ArticlePDF Available

Evidence of oxidative damage and inflammation associated with low glutathione redox status in the autism brain


Abstract and Figures

Despite increasing evidence of oxidative stress in the pathophysiology of autism, most studies have not evaluated biomarkers within specific brain regions, and the functional consequences of oxidative stress remain relatively understudied. We examined frozen samples from the cerebellum and temporal cortex (Brodmann area 22 (BA22)) from individuals with autism and unaffected controls (n=15 and n=12 per group, respectively). Biomarkers of oxidative stress, including reduced glutathione (GSH), oxidized glutathione (GSSG) and glutathione redox/antioxidant capacity (GSH/GSSG), were measured. Biomarkers of oxidative protein damage (3-nitrotyrosine; 3-NT) and oxidative DNA damage (8-oxo-deoxyguanosine; 8-oxo-dG) were also assessed. Functional indicators of oxidative stress included relative levels of 3-chlorotyrosine (3-CT), an established biomarker of a chronic inflammatory response, and aconitase activity, a biomarker of mitochondrial superoxide production. Consistent with previous studies on plasma and immune cells, GSH and GSH/GSSG were significantly decreased in both autism cerebellum (P<0.01) and BA22 (P<0.01). There was a significant increase in 3-NT in the autism cerebellum and BA22 (P<0.01). Similarly, 8-oxo-dG was significantly increased in autism cerebellum and BA22 (P<0.01 and P=0.01, respectively), and was inversely correlated with GSH/GSSG in the cerebellum (P<0.01). There was a significant increase in 3-CT levels in both brain regions (P<0.01), whereas aconitase activity was significantly decreased in autism cerebellum (P<0.01), and was negatively correlated with GSH/GSSG (P=0.01). Together, these results indicate that decreased GSH/GSSG redox/antioxidant capacity and increased oxidative stress in the autism brain may have functional consequence in terms of a chronic inflammatory response, increased mitochondrial superoxide production, and oxidative protein and DNA damage.
Content may be subject to copyright.
Evidence of oxidative damage and inflammation
associated with low glutathione redox status in the
autism brain
S Rose, S Melnyk, O Pavliv, S Bai, TG Nick, RE Frye and SJ James
Despite increasing evidence of oxidative stress in the pathophysiology of autism, most studies have not evaluated
biomarkers within specific brain regions, and the functional consequences of oxidative stress remain relatively understudied.
We examined frozen samples from the cerebellum and temporal cortex (Brodmann area 22 (BA22)) from individuals with
autism and unaffected controls (n¼15 and n¼12 per group, respectively). Biomarkers of oxidative stress, including
reduced glutathione (GSH), oxidized glutathione (GSSG) and glutathione redox/antioxidant capacity (GSH/GSSG), were
measured. Biomarkers of oxidative protein damage (3-nitrotyrosine; 3-NT) and oxidative DNA damage (8-oxo-deoxyguanosine;
8-oxo-dG) were also assessed. Functional indicators of oxidative stress included relative levels of 3-chlorotyrosine (3-CT),
an established biomarker of a chronic inflammatory response, and aconitase activity, a biomarker of mitochondrial superoxide
production. Consistent with previous studies on plasma and immune cells, GSH and GSH/GSSG were significantly decreased
in both autism cerebellum (Po0.01) and BA22 (Po0.01). There was a significant increase in 3-NT in the autism cerebellum
and BA22 (Po0.01). Similarly, 8-oxo-dG was significantly increased in autism cerebellum and BA22 (Po0.01 and
P¼0.01, respectively), and was inversely correlated with GSH/GSSG in the cerebellum (Po0.01). There was a significant
increase in 3-CT levels in both brain regions (Po0.01), whereas aconitase activity was significantly decreased in autism
cerebellum (Po0.01), and was negatively correlated with GSH/GSSG (P¼0.01). Together, these results indicate that decreased
GSH/GSSG redox/antioxidant capacity and increased oxidative stress in the autism brain may have functional consequence in
terms of a chronic inflammatory response, increased mitochondrial superoxide production, and oxidative protein and DNA
Translational Psychiatry (2012) 2, e134; doi:10.1038/tp.2012.61; published online 10 July 2012
Autism is a complex, behaviorally defined neurodevelopmen-
tal disorder characterized by significant impairments in social
interaction, verbal and non-verbal communication, and by
restrictive, repetitive and stereotypic patterns of behavior. The
Centers for Disease Control estimates that the current
prevalence of autism spectrum disorders in the United States
is 1 in 110 children.
A number of studies have shown
abnormalities in the autism brain, including the prefrontal
cortex, temporal lobe, amygdala and cerebellum, as well as
differences in total brain volume and growth trajectories.
However, despite numerous structural and functional neuro-
imaging studies, as well as post-mortem investigations, the
underlying neurobiological basis of autism continues to
remain elusive.
Abnormalities in the cerebellum are among the most
reproducibly reported alterations in the autism brain.
Neuroimaging studies have reported reduced vermis
volume as well as total cerebellar volume, whereas post-
mortem analyses have revealed a significant reduction in
the number of Purkinje cells in individuals with autism.
Evidence of neuroimmune involvement in the cerebellum
includes the presence of activated neuroglia and elevated
cytokine levels, as well as autoantibodies to cerebellar
Abnormalities in the superior temporal gyrus (STG) are
thought to be relevant in autism because of its important role in
processing sounds and speech development.
contains Brodmann area 22 (BA22), which in the left hemi-
sphere corresponds to Wernicke’s area, a region involved in
speech processing. Neuroimaging analyses have revealed
increased right STG volume in subjects with autism, consistent
with a recent finding of a more rightward asymmetry of the
STG in individuals with autism.
A reduction in neuronal cell
density and increased glial cell density was reported in a
microscopic analysis of BA22 in individuals with autism.
Neuroimmune activation in the autism STG has been reported
in a single study, finding increased transcript levels of multiple
immune-related genes.
Biomarkers of oxidative stress have been reported in many
neurological and psychiatric disorders, including Alzheimer’s
Parkinson’s disease,
Received 19 March 2012; revised 9 May 2012; accepted 31 May 2012
Department of Pediatrics, University of Arkansas for Medical Sciences, Arkansas Children’s Hospital Research Institute, Little Rock, AR, USA
Correspondence: Dr S Rose, Department of Pediatrics, University of Arkansas for Medical Sciences, Arkansas Children’s Hospital Research Institute, 13 Children’s Way,
Slot 512-41B, Little Rock, AR 72202, USA.
Keywords: aconitase; mitochondria; neuroinflammation; oxidative stress; 3-chlorotyrosine; 3-nitrotyrosine
Citation: Transl Psychiatry (2012) 2, e134, doi:10.1038/tp.2012.61
2012 Macmillan Publishers Limited All rights reserved 2158-3188/12
and alcoholism,
and may reflect a common
underlying pathophysiological mechanism. Numerous indica-
tors of oxidative stress have been documented previously in
the blood from children with autism, including decreased
antioxidant enzyme activities, elevated lipid peroxidation and
accumulation of advanced glycation end products.
three independent case/control cohorts, children with autism
were shown to exhibit abnormal plasma levels of metabolites in
the pathway of glutathione redox metabolism.
In these
studies, the mean concentration of reduced glutathione (GSH),
the primary intracellular antioxidant and redox buffer, was
found to be significantly decreased, whereas oxidized glu-
tathione disulfide (GSSG) was significantly increased, result-
ing in a decrease in the glutathione redox ratio (GSH/GSSG) in
both plasma and primary immune cells from children with
Taken together, accumulating evidence suggests
that children with autism have a more oxidized extracellular
(plasma) and intracellular immune cell microenvironment than
age-matched unaffected control children.
Oxidative stress and damage occurs when antioxidant
defense mechanisms fail to effectively counter endogenous or
exogenous sources of reactive oxygen species. Glutathione is
the primary antioxidant responsible for maintaining the
reducing intracellular microenvironment that is essential for
normal cellular function and viability. GSH/GSSG is a reliable
indicator of cellular redox status, and a chronic reduction in
GSH/GSSG reflects a reduced antioxidant capacity and
increased vulnerability to oxidative damage.
Recently, an
increase in oxidative protein and DNA damage was asso-
ciated with the decrease in intracellular and plasma GSH/
GSSG in children with autism, suggesting that the reduced
antioxidant defense capacity in these children may have
functional consequence in terms of overt oxidative damage.
Although there is growing evidence that biomarkers of
increased oxidative stress are present in the blood of children
with autism, the presence of oxidative stress and glutathione
deficit in the autism brain has remained relatively
understudied. In the cerebellum of children with autism,
Sajdel-Sulkowska et al.
found significantly increased 3-
nitrotyrosine (3-NT), a marker of oxidative protein damage,
while noting a trend of increased 8-oxo-deoxyguanosine (8-
oxo-dG), a marker of oxidative DNA damage. Another group
reported a significant increase in lipid hydroperoxides in the
cerebellum and temporal cortex in autism cases compared
with controls.
