pubs.acs.org/JAFCPublished on Web 06/30/2010
©2010 American Chemical Society
8460 J. Agric. Food Chem. 2010, 58, 8460–8466
Thionate versus Oxon: Comparison of Stability, Uptake, and
Cell Toxicity of (14CH3O)2-Labeled Methyl Parathion and
Methyl Paraoxon with SH-SY5Y Cells
SANDIP B. BHARATE,†JOHN M. PRINS,†KATHLEEN M. GEORGE,†AND
CHARLES M. THOMPSON*,†,§
†The Center for Structural and Functional Neuroscience, Department of Biomedical and Pharmaceutical
Sciences, The University of Montana, Missoula, Montana 59812, and§ATERIS Technologies LLC,
901 North Orange Street, Missoula, Montana 59802
The stability, hydrolysis, and uptake of the organophosphates methyl parathion and methyl paraoxon
were investigated in SH-SY5Y cells. The stabilities of (14CH3O)2-methyl parathion (14C-MPS) and
(14CH3O)2-methyl paraoxon (14C-MPO) at 1 μM in culture media had similar half-lives of 91.7 and
101.9 h, respectively. However, 100 μM MPO caused >95% cytotoxicity at 24 h, whereas 100 μM
MPS caused 4-5% cytotoxicity at 24 h (∼60% cytotoxicity at 48 h). Greater radioactivity was detected
inside cells treated with MPO as compared to MPS, although >80% of the total MPO uptake was
primarily dimethyl phosphate (DMP). Maximum uptake was reached after 48 h of14C-MPS or14C-
MPO exposure with total uptakes of 1.19 and 1.76 nM/106cells for MPS and MPO, respectively.
The amounts of MPS and MPO detected in the cytosol after 48 h of exposure time were 0.54 and
0.37 nM/106cells, respectively.
KEYWORDS: Uptake; radiolabel; methyl parathion; methyl paraoxon; SH-SY5Y human neuroblastoma
cells; hydrolysis; MTT
Organophosphorus (OP) insecticides 1 are a relatively safe
group of agricultural chemicals used extensively in plant and
crop protection and include malathion, chlorpyrifos, parathion,
methyl parathion, and diazinon (Figure 1). Most parent OP
insecticides contain the phosphorothionate group (PdS), which
renders the structure relatively safe to mammals owing to its
poor reactivity with target enzymes and other biomolecules.
The thionate linkage also confers hydrolytic stability owing to
increased electron density at phosphorus. OP insecticides can be
converted from thionates 1 to oxons 2 (Figure 1) either in the
environment orinvivoand becomereactiveandpotentiallytoxic
following occupational or incidental exposures by direct contact
the PdO linkage (oxons), are highly reactive, and induce rapid
The primary mechanism of action of OP insecticides is based
as the reactive oxon metabolite form 2 (Figure 1) (2). Methyl
parathion (O,O-dimethyl O-4-nitrophenyl phosphorothioate) is
representative of the thionate (PdS) class of insecticides with
insect and mammal toxicity due to inhibition of AChE (2, 3).
Parathion itself is an inherently weak cholinesterase inhibitor
with an IC50in the range of 10-4-10-5M (4), but its biotrans-
formation to the reactive metabolite paraoxon (5-7) affords a
potent inhibitor of AChE (IC50=10-8M) (8). Inhibition of
AChE by paraoxon, as for most reactive OPs, occurs mechan-
istically with loss of the Z group (Figure 1) and results in
accumulation of acetylcholine in cholinergic synapses and ex-
cessive stimulation of cholinergic pathways in the central and
peripheral nervous systems (9-11). Although AChE is the
primary target and the principal mechanism underlying toxic
action, highly reactive small molecules such as methyl paraoxon
and ethyl paraoxon can potentially modify a number of bio-
molecules. Paraoxon toxicity at the cellular level has been shown
in immortal cell lines in vitro (12-17). Subcellular targets for the
initiation of cytotoxicity have not been fully elucidated, but
nuclear (18), enzymatic (14), cytoskeletal (19), and plasma
membrane (20) alterations have been described, and a number
of alternative protein targets have been identified (21,22).
