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The effect of glutathione monoethyl ester on the potentiation of the acute toxicity of methyl parathion, methyl paraoxon or fenitrothion by diethyl maleate in the mouse
Toxicology Letters, 55 (1991) 17-83 Elsevier TOXLET
02492
The effect of glutathione monoethyl ester on the potentiation of the acute toxicity of methyl parathion, methyl paraoxon or fenitrothion by diethyl maleate in the mouse
Lester G. Sultatos, Guo-jie Huang, Olin Jackson, Kenneth Reed and Thomas M. Soranno Department
of Pharmacology
and Toxicology,
University of Medicine and Dentistry
of New Jersey,
Newark, NJ (U.S.A.) (Received
15 December
(Accepted
6 August
1989)
1990)
Key words: Methyl parathion; Organophosphate
Methyl paraoxon;
Fenitrothion;
Diethyl maleate;
Glutathione;
biotransformation
SUMMARY Depletion
of hepatic
to potentiate er, certain
glutathione
the acute toxicities studies
in the mouse by pretreatment
of many dimethyl-substituted
have raised doubts
methyl parathion
regarding
of this insecticide.
The present
study evaluates
methyl paraoxon,
One hour following
pretreatment
tration
monoethyl
of glutathione
more,
glutathione,
paraoxon,
of glutathione
or fenitrothion.
methyl paraoxon
and Dentistry
0378-4274/91/%
that DEM potentiates
of animals
indicate
Lester G. Sultatos, of New Jersey,
of the lethality
to a challenge
that DEM
potentiates
unrelated
to hepatic
Department
185 South Orange
3.50 @ 1991 Elsevier Science Publishers
depletion.
was markedly
depleted Adminis-
mice attenuated
DEM-deglutathione
of these insecticides. hepatic
Further-
glutathione
dose of methyl parathion, the toxicity glutathione
Newark,
B.V. (Biomedical
of methyl
levels, methyl
parathion,
content.
and Toxicology, NJ 07103-2757,
Division)
of
potentiation
the acute toxicities
levels. However,
of Pharmacology Avenue,
Howev-
were potentiated.
ester to naive mice increased
succumbing
by a mechanism
and fenitrothion
at or above control
potentiation
is known
other than glutathione
p.o.) to DEM-pretreated
glutathione
monoethyl
These data
or fenitrothion
Address for correspondence: of Medicine
ester (20 mmol/kg
or maintained
but did not affect the percentage
of action of DEM-induced
by a mechanism
methyl paraoxon
ester did not alter the DEM-induced
administration
the hypothesis
(DEM)
insecticides.
in the detoxification
of mice with DEM (0.75 ml/kg i.p.), glutathione
of methyl parathion,
pletion of hepatic
maleate
of glutathione
mechanism
and fenitrothion
and the acute toxicities
monoethyl
the participation
in the mouse, and hence the putative
of methyl parathion,
with diethyl
organothiophosphate
University U.S.A.
78
INTRODUCTION
Pretreatment of mice with diethyl maleate (DEM) is known to markedly reduce hepatic glutathione levels and to potentiate the acute toxicity of certain dimethyl-substituted organothiophosphate insecticides like methyl parathion and methyl chlorpyrifos [ 1,2]. Additionally these insecticides have been shown to undergo biotransformation by glutathione-dependent transferases in vitro [3]. However, several studies have raised doubts regarding the participation of glutathione in the detoxification of certain dimethyl-substituted organothiophosphate insecticides in vivo, and hence the putative mechanism by which DEM potentiates the toxicities of these chemicals. For example, depletion of glutathione in mice by pretreatment with acetaminophen had no effect on the acute toxicities of the dimethyl-substituted organothiophosphates fenitrothion, dichlorvos and methyl chlorpyrifos [4,5]. Similarly reduction of hepatic glutathione by pretreatment with buthionine sulfoximine did not alter the acute toxicities of methyl parathion or azinphos-methyl [6]. The present report extends these observations by evaluating the effects of the reduction of hepatic glutathione by treatment with buthionine sulfoximine on the acute toxicities of methyl parathion, methyl paraoxon and fenitrothion. These insecticides were chosen since numerous investigators have documented glutathione-dependent detoxification of these compounds in vitro [3]. Moreover, since DEM is known to exert many effects other than glutathione depletion [&8] the present study examines the hypothesis that DEM pretreatment of mice potentiates the acute toxicity of these insecticides by a mechanism other than depletion of hepatic glutathione. METHODS