In addition, a greater number of cells contain-
ing lipofuscin, a matrix of lysosomal degradation products and
a marker for oxidative stress, was found in BA22 in autism
compared with control samples.
Whether the pro-oxidant
glutathione redox imbalance previously observed in plasma
and immune cells from children with autism is also present in
the autism brain has not been investigated.
We hypothesized that low glutathione redox status would
be associated with elevated markers of oxidative protein and
DNA damage, inflammation and mitochondrial superoxide
production in two regions that have been reported to be
abnormal in autism, cerebellum and BA22. To this end, we
measured two stable post-translational modifications of
protein tyrosine residues, 3-NT and 3-chlorotyrosine (3-CT).
The 3-NT, a marker of protein oxidative damage, is formed
from peroxynitrite, a highly reactive free radical generated
from nitric oxide (NO) and superoxide. The tyrosine derivative,
3-CT, is a stable marker of inflammation that is generated
from hypochlorous acid, a potent chlorinating oxidant derived
from myeloperoxidase in activated immune cells during an
inflammatory response.
In addition, we measured 8-oxo-dG,
a commonly used biomarker for assessing oxidative DNA
damage during inflammatory and pro-oxidant exposures.
Finally, to assess a functional indicator of oxidative damage
and mitochondrial reactive oxygen species production, the
activity of aconitase, a redox-sensitive enzyme in the tri-
carboxylic acid cycle, was measured. Aconitase is highly
sensitive to inactivation by superoxide because of its labile
iron atom and its proximity to the superoxide-generating
electron transport chain.
As 85% of aconitase in the brain is
the mitochondrial isozyme, a decrease in brain aconitase
activity is considered a sensitive indicator of excess mito-
chondrial superoxide production.
Taken together, the findings of this study support our
hypothesis and a role for glutathione redox imbalance and
oxidative stress in the neuropathology of autism. Further, this
study provides new evidence that mitochondrial superoxide
production may be elevated in certain brain regions and that a
neuroinflammatory process may promote oxidative stress and
damage in affected cells.
Materials and methods
Post-mortem brain samples. Frozen post-mortem tissues
were obtained from the NICHD Brain and Tissue Bank for
Developmental Disorders at the University of Maryland,
Baltimore, MD, USA, and from the Autism Tissue Program at
the Harvard Brain Tissue Resource Center, Belmont, MA,
USA. A total of 15 autism and 15 control tissues from
cerebellar cortex, and 12 autism and 12 control tissues from
BA22 were evaluated. Diagnosis of autism was confirmed by
the Autism Tissue Program, using the Autistic Diagnostic
Interview Revised. Autism and control groups were perfectly
matched for gender, and were matched as closely as
possible for post-mortem interval, age, race and cause of
death. The phenotypic description of the autism case and
control tissues from the cerebellum and BA22 are presented
in Supplementary Tables 1 and 2, respectively. Although
behavioral scores from the Autistic Diagnostic Interview
Revised were available on a few samples through the Autism
Tissue Program database, there were insufficient numbers to
perform valid correlations between biomarkers and behavior.
Aconitase activity. To evaluate a functional consequence
of oxidative stress, the activity of the redox-sensitive enzyme,
aconitase, was measured using the Aconitase Assay Kit
(Cayman Chemical, Ann Arbor, MI, USA) following the
manufacturer’s instructions. The assay is based on the
conversion of citrate to isocitrate, to a-ketoglutarate, which
results in the production of NADPH. The assay measures the
increase in absorbance monitored at 340 nm associated with
the formation of NADPH, which is proportional to the
aconitase activity. Approximately 100 mg of frozen tissue
was minced in 500 ml ice-cold homogenization buffer and
homogenized in a Dounce homogenizer. Homogenates were
Oxidative stress and inflammation in the autism brain
S Rose et al
Translational Psychiatry
centrifuged at 800 gfor 10min at 4 1C, and the supernatant
was sonicated on ice for 20 s before protein quantification
(BCA protein assay, Pierce, Rockford, IL, USA). Samples
were diluted to 0.5 mg ml
in assay buffer immediately
before the assay.
High-performance liquid chromatography quantification
of GSH/GSSG, 3-NT and 3-CT. To evaluate biomarkers of
oxidative stress and damage by high-performance liquid chro-
matography (HPLC), approximately 200 mg of tissue was
minced and homogenized in 500 ml of ice-cold phosphate-
buffered saline. To precipitate proteins, 150ml of 10% meta-
phosphoric acid was added to 100 ml of tissue homogenate
and incubated for 30 min on ice. The samples were then
centrifuged at 18 000 gat 4 1C for 15 min, and 20mlofthe
resulting supernatants was injected into the HPLC column for
metabolite quantification, while the pellet was used for protein
analysis using the BCA protein assay. The methodological
details for HPLC elution and electrochemical detection of free
unbound GSH, GSSG, 3-NT and 3-CT have been described
The results are expressed as per mg protein.
HPLC/mass spectrometry quantification of 8-oxo-
dG. DNA was extracted from brain tissues using standard
phenol chloroform methodology for which the methodological
details have been published previously.
Briefly, to B1mg
DNA, RNase A (Sigma, St Louis, MO, USA) was added to a
final concentration of 0.02 mg ml
and incubated at 37 1Cfor
15 min. The purified DNA was digested into component
nucleotides using nuclease P
, snake venom phospho-
diesterase and alkaline phosphatase as previously described
in detail.
Quantification of 8-oxo-dG in DNA was performed
using mass spectrometry/liquid chromatography on a LCQ
Advantage MAX system (Thermo Electron Corporation,
Waltham, MA, USA) and expressed as pmol per mg DNA.
Statistical analyses. Within each region, biomarkers were
evaluated using a multiple linear regression to test the group
difference between cases and controls. Age, sex and post-
mortem interval were included as covariates, and controlled
for in the regression analysis. The means, s.d. and associated
P-values for case and control samples are reported.
Spearman’s correlation coefficients were computed to
determine inter-metabolite correlations. All data were
analyzed using SAS 9.2 (SAS Institute, Cary, NC, USA). All
statistical tests used a significance level of 0.05.
Demographics of tissue donors. There were no
differences in mean age, gender or post-mortem interval
between the autism and control groups for either cerebellum
or BA22. Race and cause of death were matched as closely
as possible between available case and control samples;
however, in a few subjects, the race and cause of death were
unknown (Supplementary Tables 1 and 2).
GSH, GSSG, 3-NT, 3-CT and 8-oxo-dG. Figure 1 presents
the relative concentrations of GSH, GSSG, 3-NT, 3-CT and
8-oxo-dG in the autism and control cerebellum, and the BA22
samples. All measured metabolites were significantly altered
in autism compared with matched controls in both brain
regions analyzed. GSH was decreased in autism cerebellum
by 43% compared with control cerebellum (Po0.01).
Similarly, in BA22, GSH was 32% decreased in autism
cases compared with controls (Po0.01). The concentration
of GSSG was 18% higher (P¼0.02) and the resulting GSH/
GSSG redox ratio was 52% lower (Po0.01) in the
cerebellum from autism cases compared with controls. In
BA22, the autism samples exhibited 19% higher
concentration of GSSG (Po0.01) and 43% lower GSH/
GSSG (Po0.001) redox ratio compared with controls. The
concentration of 3-NT was elevated by 42 and 72% in autism
cerebellum (Po0.01) and BA22 (Po0.01), respectively,
compared with control samples. The level of 3-CT was
significantly increased by 95 and 38% in autism cerebellum
(Po0.01) and BA22 (Po0.01), respectively, compared with
control samples. The mean concentration of 8-oxo-dG was
increased 27% in cerebellum (Po0.01) and 21% in BA22
(P¼0.01) in autism compared with control tissues. Age was
not significantly correlated with any of the measured
biomarkers; however, post-mortem interval was negatively
associated with GSSG in the combined case/control cohort
of samples from BA22 (r¼0.57; P¼0.004) and in the
autism BA22 samples (r¼0.63; P¼0.03).
Aconitase activity. Figure 2a presents the aconitase activity
(nmol min
) measured in both cerebellum and BA22
autism and control samples. The mean aconitase activity in
the cerebellum from the autism cases was significantly lower
than in the cerebellum tissues from controls (45.3%; Po0.01).
There was a trend of decreased aconitase activity in BA22
from the autism cases compared with controls; however, it
failed to reach statistical significance (P¼0.1). A positive
association between aconitase activity and GSH/GSSG was
found in both cerebellum (P¼0.01) and BA22 (P¼0.03) in
our combined case and control cohort (correlation
coefficients ¼0.46 and 0.45, respectively). Figures 2b and c
illustrate this relationship and demonstrate that the autism
cases are clustered in the lower left quadrant of the graphs
with decreased GSH/GSSG and aconitase activity relative to
8-oxo-dG is associated with GSH/GSSG in the
cerebellum. Figure 3 illustrates the significant negative
association found between 8-oxo-dG and GSH/GSSG in the
cerebellum in the combined case and control cohort
(correlation coefficient ¼0.78; Po0.0001). Although the
association was not significant within the autism cohort, the
clustering of the autism cases in the bottom right quadrant with
significantly higher 8-oxo-dG levels and lower GSH/GSSG is
In the present investigation, we demonstrate for the first time
that the decreased glutathione-mediated redox/antioxidant
capacity previously observed in plasma and immune cells
from children with autism is also significantly decreased in two
Oxidative stress and inflammation in the autism brain
S Rose et al
Translational Psychiatry
brain regions previously shown to be affected in autism, the
cerebellum and BA22. Our findings also confirm previous
preliminary reports that markers of oxidative damage (3-NT and
8-oxo-dG) are increased in these two brain regions in individuals
with autism.
