67
Biochimica et Biophysica Acta, 582 (1979) 67--78
© Elsevier]North-Holland Biomedical Press
BBA 28745 LEVELS OF GLUTATHIONE, GLUTATHIONE REDUCTASE AND GLUTATHIONE S-TRANSFERASE ACTIVITIES IN RAT LUNG AND LIVER
MARIA S. MORON, JOSEPH W. DEPIERRE and BENGT MANNERVIK Department o f Biochemistry, Arrhenius Laboratory, University o f Stockholm, S-106 91 Stockholm (Sweden)
(Received April 17th, 1978) (Revised manuscript received August 4th, 1978) Key words: Glutathione; Glutathione reductase ; Glutathione S-transferase ; (Rat lung)
Summary Levels of glutathione, glutathione reductase and glutathione S-transferase activities in rat lung and liver have been investigated. After perfusing the lung to remove contaminating blood, this organ was f o u n d to have an apparent concentration of glutathione (2 mM) which is approx. 20% of that found in the liver. Both organs contain very low levels of glutathione disulfide. Neither phenobarbital nor methylcholanthrene had a significant effect on the levels of reduced glutathione in lung and liver. In addition, the activities of some glutathione-metabolizing e n z y m e s - glutathione reductase and glutathione S-transferase activity assayed with four different s u b s t r a t e s - were observed to be 5to 60-fold lower in lung tissue than in the liver. Introduction Glutathione is present in all types of living cells [ 1]. The intracellular level of this tripeptide varies with the growth, nutritional state, and hormonal balance of the organism [2,3]. Tissues such as mammalian liver normally contain high levels of reduced glutathione [1]. The functions of glutathione in biological systems are n o t y e t fully understood. It has been suggested t h a t this c o m p o u n d protects thiol groups in protein from oxidation [4], functions as an intracellular redox buffer (see ref. 5) and serves as a reservoir of cysteine [6]. Glutathione also serves as a coenzyme for certain enzymes [7] {e.g., glyoxalase I, maleylacetoacetate isomerase, formaldehyde dehydrogenase, and indolylpyruvic acid ketoenol tautomerase) and as a substrate for others (e.g., glutathione peroxidase, which protects the cell from H20~ and organic hydroperoxides by reduction with concomitant oxida-
68 tion of reduced glutathione; and thioltransferases [8] which use reduced glutathione for the reduction of disulfides). An important group of enzymes for which glutathione is one of the substrates are the glutathione S-transferases. These enzymes are widely distributed among eukaryotes and are often present in relatively high concentrations intracellularly [9]. They are thought to play an important role in the biotransformation of many xenobiotics; the glutathione S-transferases conjugate a variety of pharmacologically active c o m p o u n d s and especially potential alkylating agents, as well as reactive intermediates formed during the metabolism of certain xenobiotics, with glutathione [10]. In general, a glutathione adduct or a mercapturic acid derivative formed from such an adduct is considerably less toxic than the unconjugated precursor c o m p o u n d [ 10]. In addition, it has been postulated that the covalent binding of certain reactive substances to the glutathione S-transferases is a detoxification mechanism [9]. These enzymes, and especially glutathione S-transferase B (also called ligandin), bind certain inducers of c y t o c h r o m e P-450 [11] and thus may have a role in the induction of this c y t o c h r o m e which results from treatment of animals with polycyclic hydrocarbons. Finally, glutathione S-transferases have recently been reported to have glutathione peroxidase activity [12]. To date, glutathione and the glutathione S-transferases have been studied most extensively in the liver. It is important to an understanding of the functioning of other organs, and especially of the relative susceptibility of different organs to the toxic and carcinogenic effects of xenobiotics, to study these cellular components in other tissues as well. Here we have examined glutathione and different glutathione S-transferase activities in rat lung. In addition, since glutathione carries o u t a majority of its functions in the reduced form and since glutathione reductase effects its reduction, we have compared the levels of this enzyme in rat lung and liver. A preliminart report of these results has been made [13]. Materials and Methods Chemicals
[7-3H]Styrene oxide was obtained from the Radiochemical Centre, Amersham, U.K.; unlabeled styrene oxide and 3,4~dichloro-l-nitrobenzene from Schuchardt, Munich, F.G.R.; 1-chloro-2,4-dinitrobenzene from Fluka AG Chemische Fabrik, Buchs, Switzerland; 1,2~epoxy-3-(p-nitrophenoxy)propane from Eastman Kodak Co., Rochester, New York; and glutathione and glutathione disulfide from Boehringer-Mannheim, F.G.R. All other chemicals were also obtained from commercial sources and were of reagent grade. Glutathione reductase from yeast was obtained from Sigma Chemical Company, St. Louis, Mo. Glutathione S-transferase C ("aryltransferase I") was prepared as described earlier [ 14]. A n i m a l s and i n d u c t i o n
Male Sprague-Dawley rats weighing approximately 180--200 g were used. The animals had free access to food and water and were not starved before being killed by decapitation. In several experiments the animals were given
69 intraperitoneal injections of methylcholanthrene (20 mg/kg b o d y weight in corn oil, 5, 3 and 1 day before killing) or phenobarbital (80 mg/kg b o d y weight in isotonic saline solution, once daily for 5 days preceding killing). In these experiments control animals were injected with the same volumes of corn oil or of isotonic saline, respectively.
