ABSTRACT
BACKGROUND: Oxidative damage to the cell, including the formation of 8-oxoG, has been regarded as a significant factor in carcinogenesis and aging. An inbred prematurely aging rat strain (OXYS) is characterized by high sensitivity to oxidative stress, lipid peroxidation, protein oxidation, DNA rearrangements, and pathological conditions paralleling several human degenerative diseases including learning and memory deterioration. METHODS: We have used monoclonal antibodies against a common pre-mutagenic base lesion 8-oxoguanine (8-oxoG) and 8-oxoguanine DNA glycosylase (OGG1) in combination with indirect immunofluorescence microscopy and image analysis to follow the relative amounts and distribution of 8-oxoG and OGG1 in various cells of different brain regions from OXYS and control Wistar rats. RESULTS: It was shown that 8-oxoG increased with age in mature neurons, nestin- and glial fibrillary acidic protein (GFAP)-positive cells of hippocampus and frontal cortex in both strains of rats, with OXYS rats always displaying statistically significantly higher levels of oxidative DNA damage than Wistar rats. The relative content of 8-oxoG and OGG1 in nestin- and GFAP-positive cells was higher than in mature neurons in both Wistar and OXYS rats. However, there was no significant interstrain difference in the content of OGG1 for all types of cells and brain regions analyzed, and no difference in the relative content of 8-oxoG between different brain regions. CONCLUSIONS: Oxidation of guanine may play an important role in the development of age-associated decrease in memory and learning capability of OXYS rats. GENERAL SIGNIFICANCE: The findings are important for validation of the OXYS rat strain as a model of mammalian aging.
Subject(s)
Aging, Premature/metabolism , Aging , DNA Damage , Frontal Lobe/metabolism , Guanine/analogs & derivatives , Hippocampus/metabolism , Memory , Aging, Premature/pathology , Animals , DNA Glycosylases/metabolism , Frontal Lobe/pathology , Guanine/metabolism , Hippocampus/pathology , Humans , Intermediate Filament Proteins/metabolism , Lipid Peroxidation , Nerve Tissue Proteins/metabolism , Nestin , Neurons/metabolism , Neurons/pathology , Oxidative Stress , Rats , Rats, WistarABSTRACT
Somatic mutations in mitochondrial DNA (mtDNA) are thought to play an important role in both aging and neurodegenerative diseases although their specific contributions remain a subject of intense debate. We analyzed somatic mutations in the mtDNA control regions in the liver of Wistar rats. The mutation rate was found to be high and increased with age from 5.3x10(-4) mutations per position to 4.48x10(-3) mutations per position at 3 and 12 months of age, respectively. The vast majority of nucleotide substitutions are transitions ( approximately 95%) with A:T>G:C transitions being the most frequent type of substitution (>50%). In 3-month-old Wistar rats, approximately 40% of somatic mutations in the control region of mtDNA are significantly consistent with the model of dislocation mutagenesis which is a signature of error-prone DNA synthesis by mtDNA polymerase gamma. The results are consistent with the previous hypothesis that normal intramitochondrial dNTP pool asymmetries, which have been shown to reduce the fidelity of mtDNA polymerase gamma, substantially contribute to somatic mutagenesis of the rat mtDNA.
Subject(s)
Aging , DNA, Mitochondrial , Mitochondria, Liver/genetics , Mutation , Animals , Base Sequence , Male , Molecular Sequence Data , Rats , Rats, WistarABSTRACT
Production of free radicals in animals is accompanied with a number of pathologic conditions, some of which may be manifested through DNA damage. Studies of mechanisms of oxidative DNA damage by free radicals in vivo are hindered by the lack of good animal models with significant overgeneration of or increased sensitivity to free radicals. An inbred rat strain (OXYS) is characterized by inherited overgeneration of free radicals, lipid peroxidation, protein oxidation, DNA rearrangements, and pathological conditions paralleling several human degenerative diseases. We have used monoclonal antibodies against a common pre-mutagenic base lesion 8-oxoguanine (8-oxoG) in combination with indirect immunofluorescence microscopy and image analysis to follow the relative age-dependent amounts and distribution of 8-oxoG in liver cells from OXYS and Wistar rats. 8-OxoG increased with age in both strains of rats, with OXYS rats always displaying statistically significantly higher levels of oxidative DNA damage than Wistar rats. Statistical analysis indicates that 8-oxoG does not uniformly accumulate in all cells with advancing age or increasing free radical load, but rather concentrates in a minor fraction of cells with a high damage level.
