Researcher in Developmental & Behavioral Neuroanatomy
April 10, 2009
Important autism findings have been reported by Jill James, Ph.D., and colleagues, who have described autism-altered metabolic pathways related to glutatione, oxidative stress, and methylation. Among their findings, the James group has documented atypical profiles of "reduced glutathione (GSH) and oxidized disulfide glutathione (GSSG)" and clinical responses to treatment (eg, 1-4).
Two newly published studies call attention to a different but related pathway. Ashwood and colleagues used peripheral blood mononuclear cells (PBMCs) to evaluate immune responses in children with an autism-spectrum disorder (ASD) in comparison with immune responses of children who are typically developing (TD). Each of the two sets of PBMCs were adulterated with physiologically relevant levels of PBE-47, a specific molecular form of of polybrominated diphenyl ethers (6-7; flame retardants). Then each group of PBMDs was stimulated with lipopolysaccharide (LPS), and immune profiles were recorded for each ASD and TD sample. Among several immune markers different between the two groups of PBMCs, a noteworth finding was that the ASD PBMCs pre-treated with BDE-47 developed "an increased IL-1β response to LPS" (5; Interleukin 1beta) and, at a higher but still physiologically relevant dose, the ASD PBMCs expressed a higher level if IL-8 (5).
The finding of elevated IL-1β and IL-8 response to LPS in the presence of a common PBDE at physiologically relevant levels suggests concern for a pathway related to cysteine. Consider some insights from Smita S. Iyer and colleagues at Emory University.
"Interleukin (IL)-1b is a pro-inflammatory cytokine that functions as a critical regulator of host defense in response to infection and injury. However when present in excess, IL-1b is extremely toxic..." (8)
"IL-1b activation and induction are associated with inflammation, a process with enhanced generation of reactive oxygen and nitrogen species..." (8)
"Recent advances in redox signaling mechanisms have revealed that functional pathways utilize diffusible oxidants such as peroxide and redox-sensitive thiols in specific proteins as sensors... The redox states of these sensors are controlled by rates of oxidation of specific amino acid residues and their reduction by thiol/disulfide control systems." (8)
"The thiol/disulfide control systems are compartmentalized; glutathione/glutathione disulfide (GSH/GSSG) and thioredoxin provide control mechanisms within cells, while cysteine/cystine (Cys/CySS) and GSH/GSSG control the redox state of proteins in the extracellular space and on the cell surface..." (8)
Note that many of Jill James' group's findings call attention to GSH and GSSG (1-4), whereas the new Ashwood et al findings (5) call attention to IL-1β, which Iyer et al describe as a cytokine related to cysteine pathways (8). A goodly number of peer-reviewed studies suggest the therapeutic value of N-acetylcysteine (NAC; eg, 9-13), with adverse effects a possibility (13). A recent review calls attention to NAC efficacy in several neuropsychiatric disorders (14).
1. Cellular and mitochondrial glutathione redox imbalance in lymphoblastoid cells derived from children with autism.
James SJ et al. FASEB J. 2009 Mar 23.
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.-James, S. J., Rose, S., Melnyk, S., Jernigan, S., Blossom, S., Pavliv, O., Gaylor, D.W. Cellular and mitochondrial glutathione redox imbalance in lymphoblastoid cells derived from children with autism.
2. Efficacy of methylcobalamin and folinic acid treatment on glutathione redox status in children with autism.
James SJ et al.
Am J Clin Nutr. 2009 Jan;89(1):425-30.
BACKGROUND: Metabolic abnormalities and targeted treatment trials have been reported for several neurobehavioral disorders but are relatively understudied in autism. OBJECTIVE: The objective of this study was to determine whether or not treatment with the metabolic precursors, methylcobalamin and folinic acid, would improve plasma concentrations of transmethylation/transsulfuration metabolites and glutathione redox status in autistic children. DESIGN: In an open-label trial, 40 autistic children were treated with 75 microg/kg methylcobalamin (2 times/wk) and 400 microg folinic acid (2 times/d) for 3 mo. Metabolites in the transmethylation/transsulfuration pathway were measured before and after treatment and compared with values measured in age-matched control children. RESULTS: The results indicated that pretreatment metabolite concentrations in autistic children were significantly different from values in the control children. The 3-mo intervention resulted in significant increases in cysteine, cysteinylglycine, and glutathione concentrations (P < 0.001). The oxidized disulfide form of glutathione was decreased and the glutathione redox ratio increased after treatment (P < 0.008). Although mean metabolite concentrations were improved significantly after intervention, they remained below those in unaffected control children. CONCLUSION: The significant improvements observed in transmethylation metabolites and glutathione redox status after treatment suggest that targeted nutritional intervention with methylcobalamin and folinic acid may be of clinical benefit in some children who have autism. This trial was registered at (clinicaltrials.gov) as NCT00692315.
