Assessment of Nuclear and Mitochondrial DNA, Expression of Mitochondria-Related Genes in Different Brain Regions in Rats after Whole-Body X-ray Irradiation

Abstract

Studies of molecular changes occurred in various brain regions after whole-body irradiation showed a significant increase in terms of the importance in gaining insight into how to slow down or prevent the development of long-term side effects such as carcinogenesis, cognitive impairment and other pathologies. We have analyzed nDNA damage and repair, changes in mitochondrial DNA (mtDNA) copy number and in the level of mtDNA heteroplasmy, and also examined changes in the expression of genes involved in the regulation of mitochondrial biogenesis and dynamics in three areas of the rat brain (hippocampus, cortex and cerebellum) after whole-body X-ray irradiation. Long amplicon quantitative polymerase chain reaction (LA-QPCR) was used to detect nDNA and mtDNA damage. The level of mtDNA heteroplasmy was estimated using Surveyor nuclease technology. The mtDNA copy numbers and expression levels of a number of genes were determined by real-time PCR. The results showed that the repair of nDNA damage in the rat brain regions occurs slowly within 24 h; in the hippocampus, this process runs much slower. The number of mtDNA copies in three regions of the rat brain increases with a simultaneous increase in mtDNA heteroplasmy. However, in the hippocampus, the copy number of mutant mtDNAs increases significantly by the time point of 24 h after radiation exposure. Our analysis shows that in the brain regions of irradiated rats, there is a decrease in the expression of genes (ND2, CytB, ATP5O) involved in ATP synthesis, although by the same time point after irradiation, an increase in transcripts of genes regulating mitochondrial biogenesis is observed. On the other hand, analysis of genes that control the dynamics of mitochondria (Mfn1, Fis1) revealed that sharp decrease in gene expression level occurred, only in the hippocampus. Consequently, the structural and functional characteristics of the hippocampus of rats exposed to whole-body radiation can be different, most significantly from those of the other brain regions.

Impact of High-Dose Irradiation on Human iPSC-Derived Cardiomyocytes Using Multi-Electrode Arrays: Implications for the Antiarrhythmic Effects of Cardiac Radioablation

“…The possible underlying mechanism resulting in loss-of-function of the above sodium channel and potassium channels may be related to the production of reactive oxygen species (ROS) upon irradiation. We did not measure ROS formation in the present study; however, it is well known that ROS is accumulated in cells after high-dose irradiation [ 22 , 23 , 24 ]. Furthermore, the recovery pattern after radiation-induced rapid change herein observed is identical to that of mitochondrial ROS production and recovery [ 23 ].…”

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“…All these molecules are involved in the regulation of oxidative phosphorylation. Mitochondrial genes are maternally inherited, and the majority (99%) of mitochondrial proteins are regulated by nuclear DNA (nDNA), translated in the cytoplasm and then transported to the mitochondrial compartment [ 19 ]. Mitochondria are being replaced within 10-25 days, and old mitochondria are replaced by autophagy (mitophagy).…”

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“…Recently, it was shown that after irradiation of the rat head with X-rays, DSBs nDNA in the cortical neurons persisted for a long post-radiation time [ 46 ]. We also recently reported that DNA damage repair in irradiated rats is slower in cortical tissue than in hippocampal tissue [ 18 ].…”

“…To determine the damage and repair of nDNA and mtDNA, we used the long amplicon quantitative polymerase chain reaction (LA-QPCR) method [ 17 ] taking into account our previous experience [ 18 ]. In these analyses, we used (2U/μL) KAPA Long Range Hot Start Kit (KAPA Biosystems, Humboldt County, CA, USA).…”

“…Preliminary assays were carried out to ensure the linearity of PCR amplification with respect to the number of cycles and DNA concentration. Since the amplification of a small region would be relatively independent of oxidative DNA damage (low probability), a small DNA fragment for nDNA (110 bp) and for mtDNA (117 bp) was also amplified for normalization of the data obtained with the large fragments, as described previously [ 18 , 19 ]. PCR analyses were performed in triplicate for each DNA sample.…”