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Effect of ethanol on the microsomal glutathione S-transferase activity in glutathione-depleted rat liver
Alcohol. Vol. 2, pp. 27-30. 1985. ©Ankho InternationalInc. Printed in the U.S.A.
0741-8329/85 $3.00 + .00
Effect of Ethanol on the Microsomal Glutathione S-Transferase Activity in Glutathione-Depleted Rat Liver HELMUTH
W. SIPPEL
Department o f Forensic Medicine, University o f Helsinki, SF-00280 Helsinki and Research Laboratories o f the State Alcohol Company, Alko Ltd., SF-O0101 Helsinki, Finland
SIPPEL, H. W. Effect of ethanol on the microsomal glutathione S-transferase activity in glutathione-depleted rat liver. ALCOHOL 2(1) 27-30, 1985.--Depletion of hepatic glutathione in male rats by starvation caused a significant increase in microsomal glutathione S-transferase activity, which was not affected by acute ethanol pretreatment. An additional depletion in fasted rats by diethylmaleate (0.5 g/kg) caused a further increase in the enzyme activity, but this increase was delayed in ethanol intoxicated rats. Although ethanol caused a small increase in hepatic microsomal lipid peroxidation in control animals, this effect of ethanol was not observed in diethylmaleate treated rats and thus was apparently not responsible for the delay in enzyme activation. It is suggested that the activation of microsomal glutathione S-transferase activity towards l-chioro-2,4-dinitrobenzene in glutathione-depleted rat liver may be produced by changes in thiol/disulfid ratio and/or some reactive oxygen species. Glutathione-depletion
Lipid peroxidation
Ethanol
Glutathione S-transferase
Diethylmaleate
Rat
administered 250, 500 or 750 mg/kg IP as a 10 or 20% (w/v) solution in maize oil at 0.5, 1.5, 2.0 or 2.5 hr prior to sacrifice. Control animals were given maize oil IP in equivalent volumes.
T H E glutathione S-transferases (GSH-transferase, EC 2.5.1.18) represent the b o d y ' s most important defence mechanism against metabolically generated electrophilic toxins [ 1,3]. Several forms of these enzymes are present in the soluble portion of the liver cell [9,10]. Recently, GSHtransferase activity was found in the microsomal fraction of the liver cell. The enzyme in this location is attached firmly to the microsomal membrane, and its properties are different from those of the soluble forms [13,14]. One important distinguishing characteristic is that the activity of the GSHtransferase localized within the membranes of the endoplasmic reticulum can be increased many fold by treatment with a thiol-reagent, N-ethylmaleimide (NEM), in vitro [11,12]. The ability of N E M to stimulate GSH-transferase activity was, however, reduced in microsomes isolated from rats intoxicated with ethanol, and it was suggested that an intact membrane lipid environment is necessary for optimal activity [16]. The present study examined whether the microsomal GSH-transferase can be also activated in vivo, and whether this activation is also inhibited in ethanol intoxicated animals.
Isolation of Microsomes Rats were killed by decapitation; livers were rapidly removed and homogenized in 10 mM Tris-HCI (pH 8.0) containing 0.25 M sucrose and 1.0 mM EDTA using a Teflonglass Potter-EIvehjem homogenizer. The homogenate was adjusted to 25% and centrifuged twice at 9,000 g for 20 rain, and the supernatant was centrifuged for 45 min at 105,000 g. The microsomal pellet was washed twice with 0.15 M TrisHCI (pH 8.0), and sedimented microsomes were resuspended in 10 mM phosphate buffer (pH 7.4) containing 0.15 M KCI and 1.0 mM EDTA.
Separation of Microsomal Lipids Microsomes were transferred to round-bottom tubes (about l0 mg protein/tube), and the microsomai lipids were extracted with dichloromethane/methanol by the method of Hashimoto and Recknagel [7]. The extracted lipid was redissolved in cyclohexane, and the absorbance was measured against a cyclohexane blank in a Beckman 25 spectrophotometer scanned over the range of 220 to 300 nm in cuvettes with a 1 cm path length. The absorption spectrum was normalised on the basis o f the lipid concentration (l mg/ml o f cyclohexane), which was assayed by the method of Chiang et al. [4]. The mean difference in O,D. between the control (saline) group and experimental groups was calculated.
