Award Number: DAMD17-03-1-0501
TITLE: Brain’s DNA Repair Response to Neurotoxicants
PRINCIPAL INVESTIGATOR: Juan Sanchez-Ramos, Ph.D., M.D.
CONTRACTING ORGANIZATION: University of South Florida
Tampa, Florida 33620
REPORT DATE: January 2007
TYPE OF REPORT: Final
PREPARED FOR: U.S. Army Medical Research and Materiel Command
Fort Detrick, Maryland 21702-5012
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4. TITLE AND SUBTITLE
Brain’s DNA Repair Response to Neurotoxicants
OMB No. 0704-0188
2. REPORT TYPE3. DATES COVERED (From - To)
1 Jul 2003 – 31 Dec 2006
5a. CONTRACT NUMBER
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8. PERFORMING ORGANIZATION REPORT
Juan Sanchez-Ramos, Ph.D., M.D.
7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES)
University of South Florida
Tampa, Florida 33620
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U.S. Army Medical Research and Materiel Command
Fort Detrick, Maryland 21702-5012
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13. SUPPLEMENTARY NOTES
Parkinson’s Disease (PD) is associated with death of dopaminergic (DA) neurons in the substantia nigra (SN) of the brain. Military personnel abroad are at a
greater risk of exposure to pesticides and toxins which may selectively damage DA neurons in the SN and increase the probability of development of
Parkinson’s disease (PD) later in life. The toxins of interest are mitochondrial poisons that create a bioenergetic crisis and generate toxic oxyradicals which
damage macromolecules, including DNA. We hypothesized that regulation of the DNA repair response within certain neurons of the SN (the pars compacta)
may be a critical determinant for their vulnerability to these neurotoxicants. We have measured regional differences in the brain’s capacity to increase repair
of oxidized DNA (indicated by oxyguanosine glycosylase (OGG1) activity) to three distinct chemical classes of neurotoxins (MPTP, two mycotoxins, and an
organochloriine pesticide). We have found that the temporal and spatial profile of OGG1 activity across brain regions elicited by each class of neurotoxicant is
distinct and unique. Even though all 3 toxicants caused various degrees of depletion of striatal dopamine, the temporal profile of DA depletion and OGG1
activity in striatum was distinct for each toxicant. DNA repair gene expression in response to OTA and dieldrin revealed differences in VTA and SN
compartments that may relate to differential vulnerability to oxidative stressors.
15. SUBJECT TERMS
DNA Damage, DNA Repair, Brain, Neurotoxicants, Mycotoxins
16. SECURITY CLASSIFICATION OF:
19a. NAME OF RESPONSIBLE PERSON
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c. THIS PAGE
Standard Form 298 (Rev. 8-98)
Prescribed by ANSI Std. Z39.18
Table of Contents
Key Research Accomplishments………………………………………….……….…page 24-26
Reportable Outcomes………………………………………………………………....page 26
Published and Pending Manuscripts
Sava et al., Rubatoxin-B Elicits Anti-Oxidative and DNA Repair Responses in Mouse Brain.
Gene Expression 11: 211-219, 2004------------------------------------------------------------page 29
Sava et al., Acute neurotoxic effects of the fungal metabolite ochratoxin-A
NeuroToxicology 27: 82–92, 2006--------------------------------------------------------------page 56
Sava et al., Can low level exposure to ochratoxin-A cause parkinsonism?
