Trialkyl phosphorothioates and glutathione S-transferases

Chem.-Biol. Interactions, 54 (1985) 243--256

243

Elsevier Scientific Publishers Ireland Ltd.

T R I A L K Y L P H O S P H O R O T H I O A T E S AND G L U T A T H I O N E S-TRANSFERASES

W.N. ALDRIDGE a, H. GRASDALEN b, K. AARSTAD c, B.W. STREETa and T. NORKOV c

aToxicology Unit, Medical Research Council Laboratories, Carshalton (U.K.), bInst, of Marine Biochemistry, The University of Trondheim, Trondheim-NTH and CDept. of Pharmacology and Toxicology, School of Medicine, University of Trondheim (Norway) (Received November 15th, 1984) (Accepted April 17th, 1985)

SUMMARY

Using a rat liver cytosol source of enzyme trialkyl phosphorothioates have been shown to be substrates of glutathione S-transferases. Using OSStrimethyl phosphorodithioate (OSS-Me(O)) and OOS-trimethyl phosphorothioate (OOS-Me(O)) the methyl transferred to the sulphydryl of glutathione is that attached to phosphorus via an oxygen atom. Fractionation of liver cytosol has shown that although the bulk activity is due to the three isozymes (1-1; 3-4; 1.2), OSS-Me(O) is a general substrate for glutathione S-transferases. The specific activity is low compared with the substrates 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene.

Key words: Trialkyl phosphorothioates -- Glutathione S-transferases -- Rat liver -- Organotin c o m p o u n d s -- Inhibition of glutathione S-transferase

INTRODUCTION

Glutathione S-transferases are a family of enzymes located mainly b u t not exclusively in the cytosol of cells. The highest activity is in mammalian liver cytosol but the enzyme(s) have been detected in many tissues. Many different substrates for these enzymes have been identified and amongst these are some organophosphorus c o m p o u n d s which include those in use as insecticides [1,2]. Several reviews on the metabolism of organophosphorus c o m p o u n d s by glutathione S-transferases have appeared recently [3,4]. Studies of these reactions and identification of products have shown that O-alkyl and O-aryl 0009-2797]85]$03.30 © 1985 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

244 conjugation occurs and sometimes conjugation of more complicated leaving groups from the parent organophosphorus compound. In recent years it has been known that dialkyl thionophosphorus insecticides may contain trialkyl phosphorothioates as impurities [5--7]. This group of compounds has proved to be very important in contributing substantially at low concentrations to the toxicity of malathion. Indeed OSSMe(O) is one of the most powerful potentiators of the toxicity of malathion, 0.6% increasing the toxicity of malathion 10-fold [7]. The trialkyl phosphorothioates also possess unusual toxicological properties because they cause lesions in the lung [8--10]. During the course of studies of their toxicity [8--10] and rates of metabolism and disposal in animals [19,20] it was found that OSS-Me(O) was a substrate for glutathione S-transferase(s) present in liver cytosol. This paper contains results of the study of this reaction, identification of the products and some results indicating which of the enzymes are active in catalysing this reaction. MATERIALS AND METHODS

Chemicals OSS-Me(O), OOS-Me(O), OOS-Me(S), OSS-triethyl phosphorodithioate (OS8-Et(O)), OOO-trimethylphosphate (OOO-Me(O)), OO-dimethyl phosphorothioic acid (OO-Me(O)S-), OS-dimethyl phosphorodithioic acid (OS-Me(O)S-), SS-dimethyl phosphorodithioate acid (SS-Me(O)O-), OSdimethyl phosphorothioate acid (OS-Me(O)O-), were synthesised by Drs. J.W. Miles and D.L. Mount of the Bureau of Tropical Diseases, Centre for Disease Control, Atlanta, Georgia, U.S.A. Glutathione, N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid (HEPES buffer) and 5,5'-dithiobis-(2-nitrobenzoic acid) were obtained from Sigma Chemical Co. 1-Chloro-2,4-dinitrobenzene (CDNB) and 1,2-dichloro-4-nitrobenzene (DCNB) were obtained from Aldrich Chemical Co. and recrystallised from ethanol before use. A solution of methyl glutathione was prepared by reaction of methyliodide and glutathione in the presence of rat liver cytosol [11]. Glutathione (500 pmol in 9 ml of 0.1 M HEPES buffer, pH 7.4) and 1 ml rat liver cytosol (from 33% homogenate) was incubated at 40°C with 45 ul methyliodide (720 pmol) with continuous addition of 10 M NaOH to maintain the pH above 7. The glutathione concentration was monitored and when all sulphydryl was removed the solution was extracted with 20 ml CHC13/hexane (1:2). After removal of solvent the aqueous layer was heated to 70°C for 10 min to denature protein, centrifuged and the clear supernatant stored frozen at -40°C.

