Mitochondrial Glutathione in Hypermetabolic Rats Following Burn Injury and Thyroid Hormone Administration: Evidence of a Selective Effect on Brain Glutathione by Burn Injury Johannes Mdrtensson, Cleon W. Goodwin, and Rebecca Blake Cerebral cortex, heart, skeletal muscle, and liver mitochondrial glutathione (GSH) levels in severely burned rats are decreased to between approximately 50% to 70% of sham-operated, normally fed controls. In semistarved rats, weight-matched with burned rats, mitochondrial GSH levels in these tissues are decreased to between approximately 70% to 91% of those in sham-operated rats. Total GSH levels in peripheral tissues and brain are decreased to approximately 50% to 66% of control levels in rats with burn injury and food restriction, suggesting a higher mitochondrial GSH turnover in burned rats than in semistarved rats, probably because of higher “stress hormone” levels in burned rats than in semistarved rats. Cerebral cortex mitochondrial GSH levels are unaffected by variations in thyroid hormone status, but liver mitochondrial GSH levels are decreased by triiodothyronine and increased by propylthiouracil. The present results suggest that mitochondrial GSH is not only regulated by the rate of GSH synthesis in the cytosol, but seems to be under hormonal influence as well; stress hormones and triiodothyronine may decrease mitochondrial GSH by increasing mitochondrial oxygen consumption with increased reactive oxygen species formation or by increasing GSH exchange between mitochondria and the cytosol. These findings may be of importance therapeutically in increasing antioxidative defenses to limit oxidative stress injury in hypermetabolic patients. Copyright 0 1992 by W.B. Saunders Company

G intracellular

LUTATHIONE

(GSH) is a major and ubiquitous antioxidant.“’ Mitochondrial oxidative metabolism is an important source of reactive oxygen species formation, since the rate of their formation is related to the rate of mitochondrial respiration.4 Trauma5” and increased thyroid hormone levels7.9are associated with increased mitochondrial oxygen consumption in most peripheral tissues. Liver mitochondrial GSH levels are markedly increased in hypophysectomized, hypometabolic, hypothyroidic rats, and triiodo+thyronine (T3) administered to such animals lowers liver mitochondrial GSH levels to below those of euthyroid animals.1o These changes in mitochondrial GSH levels are associated with alterations in mitochondrial volume, respiration rates, and Ca’+ release. Adult brain mitochondrial respiration seems to be insensitive to stimulation by peripherally administered thyroid hormones.7.9.” If so, brain mitochondrial reactive oxygen species and GSH levels should not change with altered thyroid hormone levels. Patients with major burns have normal or decreased thyroid hormone levels, whereas their levels of “stress hormones,” ie, catecholamines, cortisol, and glucagon, are increased.‘* Markedly increased basal metabolic rates are expressed in several organs in burns,” including the brain.1213 In unstressed rats and mice, severe mitochondrial GSH depletion induced by the administration of buthionine sulfoximine (BSO), an irreversible and selective inhibitor of GSH synthesis, “~15is associated with mitochondrial swelling and degeneration.‘6-w Mitochondrial damage leads to (1) myofiber necrosis in skeletal muscle,16 (2) swelling of lung capillary endothelial cells and type 2 cell lamellar bodies,17 and (3) intestinal epithelial degeneration.” In newborn mice and rats, BSO-induced mitochondrial GSH depletion is associated with cerebral cortex mitochondrial swelling and damageI and death.‘9.z0In moderate doses, BSO leads to lens mitochondrial GSH depletion and epithelial damage, with vacuolization, nuclear indentation and chromatin condensation, and cataract formation.20 Mitochondrial GSH levels are increased and mitochondrial degeneration and Metabolism, Vol41, No 3 (March), 1992: pp 273-277

tissue damage are prevented if a GSH monoester is given in conjunction with BSO, indicating that GSH exerts a protective effect on mitochondria. GSH monoesters (but not GSH itself) are transported into most cells and rapidly hydrolyzed to GSH.‘6-22GSH is transported into liver mitochondria of adult rats by a multicomponent system that has a high affinity for GSH and is stimulated by adenosine triphosphate (ATP)23; this mitochondrial transport system probably explains the unique ability of mitochondria to conserve GSH during GSH depletion at the expense of the cytosol. In the present study, the effects of stress hormones and thyroid hormones on mitochondrial GSH levels were determined in brain tissue and several peripheral tissues (ie, liver, heart, and skeletal muscle). The findings are discussed in relation to regulation of mitochondrial GSH. MATERIALS AND METHODS The burned rats (retired Sprague-Dawley male breeders, Charles River, Boston, MA) were kept in separate cages and allowed to adapt to an ambient temperature (26 t 2OC) for at least 1 week. The rats were without signs of infection or obvious physical deterioration during the study, apart from the bum injury and weight loss induced by the manipulations. The burn injury (covering 50% of the body surface area) was produced as follows: the anesthetized (sodium pentobarbital, 55 mg/kg body weight, intraperitoneally [IP] animals were shaved on the areas to be exposed to burn injury, given an IP injection of 25 mL of saline (1 mL/% burn area/kg body weight) to protect the viscera from injury and prevent the development of hypovolemic shock, and placed in