Chemicals Methyl parathion (O,O-dimethyl O-p-nitrophenyl phosphorothioate) and fenitrothion [O,O-dimethyl-O-(4-nitro-m-tolyl)phosphorothioate] were purchased from Chem Service, Inc. (West Chester, PA). Methyl paraoxon (O,O-dimethyl O-p-nitrophenyl phosphate) was synthesized by the method of Hollingworth et al. [9], as described by Benke et al. [lo]. Glutathione monoethyl ester was synthesized as previously described [l l] except that sulfuric acid was used in place of hydrogen chloride [ 121.Diethyl maleate and buthionine sulfoximine were-purchased from Sigma Chemical Co. (St. Louis, MO). Animals andpretreatments Male Hla:(SW)BR Swiss Webster mice (20-30g) obtained from Hilltop Lab Animals, Inc. (Scottdale, PA) were used in all experiments. They were housed under standard laboratory conditions at the Animal Care facility at the University of Medicine and Dentistry of New Jersey, and had free access to water and feed (Purina Laboratory Rodent Chow 5001). Methyl parathion was administered intraperitoneally
79
{at a dose of 15 mg/kg in 10% DMSO in corn oil) at a volume of 1 ml/kg. Methyl paraoxon was administered in an identical manner, except that the dose was 5 mg/kg. Fenitrothion was administered directly, intraperitoneally, at a dose of 800 mg/kg. Buthionine sulfoximine was administered in the drinking water for 15 days at a concentration of 20 mmol, followed by intraperitoneal supplemental injections of 8 mmol/kg as previously outlined [6]. Diethyl maleate was administered intraperitoneally at a dose of 1 or 0.75 ml/kg. Glutathione monoethyl ester was initially dissolved in the smallest volume of water possible (usually giving a final concentration of 1 g/23 ml). The pH of the solution, which was highly acidic, was adjusted to a pH of 5-7 by addition of sodium hydroxide [1 11. This final solution was administered by stomach tube to give a dose of 20 mmol/kg. In those experiments in which mice received both diethyl maleate and glutathione monoethyl ester, the ester was administered immediately after diethyl maleate. Animals receiving diethyl maleate, glutathione monoethyl ester, and insecticide, were challenged with insecticide 1 h following administration of the other two chemicals. Mice were observed for 3 days following insecticide administration since preliminary studies indicated mice that died did so within 3 days after insecticide challenge. These studies were reviewed and approved by the Institutional Animal Care and Use Committee at the University of Medicine and Dentistry of New Jersey. Glutathione determinations Total glutathione was assayed by the enzymatic recycling procedure [13] as modified by Griffith [14] and described by Anderson [15]. The rate of 2-nitro-5-thiobenzoic acid formation in each sample was monitored at 412 nm and the glutathione present was evaluated by comparison with a standard curve. 18
1
I **
1
2 TIME
3 AFTER
4 ADMINISTRATION
I
5
6
(h)
Fig. 1.The effects of BSO (V), DEM (0) or DEM + giutathione monoethyl ester (A) on hepatie glutathione content in the mouse. Controls (I) received no treatment. Each point represents the mean + SD from 4 mice. An asterisk (*) indicates a significant difference (PiO.05) from the corresponding control, whereas a double asterisk (**) indicates a significant difference (PiO.05) from the corresponding control and the corresponding DEM-treated group. Statistical anaiyses were performed by a f-way analysis of variance, followed by the Neuman-Keuls test [16].
80
100
60
x ;?i 5
60
5 4
40
8? 20
0/
Control
OEM
BSO
OEM
L
Ester
+ Ester
Fig. 2. Effects of DEM, BSO, DEM + glutathione monoethyl ester, or glutathione monoethyl ester alone, on the lethality of methyl parathion (0) methyl paraoxon (H), or fenitrothion (W) in the mouse. An asterisk (*) indicates a significant difference from the corresponding control group by the Friedman’s Block/Treatment test followed by the Two Sample Proportion test [16,17].
