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Chem. Res. Toxicol. 1989,2, 392-399
S-Oxygenation of Eptam in Hepatic Microsomes from Freshand Saltwater Striped Bass (Morone saxatilis) John R. Cashman* and Leslie D. Olsen Department of Pharmaceutical Chemistry and Liver Center, School of Pharmacy, University of California, San Francisco, California 94143-0446
Graham Young and Howard Bern Department of Zoology, University of California, Berkeley, California 94720 Received June 14, 1989
The in vitro liver microsomal oxidation of eptam (ethyl Nfl-dipropylthiocarbamate) in the presence of freshwater and salt water adapted striped bass liver microsomes was investigated. In freshwater hepatic microsomes from striped bass, eptam is S-oxygenated in a process consistent with the involvement of monooxygenase activity. In contrast, both eptam S-oxide and eptam sulfone are formed in microsomes from salt water adapted striped bass microsomes in a process that is independent of monooxygenase activity and consistent with a role of cooxidation by hydroperoxy fatty acids. The mechanism of oxidation of eptam by hydroperoxy fatty acids may involve radical species. Both eptam S-oxide and eptam sulfone are efficient carbamylating agents toward thiol nucleophiles and react with substituted thiophenols to produce thiocarbamates while eptam itself is relatively stable to trans thiocarbamylation. Monooxygenase-catalyzed Soxygenation of eptam in freshwater striped bass hepatic microsomes may represent a bioactivation route, which may explain the toxicity of thiocarbamate herbicides such as eptam toward freshwater fish.
Introduction Enormous amounts of thiocarbamate herbicides are applied to California rice fields every year ( 1 ) . While thiocarbamate herbicides are effective at controlling broadleaf grass, some of the agents enter into the Sacramento river delta and may pose a significant health hazard to fish (2). Herbicide application coincides with spawning of several commercially important fish including striped bass ( 3 ) . In recent years, the San Francisco Bay delta striped bass population has diminished (4). The large and unexplained striped bass die off has been linked to thiocarbamate herbicide application as a possible cause of mortality (5). The most striking pathological tissue finding of moribund striped bass taken from the Sacramento delta area was that of liver dysfunction and necrosis ( 6 ) . Thiocarbamate herbicides such as eptam have a low toxicity toward mammals (i.e., rat oral LDS0= 2550 mg/kg), but eptam is much more toxic to fish (i.e,, rainbow trout 96-h LCN = 19 mg/L) ( 7 ) . Metabolic oxidation activates thiocarbamates to potent carbamylating agents (8), which may explain the selective toxicity of eptam to fish. In addition, however, S-oxidation of eptam reduces the octanol/water partition coefficient, and therefore; (1)target action must be kinetically faster than excretion of eptam S-oxide and (2) cumulative, essentially irreversible, poisoning of striped bass must be involved in order to explain the hepatotoxicity of the animals if eptam is involved in the toxicity of striped bass. In mammalian systems, one of the major metabolites of eptam is the sulfoxide (9) (Scheme I). Eptam sulfoxide does not accumulate after administration to mice (10)and is further oxidized and degraded or reacts with thiols as a carbamylating agent ( 1 0 , l l ) . Eptam sulfoxide may be oxidized to eptam sulfone, which has also been implicated as a carbamylating agent (12). Eptam sulfoxidation and cleavage by the glutathione S-transferase system are
Scheme I. Overall S-Oxidative (Bi0)transformation of Eptam (1) to Eptam S-Oxide (2) and Eptam Sulfone (3)"
3
2
1
"The R group is n-propyl. Scheme 11. Chemical Reaction of Eptam S-Oxide [X = S(O)CH,CH,] or Eptam Sulfone (X = S0,CH2CHS)with p-Methoxythiophenol To Produce Thiocarbamate 4 or with m -Nitrothiophenol To Produce Thiocarbamate 5' 0
0
R2N-C-X II
-O
F$N-C--S*OCH, II 4
0
0
I1
R2N-C-X
'In all structures, R
+
R2N-C--S
= n-propyl.
postulated to be a detoxication process (8-11, 13), but S-oxidation may also lead to glutathione depletion (12,13). Chemical studies suggest that eptam sulfone is more reactive than eptam sulfoxide toward thiols (11,12)(Scheme 11), but neither the enzyme system(s) nor the molecular basis for the oxidative transformations of eptam has been established. In agreement with metabolism studies in mammalian systems, thiocarbamates are extensively metabolized in striped bass ( 1 1 ) . The major routes of metabolism of
0893-228~ f 89 f 2702-0392$01.5O/O 0 1989 American Chemical Society
E p t a m S-Oxygenation
thiocarbamates in striped bass include sulfoxidation,aliphatic hydroxylation,and mercapturic acid formation (11). The purpose of this study was to investigate the oxidative transformation of eptam by striped bass liver microsomes and to compare the reactivity of its major metabolites, eptam sulfoxide and eptam sulfone, as carbamylating agents toward model thiol nucleophiles in order to simulate the probable types of reactions that take place in fish. Comparison of the biotransformation of thiocarbamate herbicidesbetween mammalian and fish species may help clarify the selective toxicity of thiocarbamate herbicides toward fish. A thorough study of hepatic thiocarbamate biotransformationin striped bass, which migrate from salt water into freshwater for spawning, etc., could provide information that may lead to an understanding of the selective toxicity observed for striped bass of the Sacramento delta region. Experlmentai Section Chemicals. Chemicals used in this study were of the highest purity available and were purchased from Aldrich Chemical Co., Milwaukee, WI. Other reagents and solvents were purchased from Fischer Scientific or Bio-Rad, Richmond, CA. Eptam was a generous gift of Professor B. Hammock, University of California, Davis. Aminobenzotriazole was a gift of Professor P. Ortiz de Montellano and p-methoxythiophenol was a kind gift of Professor R. Ketcham of this department. Arachidonic acid was purchased from Aldrich. The methyl ester of 15-HETE' was synthesized according to the method of Baldwin et al. (14).5-HETE methyl ester was synthesized by the method of Corey et al. (15). Prostaglandin Bz was purchased from Sigma. The methyl esters were stored a t -20 "C in benzene under argon prior to hydrolysis (LiOH/THF, 1:2 v/v) and immediate use. The compounds were completely characterized by normal spectroscopictechniques, and the spectra were identical with published values. ['"C]Testosterone was purchased from New England Nuclear. Testosterone was obtained from Aldrich Chemical Co. Hydroxylated testosterone standards were obtained from the Steroid Reference Collection, Queen Mary College, London. Chromatography was performed with Silica Woelm (70-150 mesh), Fischer Scientific. Preparative thin-layer chromatography was with 20 X 20 cm Analtech Uniplate (500-pm thickness). LK5DF 5 X 20 cm channeled preadsorbent silica gel thin-layer chromatography plates were from Whatman (250-pm thickness). Instrument Analysis. 'H NMR spectra were recorded with a General Electric spectrometer operating a t 500 MHz interfaced to a Nicolet computer. 'H chemical shifts are expressed in ppm downfield from tetramethylsilane. Electron impact (EI) mass spectra were recorded with a Kratos MS 25 spectrometer at 6 kV and a source temperature of 50 OC. Liquid secondary ion mass spectrometry (LSIMS) was recorded with a Kratos MS 50 at 8 kV equipped with a cesium ion gun. Gas chromatography-mass spectrometry was done with a Kratos MS 25 interfaced to a Varian 3700 gas chromatograph. UV spectra were recorded with a Perkin-Elmer 559 A spectrometer. Infrared spectra were recorded on a Nicolet 5 DX FT-IR. Synthesis of Eptam S-Oxide (2). T o a stirred solution of eptam (100 mg, 0.53 mmol) in CHzCl (1.5 mL) a t 0 OC was added m-chloroperbenzoic acid (100 mg, 0.58 mmol) in CH2C12(1 mL). The resulting solution was stirred a t 0 "C for 10 min and brought to room temperature and stirred for 1h. Analysis by TLC showed that very little starting material was left. The crude mixture was extracted between CH2ClZand phosphate buffer (pH 7.6), dried, and chromatographed on silica gel pTLC (eluent CH30H/CH2C12, 1:99 v/v) to give eptam S-oxide, 45 mg, 42%, with an Rf value of 0.46: 'H NMR (CDC13) 6 3.68-3.34 (m, 4 H, -NCH,-), 3.06 Abbreviations: TLC, thin-layer chromatography; HPLC, highpressure liquid chromatography; HETE, hydroxyeicosatetraenoic acid; PGBZ, prostaglandin Bz; GC-MS, gas chromatography-mass spectrometry; LSIMS, liquid secondary ion mass spectrometry; EIMS, electron impact mass spectrometry; BSTFA, bis(trimethylsily1)trifluoroaebide; SBLO, soybean Iipoxygenase; TMS, trimethylsilyl; Tween 20, poly(oxyethylene) sorbitan monolaurate.
