Liver damage induced in rats by malathion impurities

Toxicology Letters, 52

(I 990)35-46

35

Elsevier

TOXLET

02336

Liver damage induced in rats by malathion impurities

Songsarkdi Keadtisukel, Watanaporn Dheranetral, Tsutomu Nakatsugawa2 and T.R. Fukuto’ ‘Department

of Entomology,

University of California, Riverside, CA and zDepartment

and Forest Biology, State University of New York, College of Environmental

of Environmental

Science and Forestry,

Syracuse, NY (U.S.A.) (Received

28 June 1989)

(Accepted

9 February

1990)

Ke.v words’ Malathion phosphorodithioate; deficiency;

impunties;

O&S-Trimethyl

Liver damage;

Hemostattc

disorders

phosphorothioate;

/I-Glucuronidase;

O,S,S-Trimethyl

Blood coagulation

in rats; Prothrombin;

Thrombin;

disorder;

Plasma

factor

Proaccelerin

SUMMARY Admimstration (OOS-Me)

of a single oral dose of the malathion

or O&S-trimethyl

e.g. prolongation

phosphorodithioate

of blood clotting,

II. V and VII was also observed. ronidase

m the blood

OOS-Me

and 40 mg/kg

electrophoresis

with a maxtmum

showed

the blood. Co-treatment tagonist

OSS-Me,

ofg-glucuromdase

and OSS-Me Analysis

of OOS-Me delayed

levels. However,

rettculum

prevented

pretreatment

phosphorothioate

hemostatic

disorders

of rats with piperonyl

with 60 mg/kg

(OOO-Me),

released

into

a potent an-

but had no effect in reduc-

butoxide

phosphorothioates

showed the highest activrty in increasing

Factors of /I-glucu-

by isoelectrofocusing

was the source of this enzyme

released into the blood. Of other O.O,S-trtalkyl

diethyl S-alkyl phosphorothioates

increases

treatment

of serum /I-glucuronidase

with 5% O,O.U-trimethyl toxicity,

following

disorders,

of coagulation

dose-dependent

observed

phosphorothioate

in hemostatic

time. Deficiency

also caused

of 15 and 31-fold

respectively.

O,O,S-trimethyl

to the rat resulted

and thrombin

that the liver endoplasmic

of OOS-Me-induced

ing p-glucuronidase

prothrombin

OOS-Me

impurity,

(OSS-Me),

reduced

the amount

examined,

/I-glucuromdase

the O,O-

levels.

INTRODUCTION

Since the discovery of the delayed toxic activity of the malathion impurities O,O,Strimethyl phosphorothioate (OOS-Me) and O,S,S-trimethyl phosphorodithioate (OSS-Me) over a decade ago [l, 21, a large amount of work has been carried out to Address for correspondence:

S. Keadtisuke,

Department

of Entomology,

University

92521, U.S.A.

0378-4274/90/S

3.50 @ 1990 Elsevier Science Publishers

B.V. (Bromedtcal

Division)

of California,

CA

36

determine the toxicological properties and mode of action of these compounds. When administered orally to rats at doses of 15-60 mg/kg OOS-Me and OSS-Me caused weight loss, red staining around the nose and mouth, diarrhea, incontinence, and delayed death occurring as late as 3 weeks after dosing [3, 41. A variety of other toxicological effects was also observed, including hyperaminoaciduria [5], dysproteinuria [6] and ancillary effects suggesting kidney proximal tubule damage as the primary mode of action. Moreover, evidence for lung damage by OSS-Me and related organophosphorus esters has also been reported [7]. In addition to those effects cited above, there is evidence to suggest the liver as a possible target organ of OOS-Me and OSS-Me. This includes in vivo and in vitro inhibition of liver carboxylesterase by OOS-Me and OSS-Me [8], reduction of tissue glutathione levels [9], and internal hemorrhaging as evidenced by staining around the nose and mouth. Since glutathione and the protein factors responsible for blood coagulation are synthesized in the liver, work was carried out to determine the effect of OOS-Me and OSS-Me on related liver biochemical parameters including examination of plasma glutamate oxaloacetate transaminase (GOT), plasma glutamate pyruvate transaminase (GPT), hemostatic disorders and serum j?-glucuronidase, and the results are presented. MATERIALS

AND METHODS

Animals

Male Sprague-Dawley rats (12&l 80 g) were purchased from Simonsen Laboratories, Gilroy, California, or bred from the same animals in the departmental facility. Rats were housed in stainless steel cages in an air-conditioned room maintained at 22°C and kept under an alternating 12 h light/dark cycle. Food and water were available ad libitum except for 16 h prior to treatment with the test chemicals when food was withdrawn. Treatment

