Chem. Res. Toxicol. 1990,3, 433-440
433
S-Oxygenation of Thiobencarb (Bolero) in Hepatic Preparations from Striped Bass (Morone saxafilis) and Mammalian Systems John R. Cashman* and Leslie D. Olsen Department of Pharmaceutical Chemistry and Liver Center, School of Pharmacy, University of California, S u n Francisco, S u n Francisco, California 94143-0446
Richard S. Nishioka, Elisabeth S. Gray, and Howard A. Bern Department of Integrative Biology, Cancer Research Laboratory and Bodega Marine Laboratory, University of California, Berkeley, California 94720 Received February 2, 1990 The in vitro S-oxygenation of thiobencarb (Bolero; p-chlorobenzyl Nfl-diethylthiocarbamate) in the presence of hepatic microsomes from freshwater- and seawater-adapted striped bass was investigated. Thiobencarb S-oxide was the principal metabolite and accounted for 98% of the total thiobencarb metabolized by striped bass liver microsomes. Studies on the biochemical mechanisms for striped bass hepatic S-oxygenation suggest that this reaction is catalyzed largely by the flavin-containing monooxygenase and to a lesser extent by cytochromes P-450. Following the short incubation period used, no thiobencarb sulfone was detected and no evidence was found for a contribution of cooxidation in the S-oxidation of thiobencarb. This conclusion was supported by studies with microsomes and purified mammalian monooxygenases which also metabolized thiobencarb without cooxidizing factors. Highly purified cytochrome P-450IIB-1 S-oxygenated thiobencarb more efficiently than highly purified hog liver flavin-containing monooxygenase. Thiobencarb S-oxide and thiobencarb sulfone were efficient carbamylating agents and reacted with thiol and amine nucleophiles, whereas thiobencarb itself was relatively stable to transthiocarbamylation. Monooxygenase-catalyzed S-oxygenation of thiobencarb by striped bass liver microsomes may represent a bioactivation process which could explain the known toxicity of thiobencarb in fish.
Introduction Thiobencarb (Bolero) (1) is a chemically persistent thiocarbamate herbicide used in the rice fields of the Sacramento Valley region of California and elsewhere for the control of grassy weeds (I). Between 1981 and 1988, peak levels of thiobencarb (approximately 10-60 pg/L) were detected in the waters of the Sacramento River during early June of each year (2). Application of thiobencarb in the months of May and June coincides with the spawning of several important fish including striped bass (Morone saxatilis) in the Sacramento Delta (3). Since about 1975, the striped bass populations in the Sacramento Delta have experienced a sharp decline (4).Accompanying this decline is the heretofore-unexplained striped bass die-off which may be linked to thiocarbamate herbicide application (5). Among the pathological findings on moribund striped bass taken from the Sacramento Delta area was severe liver dysfunction including necrosis (6). Thiocarbamates such as thiobencarb have a low toxicity in mammals (e.g., rat oral LD,, = 1300 mg/kg) (7), but thiobencarb is much more toxic to fish (e.g., rainbow trout, 96-h LCw = 1.2 mg/L) (8). Metabolic S-oxygenation of thiobencarb bioactivates the thiocarbamate moiety to produce potent carbamylating agents; this may explain at least in part the toxicity of thiobencarb in fish (8-13). In mammals, fish, and plants, major metabolites of thiocarbamate herbicides include thiocarbamate S-oxides (Scheme I) (14-19). Thiocarbamate S-oxides do not accumulate to high levels in striped bass exposed to another thiocarbamate herbicide, molinate; instead, molinate S-
* To whom correspondence should be addressed. 0893-228x /90/2703-0433$02.50 / O
Scheme I. S-Oxygenation of Thiobencarb (Bolero) (1) to Thiobencarb S-Oxide (2) and Thiobencarb Sulfone (3)"
t 0u
R,N-C-S-CH,
CI
+
1
0
2
3
R is ethyl in all cases.