Chem. Res. Toxicol., Vol. 2, No. 6,1989 393 ~ 6.9 H~ Hz, 2 H, SOCH,), (overlapping dq; J = 27.4 Hz, J C H = 1.72 (m, 4 H, -C&CH,), 1.44 (t, J = 7.4 Hz, 3 H, SOCH,CH,), 1.03 (t, J = 7.6 Hz, 3 H, NCH,CH,CH,) 1.01 (t,J = 7.6 Hz, 3 H, NCH2CH2CH3);mass spectrum (LSIMS) m/z 206 (MH+), 149 (MH+ - C3H7N),128 (MH+- CzH5SO);E1 (relative intensity) 205 (M+, l),189 (M+ - 0,131,128 (M+ - CzH5S0,61); high-resolution EIMS, calcd 128.1193, obsd 128.1075 (+0.1 ppm); UV (CH3CN) ,A, (e) 220 (3481). Synthesis of Eptam Sulfone (3). Into a stirred solution of eptam (300 mg, 1.59 mmol) in CH2C12(4 mL) a t 0 "C was added m-chloroperbenzoicacid (600 mg, 3.48 mmol) in CHzClz (10 mL) a t 0 "C. After the addition was complete, anhydrous KF was added to the reaction, and the resulting solution was brought to room temperature and stirred for 0.5 h. Analysis by TLC showed that no starting material was left. The crude mixture was filtered through a pad of Celite/silica. Preparative silica gel TLC (eluent ethyl acetate/hexane, 1:4 v/v) of this material gave eptam sulfone, 281 mg, 80%, with an RI value 0.28: 'H NMR (CDCl,) 6 3.62 (t, J = 7.4 Hz, 2 H, NCH,), 3.34 (4, J = 14.9 Hz, J = 7.4 Hz, 2 H, SO,CH&, 3.31 (t,J = 7.4 Hz,2 H, NCH,), 1.72 (m, 2 H, -CH&H3), 1.41 (t,J = 7.4 Hz, 3 H, -S02CH,CH,), 0.94 (t, J = 7.4 Hz, 3 H, NCH,CH,CH,), 0.93 (t,J = 7.4 Hz, 3 H, NCH,CH,CH,); mass spectrum (LSIMS) m/z 222 (MH+), 128 (MH+ - C,H5S02); E1 (relative intensity) 128 (M+ - C,H5S02, 83),86 (C4H8NO+,70), 43 (NHCO+, 100); high-resolution EIMS, calcd 128.1096, obsd 128.1079 (+3.3 ppm); UV (CH3CN) ,A, (e) 211 (4960). Synthesis of m -Nitrothiophenol. Into dry tetrahydrofuran (1 mL) was placed m-nitrothiophenol disulfide (300 mg, 0.97 mmol), and sodium borohydride (136 mg, 3.41 mmol) and a solution of tetrahydrofuran (1 mL) were added portionwise. The reaction was stirred a t room temperature for 3 h. Analysis by TLC showed that no starting material remained. The reaction was acidified, extracted with CH,Cl,, washed with water, and dried to give the thiol (276 mg, 91%) with an R, value of 0.38 (silica gel TLC, eluent ethyl acetate/hexane, 10:90 v/v) (16). Synthesis of Thiocarbamate Derivatives. The general procedure for the synthesis of thiocarbamates 4 and 5 is based on the following method (11).Eptam sulfone is placed in a 90% tetrahydrofuran/phosphatebuffer (pH 8.4) solution, and the thiophenol is added (in tetrahydrofuran). The resulting mixture is stirred at room temperature overnight. The product was isolated by extraction with dichloromethane and purified by alumina gel preparative TLC (eluent ethyl acetate/hexane, 1585 v/v) to give p-methoxyphenyl N,N-dipropylthiocarbamate(compound 4). p-Methoxyphenyl N,N-dipropylthiocarbamate (compound 4): 'H NMR (CDClJ 6 7.45 (d, J = 7.8 Hz, 2 H, HA), 6.96 (d,'U = 7.8 Hz,2 H, HB), 3.86 (s, 3 H, OCHd, 3.36 (m, 4 H, NCH,-), 1.80-1.60 (m, 4 H, -CH,-), 1.304.90 (m, 6 H, CH,); IR 1467,1408, ( 6 ) 240 (8400). 1250 cm-'; UV (CH,CN) ,A, m -Nitrophenyl N,N-dipropylthiocarbamate (compound 5): 'H NMR (CDCl,) 6 8.37 (s, 1 H, HA), 8.22 (bd, J = 7.7 Hz, 1 H, HB), 7.82 (bd, J = 7.7 Hz, 1 H, HD), 7.55 (t, J = 7.7 Hz, 1 H, HJ, 3.23 (t, J = 7.4 Hz, 4 H, NCH,-), 1.80-1.55 (m, 4 H, NCH,CH,-), 1.204.80 (m, 6 H, CH,); IR 3057,2966,1658,1607, 1532,1468,1405,1356,1272cm-'; W (CH,CN) A- (e) 250 (8640). Kinetics of Thiocarbamylation. The profile of products produced by the chemical reaction of eptam S-oxide or eptam sulfone with substituted thiophenols was investigated by HPLC analysis of the reaction mixture. Thiophenols were chosen because of the ease of assay of 4 and 5 and because, a t pH 8-9, greater than 99% of both thiophenols should be ionized and this should simplify the kinetic analysis. The reaction products were separated and quantitated on a Rainin instrument using a Zorbax C-8 column. The column was developed a t a flow rate of 1.5 mL/min by using an isocratic system of two solvents (A and B) set a t 50% A and 50% B where A is water/acetonitrile (66:33) and B is acetonitrile. The HPLC eluent was monitored at 212 nm for eptam S-oxide and eptam sulfone and 240 nm for the thiocarbamate products. Authentic standards coeluted with retention volumes of 4.5 mL (eptam S-oxide), 5.2 mL @-methoxythiophenol), 5.5 mL (m-nitrothiophenol), 7.9 mL (eptam sulfone), 9.3 mL (m-nitrophenyl N,N-dipropylthiocarbonate),9.8 mL @methoxyphenyl NJV-dipropylthiocarbamate),10.2 mL (m-nitrothiophenol disulfide), and 12.9 mL @-methoxythiophenyl disulfide). The thiocarbamate formed upon reaction of eptam S-oxide or eptam sulfone with a substituted thiophenol was also
394
Chem. Res. Toxicol., Vol. 2, No. 6, 1989
synthesized independently and thoroughly characterized by spectroscopic means (see above). Rate measurements were made at 30 "C in buffer/methanol mixtures by using a thermostated water bath. The buffer used was 50 mM phosphate buffer maintained at 0.4 M with KC1. Rate constants (kdwere measured under pseudc-fmborder conditions by monitoring the formation of thiocarbamate (i.e., compound 4 or 5) by HPLC as described above. Second-order rate constants were obtained from the slopes of plots of observed pseudofirst-order rate constants against the concentration of the thiophenol. The y intercepts of such plots were zero. Liver Preparations. Microsome fractions were isolated by the method described earlier (17)from homogenates of liver tissue from eight immature (1-2 year old) striped bass (2-4 lb). Freshwater striped bass were provided by the California Department of Fish and Game Central Valleys Hatchery in Elk Grove, CA, and raised in freshwater. Salt water adapted striped bass were age matched and transferred to salt water and raised in salt water for a t least 4 weeks. The microsomes were washed with KCl and resuspended in 0.05 M potassium phosphate, pH 7.4. T o minimize inactivation of microsomal monooxygenases, all steps were carried out as quickly as possible at 4 "C. Metabolic Incubations and Product Analyses. When striped bass liver microsomes