Widner et at. : Neopterin: indicator of oxidative stress and part of the cytotoxic armature

Pteridines Vol. 9,1998, pp. 91 ·· 102

N eopterin: Indicator of Oxidative Stress and Part of the Cytotoxic Armature of Activated Macrophages in Humans

Bernhard Widner, Gabriele Baier-Bitterlich, Irene Wede, Barbara Wirleitner, Helmut Wachter, Dietmar Fuchs§

Institute of Medical Chemistry and Biochemistry, University of Innsbruck, and Ludwig Boltzmann In­ stitute for AlDS-Research Fritz Pregl Strasse 3 A-6020 Innsbruck, Austria

, Received November 30, 1997)

Introduction artherosclerosis, cardiac diseases, or inflammatory bowel disease. Reactive oxygen species are formed by cells in Recent studies provide evidence that pteridine the course of biochemical redox reactions, in­ derivatives are able to interfere with redox systems \'()Iving oxygen as part of the normal metabolism. and hence may influence cell homeostasis and in­ In addition, exogeneous stimuli like UV light or tracellular signal transduction pathways. ionizing radiation lead to the production of free radical species. Furthermore, phagocytes and vari­ Neopterin ous other immunocompetent cells form and release reactive species as part of their cytotoxic re­ Neopterin is produced in large amounts by ac­ pertoire to guarantee self integrity under con­ tivated human monocytes/ macrophages (1 ). The ditions of oxidative stress. Due to overwhelming enzyme GTP-cyclohydrolase I converts the nu­ formation of reactive species, a series of antioxi­ cleotide guanosinetriphosphate (GTP) to form 7 , dative defense mechanisms is established in cells, 8-dihydroneopterintriphosphate (Fig. 1). The latt­ including low molecular mass compounds like as­ er intermediate is transformed by 6-pyruvoyltetra­ corbic acid, tocopherol, or reduced glutathion, de­ hydropterin synthase ( PTPS) and sepiapterin toxyfYing enzymes, e.g., superoxide dismutase, reductase to 5,6,7,8-tetrahydrobiopterin, an essen­ and a set of repairing enzymes, restoring damaged tial co-factor for certain mono-oxygenases (2), e . DNA. For homeostasis, cells have to maintain an g ., phenylalanine- and tyrosine hydroxylase, tryp­ appropriate balance between oxidative and antioxi­ tophan-5-hydroxylase, and for nitric oxide syn­ dative processes. Overproduction of reactive spec­ thase (NOS) (3,4). In various cells, GTP-cyclo­ ies or, respectively, diminution of the antioxida­ hydrolase I activity can be stimulated when ex­ tive repertoire causes o xidative stress which may posed to the cytokine interferon-y (IFN-y). There­ lead to cell death and tissue injury. by in most cells tetrahydrobiopterin accumulates. Large quantities of reactive compounds are pro­ In human monocytes/ macrophages, the constitu­ duced during immune reactions, hence the over­ tive activity of PTPS is low, and as a consequence, production of free radicals and, respectively, ox­ an excess of 7,8-dihydroneopterin and neopterin idative stress are implicated in a variety of quite over biopterin is produced (5). The ratio of 7,8- heterogeneous diseases, e.g. rheumatoid arthritis, dihydroneopterin and neopterin is about 3:1 in ar­ adult respiratory distress syndrome, cystic fibrosis, terial blood and in urine and about 2 : 1 in venous § Author to whom correspondence should be addressed. blood (6,7,8). * Dedicated to the 70th birthday of Prof. Wolfgang Pt1ei · IFN -y is excreted by activated NK cells and by derer, Konstanz, Germany T-helper lymphocytes, when the cellular immune

Pteridines/ Vol. 9 / No. 2 92 Widner et al.: Neopterin: indicator of o'XidclL \ c < ro:''' clIld part of the cytotoxic armature

amounts by mOI1ocnes/ macrophages, when the cellular immune s\stem is activated (9). Along this line, the concentration of neopterin and 7,8- dihydroneopterin in human body t1uids like blood and urine ref1ccts the