Enzyme preparations Liver cytosol was prepared from male Sprague--Dawley rats (200--250 g).

245 The rats were killed by decapitation, the liver rinsed in ice cold 0.25 M sucrose, homogenised in a Potter-Elvehjem homogeniser (1 g liver to 2 ml 0.25 M sucrose), and the homogenate was centrifuged at 16 000 X g for 10 min. The resulting supernatant was centrifuged in a Kontron ultracentrifuge at 100 000 X g for 1 h. This supernatant was used directly for enzyme separation studies. For enzyme measurements it was treated with Amberlite IRA-410 resin in the carbonate form to remove free glutathione [18]. A dialysed supernatant may be freeze dried and stored for a considerable time at --40°C without loss of activity. Purification and separation of glutathione S-transferases were carried out essentially by the m e t h o d of Habig et al. [1] with an additional chromatographic step using Sephadex G-100 after ammonium sulphate fractionation. Further purification of peak 'C' obtained by chromatography on CM-cellulose was achieved using hydroxyapatite. T h e hydroxyapatite column (1.2 X 18 cm) was equilibrated~with 0.01 M K-phosphate buffer (pH 6.5). After application of the transferase fraction the column was developed by a linear gradient of 400 ml from 0.01 M K-phosphate (pH 6.5) to 0.3 M K-phosphate (pH 7.0). The volume of each fraction was 5.5 ml. Gel electrophoresis (SDS-polyacrylamide) was performed according to Laemmli [17] to establish the subunit composition of the fractions (cf. Tables III and IV). The nomenclature of glutathione transferases used throughout this paper is taken from Ref. 25.

Measurement o f enzyme activity Most measurements of enzyme activity with CDNB and DCNB as substrate have been made in 0.1 M Hepes buffer adjusted to pH 7.3 with NaOH. The methods used are essentially those described by Habig [1]. The final concentration of reactants were: glutathione (5 mM), CDNB and DCNB (0.6 mM) and the progress of the reaction was monitored at 340 nm at 25°C. Activity in nmol substrate/min was derived from tl~e initial slope of the essentially zero order reaction rate. The extinction coe~fficient used for the calculation of activity for the reaction products of $1utathione with CDNB, and DCNB were 1.08 X 104 and 8.5 X 103 M -1, respectively. Enzyme activity with trialkyl phosphorothioates was measured by the determination of loss of glutathione [12]. The conditions for the most sensitive assay were: 0--1.0 ml enzyme preparation, 0.9--2.0 ml HEPES buffer (0.1 M, pH 7.3), 0.1 ml GSH (24 mM) and 12 ul OSS-Me(O) (density at 20°C, 1.284 g/ml). At various times after incubation at 25°C 0.3 ml samples were added to 30 ~1 perchloric acid (60% w/w). After centrifugation to remove precipitated protein 170 ~1 was taken for the determination of glutathione [12]. In order to neutralise the perchloric acid in the sampIe the concentration of buffer in the Ellman m e t h o d was increased to 0.3 M. The usual control contained enzyme and glutathione only. The rate of loss of glutathione was not zero order and approximated to a first order rate, k (see Results). The initial rate was therefore calculated as follows:

246

Rate (nmol/min/ml enzyme preparation) =

k × [GSH] × RM(ml) Enz (ml)

