2. Parkinson’s Disease
Characteristics of
Parkinson’s Disease (PD)
include:
Progressive and selective
dopaminergic neuronal
degeneration
Resting tremor
Bradykinesia
Rigidity
Postural instability
Three different kinds of
PD based on time of onset:
Idiopathic: >40 years old
Young-onset: 21-40 years
old
Juvenile: <20 years old
4. Dieldrin Overview
Formerly known as 1,2,3,4,10,10-hexachloro-6,7-epoxy-
1,4,4a,5,6,7,8,8a-octahydro-end,exo-1,4:5,8-di-
methanonaphthalene (HEOD)
First synthesized in 1946 by Julius Hyman & Co. in
Denver, US
Widely used as an insecticide around the world until
the 1970’s
Was restricted when it was discovered to cause cancer
Officially banned by US EPA in 1987
Classified as one of the top 20 most hazardous
substances to humans by ATSDR
5. Dieldrin Toxicity
Major symptoms of human poisoning include: headache,
nausea, vomiting, convulsion, and coma
Acute lethal dose: 1.5-5 g
Half-life in human blood: 266 days
Dieldrin attacks central nervous system by inhibiting
GABA, leading to hyperexcitation, causing large increase in
Ca2+ levels
Results in: oxidative stress, mitochondrial dysfunction,
activation of caspases, activation of pro-apoptotic signaling
molecules PKCδ and PARP, and cell death via DNA
fragmentation caused by apoptosis
7. Objective
To evaluate the underlying molecular mechanisms
involved with dieldrin-induced dopaminergic
neurodegeneration using:
MTS cell death assay
ROS Assay
Caspase-9 Assay
Caspase-3 Assay
Western blotting of PKCδ, PARP, Bcl2, and Bax
pathways
8. MTS Cell Death Assay
N27 cells were plated in 96-well plate. About 10,000 cells were
plated per well.
Cells were treated with 30μM dieldrin for 3h, 6h, 12h, 18h and
24h.
Post treatment cells were incubated for 90min with 1/5th volume
of MTS dye solution (containing PMS). After incubation, 25μl of
DMSO was added to each well to dissolve the crystals of
formazan.
The absorbance was recorded at 490nm. Also a set of reading
was recorded at wavelength of 670nm as a reference
wavelength in order to eliminate the background.
The final results were converted to % Control and plotted
using GraphPad software. The students t-test was used to
compare the statistical variance. N = 6 were used per treatment
group.
9. MTS Cell Death Assay
C o n tr o l 3 h 6 h 1 2 h 1 8 h 2 4 h
0
5
1 0
1 5
2 0
2 5
3 0
3 5
4 0
4 5
5 0
5 5
6 0
6 5
7 0
7 5
8 0
8 5
9 0
9 5
1 0 0
1 0 5
***
D ie ld rin 3 0 M
CellViability(%Control)
10. ROS Assay
N27 cells were plated in 96-well plate. About 20,000 cells
were plated per well.
Cells were treated with 30μM dieldrin for 3h, 6h, 12h, 18h
and 24h.
Post treatment cells were incubated with 10μM H2-
DCFDA dye for 45min. The experiment was carefully
done in dark after adding the dye.
After dye incubation, the plate was read at em- and ex-
The final results were converted to % Control and plotted
using GraphPad software. The students t-test was used to
compare the statistical variance. N = 6 were used per
treatment group.
11. ROS Assay
C o n tr o l 3 h 6 h 1 2 h 1 8 h 2 4 h
0
5 0
1 0 0
1 5 0
2 0 0
2 5 0
3 0 0
3 5 0
4 0 0
4 5 0
**
***
D ie ld rin 3 0 M
ROSGeneration(%Control)
12. Caspase-9 and -3 activity Assay
N27 cells were plated in 12-well plate. About 100,000 cells were
plated per well.
Cells were treated with 30μM dieldrin for 6h, 12h, 18h and 24h.
Post treatment cells were trypsinized, collected and lysed using
caspase buffer. These lysates were then incubated at 37°C for
20min followed by centrifugation. The supernatant, then, was
incubated with respective caspase substrate at 37°C in a black
well plate.
After 1h of incubation, readings were measured
The caspase values were normalized with mg protein
concentration.
The final results were converted to % Control and plotted
using GraphPad software. The students t-test was used to
compare the statistical variance. N = 4 were used per treatment
group.
13. Caspase-3 activity assay
C o n tr o l 6 h 1 2 h 1 8 h 2 4 h
0
1 0 0
2 0 0
3 0 0
4 0 0
***
D ie ld rin 3 0 M
**
Caspase9Activity(U/mgprotein)
(%Control)
14. Caspase-9 activity assay
C o n tr o l 6 h 1 2 h 1 8 h 2 4 h
0
1 0 0
2 0 0
3 0 0
4 0 0
5 0 0
6 0 0
7 0 0
8 0 0
9 0 0
***
D ie ld rin 3 0 M
*
Caspase3Activity(U/mgprotein)
(%Control)
15. Western Blotting Analysis
N27 cells were plated in T-75 flasks. About 3,000,000 cells
were plated per flask. Cells were treated with 30μM
dieldrin for 3h, 6h, 12h, 18h and 24h.
Post treatment cells harvested and lysed using RIPA buffer
for protein extraction. Protein estimation was done using
Bradford assay.
20μg of protein was added in each well and gels were ran
until the dye passed through the bottom of gels. Proteins
were then transferred on to nitrocellulose membranes.
Post transfer, membranes were blocked for 45min and
then incubated with primary antibody over night.
Followed by secondary antibody incubation for 60min.
Nitrocellulose membranes were developed using LI-COR
fluorescent membrane scanner.
18. Bcl2 and Bax
Bcl-2
(26kDa)
Bax
(23kDa)
DL 30μM
β-actin
- 3h 9h 12h 18h 24h
Gel- 15% Polyacrelamide (2 different gels, one for each)
Primary AB- Rabbit Anti-Bcl-2 (Santa Cruz) 1:1000 overnight
Rabbit Anti-Bax (Santa Cruz) 1:1000 overnight
Mouse Anti-B actin (Sigma) 1:10,000 overnight
Secondary AB- Anti-mouse and Anti-rabbit 1:10,000 for 60min
19. Conclusions
Dieldrin leads to:
Oxidative stress
Mitochondrial dysfunction
Activation of caspases
Activation of pro-apoptotic signaling molecules PKCδ
and PARP
All leading to cell death via DNA fragmentation
caused by apoptosis
