Possible ameliorative effects of antioxidants on propionic acid / clindamycin - induced neurotoxicity in Syrian hamsters
© El-Ansary et al.; licensee BioMed Central Ltd. 2013
Received: 4 September 2013
Accepted: 13 October 2013
Published: 4 November 2013
Propionic acid (PA) found in some foods and formed as a metabolic product of gut bacteria has been reported to mimic/mediate the effects of autism. The present study was undertaken to compare the effect of orally administered PA with that of clindamycin-induced PA-microbial producers in inducing persistent biochemical autistic features in hamsters. The neuroprotective potency of carnosine and carnitine supplements against PA toxicity was also investigated.
The following groups were studied. 1. Control group, which received phosphate buffered saline orally, 2. Propionic acid treated group which were given PA at a dose of 250 mg/kg body weight/day for 3 days orally, 3. Clindamycin treated group which received a single dose of the antibiotic orogastrically at a dose of 30 mg/kg on the day of the experiment, 4. Carnosine-treated group which were given carnosine at a dose of 10 mg/kg body weight/day orally for one week, 5. Carnitine treated group given 50 mg/kg body weight/day carnitine orally daily for one week. Group 6. Carnosine followed by PA, Group 7. Carnitine followed by PA. Dopamine, adrenaline and noradrenaline, serotonin and Gamma amino-butyric acid (GABA) were measured in the cortex and medulla of the nine studied groups.
PA administration caused significant decrease in the neurotransmitters in the brains of treated hamsters while clindamycin caused a significant decrease only in dopamine in hamster brains (cortex and medulla) and GABA in the cerebral cortex of the treated hamsters. Administration of carnosine and carnitine which are known antioxidants caused no significant changes in the levels of neurotransmitters when administered alone to hamsters. However when administered with PA both carnosine and carnitine restored the altered neurotransmitters to near normal levels.
Carnosine and carnitine may be used as supplements to protect against PA neurotoxicity.
KeywordsAutism Clindamycin Propionic acid Carnosine Carnitine Cortex Medulla
Propionic acid (PA) occurs naturally in a few food products; for example PA is present in low quantities in milk and relatively higher levels in dairy products such as yogurt and cheese, obviously due to bacterial fermentation, mostly by propionibacteria [1, 2]. The food sources however have a minor contribution in the PA levels in the body . In the colon, PA is produced by fermentation of polysaccharides, oligosaccharides, long-chain fatty acids, protein, peptides and glycoprotein precursors by the anaerobic colonic microbiota, although in quantitative terms undigested carbohydrates, such as dietary fibers and resistant starch, represent the major source for PA production [3, 4].
PA is a short chain fatty acid formed endogenously in the human body as an intermidiate of fatty acid metabolism and a metabolic end product of enteric gut bacteria such as clostridia and propionibacteria [3, 5–7]. Recent studies of El-Ansary et al.  have demonstrated that PA administration to rats caused an increased generation of oxidative stress which was accompanied by impairment of antioxidant defense indices.
Autism is a disorder of neural development which includes development deficiencies of language and social interaction skills, appearance of repetitive and disordered movements  hyperactivity, sensory disturbances, restricted interests and sometimes self injury [10, 11]. Intraventricular infusions of PA have been shown to cause behavioral and brain abnormalities in rats similar to those seen in humans suffering from autism [12–14]. Gut microbes have long been suspected to play a role in autism spectrum disorders (ASD) . Gut microbiota also play an important role in metabolism of xenobiotics, modification of drugs etc., . Clindamycin has been shown to cause loss of majority of normal caecal flora and induction of PA producing bacteria among which is Clostridia species . In this study hamsters were treated with the antibiotic clindamycin to see if its effects were similar to that of exogenous PA which has been implicated in autism. Attempts were also made to understand the neurochemical effects of PA and induced PA production either alone or in combination with carnosine, carnitine as potent antioxidants.
Material and methods
Propionic acid, carnosine and carnitine were of analytical grade products of Sigma-Aldrich.
Clindamycin (Cleocin phosphate) was obtained from Pharmacia Co.,Peapack, NJ, USA).