METHOD
Animals and Drug Treatment Male albino rats (about 4-months old) were fed a standard laboratory diet and water ad lib. The rats were fasted 18-24 hr before treatment except in one experiment (see Table 1) in which fed rats were used. Ethanol, 4 g/kg, was given IP as a 20% (w/v) solution in saline. Control rats received an isovolumetric dose of saline. Diethylmaleate (DEM) was
27
28
SIPPEL 2OO
TABLE 1 GLUTATHIONE LEVELS AND M1CROSOMAL GLUTATHIONE
S-TRANSFERASESPECIFICACTIVITIESOF LIVERSFROMFED AND FASTEDRATS, AND FASTEDRATSPRETREATEDWITHETHANOL 150
Treatment
Glutathione (GSH) (/.Lmol/g)
Transferase activity (nmol/min/mg)
7.4 _ 0.1 (4) 4.9 _+ 0.2 (6) 4.4 ___0.2 (4)
47.0 ± 1.0 (4)* 64.2 _ 6.0 (6)t 67.8 ± 10.3 (4)
Gs
Fed rats Fasted rats Fasted rats + ethanol
*Results are expressed as mean ~ S.E.M. of the number of determinations given in parentheses. The enzyme activity of microsomes was corrected for the cytosolic transferases which contaminate the washed microsomes [12]. tp<0.05.
100 l,z. r~ z
4.0
i
3,0 2.0 1,0
II
s' 0.5
ETOH
1.0
1.5
2.0
GSH-transferase activity towards l-chloro-2,4-dinitrobenzene (CDNB) was assayed using previously reported procedures [6]. Assays were routinely performed at 25°C, pH 6.5, and in presence of 2.5 mM GSH. CDNB was added in ethanol giving a final ethanol concentration of 5%.
2.5
DEM TIME
Enzyme Assay
(pMOL/G) 5.0
(H)
FIG. 1. Time-dependence of in vivo depletion of GSH and the consequent activation of microsomal GSH-transferase following the administration of 500 mg DEM/kg IP to rats. Ethanol. 4 g/kg IP, was given 0.5 hr before DEM-administration. Values represent means and their standard errors of 3 experiments each. ((3---(3) GSHconcentrations in control (saline) rats; (O----O)GSH-concentrations in ethanol pretreated rats; (¢r----~-) GSH-transferase activity in control rats; (*----~-) GSH-transferase activity in ethanol pretreated rats.
Determination of Liver Glutathione Liver portions (0.4-0.6 g) were homogenized in ice-cold 20 mM solution of EDTA, and the homogenate was deproteinized by addition of a 25% solution of trichloroacetic acid. After centrifugation the reduced glutathione (GSH) in the protein-free supernatant was determined by the method of Ellman [5].
