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Effect of hyperthyroidism on the biliary release of thiobarbituric acid reactants in the rat
Toxicology Letters Toxicology Letters 84 (I 996) 149- 153
ELSEVIER
Effect of hyperthyroidism on the biliary release of thiobarbituric acid reactants in the rat Virginia
Fernindez*,
Luis A. Videla
Departamento a’eBioquimica, Facultad de Medicina. Universidnd de Chile, Casilla 70086, Santiago-7, Chile IReceived 3 July 1995; revision received 13 October 1995; accepted 16 October 1995
Abstract The influence of daily doses of 0.1 mg 3,3’,5triiodothyronine (TJkg for three consecutive days on hepatic lipid peroxidation was asressed by the biliary release of thiobarbituric acid reactants (TBARS) in anesthetized rats. T, treatment elicited a significant 80% increase in the biliary eMux of TBARS compared to control values, an effect determined by increments in both the TBARS concentration in bile (44%) and in the bile flow (25%). It is concluded that hyperthyroidism-induced oxidative stress stimulates hepatic lipid peroxidation measured in an in situ liver preparation, which significantly correlates with the enhancement in the rate of TBARS production assessed in liver homogenates. Keywords: Thyroid calorigenesis; Liver oxidative stress; Lipid peroxidation
1. Introduction Hyperthyroidism has been found to induce an imbalance in the prooxidant-antioxidant equilibrium of the hepatocyte in favor of the prooxidants [1,2]. This enhancement in the oxidative stress status of the liver is conditioned by a higher rate of reactive 0, species production [3,4}, associated with the increased txllular respiration elicited, and by a diminution in some antioxidant mechanisms. The latter effect of ithyroid hormone is characterized by significant decreases in the activity of hepatic superoxide dismutase and catalase [5], and in the content of reduced glutathione [5,6].
l
Corresponding author.
One consequence of an enhanced cellular steady-state level of reactive 02 species is the oxidative damage to polyunsaturated fatty acids in phospholipids of biological membranes [7]. Accordingly, thyroid hormone-induced liver oxidative stress has been reported to increase hepatic lipid peroxidation, as evidenced by the significant increments in the content of thiobarbituric acid reactants (TBARS) in rat liver homogenates and in microsomal fractions [5], spontaneous chemiluminescence of rat liver homogenates [5], NADPH-dependent accumulation of hydroperoxides in rat liver microsomes [S], and in the Fe2+-induced light emission of rabbit liver mitochondria [9]. However, this effect of hyperthyroidism has not been observed when lipid peroxidation is assessed by the TBARS index,
03784274/96/1615.00 Q 1996 Elsevier Science Ireland Ltd. AI1 rights reserved SSDI 0378-4274(95)0?;617-T
150
V. Fermindez, L. A. Videla / Toxicology Letters 84 (19%)
either in liver homogenates [lo] or in liver microsomal fractions supplemented with NADPH, Fe’+, or NADPH plus Fe’+ 181.The discrepancy observed under in vitro conditions could be due to the fact that lipid peroxidation-derived aldehydic products may participate in other reactions than that with thiobarbituric acid, including aldol-type self-condensation, oxidation by aldehyde dehydrogenases, and/or reaction with primary amino groups in proteins, nucleic acids, and amino phospholipids, to give fluorescent and nonfluorescent adducts [l 11. In view of these considerations, the present work evaluates the lipid peroxidative response of the liver to thyroid hormone-induced oxidative stress by means of an in situ liver technique. For this purpose, the biliary release of TBARS was measured in anesthetized control rats and 3,3’,5triiodothyronine (T&treated animals, and results were correlated with the rate of TBARS production by liver homogenates.