were employed, the incubation medium contained 50 mM potassium phosphate buffer, pH 7.4, 0.5 mM NADP', 2.0 mM glucose 6-phosphate, 1 IU of glucose6-phosphate dehydrogenase (from Sigma), and 1.1mg of microsomes. After a brief temperature equilibration a t 33 "C, the incubation was initiated by the addition of eptam (200 pM) and the incubation was continued with constant shaking to maintain adequate oxygen. At various time intervals the reaction was quenched and analyzed for products by the procedures given below. The incubation medium employing soybean lipoxygenase contained 50 mM sodium borate buffer, pH 9.0,0.2 mM freshly purified arachidonic acid, and 0.01 mg (1265 IU) of soybean lipoxygenase type 1 (Sigma). After a brief temperature equilibration a t 33 "C, the incubation was initiated by the addition of eptam (200 pM) and the incubation was continued with constant shaking to maintain adequate oxygen. At various time intervals the reaction was quenched with an equal volume of methanol and reduced with trimethyl phosphite (30 mg). The profile of eptam metabolites was determined by HPLC analysis of CH2C12extracts of the reaction mixture. The metabolic products in the resulting extract were separated and quantitated on an IBM Model 9300 HPLC employing UV detection a t 220 nm fitted with a precolumn and a 5-km CRI C18 ODS chromosphere (7.7 mm X 25 cm) analytical reverse-phase column. The mobile phase consisted of an isocratic system of two solvents (A and B) set a t 70% A and 30% B a t a flow rate of 1.5 mL/min where A is water/acetonitrile ( 2 1 v/v) and B is acetonitrile. This system efficiently separates eptam sulfoxide, eptam sulfone, and eptam, which have retentions volumes of 4.0, 8.7, and 14.8 mL, respectively. The recovery of material as judged by HPLC was >95%, and 98% of this material was eptam, eptam sulfoxide, or eptam sulfone. The profile of fatty acid oxidation products was also determined by HPLC analysis. At the termination of the incubation, lipid was extracted following the general procedure of Bleigh and Dyer (18). The incubation mixture was extracted twice with 1 mL of methanol/dichloromethane (1:3 v/v) after the addition of prostaglandin B2 (PGB2) (80 ng) (to serve as an internal standard) and trimethyl phosphite (50 mg) (to serve as a reducing agent). The organic extracts were evaporated to dryness and analyzed immediately or taken up in 0.2 mL of dry benzene and stored under an atmosphere of argon at -70 "C until analyzed. Four 50-wL aliquots of each sample were applied to the loading zone of a channeled Whatman LK5DF preadsorbent silica gel TLC plate (5 X 20 cm,250-pm thickness) and dried in a ventilated hood for 5 min. In a parallel lane, 15-HETE and PGB, were applied to the TLC plate. The plate was developed in ethyl acetate/ hexane/acetic acid/methanol (49500.1:0.9 v/v). The bands (I$) corresponding to 15-HETE and PGB, were visualized by UV-vis light and scraped into separate test tubes. The materials were extracted from the silica with two 0.6-mL portions of methanol-dichloromethane (1:5 v/v) and evaporated to dryness. The
Cashman et al. recovery of materials from the TLC plate was 285% of the applied material as determined by parallel control extractions employing known quantities of 15-HETE and PGB2. For each sample, PGB2 and HETE content was determined as described previously (19). Extracts were reconstituted in chromatography eluent solvent and analyzed on RP-HPLC on a Rainin instrument using a Zorbax C-8 column (Rainin). The column was developed a t a flow rate of 1.0 mL/min by using an isocratic system of two solvents (A and B) set a t 75% A and 25% B, where A is methanol/water/ acetic acid (50:500.01) and B is methanol/water/acetic acid (90:100.01). The HPLC eluent was monitored at 269 nm for PGB2 and 235 nm for mono-HETEs. Authentic standards coeluted with retention volumes of 4.7 mL (PGB,), 9.7 mL (15-HETE), and 12.2 mL (5-HETE). The major HETE products formed in reactions catalyzed by striped bass microsomes were subjected to mass spectral analysis. The material isolated from HPLC runs (described above) was extracted into CH2C12. The combined extracts were dried over sodium sulfate and then taken to dryness. Gas chromatography-mass spectra were taken on a Kratos MS 25 operating at 8 kV fitted with a Varian 3700 gas chromatograph and DB-1 capillary column. The GC-MS of synthetic 15-HETE methyl ester BSTFA ether gave prominent ions at m/z (relative abundance) 407 (MH', 3.1), 406 (M+, 10.0), 335 (M+ - C5Hll,3.9), 316 (M' - OHTMS, 16.8), and 173 (M' - C15H21O2, loo), and this spectrum was similar to that of the major product (see below). The major eptam metabolite formed in reactions catalyzed by soybean lipoxygenase was subjected to mass spectral analysis. The material isolated from HPLC runs was extracted with CH2C12. The combined extracts were dried over sodium sulfate and taken to dryness. Gas chromatography-mass spectrometry (GC-MS) of the eptam metabolites was taken on a modified Kratos MS 25 operating a t 8 kV fitted with a Varian 3700 gas chromatograph and DB-1 capillary column (30 m X 0.25 pm i.d.) with helium carrier gas programmed a t 70-230 "C for 6 "C/min. In addition, samples were subjected to electron impact mass spectrometry (EIMS). In all cases, gas chromatography retention times and fragmentation patterns of authentic eptam sulfoxide and eptam sulfone were identical with those of the metabolites isolated. The GC-MS of synthetic eptam S-oxide gave prominent ions a t m/z (percent relative abundance) 177 (MH' - CzH5,6.1), 128 (M - C2H5S0, 23.6), 86 (C4H8NO+,14.4), and 43 (NHCO+, 100). The EIMS of eptam S-oxide gave prominent ions a t m / z 128 (M - C2H5S0, 72.8), 86 (C4H8NO+,62.5), and 43 (NHCO', 100). The GC-MS of synthetic eptam sulfone gave prominent ions a t m / z 128 (M - C,H5S0,, 83.8), 86 (C4H8NO+,73.3), and 43 (NHCO', 100). The EIMS of authentic sulfone gave prominent peaks a t m / z 206 (MH+ - 0, 6.1), 128 (M - C2H5S02, 18.1), 86 (C4HBNO+,71.0), and 