Statistical analyses Parametric analysis utilized at l- or 2-way analysis of variance followed by the Neuman-Keuls test, whereas non-parametric analysis utilized either the KruskalWallis test or the Friedman’s Block/Treatment test followed by the Two Sample Proportion test [16,17]. All analyses were performed on an IBM XT computer, using the software NCSS (NCSS, Kaysville, Utah). RESULTS
As previously reported [I], pretreatment
g
7
4 “0.00
of mice with DEM markedly depleted he-
I
0.25
DIETHYL
0.50
MALEATE
0.75
1 .oo
DOSE (ml/kg)
Fig. 3. Reduction of mouse liver wet weights by DEM (0) or by DEM + glutathione monoethyl ester (A). Each point represents the mean f SD of at least 6 mice. An asterisk (*) indicates a significant difference (P-c 0.05) from the group not receiving DEM. Statistical analyses were performed by a l-way analysis of variance followed by the Neuman-Keuls test [16].
81
patic glutathione levels and potentiated the acute toxicity of methyl parathion, methyl paraoxon and fenitrothion (Figs. 1 and 2). Additionally, liver wet weights were slightly reduced following treatment with DEM (Fig. 3). Conversely, treatment of mice with buthionine sulfoximine lowered hepatic glutathione levels (Fig. 1), but had no effect on the acute toxicities of methyl parathion, methyl paraoxon or fenitrothion (Fig. 2). One hour after co-administration of glutathione monoethyl ester (20 mmol/kg p.o.) with DEM, although hepatic glutathione levels were slightly lower than controls, they were significantly greater than glutathione levels in mice treated with DEM only (Fig. 1). Four to 6 h after co-administration of glutathione monoethyl ester and DEM levels of glutathione were significantly greater than those of control mice. It must be noted that the dose of DEM in these studies was 0.75 ml/kg, since at a DEM dose of 1.0 ml/kg treatment with glutathione monoethyl ester could not raise hepatic glutathione levels (data not shown). Therefore all experiments utilized a DEM dose of 0.75 ml/kg, even though previous studies employed DEM at a dose of 1.Oml/kg [ 1,2]. Administration of glutathione monoethyl ester to DEM-pretreated mice did not abolish the potentiation of the acute toxicities of methyl parathion, methyl paraoxon and fenitrothion, resulting from DEM exposure (Fig. 2). Likewise glutathione monoethyl ester did not prevent the reduction of liver wet weights resulting from exposure to DEM (Fig. 3). Two hours following administration of glutathione monoethyl ester to naive mice hepatic glutathione levels were increased (Fig. 4). However, glutathione monoethyl ester pretreatment did not alter the percentage of mice that succumbed to a challenge dose of methyl parathion, methyl paraoxon or fenitrothion (Fig. 2). DISCUSSION
Numerous dimethyl-substituted
organothiophosphate
TIME AFTER ADMINISTRATION Fig. 4. The effects of glutathione tathione
content. (0)
An asterisk
monoethyl
(*) indicates
by a 2-way analysis
ester ( W) administration a significant
of variance
difference
followed
insecticides and their oxy-
(h) (20 mmol/kg
p.o.) on hepatic
from the corresponding
by the Neuman-Keuls
test [16].
