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Hepatic glutathione biosynthetic capacity in hyperthyroid rat
Toxicology Letters Toxicology
Letters
89 (1996) 85-89
Hepatic glutathione biosynthetic capacity in hyperthyroid Virginia Departamento
de Bioquimica,
Received
Fernhndez”,
Facultad
1 April
de Medicina,
1996; revised
rats
Luis A. Videla
Universidad de Chile, Casilla
17 June
1996; accepted
18 June
70086, Santiago-i:
Chile
1996
Abstract The influence of hyperthyroidism on the capacity of the liver to synthesize glutathione (GSH) was evaluated as a possible mechanism of depletion of the tripeptide. For this purpose, the effect of daily doses of 0.1 mg 3,3’,5-triiodothyronine (T,)/l;g for 3 consecutive days on hepatic GSH biosynthetic capacity was assessed by a combined assay measuring gamma-glutamylcysteinyl synthase and GSH synthase simultaneously. T, treatment induced a significant 56% depletion of liver GSH in parallel with an increase in the rate of GSH synthesis, the latter effect being completely abolished by L-buthionine sulfoximine. According to these data, the fractional rate of hepatic GSH turnover exhibited a 3.2-fold enhancement in hyperthyroid rats compared to control animals. It is concluded that the enhanced GSH utilization in the liver of hyperthyroid rats previously observed [Fernandez et al., Endocrinology 129, 85-91, 19911, is accompanied by an increment in GSH synthesis that is insufficient to sustain the basal levels of the tripeptide observed in euthyroid animals, thus establishing a low steady-state content of GSH in the tissue. Keywords:
Thyroid calorigenesis; Liver glutathione
depletion; Gamma-glutamylcysteinyl
synthase; Glutathione
syn-
thase
1. Introduction Previous studies by our group have shown that hyperthyroidism in the rat leads to a calorigenic response involving an enhancement in hepatic respiration, with the development of an increased oxidative stress status of the liver [l-4]. This latter effect of thyroid hormone is established by
* Corresponding
0378-4274/96/$15.00
author.
Cl 1996 Elsevier
PIZ SO378-4274(96)0,79
l-7
Science Ireland
Ltd. All rights
the increase in the rate of reactive 0, species production [1,3] and by a derangement in some antioxidant mechanisms of the hepatocyte, namely, superoxide dismutase, catalase, and glutathione (GSH) [3]. One of the major changes contributing to oxidative stress is the drastic depletion of hepatic GSH, an important cellular water soluble antioxidant [5], observed both in experimental animals [3,6] and man [7]. Evaluation of the factors involved in this effect of thyroid calorigenesis rereserved
86
V. Fernhdez,
L.A. Videla / Toxicology Letters 89 (1996) 85-89
Table 1 Body weight, liver weight/body weight ratio, serum T, levels, parameters related to thyroid calorigenesis, and liver GSH content in control rats and T,-treated animals Parameters
Controls rats
TX-treated rats
P
Body weight (g) Liver/body weight (g/l00 g) Serum T, (ng/dl) Rectal temperature (“C) Liver 0, uptake (umol/g liver/min) Liver GSH content (umol/g liver)
220 k 10 (16) 3.71 + 0.20 (16) 66&6 (10) 37.5 +O.l (16) 1.95+0.10 (5) 7.79 * 0.34 (4)
225 + 3.85 k 276 + 38.4 k 2.49 k 3.40 k
NS NS 0.001 0.01 0.001 0.01
12 (16) 0.25 (16) 41 (6) 0.1 (16) 0.18 (5) 0.61 (4)
Values shown correspond to the means f S.E.M. for the number (n) of rats indicated in parentheses. The significance of the differences between mean values was assessed by the Student’s t-test for unpaired results. NS, not significant.