oxide undergoes degradation and/or reacts with glutathione as a carbamylating agent, leading to mercapturates (Figure 1, compound 6) (16).Other routes of metabolic biotransformation have been observed for thiocarbamates, but it is thought that S-oxygenation is responsible for increasing the hepatotoxic potential of some thiocarbamate herbicides, because chemical studies have shown that thiocarbamate S-oxides are reactive carbamylating agents (9,10,16,20). In agreement with in vivo observations of thiobencarb biotransformation (10,17,19), thiocarbamates are extensively S-oxygenated in hepatic microsomes of striped bass (20). We have previously shown that the major Soxygenase activities present in striped bass hepatic preparations (i.e., the flavin-containing monooxygenase and cytochromes P-450) have sufficient S-oxygenase activity to metabolize a model thiocarbamate herbicide like eptam to eptam S-oxide (20). 0 1990 American Chemical Society
434 Chem. Res. Toxicol., Vol. 3, No. 5, 1990
Et ,N
0 II
- C - S - CH,-CHCOOH I
NHCCH,
I1
6 Et,N-
0
0 II
C -NH-(CHz)rCH,
7 Figure 1. Chemical structure of mercapturate 6 and urea 7 arising from carbamylation of thiobencarb S-oxide.
The purpose of this study was to investigate thiobencarb S-oxygenation by hepatic microsomes of striped bass; thiobencarb is an herbicide that striped bass encounter in the Sacramento-San Joaquin Delta a t the peak of spawning. In addition, we compared the chemical reactivity of thiobencarb S-oxide with that of thiobencarb as a carbamylating agent toward model thiol nucleophiles to simulate the probable reactions occurring in fish liver. A determination of the molecular basis for thiobencarb metabolism by hepatic microsomes of striped bass could provide important information that may lead to understanding its possible role in the die-off of striped bass observed in the Sacramento Delta region.
Experimental Section Chemicals. Chemicals used in this study were of the highest purity available and were purchased from Aldrich Chemical, Milwaukee WI. Other reagents, buffers, and solvents were from Fisher Scientific. Thiobencarb (Bolero) 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 gift of Professor R. Ketcham of the Department of Pharmaceutical Chemistry, UCSF. [4-'C]Testmterone was purchased from New England Nuclear. Hydroxylated tesosterone standards were obtained from the Steroid Reference Collection, Queen Mary College, London. Chromatography was done on Silica Woelm (70-150 mesh) (Fisher Scientific). Preparative thin-layer chromatography was with 20 X 20 cm Analtech Uniplate (500-rm thickness). Instrument Analysis. 'H NMR spectra were recorded with a General Electric spectrometer operating at 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 a t 6 kV and a source temperature of 60 OC. Liquid secondary ion mass spectrometry (LSIMS) was recorded with a Kratos MS 50 set at 8 kV equipped with a cesium ion gun. UV spectra were recorded with a Perkin-Elmer 559A spectrometer. Infrared spectra were recorded on a Nicolet 5 DX Fourier transform infrared spectrometer. Synthesis of Thiobencarb S-Oxide (2). To a stirred solution of thiobencarb (30 mg, 0.13 mmol) in CHzClz(1 mL) a t 0 "C was added m-chloroperbenzoicacid (80% technical grade, 25 mg, 0.15 mmol) in CH2C12(1 mL). The resulting mixture was stirred for 10 min a t 0 OC and brought to room temperature and stirred for 1h. Analysis by TLC showed that no starting material remained. The crude mixture was chromatographed directly on silica gel preparative TLC (eluent CH2C12/CH30H,98:2) to give thiobencarb S-oxide, 25 mg, 80%, with an Rf value of 0.28 'H NMR (CDC13)6 7.33 (d, J = 7.9 Hz, 2 H, aromatic H), 7.26 (d, J = 7.9 Hz, 2 H, aromatic H), 4.25 (d, J = 12.3 Hz, 1 H, SOCH,), 4.18 (d, J = 12.3 Hz, 1 H, SOCH,), 3.37-3.05 (m, 4 H, NCH,), 1.41 (t, J = 6.6 Hz, 3 H, NCHZCHJ, 0.95 (t, J = 6.6 Hz, 3 H, NCH2CH3);IR (neat) 3051 (s), 2988 (s), 2101 (w), 1778 (w), 1730
Abbreviations: TLC, thin-layer chromatography; HPLC, high-performance liquid chromatography: EIMS, electron impact mass spectrometry: LSIMS,liquid secondary ion mass spectrometry; SDS, sodium dodecyl sulfate: FMO,flavin-containing monooxygenase.