One significant question that remains unanswered about OP
If this question could be answered, investigators could gain a
clearer understanding of the individual or interdependent role of
thionate and oxon forms of an insecticide to modify intracellular
or extracellular protein targets and alter biochemical path-
ways that lead to toxicity. Because the conversion of thionate
to oxon occurs with most thionate OP insecticides, the study of
cell membrane penetration is of broad and significant impact.
A differential study of cell penetration is further warranted to
address a number of hypotheses regarding cell culture studies
due to OP exposure likely begin with covalent modification
*Corresponding author [phone (406) 243-4643; fax (406) 243-5228;
Article J. Agric. Food Chem., Vol. 58, No. 14, 20108461
(adduction) of a protein by the oxon (21). Owing to the relatively
high reactivity and hydrolytic instability, it is presumed that OP
oxons would not be found at appreciable concentrations within
cells as compared to the thionate owing to lower lipophilicity.
Support for this supposition is indicated in calculations of parti-
tion coefficients in which methyl parathion (CLog P=2.79; Log
Po/w=3.0) (23) and methylparaoxon (CLogP=1.38;Log Po/w=
1.6 for ethyl paraoxon) (24) show a 20-fold difference, suggesting
that the thionate could more readily penetrate cell membranes by
passive diffusion mechanisms. However, alternative mechanisms
could enable oxons access to intracellular targets.
Given the reactivity, aqueous instability, and lower CLog P of
oxons as compared to thionates, it has been presumed that cell
penetration is somewhat limited. However, it is important to
thionate and oxon forms, and an understanding of cell penetra-
tion by these structures is important to validate and identify new
protein targets, their localization, and distribution. Moreover,
the fractional concentration of extracellular and intracellular
amounts of thionate and oxon may play an important role in
the mode of toxic action. In this paper, the stability, uptake, and
hydrolysis of doubly labeled methyl parathion and methyl para-
oxonwere conducted inSH-SY5Y cells sothatthe key structural
difference between OP insecticide (thionate) and its primary
metabolite (oxon) could be better understood. The rationale for
the double label was twofold: (1) placement of radiolabel at the
methoxy group ensures that the isotope remains attached to
lation; (2) two methoxy ester radiolabels were deemed important
because some proteins or biomolecules may be prone to aging
or loss of a second ester group following phosphorylation. The
SH-SY5Y cell line was chosen for study because it expresses
AChE, a number of neuron-specific enzymes and biosynthetic
pathways, dopamine hydroxylase, tyrosine hydroxylase, aromatic
L-amino acid decarboxylase, enzymes unique to catecholamine
neurons, and the nicotinic acetylcholine receptor (25).
MATERIALS AND METHODS
Perkin-Elmer, Boston, MA (NEN radiochemicals) with a radioactivity of
1 mCi (specific activity=41.65 mCi/mmol). Liquid scintillation counting
(LSC) was performed to determine the radiocarbon content of various
samples using a Tricarb 2900 liquid scintillation counter. Silica gel G254
thin layer chromatography plates (10 mm thickness; Analtech) were used
for purification. Radioactive images of TLC plates were recorded using a
phosphoimager (Fujifilm FLA3000). The MTT assay kit was obtained
from Roche Applied Science, and the absorbance was measured using
a microplate reader (Molecular Devices, Versamax).