Chemicals All chemicals were of reagent grade and obtained from common commercial sources. RESULTS AND DISCUSSION
In Vivo Depletion of Glutathione Starving an animal for 24 hr is known to reduce the hepatic GSH-concentration to 60% compared to normal fed controis [17]. The data presented in Table 1 show that starvation reduced the GSH-Ievel in the rat liver by 34% and that this was accompanied by a significant increase in the microsomal GSH-transferase activity. The results from fasted rats pretreated with 4 g/kg ethanol 2 hr before sacrifice are essentially the same as those from the fasted animals without ethanol. These results support the idea that in normal fed rats the GSH-levels are high enough to protect the cell membranes against peroxidative damage. When a certain minimal threshold concentration of GSH is reached after food deprevation, xenobiotic metabolism, oxidative stress, etc., reactive electrophilic oxygen radicals can no longer be counteracted and a more effective protection system is needed; the microsomal GSH-transferase, with GSH-peroxidase activity [15], is then activated. In order to test this hypothesis starved rats were treated with diethylmaleate (DEM). DEM was chosen for two reasons. First, it is a powerful GSH-depleting agent in the cell and its mechanism of action has been proposed to be exerted
by conjugation with the tripeptide forming a complex that is released to the extracellular medium [2]. Second, it was reported to have no toxicity of its own when given to rats, and does not induce hepatic lipid peroxidation [8,17]. The time dependence of both the GSH-depleting effect and the activation of GSH-transferase after treatment of the rat with 500 mg DEM/kg is shown in Fig. 1. In saline pretreated rats hepatic GSH-levels were depleted to 15-20% of control levels 30 rain after DEM-administration, remained maximally depleted for about 1 hr, after which they began to rise progressively. The GSH-levels in the ethanol intoxicated rats showed a much slower rate of resynthesis. As is also shown in Fig. 1, the microsomal GSH-transferase was significantly activated after DEM-treatment with a maximum at 1.5-2.0 hr. GSH-resynthesis seems to inhibit this activation and the enzyme activity at 2.5 hr had returned halfway to the base level. In the ethanol pretreated group, although the GSH-level was reduced at 0.5 hr to about the same degree as it was in the animals without ethanol, there was actually a decrease in the enzyme activity followed only later by an increase. The enzyme activity at 0.5 hr was significantly lower in the ethanol intoxicated rats than in the controls ~o<0.01). Thus, it seems that ethanol intoxicated rats had a reduced capacity to eliminate reactive electrophilic intermediates immediately after GSH-depletion. These results confirm the hypothesis that GSH-depletion gives rise to an activation of microsomal GSH-transferase, provided that an initiating factor is present. This factor may be a change in the thioi/disulfide ratio, active oxygen species or some electrophilic intermediate of an endogenous or exogenous substrate of the microsomal mixed function oxidases. A similar picture was obtained when DEM was administered in increasing doses (Fig, 2).
E F F E C T OF E T H A N O L ON G S H - T R A N S F E R A S E
29
250
I) I FFERENCE
SPECTRUM 200
II
/
0./4
.02
I i~0t D •
i I
Z
st
,~. 150 _1
it
,zz
,"
~.
~o
0.3
tl ° GSH
,"
~
.oi
•
100
~
(~MOL/G)
i11
5.0
/
/'J
u
I
I
23O
24O
J
25O
Oo 0.2
Oe
Z
• z an n. o
4.0
3.0
0.i
50 o
o o O0
oe °~e
2.0
1.0 |
O
i
250
I
500
I
i
750
220
DOSE (MG/KG)
!
! 230
!
I
i
2/40
! 250
WAVELENGTH (NM)
FIG. 2. Dose-dependence of in vivo depletion of GSH and GSHtransferase activity after DEM-administration to rats. Each point represents the mean of measurements obtained from 2 to 4 animals. (0---(3) GSH-concentration; (~r----~-) GSH-transferase activity. Determinations were performed 2 hr after the application of DEM.
FIG. 3. Ethanol-induced hepatic lipid peroxidation in vivo. The UV-absorption spectra and mean difference spectra are shown for rat liver microsomal lipids. The rats received 4 g ethanol/kg IP. Each point is the mean absorbance per mg lipid per ml cyciohexane for that wavelength of three animals. ((3) control (saline) rats; (O) ethanol rats.
Ethanol-Induced Lipid Peroxidation
activation by NEM in vitro (unpublished observations), suggests the possibility that ethanol-induced lipid peroxidation was responsible for ethanol's inhibition of GSHtransferase activation. Arguing against this explanation, however, is the finding that 0.5 hr after DEM-treatment only small and similar amounts of conjugated dienes were found in both saline and ethanol pretreated rats, and none could be detected at later times. Additional work will be needed to determine whether earlier differences in lipid peroxidation of some other effect of ethanol causes its initial inhibition of the activation.