vealed that alterations in GSH utilization for hydroperoxide metabolism and conjugation processes are not involved [6]. This view is supported by the lack of changes in the activity of glutathione peroxidase and glutathione reductase in the liver of hyperthyroid rats over control values, and by the significant diminution in the activities of glutathione-S-transferases [6]. The lack of changes in the activity of the glutathione peroxidase-reductase couple by T, treatment was found concomitantly with a 84% enhancement in the activity of the NADPH-generating enzyme glucose-6-phosphate dehydrogenase [6]. These findings suggest an adequate handling of the lipid hydroperoxides [8] and hydrogen peroxide [3] generated at higher rates in the liver of hyperthyroid animals, with the glutathione disulfide (GSSG) produced being effectively reduced to GSH. The assessment of additional processes related to GSH use in the hepatocyte showed that hyperthyroidism leads to a significant elevation in gamma glutamyltransferase activity and in the sinusoidal efflux of GSH, without altering the biliary release of this tripeptide [6]. Although these findings point to the loss of GSH from the liver into blood and intracellular catabolism as the major mechanisms responsible for the diminution in hepatic GSH content induced by hyperthyroidism [6], the latter process seems to be of limited importance, due to the relatively small concentration of gamma glutamyltransferase in rat liver [9]. In the present work, the possibility that thyroid hormone-induced liver GSH deple-
tion may also be conditioned by changes in the capacity of the hepatocyte to synthesize GSH de novo was considered. For this purpose, the activity of gamma-glutamylcysteinyl synthase and GSH synthase was measured by a combined assay in the liver of rats made hyperthyroid by ~-3,3’S_ triiodothyronine (TJ administration, in relation to the respective euthyroid animals.
2. Materials and methods Female Sprague-Dawley rats fed ad libitum received daily intraperitoneal injections of 0.1 mg of T,/kg body weight or equivalent volumes of T, diluent (0.1 N NaOH) (controls) for 3 consecutive days. Studies were carried out 24 h after the last treatment in control rats and T,-treated animals exhibiting comparable values of body weight and the respective liver weight/body weight ratios (Table 1). At this time, the rectal temperature of the animals (measured with a thermocouple model 8112-20, Cole-Parmer Instrument Co., Chicago, IL), the serum T, levels (determined by radioimmunoassay, Baxter Healthcare Corp., Cambridge, MA), and the rate of 0, uptake by the liver (measured polarographically in perfusion experiments as described elsewhere [4,6]), were determined in both experimental groups. Animals were anesthetized with sodium nembutal (50 mg/kg, intraperitoneally) and the liver was perfused in situ with 150 ml of a cold solution containing 150 mM KC1 and 5 mM Tris pH 7.4
V. Femindez,
L.A. Videla 1 Toxicology Lelters 89 (1996) 85-89
87
Table 2 Glutathione biosyntheiic capacity in the liver of control rats and hyperthyroid animals in the absence and presence of r_-buthionine sulfoximine (BSO) BSO (2.5 PM) nmol/mg of protein/mm _ + nmol/g of liver/min _ +
Control rats
T,-treated rats
Effect (%)
P
0.71 kO.11 (14) 0.20 * 0.14 (3)
1.40 * 0.22 (14) 0.10 + 0.09 (3)
97
0.05
67 k 9 (14) 20 f 14 (3)
122 + 18 (14) 11 *9 (3)
82
0.05
The hepatic glutathione biosynthetic capacity was measured as GSH production from its constituent amino acids, which assesses the activity of both gamma-glutamylcysteinyl synthase and GSH synthase [I 1,121. Values shown correspond to the means + S.E.M. for the number of rats indicated in parentheses. The significance of the differences between mean values was determined by the Student’s z-test for unpaired data. “Effect of BSO compa,red to no addition conditions both in controls and hyperthyroid rats (P
to remove blood. Liver total GSH equivalents were determined by the catalytic assay of Tietze [lo]. For the determination of the hepatic GSH biosynthetic capacity [I 1,121, liver samples were homogenized (1:4) in 150 mM KC1 containing 5 mM Tris pH 7.4 and were subjected to differential centrifugation. The postmicrosomal supernatant obtained after centrifugation of the samples at 105 000 x g for 60 min at 4”C, was used for the measurement of enzyme activity. This was carried out in a reaction medium containing 100 mM Tris pH 8, 5 mM L-cysteine, 5 mM glycine, 5 mM L-glutamate, 5 mM ATP, 10 mM MgCl,, and 0.2 mM EDTA [12], with and without the cytosolic fraction. This reaction medium was incubated at 37°C and different aliquots were taken between 0 and 10 min, followed by the addition of 1 N HClO, to stop the reaction. The acidified samples were supplemented with 52 mM glyoxylic acid and incubated at 65°C for 25 min to eliminate free cysteine [13], neutralized with 1.75 M K,PO,, and the GSH produced was measured according to Tietze [lo]. In these conditions, the reaction reached equilibrium after 4-7 min incubation, and values corrected for zero time GSH were expressed either as nmol/mg protein/min or nmol/g liver/min. Separate determinations were carried out in the presence of 2.5 uM L-buthionine sulfoximine, inhibitor of gamma-glutamylcysteinyl synthase [14]. Protein content was assayed according to Lowry et al. u51.
