Cellular localization of dieldrin and structure-activity relationship of dieldrin analogues in dopaminergic cells

Article abstract of DOI:10.1021/tx300458b

The incidence of Parkinson's disease (PD) correlates with environmental exposure to pesticides, such as the organochlorine insecticide, dieldrin. Previous studies found an increased concentration of the pesticide in the striatal region of the brains of PD

Full text of DOI:10.1021/tx300458b

Article
pubs.acs.org/crt
Cellular Localization of Dieldrin and Structure−Activity Relationship
of Dieldrin Analogues in Dopaminergic Cells
Erin M. G. Allen,† Virginia R. Florang, Laurie L. Davenport, Yunden Jinsmaa,‡ and Jonathan A. Doorn*
Division of Medicinal and Natural Products Chemistry, College of Pharmacy, The University of Iowa, Iowa City, Iowa 52242, United
States
S
* Supporting Information
ABSTRACT: The incidence of Parkinson’s disease (PD)
correlates with environmental exposure to pesticides, such as
the organochlorine insecticide, dieldrin. Previous studies found
an increased concentration of the pesticide in the striatal
region of the brains of PD patients and also that dieldrin
adversely affects cellular processes associated with PD. These
processes include mitochondrial function and reactive oxygen
species production. However, the mechanism and specific
cellular targets responsible for dieldrin-mediated cellular
dysfunction and the structural components of dieldrin
contributing to its toxicity (toxicophore) have not been fully
defined. In order to identify the toxicophore of dieldrin, a
structure−activity approach was used, with the toxicity profiles
of numerous analogues of dieldrin (including aldrin, endrin, and cis-aldrin diol) assessed in PC6-3 cells. The MTT and lactate
dehydrogenase (LDH) assays were used to monitor cell viability and membrane permeability after treatment with each
compound. Cellular assays monitoring ROS production and extracellular dopamine metabolite levels were also used. Structure
and stereochemistry for dieldrin were found to be very important for toxicity and other end points measured. Small changes in
structure for dieldrin (e.g., comparison to the stereoisomer endrin) yielded significant differences in toxicity. Interestingly, the cis-
diol metabolite of dieldrin was found to be significantly more toxic than the parent compound. Disruption of dopamine
catabolism yielded elevated levels of the neurotoxin, 3,4-dihydroxyphenylacetaldehyde, for many organochlorines. Comparisons
of the toxicity profiles for each dieldrin analogue indicated a structure-specific effect important for elucidating the mechanisms of
dieldrin neurotoxicity.
trafficking)? Where does dieldrin localize in a cell, and what are
the targets? How does dieldrin interact with proteins,
covalently or noncovalently, given that it has an epoxide?
Previous studies have assessed how structural changes to
dieldrin affect its pesticidal activity in various species of
insects.14−16 Significant alterations to insect toxicity were found
even with small changes to dieldrin’s structure, such as removal
of chlorine atoms or a carbon bridge.14−16 Such data suggest
that specific structural elements are critical for its insecticidal
properties. Of question then is how these same structural
features relate to dieldrin’s dopaminergic toxicity in humans.
Do the compounds with the most potent insecticidal activity
also demonstrate the most detrimental effects in a model of
dopaminergic cells?
INTRODUCTION
■
While the exact mechanism of neuronal loss in Parkinson’s
disease (PD) is unknown,1 a majority of PD cases are thought
to be due to exposure to environmental toxicants, such as
insecticides, herbicides, and heavy metals.2−6 The correlation
between pesticide exposure and PD has been corroborated by
an increased concentration of pesticides found in the brains of
PD patients.2,4−6 One pesticide of interest is dieldrin, as its level
was elevated in PD patients, a result not found in the brains of
patients with other neurodegenerative diseases, such as
Alzheimer’s disease, or in the brains of healthy, control
patients.4 In addition, previous studies have also shown dieldrin
to affect a number of cellular processes associated with PD. The
consequences of these effects include increased oxidative stress,
disruptions in the metabolism and trafficking of dopamine
(DA), apoptosis susceptibility, and mitochondrial dysfunc-
tion.1,7−12 Many of the reported adverse effects caused by
exposure to dieldrin, such as reactive oxygen species (ROS)
production and apoptosis, lead to selective dopaminergic cell
dysfunction and/or death.7,9,12,13 A question that remains is,
what is the mechanism via which dieldrin mediates such adverse
cellular effects (e.g., ROS formation and disruption of DA
To determine dieldrin’s structure−activity relationship, three
aspects of its structure were assessed (Figure 1: 1). The relative
3-dimensional orientation and presence of the methano bridge
(Groups 1 and 2). The identity of the polar moiety (olefin, cis-
diol, or epoxide) located opposite the chlorine atoms (Groups
Received: November 14, 2012
© XXXX American Chemical Society
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Figure 1. Structures and groupings of dieldrin analogues. Compared to dieldrin, each of the three groups have varied structure. Group 1: methano
bridge, opposite configuration (endrin) or absent (desmethylene dieldrin). Group 2: epoxide, hydrolyzed to diol (cis-aldrin diol) or absent (aldrin).
Group 3: no epoxide and opposite configuration of the methano bridge (isodrin) or no epoxide and methano bridge (desmethylene aldrin).
NMR (DMSO-d6): δ 28.3, 43.0, 51.7, 73.6, 81.0, 104.7, 130.8; MS
(EI) m/z calcd for C12H10Cl6O2 395.9; found m/z 396.1.
2 and 3). The combined effect of the 3-dimensional orientation
and polar group (Group 3). The effects of these compounds on
cell viability, DA metabolism, and ROS production were
investigated.
Comparison of these features and their activity will provide
valuable information about the cellular targets of these
compounds and will be used to gain valuable insight into the
mechanistic link between dieldrin exposure and the develop-
ment of PD. Ultimately, how does dieldrin exposure predispose
individuals to PD? The selectivity of dieldrin toward
dopaminergic neuronal death in vivo is predicted to facilitate
the development of PD. Therefore, it is of high importance that
the mechanism of dopaminergic toxicity from dieldrin exposure
is understood.
Desmethylene Aldrin (1,8,9,10,11,11-Hexachloro-2,3-7,6-
endotricyclo[6.2.1.02,7]undeca-4,9-diene). Using methods adap-
ted from literature methods,14,19−21 we placed hexachlorocyclopenta-
diene (1.0 g, 0.004 mol, 0.6 mL) and 1,4-cyclohexadiene (1.3 g, 0.016
mol, 1.51 mL) under argon and stirred them at 110 °C for 44 h. The
product was purified by column chromatography on silica gel with
hexanes as mobile phase, resulting in a 25% yield. 1H NMR (CDCl3):
δ 1.90−1.99 and 2.35−2.44 (m, 4H, CH2), 2.98−3.09 (m, 2H, CH),
5.86 (m, 2H, HCCH). 13C NMR (CDCl3): δ 21.9, 45.8, 82.5, 103.2,
126.7, 131.6; MS (EI) m/z calculated for C11H8Cl6 349.9; found m/z
349.9.