We further extend these findings by examining
a larger sample of carefully selected tissues for multiple markers
of oxidative protein/DNA damage (3-NT, 8-oxo-dG), as well as
functional biomarkers of inflammation (3-CT) and mitochondrial
superoxide production (aconitase activity).
A relative decrease in aconitase activity in the autism
cerebellum is an important new finding, suggesting a functional
consequence of oxidative stress in this region. In brain tissue,
aconitase is located primarily in the mitochondria where it
functions as an enzyme in the tricarboxylic acid cycle.
Mitochondrial aconitase is highly sensitive to oxidative
inactivation by superoxide radicals that are produced in close
proximity by the electron transport chain (ETC). Thus, in
addition to being a marker of oxidative protein damage, a
decrease in aconitase activity is considered to be a sensitive
indicator of elevated mitochondrial superoxide production.
The labile iron–sulfur (Fe-S) cluster present in the active
site of aconitase is a major target of excessive mitochondrial
superoxide. In the presence of sufficient reducing agents,
such as GSH or NADPH, aconitase can be restored to its
active form;
however, in the autism cerebellum, the
observed decrease in GSH concentration relative to controls
indicates a chronic deficit of reducing equivalents in this
region. As depicted in Figure 4, unscavenged superoxide
inactivates aconitase by displacing Fe
from the Fe-S
cluster, which then promotes the formation of the damaging
hydroxyl radical via reaction with H
and Fenton chemistry.
A fragile redox state within the mitochondria of individuals with
autism has been previously reported,
and may reflect a self-
amplifying cycle of antioxidant depletion and aconitase
inactivation. Several enzymes involved in ATP production
contain Fe-S clusters in the active site and are subject to
similar inactivation by superoxide, including oxoglutarate
dehydrogenase of the tricarboxylic acid cycle, as well as
ETC complexes I–III.
A deficit in the tricarboxylic acid cycle
and ETC function under conditions of excessive superoxide
production in the brain would be expected to result in a
reduced ability to maintain adequate levels of ATP required for
normal neuronal and synaptic functioning.
Aconitase inactivation and oxidative stress have been
noted in other neuropsychiatric and neurodegenerative
disorders with known mitochondrial involvement, including
Huntington’s disease
and Parkinson’s
One study of mitochondrial dysfunction in the
autism brain found decreased protein levels of multiple ETC
complexes in the cerebellum, frontal and temporal cortex.
There is mounting evidence that mitochondrial dysfunction
may be present in a significant subset of children with autism
GSH (nmol/mg protein)
15 **
GSSG nmol/mg protein
2.0 #*
20 **
3-NT pmol/mg protein
3-CT pmol/mg protein
100 * *
8-oxo-dG pmol/mg DNA
100 * **
Figure 1 Glutathione redox imbalance and increased biomarkers of oxidative stress in autism cerebellum (CB) and Brodmann area 22 (BA22). High-performance liquid
chromatography (HPLC) and HPLC/mass spectrometry were used to measure biomarkers in autism and control tissue samples from CB and BA22(n¼15 and 12 cases and
controls/group, respectively), and normalized for protein content. The concentrations of reduced glutathione (GSH; a), oxidized glutathione (GSSG; b), glutathione redox/
antioxidant capacity (GSH/GSSG; c), 3-nitrotyrosine (3-NT; d), 3-chlorotyrosine (3-CT; e), 8-oxo-deoxyguanosine (8-oxo-dG; f) are presented as mean±s.d. *Po0.01;
Oxidative stress and inflammation in the autism brain
S Rose et al
Translational Psychiatry
and contribute to the multisystem abnormalities seen in some
autistic children.
The significant decrease in aconitase
activity warrants continued investigation into interactions
between mitochondrial dysfunction, superoxide production
and altered ETC complex activity in autism.
The oxidized protein tyrosine derivative, 3-NT, provides a
stable biochemical footprint of oxidative protein damage and
has been found to be elevated in plasma of children with
autism in a previous study.
Elevated levels of 3-NT have
been described in a number of diseases with an oxidative
stress pathology, including alcoholism, smoking, diabetes,
atherosclerosis and cystic fibrosis.
The tyrosine derivative,
3-NT, is formed primarily from peroxynitrite, a damaging free
radical generated from superoxide and NO. Thus, the signi-
ficant increase in levels of 3-NT observed in the autism cere-
bellum and BA22 was not unexpected and is consistent with
elevated superoxide production and aconitase inactivation.
In addition to being a classic marker of oxidative protein
damage, elevated levels of 3-NT indicates elevated NO
production. Excessive NO competes with the antioxidants,
MnSOD and GSH, for superoxide and promotes the genera-
tion of peroxynitrite.
NO can reversibly inhibit mitochondrial
respiration at complex IV, whereas the more damaging
peroxynitrite can permanently inactivate complexes I, III
and V.
In a previous study, we demonstrated the increased
sensitivity of autism lymphoblastoid cells to acute NO-induced
mitochondrial membrane depolarization, and others have
reported elevated plasma and red blood cells levels of nitrites
in children with autism.
Neuroglial cells express iNOS
(inducible NO synthase) and produce high quantities of NO
when activated by cytokines.
Interestingly, the presence of
proinflammatory cytokines and activated neuroglia have been
reported in the autism cerebellum among other regions,
suggesting that activated neuroglia produce excess NO and
may contribute to the peroxynitrite formation and increased
3-NT protein damage observed in the present study.
A significantly elevated level of 3-CT in the autism
cerebellum and BA22 is a novel finding indicative of a chronic
neuroinflammatory state in these regions. Activated phago-
cytic cells produce hypochlorous acid, the product of
myeloperoxidase (MPO) activity that is stimulated during
Aconitase Activity
Figure 2 Aconitase activity is decreased in autism cerebellum (CB) and associated with glutathione redox/antioxidant capacity (GSH/GSSG) in control and autism CB, and
Brodmann area 22 (BA22). (a) Aconitase activity was measured in frozen post-mortem autism and control tissue samples from CB and BA22 (n¼15 and 12 cases and
controls/group, respectively), and normalized for protein content. Data are presented as mean±s.d. Aconitase activity was significantly decreased in autism CB (3.99±2.34)
compared with control CB (7.29±1.85). The difference in aconitase activity between autism BA22 (3.30±1.88) and control BA22 (5.47±3.78) did not reach significance.
(b) In the combined case and control cohort of samples from the CB, aconitase activity was significantly associated with GSH/GSSG (P¼0.01). (c) Within BA22 in the
combined case and control samples, aconitase activity was similarly significantly associated with GSH/GSSG (P¼0.03). Although the significance of the correlations does not
hold within the autism samples, the sample-specific clustering of case values (in open circles) in the bottom left quadrant of each graph is apparent. *Po0.01.
Figure 3 The 8-oxo-deoxyguanosine (8-oxo-dG) is associated with glutathione
redox/antioxidant capacity (GSH/GGSG) in the cerebellum. In the combined
case and control cohort of samples from the cerebellum, 8-oxo-dG was signifi-
cantly associated with GSH/GSSG (Po.0001). Although the significance of the
correlations does not hold within the autism samples, the sample-specific
clustering of case values (in open circles) in the bottom left quadrant of each
graph is apparent.
Oxidative stress and inflammation in the autism brain
S Rose et al
Translational Psychiatry
immune activation, resulting in the 3-CT derivative.
vated expression of MPO has previously been demonstrated
in chronic neurological disease states, such as Alzheimer’s
Parkinson’s disease
and multiple sclerosis.
The observed increase in 3-CT in the autism cerebellum and
BA22 samples is the first indication of elevated MPO
expression in the autism brain, and supports previous reports
of microglial activation and inflammatory cytokines in autism
The role of inflammation and microglial activa-
tion in the neuropathology of autism warrants further
investigation and confirmation.
In addition to markers of protein oxidative damage, 8-oxo-
dG, a marker of DNA oxidative damage, was significantly
elevated in both cerebellum and BA22 from the autism cases
relative to controls. The 8-oxo-dG adduct in the mitochondrial
and nuclear DNA is a pre-mutagenic lesion formed primarily
by an attack by the hydroxyl radical (
OH). It has been
associated with oxidative DNA damage in conditions such as
aging, cancer and pro-oxidant environmental exposures.
the cerebellum, 8-oxo-dG was negatively associated with
GSH/GSSG in the combined case and control cohort
(Figure 3); however, this association failed to reach signifi-
cance in the BA22 region. The superoxide-mediated release
of Fe
associated with mitochondrial aconitase inactivation
has been shown to be a significant source of OH radical
formation through Fenton chemistry.
