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Mercapturic acid formation from lindane in rats
PESTICIDE
BIOCHEMISTRY
AND
PHYSIOLOGY
Mercapturic NORIO *Radioisotope
10,
Acid Formation
KIJRIHARA,*
Research
137-150 (1979)
Center
from
KEIJJ TANAKA,? and
TDepartment Kyoto,
Lindane
AND MINORU
of Agricultural Japan
in Rats’ NAKAJIMA?
Chemistry,
Kyoto
University.
Received February 6, 1978 4-Chloro-, 2,4-dichloro-, 3,4-dichloro-, 2,3,5-trichloro-, and 2,4,5-trichlorophenylmercapturic acids were identified as main metabolites of lindane, y-isomer of 1,2,3,4,5,6-hexachlorocyclohexane, in rat urine. Pathways to these metabolites were shown to include (36/45)hexachlorocyclohexene as the most important intermediary metabolite. (346/5)-Pentachlorocyclohexene and (346/5)-tetrachlorocyclohexene also seem to be involved in these pathways, while (36/45)-pentachlorocyclohexene plays a minor role in the pathway. Glutathione conjugation, using the rat liver soluble fraction, occurred directly on the polychlorocyclohexenes, not on their further transformed products. In in vivo biodegradation, (36/45)-hexachtorocyclohexene may be dechforinated and dehydrochlorinated at the endoplasmic reticulum before it undergoes the glutathione conjugation in cytosol, although other polychlorocyclohexenes generally react in a manner similar to that in the in vitro reaction. INTRODUCTION
While considerable information on the oxidative biodegradation of lindane (y-BHC or ( 1245/36)-hexachlorocyclohexane)2 in mammals has been accumulated (l-3), much remains to be elucidated about another important type of biodegradation, mercapturic acid formation. In insects, glutathione conjugation, which should be the first step of mercapturic acid formation in mammals, is one of the major metabolic pathways of lindane, on which a great deal of information is now available from the investigations by Clark and his co-workers (4, 5), Bradbury and Standen (6), and Ishida and Dahm (7). Using the isotope dilution technique, the 2,4-, 2,5-, 2,6-, and 3,4’ Studies on BHC isomers and related compounds, XXIV. ‘The notation of the stereochemistry of the compounds is based on (12). The following abbreviations are used in the text. BHC, benzene hexachloride or 1,2,3,4,5,6-hexachlorocyclohexane; BTC, benzene tetrachloride or 3,4,5,6-tetrachlorocyclohexene; PCCHE, 1,3,4,5,6-pentachlorocyclohexene; HCCHE, 1,2,3,4,5,6-hexachlorocyclohexene; TFA, trifluoroacetyl (or -acetic); glc, gas-liquid chromatograph(y); ecd, electron capture detector; fpd, flame photometric detector; tid, flame ionization detector; gc-ms, gas chromatography-mass spectrometry.
isomers of dichlorothiophenol were identified in the alkaline degradation products of metabolites in the house fly, and the corresponding S-dichlorophenyl-glutathione isomers have been suggested to be the major metabolites of lindane and CX-BHC (6). In mammals, 2,4-dichlorophenyl-mercapturic acid has been identified as a urinary metabolite of (36/45)-PCCHE in rats (8). This isomer of PCCHE is the transdehydrochlorination product of lindane and is one of its intermediary metabolites (l-3, 9). Thus, 2,4-dichlorophenyl-mercapturic acid is also believed to be a metabolite of lindane in mammals, and a paper chromatographic examination supported this assumption (8), though there has not been any clear-cut evidence published. The paper chromatographic behaviors of the dichlorophenyl-mercapturic acid isomers are very similar, therefore their identification procedures should be rechecked. Recently, Koransky and his co-workers (IO) reported the presence of the 2,3-, 2,4-, 2,5-, and 2,6-isomers of dichlorothiophenol as a result of glc after alkaline treatment of the urinary metabolites of a-BHC in rats. This work seems to present clear evidence of the presence of several isomers of dichloro137 0048-3575/79/020137-14$02.00/O Copyright @ 1979 by Academic Ress. Inc. All rights of reproduction in any form reserved.
138
KURIHARA,
TANAKA,
phenyl-mercapturic acid as metabolites of a-BHC in mammals. However, lindane should be separately studied, with a nonalkaline pretreatment procedure, since alkaline treatment might give rise to unwanted aromatization if there are alicyclic polychlorinated products. Also the biochemical steps to produce mercapturic acids from lindane and the BHC isomers, especially the glutathione conjugation step, and related in vitro reactions should be studied, since the mercapturic acidformation pathways from lindane are still obscure. Here we report the presence of some unreported mono- and poly-chlorophenylmercapturic acids as the in vivo metabolites of lindane, and the nature of the in vitro glutathione conjugation reaction of the polychlorocyclohexene isomers using rat liver soluble enzymes. We also discuss the possible intermediary steps in the pathway from lindane to the mercapturic acids, based on the experimental results obtained. A short communication on this investigation has been published (11). 3 MATERIALS
AND
METHODS
Chemicals. All commercially available reagents were usually used without further purification. BTC, PCCHE, and HCCHE isomers were prepared by the methods described in previous papers (12- 14). These were purified by repeated recrystallizations and by chromatography, and were more than 99% pure when examined by glc. [V14C](36/45)-PCCHE was prepared by the dehydrochlorination of [U-14C]lindane (45 mCi/mmol, RCC Amersham) with Sorensen 3 After the completion of this investigation, we were informed by Dr. J. Portig (Philipps Universitat, Marburg, Germany) that his group had also identified various mono-, di-, and trichlorophenyl-mercaptmic acids and an isomer of tetrachlorophenyl-mercapturic acid in the urinary metabolites of o-, y-(lindane), and 6BHC using the rat. They also studied in vitro reactions using glutathione and rat liver soluble fraction and obtained results similar to ours. Their procedures are composed of an alkaline treatment of the metabolites to form thiophenols, methylation with diazomethane and glc-ecd as well as gc-ms analysis.
AND
NAKAJ’MA
buffer at pH 12 (15). Purification was performed using preparative thin-layer chromatography (silica gel, hexane). Its high purity was confirmed by thin-layer chromatography and glc with a radiodetector (Yanaco G-80 TR) (1.5% NPGS 2.25 m, i.d. 4 mm, 18O”C, proportional counter in the column oven). [d,](36/45)-PCCHE was prepared as reported before (16). (34/5)1,3,4,ITetrachlorocyclohexene was synthesized in a procedure similar to 3,4,5-trichlorocyclohexenes (17). S-Dichlorophenyl-glutathione mixtures were prepared according to the reported method (4) using liquid ammonia, metallic sodium, the reduced form of glutathione, and powdered lindane. The product was crystalline, mp 229°C. Elemental analysis-Anal. Calcd for C,,H,,N,O,Cl,S: C, 42.47; H, 4.20%. Found: C, 42.35; H, 4.28%. Although the product showed one spot on thin-layer chromatography [nBuOH/AcOH/H,O 5: 1:4 (v/v) organic phase; and n-PrGHLZV-Ammonia/H,0 2: 1: 1 (v/v)], it was shown to be a mixture of S-dichlorophenyl-glutathione isomers: 2,5(71%), 3,5- (26%), and 2,3- (3%), using the glc of the N-TFA derivative of the derived S-dichlorophenyl-cysteine butyl ester. The derivatization procedures are according to Gehrke and his co-workers (18). A dry hydrolyzed sample was heated with n-BuOHl HCl at lOO- 120°C for 1 hr, and the resulting butyl ester was dried and treated with TFA anhydride in CHzClz at 90-100°C in a tightly capped thick-wall vial for l-5 min. The S-mono-, -di-, and -trichlorophenylcysteine isomers were prepared from the corresponding chloroanilines according to the methods described by Parke (19). The melting points (“C) of the known chlorophenyl-cysteines were as follows: 2- 177” (lit. (19), 184”); 3- 195” (lit. 183”); 4- 203” (lit. 190”); 2,3- 195” (lit. 184”); 2,4- 181” (lit. 180”); 2,5- 180” (lit. 178”); 2,6 184” (lit. 175”); 3,4 179” (lit. 177”); 3,5- 185” (lit. 1810). Microanalyses for C and H of these compounds were in good agreement with those expected. Unreported trichlorophenyl-
MERCAPTURIC
ACIDS
cysteines had the following melting points (“C) and analytical values for C and H: 2,3,4- 201”, C 35.87, H 2.72; 2,3,5- 190”, C 35.74, H 2.53, 2,4,5- 187”, C 36.06, H 2.73; 2,4,6- 206”, C 37.06, H 3.00; 3,4,5- 187”, C 35.33, H 2.85. Anal. Calcd for CSHsN02SC13: C, 35.96; H, 2.67%. For the glc standard samples, the above S-chlorophenyl-cysteines were esterified with n-BuOHIHCl then trifluoroacetylated as described above. For some compounds, crystalline derivatives were obtained in yields of 70-95%, and were recrystallized from hexane. The melting points (“C) and microanalytical data of these unreported derivatives were as follows: 2,4- 65”, C 43.15, H 3.54%; 2,5- 65.5”, C 43.20, H 4.00%; 2,6- 92”, C 43.28, H 4.08%; 3,4- 68”; 3,5- 97”, C 43.52, H 3.65%. Anal. Calcd for C,,H,,NO,SCI,F,: C, 43.06; H, 3.83%. S-trichlorophenyl-cysteine derivatives: 2,3,4- 95.5”, C 39.55, H 3.22%: 2,4,5- 88”, C 39.59, H 3.16%; 2,4,6- 117” C 38.84, H 3.41% Anal. Calcd for C,,H,,NO,SCl,F,: C, 39.78; H, 3.31%. Purity of these compounds and noncrystalline derivatives was examined by glc-fpd and glc-ecd, and the structure was confirmed by gc-ms. In Fig. 1, an example of the mass spectrum was shown. Retention times under the various gas chromatographic conditions are listed in Table 1. I M’225
FROM
139
LINDANE
Dichlorothiophenols were prepared from the corresponding dichlorophenols according to the methods described by Newman and Karnes (20). The resulting dichlorothiophenols were not purified further but were dissolved in hexane and diluted for the glc and gc-ms analyses (Fig. 2). Storage at -20°C excluding air prevented autooxidation for at least 1 month. Some of the retention times in the glc of the dichlorothiophenols are listed in Table 2. The separation of these isomers by glc was not satisfactory, but the procedure can be used for rapid monitoring of the conjugate formation. In vivo experiments. Male Wistar rats (each about 150 g) were intraperitoneally administered lindane or related compounds in Ringer’s solution or in olive oil (0.5 ml). To make an emulsion in Ringer’s solution, compounds were dissolved in a minimal amount of acetone or benzene, mixed with Tween-80 (0.1 ml), and then the organic solvent was evaporated. Four tenths of a milliliter of Ringer’s solution was added to the syrupy residue and mixed thoroughly. Urine was collected over a period of 48 hr then acidified to pH 3 with HCl and washed with hexane, after which it was continuously extracted with ether for 20 hr. The hexane extract was briefly examined using glc-ecd, while the ether extract was hyCl 4:
CHCOOC,HJ h-CoCF,
+cl
SCH&HCOOC,H, NHCOCF,
II ISH,SCI, 176 I
I 1338-
\
IM’-CF3CONH,l 338
282,
II 211
c,yt
237 i
FIG. 1. Mass spectrum of the N-TFA-S-2,4,5-trichlorophenyi-cysteine bury1 ester. Other isomers showed the same spectrum. (Instrument: Shimadzu-LKB-9000 and JEOL D-300.) Isomers of the NTFA-S-dichlorophenylcysteine bury1 ester showed the molecular peak at mle 417, and peaks of fragment ions at 304 (M+-CF,CONH,), 248(304-C,H8), 19l(M+-CF,CONHCHCOOC&,). and 177 (Ca$CI,). Isomers of the N-TFA-S-monochlorophenylcysteine bury1 ester showed the following peaks: mle 383 CM’). 270 (M+-CF,CONH,), 214(27O<*H,). and 157 (M+-CF,CONHCHCOOC,H,).
