BACKGROUND: Faba bean (Vicia faba) vicine and convicine (V-C) aglycones (divicine and isouramil respectively) provoke an acute hemolytic anemia called favism in individuals with a glucose-6-phosphate dehydrogenase (G6PD) enzyme defect in their red blood cells. Geneticists/plant breeders are working with faba bean to decrease V-C levels to improve public acceptance of this high-protein pulse crop. Here, we present a fast and simple ex vivo in vitro bioassay for V-C toxicity testing of faba bean or faba bean food products. RESULTS: We have shown that 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU)-treated (i.e., sensitized) normal red blood cells, like G6PD-defective blood, displayed (i) continuous glutathione (GSH) depletion with no regeneration as incubation time and the dose of aglycones increased, (ii) progressive accumulation of denatured hemoglobin products into high molecular weight (HMW) proteins with increased aglycone dose, (iii) both band 3 membrane proteins and hemichromes, in HMW protein aggregates. We have also demonstrated that sensitized red blood cells can effectively differentiate various levels of toxicity among faba bean varieties through the two hemolysis biomarkers: GSH depletion and HMW clumping. CONCLUSION: BCNU-sensitized red blood cells provide an ideal model for favism blood, to assess and compare the toxicity of faba bean varieties and their food products. © 2018 Society of Chemical Industry.
Biological Assay/methods , Glucosides/analysis , Pyrimidinones/analysis , Uridine/analogs & derivatives , Vicia faba/chemistry , Erythrocytes/chemistry , Erythrocytes/drug effects , Erythrocytes/enzymology , Favism/blood , Favism/enzymology , Glucosephosphate Dehydrogenase/chemistry , Glucosides/toxicity , Hemolysis/drug effects , Humans , Pyrimidinones/toxicity , Uridine/analysis , Uridine/toxicity , Vicia faba/toxicity
Favism is an acute hemolytic syndrome caused by the ingestion of fava bean (FB) in glucose 6-phosphate dehydrogenase (G6PD) deficient individuals. However, little is known about the global transcripts alteration in liver tissue after FB ingestion in G6PD-normal and -deficient states. In this study, deep sequencing was used to analyze liver genes expression alterations underlying the effects of FB in C3H (Wild Type, WT) and G6PD-deficient (G6PDx) mice and to evaluate and visualize the collective annotation of a list of genes to Gene Ontology (GO) terms associated with favism. Our results showed that FB resulted in a decrease of glutathione (GSH)-to-oxidized glutathione (GSSG) ratio and an increase of malondialdehyde (MDA) both in the G6PDx and WT-control check (CK) mice plasma. Significantly, liver transcript differences were observed between the control and FB-treated groups of both WT and G6PDx mice. A total of 320 differentially expressed transcripts were identified by comparison of G6PDx-CK with WT-CK and were associated with immune response and oxidation-reduction function. A total of 149 differentially expressed genes were identified by comparison of WT-FB with WT-CK. These genes were associated with immune response, steroid metabolic process, creatine kinase activity, and fatty acid metabolic process. A total of 438 differential genes were identified by comparing G6PDx-FB with G6PD-CK, associated with the negative regulation of fatty acid metabolic process, endoplasmic reticulum, iron binding, and glutathione transferase activity. These findings indicate that G6PD mutations may affect the functional categories such as immune response and oxidation-reduction.
Favism/genetics , Glucosephosphate Dehydrogenase Deficiency/genetics , Liver/drug effects , Transcriptome , Vicia faba/toxicity , Animals , Favism/complications , Favism/immunology , Favism/pathology , Gene Expression Profiling , Gene Expression Regulation , Gene Ontology , Glucosephosphate Dehydrogenase Deficiency/complications , Glucosephosphate Dehydrogenase Deficiency/immunology , Glucosephosphate Dehydrogenase Deficiency/pathology , Glutathione/blood , High-Throughput Nucleotide Sequencing , Immunity, Innate , Liver/metabolism , Male , Malondialdehyde/blood , Mice , Mice, Inbred C3H , Mice, Knockout , Molecular Sequence Annotation , Oxidation-Reduction , Oxidative Stress/drug effects , Plant Extracts/toxicity , Vicia faba/chemistry
In spite of its positive repercussions on nutrition and environment, faba bean still remains an underutilized crop due to the presence of some undesired compounds. The pyrimidine glycosides vicine and convicine are precursors of the aglycones divicine and isouramil, the main factors of favism, a genetic condition which may lead to severe hemolysis after faba bean ingestion. The reduction of vicine and convicine has been targeted in several studies but little is known about their degradation. In this study, the hydrolysis kinetics of vicine and convicine and their derivatives during fermentation with L. plantarum DPPMAB24W was investigated. In particular, a specific HPLC method coupled to ESI-MS and MS/MS analysis, including the evaluation procedure of the results, was set up as the analytical approach to monitor the compounds. The degradation of the pyrimidine glycosides in the fermented flour was complete after 48 h of incubation and the aglycone derivatives could not be detected in any of the samples. The toxicity of the fermented faba bean was established through ex-vivo assays on human blood, confirming the experimental findings. Results indicate that mild and cost effective bioprocessing techniques can be applied to detoxify faba bean also for industrial applications.
Barbiturates/metabolism , Flour/analysis , Glucosides/metabolism , Lactobacillus plantarum/metabolism , Pyrimidinones/metabolism , Uridine/analogs & derivatives , Vicia faba/metabolism , Barbiturates/toxicity , Biotransformation , Fermentation , Food Technology/methods , Glucosides/toxicity , Humans , Hydrogen-Ion Concentration , Hydrolysis , Pyrimidinones/toxicity , Spectrometry, Mass, Electrospray Ionization , Uridine/metabolism , Uridine/toxicity , Vicia faba/toxicity
