Chem. Res. Toxicol. 1995,8, 1063-1069
1063
S-Methylation as a Bioactivation Mechanism for Monoand Dithiocarbamate Pesticides as Aldehyde Dehydrogenase Inhibitors Richard E. Staub, Susan E. Sparks, Gary B. Quistad, and John E. Casida" Environmental Chemistry and Toxicology Laboratory, Department of Environmental Science, Policy and Management, University of California, Berkeley, California 94720-3112 Received April 26, 1995@ S-Methylation is a new bioactivation mechanism for metam and metabolites of methyl isothiocyanate and dazomet in mice. These soil fumigants are converted to 5'-methyl metam [MeNHC(S)SMel which reaches peak levels in liver, kidney, brain, and blood 10-20 min after intraperitoneal (ip) treatment. The half-life of 5'-methyl metam administered ip is 8-12 min in each of these tissues. S-Methyl metam-oxon [MeNHC(O)SMelis also detected a s a metabolite of each of these soil fumigants on analysis by gas chromatography/mass spectrometry with chemical ionization. The conversion of methyl isothiocyanate to S-methyl metam and its oxon probably involves conjugation with glutathione, hydrolysis to 5'-(N-methylthiocarbamoy1)cysteine, cleavage by cysteine conjugate ,&lyase to release metam, and finally methylation and oxidative desulfuration. Metam and dazomet are converted to S-methyl metam by mouse liver microsomes on fortification with S-adenosylmethionine. Metam, methyl isothiocyanate, dazomet, and three metabolites (metam-oxon [MeNHC(O)SHl, MeNHC(S)SMe, and MeNHC(0)SMe) administered ip to mice a t 40 mg/kg inhibit low-K, liver mitochondrial aldehyde dehydrogenase and elevate ethanol-dependent blood and brain acetaldehyde levels. Several fungicides including the dialkyldithiocarbamates as the disulfide (thiram and the related alcohol-abuse drug disulfiram) and metal salts (ziram) also yield 5'-methyl thiocarbamate metabolites. Eight S-alkyl and S-(chloroallyl) thiocarbamate herbicides (EPTC, molinate, butylate, vernolate, pebulate, diallate, sulfallate, and triallate), but not their S-chlorobenzyl analog (thiobencarb), undergo sequential liberation of the thiocarbamic acid and then 5'-methylation, forming the S-methyl thiocarbamates which are new metabolites and potential aldehyde dehydrogenase inhibitors. The S-methyl mono- and dithiocarbamate metabolites of these herbicides and fungicides are easily identified by retention time on gas chromatography and by mass spectrometry giving [MHl+ plus [RlRzNCO]+or [RlRzNCS]+,respectively, as the two major ions.
Introduction A large number and variety of pharmaceuticals and agrochemicals contain thiono, thiolo, and thioether substituents important in their metabolism and action. Many of them undergo metabolic activation involving sulfoxidation or S-methylation or both. A recent interesting example is the metabolic activation of the alcoholaversion drug disulfiram (Antabuse) [EtZNC(S)SSC(S)NEtzl as an inhibitor of low-K, liver aldehyde dehydrogenase (ALDH)l via a four-step sequence involving disulfide cleavage, S-methylation, oxidative removal of the thiono sulfur, and sulfoxidation to form Et2NC(O)S(O)Me as the ultimate ALDH inhibitor ( I ) . The fungicide thiram [Me2NC(S)SSC(S)NMez]also inhibits ALDH (2) probably through the same bioactivation sequence to