where k(min -I) = 1st order rate constant determined graphically; [GSH] = concentration of GSH in nmol/ml; RM(ml) = volume of reaction mixture; Enz(ml) = volume of enzyme preparation. The extinction coefficient derived from the reaction of glutathione with 5,5'-dithiobis-(2-nitrobenzoic acid) used for the calculation of activity is 1.32 × 10 -4. N M R studies IH-NMR spectra were recorded at 100 MHz on a JEOL FX-100 spectrometer at 297 K on samples in D20 (0.4 ml), using 8K data-points, 1000 Hz spectral width, a 60 ° pulse, a pulse repetition time of 8 s, and, typically, 20--100 scans. The signal from the CH3-protons appears as a doublet in the NMR spectrum of the methyl derivatives due to spin-spin coupling with the phosphorus atom in the diester. In the spectra of ethyl derivatives, only the quartet of the --CH2-- protons is split by the phosphorus atom (Table I). The chemical shift difference between CH30 and CH3S (1.4--1.5 ppm} and between O--CH2-- and S--CH2-- (1.3 ppm) is quite large (Table I). Since the oxygen is more electronegative than the sulphur the O containing groups are more shielded. From the spectrum of a mixture of all the diesters in one sample both the CH3S-- and the CH30 signals from the different c o m p o u n d s can be distinguished. Therefore ~H-NMR has been used to identify the reaction products and in some cases to provide a semi-quantitative estimate of enzyme activity to compare with the decrease in glutathione concentration. The signal from the CH3S-protons in methylglutathione did n o t overlap with those from CH3S-protons in diesters and has served to identify the other p r o d u c t of the reaction. The solution after the enzymic reaction had taken place was extracted with approx. 3 vol. of solvent (CHC13/hexane, 1:2}, the upper solvent layer removed and the extraction repeated 2 times. The distribution coefficient of OSS-Me(O) between this solvent and HEPES buffer was mesured [13] and shown to be 7.6 (solvent/buffer). By this procedure at least 99.9% o f the substrate should be removed. The aqueous layer was heated to 70°C for 10 min to coagulate proteins, the resulting suspension centrifuged and the clear supernatant freeze-dried. In the 1H-NMR spectra of freeze dried reactant products dissolved in D20 (0.4 ml) the signals from the diesters and methylglutathione showed up as narrow peaks (AI~ ~ 1 Hz) and were well separated from buffer signals. For each spectrum the areas of these resonances, as measured by a planimeter in spectra recorded on an expanded scale, were compared with the areas of the resonances arising from an external standard of the corresponding diester in D20 (2 mg/0.4 ml) in a separate NMR tube. By running the

247

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248 spectra consecutively under exactly the same instrumental settings, and taking the memory parameters of the computer into account, the concentrations of the various diesters and methylglutathione in the reactant products could be related to that in the standard. A control was also made by adding to one sample known amounts of the diester found and checking that the intensity of the NMR lines increased proportionally. Special care was taken to ensure than none of the resonances was saturated. Under the conditions used, the areas of the resonances were directly proportional to the concentration of the protons producing them. RESULTS

Liver cytosol Glutathione and OSS-Me(O) do not react together at a measurable rate at pH 7.4 and 37°C at 5--10 mM concentrations. When cytosol from rat liver is added the reaction rate is increased. These results show that with 10 mM OSS-Me(O) the loss of glutathione is not linear with time {Fig. 1A). A logarithmic plot (Fig. 1B) gives a more linear plot and the apparent 1st

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Fig. 1. K i n e t i c s o f the reaction o f g l u t a t h i o n e and O S S - M e ( O ) in the p r e s e n c e o f liver

cytosol.

A: loss o f g l u t a t h i o n e as a f u n c t i o n o f e n z y m e c o n c e n t r a t i o n . B: data f r o m 1st order c o n s t a n t k and e n z y m e c o n c e n t r a t i o n . D: l i n e w e a v e r - B u r k e p l o t s h o w i n g 1st order rate c o n s t a n t s as a f u n c t i o n o f substrate c o n c e n t r a t i o n . Fig. 1 A p l o t t e d logarithmically. C: relation b e t w e e n

