This study aimed to highlight the molecular and biochemical changes induced by methylglyoxal (MGO) exposure in SH-SY5Y human neuroblastoma cells, and to explore how these changes contribute to its neurotoxicity, utilizing an integrated proteomics and metabolomics approach. Using label-free quantitative nanoLC-MS/MS proteomics and targeted LC-TQ-MS/MS-based metabolomics, the results revealed that MGO exposure, particularly at cytotoxic levels, significantly altered the proteome and metabolome of SH-SY5Y cells. Analysis of proteomics data showed significant alterations in cellular functions including protein synthesis, cellular structural integrity, mitochondrial function, and oxidative stress responses. Analysis of metabolomics and integration of metabolomics and proteomics data highlighted significant changes in key metabolic pathways including arginine biosynthesis, glutathione metabolism, cysteine and methionine metabolism, and the tricarboxylic acid cycle. These results suggest that MGO exposure induced both toxic effects and adaptive responses in cells. MGO exposure led to increased endoplasmic reticulum stress, disruptions in cellular adhesion and extracellular matrix integrity, mitochondrial dysfunction, and amino acid metabolism disruption, contributing to cellular toxicity. Conversely, cells exhibited adaptive responses by upregulating protein synthesis, activating the Nrf2 pathway, and reprogramming metabolism to counteract dicarbonyl stress and maintain energy levels. Furthermore, a set of key proteins and metabolites associated with these changes were shown to exhibit a significant concentration-dependent decrease or increase in their expression levels with increasing MGO concentrations, suggesting their potential as biomarkers for MGO exposure. Taken together, these findings provide insight into the molecular mechanisms underlying MGO-induced neurotoxicity and potential targets for therapeutic intervention.

α-Dicarbonyl compounds, particularly methylglyoxal (MGO), glyoxal (GO), and 3-deoxyglucosone (3-DG), are highly reactive precursors for the formation of advanced glycation end products (AGEs). They are formed in vivo and during food processing. This study aimed to investigate the role of intracellular glutathione (GSH) levels in the induction of Nrf2-mediated gene expression by α-dicarbonyl compounds. The reactions between α-dicarbonyl compounds (MGO, GO, and 3-DG) and GSH were studied by LC-MS in a cell-free system. It was shown that these three α-dicarbonyl compounds react instantaneously with GSH, with the GSH-mediated scavenging decreasing in the order MGO > GO > 3DG. Furthermore, in a cell-based reporter gene assay MGO, GO, and 3-DG were able to induce Nrf2-mediated gene expression in a dose-dependent manner. Modulation of intracellular GSH levels showed that the cytotoxicity and induction of the Nrf2-mediated pathway by MGO, GO and 3-DG was significantly enhanced by depletion of GSH, while a decrease in Nrf2-activation by MGO and GO but not 3-DG was observed upon an increase of the cellular GSH levels. Our results reveal subtle differences in the role of GSH in protection against the three typical α-dicarbonyl compounds and in their induction of Nrf2-mediated gene expression, and point at a dual biological effect of the α-dicarbonyl compounds, being reactive toxic electrophiles and -as a consequence- able to induce Nrf2-mediated protective gene expression, with MGO being most reactive.

The fungal secondary metabolite deoxynivalenol (DON) that can contaminate cereal-based food products not only induces inflammation but also reduces bile acid absorption by a healthy human intestine. Bile acid malabsorption is commonly observed in individuals with an inflamed intestine. Here we studied the effects of DON on inflammation and primary bile acid transport using an in vitro model for an inflamed intestine. An inflamed intestinal in vitro model was established by co-culturing a Caco-2 cell-layer and LPS-pre-stimulated THP-1 macrophages in Transwells. We observed a decreased transport of 5 primary bile acids across inflamed co-cultures compared to healthy co-cultures but not of chenodeoxycholic acid. DON exposure further reduced the transport of the affected primary bile acids across the inflamed co-cultures. DON exposure also enhanced the secretion of pro-inflammatory cytokines in the inflamed co-cultures, while it did not increase the pro-inflammatory cytokines secretion from LPS-pre-stimulated THP-1 monocultures. Exposure of Caco-2 cell-layers to pro-inflammatory cytokines or THP-1 conditioned media partly mimicked the DON-induced effects of the co-culture model. Local activation of intestinal immune cells reinforces the direct pro-inflammatory effects of DON on intestinal epithelial cells. This affects the bile acid intestinal kinetics in an inflamed intestine.