The data presented in Fig. 3 show that acute ethanol treatment at a dose of 4 g/kg IP administered 2 hr before sacrifice produced a small but significant increase in the conjugated dienes in the microsomal fraction of liver. This increase could only be seen after fasting (18--24 hr) in the rat. It is probable that the hepatic GSH-level had to be lowered before acute ethanol administration could induce lipid peroxidation. The present studies confirmed that DEM activates microsomal GSH-transferase in vivo. as NEM had been found to do in vitro [11, 12, 16], and that the activation in vivo was also delayed in ethanol pretreated rats. The fact that ethanol alone induced lipid peroxidation and that a lipid peroxidation generating system (NADPH-chelated iron) prevented the
ACKNOWLEDGEMENTS
1 would like to thank David Sinclair, Ph.D. for valuable discussion.
REFERENCES 1. Boyland, E. and L. F. Chasseaud. The role of glutathione and glutathione S-transferase in mercapturic acid biosynthesis. Adv Enzymol 32: 173-219, 1969. 2. Chasseaud, L. F. Conjugation with glutathione and mercapturic acid excretion. In: Glutathione: Metabolism and Function, edited by I. M. Arias and W. B. Jakoby. New York: Raven Press, 1976, pp. 77-114. 3. Chasseaud, L. F. The role of glutathione and glutathione S-transferases in the metabolism of chemical carcinogens and other electrophilic agents. Adv Cancer Res 29: 175-274, 1979. 4. Chiang, S. P., C. F. Gessert and O. H. Lowry. Colorimetric determination of extracted lipids. An adaptation for microgram amounts obtained from cerumen. Current List of Medical Literature, Air Universio, School of Aviation Medicine. USAF, Res. Rep. 56: 113-118, 1957.
5. Ellman, G. L. Tissue sulphydryl groups. Arch Biochem Biophys 82: 70-77, 1959. 6. Habig, W. H., M. J. Pabst and W. B. Jakoby. Glutathione S-transferase. The first enzymatic step in mercapturic acid formation. J Biol Chem 249: 7130-7139, 1974. 7. Hashimoto, S. and R. O. Recknagel, No chemical evidence of hepatic lipid peroxidation in acute ethanol toxicity. Exp Mol Pathol 8" 225-242, 1968. 8. H6gberg, J. and A. Kristoferson. A correlation between glutathione levels and cellular damage in isolated hepetocytes. Fur J Biochem 74: 77-82, 1977. 9. Jakoby, W. B. The glutathione S-transferases: A group of multifunctional detoxification proteins. Adv Enzymol 46: 383-414, 1978.
30 I0. Jakoby, W. B. and W. H. Habig. Glutathione transferases. In: Enzymatic Basis ofDetoxication, vol 2, edited by J. W. Jakoby. New York: Academic Press, 1980, pp. 63-94. 11. Morgenstern, R., J. W. DePierre and L. Ernster. Activation of microsomai glutathione S-transferase activity by sulfhydryl reagents. Biochem Biophys Res Commun 87: 657-663, 1979. 12. Morgenstern, R., J. Meijer, J. W. DePierre and L. Ernster. Characterization of rat-liver microsomal glutathione S-transferase activity. Eur J Biochem 104: 16%174, 1980. 13. Morgenstern, R., C. Guthenberg and J. W. DePierre. Microsomai glutathione S-transferase. Purification, initial characterization and demonstration that it is not identical to the cytosolic glutathione S-transferases A, B and C. Fur J Biochem 128: 243-248, 1982. 14. Morgenstern, R. and J. W. DePierre. Microsomal glutathione transferase. Purification in unactivated form and further characterization of an activation process, substrate specificity and amino acid composition. Ear J Biochem 134: 591-597, 1983.
