Physical Exercise and Thiol Homeostasis: Possible Implications

Oxygen Toxicity in Exercise

Oxygen, as a metabolic fuel, allows an attractive yield of energy-rich phosphates
per unit substrate, and oxidative metabolism avoids the formation of lactic acid
–a significant factor in the development of muscle fatigue during exercise.
Recent advances in free radical biochemistry have made it clear that not all
of the oxygen consumed by live cells is completely (tetravalently) reduced. An
estimated 2-8 percent of the total oxygen consumed may escape from the metabolic
path after being partly reduced. Such incompletely reduced forms of oxygen and
their byproducts, collectively referred to as reactive oxygen species, are
implicated in a wide variety of reactions that may adversely affect our state of
health and longevity. During physical exercise O₂ flux through the body is
remarkably enhanced. The rate of oxygen uptake by the body increases by tenfold
to fifteenfold and is accompanied by more than a hundredfold increase in O₂ flux
in the active skeletal muscles.

A considerable body of experimental evidence has accumulated in recent years suggesting
that physical exercise, under certain circumstances, induces oxidative stress.^1,2
This is an area of critical concern because exercising is not only a
recreational activity but also has well-established therapeutic value. For
patients suffering from disorders that are known to have an oxidative
stress-related etiology, exercise-induced oxidative stress should be treated
with particular concern. For example, oxidative modification of low density
lipoprotein is known to play a major role in the pathogenesis of
atherosclerosis. Recently, Shern et al.³ reported that the susceptibility
of low density lipoprotein to oxidation was higher in exercising humans (n = 22 per group) than in their relatively sedentary counterparts. This suggests that we should consider the exercise-induced oxidative stress factor when designing exercise regimes for such patient groups.

Physical training enhances tissue antioxidant defenses, but such added protection may not be sufficient to defend against oxidative stress during long, strenuous exercise. Habitual physical exercise is a precious therapeutic tool in preventive medicine. A thorough understanding of the complications associated with exercise-induced oxidative stress is necessary for effective handling of this undesired effect. Such knowledge will help in designing exercise and nutrition protocols that will better serve our interests.

Defense Against Oxidative Stress

In biological systems, an imbalance in the pro- and anti-oxidant forces in favor of the former is referred to as oxidative stress. Processing of the one-electron reduction product of molecular oxygen, superoxides, to hydrogen peroxide is catalyzed by the enzyme family superoxide dismutases. Decomposition of tissue hydrogen peroxide can be catalyzed by the enzymes catalase or glutathione peroxidase. Glutathione peroxidase activity requires glutathione as a substrate and selenium as a cofactor. Vitamin E serves as a major lipid phase antioxidant that protects against oxidative lipid damage. Vitamin C, in the absence of free transition metal ions, may serve as an effective antioxidant.

Oxidant-antioxidant interaction involves electron transfer from the antioxidant to the electron-seeking oxygen-free radical. As a result, during such radical neutralization interaction, antioxidants are transformed to an oxidized state that is no longer able to detoxify reactive oxygen. In this situation, it becomes necessary to recycle this oxidized antioxidant to its potent reduced form. In tissues, several antioxidants may act in concert to let such recycling happen. Thus, the strength of antioxidant defense is not dependent on a single antioxidant but on the efficacy with which several antioxidants cooperate to let the antioxidant chain reaction function.^2,4

Thiols Play a Central Role in Antioxidant Recycling

Functional sulfhydryl (SH) residue-rich protein and nonprotein agents in the biological system, commonly referred to as biothiols, have multifaceted functions including protein synthesis, detoxification, cell division, and regulation of intracellular signal transduction. In addition to its other major functions, the abundant nonprotein thiol glutathione has proved to be a master physiological antioxidant. Glutathione itself, and other pro-glutathione agents such as a-lipoic acid,⁵ play a crucial role in the antioxidant chain reaction by helping to maintain favorable redox states of other major antioxidants such as vitamins C and E.²