Desmethylene Dieldrin (1,8,9,10,11,11-Hexachloro-4,5-
epoxy-2,3-7,6-endotricyclo[6.2.1.02,7]undec-9-ene). Using meth-
ods adapted from literature methods,14,19,21,22 we added 3-
chloroperbenzoic acid (mCPBA) (0.08 g, 0.5 mmol) in 2.0 mL of
dichloromethane dropwise to desmethylene aldrin (0.1 g, 0.3 mmol)
in 2.0 mL of dichloromethane and stirred at room temperature for 2 h.
The reaction mixture was extracted twice with water. The organic layer
was then dried with magnesium sulfate and the product purified by
column chromatography on silica gel with a mobile phase of 4:1
EXPERIMENTAL PROCEDURES
■
Caution: The following compounds are hazardous and
should be handled carefully: dieldrin, aldrin, endrin, isodrin,
cis-aldrin diol, desmethylene dieldrin, and desmethylene aldrin.
All are highly lipophilic and contact with skin, eyes, and clothing
should be avoided. Chemicals. Dieldrin, aldrin, endrin, and isodrin
were purchased from Chem Service (West Chester, PA). All other
reagents, unless otherwise noted, were purchased from Sigma Aldrich
(St. Louis, MO) without further purification. Organochlorine
analogues were named based on the von Baeyer/IUPAC system for
polycyclic compounds and as discussed previously.17
1
hexanes to acetone, resulting in a 50% yield. H NMR (CDCl3): δ
1.59−1.67 and 2.40−2.48 (m, 4H, CH2), 2.91−2.99 (m, 2H, CH),
3.20−3.24 (m, 2H, CH−O). 13C NMR (CDCl3): δ 22.2, 42.2, 49.1,
82.3, 102.8, 132.0; MS (EI) m/z calculated for C11H8Cl6O 365.9;
found m/z 365.9.
Thiol and Amine Reactivity. Thiol reactivity of dieldrin was
determined using Ellman’s reagent (5,5′-dithiobis(2-nitrobenzoic acid)
(DTNB)) and a procedure adapted from the literature.23,24 N-Acetyl
Cys (100 μM) was incubated with dieldrin (0−2.5 mM) overnight at
37 °C in DMSO. DTNB (0.5 mM) in DMSO was added along with
50 mM sodium phosphate buffer, pH 7.4 (25% v/v), and absorbance
monitored at 412 nm using a Molecular Devices SpectraMax plate
reader. The free thiol concentration in each well was determined by
comparing the absorbance to a standard curve of DTNB reacted with
varying concentrations of N-acetyl Cys.
The reactivity of dieldrin with primary amines was assessed using N-
acetyl Lys. Dieldrin (0−100 μM in DMSO) was incubated with N-
acetyl Lys (100 μM in DMSO) at 37 °C for 24 h. The concentration
of dieldrin was monitored via an HPLC method adapted from Mowafy
et al.25 Briefly, the samples were analyzed on an Agilent 1100 Series
cis-Aldrin Diol (1,8,9,10,11,11-Hexachloro-4,5-(exo)cis-dihy-
droxy-2,3-7,6-endo-2,1-7,8-exotetracyclo[6.2.1.13,6.02,7]dodec-
9-ene). cis-Aldrin diol was synthesized using a method adapted from
the literature.18,19 Briefly, aldrin (86 mg, 0.24 mmol) was combined
with dry ether (6.4 mL), pyridine (0.15 mL), and osmium tetroxide
(50 mg, 0.20 mmol). The reaction was stirred for 2 h at room
temperature and then allowed to stand overnight in the dark. The
ether was evaporated with nitrogen, giving a brown residue that was
then dissolved in dichloromethane (10 mL) and stirred vigorously
overnight with a solution of water (6.5 mL), potassium hydroxide (71
mg, 1.3 mmol), and D-mannitol (630 mg, 3.46 mmol). The
dichloromethane layer was removed, washed with water, and dried
with sodium sulfate, giving the product in 90% yield. 1H NMR
(DMSO-d6): δ 1.12 (d, 1H, CH2), 1.60 (d, 1H, CH2), 2.06 (m, 2H,
CH), 2.60 (m, 2H, CH), 3.57 (m, 2H, CH), 4.93 (m, 2H, OH); 13C
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Capillary HPLC with a Phenomenex C18 Jupiter column (5 μm, 300
Å, 150 × 1.0 mm) using isocratic conditions of water (25%) and
methanol (75%), a flow rate of 50 μL/min, and the absorbance
measured via photodiode array at 214 and 234 nm. Differences in peak
area between dieldrin incubated with and without N-acetyl Lys are
predicted to indicate reactivity between these two compounds, i.e.,
dieldrin and the dieldrin-Lys conjugate.
Cell Culture. PC6-3 cells, a generous gift from Stefan Strack
(University of Iowa), were cultured in RPMI1640 medium (GIBCO,
Invitrogen, Carlsbad, CA) supplemented with heat-inactivated horse
serum (10%) (HyClone, Thermo Scientific, Rockford, IL), fetal bovine
serum (5%) (GIBCO), penicillin (10 IU/mL), and streptomycin (10
mg/mL) and were grown in 100 mm2 tissue culture dishes at 37 °C
with 5% CO2. Cells (3 × 104 cells/well or 6 × 104 cells/well for DA-
supplemented metabolism experiments) were seeded into six-well
plates and allowed to grow for four days at 37 °C with 5% CO2. The
cells were then differentiated with 2.5S nerve growth factor (NGF)
(BD Biosciences, Bedford, MA) at 50 ng/mL for 4 days. The cells
used in these experiments are a subline of PC12 cells, an established
cellular model for dopaminergic neurons.26−29 Previous work has
demonstrated that PC6-3 cells express enzymatically active tyrosine
hydroxylase and metabolize dopamine, thereby demonstrating their
utility as a cellular model for dopaminergic neurons.30−32
Treatment of Cells with Dieldrin and Analogues. Media were
removed from cells and replaced with HEPES-buffered saline
containing 115 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.8 mM
MgSO4, 5.5 mM glucose, 1.0 mM NaH2PO4, and 15 mM HEPES (pH
7.4). The cells were treated with the compounds (dieldrin, aldrin,
endrin, isodrin, cis-aldrin diol, desmethylene aldrin, or desmethylene
dieldrin) at 0−900 μM in dimethylsulfoxide (DMSO, 0.6%) for 4 h.
