A total of (500) mice of Swiss albino strain, ranged in age between (8-12) weeks old and weighed about (23-25) grams were used in this study which was divided into four parts.
- In the first part, the determination of the experimental mice females’ estrous cycle was included for the establishment of mating colonies and the production of embryos used in the initiation of the fibroblast cell cultures.Embryos at different ages of (9, 11 and 13) days old were used, and the results of the morphological examinations showed that, an embryo age of (11-11.5) days old was considered to be suitable for the production of successful fibroblast cell cultures which were designated as (the fibroblast – like AME 11D1/2 cell cultures).
- The second part of this investigation dealt with studying the biological performance of three different media including: (RPMI 1640 medium, Eagle’s minimal essential medium and medium 199) on supporting the growth of (the fibroblast – like and the malignant plasmacytoma SU99 cell cultures). Results of seeding efficiency values for the two types of cells grown in the three different media had shown that, the RPMI 1640 medium was the most suitable culture medium under the experimental conditions of this study in supporting the maximum growth of the examined cell cultures.
- The in vitro effect of 5-azacytidine on the growth and development of the fibroblast – like and the plasmacytoma SU99 cell cultures was investigated in the third part of this study.A cytotoxic effect on the growth of the two examined cell cultures was detected, expressed by increasing the values of the optical density measurements in the short-term viability assay and by decreasing the values of the plating efficiency of the growing cells in the long-term survival assay where the fibroblast – like cell cultures appeared to be more sensitive to the cytotoxic effect exerted by the 5-azacytidine than the plasmacytoma SU99 cell cultures.In addition, the phenotype of the treated cells was also affected by the 5-azacytidine treatment, where the fibroblast – like cells appeared single with different sizes other than uniform monolayers. While, the treatment of the plasmacytoma SU99 cells with 5-azacytidine induced the reversion at a high rate of an enzyme deficient malignant subclones called the (HPRT-positive cells). These cells were able to grow at HAT- medium and their reversion rate was dependent on the concentration used of 5-azacytidine in the culture medium.
- The forth part of this study was on the in vivo effects of 5-azacytidine and folic acid on mice body weight, male reproductive organs, embryogenesis, chromosomes of the bone marrow and gene expression on the levels of genomic DNA and whole protein content of the liver.
Results showed that, treatment of mice with 5-azacytidine caused a highly significant decrease (p < 0.001) in the final recorded body weight values. While, six weeks exposure of male mice to (8 mg/Kg body weight) of 5-azacytidine resulted in severe abnormalities in the seminiferous tubule, including: degeneration of the tubule, sloughing of immature germ cells into the lumen, and giant cell formation. In addition, a decrease in the fertility rate was also detected in the treated male mice revealed by the deleterious effects on some sperm’s functions, including: a decrease in the motility, concentration and viability with an increase in the percentage of morphologically abnormal sperms.Moreover, the administration of 5-azacytidine to pregnant females caused teratogenic effects where an increase in the preimplantation losses and a decrease in the pregnancy out come were detected.Furthermore, a significant increase (p <0.05) in the mitotic index with some chromosomal aberrations were also detected in bone marrow preparations from mice treated with 5-azacytidine.On the other hand, results obtained following treatment of mice with (0.1mg/Kg body weight) of folic acid showed a non - significant increase (p>0.05) in the treated animals body weights.Moreover, it was found that, the oral administration of folic acid had no detectable effects on the treated mice males’ reproductive organs or on the bone marrow chromosomes. Whereas, results obtained following treatment of pregnant females with folic acid had suggested that, prenatal supplementation of folic acid could reduce embryo lethality induced by heat exposure and sufficient folic acid intake during early pregnancy was recommended to avoid malformed embryos Frthermore, evaluation of the possible effect of 5-azacytidine and folic acid on the genomic DNA methylation pattern and the cytoplasmic protein content of the treated mice livers was investigated by means of DNA predigestion with methylation sensitive and non- sensitive restriction enzymes (HpaII and MspI), then amplifying the digested fragments using PCR with randomly selected primers and studying the effect on the gene expression using SDS-PAGE technique.Results of agarose gel electrophoresis analysis of the predigested liver DNA showed similar pattern of fragments distribution between the DNA lanes of the (control and folic acid) liver samples. While, a different pattern of fragments distribution was recognized in the DNA lane of the 5-azacytidine liver sample.Moreover, a different pattern of fragments distribution was identified in the lanes of DNA samples digested either with HpaII or MspI restriction enzymes for the DNA’s of the (control and folic acid) liver samples. Whereas, a similar distribution pattern was recognized in the DNA lane of the 5-azacytidine liver sample.On the other hand, results of RAPD-PCR amplification of the predigested DNA using the primer OPE-7 showed a similar pattern of fragment distribution in DNA lanes of the (control and folic acid) liver samples. While, a polymorphism was recognized in the DNA lanes of the 5-azacytidine liver sample.Furthermore, a possible effect on the cytoplasmic proteins of mice liver might be detected using the sodium dodecylsulphate - polyacrylamide gel electrophoresis, where the protein sample of the 5-azacytidine treated liver showed a polymorphism in the calculated (Rm- value)of the migrating proteins.Whereas, similar values were calculated for the proteins of the control and folic acid samples.