149-153
weight/body weight ratios (Table 1). Before biochemical studies, the rectal temperature of the rats was measured with a thermocouple (model 811220, Cole-Parmer Instrument Co., Chicago, IL), and blood samples were taken from the tail vein for the determination of serum T, levels by radioimmunoassay (Baxter Healthcare Corp., Cambridge, MA). The rate of O2 consumption by the liver was measured polarographically in perfusion experiments, as previously described [6,12]. Animals were anesthetized with sodium pentobarbital (50 mgikg, intraperitoneally) and the peritoneal cavity was opened to cannulate the bile duct with a PE-10 polyethylene tubing. The abdominal cavity was covered with a cheesecloth humidified with 0.9% w/v NaCl and bile was collected for 15 min in animals kept in an environmental temperature of 28-30°C. The concentration of TBARS in bile samples was immediately determined as described by Buege and Aust [13]. At the end of this procedure, the livers were perfused in situ with 200 ml of a cold solution containing 140 mM KC1 and 10 mM potassium phosphate buffer, pH 1.4, to remove blood, and weighed to express the bile flow in fig liver/mm. The biliary efflux of TBARS was calculated by multiplying their concentration in the bile by the respective bile flows. The rate of TBARS formation [ 131 was assayed in liver homogenates (1:4) prepared in 140 mM KC1 containing 10 mM potassium phosphate buffer, pH 7.4, after centrifuga-
2. Materials and methods Female Sprague-Dawley rats fed ad libitum received daily intraperitoneal injections of 0.1 mg of T&g body weight or equivalent volumes of T3 diluent (0.1 N NaOH) (controls) for 3 consecutive days, and studies were performed 24 h after the last treatment. In these conditions, control rats and Trtreated animals exhibited comparable values of body weight and the respective liver
Table 1 Body weight, liverlbody weight ratio, serum T3 levels, and parameters related to thyroid calorigenesis in control rats and T,treated animals Parameters
Control rats
Ts-treated rats
P-value
Body weight (g) (n = 13) Liver/body weight (g/100 g) (n = 13) Serum Ts (ng/dl) (n = 6) Rectal temperature (“C) (n = 13) Liver O2 consumption (runol/g liver/mm) (n = 6)
203 f 4
210
NS
3.51 f 0.09
3.62 f 0.12
NS
70 f 7
314 f 23
0.001
37.4 f 0.1
38.5 f 0.1
0.001
1.88 f 0.95
2.55 f 0.10
0.001
l
8
Values shown represent the means f S.E.M. for the indicated number of rats per experimental group. The significance of the differences between mean values was carried out by the Student’s r-test for unpaired data. NS, not significant.
V. Fernhdez. L.A. Videla/ Toxicology Letters 84 (19%) 149-153
tion at 900 x g for 20 min at 4°C. Incubations were carried out at 37°C for 75 min, and aliquots were taken every 15 min for the assessment of maximal rates, expressed as nmohmg proteiti. The protein content of liver homogenates was measured according to Lowry et al. [14]. Chemicals and reagents used were obtained from Sigma Chemical Co. (St. Louis, MO). Values shown are means 3: S.E.M. for the number of separate experiments indicated. The statistical significance of the differences between mean values was assessed by Student’s t-test for unpaired data. 3. Rest&a and discrlssion T3 administration to fed rats resulted in a significant 3.5-fold increment in the serum level of the hormone, in concomitance with a calorigenic response evidenced by the enhancement in the rectal temperature of the animals (Table 1). In these conditions, the rate of O2 consumption by the liver increased by 3(5%(Table l), in agreement with earlier studies [6,1:2]. The liver is a major organ exhibiting an accelerated respiration in the hyperthyroid state, whiclh seems to be due to the interaction of Ts with nuclear receptors, leading to an enhanced synthesis of enzymatic systems involved in redox processes 1151. Acceleration of hepatic respiration involvles a component comprising 16%-25% of the net increase in hepatic 0, uptake, which was suggested to represent 02 equivalents related to TJ-induced oxidative stress [ 121. This respiratalry component assessed in liver perfusion studies correlates with the higher rates
Fig. I. Correlation between the rate of thiobarbituric acid reactants (TBARS) formation in liver homogenates and the respective biliary release of TBARS in control rats (0) and Ts-treated animals (0). Regression line, y = -0.0047 + 0.0918x (r = 0.91; P < 0.001). Inset: rate of TBARS formation in liver homogenates from control rats (C) and hyperthyroid animals (T’d. Values shown represent the means + S.E.M. for 7 rats per experimental group. The significance between mean values was asmssed by Student’s t-test for unpaired data.