The concentrations used in this study (10−900 μM) are higher than
the concentrations detected in the brains of Parkinson’s patients (2.4
μM, based on reported values of μg of dieldrin per g of lipid in the
striatum)2,4−6 but were chosen to represent the full range of cell death
in this model system (20−100% loss of cell viability). This range was
required to calculate the IC50 values, which were used for accurate
comparisons between compounds and were also comparable to the
concentrations used in similar studies.7,11,13,33−36
Cell Fractionation and GC/MS Analysis. Upon 4 h of treatment
of the PC6-3 cells with dieldrin (0−300 μM), the HEPES-buffered
saline used for treatment was collected (extracellular fraction) and
then lysis buffer (10 mM K2HPO4 with 0.1% Triton X-100 (v/v)) was
added to each well. The cell lysate was collected and sonicated for 5
min using a sonicator bath. The membrane/lipid fraction was
separated from the cytosol and lysed organelles (intracellular fraction)
using centrifugation at 1000g for 10 min at 4 °C (Abcam Technical
Bulletin, Plasma Membrane Protein Extraction Kit). The pellet
(membrane/lipid fraction) was resuspended in phosphate-buffered
saline, and all samples were stored at −70 °C pending analysis.
Prior to extraction, each sample was spiked with an internal
standard (endrin, 1 μg/mL). The organochlorines were extracted three
times using hexanes with a volume equal to the sample volume. If an
emulsion formed, one part ethanol was added to the sample. The
solvent was removed from the organic layer using nitrogen gas and
then the sample dissolved in ethyl acetate. Dieldrin and endrin were
separated using a Thermo Voyager gas chromatograph coupled with a
single quadrapole mass spectrometer. Flow of the gas carrier was 20
mL/min, and the temperature was 50 °C for 2 min, increased by 10°/
min to 280 °C and then held for 10 min. The mass spectrometer
monitored ionization at 79 m/z and 263 m/z. Peak area quantification
was completed using Xcaliber 2.0. Dieldrin peak area at 263 m/z,
elution time 20.28 min, was first normalized to the internal standard
(endrin at 263 m/z, elution time 19.88 min) and then compared to a
standard curve. The amount of dieldrin (μg) in each of the fractions
was compared to the amount of dieldrin extracted from treatment with
10 μM of dieldrin and are given as fold change of dieldrin. Values are
reported as the mean SD, n = 3.
tetrazolium is converted to a purple formazan salt by cellular
reductases (e.g., succinate dehydrogenase). After a 4 h treatment of
the cells with each compound, the cells were incubated for 1.25 h at 37
°C with MTT (0.5 mg/mL). The HEPES-buffered saline was then
removed from each well and centrifuged at 10,000g for 5 min. This
pellet and the formazan salt remaining in each well were dissolved in
DMSO and combined. Absorbances were then measured at 570 and
650 nm using a Molecular Devices Spectra-Max plate reader. Results
are presented as % of control.
IC50 Value Calculations. In order to calculate the IC50 values for
each compound based on their cell viability, the dose−response data
was normalized between 0 and 100%, and graphed against the
log[inhibitor]. A nonlinear curve fitting was applied based on
log[inhibitor] vs normalized response with variable slope. The
concentration at which 50% normalized activity was observed is
reported as the IC50 SEM.
Cytotoxicity Assay. The cytotoxicity of each compound was
determined using Cytotoxicity Detection KitPLUS (LDH) (Roche
Applied Science, Mannheim, Germany), according to the manufac-
turer’s specifications. Briefly, a high control for each set consisted of
cells incubated with 0.6% DMSO and then for the last 15 min of the
experiment, lysis buffer, as provided by the manufacturer. The low
control in each set was a well incubated with 0.6% DMSO and no lysis
buffer. At 4 h, an aliquot of media was removed from each well and
incubated with the reaction mixture provided for 20 min in the dark at
RT. The reaction was then stopped and the absorbance at 492 and 690
nm measured using a Molecular Devices Spectra-Max plate reader. In
order to determine the percent cytotoxicity, the value obtained for the
low control was subtracted from each treated well, then divided by the
difference between the high control and the low control. Dieldrin and
the other analogues did not interfere with the absorbance readings for
this assay.
The ability of each compound to inhibit LDH was evaluated by
incubating rabbit L-LDH (0.5 U/mL) with various concentrations of
each compound (0−900 μM, 0.6% DMSO) for 30 min in HEPES-
buffered saline. At this point, the reaction mixture containing the
necessary enzyme substrates and cofactors was added and incubated in
the dark at room temperature for 5 h. Absorbances at 492 and 690 nm
were then measured and the normalized absorbance determined by
subtracting the 690 nm absorbance from the absorbance at 492 nm to
assess enzyme activity.
Dopamine Metabolism. The effect of each compound on the
extracellular concentration of DA metabolites was monitored. Cells
were treated with 0, 10, 25, 50, 100, or 300 μM of each compound. An
aliquot of the extracellular HEPES-buffered saline was removed at 0, 1,
and 4 h and then mixed with perchloric acid (5% v/v) in order to
precipitate the proteins and terminate any reaction. The samples were
stored at −70 °C, thawed, and centrifuged at 10,000g for 5 min prior
to HPLC analysis. Samples were analyzed by an Agilent 1200 Series
Capillary HPLC with a Phenomenex C18 Luna column (1 × 150
mm). The dopamine metabolites 3,4-dihydroxyphenylacetaldehyde
(DOPAL), 3,4-dihydroxyphenylacetic acid (DOPAC), and 3,4-
dihydroxyphenylethanol (DOPET) were separated using isocratic
conditions of 0.1% trifluoroacetic acid in water with 6% ACN (v/v), a
flow rate of 50 μL/min, and detection with a photodiode array
detector (absorbance at 202 and 280 nm). The peak area was then
converted to concentration by comparison to a standard curve
calculated from metabolite standards as previously described.30,37−39
For the DA-supplemented metabolism experiments, cells were
supplemented with 100 μM DA 15 min prior to dieldrin treatment
to initiate DA metabolism.
Flow Cytometry. Differentiated PC6-3 cells were pretreated with
dihydroethidium (DHE, 10 μM, 0.2% DMSO) and 2′,7′-dichlor-
odihydrofluorescein diacetate (H2DCFDA, 50 μM, 0.2% DMSO) for
20 min at 37 °C in HEPES-buffered saline. Cells were then washed
and treated with each dieldrin analogue at a nontoxic dose (<20% cell
death according to MTT assay) for 1 h. The doses of each
organochlorine used were dieldrin (25 μM), aldrin (25 μM), cis-
aldrin diol (25 μM), desmethylene dieldrin (10 μM), isodrin (100
μM), and desmethylene aldrin (10 μM). Cells were then removed
Cell Viability. The effect of each compound on cell viability was
determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-
lium bromide (MTT) reduction assay, in which the yellow, MTT
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from the wells, washed with phosphate-buffered saline, and
resuspended in HEPES-buffered saline prior to filtration (70 μm).