SIPPEL
15. Reddy, C. C., C.-P. D. Tu, J. R. Burgess, C. Y. Ho, R. W. Scholz and E. J. Massaro. Evidence for the occurrence of selenium-independent glutathione peroxidase activity in rat liver microsomes. Biochem Biophys Res Commun 101: 970--978, 1981. 16. Sippel, H. W. Effect of an acute dose of ethanol on lipid peroxidation and on the activity of microsomal glutathione S-transferase in rat liver. Acta Pharmacol Toxicol 53: 135-140. 1983. 17. Wendel, A., S. Feuerstein and K.-H. Konz. Drug-induced lipid peroxidation in mouse liver. In: Functions o f Glutathione in Liver and Kidney, edited by H. Sies and A. Wendel. Berlin: Springer-Verlag, 1978, pp. 183-188.
0741-8329/85 $3.00 + .00
Effect of Ethanol on the Microsomal Glutathione S-Transferase Activity in Glutathione-Depleted Rat Liver HELMUTH
W. SIPPEL
Department o f Forensic Medicine, University o f Helsinki, SF-00280 Helsinki and Research Laboratories o f the State Alcohol Company, Alko Ltd., SF-O0101 Helsinki, Finland
SIPPEL, H. W. Effect of ethanol on the microsomal glutathione S-transferase activity in glutathione-depleted rat liver. ALCOHOL 2(1) 27-30, 1985.--Depletion of hepatic glutathione in male rats by starvation caused a significant increase in microsomal glutathione S-transferase activity, which was not affected by acute ethanol pretreatment. An additional depletion in fasted rats by diethylmaleate (0.5 g/kg) caused a further increase in the enzyme activity, but this increase was delayed in ethanol intoxicated rats. Although ethanol caused a small increase in hepatic microsomal lipid peroxidation in control animals, this effect of ethanol was not observed in diethylmaleate treated rats and thus was apparently not responsible for the delay in enzyme activation. It is suggested that the activation of microsomal glutathione S-transferase activity towards l-chioro-2,4-dinitrobenzene in glutathione-depleted rat liver may be produced by changes in thiol/disulfid ratio and/or some reactive oxygen species. Glutathione-depletion
Lipid peroxidation
Ethanol
Glutathione S-transferase
Diethylmaleate
Rat
administered 250, 500 or 750 mg/kg IP as a 10 or 20% (w/v) solution in maize oil at 0.5, 1.5, 2.0 or 2.5 hr prior to sacrifice. Control animals were given maize oil IP in equivalent volumes.
T H E glutathione S-transferases (GSH-transferase, EC 2.5.1.18) represent the b o d y ' s most important defence mechanism against metabolically generated electrophilic toxins [ 1,3]. Several forms of these enzymes are present in the soluble portion of the liver cell [9,10]. Recently, GSHtransferase activity was found in the microsomal fraction of the liver cell. The enzyme in this location is attached firmly to the microsomal membrane, and its properties are different from those of the soluble forms [13,14]. One important distinguishing characteristic is that the activity of the GSHtransferase localized within the membranes of the endoplasmic reticulum can be increased many fold by treatment with a thiol-reagent, N-ethylmaleimide (NEM), in vitro [11,12]. The ability of N E M to stimulate GSH-transferase activity was, however, reduced in microsomes isolated from rats intoxicated with ethanol, and it was suggested that an intact membrane lipid environment is necessary for optimal activity [16]. The present study examined whether the microsomal GSH-transferase can be also activated in vivo, and whether this activation is also inhibited in ethanol intoxicated animals.
Isolation of Microsomes Rats were killed by decapitation; livers were rapidly removed and homogenized in 10 mM Tris-HCI (pH 8.0) containing 0.25 M sucrose and 1.0 mM EDTA using a Teflonglass Potter-EIvehjem homogenizer. The homogenate was adjusted to 25% and centrifuged twice at 9,000 g for 20 rain, and the supernatant was centrifuged for 45 min at 105,000 g. The microsomal pellet was washed twice with 0.15 M TrisHCI (pH 8.0), and sedimented microsomes were resuspended in 10 mM phosphate buffer (pH 7.4) containing 0.15 M KCI and 1.0 mM EDTA.
