Zearalenone Induces Endoplasmic Reticulum Stress and Modulates the Expression of Phase I/II Enzymes in Human Liver Cells

Abstract

Zearalenone (ZEN) is a mycotoxin produced by Fusarium species; however, its mechanisms of action in human livers have not been fully elucidated. Thus, we investigated the toxic mechanisms of ZEN in human liver cells. HepG2 cells were treated with ZEN (0-40 μg/mL) for up to 24 h. A significant decrease in cell viability was observed after treatment with 20 and 40 μg/mL of ZEN, including a significant increase in apoptosis and reactive oxygen species production. ZEN increased GRP78 and CHOP, and eIF2α phosphorylation, indicating ER stress; elevated transcription of the autophagy-associated genes, beclin1 and LC3, and translation of LC3; and increased phase I metabolism by increasing PXR and CYP3A4. The protein expression level of CYP3A4 was higher with ZEN treatment up to 20 μg/mL, but remained at the control level after treatment with 40 μg/mL ZEN. In phase II metabolism, Nrf2 activation and UGT1A expression were increased with ZEN treatment up to 20 μg/mL. Treating cells with an ER stress inhibitor alleviated ZEN-induced cell death and autophagy, and inhibited the expression of phase I/II enzymes. Overall, high ZEN concentrations can modulate the expression of phase I/II enzymes via ER stress and reduced protein levels in human liver cells.

Keywords: ER stress; apoptosis; hepatotoxicity; oxidative stress; phase I/II metabolism; zearalenone.

Figure 1

Zearalenone (ZEN) induced cytotoxicity in HepG2 cells. Cells were treated with ZEN (0, 1, 5, 10, 20, and 40 μg/mL) for 24 h and cell viability was measured by the trypan blue dye exclusion test. Data represent mean ± SEM of three independent experiments. * indicates significant difference vs. the control (** p < 0.01, and *** p < 0.001).

Figure 2

Zearalenone (ZEN) induced apoptosis in HepG2 cells. (A) Cells were treated with ZEN (0, 1, 5, 10, 20, and 40 μg/mL) for 24 h and apoptotic/necrotic cells were analyzed using CytoFLEX flow cytometer after Annexin V-FITC/PI double staining. (B) The apoptotic rate was calculated using CytExpert software (The parts of Annexin V+/PI- and Annexin V+/PI+ represent early apoptotic cells and late apoptotic/necrotic cells, respectively). The images represent three independent experiments. Data represent mean ± SEM. * indicates significant difference vs. the control (** p < 0.01, and *** p < 0.001).

Figure 3

Zearalenone (ZEN) induced oxidative stress in HepG2 cells. Cells treated with ZEN (0, 1, 5, 10, 20, and 40 μg/mL) for 2 h were stained with (A) 2′,7′-dichlorofluorescin diacetate (DCFH-DA) and (C) dihydroethidium (DHE) to detect intracellular reactive oxygen species (ROS) production. The intensity of green fluorescence (general ROS production) and red fluorescence (superoxide anion production) was assessed by a fluorescence microscope at 200× magnification and a super-resolution confocal laser scanning microscope at 630× magnification, respectively. (B) The DCFH-DA positive area (green fluorescence) and (D) DHE positive area (red fluorescence) were analyzed using Image J software and graphically presented. The images shown are representatives of three independent experiments. Data represent mean ± SEM. * indicates significant difference vs. the control (*** p < 0.001).

Figure 4

Zearalenone (ZEN) induced endoplasmic reticulum (ER) stress and autophagy in HepG2 cells. mRNA level and protein expression were determined by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis, respectively. (A) The protein expression level of GRP78 was measured in cells treated with ZEN for 1 h. (B) The phosphorylation of eIF2α was evaluated in cells treated with ZEN for 2 h. (C) CHOP mRNA levels, (D) Beclin1 mRNA levels, and (E) LC3-II mRNA levels were determined in cells treated with ZEN for 4 h. (F) The protein expression level of LC3-II/LC3-I was examined in cells treated with ZEN for 24 h. Data represent mean ± SEM of three independent experiments. * indicates significant difference vs. the control (** p < 0.01, and *** p < 0.001).

Figure 5

Zearalenone (ZEN) induced the expression of PXR and CYP3A4, and ZEN-induced ER stress inhibited the protein expression of CYP3A4. mRNA level and protein expression were determined by quantitative real-time PCR (qRT-PCR) and Western blot analysis, respectively. (A) PXR mRNA levels were determined in cells treated with ZEN for 4 h. (B) CYP3A4 mRNA levels were determined in cells treated with ZEN for 8 h. (C) The protein expression level of CYP3A4 was examined in cells treated with ZEN for 24 h. Data represent mean ± SEM of three independent experiments. * indicates significant difference vs. the control (* p < 0.05, ** p < 0.01, and *** p < 0.001).

Figure 6

Zearalenone (ZEN) induced Nrf2 activation and UGT1A expression. The mRNA level and protein expression of signaling mediators were determined by quantitative real-time PCR (qRT-PCR) and Western blot analysis, respectively. (A) Nrf2 nuclear translocation was measured in cells treated with ZEN (20 and 40 μg/mL) for 6 h by nuclear fractionation and Western blotting. (B) UGT1A mRNA levels were determined in cells treated with ZEN for 16 h. Data represent mean ± SEM of three independent experiments. * indicates significant difference vs. the control (* p < 0.05, ** p < 0.01, and *** p < 0.001).

Figure 7

4-phenylbutyric acid (4-PBA) alleviated zearalenone (ZEN)-induced ER stress, autophagy, and downregulation of CYP3A4 and UGT1A. Before treatment with ZEN (20 and 40 μg/mL), cells were pretreated with 1 mM 4-PBA for 6 h. mRNA and protein expression were determined by quantitative real-time PCR (qRT-PCR) and Western blot analysis, respectively. (A) Cells were treated with ZEN for 24 h after pretreatment with 4-PBA and cell viability was measured by the trypan blue dye exclusion test. (B) Phosphorylation of eIF2α was evaluated in cells treated with ZEN for 2 h. The protein expression of (C) LC3-II/LC3-I and (D) CYP3A4 was determined in cells treated with ZEN for 24 h. (E) UGT1A mRNA levels were determined in cells treated with ZEN for 16 h. Data represent mean ± SEM of three independent experiments. * indicates significant difference vs. the control (* p < 0.05, ** p < 0.01, and *** p < 0.001). # indicates significant difference between groups (p < 0.05).