For the blood coagulation assays, OOS-Me dissolved in corn oil (20 mg/ml) was administered orally to rats at doses of 10, 20, 40 and 60 mg/kg. Animals were also treated with 60 mg/kg OOS-Me adulterated with 5% of the antagonist OOO-Me. Control rats, which received corn oil only (2 ml/kg), were treated at the same time as those with the OOS-Me and OOS-Me+OOO-Me. For the P-glucuronidase determinations, OOS-Me and OSS-Me dissolved in corn oil were administered orally to rats at doses of 20, 40 and 60 mg/kg and 10, 20 and 40 mg/kg, respectively. Rats also were pretreated orally with 50 mg/kg piperonyl butoxide 30 min prior to oral administration of 40 mg/kg OSS-Me or its analogs. Bleeding time assays

Blood samples taken from the incised tail of treated or control animals were col-

37

lected in small capillary tubes (Drummond Scientific Co.). The capillary tube was then gently broken with the aid of a small file every 5-10 s until the strands of fibrin appeared [lo]. Plasma assays

Blood samples (9 parts) taken from the tail veins of treated or control animals were collected in capillary tubes (Netelson, plain) containing one part 3.8% aqueous sodium citrate. The samples were centrifuged in a desk top centrifuge at 2500 x g for 10 min and the plasma fractions were used immediately for assays described. Prothrombin and thrombin times and plasma factors deficiency tests were determined by use of kits purchased from the Sigma Chemical Co., St. Louis, MO [I 11. Prothrombin time test

One part of plasma from OOS-Me- or corn-oil-treated rats was equilibrated for at least 3 min at 37°C. After 3 min the 1:l mixture of thromboplastin solution (Sigma) and 25 mmol/l calcium chloride was added to the plasma and the clotting time was recorded. Fibrinogen time test

Preparation of 1:5 dilutions of OOS-Me- or corn-oil-treated plasma with 28 mmol/l barbital buffer (pH 7.4), containing 1k bovine albumin and 125 mmol/l sodium chloride was used for the fibrinogen time test. Two parts of a 1:5 dilution of test samples were incubated at 37°C for 1 min and one part of 100 NIH units/ml thrombin was then added to the test sample. The clotting times were recorded. Plasma deJicient factor assays

One part of a 1:10 dilution of plasma obtained from rats treated with 30 mg/kg OOS-Me- or corn-oil-treated plasma, one part of thromboplastin solution and one part of factor-deficient plasma were mixed well at 37°C. One part of 25 mmol/l calcium chloride was then added to the reaction mixture and the clotting time was recorded. The serial dilutions of control plasma were assayed in the same manner and calibration curves were constructed by plotting log % ‘P and P’ (plasma dilutions in percentage) activities against log clotting times. The percentage of plasma deficient factors was calculated. Serum P-glucuronidase

Serum P-glucuronidase levels were also determined by means of a Sigma kit which used phenolphthalein-mono-B-glucuronic acid as substrate [12]. The intensity of the red color of the liberated phenolphthalein in standard AMP (2-amino-2-methyl-lpropanol) buffer (pH 11) was measured at 550 nm in a Spectronic lOOI-plus spectrophotometer. /I-Glucuronidase isoenzyme determination

Blood samples from control rats and rats treated orally with 60 mg/kg OOS-Me or 40 mg/kg OSS-Me were obtained 2 h after dosing. The serum fraction (10 pl),

38

diluted with an equal volume of 9.5 M urea in 5 mM Tris-HCl buffer (pH 7.5), was transferred to a well of a polyacrylamide gel consisting of 3.446 polyacrylamide and 1.25% amphiline (pH 5-7) in 6 M urea. The lower electrode solution was made up of 6 M urea in 0.1 M sulfuric acid and the upper electrode solution consisted of 0.25 M sodium hydroxide. Isoelectric focussing electrophoresis of the serum /J-glucuronidase was carried out at a constant voltage of 300 V for 2 h. Serial sections of the edge of the gel were eluted with minimum amounts of water for pH determinations [13-151 and the pH values were related to the position of the /I-glucuronidase isoenzymes. Plasma glutamate oxaloacetate transaminase (GOT) transaminase (GPT) assays