7.8-

0 Nr):°H CH,OH Reactive species HN '" :): HN~~~CH3 A I"" OH A)L j 6H H2N N N H,N N N H The term 'reactive species comprises small ox­ neopterin 5.6,7.8-tetrahydrobiopterin ygen-, chlorine-, and nitrogen-containing molec­ Bigure 1. The pathway to the formation of neopterin ules, most of which possessing cytotoxic capacity, derivatives in monocytes/macrophages. Interferon-y ac­ e.g., hydrogen peroxide (H20 2 ), superoxide anion tivates GTP-cyclohydrolase I. In human monocytes/ma­ (0 ), nitric oxide (NO), peroxynitrite (ONOO), crophages, constitutive activities of the subsequent en­ 2 or hypochlorite (OCl) (Table 2, Refs. 14,15). zymes 6-pyruvoyltetrahydropterin synthase and sepi­ apterin reductase are low, leading to overwhelming for­ Reactive species are formed by various cell-types, mation of neopterin. including phagocytes (16), vascular endothelial cells (17,18,19), fibroblasts (20), lymphocytes (21), and macrophages (221, mostly as secondary products of system is stimulated. Together with other cyto­ cellular metabolism, and they may be regarded as a kines, e.g., tumor necrosis factor alpha (TNF-a), consequence from the utilization of molecular ox­ or interleukin 2 (IL-2), IFN -y acts synergistically, ygen by aerobic organisms. However, more im­ enhancing cellular immune response. As a conse­ portantly, large quantities of reactive molecules, in quence, neopterin derivatives are excreted in large particular superoxide anion and nitric oxide, are

Table 1. Neopterin concentrations in diseases linked with oxidative stress Increased neopterin production allograft rejection autoimmune diseases e.g. rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel diseases, multiple sclerosis malignant diseases e .g. gynaecological carcinoma, ovarian carcinoma, haematological neoplasias, urogenital tract cancer, cancer of the prostata, hepatocellular carcinoma, lung cancer, gastointestinal cancer, hepatic cancer infectious diseases e.g. viruses (hepatitis,HIV-infection), intracellular protozoa (malaria, schistosomiasis mansoni), intracellular bacteria (pulmonary tubercolosis, leprosy, melioidosis) cardiac disorders myocardial infarction, congestive heart failure, acute rheumatic fever neurodegenerative disorders e.g. Alzheimer's disease

Normal neopterin production

Duchenne's muscular dystrophy motor neuron disease

Pteridines/ Vol. 9 / No. 2 Widner et at.: Neopterin: indicator of oxidative stress and part of the cytotoxic armature 93

Table 2. Reactive oxygen and nitrogen species as part of cytotoxic armature and antioxidative defense Radicals Non-radicals

HydroA),1 on Hydrogen peroxide H 20 2 Alkoxyl RO Hypochlorous acid HOCI

Hydroperoxyl H02 Ozone 0, Peroxyl R02 Singlet oxygen l~g O 2

Superoxide anion O 2 Peroxynitrite ONOO' Nitric oxide NO Nitrosyl NO'