* To whom correspondence should be addressed at the Environmental Chemistry and Toxicology Laboratory, Department of Environmental Science. Policv and Management. 114 Wellman Hall. University of California, Berkeley, CA 97720-3112. Phone: (510) 642: 5424: . .~~ . ~ . . , FAX: (510) 642-6497. @Abstractpublished in Advance ACS Abstracts, October 15, 1995. Abbreviations: ALDH, aldehyde dehydrogenase; CI, chemical ionization; DMTU, dimethylthiourea; DTD, 2,4-dimethyl-l,2,4-thiadiazolodone-3,5-dithiane;GST, GSH S-transferase; MIC, methyl isocyanate; MITC, methyl isothiocyanate; MMDT, 4-methyl-5-(methylimino)-1,2,4-dithiazolidine-3-thione; RA, relative abundance; S A M , S-adenosylmethionine;SIM, selected ion monitoring; t ~retention , time; Me, methyl; Et, ethyl; nPr,n-propyl; iPr, isopropyl; nBu, n-butyl; iBu, isobutyl. ~~
~
form MezNC(O)S(O)Me(3).These findings indicate that S-methylation may be an important aspect of the bioactivation of thio- and dithiocarbamate agrochemicals and of their inhibition of ALDH. This is an unexplored possibility for several fumigants, fungicides, and herbicides indicated below. Metam (metham), methyl isothiocyanate (MITC) and dazomet are major soil fumigants (4);eg., '57 million pounds of metam were used in 1992 (5).They are also applied at very high rates (eg., up to 320 lblacre for metam) (6). Metam gained notoriety in 1991 when a spill into the Sacramento River in California resulted in human exposure and an environmental disaster (7). There are two proposals for the mechanism of toxic action of these fumigants. The first involves decomposition or metabolism of metam and dazomet to MITC as the activation product which derivatizes critical biological thiols and amines (4,8-10). Thus, metam, MITC, and dazomet are metabolized by rats and mice to the mercapturate, N-acetyl-S-(N-methylthiocarbamoy1)cysteine (11-13) via the GSH conjugate, which serves as a potential carrier for the later release of MITC (14).As an alternative, metam is very sensitive to oxidation, forming reactive sulfenic and sulfinic acids which might contribute to its toxic action (15).S-Methylationprovides a third alternative in which metam might be methylated and MITC and dazomet metabolized to S-methyl metam
0893-228x/95/2708-1063$09.0010 0 1995 American Chemical Society
1064 Chem. Res. Toxicol., Vol. 8, No. 8, 1995
-
pesticide -
thiocarbamic acid
dithiocarbamate R,R2NC(S)SR3
I thiocarbamate
RlR2NC(0)SR3
---+
S-methyl conjugate
RIRzNC(S)SH
1
RlRzNC(0)SH
Staub et al.
R,RzNC(S)SMe
-
1 RlRZNC(0)SMe
Figure 1. Hypothetical pathways for metabolism of dithiocarbamate herbicides or fungicides and monothiocarbamate herbicides via thiocarbamic acids to their S-methyl conjugates. The oxidation reactions are carried out by microsomal flavinadenine dinucleotide-containing monooxygenases or cytochrome P450 in the presence of NADPH. The methylation reactions involve mitochondrial thiol S-methyl transferase and S A M .R1 and R2 = alkyl. R3 = alkyl, chloroallyl, or 4-chlorobenzyl.
I
I
s
I M e
i
in mice (3, 16). Their metabolism may involve Smethylene hydroxylation (17)or cleavage of their respective cysteine conjugates (18) as two pathways which liberate the thiocarbamic acid for potential methylation, although this has not been observed in the past. This study considers S-methylation as a potential bioactivation mechanism (Figure 1) for metam and related fumigants (Figure 21, dialkyldithiocarbamate fungicides (structures in Materials and Methods), and thiocarbamate herbicides (Table 1)that could conceivably yield S-methyl thiocarbamates as metabolites. It considers a wide range of agrochemicals and the activity of selected pesticides and their metabolites as inhibitors of liver mitochondrial low-& ALDH and enhancers of ethanol-dependentacetaldehyde levels in blood and brain of mice.
