The aim of this study was to initiate callus from black seed Nigella sativa to extract thymol substance and then study its potency to inhibit the cytogenetic effects of mitomycin-C (MMC) on laboratory mice (in vivo). Establishment of Nigella sativa (black seed) seedlings in tissue culture was achieved in our laboratory by using Murashige and Skoog medium, (1962) (MS) and optimization of cultural medium using a combination of 2,4-D and Kin concentrations The seeds were cultured in sterilized distilled water (after their sterilization) at different concentrations of sodium hypochlorite. Then different explants (leaves, stems and roots) were cultured on MS medium supplied with different concentrations of growth hormons including 2,4-D at (0.0, 1.0, 2.0, 3.0 and 4.0 mg/L) and Kin at (0.0, 1.0, 1.5, 2.0, 2.5, 3.0 and 5.0 mg/L). Data and observations were recorded on callus growth through measuring its mean fresh weight under light conditions. Results revealed that best callus initiation was observed on explants cultured on 1mg/L of 2,4-D and 1.5 mg/L of Kin. Leaves were superior in callus production as compared to stem and root explants. Significant differences were observed among the explants in their response to callus production.Thymol was extracted and detected and semipurified by using HPLC (High Performance Liquid Chromatography) method to measure its concentration in callus induced from leaves after 75 days in culture, which observed that retention time of standard thymol was identical with retention time of thymol substance and it was (5.3 and 5.5 min.) respectively. The concentration of thymol substance in callus was 30.5 mg/ml. While, the cytogenetic study was aimed to investigate the role of the isolated thymol in reducing the cytogenetic effect of MMC in mice. Mitotic index, chromosomal aberrations and micronucleus assay in mouse bone marrow were examined. The cytogenetic effects of the drug and callus extract were investigated 7 days after treatment of mice with 5 different doses of thymol (0.5, 1.0, 2.0, 4.0 and 8.0 mg/Kg) and 2 mg/Kg for MMC. An interaction between thymol and MMC was carried out through two types of treatment (before and after treatment thymol) by determine the activity of Nigella sativa leaf callus extract in preventing or reducing the drug side effects in vivo. The results indicated that MMC has clear effects in reducing mitotic activity, increased spontaneous chromosomal aberration and increased micronucleus in mouse bone marrow cells (in vivo), these effects suggested that the drug has a genotoxic effect. Thymol had genotoxic effects at high doses over 1 mg/Kg in mouse bone marrow cells (in vivo), while at dose 1 mg/Kg show a protective value against the genotoxic effect of MMC. In mouse bone marrow, this effect was more observed in pre-treatment than in post-treatment. Thymol extracted from initiated leaf callus was considered as desmutagen in the first order and biomutagen in the second order, as a result for its ability to increase mitotic activity, decrease micronucleus and repair chromosomal aberration in mouse bone marrow cells.
