Possible mechanism of nitric oxide production from NG-hydroxy-l -arginine or hydroxylamine by superoxide ion

hr. J. Biochem. Cdl Bid. Vol. 28, No. 12, pp. 1311-1318, 1996

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Possible Mechanism of Nitric Oxide Production from NG-hydroxy+arginine or Hydroxylamine by Superoxide Ion PETR VETROVSKY,’ JEAN-CLAUDE STOCLET,’ GUSTAV ENTLICHER’” ‘Department of Biochemistry, Faculty of Sciences, Charles University, Albertov 2030, 12840 Praha 2, Czech Republic and ‘Laboratoire de Pharmacologic et de Physiopathologie Cellulaires, CNRS URA 0600, Universitt Louis Pasteur de Strasbourg, Strasbourg, France It has been speculated that NC-hydroxy+-argluine(OH-L-Arg), which is an intermediate in NO production from L-argluine,may be converted to NO by superoxide ion. However, there is still no direct evidence for this conversion. In the present study this was investigated using superoxide ion generated either in acellular or cellular systems. It was found that OH-L-Arg and hydroxylamine were converted to nitrite and nitrate apparently via NO by superoxide ion in aqueous solution. Arginine remained unaffected. These changes were observed during reaction of chemical substances as well as in a biological system (zymosan-activated macrophages in culture). Superoxide dismutase prevented this transformation. OH-L-Arg was also spontaneously hydrolysed to hydroxylamlne and L-citrulllne,however this occurred at pH > 9 only. Activated microsomes (containing different isoforms of cytochrome P450) were unable to replace NO-synthase in its ability to produce OH-L-Arg from L-arginine.These data support the hypothesis that a pathway alternative to the well-known synthesis of NO by NO-synthase via OH-L-Arg exists. This pathway may involve the production of OH-L-Arg by NO-syuthaseand decomposition of OH-L-Arg to NO by the action of superoxide ion. Alternatively, hydrolysis of OH-L-Arg to hydroxylamine may occur followed by its oxidation to NO, again by superoxide ion. Copyright 0 1996 Elsevier Science Ltd Keywords: NC-hydroxy+argiuine Superoxlde ion Hydroxylamine Nitric oxide Macrophages Znt. J. Biochem. Cell Biol. (1996) 28, 1311-1318

INTRODUCTION

Nitric oxide (NO), which has been found to be a very important molecule in mammalian cardiovascular, nervous and immune systems (Moncada et al., 1991), is generated from the *To whom all correspondence should be addressed. Abbreviations: OH-L-Arg, No-hydroxy-L-arginine; NOS, NO-synthase; Hb, haemoglobin; metHb, methaemoglobin; CO-Hb, carbonyl haemoglobin; SOD, superoxide dismutase; L-NAME, NC-nitroL-arginine methyl ester; L - L y s , L-lysine; D P I , diphenyliodonium chloride; NOx, nitrite and nitrate; cGMP, 3’,5’-cyclic guanosine monophosphate. Received 5 January 1996; accepted 31 July 1996. 1311

amino acid L-arginine by NADPH-dependent flavoprotein enzymes called NO-synthases (NOS). NG-hydroxy-L-arginine (OH-L-Arg) is the stable intermediate in this reaction (Stuehr et al., 1991a). The hypothesis has been proposed that OH-L-Arg can be transformed to NO and L-citrulline independently of NO-synthase, e.g. by cytochromes P450 or some of their products (Boucher et al., 1992; Renaud et al., 1993; Schott et al., 1994). Superoxide ion produced by cytochrome P450 or other oxidases could be involved in this transformation (Mansuy et al., 1995). Furthermore, DeMaster et al. (1989) devised a theory (which has not been either verified or rejected) that OH-L-Arg could be

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Petr Vetrovsky ef ul

hydrolysed yielding L-citrulline and hydroxylamine, with the following oxidation of the latter one to NO. Indeed, the oxidation of hydroxylamine to NO by catalase has already been described (Nicholls, 1964; Craven et al., 1979). In the present work we studied OH-L-Arg stability in aqueous solution under different pHs and the effect of superoxide ion produced either chemically or biologically on L-arginine, OH-LArg and hydroxylamine. Finally, because Clement et al. (1993) found cytochrome P450-dependent N-hydroxylation of a guanidine we also investigated the possibility of cytochrome P450-dependent hydroxylation of the guanidine moiety of L-arginine yielding OH-L-Arg in an endeavour to find an alternative way for the first step of the reaction leading to NO generation by NO-synthase. MATERIALS AND METHODS

