Epigenetic effects of casein-derived opioid peptides in SH-SY5Y human neuroblastoma cells

Malav S. Trivedi, Nathaniel W. Hodgson, Stephen J. Walker, Geert Trooskens, Vineeth Nair, Richard C. Deth

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. 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). 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. 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".