S-methylation of O,O-dialkyl phosphorodithioic acids: O,O,S-trimethyl phosphorodithioate and phosphorothiolate as metabolites of dimethoate in mice

Article abstract of DOI:10.1021/tx9600715

O,O,S-Trimethyl phosphorodithioate and phosphorothiolate [(MeO)₂P(S)SMe and (MeO)₂P-(O)SMe, respectively] are known from earlier studies to be impurities, delayed toxicants, and detoxication inhibitors in several major O,O-dimethyl phosphorodithioate insecticides. Our recent studies show extensive S-methylation of mono- and dithiocarbamic acids in mice, suggesting the possibility that phosphorodithioic acids such as (MeO)₂P(S)SH might also undergo S-methylation. This possibility was examined in ip-treated mice with emphasis on the metabolites of dimethoate [(MeO)₂P(S)SCH₂C(O)NHMe], one of the most important organophosphorus insecticides. The urinary metabolites of dimethoate, which contains no P-SMe substituent, were found to include four compounds with P-SMe moieties identified by ³¹P NMR spectroscopy as MeO(HS)P(O)SMe, MeO(HO)P(O)SMe, (MeO)₂P(S)SMe, and (MeO)₂P-(O)SMe; the latter two compounds are also established by GC-MS as dimethoate metabolites in mouse urine, liver, kidney, and lung. Several approaches verified unequivocally that the previously unknown P-SMe metabolites in urine and tissues are due to in vivo S-methylation rather than to impurities. Studies with other O,O-dimethyl and O,O-diethyl phosphorodithioate insecticides established the analogous S-methylation pathway for ethion, malathion, phenthoate, phosalone, and phosmet in mice. Thus, metabolism of O,O-dialkyl phosphorodithioate insecticides in mammals is shown here for the first time to yield S-methyl phosphorodithioates and phosphorothiolates from in vivo S- methylation of the intermediate O,O-dialkyl phosphorodithioic acids.

Full text of DOI:10.1021/tx9600715

1202
Chem. Res. Toxicol. 1996, 9, 1202-1206
S-Meth yla tion of O,O-Dia lk yl P h osp h or od ith ioic Acid s:
O,O,S-Tr im eth yl P h osp h or od ith ioa te a n d
P h osp h or oth iola te a s Meta bolites of Dim eth oa te in Mice
Mahmoud Mahajna, Gary B. Quistad, and J ohn E. Casida*
Environmental Chemistry and Toxicology Laboratory, Department of Environmental Science, Policy,
and Management, University of California, Berkeley, California 94720-3112
Received April 22, 1996X
O,O,S-Trimethyl phosphorodithioate and phosphorothiolate [(MeO)2P(S)SMe and (MeO)2P-
(O)SMe, respectively] are known from earlier studies to be impurities, delayed toxicants, and
detoxication inhibitors in several major O,O-dimethyl phosphorodithioate insecticides. Our
recent studies show extensive S-methylation of mono- and dithiocarbamic acids in mice,
suggesting the possibility that phosphorodithioic acids such as (MeO)2P(S)SH might also
undergo S-methylation. This possibility was examined in ip-treated mice with emphasis on
the metabolites of dimethoate [(MeO)2P(S)SCH2C(O)NHMe], one of the most important
organophosphorus insecticides. The urinary metabolites of dimethoate, which contains no
P-SMe substituent, were found to include four compounds with P-SMe moieties identified by
31P NMR spectroscopy as MeO(HS)P(O)SMe, MeO(HO)P(O)SMe, (MeO)2P(S)SMe, and (MeO)2P-
(O)SMe; the latter two compounds are also established by GC-MS as dimethoate metabolites
in mouse urine, liver, kidney, and lung. Several approaches verified unequivocally that the
previously unknown P-SMe metabolites in urine and tissues are due to in vivo S-methylation
rather than to impurities. Studies with other O,O-dimethyl and O,O-diethyl phosphorodithioate
insecticides established the analogous S-methylation pathway for ethion, malathion, phen-
thoate, phosalone, and phosmet in mice. Thus, metabolism of O,O-dialkyl phosphorodithioate
insecticides in mammals is shown here for the first time to yield S-methyl phosphorodithioates
and phosphorothiolates from in vivo S-methylation of the intermediate O,O-dialkyl phosphoro-
dithioic acids.