The advanced glycation endproduct carboxymethyllysine and its precursor fructoselysine are present in heated, processed food products and are considered potentially hazardous for human health. Upon dietary exposure, they can be degraded by human colonic gut microbiota, reducing internal exposure. Pronounced interindividual and intraindividual differences in these metabolic degradations were found in anaerobic incubations with human fecal slurries in vitro. The average capacity to degrade fructoselysine was 27.7-fold higher than that for carboxymethyllysine, and degradation capacities for these two compounds were not correlated (R² = 0.08). Analysis of the bacterial composition revealed that interindividual differences outweighed intraindividual differences, and multiple genera were correlated with the individuals' carboxymethyllysine and fructoselysine degradation capacities (e.g., Akkermansia, Alistipes).

Hydroxyanthraquinones that can be present in traditional Chinese medicine (TCM) and herbal extracts have claimed beneficial intestinal effects. We examined the ability of a panel hydroxyanthraquinones, and methanolic extracts from selected TCM and herbal granules to activate Nrf2-EpRE mediated gene expression using a reporter-gene assay. The results indicate that purpurin, aloe-emodin, 2-hydroxy-3-methylanthraquinone and rhein induced Nrf2 mediated gene expressions with a high induction factor (IFs>10), with BMCL₁₀ values (the lower confidence limit of the concentration giving 10% added response above background) of 16 μM, 1.1 μM, 23 μM and 2.3 μM, respectively, while aurantio-obtusin, obtusifolin, rubiadin 1-methyl ether and emodin were less potent (IFs<5), with BMCL₁₀ values for added response above background level of 4.6 μM, 15 μM, 9.8 μM and 3.8 μM, respectively. All TCM extracts and the herbal extracts of Aloe Vera, Polygonum multiflorum, Rubia (cordifolia) and Rheum officinale activated the Nrf2-EpRE pathway. Of the TCM extracts, Chuan-Xin-Lian-Kang-Yan-Pian was the most potent Nrf2-inducer. LC-MS/MS analysis indicated the presence of selected hydroxyanthraquinones in the extracts and herbs, in part explaining their Nrf2-EpRE mediated activity. In conclusion, different hydroxyanthraquinones have different potencies of Nrf2 activation. The Nrf2 activation by extracts from TCM and herbs can be partially explained by the presence of selected hydroxyanthraquinones.

Purpurin is a major anthraquinone present in the roots of Rubia cordifolia (madder). Purpurin is known to activate Nrf2 (Nuclear transcription factor erythroid 2-related factor 2) EpRE (electrophile responsive element) mediated gene expression as a potential beneficial effect. This study aimed to elucidate the balance between the electrophilicity or pro-oxidant activity of purpurin underlying the Nrf2 induction. For this, Nrf2 activation with modified intracellular glutathione (GSH) levels was measured in an Nrf2 CALUX reporter gene assay. In addition, both cell-free and intracellular ROS formation of purpurin with modified (intracellular) GSH levels at different pH were quantified using the DCF-DA assay. GSH adduct formation was evaluated by UPLC and LC-TOF-MS analysis. GSH and GSSG levels following purpurin incubations were quantified by LC-MS/MS. We show that Nrf2 induction by purpurin was significantly increased in cells with buthionine sulfoximine depleted GSH levels, while Nrf2 induction was decreased upon incubation of the cells with N-acetylcysteine being a precursor of GSH. In cell-free incubations, ROS formation increased with increasing pH pointing at a role for the deprotonated form of purpurin. Upon incubations of purpurin with GSH at physiological pH, GSH adduct formation appeared negligible (<1.5% of the added purpurin). The addition of GSH resulted in conversion of GSH to GSSG and significantly reduced the ROS formation. Together these results demonstrate that Nrf2 induction by purpurin originates from intracellular ROS formation and not from its electrophilicity, which becomes especially relevant when intracellular GSH levels can no longer scavenge the ROS. The present study demonstrated that the efficiency of intracellular Nrf2 activation by purpurin and related anthraquinones will depend on (i) their pKa and level of deprotonation at the intracellular pH, (ii) the oxidation potential of their deprotonated form and (iii) the intracellular GSH levels. Thus, the Nrf2 induction by purpurin depends on its pro-oxidant activity and not on its electrophilicity.