and plasma glutamate pyruvate

Plasma GOT and GPT levels were determined by means of a Sigma kit. Chemicals

O,O,S-Trimethyl phosphorothioate 1 (OOS-Me), O,O,O-trimethyl phosphorothioate (OOO-Me), O&S-trimethyl phosphorodithioate 2 (OSS-Me) [16], O,O-dimethyl S-ethyl phosphorothioate 3, O,O-dimethyl S-i-propyl phosphorothioate 4, O,O-diethyl S-methyl phosphorothioate 5, O,O-diethyl S-n-propyl phosphorothioate 6, O,O-diethyl S-i-propyl phosphorothioate 7, O,O-diethyl S-t-butyl phosphorothioate 8, and O,O,S-tri-n-propyl phosphorothioate 9 were available from earlier studies [17]. Piperonyl butoxide was purchased from the Aldrich Chemical Co. and atropine from the Sigma Chemical Co. RESULTS

Hemostasis

Dose-response data showing the effects of OOS-Me and OOS-Me containing 5% antagonist OOO-Me in inducing hemostatic disorders in rats after a single oral dose are presented in Table I. Prolongation of blood clotting, prothrombin and thrombin times were observed after the animals were orally administered 40, 30,20 and 10 mg/ kg OOS-Me. Hemostatic disorders were dose-dependent with maximum effects observed at 40 mg/kg, the highest dose tested. At this dose, bleeding, prothrombin and thrombin times were 1.35-, 1.72- and 2.1-fold greater than that observed for control rats. As the results in Table I indicate, except for those animals that died, all hemostatic parameters returned to normal 9 days after dosing. Although data are not presented here, prolongation of bleeding time of greater than 2-fold was observed when rats were treated with 40 mg/kg OSS-Me, the phosphorodithioate analog of OOSMe. OOO-Me, the phosphorothioate isomer of OOS-Me previously shown to be a potent antagonist of the delayed toxic action of OOS-Me [l, 3,4] also showed protective action in preventing prolongation of bleeding, prothrombin and thrombin times. The

39 TABLE

I

EFFECT BLOOD

OF

MALATHION

HEMOSTATIC

IMPURITIES,

PARAMETERS

00%Me

BY USING

AND

ITS

ONE-STAGE

ANTAGONIST CLOTTING

OOO-Me ASSAYS

ON

(IN SEC-

ONDS) Days

Blood clotting

Prothrombin

Fibrinogen

after treatment

time

time

time

Corn oil

41.7kO.6

16.3kO.5

18.8kO.8

2 ml/kg

4l.lkO.4

16.3kO.5

18.5+0.9

41.5kO.3

16.3kO.5

18.3kO.5

Treatment

00%Me

56.2+

32.8k2.2

39.4k4.0

40 mg/kg

died

died

died

died

died

died

28.0& 3.9

32.8 +4.0

21.2k2.8

31.4k2.5

41.6+0.5

16.3 +0.7

18.4kO.6

OOS-Me

48.3kl.5

30 mg/kg

44.8+

46.4k2.3

28.Ok4.5

29.Ok5.4

45.0+4.0

24.4k2.5

32.4k2.7

9

41.8+0.5

17.0+_0.7

18.6kO.5

43.9kO.9

19.8kl.l

21.2k3.1

6

42.6fO.l

18.6kO.9

20.2kO.5

9

41.5kO.3

16.4kO.6

18.2kO.8

41.6kO.2

16.7kO.5

18.6+0.6

6

41.3kO.6

16.OkO.7

18.2kO.5

9

41.1 kO.5

16.OkO.7

16.9kO.7

4l.lkO.5

16.7kO.7

18.4kO.6

OOS-Me

10w/k OOO-Me 3 mglkg

00-Me

1.4

6

OOS-Me 20 mg/kg

1.6

3 mg/kg

+ OOS-Me

6

41.2kO.4

15.9kO.2

17.9kO.7

40 mg/kg

9

41.2kO.6

15.4kO.6

16.5* 1.2

Values are means + 2 SE (n = 5 for both control

and treatment).

data in Table I reveal that the hemostatic parameters for blood sampled from animals treated with 40 mg/kg OOS-Me contaminated with 5% OOO-Me were virtually identical to those of blood from control animals. Other hemostatic parameters examined were the plasma factors required for blood coagulation (Table II). Compared to control values, the results obtained from 30 mg/ kg OOS-Me-treated rats showed significant increases in blood coagulation time from 14.8 to 18.2 s (33% Factor V deficiency) and 18.2 to 24.0 s (34% Factor II and VII deficiency) when Factor V (proaccelerin) was tested alone or Factor II (prothrombin) and Factor VII (proconvertin) were tested in combination, respectively. While small increases in blood coagulation times were observed when Factor VII was tested alone (6% deficiency) or in combination with Factor II and X (12% deficiency), the differences between control values and those obtained with the blood of OOS-Me-treated animals were not significant. Since the proteins associated with Factor II and Factor