Materials and Methods
1 M cysteine e N H iconlugate (S)-SL
MeN=C=O MIC
/
I
MeNHC(0)SMe S-methyl metam-oxon
-
MeNHC(0)SH metam-oxon
MeNHC(0)-SG + -+ MeNHC(O)-SCy5 GSH conjugate cysteine conjugate
Figure 2. Proposed pathways for metabolism of metam, MITC, and dazomet to S-methyl metam and S-methyl metam-oxon. See the legend of Figure 1 for enzymes and cofactors involved i n the oxidation and methylation reactions. The GSH conjugation reaction is nonenzymatic with MITC and MIC but involves GST with metam. The cysteine conjugates are cleaved by cysteine conjugate P-lyase.
and its oxon as potential ALDH inhibitors. The Smethylation pathway for metabolic activation might also apply to dialkyldithiocarbamate fungicides. Finally, several thiocarbamate herbicides with S-ethyl, S-propyl, S-(chlorobenzyl), and S-(chloroallyl) substituents (-50 million lb used per year) (5)also inhibit ALDH in vivo
Chemicals. Biochemicals were purchased from Sigma Chemical Co. (St. Louis, MO). The mono- and dithiocarbamates and related compounds examined (including [13Meldazomet and metam impurities) were available from previous studies in this laboratory or were obtained from Chem Service (West Chester, PA). Structures for the dialkyldithiocarbamates studied are as follows: [RzNC(S)S12,R = Me for thiram and E t for disulfiram; [Me2NC(S)S]zZn for ziram. Metam-oxon was synthesized by bubbling carbonyl sulfide for 1h into a solution of methylamine (30% w/v) and potassium hydroxide (25% w/v) in ethanol by the general procedure of Johansson et al. (19). Metam was used as the dihydrate sodium salt unless indicated otherwise. S-Methyl metam and 5'-methyl metam-oxon were prepared by reacting metam (purified by washing with dichloromethane) (15) or metam-oxon with equimolar iodomethane in a n acetone solution saturated with potassium carbonate and purifying by preparative TLC (silica gel, 7:3 hexanelacetone (v/v), R,f 0.32 and 0.31, respectively). The purities of S-methyl metam and S-methyl metam-oxon were estimated to be '95% based on GC/MS and HPLC as below. The GSH and N-acetylcysteine conjugates of MITC and the carbamoyl moiety of molinate were prepared by the general procedure of Hubbell and Casida (20).MITC (100 mg) was stirred with GSH or N-acetylcysteine (0.5 molar equivalent), and molinate sulfoxide (19 mg) was treated with
Table 1. GC/MS-CI Characterization Data for S-Methyl Mono- and Dithiocarbamates as Metabolites of Mono- and Dithiocarbamate Pesticides mlz values metabolite or herbicide MeNHC(0)SMe" MezNC(0)SMe MeNHC(S)SMe" MeZNC(S)SMe EtZNC(0)SMe EtZNC(S)SMe
parent pesticide t R , min [MHl+ S-Methyl Mono- and Dithiocarbamates as Metabolites metam, dazomet, MITC 4.06 106 thiram 4.56 120 metam, dazomet, MITC 7.21 122 thiram, ziram 7.95 136 disulfiram 6.07 148 disulfiram, sulfallate 9.28 164
CHz(CHz)5NC(O)SMe nPrzNC(0)SMe" nBu(Et)NC(O)SMe iPrzNC(0)SMe iBuzNC(0)SMe
molinate EPTC pebulate diallate, triallate butylate
-
CHz(CH2)5NC(O)SEt nPrzNC(0)SEt nPrzNC(0)SnPr nBu(Et)NC(O)SnPr iBuzNC(0)SEt EtzNC(S)SCHzCCl=CH2 EtzNC(O)SCHzCsH4-CI-4 iPrzNC(O)SCHZCCl=CHCl iPrzNC(O)SCH~CCl=CClZ a
10.43 8.15 8.34 6.90 9.12
[RR"COl+ or [RR'NCSl+ 58 72 74 88 100
116
174 176 176 176 204
126 128 128 128 156
188 190 204 204 218 224 258 270 306
126 128 128 128 156 116 100 128 128
Mono- and Dithiocarbamate Pesticides molinate EPTC vernolate pebulate butylate sulfallate thiobencarb diallate triallate
Metabolite compared with standard compound. El2 isomers.