In tr od u ction
The safety of several O,O-dimethyl phosphorodithioate
insecticides is compromised when they contain small
amounts of (MeO)2P(S)SMe and (MeO)2P(O)SMe because
these impurities synergize the toxicity of the insecticide
to mammals and are delayed toxicants in themselves (1-
5). Thus, the rat oral LD50 of malathion decreases from
12 500 to 4100 to 2900 mg/kg when (MeO)2P(O)SMe is
present at 0%, 0.2%, and 1%, respectively (2, 5). The oral
LD50 of (MeO)2P(O)SMe for rats is 20-60 mg/kg with
deaths up to 22 days after treatment (4, 5); this delayed
toxicity is antagonized or prevented by coadministration
of small amounts of (MeO)2P(S)SMe or (MeO)3P(S) (5-
8). To avoid toxicity problems, the manufacturing pro-
cesses are designed to keep these O,O,S-trimethyl im-
purities at minimal levels.
The relationship of (MeO)2P(S)SMe and (MeO)2P(O)-
SMe as impurities and possible detoxication inhibitors
in commercial O,O-dimethyl phosphorodithioate insecti-
cides such as dimethoate and malathion is reexamined
here in a new context that these O,O,S-trimethyl com-
pounds might also be formed as metabolites by S-
methylation. We recently reported that mono- and
dithiocarbamic acid fungicides and herbicides or their
F igu r e 1. S-Methylation pathway for O,O-dialkyl phosphoro-
dithioate insecticides. Reactions: (a) cleavage of S-R′ bond; (b)
methylation of P-SH substituent; (c) oxidative desulfuration.
metabolites are readily methylated in mice and that the
S-methyl compounds are a contributing factor in their
overall toxicology (9, 10). The availability of (MeO)2P-
(S)SH as a metabolic precursor for S-methylation is well
established since it is a urinary metabolite used for
biological monitoring of human exposure to O,O-dimethyl
phosphorodithioate insecticides, including dimethoate
and malathion (11, 12). The present study considers the
possibility that the phosphorodithioic acid metabolites of
O,O-dialkyl phosphorodithioate insecticides undergo in
vivo S-methylation in mice to give O,O-dialkyl S-methyl
phosphorodithioates and phosphorothiolates (Figure 1).
It first compares the urinary metabolites in mice of
dimethoate and (MeO)2P(S)SMe for common P-SMe
products, indicating that dimethoate is cleaved and the
P-SH metabolite(s) is methylated. It then examines the
tissues for the presence and persistence of (MeO)2P(S)-
SMe and (MeO)2P(O)SMe.
* To whom correspondence should be addressed, at the Environ-
mental Chemistry and Toxicology Laboratory, Department of Envi-
ronmental Science, Policy and Management, 114 Wellman Hall,
University of California, Berkeley, CA 94720-3112. Phone: (510) 642-
5424; FAX: (510) 642-6497; e-mail: ectl@nature.berkeley.edu.
X Abstract published in Advance ACS Abstracts, October 1, 1996.
S0893-228x(96)00071-9 CCC: $12.00 © 1996 American Chemical Society

S-Methylation of Dialkyl Phosphorodithioic Acids
Chem. Res. Toxicol., Vol. 9, No. 7, 1996 1203
Exp er im en ta l P r oced u r es
Ca u tion . The OP compounds described herein include known
or suspected acute and/ or delayed neurotoxicants. Their use
should be under careful containment conditions.
