FREE RADICALS AND NATURAL SUBSTANCES IN PELOIDS 75 absence of mycoplasma were checked at regular intervals. Stocks of the L5178Y cells were stored in liquid nitrogen, and subcultures were prepared from the frozen stocks for ex- perimental use. Cells were grown in RPMI 1640 supplemented with 10% heat-inactivated horse serum, 2 mM L -glutamine, and antibiotics (100 IU/ml penicillin and 100 IU/ml strep- tomycin) and incubated at 37°C in a 5% carbon dioxide (CO2) atmosphere and 100% nominal humidity. Chinese hamster ovary (CHO) cells were obtained from Prof. A. T. Natarajan (State University of Leiden, the Netherlands). The cell lines were derived from the CHO isolated from an explant of the ovary of the Chinese hamster (Cricetulus griseus, 2n = 22). The CHO cell line is particularly useful for this kind of studies because of its stable karyotype (modal number is 21 chromosomes), short cell cycle (12–14 h), and its high plating effi ciency. Stocks of CHO cells were stored in liquid nitrogen, and subcul- tures were prepared from these stocks for experimental use. Cultures were grown as monolayer cultures in Ham’s F-10 medium (Gibco BRL, Thermo Fisher, Milan, Italy) supplemented with 15% fetal bovine serum, 4 mM L -glutamine, and antibiotics (50 IU/ml penicillin and 50 IU/ml streptomycin). All incubations were at 37°C in 5% CO2 atmo- sphere and 100% nominal humidity. Cytotoxicity evaluation. Approximately 24 h before treatment, exponentially growing cells were detached by trypsin action, and an appropriate number of 75-cm2 plastic cell culture fl asks containing 15 ml of complete culture medium were individually inocu- lated with 2.0 × 106 cells. Test compounds were dissolved in dimethyl sulfoxide (DMSO) immediately before treatment and added to the culture medium such that the fi nal concentration of solvent did not exceed 1%. The assay was performed using a set of at least six dose levels for each test compound spaced by a factor of 2 (1.0–526 μg/ml), and cell cultures were treated for 3 h. At the end of the treatment, the cultures were washed twice with phosphate-buffered saline, trypsinized, and diluted to obtain an estimated number of 2 × 103 cells/ml. A volume of 100 μl of each cell suspension was plated in each of three 60-mm tissue culture petri dish to assess the viability of the cells. Plates were incubated for at least 6 days before scoring. After incubation, colonies were stained with a 10% aqueous Giemsa solution, and the number of colonies were scored by hand. Comet assay. Cultures of mouse lymphoma cells at a concentration of 1 × 106 cells/ml were treated for 3 h at 37°C in 5% CO2 atmosphere and 100% nominal humidity, with each sample at a single dose level selected as the dose levels that reduced the relative cloning effi ciency (RCE) of CHO cells to approximately 50% over the concurrent vehicle control values of cultures (Table IV). At the end of the treatment, 10 μl of each cell suspension was added to 65 μl of 0.7% (w/v) low melting point agarose (Bio-Rad Laboratories, Milan, Table IV Dose Levels for Different Fractions Compound Dose level (μg/ml) WM-Acetone 16.0 DM-Acetone 8.0 WM-EtOAc 8.0 DM-EtOAc 8.0 WM-MeOH 128.0 DM-MeOH 64.0

JOURNAL OF COSMETIC SCIENCE 76 Italy) and sandwiched between a lower layer of 1% (w/v) normal melting agarose (Bio- Rad Laboratories, Milan, Italy). For each compound, two sets of three slides each were prepared. An aliquot of 50 μl of H2O2 (0.25 μM) was added to one set, while PBS was added to the parallel set. The slides were kept at 4°C for 5 min and then immersed in lysing solution (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris, pH 10) containing 10% DMSO and 1% Triton X-100 (ICN Biomedicals Inc., Irvine, CA) at 4°C overnight. Slides were then randomly placed in a horizontal gel electrophoresis apparatus with fresh alkaline electrophoresis buffer (300 mM NaOH, 1 mM Na2EDTA, pH 13) and incu- bated for 25 min at 4°C to allow for DNA unwinding and expression of alkali-labile sites. Electrophoresis was performed at 4°C for 15 min at 30 V (1 V/cm) and 300 mA. After electrophoresis, slides were immersed in 0.3-M sodium acetate in ethanol for 30 min. Slides were then dehydrated in an alcohol series (2 min at 70%, 85%, and 100%) and air-dried. Slides stained with 20 μg/ml ethidium bromide in the presence of antifade re- agent immediately before analysis were examined at 40× magnifi cation using an auto- mated image analysis system (Comet Assay III Perceptive Instruments, Suffolk, UK) connected to a fl uorescence microscope (Zeiss Axioskop 2, Milan, Italy). DNA damage was quantifi ed from the tail moment (TM) values. A number of 50 cells from each slide (150 cells in total) were analyzed per experimental point. RESULTS MINERALOGICAL COMPOSITION Four samples of DM and three samples of WM (namely, FS I–IV and FC II–IV, respec- tively) were collected from the fl oor of the pool Bagnaccio during four representative periods of the year: winter (sample I), spring (sample II), summer (sample III), and au- tumn (sample IV). The particle size was determined by wet sieving and sedimentation methods (21), while the triangle of Shepard was used for the particle size classifi cation (22). The inorganic constituents were identifi ed by liquid ion chromatography, and mi- nor and trace elements were detected by quadrupole ICP-MS. The mineralogical compo- sition was defi ned by both diffraction analysis (Goniometer Seifert MZIV q/2q) and chemical analysis (ICP-MS). Results for DM and WM peloids are given in Tables V and VI, respectively. Samples FS II and FS IV were silty sands, while FS I and FS III were silt and sand, respectively (Table V). The percentage of clay was quite homogeneous with a standard deviation lower than that calculated for the surroundings sand and silt. The grain size composition of WM was rather heterogeneous (Table VI), with a slight predominance of sand and silt fractions (sandy silt, silty sand, sandy silt clay). The diagram of Shepard, percentages of sand, silt, and clay, respectively, are given in Supplemental #2 (Supplemental Figures 9–12). The cumulative curves (Supplemental #2, Supplemental Figure 13) of WM and DM were well sorted, in agreement with values lower than 100 of the uniformity coeffi cient U, given by the ratio between the particle diameter at 60% and the diameter at 10% (Table VII). The DM peloid consists of alumina silicates of kaolinite and montmorillonite type, while WM peloid of microcrystalline calcium sulfate. Among minor elements, relatively high