11.15 8.82 9.97 10.16 9.77 13.13 15.85 12.85113.04b 14.44
S-Methylation of Thiocarbamate Pesticides GSH (1.5 molar equivalent), i n each case in methanol (15 mL) and triethylamine (5 mL) for 18 h at 25 "C. The GSH conjugate of methyl isocyanate (MIC) was prepared by the method of H a n et al. (21),involving addition of MIC (70 mg) in acetone (2 mL) to GSH (185 mg) in acetonitrile/water (7/3) (10 mL), stirring for 30 min, filtering, and washing the conjugate with acetone. The conjugates were purified by solvent extraction (derivatives of MIC and molinate sulfoxide) or by preparative HPLC (the other adducts). Structures of the GSH conjugates of MITC and molinate were verified by fast atom bombardment mass spectrometry. The NMR spectra for S-(N-methylthiocarbamoy1)-Nacetylcysteine and S-(N-methylcarbamoy1)-GSHwere the same as those previously reported by Lam et al. (13)and Han et al. (211, respectively. Reversed-phase HPLC with UV detection for purity analysis was performed as follows: Hewlett-Packard 1050 solvent delivery system and 1040M Series I1 photodiode array detector at 220 and 260 nm; Merck 100RP-18 column (5 pm, 0.4 x 12.5 cm); 0.1% trifluoroacetic acid for 5 min, then a linear gradient of 0-50% methanol in water with constant 0.1% trifluoroacetic acid over 20 min, and finally a linear gradient to 100% methanol over an additional 10 min, each a t 1.5 m u m i n . Retention times ( ~ R s ) were 10.5 and 6.0 min for S-methyl metam and S-methyl metam-oxon, respectively, while the ~ R Sfor the GSH and N-acetylcysteine conjugates fell in t h e range of 6.2-14.0 min, and each was ' 8 8 % pure. The N-acetylcysteine conjugate with MITC chromatographed by HPLC as two poorly-resolved peaks, attributable to the conformers created by the partial double bond character and restricted rotation about the N-C bond (13) as confirmed here by lH NMR showing t h a t the two methyl resonances coalesce to single signals at 60 "C. Animals and Treatment Protocols. Male albino SwissWebster mice (20-25 g) from Simonsen Laboratories (Gilroy, CAI were administered the test compounds ip at 5-430 m g k g using water (for metam, metam-oxon, ziram, and conjugates of N-acetylcysteine and GSH) or MezSO (for the other compounds) a s the carrier vehicle (25-100 pL). At appropriate times, the animals were sacrificed by cervical dislocation and immediately dissected to obtain the liver, kidney, brain, and blood (taken from the heart by syringe). The tissues were analyzed immediately as indicated below. Extraction and Analysis of Tissues. Fresh samples of liver (0.9-1.7 g) and kidney (0.3-0.5 g) from individual mice were homogenized in water (2-3 mL), which was then partitioned twice with dichloromethane (2 mL). Similarly, brain (0.3-0.5 g) and blood (0.1-0.6 g) were homogenized i n water (1 mL) and extracted with dichloromethane (2 x 1 mL). GC/MS with chemical ionization (CI) and selected ion monitoring (SIM) was performed on a Hewlett-Packard 5890 gas chromatograph connected to a 5971A mass spectrometer: DB-5 fused-silica capillary column, 30 m x 0.25 mm i.d. (J & W Scientific, Folsom, CA); 70-250 "C over 20 min. The analysis involved a 1.0-pL aliquot injected with the HP7673A automatic sample injector. S-Methyl metam and S-methyl metam-oxon were analyzed by monitoring the major ions at m l z = 74 and 58, representing [MeNHCS]+ and [MeNHCOl+, respectively. Quantitation of 5'-methyl metam as a metabolite involved comparison with the authentic compound from synthesis as a standard curve. Additionally, an S-methyl metam external standard was analyzed before, during, and after tissue samples to monitor GC/MS performance. Instrument response was linear (regression coefficient = 0.993 f 0.003) over the range of S-methyl metam levels observed. The data presented are minimal values for S-methyl metam in tissues since they are not corrected for recoveries (e.g., 84% from fortified liver). Identification of the S-methyl derivatives in liver extracts (dried with anhydrous sodium sulfate and concentrated by evaporation to -0.1 mL) utilized GCiMS in the scan mode, surveying all ions between 50 and 300 amu. Enzymatic Methylation. Metam (0.9 mM final concentration), metam-oxon (0.9 mM), or dazomet (0.02 mM) was incubated with S-adenosylmethionine (SAM)(1.1mM), NADPH (00.12 mM), and microsomes (1mg of protein) in 100 mM sodium