Nitrogen dioxide N02 Nitroxide NO + NitroniuJ11 N02

Nitrous acid HN02 Nitrogen oxides N,Ov released from activated immunocompetent cells as is generated by consecutive reactions of the above­ part of their cytotoxic armature. For example, upon mentioned species, including nearly all oxidation IFN-y stimulation, macrophages are primed to pro­ states of oxygen and nitrogen (41). Due to the duce reactive oxygen species (ROS), obviously fast reaction rates, it is difficult to examine the \\'ithin the scope of a cellular immune response chemical nature of all these reactive species which · 23). Superoxide anion is generated in vivo, when are involved in cytotoxic processes. fundamental molecules in metabolism, including ca­ Peroxynitrite is formed in a very fast reaction techolamines, tetrahydrofOlates, reduced Havines, or from nitric oxide and superoxide anion (42). Its .xanthine, are directly aflected by oxygen. Mono-ox­ half life at physiological conditions is 1 s. When \'genases are involved in these, partly auto-oxidative peroxynitrite is protonated, a series of extremly processes, as released superoxide anion is applied reactive species is formed intermediately (43,44), for further oxidation. Interestingly, the formation and aromatic structures (45,46), thiol groups (47), of superoxide also seems to be connected with desoxyribose (48), and lipids (49) are rapidly af­ 'leakages' in mitochondrial electron transport chains fected. At physiological pH of 7.4, the nitrating 24). A series of authors described toxic eflects of potential of peroxynitrite has been found to be superoxide anion on biologic systems (25-30). maximum; the reactive intermediate involved is Nitric oxide is enzymatically formed by many described as nitronium-like ion, NO/ (50). Ac­ ..:ell types. Nitric oxide synthase utilizes molecular tually, 3-nitrotyrosine, the nitration product of oxygen to oxidize the side chain of arginine, form­ tyrosine, has been found in diseases, when the im­ i ng citrullin and nitric oxide (31). Nitric oxide rad­ mune system is activated [e.g., in rheumatoid ical plays an important physiological role in signal arthritis (51), severe lung injuries (52), or coro­ transduction, controlling blood pressure by vaso­ nary heart diseases (53)], indicating excessive per­ dilation (32,33), and influencing neurotransmiss­ oxynitrite activity in vivo. ion (34,35). Hypochlorite is formed in neutrophil granulo­ Antioxidative defense ..:\'tes from hydrogen peroxide and chloride by the ~nzyme myeloperoxidase (36-38). In order to protect healthy tissue from ox­ Reactive species formed by non-enzymatic reac­ idative assault, cells have developed a series of tions seem to have more cytotoxic character than mechanisms to encounter formation of cytotoxic superoxide anion or nitric oxide. The short lived molecules, as ROS are continuously produced. In

hydroxyl radical is formed by fission of H 2 0 und­ the first line of antioxidative defense, there are er influence of ionizing radiation (39). Also the low mass molecules, scavenging free radicals and oxidative cleavage of hydrogen peroxide by iron highly oxidative species. These radical trapping (II) leads to the formation of hydroxyl radical and molecules comprise, e.g., a-tocopherol, ascorbic hydroxid anion according to Fenton IS reaction acid, p-caroten, uric acid, bilirubin, and reduced ( 40,41): glutathion. Other mechanisms prevent the for­ mation of highly reactive species, for example, su­ peroxide dismutase (SOD) catalyzes the reaction Hydroxyl radical has a highly oxydizing poten­ of superoxide to hydrogen peroxide, which, on tial, and it damages nearly all targets in the sur­ the one hand, is a stable molecule and can even rounding of the generation site (39). A whites­ pass through cell membranes (14). On the other pread series of other radicals and reactive species hand, as mentioned, hydrogen peroxide is able to