Separation of Microsomal Lipids Microsomes were transferred to round-bottom tubes (about l0 mg protein/tube), and the microsomai lipids were extracted with dichloromethane/methanol by the method of Hashimoto and Recknagel [7]. The extracted lipid was redissolved in cyclohexane, and the absorbance was measured against a cyclohexane blank in a Beckman 25 spectrophotometer scanned over the range of 220 to 300 nm in cuvettes with a 1 cm path length. The absorption spectrum was normalised on the basis o f the lipid concentration (l mg/ml o f cyclohexane), which was assayed by the method of Chiang et al. [4]. The mean difference in O,D. between the control (saline) group and experimental groups was calculated.
METHOD
Animals and Drug Treatment Male albino rats (about 4-months old) were fed a standard laboratory diet and water ad lib. The rats were fasted 18-24 hr before treatment except in one experiment (see Table 1) in which fed rats were used. Ethanol, 4 g/kg, was given IP as a 20% (w/v) solution in saline. Control rats received an isovolumetric dose of saline. Diethylmaleate (DEM) was
27
28
SIPPEL 2OO
TABLE 1 GLUTATHIONE LEVELS AND M1CROSOMAL GLUTATHIONE
S-TRANSFERASESPECIFICACTIVITIESOF LIVERSFROMFED AND FASTEDRATS, AND FASTEDRATSPRETREATEDWITHETHANOL 150
Treatment
Glutathione (GSH) (/.Lmol/g)
Transferase activity (nmol/min/mg)
7.4 _ 0.1 (4) 4.9 _+ 0.2 (6) 4.4 ___0.2 (4)
47.0 ± 1.0 (4)* 64.2 _ 6.0 (6)t 67.8 ± 10.3 (4)
Gs
Fed rats Fasted rats Fasted rats + ethanol
*Results are expressed as mean ~ S.E.M. of the number of determinations given in parentheses. The enzyme activity of microsomes was corrected for the cytosolic transferases which contaminate the washed microsomes [12]. tp<0.05.
100 l,z. r~ z
4.0
i
3,0 2.0 1,0
II
s' 0.5
ETOH
1.0
1.5
2.0
GSH-transferase activity towards l-chloro-2,4-dinitrobenzene (CDNB) was assayed using previously reported procedures [6]. Assays were routinely performed at 25°C, pH 6.5, and in presence of 2.5 mM GSH. CDNB was added in ethanol giving a final ethanol concentration of 5%.
2.5
DEM TIME
Enzyme Assay
(pMOL/G) 5.0
(H)
FIG. 1. Time-dependence of in vivo depletion of GSH and the consequent activation of microsomal GSH-transferase following the administration of 500 mg DEM/kg IP to rats. Ethanol. 4 g/kg IP, was given 0.5 hr before DEM-administration. Values represent means and their standard errors of 3 experiments each. ((3---(3) GSHconcentrations in control (saline) rats; (O----O)GSH-concentrations in ethanol pretreated rats; (¢r----~-) GSH-transferase activity in control rats; (*----~-) GSH-transferase activity in ethanol pretreated rats.
Determination of Liver Glutathione Liver portions (0.4-0.6 g) were homogenized in ice-cold 20 mM solution of EDTA, and the homogenate was deproteinized by addition of a 25% solution of trichloroacetic acid. After centrifugation the reduced glutathione (GSH) in the protein-free supernatant was determined by the method of Ellman [5].
