Role of Cysteine and Glutathione in HIV Infection and Cancer Cachexia: Therapeutic Intervention with N-Acetylcysteine

Wulf Droge Andrea Gross Volker Hack Ralf Kinscherf Michael Schykowski Michael Bockstette Sabine Mihm Dagmar Galter Division of lmmunochemistry Deutsches Krebsforschungszentrurn D-69 I20 Heidelberg, Germany

Role of Cysteine and Glutathione in HIV Infection and Cancer Cachexia: Therapeutic Intervention with N-Acetylcysteine

1. Introduction Massive loss of skeletal muscle mass (cachexia) is one of the hallmarks of HIV infection (Kotler et al., 1985; Grunfeld, 1991), sepsis, and trauma (Long et al., 1976; Brennan, 1977) and is the single, most common cause of death among cancer patients (Warren, 1932; Harnett, 1952; Lawson et al., 1982; Friedman, 1987; Pisters and Pearlstone, 1993). The pathogenetic mechanism of the skeletal muscle catabolism in HIV infection, cancer, and sepsis is largely unknown, and there is no satisfactory treatment for these catabolic conditions. Although the work of many laboratories on the pathogenetic mechanisms has provided a large body of phenomenological findings and inspired useful working hypotheses, the demonstration of cause-andeffect relationships in vivo has been difficult. Nevertheless, the analysis of the cachectic mechanisms is a tremendous challenge in view of the enormous medical, socioeconomical, and psychological implications of this debilitating condition. Advances in Phumcology, Volume 38 Copyright 0 1997 by Academic Press, Inc. All rights of reproductmn In any form reserved.

58 I

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A significant impairment of immunological functions is often associated with the catabolic process. In H N infection, the immunological dysfunction develops progressively into a severe condition called acquired immunodeficiency syndrome (AIDS) (Pantaleo et al., 1993). The relatively milder form of immunological dysfunction in cancer patients has been demonstrated among others by mitogenic stimulation in uitro (reviewed in Droge et al., 1988a). Increasing evidence suggests that an abnormal cysteine and glutathione metabolism plays a decisive role in the development of catabolic conditions and associated immunological dysfunctions. This chapter gives a conceptual overview and a guideline for further experimentation. In this context, this chapter also discussesthe chances for a therapeutic intervention with cysteine derivatives such as N-acetylcysteine (NAC). At this moment, there is no other satisfactory treatment available for these disease processes.

B. Methodological Aspects Studies on biochemical and immunological parameters in patients yield mainly phenomenological findings. Certain diseases and conditions such as sepsis, chronic fatigue syndrome (CFS), and amyotrophic lateral sclerosis (ALS)were found to share some of the typical biochemical and immunopathological changes associated with HIV infection and cancer and can thus provide useful comparative information. A correlation between unrelated parameters cannot prove a cause-and-effect relationship but is often useful in encouraging or discouraging further experimentation. The formal proof of a cause-and-effectrelationship, however, can rarely be obtained in studies on patients. Investigations on patients have, therefore, been complemented by studies in animal models, including tumor-bearing mice. The simultaneous analysis of liver and skeletal muscle tissue samples, for example, has been possible in this animal model. Despite some species-relateddifferences, it was satisfying to see that the basic pathological changes in the various systems are similar, and that tumor-bearing mice are indeed a useful experimental model for studies on certain aspects of the cachectic process. One may safely predict that the mechanisms of the cachectic process and its immunopathological implications will be unraveled only in small steps, with each step being almost irrelevant by itself and important only in the context. The major steps in the agenda are To identify abnormal biochemical patterns in different catabolic diseases and conditions, To identify abnormal biochemical parameters that are significantly correlated with mortality, disease progression, and weight loss, To prove cause-and-effect relationships by experimental intervention,

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To develop and optimize interventive strategies that may be suitable for clinical therapy, and To test new therapeutic strategies in clinical trials.

