Article
J. Agric. Food Chem., Vol. 58, No. 14, 2010 8465
uptake, the ratio of 14C-MPS to that of degradation products
inside cells was 46:38:16 (MPS/MPO/DMTP) at 48 h of exposure
time, whereas the uptake of MPO showed an MPO/DMO ratio of
21:79 (MPO/DMP) (Table 1).
(1 μM), it is noteworthy that uptake was concentration dependent
as observed from the results of lower concentration (100 and
10 nM) uptake experiments.
The cytotoxicity induced in the 14C-MPS-treated cells followed
a delayed trend similar to that in the 14C-MPO-treated cells after
24 h of exposure likely due to a rate-dependent oxidative conver-
sion. The highest amount of cytotoxicity (90%) exhibited by MPS
was at 100 μM concentration after 72 h of exposure time.
Although very little 14C-MPO (7-10%, Table 2) was found in
the media, a significant amount of 14C-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
cytotoxicity (16).
The amounts of 14C-MPS and 14C-MPO determined inside
cells at various exposure times (Figure 6D) show that a larger
amount of 14C-MPS entered cells than of 14C-MPO at each time
point and only a negligible amount of 14C-MPS or 14C-MPO was
found to be associated with the membrane fraction. The percent
total recoveries of all label in the uptake experiments for 14C-MPS
and 14C-MPO were found to be >92 and >95%, respectively.
The cells-media partition coefficients (Kd) were calculated to be
6.3 ꢀ 10-4 and 5.9 ꢀ 10-4 with uptake rate constants of 0.054 and
0.025 h-1 for MPS and MPO, respectively.
In conclusion, differences in the stability, uptake, and cyto-
toxicity of 14C-MPS and 14C-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.
Data obtained in this work suggest that the thionate MPS and the
oxon MPO access cells at different rates and to different extents
that could yield distinct differences in protein responses.
DISCUSSION
In this study, the stability, uptake, and cytotoxicity of 14C-methyl
parathion and 14C-methyl paraoxon were tested in a SH-SY5Y
human neuroblastoma cell line. The radiolabel was placed at both
methoxy groups so 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
incorporated at the p-nitrophenoxy group (26,28-30). Synthesis of
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 either 14C-MPS or 14C-MPO remained in the media after
72 h (Figure 4). The degradation pathway expected for 14C-MPS
was oxidation/hydrolysis, and for 14C-MPO, hydrolysis was anti-
cipated (Figure 7). However, we did not observe oxidation of
14C-MPS to 14C-MPO in culture media. Thus, hydrolysis, or loss
of the p-nitrophenoxy group, to yield dimethyl thiophosphate
(DMTP) was the major degradation route for 14C-MPS in the
culture media. Direct formation of DMTP from MPS has been
previously reported (26, 27, 31-33). In contrast, a significant
amount of 14C-MPO was found in cell lysates following 14C-MPS
treatment, indicative of the greater conversion of MPS to MPO in
cells (Figure 6B).
ABBREVIATIONS USED
MPO, methyl paraoxon; MPS, methyl parathion; DMP,
dimethyl phosphate; DMTP, dimethyl thiophosphate.
SAFETY
Organophosphates are toxic reagents and should be handled in
a well-ventilated hood. Methyl parathion and methyl paraoxon
may be rendered safe by stirring with 1 N NaOH overnight at
room temperature.
ACKNOWLEDGMENT
We thank Sarah Ulatowski for her help with the cell experiments.
Supporting Information Available: Radioactivity counts of
OP and their degradation products in cell cytosol. This material is
To ensure that the uptake studies were conducted with con-
tinued viability during the course of the exposure, the cytotoxicity
was next examined. As expected, the more reactive phosphoryl-
ating agent 14C-MPO was more cytotoxic than 14C-MPS at con-
centrations >10 μM (Figure 3). As a result of these data, we chose
a concentration of MPS and MPO (1 μM) that was below the
cytotoxic threshold to examine differences in uptake of these OPs
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
overall amount of radioactivity inMPO-treated cells as compared
with MPS-treated cells, indicating relatively equal access by MPO
and MPS. However, a greater overall amount of MPS was found
inside cells compared with MPO. The greater amount of MPS
than MPO found in cells could be the result of the greater stability
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-
sion across the cell membrane (34). Although a maximum cellular
uptake of OPs was not more than 5% of total OP exposure
LITERATURE CITED
(1) Bajgar, J. Organophosphates/ nerve agent poisoning: mechanism of
action, diagnosis, prophylaxis, and treatment. Adv. Clin. Chem.
2004, 38, 151–216.
(2) Costa, L. G. Current issues in organophosphate toxicology. Clin.
Chim. Acta 2006, 366, 1–13.
(3) DuBois, K. P.; Doull, J.; Salerno, P. R.; Coon, J. M. Studies on the
toxicity and mechanisms of action of p-nitrophenyl diethyl thiono-
phosphate (parathion). J. Pharmacol. Exp. Ther. 1949, 95, 79–91.
(4) Forsyth, C. S.; Chambers, J. E. Activation and degradation of the
phosphorothionate insecticides parathion and EPN by rat brain.
Biochem. Pharmacol. 1989, 10, 1597–1603.
(5) Gage, J. C. A cholinesterase inhibitor derived from O,O-diethyl
O-p-nitrophenyl thiophosphate in vivo. Biochem. J. 1953, 54, 426–430.
(6) Levi, P. E.; Hollingworth, R. M.; Hodgson, E. Differences in
oxidative dearylation and desulfuration of fenitrothin by cyto-
chrome P-450 isoenzyme and in the subsequent inhibition of mono-
oxygenase activity. Pestic. Biochem. Physiol. 1988, 32, 224–231.
(7) Sultatos, L. G.; Minor, L. D. Biotransformation of paraoxon and
p-nitrophenol by isolated perfused mouse livers. Toxicology 1985,
36, 159–169.
(8) Wang, C.; Murphy, S. D. Kinetic analysis of species difference in
acetylcholinesterase sensitivity to organophosphate insecticides.
Toxicol. Appl. Pharmacol. 1982, 66, 409–419.
(9) Ballantyne, B.; Marrs, T. Clinical and Experimental Toxicology of
Organophosphates and Carbamates; Butterworth Heinemann: Boston,
MA, 1992.