of NADPH-dependent superoxide radical (Or’-) generation observed in liver microsomes [3], as well as with the NADH- or succinate-supported O2 .- production in liver submitochondrial particles [4]. In the latter system, Ts treatment led to hydrogen peroxide release at higher rates than
Table 2 Biliary release of thiobarbituric acid reactants (TBARS) in control rats and T,-treated animals Parameters
Control rats (n = 7)
T,-treated rats (n = 7)
Change (W
P
Bile flow @l/g liver/mm) Biliary TBARS concentration (~mol/rnl bile) Biliary release of TBARS (nmohg liver/mitt)
1.01 l 0.09 5.36 f 0.40
1.26 f 0.05 7.73 f 0.26
25 44
0.05 0.001
5.41
9.74 + 0.31
80
0.001
l
0.45
Determinations were caaried out in anesthetized control rats and hyperthyroid animals kept at an environmental temperature of 28-304C, as described in Materials and methods. Results are means f S.E.M. for the number (a) of rats indicated. The signiticance of the differences between mean values was assessed by Student’s t-test for unpaired results.
152
K Fermindez. L.A. Videla / Toxicology Letters 84 (19%)
control values, both under basal conditions and in the succinate- or urate-supported processes [4]. Under conditions of an enhanced liver free radical activity, hyperthyroidism elicited a significant 80% increase in the biliary release of TBARS, which results from the enhancement in the biliary concentration of TBARS and in the bile flow over control values (Table 2). Thyroid hormone is known to alter bile secretory functions by influencing both the rate of bile production and the synthesis and biliary release of bile acids, primarily increasing the bile acid-independent flow [ 16,171. The increment in the concentration of biliary TBARS suggests an increased production in the liver, which is likely to occur under conditions of enhanced oxidative stress status of the tissue induced by thyroid calorigenesis. In agreement with this view, hyperthyroidism elicited a 72% increase in the rate of TBARS formation by liver homogenates (Fig. 1, inset), which significantly correlates with the biliary release of TBARS (Fig. 1). It is concluded that thyroid hormone-induced liver oxidative stress stimulates cellular lipid peroxidation as assessed by the biliary eftlux of TBARS in the anesthetized rat, a finding that correlates with the enhanced chemiluminescent response of the rat liver in situ observed upon Tj treatment [S]. Apart from hyperthyroidism (Table 2; Fig. l), enhancement in the biliary release of TBARS has also been observed in other conditions of increased free radical activity in the liver, including iron overload, and acute ethanol intoxication [18]. This technique allows a closer examination of in vivo TBARS production since liver architecture remains intact. Acknowledgements
This work was supported by grant 1940312 from FONDECYT. The technical assistance of Carmen Almeyda and Manuel Suarez is kindly acknowledged. References [l] Femrlndez, V. and Videla, L.A. (1985) Thyroid hormone, active oxygen, and lipid peroxidation. In: J. Miquel, A.T.
149-153
Quintanilha and H. Weber (Eds.), Handbook of Free Radicals and Antioxidants in Biomedicine, CRC Press, Boca Raton, FL, pp. 105-l 15. 121Videla, L.A. and Fem&ndez, V. (1994) Thyroid calorigenesis and oxidative stress: modification of the respiratory burst activity in polymorphonuclear leukocytes. Brazilian J. Med. Biol. Res. 27, 2331-2342. [31 Femindez, V., Barrientos, X., Kipreos, K., Valenzuela, A. and Videla, L.A. (1985) Superoxide radical generation, NADPH oxidase. activity, and cytochrome P450 content of rat liver microsomal fractions in an experimental hyperthyroid state: relation to lipid peroxidation. Endocrinology I 17, 496-501. I41 Fern&&z, V. and Videla, L.A. (1993) Influence of hyperthyroidism on superoxide radical and hydrogen peroxide production by rat liver submitochondrial particles. Free Rad. Res. Commun. 18, 329-335. PI Femandez, V., Llesuy, S., Solari, L., Videla, L.A. and Boveris, A. (1988) Chemiluminescent and respiratory responses related to thyroid hormone-induced liver oxidative stress. Free Rad. Res. Commun. 5, 77-84. WI Femindez, 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, eRIux and tumover. Endocrinology 129, 85-91. 171 Sies, H. (1986) Biochemistry of oxidative stress. Angew. Chem. Int. Ed. En@. 25, 1058-1071. P31 Landriscina, C., Petragallo, V., Morini, P. and Marcotrigiano, G.O. (1988) Lipid peroxidation in rat liver microsomes. I. Stimulation of the NADPH+ytochrome P4% reductasedependent process in hyperthyroid state. B&hem. Int. 17, 385-393. [91 Manoev, AI., Kozlov, A.V., Andryuschenko, A.P. and Vladimirov, Y.A. (1982) Activation of lipid peroxidation in liver mitochondria of hyperthyroid rabbits. Bull. Exp. Biol. Med. 93, 269-272. WI Asayama, K., Dobashi, K., Hayashibe, H., Megata, Y. and Kato, K. (1987) Lipid peroxidation and free radical scavengers in thyroid dysfunction in the rat: A possible mechanism of injury to heart and skeletal muscle in hyperthyroidism. Endocrinology 121, 2112-2118. IIll Janero, D.R. (1990)Malondialdehyde and thiobarbituric acid-reactivity as diagnostic indices of lipid peroxidation and peroxidative tissue injury. Free Rad. Biol. Med. 9, 515-540. WI Femandez, V. and Videla, L.A. (1993) 3,3’,5Triiodothyronine-induced hepatic respiration: effects of desferrioxamine and allopurinol in the isolated perfused rat liver. Toxicol. Lett. 69, 205-210. 1131 Buege, J.A. and Aust, S.A. (1987) Microsomal lipid peroxidation. Methods Euzymol. 52, 302-310. 1141 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265-275. WI Sterling, K. (1986) Thyroid hormone action at the cellular level. In: S.H. Ingbar and L.B. Bravermann