Chemicals and reagents used were obtained from Sigma Chemical Co. (St. Louis, MO). Values shown correspond to the means f S.E.M. for the number of separate experiments indicated. The statistical significance of the differences between mean values was carried out by the Student’s t-test for unpaired results.
3. Results and discussion The administration of T, to fed rats resulted in a significant increase in the serum levels of the hormone, concomitantly with an enhancement in the rectal temperature of the animals (Table l), thus evidencing thyroid calorigenesis. Thyroid calorigenesis induced in the rat by T, administration was accompanied by an enhanced rate of O2 consumption by the liver (Table l), known to involve higher rates of reactive 0, species production at microsomal, mitochondrial, and peroxisoma1 sites [1,3]. Concomitantly, the content of hepatic GSH was reduced by 56% compared to control values (Table l), thus establishing a condition of increasing susceptibility to oxidative stress. In these conditions, the GSH-biosynthetic capacity of the liver of hyperthyroid rats was significantly increased over that in control animals, expressed either as nmol/mg protein/min or nmol/ g liver/min (Table 2). Furthermore, all the increment in the rate of GSH synthesis elicited by hyperthyroidism was abolished by L-buthionine
88
V. Fermindez,
L.A.
Videia / Toxicology
sulfoximine (Table 2), a selective inhibitor of gamma-glutamylcysteinyl synthase, enzymatic activity that initiates the biosynthesis of the tripeptide [14]. Using the respective rates of GSH synthesis (Table 2) and the size of the hepatic GSH pools (Table l), the corresponding fractional rates of GSH turnover were calculated to be 8.60 x 10V3 min’ (0.067 [umol/g/min]/7.79 [umol/g]) in control rats and 3.59 x lo-* min ’ (0.122 [umol/g/min]/3.4 [umol/g]) in TX-treated animals. However, in order to maintain basal levels of hepatic GSH of 7.79 umol/g (Table 1) in the hyperthyroid state, the rate of synthesis of the tripeptide should have increased to 0.28 umol/g/ min, considering the fractional rate of GSH turnover of 3.59 x 10 ~ * min ~ ’ . These data indicate that thyroid hormone-induced liver GSH depletion under oxidative stress conditions is accompanied by an enhancement in the rate of GSH synthesis assessed in vitro, leading to a higher GSH turnover rate that agrees with the value obtained in vivo after a pulse of [35S]cysteine [6]. Since the increment in GSH synthesis is insufficient to account for the drastic increase in the rate of sinusoidal efflux of the tripeptide previously observed [6], a low steadystate level of GSH is established in the tissue (Table 1). In agreement with the data presented in this work, liver GSH depletion induced by shortterm fasting [ 161, diethylmaleate [ 161, chronic ethanol administration [17], or acute lindane intoxication [18] has been shown to increase the rate of synthesis of GSH, which is probably related to a lower feedback inhibition of GSH synthesis imposed by the increased consumption of the tripeptide [9]. Although the liver has the capacity to synthesize GSH in large amounts at a rapid rate under normal conditions [9], the maintenance of adequate GSH levels under stressed conditions such as hyperthyroidism may depend on additional factors. These include the availability of the rate-limiting precursor cysteine, changes in the content of the enzymes involved in GSH synthesis, and/or the production of an inhibitor of GSH synthesis which may act on gamma-glutamylcysteinyl synthase or GSH synthase. These aspects, however, remain to be elucidated in the liver of hyperthyroid animals.