Journal of the Neurological Sciences 249: 68–75, 2006------------------------------------page 67
Sava et al., Neuroanatomical mapping of DNA repair and antioxidative responses
in mouse brain: Effects of a single dose of MPTP
NeuroToxicology 27:1080–1093, 2006---------------------------------------------------------page 75
Sava et al., Dieldrin Elicits a Widespread DNA Repair and Anti-Oxidative Response in Mouse
Brain . Submitted to Journal of Biochemical and Molecular Toxicology-----------------page 89
List of Published Abstracts-----------------------------------------------------------------------page 116
Parkinson’s Disease (PD) is associated with death of dopaminergic (DA) neurons in the substantia nigra
(SN) of the brain (1-3). Military personnel abroad are at a greater risk of exposure to pesticides and toxins
(4) some of which may selectively damage DA neurons in the SN and increase the probability of
development of Parkinson’s disease (PD) later in life. The toxins of interest are mitochondrial poisons
that create a bioenergetic crisis and generate toxic oxyradicals which damage macromolecules, including
DNA. We hypothesize that the DNA repair response within certain neurons of the SN (the pars
compacta) may be a critical determinant for their vulnerability to these neurotoxicants. The technical
objectives were to measure regional and cellular differences in the brain’s DNA repair response to three
neurotoxins known to interfere with mitochondrial function (the mycotoxin ochratoxin-A; the pesticide
dieldrin, and the classic dopaminergic neurotoxin, MPTP). An improved understanding of the DNA
repair response to neurotoxicants and development of methods to enhance DNA repair will form the basis
for potential preventive measures against the effects of military threat agents and military operational
hazards, and also lead to treatment interventions for Parkinson’s disease
STATEMENT OF WORK: The Brain’s DNA Repair Response to Neurotoxicants.
We propose to test the hypothesis that differences in DNA repair responses determine intrinsic neuronal
susceptibility to exogenous or endogenous neurotoxicants. Corollaries of this hypothesis are:
a) The DNA repair response, in particular the ability to upregulate the activities of 8-oxoguanine
glycosylase-1 (Ogg1) and redox factor-1 (Ref-1), will determine whether a neuron will survive
exposure to neurotoxicological insults.
b) Overactivation of poly(ADP-ribose) polymerase-1 (PARP-1) in response to oxidative stress will
exacerbate the toxicity of xenobiotics and lead to degeneration of neurons.
c) Agents that increase Ogg1 and Ref-1 activity and expression or inhibit PARP-1 will provide
protection against neurotoxicants.
Task 1: To determine the differences in oxidative DNA damage and DNA repair responses elicited
by mycotoxins (ochratoxin-A; rubratoxin-B), an organochlorine pesticide (dieldrin), and the
classical DA neurotoxicant, MPTP
Task 2: To measure the effects of chronic low dose administration of a mycotoxin and a pesticide
on brain region oxidative DNA damage and DNA repair
Task 3: To determine whether exposure to agents that up-regulate Ogg1 and Ref-1 DNA repair or
inhibit PARP-1 will protect against the neurotoxicity elicited by a mycotoxin and a pesticide
Task 4: To measure the effects of neurotoxicant exposure on the DNA repair response in DA
neurons from two specific sub-populations of the midbrain, the SN-pars compacta and the ventral
tegmental area (VTA)
SUMMARY OF RESULTS FROM TASK 1
a) Acute Effects of Rubratoxin
Rubratoxin-B (RB) is a mycotoxin with potential neurotoxic effects that have not yet been characterized.
Based on existing evidence that RB interferes with mitochondrial electron transport to produce oxidative
stress in peripheral tissues, we hypothesized that RB would produce oxidative damage t o
macromolecules in specific brain regions. Parameters of oxidative DNA damage and repair, lipid
peroxidation and superoxide dismutase (SOD) activity were measured across 6 mouse brain regions 24
hrs after administration of a single dose of RB. Lipid peroxidation and oxidative DNA damage was either
unchanged or decreased in all brain regions in RB-treated mice compared to vehicle-treated mice.
Concomitant with these decreased indices of oxidative macromolecular damage, SOD activity
was significantly increased in all brain regions. Oxyguanosine glycosylase activity (OGG1),
a key enzyme in the repair of oxidized DNA, was significantly increased in three brain regions
cerebellum (CB), caudate/putamen (CP), and cortex (CX) but not hippocampus(H), midbrain(MB), and
pons/medulla(PM). The RB-enhanced OGG1 catalytic activity in these brain regions was not due to
increased OGG1 protein expression, but was a result of enhanced catalytic activity of the enzyme. In
conclusion, specific brain regions responded to an acute dose of RB by significantly altering SOD and
OGG1 activities to maintain the degree of oxidative DNA damage equal to, or less than, that of normal
steady-state levels. Details of this study have been published (5). The report can be found in the
b) Acute Effects of Ochratoxin-A
Ochratoxin-A (OTA) is a fungal metabolite with potential toxic effects on the central nervous system.