Supported by grants from the Throne-Ho& Foundation, the Swedish Medical Society, the AGA AB Fund for Medical Research, the Trygg-Hansa AB Research Foundation, the Draco AB Fund for Medical Research, and the Killough Trust (J.M.). Partly supported by Grant No. GM26145from the National Institutes of Health (C. W.G.). Address reprint requests to Johannes MBrtensson MD, PhD, Associate Professor, Department of Biochemistry, Cornell University Medical College, 1300 YorkAve, NewYork, NY10021. Copyright 0 I992 by W. B. Saunders Company 00260495/92/4103-0008$03.00/0

273

274

MARTENSSON,

a template.*’ Each rat was then immersed in water at 99”C, so that the back was exposed for 10 seconds first and then the abdomen was exposed for 3 seconds. All animals were fed Purina Chow (Ralston-Purina, St Louis, MO) ad libitum (except the pair-fed semistarved controls, see below) and had free access to drinking water. To correct for the weight loss in burned animals induced by hypermetabolism, a group of pair-fed rats was given an amount of food (on a daily basis) that resulted in the same amount of weight loss seen in the burned rats on the previous day. The mean body weight of burned and semistarved rats decreased by 13% and 12%, respectively, as compared with sham-operated controls, whose mean body weight started at 6228 and increased by 0.2% during the study. A third group of rats were treated in the same way as the burned rats, except for the omission of the shaving and hot water exposure (“unburned,” sham-operated controls). In a separate experiment, male Sprague-Dawley rats (Charles River) with an initial weight of approximately 50 g (6 weeks old) were made hypothyroid by feeding with a diet containing 0.1% (wt/wt) propylthiouracil (PTU)T5 The weights of these animals never exceeded 250 g, their thyroid glands were enlarged, and their serum thyroxine levels were markedly decreased. Hyperthyroidism was induced in some of the hypothyroid rats by injections of T, (100 pg/kg body weight, IP) for 6 day?; their mean body-weight was unchanged by this procedure. The mean body weight of control rats was 850 g.

Preparation of Tissue and Mitochondria After an overnight fast, rats were decapitated, and heart, skeletal muscle (quadriceps),16 liver,” and brain cortex (the brain was freed from its meninges)2h were excised. For tissue removal, the animals were first perfused with 10 mL of cold saline through the left ventricle. Then, a piece of tissue was excised, rinsed in saline, blotted, immerskd in liquid nitrogen, homogenized in 5 vol (wt/vol) of 5% sulfosalicyclic acid containing 3.5 mmol/L Na,EDTA, and centrifuged for 5 minutes at 4”C, with the supernatant immediately used for GSH analysis. For mitochondria, the tissue was minced on ice, rinsed in cold saline, and blotted. Then, it was homogenized manually in a Dounce homogenizer (Cole-Palmer, Chicago, IL) by 10 strokes in 5 to 10 vol of a cold medium containing 220 mmol/L mannitol, 70 mmol/L sucrose, 0.1 mmol/L sodium EDTA, 0.1% (wtivol) albumin (fatty acid-free), and 5 mmol/L TRIS-HCI (pH 7.2). The homogenization buffer for heart and skeletal muscle also contained 100 U/mL of heparin sulfate to prevent gelatinous transformation of the homogenate.‘” Mitochondria were isolated by differential centrifugation as described previously.‘a’“~2h To the final mitochondrial pellet, 100 FL of 4.3% (wt/vol) 5-sulfosalicylic acid was added; after freezing, thawing, and centrifuging the acid-treated sample at 10,000 x g for 5 minutes at 4”C, the supernatant was used for GSH determinations. The purity of the