Synthesisof14C-Methyl Paraoxon (14C-MPO).14C-Methylpara-
oxon (14C-MPO) was prepared by oxidation of14C-methyl parathion
(14C-MPS). To a solution of14C-MPS (1 mCi with a specific activity
of 41.65 mCi/mmol) in dry methylene chloride (3 mL) was added
m-chloroperoxybenzoic acid (1.5 mmol) at 0 ?C, and the reaction mixture
confirmed by TLC. The reaction mixture was loaded onto a preparative
14C-Methyl parathion (14C-MPS) was purchased from
silica gel G254TLC plate and eluted with EtOAc/hex (1:1). The band of
14C-MPO at Rf0.15-0.20 (Rfof14C-MPS is 0.60-0.65) was correlated
with the migration of cold paraoxon standard, removed, extracted with
methylene chloride (5 mL ? 3), and filtered, and the solvent was
evaporated to afford the radiolabeled product (0.360 mCi with a specific
activity of 41.65 mCi/mmol). Total counts (cpm) were converted to dpm
using dpm=cpm/detector efficiency. The radiochemical yield of synthe-
sized14C-MPO was determined using this relationship: 1 mCi=2.2 ?
109dpm. Chemical and radiochemical purities for both14C-methyl para-
the TLC plate (Figure 2).
SH-SY5Y Cell Culture. SH-SY5Y cells (a human neuroblastoma
cell line) were obtained from American Type Culture Collection
(Rockville, MD) and cultured in DMEM/F12 medium (GIBCO BRL,
CO2incubator maintained at 5% CO2and 37 ?C. The medium was
changed every 2 days. Cells were allowed to reach 80% confluence before
exposure to14C-MPS or14C-MPO.
(MTT) Viability Assay. Approximately, 0.25 ? 105cells/well were
seeded into 96-well plates and exposed for 24, 48, or 72 h to MPS and
MPO at concentrations from 10 nM to 100 μM prepared as solutions in
acetonitrile(0.1%v/v).A 2%TritonX-100 solutioninassay mediumwas
used for a positive control (n=8 for each MPS and MPO concentration
and Triton X-100). Following exposure, cells were rinsed several times
with culture medium prior to MPS and MPO exposure. Culture medium
MPO concentrations or Triton X-100 was added to each well. After
incubation for appropriate time points, 10 μL of MTT labeling reagent
was added to each well. Plates were incubated with MTT labeling reagent
for 4 h, and then 100 μL of solubilizing solution was added to each well
and incubated overnight. Absorbance of samples was measured using
a microplate reader at 575 nm (formazan) using a reference wavelength
at 675 nm. Viability was determined by comparing the absorbance read-
ings of the wells containing the OP-treated cells with those of the vehicle
(0.1% acetonitrile)-treated cells (Figure 3).
Stability of14C-MPS and14C-MPO in Culture Media. A 1 μM
bovine serum (Hyclone; 100 U/mL penicillin, 100 μg/mL streptomycin,
points(0-120 h),samples(50 μL ? 3) were loadedonto preparative silica
gel G254TLC plates and developed with EtOAc/hex (1:1). Cold MPS and
MPO were spotted as elution standards at the corner of the plate for
reference. For14C-MPS, three bands (Rf=0.65, 0.18, and <0.1), and for
14C-MPO, two bands (Rf=0.18 and <0.1), were isolated and the total
14C-MPO in DMEM/F12 medium 1% fetal
Figure 1. Structuresofrepresentativephosphorothionateinsecticidesand
the corresponding oxons: malathion (R = Me, Z = -SCH(CO2Et)CH2-
CO2Et)); parathion (R=Et, Z=-OPh-p-NO2); methyl parathion (R=Me,
Z=-OPh-p-NO2); chlorpyrifos (R=Et, Z=-O-[3,5,6-trichloro-2-pyridyl]);
and diazinon (R=Et, Z=-O-[2-isopropyl-6-methyl-4-pyrimidinyl]).
Figure 2. Thinlayerchromatographicanalysesof14C-MPSand14C-MPO
Rfof MPO = 0.18.
8462J. Agric. Food Chem., Vol. 58, No. 14, 2010Bharate et al.
counts (cpm) recorded. The percentage of14C-MPS or14C-MPO at each
time point was calculated and plotted against incubation time (Figure 4).
