title: Epigenetic effects of casein-derived opioid peptides in SH-SY5Y human neuroblastoma cells.
abstract: BACKGROUND
  Casein-free, gluten-free diets have been reported to mitigate some of the inflammatory gastrointestinal and behavioral traits associated with autism, but the mechanism for this palliative effect has not been elucidated. We recently showed that the opioid peptide beta-casomorphin-7, derived from bovine (bBCM7) milk, decreases cysteine uptake, lowers levels of the antioxidant glutathione (GSH) and decreases the methyl donor S-adenosylmethionine (SAM) in both Caco-2 human GI epithelial cells and SH-SY5Y human neuroblastoma cells. While human breast milk can also release a similar peptide (hBCM-7), the bBCM7 and hBCM-7 vary greatly in potency; as the bBCM-7 is highly potent and similar to morphine in it's effects. Since SAM is required for DNA methylation, we wanted to further investigate the epigenetic effects of these food-derived opioid peptides. In the current study the main objective was to characterize functional pathways and key genes responding to DNA methylation effects of food-derived opioid peptides.
  
  
  METHODS
  SH-SY5Y neuroblastoma cells were treated with 1 μM hBCM7 and bBCM7 and RNA and DNA were isolated after 4 h with or without treatment. Transcriptional changes were assessed using a microarray approach and CpG methylation status was analyzed at 450,000 CpG sites. Functional implications from both endpoints were evaluated via Ingenuity Pathway Analysis 4.0 and KEGG pathway analysis was performed to identify biological interactions between transcripts that were significantly altered at DNA methylation or transcriptional levels (p < 0.05, FDR <0.1).
  
  
  RESULTS
  Here we show that hBCM7 and bBCM7, as well as morphine, cause epigenetic changes affecting gene pathways related to gastrointestinal disease and inflammation. These epigenetic consequences exhibited the same potency order as opiate inhibition of cysteine uptake insofar as hBCM7 was less potent than bBCM7, which was less potent than morphine.
  
  
  CONCLUSIONS
  Our findings indicate that epigenetic effects of milk-derived opiate peptides may contribute to GI dysfunction and inflammation in sensitive individuals. While the current study was performed using SH-SY5Y neuronal cellular models, similar actions on other cells types might combine to cause symptoms of intolerance. These actions may provide a potential contributing mechanism for the beneficial effects of a casein-free diet in alleviating gastrointestinal symptoms in neurological conditions including autism and other conditions. Lastly, our study also contributes to the evolving awareness of a "gut-brain connection".
authors.affiliation: Department of Pharmaceutical Sciences, Nova Southeastern University, Rm # 3103, HPD building, Fort Lauderdale, FL USA.
authors.forename: Malav S
authors.initials: MS
authors.lastname: Trivedi
authors.affiliation: Department of Molecular and Cellular Biology, Harvard Medical School, Boston, MA USA.
authors.forename: Nathaniel W
authors.initials: NW
authors.lastname: Hodgson
authors.affiliation: Wake Forest Institute for Regenerative Medicine, Wake Forest University Health Sciences, Winston Salem, NC USA.
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Walker
authors.affiliation: Department of Mathematical Modelling, Statistics and Bioinformatics, University of Ghent, Ghent, Belgium.
authors.forename: Geert
authors.initials: G
authors.lastname: Trooskens
authors.affiliation: Department of Pharmaceutical Sciences, Nova Southeastern University, Rm # 3103, HPD building, Fort Lauderdale, FL USA.
authors.forename: Vineeth
authors.initials: V
authors.lastname: Nair
authors.affiliation: Department of Pharmaceutical Sciences, Nova Southeastern University, Rm # 3103, HPD building, Fort Lauderdale, FL USA.
authors.forename: Richard C
authors.initials: RC
authors.lastname: Deth
keywords: Autism; Epigenetics; Gastrointestinal; Glutathione; Gluten free casein free diet; Inflammation