43 (NHCO+, 100). Fish Liver Microsomal Metabolism of Testosterone. The metabolism of [14C]testosteronewas determined by HPLC in a system capable of separating 12 of the known metabolites of testosterone (20). [14C]Testosterone (0.4 pCi, 500 pM) was incubated with hepatic microsomes (1.0 mg of protein) in the presence of 0.5 mM NADPH, 0.5 mM glucose &phosphate, 1 IU of glucose-6-phosphate dehydrogenase, and 50 mM phosphate buffer (pH 7.4) at 33 "C for 10 min. The reaction was terminated by the addition of 4 volumes of cold ethyl acetate and fixed amounts of nonradiolabeled hydroxylated testosterone metabolites (provided by Professor D. N. Kirk, Steroid Reference Collection, Queen Mary College, London). The organic extracts were dried and dmolved in 2% acetonitrile/methanol and analyzed by HPLC on a Rainin Microsorb C-18 reverse-phase column (5 pm, 150 X 4.6 mm i.d.). The column was developed a t a flow rate of 1.0 mL/min by using a gradient of two solvents (A and B) set at 80% A and 20% B, where A is methanol/acetonitrile (43:l.l) and B is methanol/acetonitrile (75:l.g). The gradient was developed a t 3 min by a progressive increase in solvent B to 37.5,60, and 100% for 15, 8, and 1.5 min, respectively, before returning to equilibration conditions. Under these conditions 7a-, Sp-, IS@-, and 2a-hydroxtestosterone gave retention volumes of 11.5, 12.7, 18.4, and 22.3 mL, respectively. The metabolites were individually collected and quantitated by scintillation counting. Other Analytical Methods. Heat inactivation of striped bass liver microsomes was accomplished by purging the protein suspended in buffer, pH 7.4, with argon and placing the microsomal
Eptam S-Oxygenation Table I. Second-Order Rate Constants for Reaction of Eptam, Eptam Sulfoxide, and Eptam Sulfone with Thiophenols' thiol k, h-' eptam derivative condition P-CH~O-C~H~SH 0.07 eptam pH 8.4 pH 8.4 m-N0&6H,SH 0.06 eptam P-CH~O-C~HISH pH 8.4 33.0 eptam S-oxide pH 9.0 p-CHsO-C6H,SH 106.6 eptam S-oxide pH 8.4 m-NOZ-CBH,SH 4.8 eptam S-oxide p-CH3O-C6H,SH 44.0 pH 8.4 eptam sulfone pH 9.0 P-CH~O-C~H~SH 154.0 eptam sulfone m-NO&H,SH 5.6 pH 8.4 eptam sulfone
'Reactions were performed as described under Experimental Section. Rate constants are the average of two to three determinations. protein in a bath of water (55 "C) for 60 s in the absence of NADPH. This procedure has been shown to completely destroy hog liver microsomal flavin-containing monooxygenase while preserving 85100% of cytochrome P-450mediated Ndealkylation (21, 22). Employing sulfide-containing substrates for the flavin-containing monooxygenase, we have validated that this procedure also abolishes flavin-containing activity in striped bass liver microsomes (data not shown). The concentration of protein was determined by the method of Bradford et al. (23).
Results Reaction of Eptam with m -ChloroperbenzoicAcid. In dichloromethane, the reaction of eptam with 1.1equiv of m-chloroperbenzoic acid gave the corresponding sulfoxide in reasonable isolated yield. The sulfoxide was the only detectable product although it was not indefinitely stable and it too oxidized to the sulfone or decomposed to other products (Scheme I). No attempt was made to determine the identity of the minor decomposition products. Eptam S-oxide was completely characterized by spectral means. The reaction of eptam with 2 equiv of m-chloroperbenzoic acid gave eptam sulfone in good yield. Eptam sulfone was completely characterized by spectral means (see Experimental Section). Chemical Reaction of Eptam Sulfoxide and Eptam Sulfone with Thiophenols. The effect of thiophenols on the decomposition of eptam, eptam S-oxide, and eptam sulfone at basic pH and in dichloromethane was determined by product analysis. The reaction of thiophenols with eptam is an extremely slow process (Table I). The reactions of eptam S-oxide with p-methoxythiophenol and m-nitrothiophenol were found to cleanly produce the corresponding thiocarbamates (Scheme 11). Thus, in dichloromethaneor phosphate buffer (pH 7.4) the only major detectable product formed between p-methoxythiophenol and m-nitrothiophenol and eptam S-oxide was 4 and 5, respectively. When the reaction was performed in phosphate buffer (pH 8.4), the reaction was much faster and the yield of thiocarbamate formed was much greater (Table I). In dichloromethane, the reaction of m-nitrothiophenol with eptam S-oxide produced thiocarbamate 5. This reaction is surprisingly clean, although the yield is low (i.e., only the starting material and the product thiocarbamate were detected at the termination of the reaction). The reaction of eptam sulfone with excess p-methoxythiophenol or m-nitrothiophenol was performed in phosphate buffer (pH 8.4). The corresponding thiocarbamates 4 and 5 were cleanly formed. The product thiocarbamates were fully characterized by spectral means. Control experiments demonstrated that little (i.e., less than 5%) S-oxide or sulfone decomposed during the reaction conditions employed (i.e., phosphate buffer or CH2C12).The
Chem. Res. Toxicol., Vol. 2, No. 6, 1989 395 Table 11. S-Oxygenation of Eptam in Liver Microsomes from Freshwater and Salt Water AdaDted Striped Bass microsomes, nmol of product/ (minsmg of protein) freshwater salt water incubation condition S-oxide sulfone S-oxide sulfone 6.0 f 0.5 NDb 4.5 f 0.6 0.1 f 0.1 complete system' ND 3.5 f 0.2 0.2 f 0.1 -NADPH ND +heat inactivation 0.04' ND 1.8 f 0.4 0.1 f 0.1 5.8 f 0.4 0.2' +n-octylamine (5 mM) 2.7 f 0.2 ND +thiourea (500 pM) 3.8 f 0.7 ND 3.4 f 0.1 0.3 f 0.1 3.4 f 0.8 0.5 f 0.2 6.5d ND +aminobenzotriazole (500 rM) The complete system contained 200 pM eptam, 0.5 mM NADP, 0.5 mM glucose 6-phosphate, 1 IU of glucose-6-phosphate dehydrogenase, 1.2 mg of microsomal protein, and 50 mM phosphate buffer, pH 7.8. Results are the average of four incubations f SD, as determined by HPLC. *Not detectable. 'The range of values was 0-0.06 nmol/(min-mg of protein). dThe range of values was 0-0.02 nmol/(min-mg of protein).