II. Elevated Venous Plasma Glutamate Levels as an Early and Possibly Universal Marker for Catabolic and Precatabolic Conditions A. Evidence for an Impairment of Muscular Membrane Transport Activities Virtually all diseases and conditions that are associated with skeletal muscle catabolism and that have been studied in this regard, including advanced malignancies (Droge et al., 1988a), HIV/SIV infection (Droge et al., 1988b; Eck et al., 1989, 1991), sepsis (Roth et al., 1985), and amyotrophic lateral sclerosis (Plaitakis and Caroscio, 1987), are associated with elevated plasma glutamate levels. At least in cancer, HIV, and SIV infection, the venous plasma glutamate levels were shown to increase before weight loss becomes detectable, i.e., in the precatabolic condition. In rhesus macaques, glutamate levels were shown to increase significantly within 2 weeks after (Eck et al., 1991). inoculation of the simian immunodeficiency virus SIVZSlmac The elevation of venous plasma glutamate levels may therefore be one of the earliest manifestations of the precachectic process. Amino acid exchange studies on well-nourished (i.e., not yet overtly cachectic) cancer patients revealed that the increase of the postabsorptive venous glutamate level reflects the failure of the muscle tissue to take up glutamate from the circulation and is, in addition, associated with an impaired transport activity of the skeletal muscle tissue for other important metabolites such as glucose and ketone bodies (Hack et at., 1996b).

B. Suggestive Evidence for an Abnormal Cysteine Catabolism and Glutathione Level in Skeletal Muscle Tissue The major glutamate transport system in skeletal muscle cells is the Na+-dependent XAG-system that is shared by aspartate (Horn, 1989; Low et al., 1994; McGivan and Pastor-Anglada, 1994). As this membrane, transport is energetically driven by the electrochemical Na+ gradient and as this, in turn, is linked via the Na+/H+ antiport to the intracellular pH, this glutamate transport activity is subject to inhibition by proton-generating processes. Another factor contributing to its pH dependency is the fact that this Na+-dependent transport system also requires an antiport of OH- or HCO; (Bouvier et al., 1992; Kanner, 1993). It is therefore reasonable to

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Wulf Droge et a/.

assume that the decreased glutamate transport activity is related to the increased glycolytic activity and lactate production that has been demonstrated in several different catabolic conditions, including cancer cachexia (Shaw et al., 1985; Shaw and Wolfe, 1987; Tayek, 1992) and sepsis (Roth et al., 1982; Roth, 1985). The glycolytic production of lactate and pyruvate causes an acidification of the skeletal muscle tissue. This acidification may be ameliorated by the fact that lactate and pyruvate are cotransported with protons across the plasma membrane and thereby remove the equivalent amount of protons generated. The acidification is aggravated, however, by the fact that the temporary increase of intracellular pyruvate causes an increased rate of cysteinefpyruvate transamination and consequently an increased cysteine catabolism into sulfate and protons. Increased intracellular sulfate levels indicative for an increased generation of protons have indeed been found in the skeletal muscle tissue of tumor-bearing mice (Hack et al., 1996a). This was associated with a decrease of the glutathione level in the skeletal muscle tissue, indicating that the cysteine catabolism was increased at the expense of glutathione biosynthesis in this tissue. There is, therefore, a possibility that the increase of the postabsorptive venous plasma glutamate level in cancer patients or HIV-infected persons may be an early indicator for an abnormal cysteine catabolism, a decrease of intracellular glutathione in the skeletal muscle tissue, and an impaired transport activity. Moreover, as the glycolytic pathway is suppressed by adequate levels of ATP and is generally not active in cells with an adequate mitochondria1 oxidative metabolism, there is also a possibility that the elevated plasma glutamate level in the precachectic condition is indicative for a failure of the mitochondria to meet the energy requirements of the muscle tissue. In support of this hypothesis, a decreased ATP level has been found in the skeletal muscle tissue of patients with sepsis (Roth, 1985). An increased venous plasma glutamate level has also been found in healthy human subjects after a 4week period with intensive anaerobic physical exercise programs which per definition exceeded the capacity of the oxidative metabolism and were therefore associated with high systemic lactate levels (Kinscherf et al., 1996).

C. Pathological Significance of Decreased Membrane Transport Activity and Elevated Venous Plasma Glutamate Levels Whether the decrease of the skeletal muscle glutathione levels and/or the increase of the cysteine catabolism and its effect on the intracellular pH may play a causative role in the catabolic process remains to be determined. The impairment of the glutamate transport activity may be responsible, at least partly, for the decreased intracellular glutamate and/or glutamine levels that have been found in the skeletal muscle tissue of catabolic patients (Fiirst et al., 1979; Askanazi et al., 1980; Roth et al., 1982). The uptake of glutamate from the circulation, the intracellular conversion of glutamate

