Comparing methods for detecting mitochondrial DNA damage using real-time polymerase chain reaction and buccal epithelial micronucleus assay to assess genetic homeostasis in humans

BACKGROUND: Various environmental factors that can affect the stability of the genetic material in humans, using a range of cytological and molecular genetic methods, are being studied actively.

AIM: This work aimed to identify and analyze the associations between the buccal epithelial micronucleus assay results and mitochondrial DNA damage detected using real-time polymerase chain reaction (RT-PCR).

METHODS: The study was conducted in Voronezh; 25 and 10 men and women, respectively, aged 20–24 years, were selected as participants. The buccal epithelial micronucleus assay and mitochondrial DNA damage assessment using RT-PCR were applied. In each sample, ≥2000 cells were examined to determine the number of cells with micronuclei, perinuclear vacuoles, notches, and protrusions. DNA repair and cytogenetic damage accumulation indices were calculated. The DNA was extracted using the CTAB buffer. RT-PCR employing the Encyclo polymerase and selected primers amplified two fragments (short and long) within the D-loop region of mitochondrial DNA. The number of DNA lesions was calculated based on the Ct values by applying a specific formula. Statistical analysis of the data was conducted with the Stadia software package.

RESULTS: The frequency of mitochondrial DNA damage and the occurrence of nuclear abnormalities in the buccal epithelial cells of the study groups were determined. A higher degree of variation in the parameters studied was observed in females compared to males. Correlations were established between mitochondrial DNA damage and the frequency of nuclear abnormalities. Similar patterns of change were observed in mitochondrial DNA damage frequencies and nuclear aberrations.

CONCLUSION: The common patterns of variation identified in the cytogenetic and molecular genetic indicators of genomic stability, as well as the correlations between the frequency of nuclear abnormalities in buccal epithelial cells and mitochondrial DNA damage, suggest a shared etiology of molecular and cell genetic lesions. These findings indicate the potential of utilizing these parameters to predict and refine the values of each other.