40

V are synthesized in the liver, the results point out the damage of the liver endoplasmic reticulum as a possible secondary site of action of OOS-Me. B-Glucuronidase

The effect of different oral doses of OOS-Me (20, 60 mg/kg) and OSS-Me (20, 40 mg/kg) in inducing the increase of plasma /3-glucuronidase at different time intervals following oral administration is presented graphically in Figure l(A, B). The figures clearly reveal a rapid, dose-dependent effect in the release of b-glucuronidase into the blood with maximum increases of 15- and 3 l-fold observed following treatment with 60 mg/kg OOS-Me and 40 mg/kg OSS-Me, respectively. These increases were observed 4 h following treatment with OOS-Me and 1 h following OSS-Me. Drop-off in p-glucuronidase levels in both cases was less rapid with a gradual decline, reaching control values 24 h after dosing. Oral pretreatment of rats with 100 mg/kg piperonyl butoxide, a known inhibitor of the mixed-function oxidases, 30 min prior to treatment with 40 mg/kg OSS-Me resulted in a substantial lowering of the maximum release of /I-glucuronidase into the blood. In contrast to its marked effect in reducing hyperaminoaciduria when administered in conjunction with OOS-Me [5], OOO-Me had relatively little effect in reducing serum /?-glucuronidase levels when it was co-administered with OOS-Me (Table III). Although there appears to be a difference in /I-glucuronidase levels at the 4-h period when OOS-Me is used alone and when it contains 596OOO-Me, the difference is inTABLE EFFECT

II OF OOS-Me

ON ONE STAGE

PROTHROMBIN

V, VII, II AND VII, AND II, VII AND X PLASMA Treatment

Days after

Clotting

CLOTTING

ASSAYS

FOR

FACTORS

DEFICIENCY

times (s) for plasma

deficient factors

treatment V

VII

II,VII

II,VII,X

19.6kO.9

24.Ok2.6

63.258.7

OOS-ME 30 mg/kg

3

1s.2+

3

33.0%

6.0%

34.0%

12.0%

3

14.8kO.5

18.8+0.5

18.2kO.3

56.6& 1.5

0%

0%

0%

0%

1.1

OOS-Me* 30 mg/kg plasma deficient factors Corn oil 2 d/kg Corn oil plasma* deficient factors *The percentage

of plasma

deficiency

% ‘P and P’ activities were plotted Values are means & 2 SE (n = 5).

of the treatment

against

log clotting

was obtained times.

from calibration

curves which log

41

significant compared to the effect of OOO-Me in antagonizing 00%Me-induced aminoaciduria. Data showing the effect of other O,O,S-trialkyl phosphorothioates in increasing serum /I-glucuronidase levels in rats are given in Table IV. While the data reveal wide variations in /I-glucuronidase levels, among those compounds examined the highest activity was observed with the O,O-diethyl S-alkyl phosphorothioates. This is in contrast to the delayed toxic activity of these compounds where the highest activity was found among the O,O-dimethyl S-alkyl phosphorothioates. Serum /I-glucuronidase obtained from rats treated with either OOS-Me or OSS-Me were examined by means of isoelectrofocussing electrophoresis and the results are

I 10

0

20

30

Hours

1

4

12

24 Hours

Fig. 1. (A) The levels of serum ,!-glucuronidase 40 mg/kg

OSS-Me+

p-glucuronidase

100 mg/kg piperonyl in rats treated

in rats treated

butoxide

with 20 mg/kg (+),

(W) and 2 ml/kg (0)

40 mg/kg (0)

OSS-Me,

corn oil. (B) The levels of serum

with 20 mg/kg ( q), 40 mg/kg ( n ) OOS-Me

and 2 ml/kg (0)

corn oil.