249 order constants derived from these results are linearly related to the a m o u n t of enzyme (Fig. 1C). Other experiments have shown that the reaction is n o t strictly 1st order and under the experimental conditions when the concentration of both reactants is decreasing appreciably and products are increasing the kinetic picture is very complicated and requires further study. However the enzyme assay system can be used on the basis indicated in Fig. 1C. Throughout this paper enzyme activity is presented as the initial rate in nmol/unit of enzyme/min derived from the apparent first order constant. Kinetics of the reaction As previously described activity of liver cytosol decreases progressively as the glutathione concentration falls. This could be due to several causes, (1) the enzyme is unstable under the conditions of the incubation, (2) the products of the reaction are inhibiting the reaction and/or (3) the OSSMe(O) substrate is activated by combination with the enzyme and reacts with glutathione in solution. The first option is eliminated by the fact that activity is retained if liver cytosol is preincubated for 2 h with OSS-Me(O) prior to the addition of glutathione. The third option would imply that other thiols would react and experiments have shown that cysteine is completely inactive. Enzyme activity measured as described in Methods and Materials and using OSS-Me(O) as substrate indicate that over a range of glutathione concentrations from 1--10 mM the same initial activity is obtained. Experiments have been carried out with glutathione (1--4 mM) and methyl-S-glutathione (1--8 mM) concentrations. The reaction is inhibited by methyl-Sglutathione and is mainly non-competitive with an approximate Ki of 4--5 mM. Using a crude liver cytosol the Km for OSS-Me(O) is 30 mM (Fig. 1D). As shown later more than one glutathione S-transferase can accept this substance as a substrate. Substrate specificity The activity of liver cytosol has been examined using several different substrates. The results show that amongst the c o m p o u n d s examined OSSMe(O) is the best substrate (Table II). The enzyme activity with other trialkylphosphorothioates as substrate relative to OSS-Me(O) (100) are: OOSMe(O), 26--32; OOS-Me(S), 8--10; SSS-Me(O), 2--5; OSS-Et(O), 1--4 (Table II). The activity with the last two c o m p o u n d s are low and near the lower limit of detection. The substrates have been checked for purity by gas chromatography and mass-spectrometry and we conclude that this low activity is unlikely to be produced by impurities. For SSS-Me(O), unchanged substrate was extracted from the reaction mixture after incubation for 60 min and it was demonstrated that isomerisation to other products, e.g. OSS-Me(S) had not taken place. For comparison the rate of the transfer of a methyl group from methyl iodide [11] is included in Table II.

250 TABLE

II

RELATIVE RATES OF REACTION GLUTATHIONE IN T H E P R E S E N C E

OF TRIALKYL PHOSPHOROTHIOATES OF RAT LIVER CYTOSOL

AND

The rat liver cytosol was derived from a 3 0 % homogenate in a m e d i u m containing 0.15 M sucrose and 0.10 M H E P E S buffer (pH 7.3). The rates are calculated from the 1st order constants for an initialconcentration of glutathione of 1.2 m M and are corrected for any loss of glutathione in a control containing enzyme and glutathione with no trialkylphosphorothioate substrate. The m e a n initialrate of loss of glutathione from the control tubes containing glutathione and enzyme only was 2.5 + 0.6 [12] nmol/min/2 ml of reaction medium. The trialkylphosphorothioate substrates used do not react at a measurable rate with glutathione and the m e a n rate of loss from tubes containing substrate and glutathione was 2.0 + 0.3 [5 ] nmol/min/2 ml reaction medium. Expt. no.

1 2 3 4 5 6

Glutathione 5-transferase (nmol/min/ml

enzyme)

OSS-Me(O) (45 mM)

OOS-Me(O) (45 raM)

OOS-Me(O) (11.2 raM)

SSS-Me(O) (45 raM)

OSS-Et(O) (45 raM)

MeI (10 raM)

250 211 199 151 143 229

-56 62 -46 .

-16.2 19.9 -. .

5.3 --8.2 .

2.7 --5.9

-----

.

. .

. 1421

Fractionation o f rat liver cy tosol The results of fractionation of liver cytosol are shown in Fig. 2. The fractions are designated by the letters assigned by Habig et al. [1]. The fractionation shows three distinct peaks of protein showing activity catalysing the reaction between CDNB and glutathione. Two of these peaks show substantial activity with DCNB as a substrate (peaks 'C' and A). Peaks 'C' and B are active with OSS-Me(O) as substrate while the results for peak A show a rather low activity compared with the activity of samples taken from troughs between peaks. The ratio of activities CDNB/OSS-Me(O) for peaks 'C' and B are approx. 2000 and 900, respectively. In several experiments there was some a s y m m e t r y of the CDNB, DCNB activity and protein in the fraction designated 'C' transferase. Upon further chromatography of this fraction on a hydroxyapatite column it was resolved into t w o fractions. The activity of these two fractions and others resolved on the first fractionation on CM-cellulose are shown in Table III. The distribution of enzyme activity with OSS-Me(O) as substrate is essentially as shown in Fig. 2 and the t w o peaks subfractionated from fraction 'C' are both active. These t w o fractions were perfectly symmetrical with respect to CDNB and DCNB activity. When specific activities were measured using rather more concentrated fractions it is clear that the highest values are obtained for those fractions containing transferase 1 and 2 subunits (Table IV). In terms of enzyme

251 1.8 1.6

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(Nos.)