OTA has complex mechanisms of action that include evocation of oxidative stress, bioenergetic
compromise, inhibition of protein synthesis, production of DNA single-strand breaks and formation of
OTA–DNA adducts. The time course of acute effects of OTA were investigated in the context of DNA
damage, DNA repair and global oxidative stress across six brain regions. Oxidative DNA damage, as
measured with the ‘‘comet assay’’, was significantly increased in the six brain regions at all time points
up to 72 h, with peak effects noted at 24 h in midbrain (MB), CP (caudate/putamen) and HP
(hippocampus). Oxidative DNA repair activity (oxyguanosine glycosylase or OGG1) was inhibited in all
regions at 6 h, but recovered to control levels in cerebellum (CB) by 72 h, and showed a trend to recovery
in other regions of brain. Other indices of oxidative stress were also elevated. Lipid peroxidation and
superoxide dismutase (SOD) increased over time throughout the brain. In light of the known
vulnerability of the nigro-striatal dopaminergic neurons to oxidative stress, levels of striatal dopamine
(DA) and its metabolites were also measured. Administration of OTA (0–6 mg/kg i.p.) to mice resulted
in a dose-dependent decrease in striatal DA content and turnover with an ED50 of 3.2 mg/kg. A single
dose of 3.5 mg/kg decreased the intensity of tyrosine hydroxylase immunoreactivity (TH+) in fibers of
striatum, TH+ cells in substantia nigra (SN) and TH+ cells of the locus ceruleus. TUNEL staining did not
reveal apoptotic profiles in MB, CP or in other brain regions and did not alter DARPP32
immunoreactivity in striatum. In conclusion, OTA caused acute depletion of striatal DA on a background
of globally increased oxidative stress and transient inhibition of oxidative DNA repair. Details of this
study have been published and can be found in the Appendix (6).
c) Acute Effects of MPTP
The primary objective of this study was to map the normal distribution of the base excision enzyme
oxyguanosine glycosylase (OGG1) across mouse-brain regions as a prelude to assessing the effects of
various neurotoxicants, ranging from highly selective molecules like MPTP to more global toxic agents,
including the mycotoxin OTA and the pesticide dieldrin. This research is based on the hypothesis that
regional brain vulnerability to a toxicant is determined, in part, by variation in the intrinsic capacity of
cellular populations to successfully repair oxidative DNA damage. After mapping the normal distributions
of OGG1 and superoxide dismutase (SOD) across 44 loci dissected from mouse brain, MPTP, a
mitochondrial toxicant with selective dopamine (DA) neuron cytotoxicity was used to elicit focal
oxidative stress and DNA repair responses. A single dose of MPTP (20 mg/kg, i.p.) elicited time- and
regiondependent changes in both SOD and OGG1, with early increases in DNA repair and anti-oxidant
activities throughout all regions of brain. In some sampled loci, notably the substantia nigra (SN) and
hippocampus, the heightened DNA repair and antioxidant responses were not maintained beyond 48 h.
Other loci from cerebellum, cerebral cortex and pons maintained high levels of activity up to 72 h. Levels
of dopamine (DA) were decreased significantly at all time points and remained below control levels in
nigro-striatal and mesolimbic systems (ventral tegmental area and nucleus accumbens). Assessment of
apoptosis by TUNEL staining revealed a significant increase in number of apoptotic nuclei in the
substantia nigra at 72 h and not in other loci. The marked degree of apoptosis that became evident in SN
at 72 h was associated with large decreases in SOD and DNA repair activity at that locus. In conclusion,
MPTP elicited global effects on DNA repair and antioxidant activity in all regions of brain, but the most
vulnerable loci were unable to maintain elevated DNA repair and antioxidant responses. The full report
has been published and can be found in the Appendix (7).