Materials

NG-hydroxy-L-arginine was purchased from Tocris Cookson Ltd (Bristol, U.K.), superoxide dismutase (EC 1.15.1 .l) from bovine erythrocytes from Sigma (St Louis, MO, U.S.A.), LPS (E. coli 055:B5, LD,, 32.76 mg/kg) from Difco L-lysine from U.S.A.), (Detroit, MI, Calbiochem (San Diego, CA, U.S.A.), diphenyliodonium chloride from Aldrich (Steinheim, Germany) and methaemoglobin from Boehringer (Mannheim, Germany). Labelled succinyl-cyclic GMP tyrosylmethylester and anti-cyclic GMP specific antibodies were supplied by Dr B. Lutz-Bucher (Laboratoire de Physiologie, Universitt Louis Pasteur de Strasbourg, France). All the other chemicals were obtained from Sigma (St Louis, MO, U.S.A.) or Lachema (Brno, Czech Republic) and were of the highest purity commercially available. Potassium superoxide as a source oj’superoxide ion (Forman and Fridowich, 1973)

Potassium superoxide was dissolved in waterfree dimethyl sulphoxide (DMSO) in a convenient concentration. The reaction was started simply by adding 0.1 ml of the above described potassium superoxide solution to 0.9 ml of an aqueous solution of tested compounds (being in various combinations, as indicated, and buffered by 0.1 M phosphate, pH 7.4) and mixed. Five minutes later nitrite and nitrate were measured.

Preparation of carbonyl haemoglobin (CO-Hb)

Methaemoglobin (2 ml of 200 PM, metHb) was reduced with sodium dithionite giving haemoglobin (Hb). It was then bubbled for 60 set with carbon monoxide (CO) produced by the reaction between potassium ferrocyanide and concentrated sulphuric acid. The presence of CO-H\, was verified spectrophotometrically (maximal absorbance A,,, for metHb = 405.5 nm; Hb = 429 nm; COHb = 408 nm). Cell culture

The mouse monocyte/macrophage cell line RAW 264 (ECACC catalogue No. 85062803) was maintained in continuous culture in 80 cm* tissue culture flasks (Nunclon) in DMEM (Gibco ref. 041-01885)/RPMI 1640 medium (Gibco ref. 041-01875) l/l supplemented with penicillin (100 units/ml), streptomycin (0.1 mg/ ml), 10% foetal calf serum (FCS) and placed at 37°C in a humidified incubator, gassed with 95% sir/5% CO,. Cells were harvested by gentle scraping and passaged every 3-6 days by a dilution of the cell suspension 1:6 with fresh medium. Incubation of cells

Cells were plated at a density of lo6 cells per well in 24-well clusters (Costar) and allowed to adhere for 2 hr. The medium was then replaced by fresh Eagle’s minimum essential medium without L-arginine (MEM, Gibco) supplemented with 0.5% FCS. Cells were then incubated for 2 hr in the absence or presence of OH-L-Arg, L-Arg or hydroxylamine, with or without L-NAME, L-Lys, DPI, SOD or zymosan A alone or in various combinations, as indicated. At the end of the incubation period, nitrite and nitrate concentrations and cGMP content were determined. Nitrite and nitrate assay

Nitrite concentrations were determined with Griess reagent (Green et al., 1982): 1% sulphanilamide, 0.1% naphtylethylendiamine, 2.5% orthophosphoric acid. Griess reagent (0.1 ml) was mixed with an equal volume of medium at room temperature. Absorbance was measured at 540 nm 10 min later against blank in MEM or in water using sodium nitrite (dissolved in MEM or in water) as standard. Nitrate concentrations were determined using the method described by Gilliam et al. (1993).

Possible mechanism of nitric oxide production

This method is based on the measurement of changes in absorbance at 340 nm during reduction of nitrate by NADPH-dependent enzyme nitrate reductase from Aspergillus.