Perfusion o f the lung and tissue preparation For some unexplained reason, reduced glutathione in rat lung homogenates seems to be oxidized more rapidly than in liver or blood homogenates. The procedures described below should be carried out rapidly enough so that the time elapased between beginning the perfusion of the lung and reading the absorption at 412 nm is not longer than an hour for lung. We found that the lung contains such large amounts of blood and such relatively small amounts of glutathione that correction had to be made for the contribution by contaminating blood. In unperfused lung this correction was as much as 50% of the total value obtained, so that the results were unreliable. Consequently, we perfused the lung in situ through the right ventricle of the heart with 40--60 ml 0.15 M NaC1 before beginning determination of glutathione. This resulted in a correction for blood of 10% or often less. Perfusion could be carried out reasonably successfully in rats whose necks had been dislocated, but it was much easier and more complete after anesthetizing the animals with a lethal dose of nembutal. Table I shows that nembutal had no effect on the levels of reduced glutathione in lung, liver, and blood. This anesthetic was subsequently used routinely. The perfused lung was diced with scissors, homogenized with ten up-anddown strokes at 440 rev./min in a Potter-Elvehjem homogenizer, and diluted to a concentration of approximately 1 g wet weight/ml with water. This homogenate was then sonicated for 1 min. If glutathione was to be determined, enough 25% trichloroacetic acid was added to the sonicate to give a final concentration of 5% (to precipitate protein and particles) and the supernatant obtained by centrifuging in a desk centrifuge for 7 min was used. If glutathione S-transferases or hemoglobin was to be measured, the sonicate was centrifuged at 105 000 × g for 90 min and the supernatant was used. Since the lung was found to contain relatively small amounts of glutathione,
TABLE I LACK OF EFFECT
OF NEMBUTAL
ANESTHESIA
ON THE LEVELS
(GSH) IN LUNG, LIVER AND BLOOD n is t h e n u m b e r o f a n i m a l s ; G S H v a l u e s a r e m e a n s + S.D. Tissue
Treatment
n
GSH nmol/mg soluble protein
Lung
Nembutal Control Nembutal Control Nembutal Control
7 4 3 4 5 1
71.3 69.9 92.4 96.5 15.8 12.6
Liver Blood
-+ 1 2 . 6 +- 1 0 . 4 -+ 1 1 . 7 + 8.2 -+ 2 . 8
OF REDUCED
GLUTATHIONE
70 it was feared that something might be happening to this c o m p o u n d during the preparative procedure. However, when enough glutathione (reduced or oxidized) was added to the lung homogenate to give a 1 mM concentration, all of this glutathione could later be recovered. Thus, reduced glutathione is not oxidized during this procedure, glutathione is not co-precipitating during the trichloroacetic acid step, and nothing else unexpected seems to be happening. Preparation of liver and blood for the determination of glutathione and hemoglobin content and enzyme activities was carried out in essentially the same manner w i t h o u t the perfusion step. Usually a 20% homogenate in water was used.