42

TABLE

III

EFFECT

OF OOS-Me

AND ITS ANTAGONIST

ELS AT 1,4 AND 8 HOURS

AFTER

OOO-Me

ON SERUM

B-GLUCURONIDASE

LEV-

TREATMENT

fi-Glucuronidase

Treatment

(Sigma units/ml)

OOS-ME

60 mg/kg

OOS-Me

60 mg/kg

+OOO-Me

lh

4h

8h

202.6k78.9

1465.2 + 200.2

1095.6*

210.5 k 38.5

1836.8k291.1

172.5

(5%)

3 mg/kg

906.8k210.7

Values are means & 2 SE (n = 5). TABLE SERUM

IV /3-GLUCURONIDASE

40 mg/kg OOS-Me,

OSS-Me

ACTIVITY AND THEIR

Compounds

AFTER

1.4 AND 8 h OF A SINGLE

ORAL

DOSE OF

ANALOGS

p-Glucuronidase (Sigma units/ml) lh

4h

8h

0

II 2. (CH,O)rP-SCH,

146.6

+

59.4

1270.0

+ 242.8

903.0

f 169.0

833.0

k295.1

(OOS-Me) 0 2. CH30P-(SCH&

3115.5

k698.1

1165.OOk395.2

(OOS-Me) 0 3. (CH,O)j’-SC,H,

153.0

& 36.8

0 II 4. (CHj0)2P-S-i-C1H,

517.5

k236.4

332.7

& 123.1

335.7

& 144.2

1894.2

k291.5

1946.6

k259.2

744.0

k 124.7

6. (C2HS0)2P-S-n-C,H,

4646.4

k 590.6

2600.3

+ 706.5

672.0

k209.5

0 II 7. (C2HS0)2P-S-i-CxH,

4063.2

+ 597.0

1283.4

k316.5

484.8

k 239.0

0 II 8. (CZHS0)2P-S-z-C4H9

2577.6

k 784.07

1105.0

*255.47

384.0

+ 158.29

1969.9 107.1

k493.4 + 21.4

1016.4

k220.6

316.8

k220.6

88.5Ok

24.9

97.80*

35.7

0 II

5. (CrH30)$‘-SCH, 0

0 I, 9. (nC,H,0)2P-S-n-C,H, 10. Corn oil 2 ml/kg Values are means f 2 SE. Compounds

1-9, n = 5; compound

10, n = 12.

43

shown in Figure 2. The isofocussing points (PI) for the multiple forms of serum /Iglucuronidase were localized within the region of pH 6.4-6.8, suggesting that the source of this enzyme was the endoplasmic reticulum [18, 191. Plasma glutamate oxaloacetate transaminase (GPT)

transaminase

(GOT)

and plasma glutamate

pyruvate

Table V summarizes the effects of OSS-Me and OOS-Me on plasma transaminase activities at different time intervals. Slight but statistically significant increases of GOT and GPT were observed after a single oral dose of 40 mg/kg OSS-Me or 60 mg/kg OOS-Me. However, the changes in GOT and GPT levels induced by 00%Me or OSS-Me were minor compared to the changes observed in serum /3-glucuronidase levels.

c

+

Fig. 2. Gel isoelectric

focussing

of Serum p-glucuronidase

Me (Lane A) and 60 mg/kg OOS-Me

obtained

from rats treated

pH 6.8

pH6.4

with 40 mg/kg OSS-

(Lane B) having pI range of 6.4-6.8.

44

DISCUSSION

The hemostatic disorders observed, i.e. prolongation of bleeding, prothrombin and thrombin times, and decreases in plasma protein factors for blood coagulation (Factors II, V and VII), following a single oral dose of either OOS-Me or OSS-Me provided a reasonable explanation for the red staining around the mouth and nose and the gastrointestinal hemorrhaging noted in rats treated with these compounds. Further, since these plasma protein factors originate in the liver endoplasmic reticulum, the blood coagulating abnormalities observed suggest this subcellular organelle to be one of the target sites of these organophosphorus impurities. A slight increase in levels of plasma glutamate oxaloacetate transaminase (GOT) and plasma glutamate pyruvate transaminase (GTP) was noted following treatment with either OOS-Me and OSS-Me. In contrast, serum p-glucuronidase levels in rats treated with OOS-Me or OSS-Me were markedly different from control values with maximum differences of 15 and 3 1-fold, respectively. Massive, selective increases in serum /I-glucuronidase following intravenous administration of the potent phosphorylating agents paraoxon (diethyl p-nitrophenyl phosphate) and DFP (diisopropyl phosphorofluoridate) to rats have been reported and evidence has been presented to indicate the liver as the source of this enzyme [18]. Multiple forms of this enzyme are present in the lysosome and endoplasmic reticulum which have been separated by isoelectrofocussing electrophoresis, with the endoplasmic reticulum p-glucuronidase having an isofocussing point (PI) of 6.7 and the lysosomal isozymes displaying a multiplicity over a pH range of 5.6-6.1 [14, 181. Since the serum /I-glucuronidase obtained from rats treated with OOS-Me or OSSMe showed a p1 range of 6.4-6.8, well out of the lysosomal /I-glucuronidase range, TABLE

