Preparation of stigmasteryl esters
Stigmasterol—ST (≥ 95%), oleic acid—OA (≥ 99%), and linoleic acid—LA (≥ 99%) standards were purchased from Sigma-Aldrich (St. Louis, MO, USA). ST-OA and ST-LA were obtained by chemical esterification based on the Neises and Steglich method42 according to the protocol described by Kasprzak et al.25. ST (500 mg) dissolved in dichloromethane (30 mL) was placed in a three-necked flask. The air in the flask was replaced with argon. The catalyst: N,N′-Dicyclohexylcarbodiimide (500 mg) and 4-Dimethylaminopyridine (15 mg), and fatty acid (OA or LA) (600 mg) were then added to the flask. Esterification was performed at room temperature for 24 h in the dark. The reaction mixture was transferred to a separatory funnel and extracted in triplicate with distilled water (10 mL), with lower layers collecting. The fractions collected were concentrated under a vacuum at 30 °C, and the residue was dissolved in hexane (20 mL). The esterification mixture was purified on a silica gel column (45 × 2.5 cm). The ester fraction was eluted with hexane:ethyl acetate (9:1) mixture (450 mL). TLC was used to check the ester fraction purity.
ST, ST-OA, and ST-LA (50 mg each) were placed separately in glass vials and heated at 180 °C for 8 h under an oxygen atmosphere. After thermo-oxidative treatment, the tested compounds were stored at − 20 °C until chemical and biological analyses. The non-heated and heated ST and its esters were analyzed to detect degradation products and oxyderivatives. Analytical methods were described in the article published previously25. Table S1 presents the composition of thermo-oxidative degradation products of ST, ST-OA, and ST-LA.
Compounds preparation for cytotoxicity and genotoxicity experiments
ST, ST-LA, and ST-OA were dissolved in acetone and then diluted in this solvent to obtain the concentrated stock solutions of the analyzed compounds at each dose tested. Each concentration was prepared by 200-fold diluting the appropriate stock solution in a culture medium.
The normal human diploid CCD 841 CoN cell line (ATCC® CRL-1790™) isolated from colon mucosa was obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma-Aldrich) supplemented with fetal bovine serum (FBS; Gibco BRL, Grand Island, NY, USA) to a final concentration of 10% and maintained at 37 °C in a humidified atmosphere with 5% CO2.
In the cytotoxicity experiments, CCD 841 CoN cells were seeded in 96-well plates at an initial density of 2.0 × 104 cells/cm2 and incubated for 24 h. Then, the cells were exposed to the non-heated and heated ST, ST-OA, and ST-LA at concentrations of 1.25, 2.5, 5, 10, 20, and 40 μg/mL for 48 h under standard culture conditions. Control culture contained the vehicle at the amount corresponding to the sample analyzed.
The Multitox-Fluor Multiplex Cytotoxicity Assay (Promega GmbH, Mannheim, Germany) was applied to determine the relative number of live and dead cells in the CCD 841 CoN cell cultures treated with the analyzed compounds. The assay determined live- and dead-cell protease activities using two fluorogenic peptide substrates, cell-permeant (glycyl-phenylalanyl-amino fluorocoumarin; GF-AFC) and cell-impermeant (bis-alanyl-alanyl-phenylalanyl-rhodamine 110; bis-AAF-R110). After treatment, the cells were incubated with GF-AFC and bis-AAF-R110 substrates for 60 min at 37 °C. The live- and dead-cell proteases produced AFC and R110 products, measured using a Tecan M200 Infinite microplate reader (Tecan Group Ltd., Männedorf, Switzerland) at different excitation (400 nm and 485 nm) and emission (505 nm and 520 nm) spectra. The assay was conducted according to the manufacturer’s instructions.