Taken together, these
data suggest the reduced GSH/GSSG antioxidant capacity is
insufficient to counter excessive
OH production, and that
OH can reach the nucleus to create the oxidative
DNA adduct, 8-oxo-dG.
The hypothesized interactions between each of the
measured biomarkers of oxidative stress and damage,
mitochondrial dysfunction and inflammation are diagrammed
in Figure 5. Elevated superoxide generated from dysfunc-
tional mitochondria promotes the formation of excessive
, the substrate for MPO-mediated hypochlorous acid
synthesis and the generation of the inflammatory biomarker,
3-CT. Elevated superoxide can combine with NO, resulting in
the formation of the peroxynitrite radical and the protein
oxidative damage biomarker, 3-NT. The hydroxyl radical is
generated by both aconitase inactivation and MPO activity,
and promotes the formation of 8-oxo-dG. Chronic elevation of
these free radicals will deplete GSH/GSSG redox/antioxidant
capacity, allowing unopposed free-radical generation and a
self-perpetuating cycle, leading to chronic oxidative stress
and damage. The lack of a correlation between age and the
biomarkers suggests that oxidative stress is a chronic
condition in autism, because the same pattern of elevated
biomarkers is seen over such a wide age range.
In summary, we show for the first time that peripheral
markers of oxidative stress and damage previously observed
in plasma and immune cells are similarly elevated in two
affected brain regions in autism, cerebellum and BA22.
Together, these observations suggest that a pro-oxidant
environment and oxidative stress are pervasive and systemic
in individuals with autism. The negative association between
GSH/GSSG, and oxidative protein and DNA damage suggest
that decreased glutathione redox capacity in the autism brain
may have functional consequence in terms of increased
mitochondrial superoxide production and a chronic inflamma-
tory state. Nonetheless, because autism is influenced by
multiple interacting genetic and environmental factors that are
case-specific and inherent limitations in post-mortem brain,
these observations will require confirmation in subsequent
Figure 4 Mechanism of mitochondrial aconitase inactivation. Mitochondrial
aconitase is a tricarboxylic acid (TCA) cycle enzyme that catalyzes the conversion
of citrate to isocitrate. It contains an iron-sulfur cluster ([4Fe-4S]) in its active site
that is inactivated by superoxide (O
) produced in close proximity as a byproduct
of the electron transport chain. This results in the release of a Fe
and a molecule
of hydrogen peroxide (H
), which, through the Fenton reaction, can react
to produce a hydroxyl radical (
OH). The glutathione redox capacity (GSH/GSSG)
will decrease as a result of the elevated free-radical production and will allow
more superoxide inactivation of aconitase, creating a self-amplifying cycle if left
Figure 5 Proposed interactions between measured biomarkers and oxidative
stress. Elevated superoxide generated from dysfunctional mitochondria promotes
the formation of excess H
, the substrate for myeloperoxidase (MPO)-mediated
hypochlorous acid (HOCl) synthesis and the generation of the inflammatory
biomarker, 3-chlorotyrosine (3-CT). An elevation in nitric oxide (NO) combined with
elevated superoxide levels results in the formation of the peroxynitrite radical and
the protein oxidative damage biomarker, 3-nitrotyrosine (3-NT). The hydroxyl radical
is generated by both aconitase inactivation and MPO, and promotes the formation of
8-oxo-deoxyguanosine (8-oxo-dG). Chronic elevation of these free radicals will
deplete the glutathione redox/antioxidant capacity (GSH/GSSG), allowing
unopposed free-radical generation and a self-perpetuating cycle, leading to chronic
oxidative stress.
Oxidative stress and inflammation in the autism brain
S Rose et al
Translational Psychiatry
Conflict of interest
The authors declare no conflict of interest.
Acknowledgements. We would like to thank the families of individuals
with autism for the thoughtful donation of post-mortem tissues to the Autism Tissue
Program at the Harvard Brain Tissue Resource Center, Belmont, MA,USA, and the
NICHD Brain and Tissue Bank for Developmental Disorders at the University of
Maryland, Baltimore, MD, USA. This work was supported, in part, by the National
Institute of Child Health and Development (RO1 HD051873 to SJJ) and the Jane
Botsford Johnson Foundation.
1. CDC. Prevalence of autism spectrum disorders - Autism and Developmental Disabilities
Monitoring Network, United States, 2006. MMWR Surveill Summ 2009; 58: 1–20.
2. Amaral DG, Schumann CM, Nordahl CW. Neuroanatomy of autism. Trends Neurosci 2008;
31: 137–145.
3. Brambilla P, Hardan A, di Nemi SU, Perez J, Soares JC, Barale F. Brain anatomy
and development in autism: review of structural MRI studies. Brain Res Bull 2003; 61:
4. Pardo CA, Eberhart CG. The neurobiology of autism. Brain Pathol 2007; 17: 434–447.
5. Whitney ER, Kemper TL, Bauman ML, Rosene DL, Blatt GJ. Cerebellar Purkinje cells are
reduced in a subpopulation of autistic brains: a stereological experiment using calbindin-
D28k. Cerebellum 2008; 7: 406–416.
6. Scott JA, Schumann CM, Goodlin-Jones BL, Amaral DG. A comprehensive volumetric
analysis of the cerebellum in children and adolescents with autism spectrum disorder.
Autism Res 2009; 2: 246–257.
7. Courchesne E, Karns CM, Davis HR, Ziccardi R, Carper RA, Tigue ZD et al. Unusual brain
growth patterns in early life in patients with autistic disorder: an MRI study. Neurology 2001;
57: 245–254.
8. Kemper TL, Bauman ML. The contribution of neuropathologic studies to the under standing
of autism. Neurol Clin 1993; 11: 175–187.
9. Vargas DL, Nascimbene C, Krishnan C, Zimmerman AW, Pardo CA. Neuroglial activation
and neuroinflammation in the brain of patients with autism. Ann Neurol 2005; 57: 67–81.
10. Singer HS, Morris CM, Williams PN, Yoon DY, Hong JJ, Zimmerman AW. Antibrain
antibodies in children with autism and their unaffected siblings. J Neuroimmunol 2006; 178:
11. Goines P, Haapanen L, Boyce R, Duncanson P, Braunschweig D, Delwiche L et al.
Autoantibodies to cerebellum in children with autism associate with behavior. Brain Behav
Immun 2011; 25: 514–523.
12. Bigler ED, Mortensen S, Neeley ES, Ozonoff S, Krasny L, Johnson M et al. Superior
temporal gyrus, language function, and autism. Dev Neuropsychol 2007; 31: 217–238.
13. Gage NM, Juranek J, Filipek PA, Osann K, Flodman P, Isenberg AL et al. Rightward
hemispheric asymmetries in auditory language cortex in children with autistic disorder: an
MRI investigation. J Neurodev Disord 2009; 1: 205–214.
14. Jou RJ, Minshew NJ, Keshavan MS, Vitale MP, Hardan AY. Enlarged right superior
temporal gyrus in children and adolescents with autism. Brain Res 2010; 1360: 205–212.
15. Lopez-Hurtado E, Prieto JJ. A micros copic study of language-related cortex in autism. Am J
Biochem Biotechnol 2008; 4: 130–145.
16. Garbett K, Ebert PJ, Mitchell A, Lintas C, Manzi B, Mirnics K et al. Immune transcriptome
alterations in the temporal cortex of subjects with autism. Neurobiol Dis 2008; 30: 303–311.
17. Butterfield DA, Drake J, Pocernich C, Castegna A. Evidence of oxidative damage in
Alzheimer’s disease brain: central role for amyloid beta-peptide. Trends Mol Med 2001; 7:
18. Martin HL, Teismann P. Glutathione–a review on its role and significance in Parkinson’s
disease. FASEB J 2009; 23: 3263–3272.
19. Nishioka N, Arnold SE. Evidence for oxidative DNA damage in the hippocampus of elderly
patients with chronic schizophrenia. Am J Geriatr Psychiatry 2004; 12: 167–175.
20. Yao JK, Leonard S, Reddy R. Altered glutath ione redox state in schizophrenia. Dis Markers
2006; 22: 83–93.
21. Andreazza AC, Kauer-Sant’anna M, Frey BN, Bond DJ, Kapczinski F, Young LT et al.
Oxidative stress markers in bipolar disorder: a meta-analysis. J Affect Disord 2008; 111:
22. Nordmann R. Oxidative stress from alcohol in the brain. Alcohol Alcohol Suppl 1987; 1:
23. Chauhan A, Chauhan V, Brown WT, Cohen I. Oxidative stress in autism: increased lipid
peroxidation and reduced serum levels of ceruloplasmin and transferrin–the antioxidant
proteins. Life Sci 2004; 75: 2539–2549.
24. Sogut S, Zoroglu SS, Ozyurt H, Yilmaz HR, Ozugurlu F, Sivasli E et al. Changes in nitric
oxide levels and antioxidant enzyme activities may have a role in the pathophysiological
mechanisms involved in autism. Clin Chim Acta 2003; 331: 111–117.