A total of 54 young male golden Syrian hamsters weighing about 80–100 grams were used in the present study. Animals were randomly into seven groups, each consisting of 6 animals. The groups studied included 1. Control group, which received phosphate buffered saline orally, 2. Propionic acid treated group which were given PA at a dose of 250 mg/Kg body weight/day for 3 days orally , 3. Clindamycin treated group which received a single dose of the antibiotic orogastrically at a dose of 30 mg/kg on the day of the experiment, 4. Carnosine-treated group were given carnosine at a dose of 10 mg/kg body weight/day orally for one week, 5. Carnitine treated group were given 50 mg/kg body weight/day carnitine orally daily for one week. The protected groups included Group 6. Carnosine followed by PA, Group 7. Carnitine followed by PA. The carnosine and carnitine protected group were given the same doses of the respective compounds for one week followed by PA for three days. All the groups were kept at controlled temperature (21 ± 1°C) with ad libtium access to food and water. The experiments were performed in accordance with national animal care guidelines approved by the Animal Ethics Committee, King Saud University, Riyadh.
Brain tissue preparation
At the end of experiment, hamsters were killed by carbon dioxide asphyxiation. The brains were dissected out and washed in saline. Cortex and medulla were separated. The tissues were homogenized in double distilled water and the homogenates were stored at −80°C until used.
Dopamine, adrenaline and noradrenaline were extracted from rat brain by using a cis-diol-specific affinity gel, acylated and then derivatized enzymatically. Quantitavive assay of the three neurotransmitters were performed using ELISA kit, a product of Immuno Biological Laboratories (IBL) using dopamine, adrenaline and noradrenaline antiserum provided. Sensitivities for adrenalin of 0.3 ng/mL, for noradrenaline of 0.6 ng/mL and for dopamine of 5 ng/mL for diluted sample were recorded.
Assay of serotonin
Serotonin was measured using an ELISA kit, a product of Immunology Biological Laboratories (IBL). Brain homogenate (derivatization of serotonin to N-acylserotonin) was done by sample dilution and incubation of the respective sample with the acylation reagent. The assay procedure followed the basic principle of competitive ELISA whereby there is competition between a biotinylated and a non-biotinylated antigen for a fixed number of antibody binding sites. The amount of biotinylated antigen bound to the antibody is inversely proportional to the N-acylserotonin concentration of the sample. When the system is in equilibrium, the free biotinylated antigen is removed by a washing step and the antibody bound biotinylated antigen is determined by use of anti-biotin alkaline phosphatase as marker and p-nitrophenyl phosphate as substrate. Quantification of unknowns was achieved by comparing the enzymatic activity of samples with a response curve prepared by using known standards.
Assay of gamma amino-butyric acid (GABA)
Quantitative determination of GABA was done using ELISA immunoassay kit, a product of ALPCO. 300 μL of diluted standards, controls and undiluted samples were placed into the appropriate wells of the extraction plate. 300 μL of the diluent was added to all wells, covered with adhesive foil and shook for 30 min at room temperature (20-25°C) on a shaker (600 rpm). Two washing cycles were performed, after which 250 μl elution buffer was placed into the appropriate wells of the extraction plate, covered and shook followed by addition of 100 μL of the extract for subsequent derivatization. 10 μL of NaOH was added to all the wells followed by 50 μL of the equalizing reagent (fresh prepared before assay) and shook for 1 min on a shaker (600 rpm). 10 μL of the D-reagent was added into all wells, incubated for 2 hours at (20-25°C) and then 150 μL Q-buffer was added into all wells, incubated for 10 min at RT (20-25°C) on a shaker (approx. 600 rpm). 25 μL of the derivative was used for the subsequent ELISA.
A computer SPSS program was used, and the results were expressed as mean ± SD (n = 6). Comparisons were made by the one-way ANOVA between the control and treated groups. Dunnett’s test was used to compare between the groups. Receiver operating characteristic (ROC) analysis was done. Area under the curve, specificity and sensitivity were calculated.
Results and discussion
Autism spectrum disorders (ASD) comprise a complex and heterogeneous group of conditions that include autism, Rett and Asperger syndromes, and pervasive developmental disorder-otherwise non specified . The main clinical features of ASD are stereotypic behaviors and marked impairment in communication, social skills and cognition [20, 21]. Animal models help us understand the mechanism of disease process and the environmental factors which serve as trigger for the disease process. Studies of MacFabe et al.  have demonstrated PA injection in rats provides a suitable animal model to study ASD. Moreover, there are a series of inherited and acquired conditions which lead to elevations of PA and other short chain fatty acids and these are related to developmental delay, seizures and gastrointestinal symptoms, resembling some aspects of autism [22, 23]. Thus, PA may be a putative link between dietary or enterobacterially derived metabolites along with genetic predisposition and subsequent features of autism .