3. Metabolic endophenotype and related genotypes are associated with oxidative stress in children with autism.
James SJ et al.
Am J Med Genet B Neuropsychiatr Genet. 2006 Dec 5;141B(8):947-56. [free online]
Autism is a behaviorally defined neurodevelopmental disorder usually diagnosed in early childhood that is characterized by impairment in reciprocal communication and speech, repetitive behaviors, and social withdrawal. Although both genetic and environmental factors are thought to be involved, none have been reproducibly identified. The metabolic phenotype of an individual reflects the influence of endogenous and exogenous factors on genotype. As such, it provides a window through which the interactive impact of genes and environment may be viewed and relevant susceptibility factors identified. Although abnormal methionine metabolism has been associated with other neurologic disorders, these pathways and related polymorphisms have not been evaluated in autistic children. Plasma levels of metabolites in methionine transmethylation and transsulfuration pathways were measured in 80 autistic and 73 control children. In addition, common polymorphic variants known to modulate these metabolic pathways were evaluated in 360 autistic children and 205 controls. The metabolic results indicated that plasma methionine and the ratio of S-adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH), an indicator of methylation capacity, were significantly decreased in the autistic children relative to age-matched controls. In addition, plasma levels of cysteine, glutathione, and the ratio of reduced to oxidized glutathione, an indication of antioxidant capacity and redox homeostasis, were significantly decreased. Differences in allele frequency and/or significant gene-gene interactions were found for relevant genes encoding the reduced folate carrier (RFC 80G > A), transcobalamin II (TCN2 776G > C), catechol-O-methyltransferase (COMT 472G > A), methylenetetrahydrofolate reductase (MTHFR 677C > T and 1298A > C), and glutathione-S-transferase (GST M1). We propose that an increased vulnerability to oxidative stress (endogenous or environmental) may contribute to the development and clinical manifestations of autism. (c) 2006 Wiley-Liss, Inc.
4. Metabolic biomarkers of increased oxidative stress and impaired methylation capacity in children with autism.
James SJ et al.
Am J Clin Nutr. 2004 Dec;80(6):1611-7. [free online]
BACKGROUND: Autism is a complex neurodevelopmental disorder that usually presents in early childhood and that is thought to be influenced by genetic and environmental factors. Although abnormal metabolism of methionine and homocysteine has been associated with other neurologic diseases, these pathways have not been evaluated in persons with autism. OBJECTIVE: The purpose of this study was to evaluate plasma concentrations of metabolites in the methionine transmethylation and transsulfuration pathways in children diagnosed with autism. DESIGN: Plasma concentrations of methionine, S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), adenosine, homocysteine, cystathionine, cysteine, and oxidized and reduced glutathione were measured in 20 children with autism and in 33 control children. On the basis of the abnormal metabolic profile, a targeted nutritional intervention trial with folinic acid, betaine, and methylcobalamin was initiated in a subset of the autistic children. RESULTS: Relative to the control children, the children with autism had significantly lower baseline plasma concentrations of methionine, SAM, homocysteine, cystathionine, cysteine, and total glutathione and significantly higher concentrations of SAH, adenosine, and oxidized glutathione. This metabolic profile is consistent with impaired capacity for methylation (significantly lower ratio of SAM to SAH) and increased oxidative stress (significantly lower redox ratio of reduced glutathione to oxidized glutathione) in children with autism. The intervention trial was effective in normalizing the metabolic imbalance in the autistic children. CONCLUSIONS: An increased vulnerability to oxidative stress and a decreased capacity for methylation may contribute to the development and clinical manifestation of autism.