Pteridines/ Vol. 9 / No. 2 Widner et at.: ~ e() rt e rin: indicator of oxidative stress and pan of the cytotoxic armature generate other reactive species, e.g. by myelo­ monstrated related to physiological environments peroxidase or by Fenton's reaction. Thus, with and also in cell cultures examining interactions of the help of detoxifYing enzymes, reactivity even neopterin derivatives with intracellular signal-trans­ can be transported from the site of generation to duction pathways. sites elsewhere, where cytotoxic potential is re­ quired. Neopterin derivatives catalyze chemical reactions Several mechanisms lower the concentration of hydrogen peroxide: catalase, for example, gen­ Neopterin was revealed to interfere with ROS­ erates a redox disproportion of hydrogen peroxide, mediated processes when luminol chemilumines­ leading to O 2 and H 20. Also glutathion perox­ cence was examined: coincubation of hydrogen per­ idase encounters toxicity by converting hydrogen oxide with neopterin diminishes chemiluminescence peroxide and lipid peroxides to harmless molec­ (65). In contrast, at neutral or slightly alkaline pH ules. In particular, lipid perioxides are able to in­ and in the presence of iron chelator complexes, itialize radical chain reactions, leading to mem­ neopterin significantly enhances chemiluminescence bran disrupture and cell death (54,55). in a concentration dependent way, whereas 7,8- Another way to control the deleterious effects dihydroneopterin acts as scavanger, extinguishing of ROS is to withdraw radical forming ions from light emission independent from environmental con­ the sites of ROS production. For example, metal ditions (66-68). It has to be pointed out that tlle binding proteins like ferritin or ceruloplasmin lim­ addition of iron to h ydrogen peroxide per se di­ 2 it the availability of unbound Fe + and hence may minishes luminol reactio n compared to the iron­ help to prevent the formation of hydroxyl radical free situation. This diminution can be overcomed (56-58). The back bone of antioxidative defense when neopterin is added. Since iron catalyzes the finally is supplied by repairing enzymes, restoring rapid cleavage of hydrogen peroxide to form hy­ cell structures, mainly DNA, which has been da­ droxyl radical (see above), the data may suggest maged by excessive oxidative action (59). Along that other reactive species formed from hydrogen this line, dietary uptake of antioxidants (vitamin E, peroxide, e.g., superoxide anion are stabilized by ascorbate) may play an important role for pro­ neopterin. Neopterin is also an enhancer and 7,8- tection from oxidative stress (60) and may there­ dihydroneopterin a scavanger of chloramin-T and b~ ' also reduce the susceptibility for certain human hypochlorite-induced chemiluminescence (66,69,70), diseases, e.g. cardiovascular disease (61,62). De­ and these effects are independent from iron a- fective absorption of vitamin E has also been found vailability. . to be associated with neurodegeneration (63). Neopterin is unaffected in these experiments, whereas 7,8-dihydroneopterin is oxidized to xan­ Effects of neopterin and 7,8-dihydroneopterin on thopterin. These findings suggest a catalytic mec­ redox-mediated processes hanism of neopterin to enhance oxidative pro­ cesses, in contrast to 7,8-dihydroneopterin, the an­ Hitherto the physiologic role of neopterin pro­ tioxidant capacity of which is concomitant \vith duction by monocytes/ macrophages has rema­ stoichiometrical consumption ( 68). Interestingly, ined unclear. In contrast to 5,6,7,8- tetrahydro­ with hydrogen peroxide a high increase of the 7,8- biopterin, no specific binding sites of neopterin dihydroneopterin concentration reverses the an­ or, respectively, 7,8-dihydroneopterin on enzymes tioxidative potential: concentrations > 0.3 mmol/ could have been found. Yet, evidences have ac­ L of 7,8-dihydroneopterin enhance chemilumines­ cumulated that neopterin and its reduced form cence, and this is true in the absence of iron che­ have impact on redox-dependent processes. The lators (71,68). Accordingly, there is data in litera­ amount of neopterin released from activated ma­ ture, describing the formation of free radicals, crophages was found to correlate well with the when O 2 reacts with reduced pteridines (72). In capacity of these cells to produce reactive species this connection, superoxide formed by large amo­ within the scope of the respiratory burst (64). Sev­ unts of 7,8 -dihydroneopterin may preferentiall y eral recent examinations proved the potential role contribute to toxicity. of neopterin derivatives to impair the sensitive bal­ Also an enhancing effect of neopterin on NO­ ance 10 cells benveen oxidative force and antio­ mediated oxidative mechanisms was demonstrated xidative defense and to modulate, at first glance, by measurement of chemiluminescence, when lu­ diverse physiological pathways. The impact of minol was incubated with nitrite plus hydrogen ncopterin derivatives on redox systems was de- p eroxide. Again, 7,8-dihydroneopterin effectively

