Parents, support groups and several clinical studies report improvement in behavioral symptoms when autistic children are treated with a gluten-free/casein-free dietary intervention. Opiod derivatives of these food products, namely β-casomorphin (β-CM) and gliadinomorphin (GM) are absorbed from a “leaky gut” and may activate opiate receptors in brain. Morphine has been linked with oxidative imbalances during the development of addiction, and oxidative stress is believed to be a significant etiological factor for autism. We therefore hypothesized that opiates and food-derived opiate peptides might promote oxidative stress in neurons, leading to exacerbated behavioral symptoms.
Objectives:
1. To observe the acute and chronic effects of morphine and food-derived opiate peptides, as well as the opiate antagonist naltrexone, on cysteine uptake in cultured human neuronal cells.
2. To measure the effect of these drugs on cellular levels of sulfur-containing metabolites, including glutathione (GSH), a major antioxidant.
Methods:
Cellular [35S]-Cysteine Uptake:
SH-SY5Y neuroblastoma cells were grown to confluence in six-well plates in α-MEM media containing 10% FBS and 1% penicillin-streptomycin. Cells were pre-treated with drugs for the specified time and [35S]-cysteine uptake was measured. Non-transported [35S]-cysteine was subtracted from total CPM for each sample and values were normalized to protein content.
HPLC Determination of Intracellular Thiols:
SH-SY5Y cells were pre-treated with drugs prior to addition of ice-cold perchloric acid. After sonication, centrifugation and filtration, the protein-free cell extract was analyzed via HPLC with electrochemical detection to measure thiol levels. Results were normalised to protein content.
Results:
Acute treatment with morphine decreased [35S]-cysteine uptake by 70% (p<0.001), while β-CM and GM decreased uptake by 40% (p<0.05). Naltrexone blocked both of these effects, indicating involvement of opiate receptors. Long-term treatment with each of the opiates caused a complex pattern of inhibition and recovery of [35S]-cysteine uptake activity, ultimately resulting in a sustained inhibition at 24 and 48 hrs.
β-CM and GM acutely lowered the cellular glutathione level (52% and 62%, respectively), while the level of homocysteine was acutely increased (50% and 44%, respectively). Glutathione levels recovered during the ensuing 8 hrs, although homocysteine levels decreased. Both glutathione and homocysteine levels were reduced at 24 and 48 hrs.
Together these results indicate that opiates acutely inhibit cysteine uptake in neuronal cells, resulting in an increase of homocysteine transsulfuration to cysteine and glutathione. However, this pathway is gradually exhausted, leading to decreased levels of intracellular levels of thiol metabolites, including glutathione. The long-term decrease in glutathione can contribute to oxidative stress.
Conclusions:
This is the first study to demonstrate inhibitory effects of food-derived opiod peptides on redox status and provides mechanistic support for the “Gut-Brain Hypothesis”. It reveals a rationale for the beneficial effect of a GF/CF dietary intervention in the treatment of autistic children, and may have general relevance for inflammatory bowel disorders in which gluten and/or casein intolerance plays a role.