1. ABSTRACT METHODS, CONT’D
LCLs were cultured in RPMI-1640 supplemented with 15% FBS, 1%
penicillin/streptomycin and incubated in a humidified atmosphere with 5% CO2 at 37o
C. The
cells were cultured in suspensions in T-25 flasks for 9 days before experiments began.
First, intracellular generation of free radicals was measured using carboxy-H2DCFDA
(DCF), which is a membrane permeable ROS/RNS sensitive probe that remains non-
fluorescent until oxidized. 1.5 x 105
cells per LCL were collected, washed with 1X PBS and
stained with 20 uM DCFDA for 30 minutes at 37o
C. The cells were then seeded in a 96-well
plate and fluorescence was measured by a multi-mode plate reader at an excitation
wavelength of 485nm and an emission wavelength of 528nm. The fluorescence intensity
was measured in both the autistic and control LCLs as mean ± SEM.
Next, the effect of DMNQ (2,3-dimethoxy-1,4-naphthoquinine) on cell viability was
assessed using Annexin-V/7-aminoactinomycin D (7-AAD) kit. 50 ug/mL of DMNQ was
added to each well and its effects were measured over a period of 24 hours. PE Annexin-V,
a phospholipic binding protein with a high affinity for phophatidylserine (PS) exposure, and
7-AAD, which binds to nucleic acids when the membrane integrity is breached in late stage
apoptosis and in necrosis, were added to each sample. A total of 10,000 events were
analyzed using a BD C6 Accuri Flow Cytometer and students’ t-tests were used with
significance set at α<0.05.
CONCLUSIONS
 LCLs from children with autism experience more detrimental
effects under the same oxidative stress levels as the LCLs
from unaffected children, suggesting variations in apoptosis
and necrosis mechanisms between groups.
 Children with autism are more susceptible to oxidative stress
than controls, leading to late stage apoptosis/necrosis and
therefore cell death much earlier. After insult, more time was
needed for the autistic LCLs to recover, and their recovery was
less complete in terms of cell viability.
 Controls respond to oxidative insult with higher levels of
apoptosis than was seen in those with autism and this
difference could potentially be related to glutathione and ATP
levels. Low glutathione levels, previously observed in children
with autism [2-3], could be associated with higher levels of
necrosis and later stage apoptosis/necrosis.
 Caspase 3 activity [4] should be further studied in children with
autism to determine if this is the key in causing more necrosis,
accelerated cell death and how this can be rescued.
Figure 2. Relative rate of intracellular free radical production in autistic and control cell lines,
where indicates p<0.05.
Figure 3. Average rate of ROS generation in autistic LCLs (n=2) and controls (n=2) at baseline
and after treatment with 15uM hydrogen peroxide.
RESULTS
I. Free Radical Production
The level of intracellular free radicals was measured in LCLs from children with autism
(n=6) and unaffected control children (n=4), using DCF. Figure 2 presents DCF fluorescence
measured in LCLs at baseline, where autistic LCLs exhibited significantly higher amounts of
ROS. DCF fluorescence increased from baseline in both groups after treatment with
hydrogen peroxide as a positive control (Figure 3). A greater increase in free radical
production was seen in the autistic LCLs.
II. Cell Viability After Treatment with DMNQ
Cell viability assays on autistic LCLs and control LCLs were performed at baseline and
after treatment with DMNQ using flow cytometry (Figure 4). The mean ± SEM from the
autistic (n=6) and controls (n=4) were obtained at baseline and after treatment for 4–24
hours. The autistic cell lines had a greater change in cell viability as compared to the
controls as seen in Figure 5, most notably at 16 hours (p<0.05). Also, the autistic LCLs
steadily increased in cell death from 12–24 hours at a steeper rate than controls, showing
their inability to combat oxidative stress (Figure 6). Moreover, minor differences in necrosis
rates were observed, as shown in Figure 7.
Table 1. Autism and Control LCL groups with ages.
REFERENCES
1. C.D.C. Investigators. Prevalence of autism spectrum disorders –
autism and developmental disabilities monitoring network, 14 sites,
United States, 2010. MMWR Surveill Summ 2014;63(2):1–19.
2. Main P E, Thomas P, Esterman A, Fenech MF. Necrosis is increased
in lymphoblastoid cell lines from children with autism compared with
their non-autistic siblings under conditions of oxidative and nitrosative
stress. Mutagenesis. 2013;28(4):475–484.
3. James SJ, Cutler P, Melnyk S, et al. Metabolic biomarkers of
increased oxidative stress and impaired methylation capacity in
children with autism. Am J Clin Nutr. 2004;80(6):1611–1617.
4. Vairetti M, Ferrigno A, Bertone R, Richelmi P, Bertè F, Freitas I.
Apoptosis vs. necrosis: glutathione-mediated cell death during
rewarming of rat hepatocytes. Biochim Biophys Acta.
2005;1740(3):367–374.
Figure 6. Proportion of dead LCLs in autistic and control LCLs after DMNQ treatment.
denotes p<0.05 at 12 hours and denotes p<0.1 at 16 hours, 20 hours and 24 hours.
Figure 7. Proportion of necrotic LCLs in autistic and control LCLs after treatment with DMNQ. At
16 hours, autistic LCLs had significantly greater change from baseline than controls (p<0.05) .
METHODS
ACKNOWLEDGEMENTS
We gratefully acknowledge the resources provided by the Autism Genetic
Resource Exchange (AGRE) Consortium* and the participating AGRE
families. The Autism Genetic Resource Exchange is a program of Autism
Speaks and is supported, in part, by grant 1U24MH081810 from the
National Institute of Mental Health to Clara M. Lajonchere (PI).