second-order rate constant values for the reaction of eptam, eptam S-oxide, or sulfone with the thiophenols are listed in Table I. The data of Table I suggest that eptam sulfone is more reactive toward thiols than is the sulfoxide and that the reaction is pH-dependent (10). In Vitro Metabolism of Eptam. The (bio)transformation of eptam was studied in vitro in order to characterize the oxidative metabolites in terms of the metabolites formed and the enzymes or oxidants involved. Fresh- and saltwater striped bass hepatic microsomes were chosen because fish are much more sensitive to the hepatotoxic effects of thiocarbamate herbicides (24) than mammals and previous studies have demonstrated that striped bass contain significant amounts of microsomal monooxygenase enzyme activity (11). The biotransformation of eptam was studied in vitro. When striped bass liver microsomes supplemented with an NADPH-generating system were used, the major initial metabolite of eptam observed was eptam S-oxide. Preliminary studies demonstrated that eptam S-oxide formation was linearly dependent upon incubation time for at least 10 min and on protein concentration (1-4.5 mg/ mL). Under all incubation conditions no other metabolites could be detected. A small but significant amount of eptam sulfone could be detected during the in vitro metabolism of eptam, but this was always associated with hepatic microsomes from salt water equilibrated striped bass. Data from Table I1 show that the S-oxygenation of eptam in hepatic microsomes from freshwater striped bass is dependent upon NADPH and active microsomal protein. Microsomes treated with n-octylamine catalyze the formation of eptam S-oxide, but in the presence of this primary amine S-oxygenation is significantly inhibited. This result may be a consequence of the inhibition of cytochrome P-450 since n-octylamine is a known inhibitor of this enzyme (25). The inhibition of S-oxygenation by n-octylamine may also be a consequence of its effect as an alternative substrate competitive inhibitor since in some tissues from some species n-octylamine has been shown to be a substrate (26, 27) instead of an activator (21) of the flavin-containing monooxygenase. To investigate this further, the effects of inhibitors of the flavin-containing monooxygenase or cytochrome P-450 (listed in Table 11) were examined. Aminobenzotriazole, a potent mechanism-based inhibitor of cytochrome P-450 (28), did not inhibit S-oxygenation. Conversely, thiourea, a well-documented alternate substrate for the hepatic flavin-containing monooxygenase (21), significantly inhibited the eptam S-oxygenation. Most likely, the action of n-octyl-
396 Chem. Res. Toxicol., Vol. 2, No. 6, 1989
amine is to inhibit the contribution of cytochrome P-450 to eptam S-oxygenation, while concurrently stimulating flavin-containing monooxygenase activity, albeit only slightly. In the presence of heat-inactivated microsomes under conditions where W90%of mammalian cytochrome P-450 activity is retained but almost all mammalian flavin-containing monooxygenase activity is abolished (21,22), almost no S-oxygenation of eptam is observed (Table 11). These results suggest that monooxygenase activity similar to the mammalian flavin-containing monooxygenase may be responsible, at least in part, for the S-oxygenation of eptam in liver microsomes from freshwater striped bass. In contrast to the results for freshwater microsomes, the results obtained from microsomes from salt water adapted striped bass do not show a typical pattern of cytochrome P-450 or flavin-containing monooxygenase involvement in the S-oxygenation of eptam. Thus, in the presence of hepatic microsomes from salt water equilibrated striped bass, little dependence upon incubation conditions and rate of eptam S-oxygenation is observed. In the absence of NADPH or in the presence of heat-inactivated microsomes significant amounts of eptam S-oxide are observed. Addition of various specific inhibitors for the monooxygenase systems had practically no effect on eptam S-oxide formation. In marked contrast to the results obtained for eptam metabolism in the presence of hepatic microsomes from freshwater striped bass (Table 11),the metabolism results using saltwater striped bass liver microsomes suggest that eptam S-oxygenation is independent of microsomal monooxygenase activity (Table 11). Lipid Peroxidation in the Microsomal Oxidation of Eptam. Because eptam is readily oxidized by excess peroxides and peracids (10, 13), experiments were conducted to determine a role of autoxidation in the microsomal oxidation of eptam. If lipid hydroperoxides were involved in the nonenzymic S-oxygenation of eptam, then the reduction products of lipid hydroperoxides (Le., hydroxyeicosatetraenoic acids, HETEs) should be detectable and there should be a correlation between levels of HETEs observed and rates of eptam S-oxygenation. The S-oxygenation of eptam was studied in vitro with liver microsomes from freshwater and salt water adapted striped bass, along with soybean lipoxygenase supplemented with arachidonic acid and authentic synthetic alkyl hydroperoxides as a standard for comparison. As shown in Table 111, soybean lipoxygenase efficiently converts arachidonic acid to 15-HETE as determined by the procedure described under Experimental Section (29). In the presence of soybean lipoxygenase and eptam, the extent of 15-HETE formation is reduced but a large amount of eptam S-oxide and eptam sulfone is produced (Table 111). The products detected in dichloromethane extracts of reactions catalyzed by soybean lipoxygenase eluted on HPLC with retention volume identical with that of authentic l&HETE, eptam S-oxide, and eptam sulfone. The mass spectrum of each material gave mass spectra virtually identical with that of synthetic standards. These results suggest that, in the presence of soybean lipoxygenase, arachidonate-derived hydroperoxides or reactive peroxyl radicals convert eptam to eptam S-oxide and eptam sulfone. A further experiment was conducted to investigate the apparent cooxidation of eptam that was observed. Eptam (50 pM) was incubated for 10 min in the presence of 15hydroperoxyeicosatetraenoic acid (15-HPETE, 50 pM), hematin (10 pM), and Tween 20 (30 pM) in a phosphate buffer (pH 7.4) for 10 min. The reaction mixture was
Cashman et al. Table 111. Lipid Peroxidation Products of Eptam products, ng/(min.mg of protein) eptam eptam incubation condition 15-HETEb S-oxide sulfone SBLO' 12440 SBLO + eptamd 1920 lolle 630e saltwater microsomes 20.3 +eptam 14.8 450 115 +eptam + NADPH GS 15.8 312 90 freshwater microsomes 3.27 +eptam 1.95 3.7 ND +eptam + NADPH GS 0.82 357 ND "The incubation conditions consisted of 1.1 mg of microsomal protein, eptam (200 pM), and 0.5 mM NADPH-generating system (GS), in phosphate buffer (pH 7.8), for 10 min at 33 "C. Each value is the average of three determinations. bHETE = hydroxyeicosatetraenoic acid; minor amounts of other HETEs and diHETEs were also observed but were not quantitated. 'Soybean lipoxygenase (0.01 mg) was incubated with 200 pM arachidonic acid in borate buffer (pH 9.0) for 15 min as described under Experimental Section. Same conditions as in footnote c, but 200 pM eptam was present. The significant degree of lipoxygenase activity inhibition by sulfur-containing compounds has been noted previously (41). eNanograms of product per minute.