25. Boso M, Emanuele E, Minoretti P, Arra M, Politi P, Ucelli di Nemi S et al. Alterations of
circulating endogenous secretory RAGE and S100A9 levels indicating dysfunction of the
AGE-RAGE axis in autism. Neurosci Lett 2006; 410: 169–173.
26. James SJ, Cutler P, Melnyk S, Jernigan S, Janak L, Gaylor DW et al. Metabolic biomarkers
of increased oxidative stress and impaired methylation capacity in children with autism.
Am J Clin Nutr 2004; 80: 1611–1617.
27. James SJ, Melnyk S, Fuchs G, Reid T, Jernigan S, Pavliv O et al. Efficacy of
methylcobalamin and folinic acid treatment on glutathione redox status in children with
autism. Am J Clin Nutr 2009; 89: 425–430.
28. James SJ, Melnyk S, Jernigan S, Cleves MA, Halsted CH, Wong DH et al. Metabolic
endophenotype and related genotypes are associated with oxidative stress in children with
autism. Am J Med Genet B Neuropsychiatr Genet 2006; 141B: 947–956.
29. Rose S, Melnyk S, Trusty TA, Pavliv O, Seidel L, Li J et al. Intracellular and extracellular
redox status and free radical generation in primary immune cells from children with autism.
Autism Res Treatment 2011; 2012.
30. Jones DP. Extracellular redox state: refining the definition of oxidative stress in aging.
Rejuvenation Res 2006; 9: 169–181.
31. Melnyk S, Fuchs GJ, Schulz E, Lopez M, Kahler SG, Fu ssell JJ et al. Metabolic imbalance
associated with methylation dysregulation and oxidative damage in children with autism.
J Autism Dev Disord 2012; 42: 367–377.
32. Sajdel-Sulkowska EM, Xu M, Koibuchi N. Increase in cerebellar neurotrophin-3 and
oxidative stress markers in autism. Cerebellum 2009; 8: 366–372.
33. Sajdel-Sulkowska EM, Xu M, McGinnis W, Koibuchi N. Brain region-specific changes in
oxidative stress and neurotrophin levels in autism spectrum disorders (ASD). Cerebellum
2011; 10: 43–48.
34. Chauhan A, Gu F, Essa MM, Wegiel J, Kaur K, Ted Brown W et al. Brain region-specific
deficit in mitochondrial electron transport chain complexes in children with autism.
J Neurochem 2011; 117: 209–220.
35. Heinecke JW, Hsu FF, Crowley JR, Hazen SL, Leeuwenburgh C, Mueller DM et al.
Detecting oxidative modification of biomolecules with isotope dilution mass spectrometry:
sensitive and quantitative assays for oxidized amino acids in proteins and tissues. Methods
Enzymol 1999; 300: 124–144.
36. Gardner PR, Fridovich I. Superoxide sensitivity of the Escherichia coli aconitase. J Biol
Chem 1991; 266: 19328–19333.
37. Koen AL, Goodman M. Aconitate hydratase isozymes: subcellular location, tissue
distribution and possible subunit structure. Biochim Biophys Acta 1969; 191: 698–701.
38. Fridovich I. Superoxide anion radical (O2-.), superoxide dismutases, and related matters.
J Biol Chem 1997; 272: 18515–18517.
39. Gardner PR, Fridovich I. Inactivation-reactivation of aconitase in Escherichia coli.
A sensitive measure of superoxide radical. J Biol Chem 1992; 267: 8757–8763.
40. Melnyk S, Pogribna M, Pogribny I, Hine RJ, James SJ. A new HPLC method for the
simultaneous determination of oxidized and reduced plasma aminothiols using coulometric
electrochemical detection. J Nutr Biochem 1999; 10: 490–497.
41. Melnyk S, Pogribna M, Pogribny IP, James SJ. Measurement of plasma and intracellular
S-adenosylmethionine and S-adenosylhomocysteine utilizing coulemetric electrochemical
detection: alteration with plasma homocysteine and pyridoxal 50-phosphate concentrations.
Clin Chem 2000; 46: 265–272.
42. Chango A, Abdel Nour AM, Niquet C, Tessier FJ. Simultaneous determination of genomic
DNA methylation and uracil misincorporation. Med Princ Pract 2009; 18: 81–84.
43. Helbock HJ, Beckman KB, Shigenaga MK, Walter PB, Woodall AA, Yeo HC et al. DNA
oxidation matters: the HPLC-electrochemical detection assay of 8-oxo-deoxyguanosine
and 8-oxo-guanine. Proc Natl Acad Sci USA 1998; 95: 288–293.
44. Sajdel-Sulkowska EM, Lipinski B, Windom H, Audhya T, McGinnis W. Oxidative stress in
autism: cerebellar 3 nitrotyrosine levels. Am J Biochem Biotechnol 2008; 4: 73–84.
45. Liang LP, Ho YS, Patel M. Mitochondrial superoxide production in kainate-induced
hippocampal damage. Neuroscience 2000; 101: 563–570.
46. Liochev SI, Fridovich I. The role of O2.- in the production of HO: in vitro and in vivo.Free
Radic Biol Med 1994; 16: 29–33.
47. Vasquez-Vivar J, Kalyanaraman B, Kennedy MC. Mitochondrial aconitase is a source
of hydroxyl radical. An electron spin resonance investigation. J Biol Chem 2000; 275:
48. James SJ, Rose S, Melnyk S, Jernigan S, Blossom S, Pavliv O et al. Cellular and
mitochondrial glutathione redox imbalance in lymphoblastoid cells derived from children
with autism. FASEB J 2009; 23: 2374–2383.
49. Welter R, Yu L, Yu CA. The effects of nitric oxide on electron transport complexes. Arch
Biochem Biophys 1996; 331: 9–14.
50. Bubber P, Hartounian V, Gibson GE, Blass JP. Abnormalities in the tricarboxylic acid
(TCA) cycle in the brains of schizophrenia patients. Eur Neuropsychopharmacol 2011; 21:
51. Prabakaran S, Swatton JE, Ryan MM, Huffaker SJ, Huang JT, Griffin JL et al. Mitochondrial
dysfunction in schizophrenia: evidence for compromised brain metabolism and oxidative
stress. Mol Psychiatry 2004; 9: 684–697, 643.
52. Sorolla MA, Reverter-Branchat G, Tamarit J, Ferrer I, Ros J, Cabiscol E. Proteomic and
oxidative stress analysis in human brain samples of Huntington disease. Free Radic Biol
Med 2008; 45: 667–678.
53. Liang LP, Patel M. Iron-sulfur enzyme mediated mitochondrial superoxide toxicity in
experimental Parkinson’s disease. J Neurochem 2004; 90: 1076–1084.
54. Pitkanen S, Robinson BH. Mitochondrial complex I deficiency leads to increased
production of superoxide radicals and induction of superoxide dismutase. J Clin Invest
1996; 98: 345–351.
Oxidative stress and inflammation in the autism brain
S Rose et al
Translational Psychiatry
55. Frye RE, Rossignol DA. Mitochondrial dysfunction can connect the diverse medical
symptoms associated with autism spectrum disorders. Pediatr Res 2011; 69(5 Part 2):
56. Rossignol DA, Frye RE. Mitochondrial dysfunction in autism spectrum disorders: a
systematic review and meta-analysis. Mol Psychiatry 2012; 17: 290–314.
57. Giulivi C, Zhang YF, Omanska-Klusek A, Ross-Inta C, Wong S, Hertz-Picciotto I et al.
Mitochondrial dysfunction in autism. JAMA 2010; 304: 2389–2396.
58. Mohiuddin I, Chai H, Lin PH, Lumsden AB, Yao Q, Chen C. Nitrotyrosine and
chlorotyrosine: clinical significance and biological functions in the vascular system. J Surg
Res 2006; 133: 143–149.
59. Bishop A, Anderson JE. NO signaling in the CNS: from the physiological to the
pathological. Toxicology 2005; 208: 193–205.
60. Brown GC. Nitric oxide and mitochondrial respiration. Biochim Biophys Acta 1999; 1411:
61. Zoroglu SS, Yurekli M, Meram I, Sogut S, Tutkun H, Yetkin O et al. Pathophysio-
logical role of nitric oxide and adrenomedullin in autism. Cell Biochem Funct 2003; 21:
62. Sweeten TL, Posey DJ, Shankar S, McDougle CJ. High nitric oxide production in autistic
disorder: a possible role for interferon-gamma. Biol Psychiatry 2004; 55: 434–437.
63. Bolanos JP, Herrero-Mendez A, Fernandez-Fernandez S, Almeida A. Linking glycolysis
with oxidative stress in neural cells: a regulatory role for nitric oxide. Biochem Soc Trans
2007; 35: 1224–1227.
64. Brown GC, Bolanos JP, Heales SJ, Clark JB. Nitric oxide produced by activated astrocytes
rapidly and reversibly inhibits cellular respiration. Neurosci Lett 1995; 193: 201–204.