Chem. Res. Toxicol., Vol. 8, No. 8, 1995 1065 phosphate buffer (pH 7.4, 0.5 mL total volume) for 30-60 min at 37 "C. For controls, S A M was not added, or heat-denatured microsomes (80 "C, 10 min) were used. Each reaction mixture was partitioned with dichloromethane (1mL) to terminate the reaction and analyzed by G C N S with SIM as above. ALDH Activity and Acetaldehyde Analyses. The test compounds were administered ip at 40 m g k g followed after 2 h by either sacrifice for liver enzyme assay or treatment with ethanol ip at 1000 m g k g for sacrifice 30 min later and analysis of acetaldehyde levels in brain and blood. Controls received carrier solvent only (enzyme assay) o r carrier solvent and ethanol (acetaldehyde assay). The procedure for ALDH activity assay described for mitochondrial low&, enzyme from rat liver (22)was adapted for mice (3)using 250 p g of protein per assay in the present investigation. Ethanol-dependent acetaldehyde levels in brain and blood were quantitated by G C M S with SIM for the 0-benzyloxime ether derivative (3). Toxicity in Mice. Mice were treated ip with the test compounds in MezSO as above in studies with a dose differential of 2-fold and 4-6 mice per dose. LD50 values were determined after 1 week with daily observation.
Results GUMS-CIIdentificationof Metabolically-Formed S-Methyl Mono- and Dithiocarbamates (Table 1). MeNHC(S)SMe, MeNHC(O)SMe, and nPrzNC(0)SMe are identified as metabolites by comparison with authentic standards from synthesis with respect to their tR values and [MH]+ and [RR"CO]+ or [RR'NCS]' ions. In other cases, where authentic standards were not available, the GC/MS-CI assignments of structures for metabolites are made on the basis of relative tR values and diagnostic ions for the metabolites themselves. The tR values generally increase with molecular weight, and the tR ratios for SMe:SEt:SnPr are almost constant within each group (1.00:1.07:1.22) and therefore diagnostic for assigning the SMe derivatives when a standard is not available. The two major ions in every case are [MHIand either [RR'NCOl+or [RR'NCSl+ as the main criteria for identification. Figure 3 illustrates the analysis of S-methyl metam-oxon and S-methyl metam as metabolites of MITC and dazomet, respectively. Metabolic Methylation of Metam and Metamoxon. Metam (ip, 100 mgkg) is rapidly methylated to S-methyl metam, which reaches a maximum tissue level at 10-20 min after treatment of -10, 3.5, 1.2, and 1.1 ppm in liver, kidney, brain, and blood, respectively, and much lower levels by 60 min (Figure 4). The level of S-methyl metam in liver 10 min after dosing increases almost proportionate to the dose of metam from 5 up to 210 but not 425 mgkg (Figure 5). S-Methyl metam-oxon is detected in liver extracts from mice 10-60 min after treatment with metam or metamoxon a t 100 mgkg (data not shown). For mice treated with metam, the level of S-methyl metam-oxon is barely detectable by GC/MS SIM analysis in most extracts. Metabolic Conversion of MITC and Conjugates of Both MITC and MIC to S-Methyl Metam andor S-Methyl Metam-oxon. MITC (ip, 50 mg/kg) yields 1-3 ppm S-methyl metam in liver and kidney 10-60 min after treatment with lower levels at 5 min (Figure 4). Maximal blood and brain levels of S-methyl metam are 0.5-0.7 ppm. S-Methyl metam-oxon is also detected in extracts of all analyzed tissues from mice 20-60 min after treatment but at much lower levels than S-methyl metam (data not shown). Potential intermediates in the metabolic conversion of MITC to S-methyl metam and S-methyl metam-oxon
Staub et al.