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into glutamine, and the export of glutamine into the circulation are some of the most important biochemical functions of skeletal muscle tissue (Newsholme and Parry-Billings, 1990). In addition, the elevated venous glutamate levels may have a direct effect on the cysteine supply to cells of the immune system. Elevated extracellular concentrations of glutamate in the pathologically relevant range were found to inhibit the cellular uptake of cystine and to decrease intracellular cysteine and glutathione levels in macrophages and peripheral blood mononudear cells (Eck and Droge, 1989; Gmiinder et al., 1991). It has therefore been proposed that an increased plasma glutamate level is a pathogenetic factor in its own right and is responsible for the decreased intracellular glutathione levels in the peripheral blood lymphocytes of HIV-infected patients (Eck et al., 1989) and for the decreased lymphocyte functions in advanced malignancies (Droge et al., 1988a).

111. “Push” and “Pull” Mechanisms in Catabolic and Precatabolic Processes A. Two Principal Mechanisms in the Catabolic Process The hallmark of cachectic processes is an excessive urea production that proceeds at the expense of the skeletal muscle protein and which results in a negative nitrogen balance (Brennan, 1977; Shaw et al., 1985, 1988; Shaw and Wolfe, 1987). Because urea production occurs mainly in the liver, and catabolic processes are associated with biochemical changes in skeletal muscle tissue and liver, it has been an intriguing question whether the excessive urea production is primarily the consequence of a hepatic dysfunction that may drain indirectly the amino acid pool of the skeletal muscle tissue (i.e., a “pull” mechanism) or the consequence of a biochemical dysfunction in the skeletal muscle tissue (i.e., a “push” mechanism). Conditions with subnormal plasma amino acid levels should be indicative, by definition, for the “pull” mechanism, whereas conditions with higher than normal plasma amino acid levels should be indicative for the “push” mechanism. The available evidence suggests that both mechanisms are operating and may occur sequentially in the same disease process. As a rule, the “pull” condition is found in seemingly well-nourished patients with potentially cachectic diseases, i.e., in a stage that can be described as “precachectic.” Although this condition may seem relatively benign, there is reason to believe that it triggers the progression to overt cachexia and, in the case of HIV infection, the breakdown of the immunological defense against the virus as discussed later. 6. Decreased Plasma Cystine Levels and Evidence for “Push” and “Pull” Mechanisms in Sepsis

Sepsis is a particularly well-studied model of cachexia. Patients with sepsis pass through a stage with a significant decrease o€total plasma amino

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acid levels, including a decrease of plasma cystine, glutamine, and arginine levels (Roth et al., 1985). In addition, significantly decreased glutamine levels have been found in skeletal muscle tissue by various authors (Fiirst et al., 1979; Askanazi et al., 1980; Roth et al., 1982). All of these findings are indicative for a “pull” mechanism. However, patients in the phase of severe skeletal muscle catabolism ( “autocannibalism”) were found to have increased plasma amino acid concentrations (Siege1 et al., 1979; Cerra et al., 1980), indicative for a “push” mechanism. In either case, the amino acids glutamine and alanine were found to account for the majority of the amino acid nitrogen that was released from the skeletal muscle tissue and taken up by the splanchnic bed (Aulick and Wilmore, 1979; Duff et al., 1979; Wilmore et al., 1980; Souba and Wilmore, 1983). C. Decreased Plasma Cystine and Cysteine Levels and Evidence for “Push” and “Pull” Mechanisms in HIV Infection

A significant decrease of plasma cystine and cysteine levels has also been found in HIV infection (Droge et al., 1988b; Eck et al., 1989; Hortin et al., 1994). A more detailed analysis of our data revealed that the most dramatic decrease of plasma cystine levels occurred in asymptomatic HIV-infected persons with CD4+ counts C400 mm-3, i.e., in the phase with the strongest decrease of the absolute CD4+ T-cell number but without obvious weight loss (i.e., in the precachectic stage) (Table I). Using bioelectrical impedance analysis, Siittmann et al. (1991) found that the loss of body cell mass starts already in the asymptomatic stage, i.e., well before weight loss becomes detectable. The decrease of plasma cystine was associated with plasma glutamine and arginine levels significantly below the mean of healthy control subjects. The plasma cystine levels of symptomatic HIV-infected patients were, on the average, still lower than those of uninfected controls or infected persons with >400 mm-3 CD4+ cells, but higher than the levels found in TABLE I Plasma Amino Acid Levels in HIV-Infected Persons