Determination o f contamination by blood The extent to which the supernatant fractions from lung and liver were contaminated by blood was determined by measuring the hemoglobin content of these fractions (by the absorption at the Soret band) and relating this value to the hemoglobin content of a known volume of blood. Enzyme assays Glutathione S-transferase activity was assayed spectrophotometrically with 3,4
Biochimica et Biophysica Acta, 582 (1979) 67--78
© Elsevier]North-Holland Biomedical Press
BBA 28745 LEVELS OF GLUTATHIONE, GLUTATHIONE REDUCTASE AND GLUTATHIONE S-TRANSFERASE ACTIVITIES IN RAT LUNG AND LIVER
MARIA S. MORON, JOSEPH W. DEPIERRE and BENGT MANNERVIK Department o f Biochemistry, Arrhenius Laboratory, University o f Stockholm, S-106 91 Stockholm (Sweden)
(Received April 17th, 1978) (Revised manuscript received August 4th, 1978) Key words: Glutathione; Glutathione reductase ; Glutathione S-transferase ; (Rat lung)
Summary Levels of glutathione, glutathione reductase and glutathione S-transferase activities in rat lung and liver have been investigated. After perfusing the lung to remove contaminating blood, this organ was f o u n d to have an apparent concentration of glutathione (2 mM) which is approx. 20% of that found in the liver. Both organs contain very low levels of glutathione disulfide. Neither phenobarbital nor methylcholanthrene had a significant effect on the levels of reduced glutathione in lung and liver. In addition, the activities of some glutathione-metabolizing e n z y m e s - glutathione reductase and glutathione S-transferase activity assayed with four different s u b s t r a t e s - were observed to be 5to 60-fold lower in lung tissue than in the liver. Introduction Glutathione is present in all types of living cells [ 1]. The intracellular level of this tripeptide varies with the growth, nutritional state, and hormonal balance of the organism [2,3]. Tissues such as mammalian liver normally contain high levels of reduced glutathione [1]. The functions of glutathione in biological systems are n o t y e t fully understood. It has been suggested t h a t this c o m p o u n d protects thiol groups in protein from oxidation [4], functions as an intracellular redox buffer (see ref. 5) and serves as a reservoir of cysteine [6]. Glutathione also serves as a coenzyme for certain enzymes [7] {e.g., glyoxalase I, maleylacetoacetate isomerase, formaldehyde dehydrogenase, and indolylpyruvic acid ketoenol tautomerase) and as a substrate for others (e.g., glutathione peroxidase, which protects the cell from H20~ and organic hydroperoxides by reduction with concomitant oxida-
68 tion of reduced glutathione; and thioltransferases [8] which use reduced glutathione for the reduction of disulfides). An important group of enzymes for which glutathione is one of the substrates are the glutathione S-transferases. These enzymes are widely distributed among eukaryotes and are often present in relatively high concentrations intracellularly [9]. They are thought to play an important role in the biotransformation of many xenobiotics; the glutathione S-transferases conjugate a variety of pharmacologically active c o m p o u n d s and especially potential alkylating agents, as well as reactive intermediates formed during the metabolism of certain xenobiotics, with glutathione [10]. In general, a glutathione adduct or a mercapturic acid derivative formed from such an adduct is considerably less toxic than the unconjugated precursor c o m p o u n d [ 10]. In addition, it has been postulated that the covalent binding of certain reactive substances to the glutathione S-transferases is a detoxification mechanism [9]. These enzymes, and especially glutathione S-transferase B (also called ligandin), bind certain inducers of c y t o c h r o m e P-450 [11] and thus may have a role in the induction of this c y t o c h r o m e which results from treatment of animals with polycyclic hydrocarbons. Finally, glutathione S-transferases have recently been reported to have glutathione peroxidase activity [12]. To date, glutathione and the glutathione S-transferases have been studied most extensively in the liver. It is important to an understanding of the functioning of other organs, and especially of the relative susceptibility of different organs to the toxic and carcinogenic effects of xenobiotics, to study these cellular components in other tissues as well. Here we have examined glutathione and different glutathione S-transferase activities in rat lung. In addition, since glutathione carries o u t a majority of its functions in the reduced form and since glutathione reductase effects its reduction, we have compared the levels of this enzyme in rat lung and liver. A preliminart report of these results has been made [13]. Materials and Methods Chemicals
[7-3H]Styrene oxide was obtained from the Radiochemical Centre, Amersham, U.K.; unlabeled styrene oxide and 3,4~dichloro-l-nitrobenzene from Schuchardt, Munich, F.G.R.; 1-chloro-2,4-dinitrobenzene from Fluka AG Chemische Fabrik, Buchs, Switzerland; 1,2~epoxy-3-(p-nitrophenoxy)propane from Eastman Kodak Co., Rochester, New York; and glutathione and glutathione disulfide from Boehringer-Mannheim, F.G.R. All other chemicals were also obtained from commercial sources and were of reagent grade. Glutathione reductase from yeast was obtained from Sigma Chemical Company, St. Louis, Mo. Glutathione S-transferase C ("aryltransferase I") was prepared as described earlier [ 14]. A n i m a l s and i n d u c t i o n
Male Sprague-Dawley rats weighing approximately 180--200 g were used. The animals had free access to food and water and were not starved before being killed by decapitation. In several experiments the animals were given
69 intraperitoneal injections of methylcholanthrene (20 mg/kg b o d y weight in corn oil, 5, 3 and 1 day before killing) or phenobarbital (80 mg/kg b o d y weight in isotonic saline solution, once daily for 5 days preceding killing). In these experiments control animals were injected with the same volumes of corn oil or of isotonic saline, respectively.