stopped by the addition of dichloromethane. Analysis of the dichloromethane extract with HPLC (as described under Experimental Section) revealed that this system was competent to produce 7.7 f 1.4 nmol of eptam S-oxide and traces of sulfone ( n = 3). In contrast, the reaction of eptam with a slight excess of tert-butyl hydroperoxide or 15hydroperoxyeicosatetraenoicacid in dichloromethane in the absence of iron produced eptam S-oxide but only at a very slow rate (data not shown). In a parallel experiment, the metabolism of eptam and the presence of HETEs were examined concurrently in liver microsomes from freshwater and salt water adapted striped bass. As shown in Table 111,15-HETEwas present in liver microsomes from salt water adapted striped bass in amounts 6- to 20-fold greater than those from freshwater-adapted striped bass. The HETE products detected by HPLC from incubations of eptam in the presence of saltwater microsomes were collected, extracted, and subjected to GC-MS. Of three materials that coeluted on HPLC with retention times similar to 5-, 12-, and 15-HETE, only sufficient quantities of 15-HETE were available for mass spectrometric analysis. The major HETE product isolated from HPLC runs was treated with diazomethane and BSTFA, and the GC-MS spectra were obtained. The GC-MS of this material gave prominent ions at m/z (relative abundance) 407 (MH+,0.9), 406 (M+,2.9), 335 (M+- C5Hll, 1.5), 316 (M+ - OHTMS, 5.4), and 173 (M+ - C15H2102,100.0). The GC-MS and HPLC behavior of this material is virtually identical with that of the TMS ether of authentic 15-HETE methyl ester. In the presence of salt water adapted striped bass liver microsomes, large amounts of eptam S-oxide and eptam sulfone were formed and formation of these materials was largely independent of the presence of NADPH. In the presence of hepatic microsomes from freshwater-adapted striped bass, no detectable amount of eptam sulfone was observed and formation of eptam sulfoxide was almost completely dependent upon NADPH. The results of eptam metabolism in microsomes from freshwater striped bass suggest that eptam Soxygenation is almost entirely due to an NADPH-dependent monooxygenase reaction. In contrast, formation of eptam S-oxide and eptam sulfone in the presence of striped bass microsomes from salt water adapted fish is due in large part to lipid peroxide mediated S-oxygenation.
Eptam S-Oxygenation Table IV. Striped Bass Microsomal Testosterone Hydroxylase Activity testosterone metabolite: pmol/(min.mg of protein) DreDaration 2a 619 7a 16B 5.7 305 11.9 2.6 freshwater microsomes 4.1 129 2.9 1.3 saltwater microsomes 'Incubations were performed in the presence of 500 pM [14C]testosterone, NADPH, 1.0 mg of microsomal protein, and 50 mM phosphate buffer, pH 7.4. Results are the average of three determinations, and quantitation was performed by the HPLC method as outlined under Experimental Section.
Testosterone Metabolism in Striped Bass Hepatic Microsomes. In mammalian systems, steriods may be hydroxylated in the liver and excreted. Hydroxylation may also result in the formation of sterols that have specific physiological functions not presently recognized (20,30). The identification of specific steroid metabolites that are diagnostic of particular cytochrome P-450 isozymes in mammalian tissue may be used to assess the contribution of cytochrome P-450 isozymes to xenobiotic metabolism in the liver of striped bass (20, 30). [14C]Testosterone hydroxylase activity in microsomes from striped bass was investigated in order to determine the identity of the isozymes and the level of cytochrome P-450 activity present. Liver microsomes prepared from freshwater and salt water adapted striped bass were incubated with [ 14C]testosterone, and the monohydroxytestosterones produced were quantitated with HPLC (20). Both freshwater and salt water adapted striped bass microsomes possess significant 60-hydroxylase activity. In addition, minor but detectable levels of 2a-, 160-, and 7ahydroxylase activity were observed (Table IV).
Discussion The reaction of eptam with m-chloroperbenzoic acid was investigated in order to identify the products and characterize the chemical reactivity of the products formed (IO). The oxidant m-chloroperbenzoic acid was chosen because previous studies have demonstrated that mchloroperbenzoic acid mimics the microsomal monooxygenase system (31). In good agreement with previous studies we determined that the major product of eptam oxidation with m-chloroperbenzoic acid was eptam S-oxide (IO). Eptam S-oxide is not indefinitely stable and is slowly converted to other products presumably due to acylation of water and/or other reactions. The conversion of eptam to eptam sulfone is a facile process. Product studies showed that eptam sulfone was readily isolated in good yield. That eptam S-oxideand eptam sulfone are powerful acylating agents is borne out by kinetic studies. In the presence of thiophenols, eptam S-oxide or eptam sulfone is cleanly transformed to their corresponding thiocarbamates (Scheme 11). The relative rates of thiocarbamate formation confirm the greater electrophilic nature of sulfone 3 compared to sulfoxide 2. In strong contrast, trans thiocarbamylation of eptam by either pmethoxybhiophenol or m-nitrothiophenolis extremely slow (Table I). S-Oxygenation of eptam markedly increases the rate of nucleophilic attack by thiophenols on S-oxygenated derivatives of eptam. The rates of nonenzymatic thiol attack on electrophilic eptam S-oxide or eptam sulfone suggest that similar thiocarbamylation reactions might be expected to occur under in vivo conditions. The pH dependence of the reaction of thiophenols with S-oxygenated derivatives of eptam suggests that at high pH other mechanisms are contributing to formation of trans thiocarbamylation products. Under physiological conditions
Chem. Res. Toxicol., Vol. 2, No. 6, 1989 397 0
II
Et2NCSCH2CHCOOH I
~HCCH,
II
0
Figure 1. Mercapturate 6 arising from carbamylation of a thiocarbamate herbicide (Bolero; see ref 10).