Cashman et al. (m), 1701 (s), 1666 (s), 1595 (m), 1490 (m), 1420 (s), 1279 (s), 1601 (s) cm-'; mass spectrum (EI)m/z (relative intensity) 257 (M+ 0, 4.0), 229 (M+ - C3H8, 2.0), 155 (ClCBH4CHZS+, 19.0), 125 (C1C6H4CH2+,81), 100 (M+ - C1C&&H2SO, loo), 89 (C7H5+,4.0), 72 (C4HloN+,87.0); mass spectrum (LSIMS) m/z (relative intensity) 274 (MH', 20.0); UV (CH3CN) ,A, (c) 222 (5200). Synthesis of Thiobencarb Sulfone (3). Into a stirred solution of thiobencarb (30 mg, 0.13 mmol) in CHZCl2(1 mL) at 0 "C was added m-chloroperbenzoic acid (50 mg, 0.3 mmol) in CHzC12(1.5 mL). Then the resulting mixture was brought to room temperature and stirred for 20 h. Analysis by TLC showed that no starting material was left. The crude mixture was chromatographed directly on silica gel preparative TLC (eluent CH2C12/CH30H,991) to give thiobencarb sulfone, 23 mg, 60%, with an R, value of 0.83: 'H NMR (CDCl,) 6 7.44 (d, J = 8.6 Hz, 2 H, aromatic H), 7.41 (d, J = 8.6 Hz, 2 H, aromatic H), 4.62 (s, 2 H, SO2CHz), 3.75 (9,J = 14.2 Hz, J = 7.3 Hz, 2 H, NCH,), 3.44 (4, J = 14.2 Hz, J = 7.3 Hz, 2 H, NCH,), 1.30 (t, J = 7.3 Hz, 3 H, NCH,CH,), 1.26 (t,J = 7.3 Hz, 3 H, NCH2CH3);IR (neat) 3079 (s), 2495 (m), 1905 (w), 1687 (s), 1595 (m), 1490 (m), 1426 (m), 1314 (m), 1272 (s), 1208 (m), 1110 (m), 842 (w) cm-'; mass spectrum (EI) m / t (relative intensity) 156 (C1C6H4CHS+,32.0), 125 (ClC&CH2+, 50.0), 111 (Clc&+, 71.0), 100 (M+- ClC6H4CH2S0, 72.0); mass spectrum (LSIMS) m/z (relative intensity) 290 (MH', 100.0), 124 (ClCTHE+, 91.0); UV (CHSCN) ,A, (c) 222 (6500). Synthesis of Thiocarbamate Derivatives. The general procedure for the synthesis of thiocarbamates 4 and 5 was that of Tjeerdema and Crosby (16). The required thiophenols were synthesized as previously described (20). Thiobencarb sulfone (17 mg, 0.06 mmol) was placed in tetrahydrofuran/phosphate buffer (pH 8.4) solution (2 mL) with the required thiophenol(O.15 mmol), and the resulting mixture was stirred at room temperature. Analysis by TLC showed that the reaction was completed after 14 h. The reaction mixture was extracted with CH2C12,evaporated to dryness, and purified by silica gel preparative TLC (eluent ethylacetate/hexane, 10:90 v/v) to give the phenyl N,N-diethylthiocarbamate (compounds 4 and 5 ) . m -Nitrophenyl N,N-diethylthiocarbamate (compound 4): lH NMR (CDCL,) d 8.39 (s, 1 H, aromatic H), 8.24 (bd, J = 7.4 Hz, 1H, aromatic H), 7.84 (bd, J = 7.4 Hz, 1 H, aromatic H), 7.57 (bt, J = 8.0 Hz, 1 H, aromatic H), 3.44 (m, 4 H, NCH,), 1.61-1.30 (m, 6 H, 2 CH,); IR (neat) 3058 (s), 2980 (w), 2938 (w), 1665 (s), 1532 (s), 1405 (m), 1349 (s), 1257 (s), 1117 (m) cm-'. p -Methoxyphenyl NJV-diethylt hiocarbamate (compound 5): 'H NMR (CDCl,) d 7.46 (d, J = 8.2 Hz, 2 H, aromatic H), 6.96 (d, J = 8.2 Hz, 2 H, aromatic H), 3.86 (s, 3 H, OCH3), 3.47 (m, 4 H, N-CH,), 1.40-1.10 (m, 6 H, 2 CH,); IR (neat) 3064 (m), 2987 (s), 1721 (w), 1659 (s), 1595 (m), 1497 (m), 1461 (w), 1405 (m), 1251 (s), 1117 (m), 1033 (m) cm-'. Liver Preparations. Microsome fractions were isolated according to the method described before (20,21)from liver homogenates from immature striped bass. Freshwater striped bass were obtained from the California Department of Fish and Game Central Valley Hatchery in Elk Grove, CA, and raised in fresh water a t the Bodega Marine Laboratory. Freshwater-reared fish were transferred and raised in seawater for a t least 4 weeks. Hog and rat liver microsomes were isolated by the methods described previously ( 2 1 , 2 3 ) . Dexamethasone-pretreated rat microsomes were a generous gift of Prof. Almira Correia, Department of Pharmacology, UCSF, and exhibited characteristically high 6ptestosterone hydroxylase activity [ 15-20 nmol of product/(minmg of protein)]. Hog liver microsomes were a generous gift of Prof. D. M. Ziegler, University