Letters
89 (1996) 85-89
Acknowledgements This work was supported by grant 19403 12 from FONDECYT (Chile). The technical assistance of C. Almeyda and M. Suarez is kindly acknowledged.
References 111Fernandez,
V., Barrientos, X., Kipreos, K., Valenzuela, A. and Videla, L.A. (1985) Superoxide radical generation, NADPH oxidase activity, and cytochrome P-450 content of rat liver microsomal fractions in an experimental hyperthyroid state: relation to lipid peroxidation. Endocrinology 117, 4966501. PI Fernandez, V., Llesuy, S., Solari, L., Kipreos, K., Videla, L.A. and Boveris, A. (1988) Chemihtminescent and respiratory responses related to thyroid hormone-induced liver oxidative stress. Free Radic. Res. Commun. 5, 7784. [31 Fernandez, V. and Videla, L.A. (1993) Influence of hyperthyroidism on superoxide radical and hydrogen peroxide production by rat liver submitochondrial particles. Free Radic. Res. Commun. 18, 329-335. [41 Fernandez, V. and Videla, L.A. (1993) 3,3’,5_Triiodothyronine-induced hepatic respiration: effects of desferrioxamine and allopurinol in the isolated perfused rat liver. Toxicol. Lett. 69, 2055210. toxicological implications. [51 Reed, D.J. (1990) Glutathione: Annu. Rev. Pharmacol. Toxicol. 30, 603-631. PI Fernandez, V., Simizu, K., Barros, S.B.M., Azzalis, L.A., Pimentel, R., Junqueira, V.B.C. and Videla, L.A. (1991) Effects of hyperthyroidism on rat liver glutathione metabolism: related enzymes’ activities, efflux, and turnover. Endocrinology 129, 85-91. 171Sir, T., Wolff, C., Soto, J.R., Perez, G. and ArmasMerino, R (1987) Relationship between hepatic levels of glutathione and sulphobromophthalein retention in hyperthyroidism. Clin. Sci. 73, 235-237. PI Landriscina, C., Petragallo, V., Morini, P. and Marcotrigiano, G.O. (1988) Lipid peroxidation in rat liver microsomes. I. Stimulation of the NADPH cytochrome P-450 state. reductase-dependent process in hyperthyroid Biochem. Int. 17, 3855393. [91 Kaplowitz, N., Aw, T.Y. and Ookhtens, M. (1985) The regulation of hepatic glutathione. Annu. Rev. Pharmacol. Toxicol. 25, 7155744. deter[lOI Tietze, F. (1969) Enzymic method for quantitative mination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Anal. Biochem. 27, 5022522. u11 Guerri, C. and Grisolia, S. (1989) Changes in glutathione in acute and chronic alcohol intoxication. Pharmacol. Biochem. Behav. 13, 53361.
V. Femindez,
L.A. Videla / Toxicology Letters 89 (1996) 85-89
[12] Morton, S. and Mitchell, M.C. (1985) Effects of chronic
ethanol feeding on glutathione turnover in the rat. Biochem. Pharmacol. 34, 1559- 1563. [13] Ball, C.R. (1966) IEstimation and identification of thiols in rat spleen after lcysteine or glutathione treatment: relevance to protection against nitrogen mustards. Biochem. Pharmacol. 15, 80!)-816. [14] Meister, A. (1984) New aspects of glutathione biochemistry and transport: selective alteration of glutathione metabolism. Fed. Proc. 43, 3031-3042. [I 51 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randal1,R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275.