Rateconstantsforthe degradation ofMPSandMPO weredeterminedby
calculating the negative slope of ln(% of MPS/MPO remaining) versus
Uptake of14C-MPS and14C-MPO in SH-SY5Y Cells.14C-MPS
and14C-MPO stock solutions were prepared in acetonitrile (500 μM) and
stored at 0 ?C.14C-MPS and14C-MPO treatment concentrations (1 μM)
were prepared by dilution of the stock solution in DMEM/F12 medium
1% fetal bovine serum (Hyclone), 100 U/mL penicillin, 100 μg/mL
streptomycin, and 2 mM L-glutamine (400 μL of stock solution into
200 mL of media to make 1 μM14C-MPS/14C-MPO media solution).
In preliminary experiments, cells were exposed to 1 μM
14C-MPO at 37 ?C for 0, 8, 16, 24, 48, and 72 h in a 96-well plate (2 μL
of medium in each well). At the determined time points, culture medium
was removed and the cells were washed with ice-cold PBS (3 ? 1 mL) to
remove residual14C-MPS or14C-MPO and placed in a scintillation vial.
To the remaining washed cells was added 5 mL of ice-cold PBS, and the
cellswereremovedusing acellscraper andtransferredtocentrifugetubes.
Each well was washed an additional time with PBS to remove residual
scintillation vial. Cells were treated with lysis buffer containing 20 mM
Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1%
and 1 mM Na3VO4(Cell Signaling Technology, Beverly, MA). Additions
deoxycholate, 0.5% SDS, 1 μM okadaic acid, 1 mM phenylmethanesul-
fonyl fluoride (PMSF), 0.1 mg/mL benzamidine, 8 μg/mL calpain in-
hibitors I and II, and 1 μg/mL eachleupeptin, pepstatin A, and aprotinin.
Lysis buffer (500 μL) was added to the cell pellet and vortexed. After
30min, thecell lysate was centrifuged,and thesupernatant containingthe
cytosolic fraction was separated. Scintillation fluid was added to all
samples to obtain a standard volume and total counts (cpm) were
recorded. Data were collected in triplicate for each time point. The total
was plotted against OP exposure time (Figure 5).
total amount of OP inside cells ðnMÞ
¼ ½total radioactivity inside cells ðcpmÞ
? initial OP concentration ðnMÞ?=
½total radioactivity in medium ðcpmÞ
þtotal radioactivity inside cells ðcpmÞ?
Fate of14C-MPS and14C-MPO in Uptake Experiments. To
determine the fate of14C-MPS and14C-MPO in media during uptake
experiments and the amounts of MPS/MPO and their degradation
72 h in a Petri dish plate (20 mL of medium in each plate; 1 μM
14C-MPS or14C-MPO) using a protocol similar to that described in the
previous section. After cell harvesting, total cell numbers were calculated
and was loaded on preparative silica gel G254TLC plate, and reference
standards of cold MPS and MPO were spotted at the corner of the plate.
identified by correlation with standards, removed, and collected in
individual liquid scintillation vials. Similarly, 100 μL of medium was also
Figure 3. Cytotoxiceffectof14C-MPSand14C-MPOonSH-SY5Ycells:(squares)SH-SY5Ycellviabilityfollowing24hexposureto14C-MPSand14C-MPO;
(triangles) SH-SY5Ycellviabilityfollowing48hexposureto14C-MPSand14C-MPO;(circles) SH-SY5Ycellviabilityfollowing72hexposureto14C-MPSand
14C-MPO. Cells were treated with various concentrations of14C-MPS and14C-MPO for 24, 48, and 72 h. Cell viability was determined by 3-[4,5-
dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. Results are presented as percent of vehicle control, determined by comparing the
absorbance readings of the wells containing the OP-treated cells withthoseofthe vehicle(0.1% acetonitrile)-treated cells.Datarepresent the mean( SEM
(n = 8). Triton X-100 was used as a positive control (= 100% cell death). Solid symbols are used for MPS and open symbols for MPO.
