Carlucci_et_al_Sterylglucosides_toxicity_cells_mice.tab

Solubility and MTT result are resumed for Acetone and DMSO vehicles in Sheet 1. Raw UV-vis data and viability percentages for MTT assays for both control and SGs are described in Sheets 2-5. Mice body weights (BW) and liver weights (LW) results are described in Sheet 6. Mice plasma and tissue metabolic assays results (raw data included) are described in Sheets 7-11 (i.e., TG & cholesterol, glycemia, GOT, GPT and WP MTP, respectively)
To determine the toxicity of isolated mixture Huh7 cell line (obtained from ATCC, Manassas, VA, USA) was treated with 4 different concentrations of SGs (0.5-50 ppm) in two vehicles (acetone and DMSO). Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum (FBS), 100 IU/ml penicillin, and 100 mg/ml streptomycin at 37°C in a humidified atmosphere of 95% O2 and 5% CO2. For cell detachment, cells were washed twice with phosphate-buffered saline (PBS, 1X: 80 mM Na2HPO4, 20 mM KH2PO4, and 100 mM NaCl; pH 7.5) and treated with trypsin-EDTA (0.01 mg/cm² and 4 µg/cm² of culture surface, respectively) (Gibco). The trypsinization process was halted by the addition of DMEM 10% FBS. Cell density was determined using a Neubauer chamber (Boeco, Hamburg, Germany) before seeding the required number of cells for further maintenance or experimental use.
Independent stock solutions of SGs were prepared in acetone and DMSO by sonication at 40°C for 10 min. For the acetone solution, 1.1 mg SGs were weighed into a screw-capped tube, followed by the addition of 11 mL of acetone. The mixture was sonicated until a clear solution was obtained. For the DMSO solution, 11.4 mg SGs were weighed into a screw-capped tube, and 2 mL DMSO were added. The mixture was also sonicated until a clear solution was achieved.
Huh7 cells were seeded and incubated overnight to ensure proper adhesion to the plates. The following day, the culture medium was replaced with fresh medium containing various concentrations of SGs. Control groups received only the vehicle (acetone or DMSO), corresponding to the solvent used for preparing the SGs stock solutions.
Cell viability was assessed using the MTT assay, which measures metabolic activity through absorbance. MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide), a water-soluble yellow dye, is reduced by mitochondrial dehydrogenases in viable cells to form insoluble purple formazan crystals. The quantity of formazan produced is directly proportional to the number of viable cells with functional mitochondria.20
Cells were seeded in 96-well plates at a density of 3,000 cells per well. A 1/100 dilution was prepared from the SGs stock solutions, followed by serial dilutions in the medium. The concentrations used were specified for each experiment. After 24 h, cells were treated with various doses of the SGs solutions for 72 h. Following treatment, MTT reagent was added to the culture medium at a final concentration of 0.5 mg/mL. After a 2 h incubation at 37°C, the cells were lysed, and the formazan crystals were dissolved by adding 200 µL of DMSO. Absorbance was measured at 540 nm, with a reference filter at 650 nm, using a DTX 880 microplate reader (Beckman Coulter Inc., Fullerton, CA, USA). Results were expressed as a percentage of control cell absorbance, and dose-response curves were generated.
Male C57BL/6 mice, 7-8 weeks of age, received human care according to criteria outlined in the “Guide for the Care and Use of Laboratory Animals” (National Research Council, Washington D.C.: National Academy Press, 1996). All the experimental protocols were performed according to the Regulation for the Care and Use of Laboratory Animals and approved by the Institutional Animal Use Committee of the National University of Rosario, Argentina (CICUAL) of the National University of Rosario (UNR).
The animals were housed under controlled conditions (20–22°C, 12-h light/dark cycles) with free access to water and a standard commercial diet. Mice were randomly divided into five groups for treatment. The treated groups received SGs by gastric gavage at doses of 25, 50, 100, or 200 mg/kg/day for 3 weeks. The control group was administered the vehicle (0.5% carboxymethylcellulose).
At the end of the treatment period, mice were anesthetized via intraperitoneal injection of a ketamine (100 mg/kg) and xylazine (3 mg/kg) mixture, followed by euthanasia through cardiac puncture to collect blood for plasma preparation. The livers and intestines (duodenum and jejunum) were excised, inspected, flushed with PBS and weighed. Tissue samples were either frozen in liquid nitrogen and stored at -70°C for future studies, processed for biochemical analyses, or prepared for histological examination.
Plasma levels of TAGs, total cholesterol, low-density lipoprotein (LDL)-cholesterol, glycemia, activities of alanine transaminase (ALT/GPT) and aspartate transaminase (AST/GOT) were determined using commercial kits according to the manufacturer's protocols (Weiner Lab, Rosario, Argentina).
Tissues stored at -70°C were used to prepare total homogenates 20% (w/v). The tissues were mechanically homogenized in a buffer consisting of 50 mM Tris-HCl (pH 7.4), 250 mM sucrose, and 5 mM EDTA, supplemented with protease and phosphatase inhibitors (PMSF, sodium orthovanadate, and sodium fluoride). The resulting total homogenates were aliquoted and stored at -70°C for further analysis.
Protein concentration of the prior homogenates was quantified using the Lowry method. 21 Bovine serum albumin was used as a control.
To determine TAG and cholesterol levels in liver and intestinal tissues, lipids were extracted using the Bligh and Dyer method. Following extraction, TAG and cholesterol levels were quantified as described previously.