Exogenous glutathione increases endurance to muscle effort in mice

Pharmacological Research, Vol. 23, No. 2, 1991

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EXOGENOUS GLUTATHIONE INCREASES ENDURANCE TO MUSCLE EFFORT IN MICE G. PAOLO NOVELLI, SILVIA FALSINI and GIOVANNA BRACCIOTTI

Institute of Anaesthesiology and Intensive Care, Poficlinico Careggi, University of Florence, Italy Received in final form 25 May 1990

SUMMARY Many data suggest an involvement of toxic oxygen radicals in the termination of endurance to muscle fatigue. Being reduced glutathione (GSH), an efficient intracellular physiological antioxidant, experiments have been performed to discover whether exogenous GSH modifies endurance to exhaustive swimming in mice. GSH was administered to mice as a single dose (250, 500, 750 or 1000 mg/kg i.p.) or as repeated doses (250 mg/kg i.p. once a day during 7 days) 10 min before a swimming test to exhaustion. GSH 500, 750 and 1000mg/kg, increased endurance to swimming by respectively 102.4%, 120.0% and 140.7%. GSH 250mg/kg did not affect endurance when injected in a single dose but increased it by 103.7% when injected once a day for 7 days.

KEYWORDS:glutathione, antioxidant, exercise, muscle fatigue. INTRODUCTION

The breakdown to strenuous muscle effort is commonly related to a lack of energy or to accumulation of lactic acid and other metabolites into muscles [1, 2]. However, there is the hypothesis of an involvement of toxic oxygen radicals overgenerated in mitochondria in front of a relative hypoxia consequent to an increase of muscular oxygen consumption largely greater than oxygendelivery. The oxygen radical overgeneration could also be attributed to xanthine-oxidase [3]. This hypothesis is supported by many data. In fact Davies et al. [4] reported that exercise to exhaustion was accompanied by decreased mitochondrial respiratory control and increased lipid peroxidation; moreover, an electron spin resonance (ESR) signal indicative of a free radical generation was identified in muscles of exhausted rats. Correspondence to: Prof. GiE Novelli, Institute of Anaesthesiology and Intensive Care, Policlinico di Careggi, Viale Morgagni, 50134 Firenze, Italy. 1043-6618/91/020149-07/S03.00/0

© 1991 The Italian Pharmacological Society

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Increased biogenesis of mitochondria [5] and increased superoxide dismutase activity [6] were also observed in muscles of rats trained to running. Alkane exhalation was reported to be increased during physical exercise both in untrained rats [7] and in men [8]. Moreover, an elevation of lipid peroxidation byproducts was reported during running exercise in the blood of sedentary rats but not in that of trained ones [9]. Some signs of oxygen radical generation related to physical effort was .also observed in the liver [4]. Therefore, toxic oxygen radicals seem to be produced during muscle effort. To demonstrate the role of toxic oxygen radicals in endurance to muscle effort experiments were performed by use of scavenging drugs. Large daily doses of vitamin E during a 15-day period decreased penthane exhalation in men during exercise [8]. In contrast, vitamin E deficiency lessened endurance to physical effort in rats [10] and increased the ESR signal in both muscles and liver [4]. Vitamin E administration increased endurance to physical effort in mice. A similar relationship between vitamin C and muscle effort has also been suggested [3]. In a paper from this laboratory a highly significant increased endurance to swimming was reported in mice injected with spin-trapping drugs, whose action is that of trapping oxygen radicals or their secondary radicals [11]. Reduced glutathione (GSH) is the substrate for glutathione-peroxidases whose role in protecting cell membranes against peroxidative damage is well accepted. Some GSH mobilization and consumption during muscle effort in rats having been hypothesized, [3], experiments were performed on mice to search for changes in endurance to an exhaustive physical effort consequent to single or repeated injections of exogenous GSH.