Noninfected persons HIV+ without symptoms CD4+ cells > 400 mm-3 HIV’ without symptoms CD4’ cells < 400 mm-’ HIV with symptoms CD4+ cells < 400 mm-3

Cystine

Glutamine

Arginine

Glutamate

n

(PM)

(PM)

(PM)

(PM)

145 26

56.8 ? 1.4 61.2 i 3.4

614 ? 11 485 2 27

89.7 2 2.9 60.2 i 3.0

26.9 81.0

14

40.3 5 1.7

468

26

70.8 i 5.7

53.0 i 5.7

71

52.9 t 1.9

468 2 15

76.1 t 8.0

70.0

5

? ?

?

1.2 9.6

0.2

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the intermediate asymptomatic stage with <400 mm-3 CD4+ T cells. This increase in the later stages of the disease suggests the possibility that the catabolic process may shift, at least partly, from a “pull” to a “push” mechanism.

D. Decreased Plasma Cystine Levels and Evidence for “Push” and “Pull” Mechanisms in Malignant Diseases Advanced cancer patients with massive cachexia have abnormally high plasma glutamate levels and often also relatively high plasma cystine, glutamine, and arginine levels indicative for a “push” mechanism of the catabolic process. This pattern was seen among others in a group of patients with liver cancer (Zhang and Pang, 1992) and in a group of advanced and strongly catabolic patients with gastrointestinal cancer, lung cancer, or cancer of the pancreas (V. Hack and W. Droge, unpublished observation). However, at least one study on well-nourished patients with lung or breast cancer, i.e., on patients at a relatively early stage of the malignant disease, revealed that cystine, glutamine, and arginine levels were markedly decreased in comparison with healthy controls (Zhang and Pang, 1992). These findings suggest that “push” and “pull” mechanisms may also both be operating in malignant diseases and that the “pull” mechanism may be typical of the precatabolic stage. Significantly decreased plasma cystine levels have also been found in C57BL/6 mice bearing the syngeneic MCA 105 fibrosarcoma (Hack et al., 1996a). E. Abnormally Low Plasma Cystine and Glutamine Levels in Patients with Chronic Fatigue Syndrome (CFS) The etiology and the pathogenetic mechanisms of CSF are poorly understood. By definition, these patients show a severe skeletal muscle dysfunction (fatigue), and they were also found to have significantly decreased plasma cystine and glutamine levels (Aoki et al., 1993). In addition, they express an immunological dysfunction as discussed in Section IV,A. F. Hypothetical Mechanism of the “Pull” Condition: Evidence for an Abnormal Cysteine and Glutathione Metabolism in Liver Whereas the “push” mechanism of the catabolic process is still obscure, there are some important clues regarding the “pull” mechanism. Studies on patients with sepsis have already shown that the decrease of plasma cystine levels was associated with a decrease of hepatic cystine and taurine levels (Roth et al., 1985). More recent studies on C57BL/6 mice bearing the

588

Wulf Drage e t a / .

syngeneic MCA 105 fibrosarcoma have shown that the decrease of the plasma cystine level was associated with a significant decrease of the intrahepatic sulfate level, suggesting that the proton-generating catabolism of cysteine into sulfate may be markedly decreased in the liver of the tumor-bearing host (Hack et al., 1996a). As the intrahepatic pH is normally maintained by the ratio of hepatic urea production versus glutamine biosynthesis (Haussinger and Gerok, 1985), it is reasonable to assume that a decreased generation of protons will lead to an increased formation of carbamoylphosphate and urea biosynthesis at the expense of the glutamine pool (see scheme, Fig. 1). This hypothetical mechanism is supported by the experimental observation that the decrease of the hepatic sulfate levels in the tumor-bearing mice was associated with an increased hepatic urea level and with increased urea/ glutamine and glutamate/glutamine ratios (Hack et al., 1996a). To prove the cause-and-effect relationship between cysteine catabolism and hepatic urea levels, some of the tumor-bearing mice were treated with daily injections of cysteine. The results showed that the administration of cysteine reconstituted not only the hepatic sulfate level, but also decreased the hepatic urea level and normalized the urea/glutamine and glutamate/glutamine ratios. Incidentally, the tumor-bearing mice showed a significant increase of the y-glutamylcysteine synthetase activity, i.e., the first rate-limiting enzyme

fat 4 glucose

-

amino acids

a-ketoglutarate glutamate

carbamoyl-phosphateI @ glutamate-a-KG-transaminase @ L-glutamate dehydrogenase @ glutamine synthetase I glutaminase @ carbamoylphosphate synthase @ carknic anhydrase

