Increased Oxidative Damage in Lymphoblasts from Autism: Enhanced Free Radical Generation Coupled with Reduced Antioxidant Status

Background: Recent evidence suggests that autistic subjects are under oxidative stress. Most of these studies were done with serum, plasma or erythrocytes. The studies with cell cultures are lacking in autism. Under normal conditions, a dynamic equilibrium exists between the production of free radicals (reactive oxygen species - ROS) and the anti-oxidant capacity of the cell. The free radicals are highly reactive, and their elevated levels lead to oxidative stress and cell death. Antioxidants enzymes scavenge the free radicals and play vital role in the intracellular defense mechanism against oxidative damage.

Objectives: The aim was to compare the status of oxidative stress and antioxidant potential in lymphoblasts from autistic subjects (n=11) and control subjects (n=11) by analyzing lipid peroxidation, generation of free radicals (ROS), extent of membrane damage and the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR).

Methods: The lymphoblasts from autistic and control subjects were obtained from Autism Genetic Resources Exchange Program, and the cell lysates were prepared. Lipid peroxidation was assessed by measuring malonyldialdehyde, an end product of fatty acid oxidation. ROS levels (basal and upon induction by Fenton reaction) were determined by using dichlorofluorescin-diacetate (DCFH-DA) as a fluorescent  probe. Damage of the plasma membrane was evaluated by measuring the amount of intracellular lactate dehydrogenase (LDH) that was released into the conditioned medium. SOD activity was measured by using assay kit from Calbiochem. CAT activity was measured spectrophotometrically by analyzing complex of ammonium molybdate with H2O2.  Measurement of GPx activity was based on the oxidation of GSH to oxidized glutathione (GSSG) catalyzed by GPx. Activity of GR was measured by the method based on the reduction of GSSH by NADPH.

Results: Lipid peroxidation and ROS levels were significantly increased in lymphoblasts from autistic subjects as compared with control lymphoblasts. The release of LDH, a marker of membrane damage, into the medium was also significantly increased in lymphoblasts from autistic subjects as compared with controls, suggesting that membrane integrity is affected in autism. The activities of SOD, CAT and GR, which scavenge free radicals and participate in antioxidant defense were significantly reduced in lymphoblasts from autistic subjects as compared with controls, suggesting that intracellular defense mechanisms against oxidative damage is impaired in autism.  A trend towards decrease in the activity of GPx was also observed, but it was not significant.

Conclusions: Our results indicate that autism is associated with increased  ROS generation along with a reduction in the activities of antioxidant enzymes, which may lead to increased oxidative damage and membrane damage in autism.