Antibiotics are commonly used pharmaceuticals. While most of them are well tolerated, they do have side effects the most common being diarrhea. The neurotoxic effects of antibiotics are not well recognized . Therefore there are chances of neurotoxic effects of antibiotics being confused with different neurological conditions .
Clindamycin administration has previously been shown to markedly suppress the anaerobic microbial community including members of the Bacteroides group, which account for ∼ 30% of the intestinal bacterial flora [2, 27, 28]. The change in the intestinal flora may interfere with intestinal enzyme DPP-IV (dipeptydal peptidase), which catalyzes the breakdown of proteins into amino acids, and has been hypothesized as a contributing factor to autism . This is due to the fact that this enzyme is necessary to breakdown peptides into amino acids which are precursors of neurotransmitters.
Though clindamycin is not known have major neurotoxic effects  in the present study treatment of hamsters with antibiotic alone caused a significant decrease in dopamine by 15% (P >0.05) and GABA by 16% (P <0.05) in the cortex when compared to the cerebral cortex of control group of rats. Though the exact cause of decrease of these neurotransmitters is not known, it may be due to inhibition of monoamine oxidase . Carnosine is a an antioxidant dipeptide consisting of amino acids histidine and alanine. L-Carnosine is a highly effective anti-aging nutrient that it possesses powerful antioxidant, free radical scavenging and neurotransmitter properties. Carnosine inhibits the formation of carbonyl groups, thereby reducing the formation of abnormal proteins. L-Carnosine extends maximum cell division capacity, protects against DNA oxidation, blocks glycosylation and reduces Advanced Glycation Endproducts, as well as acts as a cell membrane stabilizer and an intracellular buffer . L-Carnosine has recently been shown to possess a tremendous potential for improving language and behavior in children diagnosed with ASDs . In the present study carnosine treatment caused no significant change in the levels of neurotransmitters studied (P >0.05). Similarly carnitine treatment alone caused no significant change in the levels of neurotransmitters studied (P >0.05). Earlier studies [32, 33] have found that acetyl-L-carnitine treatment increases noradrenalin and serotonin levels in brains of treated mice. This was accompanied by decrease in the levels of GABA in the brains of treated mice . Co administration of propionic acid and carnosine to hamsters caused an increase in adrenalin by 60% (P <0.05), dopamine by 8% (P <0.05) and in GABA by 35% (P <0.01) when compared to the group treated with propionic acid alone in the cortex of treated hamsters.
Treatment of propionic acid and carnitine also caused an increase in adrenalin by 23% (P < 0.01), noradrenalin by 20% (P < 0.01), dopamine by 7% (P <0.05) and GABA by 120% (P <0.01) when compared to the group treated with propionic acid alone.
ROC analysis showed satisfactory values of area under the curve, sensitivity and specificity. This helps to suggest that the measured neurotransmitters could be used as biomarkers for PA and bacterial overgrowth neurotoxicity and as predictive markers to follow the efficacy of both carnosine and carnitine in ameliorating the toxic effects related to PA.
The results of the present study lead us to speculate that PA may play a role in ASD by interfering with the neurotransmitters. Carnosine and carnitine which are known antioxidants cause no significant changes in the levels of neurotransmitters when administered alone to hamsters. However when administered with PA both carnosine and carnitine tended to restore the altered levels of neurotransmitters to near normal levels. Therefore carnosine and carnitine may be used as antioxidants as supplements to protect against PA neurotoxicity.
This research project was supported by a grant from the Research Center of the Center for Female Scientific and Medical Colleges in King Saud University.