Figure 4. StabilityofradiolabeledMPSorMPO in1% FBSculturemedia:
(A)14C-MPS; (B)14C-MPO. Data represent mean ( SEM (n = 3).
14C-DMP are primary metabolites of
ArticleJ. Agric. Food Chem., Vol. 58, No. 14, 20108463
loaded on TLC plate, and bands were collected. Scintillation fluid was
added to all samples to obtain a standard volume, and total counts (cpm)
were recorded. Data were collected in triplicate for each time point.
Concentrations of OP in cell cytosol per million cells were calculated
using the following equation and plotted against OP exposure time
nM OP in cytosol=106cells
¼ ½radioactivity for OP or its degradation product in cytotosol ðcpmÞ
? initial OP concentration ðnMÞ?=f½total radioactivity in media ðcpmÞ
þrotal radioactivity inside cells ðcpmÞ?? number of cells ðin millionsÞg
The partition coefficient values (media/cell) for the OPs and their
degradation products were calculated by dividing the amount of
14C-MPS/14C-MPO in cell cytosol by the amount in the medium at 48 h
of exposure time. The uptake rate constant was calculated from the
negative slope of the plot between ln(nM increment of OP in cytosol)
versus OP exposure time (h).
parathion (1 mCi) with a specific activity of 41.65 mCi/mmol
was oxidized with m-chloroperoxybenzoic acid (m-CPBA) in
methylene chloride to yield
The radioactive yield of14C-MPO was 0.360 mCi, and the radio-
chemical purities of both14C-MPS and14C-MPO were >98%
were characterized by NMR and MS.
Viability of SH-SY5Y Cells Exposedto14C-MPS and14C-MPO.
SH-SY5Y cells were treated with14C-MPS or14C-MPO ranging
from 10 nM to 100 μM, and the cell viability was assessed by
MTT assay.Some cytotoxicitywas observedwithbothMPS and
MPO at 50 μM at time points of 48 and 72 h, and at least 60%
cytotoxicity occurred at all time points when the cells were
exposed to 100 μM except MPS treatment for 24 h (Figure 3),
which showed no significant decrease in viability. At 100 μM
concentration, MPS showed 4% (24 h), 60% (48 h), and 90%
(72 h) cytotoxicity, respectively, whereas MPO showed >80%
cytotoxicity after just 24 h.
The medium was analyzed at various time points by prep-TLC,
radioactivity counting of isolated bands. The major degradation
pathways for14C-MPS and14C-MPO are oxidation/hydrolysis
14C-Methyl Paraoxon (14C-MPO).
14C-MPO. Purification was con-
Figure 5. Uptake of14C-MPS or14C-MPO intoSH-SY5Y cells at different
(n = 3).
Figure 6. Uptake of14C-MPS or14C-MPO in SH-SY5Y cells at different exposure times: (A) total radioactivity (OP and their degradation products) found
inside cells following exposure to 1 μM MPS or MPO; (B) uptake of14C-MPS (1 μM) and its degradation products; (C) uptake of14C-MPO (1 μM) and its
degradation product; (D) Actualamountof14C-MPS/14C-MPOfound insidecells at1 μM. Cellnumbersperexperiment werenormalized anduptakevalues
are indicated for 106cells. Data represent mean ( SEM (n = 3).14C-DMTP and14C-DMP are primary metabolites of14C-MPS and14C-MPO hydrolysis.
8464J. Agric. Food Chem., Vol. 58, No. 14, 2010 Bharate et al.
and hydrolysis, respectively(Figure7), forming the water-soluble
DMTP and DMP as primary metabolites. The formation of
DMTP and DMP from paraoxon and like compounds is well
documented (26, 27).14C-MPS and14C-MPO showed similar
stability profiles. After 72 h of incubation in medium, >50% of
14C-MPS (Figure 4A) and14C-MPO (Figure 4B) were recovered
from the medium unchanged. Both
thiophosphoric acid (thiophosphate) and dimethylphosphate
(phosphoric acid) (Figure 7), respectively, as determined by
coelution with standards.14C-MPS was found to be relatively
stable to oxidation in media and formed <1%14C-MPO after
amount of14C-MPS and14C-MPO were recovered. The rate
werecalculatedtobe7.0? 10-3and6.0? 10-3h-1,respectively.
