An Analysis of Dieldrin in Parkinson’s Disease

Rachel A. SchroederPsychology, Iowa State University

Parkinson’s DiseaseCharacteristics of Parkinson’s Disease (PD) include: Progressive and

selective dopaminergic neuronal degeneration

Resting tremorBradykinesiaRigidityPostural instability

Three different kinds of PD based on time of onset:Idiopathic: >40

years oldYoung-onset: 21-40

years oldJuvenile: <20 years

old

Environmental Contributors

Dieldrin OverviewFormerly 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 cancerOfficially banned by US EPA in 1987Classified as one of the top 20 most hazardous

substances to humans by ATSDR

Dieldrin ToxicityMajor symptoms of human poisoning include:

headache, nausea, vomiting, convulsion, and comaAcute lethal dose: 1.5-5 g Half-life in human blood: 266 daysDieldrin 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

Dieldrin Exposure

ObjectiveTo evaluate the underlying molecular mechanisms involved with dieldrin-induced dopaminergic neurodegeneration using:MTS cell death assayROS AssayCaspase-9 AssayCaspase-3 AssayWestern blotting of PKCδ, PARP, Bcl2, and

Bax pathways

MTS Cell Death AssayN27 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.

MTS Cell Death Assay

Control 3h 6h 12h 18h 24h05

101520253035404550556065707580859095

100105

***

Dieldrin 30M

Cel

l Via

bilit

y (%

Con

trol

)

ROS AssayN27 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.

ROS Assay

Control 3h 6h 12h 18h 24h0

50

100

150

200

250

300

350

400

450

**

***

Dieldrin 30M

RO

S G

ener

atio

n (%

Con

trol

)

Caspase-9 and -3 activity AssayN27 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 measuredThe 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.

Caspase-3 activity assay

Control 6h 12h 18h 24h0

100

200

300

400***

Dieldrin 30M

**

Cas

pase

9 A

ctiv

ity (U

/mg

prot

ein)

(% C

ontr

ol)

Caspase-9 activity assay

Control 6h 12h 18h 24h0

100

200

300

400

500

600

700

800

900***

Dieldrin 30M

*

Cas

pase

3 A

ctiv

ity (U

/mg

prot

ein)

(% C

ontr

ol)

Western Blotting AnalysisN27 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.

PKCδ - 3h 9h 12h 18h 24h

DL 30μM

β-actin

Native(78kDa)

Cleaved(38, 42kDa)

PKCδ

Gel- 10% PolyacrelamidePrimary AB- Rabbit Anti-PKCδ (Santa Cruz) 1:1000 overnight

Mouse Anti-B actin (Sigma) 1:10,000 overnightSecondary AB- Anti-mouse and Anti-rabbit 1:10,000 for 60min

PARP

Native(116kDa)

Cleaved(89kDa)

DL 30μM

β-actin

- 3h 9h 12h 18h 24h

PARP

Gel- 10% PolyacrelamidePrimary AB- Rabbit Anti-PARP (Cell Signaling) 1:1000 overnight

Mouse Anti-B actin (Sigma) 1:10,000 overnightSecondary AB- Anti-mouse and Anti-rabbit 1:10,000 for 60min

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 overnightMouse Anti-B actin (Sigma) 1:10,000 overnight

Secondary AB- Anti-mouse and Anti-rabbit 1:10,000 for 60min

ConclusionsDieldrin leads to:Oxidative stress Mitochondrial dysfunctionActivation of caspasesActivation of pro-apoptotic signaling

molecules PKCδ and PARPAll leading to cell death via DNA fragmentation caused by apoptosis