FIGURE I Role of cysteine in the feedback control of nitrogen disposal: A hypothetical model. The hypothesis states that the hepatic cysteine level determines the rate of its catabolic conversion into sulfate and protons. This, in turn, inhibits the conversion of amino groups into urea ( J ) through carbarnoylphosphate synthesis ( J ) and the urea cycle. H+-generating into glutamine ( 7 ). Accordingly, the cysteine processes thus favor the conversion of catabolism plays a role in conserving the glutarnine pool. The hypothesis predicts that the availability of cysteine sets the threshold at which amino acids are being converted into glucose and fat. This threshold determines decisively the loss of nitrogen and contributes to the control of body cell mass and body fat.

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of glutathione biosynthesis, suggesting that more cysteine is drained into the glutathione pool. The simplest interpretation for the enhanced production of urea in the “pull” condition is therefore that a decreased availability of cysteine in the liver leads to a decreased cysteine catabolism into sulfate and protons which, in turn, enhances urea biosynthesis at the expense of glutamine. The hepatic cysteine deficiency may be explained partly by an increased hepatic glutathione biosynthesis and partly by the increased cysteine catabolism in the skeletal muscle tissue (see Section 11,B). The liver was previously shown to export glutathione disulfide mainly into the bile canaliculus (Akerboom and Sies, 1994a,b). Our own studies on the tumorbearing mice revealed that the glutathione disulfide levels in the bile were significantly increased in comparison to the healthy controls. This suggests that cysteine is, at least partly, lost by an increased export of glutathione disulfide into the bile.

IV. Immunological Implications A. Cysteine Deficiency and Immunological Dysfunction In view of the important role of the cysteine supply and the intracellular glutathione level for lymphocyte functions, it has been hypothesized that the cysteine and glutathione deficiency in HIV-infected patients may contribute to the immunopathology of the disease (Droge et al., 1988b, 1992; Eck et al., 1989). The almost complete clearance of the virus within a few weeks after primary infection (see Borrow et al., 1994) and the relatively long asymptomatic period with relatively normal CD4+ T-cell counts (Pantaleo et al., 1993), plus the phenomenon of the long-term nonprogressors (Buchbinder et al., 1994; Klein and van Baalen, 1995; Rinaldo et al., 1995), are striking examples for the ability of the immune system to control this virus infection, at least in principle. The fact that the most profound decrease of plasma cystine levels coincides with the decrease of the CD4’ T-cell numbers in asymptomatic HIV-infected persons (Table I), therefore, suggests the possibility that this decrease of the cystine levels may weaken the immune system so that the delicate balance between the virus and the immune system is tilted in favor of the virus. Other conditions with abnormally low cystine levels, including cancer and CFS, show clearly demonstrable immunological dysfunctions. It is a common finding that peripheral blood lymphocytes from patients with malignant diseases exhibit diminished proliferative responses to mitogens in comparison with healthy controls (Watkins, 1973; Wanebo et al., 1975; Braun et al., 1980; Collins et al., 1980; Muller et al., 1984; Yron et al., 1986; Droge et al., 1988a). An even more profound suppression has been observed in various in vitro assays of the reactivity of tumor-infiltrating lymphocytes (Vose et al., 1977; Totterman et al., 1980;

590

Wuif Drage et al.

