Unscheduled DNA synthesis in various types of cells of the mouse brain in vivo

Abstract

Very low incorporation of 3H-thymidine (TdR) into neurons and non-proliferating glial and endothelial cells in various brain areas of the adult mouse after 3H-TdR injection and subsequent X-irradiation of the head with 45 Gy has been demonstrated autoradiographically after exposure times of 250 days. In accordance with biochemical studies this incorporation of 3H-TdR represents DNA repair synthesis or UDS (unscheduled DNA synthesis). However, 3H-TdR incorporation into nuclear DNA of non-proliferating cells in the brain was not only found in X-irradiated but also in sham-irradiated mice. This suggests that spontaneous UDS also occurs. Up to now spontaneous UDS has been shown only in HeLa cells in vitro. Nearly all the various types of brain cells tested exhibited UDS after X-irradiation as well as spontaneous UDS. After correcting the mean grain numbers per nucleus not only for background but also for beta-self-absorption, substantial differences became apparent in the extent of UDS between the individual types of cells. After X-irradiation, UDS was highest in Purkinje cells and hippocampal granular cells but comparable UDS was also found in endothelial cells, regardless of the different brain areas studied. The extent of spontaneous UDS is also quite different in the various cell types, being highest in neurons of different sites and considerably lower in endothelial and glial cells.

Accumulation of nuclear DNA damage or neuron loss: Molecular basis for a new approach to understanding selective neuronal vulnerability in neurodegenerative diseases

“…However, studies describing the relative amount of long patch repair events and single nucleotide repair events during BER and SSBR in vivo have not been published. Incorporation of [ 3 H]TdR into the nDNA can then be detected by liquid scintillation (see, e.g., [79]) and, as established by Korr et al [20,21,39,[80][81][82], with autoradiography on tissue sections after injection of [ 3 H]TdR in animals. The latter provides a quantitative readout of UDS in a cell-type-specific manner.…”

“…However, due to the fact that the silver grains in the autoradiographs are generated as the product of 3 H-activity multiplied by the autoradiographic exposure time, very small amounts of 3 H-activity incorporated into the nDNA can also be demonstrated provided the exposure time is extended to several months (see [50] for details). As such, an exposure time of 250 days (approximately 9 months) is suitable to demonstrate UDS under physiological conditions in the brain of rodents [20,21,39,[80][81][82]. Then single silver grains over the cell nuclei can be counted and, after correction for autoradiographic background, β-self-absorption, and nuclear size, the results obtained on different cell types can be directly compared to each other (for details see [21,50,80,81]).…”

“…As such, an exposure time of 250 days (approximately 9 months) is suitable to demonstrate UDS under physiological conditions in the brain of rodents [20,21,39,[80][81][82]. Then single silver grains over the cell nuclei can be counted and, after correction for autoradiographic background, β-self-absorption, and nuclear size, the results obtained on different cell types can be directly compared to each other (for details see [21,50,80,81]). Several studies have addressed the validity of this method (see, e.g., [83][84][85]).…”

“…The use of different methods for detecting nDNA damage and nDNA repair as well as differences between experimentally induced nDNA damage and nDNA damage occurring in physiological conditions might have contributed to these discrepancies. It was the introduction of autoradiographic methods by Korr et al [10,20,21,39,47,[80][81][82] to study quantitatively the relative amounts of nDNA damage and nDNA repair on tissue sections that made it possible to test Gensler and Bernstein's [86] hypothesis about the aging brain in a cell-type-specific manner under physiological conditions. Interestingly, it turned out that the situation in the aging brain is more complex than predicted by Gensler and Bernstein [86].…”

Brain's DNA Repair Response to Neurotoxicants

“…The accuracy of overall DNA replication during proliferation is assured by several mechanisms that include nucleotide selection and exonuclease proof-reading activity with DNA polymerases and post-replicative methylation-instructed mismatch repair system ). Post-mitotic cells contain lower levels of DNA repair enzymes and repair DNA slower than proliferating cells (Alexander 1967;Korr and Schultz 1989;Stedeford et al 2001) Repair of oxo 6 -alkylguanine lesions as well as the damage from UV and ionizing radiations is slower in neurons than in other cultured cell types . The decline in DNA repair capacity of post-mitotic cells may be related to the development and achievement of their terminal differentiated state.…”