140
KURIHARA,
TANAKA,
AND
TABLE Gas Chromatographic
Data
NAKAJ’MA
1
of N-TFA-S-Chlorophenyl-Cysteine Retention
3% ov-17 (2 m, 200°C)”
time
Butyl
Ester
(min)
1.5% NPGS (2 m, 200°C)
5% DEGS (2 m, 210°C)
8.5 8.0 8.0
(0.65)* (0.615) (0.615)
10.1 (0.60) 10.6 (0.625) 10.9 (0.65)
5.4 (0.68) 5.3 (0.67) 5.4 (0.68)
2,32,42,52,63,43,5-
16.15 13.0 13.4 15.1 14.3 11.5
(1.24) (1.00) (1.03) (1.16) (1.095) (0.88)
21.6 16.9 18.3 15.4 21.3 16.9
(1.28) (1.00) (1.085) (0.91) (1.26) (1.00)
11.3 8.0 8.6 7.8 9.8 7.2
(1.33) (1.00) (1.08) (0.98) (1.23) (0.90)
2,3,42,3,52,4,5-
27.2 21.8 21.2 20.1 -
(2.09) (1.68) (1.63) (1.55) (1.84)’
37.7 30.1 28.8 19.5 -
(2.23) (1.78) (1.70) (1.15)
17.3 13.1 12.6 8.8 -
(2.16) (1.64) (1.58) (1.10)
234-
W63,4,5-
0 Column length and temperature. * Relative retention time in parentheses. c This datum is based on the retention
times
on the column
drolyzed with 2N HCl for 2 hr at 100°C. The hydrolyzed product was processed as described under Identification of Metabolites. Preparation
of the crude soluble fraction.
1% OV-17,
1 m, 190°C.
g, 30 min, was further centrifuged at 105,OOOg for 90 min (Hitachi 65-P ultracentrifuge). Supernatant was used as the crude soluble enzyme preparation without further purification, although, in some reactions, the enzyme preparation was used after it was dialyzed against three changes of a total volume of 1.5 liters of the same buffer solution for 24 hr.
A male Wistar rat (about 150 g), which had been starved for 1 day, was decapitated and its liver (5-8 g) was quickly removed. All subsequent steps were performed between 0 and 5°C. The liver was homogenized (1:4, w/v) in 50 mM potassium phosphate, pH 6.7 Partially purified glutathione S-transor 7.4, or 50 mM Tris-HCI, pH 7.4, using a ferases. The procedures for the partial Potter-Elvehjem type glass homogenizer purification of the glutathione S-transequipped with a Teflon pestle. The supematant obtained after centrifugation at 12,000 TABLE
(%) 100
Gas Chromatographic Dichlorothiophenol
178( t-i’) 143 (M-Cl)’
Retention
“‘Q,, SH
3% ov-17” 2,32,42,5-
”
”
5b
lb0
140
26
200 (m/e)
FIG. Other ment:
2. Mass isomers Hitachi
spectrum showed the RMS-4.)
of 2,5-dichlorothiophenol. same
spectrum.
2
(Instru-
3,43,5-
5.60 4.50 4.55 4.65 5.55 4.45
(l.24)b (1.00) (1.01) (1.03,’ (1.23) (0.99)
Data of Isomers time (min)
1.5% NFGS” 4.2 (1.27) 3.3 (1.00) 3.4 (1.03) 2.9 (0.88) 4.8 (1.45) 3.8 (1.15)
o Column length, 2 m; temperature, 140°C. * Relative retention time in parentheses.
3% QF-1” 1.5 1.3 1.25 1.35
(1.15) (1.00) (0.71) (1.04)
MERCAPTURIC
ACIDS
ferases were essentially identical with those reported by Jakoby and his coworkers (21,22). Column chromatography with DE-52 and then CM-52 was performed. Three peaks of transferase activity, which was monitored with 2,4-dinitrochlorobenzene as a substrate, were eluted from CM-cellulose, and were designated CM-I, CM-II, and CM-III according to the elution order. Concentration of the each fraction was achieved by precipitation with ammonium sulfate. In vitro reaction with polychlorocyclohexenes. The crude soluble enzyme preparation (0.5 ml) was diluted with 1.4 ml of 50 mM potassium phosphate (pH 7.4) or 50 mM Tris-HCl (pH 7.4), after which0.1 ml of a neutralized glutathione solution was added. After preincubation for 1 to 2 min at the reaction temperature, the reaction was initiated by adding an ethanol solution (25 ~1) of the substrate. The resulting concentrations were: substrate, 10m5M ( 10e3 M for preparative purpose), glutathione, 6.25 x 10m3M, and protein 5 t 0.5 mg/ml. Protein concentrations were determined by the method of Lowry et al. (23). The reaction was performed at 25°C or 37°C with shaking. After a definite period, the mixture was chilled in ice and hexane (usually 2 vol) was added followed by vigorous shaking for at least 1 min. The hexane layer was appropriately diluted and analyzed using glc-ecd to determine the amount of the remaining substrate in order to obtain kinetic data. A solution containing the buffer solution instead of the enzyme solution was treated in the same way as a blank. Ethanol (4 vol) was added to the aqueous layer and the precipitate formed was centrifuged off (3000 t-pm, 30 mitt). The supernatant was passed through a column of Biogel P-2 (in water) or Sephadex G-15 (in 5% aqueous AcOH) to remove any remaining protein. The synthetic glutathione conjugates described above were used as the model mixture of the products. The metabolite fractions were collected and concentrated. If necessary, further purification
FROM
LINDANE
141
with preparative thin-layer chromatography was performed using silica gel G, 0.5 mm thick, with n-BuOH/AcOWH,O 5: 1:4 (v/v) (organic phase). The recovered glutathione and oxidized glutathione had much smaller Rf values than that of the synthetic conjugate mixture. The products on chromatogram were partially visualized by ninhydrin spraying and were extracted with n-BuOH/AcOH/H,O 1:1:1 (v/v). The concentrated metabolite fraction was then hydrolyzed with 2 N HCl at 100°C for lo- 12 hr. The hydrolysate was processed as described under Identification of Metabolites. The conjugation reaction was similarly performed using partially purified enzyme preparations, but to prevent undesired dehydrochlorination, the pH was maintained at 6.5, which was more acidic than the optimum, pH 7.4, for the reaction. Identification of metabolites. After the acid hydrolysate of the metabolites was thoroughly dried in the desiccator, n-BuOHIHCl was added and the mixture was heated for 2 hr at 1 lo- 120°C with continuous stirring. The resulting solution was again evaporated to dryness. To the dried residue, CH,Cl, (0.5 ml) and TFA anhydride (50 4) were added, and the mixture was briefly heated at 100°C in a tightly capped vial. The resulting solution was appropriately diluted with CHQ, and hexane and examined by glc-fpd. The remaining impurities at this stage could be removed by silicic acid column chromatography using hexane and hexane/CH,Cl, (2:l v/v) as the eluents. The trichlorophenyl-cysteine derivatives appeared first, followed by the dichloro and monochloro derivatives, in this order. The glc-ecd examination was performed at this purification stage. From the retention times in three different glc conditions (Table l), the derivatized metabolites were identified. Further identifications were made by gc-ms analyses, as described below. Glc and gc-ms analysis. For glc, a Shimadzu GC4BM (glc-fpd and glc-tid) and a Yanaco G-80 (glc-ecd) were used. A
142
KURIHARA,
TANAKA,
Shimadzu GC-6AM equipped with a wallcoated capillary column (50 m, i.d. 0.25 mm, OV-17) was also used. Column conditions are described in the Tables 1 and 2, and in the captions of Figs. 3 and 4. Carrier gas was N2 for glc-fpd and glc-ecd. Helium gas was used for glc-fid. Electronimpact ionization mass spectra were recorded with a Shimadzu-LKB 9000, a Jeol D-300, or a Hitachi RMS-4 mass spectrometer. Electron energy was 70 or 20 eV. The gas chromatographic portion of these mass spectrometers was equipped with a
AND
NAKAJIMA
column 1 m or 2 m long and i.d. 3 mm packed with 3% SE-52 or 3% OV-17. In vitro study with [U-14C](36/45)PCCHE. In the study using [U-14C](36/ 45)-PCCHE, the analysis was made by liquid scintillation counting of an appropriate aliquot. The counters used were the Searle-Isocap-300 and the Packard 23 11. The scintillator solution was the customary dioxane -naphthalene - PPO mixture. Purification of the radioactive metabolites was performed as in the cold runs, except for the thin-layer chromatography, in which
FIG. 3. Gas chromatograms of the N-TFA-S-chlorophenylcysteine butyl esters obtained from the mercapturic acids in rat urine. The rat had been intraperitoneally administered lindane or (36/45)HCCHE. See Materials and Methods for procedures of administration, extraction, and derivatization. Gas chromatographic condition: I ..5% NPGS on Chromosorb W AW DMCS 2m, 22O”C, N2 35 mllmin., fpd. (Instrument: Shimadzu GC-4BM.)
MERCAPTURIC
ACIDS
FROM
LINDANE
143
column showed a better separation of the peaks (Fig. 4) than the usual packed OV-17 column. About 15% of the administered dose was recovered in the mercapturic acid fraction. The formation of the mono- and trichlorophenyl-mercapturic acids from lindane as well as the previously reported dichlorophenyl-mercapturic acids is remarkable. In the first hexane washing of the urine before the ether extraction, various polychlorophenols, polychlorobenzenes, and polychlorocyclohexenols were shown to be present using glc-ecd as described by Chadwick and Freal (2, 24).
-
I
~&----~.-.& *
j’;c.‘ I
FIG. 4. Gas chromatograms of the samples shown in Fig. 3 using a 50 m OV-17-wall-coated glass capillary column. 195”C, fid. (Instrument: Shimadzu GC6 AM).
the radioactive spots or bands were detected with either a chromatogram-radioscanner (Aloka TLC-IOl), or with autoradiography using Sakura N-type X-ray film. A beta camera (BF-290: equipped with a spark chamber and a Polaroid camera) was also utilized for rapid detection. RESULTS
In Vivo Metabolites The mercapturic acids in rat urine formed from polychloro compounds are listed in Table 3. Some examples of the glc examination are shown in Fig. 3. A glass capillary
In Vitro Reactivity The rates of disappearance of the isomers of PCCHE at a fixed initial substrate concentration in the presence of excess glutathione and the crude soluble enzyme preparation are summarized in Table 4. The reactivity of lindane in this system was too low to afford any glutathione conjugates detectable at 5 hr at 37°C. The results shown are obtained using enzyme in 50 mM phosphate buffer, pH 7.4. Similar reaction trends were observed for the differently prepared soluble fractions and under different reaction conditions described under Materials and Methods. Reactions at 37°C occurred faster than at 25°C. Pentadeuterated (36/45)PCCHE essentially had a rate of disappearance identical with that of the nondeuterated equivalent. Only (34/56)- and (36/45)-PCCHE were studied for glutathione conjugation of the partially purified enzyme preparations. The specific activities for these compounds were as follows: (34/56)-PCCHE at 25°C: 1.5 (CM-I), 2.3 (CM-II), and 1.9 (CM-III) (nmol/min/mg protein); (36/45)-PCCHE at 37°C: 1.7 (CM-I), 2.3 (CM-II), and 1.7 (CM-III) (X IO-” moV3 hr/mg protein). Those for 2,4-dinitrochlorobenzene at 25°C were: 1.2 (CM-I), 5.6 (CM-II), and 1.9 (CM-III) (pmole/min/mg protein). The (34/ 56)-isomer was degraded much faster than the (36/45)-isomer as in the reaction using the crude enzyme.
