In vivo Study of Mitochondrial DNA and Cytogenetic Changes in Mice Treated with Mitomycin C as a Mutagenic Agent

 Six to eight weeks old white mice sharing the same mother and father were injected intraperitoneally with different dosages 0.1, 0.2 and 0.5mg/kg body weight (bw) of the chemotherapeutic antitumor drug Mitomycin C (MMC). The effect of the drug on mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) was investigated by molecular techniques and cytogenetic analyses were performed on spleen and bone marrow cells.
 MtDNA was isolated from spleen tissues and molecular analysis was preformed by Random Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) assay and Specific PCR directed to the non coding control region or displacement loop (D-loop) in mtDNA of mice. Restriction endonuclease analysis was performed on the specific PCR product. In RAPD-PCR, ten different decamer primers chosen randomly were employed to analyze mtDNA of mice treated with 0.5mg/kg bw MMC in addition to the control animals. RAPD-PCR assay revealed polymorphisms ranging between 7.7 -100%. The high percentage of polymorphism observed might be due to exposure to MMC. The D-loop region of mice progeny from the same mother and father treated with different dosages of the drug MMC (0.1, 0.2 and 0.5mg/kg bw was amplified by the PCR using specific primers. The PCR product was then subjected to four restriction enzymes (BamHI, HaeIII, HindIII and Sau3A) to examine induced potential variation within the region. Restriction enzyme analysis of D-loop region amplified by PCR did not detect any DNA variation within this region between mice individuals treated with all of the three doses of MMC in comparison to the control animals.  The mitomycin C effect was tested cytogenetically in both spleen and bone marrow cell suspensions from the same animals. Direct short term culture was used for chromosomal preparation and examination for both bone marrow and spleen cells in 0.1, 0.2 and 0.5mg/kg bw MMC treated animals and non treated control mice. Results showed that a negative relationship was observed between the mean values of mitotic index (MI) and blast index (BI) to higher doses of MMC in both bone marrow and
spleen cells while a positive correlation was observed in total number of chromosomal aberrations (CA) and higher doses of MMC in both bone marrow and spleen cells.
 Molecular technique of RAPD-PCR was able to detect mtDNA variations between 0.5mg/kg bw treated animal in comparison to the control. While analysis of the D-loop region of mice treated with 0.1, 0.2 and 0.5mg/kg bw by restriction analysis did not detect DNA alterations in reference to the control animals. The inhibition of proliferation rate and the increase in CA observed in both bone marrow and spleen cells, together with
the inhibition of blast percentage in bone marrow cells confirm the positive
correlation between MMC and DNA damage in mice somatic cells.