5. Preliminary evidence of the in vitro effects of BDE-47 on innate immune responses in
children with autism spectrum disorders
Paul Ashwood et al. Journal of Neuroimmunology 208 (2009) 130–135.
Autism spectrum disorders (ASD) are complex neurodevelopmental disorders that manifest in childhood. Immune dysregulation and autoimmune reactivity may contribute to the etiology of ASD and are likely the result of both genetic and environmental susceptibilities. A common environmental contaminant, 2,2′,4,4′- tetrabrominated biphenyl (BDE-47), was tested for differential effects on the immune response of peripheral blood mononuclear cells (PBMC) isolated from children with ASD (n=19) and age-matched typically developing controls (TD, n=18). PBMC were exposed in vitro to either 100 nM or 500 nM BDE-47, before challenge with bacterial lipopolysaccharide (LPS), an innate immune activator, with resultant cytokine production measured using the Luminex™multiplex platform. The cytokine responses of LPS stimulated PBMC from ASD and TD subjects diverged in the presence of 100 nM BDE. For example, cells cultured from the TD group demonstrated significantly decreased levels of the cytokines IL-12p40, GM-CSF, IL-6, TNFα, and the chemokines MIP-1α and MIP-1β following LPS stimulation of PBMC pretreated with 100 nMBDE-47 compared with samples treated with vehicle control (pb0.05). In contrast, cells cultured from subjects with ASD demonstrated an increased IL-1β response to LPS (p=0.033) when pretreated with 100 nM BDE-47 compared with vehicle control. Preincubation with 500 nM BDE-47 significantly increased the stimulated release of the inflammatory chemokine IL-8 (pb0.04) in cells cultured from subjects with ASD but not in cells from TD controls. These data suggest that in vitro exposure of PBMC to BDE-47 affects cell cytokine production in a pediatric population. Moreover, PBMC from the ASD subjects were differentially affected when compared with the TD controls suggesting a biological basis for altered sensitivity to BDE-47 in the ASD population. © 2009 Elsevier B.V. All rights reserved.
6. Polybrominated diphenyl ethers
7. Polybrominated diphenylethers (PBDEs)
8. Cysteine redox potential determines pro-inflammatory IL-1beta levels.
Iyer SS et al. PLoS ONE. 2009;4(3):e5017. Epub 2009 Mar 27. [free online]
BACKGROUND: Cysteine (Cys) and its disulfide, cystine (CySS) represent the major extracellular thiol/disulfide redox control system. The redox potential (E(h)) of Cys/CySS is centered at approximately -80 mV in the plasma of healthy adults, and oxidation of E(h) Cys/CySS is implicated in inflammation associated with various diseases. METHODOLOGY/PRINCIPAL FINDINGS: The purpose of the present study was to determine whether oxidized E(h) Cys/CySS is a determinant of interleukin (IL)-1beta levels. Results showed a 1.7-fold increase in secreted pro-IL-1beta levels in U937 monocytes exposed to oxidized E(h) Cys/CySS (-46 mV), compared to controls exposed to a physiological E(h) of -80 mV (P<0.01). In LPS-challenged mice, preservation of plasma E(h) Cys/CySS from oxidation by dietary sulfur amino acid (SAA) supplementation, was associated with a 1.6-fold decrease in plasma IL-1beta compared to control mice fed an isonitrogenous SAA-adequate diet (P<0.01). Analysis of E(h) Cys/CySS and IL-1beta in human plasma revealed a significant positive association between oxidized E(h) Cys/CySS and IL-1beta after controlling for age, gender, and BMI (P<0.001). CONCLUSIONS/SIGNIFICANCE: These data show that oxidized extracellular E(h) Cys/CySS is a determinant of IL-1beta levels, and suggest that strategies to preserve E(h) Cys/CySS may represent a means to control IL-1beta in inflammatory disease states.
9. N-acetylcysteine prevents LPS-induced pro-inflammatory cytokines and MMP2 production in gingival fibroblasts.
Kim do Y et al. Arch Pharm Res. 2007 Oct;30(10):1283-92.