V

EFFECT

OF MALATHION

PLASMA

ENZYMES,

PYRUVATE Treatment

IMPURITIES,

GLUTAMATE

TRANSAMINASE Plasma

OSS-Me-

OXALOACETATE

AND

OOS-Me-INDUCED

TRANSAMINASE

ELEVATION AND GLUTAMATE

IN RAT Plasma

GOT (units/ml)

GPT (units/ml)

2h

4h

8h

2h

4h

8h

112.4_+25.4

113.6k26.2

113.2k28.6

17.6_+ 5.6

22.4*

4.3

17.0*

3.1

40.2*

16.6

OSS-Me 40 mglkg Corn oil

94.0*

3.7

27.8_+

8.7

35.8+

13.2

00%Me 60 mg/k

108.0+21.97

142.2k12.0

Values are means + 2 SE (n = 5).

130.0+35.18

32.2+

17.3

IN

45

the enzyme released by these organophosphorus impurities probably originates in the endoplasmic reticulum. The results, therefore, clearly point to damage of the endoplasmic reticulum by OOS-Me and OSS-Me. The observation that plasma GOT and GPT levels are only slightly increased indicates a specific effect by these compounds on the endoplasmic reticulum with little effect on the cytoplasm. The data in Table IV, which shows the effect of different O,O,S-trialkyl phosphorothioates on serum p-glucuronidase levels, is of interest. Compared to the diethyl S-alkyl phosphorothioates, lower levels of serum /%glucuronidase were found following treatment with the dimethyl esters and the dimethyl S-ethyl analog (3) had virtually no effect in raising /?-glucuronidase levels. This was somewhat surprising since 3, with a rat oral LDso of 18 mg/kg, is highly toxic to rats with delayed toxic properties similar to those of OOS-Me [ 161.The highest activity in increasing serum p-glucuronidase levels was found with the diethyl S-n-propyl and S-i-propyl phosphorothioates (6 and 7) where greater than 40-fold increases were observed. These compounds, with respective rat LDso values of 49 and 179 mg/kg [ 171, are substantially less toxic to rats with little or no delayed toxic activity. Thus, while serum /3glucuronidase may be useful in estimating endoplasmic reticulum damage [15, 18, 191,it appears to be less useful as a marker for delayed toxic activity. There is ample evidence to indicate the kidney as the primary target organ of OOSMe and OSS-Me [20]. Based on the signs of poisoning elicited by OOS-Me or OSSMe and the direct correlation observed between these poisoning signs and toxicological effects associated with kidney proximal tubule damage, the primary target for intoxication remains the kidney. However, the results presented here show that the liver, particularly its endoplasmic reticulum, is also adversely affected by these compounds. ACKNOWLEDGEMENT

This investigation was supported by U.S. Public Health Service Grant ES-002225. REFERENCES 1 Umetsu, N., Grose, F.H., Allahyari, R., Abu-El-Haj, S. and Fukuto, T.R. (1977) Effect of impurities on the mammalian toxicity of technical malathion and acephate. J. Agric. Food Chem., 25946-953. 2 Hammond, P.S., Badawy, S.M.A., March, R.B. and Fukuto, T.R. (1982) Delayed acute toxicity of 0,&S-trimethyl phosphorodithioate and O,O,S-trimethyl phosphorothioate to the rat. Pestic. Biothem. Physiol., 18,90-100. 3 Umetsu, N., Mallipudi, N.M., Toia, R.F., March, R.B. and Fukuto, T.R. (1981) Toxicological properties of phosphorothioate and related esters present as impurities in technical organophosphorus insecticides, J. Toxicol. Environ, Health, 7,481497. 4 Umetsu, N., Toia, R.F., Mallipudi, N.M., March, R.B. and Fukuto, T.R. (1979) A novel antagonistic effect to the toxicity in the rat of O,O,S-trimethyl phosphorothioate by its phosphorothioate isomers. J. Agric. Food Chem. 27,1423-1425. 5 Keadtisuke, S. and Fukuto, T.R. (1986) Hyperaminoaciduria induced in rats by O,O,S-trimethyl phosphorothioate, Pestic. Biochem. Physiol. 26, 375-381.

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