Pteridines/ Vol. 9/ No. 2 Widner et al.: Neopterin: indicator of oxidative stress and part of the cytotoxic armature 95 scavanges this reaction. ion. The nitrating assault of peroxynitrite was ex­ In parallel to the chemiluminescence experi­ amined by nitration of L-tyrosine. Neopterin turn­ ments discussed, neopterin also enhances tOXIClt)' ed out to increase the nitration rate of tyrosine of chloramine-Ton Escherichia coli cultures (66). significantly in a slightly acidic environment, when The number of colonies of E. coli surviving after the conditions of the nitrating reaction are su­ 30 min incubation with chloramine-T and neopte­ boptimum (77). This circumstance may become rin is drastically reduced when neopterin is added. even more relevant by the fact that the pH de­ In contrast, 7,8 -dihydroneopterin diminishes tox­ clines in inflamed tissues. A catalytic interaction of icity by scavanging the effects of reactive in­ neopterin on the nitration of tyrosine by perox­ termediates. Accordingly, the number of surviving ynitrite can be assumed. In contrast, 7,8-dihy­ colonies was significantly higher than in the con­ droneopterin lowers the rate of t)'rosine nitration. trol preparation. Obviously, the radical-scavenging attributes of the There are also effects of pteridines on specific reduced pteridines also affect nitrating agents, and enzyme reactions: xanthine oxidase (XOD) is an this may demonstrate the broad range of reactive iron and molybdopterin containing flavoprotein compounds which can be modified by neopterin \\'hich catalyzes the oxidation of hypoxanthine to derivatives. :\anthine and of xanthine to urate. Reduced pter­ All these data show that neopterin derivatives idines (e.g., dihydroneopterin, tetrahydrobiopterin) have potential impact on redox-controlled pro­ .lS well as oxidized forms, neopterin and biopterin, cesses. However, it remains unclear so far, by significantly decrease XOD activity (73,74). Re­ which extent neopterin derivatives may influence duced pteridines are supposed to inhibit XOD redox-sensitive reactions in. vivo, because of the ~ompetitively by blocking the hypoxanthine bind­ complex interactions during immune response, im­ ing site, whereas the effect of neopterin may be plicating cytokines, ROS, and antioxidative de­ distinct from the reduced derivative. It is possible fense mechanisms. It has to be kept in mind that that neopterin interacts with superoxide anion \\'hich is accumulating during the XOD reaction. It Activated cell-mediated immune (Th 1) response has been described that superoxide inhibits the en­ Cytokines z,'me by a kind of a negative feedback regulatory mechanism (75), and hence neopterin could enha­ ~'FN-" TNF~ n~e the effect of superoxide anion on XOD. Further evidence for the influence of neopterin derivatives on the toxicity of peroxynitrite stems e.g from studies of mitochondrial respiratory enzymes endothelial cells, isolated from rat brains (76). Both, neopterin and granulocytes 7,8-dihydroneopterin, decrease the activit)T of en­ z"me complex II/ III (succinat cytochome c re­ ductase) of the mitochondrial respiratory chain, (:ontaining succinat dehydrogenase and catalyzing electron transfer to coenzyme Qj. The activities ~I o f complex I (NADH CoQ] reductase) and IV Reactive Species 0 ,-. ONOO', H20 " OCI' etc , cytochrome c oxidase) remained unaltered. Treat­ Neopterin + ment of the respiratory chain enzymes with perox­ 7,8-Dihydroneopterin -=------"nitrite diminished the activity of complex I, II/ low concentrations ---",.- III, and of citrate synthase, but not of complex + IV. The toxic effect of peroxynitrite on these en­ 7 ,8-Dihydroneopterin high c(lncentrations zyme complexes can be increased by addition of neopterin, whereas 7,8-dihydroneopterin has de­ Toxicity toxifYing effects on peroxynitrite-mediated en­ Figure 2. Neopterin and 7 ,8-dihydroneopterin influence zyme inhibition. cytotoxicity mediated hy reactive species. During cellular Recent chemical studies confirm that neopterin imnlune response, among other cytokines interferon-y (IFN-y) is released which triggers production of neopte­ derivatives interact with reactive nitrogen species, rin derivatives in human ll1onocytes/ macrophagcs. At in particular, with peroxynitrite. Peroxynitrite is a the saIne time, variolls reactive species are fornled by toxic product from nitric oxide which is formed in. these cells and hy various immunocompetent cells in the vivo, when nitric oxide heads with superoxide an- microenvironment.