144
KURIHARA,
TANAKA,
TABLE Composition of the ChlorophenyCMercapturic
Substrate
Dose @noI)
Lindane
34.4 17.2
(36/45)-HCCHE
34.6 (BR) 17.3 (0)’ 10.4 (0)
(36/45)-PCCHE (346/T)-PCCHE
(346/T)-BTC
(BR)’ (ARP’
AND
NAKAJ’MA
3
Acids Formed from Lindane and Related Compounds in Vivo”
CPMAb
DCPMAC
2-
3-
4-
t’ t
t t
t t t
2,3-
2,4-
2,5-
3,4-
2,3,5-
2,4,5-
15 10
4 t
9 9
6 2
33 36
17 21
16 23
0”
3
4
0 0
t 0
t t
16 12 14
5 t
1
24 36 34
22 24 25
26 28 27
2 3
0 0
t t
t t
16 6 24
57 85 56
t 0 t
t 0 t
0 0
0 0
(BR)
0
0
t
0
97
(0)
0
0
t
0
95
(BR) (AR) 1.0 (AR)
0 t 0
0 0 0
20 1 t
0 0 0
7
(AR) (AR)
0 0
0 0
100 100
0 0
39.2 39.2 11.8 5.5 45.4 4.5
TCPMAd
8 20
0 0
0 0
0 0
a Intraperitoneally administered to rat. Percentages in the mercapturic acid fraction are shown. The standard error in the glc determination was estimated as less than 5% of each value. The compositions in this table are somewhat different from those in the preliminary report by us (11). The differences are mostly due to the errors in the amount estimation of N-TFA-3,4-dichlorophenyl-cysteine butyl ester, which showed sometimes peculiar dose-peak height responses in the glc-fpd. b (CPMA) Chlorophenyl-mercapturic acid. c (DCPMA) Dichlorophenyl-mercapturic acid. d (TCPMA) Trichlorophenyl-mercapturic acid. e (BR) Administered as an emulsion in Ringer’s solution made from benzene solution. ‘(t) Trace amount; less than 1%. g (AR) Administered as an emulsion in Ringer’s solution made from acetone solution. * (0) Not detectable. i (0) Administered as an olive oil solution.
In Vitro Metabolites The metabolite compositions from the polychlorocyclohexenes in the reaction using the crude enzyme preparation are TABLE
4
In Vitro Reactivity of PCCHE Isomers with Giutathione in the Presence of the Crude Soluble Enzyme Preparation’ Isomer (36/45) (35/46) (35W4) (3460 (34l56)
Specific activity (motiminlmg protein) 1.57 x lo-“* a.77 14.3 la.7 170
Relative rate 1 5.6 9.1
11.9
108
a Initial substrate concentration: lOen mokmt; glutathione: 6.25 ~mol/ml; protein: 5.3 ~&II; at WC, pH 7.4 (50 mM phosphate). For other conditions, seeMaterials and Methods. b (36/45)-HCCHE exhibits a less specific activity than this value, and the rate relative to that of (36/45kPCCHE determined at 37°C was 0.36.
summarized in Table 5. When one of the CM-fractions (I, II, and III) was used instead of the crude enzyme, (34/56)-PCCHE afforded S-2,5-dichlorophenyl-glutathione accompanied by smaller amounts of the 2,4and 3 ,Cisomer. Recovery Ratio of the in Vitro Metabolites The recovered radioactivity in the in vitro metabolites produced from the radioactive (36/45)-PCCHE was examined. The TFA derivative for the glc analysis retained 75-85% of the radioactivity of the reacted substrate. DISCUSSION
Intermediary Metabolites in the Pathways from Lindane to Mercapturic Acids As shown in Table 3, the mercapturic acids formed from lindane in vivo include mono- and trichlorophenyl-mercapturic
MERCAPTURIC
Composition
PCCHE (36/45) (35/46) (35614) (34615) (34/56)
Substrate (35/46)-BTC (346/5)-BTC (36/45)-BTC (34/5)-1,3,4,5-Tetrachlorocyclohexene (36/45)-HCCHE (35/46)-HCCHE (346/5)-HCCHE
ACIDS
FROM
TABLE
5
of the S-Chlorophenyl-Glutathione
Formed
from
Polychlorocyclohexenes
in Vitro”
DCPG” formed (sum = 100)
No. of experiments 10 4 I 4 5
145
LINDANE
2,4-
2,5-
2,6-
3.4-
95 k 2’ 67 t 3 44 14 2 2 33 k 3
0 0 2 13 + 2 60 t 3
31-l 0 20 0 221
321 33 + I 34 73 t 5 5x2
No. of experiments
Product
2 2 2
4-CPGd (almost exclusively) 4-CPG (almost exclusively) 4-CPG (almost exclusively)
1 4 1 1
3-CPG (almost exclusively) 2,4,6-TCPG’ and others One unknown (2,3,6- or 3,4,5-TCPG) > 2,3,4-TCPG 2,3,4-TCPG > 2,3,5-TCPG > others
n Temperature: 37°C. See Materials and Methods for other reaction conditions and procedures. See footnote to the text for abbreviations of substrates. DNeither the 2,3- nor the 3,5-isomer was produced from any of the PCCHE isomers tested. (DCPG) S-Dichlorophenyl-glutathione. ’ Average values 2 SE. d ( CPG) S-Chlorophenyl-glutathione r (TCPG) S-Trichlorophenyl-glutathione.
acids as well as dichlorophenylmercapturic acids. The 2,4-isomer of dichlorophenylmercapturic acid has long been believed to be the most important one in the sulfurcontaining metabolites of lindane, and the route: lindane * (36/45)-PCCHE * S-2,4dichlorophenyl-glutathione + 2,4-dichlorophenyl-mercapturic acid has been proposed as the major mercapturic acidforming pathway (8, 25). However, the present in vivo results required other important pathways which could explain the formation of 4-chloro-, 3,4-dichloro-, and 2,3,5- and 2,4,5-trichlorophenyl-mercapturic acid. (36/45)-HCCHE, the cis-dehydrogenation product of lindane, afforded 2,4- and 3,4dichloro- and 2,3,5- and 2,4,5trichlorophenyl-mercapturic acid along with 2,3- and 2,5-dichlorophenyl-mercapturic acid in the in vivo experiments. The 2- and 4-chloro-
phenyl-mercapturic acid were also produced, though in much smaller amounts (Table 3 and Figs. 3 and 4). All the important mercapturic acids from lindane are involved in the products, and the product composition is similar to that of lindane, although the percentage of 2,4-dichlorophenyl-mercapturic acid (or the ratio of 2,4and 3,4-isomer) is higher and that of 4-chlorophenyl-mercapturic acid is much smaller than when derived from lindane. The general similarity in metabolite composition and the fact that this HCCHE is an important oxidative metabolite of lindane (l-3) strongly suggest that it is an important intermediary compound in the in viva metabolic pathways from lindane to the mercapturic acids. The formation of mercapturic acids such as 3,4-dichloroand 4-chlorophenyl-mercapturic acid from lindane might require
146
KIJRIHARA,
TANAKA,
other intermediates, of which most plausible candidates are (346/5)-PCCHE and (346/5)-BTC, respectively. The former is the cis-dehydrochlorination product and the latter is the trans-dechlorination product of lindane. (346/5)-PCCHE was one of the in vitro metabolites in the reaction using house fly microsomes (16), and (346/ 5)-BTC has been produced from lindane in an anaerobic reaction using a rat liver microsomal fraction (2, 26). Thus, the previous held conception of the mercapturic acid-forming pathway should be largely corrected to one, which includes (36/45)-HCCHE and probably (346/5)PCCHE and (346/5)-BTC. The role of (361 45)-PCCHE in this pathway seems to be a minor one. Glutathione Conjugation Step The in vitro study elucidated the features of the glutathione conjugation step in mercapturic acid formation. The very low or no glutathione conjugation of lindane itself with a rat liver supernatant fraction and the rather facile conjugation of polychlorocyclohexenes (Table 4) support the view anticipated in the preceding section that hydrogen abstraction and other desaturation reactions operate in the first step for mercapturic acid formation from lindane . Since the in vitro reaction using a soluble enzyme preparation corresponds to the in vivo reaction in cytosol, the desaturation reactions should take place at the endoplasmic reticulum before the transport of the molecule into the cytosol. Precise informations on the glutathioneattack step and on its mode can be obtained from in vitro experimental results. The absence of deuterium isotope effects in the metabolic degradation of PCCHE using glutathione and the soluble fraction suggests that, at least in the in vitro reactions, the glutathione molecule attacks these polychlorocyclohexenes directly and nucleophilically, not after they have been further transformed, such as to dehydrochlorinated products. The metabolite compositions shown in Table 5 are consistent
AND
NAKAJIMA
with this view. For example, the production of 2,6-dichlorophenyl-glutathione in these reactions requires an SN2- or SN2’-like attack of glutathione on the PCCHE isomers. When glutathione attacks at the C-6 position of the PCCHE molecule, subsequent dehydrochlorinations can form the 2,6isomer as well as the 2,4- and 2,5-isomers (see Fig. 5a). If the attack of glutathione occurs at the C-2 position, the vinylic carbon which bears no chlorine atom and is sterically unhindered, the 2,4-, 2,5-, and 2,6-dichlorophenyl-glutathione isomers can be produced after the subsequent dehydrochlorinations (Fig. 5b). The 3,4-isomer, an important in vitro metabolite from most of the PCCHE isomers (Table 5), is produced by glutathione attack on the C-3 position of a PCCHE molecule (Fig. 5~). Some stereochemical considerations favor the above described mode for the in vitro glutathione conjugation reaction (Fig. 6). The 3-chlorine of (346/5)-PCCHE is pseudo-axial and should be more reactive than any of the other chlorines in the molecule; thus the nucleophilic attack by glutathione should be most favorable at this site, accounting for the abundant formation of 3 ,Cdichloro-compound from this isomer (Fig. 6a). The almost exclusive production of the 2,4-isomer from (36/45)-PCCHE re-
4 3 e 5
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J
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-
FIG. 5. Modes chlorocyclohexene pheny&lutathione.