control
glugroup
82
gen analogs are biotransformed in vitro by glutathione-dependent transferases [3]. In contrast, however, the present report suggests that methyl parathion, methyl paraoxon and fenitrothion do not undergo biotransformation by these enzymes in vivo, since lowering hepatic glutathione by buthionine sulfoximine had no effect on their acute toxicities (Figs. 1 and 2). Similar results were obtained with these and other insecticides previously, although glutathione was depleted by pretreament of mice with acetaminophen [4,5]. If glutathione does not detoxify dimethyl-substitued organothiophosphates in vivo, it must be hypothesized that DEM should potentiate the toxicity of these insecticides even in the presence of hepatic glutathione. This hypothesis was tested by use of glutathione monoethyl ester to replenish hepatic glutathione levels. Anderson et al. [l l] have demonstrated the administration of glutathione monoesters to be an effective means of increasing intracellular levels of glutathione since monoesters of glutathione are effectively taken up by numerous cells while glutathione itself is not [18]. In particular, glutathione monoethyl ester is transported into cells of the liver, kidney, spleen, pancreas, heart and lungs in the mouse, and is subsequently hydrolyzed to form glutathione and ethanol [l 11. Consequently, administration of glutathione monoethyl ester to mice can afford protection against certain toxic chemicals which are detoxified by glutathione [12,18]. In the present study, treatment of mice with glutathione monoethyl ester simultaneously with DEM attenuated the depletion of hepatic glutathione levels 1 h after administration, and increased glutathione content, compared to controls, 4-6 h after treatment (Fig. 1). However, these alterations in hepatic glutathione had no effect on DEM-induced potentiation of the acute toxicity of methyl parathion, methyl paraoxon or fenitrothion (Fig. 2). Consequently, just as Dorough [4] demonstrated that the chemical methyl iodide potentiated the acute toxicity of fenitrothion by a mechanism other than glutathione depletion, the present study suggests that depletion of hepatic glutathione is not the mechanism of DEMinduced potentiation of the acute toxicity of methyl parathion, methyl paraoxon or fenitrothion in the mouse. Additionally, that the acute toxicities of methyl parathion, methyl paraoxon and fenitrothion in the presence of elevated hepatic glutathione levels are unchanged (Figs. 2 and 4) lends support to the conclusion that glutathionedependent detoxification following lethal doses of certain dimethyl-substituted organothiophosphate insecticides does not occur to any significant extent in vivo in the mouse [6]. Although the present study offers no explanation for the potentiation of the acute toxicity of methyl parathion, methyl paraoxon or fenitrothion by DEM, several previous studies have documented numerous biological effects of DEM in addition to the depletion of GSH [68, 191. As a result it is not unreasonable to assume that DEM could exert other, as yet undetermined, effects which could potentiate the toxicity of these insecticides. The decreased hepatic wet weight observed in DEM-pretreated mice (Fig. 3) further demonstrates the non-selective nature of this chemical. Moreover, this effect on liver weight is unrelated to hepatic glutathione levels since
83
the reduced liver weights persisted following administration ethyl ester (Fig. 4).
of glutathione mono-
ACKNOWLEDGEMENTS
This work was supported by NIEHS Grant ES04335 REFERENCES 1 Mirer,
F.E., Levine,
tabolism
B.S. and Murphy,
in piperonyl
2 Sultatos,
butoxide
L.G., Costa,
SD.
L.G. and Murphy,
ties of the insecticides
(1977) Parathion
and diethyl maleate
chlorpyrifos
pretreated
S.D. (1982) Factors
and methyl
chlorpyrifos
and methyl parathion mice. Chem.-Biol. involved
toxicity
Interact.
in the differential
in mice. Toxicol.
Appl.
and me-
17,99-l
12.
acute toxici-
Pharmacol.
65,
144152. 3 Fukami,
J. (1980) Metabolism
10,473-5
of several insecticides
by glutathione
S-transferase.
Pharmacol.
Ther.
14.
4 Dorough,
H.W. (1983) Toxicological
significance
of pesticide conjugates.
J. Toxicol.
Clin. Toxicol.
19,
6377659. 5 Costa,
L.G. and Murphy,
mice. Res. Commun. 6 Sultatos,
S.D. (1984) Interaction
Chem. Pathol.
L.G. and Woods,
methyl parathion 7 Plummer,
between
acetaminophen
L. (1988) The role of glutathione
and azinphos-methyl
Enzymol. Pharmacol.
9 Hollingworth,
in the mouse. Toxicol.
in the detoxification Appl. Pharmacol.
of the insecticides
96, 1688174.
depletion
of glutathione
in vivo.
and enhancement
of microsomal
drug metabolism
by diethyl maleate.
27, 1098-l 101.
R.M.,
methyl parathion.
in
77, 51-59.
M.W. (1978) Inhibition
Biochem.
and organophosphates
44, 389400.
J.L., Smith, B.R., Sies, H. and Bend, J.R. (1981) Chemical
Methods 8 Anders,
Pharrnacol.
