TEST FOR GENOTOXIC POTENTIAL BY MICRONUCLEI ASSAY 31 types, chromosome and chromatid. Since the majority of genotoxic compounds are mu- tagens, the aberrations are normally of the chromatid type, but the chromosome type may also occur. Mutations in chromosomes and events that are mutation related are the cause of numerous human genetic disorders such as cystic fi brosis, hemophilia, and sickle cell anemia (5). In addition, there is evidence that events causing modifi cations in oncogenes and tumor suppressor genes are involved in the induction of cancer. While this protocol requires cytochalasin B to block cytokinesis, the method utilized in this study does not require it because it uses CHO-K1 cells lines and the OECD Guideline number 487 protocol states that use of cytochalasin B is not necessary for cell lines if proof of cell pro- liferation is provided. The principle of this method is the exposure of a mammalian cell culture to a test sub- stance with and without metabolic activation (6). Metabolic activation is required to as- sess potential genotoxic effects in a case where enzymatic transformation occuring in vivo leads to a toxic metabolite. At predetermined time intervals, the cells are treated with a test substance, harvested, and stained, and metaphase cells are analyzed microscopically for micronuclei formation. MATERIALS AND METHODS MATERIALS The chemicals for the control group were purchased from Sigma Chemical Co. (St. Louis, MO). These include cyclophosphamide (CAS no. 50-18-0), ethyl methanesulfonate (CAS no. 62-50-0), and dimethyl sulfoxide (DMSO). The test substance, OB, was a product from Lipo Chemicals Inc. (Paterson, NJ). Nicotinamide adenine dinucleotide phosphate (NADP) was purchased from Fisher Scientifi c Co. (Suwannee, GA). Sodium phosphate buffer was obtained from Moltox (Boone, NC). Glucose 6 phosphate and Tween 20 were received from Sigma Aldrich (St Louis, MO), and 4′,6- diamidino-2-phenylindole dihy- drochloride (DAPI) was purchased from Sigma. PREPARATION OF CHEMICALS Cyclophosphamide (density: 1.479 g/cm3), and ethyl methanesulfonate (EMS) were used as positive controls to analyze the formation of micronuclei. One gram of cyclophospha- mide was dissolved in 100 ml of water to give a concentration of 10 mg/ml. Five micro- liters of this solution was added to a volumetric fl ask and water was added to give the required fi nal concentration of 10 μg/ml in the fl ask (2). An amount of 0.1 ml of EMS (density 1.15 g/cm3) was diluted to 1.0 ml with dimethyl sulfoxide (DMSO). A measured 17.5 μl of this solution was added to a 5-ml fl ask and DMSO was added to give a fi nal concentration of 400 μg/ml. (2). The OB powder was dispersed at 10 mg/ml in DMSO. An amount of 50 mg of this sus- pension was added to 5 ml of DMSO, subjected to ultrasonifi cation for 30 minutes, and centrifuged at 2000 rpm for 15 minutes. Volumes of 100 μl and 150 μl of the superna- tant were added to fl asks of 25 cm2 base surface area containing 5 ml of media, to give fi nal concentrations of 0.2 mg/ml and 0.3 mg/ml, respectively. These doses were selected
JOURNAL OF COSMETIC SCIENCE 32 in preliminary experiments based on the concentrations of DMSO that are nontoxic for the types of cells used. To prepare the stock solution for cell staining according to the protocol for this assay (7), 5 mg of DAPI was dissolved in 1 ml of dimethylformamide and allowed to stand until the dissolution was complete. For the working solution, 4 μl of the stock solution was added to 50 ml of phosphate buffer solution (PBS) and stored at 4°C, protected from light before the time of staining. METABOLIC ACTIVATION SYSTEM Inclusion of a metabolic activation system in the genotoxicity assay enables the detection of mutagenic activity for carcinogens and/or mutagens that require such transformation (i.e., cyclophosphamide). The metabolic activation system was prepared according to the method described in references (7) and (8). Aroclor-1254-induced rat liver S9 fraction was purchased from Moltox. The following chemicals were added in the order listed to get a total volume of 3 ml of S9 mix: sterile double-distilled H2O (840 μl) sodium phosphate buffer (0.1 M), pH 7.4 (1.5 ml) 4 mM NADP (150 μl) 120 mM glucose-6-phosphate (22 μl) potassium magnesium salt solution, 8 mM–33 mM (60 μl) and rat liver fraction (3.00 μl) to give a fi nal concentration of 10% (v/v). The fi nal concentration of the meta- bolic activator used for each test fl ask was 1% (v/v). In contrast to cyclophosphamide, EMS does not require metabolic activation and therefore the metabolic activation system was not used in the experiments involving EMS. CELL LINE Chinese hamster ovary (CHO-K1) cells were used as recommended in the OECD protocol (3) and were bought from American Type Culture Collection (ATCC, Manassas, VA). Cells were cultured in ATCC-formulated F-12K medium supplemented with 10% fetal bovine serum (Fisher Chemicals, Fairlawn, NJ) and penicillin-streptomycin (100 UI/ ml–100 ug/ml). Cells were grown at 37°C in a humidifi ed atmosphere of 5% CO2 (v/v) in air. All experiments were performed on cells in the exponential growth phase. EXPERIMENTAL SERIES AND CONDITIONS About 300,000 cells were cultured with the media in each 25-cm2 fl ask and held 24 hours before treatment. They were then incubated with the substances as indicated in Figure 1. For the series with metabolic activation, the cells were treated for three hours after which the media were replaced with fresh media and the cells were incubated for 24 hours. For the groups without S9 activation, the cells were incubated for 24 hours. All the cells were harvested at the end of 24 hours and stained to detect the presence of micronuclei. The following series of experiments was carried out: (1) media only (negative control) (2) DMSO (100 μl) (negative control) (3) cyclophosphamide + S9 mix (10 μg/ml) (positive control) (4) ethyl methanesulfonate (400 μg/ml) (positive control) (5) OB (0.2 mg/ml) (6) OB (0.3 mg/ml) (7) OB (0.2 mg/ml) + S9 mix and (8) OB (0.3 mg/ml) + S9 mix.
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