65. Green PS, Mendez AJ, Jacob JS, Crowley JR, Growdon W, Hyman BT et al. Neuronal
expression of myeloperoxidase is increased in Alzheimer’s disease. J Neurochem 2004;
90: 724–733.
66. Choi DK, Pennathur S, Perier C, Tieu K, Teismann P, Wu DC et al. Ablation of the
inflammatory enzyme myeloperoxidase mitigates features of Parkinson’s disease in mice.
J Neurosci 2005; 25: 6594–6600.
67. Nagra RM, Becher B, Tourtellotte WW, Antel JP, Gold D, Paladino T et al.
Immunohistochemical and genetic evidence of myeloperoxidase involvement in multiple
sclerosis. J Neuroimmunol 1997; 78: 97–107.
68. Pilger A, Rudiger HW. 8-Hydroxy-20-deoxyguanosine as a marker of oxidative DNA
damage related to occupational and environmental exposures. Int Arch Occup Environ
Health 2006; 80: 1–15.
Translational Psychiatry is an open-access journal
published by Nature Publishing Group. This work is
licensed under the Creative Commons Attribution-Noncommercial-No
Derivative Works 3.0 Unported License. To view a copy of this license,
Supplementary Information accompanies the paper on the Translational Psychiatry website (
Oxidative stress and inflammation in the autism brain
S Rose et al
Translational Psychiatry
... The biomarkers of interest included: (1) 8-oxo-deoxyguanine (8-OHdG), an oxidative DNA adduct (Valavanidis et al., 2009); (2) Glutathione (GSH), the primary intracellular ROS antioxidant and detoxification mechanism in the body (Kim et al., 2015); (3) Glutathione disulfide (GSSG), the oxidized counterpart to GSH and a marker of extra-cellular oxidative stress; (4) the ratio of GSH:GSSG, which acts as an indicator of redox balance (Jones, 2002); and (5) 3-nitrotyrosine (nitrotyrosine), an amino acid modified by however, left unregulated, OS can induce DNA damage, protein oxidation, and lipid peroxidation (Birben et al., 2012;Thompson & Al-Hasan, 2012). The brain is particularly susceptible to OS due to its demand for oxygen and high lipid cell content, particularly during fetal development (Kim et al., 2015;Rose et al., 2012). OS has been hypothesized as a mechanism in ASD etiology, given the susceptibility of the fetus to ROS (Dennery, 2010; Thompson & Al-Hasan, 2012), and the links between multiple risk factors for ASD and OS, including low birth weight, maternal conditions such as preeclampsia, as well as environmental exposures such as air pollution and pesticides (Shelton et al., 2012;Lopez-Tinoco et al., 2013;Matsubasa et al., 2002;Olsson et al., 2010;Atamer et al., 2005). ...
... Some prior work has suggested associations between maternal OS and broader neurodevelopmental outcomes (Rommel et al., 2020;Wells et al., 2016;Wells et al., 2009). Direct links between OS and ASD specifically have primarily come from studies examining levels of ROS in children diagnosed with ASD, suggesting increased OS in cases vs. controls in plasma and post-mortem brain samples (James et al., 2004(James et al., , 2006Melnyk et al., 2012;Rose et al., 2012). However, retrospective studies in children already diagnosed with ASD cannot provide evidence as to whether OS differences are a cause or a consequence of ASD. ...
Full-text available
We examined associations between prenatal oxidative stress (OS) and child autism-related outcomes. Women with an autistic child were followed through a subsequent pregnancy and that younger sibling’s childhood. Associations between glutathione (GSH), glutathione disulfide (GSSG), 8-oxo-deoxyguanine (8-OHdG), and nitrotyrosine and younger sibling Social Responsiveness Scale (SRS) scores were examined using quantile regression. Increasing GSH:GSSG (suggesting decreasing OS) was associated with minor increases in SRS scores (50th percentile β: 1.78, 95% CI: 0.67, 3.06); no other associations were observed. Results from this cohort with increased risk for autism do not support a strong relationship between OS in late pregnancy and autism-related outcomes. Results may be specific to those with enriched autism risk; future work should consider other timepoints and biomarkers.
... Upon inflammatory stimuli, astrocytes not only secrete cytokines but also upregulate adhesion proteins such as ICAM-1 [18]. The provision of GSH is also altered in many pathological conditions [19][20][21][22][23]. ...
... Astrocytes synthesize and secrete GSH [63,64], shielding CNS cells from oxidative stress, which is also reflected in the high astrocytic GSH content of ~ 8 mM [65]. Interestingly, GSH availability is altered in traumatic brain injury [19] and autism [20,21], while GSH efflux is impaired in AD [22,23]. A largely unexplored astrocytic aspect in stem cell-derived astrocytes is the capacity of astrocytes in vivo to protect the CNS against oxidative stress through GSH. ...
Full-text available
The generation of astrocytes from human induced pluripotent stem cells has been hampered by either prolonged differentiation—spanning over two months—or by shorter protocols that generate immature astrocytes, devoid of salient mature astrocytic traits pivotal for central nervous system (CNS) modeling. We directed stable hiPSC-derived neuroepithelial stem cells to human iPSC-derived Astrocytes (hiAstrocytes) with a high percentage of star-shaped cells by orchestrating an astrocytic-tuned culturing environment in 28 days. We employed RT-qPCR and ICC to validate the astrocytic commitment of the neuroepithelial stem cells. To evaluate the inflammatory phenotype, we challenged the hiAstrocytes with the pro-inflammatory cytokine IL-1β (interleukin 1 beta) and quantitatively assessed the secretion profile of astrocyte-associated cytokines and the expression of intercellular adhesion molecule 1 (ICAM-1). Finally, we quantitatively assessed the capacity of hiAstrocytes to synthesize and export the antioxidant glutathione. In under 28 days, the generated cells express canonical and mature astrocytic markers, denoted by the expression of GFAP, AQP4 and ALDH1L1. In addition, the notion of a mature phenotype is reinforced by the expression of both astrocytic glutamate transporters EAAT1 and EAAT2. Thus, hiAstrocytes have a mature phenotype that encompasses traits critical in CNS modeling, including glutathione synthesis and secretion, upregulation of ICAM-1 and a cytokine secretion profile on a par with human fetal astrocytes. This protocol generates a multifaceted astrocytic model suitable for in vitro CNS disease modeling and personalized medicine. Graphical abstract
... Redox homeostasis is a balance between the production of oxidants and endogenous antioxidant defense mechanisms and its disturbance leads to oxidative stress that has deteriorating effects on cells [16]. Oxidative stress, clinically defined by a decrease in the GSH/GSSG ratio, has been described in many neurobehavioral disorders, including autism [13,37,39,61]. Indeed, as mentioned above, multiple studies, including ours, reported lower GSH levels and a lower GSH/GSSG ratio in patients with autism in comparison to healthy controls [16,18,23,26], which were elevated during methylcobalamin treatment [31,37,44]. ...
Full-text available
(1) Background: Autism, also known as autism-spectrum disorder, is a pervasive developmental disorder affecting social skills and psychological status in particular. The complex etiopathogenesis of autism limits efficient therapy, which leads to problems with the normal social integration of the individual and causes severe family distress. Injectable methylcobalamin was shown to improve the clinical status of patients via enhanced cell oxidative status and/or methylation capacity. Here we tested the efficiency of a syrup form of methylcobalamin in treating autism. (2) Methods: Methylcobalamin was administered daily at 500 µg dose to autistic children and young adults (n = 25) during a 200-day period. Clinical and psychological status was evaluated by parents and psychologists and plasma levels of reduced and oxidized glutathione, vitamin B12, homocysteine, and cysteine were determined before the treatment, and at day 100 and day 200 of the treatment. (3) Results: Good patient compliance was reported. Methylcobalamin treatment gradually improved the overall clinical and psychological status, with the highest impact in the social domain, followed by the cognitive, behavioral and communication characteristics. Changes in the clinical and psychological status were strongly associated with the changes in the level of reduced glutathione and reduced/oxidized glutathione ratio. (4) Conclusion: A high dose of methylcobalamin administered in syrup form ameliorates the clinical and psychological status of autistic individuals, probably due to the improved oxidative status.
... For example, Silva et al. [41] studied β s -haplotypes and Hb F levels of oxidative stress markers in sickle cell anemia in Brazil. Rose et al. [42] examined the levels of biomarkers for oxidative stress in the cerebellum and temporal cortex from autistic patients and unaffected controls. Mice were tested for oxidative damage to the liver caused by morphine [43]. ...