Measurement of cGMP content

The incubation medium was replaced by 500 ~1 cold hydrochloric acid (0.1 M) and the cells were scraped and harvested. The cell suspension was sonicated (UltrasonAnnemasse, type 75 TS, France) for 1.5 set and centrifuged at 10 000 g for 5 min. The cyclic GMP content was determined in supernatant using a radioimmunoassay as described previously (Cailla et al., 1973), modified by separation of free cyclic GMP with activated carbon (Koch and Lutz-Bucher, 1991). DNA was determined in the pellet according to Setaro and Morley (1976) and the cyclic GMP content was calculated as fmol/pg DNA.

Preparation of rat liver microsomes

Microsomes were prepared from the liver of premeditated male Wistar rats (ethanol 10% in drinking water for 10 days; phenobarbital 0.1% in drinking water for 7 days; dexamethasone 50 mg/kg i.p. in corn oil, once daily for 4 days). The differential sedimentation method of Coon et al. (1978) was used. Characterization of microsomes (cytochrome P450, cytochrome b5 and protein content) and determination of induced isoforms (SDS electrophoresis) was performed as described previously (Estabrook and Werringloer, 1978; Omura and Sato, 1964; Lowry et al., 1951; Laemli, 1970).

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Incubation procedure with microsomes

The reactions were performed at 37”C, pH 7.5. The incubation mixture contained (in total volume 1.0 ml) 10 mM L-arginine, 2 mM NADPH and 3 mg of microsomal protein. After 60 min of incubation, the reaction was stopped by cooling down to 0°C. Determination of amino acids

The amino acids present in the reaction mixtures described above were determined using Automatic Amino Acid Analyser T 339M (Mikrotechna-Praha, Czech Republic). RESULTS

Stability of OH-L-Arg in aqueous solution at different pHs

Spontaneous hydrolysis of OH-L-Arg towards L-citrulline and hydroxylamine was studied at room temperature in borate buffer at different pHs. OH-L-Arg was quite stable in a pH range from 1 to 8. Further increase of pH, however, led to small but significant hydrolysis within 24 hr at pH 9, complete hydrolysis within 24 hr at pH 10 and finally complete hydrolysis within 1 hr at pH 11. The e#ect of superoxide ion on the transformation of hydroxylamine, OH-L-Arg and Larginine to nitrite and nitrate

Superoxide ion (.O; ), generated chemically by the reaction of potassium superoxide with water, attacked both hydroxylamine and OH- LArg (both O-200 PM) with the production of NOx (Fig. 1). By contrast, neither nitrite nor nitrate was found when L-arginine (up to 1 mM) was exposed to superoxide ion under the same (b)

(a)

5: ,3 15 G 2 7

2 10 8 c 8 K

5

E 50

100

1.50

OH-L-Arg concentration

200

(FM)

I

250

0

50

100

150

Hydroxylamine concentration

200

250

(PM)

Fig. 1. The effect of superoxide [(O) 0.1 mM; (0) 0.2 mM] and the effect of SOD in the presence of 0.1 mM superoxide [(A) 100 U/ml] on the decomposition of (A) OH-L-Arg and (B) hydroxylamine to NOx. Symbols are means f SEM (vertical bars), n = 5.

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Petr Vetrovsky et al.

conditions (not shown). Amino acid analysis confirmed that L-arginine remained unchanged during the reaction time. In the case of OH-L-Arg, L-citrulline was the second product of the reaction, as shown by amino acid analysis. Under all conditions studied, OH-L-Arg completely disappeared from the reaction medium. NOx production was inhibited by superoxide dismutase (100 U/ml), Fig. 1, giving clear evidence that superoxide ion was really involved in the decomposition. The inability of SOD entirely to inhibit NOx production is probably because of instant but large superoxide production which exceeded SOD capacity. NOx production increased linearly with hydroxylamine (up to 200 PM) and OH-L-Arg (up to 100 PM) concentrations, independently of the concentration of added potassium superoxide or the additional presence of SOD (Fig. 1). Thus, hydroxylamine or OH-L-Arg concentration was the limiting factor under these conditions. The amount of NOx produced by the reaction remained smaller than OH-L-Arg or hydroxylamine used, even in the presence of excess superoxide ion (linear part of the curve). This may be due to the loss of NOx precursor (probably NO) in the atmosphere, or to the formation of an adduct of NO with OH-L-Arg (Hecker et al., 1995a) or hydroxylamine (Bonner et al., 1978). Alternatively, products other than NOx might be formed during the reaction. It should also be noted that hydroxylamine gave about four times less NOx than OH-L-Arg at equimolar concentrations. This indicates that OH-L-Arg is more sensitive to superoxide attack.