V. Femindez.
L.A. Viakla / Toxicology Letters 84 (19%)
(Eds.), The Thyrolld, a Fundamental and Clinical Text, J.B. Lippincott, Philadelphia, pp. 219-237. [16] Layden, T.J. and Bayer, J.L. (1976) The effect of thyroid hormone on bile salt-independent bile flow and Na+, K+-ATPase activity in liver plasma membranes enriched in bile canaliculi. J. Clin. Invest. 57, 1009-1018. 1171 Keeffe, E.B., Sch~srschmidt, B.F., Blankenship, N.M. and Ockner, R.K. (1979) Studies of relationships among
149-153
153
bile flow, liver plasma membrane NaK-ATPase, and membrane microviscosity in the rat. J. Clin. Invest. 64, 1590-1598. [18] Videla, L.A., Femtidez, V. and Valenmela, A. (1985) Effect of ethanol and iron on the hepatic and biliary levels of glutathione and lipid peroxidative indexes. Alcohol 2, 457-462.
ELSEVIER
Effect of hyperthyroidism on the biliary release of thiobarbituric acid reactants in the rat Virginia
Fernindez*,
Luis A. Videla
Departamento a’eBioquimica, Facultad de Medicina. Universidnd de Chile, Casilla 70086, Santiago-7, Chile IReceived 3 July 1995; revision received 13 October 1995; accepted 16 October 1995
Abstract The influence of daily doses of 0.1 mg 3,3’,5triiodothyronine (TJkg for three consecutive days on hepatic lipid peroxidation was asressed by the biliary release of thiobarbituric acid reactants (TBARS) in anesthetized rats. T, treatment elicited a significant 80% increase in the biliary eMux of TBARS compared to control values, an effect determined by increments in both the TBARS concentration in bile (44%) and in the bile flow (25%). It is concluded that hyperthyroidism-induced oxidative stress stimulates hepatic lipid peroxidation measured in an in situ liver preparation, which significantly correlates with the enhancement in the rate of TBARS production assessed in liver homogenates. Keywords: Thyroid calorigenesis; Liver oxidative stress; Lipid peroxidation
1. Introduction Hyperthyroidism has been found to induce an imbalance in the prooxidant-antioxidant equilibrium of the hepatocyte in favor of the prooxidants [1,2]. This enhancement in the oxidative stress status of the liver is conditioned by a higher rate of reactive 0, species production [3,4}, associated with the increased txllular respiration elicited, and by a diminution in some antioxidant mechanisms. The latter effect of ithyroid hormone is characterized by significant decreases in the activity of hepatic superoxide dismutase and catalase [5], and in the content of reduced glutathione [5,6].
l
Corresponding author.