Hoechst 33258 (4 μg/mL) was added and the absorbance within the
cells measured using a Becton Dickinson LSR II flow cytometer with
UV and 440/40 band-pass (BP) (Hoechst 33258), 530/30 BP
(H2DCFDA), and 610/20 BP (DHE). Data were processed using
CellQuest (BD Bioscience), and results were reported as the
geometric mean SEM.
Statistics. All statistics and curve fittings were calculated using
GraphPad Prism, version 5.01c (GraphPad Software, San Diego, CA).
Rules of error propagation were used to determine the standard
deviation of the fold change of dieldrin concentration for the cellular
localization experiments. Significance from control for the thiol and
amine reactivity was determined using a two-tailed t test (p < 0.05). All
other statistical analyses were calculated using one-way ANOVA with
Newman-Keuls post-test, unless otherwise noted.
RESULTS
■
Reactivity of Dieldrin (Epoxide) with Cys and Lys. To
determine the reactivity of dieldrin, specifically the epoxide,
toward the protein nucleophiles Cys and Lys, dieldrin was
incubated with N-acetyl Lys and Cys and the reaction
monitored. The thiol reactivity of dieldrin was assessed using
N-acetyl Cys, and the concentration of free thiols was
monitored via DTNB. No decrease in thiol concentration was
observed.
In addition, dieldrin was incubated with N-acetyl Lys and the
concentration of dieldrin remaining over time measured using
HPLC. No significant decrease in dieldrin concentration was
observed upon addition of N-acetyl Lys, indicating no reaction
under these conditions. These results indicate that dieldrin does
not covalently modify target proteins under the conditions used
and that any observed protein interactions are most likely
noncovalent, despite the presence of an epoxide on the
organochlorine.
Localization of Dieldrin in Dopaminergic PC6-3 Cells.
To determine the cellular localization of dieldrin following the
exposure of PC6-3 cells to the organochlorine, cells were
fractionated and dieldrin measured using GC/MS. Upon
treatment with dieldrin (0−300 μM), the PC6-3 cells were
fractionated giving extracellular, membrane/lipid, and intra-
cellular fractions. Representative chromatograms of the extract
of a cellular fraction are shown in Supporting Information,
Figure 1. As depicted in Figure 2A, the amount of dieldrin in
the membrane/lipid fraction increased with the dose of
dieldrin. Cells treated with 3.8 μg (10 μM), 9.5 μg (25 μM),
19 μg (50 μM), 38 μg (100 μM), and 114 μg (300 μM)
dieldrin had measured lipid/membrane organochlorine
amounts of 0.022 μg, 0.16 μg, 0.89 μg, 3.1 μg, and 17 μg,
respectively. When dieldrin in the intracellular fraction was
quantified (Figure 2B), it was found that the amount of dieldrin
increased with dosage through 50 μM treatment but not to the
same extent as that observed for the membrane/lipid fraction.
Cells treated with 3.8 μg (10 μM), 9.5 μg (25 μM), 19 μg (50
μM), 38 μg (100 μM), and 114 μg (300 μM) dieldrin had
measured intracellular organochlorine amounts of 0.34 μg, 0.83
μg, 1.3 μg, 0.77 μg, and 2.3 μg, respectively. Such results
indicate the limited capacity of the intracellular fraction for
dieldrin and that the majority of the organochlorine remains in
the membrane/lipid component.
Figure 2. Amount of dieldrin, normalized to internal standard and
standard curve, following hexane extraction of cellular fractions from
cells treated with 0−300 μM dieldrin. Each bar represents the fold
change in dieldrin concentration compared to the amount extracted
after treatment of cells with 10 μM dieldrin ( SD, n = 3). (A)
membrane/lipid fractions; (B) intracellular fractions.
depicted for dieldrin in Figure 3A, Group 1 analogues in Figure
3B and C, Group 2 in Figure 3D and E, and Group 3 shown in
Figure 3F and G. All of the compounds, except for endrin,
displayed a dose-dependent decrease in cell viability in the
PC6-3 cells after 4 h. On the basis of these results, IC50 values
were calculated in order to easily compare the potency of each
compound (Table 1).
The parent compound dieldrin showed a dose-dependent
increase in toxicity as determined via the decrease in cell
viability, with an IC50 value of 293 μM. Endrin did not exhibit a
dose-dependent increase in toxicity (Figure 3B); therefore, no
IC50 value could be calculated. When the methano bridge was
removed, yielding a more flexible structure (desmethylene
dieldrin), the compound was substantially more toxic than
dieldrin (Figure 3C) with an IC50 value of 22 μM.
When dieldrin was compared to Group 2, compounds with
changes to the polar moiety, it was found that the substitution
of the epoxide for an olefin (aldrin, Figure 3D) or a cis-diol (cis-
aldrin diol, Figure 3E) both resulted in decreased cell viability
(IC50 values of 46 and 36 μM, respectively), as compared to
that for dieldrin. Such a result implies that a moiety capable of
being a hydrogen bond donor (i.e., cis diol) enhances the
toxicity, as does the presence of an olefin.
The last group of compounds takes into account the
presence of the olefin, already shown to enhance toxicity, as
well as the 3-dimensional orientation of the compound. The
isomer of aldrin (isodrin, Figure 3F) showed very little toxicity
(IC50 value was 578 μM). When the desmethylene analogue of
aldrin was assessed (desmethylene aldrin, Figure 3G), it
resulted in the most substantial decrease of cell viability of all
of the analogues with an IC50 value of 16 μM. These results
imply that the presence of the olefin augments the toxicity of
dieldrin, and when this modification is coupled with the
removal of the methano bridge, the toxicity is further enhanced.
Cell Viability. To assess the structure−activity relationship
of dieldrin toxicity, the viability of PC6-3 cells was monitored
after 4 h of treatment with various concentrations of each
dieldrin analogue via the MTT assay (Figure 4). Results are
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Figure 3. Cell viability of PC6-3 cells treated with dieldrin, Group 1,
Group 2, or Group 3 compounds (% control SD, n = 4−6) obtained
from MTT assay after 4 h of incubation with (A) dieldrin, (B) endrin,
(C) desmethylene dieldrin, (D) aldrin, (E) cis-aldrin diol, (F) isodrin,
and (G) desmethylene aldrin. The difference between each pair of
treated groups is significant (p < 0.05) unless otherwise noted (NS).
Figure 4. Percent cytotoxicity by lactate dehydrogenase release from
PC6-3 cells after 4 h of incubation with dieldrin, Group 1, Group 2, or
Group 3 compounds (% cytotoxicity SD, n = 4−6). (A) Dieldrin,
(B) endrin, (C) desmethylene dieldrin, (D) aldrin, (E) cis-aldrin diol,
(F) isodrin, and (G) desmethylene aldrin. The difference between
each pair of treated groups is significant (p < 0.05) unless otherwise
noted (NS).
