El-Ansary et al. Lipids in Health and Disease 2011, 10:142 http://www.lipidworld.com/content/10/1/142 RESEARCH Open Access On the protective effect of omega-3 against propionic acid-induced neurotoxicity in rat pups Afaf K El-Ansary*, Sooad K Al-Daihan and Amina R El-Gezeery Abstract Backgrounds: The investigation of the environmental contribution for developmental neurotoxicity is very important. Many environmental chemical exposures are now thought to contribute to the development of neurological disorders, especially in children. Results from animal studies may guide investigations of human populations toward identifying environmental contaminants and drugs that produce or protect from neurotoxicity and may help in the treatment of neurodevelopmental disorders. Objective: To study the protective effects of omega-3 polyunsaturated fatty acid on brain intoxication induced by propionic acid (PPA) in rats. Methods: 24 young male Western Albino rats were enrolled in the present study. They were grouped into three equal groups; oral buffered PPA-treated group given a nuerotoxic dose of 250 mg/Kg body weight/day for 3 days; omega-3 - protected group given a dose of 100 mg/kg body weight/day omega-3 orally daily for 5 days followed by PPA for 3 days, and a third group as control given only phosphate buffered saline. Tumor necrosis factor-a, caspase-3, interlukin-6, gamma amino-buteric acid (GABA), serotonin, dopamine and phospholipids were then assayed in the rats brain’s tissue of different groups. Results: The obtained data showed that PPA caused multiple signs of brain toxicity as measured by depletion of gamaaminobyteric acid (GABA), serotonin (5HT) and dopamine (DA) as three important neurotransmitters that reflect brain function. A high significant increase of interlukin-6 (Il-6), tumor necrosis factor-a (TNF-a) as excellent markers of proinflammation and caspase-3 as a proapotic marker were remarkably elevated in the intoxicated group of rats. Moreover, brain phospholipid profile was impaired in PPA-treated young rats recording lower levels of phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylcholine (PC). Conclusions: Omega-3 fatty acids showed a protective effects on PPA - induced changes in rats as there was a remarkable amelioration of most of the measured parameters (i.e. higher GABA, 5HT, DA, PE, PS and PC) and lower Il-6, TNF-a and caspase-3. Keywords: Propionic acid, Omega-3, Neurotransmitters, Caspase-3, Cytokines, Phospholipids, Autism Introduction Environmental chemical exposures are now thought to There are several reasons to study and test environmen- contribute to the increases in attention deficit hyperac- tal exposures for developmental neurotoxicity: [1] Five tivity disorder, cognitive problems, and autism spectrum thousand new chemicals are produced each year, and disorders that have been documented in the last 5-10 according to estimates by the Environmental Protection years [4]. Results from animal studies could be helpful Agency, about 25% of them may be neurotoxic, but only in identifying environmental toxins that related to neu- 10% of them will ever be tested for neurotoxicity [2]. rodevelopmental disorders as autism [1]. The developing brain is highly vulnerable and more sus- Neurotransmitters play unique trophic roles in brain ceptible to neurotoxic insults than is the adult brain [3]. development. Accordingly, drugs and environmental toxicants that promote or interfere with neurotransmit- * Correspondence: [email protected] ter function evoke neurodevelopmental abnormalities by Biochemistry Department, Science College, King Saud University, P.O box disrupting the intensity of neuro trophic actions [2]. 22452, zip code 11495, Riydh, Saudi Arabia © 2011 El-Ansary et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. El-Ansary et al. Lipids in Health and Disease 2011, 10:142 Page 2 of 10 http://www.lipidworld.com/content/10/1/142 The brain phospholipids are exceptionally rich in This information initiates our interest to study the highly unsaturated fatty acids (HUFAs) [3,4] and in con- neuroprotective effects of omega-3 on PPA-induced trast to other bodily tissue, a unique feature of neurons neurotoxicity through the measurement of neurotrans- is the smaller amounts of the precursors LA(18:2, n-6) mitters, cytokines, caspase-3 and phospholipids in con- and ALA(18:3, n-3) and the higher amounts of their trol, PPA-treated and omega-3-protected-PPA metabolites: AA(20:4, n-6) and DHA(22:6, n-3) [3-5]. intoxicated rat pups. Propionic acid (PPA) is a weak organic acid that can cross the lipid bilayer of neuronal membranes and cause Materials and methods mild, reversible intracellular acidification, which can Animals produce wide spread effects on neurotransmitter release A total of 24 young male rats weighed about 45-60 involving glutamate, dopamine, and serotonin, each of (approximately 21 days old) were used in the