Gen er a l. 1H and 31P NMR spectra were recorded with a
Bruker WM-300 spectrometer at 300 and 121.5 MHz, respec-
tively. Chemical shifts are referenced to internal sodium
3-(trimethylsilyl)propionate-2,2,3,3-d4 in D2O or external tri-
methyl phosphate in D2O for 1H and 31P spectra, respectively.
GC-chemical ionization-mass spectrometry (CI-MS)1 with
selected ion monitoring (SIM) involved the following: Hewlett
Packard 5890 gas chromatograph coupled to a 5971A mass
spectrometer; DB-5 fused-silica capillary column, 30 m × 0.25
mm i.d. (J and W Scientific, Folsom, CA); injection port
temperature 250 °C; temperature program 70-250 °C over 18
min.
Or ga n op h osp h or u s Com p ou n d s. Chemicals designated
by common name only are identified by structure in The
Pesticide Manual (13). Dimethoate (>99% pure) was obtained
from Chem Service (West Chester, PA), malathion (95% pure)
and the dimethoate metabolite (MeO)2P(S)SCH2C(O)OH were
from American Cyanamid Co. (Wayne, NJ ), and MeO(NH2)P-
(O)SMe and MeO(NH2)P(O)SNa were from Chevron Chemical
Co. (Richmond, CA). The sample of dimethoate contained
<0.05% (MeO)2P(S)SMe or (MeO)2P(O)SMe based on both 31P
NMR and GC-CI-MS analysis. Malathion was purified by
crystallization and four recrystallizations from methanol at -50
°C (2). Ethion, fenthion, phenthoate, phosalone, and phosmet
(obtained from Chem Service) and the purified malathion
contained <0.05% of P-SMe impurities analyzed by GC-CI-MS.
(MeO)2P(S)SMe was prepared as described by Lin et al. (14)
and (MeO)2P(O)SMe according to Norman et al. (15). (EtO)2P-
(S)SMe was synthesized from (EtO)2P(S)SK by reacting with
excess MeI in refluxing methanol. Other organophosphorus
compounds used were available from previous syntheses in this
F igu r e 2. 31P NMR spectra of urinary metabolites of (MeO)2P-
(S)SMe and dimethoate. The 0-24 h urine is examined following
an ip dose of 100 mg/kg. Metabolites with P-SMe substituents
(a-d) as cleavage products of (MeO)2P(S)SMe and from methyl-
ation of dimethoate metabolites are identified by comparison
with standards. Major metabolites of both (MeO)2P(S)SMe and
dimethoate and their chemical shifts (δ ppm) are as follows:
(MeO)2P(S)SH (112.7), (MeO)2P(O)SH (55.2), and (MeO)2P(O)-
OH (0.1). Additional metabolites of dimethoate are as follows:
(MeO)2P(S)SCH2C(O)OH (96.5) and MeO(HS)P(O)SCH2C(O)-
NHMe (70.8), assigned by comparison with standards; MeO-
(HS)P(O)SCH2C(O)OH (71.6) and MeO(HO)P(O)SCH2C(O)OH
(17.6), tentatively proposed based on chemical shifts. Phosphoro-
monothioic acids are shown in the P-SH form.
analyzed immediately by CI-MS-SIM, monitoring MH+ ions. The
analysis involved a 1-µL aliquot injected with the HP7673A
automatic sample injector. Retention times (min) for the
products were as follows: (MeO)2P(S)SMe, 7.16; (MeO)2P(O)-
SMe, 6.79; (EtO)2P(S)SMe, 8.64; (EtO)2P(O)SMe, 8.26. Quan-
titation of (MeO)2P(S)SMe, (MeO)2P(O)SMe, and dimethoate
involved comparison to standard curves prepared with the
authentic compounds using (MeO)3P(O) as the internal stan-
dard. Identification of (MeO)2P(S)SMe and (MeO)2P(O)SMe in
liver extracts (concentrated by evaporation to 0.1 mL) utilized
CI-MS in the scan mode, surveying all ions between 50 and 300
amu.