Chemicals All chemicals were of reagent grade and obtained from common commercial sources. RESULTS AND DISCUSSION
In Vivo Depletion of Glutathione Starving an animal for 24 hr is known to reduce the hepatic GSH-concentration to 60% compared to normal fed controis [17]. The data presented in Table 1 show that starvation reduced the GSH-Ievel in the rat liver by 34% and that this was accompanied by a significant increase in the microsomal GSH-transferase activity. The results from fasted rats pretreated with 4 g/kg ethanol 2 hr before sacrifice are essentially the same as those from the fasted animals without ethanol. These results support the idea that in normal fed rats the GSH-levels are high enough to protect the cell membranes against peroxidative damage. When a certain minimal threshold concentration of GSH is reached after food deprevation, xenobiotic metabolism, oxidative stress, etc., reactive electrophilic oxygen radicals can no longer be counteracted and a more effective protection system is needed; the microsomal GSH-transferase, with GSH-peroxidase activity [15], is then activated. In order to test this hypothesis starved rats were treated with diethylmaleate (DEM). DEM was chosen for two reasons. First, it is a powerful GSH-depleting agent in the cell and its mechanism of action has been proposed to be exerted
by conjugation with the tripeptide forming a complex that is released to the extracellular medium [2]. Second, it was reported to have no toxicity of its own when given to rats, and does not induce hepatic lipid peroxidation [8,17]. The time dependence of both the GSH-depleting effect and the activation of GSH-transferase after treatment of the rat with 500 mg DEM/kg is shown in Fig. 1. In saline pretreated rats hepatic GSH-levels were depleted to 15-20% of control levels 30 rain after DEM-administration, remained maximally depleted for about 1 hr, after which they began to rise progressively. The GSH-levels in the ethanol intoxicated rats showed a much slower rate of resynthesis. As is also shown in Fig. 1, the microsomal GSH-transferase was significantly activated after DEM-treatment with a maximum at 1.5-2.0 hr. GSH-resynthesis seems to inhibit this activation and the enzyme activity at 2.5 hr had returned halfway to the base level. In the ethanol pretreated group, although the GSH-level was reduced at 0.5 hr to about the same degree as it was in the animals without ethanol, there was actually a decrease in the enzyme activity followed only later by an increase. The enzyme activity at 0.5 hr was significantly lower in the ethanol intoxicated rats than in the controls ~o<0.01). Thus, it seems that ethanol intoxicated rats had a reduced capacity to eliminate reactive electrophilic intermediates immediately after GSH-depletion. These results confirm the hypothesis that GSH-depletion gives rise to an activation of microsomal GSH-transferase, provided that an initiating factor is present. This factor may be a change in the thioi/disulfide ratio, active oxygen species or some electrophilic intermediate of an endogenous or exogenous substrate of the microsomal mixed function oxidases. A similar picture was obtained when DEM was administered in increasing doses (Fig, 2).
E F F E C T OF E T H A N O L ON G S H - T R A N S F E R A S E
29
250
I) I FFERENCE
SPECTRUM 200
II
/
0./4
.02
I i~0t D •
i I
Z
st
,~. 150 _1
it
,zz
,"
~.
~o
0.3
tl ° GSH
,"
~
.oi
•
100
~
(~MOL/G)
i11
5.0
/
/'J
u
I
I
23O
24O
J
25O
Oo 0.2
Oe
Z
• z an n. o
4.0
3.0
0.i
50 o
o o O0
oe °~e
2.0
1.0 |
O
i
250
I
500
I
i
750
220
DOSE (MG/KG)
!
! 230
!
I
i
2/40
! 250
WAVELENGTH (NM)
FIG. 2. Dose-dependence of in vivo depletion of GSH and GSHtransferase activity after DEM-administration to rats. Each point represents the mean of measurements obtained from 2 to 4 animals. (0---(3) GSH-concentration; (~r----~-) GSH-transferase activity. Determinations were performed 2 hr after the application of DEM.
FIG. 3. Ethanol-induced hepatic lipid peroxidation in vivo. The UV-absorption spectra and mean difference spectra are shown for rat liver microsomal lipids. The rats received 4 g ethanol/kg IP. Each point is the mean absorbance per mg lipid per ml cyciohexane for that wavelength of three animals. ((3) control (saline) rats; (O) ethanol rats.
Ethanol-Induced Lipid Peroxidation
activation by NEM in vitro (unpublished observations), suggests the possibility that ethanol-induced lipid peroxidation was responsible for ethanol's inhibition of GSHtransferase activation. Arguing against this explanation, however, is the finding that 0.5 hr after DEM-treatment only small and similar amounts of conjugated dienes were found in both saline and ethanol pretreated rats, and none could be detected at later times. Additional work will be needed to determine whether earlier differences in lipid peroxidation of some other effect of ethanol causes its initial inhibition of the activation.