MATERIALS AND METHODS

Experiments were performed on male Swiss mice (weight 31.7 +_0.2 g) fed on a commercial balanced diet. Weight was measured at regular intervals during the whole experimental period, because differences in weight would be putatively related to differences in endurance. To measure the endurance to muscle exercise a swimming test [12] was used. Mice were put to swim in a bath (33 x 54 cm) of fresh water where waves were produced by an exact amount of gas flowing from the centre of the bottom. Temperature of the water was maintained constant (20°C) as it was demonstrated that its changes affect the endurance to swimming [12]. Fasted (6 h) mice were put in the bath in groups of four together after fixing a weight (1 g) on their tail at 2 cm from the root, so as to increase the effort and thereby accelerate exhaustion. The interval between the introduction of the mouse into the water and the third consecutive submersion of the head within a few seconds was accepted as maximal endurance to swimming. All native mice were first submitted to three swimming tests (every second day) in order to determine their endurance to exercise and to exclude from experiments the good swimmers (more than 120 s) and the bad swimmers (less than 70 s). In this way the basal value of endurance to swimming was obtained. Mice were then

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submitted to swimming tests (every second day) after the injection of GSH (two doses alone per mouse) or of saline. At the end of each series of experiments the test was repeated after saline alone. All the tests (except the last one with saline) were performed in duplicate, in separate sessions. .After these numerous tests all mice were sacrificed on humane grounds for fear of the consequences of repeated immersions in cold water and of repeated intraperitoneal injections. In a first series of experiments mice were randomly assigned to a group receiving GSH 250 mg/kg or 500 mg/kg i.p. in 0.1 ml of saline, or saline alone 0.1 ml i.p. Injections were performed 10 min before the swimming test. In a second series of experiments mice received GSH 750 mg/kg or 1000 mg/kg i.p. in 0.1 ml of saline or the same volume of saline alone. A third series of experiments was performed to search for a cumulative effect of repeated administrations. GSH 250 mg/kg or saline were injected i.p. every day for 7 days. The swimming test was performed only before and at the end of the treatment. A fourth group was caged in parallel to the third one and submitted to the swimming test before and after injections of saline alone. Results were statistically evaluated by the Student's t-test, comparing endurances to swimming (s) in various experimental conditions to the initial endurance.

RESULTS No apparent untoward effect related to the administration of GSH was evident. No differences in weight increase between the different experimental and control groups were observed (Table I). Endurance to swimming was unmodified after GSH 250 mg/kg but it was largely increased by 500mg/kg (184.03+6.95 s an increase from the initial value of 90.89 _+4.29 s, equivalent to 102.4%), by 750 mg/kg (211.04_+ 5110 s an increase from the initial value of 95.93 _+5.71 s, equivalent to 120.0%) and by 1000 mg/kg (230.91 +6.29 s an increase from the initial value of 95.93 + 5.71 s, equivalent to 140.7%). All the differences were highly significant (P<0.0005). The endurance always returned to normal values in the last test performed with saline (Tables II and III).

Table I Weight (g) of mice of each group during the experimental period Group ( 1) 250 and 500 mg/kg (2) 750 and 1000 mg/kg (3) 250 mg/kg x 7 days (4) Saline

Initial 31.7 + 0.2 31.6 + 0.2 31.7 + 0.3 31.6 + 0.2

lOth day 32.7 ± 0.2 32.6 + 0.2 31.9 ± 0.2 32.9 + 0.3

End of experiments 32.9 ± 0.2 32.8 + 0.2 32.2 _+0.2 32.7 + 0.2

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Table II Endurance to exhaustive swimming in mice injected with 250, 500 mg/kg i.p. of GSH or with saline 10 min before the test

n Mean (s) sE

Initial

250 mg/kg

500 mg/kg

Saline

36 90.89 _ 4.29

36 88.25 -+ 5.19

36 184.03 + 6.95

36 89.58 _+8.47

L,

[

Ns

v< o.0005 I I

NS

Table III Endurance to exhaustive swimming in mice injected with 750, 1000 mg/kg i.p. of GSH or with saline 10 rain before the test Initial

750 mg/kg

1000 mg/kg

Saline

n Mean (s) sE

20 95.93 + 5.71

20 211.04 + 5.10

20 230.91 + 6.29

20 77.78 _+10.28

P

[......

1

U °°°°5.1 P< 0.0005 [

j

Ns I

A dose-effect relationship between GSH given and increase in endurance to swimming was evident (Fig. 1). The endurance remained unmodified in control experiments performed on mice submitted to the whole series of tests after saline alone (Table IV). After seven daily injections of GSH 250 mg/kg the increase (189.38 + 19.26 s from the initial value of 93.08 +_5.88 s, equivalent to 103.4%) was highly significant (P< 0.0001)(Table V). After seven daily injections of saline, endurance to swimming was increased but the increase (102.00 +_ 15.34 s from the initial value of 90.13 + 17.53 s) was not significant (Table V).