GOODWIN,

AND BLAKE

mitochondrial preparations was assessed by electron microscopy as previously described.” The total and mitochondrial GSH (plus any oxidized GSH: GSSG) concentrations were determined by the enzymatic GSSGi 5,5’dithiobis2+itro benzoic acid (DTNB) recycling method as described previously.” The assay was performed immediately following preparation; occasionally, samples were stored at -70°C for not more than 1 week (during which time GSH is known to be stable’“) pending analyses. However, the absolute GSH levels in mitochondria may vary depending on the composition of the isolation media. Thus, mitochondrial GSH levels are approximately 30% lower if the isolation medium does not contain EDTA, or if they are washed more extensively (data not shown). EDTA is known to stabilize GSH in solution.“,‘x It is suggested that mitochondria prepared without EDTA in the medium loses GSH from the intermembranous space, since this mitochondrial pool of GSH is the most labile one in mitochondria.” Protein was determined by the bicinchoninic acid method,‘” using bovine serum albumin as a standard. Statistical analyses were performed by the one-way ANOVA, followed (post hoc) by the Tukey test for multiple comparisons. RESULTS Mitochondrial GSH levels in several brain regions (cerebral cortex, midbrain, rostra1 part of hindbrain, and cerebellum) were determined and showed no significant variations; levels in the cerebral cortex were 8.24 2 0.49 (SD) nmol/mg protein (n = 3), in the midbrain 7.72 2 0.47 nmolimg protein (n = 3), and in the cerebellum 7.63 2 0.73 nmol/mg protein (n = 3). These levels were comparable to that in whole brain (8.00 2 0.24 nmolimg protein, n = 3). Severely burned rats were studied on day 12 after injury, since this day coincides with the period of maximal hypermetabolism and mitochondrial oxygen consumptior?; this will result in increased reactive oxygen species formation.” Burn injury leads to severe tissue catabolism and weight loss, possibly because of increased stress hormone levels.” Weight-matched (to burned rats) semistarved rats were studied in parallel to burned rats, to distinguish between weight loss-induced GSH depletion and any additional stress hormone-related changes in GSH levels more specifically related to burn injury. Mitochondrial GSH levels were decreased in cerebral cortex, heart, skeletal muscle, and liver tissue following burn injury (Table 1). Decreased food intake also resulted in decreased mitochondrial GSH levels in ail tissues,

Table 1. Effect of Burn Injury and Restricted Food Intake on Mitochondrial GSH Levels in Brain, Heart. Skeletal Muscle, and Liver in Adult Male Rats Mitochondrial Brain

Heart

GSH Levels MllS&

Liver

Sham group

8.24 + 0.49

7.48 + 0.31

Weight-matched group

7.48 + 0.31*

4.54 + 0.18t

4.14 + 0.17t

4.44 2 0.47t

5.8 + 0.43t

3.60 + 0.19t

3.84 -c 0.19t

3.23 of:0.21t

Burned group

NOTE. Mitochondrial GSH levels (mean 2 SD, nmob’mg protein) were determined

6.0 z!z0.29

6.0 t 0.33

in cerebral cortex (n = 5 in all groups], heart (n = 5 in all

groups), skeletal muscle (n = 4 in all groups), and liver (n = 4 in all groups) from mitochondria isolated in a medium containing EDTA. The mitochondria were isolated from samples taken between 11 AM and 12 noon after an overnight fast. Food intake of the rats in the weight-matched (semistarved) group were adjusted to that of burned animals on the basis of weight gain. Statistically significant differences: semistarved§ rats.

*P < .05, tP

< ,001 were found in comparison with unburned sham-operated*

and weight-matched

275

EFFECT ON BRAIN GSH BY BURN INJURY

including the brain, although the decreases, apart from skeletal muscle, were less pronounced than those found in burned rats. Notably, total tissue GSH levels were decreased similarly in burned and semistarved rats; in contrast, liver GSH levels were more decreased by burns (Table 2). The adult brain is normally exposed to thyroid hormones.” However, in hyperthyroid rats, brain mitochondrial oxygen consumption is unaffected by increased thyroid hormone levels.5~s~”Our results showed that cerebral cortex mitochondrial GSH levels were also unchanged in rats with abnormal thyroid hormone levels (Table 3). Liver mitochondrial GSH levels are increased (compared with controls) regardless of whether hypothyroidism is induced by PTU (present study) or by hypophysectomy,1° whereas total liver GSH levels decreased from 7.58 kmol/g to 6.17 kmol/g (mean value, based on a liver protein content of 15%) following hypophysectomy.” Hypothyroidism produced by PTU resulted in markedly increased liver mitochondrial GSH levels, but these were reduced to well below control levels after T3 administration (Table 3), as earlier reported in hypophysectomized rats given T,.“’ The low total liver GSH level in hypophysectomized rats was not further decreased by giving T3.10Thus, the present and previous5,9-” results suggest that a reciprocal relationship exists between mitochondrial oxygen consumption (and reactive species formation) and mitochondrial GSH levels. DISCUSSION

