Anti-proliferative potential and oxidative reactivity of thermo-oxidative degradation products of stigmasterol and stigmasteryl esters for human intestinal cells

Abstract

Stigmasterol in free and esterified form is incorporated in LDL cholesterol-lowering food products, intended for direct consumption and cooking, baking, and frying. Under thermal treatment, stigmasterol compounds may constitute a source of thermo-oxidative degradation products and oxyderivatives with potentially adverse health effects. This study aimed to analyze the anti-proliferative potential and genotoxicity of thermo-oxidatively treated stigmasterol (ST), stigmasteryl linoleate (ST-LA), and oleate (ST-OA). The effects on cell viability and proliferation, cell cycle progression, intracellular reactive oxygen species (ROS) generation, and DNA damage were analyzed in normal human intestinal cells. The mutagenic potential was assessed in a bacterial reverse mutation test using Salmonella enterica serovar Typhimurium strains involving metabolic activation. Stigmasteryl esters showed a significantly lower potential to affect intestinal cell viability and proliferation than non-esterified ST, regardless of heating. Thermo-oxidatively treated ST suppressed intestinal cell proliferation by arresting the cell cycle in the G₂/M phase and DNA synthesis inhibition. The enhanced intracellular ROS generation and caspase 3/7 activity suggest targeting intestinal cells to the apoptosis pathway. Also, heated ST-LA intensified ROS production and elicited pro-apoptotic effects. Thermo-oxidative derivatives of ST and ST-LA may evoke harmful gastrointestinal effects due to their high oxidative reactivity towards intestinal cells.

Figure 1

Structure of stigmasterol (ST) and its esters with oleic (ST-OA) and linoleic (ST-LA) acid.

Figure 2

Effect of stigmasterol (ST) (a,d), stigmasteryl linoleate (ST-LA) (b,e), and stigmasteryl oleate (ST-OA) (c,f) non-heated (a–c) and heated at 180 °C for 8 h (d–f) on the colon CCD 841 CoN cell viability after 48-h treatment with the analyzed compounds at concentrations ranging from 1.25 to 40 μg/mL. Cell viability (live cell fluorescence) and cytotoxicity (dead cell fluorescence) were determined using MultiTox-Fluor Multiplex Cytotoxicity Assay. The values represent the means (n = 3) ± SD. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001 relative to vehicle-treated control cells (Tukey’s post hoc test).

Figure 3

Dose-dependent inhibition of DNA synthesis in the colon mucosa CCD 841 CoN cells by stigmasterol (ST) (a) and stigmasteryl esters (ST-LA, ST-OA) (b) non-heated (a,b) and heated at 180 °C for 8 h (c,d). Values represent the means ± SD (n = 3). ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001 relative to vehicle-treated control cells (Tukey’s post hoc test).

Figure 4

Cell distribution in different cell cycle phases (a), caspase 3/7/ activity (b), and intracellular reactive oxygen species (ROS) generation (c) in the colon mucosa CCD 841 CoN cells treated with non-heated and heated (180 °C, 8 h) stigmasterol (ST), stigmasteryl linoleate (ST-LA) and stigmasteryl oleate (ST-OA) at a concentration of 40 μg/mL. Camptothecin (CMPT, 50 nM) and H₂O₂ (OXIDANT, 50 μM) were positive controls. Values represent the means ± SD (n = 3). ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001 relative to vehicle-treated control cells (Student’s t-test).

Figure 5

DNA strand breaks in the colon mucosa CCD 841 CoN cells induced by stigmasterol (ST) and its esters (stigmasteryl linoleate, ST-LA, and stigmasteryl oleate, ST-OA) non-heated and heated at 180 °C for 8 h. All compounds were applied at a dose of 40 μg/mL. An oxidant (H₂O₂, 100 μM) was used as a positive control in the experiments. Based on the DNA content in the tails, comets were classified into 5 categories: class 0 (no damage), < 1%; class 1 (low damage), 1–25%; class 2 (medium damage), > 25–45%; class 3 (high damage), > 45–70%; class 4 (very high damage), > 70%. Comet class distribution is presented in figure (a). Total comet score (TCS), calculated as TCS = 0(n) + 1(n) + 2(n) + 3(n) + 4(n), where “n” means the number of cells in each comet class, is shown in figure (b). Values represent the means ± SD (n = 3). ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001 relative to vehicle-treated control cells (Student’s t-test). Photos present CCD 841 CoN cells treated with non-heated and heated ST, ST-LA, and ST-OA at 40 μg/mL concentration and analyzed in the comet assay. Photos were taken at a magnification of 100 ×.

Figure 6

The mutagenic potential of stigmasterol (ST), stigmasteryl linoleate (ST-LA), and stigmasteryl oleate (ST-OA) non-heated and heated at 180 °C for 8 h, applied at a dose of 40 μg/mL in the treatment of three Salmonella typhimurium TA102 (a–c), TA100 (d–f) and TA98 (g–i) strains cultured without or with a microsomal fraction (− S9 or + S9) to induce metabolic activation. Values represent the means of the number of revertants (CFU/plate) ± SD (n = 3). ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001 relative to vehicle-treated control group (Student’s t-test).