Fukuto,
Influence
T.R. and Metcalf, of structure
R.L. (1967) Selectivity
on anticholinesterase
activity.
of sumithion J. Agric.
compared
with
Food Che’m. 15, 235-
241. 10 Benke, G.M., Cheever, terase action Pharmacol.
M.E., Powrie,
tion, uptake
parathion
S.D. (1974) Comparative
and parathion
F., Puri, R.N. and Meister,
by tissues, and conversion
R.K., Anderson,
mium toxicity.
A. (1985) Glutathione
to glutathione.
M.E. and Meister,
Arch. Biochem.
A. (1987) Glutathione,
glutathione.
15 Anderson, 16 Winer,
anticholines-
monoethyl
Biophys.
Appl.
ester: prepara-
239, 538-548.
a first line of defense against
method
for quantitative
Anal. Biochem.
cad-
determination
of nanogram
M.E. (1985) Determination Enzymol.
amounts
of total and
27, 502-522.
14 Griffith, O.W. (1980) Determination of glutathione and glutathione tase and 2-vinylpyridine. Anal. Biochem. 106,207-212. Methods
toxicity,
in sunfish and mice. Toxicol.
FASEB J. I, 22&223.
13 Tie&e, F. (1969) Enzymatic oxidized
of methyl
28,97-109.
II Anderson, 12 Singhal,
K.E., Mirer, FE. and Murphy,
and metabolism
of glutathione
disulfide
and glutathione
using glutathione
disulfide
in biological
reducsamples.
113,548-555.
B.J. (1971) Statistical
Principles
in Experimental
Design,
2nd edn. McGraw-Hill
Book Com-
pany, New York, pp. 191-195,309-342,848-853. 17 Hintze, J.L. (1987) Number pp. 167-211. 18 Puri,
R.N. and Meister,
liver and kidney. 19 Meister, tathione
A.
Cruncher
Statistical
A. (1983) Transport
System, Version of glutathione,
as y-glutamylcysteinylglycyl
UT,
ester, into
Proc. Natl. Acad. Sci. USA, 80, 5258-5260.
(I 985) Methods for the selective modification
transport.
5.01. Jerry L. Hintze, Kaysville,
Methods
Enzymol.
113, 571-583.
of glutathione
metabolism
and study of glu-
02492
The effect of glutathione monoethyl ester on the potentiation of the acute toxicity of methyl parathion, methyl paraoxon or fenitrothion by diethyl maleate in the mouse
Lester G. Sultatos, Guo-jie Huang, Olin Jackson, Kenneth Reed and Thomas M. Soranno Department
of Pharmacology
and Toxicology,
University of Medicine and Dentistry
of New Jersey,
Newark, NJ (U.S.A.) (Received
15 December
(Accepted
6 August
1989)
1990)
Key words: Methyl parathion; Organophosphate
Methyl paraoxon;
Fenitrothion;
Diethyl maleate;
Glutathione;
biotransformation
SUMMARY Depletion
of hepatic
to potentiate er, certain
glutathione
the acute toxicities studies
in the mouse by pretreatment
of many dimethyl-substituted
have raised doubts
methyl parathion
regarding
of this insecticide.
The present
study evaluates
methyl paraoxon,
One hour following
pretreatment
tration
monoethyl
of glutathione
more,
glutathione,
paraoxon,
of glutathione
or fenitrothion.
methyl paraoxon
and Dentistry
0378-4274/91/%
that DEM potentiates
of animals
indicate
Lester G. Sultatos, of New Jersey,
of the lethality
to a challenge
that DEM
potentiates
unrelated
to hepatic
Department
185 South Orange
3.50 @ 1991 Elsevier Science Publishers
depletion.
was markedly
depleted Adminis-
mice attenuated
DEM-deglutathione
of these insecticides. hepatic
Further-
glutathione
dose of methyl parathion, the toxicity glutathione
Newark,
B.V. (Biomedical
of methyl
levels, methyl
parathion,
content.
and Toxicology, NJ 07103-2757,
Division)
of
potentiation
the acute toxicities
levels. However,
of Pharmacology Avenue,
Howev-
were potentiated.
