Two overlapping pathways in Autism: glutathione and cysteine

Teresa Binstock
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  ( 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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