d) Acute Effects of Dieldrin
Dieldrin, an organochlorine pesticide, has several molecular characteristics that make it a
potential etiological agent for Parkinson’s Disease. The half life of dieldrin in soil is approximately 5
years. This persistence, combined with high lipid solubility, provides the necessary conditions for
dieldrin to bioconcentrate and biomagnify in organisms. Dieldrin appears to be retained for life in lipid-
rich tissue and has been measured in human brain. It was found at high concentrations in caudate nucleus
from post-mortem brain of idiopathic Parkinson’s Disease (IPD) cases. Dieldrin has toxic effects for
dopaminergic (DA) and monoaminergic neurons in many species, both in vitro and in vivo. Like
rotenone and the dopaminergic neurotoxin 1-methyl-4-phenyl-pyridinium (MPP+), dieldrin interferes
with mitochondrial oxidative phosphorylation. Insights derived from studies of 1-methyl-4-phenyl-
1,2,3,6-tetrahydropyridine (MPTP) led to the observation that mitochondrial function appears to be
compromised in brain and peripheral tissues from PD patients.
The present study was designed to test the hypothesis that the DNA repair response to dieldrin is
a determinant of the vulnerability of DA neurons of the nigro-striatal system. The activity of the
mammalian base excision repair enzyme oxyguanosine glycosylase (Ogg1) was utilized as the index of
DNA repair. Other measures of oxidative stress were also studied, including the regional distribution of
lipid peroxidation and superoxide dismutase (SOD) activity. The primary objectives of this component of
the study were to determine the effects of acute and slow infusion of dieldrin on a) DA and its
metabolites in the striatum and b) to measure the regional distribution of the brain’s DNA repair response
and parameters of oxidative stress. Secondary objectives were to note observable changes in motor
behavior and to measure whole body tremor elicited by dieldrin administration.
Effects of Dieldrin on Striatal DA and metabolites
Four groups (6-8 mice per group) of mice were injected with dieldrin i.p. (6 mg/kg or 30 mg/kg).
Animals were euthanatized at 6, 24 and 72 hrs after injections. Brains were dissected and striatal tissue
was harvested for assay of DA and metabolites. Striatal DA levels were transiently decreased at 6 hrs, but
recovered to levels equal to or greater than baseline by 72 hrs (Figs 1, 2 ). In the group of mice that
received the high dose of dieldrin (30 mg/kg) the levels at 72 hrs far exceeded baseline levels. Striatal DA
turnover was initially increased but by 72 hrs was significantly diminished (Fig 2).
Fig 1. Acute effects of dieldrin (6 mg/kg i.p) on striatal dopamine and metabolites.
A. Striatal DA was initially decreased at early time points and returned to levels above
baseline at 72 hrs. B. Dieldrin had no effect on HVA at early time points and only was
significantly increased at 72 hrs. C. Dieldrin decreased DOPAC levels significantly at
24 hrs but levels returned to baseline by 72 hours. D. DA turnover One-way ANOVA
showed that the DA, DOPAC and DA turnover means were significantly different (p <
0.05) and Dunnett’s multiple comparison test showed significant differences in striatal
DA, DOPAC and DA turnover at times indicated by asterisks.
Fig 2. Acute effects of dieldrin (30 mg/kg i.p.). A. Striatal DA was initially decreased at 6 hrs to half the
baseline levels and then was elevated significantly above baseline at 24 and 72 hrs. B. Dieldrin had no
effect on HVA at all time points. C. Dieldrin decreased DOPAC levels significantly at 6 and 24 hours. D.
DA turnover was decreased significantly at 24 and 72 hrs. One-way ANOVA showed that the DA,
DOPAC and DA turnover means were significantly different (p < 0.05) and Dunnett’s multiple
comparison test showed significant differences in striatal DA, DOPAC and DA turnover at times
indicated by asterisks.