101.9 h, respectively.
Neuroblastoma Cells. Using the cell viability and media stability
data, timepointsupto 96h and OP concentrationsof1 μM were
chosen for uptake studies because little to no loss of cells was
observed under these conditions. In preliminary uptake experi-
ments, cells were exposed to 1 μM14C-MPS and14C-MPO for
different exposure times, namely, 0, 8, 12, 24, 48, 72, and 96 h. A
comparative plot of uptake for14C-MPS and14C-MPO is shown
in Figure 5, indicating that MPO achieved a maximum level of
46 nM at the 48 h time point and MPS 13 nM maximum at 24 h.
14C-MPO in SH-SY5Y Human
ing Information) of the total applied radioactivity (170,000 cpm)
of either MPS or MPO entered the cells.
To determine the fate of14C-MPS and14C-MPO in media
during the uptake experiments and the total amount of MPS,
MPO, and/or the degradation products in cell cytosol, uptake
loaded on a preparative TLC plate and the OPs separated and
analyzed to determine the stability of14C-MPS or14C-MPO and
formation of the hydrolysis products during the uptake experi-
ment (Tables 1 and 2).14C-MPS and14C-MPO were stable in
media throughout the uptake experiment. The cell counts for
both experiments were normalized, and values of14C-MPS and
the preliminary uptake experiment (Figure 5), the total amount
of MPO uptake (sum of MPO and its degradation product)
was greater than the total MPS uptake (sum of MPS and its
degradation products) (Figure6A). Itwasinterestingtonotethat
in case of14C-MPO uptake, a large amount of the hydrolyzed
product, dimethyl phosphate (80-90% of total radioactivity
inside cells) (Figure 6C), was found in cells, whereas a greater
amount of14C-MPS was found inside cells compared with its
degradation products, MPO and DMTP (Figure 6B).
The maximum radioactivity inside cells was reached after 48 h
of14C-MPS/14C-MPO exposure with total uptakes of 1.19 and
1.76 nM/106cells for MPS and MPO, respectively, whereas the
actual amounts of MPS and MPO inside cells after 48 h of
exposure time were 0.54 and 0.37 nM/106cells (Table 1), respec-
tively. On the basis of TLC analysis, in the case of14C-MPS
Figure 7. Degradation (hydrolysis) of
methyl paraoxon in media.
14C-methyl parathion and
Table 2. Relative Ratios of MPS, MPO, DMTP,and/or DMP in Culture Media
after Exposure of Cells to 1 μM OP (MPS or MPO)a
incubation time (h)
MPS MPODMP and/or DMTP
aData represent mean ( SEM (n = 3).bRatios of OP and their degradation
products were determined by preparative TLC analysis.
Table 1. Percentage of MPS, MPO, DMTP, and/or DMP in Cell Cytosol after Exposure of Cells to 1 μM OP (MPS or MPO)a
percentage of MPS, MPO, DMTP, and/or DMP in cell cytosolb(per 106cells)
incubation time (h)
MPSMPODMP and/or DMTPMPS MPODMP and/or DMTP
aData represent mean ( SEM (n = 3).bRatios of OP and their degradation products were determined by preparative TLC analysis.
ArticleJ. Agric. Food Chem., Vol. 58, No. 14, 20108465
uptake, the ratio of14C-MPS to that of degradation products
21:79 (MPO/DMP) (Table 1).
The amounts of14C-MPS and14C-MPO determined inside
cells at various exposure times (Figure 6D) show that a larger
amount of14C-MPS entered cells than of14C-MPO at each time
found to be associated with the membrane fraction. The percent
and14C-MPO were found to be >92 and >95%, respectively.