of Texas, Austin. Flavin-containing monooxygenase was isolated and purified from hog liver microsomes by a modification of a procedure previously described (23). To minimize inactivation of flavin-containing monooxygenase activity, all steps were carried out as quickly as possible a t 4 "C. Rat liver cytochrome P-450IIB-1, cytochrome b5, and NADPHcyrochrome P-450 reductase were purified to homogeneity by a method previously described (24). The major phenobarbital-inducible cytochrome P-450 (P-450 IIB-1) from rat liver exhibited characteristically high pentoxyresorufii 0-dealkylase and 16P-testosteronehydroxylase activities [6 and 3 nmol of product/(min-nmol of cytochrome P-450), respectively] shown to be characteristicof the phenobarbital-induced isozymes and was judged to be homogeneous by SDS-poly-
Chem. Res. Toxicol., Vol. 3, No. 5, 1990 435
S-Oxygenation of Thiobencarb acrylamide gel electrophoresis. Rat liver cytochrome P-450IIB-1 had a specific content of 19.5 nmol of P-450/mg of protein. The specific content of cytochrome b5 was 32.8 nmol of cytochrome b5/mg of protein, and the reductase preparation had an activity of 66 gmol of cytochrome c/(min-mg of protein) in 0.3 M potassium phosphate buffer (pH 7.7) at 25 O C . Enzyme Assays. The basic incubation medium for microsomal assays contained 50 mM potassium phosphate buffer (pH 8.4), 0.5 mM NADP+,2.0 mM glucose 6-phosphate, and 1 IU of glucose-6phoephatedehydrogenase. In incubationswith striped bass, rat, and hog liver microsomes, 0.8-1.6 mg of protein was used. In incubations with the purified hog liver flavin-containing monooxygenase, 50-600 of enzyme was used (pH 8.4) (20,W). The reaction was initiated by the addition of substrate and incubated at 33 O C . At various time intervals, the reaction was stopped by the addition of cold CHzCll and analyzed for products by the procedure given below. CytochromeP-4501IB-1was reconstituted as follows: 50 pg of dilauroylphosphatidylcholine was combined with 300 units of rat liver cytochrome P-450 reductase and 100 pmol of cytochrome P-450IIB-1(26). For some incubations, 100 pmol of cytochrome b5 was added. After a 10-minequilibration at 4 “C, the mixture was diluted with potassium phosphate buffer (pH 7.4) and substrate was added (400 gM final concentration in 10 pL of methanol). The reaction was immediately initiated by the addition of a 0.5 mM NADPH-generatingsystem to a final volume of 0.5 mL. After a 10-min incubation at 33 O C with constant shaking in air, the reaction was stopped by addition of 2 volumes of cold CHzC12and prepared for HPLC as previously described (25, 26). The profile of thiobencarb metabolites was determined by HPLC analyis of CH2Clzextracts of the reaction mixture. The metabolic products from the extract were separated and quantified by an IBM Model 9533 HPLC interfaced to a HP Model 3392A integrator with a UV detector set at 220 nm, fitted with a precolumn and a 5-rm CRI CI8 ODS chromosphere (7.7 mm X 25 cm) analytical reverse-phasecolumn. The mobile phase consisted of an isocratic system of two solvents (A and B) set at 66% A and 34% B at a flow rate of 1.5 mL/min, where A is water/acetonitrile (21 v/v) and B is acetonitrile. This system efficiently separates thiobencarb S-oxide,thiobencarb sulfone, and thiobencarb,which have retention volumes of 4.8, 9.2, and 30.3 mL, respectively. Metabolites were quantified by comparing the metabolite and substrate peak areas of the chromatagram after taking into account the extinction coefficient difference of each material at 220 nm. The recovery of material as judged by HPLC was >90%, and 95% of this material was thiobencarb or thiobencarb S-oxide. The metabolism of [“C]testosterone by striped bass hepatic microsomes was determined by HPLC as described previously (20).