Pteridines/ Vol. 9 / No. 2 96 Widner et at.: Neopterin: indicator of oxidative stress and part of the cytotoxic armature neopterin and 7,8-dihydroneopterin may increase 84), decompartmentalization of catalytic metal or decrease ROS-mediated effects during immune Ions (85), damage of membrane ion transporters, response since both compounds are always releas­ e.g., K + (86 ), and, in general, inactivation of es­ ed in parallel (Fig. 2 ). The environmental con­ sential enzymes (25,87). In the course of an ex­ ditions may decide whether the enhancing or tended immune response, formation and ex­ scavening impact of neopterin derivatives will be­ cretion of ROS increase during the oxidative come more important. burst (88,89). The incidence of oxidative burst in The finding that neopterin derivatives have po­ the course of inflammation and immune ac­ tential to interfere with redox -sensitive reactions tivation is closely correlated with the expression of and may be part of the defensive armature of the TNF-a. (90-92), a cytokine preferentially released activated macrophage, raises the question, why fi-om activated monocytic cells. Also the excretion human/ primate macrophages may have develo­ of neopterin correlates with the occurence of ox­ ped this additional strategy. As human mono­ idative burst, suggesting neopterin as indirect mar­ cytes/ macrophages in contrast to non-human ker for ROS production in humans. Oxidative cells are hardly to be stimulated by IFN-y to pro­ burst leads to a strong disturbance of the redox duce nitric oxide, neopterin may be directed to balance. compensate the relative deficiency of human ma­ Various intracellular signal transduction path­ crophages to produce physiologically necessary ni­ ways have been demonstrated to be influenced by tric oxide (78,79 ). redox imbalances (Table 3). Reactive species in­ fluence the expression of the nuclear factor (NF)­ Oxidative stress and intracellular signal transduc­ KB (93) and cause accumulation of p53 (94), a tion pathways signaling protein which is associated with pro­ grammed cell death, the so-called apoptosis. In Cellular survival depends on a delicate balance contrast to necrosis, apoptosis is an active biolog­ between oxidative forces and antioxidative defense ical process, following a distinct genetically det­ mechanisms (80). Overwhelming production of ermined program. Apoptosis takes part to balance reactive species and/or depletion of antioxidants growth and death rates in a cell population and is result in oxidative stress. Whereas mild oxidative characterized by internucleosomal cleavage of stress can be compensated by diverse antioxidants, DNA strands, cell shrinkage with a distinct mor­ restoring the redox balance, overwhelming and phologic form, cell membran disrupture, and chro­ chronic formation of reactive species may lead to matin condensation (95). Oxidative stress is a po­ irreversible injuries, including DNA strand break­ tent trigger of signal transduction pathway for age, base and sugar modifications (81), lipid apoptosis (96-98); hence an equlilibrium of redox­ 2 peroxidation (19,82), rise of intracellular Ca + (83, active agents seems to be necessary for maintai-

Table 3. Intracellular events sensitive to oxidative stress and neopterin influence influe nced by neopterin and 7,8- influenced by oxidative stress dihydroneopterin 2 Ca • -signalling pathway + ( 116) + (117) stimulation of protein kinases not done + (118, 119) stimulation of phospholipase A z not done + ( 118, 120) HTLV transactivation + (121) + (121) HIV promotor + (122) + (121, 124, 135) IFN-y + ( 113) not done apoptosis + ( 71) + ( 123) erythropoietin + (109) + ( 136, 137) stimulation of transcription factors: TNF-a, NF-KB + ( ll5) + (90, 124, 125) c-fos, c-jun proto-oncogene + (lll ) + (126-128) interleukin 113 not done + ( 124, 129) transforming growth factor-131 not done + (130) platelet derived growth factor not done + ( 131, 132) insulin production not done + (133) angiotensin II not done + ( 132, 134) vitamin D3 not done + ( 129) parathyroid hormone not done + ( 129)