Full-text available
The imbalance of oxidation and antioxidant systems in the biological system can lead to oxidative stress, which is closely related to the pathogenesis of many diseases. Substances with antioxidant capacity can effectively resist the harmful damage of oxidative stress. How to measure the antioxidant capacity of antioxidants has essential application value in medicine and food. Techniques such as DPPH radical scavenging have been developed to measure antioxidant capacity. However, these traditional analytical techniques take time and require large instruments. It is a more convenient method to evaluate the antioxidant capacity of antioxidants based on their electrochemical oxidation and reduction behaviors. This review summarizes the evaluation of antioxidants using electrochemical sensors by bibliometrics. The development of this topic was described, and the research priorities at different stages were discussed. The topic was investigated in 1999 and became popular after 2010 and has remained popular ever since. A total of 758 papers were published during this period. In the early stages, electrochemical techniques were used only as quantitative techniques and other analytical techniques. Subsequently, cyclic voltammetry was used to directly study the electrochemical behavior of different antioxidants and evaluate antioxidant capacity. With methodological innovations and assistance from materials science, advanced electrochemical sensors have been fabricated to serve this purpose. In this review, we also cluster the keywords to analyze different investigation directions under the topic. Through co-citation of papers, important papers were analyzed as were how they have influenced the topic. In addition, the author’s country distribution and category distribution were also interpreted in detail. In the end, we also proposed perspectives for the future development of this topic.
... Autism pathogenesis is characterized by elevated oxidative stress markers [87,88]. Autistic patients have increased glutathione oxidation, decreased glutathione redox ratios in the cerebellum and temporal cortices and decreased Nrf2 expression along with increased inflammation and nitrative stress [90,91]. The activation of the JAK-STAT signalling pathway by hydroxyl (OH) free radicals causes an increase in oxidative stress. ...
Full-text available
The Janus-kinase (JAK) and signal transducer activator of transcription (STAT) signalling pathways regulate gene expression and control various factors involved in normal physiological functions such as cell proliferation, neuronal development, and cell survival. JAK activation phosphorylates STAT3 in astrocytes and microglia, and this phosphorylation has been linked to mitochondrial damage, apoptosis, neuroinflammation, reactive astrogliosis, and genetic mutations. As a regulator, peroxisome proliferator-activated receptor gamma (PPAR-gamma), in relation to JAK-STAT signalling, prevents this phosphorylation and aids in the treatment of the above-mentioned neurocomplications. Changes in cellular signalling may also contribute to the onset and progression of autism. Thus, PPAR-gamma agonist upregulation may be associated with JAK-STAT signal transduction downregulation. It may also be responsible for attenuating neuropathological changes by stimulating SOCS3 or involving RXR or SMRT, thereby reducing transcription of the various cytokine proteins and genes involved in neuronal damage. Along with JAK-STAT inhibitors, PPAR-gamma agonists could be used as target therapeutic interventions for autism. This research-based review explores the potential involvement and mutual regulation of JAK-STAT and PPAR-gamma signalling in controlling multiple pathological factors associated with autism.
Epidemiological studies increasingly associate air pollution with neurodevelopmental disorders, such as autism spectrum disorder, attention deficit hyperactivity disorder, and schizophrenia, all male‐biased disorders, with such effects reported in different cohorts and in different countries. Studies in animal models are providing biological plausibility for these reported associations. Multiple mechanisms may underlie these impairments, such as microglial activation, inflammation, and alterations in brain neurochemistry, including excitatory/inhibitory balance. Future studies of air pollution exposures, specifically ambient exposures, will be critical to further understanding how critical periods of brain development influence outcome, as well as the basis for sex differences in the neurotoxicity produced by developmental exposures. Such studies may be assisted by assessment of the role of specific contaminants of air pollution, e.g., metals and trace elements, in producing neurotoxicity.
Autism spectrum disorder is an increasingly prevalent neurodevelopmental disorder in the world today, with an estimated 2% of the population being affected in the USA. A major complicating factor in diagnosing, treating, and understanding autism spectrum disorder is that defining the disorder is solely based on the observation of behavior. Thus, recent research has focused on identifying specific biological abnormalities in autism spectrum disorder that can provide clues to diagnosis and treatment. Biomarkers are an objective way to identify and measure biological abnormalities for diagnostic purposes as well as to measure changes resulting from treatment. This current opinion paper discusses the state of research of various biomarkers currently in development for autism spectrum disorder. The types of biomarkers identified include prenatal history, genetics, neurological including neuroimaging, neurophysiologic, and visual attention, metabolic including abnormalities in mitochondrial, folate, trans-methylation, and trans-sulfuration pathways, immune including autoantibodies and cytokine dysregulation, autonomic nervous system, and nutritional. Many of these biomarkers have promising preliminary evidence for prenatal and post-natal pre-symptomatic risk assessment, confirmation of diagnosis, subtyping, and treatment response. However, most biomarkers have not undergone validation studies and most studies do not investigate biomarkers with clinically relevant comparison groups. Although the field of biomarker research in autism spectrum disorder is promising, it appears that it is currently in the early stages of development.
Background: Increasing evidence exists that higher levels of androgens can be found in individuals with autism. Evidence yields to a susceptible role of Cytochrome P450 17A1 (CYP17A1) with its catalyzation of the two distinct types of substrate oxidation by a hydroxylase activity (17-alpha hydroxylase) and C17/20 lyase activity. However, to what extent steps are altered in affected children with autism versus healthy controls remains to be elucidated. Methods: Urine samples from 48 boys with autism (BMI 19.1 ± 0.6 kg/m2, age 14.2 ± 0.5 years) and a matched cohort of 48 healthy boys (BMI 18.6 ± 0.3 kg/m2, 14.3 ± 0.5 years) as well as 16 girls with autism (BMI 17.5 ± 0.7 kg/m2, age 13.8 ± 1.0 years) and a matched cohort of 16 healthy girls (BMI 17.2 ± 0.8 kg/m2, age 13.2 ± 0.8 years) were analyzed for steroid hormone metabolites by gas chromatography-mass spectrometry. Results: The activity of 17-alpha Hydroxylase increased by almost 50%, whereas activity of 17/20 Lyase activity increased by around 150% in affected children with autism. Furthermore, the concentration of Cortisol was higher as compared to the average increase of the three metabolites TH-Corticosterone, 5α-TH-Corticosterone and TH-11β-DH-Corticosterone, indicating, in addition, a stimulation by the CRH-ACTH system despite a higher enzymatic activity. Discussion: As it was shown that oxidative stress increases the 17/20-lyase activity via p38α, a link between higher steroid hormone levels and oxidative stress can be established. However, as glucocorticoid as well as androgen metabolites showed higher values in subjects affected with autism as compared to healthy controls, the data indicate, despite higher CYP17A1 activity, the presence of increased substrate availability in line with the Cholesterol theory of autism.
Full-text available
Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by three core symptoms, specifically impaired social behavior, stereotypic/repetitive behaviors, and sensory/communication deficits. Although the exact pathophysiology of ASD is still unknown, host genetics, oxidative stress, and compromised blood brain barrier (BBB) have been implicated in predisposition to ASD. With regards to genetics, mutations in the genes such as CNTNAP2 have been associated with increased susceptibility of developing ASD. Although some studies observed conflicting results suggesting no association of CNTNAP2 with ASD, other investigations correlated this gene with autism. In addition, CNTNAP2 mediated signaling is generally considered to play a role in neurological disorders due to its critical role in neurodevelopment, neurotransmission, and synaptic plasticity. In this investigation, we studied BBB integrity and oxidative stress in Cntnap2−/− rats. We observed that the BBB permeability was significantly increased in Cntnap2−/− rats compared to littermate wild-type (WT) animals as determined by FITC-dextran and Evans blue assay. High levels of thiobarbituric acid reactive substances and lower amounts of reduced glutathione were observed in brain homogenates of Cntnap2−/− rats, suggesting oxidative stress. Brain sections from Cntnap2−/− rats showed intense inducible nitric oxide synthase immunostaining, which was undetectable in WT animals. Quantification of nitric oxide in brain homogenates revealed significantly high levels in Cntnap2−/− rats compared to the control group. As increased permeability of the BBB and oxidative stress have been observed in ASD individuals, our results suggest that Cntnap2−/− rats have a high construct and face validity and can be explored to develop effective therapeutic modalities.
Full-text available
The modulation of the redox microenvironment is an important regulator of immune cell activation and proliferation. To investigate immune cell redox status in autism we quantified the intracellular glutathione redox couple (GSH/GSSG) in resting peripheral blood mononuclear cells (PBMCs), activated monocytes and CD4 T cells and the extracellular cysteine/cystine redox couple in the plasma from 43 children with autism and 41 age-matched control children. Resting PBMCs and activated monocytes from children with autism exhibited significantly higher oxidized glutathione (GSSG) and percent oxidized glutathione equivalents and decreased glutathione redox status (GSH/GSSG). In activated CD4 T cells from children with autism, the percent oxidized glutathione equivalents were similarly increased, and GSH and GSH/GSSG were decreased. In the plasma, both glutathione and cysteine redox ratios were decreased in autistic compared to control children. Consistent with decreased intracellular and extracellular redox status, generation of free radicals was significantly elevated in lymphocytes from the autistic children. These data indicate primary immune cells from autistic children have a more oxidized intracellular and extracellular microenvironment and a deficit in glutathione-mediated redox/antioxidant capacity compared to control children. These results suggest that the loss of glutathione redox homeostasis and chronic oxidative stress may contribute to immune dysregulation in autism.
