In situ hybridization with wcp and cep for chromosomes 7, 18 and X was used to analyze the presence of specific chromosomal material in MN induced in human lymphocytes by MMC. Mitomycin C is often used as a positive control in studies with MN tests [20] and is reported to induce breakage mainly in the pericentromeric heterochromatin [21]. The results obtained are shown in Figure 1 and summarized in Table 1. We found that in the female cells a total of 4.16% MN contain centromere-specific signals and 8.41% MN contain wcp signals from the three selected chromosomes. X-linked material with wcp signals were detected twice as often (4.25%) in a MN as that of chromosomes 7 and 18 (2.07% for both). Also, the representation of centromeric signals for chromosome X in MN (2.16%) was higher than for chromosomes 18 (1.24%) and 7 (0.77%). While the frequency of wcp signals in MN is the same for both chromosomes 7 and 18, the frequency of MN containing centromere signals for chromosome 18 is 1.7-times higher than for chromosome 7. We found that only nine of the male MN contained wcp X-positive material, i.e., 0.99%.            

   Therefore, on the basis of the results obtained in this study, different frequency of involvement of selected chromosomes in MMC-induced MN formation has been demonstrated. It was shown that chromosomes 7, 18 and X with the analogous peripheral localization in the nucleus [12] were involved in the formation of MN with different rates. To answer the question if a correlation is to be suggested for MN formation and the chromosomal position within the interphase nucleus further studies are necessary. In case of a direct correlation of chromosome size and participation in MN formation a distribution, as shown in Figure 2 (red bars), would have to be expected. For chromosomes X (in females) and 18, such a correlation could exist, although we did not observe it for chromosomes 7 and X (in males). Thus, chromosome size could be important but not the single factor for chromosome-specific MN formation. The obvious difference between male- and female-derived MN contained wcp X-positive material (0.99 versus 4.25%) has been established. Even though the dosage of the X-chromosome is only half in male, an additional effect seems to drive a more preferred inclusion of (at least) one X-chromosome in female. Thus, an effect of increased probability of damage of inactive X-chromosome in female has here at least to be discussed (see Figure 2). Finally, MMC-induced MN could involve other chromosomes and/or chromosomal parts than MN induced by other chemicals. Better knowledge of MN formation would enable the use of appropriate FISH probes, and improve the efficiency and value of this test [22].