Measurement of DNA synthesis
CCD 841 CoN cells were grown in black 96-well plates at an initial cell density of 2 × 104 cells/cm2 for 24 h under standard culture conditions. The cells were treated with ST (2.5, 5, 10, 20, and 40 μg/mL) and its esters ST-OA and ST-LA (10, 20, and 40 μg/mL) for 48 h. DNA synthesis was measured by incorporating thymidine analog 5-bromo-2′-deoxyuridine (BrdU) into newly synthesized DNA using the Cell Proliferation ELISA kit according to the protocol recommended by the manufacturer (Roche Diagnostics GmbH, Mannheim, Germany). Briefly, BrdU was added to the treated cells 24 h before the end of the exposure to the ST, ST-OA, and ST-LA (final BrdU concentration of 10 μM). After treatment, BrdU-labeled DNA was denatured (30 min, 20 °C). Then, BrdU was bonded with a peroxidase-conjugated anti-BrdU antibody (90 min, 20 °C) and reacted with the peroxidase substrate. The absorbance measurement was done with a stop solution (1 M H2SO4) at 450 nm with reference wavelength 690 nm using a Tecan M200 Infinite microplate reader.
Cell cycle analysis
CCD 841 CoN cells were grown in 6-well plates at an initial cell density of 2 × 104 cells/cm2 for 24 h under standard culture conditions. The cells were treated for 24 h with the analyzed compounds at a concentration of 40 μg/mL; the high dose was applied because of the relatively low cytotoxic potential of ST-OA and ST-LA as determined in cytotoxicity studies. In addition, the cells were exposed to 0.05 μM camptothecin (Sigma-Aldrich) as a positive reference compound known to modulate cell cycle progression.
After treatment, the cells were harvested by trypsinization, washed in PBS, and fixed in 70% ethanol. Then, the cells were stained with 50 μg/mL propidium iodide in the presence of 100 μg/mL RNase (Sigma-Aldrich). The sample preparation method and staining protocol were described previously43. The cycle phase distribution was analyzed with an Amnis™ FlowSight™ flow cytometer (Luminex Corporation, TX, USA).
Caspase 3/7 activity assay
CCD 841 CoN cells were grown in black 96-well plates at an initial cell density of 2 × 104 cells/cm2 for 24 h under standard culture conditions. The cell cultures were treated for 24 h with ST, ST-OA, and ST-LA at a 40 μg/mL dose. After treatment, caspase-3/7 activity was determined using the APO-One Homogeneous Caspase-3/7 Assay (Promega Corporation, Wisconsin, USA), which is based on the proteolytic cleavage of C-terminal side of aspartate residue in the DEVD peptide substrate by caspase 3/7 into fluorescent rhodamine 110 (R110). Analysis of caspase 3/7 activity was carried out following the manufacturer’s instructions. Briefly, the cells after the treatment were incubated at room temperature with Apo-ONE® Caspase-3/7 Reagent—bifunctional cell lysis/caspase activity buffer combined with the pro-fluorescent caspase-3/7 substrate Z-DEVD-R110. After 4-h incubation, fluorescence was measured at an excitation wavelength of 485 nm and an emission wavelength of 530 nm using a Tecan M200 Infinite microplate reader. Data obtained were normalized to cellular protein content, quantified by BCA assay (Pierce® BCA Protein Assay Kit, Thermo Scientific Inc., USA) according to the manufacturer’s protocol.
Intracellular ROS measurement
CCD 841 CoN cells were grown in 6-well plates at an initial cell density of 2 × 104 cells/cm2 for 24 h under standard culture conditions. The cell cultures were treated for 24 h with the analyzed compounds at a 40 μg/mL concentration. After treatment, the cells were harvested by trypsinization, washed with Hank’s Balanced Salt Solution, and incubated with 10 μM 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) (Life Technologies, Carlsbad, CA, USA) at 37 °C for 30 min. Intracellular DCF fluorescence (λex/em = 488/530 nm) was examined by flow cytometry (Amnis™ FlowSight™ flow cytometer).