- Fernandez-Garcia E, McGregor JU: Determination of organic acids during the fermentation and cold storage of yogurt, J. Dairy Sci. 1994, 77: 2934-2939. 10.3168/jds.S0022-0302(94)77234-9.View Article
- Leigh DA, Simmons K: Effect of clindamycin and lincomycin therapy on faecal flora. J Clin Pathol. 1978, 31: 439-443. 10.1136/jcp.31.5.439.PubMed CentralView ArticlePubMed
- Al-Lahham SH, Peppelenbosch MP, Roelofsen H, Vonk RJ, Venema K: Biological effects of propionic acid in humans;metabolism,potential applications and underlying mechanisms. Biochim Biophys Acta. 2010, 1801: 1175-1183. 10.1016/j.bbalip.2010.07.007.View ArticlePubMed
- Macfarlane S, Macfarlane GT: Regulation of short-chain fatty acid production. Proc Nutr Soc. 2003, 62: 67-72. 10.1079/PNS2002207.View ArticlePubMed
- Nyhan WL, Bay C, Beyer EW, Mazi M: Neurological nonmetabolic presentation of propionic acidemia. Arch Neurol. 1999, 56: 1143-1147. 10.1001/archneur.56.9.1143.View ArticlePubMed
- Thompson GN, Walter JH, Bresson JL, Ford GC, Lyonnet SL, Chalmers RA: Sources of propionate in inborn errors of propionate metabolism. Metabolism. 1990, 39: 1133-1137. 10.1016/0026-0495(90)90084-P.View ArticlePubMed
- Zarate G, Gonzalez S, Chaia AP: Assessing survival of dairy propionibacteria in gastrointestinal conditions and adherence to intestinal epithelia. Methods Mol Biol. 2004, 268: 423-432.PubMed
- Afaf K, El-Ansary , Abir Ben Bacha , Malak Kotb : Etiology of autistic features: the persisting neurotoxic effects of propionic acid. J Neuroinflammation. 2012, 9: 74-10.1186/1742-2094-9-74.View Article
- Nayate A, Bradshaw JL, Rinehart NJ: Autism and Asperger's disorder: are they movement disorders involving the cerebellum and/or basal ganglia?. Brain Res Bull. 2005, 67: 327-334. 10.1016/j.brainresbull.2005.07.011.View ArticlePubMed
- Andres C: Molecular genetics and animal models in autistic disorder. Brain Res Bull. 2002, 57: 109-119. 10.1016/S0361-9230(01)00642-6.View ArticlePubMed
- Zwaigenbaum L, Bryson S, Rogers T, Roberts W, Brian J, Szatmari P: Behavioral manifestations of autism in the first year of life. Int J Dev Neurosci. 2005, 23: 143-152. 10.1016/j.ijdevneu.2004.05.001.View ArticlePubMed
- MacFabe DF, Cain DP, Rodriguez-Capote K, Franklin AE, Hoffman JE, Boon F, Taylor AR, Kavaliers M, Ossenkopp KP: Neurobiological effects of intraventricularpropionic acid in rats: possible role of short chain fatty acids on thepathogenesis and characteristics of autism spectrum disorders. Behav Brain Res. 2007, 176: 149-169. 10.1016/j.bbr.2006.07.025.View ArticlePubMed
- Shultz SR, MacFabe DF, Ossenkopp KP, Scratch S, Whelan J, Taylor R, Cain DP: Intracerebroventricular injection of propionic acid, an enteric bacteria metabolic end-product, impairs social behavior in the rat: implications for an animal model of autism. Neuropharmacology. 2008, 54: 901-911. 10.1016/j.neuropharm.2008.01.013.View ArticlePubMed
- Shultz SR, Macfabe DF, Martin S, Jackson J, Taylor R, Boon F, Ossenkopp KP, Cain DP: Intracerebroventricular injections of the enteric bacterial metabolic product propionic acid impair cognition and sensorimotor ability in the Long- Evans rat: further development of a rodent model of autism. Behav Brain Res. 2009, 200: 33-41. 10.1016/j.bbr.2008.12.023.View ArticlePubMed
- Parracho HM, Bingham MO, Gibson GR, McCartney AL: Differences between the gut microflora of children with autistic spectrum disorders and that of healthy children. J Med Microbiol. 2005, 54: 987-991. 10.1099/jmm.0.46101-0.View ArticlePubMed
- Gonzalez A, Stombaugh J, Lozupone C, Turnbaugh PJ, Gordon JI, Knight R: The mind-body-microbial continuum. Dialogues Clin Neurosci. 2011, 13: 55-62.PubMed CentralPubMed
- Merrigan M, Sambol S, Johnson S, Gerding DN: Susceptibility of hamsters to human pathogenic Clostridium difficile strain B1 following clindamycin, ampicillin or ceftriaxone administration. Anaerobe. 2003, 9 (2): 91-95. 10.1016/S1075-9964(03)00063-5.View ArticlePubMed
- Wyatt I, Farnworth M, Gyte AJ, Lock EA: L-2-chloropropionic acid metabolism and disposition in male rats: relevance to cerebellar injury. Arch Toxicol. 1997, 71: 668-10.1007/s002040050443.View ArticlePubMed
- American Psychiatric Association: Diagnostic and Statistical Manual of Mental Disorders, IV. Washington, DC: American Psychiatric Association, 1994.
