A multi-omics approach to elucidate okadaic acid-induced changes in human HepaRG hepatocarcinoma cells.

Okadaic acid (OA), a prevalent marine biotoxin found in shellfish, is known for causing acute gastrointestinal symptoms. Despite its potential to reach the bloodstream and the liver, the hepatic effects of OA are not well understood, highlighting a significant research gap. This study aims to comprehensively elucidate the impact of OA on the liver by examining the transcriptome, proteome, and phosphoproteome alterations in human HepaRG liver cells exposed to non-cytotoxic OA concentrations. We employed an integrative multi-omics approach, encompassing RNA sequencing, shotgun proteomics, phosphoproteomics, and targeted DigiWest analysis. This enabled a detailed exploration of gene and protein expression changes, alongside phosphorylation patterns under OA treatment. The study reveals concentration- and time-dependent deregulation in gene and protein expression, with a significant down-regulation of xenobiotic and lipid metabolism pathways. Up-regulated pathways include actin crosslink formation and a deregulation of apoptotic pathways. Notably, our results revealed that OA, as a potent phosphatase inhibitor, induces alterations in actin filament organization. Phosphoproteomics data highlighted the importance of phosphorylation in enzyme activity regulation, particularly affecting proteins involved in the regulation of the cytoskeleton. OA's inhibition of PP2A further leads to various downstream effects, including alterations in protein translation and energy metabolism. This research expands the understanding of OA's systemic impact, emphasizing its role in modulating the phosphorylation landscape, which influences crucial cellular processes. The results underscore OA's multifaceted effects on the liver, particularly through PP2A inhibition, impacting xenobiotic metabolism, cytoskeletal dynamics, and energy homeostasis. These insights enhance our comprehension of OA's biological significance and potential health risks.

How processing affects marker peptide quantification - A comprehensive estimation on bovine material relevant for food and feed control.

Processing food and feed challenges official control e.g. by modifying proteins, which leads to significant underestimation in targeted, MS-based protein quantification. Whereas numerous studies identified processing-induced changes on proteins in various combinations of matrices and processing conditions, studying their impact semi-quantitatively on specific protein sequences might unveil approaches to improve protein quantification accuracy. Thus, 335 post-translational modifications (e.g. oxidation, deamidation, carboxymethylation, Amadori, acrolein adduction) were identified by bottom-up proteomic analysis of 37 bovine materials relevant in food and feed (meat, bone, blood, milk) with varying processing degrees (raw, spray-dried, pressure-sterilized). To mimic protein recovery in a targeted analysis, peak areas of marker and reference peptides were compared to those of their modified versions, which revealed peptide-specific recoveries and variances across all samples. Detailed analysis suggests that incorporating two modified versions additionally to the unmodified marker may significantly improve quantification accuracy in targeted MS-based food and feed control in processed matrices.

Pregnane X receptor activation remodels glucose metabolism to promote NAFLD development in obese mice.

OBJECTIVE: Both obesity and exposure to chemicals may induce non-alcoholic fatty liver disease (NAFLD). Pregnane X Receptor (PXR) is a central target of metabolism disrupting chemicals and disturbs hepatic glucose and lipid metabolism. We hypothesized that the metabolic consequences of PXR activation may be modified by existing obesity and associated metabolic dysfunction. METHODS: Wildtype and PXR knockout male mice were fed high-fat diet to induce obesity and metabolic dysfunction. PXR was activated with pregnenolone-16α-carbonitrile. Glucose metabolism, hepatosteatosis, insulin signaling, glucose uptake, liver glycogen, plasma and liver metabolomics, and liver, white adipose tissue, and muscle transcriptomics were investigated. RESULTS: PXR activation aggravated obesity-induced liver steatosis by promoting lipogenesis and inhibiting fatty acid disposal. Accordingly, hepatic insulin sensitivity was impaired and circulating alanine aminotransferase level increased. Lipid synthesis was facilitated by increased liver glucose uptake and utilization of glycogen reserves resulting in dissociation of hepatosteatosis and hepatic insulin resistance from the systemic glucose tolerance and insulin sensitivity. Furthermore, glucagon-induced hepatic glucose production was impaired. PXR deficiency did not protect from the metabolic manifestations of obesity, but the liver transcriptomics and metabolomics profiling suggest diminished activation of inflammation and less prominent changes in the overall metabolite profile. CONCLUSIONS: Obesity and PXR activation by chemical exposure have a synergistic effect on NAFLD development. To support liver fat accumulation the PXR activation reorganizes glucose metabolism that seemingly improves systemic glucose metabolism. This implies that obese individuals, already predisposed to metabolic diseases, may be more susceptible to harmful metabolic effects of PXR-activating drugs and environmental chemicals.

Bioactivation of estragole and anethole leads to common adducts in DNA and hemoglobin.

Estragole and anethole are secondary metabolites occurring in a variety of commonly used herbs like fennel, basil, and anise. Estragole is genotoxic and carcinogenic in rodents, which depends on the formation of 1'-sulfoxyestragole after hydroxylation and subsequent sulfoconjugation catalyzed by CYP and SULT, respectively. It was hypothesized recently that anethole may be bioactivated via the same metabolic pathways. Incubating estragole with hepatic S9-fractions from rats and humans, specific adducts with hemoglobin (N-(isoestragole-3-yl)-valine, IES-Val) and DNA (isoestragole-2'-deoxyguanosine and isoestragole-2'-deoxyadenosine) were formed. An isotope-dilution technique was developed for the quantification of IES-Val after cleavage with fluorescein isothiocyanate (FITC) according to a modified Edman degradation. The same adducts, albeit at lower levels, were also detected in reactions with anethole, indicating the formation of 3'-hydroxyanethole and the reactive 3'-sulfoxyanethole. Finally, we conducted a pilot investigation in which IES-Val levels in human blood were determined during and after the consumption of an estragole- and anethole-rich fennel tea for four weeks. A significant increase of IES-Val levels was observed during the consumption phase and followed by a continuous decrease during the washout period. IES-Val may be used to monitor the internal exposure to the common reactive genotoxic metabolites of estragole and anethole, 1'-sulfoxyestragole and 3'-sulfoxyanethole, respectively.

Probing glycation potential of dietary sugars in human blood by an integrated in vitro approach.

Glycation is referred to as the interaction of protein amino and guanidino groups with reducing sugars and carbonyl products of their degradation. Resulting advanced glycation end-products (AGEs) contribute to pathogenesis of diabetes mellitus and neurodegenerative disorders. Upon their intestinal absorption, dietary sugars and α-dicarbonyl compounds interact with blood proteins yielding AGEs. Although the differences in glycation potential of monosaccharides are well characterized, the underlying mechanisms are poorly understood. To address this question, d-glucose, d-fructose and l-ascorbic acid were incubated with human serum albumin (HSA). The sugars and α-dicarbonyl intermediates of their degradation were analyzed in parallel to protein glycation patterns (exemplified with hydroimidazolone modifications of arginine residues and products of their hydrolysis) by bottom-up proteomics and computational chemistry. Glycation of HSA with sugars revealed 9 glyoxal- and 14 methylglyoxal-derived modification sites. Their dynamics was sugar-specific and depended on concentrations of α-dicarbonyls, their formation kinetics, and presence of stabilizing residues in close proximity to the glycation sites.