Cellular and mitochondrial glutathione redox imbalance in lymphoblastoid cells derived from children with autism

Abstract

Research into the metabolic phenotype of autism has been relatively unexplored despite the fact that metabolic abnormalities have been implicated in the pathophysiology of several other neurobehavioral disorders. Plasma biomarkers of oxidative stress have been reported in autistic children; however, intracellular redox status has not yet been evaluated. Lymphoblastoid cells (LCLs) derived from autistic children and unaffected controls were used to assess relative concentrations of reduced glutathione (GSH) and oxidized disulfide glutathione (GSSG) in cell extracts and isolated mitochondria as a measure of intracellular redox capacity. The results indicated that the GSH/GSSG redox ratio was decreased and percentage oxidized glutathione increased in both cytosol and mitochondria in the autism LCLs. Exposure to oxidative stress via the sulfhydryl reagent thimerosal resulted in a greater decrease in the GSH/GSSG ratio and increase in free radical generation in autism compared to control cells. Acute exposure to physiological levels of nitric oxide decreased mitochondrial membrane potential to a greater extent in the autism LCLs, although GSH/GSSG and ATP concentrations were similarly decreased in both cell lines. These results suggest that the autism LCLs exhibit a reduced glutathione reserve capacity in both cytosol and mitochondria that may compromise antioxidant defense and detoxification capacity under prooxidant conditions.

Figure 1.

Relative rate of intracellular free radical production in autistic and control cell lines is presented as mean ± sd DCF fluorescence measured in 6 different cell lines/group before (baseline) and after the addition of increasing doses of thimerosal.

Figure 2.

Baseline concentrations without the addition of thimerosal and after a 3-h exposure to increasing concetrations of 0.15–2.5 μM thimerosal. A) Intracellular free glutathione. B) Intracellular GSSG. C) Extracellular (medium) GSSG. D) Intracellular GSH/GSSG redox ratio. Inset: GSH/GSSG ratio after 24 h exposure to 10–160 nM concentrations of thimerosal.

Figure 3.

Percentage decrease in JC-1 red/green fluorescence is proportional to the degree of mitochondrial membrane depolarization. A) Mean ± sd shift in percentage JC-1 red/green fluorescence after 30 min exposure to nitric oxide generated by 0.5 mM SNAP (6/group). B) Percentage decrease in JC-1 red/green fluorescence in two autism and control cell pairs at baseline and after SNAP exposure as measured by flow cytometry.

Figure 4.

Intracellular ATP concentration (nmol/10⁶ cells) measured after 30-min exposure to PBS or nitric oxide generated by 1.0 mM SNAP.