Ur in a r y Meta bolites. The mice were treated ip with the
test compounds, the urine was collected for 24 h, filtered (Millex-
HV filter unit, 0.45 µm, nonsterilized, Millipore Products
Division, Bedford, MA), and lyophilized by a Speed Vac concen-
trator (SVC 100, Savant Instruments, Farmington, NY), and
the viscous liquid obtained was dissolved in D2O (0.5 mL).
Phosphorus-containing urinary products were analyzed by 31P
NMR. Organic extracts of the samples (1 mL of methylene
chloride) were analyzed by CI-MS-SIM, monitoring MH+ ions
of the triesters.
laboratory. The structure of each compound was verified by 31
NMR and GC-CI-MS (when appropriate).
P
Four candidate metabolites were prepared by O-demethyla-
tion of the appropriate O,O,O- or O,O,S-trimethyl derivatives.
MeO(KS)P(O)SMe was obtained by treating (MeO)2P(S)SMe (10
mmol) with (EtO)2P(S)SK (10 mmol) in dry acetone at reflux
for 4 h: mp 109-112 °C (Et2O); 1H NMR δ 3.75 (3H, d, J )
14.8 Hz, OCH3), 2.31 (3H, d, J ) 13.8 Hz, SCH3); 31P NMR δ
73.1. For characterization by GC/MS the salt was acidified and
treated with diazomethane to give MeO(MeS)P(O)SMe (MH+
) 173). For the preparation of MeO(NaO)P(O)SMe, (MeO)2P-
(O)SMe (10 mmol) was refluxed with NaI (15 mmol) in dry
acetone for 4 h, resulting in precipitation of the desired
product: 1H NMR δ 3.59 (3H, d, J ) 12.8 Hz, OCH3), 2.13 (3H,
d, J ) 13.8 Hz, SCH3); 31P NMR δ 21.13. Esterification of the
acidified product with diazomethane gave (MeO)2P(O)SMe
characterized by GC/MS (MH+ ) 157). On a comparable basis,
(MeO)2P(O)SH and (MeO)2P(O)OH were prepared by demeth-
ylation of (MeO)2P(S)OMe and (MeO)3P(O).
An im a ls a n d Tr ea tm en ts. Male albino Swiss-Webster mice
(23-27 g) from Simonsen Laboratories (Gilroy, CA) were
administered the test compounds ip or orally, at 100 mg/kg,
using triethylene glycol monomethyl ether as the carrier (30
µL for each treatment); as exceptions, (MeO)2P(S)SMe was
administered ip at 10 as well as 100 mg/kg in the same carrier
and MeO(NH2)P(O)SNa ip at 500 mg/kg with water as the
vehicle. At appropriate times the mice were sacrificed by
cervical dislocation and dissected to obtain the liver, kidney,
and lung which were analyzed immediately as indicated below.
Extr a ction a n d An a lysis of Tissu es. Fresh samples of
liver (1 g), kidney (0.3-0.4 g), and lung (0.1 g) from individual
mice were homogenized in methylene chloride (2 mL), and the
homogenate was centrifuged at 5000g for 10 min. The separated
organic layer was dried over sodium sulfate (anhydrous) and
En zym a tic Meth yla tion . (MeO)2P(S)SH (0.12 mM final
concentration) was incubated with S-adenosylmethionine (SAM)
(Sigma Chemical Co., St. Louis, MO) (0.58 mM) and mouse liver
microsomes (10) (1 mg of protein) in 100 mM sodium phosphate
buffer (pH 7.4, 1 mL) for 60 min at 37 °C. For controls, SAM
was not added or microsomes were denatured (85 °C, 15 min).
Each reaction was partitioned with methylene chloride (1 mL)
and analyzed by CI-MS-SIM as above.
Resu lts
Ur in a r y Meta bolites of (MeO)2P (S)SMe a n d Di-
m eth oa te (F igu r e 2). (MeO)2P(S)SMe treatment gives
seven 31P NMR signals in the urine, six of which are
identified by coincidence with those resulting from ad-
dition of the appropriate authentic standard. These
compounds in order of decreasing peak heights are:
MeO(HS)P(O)SMe, (MeO)2P(O)SH, (MeO)2P(O)OH, MeO-
(HO)P(O)SMe, (MeO)2P(S)SH, and (MeO)2P(S)SMe.