Acute Effects of Dieldrin on Regional DNA Repair (OGG1 activity)
Four groups of mice (n=6 per group) were injected with 6 or 30 mg/kg of dieldrin i.p. or vehicle. Groups
were euthanatized at 6, 24 and 72 hrs after injection. (Data from the low dose is not shown but was
similar to the effects of the high dose in the time-course and brain regional pattern). Dieldrin elicited a
significant time and brain-region dependent increase in OGG1 activity (Fig 3). The greatest extent of
increased activity was measured in MB (5 fold), followed closely by PM (4.3 fold) and CP (4.2 fold).
These three regions have high levels of monoaminergic neuronal activities. Notably all regions of brain
exhibited at least a 2.5 fold increase in OGG1 activity at 72 hrs after dieldrin injection.
Fig 3 Acute effects of 30 mg/kg dieldrin on OGG1 activity. Left panel: OGG1 activity plotted against
brain region reveals a brain and time-dependent increase in OGG1 activity. Two-way ANOVA showed
that time contributed 72% of total variance (p < 0.0001); brain regions accounted for 4% of total variance
(p< 0.01) and the interaction with brain region accounted for 3% of total variance. Asterisks indicate
significant differences from control values based on post-hoc t-tests with Bonferroni corrections for
multiple comparisons. Right panel: Fold Increase of OGG1 activity (ratio of values at 72 hrs to control
values) plotted against brain regions. The MB showed the greatest increase in DNA repair activity,
followed by PM and CP. CB=cerebellum; MB= midbrain; PONS=pons; MD=medulla;
T/HT=thalamus/hypothalamus; HP=hippocampus; CP= caudate/putamen; CX= cerebral cortex.
SUMMARY OF RESULTS FROM TASK 2
a) Effects of slow infusion of OTA via osmotic minipump over two weeks
The effects of chronic low dose OTA exposure on regional brain oxidative stress and striatal DA
metabolism was studied and a manuscript summarizing the results has been published (8). (A reprint of
the manuscript is found in the Appendix). The continuous subcutaneous administration of OTA at low
doses over a period of 2 weeks caused small, but significant depletion of striatal DA. OTA also caused
pronounced global oxidative stress, evoking a strong antioxidative and DNA repair response across the
entire brain. Even though the depletion of striatal DA did not cause overt parkinsonism in these mice, it
is important to consider that the superimposition of normal age-related decline in striatal DA could
ultimately result in signs of parkinsonism such as slowness of movement and rigidity in the mice.
Without completing the understanding why DA terminals in striatum are especially vulnerable to OTA, it
is likely that a toxic insult to the nigro-striatal system will increase the risk of developing Parkinson's
Disease at an earlier age than normal. This hypothesis can be tested by studying the long term
consequences of episodes of OTA exposure in mice during the aging process. In the real world, it will be
important to monitor the neurological status of Gulf War veterans as they age.
b) Effects of slow infusion of dieldrin on striatal DA and metabolites
Slow sub-cutaneous infusion of dieldrin with an ALZET osmotic pump over 2 wks (50 mg/kg cumulative
dose) resulted in significantly increased levels of striatal DA and HVA but not DOPAC (Fig 4). DA
turnover was significantly decreased at 14 days (Fig 4 ).
Fig 4 Effects of slow infusion of dieldrin on striatal DA and metabolites. Left panel: Striatal DA and
metabolites following 2 wks of infusion of dieldrin by osmotic pump (cumulative dose 50 mg/kg). Right
panel: Striatal DA turnover at 14 days compared to control turnover. Asterisks denote significant
difference between values at baseline and 14 days (unpaired t-tests)
c) Effects of dieldrin infusion over 2 weeks on DNA Repair (OGG1)
Six groups of mice (n=8 per group) were implanted with osmotic pumps loaded with dieldrin and
calibrated to deliver 3, 6, 12, 24 and 48 mg/kg over a period of 2 weeks. After euthanasia and rapid
dissection of brain, OGG1 activities were determined. Dieldrin infusion elicited a dose dependent increase
of OGG1 activities in all brain regions, with maximum effects reaching a plateau between 24 and 48
mg/kg (Fig 5). The distribution of OGG1 activity across brain regions was fairly homogenous. However,
at the 24mg/kg cumulative dose, there was a more heterogeneous distribution of activity, with pons
exhibiting significantly greater activity than striatum and cerebral cortex (Fig 5).