et al. (1995), we speculate that the remaining

(for metHb reduction non consumed) dithionite, as a strong reducing compound, protects NO against immediate oxidation by 0, + H,Oz mixture. This mixture rises from the reaction of potassium superoxide with water (Weltzien, 1866), which runs parallel to the reaction between potassium superoxide and OH-L-Arg or hydroxylamine. On the contrary, metHb, a compound known to be unable to trap NO, did not influence NOx production. As a control, we verified that CO-Hb (20 PM) did not interfere with either nitrite or nitrate determination. In zymosan-activated macrophages OH-L-Arg and hydroxylamine cause the accumulation of nitrite in the incubation medium and an increase of cGMP content

The incubation of the macrophage RAW 264 cell line with zymosan A and either hydroxylamine or OH-L-Arg resulted in a significant production of nitrite (Fig. 2). Nitrate was not produced. There was an increase in intracellular cGMP levels (Fig. 3). Zymosan A is known to stimulate superoxide production but not NOS expression (Assreuy et al., 1994). Nitrite production returned to a basal level when superoxide dismutase (100 U/ml) or diphenyliodonium chloride (DPI, 30 PM) was added to the cultivation medium. Diphenyliodonium chloride is an analogue of diphenyleneiodonium [which was found to be

NO is apparently an intermediate in the decomposition of both hydroxylamine and OH-L Arg to NOx

To verify that nitric oxide was a precursor of nitrite and nitrate, we used CO-Hb (20 PM), a known trapper of nitric oxide. This compound suppressed NOx production from both compounds below the detection limit in the whole concentration range. Also, the EPR spectrum proved the production of a NO-haemoglobin complex (not shown), but only when an excess of dithionite was used to prepare Hb. When an equimolar amount (compared with haem groups) of dithionite was used to reduce metHb toward Hb, no NO-haemoglobin signal appeared. According to the findings of Pufahl

0

ZYM ZYM+L-Lys ZYM+DPI ZYM + L-NAME ZYM + SOD Control

Fig. 2. The effect

of L-NAME (300 FM), L-Lys (3 mM), superoxide dismutase (SOD, 100 U/ml) or DPI (30 PM) in combination with zymosan A (50 pg/ml) o n n i t r i t e production from OH-L-Arg (300 PM, hatched columns) and hydroxylamine (300 PM, cross-hatched columns) by macrophage RAW 264 cell culture. Control means cells without activation by zymosan A. Nitrite production is expressed per IO6 cells/ml. Symbols are means + SEM (vertical bars), n = 5.