One consequence of an enhanced cellular steady-state level of reactive 02 species is the oxidative damage to polyunsaturated fatty acids in phospholipids of biological membranes [7]. Accordingly, thyroid hormone-induced liver oxidative stress has been reported to increase hepatic lipid peroxidation, as evidenced by the significant increments in the content of thiobarbituric acid reactants (TBARS) in rat liver homogenates and in microsomal fractions [5], spontaneous chemiluminescence of rat liver homogenates [5], NADPH-dependent accumulation of hydroperoxides in rat liver microsomes [S], and in the Fe2+-induced light emission of rabbit liver mitochondria [9]. However, this effect of hyperthyroidism has not been observed when lipid peroxidation is assessed by the TBARS index,
03784274/96/1615.00 Q 1996 Elsevier Science Ireland Ltd. AI1 rights reserved SSDI 0378-4274(95)0?;617-T
150
V. Fermindez, L. A. Videla / Toxicology Letters 84 (19%)
either in liver homogenates [lo] or in liver microsomal fractions supplemented with NADPH, Fe’+, or NADPH plus Fe’+ 181.The discrepancy observed under in vitro conditions could be due to the fact that lipid peroxidation-derived aldehydic products may participate in other reactions than that with thiobarbituric acid, including aldol-type self-condensation, oxidation by aldehyde dehydrogenases, and/or reaction with primary amino groups in proteins, nucleic acids, and amino phospholipids, to give fluorescent and nonfluorescent adducts [l 11. In view of these considerations, the present work evaluates the lipid peroxidative response of the liver to thyroid hormone-induced oxidative stress by means of an in situ liver technique. For this purpose, the biliary release of TBARS was measured in anesthetized control rats and 3,3’,5triiodothyronine (T&treated animals, and results were correlated with the rate of TBARS production by liver homogenates.
149-153
weight/body weight ratios (Table 1). Before biochemical studies, the rectal temperature of the rats was measured with a thermocouple (model 811220, Cole-Parmer Instrument Co., Chicago, IL), and blood samples were taken from the tail vein for the determination of serum T, levels by radioimmunoassay (Baxter Healthcare Corp., Cambridge, MA). The rate of O2 consumption by the liver was measured polarographically in perfusion experiments, as previously described [6,12]. Animals were anesthetized with sodium pentobarbital (50 mgikg, intraperitoneally) and the peritoneal cavity was opened to cannulate the bile duct with a PE-10 polyethylene tubing. The abdominal cavity was covered with a cheesecloth humidified with 0.9% w/v NaCl and bile was collected for 15 min in animals kept in an environmental temperature of 28-30°C. The concentration of TBARS in bile samples was immediately determined as described by Buege and Aust [13]. At the end of this procedure, the livers were perfused in situ with 200 ml of a cold solution containing 140 mM KC1 and 10 mM potassium phosphate buffer, pH 1.4, to remove blood, and weighed to express the bile flow in fig liver/mm. The biliary efflux of TBARS was calculated by multiplying their concentration in the bile by the respective bile flows. The rate of TBARS formation [ 131 was assayed in liver homogenates (1:4) prepared in 140 mM KC1 containing 10 mM potassium phosphate buffer, pH 7.4, after centrifuga-
2. Materials and methods Female Sprague-Dawley rats fed ad libitum received daily intraperitoneal injections of 0.1 mg of T&g body weight or equivalent volumes of T3 diluent (0.1 N NaOH) (controls) for 3 consecutive days, and studies were performed 24 h after the last treatment. In these conditions, control rats and Trtreated animals exhibited comparable values of body weight and the respective liver
Table 1 Body weight, liverlbody weight ratio, serum T3 levels, and parameters related to thyroid calorigenesis in control rats and T,treated animals Parameters
Control rats
Ts-treated rats
P-value
Body weight (g) (n = 13) Liver/body weight (g/100 g) (n = 13) Serum Ts (ng/dl) (n = 6) Rectal temperature (“C) (n = 13) Liver O2 consumption (runol/g liver/mm) (n = 6)
203 f 4
210
NS
3.51 f 0.09
3.62 f 0.12
NS
70 f 7
314 f 23
0.001
37.4 f 0.1
38.5 f 0.1
0.001
1.88 f 0.95
2.55 f 0.10
0.001
l
8
Values shown represent the means f S.E.M. for the indicated number of rats per experimental group. The significance of the differences between mean values was carried out by the Student’s r-test for unpaired data. NS, not significant.
