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).
References:
1. Cellular and mitochondrial glutathione redox imbalance in
lymphoblastoid cells derived from children with autism.
James SJ et al. FASEB J. 2009 Mar 23.
http://www.fasebj.org/cgi/rapidpdf/fj.08-128926v1
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.
http://www.ajcn.org/cgi/content/full/89/1/425
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]
http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=2610366&blobtype=pdf
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]
http://www.ajcn.org/cgi/content/full/80/6/1611
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.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2692510/pdf/nihms108073.pdf
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
http://en.wikipedia.org/wiki/Polybrominated_diphenyl_ethers
7. Polybrominated diphenylethers (PBDEs)
http://www.epa.gov/oppt/pbde/
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]
http://tinyurl.com/c6m8mj
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.
http://erj.ersjournals.com/cgi/reprint/16/5/933
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.
http://www.informapharmascience.com/doi/pdf/10.1517/14728220802517901
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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