Physical Activity Influences Tissue Glutathione Level

Studies from several laboratories have suggested that physical training may
upregulate the activities of glutathione-dependent antioxidant enzymes in some
tissues. Higher glutathione content in skeletal muscle of exercise-trained
animals also have been reported from our laboratory and others. Somani has
reported that physical training may improve the redox state of glutathione
in different regions of the brain.¹ In our experiments with beagle dogs that
were endurance-trained on a treadmill for 55 weeks or activity-restricted by fiber-cast immobilization of one of the pelvic limbs, we observed that the state of physical activity is an important determinant of skeletal muscle glutathione content. Endurance training increased and activity restriction decreased red gastrocnemius muscle glutathione content.

N-Acetylcysteine Spared Blood Glutathione Oxidation in Humans

Physical exercise is followed by rapid oxidation of blood glutathione, mainly in
the erythrocytes. Exercise-induced tissue glutathione oxidation has been
consistently observed in our laboratory as well as in several others. In a
recent study of young healthy men, we observed that blood glutathione oxidation
induced by maximal bicycle ergometric test (mean duration = 14 minutes) could
be spared in subjects who took 200 mg of N-acetylcysteine four times daily for
2 days and an additional 800 mg 2 hours before the exercise test.
N-acetylcysteine is a common mucolytic drug that has been safely used in the
clinic over a long period of time. A few months after our report was published,
Reid et al.⁶ reported that N-acetylcysteine inhibits muscle fatigue in humans. In a later rat study, we observed that N-acetylcysteine supplementation may also spare exercise-induced glutathione oxidation in the lung-an organ in which this drug is known to be bioavailable.

Enhancing Tissue Glutathione by use of Nutritional Supplements

When ingested, antioxidants such as vitamins C and E are bioavailable to a
certain extent. However, enhancing tissue glutathione level by nutritional
supplementation is a challenging task. A few reports have claimed that an oral
or intraperitoneal supply of glutathione may remarkably enhance endurance during
exercise. These studies were brief and did not follow any biochemical parameter
to explain the results. We conducted a more thorough study to evaluate the
significance of endogenous and exogenously supplied glutathione with respect
to exercise-induced oxidative stress. Glutathione-deficient rats in which
tissue glutathione synthesis was arrested had a remarkably lower endurance
(treadmill run) before exhaustion. This suggested that endogenous glutathione
is an important factor in exercise performance. This is understandable because
glutathione not only functions as a antioxidant but also has several other
critical functions, including delivery of cysteine for protein synthesis.
When a lower exercise intensity was selected for mice, Ji and associates
did not observe any effect of glutathione deficiency on endurance. In our
study, exogenously supplied (intraperitoneal) glutathione was poorly available to most tissues and did not influence endurance.

Availability of cysteine, in its reduced form, in the cell is a limiting step
in intracellular glutathione synthesis. Several agents have been tested for their
efficacy in this respect. Among the ones that hold clinical promise are
N-acetylcysteine and a-lipoate.⁵ Both agents have proved to be safe for human
use. a-Lipoate has been recently introduced in the United States as a dietary supplement. It has been used in Germany for a long time for the treatment of diabetic polyneuropathies. Recently the laboratory of Klip has reported that a-lipoate also stimulates glucose uptake by cultured skeletal muscle cells.

Thiols As Critical Determinants of Cell Function and Response to Stress

To develop a better understanding of the exact mechanisms that underlie reactive
oxygen species-dependent disorders in biological systems, recent studies have
focused on the regulation of gene expression by oxidants, antioxidants, and other
determinants of the intracellular reduction-oxidation (redox) state.^7-9 At least
two well-defined transcription factors, nuclear factor (NF)-kB and activator
protein (AP)-I are regulated by the intracellular redox state. One major
clinical significance of NF-KB activation is that it enhances HIV gene
expression. The long terminal repeat of HIV-I has been shown to contain two
NF-kB binding sites that may be crucial in regulating AIDS latency. AP-1 is an
important mediator of tumor promotion, and is thus a focal point in cancer
research.