1
Abbreviations: CI-MS, chemical ionization-mass spectrometry;
SAM, S-adenosylmethionine; SIM, selected ion monitoring; Me, methyl;
Et, ethyl.

1204 Chem. Res. Toxicol., Vol. 9, No. 7, 1996
Mahajna et al.
F igu r e 3. GC-CI-MS chromatograms showing the parent compound and two metabolites in the liver of mice 1 h after ip administration
of (MeO)2P(S)SMe, dimethoate, or phosalone. The relative sensitivity is 3 times greater for (MeO)2P(O)SMe than (MeO)2P(S)SMe.
Phosalone does not elute within 23 min under the standard conditions.
F igu r e 4. Metabolites and persistence of (MeO)2P(S)SMe, (MeO)2P(O)SMe, and dimethoate in the liver of mice 15-360 min after
ip treatment at 100 mg/kg. The treatment with (MeO)2P(O)SMe was compared alone and with 5% (MeO)2P(S)SMe.
Dimethoate gives four urinary metabolites with P-SMe
substituents in decreasing 31P NMR peak heights as
follows: (MeO)2P(O)SMe, MeO(HS)P(O)SMe, and MeO-
(HO)P(O)SMe plus (MeO)2P(S)SMe in a signal also
containing (MeO)2P(S)SCH2C(O)OH. The presence of
(MeO)2P(S)SMe and (MeO)2P(O)SMe as urinary metabo-
lites of dimethoate is confirmed by GC-MS-SIM. Other
identified metabolites are (MeO)2P(S)SCH2C(O)OH, MeO-
(HS)P(O)SCH2C(O)NHMe, and three acids also obtained
with (MeO)2P(S)SMe, i.e., (MeO)2P(O)SH, (MeO)2P(O)-
OH, and (MeO)2P(S)SH in decreasing peak heights.
Tentatively-identified metabolites are the O-demeth-
ylated derivatives MeO(HS)P(O)SCH2C(O)OH and MeO-
(HO)P(O)SCH2C(O)OH. These products are metabolites
of dimethoate not impurities or degradation products
formed during analysis as they are completely absent in
two types of controls analyzed by 31P NMR: the dimeth-
oate sample used for this study; urine spiked with
dimethoate for 24 h then the spectra recorded for a
further 24 h at room temperature.
Meta bolites a n d P er sisten ce of (MeO)2P (S)SMe in
Liver (F igu r es 3 a n d 4). The parent compound and two
metabolites are detected in liver extracts of mice dosed
at 100 mg/kg with (MeO)2P(S)SMe, i.e., (MeO)2P(O)SMe
(MH+ ) 157) and Me2SO2 (MH+ ) 95). The level of
parent compound peaks at ∼25-30 ppm at 30-60 min
then progressively drops to 8 ppm at 360 min. The
metabolite (MeO)2P(O)SMe remains at 2-4 ppm through-
out the period of 30-360 min (but neither the parent
compound nor the metabolite is detected at 14 h). In
another study (not shown) with (MeO)2P(S)SMe treat-
ment at 10 mg/kg and analysis 1 h after dosing, the
parent compound is not detected in liver and (MeO)2P-
(O)SMe is found at 0.4 ppm whereas at a 5 mg/kg dose
even the oxidized metabolite is not detected.
P er sisten ce of (MeO)2P (O)SMe in Liver (F igu r e
4). The level of (MeO)2P(O)SMe in liver was examined
as a function of time after an ip dose of 100 mg/kg of
(MeO)2P(O)SMe alone or containing 5% (MeO)2P(S)SMe
(i.e., 5 mg/kg of the phosphorodithioate). The phosphoro-
thiolate alone decreases from 50 to 10 to 3 ppm at 30,
60, and 120 min posttreatment and <0.05 ppm by 150
min, whereas with the small amount of dithioate present
it is greatly stabilized with levels of >20 ppm persisting
for 150 min and still with detectable levels at 360 min.