3,4-DCPG
etc
of glutathione attack on pentaisomers. (DCPG) S-Dichloro-
MERCAPTURIC
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.>QG
-
ACIDS
so
FROM
147
LINDANE
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6. Proposed stereochemical pathways for glutathione conjugation with various polychlorocyclohexenes. (CPG, DCPG, and TCPG) S-Chloro-, S-dichloro-, and S-trichlorophenyl-glutathione. respectively. (TCCHE) Tetrachlorocyclohexene. (0) Show the attacking glutathione molecules. For abbreviations of other compounds, see the footnote to the text. FIG.
fleets the low reactivity of the allylic positions of this isomer (13). The glutathione attack, therefore, seems to occur at the C-2 position followed by 1,2-dehydrochlorination which eliminates the labile proton at the attacked site. When one more mole of HCI is eliminated, the resulting dichlorophenyl-glutathione should be the 2,4-isomer, since the chlorine atom at the m-position from the glutathione group becomes labile due to the electron donating power of sulfur (Fig. 6b). Although the trichlorophenyl-glutathione isomers from the HCCHE isomers have yet to be completely identified, results are consistent with the above discussions. For example, (36/45)-HCCHE has the same configuration of chlorine substituents as the (36/45)-PCCHE discussed above, and its allylic positions are relatively unreactive. As with (36/45)-PCCHE, the glutathione attack on a vinylic position of this HCCHE would explain the formation of the 2,4,6-isomer (Fig. 6~). Each examined BTC isomer gave 4-chlorophenyl-glutathione as a major product, the formation of which can be explained by an SN2 and/or SN2’ type of
glutathione attack on these substrates. (341 5)-1,3,4,5-Tetrachlorocyclohexene produced 3-chloro-isomer, almost exclusively. This reaction is obviously initiated by the glutathione attack on the C-3 position, the only allylic carbon bearing a chlorine atom. Subsequent elimination of 2 mol of HCl necessarily forms the 3-isomer of monochlorophenyl-glutathione (Fig. 6d). Table 4 shows the relative reactivities among the PCCHE isomers. The higher reactivities of (34/56)-(aeeu-conformation of Cl) and (346/S)-(aeee)-PCCHE as compared to the (35/46)-(eeee)-isomer reflect the higher reactivity of the pseudo-axial chlorine substituent as compared to the pseudo-equatorial one. The lowest reactivity of the (36/45)-isomer is consistent with the above stereochemical considerations which assumed the low reactivity of the allylic positions in this PCCHE was enforcing the glutathione attack on a vinylic carbon. A brief reaction of (34/56)-PCCHE with glutathione in the presence of a partially purified enzyme afforded only aromatic glutathione conjugates as in the reaction with the crude enzyme, although it was expected that this PCCHE (the most reactive
148
KURIHARA,
TANAKA,
AND
NAKAJIMA
sequent glutathione attack would produce S-dichlorophenyl-glutathione and then dichlorophenyl-mercapturic acid. Thus, the first type of difference can be interpreted by reductive dechlorination in the endoplasmic reticulum before the glutathione conjugation in cytosol. Differences in Composition between the in The other type of difference is found in Vivo and in Vitro Products the composition of the product isomers. AlSome differences in metabolite composithough the 2,3,5- and 2,4,5-isomers of trition were observed between the in vivo and chlorophenyl-glutathione were not formed in vitro studies of the same compounds. In in vitro from (36/45)-HCCHE, they were particular, (36/45)-HCCHE showed differ- major structures in the mercapturic acids ent isomer distributions in its three found in the urine of the rat administered chlorine-containing metabolite molecules this HCCHE isomer. This difference should for these two types of biodegradation, and also be explained by some other reactions produced a significant amount of dichlorowhich occur in the in vivo system. If we compounds in vivo. assume that one dehydrochlorination ocThe differences between in vivo and in curs, e.g., in the endoplasmic reticulum bevitro results deserve consideration and may fore the glutathione attack, it would probe classified into two types. One is the dif- duce a 1 ,Cdiene: 1,2,3,4,6-pentachloroference in the numbers of chlorine atoms in cyclohexadiene- 1,4. The glutathione attack the product molecules. In the in vitro reac- on the less hindered allylic position (C-6) tions, HCCHE, PCCHE, and BTC isomers and the less hindered vinylic position (C-5) always produced three chlorine-less prodin the resulting diene would necessarily ucts: trichloro-, dichloro-, and monchloroproduce the respective 2,3,5-and 2,4,5phenyl-glutathione, respectively, as ex- conjugates. These conjugates might be also pected from the sequence of glutathione produced from 1,2,4-trichlorobenzene. The substitution of one chlorine atom then the latter is one of the most important in vitro elimination of 2 mol of HCl. However, in metabolites of (36/45)-HCCHE in aerobic vivo, (36/45)-HCCHE gave dichloro- and (31) and in anaerobic condition.4 monochlorophenyl-mercapturic acids as With these considerations, we need to aswell as trichloro-compounds; and (346/5)- sume the in vivo occurrence of such transPCCHE gave 4-chlorophenyl-mercapturic formations as dechlorination and dehydroacid, although distinctly in one case. This chlorination before glutathione conjugation, type of product formation in vivo occurs at least with (36/45)-HCCHE. Further study when the substrates suffer reductive is needed before the assumed mechanistic dechlorination(s) as well as glutathione con- details can be confirmed. jugation and dehydrochlorination. ReducIn summary, in the rat, lindane suffers tive dechlorination can occur in the endo- desaturation in the endoplasmic reticulum plasmic reticulum depending on cytochrome producing (36/45)-HCCHE, (346/5)- and P-450 (2,26-30). When 1 mol of chlorine is (36/45)-PCCHE, and (346/5)-BTC (1, 2, 16, eliminated4 from (36/45)-HCCHE, the sub- 26). Thereafter, glutathione conjugation occurs on these compounds. It may be a attack of glutathione on these 4The occurrence of dechlorination is observed in direct the in vitro reactions: (36/45)-HCCHE gives 1,2,4- polychlorocyclohexenes, but for (36/45)trichlorobenzene when incubated with NADPH and HCCHE some other transformations such the rat liver microsomal fraction [(3 1) and unpublished as dechlorination and dehydrochlorination observation under an anaerobic condition]. The forseem to precede it. In all cases, further mation of 1,2-dichlorobenzene is also observed (undehydrochlorination produces various published). one) would give, at least partly, S-tetrachlorocyclohexenyl-glutathione as an intermediate without suffering subsequent aromatization. The supposed nonaromatic primary conjugates seem to be too unstable to be isolated.
MERCAPTURIC
Endoplasmic
ACIDS
FROM
149
LINDANE
reticulum
2.3.FTCPG : 36/45)
-HCCHE
(346/5)
-PCCHE
2,4,5-TCPG 3,4-DCPG 2,5-DCPG
FIG. 7. Proposed pathways for the formation of glutathione conjugates from lindane in the rat. as the primary step of mercapturic acid formation. (+) Biochemical transformation; f-D) transport. (TCB) Trichlorobenzene andlor trichlorobenzene epoxide. (CPG, DCPG, and TCPGJ S-Chloro-, S-dichloro-, and S-trichlorophenyl-glutathione, respectively. For abbreviations of other compounds, see the footnote to the text. The structure of dienes: 1,4-Diene (C/s) is 1.2.3,4,6-pentachlorocyclohexadiene-1,4; and 1,3-diene (Cl*) is 1,2,5,6-tetrachlorocyclohexadiene-I $3. Both of them are tentatively assumed intermediary metabolites.
S-chlorophenyl-glutathiones, then the mercapturic acids. Thus, the pathways formercapturic acid formation from lindane can be summarized as shown in Fig. 7. ACKNOWLEDGMENTS
We wish to express our sincere thanks to Dr. Toshio Fujita, the Department of Agricultural Chemistry, Kyoto University, for his helpful advice and discussions; to Dr. Tamio Ueno, the Pesticide Research Institute, Kyoto Univsersity, Dr. Tetsuya Suzuki, the Research Institute for Food Science, Kyoto University, and Drs. Yasuhiro Itagaki and Eiji Kubota, JEOL Ltd., for the gc-ms analyses; and to Dr. Tokuzo Nishino, the Department of Chemistry, Kyoto University, for his courtesy in allowing the use of their glc equipped with a capillary column. Some S-chlorophenyl-cysteines were prepared by Miss A. Ueno. This work was supported in part by a grant from the Ministry of Education of Japan. REFERENCES
1. K. Tanaka, N. Kurihara, and M. Nakajima, Pathways of chlorophenol formation in oxidative biodegradation of BHC, Agr. Biol. Chem. 41, 723 (1977). 2. R. W. Chadwick, L. T. Chuang, and K. Williams, Dehydrogenation: a previously unreported pathway of lindane metabolism in mammals, Pestic. Biochem. Physiol. 5, 575 (1975). 3. K. Stein, J. Portig, and W. Koransky, Oxidative transformation of hexachlorocyclohexane in rats and with rat liver microsomes, NaunynSchmiedeberg’s Arch. Pharmacol. 298, 115 (1977). 4. A. G. Clark, M. Hitchcock, and J. N. Smith, Metabolism of gammexane in flies, ticks and locusts, Nature (London) 183, 103 (1966).
5. A. G. Clark, S. Murphy, and J. N. Smith, The metabolism of hexachlorocyclohexanes and pentachlorocyclohexenes in flies and grubs, Biochem. J. 113,89 (1969). 6. F. R. Bradbury and H. Standen, Metabolism of benzene hexachloride by resistant houseflies Musca domestica, Nature (London) 183, 983 (1959). 7. M. Ishida and P. A. Dahm, Metabolism of benzene hexachloride isomers and related compounds in vitro, J. Econ. Entomol. 58, 383 (1965). 8. P. L. Grover and P. Sims, The metabolism of y2,3,4,5,6-pentachlorocyclohex-1-ene and yhexachlorocyclohexane in rats, Biochem. J. %, 521 (1%5). 9. W. T. Reed and A. J. Forgash, Metabolism of lindane to organic-soluble products by houseflies, J. Agr. Food Chem. 18, 475 (1970). 10. W. Koransky, G. Munch, G. Noack, J. Portig, S. Sodomann, and M. Wirsching, Biodegradation of a-hexachlorocyclohexane, V. Characterization of the major urinary metabolites, NaunynSchmiedeberg’s Arch. Pharmacol. 288, 6.5 (1975). 11. N. Kurihara, K. Tanaka, and M. Nakajima, Pathways of chlorophenyl-mercapturic acid formation in biodegradation of lindane, Agr. Biol. Chem. 41, 1317 (1977). 12. N. Kurihara, Y. Sanemitsu, T. Kimura, M. Kobayashi, and M. Nakajima, Stepwise synthesis of 3,4,5,6-tetrachlorocyclohexene-1 (BTC) isomers, Agr. Biol. Chem. 34, 784 (1970). 13. N. Kurihara, S. Wakamura, T. Nakamura, and M. Nakajima, Isomerization of 1,3,4,5,6pentachlorocyclohexene- 1 isomers in dimethyl sulfoxide, Agr. Biol. Chem. 38, 1717 (1974). 14. N. Kurihara, K. Tanaka, and M. Nakajima. Isom-
150
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16.
17.
18.
19. 20. 21.
22. 23.