Fig 5 Effects of 2 wk infusion of dieldrin on DNA repair (OGG1 activity) Left panel depicts OGG1
activity as a function of the cumulative dose of dieldrin. The increase in OGG1 activity was significantly
dependent on the cumulative dose delivered but did not vary significantly as a function of brain region.
Two-way ANOVA revealed that cumulative concentration of dieldrin accounted for 79% of total
variance (p< 0.0001) and brain regions accounted for 1.84% of total variance (p=0.61). Post-hoc t-tests
with Bonferroni corrections for multiple comparisons showed OGG1 activities in the pons were
significantly higher than in the CP and CX following a cumulative dose of 24 mg/kg (right panel).
CB=cerebellum; MB= midbrain; PONS=pons; MD=medulla; T/HT=thalamus/hypothalamus;
HP=hippocampus; CP= caudate/putamen; CX= cerebral cortex.
d) Effects of slow infusion of dieldrin on lipid peroxidation:
Slow infusion of dieldrin resulted in a dose-dependent increase in oxidative stress across all brain
regions as indicated by measurements of lipid peroxidation (Fig 6). This curve resembled the DNA repair
response shown in Fig 6. The maximum effect was produced following infusion of 48 mg/kg over 2
weeks. The increase in lipid peroxidation was significantly dependent on dose and did not vary
significantly with brain region similar to the effects on OGG1. However, post-hoc t-tests revealed that
lipid peroxidation was significantly higher in CB than in MB following a dose of 12 mg/kg (p<0.05).
Similarly, lipid peroxidation was greater in CB than in the PONS following 24 mg/kg.
Fig 6 Effects of 2 wk infusion of dieldrin (with pump) on lipid peroxidation (TBARS units). The
increase in TBARS was significantly dependent on the cumulative dose delivered but did not vary
significantly as a function of brain region. Two-way ANOVA revealed that cumulative concentration of
dieldrin accounted for 76% of total variance (p< 0.0001) and brain regions accounted for 0.9 4% of total
variance (p=0.36). * Posthoc t-tests with Bonferroni corrections for multiple comparisons showed
TBARS in the CB were significantly higher than in the MB (p < 0.05) following a cumulative dose of 12
mg/kg; TBARS in CB were also significantly higher than in the PONS (p<0.05) following 24 mg/kg.
SUMMARY OF RESULTS FROM TASK 3
Task 3: To determine whether exposure to agents that up-regulate Ogg1 and APEX DNA repair
will protect against the neurotoxicity elicited by a mycotoxin and a pesticide
Measurement of DNA Damage with a Modification of the PARP Assay.
Poly(ADP-ribose) polymerase (PARP) is nuclear protein of 116 kDa present at approximately 1 x 106
copies in somatic cells. PARP undergoes a conformational change on binding to damaged DNA via a zinc
finger domain. This activated PARP converts NAD to nicotinamide and polymers of ADP-ribose. The
PARP assay allows determining PARP activity by measuring the incorporation of radiolabeled NAD in
presence of activated DNA. Quantitative values are determined from scintillation counting. The assay
may be also used for indirect quantitative measure of DNA damage in cell extracts without addition of
exogenous activated DNA. Carrying out reaction in presence of exogenous PARP enzyme allows
incorporation of radiolabeled NAD in extent that reflects the degree of DNA damage in cell extract.