Subject(s)
Aging/genetics , Aging/metabolism , DNA Damage , Guanine/analogs & derivatives , Animals , Fluorescent Antibody Technique, Indirect , Free Radicals/metabolism , Guanine/metabolism , Hepatocytes/metabolism , Male , Rats , Rats, Inbred Strains , Rats, WistarABSTRACT
The objective of this study was to investigate the CYP1A1 and CYP1A2 mRNAs and enzyme activities in mouse liver during induction with o-aminoazotoluene (OAT) as well as the capability of the hepatic S9-fraction from OAT-treated mice to induce its own activation to mutagens in the Ames test using S. typhymurium strain TA98. The data obtained indicate that when used at appropriate doses, OAT is a PAH-type inducer of mouse hepatic microsomal monooxygenases, which activity is not less than that of the known inducer 3,4-benzo[alpha]pyrene. In the absence of S9-fraction enzymes no OAT-mediated mutagenicity was observed in the Ames test. In the presence of the S9-fraction from OAT-pretreated mice, OAT induced as high revertant numbers, as it did in the presence of the S9 fraction from the liver of Aroclor 1254-treated mice. Thus, OAT does induce the enzymes of its own mutagenic activation in mouse liver.
Subject(s)
Enzyme Induction/drug effects , Liver/enzymology , Mutagens/metabolism , Mutagens/toxicity , o-Aminoazotoluene/pharmacology , Animals , Benzo(a)pyrene/metabolism , Biotransformation/drug effects , Cytochrome P-450 CYP1A1/biosynthesis , Cytochrome P-450 CYP1A2/biosynthesis , DNA, Complementary/biosynthesis , DNA, Complementary/isolation & purification , Hydroxylation , Liver/drug effects , Male , Mice , Mice, Inbred C57BL , Mutagenicity Tests , RNA/biosynthesis , RNA/isolation & purification , Reverse Transcriptase Polymerase Chain Reaction , Subcellular Fractions/drug effects , Subcellular Fractions/enzymology , o-Aminoazotoluene/metabolism , o-Aminoazotoluene/toxicityABSTRACT
BACKGROUND: Oxygen free radicals have been hypothesized to play an important role in the process of aging. MATERIAL/METHODS: To investigate the correlation between oxidative stress and accumulation of DNA damage we determined age-dependent levels of activities eliminating 8-oxoguanine, hypoxanthine and uracil from DNA in liver cells from OXYS rats, which are characterized by inherited overgeneration of free radicals, in comparison with those of control Wistar rats. RESULTS: A pronounced difference in the specificity of mitochondrial and nuclear 8-oxoguanine DNA glycosylase/AP lyase activities were revealed in both cases. Our results suggest the induction of an 8-oxoG-, uracil- and hypoxanthine-specific repair pathway with age in both types of rats. The levels of 8-oxoguanine DNA glycosylase/AP lyase activities in nuclear extracts from both strains of rats are comparable and approximately tenfold higher than in mitochondrial extracts. On the contrary, 8-oxoguanine DNA glycosylase/AP lyase activity in OXYS mitochondrial extracts was remarkably higher than that from old Wistar rats, and a significant increase of this activity occurs earlier in OXYS than in Wistar rats. CONCLUSIONS: Our results are consistent with the shorter life-span of OXYS rats, and with the mitochondrial theory of aging, which postulates that the accumulation of DNA damage in mitochondrial genomes leads to mitochondrial dysfunction and accelerates the process of aging.