The cells-media partition coefficients (Kd) were calculated to be
0.025 h-1for MPS and MPO, respectively.
parathion and14C-methyl paraoxon were tested in a SH-SY5Y
human neuroblastoma cell line. The radiolabel was placed at both
methoxy groupsso that attachment of the OP to biomolecules and
the formation of hydrolysis products could be tracked more
efficiently as compared to studies in which the radiolabel was
radiolabeled paraoxon from (14CH3O)2-parathion was accom-
plished in >98% chemical and radiochemical purity. The specific
activity was 41.65 mCi/mmol as determined by chromatography.
Stability studies in culture media indicated that >50%
of either14C-MPS or14C-MPO remained in the media after
72 h (Figure 4). The degradation pathway expected for14C-MPS
cipated (Figure 7). However, we did not observe oxidation of
14C-MPS to14C-MPO in culture media. Thus, hydrolysis, or loss
of the p-nitrophenoxy group, to yield dimethyl thiophosphate
(DMTP) was the major degradation route for14C-MPS in the
culture media. Direct formation of DMTP from MPS has been
previously reported (26, 27, 31-33). In contrast, a significant
cells (Figure 6B).
To ensure that the uptake studies were conducted with con-
was next examined. As expected, the more reactive phosphoryl-
ating agent14C-MPO was more cytotoxic than14C-MPS at con-
a concentration of MPS and MPO (1 μM) that was below the
in viable cells.
Although a 20-fold difference in the lipophilicity of MPS and
MPO exists, this structural difference did not result in equally
dramatic change in cellular uptake; in fact, our initial uptake
results showed that uptake was not related to the lipophilicity.
Our experiments showed that the hydrolysis product of MPO,
dimethyl phosphate (DMP), actually accounted for a higher
and MPS. However, a greater overall amount ofMPS was found
inside cells compared with MPO. The greater amount of MPS
of MPS once inside the cell and/or rapid breakdown of MPO in
cells to DMP, thereby reducing the net amount of MPO found.
The higher amount of DMP found in cells could be due to the
intracellular hydrolysis and metabolism of MPO and/or extra-
cellular (media-mediated) metabolism followed by passive diffu-
uptake of OPs was not more than 5% of total OP exposure
as observed from the results of lower concentration (100 and
10 nM) uptake experiments.
a delayed trend similar to that in the14C-MPO-treated cells after
24 h of exposure likely due to a rate-dependent oxidative conver-
was at 100 μM concentration after 72 h of exposure time.
Although very little14C-MPO (7-10%, Table 2) was found in
the media, a significant amount of14C-MPO (33-37%, Table 1)
was found in the cytosol in MPS-treated cells. Therefore, it is
likely that conversion from MPS to MPO contributed to the
In conclusion, differences in the stability, uptake, and cyto-
toxicity of14C-MPS and14C-MPO in SH-SY5Y human neuro-
blastoma cells were found. The use of dual-labeled MPS and
MPO allowed for precise intracellular measurements of parent
and hydrolyzed product and for determining rate comparisons.
oxon MPO access cells at different rates and to different extents
that could yield distinct differences in protein responses.
MPO, methyl paraoxon; MPS, methyl parathion; DMP,
dimethyl phosphate; DMTP, dimethyl thiophosphate.
a well-ventilated hood. Methyl parathion and methyl paraoxon
may be rendered safe by stirring with 1 N NaOH overnight at
Supporting Information Available: Radioactivity counts of
available free of charge via the Internet at http://pubs.acs.org.
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Received for review March 16, 2010. Revised manuscript received June
13, 2010. Accepted June 16, 2010. We thank the NIH for grant support
ES016392 and U44 NS058229). Support from the Core Laboratory for
Neuromolecular Production (NIH P30-NS055022) and the Center for
Structural and Functional Neuroscience (NIH P20-RR015583) is