DISCUSSION Experimental and clinical data suggest the involvement of toxic oxygen radicals in limiting endurance to muscle fatigue. The sites of production of these radicals are largely unknown, putative sites being mostly mitochondria but also granulocytes and xanthine-oxidase, all activated

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250

20C

150

I00

5(:

I

I

I

I

250

500

750

I000

mg/kg

Fig. 1. Cumulative curve of experiments in mice on endurance to exhaustive swimming (s + sE) after treatments with various doses of GSH. ***P< 0.0005.

Table IV E n d u r a n c e to e x h a u s t i v e s w i m m i n g of mice after saline a l o n e Initial

Saline

Saline

Saline

n Mean (s) sE

21 94.48 + 6.18

21 109.93 + 8.51

21 110.01 -+ 7.99

P

I I i

21 107.00 + 8.31 NS[

NSl NSI

The swimming tests were performed in the same days as the GSH-treated mice. Table V E n d u r a n c e to s w i m m i n g of m i c e receiving G S H or saline solution for 7 days

n Mean (s) SE P

Initial

Saline x 7 days

Initial

GSH 250 mg/kgx 7 days

5 90.13 _+7.53 !

5 102.00 ___15.54 NS I

8 93.08 --+5.88 l

8 189.38 + 19.26 P
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by the ischaemia consequent to the imbalance between oxygen consumption and oxygen delivery during strenuous effort in untrained muscles. Whichever the case, vitamin E and true scavengers like spin-trappers were reported to increase endurance to exhaustive fatigue [11]. The experiments presented here show that intraperitoneal injection of GSH in mice is followed within a few minutes by a highly significant increase in endurance to exhaustive physical effort. The increase was dose-related with the GSH injected. The effect of GSH on endurance to physical effort was of brief duration, being completely absent on the second day after the last GSH injection. However, a slow cumulative effect was present because after seven daily injections of an otherwise ineffective dose of GSH, a significant increase of endurance to swimming was observed. The interference of training was excluded by control experiments as was any interference of ageing and of weight increase. GSH is a low-weight peptide, ubiquitously localized, that acts as an intracellular antioxidant, being the substrate of peroxidases, or possibly in plasma maintaining the redox states of the extracellular compartment [13]. An inverse relationship between tissue level of GSH and tissue damage due to toxic oxygen radicals has been demonstrated in renal ischaemia reperfusion [14] or in Paraquat or Adriamycin induced damage on isolated cells [15, 16]. An increase of intracellular level of GSH in muscles within a few minutes after intraperitoneal injection so as to improve endurance to physical fatigue seems improbable. In fact, the mechanisms of transport of GSH through cell membranes are not so active as to affect intracellular level in a few minutes mostly in skeletal muscle where a very low activity of gammaglutamyl-transpeptidase has been reported [17]. Moreover, the administration of exogenous GSH (but not of the glutathione monoester) on GSH-depleted mice has been proved to be quite ineffective in restoring muscle levels of the peptide even over some days [17]. However, an elevated tissue level seems to be possible as the result of the repeated daily injections of GSH, so explaining the increase in endurance to swimming afforded by seven injections of a close of GSH otherwise ineffective as a single dose. To explain the improvement in endurance to physical fatigue observed a few minutes after an injection of GSH an extracellular action of the reduced and oxidized thiols would be considered. In fact, although plasma levels of GSH are many times lower than the intracellular ones, significant amounts of GSH occur in the circulation as the result of the interorgan cycle and cooperation [13]. This circulating GSH appears to be the mechanism by which plasma proteins and cell surface membrane components are protected from oxidative stress and the redox state is maintained [13, 18]. To test this hypothesis other experiments will be made, to discover whether the effects of exogenous GSH would be reproduced by other thiols. Whichever the mechanism, the experiments reported here demonstrate that a single injection of GSH on mice is followed within a few minutes by a highly significant increase in endurance to exhaustive physical effort. This may be useful in clinical practice; in every case toxic oxygen radicals and their effect cannot be proposed as the only mechanism of regulation of muscle effort but other more customary mechanisms should also be considered.

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ACKNOWLEDGEMENTS Glutathione (Tationil) was kindly purchased by Boehringer Mannheim S.p.A. (Milano).

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