Ellagic Acid Triggers the Necrosis of Differentiated Human Enterocytes Exposed to 3-Nitro-Tyrosine: An MS-Based Proteomic Study

Abstract

To study the molecular basis of the toxicological effect of a dietary nitrosated amino acid, namely, 3-nitrotyrosine (3-NT), differentiated human enterocytes were exposed to dietary concentrations of this species (200 μM) and analyzed for flow cytometry, protein oxidation markers and MS-based proteomics. The possible protective role of a dietary phytochemical, ellagic acid (EA) (200 μM), was also tested. The results revealed that cell viability was significantly affected by exposure to 3-NT, with a concomitant significant increase in necrosis (p < 0.05). 3-NT affected several biological processes, such as histocompatibility complex class II (MHC class II), and pathways related to type 3 metabotropic glutamate receptors binding. Addition of EA to 3-NT-treated cells stimulated the toxicological effects of the latter by reducing the abundance of proteins involved in mitochondrial conformation. These results emphasize the impact of dietary nitrosated amino acids in intestinal cell physiology and warn about the potential negative effects of ellagic acid when combined with noxious metabolites.

Keywords: 3-Nitro-L-tyrosine; MHC class II; ellagic acid; flow cytometry; mitochondria; protein oxidation; proteomics.

Figure 1

Percentages of live cells (Hoechst +), apoptotic cells (Caspase 3+) and necrotic cells (Ethidium homodimer +) on human enterocytes as affected by exposure to 200 μM 3-NT for 72 h compared to control counterparts (A). Relative fluorescence units (r.f.u.) of ROS occurrence (Cell Rox +) on human enterocytes as affected by exposure to 200 μM 3-NT for 72 h compared to control counterparts (B). Percentages of live cells (Hoechst +), apoptotic cells (Caspase 3+) and necrotic cells (Ethidium homodimer +) on differentiated human enterocytes as affected by exposure to 200 μM 3-NT+ 200 μM EA for 72 h compared to 200 μM 3-NT treated cells (C). Relative fluorescence units (r.f.u.) of ROS occurrence (Cell Rox +) on human enterocytes as affected by exposure to 200 μM 3-NT+ 200 μM EA for 72 h compared to control counterparts (D). Percentages of live cells (Hoechst +) (E), relative fluorescence units (r.f.u.) of ROS occurrence (Cell Rox +) (F) and relative fluorescence units (r.f.u.) of apoptotic events (Caspase 3+) (G) on human enterocytes as affected by exposure to 200 μM EA for 72 h compared to control counterparts. Results are expressed as means ± standard deviations. Asterisk on top of bars denote significant differences (* p < 0.05; ** p < 0.01) between paired group of samples (control vs. 3-NT; 3-NT vs. 3-NT). Ns: no significant differences.

Figure 2

Protein carbonyls (nmol/mg protein of α-AS and γ-GS) (black bars) and advanced oxidation protein products (Pentosidine; fluorescence intensity) (grey bars) on differentiated human enterocytes upon exposure to 200 μM 3-NT and 200 μM 3-NT+200 μM EA for 72 h. Asterisks on top of bars denote significant differences between group of samples in ANOVA (*** p < 0.001). (a–c) Different letters on top of bars denote significant differences between means in post-hoc Tukey tests (p < 0.05).

Figure 3

Antioxidant activity of catalase (black bars) and superoxide dismutase (grey bars) on differentiated human enterocytes upon exposure to 200 μM 3-NT and 200 μM 3-NT+200 μM EA for 72 h. Asterisks on top of bars denote significant differences between group of samples in ANOVA (* p<0.05; *** p < 0.001).(a–b) Different letters on top of bars denote significant differences between means in post-hoc Tukey tests (p < 0.05).

Figure 4

Proposal of underlying molecular mechanisms of the toxicological effects of 200 μM 3-NT and 200 μM 3-NT+200 μM EA for 72 h on differentiated human enterocytes. Upstream head blue arrows indicate higher concentration of proteins/upregulated biological processes in treated enterocytes. Downstream head red arrows indicate lower concentration of proteins/downregulated biological processes in treated enterocytes. Blunt head connectors indicate an inhibited biological process or metabolic pathway. Effect of EA is specifically denoted by green arrows. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article). The mechanisms and routes depicted in this Figure are proposed based on the interpretation of the data from cytometry, proteomics and accretion of protein oxidation (oxidative stress). Confirmation of the impairment of all these downstream paths and interconnections of complex cellular mechanisms requires further specific studies.