The enantioselective S-oxygenation of @-methoxypheny1)1,3-dithiolane was performed as previously described (25, 27). Other Analytical Methods. Heat inactivation of striped bass and hog liver microsomes was accomplished as previously described (22,27). Before initiating the reaction employing heatinactivated microsomes, catalase was added to remove any hydrogen peroxide. The concentration of protein used was determined by the method of Bradford (28).
Results Reaction of Thiobencarb with m -Chloroperbenzoic Acid. In dichloromethane, the reaction of thiobencarb with 1.1equiv of m-chloroperbenzoic acid gave the corresponding sulfoxide in reasonable isolated yield. The sulfoxide was not indefinitely stable, and it too oxidized to the sulfone or decomposed to other products (Scheme I). Thiobencarb S-oxide was completely characterized by spectral means (see Experimental Section). The reaction of thiobencarb with 2 equiv of m-chloroperbenzoic acid gave thiobencarb sulfone in good yield. Thiobencarb sulfone was completely characterized by spectral means (see Experimental Section). Chemical Reaction of Thiobencarb Sulfoxide and Thiobencarb Sulfone with Thiophenols. To investigate the relative electrophilicity of thiobencarb S-oxide and
Scheme 11. Chemical Reaction of Thiobencarb S-Oxide [X = S(0)CH,C6H4-p-Cl]or Thiobencarb Sulfone [X = S(0)zCHzC6H4-p-Cl] with p-Methoxythiophenol To Produce Thiocarbamate 4 or with m -NitrothiophenolTo Produce Thiocarbamate 5 O 0
R2N- C-X II
-+
R,N-
0
{ -S 4
0
It
R2N- C - X
a -
0
+
=OW,
R2N- &!-S
5
\No2
R is ethyl in all cases.
thiobencarb sulfone, the effect of thiophenols on the decomposition of thiobencarb, thiobencarb S-oxide, and thiobencarb sulfone was determined by product analysis. The reaction of thiophenols (20) or other thiols (10, 16) with thiocarbamates was a slow process. No trans-thiocarbamylation reaction product were detected following a 3-day reaction of thiobencarb with a substituted thiophenol. However, the reactions of thiobencarb S-oxide with p-methoxythiophenol and m-nitrothiophenol produced the corresponding thiocarbamates (Scheme 11). In the presence of a slight excess of thiophenol, thiobencarb sulfoxide and thiobencarb sulfone were transformed into 4 and 5 in 7 0 4 0 % isolated yield within 15 h. S-Oxygenation of Thiobencarb by Striped Bass Hepatic Microsomes. The biotransformation of thiobencarb was studied in vitro to characterize the oxidative metabolites formed and the enzymes involved. Hepatic microsomes from freshwater and seawater striped bass were used because fish are much more sensitive to the hepatotoxic effects of thiocarbamate herbicides than mammals (29),and we postulate that there is a metabolic basis for this observation. For comparison we investigated the oxidative biotransformation of thiobencarb with hog and rat liver microsomes and highly purified hepatic mammalian monooxygenases. Although thiobencarb has been shown to undergo other routes of metabolism in fish, we did not detect the formation of other metabolites during the short incubation periods used. The formation of the S-oxide was a linear function of protein concentration (0.2-1.2 mg of protein) and of incubation time for a t least 7 min. In contrast to what we have observed for other thiocarbamates (20), no detectable thiobencarb sulfone was observed during the short metabolic incubations (Table I). Data from Table I show that the S-oxygenation of thiobencarb in hepatic microsomes from freshwater and seawater striped bass was dependent upon NADPH and active microsomal protein. Thus, in the presence of heat-inactivated microsomes under conditions which preserved cytochrome P-450 activity and which inactivated flavin-containing monooxygenase activity, the Soxygenation of thiobencarb was almost completely abolished. These results suggest that S-oxygenation of thiobencarb is catalyzed largely by a striped bass hepatic monooxygenase and not through some lipid peroxidation mediated cooxidation (20). The molecular basis for formation of the S-oxide was further examined by measuring the effects of two monooxygenase inhibitors on S-oxide formation. Thiourea, a well-documented alternate substrate competitive inhibitor of the flavin-containing monooxygenase (22),significantly inhibited thiobencarb S-