Miescher etal., 1986).Persons with CFS were found to have, on the average, not only decreased plasma cystine and glutamine, but also a significantly activity and relatively low numbers of CD16+ decreased natural killer (NK) cell numbers (Fc-y receptor+ cells) (reviewed in Aoki et al., 1993). The decrease of NK activity was correlated with a decrease of antibody-dependent cell-mediated cytotoxicity (ADTC). In addition, a low CD4+/CD8 ratio has been observed in 20% or more of CFS patients (reviewed in Aoki et al., 1993). These changes are indeed reminiscent of the cellular dysfunctions that are observed in HIV-infected patients before the CD4+ Tcell numbers start to decline (Miedema et al., 1988; Giorgi and Detels, 1989; Rosenberg and Fauci, 1989). Poli et al. (1985) have shown-that NK cells of HIV-infected persons are phenotypically and numerically normal but functionally defect. A qualitative defect of NK cells and LAK cells has also been reported (Fontana et al., 1986; Ullum et al., 1995; see also the review by Sirianni et al., 1990). The loss of NK cell function may well be a decisive event in the switch from stable to progressive disease because NK cells were previously shown to play an important role in the defense against virus infections (Herberman and Ortaldo, 1981). Finally, in a study on healthy human subjects, Kinscherf et at. (1994) found that persons with relatively low intracellular glutathione levels in the peripheral blood mononuclear cells ( <20 nmol/mg protein) had significantly lower CD4+ T-cell numbers than persons with approximately median levels of intracellular glutathione (20-30 nmol/mg protein). All of these phenomena, i.e., the immunological dysfunctions in cancer or CSF patients and the conspicuously low CD4+ T-cell counts in persons with relatively low intracellular glutathione levels, may not seem very impressive in comparison with the severe destruction of the immune system in HIV infection (Pantaleo et al., 1993). It would, therefore, be unreasonable to assume that the cysteine deficiency accounts for all aspects of the HIV-mediated immunopathology. However, even a relatively moderate impairment of immunological functions may be sufficient to have disastrous consequences in the course of the HIV disease by interfering with the initially successful battle between the host immune system and the virus. If so, this disastrous process may be prevented if the plasma cystine levels are corrected in time, as discussed in Section V. +

B. Abnormal Glutathione Levels in HIV Infection The cysteine-containing tripeptide glutathione is a limiting factor for the immune system to the extent that certain T-cell functions can be potentiated in vivo by the administration of glutathione (Droge et al., 1986). In a number of different experimental systems, the intracellular glutathione level of lymphocytes was shown to determine decisively the magnitude of immunological functions (reviewed in Droge et al., 1994). Because cysteine is a rate-limiting precursor for the glutathione biosynthesis in lymphoid cells

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(Meister and Anderson, 1983), it was not unexpected to see that the decreased plasma cysteine and cystine levels of HIV-infected patients (Droge et al., 1988b; Eck et al., 1989)were associated with a decrease of intracellular glutathione levels in the peripheral blood mononuclear cells, blood plasma, and epithelial lining fluid of the lung (Buhl et al., 1989; Eck et al., 1989; Roederer et al., 1991).More recently, Aukrust et al. (1995)found abnormal glutathione/glutathione disulfide ratios but normal absolute glutathione levels in peripheral blood mononuclear cells of HIV-infected patients. The apparent discrepancy may be explained by the fact that the earlier findings were obtained mainly with patients at relatively early stages of the disease (Eck et al., 1989), whereas Aukrust et al. (1995) may have studied more advanced patients, where the massive decrease of the plasma cystine level may have been largely reversed (see Section 111,C). C. Redox Regulation by Glutathione and Glutathione Disulfide The changes of the intracellular glutathione and/or glutathione disulfide levels in HIV-infected patients (Eck et al., 1989; Roederer et al., 1991; Aukrust et al., 1995) raise the question of how these changes may affect lymphocyte functions. The redox regulation of signal cascades and gene transcription is a relatively young area of research and is still not completely understood at the molecular level. It is clear, however, that glutathione disulfide has a profound effect on the DNA-binding activity of the nuclear factor-KB (NF-KB)in both intact cells and cell-free systems (Galter et al., 1994; Mihm et al., 1995). The transcription factor NF-KB is involved in the inducible transcription of several immunologically important genes, including those of the interleukin-2 receptor a-chain, tumor necrosis factora (TNF-a),major histocompatibility complex antigens, and c-fos (reviewed in Ullmann et al., 1990; Baeuerle and Baltimore, 1991). The DNA-binding activity of NF-KBis inhibited in cell-free systems by physiologically relevant concentrations of GSSG even in the presence of a large excess of thiols. Moreover, three independent procedures that elevate GSSG levels in intact cells were found to inhibit DNA-binding activity and transactivating activity in intact cells (Galter et al., 1994; Mihm et al., 1995).Whereas NF-KBDNAbinding activity is inhibited by GSSG, it is enhanced by thiols, including dithiothreitol, cysteine, glutathione, and reduced thioredoxin (Matthews et al., 1992; Okamoto et al., 1992; Galter et al., 1994). The latter is by far the most effective and possibly the only physiologically relevant thiol compound in this context. Thioredoxin was found to restore the DNAbinding activity of oxidized NF-KBin vitro and to augment gene expression from HIV-LTR in intact cells as shown with a corresponding reporter gene construct (Matthews et al., 1992; Okamoto et al., 1992). The induction of

592

Wulf Droge et ol.