Possible mechanism of nitric oxide production

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non-specific inhibitor of several nucleotide-re2B or 3A, respectively (as verified by SDS quiring flavoproteins (Holland et al., 1973) electrophoresis), produced no detectable including NADPH-oxidase (Cross and Jones, amount (i.e. less than 0.05 nmol) of OH-L-Arg. 1986, Hancock and Jones, 1987) with similar As we did not find any L-citrulline as a product potency (Stuehr et al., 1991b). Neither inhibitor of this reaction and as L-citrulline was not of NO-synthase L-NAME (1 mM) nor L-Lys recyclysed back to L-arginine by the microsomes [3 mM; inhibitor of OH-L-Arg entry into the used, we exclude the possibility that the cells (Schott et al., 1994, Chenais et al., 1993)] theoretically produced OH-L-Arg was immediaffected nitrite production. As increased cyclic ately cleaved to L-citrulline. We therefore GMP levels in cells are generally accepted as a conclude that none of the tested inducers of mark for the involvment of NO, these results P450 isoforms are able to stimulate de nova indicate that nitrite production apparently synthesis of such an isoform of P450 which involved NO as a precursor. Further, they could metabolize L-arginine into OH-L-Arg. suggest that nitrite production was induced by superoxide (possibly produced by NADPH-oxiDISCUSSION dase) but not by NOS and that the reaction between superoxide and OH-L-Arg resulting in The production of NO from L-arginine, with nitrite production took place in the extracellular the formation of OH-L-Arg as an intermediate, space. is a well documented reaction which is catalysed As in the experiments with chemically by NO-synthases (Stuehr et al., 1991a). NOgenerated superoxide ion, NOx production synthases recognize L-arginine with a very high from L-arginine (300 PM) was not found under specificity and they are the only enzymes which these conditions. cGMP content was not altered are able to oxidize the N-guanidino moiety of (Fig. 3). L-arginine into NO. L-citrulline is the second product of the reaction. Recently, it has been Activated rat liver microsomes are unable to suggested that cytochrome P450 monooxygenases may be able to catalyse at least the second metabolize L-arginine to OH-L-Arg Rat liver microsomes were activated by three step of the reaction, i.e. conversion of OH-L-Arg different cytochrome P450 inducers: ethanol, into NO and L-citrulline (Boucher et al., 1992; phenobarbital and dexamethasone (stimulating Renaud et al., 1993; Schott et al., 1994). induction of isoform 2E1, 2B and 3A, Moreover, Mansuy et al. (1995) proposed that superoxide ion, generated by these monooxyrespectively). Incubation of 10 mM L-arginine with microsomes containing either isoform 2E1, genases, participates in the OH-L-Arg conversion into NO. Indeed, oxidation of compounds containing > C = N(OH) moiety by superoxide in non-aqueous system providing NOx has already been found (Sennequier et al., 1995). =‘ 500 The present findings show that superoxide ion is g able to interact with OH-L-Arg to produce NOx 2 400 and L-citrulline, probably via NO, but they do ,” 0 not show any evidence for the oxidation of 5 300 L-arginine into OH-L-Arg either by superoxide z ion or by cytochromes P450. Thus, NO-syn2 200 8 thases remain the only enzymes which are able g 100 to catalyse OH-L-Arg formation and therefore 0 to trigger the subsequent NO formation. 0 The various pathways which may possibly L-arginine Hydroxylamine OH-L-Arg lead to NO production from OH-L-Arg are illustrated in Fig. 4. It has previously been Fig. 3. Intracellular cGMP level in macrophages RAW 264 exposed to zymosan A (50 pg/ml) in the presence of either speculated that OH-L-Arg might be hydrolysed L-arginine (300 ,uM, shadow columns), OH-L-Arg (300 PM, into hydroxylamine and L-citrulline (DeMaster hatched columns) or hydroxylamine (300 PM, crosshatched columns). Data are means k SEM (n = 10) and et al., 1989). The results reported above show that this reaction occurs non-enzymatically at are expressed as per cent of controls measured in high pH ( 2 9). The enzyme(s) which would be non-activated macrophages. Significance was determined by Student’s r-test for independent means. *P < 0.01. able to catalyse this hydrolysis in tissues are

Petr Vetrovsky et al.

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[NO] ---) Nitrite ‘Oiattack i/

Hydroxylamine Catalase

Hydrolysis

\

/

L-arginine

NOS NADPH,

0,

/ * NC-hydroxy-L-arginine

NOS

NO

* NO - N i t r i t e ’

2

ygy\ 2

[NO] - Nitrite Fig. 4. Scheme showing classical L-arginine/NO pathway with proposed alternative pathways for NO biosynthesis from OH-L-Arg including hydrolysis of OH-L-Arg as well as the attack by superoxide ion.

unknown. Arginase can not be involved as it is strongly inhibited by OH-L-Arg (Boucher et al., 1994, Daghigh et al., 1994) and would not produce hydroxylamine anyway. The next step, enzymic production of NO from hydroxylamine by catalase, was reported many years ago (Nicholls, 1964, Craven et al., 1979). We show here that non-enzymic attack of hydroxylamine by superoxide ion can obviously also account for the production of NO via hydroxylamine. The results reported above clearly show that not only hydroxylamine but also OH-L-Arg can interact with superoxide ion to produce nitrite and nitrate, which are stable breakdown products of NO in water. L-citrulline was simultaneously formed from OH-L-Arg. The use of carbonyl-haemoglobin, a known scavenger of NO (Martin et al., 1985, Ignarro et al., 1987) supports the view that NO was produced as an intermediate in this reaction although the EPR spectrum of NO-haemoglobin was found only in the presence of dithionite excess. This can be explained by two reactions running simultaneously: NH,OH(or OH- L -Arg) + KO,-+ NO + KOH + H,O(or 2H,O+2KO,+2KOH

L

- citrulline) + 0, + H,O,.