KURIHARA,
TANAKA,
erization of hexachlorocyclohexene, Agr. Biol. Chem. 40, 411 (1976). M. Nakajima, T. Okubo, and Y. Katsumura, The alkaline dehydrochlorination of the benzene hexachloride isomers, I, Boryu-kagaku, 14, 10 (1949). K. Tanaka, N. Kurihara, and M. Nakajima, Metabolism of lindane in house flies: Metabolic desaturation, dehydrogenation and dehydrochlorination, and conjugation with glutathione, Pestic. Biochem. Physiol. 6, 392 (1976). K. Tanaka, M. Kiso, M. Yoshida, N. Kurihara, and M. Nakajima, Syntheses and PMR studies of 1,2,3,4&pentachlorocyclohexane isomers, tetrachloromonomethoxycyclohexane isomers and related compounds, Agr. Biol. Chem. 39, 229 (1975). W. M. Lamkin and C. W. Gehrke, Quantitative gas chromatography of amino acids, Preparation of n-butyl N-trifluoroacetyl esters, And. Chem. 37, 383 (1965). D. V. Parke, Studies in detoxication 64. The synthesis of the isomeric dichlorophenylmercaptutic acids, Biochem. J. 59, 422 (1955). M. S. Newman and H. A. Karnes, The conversion of phenols to thiophenols via dialkylthiocarbamates, J. Org. Chem. 31, 3980 (1966). W. H. Habig, M. J. Pabst, and W. B. Jakoby, Glutathione S-transferases, the first step in mercapturic acid formation, J. Biol. Chem. 249, 7130 (1974). W. H. Habig, M. J. Pabst, and W. B. Jakoby, Glutathione S-transferase AA from rat liver, Arch. Biochem. Biophys. 175,710 (1976). 0. H. Lowry, N. J. Rosebrough, A. L. Farr, and
AND
24.
25. 26.
27.
28.
29. 30.
31.
NAKAJIMA
R. J. Randall, Protein measurement with Folin phenol reagent, J. Biol. Chem. 193,265 (1951). R. W. Chadwick and J. J. Freal, The identification of five unreported lindane metabolites recovered from rat urine, Bull. Environ. Contam. Toxicol. 7, 137 (1972). G. T. Brooks, “Chlorinated Insecticides,” Vol. 2, pp. 80-84, CRC Press, Cleveland, 1974. K. Tanaka, N. Kurihara, and M. Nakajima, Oxidative metabolism of lindane and its isomers with microsomes from rat liver and house fly abdomen, Pestic. Biochem. Physiol. 10, 96 (1979). J. R. Gillette, J. J. Kamm and H. A. Sasame, Mechanism of p-nitrobenzoate reduction in liver: the possible role of cytochrome P-450 in liver microsomes, Mol. Pharmacol. 4, 541 (1968). K. A. Hassall, Reductive dechlorination of DDT: the effect of some physical and chemical agents on DDD production by pigeon liver preparations, Pestic. Biochem. Physiol. 1, 259 (1972). C. H. Walker, Reductive dechlorination of pp’DDT by pigeon liver microsomes, Life Sci. 8, 1111 (1969). M. Sugiura, K. Iwasaki, and R. Kato, Reduction of tertiary amine N-oxides by liver microsomal cytochrome P-450, Mol. Pharmacol. 12, 322 (1976). K. Tanaka, N. Kurihara, and M. Nakajima, Oxidative metabolism of tetrachlorocyclohexenes, pentachlorocyclohexenes, and hexachlorocyclohexenes with microsomes from rat liver and house fly abdomen, Pestic. Biochem. Physiol. 10, 79 (1979).
BIOCHEMISTRY
AND
PHYSIOLOGY
Mercapturic NORIO *Radioisotope
10,
Acid Formation
KIJRIHARA,*
Research
137-150 (1979)
Center
from
KEIJJ TANAKA,? and
TDepartment Kyoto,
Lindane
AND MINORU
of Agricultural Japan
in Rats’ NAKAJIMA?
Chemistry,
Kyoto
University.
Received February 6, 1978 4-Chloro-, 2,4-dichloro-, 3,4-dichloro-, 2,3,5-trichloro-, and 2,4,5-trichlorophenylmercapturic acids were identified as main metabolites of lindane, y-isomer of 1,2,3,4,5,6-hexachlorocyclohexane, in rat urine. Pathways to these metabolites were shown to include (36/45)hexachlorocyclohexene as the most important intermediary metabolite. (346/5)-Pentachlorocyclohexene and (346/5)-tetrachlorocyclohexene also seem to be involved in these pathways, while (36/45)-pentachlorocyclohexene plays a minor role in the pathway. Glutathione conjugation, using the rat liver soluble fraction, occurred directly on the polychlorocyclohexenes, not on their further transformed products. In in vivo biodegradation, (36/45)-hexachtorocyclohexene may be dechforinated and dehydrochlorinated at the endoplasmic reticulum before it undergoes the glutathione conjugation in cytosol, although other polychlorocyclohexenes generally react in a manner similar to that in the in vitro reaction. INTRODUCTION
While considerable information on the oxidative biodegradation of lindane (y-BHC or ( 1245/36)-hexachlorocyclohexane)2 in mammals has been accumulated (l-3), much remains to be elucidated about another important type of biodegradation, mercapturic acid formation. In insects, glutathione conjugation, which should be the first step of mercapturic acid formation in mammals, is one of the major metabolic pathways of lindane, on which a great deal of information is now available from the investigations by Clark and his co-workers (4, 5), Bradbury and Standen (6), and Ishida and Dahm (7). Using the isotope dilution technique, the 2,4-, 2,5-, 2,6-, and 3,4’ Studies on BHC isomers and related compounds, XXIV. ‘The notation of the stereochemistry of the compounds is based on (12). The following abbreviations are used in the text. BHC, benzene hexachloride or 1,2,3,4,5,6-hexachlorocyclohexane; BTC, benzene tetrachloride or 3,4,5,6-tetrachlorocyclohexene; PCCHE, 1,3,4,5,6-pentachlorocyclohexene; HCCHE, 1,2,3,4,5,6-hexachlorocyclohexene; TFA, trifluoroacetyl (or -acetic); glc, gas-liquid chromatograph(y); ecd, electron capture detector; fpd, flame photometric detector; tid, flame ionization detector; gc-ms, gas chromatography-mass spectrometry.
isomers of dichlorothiophenol were identified in the alkaline degradation products of metabolites in the house fly, and the corresponding S-dichlorophenyl-glutathione isomers have been suggested to be the major metabolites of lindane and CX-BHC (6). In mammals, 2,4-dichlorophenyl-mercapturic acid has been identified as a urinary metabolite of (36/45)-PCCHE in rats (8). This isomer of PCCHE is the transdehydrochlorination product of lindane and is one of its intermediary metabolites (l-3, 9). Thus, 2,4-dichlorophenyl-mercapturic acid is also believed to be a metabolite of lindane in mammals, and a paper chromatographic examination supported this assumption (8), though there has not been any clear-cut evidence published. The paper chromatographic behaviors of the dichlorophenyl-mercapturic acid isomers are very similar, therefore their identification procedures should be rechecked. Recently, Koransky and his co-workers (IO) reported the presence of the 2,3-, 2,4-, 2,5-, and 2,6-isomers of dichlorothiophenol as a result of glc after alkaline treatment of the urinary metabolites of a-BHC in rats. This work seems to present clear evidence of the presence of several isomers of dichloro137 0048-3575/79/020137-14$02.00/O Copyright @ 1979 by Academic Ress. Inc. All rights of reproduction in any form reserved.
138
KURIHARA,
TANAKA,
phenyl-mercapturic acid as metabolites of a-BHC in mammals. However, lindane should be separately studied, with a nonalkaline pretreatment procedure, since alkaline treatment might give rise to unwanted aromatization if there are alicyclic polychlorinated products. Also the biochemical steps to produce mercapturic acids from lindane and the BHC isomers, especially the glutathione conjugation step, and related in vitro reactions should be studied, since the mercapturic acidformation pathways from lindane are still obscure. Here we report the presence of some unreported mono- and poly-chlorophenylmercapturic acids as the in vivo metabolites of lindane, and the nature of the in vitro glutathione conjugation reaction of the polychlorocyclohexene isomers using rat liver soluble enzymes. We also discuss the possible intermediary steps in the pathway from lindane to the mercapturic acids, based on the experimental results obtained. A short communication on this investigation has been published (11). 3 MATERIALS
AND
METHODS
Chemicals. All commercially available reagents were usually used without further purification. BTC, PCCHE, and HCCHE isomers were prepared by the methods described in previous papers (12- 14). These were purified by repeated recrystallizations and by chromatography, and were more than 99% pure when examined by glc. [V14C](36/45)-PCCHE was prepared by the dehydrochlorination of [U-14C]lindane (45 mCi/mmol, RCC Amersham) with Sorensen 3 After the completion of this investigation, we were informed by Dr. J. Portig (Philipps Universitat, Marburg, Germany) that his group had also identified various mono-, di-, and trichlorophenyl-mercaptmic acids and an isomer of tetrachlorophenyl-mercapturic acid in the urinary metabolites of o-, y-(lindane), and 6BHC using the rat. They also studied in vitro reactions using glutathione and rat liver soluble fraction and obtained results similar to ours. Their procedures are composed of an alkaline treatment of the metabolites to form thiophenols, methylation with diazomethane and glc-ecd as well as gc-ms analysis.