ester to naive mice increased
succumbing
by a mechanism
and fenitrothion
at or above control
potentiation
is known
other than glutathione
p.o.) to DEM-pretreated
glutathione
monoethyl
These data
or fenitrothion
Address for correspondence: of Medicine
ester (20 mmol/kg
or maintained
but did not affect the percentage
of action of DEM-induced
by a mechanism
methyl paraoxon
ester did not alter the DEM-induced
administration
the hypothesis
(DEM)
insecticides.
in the detoxification
of mice with DEM (0.75 ml/kg i.p.), glutathione
of methyl parathion,
pletion of hepatic
maleate
of glutathione
mechanism
and fenitrothion
and the acute toxicities
monoethyl
the participation
in the mouse, and hence the putative
of methyl parathion,
with diethyl
organothiophosphate
University U.S.A.
78
INTRODUCTION
Pretreatment of mice with diethyl maleate (DEM) is known to markedly reduce hepatic glutathione levels and to potentiate the acute toxicity of certain dimethyl-substituted organothiophosphate insecticides like methyl parathion and methyl chlorpyrifos [ 1,2]. Additionally these insecticides have been shown to undergo biotransformation by glutathione-dependent transferases in vitro [3]. However, several studies have raised doubts regarding the participation of glutathione in the detoxification of certain dimethyl-substituted organothiophosphate insecticides in vivo, and hence the putative mechanism by which DEM potentiates the toxicities of these chemicals. For example, depletion of glutathione in mice by pretreatment with acetaminophen had no effect on the acute toxicities of the dimethyl-substituted organothiophosphates fenitrothion, dichlorvos and methyl chlorpyrifos [4,5]. Similarly reduction of hepatic glutathione by pretreatment with buthionine sulfoximine did not alter the acute toxicities of methyl parathion or azinphos-methyl [6]. The present report extends these observations by evaluating the effects of the reduction of hepatic glutathione by treatment with buthionine sulfoximine on the acute toxicities of methyl parathion, methyl paraoxon and fenitrothion. These insecticides were chosen since numerous investigators have documented glutathione-dependent detoxification of these compounds in vitro [3]. Moreover, since DEM is known to exert many effects other than glutathione depletion [&8] the present study examines the hypothesis that DEM pretreatment of mice potentiates the acute toxicity of these insecticides by a mechanism other than depletion of hepatic glutathione. METHODS
Chemicals Methyl parathion (O,O-dimethyl O-p-nitrophenyl phosphorothioate) and fenitrothion [O,O-dimethyl-O-(4-nitro-m-tolyl)phosphorothioate] were purchased from Chem Service, Inc. (West Chester, PA). Methyl paraoxon (O,O-dimethyl O-p-nitrophenyl phosphate) was synthesized by the method of Hollingworth et al. [9], as described by Benke et al. [lo]. Glutathione monoethyl ester was synthesized as previously described [l l] except that sulfuric acid was used in place of hydrogen chloride [ 121.Diethyl maleate and buthionine sulfoximine were-purchased from Sigma Chemical Co. (St. Louis, MO). Animals andpretreatments Male Hla:(SW)BR Swiss Webster mice (20-30g) obtained from Hilltop Lab Animals, Inc. (Scottdale, PA) were used in all experiments. They were housed under standard laboratory conditions at the Animal Care facility at the University of Medicine and Dentistry of New Jersey, and had free access to water and feed (Purina Laboratory Rodent Chow 5001). Methyl parathion was administered intraperitoneally
79
{at a dose of 15 mg/kg in 10% DMSO in corn oil) at a volume of 1 ml/kg. Methyl paraoxon was administered in an identical manner, except that the dose was 5 mg/kg. Fenitrothion was administered directly, intraperitoneally, at a dose of 800 mg/kg. Buthionine sulfoximine was administered in the drinking water for 15 days at a concentration of 20 mmol, followed by intraperitoneal supplemental injections of 8 mmol/kg as previously outlined [6]. Diethyl maleate was administered intraperitoneally at a dose of 1 or 0.75 ml/kg. Glutathione monoethyl ester was initially dissolved in the smallest volume of water possible (usually giving a final concentration of 1 g/23 ml). The pH of the solution, which was highly acidic, was adjusted to a pH of 5-7 by addition of sodium hydroxide [1 11. This final solution was administered by stomach tube to give a dose of 20 mmol/kg. In those experiments in which mice received both diethyl maleate and glutathione monoethyl ester, the ester was administered immediately after diethyl maleate. Animals receiving diethyl maleate, glutathione monoethyl ester, and insecticide, were challenged with insecticide 1 h following administration of the other two chemicals. Mice were observed for 3 days following insecticide administration since preliminary studies indicated mice that died did so within 3 days after insecticide challenge. These studies were reviewed and approved by the Institutional Animal Care and Use Committee at the University of Medicine and Dentistry of New Jersey. Glutathione determinations Total glutathione was assayed by the enzymatic recycling procedure [13] as modified by Griffith [14] and described by Anderson [15]. The rate of 2-nitro-5-thiobenzoic acid formation in each sample was monitored at 412 nm and the glutathione present was evaluated by comparison with a standard curve. 18