The mixture 0, + H,O, generated in the latter reaction (Weltzien, 1866) thus can immediately oxidize NO toward NOx (Pufahl et al., 1995). Dithionite, a strong reducing compound, can probably protect this NO oxidation. Alternatively, the CO-Hb was quickly oxidized to metHb by superoxide ion and dithionite surplus can prevent this reaction. Futhermore, the inhibitory effect of SOD confirmed the involvement of superoxide ion. The production of hydroxylamine from OH- L-

Arg as an intermediate is not likely under the experimental conditions, as NOx production from hydroxylamine was lower than NOx production from OH-L-Arg. In addition to the attack of OH-L-Arg and hydroxylamine, superoxide ion can react with NO leading to peroxynitrite (Blough and Zafinou, 1985; Ignarro, 1991) and further breakdown products. This may explain the presence of nitrate in the reaction medium, derived from peroxynitrite, in addition to nitrite formed by oxidation of NO in aqueous solution. The observation that NOxs were not the only nitrogen products derived from OH-L-Arg or hydroxylamine (on the basis of consumed substrate and L-citrulline formation) probably corresponds to the complexity of redox reactions of NO (Stamler et uf., 1992). Alternatively, some NO could be linked to hydroxylamine (Bonner et al., 1978) or OH-L-Arg (Hecker et al., 1995a) leading to undetected adducts, or be lost in the atmosphere. Superoxide ion is produced in many tissues, especially by inflammatory cells. Inflammatory stimuli cause induction of iNOS expression in many cells leading to subsequent OH-L-Arg release from cells into the extracellular space (Chenais et al., 1993; Hecker et al., 1995b). Thus, reaction between superoxide ion and OH-L-Arg is quite possible. This mechanism would lead to the formation of NO and derivatives, very likely including peroxynitrite. Our findings with zymosan-activated macrophages (which produce superoxide apparently via NADPH-oxidase) suggest that NO and its breakdown products can indeed be formed from OH-L-Arg by cells producing superoxide ion. Whether this mechanism is protective or harmful for cells is unknown, as there are opposing views on cytotoxicity (Beckman et al..

Possible mechanism of nitric oxide production

1990) or lack of toxic effect (Assreuy et al., 1994) of peroxynitrite. Scavenging of superoxide ion by NO may protect cells against oxidative stress. As a conclusion, we report here that superoxide ion is able to react with OH-L-Arg and hydroxylamine to produce NOx most probably via NO. This non-enzymic NOx formation may explain tissue formation of nitrite from OH-L-Arg, previously reported in vascular smooth muscle cells (Schott et al., 1994). The physiological or pathophysiological significance of this finding is obvious. As mentioned already, both superoxide ion formation and OH-L-Arg release are induced by endotoxin (Hecker et al., 1995b) and other inflammatory stimuli (Chenais et al., 1993). The reaction between these two compounds producing NO can therefore contribute to increased availability of NO for different actions, e.g. bacteria killing. Alternatively, OH-L-Arg and also hydroxylamine can serve as a NO precursor for such cells which do not possess the r.-Arg/NO pathway but generate a large quantity of superoxide via NADPH-oxidase, e.g. human monocytes (Martin and Edwards, 1993). Thus the mechanism described above may be important during endotoxaemia and other inflammatory diseases. Acknowledgements-The

authors are deeply indebted to Drs Jean-Luc Boucher and Daniel Mansuy for valuable discussions before starting this work and to Dr Andrey Kleschyov (visiting scientist of Universite Louis Pasteur de Strasbourg from Russia Medical Academy, Irkutsk) for EPR spectroscopy measurements. REFERENCES

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