V. Fernhdez. L.A. Videla/ Toxicology Letters 84 (19%) 149-153
tion at 900 x g for 20 min at 4°C. Incubations were carried out at 37°C for 75 min, and aliquots were taken every 15 min for the assessment of maximal rates, expressed as nmohmg proteiti. The protein content of liver homogenates was measured according to Lowry et al. [14]. Chemicals and reagents used were obtained from Sigma Chemical Co. (St. Louis, MO). Values shown are means 3: S.E.M. for the number of separate experiments indicated. The statistical significance of the differences between mean values was assessed by Student’s t-test for unpaired data. 3. Rest&a and discrlssion T3 administration to fed rats resulted in a significant 3.5-fold increment in the serum level of the hormone, in concomitance with a calorigenic response evidenced by the enhancement in the rectal temperature of the animals (Table 1). In these conditions, the rate of O2 consumption by the liver increased by 3(5%(Table l), in agreement with earlier studies [6,1:2]. The liver is a major organ exhibiting an accelerated respiration in the hyperthyroid state, whiclh seems to be due to the interaction of Ts with nuclear receptors, leading to an enhanced synthesis of enzymatic systems involved in redox processes 1151. Acceleration of hepatic respiration involvles a component comprising 16%-25% of the net increase in hepatic 0, uptake, which was suggested to represent 02 equivalents related to TJ-induced oxidative stress [ 121. This respiratalry component assessed in liver perfusion studies correlates with the higher rates
Fig. I. Correlation between the rate of thiobarbituric acid reactants (TBARS) formation in liver homogenates and the respective biliary release of TBARS in control rats (0) and Ts-treated animals (0). Regression line, y = -0.0047 + 0.0918x (r = 0.91; P < 0.001). Inset: rate of TBARS formation in liver homogenates from control rats (C) and hyperthyroid animals (T’d. Values shown represent the means + S.E.M. for 7 rats per experimental group. The significance between mean values was asmssed by Student’s t-test for unpaired data.
of NADPH-dependent superoxide radical (Or’-) generation observed in liver microsomes [3], as well as with the NADH- or succinate-supported O2 .- production in liver submitochondrial particles [4]. In the latter system, Ts treatment led to hydrogen peroxide release at higher rates than
Table 2 Biliary release of thiobarbituric acid reactants (TBARS) in control rats and T,-treated animals Parameters
Control rats (n = 7)
T,-treated rats (n = 7)
Change (W
P
Bile flow @l/g liver/mm) Biliary TBARS concentration (~mol/rnl bile) Biliary release of TBARS (nmohg liver/mitt)
1.01 l 0.09 5.36 f 0.40
1.26 f 0.05 7.73 f 0.26
25 44
0.05 0.001
5.41
9.74 + 0.31
80
0.001
l
0.45
Determinations were caaried out in anesthetized control rats and hyperthyroid animals kept at an environmental temperature of 28-304C, as described in Materials and methods. Results are means f S.E.M. for the number (a) of rats indicated. The signiticance of the differences between mean values was assessed by Student’s t-test for unpaired results.
152
K Fermindez. L.A. Videla / Toxicology Letters 84 (19%)
control values, both under basal conditions and in the succinate- or urate-supported processes [4]. Under conditions of an enhanced liver free radical activity, hyperthyroidism elicited a significant 80% increase in the biliary release of TBARS, which results from the enhancement in the biliary concentration of TBARS and in the bile flow over control values (Table 2). Thyroid hormone is known to alter bile secretory functions by influencing both the rate of bile production and the synthesis and biliary release of bile acids, primarily increasing the bile acid-independent flow [ 16,171. The increment in the concentration of biliary TBARS suggests an increased production in the liver, which is likely to occur under conditions of enhanced oxidative stress status of the tissue induced by thyroid calorigenesis. In agreement with this view, hyperthyroidism elicited a 72% increase in the rate of TBARS formation by liver homogenates (Fig. 1, inset), which significantly correlates with the biliary release of TBARS (Fig. 1). It is concluded that thyroid hormone-induced liver oxidative stress stimulates cellular lipid peroxidation as assessed by the biliary eftlux of TBARS in the anesthetized rat, a finding that correlates with the enhanced chemiluminescent response of the rat liver in situ observed upon Tj treatment [S]. Apart from hyperthyroidism (Table 2; Fig. l), enhancement in the biliary release of TBARS has also been observed in other conditions of increased free radical activity in the liver, including iron overload, and acute ethanol intoxication [18]. This technique allows a closer examination of in vivo TBARS production since liver architecture remains intact. Acknowledgements
This work was supported by grant 1940312 from FONDECYT. The technical assistance of Carmen Almeyda and Manuel Suarez is kindly acknowledged. References [l] Femrlndez, V. and Videla, L.A. (1985) Thyroid hormone, active oxygen, and lipid peroxidation. In: J. Miquel, A.T.