Table 1. IC50 Values (μM SEM) for Each Compound
Based on the Concentration of Each Inhibitor at 50% of the
Normalized Cell Viability
a
analogues being the most potent, followed by both compounds
in Group 2 (aldrin and cis-aldrin), then dieldrin. Isodrin and
endrin were the least toxic.
compd
IC50 (μM)
dieldrin
endrin
293 1.08
b
ND
In the LDH results, a deviation from the dose-dependent
trend was observed at higher concentrations of organochlorine,
particularly with the more toxic derivatives. Each compound
showed increased release of LDH until the concentration at
which roughly 65% cytotoxicity was achieved. At organo-
chlorine concentrations that would be expected to yield greater
than 65% cytotoxicity, the values representing LDH release and
activity remained constant (desmethylene dieldrin, Figure 4C)
or decreased (cis-aldrin diol, Figure 4E). This deviation from
the expected dose−response was not seen with the MTT
results. It was determined that the compounds did not absorb
at the wavelengths monitored or react with the reagents used in
this experiment.
Since this experiment was based on enzyme activity, the
compounds were then tested for inhibition of the enzyme,
LDH. When each compound was incubated with rabbit L-LDH
at higher concentrations, a decrease in enzyme activity was
observed as shown in Supporting Information, Figure 2. It can
be inferred that when the cells were treated with higher
concentrations of the dieldrin analogues, the outer membrane
of the cells was disrupted, causing a release of LDH. However,
desmethylene dieldrin
aldrin
22.3 1.05
46.4 1.08
36.9 1.11
578 1.22
15.8 1.05
cis-aldrin diol
isodrin
desmethylene aldrin
a
b
Values represent the mean SEM. Not determined. The measured
toxicity was not dose dependent.
Release of Lactate Dehydrogenase. To further assess
the structure−activity relationship of dieldrin toxicity, the
viability of PC6-3 cells was monitored after 4 h of treatment
with various concentrations of each dieldrin analogue via the
LDH assay. For each of the compounds, a dose-dependent
decrease in cell viability with respect to that of the control was
observed, except for endrin, which showed no cytotoxicity, even
at high concentrations (900 μM) (Figure 4). The results from
the LDH experiment correlate well to those observed using
MTT to monitor cell viability (Figure 3) and data from a
previous study.7 The relative order of toxicity between each
analogue was also comparable, with the desmethylene
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a
Scheme 1. DA Metabolism
a
DA is metabolized by monoamine oxidase (MAO) to the aldehyde, DOPAL. This intermediate is oxidized by aldehyde dehydrogenase (ALDH) to
the acid metabolite (DOPAC) or reduced by aldehyde reductase (AR) to an alcohol, DOPET.
a
Table 2. Concentration of Dopamine Metabolites (DOPAL, DOPAC, or DOPET)
b
compd
control
group
[DOPAL] (μM)
[DOPAC] (μM)
[DOPET] (μM)
0.76 0.21
2.94 0.87
1.67 0.32
dieldrin
1.53 0.25NS
0.60 0.09NS
7.79 2.49***
7.75 1.61***
10.7 5.48***
1.00 0.19NS
7.02 2.19***
2.87 1.36NS
3.51 0.19NS
0.54 0.15***
1.15 0.20**
0.53 0.11***
2.76 0.95NS
0.23 0.20***
1.58 0.49NS
1.78 0.14NS
1.16 0.27NS
1.24 0.11NS
0.58 0.06***
2.12 0.44NS
0.85 0.06**
endrin
1
1
2
2
3
3
des dieldrin
aldrin
cis-aldrin diol
isodrin
des aldrin
a
Concentrations of dopamine metabolites ( SD, n = 3, except for controls where n = 21) after 4 h of treatment with 300 μM of each compound or
vehicle control (0.6% DMSO). Difference from vehicle control was significant with p < 0.001 (**) or p < 0.0001 (***), or not significant with p >
0.05 (NS). Statistical analysis was performed via ANOVA with Newman-Keuls post-test. The elevation of DOPAL following dieldrin treatment was
b
significantly different from that of the control when analyzed by a two-tailed t test (p < 0.0001). See Figure 1 for group assignments and structures.
due to the substantial amount of the dieldrin analogue still
remaining in the extracellular media or that was released from
the cell during apoptosis, the LDH was inhibited by the
compound. This resulted in the decreased enzyme activity
corresponding to the observed deviation from the dose-
dependent increase in cytotoxicity for each analogue at higher
concentrations (Figure 4).
Dopamine Catabolism. To determine the effect of
dieldrin and analogues on DA catabolism, which is a process
capable of generating reactive and toxic intermediates, PC6-3
cells were incubated with the organochlorines and extracellular
DA metabolites measured, including DOPAL and DOPAC.
Extracellular DA metabolites (Scheme 1) were quantified by
HPLC, with the results presented in Table 2 and Figures 5 and
6. The concentration of extracellular DOPAL after 4 h of
treatment is shown for the control (vehicle-treated) and all
organochlorine compounds in Figure 5. When the cells were
treated with dieldrin, a time- and concentration-dependent
increase in DOPAL was observed. Small changes in the levels
for the other DA metabolites, DOPAC and DOPET, were also
observed (Figure 6 and Table 2). Such findings were also noted
following treatment of the cells with the various isomers of
dieldrin.
When endrin, the isomer of dieldrin, was analyzed, a slight
decrease in DOPAL and an increase in DOPAC were observed.
For desmethylene dieldrin (300 μM), the analogue of dieldrin
lacking the methano bridge, the extracellular DOPAL
concentration increased 10-fold. The concentration of
Figure 5. Quantification of the extracellular concentration of DOPAL
from PC6-3 cells incubated for 4 h with vehicle control (0.6%
DMSO), dieldrin, and Group 1 compounds (endrin and desmethylene
dieldrin, A), Group 2 compounds (aldrin and cis-aldrin diol, B), or
DOPAC was significantly reduced from the control following
treatment with this analogue (Figure 6A).
Group 3 compounds (isodrin and desmethylene aldrin, C). The values
Group 2, with changes to the polar group opposite of the
chlorine atoms, also showed a profound effect on the
extracellular concentration of DOPAL in comparison to
dieldrin, with both aldrin and cis-aldrin diol resulting in
shown represent the mean SD (n = 3, except for controls where n =
21), where peak area was converted to concentration using a standard
curve.
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Figure 7. Percent control of the concentration of DOPAL released
from PC6-3 cells pretreated with 100 μM dopamine prior to 4 h of
exposure to 10, 30, 100, or 300 μM dieldrin. The values shown
represent the mean SEM (n = 3 or 4), normalized to the percent
control of DOPAL release. The percent control of DOPAL release
after treatment with 100 and 300 μM dieldrin was significantly
different from that of the control (p < 0.05).