Pteridines/Vol. 9/No. 2 Widner et at.: Neopterin: indicator of oxidative stress and part of the cytotoxic armature 97 nance of cell homeostasis. In particular, low doses to induce apoptosis was similar to that of the cy­ of hydrogen peroxide have been found to induce tokines IFN-y and TNF-a, moreover co-incuba­ apoptosis (71,99,100). Regulatory poteins (e.g., tion of the cytokines TNF-a and IFN-y with protein kinase C) (101,102) and/or intracellular neopterin further enhances the rate of apoptosis Ca2+ -levels (103,104) controlled by the redox bal­ (100). Furthermore, it has been found that ance may crucially trigger apoptosis. In contrast, neopterin activates TNF-a, which per se induces antioxidant defense mechanisms, e.g. SOD, ca­ immune response, and oxidative burst (112). In­ talase or certain cellular genes, like Bel -2 (105, terference of neopterin derivatives in signal trans­ 106) reduce the rate of apoptosis. duction pathways may be assumed. Considering An augmentation of NF-K13 production in peri­ that ROS are apoptosis-inducing agents and, ad­ pheral blood mononuclear cells and macrophages ditionally, neopterin derivatives are known to in­ has been described under influence of oxidative terfere with reactive oxygen intermediates, the stress (93,107). Moreover, activation of NF-K13 conclusion seems conceivable that neopterin and may itself contribute to enhance oxidative stress 7,8-dihydroneopterin exert impact on the cell because cytokines like TNF-a, and also inducible homeostasis via reactive species, which may be nitric oxide synthase will be upregulated, e.g., the produced in, e.g., alveolar cells under subop­ promotor of the latter enzyme contains two pu­ timum conditions in cell culture (7S). The in­ tative binding sites for NF-l(B (lOS). In com­ fluence of neopterin derivatives on apoptosis sup­ bination with IFN-y as prosthetic group for iNOS, ports the hypothesis of a complex control mechan­ ~F-KB seems to crucially contribute to NO-medi­ ism of proliferation and cell death in the course of ated cell toxicity in the case of cellular immune ac­ immune activation (SO). Thus, neopterin may be tivation. As nitric oxide may enfold reasonable ox­ regarded as link in a complex signaling network idative potential via peroxynitrite, NF-lCB may be within the scope of immune response (113). a mediator of an enhancing signal transduction Neopterin is also an activator of inducible nitric cascade, which in sum aims to immune activated oxide synthase (iNOS) in rat vascular smooth mus­ states and, respectively, promotes apoptotic path­ cle cells (114), whereas 7,8 -dihydroneopterin in­ ways. hibits the induction of iNOS gene expression. Nso an activation of the nuclear factor NF-K13 Effects of neopterin derivatives on intracellular sig­ with neopterin stimulation has been described (12, nal transduction pathways 115) which may explain the effect of neopterin to induce iNOS, because iNOS gene expression is Several redox-sensitive intracellular signal trans­ presumably linked with NF-l(B (see above). No­ duction pathways can be influenced by neopterin teworthy, the impact of neopterin on NF-l(B in­ derivatives. Neopterin inhibits the gene expression duction can be blocked by the reductant pyr­ for the hormon erythropoietin (109). This finding rolidine dithiocarbamate. Thus, the effect of neo­ corresponds perfectly with correlations found ear­ pterin is most likely due to enhancement of ox­ lier between increased neopterin and decreased idative stress. The induction of NF-l(B is ad­ hemoglobin levels in various chronic disorders like ditionally of great interest, since expression of vari- infections and cancer (110). N eopterin and 7 ,S­ 0us cytokines and virus transactivation of, e .g. dihydroneopterin induce the proto-oncogene c-fos HIV, is regulated via this nuclear factor. Similarly, ( 111), a well known promotor of tumor develo­ 7,8-dihydroneopterin was found to activate the pment and growth. This observation sheds some HIV promotor (12). new light on the earlier findings that increased In conclusion, neopterin can be regarded as a neopterin in patients with cancer is associated sensitive marker for oxidative stress in the human with more rapid disease progression and earlier organism. The present data also provides evidence death (13). Enhanced oxidative stress may sup­ that neopterin derivatives may interfere with port malignant transformation and growth. redox-sensitive systems and hence may modulate In a monocytic cell line, U937, lower concen­ the balance of pro-oxidative assault and antioxida­ trations « 300 /lM) of 7,S-dihydroneopterin de­ tive defense. Other pteridines may have similar ef­ creases TNF-a-mediated apoptosis, but 5 mM of fects, but as neopterin and 7,8-dihydroneopterin the reduced pteridine superinduces TNF-a-medi­ are produced in high concentrations during cell ated apoptosis (71). In addition, neopterin and 7, mediated immune response in humans, it appears 8-dihydroneopterin were shown to induce apop­ conceivable that neopterin derivatives are part of a tosis in rat alveolar cells. The effect of neopterin controlling mechanism provided by immuno-