89
[16] Lauterburg, B.H. and Mitchell, J.R. (1981) Regulation of hepatic glutathione turnover in rats in vivo and evidence for kinetic homogeneity of the hepatic glutathione pool. J. Clin. Invest. 67, 1415-1424. [17] Femandez-Checa, J.C., Ookhtens, M. and Kaplowitz, N. (1987) Effect of chronic ethanol feeding on rat hepatocytic glutathione. Compartmentation, eftlux, and response to incubation with ethanol. J. Clin. Invest. 80, 57-62. [18] Junqueira, V.B.C., Barros, S.B.M., Simizu, K., Fernandez, V., Carrion, Y., Pimentel, R., Azzalis, L.A. and Videla, L.A. (1993) Turnover of hepatic giutathione after acute lindane intoxication. Toxicol. Lett. 69, 21 l-216.
Letters
89 (1996) 85-89
Hepatic glutathione biosynthetic capacity in hyperthyroid Virginia Departamento
de Bioquimica,
Received
Fernhndez”,
Facultad
1 April
de Medicina,
1996; revised
rats
Luis A. Videla
Universidad de Chile, Casilla
17 June
1996; accepted
18 June
70086, Santiago-i:
Chile
1996
Abstract The influence of hyperthyroidism on the capacity of the liver to synthesize glutathione (GSH) was evaluated as a possible mechanism of depletion of the tripeptide. For this purpose, the effect of daily doses of 0.1 mg 3,3’,5-triiodothyronine (T,)/l;g for 3 consecutive days on hepatic GSH biosynthetic capacity was assessed by a combined assay measuring gamma-glutamylcysteinyl synthase and GSH synthase simultaneously. T, treatment induced a significant 56% depletion of liver GSH in parallel with an increase in the rate of GSH synthesis, the latter effect being completely abolished by L-buthionine sulfoximine. According to these data, the fractional rate of hepatic GSH turnover exhibited a 3.2-fold enhancement in hyperthyroid rats compared to control animals. It is concluded that the enhanced GSH utilization in the liver of hyperthyroid rats previously observed [Fernandez et al., Endocrinology 129, 85-91, 19911, is accompanied by an increment in GSH synthesis that is insufficient to sustain the basal levels of the tripeptide observed in euthyroid animals, thus establishing a low steady-state content of GSH in the tissue. Keywords:
Thyroid calorigenesis; Liver glutathione
depletion; Gamma-glutamylcysteinyl
synthase; Glutathione
syn-
thase
1. Introduction Previous studies by our group have shown that hyperthyroidism in the rat leads to a calorigenic response involving an enhancement in hepatic respiration, with the development of an increased oxidative stress status of the liver [l-4]. This latter effect of thyroid hormone is established by
* Corresponding
0378-4274/96/$15.00
author.
Cl 1996 Elsevier
PIZ SO378-4274(96)0,79
l-7
Science Ireland
Ltd. All rights
the increase in the rate of reactive 0, species production [1,3] and by a derangement in some antioxidant mechanisms of the hepatocyte, namely, superoxide dismutase, catalase, and glutathione (GSH) [3]. One of the major changes contributing to oxidative stress is the drastic depletion of hepatic GSH, an important cellular water soluble antioxidant [5], observed both in experimental animals [3,6] and man [7]. Evaluation of the factors involved in this effect of thyroid calorigenesis rereserved
86
V. Fernhdez,
L.A. Videla / Toxicology Letters 89 (1996) 85-89
Table 1 Body weight, liver weight/body weight ratio, serum T, levels, parameters related to thyroid calorigenesis, and liver GSH content in control rats and T,-treated animals Parameters
Controls rats
TX-treated rats
P
Body weight (g) Liver/body weight (g/l00 g) Serum T, (ng/dl) Rectal temperature (“C) Liver 0, uptake (umol/g liver/min) Liver GSH content (umol/g liver)
220 k 10 (16) 3.71 + 0.20 (16) 66&6 (10) 37.5 +O.l (16) 1.95+0.10 (5) 7.79 * 0.34 (4)
225 + 3.85 k 276 + 38.4 k 2.49 k 3.40 k
NS NS 0.001 0.01 0.001 0.01
12 (16) 0.25 (16) 41 (6) 0.1 (16) 0.18 (5) 0.61 (4)
Values shown correspond to the means f S.E.M. for the number (n) of rats indicated in parentheses. The significance of the differences between mean values was assessed by the Student’s t-test for unpaired results. NS, not significant.