The liver is the main source of plasma GSH for interorgan GSH translocation’; plasma GSH utilization is regulated by membrane-bound y-glutamyltranspeptidase 3 and is most expressed in kidney3 and lung” tissue. In this process, GSH is first cleaved extracellularly, forming a y-glutamyl amino acid and cysteinylglycine; the constituent amino acids are then taken up and resynthesized to GSH intracellularly. Epinephrine has been shown to increase the efflux of GSH across the sinusoidal plasma membrane in an isolated perfused liver preparation,3” suggesting that the process regulating GSH excretion by the liver is hormonesensitive. Brain tissue utilizes plasma GSH,lY,” and plasma GSH is probably of importance for brain GSH homeostasis. Although direct transport of GSH into brain tissue from plasma has been suggested,)’ most probably this process is regulated by y-glutamyl transpeptidase; in lCday-old rats that were depleted of GSH by the administration of BSO, cerebral cortex GSH levels did not increase by infusing

Table 3. Effects of PTU-Induced Hypothyroidism TJnduced

Hyperthyroidism

and PTU Plus

on Liver and Brain Miochondrial

GSH

Levels in Adult Male Rats Mitochondrial

GSH Levels

Liver

Brain

6.35 + 0.29

Control group

a.25 + 0.48

PTU group

10.9 + 1.16’

8.10 f 0.40

PTU + T, group

2.78 + 0.32t*=

8.45 + 0.82

NOTE. Mitochondrial were determined

GSH levels (mean + SD, nmol/mg

protein)

in liver and brain mitochondria isolated in a media

containing EDTA (controls; n = 6 for liver and brain (mitochondria isolated from cerebral cortex), PTU rats; n = 3 for liver and cerebral cortex, PTU + TA rats; n = 4for liver and cerebral cortex). Statistically significant differences

(f < .OOl) were obtained with

respect to controlt and PTU** rats.

GSH IP (increasing plasma GSH to 5 mmol/L, or 250 to 500 times the level found in controls).‘9 The low mitochondrial GSH level, presently found in all tissues of burned rats, suggests that the utilization of plasma GSH by brain and peripheral tissues may be increased by stress hormones; however, this hypothesis requires further studies. Decrease in cerebral cortex and peripheral tissue mitochondrial GSH levels was less pronounced in semistarved rats than in rats subjected to burn injury; semistarvation can only partly explain the decreased mitochondrial GSH levels found in burned rats. The catabolic condition in burned rats is associated with increased plasma catecholamine levels.32 Increased noradrenergic and dopaminergic neuronal activity of the central nervous system (CNS) probably occurs in parallel to the stress response in peripheral tissues in major burns,“,” either by direct stimulation through local release of hormones (neurotransmittors), or indirectly, by uptake of peripherally secreted catecholamines through the bloodbrain barrier. Accumulation of catecholamines may generate increased amounts of reactive oxygen species, deplete GSH, and thereby cause neuronal damage in the CNS.33,34 Notably, loss of brain GSH has been suggested to precipitate Parkinson’s disease,‘3 and brain GSH is decreased in ischemic brain injury.“4 Severe mitochondrial GSH depletion and damage in unstressed adult animals can experimentally be induced by giving BSO (6 to 8 mmol/kg) for 2 to 3 weeks’6-‘8(or for 5 days in newborn rats and mice).“~*’ The present results show that mitochondrial GSH depletion also can develop during hypermetabolism, most likely by increasing mitochondrial oxygen consumption and thereby increasing reactive

Table 2. Effect of Burn Injury and Food Restriction on Tissue GSH Levels in Adult Rats Totat GSH Brain

Sham group Weight-matched

group

Burned group

Hean

MUSCk

Liver

1.47 * 0.17

1.62 2 0.10

0.74 f 0.05

5.04 z 0.39

1.oo -t 0.14ll

1.04 + 0.16$

0.45 + 0.06t

3.66 * 0.45$

1.03 + 0.221

1.12 r 0.21*

0.41 ? 0.08t

2.48 + 0.21t5

NOTE. Total GSH levels (pmol/g, mean + SD, n = 3-5). Samples were taken at 11 AM after an overnight fast. Animals were treated identically to those in Table 1. Corresponding GSH levels in fed rats are in brain 1.20 2 0.05 ~mol/g,‘9 heart 1.52 r 0.06 rmol/g, Fmol/g,‘6 and liver 8.12 2 0.25 pmol/g.”

skeletal muscle 0.64 f 0.03

Liver GSH levels are known to decrease during a 24-hourfast.49

Statistically significant differences were determined with respect to sham-operated rats: llf < ,025, Sf < ,005, tP < ,001; and to weight-matched (semistarved) rats: §P < ,005.