CDNB activity 10-4x tnmol/min)

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1.53

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DCNB activity 10 -3 x ( nmol / min)

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11.8

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0.66

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4.64

2.43

OSS -Me (0) activity nmol GSH/min)

33

106

4.4

82

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10

7.2

Fig. 2. Fractionation of glutathione S-transferases from rat liver. Activity with different substrates, absorbance wavelength, volume taken for analysis and incubation times are as follows: *, (protein; A2~0m,a; 3.0 ml): o, (CDNB; A340m~; 25 ~l; 1 min) o, (DCNB; A340nm; 0.5 ml; 1 min). The fractionation was using CM-cellulose and a KCI gradient according to the procedure of Habig et al. [1] after preliminary separations described in Methods. Using the most recent nomenclature [25] fraction A is transferase 3-3, fraction B is transferase 1-2 and fraction 'C' is a mixture of transferases 3-4 and 1-1.

activity in liver cytosol the major part of the measured activity is due to glutathione transferases 1-2, 3-4 and 1-1 (Table III).

Inhibition by organotin compounds Glutathione S-transferases are inhibited by triorganotin compounds [21--23], although there are major differences (102--103-fold) in sensitivity to the action of triethyltin [21,23].

252 T A B L E III FRACTIONATION CYTOSOL

OF GLUTATIONE

S-TRANSFERASE

FROM

RAT LIVER

Fractionation was carried out as in Methods with transferase 2-2 in fractions 100-110, transferase 3-3 in fractions. 82-92, transferase 1-2 in fractions 73-81 and transferase 'C' in fractions 55-68 (cf. Fig. 2). Transferase 'C' was refractionated on a hydroxyapatite column to yield transferases 3-4 and 1-1. The subunit composition was established by SDS-electrophoresis. Enzyme activity was measured with OSS-Me(O) as substrate. Transferase (Ref. 25)

Volume of bulked fraction (ml)

Enzyme activity (nmol/min/ml)

Enzyme activity (nmol/min/fraction)

2-2 3-3 1-2 ,C,{3-4 1-1

39 45 39 40 30

1.35 0.80 5.43 2.87 4.76

52.6 36.0 212 115 4 143} 257('C')

E n z y m e activity o f rat liver c y t o s o l with OSS-Me(O) as s u b s t r a t e is n o t sensitive t o i n h i b i t i o n b y several o r g a n o t i n c o m p o u n d s (Table V). It is clear t h a t t h e r e are large d i f f e r e n c e s in t h e sensitivity o f g l u t a t h i o n e S-transferase activity w i t h t h e t h r e e substrates, OSS-Me(O), C D N B a n d DCNB.

Reaction mechanism T h e diester p r o d u c t s o f the r e a c t i o n o f OSS-Me(O) a n d g l u t a t h i o n e m a y be i d e n t i f i e d a n d d e t e r m i n e d b y p r o t o n NMR. In Table V I are s h o w n t h e results f r o m several e x p e r i m e n t s i n d i c a t i n g t h a t w h e n OSS-Me(O) or OOS-Me(O) are substrates the m e t h y l g r o u p is t r a n s f e r r e d f r o m t h e m e t h o x y g r o u p in each case. N o S- c o n t a i n i n g diester was f o u n d . In e a c h case t h e TABLE IV FRACTIONATION OF GLUTATHIONE S-TRANSFERASE-ISOZYMES FROM RAT LIVER AND MEASUREMENT OF THEIR SPECIFIC ACTIVITY WITH OSS-Me(O) AND CDNB AS SUBSTRATES See Table III and Methods for fractionation procedures. Transferases (Ref. 25)

2-2 3-3 1-2 3-4 1-1

Protein (mg/ml)

0.28 1.20 1.58 2.46 1.66

Enzyme activity (nmol/min/mg protein)

OSS-Me(O)

CDNB

17.8 3.4 10.9 2.45 9.17

4090 4780 7310 4990 10070

Ratio CDNB/ OSS-Me(O) 230 1420 671 2034 1098

253 TABLE V INHIBITION OF GLUTATHIONE S-TRANSFERASES OF RAT LIVER CYTOSOL BY TRIORGANOTIN COMPOUNDS All reactions were measured with 5 raM glutathione except those marked* which were with 1.0--1.2 mM. If a sufficient range of concentrations of triorganotin were examined, the I50 is quoted. Otherwise uM concentration (% inhibition) is given. Organotin

Inhibition with various substrates

Triethyltin Diethylphenyltin

OSS-Me(O)

CDNB

DCNB

800 (0%) 800 (66%)* 10 (15%)

