Contribution of de novo point mutations to the overall mutational burden in mitochondrial DNA of adult rats M. Khaidakov * , N. Chavannes-Turesky, C.A. Cooney, E.E. Dupont-Versteegden, R.H. Kennedy, E.R. Siegel, G. Khaidakova, R.J. Shmookler Reis Departments of Geriatrics, Biochemistry and Molecular Biology, Pharmacy and Biostatistics, University of Arkansas for Medical Sciences, John McClellan VA Hospital, 4300 West 7th Street, Little Rock, AR 72205, USA Received 17 November 2004; received in revised form 10 February 2005; accepted 16 February 2005 Available online 28 March 2005 Abstract This study analyzed the incidence of point mutations in mitochondrial DNA of brain and muscle tissues from young (6-month) and old (24-month) male F344 rats. Coding sequence mutations in subunit 5 of the NADH dehydrogenase gene were detected after high-fidelity PCR amplification and cloning by denaturing gradient gel electrophoresis (DGGE) assay followed by sequencing of detected mutants. In total, almost a thousand individual clones were analyzed both in brain and muscle samples. On average, mtDNA from brain tissue showed a 66% increase with age in mutation frequencies (2.3G1.9 vs. 3.8G4.5!10 K4 mutations/bp, meanGSD), which failed to reach statistical significance (pZ0.45). Muscle tissues yielded substantially fewer mutants with average mutant frequencies for both young and old rats almost 10 times lower than the corresponding values in the brain tissue (0.3G0.4 and 0.5G0.6!10 K4 , respectively). The difference in mutation accumulation between muscle and brain was highly significant in both the younger group (Chi-squaredZ9.7, p%0.01) and in older animals (Chi-squaredZ10.9, p%0.001). Molecular analysis of the mutated sequences revealed that almost half were identical sequences recurring in different samples and tissues. Our findings indicate that the process of mutation accumulation in mitochondria begins in the germ-line and/or during earlier stages of life, contributing up to half of the total mutational burden, whereas de novo spontaneous formation of point mutations in adulthood is far less than was anticipated. q 2005 Elsevier Inc. All rights reserved. Keywords: Mitochondrial DNA; Point mutations; Aging; Rats; Brain; Muscle 1. Introduction Mitochondrial theories of aging have been proposed in which senescence is attributed to a vicious cycle created by the elevated exposure of mitochondria, and in particular, mitochondrial DNA (mtDNA), to free radicals leaked from the electron transport chain (Harman, 1972; Miquel et al., 1980). A portion of oxidative DNA damage is converted into mutations leading to synthesis of defective proteins, which in turn causes ever-increasing production of oxidants. There are several lines of evidence indicating that these organelles may be a weak link in terms of aging. Mitochondria possess several unique features setting them apart from other organelles. Functionally, mitochondria are responsible for production of ATP via oxidative phosphorylation, regulation of apoptosis, and calcium signaling. They are the main site of oxygen consumption and reactive oxygen species (ROS) production. Mitochondria harbor a second genome, which codes for key components of the electron transport system (ETS). By virtue of its close proximity to the ETS machinery and its asymmetric replication mechanism, mitochondrial DNA may be subjected to considerably higher oxidative damage (Richter et al., 1988; Mecocci et al., 1993) repaired less efficiently than similar lesions in the nuclear DNA (Croteau et al., 1999). These factors combine to produce mutation accumu- lation in mitochondrial DNA (mtDNA) that is dramatically more severe than in the nucleus. A considerable fraction of the mtDNA collected from old individuals comprised of a variety of truncated molecules (Kovalenko et al., 1998). Experimental Gerontology 40 (2005) 396–402 www.elsevier.com/locate/expgero 0531-5565/$ - see front matter q 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.exger.2005.02.007 * Corresponding author. Address: GB-100, McClellan VA Hospital, 4300 West 7th Street, Little Rock, AR 72205, USA. Tel.: C1 501 257 5562; fax: C1 501 257 4822. E-mail address: khaidakovmagomed@uams.edu (M. Khaidakov).