436 Chem. Res. Toxicol., Vol. 3, No. 5, 1990 Table I. S-Oxygenation of Thiobencarb in Microsomes from Striped Bass nmol/(min.ma of motein) seawater freshwater microsomes microsomes condition S-oxide sulfone S-oxide sulfone ND 3.1 f 0.3 ND complete" 2.7 f 0.2 NDb ND 0.03( ND -NADPH-GS ND 0.1 f 0.1 ND +heat inactivation 0.6 f 0.2 ND 1.1 f 0.1 ND +thiourea (0.5 mM) 0.5d ND 2.0 f 0.4 ND +aminobenzotriazole 2.4 f 0.2 (0.5 mM)
" The complete system contained 200 FM thiobencarb, the NADPH-generating system (GS), 0.5 mM NADP, 0.5 mM glucose 6-phosphate, 1 IU of glucose-6-phosphate dehydrogenase, 1.0 mg of microsomal protein, and 50 mM phosphate buffer, pH 8.4. Results are the average of 3-4 incubations f SD, as determined by HPLC. bNot detectable; the limit of detection was 0.003 nmol/(min.mg of protein). (The range of values was 0.02-0.06 nmol/(min.mg of protein). dThe range of values was 0.48-0.52 nmol/(min.mg of protein). Table 11. S-Oxygenation of Thiobencarb in Microsomes from Rat and Hog Liver nmol/(min.mg of protein) hog rat condition S-oxide sulfone S-oxide sulfone NDb 1.0 f 0.1 ND completen 2.2 f 0.2 ND 0 ND -NADPH-GS ND ND +heat inactivation 0.3 f 0.1 ND 0.2d ND +thiourea (0.5 mM) L O c ND 0.6' ND +aminobenzotriazole 1.7 f 0.1 (0.5 mM)
"The complete system is as described in Table I, except that 0.86 (hog) or 1.5 mg (rat) of protein was used. *Not detectable; the limit of detection was 0.003 nmol/(min.mg of protein). (The range of values was 0.97-1.03 nmol/(min.mg of protein). dThe range of values was 0.14.3 nmol/(min.mg of protein). 'The range of values was 0.52-0.64 nmol/(min-mg of protein).
oxygenation. Conversely, aminobenzotriazole, a mechanism-based inhibitor of cytochrome P-450 (30), only slightly inhibited thiobencarb S-oxygenation. S-Oxygenation of Thiobencarb by Rat and Hog Liver Microsomes. We next investigated the biotransformation of thiobencarb in the presence of rat and hog liver microsomes (Table 11). The mirosomes used have been shown to possess significant amounts of microsomal monooxygenase enzyme activity (22,31). During the short incubation period employed, thiobencarb S-oxide was the only product observed. Thiobencarb sulfone was not formed in any detectable amount. The formation of thiobencarb S-oxide was a linear function of protein concentration (0.3-1.6 mg) and with incubation time for at least 7 min. Thiobencarb S-oxide was recovered essentially unchanged after incubation in the presence of inactive protein. Data from Table I1 show that the S-oxygenation of thiobencarb in hepatic microsomes from rat and hog liver was dependent upon NADPH and active microsomal protein. Treatment of hog liver microsomes with heat under conditions that completely inactivated the flavincontaining monooxygenase and preserved 80-85 % of the cytochrome P-450 activity almost completely abolished thiobencarb S-oxygenation. Heat inactivation was not attempted with rat liver microsomes because the major dexamethasone-inducible form of rat liver cytochrome P-450 is extremely heat labile. The enzymatic basis for the formation of thiobencarb S-oxide was further examined by measuring the effects of various monooxygenase in-
Cashman et al. Table 111. 5-Oxygenation of Thiobencarb and (p-Methoxyphenyl)-l,3-dithiolane by Highly Purified Cytochrome P-450IIB-1 and Flavin-Containing Monooxygenases cytochrome flavin-containing P-4501IB-1, monooxygenase, nmol/(min.nmol nmol/(min-mg substrate description of P-450IIB-1) of protein) thiobencarb 108.5 f 5 complete system" 85.9 f 3 -enzyme NDb ND -NADPH ND ND 74.9 f 4 +Cyt b6 (p-methoxypheny1)1,3-dithiolane' complete system 7.9 f 2 570 f 11