AND
NAKAJ’MA
buffer at pH 12 (15). Purification was performed using preparative thin-layer chromatography (silica gel, hexane). Its high purity was confirmed by thin-layer chromatography and glc with a radiodetector (Yanaco G-80 TR) (1.5% NPGS 2.25 m, i.d. 4 mm, 18O”C, proportional counter in the column oven). [d,](36/45)-PCCHE was prepared as reported before (16). (34/5)1,3,4,ITetrachlorocyclohexene was synthesized in a procedure similar to 3,4,5-trichlorocyclohexenes (17). S-Dichlorophenyl-glutathione mixtures were prepared according to the reported method (4) using liquid ammonia, metallic sodium, the reduced form of glutathione, and powdered lindane. The product was crystalline, mp 229°C. Elemental analysis-Anal. Calcd for C,,H,,N,O,Cl,S: C, 42.47; H, 4.20%. Found: C, 42.35; H, 4.28%. Although the product showed one spot on thin-layer chromatography [nBuOH/AcOH/H,O 5: 1:4 (v/v) organic phase; and n-PrGHLZV-Ammonia/H,0 2: 1: 1 (v/v)], it was shown to be a mixture of S-dichlorophenyl-glutathione isomers: 2,5(71%), 3,5- (26%), and 2,3- (3%), using the glc of the N-TFA derivative of the derived S-dichlorophenyl-cysteine butyl ester. The derivatization procedures are according to Gehrke and his co-workers (18). A dry hydrolyzed sample was heated with n-BuOHl HCl at lOO- 120°C for 1 hr, and the resulting butyl ester was dried and treated with TFA anhydride in CHzClz at 90-100°C in a tightly capped thick-wall vial for l-5 min. The S-mono-, -di-, and -trichlorophenylcysteine isomers were prepared from the corresponding chloroanilines according to the methods described by Parke (19). The melting points (“C) of the known chlorophenyl-cysteines were as follows: 2- 177” (lit. (19), 184”); 3- 195” (lit. 183”); 4- 203” (lit. 190”); 2,3- 195” (lit. 184”); 2,4- 181” (lit. 180”); 2,5- 180” (lit. 178”); 2,6 184” (lit. 175”); 3,4 179” (lit. 177”); 3,5- 185” (lit. 1810). Microanalyses for C and H of these compounds were in good agreement with those expected. Unreported trichlorophenyl-
MERCAPTURIC
ACIDS
cysteines had the following melting points (“C) and analytical values for C and H: 2,3,4- 201”, C 35.87, H 2.72; 2,3,5- 190”, C 35.74, H 2.53, 2,4,5- 187”, C 36.06, H 2.73; 2,4,6- 206”, C 37.06, H 3.00; 3,4,5- 187”, C 35.33, H 2.85. Anal. Calcd for CSHsN02SC13: C, 35.96; H, 2.67%. For the glc standard samples, the above S-chlorophenyl-cysteines were esterified with n-BuOHIHCl then trifluoroacetylated as described above. For some compounds, crystalline derivatives were obtained in yields of 70-95%, and were recrystallized from hexane. The melting points (“C) and microanalytical data of these unreported derivatives were as follows: 2,4- 65”, C 43.15, H 3.54%; 2,5- 65.5”, C 43.20, H 4.00%; 2,6- 92”, C 43.28, H 4.08%; 3,4- 68”; 3,5- 97”, C 43.52, H 3.65%. Anal. Calcd for C,,H,,NO,SCI,F,: C, 43.06; H, 3.83%. S-trichlorophenyl-cysteine derivatives: 2,3,4- 95.5”, C 39.55, H 3.22%: 2,4,5- 88”, C 39.59, H 3.16%; 2,4,6- 117” C 38.84, H 3.41% Anal. Calcd for C,,H,,NO,SCl,F,: C, 39.78; H, 3.31%. Purity of these compounds and noncrystalline derivatives was examined by glc-fpd and glc-ecd, and the structure was confirmed by gc-ms. In Fig. 1, an example of the mass spectrum was shown. Retention times under the various gas chromatographic conditions are listed in Table 1. I M’225
FROM
139
LINDANE
Dichlorothiophenols were prepared from the corresponding dichlorophenols according to the methods described by Newman and Karnes (20). The resulting dichlorothiophenols were not purified further but were dissolved in hexane and diluted for the glc and gc-ms analyses (Fig. 2). Storage at -20°C excluding air prevented autooxidation for at least 1 month. Some of the retention times in the glc of the dichlorothiophenols are listed in Table 2. The separation of these isomers by glc was not satisfactory, but the procedure can be used for rapid monitoring of the conjugate formation. In vivo experiments. Male Wistar rats (each about 150 g) were intraperitoneally administered lindane or related compounds in Ringer’s solution or in olive oil (0.5 ml). To make an emulsion in Ringer’s solution, compounds were dissolved in a minimal amount of acetone or benzene, mixed with Tween-80 (0.1 ml), and then the organic solvent was evaporated. Four tenths of a milliliter of Ringer’s solution was added to the syrupy residue and mixed thoroughly. Urine was collected over a period of 48 hr then acidified to pH 3 with HCl and washed with hexane, after which it was continuously extracted with ether for 20 hr. The hexane extract was briefly examined using glc-ecd, while the ether extract was hyCl 4:
CHCOOC,HJ h-CoCF,
+cl
SCH&HCOOC,H, NHCOCF,
II ISH,SCI, 176 I
I 1338-
\
IM’-CF3CONH,l 338
282,
II 211
c,yt
237 i
FIG. 1. Mass spectrum of the N-TFA-S-2,4,5-trichlorophenyi-cysteine bury1 ester. Other isomers showed the same spectrum. (Instrument: Shimadzu-LKB-9000 and JEOL D-300.) Isomers of the NTFA-S-dichlorophenylcysteine bury1 ester showed the molecular peak at mle 417, and peaks of fragment ions at 304 (M+-CF,CONH,), 248(304-C,H8), 19l(M+-CF,CONHCHCOOC&,). and 177 (Ca$CI,). Isomers of the N-TFA-S-monochlorophenylcysteine bury1 ester showed the following peaks: mle 383 CM’). 270 (M+-CF,CONH,), 214(27O<*H,). and 157 (M+-CF,CONHCHCOOC,H,).
140
KURIHARA,
TANAKA,
AND
TABLE Gas Chromatographic
Data
NAKAJ’MA
1
of N-TFA-S-Chlorophenyl-Cysteine Retention
3% ov-17 (2 m, 200°C)”
time
Butyl
Ester
(min)
1.5% NPGS (2 m, 200°C)
5% DEGS (2 m, 210°C)
8.5 8.0 8.0
(0.65)* (0.615) (0.615)
10.1 (0.60) 10.6 (0.625) 10.9 (0.65)
5.4 (0.68) 5.3 (0.67) 5.4 (0.68)
2,32,42,52,63,43,5-
16.15 13.0 13.4 15.1 14.3 11.5
(1.24) (1.00) (1.03) (1.16) (1.095) (0.88)
21.6 16.9 18.3 15.4 21.3 16.9
(1.28) (1.00) (1.085) (0.91) (1.26) (1.00)
11.3 8.0 8.6 7.8 9.8 7.2
(1.33) (1.00) (1.08) (0.98) (1.23) (0.90)
2,3,42,3,52,4,5-
27.2 21.8 21.2 20.1 -
(2.09) (1.68) (1.63) (1.55) (1.84)’
37.7 30.1 28.8 19.5 -
(2.23) (1.78) (1.70) (1.15)
17.3 13.1 12.6 8.8 -
(2.16) (1.64) (1.58) (1.10)
234-
W63,4,5-
0 Column length and temperature. * Relative retention time in parentheses. c This datum is based on the retention
times
on the column
drolyzed with 2N HCl for 2 hr at 100°C. The hydrolyzed product was processed as described under Identification of Metabolites. Preparation
of the crude soluble fraction.
1% OV-17,
1 m, 190°C.
g, 30 min, was further centrifuged at 105,OOOg for 90 min (Hitachi 65-P ultracentrifuge). Supernatant was used as the crude soluble enzyme preparation without further purification, although, in some reactions, the enzyme preparation was used after it was dialyzed against three changes of a total volume of 1.5 liters of the same buffer solution for 24 hr.
A male Wistar rat (about 150 g), which had been starved for 1 day, was decapitated and its liver (5-8 g) was quickly removed. All subsequent steps were performed between 0 and 5°C. The liver was homogenized (1:4, w/v) in 50 mM potassium phosphate, pH 6.7 Partially purified glutathione S-transor 7.4, or 50 mM Tris-HCI, pH 7.4, using a ferases. The procedures for the partial Potter-Elvehjem type glass homogenizer purification of the glutathione S-transequipped with a Teflon pestle. The supematant obtained after centrifugation at 12,000 TABLE
(%) 100
Gas Chromatographic Dichlorothiophenol
178( t-i’) 143 (M-Cl)’
Retention
“‘Q,, SH
3% ov-17” 2,32,42,5-
”
”
5b
lb0
140
26
200 (m/e)
FIG. Other ment:
2. Mass isomers Hitachi
spectrum showed the RMS-4.)
of 2,5-dichlorothiophenol. same
spectrum.
2
(Instru-
3,43,5-
5.60 4.50 4.55 4.65 5.55 4.45
(l.24)b (1.00) (1.01) (1.03,’ (1.23) (0.99)
Data of Isomers time (min)
1.5% NFGS” 4.2 (1.27) 3.3 (1.00) 3.4 (1.03) 2.9 (0.88) 4.8 (1.45) 3.8 (1.15)
o Column length, 2 m; temperature, 140°C. * Relative retention time in parentheses.
3% QF-1” 1.5 1.3 1.25 1.35
(1.15) (1.00) (0.71) (1.04)
MERCAPTURIC
ACIDS
ferases were essentially identical with those reported by Jakoby and his coworkers (21,22). Column chromatography with DE-52 and then CM-52 was performed. Three peaks of transferase activity, which was monitored with 2,4-dinitrochlorobenzene as a substrate, were eluted from CM-cellulose, and were designated CM-I, CM-II, and CM-III according to the elution order. Concentration of the each fraction was achieved by precipitation with ammonium sulfate. In vitro reaction with polychlorocyclohexenes. The crude soluble enzyme preparation (0.5 ml) was diluted with 1.4 ml of 50 mM potassium phosphate (pH 7.4) or 50 mM Tris-HCl (pH 7.4), after which0.1 ml of a neutralized glutathione solution was added. After preincubation for 1 to 2 min at the reaction temperature, the reaction was initiated by adding an ethanol solution (25 ~1) of the substrate. The resulting concentrations were: substrate, 10m5M ( 10e3 M for preparative purpose), glutathione, 6.25 x 10m3M, and protein 5 t 0.5 mg/ml. Protein concentrations were determined by the method of Lowry et al. (23). The reaction was performed at 25°C or 37°C with shaking. After a definite period, the mixture was chilled in ice and hexane (usually 2 vol) was added followed by vigorous shaking for at least 1 min. The hexane layer was appropriately diluted and analyzed using glc-ecd to determine the amount of the remaining substrate in order to obtain kinetic data. A solution containing the buffer solution instead of the enzyme solution was treated in the same way as a blank. Ethanol (4 vol) was added to the aqueous layer and the precipitate formed was centrifuged off (3000 t-pm, 30 mitt). The supernatant was passed through a column of Biogel P-2 (in water) or Sephadex G-15 (in 5% aqueous AcOH) to remove any remaining protein. The synthetic glutathione conjugates described above were used as the model mixture of the products. The metabolite fractions were collected and concentrated. If necessary, further purification
FROM
LINDANE
141
with preparative thin-layer chromatography was performed using silica gel G, 0.5 mm thick, with n-BuOH/AcOWH,O 5: 1:4 (v/v) (organic phase). The recovered glutathione and oxidized glutathione had much smaller Rf values than that of the synthetic conjugate mixture. The products on chromatogram were partially visualized by ninhydrin spraying and were extracted with n-BuOH/AcOH/H,O 1:1:1 (v/v). The concentrated metabolite fraction was then hydrolyzed with 2 N HCl at 100°C for lo- 12 hr. The hydrolysate was processed as described under Identification of Metabolites. The conjugation reaction was similarly performed using partially purified enzyme preparations, but to prevent undesired dehydrochlorination, the pH was maintained at 6.5, which was more acidic than the optimum, pH 7.4, for the reaction. Identification of metabolites. After the acid hydrolysate of the metabolites was thoroughly dried in the desiccator, n-BuOHIHCl was added and the mixture was heated for 2 hr at 1 lo- 120°C with continuous stirring. The resulting solution was again evaporated to dryness. To the dried residue, CH,Cl, (0.5 ml) and TFA anhydride (50 4) were added, and the mixture was briefly heated at 100°C in a tightly capped vial. The resulting solution was appropriately diluted with CHQ, and hexane and examined by glc-fpd. The remaining impurities at this stage could be removed by silicic acid column chromatography using hexane and hexane/CH,Cl, (2:l v/v) as the eluents. The trichlorophenyl-cysteine derivatives appeared first, followed by the dichloro and monochloro derivatives, in this order. The glc-ecd examination was performed at this purification stage. From the retention times in three different glc conditions (Table l), the derivatized metabolites were identified. Further identifications were made by gc-ms analyses, as described below. Glc and gc-ms analysis. For glc, a Shimadzu GC4BM (glc-fpd and glc-tid) and a Yanaco G-80 (glc-ecd) were used. A
142
KURIHARA,
TANAKA,
Shimadzu GC-6AM equipped with a wallcoated capillary column (50 m, i.d. 0.25 mm, OV-17) was also used. Column conditions are described in the Tables 1 and 2, and in the captions of Figs. 3 and 4. Carrier gas was N2 for glc-fpd and glc-ecd. Helium gas was used for glc-fid. Electronimpact ionization mass spectra were recorded with a Shimadzu-LKB 9000, a Jeol D-300, or a Hitachi RMS-4 mass spectrometer. Electron energy was 70 or 20 eV. The gas chromatographic portion of these mass spectrometers was equipped with a
AND
NAKAJIMA
column 1 m or 2 m long and i.d. 3 mm packed with 3% SE-52 or 3% OV-17. In vitro study with [U-14C](36/45)PCCHE. In the study using [U-14C](36/ 45)-PCCHE, the analysis was made by liquid scintillation counting of an appropriate aliquot. The counters used were the Searle-Isocap-300 and the Packard 23 11. The scintillator solution was the customary dioxane -naphthalene - PPO mixture. Purification of the radioactive metabolites was performed as in the cold runs, except for the thin-layer chromatography, in which
FIG. 3. Gas chromatograms of the N-TFA-S-chlorophenylcysteine butyl esters obtained from the mercapturic acids in rat urine. The rat had been intraperitoneally administered lindane or (36/45)HCCHE. See Materials and Methods for procedures of administration, extraction, and derivatization. Gas chromatographic condition: I ..5% NPGS on Chromosorb W AW DMCS 2m, 22O”C, N2 35 mllmin., fpd. (Instrument: Shimadzu GC-4BM.)