Cell lines identified with numbers beginning with ”GM“ were obtained
from the NIGMS Human Genetics Cell Repository at the Coriell Institute
for Medical Research.
Work was supported in part by a grant from the Health Professions
Division of Nova Southeastern University.
Control LCLs have higher levels of apoptosis as compared to
autistic LCLs, but autistic LCLs showed a greater percent change
in apoptosis from their baseline levels, as seen in Figures 9 and
10. Autistic LCLs slightly increased their level of apoptosis
following treatment. Both groups had the highest amounts of
apoptotic cells at 16 hours post treatment.
Figure 4: Flow cytometry plots of representative control LCL (F01) and autistic LCLs (C10)
after incubation with 50uM DMNQ for 12 hours.
Figure 5: Cell viability decreases after DMNQ treatment in both autistic and control LCLs,
indicates p<0.05 and denotes p<0.1.
Autistic LCL Age Control LCL Age
AU3964302 3.8 GM09659 4
AU1157303 3.1 GM08336 3
AU055104 5 GM11898 5
AU2140305 5.9 GM09380 6
AU3907302 4.4 GM09659 4
AU3912302 4.9 GM11898 3
In this study, we have used in vitro models to determine the
differences in cell viability/death after exposure to an
oxidative reagent in children with autism as compared to
controls. Six LCLs from children with this disorder were
obtained from the Autism Genetic Resource Exchange
(AGRE; Los Angeles, CA, USA) and paired with four LCLs
from unaffected children from Coriell Cell Repository
(Camden, NJ, USA) as seen in Table 1. All lines were derived
from Caucasian males within the ages of 3-6 years old.
Figure 9. Proportions of apoptosis before and after treatment with
50uM DMNQ analyzed every 4 hours. Significant differences arise at
baseline (p<0.05), 4, 8, 16, 20 and 24 hours (p<0.1).
Figure 10. Change in apoptotic levels from baseline in autistic and
control LCLs. There are significant differences in the rate of change
between the two groups at 4 hours (p<0.1), 8 hours (p<0.05), 12
hours (p<0.05) and 16 hours (p<0.1).
CELL DEATH DURING OXIDATIVE STRESS IN
LYMPHOBLASTOID CELL LINES FROM AUTISTIC CHILDREN
Jordan A. Spaw1
, Stephen G. Grant2
and Ana M. Castejon1
.
1
College of Pharmacy, and
2
Public Health Program, College of Osteopathic Medicine,
Nova Southeastern University, Fort Lauderdale, FL 33301.
Numerous studies have suggested oxidative stress plays
a role in the pathogenesis of autism. Oxidative stress results
from an imbalance between the production of reactive
oxygen species (ROS) and a decrease in either the
efficiency of the endogenous antioxidant defense
mechanisms or the ability to effectually scavenge free
radicals. We propose that there is a direct link between
oxidative stress and cell death in children with autism, with
deficient glutathione levels as the underlying mechanism. In
this study, we evaluated the susceptibility of autistic children
in vitro to various oxidative stressors as compared with to
unaffected, age-matched controls. In order to test this
hypothesis, lymphoblastoid cell lines (LCLs) from affected
and control children from the Autism Genetic Research
Exchange (AGRE) and Coriell Cell Repository were treated
with the pro-apoptotic agent, DMNQ, for various lengths of
time. Cell viability, cell death, apoptosis, and necrosis rates
were analyzed in these treated cell lines, both at baseline
and in the presence of oxidative stressors, using flow
cytometry. In addition, the formation of ROS was quantified
using fluorescence. Preliminary results have shown
increased levels of ROS in those LCLs from children with
autism as compared to controls at baseline conditions.
Significantly higher apoptotic rates were found in the control
LCLs (p<0.05) at almost all time points, whereas the autistic
LCLs had higher proportions of cell death (p<0.1). Overall,
the results from this study will provide a better understanding
of the underlying molecular mechanisms of the pathogenesis
of autism, which can aid in the development of laboratory
tests and personalized treatments for this disorder.
INTRODUCTION
Autism is one of the most common developmental
disabilities, occurring in every race, ethnic group, and
socioeconomic background. In the last two years, there has
been a 30% increase in the prevalence of autism spectrum
disorders (ASD) in the United States, where it now affects 1
out of every 68 children [1]. Autism is clinically characterized
by a range of qualitative deficiencies in social interaction,
communication and restricted/stereotyped patterns of
behavior. Autism is diagnosed with variety of subjective
behavioral assessments, usually around age 3. The cause(s)
of autism is still unknown, hindering the development of
definitive diagnostic tests and innovative treatments. There
is accumulating evidence that oxidative stress plays a
causative role in this disorder and may serve as a link
between known and postulated genetic and environmental
factors. Glutathione is reliably used as an overall measure of
oxidative stress; it also modulates cell survival, cell death
and a variety of signal transduction pathways. Glutathione
levels are decreased in children with autism [2], but have
rarely been linked to altered cell death mechanisms within
the disorder. Also, this impaired antioxidant capacity,
potentially leading to oxidative damage in various brain
regions, supports a relationship between glutathione
concentrations and neurocognitive function. Previous
research has suggested children with autism have lower
levels of glutathione and therefore have an impaired ability to
combat oxidative stress creating damage on the cellular
level [2-3]. Cell death occurs in extreme circumstances of
long term stress and can be mediated by low glutathione
levels [4]. We believe there will be differences in how each
group combats oxidative stress and the underlying death
mechanisms they take in these stressful conditions. Since
apoptosis requires ATP and is related to glutathione levels,
we propose that autistic children will have lower rates of this
type of cell death as compared to controls.
Control Autistic