276

M#RTENSSON,

oxygen species formation.4.35 Decrease in mitochondrial GSH levels after burn injury is more generalized than after triiodothyronine administration; brain GSH levels are decreased by stress hormones, but they seem resistant to thyroid hormones. These results strongly support tissuespecificity in hormonal regulation of GSH, possibly involving cell membrane differentiation at the receptor level. Notably, experimentally induced hypothyroidism (by thyroidectomy’6 or PTU”) protects against free radicalinduced damage in kidney (eg, after ischemic renal failure)‘” and lung tissue (eg, during hyperoxia),” probably by decreasing mitochondrial oxygen metabolism, leading to decreased consumption of kidney and lung mitochondrial GSH. However, the normal kidney GSH level reported36 (-57 Fmol/g, based on a protein content of 13% of wet weight) was too high; the normal kidney GSH level is approximately 2.5 kmol/g in adult rats.’ The present study supports the concept of the sparing effect of hypothyroidism on mitochondrial GSH.” Hyperthyroidism accelerates the development of lung damage in hyperoxic rat? and is associated with decreased liver mitochondrial GSH (present study)” and increased malondialdehyde formation (lipid peroxidation). Mitochondrial GSH is depleted more slowly than cytosolic GSH during BSO administration,‘6-20~3Yprobably because it is regulated by a high-affinity, multicomponent transport system for GSH that is located on the inner mitochondrial membrane and is stimulated by ATP and inhibited by glutamate and ophthalmic acid.‘3 Thus, substantial mitochondrial GSH depletion is not seen before total (ie, mainly cytosolic) GSH levels are depleted below approximately 50% of control levels.“Z In burned and hyperthyroid rats, mitochondrial GSH depletion was more pronounced than in semistarved animals, although total GSH depletion was similar in all groups. This suggests that hypermetabolism specifically decreases mitochondrial GSH, possibly by altering the affinity of GSH for the GSH transporter, possibly by decreasing cellular ATP? or by increasing mitochondrial GSH efflux. Thyroxine, epinephrine, and phenylephrine not only

GOODWIN, AND BLAKE

influence mitochondrial GSH levels by changing mitochondrial oxygen consumption and reactive oxygen species formation, but may also alter mitochondrial GSH by changing mitochondrial efflux of Ca” and pyrophosphate.40~4’ Thus, data from the present study and earlierZ.25,a-4’studies lead one to speculate that there may be interactions between mitochondrial respiration and mitochondrial GSH and Ca’+ (and perhaps NADHINADPH) homeostasis. However, the mechanisms underlying these interactions have yet to be clarified. Plasma, leukocyte “huffy coats,” and tissue ascorbate levels decrease during hypermetabolism43.44; GSH, which is decreased during hypermetaboiism (according to both the present study and to the work of Rapuano and Maddaiah”‘), is required for reduction of dehydroascorbate to ascorbate,45.“6 a reaction catalyzed by glutaredoxin or protein disulfide isomerase.45 By combining ascorbate (but not dehydroascorbate) with BSO, mitochondrial GSH is spared.46.‘” This suggests that ascorbate can serve as an antioxidant during GSH depletion, and that a major function of GSH is to maintain tissue ascorbate levels. Therapy aimed at increasing mitochondrial GSH levels in hypermetabolic patients could be important in reducing the potential risk of oxidative stress injury induced by the hormonal surge following trauma. Mitochondrial GSH levels may be increased by giving cysteine or cysteine precursors, since cysteine is the limiting amino acid in GSH synthesis, in most tissues.l.‘O~‘OPrevious studies on unstressed animals during BSO-induced GSH depletion demonstrate that administration of GSH monoesters’@‘” or ascorbate 46-48increase mitochondrial GSH levels in most tissues and protect against oxidative cell damage and associated lethality; their therapeutic effects should be explored further. ACKNOWLEDGMENT

The authors wish to express their gratitude to Dr R. Greif for the kind gift of rats with deranged thyroid status and to Drs R. Greif, A. Meister, and J.C.K. Lai for support and advice during preparation of the manuscript.

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EFFECT ON BRAIN GSH BY BURN INJURY

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