MERCAPTURIC
ACIDS
FROM
LINDANE
143
column showed a better separation of the peaks (Fig. 4) than the usual packed OV-17 column. About 15% of the administered dose was recovered in the mercapturic acid fraction. The formation of the mono- and trichlorophenyl-mercapturic acids from lindane as well as the previously reported dichlorophenyl-mercapturic acids is remarkable. In the first hexane washing of the urine before the ether extraction, various polychlorophenols, polychlorobenzenes, and polychlorocyclohexenols were shown to be present using glc-ecd as described by Chadwick and Freal (2, 24).
-
I
~&----~.-.& *
j’;c.‘ I
FIG. 4. Gas chromatograms of the samples shown in Fig. 3 using a 50 m OV-17-wall-coated glass capillary column. 195”C, fid. (Instrument: Shimadzu GC6 AM).
the radioactive spots or bands were detected with either a chromatogram-radioscanner (Aloka TLC-IOl), or with autoradiography using Sakura N-type X-ray film. A beta camera (BF-290: equipped with a spark chamber and a Polaroid camera) was also utilized for rapid detection. RESULTS
In Vivo Metabolites The mercapturic acids in rat urine formed from polychloro compounds are listed in Table 3. Some examples of the glc examination are shown in Fig. 3. A glass capillary
In Vitro Reactivity The rates of disappearance of the isomers of PCCHE at a fixed initial substrate concentration in the presence of excess glutathione and the crude soluble enzyme preparation are summarized in Table 4. The reactivity of lindane in this system was too low to afford any glutathione conjugates detectable at 5 hr at 37°C. The results shown are obtained using enzyme in 50 mM phosphate buffer, pH 7.4. Similar reaction trends were observed for the differently prepared soluble fractions and under different reaction conditions described under Materials and Methods. Reactions at 37°C occurred faster than at 25°C. Pentadeuterated (36/45)PCCHE essentially had a rate of disappearance identical with that of the nondeuterated equivalent. Only (34/56)- and (36/45)-PCCHE were studied for glutathione conjugation of the partially purified enzyme preparations. The specific activities for these compounds were as follows: (34/56)-PCCHE at 25°C: 1.5 (CM-I), 2.3 (CM-II), and 1.9 (CM-III) (nmol/min/mg protein); (36/45)-PCCHE at 37°C: 1.7 (CM-I), 2.3 (CM-II), and 1.7 (CM-III) (X IO-” moV3 hr/mg protein). Those for 2,4-dinitrochlorobenzene at 25°C were: 1.2 (CM-I), 5.6 (CM-II), and 1.9 (CM-III) (pmole/min/mg protein). The (34/ 56)-isomer was degraded much faster than the (36/45)-isomer as in the reaction using the crude enzyme.
144
KURIHARA,
TANAKA,
TABLE Composition of the ChlorophenyCMercapturic
Substrate
Dose @noI)
Lindane
34.4 17.2
(36/45)-HCCHE
34.6 (BR) 17.3 (0)’ 10.4 (0)
(36/45)-PCCHE (346/T)-PCCHE
(346/T)-BTC
(BR)’ (ARP’
AND
NAKAJ’MA
3
Acids Formed from Lindane and Related Compounds in Vivo”
CPMAb
DCPMAC
2-
3-
4-
t’ t
t t
t t t
2,3-
2,4-
2,5-
3,4-
2,3,5-
2,4,5-
15 10
4 t
9 9
6 2
33 36
17 21
16 23
0”
3
4
0 0
t 0
t t
16 12 14
5 t
1
24 36 34
22 24 25
26 28 27
2 3
0 0
t t
t t
16 6 24
57 85 56
t 0 t
t 0 t
0 0
0 0
(BR)
0
0
t
0
97
(0)
0
0
t
0
95
(BR) (AR) 1.0 (AR)
0 t 0
0 0 0
20 1 t
0 0 0
7
(AR) (AR)
0 0
0 0
100 100
0 0
39.2 39.2 11.8 5.5 45.4 4.5
TCPMAd
8 20
0 0
0 0
0 0
a Intraperitoneally administered to rat. Percentages in the mercapturic acid fraction are shown. The standard error in the glc determination was estimated as less than 5% of each value. The compositions in this table are somewhat different from those in the preliminary report by us (11). The differences are mostly due to the errors in the amount estimation of N-TFA-3,4-dichlorophenyl-cysteine butyl ester, which showed sometimes peculiar dose-peak height responses in the glc-fpd. b (CPMA) Chlorophenyl-mercapturic acid. c (DCPMA) Dichlorophenyl-mercapturic acid. d (TCPMA) Trichlorophenyl-mercapturic acid. e (BR) Administered as an emulsion in Ringer’s solution made from benzene solution. ‘(t) Trace amount; less than 1%. g (AR) Administered as an emulsion in Ringer’s solution made from acetone solution. * (0) Not detectable. i (0) Administered as an olive oil solution.
In Vitro Metabolites The metabolite compositions from the polychlorocyclohexenes in the reaction using the crude enzyme preparation are TABLE
4
In Vitro Reactivity of PCCHE Isomers with Giutathione in the Presence of the Crude Soluble Enzyme Preparation’ Isomer (36/45) (35/46) (35W4) (3460 (34l56)
Specific activity (motiminlmg protein) 1.57 x lo-“* a.77 14.3 la.7 170
Relative rate 1 5.6 9.1
11.9
108
a Initial substrate concentration: lOen mokmt; glutathione: 6.25 ~mol/ml; protein: 5.3 ~&II; at WC, pH 7.4 (50 mM phosphate). For other conditions, seeMaterials and Methods. b (36/45)-HCCHE exhibits a less specific activity than this value, and the rate relative to that of (36/45kPCCHE determined at 37°C was 0.36.
summarized in Table 5. When one of the CM-fractions (I, II, and III) was used instead of the crude enzyme, (34/56)-PCCHE afforded S-2,5-dichlorophenyl-glutathione accompanied by smaller amounts of the 2,4and 3 ,Cisomer. Recovery Ratio of the in Vitro Metabolites The recovered radioactivity in the in vitro metabolites produced from the radioactive (36/45)-PCCHE was examined. The TFA derivative for the glc analysis retained 75-85% of the radioactivity of the reacted substrate. DISCUSSION
Intermediary Metabolites in the Pathways from Lindane to Mercapturic Acids As shown in Table 3, the mercapturic acids formed from lindane in vivo include mono- and trichlorophenyl-mercapturic
MERCAPTURIC
Composition
PCCHE (36/45) (35/46) (35614) (34615) (34/56)
Substrate (35/46)-BTC (346/5)-BTC (36/45)-BTC (34/5)-1,3,4,5-Tetrachlorocyclohexene (36/45)-HCCHE (35/46)-HCCHE (346/5)-HCCHE
ACIDS
FROM
TABLE
5
of the S-Chlorophenyl-Glutathione
Formed
from
Polychlorocyclohexenes
in Vitro”
DCPG” formed (sum = 100)
No. of experiments 10 4 I 4 5
145
LINDANE
2,4-
2,5-
2,6-
3.4-
95 k 2’ 67 t 3 44 14 2 2 33 k 3
0 0 2 13 + 2 60 t 3
31-l 0 20 0 221
321 33 + I 34 73 t 5 5x2
No. of experiments
Product
2 2 2
4-CPGd (almost exclusively) 4-CPG (almost exclusively) 4-CPG (almost exclusively)
1 4 1 1
3-CPG (almost exclusively) 2,4,6-TCPG’ and others One unknown (2,3,6- or 3,4,5-TCPG) > 2,3,4-TCPG 2,3,4-TCPG > 2,3,5-TCPG > others
n Temperature: 37°C. See Materials and Methods for other reaction conditions and procedures. See footnote to the text for abbreviations of substrates. DNeither the 2,3- nor the 3,5-isomer was produced from any of the PCCHE isomers tested. (DCPG) S-Dichlorophenyl-glutathione. ’ Average values 2 SE. d ( CPG) S-Chlorophenyl-glutathione r (TCPG) S-Trichlorophenyl-glutathione.