The data presented in Fig. 3 show that acute ethanol treatment at a dose of 4 g/kg IP administered 2 hr before sacrifice produced a small but significant increase in the conjugated dienes in the microsomal fraction of liver. This increase could only be seen after fasting (18--24 hr) in the rat. It is probable that the hepatic GSH-level had to be lowered before acute ethanol administration could induce lipid peroxidation. The present studies confirmed that DEM activates microsomal GSH-transferase in vivo. as NEM had been found to do in vitro [11, 12, 16], and that the activation in vivo was also delayed in ethanol pretreated rats. The fact that ethanol alone induced lipid peroxidation and that a lipid peroxidation generating system (NADPH-chelated iron) prevented the
ACKNOWLEDGEMENTS
1 would like to thank David Sinclair, Ph.D. for valuable discussion.
REFERENCES 1. Boyland, E. and L. F. Chasseaud. The role of glutathione and glutathione S-transferase in mercapturic acid biosynthesis. Adv Enzymol 32: 173-219, 1969. 2. Chasseaud, L. F. Conjugation with glutathione and mercapturic acid excretion. In: Glutathione: Metabolism and Function, edited by I. M. Arias and W. B. Jakoby. New York: Raven Press, 1976, pp. 77-114. 3. Chasseaud, L. F. The role of glutathione and glutathione S-transferases in the metabolism of chemical carcinogens and other electrophilic agents. Adv Cancer Res 29: 175-274, 1979. 4. Chiang, S. P., C. F. Gessert and O. H. Lowry. Colorimetric determination of extracted lipids. An adaptation for microgram amounts obtained from cerumen. Current List of Medical Literature, Air Universio, School of Aviation Medicine. USAF, Res. Rep. 56: 113-118, 1957.
5. Ellman, G. L. Tissue sulphydryl groups. Arch Biochem Biophys 82: 70-77, 1959. 6. Habig, W. H., M. J. Pabst and W. B. Jakoby. Glutathione S-transferase. The first enzymatic step in mercapturic acid formation. J Biol Chem 249: 7130-7139, 1974. 7. Hashimoto, S. and R. O. Recknagel, No chemical evidence of hepatic lipid peroxidation in acute ethanol toxicity. Exp Mol Pathol 8" 225-242, 1968. 8. H6gberg, J. and A. Kristoferson. A correlation between glutathione levels and cellular damage in isolated hepetocytes. Fur J Biochem 74: 77-82, 1977. 9. Jakoby, W. B. The glutathione S-transferases: A group of multifunctional detoxification proteins. Adv Enzymol 46: 383-414, 1978.
30 I0. Jakoby, W. B. and W. H. Habig. Glutathione transferases. In: Enzymatic Basis ofDetoxication, vol 2, edited by J. W. Jakoby. New York: Academic Press, 1980, pp. 63-94. 11. Morgenstern, R., J. W. DePierre and L. Ernster. Activation of microsomai glutathione S-transferase activity by sulfhydryl reagents. Biochem Biophys Res Commun 87: 657-663, 1979. 12. Morgenstern, R., J. Meijer, J. W. DePierre and L. Ernster. Characterization of rat-liver microsomal glutathione S-transferase activity. Eur J Biochem 104: 16%174, 1980. 13. Morgenstern, R., C. Guthenberg and J. W. DePierre. Microsomai glutathione S-transferase. Purification, initial characterization and demonstration that it is not identical to the cytosolic glutathione S-transferases A, B and C. Fur J Biochem 128: 243-248, 1982. 14. Morgenstern, R. and J. W. DePierre. Microsomal glutathione transferase. Purification in unactivated form and further characterization of an activation process, substrate specificity and amino acid composition. Ear J Biochem 134: 591-597, 1983.
SIPPEL
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