DNA damage detection
The cells were grown at 6-well plates at the established density and standard culture conditions and exposed to the non-heated and heated ST, ST-OA, and ST-LA for 48 h. The non-treated cells and cells treated with H2O2 (100 μM, 30 min) to induce oxidative DNA damage constituted the negative and positive controls, respectively. After treatment, cells were analyzed for DNA damage using a single cell gel electrophoresis (SCGE) called comet assay, described in detail previously44. Briefly, harvested cells suspended in low melting point agarose were put onto microscope slides pre-coated with normal melting point agarose and subjected sequentially to lysis, alkaline electrophoresis (pH > 13), and neutralization (pH 10). The cells were stained with SYBRGold (Molecular Probes), viewed under a fluorescence microscope (Axiovert 200, Zeiss, Carl Zeiss, Gottingen, Germany), and analyzed using CometScore™ software (TriTek Corp., Sumerduck, VA, USA). At least 100 cells were analyzed on a microscope slide; 300 cells were considered for DNA damage detection in one sample. Data were expressed as the mean percentage of DNA content in the comet tail. Moreover, depending on DNA content in comet tails, comets were classified into five categories: class 0 (< 1%, no damage), class 1 (1–25%; low damage), class 2 (> 25–45%; medium damage), class 3 (> 45–70%; high damage), class 4 (> 70%; very high damage). Based on the comets’ categorization, the total comet score (TCS) was calculated according to the formula: TCS = 0(n) + 1(n) + 2(n) + 3(n) + 4(n), where “n” is the number of cells in each comet class (0–4)45.
Potential mutagenicity or pro-mutagenicity of non-heated and heated ST, ST-LA, and ST-OA was determined using the bacterial reverse mutation assay (Ames test) with Salmonella enterica subsp. enterica ser. typhimurium tester strains TA98, TA100, and TA102, which were obtained from the Polish Collection of Microorganisms of the Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences in Wroclaw.
Mutagenicity experiments were performed in the liquid pre-incubation assay, without and with metabolic activation by supplying Aroclor 1254-induced rat liver microsomal fraction (S9, Sigma-Aldrich) with NADP and cofactors for NADPH-supported oxidation, according to the procedure described by Mortelmans and Zeiger39. Briefly, reaction mixtures, consisting of 12-h bacterial culture, non-heated and heated compound (ST, ST-LA, ST-OA) at a concentration of 40 μg, 0.2 M phosphate buffer (pH 7.4), and alternatively S9 activating mixture, were pre-incubated at 37 °C for 20 min. The mutagens, 2-aminofluorene (100 μg), sodium azide (1 μg), and tert-butyl-hydrogen peroxide (tert-butyl-H2O2) (50 μM) (all supplied by Sigma-Aldrich), were used as positive controls to reverse mutation in the TA98, TA100, and TA102 strain, respectively. The mutagen, 2-aminoanthracene (5 μg) (Sigma-Aldrich), was a positive control in pro-mutagenicity experiments. After pre-incubation, the reaction mixtures were added to the top agar supplemented with traces of histidine and biotin and poured onto the plates covered with minimum glucose agar. The cultures were incubated for 48 h at 37 °C, and the number of revertants (His+) colonies was counted manually.
The mutagenic index (MI), which reflects the number of induced revertants (Ri) with mutagen or compound tested divided by the number of spontaneously induced revertants (Rs), was applied to express mutagenic activity. Compounds with MI ≥ 2 can be recognized as potentially mutagenic46. The MI values calculated for reference mutagens applied to induce reverse mutation in tester strains ranged from 2.51 to 13.55 (Table 1).
Data are presented as means ± SD from three independent replications. Statistical analysis was performed using STATISTICA version 13.3 software (Statsoft, Inc., Tulsa, OK, USA). A Student’s t-test was used to compare two groups of data. One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was performed to determine the differences between the mean values of multiple groups. P ≤ 0.05 was the cut-off point for a significant difference.