Matthews, Z. | Search bioRxiv

bioRxiv2018

The in vitro toxicity of nitrile and epithionitrile derivatives of glucosinolates from rutabaga in human and bovine liver cells.

Previous evidence suggests that select nitrile and epithionitrile derivatives of glucosinolates can cause liver disease in cows grazing on brassica forage crops. A toxic incidence in New Zealand in cattle grazing brassica led us to investigate the direct in vitro hepatotoxicity and possible inhibition of the ABCG2 transporter of five nitrile compounds. In this study, we investigated 1-cyano-2-hydroxy-3-butene (CHB, epithionitrile derivative of progoitrin), 1-cyano-2-hydroxy-3,4-epithiobutane (CHEB, nitrile derivative of progoitrin), 3-butenenitrile (nitrile from sinigrin), 4-pentenenitrile (nitrile from gluconapin), and 5-hexenenitrile (nitrile from glucobrassicanapin). Cell viability was assessed following 24- and 72-hr treatments with the 5 different compounds using the MTT assay (HepG2 cells and bovine primary liver cells). Additionally, ABCG2 transporter function was assessed. The results showed that none of the tested compounds caused cytotoxicity at concentrations up to 2 mM for 24hr. Over 72-hr the maximum concentration was 20 M but no reduction in cell viability was observed. No inhibition of the ABCG2 transporter occured at concentrations up to 1 mM. Overall this study suggests that direct or secondary toxicity due to selected nitrile or epithionitrile derivatives of these glucosinolates was not the cause of the toxic event in cattle.

pharmacology and toxicology↗

bioRxiv2018

Integrative analysis of Paneth cell proteomic data from intestinal organoids reveals functional processes affected in Crohn’s disease due to autophagy impairment

Summary statementUsing an integrative approach encompassing intestinal organoid culture, proteomics, transcriptomics and protein-protein interaction networks, we list Paneth cell functions dependent on autophagy.\n\nAbstractPaneth cells are key epithelial cells providing an antimicrobial barrier and maintaining integrity of the small intestinal stem cell niche. Paneth cell abnormalities are unfortunately detrimental to gut health and often associated with digestive pathologies such as Crohns disease or infections. Similar alterations are observed in individuals with impaired autophagy, a process which recycles cellular components. The direct effect of autophagy-impairment on Paneth cells has not been analysed. To investigate this, we generated a mouse model lacking Atg16l1 specifically in intestinal epithelial cells making these cells impaired in autophagy. Using 3D intestinal organoids enriched for Paneth cells, we compared the proteomic profiles of wild-type (WT) and autophagy-impaired organoids. We used an integrated computational approach combining protein-protein interaction networks, autophagy targeted proteins and functional information to identify the mechanistic link between autophagy-impairment and disrupted pathways. Of the 284 altered proteins, 198 (70%) were more abundant in autophagy-impaired organoids, suggesting reduced protein degradation. Interestingly, these differentially abundant proteins comprised 116 proteins (41%), predicted targets of the selective autophagy proteins p62, LC3 and ATG16L1. Our integrative analysis revealed autophagy-mediated mechanisms degrading proteins key to Paneth cell functions, such as exocytosis, apoptosis and DNA damage repair. Transcriptomic profiling of additional organoids confirmed that 90% of the observed changes upon autophagy alteration affect protein level and not gene expression. We performed further validation experiments showing differential lysozyme secretion, confirming our computationally inferred down-regulation of exocytosis. Our observations could explain how protein level alterations affect Paneth cell homeostatic functions upon autophagy impairment.

systems biology↗