1
I **
1
2 TIME
3 AFTER
4 ADMINISTRATION
I
5
6
(h)
Fig. 1.The effects of BSO (V), DEM (0) or DEM + giutathione monoethyl ester (A) on hepatie glutathione content in the mouse. Controls (I) received no treatment. Each point represents the mean + SD from 4 mice. An asterisk (*) indicates a significant difference (PiO.05) from the corresponding control, whereas a double asterisk (**) indicates a significant difference (PiO.05) from the corresponding control and the corresponding DEM-treated group. Statistical anaiyses were performed by a f-way analysis of variance, followed by the Neuman-Keuls test [16].
80
100
60
x ;?i 5
60
5 4
40
8? 20
0/
Control
OEM
BSO
OEM
L
Ester
+ Ester
Fig. 2. Effects of DEM, BSO, DEM + glutathione monoethyl ester, or glutathione monoethyl ester alone, on the lethality of methyl parathion (0) methyl paraoxon (H), or fenitrothion (W) in the mouse. An asterisk (*) indicates a significant difference from the corresponding control group by the Friedman’s Block/Treatment test followed by the Two Sample Proportion test [16,17].
Statistical analyses Parametric analysis utilized at l- or 2-way analysis of variance followed by the Neuman-Keuls test, whereas non-parametric analysis utilized either the KruskalWallis test or the Friedman’s Block/Treatment test followed by the Two Sample Proportion test [16,17]. All analyses were performed on an IBM XT computer, using the software NCSS (NCSS, Kaysville, Utah). RESULTS
As previously reported [I], pretreatment
g
7
4 “0.00
of mice with DEM markedly depleted he-
I
0.25
DIETHYL
0.50
MALEATE
0.75
1 .oo
DOSE (ml/kg)
Fig. 3. Reduction of mouse liver wet weights by DEM (0) or by DEM + glutathione monoethyl ester (A). Each point represents the mean f SD of at least 6 mice. An asterisk (*) indicates a significant difference (P-c 0.05) from the group not receiving DEM. Statistical analyses were performed by a l-way analysis of variance followed by the Neuman-Keuls test [16].
81
patic glutathione levels and potentiated the acute toxicity of methyl parathion, methyl paraoxon and fenitrothion (Figs. 1 and 2). Additionally, liver wet weights were slightly reduced following treatment with DEM (Fig. 3). Conversely, treatment of mice with buthionine sulfoximine lowered hepatic glutathione levels (Fig. 1), but had no effect on the acute toxicities of methyl parathion, methyl paraoxon or fenitrothion (Fig. 2). One hour after co-administration of glutathione monoethyl ester (20 mmol/kg p.o.) with DEM, although hepatic glutathione levels were slightly lower than controls, they were significantly greater than glutathione levels in mice treated with DEM only (Fig. 1). Four to 6 h after co-administration of glutathione monoethyl ester and DEM levels of glutathione were significantly greater than those of control mice. It must be noted that the dose of DEM in these studies was 0.75 ml/kg, since at a DEM dose of 1.0 ml/kg treatment with glutathione monoethyl ester could not raise hepatic glutathione levels (data not shown). Therefore all experiments utilized a DEM dose of 0.75 ml/kg, even though previous studies employed DEM at a dose of 1.Oml/kg [ 1,2]. Administration of glutathione monoethyl ester to DEM-pretreated mice did not abolish the potentiation of the acute toxicities of methyl parathion, methyl paraoxon and fenitrothion, resulting from DEM exposure (Fig. 2). Likewise glutathione monoethyl ester did not prevent the reduction of liver wet weights resulting from exposure to DEM (Fig. 3). Two hours following administration of glutathione monoethyl ester to naive mice hepatic glutathione levels were increased (Fig. 4). However, glutathione monoethyl ester pretreatment did not alter the percentage of mice that succumbed to a challenge dose of methyl parathion, methyl paraoxon or fenitrothion (Fig. 2). DISCUSSION
Numerous dimethyl-substituted
organothiophosphate
TIME AFTER ADMINISTRATION Fig. 4. The effects of glutathione tathione
content. (0)
An asterisk
monoethyl
(*) indicates
by a 2-way analysis
ester ( W) administration a significant
of variance
difference
followed
insecticides and their oxy-
(h) (20 mmol/kg
p.o.) on hepatic
from the corresponding
by the Neuman-Keuls
test [16].