NF-KBactivation and nuclear translocation, in contrast, is strongly inhibited by thioredoxin (Schenk et al., 1994). In contrast, the expression of a reporter gene under the control of the transcription factor activator protein-1 (AP-1), i.e., another immunologically important transcription factor, was shown to be strongly increased by procedures that elevate intracellular GSSG levels, indicating that the inhibitory effect of glutathione disulfide is selective (Galter et al., 1994). In contrast to NF-KB,AP-1 is almost exclusively localized in the nucleus, which contains considerably lower concentrations of GSSG. Because the DNA-binding activity of AP-1 in cell-free systems is not increased but rather decreased by GSSG, this finding suggests that GSSG modulates one of the components in the upstream signal cascade that induces AP-1 activity. Details of this regulatory effect, including the identification of the redox-sensitive component of the signal cascade and its biochemical modification by the oxidizing agent, remain to be investigated. Because glutathione disulfide levels are determined largely by the availability of cysteine (Droge et al., 1995), it makes sense that T lineage cells have generally a tightly controlled cysteine supply that is limited by an extremely weak membrane transport activity for cystine. Cystine is the quantitatively most important source of cysteine in blood plasma and in standard cell culture medium, and thus the limiting precursor for the biosynthesis of glutathione and GSSG. In T cells, the transport activity-of cystine is more than 10-fold lower than that of cysteine, alanine, or arginine (Ishii etal., 1987, Gmunder et af., 1991, Lira et al., 1992). It is therefore conceivable that the delicate balance of the cysteine supply and of the intracellular glutathione and glutathione disulfide levels may be disturbed in HIV infection. HIV-infected persons were shown to have an increased expression of NF-KBcontrolled genes, including the genes of TNF-a, soluble IL-2 receptor (i.e., a truncated form of the IL-2 receptor a-chain), and &-microglobulin (reviewed in Droge et al., 1992).The abnormal cytokine expression in HIVinfected persons is thus potentially a consequence of the abnormal redox regulation in these patients.

V. Therapeutic Intervention with a Cysteine Derivative: Effects of Long-Term Treatment with N-Acetylcysteine

-

Section IV,A discussed the hypothesis that the decrease of plasma cystine levels in HIV-infected patients may cause a (moderate) immunological dysfunction that tilts the balance between immune system and virus in favor of the virus and starts the catastrophic progression of the disease. Moreover, Section II1,G described the hypothesis that the availability of cysteine may also regulate the loss of nitrogen from the amino acid reservoir in the (pre)cataboliccondition. To test these hypotheses, we have looked for strate-

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gies to reconstitute the plasma cystine levels in these patients. Since 1988, we have collected longitudinal data on HIV-infected patients treated with the cysteine derivative N-acetylcysteine for periods up to 4 years. Data show that the plasma cystine and glutamine levels can indeed be substantially increased by this treatment in comparison with pretreatment values and with a large group of untreated HIV-infected patients (Figs. 2 and 3 ) . CD4+ T-cell numbers did, on the average, not change significantly after Nacetylcysteine treatment but remained essentially stable. This effect remains to be confirmed in a larger study. Excessive doses of N-acetylcysteine, however, may lead to higher than normal plasma cystine and cysteine levels and impose certain risks. The dose required to raise cystine and glutamine levels close to the mean of healthy individuals was found to vary in the course of the disease, suggesting a variable loss of cyst(e)ine. Data, therefore, suggest that HIV-infected patients should be treated with a cysteine derivative, but that the therapeutic dose may have to be adjusted according to individual needs. This requirement is analogous to the variable needs of diabetic patients with respect to insulin. Section IV,A mentioned already the study of Kinscherf et al. (1994) on healthy human individuals showing that persons with intracellular glutathione levels of 20-30 n m o l h g protein had significantly higher numbers of

1

PM plasma cystine

PM

h s m a glutamine

uM ,lasma arginine

Pk

hasma glutamate

p<.003

HIVHIV+ pre-post treatment

HIVHIV+ pre-post treatment

HIVHIV+ pre-post treatment

HIVHIV+ pre-post treatment

FIGURE 2 Effect of N-acetylcysteine (NAC) treatment on plasma amino acid patterns in HIV-infected individuals. Data show the mean plasma cystine, glutamine, arginine, and glutamate levels of 145 randomly selected healthy human subjects (HIV-), 11 1 randomly selected HIV-infected persons without NAC treatment (HIV+), and 4 HIV-infected persons before and after NAC treatment. Posttreatment data show the mean of a total of 45 longitudinal measurements of the 4 patients. Pretreatment data as well as the HIV- and HIV' data represent the mean of single measurements per person.