Figure 6. Quantification of the extracellular concentration of DOPAC
from PC6-3 cells incubated for 4 h with vehicle control (0.6%
DMSO), dieldrin, and Group 1 compounds (endrin and desmethylene
dieldrin, A), Group 2 compounds (aldrin and cis-aldrin diol, B), or
Group 3 compounds (isodrin and desmethylene aldrin, C). The values
shown represent the mean SD (n = 3, except for controls where n =
21), where peak area was converted to concentration using a standard
curve.
significant and substantial increases in DOPAL levels, as
compared to the control (Figure 5B). This observation was
accompanied by a marked decrease in the production of
DOPAC (Figure 6B).
For the third group of compounds, isodrin and desmethylene
aldrin, results similar to those of endrin and desmethylene
dieldrin were observed, respectively. Isodrin showed only a
small change from the control for both DOPAL (slight
increase) and DOPAC (slight decrease), whereas desmethylene
aldrin showed a marked increase in DOPAL concentration and
a corresponding decrease in DOPAC (Figures 5C and 6C).
Because the PC6-3 cells released low levels of DA
metabolites, the cells were spiked with 100 μM DA 15 min
prior to dieldrin exposure in order to more accurately
determine the influence of dieldrin on DA metabolism (Figure
7). DOPAL levels were measured as previously described and
were expressed as % control [DOPAL] for treatment with 10,
30, 100, or 300 μM dieldrin. A dose-dependent trend was
observed, with concentrations as low as 10 μM dieldrin
generating a nearly 200% increase in extracellular DOPAL
levels.
Reactive Oxygen Species. To determine the effect of
nontoxic concentrations (<20% cell death) of each dieldrin
analogue on the production of ROS, cells were incubated with
the organochlorines, and ROS was measured via flow
cytometry. General ROS production was determined using
H2DCFDA, and a significant increase in oxidative stress was
observed upon treatment with each of the compounds studied
(endrin was not included in this experiment) (Figure 8A).
Figure 8. Production of ROS monitored via flow cytometry after 1 h
of treatment with nontoxic concentrations of each compound (10 μM
for desmethylene aldrin and desmethylene dieldrin, 25 μM for
dieldrin, aldrin, and cis-aldrin diol, and 100 μM for isodrin). Results
depicted as geometric mean ( SD, n = 3) for (A) H2DCFDA (general
ROS) or (B) DHE (O2•−). Change in geometric mean upon treatment
with each compound in A for H2DCFDA fluorescence was
significantly different from that of the control (p < 0.0001), except
for desmethylene aldrin; whereas in B, for DHE fluorescence, no
significant change was observed with any of the compounds (p >
0.05).
When levels of superoxide anion were monitored using DHE,
no significant change from the control was observed (Figure
8B), indicating that the accumulation of ROS from dieldrin
analogue exposure is most likely from H2O2 in the PC6-3 cells.
DISCUSSION
■
The goal of this study was to elucidate the interaction of the
dieldrin with components of dopaminergic cells and determine
structure−activity relationships in order to better understand
the mechanism of toxicity for this organochlorine pesticide. To
complete this goal, a series of experiments were conducted to
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determine the protein reactivity, localization, effect on DA
metabolism, and structure−activity relationship of dieldrin for
toxicity using dopaminergic cells. Such work is significant given
that the structure−activity relationship of dieldrin is not well-
defined in mammals.40
(aldrin) or a cis-diol (cis-aldrin diol). Aldrin was widely used as
a pesticide around the same time as dieldrin but is slightly less
toxic in various species of insects.14,46 Aldrin has a shorter half-
life than dieldrin, mostly due to increased volatility and rate of
metabolism in the environment and in mammals.47 Despite
these characteristics limiting the adverse effects of aldrin
exposure, it is still listed as #25 on the CERCLA priority list of
hazardous substances48 and has been detected in the post-
mortem brains of Parkinson’s disease patients.2,4−6 cis-Aldrin
diol is a metabolite of dieldrin that can then be epimerized to
trans-diol in mammals.49 This compound is less toxic to insects,
Dieldrin contains an epoxide, a moiety that is typically
electrophilic and highly reactive toward nucleophiles.41 It is
conceivable that the nucleophilic sites on proteins (i.e., Cys or
Lys residues) would be able to react with the epoxide, causing
the opening of this three-membered ring and protein
modification.41 Previous studies have shown that dieldrin
“binds” to proteins, such as those found in spinal cord
homogenate from rats and cockroaches and, to a lesser extent,
albumin.42,43 While isolated amino acids cannot completely
model the unique environment of these amino acids in each
possible protein target, they can provide a valuable reactivity
profile. In the current study, it was determined that dieldrin was
not capable of covalently modifying Lys or Cys residues,
supporting the previous assumptions of noncovalent inter-
actions. The dieldrin epoxide could be stabilized due to the
rigid polycyclic structure, thereby limiting the likelihood of
nucleophilic attack by amino acids such as Lys and Cys.
The cellular localization of dieldrin in PC6-3 cells was
investigated using cell fractionation and GC/MS. On the basis
of the results (Figure 2), dieldrin was found to accumulate in
the cell, particularly in the membrane and lipid fraction (Figure
2A). Saturation of the membrane fraction was not observed
with organochlorine concentrations used, and dieldrin levels
increased as treatment levels increased. The amount of dieldrin
in the intracellular fraction increased as organochlorine
treatment concentration (up to 50 μM) increased. At higher
treatment concentrations (>50 μM), the amount of dieldrin in
the intracellular fraction began to level out, indicating decreased
diffusion or saturation of binding sites. From the results in
Figure 2, it may be concluded that dieldrin readily enters PC6-3
cells and accumulates in the membrane and lipid-containing
portion; however, binding to intracellular components can be
saturated at low micromolar levels. This trend is most likely due
to the lipophilicity of the organochlorine.
The adverse cellular effects of dieldrin have been previously
investigated, particularly with respect to the effect of dieldrin on
various aspects of mitochondrial activity.7,11,44,45 Bergen et al.44
demonstrated dieldrin’s ability to disrupt respiration in rat liver
mitochondria and inhibit electron transport. In a dopaminergic
system similar to that of the PC6-3 cells (i.e., PC12 cells),
dieldrin has been shown to cause a 50% decrease in cell viability
(MTT) at 143 μM at 1 h in PC12 cells.7 Even with variations in
cell line, cell preparation, and time frame, this value is
comparable with the IC50 value of 293 μM obtained using
the MTT assay in the current study.
