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

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”.

Keywords: Epigenetics, Gluten free casein free diet, Autism, Glutathione, Gastrointestinal, Inflammation