149-153
Quintanilha and H. Weber (Eds.), Handbook of Free Radicals and Antioxidants in Biomedicine, CRC Press, Boca Raton, FL, pp. 105-l 15. 121Videla, L.A. and Fem&ndez, V. (1994) Thyroid calorigenesis and oxidative stress: modification of the respiratory burst activity in polymorphonuclear leukocytes. Brazilian J. Med. Biol. Res. 27, 2331-2342. [31 Femindez, V., Barrientos, X., Kipreos, K., Valenzuela, A. and Videla, L.A. (1985) Superoxide radical generation, NADPH oxidase. activity, and cytochrome P450 content of rat liver microsomal fractions in an experimental hyperthyroid state: relation to lipid peroxidation. Endocrinology I 17, 496-501. I41 Fern&&z, V. and Videla, L.A. (1993) Influence of hyperthyroidism on superoxide radical and hydrogen peroxide production by rat liver submitochondrial particles. Free Rad. Res. Commun. 18, 329-335. PI Femandez, V., Llesuy, S., Solari, L., Videla, L.A. and Boveris, A. (1988) Chemiluminescent and respiratory responses related to thyroid hormone-induced liver oxidative stress. Free Rad. Res. Commun. 5, 77-84. WI Femindez, 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, eRIux and tumover. Endocrinology 129, 85-91. 171 Sies, H. (1986) Biochemistry of oxidative stress. Angew. Chem. Int. Ed. En@. 25, 1058-1071. P31 Landriscina, C., Petragallo, V., Morini, P. and Marcotrigiano, G.O. (1988) Lipid peroxidation in rat liver microsomes. I. Stimulation of the NADPH+ytochrome P4% reductasedependent process in hyperthyroid state. B&hem. Int. 17, 385-393. [91 Manoev, AI., Kozlov, A.V., Andryuschenko, A.P. and Vladimirov, Y.A. (1982) Activation of lipid peroxidation in liver mitochondria of hyperthyroid rabbits. Bull. Exp. Biol. Med. 93, 269-272. WI Asayama, K., Dobashi, K., Hayashibe, H., Megata, Y. and Kato, K. (1987) Lipid peroxidation and free radical scavengers in thyroid dysfunction in the rat: A possible mechanism of injury to heart and skeletal muscle in hyperthyroidism. Endocrinology 121, 2112-2118. IIll Janero, D.R. (1990)Malondialdehyde and thiobarbituric acid-reactivity as diagnostic indices of lipid peroxidation and peroxidative tissue injury. Free Rad. Biol. Med. 9, 515-540. WI Femandez, V. and Videla, L.A. (1993) 3,3’,5Triiodothyronine-induced hepatic respiration: effects of desferrioxamine and allopurinol in the isolated perfused rat liver. Toxicol. Lett. 69, 205-210. 1131 Buege, J.A. and Aust, S.A. (1987) Microsomal lipid peroxidation. Methods Euzymol. 52, 302-310. 1141 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265-275. WI Sterling, K. (1986) Thyroid hormone action at the cellular level. In: S.H. Ingbar and L.B. Bravermann
V. Femindez.
L.A. Viakla / Toxicology Letters 84 (19%)
(Eds.), The Thyrolld, a Fundamental and Clinical Text, J.B. Lippincott, Philadelphia, pp. 219-237. [16] Layden, T.J. and Bayer, J.L. (1976) The effect of thyroid hormone on bile salt-independent bile flow and Na+, K+-ATPase activity in liver plasma membranes enriched in bile canaliculi. J. Clin. Invest. 57, 1009-1018. 1171 Keeffe, E.B., Sch~srschmidt, B.F., Blankenship, N.M. and Ockner, R.K. (1979) Studies of relationships among
149-153
153
bile flow, liver plasma membrane NaK-ATPase, and membrane microviscosity in the rat. J. Clin. Invest. 64, 1590-1598. [18] Videla, L.A., Femtidez, V. and Valenmela, A. (1985) Effect of ethanol and iron on the hepatic and biliary levels of glutathione and lipid peroxidative indexes. Alcohol 2, 457-462.