In an effort to assess the structure−activity relationship of
dieldrin, as it relates to its toxicity in dopaminergic cells, six
analogues were used (Figure 1). These compounds were
chosen based on their chemical structure, allowing for the
assessment of three structural characteristics: (1) the
importance of the 3-dimensional orientation and presence of
the methano bridge (Group 1, endrin and desmethylene
dieldrin), (2) the effect of the polar group opposite of the
chlorine atoms (Group 2, aldrin and cis-aldrin diol), and (3) the
interplay between both the polar moiety and the 3-dimensional
orientation (Group 3, isodrin and desmethylene aldrin).
Dieldrin contains an epoxide opposite of the chlorine atoms,
whereas the compounds in Group 2 contain either an olefin
50
as compared to dieldrin.
The third group aimed to assess the combined effects of
altering the polar moiety (epoxide to olefin) along with the 3-
dimensional structure. The two compounds in this group are
based on aldrin, with isodrin being its isomer, and des-
methylene aldrin lacking the methano bridge found in aldrin
and dieldrin. Isodrin was never used commercially as a
pesticide, but in Musca domestica, it is only slightly less toxic
than endrin.14 The toxicity profile of desmethylene aldrin has
not yet been investigated. These organochlorines (Groups 1, 2,
and 3) were chosen based on their environmental and
metabolic relevancy and also to assess the role of dieldrin’s
epoxide.
The order of potency for each group of compounds on cell
viability was assessed by LDH and MTT assays, both showing
similar results. Group 2, aldrin and cis-aldrin diol, showed
moderate toxicity, the dieldrin isomers (endrin and isodrin)
evinced little to no toxicity, and the desmethylene compounds
were the most toxic in the PC6-3 cells. Dieldrin, while
substantially more toxic than endrin and isodrin, was markedly
less toxic than the Group 2 analogues. Orientation of the bridge
did greatly influence toxicity (the structural difference between
dieldrin and endrin and between aldrin and isodrin), but its
absence also increased toxicity as the desmethylene compounds
were the most toxic. These results demonstrate the importance
of the three-dimensional structure in the cytotoxic potency of
the cyclodiene pesticides in a dopaminergic model. This
structure−activity relationship differs significantly from the
structural features found to be important for insect toxicity. The
toxicities of many organochlorines have been assessed in
various species of insects, including Musca domestica (house
fly), Anopheles stephensi (mosquito), Glossina austeni (tsetse fly),
and Periplaneta americana (American cockroach).14−16,46,50
When considering only the compounds investigated in the
current study, previous work has shown dieldrin to be the most
toxic in insects, usually followed by aldrin, endrin, and then
isodrin. cis-Aldrin diol, desmethylene aldrin, and desmethylene
dieldrin were all found to have minimal toxicity in the insect
species investigated. There were many other compounds also
investigated in these studies (>51 compounds in total), and
some species differences, in terms of the relative potency of
each compound, were observed.14−16
The general characteristics of the structural features
important for toxicity in both insects and dopaminergic cells
are similar.14−16 These characteristics include the importance of
the 3-dimensional structure and the identity of electronegative/
polar moiety opposite of the chlorine atoms (i.e., epoxide or cis-
diol). For example, while dieldrin and endrin are stereoisomers
of each other differing only the position/orientation of the
epoxide and methylene bridge (Figure 1), the measured toxicity
is significantly different. Similar results were found for the
stereoisomers aldrin and isodrin. The mechanistic basis for such
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differences in toxicity, e.g., dieldrin versus endrin, are unknown
at this point but might include diversity in regards to cellular
targets, metabolism, and disposition.51,52 In regards to the
latter, it is known that unlike dieldrin, endrin is rapidly excreted
in humans and does not bioaccumulate.51,52
DOPET). Additionally, pretreatment of the cells with DA
demonstrated an even greater increase in DOPAL production
following dieldrin treatment as compared to the control (Figure
7). While there appears to be a correlation between toxicity and
extracellular levels of DOPAL, it is likely that the generation of
DOPAL is a secondary effect of cell death in the current study
given the full range of organochlorine compounds used with
some concentrations higher than the measured LC50. Further
experiments should investigate the correlation between organo-
chlorine treatment and generation of DOPAL, especially for
chronic low-level dieldrin exposure.
Of question is how treatment of dopaminergic cells with
dieldrin or other organochlorines at concentrations lower than
needed for toxicity yield elevated DOPAL. As noted above,
exposure of PC12 cells to dieldrin resulted in increased
extracellular DA and DOPAC, indicative of organochlorine-
mediated release of vesicular DA.7 One other mechanism that
could account for elevated DOPAL would be the impairment of
aldehyde metabolism, and previous works have demonstrated
aldehyde dehydrogenases and reductases to be inhibited by low
levels of products of oxidative stress.30,38 Further work is
needed to determine whether either or both pathways (i.e.,
release of vesicular DA or oxidative stress products) are
responsible for elevated DOPAL.
In addition to DA metabolism, the current study also
investigated the effect of dieldrin and its analogues on the
production of ROS. It was found that treatment of PC6-3 cells
with nontoxic doses of each of the organochlorines resulted in a
significant increase in H2O2 levels. This increased production of
ROS was not necessarily indicative of toxicity, such as in the
case of isodrin which caused the largest increase in ROS but
showed the least toxicity. A similar trend has been observed for
the organophosphates, another class of synthetic pesticides that
includes compounds such as chlorpyrifos and malathion.68,69 It
was initially thought that all of the organophosphates had the
same mechanism of action: irreversible inhibition of acetylcho-
linesterase. While there were some observed differences in
relative potency, these could be easily attributed to structural
changes.68 It was later determined that there were exceptions to
this original hypothesis and observed that organophosphates
could produce similar levels of oxidative stress, but the cellular
compensations and/or reactions to this insult were different
depending on the toxicant.34,36,70−76 Therefore, the same
degree of initial insult can result in a varied degree of toxicity,36
as observed with the dieldrin analogues investigated here.
At this point, it is not clear why elevated hydrogen peroxide
but not superoxide anion was observed following organo-
chlorine treatment. However, exposure of the cells to dieldrin
and other analogues resulted in increased DOPAL, indicative of
higher DA metabolism via monoamine oxidase, which would
produce hydrogen peroxide as a byproduct.61
The information obtained regarding the reactivity and
localization of dieldrin is important to better understand the
interaction of this pesticide with proteins in dopaminergic cells.
The major findings of this study are the following: (1) dieldrin
readily diffuses into cells and accumulates in the membrane
fraction of PC6-3 cells, while the intracellular solution
demonstrated saturation. (2) The epoxide of dieldrin is not
reactive toward Cys or Lys nucleophiles under conditions used.