acids as well as dichlorophenylmercapturic acids. The 2,4-isomer of dichlorophenylmercapturic acid has long been believed to be the most important one in the sulfurcontaining metabolites of lindane, and the route: lindane * (36/45)-PCCHE * S-2,4dichlorophenyl-glutathione + 2,4-dichlorophenyl-mercapturic acid has been proposed as the major mercapturic acidforming pathway (8, 25). However, the present in vivo results required other important pathways which could explain the formation of 4-chloro-, 3,4-dichloro-, and 2,3,5- and 2,4,5-trichlorophenyl-mercapturic acid. (36/45)-HCCHE, the cis-dehydrogenation product of lindane, afforded 2,4- and 3,4dichloro- and 2,3,5- and 2,4,5trichlorophenyl-mercapturic acid along with 2,3- and 2,5-dichlorophenyl-mercapturic acid in the in vivo experiments. The 2- and 4-chloro-
phenyl-mercapturic acid were also produced, though in much smaller amounts (Table 3 and Figs. 3 and 4). All the important mercapturic acids from lindane are involved in the products, and the product composition is similar to that of lindane, although the percentage of 2,4-dichlorophenyl-mercapturic acid (or the ratio of 2,4and 3,4-isomer) is higher and that of 4-chlorophenyl-mercapturic acid is much smaller than when derived from lindane. The general similarity in metabolite composition and the fact that this HCCHE is an important oxidative metabolite of lindane (l-3) strongly suggest that it is an important intermediary compound in the in viva metabolic pathways from lindane to the mercapturic acids. The formation of mercapturic acids such as 3,4-dichloroand 4-chlorophenyl-mercapturic acid from lindane might require
146
KIJRIHARA,
TANAKA,
other intermediates, of which most plausible candidates are (346/5)-PCCHE and (346/5)-BTC, respectively. The former is the cis-dehydrochlorination product and the latter is the trans-dechlorination product of lindane. (346/5)-PCCHE was one of the in vitro metabolites in the reaction using house fly microsomes (16), and (346/ 5)-BTC has been produced from lindane in an anaerobic reaction using a rat liver microsomal fraction (2, 26). Thus, the previous held conception of the mercapturic acid-forming pathway should be largely corrected to one, which includes (36/45)-HCCHE and probably (346/5)PCCHE and (346/5)-BTC. The role of (361 45)-PCCHE in this pathway seems to be a minor one. Glutathione Conjugation Step The in vitro study elucidated the features of the glutathione conjugation step in mercapturic acid formation. The very low or no glutathione conjugation of lindane itself with a rat liver supernatant fraction and the rather facile conjugation of polychlorocyclohexenes (Table 4) support the view anticipated in the preceding section that hydrogen abstraction and other desaturation reactions operate in the first step for mercapturic acid formation from lindane . Since the in vitro reaction using a soluble enzyme preparation corresponds to the in vivo reaction in cytosol, the desaturation reactions should take place at the endoplasmic reticulum before the transport of the molecule into the cytosol. Precise informations on the glutathioneattack step and on its mode can be obtained from in vitro experimental results. The absence of deuterium isotope effects in the metabolic degradation of PCCHE using glutathione and the soluble fraction suggests that, at least in the in vitro reactions, the glutathione molecule attacks these polychlorocyclohexenes directly and nucleophilically, not after they have been further transformed, such as to dehydrochlorinated products. The metabolite compositions shown in Table 5 are consistent
AND
NAKAJIMA
with this view. For example, the production of 2,6-dichlorophenyl-glutathione in these reactions requires an SN2- or SN2’-like attack of glutathione on the PCCHE isomers. When glutathione attacks at the C-6 position of the PCCHE molecule, subsequent dehydrochlorinations can form the 2,6isomer as well as the 2,4- and 2,5-isomers (see Fig. 5a). If the attack of glutathione occurs at the C-2 position, the vinylic carbon which bears no chlorine atom and is sterically unhindered, the 2,4-, 2,5-, and 2,6-dichlorophenyl-glutathione isomers can be produced after the subsequent dehydrochlorinations (Fig. 5b). The 3,4-isomer, an important in vitro metabolite from most of the PCCHE isomers (Table 5), is produced by glutathione attack on the C-3 position of a PCCHE molecule (Fig. 5~). Some stereochemical considerations favor the above described mode for the in vitro glutathione conjugation reaction (Fig. 6). The 3-chlorine of (346/5)-PCCHE is pseudo-axial and should be more reactive than any of the other chlorines in the molecule; thus the nucleophilic attack by glutathione should be most favorable at this site, accounting for the abundant formation of 3 ,Cdichloro-compound from this isomer (Fig. 6a). The almost exclusive production of the 2,4-isomer from (36/45)-PCCHE re-
4 3 e 5
Cc)’
J
(a)
(a)-!kLtitutcm
2.4;
2.5,
by
a-
6
‘r-1
6
(a) Q,
SG
8
26-DCPG
(b) -0’GS
/
-
FIG. 5. Modes chlorocyclohexene pheny&lutathione.
3,4-DCPG
etc
of glutathione attack on pentaisomers. (DCPG) S-Dichloro-
MERCAPTURIC
6~)
.>QG
-
ACIDS
so
FROM
147
LINDANE
--
3,4-DCPG
~346215AxE
(b)
;@I
-
0
-
-
@
+
G50
-
G@
-
2.4-DCPG
-
GWCPG
(36/45)-Pt~~~
(c)
$-$+ .
(d’
(36145)
i;
HCCHE
.)o
-
3-CPG
(34/5)-1,3,4,5-TCCHE
6. Proposed stereochemical pathways for glutathione conjugation with various polychlorocyclohexenes. (CPG, DCPG, and TCPG) S-Chloro-, S-dichloro-, and S-trichlorophenyl-glutathione. respectively. (TCCHE) Tetrachlorocyclohexene. (0) Show the attacking glutathione molecules. For abbreviations of other compounds, see the footnote to the text. FIG.
fleets the low reactivity of the allylic positions of this isomer (13). The glutathione attack, therefore, seems to occur at the C-2 position followed by 1,2-dehydrochlorination which eliminates the labile proton at the attacked site. When one more mole of HCI is eliminated, the resulting dichlorophenyl-glutathione should be the 2,4-isomer, since the chlorine atom at the m-position from the glutathione group becomes labile due to the electron donating power of sulfur (Fig. 6b). Although the trichlorophenyl-glutathione isomers from the HCCHE isomers have yet to be completely identified, results are consistent with the above discussions. For example, (36/45)-HCCHE has the same configuration of chlorine substituents as the (36/45)-PCCHE discussed above, and its allylic positions are relatively unreactive. As with (36/45)-PCCHE, the glutathione attack on a vinylic position of this HCCHE would explain the formation of the 2,4,6-isomer (Fig. 6~). Each examined BTC isomer gave 4-chlorophenyl-glutathione as a major product, the formation of which can be explained by an SN2 and/or SN2’ type of
glutathione attack on these substrates. (341 5)-1,3,4,5-Tetrachlorocyclohexene produced 3-chloro-isomer, almost exclusively. This reaction is obviously initiated by the glutathione attack on the C-3 position, the only allylic carbon bearing a chlorine atom. Subsequent elimination of 2 mol of HCl necessarily forms the 3-isomer of monochlorophenyl-glutathione (Fig. 6d). Table 4 shows the relative reactivities among the PCCHE isomers. The higher reactivities of (34/56)-(aeeu-conformation of Cl) and (346/S)-(aeee)-PCCHE as compared to the (35/46)-(eeee)-isomer reflect the higher reactivity of the pseudo-axial chlorine substituent as compared to the pseudo-equatorial one. The lowest reactivity of the (36/45)-isomer is consistent with the above stereochemical considerations which assumed the low reactivity of the allylic positions in this PCCHE was enforcing the glutathione attack on a vinylic carbon. A brief reaction of (34/56)-PCCHE with glutathione in the presence of a partially purified enzyme afforded only aromatic glutathione conjugates as in the reaction with the crude enzyme, although it was expected that this PCCHE (the most reactive
148
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TANAKA,
AND
NAKAJIMA
sequent glutathione attack would produce S-dichlorophenyl-glutathione and then dichlorophenyl-mercapturic acid. Thus, the first type of difference can be interpreted by reductive dechlorination in the endoplasmic reticulum before the glutathione conjugation in cytosol. Differences in Composition between the in The other type of difference is found in Vivo and in Vitro Products the composition of the product isomers. AlSome differences in metabolite composithough the 2,3,5- and 2,4,5-isomers of trition were observed between the in vivo and chlorophenyl-glutathione were not formed in vitro studies of the same compounds. In in vitro from (36/45)-HCCHE, they were particular, (36/45)-HCCHE showed differ- major structures in the mercapturic acids ent isomer distributions in its three found in the urine of the rat administered chlorine-containing metabolite molecules this HCCHE isomer. This difference should for these two types of biodegradation, and also be explained by some other reactions produced a significant amount of dichlorowhich occur in the in vivo system. If we compounds in vivo. assume that one dehydrochlorination ocThe differences between in vivo and in curs, e.g., in the endoplasmic reticulum bevitro results deserve consideration and may fore the glutathione attack, it would probe classified into two types. One is the dif- duce a 1 ,Cdiene: 1,2,3,4,6-pentachloroference in the numbers of chlorine atoms in cyclohexadiene- 1,4. The glutathione attack the product molecules. In the in vitro reac- on the less hindered allylic position (C-6) tions, HCCHE, PCCHE, and BTC isomers and the less hindered vinylic position (C-5) always produced three chlorine-less prodin the resulting diene would necessarily ucts: trichloro-, dichloro-, and monchloroproduce the respective 2,3,5-and 2,4,5phenyl-glutathione, respectively, as ex- conjugates. These conjugates might be also pected from the sequence of glutathione produced from 1,2,4-trichlorobenzene. The substitution of one chlorine atom then the latter is one of the most important in vitro elimination of 2 mol of HCl. However, in metabolites of (36/45)-HCCHE in aerobic vivo, (36/45)-HCCHE gave dichloro- and (31) and in anaerobic condition.4 monochlorophenyl-mercapturic acids as With these considerations, we need to aswell as trichloro-compounds; and (346/5)- sume the in vivo occurrence of such transPCCHE gave 4-chlorophenyl-mercapturic formations as dechlorination and dehydroacid, although distinctly in one case. This chlorination before glutathione conjugation, type of product formation in vivo occurs at least with (36/45)-HCCHE. Further study when the substrates suffer reductive is needed before the assumed mechanistic dechlorination(s) as well as glutathione con- details can be confirmed. jugation and dehydrochlorination. ReducIn summary, in the rat, lindane suffers tive dechlorination can occur in the endo- desaturation in the endoplasmic reticulum plasmic reticulum depending on cytochrome producing (36/45)-HCCHE, (346/5)- and P-450 (2,26-30). When 1 mol of chlorine is (36/45)-PCCHE, and (346/5)-BTC (1, 2, 16, eliminated4 from (36/45)-HCCHE, the sub- 26). Thereafter, glutathione conjugation occurs on these compounds. It may be a attack of glutathione on these 4The occurrence of dechlorination is observed in direct the in vitro reactions: (36/45)-HCCHE gives 1,2,4- polychlorocyclohexenes, but for (36/45)trichlorobenzene when incubated with NADPH and HCCHE some other transformations such the rat liver microsomal fraction [(3 1) and unpublished as dechlorination and dehydrochlorination observation under an anaerobic condition]. The forseem to precede it. In all cases, further mation of 1,2-dichlorobenzene is also observed (undehydrochlorination produces various published). one) would give, at least partly, S-tetrachlorocyclohexenyl-glutathione as an intermediate without suffering subsequent aromatization. The supposed nonaromatic primary conjugates seem to be too unstable to be isolated.
MERCAPTURIC
Endoplasmic
ACIDS
FROM
149
LINDANE
reticulum
2.3.FTCPG : 36/45)
-HCCHE
(346/5)
-PCCHE
2,4,5-TCPG 3,4-DCPG 2,5-DCPG
FIG. 7. Proposed pathways for the formation of glutathione conjugates from lindane in the rat. as the primary step of mercapturic acid formation. (+) Biochemical transformation; f-D) transport. (TCB) Trichlorobenzene andlor trichlorobenzene epoxide. (CPG, DCPG, and TCPGJ S-Chloro-, S-dichloro-, and S-trichlorophenyl-glutathione, respectively. For abbreviations of other compounds, see the footnote to the text. The structure of dienes: 1,4-Diene (C/s) is 1.2.3,4,6-pentachlorocyclohexadiene-1,4; and 1,3-diene (Cl*) is 1,2,5,6-tetrachlorocyclohexadiene-I $3. Both of them are tentatively assumed intermediary metabolites.
S-chlorophenyl-glutathiones, then the mercapturic acids. Thus, the pathways formercapturic acid formation from lindane can be summarized as shown in Fig. 7. ACKNOWLEDGMENTS
We wish to express our sincere thanks to Dr. Toshio Fujita, the Department of Agricultural Chemistry, Kyoto University, for his helpful advice and discussions; to Dr. Tamio Ueno, the Pesticide Research Institute, Kyoto Univsersity, Dr. Tetsuya Suzuki, the Research Institute for Food Science, Kyoto University, and Drs. Yasuhiro Itagaki and Eiji Kubota, JEOL Ltd., for the gc-ms analyses; and to Dr. Tokuzo Nishino, the Department of Chemistry, Kyoto University, for his courtesy in allowing the use of their glc equipped with a capillary column. Some S-chlorophenyl-cysteines were prepared by Miss A. Ueno. This work was supported in part by a grant from the Ministry of Education of Japan. REFERENCES
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