vealed that alterations in GSH utilization for hydroperoxide metabolism and conjugation processes are not involved [6]. This view is supported by the lack of changes in the activity of glutathione peroxidase and glutathione reductase in the liver of hyperthyroid rats over control values, and by the significant diminution in the activities of glutathione-S-transferases [6]. The lack of changes in the activity of the glutathione peroxidase-reductase couple by T, treatment was found concomitantly with a 84% enhancement in the activity of the NADPH-generating enzyme glucose-6-phosphate dehydrogenase [6]. These findings suggest an adequate handling of the lipid hydroperoxides [8] and hydrogen peroxide [3] generated at higher rates in the liver of hyperthyroid animals, with the glutathione disulfide (GSSG) produced being effectively reduced to GSH. The assessment of additional processes related to GSH use in the hepatocyte showed that hyperthyroidism leads to a significant elevation in gamma glutamyltransferase activity and in the sinusoidal efflux of GSH, without altering the biliary release of this tripeptide [6]. Although these findings point to the loss of GSH from the liver into blood and intracellular catabolism as the major mechanisms responsible for the diminution in hepatic GSH content induced by hyperthyroidism [6], the latter process seems to be of limited importance, due to the relatively small concentration of gamma glutamyltransferase in rat liver [9]. In the present work, the possibility that thyroid hormone-induced liver GSH deple-
tion may also be conditioned by changes in the capacity of the hepatocyte to synthesize GSH de novo was considered. For this purpose, the activity of gamma-glutamylcysteinyl synthase and GSH synthase was measured by a combined assay in the liver of rats made hyperthyroid by ~-3,3’S_ triiodothyronine (TJ administration, in relation to the respective euthyroid animals.
2. Materials and methods Female Sprague-Dawley rats fed ad libitum received daily intraperitoneal injections of 0.1 mg of T,/kg body weight or equivalent volumes of T, diluent (0.1 N NaOH) (controls) for 3 consecutive days. Studies were carried out 24 h after the last treatment in control rats and T,-treated animals exhibiting comparable values of body weight and the respective liver weight/body weight ratios (Table 1). At this time, the rectal temperature of the animals (measured with a thermocouple model 8112-20, Cole-Parmer Instrument Co., Chicago, IL), the serum T, levels (determined by radioimmunoassay, Baxter Healthcare Corp., Cambridge, MA), and the rate of 0, uptake by the liver (measured polarographically in perfusion experiments as described elsewhere [4,6]), were determined in both experimental groups. Animals were anesthetized with sodium nembutal (50 mg/kg, intraperitoneally) and the liver was perfused in situ with 150 ml of a cold solution containing 150 mM KC1 and 5 mM Tris pH 7.4
V. Femindez,
L.A. Videla 1 Toxicology Lelters 89 (1996) 85-89
87
Table 2 Glutathione biosyntheiic capacity in the liver of control rats and hyperthyroid animals in the absence and presence of r_-buthionine sulfoximine (BSO) BSO (2.5 PM) nmol/mg of protein/mm _ + nmol/g of liver/min _ +
Control rats
T,-treated rats
Effect (%)
P
0.71 kO.11 (14) 0.20 * 0.14 (3)
1.40 * 0.22 (14) 0.10 + 0.09 (3)
97
0.05
67 k 9 (14) 20 f 14 (3)
122 + 18 (14) 11 *9 (3)
82
0.05
The hepatic glutathione biosynthetic capacity was measured as GSH production from its constituent amino acids, which assesses the activity of both gamma-glutamylcysteinyl synthase and GSH synthase [I 1,121. Values shown correspond to the means + S.E.M. for the number of rats indicated in parentheses. The significance of the differences between mean values was determined by the Student’s z-test for unpaired data. “Effect of BSO compa,red to no addition conditions both in controls and hyperthyroid rats (P
