TEST FOR GENOTOXIC POTENTIAL BY MICRONUCLEI ASSAY 35 Structural aberrations may be of the chromosome or chromatid type. The induction of generation of two or more homologous sets of chromosomes, called polyploidy, may be an indication that a substance carries the potential to induce numerous aberrations that can lead to the initiation of mutations that transform the cells to the cancerous stage. The identifi cation of aberrations in this study is normally conducted by obser- vational assessment of the morphological changes in the nucleus. It therefore requires an experienced individual who is capable of differentiating between a state that is unusual and a state wherein the cell was arrested in the course of normal division. Therefore, we also added quantifi cation of the data to corroborate the microscope observations. MICRONUCLEI FORMATION The in vitro micronucleus assay is a mutagen test system for the detection of the chemi- cally induced formation of small membrane-bound DNA fragments, i.e., micronuclei in the cytoplasm of cells (13,14). These micronuclei may originate from acentric fragments (chromosome fragments lacking a centromere) or whole chromosomes that are unable to migrate with the rest of the chromosomes during the anaphase of cell division. The mi- cronucleus assay is widely used for monitoring genetic damage caused by different sub- stances. Typical images of cells with stained nuclei and quantitative analysis of micronuclei in cell populations are presented in Figures 2 and 3, respectively. It can be clearly seen that both positive controls induced substantial genetic damage leading to the formation of 140–170 micronuclei per 1000 cells. In contrast, cellular nuclei appear in- tact after the incubation with OB at both test concentrations and the average number of micronuclei per 1000 cells was close to that of the control cells. CELLULAR VIABILITY AND PROLIFERATION A cell that had gone through mutational changes can either go into senescence or apo- ptosis, or it may survive. If it survives and mutations are not corrected by DNA repair enzymes, these mutations can possibly lead to unregulated cell division and the cre- ation of cancerous tissue. Cancerous cells, therefore, do not obey the normal apoptotic paths that are typical of normal cells. In fact, in cancer cells activation of biochemical substances such as cytokines and other mediators may enhance cell proliferation and viability markers may increase as the cell becomes more sensitive in response to pro- moters that are involved in the induction of cell division. In addition, in these cells metabolic activity may be accelerated. The MTT ((3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide, a tetrazole) test detects the activity of mitochondrial enzymes in viable cells and is an indication of cell viability (15). We tested cell viabil- ity in an attempt to draw additional data that will further validate our fi ndings related to chromosomal changes (Figure 4) It was previously shown that a positive cell viabil- ity assay response has a strong probability of predicting carcinogenicity in vivo (16). In other words, if a compound (such as the positive controls in this study) is generating both micronuclei formation and accelerating cell proliferation and/or metabolism, there are two related pieces of evidence that point to its potential of being a carcinogen. The cell-counting studies also show that the proliferation of the cells incubated with the studied compound are similar to the proliferation of the cells incubated with fresh media (Figure 5).
JOURNAL OF COSMETIC SCIENCE 36 Figure 2. Typical light and fl uorescent images of CHO-K1 cells incubated for 24 hours with the following substances: (1) Media (negative control) (2) DMSO (negative control) (3) Cyclosphosphamide + S9 mix (positive control) (4) Ethyl methyl sulfonate (positive control) (5) OB, 0.2 mg/ml in DMSO (6) OB, 0.3 mg/ml in DMSO (7) OB, 0.2 mg/ml in DMSO + S9 mix and (8) OB, 0.3 mg/ml in DMSO + S9 mix. The cells were stained with DAPI nuclear dye.
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