present which can influence locomotion and other behaviors study. Rats were randomly allocated to the following [6-8]. groups. Oral buffered PPA-treated group given a nuero- Dependant mechanism may be important for effects of toxic dose of 250 mg/Kg body weight/day for 3 days as, PPAonbehaviororcognition.Inaddition,PPAis n = 8 [23] (omega-3 - protected group given a dose of thought to affect mitochondrial fatty acid metabolism by 100 mg/kg body weight/day omega-3 orally daily for 5 binding to propionyl coenzyme A and by sequestering days followed by PPA for 3 days, n = 8. A third group carnitine [[9,10], El-Ansary A, Nounou H: On the effec- of 8 rats were used as control given only phosphate buf- tiveness of creatine and cyclosporine to protect against fered saline. All groups were kept at controlled tempera- propionic acid - induced neurotoxicity, submitted). Aut- ture (21 ± 1°C) with ad libtium access to food and ism has been suggested to be a mitochondrial disorder water. All experiments were performed in accordance of impaired fatty acid metabolism [11,12]. Recently, with national animal care guidelines and were pre- Macfabe et al (2011) [13] reported that rats treated with approved by the faculty ethics committee, King Saud PPA displayed some autistic features as restricted beha- University. vioral interest to a specific object among a group of objects, impaired social behavior, and impaired reversal Tissue preparation in a T-maze task compared to controls given phosphate At the end of experiment, rats were anesthetized with buffered saline. Moreover the reactive astrogliosis and carbon dioxide. The brain was removed from the skull activated microglia, indicating an innate neuroinflamma- and was cut into small pieces. Brain tissues of the three tory response in PPA-treated rats. groups of rats were homogenized in 10 times w/v bidis- Despite great efforts are directed towards the develop- tilled water and were kept at -80°C until use. ment of effective treatment for autism, only few strate- gies have been offered to autistics thus far. The Biochemical analyses identification of an environmental risk factor related to - Assay of tumor necrosis factor-a autism could help to develop a neuroprotective strategy Tumor necrosis factor -a was measured using ELISA which could delay the onset of the disease. Several stu- kit, a product of Hycult Biotech. The antibody reacts dies now recognize the central role of omega-3 polyun- with rat natural TNF -a and recognizes both membrane saturated fatty acids (n-3 PUFAs) in brain health. and receptor bound. The TNF-a trimer interacts with Docosahexaenoic acid (DHA; 22:6n-3) is the most com- either of the two types of TNF-R leading to receptor mon n-3 PUFA within phospholipids of brain cell mem- cross-linking. One unit of Hycult Biotech Mouse Tumor branes, while eicosapentaenoic acid (EPA; 20:5n-3) is Necrosis Factor -a approximates the bioactivity of 16 usually found in very limited amounts in the central units of the human Tumor Necrosis factor -a standard nervous system (CNS) [14,15]. The main dietary sources prepared by WHO as determined in L929 cytotoxic of long chain PUFA, namely EPA and DHA, remain assay. fatty fish consumption. Besides their critical role in cell - Assay of Caspase-3 membrane fluidity, PUFAs can be released from the Caspase-3 was measured using an ELISA kit, a product hydrophobic moiety of phospholipids via the enzyme of Cusabio. The microtiter plate provided in this kit has phospholipase A2 (PLA2) from which they can be soli- been pre-coated with an antibody specific to Casp-3. cited in several intracellular events [16,17]. Although Standards or samples are then added to the appropriate specific mechanisms of action through which n-3 microtiter plate wells with a biotin-conjugated antibody PUFAs act remain unknown, some pathways have been preparation specific for Casp-3 and Avidin conjugated suggested and studied in vitro as well as in vivo. These to Horseradish Peroxidase (HRP) is added to each include anti-inflammatory [18,19], anti-oxidant microplate well and incubated. Then a TMB (3,3’,5,5’ [18,20,21] and anti-apoptotic properties [22]. tetramethyl-benzidine) substrate solution is added to El-Ansary et al. Lipids in Health and Disease 2011, 10:142 Page 3 of 10 http://www.lipidworld.com/content/10/1/142 each well. Only those wells that contain Casp-3, biotin- N-acylserotonin) is part of the sample dilution and is conjugated antibody and enzyme-conjugated Avidin will achieved by incubation of the respective sample with the exhibit a change in color. The enzyme-substrate reaction Acylation Reagent. The assay procedure follows the is terminated by the addition of a sulphuric acid solu- basic principle of competitive ELISA whereby there is tion and the color change is measured spectrophotome- competition between a biotinylated and a non-biotiny- trically at a wavelength of 450 nm ± 2 nm.