1066 Chem. Res. Toxicol., Vol. 8, No. 8, 1995 1-
metam
metam
*
liver
+
blood
8-
7.06 min
4-
-
0-r
I.o
RA
0.5
I 58m/z
: i : I
\
MeNHC(0) SMe
2-
1-
metabolite of [13Me\12Me]da~~met mixture
O+0
1.0
1
40
20
60
I
dazomet 13.26min
S-methyl 7.24 min
kidney brain
4-
1.0
~
I 74175 m/z
" 0
! RA
0.5
40
20
60
min Figure 4. S-Methyl metam levels in tissues of mice 5-60 min after ip treatment with metam, MITC, and dazomet. Error bars represent the standard error of the mean.
Figure 3. Metabolites of MITC and dazomet in liver of mice 15 and 10 min, respectively, after treatment showing GC traces and MS spectra. Dazomet was isotopically diluted with [l3Me1dazomet to yield the characteristic increase of 1 m l z to unambiguously distinguish metabolites derived from dazomet from coextractives. Retention times are given for the principal metabolites. RA refers to relative abundance.
were examined by injecting mice with the GSH or N-acetylcysteine conjugates of MITC (200 mgkg) 20 min prior to sacrifice for analysis of the liver extract (data not shown). The GSH conjugate yielded a significant peak for S-methyl metam while the N-acetylcysteine conjugate gave even larger amounts of both S-methyl metam and S-methyl metam-oxon, as determined by GCI MS in the scan mode. In addition, comparable experiments with the GSH conjugate of MIC gave detectable S-methyl metam-oxon 30 min after treatment. Metabolic Conversion of Dazomet to S-Methyl Metam and S-Methyl Metam-oxon. Dazomet (ip, 50
metam (mg/kg)
Figure 5. S-Methyl metam levels in liver of mice 10 min after ip treatment with metam at 5-425 mgkg. Error bars represent the standard error of the mean.
m a g ) yields S-methyl metam in liver, kidney, brain, and blood a t 2-7 ppm over a period of 10-40 min after treatment and rapidly decreasing by 60 min to -1 ppm (Figure 4). The highest level achieved is 11ppm in liver
Chem. Res. Toxicol., Vol. 8, No. 8, 1995 1067
S-Methylation of Thiocarbamate Pesticides S-methyl metam, 15 mg/kg
a
h
6
ka
v
s
c)
a,
E
4
20
40
60
L
(A 2
0
0
40
20
60
min Figure 6. S-Methyl metam levels in tissues of mice 5-60 min after ip treatment with S-methyl metam. The inset shows by a semilogarithmetic plot the similar rates for residue dissipation in the four tissues. Error bars represent the standard error of the mean. Results a t 5 min are shown only as the mean due t o the large variations encountered, Le., 6.4 f 6.2, 3.8 k 2.4, 2.7 & 1.8, and 3.2 rf 3.0 ppm for the liver, kidney, brain, and blood, respectively.