to remove blood. Liver total GSH equivalents were determined by the catalytic assay of Tietze [lo]. For the determination of the hepatic GSH biosynthetic capacity [I 1,121, liver samples were homogenized (1:4) in 150 mM KC1 containing 5 mM Tris pH 7.4 and were subjected to differential centrifugation. The postmicrosomal supernatant obtained after centrifugation of the samples at 105 000 x g for 60 min at 4”C, was used for the measurement of enzyme activity. This was carried out in a reaction medium containing 100 mM Tris pH 8, 5 mM L-cysteine, 5 mM glycine, 5 mM L-glutamate, 5 mM ATP, 10 mM MgCl,, and 0.2 mM EDTA [12], with and without the cytosolic fraction. This reaction medium was incubated at 37°C and different aliquots were taken between 0 and 10 min, followed by the addition of 1 N HClO, to stop the reaction. The acidified samples were supplemented with 52 mM glyoxylic acid and incubated at 65°C for 25 min to eliminate free cysteine [13], neutralized with 1.75 M K,PO,, and the GSH produced was measured according to Tietze [lo]. In these conditions, the reaction reached equilibrium after 4-7 min incubation, and values corrected for zero time GSH were expressed either as nmol/mg protein/min or nmol/g liver/min. Separate determinations were carried out in the presence of 2.5 uM L-buthionine sulfoximine, inhibitor of gamma-glutamylcysteinyl synthase [14]. Protein content was assayed according to Lowry et al. u51.
Chemicals and reagents used were obtained from Sigma Chemical Co. (St. Louis, MO). Values shown correspond to the means f S.E.M. for the number of separate experiments indicated. The statistical significance of the differences between mean values was carried out by the Student’s t-test for unpaired results.
3. Results and discussion The administration of T, to fed rats resulted in a significant increase in the serum levels of the hormone, concomitantly with an enhancement in the rectal temperature of the animals (Table l), thus evidencing thyroid calorigenesis. Thyroid calorigenesis induced in the rat by T, administration was accompanied by an enhanced rate of O2 consumption by the liver (Table l), known to involve higher rates of reactive 0, species production at microsomal, mitochondrial, and peroxisoma1 sites [1,3]. Concomitantly, the content of hepatic GSH was reduced by 56% compared to control values (Table l), thus establishing a condition of increasing susceptibility to oxidative stress. In these conditions, the GSH-biosynthetic capacity of the liver of hyperthyroid rats was significantly increased over that in control animals, expressed either as nmol/mg protein/min or nmol/ g liver/min (Table 2). Furthermore, all the increment in the rate of GSH synthesis elicited by hyperthyroidism was abolished by L-buthionine
88
V. Fermindez,
L.A.
Videia / Toxicology
sulfoximine (Table 2), a selective inhibitor of gamma-glutamylcysteinyl synthase, enzymatic activity that initiates the biosynthesis of the tripeptide [14]. Using the respective rates of GSH synthesis (Table 2) and the size of the hepatic GSH pools (Table l), the corresponding fractional rates of GSH turnover were calculated to be 8.60 x 10V3 min’ (0.067 [umol/g/min]/7.79 [umol/g]) in control rats and 3.59 x lo-* min ’ (0.122 [umol/g/min]/3.4 [umol/g]) in TX-treated animals. However, in order to maintain basal levels of hepatic GSH of 7.79 umol/g (Table 1) in the hyperthyroid state, the rate of synthesis of the tripeptide should have increased to 0.28 umol/g/ min, considering the fractional rate of GSH turnover of 3.59 x 10 ~ * min ~ ’ . These data indicate that thyroid hormone-induced liver GSH depletion under oxidative stress conditions is accompanied by an enhancement in the rate of GSH synthesis assessed in vitro, leading to a higher GSH turnover rate that agrees with the value obtained in vivo after a pulse of [35S]cysteine [6]. Since the increment in GSH synthesis is insufficient to account for the drastic increase in the rate of sinusoidal efflux of the tripeptide previously observed [6], a low steadystate level of GSH is established in the tissue (Table 1). In agreement with the data presented in this work, liver GSH depletion induced by shortterm fasting [ 161, diethylmaleate [ 161, chronic ethanol administration [17], or acute lindane intoxication [18] has been shown to increase the rate of synthesis of GSH, which is probably related to a lower feedback inhibition of GSH synthesis imposed by the increased consumption of the tripeptide [9]. Although the liver has the capacity to synthesize GSH in large amounts at a rapid rate under normal conditions [9], the maintenance of adequate GSH levels under stressed conditions such as hyperthyroidism may depend on additional factors. These include the availability of the rate-limiting precursor cysteine, changes in the content of the enzymes involved in GSH synthesis, and/or the production of an inhibitor of GSH synthesis which may act on gamma-glutamylcysteinyl synthase or GSH synthase. These aspects, however, remain to be elucidated in the liver of hyperthyroid animals.