- Lord C, Cook EH, Leventhal BL, Amaral DG: Autism spectrum disorders. Neuron. 2000, 28: 355-363. 10.1016/S0896-6273(00)00115-X.View ArticlePubMed
- Rapin I: Autism. N Engl J Med. 1997, 337: 97-104. 10.1056/NEJM199707103370206.View ArticlePubMed
- Burlina AB, Bonafe L, Zacchello F: Clinical and biochemical approach to the neonate with a suspected inborn error of amino acid and organic acid metabolism. Semin Perinatol. 1999, 23: 162-10.1016/S0146-0005(99)80048-2.View ArticlePubMed
- Wajner M, Latini A, Wyse AT, Dutra-Filho CS: The role of oxidative damage in the neuropathology of organic acidurias: insights from animal studies. J Inherit Metab Dis. 2004, 27: 427-View ArticlePubMed
- DeCastro M, Nankova BB, Shah P, Patel P, Mally PV, Mishra R, La Gamma EF: Short chain fatty acids regulate tyrosine hydroxylase gene expression through a cAMP-dependent signaling pathway. Brain Res Mol Brain Res. 2005, 142 (1): 28-38. 10.1016/j.molbrainres.2005.09.002.View ArticlePubMed
- Lam KS, Aman MG, Arnold LE: Neurochemical correlates of autistic disorder: a review of the literature. Res Dev Disabil. 2006, 27: 254-289. 10.1016/j.ridd.2005.03.003.View ArticlePubMed
- Grill MF, Maganti RK: Neurotoxic effects associated with antibiotic use: management considerations. Br J Clin Pharmacol. 2011, 72 (3): 381-393. 10.1111/j.1365-2125.2011.03991.x.PubMed CentralView ArticlePubMed
- Sullivan A, Barkholt L, Nord CE: Lactobacillus acidophilus, Bifidobacterium lactis and Lactobacillus F19 prevent antibiotic-associated ecological disturbances ofBacteroides fragilis in the intestine. J Antimicrob Chemother. 2003, 52: 308-311. 10.1093/jac/dkg346.View ArticlePubMed
- Jernberg C, Sullivan A, Edlund C: Monitoring of antibiotic-induced alterations in the human intestinal microflora and detection of probiotic strains by use of terminal restriction fragment length polymorphism. Appl Environ Microbiol. 2005, 71: 501-506. 10.1128/AEM.71.1.501-506.2005.PubMed CentralView ArticlePubMed
- http://gailbuck.hubpages.com/hub/autism-biochemistry. Accessed from the internet on 24/8/2013
- Flanagan S, Bartizal K, Minassian SL, Fang E, Prokocimer P: In Vitro, In Vivo,and Clinical Studies of Tedizolid To Assess the Potential for Peripheral or Central Monoamine Oxidase Interactions. Antimicrob Agents Chemother. 2013, 57 (7): doi:10.1128/AAC.00431-13
- Improving language and behavior in children diagnosed with autistic spectrum disorder. http://nutritionreview.org/2013/04/lcarnosine-autism/ accessed from the internet on 24/8/2013
- Chez MG, Buchanan CP, Aimonovitch MC, Becker M, Schaefer K, Black C, Komen J: Double-blind, placebo-controlled study of L-carnosine supplementation in children with autistic spectrum disorders. J Child Neurol. 2002, 17: 833-7. 10.1177/08830738020170111501.View ArticlePubMed
- Smeland OB, Meisingset TW, Borges K, Sonnewald U: Chronic acetyl-L-carnitine alters brain energy metabolism and increases noradrenaline and serotonin content in healthy mice. Neurochem Int. 2012, 61 (1): 100-7. 10.1016/j.neuint.2012.04.008.View ArticlePubMed
- Carlson NR: Physiology of behavior. 2001, Boston: Allyn and Bacon, 96-129. 7
- Kandel ER, Schwartz JH, Jessell TM: Essentials of neural science and behavior. 1995, Norwalk, Connecticut: Appleton and Lange, 296-297.
- Fatemi SH, Halt AR, Stary JM, Kanodia R, Schulz SC, Realmuto GR: Glutamic acid decarboxylase 65 & 67 kDa proteins are reduced in autistic parietal & cerebellar cortices. Biol Psychiatry. 2002, 52: 805-10. 10.1016/S0006-3223(02)01430-0.View ArticlePubMed
- El-Ansary A, Shaker GH, Amina R, El-Gezeery AR, Al-Ayadhi L: The neurotoxic effect of clindamycin – induced gut bacterial imbalance and orally administered propionic acid on DNA damage assessed by the comet assay: protective potency of carnosine and carnitine. Gut Pathogens. 2013, 5: 9-10.1186/1757-4749-5-9.PubMed CentralView ArticlePubMed
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