Certain intracellular protein and nonprotein thiols are known to act as “redox
sensors” that signal for much of the activity of the aforementioned transcription
factors.⁷ Under conditions of oxidative stress, certain thiols
such as glutathione and thioredoxin are transformed from a reduced sulflhydryl
(-SH) state to an oxidized disulfide (-S-S-) state. This change serves as a
signal for redox-sensitive transactivation to start. In the nucleus, these
transcription factors are known to require a reducing atmosphere to be able
to bind with the consensus DNA sites and initiate transactivation. Again,
certain protein thiols in the nucleus regulate this DNA binding. In brief,
subtle changes in intracellular thiol-disulfide status have an important
bearing on the molecular events associated with cellular response to the
stress. For example, it is suggested that elevated GSSG/GSH in the cytosol
may be implicated in NF-kB activation.^7,10 A number of studies have shown
that physical exercise may increase the tissue GSSG/GSH ratio, but does this
lead to NF-kB activation? We are currently conducting a pilot human study to
address this issue. If indeed physical exercise induces NF-kB activation,
does this mean that the rate of progression of AIDS may be accelerated in
strenuously exercising HIV-positive individuals? This is one of many
exercise-induced oxidative stress-related issues that deserve careful
attention. Activation of NF-KB may also upregulate the expression of
adhesion and other molecules that are known to be implicated in the
etiology of atherosclerotic and diabetic complications.

So, is exercising bad? Certainly not. A physically active lifestyle coupled with
well-balanced nutrition is of great help. However, antioxidant defenses of active
tissues can be overwhelmed by excess reactive oxygen generated during exercise.
A vivid understanding of the possible mechanisms that may contribute to
exercise-induced oxygen toxicity, associated physiological response, and
the design of appropriate measures to circumvent or minimize such toxicity is
fundamental to (1) enhancing the effectiveness of physical exercise as a
preventive and therapeutic tool in clinical practice and (2) controlling exercise-induced oxygen toxicity-dependent tissue damage and augmentation of other possible health risks.

1. Sen CK, Packer L, Hanninen O. eds. Exercise and oxygen toxicity. Amsterdam: Elsevier, 1994:536.

2. Sen CK. Oxidants and antioxidants in exercise (review). J Appl Physiol 1995;79:675-86.

3. Shern R. Santanam N. White-Welkley J. Parthasarathy S. Enhanced rate of oxidation seen in low density lipoprotein isolated from chronic exercisers. A cause for concern? (abstract). The annual meeting of The Oxygen Society 1995, Pasadena, California, p. 109.

4. Constantinescu A, Han D, Packer L. Vitamin E recycling in human erythrocyte membranes. J Biol Chem 1993;268: 10906-13.

5. Packer L, Witt EH, Tritschler HJ. a-Lipoic acid as a biological antioxidant (review). Free Radic Biol Med 1995;19:227-50.

6. Reid MB, Stokic DS, Koch SM, Khawli FA. N-acetylcysteine inhibits muscle fatigue in humans. J Clin Invest 1994;94:2468-74.

7. Sen CK, Packer L. Antioxidant and redox regulation of gene transcription. FASEB J. May 1996, in press.

8. Sen CK, Traber K, Packer L. Inhibition of NF-KB activation in human T-cell lines by anetholdithiolthione. Biochem Biophys Res Commun 1996;218:148-53.

9. Sen CK, Roy S. Packer L. Involvement on intracellular Ca2+ in oxidant induced NF-KB activation. FEBS Lett 1996, in press.

10. DrogeW. Scl~ulze Os~off K, Mihm S. Galter D, Schlock H. Eck HP, et al. Functions of glutathione and glutathione disulfide in immunology and immunopathology (review). FASEB J 1994;8:1131-8.