Approximately 50 mg of tissue was sonicated in 450 μL of Extraction buffer for about 10 s following
centrifugation at 3,000 g for 5 min at 4 ˚C. Supernatant was transferred in pre-chilled test tube and
concentration of protein was adjusted to 1 μg/μL. Core Reaction Mixture (CRM) was prepared by mixing
together: 2·(n+1) μL of 32 P-NAD; 10·(n+1) μL of Histone H1 (1mg/ml); 10·(n+1) μL of NAD (1 mM)
and 10·(n+1) μL of 10 x buffer (n - is a number of reactions). The following components were dispensed
into test tubes contained 20 μL of tissue extract: 32 μL of CRM; 1 μL of PARP enzyme and 47 μL of
distilled water. Additional set of test tubes using for measure of background contained 20 μL of tissue
extract; 32 μL of CRM; 1 μL of PARP enzyme; 6 μL of aminobenzamide (an inhibitor of PARP) and 41
μL of distilled water. Tubes were incubated for 10 min at room temperature and were transferred on ice.
900 μL of ice cold 20% TCA was added followed by centrifugation at 12,000 g for 10 min at room
temperature. Supernatant was removed and 1mL of liquid scintillation cocktail was added to each tube.
Each tube was vortexed for about 1 min to solubilize the protein pellet. Tubes were placed in a standard
scintillation vial and counted for 32P. Background was subtracted from each measurement to calculate the
degree of DNA damage.
Measurement of DNA Repair Activities (OGG1, PARP)
Measurement of OGG1 enzymatic activy was performed as previously described (6, 8). PARP
enzymatic activity was measured with the Poly(ADP-Ribose) Polymerase Assay Kit (Trevigen,
Design of the studies on Pre-conditioning (Effects of DEM pretreatment on neurotoxicant-induced
damage (Fig 7)
DEM (1 mmol/kg, ip) dissolved in DMSO
OTA (4 mg/kg, ip) dissolved in NaHCO3
0 h 6 h 72 h
DEM NaHCO3 Tissue collection
0 h 6 h 72 h
DMSO OTA Tissue collection
0 h 6 h 72 h
DEM OTA Tissue collection
Fig 7 Experimental design for the assessment of the effect of pre-conditioning with a mild pro-oxidant
DEM on OTA toxicity. DEM (1 mmol/kg, ip) was dissolved in DMSO and OTA (4 mg/kg, ip) was
dissolved in.NaHCO3 Animals were euthanatized 72 hrs after treatment and midbrains were harvested
0 h 6 h 72 h
Three groups of mice (n=6) were injected with single doses of OTA (4 mg/kg), Dieldrin ( 16
mg/kg) or MPTP ( 20 mg/kg) and extent of DNA damage in total midbrain was assayed. Each of the
toxicants caused DNA damage, but the effect of dieldrin was much greater than the other two (Fig 8).
15 Download full-text
OTA Dieldrin MPTP
DNA damage relative to control
Fig. 8. Relative oxidative DNA damage in midbrain caused by different neurotoxicants (OTA= 4 mg/kg;
Dieldrin = 6 mg/kg; MPTP = 20 mg/kg). Bars illustrate mean "SD (n=6). DNA damage was evaluated
with Poly(ADP-ribose) Polymerase Assay Kit (Trevigen, Gaithersburg, MD).
To assess the effects of pre-conditioning, four groups of mice (n= 6 per group) were injected i.p.
with DEM (1 mmol/kg), the specific neurotoxicant (OTA, dieldrin or MPTP) or a combination of both
according to the experimental design illustrated in Fig 7. DEM was dissolved in DMSO and OTA (4
mg/kg, ip) was dissolved in NaHCO3 In the pre-conditioning group, mice were injected with DEM 6 hrs
before the toxicant. All animals were euthanatized 72 hrs after the first injection. Entire midbrain and
micro-punches of midbrain (SN and VTA) from another set of mice were harvested.
Total oxidative DNA damage in midbrain samples was estimated and shown to increase following
administration of the toxicant (Fig 9). OTA, but not DEM caused a significant increase in total midbrain
DNA damage. Pretreatment with DEM 6 hrs before administration of OTA potentiated the total DNA
damage caused by OTA (Fig 9, top panel).