5.0 a

0.5

--

0.015

aA very fiat inhibition curve over a range of concentration (10-3--10 -7 M). yield of methyl-S-glutathione approximates to that expected from the Odiester formed. F r o m t h e t w o c h e m i c a l e x p e r i m e n t s in the a b s e n c e of e n z y m e , experim e n t 1 shows t h a t the m e t h y l - S - g l u t a t h i o n e can be d e t e c t e d after the r e a c t i o n w i t h m e t h y l i o d i d e . E x p e r i m e n t 2 is a n a t t e m p t t o s p e e d u p t h e reaction of glutathione and OSS-Me(O) by using higher concentrations of TABLE VI DETERMINATION BY NMR OF THE PRODUCTS OF THE REACTION OF RAT LIVER GLUTATHIONE S-TRANSFERASE AND TRIALKYL PHOSPHOROTHIOATES ND, not done. For details of fractionated liver cytosol cf. Fig. 2. R = methyl or ethyl. Values expressed as umol. No.

Reactants

SS-R(O)O-

OS-R(O)O-

GSMe

--

--

6.6

12.8

--

6.2

--1.6 1.8 --

6.3 3.8 2.2 ND --

Chemical reaction 1. 2.

MeI (9.9 raM) GSH (5.9 mM) OSS-Me(O) (476 mM), GSH (56 raM) (pH 9)

Enzymic reaction (liver cytosol) 3. 4.

OSS-Me(O) OSS-Me(O)

7.2 3.9

5.

OOS-Me(O)

--

6. 7.

OOS-Me(O) OSS-Et(O)

--

--

Enzymic reaction (fractionated liver cytosol) 8. 9.

OSS-Me(O) (peak 'C'; fractions 48--51) OSS-Me(O) (peak B; fraction 60,62)

3.3

3.1

3.6

3.3

254 both reactants and raising the pH. Both methyl-S-glutathione and O- diester were found. The concentration of O- diester found was more than twice that of methyl-S-glutathione, probably indicating that t w o reactions were taking place -- one the reaction of glutathione and OSS-Me(O) and the other hydrolysis of OSS-Me(O) at pH 9 both yielding the O- diester. When glutathione S-transferase fractions 'C' and B were examined (cf. Fig. 2), the expected products were found and only the O- diester from OSS-Me(O) was produced. In one experiment using OSS-Et(O) no reaction products were detected, consistent with the low reaction rates shown in Table II. The sensitivity of the method is also inadequate to determine the products of the very slow reaction with SSS-Me(O) as substrate (Table II). DISCUSSION The results in this paper establish that trialkylphosphorothioates are substrates for at least t w o glutathione S-transferases present in rat liver cytosol. The reaction products have been identified by NMR and indicate that the reaction between glutathione and OSS-Me{O) is: O

O

II

II

CH3S--P--SCH3 + GSH I

O

,

-

CH3S--P--SCH3 + GSMe + H÷ I

O-

f CH3

For both substrates OSS-Me(O) and OOS-Me(O), the methyl group attached to phosphorus via oxygen is transferred to glutathione sulphydryl group (Table VI). The trialkylphosphorothioates are inhibitors of esterases [14--16]. The differing nature of the mechanisms of this reaction, when compared to the transferase reaction, is well illustrated by the fact that the leaving group for esterase inhibition is--SMe [ 15 ]. From the studies reported in this paper the OSS-Me(O), OOS-Me(O) and OOS-Me(S) are shown to be substrates whereas activity with OSSEt(O) is very low. This preference for methyl rather than ethyl group transfer is c o m m o n [3,16] although recent results indicate that this generalisation should be used with caution [24]. The low activity measured with SSS-Me(O) cannot be explained by impurities nor isomerisation during incubation to measure enzyme activity. This result would seem to indicate that a methyl group can be transferred from MeS. However the results obtained with MeO containing c o m p o u n d s (Table II) therefore indicate a marked preference for methyl transfer from this group. The fractionation of rat liver cytosol (Fig. 2, Tables III and IV) show that the predominant contribution to the activity is due to ligandin (1-1) and

255 transferases 1-2 and 3-4 (Table III). However the specific activities of several other glutathione S-transferases indicate that OSS-Me(O) is a rather general substrate for these enzymes (Table IV). The results previously published on the inhibition o f glutathione-S-transferases by triethyltin [21,23] suggest that the subunits 2 and 3 are inhibited by low concentrations ( 1 0 pm). Since the major activity in rat liver cytosol is due to 1-1, 3-4 and 1,2, it is possible that the activity in rat liver cytosol due to OSS-Me(O) is mainly catalysed by subunits 1 and 4. ACKNOWLEDGEMENTS

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