Periodontitis is an inflammatory process that ultimately results in tooth loss. Although the primary etiologic agent for periodontitis is bacteria, the majority of periodontal tissue destruction is thought to be caused by an inappropriate host response. Reactive oxygen species (ROS) have been known to be involved in periodontal tissue destruction. We treated human gingival fibroblasts with lipopolysaccharide (LPS) obtained from E. coli and the periodontopathogens Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis, and examined their inflammatory responses in the presence and absence of the antioxidant N-acetylcysteine (NAC). LPS enhanced ROS production, as well as, expression of pro-inflammatory cytokines such as interleukin-1beta, interleukin-6, interleukin-8 and tumor necrosis factor-alpha, and the production and activation of MMP2. NAC suppressed all LPS-induced inflammatory responses examined, suggesting that LPS-induced ROS may play a major regulatory role in these responses in gingival fibroblasts. In addition, NAC prevented LPS-induced activation of p38 MAPK and JNK but not phosphorylation and subsequent degradation of IkB. These results indicate that NAC exerts anti-inflammatory effects in LPS-stimulated gingival fibroblasts, functioning at least in part via down-regulation of JNK and p38 MAPK activation. Furthermore, this work suggests that antioxidants may be useful in adjunctive therapies that complement conventional periodontal treatments.
10. Effects of N-acetylcysteine against systemic and renal hemodynamic effects of endotoxin in healthy humans.
Schaller G et al. Crit Care Med. 2007 Aug;35(8):1869-75.
OBJECTIVE: Systemic inflammation causes vasodilation and impairs the vascular response to catecholamines. There is evidence that altered vasoreactivity is associated with increased production of free radicals. We studied the influence of systemic doses of the antioxidant N-acetylcysteine on inflammatory cytokines and renal plasma flow and on the systemic pressor response to norepinephrine during experimental endotoxemia. DESIGN: A double-blind, placebo-controlled crossover study. SETTING: Medical University of Vienna, Clinical Pharmacology, Vienna General Hospital, AKH. SUBJECTS: Eight healthy, male humans. INTERVENTIONS: Intravenous administration of Escherichia coli endotoxin (lipopolysaccharide, 20 IU/kg) on two separate study days with concomitant intravenous infusion of placebo or N-acetylcysteine (150 mg/kg loading dose; 15 mg/kg/hr continuous infusion), respectively. MEASUREMENTS AND MAIN RESULTS: Measurements of inflammatory cytokines, of renal plasma flow by the para-aminohippurate-clearance method, and of the systemic pressor response to norepinephrine were taken at baseline and after endotoxin. Lipopolysaccharide increased body temperature and plasma concentrations of tumor necrosis factor-alpha, which was mitigated during N-acetylcysteine infusions. Likewise, the lipopolysaccharide-induced increases in renal plasma flow and decreases in blood pressure were attenuated, and the hyporeactivity of pulse rate to norepinephrine 4 hrs after lipopolysaccharide was improved by N-acetylcysteine. CONCLUSION: High doses of N-acetylcysteine might exert protective effects on systemic hemodynamics and on the reactivity to catecholamines in humans challenged by lipopolysaccharide. This action of the antioxidant N-acetylcysteine is paralleled by humoral anti-inflammatory mechanisms and may be useful in patients with systemic inflammation.
11. Astroglial CYP1B1 up-regulation in inflammatory/oxidative toxic conditions: IL-1beta effect and protection by N-acetylcysteine.
Malaplate-Armand C et al.
Toxicol Lett. 2003 Mar 3;138(3):243-51.
The present work aims to determine the relevance of an astrocytoma cell line U373 MG, for assessing the role of some astroglial cytochrome P450 in neurotoxicity and neuroprotection. CYP1B1, CYP2C8, CYP2C9, CYP2D6, CYP2J2, CYP2E1 and CYP4A11 mRNA were detected by reverse transcriptase-polymerase chain reaction in control U373 MG cell cultures. Among them we focused on CYP1B1 expression. After 48 h treatment with a range of concentrations of interleukin-1beta (1, 5, 10 ng/ml) used to simulate stress conditions, CYP1B1 mRNA expression was enhanced in a dose-dependent way. This increased expression was followed 24 h later by an increase in protein level, determined by Western-blot. N-acetylcysteine (NAC) partially inhibited this effect both on the mRNA and protein levels. As CYP1B1 activates procarcinogenic compounds to reactive metabolites, an increase in this P450 isoform will participate to toxic consequences of an inflammatory/oxidative stress. NAC will prevent this deleterious effect. Copyright 2002 Elsevier Science Ireland Ltd.