control
glugroup
82
gen analogs are biotransformed in vitro by glutathione-dependent transferases [3]. In contrast, however, the present report suggests that methyl parathion, methyl paraoxon and fenitrothion do not undergo biotransformation by these enzymes in vivo, since lowering hepatic glutathione by buthionine sulfoximine had no effect on their acute toxicities (Figs. 1 and 2). Similar results were obtained with these and other insecticides previously, although glutathione was depleted by pretreament of mice with acetaminophen [4,5]. If glutathione does not detoxify dimethyl-substitued organothiophosphates in vivo, it must be hypothesized that DEM should potentiate the toxicity of these insecticides even in the presence of hepatic glutathione. This hypothesis was tested by use of glutathione monoethyl ester to replenish hepatic glutathione levels. Anderson et al. [l l] have demonstrated the administration of glutathione monoesters to be an effective means of increasing intracellular levels of glutathione since monoesters of glutathione are effectively taken up by numerous cells while glutathione itself is not [18]. In particular, glutathione monoethyl ester is transported into cells of the liver, kidney, spleen, pancreas, heart and lungs in the mouse, and is subsequently hydrolyzed to form glutathione and ethanol [l 11. Consequently, administration of glutathione monoethyl ester to mice can afford protection against certain toxic chemicals which are detoxified by glutathione [12,18]. In the present study, treatment of mice with glutathione monoethyl ester simultaneously with DEM attenuated the depletion of hepatic glutathione levels 1 h after administration, and increased glutathione content, compared to controls, 4-6 h after treatment (Fig. 1). However, these alterations in hepatic glutathione had no effect on DEM-induced potentiation of the acute toxicity of methyl parathion, methyl paraoxon or fenitrothion (Fig. 2). Consequently, just as Dorough [4] demonstrated that the chemical methyl iodide potentiated the acute toxicity of fenitrothion by a mechanism other than glutathione depletion, the present study suggests that depletion of hepatic glutathione is not the mechanism of DEMinduced potentiation of the acute toxicity of methyl parathion, methyl paraoxon or fenitrothion in the mouse. Additionally, that the acute toxicities of methyl parathion, methyl paraoxon and fenitrothion in the presence of elevated hepatic glutathione levels are unchanged (Figs. 2 and 4) lends support to the conclusion that glutathionedependent detoxification following lethal doses of certain dimethyl-substituted organothiophosphate insecticides does not occur to any significant extent in vivo in the mouse [6]. Although the present study offers no explanation for the potentiation of the acute toxicity of methyl parathion, methyl paraoxon or fenitrothion by DEM, several previous studies have documented numerous biological effects of DEM in addition to the depletion of GSH [68, 191. As a result it is not unreasonable to assume that DEM could exert other, as yet undetermined, effects which could potentiate the toxicity of these insecticides. The decreased hepatic wet weight observed in DEM-pretreated mice (Fig. 3) further demonstrates the non-selective nature of this chemical. Moreover, this effect on liver weight is unrelated to hepatic glutathione levels since
83
the reduced liver weights persisted following administration ethyl ester (Fig. 4).
of glutathione mono-
ACKNOWLEDGEMENTS
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