We and others have found that physiological/pathological levels of nitric oxide (NO) reversibly inhibit mitochondrial respiration, by binding to cytochrome oxidase in competition with oxygen. We measured O2 and NO concentrations simultaneously using specific electrodes, and showed that nanomolar NO reversibly inhibits oxygen consumption of isolated cytochrome oxidase, mitochondria, synaptosomes, and cells. Oxygen consumption of nerve terminals isolated from brain (synaptosomes) was half inhibited by 270nM NO when the oxygen concentration is 150μM, but half inhibited by 60nM NO when the oxygen concentration was 30μM (a roughly physiological level of oxygen). Cultured astrocytes, activated to express the inducible form of NO synthase produced up to 1μM which strongly and reversibly inhibited cellular respiration via the inhibition of cytochrome oxidase. Mitochondria caused rapid NO breakdown, which may be due to reaction with cytochrome oxidase and/or mitochondrial superoxide. Long-term exposure to NO can cause irreversible damage to mitochondria and cells, which may be mediated by reactive oxygen species or peroxynitrite. We have found that NO reversibly inhibits catalase, with a Ki of 0.3μM NO, and that superoxide dismutase catalyses the reaction of NO and H2O2 to produce peroxynitrite. These reactions might contribute to the cytotoxicity of NO.
Problem/Condition: Autism spectrum disorders (ASDs) are a group of developmental disabilities characterized by atypical development in socialization, communication, and behavior. ASDs typically are apparent before age 3 years, with associated impairments affecting multiple areas of a person's life. Because no biologic marker exists for ASDs, identification is made by professionals who evaluate a child's developmental progress to identify the presence of developmental disorders. Reporting Period: 2006. Methods: Earlier surveillance efforts indicated that age 8 years is a reasonable index age at which to monitor peak prevalence. The identified prevalence of ASDs in U.S. children aged 8 years was estimated through a systematic retrospective review of evaluation records in multiple sites participating in the Autism and Developmental Disabilities Monitoring (ADDM) Network. Data were collected from existing records in 11 ADDM Network sites (areas of Alabama, Arizona, Colorado, Florida, Georgia, Maryland, Missouri, North Carolina, Pennsylvania, South Carolina, and Wisconsin) for 2006. To analyze changes in identified ASD prevalence, CDC compared the 2006 data with data collected from 10 sites (all sites noted above except Florida) in 2002. Children aged 8 years with a notation of an ASD or descriptions consistent with an ASD were identified through screening and abstraction of existing health and education records containing professional assessments of the child's developmental progress at health-care or education facilities. Children aged 8 years whose parent(s) or legal guardian(s) resided in the respective areas in 2006 met the case definition for an ASD if their records documented behaviors consistent with the Diagnostic and Statistical Manual of Mental Disorders, 4th edition, text revision (DSM-IV-TR) criteria for autistic disorder, pervasive developmental disorder--not otherwise specified (PDD NOS), or Asperger disorder. Presence of an identified ASD was determined through a review of data abstracted from developmental evaluation records by trained clinician reviewers. Results: For the 2006 surveillance year, 2,757 (0.9%) of 307,790 children aged 8 years residing in the 11 ADDM sites were identified as having an ASD, indicating an overall average prevalence of 9.0 per 1,000 population (95% confidence interval [CI] = 8.6--9.3). ASD prevalence per 1,000 children aged 8 years ranged from 4.2 in Florida to 12.1 in Arizona and Missouri, with prevalence for the majority of sites ranging between 7.6 and 10.4. For 2006, ASD prevalence was significantly lower in Florida (p<0.001) and Alabama (p<0.05) and higher in Arizona and Missouri (p<0.05) than in all other sites. The ratio of males to females ranged from 3.2:1 in Alabama to 7.6:1 in Florida. ASD prevalence varied by type of ascertainment source, with higher average prevalence in sites with access to health and education records (10.0) compared with sites with health records only (7.5). Although parental or professional concerns regarding development before age 36 months were noted in the evaluation records of the majority of children who were identified as having an ASD, the median age of earliest documented ASD diagnosis was much later (range: 41 months [Florida]--60 months [Colorado]). Of 10 sites that collected data for both the 2002 and 2006 surveillance years, nine observed an increase in ASD prevalence (range: 27%--95% increase; p<0.01), with increases among males in all sites and among females in four of 11 sites, and variation among other subgroups. Interpretation: In 2006, on average, approximately 1% or one child in every 110 in the 11 ADDM sites was classified as having an ASD (approximate range: 1:80--1:240 children [males: 1:70; females: 1:315]). The average prevalence of ASDs identified among children aged 8 years increased 57% in 10 sites from the 2002 to the 2006 ADDM surveillance year. Although improved ascertainment accounts for some of the prevalence increases documented in the ADDM sites, a true increase in the risk for children to develop ASD symptoms cannot be ruled out. On average, although delays in identification persisted, ASDs were being diagnosed by community professionals at earlier ages in 2006 than in 2002. Public Health Actions: These results indicate an increased prevalence of identified ASDs among U.S. children aged 8 years and underscore the need to regard ASDs as an urgent public health concern. Continued monitoring is needed to document and understand changes over time, including the multiple ascertainment and potential risk factors likely to be contributing. Research is needed to ascertain the factors that put certain persons at risk, and concerted efforts are essential to provide support for persons with ASDs, their families, and communities to improve long-term outcome.
Objective: The authors investigated the idea that some patients with schizophrenia exhibit a chronic, deteriorating course without showing Frank neurodegeneration at postmortem examination. Oxidative damage is one form of cellular injury that may cause cellular dysfunction without necessarily leading to neuron death. Methods: Authors obtained postmortem hippocampi from 13 elderly non-psychiatric comparison cases and 10 patients with 'Poor-outcome" schizophrenia. The groups were compared for the presence of neuronal 8-hydroxy, 2' deoxyguanosine (8-OhHdG), a robust marker of oxidized DNA, and neuronal Ki-67, a marker of cell-cycle activation, as well as neurofibrillary tangles, amyloid-beta senile plaques, and astrocytosis. Results: The mean proportion of neurons exhibiting 8-OHdG immunoreactivity was 10 times higher in schizophrenia than in comparison cases. Ki-67 was similarly elevated and was correlated with 8-OHdG in the schizophrenia group. There were no significant between-group differences for densities of neurofibrillary tangles, amyloid-beta Plaques, or astrocytes. Conclusions: Our delta provide evidence for oxidative DNA damage and coordinated cell-cycle activation in elderly ''Poor-outcome" schizophrenia. These findings could have important implications for cellular metabolism, gene expression, and membrane functioning in schizophrenia.
A new method has been developed that is capable of providing a complete profile of the most common monothiols and disulfides present in plasma or tissue extracts. The method utilizes reversed phase ion-pairing high performance liquid chromatography coupled with coulometric electrochemical detection to simultaneously quantify free oxidized and reduced aminothiols or total aminothiols after chemical reduction. The method is extremely sensitive, with limits of detection in the 5 fmol/mL range for monothiols and 50 fmol/mL for dithiols. The interassay and intraassay coefficients of variation for total and free aminothiols ranged between 1.2 and 5.8%. The mean recoveries for total and plasma aminothiols ranged between 97.1 and 102.8%. The aminothiols are quantified directly, without derivatization, and include methionine, homocysteine, homocystine, cystathionine, cysteine, cystine, cysteinylglycine, and oxidized and reduced glutathione. Because a complete aminothiol profile of metabolites in both the remethylation (anabolic) and transulfuration (catabolic) pathways of homocysteine metabolism can be determined simultaneously, this new method should be useful in determining the metabolic etiology of homocysteinemia and in designing appropriate nutritional intervention strategies. Basic research applications of this method should lead to an increased understanding of the metabolic pathology of aminothiol imbalance.
Article abstract—Objective: To quantify developmental abnormalities in cerebral and cerebellar volume in autism. Methods: The authors studied 60 autistic and 52 normal boys (age, 2 to 16 years) using MRI. Thirty autistic boys were diagnosed and scanned when 5 years or older. The other 30 were scanned when 2 through 4 years of age and then diagnosed with autism at least 2.5 years later, at an age when the diagnosis of autism is more reliable. Results: Neonatal head circumferences from clinical records were available for 14 of 15 autistic 2- to 5-year-olds and, on average, were normal (35.1 6 1.3 cm versus clinical norms: 34.6 6 1.6 cm), indicative of normal overall brain volume at birth; one measure was above the 95th percentile. By ages 2 to 4 years, 90% of autistic boys had a brain volume larger than normal average, and 37% met criteria for developmental macrencephaly. Autistic 2- to 3-year-olds had more cerebral (18%) and cerebellar (39%) white matter, and more cerebral cortical gray matter (12%) than normal, whereas older autistic children and adolescents did not have such enlarged gray and white matter volumes. In the cerebellum, autistic boys had less gray matter, smaller ratio of gray to white matter, and smaller vermis lobules VI-VII than normal controls. Conclusions: Abnormal regulation of brain growth in autism results in early overgrowth followed by abnormally slowed growth. Hyperplasia was present in cerebral gray matter and cerebral and cerebellar white matter in early life in patients with autism. NEUROLOGY 2001;57:245-254