Therefore, dieldrin most likely associates with proteins
noncovalently. (3) Minor structural changes can greatly alter
toxicity for dieldrin. The most toxic analogues of dieldrin were
the desmethyl isomers and the cis-diol metabolite. Interestingly,
In addition, it seems the minor structural changes that make
a molecule less toxic toward insects increase toxicity toward
dopaminergic cells (i.e., orientation and presence of the
methano bridge). There are significant differences in the
model systems used between these studies, whole insect versus
immortalized cell line, that affect the metabolism, localization,
and absorption of these compounds. However, based on
findings for the organochlorine isomers used in the current
study (e.g., dieldrin and endrin), it may still be concluded that
there are significant differences in the cellular targets that
warrant further investigation. It is these differences that will be
useful in developing new, potent insecticides that do not
adversely affect the dopaminergic system of humans.
The cis-diol metabolite of dieldrin resulting from cleavage of
the epoxide was found to be significantly more toxic than the
parent compound dieldrin (Table 1).49 Such a finding raises the
question of whether dieldrin or its metabolites are the toxic
insults relevant to neurodegenerative disease development.
Dieldrin has been associated with the disruption of many key
processes in dopaminergic neurons, including the metabolism
of the neurotransmitter, DA; mitochondrial function; and the
balance of oxidative stress. Dysregulation of these processes and
exposure to dieldrin have been correlated to PD.4−6,53−57
Previous studies have shown that exposure to dieldrin causes
depletion of DA in both ring doves and mallards.58,59 This
effect has also been shown to be specific to DA, with no
corresponding inhibition of GABA.60 When this effect was
further investigated in mice dosed with dieldrin, a decrease in
DOPAC, the carboxylic acid metabolite of DA, was detected,
along with a corresponding increase in cysteinyl-DA, cysteinyl-
DOPAC, and elevated levels of protein carbonyls. This
indicates a disruption in DA metabolism and trafficking, but
instead of seeing elevated levels of the DA metabolites,
increased protein modification was observed.9
Dieldrin has also been found to cause the release of striatal
DA and DOPAC in mice.12 Developmental exposure to
dieldrin has also been shown to have long-term, adverse effects
on murine DA metabolism. An increase in DOPAC was
observed in the striatal region of these mice after they were
aged to 12 weeks, and they were much more sensitive to MPTP
toxicity.10 In dopaminergic PC12 cells, treatment with dieldrin
resulted in decreased intracellular DA and a corresponding
increase in extracellular DA and DOPAC.7
Higher extracellular DA and DOPAC levels suggest release of
vesicular DA and elevated DA turnover/metabolism to
DOPAC. The metabolism of DA to DOPAC proceeds via
the activity of monoamine oxidase and involves production of
an aldehyde intermediate, DOPAL.61 DOPAL is an endoge-
nous neurotoxin, capable of covalently modifying pro-
teins.30,38,39,62,63 Aberrant levels of this reactive compound
have been associated with an increased risk for Parkinson’s
disease.62−67 In the current study, the compounds that
exhibited the greatest toxicity caused the largest increase in
extracellular levels of DOPAL (desmethylene aldrin, des-
methylene dieldrin, and cis-aldrin diol). The least toxic
compounds (endrin and isodrin) showed the least effect on
DOPAL concentration, and minimal effect on the concen-
trations of the other DA metabolites quantified (DOPAC and
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the cis-diol analogue is a metabolite of dieldrin, which raises a
question as to whether dieldrin or a metabolite is the insulting
species. (4) Treatment of PC6-3 cells with dieldrin and
organochlorine analogues yielded elevated levels of the toxic
DA metabolite DOPAL. (5) Exposure of PC6-3 cells to dieldrin
and analogues resulted in elevated hydrogen peroxide but not
superoxide anion. Given that minor structural changes to
dieldrin resulted in significant alterations in toxicity and
dopamine catabolism implies specificity in regards to molecular
targets. Work is in progress to better understand the interaction
of the pesticide dieldrin with dopaminergic cells, focusing on
mechanisms for disruption of cell processes and identification
of novel intracellular targets.
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ASSOCIATED CONTENT
* Supporting Information
■
S
Chromatographs from GC separation of extracted dieldrin and
endrin and inhibition of rabbit LDH by organochlorines. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*Tel: +1-319-335-8834. Fax: +1-319-335-8766. E-mail:
jonathan-doorn@uiowa.edu.
Present Addresses
†E.M.G.A: Department of Pharmacology, Case Western
Reserve University, Cleveland, Ohio.
‡Y.J.: National Institute for Neurological Disease and Stroke,
National Institutes of Health, Bethesda, Maryland.
Funding
This work was supported by grants NIH R01 ES15507 (to
J.A.D.), NIH T32 GM067795 (to E.M.G.A. and L.L.D.), and
NIH T32 DK007319 (to E.M.G.A.), fellowships through the
American Foundation for Pharmaceutical Education (to
E.M.G.A. and L.L.D.), a scholarship from the Marshall H.
and Nellie Alworth Memorial Fund (to L.L.D.), and University
of Iowa College of Pharmacy Dissertation Fellowships (to
E.M.G.A. and L.L.D.).
(12) Sava, V., Velasquez, A., Song, S., and Sanchez-Ramos, J. (2007)
Dieldrin elicits a widespread DNA repair and antioxidative response in
mouse brain. J. Biochem. Mol. Toxicol. 21, 125−135.
(13) Sanchez-Ramos, J., Facca, A., Basit, A., and Song, S. (1998)
Toxicity of dieldrin for dopaminergic neurons in mesencephalic
cultures. Exp. Neurol. 150, 263−271.
Notes
The authors declare no competing financial interest.
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(1981) The toxicities of some analogs of dieldrin, endosulfan and
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ACKNOWLEDGMENTS
■
The GC/MS data were acquired by Dr. Lynn M. Teesch in the
High Resolution Mass Spectrometry Facility at The University
of Iowa.
ABBREVIATIONS
■
DCFDA, 2′,7′-dichlorodihydrofluorescein diacetate; MTT, 3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;
DOPAL, 3,4-dihydroxyphenylacetaldehyde; DOPAC, 3,4-dihy-
droxyphenylacetic acid; DOPET, 3,4-dihydroxyphenylethanol;
mCPBA, 3-chloroperbenzoic acid; DTNB, 5,5′-dithiobis-2-
nitrobenzoic acid; ALDH, aldehyde dehydrogenase; ALR,
aldehyde reductase; Adn, aldrin; CAD, cis-aldrin diol; dAdn,
desmethylene aldrin; dDI, desmethylene dieldrin; DI, dieldrin;
DHE, dihydroethidium; DMSO, dimethylsufloxide; DA,
dopamine; End, endrin; GC/MS, gas chromatography tandem
mass spectrometer; Idn, isodrin; LDH, lactate dehydrogenase;
NGF, nerve growth factor; PD, Parkinson’s disease; ROS,
reactive oxygen species; SOD, superoxide dismutase
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