Got milk [opioid peptides]?

"Bovine and human casein-derived opioid peptides increased genome-wide DNA methylation in the transcription start site region with a potency order similar to their inhibition of cysteine uptake".

Those were the findings from the paper by Malav Trivedi and colleagues [1] (open-access) including Richard Deth on the authorship team. I was really interested to read this paper having met Malav and Dick a few months back and listened very attentively to some potentially important investigations on-going in their lab. Indeed, such results potentially cast a whole new light on some of my own autism research interests looking at the use of a gluten- and casein-free (GFCF) diet (see here) and more latterly, the reformulation of a great opioid antagonist naltrexone, into a handy cream (see here).

Call me a liar? @ Wikipedia 

A few starting details might be useful:

• Without hopefully just regurgitating what the authors said, the focus of this study was to look at whether "morphine and food-derived opioid-peptides can induce alterations in the cellular redox status, DNA methylation... and transcription process, by altering cysteine uptake in SH-SY5Y cells". As far as I see it, there are 3 important elements to explain, all with an autism slant...
• Opioid peptides - short chains of amino acids with opiate-like activity - have been the topic of quite a bit of discussion when it comes to autism. No-one really disputes the fact that when we eat foods containing gluten and casein, the respective proteins are broken down into peptides, some of which have an affinity to our own opioid (morphine) receptors. The controversy when it comes to autism is the suggestion that said opioid peptides might be part and parcel of the effect noted when some people with autism embark on a diet devoid of gluten and casein - the GFCF diet - and might be detectable in certain biofluids (see here). 
• Redox and oxidative stress also have some research form when it comes to autism (see here) and in particular the quite consistent literature on the antioxidant glutathione being perturbed in quite a few cases of autism (see here). No-one really knows why levels of glutathione (GSH) are coming out so consistently low in relation to autism but one suggestion is that a transporter called EAAT3 involved in the uptake of cysteine, an important building block of glutathione, might be misbehaving. This authorship team together with a few other notable names have talked about this process in relation to autism [2] previously which was the topic of a past post on this blog (see here). 
• DNA methylation is also something talked about quite a bit on this blog. Part of that rising star discipline known as epigenetics, the idea is that adding a methyl group to specific sites on DNA has the ability to modify the expression of certain genes. Hypermethylation is generally taken to mean gene silencing.

And then to the paper itself bearing in mind it is open-access:

• This was a study carried out using "SH-SY5Y and Caco2 cancer cell lines". Said cell lines were subjected to the addition of morphine, bovine [cow] beta-casomorphin 1-7, human beta-casomorphin 1-7 or gliadinomorphin 7 at varying doses. The casomorphins are derived from casein, the milk protein and the gliadinomorphin from the gluten protein.
• Radio-labelled cysteine uptake was monitored in cells and was found to be inhibited by the addition of morphine and the various food-derived opioid peptides. Morphine was most effective at inhibiting cysteine uptake followed by bovine beta-casomorphin 1-7 then human beta-casomorphin 1-7 and finally the opioid peptide derived from gliadin. Interestingly, this effect was "blocked by naltrexone".
• Levels of "metabolic intermediaries involved in transsulfuration and methionine methylation cycle pathways" were also examined. This included looking directly at levels of cysteine and GSH alongside other important compounds in this pathway such as methionine and homocysteine (the big H). Again, changes were noted to these compounds as a function of the addition of morphine and opioid peptides: "consistent with decreased GSH synthesis, decreased activity of methionine synthase, and increased transsulfuration of homocysteine to cystathione". Methionine synthase is again, something already talked about by this authorship group (see here).
• Global DNA methylation status was also examined as a function of morphine and opioid peptide addition to the SH-SY5Y cell line. "A 4 h[our] treatment with morphine or milk-derived opioid peptides... caused a significant shift towards increased methylation (i.e. promoter hypermethylation) in the immediate TSS [transcription start site] region". Morphine was once more most effective at increasing genome-wide methylation followed by bovine and then human beta-casomorphin. Ergo, "opioid-induced changes in cysteine uptake, redox status... are associated with significant genome-wide changes in DNA methylation levels".

I find these results to be absolutely fascinating. Of course, as the authors point out, this was a study of cell lines and hence "need to be replicated in other systems before they can be confidently extrapolated to clinical manifestations". But the possibility that elements of food may have such profound knock-on effects pertinent to the way we 'detox' (yes, it is a real concept) and the expression of some of our genes has endless connotations. I might add that the reported effects of human beta-casomorphin for example, on the processes described may also indicate that not all effects are going to be adverse from an evolutionary perspective.

From the point of view of the use of a GFCF diet in cases of autism, this work potentially adds another string to the various explanations as to why diet may be useful for some on the spectrum. So, outside of other ideas on effect including: lactose intolerance (see here), coeliac disease (see here), non-coeliac gluten sensitivity (see here), altering intestinal hyperpermeability (see here), folate receptor autoantibodies (see here), FPIES (see here), et al, linking gluten and/or casein opioid peptides to something like glutathione production or methylation capacity provides even more research fodder for any interested parties.

Finally(!) there is one small additional point to mention about the Trivedi paper. Tucked in at the end of a sentence in the discussion section are the words: "It should be noted that bovine form of BCM7 [beta-casomorphin 1-7] is only released from cows with the A1 genotype and not A2 genotype cows". I've kinda alluded to the concept that not all mammalian forms of casein are the same in a previous post talking about camel milk and autism (see here). As bizarre as it might sound, there is a growing appreciation that different animals (including humans) might not necessarily produce the same kind of milk [3] and that some types of milk might be advantageous over others. The paper by Barnett and colleagues [4] hinted at one effect - gastrointestinal transit time - when comparing A1 and A2 milks. At the moment however, the science is still a little hazy for suggesting that the differences between the various types of milk might make some more useful for some groups/individuals over others. But certainly there is a potentially intriguing hypothesis requiring some experimental testing there...

Music to close. Paolo Nutini and Pencil Full of Lead.

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[1] Trivedi MS. et al. Food-derived opioid peptides inhibit cysteine uptake with redox and epigenetic consequences. Journal of Nutritional Biochemistry. 2014. June 9.

[2] Waly MI. et al. Prenatal and Postnatal Epigenetic Programming: Implications for GI, Immune, and Neuronal Function in Autism. Autism Res Treat. 2012;2012:190930.

[3] Kamiński S. et al. Polymorphism of bovine beta-casein and its potential effect on human health. J Appl Genet. 2007;48(3):189-98.

[4] Barnett MP. et al. Dietary A1 β-casein affects gastrointestinal transit time, dipeptidyl peptidase-4 activity, and inflammatory status relative to A2 β-casein in Wistar rats. Int J Food Sci Nutr. 2014 Mar 20.

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Trivedi, M., Shah, J., Al-Mughairy, S., Hodgson, N., Simms, B., Trooskens, G., Van Criekinge, W., & Deth, R. (2014). Food-derived opioid peptides inhibit cysteine uptake with redox and epigenetic consequences The Journal of Nutritional Biochemistry DOI: 10.1016/j.jnutbio.2014.05.004