Meta bolites of Dim eth oa te in Tissu es (F igu r es 3
a n d 4). The dimethoate level in liver is maximal at 21
ppm at 30 min after ip treatment at 100 mg/kg. (MeO)2P-
(S)SMe and (MeO)2P(O)SMe are easily detected in the
liver for 3 h after treatment at a ratio of ∼4:1, respec-
tively, with the highest level of both appearing at 60 min
(0.7 ppm in liver, 0.2 ppm in kidney, and <0.1 ppm in
lung). The levels of (MeO)2P(S)SMe and (MeO)2P(O)SMe
are similar following oral administration of dimethoate
(60 min, 100 mg/kg, combined metabolites 0.6 ppm in
liver), and at 60-120 min the amounts of the two
metabolites combined are ca. 4% of the dimethoate level
in liver.
S-Met h yla t ion of Ot h er P h osp h or od it h ioa t es.
(MeO)2P(S)SMe is detected in liver 60 min after a 100
mg/kg ip dose of the dimethyl phosphorodithioate insec-
ticides dimethoate, phosmet, phenthoate, and malathion,
decreasing in that order. In similar experiments with
diethyl phosphorodithioate insecticides, (EtO)2P(S)SMe

S-Methylation of Dialkyl Phosphorodithioic Acids
Chem. Res. Toxicol., Vol. 9, No. 7, 1996 1205
This conclusion extends to an important phosphorami-
dothiolate insecticide (i.e., methamidophos) where the
MeO(NH2)P(O)SH metabolite does not appear to be an
acceptable substrate for methylation.
O- and S-demethylation are prominent metabolic reac-
tions for (MeO)2P(O)SMe in mice, but it is not known if
this involves sulfoxidation or is catalyzed by GSH S-
transferase (6). However, the prolonged persistence in
this study of (MeO)2P(O)SMe in the presence of a small
amount of (MeO)2P(S)SMe, as noted earlier with (MeO)3P-
(S) (6), is more easily attributed to inhibition of cyto-
chrome P450. The mechanism of toxic action for (MeO)2P-
(O)SMe is unknown (20) but thought to involve a sulfoxide
intermediate (6, 21) which covalently derivatizes tissues
(22). (MeO)2P(S)SMe and (MeO)2P(O)SMe are poor
inhibitors of cholinesterase (4, 5) and poor alkylating
agents (23, 24). Several studies have focused on the lung
as a primary site of toxicity for (MeO)2P(O)SMe (7, 8, 25-
27). (MeO)2P(S)SMe is not a lung-specific toxin, and
when administered to rats in small amounts, it prevents
the lung damage caused by analogs of (MeO)2P(O)SMe
(8, 28). (MeO)2P(O)SMe requires activation by the cyto-
chrome P450 system before it becomes toxic to the lung
(28), and (MeO)2P(S)SMe has been shown to be a specific
inhibitor of pulmonary cytochrome P450 2B1 activity
(29).
The contribution of metabolically-generated (MeO)2P-
(S)SMe and (MeO)2P(O)SMe to the mammalian toxicity
of dimethoate, malathion, and related phosphorodithioate
insecticides is unclear. The delayed neurotoxic poisoning
symptoms after high doses of dimethoate in humans (30)
are conceivably due in part to (MeO)2P(O)SMe as a
metabolite, a possibility that has not been examined. This
study demonstrates that O,O,S-trimethyl phosphorodithio-
ate and phosphorothiolate are not only impurities in
technical phosphorodithioate insecticides but also poten-
tially toxic metabolites formed in tissues by a previously
unrecognized pathway for mammals.
