Apoptosis and Oxidative Stress in Human Intestinal Epithelial Caco-2 Cells Caused by Marine Phycotoxin Azaspiracid-2

Abstract

When humans consume seafood contaminated by lipophilic polyether phycotoxins, such as azaspiracids (AZAs), the toxins are mainly leached and absorbed in the small intestine, potentially causing intestinal damage. In this study, human intestinal epithelial Caco-2 cells were used to investigate the adverse effects of azaspiracid-2 (AZA-2) on human intestinal epithelial cells. Cell viability, apoptosis, oxidative damage and mitochondrial ultrastructure were investigated, and ribonucleic acid sequence (RNA-seq) analysis was applied to explore the potential mechanisms of AZA-2 toxicity to Caco-2 cells. Results showed that AZA-2 significantly reduced the proliferation of Caco-2 cells in a concentration-dependent response, and the 48 h EC₅₀ of AZA-2 was 12.65 nmol L^-1. AZA-2 can induce apoptosis in Caco-2 cells in a dose-dependent manner. Visible mitochondrial swelling, cristae disintegration, membrane rupture and autophagy were observed in Caco-2 cells exposed to AZA-2. Reactive oxygen species (ROS) and malondialdehyde (MDA) content were significantly increased in Caco-2 cells after 48 h of exposure to 1 and 10 nmol L^-1 of AZA-2. Transcriptome analysis showed that KEGG pathways related to cellular oxidative damage and lipid metabolism were affected, mainly including mitophagy, oxidative phosphorylation, cholesterol metabolism, vitamin digestion and absorption, bile secretion and the peroxisome proliferator-activated receptor signaling pathway. The cytotoxic effects of AZA-2 on Caco-2 cells may be associated with ROS-mediated autophagy and apoptosis in mitochondrial cells. Results of this study improve understanding of the cytotoxicity and molecular mechanisms of AZA-2 on Caco-2 cells, which is significant for protecting human health.

Keywords: Caco-2 cells; azaspiracids; cell viability; mitophagy; oxidative damage.

Figure 1

Viability of Caco-2 cells exposed to different concentrations of AZA-2 for 48 h. Data are shown as mean values ± standard error (n = 3).

Figure 2

Fluorescence microscopy images of Caco-2 cells stained with AO/PI and exposed to various concentrations of AZA-2 for 48 h. (scale bar: 200 μm).

Figure 3

Effects of AZA-2 on the mitochondrial ultrastructure of Caco-2 cells monitored by a transmission electron microscope (TEM). Pictures in the second row indicate zooming in the area in the red box. “N” indicates the cell nucleus. “M” in red color indicates normal mitochondria; “M” in blue color indicates impaired mitochondria. “A” in red color indicates fusion of autophagosomes and lysosomes.

Figure 4

Relative fluorescence intensity (A) and laser scanning confocal microscope images (B) of ROS and the content of MDA (C) in Caco-2 cells exposed to different concentrations of AZA-2 for 48 h. Data are shown as mean values ± standard error (n = 3), and significant differences between AZA-2 exposed and control groups are indicated as *** p < 0.001.

Figure 5

Volcano plot of the differential expressed genes (DEGs) of the Caco-2 cell line between 0.1 nmol L⁻¹ (A) and 0.01 nmol L⁻¹ (B) AZA-2 exposed groups and the control group, respectively, and the top 20 KEGG pathways enriched by DEGs (|fold change|> 2) in Caco-2 cells in the 0.1 nmol L⁻¹ AZA-2 exposure and control groups (C).

Figure 6

The autophagy and mitophagy KEGG pathways enriched by DEGs. Orange outline: up-regulation. Blue outline: down-regulation.