" The complete system is as described under Experimental Section. Results are the average of 3 incubations f SD. bNot detectable; the limit of detection was 0.005 nmol/(min.mg of protein). 'The incubation was carried out as described in refs 26 and 27. hibitors on the rate of S-oxide formation (Table 11). Thiourea significantly reduced the rate of rat and hog liver microsome catalyzed thiobencarb S-oxygenation, and aminobenzotriazole demonstrated a modest degree of inhibition of thiobencarb S-oxygenation. From the data presented in Tables I and 11, it is likely that the flavincontaining monooxygenase in large part, and to a lesser extent cytochromes P-450, contributes to the Soxygenation of thiobencarb by striped bass microsomes as well as by rat and hog liver microsomes. In addition to the use of monooxygenase inhibitors, studies of pH optima can be used to investigate the relative contribution of cytochrome P-450 and flavin-containing monooxygenase to thiobencarb S-oxygenation,because the pH optimum for hepatic cytochrome P-450 is pH 7.4 and that of the flavin-containing monooxygenase is generally at least pH 9.0. The S-oxygenation of thiobencarb in the presence of either freshwater or seawater striped bass hepatic microsomes exhibited a bimodal pH-S-oxygenase activity profile with maxima between pH 7 and 7.4 and at pH 9.0 (data not shown). To investigate this further, we examined the S-oxygenationof thiobencarb with highly purified preparations of rat liver cytochrome P-450IIB-1 and highly purified flavin-containing monooxygenase from hog liver. These enzymes were chosen as representative S-oxygenase monooxygenases present in mammalian (and possibly striped bass) liver microsomes. S-Oxygenation of Thiobencarb by Highly Purified Monooxygenases. As shown in Table 111, thiobencarb is an excellent substrate for the purified rat liver cytochrome P-450IIB- 1. Following a 10-min incubation period, no sulfone metabolite could be detected. Formation of thiobencarb S-oxide was dependent on active cytochrome P-450 and a NADPH-generating system. Addition of cytochrome b5 to the reaction mixture decreased the rate of thiobencarb S-oxidation by 13%. Kinetic constants for the S-oxygenation of thiobencarb catalyzed by rat liver cytochrome P-450IIB-1 were calculated from the rate of 5'-oxide formation at variable substrate concentrations by the HPLC procedure described under Experimental Section. The K , and V,, values obtained from double-reciprocal plots of velocity versus substrate (n = 2) were 61 ~LM and 36.7 nmol/(min.nmol of cytochrome P-450IIB-1), respectively. In a parallel experiment, the cytochrome P-450IIB-1 catalyzed S-oxygenation of (p-methoxyphenyl)-l,3-dithiolane was also examined. From the data shown in Table 111, it is apparent that thiobencarb is S-oxidized more
Chem. Res. Toxicol., Vol. 3, No. 5, 1990 437
S-Oxygenation of Thiobencarb
Table IV. Striped Bass Microsomal Testosterone Hydroxylase Activity testosterone metabolite,” pmol/ (min-mg of protein) preparation freshwater microsomes seawater microsomes
15a 3.2
2ab
5.7 4.1
16a 1.9 1.5
7ab
11.9 2.9
6a 18 1.7
16Ob 2.6 1.3
A4-A 49 29
28 305 129
11
4.3
aIncubations were performed in the presence of 500 pM [“Cltestosterone, NADPH, 1.0 mg of microsomal protein, and 50 mM phosphate buffer, pH 7.4. Results are the average of 3 determinations, and quantification was performed by the HPLC method outlined in ref 20. bThese data are taken from ref 20. Table V. S-Oxygenation of (p-Methoxyphenyl)-l,3-dithiolane by Microsomes from Striped Bassa % enantioselectivityb % diastereoselectivity trans-S-oxide cis-S-oxide microsome condition trans-S-oxide cis-S-oxide 1s,2s 1R.2R 1S.2R 1R.2S freshwater >98 43.7 f 1.7 56.3 f 1.7 seawater 61.8 f 1.5 38.2 f 1.5 30.5 f 2.1 31.3 f 0.7 13.4 f 2.7 24.8 1.6 ~
~~
nIncubations were performed as described in Table VI. Results are the average of 3 determinations f SD. bEnantioselective Soxygenation was determined by HPLC as described before (26, 27). Scheme 111. Enantioselective and Diastereoselective S-Oxygenation of (p-Methoxyphenyl)-l,3-dithiolane