F igu r e 5. Partial metabolic pathways for dimethoate in mice
with emphasis on S-methylation products and further metabo-
lites of (MeO)2P(S)SMe. All metabolites shown are detected in
urine and the triesters also in liver.
is detected from phosalone (Figure 3 which also shows
(EtO)2P(O)SMe as a metabolite), ethion, and the hydroly-
sis product (EtO)2P(S)SH in decreasing amounts. There
is no detectable (EtO)2P(O)SMe with (EtO)2P(O)SR com-
pounds when R ) n-propyl, sec-butyl, and tert-butyl, nor
with (EtO)2P(S)OEt or (EtO)2P(S)OC6H5. There is also
no detectable (MeO)2P(O)SMe formed via (MeO)2P(O)-
SH on metabolism of the (MeO)2P(S)O-aryl insecticide
fenthion. Finally, the urine of mice receiving an ip dose
(500 mg/kg) of MeO(NH2)P(O)SNa does not contain MeO-
(NH2)P(O)SMe (methamidophos) detected by 31P NMR
examination nor is any toxicity observed.
En zym a tic Meth yla tion . (MeO)2P(S)SMe is easily
detected by CI-MS-SIM of incubation mixtures of (MeO)2P-
(S)SH in the enzymatic S-methylation system but not in
two types of controls, i.e., the peak height with mi-
crosomes and SAM is >17-fold greater than with incuba-
tions lacking this cofactor or when the microsomes are
denatured (85 °C, 15 min).
Discu ssion
Figure 5 shows the new S-methylation pathway for
dialkyl phosphorodithioate insecticides as illustrated
primarily with dimethoate giving rise to (MeO)2P(S)SMe
and its further metabolites in mice including the toxicant
(MeO)2P(O)SMe and its cleavage products. Identification
of the four P-SMe urinary metabolites of dimethoate
[which are not reported in earlier studies (refs 16-18 and
references cited therein)] constitutes the strongest evi-
dence for in vivo S-methylation, but GC-CI-SIM analysis
also supports this observation. It is difficult to establish
the overall yield of (MeO)2P(S)SMe and (MeO)2P(O)SMe
from dimethoate since they are transient intermediates
except for that portion removed from the metabolic pool
by excretion. The P-SMe metabolites of dimethoate are
not artifacts due to impurities based on rigorous controls
and the identification of (EtO)2P(S)SMe from phosalone
that in itself contains no methyl group, further validating
the conclusion that S-methylation is metabolic and not
artifactual. In comparing the 31P NMR spectra of
dimethoate metabolites in rat urine with those in mouse
urine, we observe (data not shown) a preference for
cleavage of the PS-C bond in mice and the C(O)-NHMe
substituent in rats, consistent with known species dif-
ferences in cleavage sites and rates (19) and perhaps with
the higher toxicity of dimethoate to mice than to rats (13).
The S-methylation of metabolites from dimethoate and
related compounds (ethion, malathion, phenthoate, phos-
alone, and phosmet) appears to be a general pathway for
O,O-dialkyl phosphorodithioate pesticides with SAM a
likely cofactor as demonstrated in vitro with liver and
(MeO)2P(S)SH. (RO)2P(S)SH is methylated, whereas the
more acidic (RO)2P(O)SH is not observed as its meth-
ylated derivative, in contrast to R1R2NC(S)SH and R1R2-
NC(O)SH both of which undergo S-methylation (9, 10).
Ack n ow led gm en t. The project described was sup-
ported by Grant PO1 ES00049 from the National Insti-
tute of Environmental Health Sciences, NIH. Our labo-
ratory colleague Susan Sparks helped treat the mice.
Refer en ces
(1) Pellegrini, G., and Santi, R. (1972) Potentiation of toxicity of
organophosphorus compounds containing carboxylic ester func-
tions toward warm-blooded animals by some organophosphorus
impurities. J . Agric. Food Chem. 20, 944-950.
(2) Umetsu, N., Grose, F. H., Allahyari, R., Abu-El-Haj, S., and
Fukuto, T. R. (1977) Effect of impurities on the mammalian
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