significant amounts of mercapturates (Figure 1)presumably arising from S-oxide metabolites of thiocarbamate herbicides have been observed in vivo in striped bass (ll), which tends to confirm the electrophilic nature of thiocarbamate metabolites in vivo. Presumably, this process constitutes a detoxification step (IO). Although eptam sulfone and, to a lesser extent, eptam sulfoxide are chemically reactive toward nucleophiles (i.e., glutathione and other thiols), they are nevertheless sufficiently stable to be isolated and quantitated. Below, we discuss our results concerning eptam S-oxidation in hepatic microsomal homogenates of striped bass and provide some evidence for the molecular basis for the formation of these S-oxidized metabolites. There have been numerous reports that the toxicity of thiocarbamate herbicides results at least in part from products of their oxidative biotransformation rather than from the parent compounds ( g I 3 ) . Efficient oxidation of eptam to eptam S-oxide is well documented to occur both in vivo (9, I O ) and in vitro (IO). However, the molecular basis for the S-oxidation of eptam has not been completely established. In the case of freshwater-adapted striped bass liver microsomes, S-oxidation of eptam may be catalyzed largely by an enzymatic activity that is analogous to the mammalian flavin-containing monooxygenase. The data do not support a major role of cytochrome P-450 in the S-oxygenation of eptam, but neither do they conclusively rule out such a role. The apparent paradox that n-octylamine, a known inhibitor of cytochrome P-450 (25) and a positive effector for some but not all mammalian flavin-containing monooxygenase activity, inhibits eptam S-oxygenation can be rationalized by suggesting that n-octylamine serves as a substrate for striped bass liver flavin-containing monooxygenase activity, as has been reported for the rabbit lung enzyme (26,27),or that it stimulates flavin-containing monooxygenase while simultaneously inhibiting cytochrome P-450 mediated eptam S-oxygenation. That thiourea, a good alternate substrate competitive inhibitor for flavin-containing monooxygenase activity (21),actually inhibits eptam S-oxygenation and aminobenzotriazole, a potent mechanism-based inactivator of cytochrome P-450 (28),does not have any inhibitory effect is consistent with a role of flavin-containing monooxygenase-like activity in the S-oxygenation of eptam. The regioselectivity and stereoselectivity of testosterone hydroxylation catalyzed by mammalian hepatic NADPHdependent cytochrome P-450 have been successfully used to examine the levels of cytochrome P-450 isozymes in various tissues (20,30). Results of testosterone hydroxylation in striped bass liver microsomes demonstrate that a number of cytochrome P-450 isozymes are present. As shown in Table IV, significant amounts of 6P-hydroxytestosterone are formed, and this is consistent with the presence of cytochrome P-450 P (or 2a) in liver microsomes from freshwater and salt water adapted striped bass. Likewise, the formation of 2c~-hydroxytestosterone,7ahydroxytestosterone, and 16/3-hydroxytestosterone is consistent with the presence of cytochrome P-450 H (or 2c), cytochrome P-450 A (or 3), and cytochrome P-450 B (or 4) activity, respectively. The presence of cytochrome
398 Chem. Res. Toxicol., Vol. 2, No. 6, 1989
P-450 activity in striped bass liver microsomes reported here is in agreement with reports of oxidative metabolism reported elsewhere for striped bass (11)and for the hydroxylation of steroids in liver microsomes from other fish (32-34). Although the apparent activities of cytochrome P-450 isozymes of freshwater and salt water adapted striped bass are comparable, further studies are required to distinguish this point. A major contribution of hepatic striped bass cytochrome P-450 to the oxidative biotransformation of eptam cannot be established, but neither can it be completely ruled out. It should be pointed out that the levels of cytochrome P-450 activity are considerably lower than those generally reported for mammalian hepatic cytochrome P-450 (20,30)but are in reasonable agreement with cytochrome P-450 activity reported for fish (32-34). The large amount of eptam S-oxide products that are observed during the biotransformation of eptam with liver microsomes from freshwater striped bass is consistent with the involvement of a second monooxygenase system, possibly analogous to the mammalian flavin-containing monooxygenase. The observation that eptam is S-oxidized to reactive S-oxide and sulfone metabolites in vitro is consistent with the postulate that S-oxidative bioactivation is required for the expression of toxicity of eptam in fish. The oxidation of eptam and other herbicides to reactive metabolites having sufficient reactivity to undergo covalent binding to cellular nucleophiles has been demonstrated by our chemical studies of the reaction of thiol nucleophiles with eptam S-oxide. That mercapturate 6 has been isolated by others (11)from striped bass administered thiocarbamate herbicides suggests that the process of glutathione conjugation may constitute a detoxification step in vivo (Figure 1). In addition, mercapturate formation may also suggest that glutathione depletion may be participating in the overall manifestation of hepatic toxicity of thiocarbamates in fish. The cooxidation of sulfur-containing compounds has not received as much attention as that of other organic chemicals and drugs (35). However, it has been observed that arachidonate-derived peroxides (36) as well as other systems (37)convert sulfides to sulfoxides. Our observation that lipid hydroperoxides catalyze the oxidation of sulfur-containing environmentally persistent chemicals such as thiocarbamates to thiocarbamate S-oxides may have important consequences, especially for tissues from species such as fish which are highly enriched in polyunsaturated fatty acids. The mechanism of eptam S-oxide and eptam sulfone formation in the presence of hepatic microsomes from salt water adapted striped bass probably involves reduction of oxidation products of fatty acids or radicalderived materials by eptam to form eptam oxidation products (38-40). In the presence of excess fatty acid hydroperoxides a second mechanism involving nucleophilic attack of thiocarbamate sulfur on electrophilic peroxide may also contribute to eptam S-oxide formation. However, in the absence of iron, reactions of eptam with alkyl hydroperoxides are slow and probably only make a minor contribution to S-oxidation. Oxidation studies of eptam conducted in the presence of hematin and equimolar amounts of fatty acid hydroperoxides demonstrated good conversion of eptam to eptam S-oxide. If direct bimolecular interaction of eptam with fatty acid hydroperoxides was the sole mechanism of eptam S-oxide formation, then the relatively slow stoichiometric peroxide reaction would have yielded much less eptam S-oxide. A more likely mechanism for eptam S-oxide and especially eptam sulfone formation probably involves peroxyl radicals.
Cashman et al.
For S-oxygenation of S-oxides, generally stronger oxidizing agents are required than for S-oxygenation of the parent sulfide. In the present study, under conditions where eptam sulfone metabolites have been detected, the involvement of stronger oxidizing agents like peroxyl radicals is likely. Thus, in the presence of soybean lipoxygenase, hematin/ hydroperoxide, and the NADPHgenerating system in the presence of saltwater striped bass hepatic microsomes, conditions that generate lipid peroxyl radicals, eptam is converted to eptam S-oxide and eptam sulfone. In contrast, under conditions of much milder oxidizing agents (e.g., m-chloroperbenzoic acid) eptam sulfoxide is formed almost exclusively. At present, we are unable to explain the apparent uncoupled peroxide tone that we have observed in hepatic microsomes from saltwater striped bass. High levels of fatty acid hydroperoxides are not present in hepatic microsomes from freshwater striped bass. The high levels of fatty acid hydroperoxides present are not due to glutathione levels since we have determined that large and equivalent amounts of glutathione are present in microsomes from both types of striped bass (C. Lambert, unpublished observation).