12. Inhibition of polymethylmethacrylate particle-induced monocyte activation and IL-1beta and TNF-alpha expression by the antioxidant agent N-acetylcysteine.
Mulhall KJ et al. Acta Orthop Scand. 2002 Apr;73(2):206-12.
We investigated the effectiveness of an antioxidant agent, N-acetylcysteine (NAC), in suppressing macrophage activation and mediator release in response to particulate debris. Polymethylmethacrylate (PMMA) particle-stimulated monocyte-macrophages were cultured alone and with varying concentrations of NAC. Tumor necrosis factor alpha (TNFalpha) and interleukin-1beta (IL-1beta) expression in the resultant cultures were measured using enzyme-linked immunosorbant assays. The ultrastructural effect of treatment was also assessed by electron microscopy. Cell viability in the various cultures was measured to rule out an effect of cytotoxicity. NAC treatment reduced TNFalpha and IL-1beta expression by the monocyte-macrophages. Culturing with NAC was also associated with less ultrastructural activation of the monocytes. Furthermore, NAC was not associated with any adverse effect on cell viability in the concentrations used. Our findings demonstrate the effectiveness of the antioxidant N-acetylcysteine in suppressing the cell activation and TNFalpha release seen on exposure to wear debris. This represents a novel potential therapeutic method in the prevention or treatment of periprosthetic osteolysis.
13. Regulation of lipopolysaccharide-mediated interleukin-1beta release by N-acetylcysteine in THP-1 cells.
Parmentier M et al. Eur Respir J. 2000 Nov;16(5):933-9.
Increased levels of inflammatory cytokines such as interleukin (IL)-1 and IL-8 occur in the bronchoalveolar lavage fluid in various lung diseases. Cytokine gene expression is controlled by transcription factors such as nuclear factor-kappaB (NF-kappaB) which can be activated by a number of stimuli including the oxidants prevent. It was hypothesized that lipopolysaccharide (LPS)-induced IL-1beta secretion may be modulated by the intracellular thiol redox status of the cells. The effect of the antioxidant compound, N-acetyl-L-cysteine (NAC), on IL-1beta release and regulation of NF-kappaB in a human myelo-monocytic cell line (THP-1) differentiated into macrophages was studied. LPS (10 microg x mL(-1)) increased IL-1beta release at 24 h compared to control levels (p<0.001). NAC (5 mM) also enhanced LPS-induced IL-1beta release from THP-1 cells (p<0.001). In addition, treatment of cells with cycloheximide, an inhibitor of protein synthesis, inhibited the NAC-mediated IL-1beta release. Under the same conditions, NF-kappaB binding was activated by LPS and NAC increased this LPS-mediated effect. Western blot analysis revealed that NAC treatment leads to an increase in p50 and p65 protein synthesis. These data indicate that N-acetyl-L-cysteine modulates interleukin-1kappa release by increasing levels of the homo- and heterodimeric forms of nuclear factor-kappaB.
14. N-acetylcysteine for antioxidant therapy: pharmacology and clinical utility.
Dodd S et al. Expert Opin Biol Ther. 2008 Dec;8(12):1955-62.
BACKGROUND: Glutathione is an endogenous antioxidant and has a ubiquitous role in many of the body's defences. Treatment with N-acetylcysteine (NAC) has been shown to increase levels of glutathione. NAC has been proposed as a treatment for several illnesses. OBJECTIVES: The efficacy and tolerability of NAC was examined across a range of conditions to evaluate the evidence supporting the use of NAC for each indication. METHODS: A literature search was conducted using PubMed. Information was also collected from other online sources including the websites of the Therapeutic Goods Administration of Australia and the FDA. RESULTS: Reports ranged from case studies to clinical trials. There is strong evidence to support the use of NAC for the treatment of paracetamol overdose and emerging evidence suggesting it has utility in psychiatric disorders, particularly schizophrenia and bipolar disorder. NAC is safe and well tolerated when administered orally but has documented risks with intravenous administration.
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