Perfusion o f the lung and tissue preparation For some unexplained reason, reduced glutathione in rat lung homogenates seems to be oxidized more rapidly than in liver or blood homogenates. The procedures described below should be carried out rapidly enough so that the time elapased between beginning the perfusion of the lung and reading the absorption at 412 nm is not longer than an hour for lung. We found that the lung contains such large amounts of blood and such relatively small amounts of glutathione that correction had to be made for the contribution by contaminating blood. In unperfused lung this correction was as much as 50% of the total value obtained, so that the results were unreliable. Consequently, we perfused the lung in situ through the right ventricle of the heart with 40--60 ml 0.15 M NaC1 before beginning determination of glutathione. This resulted in a correction for blood of 10% or often less. Perfusion could be carried out reasonably successfully in rats whose necks had been dislocated, but it was much easier and more complete after anesthetizing the animals with a lethal dose of nembutal. Table I shows that nembutal had no effect on the levels of reduced glutathione in lung, liver, and blood. This anesthetic was subsequently used routinely. The perfused lung was diced with scissors, homogenized with ten up-anddown strokes at 440 rev./min in a Potter-Elvehjem homogenizer, and diluted to a concentration of approximately 1 g wet weight/ml with water. This homogenate was then sonicated for 1 min. If glutathione was to be determined, enough 25% trichloroacetic acid was added to the sonicate to give a final concentration of 5% (to precipitate protein and particles) and the supernatant obtained by centrifuging in a desk centrifuge for 7 min was used. If glutathione S-transferases or hemoglobin was to be measured, the sonicate was centrifuged at 105 000 × g for 90 min and the supernatant was used. Since the lung was found to contain relatively small amounts of glutathione,
TABLE I LACK OF EFFECT
OF NEMBUTAL
ANESTHESIA
ON THE LEVELS
(GSH) IN LUNG, LIVER AND BLOOD n is t h e n u m b e r o f a n i m a l s ; G S H v a l u e s a r e m e a n s + S.D. Tissue
Treatment
n
GSH nmol/mg soluble protein
Lung
Nembutal Control Nembutal Control Nembutal Control
7 4 3 4 5 1
71.3 69.9 92.4 96.5 15.8 12.6
Liver Blood
-+ 1 2 . 6 +- 1 0 . 4 -+ 1 1 . 7 + 8.2 -+ 2 . 8
OF REDUCED
GLUTATHIONE
70 it was feared that something might be happening to this c o m p o u n d during the preparative procedure. However, when enough glutathione (reduced or oxidized) was added to the lung homogenate to give a 1 mM concentration, all of this glutathione could later be recovered. Thus, reduced glutathione is not oxidized during this procedure, glutathione is not co-precipitating during the trichloroacetic acid step, and nothing else unexpected seems to be happening. Preparation of liver and blood for the determination of glutathione and hemoglobin content and enzyme activities was carried out in essentially the same manner w i t h o u t the perfusion step. Usually a 20% homogenate in water was used.
Determination o f contamination by blood The extent to which the supernatant fractions from lung and liver were contaminated by blood was determined by measuring the hemoglobin content of these fractions (by the absorption at the Soret band) and relating this value to the hemoglobin content of a known volume of blood. Enzyme assays Glutathione S-transferase activity was assayed spectrophotometrically with 3,4