Summary In summary, we have demonstrated that eptam is efficiently oxidized in hepatic microsomes from freshwater and salt water adapted striped bass. For hepatic microsomes from freshwater-adapted striped bass, eptam Soxygenation is mediated by an NADPH-dependent monooxygenase system. Due to the relatively low levels of cytochrome P-450 activity present in these microsomes we attribute the S-oxygenation to a second monooxygenase system, possibly related to the flavin-containing monooxygenase. In contrast to freshwater striped bass, hepatic microsomes isolated from salt water adapted striped bass produce eptam S-oxide and eptam sulfone in a process that is largely independent of an NADPH-dependent monooxygenase system. We attribute the formation of eptam S-oxide and eptam sulfone to lipid hydroperoxide mediated free-radical-derivedprocesses. In support of this work we have shown that, in the presence of arachidonic acid/soybean lipoxygenase and hematin/ 15-hydroperoxyeicosatetraenoic acid, these agents catalyze the formation of eptam S-oxide and sulfone from eptam. This is analogous to work reported by others where iron-containing systems function with alkyl hydroperoxides to form peroxyl radicals to oxidize drugs or chemicals (35-40). An analogous eptam oxidation system must be at work in the hepatic microsomes from salt water adapted striped bass. That the oxidation of eptam leads to metabolites that have sufficient chemical reactivity to undergo spontaneous covalent binding to cellular nucleophiles has been demonstrated by our model chemical studies with thiophenols. Thus, it appears that the toxicity associated with the thiocarbamate herbicide eptam may be correlated with reactive S-oxide and especially eptam sulfone formation and subsequent covalent binding and cellular impairment. Acknowledgment. We thank Drs. R. Nishioka and C. Brown (University of California, Berkeley) and Dr. Philip S. Magee (University of California, San Francisco) for interesting discussions. The generous help of the UCSF Bioorganic Biomedical Mass Spectrometry Resource (A. L. Burlingame, Director) is gratefully acknowledged (supported by NIH Division of Research Resources Grant RR01614) as is the support of striped bass research by the California Department of Fish and Game, Contract 8026. We thank Dr. C. Lambert for preliminary testosterone hydroxylase studies. We thank Andrea Maze1 for her ex-
E p t a m S-Oxygenation pert typing and Brian Williams for his expert assistance. Registry No. 1, 759-94-4; 2, 51892-56-9; 3, 51892-58-1; 4, 123489-71-4; 5, 123489-72-5; p-MeOCeH4SH, 696-63-9; mN02C6H4SH,3814-18-4; testosterone hydroxylase, 42616-24-0.
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Use Report, 1984. Sacramento, CA, p 109. (2) California Department of Fish and Game (1983) Rice Herbicide Concentrations in Sacramento River and Associated Agricultural Drains, 1982. Report No. 83-5, Sacramento, CA, pp 16-33. (3) Moyle, P. B. (1976) in Inland Fisheries of California, p 405, University of California Press, Berkeley, CA. (4) Stevens, D. E., Kohlhorst, D. W., Miller, L. W., and Kelley, D. W. (1985) The decline of striped bass in the Sacramento-San Joaquin estuary. Trans. Am. Fish. SOC. 114, 12-30. (5) California State Water Resources Control Board (1984) Rice Herbicides: Molinate (Ordam) and Thiobencarb (Bolero), Special Projects Report. No. 94-4 sp, Sacramento, CA, p 176. (6) Brown, C. L., Young, G., Nishioka, R. S., and Bern, H. A. (1987) Preliminary report on the physiological status of striped bass in the Carquinez Strait Die-off. Fish. Res. 6, 5-16. (7) Worthing, C. R., and Walker, S. B. (1987) in The Pesticide Manual, BCPC, Thornton Heath, U.K. (8) Lay, M. M., and Casida, J. E. (1978) Involvement of GSH and GSH-transferases in the action of dichloroacetamide antidotes for thiocarbamate herbicides. In Chemistry and Action of Herbicide Antidotes (Pallos, F. M., and Casida, J. E., Eds.) .. pp 151-160, Academic Press, New York. (9) , , Chen. S. Y.. and Casida. J. E. (1978) . . Thiocarbamate herbicide metabolism: microsomal oxygenase metabolism of EPTC involving mono- and dioxygenation at the sulfur and hydroxygenation at each alkyl carbon. J. Agric. Food Chem. 26, 263-267. (10) Casida, J. E., Kimmel, E. C., Ohkawa, H., and Ohkawa, R. (1975) Sulfoxidation of thiocarbamate herbicides and metabolism of thiocarbamate sulfoxides in living mice and liver enzyme systems. Pestic. Biochem. Physiol. 5, 1-11. (11) Tjeerdema, R. S., and Crosby, D. G. (1987) The biotransformation of molinate (Ordram) in the striped bass, Morone saxatilis. Aquat. Toxicol. 9, 305-317. (12) Lay, M. M., and Menn, J. J. (1979) Mercapturic acid occurrence in fish bile. A terminal product of metabolism of the herbicide molinate. Xenobiotica 9, 669-673. (13) Casida, J. E., Gray, R. A., and Tilles, H. (1974) Thiocarbamate sulfoxides: potent, selective, and biodegradable herbicide. Science 184,573-574. (14) Baldwin, J. E., Davies, D. I., Hughes, L., and Gutteridge, N. J. A. (1979) Synthesis from arachidonic acid of potential prostaglandin precursors. J. Chem. SOC.,Perkin Trans. 1, 115-121. (15) Corey, E. J., Albright, J. O., Barton, A. E., and Hashimoto, S. (1980) Chemical and enzymatic syntheses of 5-HEPE, a key biological precursor of slow-reacting substance of anaphylaxis (SRS) 102, 1435-1437. and 5-HETE. J. Am. Chem. SOC. (16) Brown, H. C., and Rao, B. C. S. (1956) A new powerful reducing agent-sodium borohydride in the presence of aluminum chloride and other polyvalent metal halides. J. Am. Chem. SOC. 78, - 2582-2588. (17) Cashman. J. R.. and Hanzlik. R. P. 11981) Microsomal oxidation of thiobenzamide. A photometric assay for the flavin-containing monooxygenase. Biochem. Biophys. Res. Commun. 98,147-153. (18) Bleigh, E. G., and Dyer, W. J. (1959) A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911-917. (19) Cashman, J. R., Lambert, C., and Sigal, E. (1988) Inhibition of human leukocyte 5-lipoxygenase by 15-HPETE and related eicosanoids. Biochem. Biophys. Res. Commun. 155, 38-44. (20) Waxman, D. J., KO,A., and Walsh, C. (1983) Regioselectivity and stereoselectivity of androgen hydroxylations catalyzed by cytochrome P-450 isozymes purified from phenobarbital-induced rat liver. J. Biol. Chem. 250, 11937-11947. (21) Ziegler, D. M. (1980) Microsomal flavin-containing monooxygenase: oxygenation of nucleophilic nitrogen and sulfur com~~
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