer Center and Department of Pharmacology and Toxicology, University of Arizona, Tucson, AZ 85724, USA
Douglas R. Spitz
Free Radical and Radiation Biology Program, Department of Radiation Oncology, Carver College of Medicine and the Holden Comprehensive Cancer Center, The University of Iowa, Iowa City, IA 52242, USA
Frederick E. Domann
Free Radical and Radiation Biology Program, Department of Radiation Oncology, Carver College of Medicine and the Holden Comprehensive Cancer Center, The University of Iowa, Iowa City, IA 52242, USA
Manganese superoxide dismutase, encoded by the Sod2 gene, is a ubiquitously expressed mitochondrial antioxidant enzyme that is essential for mammalian life. Mice born with constitutive genetic knockout of Sod2 do not survive the neonatal stage, which renders the longitudinal study of the biochemical and metabolic effects of Sod2 loss difficult. However, multiple studies have demonstrated that tissue-specific knockout of Sod2 in murine liver yields no observable gross pathology or injury to the mouse. We hypothesized that Sod2 loss may have sub-pathologic effects on liver biology, including the acquisition of reactive oxygen species-mediated mitochondrial DNA mutations. To evaluate this, we established and verified a hepatocyte-specific knockout of Sod2 in C57/B6 mice using Cre-LoxP recombination technology. We utilized deep sequencing to identify possible mutations in Sod2−/− mitochondrial DNA as compared to wt, and both RT-PCR and traditional biochemical assays to evaluate baseline differences in redox-sensitive pathways in Sod2−/− hepatocytes. Surprisingly, no mutations in Sod2−/− mitochondrial DNA were detected despite measurable increases in dihydroethidium staining in situ and concomitant decreases in complex II activity indicative of elevated superoxide in the Sod2−/− hepatocytes. In contrast, numerous compensatory alterations in gene expression were identified that suggest hepatocytes have a remarkable capacity to adapt and overcome the loss of Sod2 through transcriptional means. Taken together, these results suggest that murine hepatocytes have a large reserve capacity to cope with the presence of additional mitochondrial reactive oxygen species.