5 94

Wulf Droge et a/.

NAC dose

Patient 6 start 25.4.88, age 39

Patient D ,start 3.12.91. age 50

mglweek

rh

>90 90

z -z 2,:s

.-

coal

E ‘25

-am v)

3 .-?

5

c

3

E m

E,

S a

80

70 60 50 40 700 600

500

1.7.1.7. 1.1. 1988 1989 1990

1.1.

1991

1.1.

1992

1.1.

1993

1.1.

1.1.

1.1.

1994 1995 1996 FIGURE 3 Longitudinal analysis of the effects of N-acetylcysteine (NAC) over >3-year

periods. Longitudinal data include plasma amino acid levels, T-cell counts, body weight, and body cell mass from two HIV-infected persons observed over 4-year periods. The periods of NAC treatment and the doses used are indicated on the top of the figure. NAC treatment can cause a substantial increase of plasma cystine, glutamine, and arginine levels. However, the doses required to maintain plasma cystine, glutamine, and arginine levels close to the mean of healthy human subjects (i.e., > 50,> 600, and > 70 p M , respectively) may change considerably during the course of infection. The therapeutic dose of N-acetylcysteine may therefore have to be adjusted according to the individual needs of a given patient at a given time.

NAC Therapeutic Intervention

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CD4+ T cells than persons with lower glutathione levels. This study also showed that persons who changed during a 4-week observation period from the optimal to the suboptimal range (10-20 nmoUmg protein) experienced, on the average, a 30% decrease of CD4+T-cell numbers. This decrease was prevented by treatment with N-acetylcysteine (Kinscherf et al., 1994).

VI. Concluding Remarks The mechanisms of cachectic processes as they occur in HIV infection, cancer, and sepsis are complex and appear to involve at least two distinct mechanisms that can be described as “push” and “pull” mechanisms. The “push” mechanism appears to prevail in the phase of massive muscle cell catabolism, i. e., in the relatively late phase of the catabolic process. This process is believed to flood the system with amino acids and is therefore associated with normal or even elevated plasma amino acid levels. The “pull” mechanism, in contrast, is determined by a dysregulation at the site of the liver. Evidence suggests that an abnormally low cysteine level in the liver may cause an increased production of urea at the expense of the glutamine pool. The “pull” condition is therefore characterized by abnormally low plasma cystine and glutamine levels. The decrease of the plasma cystine level is explained partly by an increased rate of glutathione biosynthesis in the liver and partly by an increased cysteine catabolism in the skeletal muscle tissue. In the major catabolic diseases, including sepsis, HIV infection, and cancer, the “pull” mechanism appears to precede the “push” mechanism. Because the decrease of plasma cystine levels in chronic fatigue syndrome and cancer patients is associated with a relatively mild immunological dysfunction, it seems unlikely that the cystine deficiency accounts for all aspects of the immunopathology of HIV infection. Nevertheless, even a relatively moderate impairment of lymphocyte functions may have disastrous consequences for the progression of the disease, if it tilts the initially stable balance between the immune system and the virus in favor of the virus. Preliminary studies on N-acetylcysteine-treated HIV-infected patients indicate that the decrease of plasma cystine, glutamine, and arginine levels can be corrected by this agent. Anecdotal data also suggest that this strategy may slow or even prevent the progression of the disease. This possibility needs to be confirmed in a larger trial. The dose of N-acetylcysteine, however, may have to be adjusted according to the individual needs as judged by the plasma levels of cystine, glutamine, and acid-soluble thiol (i. e., cysteine). Placebo-controlled, double-blind trials with a constant and arbitrarily chosen dose of NAC may, therefore, not be adequate to evaluate the therapeutic potential of this drug.

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Acknowledgment The assistance of Mrs. I. Fryson in the preparation of this manuscript is gratefully acknowledged.

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