at 5 min. S-Methyl metam-oxon is also observed, but at barely detectable levels, in extracts of all analyzed tissues from mice 30 min after treatment with dazomet at 50 mg/kg (data not shown). Rate of S-Methyl Metam Degradation in Mice. S-Methyl metam (ip, 15 mgkg) yields peak tissue levels 10 min after treatment, with those in liver and kidney twice as high as those in brain and blood (Figure 61, and in each case with a t l / z of about 5-7 min (Figure 6, semilogarithmetic plot). Although not shown, S-methyl metam-oxon is also detected in extracts of all analyzed tissues from these mice 10-40 min after treatment. Enzymatic Methylation. Metam is readily converted to S-methyl metam in vitro by microsomes fortified with S A M under conditions in which little S-methyl metamoxon is detected from metam-oxon (data not shown). The yield of S-methyl metam is limited by the amount of microsomal protein. Dazomet is converted in small amounts to S-methyl metam, but fortification with NADPH over and above SAM does not significantly increase the yield. These methylated derivatives are not detected if the microsomes are denatured (80 "C, 15 min) or if S A M is not added to the reaction mixture. Metabolic Conversion of Dialkyldithiocarbamate Fungicides to Their S-Methyl Derivatives. Disulfiram, ziram, and thiram (ip, 100-200 mgkg) are converted in mice to their S-methyl derivatives detected in liver 10 min after treatment; the corresponding oxons are also observed with disulfiram and thiram (Table 1). Metabolic Conversion of Thiocarbamate Herbicides and Their Thiol Cleavage Products to SMethyl Derivatives. Several thiocarbamate herbicides (ip, 200 mg/kg) yield S-methyl thiocarbamate metabolites detected in liver extracts 15-30 min following treatment with molinate, EPTC, butylate, vernolate, pebulate, sulfallate, diallate, and triallate, but not for those treated
with thiobencarb (Table 1).S-Methyl molinate (Table 1) is detected in liver extracts from mice 20 min after treatment not only with molinate but also with its GSH conjugate (ip, 200 mg/kg). The levels of S-methyl derivatives of the thiocarbamate herbicides were much less than those of S-methyl metam derived from metam, MITC, and dazomet. The mercaptans released on oxidative or hydrolytic cleavage of thiobencarb, diallate, and triallate are observed in liver extracts as the methyl derivatives and their sulfones. Thus, both 4-chlorobenzyl methyl thioether ( t 9.2 ~ min, m / z = 173 [MHl+ and 125 [ClPhCH21') and the corresponding sulfone ( t R 13.4 min, m l z = 205 [MHI' and 125 [ClPhCH#) are detected in liver extracts from mice treated with thiobencarb. 2,3-Dichloroallyl methyl thioether ( t R 4.6 min, m / z = 157 [MHl+ and 109 [ClCH=CClCH21+) and the corresponding sulfone ( t 8.9 ~ min, m l z = 189 [MH]' and 109 [ClCH=CClCH2]') are found in mice treated with diallate, and 2,3,3-trichloroallyl methyl sulfone ( t 10.7 ~ min, m l z = 223 [MH]+and 143 [C12C=CC1CH21+) is detected following triallate treatment. ALDH Inhibition in Vivo (Table 2). Metam, MITC, dazomet, metam-oxon, S-methyl metam, S-methyl metamoxon, and metam disulfide inhibit liver mitochondrial low-K, ALDH activity by 36-49% 2 h after ip administration at 40 mgkg. Three decomposition products of metam (DMTU, DTD, and MMDT) (23) do not significantly inhibit this ALDH activity in the liver. Acetaldehyde Levels in the Blood and Brain following Ethanol Administration (Table 2). Metam, MITC, dazomet, and four of their derivatives or related compounds administered at 40 mgkg, followed after 2 h by ethanol at 1000 mgkg, greatly elevate the acetaldehyde levels in blood and brain. S-Methyl metam-oxon gives the highest elevation 30 min after the ethanol, at 418-458% in the blood and brain. Two of the metam impurities (DMTU and MMDT) do not significantly elevate acetaldehyde levels in the brain and blood when administered a t 40 mgkg, whereas a third (DTD) may have a minor effect. Toxicity in Mice (Table 2). Metam-oxon is more acutely toxic than metam or their S-methyl derivatives, and it also acts more rapidly, with death in