Letters
89 (1996) 85-89
Acknowledgements This work was supported by grant 19403 12 from FONDECYT (Chile). The technical assistance of C. Almeyda and M. Suarez is kindly acknowledged.
References 111Fernandez,
V., Barrientos, X., Kipreos, K., Valenzuela, A. and Videla, L.A. (1985) Superoxide radical generation, NADPH oxidase activity, and cytochrome P-450 content of rat liver microsomal fractions in an experimental hyperthyroid state: relation to lipid peroxidation. Endocrinology 117, 4966501. PI Fernandez, V., Llesuy, S., Solari, L., Kipreos, K., Videla, L.A. and Boveris, A. (1988) Chemihtminescent and respiratory responses related to thyroid hormone-induced liver oxidative stress. Free Radic. Res. Commun. 5, 7784. [31 Fernandez, V. and Videla, L.A. (1993) Influence of hyperthyroidism on superoxide radical and hydrogen peroxide production by rat liver submitochondrial particles. Free Radic. Res. Commun. 18, 329-335. [41 Fernandez, V. and Videla, L.A. (1993) 3,3’,5_Triiodothyronine-induced hepatic respiration: effects of desferrioxamine and allopurinol in the isolated perfused rat liver. Toxicol. Lett. 69, 2055210. toxicological implications. [51 Reed, D.J. (1990) Glutathione: Annu. Rev. Pharmacol. Toxicol. 30, 603-631. PI Fernandez, V., Simizu, K., Barros, S.B.M., Azzalis, L.A., Pimentel, R., Junqueira, V.B.C. and Videla, L.A. (1991) Effects of hyperthyroidism on rat liver glutathione metabolism: related enzymes’ activities, efflux, and turnover. Endocrinology 129, 85-91. 171Sir, T., Wolff, C., Soto, J.R., Perez, G. and ArmasMerino, R (1987) Relationship between hepatic levels of glutathione and sulphobromophthalein retention in hyperthyroidism. Clin. Sci. 73, 235-237. PI Landriscina, C., Petragallo, V., Morini, P. and Marcotrigiano, G.O. (1988) Lipid peroxidation in rat liver microsomes. I. Stimulation of the NADPH cytochrome P-450 state. reductase-dependent process in hyperthyroid Biochem. Int. 17, 3855393. [91 Kaplowitz, N., Aw, T.Y. and Ookhtens, M. (1985) The regulation of hepatic glutathione. Annu. Rev. Pharmacol. Toxicol. 25, 7155744. deter[lOI Tietze, F. (1969) Enzymic method for quantitative mination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Anal. Biochem. 27, 5022522. u11 Guerri, C. and Grisolia, S. (1989) Changes in glutathione in acute and chronic alcohol intoxication. Pharmacol. Biochem. Behav. 13, 53361.
V. Femindez,
L.A. Videla / Toxicology Letters 89 (1996) 85-89
[12] Morton, S. and Mitchell, M.C. (1985) Effects of chronic
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