Pteridines/ Vol. 9 / No. 2 98 Widner et at.: Neopterin: indicator of oxidative stress and part of the cytotoxic armature competent cells to affect the cytotoxic potential of terferon-gamma, 6 -pyruvoyl tetrahydropterin syn­ ROS and certain reactive nitrogene species, e.g. thase and sepiapterin reductase are constitutively peroxynitrite. At the site of inflammation, high present. J BioI. Chcm. 1990; 265: 3180-3192. 6. Fuchs, D., Milstien, S., Kramer, A., Reibnegger, G., amounts of neopterin plus dihydroneopterin are Werner, E .R., Goedert, J.J., Kaufman, S ., Wachter, released in parallel to short-lived reactive species. H . Urinary neopterin concentrations vs total neopte­ Consequently, neopterin derivatives may ef­ rins for clinical utility. Clin. Chem. 1989; 35: 2305- fectively modulate the function of cell-damaging 2307. ROS. This assumption carries weight, regarding 7. Howells, D.W., Smith, 1., Hyland, K. Estimation of that neopterin derivatives are exclusively produced tetrahydrobiopterin and other pterins in cerebros­ by human and primate macrophages/monocytes pinal fluid using reversed-phase high-performance li­ upon stimulation with IFN -y which, on the other quid chromatography with electrochemical and flu­ orescence detection. J. Chromatogr. 1986; 381: 285- hand, is obviously not able to stimulate suffic­ 294. iently iNOS in human macrophages. Neopterin is 8. Weiss, G., Glaser, K., Kronberger, P., Ambach, E., capable of modifYing the cytotoxic effects of con­ Fuchs, D., Bodner, E., Wachter, H . Distinct dis­ stitutively produced NO, and hence the cytotoxic tributions of D-erythro-neopterin in arteries and function of NO in humans is apparantly upre­ veins and its recovery by all. enterohepatic circulation. gulated via neopterin in contrast to other ver­ BioI. Chem. Hoppe Seyler 1992; 373: 289-294. tebrates where the quantity of NO, mediated by 9. Fuchs, D ., Hausen, A., Reibnegger, G., Werner, E .R ., Dierich, M.P., Wachter, H. Neopterin as a marker IFN-y stimulation, is crucial for its toxic effect. for activated cell-mediated immunity: Application in This conclusion is further supported by the close HIV infection. Immunol. Today 1988,9, 150-155. relationship between increased neopterin con­ 10. Fuchs, D., Weiss, G., Reibnegger, G., Wachter, H. centrations and the activity of diseases in which The role of neopterin as a monitor of cellular im­ oxidative stress is considered to play an important mune activation in transplantation, inflammatory, in­ pathogenetic role like autoimmune disorders, neu­ fectious and malignant diseases. Crit. Rev. Clin. Lab. rode generative diseases and malignancies. Sci. 1992; 29: 307-341. 11. Fuchs, D., Hausen, A., Reibnegger, G., Reissigl H ., Schonitzer, D . Spira, T .J., Wachter, H. Urinary neo­ Acknowledgement pterin in the diagnosis of aquired immune deficiency syndrome. Eur. J. Clin. Microbiol. 1984; 3: 70-7l. This work was financially supported by the 12. Baier-Bitterlich, G., Wachter, H., Fuchs, D. Role of 'Austrian Ministry of Science and Research, Sek­ neopterin and 7,8-dihydroneopterin in human im­ tion Forschung' . munodeficiency virus infection: marker for disease progression and pathogenic link. J. Aquir. Immune References Defic. Syndr. 1996; 13: 184-93. 13. Reibnegger, G., Fuchs, D., Fuith, L.C., Hausen, A., 1. Huber, C., Batchelor, J.R., Fuchs, D., Hausen, A ., Werner, E .R ., Werner-Felmayer G., Wachter H. Lang, A., Niederwieser, D., Reibnegger, G., Swetly, Neopterin as a marker for activated cell mediated im­ P., Troppmair, J., Wachter, H. Immune response-as­ munity: application in malignant disease. Cancer De­ sociated production of neopterin. Release from ma­ tect. Prevent. 1991; 15: 483-490. crophages primarily under control of interferon gam­ 14. Halliwell, B. Free radicals and antioxidants: a per­ ma. J. Exp. Med. 1984; 160: 310-316. sonal view. 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